p-issn: 1978-3728 e-issn: 2442-9740 volume 54, number 2, june 2021 editorial team of dental journal (majalah kedokteran gigi) sk: 17/un3.1.2/2021 january 4 – december 31, 2021 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: muhammad dimas aditya ari, drg., m.kes (department of prosthodontics, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); beshlina fitri widayanti (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers arlette suzy setiawan (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); irna sufiawati (deparment of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); retno widayati (deparment of orthodontics, faculty of dentistry, universitas indonesia, indonesia); ananto ali alhasyimi (deparment of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); masniari novita (deparment of forensic, faculty of dentistry, universitas jember, indonesia); sianiwati goenharto (vocational faculty, universitas airlangga, indonesia); diah savitri ernawati (deparment of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biologyy, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); maretaningtyas dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); ratri maya sitalaksmi (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); dini setyowati (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); ninuk hariyani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 54, number 2, june 2021 p-issn: 1978-3728 e-issn: 2442-9740 1. the effect of 3% binahong leaf extract gel on the wound healing process of post tooth extraction olivia avriyanti hanafiah, diana sofia hanafiah and rahmi syaflida ..................................... 57–62 2. the effectiveness of the combination of moringa oleifera extract and propolis on porphyromonas gingivalis biofilms compared to 0.7% tetracycline hansen kurniawan, widyastuti and mery esterlita hutapea .................................................... 63–67 3. bone remodeling using a three-dimensional chitosan hydroxyapatite scaffold seeded with hypoxic conditioned human amnion mesenchymal stem cells michael josef kridanto kamadjaja ............................................................................................... 68–73 4. the severity and direction prevalence rate of patients with a mandible deviation compared to cobb’s angle kimberly clarissa oetomo, i gusti aju wahju ardani, thalca hamid and komang agung irianto ............................................................................................................ 74–77 5. effect of formula milk on the roughness and hardness of tooth enamel amaliyah nur irianti, sri kuswandari and al supartinah santoso ........................................... 78–81 6. the antifungal susceptibility of candida albicans isolated from hiv/aids patients sri rezeki, siti aliyah pradono, gus permana subita, yeva rosana, sunnati and basri a. gani ............................................................................................................................. 82–86 7. pamegranate (punica granatum l.) gel extract as an antioxidant on the shear bond strenght of a resin composite post-bleaching application with 40% hydrogen peroxide indes rosmalisa suratno, irfan dwiandhono and ryana budi purnama ................................. 87–91 8. occlusion and occlusal characteristics of the primary dentition in emirati schoolchildren vivek padmanabhan, bayan madan and sundus shahid ........................................................... 92–95 9. facial height proportion based on angle's malocclusion in deutero-malayids aulia rohadatul aisy, avi laviana and gita gayatri ................................................................. 96–101 contents page 10. space maintainer 'y model' as a preventive orthodontic treatment for paediatric patients: a case report laelia dwi anggraini, sunarno, rinaldi budi utomo, dibyo pramono ..................................... 102–107 case report 11. the role of family history as a risk factor for non-syndromic cleft lip and/or palate with multifactorial inheritance agung sosiawan, mala kurniati, coen pramono danudiningrat, dian agustin wahjuningrum and indra mulyawan ........................................................................................... 108–112 review article original articles 161 the correction of occlusal vertical dimension on tooth wear rostiny department of prosthodontic faculty of dentistry airlangga university surabaya indonesia abstract the loss of occlusal vertical dimension which is caused by tooth wear is necessarily treated to regain vertical dimension. correctional therapy should be done as early possible. in this case, simple and relatively low cost therapy was performed. in unserve loss of occlusal vertical dimension, partial removable denture could be used and the improvement of lengthening anterior teeth using composite resin to improve to regain vertical dimensional occlusion. key words: vertical dimension occlusion, tooth wear correspondene: rostiny, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction worn teeth due to tooth wear could be pathologically caused by endogen and exogen factors. tooth wear could be caused by parafunction abnormality and its occurrence is estimated three times faster than physiological tooth wear. 1 the etiology of tooth wear could be classified as mechanical and chemical. mechanical tooth wear are attrition and abrasion, while chemical tooth wear is erotion.2 another factor which has essential role on causing tooth wear is the prolonged posterior teeth loss which are not replaced. thus the patient tends to chew using anterior teeth, which resulting the anterior teeth worn. it makes the patient loss the occlusal vertical dimension. the loss of posterior teeth could generally cause abrasion and attrition in anterior teeth. it makes replacement of mandible to anterior position and the mastication force of anterior teeth become heavier. the anterior teeth might worn and mobile. the loss of occlusal vertical dimension because of physiological factor could be compensated by teeth eruption and continuous growth of alveolar bone.3 severe tooth wear could cause morphological change of occlusal tooth, decrease the vertical dimension, pulp pathology, occlusal disharmony and change the masticatory function. in this condition, more complex therapies are needed such as: endodontics, periodontics, and full coronal converage. to establish the diagnosis, it is necessary to understand the etiology of tooth wear such as: bruxism, diet, gastro esophageal disease, eating disorders, xerostomia, amelogenesis and dentinogenesis imperfecta.4,5 besides the etiology, clinical parameter and classification or type of tooth wear are necessary to be considered. several methods could be applied to determine the occlusal vertical dimension. the first method is niswonger, occlusal vertical dimension could be achieved from vertical jaw dimension in rest position subtracted free way space (2–4 mm).6 the second method is willis, the distance between the pupil of the eye and the rima oris is equal to the distance between nasal base to the point below the chin when the teeth or bite wall contacted.7 the third method is silverman (phonetics): utter the phonetic consisting “s” (e.g. “yess”), the distance from incisal edge to maximum occlusal contact is recorded while saying it. the incisal gap is approximately 2–4 mm identical with freeway space. prolonged loss of occlusal vertical dimension would influence face appearance. the face looks older and in severe condition might cause angular cheilitis. in case of occlusal vertical dimensional loss due to tooth wear, therapy is needed to regain the vertical dimension. the therapies such as: lengthening the crown, orthodontical tooth movement and reposition therapy are efficient and effective to correct dimensional vertical loss. the aim of this study was to reported that loss of occlusal vertical dimension due to tooth wear is necessary to be treated as early as possible by simple and relative cheap therapy. case a 44 year old female patient came to dental clinic of faculty of dentistry airlangga university to have partial denture on upper and lower jaw, to regain masticatory and esthetic function. some caries teeth had been extracted prior the visit and the last extraction was done two years ago. the patient never use denture before. intra oral examination: the loss teeth were: 15, 17, 22, 23, 24, 36, 37, 43, and 47; gangrene radix: 16, 27, 35, 45, 46; caries on: 26, 31, 32; worn teeth 34, 33, 32, 31, 41, 42, 44 (figure 1–4), deep bite anterior relation. dimensional vertical occlusion examination was done by using niswonger and willis method with attention given to the facial appearance.6,7 vertical dimension of occlusion 162 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 161-164 figure 1. panoramic rontgenographic of patient. figure 4. the teeth condition in upper jaw. measurement result was: 63 mm, in rest position was 69 mm. the measurement result showed loss of occlusal vertical dimension. based on the estimation, rest position occlusion was 69–63 mm = 6 mm, then subtracted by freeway space and the result was 6–4 mm = 2 mm. case management vertical occlusal correction was done by increasing the dental crown length 2 mm at 34, 33, 32, 31, 41, 42, and 44 using resin composite (a3) and inserting acrylic partial denture on upper and lowerjaw (figure 5, 6, 8). evaluation was done 2 weeks after correction no complaint of pain in temporo mandibular joint was reported. teeth extraction in 16, 27 35, 45, and 46. rest occlusal preparation on distal 14, mesial 25, 34 and mesial 44. lengthening teeth crown, the material, long life the material and the possibility of color alteration, schedule of visiting and financial were discussed with the patient. for individual tray preparation, the jaw was impressed with stock tray using irreversible hydrocolloid impression material. the impression result was filled with gypsum type 1 to make diagnostic model and used to make individual tray using self cured acrylic. the lower jaw were impressed using individual tray with elastomer impression material. the impression was filled with gypsum type iii and used as working model. the working model was surveyed to decide maximum contour where clamer should be placed. bite wall was made in master model and horizontal jaw relation was done (figure 7). the master model was mounted in semi adjustable articulator, teeth arrangement was performed and further, the patient was recommended to try the denture wax. in this case, the denture construction was partially removable using acrylic. three finger wrought wire in 14 and 24 and two finger wrought wire with rest mesial in 34 and 44 after the patient agreed and continued by acrylic process during the trial of partial removable acrylic denture, occlusal correction was done using articulating paper to perform adjustment to achieve stable occlusion, then the removable denture was inserted. discussion the patient did not realize that tooth wear without therapy could develop to be mandibular disturbance and occlusion, there fore early therapy is necessarily done to keep the rest of the teeth has long as possible in oral cavity. correction with the increasing of teeth occlusal to regain occlusal vertical dimension could result less stable occlusion. it is not recommended to change the figure 2. the teeth in occlusion stage in initial examination. figure 3. the teeth condition in lower jaw. 163rostiny: the correction of occlusal vertical dimension on tooth wear figure 6. occlusion of teeth in lower and upper jaw after restoration. figure 7. mounting master model and teeth arrangement in semi adjustable articulator. figure 8. insertion of the denture.figure 5. the teeth in lower jaw after restoration was made. patient’s pattern of dynamic occlusion. it is advisable that the addition of occlusion is about 1–1.5 mm during the restoration to improve occlusal vertical dimension, if it is higher resulting in unstable occlusion. to obtain the association between proper occlusion and the stability of partial removable denture, occlusion contact should occur simultaneously with working contact of natural teeth so stress/load would be spread equally on the whole surface.10 this condition could be obtained by doing occlusal adjustment on the patient. if the correction of occlusal vertical done periodically. in this case, plate could be used to increase the occlusal vertical dimension. basically, the correction of occlusal vertical dimension should be done according to the estimation of occlusal vertical dimension loss is not accurate. the increasing of occlusal vertical dimension would possibly occur. excessive increase of occlusal vertical dimension could result freeway space loss, inflammation of tissue under the removable denture, muscle pain, resorption in residual alveolar bone, horse sound, p, b, m sound would be disturbed because the lips are difficult to close, the increase symptom of temporo mandibular joint syndrome due to the load on the tissue associated with the joint. 11 in the case of occlusal vertical dimensional correction, is advisable to be done using adjustable articulator or semi adjustable. this type of articulator has advantage such as: the distance between the condile of lower jaw and upper jaw could be moved with the same distance in articulator. by using articulator it could be determined anatomical artificial teeth, the inclination of occlusion plane and lateral movement in which it is almost the same with the patient could be determined. if adjust is needed, there would be small mistake found or even no mistake at all when trying denture. occlusal vertical dimension correction with the increase of occlusal needs examination, evaluation and periodical occlusal adjustment until stable occlusion is achieved are necessary. removable denture could be used to restore and to improve occlusal functional occlusal comfort, improve aesthetic, stabilize and relieve tmj syndrome12 and improve occlusal damage due to tooth wear.8,13 164 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 161-164 as conclusion partial removable denture could be used in mild occlusal vertical dimensional loss and the improvement the height of incisal anterior teeth to regain dimension occlusion vertical. references 1. xhonga fa. bruxism and its effect on the teeth. j oral rehabil 1997; 4:65–67. 2. glossary of prosthodontic terms. j prosthet dent 1999; 81: 39–110. 3. verret rg. analyzing the etiology of an extremely worn dentition. j prosthodont 2001; 10:224–33. 4. smith bg, bartlett dw, robb ng. a comparison of patterns on tooth wear with aetiological factors. j prosthet dent 1997; 78:367–72. 5. ganddini mr, mardini m, graser gn, almog d. maxillary and mandibular overlay removable partial dentures for the restoration of worn teeth. j prosthet dent 2004; 91:210–4. 6. hickey jc, zarb ga, bolender cl. boucher’s prosthodontic treatment of edentulous patients. 10th ed. st louis: the cv mosby co; 1990. p. 237–76. 7. sharry jj. complete denture prosthodontics. 3rd ed. new york: mcgraw-hill book co; 1974. p. 211–3. 8. dawson pe. evaluation, diagnosis and treatment of occlusal problems. saint louis: the cv mosby co; 1974. p. 275–85. 9. turner ka, missirlian dm. restoration of the extremely worn dentition. j prosthet dent 1984; 52:467-74. 10. haryanto ag. buku ajar ilmu geligi tiruan sebagian lepasan. jilid i. jakarta: hipokrates; 1991. p. 52–55. 11. grant aa, johnson w. removable denture prosthodontics. 2nd ed. edinburgh, london, madrid, melbourne, new york, tokyo: churchill livingstone; 1992. p. 71–7. 12. pudjirochani e. over closure dan permasalahannya di bidang prostodonsia. majalah kedokteran gigi (dental journal) 2001 october–desember; 34(4):161–63. 13. ash mm, ramfjord s. occlusion. 4th ed. philadelphia: wb saunders co; 1995. p. 1–75. � a comparison of three dimensional change in maxillary complete dentures between conventional heat polymerizing and microwave polymerizing techniques shinsuke sadamori,* toshiya ishii,* taizo hamada* and arifzan razak** * department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, hiroshima, japan ** department of prosthodontics, faculty of dentistry, airlangga university, surabaya, indonesia abstract the purpose of this study was to measure and compare two different polymerizing processes, heat polymerizing (hp) and microwave polymerizing (mp), on the three dimensional changes in the fitting surface and artificial teeth of maxillary complete dentures. a threedimensional coordinate measurement system was used to record distortion of the specimens. the distortion of the fitting surface was measured from the reference plane on the fitting side from which a coordinate system was set, and the movement of the artificial teeth and the distortion of the polished surface was measured from the reference plane of the artificial tooth side, from which a coordinate system was set. it was clearly showed that various distortions of denture specimens after polymerization process can be measured with this three-coordinate measuring machine. the study showed that the overall distortion of the fitting surface in hp specimens was shown to be larger than in mp ones. key words: maxillary complete denture, three dimensional change, microwave polymerizing correspondence: dr shinsuke sadamori, department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, 1-2-3 kasumi, minami-ku, hiroshima city, 734-8553, japan, tel: +81-82-257-5681, fax: +81-82-257-5684, e-mail:tsada@hiroshima-u.ac.jp introduction it is now widely recognized that natural teeth can be retained for life. the number of people retaining their natural teeth is growing, and the absolute number of persons over the age of 65 is increasing rapidly. however, the actual number of patients requiring complete dentures remains almost constant.1 with the increasing percentage of population over the age of 65, the number of individuals with dementia will also grow. it constitutes a serious problem. however, a two-year follow-up study in a dementia ward in a mental hospital suggested that denture wearing has an effect on some activities of daily life.2 the dimensional accuracy of heat-cured denture resin was evaluated by comparing the distance between reference points in the deflasked specimen and the master model after the deflasked specimen was adapted to the master model,3–5 or by comparing the distance between marked points in the deflasked specimen and the master model.6–8 other studies examined changes in the position of teeth in complete dentures.9,10 several papers have investigated the accuracy of three-dimensional coordinate measurement on the mucosal surface of dentures.11–14 in this study, the three dimensional change in the fitting surface and artificial teeth of maxillary complete dentures was measured and compared for two different polymerizing processes. materials and methods specimen preparation to fabricate the working cast, a die of an edentulous maxillary arch (402u, nisshin, kyoto, japan) was duplicated using silicone elastomer (gc, tokyo, japan). a master cast was made of dental stone (new fujirock, gc, tokyo, japan) according to the manufacturer’s recommendation. the measuring points on the master cast consisted of 12 points on the alveolar ridge related to each second molar and first premolar tooth and the midway point of the incisor teeth, and on the denture flange in the molar, premolar and anterior regions. in addition, we set three datum-points (reference points) to set the coordinate system in the palate. the measuring points were set with steel balls with a diameter of 2 mm, and the datum-points were steel balls with a diameter of 4 mm set on the model using self-curing resin. the master cast was duplicated and six master casts were made. a wax denture was made on one of the six working casts. one thickness (approximately 1.5 mm) of base plate wax (paraffin wax, gc, tokyo, japan) was adapted to the working cast and the artificial teeth (gc duradent: anterior teeth c3, posterior teeth 30m, gc, tokyo, japan) were positioned in the usual manner. the core of the wax denture was made with dental stone and silicon impression material. after the working casts were measured, three steel balls 4 mm in diameter were 7sadamori: a comparison of three dimensional change set in the appointed positions as datum-points, and the wax dentures were made from the core. the measurement points were prepared on mesiolingual cusps of the right and left second molar tooth, the lingual cusps of the right and left first premolar tooth, and the mesial edge of the right incisor with a diamond point 1.8 mm in diameter (diamond point fg regular 340, shofu, kyoto, japan). polymerizing process two acrylic resins were used: bio resin (shofu, kyoto, japan) and acron mc (gc, tokyo, japan) (table 1). the bio resin was mixed using 4.5 ml liquid to 10 g powder. the polymerizing process followed japan industrial standard’s (jis) recommendation: an initial 90 minutes at 70 °c followed by 30 minutes at 100 °c (hp). the acron mc was mixed using 4.3 ml liquid to 10 g powder. specimens (mp) were processed for 3 minutes in a 500 w microwave oven (em-m 535 t, sanyo electric, osaka, japan). ten standardized denture specimens were fabricated: five using the conventional technique and five using the microwave technique. after polymerization, flasks were allowed to cool at room temperature for over 12 hours and deflasked. measuring method dimensional change was measured using a threecoordinate measuring machine (tristation, tst 600-fc, nikon corp., tokyo, japan) graduated to an accuracy of 0.5 m or less at 20 °c with a ball stylus measuring 0.5 mm in diameter (figure 1). a device made of plaster was put on the surveyor to provide fixation for the denture specimen when measuring by probe. two types of fixation devices were prepared to allow measurements to be taken on the fitting surface and the occlusal side. the measurements were performed on working casts, wax dentures, and dentures after deflasking. the probe interfaces with the computer to measure and record point locations in the x-, y-, and z-axes. such figure 1. the measuring machine used in this study (tristation). processing method brand polymerizing cycle processing technique powder-to-liquid ratio (g/ml) manufacturer heat polymerizing (hp) bio resin 90 min at 70 °c 30 min at 100 °c hot water bath 10/4.5 shofu kyoto, japan microwave polymerizing (mp) acron mc 3 min microwave 500w 10/4.3 gc tokyo, japan tabel 1. materials used in this study figure 3. probing to detect measuring point.figure 2. probing to detect reference point. � dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 6–10 figure 6. dimensional change after polymerization (artificial teeth). the cross point of the axes indicates the center of the three reference points. a digitizing system requires a reference plane to standardize the three-dimensional measurements. in this study, the reference plane (x-y plane) was established as the plane linking the centers of the hemispherical elevations 4 mm in diameter placed at three points on the palate of the denture, on both the polishing side and the fitting side. the center was measured by probing the surface of each reference hemisphere ten times (figure 2). the measurement points consisted of 12 points on the cast and five points on the artificial teeth in the form of hemispherical hollows 2 mm in diameter. each measurement point was measured three times, and the average value was used (figure 3). results measurement accuracy the measurement accuracy of each method was determined by calculating the square root of the standard deviation of each measured value.15 the measurement accuracy on the stone casts was x-axis (5.6 m), y-axis (2.9 m) and z-axis (4.7 m). on artificial teeth, the measurement accuracy was x-axis (5.6 m), y-axis (7 m) and z-axis (7.5 m). the official measurement accuracy of the three-dimensional coordinate measuring machine was 5 m, so the above accuracies were considered to be adequate. dimesional changes figure 4, 5, and 6 show the dimensional changes after each polymerizing process for both the fitting surface and the polishing surface. the cross point of the x and y axes of the reference plane is the center of the three reference points, and this point is the reference point for measurement in this study. the distortion of the fitting surface of the alveolar ridge in both hp and mp occurred in a horizontal direction towards the center of the palate. the fitting surface of the alveolar ridge of the posterior section of mp moved slightly towards the alveolar mucosa compared to hp. the amount of distortion at the fitting surface of the alveolar ridge was similar in both polymerizing methods (figure 7). the distortion of flanges in both polymerizing hp hp mp mp lateral view posterior view figure 5. dimensional change after polymerization. the cross point of the axes indicates the center of the three reference points. figure 4. dimensional change after polymerization. the cross point of the axes indicates the center of the three reference points. arrows indicate the direction of the change. hp occlusal view fitting surface view reference point lateral view posterial view hp mp mp hp 10 mm �sadamori: a comparison of three dimensional change methods involved horizontal movement towards the center of the palate. the center and posterior border of the palate in both polymerizing methods were displaced in the original direction, and the palate center in hp was distorted to a greater degree than in mp (figure 7). the artificial teeth moved slightly backwards from the origin in a horizontal direction in specimens polymerized by both methods; however, the artificial teeth polymerized by mp tended to move more than those polymerized by hp (figure7). the dimensional changes in the lateral and posterior views are shown in figure 5 and 6. in the vertical direction, the anterior flange of the hp denture moved slightly, but the distortion of the flange was larger in the premolar region, and larger again in the molar region. in the mp denture, the vertical distortion was similar over the entire surface of the denture, and there was less distortion than in the hp denture (figure 7). discussion we revealed the influence of thickness on the linear dimensional change of a denture base resin.16 developments in coordinated measuring systems, such as the nikon 6000 (figure 1), which was originally developed for applications in engineering, would appear to have considerable potential in studies on the dimensional accuracy and stability of prosthetic appliances. this system offers the advantages of three dimensional measuring linked with sophisticated computer technology. in this study, denture specimens were not polished because the generation of excessive heat during finishing and polishing with burs and arbor bands may have contributed to stress release.17–20 a three-dimensional coordinate measurement system that situates a coordinate system on the denture specimens was used to record distortion of the specimens in this study. the coordinate system used a hemisphere form of datumpoint. the distortion of the fitting surface was measured from the reference plane on the fitting side from which a coordinate system was set, and the movement of the artificial teeth and the distortion of the polishing surface were measured from the reference plane of the artificial tooth side, from which a coordinate system was set. the coordinate system of the fitting surface and the artificial teeth and polishing surface is not the same because of the measuring error created by the 4 mm standard balls. however, it is considered acceptable for comparison of the measured values between both surfaces. the vertical movement of the artificial teeth was measured using the standard plane set by the polishing surface. takahashi11 recorded the distortion in the vector of the fitting surface of specimens made by the similar model used in this study. this study used a different measurement method, but there was little difference between them in the measurements on the fitting surface. some studies found that the distortion of the fitting surface and the movement of the artificial teeth were indicated by the vector. our study found that the overall distortion of the denture occurred as a displacement into the center of the denture after polymerization. the fitting surface in the hp denture showed more vertical displacement after polymerization than the mp denture and this tendency was most sifnificant in the flange. in the hp denture, the heat is conducted from the surface to the center of the flask, so this process might be the cause of the vertical distortion of the flange in denture specimens. the amount of movement of the fitting surface in the mp denture was smaller than that in the hp figure 7. amount of distortion. �0 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 6–10 denture, especially in the flange. these results are consistent with several reports.21,22 nelson et al.23 also found that the amount of movement of artificial teeth in mp specimens tended to be greater than in hp specimens. our results might be similar to their results. this may be due to the difference of thermal conductivity of both polymerizing process because microwave energy is independent of thermal conductivity. this study clearly showed that various distortions of denture specimens after polymerization can be measured with a three-coordinate measuring machine. the overall distortion of the fitting surface in hp specimens was shown to be larger than in mp specimens. a more complete understanding of the distortion caused by different curing methods might improve the outcome for patients requiring complete dentures by shortening the adjustment period. acknowledgement this study was supported in part by a grant-in-aid for scientific research (16390617) from japan society for the promotion of science (jsps). references 1. budtz-jorgensen e. treatment of edentulous patients. in: prosthodontics for the elderly: diagnosis and treatment. chicago, il: quintessence; 1999, p. 203–28. 2. sadamori s, hamada t, nakai n, nishimura m. influence of denture wearing on the stage of dementia and adl of the elderly with severe dementia a two-year follow-up study in a dementia ward in a mental hospital. dentistry in japan 2004; 40:163–7. 3. kraut ra. a comparison of denture base accuracy. j am dent assoc 1971; 83:352–7. 4. laughlin ga, eric jd, glaros ag, young l, moore dj. a comparison of palatal adaptation in acrylic resin denture bases using conventional and anchored polymerization techniques. j prosthodont 2001; 10:204–11. 5. consani rl, domitti ss, rizzatti barbosa cm, consani s. effect of commercial acrylic resins on dimensional accuracy of the maxillary denture base. brazilian dent j 2002; 13:57–60. 6. huggett r, brooks sc, bates jf. the effect of different curing cycles on the dimensional accuracy of acrylic resin denture base materials. quint dent tech 1984; 8:81–5. 7. jagger rg. dimensional accuracy of thermoformed poly-methyl methacrylate. j prosthet dent 1996; 76:573–5. 8. wong dm, cheng ly, chow tw, clark rk. effect of processing method on the dimensional accuracy and water sorption of acrylic resin dentures. j prosthet dent 1999; 81:300–4. 9. mainieri et, boone me, potter rh. tooth movement and dimensional change of denture base materials using two investment methods. j prosthet dent 1980; 44:368–73. 10. garfunkel e. evaluation of dimensional changes in complete dentures processed by injection-pressing and the pack and press technique. j prosthet dent 1983; 50:757–61. 11. takahashi y. three dimensional changes of the denture base of the complete denture following polymerization. j jpn prosthodont 1990; 34:136–48. 12. turck md, lang br, wilcox de, meiers jc. direct measurement of dimensional accuracy with three denture-processing techniques. int j prosthodont 1992; 5:367–72. 13. jackson ad, lang br, wang rf. the influence of teeth on denture base processing accuracy. int j prosthodont 1993; 6:333–40. 14. nogueira s, ogle r, davis el. comparison of accuracy between compressionand injection-molded complete dentures. j prosthet dent 1999; 82:291–300. 15 takahashi y, takeuchi t, sawamura n, inanaga a, habu t. improved denture measuring method using the three dimensional measurement system. j jpn prosthodont 1988; 32: 1358–1362. 16. sadamori s, ishii t, hamada t. influence of thickness on the linear dimensional change, warpage, and water uptake of a denture base resin. int j prosthodont 1997; 10:35–43. 17. woelfel jb, paffenbarger gc. dimensional changes occurring in artificial dentures. int dent j 1959; 9:451–60. 18. woelfel jb, paffenbarger gc, sweeney wt. dimensional changes occurring in dentures during processing. j am dent assoc 1960; 61:413–30. 19. woelfel jb, paffenbarger gc, sweeney wt. clinical evaluation of complete dentures made of eleven different types of denture base materials. j am dent assoc 1965; 70:1170–88. 20. lorton l, phillips rw. heat-released stress in acrylic dentures. j prosthet dent 1979; 42:23–6. 21. takamata t, setcos jc, phillips rw, boone me. adaptation of acrylic resin dentures as influenced by the activation mode of polymerization. j am dent assoc 1989; 119:271–6. 22. wallance pw, graser gn, myers ml, proskin hm. dimensional accuracy of denture resin cured by microwave energy. j prosthet dent 1991; 66:403–9. 23. nelson mw, kotwal kr, sevedge sr. changes in vertical dimension of occlusion in conventional and microwave processing of complete dentures. j prosthet dent 1991; 65:306–8. 211 volume 47, number 4, december 2014 daya antibakteri obat kumur chlorhexidine, povidone iodine, fluoride suplementasi zinc terhadap, streptococcus mutans dan porphyromonas gingivalis (antibacterial effect of mouth washes containing chlorhexidine, povidone iodine, fluoride plus zinc on streptococcus mutans and porphyromonas gingivalis) betadion rizki sinaredi, seno pradopo, dan teguh budi wibowo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: dental caries and periodontal disease prevalence in indonesian children are still high. some efforts can be done to overcome the problem; one of them is the use of mouthwash to decrease pathogen microorganisms. the mouthwashes that commercially available in market are chlorhexidine, povidone iodine and fluoride with zinc supplementation. purpose: the purpose of this study was to examine the anti bacterial effect of the mouthwashes chlorhexidine, povidone iodine and fluoride with zinc supplementation against mix bacteria that found in the plaque, streptococcus mutans and porphyromonas gingivalis. methods: the antibacterial effect was measured using disk diffusion test. the bacteria samples (plaque polybacteria, s.mutans and p. gingivalis) were inoculated and spread in the petridish containing mha. paper discs containing the mouthwashes were placed in the petridish and incubated for 24 hours at 37oc (anaerobe for p. gingivalis, aerobe for s. mutans and polybacteria). the diameter of inhibition zone surrounding the paper discs were measured and compared between each active ingredient contained in mouthwash. results: chlorhexidine had the strongest antibacterial effect than povidone iodine and fluoride. chlorhexidine was more effective to inhibited the growth of s. mutans than to polybacteria or p.gingivalis, while povidone iodine and fluoride were more effective to inhibited the growth of polybacteria. conclusion: the mouthwash chlorhexidine was more effective to inhibit the growth of plaque polybacteria, streptoccous mutans and porphyromonas gingivalis compared with povidone iodine and fluoride with zinc supplementation. key words: mouthwash, chlorhexidine, fluoride, povidone iodine, streptococcus mutans, porphyromonas gingivalis abstrak latar belakang: prevalensi karies gigi dan penyakit periodontal masih tinggi pada anak indonesia. usaha mengatasi hal tersebut antara lain melalui melalui penggunaan obat kumur untuk mengurangi jumlah kuman pathogen. kandungan obat kumur yang beredar di pasar diantaranya adalah chlorhexidine, povidone iodine dan fluoride dengan suplementasi zinc. tujuan: penelitian ini bertujuan untuk meneliti efek antibakteri dari obat kumur berbahan aktif chlorhexidine, povidone iodine dan fluoride dengan suplementasi zinc terhadap bakteri campur plak, s. mutans dan p. gingivalis. metode: pengukuran efek antibakteri dilakukan dengan metode disk diffusion. bakteri sampel (bakteri campur plak, streptococcus mutans dan porphyromonas gingivalis) ditanam secara merata pada cawan petri dengan medium mha. cakram kertas yang mengandung obat kumur diletakkan di tengah cawan petri dan diinkubasi selama 24 jam pada suhu 37o c (anaerob untuk p. gingivalis, aerob untuk s.mutans dan bakteri campur). diameter zona hambat bakteri yang mengelilingi cakram kertas diukur dan dibandingkan antara masing-masing bahan aktif yang terkandung dalam obat research report 212 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 211–214 kumur. hasil: chlorhexidine mempunyai efek antibakteri paling kuat dibanding povidone iodine dan fluoride. chlorhexidine lebih ampuh menghambat pertumbuhan bakteri s.mutans dibanding terhadap bakteri p.gingivalis dan bakteri campur dalam plak, sedang povidone iodine dan fluoride lebih efektif menghambat pertumbuhan bakteri campur. simpulan: obat kumur chlorhexidine lebih efektif dalam menghambat pertumbuhan bakteri campur dari plak, streptococcus mutans dan porphyromonas gingivalis dibanding povidone iodine dan fluoride dengan suplementasi zinc. kata kunci: obat kumur, chlorhexidine, fluoride, povidone iodine, streptococcus mutans, porphyromonas gingivalis korespondensi (correspondence): betadion rizki sinaredi, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: betadion@yahoo.com pendahuluan masalah kesehatan rongga mulut pada anak di indonesia masih merupakan suatu masalah yang belum teratasi dengan baik. hasil riset kesehatan dasar (rikesdas) tahun 2007 yang diselenggarakan departemen kesehatan, prevalensi nasional karies aktif adalah 43,4%. berdasarkan skrt 2004, tingginya prevalensi karies di indonesia mencapai 90,05%. berbagai metode telah diterapkan untuk mengatasi masalah terjadinya penyakit di rongga mulut, diantaranya adalah dengan penggunaan obat kumur. penyakit pada rongga mulut terjadi akibat adanya akumulasinya bakteri, termasuk diantaranya adalah bakteri penyebab karies gigi (streptococcus mutans) dan penyebab penyakit periodontal (p. gingivalis).1 bahan anti mikroba yang biasa digunakan dalam obat kumur adalah chlorhexidine, fluoride, dan povidone iodine. beberapa penelitian telah dilakukan terhadap bahan anti bakteri ini, diantaranya oleh baker,2 mengenai penggunaan obat kumur sebagai tindakan prefentif dan kuratif terhadap penyakit periodontal. chlorhexidine dipercaya sebagai obat kumur yang mampu mengurangi pembentukan plak, menghambat pertumbuhan plak dan mencegah terjadinya penyakit periodontal.1 hal ini dikarenakan sifat dari chlorhexidine sendiri, yaitu bakterisid dan bakteriostatik terhadap berbagai macam bakteri, termasuk bakteri yang berada di dalam plak. fluoride dan zinc memiliki karakteristik mampu bekerja dengan cara menghambat metabolisme bakteri plak yang dapat menyebabkan kematian bakteri pada plak, sedangkan povidone iodine memiliki kemampuan sebagai bahan bakterisidal maupun fungisidal.3 penelitian ini bertujuan meneliti efektifitas ketiga bahan antimikroba yang terdapat didalam obat kumur di pasaran, yaitu chlorhexidine, fluoride dengan suplementasi zinc, dan povidone iodine dalam menghambat pertumbuhan bakteri campur pada plak, s. mutans dan p. gingivalis. bahan dan metode penelitian ini adalah penelitian eksperimental laboratoris (in vitro) dengan desain post test only. subyek penelitian adalah enam pasien anak usia 7-12 tahun, dengan kondisi umum baik dan terdapat plak pada permukaan geliginya. subyek tidak sedang mengkonsumsi antibiotik maupun menggunakan zat antimikroba lainnya dalam jangka waktu dua minggu sebelum dilakukan pengambilan plak. bakteri campur plak didapat dengan tehnik swab pada daerah yang terdapat plak kemudian dimasukkan ke dalam tabung reaksi yang telah berisi brain heart infusion broth (bhib), selanjutnya diinkubasi selama 24 jam pada suhu 37o c. s. mutans didapat dari sediaan bakteri s. mutans yang telah diisolasi sebelumnya dari pasien, ditanam dalam media tyc, kemudian diinkubasi selama 48 jam dalam suasana anaerob dengan suhu 37oc. p. gingivalis didapat dari sediaan bakteri p.gingivalis yang telah tersedia (atcc no.33277) dimasukkan ke dalam tabung reaksi berisi bhib. diinkubasi selama 48 jam dalam suasana anaerob dengan suhu 37o c. semua bakteri diusap secara merata pada media mha dengan osse bulat pada cawan petri. dasar cawan petri dibalik dan dibagi menjadi tiga bagian, untuk menentukan batas daerah tiap perlakuan pada media. tiga kertas saring berbentuk cakram dengan diameter 4 mm masing-masing diberi larutan zat antimikroba chlorhexidine 0,2%, fluoride 0,2%, povidone iodine1% dengan cara diteteskan sebanyak 0,5 ml. kemudian diletakkan dalam media mueller hinton agar (mha) dan diinkubasi selama 24 jam pada suhu 37o c. daya antibakteri dilihat dari zona hambat pertumbuhan yang terjadi pada semua sampel. pengukuran zona hambat dilakukan dengan mengukur diameter zona hambat yang terjadi menggunakan jangka sorong. dilakukan sebanyak tiga kali untuk masing–masing sampel kemudian diambil rata-ratanya. hasil hasil penelitian menunjukkan bahwa chlorhexidine mempunyai efek antibakteri paling kuat dibanding povidone iodine dan fluoride. chlorhexidine lebih ampuh menghambat pertumbuhan bakteri s.mutans dibanding terhadap bakteri p.gingivalis dan bakteri campur dalam plak. povidone iodine dan fluoride lebih efektif menghambat pertumbuhan bakteri campur (tabel 1). pada tabel 2 terlihat bahwa hampir seluruh perbandingan didapatkan nilai signifikansi p<0.05; hal tersebut menjelaskan bahwa terdapat perbedaan yang signifikan pada perbandingan antar masing-masing kelompok perlakuan, sedangkan pada perbandingan antara 213sinaredi, et al.: daya antibakteri obat kumur chlorhexidine, povidone iodine, fluoride povidone iodine dengan fluoride dengan suplementasi zinc terhadap s. mutans, tidak didapatkan perbedaan yang signifikan (p > 0,05). pembahasan pada kelompok bakteri s. mutans, chlorhexidine memiliki rerata diameter zona hambat lebih besar daripada povidone iodine serta fluoride dengan suplementasi zinc. perbedaan penurunan jumlah koloni s. mutans dapat terjadi pada percobaan yang dilakukan secara in vivo (saliva dalam rongga mulut) dengan in vitro. perbedaan tersebut dapat diakibatkan oleh beberapa faktor, diantara faktor yang berpengaruh adalah kondisi saliva dan jumlah bakteri awal. saliva memiliki berbagai macam komponen yang berfungsi untuk pertahanan tubuh melawan infeksi mikroorganisme, diantaranya adalah berbagai macam protein seperti lyzozyme,bactericidal/ permeability increasing protein (bpi), peroxidise, iga serta igg yang berbeda konsentrasinya pada tiap individu.4 kadar keasaman (ph) saliva juga berpengaruh terhadap efektifitas obat kumur, saliva yang bersifat asam (ph rendah) akan mengurangi efektifitas chlorhexidine,5 sedangkan fluoride tidak terganggu efektifitasnya pada ph saliva yang rendah. jumlah bakteri akan berpengaruh terhadap daya kerja povidone iodine, dimana jumlah iodine bebas yang dilepas sebagai bahan aktif bakterisidal akan berkurang efektifitasnya dengan jumlah bakteri awal yang tinggi.6 poviodone iodine memiliki sifat anti bakteri utamanya melalui mekanisme dimana povidone membawa senyawa iodine bebas masuk menembus membran sel. senyawa iodine memiliki sifat yang sitotoksik sehingga mampu membunuh sel bakteri.7 povidone iodine dapat merubah struktur dan fungsi dari protein dan enzim sel dan merusak fungsi sel bakteri dengan jalan menghambat perlekatan hidrogen dan merubah struktur membran sel,8 selain itu juga menghambat terjadinya sintesis protein oleh bakteri melalui proses oksidasi thiol di dalam asam amino sistein.9 salah satu keuntungan povidone iodine adalah mampu menghambat sintesis glucosyltransferase (gtf) dan fructosyltransferase (ftf) oleh s.mutans. gtf dan ftf merupakan enzim ekstraseluler yang mensintesis polisakarida glucans dan fructans yang berperan penting dalam proses perlekatan s. mutans dan pembentukan biofilm pada permukaan gigi.10 mekanisme daya anti bakteri fluoride adalah dengan jalan menghambat kinerja dua sistem enzim dalam proses glikolisis, yaitu enzim enolase dan enzim active protontransport atp-ase. fluoride menghambat pemecahan tabel 1. rerata dan standar deviasi nilai diameter zona hambat masing-masing kelompok penelitian bakteri kelompok rerata (mm) standar deviasi p. gingivalis chlorhexidine 4,0833 0,73598 povidone iodine 1,2500 0,52440 fluoride 0,3333 0,40825 s. mutans chlorhexidine 16,0833 0,58452 povidone iodine 1,0833 0,58452 fluoride 0,7500 0,41833 campur chlorhexidine 12,7500 1,08397 povidone iodine 2,6667 0,51640 fluoride 1,5000 0,89443 tabel 2. nilai signifikansi uji beda antar masing-masing kelompok penelitian pada setiap bakteri uji bakteri kelompok chlorhexidin povidone iodine fluoride p. gingivalis chlorhexidine 0,000* 0,000* povidone iodine 0,014* fluoride s. mutans chlorhexidine 0,000* 0,000* povidone iodine 0,297 fluoride campur chlorhexidine 0,000* 0,000* povidone iodine 0,034* fluoride *p< 0,05 = terdapat perbedaan yang bermakna 214 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 211–214 glukosa menjadi asam piruvat dengan menghambat enzim enolase. enolase merupakan enzim yang paling sensitif terhadap fluoride dalam jalur glikolisis. hal ini kemudian menyebabkan berkurangnya produksi asam piruvat dan atp oleh bakteri. berkurangnya sintesis asam piruvat menyebabkan berkurangnya sintesis asam laktat dan menghambat transport glukosa pada sistem phosphoenolpyruvate-phosphotransferase sehingga sangat menghambat aktifitas metabolisme secara keseluruhan dari bakteri.11 fluoride juga memiliki mekanisme kerja lain, yaitu mempengaruhi terjadinya akumulasi proton pada sel bakteri sehingga mengurangi toleransi bakteri untuk tumbuh dan mengurangi laju metabolismenya pada lingkungan yang bersifat asam.10 akumulasi proton secara intraselular akan mengurangi nilai ambang ph untuk proses katabolisme dan biosintesis enzim oleh sel bakteri.12 zinc bersinergi dengan fluoride, zinc memiliki daya antibakteri dengan mekanisme kerja menghambat proses glikolisis bakteri sehingga zinc secara signifikan mengurangi pertumbuhan bakteri anaerob dan streptococcus.13 chlorhexidine memiliki rerata diameter zona hambat terbesar pada tiga kelompok penelitian (bakteri campur, s. mutans dan p. gingivalis). hal ini berarti daya antibakteri chlorhexidine lebih besar dibandingkan dengan fluoride dengan suplementasi zinc maupun povidone iodine. mekanisme kerja dari chlorhexidine efektif untuk menghambat pertumbuhan maupun membunuh bakteri gram positif dan gram negatif, tergantung dari konsentrasi yang digunakan. molekul chlorhexidine memiliki muatan positif (kation) dan sebagian besar muatan molekul bakteri adalah negatif (anion). hal ini menyebabkan perlekatan yang kuat dari chlorhexidine pada membran sel bakteri. chlorhexidine akan menyebabkan perubahan pada permeabilitas membran sel bakteri sehingga menyebabkan keluarnya sitoplasma sel dan komponen sel dengan berat molekul rendah dari dalam sel menembus membran sel sehingga menyebabkan kematian bakteri. mekanisme ini berbeda dengan fluoride dengan suplementasi zinc yang berfokus pada berkurangnya enzim atp-ase maupun pada povidone iodine yang molekul iodine bebasnya masuk menembus membran sel kemudian membunuh sel bakteri. chlorhexidine lebih efektif terhadap bakteri gram positif (s. mutans) merupakan bakteri gram positif) dibandingkan terhadap bakteri gram negatif (p. gingivalis). hal ini terlihat dari rerata diameter zona hambat bakteri pada kelompok penelitian s. mutans sebesar 16,0833 mm dibandingkan pada kelompok penelitian p. gingivalis sebesar 4,0833 mm. terdapat perbedaan jenis dinding sel pada bakteri gram positif dimana bakteri gram positif tidak memiliki lipopolisakarida sedangkan bakteri gram negatif memiliki lipopolisakarida. lipopolisakarida mampu untuk menahan molekul kationik dari chlorhexidine sehingga membatasi mengurangi efektifitas kerjanya.14 selain itu, membran luar dari bakteri gram negatif, bertindak sebagai penghalang terhadap zat anti bakterial yang bersifat kationik seperti chlorhexidine.15 penelitian ini menunjukkan bahwa chlorhexidine lebih efektif dibanding povidone iodine dan fluoride dengan suplementasi zinc dalam menghambat pertumbuhan bakteri campur dari plak, streptococcus mutans dan porphyromonas gingivalis. daftar pustaka 1. carranza fa, newman, m. carranza’s clinical periodontology. 9th ed. philadelphia: wb. saunders; 2002. 2. baker k. mouthrinses in the prevention and treatment of periodontal disease. curr opin periodontol 1993; 89-96. 3. demir a. effects of clorexidine and povidone iodine mouth rinses on the bond strength of an orthodontic composite. angle orthod j 2005; 75(3): 392-6. 4. fábián tk, hermann p, beck a, fejérdy p, fábián g. salivary defense proteins: their network and role in innate and acquired oral immunity. int j mol sci 2012; 13(4): 4295-320. 5. russel ad. chlorhexidine: anti bacterial action and resistance. infection 1986; 14(5): 212-8. 6. ferguson aw, scott ja, mcgavigan j, elton ra, mclean j, schmidt u, kelkar r, dhillon b. comparison of 5% povidone-iodine solution against 1% povidone-iodine solution in preoperative cataract surgery antisepsis: a prospective randomised double blind study. br j ophthalmology 2003; 87: 163–7. 7. lacey, rw., catto, a. action of povidone-iodine against methicillinsensitive a nd -resista nt cultures of staphylococcus aureus. postgraduate medical journal 1993; 69 : p 78–83. 8. schreier h, erdos g, reimer k. molecular effects of povidoneiodine on relevant micro-organisms: an electron-microscopic and biochemical study. dermatology 1997; 195: 111-6. 9. thornton spann c, taylor sc, weinberg jm. topical antimicrobial agents in dermatology. clin dermatol 2003; 21(1): 70-7. 10. avshalom t, moshe s, uri w, amnon s, doron s. effect of different iodine formulations on the expression and activity of streptococcus mutans glucosyltransferase and fructosyltransferase in biofilm and planktonic environments. j antimicrobial chemotherapy 2006; 57: 865–71. 11. nouri m, titley c. pediatrics: a review of the antibacterial effect of fluoride. 2003. available from: http://www.oralhealthgroup.org/. accessed january20, 2013. 12. ha m ilton i r. g rowth, metabolism a nd acid production by streptococcus mutans. in: hamada s, michalek sm, kiyono h, menaker l, mcghee jr, eds. molecular microbiology and immunobiology of streptococcus mutans. amsterdam: elsevier science publishers; 1986. p. 145-55. 13. sreenivasan pk, furgang d, markowitz k, mckiernan m, tischiobereski d, devizio w, fine d.. clinical anti-microbial efficacy of a new zinc citrate dentifrice. clin oral investig 2009; 13(2): 195–202. 14. cheung hy, wong mm, cheung sh, liang ly, lam yw, chiu sk. differential actions of chlorhexidine on the cell wall of bacillus subtilis and escherichia coli. plos one 2012; 7(5): e36659. 15. nikaido h, vaara m. molecular basis of bacterial outer membrane permeability. microbiological review 1985; 49: 1–32. vol 50 no 4 desember 2017.indd 226 dental journal (majalah kedokteran gigi) 2017 desember; 50(4): 226–229 research report a comparison of the adhesive strength of zinc phosphate and self-adhesive resin cement as fiber post cementation materials setyawan bonifacius, deddy firman, and hasna djiab department of prosthodontics, faculty of dentistry, universitas padjajaran, bandung – indonesia abstract background: the use of fiber post has become commonplace among dental practitioners due to its several advantages. in accordance with the intended use of post which provides retention for coronal restoration, a cement is used that can provide high quality adhesion. conventional resin cement has long been adopted as a cementation material for consumer fiber post. however, allowing attachment fiber post failure due to errors in the cementing procedure leads to complications. purpose: this study aimed to compare the adhesion strength of zinc phosphate cement and self-adhesive resin cement as fiber post cementation material. both consumer cements were easy to use and cheap. methods: the samples used numbered up to 20 and were divided into two groups. group 1 used zinc phosphate cement, while group 2 used self-adhesive resin cement. results: the value of the average adhesion strength of group 1 (zinc phosphate) was 82.65 n, whereas that of group 2 (self-adhesive resin) was 402.81 n. conclusion: this study concluded that the adhesive strength of self-adhesive resin cement as fiber post cementation material was higher than that of zinc phosphate cement. keywords: adhesive strength; zinc phosphate; self-adhesive resin; fiber post correspondence: setyawan bonifacius, department of prosthodontics, faculty of dentistry, universitas padjajaran. jl. sekeloa selatan i bandung 40132, indonesia. e-mail: setyawan.bonifacius@fkg.unpad.ac.id introduction the increasing demand for aesthetic post and core crowns stimulated the development of a non-metallic post core system, especially the use of a translucent fiber post.1 prefabricated posts, especially those made from fiber glass, represent the preferred choice of many dentists. in addition to its relative ease-of-use, these types of post add high aesthetic value, especially when combined with other ceramic restorations. clinical studies confirm the success rate of dental restoration using fiber-reinforced post as ranging from 95% to 99%. frequent cases of failure in this study occurred because the restoration was discontinued as a result of poor retention of the fiber post in the root canals. it is further argued that such retention depends on the adhesive forces between the post material and the resin luting agent, as well as the inherent strength of the bond between the resin luting agent and the root canal wall.2 various types of resin luting cement and associated bonding systems are recommended for the cementation of fiber post. cementing techniques with resin cement require precision and are very complicated for most dental practitioners. the price of resin cement is relatively high when compared with zinc phosphate cement or glass ionomer cement, both of which are often used by dental practitioners. zinc phosphate cement, first developed in the early 1900s, is widely used in dentistry and has enjoyed success as a permanent luting agent becoming the standard for comparison in the future development of cement. burgess and ghumann developed a list of directions for the use of various luting agents, including zinc phosphate cement, which is recommended for permanent cementing of restorations with posts, both cast posts and composite fiber posts.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p226–229 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p226-229 mailto:setyawan.bonifacius@fkg.unpad.ac.id 227227bonifacius, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 226–229 resin cement is divided into two subgroups, depending on the adhesive system used to prepare the tooth before cementation. the first group uses etch-and-rinse adhesive systems (e.g. variolink, variolink ii, ivoclar-vivadent, schaan, lichtenstein, calibra, dentsply caulk, milford, de, usa; nexus, kerr, orange, ca, usa). in the second group, enamel and dentine are prepared using primary self-etching (e.g. panavia 21, panavia f and panavia f 2.0, kuraray medical inc., tokyo, japan; multilink, ivoclarvivadent).4 self-adhesive resin cement was introduced in 2002 as a new subgroup of resin cement (e.g. relyx unicem, 3m espe, st paul, mn, usa) designed to overcome certain deficiencies of conventional cement (zinc phosphate, polycarboxylic and glass ionomers) and resin cement, as well as combine favorable characteristics from various classes of cement in a single product. self-adhesive resin cement is easy to apply which fulfills clinicians’ desire for a straightforward cementation technique. compared to the more complex cement of resin etch-and-rinse and selfetching procedures, self-adhesive resin cement provides a more promising cementation result because the possibility of error during the process is extremely limited or even absent.4 the purpose of this research was to identify differences in the attachment attributes of fibers cemented with zinc phosphate cement and those using self-adhesive resin cement. materials and methods the research sample was drawn from a number of maxillary first incisors collected from various dental practices and clinics in bandung. selection of its subjects was based on the criteria of their being almost equal in size and free of caries, restorations, root canal treatment and fractures. the length of each tooth was measured using a sliding thread from the apex to a point in the labial center of the cementoenamel junction. the teeth were then cut horizontally with the point of incision 2mm from the cementoenamel junction of the proximal and treated root canals. root canal preparation was performed by means of protaper (dentsply) hand use up to f1 size. root canal filling used roekoseal paste (roekoseal endodontic sealer, coltene whaledent) as a sealant with obturation involving the use of gutaperca point (protaper, dentsply) size f1. from the examination results obtained, a sample of 20 teeth that had undergone root canal treatment was produced which was subsequently divided into two groups. the first consisted of ten first maxillary incisors to be mounted on fiber posts with zinc phospate cementation (group 1). the second comprised ten maxillary first incisors mounted on fiber post with self-adhesive resin cementation (group 2). root canal preparation was performed on all samples using a 1.5 mm diameter drill included in the fiber stick kit (fiberkleer 4x smooth surface parallel post system kit 1.5 mm diameter, pentron) to a depth of 9 mm (figure 1). all posts in group 1 were cemented with zinc phosphate (elite cement 100, gc japan). the cement was stirred according to the manufacturer’s instructions before being inserted in the root canal with a lentulo spiral and applied to the surface of the fiber post. all fiber posts were inserted into the root canals using tweezers, with any excess cement being cleaned away. the tooth sample was placed on a preprepared sample holder tube and then inserted in the press before being subjected to a 1kg load for one minute. in the group 2 sample, the fiber posts were cemented by means of self-adhesive dual cure resin cement (breeze, pentron) according to the manufacturer’s instructions and the fiber insertion application and insertion were then performed as with group 1. light was activated using a light curing unit (litec) for 40 seconds. a tapered drill was used to produce retention grooves on the root surface of every tooth in each sample group before they were placed in cylindrical self-curing acrylic molds of equal size and stored until the hardening process was complete (figure 2). the coronal part of the fiber post was also placed in a molded tube filled with self-curing acrylic. after the acrylic had completely hardened, a hole was made at both ends of the gear tube for the insertion of the sample holder’s metal rod. samples already embedded in the resin were mounted on an additional tool made specifically for this study. the auxiliary device was then attached to the instron instrument clip (instron lrx plus, lloyd instrument ltd) located at both the base and upper section of the sample holder (figure 3). the test apparatus was activated and the top holder moved to exert continuous tension until the fiber post was detached from the root canal wall. the test result expressed the magnitude of the tensile force as kgf. the magnitude of the tensile force causing separation of the fiber post from the root canal wall was recorded and then calculated statistically using a t-test. figure 1. fiberkleer 4x fiber posts dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p226–229 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p226-229 228 bonifacius, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 226–229 results after studying two groups of samples; a group of ten maxillary first incisors mounted with fiber post using zinc phospate cementation (group 1) and a second such group of ten maxillary mounted with fiber posts using self-adhesive resin cementation (group 2), the mean value of fiber attachment strength was calculated (table 1). it confirmed the average values of the adhesion strength of fiber posts that had been cemented with zinc phosphate cement and self-adhesive resin cement; 82.65 n and 402.81 n respectively. the study result data was presented in tabulated form, then tested by equality 2 test average using t student statistic. the result of the statistical test confirmed there to be a contrast in adhesive strength between selfadhesive resin cement and zinc phosphate cement where the self-adhesive resin cement strength (423.92 n) was higher than that of zinc phosphate (82.65 n) cement (p < 0.05). discussion posts are made for the purpose of providing retention for coronal restorations. there is increasing use of fiber posts due to their desirable qualities such as modulus elasticity which is similar to the modulus of dentine and its aesthetics in terms of color. an effective cement capable of attaching the fiber post to the preparation of the root canal and possessing strong retention force is required. this study aimed to compare the two types of cement adhesive strength to fiber posts. it did not examine the failure of cement adhesion, the lack of effective cohesive cement and cement adhesion, or the adhesion of fiber posts and cement to the canal walls. in this research, the strength test of selfadhesive resin cement and zinc phosphate cement employed a pull-out bond strength evaluation methodology. while this methodology has been used by certain researchers, it is not representative of the actual clinical situation.5 the self-adhesive resin cement form of breeze (pentron clinical technologies, wallingford, ct, usa) consists of two pastes, namely: base and catalyst. the cement was mixed using the auto-mixing tip on the glass pad and then inserted into the root canal using a lentulo spiral. its employment during the cementation process was also carried out on the group 1 (zinc phosphate cement). the use of lentulo spiral is very important in obtaining a uniform cement layer free of air bubbles.5 the fiber post surfaces in both sample groups were also smeared with cement to ensure its being in contact with the entire surface of the fiber post. in this study, the adhesive strength of resin cement was higher compared to that of zinc phosphate cement. this was in accordance with the results of a previous study which showed the adherence strength of zinc phosphate cement to be greater than that of calibra and relyx arc resin, despite the highest adhesion force being found in the fiber posts cemented with relyx unicem self-adhesive resin cement.6 this low strength of zinc phosphate cement bonding is due to the zinc phosphate cement bonding mechanism that relies on friction. phosphoric acid of zinc phosphate cement causes the surface of the eroded teeth to increase in roughness and wettability. however, this cement does not have the ability to attach to dentine or enamel as with resin cement. therefore, the zinc phosphate cement is also called “frictional prototype” cement as opposed to “adhesive” resin cement.7 the brittle physical properties of zinc phosphate cement may also be the cause of the low adhesive strength values in this study, since this brittleness figure 2. the tooth with fiber post which is placed in the tooth holder figure 3. samples ready for testing on the instron machine table 1. mean value of zinc phosphate cement bonding strength and cement resin self-adhesive as fiber post cementation material expressed in newton units. group 1 zinc phosphate cement group 2 self-adhesive resin cement average (mean) 82.65 n 402.81 n standard deviation (std. dev.) 23.212 n 56.719 n number of samples (n) 10 10 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p226–229 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p226-229 229229bonifacius, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 226–229 tends to cause the cohesive bonds of zinc phosphate cement to break easily.8 this zinc phosphate cement may be used if the preparation forms have a limited path of withdrawal cast restoration in one direction and if the restoration fits its preparation.9 the test performed on the ten first maxillary upper incisors mounted with fiber posts with zinc phospate cementation (group 1) resulted in a much smaller mean value of zinc phosphate cement bonding strength than the adhesive cement adhesive resin strength. this finding is not in accordance with that of the previous study.6 this may also be due to poor adaptation of the fiber post used in this study to the root canal walls resulting from differences in the diameter of the fiber post and the drill diameter included in the fiberkleer kit from the plant. according to the data, the diameter of the fiber post on the kit is 1.5 mm, while the size of the drill diameter included in the fiberkleer kit according to the measurements made with ats measurement tools diameter is 1.62 mm. consequently, there will be a gap between the fiber post with a root canal wall of 0.12 mm. the gap between the posts and the wall of the root canal is wide enough to cause the layer of cement around the post to thicken. the gap for ideal luting cement materials should be kept to a minimum by improving the adaptation of the restoration.7 while there is no definitive measure of the thickness of a cement luting, one of 50–100 μm is considered to be ideal. american dental association specification no. 8 states that the thickness of the zinc phosphate cement layer should be between 25 μm to 40 μm.7 a high degree of adaptation between the post and root canal preparation is required if zinc phosphate cement is used as a cementation material for the post.10 the results of other studies have shown that the root canal preparation diameter of prefabricated post placement has no effect on push-out bond strength.11 in conclusion, the adhesion strength of self-adhesive resin cement as fiber post cementation material was higher than that of zinc phosphate cement. references 1. erdemir u, sar-sancakli h, yildiz e, ozel s, batur b. an in vitro comparison of different adhesive strategies on the micro push-out bond strength of a glass fiber post. med oral patol oral cir bucal. 2011; 16(4): e626–34. 2. mosharraf r, haerian a. push-out bond strength of a fiber post system with two resin cements. dent res j. 2011; 8: s88–93. 3. burgess jo, ghuman t. a practical guide to the use of luting cements. 2011. p. 1–11. available from: https://www.dentalacademyofce.com/ courses/1526/pdf/apractical guide.pdf. accessed 2012 mar 15. 4. radovic i. different aspects of related to luring fiber posts. dissertation. siena: university of siena; 2009. p. 91–114. 5. aleisa ki. bond strengths of custom cast and prefabricated posts luted with two cements. quintessence int. 2011; 42(2): e31–8. 6. yahya na, lui jl, chong kwa, lim cm, abu kasim nh, radzi z. effect of luting cement to push-out bond strength of fiber reinforced post. ann dent univ malaya. 2008; 15(1): 11–9. 7. de la macorra j, pradíes g. conventional and adhesive luting cements. clin oral investig. 2002; 6(4): 198–204. 8. cheylan j-m, gonthier s, degrange m. in vitro push-out strength of seven luting agents to dentin. int j prosthodont. 2002; 15(4): 365–70. 9. hill e, lott j. a clinically focused discussion of luting materials. aust dent j. 2011; 56: 67–76. 10. schmage p, pfeiffer p, pinto e, platzer u, nergiz i. influence of oversized dowel space preparation on the bond strengths of frc posts. oper dent. 2009; 34(1): 93–101. 11. perdigão j, gomes g, augusto v. the effect of dowel space on the bond strengths of fiber posts. j prosthodont. 2007; 16(3): 154–64. figure 4. the mean value of adhesion strength of fiber posts cemented with zinc phosphate cement and self-adhesive resin cement. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p226–229 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p226-229 vol 38-no 1-2005 1 the advantage of the three dimensional computed tomographic (3 d-ct) for ensuring accurate bone incision in sagittal split ramus osteotomy coen pramono d department of oral surgery faculty of dentistry airlangga university surabaya indonesia abstract functional and aesthetic dysgnathia surgery requires accurate pre-surgical planning, including the surgical technique to be used related with the difference of anatomical structures amongst individuals. programs that simulate the surgery become increasingly important. this can be mediated by using a surgical model, conventional x-rays as panoramic, cephalometric projections and another sophisticated method such as a three dimensional computed tomography (3 d-ct). a patient who had undergone double jaw surgeries with difficult anatomical landmarks was presented. in this case the mandible foramens were seen highly relatively related to the sigmoid notches. therefore, ensuring the bone incisions in sagittal split was presumed to be difficult. a 3d-ct was made and considered to be very helpful in supporting the pre-operative diagnostic. key words: three dimensional computed tomography (3d-ct), sagittal split osteotomy, accuration of bone incision korespondensi (correspondence): coen pramono d, c/o: bagian bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. specifically in the area located between the sigmoid notch and mandible foramen, therefore identification of this location is very important. adequate space in that area should be available to ensure this procedure, in order to predict the amount of bone. an accurate imaging technique might be required to give a detailed form of the mandible including the position of the mandible foramens in relation to the sigmoid notches. a poor radiography result, such as shift of the x-ray apparatus tube, low sharpness or poor contrast may influence in the prediction during the surgical plan. introduction various types of surgical techniques for correction of mandible progeny have been published. since the introduction of the sagittal split ramus osteotomy (ssro) of the mandible by trauner and obwegeser in 1957,1 dal pont in the year 1961,2 and obwegeser 1964,3 the surgical combination technique of obwegeser-dal pont has become the most popular method and is commonly used (figure 1). this surgical technique has a very important step of a horizontal bone incision in the ascending ramus, figure 1. technique for mandibular sagittal split osteotomy by obwegeser-dal pont: a). this procedure requires precession in all aspects. the medial bony incision is made from anterior border to posterior border halfway through the mediolateral thickness of vertical ramus located between the sigmoid notch and mandible foramen; b). the ramus split with osteotomy; c) the split ramus is separated so that the inferior alveolar neurovascular bundle may be desected from proximal fragment.4 2 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 1–3 in the majority of cases a panoramic radiograph is usually sufficient. in some cases, where a more precise location of the mandible foramens needs to be clearly identified, the aid of more sophisticated radiography technique may be required, such as computed tomography (ct). three-dimensional (3-d) studies in medicine began in the early 1970s presented by ferencz and graco.5 a computed tomography (ct) and 3-d reconstruction virtual reality surgical planning has been used in orthognathic surgery for various purposes, e.g. 3-d assisted simulation combining facial skeleton with facial morphology,6 3-d graphic demonstration of facial soft tissue changes in mandible progeny patients after a mandible sagittal split ramus osteotomy.7 a 3-d visualization may provide a surgeon with reality recognizable of shape of bone for precise surgical planning and simulation.5-13 case a problem arose in a case of a 20-year-old female presented with dento-facial deformation and for surgical correction. her primary concerns included an unaesthetic face due to mandible progeny as well as pain in her tmjoint. clinical and cephalometric evaluations by an orthodontist concluded a case of class iii malocclusion and a maxillary hypoplasia. a routine surgical procedure was planned and a panoramic radiography was used guiding the surgical judgment. the mandible evaluation of the medial ascending ramus showed unavailable spaces between both sigmoid notches and the mandible foramens (figure 2a). the spaces available in the area between foramens and sigmoid notches are necessary, as the bone incision for performing a bilateral-ssro (bssro) are took place in these areas. the foramens and sigmoid notches. in this projection showed differ with that what given in panoramic radiograph, a narrow spaces located in between the foramens and the sigmoid notches in both ascending ramus, were seen enough for performing a bone incision (figure 2b). pre-operative surgical orthodontic treatment was done a period of one year. pre-surgical cephalometric and clinical analysis concluded a le fort i osteotomy for reposition of the maxilla, 5 mm forward, and a mandible set back of 6 mm were necessary. a surgical model analysis and acrylic splint for occlusal guidance were also made to ensure the position of both upper and lower jaws. figure 2a. panoramic radiograph show narrow spaces between the sigmoid notch and mandible foramen in both sites. surgical treatment included a le fort i osteotomy as proposed by some authors was done.13-17 a bssro of the mandible using a combination technique of obwegeserdal pont were performed after a secure location for the first horizontal bone incision was found by a 3-d ct guidance.1-4 difficulties were also present during the surgery in located of the area between sigmoid notch and mandible foramen in both sites due to the narrow spaces available in that areas. the surgery was done with no complication as it was guided by the 3d-ct. discussion in bssro cases, a panoramic radiograph is usually used for assisting the surgical procedure. more detailed anatomical landmarks sometimes require to be displayed at a high resolution of radiograph as this allowing a better understanding of the anatomical landmarks. in this case, a conventional radiograph was not adequate and sufficient for pre-surgical planning guidance. a poor position of the head or the shifting of the x-ray tube apparatus during taken the panoramic photograph may result in the mandible foramens being seen very high on the ascending ramus. in this case, the mandible foramens was predicted to be high in relation to the sigmoid notches, therefore a 3-d ct was made. through this medium the mandible bone surfaces and structures can be displayed clearly. figure 2b. the result of 3d-ct presented available spaces for bony incision line between sigmoid notches and mandible foramens in both sites. case management taking account of the panoramic radiography, a bssro with the surgical modification technique from obwegeser-dal pont, seemed impossible to ensure. a 3-d ct was made to ensure a precise location between 3pramono: the advantage of the three dimentional computed thomographic precise diagnosis and treatment planning of patients who need orthognatic surgery is necessary. careful consideration of the anatomical landmarks should be noted as to the possibility of a different anatomical structure amongst different individuals. panoramic radiographs analysis integrated with a 3dct reconstruction proved have an advantage to quantify the amount of space between the mandible foramens and the sigmoid notches. this procedure had tremendous potential for aiding in planning the surgical procedure more accurately, and thus the risk of alveolar nerve injury was reduced. in conclusion, in certain cases of patients who need for undergo bssro a standard projection of radiography might not enough in supporting the surgical planning, and more sophisticated radiography technique might be needed. references 1. trauner r, obwegeser h. the surgical correction of mandibular prognatism and retrognathia with concideration of genioplasty: surgical procedures to correct mandibular prognathism and reshaping the chin. oral surg oral med oral pathol 1957; 10: 671–92. 2. obwegeser hl indication for surgical correction of mandibular deformity by sagittal spiltting technique. br j oral surg 1964; 50: 157. 3. dal pont. retromolar osteotomy for correction of prognathism. j oral surg 1961; 19: 42–7. 4. kruger ok. textbook of oral surgery. 4th ed. st louis: cv mosby co; 1974. p. 491. 5. xia j, samman n, yeung rwk, shen sg, wang d, ip hhs, tideman h. three dimensional virtual reality surgical planning and simulation workbench for orthognathic surgery. the int j adult orthod and orthognath surg 2000; 15: 265–82. 6. liang hc, wen hc. three-dimensional computer-assisted simulation combining facial morphology for orthognathic surgery. int j adult orthod orthognath surg 1999; 14(2): 140–5. 7. techalertpaisarn p, kuroda t. three-dimensional computergraphic demonstration of facial soft tissue changes in mandibular prognathic patients after mandibular sagittal ramus osteotomy. int j adult orthod orthognath surg 1998; 13(3): 217–25. 8. altobelli de, kikinis r mulliken jb, cline h, lorensen w, jolesz f. computer-assisted three dimensional planning in craniofacial surgery. plast reconstr surg 1993; 92: 576–85. 9. carls fr, schuknecht b, sailer hf. value of three-dimensional computed tomography in craniomaxillofacial surgery. j craniofac surg 1994; 5: 282–88. 10. fuhrmann ra, frohberg u, diedrich pr. treatment prediction with three-dimensional computer tomographic skull models. am j orthod dentofac orthop 1994; 106: 156–60. 11. mccance am, moss jp, fright wr, linney ad, james dr. threedimensional analysis techniques-part 1: three dimensional softtissue analysis of 24 adult cleft palate patients following le fort i maxillary advancement: a preliminary report. cleft palate craniofac j 1997; 34: 36–45. 12. southard te, morris jh, couthard ka, zeitler dl. a threedimensional system for planning orthognathic surgery. j am dent assoc 1994; 125: 452–60. 13. vannier mw, marsh jl, warren jo. three-dimensional ct reconstruction images for craniofacial planning and evaluation. radiology 1984; 150: 179–84. 14. bell wh, mannai ch, luhr hg. art and science of the le fort i downfracture. international journal of adult orthodontics and orthognathic surgery 1988; 1: 23–52. 15. reyneke jp, masureik cj. treatment of maxillary deficiency by a le fort i osteotomy downsliding technique. j oral maxillofacial surg 1985; 43: 914–6. 16. perssonn g, hellem s, nord pg. bone-plates for stabilizing le fort i osteotomies. j maxillofacial surgery 1986; 14: 69–73. 17. profit wr, phillips c, prewit jw, turvey ta. stability after surgical orthodontic correction of skeletal class iii malocclusion. ii. maxillary advancement. int j adult 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 173 volume 47, number 3, september 2014 research report perubahan suhu transisi kaca dan massa resin akrilik heat cured akibat kelembaban dan lama penyimpanan (changes in glass transition temperature and heat cured acrylic resin mass due to moisture and storage time) sherman salim departemen prostodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: acrylic resins, especially poly methyl methacrylate (pmma) was introduced in 1937. acrylic resin has favorable properties, among others, aesthetic, color and texture similar to that of the gingival aesthetic in the mouth, relatively low water absorption and dimensional changes. however, some studies suggest that the duration of storage of acrylic resin will affect the changes in the glass transition temperature and the mass of acrylic resin. purpose: the objective of this research was to study the effect of humidity and storage time led to changes in the glass transition temperature and the mass of the acrylic resin. methods: the research method is experimental laboratory. acrylic resin specimens are kept in conditions of humidity of 90%, 70%, 40% and 30% for 24 hours, one week, one month and two months. in this study used three methods of curing, namely conventional jis, 24-hour curing at 70 °c and using the microwave. results: low humidity causes changes in the glass transition temperature and the mass of acrylic resin. longer storage of acrylic resins in low humidity, can affect change greater than the glass transition temperature and the mass of acrylic resin. conclusion: it can be concluded that the humidity and longer storage of acrylic resins can affect the glass transition temperature and a change in mass. key words: acrylic resin, mass change, glass transitional temperature abstrak latar belakang: resin akrilik terutama poli metil metakrilat (pmma) telah diperkenalkan pada tahun 1937. resin akrilik memiliki sifat yang menguntungkan antara lain estetis, warna dan tekstur mirip dengan gingiva sehingga estetik di dalam mulut baik, daya serap air relatif rendah dan perubahan dimensi kecil. akan tetapi, dari beberapa penelitian menyatakan bahwa lamanya waktu penyimpanan resin akrilik akan berpengaruh pada perubahan suhu transisi kaca dan massa resin akrilik. tujuan: tujuan dari penelitian ini adalah untuk mempelajari pengaruh kelembaban dan waktu penyimpanan yang menyebabkan perubahan suhu transisi kaca dan massa pada resin akrilik. metode: metode penelitian adalah eksperimen laboratoris. spesimen resin akrilik disimpan dalam kondisi kelembaban 90%, 70%, 40% dan 30% selama 24 jam, satu minggu, satu bulan dan dua bulan. dalam penelitian ini digunakan tiga metode curing, yaitu konvensional jis, 24 jam curing pada suhu 70 °c dan menggunakan microwave. hasil: kelembaban rendah menyebabkan perubahan suhu transisi kaca dan massa resin akrilik. penyimpanan lebih lama dari resin akrilik dalam kelembaban rendah, dapat 174 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 173–177 mempengaruhi perubahan yang lebih besar dari suhu transisi kaca dan massa dari resin akrilik. simpulan: dapat disimpulkan bahwa kelembaban dan penyimpanan yang lebih lama dari resin akrilik dapat mempengaruhi suhu transisi kaca dan perubahan massa. kata kunci: resin akrilik, perubahan massa, suhu transisi kaca korespondensi (correspondence): sherman salim, departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: sherman.salim@yahoo.com pendahuluan di bidang prostodonsia, resin akrilik terutama poli metil metakrilat (pmma) merupakan bahan pilihan utama dalam pembuatan basis gigi tiruan. pada tahun 1937, pmma telah diperkenalkan dan dengan cepat menggantikan bahan sebelumnya. resin akrilik memiliki sifat yang menguntungkan yaitu estetis, warna dan tekstur mirip dengan gingiva sehingga estetik di dalam mulut baik, daya serap air relatif rendah dan perubahan dimensi kecil.1,2 beberapa penelitian mengenai gigi tiruan resin akrilik didapatkan lamanya waktu penyimpanan berpengaruh pada perubahan massa. perubahan terbesar pada gigi tiruan terjadi selama 1 bulan pertama, setelah 2 bulan kemudian perubahan massa yang terjadi sangat kecil.3 resin akrilik yang direndam air selama 1 bulan mengalami ekspansi, tetapi perubahan massa yang terjadi secara klinis tidak bermakna.4,5 perbedaan penyimpanan berpengaruh pada gigi tiruan resin akrilik, hal ini disebabkan oleh sifat kimia dan fisik yang dimiliki resin akrilik yaitu bersifat menyerap air. dengan perkataan lain bahwa resin akrilik dapat juga menyerap air dan udara, terutama di tempat yang lembab dengan tingkat kelembababan yang tinggi. berdasarkan latar belakang tersebut, maka dilakukan penelitian untuk melihat apakah semakin tinggi kelembaban udara pada penyimpanan resin akrilik akan menyebabkan semakin kecilnya perubahan massa dan suhu transisi kaca selain itu untuk mengetahui apakah penyimpanan resin akrilik yang lama pada kelembaban rendah akan menyebabkan perubahan massa dan suhu transisi kaca semakin besar. bahan dan metode batang atau lempeng uji dibuat dari bahan resin akrilik heat curing dan microwave curing. perbandingan air dan gipsum yaitu air 24 ml dan gipsum 10 gram. gipsum diaduk di atas vibrator, kemudian dimasukkan ke dalam kuvet. batang uji diletakkan di tengah kuvet dan didiamkan sampai mengeras. setelah gipsum mengeras permukaannya diulasi dengan cold mould seal sebagai separator dan kuvet diisi dengan adonan gipsum di atas vibrator. selanjutnya batang uji yang telah di tanam dengan gipsum pada kuvet dibiarkan selama satu malam (24 jam). pada heat curing resin bubuk dan cairan di campur dengan perbandingan 10 gram dan 4.5 ml sesuai dengan petunjuk pabrik. perbandingan bubuk dan cairan resin microwave ialah 10 gram dan 4.3 ml juga sesuai dengan petunjuk pabrik. setelah 20 menit adonan resin dimasukkan ke dalam cetakan yang sebelumnya diulasi dengan cold mould seal. kuvet ditutup dan dipres dengan hydraulic press (yoshida, jepang) perlahan-lahan, kuvet dibuka dan kelebihan akrilik dipotong kemudian kuvet ditutup kembali dan dipres sampai tekanan 2200 psi atau 50 kg/ cm2. prosedur ini diulang tiga kali kemudian dipindahkan pada klem.6 batang uji resin akrilik dengan berbagai cara kuring dan ukuran yang diperlukan untuk masing-masing penelitian, kemudian dibagi menjadi lima kelompok. kelompok i, direndam air pada suhu ± 24 °c. kelompok ii, diletakkan di udara pada kelembaban 90% (desikator + h2o), setelah direndam air selama 3 bulan. kelompok iii, diletakkan di udara pada kelembaban 70% (desikator + nabr), setelah direndam air selama 3 bulan. kelompok iv, diletakkan di udara pada kelembaban 40% (desikator + cacl2), setelah direndam air selama 3 bulan. kelompok v, diletakkan di udara pada kelembaban 30% (desikator + silica gel), setelah direndam air selama 3 bulan. semua kelompok pada keadaan suhu ± 24 °c. kemudian dilakukan pengukuran secara periodik setelah proses kuring selesai. untuk pengukuran perubahan massa maka batang uji resin akrilik harus direndam air sampai jenuh (satured). kemudian dilakukan pengukuran dengan cara ditimbang/ balance dengan alsep electronic balance ex-200a (japan) secara periodik. batang uji resin akrilik berbentuk persegi empat dengan ukuran 60 mm × 25 mm × 1.5 mm. pada penelitian ini, suhu transisi kaca diperiksa dengan differensial thermal analysis. untuk uji sifat termal polimer, dilakukan pemeriksaan suhu transisi kaca dengan menggunakan alat thermal analysis dt-30b (shimadzu, japan). dalam pemeriksaan ini digunakan probe dengan diameter 1 mm2 dengan beban 22.1 g, rata-rata kenaikan suhu 5 °c/ menit dan kepekaan 100 m serta kecepatan keras perekam 5 mm/menit. data hasil pengukuran dikumpulkan dan ditabulasi menurut kelompok masing-masing kemudian dilakukan analisis statistik multivariate dan analisis ragam untuk menguji hipotesis nihil. hasil untuk menentukan perubahan massa resin akrilik dilakukan dengan cara di ukur dengan alat alsep electronic balance setelah semua resin akrilik selesai polimerisasi dan 175salim: perubahan suhu transisi kaca dan massa resin akrilik heat cured direndam di dalam air selama tiga bulan yang kemudian di tempatkan pada kelembaban 90%, 70%, 40%, 30%. hasil analisis ragam pemeriksaan perubahan massa dilihat dari harga f menunjukkan bahwa perubahan massa resin akrilik sangat dipengaruhi oleh kelembaban (f=6705.834) dan lamanya waktu penyimpanan (f=3482.580) serta interaksi kelembaban-waktu. untuk mengetahui pengaruh perbedaan kelembaban dan lama penyimpanan terhadap perubahan massa resin akrilik maka dilakukan uji lsd. terdapat perbedaan yang bermakna pada perubahan massa resin akrilik menurut perbedaan waktu yaitu pengukuran setelah direndam air selama 3 bulan dan dalam kondisi kelembaban yang berbeda (p≤0,05). perubahan massa resin akrilik dengan metode kuring konvensional jis, pemanasan air selama 24 jam pada suhu 70 °c dan microwave menurut perbedaan a 8 gambar 1. grafik perubahan massa resin akrilik konvensional jis: a) menurut waktu; b) menurut kelembaban. a b b 8 gambar 1. grafik perubahan massa resin akrilik konvensional jis: a) menurut waktu; b) menurut kelembaban. a b gambar 1. grafik perubahan massa resin akrilik konvensional jis: a) menurut waktu; b) menurut kelembaban. a 9 gambar 2. grafik perubahan massa resin akrilik pemanasan 24 jam suhu 70℃: a) menurut waktu; b) menurut kelembaban. a b b 9 gambar 2. grafik perubahan massa resin akrilik pemanasan 24 jam suhu 70℃: a) menurut waktu; b) menurut kelembaban. a b gambar 2. grafik perubahan massa resin akrilik pemanasan 24 jam suhu 70°c: a) menurut waktu; b) menurut kelembaban. a 10 gambar 3. grafik perubahan massa resin akrilik microwave: a) menurut waktu; b) menurut kelembaban. tabel 1. uji lsd suhu transisi kaca resin akrilik menurut kelembaban kelembaban kuring 3 bln (h2o) 5 bln (h2o) 90% (2 bln) 70% (2 bln) 40% (2 bln) 30% (2 bln) kuring * * * * * 3 bln (h2o) * * * * * 5 bln (h2o) * * * * 90% (2 bln) * * * 70% (2 bln) * * 40% (2 bln) 30% (2 bln) *: secara statistik perbedaan bermakna (α = 0.05) a b b 10 gambar 3. grafik perubahan massa resin akrilik microwave: a) menurut waktu; b) menurut kelembaban. tabel 1. uji lsd suhu transisi kaca resin akrilik menurut kelembaban kelembaban kuring 3 bln (h2o) 5 bln (h2o) 90% (2 bln) 70% (2 bln) 40% (2 bln) 30% (2 bln) kuring * * * * * 3 bln (h2o) * * * * * 5 bln (h2o) * * * * 90% (2 bln) * * * 70% (2 bln) * * 40% (2 bln) 30% (2 bln) *: secara statistik perbedaan bermakna (α = 0.05) a b gambar 3. grafik perubahan massa resin akrilik microwave: a) menurut waktu; b) menurut kelembaban. tabel 1. uji lsd suhu transisi kaca resin akrilik menurut kelembaban kelembaban kuring 3 bln (h2o) 5 bln (h2o) 90% (2 bln) 70% (2 bln) 40% (2 bln) 30% (2 bln) kuring * * * * * 3 bln (h2o) * * * * * 5 bln (h2o) * * * * 90% (2 bln) * * * 70% (2 bln) * * 40% (2 bln) 30% (2 bln) *: secara statistik perbedaan bermakna (α = 0.05) 176 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 173–177 kelembaban dan lama penyimpanan dapat dilihat pada gambar 1, 2, dan 3. untuk menentukan adanya perubahan pada suhu transisi kaca resin akrilik maka digunakan alat analisis termomekanikal. dari hasil analisis ragam pemeriksaan suhu transisi kaca dilihat dari harga f menunjukkan bahwa suhu transisi kaca sangat dipengaruhi oleh kelembaban (f=401.049). selanjutnya dilakukan uji lsd untuk mengetahui manakah yang memberikan perbedaan (tabel 1). dapat diamati bahwa menurut kondisi penyimpanan, terdapat perbedaan yang bermakna pada suhu transisi kaca resin akrilik (p≤0,05) setelah kuring dan direndam air selama tiga bulan dengan direndam air lima bulan dan dua bulan pada kelembaban 90%, 70%, 40%, 30%. selain itu, didapatkan perbedaan yang bermakna antara lima bulan direndam air dengan dua bulan pada kelembaban 90%, 70%, 40%, 30% dan kelembaban 90% dengan 70%, 40%, 30% serta kelembaban 70% dengan 40% dan 30% (tabel 1). suhu transisi kaca resin akrilik menurut metode dan kelembaban dapat dilihat pada gambar 4 dan 5. pembahasan resin akrilik heat cured digunakan sebagai bahan penelitian oleh karena resin akrilik tipe ini yang umum digunakan khususnya di indonesia. pemanasan dengan oven microwave akhir-akhir ini semakin khas penggunaannya dan waktu yang diperlukan untuk polimerisasi sangat singkat yaitu hanya tiga menit. pada penelitian ini dipilih kondisi kelembaban 90%, 70%, 40%, 30% karena ratarata kondisi kelembaban di indonesia sekitar 70%-90%, kemudian dengan asumsi di negara yang mempunyai empat musim keadaan kelembaban yang terendah sekitar 30% dan 40%. dari analisis ragam dan uji lsd perubahan massa resin akrilik dapat disimpulkan bahwa kelembaban dan waktu penyimpanan memberikan perbedaan perubahan massa. fenomena penyerapan air oleh resin akrilik selalu disertai perubahan dimensi, dapat disimpulkan bahwa perubahan dimensi erat kaitannya dengan perubahan massa air pada resin akrilik.7,8 pada penelitian ini didapatkan perubahan massa yang lebih besar pada cara kuring 24 jam pada suhu 70 °c dibandingkan dengan cara konvensional jis dan microwave. yang dapat memberikan perbedaan perubahan massa selain factor perbedaan cara kuring adalah keadaan awal atau kapasitas kandungan air yang terdapat pada resin akrilik. demikian juga pengaruh kelembaban dan lama waktu pada perubahan massa resin akrilik sama dengan yang terjadi pada perubahan dimensi. pada kelembaban rendah (30% dan 40%) didapatkan perubahan massa yang besar dibandingkan dengan resin akrilik yang ditempatkan pada kelembaban tinggi (70% dan 90%). begitu juga dengan waktu, didaptkan perubahan massa yang besar setelah satu minggu ditempatkan pada kelembaban rendah (30% dan 40%). perubahan massa yang terjadi setelah satu minggu, satu bulan dan dua bulan hampir sama sehingga dapat dikatakan bahwa dalam waktu satu minggu telah tercapai keseimbangan air pada resin akrilik. adapun proses maupun mekanisme perubahan massa resin akrilik dapat dijelaskan sebagai berikut: pada dasarnyaperbuhan massa air terjadi karena resin akrilik mempunyai sifat yang dapat menyerap dan melepaskan air yang berperan dalam proses ini adalah perpindahan massa air. beberapa penelitian menyatakan bahwa perpindahan massa berdasarkan perpindahan bahan dari satu fase ke fase lain yang berbeda dengan pemisahan yang semata-mata berdasar mekanik.4,7,9 terjadinya perpindahan massa karena adanya perbedaan tekanan uap atau perbedaan kelarutan. gaya dorong yang timbul pada perpindahan massa karena adanya perbedaan konsentrasi atau gradient concentration. dari hasil analisis ragam uji dan uji lsd suhu transisi kaca dapat disimpulkan bahwa perbedaan kelembaban berpengaruh pada suhu transisi kaca. makin rendah kelembaban maka suhu transisi kaca makin tinggi. hal ini menunjukkan jumlah kandungan air dalam resin akrilik mempengaruhi suhu transisi kaca. sejalan dengan hal tersebut pada kelembaban rendah kekuatan resin akrilik juga meningkat. sedangkan perbedaan metode kuring memberikan perbedaan suhu transisi kaca pada resin akrilik. selain itu, suhu transisi kaca menentukan sifat fisik dan 11 gambar 4. grafik suhu transisi kaca resin akrilik menurut metode kuring. gambar 5. grafik suhu transisi kaca resin akrilik menurut kelembaban. gambar 4. grafik suhu transisi kaca resin akrilik menurut metode kuring. 11 gambar 4. grafik suhu transisi kaca resin akrilik menurut metode kuring. gambar 5. grafik suhu transisi kaca resin akrilik menurut kelembaban. gambar 5. grafik suhu transisi kaca resin akrilik menurut kelembaban. 177salim: perubahan suhu transisi kaca dan massa resin akrilik heat cured mekanik resin akrilik. resin akrilik dengan suhu transisi kaca yang tinggi akan mempunyai sifat fisik dan mekanik yang optimum. suhu transisi kaca juga berhubungan langsung dengan berat molekul, bila berat molekul tinggi maka suhu transisi kaca juga tinggi dan kekuatan resin akrilik juga meningkat. 10,11,12 dapat disimpulkan bahwa perubahan massa dan suhu transisi kaca resin akrilik sangat dipengaruhi oleh kelembaban dan lama waktu penyimpanan. daftar pustaka 1. craig rg, powers jm, wataha jc. dental materials: properties and manipulation. 7th ed. india: mosby; 2000. p. 257-70. 2. noort rv. introduction to dental material. london: mosby inc; 2007. p. 219-22. 3. ghani f, moosa r. effect of curing methods and temperature on porosity in acrylic resin denture bases. jpda 2012; 21(03): 127-35. 4. al-nori ak, hussain ama, rejab lt. water sorption of heat-cured acrylic resin. al-rafidain dent j 2007; 7(2): 186-94. 5. widad a, reta m, abd alwahid k. filler reinforced acrylic denture base material. part 2– effect of water sorption on dimensional changes and transverse strength. j coll dentistry 2005; 17(1): 6–10. 6. salim s. the differences of acrylic resin residual monomer levels with various polymerization method. dent j (majalah kedokteran gigi) 2011; 44(4): 196-9. 7. gurbuz o, unalan f, dikbas i. comparison of the transverse strength of six acrylic denture resins. ohdmbsc 2010; 10(1): 21-4. 8. bettencourt af, neves cb, de almeida ms, pinheiro lm, oliveira sa, lopes lp, castro mf. biodegradation of acrylic based resins: a review. dent mater 2010; 26(5): e171-80. 9. meloto cb, silva-concilio lr, machado c, riberio mc, joia fa, rizatti-barbosa cm. water sorption of heat-polymerized acrylic resins processed in mono and bimaxillary flasks. braz dent j 2006; 17(2): 122-5. 10. celebi n, yuzugullu b, canay s, yucel u. effect of polymerization methods on the residual monomer level of acrylic resin denture base polymers. polym adv technol 2008; 19: 201-6. 11. kassim n, wahab ms, yusof y, rajion za, sahar aj. physical properties and fracture surface of acrylic denture bases processed by conventional and vacuum casting fabrication technique. proceeding of the 12th international conference on qir (quality in research), 2011; p. 1001-6. 12. bayra k ta r g, guvener b, bura l c, uresin y. i n f luence of polymerization method, curing process, and length of time of storage in water on the residual methyl methacrylate content in dental acrylic resins. j biomed mater res b appl biomater 2006; 76(2): 340-5. 110 volume 47, number 2, june 2014 topical applications effect of casein phospho peptide-amorphous calcium phosphate and sodium fluoride on salivary mutans streptococci in children fajriani and aini dwi handini department of pediatric dentistry faculty of dentistry, universitas hasanuddin makassar – indonesia abstract background: dental caries is one of the major human diseases caused by mutans streptococci (ms). topical application casein phosphopeptide–amorphous calcium phosphate (cpp-acp) dan sodium fluoride are often use in children and play a role in the caries prevention. purpose: the aim of study was to determine the effect of casein phosphopeptide–amorphous calcium phosphate (cpp-acp) and sodium fluoride topical applications to the number of salivary ms colonies in children. methods: this study using cross-over design with quase experiment time-series. the subjects were 30 children in range of age 6-12 years old that obtained with simple random sampling. the saliva samples of subjects were collected 3 times. first, saliva samples were taken before the treatment; second, after cpp-acp topical application; third, after sodium fluoride topical application. between the cpp-acp and sodium fluoride treatments there was a one week wash-out period. after each treatment, saliva samples were taken twice, 15 and 30 minutes after topical applications respectively. after cultivation on specific agar, the colony number of salivary ms was determined by colony counting (colony forming units-cfu). results: there was no significant difference between topical application casein phosphopeptide – amorphous calcium phosphate (cpp-acp) and sodium fluoride in reducing the number of streptococcus mutans. but topical application of sodium fluoride tended to show more reduction than cpp-acp. conclusion: the topical application of cpp-acp and sodium fluoride could reduce the number of salivary ms in children. the effect of sodium fluoride was somewhat greater than cpp-acp. key words: casein phosphopeptide–amorphous calcium phosphate, sodium fluoride, streptococcus mutans, saliva, children abstrak latar belakang: karies gigi merupakan salah satu penyakit manusia utama yang disebabkan oleh streptococcus mutans (ms). topikal aplikasi kasein phosphopeptide amorf kalsium fosfat (cpp-acp) dan sodium fluoride sering digunakan pada anak-anak dan berperan dalam pencegahan karies. tujuan: penelitian ini bertujuan untuk mengetahui pengaruh aplikasi kasein phosphopeptide amorf kalsium fosfat (cpp-acp) dan sodium fluoride secara topikal terhadap jumlah koloni ms pada saliva anak. metode: penelitian ini menggunakan desain cross-over dengan percobaan quase time-series. subjek penelitian ini adalah 30 anak dalam rentang usia 6-12 tahun yang diperoleh dengan simple random sampling. sampel saliva subjek dikumpulkan 3 kali. pertama, sampel saliva diambil sebelum perlakuan; kedua, setelah aplikasi topikal cpp-acp; ketiga, setelah aplikasi topikal sodium fluoride. antara aplikasi cppacp dan sodium fluoride ada periode wash-out satu minggu. setelah perlakuan, sampel saliva yang diambil dua kali, 15 dan 30 menit setelah masing-masing aplikasi topikal. setelah ditumbuhkan pada media agar yang spesifik, jumlah koloni ms ditentukan dengan cara hitung koloni (colony forming unit-cfu). hasil: tidak ada perbedaan yang signifikan antara topikal aplikasi kasein phosphopeptideamorf kalsium fosfat (cpp-acp) dan sodium fluoride dalam mengurangi jumlah streptococcus mutans . tetapi aplikasi topikal sodium research report 111fajriani and handini: topical applications effect of casein phospho peptide-amorphous calcium phosphate fluoride cenderung menunjukkan penurunan lebih banyak dari cpp-acp. simpulan: aplikasi topikal dari cpp-acp dan sodium fluoride dapat mengurangi jumlah ms pada saliva anak. pengaruh sodium fluoride sedikit lebih besar dari cpp-acp. kata kunci: casein phosphopeptide -amorf kalsium fosfat, sodium fluoride, streptococcus mutans, saliva, anak correspondence: fajriani, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas hasanuddin. jl. perintisjl. perintis kemerdekaan km 10 kampus tamalanrea ujung pandang, indonesia. e-mail: fajriani_fkg@yahoo.comujung pandang, indonesia. e-mail: fajriani_fkg@yahoo.com introduction the prevalence of dental caries in children remains a significant clinical problem. in 2004 the caries rate in indonesia is 90.05% of population, and in 2011 the most significant incidence occurs in children aged 3-5 years (81.2%) which mean most of the case are left untreated.1,2 so in indonesia with population more than 225 million people the prevention is at top priority. therefore, prevention efforts should be made as early as possible using simple and effective dental caries prevention method which affordable by all levels of society. topical application of sodium fluoride is widely used as dental caries prevention. fluoride compound has beenfluoride compound has been applied extensively, and its efficacy has been recognized by researchers and dentists. besides of sodium fluoride recently there are materials that have role in preventing dental caries. one of them is the agent that has the casein phosphopeptideamorphous calcium phosphate (cppacp). several studies on cpp-acp showed not only the increase of enamel remineralization, but also prevent bacterial adhesion of streptococcus mutans on tooth surfaces.3 the aim of study was to determine the effecte aim of study was to determine the effect of casein phosphopeptide–amorphous calcium phosphate (cpp-acp) and sodium fluoride topical applications to the number of salivary streptococcus mutans colonies in children. materials and methods this study using cross-over design with quase experiment time-series. the subjects were 30 children in range of age 6-12 years old that obtained with simple random sampling. oral hygiene examination in children aged 6-12 years were done by using the ohi-s index with the criteria sample of very bad ohi-s; did not have allergies or certain systemic diseases; and were not taking antibiotics. sampling was conducted in sdi tamalanrea of universitas hasanuddin, while laboratory procedure was conducted at the microbiology laboratory faculty of medicine universitas hasanuddin. the saliva samples of subjects were collected 3 times. first, saliva samples were taken before the treatment; second, after cpp-acp topical application; third, after sodium fluoride topical application. between the cpp-acp and sodium fluoride treatments there was a one week wash-out period. after each treatment, saliva samples were taken twice, 15 and 30 minutes after topical applications respectively. after 10-3 dilution, the saliva samples were cultured on glucose nutrient agar (gna). after the anaerob incubation 37° c for 24 hours, the colony number of salivary ms was determined by colony counting (colony forming units-cfu). topical application of cpp-acp (first treatment) and topical application of sodium fluoride (second treatment) were given to subjects with following manner: (a) dry the entire surface of the tooth samples by using cotton or cotton pellet; (b) topical application of cpp-acp (gc tooth mousse®-recaldent) approximately 1 mg and 0.5 ml sodium fluoride 5% (floucal solute®septodont) were applied to the entire surface of the teeth; (c) after 15-minute the application of topical material, the second saliva collection was carried out; (d) 30 minutes later, the third saliva sample was taken; (e) all saliva samples were brought to the laboratory for evaluation of the number of ms colonies. data obtained by the calculation of the bacteria and then noted in table form and subsequently statistically tested by using anova and t-test pairs. results the subject distribution was 18 males (60%) and 12 females (40%) with mean age was 8.56 ± 2.02 years. the value of oral hygiene index was 3.53 ± 0.38 (table 1). topical applications of cpp-acp and sodium fluoride showed the significant reduction of salivary mutans streptococci colonies in 15 and 30 minutes after treatment respectively (table 2), based on anova test results, the value of p = 0.000 (p<0.05). the further test results for the number of salivarysalivary mutans streptococci colonies by interval of time after the intervention of topical application of cpp-acp and sodium fluoride materials described the differences of each bacterial colony count (table 3). the colonies number of ms from the pretest to 30 minutes after of application topical of cpp-acp and sodium fluoride decreased up to approximately 45 colonies. in addition, the decrease of colonies number based on each intervals showed significant results. in the cpp-acp group of materials, the number of colonies decreased to 40.00 cfu/ ml, whereas the number of colonies sodium fluoride group decreased to 35.73 cfu/ ml. the result of statistical tests showed p = 0214 (p > 0.05), it means that the difference was not significant (table 4). meanwhile, after 30 minutes sodium fluoride 112 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 110–114 table 1. characteristics distribution of the research sample characteristics of research sample frequency (n) percentage (%) mean ± sd sex male 18 60 female 12 40 age 8.56 ± 2.02 value of oral hygiene (ohi-s) 3.53 ± 0.38 number of colonies of s. mutans before (pre-test) 83.40 ± 22.63 cpp-acp group 30 100 number of colonies in 15 minutes (1st post-test) 40.00 ± 9.57 number of colonies in 30 minutes (2nd post-test) 18.73 ± 9.87 sodium fluoride group 30 100 number of colonies in 15 minutes (1st post-test) 35.73 ± 9.83 number of colonies in 30 minutes (2nd post-test) 12.93 ± 7.86 table 2. the differences effect between cpp-acp and sodium fluoride based on times interval topical application material group cfu pre-test cfu 15 minutes cfu 30 minutes p-value mean ± sd mean ± sd mean ± sd cpp-acp 83.40 ± 22.63 40.00 ± 9.57 18.73 ± 9.87 0.000* sodium fluoride 83.40 ± 22.63 35.73 ± 9.83 12.93 ± 7.86 0.000* *repeated analysis of variance (anova) test: p < 0.05; significants table 3. further test results based on the number of colonies of s. mutans time intervals after giving topical application materials cpp-acp and sodium fluoride topical application material type cfu s. mutans comparator mean difference p-value cpp-acp cfu pre-test cfu 15 minutes 43.400 0.000* cfu 30 minutes 64.666 0.000* cfu 15 minutes cfu 30 minutes 21.266 0.000* sodium fluoride cfu pretest cfu 15 menit 47.666 0.000* cfu 30 menit 70.466 0.000* cfu 15 menit cfu 30 menit 22.800 0.000* *pos hoc test: least significant difference (lsd) test: p<0.05: significant table 4. the differences effect of topical application of cpp-acp and sodium fluoride materials in time intervals 15 minutes and 30 minutes after treatment topical application material group cfu pre-test cfu 15 minutes p-value cfu 30 minutes p-value mean ± sd mean ± sd mean ± sd cpp-acp 83.40 ± 22.63 40.00 ± 9.57 0.214** 18.73 ± 9.87 0.611* sodium fluoride 83.40 ±22.63 35.73 ± 9.83 12.93 ± 7.86 *paired sample t-test: p>0.05; not significant 113fajriani and handini: topical applications effect of casein phospho peptide-amorphous calcium phosphate materials reduced the colony number of ms to 12.93, while the material of cpp-acp reduced to 18.73. based on the result of statistical tests, the value of p = 0611 (p > 0.05), which means that there was no significant difference. discussion the study used topical application of gc tooth mousse product of recaldent® which contain derivate calsium phosphate compound or cpp-acp that have been applied for approximately 1 mg. the study used container of topical application because it has already tested and reported by several literatures. dr. santosh4 from india stated that consuming products that had the same anti-caries cppacp can give good effect on controlling caries at the age of children and adults. as for the comparison the study used 0.5 ml floucal solute -septodont® that contain sodium fluoride compounds. it is based on the results of research on the role of fluorine reduces the ability of bacteria to form acid. in addition it also functions in the formation of fluorine mineral that will stop the caries process.4, 5 the result showed the difference in the colony number of ms after topical treatments which were given to subjects after 15 and 30 minutes. the results between each treatment compared with each other to analyze which one was more effective in reducing the number of colonies of s. mutans. the data revealed that both topical application materials were indeed effective in reducing the number of s. mutans.4,5,6 research by reynolds in 2006 attracted public attention by revealing the fact that phosphopeptida casein amorphous calcium phosphate, which was one of the derivatives casein was able to get into the enamel surface and affect the caries process. cpp-acp prevents the vicinity of ms to the tooth surface. phosphopeptida bond casein (cpp) containing sequence group nano-complex chain was ser (p)-ser (p)-ser (p)-glu-glu had an ability to prevent bacteria. phosphopeptida casein chain arrangement (cpp) binds to amorphous calcium phosphate (acp) which can prevent the development of bacteria.4, 6,7 in addition to preventing the vicinity of ms, cppacp also assist in the remineralization of tooth enamel. the effectiveness of a paste containing 10% cpp-acp on the enamel surface in vitro, revealed that 10% cpp-acp has a positive effect on enamel remineralization. other research suggests the use of cpp-acp with 0.1% mg/ ml significantly reduced caries activity by 14%, whereas the levels of cpp-acp with 1% mg/ ml could reduce 55% of caries activity.8,9 effectiveness of topical application of fluoride in reducing the number of colonies of ms has also been proved in the literatures and research. fluoride works by inhibiting the metabolism of plaque bacteria that can ferment carbohydrates through changes hydroxyl apatite in enamel to fluoride apatite. fluorine chemical reactions: ca10(po4)6.(oh)2 + f  ca10(po4)6.(ohf) (ohf) produces the enamel more resistant to acid that can inhibit demineralization and enhance remineralization processes that trigger repair and termination of carious lesions.1,10 this study also showed a difference in the effectiveness of topical application of cpp-acp materials and sodium fluoride at time intervals of 15 minutes and 30 minutes after give the materials. at 15 minutes after the intervention materials, group topical application of cpp-acp materials, number of colony decreased to 40.00 cfu/ ml, while in the topical application of sodium fluoride materials decreased to 35.73 cfu/ ml. after 30 minutes, the topical application of cpp-acp materials capable of reducing the number of colonies to 18.73 cfu/ ml, whereas topical application of sodium fluoride materials have reduced to 12.93 cfu/ ml.12.93 cfu/ ml. this shows that there is no significant difference between the effects of topical application of cpp-acp materials and sodium fluoride to decrease the number of colonies of s. mutans in the 15th minutes and 30 after give topical materials. the study suggested that the two materials are effective in lowering the number of colonies of s. mutans, but the topical application of sodium fluoride materials showed a greater decrease compared with cpp-acp materials at intervals of 15 and 30 minutes after give topical material in saliva children aged 6-12 years. however, there was no significant difference between topical application of cpp-acp and sodium fluoride at 15 and 30 minutes after providing topical material. fluoride when consumed in large amounts can give side effects to the body, i.e. acute toxicity, as well as the occurrence of fluorosis (mottled enamel). it is therefore not recommended for use at home, and would be better if applied by professionals in order to prevent that side effect.10,11 while topical application of cpp-acp material is derived from casein (milk) safe for children. the study suggested that topical application of cppacp and sodium fluoride could reduce the number of salivary ms in children. the effect of sodium fluoride was somewhat greater than cpp-acp. references 1. angela a. pencegahan primer pada anak yang berisiko karies tinggi. maj ked gigi (dent j) 2005; 38(3): 130-4. 2. martha m. formula kesehatan gigi & mulut, cara mudah untuk, cara mudah untuk merdeka dari masalah gigi dan malu. available form :alu. available form :available form : http://www. klikdokter.com/gigimulut/read/2010/09/27/291/cara-mudah-untukmerdeka-dari-masalah-gigi-dan-mulut. accessed on december 19, 2012. 3. ravinshankar tl, yadav v, tanged ps, tirth a, chaitra tr. effect of consuming different dairy products on calcium, phosphorus and ph levels of human dental plaque; a comparative study. eur arc of ped dent 2012; 13(issue 3): 144-6. 4. bp santhosh, jethmalani p, shashibhushan kk, subba reddy vv. effect of phosphate containing chewing gum on salivary concentration of calcium and phosphorus: an in vivo study. j indian soc pedod prev dent 2012 30(2): 146-50. 5. jones s, burt ba, petersen pe, lennon ma. the effective use of fluorides in public health. bulletin of the world organization 2005; 83(9): 670-6. 114 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 110–114 6. beerens mw, van der veen mh, van beek h, cate jm. effects of casein phosphopeptide amorphous calcium fluoride phosphate paste on white spot lessions and dental plaque after orthodontic treatment: a 3 month follow up. eur j oral sci 2010; 118: 610-7. 7. reynold ec, cai f, cocharane nj, shen p, walker gd, morgan mv. fluoride and calcium phosphopeptide – amorphous calcium phospate; a research reports. j dent res 2008; 87(4): 344-8. 8. oshiro m, yamaguchi k, takamizawa t, inage h, watanabe t, irokawa a, ando s, miyazaki m. effect of cpp-acp paste on tooth mineralization: an fem – sem study. j of oral science 2007; 2(49): 115-20. 9. reynold ec. casein phosphopeptide–amorphous calcium phosphate: the scientific evidence adv dent res 2009; 21(1): 25-9. 10. emmerling h, standley e. current fluoride modalities for reduction of dental caries. a peer-reviewed publication. february, 2013. p. 63-9. 11. liaison. policy on use of fluoride. a review council. american academy of pediatric dentistry. oral health police. revised from 1978-2012. �� volume 47, number 1, march 2014 research report the effect of soda immersion on nano hybrid composite resin discoloration m. chair effendi,1 yuli nugraeni2 and rizki widya pratiwi2 1 department of pediatric dentistry, school of dentistry faculty of medicine, universitas brawijaya 2 department of conservative and endodontic, school of dentistry faculty of medicine, universitas brawijaya malang indonesia abstract background: composite resin is the tooth-colored restorative material which most of the people are fond of due to their aesthetic value. the composite resin discoloration may happen because of the intrinsic and extrinsic factors. soda water is one of the beverages which can cause the composite resin discoloration. purpose: the study was aimed to determine the effect of soda immersion on nano hybrid composite resin discoloration. methods: the study was an experimental laboratory study using 100 shade a3 nano hybrid composite resin specimens with the diameter of 5 mm and density of 2mm. the samples were divided into 5 groups, each group was immersed in different beverages. the beverages were mineral water; lemon-flavored soda; strawberry-flavored soda; fruit punch-flavored soda; and orange-flavored soda for 3, 7, 14 and 21 days respectively, in the temperature of 37o c. the discoloration measurement utilizes spectrophotometer, vita easy shade, and uses ciel*a*b* method. results: the result showed that the duration of immersion in soda had an effect on the nano hybrid composite resin discoloration. strawberry and fruit punchflavored soda were the most influential components toward the discoloration. nevertheless, the generally-occurred discoloration was clinically acceptable (∆e ≤ 3,3). conclusion: the study suggested that the soda immersion duration has effect on nano hybrid composite resin discoloration. key words: nano hybrid composite, discoloration, soda beverages abstrak latar belakang: resin komposit adalah material sewarna gigi yang diminati masyarakat karena memiliki nilai estetik yang baik. perubahan warna resin komposit dapat terjadi karena faktor intrinsik dan ekstrinsik. minuman soda merupakan salah satu minuman yang dapat menyebabkan perubahan warna pada resin komposit. tujuan: tujuan dari penelitian ini untuk meneliti perubahan warna resin komposit nanohibrida akibat perendaman dalam minuman soda. metode: metode yang digunakan pada penelitian ini adalah eksperimental laboratorik dengan menggunakan 100 spesimen resin komposit nanohibrida shade a3 berdiameter 5 mm dan tebal 2 mm. sampel dibagi dalam 5 kelompok, masing-masing kelompok direndam dalam minuman yang berbeda, yaitu direndam dalam air mineral, soda lemon, soda strawberi, soda fruitpunch, dan soda jeruk selama 3, 7, 14 dan 21 hari, dalam suhu 37o c. pengukuran perubahan warna menggunakan spectrophotometer, vita easy shade dengan metode ciel*a*b*. hasil: hasil penelitian menunjukkan bahwa lama perendaman pada minuman soda berpengaruh terhadap perubahan warna resin komposit nanohibrida. soda yang paling berpengaruh terhadap perubahan warna adalah soda strawberi dan soda fruitpunch. namun perubahan warna yang terjadi secara umum masih dapat diterima secara klinis (∆e ≤ 3,3). simpulan: hasil penelitian ini menunjukkan bahwa lamanya waktu perendaman mempengaruhi perubahan warna pada resin komposit nanohibrida. kata kunci: komposit nanohibrida, perubahan warna, minuman soda corespondence: m. chair effendi, c/o: departemen ilmu kedokteran gigi anak, program studi pendidikan dokter gigi. universitas brawijaya. jl. veteran malang 65145, indonesia. e-mail: chaireffendi@gmail.com �8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 37–40 introduction soda is the carbonated water which has various flavorsand colors that can attract people to consume.1 it is proven by american academy of pediatrics that reported 56-85% of students consume soda at least once a day. this percentage increases on teenage boys, 20% of them consume it at least four times a day.2 people’s habitto consume soda,which actually contains various dye substances, can affect the discoloration oftheir composite resinrestoration.3 in his previous experiment, alshalan4 proposed that there is composite resin discoloration after immersing intothe coffee, tea, and wine. it is supported by chong et al.2 that consuming coffee, tea, soft drink (soda water), juice, and liquor may influence composite resin aesthetic and physical quality. wan bakar et al.5 once noted that the discoloration may be caused by the absorption from staining substances such as wine, coffee, tea, and cola (soda). composite resin is the often used as restorative materials in dentistry due to its high aesthetic value. composite resin are able to produce restoration color which is identical to original tooth color.5 tyas6 reported the survey result toward 100 restoration users-55% use composite resins, 28% use amalgam, 15% use gic, and 2% use polyacidmodified composite resin. nano hybrid composite resin has better physical and mechanical value rather than the conventional composite resin. according to al-shalan,4 nano hybrid composite resin have many advantages, that they can be used for anterior and posterior tooth restoration and reduce the shrinking during polymerization. moreover, they also have smoother surface due to their nano particles, better mechanical and aesthetic quality, and they are also suitable for restoration on anterior teeth. as according to wan bakar et al.,5 the lack of composite resin is that their color is changed while exposed to other dye substances; whereas the most important roleof composite resin as the high aesthetic valued-restorationdepends on the color suitability among others.7 the discoloration may occur due to the intrinsic and extrinsic factors. the intrinsic factor derived from the composite resin it self, which occurs in the matrix resins or between the matrix gap and filler, while the extrinsic factor comes from plaque accumulation and staining caused by color substances penetration and exogenous contamination. the degree of exogenous discoloration is influenced by oral hygiene, food and beverages consumed, and smoking habit. furthermore, discoloration may occur chemically, which relates to the alteration or oxidation on amine accelerator, oxidation of polymer matrix structure and methacrylate group.8 soda not only contains dye substance but also contains sugar and has low ph. the sugar in it can increase the color change in composite resinrestoration material, while low ph (2.7) can influence the softening matrix in composite resin, so that it will affect on composite resin’s integrity.3 the existence of microcracks and microvoids between the filler and matrix resin can be the entrance for the dye substance to penetrate into the composite resin.4 toksoy et al.9 supported that dye substance penetration causes the discoloration of composite resin from yellow to brownish. more than 80% of patients are anxious about discoloration in the composite resin restoration they use. this discoloration is one of the reasons to replace the composite resin restoration. the previous study reported that the discoloration starts from the 1st up to the 2nd week since the composite resin is exposed to the beverages.4 nevertheless, the discoloration has not been examined up to the 3rd week. the study was aimed to determine the effect of soda immersion on nano hybrid composite resin discoloration. materials and methods this study was an experimental laboratory using 100 plates of nano hybrid composite resin mono ceram x dentsply® shade a3 which were immersed in mineral water and various kinds of flavored soda (lemon, strawberry, fruit punch, and orange) for 3, 7, 14 and 21 days in the temperature of 370 c. the discoloration is measured using ciel *a*b method. the tool used to measure the discoloration was vita easy shade guide, which results in numbers to present the colors. colors component measured were as follows: (1) l* or lightness, the range of value 0 (black) to 100 (white); (2) a* was a chroma component, which the discoloration was indicated by +a* to show reddish color and –a* to show greenish color; (3) b* was chroma component, which indicates +b* as yellowish and –b* as bluish. the formula of discoloration: ∆e= {(∆l*)2+(∆a*)2+(∆b*)2}1/2 note: ∆e : discoloration ∆l : black-white (l2 – l1) ∆a : color differences from red-green (a2 – a1) ∆b : color differences from yellow-blue (b2 – b1) the straws were cut into 2 mm in height to make composite resin plates. after that, apply nano hybrid composite resin using plastic filling. then, it was polymerized using the light cured for 20 seconds. all the specimens were immersed in distilled water for 24 hours in order to be perfectly polymerized in the temperature of 370 c to stimulate the mouth cavity temperature. after being immersed by the distilled water, the specimens were dried with tissue. then, the initial measurement was done to the composite resin using white paper as the base. specimens were immersed in each flavored-beverages and mineral water for 3, 7, 14 and 21 days respectively. the flavored��effendi, et al.: the effect of immersion in soda on nano hybrid composite resin discoloration beverages (lemon, strawberry, orange, and fruit punch soda) were changed daily. measure specimens’ colors on the 3rd, 7th 14th and 21st day after the first immersion. results the effect of immersion duration toward the composite resin discoloration after being immersed in various colored and flavored soda was evaluated using statistical analysis repeated measures anova with significance level of 0.05 (p = 0,05) and confidence level of 95% (α = 0.05). the difference of nano hybrid composite resin discoloration due to the consumption of various colored and flavored soda water was evaluated using one way anova statistical technique with significance level of 0.05 and confidence level of 95% (α = 0.05). the result of repeates measures anova test showed that the significance level got from the calculating process was lesser than α = 0,05. so, it could be concluded that the duration of immersion in various colored and flavored soda water (mineral water, lemon, strawberry, fruit punch, and orange soda water) influences nano hybrid composite resin discoloration significantly. the result of one way anova test shows that the significance level obtained from the calculating process was less than α = 0,05. there was nano hybrid composite resin discoloration as the effect of consuming various colored and flavored soda. strawberry and fruit punchflavored soda were the most influential components toward the discoloration. discussion ∆e* result on control group (immersed in mineral water) from days 3-21 indicates a range of value between 0.62-1.36. the discoloration increases in accordance with immersion duration, yet the discoloration on the 14th and 21st day did not differ significantly. it is corresponds to alshalan4 that the discoloration occurs from the first week of exposure and increases until the second week. ∆l* result on control group day 3 until 21 indicates the blackened composite resin which is marked by the decreasing of white spectrum (l*) degree during 21 days. the decreasing of color spectrum also occurs on ∆a* dan ∆b*. the immersion in mineral water only visually noticeable changes started from the 14th day, which is ∆e ≥ 1. yet, it was still clinically acceptable. it is also happened in fontes et al.10 that stated that after nano filler resin immersion in water for a week did not indicate any significant discoloration or it was still clinically acceptable. the discoloration may occur chemically related to the change or oxidation of amine accelerator, oxidation of polymer matrix structure, and oxidation of methacrylate group. the immersion in lemon soda indicates an increase in discoloration (∆e*), which is in accordance with immersion duration. the increasing of discoloration in lemon soda indicates the different result among the other immersion group. composite resin immersion in lemon soda causes an increase in degree of white color significantly (visually noticeable) begun on day 7 to 21 (∆l*= 1.05 – 1.79). it is due to the acid properties which have erosive effect to polymer material degradation and color pigment on composite resin (ferric oxide). the acid properties of soda water can dissolve polymer matrix of composite resin which causes white color appearance comes from silica powder contained in composite resin. likewise in ∆a* dan ∆b*, there is an increase in accordance with immersion duration, the composite resin is heading to red and yellow. the range of composite resin discoloration caused by immersion in lemon soda is 0.62-1.79. the experiment result shows the quite significant discoloration in each immersion duration, however, the discoloration is small and still clinically acceptable (∆e ≤ 3.3). the discoloration caused by strawberry soda can be seen from ∆e which is noticeable on 3rd, 7th, 14th, and 21st day with value range of 1.02-3.52. color spectrum change that mostly affects ∆e value showed by ∆a* value (reddish) which is higher rather than ∆l* and ∆b* value from 1st until 21st day. this change indicates an increase of red spectrum degree which is caused by red pigment in strawberry soda. the nano hybrid composite resin discoloration may berelatedto resin’ ability to absorb or adsorb color pigment in assorted soda colors and flavors.11 it is also concluded by burrow and makinson that composite resin discoloration is more visible due to absorption from colored-food rather than water. based on the experiment result, there is a significant change in eachimmersion duration, yetthere are only 2 samples at the 21st day which the changes are clinically acceptable, which is more than clinically acceptable maximum criteria(∆e ≥ 3.3). discoloration in strawberry soda is more than other soda water since it has very condensed color pigment. it can be proven from the left red stain in lips and tongue after consuming strawberry soda. nano hybrid composite resin discoloration range (∆e) after being immersed in fruit punch soda is 0.9-3.39. this discoloration ∆e* is highly influenced by ∆l*, ∆a*, dan ∆b* values which continuously decrease after immersion. this condition indicates the discoloration proven by the figure 1. graphic of discoloration. water lemon soda strawberry soda fruitpunch soda orange soda days days days days �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 37–40 decreasing of white, red, and yellow spectrum degree in the nano hybrid composite resin to the dark colorsgreen and blue. based on the experiment, there is a still clinically acceptable significant discoloration of each immersion duration. there is only 1 sample at the 21st day which experiences discoloration more than 3.3. this is caused by the concentrated green pigment in fruit punch soda. it is proven by the left green stain in lips and tongue after consuming it. generally, composite resin’s color stability is affected by various factors such as matrix resin, initiator concentration, oxidation of unreacted monomers, filler content, and pigment.11 discoloration ∆e* in orange soda is tremendously affected by ∆l* value decreasing, while ∆a* dan ∆b* increase after immersion. this condition represents discoloration evidenced by degree decreasing of white spectrum and increasing of red and yellow spectrum in nano hybrid composite resin specimens. discoloration range on orange soda is 1.02-1.92. it can be known from the experiment that the discoloration is still clinically acceptable despite the significant discoloration of each immersion duration, which keep increasing during 3, 7, 14 and 21 days.this change is caused by orange pigment in orange soda. it is concluded that the increasing of discoloration along with immersion duration indicates pigment exposure toward nano hybrid composite resin, resulted on the clearer discoloration. all groups of composite resins immersed in assorted soda colors and flavors undergo quite significant discoloration in accordance with immersion duration. still, commonly, the discoloration cannot be clinically accepted even though some samples immersed in strawberry and fruit punch show ∆e ≥ 3.3. the immersion in mineral water, lemon soda, orange soda, fruit punch soda, and strawberry soda indicates the discoloration sequences from the least to the most. although nano hybrid composite resin has a good color stability, this experiment proves that there was a significant discoloration from the average value of ∆e* at 3rd until 21st day. some researchers stated that ∆e* > 1 in composite resin shows visually noticeable discoloration, however, up to ∆e* ≤ 3,3 can be clinically acceptable according to the experiment result, there is a significant discoloration along with nano hybrid composite resin immersion, yet this experiment indicates the smaller ∆e* value than the previous experiment of micro hybrid composite resins done by toksoy fulya in 2009. the study suggested that the soda immersion duration has effect on nano hybrid composite resin discoloration. however, the discoloration caused by immersion in lemon and orange soda is still clinically acceptable. since soda can cause discoloration, the patients who are using composite resins are suggested not to consume too much soda water. references 1. zakwan. pengaruh konsentrasi karbondioksida terhadap mutu minuman ringan rosela. usu2011; h. 7-15. 2. chong sy, seow ll, lau mn, tiong sg, yew cc. effect of beverages and food source on wear resistance of composite resins. malaysian dental j 2008; 34-9. 3. padiyar n, pragati k. colour stability: an important physical property of esthetic restorative materials. dental college and hospital 2010; p. 81-4. 4. al-shalan ta. in vitro staining of nanocomposite exposed to a cola beverage. pakistan oral and dental j 2009; 79-84. 5. wan bakar wz, mior a, adam h. a comparison of staining resistant of two composite resins. school of dental science 2009; 13-6. 6. tyas mj. placement and replacement of restoration by selected practitioners. aust dent j 2005; 50(2): 81-9. 7. aleixo p, patricia p, ana l, regina g. composite resin color stability: influence of light sources and immersion media. j appl oral sci 2011; 19(3): 204-11. 8. ghinea r, ugarte-alvan l, yebra a, pecho oe, paravina rd, perez mdel m. influence of surface roughness on the color of dental-resin composite. j zhejiang univ sci b 2011; 12(7): 552-62. 9. toksoyft, sahinkesen g, yamanel k, erdemir u, oktay ea, ersahan s. influence of different drinks on the colour stability of dental resin composites. eur j dent 2009; 3(1): 50-6. 10. nasim i, neelakantan p, sujeer r, subbarao cv. color stability of microfilled, microhybrid, and nanocomposite resin–an in vitro study. j dent 2010; 38 (suppl 2): e137-42. 11. park jk, tae-hyong kim, ching-chang ko, franklin garcia-godoy, hyung-il kim, yong hoon kwon. effect of staining solutions on discoloration of resin nanocomposites. nih public access 2010; 1-6. 215215 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 215–218 research report minimum inhibitory concentration of cocoa pod husk extract in enterococcus faecalis extracellular polymeric substance biofilm thickness tamara yuanita, latief mooduto, reinold christian lina, fajar agus muttaqin, ika tangdan, revina ester iriani marpaung and yulianti kartini sunur department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: root canal treatment constitutes a treatment sequence for infected pulp to eliminate the etiological factors of pulp necrosis and periapical lesion. enterococcus faecalis (e. faecalis) is an organism commonly found in a high proportion of root canal failure because of its ability to form biofilm. degradation of extracellular polymeric substance (eps) by oxidizing agents such as sodium hypochlorite is the first step in removing biofilm. however, the toxicity of sodium hypochlorite constitutes the main concern and, therefore, the safest alternative irrigants possible are required. the use of fruits, herbs and plants is widespread, especially in the fields of medicine and dentistry. food crops are known to be rich in bioactive compounds, especially polyphenols, which have antioxidant and antimicrobial properties. cocoa pod husk extract can, therefore, represent an alternative irrigant. purpose: this study aimed to determine the minimum inhibitory concentration of cocoa pod husk extract in relation to the thickness of e. faecalis eps biofilm. methods: four groups of e. faecalis cultured biofilm samples were analysed: group one contained e. faecalis without cocoa pod husk as a positive control; group two contained e. faecalis with 1.56% cocoa pod husk extract; group 3 contained e. faecalis with 3.125% cocoa pod husk extract; and group 4 contained e. faecalis with 6.25% cocoa pod husk extract. the biofilm thickness of all groups was measured by confocal laser scanning microscopy with statistical analysis subsequently undertaken by means of a post hoc test and tukey hsd. results: the average values of eps biofilm thickness were as follows: group 1: 9500 nm; group 2: 8125 nm; group 3: 8000 nm; and group 4: 6375 nm. a post hoc tukey hsd test indicated a significant difference between group 1 and group 4, while in group 2 and group 3 compared to group 1, there were no significant differences with the values of each being p = 0.340 and p = 0.267 (p>0.05). conclusion: 6.25% cocoa pod husk extract reduces e. faecalis eps biofilm thickness. keywords: cocoa pod husk extract; endodontic; enterococcus faecalis; extracellular polymeric substance biofilm correspondence: tamara yuanita, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: tamara-y@fkg.unair.ac.id introduction the pathology of pulp tissue and periapical tissue is directly or indirectly related to microorganisms. microbes can be removed and minimized by root canal treatment whose success is influenced by several interrelated factors1, including; effective diagnosis, aseptic action, knowledge of dental anatomy, chemical-mechanical preparation, threedimensional obturation and the use of root canal dressing. all of these factors relate to one key point, specifically; root canal decontamination.1 the effectiveness of root canal preparation can be increased by the use of irrigation solutions such as sodium hypochlorite (naocl), chlorhexidine, and ethylenediaminetetraacetic acid (edta). naocl represents the gold standard for root canal irrigation solutions because, at present, no other solution has demonstrated similar effectiveness. however, the drawback of naocl is its dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p215–218 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p215-218 216 yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 215–218 cytotoxicity which can cause acute injury if it infiltrates the periapical region. if naocl comes into contact with tissue, it rapidly oxidizes the surrounding living tissue and triggers rapid hemolysis, inhibits neutrophil migration, and damages endothelial and fibroblast cells.2 the higher the concentration of naocl, the higher the anti-bacterial effect and tissue dissolution, and also the higher the toxicity.3 moreover, the very low concentrations of naocl (>0.01%) present in in vitro cell culture cause the death of human fibroblast cells.4 root canal treatment can fail due to the absence of an effective coronal seal, microleakage, failure of chemicalmechanical preparation, or poor quality root canal filling with the result that certain microorganisms survive or reinfection ensues. of the microorganisms associated with the failure of root canal treatment, one of the most common is enterococcus faecalis (e. faecalis).5 this is due to its ability to survive in environmental conditions low in nutrition and to form biofilms which renders e. faecalis 1000 times more resistant to phagocytic cells, antibodies and antimicrobials compared to those organisms unable to manufacture biofilm.1,6 biofilms are defined as multicellular microbial communities characterized by cells that attach strongly to the surface and produce a matrix extracellular polymeric substance (eps).7 eps consists of bacterial proteins, nucleic acids, polysaccharides and fats. microbes that form biofilms are thought to be the cause of 80% of infections.8 herbs, fruits and plants are widely used, especially in the fields of medicine and dentistry. food crops are known to be rich in bioactive compounds, especially polyphenols, which possess antioxidant and antimicrobial properties. one of the food plants rich in antioxidants whose pod husk has antimicrobial properties is cocoa.9–12 the cocoa pod husk contains unsaturated fatty acids and epakitin polymers which promote antibacterial and antiglucosyltransferase activity, whereas the coco pods consist mainly of polysaccharides (cellulose and hemicellulose), lignin and small quantities of phenolic compounds, tannin, purine alkaloids and cocoa butter.10 the minimum concentration that can inhibit e. faecalis biofilm formation is one of 3.125%.13 to the best of the authors’ knowledge, no studies evaluating the effect of cocoa pod husk on the thickness of e. faecalis eps biofilm have, to date, been conducted. therefore, the purpose of this research was to determine the minimum inhibitory concentration of cocoa pod husk extract on e. faecalis eps biofilm thickness. materials and methods the ingredients used in this study consisted of forastero type cocoa fruit (theobroma cacao l.) extract at concentrations of 1.56%, 3.125%, and 6.25%. the cocoa pods obtained from the coffee research center and cocoa jember were of the forestero type with a yellow mark when picked which had been cooked. before processing, the picked pods were left for approximately five days to facilitate the release of their entire contents, including the seeds, from the cocoa husk. this is a process known as maceration.13 the 6 kg of fresh cocoa pod husk used in this study was cut and aerated. half-dried pod husk was further dehydrated in an oven at a temperature of 50° c, producing 1 kg of desiccated husk which was subsequently milled, macerated with 70% ethanol for 24 hours and filtered. this process produced filtrate and dregs which were soaked and filtered a second time. the maceration and filtration processes were repeated until a clear filtrate had been obtained. at this point, ethanol evaporation was conducted by means of a rotary evaporator at a temperature of 50° c to obtain cocoa pod husk extract thick in texture. five liters of ethanol were required during the solvation process which produced 134 grams of cocoa pod husk extract. this study used 32 samples divided into four treatment groups, namely; group 1 (control group) consisting of e. faecalis without cocoa pods husk extract; group 2 containing e. faecalis bacterial culture with 1.56% cocoa pod husk extract; group 3 containing e. faecalis bacterial culture with 3.125% cocoa pod husk extract; and group 4 containing e. faecalis bacterial culture with 6.25% cocoa pod husk extract. e. faecalis bacteria stock was diluted in accordance with standard mcfarland 0.5 or 1.5 x 10 cfu/ml to obtain a density of 106 cfu/ml. the stock was then cultured in tsb media in a flat button 24 well microtiter plate before being incubated for 3 x 24 hours at 35ºc.13,14 after the biofilm formation process, cocoa pod husk extract was applied to each titer at concentrations of 1.56%, 3.125%, and 6.25% and then incubated again at 35º c for 24 hours. at this point, the contents of each microtiter plate were aspirated, washed four times with 0.2 ml of phosphatebuffered saline (ph 7.3), cleared of planktonic bacteria by means of a pipette and, finally, dried. the biofilms attached to the microtiter plate were stained with 1ml of alexa dextran (thermo fisher scientific, singapore) stored in dark conditions for thirty minutes and rinsed with aquadest to remove any dyestuffs present. following the staining procedure, the appropriate specimens were immediately examined with a confocal laser scanning microscope (clsm) at 400x magnification (olympus, tokyo, japan). preliminary research was conducted to obtain the minimum concentration using the calculation of bacterial density in biofilms conducted by an optical density (od) unit incorporating an elisa reader. the difference between the treatment group and the control group was determined by completion of a post hoc test (p = 0.05). a tukey hsd was used to assess the significance of the differences between the treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p215–218 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p215-218 217yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 215–218 results figure 1 shows the intensity values of eps biofilms reviewed through 3d slices. the brighter color of the graph indicates the remaining eps biofilms with dyes that remain attached to this eps and their higher intensity values. from the image, it is evident that there is a difference in intensity value between the eps biofilm of the control group (group 1) and that of the treatment groups (group 2, group 3 and group 4). the 6.25% cocoa pod husk has the lowest intensity, while the control group has the highest intensity. the mean and standard deviation of each sample group used to quantify the value of e. faecalis eps biofilm thickness is shown in the table 1. the results of the post hoc tukey hsd test (table 2) confirmed a significant difference between group 1 and group 4. in contrast, when group 2 and group 3 are compared to group 1, there were no significant differences between the values of each, viz; p=0.340 and p=0.267 (p>0.05). discussion this study aims to establish the concentration of inhibitory formation e. faecalis eps biofilm following exposure to cocoa extract (theobroma cacao) which represents a potential alternative material for root canal irrigation. this study used cocoa pod husk extract at concentrations of 1.56%, 3.125% and 6.25%. in this study there was a significant difference between the control group (group 1) and the 6.25% cocoa pod husk extract (group 4), whereas in the 1.56% cocoa pod husk extract group (group 2) and the 3.125% cocoa pod husk extract group (group 3) no significant difference was evident when compared to the control group (group 1). it can be seen that the level of concentration of cocoa pod husk extract influences the inhibition of e. faecalis eps biofilm formation which reached the minimum inhibitory concentration of 6.25%. this is due to the fact that cocoa pod husk contains alkaloid, flavonoid, tannin and saponin all of which possess antibacterial properties.14 a b c d figure 1. fluorescence color intensity chart and eps thickness. (a) group 1 (b) group 2 (c) group 3 (d) group 4. table 1. mean and standard deviation of e. faecalis eps biofilm thickness group n mean(nm) sd (nm) group 1 8 9500.00 1195.23 group 2 8 8125.00 1727.89 group 3 8 8000.00 2000.00 group 4 8 6375.00 1408.00 note: n = number of samples; mean = average; sd = standard deviation table 2. difference test between treatment groups (tukey hsd test) group 1 group 2 group 3 group 4 group 1 0.340 0.267 0.03* group 2 0.999 0.156 group 3 0.206 group 4 note: * there is a significant difference (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p215–218 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p215-218 218 yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 215–218 the mechanism of tannin inhibiting the formation of eps biofilms is that of binding and precipitating proteins in eps. in addition, tannins are able to bind to carbohydrates where the greater the molecular weight, the stronger the interaction. tannin is also a chelating agent because it is able to form bonds with iron ions which will result in a rupturing of the eps matrix bond.15 the mechanism of saponin as antibiofilm involves reducing the bacterial extracellular dna component resulting in decreased biofilm formation. bioactive fractions that are rich in saponins can also inhibit the formation of biofilms by preventing the initial cell-surface attachment of bacteria.16 from the explanation above, it can be concluded that the presence of compounds found in the extract of cocoa pod husk can inhibit the formation of e. faecalis eps biofilms. the extract of 6.25% cocoa pod husk is at a concentration that can reduce the thickness of e. faecalis eps biofilm. references 1. hargreaves km, berman lh, rotstein i. cohen’s pathways of the pulp. 11th ed. st. louis: mosby elsevier; 2015. p. 621–5. 2. guivarc’h m, ordioni u, ahmed hma, cohen s, catherine jh, bukiet f. sodium hypochlorite accident: a systematic review. j endod. 2017; 43(1): 16–24. 3. spencer hr, ike v, brennan pa. review: the use of sodium hypochlorite in endodontics potential complications and their management. br dent j. 2007; 202(9): 555–9. 4. zhu wc, gyamfi j, niu ln, schoeffel gj, liu sy, santarcangelo f, khan s, tay kcy, pashley dh, tay fr. anatomy of sodium hypochlorite accidents involving facial ecchymosis a review. j dent. 2013; 41(11): 935–48. 5. murad cf, sassone lm, faveri m, hirata r, figueiredo l, feres m. microbial diversity in persistent root canal infections investigated by checkerboard dna-dna hybridization. j endod. 2014; 40(7): 899–906. 6. jhajharia k, mehta l, parolia a, shetty kv. biofilm in endodontics: a review. j int soc prev community dent. 2015; 5(1): 1. 7. mohammadi z, palazzi f, giardino l, shalavi s. microbial biofilms in endodontic infections: an update review. biomed j. 2013; 36(2): 59–70. 8. kreth j, herzberg mc. molecular principles of adhesion and biofilm formation. in: chávez de paz le, sedgley cm, kishen a, editors. the root canal biofilm. berlin, heidelberg: springer; 2015. p. 23–53. 9. matsumoto m, tsuji m, okuda j, sasaki h, nakano k, osawa k, shimura s, ooshima t. inhibitory effects of cacao bean husk extract on plaque formation in vitro and in vivo. eur j oral sci. 2004; 112(3): 249–52. 10. byung yc, cho jy, seung sl, nishiyama y, matsumoto y, iiyama k. the relationship between lignin and morphological characteristics of the tracheary elements from cacao (theobroma cacao l.) hulls. j plant biol. 2008; 51(2): 139–44. 11. mulyatni as, budiani a, taniwiryono d. aktivitas antibakteri ekstrak kulit buah kakao (theobroma cacao l.) terhadap escherichia coli, bacillus subtilis, dan staphylococcus aureus. menara perkeb. 2012; 80(2): 77–84. 12. sartini, djide mn, alam g. ekstraksi komponen bioaktif dari limbah kulit buah kakao dan pengaruhnya terhadap aktivitas antioksidan dan antimikroba. maj obat tradis. 2012; 14(4): 47–54. 13. yuanita t, vergeina d, rukmo m, zubaidah n, wahjuningrum da, kunarti s. antibiofilm power of cocoa bean pod husk extract (theobroma cacao) against entercoccus faecalis bacteria (in vitro). in: international medical device and technology conference. johor bahru: universiti teknologi malaysia; 2017. p. 129–31. 14. rachmawaty, mu’nisa a, hasri. analisis fitokimia ekstrak kulit buah kakao (theobroma cacao l.) sebagai kandidat antimikroba. in: proceedings of national seminar. makassar: research and community service institute universitas negeri makassar; 2017. p. 667–70. 15. daglia m. polyphenols as antimicrobial agents. vol. 23, current opinion in biotechnology. 2012. p. 174–81. 16. kanaan h, el-mestrah m, sweidan a, as-sadi f, bazzal a al, chokr a. screening for antibacterial and antibiofilm activities in astragalus angulosus. j intercult ethnopharmacol. 2017; 6(1): 50–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p215–218 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p215-218 173173 allergic contact cheilitis due to lipstick yatty ravitasari, desiana radithia, and priyo hadi department of oral medicine faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: cheilitis is a common problem of unknown etiology. a possible cause of cheilitis is contact allergy. drugs, lipsticks, sunblock and toothpaste are the most common implicated allergens. allergic contact cheilitis is a chronic superficial inflammatory disorder of the vermilion borders characterized by desquamation due to delayed-type hypersensitivity reaction. purpose : we report a management of allergic contact cheilitis due to lipsticks. case: a 21-year-old woman had a history of atopic allergy to eggs, milk, and chicken presented with sore, dry, fissured, scaled and sometimes bleeding lip, over a 3-month period after application of a lipstick. her symptoms persisted despite treatments with hydrocortisone cream. the patient provided a detailed history and underwent physical examination and patch tests to cosmetic components and patch test to her own lipstick. the patient had strongly-positive result to the tested lipstick. a diagnosis of allergic contact cheilitis was made based on the history and clinical findings. case management: patient was advised to avoid wearing lipstick. to relieve symptoms, treatment was initiated with combined topical corticosteroid, antibiotic, and moisturizer. conclusion: contact allergy patients should be tested for both cosmetic component series and their own lipsticks to exclude exfolliative cheilitis, infection, or light actinic cheilitis as causal agents. keywords: allergic contact cheilitis; delayed-type hypersensitivity; lipstick allergen correspondence: yatty ravitasari, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: yravitasari@gmail.com case report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 173–176 introduction in normal circumstances, body’s defense mechanism, both humoral and cellular, is dependent on the activation of b cells and t cells. however, the excessive activation by antigens or interruption of this mechanism will trigger undesirable immunopathology condition since it can cause tissue damage to fatal circumstances, such as death called hypersensitivity reaction.1 hypersensitivity reaction, first introduced by von pirquet in 1906, is a change in immune activity induced by antigens.2 hypersensitivity reaction outlined by gell and coombs can be divided into four types based on speed and immune mechanism of hypersensitivity reaction. hypersensitivity reaction may occur through one type of reaction, but clinically two or more types of these reactions may occur in the same time.3 allergic contact cheilitis is one of allergy types classified into delayed hypersensitivity arising after mucosal contact with certain substances and 24-72 hours after exposure to antigens. those substances, for example, are drugs, cosmetics, metal, and other substances. at the time of first contact with the skin, mucous substances will penetrate into the bottom layer of the epidermis, then bind to protein carriers, and change into immunogenic. after that, it will trigger a hypersensitivity reaction characterized by the presence of erythema and edema. this manifestation sometimes is followed with the presence of vesicles on more severe condition.1,4 delayed hypersensitivity does not involve antibodies, but involves t lymphocytes. this reaction occurs because t lymphocytes synthesized will react specifically with a certain antigen, leading to an immune reaction.3,5 cheilitis is a term commonly used to describe an inflammation in the vermillion border of the lips. vermilion border is the boundary between mucosa and skin. in this area, there is a thin layer of the epithelium and quite a dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p173-176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p173-176 174 ravitasari, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 173–176 lot capillaries supplying blood in order to give red color in lips.3,6 cheilitis can be considered as an inflammatory reaction that occurs in the mouth caused by exogenous and endogenous factors. exogenous factors, for instance, are lipstick, lip balm, sunscreen material, and dental materials. contact cheilitis can be classified into several types, namely allergic contact cheilitis, irritant contact cheilitis, and atopic cheilitis.6,7 15-20% of allergic contact cheilitis cases are caused by cosmetics, especially lipstick and powder beauty, and mostly occur in women. lipstick is made of various substances, such as lanolin, perfume, and some metals (metal cadmium, lead, and nickel) needed to make the color more resistant and to make the packaging of the lipstick. patch test examination on 196 cases of allergic contack cheilitis showed that 16% of positive relation to cosmetic products used by the patients.5,6 we report a management of allergic contact cheilitis due to lipsticks. case this case was about a female student aged 21 years who visited the clinic of mouth disease, faculty of dental medicine, universitas airlangga on june 4, 2015 with complaints of pain, stiff, dry, cracked, and bleeding easily on the upper and bottom lips since 3 months earlier. those complaints arose after using a new lipstick obtained from friends. this patient had never worn this brand of lipstick before. she actually had ever visited a dermatologist since her lips was getting hot and sore. she was then asked to take hydrocortisone ointment regularly twice a day for 2 weeks. during using the ointment, she felt more comfortable since her complaints reduced. when the ointment was up, she felt her lips more dried, cracked, sored, and bleeding easily. since her lips were dried and chapped, she started to try to peel them and given lip balm. in addition, she also tried to smear her lips with honey, but the condition of her lips did not change. based on her medical history, she had allergies to chicken, eggs, milk, and shrimp. she also suffers from asthma and gastritis. drugs that had been undertaken were cetrizin®, but only when allergy recurrence. her sister also has allergies to dust the results of clinical examination conducted were multiple fissure, easy bleeding, exfoliative condition, pain in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip as shown in figure 1. case management in the first visit, the patient was given oxyfresh and ascribed to have complete hematological laboratory examination, total ige, and patch test. the patient was encouraged to use the drug regularly, avoid using lipstick brands that cause allergies and other brands, avoid eating foods that can trigger allergies, and control after obtaining the examination results. in the second visit, on the fourth day after the first visit, the patient felt better since her complaints diminished, but she still felt her lips little stiff, dry, and fissure bleeding easily. the last dose of the drug recommended was taken in that morning. however, she admitted that she ate chicken two days earlier despite knowing she has allergy to chicken. she then took cetrizin® since she felt itchy after eating chicken. during clinical examination, there were red areas, multiple fissures bleeding easily, and desquamation at the vermillion border of the upper and lower lips found. she came with the results of the laboratory tests. the results showed that her neutrophil count was low as well as her lymphocytes, monocytes, and reticulocytes, but her erythrocyte sedimentation rate was higher than the normal one. during this second visit, she was given compounded prescription, including hydrocortisone 0.125 g, kemicitine 0.125 gr, lanolin 0.25 g, and 5 g of petroleum jelly in the 8 figure 1. the condition of the patient’s mouth, namely multiple fissure, easy bleeding, exfoliative condition in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip. figure 2. (a) the condition of the patient's lips in the second visit on day 4; (b) the condition of the patient's lips in the third visit on day 11. figure 3. the condition of the patient's lips in the fourth visit on the 14th day. there was no abnormality in her upper and lower lips. the patient was declared cured. figure 1. the condition of the patient’s mouth, namely multiple fissure, easy bleeding, exfoliative condition in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip. 8 figure 1. the condition of the patient’s mouth, namely multiple fissure, easy bleeding, exfoliative condition in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip. figure 2. (a) the condition of the patient's lips in the second visit on day 4; (b) the condition of the patient's lips in the third visit on day 11. figure 3. the condition of the patient's lips in the fourth visit on the 14th day. there was no abnormality in her upper and lower lips. the patient was declared cured. a 8 figure 1. the condition of the patient’s mouth, namely multiple fissure, easy bleeding, exfoliative condition in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip. figure 2. (a) the condition of the patient's lips in the second visit on day 4; (b) the condition of the patient's lips in the third visit on day 11. figure 3. the condition of the patient's lips in the fourth visit on the 14th day. there was no abnormality in her upper and lower lips. the patient was declared cured. b figure 2. (a) the condition of the patient’s lips in the second visit on day 4; (b) the condition of the patient’s lips in the third visit on day 11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p173-176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p173-176 175175ravitasari, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 173–176 form of a topical drug applied to the lips four times a day. she was also encouraged to use the drugs on a regular basis, and avoid using all brands of lipstick either causing allergies or not. she was stressed to avoid eating foods triggering allergies and asked to come to control seven days later. in the third visit on the eleventh day, the patient felt better without any complaints of pain in her upper and lower lips. her lips were not dry anymore, yet still little stiff. she had taken the drugs on a regular basis in accordance with the doctor’s instructions. she also had avoided eating food causing allergens and using any brand of lipstick. during clinical examination, her upper lip did not seem to have any abnormalities, but her lower lip seemed to have multiple minor erosion. she was then instructed to continue the treatment and avoid eating foods considered as the originator of her allergy. she was also encouraged to use the drug on a regular basis, and avoid using all brands of lipstick, lip gloss, or lip cosmetic products. in the fourth visit on the fourteenth day, she had no complaints. she felt comfortable and had no pain anymore on her lips. the topical drug concoction was up, and the last one was used in that morning. during clinical examination, there was no abnormality on her upper and lower lips (figure 3). the patient was instructed to discontinue using topical medication concoction. she also asked to avoid eating foods triggering allergies and also using any brand of lipstick. she was then referred to do a patch test in dermatology section of hospital rs. dr sutomo, surabaya to determine the cause of the allergy. she came again without any complaints with the results of the patch test. she had not used any brand of lipstick and not eaten food causing allergy two weeks before the allergy test. she followed those instructions well. based on the results of the patch test, it was concluded that the patient was allergic to lipstick. on this visit, the treatment was complete, and the patient was advised to maintain oral hygiene and avoid consuming certain ingredients triggering allergies. she was also advised to avoid using of lipstick causing allergens and to perform a skin test on a new lipstick that will be used. discussion lipstick is one of the most frequent cosmetics that can cause allergic contact cheilitis, and often occurs in women. allergic contact cheilitis is usually caused by the use of lipstick because of materials contained in lipstick. the main materials composed of the lipstick are metal, dyes, and fragrances, and patients are often allergic to one or three ingredients.5,7 patch test was performed on the materials contained in any lipstick and the lipstick worn by the patient because the patient might also not be allergic to the ingredients composing the lipsticks, but allergic to the lipstick used. chemical substances contained in lipstick can bind to carrier proteins in the body in order to be immunogenic. immediately after contacting with the mucosa, the carrier proteins will bind these ions and become immunogenic antigens. the protein molecules will then become phagocyte by macrophage functioning as a cellular immune response and as antigen presenting cells (apc). through major histocompatibility complex (mhc), the class 2 proteins were presented on the cell surface of the apc in the form of fragments in order to be recognized by t lymphocytes and activate t-helper and tc memory lymphocytes. on the next contact, t memory cells will recognize antigens and activate th1 and th2, known as cd4 + and cd8 + t considered as cytotoxic t set. lymphocytes activated will then secrete cytokines including interleukin-2 (il-2) and interferon-gamma (ifn-γ) which are signaling proteins that have strong chemottraction to attract eosinophils, basophils, and macrophages that can cause inflammatory responses and tissue damage manifesting on lips.1,2,7 based on history, clinical examination, and patch test, the patient was diagnosed with allergic contact cheilitis because of inflammation in mouth after contacting with materials suspected to trigger allergic reactions. allergic contact cheilitis is a contact allergic reaction on lips. in this case, it was caused by chemical substances contained in the lipstick worn by the patient. this reaction had caused inflammation in the lip area. the symptoms, however, depend on the frequency and duration of contact with allergens. contact allergies can be classified into type 4 hypersensitivity reactions or delayed-type hypersensitivity. this reaction occurs in 24-72 hours after exposed to an allergen lasting until the next 2-3 days. in this type, there is no involvement of immunoglobulin and t-cell mediation (tcell-mediated hypersensitivity). this type of allergic reaction can only take place if no prior contact with allergens, and also become sensitized. subsequent exposure to the allergens will cause several symptoms, such as: exzema-like lesions, fissures, erythema, burning sensation, as well as itching, and in more severe conditions it can trigger vesicles and crusting.2,4,7,8 8 figure 1. the condition of the patient’s mouth, namely multiple fissure, easy bleeding, exfoliative condition in the vermillion border of the upper lip, and multiple minor erosion on the vermillion border of the lower lip. figure 2. (a) the condition of the patient's lips in the second visit on day 4; (b) the condition of the patient's lips in the third visit on day 11. figure 3. the condition of the patient's lips in the fourth visit on the 14th day. there was no abnormality in her upper and lower lips. the patient was declared cured. figure 3. the condition of the patient’s lips in the fourth visit on the 14th day. there was no abnormality in her upper and lower lips. the patient was declared cured. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p173-176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p173-176 176 ravitasari, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 173–176 patients are treated with topical hydrocortisone lips, lanolin, kemicitine, and vaseline. hydrocortisone is classified into the group of corticosteroids that are anti-inflammatory to reduce inflammation of the lips. kemicitine is a broad-spectrum antibiotic with another name, chloramphenicol. kemicitine is sensitive against gram positive negative bacteria, thus, additional administration of antibiotics in these patients is aimed to prevent secondary infection because of their open wounds, such as deep fissure and bleeding easily. lanolin is added as a natural moisturizer needed to alleviate the symptoms of dry mouth. vaseline is an additional material as an ointment base.9 in this visit, the administration of those drugs was aimed to deliver treatment in order to immediately reduce complaints in patient. patch test only could be performed after the patient had been declared cured since to perform this test, the patient should be free from the use of medications, especially corticosteroids, and also free of lesions that may increase due to a hypersensitivity reaction and can interfere with the reading of the results of the patch test. reading was taken at 48 hours, 72 hours and 7 days after the test. based on the results of the patch test, the patient was allergic to the lipstick worn. management in this case consisted of tracing the history of allergy on the patient and her family, and conducting patch test procedure to determine definitely allergens contacting with the patient and causing allergic reactions. avoiding allergens is the key to the success of the treatment in addition to the use of drugs needed to suppress inflammation and reduce the patient’s complaints. 10,11 in this case management, the patient was then diagnosed with allergic contact cheilitis with lipstick as contributing factors. diagnosis was made based on the history of allergies and clinical features paired with the patch test as a primary screening contact allergy. differential diagnosis was exfoliative cheilitis because it clinically had desquamation description on the vermillion borders of the lips. the main treatment was to avoid the cause and the use of steroid drugs to suppress inflammation. in conclusion, patients with allergic contact cheilitis against cosmetic products are advised to be more careful in choosing and using cosmetics, especially lipstick. finally, it is recommended for skin testing prior to cosmetic use. references 1. munasir z, suyoko emd. reaksi hipersensitivitas. in: akib a, kurniati n, editors. buku ajar alergi-imunologi anak. 2nd ed. jakarta: idai; 2010. p. 115-31. 2. holgate st, church mk, broide dh, martinez fd. allergy. 4th ed. edinburg: saunders elsevier limited; 2012. p. 112-4. 3. baratawidjaja kg, rengganis i. imunologi dasar. in: alergi imunologi. jakarta: badan penerbit idai; 2010; p. 241-3. 4. delong l, burkhart wn. general and oral pathology for the dental hygienist. philidelphia: lippincot williams & wilkins; 2008. p. 49, 61-8. 5. otto s, zirwas mj. toothpaste allergy diagnosis and management. j clinical and aestetic dermatology 2010; 5(1): 1-7. 6. draelos, diana md. a cosmetic approach to cheillitis. j cosmetic dermatology 2005; 18(10): 709-11. 7. goossens a. new cosmetic contact allergens. j cosmetics 2015; 2(1): 22-32. 8. goossens a, rance f. contact allergic reactions to cosmetics. j allergy 2011; 2(11): 1-6. 9. lokesh p, rooban t, elizabeth j, umadevi k, ranganathan k. allergic contact stomatitis: a case report and review of literature. indian journal of clinical practice 2012; 22 (9): 458-62. 10. shetty sr, rangare a, babu s, rao p. contact allergic cheilitis secondary to latex gloves: a case report. j oral and maxillofacial research 2011; 2(1): 1-5. 11. pigatto p, martelli a, marsili c, fiocchi a. contact dermatitis in children. italian journal of pediatrics 2010; 36(2): 1-6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p173-176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p173-176 vol 52 no 1 jan-mar 2019_new.indd 4141 the difference in microleakage levels of nanohybrid composite resin using eighth-generation ethanol and isopropanol solvent bonding materials under moist and dry conditions (in vitro study) irfan dwiandhono, setiadi w. logamarta, and taura dhanurdara faculty of medicine, universitas jenderal soedirman, purwokerto – indonesia abstract background: microleakage during restoration causes secondary caries. the shrinkage of nanohybrid composite resin can occur during the polymerization process, affected by both the selection of bonding materials featuring different solvents, such as ethanol and isopropanol, as well as contrasting conditions such as moist and dry. purpose: this study aimed to determine and analyze the differences of microleakage level of nanohybrid composite resin using the eighth-generation bonding materials made from ethanol and isopropanol solvents under moist and dry conditions. methods: this research constituted an experimental laboratory study. the samples were divided into four groups. group i used a bonding material produced from ethanol under dry conditions. group ii used a bonding material produced from ethanol under moist conditions. group iii used a bonding material produced from isopropanol under dry conditions. group iv used a bonding material produced from isopropanol under moist conditions. the levels of microleakage were subsequently tested using a stereo microscope. results: microleakage examination was performed by means of a stereo microscope to observe the methylene blue color penetration with assessment subsequently being performed on a scale of 0 to 3. the statistical results of a kruskal-wallis test showed that no significant differences occurred in any of the treatment groups (sig = 0.141, p<0.05). conclusion: there was no difference in the microleakage levels of nanohybrid composite resin using eighth-generation bonding materials produced from ethanol and isopropanol solvents under moist and dry conditions. keywords: eighth-generation bonding; moist; dry; ethanol; isopropanol correspondence: irfan dwiandhono, faculty of medicine, universitas jenderal soedirman. jl. dr. soeparno karangwangkal, purwokerto, jawa tengah 53123, indonesia. e-mail: irfandrg@gmail.com dental journal (majalah kedokteran gigi) 2019 march; 52(1): 41–44 research report introduction eighth-generation bonding materials are known to be universal adhesive materials which can be applied not only using total-etch and self-etch techniques, but also by means of a selective-etch technique.1 eighth-generation bonding has several advantages including its applicability to moist and dry cavities, since it can be made from solvents such as ethanol (c2h5oh) and isopropanol (c3h7oh) which can absorb excess water relatively effectively.2 isopropanol demonstrates a greater ability to bind water and a higher viscosity than ethanol. the evaporation rate of isopropanol is lower than that of ethanol with the result that it can absorb water more effectively and the smear layer below the liquid can be modified and infiltrated into the dentinal tubules that then bind to collagen fibers.2 under moist conditions, the bonding technique produces strong bonds between dentine and composite resin, while excessively moist dentin conditions can induce the trapped water to interfere with the diffusion and polymerization of monomer resins. during this process, porous layers can be formed with the result that the bond decreases both in vivo and in vitro. meanwhile, under dry dentine conditions, the application of bonding will result in obstruction of monomer diffusion due to collagen matrix collapse.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p41–44 mailto:irfandrg@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p41-44 42 dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 41–44 a previous study compared the shear bond strength of the attachment between ethanol and acetone solvents using one step self-etch bonding technique under dry and moist dentine conditions. this previous study demonstrated that bonding material made from ethanol solvent possessed higher shear bond strength under dry conditions than ethanol solvent under moist conditions. moreover, this previous study also showed that bonding material made from acetone solvent possessed a higher adhesive shear strength under moist conditions than did such materials under dry conditions.4 nanohybrid composite resin contains nano-sized particles, namely; 0.005-0.020 μm in the resin matrix.5 it also contains additional components of the resin matrix in the form of nanoparticles and nanoclusters6, the combination of which can reduce the interstitial distance between particles, thereby increasing resistance filler, physical properties, mechanical properties and retention.7,8 microleakage can be caused by shrinkage during the polymerization process and the lack of adhesive material attachments to enamel and dentin. emergent cracks are still considered restoration-related problems because fluids, ions and bacteria can pass through them, causing other problems such as secondary caries, discoloration of ridge edges, pulp irritation and sensitive teeth. therefore, the eighthgeneration bonding that combines etching, primer and adhesive components is expected to reduce the weakness of previous generation bonding with the result that it can be applied not only to moist and dry cavity conditions, but can also support dentists in their application to achieve the success of restoration by causing more limited microleakage.8 hence, this study aimed to determine the difference of microleakage levels of nanohybrid composite resin using the eighth-generation bonding materials made from ethanol and isopropanol as solvents under moist and dry conditions. materials and methods this study, which constituted an experimental laboratory research involving 36 maxillary permanent premolars, received ethical approval from the health research ethics commission of the faculty of medicine, universitas jenderal soedirman (number: 272/kepk/x/2018). a rectangular grade v cavity on the buccal side 1mm above the cervical line of the tooth was created in each sample. the resulting cavities had a mesiodistal length of 3 mm, an occluso-gingival width of 2 mm and a buccolingual direction depth of 2 mm.9 a self-etch technique was employed to apply the eighth-generation bonding materials to those cavities which were then filled with nanohybrid composite. thereafter, the samples were divided into four groups, namely: group i ethanol-based bonding applied under dry conditions, group ii ethanol-based bonding applied under moist conditions, group iii isopropanolbased bonding applied under dry conditions and group iv isopropanol-based bonding applied under moist conditions. in groups i and iii, application of the eighth-generation bonding material under dry conditions was performed after the cavities had been cleaned and dried with a wind spray for ten seconds. meanwhile, in groups ii and iv, the application of the eighth-generation bonding material under moist conditions was conducted after the cavities had been cleaned with water and rubbed for three seconds using an absorbent pad without contact being made with the bases of the cavities. the eighth-generation bonding material produced from ethanol solvent was applied to groups i and ii for 20 seconds, while the cavities in those groups were scrubbed. similarly, the eighth-generation bonding material manufactured from isopropanol was also applied to groups iii and iv for 20 seconds, while the cavities in those groups were being scrubbed. after application of the bonding materials, the cavities were dried with a wind spray for ten seconds and a curing light for 20 seconds. nanohybrid composite resin was then applied to the cavities until they were completely closed.10 the samples were soaked in receptacles containing distilled water, placed in an incubator at 37oc for 24 hours and dried a with paper towel. the entire surface of the samples up to 1 mm around the restoration areas was subsequently coated with transparent nail polish before being immersed in a methylene blue solution and incubated for four hours at 37oc. after each group had been treated, all the teeth were washed with water to remove the remaining methylene blue and dried with a paper towel. finally, the samples were cut vertically to enable the mesial and distal portions to be extracted.11 each pair of tooth halves was examined for signs of microleakage around the edge of the teeth with a stereo microscope (olympus sz61). one side of the tooth halves in each sample which had the deepest penetration of methylene blue 0.5% was then selected for scoring. the scores for microleakage assessment comprised: score 0 which indicated no color penetration of the cavity; score 1 which related to penetration of less than or equal to half the cavity wall; score 2 which indicated color penetration of more than half the cavity wall, but not extending to the base of the cavity and score 3 which related to color penetration reaching the base of the cavity.12 the results of the data obtained were in the form of ordinal data scales. the data was subsequently analyzed using a non-parametric statistical test, the kruskal wallis test, to determine differences in microleakage levels between groups. results microleakage examination was performed by means of a stereo microscope to observe the methylene blue color penetration with assessment subsequently being performed on a scale of 0 to 3. the mean score of microleakage levels in each group can be seen in figure 1 which shows that the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p41–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p41-44 43dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 41–44 respective mean microleakage scores in the various groups was as follows: group i 2.78 ± 0.441, group ii 2.67 ± 0.707, group iii 2.33 ± 0.707 and group iv 1.89 ± 1.054. the microleakage test-produced data of all groups were then statistically analyzed with a kruskal-wallis test to determine the effects of the overall bonding solvents on microleakage levels under different conditions (see table 1). according to the kruskal-wallis statistical test results, the p-value of the microleakage level between groups was 0.141. this indicates that there was no significant difference in the levels of microleakage between groups (p<0.05). discussion the results of this study showed that there was no significant difference between the application of the eighth-generation bonding material made from ethanol solvent under moist and dry conditions. the low level of viscosity characteristic of ethanol enables it to facilitate penetration of bonding material into dentinal tubules. ethanol also has a vapor pressure of 5.95 kpa with the result that the high evaporation rate will cause the solvent to vaporize more rapidly. as a result, the eighth-generation bonding materials, which still require longer to penetrate the dentinal tubules, can evaporate faster.13 the results also indicated that the dry and moist conditions of cavities does not affect the bonding materials when the self-etch technique is employed. moreover, because it is not applied to the etching separately, it neither removes the smear layer nor open ups collagen fibers. under moist conditions, the water molecules remain present in the dentine. if ethanol-based bonding material is applied under moist conditions, these cannot vaporize entirely because of the high level of ethanol evaporation resulting in disruption of the remaining water molecules and penetration of the bonding material. meanwhile, under dry conditions, no water molecules are present in the dentine with the result that the bonding material can penetrate the dentinal tubules. high levels of ethanol evaporation can accelerate bonding material evaporation before completely penetrating the dentinal tubules resulting in a low hybrid layer.14 acid in the eighth-generation bonding derived from ethanol and isopropanol solvents will produce areas of demineralization. however, because of the presence of acid in the eighth-generation bonding material with a ph higher than 2.5, the resulting demineralization is shallow. the dissolved smear layer will then emerge around the collagen fibers that form a low hybrid layer because of the area of low demineralization.11 the low hybrid layer will result in a weak bond between the tooth structure and the composite leading to microleakage. the enamel bonding mechanism will subsequently experience chemical interactions between the 10-mdp functional monomers which produce low microtags with the result that the self-etch bonding technique does not address all problems associated with enamel attachment.15 this finding is consistent with that of research conducted by usha et al. (2017),15 and choi et al. (2017),16. for example, usha et al. (2017),15 showed that high evaporation rates in ethanol trigger more rapid evaporation of bonding materials, while the results of a study conducted by choi et al. (2017),16 showed that the application of bonding material under dry dentin conditions will produce a low hybrid layer. in addition, the results of this study also found that there was no significant difference between the application of the eighth-generation bonding material made from isopropanol solvent under moist conditions and the application of such material under dry conditions. the viscosity level of isopropanol was high with the result that the bonding material will immediately dissolve the smear layer and open the dentinal tubules. high viscosity makes it difficult for bonding material to pass into dentinal tubules and expose collagen fibers.17 isopropanol also has a vapor pressure of 1.99 kpa. low vapor pressure will cause the solvent to evaporate over a longer period with the result that the bonding material still has time to penetrate the dentinal tubules to produce deeper dentine demineralization.18 if the bonding material made from isopropanol is applied under moist conditions, the the water molecules will be evaporated by isopropanol. the high viscosity of the bonding material made from isopropanol solvent renders the penetration of the bonding material shallow. similarly, under dry conditions, isopropanol does not cause water molecules to evaporate. however, the high viscosity of the bonding material made from isopropanol solvent triggers the formation of the low hybrid layer.14 finally, it can be concluded that the application of bonding materials made from ethanol and isopropanol solvents under moist and dry table 1. microleakage level results of kruskal-wallis test. sig.mean rankgroup 22.28i 0.141 22.50ii 16.67iii 13.56iv 2.78 2.67 2.33 1.89 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 group ivgroup i group ii group iii t he m ic ro le ak ag e le ve l figure 1. microleakage levels dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p41–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p41-44 44 dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 41–44 conditions still induces microleakage due to differences in evaporation and viscosity levels resulting in low hybrid layers. hence, it can be stated that there is no difference in the microleakage levels of nanohybrid composite resin between the eighth-bonding materials made from ethanol and isopropanol under both moist and dry conditions. references 1. hanabusa m, mine a, kuboki t, momoi y, van ende a, van meerbeek b, de munck j. bonding effectiveness of a new ‘multimode’ adhesive to enamel and dentine. j dent. 2012; 40(6): 475–84. 2. dos santos lgp, felippe wt, teixeira cs, bortoluzzi ea, felippe mcs. endodontic re-instrumentation enhances hydroxyl ion diffusion through radicular dentine. int endod j. 2014; 47(8): 776–83. 3. ayar mk, yildirim t, yesilyurt c. nanoleakage within adhesivedentin interfaces made with simplified ethanol-wet bonding. j adhes sci technol. 2016; 30(22): 2511–21. 4. al qahtani mq, al shethri se. shear bond strength of one-step self-etch adhesives with different co-solvent ingredients to dry or moist dentin. saudi dent j. 2010; 22(4): 171–5. 5. hatrick cd, eakle ws. dental materials: clinical applications for dental assistants and dental hygienists. 3rd ed. st. louise: elsevier saunders; 2015. p. 65–9. 6. ferooz m, basri f, negahdari k, bagheri r. fracture toughness evaluation of hybrid and nano-hybrid resin composites after ageing under acidic environment. j dent biomater. 2015; 2(1): 18–23. 7. jain n, wadkar a. effect of nanofiller technology on surface properties of nanofilled and nanohybrid composites. int j dent oral heal. 2015; 1(1): 1–5. 8. sakaguchi rl, powers jm. craig’s restorative dental materials. 13th ed. philadelphia: elsevier mosby; 2012. p. 73, 83, 117–8, 164–7. 9. sooraparaju sg, kanumuru pk, nujella sk, konda kr, reddy kbk, penigalapati s. a comparative evaluation of microleakage in class v composite restorations. int j dent. 2014; 2014: 1–4. 10. costa dm, somacal dc, borges ga, spohr am. bond capability of universal adhesive systems to dentin in self-etch mode after shortterm storage and cyclic loading. open dent j. 2017; 11: 276–83. 11. syafri m, nugraheni t, untara te. perbedaan kebocoran mikro resin komposit bulkfill vibrasi sonic dan resin komposit nanohibrid pada kavitas kelas i. j kedokt gigi. 2014; 5(2): 158–68. 12. ekambaram m, yiu cky, matinlinna jp. an overview of solvents in resin-dentin bonding. int j adhes adhes. 2015; 57: 22–33. 13. fleming ps, johal a, pandis n. self-etch primers and conventional acid-etch technique for orthodontic bonding: a systematic review and meta-analysis. am j orthod dentofac orthop. 2012; 142: 83–94. 14. ritter a v., boushell lw, walter r, sturdevant cm. sturdevant’s art and science of operative dentistry. 7th ed. st. louise: elsevier mosby; 2018. p. 149–50. 15. usha c, ramarao s, john bm, rajesh p, swatha s. evaluation of the shear bond strength of composite resin to wet and dry enamel using dentin bonding agents containing various solvents. j clin diagnostic res. 2017; 11: zc41–4. 16. choi a-n, lee j-h, son s-a, jung k-h, kwon y, park j-k. effect of dentin wetness on the bond strength of universal adhesives. mater. 2017; 10(11): 1–13. 17. monteiro ts, kastytis p, gonçalves lm, minas g, cardoso s. dynamic wet etching of silicon through isopropanol alcohol evaporation. micromachines. 2015; 6(10): 1534–45. 18. nair m, paul j, kumar s, chakravarthy y, krishna v, shivaprasad. comparative evaluation of the bonding efficacy of sixth and seventh generation bonding agents: an in-vitro study. j conserv dent. 2014; 17: 27–30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p41–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p41-44 109109 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 109–112 xerostomia severity difference between elderly using alcohol and non alcohol-containing mouthwash hendri susanto department of oral medicine faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: there are alcohol and non alcohol-containing mouthwash available in the market. alcohol-containing mouthwash may have side effects which induced by alcohol in the mouthwash. dry mouth/xerostomia may be a potential side effect of alcoholcontaining mouthwash when used by elderly person who has a tendency to have dry mouth. the evidence of xerostomia induced by alcohol-containing mouthwash used by elderly is not available yet. purpose: the aim of this study is to know the differences of xerostomia severity between elderly use alcohol-containing mouthwash and non alcohol-containing mouthwash. method: this study was performed in elderly with age above sixty who do not have systemic diseases based on anamnesis, do not have oral diseases, and do not have allergy to one of mouthwash components, do not use denture. of total, thirty elderly participated in this study. the first group consists of elderly who use alcohol-containing mouthwash (am) and the second group consists of elderly who use non alcoholcontaining mouthwash (nam). both groups use mouthwash for seven days (one week) twice a day. xerostomia severity was assessed by vas questionnaire. the mean score of the visual analogue score (vas) xerostomia each group in day one (baseline) and day eight (post treatment) was analyzed by the wilcoxon sign ranked test and mann whitney u test with 95% confidence level. result: the vas score of xerostomia post treatment (mean+sd/19.47+8.33) higher than baseline (mean+sd/15.87+8.91) in am group (p<0.05), but, there is no significant difference of vas score of xersotomia between post treatment (mean+sd/23.53+10.81) and baseline (mean+sd/23.67+11.82) in nam group (p>0.05). the mean difference of vas score of post-treatment and baseline between am and nam group was not significant (p>0.05). conclusion: the conclusion is no significance difference of xerostomia severity between alcohol-containing mouthwash and non alcohol-containing mouthwash in elderly. keywords: alcohol-containing mouthwash; elderly; non alcohol-containing mouthwash; xerostomia severity correspondence: hendri susanto, c/o: bagian ilmu penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta sekip utara no. 1 yogyakarta 55281, indonesia. e-mail: drghendri@ugm.ac.id. telp: +62274515307 introduction it has been known that mouthwash was recommended as additional routine oral health care after toothbrush. people like to use mouthwash because the advantages of mouthwash can reach all surfaces of the oral cavity which may not be achieved by a toothbrush and used mouthwash used within a short time. the campaign through the media, the availability of products of various brands and the content of mouthwash in stores or supermarkets, make people easier to choose mouthwash for maintaining oral health. sometimes, people use mouthwash excessively and do not use it according to the indications or suggested manufacturer’s instructions.1 there are two types of mouthwash available in the market today, alcohol-containing mouthwash and non alcoholcontaining mouthwash. basically, both types of mouthwash use for oral antiseptic and may have similar indications such as for reducing dental plaque, relieve inflammation of the periodontal tissues, especially gingivitis, reducing halitosis and also to prevent caries due the presence of the fluoride content in the mouthwash.2,3 the concentration of alcohol mouthwashes are commonly up to 21.6% with dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p109-112 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p109-112 110 susanto/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 109–112 the main content of essential oils. the alcohol-containing mouthwashes are available globally over the world. the containing of alcohol in mouthwash more than 10% has been suggested may cause discomfort in the oral cavity.4,5 although it is still controversial, the alcohol in mouthwash may be a risk factor for oral cancer.6,7 in addition, some evidence suggests that the use of alcohol in the mouthwash may cause complaints of dry mouth (xerostomia).6,8 whether the evidence of alcohol-containing mouthwash may give serious side effects in oral cavity or not, a nonalcohol-containing mouthwash has been developed. some products of non alcohol mouthwash have been already available in the market in indonesia.2,9 a study of kerr et al.10 showed that there is no difference of xerostomia and salivary secretion significantly between groups of adult subjects who use mouthwash containing alcohol (listerine) and non alcohol mouthwash (mint act). the study was conducted in adult subjects (mean age 40 years) with healthy and do not have systemic diseases, but, the mouthwash (mint act) used in that study was not available in indonesia. it is important to know the effect of alcohol containing mouthwash on xerostomia in elderly because there is not a study of the difference of xerostomia using mouthwash containing alcohol and non alcohol in the elderly yet since the pupulation of elderly relatively increased in indonesia. it is suggested that the decreased salivary secretion and xerostomia found in the elderly, although aging as a cause of the decreased salivary secretion and xerostomia in the elderly is still debated.11 most causes of decreased salivary secretion and xerostomia in the elderly are mainly by systemic diseases and drugs.12,13 xerostomia and decreased salivary flow rate (hyposalivation) are not similar. hyposalivation may cause xerostomia if the saliva production less than half of normal saliva production and xersotomia may be the first symptoms which will be found and told by patients in the clinic because the hyposalivation may be only assessed by salivary flow rate examination.14 the use of mouthwash may affect oral condition, and xersotomia may be the complaint of subjetcs who used mouthwash. alcohol-containing mouthwash may increase xerostomia severity in elderly. the aim of this study is to investigate the difference of xerostomia severity between elderly use non-alcohol mouthwash and alcohol mouthwash. material and methods this study was a pre-post study and performed in elderly who live in yogyakarta, indonesia. the inclusion criteria of this study is man or woman with age above 60 years old who do not have systemic diseases based on anamnesis such as diabetes mellitus (dm) etc, do not have acute oral diseases, and do not have allergy to one of mouthwash components, do not use denture, at least have four teeth in the mouth. only subjects who meet the inclusion criteria and sign the informed consent involve in this study. the exclusion criteria of this study are elderly who have motoric control problem, under dentist or physician treatment, have acute oral problems and refuse to involve in this study. this study divided into two groups. the first group consist elderly who use alcohol-containing mouthwash (listerie coll minttm) and the second group consists of elderly who use non alcohol-containing mouthwash (hexadoltm). all subjects do not know the mouthwash they received to use in this study. both groups use mouthwash for 7 days (one week) twice a day with each rinse 15 ml (30 ml for a day). before using mothwash (baseline), xerostomia severity was assessed by vas questionnaire15 and xerostomia severity reevaluate in day eight after routinely use mouthwash for seven days (post-treatment). the xersotomia severity assessed in the morning between 9.00-11.00 am. the mean score of the visual analogue score (vas) xerostomia each group in baseline and post-treatment was analyzed by the wilcoxon sign ranked test, and mann whitney u test with 95% confidence level using spss 16.0 statistical program (spss incorporation, chicago, usa). this study approved by ethical committee of faculty of dentistry, universitas gadjah mada. results there were 30 volunteer elderly who meet inclusion criteria participated in this study. fifteen elderly in the first group used non alcohol mouthwash and the other fifteen elderly in the second group used alcohol-containing mouthwash. the characteristic of subjects of this study present in table 1. the mean ages of subjects in non alcohol mouthwash (nam) was 67 year old, and the mean age in alcohol mouthwash (am) was 66 year old. female subjects were less in nam (40%) than 53% female in am group. all subjects in both group was retired and 100 % education level is middle education level graduate. almost all subjects were javanese. the vas score of xerostomia post treatment ( m e a n + s d / 1 9 . 4 7 + 8 . 3 3 ) h i g h e r t h a n b a s e l i n e (mean+sd/15.87+8.91) in am group (p<0.05), but, there is no significant difference of vas score of xersotomia between post treatment (mean+sd/23.53+10.81) and baseline (mean+sd/23.67+11.82) in nam group (p>0.05). the mean difference of vas score of post-treatment and baseline between am and nam group was not significant (p>0.05). discussion this study is the first study which comparing xerostomia severity between elderly using non alcohol-containing mouthwash and alcohol-containing mouthwash in indonesia. this study showed the increase of xerostomia severity of elderly who used alcohol-containing mouthwash dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p109-112 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p109-112 111111susanto/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 109–112 was higher than non alcohol-containing mouthwash which indicated by the high vas score after seven days using mouthwash. on the contrary, the difference of vas score of xerostomia severity of elderly who used non alcoholcontaining mouthwash between baseline and post treatment were relatively similar (table 2). however the mean vas scores of xerostomia in both pre and post treament in alcohol containing mouthwash subjetc were lower than the mean vas scores of xerostomia in non alcohol-containing mouthwash. this difference of mean score of vas in both groups may be caused by the effect of the other contents of alcohol-containing mouthwash (listerine) such as the essential oils which may mask dry sensation of alcohol in mouthwash in oral cavity. according to analysis, there was a significance differences of vas score of xerostomia between baseline (before using mouthwash) and post treatment (after using mouth for seven days) in elderly using alcohol mouthwash but there is no significant difference of vas score in nonalcohol mouthwash group between baseline and post-treatment (p<0.05). this study only assessed subjective feeling of xerostomia due the xerostomia may have more clinical relevance to assess salivary gland dysfunction or impairment rather than objective measurement using salivary flow rate. the measurement of salivary flow rate also tends to be influenced by the collection time of salivary flow rate and circadian rhythm.16 this result showed that there is no significant difference between mean difference of pre and post of alcohol-containing mouthwash and non alcoholcontaining mouthwash. this result was in line with kerr et al.17 study that showed there was not a significant difference of xerostomia between non alcohol-containing mouthwash and alcohol-containing mouthwash (table 2). we used the same alcohol-containing mouthwash (listerine) with alcohol concentration approximately to 21.6% as kerr et al. study.17 the difference between this study and kerr et al study were the non alcohol-containing mouthwash and the method of study. our study used hexadol (hexetidine 0.1%) which is one of non alcohol-containing mouthwashes available in market in indonesia and the study of kerr et al.,17 use min act total care mouthwash. hexetidine has been proved having an ability to inhibit of dental plaque attachment, and treat oral ulcers like the aphthous stomatitis. however, hexetidine with concentrations exceeding 0.1% may cause ulcers in the oral mucosa and erosion of the enamel.18 the duration of mouthwash used by elderly is the consideration in our study method, since the elderly were vulnerable person, so we performed in elderly only in seven days using mouthwash. besides, there is a possibility that topical adverse side effect of alcoholcontaining mouthwash to oral tissue in elderly.7 xerostomia does not always correlate with decrease salivary flow rate, people with xerostomia may not have salivary secretion impairment or in other way people with decreased salivary flow rate may not have xerostomia feeling.19 although aging may alter salivary gland tissue table 1. demography of study subjects variable elderly with non-alcohol mouthwash/nam (hexadol) elderly with alcohol mouthwash/am (listerine cool mint) n sex : n (%) female male ages : mean (sd) education level : n (%) middle (junior & high school) ethnicity: n (%) javanese 15 6 (40) 9 (60) 67.93 (4.95) 100% 93% 15 8 (53) 7 (47) 66.80 (44.04) 100% 100% n : number; sd : standard deviation table 2. comparison of xerostomia severity between non-alcohol mouthwash (nam) and alcohol mouthwash (am) group treatment vas score mean (sd) p| vas score mean (sd) diffference p# non-alcohol mouthwash (hexadol) alcohol-mouthwash (listerine cool mint) baseline post therapy baseline post therapy 23.67 (11.82) 23.53 (10.81) 15.87 (8.91) 19.47 (8.33) >0.05 <0.05 -0.13 (5.05) 3.60 (4.64) >0.05 sd : standard deviation; p : significance with p<0.05; | : wilcoxon sign rank t test; # mann whitney u test between mean difference post treatment and baseline dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p109-112 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p109-112 112 susanto/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 109–112 by replacing acinar cell by fibrous and adipose tissue, but there is not an agreement yet that aging may independent factor which may cause decreased salivary flow rate (hyposalivation). salivary flow rate were reduced with the increase age.20 most elderly may have low salivary flow rate because of multi factorial. the most factors that may cause xerostomia in elderly are medications and systemic diseases.11,21 in regard of the method which only a cross sectional study, this study showed that the alcohol may increase xersotomia severity in elderly, so, it did not showed cause and effect relationship. we use vas questionnaire to assess the severity of xerostomia. this vas quetioonaire has been used in our previous study which show the xerostomia severity correlate with serum inflammatory marker c-reactive protein (crp) in indonesian type 2 dm patient. the exact mechanism of xerostomia induced by alcohol containing mouthwash is still not available. alcohol may constrict small blood vessel in minor salivary glands in oral mucosal. the constriction of small blood vessel will reduce only 7-8% salivary flow rate of total flow rate because there are aprooximatley 600-1000 minor salivary glands in oral mucosa which depends on vascularization.22 the xerostomia may present if only hyposalivation under 50% of normal saliva productions.23 the probably explanation of the local effect of alcohol in oral mucosa that could apply for our current study that the alcohol may cause dehydration effects of oral mucosal through transdermal/ transmucosal water loss.4 alcohol in mouthwash may induce the contristion of minor salivary glands and activation of vannilloid receptor-1 which result in hydration of oral mucosal.24 this xerostomia effect of alcohol containing mouthwash may be temporary and it is a dose-response association. it means that the xerostomia effects of alcohol mouthwash will not longer when the alcohol containing mouthwash used in short periods and low dosage. in conclusion, there was not a significant difference of xerostomia severity between elderly used non alcohol-containing mouthwash and alcohol-containing mouthwash. acknowledgement this study was funded by faculty of dentistry, universitas gadjah mada yogyakarta, indonesia. references 1. asadoorian j. cdha position paper on commercially available over the counter oral rinsing products. cjdh 2006; 40(4): 1-13. 2. lemos ca, villoria gem. reviewed evidence about the safety of the daily use of alcohol-based mouthrinses. braz oral res 2008; 22 (suppl 1): 24-31. 3. o’reilly m. oral care of the critically ill: a review of the literature and guidelines for practice. aust crit care 2003; 16(3): 101-10. 4. lachenmeier dw. safety evaluation of topical applications of ethanol on the skin and inside the oral cavity. j occup med toxicol 2008; 3: 26. 5. satpathy a, ravindra s, porwal a, das ac, kumar m, mukhopadhyay i. effect of alcohol consumption status and alcohol concentration on oral pain induced by alcohol-containing mouthwash. j oral sci 2013; 55(2): 99-105. 6. wigmore jg, bugyra lm. decreasing the mouth alcohol effect by increasing the salivary glow rate decreasing the mouth alcohol effect by icreasing the salivary flow rate. can soc forens sci j 2003; 36(4): 211-6. 7. mccullough mj, farah cs. the role of alcohol in oral carcinogenesis with particular reference to alcohol-containing mouthwashes. aust dent j 2008; 53(4): 302-6. 8. gagari e, kabani s. adverse effects o f mouthwash use. a review. oral surg oral med oral pathol oral radiol endod 1995; 80(4): 432-9. 9. adams d, addy m. mouthrinses. adv dent res 1994; 80(2): 291301. 10. kerr a r, katz rw, ship j a. a comparison of the effects of 2 commercially available nonprescription mouthrinses on salivary flow rates and xerostomia. quintessence int 2007; 38(8): e440e447. 11. gupta a, epstein jb, sroussi h. hyposalivation in elderly patients. j can dent assoc 2006; 72(9): 841-6. 12. daelemans m, sternon j. drug-induced dry mouth. rev med brux 1985; 6(8): 575-7. 13. yuan a, woo s. adverse drug events in the oral cavity. oral surgery, oral med oral pathol oral radiol 2015; 119(1): 35-47. 14. dodds mwj, johnson da, yeh ck. health benefits of saliva: a review. j dent 2005; 33(3): 223-33. 15. pai s, ghezzi em, ship ja. development of a visual analogue scale questionnaire for subjective assessment of salivary dysfunction. oral surg oral med oral pathol oral radiol endod 2001; 91(3): 311-6. 16. flink h, tegelberg å, lagerlöf f. inf luence of the time of measurement of unstimulated human whole saliva on the diagnosis of hyposalivation. arch oral biol 2005; 50(6): 553-9. 17. kerr ar, corby pm, kalliontzi k, mcguire ja, charles ca. comparison of two mouthrinses in relation to salivary flow and perceived dryness. oral surg oral med oral pathol oral radiol 2015; 119(1): 59-64. 18. chadwick b, addyt m, walkers dm, wade wg. short communication effect of a 0 . 1 per cent hexetidine mouthwash on the microflora in aphthous ulceration. microb ecol health dis 1991; 4: 181-6. 19. napeñas jj, brennan mt, fox pc. diagnosis and treatment of xerostomia (dry mouth). odontology 2009; 97(2):76-83. 20. takeuchi k, furuta m, takeshita t, shibata y, shimazaki y, akifusa s, ninomiya t, kiyohara y, yamashita y. risk factors for reduced salivary flow rate in a japanese population : the hisayama study. biomed res int 2015; 2015: 381821. 21. beirne or. advances in dental research. j am dent assoc 1990; 121(6): 672, 674. 22. holsinger f, bui d. anatomy, function, and evaluation of the salivary glands. salivary gland disord pittsburgh springer; 2007. p. 1-15. 23. kranthikumar r, ashwinmkumar k, kiran kumar d, karthikeyan d,a review buccal mucosa and buccal formulations. ijipsr 2014; 2(9): 1957-1987. 24. haq mw, batool m, ahsan sh, qureshi nr. alcohol use in mouthwash and possible oral health concerns. j pak med assoc 2009; 59(3): 186-90. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p109-112 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p109-112 �5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �5 the decreasing of nfκb level in gingival junctional epithelium of rat exposed to porphyromonas gingivalis with application of �% curcumin on gingival sulcus agung krismariono department of periodontics faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: periodontal disease is a chronic, multi-factorial disease. chronic periodontitis is one of the main causes of tooth loss. chronic periodontitis is usually caused by porphyromonas gingivalis (p. gingivalis). p. gingivalis can induce nfκb activation resulting in the increasing of periodontal extracellular matrix degradation. curcumin can inhibit nfκb activation and reduce the severity of periodontal degradation. purpose: this research was aimed to observe level of nfκb in gingival junctional epithelium of rat exposed to porphyromonas gingivalis with local administration of curcumin. methods: sixteen wistar rat were divided into two groups. group 1 (treatment) consisted of eight rat given 2 x 106 cfu/ml p. gingivalis and 1% curcumin. meanwhile, group 2 (control) consisted of eight rat given 2 x 106 cfu/ml p. gingivalis only. gcf samples were collected from gingival sulcus. the samples were biochemically analyzed with elisa method. data were then analyzed statistically by using independent t-test (α=0.05). results: the examination of nfκb level showed that there was significant difference between treatment group and control group (p<0.05). the level of nfκb in the treatment group was significantly lower than the control group. conclusion: it can be concluded that 1% curcumin application can reduce nfκb level in gingival junctional epithelium of rat exposed to p. gingivalis. keywords: nfκb; junctional epithelium gingiva; curcumin correspondence: agung krismariono, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: agungkr@yahoo.com introduction periodontal disease is irreversible so that if there is no optimal treatment, most patients will potentially have lifelong disorder. however, this disease is likely painly, so people are not aware, as a result, it is generally found in the advanced level.1 chronic periodontitis is one of the types of periodontal disease widely spread in community. porphyromonas gingivalis (pg) bacteria are a major cause of this disease.2 in person with poor oral hygiene, the presence of pg bacteria in the gingival sulcus can potentially damage junctional epithelium, causing periodontitis characterized by loss of attachment. loss of attachment to the junctional epithelium may be caused by the invasion of pg bacteria. pg bacteria causing chronic periodontitis can produce certain endotoxin, namely lps and proteases, that can activate neutrophils and macrophages, which then will activate the transcription factor of nuclear factor-kappa b (nfκb). the activation of nfκb can trigger transcription and translation processes producing mediators that play a role in inflammation process.3 a research in rat showed that lps in 10 days can cause inflammation in periodontal tissue.4 inflammatory mediators in the optimal number actually can serve to neutralize antigen. if the number of inflammatory mediators produced are excessive, the extracellular matrix research report dental journal (majalah kedokteran gigi) 20�5 march; 48(�): �5–�8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �6 krismariono/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 35–38 will be damaged, so the periodontal tissues then can be damaged.5 various attempts have been made to treat chronic periodontitis, such as by scaling and root planning as well as by giving antibiotics and anti-inflammatory either locally or systemically. anti-inflammatory is required in the treatment of chronic periodontitis since it can neutralize the effects caused by the increasing of pro-inflammatory mediators. anti-inflammatory commonly used in the treatment of chronic periodontitis is administered systemically. systemic administration has few adverse side effects. therefore, it needs proper efforts that are effective, efficient and safe for treating this disease. one of those is by using herbal ingredients, which have medicinal properties and can be administered locally, such as turmeric (curcuma longa).6 the main active ingredient contained in turmeric is curcuminoid which main content is curcumin. curcumin has useful properties, such as anti-inflammatory, antioxidant, antibacterial, antitumor and antihepatotoxic. curcumin is also considered as a potent inhibitor of transcription factor nfκb.6,7 nevertheless, the role of curcumin against nfκb associated with periodontal disease, especially chronic periodontitis with indicators taken from gcf has never been proven. based on those facts, curcumin is expected to reduce the degree of inflammation in the gingival junctional epithelium caused by bacterial pg invasion. curcumin is also expected to inhibit the increasing of nfκb level so that pro-inflammatory mediators produced by nfκb that cause periodontitis can be decreased. the purpose of this research is to investigate nfκb level in gingival junctional epithelium of rat exposed to porphyromonas gingivalis with local administration of curcumin. materials and methods this research can be considered as an in vivo experiment using male rat (rattus norvegicus) wistar aged 5-6 months old and weighed 250-300 grams. the choosing of the age was based on the fact that the size of the jaw and gingival of the rat in that age is big enough to be applied with the medicines. this research was conducted at laboratory of biology in faculty of natural science and mathematics, laboratory of microbiology, and laboratory of biochemistry in universitas brawijaya. the samples of this research were selected randomly with the sample size determined through trial. based on a sample size formula the representative number of samples must be eight.7 curcumin powder was prepared in solution by using corn oil solvent to achieve a concentration of 1%. the concentration of 1% was determined based on a previous research conducted by suhag.9 meanwhile, to stimulate periodontitis disease in those rat, pg bacteria (atcc 33 277) were given and grown in a medium containing tryptic soy broth (tsb). they were incubated in an anaerobic atmosphere for 24 hours. they were grown in blood agar containing 10% sheep blood, 0.4 ml/ml vitamin k1 and 5 ml/ml hemin and then put into an anaerobic incubator with a composition of 80% n2, 10% h2, and 10% co2 for 24 hours at a temperature of 370 c. the largest colonies were transferred into liquid medium containing thioglicolat, and then incubated for 24 hours at 370 c in an anaerobic atmosphere. after pbs was given, spectrophotometry with 624 nm wavelength was used to make bacterial concentration as many as 1 x 106 cfu.10 rat used as experimental animals were adapted for 1 week before the research, by conditioning them in a cage with feeding (certain concentrate) and beverages (distilled water) according to standards of animal care in biological laboratory, faculty of natural science and mathematics, universitas brawijaya. those rat were then locally given 0.03 ml of live pg atcc 33 277 with the concentration of 2 x 106 cfu/ ml in the gingival sulcus bottom of their left and right mesial incisor teeth. the same procedure was then repeated every three days for two weeks. this procedure was conducted to make the animals get periodontitis. in the treatment group, 1% curcumin in corn oil as much as 0.03 ml was administered locally in the same area as the provision of bacteria every day for two weeks to provide a therapeutic effect. meanwhile, in the control group, only corn oil was administered every day for two weeks in the same area as the provision of bacteria. nfκb level was measured on day 14 by using periopaper put into the gingival sulcus for 30 seconds to get gingival crevicular fluid (gcf).11 the examination was conducted two weeks after the treatment. periodontitis can be occurred in the past 10 days.4 samples derived from gcf (in periopaper) put into eppendorf tubes and stored at -800 c until all the samples were collected and ready for examination. after all the samples were collected from the gcf, then the preparation of elisa examination was conducted. each eppendorf tube containing periopaper was added with 0.5ml, 0.02m pbs with ph 7.0 to 7.2 as the solvent, and then centrifugation was conducted at 3000 rpm for 20 minutes. finally, the supernatant of all of those samples was taken for examination in accordance with elisa procedure using specific rat monoclonal antibody for nfκb. results table 1 and figure 1 show that nfκb levels in both the control group (bacteria and corn oil) and the treatment group (bacteria and curcumin in corn oil) on day 14. the results of kolmogorov-smirnov goodness of fit test showed the value of p was 0.954 (p>0.05). it indicates that the survey data were normally distributed. based on the analysis of independent t-test results between the treatment group and the control group, it is known that there were significant differences between the control group and the treatment group with p=0.000 (p<0.05). it is also showed that nfκb level in the treatment �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��krismariono/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 35–38 group given 1% curcumin was lower than that in the control group. discussion this research was conducted to find an alternative solution for healing chronic periodontitis. the data from previous researches indicate that oral hygiene is the main cause of pathogen bacteria accumulation. this accumulation of bacteria causes periodontal tissue damage characterized by loss of attachment and migration of gingival junctional epithelium to apical. the results of this research showed that the group of rat not given curcumin had higher nfκb level than the group given curcumin. it means that curcumin has an effect in inhibiting nfκb activity in gingival junctional epithelium cells of those rat. curcumin can inhibit the phosphorylation of iκb kinase, so the translocation of nfκb into the nucleus of the cell is inhibited, resulting the decreasing of nfκb activity so that the expression of pro-inflammatory cytokines is also decrased.7 the inhibition of the phosphorylation of the inhibitor factor of iκb kinase in the cytoplasm can occur because curcumin is lipophilic13 so it could penetrate cell membranes and be located in either the cytoplasm or nucleus.14 curcumin can inhibit nfκb activation prior to its translocation into the cell nucleus. nfκb activation in patients with periodontitis has increased about 90%, while that in healthy tissue is only 30%. the expression of iκb as an nfκb inhibitor in patients with periodontitis is only 5%.15 curcumin as an anti-inflammatory is a potential inhibitor of nfκb. nfκb determines the expression of genes encoding cytokines, chemokine, growth factors, cell adhesion molecules, some acute protein phases, such as inos and cox-2, and receptors on the cell membrane, such as tlr.16 the activation of nfκb can be caused by a wide variety of triggers, such as bacteria, viruses, cytokines, free radicals and toxic environments. the activation of nfκb is associated with inflammatory process found in diseases, such as periodontitis, arthritis, atherosclerosis, and other degenerative diseases.17 on the other hand, the complex and persistent inhibition of nfκb is also connected directly with apoptosis, impaired immune cell development, and cell growth inhibition.16 chronic periodontitis disease is generally caused by pg bacteria.2 the increasing of nfκb is generally caused by products produced by pg bacteria, such as protease, lps, and fimbriae.18 protease produced by pg bacteria can trigger a series of immune response. arg-gingipain (rgp), protease produced by pg bacteria, is a potential activator for protease-activated receptor-2 (par-2).19 it means that protease is capable of breaking peptide in n-terminal of par-2 molecule, resulting in a new amino acid (neo-ligand) binding to the trans-membrane domain triggering par-2 activation. par-2 will further stimulate signals to nfκb, so nfκb is activated and trans-located into the nucleus.20 in addition to proteases, lps produced by pg bacteria is also a potent stimulator to produce pro-inflammatory cytokines. it means that in periodontal tissue exposed to lps, monocytes will be activated, and pro-inflammatory cytokines will be secreted. lps may also activate receptors on the cell membrane, such as tlr-2, tlr-4 and tlr-7. the activation of tlr causes intracellular signals passing through myd88 pathway that induces p38 mapk and subsequently activates nf-κb.21 fimbriae contained on live pg can also be considered as an effective inducer that can activate nfκb due to signals from tlr-2. tlr-2 can enhance the activity of nfκb due to fimbriae of pg bacteria.21 the fimbriae of pg bacteria can stimulate tlr-2 which signals will further activate nfκb.22 in other words, the activation of nfκb is stimulated by tlr-2 signals more dominantly activating cytokines induced by tnf in acute responses of osteoclastogenesis process.23 the increasing of osteoclasts then will cause damage to the periodontal tissues. in addition to extracellular signals, the increasing of nfκb level may also be caused by intracellular signals derived from virulence factor of the structure of pg bacteria, ie peptidoglycan. peptidoglycan, a bacterial cell wall, can be recognized by tlr-2 located on the cell membrane or by nod1 and nod2 located in the cytoplasm. this synergism between tlr and nod-2 then can activate nfκb.24 table 1. the mean and standard deviation levels of nfκb nfκb treatment group x + sd control group x + sd 63,79 + 8,049 pg/ml 130,48 + 22,378 pg/ml figure1. nfκb level in the control group and the treatment group 0 20 40 60 80 100 120 140 control group treatment group figure1. nfκb levels in the control group and the treatment group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �8 krismariono/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 35–38 the focus of this research is to analyze the activity of the transcription factor nfκb used as a strategy for the prevention and treatment of periodontal disease, especially chronic periodontitis. from the results of this research, it is known that the administration of 1% curcumin can make nfκb level in the treatment group lower than that in the control group. this means that inflammation is the problem of tissue damage in chronic periodontitis disease, but its severity can be reduced by administration of curcumin. it can be concluded that 1% curcumin locally given in gingival sulcus can reduce the degree of inflammation in the periodontal tissues, especially gingival junctional epithelium, by decreasing nfκb level. references 1. deschner j, eick s, damanaki a, nokhbehsaim m. the role of adipokines in periodontal infection and healing. mol oral microbiol 2014; 29(6): 258-69. 2. mysak j, podzimek s, sommerova p, lyuya-mi y, bartova j, janatova t, prochazkova j, duskova j. porphyromonas gingivalis: major periodontopathic pathogen overview. j immunology res 2014; 1–8. 3. lisboa, assis r, andrade, vinícius m, melo c, renan j. toll-like receptor activation and mechanical force stimulation promote the secretion of matrix metalloproteinases 1, 3 and 10 of human periodontal fibroblasts via p38, jnk and nf-kb. archives of oral biology 2013; 58 (6): 731–9. 4. feng z, weinberg a. role of bacteria in health and disease of periodontal tissues. periodontology 2000, 2006; 40: 50–76. 5. aggarwal bb, bhatt id, ichikawa h, ahn ks, sethi g, sandur sk, natarajan c, seeram n, shishodia s. curcumin: biological and medicinal properties. 2006; 297-340. 6. jurenka js. anti-inflammatory properties of curcumin, a major constituent of curcuma longa: a review of preclinical and clinical research. altern med rev 2009; 14(2): 141–53. 7. kuntoro. metode sampling dan penentuan besar sampel. surabaya: pustaka melati; 2008. p. 211–20. 8. suhag a, dixit j, dhan p. role of curcumin as a subgingival irrigant: a pilot study. qiuntessence j 2007; 4(2): 115–21. 9. polak d, wilensky a, shapira l, halabi a, goldstein d, weiss ei, haddad yh. mouse model of experimental periodontitis induced by porphyromonas gingivalis/ fusobacteriumnucleatum infection: bone loss and host response. j clin periodontol 2009; 36: 406–10. 10. kaya fa, arslan sg, kaya ca, arslan h, hamam o. the gingival crevicular fluid levels of il-1β, il-6 and tnf-α in late adult rat. int dent res 2011; 1: 7–12. 11. chen d, nie m, fan mw, bian z. anti-inflammatory activity of curcumin in macrophages stimulated by lipopolysaccharides from porphyromonas gingivalis. pharmacology 2008; 82(4): 264–9 . 12. minear s, o’donnell af, ballew a, giaever g, nislow c, stearns t, cyert ms. curcumin inhibits growth of saccharomyces cerevisiae through iron chelation. eukaryotic cell 2011; 10(11): 1574–81. 13. kunwar a, barik a, mishra b, rathinasamy k, pandey r, priyadarsini ki. quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells. biochim biophys acta 2008; 1780(4): 673-9. 14. ambili r, santhi ws, prasanthila j, nandakumar k, pillai r. expression of activated transcription factor nuclear factor-κb in periodontally diseased tissues. j periodontol 2005; 76(7): 1148– 53. 15. balistreri cr, candore g, accardi g, colonna-romano g, lio d. nf-κb pathway activators as potential ageing biomarkers: targets for new therapeutic strategies. immunity & ageing 2013; 10(24): 1–16. 16. arabaci t, cicek y, canakci v, canakci cf, ozgoz m, albayrak m, keles on. immunohistochemical and stereologic analysis of nf-κb activation in chronic periodontitis. eur j dent 2010; 4(4): 454–61. 17. yu wh, hu h, zhou q, xia y, amar s. bioinformatics analysis of macrophages exposed to porphyromonas gingivalis: implications in acute vs. chronic infections. plos one 2010; 5(12): 1–7. 18. holzhausen m, spolidorio lc, ellen rp, jobin mc, steinhoff m, gordon pa, vergnolle n. protease-activated receptor-2 activation a major role in the pathogenesis of porphyromonas gingivalis infection. am j pathol 2006; 168: 1189–99. 19. rallabhandi p, nhu qm, toshchakov vy, piao w, medvedev ae, hollenberg md, fasano a, vogel sn. analysis of proteinaseactivated receptor 2 and tlr4 signal transduction. a novel paradigm for receptor cooperativity j biol chem 2008; 283(36): 24314–25. 20. zhou q, amar s. identification of signaling pathways in macrophage exposed to porphyromonas gingivalis or to its purified cell wall components. j immunol 2007; 179(11): 7777–90. 21. krachler am, woolery ar, orth k. manipulation of kinase signaling by bacterial pathogens. j cell biol 2011; 195(7):1083-92. 22. kanaya s, nemoto e, ogawa t, shimauchi h. porphyromonas gingivalis fimbriae induce unique dendritic cell subsets via toll-like receptor 2. j periodontal res 2009; 44(4): 543–9. 23. sugawara y, uehara a, fujimoto y, kusumoto s, fukase k, shibata k, sugawara s, sasano t, takada h. toll-like receptors, nod1, and nod2 in oral epithelial cells. j dent res 2006; 85(6): 524–9. 92 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 92–95 original article occlusion and occlusal characteristics of the primary dentition in emirati schoolchildren vivek padmanabhan, bayan madan and sundus shahid pediatric and preventive dentistry, rak college of dental sciences, rak medical and health sciences university, ras al khaimah, united arab emirates abstract background: the prevalence of occlusion and various occlusal characteristics differ between populations. major contributions to these different types of occlusion and occlusal features include ethnic, genetic and environmental factors. purpose: the objective of the study was to understand the type and prevalence of terminal plane relationships and other occlusal traits, including physiological spacing and primate spacing, in emirati schoolchildren. methods: a cross-sectional study was conducted involving 458 participants in the age range of 3–6 years. a clinical evaluation was performed to record other occlusal characteristics. the data was then subjected to statistical analysis. results: the present study revealed that the bilateral flush terminal plane was seen in 40.8% of the examined children, the bilateral mesial step in 37.3% and the bilateral distal step in 1.7%. it was found that 44.5% of the examined children had physiologic space in both the upper and lower arches, while 14.19% of them had physiologic space only in the upper arch, 2.18% had it only in the lower arch, and 39% of them had no physiologic space. primate space was found to be present in both the upper and lower arches in 46% of the examined children. conclusions: the bilateral flush terminal plane relationship was the most common, and the bilateral distal step was the least common of the terminal plane relationships. in addition, primate spacing had a lower prevalence when compared to other studies. keywords: occlusion; occlusal characteristics; primary dentition; terminal plane relationships correspondence: vivek padmanabhan, pediatric and preventive dentistry, rak college of dental sciences, rak medical and health sciences university, ras al khaimah, united arab emirates. email: vivek.padmanabhan@rakmhsu.ac.ae introduction occlusion in dentistry is a term that describes the relationship between the teeth of the upper and lower jaws.1 correct dental occlusion plays a very important role in oral functions, including mastication, swallowing, speech and respiration, which greatly affect quality of life.2–6 in the primary dentition, dental occlusion is usually established by the age of three years; by then, all the deciduous teeth have usually erupted, and this lasts until the age of six years when the first permanent tooth starts to erupt.2 occlusal relationship traits vary among different populations depending on multiple factors, including ethnicity, genetics and environment. 1–3,5,6 the status of occlusion in the deciduous dentition acts as a mirror that reveals a prospective picture of the occlusal conditions in the permanent dentition.2,6 the following are the characteristics of normal occlusion in the primary dentition: spacing between anterior teeth, primate spaces, flush terminal plane molar relations, and ovoid arch forms.2 each characteristic has a specific indication for occlusal relationship traits in the permanent dentition. the presence of spacing in deciduous teeth denotes the possible proper alignment of the permanent dentition and the absence of crowding, while canine relationships in permanent teeth can be predicted by evaluating the primate spaces, which are present mesial to the maxillary canines and distal to the mandibular canines in the deciduous dentition.2,3 the relationship between the distal surfaces of the upper and lower primary second molars is the major predictor of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p92–95 mailto:vivek.padmanabhan@rakmhsu.ac.ae https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p92-95 93padmanabhan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 92–95 the permanent molar relationship, which is categorised into three different types: the flush terminal plane, the mesial step and the distal step.6,7 thus, evaluations of occlusal characteristics in the primary dentition play a crucial role in the early detection and prevention of malocclusion development in the permanent dentition.1,7 the development of malocclusion can be considered a disorder of the craniofacial complex that affects the development of the dental maxillofacial region and the masticatory function, leading to compromised physiological and psychological health.6 there is a paucity of literature available with regards to occlusal characteristics in pre-school children in the united arab emirates (uae). the objective of the study was to understand the type and prevalence of terminal plane relationships and other occlusal factors, such as physiological spacing and primate spacing, in emirati schoolchildren. materials and methods this was a cross-sectional study conducted at ras al khaimah college of dental sciences (rakcods), ras al khaimah medical and health sciences university (rakmhsu), ras al khaimah (rak), the united arab emirates (uae). this research was approved by the research and ethics committee of the university and the rak research and ethics committee, ministry of health (proposal number: rakmhsu-rec-108-2018-ug-d). prior to the commencement of the study, consent forms were provided to the children’s parents through the school authorities. the parents were given two weeks’ time to return the consent forms. children whose parents had given their consent were recruited for the study. children aged between 3 and 6 years were included in the study. children who had any medical conditions were not included in the study to prevent any bias in the findings. children with decayed teeth and children with any permanent teeth were excluded from the study. request letters were sent to the schools, and approvals were received before going to the schools. eight schools agreed to participate in the study, and they had a total population of 780 children in the selected age group. considering a margin of error of 5% and a confidence level of 90%, the appropriate sample size was calculated to be 258. however, during the school visits, all the students whose parents had given their consent were examined to cover as much of the population as possible. 458 emirati children (233 girls and 225 boys) aged 3–6 years were finally included and examined. their basic demographic data was recorded, and then the data sheet was used to evaluate and record the terminal plane relationships in the selected sample. the observation form was also used to determine the prevalence of physiological and primate spacing in the children’s teeth. a blinded evaluator was responsible for determining the scores. the supervising faculty had trained the evaluator to correctly diagnose and report the findings. the evaluations of the children were completed in 20 days. each day only 25–30 children were examined to ensure the evaluator was not fatigued, which could otherwise have resulted in unintended mistakes while recording the details. the examination was conducted in optimal natural light with the aid of a mouth mirror, an explorer and a cheek retractor. the occlusal characteristics and terminal plane relationships were assessed with the children in a centric relation position. this position was achieved by requesting each child to close their mouth as they swallowed. this step also allowed the process to be standardised. the prevalence of malocclusion was reported by age and gender and in total. the prevalence rates of the terminal plane relationships and other occlusal characteristics were reported in percentages. results the present study evaluated the occlusal traits of 458 emirati schoolchildren aged 3–6 years. most of the children included were aged 4 or 5 years (figure 1). the study population had 50.9% females and 49.1 % males (figure 2). we assessed the prevalence of occlusal traits, including terminal plane relationships, physiological spacing, primate spacing and deep bite in the primary dentition. all of these traits are normal occurrences in the (0.2%) 1 (47.2%) 216 (48%) 220 (4.6%) 21 0 50 100 150 200 250 3 4 5 6 n u m b er o f ch ild re n age groups (in years) age groups female 50.9% male 49.1% female male gender figure 2. percentages of children by gender included in the figure 1. age groups included in the study. study. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p92–95 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p92-95 94 padmanabhan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 92–95 primary dentition, and their presence suggests possible normal adult occlusion in the future. in terms of the prevalence of terminal plane relationships, the results revealed that the bilateral flush terminal plane was seen in 40.8% of the examined children, the bilateral mesial step in 37.3% and the bilateral distal step in 1.7%. right flush and left mesial was seen in 14.19%, right mesial and left flush in 4.14%, right distal and left mesial, and right flush and left distal were found to have an equal percentage of 0.7%, followed by right distal and left flush, and right mesial and left distal in 0.21% of the cases (table 1). when evaluated, 44.5% of the examined children were found to have physiologic space in both the upper and lower arches, while 14.19% of them had physiologic space only in the upper arch, 2.18% of them had physiologic space only in the lower arch, and 39% of them were found to have no physiologic space (table 2). primate space was found to be present in both the upper and lower arches in 46% of the examined children, while it was present only in the upper arch in 27.2% of the cases and only in the lower arch in 1.31% of them. however, 25.3% of the children presented with no primate space in either the upper or lower arch (table 3). the last trait observed was the prevalence of a deep bite, which was seen in 40.8% of the children and absent in 59.1% of them (table 4). discussion the dynamic nature of the primary dentition is wellknown, and the significance of spacing in this age group cannot be underestimated. the presence of spacing in the primary dentition is a good predictor of a healthy and well-aligned permanent dentition.8 early intervention in terms of interceptive treatment is a major role that paediatric dentists play. for this, a thorough understanding of the anteroposterior changes that occur in the occlusion between the primary and the permanent dentition is crucial.9 as a consequence, it is of paramount importance that any condition in the primary dentition is identified early to prevent a possible malocclusion in the permanent dentition.10 in the present study, when terminal plane relationships were evaluated, a 40.8% prevalence of the bilateral flush terminal plane relationship was seen, compared to a 37.3% prevalence of the bilateral mesial step terminal plane relationship and a tiny 1.7% prevalence of the bilateral distal step terminal plane relationship. in a previous study done in northern india, it was found that there was a higher prevalence of the flush terminal plane relationship when compared to the mesial and distal step terminal plane relationships. the results were similar to the present study.11 another study done in southern india also reported similar results, with a higher prevalence of the flush terminal plane relationship.12 however, studies from jordan and turkey have revealed a higher prevalence of the mesial step when compared to the flush terminal plane relationship.13,14 generally, it is seen throughout various populations that the flush terminal plane has a higher prevalence than the other types of terminal plane relationships, as reflected in the present study.11,12 in the present study, when the population was assessed for physiological space, it was seen that 44.5% of the examined children had physiologic space in both the upper and lower arches, while 14.19% of them had physiologic space only in the upper arch, 2.18% had it only in the lower arch, and 39% of them were found to have no physiologic space (table 2). in studies worldwide, varying results have been reported.15–18 it has generally been observed that the prevalence of physiological spacing is higher in european children compared to the rest of the world population. the rates reported have ranged from 55% to 98% in european children.15,16 however, some studies done in parts of europe have found lower percentages of physiological space.17,18 in asian countries like india, the prevalence rates of physiologic space vary from 69% to 85%.11,19,20 it can be understood that ethnic factors play a significant role in determining the prevalence rates of physiological spacing in the primary dentition within various populations worldwide. in the present study, it was seen that a large percentage of the population (39%) had no spacing present. it is likely that these children will have a definite crowding in their permanent dentition unless there is a timely intervention. table 1. prevalence of terminal plane relationships code no. types of terminal plane relationship number of children percentage (%) 1 bilateral distal 8 1.7 2 bilateral flush 187 40.8 3 bilateral mesial 171 37.3 4 right distal, left flush 1 0.2 5 right distal, left mesial 3 0.7 6 right flush, left distal 3 0.7 7 right flush, left mesial 65 14.2 8 right mesial, left distal 1 0.2 9 right mesial, left flush 19 4.1 table 2. prevalence of physiologic spacing physiologic spacing number of children percentage (%) absent 179 39.1 lower 10 2.2 upper 65 14.2 upper & lower 204 44.5 table 3. prevalence of primate spacing primate spacing number of children percentage (%) absent 116 25.3 lower 6 1.3 upper 125 27.3 upper & lower 211 46.1 table 4. prevalence of a deep bite deep bite number of children percentage (%) absent 271 59.2 present 187 40.8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p92–95 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p92-95 95padmanabhan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 92–95 in the present study, when primate spacing was assessed, it was found to be present in 46% of cases in both the upper and lower arches, while it was present only in the upper arch in 27.2% of cases and only in the lower arch in 1.31% of them. however, 25.3% of the children presented with no primate spacing in either the upper or lower arch. the results of the present study show a lower prevalence of primate spacing when compared to previous studies done worldwide. studies done in europe and asia have reported prevalence rates ranging from 60% to 90%.21,22 it can be concluded from the present study that the prevalence of the bilateral flush terminal plane relationship was the most common, and the bilateral distal step terminal plane relationship was the least common. the results of the present study also suggest that physiologic spacing and primate spacing have a lower prevalence when compared to populations elsewhere in the world. the results suggest that this population will need regular observation to ensure that the permanent dentition is free of crowding and other types of malocclusion. references 1. baral p, budathoki p, bhuju kg, koirala b. prevalence of occlusal traits in the deciduous dentition of children of kaski district, nepal. j nepal med assoc. 2014; 52(195): 862–5. 2. vegesna m, chandrasekhar r, chandrappa v. occlusal characteristics and spacing in primary dentition: a gender comparative crosssectional study. int sch res not. 2014; 2014: 512680. 3. bahadure rn, thosar n, gaikwad r. occlusal traits of deciduous dentition of preschool children of indian children. contemp clin dent. 2012; 3(4): 443–7. 4. zhou x, zhang y, wang y, zhang h, chen l, liu y. prevalence of malocclusion in 3to 5-year-old children in shanghai, china. int j environ res public health. 2017; 14(3): 328. 5. bhayya dp, shyagali tr, dixit ub, shivaprakash. study of occlusal characteristics of primary dentition and the prevalence of maloclusion in 4 to 6 years old children in india. dent res j (isfahan). 2012; 9(5): 619–23. 6. fernandes s, gordhanbhai patel d, ranadheer e, kalgudi j, santokì j, chaudhary s. occlusal traits of primary dentition among preschool children of mehsana district, north gujarat, india. j clin diagn res. 2017; 11(1): zc92–6. 7. bhat ss, rao ha, hegde ks, kumar bk. characteristics of primary dentition occlusion in preschool children: an epidemiological study. int j clin pediatr dent. 2012; 5(2): 93–7. 8. facal-garcía m, suárez-quintanilla d, de nova-garcía j. diastemas in primary dentition and their relationships to sex, age and dental occlusion. eur j paediatr dent. 2002; 3(2): 85–90. 9. moslemi m, nadalizadeh s, sarsanghizadeh s, sadrabad zk, shadkar s, s sm. evaluation of dental occlusion in 3-5 year-old children. int j multidiscip res inf. 2015; 1(1): 48–53. 10. malandris m, mahoney ek. aetiology, diagnosis and treatment of posterior cross-bites in the primary dentition. int j paediatr dent. 2004; 14(3): 155–66. 11. khan r, singh n, govil s, tandon s. occlusion and occlusal characteristics of primary dentition in north indian children of east lucknow region. eur arch paediatr dent. 2014; 15(5): 293–9. 12. reddy bp, rani ms, santosh r, shailaja am. incidence of malocclusion in deciduous dentition of bangalore south populationindia. int j contemp dent. 2010; 1(1): 20–3. 13. abu alhaija esj, qudeimat ma. occlusion and tooth /a rch dimensions in the primary dentition of preschool jordanian children. int j paediatr dent. 2003; 13(4): 230–9. 14. kirzioglu z, simsek s, yilmaz y. longitudinal occlusal changes during the primary dentition and during the passage from primary dentition to mixed dentition among a group of turkish children. eur arch paediatr dent. 2013; 14(2): 97–103. 15. randall le, beck fm, huja ss. bone remodeling surrounding primary teeth in skeletally immature dogs. angle orthod. 2011; 81(6): 931–7. 16. góis eg, vale mp, paiva sm, abreu mh, serra-negra jm, pordeus ia. incidence of malocclusion between primary and mixed dentitions among brazilian children. a 5-year longitudinal study. angle orthod. 2012; 82(3): 495–500. 17. bhayya dp, shyagali tr. gender influence on occlusal characteristics of primary dentition in 4to 6-year-old children of bagalkot city, india. oral health prev dent. 2011; 9(1): 17–27. 18. anderson aa. the dentition and occlusal development in children of african american descent. angle orthod. 2007; 77(3): 421–9. 19. vinay s, keshav v, sankalecha s. prevalence of spaced and closed dentition and its relation to malocclusion in primary and permanent dentition. int j clin pediatr dent. 2012; 5(2): 98–100. 20. shavi gr, hiremath n v, shukla r, bali pk, jain sk, ajagannanavar sl. prevalence of spaced and non-spaced dentition and occlusal relationship of primary dentition and its relation to malocclusion in school children of davangere. j int oral heal. 2015; 7(9): 75–8. 21. janiszewska-olszowska j, stepien p, syrynska m. spacing in deciduous dentition of polish children in relation to tooth size and dental arch dimensions. arch oral biol. 2009; 54(5): 397–402. 22. hegde s, panwar s, bolar dr, sanghavi mb. characteristics of occlusion in primary dentition of preschool children of udaipur, india. eur j dent. 2012; 6(1): 51–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p92–95 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p92-95 4545 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 45–49 research report the effects of zinc oxide non-eugenol and cellulose as periodontal dressings on open wounds after periodontal surgery yoeliani budisidharta, ahmad syaify and sri pramestri lastianny department of periodontics, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: periodontal surgery forms a part of periodontal treatment that can sometimes cause open wounds, such as gingivectomy and depigmentation. unfortunately, the healing process of open wounds can be inhibited due to bacterial infections and systemic factors. thus, after surgery, the open wounds need to be closed with periodontal dressing. purpose: this study aims to reveal the differences between using zinc oxide non-eugenol and cellulose periodontal dressings on open wounds after periodontal surgery. methods: thirty-two samples were divided into two groups. group i consisted of 16 samples where zinc oxide non-eugenol was applied as a periodontal dressing. similarly, group ii consisted of 16 samples where cellulose was applied as a periodontal dressing. the dressings were applied to open wounds after periodontal surgery using the split-mouth technique. hence, zinc oxide non-eugenol was applied on the right side and cellulose was applied on the left side of the mouth. the patients’ healing index (hi) score was measured on day seven and their wound healing index (whi) score was measured on days seven and twenty-one. results: the day-seven hi score of the wounds applied with cellulose was higher than those applied with zinc oxide non-eugenol. meanwhile, the whi of the cellulose group was lower than that of the zinc oxide non-eugenol group, except on day twenty-one. both the hi and whi scores then were analysed using mann whitney. conclusion: the application of cellulose is better than zinc oxide non-eugenol on the healing of open wounds after periodontal surgery. keywords: periodontal dressing; healing index; open wound; periodontal surgery; wound healing index correspondence: ahmad syaify, department of periodontics, faculty of dentistry, universitas gadjah mada, jl. denta no. 1, sekip utara, yogyakarta 55281, indonesia. email: ahmad.syaify@ugm.ac.id introduction periodontal treatment is generally divided into four phases: phase i (non-surgical), phase ii (surgical), phase iii (restoration) and phase iv (maintenance). in phase ii (surgical), some procedures are needed, such as incisions or cutting gingival tissue, to provide access and visual field as well as repair anatomic and morphological damage. however, they still can cause plaque accumulation and pocket formation. also, some of the procedures of periodontal surgery, as part of periodontal treatment in phase ii, can cause open wounds such as gingivectomy and depigmentation.1 when a wound occurs, the body will naturally protect and prevent itself from infection; this is considered part of the healing process.2 there are four phases of the wound-healing process: haemostasis, inflammation, proliferation and remodelling.3 unfortunately, the wound-healing process can be inhibited due to bacterial infections or systemic factors.4 therefore, in periodontal surgical procedures, wound closure is required using periodontal dressing material. a periodontal dressing is a physical barrier that protects wounds from compressive mastication and provides tissue with the opportunity to adapt to the wound-healing process.5 the periodontal dressing has no curative function, but it still can accelerate the healing process by protecting the wound while minimising the possibility of infection and postoperative bleeding.6 thus, the use of periodontal dressing on the wound surface aims to provide comfort to the patient, protect the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p45–49 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p45-49 46 budisidharta, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 45–49 wound, minimise the occurrence of infection and reduce postoperative bleeding.5 it also supports the healing process by preventing trauma caused by any contact between the wound, tongue and food during mastication. zinc oxide non-eugenol is a material widely used for periodontal dressing in dentistry. it has an antibacterial reaction from metal oxide and fatty acids that can be used as a barrier to protect wounds.5 however, it has been reported lately that the weakness of zinc oxide non-eugenol is its higher toxicity to osteoblasts and fibroblasts. through in vitro research, it was found that the rosin content in zinc oxide non-eugenol can trigger increased inflammatory reactions characterised by high polymorphonuclear neutrophilic leukocytes (pmn).6 it was also reported that zinc oxide non-eugenol can inhibit the wound-healing process, characterised by inflammation, until day seven after it has been applied.7 one of the periodontal dressing materials containing neither zinc oxide nor eugenol is cellulose, which can dissolve in 30 hours without leaving a residue. the content of cellulose does not interfere with the formation of fibroblasts so that the healing process occurs normally and does not trigger inflammation. cellulose is not toxic to the cells that play a role in healing so the wound-healing process is not interrupted. this study aims to reveal the differences between the use of zinc oxide non-eugenol and cellulose as a periodontal dressing on open wounds after periodontal surgery. hence, the results of this study are expected to help dentists determine what periodontal dressing material to use after gingivectomy and gingival depigmentation. materials and methods this study is quasi-experimental and involves two researchers. the research was conducted at the periodontics specialist clinic at prof. soedomo dental and oral hospital in universitas gadjah mada, yogyakarta. it was approved by the research ethics commission of the faculty of dentistry, universitas gadjah mada, no. 001389/kkep/ fkg-ugm/ ec/2018. sixteen patients were involved in the study, consisting of fourteen people who experienced hyperpigmentation and two people with gingival enlargement in the anterior region of their lower jaw. the 16 subjects were divided into two groups. in group i, open wounds were closed using zinc oxide non-eugenol coe-paktm (gc america, illinois, usa), while those in group ii were closed using cellulose reso-pac® (hager & werken gmbh & co. kg, germany). the selection of subjects was based on certain criteria such as suffering depigmentation or indications of gingivectomy, non-smokers, without systemic disease and willing to sign informed consent. periodontal surgical treatment was performed by gingival depigmentation or gingivectomy with a conventional technique using scalpels no. 11 and 14 (swan morton limited, england). open wounds were then irrigated with saline and distilled water and were dried using sterile gauze (pt. ahmadharis, indonesia). the wound areas were covered using a splitmouth technique. thus, zinc oxide non-eugenol using coe-paktm was applied on the right side, while cellulose was applied on the left side using reso-pac®. subsequently, during post-periodontal surgical treatment, the patients were instructed not to brush their teeth in the area of the surgery. once the periodontal dressing was applied, patients had to rinse with clean water and then take 500 mg amoxicillin antibiotic (pt. kalbe farma tbk, bekasi, indonesia), every eight hours for five days; they were advised to take 500 mg mefenamic acid analgesics (pt. hexpharm jaya, bekasi, indonesia) if they experienced pain. on the seventh day after post-periodontal surgery, the periodontal dressing using coe-paktm (gc america, illinois, usa) was removed and oral hygiene control and wound-healing procedures were carried out once a week for four weeks. the healing index (hi) and wound healing index (whi) on days seven and twenty-one were examined and evaluated. hi was based on an index from landry et al. describing post-surgical levels of clinical healing.2 a score of 1 was very bad if there was more than 50% red-coloured gingival wounds, palpation bleeding, granulation tissue and no epithelialisation, with epithelial loss beyond the incisional limit. score 2 was poor if there was more than 50% red gingiva, bleeding when palpated, granulation and open connective tissue because there was no epithelialisation. score 3 was good if there was 25−50% red gingiva, no palpation bleeding, no granulation tissue and no open connective tissue. score 4 was very good if there was less than 25% red gingiva, no palpation bleeding, no granulation tissue and no open connective tissue. whi was based on sharon et al.’s index evaluating post-surgical wound healing with epithelialisation parameters through toluidine blue staining. first, the post depigmentation and gingivectomy wounds were smeared with toluidine blue before clinical photographs were taken using a nikon d7100 digital camera (nikon corp japan, thailand). for whi evaluation, a score of 1 meant perfect epithelialisation if staining with toluidine blue was negative. score 2 was imperfect epithelialisation if the gingiva was bluish. score 3 indicated an ulcer if the colour was yellowish or white. score 4 indicated necrosis if the gingiva was blackish. next, data obtained from the hi and whi observations was qualitative with ordinal scale. the hi and whi data of both groups on the seventh and twenty-first days were statistically analysed with the non-parametric mann whitney test. all statistical analysis calculations were performed using spss version 22.0 (ibm, new york, usa) for windows with an error rate of 5%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p45–49 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p45-49 47budisidharta, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 45–49 results figure 1 shows the post-periodontal surgical open wound. figure 2 shows the post-periodontal surgical wound after it was closed with periodontal dressing using the splitmouth technique. hi observation was carried out on day seven to describe the level of clinical healing after periodontal surgery (figure 3; table 1). the data in table 1 shows the results of hi assessment conducted on day seven. in group i (zinc oxide non-eugenol) 16 samples scored 2 (poor) and indicated as much as ≥ 50% reddish tissue colour and bleeding during palpation. meanwhile, in group ii (cellulose), two samples scored 2 (poor) and 14 samples scored 3 (good); they indicated as much as 25−50% reddish tissue colour and no bleeding during palpation. table 2 shows the difference in hi score on day seven between the zinc oxide non-eugenol and cellulose groups. based on the results of the mann whitney non-parametric statistical tests, there was a significant difference (p <0.05) between the non-eugenol zinc oxide and cellulose groups. on the other hand, whi on days seven and twenty-one was observed by applying a toluidine blue liquid. the observation showed a bluish-purple colour, indicating inflammation. the clinical pictures can be seen in figures 4 and 5. table 3 shows the whi assessment results on day seven; in group i, the zinc oxide non-eugenol group, 16 samples figure 1. the open wound after the surgical periodontal treatment of the mandibular ridge, considered as a depigmentation case. figure 2. the postoperative periodontal dressing application using the split-mouth technique, coe-paktm (a) on the right side and reso-pac® (b) on the left side. figure 3. clinical evaluation on the seventh day after the coepaktm was released and hi was observed. figure 4. observation of the wound healing index (whi) on day seven by applying a toluidine blue liquid indicated a positive result with a bluish-purple colour. table 1 hi results on day seven periodontal dressing hi on day 7 score 2 score 3 zinc oxide non-eugenol 16 cellulose 2 14 table 2. hi median score on day 7 periodontal dressing median zinc oxide non-eugenol score 2 (2−2) cellulose score 3 (2−3) figure 5. observation of the wound healing index (whi) on day 21 by applying a toluidine blue liquid indicated a negative result with no bluish-purple colour. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p45–49 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p45-49 48 budisidharta, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 45–49 scored 2 (imperfect epithelialisation). meanwhile, in group ii, the cellulose group, 15 samples scored 1 (perfect epithelialisation) and one sample scored 2 (imperfect epithelialisation). based on whi assessment results on day twenty-one, all 16 samples in both groups scored 1 (complete epithelialisation). table 4 shows a difference in whi score on the seventh day between the zinc oxide non-eugenol and cellulose group, but there was no difference in whi score on the twenty-first day. based on the results of the mann whitney non-parametric statistical test on whi scores, there was a significant difference (p <0.05) between the zinc oxide non-eugenol and cellulose groups on day seven. meanwhile, there was no significant difference (p> 0.05) between the zinc oxide non-eugenol and cellulose groups on day twenty-one. discussion hi’s descriptive data showed a higher score in the cellulose group than in the zinc oxide non-eugenol group on day seven, therefore, there was a significant difference after data analysis was performed with the mann whitney test. in wounds closed with zinc oxide non-eugenol, prolonged inflammatory reactions can occur due to the side effects of rosin content.6 the acidic nature of rosin will stimulate polymorphonuclear (pmn) cells so that it triggers inflammation, which will inhibit the formation of fibroblasts resulting in an inhibited wound-healing process. fibroblasts play a role in the early stages of wound healing to regenerate new tissue.7 this condition is characterised by bleeding when palpated on the seventh day on an open wound covered with zinc oxide non-eugenol. according to sachs et al., when zinc oxide non-eugenol is applied, it has rigid physical properties and changes in dimensions can harbour food scraps and accumulate plaque.9 this can trigger the invasion of bacteria into the wound so that inflammation occurs as a form of self-defence so that the bacteria and endotoxin do not spread into other tissues. inflammation can be caused by haemolysis in mucosal tissue due to the high toxicity of zinc oxide noneugenol against osteoblasts and gingival fibroblasts. cellulose content is biocompatible with mucosal tissue so that it does not interfere with the healing process of open wounds after surgery. this is because cellulose does not affect tissue epithelialisation, angiogenesis and vascularisation and does not trigger excessive inflammatory reactions in the wound healing process.10 furthermore, cellulose only lasts for 30 hours before dissolving in saliva, so it does not disrupt the oxygen supply needed for angiogenesis and does not become a place for debris retention and plaque accumulation.11 the descriptive data of whi on day seven showed that the cellulose group had a higher whi score than the zinc oxide non-eugenol group. in group i, the zinc oxide non-eugenol produced a positive (+) bluish colour when stained using toluidine blue. this is due to inflammation in the open wound. during the inflammation process, mast cells containing granules will absorb the colour and turn purplish-blue when smeared with toluidine blue.12 the combination of colophony and zinc found in zinc oxide non-eugenol also causes a cytotoxic effect on fibroblasts that have been investigated in vitro, thereby inhibiting the formation of new tissue in the wound-healing process.13 open wounds that are closed with zinc oxide noneugenol for seven days can experience a disruption to their oxygen supply. oxygen plays an important role in the process of angiogenesis, the function of fibroblasts, the synthesis of collagen, the production of growth factors, the production of reactive oxygen species (ros) and the prevention of anaerobic bacterial infections arising from plaque accumulation.14 the wound-healing stage on day seven is still in the process of epithelialisation, angiogenesis and matrix formation so the interruption of oxygen supply can inhibit the process. in contrast, on day seven, group ii indicated negative results (-) after staining with toluidine blue as there was no inflammation, so the colour was not absorbed into the wound area. lee et al. state that cellulose content is not cytotoxic, so it does not cause lysis of cells. wounds closed with cellulose do not interfere with microvascular oxygen supply.15 in the early stages of the woundhealing process, oxygen plays an important role in cell metabolism to produce adenosine triphosphate (atp), prevent infection in the wound, stimulate angiogenesis, increase the differentiation of keratinocytes, migration and re-epithelialisation, as well as increase fibroblast cell proliferation and collagen synthesis.16 observation of hi on day twenty-one showed no difference between the scores of the two treatment groups. the administration of toluidine blue in the post-operative periodontal wound areas indicated negative (-) scores because inflammation and the wound-healing process did table 3 whi scores on the 7th and 21st days periodontal dressing day 7 whi score day 21 whi score 1 2 1 2 zinc oxide noneugenol 16 16 cellulose 15 1 16 table 4. whi median scores for each group on the 7th and 21st days periodontal dressing day 7 median score day 21 median score zinc oxide noneugenol score 2 (2−2) score 1 (1−1) cellulose score 2 (1−2) score 1 (1−1) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p45–49 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p45-49 49budisidharta, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 45–49 not occur on the twenty-first day and it had already reached the maturation and remodelling stages of collagen tissue and matrix deposition. open wounds had been epithelialised completely and replaced with new tissue.17 finally, the results of this study prove that the hi is higher in cellulose periodontal dressing than in zinc oxide non-eugenol periodontal dressing. however, the whi is lower in cellulose periodontal dressing, except on day twenty-one. it can be concluded that the effects of cellulose periodontal dressing are better than those of zinc oxide non-eugenol dressing during the healing process of open wounds after periodontal surgery. this is in agreement with a previous study by kadkhodazadeh et al.,6 who compared the effects of reso-pac® and coe-packtm in vitro although, unlike the previous study, this study focused on the effects of both dressing ingredients clinically. consequently, the results of this study are expected to help dentists choose an appropriate dressing material for post-periodontal surgery treatment. references 1. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. st. louis: saunders elsevier; 2015. p. 408–10. 2. pippi r. post-surgical clinical monitoring of soft tissue wound healing in periodontal and implant surgery. int j med sci. 2017; 14(8): 721–8. 3. gupta a, kumar p. assessment of the histological state of the healing wound. plast aesthetic res. 2015; 2(5): 239. 4. kathariya r, jain h, jadhav t. to pack or not to pack: the current status of periodontal dressings. j appl biomater funct mater. 2015; 13(2): e73–86. 5. david k, neetha sj, swati p. periodontal dressings: an informed view. j pharm biomed sci. 2013; 26(26): 269–72. 6. kadkhodazadeh m, baghani z, torshabi m. in vitro comparison of biological effects of coe-pak and reso-pac periodontal dressings. j oral maxillofac res. 2017; 8(1): e3. 7. savitha an, sunil c, bose s. reso pac tm a novel periodontal dressing in comparison with coe-pak : a clinical study. int j prev clin dent res. 2015; 2(1): 32–7. 8. grover h, dadlani h, bhardwaj a, yadav a, lal s. evaluation of patient response and recurrence of pigmentation following gingival depigmentation using laser and scalpel technique: a clinical study. j indian soc periodontol. 2014; 18(5): 586–92. 9. madan e, bharti v, chaubey kk, arora vkr, thakur rk, nirwal a. light-cured resin “barricaid”-an aesthetic and biocompatible dressing: a step ahead. j indian soc periodontol. 2013; 17(6): 753–6. 10. petelin m, pavlica z, batista u, štiblar-martinčič d, skaleric u. effects of periodontal dressings on fibroblasts and gingival wound healing in dogs. acta vet hung. 2004; 52(1): 33–46. 11. gautami sp, ramya tg, anudeep m, chaitanya a. evaluation of post operative healing response and patient comfort with two periodontal dressingsresopac and coepak following periodontal flap surgerya comparative clinical study. j biomed pharm res. 2017; 6(2): 66–71. 12. sridharan g, shankar aa. toluidine blue: a review of its chemistry and clinical utility. j oral maxillofac pathol. 2012; 16(2): 251–5. 13. sunzel b, söderberg ta, johansson a, hallmans g, gref r. the protective effect of zinc on rosin and resin acid toxicity in human polymorphonuclear leukocytes and human gingival fibroblasts in vitro. j biomed mater res. 1997; 37(1): 20–8. 14. gottrup f. oxygen in wound healing and infection. world j surg. 2004; 28(3): 312–5. 15. lee ls, lee su, che cy, lee je. comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells. int j ophthalmol. 2015; 8(2): 215–21. 16. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219–29. 17. stephen-haynes j, callaghan r, stephens c. evaluating the performance of a new carboxymethyl cellulose dressing in the community setting. br j nurs. 2017; 26(6): s36–41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p45–49 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p45-49 vol 51 no 4 okt-des 2018.indd 200200 research report dental journal (majalah kedokteran gigi) 2018 december; 51(4): 200–204 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg contrasting perceptions of male and female dental students regarding smile aesthetics based on their gingival display yessy josephine sijabat, c. christnawati, and dyah karunia department of orthodontics faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: perception consists of personal opinion in relation to an object. in terms of aesthetics, perception normally differs from one individual to another based on several factors such as gender. when expressing emotion, a smile is the most important facial expression whose aesthetics are constructed from a number of components, including gingival display. purpose: this study aimed to establish the comparative perceptions of the smile aesthetics of male and female dental students based on their gingival display. methods: 36 dental students, divided equally according to gender, were enrolled in this study. photographic images of the smile of each subject were taken from a frontal direction with a canon eos 700d digital camera and subsequently printed. assessments were conducted by comparing the photographs of subjects from the perspective of smile references based on the gingival display, followed by subject scoring on the basis of smile classification. assessments were conducted twice within a two-week period to confirm test reliability. the data collected was analyzed by means of kappa statistic and u-mann whitney tests. results: the test results indicated that all subjects demonstrated a coincidence in their analysis (κ=0.84). statistical analysis showed that a score of 0.902 (p>0.05) had been produced by a u-mann whitney test. conclusion: it can be concluded that no difference exists between male and female students in the perception of smile aesthetics based on the gingival display . keywords: gingival display; perception of aesthetic; smile aesthetics correspondence: christnawati, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: christnawati_fkg@ugm.ac.id introduction the analysis of a smile in relation to the surrounding soft tissues is has assumed a major role in the transformation of the aesthetics paradigm within the field of orthodontics. having conducted several studies of facial alteration affecting aesthetic quality, orthodontists have found that a balanced smile is frequently adopted as a guideline in orthodontic treatment.1 therefore, it is crucial to identify an instrument reliable in overcoming subjectivity when evaluating aesthetics. in the field of orthodontics, it is important to recognize the factors which disrupt a smile and influence the diagnosis of potential abnormalities when deciding on the treatment plan.2 orthodontic treatment is the branch of dentistry intended to improve the structure of the teeth in order to enhance mastication, phonetics and aesthetics.3 when seeking orthodontic treatment, patients normally cite facial aesthetic factors, both highly individual and subjective in nature, as their main motivation.4,5 an aesthete aims to create beauty and attraction to improve self-esteem and satisfaction with specific parts of their own body in order to experience greater confidence that he/she will be more appreciated by society.6 it is recognized that each individual can possess a specific mechanism to assess him/herself as well as others in terms of appearance and aesthetics.5 perception constitutes the opinion or response of an individual to an object which strongly affects his/her character and behaviour in relation to it. perceptions of certain stimuli will differ from one individual to another.7 in terms of the perception of aesthetics, it is very possible that individual experiences and socio-cultural environment play a major role. gender, socio-economic background and age are known to be factors influencing perceptions doi: 10.20473/j.djmkg.v51.i4.p200–204 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p200-204 201 sijabat, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 201–204 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p200–204 of tooth display.8 it has been found that females tend to be less satisfied with their smiles than males, rendering them generally more aware, sensitive and concerened when it comes to appearance. it is, thus, proven that females pay more attention to aesthetics.6 it is widely accepted that the smile plays an important role in both facial expression and appearance. there are three aspects of smile aesthetics, namely; gingival display, visibility of the smile curve line, and the buccal corridor.9 gingival display is the relationship between lips and the visibility of the gingiva tissues and teeth.10 it is accepted that a smile with minimal gingival display is considered to be more aesthetically pleasing compared to excessive gingival display or a gummy smile.9 the results of research conducted in 2010 by the school of dentistry at the federal university of pelotas, brazil shows that, in terms of self-perception of their smile aesthetics, females tend to be less satisfied than males. it shows that gender influences the perception of an individual regarding the appearance of his/her dental aesthetics.6 however, research on the same subject based on buccal corridors and the smile curve line conducted at the university of jordan showed that there is no difference between males and females in terms of perception when assessing smile aesthetics.1 the present study was carried out to compare the perceptions of male and female dental students with regard to the aesthetics of smiles based on gingival display. materials and methods this research falls within the descriptive analytical category featuring 36 subjects divided into two groups, female and male. the number of research subjects in each group was adjusted according to their availability, namely 18 male and 18 female participants who attended the faculty of dentistry at universitas gadjah mada during the 2014-15 academic year. it was confirmed that none of the research subjects were either currently undergoing or had previously undergone any of the following forms of orthodontic treatment: class i angle malocclusion, overbite and overjet ranging between 2–4 mm and a mild crowding and that they were all willing study participants. a canon digital camera (canon eos 700d 18.0 megapixels, japan), a tripod (excell promos, china), a laptop (hp®pav14, japan) and a printer (canon e510, vietnam) were employed during the conduct of this research. the research was approved by the ethics sub-committee of the universitas gadjah mada ethics commission which assigned the number 00959/kkep/fkg-ug/ec/2017. research subject selection had been agreed by the ethics committee, faculty of dentistry, universitas gadjah mada. the research procedure was explained to the selected participants who confirmed their understanding by signing letters of informed consent. during the photo shoot session, a canon eos 700d was positioned on a tripod 91 cm from the subjects, as recommended by the american academy of cosmetic dentistry photographic accreditation review.11 the height of the camera lens was at the eye level of the subject on whom it was focused with the setting on autofocus at iso 200. subjects posed in an upright seated position in a backless chair, looking at the camera with their centrical occlusal teeth parallel to the floor and their facial muscles relaxed. subjects were trained to smile broadly before a frontal photograph of the extraoral was taken. the photo shoot session results were transferred from the camera to an hp®pav14 laptop where they were placed adjacent to the smile reference pictures featuring the gingival display before being printed on a canon a510 printer. each subject assessed all the smile photographs (printed forms) by comparing the photographs of the subjects with the smile reference pictures based on the criterion of gingival display. subjects chose one of the reference pictures based on the gingival display which they considered most similar to the picture of their own smile. subjects subsequently made an assessment on a scale of 1 to 4 based on the smile classification (figure 1), namely; 1) very high smile indicated by the width of the smile margin of the gingival upper jaw or apical to cement-enamel junction being more than two millimetres, 2) high smile line with the gingival upper jaw margin (apical to the cementoenamel junction) measuring more than two millimetres, 3) average smile line in which only a gingival embrasure is visible, 4) low smile line in which the gingival embrasure and cementoenamel junction is not visible.12 the research subjects underwent re-assessment after two weeks to establish whether any difference existed between the first and the second assessment. in cases where a difference existed, a re-assessment process was undertaken two weeks after the second assessment with the average being calculated from the data collected. the data was subsequently analyzed by means of kappa-statistic and u-mann whitney tests. p-values <0.05 were considered statistically significant. results the results provided by the subjects formed two sets of assessment data, perception assessments i and ii, relating to their perceptions of an ideal, gingival display-based smile. the kappa statistic was employed in order to establish the reliability between the intra-examiner and inter-examiner results produced by assessment i and ii, calculated by dividing the number of agreement scores by the total number of scores. all examiners demonstrated an extremely high level of agreement in both their intra-examiner and inter-examiner analysis (κ = 0.84). the results confirmed that there was no difference between the first and the second assessments. thereafter, a u mann-whitney test was conducted to establish whether any contrast existed between male and female perceptions of their gingival http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p200-204 202sijabat, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 201–204 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p200–204 display. the u mann-whitney test value was 0.902, signifying the absence of male-female difference (table 1). this result, in turn, implied that no difference existed in the perceptions of members of either gender. based on the contents of table 1, the male responses were similar to those of females in that their assessment of the smiles of subjects in classification 3 was 44.44%, while that of their female counterparts was 41.67%. discussion the kappa statistic results indicate the lack of difference between perception assessments i and ii. the test results were potentially influenced by several factors, including: 1) all subjects were given a comprehensive explanation of how to assess a smile based on gingival display before conducting an assessment, 2) all subjects were current dentistry program students, thereby ensuring shared background knowledge of smile aesthetics. examination of the results indicated that males and females demonstrated similarity with regard to frequency and test percentages; the highest frequency and percentage occurring in classification three (average smile line), while the lowest frequency and percentage related to classification four (very high smile line). the results reported here are supported by research on smile aesthetics perception conducted by the students of the faculty of dentistry, university of valencia, spain which concluded that no difference existed between males and females when assessing smile aesthetics. both genders considered that the most attractive smile aesthetic to be one in which the gingival display is no longer than 2 mm, while the least appealing is a smile with a gingival display greater than 2 mm in length.13 the similarity between the male and female assessments can, potentially, be influenced by their shared background knowledge, as dentistry students, of the subjects. it is also generally accepted that dentistry students pay more attention to the factors influencing smile aesthetics and that their high assessment of smile aesthetics is possibly influenced by their background knowledge, both clinical and theoretical, related to tooth aesthetics and their thorough understanding of factors influencing smile aesthetics.14 the mode test data indicated that the mode of the male and female groups is similar in classification 3. it is possible that this comparability is influenced by the shared background knowledge of the subjects as dentistry students in the area of smile aesthetics. it is also supported by research on the comparison of the smile aesthetics perception of orthodontists and dentistry students based on gingival display at the faculty of dentistry, kyushu university, japan. the results show that no difference existed between the aesthetic assessment of the male and female members of each group, suggesting that dentistry students tend to be less accepting of gingival display. the study also reported that, in their opinion, the most interesting smile was one in which two millimetres of the upper lip covers the upper jaw incisivus (average smile line).15 there is a difference in the aesthetic perception of males and females. female teenagers pay more attention figure 1. smile classification based on gingival display: (1) very high smile line, (2) high smile line, (3) average smile line, (4) low smile line table 1. percentage of buccal corridor value for each group and a comparison of u mann-whitney test results of the two groups tested classification of gingival display number of choice (percentage of classification of gingival display) p -value femalemale 4 (11.11)5 (13.89)1 0.90210 (27.78)9 (25)2 15 (41.67)16 (44.44)3 7 (19.44)6 (16.67)4 36 (100)36 (100)total notes: 1) very high smile line 2) high smile line 3) average smile line 4) low smile line. http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p200-204 203 sijabat, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 201–204 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p200–204 to aesthetics, including healthy teeth, as a result of their considering themselves to have an unsatisfactory facial shape.16 females tend to be less satisfied with their smile compared to males which strongly suggests that they are more self-conscious about their appearance.6 a body of research shows that females tend to value aesthetics more than males,17 while also highlighting the fact that there is no difference in either test frequency or assessment mode between groups. the similarity of the assessment suggests that females are not more aware of, sensitive to or concerned about aesthetic considerations compared to males. the attention paid to aesthetics is also related to the satisfaction of subjects regarding this aspect. the major factor influencing research is the background knowledge of subjects as faculty of dentistry students who tend to pay more attention to dental aesthetics during their university studies, a fact obviously affecting their perception of facial aesthetics.8 this conclusion is also supported by research into the perception and level of satisfaction regarding dental aesthetics on the part of students in saudi arabia which showed that, based on a satisfaction assessment index, no differences exist between satisfaction levels in males and females regarding their dental aesthetics.18 age control and orthodontic treatment experiences also influence the assessment result of the subject. young people appear to pay more attention to dental aesthetics compared to their elders.6 in this research, the subjects of the study were 18-21 year old students. the extremely limited age range made it possible that insignificant assessment differences would exist between the female and the male groups.19 other possible factors influencing the assessment of the subject included individual experiences of which orthodontic treatment is an example. it is believed that orthodontics treatment affects the perception of aesthetics harboured by an individual. as for the subjects of this study, it was confirmed that all participants were neither undergoing orthodontic treatment, nor had any experience of such previous treatment. therefore, it was possible that both female and male groups had the same perception of assessing aesthetics.20 the result of the study conducted here is supported by related research which confirmed no difference between males and females when assessing smile aesthetics based on their buccal corridor.7 the weaknesses of the study are also presented here, including: the clinical crown lengths of the incisors of the subjects being neglected and the smile aesthetics only being observed and examined based on the gingival display. the assessment of smile aesthetics based on the gingival display in this piece of research took account of several factors such as disproprotionate vertical maxillary growth, excessive upper lip muscle and the clinical crown height of the maxillary incisive teeth.21 one important parameter in assessing smile aesthetics based on gingival display is the maxillary incicivus length and width ratio.22 in cases where the clinical crown length of the incisors appears shorter, possible causes include attrition or an excessive gingiva. smile aesthetics are examined on the basis of many factors and smile with excessive gingival display is not the only category used to determine the presence or otherwise of smile aesthetics.23 it is important to examine smile aesthetics from several perspectives in order to best decide what treatment should be undertaken based on aetiological causes. it can be concluded that male and female dental students share the same perception of an aesthetic smile with regard to its gingival display. references 1. badran sa, mustafa m. perception of smile attractiveness by laypeople – influence of profession and treatment experience. br j med med res. 2014; 4(20): 3777–86. 2. câmara ca. aesthetics in orthodontics: six horizontal smile lines. dental press j orthod. 2010; 15(1): 118–31. 3. avriliyanti f, suparwitri s, alhasyimi aa. rinsing effect of 60% bay leaf (syzygium polyanthum wight) aqueous decoction in inhibiting the accumulation of dental plaque during fixed orthodontic treatment. dent j (maj ked gigi). 2017; 50(1): 1–5. 4. prabhu s, divya m, sneha k v, veena n. prevalence of malocclusion, aesthetic self-perception and their correlation among 18 to 24 years old college students in chennai. j oral hyg heal. 2017; 5(2): 1–4. 5. musskopf ml, rocha jm da, rosing ck. perception of smile esthetics varies between patients and dental professionals when recession defects are present. braz dent j. 2013; 24(4): 385–90. 6. silva gc, castilhos ed, masotti as, junior sar. dental esthetic self-perception of brazilian dental students. rsbo. 2012; 9(4): 375–81. 7. nurfitrah a, christnawati c, alhasyimi aa. comparison of esthetic smile perceptions among male and female indonesian dental students relating to the buccal corridors of a smile. dent j (maj ked gigi). 2017; 50(3): 127–30. 8. abidia rf, azam a, el –hejazi aa, al-mugbel k. k, haider ms, al-owaid nm. female dental student’s perception of their dental aesthetics and desired dental treatment. eur sci j. 2017; 13(3): 171–81. 9. ioi h, nakata s, counts al. effects of buccal corridors on smile esthetics in japanese. angle orthod. 2009; 79(4): 628–33. 10. gaddale r, desai sr, mudda ja, karthikeyan i. lip repositioning. j indian soc periodontol. 2014; 18(2): 254–8. 11. schabel bj, baccetti t, franchi l, mcnamara ja. clinical photography vs digital video clips for the assessment of smile esthetics. angle orthod. 2010; 80(4): 678–84. 12. sepolia s, sepolia g, kaur r, gautam dk, jindal v, gupta sc. visibility of gingiva an important determinant for an esthetic smile. j indian soc periodontol. 2014; 18(4): 488–92. 13. españa p, tarazona b, paredes v. smile esthetics from odontology students’ perspectives. angle orthod. 2014; 84(2): 214–24. 14. omar h, tai y. perception of smile esthetics among dental and nondental students. j educ ethics dent. 2014; 4(2): 54–60. 15. ioi h, nakata s, counts al. influence of gingival display on smile aesthetics in japanese. eur j orthod. 2010; 32(6): 633–7. 16. prasanti aa, santosa o. perbedaan indeks periodontal dan skor pembesaran gingiva kelompok pemakai dan bukan pemakai pesawat ortodonti cekat. j kedokt diponegoro. 2016; 5(1): 1–8. 17. kiani h, bahir u, durrani ok, zulfiqar k. comparison of difference in perception between orthodontists and laypersons in terms of variations in buccal corridor space using visual analogue scale. poj. 2013; 5(2): 67–72. 18. bilal r. self perception and satisfaction with dental esthetics in dental students of qassim region of saudi arabia. pakistan oral dent j. 2016; 36(3): 399–403. 19. handayani s, hardjajani t, yuliadi i. perbedaan perilaku seksual mahasiswa laki-laki uns yang tinggl di kos dan tidak tinggal di kos http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p200-204 204sijabat, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 201–204 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p200–204 ditinjau dari interaksi dengan teman sebaya. j ilm psikol candrajiwa. 2013; 2(4): 70–82. 20. an s-m, choi s-y, chung y-w, jang t-h, kang k-h. comparing esthetic smile perceptions among laypersons with and without orthodontic treatment experience and dentists. korean j orthod. 2014; 44(6): 294–303. 21. suzuki l, machado aw, bittencourt mav. an evaluation of the influence of gingival display level in the smile esthetics. dental press j orthod. 2011; 16(5): 1–10. 22. zawawi kh, malki ga, al-zahrani ms, alkhiary ym. effect of lip position and gingival display on smile and esthetics as perceived by college students with different educational backgrounds. clin cosmet investig dent. 2013; 5: 77–80. 23. kallidass p, srinivas s, charles a, davis d, sushil charravarthi nc. smile characteristics in orthodontics: a concept review. int j orofac res. 2017; 2(1): 1–4. http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p200-204 221221 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 221–226 original article bioinformatic approach of propolis as an inhibitor of peptidoglycan glycosyltransferase to improve antibacterial agent: an in-silico study imelia arifatus sani, siska maulidina cahyani, safira fariha, oliresianela and diah department of periodontics, faculty of dentistry, brawijaya university, malang, indonesia abstract background: in indonesia, the prevalence of dental and oral problems is still high at 57.6% in 2018, especially periodontitis at 74.1%. peptidoglycan is an essential component of the bacterial cell wall. peptidoglycan glycosyltransferase (pgt) is a protein target that plays a role in transferring lipid disaccharides ii to growing glycan chains for bacterial cell wall synthesis. propolis is a natural ingredient produced by bees and has anti-inflammatory, antibacterial, antiviral and antioxidant properties so that it has the potential to be a natural mouthwash ingredient. one of the antibacterial properties of propolis is to be able to kill and reduce the number of bacteria that cause periodontitis. purpose: this study aims to investigate the potential of a specific compound of propolis as an inhibitor of protein peptidoglycan glycosyltransferase through bonding interactions. methods: the method used is an in-silico test in molecular docking with computational software, namely molegro virtual docker and discovery studio visualizer. results: this study showed the types of bonds between the four compounds, and chlorhexidine as a control showed similar types of bonds, including hydrogen bonds, hydrophobic interactions and unfavourable bonds. the binding energy values of each of the five compounds were pinocembrin -222.166 kj/mol, hesperetin -230.144 kj/mol, chrysin -219.45 kj/mol, caffeic acid phenethyl ester -266.64 kj/mol and chlorhexidine -362.71 kj/mol. conclusion: caffeic acid phenethyl ester (cape) is the most significant potential as an inhibitor of protein peptidoglycan glycosyltransferase and chlorhexidine has the highest binding affinity than the four propolis compounds, followed by caffeic acid phenethyl ester in propolis in silico. keywords: oral hygiene; peptidoglycan glycosyltransferase; propolis correspondence: imelia arifatus sani, department of periodontics, faculty of dentistry, brawijaya university. jl. veteran, malang, 65145, indonesia. email: melikaliraaa@gmail.com introduction oral health is an overall component of general health.1 however, there are still many indonesian people who do not understand the importance of maintaining oral health. it is known from the national prevalence of dental and oral problems in 2013, which reached 25.9%, with 14 provinces having a prevalence above the national figure. the prevalence increased to 57.6% in 2018.2 one of the dental and oral diseases whose prevalence is still high in indonesia is periodontitis. according to basic health research (riskesdas), 2018,2 the prevalence of periodontitis in indonesia reached 74.1%. periodontitis is a disease of the oral cavity, a complex infection with several etiologic factors and contributing factors. aggregatibacter actinomycetemcomitans and porphyromonas gingivalis are pathogens that play an essential role in periodontitis.3 harvey explains that periodontitis is caused by a mixed bacterial infection resulting in damage to the supporting tissues of the teeth.4 in this study, peptidoglycan (pg) is an essential component of the bacterial cell wall. peptidoglycan glycosyltransferase (pgt) of the 51 families is an essential enzyme for synthesizing bacterial cell wall glycan chains. this enzyme is considered a potential antibacterial target.5 peptidoglycan glycosyltransferase transfers lipid ii disaccharides to growing glycan chains for bacterial cell wall synthesis. lipid ii (pgt substrate) is a large amphiphilic dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 mailto:melikaliraaa@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 222 sani et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 221–226 molecule containing a hydrophilic peptide disaccharide head and a hydrophobic undecaprenyl diphosphate tail as a barrier to the cell membrane polymerization. the pgt structure also exhibits a hydrophobic region close to the active site, most likely interacting with the substrate lipid chain upon entering the active site. pgt inhibitor with broad-spectrum antibacterial activity guards against grampositive and gram-negative bacteria.6 a natural ingredient, namely propolis has received much attention in the medical field because it has antiinflammatory, antibacterial, antioxidant, antifungal, antiviral, and anticancer properties.7 propolis has a high flavonoid content, which is 50% of the total composition in the resin.8 propolis enhances the immune response and has the potential as a mouthwash with minimal cytotoxic properties compared with chlorhexidine.9,10 one of the properties of propolis is antibacterial, so it is expected to be able to kill and reduce the number of bacteria that cause periodontitis. the antibacterial properties were tested by molecular docking. the rapid development of science and technology has encouraged various research in the health sector, one of which is an in silico-based research approach. in silico is a research term through computer simulations supported by the availability of information from a database.11 one of the goals of in silico-based research is drug discovery and development. drug design aims to obtain a new type of drug with better activity and lower toxicity. through an in-silico approach, it is possible to identify targets and compounds from the existing database to increase the efficiency of drug discovery and development. the method used in silicobased research is molecular docking, which is used for drug discovery. furthermore, this study aims to investigate the potential of a specific compound of propolis as an inhibitor of protein peptidoglycan glycosyltransferase through bonding interactions in silico. materials and methods the method used in this paper was molecular docking, an in-silico study in nature. the duration of the research was four months. this research was conducted online or in a network supported by hardware and software. molecular docking is a method based on in silico studies and is widely used for drug discovery. molecular docking allows identifying new therapeutically related compounds and predicts ligand-target interactions at the molecular level.12 the hardware used was a set of computers or laptops with the recommended minimum specifications according to khaerunnisa et al.,11 namely a minimum of core i and 2 gb ram, operating systems (os) windows, mac, and linux, and software included molegro virtual docker and discovery visualizer studio version 21.1.1. in determining protein and ligands, several databases, such as pubchem and protein data bank (pdb), were used. the 3d structures were obtained from secondary data in the pubchem database to download compounds and the pdb to download protein structures. the target antibacterial protein is pgt with id 3fwl. the control ligands selected were chlorhexidine with cid (cid_9552079), and the comparison ligands were four active compounds from propolis (table 1). protein preparation was carried out using molegro virtual docker software to remove solvent and native ligands attached to the protein structure. compound or ligand preparation was done directly by entering the compound file and then clicking import. docking on the five target compounds was carried out using molegro virtual docker software. the target protein cavity was identified (the native ligand-binding area) and docked in the cavity area. docking parameters included: cavity volume 1184.77a; surface 2833.92a with grid x 40.59; y 78.00; z -37.29; moldock score [grid] 0.30a; number of running 10; binding pose maximum 5, and rmsd maximum 2. visualization of the interaction of each protein with the ligand was presented using the discovery studio visualizer version 21.1.1 software. this device aims to determine the binding area of protein with a specific ligand along with the constituent atoms of the compound, the amino acids that interact with the ligand and the type of bond. binding energy is obtained by summing the moldock score, moldock grid score and rerank score, which is averaged from 5 replications. results the 3d view shows pinocembrin, hesperetin, chrysin, caffeic acid phenethyl ester (cape), and chlorhexidine compounds bound to protein transglycosylase at various amino acid residues (table 2). the benefits of propolis components are listed in table 3. the types between the four compounds and chlorhexidine as a control show similar hydrogen and hydrophobic interactions. in addition, it also shows poor binding to the fifth complex ligand – pgt protein. pinocembrin binds to peptidoglycan glycosyltransferase protein via hydrogen at amino acid residues leu584, tyr517, ala520, leu521, leu565, thr577 and also binds to leu580, gly581 and ala583 with an unfavourable bond (figure 1). the energy of pinocembrin is -222.166 kj/mol. hesperetin has an energy value of -230.144 kj/mol at amino acid residues gln675, table 1. list of propolis active compounds. compounds cid pinocembrin 68071 hesperitin 72281 chrysin 5281607 caffeic acid phenethyl ester 5281787 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 223sani et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 221–226 table 2. interaction of pinocembrin, hesperetin, chrysin, caffeic acid phenethyl ester and chlorhexidine compounds on protein transglycosylase ligands/ binding energy (kj/mol) interactions category types pinocembrin/ -222.166 leu584, tyr517, ala520, leu521, leu565, thr577 hydrogen bond conventional hydrogen bond leu580, gly581, ala583 unfavorable unfavorable bump hesperitin/ -230.144 gln675, tyr517, gly697, ser572 lys698 hydrogen bond conventional hydrogen bond tyr517, leu671, ile713, phe625, pro514, leu569, val677, lys698, ala568 hydrophobic pi-pi stacked leu565, val566, ala568, leu569, gly697 unfavorable unfavorable bump chrysin/ -219.45 leu584 hydrogen bond conventional hydrogen bond ala520, tyr527, tyr517, ile533, hydrophobic pi-sigma leu584, leu521, leu565, thr577, leu580, gly581, leu584 hydrophobic pi-alkyl caffeic acid phenethyl ester (cape)/ -266.64 gly697, leu671 hydrogen bond conventional hydrogen bond leu521, leu529, val566, leu569, pro514, val677, ala696, lys698 hydrophobic pi-alkyl leu565, val566, leu569, gln675 unfavorable unfavorable bump chlorhexidine/ -362.71 val566, thr570, tyr517 hydrogen bond conventional hydrogen bond val563, lys513, pro514 ala568 ile533 tyr517 trp532, leu565 ala568 hydrophobic pi-sigma val566, leu565, asp567 ala568 leu569 thr570 pro576 arg571 ser572 unfavorable unfavorable bump table 3. benefits of propolis components component benefit caffeic acid phenethyl esters (cape) ho1 regulation can counteract oxidative stress and inflammation involving the p38 mapk signal.13 inhibits nf-kβ p65 subunit so that it can control anti-inflammatory activity in periodontitis.14 inhibits bacterial rna-polymerase.15other pinocembrin inhibits bacterial rna-polymerase.15 chrysin downregulation of pro-inflammatory cytokine expression.16 hesperetin inhibits the ace-ii enzyme.17 tyr517, gly697, ser572 lys698, tyr517, leu671, ile713, phe625, pro514, leu569, val677, lys698, ala568, leu565, val566, ala568, leu569 and gly697. the types between hesperetin and pgt proteins are hydrogen, hydrophobic and are unfavorable (figure 2). chrysin interacts with pgt protein at the amino acid residues leu584, ala520, tyr527, tyr517, ile533, leu584, leu521, leu565, thr577, leu580, gly581 and leu584 with an energy of -219.45 kj/mol. the type between chrysin and pgt protein is hydrogen and hydrophobic (figure 3). cape has the lowest energy compared with the other three active compounds, -266.64 kj/mol. caffeic acid phenethyl ester binds to pgt protein via hydrogen at amino acid residues gly697 and leu671, hydrophobic acid at amino residues leu521, leu529, val566, leu569, pro514, val677, ala696, and figure 1. 3d and 2d models of docking between pinocembrin ligand and peptidoglycan glycosyltransferase protein. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 224 sani et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 221–226 figure 3. 3d and 2d models of docking between chrysin ligand and peptidoglycan glycosyltransferase protein. figure 4. 3d and 2d models of docking results between cape ligand and peptidoglycan glycosyltransferase protein. lys698, and via unfavorable bonds in amino residues leu565, val566, leu569 and gln675 (figure 4). chlorhexidine which is the control compound has the lowest energy compared with for pro-specific compounds, namely -362.71 kj/mol and binds to amino acid residues val566, thr570, tyr517 via hydrogen, val563, lys513, pro514, ala568, ile533, tyr517, trp532, leu565, and ala568 via hydrophobic bond and val566, leu565, asp567, ala568, leu569, thr570, pro576, arg571, ser572 via unfavorable bond (figure 5). figure 2. 3d and 2d models of docking between hesperitin ligand and peptidoglycan glycosyltransferase protein. figure 5. 3d and 2d models of docking between chlorhexidine ligand and peptidoglycan glycosyltransferase protein. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 225sani et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 221–226 discussion several residues of the active site of the four compounds identified were found on the active site of chlorhexidine as a control, including tyr517, pro514, val566, leu565, ala568, leu565 and leu569. it indicates that the four compounds have inhibitory potential as antibacterial, almost the same as chlorhexidine as a control. the docking results showed that chlorhexidine had the lowest bond energy as a control, which was -362.71 kj/mol. the lower ligand and protein interaction, the stronger bond between the ligand and protein complex. therefore, chlorhexidine has the strongest binding to pgt protein. however, the caffeic acid phenethyl ester compound had the lowest bond energy of -266.64 kj/mol compared with the three other specific propolis compounds, namely pinocembrin, hesperetin and chrysin. research states that one type of propolis, namely tetragonula sapiens, has good anti-inflammatory properties at a concentration of 120 mg/ml.18 the cytotoxic test is safe to use in the concentration range of 6.25-200 g/ml. one of the most essential ingredients in propolis that is quite large is flavonoids. flavonoids in propolis are stored in large amounts of resin, which is about 50% of the total content. other propolis content is about 30% wax, 10% aromatic compounds and oils and 5% pollen.8 the composition of propolis may vary based on the propolis-producing region, but previous studies have shown that different propolis samples contain flavonoids such as luteolin, chrysin, pinocembrin, hesperetin and rutin. the most abundant components are chrysin, hesperetin and pinocembrin. in addition, one of the phenolic bonds in propolis, namely cape, reaches 50% of the total components.17 propolis has antibacterial properties by eliminating the permeability of porphyromonas gingivalis bacterial cells by lysing bacteria through protein binding.19 the flavonoids in propolis also inhibit the biofilm by reducing the number of polysaccharides in the biofilm and bacterial adhesion.20 antibacterial compounds have several mechanisms to inhibit bacterial growth. these include the destruction of cell walls by changing cell walls after they are formed, inhibiting the synthesis of new cell walls, changing the cytoplasmic membrane so that the core material inside the cell comes out, inhibition of enzyme action, inhibition of nucleic acid and protein formation, as well as changes in protein molecules 21 in molecular docking, the target protein used is peptidoglycan glycosyltransferase. protein peptidoglycan glycosyltransferase is a protein that plays a role in transferring disaccharide peptides from lipid ii to the glycan chain in bacterial wall synthesis.6 based on the research conducted, it was found that the four specific propolis compounds, including pinocembrin, hesperetin, chrysin and cape, and the control compound chlorhexidine, could inhibit protein peptidoglycan glycosyltransferase. the inhibition of the four propolis and chlorhexidine compounds occurred at the active site for disaccharide transfer so that the disaccharide transfer activity in pgt decreased. if the transfer of disaccharides decreases, the cell wall synthesis will be disrupted and this results in the inhibition of bacterial growth.22 based on this research, it can be concluded that cape is a propolis compound with the most significant potential as an inhibitor of protein peptidoglycan glycosyltransferase because it has the strongest bond to protein compared with the other three specific propolis compounds. however, chlorhexidine had the most robust binding to the inhibition of pgt protein. unfortunately, this research did not show the toxicity of propolis components such as pinocembrin, hesperitin, chrysin and cape as an antibacterial agent, so it requires further treatment to evaluate the toxicity of each component. further research is needed to identify and analyse the primary molecular candidates through in vitro or in vivo studies using physicochemical parameters.23 acknowledgements the researchers thank the faculty of dentistry at brawijaya university and direktorat jenderal pembelajaran dan kemahasiswaan kementerian riset, teknologi, dan pendidikan tinggi republik indonesia for supporting this research. references 1. wulandari nnf, handoko sa, kurniati dpy. determinan perilaku perawatan kesehatan gigi dan mulut pada anak usia 12 tahun di wilayah kerja puskesmas i baturiti. intisari sains medis. 2018; 9(3): 55–8. 2. badan penelitian dan pengembangan kesehatan. hasil utama riset kesehatan dasar. jakarta: kementerian kesehatan republik indonesia; 2018. p. 1–200. 3. slots j. periodontitis: facts, fallacies and the future. periodontol 2000. 2017; 75(1): 7–23. 4. harvey jd. periodontal microbiology. dent clin north am. 2017; 61(2): 253–69. 5. dahmane i, montagner c, matagne a, dumbre s, herdewijn p, terrak m. peptidoglycan glycosyltransferase-ligand binding assay based on tryptophan fluorescence quenching. biochimie. 2018; 152: 1–5. 6. wang y, cheong wl, liang z, so ly, chan kf, so pk, chen yw, wong wl, wong ky. hydrophobic substituents on isatin derivatives enhance their inhibition against bacterial peptidoglycan glycosyltransferase activity. bioorg chem. 2020; 97(february): 103710. 7. nazeri r, ghaiour m, abbasi s. evaluation of antibacterial effect of propolis and its application in mouthwash production. front dent. 2019; : 1–12. 8. pobiega k, kraśniewska k, derewiaka d, gniewosz m. comparison of the antimicrobial activity of propolis extracts obtained by means of various extraction methods. j food sci technol. 2019; 56(12): 5386–95. 9. berretta aa, silveira mad, cóndor capcha jm, de jong d. propolis and its potential against sars-cov-2 infection mechanisms and covid-19 disease: running title: propolis against sars-cov-2 infection and covid-19. biomed pharmacother. 2020; 131(august): 110622. 10. khurshid z, naseem m, zafar ms, najeeb s, zohaib s. propolis: a natural biomaterial for dental and oral healthcare. j dent res dent clin dent prospects. 2017; 11(4): 265–74. 11. khaerunnisa s, suhartati, awaluddin r. penelitian in silico untuk pemula. surabaya: airlangga university press; 2020. p. 112. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 226 sani et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 221–226 12. pinzi l, rastelli g. molecular docking: shifting paradigms in drug discovery. int j mol sci. 2019; 20(18): 4331. 13. stähli a, maheen cu, strauss fj, eick s, sculean a, gruber r. caffeic acid phenethyl ester protects against oxidative stress and dampens inflammation via heme oxygenase 1. int j oral sci. 2019; 11(1): 6. 14. huang yk, tseng kf, tsai ph, wang js, lee cy, shen my. il-8 as a potential therapeutic target for periodontitis and its inhibition by caffeic acid phenethyl ester in vitro. int j mol sci. 2021; 22(7): 3641. 15. salvatori c, bernardo m, fra o, san gbo, hospital pfbf, gargari m. effectiveness of a standardized propolis extract in non-surgical periodontal therapy. res sq. 2021; : 1–15. 16. zulhendri f, felitti r, fearnley j, ravalia m. the use of propolis in dentistry, oral health, and medicine: a review. j oral biosci. 2021; 63(1): 23–34. 17. guler hi, tatar g, yildiz o, belduz ao, kolayli s. investigation of potential inhibitor properties of ethanolic propolis extracts against ace-ii receptors for covid-19 treatment by molecular docking study. arch microbiol. 2021; 203(6): 3557–64. 18. sahlan m, mahira kf, pratami dk, rizal r, ansari mj, al-anazi km, farah ma. the cytotoxic and anti-inflammatory potential of tetragonula sapiens propolis from sulawesi on raw 264.7 cell lines. j king saud univ sci. 2021; 33(2): 101314. 19. kurniawan h, widyastuti w, hutapea me. the effectiveness of the combination of moringa oleifera extract and propolis on porphyromonas gingivalis biofilms compared to 0.7% tetracycline. dent j (majalah kedokt gigi). 2021; 54(2): 63–7. 20. marhamah, achmad h, mardiana, handayani h, fajriani, amin a, oktawati s. mouthwash product development based on ethanol extract of white rice bran (oryza sativa l.) as antibacterial of streptococcus mutans and porphyromonas gingivalis. j int dent med res. 2019; 12(3): 985–90. 21. alhaddad za, wahyudi d, tanod wa. bioaktivitas antibakteri dari ekstrak daun mangrove avicennia sp. j kelaut. 2019; 12(1): 12–22. 22. mesleh mf, rajaratnam p, conrad m, chandrasekaran v, liu cm, pandya ba, hwang ys, rye pt, muldoon c, becker b, zuegg j, meutermans w, moy ti. targeting bacterial cell wall peptidoglycan synthesis by inhibition of glycosyltransferase activity. chem biol drug des. 2016; 87(2): 190–9. 23. jain ak, thareja s. in vitro and in vivo characterization of pharmaceutical nanocarriers used for drug delivery. artif cells, nanomedicine, biotechnol. 2019; 47(1): 524–39. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p221–226 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p221-226 �� the effect of psidium guajava linn leaf extract on candida albicans adherence and the transversal strength of acrylic resin amiyatun naini1 and sherman salim2 1 department of prosthodontic, faculty of dentistry jember university, jember indonesia 2 department of prosthodontic, faculty of dentistry airlangga university, surabaya indonesia abstract denture stomatitis is an inflammation of oral cavity due to removable denture wearing. prevention of denture stomatitis can be effectively done by using mouth rinsing. currently, indonesian government is actively promoting traditional herbal medicine as an alternative medicine such as psidium guajava linn leaf which has an anti bacterial and anti fungal ability. the purpose of this study was to know the effective concentration and soaking duration to reduce candida albicans without lowering transversal strength of acrylic resin. this experimental laboratory study was using heat cured acrylic resin plate without surface polishing. the concentration of psidium guajava linn leaf extract used in this study were 32%, 34%, 36%, and 38% respectively with 15 minutes, 30 minutes, 1 hour and 8 hours soaking duration. the transversal strength was measured in the same concentration with 2 days, 10 days and 60 days soaking duration. sterile aquadest was used as control. two direction anova and lsd test were used in data analysis. the result showed significant difference in the number of candida albicans colony among concentrations and soaking durations. significant difference was also found in transversal strength among concentrations and soaking durations. it is concluded that the extract of psidium guajava linn leaf in 38% concentration with 8 hours soaking duration will lower the candida albicans colony, whereas 38% concentration with 60 days soaking duration will lower the transversal strength but it is still above the standard value. key words: psidium guajava linn, acrylic resin, candida albicans, transversal strength correspondence: amiyatun naini, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas jember. jln. kalimantan no. 37 jember 68121, indonesia. e-mail: amiyatunnaini_drg@yahoo.co.id introduction in dentistry, up to the present time, denture base acrylic resin is still widely used besides denture base metal due to the relatively cheap price, easily manipulated, non toxic, easily reparation process when it is broken, and the small dimensional changes,1,2 while the disadvantages of acrylic resin are: porous, water absorption ability, easily fracture and broken.1 denture is a good place for food debris accumulation. denture base acrylic resin can be the place of stain, tartar and plaque accumulation. accumulative plaque and food debris will increase the number of candida albicans, therefore the endotoxin product can penetrate into mucous membrane and cause inflammation which is called denture stomatitis.3,4 it is found that 65% of elderly population are wearing denture, two thirds of 65% of denture users suffer from denture stomatitis.5-7 prevention of denture stomatitis including pathogenic candida albicans decreasing is very important. candida albicans infection can be prevented by good cleaning. removable denture cleaning can be done in two ways i.e mechanically and chemically. mechanically cleaning can be done by brushing or using ultrasonic cleaner. chemically cleaning can be done by soaking the denture into cleaning solution. the soaking duration in cleaning solution can be done for the whole night, 1 hour, 30 minutes or 15 minutes depending on the type of material.3 denture cleaning solution is classified into five kinds i.e: alkaline peroxide, alkaline hypochlorite, inorganic acid, disinfectant and enzyme.8 due to relatively expensive price of chemical cleaning solution, traditional herb can be used as alternative cleaning solution for denture base acrylic resin. some traditional herbal medicines such as leaf of psidium guajava linn9 can be used as denture cleaning solution and has an antiseptic and disinfectant function. the simple name of psidium guajava linn is psidii folium. the chemical contents of the leaf are tannin, atsiri oil, cuercetine, 3–arabino piranosida, guayaverin, leukocianidine, anritoxide, avicularin, galate acid. the data taken from fitochemical laboratory showed that the mean concentration of galate acid was equivalent to 7.71%, and 2.33% cuercetine of 96% ethanol extract. the result of pharmacological study showed that extract of psidium guajava linn leaf has an antimicrobial effect against staphylococcus sp.10 the use of tannin is as an astringent, whereas atsiri oil and other active material as an antibacterial substance. galate acid functions as antibacterial, antiviral, antifungal, anti inflammation, anti tumor, anti anaphilactic, anti mutagenic and it is usually used as astringent.11 galate acid is including phenol group. phenol can kill vegetative cell, fungi and bacteria forming spores by protein denaturation and lowering the surface tension, so that bacterial permeability will increase.12 if �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 25-29 phenol group is used for acrylic resin soaking, either as cleaning or anti bacterial solution, the physical characteristic of denture base acrylic resin will be changed resulting in transversal strength decrease.13 according to department of health, republic of indonesia, extract of psidium guajava linn leaf in 30%, 40%, 50%, 60% concentration will inhibit the growth of s. typhi, s. paratyphi a and s. paratyphi b.14 up to now there is no study on mycological effect of extract of psidium guajava linn leaf against candida albicans, meanwhile acrylic resin can be adversely affected by phenol. based on the above issue, a study on soaking duration of acrylic resin plate in various concentrations of psidium guajava linn leaf extract against candida albicans and transversal strength is necessarily done. material and method this study was experimental laboratory using the post test–only control group design. the samples were: 10 × 10 × 1 mm acrylic resin plate for candida albicans test and 65 × 10 × 2.5 mm for transversal strength.15 the study was taken place in laboratorium dasar bersama (ldb), laboratory of dental technology, fitochemical of pharmacy faculty, laboratory of microbiology faculty of dentistry airlangga university. the acrylic resin plates were prepared using 10 × 10 × 1 mm stippled wax. samples for candida albicans test were not polished. brass plate master model, 65 × 10 × 2.5 mm in size was prepared for transversal strength test, then hard gypsum mold was made by mixing 100 gr of gypsum powder and 24 ml of water (based on manufacturer's recommendation). the mixture was manually steered for 15 seconds and put into the cuvet on the vibrator. stippled wax was put on the gypsum and left for 15 minutes. the gypsum surface was covered by vaseline, then the upper cuvet was filled by gypsum on the vibrator. the wax was removed by pouring hot water until it was clean. the next step, mould was filled by acrylic resin i.e powder and acrylic resin liquid in 5.75 gr : 2.5 ml (based on manufacturer's recommendation) was prepared in porcelain bowl and steered at room temperature until it reached dough phase. the mould which was already covered by cold mould seal filled by acrylic resin dough. cuvet was closed and pressed by hydrolic press in 22 kg/cm2 hg pressure and then put into the curing unit. the process was done at 75° c for 90 minutes and continued at 100° c for 30 minutes. the curing process was completed and it was left to let it cool, finally the sample was removed from the cuvet. the leaves for making psidium guajava linn extract were taken from a ten year old tree, located in jl. kawi 8, jenggawah, jember, and identified at the laboratory of botany pharmacy–pharmacognocy of pharmacy faculty, airlangga university. first, fresh psidium guajava linn leaf was cleaned and dried in the room. weighed 500 gr and put into a crusher and given 1000 ml of ethanol, being maserated for 72 hours and then filtered using buchener funnel. the filtrate was evaporated using vacuum evaporator. finally, 75 gr extract was obtained and this was 100% extract. the extract of psidium guajava linn leaf was weighed in 3.2 gr, 3.4 gr, 3.6 gr, 3.8 gr then dissolved in 10 ml of sterile aquadest until 32%, 34%, 36%, 38% concentration of extract leaf was reached. the acrylic resin plates were washed in running water for 48 hours, sterilization was done using autoclave at 121° c for 18 minutes, soaked in sterile saliva for 1 hour and washed by pbs twice, then, soaked in extract of psidium guajava linn leaf in 4 kinds of soaking duration (15 minute, 30 minute, 1 hour, 8 hour). in every concentration the soaking was repeated 7 times and aquadest was as control. the samples were washed by pbs twice, put into sabouraud’s broth followed by 30 seconds vortexed. incubation was done in saboroud’s dextrose at 37° c for 48 hours, continued by counting the number of candida albicans colony (cfu/ml). for transversal strength test, all samples of acrylic resin plates (65 × 10 × 2.5 mm in size) soaked in aquadest for 48 hours, followed by soaking in extract of psidium guajava linn leaf 32%, 34%, 36%, 32%, 38% concentration for 2 days, 10 days, 30 days and 60 days. the replacement of soaking solution was done in every 24 hours, then washed by aquadest. the samples were dried before transversal strength test was done using autograph ag-10te in cross head speed 1/10 mm/second. the distance between the two holders was 50 mm. statistical analysis was done using two-way anova test, followed by lsd test. result table 1 shows the occurrence of candida albicans on the surface of acrylic resin plate after being soaked in 32%, 34%, 36%, 38% concentration of psidium guajava linn leaf extract for 15 minutes, 30 minutes, 1 hour and 8 hours. kolmogorof smirnov test showed p > 0.05 which means the data had normal distribution. levene test score was 1.411 and level of significance = 0.134 (p > 0.05) for concentration and soaking duration, which means the data was homogenous, therefore the test was continued by twoway anova test. anova test showed a significant difference among the number of candida albicans colony in acrylic resin plate which was soaked in 32%, 34%, 36%, 38% concentration of psidium guajava linn leaf extract in 15 minutes, 30 minutes, 1 hour and 8 hours (p < 0,05). there is an interaction of significant difference in the number of candida albicans colony between concentration and soaking duration. to know further about the difference among the sample groups, lsd test was done and the result can be seen in table 2 and 3. ��naini and salim: the effect of psidium guajava linn leaf extract there is significant difference among every concentration (32%, 34%, 36%, 38% and control). the increase of concentration will influence the number of candida albicans colony and also the increase of soaking duration will show significant difference. the result of transversal strength acrylic resin plate will be shown in table 4. kolmogorof smirnov test showed p > 0.05 which means the data had normal distribution. levene test score was 1.477 with level of significance 0.106 (p > 0.05) for concentration and soaking duration. it means the data was homogeneous. therefore data analysis was done using two-way anova test. anova test showed significant difference in transversal strength of acrylic resin plate soaked in 32%, table 2. lsd test on the number of candida albicans on the acrylic resin plate after being soaked in 32%, 34%, 36%, 38% concentration of psidium guajava linn leaf extract and control (cfu/0.1 ml) group 32% 34% 36% 38% control control 32% 34% 36% 38% s s s s s s s s s s note: s = significant table 3. lsd test on the number of candida albicans on the acrylic resin plate after being soaked in psidium guajava linn leaf extract for 15 minutes, 30 minutes, 1 hour, 8 hours (cfu/0.1 ml) group 15 minutes 30 minutes 1 hour 8 hours 8 hours 1 hour 30 minutes 15 minutes s s s s s s note: s = significant table 5. lsd test on transversal strength of acrylic resin soaked in 32%, 34%, 36%, 38% concentration of psidium guajava linn leaf extract and control (n/mm2) group 32% 34% 36% 38% control control 32% 34% 36% 38% s s ns ns s ns ns s ns ns note: s = significant, ns = not significant table 1. the mean of candida colony on the surface of acrylic resin plate after being soaked in extract of psidium guajava linn leaf (cfu/0.1 ml) group soaking duration 15 minutes 30 minutes 1 hour 8 hours x sd x sd x sd x sd control 32% 34% 36% 38% 803.29 776.57 663.71 545.57 396.43 1.98 1.90 2.29 3.90 2.82 975.71 629.71 524.14 400.86 247.14 2.56 4.19 2.73 4.26 5.84 1021.43 565.00 496.29 281.27 126.29 5.22 3.21 1.49 3.40 3.82 1392.29 67.33 52.71 36.86 8.86 3.39 2.99 2.87 5.55 2.60 note: x = mean, sd = standard deviation table 4. mean of transversal strength of acrylic resin plate soaked in psidium guajava linn leaf extract (n/mm2) group soaking duration 2 days 10 days 30 days 60 days x sd x sd x sd x sd control 32% 34% 36% 38% 182.31 176.06 170.06 168.94 168.26 9.76 14.13 7.36 8.89 9.92 167.14 166.71 166.03 164.91 163.80 4.17 10.96 5.89 9.48 6.37 163.80 162.69 161.57 159.34 158.23 6.36 11.42 10.77 8.84 9.78 159.34 158.23 157.11 154.89 151.54 9.92 8.68 11.42 10.49 7.61 note: x = mean, sd = standard deviation �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 25-29 table 6. lsd test on transversal strength of acrylic resin plate soaked in psidium guajava linn leaf extract for 2 days, 10 days, 30 days, and 60 days (n/m2) group 2 days 10 days 30 days 60 days 60 days 30 days 10 days 2 days s s s s s s note: s = significant 34%, 36%, 38% concentration of psidium guajava linn leaf extract and control with soaking duration 2 days, 10 days, 30 days and 60 days (p < 0.05). there is interaction between concentration and soaking duration and lsd test was done (table 5). significant difference was found among groups of 32%, 34%, 36%, 38% concentration and control. the increase of concentration of psidium guajava linn leaf extract would decrease the transversal strength and also significant difference was found in soaking duration. the increase of soaking duration would decrease the transversal strength. discussion in this study, we used one of family herbal medicine plants which is frequently planted in the garden and easily grows i.e. psidium guajava linn (guava) which contains galate acid and atsiri oil as anti fungal.16 one of the forces received by acrylic denture base is transversal strength. soaking of acrylic resin plate either in cleaning or anti bacterial solution can alter the basic character of denture base acrylic resin such as transversal strength.14 statistical analysis two-way anova test followed by lsd test shows that the higher the concentration of the extract and the longer the soaking duration resulting significant decreasing of the number of candida albicans. on the contrary the lower concentration and the shorter the soaking duration, the number of candida albicans will significantly increase. it is because extract of psidium guajava linn contains 96% of mean total of 7.71% galate acid. by the increase concentration, galate acid content would also increase including phenol in the extract solution so the anti candida albicans will also increase. the duration of soaking was 15 minutes, 30 minutes, 1 hour, and 8 hours in extract of psidium guajava linn leaf will also decrease the number of candida albicans colony because the increase of contact duration will increase the affectivity of anti microbial agent. the affectivity of material will be influenced by the concentration, duration and temperature.17 galate acid including phenol group is used as anti bacterial, anti fungal, and can also be used as astringent.11 chemical substance of galate acid has anti fungal activity against candida albicans.16 based on anti microbial function, phenol can kill cell of vegetative fungi and bacteria forming spore by protein denaturation and decrease the surface tension so the permeability of bacteria and fungi will increase.12 transversal strength of acrylic resin plate resulted from two-way anova and lsd test shows the higher the concentration and the lower the soaking duration, the transversal strength of acrylic resin plate will significantly decrease. on the contrary, the lower the concentration and the shorter the soaking duration, the transversal strength of acrylic resin plate will significantly increase. long polyester polymer consisting of repeated methyl metacrilate unit with high polarity is the possible cause of acrylic resin degradation resulting in transversal strength decrease. ester in acid environment will be hydrolyzed. if polymer is hydrolized, degradation will occur resulting in transversal strength decrease.2 phenol content in guava leaf will cause stretching of intra molecular bind in which sooner or later can break intra molecular bind, so the transversal strength will decrease.18 the mean of the lowest transversal strength is in 60 day–soaking duration (151.54 n/mm2). this value is much higher than recommended transversal strength of heat cured acrylic resin that is not less than 55 n/mm2.16 the acrylic 2 days, 10 days, 30 days, and 60 days soaking duration of the acrylic resin plate is equal to 6 days, 1 month, 3 months, and 6 months use. it is concluded that 38% concentration and 8 hour soaking duration will decrease the number of candida albicans in acrylic resin plate. thirty eight percent concentration and 60 days soaking duration will decrease the transversal strength of acrylic resin plate but it was still above the standar value that is not less than 55 n/mm2. references 1. reisbick mh. dental material in clinical dentistry. london: john wright; 1982. p. 309–23. 2. combe ec. notes on dental material. 6th ed edinburg: churchil livingstone; 1992. p. 79–120. 3. jorgensen be. material and method for cleaning denture. j prosthet dent 1979; 42: 619–22. 4. samaranayake lp, mc courtie j, mac fartene tw. factor affecting the in vitro adherence of candida albicans to acrylic surface. arch oral biol 1980; 25:611–5. 5. segal e, lehrman o, dayan d. adherence in vitro of various candida spesies to acrylic surface. oral surg oral med oral pathol 1988; 66:670–3. 6. lapocino am, wathen wf. oral candida infection and denture stomatitis: a comprehensive review. j am dent assoc 992; 123:446–51. 7. kulak y, arikan a. aetiology of denture stomatitis. j of mamara university dental faculty 1993; 1:307–14. 8. davenport jc. the oral distribution of candida albicans in denture stomatitis. brit dent j 1970; 129:151–7. 9. moore tc, de smith, kenny ge. sanitazion of denture by severalsanitazion of denture by several denture hygiene methods. j prosthet dent 1984; 52:158–63. 10. departemen kesehatan ri. pemanfatan tanaman obat. edisi iii.edisi iii. jakarta: dep. kes. ri; 1983. p. 82–3. 11. bambang. tampil percaya diri dengan ramuan tradisional. edisi ii.edisi ii. jakarta: penebar swadaya; 2000. p. 70–1. 12. jeffrey bh, herbert b, gerard pm. phytochemical. dictionary. 2dictionary. 2nd ed. france: taylor & francis ltd; 1999. p. 501. ��naini and salim: the effect of psidium guajava linn leaf extract 13. shen c, javid ns, colaizzi fa. the effect of glutaraldehyde basethe effect of glutaraldehyde base disinfectants on denture base resin. j prosthet dent 1989; 61:583-9. 14. departemen kesehatan ri. penelitian tanaman obat di beberapa perguruan tinggi di indonesia, badan penelitian dan pengembangan kesehatan pusat penelitian dan pengembangan farmasi. jakarta. 2000; p. 240. 15. devi r. the transverse strength of acrylic after coleus amboinicus, lour extract solution immersion. dent j (majalah kedokteran gigi) 2006; 39(4):156–60. 16. asad t, watkinson ac, huggest r. the effect of desinfection procedures on flexural properties of denture base acrylic resins. j prosthet dent. 1992; 68:194–5. 17. li xc, jacob mr, pasco ds, elsobly hn, nimrod ac, walker la, clark am. phenolic compounds from miconia myriantha inhibiting candida aspartic proteases. j nat pred 2001; 64(10):1282–5. 18. siswandono, soekarjo b. kimia medisinal. cetakan i. surabaya: airlangga university press; 1995. p. 247–8. 122122 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 122–126 original article changes in the corrosion rate and microstructure of beta titanium wire using kiwi peel extract hilda fitria lubis, hanifa natarisya department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: beta titanium orthodontic wire is known to have good corrosion resistance but is weak in acidic environments, which advance the corrosion rate. one natural inhibitor that can be used to decrease corrosion is kiwi peel extract, which has a high antioxidant level. purpose: this study aims to examine the ability of the extract to decrease the corrosion rate and microstructural changes of beta titanium at an acidic ph (ph 5). methods: the samples used were beta titanium with a diameter of 0.016 x 0.022 in and a length of 6 cm. a total of 28 samples (n=28) were divided into four groups—a control group immersed in ph 5 artificial saliva and three treatment groups immersed in kiwi peel extract at concentrations of 400, 500, and 600 ppm (n=7), respectively. the samples were immersed for seven days at 37oc in an incubator. the corrosion rate was tested using the weight-loss method and microstructure change was analyzed using a scanning electron microscope (sem). results: one-way anova showed that there are significant differences in corrosion rates between beta titanium immersed in artificial saliva and beta titanium immersed in kiwi peel extract with p=0.01 (p<0.05). sem analysis results showed that the group with the least surface changes was the one immersed in 400 ppm of kiwi peel extract. conclusion: weight-loss and sem methods show similar results. kiwi peel extract proved to decrease the corrosion rate and changes in the microstructure of the wire most effectively at a concentration of 400 ppm. keywords: beta titanium; corrosion rate; kiwi peel extract; medicine; dentistry article history: received 18 may 2022; revised 20 august 2022; accepted 31 august 2022 correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no 2, medan 20155, indonesia. email: hilda.fitria@usu.ac.id introduction beta titanium orthodontic wire, also known as titanium molybdenum alloy (tma), is a type of orthodontic wire developed by burstone and goldberg in 1980. this wire is composed of 78% titanium, 11.5% molybdenum, 6% zirconia, and 4.5% tin.1,2 beta titanium orthodontic wire has a rough surface with irregular grooves that affect plaque accumulation and increase friction.3 orthodontic wire alloy characteristics and surface roughness play an important role and can change the behavior of the wire. studies show that surface characteristics of orthodontic wires affect their performance and biocompatibility. the surface topography can also critically modify the aesthetics, the performance efficiency of orthodontic components, and the corrosion rate. plaque accumulation is affected by surface roughness and plays an important role in other properties of orthodontic wires. surface roughness can also change the coefficient of friction.4 increased friction due to corrosion will cause non-optimal static or dynamic friction movement. friction can reduce available forces up to 40%, which causes loss of anchor.5–7 corrosion in the beta titanium wire can also cause titanium ion release that can cause hypersensitivity reactions in some individuals. studies report that sensitivity to titanium is between 0.6% and 5% in the general population.8 corrosion is unavoidable, but the rate of corrosion can be reduced. one way to prevent corrosion is to add an inhibitor. corrosion inhibitors are chemicals that will effectively reduce the corrosion rate when added. chemical corrosion inhibitors can be inorganic or organic, but since inorganic corrosion inhibitors have toxicity, organic inhibitors are preferred for their nontoxic properties. various types of organic inhibitors have been studied for their ability to inhibit corrosion, but their effectiveness and potential depends on their being used properly.9 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p122–126 mailto:hilda.fitria@usu.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p122-126 123 lubis and natarisya. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 122–126 kiwi (actinidia deliciosa) is commonly consumed worldwide. kiwi peel has a high level of antioxidants, such as flavonoids, carotenoids, polyphenols, and minerals, which form a protective layer and reduce corrosion rate. flavonoids are one of the most common secondary metabolic compounds found in plant tissues and play important roles in plant biochemistry and physiology, acting as antioxidants, enzyme inhibitors, precursors of toxic substances, and pigments. flavonoids belong to a group of phenolic compounds with a c6-c3-c6 chemical structure. quercetin is one of the most commonly occurring natural flavonoids and is a secondary plant metabolite with anti-inflammatory, antibacterial, antiviral, and antioxidant activities. kiwi peel has a 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging rate of 95.16%, which is very effective in inhibiting corrosion.10–14 the adsorption of organic corrosion inhibitors on the surface of the wire begins with a replacement of water molecules on the metal surface with inhibitor molecules (inh). the molecular shift occurs because the water tension is higher than the surface tension of the inhibitor. the inhibitor binds to the ions on the metal surface to form a metal-inhibitor complex compound. this is necessary to resist and limit direct interaction between the metal and the corrosive solution.15 kiwi peel extract is thought to decrease corrosion and microstructural changes of beta titanium orthodontic wires due to its high antioxidant levels, which play a role in forming a protective layer and reducing the corrosion rate. currently, very little research has been done on the use of kiwi peel extract as a corrosion inhibitor for beta titanium wire. therefore, investigated the differences in the corrosion rate and microstructure of beta titanium wire after immersion in ph 5 artificial saliva and in macerated kiwi peel extract at concentrations of 400, 500, and 600 ppm. this study is expected to provide a reference for dentists considering the use of kiwi peel extract as an alternative mouthwash during orthodontic treatment to decrease the corrosion of beta titanium orthodontic wires. materials and methods the experiment has a preand post-test design and includes a control group. twenty-eight beta titanium orthodontic wires from american orthodontics (sheboygan, wisconsin, us), each with a diameter of 0.016 x 0.022 in and a length of 6 cm, were used. each wire was weighed before immersion using an analytical scale. the wires were then divided into four groups. group 1 was a control group immersed in artificial saliva with ph 5, while groups 2–4 were treatment groups immersed in 400, 500, and 600 ppm of kiwi peel extract, respectively (figure 1). making kiwi peel extract began with peeling the skin of the kiwi fruit that had been washed thoroughly. it was then weighed to 500 g and placed in a drying cabinet for two days until the whole kiwi peel dried or became simplicia. the simplicia was weighed again to 95 g and then gradually blended into a smaller form. the blended simplicia was put into a closed container and dissolved in a liter of 70% ethanol, stirred, and soaked for 3 x 24 hours. after that, the solution was filtered to obtain a liquid extract of kiwi peel, or macerate i. the pulp extraction process was repeated using 70% ethanol to obtain macerate ii. then evaporation was carried out to obtain a thick extract of 100% pure kiwi peel. concentrations of 400, 500, and 600 ppm of kiwi peel extract were obtained by mixing 0.04, 0.05, and 0.06 g of the thick extract with distilled water to a total volume of 100 ml, respectively. each group was stored in an incubator for seven days at 37oc. the wires were then taken out, rinsed, and dried. they were weighed again after immersion using the same analytical scale. the corrosion rates of the wires were calculated using the weight-loss method based on american standard testing and material (astm) g i “practice for preparing, cleaning, and evaluating corrosion test specimens” with the following formula: 𝐶𝑜𝑟𝑟𝑜𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 = � × � �×�×� where: k = constant (3.45 x 106) t = time of exposure (h) a = surface area (cm2) w = mass loss (g) d = density (g/cm3). samples were analyzed with a scanning electron microscope (sem) to determine microstructure changes in the orthodontic wire surface after immersion. statistical analysis was carried out using the statistical package for the social sciences (spss) with the shapiro-wilk normality test. the one-way anova parametric test was then used with a confidence interval of 95%. results the shapiro-wilk normality test results showed that the average corrosion rates of groups 1, 2, and 3 had significance values of p=0.771 (≥0.05), p=0.738 (≥0.05), a b figure 1. beta titanium orthodontic wire with a diameter of 0.016 x 0.022 in and a length of 6 cm (a), beta titanium orthodontic wires immersed according to the control and treatment group protocols (b). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p122–126 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p122-126 124lubis and natarisya. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 122–126 table 1. differences between the control and treatment groups using the lsd test group control 400 ppm kiwi peel extract 500 ppm kiwi peel extract 600 ppm kiwi peel extract control 0.004* 0.006* 0.060 400 ppm kiwi peel extract 0.004* 0.837 0.222 500 ppm kiwi peel extract 0.006* 0.837 0.307 600 ppm kiwi peel extract 0.060 0.222 0.307 * there is a significant difference. table 2. corrosion rate differences between the control and treatment group beta titanium orthodontic wires after immersion group mils per year (mpy) mean ± sd p control 0.63±0.422 0.014** 400 ppm kiwi peel extract -0.16±0.263* 500 ppm kiwi peel extract -0.11±0.602* 600 ppm kiwi peel extract 0.15±0.336 * (-) indicates that the corrosion rate obtained per year (mils per year) after being given a corrosion inhibitor is below 0. ** p=0.014 (< 0.05) which means that there is a significant difference between groups. a b c d figure 2. the beta titanium orthodontic wire of the control group shows a wire surface with many round and oval grooves with a black base (a), the 400-ppm kiwi peel extract wire surface shows a long groove shape that is more regular and more closely resembled unused beta titanium orthodontic wire (b), the 500-ppm kiwi peel extract shows a wire surface with a regular groove shape, but there is also a small round groove around the wire surface (c), the 600-ppm kiwi peel extract shows a surface with many round and oval grooves on a black base (d). 1000x magnification. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p122–126 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p122-126 125 lubis and natarisya. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 122–126 and p=0.583 (≥0.05), respectively, while the control group had a significance value of p=0.120 (≥0.05). the results showed that all treatment group data met the assumption of normality or were normally distributed because all groups had a significance value of p≥0.05. table 1 shows the differences between the treatment groups using the least significant difference (lsd) test. the basis for decision-making was as follows: looking at the probability numbers, if the p-value was <0.05, the difference between the two treatment groups was taken as significant. table 2 shows the changes in the corrosion rates of beta titanium orthodontic wire after immersion in either artificial saliva or kiwi peel extract (actinidia deliciosa) at 400, 500, and 600 ppm concentrations. one-way anova results showed a p-value of 0.014 (p<0.05), which means that there is a significant difference between the groups. these results also indicate that the alternative hypothesis (h1) is accepted and kiwi peel extract has an inhibiting effect on the corrosion rates of beta titanium orthodontic wires and on changes in the microstructure of the wires. sem analysis of each group, seen in figure 2, showed that the smoothest wire surface microstructure was seen at 400 ppm of kiwi peel extract, and the smoothness decreased with increasing extract concentrations. the results of the sem test in the control group showed a wire surface with many round and oval grooves with a black base; the normal structure of the beta titanium orthodontic wire was also grooved. sem analysis of the 400-ppm kiwi peel extract showed a wire surface with a long groove shape that was more regular and more nearly resembled unused beta titanium orthodontic wire. sem test of the 500-ppm kiwi peel extract showed that the wire surface had a regular groove shape, but additionally, there was a small round groove all around the wire surface. the results of the sem test on the 600-ppm kiwi peel extract showed that the wire surface had many round and oval grooves on a black base. discussion beta titanium orthodontic wire immersed in kiwi peel extract had a lower corrosion rate compared to the control group. this result was in line with dehghani et al.16 and arias-montoya et al.,17 who stated that the addition of kiwi peel extract can decrease the corrosion rate of wires. organic corrosion inhibitors are usually used in low concentrations. they can decrease the reaction between the metal and its environment. kiwi peel extract is effective in decreasing corrosion rate due to the mixture of phytochemicals it contains. this mixture has various functional groups that can adsorb on the surface of the wire.18 compounds such as flavonoids, steroids, tannins, and terpenoids can adsorb on the surface of the wire. flavonoids present in kiwi peel act as antioxidants by donating hydrogen atoms, by their ability to adhere to metals as glucosides (containing a glucose side chain), or in a free form called aglycone. a common natural flavonoid occurring as a plant secondary metabolite is quercetin. as synthetic flavonoid production is still uncommon, plants are the only source of quercetin. vegetables and fruits are composed of various groups of flavonoids in different amounts.11,12,18 in this study, kiwi peel extract at a concentration of 400 ppm had the best inhibition strength compared to higher concentrations of 500 ppm and 600 ppm. increasing the concentration of organic inhibitors can increase the complex compounds formed and the corrosion rate is lower due to the inhibitor being adsorbed on the metal surface. however, a high concentration of the corrosion inhibitor can cause the inhibitor molecules on the metal surface to be replaced by other molecules, thus reducing the protective effect of the corrosion inhibitor.19 titanium is the highest component of the beta titanium orthodontic wire with a percentage of 78%. it can withstand mechanical pressure during mastication and has very good chemical stability due to a highly protective titanium dioxide layer (tio2) on its surface. titanium can withstand corrosion. however, if the stable oxide layer on its surface is gone or cannot be regenerated, corrosion can occur. disintegration in metal alloys can occur due to moisture, acid or base solutions, or certain chemicals.20 the surface structure of the wire depends on the alloy used, the complex manufacturing process, and the final treatment of the wire surface. beta titanium orthodontic wire has a surface microstructure that looks rough compared to other types of wires. it has a surface structure with large pores that are evenly distributed and also has a deep groove. the surface roughness of beta titanium wire is due to adherence or cold welding of titanium to the mold during processing.21 sem results of beta titanium orthodontic wire immersed in ph 5 artificial saliva in this study were in line with pataijindachote et al.,22 who investigated four types of orthodontic wires immersed in ph 2.5 and ph 6 for 90 days. their results showed that the mean corrosion rate at ph 2.5 was higher than at ph 6, and the mean corrosion rate for 90 days of immersion was higher compared to untreated wire. the sem image of the beta titanium wire after immersion for 90 days at ph 2.5 showed a widened beta titanium normal groove with additional grooves, while the wire at ph 6 showed smaller grooves.22 these results indicate that the more acidic the ph in contact with the wire, the greater the damage to the wire surface due to corrosion and the more different it is from the normal structure of the beta titanium orthodontic wire. corrosion rates tested using the weight-loss method and microstructure changes of beta titanium orthodontic wire observed using sem showed significant differences between the groups. the lowest corrosion rate was found in the 400-ppm kiwi peel extract group, with a wire surface microstructure that was most similar to the wire image before use, followed by the 500-ppm kiwi peel extract group, with a wire surface microstructure that had an copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p122–126 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p122-126 126lubis and natarisya. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 122–126 additional small round groove around the wire surface. the kiwi peel extract group with a concentration of 600 ppm had a microstructure depiction of the wire surface with many round and oval grooves with a black base. the control group wire had a normal structure, though it was covered with additional grooves due to corrosion. references 1. castro sm, ponces mj, lopes jd, vasconcelos m, pollmann mcf. orthodontic wires and its corrosion—the specific case of stainless steel and beta-titanium. j dent sci. 2015; 10(1): 1–7. 2. huda mm, siregar e, ismah n. slot deformation of various stainless steel bracket due to the torque force of the beta-titanium wire. j phys conf ser. 2017; 884: 012105. 3. alsabti n, talic n. comparison of static friction and surface topography of low friction and conventional tma orthodontic arch wires: an in-vitro study. saudi dent j. 2021; 33(5): 268–75. 4. syahdinda mr, lucynda l, triwardhani a, hamid t. comparison of frictional coefficient and surface roughness between three different active self-ligating brackets: an experimental in vitro study. j int oral heal. 2020; 12(6): 551–5. 5. syahdinda mr, nugraha ap, triwardhani a, noor tne binti ta. management of impacted maxillary canine with surgical exposure and alignment by orthodontic treatment. dent j. 2022; 55(4): 235–9. 6. singh v, acharya s, patnaik s, nanda sb. comparative evaluation of frictional forces between different archwire-bracket combinations. orthod j nepal. 2014; 4(1): 22–8. 7. monteiro mrg, da silva le, elias cn, vilella o de v. frictional resistance of self-ligating versus conventional brackets in different bracket-archwire-angle combinations. j appl oral sci. 2014; 22(3): 228–34. 8. babu cs, pasha mf, satpathy s, gowda g. titanium hypersensitivity: a clinical study. int j oral implantol clin res. 2014; 5(1): 8–11. 9. devi i, sufarnap e, finna, pane erp. chitosan’s effects on the acidity, copper ion release, deflection, and surface roughness of copper-nickel-titanium archwire. dent j. 2023; 56(1): 41–7. 10. salama zeinab a, aboul-enein ahmed m, gaafar alaa a, faten a-e, aly hanan f, asker mohsen s, ahmed habiba a. active constituents of kiwi (actinidia deliciosa planch) peels and their biological activities as antioxidant, antimicrobial and anticancer. res j chem environ. 2018; 22(9): 52–9. 11. alim a, li t, nisar t, ren d, zhai x, pang y, yang x. antioxidant, antimicrobial, and antiproliferative activity-based comparative study of peel and flesh polyphenols from actinidia chinensis. food nutr res. 2019; 63: 1577. 12. subekti n, soedarsono jw, riastuti r, sianipar fd. development of environmental friendly corrosion inhibitor from the extract of areca flower for mild steel in acidic media. eastern-european j enterp technol. 2020; 2(6 (104)): 34–45. 13. ozgen s, kilinc ok, selamoğlu z. antioxidant activity of quercetin: a mechanistic review. turkish j agric food sci technol. 2016; 4(12): 1134. 14. wang y, li l, liu h, zhao t, meng c, liu z, liu x. bioactive compounds and in vitro antioxidant activities of peel, flesh and seed powder of kiwi fruit. int j food sci technol. 2018; 53(9): 2239–45. 15. dariva cg, galio af. corrosion inhibitors – principles, mechanisms and applications. in: aliof khazraei m, editor. developments in corrosion protection. intechopen; 2014. p. 366–78. 16. dehghani a, bahlakeh g, ramezanzadeh b. kiwi fruit shell extract; potent, and low cost green inhibitor for mid steel in 1 m hcl media. in: national conference on pigments, environment and sustainable development. science and technology research institute of paint and coatings; 2019. p. 1–2. 17. arias-montoya mi, dominguez-patiño gf, gonzalez-rodriguez jg, dominguez-patiño ja, dominguez-patiño ml. corrosion inhibition of carbon steel in acidic mediaby using actinidia deliciosa (kiwifruit) extract. adv mater phys chem. 2015; 05(11): 447–57. 18. fazal br, becker t, kinsella b, lepkova k. a review of plant extracts as green corrosion inhibitors for co2 corrosion of carbon steel. npj mater degrad. 2022; 6(1): 5. 19. banjang lg, wibowo d, kurniawan fkd. the analysis of wuluh starfruit leaf extract (averrhoa blimbi linn) as inhibitor on corrosion rate of stainless steel orthodontic wire. dentino j kedokt gigi. 2018; 3(2): 138–42. 20. ardhy s, gunawarman, affi j. perilaku korosi titanium dalam larutan modifikasi saliva buatan untuk aplikasi ortodontik. j mek. 2015; 6(2): 585–93. 21. isac j, chandrashekar b, mahendra s, mahesh c, shetty b, arun a. effects of clinical use and sterilization on surface topography and surface roughness of three commonly used types of orthodontic archwires. indian j dent res. 2015; 26(4): 378–83. 22. pataijindachote j, ju ntavee n, viwat tanatipa n. cor rosion analysis of or thodontic wires: an interaction st udy of wire type, ph and immersion time. adv dent oral heal. 2018; 10(1): 555780. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p122–126 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p122-126 200 vol. 44. no. 4 december 2011 research report stimulation of osteoblast activity by induction of aloe vera and xenograft combination utari kresnoadi1 and retno pudji rahayu2 1department of prosthodontics 2department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: tooth extraction is generally followed by alveolar ridge resorption that later can cause flat ridge. aloe vera have biogenic stimulator and hormone activities for wound healing. purpose: this study was aimed to know osteoblast activities in alveolar bone after induction of aloe vera and xcb combination. methods: fifty four of cavia cabaya were divided into three main groups. group i was control group. group ii was filled with xenograft concelous bovine (xcb) and group iii was filled with the combination of aloe vera gel and xcb. then, each group was divided into three sub groups according to timing, they are 14, 30, and 60 days after tooth extraction and application. histology and morphology examination were performed on the harvested specimens. results: there were significant differences between the control group and the other groups filled with the combination of aloe vera and xcb. conclusion: in conclusion, the application of aloe vera gel and xenograft combination decrease the number of osteoclast and increase the number of osteoblast in post tooth extraction alveolar bone structure indicating the new growth of alveolar bone. key words: osteoblast, osteoclast, aloe vera, xenograft concelous bovine, tooth extraction socket abstrak latar belakang: pencabutan gigi pada umumnya selalu diikuti resopsi tulang alveolar, sehingga bila terjadi dalam waktu yang lama ridge akan menjadi flat. aloe vera adalah bahan stimulasi biogenik dan mempunyai aktivitas hormon untuk proses penyembuhan luka. tujuan: tujuan dari penelitian ini adalah untuk mengetahui aktivitas osteoblas pada tulang alveol dengan pemberian kombinasi aloe vera gel dan xenograft concelous bovine (xcb). metode: lima puluh empat ekor cavia cabaya, dibagi menjadi 3 kelompok besar, kelompok pertama adalah kelompok kontrol yaitu hanya dilakukan pencabutan saja tanpa perlakuan, kelompok ke-2 yaitu kelompok yang setelah dicabut diberi xcb saja dan kelompok ke-3 yaitu kelompok yang setelah pencabutan diberi kombinasi aloe vera gel dengan xcb pada luka bekas pencabutan gigi. kemudian masing-masing kelompok besar ini dibagi lagi berdasarkan waktu menjadi 3 sub kelompok yaitu setelah 14, 30 dan 60 hari. kemudian dilakukan pemeriksaan histology dan morfologi pada specimen hewan coba. hasil: terdapat perbedaan bermakna antara kelompok kontrol dan kelompok yang diberi kombinasi aloe vera dan xcb. kesimpulan: �isimpulkan bahwa pemberian kombinasi aloe vera gel dan xenograft menyebabkan penurunan jumlah osteoklas dan peningkatan jumlah osteoblas pada struktur tulang alveol pasca pencabutan gigi yang menunjukkan adanya pertumbuhan tulang alveol baru. kata kunci: osteoblas, osteoklas, aloe vera, xenograft concellous bovine, soket pencabutan gigi correspondence: utari kresnoadi, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: utari.kresnoadi@yahoo.com 201kresnoadi and rahayu: stimulation of osteoblast activity by induction of aloe vera introduction the preparation of complete denture will be successful if the denture is retentive, stable, and comfortable for patients. however, the success of the denture is also related to other factors, such as the skills of operators as well as anatomical factors of supporting tissues, one of which is the alveolar bone. patients with longer tooth loss which is not replaced by artificial tooth or dentures may have alveolar bone resorption or residual ridge resorption, and later can cause the prevalence of increased alveolar bone resorption. the initial phase of residual ridge is actually started from tooth loss though periodontal membrane which has the ability to form bone. it is because if the alveolar bone loss occurs in labio and vertical lingual, the residual ridge will become narrower. in some cases, it can even be sharp as a knife edge and retracted. these conditions will not only cause alveolar process disappeared, but can also cause lower residual ridge, rounded, or flat. if this resorption condition continuously occurs, basal bone can disappear, so the shortening of ridge occurs in mouth.1 generally, dento alveolar component is a system showing degree of bone health that is able to withstand constant strength and strain. tooth extraction, on the other hand, can not only lead to the existence of narrow residual margin, but can also lead to shortening and jawbone atrophy.2,3 if this condition is not solved, then it can affect the manufacture of artificial teeth that are not adequate. thus, to prevent alveolar bone resorption, it requires the development of an innovative technique of alveolar ridge augmentation elevation.2 therefore an effort is needed to prevent post-tooth-extraction sockets from resorption, so periodical relining action is not required. the compositions of bone actually consists of mineral, organic matrix, cells, and water at a ratio of 65% minerals and 35% both organic matrix, such as osteoblasts, osteocytes and osteoclasts, and water. organic matrix, about 35% of bone weight in a dry state, consists of 90% collagen, the highest bone protein, and non-collagen bone protein, such as osteonectin, osteocalcin, osteopontin and, sialoprotein. osteoblasts produce bone matrix proteins. osteoblasts also synthesize other proteins in the bone matrix, such as osteonectin and osteocalsin, about 40-50% of bone non-collagen protein.4 canalis cit. lindawati4 even said that other bone protein produced by osteoblasts are glycosaminoglycan, osteopontin, sialoprotein, fibronectin, vitronectin, and trombospondin that serve as glue interacting with integrins. the most common result of osteoblasts is collagen type 1, which will form collagen fibrils. 4 the use of bovine graft in this study is aimed to repair bone defect or augmentation oftenly performed in the fields of general surgery and oral surgery that still requires an innovative modification of bio-products to produce maximal bone growth. therefore an innovation of material modification used as biogenic stimulators in order to stimulate alveolar bone and to accelerate the growth of the bone is needed. aloevera is considered to have biogenic stimulator and hormone activities for wound healing. liquid derived from aloe vera can prevent scar tissue ocured at the time of incision, thus, when aloe vera gel is used after surgery, the incision will heal quickly.5,6 aloe vera contains with two liquid, clear and yellow-colored liquid. the clear liquid, jelly-like liquid, contains with anti-bacterial and anti fungal stimulating fibroblasts or skin cells that serve to heal wounds, while the yellow-colored liquid contains aloin derived from latex skin of aloe vera.7 aloevera gel is a gel made from the meat leaves of spama simplicia plants, such as aloe, succus aloe insipissatus from familia liliaceae, containing chemical aloin and aloe emodine that have anti-inflammatory properties.5,6 aloe vera can not only be used as an antiinflammatory, antibacterial, antifungal, and antiallergy, but can also increase immunity and accelerate the process of wound healing by increasing cell regeneration. aloevera is a natural material that is necessary to be tested for the safe extract dose used for this study.8 it is needed to know whether the use of aloe vera gel and xenograft cancellous bovine (xcb) combination on post tooth extraction sockets can accelerate the growth of alveolar bone, thus, it can prevent the occurrence of alveolar bone resorption. as a result, the making of artificial tooth can be conducted well. therefore, this study is aimed to examine osteoblast activities in alveolar bone after introduction of aloe vera and xcb combination. materials and methods this study was conducted on experimental animals, cavia cabaya, with randomized post test only control group design. the number of samples of each group is 6 from the total number of 54 cavia cabaya with inclusion criteria: male, 3 months old, 300–350 grams, healthy and active. the samples were divided into nine treatment groups. group i, ii, iii consisted of cavia cabaya which teeth were extracted, but without treatment. meanwhile, groups iv, v, vi consisted of cavia cabaya which teeth were extracted, but treated with xenograft concelous bovine only. group vii, viii, ix consisted of cavia cabaya which teeth were extracted, and treated with the combination of aloe vera gel and xcb. all of them were examined 14, 30, and 60 days after the tooth extraction and treatment. materials used in this study were aloe vera gel, this gel was produced from 1000 grams of aloe vera leaves blending and made into extract. this aloe vera extract was freeze dried and stored in special tube. one gram of this extract was mixed with 1 ml sterile aquadest, filtered and centrifuged at 4000 rpm. the result was filtered using milipor 0.045 μml to get 0.5 ml. aloe vera extract was done in 70%, 85% and 100%. these samples were sterilized with ultraviolet for 15 minutes before testing. aloe vera toxicity testing was conducted in 70%, 85% and 100% concentration on fibroblast cells using mtt assay. 202 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 200–204 combination of aloe vera gel and xcb was produced by mixing 0.5gram of aloe vera from freeze drying with xcb, and mixed with 99ml polyethylene glycol (peg 4000 and peg 400). this mixture was put into sterile container for application. this gel was made to ease application into tooth sockets. cavia cabaya was taken and anaesthetized using inhalation anesthetic ether, with 1 ml/1kg weight dose,8 then the incisive tooth was extracted. post tooth extraction sockets were filled according to grouping and then stitched. after 14, 30, and 60 days, cavia cabaya were harvested, and the jaws were cut to make paraffin block preparation after decalcification process with fixation material of 2% nitric acid, for a week. after that, the paraffin block was cut, mounted on its slide, and coloured with hematoxylineosin (he) staining. next, the checking and calculating processes were conducted separately with double blind technique by 2 (two) different examiners. each slide was examined with 1000× magnification for about ten fields of view. after that, the results were recorded, and the mean value per field of view was calculated. the calculation results were recorded and then tabulated. afterwards, statistical analysis was conducted by using analysis of variance (anova) test, and then tukey-hsd test was also conduceted to compare among the best groups. results the mean results of toxicity test of aloe vera extract with concentrations of 70%, 85%, and 100% on day 1, 2 and 3 can be seen in table 1. table 1. the mean results of toxicity test of aloe vera extracts with concentrations of 70%, 85% and 100% on the first, second and third day in optical density concentration/day first day second day third day 70% aloe vera 85% aloe vera 100% aloe vera control 1.90 1.91 1.90 1.814 1.87 1.91 1.81 1.838 1.90 1.814 1.925 1.820 through one-way anova statistical analysis, it is known that on the first day value of p > 0.05, while on the second day value p > 0.05. it means that there was no significant difference. meanwhile, on the third day there was significant difference, value of p < 0.05. it means that there was significant difference in toxicity tests by using mtt assay. based on the results of multiple comparison tukey hsd test, it is also known that on the first day, the value of p > 0.05. it means that there was no significant difference between the three aloe vera extract groups and the control groups. it is also known that on the second day the value of p > 0.05, means that there was also no significant difference between the three aloe vera extract groups and the control groups. on the third day, the value of p > 0.05, means that there was no significant difference between the control groups and the three aloe vera extract groups with concentrations 70%, 85%, and 100%. thus, it can be indicated that aloe vera extract with concentration 100% can only be used on the first day and the second day, while on the third day it should not be used. histological examination was conducted by observing the morphology of alveolar bone structure, it can also be known that the descriptions of osteoclasts and osteoblasts can be seen in figure 1. 0 5 10 15 20 25 30 i ii iii iv v vi vii viii ix kelompokgroups o st eo cl as t figure 1. the number of osteoclast from histological examination. there is an increase mean of osteoclast number from control group i, ii, iii after extraction, but on group iv, v, vi with xcb only there is increased number of osteoclast even though not significant. on group vii, viii, ix with aloe vera and xcb there is significant decrease of osteoclast number after extraction. statistical anova analysis on osteoclasts among group i-ix showed significant differences on osteoclast number (p < 0.05). multiple comparison growth of osteoclasts showed no significant difference among 14 day groups after the tooth extraction, which were between group i (control) and group iv (with xcb application), with significance of p > 0.005, and between group i (control) and group vii (with aloe vera and xcb application), with significance p > 0.005. in the 30 day groups, after the tooth extraction, there was significant difference between group ii (control) and group v (with xcb application only), with significance p < 0.05. similarly, there was also significant difference between group ii (control) and group viii (with aloe vera and xcb application), with significance p < 0.05. in the 60 day groups, furthermore, the tooth extraction there was significant difference between group iii (control) and group vi (with xcb application only) with significance p < 0.05. similarly, there was significant difference between group iii (control) and group ix (with the administration of aloe vera and xcb application), with significance p < 0.05. decrease number of osteoblast on group i, ii, iii which were 14 days after tooth extraction (figure 2). in group iv, v, vi (with xcb application only) there were increased number of osteoblast. in group vii,viii, ix (with aloe vera and xcb application) there were significant increase of 203kresnoadi and rahayu: stimulation of osteoblast activity by induction of aloe vera osteoblast number in group vii eventhough in group viii and ix there were slight decrease of osteoblast number but if compared to control group there were still significant increase. statistical anova analysis on osteoblasts among group i-ix showed significant differences on osteocblast number (p < 0.05). 0 5 10 15 20 25 30 35 i ii iii iv v vi vii viii ix groups o st eo bl as t i groups ii iii iv v vi vii viii ix o st eo bl as t 35 30 25 20 15 10 5 0 figure 2. the number of osteoblasts from histological examination. multiple comparison calculation of osteoblasts showed significant differences among 14 day groups after the tooth extraction, which was between group i (control) and group iv, with significance p < 0.005, and also between group i (control) and group vii, with significance p < 0.005. in the 30 day groups, after the tooth extraction there were significant differences between group ii (control) and group v with significance p < 0.05, as well as between group ii and group viii with significance p < 0.05. then, in the 60 day groups, after the tooth extraction there were also significant differences between group iii (control), group vi, and group ix with significance p < 0.05. discussion in this research, mtt assay toxicity testing was performed to find out aloe vera’s toxicity. the result was there was no significant difference on one-way anova analysis on the first and second day but on the third day there was significant difference. multiple comparison test with tukey-hsd showed no significant difference among the first, second and third day. therefore 70%, 85% and 100% aloe vera extract are safe to use against fibroblast cells. aloe vera is a plant with anti inflammatory effect, promote wound healing, and increase blood supply on wounds. 6 histological finding on figure 1 and 2 showed that there are decrease number of osteoclast and significant increase number of osteoblast. this finding showed that aloe vera is a biogenic stimulator which can promote xcb to activate inside alveolar bone socket. this research is in accordance to some opinions that aloe is a widely used traditional medical plant with various conditions. aloe is also called barbaloin is a yellow crystal with bitter taste and derivative of c-glycoside from anthraquinone. a c-glycoside if hydrolyzed will form aloe-emodin, anthrone which can auto-oxidated forming quinine, aloe emodin. aloin and aloe-emodin has not only laxative effect but also anti bacterial, anti virus, hepatoprotective and anti cancer effects. aloin and aloe-emodin contains polyphenol which has anti inflammatory effect.11 traumatic tooth extraction can lead to inflammation causing osteoclastogenesis, the growing process of osteoclasts induced with inflammation due to traumatic tooth extraction. the mature osteoclasts have a capacity for bone resorption.12 in the process of osteoclastogenesis, table 2. multiple comparison growth of osteoclasts (hpa) groups i ii iii iv v vi vii viii ix i ii iii iv v vi vii viii ix * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * note: group i: 14 days after the tooth extraction without any treatment (control); group ii: 30 days after the tooth extraction without any treatment (control); group iii: 60 days after the tooth extraction without any treatment (control); group iv: 14 days after the tooth extraction with filled in xcb; group v: 30 days after the tooth extraction with filled in xcb; group vi: 60 days after the tooth extraction with filled in xcb; group vii: 14 days after the tooth extraction with filled in aloe vera + xcb; group viii: 30 days after the tooth extraction with filled in aloe vera + xcb; group ix: 60 days after the tooth extraction with filled in aloe vera + xcb; *: significance table 3. multiple comparison calculation of osteoblasts (hpa) groups i ii iii iv v vi vii viii ix i ii iii iv v vi viivii viii ix * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * note: group i: 14 days after the tooth extraction without any treatment (control); group ii: 30 days after the tooth extraction without any treatment (control); group iii: 60 days after the tooth extraction without any treatment (control); group iv: 14 days after the tooth extraction with filled in xcb; group v: 30 days after the tooth extraction with filled in xcb; group vi: 60 days after the tooth extraction with filled in xcb; group vii: 14 days after the tooth extraction with filled in aloe vera + xcb; group viii: 30 days after the tooth extraction with filled in aloe vera + xcb; group ix: 60 days after the tooth extraction with filled in aloe vera + xcb; *: significance 204 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 200–204 the differentiation of osteoclasts and the activation of the combination of macrophage colony-stimulating factor (m-csf) and receptor activator of nuclear factor ligand (rankl) occur. precusors of osteoclasts replicate and induce rank in inflammatory condition produced by b and t lymphocyte. the role of aloe vera gel, on the other hand, is to prevent inflammation that osteoclastogenesis can be decreased, so bone cell resorption does not occur.12 this is in accordance with the opinion of kyung et al.,13 that osteoclasts are cells responsible for bone resorption process, which is formed from haemopoetic stem cell. the regulation of osteoclastogenesis in inflammation, such as arthritis, proinflammatory cytokines (il1, il 6 and tnf-a), and rankl, can stimulate osteoclasts and bone damage. similarly, the opinion of regina14 on her research on scar removal stated that by adding dbm (dimineralized bone matrix), the allograft dbm powder can not only reduce the expression of gelatinase a, but can also increase the expression of timp-2 so that the dbm stimulates the healing process of gingival wound. 14 aloe vera and xcb combination can reduce osteoclast because aloe vera has antiinflammatory effect from antraguinon (aloe, aloe emodin) substance and saponin hormone which faster extraction wound healing and osteoblast formation (biogenic stimulator). xcb is an osteoinduction substance which can induce osteoblast in new bone formation. therefore, the combination of aloe vera and xcb can not only accelerate the healing of scar removal, but can also enable the growth of bone cells by osteoblast cells. finally, it then can be concluded that the addition of the combination of aloe vera and xcb on post tooth extraction socket in this study not only can increase osteoblasts, but also can decrease osteoclasts indicating the new growth of alveolar bone. references 1. nishimura i, hosokawa r, attwood da. knive edge tendency inknive edge tendency in mandibular ridge in women. j prosthed dent 1992; 67: 820–6. 2. oktavia r. tehnik bedah peninggian lingir alveolar sebagai salah satu penunjang keberhasilan di bidang prosthetic. karya tulis akhirkarya tulis akhir pendidikan dokter gigi spesialis. sumatera utara: fkg usu; 2005. p. 28 3. gupta a, tiwari b, goel h, shekawat h. residual ridge resorption: a review. indian j dent sci 2010; 2(2): 7–11. 4. khudhany ls. penentuan indeks densitas tulang mandibula perempuan pasca maenopause dengan memperhatikan beberapa faktor risiko terjadinya osteoporosis. disertation. jakarta: pascasarjana universitas indonesia; 2003. 5. sudibyo m. alam, sumber kesehatan, manfaat dan kegunaan. jakarta:jakarta: penerbit balai pustaka; 1998. p. 250. 6. rostita, tim redaksi qanita. lidah buaya. cetakan i. bandung: pt mizan pustaka; 2008. p. 19–38. 7. marthanthi r. uji sitoksisitas aloe vera gel 100% metode freze drying terhadap sel fibroblas ditinjau dari waktu inkubasi. skripsi. surabaya:skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2007. p. 7–10. 8. ariyani e, khoswanto c. the use 90% aloe vera frezze drying as modulator of collagen density in exctraction socket of incisivus in cavia cabaya. maj ked. gigi (dent j) 2008; 41(2): 74–6. 9. kusumawati d. bersahabat dengan hewan coba. cetakan pertama. jogjakarta: gajah mada university press; 2004. p. 67. 10. kurniawati r, marminah mt. efek anti imflamasi gel lidah buaya (aloe vera linn) terhadap tikus putih. solo: prosiding seminar nasional tumbuhan obat indonesia xxix; 2006. p. 82–89. 11. park my, kwon hj, sun mk. evaluation of aloin and aloe emodinevaluation of aloin and aloe emodin as anti inflammatory agents in aloe by using murin macrophage. j of biosci biotech biochem 2009; 73(4): 828–32. 12. lorenzo j. osteoimmunology: interaction of the bone and immune system. j of endocrine review 2008; 29(4): 403–40. 13. kyung tw, lee je, shin hh, choi hs. rutin inhibits osteoclast formation by decresing reactivee oxygen species and tnf-a by inhibiting activation of nfkb. j experiment and molecular medicine 2008; 40(1): 52–8. 14. tandelilin rtc. dbm augmentation on mandibular incisivus extraction wound healing. j of dentistry 2006; 13(3): 190–6. 190 volume 46, number 4, december 2013 respon inflamasi pulpa gigi tikus sprague dawley setelah aplikasi bahan etsa ethylene diamine tetraacetic acid 19% dan asam fosfat 37% (dental pulp inflammatory response of sprague dawley rats after etching application of 19% ethylene diamine tetraacetic acid and 37% phosphoric acid) nadie fatimatuzzahro,1 tetiana haniastuti2 dan juni handajani2 1 bagian biomedik, fakultas kedokteran gigi, universitas jember, jember-indonesia 2 bagian biologi mulut, fakultas kedokteran gigi, universitas gadjah mada, yogyakarta indonesia abstract background: etching agents such as ethylene diamine tetraacetic acid (edta) and phosphoric acid which are widely used in adhesive restoration system, are aimed to increase retention of restorative materials; however, these agents may induce inflammation of dental pulp. the major function of the inflammatory response is to remove invading pathogens or damaged tissue/ cells and therefore, initiate repair. neutrophils and macrophages are motile phagocytes that constitute the body's first line of defense. purpose: the purpose of the present research was to study the effect of 19% edta and 37% phosphoric acid for etching application agents on the inflammatory response of the dental pulp. methods: forty-five male sprague dawley rats were divided into 3 groups. cavity preparation was made on the occlusal surface of maxillary first molar using a round diamond bur. nineteen percent of edta, 37% phosphoric acid, and distilled water were applied on the surface of the cavity of the teeth in group i, ii and iii respectively. the rats were sacrified at 1, 3, 5, 7, and 14 days after the application (n=3 for each day). the specimens were then processed histologically and stained with hematoxylin eosin. results: anova showed a significant difference (p<0.05) among treatment groups, indicating that etching agents application induced neutrophils, macrophages and lymphocytes infiltration in the dental pulp. tuckey hsd test showed that application of 37% phosphoric acid increased higher number of neutrophils, macrophages and lymphocytes significantly than 19% edta (p<0.05). conclusion: the study suggested that 37% phosphoric acid induced higher number of the inflammatory cells than 19% edta. key words: 19% edta, 37% phosphoric acid, inflammation, dental pulp, sprague dawley rats abstrak latar belakang: penggunaan bahan etsa seperti ethylene diamine tetraacetic acid (edta) dan asam fosfat pada sistem restorasi adhesif bertujuan untuk meningkatkan retensi bagi bahan restorasi, namun penggunaan bahan-bahan tersebut dapat menginduksi inflamasi pada pulpa. respon inflamasi berfungsi untuk menghilangkan patogen, sel-sel atau jaringan yang rusak dan menginisiasi perbaikan. netrofil dan makrofag adalah sel fagosit yang merupakan garis pertama pertahanan tubuh. tujuan: penelitian ini bertujuan untuk meneliti efek edta 19% dan asam fosfat 37% sebagai bahan etsa terhadap respon inflamasi pada pulpa gigi. metode: empat puluh lima ekor tikus sprague dawley jantan dibagi menjadi 3 kelompok. permukaan oklusal gigi molar satu rahang atas dipreparasi menggunakan diamond round bur. pada kelompok i kavitas diaplikasikan edta 19%, kelompok ii diaplikasikan asam fosfat 37% dan kelompok iii diaplikasikan akuades. hewan coba dikorbankan pada hari ke-1, 3, 5, 7 dan 14 setelah aplikasi bahan etsa (n=3). spesimen diproses secara histologis dan dicat dengan hematoksilin eosin. hasil: hasil anova menunjukkan perbedaan yang bermakna research report 191fatimatuzzahro, et al.,: respon inflamasi pulpa gigi tikus sprague dawley (p<0,05) antar kelompok perlakuan, mengindikasikan bahwa aplikasi bahan etsa menyebabkan infiltrasi sel inflamasi pada pulpa, baik netrofil, makrofag dan limfosit. hasil uji tuckey hsd menunjukkan bahwa asam fosfat 37% menstimulasi infiltrasi sel netrofil, makrofag dan limfosit signifikan (p<0,05) lebih banyak dibanding edta 19%. simpulan: penelitian ini menunjukkan bahwa asam fosfat 37% menyebabkan infiltrasi sel inflamasi yang lebih banyak dibanding edta 19%. kata kunci: edta 19%, asam fosfat 37%, inflamasi, pulpa gigi, tikus sprague dawley korespondensi (correspondence): nadie fatimatuzzahro, bagian konservasi gigi, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: nadiefatima@gmail.com pendahuluan karies adalah penyakit infeksi mikroba pada gigi yang menyebabkan terurai dan rusaknya jaringan keras gigi. destruksi gigi oleh karena karies atau faktor lain, membutuhkan restorasi untuk menggantikan substansi gigi yang hilang sehingga akan mengembalikan bentuk, fungsi dan estetika.1 restorasi adhesif menggunakan resin komposit digunakan oleh para dokter gigi karena memiliki estetik yang baik. desain preparasi kavitas untuk bahan restorasi adhesif tidak memerlukan pembuatan retensi seperti undercut, sehingga meminimalkan pembuangan jaringan dan mengurangi terbukanya tubulus dentin.2 penggunaan asam sebagai dentin kondisioner atau bahan etsa pada sistem restorasi adhesif bertujuan untuk menghilangkan smear layer dan mempersiapkan permukaan dentin untuk menerima bahan adhesif. prosedur etsa menyebabkan demineralisasi komponen anorganik gigi sehingga terbentuk retensi berupa mikroporositas yang akan terisi oleh bahan adhesif.3 bahan etsa asam fosfat konsentrasi 32-37% paling banyak digunakan pada sistem restorasi adhesif. bahan ini merupakan asam kuat yang aktif pada ph rendah. asam fosfat tidak hanya mampu menghilangkan smear layer, tetapi juga dapat menyebabkan demineralisasi dan membuka tubulus dentin.4 penelitian terdahulu menunjukkan larutnya sebagian besar hidroksi apatit dan terbukanya kolagen setelah aplikasi asam fosfat pada dentin.3 ethylene diamine tetraacetic acid (edta) merupakan agen khelasi yang bekerja pada ph netral dan efektif menghilangkan smear layer sebanding dengan bahan etsa lain yang mempunyai ph rendah.4 penggunaan edta untuk menghilangkan smear layer tidak mempengaruhi kekerasan dentin sehingga tidak menyebabkan dentin menjadi rapuh.5 aplikasi 0,5 m edta (setara dengan edta konsentrasi 19%) selama 30 detik terbukti meningkatkan kekuatan pelekatan bahan adhesif dengan dentin dibandingkan asam fosfat 37%6 dan dapat meminimalkan kebocoran mikro pada tepi pelekatan antara komposit dan dentin.7 kompleks dentin-pulpa bereaksi terhadap semua rangsangan yang mengenai gigi. rangsangan dapat berupa karies, trauma, maupun semua tindakan dalam prosedur penumpatan, mulai dari preparasi, pembersihan dan pengeringan kavitas, serta penumpatan dan pemolesannya.8 berbagai zat yang digunakan untuk sterilisasi seperti fenol dan eugenol, pembersih dentin seperti larutan asam, pelapis kavitas serta zat yang terdapat pada bahan tambal, merupakan rangsangan kimiawi yang dapat menyebabkan inflamasi pada pulpa.9 inflamasi merupakan respon perlindungan inang yang bertujuan untuk menghilangkan penyebab jejas serta sel-sel dan jaringan nekrotik, sehingga terjadi proses penyembuhan dan perbaikan jaringan. pada awal terjadinya inflamasi, netrofil merupakan sel pertahanan tubuh pertama terhadap jejas atau infeksi, kemudian makrofag akan membantu proses eliminasi infeksi dan jaringan yang rusak melalui proses fagositosis. selanjutnya sel limfosit t berperan pada respon inflamasi kronis.10 penelitian ini bertujuan untuk meneliti efek edta 19% dan asam fosfat 37% sebagai bahan etsa terhadap jumlah infiltrasi sel inflamasi (netrofil, makrofag dan limfosit) pada pulpa gigi tikus sprague dowley. bahan dan metode jenis penelitian ini merupakan penelitian eksperimental laboratoris, yang dilakukan di laboratorium farmakologi dan laboratorium histologi fakultas kedokteran universitas gadjah mada yogyakarta. seluruh prosedur penelitian ini telah mendapat persetujuan dari komisi etik fakultas kedokteran gigi universitas gadjah mada yogyakarta. ethylene diamine tetraacetic acid 19% dibuat dengan melarutkan 19 gram serbuk edta (berat molekul 372,24 gram/mol) dalam 100 cc akuades. larutan kemudian disaring dengan kertas saring dan ph disesuaikan hingga 7,4. penelitian ini menggunakan tikus sprague dawley jantan sebanyak 45 ekor. tikus dianastesi secara intramuskular dengan ketamine hcl 0,2 ml/200 gram berat badan sebelum dilakukan preparasi kavitas. gigi molar satu rahang atas dipreparasi pada permukaan oklusal menggunakan diamond round bur 0,9 (edenta, switzerland) dengan kedalaman 0,5 mm. tikus dibagi menjadi 3 kelompok perlakuan secara acak. pada kelompok i, edta 19% diaplikasikan pada kavitas selama 30 detik menggunakan microbrush, selanjutnya bilas dengan akuades selama 30 detik.11 kelompok ii, gel asam fosfat 37% (dentamerica, usa) 192 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 190–195 diaplikasikan pada kavitas selama 20 detik, selanjutnya bilas dengan akuades selama 30 detik. kelompok iii, kavitas hanya dibilas akuades selama 30 detik. kavitas dikeringkan dengan cotton pellet kemudian ditumpat dengan semen ionomer kaca fuji ix-gc. pada hari ke-1, 3, 5, 7 dan 14 setelah perlakuan (n=3), tikus dikorbankan dengan cara dekapitasi. rahang atas pada bagian gigi molar yang telah diberi perlakuan difiksasi dengan buffered formalin 10% selama 24 jam. spesimen kemudian didekalsifikasi menggunakan edta 10% ph 7,4 selama 4 minggu pada suhu 4°c. setelah lunak, spesimen ditanam dalam parafin, dan dipotong dengan ketebalan 5 µm untuk dilakukan pengecatan hematoksilin eosin (he). sel inflamasi yaitu netrofil, makrofag dan limfosit diamati menggunakan mikroskop dengan pembesaran 400×××x pada area di bawah preparasi kavitas. jumlah infiltrasi sel inflamasi dihitung dari 3 lapang pandang yang berbeda. data selanjutnya dianalisis dengan menggunakan anova dan tuckey hsd. hasil pada semua kelompok, jumlah netrofil paling banyak ditemukan pada hari ke-1 setelah perlakuan dan jumlahnya semakin berkurang seiring dengan bertambahnya hari pengamatan (gambar 1). makrofag sudah dapat diamati pada hari ke-1 setelah perlakuan. jumlah makrofag semakin meningkat, mencapai puncaknya pada hari ke-5 setelah perlakuan, dan menurun sesudahnya. rerata jumlah infiltrasi makrofag pada pulpa gigi tikus setelah aplikasi edta 19%, asam fosfat 37% dan akuades ditunjukkan pada gambar 2. limfosit juga dapat diamati mulai hari ke-1 setelah perlakuan. jumlah limfosit semakin meningkat dan paling banyak ditemukan pada hari ke-7 setelah perlakuan. rerata jumlah infiltrasi limfosit pada pulpa gigi tikus setelah aplikasi edta 19%, asam fosfat 37% dan akuades ditunjukkan pada gambar 3. hasil anova menunjukkan bahwa jumlah netrofil, makrofag dan limfosit berbeda bermakna (p<0,05) antar kelompok. hal ini menunjukkan bahwa aplikasi bahan etsa berpengaruh bermakna terhadap infiltrasi sel inflamasi. hasil uji tuckey hsd menunjukkan bahwa terdapat perbedaan yang bermakna jumlah infiltrasi sel netrofil, makrofag dan limfosit antara kelompok dengan aplikasi edta 19% maupun akuades bila dibandingkan dengan asam fosfat 37% (p<0,05). tidak terdapat perbedaan yang bermakna antara kelompok dengan aplikasi edta 19% dibandingkan akuades (p>0,05). hal ini mengindikasikan bahwa asam fosfat 37% menyebabkan infiltrasi sel netrofil, makrofag dan limfosit yang lebih banyak dibandingkan edta 19% maupun akuades. hasil pengamatan histologis pulpa gigi setelah aplikasi edta 19%, asam fosfat 37% dan akuades ditunjukkan pada gambar 4. pembahasan hasil penelitian ini menunjukkan bahwa aplikasi asam fosfat 37% menyebabkan infiltrasi sel inflamasi lebih banyak dibandingkan edta 19%. hal ini kemungkinan karena ph asam fosfat 37% sangat rendah yaitu 0,26, sedangkan edta 19% yang digunakan memiliki ph 7,4. aplikasi asam fosfat pada kavitas akan menyebabkan demineralisasi yang berlebihan sehingga akan meningkatkan permeabilitas dentin. akibatnya, terjadi penetrasi lebih banyak dari bahan gambar 3. rerata dan simpangan baku jumlah limfosit. infiltrasi sel limfosit pada kelompok setelah aplikasi edta 19% dan akuades lebih sedikit dibanding asam fosfat 37%. gambar 1. rerata dan simpangan baku jumlah netrofil. infiltrasi sel netrofil lebih banyak pada kelompok yang diaplikasikan asam fosfat 37% dibandingkan edta 19% dan akuades. gambar 2. rerata dan simpangan baku jumlah makrofag. jumlah infiltrasi sel makrofag lebih sedikit pada kelompok setelah aplikasi edta 19% dan akuades dibanding asam fosfat 37%. 193fatimatuzzahro, et al.,: respon inflamasi pulpa gigi tikus sprague dawley etsa melalui tubulus dentin ke dalam pulpa.12 bahan etsa asam dengan ph rendah menyebabkan lingkungan di luar sel bersifat hipertonik, sehingga cairan di dalam sitoplasma akan tertarik ke luar dan sel akan mengkerut. hal ini dapat memicu kerusakan permanen pada sel odontoblas dan menyebabkan reaksi inflamasi. pada inflamasi akan terjadi infiltrasi sel-sel inflamasi ke daerah jejas untuk mengeliminasi iritan dan debris seluler.13 pada kondisi normal, terdapat sel makrofag dan sel dendritik sebagai antigen precenting cell (apc) pada pulpa yang akan mengenali iritan dan debris seluler.14 iritan dan kerusakan sel/jaringan merupakan sinyal bagi makrofag sehingga teraktivasi dan akan mensekresi sitokin antara lain il-1 dan tnf-α. interleukin-1 dan tnf-α akan menginduksi sel endotel untuk menghasilkan e-selektin dan intercellular adhesion molecule-1 (icam-1). kedua molekul adhesi tersebut akan berikatan dengan ligan pada netrofil sehingga menyebabkan netrofil teraktivasi dan melekat pada dinding endotelium. langkah berikutnya dalam proses ini adalah migrasi netrofil melalui endotelium menuju lokasi jejas.10 pada hari ke-1 setelah perlakuan, pada semua kelompok terjadi infiltrasi sel inflamasi terutama netrofil, dan gambar 4. pada hari ke-1 setelah perlakuan, tampak infiltrasi sel inflamasi terutama netrofil. infiltrasi sel inflamasi pada kelompok dengan aplikasi edta 19% tampak lebih sedikit dibandingkan asam fosfat 37%. jumlah makrofag paling banyak ditemukan pada hari ke-5 setelah perlakuan. pada hari ke-14 semua kelompok perlakuan tampak memberikan gambaran pulpa normal. sel netrofil ( ), makrofag ( ), limfosit ( ). d= dentin, pd=predentin, o=odontoblas, f=zona bebas sel, r=zona kaya sel. jumlahnya semakin menurun dengan bertambahnya hari pengamatan. netrofil adalah sel fagosit yang merupakan pertahanan tubuh pertama terhadap infeksi atau benda asing, berperan pada respon inflamasi akut dan hanya berumur pendek (24-36 jam). netrofil akan mati setelah menghancurkan iritan serta jaringan yang rusak melalui proses fagositosis.14 penetrasi bahan etsa merupakan iritan kimia bagi sel-sel pada pulpa, dan sinyal bagi makrofag sehingga teraktivasi dan mensekresi sitokin antara lain il-1 dan tnf-α. tumor necrosis factor-α dapat menginduksi sel endotel dan sel dendritik untuk menghasilkan monocyte chemotactic protein-1 (mcp-1) yang akan menyebabkan monosit keluar dari pembuluh darah menuju ke daerah inflamasi. di jaringan, monosit akan berdiferensiasi menjadi makrofag. infiltrasi makrofag paling banyak ditemukan pada hari ke-5 setelah perlakuan. hal ini disebabkan setelah monosit bermigrasi ke jaringan, dibutuhkan waktu 48-72 jam untuk berdiferensiasi menjadi makrofag pada area jejas.14 makrofag berperan penting dalam sistem imun untuk mengeliminasi antigen dengan aktivitas fagositosis.15 sel dendritik merupakan apc utama yang terlibat pada respon imun pulpa. sel dendritik akan bergerak memasuki 1 edta 19% asam fosfat 37% akuades h ar i k e1 h ar i k e5 h ar i k e14 gambar 4. pada hari ke-1 setelah perlakuan, tampak infiltrasi sel inflamasi terutama netrofil. infiltrasi sel inflamasi pada kelompok dengan aplikasi edta 19% tampak lebih sedikit dibandingkan asam fosfat 37%. jumlah makrofag paling banyak ditemukan pada hari ke-5 setelah perlakuan. pada hari ke-14 semua kelompok perlakuan tampak memberikan gambaran pulpa normal. sel netrofil ( ), makrofag ( ), limfosit ( ). d= dentin, pd=predentin, o=odontoblas, f=zona bebas sel, r=zona kaya sel. 194 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 190–195 jaringan limfoid setelah menangkap dan memproses antigen untuk selanjutnya dipresentasikan pada limfosit t melalui molekul major histocompatibility complex (mhc) klas ii. hal ini merupakan sinyal bagi aktivasi limfosit untuk berproliferasi dan berdiferensiasi.16,17 molekul mhc klas ii juga diekspresikan pada permukaan sel makrofag yang akan mempresentasikan antigen pada limfosit t. makrofag akan mensekresi il-1 dan il-12 setelah memproses antigen. interleukin-1 akan memberi sinyal kepada limfosit t helper untuk berikatan dengan molekul mhc klas ii pada makrofag dan il-12 berperan pada aktivasi limfosit.18 infiltrasi sel limfosit paling tinggi ditemukan pada hari ke-7 dan menurun pada hari ke-14 setelah perlakuan. waktu yang dibutuhkan oleh limfosit untuk produksi dan diferensiasi menjadi sel efektor sekitar 3 sampai 5 hari, dan selanjutnya limfosit akan keluar dari vaskularisasi menuju ke jaringan.14 mekanisme lain yang dapat menjelaskan terjadinya infiltrasi sel inflamasi setelah aplikasi bahan etsa, yaitu bahwa penggunaan asam fosfat dan edta dapat menyebabkan terlepasnya tgf-β1 yang terdapat pada dentin. selama dentinogenesis, odontoblas mensekresi growth factor tersebut kemudian termineralisasi dalam matriks dentin. demineralisasi jaringan gigi akibat penggunaan bahan etsa dapat menyebabkan terlepasnya tgf-β1.14 setelah dilepaskan, tgf-β1 akan berpenetrasi melalui tubulus dentinalis menuju pulpa, berperan pada respon pulpa terhadap jejas.19 hasil penelitian in vitro menunjukkan bahwa tgf-β1 menyebabkan peningkatan ekspresi il-1 dan il-8 pada kultur sel odontoblas dan jaringan pulpa.20 interleukin-8 merupakan kemoatraktan dan berperan pada aktivasi netrofil. transforming growth factor-β1 juga mampu menstimulasi ekspresi il-1 dan tnf-α oleh makrofag yang berperan penting pada respon inflamasi pulpa.21 hasil penelitian ini juga menunjukkan terjadinya infiltrasi sel inflamasi pada tikus yang diaplikasi akuades. hal ini kemungkinan disebabkan oleh preparasi kavitas yang dilakukan. sesuai dengan penelitian yang dilakukan oleh feng mei dkk. bahwa gesekan dan panas yang dihasilkan akibat penggunaan rotary instrument menyebabkan peningkatan ekspresi nitric oxide (no) pada sel odontoblas.22 nitric oxide merupakan radikal bebas yang dihasilkan dari enzim nitric oxide synthase (nos). radikal bebas tersebut berperan pada proses inflamasi dan kerusakan jaringan serta dapat menyebabkan vasodilatasi pembuluh darah sehingga sel inflamasi bermigrasi dari pembuluh darah menuju ke jaringan.15 pada hari ke-14 setelah perlakuan, jumlah infiltrasi sel inflamasi semakin sedikit dibandingkan hari-hari sebelumnya dan memberikan gambaran pulpa normal. hal ini mengindikasikan bahwa sel imun berhasil menghilangkan iritan dan debris seluler sehingga inflamasi tidak berlanjut. selain itu kompleks dentin pulpa memiliki mekanisme perlindungan diri dalam membatasi penetrasi bahan-bahan yang membahayakan pulpa. ion kalsium, fosfat dan cairan yang terdapat dalam tubulus dentin mempunyai kapasitas buffer untuk menetralkan asam dari bahan etsa.14 penggunaan glass ionomer sebagai bahan tumpatan dalam penelitian ini bertujuan untuk meminimalkan iritasi pada pulpa bila dibandingkan dengan resin komposit.23 pada penelitian ini tidak digunakan bahan tumpatan sementara oleh karena daya tahan yang rendah dalam rongga mulut. apabila tumpatan sementara lepas akan terjadi deposit debris dan sisa makanan yang dapat menyebabkan inflamasi pulpa sehingga mempengaruhi hasil penelitian.24 penelitian menunjukkan bahwa asam fosfat 37% menyebabkan infiltrasi sel netrofil, makrofag dan limfosit yang lebih banyak dibanding edta 19%. daftar pustaka 1. roberson tm, harald oh, edward js. sturdevant’s art and science of operative dentistry. 5th ed. st. louis: mosby elsevier; 2006. p. 68-9, 283-5. 2. walmsley ad, walsh tf, lumley pj, burke fjt, shortall ac, hayes-hall r, pretty ia. restorative dentistry. 2nd ed. philadelphia: elsevier; 2007. p. 76-8. 3. summit jb, robbins jw, hilton t, schwartz r. fundamentals of operative dentistry. a contemporary approach. 3rd ed. illinois: quintessence publishing co., inc.; 2006. p. 188-92. 4. cederlund a, jonsson b, blomlöf j. shear strength after ethylened iaminetetraacetic acid conditioning of dentin. acta odontol scand 2001; 59(6): 418-22. 5. swift ej jr, perdigão j, wilder ad jr, heymann ho, sturdevant jr, bayne sc. clinical evaluation of two one-bottle dentin adhesives at three years. j am dent ass 2001; 132(8): 11-7. 6. jacques p, hebling j. effect of dentin conditioners on mocrotensile bond strength of a conventional and a self-etching primer adhesive system. dent mater 2005; 21: 103-9. 7. shafiei f, mema r pour m. effect of edta conditioning on mikroleakage of four adhesive system in composite restorations. j dent 2008; 5(3): 150-5. 8. sundoro eh. serba-serbi ilmu konservasi gigi. jakarta: universitas indonesia press; 2005. h. 115-29. 9. walton r, torabinejad m. 20 01. pr inciples and practice of endodontics. sumawinata n, editor. prinsip dan praktek ilmu endodonsi. edisi 3. jakarta: egc; 2008. h. 10-20, 30-5. 10. kumar v, abbas a, fausto n, robbins sl, cotran r. pathologic basis of disease. 8th ed. philadelphia: saunders; 2006. p. 48-85. 11. stape th, menezes ms, barreto bc, aguiar fh, martins lr, quagliatto ps. influence of matrix metalloproteinase synthetic inhibitors on dentin microtensile bond strength of resin cements. op dent 2012; 37(4): 386-96. 12. amaral kf, rogero mm, fock ra, borelli p, gavini g. cytotoxicity analysis of edta and citric acid applied on murine resident macrophages culture. int endod j 2007; 40(5): 338-43. 13. nemeth l, erman a, stiblar-martincic d. early odontoblastic layer response to cavity preparation and acid etching in rats. folia biol (krakow) 2006; 54(1-2): 23-30. 14. hargreaves mk, goodis he. seltzer and bender’s dental pulp. carlos stream: quintessence publishing co., inc; 2002. p. 13, 42, 54, 65, 69, 95-6, 137-9. 15. roitt im. 1996. imunologi-essential immunology. edisi 8. harahap a, dkk, editor. jakarta: widya medika; 2002. h. 233. 16. lo hahn c, liewehr fr. innate immune responses of the dental pulp to caries. j endod 2007; 33(a): 643–51. 17. lo hahn c, liewehr fr. update on the adaptive immune responses of the dental pulp. j endod 2007; 33(b):773–81. 18. raven, johnson, mason, singer. biology. 9th ed. new york: mosby elsevier; 2007. p. 1156-8. 195fatimatuzzahro, et al.,: respon inflamasi pulpa gigi tikus sprague dawley 19. murray pe, windsor lj, smyth tw, hafez aa, cox cf. analysis of pulpal reactions to restorative procedures, materials, pulp capping, and future therapies. crit rev oral biol med 2002; 13(6): 509-20. 20. pääkkönen v, vuoristo j, salo t, tjäderhane l. effects of tgf-β1 on interleukin profile of human dental pulp and odontoblasts. cytokine 2007; 40(1): 44-51. 21. cooper pr, takahashi y, graham lw, simon s, imazato s, smith aj. inflammation-regeneration interplay in the dentin-pulp complex. j dent 2010; 38(9): 687-97. 22. feng mei y, yamaza t, atsuta i, danjo a, yamashita y, kido ma, goto m, akamine a, tanaka t. sequential expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, and nitrotyrosine in odontoblasts and pulp cells during dentin repair after tooth preparation in rat molars. cell tissue res 2007; 328: 117–27. 23. rendjova v, gjorgoski i, ristoski t, apostolska s. in vivo study of pulp reaction to glass ionomer cements and dentin adhesives. prilozi 2012 ; 33(1): 265-77. 24. schmalz g, arenholt-bindslev d. biocompatibility of dental materials. berlin: springer; 2009. p. 151. vol 52 no 1 jan-mar 2019_new.indd 36 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 36–40 research report comparative in vitro study of the cytotoxicity of gelatine and alginate to human umbilical cord mesenchymal stem cells nike hendrijantini department of prosthodontics faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: mesenchymal stem cells (mscs) and scaffold combination constitute a promising approach currently adopted for tissue engineering. umbilical cord-derived mesenchymal stem cells (huc-mscs) are easily obtained and non-invasive. gelatine and alginate constitute a biocompatible natural polymer scaffold. at present, a cytotoxicity comparison of gelatine and alginate to huc-mscs is not widely conducted. purpose: this study aimed to compare the cytotoxicity of gelatine and alginate in huc-mscs in vitro. methods: isolation and culture were performed on huc-mscs derived from healthy full-term neonates. flow cytometry cd90, cd105 and cd73 phenotype characterization was performed in passage 4. 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (mtt) colorimetric assay was performed to measure the cytotoxicity. the three sample groups were: (t1) huc-mscs with α-mem (alphaminimum essential medium) solution as control; (t2) huc-mscs with gelatine; (t3) huc-mscs with alginate. results: flow cytometry of huc-mscs displayed positive cd90, cd105 and cd73 surface markers. gelatine and alginate had no effect on the viability of huc-mscs and no statistically significant difference (p>0.05) of cytotoxicity between gelatine and alginate to huc-mscs. conclusion: gelatine and alginate proved to be non-toxic to huc-mscs in vitro. keywords: alginate; gelatine; mesenchymal stem cells; umbilical cord correspondence: nike hendrijantini, department of prosthodontics, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo 47, surabaya 60132, indonesia. e-mail: nike-h@fkg.unair.ac.id introduction stem cell research has increased due to the realisation that stem cell-based therapies have the potential to repair bone or tooth loss caused by trauma, fractures, surgery, tumour resection, congenital malformation, dental implant failureassociated osteoporosis and periodontitis in dentistry.1 tissue construction which consists of stem cells and scaffold combination represents a promising approach to bone tissue engineering. mesenchymal stem cells (mscs) have considerable potential for the field of regenerative medicine due to their self-renewing capacity, multilineage differentiation potential and immunosuppressive properties.2 although human bone marrow mesenchymal stem cells (hbm-mscs) are the most common and best characterized stem cell source, umbilical cord derived stem cells (hucmscs) provide a novel source of mscs3 and, recently, hucmscs have been shown to possess significant osteogenic differentiation potential.4 isolation of hbm-mscs requires an invasive procedure that may cause aspiration site morbidity, while huc-mscs are easily obtained through a non-invasive process that does not result in morbidity.5 moreover, hucmscs can be less immunogenic than hbm-mscs.3 the ideal scaffold is able to facilitate adhesion, migration, proliferation and cellular organization in three-dimensional fashion from a cell population required for tissue engineering. high porosity and ideal pore size facilitate the diffusion of nutrients, oxygen and waste products from cellular metabolism.6,7 biodegradability allows scaffold to be absorbed by the body. the time required for degradation ideally matches that of new engineered tissue formation.6 biocompatible and nontoxic properties represent prerequisites to avoiding an inflammatory reaction and toxicity.7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p36–40 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p36-40 37hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 36–40 biomaterial scaffold is broadly divided into two categories: natural polymer such as gelatine, alginate, dextran, chitosan and synthetic polymer of which poly (lactic) acid (pla), poly(glycolic) acid (pga), poly (urethanes) are examples. the biocompatibility of natural polymer is greater.8 however, synthetic polymer demonstrate superior mechanical properties, but induce an inflammatory response in the body of the host, both acute and chronic.9 gelatine and alginate are natural polymers both of which can be processed into injectable scaffold that easily fill any irregularly-shaped defect.10 alginate originates from algae which requires an extensive purification process in order to avoid an immune response after implantation. the advantages of alginate are those of lower low toxicity and higher biocompatibility. nevertheless, the mechanical strength and biodegradability of this material is low and less capable of accommodating cell adhesion.8 disadvantages of using alginate include large batch-to-batch variations, the high cost of biosynthesis and the hydrophilic properties that render it ineffective in protein adsorption. therefore, alginate scaffold must be modified to carry out cellular function.11 gelatine, the result of protein denaturation from partial hydrolysis of collagen, is considered a choice polymer that can ideally be used in bone tissue engineering. this material is non-toxic, biocompatible and biodegradable both in vitro and in vivo. as a collagen derivative, gelatine contains cell binding motifs such as arginine-glycineaspartic acid sequences (rgd) which play a role in the processes of adhesion, proliferation, cell differentiation and matrix metalloproteinase (mmp) which influences biodegradation.10 gelatine is cost effective and can be processed to resemble the structure of collagen as the major organic protein of the bone matrix. particulate leaching, gas foaming and freeze drying all represent processing methods that have been adopted in the preparation of gelatine porous scaffold. the majority of the fabrication methods are simple and economical.12 it is hoped that gelatine and alginate possess properties non-toxic to huc-mscs in order that one can replace the other if either is unavailable. the major challenge to the development of optimum bone scaffold is its biocompatibility. our previous study demonstrated that 2% of gelatine solvent was non-toxic for huc-mscs in vitro.13 an in vivo study showed that 2% of alginate was safe for bone marrow mesenchymal stem cells.14 cytotoxicity comparison of gelatine and alginate to huc-mscs has not been widely studied. the aim of the research reported here was, therefore, to compare the cytotoxicity of gelatine and alginate to huc-mscs in vitro. materials and methods a caesarean section was performed on a healthy full-term neonate. ethical approval was granted by the research ethics committee of soetomo public hospital in surabaya. (547/panke.kke/ix/2017). this isolation and culture procedure was performed using stem cell laboratory protocols at the stem cell research and development centre, universitas airlangga, surabaya, indonesia. the section of umbilical cord was cut to a length of approximately 1 cm., with the artery, vein and adventitia being separated. wharton’s jelly was subsequently immersed in a tube containing 0.25% trypsin at 37ºc for 40 minutes and centrifuged in order to separate the supernatant. samples were immersed in phosphate buffered saline (pbs) (1x, ph 7.4), containing 0.75 mg/ ml of collagenase type iv (sigma-aldrich, st. louis, mo, usa) and 0.075 mg/ ml of dnase i (takara bio, shiga, japan) prior to incubation at 37°c for 60 minutes. filtering was carried out using a cell strainer. one cc of fetal bovine serum (fbs) was added and agitated for 10 minutes after which the samples were filtered using sterile gauze on becker glass and centrifuged for ten minutes at 1600 rpm. the resulting pellets were resuspended in dulbecco’s modified eagle’s medium (dmem). solutions containing the single cell were transferred to a petri dish and incubated at 37ºc and in 5% co2. replacement of the medium was performed every three days, with passage being carried out after confluence had occurred for approximately 21 days. cells from passage 4 were harvested and evaluated for phenotypic characterization. characterization of mscs phenotype in huc-mscs cultures was performed by means of flow cytometry. in passage 4, hucmscs were seeded in wells with alpha minimum essential medium (αmem) (sigma-aldrich, st. louis, mo, usa) before being washed with pbs (1x, ph 7.4) and fixed with 10% formaldehyde for ten minutes. the cells were then incubated at 37°c using the human mscs analysis kit (bd bioscience, usa) with the addition of a cd90, cd105 and cd73 and negative cd45 cocktail of primary antibodies and washed with pbs (1x, ph 7.4). the primary antibody was labelled using fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody (sigma-aldrich, st. louis, mo, usa) and incubated for 30 minutes. the cells were subsequently viewed and analysed by fluorescence assisted cell sorting (facs) using a calibur flow cytometer (bd bioscience, usa). this study used 2% gelatine (rousselot, vion company, guangdong, china) dissolved in a solution of 0.15 m sodium chloride and 25 m hepes buffer solution (sigma-aldrich, st. louis, mo, usa) at ph 7.0 which had been sterilized by autoclaving (at 121°c for 15 minutes), a process described by hendrijantini et al.13 a 2% alginate solution, as described earlier by wang,14 was prepared by dissolving sodium alginate (sigma-aldrich, st. louis, mo, usa) in distilled water at room temperature with vigorous agitation continuing until complete uniform dispersion had been achieved. the dispersion was heated to 80°c in a water bath and maintained at this temperature for 30 minutes. hydrochloride acid of 0.1 m was used to adjust the solution to ph 7.0. a 15-minute autoclaving process at 121°c was then undertaken.14 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p36–40 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p36-40 38 hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 36–40 colorimetric assay of 3-(4,5-dimethythiazol2-yl)-2,5diphenyl tetrazolium bromide (mtt) was performed to measure the cytotoxicity of the gelatine and alginate solvent against huc-mscs. three sample groups were prepared: (t1) huc-mscs with α-mem solution; (t2) huc-mscs with 2% of gelatine and (t3) huc-mscs with 2% of alginate. each group consisted of three samples with all groups being prepared in 96-well plates containing a final volume of 200μl and density of 5000 cells per well. after incubation, 10 μl of the mtt reagent was added to each well and subsequently serially diluted and incubated for 2-4 hours at 37°c. the living cells converted the mtt into purple formazan crystals, the sum of which was calculated using an elisa reader at a wavelength of 595 nm. the data obtained was described as a mean value and standard deviation. the data underwent statistical analysis using a shapiro wilk test to obtain the distribution of data and followed by a mann-whitney test to identify the differences between groups using r. version 3.4.0 software. (gnu, auckland, new zealand). a value of p<0.05 were considered statistically significant. results in passage 4, huc-mscs expressed 80.48 % cd90, 86.33% cd105 and 84.34% cd73. the result of flow cytometry is shown in figure 1, while the phenotype characteristics of huc-mscs can be seen in figure 2. the photograph of mtt assay of huc-mscs on gelatine and alginate can be seen in figure 3. the optical density was then calculated and is shown in table 1. based on statistical analysis, it can be concluded that the gelatine and alginate solvent did not affect the viability of huc-mscs and that no significant statistical difference (p>0.05) of cytotoxicity existed between gelatine, alginate and huc-mscs. discussion in this study, umbilical cord-derived stem cells were considered to be mesenchymal stem cells (mscs) due to having positive surface antigen for mscs (cd90, cd105 and cd73).15 an immunosuppressive mechanism was demonstrated by mscs, while the immunoregulation molecule of mscs constituted hla (human leucocyte antigen) class 1 which suppresses t cell proliferation. this molecule was able to inhibit lysis of mscs mediated by nk (natural killer) cells, as well as their secretion of ifn-γ (interferon gamma). the addition of mscs to mixed lymphocyte culture suppressed the production of immunoglobulins (igm, igg and iga) in vitro.16 in a xenograft model, t-cell proliferation as adaptive immunity was effectively suppressed by huc-mscs as seen in the negative expression of cd40, cd80 and cd86 which played a role in t-cell activation. humeral immune figure 1. flow cytometry cd90, cd105 and cd73 of hucmscs. figure 2 phenotype characteristic expression in huc-mscs.. alginategelatincontrol figure 3. mtt assay on control (a), gelatine (b), and alginate (c) in huc-mscs. table 1. mtt assay huc-mscs on gelatine and alginate mscs source p valuesdmeangroup hucmscs 0.009control 0.616 0.110.01470.578gelatin 0.578alginate 0.626 data presented as mean ± sd (n=12) cd90 cd105 cd73 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p36–40 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p36-40 39hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 36–40 acknowledgements the author would like to thank the stem cell research and development centre, universitas airlangga, surabaya, indonesia. references 1. jimi e, hirata s, osawa k, terashita m, kitamura c, fukushima h. the current and future therapies of bone regeneration to repair bone defects. int j dent. 2012; 2012: 1–7. 2. wa ng m , yu a n q, x ie l . me s enchy m a l st em c el l-ba s e d immunomodulation: properties and clinical application. stem cells int. 2018; 2018: 1–12. 3. el omar r, beroud j, stoltz j-f, menu p, velot e, decot v. umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies? tissue eng part b rev. 2014; 20(5): 523–44. 4. shen c, yang c, xu s, zhao h. comparison of osteogenic differentiation capacity in mesenchymal stem cells derived from human amniotic membrane (am), umbilical cord (uc), chorionic membrane (cm), and decidua (dc). cell biosci. 2019; 9: 17. 5. malgieri a, kantzari e, patrizi mp, gambardella s. bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. int j clin exp med. 2010; 3(4): 248–69. 6. patel h, bonde m, srinivasan g. biodegradable polymer scaffold for tissue engineering. trends biomater artif organs. 2011; 25: 20–9. 7. saber sem. tissue engineering in endodontics. j oral sci. 2009; 51(4): 495–507. 8. tayebi l, moharamzadeh k. biomaterials for oral and dental tissue engineering. sheffield: woodhead publishing; 2017. p. 29–30. 9. chan g, mooney dj. new materials for tissue engineering: towards greater control over the biological response. trends biotechnol. 2008; 26(7): 382–92. 10. chang b, ahuja n, ma c, liu x. injectable scaffolds: preparation and application in dental and craniofacial regeneration. mater sci eng r reports. 2017; 111: 1–26. 11. park h, kang s-w, kim b-s, mooney dj, lee ky. shear-reversibly crosslinked alginate hydrogels for tissue engineering. macromol biosci. 2009; 9(9): 895–901. 12. liao h-t, shalumon kt, chang k-h, sheu c, chen j-p. investigation of synergistic effects of inductive and conductive factors in gelatinbased cryogels for bone tissue engineering. j mater chem b. 2016; 4(10): 1827–41. 13. hendrijantini n, kresnoadi u, salim s, agustono b, retnowati e, syahrial i, mulawardhana p, wardhana mp, pramono c, rantam fa. study biocompatibility and osteogenic differentiation potential of human umbilical cord mesenchymal stem cells (hucmscs) with gelatin solvent. j biomed sci eng. 2015; 8(7): 420–8. 14. wang z, goh j, das de s, ge z, ouyang h, chong jsw, low sl, lee eh. efficacy of bone marrow – derived stem cells in strengthening osteoporotic bone in a rabbit model. tissue eng. 2006; 12(7): 1753–61. 15. arutyunyan i, elchaninov a, makarov a, fatkhudinov t. umbilical cord as prospective source for mesenchymal stem cell-based therapy. stem cells int. 2016; 2016: 1–17. 16. machado c de v, telles pd da s, nascimento ilo. immunological characteristics of mesenchymal stem cells. rev bras hematol hemoter. 2013; 35: 62–7. 17. garate a, murua a, orive g, hernández rm, pedraz jl. stem cells in alginate bioscaffolds. ther deliv. 2012; 3(6): 761–74. 18. liu j, zhou h, weir md, xu hhk, chen q, trotman ca. fastdegradable microbeads encapsulating human umbilical cord stem cells in alginate for muscle tissue engineering. tissue eng part a. 2012; 18(21–22): 2303–14. 19. kumbhar sg, pawar sh. synthesis and characterization of chitosanalginate scaffolds for seeding human umbilical cord derived mesenchymal stem cells. biomed mater eng. 2016; 27(6): 561–75. response and b cell proliferation were inhibited by hucmscs.3,15 human leukocyte antigen hla-g6 that inhibits natural killer nk cells cytolytic activity was produced by huc-mscs as well as anti-inflammatory cytokines.15 these features of huc-mscs might have contributed to cell viability in the study reported here. the mtt assay results proved that the alginate and gelatine were non-toxic to huc-mscs. this finding was consistent with studies conducted on both gelatine and alginate scaffold cytotoxicity to huc-mscs. a previous study of mtt assay demonstrated that gelatine solvent was non-toxic for huc-mscs.13 alginate scaffold provides an environment that supports cellular activity and the viability of huc-mscs.17 the results of this study of alginate cytotoxicity in huc-mscs were similar to those of a previous one indicating that huc-mscs capsulated in alginate-fibrin microbeads significantly enhanced cell viability.18 alginate and chitosan combined scaffold showed strong cytocompatibility features in huc-mscs in vitro using mtt assay.19 furthermore, huc-mscs cultured on scaffold consisting of gelatine, alginate and beta-tricalcium-phosphate demonstrated cell viability, metabolic activity and proliferation.20 this study revealed no significant statistical difference in mtt assay results between gelatine and alginate to huc-mscs. this may be due to the hydrophilic nature of gelatine and alginate as biomaterial scaffold that facilitates cell attachment and water absorption in order to provide cell nutrition and metabolism activity.21,22 another factor that influenced cell metabolism in both biomaterial scaffolds was the pore size of the porous scaffold. a pore size of 100-300nm provided an environment conducive to cell metabolism.23 gelatine solvent possessed a pore size between 58nm and 475nm.24 a pore 5-200nm in diameter was identified in the alginate solvent.18 gelatine contained high levels of amino acids such as glycine 26-34% and arginine 8-9%. glycine signalling reduced cell apoptosis.25 arginine was consumed by human mscs during cell culture to maintain cellular metabolis26, thereby implying that gelatine supports cell viability. moreover, proliferation of mscs was enhanced by arginine.27 alginate contained blocks of (1,4)-linked β-dmannuronate (m) and α-l-guluronate (g) residues. high m-block content alginates were immunogenic and more potent in inducing production of cytokine compared to high g-block alginates. alginate extracted from laminaria spp. contained 60% g-block.22 therefore, this study confirmed that alginate was non-toxic to huc-mscs. the cell culture condition in huc-mscs influenced cell viability. the cell culture in this study was carried out at standard neutral ph 7. the acidity of cell culture significantly inhibited cell proliferation, increased cell apoptosis and decreased cell viability.28 finally, it can be concluded that gelatine and alginate scaffold were non-toxic to huc-mscs in vitro. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p36–40 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p36-40 40 hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 36–40 20. soleimani m, khorsandi l, atashi a, nejaddehbashi f. chondrogenic differentiation of human umbilical cord blood-derived unrestricted somatic stem cells on a 3d beta-tricalcium phosphate-alginategelatin scaffold. cell j. 2014; 16: 43–52. 21. azizian s, khatami f, modaresifar k, mosaffa n, peirovi h, tayebi l, bahrami s, redl h, niknejad h. immunological compatibility status of placenta-derived stem cells is mediated by scaffold 3d structure. artif cells, nanomedicine, biotechnol. 2018; 46(sup1): 876–84. 22. l ee ky, mooney dj. a lginate: proper ties a nd biomedica l applications. prog polym sci. 2012; 37: 106–26. 23. utomo dn, mahyudin f, wardhana th, purwati p, brahmana f, gusti awr. physicobiochemical characteristics and chondrogenic differentiation of bone marrow mesenchymal stem cells (hbmmscs) in biodegradable porous sponge bovine cartilage scaffold. int j biomater. 2019; 2019: 1–11. 24. chen s, zhang q, nakamoto t, kawazoe n, chen g. gelatin scaffolds with controlled pore structure and mechanical property for cartilage tissue engineering. tissue eng part c methods. 2016; 22(3): 189–98. 25. bekri a, drapeau p. glycine promotes the survival of a subpopulation of neural stem cells. front cell dev biol. 2018; 6: 1–11. 26. higuera ga, schop d, spitters twgm, van dijkhuizen-radersma r, bracke m, de bruijn jd, martens d, karperien m, van boxtel a, van blitterswijk ca. patterns of amino acid metabolism by proliferating human mesenchymal stem cells. tissue eng part a. 2012; 18(5–6): 654–64. 27. huh je, choi jy, shin yo, park ds, kang j, nam d, choi dy, lee jd. arginine enhances osteoblastogenesis and inhibits adipogenesis through the regulation of wnt and nfatc signaling in human mesenchymal stem cells. int j mol sci. 2014; 15(7): 13010–29. 28. liu j, tao h, wang h, dong f, zhang r, li j, ge p, song p, zhang h, xu p, liu x, shen c. biological behavior of human nucleus pulposus mesenchymal stem cells in response to changes in the acidic environment during intervertebral disc degeneration. stem cells dev. 2017; 26(12): 901–11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p36–40 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p36-40 113 volume 46, number 3, september 2013 research report peran kalsium sebagai prevensi terjadinya hipoplasia enamel (the role of calcium on enamel hypoplasia prevention) soegeng wahluyo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: fluoride is a trace element found in many natural and commonly consumed by humans in the form of fluoride salts such as sodium fluoride (naf). the impacts that are most often caused by the intake of fluoride is a damage of enamel tooth/enamel hypoplasi or fluorosis. the manifestations of these effects are defects in teeth with whitish colour, brown to black colour effected on the aesthetic. so that the prevention of fluorosis is required. purpose: the aim of this study was to analyze the effect of calcium as prevention against tooth enamel fluorosis in wistar rats caused by exposure to fluoride through indicators of amelogenin, calbindin28kda protein expression, the matrix of tooth enamel density and distance between ameloblast cell. methods: this was an experimental studies, post-test only control group design. this study used three groups of rats. group 1 (control) was induced by sterile destilled water, group 2 (treatment 1) was induced by fluoride and group 3 (treatment 2) was induced by combination of fluoride and calcium. each induction was done through sonde for 28 days. results: the results showed that the induction of fluoride causes the increased expression of amelogenin protein; decreased expression of calbindin-28kda protein; a decrease in the density of the enamel matrix and widen the distance between cells ameloblast, while the result of the combination induced by fluoride and calcium showed increased protein expression of calbindin-28kda and increased density of the enamel matrix. conclusion: calcium can be used as an alternative preventive against the occurrence of enamel hypoplasia due to exposure of fluoride in wistar rats. key words: fluorosis, calcium, amelogenin, calbindin-28kda, enamel matrix density abstrak latar belakang: fluorida adalah salah satu trace element yang ada dialam dan sering dikonsumsi manusia dalam bentuk garam fluorida yaitu sodium fluoride (naf). paparan fluorida biasanya berkaitan dengan asupan fluorida yang dapat membahayakan enamel gigi yaitu terjadinya hipoplasia enamel atau fluorosis. manifestasi efek ini memberikan gambaran berupa defect pada enamel gigi ditandai dengan perubahan warna dari kecoklatan hingga kehitaman dan penyebab estetik yang tidak baik, maka diperlukan usaha pencegahan fluorosis tersebut. tujuan: studi ini adalah menganalisis efek kalsium terhadap prevensi terjadinya fluorosis pada tikus wistar yang terpapar fluorida, dengan indikator ekspresi protein amelogenin, calbindin-28kda, densitas matriks enamel dan jarak antar sel ameloblas. metode: studi ini merupakan studi eksperimental dengan desain post test only control group, yang menggunakan 3 kelompok tikus. kelompok-1 (kontrol) di induksi dengan aquadest steril, kelompok-2 (treatmen-1) diinduksi dengan fluorida dan kelompok-3 (treatmen-2) diinduksi dengan kombinasi antara fluorioda dan kalsium. induksi dilakukan selama 28 hari melalui sonde. hasil: menunjukkan bahwa induksi dengan fluorida menyebabkan peningkatan ekspresi protein amelogenin dan terjadi penurunan ekspresi protein calbindin-28kda, dan penurunan kepadatan matriks enamel serta pelebaran jarak antar sel. tetapi bila diinduksi dengan kombinasi fluorida dan kalsium maka terjadi peningkatan ekspresi calbindin-28kda dan peningkatan densitas matriks enamel. 114 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 113–118 pendahuluan salah satu permasalahan di bidang kesehatan gigi adalah peningkatan prevalensi hipoplasia enamel di daerah dengan kadar fluorida yang relatif tinggi. hipoplasia enamel adalah kelainan pembentukan enamel yang tidak sempurna yang sering ditandai dengan perubahan warna gigi kekuningan, kemerahan, coklat sampai kehitaman dan pada kasus yang berat memberikan manifestasi perubahan struktur dan anatomi gigi. dental fluorosis adalah suatu gambaran hipoplasia enamel gigi yang disebabkan oleh pajanan fluorida dengan dosis diatas optimal dalam waktu yang relatif lama pada saat fase pembentukan dan kalsifikasi gigi.1 prevalensi dental fluorosis di indonesia bervariasi tergantung tinggi-rendahnya kadar fluorida yang terdapat dalam air tanah yang digunakan sebagai konsumsi air minum masyarakat. berbagai penelitian telah dilakukan tentang dental fluorosis seperti yang dilakukan oleh wondwossen,2 menyatakan bahwa penelitian yang dilakukan pada 233 anak di daerah dengan kadar 0,5 ppm dihasilkan 24,1% mengalami dental fluorosis sedang dan 75,9% dengan derajat ringan. penelitian terbaru pada tahun 20113 di daerah kecamatan asembagus kabupaten situbondo-jawa timur yang merupakan daerah endemik fluorosis memberikan hasil bahwa rerata kadar fluorida dalam air minum sebesar 2,08–2,90, prevalensi dental fluorosis sebesar 78,75–98,33% dengan rerata community fluorosis index (cfi) 0,80–1,60. penelitian terdahulu lebih terfokus pada kajian secara epidemiologis yaitu besarnya prevalensi terjadinya dental fluorosis dan beberapa aspek seperti aspek biologis dan klinis terutama yang berkaitan dengan teknik perawatannya. persoalan yang sampai saat ini belum menyentuh aspek preventif dari dental fluorosis tersebut.4-6 berbagai upaya harus diusahakan untuk melakukan usaha preventif terhadap terjadinya dental fluorosis. salah satu alternatif upaya preventif pada penelitian ini adalah dengan menggunakan induksi kalsium klorida (cacl2). 7 penggunaan cacl2 sebagai bahan untuk induksi merujuk pada national research council of united states (2005) yang menyatakan bahwa bahan tersebut digunakan sebagai campuran bahan makanan dan aman bagi kesehatan. studi ini menggunakan beberapa parameter antara lain ekspresi protein amelogenin, calbindin-28kda, densitas matriks enamel dan jarak antar sel ameloblas. tujuan studi adalah untuk meneliti dan menganalisa pengaruh induksi kalsium (cacl2) sebagai bahan alternatif untuk prevensi terhadap terjadinya hipoplasia enamel akibat pajanan fluorida. studi ini tidak dilakukan pada manusia karena beberapa alasan etika namun dilakukan pada hewan coba yaitu rattus norvegicus strain wistar. bahan dan metode studi ini merupakan penelitian eksperimental dengan desain randomized post test only control group design. studi ini menggunakan binatang coba sebagai model yaitu rattus norvegicus strain wistar jantan usia 10–11 minggu dengan berat badan 150–170 gram,8 yang terdiri dari 3 kelompok. tiap kelompok terdiri dari 8 ekor tikus yang diambil secara acak: kelompok 1: dipajan dengan aquades steril sebesar 2 ml (sebagai kontrol); kelompok 2: dipajan dengan naf (6,75 mgr dalam 2 ml aquades steril); kelompok 3: dipajan dengan naf + cacl2 (6,75 mgr + 3 mgr dalam 2 ml aquades steril). besarnya dosis fluorida yang berdampak terjadinya hipoplasia enamel (fluorosis) dan pajanan kalsium sebagai prevensi, menggunakan pendekatan beberapa referensi.9-12 analisis penelitian dilakukan setelah 28 hari induksi yang meliputi beberapa aspek sebagai bukti dan penunjang analisis antara lain ekspresi protein amelogenin dan calbindin-28kda dengan menggunakan antibodi monoklonal anti amelogenin dan anti calbindin-28kda yang diukur dengan cara menghitung jumlah sel ameloblas yang memberikan reaksi positif. ekspresi kedua protein ini dilakukan melalui teknik perwarnaan imunohistokimia yang dihitung per 10 high power field (hpf) dan diamati melalui mikroskop cahaya dengan pembesaran 400x.13,14 untuk analisis densitas matriks enamel diukur secara kualitatif berdasarkan perubahan yang terjadi dengan menggunakan scanning electron microscope (sem) dengan pembesaran 20.000x. analisis melalui sem ini menggunakan pendekatan kriteria dudea15 yang membagi gambaran hasil sem pada gigi menjadi 4 kuadran dan masing-masing kuadran dinilai sesuai dengan besarnya prosentase porositas dari kuadran tersebut (skor 1–3). unit analisis yang lainnya yang digunakan adalah pengukuran densitas enamel adalah dengan mengukur jarak antar sel ameloblas pada ketiga kelompok tersebut (soft ware: cell-d). analisis statistik yang digunakan untuk menganalisis perbedaan tiap variabel antara kelompok kontrol dan perlakuan menggunakan uji anava dan lsd, sedangkan untuk menganalisis beberapa variabel yang berpengaruh simpulan: kalsium dapat digunakan sebagai alternative terhadap terjadinya hipoplasia enamel akibat paparan fluorida pada tikus wistar kata kunci: fluorosis, kalsium, amelogenin, calbindin-28kda, kepadatan matriks enamel koresponsdensi (correspondence): soegeng wahluyo, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. 115waluyo: peran kalsium sebagai prevensi terjadinya hipoplasia enamel pada studi ini digunakan uji korelasi pearson dan uji regresi linier. hasil efek pajanan naf pada rattus norvegicus akan memberikan dampak perubahan pada gigi insisivus rahang bawah berupa hipoplasia enamel serta berpengaruh terhadap struktur sel ameloblas. perubahan tersebut seperti terlihat pada gambar 1. dampak yang terjadi setelah terpajan fluorida yaitu gigi tikus akan mengalami fluorosis seperti terlihat pada gambar 1b, sedangkan pada gambar 1c adalah gambar gigi tikus yang mengalami mineralisasi setelah dipajan kombinasi antara naf+cacl2. terjadinya hipoplasia enamel pada gigi tidak terlepas dari peran protein amelogenin yang selama proses mineralisasi akan mengalami delay proses degradasi sehingga menghasilkan struktur enamel yang terisi oleh protein tersebut. sebagai akibat keadaan ini akan terjadi peningkatan ekspresi amelogenin pada kelompok yang dipajan fluorida seperti terlihat pada tabel 1. peran kalsium pada studi ini sangat penting. sebagai akibat induksi kalsium (cacl2) maka terjadi peningkatan transportasi kalsium menuju sel target yaitu sel ameloblas yang dibuktikan melalui teknik imunohistokimia (ihc) menggunakan antibodi monoklonal anti calbindin-28kda dengan hasil terjadi peningkatan ekspresi protein calbindin28kda pada sel ameloblas yang mengalami dental fluorosis seperti pada gambar 2. setelah dilakukan pajanan dengan fluorida tanpa kalsium maka ekspresi protein calbindin-28kda akan mengalami penurunan (gambar 2b), namun setelah dilakukan pajanan kombinasi fluorida+kalsium, maka terjadi peningkatan ekspresi protein calbindin-28kda (gambar 2c). besarnya peningkatan ekspresi protein tersebut dapat dilihat pada tabel 1. hasil analisis mengenai densitas matriks enamel melalui pemeriksaan scanning electron microscopy (sem) melalui pembesaran 20.000 kali seperti tampak pada gambar 3. untuk membuktikan pengaruh induksi kalsium terhadap densitas matriks enamel dilakukan analisis jarak atau kerapatan antar sel ameloblas gigi insisivus tikus seperti tampak pada gambar 4. dari hasil analisis ekspresi protein calbindin-28kda, analisis densitas matriks enamel dengan mengunakan sem dan analisis densitas matriks enamel dengan memperhitungkan jarak antar sel ameloblas dirangkum dalam hasil analisis tersebut seperti terlihat pada tabel 1. dari tabel 1 terlihat bahwa rerata ekspresi protein calbindin-28kda pada kelompok yang dipajan fluorida lebih kecil bila dibanding kelompok yang dipajan kombinasi fluorida+kalsium, sedangkan untuk besarnya porositas enamel, paling besar terjadi pada kelompok yang dipajan kombinasi fluorida dibanding kedua kelompok lainnya. gambar 1. a) gigi incisivus normal (kelompok kontrol); b) gigi incisivus yang mengalami fluorosis (kelompok induksi naf); c) gigi incisivus yang mengalami mineralisasi (kelompok induksi naf+cacl2). gambar 2. a) ekspresi protein calbindin-28kda pada kelompok control; b) ekspresi protein calbindin-28kda kelompok yang dipajan naf; c) ekspresi protein calbindin-28kda kelompok yang dipajan kombinasi naf+cacl2 (pengecatan imunohistokimia/ihc dengan pembesaran 400x). a b c a b c 116 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 113–118 pada pengamatan jarak antar sel didapatkan hasil bahwa kerapatan atau jarak antar sel ameloblas pada kelompok yang dipajan dengan kalsium (cacl2) lebih kecil bila dibanding dengan kelompok yang dipajan dengan naf. hasil analisis statistik menunjukkan bahwa terdapat perbedaan bermakna antara kelompok yang dipajan dengan fluoride (k-2) dengan kedua kelompok lainnya (k-1 dan k-3) untuk ekspresi amelogenin, calbindin-28-kda dan densitas matriks enamel serta analisis jarak antar sel ameloblas, sedangkan antara k-2 dan k-3 tidak didapatkan perbedaan yang bermakna. analisis korelasi dan regresi memberikan hasil bahwa terdapat pengaruh dan keterkaitan antara beberapa veriabel dalam studi ini (koofisien korelasi = 0,804). pembahasan pengaruh pajanan fluorida dan kalsium akan mempengaruhi keseimbangan beberapa protein utama pada masa tumbuh-kembang gigi antara lain amelogenin dan calbindin-28kda serta berpengaruh terhadap densitas enamel. hasil penelitian menunjukkan bahwa ekspresi amelogenin kelompok kontrol (k-1) paling rendah dibanding kelompok yang dipajan fluorida yang mempunyai nilai ekspresi paling tinggi. hal ini disebabkan pada saat proses amelogenesis secara normal, amelogenin akan terdegradasi menjadi beberapa fragmen melalui proses hidrolisis oleh aktivitas matrixmetalloproteinase-20 (mmp-20)16-18 yang merupakan mediator dari remodeling gambar 3. a) gambaran densitas matriks enamel pada kelompok control; b) gambaran densitas matriks enamel kelompok yang dipajan naf; c) gambaran densitas matriks enamel kelompok yang dipajan naf+nacl2 (pengamatan menggunakan sem dengan pembesaran 20.000x). gambar 4. a) jarak antar sel ameloblas pada kelompok kontrol; b) jarak antar sel ameloblas pada kelompok yang dipajan naf; c) jarak antar sel ameloblas pada kelompok yang dipajan naf+cacl2 (pengamatan dengan mikroskop+soft ware: cell-d). tabel 1. rerata dan simpang baku ekspresi protein calbindin-28kda, besarnya porositas matrik enamel gigi dan jarak antar sel ameloblas tikus wistar yang dipajan naf dan kombinasi naf+cacl2 kelompok induksi rerata + simpang baku ekspresi amelogenin ekspresi calbindin28kda besarnya porositas enamel jarak antar sel ameloblas induksi aquades (k-1) 5,88 ± 2,031 12,13 ± 1,808 1,0625 ± 0,11573 58,7200 ±4,54538 induksi naf (k-2) 14,75 ± 2,375 3,50 ± 1,414 2,5938 ± 0,22903 111,6775±21,13876 induksi naf + cacl2 (k-3) 6,38 ± 1,061 12,00 ± 1,852 1,1875 ± 0,11565 73,3387 ± 9,10034 a b c a b c 117waluyo: peran kalsium sebagai prevensi terjadinya hipoplasia enamel matriks enamel pada ph 7,2–7,3. tetapi akibat pajanan fluorida, maka akan terjadi peningkatan ion f yang akan berikatan dengan tiga komponen asam amino yang terkandung dalam protein amelogenin yaitu glutamin dan histidin yang akan mempengaruhi sifat basa dari residu asam amino tersebut sehingga terjadi perubahan ph menjadi 5,5–5,6. perubahan ini akan menghambat aktivitas mmp20 dalam mendegradasi amelogenin sehingga terjadi delay proses reduksi amelogenin. hambatan ini akan memberikan dampak pada struktur dan komposisi matriks enamel yang akan terisi oleh sebagian besar protein amelogenin sehingga saat maturasi enamel didapatkan adanya beberapa porositas dan jarak antar sel melebar/panjang, hal ini sesuai dengan penelitian sebelumnya.19,20 pemberian kalsium bertujuan untuk meningkatkan mineralisasi dan untuk meningkatkan transportasi kalsium ke dalam matriks enamel. efektivitas kalsium menuju sel target yaitu ameloblas memerlukan media transport yaitu protein calbindin-28kda yang merupakan superfamili calmodulin dan mempunyai afinitas tinggi dalam mengikat kalsium, serta berfungsi sebagai fasilitator diffusi, sensor dan buffering kalsium. pada studi ini setelah dilakukan pajanan fluorida+ kalsium pada kelompok-3 (k-3) terjadi penurunan ekspresi amelogenin dan terjadi peningkatan ekspresi calbindin-28kda (tabel 1). hal ini seperti diungkapkan oleh peneliti terdahulu bahwa pajanan kalsium memberikan dampak meningkatnya beberapa chaperon dan beberapa reseptor dari kalsium. dalam intraseluler konsentrasi kalsium adalah 10-7 akan bergerak keluar sel dalam bentuk ikatan antara ca++ + calbindin28d.21 sebagai akibat dari keadaan tersebut maka terjadi peningkatan rangsangan sekresi mmp-20 yang selanjutnya akan mempercepat proses degradasi amelogenin. peran calbindin-28kda sangat besar sebagai transport kalsium karena protein tersebut mempunyai affinitas yang tinggi22,23 di dalam mengikat kalsium melalui empat sisi/ lengan dan jumlahnya lebih banyak bila dibanding dengan protein calbindin lainnya sehingga kontribusinya terhadap transportasi dan endapan kalsium sangat besar. efek pemberian kalsium juga terkait dengan proses pembentukan kristal pada matriks enamel, maka sejak awal proses pembentukan enamel sampai maturasi akan memberikan akses terjadinya penggabungan beberapa kristal menjadi beberapa kristal yang lebih besar dan padat. proses tersebut diikuti dengan proses degradasi protein amelogenin, karena pada proses normal akan terjadi fase ini yang selanjutnya posisi amelogenin yang terdegradasi akan digantikan oleh beberapa mineral. untuk memperkuat bukti tersebut maka studi ini dilakukan analisis dengan menggunakan sem. hasil pemeriksaan sem (dengan pembesaran 20.000x) yang sesuai hasil rujukan peneliti sebelumnya menunjukkan bahwa material organik matriks enamel (4–10%) berisi air dan protein memberikan gambaran spesifik berupa warna kehitaman sedangkan material anorganik (90%) berisikan mineral hidroksi apatit (ca10(po4)10(oh)2) akan memberikan gambaran warna cerah/putih.24 hal ini menunjukkan tingkat kepadatan dan berat molekul yang tinggi. analisis sem menunjukkan adanya perbedaan bermakna untuk densitas pada ketiga kelompok, secara setatistik dinyatakan bahwa perbedaan antara kelompok kontrol/k-1 dan kelompok yang dipajan dengan kombinasi fluorida+kalsium/k-3 memberikan hasil yang tidak signifikan (tabel 1) dan hal ini juga dibuktikan pada gambar 2-a dan 2-c, sedangkan hasil analisis sem antara kelompok yang dipajan dengan fluorida terdapat perbedaan yang bermakna dengan kedua kelompok lainnya. keadaan ini disebabkan susunan matriks enamel lebih banyak terisi oleh material organik yang didominasi oleh protein amelogenin yang tidak terdegradasi oleh mmp-20 sehingga memberikan manifestasi gambaran banyaknya porositas matriks enamel (gambar 3b). pembuktian lain yaitu melalui analisis jarak antar sel ameloblas yang menggambarkan kepadatan matriks enamel. pada kelompok yang dipajan dengan naf memberikan manifestasi jarak antar sel ameloblas yang paling besar bila dibanding dua kelompok lainnya. hal ini disebabkan akibat paparan fluorida yang selanjutnya akan menyebabkan kematian sel ameloblas. kondisi tersebut berpengaruh terhadap eksistensi sel ameloblas, sehingga pada kelompok yang dipajan dengan fluorida tampak lebih renggang jarak antar sel-nya. pada kelompok yang dipajan dengan kombinasi antara naf+cacl2 akan terjadi mineralisasi akibat fungsi kalsium yang akan meningkatkan mineralisasi matriks enamel dan menekan terjadinya kematian sel ameloblas melalui proses apoptosis dengan menekan fungsi protein caspase-3 sebagai eksekutor pada proses apoptosis. sesuai dengan fungsi calbindin-28kda yaitu sebagai protein anti apoptosis dan sebagai buffering terhadap kalsium pada tumbuh-kembang ameloblas atau osteoblas namun juga merupakan protein penghambat caspase-3 dengan cara memblokade sebagian fungsi degradasi dan sintesis substrat alami dari caspase-3.25 pada konsentrasi ca++ yang tinggi merupakan media dari perubahan morfologi sel ameloblas, karena ameloblas secara cepat akan memompa ca++atpase dan ca++ calbindin-28kda. hal ini merupakan salah satu mekanisme potensial dari ameloblas dalam meregulasi proses maturasi enamel dengan mengontrol ca++-transpor ke dalam matriks enamel. analisis korelasi dan regresi untuk beberapa variabel menunjukkan bahwa semua variabel yang terkait sangat signifikan pengaruhnya pada studi ini (koofisien korelasi = 0,804). berdasarkan kajian dan beberapa fakta dan bukti hasil penelitian ini maka dapat disimpulkan bahwa kalsium sangat berperan dalam prevensi terjadinya hipoplasia enamel akibat paparan fluorida/dental fluorosis pada binatang coba. penggunaan pada manusia diperlukan penelitian lebih lanjut tentang dosis konversi untuk manusia dan waktu yang efektif untuk pemberian kalsium. hal ini diperlukan untuk menghindari beberapa akibat yang disebabkan oleh fluorida dan kalsium terutama pada daerah endemik fluorosis. hasil penelitian ini menurut peneliti adalah merupakan temuan dan sumbangan pikiran bagi prospek masa depan untuk penanggulangan dental fluorosis. 118 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 113–118 daftar pustaka 1. mc. donald re, avery dr, dean ja. dental fluorosis in child. in: dentistry for the child and adolescent). 6th ed. saint louis-missouri: the cv mosby; 2005. p. 115-22, 230–1. 2. wo n d wo s s e n f, a s t r o m a n , bj o r v a t n k , b a r d s e n a . sociodemographic and behavoural correlates of severe dental fluorosis. inter j paediatric dent 2006; 16(2): 95–103. 3. wahluyo s, rahayu rp. beberapa sumber fluorida dalam makanan dan sumber air serta dampak konsumsi fluorida pada anak. laporan penelitian hibah pemerintah, provinsi jawa timur (bappedal); 2011. p. 1–53. 4. wa hluyo s. hubungan anta ra terjadinya hipoplasia enamel dengan kadar kalsium dan fluor dalam air minum pada anak usia 11–13 tahun. tesis. surabaya: program s-2 pascasarjana universitas airlangga; 1995. 5. aoba t, fejerskov o. dental fluorosis: chemistry and biology. sage j 2002, 13(2): 155–70. 6. fagin d. fluoride. j of scientific america 2008. available from: http://www.sciam.com. diakses tanggal 7 juli 2009. 7. wahluyo s. ekspresi bcl-2, caspase-3, amelogenin, calbindin-28kda dan densitas matriks enamel pada sel ameloblas akibat paparan sodium fluoride dengan atau tanpa tambahan kalsium klorida. disertasi. surabaya: program studi s3 kedokteran, fakultas kedokteran universitas airlangga; 2012. 8. isroi. biologi rattus norvegicus. 2010. available from: http://isroi. wordprss.com. accessed februari 26, 2011. 9. national research council of united states. mineral tolerance of animals. 2nd ed. new york–washington dc; the national academies press; 2005. p. 1–308. 10. national research council of united states. calcium chlorida chemical profile. 2005. available from: http://www.the-motivationgroup.com. accessed september 10, 2010. 11. mathieu lg, pelletier rp. a study of the oral toxicity of calcium chloride, scientific commons-beta. 2009. available from: http:// www.pubmedcentral.gov/articlevender.fegi. accessed september 9, 2010. 12. catani db, tenuta lma, andalo fa, cury ja. fluorosis in rats exposed to oscillating chronic fluoride doses. braz dent 2010; 21(1): 217–25. 13. soresi at. interleukin 6 and its soluble receptor in patients with liver chirrosis and hepatocellular carcinoma. world j gastroenterol 2006; 12(16): 2563–8. 14. taylor cr, shi sr, bar nj. techniques of immunohistochemistry: p r inciple, pitfa ll sta nda rdization. i n: dabbs d. diagnostic immunohistochemistry/theranostis, and genomic applications. saunders elsevier; 2010; p. 1–31. 15. dudea d, florea a, mihu c, câmpeanu r, nicola c, benga g. the use of scanning electron microscopy in the evaluating the effect of bleaching agent on the enamel surface (original paper). rom j morphol embryol 2009; 50(3): 435–40. 16. fincham ag. molecular mechanism of growth and development of dental enamel. critical review in oral biology–simmer and fincham 2008; 6(2): 84. 17. gibson cw, yuan za, hall b, longenecker g, chen e, thyagarajan t, sreenath t, wright jt, decker s, piddington r, harrison g, kulkarni ab. amelogenin-deficient mice display an amelogenesis imperfecta phenotype. j biol chem 2001; 276(34): 31871–5. 18. tanimoto k, le t, zhu l, witkowska he, robinson s, hall s, hwang p, denbesten p, li w. reduced amelogenin-mmp-20 interaction in amelogenesis imperfecta, research report biological. j dent res 2008; 87(5): 451–5. 19. denbesten pk, yan y, featherstone jdb, hilton jf, smith ce, li w. effect of fluoride on rat dental enamel matrix proteinase. arch oral biol 2002; 47(11): 763–70. 20. uskoković v, khan f, liu h, witkowska he, zhu l, li w, habelitz s. hydrolysis of amelogenin by matrix metalloprotease20 accelerates mineralization in vitro. arch oral biol 2011; 56(12): 1548–59. 21. chen jr, huang st, huang y, gilchrist ph, singh b, borke jl. calcium transport protein and amelogenin expression in ameloblastoma: relatinship to normal amelogenesis, taiwan. j oral med health sci 2005; 21: 85–94. 22. turnbull ci, looi k, mangum je, meyer m, sayer rj, hubbard j. calbindin independence of calcium transport in developing teeth. contradicts the calcium ferry dogma. j biol chem 2004; 279(53): 55850–4. 23. bronckers alj, lyaruu dm, denbesten pk. the impact of fluoride on ameloblasts and the mechanism of enamel fluorosis. j dent res 2009; 88(10): 877–93. 24. soares cj, moura ccg, soares pb, naves lz. scanning electric microscopy used to analyse the effect of gamma radiation on enamel and dentin. microscopy: science, technology, application and education. a mendez–vilas and j diaz 2010; 372–8. 25. bellido t, huening m, raval-pandya m, manolagas sc, christakos s. calbindin-28kd is expressed in ostoblastic cell and suppresses their apoptosis by inhibiting caspase-3 activity. j of biol chem 2008; 275(34): 26328–32. 181181 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 181–185 original article inhibitory effect of calcium hydroxide combined with nigella sativa against enterococcus faecalis myrna nurlatifah zakaria1, yusfien shabrina putri1, asih rahaju1, sri fatmawati2 and arief cahyanto3 1department of endodontology and operative dentistry, faculty of dentistry, universitas jenderal achmad yani, cimahi, indonesia 2department of chemistry, faculty of sciences, institut teknologi sepuluh nopember, surabaya, indonesia 3department of dental materials science and technology, faculty of dentistry, universitas padjadjaran, bandung, indonesia abstract background: calcium hydroxide is the gold standard medicament for root canal treatment. enterococcus faecalis, the primary cause of intraradicular persistent endodontic infection, is often identified even after endodontic treatment. thymoquinone, an active ingredient of nigella sativa, has an antimicrobial effect on both gram-negative and positive bacteria, including e. faecalis. purpose: this study aimed to evaluate the inhibitory effect of calcium hydroxide combined with nigella sativa extract and determine the best ratio for the combined material. methods: this is an experimental study comprised of six groups (n = 4 per group) based on the material and its ratio, namely; (1) calcium hydroxide; (2) nigella sativa extract; and groups of the combination of calcium hydroxide and nigella sativa extract with a ratio (3) 70:30, (4) 50:50, (5) 30:70, (6) 10:90. the inhibitory effect against e. faecalis was evaluated by the agar well diffusion method in muller–hinton agar. observation of the inhibitory zone was performed on the first, third, and seventh days. the collected data were analysed by a one-way anova and lsd post hoc test. results: calcium hydroxide has the highest inhibitory effect, and the combination of nigella sativa extract with calcium hydroxide ratio 50:50 was second. the inhibitory zone of these two groups was significantly higher than in any other group (p<0.05). conclusion: nigella sativa extract combine with calcium hydroxide did not enhance calcium hydroxide’s antimicrobial property against e. faecalis. an equal amount of nigella sativa and calcium hydroxide is the best combination ratio, with a stable effect for up to seven days. keywords: calcium hydroxide; endodontic infection; enterococcus faecalis; intracanal medicament; nigella sativa correspondence: myrna nurlatifah zakaria, department of endodontology and operative dentistry, faculty of dentistry, universitas jenderal achmad yani. jl. terusan jenderal sudirman, cimahi 40531, indonesia email: myrna.nurlatifah@lecture.unjani.ac.id introduction deep caries or traumatic injury often become a pathway for bacteria to enter the pulp and resume infection to the periapical tissue, known as endodontic infection. the endodontic infection may form as primary, secondary, or persistent infection.1 one bacterium commonly found in persistent infections is enterococcus faecalis (e. faecalis). virulence factors of e. faecalis increase the bacteria’s survival in harsh environments as it adheres to and penetrates the dentinal tubules. bacterial virulence is one of the primary factors of root canal treatment failure.2 the persistent infection commonly results in failed tissue regeneration seen as an unhealed radiolucency in the apical area or as unrelieved symptoms such as pain or ongoing sinus tract problems.3 in multiple visit endodontic treatments, the root canal system needs to be filled by an intracanal medicament to suppress the amount and inhibit the growth of bacteria, reduce pain, and provide long-term disinfection. calcium hydroxide has been the most commonly used material in endodontics since 1920, with various indications from pulp capping, apexification, apexogenesis, and root canal disinfection.4 the main mechanism of action of calcium hydroxide is due to the release of calcium and hydroxyl ions in the presence of water. the hydroxyl ion elevates the environment ph to an alkaline condition, which has a detrimental effect on bacteria. it reduces bacterial virulence and eventually reduces symptoms, assisting the healing process and tissue regeneration.5 however, several studies mentioned the limitations of calcium hydroxide in eliminating e. faecalis because of the calcium hydroxide dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p181–185 mailto:myrna.nurlatifah@lecture.unjani.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p181-185 182 zakaria et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 181–185 paste’s viscosity, penetration ability, and bacterial virulence, which can survive alkaline and starvation conditions.6,7 nigella sativa is a herb that is widely used by people, especially in the middle east. nigella sativa seeds and oils have been used as an analgetic, antibacterial, anti-allergy, anti-cancer, and anti-inflammatory agent. thymoquinone is an active ingredient in nigella sativa, which can inhibit the growth of bacteria and function as an antimicrobial agent. thymoquinone can damage the bacteria cell’s integrity, resulting in the death of bacteria cells through necrosis and apoptosis.8–10 nigella sativa extract effectively eliminates both gram-negative and positive bacteria, including e. faecalis. a previous study reported that nigella sativa is dose-dependent, in which 100% extract had better antibacterial action against enterococcus than 25%, 50%, and 75%.11 other studies mentioned that nigella sativa extract with low viscosity could penetrate and provide a good antibacterial effect, unlike the high viscosity extract.12,13 the nigella sativa’s ability to eliminate e. faecalis and the low viscosity makes them of interest as an intracanal disinfection agent. it was hoped future research of the compound could synergically improve the antimicrobial efficacy of calcium hydroxide. based on previous studies on the high potency of nigella sativa, particularly as an antimicrobial agent, we proposed using this agent as supplemental to the commonly used endodontic intracanal medicament, calcium hydroxide. as far as we know, this has not been previously studied. therefore, the proper ratio in combining the materials has yet to be determined. whether this combination can work synergistically also needs to be evaluated; one of the ways is by evaluating the inhibitory effect of both materials in different combinations. considering calcium hydroxide is a time-dependent medicament, the evaluation was done on three spesific days (days one, three and seven) to evaluate the stability of the material.14 therefore, we investigated the efficacy of the nigella sativa extract and its combination with calcium hydroxide in several ratios to evaluate the inhibitory effect against e. faecalis, and determined the best ratio for the combining material. materials and methods this is an experimental study with a posttest-only control design, consisted of six groups (n = 4 per group), paired numeric analytic with 95% confidence interval) according to the sample components and ratio: (1) calcium hydroxide; (2) nigella sativa extract; a combination of calcium hydroxide with nigella sativa (3) ratio 70:30; (4) ratio 50:50; (5) ratio 30:70; (6) ratio 10:90. to ensure the bacteria itself was not affected by the distilled water used as a solvent for the calcium hydroxide a group by using only distilled water was used as negative control. the extraction of nigella sativa was carried out by maceration using methanol as a solvent at the department of chemistry, faculty of sciences, institut teknologi sepuluh nopember, surabaya, indonesia.15 calcium hydroxide powder (merck, darmstadt, germany) was mixed with distilled water with the w/p ratio of 0.8 to provide the best consistency as intracanal medicament paste to have acceptable flowability. the total crude extract (100% concentration) and calcium hydroxide were weighed on an analytical scale to obtain the exact weight for each group according to its ratio (table 1). table 1. tested groups and weight ratio group ratio nigella sativa (milligram) calcium hydroxide (milligram) 1 0:100 260 2 100:0 260 3 70:30 182 78 4 50:50 130 130 5 30:70 78 182 6 10:90 26 234 first day third day seventh day negative control figure 1. antibacterial inhibitory zone of nigella sativa extract, calcium hydroxide and its combination, to enterococcus faecalis. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p181–185 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p181-185 183zakaria et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 181–185 table 2. antibacterial inhibitory zone of tested groups to e. faecalis group mean (mg/dl) ± sd day p calcium hydroxide 21.63 ± 0.13 first *0.001 nigella sativa 16.8 ± 1.28 ratio 70:30 17.58 ± 0.88 ratio 50:50 19.2 ± 0.98 ratio 30:70 18. ± 0.51 ratio 10:90 16.98 ± 0.67 calcium hydroxide 21.63 ± 0.13 third *0.001 nigella sativa 16.8 ± 0.91 ratio 70:30 17.83 ± 0.61 ratio 50:50 18.93 ± 0.93 ratio 30:70 17.65 ± 0.75 ratio 10:90 16.93 ± 0.77 calcium hydroxide 21.53 ± 0.15 seventh *0.006 nigella sativa 16.73 ± 0.93 ratio 70:30 17.7 ± 0.52 ratio 50:50 18.78 ± 0.93 ratio 30:70 17.65 ± 0.69 ratio 10:90 16.83 ± 0.71 *one-way anova p≤0.05 (significant difference) table 3. intergroup comparisons of the antibacterial inhibitory zone of the groups against e. faecalis group day calcium hydroxide nigella sativa ratio 70:30 ratio 50:50 ratio 30:70 ratio 10:90 negative control calcium hydroxide first nigella sativa 0.000* ratio 70:30 0.000* 0.200 ratio 50:50 0.001* 0.001* 0.012* ratio 30:70 0.000* 0.050* 0.450 0.059 ratio 10:90 0.000* 0.767 0.317 0.001* 0.088 calcium hydroxide third nigella sativa 0.000* ratio 70:30 0.000* 0.064 ratio 50:50 0.000* 0.001* 0.048 ratio 30:70 0.000* 0.119 0.740 0.025* ratio 10:90 0.000* 0.813 0.100 0.001* 0.180 calcium hydroxide seventh nigella sativa 0.001* ratio 70:30 0.040* 0.220 ratio 50:50 0.271 0.029* 0.230 ratio 30:70 0.021* 0.329 0.802 0.230 ratio 10:90 0.001* 1.000 0.220 0.029* 0.329 *post hoc lsd, significant difference (p<0.05 level) the e. faecalis atcc 2912 was reidentified and cultured to achieve a pure suspension of e. faecalis. the concentration of the bacteria was standardised by spectrophotometry based on the mcfarland standard at 0.5. the antibacterial test was carried out by preparing a bacterial suspension followed by the well diffusion test.15 mueller–hinton agar plates were prepared, and the bacteria suspension was plated in each agar plate. each agar plate was divided into four, and then a well was made with a sterilised perforator. afterwards, each sample group was placed into the well. the agar plates with the tested material were then incubated at 37o c for the evaluation interval. all work was conducted on a workbench and in a sterile protocol. observation of the inhibitory zone was performed on the first, third, and seventh days. digital callipers were used to measure the diameter of the inhibition zone around the well. the measurement was repeated three times for each inhibition zone, and the average value was taken. collected data were analysed using a one-way analysis of variance (anova) test (p<0.05) to compare the inhibitory zones between groups, and a post hoc least significant difference (lsd) test was performed to determine which specific groups were statistically significant from others (p<0.05). results the inhibition zone of nigella sativa extract, calcium hydroxide, and its combinations are depicted in figure 1. descriptively, all samples showed an inhibitory area surrounding the sample-containing well, except for the negative control group. the mean value of each group and the one-way anova test are presented in table 2. in all three intervals evaluated, calcium hydroxide has higher antibacterial efficacy than the others. the combination of nigella sativa extract with a calcium hydroxide ratio 50:50 was second after calcium hydroxide. on all observation days, the mean value of an inhibitory zone for nigella sativa extract was the lowest of all medicament groups. the inhibitory zone for all groups did not show notable differences until the seventh day of observation. comparative analysis of the study groups by the post hoc lsd test (p<0.05) showed significant differences between tested groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p181–185 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p181-185 184 zakaria et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 181–185 intergroup comparisons between groups on each day by the post hoc analysis using the lsd test are described in table 3. on the first day and third day of observation, the inhibitory zone of calcium hydroxide was significantly higher compared to all other tested groups. however, on the third, the inhibitory zone did not differ significantly between the calcium hydroxide group and the combination group of nigella sativa and calcium hydroxide. in all three evaluations, a consistent significant difference between different combination ratios of nigella sativa and calcium hydroxide extract groups compared to nigella sativa alone was only observed in the 50:50 ratio group. the 50:50 ratio had a higher inhibitory zone than nigella sativa alone, but less than the calcium hydroxide group. the addition of nigella sativa extract to calcium hydroxide did not increase the inhibitory zone of calcium hydroxide. discussion our study revealed that the calcium hydroxide group has the highest inhibition zone. however, others reported that calcium hydroxide is ineffective against e. faecalis, a prevalent bacterium in persistent infection.6,7 controversial effectiveness of calcium hydroxide against e. faecalis is influenced mainly by the different methods used in the studies. almost all studies that allow direct contact of calcium hydroxide to the bacteria resulted in high antimicrobial efficiency. it was found to be less effective in studies using root canal models or in vivo studies where the complexity of root canal morphology contributes significantly to the limitation of material to be in intimate contact with the bacteria.16–18 this is mainly caused by the high viscosity of calcium hydroxide, limiting its ability to reach small, narrow areas and it has difficulty penetrating the dentine tubules. in this study, the calcium hydroxide was in close contact with the bacteria and could diffuse through the agar plate, resulting in an enhanced inhibitory effect. based on our study, the inhibition zones of all medicament groups were formed from day one and were still stable until day seven, showing that the samples were still active during all evaluations. in contrast, a study on the antibacterial activity of nigella sativa in various germination phases against clinical bacterial strains found that the effectiveness of nigella sativa extracts was highest on the fifth and eleventh days. however, between the fifth and eleventh days, the antibacterial inhibition of nigella sativa extract decreased.15 another study also reported that the components of nigella sativa extract such as tannins, saponins, phenols, and terpenoids concentrations decreased from the third until the seventh day of the observation.17 however, nigella sativa as a single component without the addition of calcium hydroxide had a lower inhibitory zone than those with the addition of calcium hydroxide. thymoquinone is the bioactive constituent of nigella sativa, which is reported to have an antimicrobial effect.8–10 other constituents with the same effect include thymol, monoterpene, and tannin. the mechanism of action of nigella sativa depends on its ability to penetrate through the bacterial membrane and damage the integrity of the bacteria cells. gram-negative bacteria have good permeability defences due to their outer membrane structure. thus, the nigella sativa extract is more effective against grampositive bacteria.19,20 calcium hydroxide can release stable hydroxyl ions for up to 14–21 days, maintaining the environment’s alkalinity. therefore, this intracanal medicament can be used for long-term interappointment dressings, which is in line with the results of this study. however, we did not observe any enhancement of calcium hydroxide’s inhibitory effect when adding nigella sativa extract. the combination did not seem to have a synergic effect from the inhibitory zone evaluation in all ratios evaluated because all combinations had a lower inhibitory effect. also, the 50:50 ratio did not differ significantly compared to calcium hydroxide. looking at the inhibitory effect between the different combination ratios, calcium hydroxide combined with nigella sativa extract in 50:50 ratio indicates a higher inhibitory effect than any other combination ratios or nigella sativa extract alone. compared to different ratios, this combination was the only combined material with a significantly higher inhibitory zone than nigella sativa extract alone. however, compared to calcium hydroxide, this combination had a significantly lower inhibitory zone, confirming that the addition of nigella sativa extract did not improve the inhibitory property of calcium hydroxide. the addition of the extract may impede the liberation of hydroxyl ions. the effectiveness of calcium hydroxide is strongly related to the dissociation of calcium hydroxide to hydroxyl and calcium ions.21,22 in our present study, the addition of nigella sativa extract decreased the inhibitory effect of calcium hydroxide. one possibility is that the addition of extract inhibits the hydroxyl ion release and contributes to a lower ph generated by the calcium hydroxide. this can be further investigated by evaluating the hydroxyl ion release and ph of the samples. the method used in this study could affect penetration ability and contribute to the active component’s success to diffuse through the agar plate. when preparing the samples, we noticed that the combination with a ratio of 50:50 formed a homogenous paste consistency and had better flowability than other ratios, affecting the agar plate’s penetration ability. however, the conditions between in vitro and oral cavity will be different. calcium hydroxide can maintain its ph level for sustained periods in the media compared to infected teeth. it will not be influenced by the buffering effect of dentinal fluid and hydroxyapatite content. this causes the antibacterial efficacy of calcium hydroxide to survive for a long time on the agar media.20 another limitation of this study is the simplified in vitro condition on agar media compared to the complex root canal system in vivo, which will affect the antimicrobial property. therefore, further study will be needed before using the results clinically. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p181–185 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p181-185 185zakaria et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 181–185 the evaluation methods or concentration could also be contributing to the results. an in vitro study reported a moderate ability of thymoquinone to reduce biofilm formation to staphylococcus aureus and staphylococcus epidermidis. in contrast, the thymoquinone concentration needs to be doubled for the same settings to have the same effect on e. faecalis. this showed that the effectiveness of nigella sativa extract as an antibacterial agent depends on its concentration.12 according to a study, the minimum concentration of thymoquinone to inhibit e. faecalis is 256 µg/ml.13 in our present study, we use a total crude extract without evaluating different concentrations. this was based on a previous study reporting that 100% extract had better antibacterial action against enterococcus than 25%, 50% and 75%.11 however, this concentration was used for nigella sativa without combining the extract with calcium hydroxide. this conclusion could be improved by a further study using different concentrations of nigella sativa extract, such as by serial dilution of the extract. dilution may provide a more aqueous vehicle for the dissociation of calcium hydroxide ions. active component evaluation of the extract should also be conducted to confirm the concentration of thymoquinone on the extract, which could also be a contributing factor. nigella sativa extract and its combination with calcium hydroxide can provide antibacterial effects to enterococcus faecalis as a common bacterium in persistent infections. the inhibitory effect of nigella sativa extract and its combination with calcium hydroxide is not more significant than calcium hydroxide itself. in conclusion, nigella sativa extract combined with calcium hydroxide did not improve calcium hydroxide antimicrobial properties against e. faecalis. a combination of 50:50 ratio provides a better inhibitory zone compared to other combinations. acknowledgements the authors are grateful to the faculty of dentistry, universitas jenderal achmad yani (unjani), indonesia, in collaboration with the faculty of dentistry, universitas padjadjaran, indonesia, for supporting this study and also to the lppm unjani. references 1. mohammadi z, dummer pmh. properties and applications of calcium hydroxide in endodontics and dental traumatology. int endod j. 2011; 44(8): 697–730. 2. zakaria mn, takeshita t, shibata y, maeda h, wada n, akamine a, yamashita y. microbial community in persistent apical periodontitis: a 16s rrna gene clone library analysis. int endod j. 2015; 48(8): 717–28. 3. z a rga r n, ma ra sh i m a, ash ra f h, ha kopia n r , beig i p. identification of microorganisms in persistent/secondary endodontic infections with respect to clinical and radiographic findings: bacterial culture and molecular detection. iran j microbiol. 2019; 11(2): 120–8. 4. ba-hattab r, al-jamie m, aldreib h, alessa l, alonazi m. calcium hydroxide in endodontics: an overview. open j stomatol. 2016; 06(12): 274–89. 5. zakaria mn. save the pulp is the essential issues on pulp capping treatment. j dentomaxillofacial sci. 2016; 1(2): 73–6. 6. vasudeva a, sinha dj, tyagi sp, singh nn, garg p, upadhyay d. disinfection of dentinal tubules with 2% chlorhexidine gel, calcium hydroxide and herbal intracanal medicaments against enterococcus faecalis: an in-vitro study. singapore dent j. 2017; 38: 39–44. 7. lima rkp, guerreiro-tanomaru jm, faria-júnior nb, tanomarufilho m. effectiveness of calcium hydroxide-based intracanal medicaments against enterococcus faecalis. int endod j. 2012; 45(4): 311–6. 8. ahmad a, husain a, mujeeb m, khan sa, najmi ak, siddique na, damanhouri za, anwar f. a review on therapeutic potential of nigella sativa: a miracle herb. asian pac j trop biomed. 2013; 3(5): 337–52. 9. mahmoudvand h, sepahvand a, jahanbakhsh s, ezatpour b, ayatollahi mousavi sa. evaluation of antifungal activities of the essential oil and various extracts of nigella sativa and its main component, thymoquinone against pathogenic dermatophyte strains. j mycol med. 2014; 24(4): e155–61. 10. shokri h. a review on the inhibitory potential of nigella sativa against pathogenic and toxigenic fungi. avicenna j phytomedicine. 2016; 6(1): 21–33. 11. nader mi, al-thwaini an, abdul-hassan ia, ali aw. effect of nigella sativa (black seed), salvadora persica (siwak) and aluminum potassium sulphate (alum) aqueous extracts on isolated bacteria from teeth root canal. iraqi j biotech. 2010; 9(1): 99–104. 12. chaieb k, kouidhi b, jrah h, mahdouani k, ba k h rouf a. antibacterial activity of thymoquinone, an active principle of nigella sativa and its potency to prevent bacterial biofilm formation. bmc complement altern med. 2011; 11(1): 29. 13. kouidhi b, zmantar t, jrah h, souiden y, chaieb k, mahdouani k, bakhrouf a. antibacterial and resistance-modifying activities of thymoquinone against oral pathogens. ann clin microbiol antimicrob. 2011; 10: 29. 14. abbaszadegan a, sahebi s, gholami a, delroba a, kiani a, iraji a, abbott pv. time-dependent antibacterial effects of aloe vera and zataria multiflora plant essential oils compared to calcium hydroxide in teeth infected with enterococcus faecalis. j investig clin dent. 2016; 7(1): 93–101. 15. islam mh, ahmad iz, salman mt. antibacterial activity of nigella sativa seed in various germination phases on clinical bacterial strains isolated from human patients. e3 j biotechnol pharm res. 2012; 4(1): 8–13. 16. attia da, farag am, afifi ik, darrag am. antimicrobial effect of different intracanal medications on various microorganisms. tanta dent j. 2015; 12(1): 41–7. 17. dianat o, saedi s, kazem m, alam m. antimicrobial activity of nanoparticle calcium hydroxide against enterococcus faecalis: an in vitro study. iran endod j. 2015; 10(1): 39–43. 18. sangalli j, jardim júnior eg, bueno cre, jacinto rc, sivieri-araújo g, filho jeg, cintra ltâ, dezan junior e. antimicrobial activity of psidium cattleianum associated with calcium hydroxide against enterococcus faecalis and candida albicans: an in vitro study. clin oral investig. 2018; 22(6): 2273–9. 19. farkhondeh t, samarghandian s, shahri amp, samini f. the neuroprotective effects of thymoquinone: a review. dose-response. 2018; 16(2): 1–11. 20. ugur ar, dagi ht, ozturk b, tekin g, findik d. assessment of in vitro antibacterial activity and cytotoxicity effect of nigella sativa oil. pharmacogn mag. 2016; 12(47): s471–4. 21. zakaria mn, siti halimah ir, sidiqa an, artilia i, cahyanto a. antimicrobial activity of calcium hydroxide synthesized from indonesian limestone against enterococcus faecalis. mater sci forum. 2021; 1044: 171–7. 22. guerreiro-tanomaru jm, chula dg, lima rk de p, berbert flvc, tanomaru-filho m. release and diffusion of hydroxyl ion from calcium hydroxide-based medicaments. dent traumatol. 2012; 28(4): 320–3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p181–185 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p181-185 vol 49 no 1 jan-mrt 2016.indd 22 research report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 22–27 the potential of chitosan combined with chicken shank collagen as scaffold on bone defect regeneration process in rattus norvegicus fitria rahmitasari,1 retno pudji rahayu,2 and elly munadziroh3 1department of dental materials, faculty of dentistry, universitas hang tuah 2department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga 3department of dental materials, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: in the field of dentistry, alveolar bone damage can be caused by periodontal disease, traumatic injury due to tooth extraction, cyst enucleation, and tumor surgery. one of the ways to regenerate the bone defect is using graft scaffold. thus, combination of chitosan and collagen can stimulate osteogenesis. purpose: the aim of this study was to examine the potential of chitosan combined with chicken shank collagen on bone defect regeneration process. method: twelve rattus norvegicus were prepared as animal models in this research. a bone defect was intentionally created at both of the right and left femoral bones of the models. next, 24 samples were divided into four groups, namely group 1 using chitosan – collagen scaffold (50:50), group 2 using chitosan collagen-scaffold (80:20), group 3 using chitosan scaffold only, and control group using 3% cmc-na. on 14th day, those animals were sacrificed, and histopathological anatomy examination was conducted to observe osteoclast cells. in addition, immunohistochemistry examination was also performed to observe rankl expressions. result: there was a significant difference in rankl expressions among the groups, except between group 3 using chitosan scaffold only and control group (p value > 0.05). the highest expression of rankl was found in group 1 with chitosan – collagen scaffold (50:50), followed by group 2 with chitosan-collagen scaffold (80:20). moreover, there was also a significant difference in osteoclast generation, except between group 1 using chitosan – collagen scaffold (50:50) and group 2 using chitosan-collagen scaffold (80:20), p value < 0.05; and between group 3 using chitosan scaffold only and control group, p value > 0.05. less osteoclast was found in the groups using chitosan – collagen scaffold (group 1 and group 2). conclusion: combination of chitosan and chicken shank collagen scaffold can improve regeneration process of bone defect in rattus novergicus animals through increasing of rankl expressions, and decreasing of osteoclast. keywords: chitosan scaffold; chicken shank collagen; bone regeneration correspondence: fitria rahmitasari, department of dental materials, faculty of dentistry, universitas hang tuah. jln. arief rahman hakim no. 150 surabaya 60111, indonesia. e-mail: fitri.rahmitasari@gmail.com introduction in the field of dentistry, alveolar bone damage can be caused by periodontal disease, traumatic injury due to tooth extraction, post-cyst enucleation, and post-tumor surgery. biotechnology development actually has already introduced tissue engineering concept by using xenografts to help bone regeneration process since it does not require additional surgery, cause low morbidity, and reduce the risk of disease transmission.2 however, in the last few years, innovation of bone tissue engineering has been developed, as a result, biomaterials are focused on a physicochemically suitable scaffold design for cell attachment, proliferation, differentiation, and specific organ tissue formation.3 scaffold is the term for the synthesis of the extracellular matrix. scaffold also becomes a place of attachment and growth of new cells. thus, scaffold must be made of biodegradable materials that can be metabolized in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p22-26 mailto:fitri.rahmitasari@gmail.com http://e-journal.unair.ac.id/index.php/mkg 2323rahmitasari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 22–26 body and eventually disappear when the new cells have started to grow a lot, be healthy, and survive. some of the polymeric materials, which have been developed in tissue engineering, are chitosan and collagen.4 chitosan is an amino polysacharide (poly-1,4d-glucosamine), widely used as polymers in tissue engineering.5 these polymers have been regarded as a material which has many functional advantages because it has high biocompatibility, biodegradable properties, and low toxicity.6,7 chitosan is also known to stimulate the growth and differentiation of osteoblasts in cell culture. the existence of chitosan alone is not osteoconductive enough, so the ability in new bone formation is still less than optimal. approaches to overcome the weaknesses has been conducted, such as designing a composite by combining the strengths of different materials to minimize the weaknesses of two different materials.8 another organic material playing an important role in tissue engineering is collagen.9 collagen can be found in cartilage, bone, intervertebral disc, blood vessels, tendons, ligaments, skin, and a major component of the extracellular matrix. collagen can also be found in chicken shank. collagen contains rgd (arg-gly-asp) and non-rgd peptides that can bind to cell surface related to integrin, consequently, collagen can facilitate migration, adhesion, proliferation, and differentiation of cells.10 collagen, furthermore, has been used in numerous applications in tissue engineering since it has good biocompatibility and biodegradable properties, as well as low antigenicity.11,12 this material, thus, is considered as a material suitable for repairing damaged tissue and organ.9 collagen actually has many disadvantages, such as rapid degradation time and weak mechanical strength. therefore, a combination of the two polymer materials is needed to produce better material. chitosan and collagen can be combined into a new material to form a unique structure that can improve the mechanical strength and decrease the biodegradation rate of collagenase.13,14 the concentrations used to make a good combination of chitosan and collagen scaffold with good mechanical strength are 50: 5015,16 and 80: 20.17 in addition, bone healing is characterized by a series of cellular and molecular processes, as well as tissue transformation consisted of resorption and formation of hard and soft tissues. bone formation by osteoblasts and bone resorption by osteoclasts regulate skeletal remodeling.18 these processes are fundamental in maintaining bone mass and architecture.19 osteoclasts play an important role in bone resorption both physiologically and pathologically. osteoclasts require the presence of cytokine receptor activator of nuclear factor-κb ligand (rankl) as a key cytokine inducing osteoclastogenesis.20 in this research, two organic natural ingredients were used, namely chitosan combined with chicken shank collagen as a scaffold to determine the potential of those material on the healing process of bone defects in rattus norvegicus rats on day 14 with by observing rankl expressions and osteoclast count. similarly, a previous research also show that bone callus would increase on the 14th day after the administration of bone defect.21 materials and methods this research was an experimental laboratory research. extraction process of chicken shank collagen and manufacture of chitosan and collagen scaffold were performed in unit research services (ulp) of pharmacy faculty, universitas airlangga and the laboratory of human genetics-tropical disease center of universitas airlangga. treatment in experimental animals was conducted at the laboratory of biochemistry faculty of medicine, universitas airlangga. histological and immunohistochemical preparations was carried out in diagnostic center dr. soetomo hospital in surabaya. gel base for the control group was made of carboxy methyl celulose sodium (cmc na) at a concentration of 3%. chitosan used in this research was chitosan with the degree of deacetylation of >75-85%. chitosan gel was made by 200 mg of chitosan powder mixed with 5 ml of 0.1m acetic acid, and then added with 15 ml of 0.1 m naoh. after that, centrifuge at 9000 rpm was carried out to obtain pure chitosan gel. moreover, collagen was obtained from the extraction of chicken shank collagen. chicken shank was cut into small pieces and mashed. smoothed chicken shank was mixed with 250 u/ mg of trypsin enzyme powder, and then incubated at 37° c for 24 hours. glacial acetic acid was added and stored at 4° c for 48 hours. the preparations were mixed using a mixer to form a fiber, and then centrifuged to obtain a supernatant. they were centrifuged twice to obtain a pure supernatant. supernatant obtained was mixed with 0.5m acetic acid to dissolve, and then added with 5% nacl to form bands of collagen. the process was repeated three times. those collagen bands were filtered with filter paper. after that, the collagen was dialysed and centrifuged again. furthermore, manufacture of chitosan and collagen scaffold was conducted by mixing chitosan gel and chicken shank collagen gel homogeneously with ratios of 50:50 and 80:20. the gel was inserted into scaffold mold made of teflon, then frozen at -20° c for 2 hours, and was followed by freeze dry for 24 hours. scaffold then was sterilized using a clean bench uv. in addition, samples used were 12 male wistar rats (rattus norvegicus) weighed 250-300 mg. their left and right femur bone then was defected. thus, the total of samples used in this research was 24 samples. those samples were classified into four groups each of which consisted of six samples. the research groups were control group (3% cmc na), group 1 using chitosan – collagen scaffold (50:50), group 2 using chitosan collagen-scaffold (80:20), and group 3 using chitosan scaffold only. femur bone defect then was made with a diameter and height of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p22-26 http://e-journal.unair.ac.id/index.php/mkg 24 rahmitasari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 22–26 3 mm using a low-speed round bur under anesthesia condition using ketamine and xylazine. the scaffold material then was applied to the defected bone in accordance with the treatment of each group. on day 14, the defected bones in the control and treatment groups were taken under the effects of inhalation anesthetics. those bones were processed for the manufacture of histological preparations with hematoxylin eosin staining (he) to examine the number of osteoclasts. immunohistochemical examination then was carried out using rankl antibody to observe rankl expressions. finally, osteoclast count and rankl expressions were measured manually on five of the visual field examination using a light microscope with a magnification of 400x. results the results of this research showed the average values of osteoclast count and rankl expressions in the control group, the treatment group using chitosan scaffold only, the treatment group using chitosan-collagen scaffold with the ratio of 80:20, and the treatment group using chitosancollagen scaffold with the ratio of 50:50 during the healing process of those femur bone defect as shown in table 1. the most rankl expressed by osteoblasts was found in the group using chitosan-collagen scaffold with the ratio of 50:50. meanwhile, the least rankl expressed by osteoblasts was in the control group. the highest number of osteoclasts was widely obtained in the control group, while the least number of osteoclasts was in the group using chitosan-collagen scaffold with the ratio of 50:50. the results of histopathological examination then were obtained by observing the number of osteoclasts in the control and treatment groups using a light microscope with a magnification of 400x as seen in figure 1. the fewest osteoclasts were found in the group using of chitosan-collagen scaffold with the ratio of 50:50 (d), following the group using of chitosan-collagen scaffold with the ratio of 80:20 (c), the group using chitosan (b), and the control group (a) (figure 1). rankl expressions in each treatment group and the control can be seen in figure 2. the illustration photo above show the results of immunohistochemical examination that the combination of chitosan and chicken shank collagen scaffold can increase rankl expressions during the healing process of the fermur bone defect. however, the combination of chitosan-collagen scaffold with the ratio of 50:50 was more effective in increasing rankl expression than with the ratio of 80:20. table 1. the average values of osteoclast count and rankl expressions group number of samples average values (mean) osteoclast count rankl expressions control 6 4.8333 5.8333 chitosan scaffold 6 4.6667 8.1667 chitosan scaffold with the ratio of 80:20 6 2.1667 17.5000 chitosan scaffold with the ratio of 50:50 6 1.3333 28.0000 figure 1. osteoclast cells in each group. a) the control group; b) the group using chitosan scaffold only; c) the group using chitosan collagen-scaffold (80:20); d) the group using chitosan–collagen scaffold (50:50). figure 2. rankl expressions in each group. a) the control group; b) the group using chitosan scaffold only; c) the group using chitosan collagen-scaffold (80:20); d) the group using chitosan – collagen scaffold (50:50). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p22-26 http://e-journal.unair.ac.id/index.php/mkg 2525rahmitasari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 22–26 the research data of osteoclast count and rankl expressions in each group were then tested using one way anova test. before the one way anova test conducted, the results of kolmogorov smirnov test showed that the distribution of the data in this research was normal (p> 0.05). based on one way anova test, the significance value obtained was 0.000 (p<0.05). it means that there was a significant difference in osteoclast count and rankl expressions between the control group, the group using chitosan scaffold only, the group using chitosan collagenscaffold (80:20), and the group using chitosan–collagen scaffold (50:50). thus, post hoc test and tukey hsd test were performed to know differences between one group and another group. there was a significant difference in the number of osteoclast cells between one group and another group. nevertheless, there was no significant difference in the number of osteoclast cells (p>0.05) between the group using chitosan scaffold only and the control group, as well as between the group using chitosan-collagen scaffold with the ratio of 50:50 and the group using chitosan-collagen scaffold with the ratio of 80:20. in addition, there were significant differences in rankl expressions between one group and another group. however, there was no significant difference in rankl expressions (p>0.05) between the group using chitosan scaffold and the control group (p> 0.05). discussion on the 14th day, histopathological anatomy observation was conducted to examine the number of osteoclasts. osteoclasts play an important role in bone resorption, both in physiological and pathological conditions. osteoclast are derived from myeloid cells and macrophages, producing many cytokines and regulating macrophages and dendritic function. osteoclast, moreover, are located on the surface of endosteal cells in the havers channels along cortical and trabecular bones.22 osteoclasts are the largest, multinucleated, irregularly shaped cells with pale cytoplasm color.23 the least average number of osteoclasts was found in the group using chitosan – collagen scaffold (50:50), following the group using chitosan–collagen scaffold (80:20), and the group using chitosan scaffold only. the highest average number of osteoclasts was found in the control group. osteoclasts, furthermore, are known to contribute to bone resorption. as a result, the least number of osteoclasts was found in the groups using chitosan–collagen scaffold. it may indicate that bone formation is stimulated dominantly by osteoblasts during the healing process of the femur bone defect in those experimental animals. rgd (arg-glyasp) contained in the collagen can inhibit the expression of rank by blocking the integrin, αvβ3, so possibility of rankl to bind to rank is lower and inhibits the formation of mature osteoclasts.24 the statistical results, however, showed that there were significant differences between each groups, except between the group using chitosan-collagen scaffold with the ratio of 50:50 and the group using chitosan-collagen scaffold with the ratio of 80:20, as well as between the group using chitosan scaffold only and the control group (p>0.05). this is due to the fact that there was not much different in the average pore size of the scaffold between in the the group using chitosan-collagen scaffold with the ratio of 50:50 and in the group using chitosan-collagen scaffold with the ratio of 80:20 about 183 m and 123 m. thus, there was no significant effect in reducing the number of osteoclasts useful in the process of bone healing. the greater porosity size actually can trigger a better vascularization so that the healing process can be more optimal.17 both the treatment group using chitosan scaffold only and the control group, consequently, did not produce statistically significant differences in the number of osteoclasts because the chitosan alone is not enough osteoconductive. therefore, the bone healing process was less than optimal. in this case, the ability to produce osteoclasts was not much different between the control group and the treatment group using 3% cmc-na on the 14th day. the expressions of rankl by osteoblasts, moreover, were more visible in the treatment groups using chitosancollagen scaffold combination than the group using chitosan scaffold only and the control group due to a possible increase in the number of osteoblasts and the expression of osteoprotegerin (opg), playing important roles in bone formation. in other words, the combination of chitosan-collagen scaffold can stimulate the occurrence of osteogenesis by facilitating adhesion, proliferation, and differentiation of cells. collagen can also enhance osteoblast differentiation and increase bone formation by activating genes runx-2 to stimulate pre-osteoblasts into osteoblas.25 therefore, it can be said that the greater level opg expressions than the level of rankl expression may trigger bone formation. rankl is a key cytokine in stimulating osteoclastogenesis.19 rankl can stimulate differentiation, maintain viability, and activate mature osteoclasts. all those functions can be run because of the interaction between rankl and receptor activator of nuclear factorκb (rank). rank is a transmembrane protein expressed by the pre-osteoclasts. in the bone healing process, there is a protein, opg, which can inhibit osteoclast development. opg functions as a decoy receptor by binding to rankl, resulting in inhibiting rank signaling.26 besides, since rankl is expressed by osteoblasts, the high number of osteoblasts may also contribute to the high expressions of rankl. according to a research conducted by wang, rankl and opg levels can significantly increase immediately after a fracture in a bone fracture until the 4th week compared to the control group of healthy bone.27 although both increase, the presence of rankl is less than opg, thus showing that the number of osteoblasts will increase over that period and bone formation is more dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p22-26 http://e-journal.unair.ac.id/index.php/mkg 26 rahmitasari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 22–26 dominant in playing the role during the bone healing process. finally, the results of this research need to be analyzed further. further researches should reveal the location of rank and opg expressions since rank/ rankl/ opg is a signaling system that is responsive to control osteoclastogenesis. properties of chitosan itself have good mechanical strength less than in combination with other polymers so that chitosan scaffold is less optimal in facilitating osteogenesis. in this case, the ability to express rankl in bone defects was not much different from the control group on the 14th day. it can be concluded that the combination of chitosan and collagen scaffold can improve the healing process of bone defect in wistar rats through an increase in rankl expressions and a decrease in the number of osteoclasts. references 1. fokkema sj. the central role of monocytes in systemic immune effects induced by the chronic periodontal infection. wageningen: ponsen & looijen bv; 2002. p. 104-16. 2. mehta m, schmidt-bleek k, duda gn, mooney dj. biomaterial delivery of morphogens to mimic the natural healing cascade in bone. adv drug deliv rev 2012; 64(12): 1257-76. 3. niu x, fan y, liu x, li x, li p, wang j, sha z, feng q. repair of bone defect in femoral condyle using microencapsulated chitosan, nanohydroxyapatite/collagen and poly (l-lactide)-based microsherescaffold delivery system. artificial organs 2011; 35(7):119. 4. chen g, ushida t, tateishi t. scaffold design for tissue engineering. macromol biosci 2002; 2(2): 67-77. 5. amaral if, cordeiro al, sampaio p, barbosa ma. attachment, spreading, and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation. j biomaterial science polymer 2007; 18(4): 469-85. 6. ven katesan j, k im s. chitosan composites for bone tissue engineering–an overview. mar drugs 2010; 8(8): 2252-66. 7. sonia ta, sharma cp. chitosan and its derivatives for drug delivery perspective. adv polym sci 2011; 243: 23-54. 8. ariani md, matsuura a, hirata i, kubo t, kato k, akagawa y. new development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. dental materials journal 2013; 32(2): 317-8. 9. cui k, zhu y, wang xh, feng ql, cui fz. a porous scaffold from bone-like powder loaded in a collagen-chitosan matrix. journal of bioactive and compatible polymers 2004; 19(1): 17-31. 10. k ruger te, miller ah, wang j. collagen scaffolds in bone sialoprotein-mediated bone regeneration. scientificworld journal 2013; 2013: 812718. 11. ma l, gao c, mao z, zhou j, shen j, hu x, han c. collagen/ chitosan porous scaffold s with improved biostability for skin tissue engineering. biomaterials 2003; 24(26): 4833-41. 12. osidak eo, osidak ms, ak hmanova ma, domogatskii sp. collagen-a biomaterial for delivery of growth factors and tissue regeneration. russian journal of general chemistry 2014; 84(2): 368-9. 13. arpornmaeklong p, suwatwirote n, pripatnanont p, oungbho k. growth and differentiation of mouse osteoblasts on chitosancollagen sponges. int.j.oral maxillofac 2007; 36(4): 328-9. 14. yoo jh, lee mc, lee je, jeon kc, kim ym, jung my, ahn hj, seong sc, choi sj, lee jh. evaluation of chondrogenesis in collagen/chitosan/ glycosaminoglican scaffolds for cartilage tissue engineering. j korean orthop res soc 2005; 8(1): 28-40. 15. tangsadthakun c, kanokpanont s, sanchavanakit n, banaprasert t, damrongsakkul s. properties of collagen/chitosan scaffold s for skin tissue engineering. journal of metals, materials, and minerals 2006; (1): 38. 16. sionkowska a, kaczmarek b, stalinska j, osyczka am. biological properties of chitosan/collagen composites. key engineering materials 2014; 587: 205. 17. suryati, agusnar h, gea s, ilyas s. nonporous chitosan/collagen scaffold for skin tissue engineering. proceeding of the 2nd annual international conference syiah kuala university 2012; 2(2): 376. 18. bab ia, sela jj. cellular and molecular aspects of bone repair. principles of bone regeneration jerusalem: springer science + business media; 2012. p. 11-28. 19. nakashima t, hayashi m, takayanagi h. new insights into osteoclastogenic signaling mechanisms. trends in endocrinology and metabolism 2012; 23(11): 582. 20. clarke b. normal bone anatomy and physiology. clin j am soc nephrol 2008; 3 suppl 3: s131-9. 21. kon t, cho tj, aizawa t, yamazaki m, nooh n, graves d, gerstenfeld lc, einhorn ta. expression of osteoprotegerin, receptor activator of nf-kappab ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. j bone miner res 2001; 16(6): 1004-14. 22. lorenzo j, horowitz m, choi y, takayagi h. osteoimmunology. 1st edition. uk: academic press; 2011. p. 10-235. 23. baron r. anatomy and ultrastructure of bone histogenesis, growth, and remodelling. in: arnold a, editor. disease of bone and mineral metabolism. mdtext.com, south dartmouth ma. web 2010. 24. mochizuki a, takami m, miyamoto y, nakamaki t, tomoyasu s, kadono y, tanaka s, inoue t, kamijo r. cell adhesion signaling regulates rank expression in osteoclast precusors. plos one 2012; 7(11): e48795. 25. uchihashi k, aoki s, matsunobu a, toda s. osteoblast migration into type i collagen gel and differentiation to osteocyte-like cells within a self-produced mineralized matrix: a novel system for analyzing differentiation from osteoblast to osteocyte. bone 2013; 52(1): 102-10. 26. pérez-sayáns m, somoza-martín jm, barros-angueira f, rey jm, garcía-garcía a. rank/rankl/opg role in distraction osteogenesis. oral surg oral med oral pathol oral radiol endod 2010; 109(5): 679-86. 27. wang xf, zhang yk, yu zs, zhou jl. the role of the serum rankl/opg ratio in the healing of intertrochanteric fractures in elderly patients. mol med rep 2013; 7(4): 1169-72. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p22-26 http://e-journal.unair.ac.id/index.php/mkg 24 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 24–29 research report effect of various inductions of sleep deprivation stress on proinflammatory cytokine levels in gingival crevicular fluids of white male wistar strain rats (rattus novergicus) 1 2 3pratiwi nur widyaningsih, fitranto arjadi and erlina sih mahanani 1postgraduate student of biomedical study program, faculty of medicine, universitas jenderal soedirman, purwokerto indonesia 2department of anatomy, faculty of medicine, universitas jenderal soedirman, purwokerto indonesia 3department of dental biomedic, faculty of medicine and health science, universitas muhammadiyah yogyakarta, yogyakarta indonesia abstract background: stress that is induced by sleep deprivation can modulate the damage of periodontal tissue by elevating the levels of proinflammatory cytokines (i.e. il-1β and tnf-α). the effects of sleep deprivation can be resolved with sleep recovery. gingival crevicular fluid (gcf) is fluid in sulcular gingiva which acts as an oral biomarker for evaluating periodontal abnormalities. purpose: the aim of this study was to determine the effect of various induction methods of sleep deprivation stress on cytokine levels in gcf of white male wistar strain rats (rattus novergicus). methods: the study method was true experimental with a posttest-only control group design. thirty male wistar rats were randomly divided into five groups: paradoxical sleep deprivation (psd), total sleep deprivation (tsd), partial sleep deprivation with sleep recovery for five days (psd+sr), total sleep deprivation with sleep recovery for five days (tsd+sr) and a healthy control group. data were analysed via one-way anova to determine differences between groups. result: the results showed the highest level of il-1β and tnf-α was found in the psd group. one-way anova analysis showed significant differences (p<0,05) of il-1β level between psd and control groups, psd and psd+sr groups and psd and tsd+sr groups; in contrast, the analysis of tnf-α levels showed significant differences (p<0,05) between psd group to control group, psd to psd+sr group and tsd to tsd+sr group. conclusions: there is an effect of various induction methods of sleep deprivation stress on proinflammatory cytokines (il-1β and tnf-α). keywords: paradoxical sleep deprivation; proinflamatory cytokines; sleep deprivation; total sleep deprivation; sleep recovery correspondence: pratiwi nur widyaningsih, postgraduate student of biomedical study program, faculty of medicine, universitas jenderal soedirman. jl. dr soeparno purwokerto, banyumas, indonesia 53122. e-mail: pratiwinurwidyaningsih@gmail.com introduction stress is a common problem in modern life, causing a significant decrease in the number of sleep hours among adults and children. epidemiological data show that sleep disorders and short duration of sleep have negative impacts on human physical health.1 sleep deprivation causes an increase in lymphocyte activation, so that it produces more proinflammatory cytokines (il-1, il-6, il-17 and tnf-α), thereby increasing the risk of inflammation or damage to tissue, including periodontal tissue. in addition, due to an imbalance between the formation and elimination of reactive oxygen species (ros), sleep deprivation is also related to the mechanism of oxidative stress. however, the impact of sleep deprivation can be corrected by sleep recovery, which results in a decrease in lipid peroxidase and free radical productionm, thus restoring antioxidant activity in inhibiting oxidative stress.2–5 proinflammatory cytokines play an important role in inflammation and bone resorption; therefore, they are important parameters in periodontal research. the presence of il-1β in gingival crevicular fluid (gcf) can stimulate the occurrence of alveolar bone resorption. tnf-α produced by macrophages and lymphocytes has the same effect as il-1β. tumor necrosis factor-α (tnf-α) can stimulate cell proliferation and differentiation such as osteoblasts and osteclasts. the ability of tnf-α to stimulate the production dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 mailto:pratiwinurwidyaningsih@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 25widyaningsih, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 24–29 of enzymes that damage the matrix and bone recession activities plays an important role in damage caused by periodontal disease.6,7 gingival crevicular fluid is a product of cells (transudates) found in the gingival sulcus originating from post-capillary venules from the gingival plexus, and it can be influenced by an inflammatory response in the area around the gingival sulcus. cytokines detected in gcf can be used as oral biomarkers in evaluating periodontal disease conditions or in periodontal treatment results.7 research on the effect of various induction methods of sleep deprivation stress on proinflammatory cytokine levels, il-1β and tnf-α levels in gcf of white male wistar strain rats (rattus norvegicus) has never been done before; therefore, the authors are interested in exploring this topic. materials and methods -this study used a true experimental approach with a post test-only control group design for white male wistar strain rats (rattus norvegicus) aged 3–4 months with a weight of 200–300 grams. the rates were given various inductions of sleep deprivation stress with paradoxical model sleep deprivation (psd) with 20 hours given stress and four hours rest for five days; total sleep deprivation (tsd) with 24 hours given stress and no rest for five days; paradoxical sleep deprivation was continued with sleep recovery for five days (psd + sr); and total sleep deprivation was continued with sleep recovery for five days (tsd + sr). all stress models used a single platform method (spm) tank measuring 23 x 23 x 35 cm where one rat in a flowerpot was equipped with muscle atonia which gave a shock effect automatically every 10 minutes in experimental animals.8 the subjects of this study were 30 white male wistar strain rats (rattus norvegicus) aged 3–4 months with a weight of 200–300 grams obtained from the pharmacology and therapy department, faculty of medicine, universitas gadjah mada. all experiments conducted in this study have been approved by the ethics commission of the faculty of medicine, universitas jenderal soedirman no. ref. 4704/ kepk/x/2018. the experimental animals were randomly divided into five groups: psd (ki) group, tsd group (kii), psd + sr group (kiii), tsd + sr group (kiv), and healthy control group (kv); each group consisted of six white rats. all experimental animal samples were acclimatised for seven days before the induction of various sleep deprivation methods. after acclimatisation, groups i and iii were given psd stress treatment for 120 hours, and groups ii and iv were given tsd stress treatment for 120 hours, while group v was not treated with stress. groups i, ii and iii then returned to their original state while groups iv and v continued with sleep recovery for 120 hours and then returned to their original conditions. after the induction of stress deprivation was completed, gcf was taken using filter paper with a size of 2.55 mm x 14.19 mm with a thickness of 0.16 mm with the intracrevicular absorption method. previously, filtered paper was carried out by gas ethyleneoxide (eog) sterilization to prevent bacterial or fungal contamination that could affect variables. the gcf retrieval method was carried out by controlling saliva with cotton in the area around the anterior gingival of the mandibula, inserting filter paper into the gingival sulcus at a depth of 1 mm in the anterior gingival of the mandibula of the white male wistar strain rats for 30 seconds. gcf retrieval was carried out after the treatment was completed, and the extraction time was adjusted to the circadian rhythm of the rats at 07.00–10.00. filter paper containing gcf was immediately inserted into an eppendorf tube containing 200µl of phosphate buffered saline (pbs) (gibco®, usa) with a ph of 7.4; it was then homogenised using a vortex mixer. gcf was stored at -20oc or allowed to melt at 2–8oc, and then centrifuged from 2,000 to 3000 rpm for 20 seconds. measurement of il-1β and tnf-α levels was carried out using the enzyme-linked immunosorbent assay (elisa) sandwich method which was read on an elisa reader with an absorbance wave of 450 nm. data were tabulated and tested for normality by the saphiro-wilk and levene test for its homogeneous. data were then analysed by one-way anova to determine differences between groups. results the results of the study on the measurement of il-1β levels revealed mean levels of il-1β of experimental animals in each group described in figure 1. figure 1 shows that the highest mean of il-1β level experimental animals is in the psd group with a mean of 453.05 ± 48.81 pg/l. the lowest mean of il-1β levels was found in the psd + sr group: 380.40 ± 24.65 pg/l. the results of the shapiro-wilk and levene statistical analysis showed that the data of each group were normally distributed and homogeneous. the statistical test continued using the parametric one-way anova test, and the result of the significance obtained is p<0.05. these results indicate the effect of sleep deprivation treatment on il-1β levels. a post-hoc lsd test was carried out to determine if there are significant differences between each treatment in the sample groups. table 1 shows groups with significant results (p<0.05), a significant control group for the psd group and vice versa. the psd group was significant for the psd + sr group and vice versa. the tsd + sr group is significant for the psd group and vice versa. in contrast, the tsd group did not show significant results (p>0.05) for all groups. the results of the study on the measurement of tnf-α levels obtained mean levels of tnf-α in each group as described in figure 2. figure 2 shows that the highest mean of tnf-α level in experimental animals was in the psd group, with a mean of 377.89 ± 69.00 ng/ml. the lowest dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 26 widyaningsih, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 24–29 0,00 50,00 100,00 150,00 200,00 250,00 300,00 350,00 400,00 450,00 500,00 psd tsd psd+sr tsd+sr control m ea n of il -1 β (p g/ l) le ve ls 453.05 ±48.81 380.40±24.65 407.96±18.44 380.40±24.65 418.21±30.58 figure 1. mean of il-1β levels in experimental animals. 0,00 50,00 100,00 150,00 200,00 250,00 300,00 350,00 400,00 psd tsd psd+sr tsd+sr control m ea n of t n fα (n g/ m l) le ve ls 273.68 ±41.27 337.89 ±69.00 311.53 ±65.86 217.31 ±26.40 305.06 ±59.31 figure 2. mean bar chart of tnf-α levels in experimental animals. table 1. post-hoc lsd test, mean il-1β levels in sleep deprivation stress treatment in each group groups p-value control psd tsd psd+sr tsd+sr control 0.046* 0.651 0.231 0.354 psd 0.134 0.004* 0.007* tsd 0.120 0.192 psd+sr 0.788 tsd+sr notes: * p<0.05 table 2. post-hoc lsd test, mean of tnf-α levels in sleep deprivation stress treatment in each group groups p-value control psd tsd psd+sr tsd+sr control 0.317 0.842 0.016* 0.339 psd 0.420 0.002* 0.057 tsd 0.010* 0.251 psd+sr 0.108 tsd+sr notes: *p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 27widyaningsih, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 24–29 mean of tnf-α levels was found in the psd + sr group: 217.31 ± 26.40 ng/ml. the results of the shapiro-wilk and levene statistical analysis showed that the data were normally distributed and homogeneous. the statistical test continued using the parametric one-way anova test, and the results of significance obtained was p<0.05. these results indicate that there is an effect of sleep deprivation treatment on tnf-α levels. post-hoc lsd tests were carried out to determine if there are significant differences between each treatment in the sample group. table 2 shows groups with significant results (p<0.05), which is the significant control group for the psd group and vice versa. the psd group was significant for the psd + sr group and vice versa. the tsd group was significant for the psd + sr group and vice versa. in contrast, the tsd + sr group did not show significant results (p> 0.05) for all groups. discussion based on the results of the study, the mean of il-1β level was highest in the psd group, while the lowest mean of il-1β level was found in the psd + sr group. this indicates that sleep deprivation stress has an impact on il-1β levels, and the administration of sleep recovery after sleep deprivation treatment can reduce il-1β levels. when sleep deprivation occurs, endogenous and endotoxin factors will induce the production of proinflammatory cytokines, including il-1, tnf-α, and nuclear factor kappa b (nfκb). furthermore, proinflammatory cytokines along with neurochemicals play an important role in the regulation of sleep in the non-rapid eye movement sleep (nrem) phase, resulting in an increase in proinflammatory cytokines in the nrem phase and will decrease during the rapid eye movement sleep (rem) phase. this is in line with the results in this study, wherein the mean of il-1β level of the psd group was significant (p <0.05) for the control group who were not given stress exposure; hence, in the control group, there was no increase in il-1β levels.9–11 sleep deprivation can be divided into acute sleep deprivation and chronic sleep deprivation. acute sleep deprivation is a long period of an awake condition, whereas chronic sleep deprivation is an accumulation of sleep deprivation that occurs over several days. according to landolt et al. (2014),9 partial sleep deprivation (psd) is included in acute deprivation (short-term), whereas total sleep deprivation (tsd) is included in chronic sleep deprivation (long-term). stress exposure can have an effect on the immune response, where acute stress deprivation sleep can induce innate immunity, antigen presentation, antibodies and cytokine production.12,13 il-1β levels in the psd and tsd groups in this study did not show a significant difference (p> 0.05), although il-1β levels in the psd group were higher than the il-1β levels in the tsd group. this shows that sleep deprivation stress in the psd group can produce more il-1β than the tsd group. this condition is caused by the immune response in the acute phase of sleep deprivation activating toll-like receptors (tlr), which will trigger gene transcription in nf-κb and increase production of il-1β and other proinflammatory cytokines, so that il-1β levels in the psd group are higher than in tsd groups. in addition, interleukin 1 (il-1α and il-1β) is a key mediator in the acute phase response to infected hosts.9,14 some studies have suggested that sleep can play an anti-stress role through the mechanism of inhibiting the hpa axis. sleep recovery can improve the impact of sleep deprivation by returning the hpa axis interaction to normal. sleep deprivation conditions can activate the hpa axis, playing an important role in the occurrence of stress, inducing an increase in the production of il-1β, and returning to normal after sleep recovery.15–17 il-1β levels in this study had significant results (p<0.05) in the psd group against the psd + sr group. the il1β levels in the psd + sr group decreased significantly when compared with the psd group. this shows that the administration of sleep recovery in the psd + sr group succeeded in reducing levels of il-1β. sleep recovery can improve the effects of sleep deprivation by returning the hpa axis interaction to normal, reducing the hormone cortisol so that it results in a decrease of crh. sleep deprivation will stimulate the activation of the hpa axis and induce the crh hormone, which is the main regulator in the wake condition, inhibiting the non-rem (nrem) and rem sleep phase.14,18,19 this study also showed significant results (p<0.05) in tsd + sr groups for the psd group, as the mean of il-1β levels in tsd + sr group was lower than the psd group. this suggests that the administration of sleep recovery exposure in the tsd + sr group also reduced il-1β levels. the psd group was included in the acute stress category so that levels of il-1β produced were higher when compared with the tsd + sr group, which was included in the category of chronic stress to be then given additional treatment, namely sleep recovery. the administration of sleep recovery to psd and tsd treatment in the psd + sr and tsd + sr groups in this study had almost the same results, which showed a nonsignificant difference (p>0.05), although the il-1β levels in the psd + group sr show lower results than the tsd + sr group. it demonstrated that administering sleep recovery may successfully reduce levels of il-1β after treatment of psd and tsd. sleep recovery can restore body performance, especially in the rem cycle recovery, where rem sleep deprivation for more than 72 hours can cause increased levels of il-1β, il-6, il-17a, and tnf-α in rats.16,17 changes in tnf-α levels due to stress deprivation sleep have been widely demonstrated in previous studies. based on the results of this study, sleep deprivation stress has an impact on the levels of tnf-α, where the highest levels of tnf-α were highest in the psd group, and the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 28 widyaningsih, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 24–29 lowest levels of tnf-α were found in the psd + sr group. sleep deprivation stress plays a role in modulating cytokine production, one of which is tnf-α. the body’s response to stress can change the homeostasis system because stress will induce activation of the hpa axis and sympathetic nervous system. the activation of the hpa axis will cause glucocorticoid production in the adrenal cortex. stress exposure that occurs continuously has an effect on adrenaline response quickly, so glucocorticoids experience a rapid increase in exposure to acute stress, whereas, in chronic stress, the body can control these conditions so that glucocorticoid production decreases and can be lost. in chronic sleep deprivation, there is an increase in cortisol levels which causes desensitisation of glucocorticoid receptors, resulting in a decrease in glucocoticoid levels.5,14,20 this study showed a higher increase in tnf-α levels in the psd group than in the tsd group. however, the results of the post-hoc lsd analysis did not show a significant difference (p>0.05) between the two groups. this shows an increase in tnf-α levels in the psd group which can be seen when compared with the tsd group. as in the il1β pattern, stress exposure psd treatment is included in the category of acute stress induction, where the immune response in the acute phase of sleep deprivation will activate receptors such as toll-like receptors (tlr) that will transcript genes at nf-κb and increase production of tnf-α and other proinflammatory cytokines.9,21 similar to il-1β, tnf-α plays a role in the sleep regulatory mechanism in the hypothalamus and locus coeruleus (lc) in the nrem phase. the administration of sleep recovery treatment in experimental animals can reduce the activity of the hpa axis so that it can reduce tnf-α levels. this study had significant results (p <0.05), namely in the psd group for the psd + sr group, where tnf-α levels in the psd + sr group decreased after being given a sleep recovery treatment. this shows that exposure to sleep deprivation stress with the treatment of psd + sr has been successful in reducing tnf-α levels. sleep recovery can improve the effects of sleep deprivation by returning the interaction from the hpa axis to normal. a decrease in activation of the hpa axis can reduce the production of tnf-α.16,22,23 the administration of sleep recovery in the psd + sr group in this study was also significant (p<0.05) for the tsd group. the psd + sr group had lower tnf-α results when compared with the tsd group. this happens because the administration of sleep recovery in the sr psd + group can reduce levels of tnf-α. sleep can reduce substances that can stimulate tnf-α production and can inhibit the corticotropin-releasing hormone (crh). reduction in crh secretion will reduce activation of the hpa axis so that tnf-α levels decrease.15 the psd + sr and tsd + sr groups showed almost the same results so that there was no significant difference (p>0.05). tnf-α levels in the tsd + sr group were higher when compared to the psd + sr group. this happens because the psd + sr group still has time to sleep every day and also gets additional time for sleep recovery so that the levels of tnf-α in the psd + sr group are lower than the tsd + sr group. subjects who were given tsd for 24 hours and 48 hours full, and then given a sleep recovery treatment for 24 hours, will recover from the sleep deprivation by 72% for tsd for 24 hours and have 42% for tsd for 48 hours.24 in conclusion, the induction of various stress deprivation stress methods has an impact on the levels of proinflammatory cytokines namely il-1β and tnf-α in gcf white male wistar strain rats (rattus norvegicus). the administration of sleep recovery after the induction of sleep deprivation stress has an effect on levels of il-1β and tnf-α in gcf white male wistar strain rats (rattus norvegicus). acknowledgements we would like to thank to research and community service institute of jenderal soedirman university for the research funding from improvement fund research grant batch iii in 2018, research laboratory, pharmacology laboratory, and experimental animal laboratory of faculty of medicine, jenderal soedirman university for the facilites provided. references 1. cohen s, doyle wj, alper cm, janicki-deverts d, turner rb. sleep habits and susceptibility to the common cold. arch intern med. 2009; 169(1): 62–7. 2. frey dj, fleshner m, wright kp. the effects of 40 hours of total sleep deprivation on inflammatory markers in healthy young adults. brain behav immun. 2007; 21(8): 1050–7. 3. linden gj, lyons a, scannapieco fa. periodontal systemic associations: review of the evidence. j clin periodontol. 2013; 40(suppl. 14): s8-19. 4. periasamy s, hsu dz, fu yh, liu my. sleep deprivation-induced multi-organ injury: role of oxidative stress and inflammation. excli j. 2015; 14: 672–8. 5. sgoifo a, carnevali l, grippo aj. the socially stressed heart. insights from studies in rodents. neurosci biobehav rev. 2014; 39: 51–60. 6. cetinkaya b, guzeldemir e, ogus e, bulut s. proinflammatory and anti-inflammatory cytokines in gingival crevicular fluid and serum of patients with rheumatoid arthritis and patients with chronic periodontitis. j periodontol. 2013; 84(1): 84–93. 7. buduneli n, buduneli e, çetin ey, kirilmaz l, ktkçler n. clinical findings and gingival crevicular f luid prostaglandin e2 and interleukin-1-beta levels following initial periodontal treatment and short-term meloxicam administration. expert opin pharmacother. 2010; 11(11): 1805–12. 8. ruiz fs, andersen ml, martins rcs, zager a, lopes jd, tufik s. immune alterations after selective rapid eye movement or total sleep deprivation in healthy male volunteers. innate immun. 2010; 18(1): 44–54. 9. landolt h-p, sousek a, holst sc. effects of acute and chronic sleep deprivation. zurich open repos arch. 2014; : 49–61. 10. fang j, wang y, krueger jm. effects of interleukin-1 beta on sleep are mediated by the type i receptor. am j physiol. 1998; 274(3): r655-60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 29widyaningsih, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 24–29 11. izyumov ds, domnina l v, nepryakhina ok, avetisyan a v, golyshev sa, ivanova oy, korotetskaya m v, lyamzaev kg, pletjushkina oy, popova en, chernyak b v. mitochondria as source of reactive oxygen species under oxidative stress. study with novel mitochondria-targeted antioxidants--the “skulachev-ion” derivatives. biochemistry (mosc). 2010; 75(2): 123–9. 12. olayaki la, sulaiman so, anoba nb. vitamin c prevents cleep deprivation-induced elevation in cortisol and lipid peroxidation in the rat plasma. niger j physiol sci. 2015; 30(1–2): 5–9. 13. himmerich h, fischer j, bauer k, kirkby kc, sack u, krügel u. stress-induced cytokine changes in rats. eur cytokine netw. 2013; 24(2): 97–103. 14. hurtado-alvarado g, pavón l, castillo-garcía sa, hernández me, domínguez-salazar e, velázquez-moctezuma j, gómezgonzález b. sleep loss as a factor to induce cellular and molecular inflammatory variations. clin dev immunol. 2013; 2013: 1–14. 15. faraut b, boudjeltia kz, dyzma m, rousseau a, david e, stenuit p, franck t, antwerpen p van, vanhaeverbeek m, kerkhofs m. benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction. brain behav immun. 2011; 25(1): 16–24. 16. el-aziz eaa, mostafa dg. impact of sleep deprivation and sleep recovery on reproductive hormones and testicular oxidative stress in adult male rats. al-azhar assiut med j. 2012; 10(3): 160–88. 17. voderholzer u, fiebich bl, dersch r, feige b, piosczyk h, kopasz m, riemann d, lieb k. effects of sleep deprivation on nocturnal cytokine concentrations in depressed patients and healthy control subjects. j neuropsychiatry clin neurosci. 2012; 24(3): 354–66. 18. süer c, dolu n, artis as, sahin l, yilmaz a, cetin a. the effects of long-term sleep deprivation on the long-term potentiation in the dentate gyrus and brain oxidation status in rats. neurosci res. 2011; 70(1): 71–7. 19. lima ama, de bruin vms, rios erv, de bruin pfc. differential effects of paradoxical sleep deprivation on memory and oxidative stress. naunyn schmiedebergs arch pharmacol. 2014; 387(5): 399–406. 20. dhabhar fs, malarkey wb, neri e, mcewen bs. stress-induced redistribution of immune cells-from barracks to boulevards to battlefields: a tale of three hormones curt richter award winner. psychoneuroendocrinology. 2012; 37(9): 1345–68. 21. han k, park jb. evaluation of the association between sleep duration and tooth loss among korean adults: data from the korean national health and nutrition examination survey (knhanes 2012-2014). bmj open. 2018; 8(5): 1–8. 22. davis cj, zielinski mr, dunbrasky d, taishi p, dinarello ca, krueger jm. interleukin 37 expression in mice alters sleep responses to inflammatory agents and influenza virus infection. neurobiol sleep circadian rhythm. 2017; 3: 1–9. 23. irwin mr, olmstead r, carroll je. sleep disturbance, sleep duration, and inflammation: a systematic review and meta-analysis of cohort studies and experimental sleep deprivation. biol psychiatry. 2016; 80(1): 40–52. 24. mathangi dc, shyamala r, subhashini as. effect of rem sleep deprivation on the antioxidant status in the brain of wistar rats. ann neurosci. 2012; 19(4): 161–4. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p24–29 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p24-29 subject index volume 43 1% carrageenan, 113 acrylic resin, 40 plate, 201 aesthetics, 157 aggressive periodontitis, 210 alkaline phosphatase, 176 alveolar resorption, 141 bone necrosis, 151 ampicillin, 146 ankylosis, 67 anti-inflammation, 113 apical leakage, 102 aspirin, 113 assisted drainage, 97 atrophic ridge, 181 attention deficit hyperactivity disorder, 190 augmentation, 181 australomelanesoid, 81 autogenous bone graft, 11 baby bottle tooth decay syndrome, 44 bacterial colonization, 131 black tea, 201 blaz, 146 bleeding disorder, 163 bonding material, 62 bone defects, 11 bruxism, 97 bucco-lateral defect, 195 calcium hydroxide, 168 lactate, 141 candida albicans, 201 carbamazepine, 49 caries, 186 cervical end preparation design, 131 children, 17, 97 chitosan, 168 chlorhexidine, 102 chronic periodontitis, 210 citotoxicity, 62 collarless metal ceramic crown, 131 complete unilateral cleft lip, 172 compomer, 190 computer-aided system, 107 condylar fracture, 67 contact hypersensitivity, 126 curing method, 40 delayed root formation, 76 dental anxiety, 17 modifications, 81 pulp, 186 dentin surface drying technique, 54 dentist, 163 direct pulp capping, 168 effectiveness, 122 enamel defect, 91 hypoplasia, 157 endodontic therapy, 205 endo-perio lesion, 195 erythema multiforme, 49 estrogen, 117 receptor a and b, 117 etiology, 1 extraction wound, 31 fibroblast, 31 cells, 62 fluoride, 72 furcation involvement, 205 hema, 62 honey solution, 58 human leukocyte antigen, 26 hybrid prosthesis, 136 hydroxyethyl methacrylate dentin bonding agent, 54 hypersensitivity, 49 indirect veneer, 157 indonesian chronology, 81 injectable bone xenografi, 176 hydroxyapatite, 176 hydroxyapatite-chitosan, 176 interleukin-1ß, 210 intrauterine growth restriction, 91 line strength, 107 malnutrition, 6 management, 6, 195 mandible, 181 mandibular fracture location, 1 growth disturbance, 67 medicine and dentistry applications, 81 mengkudu (morinella citrifolia linn.) gel, 31 mfp implant, 72 miacalcic, 141 millard’s surgical technique, 172 mongoloid, 81 mononuclear cell, 126 mouth-rinse, 58 mozart effect, 17 mta, 102 mtt viability, 176 215 mynocycline gel, 21 nerve fibers, 186 night guard, 97 noma, 6 nostril sill, 172 odontoblast-like cells, 168 odontoma, 76 oral mucosal epithelium, 26 rinse, 21 oroantral fistula, 151 closure, 151 orthodontic treatment, 35 osteoblast, 176 osteoporosis, 107 panoramic radiographs, 107 periodontal care, 58 disease/severity, 117 pocket, 117 plaque control, 58 pocket depth, 21 porcelain, 157 precision attachment, 136 protein gene product 9.5, 186 recurrent aphthous stomatitis, 26 resin, 62 restrain, 190 retrograde filling, 102 rhinosinusitis, 97 risk factor, 44 root resection, 205 resorption, 35 rounded end bristle, 122 saline, 102 sardinella longiceps oil, 113 sharp alveolar process, 136 end bristle, 122 small for gestational age, 91 soft liner, 136 spontaneous eruption, 76 staphylococcus aureus, 146 systemic lupus erythematosus,6 tell show do, 190 tensile bond strength, 54 the frequency of bottle-feeding, 44 the incisors of white rats, 72 thrombocytopenia, 163 tissue engineering, 11, 176 tongue piercing, 126 toothbrush, 122 trabecular bone, 107 transverse strength, 40 trauma mechanisms, 1 traumatized anterior teeth, 190 vestibuloplasty, 181 vitamin c, 141 216 author index volume 43 anggani, haru s, 35 apriasari, maharani laillyza, 49 arifin, agus zainal, 107 augustina, eka fitria, 21 bachtiar ew, 176 da’at arina, yuliana mahdiyah, 117 endrajana, 67 ernawati, diah savitri, 26 fakhrurrazi, 1 haniastuti, tetiana, 186 harijadi, achmad, 76 herachakri p, ananta, 126 irna sufiawati, 6 ismiyatin, kun, 54 januar, paulus, 122 juniarti, devi eka, 157 kentjananingsih, sri, 141 khoswanto, christian, 31 kuntjoro, mefina, 181 laksmiastuti, s. ratna, 163 machmud, edy, 131 mulyawati, ema, 102 nugraeni, yuli, 195 nurul m, dewi, 58 prahasanti, chiquita, 210 pramono d, coen, 172 prananingrum, widyasri, 168 rizal, mochamad fahlevi, 44 salim, sherman, 40 saraswati, widya, 62 sari, rima parwati, 113 satari, mieke, 146 setiawan, arlette suzy, 17 setiawati, ernie maduratna, 205 soebagio, 201 sukaedi, 136 sumarta, ni putu mira, 151 suriyanto, rusyad adi, 81 syarif,, willyanti s, 91 utomo, haryono, 97 veranica, 190 vitria, evy eida, 11 widjijono, 72 217 thanks to editor in duty of dental journal (majalah kedokteran gigi) volume ��� number � march ����: 1. prof. dr. peter agus, drg., ms., sp.bm (oral & maxillofacial surgery – airlangga university) 2. achmad harijadi, drg., ms., sp.bm (oral & maxillofacial surgery – airlangga university) 3. kus harijanti, drg., ms., sp.pm (oral medicine – airlangga university) 4. dr. retno indrawati, drg., m.si (oral biology – airlangga university) 5. dr. ernie maduratna setiawati, drg., m.kes., sp.perio (periodontic – airlangga university) 6. david buntoro kamadjaja, drg., mds., sp.bm (oral & maxillofacial surgery – airlangga university) 7. hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) volume ��� number � june ����: 1. prof. widowati witjaksono, dds, ph.d (kulliyah of dentistry, international islamic university malaysia) 2. prof. dr. peter agus, drg., ms., sp.bm (oral & maxillofacial surgery – airlangga university) 3. dr. ernie maduratna setiawati, drg., m.kes., sp.perio (periodontic – airlangga university) volume ��� number �� september ����: 1. prof. dr. trijoedani widodo, drg., ms., sp.kg. (conservative dentistry – airlangga university) 2. endrajana, drg., ms., sp.bm. (oral and maxillofacial surgery – airlangga university) 3. dr. retno indrawati, drg., msi. (oral biology – airlangga university) 4. dr. indah listiana kriswandini, drg., m.kes. (oral biology – airlangga university) 5. dr. theresia indah bs, drg., m.kes. (oral biology – airlangga university) 6. hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) volume ��� number � december ����: 1. prof. dr. h. boedi oetomo roeslan, drg., mbiomed (biochemistry – trisakti university) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – airlangga university) 3. els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university) 4. endrajana, drg., ms., sp.bm. (oral and maxillofacial surgery – airlangga university) 5. ester arjani rachmat, drg., ms (oral biology – airlangga university) 6. dr. retno indrawati, drg., m.si. (oral biology – airlangga university) 7. rostiny, drg., m.kes., sp.pros(k) (prosthodontic – airlangga university) 8. hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) 218 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with the left, right, top, and bottom margin should be 2.5 cm or 1 inch length, printed on good quality a4 white paper (210 × 297mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. authors should also follow the manuscript preparation guidelines. format for research reports: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in research reports should consists of “background:”, “purpose:”, “methods:”, “results:” and “conclusion:” typed in bold within one paragraph. • key words contain 3–5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on computer, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustrations, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all research reports should have more than 10 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205–9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005.2005. available at: http://www.dephut.go.id/informasi/humas/lebah. htm. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication 219 by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8–21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 format for case reports: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. • key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. tooth nomenclature must be explained, whether using zygmondy system, world health organization system, or universal system. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all case reports should have more than 10 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005.available2005. available at: http://www.dephut.go.id/informasi/humas/lebah.htm. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8-21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 format for literature reviews: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. • key words contain 3–5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. • review contains points and detailed matters based on literature which correlates with the discussed subject, to be discussed in the discussion section. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all literature reviews should have more than 30 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205–9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005. available2005. available at: http://www.dephut.go.id/informasi/humas/lebah.htm. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8–21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (in jpeg or tiff format). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 x 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 146 volume 47, number 3, september 2014 oral health knowledge among parents of autistic child in bandung-indonesia yetty herdiyati nonong,1 arlette setiawan,1 fellani danasra dewi2 and cugati navaneetha2 1 department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, bandung indonesia 2 academy of primary and preventive dentistry, faculty of dentistry, aimst university, malaysia abstract background: autistic children as well as other special needs individual demand special care given by their parents. but there exist limited awareness among parents in indonesia society, especially with regard to their oral health. purpose: the study was aimed to assess the oral health related knowledge, attitude and behavior of the parents; and oral health status of their autistic children in comparison with non-autistic children. methods: total of 56 children (23 autistic and 23 normal) between 7-12 years was included in this study. data on parents’ knowledge, attitude, oral health practice and behavior of their children were gathered from the questionnaires. the oral health status of the children was recorded using deft and dmft caries index. results: all obtained data were analyzed using spss version 13 to correlate the index of the sample. it showed that caries index of autistic child was lower and limited oral health knowledge among parents. conclusion: there is need of greater awareness to be spread among the population of indonesia about the existing professional help for the special children and educate the parents to maintain their child’s oral health for a better quality of life. key words: parental knowledge, autism, oral health, child, dental caries abstrak latar belakang: anak autis seperti juga individu berkebutuhan khusus lainnya memerlukan perhatian khusus dari orang tuanya. namun banyak keterbatasan kesadaran orang tua dalam masyarakat indonesia, terutama berkaitan dengan kesehatan mulut anak autis mereka. tujuan: penelitian ini bertujuan untuk menguji pengetahuan kesehatan gigi, perilaku orang tua dan anak mereka yang autis. metode: data pengetahuan orang tua dikumpulkan dari kuesioner dan status kesehatan mulut anak dicatat menggunakan indeks karies dmft dan deft. sejumlah 56 anak (23 autis dan 23 non-autis sebagai kelompok kontrol) usia 7-12 tahun ikut serta dalam penelitian ini. hasil: data yang didapatkan dianalisis menggunakan spss versi 13 untuk mengkorelasikan indeks subjek. hasil menunjukkan indeks karies anak autis lebih rendah dengan pengetahuan kesehatan mulut orang tua yang terbatas. simpulan: diperlukan penyebarluasan kesadaran yang lebih tinggi di antara populasi orang indonesia mengenai mempertahankan status kesehatan mulut anak autis mereka untuk mencapai kualitas hidup yang lebih baik. kata kunci: kesehatan mulut, peran orang tua, autis, karies, anak correspondence: yetty herdiyati nonong, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: a.suzy@unpad.ac.id, arlettesuzy@yahoo.com research report 147nonong, et al.: oral health knowledge among parents of autistic child in bandung-indonesia introduction autistic disorder (ad) is an organic disorder affecting the cerebellum and limbic system of the brain, resulting in behavioral and cognitive aberrations. it is characterized by impaired interpersonal and communication skills, limited attention span, hyperactivity interests, repetitive bodily movements and a stereotype behavioral pattern, that is established in the early childhood.1 its incidence ranges from 0.2-1.5% across the globe,2,3 with a higher predilection in males (four times), but in its most oppressive form in females.4 in 2009, ministry of health in jakarta, indonesia reported the prevalence of autism as one in every 150 children and the numbers of children diagnosed with this neurodevelopmental disorder are perpetually increasing. yet, there exists a limited knowledge and awareness among parents concerning the health, habits and management of their children with autism including their oral health. autistic individuals’ exhibit severe abnormality of reciprocal social relatedness and communicative incompetence prevents them to interact, understand and follow the instructions. their sensory and auditory hyperactivity to odors, lights and sounds in the dental clinic stimulate them for an unpredicted and exaggerated response on the dental chair, making them uncooperative in the dental setting.1 heterogeneous proclamation has been reported in the literature concerning oral health status and dental needs of autistic children and young adults. where studies in the 80slate 90s have found the prevalence of caries and periodontal disease to be of no difference compare with non-autistic individuals,5-6 contradicted to some studies in recent years that have evidenced comparatively lower prevalence of caries in children with asd.8,9 it is well established that health related practices are derived from the norms, goals, values and behaviors of the family members/parents, who contribute to their children’s healthy lifestyle habits.10-11 therefore this study was aimed to determine the oral health related knowledge, attitude and behavior of the parents; and oral health status of their autistic children. materials and methods this two-phased study was conducted at bandung, west java, indonesia. ethical clearance was obtained from health research committee, universitas padjadjaran, indonesia. total of 56 children and their parents were included for this project. twenty-three children with ad were selected randomly from three institutions. besides the diagnosed of autism, all children were medically healthy and were not on any therapeutic drugs. similarly, the number, age and gender matched counterparts were chosen from one private elementary school. all the children were aged between 712 years. in the first phase of the study, parents of all the children participants were informed about the study purpose and procedures; and all the parents were provided with 12itemed questionnaire to elicit the knowledge, attitude, oral hygiene practice and behavioral habits in their children. in the second phase, informed consent was obtained from the parents to conduct oral examination on minor children using dental diagnostic instruments (single use disposable plain mouth mirror, blunt probe, tweezers), light source (flash light) and personal protective attire (disposable surgical gown, masker, gloves) under cotton roll isolation, to record deft and/ dmft index for primary and permanent dentition respectively. the questionnaire for the parents targeted on the following concern; knowledge (awareness in tooth brushing and fluoridated toothpastes), attitude (previous experience of toothache, toothache management, reason for not seeking professional care), oral hygiene practice (frequency, timing, and parental assistance of tooth brushing activity), behavior (preference and frequency of sweetened food, pocket money to buy food from venders at school, frequency of those foods). while, the dental examinations was performed in the classroom by a single examiner in a conventional method using fdi tooth numbering system; scoring decayed, extracted missing and filled tooth index (deft) for primary teeth; and decayed, missing, and filled tooth index (dmft) for permanent teeth was done using who caries criteria.12 results among the children participants in this study, 20 were males and 3 were females in both autistic and control groups. in each group, six children ranged from 7-9 years and seventeen children ranged from 10-12 years. all the parent samples involved in this study responded completely for the survey questionnaire and their reports are illustrated in table 1. it was fortunate to know that most of the parents for autistic children (82.60%) and normal children (60.86%) knew about fluoridated toothpaste. table 1 also identified that majority of normal children (82.61%) have self awareness to brush their teeth. in contrast most of parent for autistic children claimed that their children were assisted for the routine oral hygiene practice. more than 2/3 (69.56%) of the autistic children were previously experienced with episodes of toothache and 56.52% of them was seeking the treatment from dentist, but 39.13% of the children were medicated by the parents themselves. it was described by 56.52% of the parents of autistic children that, it was unnecessary to get a professional helped for child’s toothache. in contrast, all the children in the control group had pervious experience of toothache. though majority of parents did self-medication (47.82%) to their children, considerable proportion (43.47%) of them incurred dentist’s advice and treatment. cost and the fear of dentist were the main reasons among the control group for not obtaining professional consultation for toothache in their children. 148 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 146–152 table 1. parental report on knowledge, attitude, oral health practice and behavior between autistic and normal children variable name autistic children normal children n % n % demographic: sex male female age 7 – 9 years 10-12 years 20 3 6 17 86.96 13.04 26.09 73.91 20 3 6 17 86.96 13.04 26.09 73.91 question item: knowledge heard of fluoridated toothpastes yes no awareness in tooth brushing told by parent self awareness attitude experience in toothache yes no managing toothache leave it take some medication went to a dentist reason didn’t go for a dentist scared lazy unnecessary no pain cost 19 4 16 7 1 9 13 5 1 12 4 1 82.60 17.39 69.56 30.43 4.34 39.13 56.52 21.73 4.34 52.17 17.39 0.00 14 9 4 19 23 0 2 11 10 6 1 3 7 6 60.86 39.13 17.39 82.61 100 0.00 8.69 47.82 43.47 26.08 4.34 13.04 30.43 26.08 oral hygiene practice frequency of tooth brushing once twice thrice timing of tooth brushing before taking a bath after meal & before bath during take a bath & before sleeping after meal & before sleeping take a bath, after meal, before sleep assisting in brushing teeth yes no behavior pocket money to buy food from venders yes no frequency of buying food from venders at school in one week once 2-3times everyday never if parent gave the money 0 15 8 9 1 8 3 2 17 6 0.00 65.22 34.78 39.13 4.35 34.78 13.04 8.70 73.91 26.09 2 19 2 6 2 8 5 2 20 3 3 8 4 0 8 8.70 82.61 8.70 26.09 8.70 34.78 21.74 8.70 89.96 13.04 13.04 34.78 17.39 0.00 34.78 149nonong, et al.: oral health knowledge among parents of autistic child in bandung-indonesia variable name autistic children normal children n % n % sweet consumption yes no frequency sweet consumption once twice thrice and more 19 4 10 1 12 82.60 17.39 43.47 4.34 52.17 14 9 11 8 4 60.86 39.13 47.82 34.78 17.39 table 2. deft and dmft index in the study samples no sex age autistic children normal children def-t dmf-t def-t dmf-t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 m m m m m m m m m m f f m m m m m m f m m m m 7 8 8 8 8 9 10 10 10 10 10 11 11 11 11 11 11 11 12 12 12 12 12 0 2 7 3 1 3 1 2 7 0 2 0 0 0 8 4 2 4 0 0 2 6 0 0 1 0 2 2 4 3 1 1 2 1 1 5 1 1 0 4 2 0 0 3 0 2 5 6 8 7 10 8 5 4 4 5 4 0 0 4 6 2 0 4 3 0 0 0 0 3 1 3 0 1 0 2 2 4 2 4 5 7 0 1 3 4 0 4 5 2 5 1 total 28 36 85 52 table 3. mann-whitney test for the comparison for def-t index between autistic children and normal children d e f def-t d m f dmf-t mann-whitney u wilcoxon w z asymp. sig. (2-tailed) 228.500 504.500 -.876 .381 141.000 417.000 -3.087 .002 264.500 540.500 .000 1.000 197.500 473.500 -1.503 .133 221.000 497.000 -.979 .328 264.500 540.500 .000 1.000 241.500 517.500 -1.430 .153 185.500 461.500 -1.767 .077 sig: < p =0.05; *0.77/2 = 0,039 (2-tailed) 150 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 146–152 it was claimed by the parents of autistic children that 65.22% of them brushed twice a day and 34.78% brushed three times a day. while greater percentage (82.61%) of the control group brushed two times per day. more than 1/3 (34.78%) of the study population in each of the group practiced their routine habit of brushing during bathing time in the morning and prior to sleeping in the night. however majority (39.13%) of the autistic children brushed before bathing hours and 73.91% of them were assisted by the parents during the daily ritual of brushing. all the children in the control group brushed themselves. according to the parents, 82.60% of the autistic children showed their favourism towards sweet food and 52.17% of them consumed sweetened food three or more times a day. on the contrary, though 60.86% of normal children carved for sweetened food, their snacking frequency was only once a day in 47.82% and two times in a day among 34.78%. the parents of normal children were questioned regarding pocket money given to the children to buy food from venders. this was to identify the possibility of unhealthy food consumption, which in turn could increase caries index. ninety precent of the normal children were given pocket money and 34.78% bought food from the venders during school hours, for 2-3 times in a week. however, autistic children were very selective in their food preference and were forbidden to buy meals from the venders without parents’ knowledge. as an oral health status reflected by the caries index showed in table 2. three autistic children were completely free from carious attack (0). the caries experience recorded using deft/dmft index illustrates that autistic children had lesser caries incidence than normal children. in autistic individuals deft was 28 and dmft were 36; in contrast to 85 deft and 52 dmft in normal healthy children. mann-whitney test performed to determine the significance between the deft index in autistic and normal children showed no statistical difference except for the index “e” (extracted primary teeth), which shows the difference as very significant (p = 0.002 with 95% confidence level). similarly, the dmft index between autistic children and normal children showed statistical significant difference (p = 0.039 with 95% confidence level) (table 3.). chi-square test was performed to correlate the parent’s reply of the questionnaire and caries index in the samples. the dmft index in autistic children and parental practice of oral hygiene with regard to the timings of brushing (31.83 > 21.03); and parental attitude towards managing toothache (23.14 > 12.59) for their autistic children were significant correlated. whereas for deft index in the control group and the parental attitude with regard to their reasoning for not consulting the dentist (26.91 > 26.29) for their child’s toothache problem (cost, fear of dentist and unreasonable cause) was significant (table 4). contingency coefficient would be the most appropriate measure of association between the two variables. the calculated value for this statistics suggests association of timing of tooth brushing with dmft in autistic children (0.762), displaying strong correlation. likewise, strongest correlation in normal children was the reason for not sending their children to the dentist when their child’s experienced toothache with deft (0.734). table 4. correlation among the questionnaires with def-t and dmf-t index question item autistic children normal children def-t dmf-t def-t dmf-t χ2 χ2table c χ 2 χ2table c χ 2 s2table c χ 2 χ2table c frequency of tooth brushing 6.744 7.815 0.476 2.196 7.815 0.295 4.81 15.51 0.416 4.784 12.59 0.415 timing of tooth brushing 9.044 21.03 0.531 31.83 21.03 0.762 7.124 26.29 0.486 10.76 21.03 0.565 assisting in brushing teeth 2.579 7.815 0.318 4.110 7.815 0.389 awareness in tooth brushing 2.930 9.49 0.336 2.229 7.815 0.297 heard of fluoridated toothpastes 6.034 7.815 0.456 1.369 7.815 0.237 5.317 9.488 0.433 6.809 7.815 0.478 pocket money to buy food from venders at school 9.334 9.49 0.537 0.993 7.815 0.203 frequency of buying food from venders at school in one week 12.66 21.03 0.596 6.625 16.92 0.473 experience in toothache 1.746 7.815 0.266 3.598 7.815 0.368 managing toothache 2.973 12.59 0.338 23.14 12.59 0.708 5.882 15.51 0.451 13.92 12.59 0.614 reason didn’t go for a dentist 11.19 16.92 0.572 5.691 16.92 0.445 26.91 26.29 0.734 6.202 21.03 0.461 sweet consumption 6.034 7.815 0.456 1.369 7.815 0.237 5.317 9.488 0.433 6.809 7.815 0.478 frequency sweet consumption 3.925 7.815 0.382 0.901 7.815 0.194 10.58 15.51 0.561 6.202 12.59 0.461 note: chi square: χ2 ; contingency coefficient: c ; correlation significant: χ2 > χ2table 151nonong, et al.: oral health knowledge among parents of autistic child in bandung-indonesia discussion psychosocial, neurobiological and emotional disorder of autism presents with pathognomonic behavioral pattern and preference in the victim patients. they exert extreme and distinct sensitivity to varying environmental factor and are dependents for their routine activities. perhaps, they are not devoid of dental disease and through a good oral care an optimum oral health can be achieved. however, this greatly depends on the knowledge and attitude of the parents, guardians or the caretakers of these patients. a systematic assessment of varied parental factors that could influence the overall oral health of the children is very much of a necessity to undertake the schemes and, therefore to provide a comprehensive dental health in all the children uniformly. the change in food habits and current trend in food consumption pattern demonstrates its inclination towards frequent and refined carbohydrates. this has reported higher incidence of dental caries in the literature and the autistic children are not exempted from this. it is demonstrated in the literature that higher priority caries risk group for all between 11-14 years.13 year 12 being the average age is the important in conducting the survey as it is the age at which the child leaves the schools, from where the reliable source of samples can be obtained from and also this is the highest priority risk group.14-16 it was well established in this study that the parents of both groups were knowledgeable with the beneficial effect of fluorides in the toothpastes. though majority of the normal children were aware of tooth brushing and its benefit, there were not affirmative about the correct method of brushing which could have increased the caries index. considering the fact that the autistic children have problems with fine motor control and were assisted by their parents for the routine oral hygiene practice, a thorough and correct procedure has to be known by the parents. it is concluded in the literature that significant predictors of children’s favorable habits were parents’ favorable attitudes towards controlling their children’s tooth brushing and sugar snacking habit.17 autistic children being highly sensitive to taste and food consistency, early introduction to good food and oral hygiene habits may play a role in “oral perception” of the child with communicative disorder.13 it was observed in this study that the frequency of brushing was more in autistic children than the normal children with a majority of them practicing their brushing during bathing and prior to sleeping. this was in similarity with the normal children. however in three-fourth (73.91%) of the autistic children, brushing was assisted by the parents, while all the children in the control group brushed themselves, without any parental guidance, assistance or supervision. this may affect the overall oral hygiene of the child to a considerable extent, as the manual dexterity of the children is still immature in the earlier years of development and improves over time gradually, therefore affecting the oral hygiene index of the study sample. this study was also an effort to understand the beliefs and attitudes of parents towards dentists and dental health. based on the children’s oral health status, it was evident that the behavior of parents was comparably different in both the groups. though all the samples in the control group had experienced toothache, greater proportion of their parents (47.82%) self-medicated their children. this was in contrast to the autistic children group, where half (56.52%) of them seeked the professional help and the quantity of parents doing self medication were comparably less (39.13%), while only 4.34% ignored the child’s complain about toothache. this reflects the care and concern of the parents of autistic children, despite their limited knowledge about oral health. this corroborates the requirement of specially training dental professionals for treating these children and their parents. diverse reasons were attributed in this study by the parents for avoiding dental treatment and the reasons being the fear of dentist, cost issue, ignoring the complaint and not considering the problem as real serious; and therefore managing toothache by parents themselves was apathetic. though major percentage of parents of autistic children found it was unnecessary to seek professional help for their child’s dental problem, none worried about the cost. but more than 25% of the parents of normal children considered cost as one of the major issue to avoid dentist. this was followed by the fear of dentist in children preventing them from professional care, thus projecting care from an expertised specialist, who can formulate structured timings and space for better patient management.18 regarding the behavior of samples to their food habits, in this study autistic group preferred sweeter food and increased frequency of consumption in comparison with their control group; they also postulating lower deft/ dmft index, which was independent of the parents attitude and knowledge, while only ‘extracted’ primary teeth index showed significance difference in both the study sample. this indicates that the severity of the dental disease that needed extraction, implying the severity of the disease. however, there difference in the dmft index between the groups reflected statistically significant difference, which in concordance recent studies.8,9,18-20 the study culminates the caries experience in autistic children as less in comparison with their normal counterparts and was independent of the parents’ behavior, knowledge, practice and attitude. yet, there are needs moulding and modulation in parental attributes that can be achieved by schemed professional training by the general dentist or by the specialists and/ or experts in the fields of managing challenging children. it concluded that there is need of greater awareness to be spread among the population in indonesia about the existing professional help for the special children and educate the parents to maintain their oral health for betterment. a greater effort has to be implemented for community dental services with optimum and timely parental education and follow up. 152 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 146–152 references 1. weddel j, anders b, jones j. dental problems of children with disabilities. in: mcdonald r, avery d, dean j, eds. dentistry for the child and adolescent. 9th ed. st. louis: mosby; 2010. p. 540. 2. thamer a. prevalence of dental caries and oral hygiene status among autistic children in riyadh, saudi arabia. edj 2011; 57(2): 1299. 3. veenstra-vanderweele j, cook e jr, lombroso pj. genetics of childhood disorders: xlvi. autism, part 5: genetics of autism. j am acad child adolesc psychiatry 2003; 42(1): 116-8. 4. american psychiatric association. diagnostic and statistical manual for mental disorders. 5th ed. arlington va: american psychiatric association; 2013. 5. kamen s, skier j. dental management of the autistic child. spec care dentist 1985; 5(1): 20-3. 6. klein u, nowak a. characteristics of patients with autistic disorder (ad) presenting for dental treatment: a survey an chart review. spec care dentist 1999; 19(5): 200-7. 7. saphira j, mann j, tamari i, mester r, knobler h, yoeli y. oral health status and dental needs of an autistic population of children and young adults. spec care dentist 1999; 9(2): 38-41. 8. namal n, vehit h, koksal s. do autistic children have higher levels of caries? a cross-sectional study in turkish children. j indian soc pedod prev dent 2007; 25(2): 97-102. 9. loo c, graham r, hughes c. the caries experience and behavior of dental patients with autism spectrum disorder. j am dent assoc 2008; 139: 1518-24. 10. prinstein m, boergers j, spirito a. the relationship of caries with oral hygiene status and extral oral risk factors. j ayub med coll abbottabad. 2001; 20(1): 103-8. 11. christensen p. the helath-promoting family: a conceptual framework for future research. soc sci med 2004; 59(2): 377-87. 12. world health organization. health surveys. basic methods. geneva: world helath organization; 1987. 13. peres m, peres kaj, junqueira s, frazao p, narvai p. distribution of dental caries in brazilian children. rev panam salud publica 2003; 14(3): 149-57. 14. rehman m, mahmood n, rehman b. the relationship of caries with oral hygiene stauts and extra oral risk facors. j ayub med coll abbottabad 2008; 20(1): 103-8. 15. demattei r, cuvo a, maurizio s. oral assessment of children with an autism spectrum disorder. j dent hyg 2007; 81(3): 1-7. 16. adair p, pine c, burnside g, nicoll a, gillett a, anwar s. familial and cultural perceptions and beliefs of oral hygiene and dietary practices among ethnically and socio-economically diverse groups. comm dent health 2009; 21(suppl): 102-11. 17. friedlander a, yagiela j, paterno v, mahler m. the pathophysiology, medical management, and dental implications of autism. j calif dent assoc 2003; 31: 681-2. 18. jaber ma. dental caries experience, oral health status and treatment needs of dental patients with autism. j appl. oral sci 2011; 19(3): 212-7. 19. kopycka-kedzierawski dt, auinger p. dental needs and status of autistic children: results from the national survey of children’s health. pediatr dent 2008; 30(1): 54-8. 20. pilebro c, backman b. teaching oral hygiene to children with autism. int j paed dent 2005; 15(1): 1-9 106 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 106–110 research report the effect of a combination of 12% spirulina and 20% chitosan on macrophage, pmn, and lymphocyte cell expressions in post extraction wound nike hendrijantini, rostiny, mefina kuntjoro, kevin young, bunga shafira, and yunita pratiwi department of prosthodontics faculty of dental medicine, universitas airlangga surabayaindonesia abstract background: tooth extraction is the ultimate treatment option for defective teeth followed by the need for dentures. inflammation is one phase of the healing process that should be minimized in order to preserve alveolar bone for denture support. macrophage, pmn and lymphocyte cells are indicators of acute inflammation. spirulina and chitosan are natural compounds with the potential to be anti-inflammatory agents. purpose: this study aimed to determine macrophage, pmn and lymphocyte cells of animal models treated with a combination of 12% spirulina and 20% chitosan on the 1st, 2nd and 3rd post-extraction day. methods: animal models were randomly divided into control (k) and treatment (p) groups. each group was further divided into three subgroups (ki, kii, kiii and pi, pii, piii). the post-extraction sockets of the control group animals were then filled with cmc na 3%. meanwhile, the post-extraction sockets of the treatment group members were filled with a combination of 12% spirulina and 20% chitosan. subsequently, the number of pmn, macrophage and lymphocyte cells was analyzed by means of he analysis on the 1st, 2 nd and 3 rd days. statistical analysis was then performed using a t-test. results: there was a decrease in pmn cells and an increase in macrophage and lymphocyte cells on days 1, 2 and 3. conclusion: it can be concluded that a combination of 12% spirulina and 20% chitosan can not only decrease pmn cells, but can also increase macrophage and lymphocyte cells on day 1, 2 and 3 after tooth extraction. keywords: spirulina; chitosan; inflammation; pmn; macrophage; lymphocyte correspondence: nike hendrijantini, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: nike-h@fkg.unair.ac.id introduction tooth loss is one of dental health problem. its prevalence in indonesia, according to riskesdas data in 2013, was 14.51% within the 45-54 years age group, 25.02% in the 5564 years age group, and 43.79% in the >65 years age group.1 tooth loss can occur due to extraction which can increase injury to dental tissues resulting in an acute inflammatory reaction as well as inflammatory cell infiltration, such as polymorphonuclear (pmn), macrophages, and lymphocytes.2 clinical inflammatory reaction is usually indicated by edema, redness, and pain.3 the inflammatory phase commonly lasts from the point of injury to day 6 after its occurrence.4 thus, in order to maintain the alveolar bone while promoting prosthetic restoration, biomaterial is required to eliminate the inflammatory process and accelerate the post-extraction healing process. recently, the use of natural materials in accelerating the wound healing process has been widely studied since they are safer than synthetic materials. one of the natural ingredients that have many benefits for the wound healing process is spirulina a greenish-green algae containing c-phycocyanin, b-carotenoids, vitamin e, zinc, and other components useful to the human body. c-phycocianin is even considered to have an anti-inflammatory and antioxidant components.5,6 another natural ingredient in the healing process that is commonly studied is chitosan, a polymer of deacetylated dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p106–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p106-110 107107hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 106–110 chitin. chitin is a copolymer of n-acetyl-d-glucosamine and d-glucosamine bound by ß(1-4) glycosidic bonds. chitin and chitosan can both be found in aquatic and terrestrial organisms. chitosan can currently be obtained via the food industry through the processing of waste derived from shrimps, lobsters, crabs, and squids.7 chitosan is widely used as one of ingredients for drug delivery systems, wound healing processes, and orthopedic implants, while also being tknown to increase the activities of immune cells, inflammatory cells and angioendothelial cells. chitosan oligosaccharides have anti-inflammatory properties since chitosan can inhibit the production of tumor necrotizing factor-α (tnf-α) in inflammation stimulated by lipopolysaccharide (lps).6 other studies have also shown that chitosan can reduce the inflammation associated with allergic responses by inhibiting the secretion of interleukin-8 (il-8) and tnf-α.7 some cases in the field of prosthodontics, such as immediate denture and ovate pontik installation, also require a more rapid wound healing process. consequently, a shorter treatment that can lead to optimal results for patient comfort is necessary. considerable previous research into the effects of spirulina and chitosan induction on collagen, osteoblast, and osteoclast cells in animal models has been conducted. the results of past investigations have shown that a combination of 12% spirulina and 20% chitosan can not only increase collagen and osteoblast expressions, but can also decrease osteoclast expression. as a result, it can be said that spirulina and chitosan has the potential to promote the bone healing process.8 the effects of spirulina and chitosan in this regard have actually already been studied, unlike the effects of a combination of spirulina and chitosan on inflammatory cells. therefore, this research aimed to reveal the effects of a combination of 12% spirulina and 20% chitosan on pmn, macrophage and lymphocyte cells in animal models on day 1, 2 and 3 after extraction. materials and methods this investigation reported here constituted laboratory based experimental research incorporating a post test only control group design and using male cavia cobaya specimens (n = 42) weighing 300-350 grams and aged 3-3.5 months. the research passed an ethical test performed by the faculty of dental medicine, universitas airlangga (no. 110/kkepk.fkg/vii/2016). the animal specimens were subsequently divided into two groups, namely; control and treatment. the control (k) and treatment (p) groups were each sub-divided into three sub-groups referred to as ki, kii, kiii, pi, pii, and piii. the roman numerals i, ii and iii represent day 1, 2, and 3 after the specimens had been terminated. thereafter, the mandibular incisors of the members of all groups were extracted under ketamine anastesi (ketalar, pt pfizer, jakarta, indonesia) at a dose of 40 mg/kgbw. after the extraction, the sockets of the ki, kii, and kiii control groups were filled with 3% sodium-carboxymethyl cellulose natrium (cmc na). meanwhile, the sockets of the treatment groups, pi, pii, and piii, were filled with a combination of 3% cmc na, 12% spirulina and 20% chitosan using a 0.1 cc syringe. the sockets were stitched with 3/0-size silk threat. after the treatment, these animals were returned to their cages. on the first day, members of the ki and pi groups were decapitated, a process repeated for the kii and pii groups on day 2, as well as the kiii and piii groups on day 3. mandible samples were then taken and fixed. at that point, the mandibles were with 2.5% nitric acid for 2 days. thereafter, the sagittal incisive socket area of the specimens was cut and soaked in a 10% buffered formalin for 24 hours. mixed preparations were then performed by using eosin haematoxylin (he) before pmn, macrophages, and lymphocytes on one-third of the sockets’ area were observed using a light microscope (nikon h600l®, tokyo, japan) at a magnification of 400x. the data obtained was then analyzed by means of a saphiro wilk test to analyze the data distribution, followed by an independent t-test to identify the differences between the groups. results the results of the observation of pmn, macrophage, and lymphocyte cells in the control and treatment groups can be seen in table 1. moreover, the results of he staining in both groups are contained in figure 1. the results of he staining illustrate that pmn possessed a large cell picture, a nucleus featuring lobes (2-5 lobes), chromatin in a condensed nucleus and visible organelle in cytoplasm. they also depicted macrophages as having a large cell image and generally kidney-shaped, erratically located cell nuclei. furthermore, the cytoplasm cells were solid and appeared to be composed of a pink and purple granule, table 1. mean and standard deviation of pmn, macrophage, and lymphocyte expressions no groups n pmn macrophages lymphocytes 1 ki 7 41.85±5.87 1.43±0.53 3.00±0.82 2 pi 7 32.00±3.26 5.57±1.51 12.00±2.00 3 kii 7 35.29±2.81 2.00±1.15 8.28±2.28 4 pii 7 11.71±1.60 19.71±1.98 21.43±2.37 5 kiii 7 20.57±3.69 15.57±1.72 19.14±2.41 6 piii 7 15.14±2.41 20.57±2.70 22.43±3.31 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p106–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p106-110 108 hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 106–110 whereas the lymphocytes had a circular or spherical nucleus cell with dark blue chromatin and a surrounding thin, light blue cytoplasm. subsequently, before a t-test analysis was performed, normality test, saphiro wilk test was conducted. results of the saphiro wilk test showed that all data were normally distributed because p value was more than 0.05. independent t-test then was carried out. results of the independent t-test indicated that the p values of pmn cells between ki and pi groups, between kii and piii groups, and between kiii and piii groups were 0.020, 0.000, and 0.007, respectively. the results of the independent t-test also showed that the p values of macrophages between ki and pi groups, between kii and pii groups, and between kiii and piii groups were 0.000, 0.000, and 0.001, sequentially. meanwhile, the p values of lymphocytes between ki and pi groups, between kii and pii groups, and between kiii and piii groups were 0.000, 0.000, and 0.055, respectively. almost all of the independent t-test results in the control groups compared with the treatment groups indicated significant increase macrophages and lymphocytes differences (p<0.05), except between group pmn significant decrease (p>0.05). thus, it can be said that no significant difference existed between the two groups. discussion the presence of pmn cells is very important as an indicator of the wound healing process since pmn cells are the first to appear in the acute inflammatory phase.9 pmn cells are cellular defenses that play an active role in the process of bacterial destruction through endothelial adherens, chemotaxis and phagocytosis. furthermore, pmn cells are able to move actively, and in a short period of time can collect in large numbers in the wound area. pmn cells have a lifespan of 1-3 days in connective tissue.10 another cell that plays a role in the inflammatory process is the macrophage cell which carries out several functions in the wound healing process, such as producing collagenase and elastase enzymes, generating cytokines, facilitating phagocytosis and angiogenesis processes, as well as stimulating granulation tissue formation in the proliferative phase.11 macrophage cells in the inflammatory process can be distinguished by the origin of the tissue macrophage resident and monocytes undergoing differentiation.12 monocytes in the blood vessels will be transported toward inflammatory tissues due to chemotaxis resulting from the response to chemoattractant. chemoattractant consists partly of kemokin, a protein (8-14 kda) that regulates cell travel through interaction with a 7-transmembrane subset g-protein pair receptor.13 similarly, lymphocyte cells play a role in the inflammatory process. lymphocytes result in both immunemediated inflammation caused by infectious agents and non-immune inflammation. t and b lymphocytes migrate to the inflammatory area and direct neutrophils and other leukocytes.14 in the remodeling process, when the wound has been closed and the local infection has subsided, the leukocyte subtance most often found in the injured tissue is that of t cell which acts as an adaptive immune response cell.6 results of the research on day 1 revealed that the presence of pmn cells in the control group was higher than in the treatment group. this occurred because phycocyanin and b-carotene contained in spirulina can decrease the 12 figure 1. inflammatory cells in the tooth extraction sockets of cavia cobaya animals in group ki (a), group pi (b), group kii (c), group pii (d), group kiii (e), and group piii (f). (blue arrows: pmn, yellow arrows: macrophage cells, green arrows: lymphocytes). 12 figure 1. inflammatory cells in the tooth extraction sockets of cavia cobaya animals in group ki (a), group pi (b), group kii (c), group pii (d), group kiii (e), and group piii (f). (blue arrows: pmn, yellow arrows: macrophage cells, green arrows: lymphocytes). 12 figure 1. inflammatory cells in the tooth extraction sockets of cavia cobaya animals in group ki (a), group pi (b), group kii (c), group pii (d), group kiii (e), and group piii (f). (blue arrows: pmn, yellow arrows: macrophage cells, green arrows: lymphocytes). 12 figure 1. inflammatory cells in the tooth extraction sockets of cavia cobaya animals in group ki (a), group pi (b), group kii (c), group pii (d), group kiii (e), and group piii (f). (blue arrows: pmn, yellow arrows: macrophage cells, green arrows: lymphocytes). ba c ed f figure 1. inflammatory cells in the tooth extraction sockets of cavia cobaya animals in group ki (a), group pi (b), group kii (c), group pii (d), group kiii (e), and group piii (f). (blue arrows: pmn, yellow arrows: macrophage cells, green arrows: lymphocytes). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p106–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p106-110 109109hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 106–110 production of proinflammatory cytokines, tnf-α and il1b. b-carotene in spirulina also has an anti-inflammatory effect through resistance to the production of nitric oxide and prostaglandin e2. furthermore, b-carotene also inhibits the expression of inos, cox2, tnfα and il1b. suppression of inflammatory mediators is due to nf-κb inhibition that restricts the nuclear translocation of subunits nf-κb p65.15 as a result, anti-inflammatory activity of spirulina can decrease the number of pmns in the lesion site. tnf-α and il-1b can also facilitate the movement of pmn cells to the site of the lesion and spur the production of endothelial adherens.16 it is intended that pmn cells easily pass through the gap between endothelial cells in capillary blood vessels to eliminate bacteria, so the reduction of proinflammatory cytokines can, in turn, lead to a decrease in the production of pmn cells.16 in addition, on the first day after dental extraction, the average number of pmn cells in group ki was the highest compared with the other control groups (k ii and k iii) and the treatment groups on the other days (p ii and p iii) because pmn cells had already been active and assembled at a large number of lesion sites very rapidly, i.e. within hours.17 pmn cells are highly reactive to chemotactic products in the form of proteins produced by bacteria.16 moreoever, the results of calculating macrophage cells on day 1 indicated that the treatment group (p i) had a higher number of such cells than the control group (k i). this may occur because the inflammatory cells that appear immediately post-incision are not only neutrophils, but also monocytes moving into the inflammatory area. however, the number of monocytes migrating to the wound area is not as high as that of neutrophils.18 the combination of 12% spirulina and 20% chitosan also contains more synthetic c-phycocyanin components which execute a greater immunomodulatory function than when applied alone.19 furthermore, the results of the independent t-test showed there to be a significant difference in the presence of lymphocytes between the k1 and p1 groups on the first post-extraction day. this may occur because phycocyanin pigments contained in spirulina may act as an antiinflammatory by inhibiting proinflammatory cytokines, namely; tnf-α and il-1b.15 chitosan also plays a role in increasing lymphocyte cells. a previous piece of research using mice orally induced with chitosan finds that the latter can stimulate the release of il-10, il-4, and tgf-b mrna expressions in gastric mucosa, cd3 + t lymphocytes in the spleen, as well as natural killer cells (nk) in intestinal intraepithelial lymphocytes.20 in addition, the number of pmn cells in group pii, based on the results of the second day’s observation, was lower than that in group kii. the decrease in pmn cells on the second day was higher than on the first day due to the homeostasis process where the number of pmn cells produced in the bone marrow must be balanced with that having clearance which had already worked on the system within the body. the number of pmn cells produced in the bone marrow can reach a post-injury maximum within 24-48 hours. on the other hand, the process of pmn cell clearance can reach its peak 48 hours after the occurrence of the lesion. the number of pmn cells will then decrease as the chronic inflammatory phase is entered. clearance can also occur when the pmn cells extravatase into the peripheral tissues. a previous investigation into mice revealed that pmn cells can migrate back from peripheral tissue into the bloodstream through a process known as reverse transmigration.16 this suggests that the extravasation of pmn cells does not necessarily lead to the clearance of tissue. the clearance process reaches its peak on the second day. consequently, the anti-inflammatory effect of the combination of 12% spirulina and 20% chitosan will reduce the number of pmn cells on the second day to a greater extent than on the first day.16 the number of lymphocytes in group pii, based on the results of the second day of observation, was higher than that in group kii. this difference is due to spirulina being able to increase the number of lymphocytes. previous research has even shown spirulina to have an immune modulatory effect on lymphocytes by significantly increasing ifn-.2 production.21 the increased number of lymphocyte cells in the treatment group is also due to chitosan that exhibits biocompatible and biodegradable properties. the latter allow chitosan to be broken down into micromolecules so that they are easily absorbed by the body without causing toxicity, thereby enabling it to be used as an analgesic, anti-tumor, anti-microbial, anti-oxidant, and wound healing agent.22 chitosan also contains a n-acetyl-d-glucosamine unit, polysaccharide similar to glucan, which accelerates cytokine production in order to stimulate repair of affected tissue.23 the results of the third day’s observation found that the number of pmn cells in group piii was lower than that in group kiii. the number of pmn cells on day 3 was lower than that on day 2. this happened because the number of pmn cells will usually decrease between day 3 and 7.18.24 this reduction is essential to preventing further damage to healthy body tissue. the body responds to a reduction in the production of pmn cells in order not to damage other tissues because pmn cells issue anti-microbial products that can damage healthy tissue of the body. the number of macrophages in group piii, based on the results of the third day’s observation, was still higher than that in group kiii. nevertheless, there was no significant difference in the post-extraction number of macrophages between the treatment groups on day 2 and day 3. this may be due to the inflammatory process beginning to enter the resolution phase during which a reduction of chemokine by the mechanism of proteolysis and chemokine sequestration occurs.25 macrophages, as a result, begin to dominate the wound area from day 3 to day 7 after extraction. the number of lymphocyte cells in the treatment group, based on the results of the third day’s observation, appeared to increase compared with that of the control group. however, based on the results of the independent t-test, there was no significant difference in the number dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p106–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p106-110 110 hendrijantini, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 106–110 of lymphocyte cells between the control group and the treatment group on day 3. during the chronic inflammatory process, the presence of lymphocyte can usually reach a peak between day 5 and day 10.15 thus, 12% spirulina and 20% chitosan are expected to act as anti-inflammatories. this insignificant difference between the control group and the treatment group is due to the chronic inflammatory process having begun to subside and entering the maturation process which leads to regeneration. the phycocyanin and b-carotene substances contained in spirulina can be considered to be anti-inflammatory antioxidants that can accelerate the wound healing process. phycyocyanin, according to previous in vitro and in vivo research using rat-fed animals, can inhibit tnf-α inflammatory cytokine secretion and act as an antioxidant.26 this strongly suggests that the anti-inflammatory activity of spirulina may cause tnf-α and il-1b secretions to decrease.27 similarly, the antioxidants in b-carotene contained in spirulina can improve the wound healing process.6 chitin and chitosan are biopolymers that offer many benefits, including; a high level of biocompatibility, low toxicity, increased antibacterial activity and accelerated wound healing.28 chitosan can also stimulate pmn cells to chemotaxyize the wound area due to the presence of il-1, tnf-α, and bacterial products.29 in other words, spirulina and chitosan exert a synergistic effect when combined since chitosan plays a role in drug delivery, while spirulina has a therapeutic effect. spirulina and chitosan can also interact intermolecularly to increase mechanical resistance. both of these materials can even work together to provide more effective benefits than if applied alone. in addition, a high degree of chitosan acetylation will improve the hydrophobic properties of chitosan. the hydrophilic component is capable of diffusing through the chitosan polymer into the outer medium to be absorbed by the body.22 finally, based on the above discussion, it can be concluded that 12% spirulina and 20% chitosan combination can not only decrease pmn expression, but also increase macrophages and lymphocytes in cavia cobaya animals on the first, second and third days after extraction. references 1. departemen kesehatan republik indonesia. riset kesehatan dasar (riskesdas) 2013. jakarta: badan penelitian dan pengembangan kesehatan kementerian kesehatan ri; 2013. p. 143-5. 2. nanci a. ten cate’s oral histology. 8th ed. st. louis: elsevier; 2013. p. 338. 3. andersson l, kahnberg k, pogrel ma. oral and maxillofacial surgery. sussex: wiley-blackwell; 2010. p. 165-6. 4. hess ch. clinical guide to skin and wound care. 7th ed. philadelphia, usa: wolters kluwer health; 2012. p. 10. 5. gershwin me, belay a. spirulina in human nutrition and health. florida: crc press; 2007. p. 4, 127-8. 6. ma hendra j, ma hendra l, muthu j, john l, romanos ge. clinical effects of subgingivally delivered spirulina gel in chronic periodontitis cases: a placebo controlled clinical trial. j clin diagn res. 2013; 7(10): 2330-3. 7. kim s. chitin, chitosan, oligosaccharides and their derivatives. florida: crc press; 2010. p. 3, 11. 8. rostiny, kuntjoro m, sitalaksmi rm, salim s. spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket. dent j (maj ked gigi). 2014; 47(1): 19-24. 9. azzahra h, pujiastuti p, purwanto p. potensi ekstrak kulit buah ma nggis (ga rcinia ma ngosta na l.) buata n pabr ik terhadap peningkatan aktivitas mikrobisidal sel neutrofil yang dipapar streptococcus mutans. e-jurnal pustaka kesehat. 2014; 2(1): 161-6. 10. kumar v, cotran rs, robbins sl. robbins buku ajar patologi volume 1. 7th ed. jakarta: egc; 2007. p. 35-56. 11. sussman c, bates-jensen b. wound care: a collaborative practice manual for health professionals. philadelphia, usa: wolters kluwer health/lippincott williams & wilkins; 2012. p. 33-9. 12. murray pj, wynn ta. protective and pathogenic functions of macrophage subsets. nat rev immunol. 2012; 11: 723-37. 13. sánchez-martín l, estecha a, samaniego r, sánchez-ramón s, vega má, sánchez-mateos p. the chemokine cxcl12 regulates monocyte-macrophage differentiation and runx3 expression. blood. 2011; 117: 88-97. 14. port cm. essentials of pathophysiology. 4th ed. philadelphia, usa: wolters kluwer; 2015. p. 49-61. 15. quader sh, islam su, saifullah a, majumder mfu, hanna j. in vivo studies of the anti-inflammatory effects of spirulina platensis. j pharmacogn phytochem. 2013; 2(4): 70-80. 16. baratawidjaja kg. imunologi dasar. 11th ed. jakarta: badan penerbit fakultas kedokteran universitas indonesia; 2012. p. 34-46. 17. tak t, tesselaar k, pillay j, borghans ja, koenderman l. what’s your age again? determination of human neutrophil half-lives revisited. j leukoc biol. 2013; 94: 595-601. 18. rodero mp, licata f, poupel l, hamon p, khosrotehrani k, cambadiare c, boissonnas a. in vivo imaging reveals a pioneer wave of monocyte recruitment into mouse skin wounds. plos one. 2014; 9(12): 1-9. 19. madhyastha hk, radha ks, nakajima y, omura s, maruyama m. upa dependent and independent mechanisms of wound healing by c-phycocyanin. j cell mol med. 2008; 12(6b): 2691-703. 20. borges o, borchard g, de sousa a, junginger he, cordeiro-dasilva a. induction of lymphocytes activated marker cd69 following exposure to chitosan and alginate biopolymers. int j pharm. 2007; 337: 254-64. 21. karkos pd, leong sc, karkos cd, sivaji n, assimakopoulos da. spirulina in clinical practice: evidence-based human applications. evid-based complement alternat med. 2011; 2011: 1-5. 22. aranaz i, mengíbar m, harris r, panos i, miralles b, acosta n, galed g, heras a. functional characterization of chitin and chitosan. current chem biol. 2009; 3: 203-30. 23. nam ks, kim mk, shon yh. inhibition of proinf lammatory cy tok i ne-i nduced i nvasiveness of h t-29 cel ls by ch itosa n oligosaccharide. j microbiol biotechnol. 2007; 17(12): 2042-5. 24. sunarjo l, hendari r, rimbyastuti h. manfaat xanthone terhadap kesembuhan ulkus rongga mulut dilihat dari jumlah sel pmn dan fibroblast. odonto dent j. 2015; 2(2): 14-21. 25. o r tegagómez a, per ret t i m, soeh n lei n o. resolut ion of inflammation: an integrated view. embo mol med. 2013; 5: 66174. 26. pak w, takayama f, mine m, nakamoto k, kodo y, mankura m, egashira t, kawasaki h, mori a. anti-oxidative and antiinflammatory effects of spirulina on rat model of non-alcoholic steatohepatitis. j clin biochem nutr. 2012; 51(3): 227-34. 27. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219-29. 28. rudiyarjo di. pengaruh penambahan plasticies gliserol terhdap karakteristik hidrogel kitosan glutaraldehid untuk aplikasi penutup luka. j ilmiah sains. 2014; 14(1): 18-28. 29. sharma rk, john jr. role of stem cells in the management of chronic wounds. indian j plast surg. 2012; 45: 237-43. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p106–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p106-110 30 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 30–35 original article properties of nanocellulose and zirconia alumina on polymethylmethacrylate dental composite eva febrina1, angela evelyna1, andrie harmaji2, bambang sunendar3 1faculty of dentistry, maranatha christian university, bandung, indonesia 2department of metallurgical engineering, institut teknologi sains bandung, bekasi, indonesia 3department of engineering physics, institut teknologi bandung, bandung, indonesia abstract background: polymethylmethacrylate (pmma) is one of the synthetic polymers generally used for temporary jacket crown restorations because of its good translucency, making its aesthetic value higher, but its mechanical properties, such as hardness and flexural strength are lower than composite resins. hence, adding zirconia and cellulose filler is necessary to enhance its mechanical properties. purpose: this is an experimental laboratory study to make nanocomposites with pmma as a matrix with crystalline nanocellulose, zirconia, and alumina added as fillers. methods: the crystalline nanocellulose filler was synthesized by acid hydrolysis. zirconia and alumina were synthesized using the sol-gel technique and then characterized by transmission electron microscope and x-ray diffraction. the micro vickers hardness test and three-point bending tested mechanical properties. the analysis was carried out with a one-way analysis of variance, followed by a post hoc tuckey’s test with a p < 0.05 taken as statistically significant. results: the micro vickers hardness test showed the highest hardness in the group with a ratio of pmma and zirconia-alumina filler of 50%: 2%: 48% (12.73 vhn). the results of the three-point bending test showed that the highest flexural strength was found in the control group (19.4 mpa). conclusion: the addition of crystalline nanocellulose, zirconia, and alumina increase the hardness of the nanocomposite, while the flexural strength was lower than pmma without filler addition. keywords: alumina; crystalline nanocellulose; mechanical properties; pmma; zirconia article history: received 12 may 2022, revised 4 july 2022, accepted 15 july 2022 correspondence: eva febrina, faculty of dentistry, maranatha christian university. jl. surya sumantri no. 65 bandung, 40164, indonesia. email: evafebrina11@gmail.com introduction the development of science and technology in dentistry improves the quality of life in patients with teeth and mouth problems. damage to the calcified tooth structure and supporting tissues by noxious stimuli can cause pulp and peri-radicular tissue changes. noxious stimuli can be physical, chemical, or bacterial that can produce reversible or irreversible changes, depending on the duration, intensity, and pathogenesis, and the ability of the host to resist them and repair tissue damage.1 caries is a microbiological infectious disease of the teeth that causes local changes and destruction of the hard tissues of the teeth. tooth structure loss can be repaired with restoration procedures. the materials most commonly used to restore dental caries are metals, ceramics, polymers, and composites. the use of metal to restore caries has been abandoned because of aesthetic and biocompatibility issues. therefore, many have started to switch to composite restorative materials. nanocomposite has been widely developed for restorative materials today.1,2 composites are physical mixtures of metals, ceramics, and/or polymers to obtain the desired mechanical properties of each mixed material. the mixture commonly found in dental composites is a mixture of ceramic and polymer matrix. today, polymethylmethacrylate (pmma) and bisphenol a-glycidyl methacrylate are the polymers widely used as composite materials. mixing these materials is intended to obtain the desired mechanical properties of each material.1,3 composites are currently limited to restoring class iii, iv, v, and class i cavities if aesthetics are required. restoration materials should have good copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 mailto:evafebrina11@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 31febrina et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 30–35 hardness and flexural strength like enamel and ideally have good antibacterial properties to prevent secondary caries.4–6 composite materials currently being developed are often used as restorations in the anterior and posterior teeth. current products in the market have low flexural strength and hardness, which is not good enough for patients with parafunctional habits such as bruxism. the lack of strength to resist composite fractures in high-pressure areas, such as in the case of extensive preparations, including the cusp, requires reinforcing material since the composites must withstand cavity restorations under high stresses.7 hardness and flexural strength can be reduced due to consuming various types of food, especially acidic foods, and frequent mouthwash usage.8 the raw materials for manufacturing composite fillers are generally imported from other countries, even though indonesia’s abundant natural resources provide good quality raw materials for manufacturing dental materials. zirconia can be used as filler material for pmma matrix because it is chemically stable, increases fracture toughness, has good dimensional stability, a modulus of elasticity and flexural strength similar to steel, and good biocompatibility. one of the main reasons zirconia is used as a raw material for dental materials, and especially restoration materials, is because its tooth-like color increases its aesthetic value. in addition to zirconia, alumina is often used as a composite filler because it has good wear resistance, optimal hardness, good thermal conductivity, and sufficient rigidity.8,9 currently, nanocellulose is a new material being developed as a raw material for restoration materials in dentistry. nanocellulose is a natural material with unique characteristics and is synthesized from cellulose (obtained from plants, animals, and bacteria). this material has received much attention for its use as a biomedical material because of its mechanical, chemical, and biological properties (biocompatibility, biodegradability, and low toxicity). nanocellulose can be developed as a raw material for biomedical materials, especially for filler composites, because of its properties. the nanocellulose is extracted from palm kernel cake by converting large units (cm) to small units (nm) using chemical aids such as acid hydrolysis, which is commonly used to remove the amorphous part and extract the crystalline form of pure cellulose, which is essential because it can fill the void in composites fused with alumina-toughened zirconia (zro2-al2o3). 10 based on the facts stated above, this study aims to develop a nanocomposite based on pmma, crystalline nanocellulose, and zro2-al2o3 filler as a dental restoration material and analyze its morphological and mechanical properties. since the resulting products must comply with the american national standards institute and the american dental association (ada) standards, the flexural strength and hardness of the material must be analyzed. materials and methods palm kernel cake was used as a precursor for the synthesis of crystalline nanocellulose mixed with demineralized water (dm), nitric acid 3.5%, sodium nitrite, sodium hydroxide, sodium sulfite, sodium hypochlorite, and sulfuric acid (h2so4) 45%. all chemicals were from the brand sigma aldrichtm. zirconium dioxide (zro2) powder was synthesized by mixing zirconium chloride (zrcl4), calcium chloride hydrate as a stabilizer, ethanol 90%, and dm. the zrcl4 precursor (4.66 g) was mixed with 200 ml of dm and then stirred using a magnetic stirrer for 15 minutes to obtain a 0.1m zrcl4 solution. calcium chloride hydrate stabilizer 0.234 g was added to the precursor solution, mixed with a magnetic stirrer for 15 minutes until homogeneous, and added to approximately 8% of the total molarity of the precursor. the sample underwent an aging and drying process in an oven with a temperature of 120°c for 24 hours until the solvent evaporated and obtained xerogel results. after grinding, the sample was transferred to a combustion boat, then calcined in a furnace starting from room temperature to 900°c and maintained at that temperature for two hours to form metal oxide particles. the temperature was then lowered. the particles were ground with a mortar and pestle until smooth to get smaller particles. the calcined sample was dissolved in 50 ml of ethanol and then homogenized with an ultrasonic homogenizer with an amplitude of 80 for 30 minutes to produce nanometer-sized particles. the samples were dried in an oven for 24 hours to obtain zro2 in the form of calcium partially stabilized zirconia (capsz) particles. aluminum nitrate hydrate (al(no3)3.9h2o) as a precursor (11.25 g) was dissolved in 50 ml of dm, and 0.1m ammonium carbonate solution was added as a ph controller to obtain a ph of 8–9 while stirring, using a magnetic stirrer for 45 minutes. then aging was done using an ultrasonic bath for three hours without heat until two layers were formed. the top layer was clear, while the bottom layer was a gel precipitate. the precipitate was then filtered using a buchner funnel while rinsing with aqua dm until a neutral ph was attained and filtered using filter paper. the filtered residue was then dried in an oven at 100°c for one day. after the sample was dry and formed a white solid, the next step was calcination at 550°c for one hour. the calcined results were then ground into powder using a mortar and pestle to obtain aluminum oxide (al2o3) powder. the nanocomposite was synthesized by mixing pmma matrix, cellulose nanocrystalline, and zro2-al2o3 powder with three different compositions: (i) pmma without filler, (ii) pmma 50% with cellulose nanocrystalline 1% and zro2-al2o3 powder 49%, and (iii) pmma 50% with cellulose nanocrystalline 2% and zro2-al2o3 powder 48%. specimens were made according to ada specification no. 27.11 the micro vickers hardness test specimen was of ø 6 mm x 3 mm thickness. it was tested with lecojapan m-400-h1 exposed to 100g force for 15 seconds in copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 32 febrina et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 30–35 the physical metallurgy laboratory, institut teknologi bandung. the specimen for the three-point bending was a beam of 2 mm x 2 mm x 25 mm, exposed to 1 kn force and a crosshead speed of 1mm/min using the shimadzu autograph ags-5knx in the faculty of dentistry, maranatha christian university. materials for making specimens were pmma resin without filler and its monomer, zro2-al2o3 filler, and crystalline nanocellulose. transmission electron microscopy (tem) was conducted using a hitachi ht7700 (25000x magnification). x-ray diffraction (xrd) with 0–90° angle was performed using a bruker-d8 advance. both characterizations were conducted at the center of advanced science, institut teknologi bandung. results the average hardness values in test group i were 9.61 ± 1.64 vhn in test group i, 11.34 ± 0.81 vhn in test group ii, and 12.73 ± 0.6 vhn in test group iii. the data for the micro vickers hardness were tested for normality with the shapiro–wilk test. the p value of < 0.05 confirmed that the data distribution was not normal. the data for the micro vickers hardness test was then tested for homogeneity. it showed a p value of 0.756 (> 0.05), confirming that the data was homogeneous. since the data distribution was not normal, the kruskal-wallis non-parametric test was used for statistical analysis. the results show that the p value (asymp. sig.) was 0.004 (< 0.05), confirming that there was a significant difference in micro vickers hardness between the test groups. the mann–whitney non-parametric test was performed between two groups to check which differences between groups were significant. the test between groups i and ii resulted in a p value (asymp. sig. 2-tailed) of 0.016 (< 0.05), confirming that there was a significant difference in the hardness between these groups. the test between groups ii and iii resulted in a p value (asymp. sig. 2-tailed) of 0.028 (< 0.05), establishing that there was a significant difference in hardness between groups ii and iii. the test between groups i and iii produced a p value (asymp. sig. 2-tailed) of 0.009, confirming a significant difference in hardness between groups i and iii. table 1 shows the micro vickers hardness test results. the three-point bending test was carried out on 15 specimens which were divided into three groups, namely group i (pmma without the addition of filler), group ii (pmma with the addition of crystalline nanocellulose, zirconia and alumina fillers in a ratio of 50:1:49), group iii (pmma with the addition of crystalline nanocellulose, zirconia and alumina fillers in a ratio of 50:2:48) table 2 shows the results of the three-point bending test. table 2. three-point bending test result (mpa) sample group i ii iii 1 18.17 8.56 10.48 2 20.94 8.55 10.20 3 18.64 7.24 10.60 4 20.30 9.67 9.33 5 18.95 6.77 8.36 average 19.4 8.15 9.79 table 1. micro vickers hardness test results (vhn) sample group i ii iii 1 10.03 12.3 13.06 2 10.26 11.8 13.6 3 10.03 11.8 12.1 4 9.73 10.76 12.9 5 8.03 10.06 12.0 average 9.61 11.34 12.73 figure 1. results of tem characterization of crystalline nanocellulose showing fiber-shaped particles (25000x magnification). copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 33febrina et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 30–35 the data contained in table 1 were tested for normality with the shapiro–wilk test. the p value (sig.) was > 0.05 in all groups, confirming that the data distribution was normal. the homogeneity test showed a p value (sig.) of 0.742 (> 0.05), establishing that the data was homogeneous. since the data were normal and homogeneous, a one-way analysis of variance was used for statistical analysis. the result of the p value (sig.) was 0.000 (p < 0.05), indicating there was a significant difference in the flexural strength values between the test groups, and the null hypothesis was successfully rejected. tukey’s post hoc test then processed the data to find which groups had a significant difference in value. tukey’s post hoc test results showed that group i (nanocellulose content of 0.00%) significantly differed in flexural strength values compared with groups ii and iii. crystalline nanocellulose was characterized using tem to measure its particle size, while zirconia and alumina were characterized using xrd to analyze their crystal structure. the results of the characterization of crystalline nanocellulose in the form of a gel that had previously been prepared using isopropyl ethanol using tem can be seen in figure 1. the results of xrd characterization of zirconia and alumina show peaks describing the diffractogram’s crystallinity in figures 2 and 3, respectively. figure 2. xrd characterization results of zirconia powder with a calcination temperature of 900°c. figure 3. xrd characterization results of alumina powder with a calcination temperature of 550°c. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 34 febrina et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 30–35 discussion polymethylmethacrylate is often used as a resin matrix in dentistry because it has sufficient strength, good translucency, malleability, and resistance to microbial colonization. it is odorless, rigid, tasteless, and nonirritating to tissues. however, it is brittle compared to other composite resins. however, this deficiency can be overcome by adding filler.1,12 the fillers used in this study were zirconia, alumina, and crystalline nanocellulose. zirconia was synthesized with zrcl4 as a precursor and alumina with al(no3)3.9h2o as a precursor. zirconia and alumina were obtained using the sol-gel technique. crystalline nanocellulose in this study was synthesized using the acid hydrolysis method using h2so4. the temperature was lower, and the processing time was faster to dissolve lignin and hemicellulose in the precursor to obtain crystalline nanocellulose. in this study, palm kernel cake was used. as a precursor. palm kernel cake is often used as a source of crystalline nanocellulose because it has high levels of hemicellulose, cellulose, and lignin, making it suitable cellulose-forming biomass. palm kernel cake has a cellulose content of 30%, so it is produced quite a lot in the palm oil industry as a residual extraction material. due to its abundant availability, palm kernel cake was used as animal feed before being widely used as cellulose-forming biomass. palm kernel cake is environmentally friendly because it utilizes waste from palm oil extraction.13 the synthesized filler was characterized to see the particle size and morphology. the results of the tem characterization of crystalline nanocellulose showed that the crystalline nanocellulose produced using the acid hydrolysis method had particle diameter sizes ranging from 6.80 nm to 25 nm and were in the form of fibers. nanocellulose in fiber form generally has a diameter of 2–20 nm and a length of 100–600 nm. the acid hydrolysis process at 45°c typically produces crystalline nanocellulose in spherical form, but it does not rule out the possibility of forming nanocellulose as fiber. the shape of this fiber depends on the transfer of stress. if the fiber is long, the modulus of elasticity will increase, so the stress transfer will be better.14,15 the distribution of nanocellulose seen in the tem in this study was not homogeneous and experienced agglomeration. agglomeration may be caused by a preparation error during characterization when mixing isopropyl ethanol.16 the results of xrd characterization of the alumina filler contained one crystallite phase, namely, alumina with a metastable phase, identified from alumina powder calcined at a temperature of 550°c. a metastable condition is one in which a material has critical stability. external influences, such as humidity or specific temperature changes, can disrupt the stability causing the material to fall to a lower energy level. when a peak is narrower and sharper, the degree of crystallinity of the material is higher. a higher degree of crystallinity increases the mechanical properties of the material. the calcination temperature affects the crystallite structure and phases formed in a material. in this study, zirconia was calcined at 900°c for two hours so that two crystallite phases were identified, namely tetragonal and monoclinic zirconia, where the tetragonal phase was more dominant than the monoclinic. the tetragonal structure has advantages over the monoclinic and cubic structures because it has a relatively high resistance to cracking.17–19 metastable alumina is the purest form of alumina and has high porosity and surface area. due to its acidic and basic properties, it is often used as a catalyst, absorbent material, catalyst support, filler, or polymer composite component with reasonably good mechanical properties.20 based on their composition, there were three groups of nanocomposite specimens: i, ii, and iii. a comparison of the filler composition, ranging from 50–85% of the matrix, was selected.21 in this study, the ratio of the matrix and filler was 50%:50%. this ratio was chosen because it is easier to mix, even though theoretically, the more the filler, the better the mechanical strength. adding more filler than the matrix makes mixing difficult, mainly if manual mixing and agglomeration are performed. the statistical analysis between test groups i and ii showed that the p value was 0.016 (< 0.05), indicating a significant difference in hardness between groups i and ii. similarly, the results of the statistical analysis conducted between groups ii and iii resulted in a p value of 0.028 (< 0.05), indicating a significant difference in hardness between groups ii and iii. thus, adding zirconia, alumina, and crystalline nanocellulose fillers can increase the hardness of pmma because crystalline nanocellulose acts as a binding material between pmma and zirconia and alumina fillers. crystalline nanocellulose has a tensile strength of 7.5–7.7 gpa (greater than steel), and the modulus of elasticity is 150 gpa. nanocellulose is often used as a reinforcing agent because it has good mechanical strength and fibers that can transfer stress. the pmma hardness increases with the amount of crystalline nanocellulose added as filler compared with the test group without filler.22,23 table 1 shows the three-point bending test results. the average results of the test were 19.4 mpa in group i, 8.15 mpa in group ii, and 9.79 mpa in group iii. the average value was the highest in group i compared with the other groups that were given filler. this is because adding zirconia, alumina, and crystalline nanocellulose fillers in test groups ii and iii makes the compound more brittle, lowering its flexural strength as compared with group i not given a filler. this is due to the poor mixing process, whereby crystalline nanocellulose is not bonded and mixed homogeneously. in test groups ii and iii, there was an increase in flexural strength because cellulose is a natural polymer that is fibrous, strong, has good mechanical properties, and is often used as a reinforcing agent. cellulose has a tensile strength of 7.5—7.7 gpa, greater than steel, and a modulus of elasticity of 150 gpa, copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 35febrina et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 30–35 demonstrating that mechanical strength will increase the more crystalline nanocellulose is added to a material.24 the hardness value of pmma is around 20 vhn, and the flexural strength is 70 mpa.25 the hardness value of enamel is 300 vhn, and the flexural strength is 85 mpa, whereas the hardness value of dentin is 60 vhn, and the flexural strength is 15 mpa. the average hardness value was only 12.73 vhn in test group iii, and the mean flexural strength was 9.79 mpa, lower than expected. this could be due to the poor polymerization process because of the presence of impurities in the specimen. moreover, the heating was done over a stove and hot water. mixing the polymer, matrix, and monomer manually resulted in a less homogeneous and more porous compound due to air trapping. further, a coupling agent was not used. in this study, nanocomposite has been successfully synthesized from crystalline nanocellulose, zro2, and al2o3. tem results of crystalline nanocellulose showed that the fiber generally has a diameter of 2–20 nm and a length of 100–600 nm. xrd results show that zro2 has tetragonal and monoclinic forms. these phases have good resistance to cracking. alumina has a metastable phase, showing the material has critical stability. the addition of crystalline nanocellulose, zirconia, and alumina reduces the flexural strength but increases the hardness of the pmma matrix nanocomposite. the best hardness was achieved in group iii (12.73 ± 0.6 vhn), which shows an improvement compared with pmma without filler addition (9.61 ± 1.64 vhn). references 1. anusavice k, shen c, rawls hr. phillips’ science of dental materials. 12th ed. philadelphia: saunders; 2013. p. 592. 2. powers j, wataha j. dental materials: foundations and applications. 11th ed. st. louis: mosby; 2016. p. 272. 3. mccabe jf, walls awg. applied dental materials. 9th ed. oxford: wiley-blackwell; 2013. p. 320. 4. jacobsen p. restorative dentistry: an integrated approach. 2nd ed. oxford: wiley-blackwell; 2008. p. 352. 5. galvão mr, caldas sgfr, bagnato vs, de souza rastelli an, de andrade mf. evaluation of degree of conversion and hardness of dental composites photo-activated with different light guide tips. eur j dent. 2013; 7(1): 86–93. 6. scribante a, bollardi m, chiesa m, poggio c, colombo m. flexural properties and elastic modulus of different esthetic restorative materials: evaluation after exposure to acidic drink. biomed res int. 2019; 2019: 5109481. 7. gundogdu m, kurklu d, yanikoglu n, kul e. the evaluation of flexural strength of composite resin materials with and without fiber. dentistry. 2014; 4(9): 259. 8. erdemir u, yildiz e, eren mm, ozel s. surface hardness evaluation of different composite resin materials: influence of sports and energy drinks immersion after a short-term period. j appl oral sci. 2013; 21(2): 124–31. 9. özyürek t, topkara c, koçak i̇, yılmaz k, gündoğar m, uslu g. fracture strength of endodontically treated teeth restored with different fiber post and core systems. odontology. 2020; 108(4): 588–95. 10. ravikumar, prasad mss. fracture toughness and mechanical properties of aluminum oxide filled chopped strand mat e-glass fiber reinforced – epoxy composites. int j sci res publ. 2014; 4(7): 1–7. 11. sunendar b, fathina a, harmaji a, mardhian df, asri l, widodo hb. the effect of cha-doped sr addition to the mechanical strength of metakaolin dental implant geopolymer composite. in: aip conference proceedings 1887. aip publishing; 2017. p. 020020. 12. manappallil j. basic dental materials. 4th ed. new delhi: jaypee brothers medical publishers (p) ltd.; 2016. p. 630. 13. sawalha s, ma’ali r, surkhi o, sawalha m, dardouk b, walwel h, haj ahmad d. reinforcing of low-density polyethylene by cellulose extracted from agricultural wastes. j compos mater. 2019; 53(2): 219–25. 14. roszowska-jarosz m, masiewicz j, kostrzewa m, kucharczyk w, żurowski w, kucińska-lipka j, przybyłek p. mechanical properties of bio-composites based on epoxy resin and nanocellulose fibres. mater (basel, switzerland). 2021; 14(13): 3576. 15. khalil hpsa, davoudpour y, aprilia nas, mustapha a, hossain s, islam n, dungani r. nanocellulose-based polymer nanocomposite: isolation, characterization and applications. in: nanocellulose polymer nanocomposites. hoboken, nj, usa: john wiley & sons, inc.; 2014. p. 273–309. 16. sept eva n i a a, a n na ma la i pk , ma r t i n dj. sy nt hesis a nd characterization of cellulose nanocrystals as reinforcing agent in solely palm based polyurethane foam. in: aip conference proceedings 1904. aip publishing; 2017. p. 020042. 17. sasaki k, hayashi t, asakura m, ando m, kawai t, ban s. improving biocompatibility of zirconia surface by incorporating ca ions. dent mater j. 2015; 34(3): 336–44. 18. kang ms, jang hj, lee sh, lee je, jo hj, jeong sj, kim b, han d-w. potential of carbon-based nanocomposites for dental tissue engineering and regeneration. mater (basel, switzerland). 2021; 14(17): 5104. 19. asri l, septawendar r, sunendar b. zirkonia untuk aplikasi material restorasi gigi. j keramik dan gelas indones. 2016; 25(2): 79–88. 20. gafur ma, al-amin m, sarker msr, alam mz. structural and mechanical properties of alumina-zirconia (zta) composites with unstabilized zirconia modulation. mater sci appl. 2021; 12(11): 542–60. 21. faza y, hasratiningsih z, harmaji a, joni im. preparation and characterization of zirconia-alumina system via solution and solid phase mixing method. in: aip conference proceedings 1927. aip publishing; 2018. p. 030030. 22. trache d, tarchoun af, derradji m, hamidon ts, masruchin n, brosse n, hussin mh. nanocellulose: from fundamentals to advanced applications. front chem. 2020; 8: 392. 23. lin n, dufresne a. nanocellulose in biomedicine: current status and future prospect. eur polym j. 2014; 59: 302–25. 24. george j, sabapathi sn. cellulose nanocrystals: synthesis, functional properties, and applications. nanotechnol sci appl. 2015; 8: 45–54. 25. hasratiningsih z, takarini v, cahyanto a, faza y, asri latw, purwasasmita bs. hardness evaluation of pmma reinforced with two different calcinations temperatures of zro 2 -al 2 o 3 -sio 2 filler system. iop conf ser mater sci eng. 2017; 172: 012067. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p30–35 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p30-35 vol 51 no 3 jul sep 2018_pus.indd 147 o r a l l e s i o n s a s a c l i n i c a l s i g n o f s y s t e m i c l u p u s erythematosus eliza kristina m. munthe and irna sufiawati department of oral medicine faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: oral lesions represent one of the most important clinical symptoms of systemic lupus erythematosus (sle), an autoimmune disease with a high degree of clinical variability rendering it difficult to arrive at a prompt and accurate diagnosis. there are many unknown causes and multiple organ systems involved, with the result that permanent organ damage may occur before treatment commences. purpose: the purpose of this case report is to discuss the importance of recognizing the lesions related to sle which may help dentists to make an early diagnosis. case: a 17-year-old female patient was referred by the internal medicine department with a suspected case of sle. prior to admittance to the hospital, the patient was diagnosed with tuberculosis. a subsequent extraoral examination revealed ulceration with a blackish crust on the upper lip. an intraoral examination showed similar ulceration covered with a blackish crust on the labial mucosa accompanied by central erythema in the hard palate. blood tests indicated decreased levels of hemoglobin, hematocrit and platelets, but increased levels of leukocytes. a diagnosis of oral lesions associated with sle and angioedema was formulated. case management: the patient was given 1% hydrocortisone and vaseline album for extraoral lesions, while 0.2% chlorhexidine gluconate and 0.1% triamcinolone acetonide was used to treat intraoral lesions. an improvement in the oral lesions manifested itself after two weeks of treatment. conclusion: early detection of oral lesions plays a significant role in diagnosing sle. it is important for the dentist to recognize the presentation of diseases that may be preceded by oral lesions. a multidisciplinary approach and appropriate referrals are necessary to ensure comprehensive medical and dental management of patients with sle. keywords: early detection; oral lesions; systemic lupus erythematosus correspondence: irna sufiawati, department of oral medicine, faculty of dentistry, universitas padjadjaran, jl. sekeloa selatan no.1, bandung 40132, indonesia. e-mail: irna.sufiawati@fkg.unpad.ac.id dental journal (majalah kedokteran gigi) 2018 september; 51(3): 147–152 case report introduction systemic lupus erythematosus (sle) is a systemic autoimmune disorder with a broad spectrum of clinical manifestations, multi-organ inflammation and a multitude of laboratory and immunologic abnormalities. depending on the body part involved, clinical sle courses are characterized by relapse and remittance that can be mild, moderate or severe. the difficulty in identifying sle patients in the early stages of the disease stems from its complexity. the production of pathogenic autoantibodies directed against nucleic acids and their binding proteins is one symptom of the disease, reflecting global loss of self-tolerance. immune dysregulation disorder with reduced tolerance results from a combination of genetic factors, the regulation of environmental triggers and stochastic events. recent research has produced data that more than 30 genetic loci are involved in the pathogenesis of the disease.1–4 sle has an incidence rate that varies between ethnic groups and geographic locations, genders and age groups. a prevalence of approximately 20 to 150 cases per 100,000 people within the general population has been reported.4 women are estimated to be six to ten times more likely to develop sle than men with double x chromosomes and different estrogen levels which modulate the immune response possibly being associated with this event. sle mainly affects young women, with a peak incidence rate occurring between the ages of 15 and 40. the annual incidence rate of sle in adults is estimated to be 2-7.6 cases per 100,000 individuals, while that in children in the us is estimated to be 0.53-0.60 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 148munthe and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 147–152 per 100,000. individuals in addition, the onset of childhood sle has a far greater impact than that in adults due to its following the clinical course of a more severe disease. a worse prognosis is also often associated with male and elderly patients.5 a tendency to lower photosensitivity, more serious serositis, older age at the time of diagnosis and a higher 1-year mortality rate compared to women are all characteristics of sle in men.6 the mortality rate of sle patients remains two to four times higher than that of healthy people.4 the etiology of sle remains unknown. however, genetic, hormonal and environmental factors, in addition to immune disorders, have been identified as elements in its pathogenesis6,7, with sle being linked to a single gene defect. indeed, genetic factors exert a fundamental influence on disease progression, as do exposure to environmental stimuli in the form of ultraviolet light, dietary factors, certain infections and drugs, smoking, dna demethylation, and infectious or endogenous viruses or elements similar to viruses.8,9 certain gene products that interact with environmental stimuli are etiological factors that will produce a deregulated immune response. various organ system damage will be caused by sle, as a consequence of the formation and deposition of autoantibodies and immune complexes.10 the prevalence of mucosal involvement is present in approximately 9-45% of cases and accompanied by a systemic form of the disease. according to the literature, oral lesions occur more frequently in women with a female to male ratio that is 2.7 times greater.7 this report describes the case of a patient who presented early clinical features of oral lesions that constitute an important symptom in the diagnosing of sle. dentists have an important role in the early detection of sle since oral lesions represent one of the clinical symptoms of the condition. case a 17-year-old female patient was referred by the internal medicine department to the oral medicine department in the emergency unit at rs hasan sadikin bandung, the chief complaint being bleeding and blood clots on the left upper lips and labial mucosa. these caused difficulties when eating and drinking with patients also complaining of problems swallowing accompanied by general physical weakness. the first complaint manifested itself approximately four months before admittance to hospital with patches of reddish spots accompanied, at times, by itching on both arms and neck after patients had been administered with the drug. this was described as a pulmonary medicine consisting of red and yellow tablets which had been prescribed by a general practitioner. after three months of following the drug regime, the patient was referred to rotinsulu hospital and treated with five different forms of anti-tubercular medication (unfortunately, the family of the patient was unable to recall the name and shape of the drugs in question). since less than three weeks earlier, patches of reddish spots were presenton both arms and the neck extending to the chest, abdomen and back, while blackish spots were visible on the skin. the presence of similar complaints or a history of allergies or recurrent stomatitis aphthous in patients and their families were denied. however, there was a history of joint pain and hair loss commencing approximately two weeks before admittance to hospital. an examination adhering to american college of rheumatology (acr) criteria was conducted and produced four positive results in 11 criteria including: arthritis, malar rash, renal disease and hematologic abnormalities. multidisciplinary management was performed by the oral medicine, internal medicine, dermatology and venereology departments. the patient is suspected of suffering from sle with renal involvement, mucocutaneous candidiasis (cmc), hematology, acute kidney injury (aki), superimposed chronic kidney disease (ckd) et causa lupus nephritis with metabolic acidosis, uremic gastropathy, moderate dehydration et causa gastrointestinal (gi) loss and hyponatremia et causa gi loss. hematological examination showed a decrease in hemoglobin, hematocrit, platelets, and an increased in leukocytes. the thorax x-ray examination confirmed cardiomegaly, rather than active pulmonary tb. the results of hematological, blood urea and creatinine examinations performed during the patient’s treatment at hasan sadikin hospital (rshs) bandung can be seen in figure 1 and 2. figure 2. ureum and creatinin examination result.figure 1 hematological examination result.. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 149 munthe and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 147–152 of less pain and there was an accompanying improvement in the lesions on the lip, labial mucosa and palate during inpatient treatment. however, a blackish crust on the lips and ulceration of the labial mucosa were still evident. the central erythema of the palate, although showing signs of healing, was present (figure 4). the previous therapy was continued and, given the dryness of the patient’s lips, vaseline album was added three times a day and she was instructed to rinse the mouth thoroughly with 10ml of 0.2% chlorhexidine gluconate three times a day. on the third visit seven days after the initial visit, the oral lesions had largely healed, except for the ulceration of the labial mucosa (figure 5). consequently, 0.1% triamcinolone acetonide in orabase was applied to the lesions on the labial mucosa three times a day. those patients who had previously tested positive for antinuclear antibody (ana) with a homogeneous pattern were discharged from hospital. a week later, the lesions were observed to have completely healed. the pain within the oral cavity was eradicated, while the crust on the lips, ulceration on the labial mucosa and central erythema of the palate were all healed (figure 6). nevertheless, the patient was instructed to continue rinsing her mouth with 10ml of 0.2% chlorhexidine gluconate three times a day. discussion on examination of the patient in question, oral lesions were found on the lips, labial mucosa and hard palate. based on acr criteria, oral lesions are one of the clinical symptoms in the diagnosis of sle. more than 40% of patients suffering from the condition experience ulceration the patient was hospitalized for eight days during which period an initial regimen was applied by the department of internal medicine including: systemic treatment with 3-4 liters of oxygen per minute, infusion of 0.9% sodium chloride (nacl) 1800 cc every 24 hours, topical treatment of the entire body using 10% urea lotion, the application to the lips and buttocks of open compresses with 0.9 % nacl three times a day and 0.1% gentamicin sulfate cream to the buttocks twice a day. extraoral clinical examination of the patient revealed a pale conjunctiva with painful ulceration covered by a blackish crust on her left upper lip accompanied by a reddish edema on her upper lip (figure 3a). an intraoral examination revealed painful ulceration covered by a blackish crust on her upper labial mucosa (figure 3b), while central erythema was present in the hard palate (figure 3c). intraoral assessment is limited because of the condition of those patients who experience pain when opening their mouths. based on anamnestis and clinical findings, a clinical diagnosis of oral lesions associated with sle and angioedema was made. such lesions can be differentiated from other oral lesions, for example, erythema multiforme, oral lichen planus and erythematosus candidiasis. case management the oral lesions were treated with 1% hydrocortisone for those on the lips, 0.2% chlorhexidine gluconate was compressed on the labial mucosa and the palate three times a day. a 0.2% chlorhexidine gluconate was compressed on lesions in the oral cavity due to the inability of the patient to open her mouth fully or rinse it with mouthwash. on the second visit three days after the first, the patient complained figure 3. extra oral and intra oral examination on the first visit. a) ulceration covered by blackish crust on left upper lip. b) ulceration covered by blackish crust on upper labial mucosa. c) central erythema on hard palate. a b c figure 4 extra oral and intra oral examination on the second visit. a) the crust was still present on the lips. b) a blackish crust and ulceration of the labial mucosa were still present. c) central erythema of the palate was still present and showed a healing. ba c dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 150munthe and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 147–152 in the oral mucosa. another study reported the prevalence of oral lesions in patients with sle to be approximately 7-52%.11–13 early management of sle must be initiated due to the several clinical manifestations of oral lesions present when the disease is active. according to acr classification criteria, le-specific and non-le specific are typical oral lesions.14,15 the specific oral lesions associated with le consist of palatal erythematous ulcers, oral discoid lupus erythematosus, honeycomb plaques and verrucous le. the le nonspecific oral ulcers comprise aphthous ulcers, lupus cheilitis and other types of oral lesions. within the acr criteria, a typical oral ulcer which is the most specific for an sle, is a palatal erythematous ulcer usually located on the hard palate with single/multiple lesions that are painless in masticatory or mucosal keratinization. when the disease is active, this constitutes an acute symptom of the disease and is occasionally the first evidence of sle unaccompanied by skin lesions. haemorrhaging usually occurs before the early lesion developes into an ulcer. the progression of the lesion into a reticulated restricted ulcer will be preceded by the appearance of solitary erythema and a patch of hemorrhaging which is a lupus-specific lesion. non-painful lesions located on the hard palate are typical of this type. oral dle is an atrophic plaque accompanied by white radiating keratotic striae and telangiectasia located in the lining layer, covering the buccal mucosa and soft palate. a honeycomb plaque is classified as chronic and well-circumscribed with white lacy hyperkeratosis and buccal erythema. intense keratotic and raised plaques that are usually found in the mucosal lining, such as in the buccal mucosa, lips and hard palate (e.g. alveolar ridge) are characteristic features of le verucosa that may be involved.14,15 in contrast, lesions on the buccal mucosa, lips and nasal septum, which are usually painful and tend to bleed, are characteristic of nonspecific aphthous ulcers more common in juvenile than adult sle patients and usually occur when the disease is active. lupus cheilitis may turn into a painful ulcer which presents clinically as inflammation of the buccal lips including small or diffuse erythematous and edematous plaques. ulcers, usually shallow and 1-2 mm in diameter and present in approximately one third of patients, may extend to the pharynx. lower lip vermilion is often related to cheilitis (typical of lupus cheilitis). labial lesions are very common, possible affected areas being the upper and lower lips. erosion, crusting and necrosis often occur in addition to the onset of erythema and edema. a rare clinical manifestation is bullous sle whose lesions typically have clinical features which include multiple tense bullae on the face, neck and trunk.11,12,14–16 according to the results of examinations conducted by the internal medicine department, the patient recorded four positive results in 11 acr criteria including arthritis, malar rash, renal disease and hematologic abnormalities. the clinical symptoms of this patient included those of arthritis with joint pain being experienced in the two weeks before admission to hospital. nonerosive arthritis is a hallmark of sle, often being the earliest manifestation, recorded in up to 53–95% of patients suffering from the condition. the clinical symptoms may be misinterpreted as another type of inflammatory arthritis, rendering diagnosis of sle difficult.3,5,17,18 other observable clinical symptoms in these patients included irregular shaped, 0.1 x 0.1cm to 20 x 35cm sized, hyperpigmented macules with partially clear boundaries on the face, both arms, chest, abdomen and back. this complaint was first experienced less than four months ago with the figure 5. extra oral and intra oral examination on the third visit. a) the crust on the lip was heal. b) a blackish crust was heal, but there was still ulceration on the labial mucosa. c) central erythema of the palate was still present but showed a healing. a cb figure 6. extra oral and intra oral examination on the fourth visit a) the crust on the lip was healed. b) ulceration on the labial mucous was healed. c) central erythema of the palate was healed. cba dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 151 munthe and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 147–152 appearance of occasionally itchy red spots on both arms and the neck after the taking of medicine prescribed by a general practitioner who declared it to be for the treatment of lung conditions. approximately three weeks before admission to hospital, the existing red spots spread to the chest, abdomen and back turning blackish in colour and sometimes feeling itchy. one of the worst affected organs in sle patients is the skin, which may actually be the sole organ involved, as in cutaneous le.4 a study reported that the prevalence of malar rash among sle patients was high (73.5%).17 examination by the hematology laboratory following initial treatment of the patient revealed low levels of hemoglobin, hematocrit, erythrocytes and platelets, but high levels of leukocytes. the examination results also revealed anemia that was classified as normocytic-normochromic in nature based on the mean corpuscular volume and mean corpuscular hemoglobin concentration. hematologic abnormalities in sle can constitute a symptom of the presentation of sle that is common, varied and affects each cell. anemia, leukopenia, thrombocytopenia and antiphospholipid syndrome are the main clinical manifestations of sle. anemia is found in approximately half of sle patients. it is common and correlates to disease activity with pathogenesis including: chronic anemia, hemolysis (autoimmune or microangiopathy), blood loss, renal insufficiency, medications, infection, hypersplenism, myelodysplasia, myelofibrosis and aplastic anemia.3,19 the high levels of blood urea and creatinine indicated a problem with the patient’s kidney. renal involvement is common in sle patients, with an incidence rate of 40-70%, together with significant rates of morbidity and hospitalization. an extremely common characteristic of lupus-related renal disease is the occurrence of proteinuria at various levels.3 all patients who experience oral disease, be it painful or painless, must be considered to have lupus, rather than the condition being automatically associated with systemic disease. oral lesions may be the initial symptom of lupus.3 57% of the mucosal lesions studied proved painful, while other observations confirmed 82% of oral ulcers to be painless. the contrasting findings of these two studies may be due to differences in the types of lesions studied. erythematous lesions are usually painless, whereas discoid lesions often cause considerable discomfort. careful examination of the oral cavity in all lupus patients must be undertaken because of the significant proportion of asymptomatic oral lesions. at the active stage of the disease, discoid and ulcer lesions are often observed and subside with remission of the disease.11,12,14 oral lesions and the duration of the disease are closely associated, the duration of the prolonged disease being associated with fewer oral mucosal lesions. for the duration of the most active period of the disease the greatest number of oral mucosal lesions can be encountered. treatment of the disease confirms that it is under control and in an inactive phase.13,20 specific genes that interact with environmental stimuli can initiate the onset of systemic lupus erythematosus resulting in a deregulated immune response. environmental stimuli include: ultraviolet light, diet, certain infections and drugs, smoking, demethylation of dna and infection or endogenous viruses or viral-like elements. one suspected cause of sle might be that of drug use where the patient had previously been prescribed the aforementioned drug (red and yellow tablets) as pulmonary medicine for tb approximately eight months before hospitalization (five months with a general practitioner and three months in rotinsulu). various drugs have been identified as, possible, probable or definite causes of lupus. patients without a diagnosis or history of sle should be suspected of having drug-induced lupus (dil) following a positive ana examination and at least one clinical feature of lupus after the appropriate duration of a drug regime, with symptoms disappearing after discontinuation of the drug’s use.3 when the clinical and serologic manifestations of sle appear in patients with historical use of certain medications, a diagnosis of dil must be made.19 certain drugs can certainly induce autoantibodies in a large number of patients, a condition referred to as drug-induced lupus. more than 100 drugs that cause dil have been reported, including a number of new biological and antiviral agents. the best known of these drugs are procainamide, hydralazine, quinidine, phenytoin and isoni azid. the pathogenesis of dil is not fully understood, but genetic predisposition plays an important role in the case of certain drugs. gene expression in cd4+ t cells is altered by drugs with the inhibitory mechanisms of dna methylation and induction of over-expression of lymphocyte function related to antigen-1, causing autoreactivity.3,18,21 the patient’s management in this case involved the departments of internal medicine, dermatology, venereology and oral medicine. while hospitalized, her intraoral lesions gradually healed following two weeks of treatment. oral lesions associated with sle are often difficult to resolve. the same regimen used to treat the overall le process is also applied in the treatment of oral le lesions. the treatment of oral ulcers in sle includes the use of topical anti-inflammatory agents. some of the most common drugs used in sle patients are topical corticosteroids (e.g., 0.1% triamcinolone oral paste). intralesional applications of corticosteroid may also be considered. the severity of symptoms is related to the duration of corticosteroid use. if the treatment of oral lesions is refractory or there is no appropriate response to topical steroids within two weeks, then a more potent agent (for example, betamethasone or clobetasol in oral preparation) or the use of antimalarial agents and systemic drugs, including steroids, thalidomide, clofazimine and methotrexate may be needed.15,22 the oral lesions were treated with 1% hydrocortisone, 0.2% chlorhexidine gluconate and 0.1% triamcinolone acetonide in orabase which promoted healing after a 2-week period of treatment. chlorhexidine gluconate was used to disinfect the skin and cleanse traumatic wounds, being an antiseptic capable of eliminating bacteria by combining the mechanical action of an inert liquid and producing active chemical antimicrobial effects without damaging the host tissue. chlorhexidine gluconate produces an effect on the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 152munthe and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 147–152 prokaryotic cell membrane rendering it active against a broad spectrum of microbes including: gram-negative and grampositive bacteria and fungi through its effect on prokaryotic cell membranes, while exhibiting low toxicity to mammalian tissue.23 sle is a fatal disease requiring complex management because it involves multiple organs and, therefore, necessitates a multidisciplinary approach.4 while the symptoms of sle can be controlled, the condition itself cannot be cured. consequently, sle treatment is primarily associated with improved disease control and patient survival. the management objectives of sle are those of reducing inflammation and symptoms, while maintaining re mission. protection from ultraviolet light can prevent an eruption of lupus skin lesions.18,19 determining the severity of the disease, including the presence of organ dysfunction and the level of inflammation, before planning sle treatment is mandatory. high blood pressure, limited real function, low blood count and low levels of serum protein are associated with a poor prognosis.4,24 an important role is played by the dentist in arriving at a diagnosis with the aid of clinical and histopathological findings before the cutaneous lesions become apparent. to avoid infection, a thorough clinical examination is required. patients with sle can rapidly develop infection due to disease or therapy-related immunosuppression. before surgery, the results of the latest laboratory tests can be analysed to determine platelet counts, prothrombin time and international normalization ratio for blood clotting time. local measures may also prove essential to maintain hemostasis.18,20 in conclusion, oral lesions are one of the important clinical symptoms in the diagnosing of sle which are frequently found in patients suffering from the condition. a painless erythematous palate ulcer in the masticatory or keratinized mucosa, especially the hard palate, is a typical lesion. an appropriate understanding of the complex clinical features and locations of sle is very important in order that effective dental management can be provided by a dentist. detection of oral lesions plays a significant role in diagnosing sle. it is important for the dentist to recognize the symptoms of diseases to enable the definitive diagnosis and appropriate treatment to be identified, thereby enhancing the prognosis for patients. a multidisciplinary approach and appropriate referrals ensure complete dental and medical management of patients with sle. references sebastiani gd, prevete i, iuliano a, minisola g. the importance of1. an early diagnosis in systemic lupus erythematosus. isr med assoc j. 2016; 18(3–4): 212–5. choi j, kim st, craft j. the pathogenesis of systemic lupus erythe-2. matosus-an update. curr opin immunol. 2012; 24(6): 651–7. bertsias g, cervera r, boumpas dt. systemic lupus erythemato-3. sus: pathogenesis and clinical features. in: textbook on rheumatic diseases. 2nd ed. zurich: eular; 2015. p. 476–505. fortuna g, brennan mt. systemic lupus erythematosus: epidemiol-4. ogy, pathophysiology, manifestations, and management. dent clin north am. 2013; 57(4): 631–55. ben-menachem e. systemic lupus erythematosus: a review for5. anesthesiologists. anesth analg. 2010; 111(3): 665–76. maidhof w, hilas o. lupus: an overview of the disease and manage-6. ment options. p t. 2012; 37(4): 240–9. kuhn a, bonsmann g, anders h-j, herzer p, tenbrock k, schneider7. m. the diagnosis and treatment of systemic lupus erythematosus. dtsch aerzteblatt online. 2015; 112(25): 423–32. rivas-larrauri f, yamazaki-nakashimada ma. systemic lupus erythe-8. matosus: is it one disease? reumatol clínica. 2016; 12(5): 274–81. barbhaiya m, costenbader kh. environmental exposures and the9. development of systemic lupus erythematosus. curr opin rheumatol. 2016; 28(5): 497–505. 10. mackern-oberti jp, llanos c, riedel ca, bueno sm, kalergis am. contribution of dendritic cells to the autoimmune pathology of systemic lupus erythematosus. immunology. 2015; 146(4): 497–507. 11. wesley sj. oral manifestations of systemic lupus erythematosus : a case report. int j dent clin. 2014; 6(2): 35–6. 12. uva l, miguel d, pinheiro c, freitas jp, marques gomes m, filipe p. cutaneous manifestations of systemic lupus erythematosus. autoimmune dis. 2012; 2012: 1–15. 13. khatibi m, shakoorpour ah, jahromi zm, ahmadzadeh a. the prevalence of oral mucosal lesions and related factors in 188 patients with systemic lupus erythematosus. lupus. 2012; 21(12): 1312–5. 14. chiewchengchol d, murphy r, edwards sw, beresford mw. mucocutaneous manifestations in juvenile-onset systemic lupus erythematosus: a review of literature. pediatr rheumatol online j. 2015; 13: 1–9. 15. rodsaward p, prueksrisakul t, deekajorndech t, edwards sw, beresford mw, chiewchengchol d. oral ulcers in juvenile-onset systemic lupus erythematosus: a review of the literature. am j clin dermatol. 2017; 18(6): 755–62. 16. nico mms, bologna sb, lourenco s v. the lip in lupus erythematosus. clin exp dermatol. 2014; 39(5): 563–9. 17. khan a, shah mh, nauman m, hakim i, shahid g, niaz p, sethi h, aziz s, arabdin m. clinical manifestations of patients with systemic lupus erythematosus (sle) in khyber pakhtunkhwa. j pak med assoc. 2017; 67(8): 1180–5. 18. cojocaru m, cojocaru im, silosi i, vrabie cd. manifestations of systemic lupus erythematosus. mædica. 2011; 6(4): 330–6. 19. bashal f. hematological disorders in patients with systemic lupus erythematosus. open rheumatol j. 2013; 7: 87–95. 20. ranginwala am, chalishazar mm, panja p, buddhdev kp, kale hm. oral discoid lupus erythematosus: a study of twenty-one cases. j oral maxillofac pathol. 2012; 16(3): 368–73. 21. tsokos gc. systemic lupus erythematosus. n engl j med. 2011; 365(22): 2110–21. 22. nico mms, romiti r, lourenço s v. oral lesions in four cases of subacute cutaneous lupus erythematosus. acta derm venereol. 2011; 91(4): 436–9. 23. penn-barwell jg, murray ck, wenke jc. comparison of the antimicrobial effect of chlorhexidine and saline for irrigating a contaminated open fracture model. j orthop trauma. 2012; 26(12): 728–32. 24. nobee a, vaillant aj, akpaka pe, poon-king p. systemic lupus erythematosus (sle): a 360 degree review. am j clin med res. 2015; 3(4): 60–3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p147–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p147-152 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum 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(017/02.11/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 43 number 3 september 2010 issn 1978 3728 dental journal majalah kedokteran gigi 1. computer-aided diagnosis for osteoporosis based on trabecular bone analysis using panoramic radiographs agus zainal arifin, anny yuniarti, lutfiani ratna dewi, akira asano, akira taguchi, takashi nakamoto, arifzan razak, and hudan studiawan ........................................................ 107–112 2. anti-inflammation effects of sardinella longicep oil against paw oedema on rattus novergicus induced by 1% carrageenan rima parwati sari and yenny sugiharto ....................................................................................... 113–116 3. the correlation between immunoexpression of estrogen receptor and the severity of periodontal disease yuliana mahdiyah da’at arina, s. sunardhi widyaputra, and koeswadji ............................... 117–121 4. the effectiveness of sharp end and rounded end bristle toothbrush paulus januar, anastasia susetyo, and ratih widyastuti ............................................................ 122–125 5. contact hypersensitivity after tongue piercing ananta herachakri p, afrini puspita, feby aryani, and hendri susanto ................................ 126–130 6. cervical end preparation design on collarless metal ceramic crown to the decrease of bacterial colony edy machmud ................................................................................................................................. 131–135 7. treatment of sharp mandibular alveolar process with hybrid prosthesis sukaedi and eha djulaeha .............................................................................................................. 136–140 8. the combination of miacalcic, calcium lactate, and vitamin c as post-extraction alveolar bone resorption inhibitor sri kentjananingsih ........................................................................................................................ 141–145 9. the molecular phenomena of the blaz genes forming beta-lactamase enzymes structure in staphylococcus aureus resistant to beta-lactam antibiotics (ampicillin) mieke satari ..................................................................................................................................... 146–150 10. management of idiopathic alveolar bone necrosis associated with oroantral fistula after upper left first molar extraction ni putu mira sumarta ..................................................................................................................... 151–156 11. indirect veneer treatment of anterior maxillary teeth with enamel hypoplasia devi eka juniarti ............................................................................................................................. 157–161 vol 52 no 1 jan-mar 2019_new.indd 32 the different symptoms determining management of hand foot and mouth disease and primary varicella zoster infection maharani laillyza apriasari department of oral medicine, faculty of dentistry, universitas lambung mangkurat, banjarmasin, south kalimantan – indonesia abstract background: hand, foot and mouth disease (hfmd) is a medical condition endemic among children in south-east asia, including indonesia and, more specifically, banjarmasin – the capital of south sulawesi. the disease is mediated by enterovirus 71 and coxsackievirus 16 which attack the oral cavity, hands, feet, buttocks and genital areas. one differential diagnosis of this disease is primary varicella zoster infection. both diseases have similar clinical symptoms but different etiologies which can precipitate errors in the administration of therapy. purpose: to elucidate the distinction between hfmd and primary varicella zoster infection. case: an 8 year-old male sought treatment complaining of ulcers on the upper maxillary gingiva followed by the appearance of itchy and painful lesions affecting the nose, upper lip, hands and feet. the patient’s mother reported his history of 39oc fever followed by the development of red spots and ulcers on the face, hands and feet which caused itching. clinically, it is similar to primary varicella zoster infection which can affect any part of the body. the patient only used an immunomodulator once a day and was actively seeking available healthcare. case management: extraoral examination confirmed the presence of multiple erythematous vesicles and ulcers, 2 mm in diameter, which caused a sensation of itching around the nose and upper lip region. multiple painful and itchy red macules and vesicles, 3-6 mm in diameter, appeared not only on the patient’s palms, back of the hands and feet. intraoral examination of the right maxillary gingiva revealed multiple painful ulcers, 1-2 mm in diameter and yellowish in appearance, surrounded by erythema. the results of history-taking implied that no lesions appeared on other parts of the body. conclusion: while these conditions share similar clinical manifestations, their contrasting etiologies require different treatments. the ultimate diagnosis can be determined clinically by the dentist, thereby preventing errors in the administration of therapy. keywords: differential diagnosis; hand foot mouth disease; primary varicella zoster infection correspondence: maharani laillyza apriasari, department of oral medicine, faculty of dentistry, universitas lambung mangkurat, jl. veteran 128 b, banjarmasin 70122, indonesia. e-mail: maharaniroxy@gmail.com dental journal (majalah kedokteran gigi) 2019 march; 52(1): 32–35 case report introduction hfmd or singapore flu has frequently been endemic among children in the south east asia region,1 including banjarmasin, indonesia. a previous study estimated that in the city between 2014 and 2017 the prevalence of infectious diseases affecting the oral mucosal was 10.07% 2, one form of viral infection being hfmd.3,4 hfmd is an infectious disease presenting in the oral cavity and integumentum areas particularly the hands, feet and buttocks4 which is mediated by enterovirus 71 and coxsackievirus 16 infection.5,6 complications arising from viral infection may cause severe cns diseases such as meningitis and encephalitis, paralysis, pulmonary edema and death.4 previous studies have shown that close monitoring and timely management may prevent the severity of such complications and prevent death.5 clinical manifestations of hfmd include a fever followed by the development of red vesicles and ulcers in the oral cavity and integumentum system (on the hands, feet and genital area).7,8 lesions on the skin appear as red macula which progress to vesicles and ulceration. patients may also complain of a sore mouth and throat and ulcers may be present on all oral mucosal surfaces, the tongue, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p32–35 mailto:maharaniroxy@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p32-35 33apriasari/dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 32–35 gingiva, lips and cheeks. oral mucosa lesions initially develop as red maculars which develop into vesicles and rupture into ulcers.4,8 the clinical oral manifestations of hfmd demonstrate certain similarities with other viral infections, resulting in differential diagnoses for this disease such as primary herpetic gingivo stomatitis (phgs) and primary varicella zoster infection.4,9,10 hfmd is a fundamentally self-limiting disease which, consequently, requires supportive therapy to boost the immune system and prevent complications resulting from its treatment. acyclovir is not indicated for hfmd. a vaccine against coxsackie virus infection preventing the spread of hfmd is currently being developed for future research.8 this case report aims to elucidate the distinctions between hfmd and primary varicella zoster infection. clinically, both diseases exhibit similar manifestations, but further elaboration is essential to distinguish them from a variety of perspectives. they have similar clinical manifestations, but each disease has a different etiology, a fact which can cause errors in the administration of therapy. it would be advantageous for the dentist to both diagnose the disease and personally manage treatment of the patient. case an 8 year-old male sought treatment complaining of ulcers on the right maxillary gingiva accompanied by the appearance of itchy and painful wounds around the nose, upper lips, hands and feet which he had suffered for the previous three days. this condition is clinically similar to primary varicella zoster infection which can afflict any part of the body. the boy’s mother reported her son as having developed a 39oc fever accompanied by itchy red spots and ulcers on the face, hands and feet. the patient, who had no allergic history, was actively seeking available healthcare assistance and used an immunomodulator only once a day. his mother intimated that a neighbor of the family had previously been diagnosed with singapore flu. case management first visit (day 1): extraoral examination revealed the presence of multiple itchy erythematous vesicles and ulcers, 2 mm in diameter, surrounding the nose and upper lips (figure 1). the palms, back of the hands and feet demonstrated the presence of multiple erythematous macules and vesicles, 3-6 mm in diameter, which caused itchiness and pain (figure 2). intraoral examination confirmed the presence of painful multiple ulcers, 1-2 mm in diameter and yellowish in appearance, surrounded by erythema on the right maxillary gingiva (figure 3). anamnesis conducted with the patient’s mother indicated that no lesions had appeared on other parts of his body, leading to a diagnosis of hfmd. the patient was prescribed a combination of bed rest and soft liquid food and beverages figure 1. upper lips and nose developed multiple itchy e r y t h e m a t o u s v e s i c l e s a n d u l c e r s , 2 m m i n diameter. figure 3. upper right gingival developed multiple painful ulcers 1-2 mm in diameter with yellowish appearance surrounded by erythema. figure 2. palm, back of the hands and feet presented multiple itchy and painful vesicles, erythematous maculas, 3-6 mm in diameter. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p32–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p32-35 34 apriasari/dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 32–35 high in calories and protein. he was also instructed to use an aloe vera mouthwash three times a day, 250 mg of methisoprinol syrup three times a day, 250 mg of ibuprofen syrup three times a day and multivitamin b complex once a day for seven days. patient follow-up was scheduled for seven days later. second visit (day 14): the patient arrived for followup treatment of ulcers on the right maxillary gingiva. the results of history-taking confirmed the existence of a painless ulcer from day 9. the painless wounds on the nose, upper lip, hands and feet which did not itch had been present since day 12. the patient’s fever broke on day 5 and he consumed oral drugs and regularly applied mouthwash. extraoral examination of the nose, upper lip, hands and feet confirmed a marked improvement in their condition. intraoral examination indicated the presence of normal, lesion-free tissue and the patient was, therefore, declared healthy (figure 4). discussion one differential diagnosis of hfmd is that of primary varicella zoster infection.9 this case report discussed similarities and differences between both diseases from various perspectives. although both diseases generally present similar clinical symptoms frequently found in children and the presence of lesion on oral mucosa and skin, they were mediated by different forms of viral infection. both diseases have similar clinical manifestations, but contrasting etiology which necessitates different treatment. in this case, patient experienced a cough and influenza accompanied by a high fever of 39oc. hfmd and primary varicella zoster infection present similar symptoms such as fever and flu like syndrome prior to the appearance of lesions.10 as the fever resides, itchy erythematous macula, vesicles and ulceration appeared on the upper lip, nose, palms, backs of the hands and the feet. there was no lesion around buttocks and genital area. this differentiates hfmd from primary varicella zoster infection as a result of which similar lesions may appear on the entire body surface, including: the eyes, oral cavity and genital mucosa. it has been declared the first primary disease suffered during the patient’s life.4,10 the etiology of hfmd is enterovirus 71 (ev 71) large outbreaks of which in the asia pacific region over the last decade have been associated with neurological disease and mortality. enteroviruses constitute small, non-enveloped rna viruses categorized as members of the picornaviridae family.9 meanwhile, the coxsackie virus is the rna virus also belonging to the picornaviridae family. group a coxsackie viruses associated with infections of the skin and mucous membrane, acute hemorrhagic conjunctivitis and hfmd.4,8 coxsackie virus replicates in the buccal and ileal mucosa. after initial infection, the virus can be detected in the respiratory tract for up to three weeks and in faeces for as long as eight weeks. the viruses replicate in the submucosal lymph nodes within 24 hours and disseminate through the reticuloendothelial system. meanwhile, enteroviruses are transmitted primarily through the fecaloral route or fomite before replicating in the mucosa of oropharynx, small intestine and lymphoid tissue of the intestinal mucosa.8 varicella zoster virus is a pathogenic human alpha herpes virus causing varicella zoster as a primary infection which occurs in unvaccinated children. following the primary infection, this neurotropic virus becomes latent. latency primarily exists in neurons of peripheral autonomic ganglia throughout the entire neuroaxis including the dorsal root ganglia, cranial nerve ganglia such as the trigeminal ganglia and autonomic ganglia, including those in the nervous system. after several years, the latent virus can reactivate as herpes zoster, a complication of which may be post herpetic neuralgia (phn) which usually appears as a vesicle and painful mucocutaneous eruption demonstrating characteristic distribution. this viral reactivation increases in frequency with the increasing age of the patient. immunosuppressive conditions, irritation, x ray irradiation, infection and malignancy may trigger virus reactivation.11,12 varicella zoster and hfmd infection can spread via the fecal-oral route, respiratory droplets or contact with figure 4. upper lip, nose, hands and feet showed no sign of pain and itchiness. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p32–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p32-35 35apriasari/dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 32–35 vesicular liquid.5,11 no examination of this patient was conducted other than that of diagnosing the disease. this was conducted on the basis of particular clinical symptoms of hfmd such as lesions on the oral mucosa, lips, hands and feet without lesions affecting other parts of the body being considered. this caused difficulty in identifying the specific etiological virus. the diagnosis of hfmd and varicella zoster are typically clinical because the association of the disease is highly predictive in endemic regions.8,11 the virus can be isolated via serology testing, cell culture and polymerase chain reaction (pcr) testing.8,13 varicella zoster infection is mediated by the herpes virus, thus requiring prescription of acyclovir or methisoprinol. acyclovir is an antiviral drug containing essential agents rendering the normal chain capable of blocking the virus’s dna. in cases where cyclic sugars are absent from acyclovir triphosphate termination of chain elongation occurs. methisoprinol can be prescribed during the onset of the disease as a prophylaxis against reactivation of latent varicella zoster infection.12,14 the etiology of hfmd is enterovirus 71 or coxsackie virus, both of which constitute rna viruses. the case reported here indicated that hfmd can be treated with methisoprinol which contains an antiviral and immunomodulator agent, as opposed to acyclovir, because hfmd is not a herpes virus. an antiviral agent increases potency of depressed mrna protein synthesis, disturbs translational ability process and inhibits polyadenylic acid attachment to viral mesenger rna. as an immunomodulator agent, methisoprinol can enhance dysfunctional cell-mediated immunity by stimulating a th-1 response, triggering t-lymphocyte maturation and differentiation to induce lymphoproliferative responses in mitogen or antigenactivated cells. it can modulate t-lymphocyte and natural killer cell cytotoxicity, t4 helper and t8 suppressor cell functions.14,15 based on the foregoing discussion, it can be concluded that a dentist is responsible for detecting specific clinical symptoms of a disease to distinguish hfmd and primary varicella zoster infection. although these diseases share similar clinical symptoms, they have contrasting etiologies which require different treatment. varicella zoster infection can be treated by acyclovir or methisoprinol. since hfmd is not caused by the herpes virus methisoprinol, rather than acyclovir, is used to treat it. the final diagnosis can be determined clinically by the dentist in order to avoid errors in the administration of therapy. methisoprinol constitutes an antiviral which prevents viral replication by inhibiting viral dna polymerase in varicella zoster as a dna virus infection. in hfmd, methisoprinol functions as an immunomodulatory. references 1. sittisarn s, wongnuch p, laor p, inta c, apidechkul t. effectiveness of hand foot mouth disease prevention and control measures between high and low epidemic areas, northern thailand. j heal res. 2018; 32(3): 217–28. 2. hatta i, firdaus iwak, apriasari ml. the prevalence of oral mucosa disease of gusti hasan aman dental hospital in banjarmasin, south kalimantan. dentino j kedokteran gigi. 2018; 2(2): 211–4. 3. koh wm, bogich t, siegel k, jin j, chong ey, tan cy, chen mi, horby p, cook ar. the epidemiology of hand, foot and mouth disease in asia: a systemic review and analysis. pediatr infect dis j. 2016; 35(10): e285–300. 4. glick m. burket’s oral medicine. 12th ed. usa: people’s medical publishing house; 2014. p. 194–201. 5. sun bj, chen hj, chen y, an xd, zhou b sen. the risk factors of acquiring severe hand, foot, and mouth disease: a meta-analysis. can j infect dis med microbiol. 2018; 2018: 1–12. 6. chadsuthi s, wichapeng s. the modelling of hand, foot, and mouth disease in contaminated environments in bangkok, thailand. comput math methods med. 2018; 2018: 1–8. 7. repass gl, palmer wc, stancampiano ff. hand, foot, and mouth disease: identifying and managing an acute viral syndrome. cleve clin j med. 2014; 81(9): 537–43. 8. afrose t. coxsackie virus: the hand, foot, and mouth disease (hfmd). juniper online j public heal. 2017; 1(4): 1–5. 9. sarkar pk, sarker nk, tayab ma. hand, foot and mouth disease (hfmd): an update. bangladesh j child heal. 2016; 40(2): 115–9. 10. apriasari ml, baharuddin em. penyakit infeksi rongga mulut. surakarta: yuma pustaka; 2012. p. 6–8, 11–3. 11. gershon aa, breuer j, cohen ji, cohrs rj, gershon md, gilden d, grose c, hambleton s, kennedy pge, oxman mn, seward jf, yamanishi k. varicella zoster virus infection. nat rev dis prim. 2015; 1: 1–41. 12. kennedy p, gershon a. clinical features of varicella-zoster virus infection. viruses. 2018; 10(11): 1–11. 13. andric b, mijovic g, andric a. characteristics of hand foot and mouth disease. j hum virol retrovirology. 2016; 3(6): 1–5. 14. apriasari ml. methisoprinol as an immunomodulator for treating infectious mononucleosis. dent j (majalah kedokteran gigi). 2016; 49: 1–4. 15. ompico mg. methisoprinol for children with early phase dengue infection: a pilot study. paediatr indones. 2013; 53(6): 1–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p32–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p32-35 �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �� the influence of adhesin protein from aggregatibacter actinomycetemcomitans on il-8 and mmp-8 titre in aggressive periodontitis rini devijanti ridwan department of oral biology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: adhesion can actually be considered as a part of both a powerful survival mechanism and a virulence mechanism for bacterial pathogens. bacterial adhesin is an instrument for bacteria to do invasion to host. bacterial adhesin depends on ligand interaction as a signaling mediator that will influence invasion and increase pro and anti-inflammatory because of the influence of the receptors of innate immune response. aggregatibacter actimycetemcomitans has fimbriae included in type iv pili containing mostly with protein weighed 6.5 kda and at least with protein weighed 54 kda. purpose: the purpose of this research is to analyze the influence of the induction of adhesin protein derived from a. actinomycetemcomitans on il-8 and mmp-8 titre of wistar rats. methods: adhesin protein derived from a. actinomycetemcomitans weighed 24 kda was induced on the maxillary first molar sulcus of wistar rats to prove that adhesin protein could affect il-8 and mmp-8 titre. next, to determine its influence, elisa technique was conducted. results: it is known that the levels of il-8 and mmp-8 titre were increased in the group induced with adhesin protein derived from a. actinomycetemcomitans compared with the control group. conclusion: it can be concluded that adhesin protein derived from a. actinomycetemcomitans can cause alveolar bone damage through the increasing levels of il-8 and mmp-8 in aggressive periodontitis. keywords: a. actinomycetemcomitans adhesin; il-8; mmp-8; aggressive periodontitis correspondence: rini devijanti ridwan, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya 60132, indonesia. email: devi.rini@yahoo.co.id research report introduction aggressive periodontitis is a disease found on tissues supporting teeth, characterized by rapid deterioration in periodontal ligament and alveolar bone. aggressive periodontitis is also known as a process of tissue attachment loss and gingival recession four times faster than chronic periodontitis.1,2 this disease is more commonly found on patients aged under 30 years old. however, aggressive periodontitis found on young patients is still a problem in dentistry. the pathogenesis of periodontitis is affected by the interaction between host and bacteria dominated by aggregatibacter actinomycetemcomitans (a.actinomycetem comitans). the presence of the bacteria in dental plaque can be associated with the aggressiveness of periodontal tissue damage and exacerbated by both genetic and environmental factors.3 the direct contact between an infectious agent and a host cell actually begins with adhesion (attachment). therefore, the increasing of il-8 in periodontal tissues and gcf is related to the severity of periodontitis. in vitro chemokines, such as il-8, can be produced by gingival fibroblasts cells, gingival epithelial cells, and endothelial cells. il-8 produced by osteoblasts can be induced by bacterial products, inflammatory mediators, dentin protein, and debris. 4 il-8 is the body’s first defense by enhancing phagocytosis, killing bacteria, as well as secreting lysosomal enzymes and superoxide anion. matrix metalloproteinases (mmps) are major proteinase enzymes involved in periodontal tissue destruction with dental journal (majalah kedokteran gigi) 20�5 march; 48(�): ��–42 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 40 ridwan/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 39–42 the degradation of extracellular matrix molecules. mmps are also known as a group of proteolytic enzymes found in neutrophils, macrophages, fibroblasts, epithelial cells, osteoblasts, and osteoclasts, destroying the extracellular matrix molecules, such as collagen, gelatin and elastin. both mmp-8 and mmp-1 are actually collagenase. mmp8 is expressed because of the infiltration of neutrophils, whereas mmp-1 is expressed by fibroblasts, monocytes/ macrophages, and epithelial cells. in addition, it is also known that mmps are produced by bacterial pathogens causing periodontitis, such as p. gingivalis and a. actinomycetemcomitans although they are not the major factors in the aggressiveness of this disease.5 this study is aimed to know how the induction of adhesin protein derived from a. actinomycetemcomitans can affect the levels of il-8 and mmp-8 in wistar rats as an indicator of tissue and bone damage in aggressive periodontitis. materials and method the culture of a. actinomycetemcomitans was made of bacteria that had been isolated from patients with aggressive periodontitis in periodonsia clinic, faculty of dental medicine, universitas airlangga. however, those patients had to fill informed concern forms as approval first. this research was approved based on ethical test conducted at faculty of dentistry, universitas airlangga. the culture of a. actinomycetemcomitans made was about 200 ml for two groups. it means that the culture of a. actinomycetemcomitans in each group consisting of 20 rats was at least about 5 ml at a density of 108. a.actinomycetemcomitans was induced for 7 days of treatment. furthermore, there were four groups, each of which consisted of ten rats. the first group was the negative control group induced with 0.9% nacl. the second group was induced with adhesin, while the third group was induced with adhesin and whole cell a. actinomycetemcomitans. and, the fourth group was the positive control group induced with whole cell a. actinomycetemcomitans. adhesin induction in those rats was conducted by giving adhesin a.actinomycetemcomitans and whole cell a. actinomycetemcomitans about 200 ml for each with 200 pg/ml of protein levels, while the concentration of a. actinomycetemcomitans given for 7 days was 108 in order to get the real symptoms of aggressive periodontitis.6 the induction was conducted in the pockets of m1 upper right tooth based on “dumitrescu” method.7 finally, il-8 and mmp-8 were measured by using elisa technique, namely elisa kit, bg-rat11692 (novateinbio). the basic principle of elisa kit is the use of a doubleantibody sandwich elisa to analyze the levels of il-8 and mmp-8 in the samples of those alveolar bones. put standards and samples into the wells that had been coated with the rats’ osteocalcin antibody. they were added with osteocalcin antibody hrp conjugates to bind the analyte, and then incubated and washed based on the procedure to remove unbound materials. they were added with hrp substrate, and then incubated for detection. if the colour appered is blue, it means that there is a reaction. the color will change to yellow when the reaction is stopped after they are added with stopping solution (acid solution). the intensity of the yellow color can indicate the concentration of il-8 or mmp-8 in those rats. results the results of elisa test on il-8 level in the control group, in the group induced with adhesin, induced with adhesin + a. actinomycetemcomitans, and induced with a. actinomycetemcomitans can be seen in table 1. based on the results of kolmogorov-smirnov test, it is known that the distribution of il-8 level in the alveolar bones of those four groups was normal (p> 0.05, p=0.195). based on the results of anova test, it is also known that there was a significant difference among the treatment groups (p<0.05, p=0.001). based on the results of lsd test, moreover, it can be seen that there was a significant difference between the control group and all the treatment groups. furthermore, it is known that il-8 level in the group induced with a. actinomycetemcomitans was 8.1 µg/ml (8.10091± 0.621), while that in the group induced with a. actinomycetemcomitans + adhesin was 5.8 µg µg/ml (5.80317 ± 0.68502). il-8 level in the group induced with adhesin was 2.6 µg/ml (2.57854 ± 0.91238), while that in the control group amounted was 0.00017 µg/ml (0.00017 ± 0.00013). in other words, the highest level of il-8 was in the group induced with a. actionomycetemcomitans compared to the other treatment groups. the average levels of il-8 can be seen in figure 1. on the other hand, the results of elisa test on mmp8 level in the control group, in the group induced with adhesin, induced with adhesin + a. actinomycetemcomitans, and induced with a. actinomycetemcomitans can be seen in table 2. based on the results of kolmogorov-smirnov test, it is known that the distribution of mmp-8 level in the alveolar bones of those four groups was normal (p> 0.05, p=0.195). based on the results of anova test, there was a significant difference among the treatment groups (p<0.05, figure 1. the mean of the levels of il-8 in alveolar bone. c on ce nt ra ti on ( m g/ m l) control 4� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 4�ridwan/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 39–42 p=0.001). based on the results of lsd test, moreover, it can be seen that there was a significant difference between the control group and all the treatment groups. in addition, mmp-8 level in the group induced with a. actinomycetemcomitans + adhesin was 2.2 µg/ml (2.2339 ± 0.6846), while that in the group induced with a. actinomycetemcomitans was 1.7 µg/ml (1.7654 ±0.6821). il-8 level in the group induced with adhesin was 0.6 µg/ml (0.5735 ± 0.2947), while that in the control group was 0.1 µg/ml (0.1081 ± 0.2828). in other words, the highest level of mmp-8 was in the group induced with a. actinomycetemcomitans + adhesin compared to the other treatment groups. the average levels of mmp-8 can be seen in figure 2. discussion based on the results, there was a significant difference of of il-8 level in the alveolar bones between in the group induced with a. actinomycetemcomitans, in the group induced with a. actinomycetemcomitans + adhesin, in the group induced with adhesion, and in the control group. il-8 level in the group induced with a. actinomycetemcomitans was significantly increased compared to that in the group induced with a.actinomycetemcomitans + adhesin, in the group induced with adhesion, and in the control group. it indicates that the presence of adhesion can make the colonization and invasion of a. actinomycetemcomitans in periodontal tissues stimulate the activation of proinflammatory cytokines, one of which is il-8 serving as chemokines. il-8 will attract neutrophils, causing neutrophil degradation. the neutrophil degradation then can cause the expression of elastase and lactoferrin. elastase can usually cause damage to periodontal tissues and alveolar bone. the induction of a. actinomycetemcomitans + adhesin can cause il-8 level lower than the induction of a. actinomycetemcomitans only, but, the induction of adhesin can cause il-8 level higher than in the control group. it is because adhesin induced along with a. actinomycetemcomitan can serve as an inhibitory role in the process of adhesion, so the colonization and invasion of the host, a. actinomycetemcomitans, will be reduced. this condition has been confirmed in an adhesion test of a. actinomycetemcomitans on hela cells. the test result shows that the adhesion of a. actinomycetemcomitans adhesin protein with receptors can be found in hela cells. the result also shows that this adhesin protein can inhibit the attachment of a. actinomycetemcomitans on hela cell surface as indicated by the declining number of a. actinomycetemcomitans attached to the hela cells when administered with the increasing doses of adhesin protein derived from a. actinomycetemcomitans.8 m m p 8 l e v e l i n t h e g r o u p i n d u c e d w i t h a . actinomycetemcomitans + adhesin was significantly increased compared to that in the group induced with a. actinomycetemcomitans, in the group induced with adhesion, and in the control group. it indicates that the ability of a. actinomycetemcomitans and adhesin protein derived from a. actinomycetemcomitans to perform attachment to the receptors of the host, so a. actinomycetemcomitans can do table 1. the mean and standard deviation of the levels of il-8 in alveolar bone group x sd min max anova control 0.00017 0.00013 0.0000156 0.0003498 f= 300.5 a. actinomycetemcomitans 8.10091 0.621 6.8367827 8,9837638 p= 0.001 adhesin 2.57854 0.91238 1.2672389 3.9748729 a. actinomycetemcomitans + adhesin 5.80317 0.68502 4.8738794 6.8738987 table 2 the mean and standard deviation of the levels of mmp-8 in alveolar bone group x sd min max anova control 0.1081 0.2828 0.0007 0.9087 f= 35.97 a. actinomycetemcomitans 1.7654 0.6821 1.0571 2.9387 p= 0.001 adhesin 0.5735 0.2947 0.2538 0.8376 a. actinomycetemcomitans + adhesin 2.2339 0.6846 1.2839 3.0981 figure 2. the mean of the levels of mmp-8 in alveolar bone. c on ce nt ra ti on ( m g/ m l) control dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 42 ridwan/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 39–42 colonization and invasion. the high level of mmp-8 caused by the induction of a. actinomycetemcomitans + adhesin will stimulate proinflammatory cytokines, namely il-8 expressed by monocytes, keratinocytes, endothelial cells and fibroblasts which then will stimulate the expression of mmp-8 by neutrophils. mmp-8 is considered as the potential collagenase-2 playing an important role in the degradation of connective tissue in inflammation area. it means that the increasing of mmp-8 will result in damage to periodontal tissues and alveolar bone. a. actinomycetemcomitans is a powerful stimulator of mmp-8 expression, notably by ltxa as one of virulence factors.8 the situation is also in accordance with the statement of kiili10 stating that mmp-8 plays a role in inflammation and tissue destruction diseases, so the activation of mmp-8 in periodontitis has been reported to reflect the level of disease severity and activity. mmp group, such as mmp 1, 3, 7, 8, 9, 13, 25 and 26 has been widely studied in gingival tissue and gingival crevicular fluid (gcf) of patients with different periodontal diseases, and then mmps are associated with the development and progression of periodontal disease.11 it can be concluded that adhesin protein derived from a. actinomycetemcomitans with a molecular weight of 24 kda has an ability to increase the levels of il-8 and mmp-8 in the wistar rats’ alveolar bone. acknowledgment this research was supported by universitas airlangga (excellent research fund for higher education, boptn dipa, fiscal year 2014). references 1. velden v, abbas f, armand s, loos bg, timmerman mf, weijden v. java project on periodontal diseases. the natural development of periodontitis: risk factor, risk predictors and risk determinants. j clin. periodontol 2006; 33 : 540-49. 2. newman mg, takei n, klokkevold p, carranza f. carranza’s clinical periodontology. 10th ed. philadelphia, new york, london: wb saunders co; 2006. p. 168-81, 409-14, 675-88. 3. africa jwj. the microbial aetiology of periodontal diseases. periodontal diseases. a.clinician’s guide. 2012. p. 1-52. 4. silva ta, garlet gp, fukada sy, silva js, cunha fq. chemokines in oral inflamatoty diseases: apical periodontitis and periodontal diseases. critical reviews in oral biology & medicine. j dent res 2007; 86(4): 306-31. 5. nisengard rj, haake sk, newman mg, miyasaki kt. microbial interaction with the host in periodontal disease. 2009. p. 110-9. zhou q, desta t, fenton m, graves dt, amar s. lps cytokines profiling of macrophage exposed to porphyromonas gingivalis, its lipopolysachcharide, or its fima protein. infect immun 2005; 73(2): 935-43. 6. du m ist rescu a l . h istolog ica l compa r ison of p er iodont a l inflammatory changes in two models of experimental periodontitis the rat: a pilot study. tmj 2006; 56(2): 211-7. 7. devijanti r., the role of actinobacillus actinomycetemcomitans fimbrial adhesin on mmp-8 activity in aggressive periodontitis pathogenesis. dental journal 2012; 45(4) 181-6. 8. claesson r, johansson a, belibasakis g, hänstrőm, kalfas s. release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of actinobacillus actinomycetemcomitans. blackwell munksgaard ltd 2002; 37(5): 356-9. 9. kiili m, cox sw, chen hw, wahlgren j, maisi p, eley bm, salo t, sorsa t. collagenase-2 (mmp-8) and collagenase-3 (mmp-13) in adult periodontitis: molecular forms and levels in gingival crevicular fluid and immunolocalisation in gingival tissue. j clin periodontol 2002; 29: 224–32. 10. padmavati p, savita s, shivaprasad bm, kripal k, rithesh k. mrna expression of mmp-28 (epilysin) in gingival tissues of chronic and aggressive periodontitis patients. a reverse transcriptase pcr study 2013; 35(2): 113-8. vol 38 no 2-2005 64 perbedaan daya hambat terhadap streptococcus mutans dari beberapa pasta gigi yang mengandung herbal (the difference of inhibition zones toward streptococcus mutans among several herbal toothpaste) rini pratiwi bagian ilmu kesehatan gigi masyarakat fakultas kedokteran gigi universitas hasanuddin makassar – indonesia abstract prevention of dental caries and periodontal disease with health promotion has been the main goal of dentistry, since the dental plaque is a common and predominating factor leading to loss of teeth both caused by both dental caries and periodontal disease. most devices used for plaque removal and periodontal care are based on mechanical action, including toothbrushes, interdentally brushes and oral irrigators. today, plaque control is facilitated by an increasing variety of active agents based on either natural ingredients or synthetic product. alternative materials based on essential oil and plant extracts are therefore of particular interest. the aim of the present study was to investigate the different of zone inhibition of streptococcus mutans of herbal toothpastes. samples of the subjects were 4 types of herbal toothpastes and 1 herbal toothpaste as control. for inhibition zone trial toward streptococcus mutans, agar diffusion method was used and the interpretation was done by measuring the widest inhibition zone. the result of the study indicated a statistical significance different on inhibition zone among the 5th toothpastes, but toothpaste containing siwak was the widest inhibition zone. key words: zone inhibits, streptococcus mutans, herbal toothpaste korespondensi (correspondence): rini pratiwi, bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas hasanuddin. jln. perintis kemerdekaan makassar, indonesia. pendahuluan karies adalah suatu penyakit yang disebabkan oleh adanya interaksi antara bakteri plak, diet, dan gigi. tidak diragukan lagi bahwa tanpa adanya plak, maka tidak akan timbul karies. penelitian klasik keyes tahun 1960 dan fitzsgerald and keyes tahun 1960 pada binatang bebas kuman memperlihatkan bahwa plak yang didominasi oleh kuman streptococcus mutans dan lactobacillus menyebabkan terbentuknya karies.1 s. mutans dan lactobasillus merupakan kuman yang kariogenik karena mampu segera membentuk asam dari karbohidrat yang dapat diragikan. kuman tersebut dapat tumbuh subur dalam suasana asam dan dapat menempel pada permukaan gigi karena kemampuannya membuat polisakarida ekstra sel. polisakarida ekstra sel ini terutama terdiri dari polimer glukosa yang menyebabkan matriks plak mempunyai konsistensi seperti gelatin, akibatnya bakteri terbantu untuk melekat pada gigi serta saling melekat satu sama lain. plak makin lama makin tebal, sehingga akan menghambat fungsi saliva untuk melakukan aktivitas antibakterinya. banyak yang dapat dilakukan untuk mencegah karies, dengan mengetahui penyebabnya merupakan hal penting agar mengerti cara melakukan pencegahannya. 1 pencegahan karies dan penyakit periodontal dengan melakukan peningkatan kesehatan gigi telah menjadi tujuan utama dalam dunia kedokteran gigi, sejak diketahui plak gigi merupakan faktor yang mendominasi penyebab hilangnya gigi oleh karena karies dan penyakit periodontal.2 salah satu cara pencegahan karies adalah mengusahakan agar pembentukan plak pada permukaan gigi dapat dibatasi baik dengan cara mencegah pembentukannya atau dengan pembersihan plak secara teratur. pengendalian plak dapat dilakukan dengan cara pembersihan plak secara mekanis dan kemungkinan penggunaan bahan anti kuman terutama untuk menekan s. mutans. menyikat gigi membantu kontrol plak dan merupakan langkah awal untuk mengontrol karies dan penyakit periodontal baik untuk individu maupun populasi.1,2 saat ini kontrol plak dilengkapi dengan penambahan jenis bahan aktif yang mengandung bahan dasar alami ataupun bahan sintetik sebagai bahan anti kuman. bahan anti kuman tersebut tersedia dalam bentuk larutan kumur dan pasta gigi.3 pada masa lalu, pasta gigi yang digunakan bersama sikat gigi hanya bersifat sebagai alat kosmetik. tetapi dalam tahun terakhir ini banyak dibuat pasta gigi yang mempunyai efek untuk mengobati penyakit mulut dan mencegah karies gigi, sehingga sukar dibedakan dengan jelas antara pasta yang berefek kosmetik dan yang berefek 65pratiwi: perbedaan daya hambat terhadap streptococcus mutans terapi. bahan anti kuman yang umum digunakan untuk kontrol plak diantaranya adalah fenol, hexetidine, fluor dan chlorhexidine. chlorhexidine merupakan salah satu formula yang paling efektif untuk mengontrol plak, tetapi penggunaannya dalam jangka waktu lama dapat menimbulkan efek merugikan. oleh karena itu bahan alternatif dari bahan minyak esensial dan ekstrak tumbuhtumbuhan (herbal) merupakan hal yang menarik untuk dijadikan pilihan sebagai bahan anti kuman dalam pasta gigi.3 meluasnya pemakaian pasta gigi adalah karena secara komersil mudah didapatkan dan akhir-akhir ini ketertarikan akan produk dengan kandungan dasar dari bahan alami telah meningkat. di pasaran kini banyak beredar pasta gigi dengan kandungan bahan herbal antara lain: aloe vera, eucalyptus, siwak, dan daun sirih. pemakaian aloe vera di bidang kedokteran gigi telah dilaporkan oleh seorang dokter gigi di chicago yaitu melnick (1982), yang menggunakannya sebagai bahan pasta gigi untuk membantu penyembuhan pasien dengan radang gusi dan mengurangi pewarnaan akibat rokok.4 siwak sangat umum digunakan di timur tengah dan diketahui memiliki efek antiplak dan khasiat farmakologis lainnya. beberapa peneliti melaporkan adanya efek antibakteri dari siwak terhadap bakteri kariogenik dan patogen periodontal khususnya spesies bakteriodes serta menghambat pembentukan plak. di zimbabwe ditemukan bahwa anak-anak yang menggunakan siwak untuk pembersihan gigi mereka mempunyai lesi karies yang lebih sedikit dibandingkan dengan anak yang menyikat gigi mereka dengan sikat gigi konvensional dengan pasta.5 di sudan, emsile melaporkan bahwa terdapat karies yang lebih sedikit pada pengguna siwak bila dibandingkan dengan pengguna sikat gigi.5 daun sirih sudah dikenal oleh masyarakat indonesia sejak lama, yaitu sebagai bahan untuk menginang dengan keyakinan bahwa daun sirih dapat menguatkan gigi, menghentikan perdarahan gusi dan sebagai obat kumur.6 berdasarkan uraian yang telah dikemukakan, maka perlu diketahui perbedaan daya hambat terhadap s. mutans dari beberapa pasta gigi yang mengandung herbal. tujuan dari penelitian ini adalah untuk mengetahui perbedaan daya hambat terhadap s. mutans dari beberapa pasta gigi yang mengandung herbal. manfaat penelitian ini adalah untuk memberi alternatif pilihan pasta gigi yang mengandung herbal kepada masyarakat penggunanya. bahan dan metode jenis penelitian adalah ekperimen laboratoris yang dilakukan di laboratorium mikrobiologi fakultas kedokteran universitas hasanuddin makassar. sampel adalah 4 pasta gigi yang mengandung herbal dan 1 pasta gigi non herbal sebagai kontrol. pasta tersebut adalah: 1) pasta gigi herbal a, mengandung sodiummono fluorfosfat, bahan herbal eucalyptus, clove dan tea tree oil; 2) pasta gigi herbal b, mengandung bahan aktif kalsium gliserofosfat, sodiummono fluorfosfat dan triclosan, aloe vera dan daun sirih; 3) pasta gigi herbal c, mengandung siwak; 4) pasta gigi herbal d, mengandung ekstrak daun sirih dan fluoride; 5) pasta gigi e mengandung bahan aktif sodium fluoride dan triclosan. jalannya penelitian: a) pembuatan medium nutrien agar (na) sebanyak 200 ml, 0,6 gr beef extract + 1 gr pepton + 3 gr agar dimasukkan dalam erlenmeyer dan cukupkan volumenya dengan aquades 200 ml, kemudian dimasak dalam air mendidih selama 15 menit, lalu disterilkan dalam autoclave; b) pembuatan medium glukosa nutrien agar (gna) sebanyak 300 ml : 1,5 gr beef extract + 3 gr pepton + 3 gr agar dimasukkan dalam erlenmeyer. cukupkan volumenya dengan aquades hingga 250 ml, masak selama 15 menit lalu disterilkan. ambil glukosa sebanyak 10 gr dilarutkan dengan 50 ml aquades steril. keluarkan campuran dan dimasak kembali selama 15 menit lalu masukkan glukosa ke dalam campuran, dan siap digunakan; c) peremajaan isolat s. mutans yaitu dengan cara: ambil na dan masukkan dalam tabung reaksi lalu dimiringkan. s. mutans yang berasal dari stock diambil dengan ose steril lalu dimasukkan ke dalam tabung reaksi yang berisi na beku dan diinkubasi selama 24 jam; d) pembuatan sediaan pasta gigi dibuat dengan cara, diambil sebanyak 1 gr masing-masing pasta lalu diencerkan dengan 1 ml aquades; e) untuk mengetahui kepekaan s mutans terhadap pasta gigi, dilakukan hal sebagai berikut: disiapkan 10 cawan petri dan 50 paper dish dengan diameter 55 mm. paper dish direndam selama 5 menit dalam pasta gigi yang telah diencerkan (tiap pasta gigi 10 paper dish), kemudian disetiap cawan petri yang telah berisi media gna dan isolat s. mutans diletakkan 5 paper dish dari 5 macam pasta. lalu dimasukkan dalam inkubator dengan suhu 37° c selama 24 jam; f) kriteria penilaian daya hambat yaitu dengan mengukur zona bening atau zona inhibisi disekitar paper dish dengan menggunakan kaliper secara vertikal, horizontal dan diagonal, kemudian dirata-ratakan. hasil hasil penelitian yang dilakukan menemukan semua pasta gigi mempunyai daya hambat terhadap s. mutans dengan kemampuan yang berbeda (tabel 1). hasil perhitungan dengan uji anova adalah r = 0,001 jadi ho ditolak berarti ada perbedaan bermakna daya hambat terhadap s. mutans dari pasta gigi yang diuji. karena ada perbedaan kemudian dilakukan uji beda lanjut (lsd), yang hasilnya ditampilkan pada tabel 2. 66 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 64–67 tabel 1. perbedaan daya hambat (lebar zona inhibisi dalam mm) terhadap s. mutans dari pasta gigi setelah 24 jam pasta gigi n rerata simpang baku p herbal a 10 19,90 1,09 herbal b 10 23,17 1,50 herbal c 10 25,48 2,96 0,001* herbal d 10 22,03 2,15 kontrol 10 19,79 2,03 keterangan: pasta gigi herbal a = eucalyptus; pasta gigi herbal b = aloe vera dan daun sirih; pasta gigi herbal c = siwak; pasta gigi herbal d = daun sirih; pasta gigi kontrol = non herbal. pembahasan pemeliharaan kesehatan mulut yang teratur melalui kontrol plak secara mekanis dengan sikat gigi dan pasta gigi akan menjamin kesehatan gigi dan mulut, karena telah terbukti bahwa menyikat gigi merupakan metode yang efektif untuk mengeluarkan plak. hanya terdapat sedikit dari populasi yang diharapkan dapat melakukan pembersihan plak yang adekuat secara mekanis, dan plak gigi dapat terbentuk kembali setelah beberapa jam atau hari setelah plak tersebut dibersihkan. oleh karena itu pemakaian bahan pasta antimikroba sangat bermanfaat sebagai tambahan dalam pembersihan plak secara mekanis. produk herbal yang diuji dalam percobaan ini mengandung beberapa unsur yang berbeda dan semua mengandung bahan antibakteri yang secara teori dapat mengontrol plak dan penyakit periodontal. kandungan fluor dalam pasta gigi herbal a mempunyai kemampuan memacu remineralisasi karies dini dan mengurangi kemampuan bakteri untuk memproduksi asam.7 minyak eucalyptus mempunyai daya antibakteri dan digunakan sebagai antiseptik.8 clove dalam pasta gigi merupakan anestetik ringan yang bersifat sementara.9 tea tree oil mengandung sifat antiseptik ringan yang dapat mengontrol pertumbuhan bakteri.10 kandungan kalsium gliserofosfat dalam pasta herbal b dapat mencegah karies melalui aktivitas enzim yang memacu remineralisasi.11 kandungan triclosannya termasuk golongan fenol merupakan bahan anti bakteri.12,13 selain bahan aktif tersebut, pasta ini juga mengandung ekstrak daun sirih dan aloe vera. minyak atsiri daun sirih diketahui mempunyai daya antibakteri, hal ini disebabkan oleh karena adanya senyawa fenol dan turunannya yang dapat mengubah sifat protein sel bakteri. salah satu senyawa turunan itu adalah kavikol yang memiliki daya antibakteri lima kali lebih kuat dibanding fenol.6 aloe vera mengandung gugus glikosida yang memiliki daya antiseptik yang merupakan gugus aminoglikosida yang bersifat antibiotik. senyawa aminoglikosid ini akan berdifusi pada dinding sel bakteri, dan proses ini berlangsung terus-menerus dalam suasana aerobik. setelah mabuk ke dalam sel, aminoglikosida ini akan diteruskan pada ribosom yang menghasilkan protein, sehingga akan menimbulkan gangguan pada proses sintesa protein dan selanjutnya akan menyebabkan terjadinya pemecahan ikatan protein sel bakteri.4 kandungan lain aloe vera adalah gugus antrakuinon seperti barbaloin, isobar baloin, antranol dan tannin. tannin adalah salah satu bahan antibakteri yang umumnya terdapat pada tanaman berkhasiat obat yang digunakan dalam pengobatan. menurut penelitian boel, 4 aloe vera mempunyai daya antibakteri terhadap s. mutans pada konsentrasi 25%, 50% dan 100%. daya hambatnya terhadap s. mutans akan semakin besar pada konsentrasi yang lebih tinggi. kandungan siwak (salvadora persica) pasta gigi herbal c terdiri dari trimetyl amine, silica, alkaloid, chloride, fluoride, saponin, tannin, resin, sulfur, vitamin c dan sterol.5 chloride berguna dalam mengangkat stain, silica merupakan bahan pembersih gigi, tannin dan resin membentuk lapisan pelindung pada email yang mencegah masalah kerusakan gigi, vitamin c dan trimetyl amine membantu dalam menyembuhkan jaringan gingiva, trymetyl amine sendiri berfungsi dalam mengurangi kalkulus dan stain, sulfur, alkaloid dan fluor melindungi gigi dari bakteri kariogenik.5 efek terapeutik dan profilaktik dari siwak kemungkinan diakibatkan oleh adanya pembersihan mekanis, pelepasan zat kimia aktif yang terdapat didalamnya dan atau kombinasi keduanya. adanya substansi silica pada salvadora persica (siwak) ini, diduga membantu aksi mekanis siwak terhadap pembersihan plak. dalam penelitian untuk menentukan efek penggunaan tabel 2. perbedaan daya hambat terhadap s. mutans antar pasta gigi yang mengandung herbal dan pasta gigi kontrol setelah 24 jam pasta gigi herbal a herbal b herbal c herbal d kontrol herbal a (eucalyptus) − 0,001* 0,001* 0,024* 0,905 herbal b (aloe vera + daun sirih) 0,001* − 0,015* 0,218 0,001* herbal c (siwak) 0,001* 0,015* − 0,001* 0,001* herbal d (daun sirih) 0,024* 0,218 0,001* − 0,018* kontrol 0,905 0,001* 0,001* 0,018* − keterangan: * = ada perbedaan bermakna 67pratiwi: perbedaan daya hambat terhadap streptococcus mutans siwak sebagai sikat gigi dapat mempengaruhi jumlah s. mutans yang terdapat pada permukan gigi, 30 relawan diinstruksikan untuk menggunakan siwak komersial dua kali sehari sebagai tambahan pembersihan mulut secara rutin. ditemukan bahwa 6 orang (20%) dari partisipan tersebut mengalami pengurangan jumlah s. mutans lebih besar daripada cara lain.5 pasta gigi herbal d yang mengandung minyak atsiri daun sirih mempunyai daya antibakteri karena adanya fenol dan turunannya yang dapat mengubah sifat protein sel bakteri. salah satu senyawa turunan itu adalah kavikol yang memiliki daya antibakteri lima kali lebih kuat daripada fenol. adanya fenol yang merupakan senyawa toksik mengakibatkan struktur tiga dimensi protein terganggu dan terbuka menjadi struktur acak tanpa adanya kerusakan pada struktur kerangka kovalen. hal ini mengakibatkan protein berubah sifat. deret asam amino protein tersebut tetap utuh setelah berubah sifat, namun aktivitas biologisnya menjadi rusak sehingga protein tidak dapat melakukan fungsinya.6 berdasarkan penelitian hasim6 yang membandingkan aktivitas antibakteri daun sirih dan fluor ditemukan bahwa daya hambat minyak atsiri daun sirih lebih besar daripada naf pada semua konsentrasi uji. minyak atsiri daun sirih memiliki aktivitas antibakteri terhadap s. mutans lebih besar daripada fluor. dalam bentuk pasta gigi, maka pasta gigi yang mengandung fluor menunjukkan aktivitas antibakteri pada konsentrasi 0,75% sedangkan pasta gigi yang mengandung minyak atsiri daun sirih menunjukkan aktivitas antibakteri pada konsentrasi 0,1%. aktivitasnya terus meningkat dengan meningkatnya konsentrasi minyak atsiri. adanya perbedaan bermakna antara pasta gigi herbal dalam menghambat s. mutans dimungkinkan karena adanya perbedaan kandungan herbal pasta gigi tersebut. hasil penelitian yang dilakukan menyimpulkan bahwa semua pasta gigi yang diuji mempunyai daya hambat terhadap s. mutans dan berbeda secara bermakna (r < 0,05). daya hambat terbesar dimiliki oleh pasta gigi herbal c (mengandung siwak) dan terkecil pasta gigi kontrol (non herbal). daftar pustaka 1. kidd eam, joyston s. pencegahan karies dengan pengendalian plak. dalam: narlan sumawinata, safrida faruk. dasar-dasar karies: penyakit dan penanggulangannya. jakarta: egc; 1992. h. 141–54. 2. da silva dd, goncalo cs, de sousa mlr, wada rs. aggregation of plaque disclosing agent in a dentifrice. j appl oral sci 2004; 12(2): 154–8. 3. pistorius a, willershausen b, steinmeier em and kreisler m. efficacy of subgingival irrigation using herbal extract on gingival inflamation. j periodontol 2003; 74: 616–22. 4. boel t. daya antibakteri pada beberapa konsentrasi dan kadar hambat tumbuh minimal dari aloe vera. dentika dent j 2002; 7(1): 58–66. 5. almas k, al-zeid z. the immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study. j contemp dent pract 2004; 5(1): 1–8. 6. hasim. daun sirih sebagai antibakteri pasta gigi. kompas 24 september 2003. 7. sodiummonofluorfosfat. available from: url:http//scitoys.com/ ingredients/sodiummonofluorfosfat.htm. accessed may 26, 2004. 8. grieve m. eucalyptus. available from: url:http//www.botanical. com/botanical/mgmh/e/eucaly.htm. accessed april 20, 2004. 9. home toothpaste. available from: url:http//www.anglefire.com/ il2/purpleflame/herbs/toothpaste.htm. accessed may 26, 2004. 10. tea tree place. available from: url:http//www.teatreeplace.com/ naturetoothpaste.htm. accessed may 26, 2004. 11. dental hygiene toothpaste. available from: url:http// www.anewlife.co.uk. accessed may 26, 2004. 12. boel t. daya antibakteri kombinasi triklosan dan zink sitrat dalam beberapa konsentrasi terhadap pertumbuhan streptokokus mutans. dentika majalah ilmiah kedokteran gigi usu 2000; 5(1): 7–17. 13. jordan sl and taylor lt. the analysis of triclosan in toothpaste via lc/ft-ir mobile phase elimination an-20. available from: url:http//www.stjapan.co.jp/014 labconnection/014 l b a/an 20.pdf. accessed may 26, 2004. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38 no 3 2005 151 isolasi gen kariogenik gtf bc streptococcus mutans dari plak gigi anak (the isolation of streptococcus mutans cariogenic gtf bc gene from children’s tooth plaque) yetty herdiyati soemantadiredja* dan mieke hemiawati satari** ** bagian ilmu kedokteran gigi anak ** bagian mikrobiologi fakultas kedokteran gigi universitas padjadjaran bandung indonesia abstract the aim of this research was to prove that streptococcus mutans isolated from children ‘s tooth plaque was cariogenic. glf bc which is glycosiltransferase producer gene has had a role in caries development. this gene was isolated from streptococcus mutans. the specimens were analyzed using wizard genomic dna purification kit from promega. in conclusion, s. mutans isolatedfrom children’s tooth plaque truly had cariogenic gtf bc gene. key words: streptococcus mutans, gen 16s rrna, gen gtf bc korespondensi (correspondence): yetty herdiyati soemantadiredja, bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jln. sekeloa selatan i bandung 40132, indonesia. pendahuluan karies gigi merupakan suatu penyakit umum yang sering ditemukan sejak pertama terdapat sejarah kehidupan manusia. dr. wd miller (1980) merupakan orang pertama yang menggambarkan karies sebagai aksi dari asam organik terhadap kalsium fosfat pada gigi. ia memperlihatkan bila gigi diinkubasi dengan saliva dan karbohidrat, asam akan terbentuk dan menguraikan bagian gigi yang termineralisasi. ia menyimpulkan bahwa asam yang dibentuk oleh bakteri dalam saliva menguraikan gigi. dari penelitian ini ia merumuskan teori “kemo-parasitik” dari karies gigi. sejak saat itu banyak data yang mendukung teori menurunnya ph oleh produksi asam bakteri akan menghasilkan penguraian email. penelitian dr. miller telah membentuk dasar untuk teori “plak-tuan rumah-substrat” dari pembentukan karies. proses pembentukan karies gigi disebabkan oleh multifaktor, pada dasarnya dapat disederhanakan menjadi hubungan yang tidak seimbang antara daya tahan gigi dengan faktor kariogenik.1 email yang 95% terkalsifikasi merupakan struktur yang sangat termineralisasi dari tubuh manusia. mayoritas mineralnya adalah hidroksiapatit yang merupakan keluarga garam kalsium fosfat. formula umum yang diungkapkan untuk hidroksiapatit adalah ca 10(po4) 6oh 2. hidroksiapatit murni merupakan struktur yang unik, terdapat sumbu simetris enam kali lipat di sekeliling suatu sumbu simetris tiga kali lipat. kristalnya tersusun dalam konfigurasi heksagonal dengan atom kalsium dan fosfor pada kristal terluar. di bagian tengah kristal, kelompok hidroksil dikelilingi oleh tiga atom kalsium. hubungan dari masing-masing kelompok hidroksil berperan penting dalam kestabilan kristal. bahkan penggantian sejumlah kelompok hidroksil murni dengan atom dalam seperti fluoride menghasilkan peningkatan stabilitas kristal. kristal heksagonal ini dikelompokkan bersama menjadi sebuah batang. email terbentuk dari banyak batang yang dipadatkan bersama menjadi sebuah batang. batang ini terbentang dari permukaan email ke dental junction. ukuran kepadatan atau densitas dari email menunjukkan bahwa permukaan luar memiliki densitas yeng sedikit lebih tinggi daripada struktur lain. ruang antar batang email merupakan matriks organik. meskipun email kelihatannya sangat keras dan termineralisasi dengan baik, permukaannya berpori dengan adanya ion kecil seperti sodium, potassium, magnesium dan fluoride. keberadaan ruang interprismatik ini telah digunakan untuk menjelaskan permeabilitas yang terlihat pada email.1 hidroksiapatit memiliki konstanta kemampuan untuk larut yang pasti bergantung pada temperatur, ph dan kekuatan ionik dari pelarut yang mengelilingi kristal. di rongga mulut, saliva mengalami supersaturasi dalam hal kalsium dan fosfat yang menguntungkan bagi keadaan kristal email dan dengan demikian gigi tidak terurai secara bertahap seumur hidup. akibat dari sifat reaksi seimbang dalam rongga mulut, email berada dalam keadaan mineralisasi konstan dan demineralisasi pada kondisi fisiologis (ca2po4, ph 6,8). pada ph ini kemampuan untuk larut sangat kecil. meskipun demikian, jika ph diturunkan sampai sekitar 1,3 log unit sampai 5,5 maka kemampuan untuk larut meningkat sampai penguraian terjadi produksi asam dari 152 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 151–153 bakteri dan pembentukan karies gigi yang terjadi setelahnya merupakan titik puncak dari suatu proses yang sangat selektif dari perlekatan bakteri dan kolonisasi bakteri pada permukaan gigi. bila hal ini terus berlanjut akan mengarah pada pembentukan plak. plak gigi terdiri dari sekumpulan sel besar dan kecil atau sel individual dari berbagai spesies bakteri yang berbeda serta matriks interbakterial dengan komposisi heterogenous dan bervariasi. meskipun demikian, organisme asidogenik spesifik, yaitu yang berasal dari kelompok streptococcus mutans (s. mutans) saat ini secara umum dianggap memiliki peranan khusus dalam etiologi karies gigi. s. mutans merupakan salah satu pemicu karies karena bakteri ini memiliki enzim glikosiltransferase yang berperan sebagai prekursor dalam perkembangan plak gigi, namun tidak semua plak gigi dapat menyebabkan karies gigi.’ bahan dan metode rancangan penelitian adalah observasional yang bersifat eksploratif yaitu dengan uji diagnostik laboratoris untuk menentukan bahwa deteksi kuman streptococcus mutans dapat menggunakan gen 16srrna dengan pcr, dari 15 sampel yang digunakan dalam penelitian ini hanya 1 yang berhasil terdeteksi sebagai streptococcus mutans. bahan penelitian adalah plak gigi anak yang dicampur dalam bulyon kemudian campuran ini diencerkan hingga 10-3 →10-5. pembiakan dilakukan pada media tycsb kemudian diinkubasikan secara anaerob selama 3 × 24 jam. ekstraksi dna dengan metode wizard genomic dna purification kit dari promega.2,3 adapun larutan yang harus disiapkan: edta 50 mm ph 8,0; lysozim 10 mg/ml; isopropanol; etanol 70. peralatan yang digunakan berupa tabung mikro 1,5 ml; waterbath 37° c dan 80° c. cara kerja: hasil ekstrasi dna ditambahkan 1 ml overnigth kultur ketabung 1,5 ml kemudian disentrifugasi selama 2 menit pada kecepatan 13.000-16.000 × g, supernatan dibuang, selanjutnya sel di resuspensi kedalam larutan 480 μl edta 50 mm. tambahkan 60 μl lysozim l0 mg/ml dengan menggunakan pipet secara hati-hati hingga semua sel tersuspensi, kemudian di inkubasi pada suhu 80° c selama 5 menit untuk melisis sel dan selanjutnya didinginkan di dalam suhu ruang. tambahkan 3 μl larutan rnase ke dalam tabung kemudian sampel dicampur dengan menginversi tabung sebanyak 2–5 kali, setelah itu diinkubasi selama 15–60 menit pada suhu 37° c, biarkan sampel mencapai suhu ruang, selanjutnya ditambahkan larutan 200 μl protein precipitation dan vortex pada kecepatan tinggi selama 20 detik lalu di inkubasi di dalam es selama 5 menit, kemudian disentrifugasi selama 3 menit pada kecepatan 13.000– 16.000× g. supernatan yang mengandung dna dipindahkan ke tabung 1,5 ml yang mengandung 600 μl isopropanol suhu ruang, campurkan larutan dengan inversi sampai terlihat rantai dna yang seperti benang halus. lalu sentrifugasi selama 2 menit pada kecepatan 13.000-16.000× g, kemudian dengan hati-hati dekantasi supernatan dan keringkan tabung dengan membalikkan di atas kertas tissue yang bersih. tambahkan 300 μl etanol 70 temperatur ruang dan inversi tabung beberapa kali untuk mencuci dna pelet, kemudian dilakukan sentrifugasi selama 2 menit pada kecepatan 13.000–16.000× g, buang etanol dengan hati-hati. keringkan tabung dengan konsentrator, atau balikan tabung pada kertas tissue bersih selama 10–15 menit. tambahkan 50 μl larutan dna rehidrasi selanjutnya dna disimpan pada suhu 2–8° c. pcr gen l6s rrna primer yang digunakan adalah untuk universal reverse primer: uni b: 5'– ggttc(g/c) ttgttacgactt-3' dan eubacterial forward primer: bactfl: 5'– agagtttgatc (a/c)tggctac-3'. hasil optimasi pcr, dilakukan denaturasi awal pada suhu 94° c selama 2 menit, siklus amplikasi 30 siklus, kemudian di denaturasi 94° c selama 1 menit, annealing 48° c selama 1 menit, elongation 72° c selama 1 menit, pasca elongation 72° c selama 10 menit. melalui elektroforesis hasil pcr dengan menggunakan agarose dna, diperoleh sebuah larik, dengan ukuran 1400 pb. reaksi sekuensing: 16s rrna primer yang digunakan : 357 f: 5'tag ggg agg cag cag-3'; 807 f: 5' gat tag ata ccc tgg tag3'; 1114f: 5' gca acg agc gca acc a -3'; 519 r: 5' gta tta ccg cgg ctg crg-3'; 909 r: 5' ccg tca att cat ttg agt-3'. prosedur kerja: isolasi kromosom streptococcus mutans dengan menggunakan metode lisis cepat. tujuannya adalah untuk memperoleh template yang akan digunakan pada amplifikasi gen glikosltransferase dengan metode pcr. pcr gen gtf bc hasil optimasi pcr, dilakukan denatiirasi awal 94° c selama 2 menit, siklus amplifikasi 40 siklus, denaturasi 94° c selama 1 menit, annealing 50° c selama 1 menit elongasi 72° c selama 1 menit, pasca elongasi 72° c 10 menit. hasil hasil isolasi gen 16s rrna yang diperoleh memiliki panjang 1400 pb. setelah dilakukan homologi dengan streptococcus mutans ua 101 melalui program komputer dna star, maka diyakini bahwa bakteri hasil isolat gigi anak tersebut adalah streptococcus mutans, tampak pada gambar 1 dan 2. 153herdiyati: isolasi gen kariogenik gtf bc gambar 1. hasil elektroforesis dna 16s rrna. 1. marka dna puc hin f, pita pertama berukuran 1419 pb, pita kedua berukuran 517 pb. 2. hasil pcr, 16s rrna berukuran 1400 pb. gambar 3. hasil analisa sequensing gen 16s rrna gambar 2. hasil elektroforesis dna gtf bc. no. 1–3 hasil pcr gen gtf bc no. 4 marka dna puc hin f, pita pertama berukuran 1419 pb, pita kedua berukuran 517 pb. pembahasan untuk dapat mengisolasi s. mutans secara tepat maka saat ini dapat digunakan deteksi streptococcus mutans secara molekuler yaitu dengan mengamplifikasi 16s rrna. setiap bakteri memiliki 16srrna yang merupakan suatu subunit dari rna ribosom. subunit ini memiliki daerah konservatif yang berguna untuk menentukan alignment yang tepat dari suatu prokariot. disamping itu daerah ini mengandung pula sejumlah mutan yang bervariasi di tempat lain yang menjadi ciri khas untuk filogenetik suatu bakteri. gen gtf bc adalah suatu polimer glukosa ekstraseluler yang bersifat tidak larut dalam air yang dikenal sebagai salah satu faktor virulensi dalam karies. gen gtf bc adalah salah satu gen yang mengkode enzim yang bertanggung jawab untuk sintesis glukan yang tidak larut dalam air yang dikenal sebagai ig (insoluble glukan).2, 4-6 penggunaan streptococcus mutans ua 101 sebagai kontrol disebabkan bakteri ini memperlihatkan sifat kariogenik, karena memiliki gen gtf b atau gtf bc. hasil isolasi gtf bc dengan panjang 517 pb menunjukkan bahwa hasil isolat streptococcus mutans dari gigi anak ini bersifat kariogenik. streptococcus mutans secara konstitutif akan mensekresikan gtf yang dikode oleh gen gtf. gen gtf ini membentuk suatu operon yang terdiri dari gtf a, b, c, d dan yang berperan dalam pembentukan karies adalah gtf b dan gtf bc, glukan ini bersifat tidak larut dalam air, karena itu adanya kedua gen ini menunjukkan bahwa bakteri streptococcus mutans tersebut mampu menyebabkan terjadinya karies. berdasarkan penelitian ini disimpulkan bahwa, hasil isolat streptococcus mutans dari plak gigi anak menunjukkan adanya gen gtf bc yang dapat mengekspresikan glukan yang tidak larut air dan bersifat kariogenik. daftar pustaka 1. newman mg, nisengard r. oral microbiology and immunology. philadelphia: wb saunders co; 1988. p. 432–8. 2. baker gc. identification of indonesian hyperthermoghiles using 16 sr rna sequencing. bandung: ppau bioteknologi itb; 1999. 3. madigan mt, martinko jm, parker j. biology of microorganisms 9th ed. new jersey: prentice hall inc, 2000. 4. russel rrb, ivic a. the use of dna probes for the identification of oral streptococci caries. res 1989; 23:110–2. 5. chia js, yang cs, chen jy. functional analysis of a conserved region in glucocyltransferase of streptococcus mutans. j infection and immunity 1998; 66(10):4747–903. 6. yamashita y, bowen wh, kuramitsu hk. molecular analysis of a streptococcus mutans strain exhibitmg polymorphism in the tandem gtf bang gtf bc genes. j infection and immumty 1992; 60(4):1618–24. << /ascii85encodepages false /allowtransparency 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 135 volume 46, number 3, september 2013 effect of gestational diabetes mellitus on the expression of amelogenin in rat offspring tooth germ nurdiana dewi,1 ahmad syaify2 and ivan arie wahyudi3 1 study program of dental science, faculty of medicine, universitas lambung mangkurat, banjarmasin – indonesia 2 department of periodontology, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia 3 department of biomedica, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: amelogenin is a major protein constituent of the developing enamel matrix that is critical for enamel formation. mutations of amelogenin cause hypoplastic enamel phenotypes. previous research found that infant of diabetic mother has higher risk for having enamel hypoplasia. purpose: the aim of this study was to determine the effect of gestational diabetes mellitus on the expression of amelogenin in wistar rats offspring tooth germ. methods: sixteen female wistar rats, aged 2.5-3 months, body weight 150-200 g were used in this study, wistar rats were mated and divided into two groups and treated on day 0 of pregnancy. group a was dm group, consisting of 8 rats, induced by streptozotocin (stz) injection 40 mg/kg bw. group b was control group, consisting of 8 rats received citrate buffer injection. thirty-two rat pups were decapitated on day 5. immunohistochemical procedures were performed on molar tooth germ of the mandibular rat pups using antibody anti-amelx to determine the expression of amelogenin. examination carried out on the images using imagej software. all data were then statistically analyzed by mann whitney test. results: there was no significant difference in the expression of amelogenin in the dm group and control group (p>0.05). conclusion: gestational diabetes mellitus did not affect the expression of amelogenin in rat offspring tooth germ. further study is needed to examine the pattern of amelogenin expression with measurement of glucose levels of rat pups. key words: gestational diabetes mellitus, amelogenin expression, wistar rats abstrak latar belakang: amelogenin merupakan protein terbanyak pada matriks email yang berperan penting dalam pembentukan email. mutasi pada amelogenin dapat menyebabkan email menjadi hipoplastik. penelitian sebelumnya menunjukkan bahwa anak yang dilahirkan oleh ibu pengidap diabetes memiliki resiko lebih tinggi untuk mengalami hipoplasia email. tujuan: penelitian ini bertujuan untuk meneliti pengaruh diabetes mellitus gestasional terhadap ekspresi amelogenin pada benih gigi anak tikus wistar. metode: enam belas ekor tikus wistar betina, umur 2,5-3 bulan, berat badan 150-200 g digunakan dalam penelitian ini, dikawinkan kemudian dibagi menjadi dua kelompok dan diberi perlakuan pada kehamilan hari ke-0. kelompok a merupakan kelompok diabetes mellitus, terdiri atas 8 ekor tikus diberi perlakuan diabetes mellitus dengan injeksi streptozotocin (stz) 40 mg/kg bb. kelompok b merupakan kelompok kontrol, terdiri atas 8 ekor tikus diberi injeksi buffer sitrat. tiga puluh dua anak tikus yang lahir didekapitasi pada hari ke5. dilakukan prosedur imunohistokimia pada benih gigi molar rahang bawah anak tikus menggunakan antibodi anti-amelx untuk mengetahui ekspresi amelogenin. pemeriksaan dilakukan pada hasil foto menggunakan software imagej. hasil kemudian dianalisa menggunakan uji mann whitney. hasil: tidak terdapat perbedaan yang bemakna pada ekspresi amelogenin kelompok kontrol dan kelompok diabetes mellitus (p>0,05). simpulan: diabetes mellitus gestasional tidak mempengaruhi ekspresi amelogenin pada benih gigi anak tikus. diperlukan penelitian lebih lanjut untuk mengetahui pola ekspresi amelogenin dengan pengukuran kadar glukosa darah anak tikus. kata kunci: diabetes mellitus gestasional, ekspresi amelogenin, tikus wistar correspondence: nurdiana dewi c/o: program studi ilmu kedokteran gigi, fakultas kedokteran universitas lambung mangkurat. jl. veteran no. 128b banjarmasin, indonesia. e-mail: nurdianadewi@gmail.com research report 136 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 135–139 introduction diabetes mellitus (dm) is a chronic metabolic disorder of carbohydrates, lipids and proteins, which occurs because the pancreas can not produce enough insulin or because the body can not use insulin effectively resulting in increased levels of glucose in blood (hyperglycemia).1 gestational diabetes mellitus (gdm) is diagnosed when dm begins or is first detected during pregnancy.2 hyperglycemia during pregnancy can cause complications to the mother and fetus. maternal complications associated with gdm include hypertension and increased risk of developing diabetes after pregnancy. fetal complications include macrosomia, hypoglycemia, hypocalcemia, polycythemia, and hyperbilirubinemia.3,4 an infant of diabetic mothers has higher risk for having enamel hypoplasia.5 another study also showed thinner enamel in pups born to diabetic mother because of decreased secretion of enamel matrix and ultrastructural changes in the secretory ameloblast. the secretory ameloblast were shorter and the ameloblast nuclei were smaller. there were intracellular metabolic disturbances in consequence to the lack of intracellular glucose.6 amelogenin is a hydrophobic protein that is expressed by ameloblast. this is the most abundant protein of the enamel extracellular matrix, compose 80-90% of total protein, and is expressed in the secretory until post-secretory stage of ameloblast.7 amelogenin is essential for well-organized hydroxyapatite prism formation and for producing normal enamel thickness. in vivo studies of amelogenin null mice showed the occurrence of enamel hypoplasia, seen chalkywhite staining on incisivus. enamel thickness was less than 10% of normal enamel.8 amelogenin expression can be influenced by several factors, such as blood glucose and calcium levels.9,10 the result of this experiment is then expected to give information about the effect of gestational diabetes mellitus on the expression of amelogenin in rat offspring tooth germ. materials and methods sixteen female wistar rats, aged 2.5-3 months, with 150-200 g body might were adapted to the metal cages for 1 week, given the standard feed and drink ad libitum. the rats were kept on a 12-h light-dark cycle at 22–24˚ c. the day that spermatozoids appeared in vaginal smears (day 0 of pregnancy), 8 rats were intraperitoneally treated with 40 mg stz (sigma, st. louis, mo, usa)/kg bw, dissolved in 50 mm citrate buffer, ph 4.5. eight control rats were run in parallel, and received the medium. the experimental procedure was approved by the ethics and advocacy unit of the faculty of dentistry gadjah mada university. animals were weighed and blood glucose levels were measured with accu-check active (roche, germany) on day 0, 7, 14, and 19 of pregnancy. rats with fasting blood glucose levels above 120 mg/dl and showed the sign polydipsia, polyuria, poliphagia, and asthenia were considered as having diabetes.4,11 two pups from each litter were selected at random and decapitated on day 5 after birth . mandibular molar tooth germ of rat pups were taken and fixed with 4% paraformaldehide in phosphate-buffered saline (pbs formalin) for 24 hours, decalcified using 10% edta at 4° c for 14 days and embedded in paraffin. 3 μm thick cross-sectional sections were stained with immunohistochemistry. samples were deparaffinized with xylol and rehydrated with serial alcohol. after deparaffinization and hydration, the sections were treated with 0.3% h2o2 in methanol for 15 minutes to reduce endogenous peroxidise activity, then washed with distilled water and tris edta followed by administration of antigen retreaval application (in citric buffer ph 6) by heating for 15 minutes in a microwave to open antigencovered and washed with tris edta. they were then blocked with different normal serum (background snipper) at room temperature for 10 minutes followed by hatching the primary antibody and incubated at 4˚ c for 18 hours; primary antibodies anti-amelx diluted with pbs (1:1000). after washing with tris edta, the sections were incubated with secondary antibody (trekkie universal link) for 10 minutes at room temperature, washed with tris edta and treated with trekavidin-hrp label for 10 minutes. sections were then washed with tris edta and staining for peroxidase was performed with dab chromogen (1:200 in substrate) in a dark room for 3 minutes then washed with distilled water. for maximum staining, counterstain with haematoxylin meyers performed for 2 minutes and terminated by washing with water tap for 2 minutes. the section were then dehydrated with serial alcohol followed by xylol. next stage was mounting the slide. normal rat tooth germ was used as positive control. i m a g e s o f t h e c r o s s s e c t i o n a l s e c t i o n o f immunohistochemistry-stained molars were captured using a light microscope connected to camera (optilab). amelogenin expression was identified as brownish yellow spots in the cytoplasm. amelogenin expression in ameloblast was observed by measuring the density of amelogenin using imagej software. greater value stated on the imagej software showed greater density of amelogenin, and greater density of amelogenin means that amelogenin expression getting weaker. results the means of fasting blood glucose level and body weight of female rat are presented in figure 1. fasting blood glucose levels in diabetic group increased after injection of stz. the highest fasting blood glucose level was in diabetic group day 14 of pregnancy (329.00 ± 97.33 mg/dl), and the lowest was in control group day 19 of pregnancy (81.39 ± 7.05 mg/dl) (figure 1a). fasting blood glucose levels in diabetic group decreased on day 19, but 137dewi, et al.,: effect of gestational diabetes mellitus on the expression still above 120 mg/dl. there was no increase of fasting blood glucose levels in the control group. rats body weight were increased in each observation either in the control and dm group. control rats had body weight means greater than diabetic rats, with the greatest body weight mean was on control group day 19 of pregnancy (252.80 ± 19.91 g) and the lowest was in dm group day 0 of pregnancy (155.79 ± 4.64 g) (figure 1b). histological amelogenin expression can be seen in figure 2a, b, c, d. means of amelogenin density in control group greater than dm group (figure 3). the greater amelogenin density means the amelogenin expression getting weaker. this suggests that amelogenin in dm group were expressed stronger compared with control group. normality test results were 0.033 for control group and 0.102 for dm group. these results indicate that data figure 2. sections showing localization of amelogenin protein in the crossectional mandibular molar tooth germ using an immunohistochemical technique. amelogenin expression in ameloblast were marked with brownish yellow granules in the cytoplasm. a, c. control/dm group was observed at 40x magnification, b, d. control/dm group was observed at 400x magnification. em, enamel; ab, ameloblast; si, stratum intermedium; sr, stellate reticulum; am, amelogenin. figure 1. means of fasting blood glucose level (a) and body weight (b) of female rat in control and dm group. fasting blood glucose and body weight were measured on pregnancy day-0, 7, 14 and 19. a b a b c d control dm a b c d control dm 138 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 135–139 has a significance p<0.05 for control group and p>0.05 for dm group which means the control group data were not normally distributed, so it could not proceed with the parametric test. mann whitney test results showed p-value = 0.224 (p>0.05). these results indicate that there was no significant difference in the amelogenin density between control and dm group, which means maternal diabetic condition had no significant effect on the amelogenin expression. discussion results of this study indicate that there was an increase in fasting blood glucose levels of diabetic group compared with control group. diabetic group had glucose level above 120 mg/dl. increasing of fasting blood sugar levels in diabetic rats probably caused by necrosis of pancreas beta cells. streptozotocin selectively induce necrosis in pancreatic beta cells via dna methylation. dna damage is caused by free radicals that are released by stz. nitrosurea in stz causes cellular toxicity through decreased levels of nad+ and production of free radicals. streptozotocin is also able to act as a donor of nitric oxide (no) and generate reactive oxygen species (ros). necrosis of beta cells causes a decrease in the biosynthesis and secretion of insulin and blood glucose levels.12 rat also showed the signs of dm, i.e polydipsia (abnormal thrist), polyuria (increased urine volume), polyphagia (excessive hunger) and asthenia (weakness due to the inability to use glucose as a source of energy). this finding agrees with previous studied.11 in this research, the weight of rat had increased either in control and diabetic group. diabetic group weight was lower than control group, although diabetic group consumed more food and beverages. this was probably caused by metabolic disorders due to diabetic conditions.13 there were disturbances in the metabolism of carbohydrates, proteins and lipids in diabetic rats.1 low weight gain during pregnancy could be a cause of the low number of lpa foetuses in this group.13 weight gain as well as the results of abdominal palpation during pregnancy showed that the rat had been pregnant. pregnant rats was determined by palpation on the abdomen on day 13 of pregnancy. enlargement in the abdomen suggests there were multifoetuses in uterus.14 the results showed that the diabetic condition did not have a significant effect on amelogenin density. it means that diabetic condition had no effect on the expression of amelogenin significantly. the absence of significant effect is likely due to normal fasting blood glucose levels and normal serum calcium levels in rat pups. examination of blood glucose levels in rat pups was not done in this research. however, based on the results of previous studied, blood glucose levels of rat pups born to diabetic rats parent could be significantly high compared to control.15 another study showed that stz-offspring were initially hypoglycemic but became normoglycemic by weaning and remained normal up to at least 15 wk of age.16 other possible causes of the absence of significant differences in the expression of amelogenin in this study was due to the normal serum calcium levels in rat pups. serum calcium levels remained relatively constant since each cell has basic requirements for calcium. low serum calcium levels will stimulate production of parathyroid hormone. parathyroid hormone then increase resorption of bone matrix stimulate osteoblasts to release factors that increase the number and activity of osteoclasts. increased bone resorption would increase serum concentrations of calcium and phosphate. parathyroid hormone increases the absorption of calcium and decreases the absorbtion of phosphate in the kidneys causing fosfaturia. increased calcium reabsorption in the renal tubules by transport proteins (epithelial calcium channel, calbindin-d28k and plasma membrane ca2+-atpase) in children born to mothers with diabetes will normalize serum calcium levels.17 parathyroid hormone also increase the activity of 1-α-hydroxylase, resulting in increased synthesis of 1.25dihydroxyvitamin d which causes an increase in calcium absorption in the small intestine.18 it can be concluded that gestational diabetes mellitus does not affect the expression of amelogenin in wistar rat offspring tooth germ. further research is needed to examine the expression patterns of amelogenin with measurement of blood glucose and serum calcium levels in diabetic offspring. acknowledgement the authors were very grateful to direktorat jenderal perguruan tinggi kementerian pendidikan nasional and s2 study program of dental science at the faculty of dentistry gadjah mada university yogyakarta for giving the authors opportunity to conduct research. figure 3. means and standard deviations of amelogenin density in rat offspring tooth germ. amelogenin density was measured using imagej software. 139dewi, et al.,: effect of gestational diabetes mellitus on the expression references 1. hall je. insulin, glucagon, and diabetes mellitus. in: guyton and hall text book of medical physiology. 12th ed. philadelphia: elseviers saunders; 2011. p. 939–54. 2. buchanan ta, xiang ah. gestational diabetes mellitus. j clin invest 2005; 115(3): 485–91. 3. lessi il, bueno a, sinzato yk, taylor kn, rudge mv, damasceno dc. evaluation of neonatally-induced mild diabetes in rats: maternal and fetal repercussions. diabetol metab syndr 2010; 2(1): 37. 4. murthy ek, pavlic-renar i, metelko z. diabetes and pregnancy. diabetologia croatica 2002; 31(3): 131–46. 5. silva-sousa ytc, peres lc, foss mc. enamel hypoplasia in a litter of rats with alloxan-induced diabetes mellitus. braz dent j 2003; 14(2): 87–93. 6. silva-sousa ytc, peres lc, foss mc. are there structural alterations in the enamel organ of offspring in rats with alloxaninduced diabetes mellitus?. braz dent j 2003; 14(3): 162–7. 7. torres-quintana ma, gaete m, hernandez m, farias m, lobos n. ameloblastin and amelogenin expression in postnatal developing mouse molars. j oral sci 2005; 47(1): 27–34. 8. gibson cw. amelogenin-deficient mice display an amelogenesis imperfecta phenotype. j biol chem 2001; 276(34): 31871–5. 9. yeh ck, harris se, mohan s, horn d, fajardo r, chun yh, jorgensen j, macdougall m, abboud-werner s. hyperglycemia and xerostomia are key determinants of tooth decay in type 1 diabetic mice. lab invest 2012; 92(6): 868–82. 10. chen j, zhang y, mendoza j, denbesten p. calcium-mediated differentiation of ameloblast lineage cells in vitro. j exp zool (mol dev evol) 2009; 312b: 458–64. 11. carvalho en, carvalho nas, ferreira lm. experimental model of induction of diabetes mellitus in rats. acta cir braz 2003; 18: 60–4. 12. lenzen s. the mechanisms of alloxan-and streptozotocin-induced diabetes. diabetologia. 2008; 51(2): 216–26. 13. kiss aci, lima pho, sinzato yk, takaku m, takeno ma, rudge mvc, damasceno dc. animal models for clinical and gestational diabetes: maternal and fetal outcomes. diabetol metab syndr 2009; 1(1): 21. 14. ypsila nt is p, def tereos s, p rassopoulos p, si mopoulos c. ultrasonographic diagnosis of pregnancy in rats. j am assoc lab anim sci 2009; 48(6): 734–9. 15. sharma r, chauhan ss, mahmood a. modulation of intestinal brush border membrane chemical composition during postnatal development in rats: effect of gestational diabetes. indian j exp biol 2012; 50(1): 45–50. 16. han j, xu j, long ys, epstein pn, liu yq. rat maternal diabetes impairs pancreatic β-cell function in the offspring. am j physiol endocrinol metab 2007; 293(1): e228–36. 17. bond h, sibley cp, balment rj, ashton n. increased renal tubular reabsorbtion of ca and mg by the offspring of diabetic rat pregnancy. pediatr res 2005; 57(6): 890–5. 18. bass jk, chan gm. calcium nutrition and metabolism during infacy. nutrition 2006; 22(10): 1057–66. 5959 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 59–62 the correlation between rood and shehab’s radiographic features and the incidence of inferior alveolar nerve paraesthesia following odontectomy of lower third molars david b. kamadjaja, djodi asmara, and gita khairana departement of oral and maxillofacial surgery faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: odontectomy of lower third molar has a potential risk for inferior alveolar nerve impairment. paresthesia of inferior alveolar nerve has often been associated with close relationship between the apex of lower third molar and mandibular canal. rood and shehab’s category has been commonly used for radiological prediction of inferior alveolar nerve injury following third molar surgery. purpose: this study aimed to determine whether there was correlation between rood and shehab’s radiographic features and the incidence of inferior alveolar nerve paraesthesia following odontectomy of lower third molar. method: this was a retrospective cross-sectional study, using data obtained from the dental record of patients who had undergone odontectomy of lower third molars in dental hospital of universitas airlangga during 2 years period. samples were cases that, from presurgical radiograph, showed close relationship between lower third molar roots and mandibular canal. the case and non-case groups were assigned based on the presence of paraesthesia and non-paraesthesia of inferior alveolar nerves, respectively. based on rood and shehab’s category, the samples collected were then classified into two groups which were those whose relationship matched and did not match with the category, respectively. data were analyzed using chi-square correlation test. result: of 975 odontectomy cases included in this study, 80 cases were taken as study samples consisting of 15 and 65 cases assigned, respectively, as case and non-case. the 32 cases matched with the criteria of rood and shehab's category while the remainder of 48 cases did not. of 32 cases which met the criteria of rood and shehab’s relationship, only 5 cases showed paraesthesia, whereas out of 48 cases which did not met the criteria 10 cases showed paraesthesia. statistical analysis showed significance value of 0.770 (p>0.05) indicating that there was no significant correlation between relations of third molar root and mandibular canal, based on rood and shehab’s category, and the incidence of inferior alveolar nerve paraesthesia. conclusion: there was no correlation between rood and shehab’s radiographic features and the incidence of paraesthesia of inferior alveolar nerve following odontectomy of lower third molars. keywords: odontectomy; lower third molar; paraesthesia; inferior alveolar nerve; rood and shehab’s (1990) correspondence: david b. kamadjaja, department of oral and maxillofacial surgery, faculty of dental medicine universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: davidbk65@gmail.com introduction odontectomy is surgical removal of unerupted or impacted tooth which requires mucoperiosteal flap followed with removal of bone overlying the buried tooth.1 due to the close anatomic relation between lower third molars and mandibular canal, odontectomy of impacted lower third molars carry potential risk for injury of inferior alveolar nerve.2 injury of inferior alveolar nerve may be due to trauma, inflammation or infection in the periphery of the nerve manifesting clinically as reduced sensation, or paraesthesia, of lower lip and chin of the affected sides.3 various factors have been associated with the incidence which dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p59-62 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p59-62 60 kamadjaja, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 59–62 include variation of the lower third molar roots shapes and locations, less cautious procedures leading to unnecessary large trauma to the bone, the technique used by the operator, excessive pressure on the sutured tissue, and post-operative infection.4,5 study showed that inferior alveolar nerve may be afflicted in the removal of impacted lower third molar at a rate of 0.5 to 5%. in many cases the incidence of inferior alveolar nerve injury can be predicted radiographically prior to the surgery, based on the existence of close relationship between the nerve and the root.6 studies revealed that seven radiological signs could be suggested as indication for a close relationship between mandibular third molar root and mandibular canal. these signs have been used as radiological prediction of inferior alveolar nerve injury during third molar surgery.7 the aim of this study was to determine that there was correlation between the incidence of inferior alveolar nerve injury after odontectomy of lower third molar and rood & shehab’s radiographic relationship of mandibular third molar root to mandibular canal. materials and methods this was a retrospective cross-sectional study. data were collected from dental records in oral and maxillofacial surgery clinic dental hospital, universitas airlangga, surabaya during 2 years period (2014-2015). samples used in the study were cases of lower third molars odontectomy, which on pre-operative x-ray, showed a close relation between lower third molar roots and mandibular canal. included in case group were samples exhibiting sensory impairment of inferior alveolar nerve, while those which did not present with post operative nerve impairment were included in non-case group. sample inclusive criteria in this study were (1) informed consents were taken prior to surgery and (2) all the inclusive cases were operated by senior residents. samples collection was done using purposive sampling method without minimal number of sample. based on panoramic x-ray, both case and non-case samples were further assigned with rood and shehab (1990). rood and shehab (1990)7 relationship categories notified with alphabet a, b, c, d, e, f, and g (figure 1). roods and shehab’s category was as follows. relation “a” shows darkening of root indicating loss of cortical layer of the mandibular canal. relation b shows deflection of root in which roots were acutely curved on approaching mandibular canal. relation “c” shows narrowing of root in which the root tip was seen to become abruptly narrow across the mandibular canal indicating resorption of the roots. relation “d” shows dark and bifid apex of root in which root tips seemed to darken and divert usually seen when mandibular canal was passing through molar roots. relation “e” shows interruption of white line of mandibular canal. relation “f” showed diversion of canal in which mandibular canal diverted its course on approaching the tip of tooth roots. relation “g” shows narrowing of canal in which there seemed to be a decrease in diameter of mandibular canal on approaching the root tip of third molars. relation which did not fall into any of rood and shehab’s category was classified as “others”. 8 figure 1. relation of mandibular third molar roots to mandibular canal based on rood.7 note. darkening of root (a), deflection of root (b), narrowing of root (c), dark and bifid apex of root (d), interruption of white line of canal (e), diversion of canal (f) and narrowing of canal (g). table 1. incidence of paraesthesia dan non-paraesthesia in study samples associated with sex and age variable paresthesia (%) non-paresthesia (%) sex male 9 (60.00) 21 (32.30) female 6 (40.00) 44 (67.69) age group 12-16 1 (06.67) 0 (00.00) 17-25 5 (33.33) 33 (50.76) 26-35 6 (40.00) 22 (33.85) 36-45 2 (13.33) 8 (12.30) 46-55 1 (06.67) 2 (03.07) note: the study samples are 80 post odontectomy cases showing close relationship, radiographically, between lower third molar roots and mandibular canals. figure 1. relation of mandibular third molar roots to mandibular canal based on rood and shehab.7 note. darkening of root (a), deflection of root (b), narrowing of root (c), dark and bifid apex of root (d), interruption of white line of canal (e), diversion of canal (f) and narrowing of canal (g). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p59-62 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p59-62 6161kamadjaja, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 59–62 results out of 975 cases of odontectomy performed in oral and maxillofacial surgery clinic within period of january 2014 through december 2015, there were 80 cases (8.20%) showing, radiographically, close relationship between third molar roots and mandibular canal, but only 15 cases (1.5%) showed sign and symptom of inferior alveolar nerve injury following odontectomy. out of these 80 cases, only 32 cases met the criteria of rood and shehab’s category while the other 48 cases did not meet any of the rood and shehab’s criteria. out of those 32 cases which met rood and shehab’s criteria only 5 cases exhibited alveolar nerve injury paraesthesia, whereas from 48 cases of non-rood & shehab group, 10 cases showed paraesthesia of the same nerve. from the total incidence of paraesthesia in this study which were 15 cases, 60% was found in males, while non-paraesthesia was dominated by females (67%). the incidence rate of paraesthesia was highest in age group of 26-35 (40%), while the highest rate of non-paraestesia was found in age group of 17-25 (50.76%) (table 1). the result showed that the highest rate of appearance of rood and shehab’s radiographic features was relation ‘a’ (12.50%) followed by relation ‘b’ and ‘g’ (7.50%) while the lowest rate of appearance was relation ‘d’ and ‘f’ (2.50%). however, the highest rate of appearance among total samples was relation “others” (60%) which was the relation other than those of rood and shehab’s category (table 1). the result also showed that both relation ‘a’ and ‘b’ had the highest incidence rate (13.3%) of paraesthesia within rood and shehab category, while relation “others” which was the relation other than those of rood and shehab’s category showed the highest incidence rate (66.67%) among total samples (table 2). chi-square test using data analysis of the contingency table (table 3) showed that p value = 0.770 (p>0.05) indicating that there was no correlation between rood and shehab’s relation of third molar roots to mandibular canals and the incidence of inferior alveolar nerve paraesthesia. discussion the result of the study showed that male was more predominant in paraesthesia group (table 1) was consistent with the result of the study conducted by tay and go3 in which the male was higher risk factor for the incidence of inferior alveolar nerve injury. an animal study by miloro et al.,8 associating nerve injury with few risk factors such as age, sex and anatomy, showed that spontaneous nerve recovery was evident in female group. however, other studies indicated that there was no correlation between sex and inferior alveolar nerve injuries after odontectomy of lower third molar.9,10 the result showing that paraesthesia of inferior alveolar nerve was most frequently found in age group of 26-35 was in line with few previous studies which revealed that table 1. incidence of paraesthesia and non-paraesthesia in study samples associated with sex and age variable paresthesia (%) non-paresthesia (%) sex male 9 (60.00) 21 (32.30) female 6 (40.00) 44 (67.69) age group 12-16 1 (06.67) 0 (00.00) 17-25 5 (33.33) 33 (50.76) 26-35 6 (40.00) 22 (33.85) 36-45 2 (13.33) 8 (12.30) 46-55 1 (06.67) 2 (03.07) note: the study samples were 80 post odontectomy cases showing close relationship, radiographically, between lower third molar roots and mandibular canals. table 2. distribution of incidence of paraesthesia and nonparaesthesia associated with relation of lower third molar root to mandibular canal according to rood and shehab’s category relation in rood and shehab’s category paresthesia (%) no paresthesia (%) total (%) a 2 (13.33) 8 (12.30) 10 (12.50) b 2 (13.33) 4 (6.15) 6 (7.50) c 0 (0.00) 3 (4.61) 3 (3.75) d 0 (0,00) 2 (3.07) 2 (2.50) e 0 (0.00) 3 (4.61) 3 (3.75) f 0 (0.00) 2 (3.07) 2 (2.50) g 1 (6.67) 5 (7.69) 6 (7.50) others 10 (66.67) 38 (58.46) 48 (60.00) total 15 (100.00) 65 (100.00) 80 (100.00) table 3. contigency table of rood and shehab’s relation and incidence of paraeshesia relation paraesthesia (+) paraesthesia (-) total rood & shehab (+) 5 (33.3) 27 (41.5) 32 rood & shehab (-) 10 (66.7) 38 (58.5) 48 total 15 65 80 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p59-62 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p59-62 62 kamadjaja, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 59–62 age was one of the most consistent factors in determining the degree of difficulty of odontectomy associated with the increase in bone density with age. moreover, increase in age was also related with completion of root formation which may significantly increase the rate of complication related to nerve injury especially in patients above 25 years of age.6 this was supported by result of other study which showed that age group of 26-30 had the highest risk of inferior alveolar nerve injury.9 it was suggested that fully developed root tended to have close contact with nerve in mandibular canal.12 it was noted from the result above that the highest rate of appearance of rood and shehab’s radiographic features, in this study, was relation ‘a’ followed by relation ‘b’. the result was, surprisingly, consistent with rood & shehab’s study whose result showed that darkening of the root (relation a) and defection of root (relation b) had the highest rate of appearance.7 the highest incidence of inferior alveolar nerve paraesthesia, in this study, which was darkening of the root (a) and defection of root (b) groups was also somewhat consistent with the result of rood & shehab’s study which showed interruption of white line, darkening of the root and deflected root were the three highest incidence of lower lip impairment.7 few other studies revealed that darkening of the root, either isolated or in conjunction with other highrisk signs, seemed to be significant in predecting inferior alveolar nerve exposure.13,14 statistic analysis result showed no significant correlation between rood and shehab’s radiographic findings and incidence of inferior alveolar nerve paraesthesia was not consistent with few other studies suggesting that five radiographic features identified by rood and shehab were significantly related to inferior alveolar nerve injury except for narrowing of the canal and dark and bifid apex.7,13 this findings may be explained as follows. first, there were only relatively few inclusive samples, i.e. cases showing radiographic features of rood and shehab category, and the low incidence of nerve injury (1.5%) in this study, in contrast to incidence rate of 0.4-8.4% in one study by blondeu and daniel.15 second, there most likely existed other radiographic features than those of rood and shehab category which could be the significant signs of close relationship between mandibular canal and third molar roots and, therefore, could be used as predictors of inferior alveolar nerve injury. nevertheless, based on results of the study it was logical to assume that darkening of the root and defection of the root were the two significant radiographic features of rood and shehab that could be used as predictors of inferior alveolar nerve injury. further studies are required to confirm this phenomenon. the conclusion of this study was that there was no correlation between rood and shehab’s radiographic relation of lower third molar root to mandibular canal and the incidence of inferior alveolar nerve injury. references 1. sarikov r, juodzbalys g. inferior alveolar nerve injury after mandibular third molar extraction: a literature review. j oral maxillofac res 2014; 5(4): e1. 2. martini f, timmons mj, tallitsch rb. human anatomy. upper saddle river, n.j.: prentice hall; 2003. p. 195-7. 3. tay ab, go ws. effect of exposed inferior alveolar neurovascular bundle during surgical removal of impacted lower third molars. j oral maxillofac surg 2004; 62(5): 592-600. 4. dimitroulis g. handbook of third molar surgery. bristol, england: wright; 2001. p. 137-41. 5. pa l ma cc, ga rcia m b, l a r ra zaba l mc, pena r ro cha dm. radiographic signs associated with inferior alveolar nerve damage following lower third molar extraction. med oral patol oral cir bucal 2010; 15(6). e886-90. 6. pogrel ma. what are the risks of operative intervention?. j oral maxillofac surg 2012; 70(9 suppl 1): s33-6. 7. rood jp, shehab b. the radiographic prediction of the inferior alveolar nerve during third molar surgery. br j oral and maxillofac surgery 1990; 28(1): 20-5. 8. miloro, ghali m, larsen ge, waite p. peterson’s principles of oral and maxillofacial surgery. london: bc decker; 2004. p. 159-60. 9. jerjes w, upile t, shah p, nhembe f, gudka d, kafas p, mccarthy e, abbas s, patel s, hamdoon z, abiola j, vourvachis m, kalkani m, al-khawalde m, leeson r, banu b, rob j, el-maaytah m, hopper c. risk factors associated with injury to the inferior alveolar and lingual nerves following third molar surgery-revisited. oral surg oral med oral pathol oral radiol endod 2010; 109(3) :335-45. 10. gulicher d, gerlach kl. sensory impairment of the lingual and inferior alveolar nerves following removal of impacted mandibular third molars. int j oral maxillofac surg 2001; 30(4): 306-12. 11. valmaseda-castellon e, berini-aytes l, gay-escoda c. inferior alveolar nerve damage after lower third molar surgical extraction: a prospective study of 1117 surgical extractions. oral surg oral med oral pathol oral radiol endod 2001; 92(4): 377-83. 12. eyrich g, seifert b, matthews f, matthiessen u, heusser ck, kruse al, obwegeser ja, lübbers ht. 3-dimensional imaging for lower third molars: is there an implication for surgical removal?. j oral maxillofac surg 2011; 69(7): 1867-72. 13. szalma j, lempel e, jeges s, olasz l. darkening of third molar roots: panoramic radiographic associations with inferior alveolar nerve exposure. j oral maxfac surg 2011; 69(6): 1544-49. 14. szalma j, vajta l, lempel e, jeges s, olasz l. darkening of third molar roots on panoramic radiographs: is it really predominantly thinning of the lingual cortex?. int j oral maxfac surg 2013; 42(4): 483-88. 15. blondeau f, daniel ng. extraction of impacted mandibular third molars: postoperative complications and their risk factors. j can dent assoc 2007; 73(4): 325-325e. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p59-62 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p59-62 vol 49 no 1 jan-mrt 2016.indd 1717 the thickness of odontoblast-like cell layer after induced by propolis extract and calcium hydroxide irfan dwiandhono,1 ruslan effendy,2 and sri kunarti2 1faculty of medicine, universitas jenderal soedirman, purwokerto indonesia 2department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: propolis is a substance made from resin collected by bees (apis mellifera) from variety of plants, mixed with its saliva and various enzymes to build a nest. propolis has potential antimicrobial and antiinflammatory agents with some advantages over calcium hydroxide (ca(oh)2). ca(oh)2 has been considered as the “gold standard” of direct pulp-capping materials, but there are still some weakness of its application. first, it can induce pulp inflammation which last up to 3 months. second, the tissue response to ca(oh)2 is not always predictable. third, the tunnel defect can probably formed in dentinal bridge with possible bacterial invasion in that gap. purpose: this study was aimed to determine and compare the thickness of odontoblast-like cells layer after induced by propolis extract and ca(oh)2 in rat’s pulp tissue. method: class 1 preparation was done in maxillary first molar tooth of wistar mice until the pulp opened. the ca(oh)2 and propolis extract was applied to induce the formation of odontoblast-like cells, the cavity was filled with rmgic. the teeth were extracted (after 14 and 28 days of induction). the samples were then processed for histological evaluation. result: there were significant differences between the thickness of odontoblast-like cells after induced by propolis extract and ca(oh)2. conclusion: the propolis extract as the direct pulp capping agent produces thicker odontoblast-like cell layer compared to ca(oh)2. keywords: odontoblast-like cells; propolis; calcium hydroxide correspondence: irfan dwiandhono, faculty of medicine, universitas jenderal soedirman. jl. dr. gumbreg no. 1 purwokerto 53123, jawa tengah, indonesia. e-mail: irfandrg@gmail.com telp. +62 281 643744. research report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 17–21 introduction the aim of direct pulp capping treatment is to maintain the pulp’s vitality, by protecting the pulp from bacterial penetration and inducing dentinal bridge formation. the success of the pulp capping procedure greatly depends upon the capacity of the capping material to perfectly seal the tubuli, non irritative, protect the pulp from the mechanical, chemical, and bacterial irritation, induce fibroblast and odontoblast cell to form reparative dentin and dentinal bridge.1 calcium hydroxide [ca(oh)2] is the most accepted and commonly used pulp capping material. it stimulates the pulp tissue on the formation of dentinal bridge and inhibits bacterial growth. nevertheless, application of calcium hydroxide in direct pulp capping has some drawbacks. the high ph (12.5) of ca(oh)2 suspensions causes liquefaction necrosis at the surface of the pulp tissue with the formation of a necrotic layer at the material-pulp interface. the toxicity of ca(oh)2 reduced in deeper pulp layer, causing coagulation necrosis to the average degree of irritation. in this zone of coagulation necrosis, the differentiaton of odontoblast-like cells and the formation of dentinal bridge occured.2 several studies related to pulp capping materials had been conducted to determine the most appropriate pulp capping material with good biocompatibility. the success dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p17-21 18 dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 17–21 rate of calcium hydroxide as pulp capping material were 80,1% after a year, 68,0% after 5 years, and 58,7% after 9 years.3 the drawbacks of ca(oh)2 as pulp capping material are: (1) it can induce pulp inflammation which last up to 3 months, (2) unpredictable tissue response, (3) the reparative dentin formed below ca(oh)2 is irregular, the and the tunnel defect can probably formed. the defect is formed because of the tissue inclusion on the dentinal bridge. this resulted in increasing dentinal bridge’s permeability for possible bacterial invasion.4 propolis is a substance made from resin collected by bees (apis mellifera) from variety of plants, mixed with its saliva and various enzymes to be a whole different kind of resin, and used by bees to build a nest.5 the color of this whole new resin is brown to dark brown. it is sticky at room temperature, but hard and fragile at low temperature. propolis has broad-spectrum biological and pharmacological activity, anti-bacterial, anti-virus, anti-oxidant, and anti-inflammation material. propolis consists of 55% resin, 30% bee’s wax and aromatic oils, 5% bee pollen, and 10% other substances (amino acid, minerals, ethanol/alcohol, vitamin a, b complex, e, and bioflavonoid).6 many studies related to propolis extract as direct pulp capping material has been conducted, but the exact mechanism of action remains unclear. propolis extract with flavonoid was proven to reduce inflammation and to form dentinal-bridge in 4th weeks after pulp capping treatment on sprague-dawley rats.7 direct pulp capping treatment with propolis and mineral trioxide aggregate (mta) in human was succeed to form dentinal bridge on the 15th day, whereas in the group treated with ca(oh)2 the dentinal bridge was not found. 8 in other study, mta and novel endodontic cement (nec) in dog’s teeth after 8 weeks was giving better result in pulp capping and dentinal bridge formation compared to calcium hydroxide.9 the aim of this study was to analyze histological process of dentinal bridge formation by measuring the thickness of the odontoblast like cells on the pulp of wistar rats’ teeth after pulp capping treatment using propolis and ca(oh)2. materials and methods this research was an experimental laboratory study, with 42 male wistar rats as samples (healthy, weighing 200-250 gr, 8-16 month-old, with fully erupted molars). the adaptation time given was 2 weeks. the samples were divided into 6 groups as follow: group i (control group i/the pulp was perforated, filled with gic, observed on the14th day), group ii (control group ii/ the pulp was perforated, filled with gic, observed on the 28th day), group iii (the pulp was perforated, applied with propolis extract, filled with gic, observed on the 14th day), group iv (the pulp was perforated, applied with propolis extract, filled with gic, observed on the 28th day), group v (the pulp was perforated, applied with calcium hydroxide, filled with gic, observed on the 14th day), and group vi (the pulp was perforated, applied with calcium hydroxide, filled with gic, observed on the 28th day). the pulp capping material amount was 0,5 mg for each application. the euthanasia process was performed, followed by decapitation and maxilla separation. rat’s molars were sectioned parallel to the axis using microtome to 7μm. the samples were then stained with hematoxylin and eosin. assessment of the normality of the data was determined with kolmogorrov smirnov test. assessment of the homogenity of the data was determined with levene test. the statistically significant differences were determined with independent t-test. result the histological examination to see the thickness of odontoblast-like cells was done at 200x magnification. the thickness of odontoblast-like cells was defined as the thickness of the new cells found below the perforation area. the result of the thickness of the dentin can be seen in figure 1. a b d c e f figure 1. the thickness of the odontoblast-like cells in rats’ teeth (at 200x magnification). the red marked area shows the thickness of the odontoblast-like cells. a. control group on the 14th day, b. control group in 28th day, c. ca(oh)2 group on the 14 th day, d. ca(oh)2 group on the 28th day, e. propolis extract group on the 14th day, f. propolis extract group on the 28th day. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p17-21 1919dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 17–21 in the thickness of odontoblast-like cells between 14th day and 28th day of positive control group, ca(oh)2 group, and propolis extract group. kruskall-wallis revealed that there were significant differences between all groups. independent t-test revealed that there that there were significant differences in the thickness of odontoblast-like cells between 14th day and 28th day of positive control group, ca(oh)2 group, and propolis extract group. discussion the basic principle of conservation treatment is to maintain the condition and function of the dentin-pulp complex, especially in the case when the pulp is perforated. like the other connective tissues, pulp tissue has the capacity to repair themself. the characteristic of healing process of the perforated pulp tissue is the reorganization of the damaged soft tissue, fibroblast cell differentiation into odontoblast-like cells, and the reparative dentin formation on the perforated soft tissue.10 the damage in the pulp tissue will resulted in inflammation reaction to eliminate the substance that endanger the tissue or to avoid further destruction of the pulp tissue. the inflammation reaction is the early step of the healing process. at the time when tissue is injured, the fibroblasts would soon migrate toward the wound, proliferate and produce large amounts of collagen matrix that will help to isolate and repair the damaged tissue.11 growth factor plays an important role in responding to the injury and tissue repair.12 caries is estimated to trigger the odontoblast activity to increase the expression of tgf-β1. the tgf-β1 that was dissolved in matrix is released.13 tgf-β1 is an important regulator in proliferation and differentiation of the human’s pulp cells during the formation and dentin repair process.14 tgf-β1 binds to tgf-βr, the tgf-β1 receptor, which then activates the cell cycle to proliferate. tgf-β1 will regulate the proliferation process, the cell cycle running normally, and ready for the next step. the cell undergo the mitosis process to differentiate into odontoblast-like cells, characterized by table 1. mean and standard deviation of odontoblast-like cells thickness in 14th and 28th day groups mean (mm) std. deviation 14th day positive control 0,022 0,005 ca(oh)2 0,035 0,013 propolis 0,084 0,012 28th day positive control 0,032 0,005 ca(oh)2 0,056 0,015 propolis 0,104 0,012 table 2. independent t-test result of odontoblast-like cells thickness on the 14th and 28th day groups ca(oh)2 propolis 14th day 28th day 14th day 28th day positive control 14th day 0,037* 0,000* 28th day 0,004* 0,000* ca(oh)2 14 th day 0,000* 28th day 0,000* * p< 0,05 = there was signficant difference table 3. independent t-test result of odontoblast-like cells thickness on the 14th and 28th day groups positive control 28th day ca(oh)2 28th day propolis 28th day positive control 14th day 0,010* ca(oh)2 14th day 0,017* propolis 14th day 0,030* * p< 0,05 = there was signficant difference kruskall-wallis revealed that there was significant difference in the thickness of odontoblast-like cells after 14 days and 28 days of treatment (p<0,05) on table 1. independent t-test revealed that there was significant difference (p<0,05) between ca(oh)2 groups and propolis extract groups. table 2 shows that on the 14th day, there was significant difference (p<0,05) between the three groups (positive control group, ca(oh)2 group, and propolis extract group). the thickness of odontoblast-like cells in positive control group was thinner than the propolis extract group. the significant difference was also found between ca(oh)2 group and propolis extract group, it shows that the thickness of odontoblast-like cells in ca(oh)2 group was thinner than the propolis extract group independent t-test revealed that on the 28th day, there was significant difference (p<0,05) between the three groups (positive control group, ca(oh)2 group, and propolis extract group). the thickness of odontoblast-like cells in positive control group was thinner than the ca(oh)2 group and propolis extract group. the significant difference was also found between ca(oh)2 group and propolis extract group, it shows that the thickness of odontoblast-like cells in ca(oh)2 group was thinner than the propolis extract group. table 3 shows that there were significant differences dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p17-21 20 dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 17–21 the type i collagen and dentin matrix protein (dmp1), as well as an increase in activity of alkaline phospatase (alp). increased alp activity stimulates the release of minerals from the cell to start mineralization, then the process of mineral formation occurs. the hydroxy-apatite deposite and merge into collagen matrix in mineralization process. the sythesis of dmp1 increases, interact with type 1 collagen to form a sequence and undergo mineralization, and the reparative dentin formation occurs.15 the results of this study showed that the dead odontobloast was only found below the perforated area. in the intact dentin at the edge of the perforated area the damaged odontoblast was found. the growth area of the odontoblast-like cells was presumably derived from the differentiation process of fibroblasts in the pulp tissue. there are three possible sources of forming odontoblastlike cells. first, odontoblast-like cells formed by progenitor cells of odontoblast, known as undifferentiated mesenchym. these cells are located in the cell-rich zone of hohl. second, odontoblast-like cells derived from pulp fibroblasts, as fibroblasts belongs to stable cell that can differentiate when there is injury or stimuli. the third is the de-differentiation of odontoblasts. although odontoblasts belongs to postmitosis, but the possibility of differentiation is still open even if it’s small.16 all primary odontoblasts is an irreversible injury in the area of perforation. post-mitotic, a terminal cell differentiation can not proliferate to replace dead or damaged odontoblasts. as the result, this primary odontoblasts should be replaced by a new generation of cell odontoblast-like cells. new odontoblast-like cells proliferate from other pulp cells and migrate to perforated area, the place where the reparative dentin secreted. progenitor cells of new odontoblast-like cells assumed as cells from the subodontoblast layer or pulp fibroblasts.17 in the de-differentiation process, the odontoblast cell is not turned young and pluripotent, but still unipotent and just divide from the cell with the same structure and function with the related cell. there are two mechanisms of reparative dentinogenesis. first, the direct differentiation induced by dentin matrix, and second, the odontoblast-like cells differentiation through fibrodentin matrix that acts as the basal lamina for the differentiation of odontoblast-like cells for tooth repair.18,19 the thickness of the odontoblast-like cells layer in ca(oh)2 group in 14th and 28th day is thinner than the propolis extract’s. the high ph (12.5) of calcium hydroxide suspensions causes liquefaction necrosis at the surface of the pulp tissue, causing coagulation necrosis in the area between necrotic pulp and vital pulp. when the pure form of ca(oh)2 is applied on the pulp, it actually will destroy some of the pulp’s tissue, causing a persistent inflammation. moleculary, the calcium ions released by ca(oh)2 stimulates the synthesis of fibronectin and glycoproteins tenascin in dental pulp, triggering differentiation of dental pulp cells into cells forming minerals that are the main cells to form dentin bridge. ca(oh)2 also stimulates the release of adrenomedullin and tgf-β1 of human dentin matrix, both are pluripotent growth factors.4,16 propolis extract has known to have antibacterial activity, anti-inflammatory, antioxidant and immunomodulator. the prevention of infection and the promotion of cell regeneration ease the the healing process in the dental pulp that begins with the collagen fibers formation.7 flavonoids and caffeic acid in propolis extract can reduce the inflammatory response by inhibiting arachidonic acid lipoxygenase pathway. as antibacterial agent, propolis destroys the bacteria’s cell wall and prevents bacterial cell division. propolis extract was more effective to form dentin compared to calcium hydroxide or zinc oxide. 7,20 propolis extract inhibits the translation of nfkβ into the nucleus to prevent the apoptosis. the inhibition of apoptosis result in prevention of drastic decreasing amount of fibroblast in the pulp and activation of nfkβ. the activation of nfkβ inhibits the transcription of genes that secrete tnf-α. the increase of tgf-β1 expression is a host defense mechanism. dentin formation after pulp capping involves differentiation of odontoblast-like cells that form reparative dentin and the biosynthetic activity of the components around the primary odontoblast. it requires an interaction between extra cellular matrix molecules and tgf-β1, which is a growth factor that is known to play an important role in the differentiation of odontoblast-like cells. propolis extract enhances the formation of odontoblastlike cells and stimulates the production of tgf-β1.7 tgfβ1 stimulatesthe proliferation of fibroblasts, a fibrogenic substance that stimulates the fibroblasts chemotaxis to increase the formation of collagen, fibronectin, and proteoglycans.21 this ability of propolis extract to inhibit inflammation, suppress apoptosis, stimulates the production of tgf-β1 and differentiation of fibroblasts explains the result of this study, where the thickness of odontoblast-like cells layer in perforated rat’s pulp after the application of propolis extract is significantly thicker than the calcium hydroxide group. the conclusion, the propolis extract as the direct pulp capping agent produces thicker odontoblast-like cell layer compared to ca(oh)2. references 1. torabinejad m, walton re. endodontics: principles and practice. 4th ed. missouri: saunders; 2009. p. 9-14. 2. hargreaves km, berman lh, cohen s. cohen’s pathways of the pulp expert consult. 10th ed. st. louis: mosby; 2011. p. 490, 518, 626, 812. 3. willershausen b, willershausen i, ross a, velikonja s, kasaj a, blettner m. retrospective study on direct pulp capping with calcium hydroxide. quintessence int 2011; 42(2): 165-71. 4. janebodin k, horst ov, osathanon t. dental pulp response to pulp capping materials and bioactive molecules. cu dent j 2010; 33: 229-48. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p17-21 2121dwiandhono, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 17–21 5. bankova v, castro sd, marcucci m. propolis: recent advances in chemistry and plant origin. apidologie, springer verlag 2000; 31(1): 3–15. 6. almas k, dahlan a, mahmoud a. propolis as a natural remedy: an update. saudi dent j 2001; 13(1): 45-9. 7. sabir a, tabbu cr, agustiono p, sosroseno w. histological analysis of rat dental pulp tissue capped with propolis. j oral sci 2005; 47(3): 135-8. 8. parolia a, thomas ms, kundabala m, mohan m. propolis and its potential uses in oral health. int j med med sci 2010; 2(7): 210-5. 9. asgary s, eghbal mj, parirokh m, ghanavati f, rahimi h. a comparative study of histologic response to different pulp capping materials and a novel endodontic cement. oral surg oral med oral pathol oral radiol endod 2008; 106(4): 609-14. 10. tziafas d, smith aj, lesot h. designing new treatment strategies in vital pulp therapy. j dent 2000; 28(2): 77-92. 11. hargreaves km, goodis he. seltzer and bender’s dental pulp. hanover park: quintessence publishing co, inc; 2002. p. 247-79. 12. about i, bottero mj, de denato p, camps j, franquin jc, mitsiadis ta. human dentin production in vitro. exp cell res 2000; 258(1): 33-41. 13. sloan aj, perry h, matthews jb, smith aj. transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth. histochem j 2000; 32(4): 247-52. 14. shirakawa m, shiba h, nakanishi k, ogawa t, okamoto h, noshiro m, kato y. transforming growth factor-beta-1 reduces alkaline phosphatase mrna and activity and stimulates cell proliferation in cultures of human pulp cells. j dent res 1994; 73(9): 1509-14. 15. widjiastuti i, yuanita t, hutagalung j, rukmo m, safitri i. 2014. east java propolis inhibits cytokine pro-inflammatory in odontoblast-like cells human pulp. journal of agricultural science and technology a 2014; 4(1): 27-32. 16. kunarti s. pulp tissue inflammation and angiogenesis after pulp capping with transforming growth factor β1. dent j (majalah kedokteran gigi) 2008; 41(2): 88-90. 17. murray pe, windsor lj, smyth tw, hafez aa, cox cf. analysis of pulpal reactions to restorative procedures, material, pulp capping, and future therapies. crit rev oral biol med 2002; 13(6): 509-20. 18. yatim w. biologi sel lanjut. bandung: tarsito; 2003. p. 1-84. 19. goldberg m, smith aj. cell and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. crit rev oral biol med 2004; 15(1): 13-27. 20. ahangari z, naseri m, jalili m, mansouri y, mashhadiabbas f, torkaman a. effect of propolis on dentin regeneration and the potential role of dental pulp stem cell in guinea pigs. cell j 2012; 13(4): 223-8. 21. kumar, abbas, fausto. robbins & cotran dasar patologis penyakit. 7th ed. jakarta: egc; 2010. p. 101. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p17-21 � dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg � effect of casein phosphopeptide-amorphous calcium phosphate on the flexural strength of enamel-dentin complex following extracoronal bleaching diatri nari ratih and hendargo agung pribadi department of conservative dentistry faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: bleaching can affect the mechanical properties of enamel-dentin complex, such as flexural strength. casein phosphopeptide-amorphus calcium phosphate (cpp-acp) is often used following bleaching treatment to reduce hypersensitivity and to increase demineralization of tooth. purpose: the purpose of the study was to investigate the effect of cpp-acp on the flexural strength of enamel-dentin complex following extracoronal bleaching. methods: forty-eight enamel-dentin plates (size 8 x 2 x 3 mm) were randomly assigned into 6 groups, each consisted of 8 samples. group 1, no bleaching and immersed in artificial saliva. group 2, no bleaching, cpp-acp application only. group 3, bleaching using 15% carbamide peroxide. group 4, similar to group 3, except application of cpp-acp for the times between bleaching. group 5, bleaching with 40% hydrogen peroxide. group 6, similar to group 5, except application of cpp-acp for the times between bleaching. flexural strength of each enamel-dentin plate was tested by threepoint bending test using universal testing machine. results: the results showed that 15% carbamide peroxide and 40% hydrogen peroxide significantly reduced flexural strength of enamel-dentin (216.25±26.44 mpa and 206.67±32.07 mpa respectively). conversely, application of cpp-acp following both bleachings increased flexural strength (266.75± 28.27mpa and 254.58±36.59 mpa respectively). a two-way anova revealed that extracoronal bleaching agents significantly reduced flexural strength (p<0.05), while application of cpp-acp significantly increased flexural strength of bleached enamel-dentin complex (p<0.05). conclusion: extracoronal bleaching agents reduce flexural strength, whereas application of cpp-acp following bleaching either with 15% carbamide peroxide or 40% hydrogen peroxide can increase the flexural strength of enamel-dentin complex. keywords: extracoronal bleaching; hydrogen peroxide; carbamide peroxide; cpp-acp; flexural strength correspondence: diatri nari ratih, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta 1, sekip utara yogyakarta 55281, indonesia. e-mail: trinaugm@yahoo.com research report introduction tooth discoloration, especially in anterior teeth, often cause aesthetic problems. this color change can affect a patient’s self confidence; therefore patient seeks treatment to improve the color of teeth. with careful diagnosis, appropriate treatment planning and attention to technique, bleaching can be considered a conservative and safe procedure to lighten discolored teeth.1 bleaching agents, including carbamide and hydrogen peroxide, act as a poweful oxidizing agents and can give rise to agents known to be effective bleaching agents (i.e., its corresponding mono-anion (h02 -) and hydroxyl radical (oh)). moreover, carbamide peroxide also releases urea, which is rapidly decomposed into carbon dioxide and ammonia. chemical reaction of the two reagents with the organic extracellular matrix components, including pigments or chromophores, constitutes the chemical basis of tooth bleaching.2,3 although bleaching procedures are intended to be applied topically to the enamel surface, the effects of such procedures are not necessarily restricted to enamel only, dental journal (majalah kedokteran gigi) 20�5 march; 48(�): �–�� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 8 ratih and pribadi/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 7–11 but also to dentin and pulp.3 bleaching methods generally include the application of 10% to 15% carbamide peroxide or 30% to 38% hydrogen peroxide.2,4 recently, 40% hydrogen peroxide has been introduced to the market 40 a bleaching agent and become more populer than previous lower consentration of bleaching agents.4 a general concern in bleaching procedures relates to the possible weakening of tooth structure subsequent to bleaching procedures. it has been reported that changes in enamel surfaces charactheristic such as increased porosity,5 erosion,6 increased roughness,7 decreased hardness,8 demineralization of tooth structures,9 depression and superficial irregularities,10 after bleaching procedures. furthermore, alterations in the mechanical properties, including tensile strength, flexural strength, fracture toughness of dental tissues particularly in enamel and dentin following to bleaching have been reported in several studies.11-13 extracoronal bleaching can be associated with several complications, including tooth sensitivities, which has been reported in 15% to 78% of patients undergoing such procedures.10,11 various treatments have been undertaken to manage tooth sensitivity, including the use of oxalates and nitrates of potassium or a combination of potassium nitrate and fluoride, as well as the use of amorphous calcium phosphates.11 casein phosphopeptide-amorphous calcium phosphate (cpp-acp) is acp complexed with cpp, which is from the major protein of milk. cpp-acp and localizes acp in dental plaque, thereby concentrating calcium and phosphate on the tooth surface.14-16 by maintaining a high-concentration gradient of calcium and phosphate ions, cpp-acp assists to suppress demineralization and promotes remineralization of enamel by the deposition of apatite.17-19 previous studies demonstrated that cpp-acp has been able to remineralize human enamel subsurface lesions. the remineralized enamel has also been shown to be more resistant to subsequent acid attack.16,17,20,21 recently, cpp-acp has been used as a paste, in conjuction with or following treatments such as bleaching in order to decrease tooth sensitivity. the effect of applying cpp-acp to enamel surfaces on bond strength subsequent to bleaching procedures has been investigated in numerous studies.22-24 a previous study also showed a significant reduction in bovine dentin flexural strength and modulus of elasticity after a 2-week direct application of 10% and 15% carbamide peroxide.9 application of cpp-acp following bleaching treatment (in the absence of saliva) could compesate for decreased flexural strength of the bovine enamel-dentin complex.12 although several studies have evaluated the effects of cpp-acp, no study has been undertaken to evaluate the flexural strength following application of extracoronal bleaching agents on human enamel-dentin complex. therefore, the purpose of this study was to investigate the effect of cpp-acp on the flexural strength of enameldentin complex following extracoronal bleaching. materials and methods forty-eight human premolars, extracted within the previous 3 months, immediately stored in distilled water at 40 c until the study was conducted. rectangular blocks measuring 8 mm in length, 2 mm in width, and 3 mm in height were prepared from the middle part of facial surfaces of the extracted premolars using a cutting machine (microtome, leica, wetzlar, germany). the specimens were randomly assigned into 6 groups, each consisted of 8 samples. group 1, comprised the control group, which received no bleaching treatment and immersed in artificial saliva. group 2, specimens were not received bleaching treatment, but were applied with cpp-acp (tooth mousse, gc inc, tokyo, japan) for 30 minutes, twice daily for 14 days. group 3, specimens were bleached using 15% carbamide peroxide (15% opalescence pf, ultradent, south jourdan, ut., usa) for 30 minutes, twice daily for 2 consecutive weeks. group 4, similar to group 3, except specimens were applied cpp-acp for the times between bleaching (30 minutes twice daily for 2 consecutive weeks). group 5, specimens were bleached using 40% hydrogen peroxide (40% opalescence boost, ultradent, figure 1. three-point bending test using universal testing machine. 1 figure 2. the mean and standard deviation of enamel-dentin flexural strength after applicationof bleaching agents and cpp-acp (in mpa). figure 2. the mean and standard deviation of enamel-dentin flexural strength after applicationof bleaching agents and cpp-acp (in mpa). � dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �ratih and pribadi/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 7–11 south jourdan, ut, usa) for 1hour/day, twice weekly for 2 consecutive weeks. after each bleaching session, the specimens were thoroughly rinsed with distilled water for 30 seconds and stored in artificial saliva. group 6, similar to group 5, except specimens were applied cpp-acp for the times between bleaching (30 minutes, twice daily for 2 consecutive weeks). all specimens were then rinsed using distilled water and incubated in artificial saliva for 24 hours at 370 c before being tested. the specimens were subjected to a three-point bending test on a universal testing machine (type amu-5de, tokyo testing machine, mfg, co., lt, tokyo, japan) that applied force to the center of the enamel side of the specimens at a crosshead speed of 1 mm/min (figure 1). the mounting apparatus consisting of two rods, mounted parallel, with 6 mm between the centers. the maximum load supported by the specimen prior to failure was used to calculate the flexural strength value. flexural strength was determined using the following formula:10 flexural strength (in mpa)= 3pfl/2wh 2 , where pf is the measured maximum load at the time of specimen fracture, l is the distance between supports on the tension surface (6 mm), w is the mean specimen width, h is the mean height of the specimen between the tension and compression surfaces. results were subjected to statistical analysis using a two way anova and a post-hoc tukey test with 95% level of significance. results the mean and standard deviation of flexural strength values (in mpa) are depicted in figure 2. the results showed that flexural strength decreased in enamel-dentin complex, which was bleached either using 15% carbamide peroxide or 40% hydrogen peroxide (group 3 and group 5) as compared to control group, which was soaked in artificial saliva only. on the other hand, application of cpp-acp on to bleached specimens resulted in the increase of flexural strength (group 4 and group 6). specimens that were applied cpp-acp without bleaching treatment revealed the highest flexural strength (297.17±28.29 mpa) compared to the other groups. conversely, specimens which were bleached with 40% hydrogen peroxide and without application of cpp-acp showed the lowest flexural strength (206.67 ±32.07 mpa). a two way anova demonstrated that bleaching using either 15% carbamide peroxide or 40% hydrogen peroxide, as well as cpp-acp application influenced on the flexural strength of enamel-dentin complex (p<0.05). on the other hand, no interaction occured between bleaching and application of cpp-acp (p>0.05). multiple comparisons by a post-hoctukey test revealed that all groups showed significant differences (p<0.05), except between group 1and group 4, group 2 and group 4, group 4 and group 6 (p>0.05). discussion the results of this study indicated that reduction in the flexural strength values of the enamel-dentine complex occured after the extracoronal bleaching with either 15% carbamide peroxide or 40% hydrogen peroxide. the reduction in flexural strength was probable attributed to changes caused by bleaching agents in the inorganic or organic component of dentin. enamel contains 98% inorganic and 2% organic materials, while dentin consists of 70% inorganic, 20% organic materials and 10% water,24 therefore application of bleaching agents on to enameldentin complex may cause demineralization of inorganic component of enamel-dentin complex and resulting in decreasing flexural strength. in addition, the redox reaction of bleaching agents can lead to dissolution of organic and inorganic materials teeth structure and the remaining is only carbon dioxide and water.22 in other words, bleaching agent is capable to alter the ratio of ca/p of tooth structure,9 which in turns, reducing flexural strength. additionally, urea and peroxide have ben associated with proteolytic reaction, which is due to induce denaturating dentin collagen and oxidizing dentin protein, resulting in degradation of proteins.2 urea can penetrate not only the enamel surface but also interprismatic of enamel. the penetration increases the permeability of the enamel and subsequently alters the microstructural of enamel.13 bleaching agents improved access to intratubular minerals by dispersing collagen fibrils. loss of minerals leads to loss of the binding matrix. therefore, the destruction of organic and mineral parts of dentin occurs continuously, resulting in decreasing flexural strength.12 moreover, some of the structural alterations in bleached dentin have been attributed to alterations in the water, mineral, and collagen content of dentin and non-collagenous protein.3 in clinical situations, bleaching agents are usually applied to enamel rather than directly applied to dentin.21,22 however, peroxide ions are able to penetrate through enamel and dentin through a capillary rise in enamel interprismatic spaces, convective mass transfer, or classic molecular diffusion based on a molecular path, and form measurable amounts of bleach within the tooth pulp.14 thus, enamel-dentin plates were used in this study as specimens to mimic clinical condition. in this study, the application time of 15% carbamide peroxide and 40% hydrogen peroxide were 30 minutes and 1 hour respectively, since dentin in that period may be affected by bleaching agent. this result is in accordance with tam et al.3 who reported that bleaching agent diffused through enamel within 15 to 25 minutes. bleaching agents influence the enamel by affecting enamel organic matrix with the action of free radicals.14 the porosities created by the bleaching agent along the exposed area of enamel may act as stress raisers during a three-point bending tests, resulting in an early fracture during fracture resistance investigations.3 enamel could reduce the effects of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �0 ratih and pribadi/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 7–11 hydrogen peroxide on dentin.25 several factors such as the enamel thickness, the existance of enamel fractures and cracks, dentin permeability and the direction of dentinal tubules affected the diffusion of bleaching agents through dentin.18 this present study showed a significant difference in flexural strength occured between 15% carbamide peroxide and 40% hydrogen peroxide (p<0.05). nevertheless the influence of 40% hydrogen peroxide bleaching was slightly greater than that of 15% carbamide peroxide on the flexural strength of enamel-dentin complex. this difference is likely due to ph of both bleaching agents. it has been reported that 40% hydrogen peroxide having a ph that is slightly lower than the 15% carbamide peroxide. acidic ph can increase the demineralization of inorganic materials in enamel-dentin complex,23 which in turns, reduce the flexural strength of enamel-dentin complex.13 the results of this study also revealed that application of cpp-acp increased flexural strength of enamel-dentin complex, which was bleached with 15% carbamide peroxide and 40% hydrogen peroxide, although it was less than normal dentin. this material acts as a source of calcium and phosphate for enamel since cpp-acp contains calcium 13 mg/g, phosphate 5.6 mg/g, with a ph of 7.8.16,17 cpp-acp could increase enamel mineralization and prevent demineralization by buffer mechanism.15 casein is a buffer for plaque acid, and it leached amino acids that can receive proton ions. the presence of the cpp-acp on the surface of the enamel-dentin complex is able to act as a barrier that prevent protons to diffuse.4,19 in addition, casein phosphopeptide is capable to stabilize the calciumphosphate on the tooth surface, thereby maintaining the calcium-phosphate concentration remains high on the tooth surface.15 in clinical situation, application of the cpp-acp on enamel surface can also lead this material to bind with biofilms, plaque, bacteria, hydroxyapatite and surrounding soft tissues, hence it might be always provide calcium and phosphate for teeth.18 the duration of cpp-acp application and the existence of several factors such as saliva may also affect the results of the present study.4 this study used artificial saliva to simulate the clinical condition. since cpp-acp can also stimulate the flow of saliva, it might improve the effectiveness of cpp-acp in clinical conditions. immersion in artificial saliva between the applications of bleaching agents can prevent demineralization of the enamel and dentin rather than only soaked in distilled water between the times the application of bleaching agents.10 it is because saliva has buffering properties and prevents demineralization, it is also rich in calcium and phosphate and acts as a source of remineralization after bleaching procedures.15 on the contrary, the distilled water does not contain calcium and phosphate as in artificial saliva. it seems that the effect of cpp-acp might be greater in clinical conditions than in vitro study.21 cpp-acp can also reduce the hypersensitivity following bleaching treatment.17 this phenomenon is due to increase mineral crystals deposition in the enamel, hence patients who treated bleaching did not experience of dentin hypersensitivity. additionally, the application of cpp-acp did not affect the effectiveness of bleaching procedures.19 based on the results of this study, it can be concluded that extracoronal bleaching agents reduce flexural strength, whereas cpp-acp following bleaching either with 15% carbamide peroxide or 40% hydrogen peroxide can increase the flexural strength of enamel-dentin complex. acknowledgment this study was funded by universitas gadjah mada faculty of dentistry research grant. references 1. polydorou o, hellwig e, auschill tm. the effect of different bleaching agents on the surface texture of restorative materials. j op dent 2006; 31(4): 473-80. 2. goldberg m, grootveld m, lynch e. undesireable and adverse effects of tooth-whitening products: a review. clin oral invest 2010; 14(1): 1-10. 3. tam le, kuo vy, noroozi a. effect of prolonged direct and indirect peroxide bleaching on fracture toughness of human dentin. j esthet restor dent 2007; 19(2): 100-10. 4. c u n ha ag g, va sconcelos a a m, borges bc d, vitor ia no jdo, alves c, machado ct, santos ajs. efficacy of in-office bleaching techniques combined with the application of a casein phosphopeptide-amorphous calcium phosphate paste at different moments and its influence on enamel surface properties. micros res tech 2012; 1(1): 1-7. 5. ferreira ss, araujo jl, morhy on, tapety cm, youssef mn, sobral ma. the effect of flouride therapies on the morphology of bleached human dental enamel. micros res tech 2011; 74(5): 512-6. 6. martin jm, almeida jb, rosa ear, soares p, torno v, rached rn, mazur rf. effect of fluoride therapies on the surface roughness of human enamel exposed to bleaching agents. quintessence int 2010; 41(1): 71-8. 7. borges ab, samezima ly, fonseca lp, yui kck, borges als, toreres crg. influence of potentially remineralizing agents on bleached enamel microhardness. oper dent 2009; 34(5): 593-7. 8. potocnik i, kosec l, gaspersic d. effect of 10% carbamide peroxide bleaching gel on enamel microhardness, microstructure, and mineral content. j endod 2000; 26(2): 203-6. 9. tam le, lim m, khanna s. effect of direct peroxide bleaching application to bovine dentin on flexural strength and modulus in vitro. j dent 2005; 33(4): 451-8. 10. tam le, abdool r, el-badrawy w. flexural strength and modulus properties of carbamide peroxide-treated bovine dentin. j esthet restor dent 2005; 17(3): 359-68. 11. tam le, noroozi a. effects of direct and indirect bleach on dentin fracture toughness. j dent res 2007; 86(10): 1193-7. 12. khoroushi m, mazaheri h, manoochehri ae. effect of cpp-acp application on flexural strength of bleached enamel and dentin complex. oper dent 2011; 36(4): 372-9. 13. tredwin cj, naik s, lewis nj, scully j. hydrogen peroxide toothwhitening (bleaching) products: review of adverse effects and safety issues. british dent j 2006; 200(4): 371-6. 14. kowalcsyk a, botulinski b, jaworska m, kierklo a, pawinska m, dabros e. evaluation of the product based on recaldent technology in the treatment of dentin hypersensitivity. adv med sci 2006; 51 (suppl): 40-2. �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��ratih and pribadi/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 7–11 15. ratjitkar s, rodrigueez jm, kaidonis ja, richards lc, townsend gc, bartlett dw. the effect of casein phosphopeptide-amorphous calcium phosphate on erosive enamel and dentine wear by toothbrush abrasion. j dent 2009; 37(2): 250-4. 16. reynolds ec, cai f, cohrane nj, shen f, walkel gd, morgan mv. fluoride and casein phosphopeptide-amorphous calcium phosphate. j dent res 2008; 87(3): 344-8. 17. reynolds ec. casein phosphopeptide-amorphous calcium phosphate: the scientific evidence. adv dent res 2009; 21(1): 25-9. 18. azarpazhooh a, limeback h. clinical efficacy of casein derivatives: a systemic review of the literature. jada 2012; 139(7): 815-924. 19. kuma r v l , it t haga r un a, k ing n m. t he effect of casein phosphopeptide-amorphous calcium phosphates on remineralization of artificial caries-like lesion: an in vitro study. aust dent j 2008; 53(1): 34-40. 20. moule ca, angelis f. resin bonding using an all-etch or self-etch adhesives to enamel after carbamide peroxide and/or cpp-acp treatment. aust dent j 2007; 52(2): 133-7. 21. adebayo oa, burrow mf, tyas mj. dentin bonding after cpp-acp paste treatment with and without conditioning. j dent 2008; 36(12): 1013-24. 22. soldani p, amaral cm, rodrigues ja. microhardness evaluation of in situ vital bleaching and thickening agents on human dental enamel. int j periodontics restorative dent 2010; 30(2): 203-11. 23. tang b, millar bj. effect of chewing gum on tooth sensitivity following whitening. british dent j 2010; 208(5): 571-7. 24. avery jk, chiego dj. essentials of oral histology and embryology. 3th ed. st. louis: mosby elsevier; 2006. p. 55-103. 25. date rf, yue j, barlow ap, bellamy pg, prendergast mj, gerlach rw. delivery, substantivity and clinical response of a direct application percarbonate tooth whitening film. am j dent 2006; 16(1): 3b-8b. 138 dental journal (majalah kedokteran gigi) 2019 september; 52(3): 138–141 research report effects of sidestream tobacco smoke on p53 expressions in rattus novergicus tongue epithelial mucosa dian angriany,1 diah savitri ernawati,1 adiastuti endah parmadiati,1 hening tuti hendarti1 and rosnah binti zain2 1department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya – indonesia 2department of oral pathology and oral medicine, faculty of dentistry, mahsa university, bandar saujana putra – malaysia abstract background: smoking, both active and passive, has been widely recognised as toxic to the human body, since it induces several forms of cancer, including that affecting the oral cavity. benzopyrene, the carcinogen contained in tobacco smoke, can even lead to carcinogenesis which potentially affects the regulation of cell apoptosis in both active and passive smokers. purpose: this study aims to investigate the carcinogenic effects of cigarette smoke on apoptosis of rat tongue mucosae through p53 expression. to determine the risk of malignant transformation through tumor suppressor genes in the apoptotic pathway. methods: rattus norvegicus subjects were divided into four groups, namely treatment group 1 exposed to sidestream cigarette smoke for four weeks (p1), treatment group 2 exposed to sidestream cigarette smoke for eight weeks (p2), control group not exposed to sidestream cigarette smoke for four weeks (k2), and control group (k) not exposed to sidestream cigarette smoke for eight weeks (k2). the exposure process was conducted using a smoking pump and alternating exposure. four micron-thick sections of formalin were subsequently fixed together with paraffin embedded biopsy material from tongue mucosa of rattus norvegicus. the tissue sections from the treatment groups were then analyzed immunohistochemically to compare the expressions of p53 and bcl-2 proteins with those of the control groups. results: the t-test results indicated statistically significant differences in the expressions of p53 between the 4-week control group (k1) and the 4-week treatment group (p1) (p=0.01, p<0.05) as well as between the 8-week control group (k2) and the 8-week treatment group (p2) (p=0.03, p<0.05). conclusion: exposure to cigarette smoke can induce changes in tumor suppressor genes and also affect the regulation of cell apoptosis, thus changing cell structure and leading to malignancy. keywords: apoptosis; carcinogenesis; sidestream cigarette smoke; p53; tongue mucosa correspondence: diah savitri ernawati, department of oral medicine, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: diah -s-e@fkg.unair.ac.id introduction over the last 50 years, numerous studies have been conducted on the toxic chemicals contained in cigarette smoke which are regarded as carcinogens for humans. cigarette smoke is one risk factor for oral cancer in both active and passive smokers due to the presence of carcinogenic elements that can potentially induce cancer. passive smokers inhale the second-hand environmental cigarette smoke exhaled by active smokers. smoke inhaled by passive smokers can also cause health problems similar to those experienced by active smokers since it contains approximately 200 toxic substances, 69 of which are carcinogenic. these carcinogens form covalent bonds with dna (dna adducts) subsequently inducing carcinogenesis.1,2 the correlation between both active and passive smoking and carcinogenesis has actually been studied and identified to exist in several forms of cancer. a high risk of smoking-related cancers also relates to the head and neck. these include cancer of the oral cavity, pharynx and larynx, in addition to lung cancer.3 the process of carcinogenesis is a somatic event thought to be caused by accumulative genetic and epigenetic changes affecting the normal molecular control settings in cell proliferation. these genetic changes can subsequently deactivate the tumor suppressor gene, thereby triggering tumor formation.4 the tumor suppressor gene (p53 gene) is a transcription factor that activates a large number of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p138–141 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p138-141 139angriany, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 138–141 table 1. the expressions of p53 in the control groups and the treatment groups after exposure to cigarette smoke for 4 and 8 weeks groups mean + sd p k1 13.17 + 2.9 0.01* p1 7.17 + 3.8 k2 14.17 + 3.8 0.03* p2 7.5 + 1.9 13.17 7.17 14.17 7.5 0 2 4 6 8 10 12 14 16 4 weeks 4 weeks 8 weeks 8 weeks control treatment control treatment p53 expression figure 1. the graph of the average p53 expressions in the 4-week control group and the 4-week treatment group as well as between the 8-week control group and the 8-week treatment group. gene expressions involved in cell cycle regulation and apoptosis. the p53 gene is the first tumor suppressor gene identified in cancer cells whose working mechanism is normally in an inactive state. it will become active if the cell experiences stress. loss of function in the p53 gene due to mutations can affect the apoptotic mechanism involving the bcl-2 and caspase genes. the mechanism of the p53 gene induces apoptosis by stimulating mitochondria through the induction of the bax gene to release cytochrome c to the cytosol to form caspase cascade.5 in addition, carcinogenesis conditions can induce changes in cells, resulting in malignancy. therefore, this study aims to examine the carcinogenic effects of cigarette smoke exposure on the mucosa of the rat’s tongue against apoptosis through p53 expressions. materials and methods this study was received ethical approval from the ethics committee of the faculty of dental medicine, universitas airlangga, (no: 769/hrecc.fodm/xii/2019). this study constitutes experimental laboratory research involving 24 male wistar strain rats (rattus norvegicus) aged 3-week old and 170 grams in weight which had been obtained from the biochemical laboratory of universitas airlangga, surabaya. the inclusion criteria for these wistar rats comprised; a good state of health, a body weight of 160-180 grams, and a 1-week adaptation period during which they had to satisfy the requirements of being clear-eyed, having a shiny coat, being agile, and passing firm stools. this study was conducted at the biochemistry laboratory of universitas airlangga, surabaya and the biomolecular laboratory of universitas brawijaya, malang. the wistar rats were divided into four groups, namely two control groups consisting of a 4-week control group and an 8-week control group, as well as two treatment groups composed of a 4-week treatment group and an 8-week treatment group. each group contained 6 members. the subjects in the treatment groups were exposed to unfiltered clove cigarette smoke with a tar content of 34 mg and a nicotine content of 2.1 mg via a smoking pump which ensured constant exposure to a dose equivalent to that of 20 cigarettes per day.6 meanwhile, the subjects in the control groups received their usual rations of food and drink and were placed in an open space. after completion of the planned treatment, the subjects were sacrificed by means of inhalation of lethal doses of ether. those in treatment group 1 and control group 1 were sacrificed at the end of the 4th week, while their counterparts in treatment group 2 and control group 2 were sacrificed at the end of the 8th week. following excision of their tongue mucosa, histological preparations were produced. the tissue sections were immunohistochemically analyzed for the expression of p53 gene. the preparations were examined through a nikon e100 light microscope at 400x magnification in order to calculate their p53 expressions. the results of each parameter were then analyzed statistically. a shapiro-wilk normality test was carried out to determine the distribution of research data, followed by a levene’s variance homogeneity test. if the results showed normally distributed data, a paired twosample t test was subsequently performed. the analysis was carried out using spss for windows version 16 (ibm, armonk, new york, usa). results the mean and standard deviation of p53 expressions in the control groups and the treatment groups after exposure to cigarette smoke for four weeks and eight weeks are presented in table 1 and figure 1. the p53 immunohistochemical staining process results can be seen in figure 2. the t test dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p138–141 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p138-141 140 angriany, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 138–141 a b c d figure 2. a. description of wild p53 expression in the 4-week control group (k1) b. description of wild p53 expression in the 4-week treatment group (p1) c. description of wild p53 expression in the 8-week control group (k2) d. description of wild p53 expression in the 8-week treatment group (p2). p53 expression showed by red arrow. results indicated a significant difference in p53 expression between the control and treatment groups after cigarette smoke exposure for four weeks (p=0.01, p<0.05). similarly, there was also a significant difference in p53 expression between the control and treatment groups after cigarette smoke exposure for eight weeks (p=0.03, p<0.01). discussion in general, unfiltered clove cigarettes are more dangerous than their filtered counterparts due to a higher tar, nicotine and carbon monoxide (co) content. the cloves contained in such cigarettes actually constitute an additive whose effect is that the mixture of tobacco and cloves can increase the temperature of a lighted cigarette. as a result, the co and nicotine levels of clove cigarettes are three times higher than those of tobacco-only cigarettes, while their tar levels are as much as to five times higher.7 cigarette smoke can potentially induce cancer due to the presence of carcinogenic elements. consequently, it is considered a risk factor of oral cancer in both active and passive smokers, influenced by the duration and dose of exposure to it. a previous study by kushihashi et al. (2012),3 assessed the impact of the number of cigarettes smoked per day and the duration of smoking among active smokers or that of cigarette exposure among passive smokers on the occurrence of head and neck cancer. the study argued that smoking plays a significant role in the development of squamous cell carcinoma (p = 0.0338). the development of oral cancer is a complex process that can be observed through the use of animal models. their use facilitates accurate and representative descriptions of cellular and molecular changes which have been analyzed histopathologically and which arise from the initiation and development of oral cancer due to a change from normal to pathological conditions.8 employing in vivo animal models as research subjects renders the initiation, promotion, development and metastasis of cancer, including oral cancer, observable.9 therefore, this study was conducted using 3-month-old, male, wistar rats (rattus norvegicus) which represent the most widely employed animal subjects for laboratory research and which, at the age of three months, have reached biological maturity.10 male wistar rats were chosen as research subjects because their conditions are not affected by hormonal factors, such as the menstrual cycle and pregnancy.11 hormonal changes can affect the immunity of the subjects which will, in turn, affect the results of the study.12 p53 constitutes a group of tumor suppressor gene proteins playing a role in cell growth control in the nucleus, particularly with regard to the cell division cycle. this study produced significant decreases in the level of p53 expressions in the 4-week treatment group (p = 0.01, p<0.05) and the 8-week treatment group (p=0.03, p<0.05) after exposure to cigarette smoke for 4 and 8 weeks. similarly, a previous study conducted by husgafvel et al. (2000),13 focused on non-smokers to ascertain the relationship between exposure to environmental tobacco smoke (ets) and lung cancer in non-smokers and employed both frequency biomarkers and p53 mutation types as carcinogenetic biomarkers associated with tobacco use. it concluded that ets directly exhaled into the air can negatively affect the health of non-smokers without their even inhaling it since ets contains carcinogenic substances. the increase in mutant p53 in cases of lung cancer is potentially experienced to a greater extent by non-smokers who are in the immediate vicinity of smokers and, therefore, exposed to ets. this indicates that prolonged exposure to second-hand smoke increases the risk of neoplasia in non-smokers.13 other oral cancer-related studies have been carried out by inducing rats through exposure to carcinogens in the oral cavity such as those found in tobacco dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p138–141 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p138-141 141angriany, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 138–141 cigarettes.14 one such investigation was conducted by pfeifer et al. (2002),15 which found a strong correlation between smoking and oral cancer through analysis of the p53 mutation spectrum. the increased amount of carcinogenic material absorbed by mucosal epithelial cells will heighten the risk of oncogene, tumor suppressor gene (tsg) and deoxyribonucleic acid (dnarg) mutations that play a role in cell division. 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (nnk) and n'-nitrosonornicotine (nnn), together with cigarette smoke content, for example polycyclic aromatic hydrocarbons (hap), are able to modify dna nitrogen bases through methylation and hydroxylation. other ingredients, such as nitric oxide (no) and free radical compounds, can induce the accumulation of free radicals in the oral mucosa. the excessive activity of free radicals and other reactive elements can subsequently lead to oxidative stress resulting in genomic instability, such as modification of dna nitrogen bases potentially leading to gene mutations. the modification of the dna nitrogen base can promote dna adduction potentially leading to damage to the dna of oral mucosal epithelial cells. dna damage, in turn, triggers the oncogene, tsg and dnarg mutations. the gene mutations can indicate whether changes in malignant epithelial cells of the oral mucosa have been initiated.2,16 similarly, in this study, the decreased expression of p53 due to exposure to cigarette smoke shows that gene mutations can normally affect the cell cycle and initiate the risk of malignancy. in conclusion, this study argues that exposure to cigarette smoke can cause decreased expression of wild p53 leading to the cells becoming malignant. references 1. radwan lrs, grawish me, elmadawy sh, el-hawary ym. ultrasurface morphological changes in the rat tongue posterior onethird exposed to passive smoke. ec dent sci. 2016; 4(4): 846–53. 2. hang b. formation and repair of tobacco carcinogen-derived bulky dna adducts. j nucleic acids. 2010; 2010: 1–29. 3. kushihashi y, kadokura y, takiguchi s, kyo y, yamada y, shino m, kano m, suzaki h. association between head-and-neck cancers and active and passive cigarette smoking. health (irvine calif). 2012; 04(09): 619–24. 4. nurhayati s, lusiyanti y. apoptosis dan respon biologik sel sebagai faktor prognosa radioterapi kanker. bul al. 2006; 7(3): 57–66. 5. oren m. decision making by p53: life, death and cancer. cell death differ. 2003; 10(4): 431–42. 6. teague s v., pinkerton ke, goldsmith m, gebremichael a, chang s, jenkins ra, moneyhun jh. sidestream cigarette smoke generation and exposure system for environmental tobacco smoke studies. inhal toxicol. 1994; 6(1): 79–93. 7. amtha r, razak ia, basuki b, roeslan bo, gautama w, puwanto dj, ghani wmn, zain rb. tobacco (kretek) smoking, betel quid chewing and risk of oral cancer in a selected jakarta population. asian pacific j cancer prev. 2014; 15(20): 8673–8. 8. martinez car. 4nqo carcinogenesis: a model of oral squamous cell carcinoma. int j morphol. 2012; 30: 309–14. 9. ishida k, tomita h, nakashima t, hirata a, tanaka t, shibata t, hara a. current mouse models of oral squamous cell carcinoma: genetic and chemically induced models. oral oncol. 2017; 73: 16–20. 10. sengupta p. the laboratory rat: relating its age with human’s. int j prev med. 2013; 4(6): 624–30. 11. beery ak, zucker i. sex bias in neuroscience and biomedical research. neurosci biobehav rev. 2011; 35(3): 565–72. 12. khan d, ansar ahmed s. the immune system is a natural target for estrogen action: opposing effects of estrogen in two prototypical autoimmune diseases. front immunol. 2016; 6: 1–8. 13. husga fvel-p u rsia inen k, boffetta p, ka n n io a, nyberg f, pershagen g, mukeria a, constantinescu v, fortes c, benhamou s. p53 mutations and exposure to environmental tobacco smoke in a multicenter study on lung cancer. cancer res. 2000; 60(11): 2906–11. 14. snyder la, bertone er, jakowski rm, dooner ms, jenningsritchie j, moore as. p53 expression and environmental tobacco smoke exposure in feline oral squamous cell carcinoma. vet pathol. 2004; 41(3): 209–14. 15. pfeifer gp, denissenko mf, olivier m, tretyakova n, hecht ss, hainaut p. tobacco smoke carcinogens, dna damage and p53 mutations in smoking-associated cancers. oncogene. 2002; 21–48(6): 7435–51. 16. warnakulasuriya s, sutherland g, scully c. tobacco, oral cancer, and treatment of dependence. oral oncol. 2005; 41(3): 244–60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p138–141 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p138-141 205 vol. 44. no. 4 december 2011 research report the difference of dental anxiety in children based on frequency of dental appointment mia giri astri1, eka chemiawan2, and eriska riyanti2 1 dental practitioner 2 department of pediatric dentistry, faculty of dentistry, padjadjaran university bandung indonesia abstract background: problem of children’s anxiety during dental procedures is a common phenomenon. this is called dental anxiety. the anxiety children patien need to be paid a special attention, because it will affect the success of dental treatment. purpose: the purpose of this research was to find out the difference of dental anxiety degree in children aged 8 to 12 years old based on the frequency of dental visits in dental community health centre bandung. methods: the method of this research was analytical descriptive. the samples collected by using purposive sampling and the amount of sample was taken by consecutive sampling, and analysed by the u mann-whitney test. results: the result of this research was from 76 correspondence there was 12 children feel anxious and 64 children did not feel anxious. asymp.sig is less than 0.05 in children having frequent and rarely frequency of dental appointment and asymp. sig is more than 0.05 in children having frequent and normal frequency of dental appointment also in children having normal and rarely frequency of dental appointment. conclusion: in conclusion, the frequency of dental visits showed the difference of children’s dental anxiety. key words: �ental anxiety degree, dental visit frequency, children abstrak latar belakang: masalah kecemasan anak saat dilakukan perawatan gigi merupakan fenomena yang sering terjadi. kecemasankecemasan pada saat dilakukan perawatan gigi disebut juga dental anxiety. kecemasan pada penderita anak-anak perlu perhatian khusus karena akan memengaruhi keberhasilan perawatan gigi. tujuan: tujuan penelitian ini untuk mengetahui perbedaan derajat dental anxiety anak usia 8–12 tahun berdasarkan frekuensi kunjungan ke dokter gigi di balai pengobatan gigi kota bandung. metode: jenis penelitian adalah deskriptif analitik. naracoba diambil dengan metode purposive sampling dan jumlah naracoba ditentukan melalui consecutive sampling, kemudian diuji dengan u mann-whitney. hasil: hasil penelitian ini adalah dari 76 naracoba terdapat 12 anak yang merasa cemas dan 64 anak merasa tidak cemas. asymp.sig kurang dari 0,05 untuk perbandingan antara anak dengan frekuensi sering dan jarang berkunjung ke dokter gigi serta asymp.sig lebih dari 0,05 untuk perbandingan anak dengan frekuensi normal dan sering ke dokter gigi juga untuk anak dengan frekuensi normal dan jarang ke dokter gigi. kesimpulan: �isimpulkan bahwa jumlah kunjungan ke dokter gigi memberikan perbedaan pada dental anxiety anak. kata kunci: �erajat dental anxiety, frekuensi kunjungan ke dokter gigi, anak-anak correspondence: mia giri astri, c/o: praktisi kedokteran gigi. jl kertharahayu a21, denpasar bali 80224, indonesia. e-mail:mia giri astri, c/o: praktisi kedokteran gigi. jl kertharahayu a21, denpasar bali 80224, indonesia. e-mail:, c/o: praktisi kedokteran gigi. jl kertharahayu a21, denpasar bali 80224, indonesia. e-mail: miagiriastri@yahoo.co.id 206 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 205–209 introduction anxiety in facing dental treatment is a phenomenon frequently occurs in a community. the anxiety in children patient need to be paid a special attention, because it will affect the success of dental treatment and the coordination of children patient with the dentist. this anxiety can lead the patient to delay or even choose not to come to the dental appointment.1 the cooperative behavior of the children is affected by the treatment in the first appointment. negative reaction will arise if the children feel hurt in the treatment. the anxiety can come from the tooth extraction experience in their first dental appointment or the experience or anxiety of the mother or other family members.2 dental care and treatment for children by the dentist should be conducted by considering children’s feeling and their emotional conditions, therefore there will be trust and coordination of the children and the dentist in conducting the treatment. the appointment should be conducted in such a manner so that it will be an interesting experience for the children, particularly the first appointment as an introduction stage between the children and the dentist as well as the environtment. if the children feel comfortable, they will have positif behavior in facing dental treatment procedures.3 the patients who have ever had dental treatments but still show high degree of dental anxiety are due to negative experience, and the patients having routine dental appointment since they are still young age will always come to have their dental treatment although they feel anxious.4 doerr et al.,5 have conducted a survey on the frequency of dental appointment in a province. the survey was done on an adult population in united states of america. the frequency of dental appointments are classified into: frequent (more than twice a year), normal (twice a year), and rarely (less than once a year). this classification is then modified by the researcher, to make it appropriate to the children. the modification brings in the new classification: frequent (more than six times in a year), normal (three to six times in a year), and rarely (less than three times in a year). this modified result agrees with the ideas of the dentists, who suggest that children should have their tooth examined every three months.6 dmf-t index does not affect the degree of anxiety.7 children with high dmf-t index need more dental treatment procedures than children with low dmf-t index. children who have experienced many dental treatment and surgical procedures actually have the lowest anxiety degree. the same pattern was also found by murray et al.,8 who conducted a longitudinal study on the phenomenon of dental anxiety and dental appointment. an analysis of children dental medical records for more than three years indicates that the frequency of dental appointment is an important factor. children who do not have routine dental appointment and rarely have dental treatment procedures indicate the increase of anxiety. children who comes to the dentist either routinely or not and receive dental treatment along with surgical procedures indicate the highest degree of anxiety. the result of brown and murray research shows that children receiving dental treatment along with surgical procedure, but come to the dentist routinely will aid in decreasing the degree of dental anxiety.2,7,8 the degree of anxiety in children, particularly those who are 8-12 years-old is relatively easy to observe. eight to twelve years-old children are considered to have the ability in recognizing their environtment, have high curiosity, and can quickly react with the given stimulus. the age period from 8 to 12 years-old is also called as analysis period, in which children have had an ability to recognize the difference and its parts, although the connection between the parts does not entirely appear. the fantasy has decreased and change into real observation.9 the measuring of dental anxiety can be performed in many ways, for example by corah dental anxiety scale. corah dental anxiety scale is a questionnaire consisting multiple choice questions about patients’ subjective reaction to dental treatment.5 bandung city has the facility of dental community health centre distributed in all over the region. one of the visions of bandung in dental health program is: 50% of the visit to community health centre will have dental services in dental community health centre.10 the number of dental community health centre in this research are six dental community health centres, they are the dental community health centres having the greatest number of children visitor in the latest one year. the purpose of the study was to examine the children dental anxiety degree based on the frequency of dental appoitment in dental community health centres in bandung city. materials and methods this research is a descriptive analytical research, that was aimed to give description of the reality in the object studied objectively.10 the sampling method was purposive sampling, i.e. the method in taking samples based on individu or researcher consideration. the number of the samples were determined by consecutive sampling, in which the samples were selected based on the order of their visit. population criteria in this research were: children 812 years-old, healthy both physically and mentally, boys and girls, was not using fixed or removable orthodontic appliances, was not under root canal treatment, and have ever had a dental appointment. research procedures included answering corah dental anxiety scale questionnaire, filling in general information sheet, and measuring the pulse of the patients. corah dental anxiety scale used scoring method by calculate the anxiety rating. children who had moderate anxiety have 9 until 12 point, children who had high anxiety have 13 until 14 point, and severe anxiety or phobia scored up to 15 point.100 207astri, et al.,: the difference of dental anxiety the data was collected, processed, and analyzed, then was presented in the form of table of frequency and percentage distribution. results this research was conducted on 76 samples who meet the criteria. the distribution of number and percentage of the samples visiting dental community health centres in bandung city based on the frequency of dental appointment and degree of dental anxiety (table 1). table 1. distribution of samples number and percentage based on the frequency of dental appointment and degree of dental anxiety visiting frequency n anxious not feel anxious f % f % f % rare 27 35.53 7 9.21 20 26.32 normal 27 35.53 4 5.26 23 30.26 frequent 22 28.95 1 1.32 21 27.63 total 76 100 12 15.79 64 84.21 to examine the difference of dental anxiety degreedegree in children having frequent and rarely frequency of dental appointment, mann whitney test is used and themann whitney test is used and the statistical result showed that u value of mann whitney was 233.500 with z value -1.933, and asymp. sig was less than 0.05. so ho hypothesis is rejected, and it means that statistically there is a difference of dental anxiety degree in children having frequent and rarely frequency of dental appointment. to examine the difference of dental anxiety degreedegree in children having frequent and normal frequency of dental appointment, mann whitney test is used and themann whitney test is used and the statistical result showed that u value of mann whitneyshowed that u value of mann whitney is 266.500 with z value -1.169 and asymp. sig is more than 0.05. so ho hypothesis is accepted, and it means that statistically there is no difference of dental anxiety degree in children having frequent and normal frequency of dental appointment. to examine the difference of dental anxiety degreedegree in children having normal and rarely frequency of dental appointment, mann whitney test is used and the statisticalmann whitney test is used and the statisticaltatistical result showed that u value of mann whitney is 324.000howed that u value of mann whitney is 324.000 with z value -1.004 and asymp. sig is more than 0.05. so ho hypothesis is accepted, and it means that statistically there is no difference of dental anxiety degree in children having normal and rarely frequency of dental appointment. the distributions of samples number and percentage on children’s feeling when going to see the dentist, waiting in the dentist waiting room, sitting on the dental chair and waiting for the treatment and when seeing dentist holding dental instrument and taking care of children’s teeth can be seen in table 2–5. table 2. distribution of samples number and percentage baseddistribution of samples number and percentage based on children’s feeling when going to see the dentist (question 1 corah das) answer children’s feeling f (n) percentage (%) a b c d e happy not worry worry nervous very nervous 3 18 13 22 20 26.32 28.95 17.11 23.68 3.95 total 76 100 table 3. distribution of samples number and percentage baseddistribution of samples number and percentage based on children’s feeling when waiting in the dentist waiting room (question 2 corah das) (question 2 corah das) answer children’s feeling f (n) percentage (%) a b c d e happy not worry worry nervous very nervous 54 12 7 3 0 71.05 15.79 9.21 3.95 0 total 76 100 table 4. distribution of samples number and percentage based distribution of samples number and percentage based on children’s feeling when sitting on the dental chair and waiting for the treatment (question 3 corah (question 3 corah das) answer children’s feeling f (n) percentage (%) a b c d e happy not worry worry nervous very nervous 36 13 12 13 2 47.37 17.11 15.79 17.11 2.63 total 76 100 table 5. distribution of samples number and percentagedistribution of samples number and percentage based on children’s feeling when dentist holding dental instruments and taking care of children’s teeth (question 4 corah das) answer children’s feeling f (n) percentage (%) a b c d e happy not worry worry nervous very nervous 38 14 7 9 8 50.00 18.42 9.21 11.84 10.53 total 76 100 208 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 205–209 discussion the result of research on dental anxiety degree based on the frequency of dental appointment in table 1 showed a significant result. samples who feel anxious consists of 12 children (13.16%). the highest percentage were 7 children (9.21%) stated that they rarely have dental appointment, that was less than 3 times in the last one year. however, this rate is not comparable with the number of samples who do not feel anxious, either those who have frequent, normal, or rarely frequency of dental appointment. total samples who do not feel anxiety consists of 64 children (86.84%). the highest percentage were 22 children (28.95%) stated that they have dental appointment 3–6 times in the last year or have normal frequency of dental appointment, and 22 children (28.95%) stated that they have more than 6 times dental appointment in the last year, or can be classified into ‘frequent’ frequency. this indicates that the frequency of dental appointment affects children’s dental anxiety degree in facing dental treatment. children frequently come to the dentist have lower dental anxiety degree compared with those who rarely come to the dentist. this result agrees with the opinion of jay,3 who suggest that children are advisable to check up their dental health three times a year, in order to grow their selfassurance and to build comfort in undergoing dental treatment. the result of this research showed that 64 students (86.84%) or almost all the children visiting dental community health centres in bandung did not indicate high dental anxiety degree (table 1). it is supported by their answer to the questions about their feeling when they were going to have dental appointment (table 2). meanwhile 22 students (28.95%) said that they did not feel anxious and 20 students (26.32%) said that seeing the dentist is an interesting experience. based on interview result to the children who do not feel anxious in their dental appointment, the reason why they did not feel anxious were because they feel comfortable in the examination and because they often have dental examination in dental health unit in their school. the condition of waiting room also affects the anxiety degree. children answers to the question about the feeling when they are in the waiting room (table 3) is: 54 children (71.05%) or most of them feel comfortable. based on the interview, they feel comfortable because they can play and watch the television in the waiting room. the other reason is because they are accompanied by their parents. children who feel anxious can give unpleasant behavior in dental chair. they will be uncooperative and can not stay quietly in the dental chair.11 the result of the research showed that most of the children did not feel anxious in dental treatment. this result is supported by samples’ answer to the question about their feeling when sitting in the dental chair and undergoing their dental treatment (table 4). the highest percentage, 36 children (47%) or almost a half of the samples, answer that they did not feel anxious. based on the interview result, they feel comfortable when sitting in the dental chair if the parents are standing near the dental chair, waiting for them undergoing dental treatment. this result agrees with the opinion of peretz et al.,12 and folayan et al.,13 that there was a positive correlation exists between parental and children’s dental anxiety. the comfort is also as a result of the dentist who is always friendly and kind. children reaction when looking at dental instruments can affect dental anxiety degree. in this research, children respond can be found out from their answer to the question about their feeling when seeing the dentist take the instruments for dental care or treatment (table 5). 38 children (50%), or a half of the children visiting dental community health centres in bandung city, said that they do not feel anxious looking at the instruments. based on interview result, they feel comfort because they have often seen the instruments, and they frequently visit the dentist before. meanwhile 9 children (11.84%) said that they are anxious, and 8 children (10.53%) said that they are very anxious when looking at the instruments, because they rarely come to the dentist to have dental appointment, or because they are afraid that the instrument will hurt them. twelve children (15.79%), or about 1/6 of total samples, show high dental anxiety degree when undergoing dental treatment in dental community health centres in bandung (table 1). based on interview result, the anxiety of children in their dental treatment can be caused by various factors, such as trauma because they have seen their sister or brother feel pain or hurt in the dental treatment, feeling afraid of the injection, and thinking that all of dental action is painful. this result agrees with the opinion of townend,14 thatthat children’s fear was more strongly associated with subjective experience of pain and trauma than with objective dental pathology. eight to twelve years-old children are in elementary school, and this period of age is the most effective age to get knowledge on dental health and to have dental treatment. the anxiety in children comes from perception process of the children on dental treatment based on the experience, story from someone else, and based on what the read. anxiety often cause children show negative behavior towards dental treatment. dental anxiety is identical to their fear towards dental treatment. the anxiety in children is extremely affected by their experiences in previous dental treatment, their respond to the pain, and their knowledge on dental health. children’s behavior is affected by their education and experiences.15,16 from the research, it can be concluded that the frequency of dental appointment affect children’s dental anxiety, it means that children should go to the dentist as soon as possible to check their oral health conditions and children should see the dentist at least once in three months to reduce dental anxiety when visiting the dentist. 209astri, et al.,: the difference of dental anxiety acknowledgement the authors thanks to the director of rskgm, the dentists in dental community health centre where the research was conducted, and all the children participated in the research. references 1. mansky m. a simple five minute for cure dental anxiety. 1997. m. a simple five minute for cure dental anxiety. 1997. available at: http://www.dentalcyberweb.com. accessed january 20, 2008. 2. kent gg. pengelolaan tingkah laku pasien pada praktik dokter gigi. budiman ja, editor. jakarta: egc; 1994. p. 63–86. 3. jay. your child’s first dental visit. 2007. available at: http://www. drjay.com/1stvisit.com. accessed january 20, 2008. 4. joelimar fa. dental anxiety: salah satu penghambat dalam pemanfaatan jasa kedokteran gigi. kppikg fkg ui 1986; vii: 424-35. 5. doerr pa, lang wp, nyquist lv. factors associated with dental anxiety. jada 1998; 129: 1111–9. 6. woolston c. child’s first dental appointment. 2007. available at: http://healthresources.caremark.com. accessed january 20, 2008. 7. brown df, wright fa, mc murray ne. psychological and behavioural factors associated with dental anxiety in children. j behav med 1986; 9: 213–7. 8. murray p. a longitudinal study of the contribution of dental experience to dental anxiety in children between 9 and 12 years age. j behav med 1989; 12: 309–20. 9. mubin, cahyadi a. psikologi perkembangan. jakarta: ciputat press group; 2006. p. 94. 10. siegel l. daily measurements of anxiety and affect: a study among spouse caregivers. int j behav develop 1997; 4: 577–68. 11. clarke jh. norman corah dental anxiety scale. 2007. available at: http://dentalfearcentral.org. accessed december 23, 2008. 12. peretz. dental anxiety in a students’ paediatric dental clinic: children, parents and students. int j of pediatric dent 2004; 14: 192–8. 13. folayan mo. parental anxiety as a possible predispossing factor to child dental anxiety in patient seen in a suburban dental hospital in nigeria. int j of paed dent 2002; 4: 255–9. 14. townend e, diane f. a clinical study of child dental anxiety. behav research and therapy 2000; 38: 31–46. 15. priyona b, hendrertini j. pengaruh kesehatan gigi sekolah terhadap kecemasan pada perawatan gigi serta kesehatan gigi dan mulut anak sekolah dasar. prosiding lustrum viii fkg ugm, 2001; 40: 127–31. 16. budiharto. kontribusi pendidikan kesehatan gigi dan perilaku ibu terhadap radang gusi anak. jurnal kedokteran gigi universitas indonesia 1999; 6: 12–8. 215 volume 47, number 4, december 2014 uji toksisitas ekstrak bawang putih (allium sativum) terhadap kultur sel fibroblast (garlic (allium sativum) extract toxicity test on fibroblast cell culture) yulie emilda, els budipramana, dan satiti kuntari departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: previous studies have found antimicrobial effect of garlic (allium sativum). garlic has potential as sterilization material for root canal treatment. nevertheless, such material has to be non toxic and has to have adequate biocompatibility. purpose: the study was aimed to examine the toxicity of garlic (allium sativum) on fibroblast cell culture. method: toxicity test was conducted using 50%, 75%, 100% of garlic extract, and chlorphenol kamfer menthol (chkm) as control. bhk-21 cell-culture was put into microplate 96 wells with 2x105 densities and incubated in a 37oc. the garlic extracts in various concentration and chkm were then placed into the wells. mtt assay test was then use to analyze toxicity, a 50% percentage of living culture-cell was set as a parameter whether the extract is toxic or not. results: the results showed that in 50%, 75%, and 100% garlic concentration indicates a non toxic characteristic on fibroblast cell culture. the non toxic property was consistent in 72, 96, and 120 hours of observation point. conclusion: the study revealed that garlic on consentration of 50,%, 75%, 100% did not show toxic effect on fibroblast culture cell, but it needs further research for preparing it as an alternative medicament of root canal treatment. key words: garlic, allium sativum, toxicity, cell-culture, fibroblast abstrak latar belakang: penelitian sebelumnya telah menemukan efek antimikroba bawang putih (allium sativum). bawang putih memiliki potensi sebagai bahan sterilisasi pada perawatan saluran akar. namun bahan tersebut harus tidak toksik dan memiliki biokompatibilitas yang memadai. tujuan: penelitian ini bertujuan menguji toksisitas bawang putih (allium sativum) terhadap kultur sel fibroblast. metode: uji toksisitas dilakukan dengan menggunakan konsentrasi 50%, 75%, 100% dari ekstrak bawang putih, dan chlorphenol kamfer menthol (chkm) sebagai kontrol. bhk-21 kultur sel dimasukkan ke dalam microplate 96 sumuran dengan kepadatan 2x105 dan diinkubasi di suhu 37°c. ekstrak bawang putih dalam berbagai konsentrasi dan chkm kemudian ditempatkan pada sumur dipersiapkan sebelumnya. mtt assay test kemudian digunakan untuk menganalisis toksisitas, persentase 50% dari kultur sel hidup digunakan sebagai parameter apakah ekstrak beracun atau tidak. hasil: hasil penelitian ini menunjukkan pada konsentrasi bawang putih 50%, 75%, 100% menunjukkan karakteristik non toksis terhadap kultur sel fibroblast. kondisi non toksik konsisten di 72, 96, dan 120 jam pengamatan. simpulan: penelitian ini menunjukkan bahwa ekstrak bawang putih dengan konsentrasi 50,%, 75%, 100% tidak bersifat toksik terhadap kultur sel fibroblast, namun masih diperlukan pengujian lebih lanjut untuk dapat digunakan sebagai alternatif bahan sterilisasi saluran akar. kata kunci: bawang putih, allium sativum, toksisitas, kultur sel, fibroblast korespondensi (correspondence): yulie emilda, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: yulieemilda@yahoo.com. research report 216 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 215–219 pendahuluan sterilisasi saluran akar merupakan tahap yang penting dalam perawatan endodontik.1 obat sterilisasi saluran akar yang digunakan saat ini dibagi menjadi 2 golongan yaitu obat non spesifik dan preparat poliantibiotik. obat non spesifik adalah chlorphenol kamfer menthol (chkm), cresatin, cresophene, formokresol, trikresol formalin (tkf), eugenol, camphorated parachlorophenol (cmcp). preparat poliantibiotik adalah campuran beberapa macam antibiotik biasanya berupa pasta, yaitu penisilin, basitrin, streptomisin, sodium kaprilat dan kombinasi antibiotik dan kortikosteroid, contoh obat adalah ledermix, septomixine, terra cortil.2 uji biologis menyatakan bahwa formocresol sangat toksik, mudah tersebar kedalam apeks gigi dalam waktu 15 menit, menyebabkan iritasi atau bony sequestrum. demikian pula dengan camphorated parachlorophenol walaupun lebih sedikit efek toksik pada jaringan periapikal dibandingkan dengan formocresol. keduanya secara klinis diaplikasikan dalam bentuk cairan pada cotton pellet dan ditempatkan pada ruang pulpa dan akan berubah menjadi bentuk uap.3 oleh karena efek toksisitas kebanyakan obat saluran akar yang ada dipasaran, saat ini penggunaan obat mengarah pada obat ekstrak biologis dari tumbuh-tumbuhan alami untuk mengurangi berbagai aktivitas toksisitas dari bahan kimia.3 beberapa penelitian telah membuktikan bahwa bawang putih memiliki efek antimikroba, menghambat pertumbuhan bakteri gram negatif dan gram-positif seperti escherichia,salmonella, streptococcus mutans, porphyromonas gingivalis,staphylococcus, klebsiella, proteus and helicobacter pylori.4 fani et al.,5 menguji sifat antibakteri ekstrak bawang putih dengan konsentrasi 64%(w/v) terhadap streptococcus mutans. data in vitro yang diperoleh pada studi ini menyatakan bahwa ekstrak bawang putih secara signifikan dapat menghambat pertumbuhan dari s. mutans. lingga dan rustama6 melakukan uji aktivitas antibakteri ekstrak bawang putih terhadap isolate gram negatif clostridium sp, corynebacterium sp, plesiomonas sp, vibrio sp, dan isolat gram positif bacillus sp, streptococcus sp, staphylococcus sp dan erysilopethrix sp. rentang dosis yang digunakan adalah 25%, 50%, dan 75% berdasarkan perbandingan berat:berat. hasil penelitian menunjukkan bahwa ekstrak bawang putih dengan berbagai pelarut dengan pengenceran tertinggi 75% lebih memberikan pengaruh terhadap bakteri-bakteri streptococcus sp, clostridium sp dan pseidomonas sp. aktivitas antimikroba bawang putih dikaitkan dengan adanya allicin yang apabila hilang membuat bawang putih tidak aktif terhadap mikroorganisme. allicin diperoleh dengan menghancurkan atau memotong siung bawang putih. asam amino ordorless, alliin, hadir dalam siung bawang putih, dimetabolisme oleh enzim allinase (sebuah lyase sulfoxide sistein) dengan allicin dan thiosulfinates lainnya, yang memiliki efek antimikroba dan menghasilkan bau yang khas dari bawang putih. allicin bereaksi menghambat sintesis rna dan sebagian menghambat dna dan sintesa protein dimana rna merupakan target utama dari allicin.4 sel fibroblast merupakan sel utama jaringan ikat yang terletak pada lamina propria mukosa rongga mulut, merupakan sel terpenting dan komponen terbesar dari pulpa, ligamen periodontal, dan gingival.7 bahan-bahan yang masuk ke dalam rongga mulut harus bersifat tidak toksik, tidak mengiritasi dan harus mempunyai sifat biokompatibilitas atau bahan yang diproduksi tidak boleh mempunyai efek yang merugikan terhadap lingkungan biologis, baik lokal maupun sistemik.8 untuk itu diperlukan penelitian untuk menguji toksisitas suatu bahan. penelitian ini dilakukan untuk meneliti efek toksisitas larutan ekstrak bawang putih terhadap kultur sel fibroblast. uji toksisitas secara in vitro pada kultur sel bhk-21 menggunakan mtt assay. kultur cell lines digunakan karena sel ini berasal dari embrio sehingga mudah tumbuh dan mudah dilakukan sub kultur ulang. cell lines telah banyak digunakan untuk menguji toksisitas bahan dan obatobatan di bidang kedokteran gigi, antara lain sel bhk-21 yang berasal dari fibroblast ginjal bayi hamster. bahan dan metode penelitian ini merupakan eksperimental laboratoris. sampel yang digunakan dalam penelitian ini adalah ekstrak bawang putih dengan konsentrasi 50%, 75%, dan 100%, serta chkm. disiapkan pula kontrol sel sebagai kontrol positif berisi sel dalam media kultur, dianggap persentase sel hidup 100% dan kontrol media sebagai kontrol negatif berisi media kultur saja, dianggap persentase sel hidup 0%. dalam laminar flow, ekstrak bawang putih diencerkan dengam media eagle’s dan fbs sesuai dengan konsentrasi yang dibutuhkan yaitu 50%, 75%, dan 100%. ekstrak bawang putih yang telah diencerkan dan chkm dimasukkan dalam mikroplate 96 sumuran (menggunakan mikro pipet single), kemudian kultur sel diinkubasi pada masing-masing perlakuan yaitu 72 jam, 96 jam, 120 jam suhu 37° c. kemudian dilakukan uji mtt pada kultur sel. nilai densitas optic formazan dihitung dengan elisa reader pada panjang gelombang 630 nm. makin pekat warnanya, makin tinggi nilai absorbansinya maka semakin banyak jumlah sel yang hidup. persentase sel hidup dihitung dengan: 8 % sel hidup = perlakuan + media × 100% sel + media keterangan: % sel hidup : persentase jumlah sel hidup setelah pengujian perlakuan : nilai densitas optik formazan pada setiap sampel setelah pengujian media : nilai densitas optik formazan pada kontrol media sel : nilai densitas optik formazan pada kontrol sel 217emilda, et al.: uji toksisitas ekstrak bawang putih (allium sativum) data yang diperoleh dianalisa dengan independent t-test untuk melihat perbedaan persentase sel hidup pada perbandingan antar kelompok penelitian. hasil berdasarkan hasil pengamatan dan penghitungan densitas jumlah sel fibroblast terhadap penggunaan ekstrak bawang putih, yang terbagi atas 4 kelompok penelitian, yaitu kelompok konsentrasi 50%, 75%, 100%, dan chkm, didapatkan hasil sebagai berikut: ekstrak bawang putih dengan konsentrasi 50%, 75%, 100%, selama 72 jam, 96 jam, dan 120 jam tidak menunjukkan adanya toksisitas (tabel 1). hasil uji statistik tampak pada tabel 2, 3 dan 4. terlihat adanya kecenderungan peningkatan rata-rata densitas jumlah sel fibroblast yang menggambarkan jumlah sel yang hidup, pada kelompok konsentrasi ekstrak yang lebih tinggi. semakin tinggi konsentrasi ekstrak bawang putih maka semakin tinggi rata-rata densitas jumlah sel fibroblast. pada kelompok kontrol chkm didapatkan ratarata densitas jumlah sel fibroblast dengan nilai yang paling rendah (gambar 1, 2, 3). tabel 1. rata-rata dan standar deviasi jumlah sel fibroblast yang hidup pada setiap kelompok penelitian setelah 72 jam, 96 jam dan 120 jam hari (jam) kelompok rerata standar deviasi tiga (72 jam) 50% 0,97000 0,050236 75% 1,07791 0,044496 100% 1,62665 0,092523 chkm 0,86736 0,028064 empat (96 jam) 50% 0,91999 0,047930 75% 1,00943 0,039965 100% 1,49951 0,064746 chkm 0,75298 0,273543 lima (120 jam) 50% 0,91478 0,052844 75% 0,99155 0,043756 100% 1,46949 0,121242 chkm 0,86458 0,030368 tabel 2. uji beda antara masing-masing kelompok penelitian menggunakan independent t-test setelah 3 hari (72 jam) tiga hari (72 jam) 50% 75% 100% chkm 50% 0,000* 0,000* 0,000* 75% 0,000* 0,000* 100% 0,000* chkm *=terdapat perbedaan yang signifikan (p<0,05). tabel 3. uji beda antara masing-masing kelompok penelitian menggunakan independent t-test setelah 4 hari (96 jam) empat hari (96 jam) 50% 75% 100% chkm 50% 0,001* 0,000* 0,111 75% 0,000* 0,020* 100% 0,000* chkm *=terdapat perbedaan yang signifikan (p<0,05). tabel 4. uji beda antara masing-masing kelompok penelitian menggunakan independent t-test setelah 5 hari (120 jam) lima hari (120 jam) 50% 75% 100% chkm 50% 0,007* 0,000* 0,035* 75% 0,000* 0,000* 100% 0,000* chkm *=terdapat perbedaan yang signifikan (p<0,05). gambar 1. grafik rata-rata dan standar deviasi jumlah sel fibroblast yang hidup dihari ketiga (72 jam). gambar 2. grafik rata-rata dan standar deviasi jumlah sel fibroblast yang hidup dihari keempat (96 jam). 218 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 215–219 pembahasan terlihat adanya kecenderungan peningkatan rata-rata densitas jumlah sel fibroblast yang menggambarkan jumlah sel yang hidup, pada kelompok konsentrasi ekstrak yang lebih tinggi. semakin tinggi konsentrasi ekstrak bawang putih maka semakin tinggi rata-rata densitas jumlah sel fibroblast. pada kelompok kontrol chkm didapatkan rata-rata densitas jumlah sel fibroblast dengan nilai yang paling rendah. terdapat perbedaan yang signifikan di antara keempat kelompok tersebut, artinya peningkatan kepekatan ekstrak bawang putih menimbulkan perbedaan yang signifikan terhadap viabilitas sel fibroblast. tidak ada perbedaan yang signifikan antara ekstrak bawang putih konsentrasi 50% dengan chkm pada hari keempat. pada kelompok ekstrak bawang putih dengan konsentrasi 50% didapatkan persentase jumlah sel yang hidup adalah 97% pada hari ketiga, 92% pada hari keempat, dan 91,48% pada hari kelima. walaupun nilai tersebut tidak menunjukkan toksisitas, namun terdapat sel yang mengalami kematian. pengaruh yang ditimbulkan terhadap viabilitas sel fibroblast berupa perubahan permeabilitas membran sel. efek toksisitas dari sitotoksin dapat menyebabkan terjadi perubahan permeabilitas membran sel atau kerusakan integritas membran sel sehingga membran selnya bisa ditembus oleh trypan blue.7 kerusakan pada membran sel dapat menyebabkan sel menjadi non viabel, dan selanjutnya dapat menyebabkan kematian sel. pada sel yang non-viabel membran selnya bisa ditembus oleh trypan blue sedangkan sel yang viabel memiliki membran sel yang impermiabel terhadap trypan blue. jadi semakin besar pengaruh yang ditimbulkan akan mengakibatkan semakin banyak sel mati, yang juga berarti persentase kematian sel semakin meningkat. terdapat beberapa mekanisme biokimiawi yang diduga memperantarai kematian sel, hal ini dapat menjelaskan terjadinya kematian sel sehubungan dengan toksisitas suatu bahan. mekanisme biokimia tersebut antara lain: penipisan kadar adenosin triphosphate (atp) dan defek pada membran sel. enzim dehidrogenase adalah salah satu enzim yang berperan dalam pembentukan atp, yaitu suatu bentuk energi yang sangat dibutuhkan oleh sel untuk berbagai aktivitas fungsional sel. jika enzim dehidrogenase tidak aktif akibat efek sitotoksik suatu sitotoksin, maka atp berkurang, aktivitas sel terganggu, sehingga dapat mengakibatkan kematian sel. kerusakan membran atau hilangnya permeabilitas membran selektif merupakan gambaran umum jejas sel. defek ini bisa mempengaruhi mitokondria yang merupakan tempat untuk memproduksi atp.9 prinsip umum mengenai jejas sel yang memiliki kemungkinan sebagai penyebab adanya perbedaan pengaruh pada kelompok penelitian tersebut, antara lain: 1) beberapa komponen atau sistem intraseluler sel sensitif atau mudah mengalami jejas antara lain: membran sel (integritas membran sel), sistem respirasi aerob (mitokondria, enzim), komponen genetic; 2) respon seluler terhadap stimuli jejas tergantung pada jenis jejas, lama stimuli jejas dan berat ringannya stimuli jejas. jenis jejas dibedakan berdasarkan penyebab jejas sel, seperti bahan kimia dan obat-obatan, hipoksia, dan lainnya. lama stimuli jejas berhubungan dengan waktu paparan, sedangkan berat ringannya stimuli berkaitan dengan dosis ataupun konsentrasi paparan.9,10 terjadinya proliferasi sel pada ekstrak bawang putih konsentrasi 100% bisa disebabkan karena penggunaan kultur sel fibroblast yang terlalu tebal yaitu dengan kepadatan 2 x 105. persentase sel hidup yang meningkat, yang ditunjukkan dengan nilai densitas optik formazan yang meningkat menunjukkan bahwa sel fibroblast mampu mempertahankan integritas membran sel sehingga sel tidak mengalami kematian. semakin besar nilai densitas optik formazan, semakin banyak jumlah sel yang hidup. kemampuan sel mempertahankan permeabilitas sel ini besar kemungkinan disebabkan karena adanya kandungan senyawa kimia aktif yang didapatkan pada bawang putih yang dapat menstimulasi proliferasi sel fibroblast. pada penelitian ini terjadi proliferasi sel fibroblast. salah satu kandungan senyawa dalam bawang putih adalah diallyl disulfida. diallyl disulfida dalam bawang putih mampu menguraikan protein pada sel yang rusak sehingga protein tersebut mudah dicerna oleh tubuh dan mampu meningkatkan kekebalan non-spesifik melalui aktivitas fagositosis dan merangsang aktifitas sel yang berperan dalam respons imunitas.11 flavanoid yang terkandung dalam bawang putih memiliki sifat dapat menumbuhkan jaringan, menghambat respon inflamasi dengan cara menghambat siklus lipooksigenase yang menghasilkan prostaglandin.12 konsentrasi fenol yang rendah dari beberapa senyawa fenol menstimulasi proliferasi sel fibroblast pulpa manusia. fenomena ini dikenal sebagai hormesis dan berkontribusi dalam memperbaiki jaringan pulpa. penelitian ini menunjukkan bahwa ekstrak bawang putih dengan konsentrasi 50,%, 75%, 100%, tidak bersifat toksik terhadap kultur sel fibroblast, namun masih diperlukan pengujian lebih lanjut untuk dapat digunakan sebagai alternatif bahan sterilisasi saluran akar. gambar 3. grafik rata-rata dan standar deviasi jumlah sel fibroblast yang hidup dihari kelima (120 jam). 219emilda, et al.: uji toksisitas ekstrak bawang putih (allium sativum) daftar pustaka 1. grossman li, oliet s, del rio ce. preparasi saluran akar: peralatan dan teknik pembersihan,pembentukan, dan irigasi, desinfeksi saluran akar in ilmu endodontik dalam praktek. editor suryo s. 11th ed. jakarta: egc; 1995. h. 196, 248-53. 2. tarigan r. medikamen pada endodontia in perawatan pulpa gigi (endodontia). 1st ed. jakarta: widya medika; 1994, p.71-5. 3. zied sta, eissa, somaia al. comparative study on antibacterial activities of two natural plants versus three different intracanal medications. endodontic departement, faculty of oral and dental medicine, cairo university; 2011. p. 1-2. 4. eja me, asikong be, abriba c, arikpo ge, anwan ee, enyiidoh kh. a comparative assessment of the antimicrobial effects of garlic (allium sativum) and antibiotics on diarrheagenic organism. southeast asian j trop med public health 2007; 38(2): 343-8. 5. fani mm, kohanteb j, dayaghi m. inhibitory of garlic (allium sativum) on multidrugs-resistent streptococcus mutans. j indian soc pedod prevent dent 2007; 25(4): 164-8. 6. lingga me, rustama mm. uji aktivitas antibakteri dari ekstrak air dan etanol bawang putih (allium sativum) terhadap bakteri gram negatif dan gram positif yang diisolasi dari udang dogol (metapenaeus monoceros), udang lobster (panulirus sp), dan udang rebon (mysis dan acetes). available from http://pustaka. unpad.ac.id/wpcontent/uploads/2009/12/uji_aktivitas_ antibakteri. pdf. accessed march 27, 2011. 7. freshney ri. a manual of basic technique in culture of animal cells. 2th ed. new york: alan r liss inc; 1992. p. 227-45. 8. meizarini a. sitoksisitas bahan restorasi cyanoacrylate pada variasi perbandingan powder dan liquid menggunakan esei mtt. maj. ked. gigi (dent j) 2005; 38(1): 20–4. 9. cotran r, kumar v, collins t. robbins pathologic basis of disease. 6th ed. philadelphia, pennsylvania, usa: wb saunders company; 1999. p. 4-15, 102-10. 10. cowan mm. plant products as antimicrobial agents. clinical microbiology reviews 1999; 12(4): 564-82. 11. nuryati s, giri p, hadiroseyani y. efektivitas ekstrak bawang putih allium sativum terhadap ketahanan tubuh ikan mas cyprianus carpio yang diinfeksi koi herpes virus (khv). jurnal akuakultur indonesia 2008; 7(2): 139-50. 12. khayyal mt, el-ghazaly ma, el-khatib as. mechanism involved in the antiinflamatory effects of propolis extract. drugs exp clin res 1993; 19(5): 197-203. 132 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 132–136 original article analysis of soft tissue facial profiles of chinese students at w.r. supratman 1 and 2 high schools in medan using linear and angular measurements hilda fitria lubis and maureen olivia department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: soft tissue analysis is essential in treatment planning to ensure proportional, balanced, and harmonious soft tissue at the end of treatment. several factors influence the variations, such as age and gender. soft tissue profile analysis is usually studied from a lateral view. purpose: the study aimed to determine whether there were differences in the average values of linear and angular measurement of the soft tissue profiles of the middle and lower third of the face between chinese males and females high school students using lateral photometry. methods: the samples were 100 lateral photographs (50 male and 50 female) of chinese high school students at w.r. supratman 1 and 2 in medan. all samples were collected based on the inclusion and exclusion criteria obtained from secondary data. the linear and angular measurements were measured using software corel draw 2019 and analysed statistically using an independent t-test and the mann-whitney test. results: the inferior facial third, length of the lower lip, and prominence of the upper lip were, on average, greater in males, whereas in the prominence of pogonion, nasolabial angles, and nasofrontal angles were greater, on average, for females. conclusion: there were significant gender differences in chinese high school students in the inferior facial third, length of the lower lip, prominence of the upper lip, nasolabial angle, and nasofrontal angle, whereas there is no significant difference between genders in the prominence of the pogonion. keywords: chinese student; lateral photograph; soft tissue profile analysis correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: hilda.fitria@usu.ac.id introduction facial appearance affects a person’s self-esteem and selfimage, which is directly related to their quality of life.1 however, facial beauty standards vary widely and are related to the social environment.2 facial appearance could also be influenced by several factors such as age, gender, psychology, race and ethnicity.2,3 variations in the length of the nose, the protrusion of the lips, and the chin’s projection can change the alignment of the face and create a new visage, which has a different aesthetic perception.4 related to age and gender, the most significant changes in soft tissue occurs earlier in women (10 –15 years) than men (15–25 years).5 in addition, a study by torlakovic et al.6 stated the ageing of the facial profile in men is ten years slower than in women and changes in the facial profile are more significant in women than in men. the facial structure can be changed in some medical fields such as plastic surgery and orthognathics, orthodontics and prosthodontics.4 soft tissue analysis is essential in treatment planning to ensure proportional, balanced, and harmonious soft tissue at the end of treatment. the connection of the nose, lips, chin and facial soft tissue, is an important consideration. a soft tissue evaluation to better understand aesthetics can be performed from the frontal view and the profile.5 these analyses are studied from a profile or lateral perspective, either by radiography or photography.4 several studies examine facial soft tissue profiles with linear and angular measurements, such as the studies by moskelgosha et al.4 (2015) on persians, diouf et al.7 (2011) on senegalese and moroccans, leung et al.8 (2014) on southern chinese. previous studies have shown variations dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p132–136 mailto:hilda.fitria@usu.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p132-136 133lubis and olivia/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 132–136 in facial soft tissue analysis results with linear and angular measurements associated with influencing factors such as gender and ethnicity.2 indonesia is a country with a variety of cultures and ethnic groups. this variety was influenced by trade relations with indian, arab, european, dutch, portuguese and chinese peoples.9,10 the chinese originally came to indonesia, especially palembang, which was the centre of trade for the sriwijaya kingdom, for economic and trade purposes.10 therefore, this study aimed to determine whether there were differences in the average values of linear and angular measurement of the middle and lower third soft tissue profile of the face in chinese male and female high school students using lateral photometry. materials and methods the methodology used was an analysis study with a retrospective cross-sectional approach. samples were lateral photographs of chinese high school students at w.r. supratman 1 and 2 in medan, collected using a purposive sampling method based on the inclusion and exclusion criteria. the inclusion criteria were chinese ethnicity, 15–18 years old, had complete permanent teeth except for m3, had a profile photo, and had a class 1 dental occlusion. the exclusion criteria included a history of facial asymmetry or plastic surgery or corrupted photographic data. the photographic data were the secondary data taken in the natural head position (nhp). the subjects were sitting in a chair 0.75 m in front of a wall covered with a 1.2 × 1.5 m white cloth. the camera, a canon g7x, was placed 1.5 m in front of the subject’s chair. they sat facing left to show a lateral view to the camera. the same operator took all the photographs.11 this study had permission from the research ethics committee of universitas sumatera utara (number 116/kep/usu/2021). after collecting the photographs, landmarks were added using corel draw 2019 software (corel corporation, ottawa, canada), as shown in table 1.4 they were subnasal, labial superior, stomion inferior, supramental, pogonion, menton, glabella, pronasal, and columella. parameters used in this study are: the inferior facial third (figure 1a) was constructed by drawing a line from subnasal (sn) to menton (me); the length of the lower lip (figure 1b) was made by drawing a line from stomion inferior (sti) to supramental (sm); the prominence of the upper lip (figure 1c) was achieved by drawing a line from labial superior (ls) to canut’s line (subnasal (sn) to supramental (sm)); the prominence of pogonion (figure 1d) was constructed by drawing a line from pogonion (pg) to canut’s line (subnasal (sn) to supramental (sm)); the nasofrontal angle (figure 1e) was calculated by drawing an angle from nasion (n) to glabella (g) and pronasal (prn); the nasolabial angle (figure 1f) (a) (b) (c) (d) (e) (f) table 1. the landmarks were used in the middle and lower third soft tissue profile analysis in this study4 landmarks definition subnasal the point where the upper lip borders the columella labial superior the point showing the mucocutaneous border of the upper lip stomion inferior the most superior point of the lower lip supramental the innermost point of the inferior sublabial concavity pogonion the most anterior point of the chin menton the most inferior point of the inferior edge of the chin glabella the most anterior point of the midline of the forehead pronasal the most prominent point at the tip of the nose columella the most inferior and anterior point of the nose figure 1. (a) inferior facial third; (b) length of the lower lip; (c) prominence of the upper lip; (d) prominence of pogonion; (e) nasofrontal angle; (f) nasolabial angle. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p132–136 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p132-136 134 lubis and olivia/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 132–136 was made by drawing an angle from columella (cm) to subnasal (sn) and labial superior (ls). then, the lines and angles were measured using the same software. each face’s middle and lower third soft tissue profile was derived from lines and angles obtained by connecting the landmarks. results in this study, we found that the average value of the inferior facial third, the length of the lower lip, and the upper lip’s prominence was greater in males than females. in contrast, the average value of prominence of the pogonion, nasolabial angle and nasofrontal angle was more significant in females than males (table 1). when analysed statistically using the shapiro-wilk test, the data showed almost all parameters were distributed normally except the inferior facial third and upper lip length. the normally distributed data were analysed using an independent t-test, whereas the mann-whitney test was used for the data that were not normally distributed (table 2). from table 2, the results of measuring the inferior facial third, the length of the lower lip, prominence of the upper lip, nasolabial angle, and nasofrontal angle all indicated significant gender differences. in contrast, the results of measuring the prominence of pogonion showed there were no gender differences. the previous study measurements were collected as a comparative study and showed in table 3. table 2. the measurement of the middle and lower third soft tissue profile of the face on chinese male and female students at w.r. supratman 1 and 2 high schools in medan parameter gender n min max mean sd p-value a. lower third of the face inferior facial third males 50 52.54 mm 88.70 mm 68.08 mm 7.05 0.001* females 50 49.33 mm 86.63 mm 63.70 mm 8.71 length of lower lip males 50 12.01 mm 28.71 mm 17.93 mm 2.72 0.001* females 50 11.70 mm 23.34 mm 16.28 mm 2.61 prominence of upper lip males 50 3.19 mm 9.63 mm 6.01 mm 1.56 0.002* females 50 2.75 mm 8.47mm 5.07 mm 1.33 prominence of pogonion males 50 -4.72 mm 7.95 mm 1.41 mm 2.50 0.225 females 50 -1.97 mm 6.36 mm 1.97 mm 2.09 nasolabial angle males 50 72.18° 120.20° 95.46° 8.94 0.001*females 50 77.35° 114.57° 100.21° 8.58 b. middle third of the face nasofrontal angle males 50 120.85° 145.60° 131.94° 6.33 0.008* females 50 132.29° 146.04° 139.62° 3.33 *p-value = significant table 3. the measurement of the middle and lower third soft tissue profile of the face on previous studies moshkelgosha et al.4 leung et al.8 diouf et al.7 sample 110 persian females and 130 persian males south china (259 males and 255 females) 138 subjects consisting of senegalese and moroccan males and females a. lower third of the face inferior facial third males = 64.70 ± 6.36 mm, females = 61.1 ± 4 mm males (senegales) = 74.13 ± 7.78 mm, female (senegales) = 67.58 ± 5.33 mm males (moroccan) = 75.58 ± 7.10 mm, females (moroccan) = 64.46 ± 5.91 mm length of lower lip males = 18.71 ± 2.26 mm, females = 16.2 ± 1.5 mm males (senegales) = 22.89±3.30 mm, female (senegales) = 19.51±3.13mm males (moroccan) = 20.31±2.98 mm, females (moroccan) = 18.71±2.31 mm prominence of upper lip males = 3.79 ± 1.64 mm, females = 3.7 ± 1.2 mm prominence of pogonion males = 6.84 ± 1.96 mm, females = 4.6 ± 1.3mm nasolabial angle males = 107.28° ± 11.96, females = 111.2° ± 7.9 males = 99.03° ± 11.52, females = 99.05° ±10.24 b. middle third of the face nasofrontal angle males = 138.2° ± 7.86, females = 140° ± 5.1 males = 143.94°± 4.97, females = 144.68°± 4.51 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p132–136 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p132-136 135lubis and olivia/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 132–136 discussion facial appearance plays an essential role in assessing a person’s visual attractiveness and social environment.1 varied facial appearance can also be influenced by several factors such as age, gender, psychology, race, and ethnicity.2,3 soft tissue analysis is generally performed during the clinical examination. indirect measurements are also needed to record patient’s data. this analysis is, interestingly, commonly studied from a lateral view, either by radiography or photography.4 in this study, the males had a more significant inferior facial third, longer lower lip, more protrusive upper lip, a more retrusive lower lip, a more tapered nasolabial angle and nasofrontal angle than females chinese high school students at the w.r. supratman 1 and 2 in medan. this study showed different average values from the survey conducted by moshkelgosha et al.4 on 110 persian females and 130 persian males (table 3). the mean of inferior facial third in males was greater than females. compared to this study, chinese high school students have an inferior facial third larger than the persian group. the average length of the lower lip for persian males was greater than females. the average upper lip prominence in males was greater than females. the average prominence of the pogonion in males was greater than females. the average nasolabial angle and nasofrontal angle was greater in females.4 this study showed differences from the values conducted by leung et al.8 on 514 12-year-old children in south china (table 3). this study of chinese high school students had the more obtuse nasolabial angle and nasofrontal angle than the south chinese population for both males and females.8 the research conducted by diouf et al.7 on 138 subjects consisting of senegalese and moroccan males and females also showed different values from this study (table 3). the average of the inferior facial third in both senegalese and moroccan males was greater than females. the average value of the length of the lower lip in both senegalese and moroccan males was greater than females.7 compared to this study, the value of inferior facial third and length of the lower lip both senegales and moroccan was greater than chinese high school students for both males and females.7 this study presented differences in average values in all parameters from any of the studies mentioned above. ethnic factors could cause the difference in the measured values and ethnic variations that affect differences in facial soft tissue profiles in both linear and angular values. these are the primary factors that influence facial variation, in addition to genetic and environmental factors. the difference is also caused by gender, which shows differences in soft tissue growth between men and women.12 similar to the result of the survey conducted by moshkelgosha et al.4 on the persian population, which reported significant differences between men and women in the inferior facial third, the length of the lower lip, nasolabial angle, and nasofrontal angle. however, this was not in line on the prominence of the upper lip, which this study showed that there were significant differences between genders. the two studies differed on pogonion prominence where the persians exhibited significant differences between genders.4 the results of this study were different from those of leung et al.8 on 12-year-old children in southern china that showed no significant differences between genders for the nasolabial and nasofrontal angle. according to the graber and singh method, previous research conducted by hartanto and lubis11 on chinese students at w.r. supratman 1 and 2 high schools in medan showed the students had straight facial profiles. there were no differences between genders. it can be concluded from this study that there were significant differences between male and female chinese high school students in the inferior facial third, length of the lower lip, prominence of the upper lip, nasolabial angle, and nasofrontal angle. in contrast, there was no significant difference between genders in prominence of the pogonion. however, this study had limitations in determining who was chinese without taking into subgroups. at the same time, a subgroup of chinese people spread across indonesia, such as the hokkian, hakka, kanton, tiochiu and dan hainan.13 therefore, it is necessary to do further research on the soft tissue profile of the middle and lower third of the face within each chinese subgroup. since this study used lateral photographs, further research with frontal photography for comparison is advised. references 1. de oliveira wa. quality of life, facial appearance and self-esteem in patients with orthodontic treatment. rev mex ortod. 2017; 5(3): e134–5. 2. milutinovic j, zelic k, nedeljkovic n. evaluation of facial beauty using anthropometric proportions. scientificworldjournal. 2014; 2014: 428250. 3. little ac, jones bc, debr uine lm. facia l att ractiveness: evolutionary based research. philos trans r soc lond b biol sci. 2011; 366(1571): 1638–59. 4. moshkelgosha v, fathinejad s, pakizeh z, shamsa m, golkari a. photographic facial soft tissue analysis by means of linear and angular measurements in an adolescent persian population. open dent j. 2015; 9: 346–56. 5. karad a. clinical orthodontics: current concepts, goals and mechanics. 2nd ed. mumbai: elsevier india; 2014. p. 38, 49, 424–5. 6. torlakovic l, faerøvig e. age-related changes of the soft tissue profile from the second to the fourth decades of life. angle orthod. 2011; 81(1): 50–7. 7. diouf js, ngom pi, fadiga ms, badiane a, diop-ba k, ndiaye m, diagne f. vertical photogrammetric evaluation of the soft tissue profiles of two different racial groups: a comparative study. int orthod. 2014; 12(4): 443–57. 8. leung cs, yang y, wong rw, hägg u, lo j, mcgrath c. angular photogrammetric analysis of the soft tissue profile in 12-year-old southern chinese. head face med. 2014; 10: 56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p132–136 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p132-136 136 lubis and olivia/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 132–136 9. tilaar har. multikulturalisme, bahasa indonesia, dan nasionalisme dalam sistem pendidikan nasional. j dialekt. 2014; 1(2): 213–24. 10. lisminingsih s. analisis kehidupan masyarakat tionghoa suku totok dan tionghoa peranakan pada abad 17 di batavia. khasanah ilmu j pariwisata dan budaya. 2012; 3(2): 1–12. 11. hartanto w, lubis hf. facial profile analysis by photometry on w. r. supratman 1 & 2 chinese high school students in medan. in: advances in health sciences research, volume 4; 11th international dentistry scientific meeting (idsm 2017). paris, france: atlantis press; 2018. p. 245–9. 12. wen yf, wong hm, lin r, yin g, mcgrath c. inter-ethnic/ racial facial variations: a systematic review and bayesian metaanalysis of photogrammetric studies. plos one. 2015; 10(8): e0134525. 13. ch r istia n sa. identitas budaya ora ng tionghoa i ndonesia. j cakrawala mandarin. 2017; 1(1): 11–22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p132–136 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p132-136 63 volume 47, number 2, june 2014 research report evaluasi karakteristik abu sekam padi dengan kitosan molekul tinggi nanopartikel sebagai bahan dentinogenesis (characteristic evaluation of rice husk ash with chitosan high molecule nanoparticle as dentinogenesis material) pretty farida sinta silalahi,1 trimurni abidin1 dan harry agusnar2 1departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas sumatera utara 2departemen ilmu kimia, fakultas matematika dan ilmu pengetahuan alam universitas sumatera utara medan indonesia abstract background: mineral trioxide aggregate (mta) and resin modified glass ionomer cement (rmgic) are the material used for indirect and direct pulp capping due to biocompatibility, but these materials have many shortcomings. mineral trioxide aggregate contains a little amount of arsenic and has long setting time, while hema containing rmgic are cytotoxic. rice husk ash nanoparticles (rhan) is a potential source of silica. high molecular chitosan nanoparticles (hmcn) can stimulate the formation of reparative dentin. combination of these two materials is biocompatible and have good sealing ability. purpose: this study was aimed to study rhan + hmcn used as biomaterials for prevention of pulpodentinal complex by examined at the microstructure of dentin surfaces applied with rhan + hmcn. methods: twenty-four mandibular premolar teeth extracted for orthodontic purposes, were made cavity class i preparation with 3 mm depth above the cemento-enamel junction (cej). then each tooth was cut in bucco-lingual direction and each part was cut using a cervical disc bur. samples were divided into 3 groups, group i the teeth were applied with mta; group ii the teeth were applied rmgic; group iii the teeth were apllied with rhan + hmcn. characterization was done by using scanning electron microscopy (sem) on the interface between test material and dentin adjacent to the pulp to see surface microstructure. results: material microstructure of rhan + hmcn applied to the dentine showed tags like structure which was more significant than mta. rhan + hmcn showed to have better sealing ability than mta. porosity of aspn + hmcn was less than mta and rmgic. conclusion: the study suggested that the combined rhan + hmcn biomaterials could be used as an active biomaterial that can maintain the integrity of pulp dentinal complex. key words: rice husk ash nanoparticles (rhan), high molecular chitosan nanoparticles (hmcn), scanning electron microscopy (sem) abstrak latar belakang: mineral trioksida agregat (mta) dan semen ionomer kaca modifikasi resin (sikmr) adalah bahan yang digunakan untuk pulp capping langsung dan tidak langsung karena biokompatibel, namun bahan ini memiliki banyak kekurangan. trioksida mineral agregat mengandung sejumlah kecil arsenik dan setting time-nya lama, sementara hema dalam sikmr bersifat sitotoksik. abu sekam padi nanopartikel (aspn) merupakan sumber potensial dari silika. kitosan molekul tinggi nanopartikel (kmtn) dapat merangsang pembentukan dentin reparatif. kombinasi dari kedua bahan tersebut memiliki sifat biokompatibel dan memiliki kemampuan pelapisan yang baik. tujuan: penelitian ini bertujuan untuk menganalisa karakteristik mikrostruktur hubungan permukaan abu sekam padi dengan kitosan molekul tinggi nanopartikel pada jaringan dentin untuk melindungi jaringan pulpodential kompleks. metode: dua puluh empat gigi premolar mandibula yang diekstraksi untuk tujuan ortodontik digunakan sebagai sampel, gigi dibuat preparasi kavitas klas i dengan kedalaman 3 mm di atas cemento enamel junction (cej). kemudian masing-masing gigi dibelah dua 64 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 63–66 arah bucco-lingual dan setiap bagian dipotong menggunakan disc bur servikal. sampel dibagi 3 kelompok, kelompok i diaplikasikan mta, kelompok ii diaplikasikan sikmr, kelompok iii diaplikasikan aspn + kmtn. karakterisasi dilakukan dengan menggunakan scanning electron microscopy (sem) pada interface antara bahan uji dan dentin berdekatan dengan pulp untuk melihat struktur mikro permukaan. hasil: microstructure bahan aspn + kmtn yang diaplikasi pada dentin menunjukkan struktur seperti tag yang lebih signifikan daripada mta. aspn + kmtn menunjukkan kemampuan pelapisan yang lebih baik dari mta. porositas aspn + kmtn lebih sedikit dari mta dan sikmr. simpulan: penelitian ini menunjukkan bahwa kombinasi biomaterial aspn + kmtn dapat digunakan sebagai biomaterial aktif yang dapat menjaga integritas pulpa. kata kunci: abu sekam padi nanopartikel (aspn), kitosan molekul tinggi nanopartikel (kmtn), scanning electron microscopy (sem) korespondensi (correspondence): pretty farida sinta silalahi, departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas sumatera utara. jl. alumni no. 2 kampus usu, medan 20155, indonesia. e-mail: pretty_tata2001@yahoo.com pendahuluan evolusi dari produk bahan restorasi kedokteran gigi berkembang dengan cepat menghasilkan bahan-bahan yang beragam dengan sifat fisis dan mekanis yang meningkat serta mengutamakan sifat biokompatibel untuk digunakan dalam aplikasi klinis. biokompatibilitas bahan material gigi terhadap jaringan pulpodentinal kompleks merupakan hal yang penting agar dapat digunakan dalam dunia kedokteran gigi.1 masalah yang dihadapi di bidang kedokteran gigi saat ini di indonesia adalah hampir semua bahan yang dipakai dalam perawatan gigi merupakan bahan impor dan harganya mahal. khususnya di bidang konservasi gigi dalam mempertahankan jaringan pulpa tetap vital, bahan-bahan klasik yang sampai saat ini masih banyak digunakan adalah kalsium hidroksida. namun melalui sejumlah penelitian bahan ini dinyatakan kurang mampu dalam menginduksi jebatan dentin. mineral trioxide aggregate (mta) dan semen ionomer kaca modifikasi resin (sikmr) memiliki kemampuan dalam menginduksi dentinogenesis reparatif yang lebih unggul dibanding kalsium hidroksida. mineral trioxide aggregate dan sikmr masih memiliki kekurangan yaitu harga mta yang mahal, manipulasi yang sulit, waktu pengerasan yang panjang, dan sedikit mengandung arsen,2 sedangkan sikmr menggantikan sik yang mampu melepaskan ion fluor sehingga dapat mencegah karies kambuhan3 namun sikmr ini memiliki kekurangan yaitu hidroxyethyl methacrylate (hema) yang bersifat sitotoksik.4 banyak penelitian untuk mencari bahan-bahan pengganti bahan impor dengan memakai bahan-bahan yang dapat diperoleh dari lingkungan alam. indonesia kaya dengan bahan alam, contohnya abu sekam padi (asp) dan kitosan. padi merupakan produk utama pertanian di negara-negara agraris. beberapa penelitian menunjukkan bahwa asp mengandung silika dan terbukti mempunyai sifat osteoinduktif yang mampu menyebabkan terjadinya proliferasi sel osteoblast.5 kitosan (poly-β-1,4glukosamin) merupakan biopolimer alami yang banyak di jumpai di alam setelah selulosa dan merupakan hasil ndeasetilasi dari kitin. kitosan memiliki sifat biokompatibel dan biodegradabel serta mukoadhesi yang dapat menjadi keuntungan bagi aplikasi biomedis.6 pawlowska cit. petri et al.,7 mengatakan bahwa bahan primer yang telah ditambahkan dengan kitosan dan diaplikasikan pada pulpa gigi tikus dapat menyebabkan sedikit perubahan patologis reversibel. kitosan blangkas yang diuji oleh abidin tm et al.,8 menunjukkan bahwa kitosan blangkas yang mempunyai berat molekul tinggi dapat menstimulasi dentin reparatif. hasil penelitian sutrisman et al.,9 menunjukkan bahwa terjadi peningkatan viabilitas sel yang signifikan jika 0,015% b/v kitosan nano belangkas ditambahkan sikmr dan sikmrn. tujuan penelitian ini adalah menganalisa karakteristik mikrostruktur hubungan permukaan abu sekam padi dengan kitosan molekul tinggi nanopartikel pada jaringan dentin untuk melindungi jaringan pulpodentinal kompleks. bahan dan metode penelitian ini merupakan penelitian eksperimental laboratorium menggunakan dua puluh empat gigi premolar mandibular yang diekstraksi untuk keperluan ortodontik. gigi-gigi tersebut dilakukan preparasi kavitas klas i sedalam 3 mm di atas cemento enamel junction (cej) (gambar 1a). gigi tersebut dibagi tiga kelompok yaitu: kelompok i diaplikasikan mta, kelompok ii diaplikasikan semen ionomer kaca modifikasi resin (sikmr), kelompok iii diaplikasikan abu sekam padi nanopartikel (aspn) + kitosan molekul tinggi nanopartikel (kmtn). setelah mengeras, gigi tersebut dibelah dua dari arah bukal-lingual dan dipotong pada bagian servikal menggunakan bur disk (gambar 1b). sampel gigi dilekatan pada plastisin (gambar 2). asp yang berwarna merah jambu ditimbang sebanyak 20 gr. asp dimasukkan ke dalam planetary ball mills (retsch, pm 200) sehingga partikel asp menjadi nanopartikel, sekali perlakuan planetary ball mills memerlukan waktu 2 jam dengan kecepatan 250 rpm (gambar 2). gel kitosan molekul tinggi nanopartikel (kmtn) dibuat dengan melarutkan 1 gram kitosan dalam 100 ml larutan asam lemah (asam asetat 1%) lalu diaduk pada kecepatan 200 rpm selama ± 30 menit sehingga diperoleh gel. kemudian 65silalahi, et al.: evaluasi karakteristik abu sekam padi dengan kitosan molekul tinggi nanopartikel gel kitosan ditetesi dengan larutan tripoliposphat (tpp) sebanyak 20 tetes sambil diaduk. campuran larutan kitosan dengan larutan tpp diaduk kembali menggunakan stirrer selama ± 30 menit sampai berwarna keruh. penambahan tpp dilakukan agar permukaan larutan halus. larutan yang keruh tersebut dimasukkan ke dalam ultrasonic bath untuk memecahkan partikel kitosan tersebut menjadi nano. asp yang berwarna merah jambu ditimbang sebanyak 20 gram. asp dimasukkan ke dalam planetary ball mills (retsch, pm 200), dengan kecepatan 250 rpm selama 2 jam sehingga partikel asp menjadi aspn. bubuk aspn + gel kmtn sebanyak 1% berat diaduk mengunakan spatula plastik selama ± 2 menit sehingga membentuk campuran homogen. uji karakterisasi dilakukan dengan menggunakan scanning electron microscopy (sem). sem merupakan mikroskop elektron yang mampu menghasilkan gambar beresolusi tinggi dari sebuah permukaan sampel, digunakan untuk menentukan struktur permukaan morfologi gabungan bahan biomaterial kmtn + aspn dengan dentin yang mengarah ke pulpa yang dianalisa secara kualitatif (gambar 2). hasil hasil sem pada penelitian ini terlihat adanya tag like structure pada mta dan aspn+kmtn serta adanya resin tag pada sikmr, dan terlihat adanya permukaan yang porus pada ketiga sampel (gambar 3 dan 4). (a) (b) gambar 1. (a) premolar dengan kavitas klas 1; (b) gigi dibelah bucco lingual dengan bur disk. 5 gambar 2. permukaan sempel yang di-sem. hasil hasil sem pada penelitian ini terlihat adanya tag like structure pada mta dan aspn+kmtn serta adanya resin tag pada sikmr, dan terlihat adanya permukaan yang porus pada ketiga sampel (gambar 3 dan 4). (a) (b) (c) gambar 3. gambaran sem tag like structure (a) mta; (b) aspn + kmtn; (c) resin tag sikmr (pembesaran 100x). gambar 2. permukaan sempel yang di-sem. 5 gambar 2. permukaan sempel yang di-sem. hasil hasil sem pada penelitian ini terlihat adanya tag like structure pada mta dan aspn+kmtn serta adanya resin tag pada sikmr, dan terlihat adanya permukaan yang porus pada ketiga sampel (gambar 3 dan 4). (a) (b) (c) gambar 3. gambaran sem tag like structure (a) mta; (b) aspn + kmtn; (c) resin tag sikmr (pembesaran 100x). 5 gambar 2. permukaan sempel yang di-sem. hasil hasil sem pada penelitian ini terlihat adanya tag like structure pada mta dan aspn+kmtn serta adanya resin tag pada sikmr, dan terlihat adanya permukaan yang porus pada ketiga sampel (gambar 3 dan 4). (a) (b) (c) gambar 3. gambaran sem tag like structure (a) mta; (b) aspn + kmtn; (c) resin tag sikmr (pembesaran 100x). 5 gambar 2. permukaan sempel yang di-sem. hasil hasil sem pada penelitian ini terlihat adanya tag like structure pada mta dan aspn+kmtn serta adanya resin tag pada sikmr, dan terlihat adanya permukaan yang porus pada ketiga sampel (gambar 3 dan 4). (a) (b) (c) gambar 3. gambaran sem tag like structure (a) mta; (b) aspn + kmtn; (c) resin tag sikmr (pembesaran 100x). gambar 3. gambaran sem tag like structure (a) mta; (b) aspn + kmtn; (c) resin tag sikmr (pembesaran 100x). (a) (b) (c) (a) gambar 4. gambaran sem permukaan porositas (a) mta; (b) aspn + kmtn; (c) sikmr (pembesaran 100x). 6 (a) (b) (c) gambar 4. gambaran sem permukaan porositas (a) mta; (b) aspn + kmtn; (c) sikmr (pembesaran 100x). pembahasan interaksi permukaan dentin dengan bahan kedokteran gigi sangat berpengaruh dalam prosedur restorasi gigi. idealnya bahan kedokteran gigi yang bersifat biokompatibel harus dapat berinteraksi dengan jaringan lunak dan jaringan keras gigi terutama pulpodentinal kompleks.10 hasil karakteristik interaksi permukaan dentin dengan bahan biomaterial dapat dilihat melalui scanning electron microscope (sem) dengan adanya tag like structure dan porositas. tag like structure merupakan hasil dari pelepasan ion dari bahan yang mengakibatkan adanya pertumbuhan dan nukleasi pada lapisan apatit.11 tag like structure pada mta merupakan lapisan hidroksiapatit yang menyebabkan sealing ability mta yang sangat baik.12 reyes-carmona et al.,13 melaporkan bahwa tag like structure merupakan hasil dari biomineralisasi. dalam penelitian ini terlihat aspn + kmtn menghasiilkan banyak tag like structure daripada mta. hal ini disebabkan karena ukuran partikel aspn + kmtn lebih kecil dari mta. makin kecil ukuran partikel makin besar nilai absorbansinya. dengan adanya tag like structure yang lebih banyak berarti sealing ability aspn+kmtn lebih kuat dibanding mta. resin tag sikmr disebabkan proses adhesif, pada saat sikmr berpolimerisai akan dentin aspn+kmtn sikmr dentin 6 (a) (b) (c) gambar 4. gambaran sem permukaan porositas (a) mta; (b) aspn + kmtn; (c) sikmr (pembesaran 100x). pembahasan interaksi permukaan dentin dengan bahan kedokteran gigi sangat berpengaruh dalam prosedur restorasi gigi. idealnya bahan kedokteran gigi yang bersifat biokompatibel harus dapat berinteraksi dengan jaringan lunak dan jaringan keras gigi terutama pulpodentinal kompleks.10 hasil karakteristik interaksi permukaan dentin dengan bahan biomaterial dapat dilihat melalui scanning electron microscope (sem) dengan adanya tag like structure dan porositas. tag like structure merupakan hasil dari pelepasan ion dari bahan yang mengakibatkan adanya pertumbuhan dan nukleasi pada lapisan apatit.11 tag like structure pada mta merupakan lapisan hidroksiapatit yang menyebabkan sealing ability mta yang sangat baik.12 reyes-carmona et al.,13 melaporkan bahwa tag like structure merupakan hasil dari biomineralisasi. dalam penelitian ini terlihat aspn + kmtn menghasiilkan banyak tag like structure daripada mta. hal ini disebabkan karena ukuran partikel aspn + kmtn lebih kecil dari mta. makin kecil ukuran partikel makin besar nilai absorbansinya. dengan adanya tag like structure yang lebih banyak berarti sealing ability aspn+kmtn lebih kuat dibanding mta. resin tag sikmr disebabkan proses adhesif, pada saat sikmr berpolimerisai akan dentin aspn+kmtn sikmr dentin (b) (c) 66 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 63–66 pembahasan interaksi permukaan dentin dengan bahan kedokteran gigi sangat berpengaruh dalam prosedur restorasi gigi. idealnya bahan kedokteran gigi yang bersifat biokompatibel harus dapat berinteraksi dengan jaringan lunak dan jaringan keras gigi terutama pulpodentinal kompleks.10 hasil karakteristik interaksi permukaan dentin dengan bahan biomaterial dapat dilihat melalui scanning electron microscope (sem) dengan adanya tag like structure dan porositas. tag like structure merupakan hasil dari pelepasan ion dari bahan yang mengakibatkan adanya pertumbuhan dan nukleasi pada lapisan apatit.11 tag like structure pada mta merupakan lapisan hidroksiapatit yang menyebabkan sealing ability mta yang sangat baik.12 reyes-carmona et al.,13 melaporkan bahwa tag like structure merupakan hasil dari biomineralisasi. dalam penelitian ini terlihat aspn + kmtn menghasiilkan banyak tag like structure daripada mta. hal ini disebabkan karena ukuran partikel aspn + kmtn lebih kecil dari mta. makin kecil ukuran partikel makin besar nilai absorbansinya. dengan adanya tag like structure yang lebih banyak berarti sealing ability aspn+kmtn lebih kuat dibanding mta. resin tag sikmr disebabkan proses adhesif, pada saat sikmr berpolimerisai akan membentuk ikatan yang berbentuk resin tag. resin tag merupakan ikatan mikromekanikal dengan jaringan keras dan kandungannya adalah polimerisasi resin monomer.14 pada penelitian ini sem juga dapat menunjukkan porositas pada ketiga permukaan sampel, hal ini merupakan karakteristik dari semen gigi dengan mencampur bubuk dan cairan. porositas ini disebabkan bersatunya gelembung udara mikroskopis selama pencampuran. porositas dipengaruhi oleh beberapa faktor: jumlah air yang digunakan selama pencampuran, prosedur pencampuran, tekanan yang digunakan untuk pemadatan, kelembaban, lingkungan, dan suhu. porositas dan ukuran pori yang adekuat (5-10 µm) dapat memfasilitasi pemberian nutrisi bagi sel dan difusi melalui semua struktur sel-sel untuk mencapai rekonstruksi jaringan.4 porositas pada mta disebabkan bentuk mta yang amorf dan pengendapan yang terus menerus secara internal di dalam mta sehingga menyebabkan perubahan komposisi mta yang berkontak dengan dinding dentin.15 kalsium hidroksida dari hasil setting mta mempunyai sifat alkalinitas yang menyebabkan peningkatan porositas. terdapat banyak rongga dalam bentuk gelembung udara dan pori-pori pada saat mta sudah mengeras.15 silika aspn + kmtn berbentuk amorf sama seperti mta sehingga permukaan aspn + kmtn juga berporus namun aspn + kmtn ketahanan mekaniknya lebih baik dibanding mta yang lebih rapuh. hal ini disebabkan silika asp merupakan bahan hidroksiapatit, kelemahan terbesar hidroksiapatit ialah mudah rapuh saat berkontak dengan darah atau cairan tubuh sehingga sulit digunakan untuk meregenerisasi tulang. cara untuk mengatasi permasalahan ini melalui pengkombinasian hidroksiapatit dengan matriks polimer seperti kitosan untuk meningkatkan osteokonduktivitas, biodegradabilitas dan kekuatan mekaniknya. kitosan memiliki aktivitas antimikroba, biokompatibel, dan dapat meningkatkan ketahanan korosi.13 pelapisan hidroksiapatit aspn + kmtn dilakukan untuk meningkatkan ketahanan mekanik, sehingga ketahanan aspn+kmtn ini lebih baik dibanding mta karena adanya kitosan. sedangkan porositas pada sikmr terjadi pada saat bubuk dicampurkan dengan komponen cairannya dengan adanya induksi gelembung udara.16 dapat disimpulkan bahwa kemampuan adhesi bahan aspn + kmtn disebabkan karena adanya tag like structure dan porositas. sealing ability yang baik pada bahan aspn + kmtn menunjukkan kemampuannya dalam menjaga jaringan pulpodentinal kompleks sehingga diharapkan dapat memacu dentinogenesis. penelitian ini menunjukkan bahwa kombinasi biomaterial aspn + kmtn dapat digunakan sebagai biomaterial aktif yang dapat menjaga integritas pulpa. daftar pustaka 1. ghavamnasiri m, mousavinasab m, mohtahsam m. a histopathologic study on pulp response to glass ionomer cements in human teeth. j dent 2005; 2(4): 135-41. 2. monteiro bramante c, demarchi ac, de moraes ig, bernadineli n, garcia rb, spångberg ls, duarte ma. presence of arsenic in different types of mta and white and gray portland cement. oral surg oral med oral pathol oral radiol endod 2008; 106(6): 90913. 3. mccabe jf, walls awg. applied bahan kedokteran gigi. 9th ed. blackwell. 2008. p. 258-64. 4. dahl je, ǿrstavik. response of the pulp-dentin ogan to dental restorative biomaerials. endodontic topics 2010; 17: 65-71. 5. indahyani de, hamzah z, barid i. sifat osteoinduktif silika amorphous sekam padi. dentika dental jurnal 2011; 16: 116-20. 6. modena kc, casas-apayco lc, atta mt, costa ca, hebling j, sipert cr, navarro mf, santos cf. cytotoxicity and biocompatibility of direct and indirect pulp capping. materials. j appl oral sci 2009; 17(6): 544-54. 7. petri df, donegá j, benassi am, bocangel ja.. preliminary study on chitosan modified glass ionomer restoratives. dent mater 2007; 23(8): 1004-10. 8. abidin tm, agusnar h, wandania f. efek dentinogenesis kitosan dan derivatnya terhadap inflamasi jaringan pulpa gigi reversibel. laporan akhir penelitian riset pembinaan iptek kedokteran; 2006. h. 1-20. 9. sutrisman h. efek penambahan kitosan molekul tinggi nanopartikel pada semen ionomer kaca nanopartikel terhadap viabilitas sel pulpa (in vitro). tesis. medan: fkg usu; 2012. h. 9-45. 10. ferracane jl, cooper pr, smith aj. can interaction of materials with the dentin–pulp complex contribute to dentin regeneration?. odontology 2010; 98(1): 2-14. 11. chang sw. chemical characteristics of mineral trioxide aggregate and its hydration reaction. restor dent endod 2012; 37(4): 188-93. 12. bird dc, komabayashi t, guo l, opperman la, spears r. in vitro evaluation of dentinal tubule penetration and biomineralization ability of a new root-end filling material. j endod 2012; 38(8): 1093-6. 13. reyes-carmona jf, felippe ms, felippe wt. the biomineralization ability of mineral trioxide aggregate and portland cement with dentin in a phosphate –containing fluid. j endo 2009; 35: 731-6. 14. robert tm, heymann ho, swift ej. sturdevant’s art and science of operative dentistry. 5th ed. missouri: elsevier; 2006. p. 502-4. 15. marist ai. pelapisan komposit hidroksiapatit-kitosan pada logam steinless steel 316 untuk meningkatkan ketahanaan korosi. tesis. bogor: fakultas matematika dan ilmu pengetahuan alam institut pertanian bogor; 2011. h. 3-10. 16. lohbauer u. dental glass ionomer cements as permanent filling materials properties, limitations and future trends. j materials 2010; 3: 76-96. 6767 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 67–70 differences of streptococcus mutans adhesion between artificial mouth systems: a dinamic and static methods aryan morita, h. dedy kusuma yulianto, susmira d. kusdina, and nunuk purwanti department of dental biomedical sciences faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: various materials have been used for treating dental caries. dental caries is a disease that attacks hard tissues of the teeth. the initial phase of caries is a formation of bacterial biofilm, called as dental plaque. dental restorative materials are expected for preventing secondary caries formation initiated by dental plaque. initial bacterial adhesion is assumed to be an important stage of dental plaque formation. bacteria that recognize the receptor for binding to the pellicle on tooth surface are known as initial bacterial colonies. one of the bacteria that plays a role in the early stage of dental plaque formation is streptococcus mutans (s. mutans). artificial mouth system (ams) used in bacterial biofilm research on the oral cavity provides the real condition of oral cavity and continous and intermittent supply of nutrients for bacteria. purpose: this study aimed to compare the profile of s. mutans bacterial adhesion as the primary etiologic agent for dental caries between using static method and using artificial mouth system, a dinamic method (ams). method: the study was conducted at faculty of dentistry and integrated research and testing laboratory (lppt) in universitas gadjah mada from april to august 2015. composite resin was used as the subject of this research. twelve composite resins with a diameter of 5 mm and a width of 2 mm were divided into two groups, namely group using static method and group using dynamic method. static method was performed by submerging the samples into a 100µl suspension of 1.5 x 108 cfu/ml s. mutans and 200µl bhi broth. meanwhile ams method was carried out by placing the samples at the ams tube drained with 20 drops/minute of bacterial suspension and sterile aquadest. after 72 hours, five samples from each group were calculated for their biofilm mass using 1% crystal violet and read by a spectrofotometer with a wavelength of 570 nm. meanwhile, one sample from each group was taken for its surface image using scanning electron microscope (sem). result: the results showed that s. mutans biofilm mass in the group using static method was 0.34, while in the group using ams method was 0.09. the results of the statistical analysis then showed that there was a significant difference (p=0.02) in the formation of bacterial biofilm mass between those groups. sem image in the group using static method also showed that the attachment of s. mutans was more numerous and had a longer chain than in the group using ams method. conclusion: there is a difference in the profile of s. mutans bacterial adhesion between using ams method and static method. key words: biofilm; s. mutans; artificial mouth system correspondence: aryan morita, department of biomedical dentistry, faculty of dentistry, universitas gadjah mada. jl. denta i, sekip utara, yogyakarta 55281, indonesia. e-mail: drg.armorita@gmail.com introduction secondary caries could lead to a failure in restoration if occurred between the tissue of teeth and the edge of restoration. the incidence of dental caries process begins with the formation of biofilm, called as plaque. based on data from basic health research (riset kesehatan dasar) in 2013, 93.998, 727 people in indonesia suffered from dental caries.1 streptococcus mutans (s. mutans) are bacteria playing a role in the formation of dental caries. s. mutans have an ability to produce acids that play a role in the process of tooth demineralization.2 thus, a colony of s. mutans can indicate the early formation of dental plaque. these bacteria also have an ability to co-aggregate with dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p67-70 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p67-70 68 morita, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 67–70 other bacterial species in the early colonization stage of dental plaque formation, as a result, they are able to bind to different hosts of several types of molecules.3 biofilms, moreover, are colonies of bacteria protected by a matrix against anti-bacterial agents. the process of formation of biofilm begins with an attachment (adhesion) on the surface of the objects. the adhesion of bacteria, therefore, is influenced by the surface structure of the bacteria as well as the structures of the microorganisms adhering to. extracellular polymeric substances (eps) located on the surface of the bacteria in the biofilm provide mechanical stability, mediate adhesion to the material, as well as establish cohesiveness and 3-dimensional polymer bonding on biofilm.4 in the formation of biofilm using static method, the bacterial adhesions resulted tend to be higher and also have stronger interaction between the cells.5 on the other hand, artificial mouth system (ams) method is used simulate the formation of biofilms in the oral cavity as well as to evaluate the adherence of bacteria to surfaces through the dynamic conditions created.6 this study aimed to compare the profile of s. mutans bacterial adhesion as the primary etiologic agent for dental caries between using static method and using artificial mouth (ams), a dynamic method system. materials and method this study was conducted at the faculty of dentistry and integrated research and testing laboratory (lppt) in universitas gadjah mada from april to august 2015. rk disc-shaped samples were made using molds made from pvc and plastic-coated on the inside with a diameter of 5 mm and a thickness of 2 mm. plate glasses were placed on the surface of the molds that had been filled with rk, and the irradiation was performed using the led light curing unit with a wavelength of 460 nm of more than 20 seconds to enable the polymerization reaction. the surface of rk was polished using finishing and polishing dics with different levels of roughness. next, the samples were removed from the molds using the tweezers and put in a microtube wrapped in aluminum foil and stored at 37° c. the samples then were avoided from any form of contamination on the surface of rk. ams model was made as a modification of ikeda’s and rahim’s procedures.7,8 ams model consisted of two transparent tubes made of glass with a diameter of 10 cm. the first tube was used to accommodate bhi broth as a nutrient medium for bacteria, while the second tube kept rk samples in anaerobic state and also avoided rk samples from contamination. the base of the first tube was closed with a rubber stop connected with a hypodermal needle to regulate the amount of media droplets on the second tube. the bottom surface of the second tube then was given valve to remove the rest of the media in the bottom of the tube. falcon tube that had been cut and closed its upper surface with a wire was laid on the second tube. the second tube then was placed in an incubator with a thermostat set at 37° c. container of sterile distilled water was used as a rinse of the samples connected with a hipodermal needle to set the number of droplets in the second tube. the first tube and sterile distilled water were placed on a pillar with a height of approximately 50 cm from the second tube. droplets of the first tube and sterile distilled water were centered in the middle of the sample rk7. ams scheme is showed in figure 1. in the static method, rk samples were put into a sterile tube with a polished surface facing up. bacterial suspensions were 100 ml and 200 ml of bhi broth put into a sterile tube. the samples then were incubated at 37° c for 72 hours. every 24 hours media replacement was conducted as much as 100 μl.8 meanwhile, in the ams method, the samples were placed at the bottom of both rk tubes of ams with polished surface position facing upwards. the suspension of bacteria then was inserted into the tubes using a hypodermic needle. bhi broth media was inserted into the tubes with the speed of 20 drops/ minute. samples in the ams then were put in an incubator at 37° c. 7 tabel 1. optical density values on the use of static method and ams method no. sample static method ams method 1 0.25 0.11 2 0.33 0.07 3 0.37 0.12 4 0.48 0.07 5 0.28 0.08 mean ± sd 0.34 ± 0.09 0.09 ± 0.02 figure 1. schema of ams model. note: 1) incubator with a thermostat of 37° c; 2) falcon tube covered with strimin as a place to put rk samples; 3) tubes used for maintaining anaerobic conditions and avoiding contamination from rk samples; 4) sterile distilled water placed 50cm from the second tube and set at 20 drops/minute; 5) the first tube placed 50cm from the second tube and set at 20 drops/minute; 6) pillar. 1 3 2 4 5 6 figure 1. schema of ams model. note: 1) incubator with a thermostat of 37° c; 2) falcon tube covered with strimin as a place to put rk samples; 3) tubes used for maintaining anaerobic conditions and avoiding contamination from rk samples; 4) sterile distilled water placed 50cm from the second tube and set at 20 drops/minute; 5) the first tube placed 50cm from the second tube and set at 20 drops/minute; 6) pillar. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p67-70 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p67-70 6969morita, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 67–70 the images of the restoration material surface of rk were analyzed using sem, a modification of procedures conducted by fu.9 media in microtube and ams were discarded, and the surface of rk was washed using pbs to clean up the remaining bhi broth attached to the surface. one of rk samples from each group was fixed with 4% paraformaldehid solution with a ph of 7.4 for 30 minutes. rk samples that had been fixed were inserted into alcohol 70%, 80%, 95%, and absolute alcohol to the dehydration process. rk samples then were dried with aerated, and polishing was conducted using gold to be observed with sem at 10kv. afterwards, five samples of each group were washed three times using sterile distilled water. those samples then were immersed in a solution of 1% gentian violet and incubated for 20 minutes. after 20 minutes, the solution was discarded, and the samples were washed using alcoholacetone mixture with a ratio of 80:20 v/ v for 3 times. the determination of biofilms then was performed using a spectrophotometer with a wavelength of 570nm.10 data analysis were conducted in two stages. the first stage was a qualitative analysis of the results of sem imaging, while the second stage was a quantitative analysis of bacterial biofilm mass in the form of optical density (od) using spectrophotometer. in the second stage, statistical analysis were also performed using independent sample t-test. results the results of this research on the biofilm attachment of s. mutans bacteria showed the value of od as presented in table 1.table 1 shows that the mean value of od in the group using ams method was smaller than in the group using static method. the results of these calculations then were analyzed using independent sample t-test. p value obtained was 0.02 (p<0.05) indicating that there were significant differences in od between the group using ams method and the group using static method. the results of sem image in each treatment group can be seen in figure 2. a number of the colonies on the surface of the samples using ams method were less than using static method. morphology of s. mutans bacteria in the group using static method showed a longer chain than in the group using ams method (figure 2). discussion biofilm is a collection of bacteria attaching to surfaces, and its formation occurs in response to environmental changes.11 biofilm is composed of micro-colony of bacterial cells (15-20% of volume) dispersed in a matrix or glycocalyx (75-80% of the volume).12 based on dvlo theory, total interaction between the surface and the particles is a combination of van der waals bonds and coulomb interactions. the existence of charged particles in liquid environment even will cause the formation of a double electric layer because of the withdrawal of ions on the surface of the particles. the majority of the bacteria in a liquid environment have negative particles.13 consequently, based on the results of this research, od values in the group using static method (immersion) were higher than in the group using ams. it means that the biofilm mass of s. mutans bacteria in the group using static method was heavier than in the group using ams. the existence of a nutrient in a liquid environment bhi broth provides an opportunity for the adhesion of the bacteria to the surface of objects.14 in the use of ams method, the tabel 1. optical density values on the use of static method and ams method no. sample static method ams method 1 0.25 0.11 2 0.33 0.07 3 0.37 0.12 4 0.48 0.07 5 0.28 0.08 mean ± sd 0.34 ± 0.09 0.09 ± 0.02 8 figure 2. sem images of the group using static method (a) and the group using ams method (b). a b figure 2. sem images of the group using static method (a) and the group using ams method (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p67-70 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p67-70 70 morita, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 67–70 flow of nutrients and distilled water on the surface of a material actually is capable of lowering the ionic strength so that when the bacteria are trying to identify the surface, then energy barrier will be generated causing the bacteria to move away from the surface.15 sem images, moreover, showed that there were differences in the attachment of s. mutans bacterial colonies to the surface of rk between the groups. the adhesion in the group using static method was higher than in the group using ams method. in addition to the change of ionic charge on the surface, the process of the bacterial adhesions is also affected by eps in the form of mucus layer on the surface of the bacteria.4 eps matrix consists of polysaccharides and other macromolecules, such as proteins, dna, lipids, and some other substances often found in the attachment of bacteria on the surface of objects. interaction between eps and the surface is actually caused by non-covalent bonds that have weak connective power than covalent bonds.16 as a result, dynamic environment at ams can cause bonding between eps and the surface becomes weak, and its attachments are reversible so that co-aggregation of the bacteria can be prevented. thus, the use of ams on bacterial biofilm researches can evaluate both the oral cavity microbe interactions in dental plaque that are stimulated and the same biofilm, as well as monitor the physical, chemical, biological, and molecular aspects with high accuracy.6 artificial mouth system is even capable of supplying nutrients continuously interspersed with the cleaning done by sterile distilled water as the substitute of saliva. it can be concluded that there are differences in profiles of biofilm attachment of s. mutans bacteria on the use of ams method and static method. references 1. departemen kesehatan ri. riset kesehatan dasar tahun 2013. jakarta: departemen kesehatan ri; 2013. p. 187. 2. socransky ss, hafajee ad. dental biofilm: difficult therapeutic targets. periodontol 2000, 2002; 28: 12-55. 3. kolenbrander pe. oral microbial communities: biofilm, interactions, and genetic system. annu rev microbiol 2000; 54: 413-37. 4. flemming hc, wingender j. the biofilm matrix. nat rev microbiol 2010; 8(9): 623-33. 5. razak ar, othman ry, rahim zha. the effect of piper betle and psidium guajava on the cell-surface hydrophpbicity of selected early settlers on dental plaque. j oral sci 2006; 48(2): 71-5. 6. tang g, yip hk, cutress tw, samaranayake l. artificial mouth model system and their contributions to caries research: a review. j dent 2003; 31(3): 161-71. 7. ikeda m, matin k, nikaido t, foxton rm, tagami j. effect of surface characteristics on adherence of s.mutans biofilms to indirect resin composites. dent mater j 2007; 26(6): 915-23. 8. rahim zha, fathilah ar, irwan s, hasnor wiwn. an artificial mouth system (nam model) for oral biofilm research. res j microbiol 2008; 3(6): 466-73. 9. fu d, dandan p, cui h, yinchen l, xinjin d, hualing s. effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of streptococcus mutans in vitro. j jdent 2013; 41(7): 619-27. 10. pantanella f, valenti p, frioni a, natalizi t, coltella l, berlutti f. biotimer assay, a new method for counting staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm. j microbiol methods 2008; 75(3): 478 84. 11. ionescu a, brambilla e, wastl ds, giessibl fj, cazzaniga g, schneider-feyrer s, hahnel s. influence of matrix and filler fraction on biofilm formation on the surface of experimental resin-based composites. j mater sci mater med 2015; 26(1): 5372. 12. van loosdrecht mc1, lyklema j, norde w, zehnder aj. bacterial adhesion: a physicochemical approach. microb ecol 1989; 17(1): 1-15. 13. busscher hj, van de mei hc. how do bacteria know they are on a surface and regulate their response to an adhering state. plos pathogen 2012; 8(1): 1-3. 14. hori k, matsumoto s. bacterial adhesion: from mechanism to control. biochem eng j 2010; 48: 424-34. 15. per halkjær nielsen, andreas jahn, rikke palmgren. conceptual model for production and composition of exopolimers in biofilms. water sci technol 1997; 36(1): 11-9. 16. flemming hc, wingender j. relevance of microbial extracellular polymeric substances (epss)-part ii: technical aspects. water sci technol 2001; 43: 9-16. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p67-70 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p67-70 109 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 109–114 original article the potential of toothpaste containing robusta coffee bean extract in reducing gingival inflammation and dental plaque formation peni pujiastuti, neira najatus sakinah, yuliana mahdiyah da’at arina, melok aris wahyukundari, depi praharani, desi sandra sari department of periodontology, faculty of dentistry, jember university, jember, indonesia abstract background: the prevention of gingivitis using chemicals containing antibiotics and chlorhexidine can disrupt the balance of the oral microbiota and have side effects in long-term use. a recent development in the prevention of gingivitis is the use of natural ingredients. coffee is a natural ingredient that compounds several antibacterial and anti-inflammation properties. purpose: the study aimed to determine the potential of toothpaste containing robusta coffee bean extract in reducing gingival inflammation and inhibiting the formation of dental plaque. methods: twenty male rattus norvegicus were divided into four groups, namely the control group and treatment groups (tg) tg25%, tg50%, and tg75%. all groups were fitted with ligature wire on the first left molar to accumulate dental plaque. after the fourth day, the ligature wire was removed, and the tg25%, tg50%, and tg75% groups were brushed once a day using toothpaste containing various concentrations of robusta coffee extract, while the control group was brushed without using toothpaste. plaque index, gingival index, and interleukin-1 (il-1) expression were observed on the fifth day. the data was statistically tested using a one-way analysis of variance and post hoc least significant difference. results: the statistical test showed that the tg75% group had the lowest value of plaque, gingival index, and il-1 expression, while the control group had the highest (p < 0.05). conclusion: robusta coffee bean extract toothpaste has the potential to reduce gingival inflammation and dental plaque formation in a rat with gingivitis. the most effective concentration of robusta coffee bean extract toothpaste in reducing gingival inflammation and dental plaque formation was 75%. keywords: gingivitis; herbal toothpaste; medicine; robusta coffee bean extract article history: received 10 may 2022; revised 13 september 2022; accepted 3 october 2022 correspondence: peni pujiastuti, department of periodontology, faculty of dentistry, jember university. jl. kalimantan no. 37 jember, 68121, indonesia. email: peni.pujiastuti@unej.ac.id introduction periodontal disease is an inflammatory disease of the tissue around the teeth that begins with gingival inflammation and continues to damage the structure of other tooth-supporting tissue, such as cementum, periodontal tissue, and alveolar bone.1,2 based on the 2018 national basic health research report,3 in indonesia, 73.1%–75% of the population have periodontal disease. the most common periodontal disease is gingival inflammation or gingivitis, with 13.7%–14.1% of patients experiencing bleeding gums.3 gingivitis is caused by the interaction between microorganisms found in dental plaque biofilms and tissues and inflammatory cells of the host.4 dental plaque is a biofilm that contains a lot of bacteria and is found in both hard and soft tissues. dental plaque is a common etiologic factor for gingivitis.5 dental plaque accumulation is prevented by controlling plaque mechanically, namely by brushing teeth with toothpaste. currently, the use of toothpaste in the community has become a daily necessity because using toothpaste regularly can maintain dental and oral health.6 a new development in the prevention of gingivitis is the use of natural ingredients. so far, the prevention of gingivitis has been with chemicals classified as antibiotics and chlorhexidine, which can disrupt the balance of the oral microbiota and have side effects in long-term administration.7 one natural ingredient that has the potential to reduce gingivitis is coffee. many studies have stated that coffee contains active ingredients, copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 mailto:peni.pujiastuti@unej.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 110pujiastuti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 109–114 such as chlorogenic acid, flavonoids, caffeine, phenolic compounds, and trigonelline, which have antibacterial and anti-inflammatory properties.8–10 robusta coffee contains several compounds that have antibacterial properties. the antibacterial content found in robusta coffee beans includes caffeine, phenol, trigonelline, and chlorogenic acid.11 the compounds that have the most antibacterial activity in robusta coffee are flavonoids.12 the flavonoids have biological activity; they interact with bacterial cells through an adsorption process involving hydrogen bonds and then damage the cytoplasmic membrane, resulting in leakage of the bacterial cell nucleus.13,14 flavonoid compounds destroy bacterial cells through differences in polarity between the lipids that make up bacterial cells and the alcohol groups in flavonoids.15 the active ingredients of coffee can reduce inflammation through the mechanism of inhibiting nuclear factor kappa b (nf-κb) activation, thereby inhibiting the synthesis of interleukin-1 (il-1) and tumor necrosis factor alpha (tnf-α) and inhibiting vasodilation of blood vessels and capillary permeability. the adhesion of neutrophils to the blood vessel walls is inhibited and causes the infiltration of neutrophils into the tissue to decrease so that inflammation decreases.8,12,13 flavonoid compounds in robusta coffee beans might be used as an additive for plaque control agents, such as toothpaste. a previous study explained that green coffee bean extract showed a significant reduction in the streptococcus mutans colony count before and after it was used.16 therefore, green coffee bean extract as a mouthwash can be a safe and effective alternative for decreasing bacterial plaque.16 to prove the effectiveness of toothpaste containing robusta coffee bean extract as a plaque control agent, we used it in various concentrations as an antibacterial and anti-inflammation agent in gingivitis rats. we observed the anti-bacterial and anti-inflammatory potential of robusta coffee bean extract toothpaste using the plaque index, gingival index, and il-1 expression. materials and methods this research is a laboratory experimental study with a post-test-only control group design, and it has passed the ethical clearance issued by the dentistry research ethics commission of jember university with no. 1281/un25.8/ kepk/dl/2021. the sample of this research comprised 20 male wistar white rats (rattus norvegicus) aged around 12 to 14 weeks with weights of 200–250g that were divided into four groups: a control group and treatment groups (tg) tg25%, tg50%, and tg75%. all groups were fitted with ligature wire on the first left molar for three days to accumulate dental plaque and induce gingivitis. all rats were also monitored for their food and drink intake so that they consumed the same quantities.17 robusta coffee extract toothpaste was made using robusta coffee bean extract ingredients with concentrations of 25%, 50%, and 75% mixed with a placebo. the placebo paste consisted of magnesium carbonate, calcium carbonate, glycerin, propylene glycol, triethanolamine, sterile distilled water, and oleum menthae piperithae.18 on the fourth day, the ligature wire on the first left molar of all groups was removed, and the tooth was brushed once at 9 a.m. on the fourth and fifth days using an interdental brush with a roll technique. the tg25%, tg50%, and tg75% groups were brushed using toothpaste containing 25%, 50%, and 75% robusta coffee extract, respectively, while the control group was brushed without using toothpaste.18 the plaque index and gingival index were observed on the fifth day.18 the plaque was measured using the personal hygiene performance index, and the gingival inflammation was measured using the gingival index from loe & sillness.19 after the plaque index and gingival index were obtained, all samples were decapitated on the fifth day, and the left gingival tissue was taken. gingival tissue was put into a 10% formalin buffer solution for at least eight hours before decalcification so that the tissue to be observed was not damaged.18 samples were decalcified using an ethylene diamine tetra acetic acid solution. after the decalcification process, examination continued of the expression of il-1 using immunohistochemistry (ihc). the il-1 smear results were read by two pathologists using the allred method. the research data obtained was tested for normality using the shapiro–wilk test and homogeneity using the levene test. furthermore, a differences test was performed using a one-way analysis of variance (anova) and a post hoc least significant difference (lsd) test with a significance level of 95% (p = 0.05) to see which concentration of robusta coffee bean extract toothpaste was most effective in reducing inflammation and inhibiting dental plaque formation. results all groups were fitted with ligature wire on the first left molar, as shown in figure 1a, to accumulate dental plaque. after three days of placement, the ligature wire of all groups was removed. the gingiva of the ligature area showed redness compared to another gingival area without ligature wire. the redness of the gingival indicates inflamed gingiva, as shown in figure 1b. the plaque index and gingival index were observed on the fifth day. the results of plaque measurements showed a decrease in plaque value in each study group, along with a large content of robusta coffee bean extract in toothpaste. the mean and standard deviation (sd) of plaque values from each group can be seen in table 1. the values in table 1 show a decrease in average plaque value in the control group, tg25%, tg50%, and tg75%. the tg75% group had the smallest average plaque value of 0.8 ± 0.84, while the control group had the largest plaque copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 111 pujiastuti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 109–114 value of 2.2 ± 0.84. the plaque value data was analyzed using statistical tests, which showed the data was normal and homogenous. the results of the one-way anova test showed a significant difference between groups (p < 0.05), as seen in table 2. the gingival index was also measured for the presence of inflammation in the rat gingiva. the results in table 3 show a decrease in the value of the gingival index in the control group, tg25%, tg50%, and tg75%. the lowest gingival index value in the tg75% group was 0.75 ± 0.04, including the criteria for mild inflammation, while the highest gingival index value in the control group was 2.02 ± 0.23, including the criteria for severe inflammation. the data of the gingival index was analyzed using statistical tests, which showed the data was normal and homogenous. the results of the one-way anova test showed a significant difference between groups (p < 0.05), as shown in table 4. the image of the results of the il-1 examination using ihc can be seen in figure 2. from the results of the calculation of il-1 expression, it was found that the lowest average of il-1 expression was in the tg75% group, and the highest average of il-1 expression was in tg25%. the results of the examination of il-1 expression can be seen in table 5. the data on il-1 expression were analyzed using statistical tests. the statistical test showed the data was normal and homogenous. the results of the one-way anova test showed a significant difference between groups (p < 0.05), as shown in table 6. the statistical test was then continued using the lsd post hoc test to see the differences between each group. the results of the lsd post hoc test in the four groups can be seen in figure 3. the results of the lsd post hoc test of il-1 expression showed the differences between each group. the tg75% group was significantly lower than the control group (p < 0.05). however, the tg25% and tg50% groups did not significantly differ from the control group. the tg75% group is also significantly lower than the tg25% and tg50% groups. b a figure 1. (a) ligature wire placement on the first left molar (red arrows). (b) the ligature wire was removed after three days of placement, and the gingiva in the ligature area was red (blue arrows). a b c d figure 2. immunohistochemistry examination results. (a) control group, (b) tg25% group, (c) tg50% group, (d) tg75% group. il-1 expression is shown with red arrows. 400x magnification. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 112pujiastuti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 109–114 0 20 40 60 80 100 120 control tg25% tg50% tg75% ng /m l goups il-1 expression * figure 3. the results of the lsd post hoc test of il-1 expression. the tg75% group was the only group that was significantly lower than the control group. (*) significant p < 0.05. table 1. the mean and sd of plaque values on the first left molar tooth groups n mean ± sd (mm) control 5 2.2 ± 0.84 tg25% 5 2 ± 0.71 tg50% 5 1.2 ± 0.84 tg75% 5 0.8 ± 0.84 table 2. the results of statistical tests of plaque value groups normality (shapiro– wilk test) homogeneity (levene test) difference test (one-way anova) control 0.314** 0.801** 0.045* tg25% 0.325** tg50% 0.314** tg75% 0.314** (*) significant p < 0.05; (**) significant p > 0.05 table 3. the mean and sd of the gingival index on the first left molar tooth groups n mean ± sd criteria control 5 2.02 ± 0.23 severe tg25% 5 1.75 ± 0.07 moderate tg50% 5 1.5 ± 0.32 moderate tg75% 5 0.75 ± 0.04 mild table 4. the results of statistical tests of the gingival index groups normality (shapiro– wilk test) homogeneity (levene test) difference test (one-way anova) control 0.368** 0.056** 0* tg25% 0.623** tg50% 0.484** tg75% 0.325** table 5. the mean and sd of il-1 expression groups n mean ± sd control 5 63.6 ± 9.02 tg25% 5 76.2 ± 22.26 tg50% 5 60 ± 11.64 tg75% 5 36.6 ± 8.74 table 6. the results of statistical tests of il-1 expression groups normality (shapiro– wilk test) homogeneity (levene test) difference test (one-way anova) control 0.979** 0.061** 0.003* tg25% 0.894** tg50% 0.942** tg75% 0.999** (*) significant p < 0.05; (**) significant p > 0.05 discussion in this study, the first left molar was ligated using a wire so that plaque accumulation occurred. ligation aims to cause plaque accumulation that will induce gingivitis. after five days of ligation, the gingival margin was clinically reddish. this was following the second stage of gingivitis. in the second stage of gingivitis (early lesion), we found red gingiva appearing and bleeding on probing that occurred 4–7 days after plaque accumulation.20 in table 1, the results of the measurement of plaque values in the tg25%, tg50%, and tg75% groups decreased, along with the large concentration of robusta coffee bean extract in toothpaste. the results of this study are in line with prasasti et al.,18 which explains that the 75% concentration of robusta coffee bean extract found in toothpaste is effective in inhibiting the formation of dental plaque. the higher the concentration of robusta coffee bean extract, the more active chemical compounds (*) significant p < 0.05; (**) significant p > 0.05 are contained therein. the decrease in plaque value may be copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 113 pujiastuti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 109–114 due to several ingredients in robusta coffee beans, namely caffeine, flavonoids, trigonelline, and chlorogenic acid, which have antibacterial activity. each component has a different antibacterial mechanism.14,21 caffeine is one of the most important alkaloid compounds found in coffee beans. the caffeine content in robusta coffee beans is between 1.5%–2.25%.22 the ability of alkaloid compounds to be antibacterial is influenced by the active compounds of alkaloids, which are basic groups containing nitrogen. when this base group is in contact with bacteria, it will react with amino acid compounds that make up the bacterial cell wall and bacterial dna, which is the main constituent of the cell nucleus. with dna damage, bacteria will become inactive and lyse.8,23 phenolic compounds are flavonoids found in coffee beans. the flavonoid content in robusta coffee ranges from 7.3–7.5 mg/g. flavonoids damage the bacterial cell wall due to the difference in polarity between the lipids that make up bacterial dna and the alcohol groups in flavonoid compounds; these compounds can enter the bacterial cell nucleus.24 robusta coffee contains 1.18% trigonelline. trigonelline works by disrupting the stability of the bacterial cytoplasmic membrane. disturbances in the membrane will cause an imbalance in the metabolic function of bacteria, which causes bacterial growth to be inhibited.25 chlorogenic acid is the most abundant component in coffee that can neutralize free radicals in the body by maintaining normal cell structure and function. chlorogenic acid works by entering the nucleus of bacterial cells and destroying the structure of the cell wall.26 in table 3, the results of the measurement of the gingival index value in the tg25%, tg50%, and tg75% groups have a decreasing gingival index value. in the tg75% group, the lowest gingival index value is 0.75, including the category of mild inflammation. mild inflammation in the tg75% group was probably caused by the content of robusta coffee bean extract, namely flavonoids. the flavonoid content in coffee also has an anti-inflammatory effect by binding to proteins and reducing hydrophobicity in host cell membranes and bacteria.25 flavonoids can inhibit inflammation in two ways: by inhibiting arachidonic acid and the secretion of lysosomal and endothelial enzymes so that proliferation and exudation of the inflammatory process occur. the inhibition of the release of arachidonic acid from inflammatory cells leads to less availability of arachidonic substrates for the cyclooxygenase pathway and the lipoxygenase pathway.12,13,26 the results of the one-way anova test showed a significant difference in il-1 expression between groups. the statistical test was then continued using the lsd post hoc test to see the differences between each group. the lsd post hoc test result showed that the tg75% group was significantly lower than the control group (p < 0.05). however, the il-1 expression of the tg25% and tg50% groups did not significantly differ from the control group. these results indicate that toothpaste containing 75% of robusta coffee bean extract can reduce inflammation more than the other groups. pro-inflammatory il-1 is associated with gingival inflammation. according to gao et al.,27 flavonoids have the potential to inhibit the cyclooxygenase enzyme so that the formation of prostaglandins is inhibited. inhibition of prostaglandin formation can reduce inflammation. with reduced inflammation, proinflammatory cytokines, such as il-1α, il-1β, il-6, and il-8, are also reduced.27 the results of our study are also consistent with martin et al.,28 who show that the substances contained in robusta coffee bean extract play a role in inhibiting the production of tnf-α, il-1β, il-6, and cox-2 through inhibition of the nf-κb pathway. further research also explains that robusta coffee bean extract enhances osteocalcin and alkaline phosphatase expression that leads to bone regeneration in periodontal rat models.28 in conclusion, robusta coffee bean extractcontaining toothpaste has the potential to reduce gingival inflammation and dental plaque formation in a gingivitis rat. the most effective concentration of robusta coffee bean extract toothpaste in reducing gingival inflammation and dental plaque formation was 75%. acknowledgment this research was funded from the research grant of research group (keris) at the jember university in 2021. we declare that no conflicts of interest took place before, during or after this study. references 1. nugraha ap, sibero mt, nugraha ap, puspitaningrum ms, rizqianti y, rahmadhani d, kharisma vd, ramadhani nf, ridwan rd, noor tne binti ta, ernawati ds. anti-periodontopathogenic ability of mangrove leaves (aegiceras corniculatum) ethanol extract: in silico and in vitro study. eur j dent. 2022. 2. bramantoro t, zulfiana aa, amir ms, irmalia wr, mohd nor na, nugraha ap, krismariono a. the contradictory effects of coffee intake on periodontal health: a systematic review. f1000research. 2022; 11: 924. 3. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 674. 4. wulandari p, widkaja d, nasution ah, syahputra a, gabrina g. association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients. dent j. 2022; 55(1): 16–20. 5. krismariono a, setiawatie em, rachmawati ry, setiawan ya, padmarini hn, apriliyanti na. antibacterial activity of water hyacinth (eichhornia crassipes) leaf extract against bacterial plaque from gingivitis patients. j int dent med res. 2022; 15(3): 966–71. 6. budi hs, pebriani i. the protein level and molecular weight analysis in different children’s toothpaste, which probably induced hypersensitivity. contemp clin dent. 2020; 11(3): 245–8. 7. tartaglia gm, tadakamadla sk, connelly st, sforza c, martín c. adverse events associated with home use of mouthrinses: a systematic review. ther adv drug saf. 2019; 10: 204209861985488. 8. nugraha ap, ardani igaw, sitalaksmi rm, ramadhani nf, rach maya nti d, kuma la d, k ha r isma v d, ra h mada n i d, puspitaningrum ms, rizqianti y, ari mda, nugraha ap, noor copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 114pujiastuti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 109–114 tne binti ta, luthfi m. anti–peri-implantitis bacteria’s ability of robusta green coffee bean (coffea canephora) ethanol extract: an in silico and in vitro study. eur j dent. 2022. 9. naveed m, hejazi v, abbas m, kamboh aa, khan gj, shumzaid m, ahmad f, babazadeh d, fangfang x, modarresi-ghazani f, wenhua l, xiaohui z. chlorogenic acid (cga): a pharmacological review and call for further research. biomed pharmacother. 2018; 97: 67–74. 10. sari ds, sakinah n, nuri n, suswati e, widyowati r, maduratna e. chlorogenic acid fractionation in robusta green bean extract as a combination agent of dental pulp stem cells in periodontal tissue engineering. res j pharm technol. 2022; 15(11): 5005–10. 11. hakima an, ermawati t, harmono h. daya hambat ekstrak biji kopi robusta (coffea robusta) terhadap pertumbuhan fusobacterium nucleatum. stomatognaic j kedokt gigi. 2020; 17(1): 20–4. 12. jeszka-skowron m, sentkowska a, pyrzyńska k, de peña mp. chlorogenic acids, caffeine content and antioxidant properties of green coffee extracts: influence of green coffee bean preparation. eur food res technol. 2016; 242(8): 1403–9. 13. nijveldt rj, van nood e, van hoorn de, boelens pg, van norren k, van leeuwen pa. flavonoids: a review of probable mechanisms of action and potential applications. am j clin nutr. 2001; 74(4): 418–25. 14. putri s, ardhiyanto hb, permana ad. potensi kopi robusta sebagai antibakteri dan antijamur pada penyakit rongga mulut. in: prosiding the 5th dentistry scientific meeting of jember. jember: universitas jember; 2018. p. 22–31. 15. widyasari pam, aman igm, mahendra an. aktivitas antibakteri ekstrak etanol biji kopi robusta (coffea canephora) terhadap bakteri staphylococcus epidermidis atcc 12228 penyebab infeksi nosokomial. e-journal med udayana. 2021; 10(6): 74–8. 16. yadav m. effect of green coffee bean extract on streptococcus mutans count: a randomised control trial. j clin diagnostic res. 2017; 11(5): zc68–71. 17. tomina dc, petruțiu ștefan a, dinu cm, crișan b, cighi vs, rațiu ia. comparative testing of two ligature-induced periodontitis models in rats: a clinical, histological and biochemical study. biology (basel). 2022; 11(5): 634. 18. prasasti rn, sari ds, pujiastuti p. the effectiveness of using toothpaste containing robusta coffee bean extract in inhibiting the formation of dental plaque. odonto dent j. 2022; 9(1): 12–20. 19. schwartz sb, christensen jr, fields h. examination, diagnosis, and treatment planning. in: pediatric dentistry. 6th ed. elsevier; 2019. p. 419-454.e4. 20. dommisch h, kebschull m. chronic periodontitis. 12th ed. newman m, takei h, klokkevold p, carranza f, editors. carranza’s clinical periodontology expert consult. elsevier; 2014. p. 309-319.e2. 21. ranasatri aa, mahmudah n, aisyah r, sintowati r. aktivitas antibakteri ekstrak etanol 70% biji kopi robusta (coffea canephora) terhadap staphylococcus epidermidis dan salmonella typhi. biomedika. 2021; 13(2): 101–10. 22. farhaty n, muchtaridi. tinjauan kimia dan aspek farmakologi senyawa asam klorogenat pada biji kopi: review. farmaka. 2016; 14(1): 214–47. 23. hwang j-h, kim k-j, ryu s-j, lee b-y. caffeine prevents lpsinduced inflammatory responses in raw264.7 cells and zebrafish. chem biol interact. 2016; 248: 1–7. 24. surjowardojo p, susilorini t, panjaitan a. daya hambat jus kulit apel manalagi (malus sylvestris mill.) terhadap pertumbuhan bakteri staphylococcus aureus dan escherichia coli penyebab mastitis pada sapi perah. ternak trop. 2015; 16(2): 30–9. 25. maheswari ra, krismariono a, bargowo l. daya hambat ekstrak biji kopi robusta (coffea canephora) terhadap pertumbuhan bakteri plak. periodontic j. 2015; 7(2): 16–20. 26. panche an, diwan ad, chandra sr. flavonoids: an overview. j nutr sci. 2016; 5: e47. 27. gao r, yang h, jing s, liu b, wei m, he p, zhang n. protective ef fe ct of ch lorogen ic acid on l ip op olysaccha r ide -i nduce d inflammatory response in dairy mammary epithelial cells. microb pathog. 2018; 124: 178–82. 28. martin m, sari d, mantika r, praharani d. combination of dental pulp stem-cell secretome and robusta coffee bean extract (coffea canephora) in enhancing osteocalcin and alkaline phosphatase expression in periodontitis-induced wistar rats. j orofac sci. 2021; 13(2): 136–41. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p109–114 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p109-114 205205 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 205–209 original article comparison of the occlusal feature index (ofi) and dental aesthetic index (dai) in 10–14-year-old children at the universitas sumatera utara dental hospital hilda fitria lubis and arfah azriana department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: malocclusion often occurs in children due to discrepancies between primary teeth and permanent teeth. an assessment of the severity of the malocclusion is necessary for establishing the diagnosis and determining the need for treatment. the occlusal feature index (ofi) and dental aesthetic index (dai) are indices that assess the need for orthodontic treatment, but they use different weights. purpose: this study aimed to compare the need for orthodontic treatment based on the ofi and dai in 10–14-year-old children. methods: the sample in this study is secondary data in the form of 66 study models pre-treatment in children aged 10–14 years at the universitas sumatra utara (usu) dental hospital. all samples were collected based on inclusion and exclusion criteria. the measurement results were analysed statistically by the chi-square test to see the comparison between the ofi and dai. the results obtained are presented in the form of frequency and percentage. results: for the ofi, 42.4% of the samples had no orthodontic treatment needed, 31.8% were indicated to treat, and 25.8% require mandatory treatment. for the dai, 47% of samples had no/little treatment need, 25.8% had elective treatment need, 16.7% had treatment considered mandatory, and 10.6% treatment highly desirable. based on the assessment to compare the ofi and dai using the chi-square test, p=0.001 was obtained. conclusion: there was a significant difference in the need for orthodontic treatment between ofi and dai in children aged 10–14 years at the usu dental hospital. keywords: children; dai; malocclusion; ofi; orthodontic indices correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: hilda.fitria@usu.ac.id; hildadrgusu@gmail.com introduction malocclusion means dental peculiarities and occlusal properties that represent deviations from ideal occlusion.1 malocclusion will cause concerns related to dental health and quality of life due to oral health resulting from the appearance and function of teeth.1,2 in adolescence, physical appearance is considered a very important factor for physical attraction when socialising.3 therefore, children with malocclusion will experience ridicule and ostracism as well as lower self-esteem and an affected social life.4 generally, the rate of malocclusion in adolescence is high. research by adha et al.,5 found 97.9% of 8–12-year-old students with malocclusion in banjarmasin’s primary schools. research in cimahi by dayataka et al.,6 also described a high prevalence of malocclusion, amounting to 96.7% of 12–15-year-old children. orthodontic treatment is needed as an action to treat malocclusion, to correct the abnormal arrangement of teeth and jaws, as obtaining good dental function and dental aesthetics as well as a pleasant face will improve one’s psychosocial health.7 for ensuring appropriate orthodontic treatment, a uniform standard is needed to assess the severity of malocclusion to minimise subjectivity, which is known as the malocclusion index.8 malocclusion recording methods can be classified into qualitative and quantitative methods. the qualitative method describes the occlusal features and provides a descriptive classification of the teeth, but does not provide any data about the need and result of treatment. meanwhile, quantitative methods measure the complexity and severity of the problem assessed on a scale dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p205–209 mailto:hilda.fitria@usu.ac.id mailto:hildadrgusu@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p205-209 206 lubis and azriana/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 205–209 or as a proportion. this method focuses on requirement for care. its utilisation minimises the subjectivity related to the diagnosis, result, and assessment of the complexity of orthodontic treatment.9 the dental aesthetic index (dai) assesses the aesthetic component as well as the clinical component in a single score that combines the physical and aesthetic aspects of occlusion, in contrast to other indices that require separate assessments. compared to different indices, the dai is more popular, easier to use, and efficient.10 the occlusal feature index (ofi) is also an index that is easy to use, does not require complicated diagnostic equipment, and is objective. this method has proven that the severity of malocclusion indicated by an orthodontist subjectively and an assessment by a public health expert were very close or almost the same.11 both the ofi and dai can determine the severity of malocclusion and allocate orthodontic treatment needs, but the ofi is a very simple index that uses only 4 components to assess orthodontic treatment needs compared to the dai with 10 components.9 therefore, this study aimed to compare the need for orthodontic treatment based on the ofi and dai in 10–14-year-old children at the universitas sumatra utara (usu) dental hospital. materials and methods this descriptive cross-sectional study was carried out at usu dental hospital, medan. the sample size for this study was determined by the formula for the sample size of the hypothesis test for the proportion of a single population. based on the calculation results, the minimum sample for research was 60 samples, plus 10% to consider the exclusion problem, so the total sample required was 66 samples. the samples were pre-treatment study models of children, collected using a purposive sampling method based on the inclusion and exclusion criteria. the inclusion criteria were study models of children aged 10–14 years, could be measured using the ofi and dai indices, were in good condition (not broken, cracked, or porous), had good occlusion/bite, and had complete permanent teeth up to the first molar on the upper and lower jaws. the exclusion criteria were study models of patients already/ currently undergoing orthodontic treatment, and those with craniofacial anomalies of cleft lip and palate. this study had permission from the research ethics committee of universitas sumatra utara (number 532/ kep/usu/2021). after collecting the study models, measurements and scores were carried out on the study models using the ofi and dai as shown in tables 1 and 2. the scores obtained from the ofi and dai measurements on each component were included in the orthodontic treatment need category group. the treatment needs based on the ofi were classified into no needed treatment (0–3), indicated treatment (4–5), and mandatory treatment (6–9). for the dai, the total scores were categorised with no/little treatment need (≤25), treatment considered elective (26–30), treatment highly table 1. ofi components and assessment weights11 ofi components ofi score0 1 2 3 crowding anterior neat teeth the crowding is equal to half the width of the lower right first incisor the crowding is equal to the width of the lower right first incisor the crowding is bigger than the lower right first incisor interdigitation abnormalities the relationship between cusp and groove the relationship occurs between cusp and groove the relationship between cusp and cusp overbite 1/3 of the incisal part of the lower incisor is covered by the upper incisor during occlusion 2/3 of the incisal part of the lower incisor is covered by the upper incisor during occlusion 1/3 of the gingival part of the lower incisor is covered by the upper incisor during occlusion overjet 0–1.5 mm 1.5–3 mm 3 mm or more total ofi score table 2. dai components and assessment weights12 dai components dai score the number of tooth loss (incisors, canines, and premolar in maxillary and mandibular arches) 6 crowding in the incisors region (0 = no crowding; 1 = only one region with crowding; 2 = both regions with crowding) 1 spacing in the incisors region (0 = no spacing; 1 = one region with space; 2 = two region with space) 1 midline diastema (mm) 3 anterior maxillary misalignment (mm) 1 anterior mandibular misalignment (mm) 1 anterior maxillary overjet (mm) 2 anterior mandibular overjet (mm) 4 vertical anterior open bite (mm) 4 anteroposterior molar relationship (0 = normal; 1 = half cusp; 2 = one cusp; evaluated the right and left sides and only the largest deviation from normal molar relationship was recorded) 3 constant 13 total dai score dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p205–209 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p205-209 207lubis and azriana/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 205–209 desirable (31–35), and treatment considered mandatory (>36).11,12 the results of the ofi and dai measurements for the need for orthodontic treatment were compared statistically using software version 21.0 of the ibm statistical package for social science (spss) (chicago, us) with the chi-square test (p value<0.05) and presented in terms of frequency and percentage. the significance test of ofi and dai measurements was carried out to obtain valid data by performing 2 measurements on each index, where measurement 1 and measurement 2 were carried out at different times by the same researcher. the average of each group was calculated and then statistically analysed by the kappa test. the kappa test results showed no significant difference from the p value <0.05. that is, the results of the first and second examinations by the same researcher are valid and similar. results the categories for orthodontic treatment needs based on ofi and dai are shown in tables 3 and 4. according to the ofi, out of 66 examined subjects, 42.4% showed no need for treatment (ofi: 0–3), 31.8% were indicated to treat (ofi: 4–5), and 25.8% showed mandatory treatment requirement (ofi: 6–9). according to the dai, 47% of subjects showed no/little need for treatment (dai <25), 25.8% had elective treatment needs (dai: 26–30), 16.7% with treatment considered mandatory (dai: 31–35), and 10.6% with treatment highly desirable (dai >36). the comparison of orthodontic treatment needs based on the ofi and dai is shown in table 5. the chi-square test was used to assess the comparison of orthodontic treatment needs based on the ofi and dai, the result showed that p=0.001; p<0.05. there was a statistically significant difference in the need for orthodontic treatment between the two indices. regarding orthodontic treatment needs, the ofi classified 28 samples as no needed treatment, while the dai classified 25 samples as having no/little need for treatment and three samples with orthodontic treatment considered elective. according to the ofi, 21 of the samples had treatment indicated, while according to the dai, 6 of those samples had no/little treatment need, 7 samples elective treatment, 3 samples highly desirable treatment, and 5 samples with treatment considered mandatory. the results of the ofi also found that 17 samples needed mandatory treatment, but 7 of them were elective treatment needs according to the dai, 4 samples with highly desirable treatment, and 6 samples where treatment was considered mandatory. discussion malocclusion indices are a method to determine the level of treatment need or the number of deviations from normal occlusion and can be used for individual and population evaluation.13 the ofi and dai are indices to allocate patients into categories of treatment needs but with different assessment weights.9 this study conformed with research by nahusona and aprilia14 regarding the malocclusion status of dental students of hasanuddin university measured according to the ofi using 144 samples. the results of the study with the highest percentage was no treatment need at 75%, followed by treatment indicated at 21.5%, and need to treat at 3.5%.14 the results of this study also conformed with the research of simangunsong et al.,15 regarding the description of table 3. the need for orthodontic treatment based on ofi in children aged 10–14 years at the usu dental hospital ofi score treatment need n % 0–3 no need 28 42.4 4–5 indicated 21 31.8 6–9 mandatory 17 25.8 total 66 100.0 table 4. the need for orthodontic treatment based on dai in children aged 10–14 years at the usu dental hospital dai score treatment need n % ≤25 no/little need 31 47.0 26–30 elective 17 25.8 31–35 highly desirable 7 10.6 ≥36 mandatory 11 16.7 total 66 100.0 table 5. comparison of orthodontic treatment needs based on ofi and dai in children aged 10–14 years at the usu dental hospital treatment need dai p valueno/little need elective highly desirable mandatory total ofi n % n % n % n % n % no need 25 89.3 3 10.7 0 0.0 0 0.0 28 100.0 0.001 indicated 6 28.6 7 33.3 3 14.3 5 23.8 21 100.0 mandatory 0 0.0 7 41.2 4 23.5 6 35.3 17 100.0 total 31 47.0 17 25.8 7 10.6 11 16.7 66 100.0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p205–209 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p205-209 208 lubis and azriana/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 205–209 malocclusion in students at sma santo thomas 2 medan based on the dai with 50 samples. it showed the highest percentage of no/little treatment needed at 62.8%, followed by needing elective treatment at 27.4%, mandatory treatment at 7.8%, and treatment highly desirable at 2.0%.15 the number of variations in the need for orthodontic treatment is in line with the variation in the severity of the malocclusion that occurs. the occurrence of malocclusion is greatly influenced by inheritance from parents and environmental factors such as bad habits. these two factors usually manifest themselves as an imbalance in the growth and development of the dental-facial structure. the effects of these factors can directly or indirectly cause malocclusion. genetic factors have a significant impact on malocclusion, such as size, shape, and the number of teeth that are out of alignment with the mandibular arch and cause congestion.16 environmental factors such as bad habits that can cause malocclusion include thumb-sucking, sticking out the tongue, sucking or biting lips and nails, breathing from the mouth, and bruxism. habits that produce intermittent stresses or forces exceeding 4–6 hours/day on the teeth can result in permanent deformities.10,17 in this study, there was a significant difference in the comparison of the orthodontic treatment needs between the ofi and dai. comparison of several orthodontic indices has been done, but none has compared the ofi with the dai. with the exception of one of the studies on the comparison of two indices that is in line with this research, regarding the comparison of the dai and index of orthodontic treatment needs (iotn-dhc) in determining the orthodontic treatment needs of qazvin students by padisar et al.,10 indicating that there is a statistically significant difference between the dai and the dental health component (dhc). in this study, the ofi identified a greater proportion of samples in need of treatment compared with the dai. the score assessment between ofi and dai is not equal, it can be seen from the assessment of the molar relationship which shows that the ofi assesses the relationship of the upper and lower permanent first molars to be half bulge both in the mesial and distal directions higher with a score of 2 compared to the dai with a score of 1. as for the assessment for crowding anteriorly, the dai assesses the presence or absence of crowding in one jaw with a score of 1 or both jaws with a score of 2, while the ofi assessment looks at crowding only in the lower anterior by measuring the width of the position of the teeth that are crowded against the right lower first incisor. the assessment on overjet also shows that the dai can only score if the overjet is more than normal (>2 mm), while the ofi can assess overjet >1.5 mm so measurements using ofi can be higher than dai.11,12 the large number and submissions of malocclusion indices by expert researchers show the difficulty of designing a weighted, practical, valid, and reliable method to assess malocclusion with a uniform method.18 a malocclusion index must be able to identify people who do not need treatment (specificity) and those who need treatment (sensitivity).19 malocclusion indices such as the ofi and dai can be used to determine the need or priority of orthodontic treatment in epidemiological surveys.13 each malocclusion index has its own advantages and disadvantages. the ofi is a simple and objective method and does not require complicated diagnostic equipment. assessment with this method can also be done in a short time if the researcher has been trained. however, the disadvantage of the ofi is that this method only assesses the interdigitation of the cusp which examines the relationship of the right upper and lower posterior teeth. this method also requires prior training in the assessment of front lower crowding because it takes time to measure the mesiodistal width of the lower anterior teeth and measure the length of the lower front dental arch.11 the dai is internationally established by the who, which identifies occlusal properties and includes the physical and aesthetic aspects of occlusion, including patient perception. the advantage of the dai is that patients can get satisfaction from aesthetic and functional improvements because the dai considers the patient’s perception and is an effective method for prospective use in identifying the need for orthodontic treatment quantitatively and can be carried out directly in the patient’s mouth. as for the possible limitations of using the dai, this method does not identify cases with deep bite, buccal crossbite, open bite, and midline. measurements made with a millimetre gauge can cause small errors due to inaccuracy, and this method does not take into account molar loss.20,21 the availability of the number of research study models at the usu dental hospital that meets the inclusion criteria in this study is very minimal, so the number of samples obtained is small and can reduce the strength of this study. in conclusion of this study, there was a difference between these two indices. the ofi classifies the need for orthodontic treatment to be greater than the dai. the difference in the number of components and the weight of the assessment on each index greatly affects the results of this study. further study is still needed with a larger sample size and more varied analysis methods. references 1. littlewood sj, mitchell l. an introduction to orthodontics. 5th ed. oxford: oxford university press; 2019. p. 19–20. 2. setyowati p, ardhana w. perawatan maloklusi kelas iii dengan hubungan skeletal kelas iii disertai makroglosia menggunakan alat ortodontik cekat teknik begg. maj kedokt gigi indones. 2013; 20(2): 184–91. 3. nurvita v, handayani mm. hubungan antara self-esteem dengan body image pada remaja awal yang mengalami obesitas. j psikol klin dan kesehat ment. 2015; 4(1): 41–9. 4. paula df, silva ét, campos acv, nuñez mo, leles cr. effect of anterior teeth display during smiling on the self-perceived impacts of malocclusion in adolescents. angle orthod. 2011; 81(3): 540–5. 5. adha mar, wibowo d, rasyid ni. gambaran tingkat keparahan maloklusi menggunakan handicapping malocclusion assessment dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p205–209 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p205-209 209lubis and azriana/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 205–209 record (hmar) pada siswa sdn gambut 10. dentin j kedokt gigi. 2019; 3(1): 1–9. 6. dayataka rp, herawati h, darwis rs. hubungan tingkat keparahan maloklusi dengan status karies pada remaja di smp negeri 1 kota cimahi. padjadjaran j dent res students. 2019; 3(1): 43–9. 7. rahardjo p. ortodonti dasar. 2nd ed. surabaya: airlangga university press; 2012. p. 3. 8. damaryanti e, indrawati e, firdausi a. gambaran tingkat keparahan maloklusi pada pasien orthodonti antara tahun 2012-2015 dan 20152018 di rs universitas brawijaya menggunakan indeks icon. e-prodenta j dent. 2019; 3(2): 240–8. 9. gupta a, shrestha rm. a review of orthodontic indices. orthod j nepal. 2014; 4(2): 44–50. 10. padisar p, naseh r, babakhani a, jalayer s. comparing dental aesthetic index (dai) and iotn in determining the orthodontic treatment needs of qazvin students. iran j orthod. 2016; 11(2): 6231. 11. dewanto h. aspek-aspek epidemiologi maloklusi. yogyakarta: gadjah mada university press; 1993. p. 154–63. 12. bellot-arcs c, mara j, manuel j. orthodontic treatment need: an epidemiological approach. in: bourzgui f, editor. orthodontics basic aspects and cinical considerations. intechopen; 2012. p. 3–28. 13. cardoso cf, drummond af, lages emb, pretti h, ferreira ef, abreu mhng. the dental aesthetic index and dental health component of the index of orthodontic treatment need as tools in epidemiological studies. int j environ res public health. 2011; 8(8): 3277–86. 14. nahusona dr, aprilia w. status maloklusi mahasiswa fakultas kedokteran gigi universitas hasanuddin yang diukur berdasarkan occlusion feature index (ofi). malocclusion status of faculty of dentistry students in hasanuddin university measured by occlusion feature index (ofi). makassar dent j. 2017; 6(3): 91–5. 15. simangunsong sm, muttaqin z, tampubolon ia. gambaran maloklusi pada siswa/i suku batak di sma santo thomas 2 medan berdasarkan dental aesthetic index (dai). prima j oral dent sci. 2018; 1(1): 40–8. 16. wijayanti p, krisnawati k, ismah n. gambaran maloklusi dan kebutuhan perawatan ortodonti pada anak usia 9-11 tahun (studi pendahuluan di sd at-taufiq, cempaka putih, jakarta). j pdgi. 2014; 63(1): 25–9. 17. feroza na, kurniawan fkd, wibowo d. hubungan antara kebiasaan buruk bernafas melalui mulut dan tingkat keparahan maloklusi di smpn 4 banjarbaru dan sman 4 banjarbaru. dentino j kedokt gigi. 2017; 2(1): 39–43. 18. ardani igaw, nahmada ib, hamid t, sjafei a, sjamsudin j, winoto er, alida. pengantar ilmu ortodonti ii. surabaya: airlangga university press; 2017. p. 20–2. 19. borzabadi-farahani a. an insight into four orthodontic treatment need indices. prog orthod. 2011; 12(2): 132–42. 20. agarwal a, mathur r. an overview of orthodontic indices. world j dent. 2012; 3(1): 77–86. 21. utomi il, onyeaso co. relationship between two indices in the assessment of orthodontic treatment complexity and need. br j med med res. 2015; 7(6): 519–28. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p205–209 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p205-209 115 volume 47, number 2, june 2014 karakterisasi stem cell pulpa gigi sulung dengan modifikasi enzim tripsin (the characterization of stem cells from human exfoliated deciduous teeth using trypsin enzym) tri wijayanti puspitasari, tania saskianti, dan udijanto tedjosasongko departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: now a days, treatment in dentistry, using tissue regeneration that based on the stem cells from human exfoliated deciduous teeth (shed), grows rapidly. for several reason, the isolated and cultured shed is difficult to be applied in indonesia, therefore the modification is needed. this difficulties were caused by the pulp anatomy, the heterogeneous populations in the pulp chamber and the limitations of tools and materials at the laboratory. purpose: this research was aimed to examine that the modifications of isolation and culture technique of sheds for characterization by using the marker of cd105. methods: the research was experimental laboratory with the cross sectional design. the samples were the human exfoliated deciduous teeth from the children patients of pediatric dentistry department of universitas airlangga dental hospital which matched the criteria. dental pulps were isolated and cultured by using the modifications of trypsin enzymes. results: the healthy sheds could be produced from the modifications of isolation and culture and positively shown the expression of marker cd105 which were indicated by the fluorencent microscope. conclusion: shed which isolated and cultured by using the modified techniques, positively characterized by using marker cd105. key words: shed, modifications, isolated and culture techniques, characterization abstrak latar belakang: pengobatan kedokteran gigi berkembang dengan pesat terutama di bidang regenerasi jaringan berbasis stem cells from human exfoliated deciduous teeth (shed). di indonesia, isolasi dan kultur shed sulit sehingga perlu dilakukan modifikasi. kendala ini muncul karena jaringan pulpa yang kecil, heterogen dan keterbatasan alat dan bahan di laboratorium. tujuan: penelitian ini bertujuan untuk meneliti modifikasi pada cara isolasi dan kultur shed untuk karakterisasi menggunakan maker cd105. metode: jenis penelitian ini adalah eksperimental laboratoris dengan rancangan cross sectional. sampel penelitian adalah gigi sulung dari pasien anak di klinik kedokteran gigi anak, rumah sakit gigi dan mulut universitas airlangga yang telah memenuhi kriteria. pulpa gigi diisolasi dan dikultur dengan modifikasi enzim trypsin. hasil: shed yang sehat hasil dari modifikasi teknik isolasi dan kultur positif menunjukkan ekspresi marker cd105 dengan berfluoresensi berwarna hijau dilihat melalui mikroskop fluoresen. simpulan: shed yang dikultur dan diisolasi dengan teknik modifikasi positif dikarakterisasi dengan marker cd105. kata kunci: shed, modifikasi, teknik isolasi dan kultur, karakterisasi korespondensi (correspondence): tri wijayanti puspitasari, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya, 60132, indonesia. e-mail: salsabilah_tw@yahoo.co.id research report 116 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 115–119 pendahuluan pengobatan kedokteran gigi berkembang dengan pesat terutama di bidang regenerasi jaringan berbasis stem cell. stem cell dapat diisolasi dari pulpa gigi sulung manusia yang akan tanggal dan disebut stem cells from human exfoliated deciduous (shed). stem cells from human exfoliated deciduous (shed) mempunyai kelebihan apabila dibandingkan dengan stem cell jenis lain karena berasal dari jaringan yang muda sehingga mampu berdiferensiasi menjadi jenis sel yang lebih beragam, potensi proliferasi dan regenerasinya lebih besar.1,2 pulpa gigi sulung manusia secara histologis terdiri dari tiga bagian, yaitu odontoblas, cell free zone, dan cell rich zone. cell rich zone yang diisolasi dan dikultur dari pulpa gigi sulung manusia terdiri dari berbagai macam populasi sel. beberapa peneliti menemukan komponen sel-sel yang terdapat pada bagian pulpa tersebut antara lain fibroblast, sel-sel pertahanan, serat-serat kolagen, substansi dasar, pembuluh darah, limfatik, dan ujung saraf sensorik. perbandingan jumlah stem cell dari berbagai macam populasi sel lain yang ada di pulpa gigi sulung manusia adalah 1 : 1000. jumlah populasi pulpa gigi sulung yang heterogen menyebabkan stem cell sulit untuk diisolasi, dikultur dan dikarakterisasi.3 beberapa peneliti menggunakan teknik yang berbeda untuk dapat mengisolasi dan mengkultur stem cell dari pulpa gigi sulung yang heterogen. peneva dkk.4 menggunakan enzim colagenesis tipe v selama lima sampai tujuh hari di dalam dulbeccos modified eagle medium (dmem) untuk memisahkan populasi jaringan heterogen menjadi sel tunggal sehingga didapatkan inti sel pulpa gigi sulung yang mengandung stem cell.5 menggunakan enzim colagenase tipe i dan tipe ii dengan termolysin sebagai neutral protease selama 40 menit dalam inkubator pada atmosfir suhu 370c dan co2 5%. nikolic et al. 6 menggunakan enzim colagenase tipe i yang disuplementasi fetal bovine serum (fbs) selama 45 menit di dalam inkubator pada tahap isolasi dan kultur stem cell hari pertama. pada hari berikutnya, jika stem cell berhasil mengalami proliferasi dan confluent, enzim trypsin digunakan untuk proses subkultur. penelitian kultur dan isolasi primer stem cell pulpa gigi sulung belum berhasil dilakukan di indonesia. hal ini disebabkan karena populasi pulpa gigi sulung yang heterogen serta keterbatasan bahan dan alat laboratorium di indonesia. oleh karena itu, peneliti melakukan beberapa modifikasi teknik kultur dan isolasi untuk keberhasilan isolasi shed. modifikasi teknik kultur dan isolasi yang dilakukan pada penelitian ini, yaitu modifikasi waktu, alat rotomix dan enzim trypsin tanpa menggunakan enzim colagenase. uji karakterisasi stem cell pulpa gigi sulung menggunakan marker cd105 sebagai kontrol positif dan cd45 sebagai kontrol negatif dengan teknik imunositokimia. penelitian ini bertujuan untuk meneliti modifikasi enzim trypsin pada teknik isolasi dan kultur shed untuk karakterisasi dengan menggunakan marker cd105. hasil penelitian secara in vitro ini selanjutnya dapat dimanfaatkan untuk bank jaringan dan penelitian-penelitian stem cell pulpa gigi sulung yang lain. pada penelitian jangka panjang, stem cell pulpa gigi sulung diharapkan dapat digunakan untuk aplikasi pengobatan kedokteran gigi di bidang regenerasi jaringan dengan sampel penelitian yang mudah didapat di indonesia. bahan dan metode penelitian ini merupakan penelitian eksperimental laboratoris dengan rancangan penelitian cross sectional. sampel penelitian ini adalah gigi sulung yang diekstraksi dari pasien anak di rumah sakit gigi dan mulut universitas airlangga. modifikasi teknik isolasi dan kultur pulpa dilakukan pada gigi #53 yang diekstraksi dari pasien lakilaki usia 9 tahun disebut sampel pulpa i dan gigi #71 dari pasien laki-laki usia 7 tahun disebut sampel pulpa ii serta gigi #81 dari pasien tersebut disebut sampel pulpa iii. gigi sulung pada pasien anak tersebut adalah gigi yang vital, tidak terdapat karies, diekstraksi karena persistensi atau perawatan ortodonsia dan resorpsi tidak melebihi 1/3 akar. metode pengambilan sampel pada penelitian adalah purposive sampling, artinya penentuan sampel mempertimbangkan kriteria-kriteria tertentu berdasarkan tujuan penelitian. persiapan penelitian dan pembuatan sediaan pulpa gigi sulung dilakukan di rumah sakit gigi dan mulutumah sakit gigi dan mulut universitas airlangga. isolasi, kultur dan uji karakterisasi stem cell pulpa gigi sulung manusia dilakukan secara in vitro di laboratorium stem cell institute tropical disease (itd) universitas airlangga. prosedur kerja penelitian, yaitu mensterilisasikan semua alat dan ruangan untuk penelitian, kemudian dilakukan ekstraksi gigi sulung dengan hati-hati sehingga akar gigi tidak mengalami frakturhati-hati sehingga akar gigi tidak mengalami fraktur dan gigi tercabut sempurna. gigi yang telah diekstraksi dipotong membujur pada incisal-apical menggunakan bur fissure kemudian dilakukan pengambilan jaringan pulpa dan segera diletakkan dalamdiletakkan dalam medium dulbeccos modified eagle medium (sigma st. louis, mo, usa) untuk kemudian dibawa ke laboratorium. di laboratorium, pulpa gigi sulung diletakkan di dalam rotomix dan diberi perlakuan dengan enzim trypsin 0,25% (paa laboratories, linz, austria) selama 35 menit untuk memperoleh inti dari jaringan pulpa. kemudian disentrifuge 2000 rpm selama 5 menit untuk memperoleh cell pellet dan memisahkan supernatannya. setelah itu, suspensi sel dikultur pada medium dish ø35 mm yang telah diberi dmem dan disuplementasi dengan fetal bovine serum 20%, 5 ml0%, 5 ml l-gutamine, 100 u/ml penicillin-g, 100 g/ml streptomicyn dan 100 g/ml kanamycin (paa laboratories, linz, austria) dalam inkubator pada atmosfir suhu 370 c dan co2 5%. 6 sel pulpa gigi sulung hasil isolasi dan kultur yang berhasil berproliferasi dikarakterisasi melalui ekspresi marker cd105 (biosource invitrogen, camarillo, ca) 117puspitasari, et al.: karakterisasi stem cell pulpa gigi sulung dengan modifikasi teknik isolasi dan kultur sebagai kontrol positif dan cd45 (biosource invitrogen, camarillo, ca) sebagai kontrol negatif dengan teknik direct imunocytochemistry-one staining. kultur sel dibilas dengan phosphate-buffered salin (paa laboratories, linz, austria) sebanyak 3 kali dan dilakukan blocking dengan penambahan larutan blotto (paa laboratories, linz, austria) untuk mencegah terjadinya nonspecific binding yang terdiri dari 5% skim milk (paa laboratories, linz, austria) dan ditambahkan larutan tween 20/0,03% (paa laboratories, linz, austria), selanjutnya dibilas lagi dengan pbs sebanyak 3 kali.7,8 tahap berikutnya mereaksikan sel pulpa dengan marker antibodi cd105 pada satu sisi objek glass dan sisi lain dengan marker antibodi cd45 kemudian ditutup dengan deck glass. kedua marker ini berlabel fluorecent isothiocyanat (fitc) yang sudah dilarutkan dalam blotto 2 µl/ml ke dalam well. kemudian diinkubasi selama 1 jam di dalam inkubator. setelah 1 jam dilakukan pencucian ulang dengan pbs sebanyak 3 kali dan diamati melalui mikroskop fluoresen dengan pembesaran 200x di ruang gelap. populasi sel pulpa gigi sulung yang positif akan berfluoresensi berwarna hijau terhadap marker antibodi cd105 dan dikarakterisasi sebagai stem cells human exfoliated deciduous (shed).7,8 hasil sebanyak tiga sampel didapat dari pasien anak berusia 7 dan 9 tahun yang datang ke rumah sakit gigi dan mulut universitas airlangga. ketiga sampel merupakan gigi yang sesuai dengan kriteria pada metode kerja. kriteria tersebut ditentukan supaya sampel hasil modifikasi isolasi dan kultur dapat menghasilkan shed yang sehat sehingga dapat dikarakterisasi dengan marker cd105. ketiga sampel diambil pulpa giginya kemudian pada hari ke-7, sampel pulpa i, ii dan iii berhasil berproliferasi dengan morfologi berbentuk fusiform untuk pertama kalinya di pasase 0 (gambar 1). pada hari ke-20 sel pulpa yang berproliferasi mengalami 80% konfluen dan mulai dibiakkan pada dish ø35 mm yang lain di pasase 1. pada hari ke-28, pasase 4, shed yang sehat mengalami 4 kali konfluen (gambar 2). gambar 2. gambaran mikroskopik sampel pulpa i, pasase 4, nampak sel pulpa yang berproliferasi mengalami 80% konfluen pada hari ke-28 (pembesaran 100x). gambar 3. gambaran mikroskopik fluorensen sampel pulpa i, hari ke-28, hasil karakterisasi stem cells: a) sampel pulpa i nampak positif kuat terhadap marker cd105 yakni terjadi fluoresensi atau pendar cahaya berwarna hijau (tanda panah); b) sampel pulpa i nampak negatif terhadap marker cd45 yakni tidak terjadi fluoresensi atau pendar cahaya dan sel terlihat gelap (pembesaran 200x). gambar 1. gambaran mikroskopik sampel pulpa i, pasase 1, nampak morfologi sel berbentuk fusiform pada hari ke-7 (tanda panah) (pembesaran 100x). 118 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 115–119 sampel pulpa i gigi sulung yang di subkultur pada pasase 4 dilakukan karakterisasi dengan hasil positif mengandung shed ditunjukkan melalui fluoresensi atau pendar cahaya berwarna hijau terhadap marker cd105 (gambar 3a) dan tidak terjadi fluorensensi atau pendar cahaya terhadap marker cd45 (gambar 3b) dilihat melalui mikroskop fluoresen pembesaran 200x dengan teknik imunositokimia. pembahasan menurut prayogo dan wijaya9 banyak ilmuwan dari berbagai lembaga telah melakukan kultur stem cell, masingmasing dengan metode mereka sendiri. stem cell yang ditumbuhkan di berbagai laboratorium dengan berbagai teknik memiliki dua kesamaan, yaitu tumbuh di kultur sebagai sel yang melekat dengan lama hidup tertentu dan memiliki kemampuan untuk berdiferensiasi. keberhasilan modifikasi teknik isolasi dan kultur pada penelitian ini dapat dilihat dari shed yang mampu berproliferasi secara mikroskopik memiliki bentuk fusiform, fibroblast-like dan membentuk koloni pada fase pertumbuhan in vitro.10 penelitian modifikasi teknik isolasi dan kultur shed yang kami lakukan berbeda dengan penelitian peneva at al.4 yang menggunakan enzim colagenesis tipe v selama lima sampai tujuh hari di dalam dulbeccos modified eagle medium (dmem) untuk memisahkan populasi jaringan heterogen menjadi sel tunggal sehingga didapatkan inti sel pulpa gigi sulung yang mengandung stem cell. pada penelitian dengan modifikasi ini, waktu yang digunakan untuk mendapatkan sel tunggal tersebut lebih pendek, yaitu ±35 menit. tujuan memperpendek waktu adalah supaya pulpa gigi sulung tidak terkontaminasi mikroorganisme dan berbagai jenis agen lain yang terdapat di ruangan. faktor kontaminasi tersebut dinilai bisa menghambat pertumbuhan sel dan menyebabkan kegagalan kultur stem cell pulpa gigi sulung. penelitian dengan modifikasi teknik isolasi dan kultur ini juga berbeda dengan penelitian perry et al.5 yang menggunakan enzim colagenase tipe i dan tipe ii dengan termolysin sebagai neutral protease selama 40 menit dalam inkubator pada atmosfir suhu 370 c dan co2 5%. pada penelitian dilakukan modifikasi alat rotomix dan tidak menggunakan inkubator khusus untuk memisahkan sel pulpa yang heterogen karena keterbatasan alat dan bahan di laboratorium. alat rotomix diberi termometer untuk menjaga suhu tetap 370 c dan dikondisikan sedemikian rupa supaya mirip dengan keadaan yang ada di dalam tubuh sehingga stem cell pulpa gigi sulung dapat hidup dan berproliferasi. penelitian nikolic et al.6 menggunakan enzim colagenase tipe i yang disuplementasi fetal bovine serum (fbs) selama 45 menit di dalam inkubator pada tahap isolasi dan kultur stem cell hari pertama. ketika stem cell telah mengalami proliferasi dan confluent. nikolic at al.6 menggunakan enzim trypsin selama 10 menit dalam inkubator pada atmosfir suhu 370 c dan co2 5% dan diulang setiap tiga hari sekali dalam proses subkultur stem cell. berbeda dengan penelitian tersebut, pada penelitian modifikasi teknik isolasi dan kultur stem cell pulpa gigi sulung ini menggunakan enzim trypsin tanpa enzim colagenase untuk mendapatkan sel tunggal dari pulpa gigi sulung yang heterogen. enzim trypsin adalah enzim yang harganya lebih murah, tersedia dalam jumlah banyak dan mudah didapat di laboratorium. penggunaan enzim trypsin diharapkan dapat menggantikan enzim colagenase dengan kualitas yang tidak jauh berbeda sehingga stem cell pulpa gigi sulung dapat berproliferasi. keberhasilan proliferasi dari stem cell pulpa gigi sulung pada penelitian ini dipengaruhi oleh dua faktor, yaitu faktor internal dan faktor eksternal. faktor internal terdiri dari growth factor yang berperan dalam memicu terjadinya siklus sel dan nutrisi didalam sel. faktor eksternal terdiri modifikasi teknik kultur, modifikasi teknik isolasi, media, faktor kontaminasi dan suhu. media kultur yang digunakan adalah media standar dengan suplemen untuk menunjang proliferasi sel, antibiotik untuk mengatasi kontaminasi dan serum baik dari fetal bovine serum (fbs) maupun fetal calf serum (fcs). implikasinya, kultur sel in vitro harus mampu meniru kondisi yang terjadi secara in vivo untuk memastikan validitas peristiwa di laboratorium semirip mungkin dengan yang terjadi di dalam tubuh termasuk diantaranya adalah suhu.9,11 pada penelitian ini, kelima faktor eksternal dapat dilakukan dengan baik dan memenuhi syarat sehingga sel yang ditanam di dalam dish mengalami perlekatan. setelah sel mengalami perlekatan yang baik, maka satu sel dengan sel yang lain akan melakukan interaksi sehingga terjadilah komunikasi antar sel yang dilanjutkan dengan adanya migrasi sel dimana sel-sel tersebut akan saling mendekat dan berikatan satu sama lain. hasil dari ikatan sel tersebut akan menimbulkan sel baru dan memperbanyak diri sehingga jumlah sel pulpa mengalami peningkatan dan disebut sel berhasil melakukan proliferasi. pulpa yang telah berproliferasi mengandung berbagai populasi sel yang bersifat heterogen sehingga diperlukan karakterisasi untuk menguji bahwa pulpa yang diisolasi dan dikultur dengan modifikasi teknik mengandung stem cells from human exfoliated deciduous (shed). shed merupakan salah satu jenis mesenchymal stem cell. karakterisasi mesenchymal stem cell dilakukan dengan menggunakan tenik direct cytochemistry-one staining melalui marker cd105 sebagai kontrol positif dan cd45 sebagai kontrol negatif.7,12 uji karakterisasi pulpa gigi sulung yang diisolasi dan dikultur dengan modifikasi positif mengandung populasi shed. hal ini dapat diketahui melalui ekspresi positif kuat marker cd105 yaitu adanya fluoresensi atau pendar cahaya berwarna hijau dilihat melalui mikroskop dengan pembesaran 200x. fluoresensi atau pendar cahaya berwarna hijau ini disebabkan karena adanya reaksi antara 119puspitasari, et al.: karakterisasi stem cell pulpa gigi sulung dengan modifikasi teknik isolasi dan kultur antibodi 43a3 dan cd105, sebuah 180 kda glikoprotein pada permukaan sel yang merupakan disulfida bonded homodimer dari 90 kda tipe i transmembran subunit.7,11 karakterisasi pulpa gigi sulung yang diisolasi dan dikultur dengan modifikasi teknik mengandung shed dan bukan sel lain ditunjukkan melalui ekspresi negatif marker cd45 pada saat penelitian in vitro. setelah sel pulpa gigi sulung diberi perlakuan terhadap marker antibodi cd45 tidak terjadi pembentukan pendar cahaya atau fluoresensi. hal ini terjadi karena tidak adanya reaksi antara hi30 antibodi dan semua isoform cd45, suatu tipe i transmembran glikoprotein yang diekspresikan pada permukaan sel.8 stem cells from human exfoliated deciduous (shed) yang positif terhadap marker cd105 dan negatif terhadap marker cd45 pada hasil penelitian ini sama dengan hasil penelitian yang dilakukan nikolic et al.6 hal ini dikarenakan shed merupakan jenis mesenchymal stem cell yang dapat diuji karakterisasinya dengan menggunakan marker cd105 sebagai kontrol positif. marker cd45 merupakan kontrol negatif yang berfungsi untuk menguji bahwa shed hasil penelitian bukan berasal dari jenis hematopoetic cells dan sel lain yang terdapat di dalam pulpa gigi sulung.8,11 hasil penelitian menunjukkan bahwa shed yang dikultur dan diisolasi dengan modifikasi enzim trypsin positif dikarakterisasi dengan marker cd105. daftar pustaka 1. miura m, gronthos s, zhao m, lu b, fisher lw, robey pg, shi s. shed: stem cells from human exfoliated deciduous teeth. j proc natl acad sci usa 2003; 100(10): 5807-12. 2. shi s, bartold pm, miura m, seo bm, robey pg, gronthos s. the efficacy of mesenchymal stem cells to regenerate and repair dental structures. j orthod craniofac res 2005; 8(3): 191-9. 3. torneck dc, torabinejad m. biologi jaringan pulpa gigi dan jaringan periradikuler. dalam: walton re, torbinejad m, eds. prinsip dan praktik ilmu endodonsia. 3rd ed. sumawinata n. jakarta: egc; 2003. h. 4-15. 4. peneva, mitev v, ishketiev n. isolation of mesenchymal stem cells from the pulp of deciduous teeth. journal of imab-annual proceeding 2008; 2: 84-7. 5. brandon p, xiaohua z, yang fc, gabriel m, erik j, goebel ws. collection, cryopreservation and characterization of human dental pulp derived mesenchymal stem cells for banking and clinical use. j tissue engineering 2008; 2: 14. 6. kristic n, mojsilovic t, kocic j, santibanez jz, jovcic g, bugarski d. mesenchymal stem cell properties of dental pulp cells from deciduous teeth. j arch bio sce belgrade 2011; 63(4): 933-42. 7. orciani m, mariggio ma, morabito c, di bg, di primirio r. functional characterization of calcium-signaling pathways of human skinderived mesenchymal stem cells. skin pharmacol physiol 2010; 23(3): 124-32. 8. shoham s; kollet o, lapid k, schajnovitz a, goichberg p, kalinkovich a, shezen e, tesio m, netzer n, petit i, sharir a, lapidot t. cd45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules. j experimental medicine 2008; 205(10): 2381-95. 9. prayogo r, wijaya mt. kultur dan potensi stem cell dari darah tali pusat. cermin dunia kedokteran 2006; 153: 26-8. 10. kern s, eichler h, stoeve j. comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood or adipose tissue. stem cells 2006; 24: 1294-301. 11. kassem m, kristiansen m, abdallah bm. mesenchymal stem cells: cell biology and potential use in therapy. j basic clin pharmacol toxicol 2004; 95(5): 209-14. 12. yu g, wu x, dietrich ma, polk p, scott lk, ptitsyn aa, gimble jm. yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mrna analyzes. cytotherapy 2010; 12(4): 538-46. 119 volume 46, number 3, september 2013 korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg akibat paparan radiografi panoramic (the correlation of micronucleus formation and 8-oxo-dg expression due to the panoramic radiography exposure) rurie ratna shantiningsih,1 suwaldi,2 indwiani astuti3 dan munakhir mudjosemedi1 1department of dentomaxillofacial, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia 2faculty of pharmacy; universitas gadjah mada, yogyakarta – indonesia 3departement pharmacology, faculty of medicine, universitas gadjah mada, yogyakarta – indonesia abstract background: the expression of 8-oxo-dg is defined as one form of damaged dna occuring as the result of oxidation reaction due to x ray exposure. panoramic radiography exposure has been widely known to be able to increase micronucleus which are signing the early stage and as biomarker in carsinogenesis mechanism. purpose: the purpose of this research was to determine the correlation between micronucleus number and 8-oxo-dg expression as a result of panoramic radiography exposure. methods: twelve new zealand male rabbits aging 6 months were divided into 4 groups. group i were rabbits that represented 0 day, group ii represented 3th day, group iii represented 6th day, and group iv represented 9th day after the panoramic radiography exposure. respectively samples were swabbed at mandibular anterior gingival mucosa before and after the panoramic radiography exposure. the swabbed samples were coloured by using feulgen-rossenbeck modified staining for calculating the amount of micronucleus formation. expression of 8-oxodg was detected using immunohistochemical of rabbit’s gingival mucosa epithelium. statistical analysis were carried out on pearson correlation. results: there was a highest increasing of micronucleus on the 9th day after panoramic radiography exposure. meanwhile, there was significant correlation (p=0,049) of the increasing amount of micronucleus and 8-oxo-dg expression in negatively correlation (r = -0,578). the increasing of micronucleus formation on the 9th day after panoramic radiography exposure was accordance with some previous studies. the expression score of 8-oxo-dg decreases as the day goes by. conclusion: there was a correlation between the number of micronucleus and expression score of 8-oxo-dg. key words: micronucleus, 8-oxo-dg, radiography panoramic exposure, rabbit abstrak latar belakang: ekspresi 8-oxo-dg adalah suatu bentuk kerusakan dna yang terjadi akibat reaksi oksidatif dari paparan sinar x. paparan radiografi panoramik telah diketahui menyebabkan peningkatan jumlah mikronukleus yang merupakan biomarker tahap dini mekanisme karsinogenesis. tujuan: tujuan penelitian ini adalah untuk meneliti korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg akibat paparan radiografi panoramik. metode: sebanyak 12 ekor kelinci new zealand jantan usia 6 bulan dibagi menjadi 4 kelompok. kelompok i merupakan kelinci untuk mewakili hari ke-0, kelompok ii untuk mewakili hari ke-3, kelompok iii untuk mewakili hari ke-6, dan kelompok iv untuk mewakili hari ke-9 setelah paparan radiografi panoramik. terhadap seluruh kelinci dilakukan usapan pada mukosa gingiva anterior rahang bawah sebelum dan sesudah dilakukan paparan radiografi. selanjutnya terhadap sampel usapan itu dilakukan pewarnaan menggunakan teknik pewarnaan modifikasi feulgen-rossenbeck untuk menghitung jumlah mikronukleus. ekspresi 8-oxo-dg dinilai dari pemeriksaan imunohistokimia pada sel epitel mukosa gingiva kelinci. analisis statistik menggunakan uji korelasi pearson. hasil: peningkatan jumlah mikronukleus paling tinggi terjadi pada hari ke-9 setelah paparan radiografi panoramik. sementara itu, terdapat korelasi yang signifikan (p=0,049) antara peningkatan jumlah mikronukleus dan ekspresi 8-oxo-dg dengan arah korelasi yang negatif (r=-0,578). peningkatan jumlah mikronukleus yang terjadi pada hari ke-9 research report 120 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 119–123 setelah paparan radiografi panoramik ini sesuai dengan penelitian sebelumnya. dengan bertambahnya hari skor ekspresi 8-oxo-dg semakin menurun. simpulan: terdapat korelasi antara jumlah mikronukleus dan skor ekspresi 8-oxo-dg. kata kunci: mikronukleus, 8-oxo-dg, paparan radiografi panoramik, kelinci korespondensi (correspondence): rurie ratna shantiningsih, departemen bedah mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara yogyakarta 55281, indonesia. e-mail: rr_shantin@yahoo.com pendahuluan radiasi pengion, termasuk di dalamnya adalah radiografi panoramik di kedokteran gigi, dapat menyebabkan reaksi ionisasi pada objek yang dikenainya. pada teknik radiografi panoramik tersebut, mukosa mulut yang berbatasan langsung dengan saliva akan menjadi obyek pertama yang terpapar oleh radiasi pengion dan mengalami ionisasi.1 reaksi ionisasi tersebut menyebabkan terbentuknya senyawa kimia yang disebut radikal bebas. radikal bebas memiliki sifat yang sangat reaktif sehingga dapat menyebabkan oksidasi lebih lanjut pada molekul di sekitarnya. ketika radikal bebas dan hasil oksidasi bereaksi terhadap molekul kompleks dalam sel terutama kromosom, maka rantai kromosom menjadi terputus dan susunan basa nukleotida berubah. perubahan itu mengakibatkan terjadinya kerusakan pada deoxyribonucleic acid (dna).2 akibat lebih lanjut dari kerusakan dna berupa pembelahan sel yang tertunda, modifikasi dan perubahan sel secara permanen serta peningkatan kecepatan pembelahan sel yang menginduksi terjadinya tumor.3 pada sel yang mengandung radikal bebas setelah terjadinya reaksi oksidasi menyebabkan beberapa senyawa kimia bergabung bersama dna membentuk dna adduct yang mengawali terjadinya perubahan gen.4 dna adduct tersebut dapat dikenali dengan adanya ekspresi 8-hydroxy-2-deoxyguanosine (8-oxo-dg). senyawa 8oxo-dg merupakan suatu bentuk dna adduct yang terjadi akibat reaksi oksidatif antara lain akibat paparan sinar-x.5 sampai saat ini belum pernah dilakukan penelitian yang menggunakan 8-oxo-dg sebagai marker terjadinya dna adduct akibat paparan radiografi panoramik. ekspresi 8-oxo-dg sebagai dna adduct dapat terdeteksi pada sel yang terkelupas dari mukosa bukal pada hari ke-7.6 selain terbentuknya dna adduct, perubahan genetik lainnya juga dapat terjadi sebagai respons sel terhadap radiasi sinar-x, ultraviolet, dan sinar gamma yang dapat merupakan rangkaian dalam mekanisme karsinogenesis.5 penyebab terjadinya kanker yang disebut sebagai mutagen, berasal dari bahan kimia yang bersifat karsinogenik dan dapat juga akibat paparan radiasi pengion.7 peningkatan frekuensi mikronukleus pada mukosa normal di sekitar lesi berpotensi menjadi lesi prekanker akibat terjadinya ketidakstabilan kromosom merupakan mekanisme penting dalam perkembangan kanker.8 mikronukleus bersifat semipermanen karena akan menetap dalam jangka waktu sekitar 1-2 kali pembelahan. timbulnya mikronukleus sering diyakini sebagai marker pada tahap dini mekanisme karsinogenesis pada sel yang terlibat.9 pada penelitian cerqueira,10 ribeiro,11 dan popova12 menemukan bahwa radiasi sinar x dari radiografi dental menggunakan teknik panoramik menyebabkan terjadinya efek genotoksik berupa peningkatan jumlah mikronukleus pada sel epitel gingiva dan mukosa bukal. peningkatan jumlah mikronukleus tersebut maksimal terjadi pada hari ke-10 sesudah paparan dan akan mengalami penurunan pada hari ke-14 setelah paparan dihentikan. dengan adanya peningkatan jumlah mikronukleus menunjukkan terjadinya peningkatan frekuensi kerusakan kromosom dan perubahan inti sel akibat paparan radiasi dari radiografi dental teknik panoramik. berdasarkan paparan di atas, penelitian ini bertujuan untuk mengetahui korelasi antara pembentukan mikronukleus dan ekspresi 8-oxo-dg akibat paparan radiografi panoramik. bahan dan metode penelitian ini merupakan penelitian eksperimental dengan rancangan pre and post test group design, yang dilakukan di fakultas kedokteran gigi universitas gadjah mada. subyek penelitian adalah hewan coba kelinci galur new zealand, jenis kelamin jantan, umur 6 bulan, dengan berat sekitar 2-2,5 kg yang dibagi menjadi 4 kelompok. kelompok i mewakili hari ke-0, kelompok ii mewakili hari ke-3, kelompok iii mewakili hari ke-6 dan kelompok iv mewakili hari ke-9 setelah paparan radiografi panoramik. masing-masing kelinci dilakukan paparan radiografi panoramik menggunakan mesin radiografi panoramik merk yoshida panoura dengan dosis 80 kvp, 8 ma, dan 12 detik untuk satu kali paparan dengan laju dosis 47 μsv. dosis yang digunakan pada penelitian ini sama dengan yang biasa digunakan untuk paparan radiografi panoramik pada manusia walaupun densitas tulang kelinci lebih rendah dibandingkan manusia. hal ini terkait dengan ketersediaan mesin radiograf di fakultas kedokteran gigi universitas gadjah mada dan efek yang diteliti sebatas pada mukosa gingiva. hewan coba kelinci diletakkan dalam kotak kayu berukuran 20 x 25 x 20 cm dengan lubang tempat kepala sebagai tempat fiksasi hewan coba kelinci ketika dilakukan paparan sinar-x. ethical clearance untuk penelitian ini diperoleh dari komisi etik fakultas kedokteran gigi universitas gadjah mada. pengambilan sampel mikronukleus dilakukan dengan cara mengusap mukosa gingiva rahang bawah menggunakan cervical brush. selanjutnya sampel yang diperoleh dari 121shantiningsih, dkk.: korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg mukosa epitel gingiva ditempatkan di atas slide dan diberi 2 tetes larutan nacl 0,09%.10 pengambilan sampel usapan dilakukan pada saat sebelum dan sesudah paparan radiografi panoramik. untuk sampel pemeriksaan imunohistokimia (ihk) diambil dari potongan mukosa gingiva masingmasing kelinci pada hari ke-0, 3, 6 dan 9 setelah paparan radiografi panoramik. pengambilan potongan mukosa gingiva dilakukan segera setelah kelinci didekapitasi. prosedur dekapitasi hewan coba dilakukan setelah diberikan suntikan pentobarbital secara intravena sebanyak 100–150 mg/kg. pembuatan preparat untuk melihat jumlah mikronukleus diawali dengan fiksasi sampel pada slide menggunakan methanol-acetic acid (3:1), dilanjutkan perendaman dalam 5 m hcl pada suhu kamar selama 15 menit. setelah slide dicuci dengan distilled water selama 10–15 menit, dilakukan pewarnaan menggunakan metode feulgenrossenbeck dalam schiff’s reagen selama 90 menit dan dilakukan counterstain dengan fastgreen 1% selama 1 menit.10 untuk pemeriksaan ekspresi 8-oxo-dg, jaringan gingiva dimasukkan ke dalam larutan fiksatif (buffer formalin 10%), kemudian diproses untuk pembuatan blok parafin. selanjutnya blok parafin dipotong menggunakan mikrotom dengan ketebalan 4 μm. berikutnya dilakukan deparafinisasi dengan xylene dan rehidrasi menggunakan etanol kemudian dilanjutkan tahapan blocking peroxidase menggunakan 0,3% h2o2 dalam metanol dan blocking non specific binding selama 10 menit. tahap selanjutnya dilakukan pewarnaan ihk menggunakan antibodi 8-oxodg selama semalam, dilanjutkan antibodi sekunder dan pelabelan menggunakan trekavidin-hrp label. untuk pewarnaan spesifik menggunakan betazoid dab kemudian dilakukan counter stain menggunakan hematoxicillin meyers. penghitungan jumlah mikronukleus dilakukan menggunakan mikroskop cahaya yang disambungkan dengan optilab pembesaran 40x dengan menjumlahkan sel yang memiliki gambaran adanya inti tambahan berupa mikronukleus (gambar 1). mikronukleus tersebut berada di sekitar inti utama, memberikan hasil pewarnaan yang sama dengan inti utama dan berukuran lebih kecil, sekitar 1/3 diameter inti utama. perhitungan jumlah mikronukleus dilakukan sebanyak 2 kali pada saat sebelum paparan dan sesudah paparan dari masing-masing kelompok perlakuan. hasil pemeriksaan ihk diamati menggunakan mikroskop cahaya yang disambungkan dengan optilab pembesaran 10x. hasil analisis ihk ditampilkan dalam bentuk skoring untuk analisis semikuantitatif. sistem skoring digunakan dengan mengkombinasikan area yang terdeteksi positif dan intensitas warna. penilaian skoring dilakukan oleh 3 orang blind observer menggunakan standar penilaian area yang terdeteksi positif sebagai berikut: skor 0: negatif/tidak ada sel yang terwarnai; skor 1: area terwarnai jarang (<10%); skor 2: area terwarnai mencapai 10%; skor 3: area terwarnai mencapai 10-50% dan skor 4: area terwarnai >50%. selanjutnya kriteria tersebut dikombinasikan dengan standar penilaian intensitas warna yaitu skor 1: lemah; skor 2: sedang; dan skor 3: kuat. analisis statistik menggunakan korelasi pearson untuk melihat hubungan antara jumlah mikronukleus dan ekspresi 8-oxo-dg. hasil hasil penilaian jumlah mikronukleus dari kelompok i, ii, iii dan iv berdasarkan hari ditampilkan dalam gambar 2 yang nampak adanya peningkatan jumlah mikronukleus pada kelompok i, ii, iii dan iv yang mewakili hari ke-0, 3, 6 dan 9 setelah paparan radiografi panoramik. peningkatan terbesar terjadi pada hari ke-9 setelah paparan radiografi panoramik dibandingkan hari-hari sebelumnya. hari ke-0 atau sesaat setelah paparan radiografi panoramik merupakan titik awal untuk perhitungan peningkatan jumlah mikronukleus. selanjutnya, untuk melihat hasil ihk ekspresi 8-oxo-dg untuk menggambarkan adanya gambar 1. contoh gambar mikronukleus pada sel epitel mukosa gingiva kelinci yang ditunjukkan oleh anak panah. gambar 2. peningkatan jumlah mikronukleus berdasarkan penambahan hari. 122 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 119–123 dna adduct dari masing-masing kelompok ditunjukkan pada gambar 3. pada gambar 3 ekspresi 8-oxo-dg nampak paling tebal pada kelompok hari ke-0 yang menunjukkan ekspresi 8-oxo-dg dari mukosa gingiva sesaat setelah paparan radiogafi panoramik. sejalan dengan peningkatan hari ekspresi 8-oxo-dg menunjukkan penurunan ketebalan dan area yang mengekspresikan juga semakin menyempit. pada hari ke-9 tampak bahwa intensitas warna paling lemah dan area yang mengekspresikan hanya tersisa sekitar 10%. untuk melihat korelasi antara jumlah mikronukleus dan skor ekspresi 8-oxo-dg, ditunjukkan dalam gambar 4. pada gambar 4 nampak bahwa terdapat penurunan skor 8-oxo-dg berdasarkan peningkatan jumlah mikronukleus dengan nilai regresi 0,902 yang lebih besar dari r tabel (0,9000). hasil tersebut menunjukkan bahwa terdapat korelasi linear antara peningkatan jumlah mikronukleus dan penurunan skor 8-oxo-dg dengan kecepatan perubahan sebesar -100. untuk melihat korelasi antara jumlah mikronukleus dan skor ekspresi 8-oxo-dg secara statistik menggunakan analisis statistik korelasi pearson ditampilkan dalam tabel 1. berdasarkan pada hasil analisis statistik pada tabel 1 nampak adanya korelasi yang signifikan antara jumlah mikronukleus dan skor ihk 8-oxo-dg dengan kekuatan korelasi yang kuat dan arah korelasi negatif (r = -0,668). pembahasan pada penelitian sebelumnya, cerqueria 10 dan shantiningsih13 menemukan bahwa radiasi sinar-x yang dihasilkan akibat paparan radiografi panoramik dapat menyebabkan peningkatan jumlah mikronukleus secara signifikan pada sel epitel gingiva manusia. pada penelitian ini, terjadi pula peningkatan jumlah mikronukleus pada mukosa gingiva kelinci sejak hari ke-3, 6 dan 9 setelah paparan radiografi panoramik dengan laju dosis 47 μsv. peningkatan jumlah mikronukleus nampak paling tinggi pada hari ke-9 setelah paparan radiografi panoramik (gambar 2). menurut cerqueira,10 antara hari ke-9 dan 10 merupakan waktu yang dianggap paling tepat untuk mendeteksi mikronukleus, terkait dengan periode turnover dari sel epitel. periode turn over pada gingiva umumnya terjadi mulai hari ke-7 sampai dengan hari ke-16. seperti yang telah disebutkan sebelumnya bahwa sinar-x dapat menginduksi terjadinya kerusakan pada dna.2 dalam penelitian ini ditemukan bahwa paparan radiografi panoramik dapat menyebabkan terbentuknya dna adduct yang ditandai dengan munculnya ekspresi 8-oxo-dg (gambar 3). terjadinya dna adduct berkaitan pula dengan munculnya mikronukleus. kedua parameter ini menunjukkan perubahan yang berkaitan dengan preneoplastik yang diyakini sebagai paramater untuk penilaian respons terhadap agen spesifik.8. tabel 1 gambar 3. ekspresi imunohistokimia 8-oxo-dg untuk menggambarkan adanya dna adduct pada nukleus dan sitoplasma yang memberikan gambaran berwarna coklat seperti yang ditunjukkan dengan anak panah. (a) sesaat setelah paparan radiografi panoramik, (b) 3 hari setelah paparan radiografi panoramik, (c) 6 hari setelah paparan radiografi panoramik dan (d) 9 hari setelah paparan radiografi panoramik. gambar 4. kurva hubungan antara jumlah mikronukleus dan skor 8-oxo-dg dengan persamaan regresi y = -100x +5,362 dan r = 0,902. tabel 1. hasil analisis korelasi pearson antara jumlah mikronukleus dan skor ekspresi 8-oxo-dg variabel 1 variabel 2 pearson correlation sig. (2-tailed) skor ihk 8-oxo-dg mikronukleus -.658 0.020* a b c d 123shantiningsih, dkk.: korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg menunjukkan bahwa hasil analisis statistik pearson correlation memiliki korelasi negatif yang kuat (r = -0,658) antara jumlah mikronukleus dan skor 8-oxodg. dengan demikian membuktikan bahwa paparan radiografi panoramik akan mengakibatkan munculnya mikronukleus yang berkaitan dengan dna adduct yang ditunjukkan dengan ekspresi 8-oxo-dg. munculnya mikronukleus merupakan akibat kerusakan dna akibat paparan radiografi panoramik yang menimbulkan reaksi oksidatif. radiasi pengion menyebabkan lesi pada dna dan juga menyebabkan pecahnya ikatan ganda yang diyakini sebagai lesi awal dalam proses karsinogenesis akibat radiasi.14 kerusakan berupa dna adduct ini meningkatkan kemungkinan mutasi genetik dalam sel yang memicu proses karsinogenesis.4 efek paparan radiografi panoramik yang terjadi pada hewan coba kelinci kemungkinan lebih besar daripada yang terjadi pada manusia. hal itu terkait dengan dosis yang diserap oleh hewan coba kelinci lebih besar dibandingkan yang diserap oleh manusia karena densitas tulang kelinci lebih rendah daripada manusia. namun demikian terjadinya peningkatan jumlah mikronukleus pada sel epitel mukosa gingiva kelinci pada penelitian ini sejalan dengan yang ditemukan pada manusia dalam penelitian cerqueria10 dan shantiningsih13 sehingga hasil penelitian ini dapat menggambarkan kemungkinan terjadinya efek paparan dari radiografi panoramik pada manusia. dengan adanya hasil penelitian ini perlu diwaspadai untuk penggunaan radiografi panoramik harus benar-benar dilakukan berdasarkan prinsip proteksi radiasi dan dengan indikasi medis yang kuat. hasil korelasi negatif pada penelitian ini disebabkan karena munculnya mikronukleus pada epitel gingiva dapat terdeteksi setelah sel mengalami pengelupasan pada beberapa hari setelah paparan, selain itu ekspresi dna adduct yang diwakili oleh ekspresi 8-oxo-dg akan mengalami penurunan berdasarkan hari karena adanya kemampuan fisiologis tubuh dalam mengeliminasi kerusakan yang terjadi jika paparan mutagen dihentikan.3 dna adduct 8-oxo-dg merupakan single base lesion yang akan dihilangkan melalui mekanisme base excision repair (ber).15 mekanisme ber memiliki kapasitas sebagai faktor pelindung melawan toksisitas. peningkatan regulasi ber akan menurunkan sitotoksisitas akibat dari reactive oxygen species (ros).16 radiasi pengion dapat menyebabkan terbentuknya ros yang berkaitan dengan pembentukan mikronukleus.17 salah satu radiasi pengion yang digunakan di kedokteran gigi adalah paparan radiografi panoramik.1 level dari ekspresi 8-oxo-dg dan peningkatan ros yang menyebabkan sitotoksisitas akan mengalami penurunan dengan pemberian antioksidan.16 kesimpulan yang dapat diambil dari penelitian ini adalah bahwa terdapat korelasi antara jumlah mikronukleus dengan ekspresi 8-oxo-dg. jumlah mikronukleus semakin meningkat sampai dengan hari ke-9 setelah paparan radiografi panoramik. dilain pihak skor ekspresi 8oxo-dg semakin menurun sejalan dengan peningkatan jumlah mikronukleus. dari hasil tersebut disarankan untuk mengatasi peningkatan jumlah mikronukleus dan kerusakan dna akibat paparan radiografi panoramik, salah satunya dengan pemberian antioksidan sebagai agen radioprotektor. daftar pustaka 1. whaites e. essentials of dental radiography and radiology. 3rd ed. london: churchill livingstone; 2002. p. 32–40. 2. whaites e. radiography and radiology for dental care professionals. 2nd ed. london: churchill livingstone; 2009. p. 29–32. 3. alberts b, johnson a, lewis j, raff m, roberts k, walter p. molecular biology of the cell. 4th ed. new york: garland science; 2002. p. 315–45. 4. phillips dh, farmer pb, beland fa, nath rg, poirier mc, reddy mv, turteltaub kw. methods of dna adduct determination and their application to testing compounds for genotoxicity. environ mol mutagen 2000; 35(3): 222–33. 5. wulff bc, schick js, thomas-ahner jm, kusewitt df, yarosh db, oberyszyn tm. topical treatment with oggi enzyme affects uvb-induced skin carcinogenesis. photochem photobiol sci 2008; 84(2): 317–21. 6. borthakur g, butryee c, stacewicz-sapuntzakis m, bowen pe. exfoliated buccal mucosa cells as a source of dna to study oxidative stress. cancer epidem biomar 2008; 17(1): 212–9. 7. alberts b, bray d, hopkin k, johnson a, lewis j, raff m, roberts k, walter p. essential cell biology. 2nd ed. new york: garlan science; 2004. p. 726–9. 8. delfino v, casartelli g, garzoglio b, scala m, mereu p, bonatti s, margarino g, abbondandolo a. micronuclei and p53 accumulation in preneoplastic and malignant lesions of the head and neck. mutagenesis 2002; 17(1): 73–7. 9. pawitan ja. peran histologi untuk meningkatkan kesehatan masyarakat: uji mikronukleus untuk mendeteksi adanya aberasi kromosom. med j indones 2005; 12: 213–6. 10. cerqueira emm, meireles jrc, lopes ma, junqueira vc, gomesfilho is, trindade s, machado-santelli gm. genotoxic effects of x-rays on keratinized mucosa cells during panoramic dental radiography. dentomaxillofac radiol 2008; 37(7): 398–403. 11. ribeiro da, de oliveira g, de castro gm, angelieri f. cytogenetic biomonitoring in patients exposed to dental x-rays: comparison between adults and children. dentomaxillofac radiol 2008; 37(7): 404–7. 12. popova l, kishkilova d, hadjidekova vb, hristova rp, atanasova p, hadjidekova vv, ziya d, hadjidekov vg. micronucleus test in buccal epithelium cells. dentomaxillofac radiol 2007; 36(3): 168–71. 13. shantiningsih rr. the number of micronucleus between single and repeated x-rays exposure of panoramic radiography patients. yogyakarta: proceeding the 2nd international joint symposium on oral and dental sciences; 2012. p. 129–33. 14. wall bf, kendall gm, edwards aa, bouffler s, muirhead cr, meara jr. what are the risks from medical x-rays and other low dose radiation?. br j radiol 2006; 79(940): 285–94. 15. de souza-pinto nc, eide l, hogue ba, thybo t, stevnsner t, seeberg e, klungland a, bohr va. repair of 8-oxodeoxyguanosine lesion in mitochondrial dna depends on the oxoguanine dna glycosylase (ogg1) gene and 8-oxoguanine accumulates in the mitochondrial dna of ogg1-defective mice. cancer res 2001; 61(14): 5378–81. 16. preston tj, henderson jt, mccallum gp, wells pg. base excision repair of reactive oxygen species-initiated 7,8-dihydro-8-oxo-2’deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs. mol cancer ther 2009; 8(7): 2015–26. 17. choi k, kang c, cho e, kang sm, lee sb, um h. ionizing radiationincuded micronucleus formation is mediated by reactive oxygen species that are produced in a manner dependent on mitochondrial, nox1, and jnk. oncol rep 2007; 17(5): 1183–8. �� volume 47, number 1, march 2014 the role of heat shock protein �� (hsp ��) as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture sri wigati mardi mulyani,1 ernie maduratna setiawati, 2 erma safitri3 and eha renwi astuti1 1 department of dentomaxillofacial radiology, faculty of dental medicine universitas airlangga 2 department of periodontics, faculty of dental medicine universitas airlangga 3 department of reproduction, faculty of veterinary medicine universitas airlangga surabaya indonesia abstract background: the concept of stem cell therapy is one of the new hope as a medical therapy on salivary gland defect. however, the lack of viability of the transplanted stem cells survival rate led to the decrease of effectiveness of stem cell therapy. the underlying assumption in the decrease of viability and function of stem cells is an increase of apoptosis incidence. it suggests that the microenvironment in the area of damaged tissues is not conducive to support stem cell viability. one of the microenvironment is the hypoxia condition. several scientific journals revealed that the administration of hypoxic cell culture can result in stress cells but on the other hand the stress condition of the cells also stimulates heat shock protein 27 (hsp 27) as antiapoptosis through inhibition of caspase 9. purpose: the purpose of this study was to examine the role of heat shock protein 27 as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture. methods: stem cell culture was performed in hypoxic conditions (o2 1%) and measured the resistance to apoptosis through hsp 27 and caspase 9 expression of bone marrow mesenchymal stem cells by using immunoflorecence and real time pcr. results: the result of study showed that preconditioning hypoxia could inhibit apoptosis through increasing hsp 27 and decreasing level of caspase 9. conclusion: the study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing caspase 9. key words: mesenchymal stem cells, hypoxia, hsp 27, caspase-9, apoptosis abstrak latar belakang: konsep terapi stem cell merupakan salah satu harapan baru sebagai terapi medis kelainan kelenjar ludah. namun, rendahnya viabilitas stem cell yang ditransplantasikan menyebabkan penurunan efektivitas terapi. asumsi yang mendasari rendahnya viabilitas dan fungsi stem cell adalah tingginya kejadian apoptosis. hal ini menunjukkan bahwa lingkungan mikro di daerah jaringan yang rusak tidak kondusif untuk mendukung viabilitas stem cell. salah satu lingkungan mikro adalah kondisi hipoksia. beberapa jurnal ilmiah mengungkapkan bahwa kondisi hipoksia pada kultur sel dapat menyebabkan sel-sel stres, namun di sisi lain kondisi stres sel juga merangsang heat shock protein 27 (hsp 27) sebagai antiapoptosis dengan menghambat ekspresi caspase 9. tujuan: tujuan penelitian ini adalah untuk meneliti peran protein heat shock 27 sebagai inhibitor apoptosis dalam kondisi hipoksia kultur stem cell sumsum tulang. metode: kultur stem sel dilakukan dalam kondisi hipoksia (o2 1%) dan mengukur resistensi terhadap apoptosis melalui ekspresi hsp 27 dan caspase 9 stem cell mesenchymal sumsum tulang dengan menggunakan immunoflorecence dan pcr real time. hasil: hasil penelitian menunjukkan bahwa prakondisi hipoksia dapat menghambat apoptosis melalui peningkatan hsp 27 dan penurunan tingkat caspase 9. simpulan: studi ini menunjukkan bahwa prakondisi hipoksia dapat mengurangi apoptosis dengan meningkatkan jumlah protein heat shock 27 dan penurunan caspase 9. kata kunci: mesenchymal stem cells, hypoxia, hsp 27, caspase-9, apoptosis correspondence: sri wigati mardi mulyani, c/o: departemen radiologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jl. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: swigati.nina@gmail.com research report �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 41–44 introduction stem cell transplantation has been explored as a therapy to restore the function of various damaged organs like skin, cornea, heart disease etc, but some of them the probability of success to regenerate tissue using cell transplantation still remains uncertain. the possibility to transplant the cells strongly attaching and surviving in the defect area can integrate with the surrounding microenvironment or the so-called stem cell niche. niche stem cell is required to improve and maintain the viability of the transplanted stem cells in salivary gland so it has the same environment microenvironment as micro-physiological conditions and can support the cells of origin of stem cells can proliferate and differentiate into acinar like cells. however, the lack of viability in the form of the survival of the transplanted stem cells results in the effectiveness of stem cell therapy reduced. the temporary underlying assumptions of the decline in the viability and function of stem cells is an increase of incidence of apoptosis.1 the use of stem cells in myocardial infarction case indicated that more than 99% of the stem cells injected into the heart’s left ventricle of adult mice resulted in apoptosis at day 4 after injection.2,3 it suggests that the microenvironment in the damaged tissue is not conducive to support stem cell viability. one of the microenvironments is the hypoxia condition.4,5 hypoxia conditions are an important element of microenvironment but it is often neglected. in the conventional stem cell culture it is done is normoxia conditions (oxygen concentration 21%). however, contrary to the condition of in vivo stem cell requires a hypoxic environment between 1-15% depending on the type of stem cell. this shows that in vitro culture also requires the same treatment as the in vitro physiological environment, so that the success of the therapy can be achieved.6 several scientific journals stated that the administration of hypoxic cell culture can result in the stress of cells which can induce apoptosis.7 the stress condition of the cells, among others, which is triggered by hypoxia also stimulates the release of heat shock protein 27 (hsp 27). hsp 27 is known as antiapoptosis through inhibits cytochrom-c release and caspase-9.13-148,9 there is an assumption that hsp 27 inhibits apoptosis in hypoxia conditions. the purpose of this study was to examine the role of hsp 27 as an inhibitor of apoptosis in hypoxic conditions of bone marrow stem cell culture. the result of study could be served as an adaptive and survival stem cells for therapy of salivary gland defect due to ionized radiation. material and methods the experimental unit in this study was the mesenchymal stem cells (mscs) taken from the crista illiaca bone marrow of male new zealand rabbits at the age of 6 months and done through worthy of conduct research (ethical codes) in the faculty of veterinary medicine, universitas airlangga. this research was exploratory laboratory experimental studies conducted in vitro on stem cell culture. bone marrow mesenchymal stem cell were taken from bone crista illiaca new zealand rabbits were cultured in vitro hypoxic conditions (o21%) in the 3rd passage and compared with normoxia condition (o2 21%). then the incidence of apoptosis, the expression of hsp 27 was analyzed. this research was conducted at the laboratory of stem cell institute of tropical disease (itd) universitas airlangga surabaya. mononuclear cells were separated by centrifugation at 1600 rpm for 10 min at room temperature. the interface was collected and resuspended in phospate buffered saline (pbs). the cells were supended in α modified eagle medium (α mem), supplemented with 10% foetal bovine serum and penicillin-streptomycin sulfate. the cells were plated in 10 cm2 culture dish. the culture was maintaned at 37° c in humidified environment containing 5% co2. after 48 h, the non-adherent cells were removed and medium replaced. when 80-90% confluence was reached,adherentcells was trypsinised with 0.02% trypsin (hyclone, logan, ut) at 37o c for 5 min. then divided into hypoxia precondition treatment oxygen tension 1% and normoxia oxygen tension 21%. the medium was aspirated and washed with 1 ml pbs 2 times, then add 500 µl rnaiso/ well. the cells were scraped and put into the small tube, 100 µl chloroform were added and shook for several times. the supernatant (250-300 µl) was separated into a new tube by centrifugation at 12.000 rpm for 15 minutes at 4o c, then added 250 µl isopropanol and centrifuge in 12.000 rpm for 10 minutes at 4o c. the supernatant was removed from tube, leaving only rna pellet, added 500 µl 75% ethanol vortex until the pellet floating. the samples were centrifuged at 4o c-12.000 rpm for 5 minutes, the supernatant was removed from tube then dry up for 5-10 minutes, then added 25 µl water. the 1µl samples were examined the concentration of rna for measuring by spectrophotometer and acceptable range for purity of rna are 1.7–2.1. the rna sample was added with water (11µl) and the 9 µl mixtures component put into a fresh tube, then mixed with 11 µl rna samples, put it in to thermal cycle machine at 42o c 20 minutes, 99o c 5 minutes, 4o c 5 minutes, 4o c as long as possible. the 20 µl c-dna samples have prepared. data obtained are a single type only and calculated quantitatively with numerical data scale. when the distribution is normal, it can be tested by anova test ��mulyani et al.,: the role of heat shock protein 27 (hsp 27) as inhibitor apoptosis result the morphological of mscs was observed after third passage at 2 days in both cultures of normoxia (o2 21%) and hypoxia (o2 1%), the cells showed mostly have bigger cells size,less cell death and slower proliferation in hypoxia culture, meanwhile in normoxia culture have smaller cells size, more cell death and faster proliferation rate (figure 1). the identification of heat shock protein 27 and caspase9 using real time-pcr showed significantly increasing hsp 27 and decreasing caspase-9 expression in hypoxic condition (o2 1%) (p < 0.01) compared to normoxia condition as a control. discussion hypoxic condition is one of the important elements in the microenvironment of stem cells, because stem cell microenvironment is needed to protect and influence the behavior of stem cells in maintaining the self-renewal capability and viability of the stem cells. in this study on the effect of hypoxia on the incidence of apoptosis through the expression of hsp 27 indicated that the hypoxic precondition had a significant effect on apoptosis resistance. the result of an experiment conducted in a immunocytochemistry showed that in the hypoxic condition (o2 1%), cells undergoing apoptosis were only between 2.45-2.55% after the hypoxic precondition was given with the duration for 24 hours, 48 hours, and 72 hours compared to the number of cells cultured under normoxic condition (o2 21%) in which the number of cells undergoing apoptosis reached 12.5%. in addition, the results of morphological examination using by electron microscopy (magnification of 1000x) showed that the hypoxic-preconditioned stem cells had a slower proliferation, a larger cell shape and fewer dead cells, while in the normoxic condition the cell shape was smaller and flat, the proliferation was faster to make the cells confluence resulting in more dead cells. it indicated that the stem cells cultured in vitro also required the same treatment as the condition in vivo, because physiologically stem cells require a lower oxygen concentration (hypoxia) to maintain the viability and the ability to selfrenewal, depending on the type and location of the cells. the results was accordance with the study reported that the figure 1. the morphological of bmscs after third passage at 2 days in normoxia with α mem + 10% fbs medium’s cultures. cell death (arrow). a. hypoxia b. normoxia 5 hypoxia normoxia figure 1. the morphological of bmscs after third passage at 2 days in normoxia with α mem + 10% fbs medium’s cultures. cell death (arrow). the identification of heat shock protein 27 and caspase-9 using real time-pcr showed significantly increasing hsp 27 and decreasing caspase-9 expression in hypoxic condition (o2 1%) (p < 0.01) compared to normoxia condition as a control. ** p<0.01 figure 2. identification of hsp 27 by using real time-pcr showed significantly increasing hsp 27 expression (p < 0.01) in hypoxic precondition (o2 1%). ** ** ** n 5 hypoxia normoxia figure 1. the morphological of bmscs after third passage at 2 days in normoxia with α mem + 10% fbs medium’s cultures. cell death (arrow). the identification of heat shock protein 27 and caspase-9 using real time-pcr showed significantly increasing hsp 27 and decreasing caspase-9 expression in hypoxic condition (o2 1%) (p < 0.01) compared to normoxia condition as a control. ** p<0.01 figure 2. identification of hsp 27 by using real time-pcr showed significantly increasing hsp 27 expression (p < 0.01) in hypoxic precondition (o2 1%). ** ** ** n figure 2. identification of hsp 27 by using real time-pcr showed significantly increasing hsp 27 expression (p < 0.01) in hypoxic precondition (o2 1%). 6 note: ** p<0.01 figure 3. identification of caspase 9 by using rt-pcr showed significantly decreasing of caspase 9 expression (p < 0.01) in hypoxic precondition (o2 21%). discussion hypoxic condition is one of the important elements in the microenvironment of stem cells, because stem cell microenvironment is needed to protect and influence the behavior of stem cells in maintaining the self-renewal capability and viability of the stem cells. in this study on the effect of hypoxia on the incidence of apoptosis through the expression of hsp 27 indicated that the hypoxic precondition had a significant effect on apoptosis resistance. the result of an experiment conducted in a immunocytochemistry showed that in the hypoxic condition (o2 1%), cells undergoing apoptosis were only between 2.45-2.55% after the hypoxic precondition was given with the duration for 24 hours, 48 hours, and 72 hours compared to the number of cells cultured under normoxic condition (o2 21%) in which the number of cells undergoing apoptosis reached 12.5%. ** ** ** figure 3. identification of caspase 9 by using rt-pcr showed significantly decreasing of caspase 9 expression (p < 0.01) in hypoxic precondition (o2 21%). note: **p<0.01 figure 5. i d e n t i f i c a t i o n o f a p o p t o s i s e v i d e n c e u s i n g immumocytochemistry in normoxia and precondition hypoxia. (a) positive expression of apoptosis evidence in normoxia (arrow), number of apoptosis reached 12.5%; (b). hipoxia precondition 24 h, 48 h, 72 h, showed negative expression of apoptosis (arrow), have a small number of apoptosis cells (2.452.55%). 7 figure 5. identification of apoptosis evidence using immumocytochemistry in normoxia and precondition hypoxia. (a) positive expression of apoptosis evidence in normoxia (arrow), number of apoptosis reached 12.5%; (b). hipoxia precondition 24 h, 48 h, 72 h, showed negative expression of apoptosis (arrow), have a small number of apoptosis cells (2.45-2.55%). in addition, the results of morphological examination using by electron microscopy (magnification of 1000x) showed that the hypoxic-preconditioned stem cells had a slower proliferation, a larger cell shape and fewer dead cells, while in the normoxic condition the cell shape was smaller and flat, the proliferation was faster to make the cells confluentce resulting in more dead cells. it indicated that the stem cells cultured in vitro also required the same treatment as the condition in vivo, because physiologically stem cells require a lower oxygen concentration (hypoxia) to maintain the viability and the ability to selfrenewal, depending on the type and location of the cells. the results matched with the study reported that the hypoxic precondition of mscs cultured could maintain the viability and the proliferation rate and had the better ability of self-renewal.10 physiologically stem cells required o2 concentration between 1-7%, hypoxic condition is needed by the stem cells to maintain their plasticity or differentiation power.5,6 the result of an examination on the effect of hypoxia on the hsp 27 with the use of real time pcr indicated that hypoxic precondition affect the increase of the expression of hsp 27 at 24 hours, 48 hours, and 72 hours after the treatment compared to normoxic condition. it indicates that the treatment of hypoxia in cultured cells results in stress cells stimulating the release of hsp 27 as an anti-apoptosis. it also explains how hypoxia influences the decline in the incidence of apoptosis in the stem cells culture through the stimulation of the release of heat shock protein 27 as an a b 7 figure 5. identification of apoptosis evidence using immumocytochemistry in normoxia and precondition hypoxia. (a) positive expression of apoptosis evidence in normoxia (arrow), number of apoptosis reached 12.5%; (b). hipoxia precondition 24 h, 48 h, 72 h, showed negative expression of apoptosis (arrow), have a small number of apoptosis cells (2.45-2.55%). in addition, the results of morphological examination using by electron microscopy (magnification of 1000x) showed that the hypoxic-preconditioned stem cells had a slower proliferation, a larger cell shape and fewer dead cells, while in the normoxic condition the cell shape was smaller and flat, the proliferation was faster to make the cells confluentce resulting in more dead cells. it indicated that the stem cells cultured in vitro also required the same treatment as the condition in vivo, because physiologically stem cells require a lower oxygen concentration (hypoxia) to maintain the viability and the ability to selfrenewal, depending on the type and location of the cells. the results matched with the study reported that the hypoxic precondition of mscs cultured could maintain the viability and the proliferation rate and had the better ability of self-renewal.10 physiologically stem cells required o2 concentration between 1-7%, hypoxic condition is needed by the stem cells to maintain their plasticity or differentiation power.5,6 the result of an examination on the effect of hypoxia on the hsp 27 with the use of real time pcr indicated that hypoxic precondition affect the increase of the expression of hsp 27 at 24 hours, 48 hours, and 72 hours after the treatment compared to normoxic condition. it indicates that the treatment of hypoxia in cultured cells results in stress cells stimulating the release of hsp 27 as an anti-apoptosis. it also explains how hypoxia influences the decline in the incidence of apoptosis in the stem cells culture through the stimulation of the release of heat shock protein 27 as an a b �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 41–44 hypoxic precondition of mscs cultured could maintain the viability and the proliferation rate and had the better ability of self-renewal.10 physiologically stem cells required o2 concentration between 1-7%, hypoxic condition is needed by the stem cells to maintain their plasticity or differentiation ability.5,6 the result of an examination on the effect of hypoxia on the hsp 27 with the use of real time pcr indicated that hypoxic precondition affect the increase of the expression of hsp 27 at 24 hours, 48 hours, and 72 hours after the treatment compared to normoxic condition. it indicates that the treatment of hypoxia in cultured cells results in stress cells stimulating the release of hsp 27 as an anti-apoptosis. it also explains how hypoxia influences the decline in the incidence of apoptosis in the stem cells culture through the stimulation of the release of heat shock protein 27 as an antiapoptosis through inhitation of cytochrom-c and caspase 9 release. it is proven by the significant reduction of caspase 9 at 24 hours, 48 hours, and 72 hours after hypoxic precondition but in normoxic condition it increases. these results are consistent with the study resulted that the treatment of hypoxia preconditions increased stress cells, induced apoptosis and stimulating the release of heat shock protein, acting as an antiapoptosis through the barriers of cytochrom-c.7,8,9 so it can be stated that hsp 27 may act as an inhibitor of apoptosis in hypoxic conditions of cultured cells, generally in vivo hsp 27 acts as a thermo tolerance, cytoprotection and survival cells under stress conditions.11-13 the study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing capase 9. so it can strongly be attached, survive and integrate into the microenvironment of the original cells to achieve the success of therapy. acknowledgement this research was supported by a grant from upt research program funded by the ministry of educational culture–indonesia. references 1. coppess rp, stokman ma. stem cells and the repair of radiationinduced salivary gland damage. j oral disease 2011; 17(2): 14353. 2. dong z, wang jz, yu f, venkatachalam ma. apoptosis resistance of hypoxic cells: multiple factors involved and a role iap-2. am j pathol 2003; 163(2): 663-71. 3. petreaca m, green mm. the dynamic of cell-ecm ineraction. in: lanza r, langer r, vacanti j, eds. principles of tissue engineering. 3rd ed. burlington, ma: elsevier academic press; 2007. p. 81-92. 4. tang yl, tang y, zhang yc, qian k, shen l, phillips mi. improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia regulated heoxygenase1 vector. j am coll cardiol 2005; 46(7): 1339-50. 5. chow dc, wenning la, miller wm, papoutsakis et. modeling po(2) distributions in the bone marrow hametopoietic compatement. ii. modified kroghian models. biophys j 2001; 81: 685-96. 6. bizzari a, koehler h, cajlakovic m, pasic a, schaupp l, klimant i, ribitsch v. continuous oxygen monitoring in subcutaneous adipose tissue using microdialysis. analytica chimica aca 2006; 573: 4856. 7. d’ippolito g, diabira s, howard ga, roos ba, schiller pc. low oxygen tension inhibits osteogenic differentiation and enhances stemness of human miami cells. bone 2006; 39(3): 513-22. 8. yoon jh, gores gj. death receptor apoptosis and the liver. j hepatol 2002; 37(3): 400-10. 9. coppess rp, stokman ma. stem cells and the repair of radiationinduced salivary gland damage. j oral disease 2011; 17(2): 14353. 10. charette sj, lavoie jn, lambert h, landry j. inhibition of daxxmediated apoptosis by heat shock protein 27. mol cell biol 2000; 20(20): 7602-12. 11. carpenter hg, cotroneo e. salivary gland regeneration. in: tucker as, miletich i, eds. salivary gland: development, adaptations and disease. renhardt druck publisher 2010; 14: 107-28. 12. van montfort r, slingsby c, vierling e. structure and function of small heat shock protein/alpha-crystalin family of molecular chaperon. adv protein chem 2001; 59: 105-56. 13. greijer ae. the role of hypoxia inducible factor 1 (hif-1) in hypoxia induced apoptosis. j clin pathol 2012; 57: 1009-14. 104104 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 104–108 original article evaluation of bsp and dmp1 in hydroxyapatite crab shells used for dental socket preservation michael josef kridanto kamadjaja,1 sherman salim1, wiwik herawati waluyo2, tengku natasha eleena binti tengku ahmad noor3,4 1department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2resident of prosthodontics department, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3membership of faculty of dental surgery, royal college of surgeon, edinburgh university, united kingdom 4malaysian armed forces dental officer, 609 armed forces dental clinic, kem semenggo, kuching, serawak, malaysia abstract background: bone resorption due to tooth extraction leads to unpredictable bone volume for future prosthetics. crab shells were promoted as a solution to prevent bone resorption, along with an effort to reduce biological waste. purpose: this study aimed to analyze the expression of bone sialoprotein (bsp) and dentine matrix protein-1 (dmp1) in the wound healing process in tooth-extraction sockets after applying a crab shell-derived hydroxyapatite scaffold. methods: the subjects (28 cavia cobaya) were divided into control and treatment groups. the control group was left untreated, while the treatment group received a hydroxyapatite scaffold of portunus pelagicus shell in the tooth socket. the expression of bsp and dmp1 was determined by immunohistochemical staining on days 7 and 14. one-way analysis of variance and tukey’s honest significance difference test were used to find the groups with the most significant difference. results: the highest mean expression of bsp and dmp1 was in the day 14 treatment group, while the lowest was in the day 7 control group. conclusion: administering hydroxyapatite scaffold derived from the portunus pelagicus shell to the post-extraction sockets increased the expression of both bsp and dmp1. keywords: bsp; crab shell; dmp1; hydroxyapatite; medicine article history: received 13 june 2022; revised 31 august 2022; accepted 5 september 2022 correspondence: michael josef kridanto kamadjaja, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo 47, surabaya 60132, indonesia. email: michael-j-k-k@fkg.unair.ac.id introduction the changes in bone volume after tooth extraction seem to be physiological consequences, where ‘unnecessary’ bone which does not receive strain stimulus is eliminated.1 therefore, a bone graft is the solution to maintain the height of the alveolar bone for future denture prosthesis treatment.2-4 hydroxyapatite is the most widely used alloplastic bone graft material. it is well known for its osteoinductive properties in new bone regeneration since its structure is very close to normal bone and is available on the organic matrix.5,6 portunus pelagicus, a crab species, is one of indonesia’s leading fishery export commodities. the shells of the crab contain 40–70% calcium carbonate. suitably processing the calcium carbonate can turn it into calcium hydroxyapatite, which is helpful in osteogenesis. considering the value of crab shells and the amount of waste they generate, a recycling effort is carried out so that the existing waste can be controlled and utilized as well as possible. lamongan district in indonesia is one of the largest contributors to these commodities (19.4%).7 abundant hydroxyapatite sources include mammalian bone, marine or aquatic creatures, shell sources, plant or algae, and mineral sources.8 hydroxyapatite from crab shells is a new idea. the main reason behind this idea was to reduce the biological waste in the local environment. the solution offered in this study was to minimize biological waste while recycling it for use in the bone graft needed to prevent bone resorption. bone sialoprotein (bsp), one of the non-collagen proteins of extracellular matrix (ecm), is produced by osteoblasts and osteoclasts. the increase in bsp corresponds to the increase in bone mineralization. the copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p104–108 mailto:michael-j-k-k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p104-108 105 kamadjaja et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 104–108 existence of bsp is influenced by the role of runt-related transcription factor 2 and alkaline phosphatase.9 a study on mice showed that cementum decreased significantly in the absence of bsp. long bone length and bone formation rates also decreased with cortical thinning.10 dentin matrix protein-1 (dmp1) is another non collagen protein of ecm, expressed by osteoblasts, osteocytes, and hypertrophic chondrocytes. the role of dmp1 in osteogenesis is the maturation of odontoblasts, osteoblasts, and mineralization. studies have found that dmp1-deficient mice revealed severe defects in cartilage formation, such as in hereditary hypophosphatemic rickets.11 osteoblasts abundantly express bsp, especially at sites of primary bone formation. bsp is also known for its ability to promote osteoblast differentiation and increased production of mineralized matrix.12 high levels of bsp and dmp1 were observed on days 7 and 14 when the remodeling phase of wound healing begins. this study aimed to analyze the expression of bsp and dmp1 in tooth extraction sockets after applying a p. pelagicus shell-derived hydroxyapatite scaffold. bsp expression was studied to track the osteoblast differentiation around extraction sockets, while dmp1 was observed as a marker for bone ecm protein responsible for bone development. materials and methods the health research ethical clearance commission of the faculty of dental medicine, universitas airlangga, approved this study with certificate number 548/hrecc. fodm/xii/2020. the design for this study was a post-testonly control group design. twenty-eight male cavia cobaya (guinea pigs) were the subjects (the number featured in federer’s formula according to a similar study previously conducted by kresnoadi et al.).13 the requirements for the subjects were as follows: adult cavia cobaya (3–3.5 months old) in good health, weighing 300–350 grams. the c. cobaya were habituated for one week before the experiment was conducted. they received standard food pellets and water and were exposed to a 12-hour light/ dark cycle. these subjects were then randomly assigned to the following groups: control group 7 (c7), control group 14 (c14), treatment group 7 (t7), and treatment group 14 (t14). this study was carried out from january to may 2020. crab shells were obtained from a 3-month-old p. pelagicus on a beach in lamongan, east java. these shells were cleaned of soft tissue using distilled water before being soaked in a chlorine solution with a ratio of thirty ml to five liters of water. the samples were soaked in hydrogen peroxide 3% for 24 hours before being dried at room temperature. the heating process of shell calcination was as follows: the initial temperature during heating was approximately 50°c with a gradual increase of 5°c/ copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p104–108 minute to 1,000°c in a furnace. the temperature was then maintained at a stable 1,000°c for two hours and decreased naturally to approximately 100°c. a scanning electron microscope with energy-dispersive x-ray was utilized to characterize hydroxyapatite compounds. the process involved the mechanical sifting of powder to produce hydroxyapatite powder with a particle size of approximately 150–350 μm.14 five grams of gelatin were poured slowly into distilled water and mixed at 40°c for one hour. the hydroxyapatite gelatin composite was produced by adding 1.5 grams of hydroxyapatite powder to the gelatin solution and stirring it for six hours, as described previously by kamadjaja et al.15 the process was continued with centrifugation for ten minutes to isolate the water from the gel. the gel solution was transferred to a mold (2 mm in diameter and 5 mm in height), stored in a freezer for 24 hours at a temperature of -80 °c, and freeze-dried for 24 hours.16 the c. cobaya were injected 20 mg per 300 mg body weight of ketamine intramuscularly (kepro, za, denmark) for sedation and anesthesia. before tooth extraction, the left mandibular incisive tooth area was debrided. then, tooth extraction was done carefully using a sterile needle holder to prevent root fracturing. the sockets of the control group members (c7 and c14) were left untreated. those in the treatment groups (t7 and t14) were administered up to 1 ml of the gelatin-hydroxyapatite scaffold, depending on the volume of the tooth socket. simple suturing was used in all groups using polyamide monofilament ds 12 3/ 8c, 12 mm, 6/10 met, 0.7 (braun vetcare sa, rubi, spain).13 the c. cobaya were sacrificed on days 7 and 14 by administering a lethal dose of ketamine (kepro, za, denmark). the mandibles of the c. cobaya were cut medio sagitally. the mandibular samples were fixed with a 10% formalin buffer for 24 hours at 80℃ and decalcified with 2% nitric acid. dehydration was then performed using graded alcohol concentrations (decreasing from 100% to 70%), followed by clearing in xylol and embedding in paraffin. paraffin blocks were cut to a thickness of four microns and placed in an object glass.17 the tissue deparaffinization process was completed using a solution of xylol, ethanol, and alcohol. processing of the tissues was continued with a 3.3’-diaminobenzidine (dab) staining kit (pierce™ dab substrate paint kit 34002, thermofisher™, massachusetts, united states). the tissues were incubated at room temperature with primary antibodies to bsp (santacruz biotech, cat#sc7360) and dmp1 (santacruz biotech, cat#sc-73633). after adding the dab buffer solution, the antibody complex was observed under a light microscope (nikon eclipse e 100, japan). the observation area was specified as the apical third of the socket.17 the ibm statistical package for the social sciences, statistics for windows, version 24.0. (armonk, ny: ibm corp) was used in this study. the results were shown as means and standard deviations. the one-sample kolmogorov smirnov test, the levene’s test, one-way https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p104-108 106kamadjaja et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 104–108 analysis of variance, and tukey’s honest significance difference test were utilized to study the differences between groups. results the statistical analysis demonstrated a significant difference (p < 0.05) between the control and treatment groups. the control group (c7) expressed a significant difference compared with the two treatment groups (t7 and t14) (p < 0.05). similarly, the control group (c14) also showed a significant difference compared with the 14-day treatment group (t7 and t14) (p < 0.05) (tables 1 and 2, and figure 1). a surge of bsp was observed in the post-extraction sockets in more than half the subjects in the treatment groups from day 7 to day 14. the highest bsp expression was observed in the treatment group on day 14, while the lowest was in the control group on day 7. figure 1 shows the expression of bsp as indicated by the arrows. osteoblasts that synthesized bsp are marked by brown tinting of their cells. osteoblast cells in the matrix near the lining cells appear as cuboidal or polygonal cells. the control group (c7) demonstrated a significant difference as compared with the treatment groups (t7 and t14) (p < 0.05). similarly, the control group (c14) showed a significant difference compared with the treatment groups (t7 and t14) (p < 0.05) (tables 1 and 2). there was an increase in the amount of dmp1 expression in the postextraction socket in most of the subjects in the treatment table 1. mean expression of bsp and dmp1 on days 7 and day 14 group day bsp dmp1 ʃ samples mean standard deviation σ samples mean standard deviation control 7 7 6.14 1.773 7 6.43 3.35914 7 7.86 1.952 7 8.43 1.902 treatment 7 7 12.29 1.976 7 12.43 1.71814 7 14.43 2.507 7 14.71 2.812 total 28 10.18 2.052 28 10.5 2.447 table 2. tukey’s honest significance difference test results. c7: control group on day 7, c14: control group on day 14, t7: treatment group on day 7, t14: treatment group on day 14 group bsp dmp1c7 c14 t7 t14 c7 c14 t7 t14 c7 * * * * c14 * * * * t7 * * * * t14 * * * * the asterisk (*) symbol represents groups with significant differences (p < 0.05). figure 1. the black arrows point to the expression of bsp observed beneath a 1000x magnification light microscope. a: the control group on day 7 (c7). b: the control group on day 14 (c14). c: tooth extraction and crab shell hydroxyapatite application on day 7 (t7). d: tooth extraction and crab shell hydroxyapatite application on day 14 (t14). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p104–108 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p104-108 107 kamadjaja et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 104–108 groups between days 7 and 14. the highest level of dmp1 expression was detected in the treatment group on day 14, while the lowest occurred in the control group on day 7. the expression of dmp1 in the histological field is shown by the arrows (figure 2). osteoblasts that synthesized bsp are marked by brown tinting of their cells. osteoblast cells in the matrix near the lining cells appear as cuboidal or polygonal cells. discussion the groups with the hydroxyapatite scaffold from the crab shell demonstrated a significant amount of bsp and dmp1 expressions on the 14th day. the highest bsp and dmp1 expressions were found in the hydroxyapatite scaffold group on day 14. the increase in the amount of bsp and dmp1 is due to the role of hydroxyapatite in regulating bone formation. as per this theory, hydroxyapatite progressively reduces the activity of osteoclasts, decreasing bone resorption activity. on the other hand, adding hydroxyapatite can also improve the formation and differentiation of osteoblasts. osteoblasts play a role in adhering and developing effectively within bone defects, resulting in the stability of the wound due to cartilage (soft callus). in the later stage, the soft callus becomes a hard callus (bone).18 bone sialoprotein and dmp1 are members of the small integrin-binding ligand n-glycosylated family, secreted into the ecm during bone formation. mineralization of ecm facilitates the deposition of hydroxyapatite. although bsp can be found at the onset of bone formation, excess expression of bsp in osteoblasts appears to increase during mineralization.9-11 bsp can also trigger hydroxyapatite crystal nucleation and osteoblast differentiation.19 as observed in this study, levels of bsp rose significantly in the t7 and t14 groups. bsp increases slowly because the proliferation process is ongoing and the mineralization process is imperfect; hence the actual value of their increase is of no great significance. consequently, it is sufficient to conduct regular inspections until the 7th day, and further examination up to and including the 14th day is unnecessary. in addition to bsp, dmp1 is a fossilized acid ecm that binds to hydroxyapatite and mediates cell attachment through the arginyl-glycyl-aspartic acid domain. the existence of dmp1 is closely related to osteocytes and pericytes.20 in dmp1, bone is processed into fragments of 37 kda derived from the n-terminal for growth and proliferation, and fragments of 57 kda derived from the cooh-terminal (containing peptide acidic serine aspartate-rich matrix extracellular phosphoglycoproteinassociated) of the calcification and ossification zone.9-11 therefore, the expression of dmp1 rose significantly in the t7 and t14 groups compared with their c7 and c14 counterparts. however, this increase was not significant, suggesting that dmp1 increases slowly since the proliferation is ongoing while the mineralization process is imperfect. therefore, analysis up to and including the seventh day is sufficient, with further examination until the 14th day being redundant. a previous similar study using p. pelagicus shell in the tooth socket after tooth extraction examined its effect on tumor necrosis factor-alpha, osterix, receptor activator of nuclear factor kappa-β ligand, and osteoprotegerin.21,22 its effect on bone matrix mineralization has not yet been figure 2. the black arrows point to the expression of dmp1 beneath a 1000x magnification light microscope. a: the control group on day 7 (c7). b: the control group on day 14 (c14). c: tooth extraction and crab shell hydroxyapatite application on day 7 (t7). d: tooth extraction and crab shell hydroxyapatite application on day 14 (t14). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p104–108 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p104-108 108kamadjaja et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 104–108 explored. this stage is essential, particularly in future bone maturation and strength. in this study, bsp and dmp1 were increased in the treatment group from day 7 to day 14. the mean expression of the positive cells from the treatment group was significantly higher than in the control group, proving that the p. pelagicus shell induces bone matrix mineralization and density. this study could be refined more effectively later, given the exceptionally significant role of hydroxyapatite scaffold derived from crab shells in wound healing. furthermore, this research was limited and needed another marker to confirm the bone regeneration process. from the discussion above, it can be concluded that applying a hydroxyapatite scaffold derived from the p. pelagicus shell to the post-extraction sockets increases the expression of both bsp and dmp1 as documented immunohistochemically, in vivo. references 1. hansson s, halldin a. alveolar ridge resorption after tooth extraction: a consequence of a fundamental principle of bone physiology. j dent biomech. 2012; 3: 1758736012456543. 2. kamadjaja mj, tumali ba, laksono h, hendrijantini n, ariani ml, natasia, mawantari tp. effect of socket preservation using crab shell-based hydroxyapatite in wistar rats. recent adv biol med. 2020; 6(2): 1116232. 3. saskianti t, nugraha ap, prahasanti c, ernawati ds, tanimoto k, riawan w, kanawa m, kawamoto t, fujimoto k. study of alveolar bone remodeling using deciduous tooth stem cells and hydroxyapatite by vascular endothelial growth factor enhancement and inhibition of matrix metalloproteinase-8 expression in vivo. clin cosmet investig dent. 2022; 14: 71–8. 4. muthusubramanian v, harish km. alveolar bone grafting. in: oral and maxillofacial surgery for the clinician. singapore: springer nature singapore; 2021. p. 1655–73. 5. naini a, rachmawati d. physical characterization and analysis of tissue inflammatory response of the combination of hydroxyapatite gypsum puger and tapioca starch as a scaffold material. dent j. 2023; 56(1): 53–7. 6. prahasanti c, setijanto d, ernawati ds, ridwan rd, kamadjaja db, yuliati a, meizarini a, hendrijantini n, krismariono a, supandi sk, saskianti t, sitalaksmi rm, kuswanto d, putri ts, ramadhani nf, ari mda, nugraha ap. utilization of polymethyl methacrylate and hydroxyapatite composite as biomaterial candidate for porous trabecular dental implant fixture development: a narrative review. res j pharm technol. 2022; 15(4): 1863–9. 7. badan pusat statistik indonesia. data ekspor-impor 2012-2017. jakar ta; 2018. available from: https://www.bps.go.id /exim /. accessed 2021 mar 23. 8. cahyaningrum s, herdyastuty n, wiana f, devina b, supangat d. synthesis of hydroxyapatite from crab shell (scylla serrata) waste with different methods added phosphate. in: proceedings of the seminar nasional kimia national seminar on chemistry (snk 2018). paris, france: atlantis press; 2018. p. 67–9. 9. nugraha ap, narmada ib, ernawati ds, dinaryanti a, hendrianto e, ihsan is, riawan w, rantam fa. in vitro bone sialoprotein-i expression in combined gingival stromal cells and platelet rich fibrin during osteogenic differentiation. trop j pharm res. 2018; 17(12): 2341–5. 10. lin x, patil s, gao y-g, qian a. the bone extracellular matrix in bone formation and regeneration. front pharmacol. 2020; 11: 757. 11. staines ka, macrae ve, farquharson c. the importance of the sibling family of proteins on skeletal mineralisation and bone remodelling. j endocrinol. 2012; 214(3): 241–55. 12. bouet g, bouleftour w, juignet l, linossier m-t, thomas m, vanden-bossche a, aubin je, vico l, marchat d, malaval l. the impairment of osteogenesis in bone sialoprotein (bsp) knockout calvaria cell cultures is cell density dependent. plos one. 2015; 10(2): e0117402. 13. kresnoadi u, lunardhi lc, agustono b. propolis extract and bovine bone graft combination in the expression of vegf and fgf2 on the preservation of post extraction socket. j indian prosthodont soc. 2020; 20(4): 417–23. 14. raya i, mayasari e, yahya a, syahrul m, latunra ai. shynthesis and characterizations of calcium hydroxyapatite derived from crabs shells (portunus pelagicus) and its potency in safeguard against to dental demineralizations. int j biomater. 2015; 2015: 469176. 15. kamadjaja mjk, abraham jf, laksono h. biocompatibility of portunus pelagicus hydroxyapatite graft on human gingival fibroblast cell culture. med arch (sarajevo, bosnia herzegovina). 2019; 73(5): 303–6. 16. hendrijantini n, kresnoadi u, salim s, agustono b, retnowati e, syahrial i, mulawardhana p, wardhana mp, pramono c, rantam fa. study biocompatibility and osteogenic differentiation potential of human umbilical cord mesenchymal stem cells (hucmscs) with gelatin solvent. j biomed sci eng. 2015; 8(7): 420–8. 17. kresnoadi u, widjaja j, laksono h. ethanol extract of propolisbovine bone graft combination as a prospective candidate for socket preservation: enhancing bmp7 and decreasing nfatc1. saudi dent j. 2021; 33(8): 1055–62. 18. shrivats ar, alvarez p, schutte l, hollinger jo. bone regeneration. in: lanza r, langer r, vacanti j, editors. principles of tssue engineering. 4th ed. elsevier; 2014. p. 1201–21. 19. ponzetti m, rucci n. osteoblast differentiation and signaling: established concepts and emerging topics. int j mol sci. 2021; 22(13): 6651. 20. prasadam i, zhou y, shi w, crawford r, xiao y. role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis. rheumatology. 2014; 53(12): 2280–7. 21. k a madjaja m j k , sa l i m s, subia k to bds. appl icat ion of hydroxyapatite scaffold from portunus pelagicus on opg and rankl expression after tooth extraction of cavia cobaya. res j pharm technol. 2021; 14(9): 4647–51. 22. salim i, kamadjaja mjk, dahlan a. tumor necrosis factor-α and osterix expression after the transplantation of a hydroxyapatite scaffold from crab shell (portunus pelagicus) in the post-extraction socket of cavia cobaya. dent j. 2022; 55(1): 26–32. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p104–108 https://www.bps.go.id/exim/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p104-108 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with the left, right, top, and bottom margin should be 2.5 cm or 1 inch length, printed on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. authors should also follow the manuscript preparation guidelines. format for research reports: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in research reports should consists of “background:”, “purpose:”, “methods:”, “results:” and “conclusion:” typed in bold within one paragraph. • key words contain 3–5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on computer, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustrations, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all research reports should have more than 10 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205–9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005.2005. available at: http://www.dephut.go.id/informasi/humas/lebah. htm. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8–21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 format for case reports: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. tooth nomenclature must be explained, whether using zygmondy system, world health organization system, or universal system. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all case reports should have more than 10 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005. available2005. available at: http://www.dephut.go.id/informasi/humas/lebah.htm. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8–21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 format for literature reviews: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in single space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. footnotes, references, and abbreviations are not used in the abstract. abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia, with standard scientific phrase or word. • correspondence should contain details of the author in charge with detailed mailing address and e-mail (consists of full name, name of institution, mailing address, telephone number, fax number and email address). • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. • review contains points and detailed matters based on literature which correlates with the discussed subject, to be discussed in the discussion section. • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. all literature reviews should have more than 30 references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530–5.. 2. fekonja a. hypodontia in orthodontically treated children. eur j of orthod. 2005; 27: 457–60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205–9, 231–48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. citation format for electronic publications: 1. departemen kehutanan. perlebahan di indonesia. 2005. available2005. available at: http://www.dephut.go.id/informasi/humas/lebah.html. accessed december 25, 2009. 2. yu f. management of thumbs duplication. emerg infect dis (on line) available at: http://www.cdc.gov/ncidod/ eid/eid.html. 1997. accessed april 30, 2010. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. prosiding timnas v & lustrum xvi. surabaya; 2009. p. 16–20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. prosiding temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131–4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: pascasarjana universitas airlangga; 2008. p. 8–21. citation format for patents: 1. setijanto d. tusuk gigi bentuk setengah bulat. hc-h3.02. p01.012.1796/2002 all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (in jpeg or tiff format). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. �0 side effects of mercury in dental amalgam titiek berniyanti and ninuk hariyani faculty of dentistry airlangga university of dental public health department surabaya indonesia abstract dental amalgam is an alloy composed of mixture of approximately equal parts of elemental liquid mercury and an alloy powder. the popularity of amalgam arises from excellent long term performance, ease of use and low cost. despite the popularity of dental amalgam as restorative material, there have been concerns regarding the potential adverse health and environmental effects arising from exposure to mercury in amalgam. they have long been believed to be of little significance as contributors to the overall body burden of mercury, because the elemental form of mercury is rapidly consumed in the setting reaction of the restoration. in 1997, 80% of dentist in indonesia still using amalgam as an alternative material, and 60% of them treat the rest of unused amalgam carelessly. in recent years, the possible environmental and health impact caused by certain routines in dental practice has attracted attention among regulators. as part of point source reduction strategies, the discharge of mercury/amalgam-contaminated wastes has been regulated in a number of countries, even though it has been documented that by adopting appropriate mercury hygiene measures, the impact of amalgam use in dentistry is minimal. the purpose of this paper is to examine on studies that relate mercury levels in human to the presence of dental amalgams. it is concluded that even though mercury used in filling is hazardous, if normal occupational recommendations for proper mercury hygiene routines and source of reduction strategies are followed, no occupational health risk can be assumed. key words: amalgam, mercury, chemical hazard correspondence: titiek berniyanti, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jln. mayjend prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: urge01@rad.nat.id; ninuk_hariyani@yahoo.co.id. telp. 031 5018347 introduction amalgam fillings typically compose 50% pure elemental mercury, 35% silver, 13% tin, 2% copper, and a trace of zinc.1,2 the metal powders react with liquid mercury to produce an amalgam (or alloy) that provides a flexible material that can be easily packed and shaped.2,3 amalgam fillings are often called silver fillings because of its appearance and composition.2,4 the american dental association (ada) prefers the use of amalgam because the fillings are inexpensive and durable, while gold and other composite materials are more expensive and more difficult to fit. because of its flexibility, the use of amalgam arguably requires less skill. thus, dentists can usually fill a cavity in less time. other reasons behind ada’s support of amalgam may include ease of use, low cost, have excellent long-term performance,5 additional training and equipment required to use alternative materials3 and potential liability associated with acknowledging the dangers of amalgam previously used. there are many countries, including indonesia still using amalgam as an alternative material. in 1997, moetmainah6 said that around 80 % of dentist in indonesia use it. unfortunately 60% of them treat the residue carelessness. despite the ada recommendation of using amalgam filling, the medical scientific community is now in general agreement, that patient with dental amalgam filling are chronically exposed to mercury. it is clear from the subcommittee’s review that a fraction of the mercury in amalgam is absorbed by the body. people with amalgam have higher concentrations of mercury in various tissues (including blood, urine, kidney, and brain) than those without amalgam. also, a small proportion of individuals may manifest allergic reactions to these restorations. the average daily absorption of mercury from dental amalgam according to who, 1991, around 3 to 17 µl per day and that 1.25 to 6.6 times the average mercury absorption from dietary sources.7 mercury is absorbed from many sources, including food and ambient air. thus, it is not known whether the vast majority of people with amalgam experience has any clinical effect from this small additional body burden of mercury and this is the key question which must be answered in order to resolve the issue of whether amalgam poses a public health risk or not. dental technicians and patients should be carefully in using dental amalgam, because of the side effects and do some efforts to minimize it. this article will first examine the history of amalgam fillings. second, it will review available research to demonstrate the potential health hazards of mercury in amalgam filling, including clinical side-effects and environmental aspects. third, this paper will discuss some efforts which can do by medical ��berniyanti and hariyani: side effects of mercury in dental amalgam technician to minimize the potential health hazards of mercury in amalgam fillings. the history of amalgam fillings dental amalgam is an alloy composed of a mixture of approximately equal parts of elemental liquid mercury and an alloy powder.5,8 the first use of amalgam was recorded in the chinese literature in the year 659,5,9 and for the last 150 years, amalgam has been the most popular and effective restorative material used in dentistry. before the 1970s, amalgam accounted for more than 75 percent of all restorations.5,10 during the past 20 years, however, the use of amalgam has been declining, largely due to the decreasing incidence of dental caries, more frequent use of crowns and the availability of tooth-colored alternative restorative materials for certain applications.5 however, because of their strengthness, they still use as an alternative restoration until now. clinical side effects to dental amalgam it has been well-documented and referenced that classic signs of chronic mercury exposure is including gingivitis, alveolar bone loss, loosening and loss of teeth, bruxism, metallic taste, oral ulceration, and excessive salivation.6 besides that, symptoms associated with mercury toxicity can be characterized by tremor, ataxia, personality change, loss of memory, insomnia, anxiety, fatique, depression, headaches, irritability, slowed nerve conduction, weight loss, appetite loss, gastrointestinal problems, and psychological distress.5,11 mercury vapor absorption occurs through the lungs, with about 80% of the inhaled vapor being absorbed by the lungs and rapidly entering the blood circulation, so that mercury can enter and remain in certain tissues for longer periods of time, since the half-life of excretion is prolonged. two of the primary target organs of concern are the central nervous system and kidneys. there have been three recent autopsy studies relating the presence of dental amalgams in humans to mercury levels in tissue. the studies indicate that mercury vapor exposure from dental amalgams appear to contribute to tissue burdens, especially in the cns, kidneys, and certain glands.12 mercury also has side effects on oral and periodontal. trivedi and talim13 showed a histological analysis of gingival tissue adjacent to amalgam restoratives. an inflammatory reaction occurred at 62.5% of the tissue sites in contact with amalgam, and proliferation of epithelium occurred in 68.7% of the sites adjacent to amalgam. turgeon et al.14 have a clinical investigation on 30 amalgam class ii fillings that compare with contra lateral sites without amalgam fillings served as controls. they found that clinical procedures involved in restoring posterior teeth with class ii amalgam restorations caused an immediate gingival inflammation characterized by erythema and increased crevicular depth, but without significant migration of the epithelial attachment. after eight months, the experimental areas showed significantly more erythema than did the control areas. freden et al.15 biopsies gingival tissue adjacent to dental amalgam fillings and control tissue in contact with intact tooth structure. the tissues were analyzed for mercury content by flameless atomic absorption spectrophotometry. all of the biopsies which had been in contact with amalgam fillings showed markedly higher mercury contents than did the control biopsies. siblerud16 compared oral health parameters of 50 subjects with amalgam fillings with those of 51 subjects without amalgams. amalgam subjects displayed more gingival bleeding, periodontal disease, metallic taste, and foul breath than did the amalgam-free group. an additional 86 subjects were surveyed before and after amalgam removal. in this group, 86% of the oral cavity symptoms were either eliminated or improved after amalgam removal. study of the relationship between dental amalgam and oral lichen planus showed that some people with oral lichen planus may manifest allergic reactions to these mercury and the removal of their dental amalgam filling showed a total remission of the lesions and considerable improvement.7 an epidemiologic case-control study of mercury body burden and idiopathic parkinson’s disease, concluded that the body burden of mercury is strongly associated with parkinson’s disease.17 environmental aspects of dental amalgam since all dental restorative materials are foreign substances, their potential for producing adverse health effects is determined by their relative toxicity and bioavailability, as well as by host susceptibility. adverse health effects to dental restoratives may be local in the oral cavity or systemic, depending on the ability of released components to enter the body and, if so, on their rate of absorption. bindslev,18 in his study, said that professional activities performed in dental clinics result in the production of wastes, which can be divided into three major groups i.e. sharps, infectious waste and chemical wastes. in general, the amount of waste generated in dental offices is considered to be relatively small compared with that of other healthcare facilities, such as hospitals, and industry. hazardous wastes generated by the handling of dental filling materials are generally classified as chemical wastes. chemical wastes from the dental profession can be sub classified as liquids and solids (table 1). among the liquids, mercury contaminated waste water and disposal of photographic solutions are a major environmental concern. loosening and loss of teeth with amalgam, and trituring surplus during carving and burnishing of amalgam are major environmental concern among the solid. �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 30-34 table 1. categories of liquid and solid waste generated in dental offices18 solid chemical wasted liquid chemical wastes mercury and amalgam contaminated wastes lead foils disinfectants batteries metals dental materials residues drug residues mercury and amalgam contaminated wastewater photographic solutions plating solutions monomers solvents disinfectants oil acids/alkalis drug residues there have been periodic concerns regarding the potential adverse health effects arising from exposure to mercury in amalgam.7,5,19-23 the industrial discharge of mercury has been reduced markedly in several countries. subsequently, increased attention has been focused on the uncontrolled discharge of mercury waste from dental clinics. however, the relative mercury contribution from dental offices to the environmental mercury pollution is not well documented. mercury consumption for dental purposes was estimated to be 3–4% world-wide.24 national surveys have shown that mercury consumption in dentistry has considerably declined in recent years, largely due to the decreasing incidence of dental caries, more frequent use of crowns and the availability of tooth-colored alternative restorative materials for certain applications.5 figure 1 summarizes the mercury cycle in dentistry.25 according to a recent german report, around 46% of the freshly triturated amalgam will be inserted as new amalgam fillings. major amalgam particles (around 15%), surplus in trituration capsules and carved surplus, are expected to be collected for recycling. minor amalgam particles produced during carving, burnishing, and polishing procedures will be sucked up and transported by the vacuum system. a part of it will sediment in tubes and drains in the clinic. depending on the presence or absence of an amalgam separating unit in the clinic, a part of the generated amalgam-contaminated sludge will be discharged with the sewage. lost or extracted teeth with amalgam fillings and amalgam-contaminated waste as trituration capsules and cotton rolls will be discharged with the solid waste and, in most instances, will be subjected to combustion. corpses with or without amalgam fillings are cremated or buried. discussion increasing knowledge about the risk of toxic effects caused by anthropogenic mercury accumulation in ecosystems has resulted in a growing pressure for reduction of the discharge of mercury waste. consequently, the mercury waste problems of dental clinics have been given increased attention, cause they are also as a source of metal which is toxic in biological groups and difficult to distribute. as we know during handling of dental amalgam as a filling material, the dental staff may be exposed to mercury figure 1. mercury cycle in dentistry.25 ��berniyanti and hariyani: side effects of mercury in dental amalgam vapour and the restrictions on handling and discharge of contaminated waste have been established in several countries. even though, based on the present literature it can be concluded that the concentrations which may occur lie considerably below the internationally recommended limit values for occupational exposure, and far below the limits where toxic effects are described. water spray cooling and vacuum suction during amalgam removal significantly reduce the evaporation of mercury to levels far below the who threshold limit values for both short-term (stel) and long-term occupational exposure (tlv). mercury compound that is really toxic is methylmercury. they are soluble in fat, so that the highest concentration is in the brain. the damage that has happened was permanent, other than that the acut toxic effect of this compound is loss of conciousness to dead. compared to the general population, dental personnel have shown slightly elevated mercury levels in blood, urine and in certain organs. however, the levels are far below the corresponding limit values, and surveys of dental staff’s health status with regard to sensitive parameters such as fertility disorders, do not reveal any increased risk of mercury-related toxic effects. recently, it has been suggested that sensitive neurobehavioral tests may demonstrate subtle mercuryrelated effects at lower occupational exposure levels than previously observed. according to the fact above, resin-based dental materials has use as an alternative restoration materials. however, it doesn’t mean that material without side effect. resin-based dental materials and bonding agents contain several known contact allergens or may release allergenic substances (i.e., formaldehyde) by degradation. a recent danish survey including 2,208 dentists reported that allergic contact eczema caused by (di)methacrylate-containing materials was diagnosed among 0.7% but estimated by the description of symptoms to be nearly 2%. these results call for attention to the sensitizing potential of certain ingredients in resin-based dental materials and, thus, also for practicing daily routines which avoid direct skin contact with such products. to ensure proper handling and recycling, the dental team should take care that amalgam scrap is disposed of by companies that adhere to government regulations. as mercury evaporates from amalgam undergoing decomposition by heating, amalgam scrap and extracted teeth with amalgam fillings should not be disposed of in waste undergoing incineration. amalgam particles in wastewater discharged from dental clinics may theoretically accumulate in wastewater treatment plants. it has been reported that the majority of mercury entering a large modern municipal wastewater treatment plant is removed effectively from the wastewater stream and retained in the sewage sludge. the subsequent handling of the residual sludge may thus result in mercury emissions to the environment. it was recently shown that by incineration of waste water sludge, almost the entire mass of mercury removed from the waste water can be discharged to the atmosphere. sunlight-mediated emission of elemental mercury from soil amended with municipal sewage sludge has also been demonstrated. mercury accumulated in waste water treatment plants has caused concern among regulators and resulted in point source reduction strategies that include the dental profession. the relative contribution from dental clinics is, however, scarcely elucidated. water spray cooling and vacuum suction during amalgam removal significantly reduce the evaporation of mercury to the levels far below the who threshold limit for both short-term and long-term occupational exposure. the efficacy of modern amalgam separators is presently being proven in practice, and it has been shown that the outlet of amalgam particles in sewers can be reduced to at least 10% of the original mercury level. major amalgam particles from trituration surplus of those produced during the carving and burnishing of new amalgam restorations are generally collected in coarse filters and sold for refinement. in order to avoid emission of mercury vapor during storage, scrap should be stored in unbreakable containers covered by water or used x-ray solution. increasing knowledge of the risk of toxic effects to human and environment from mercury pollution in dental practices should be followed by efforts to minimize it. all hazardous wastes in dental offices must be handled according to national regulation, and the dental team should be adequately educated to collect and handle the mercury wastes. the modern dental team should be well educated to increase the professionallity and public awareness of the biocompatibility aspects of dental materials in all respects. all personnel must aware of the potential sources of mercury vapor in the dental operator. they should work in wellventilated spaces and check the dental operator atmosphere for mercury vapor periodically. use only precapsulated alloys and an amalgamator with a completely enclosed arm when we need amalgam alloys as an alternative material. all personnel must remove professional clothing before leaving the workplace if normal occupational recommendations for mercury hygiene routines (e.g., water spray coolant and high vacuum suction during removal of amalgam fillings) are followed, no health risk can be assumed to be associated with occupational handling of amalgam as a dental filling material, except in extremely rare cases of allergy. references 1. craig r, o’brien w, powers j. dental materials: properties and manipulation. 1994. p. 4. 2. royal ma. amalgam fillings: do dental patients have a right to informed consent? available from http://health.consumercide.com/ dent-infconsent.html. accessed april 18, 2007. 3. smith d, williams d. biocompatibility of dental materials. dental materials. 1982; 3:29. 4. international academy oral medicine and toxicology. special report: when your patients ask about amalgam. j am dent assoc 1990; 120:398. �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 30-34 5. american dental association. association report: dental amalgam. update on safety concerns. j am dent assoc 1998; 129:494–503. 6. moetmainah. perhatian dokter gigi terhadap uap merkuri akibat pemrosesan amalgam. maj ked gigi 1997; 30:2. 7. ziff mf. documented clinical side effects to dental amalgam. adv dent res 1992; 6:131–4. 8. phillips rw. skinner’s science of dental materials. 9th ed. philadelphia: saunders; 1991. p. 21–30. 9. li y, zhang b, christen a. dentistry in china: past and present. bull hist dent 1987; 35:21–8. 10. rupp nw. clinical use of some dental materials. amalgams. j indianaj indiana dent assoc 1973; 52(8):432–4. 11. environmental protection agency (epa), us. mercury health effects update: health issue assessment. washington dc: office of health and environmental assessment, 1984; phs publication no. epa600/8-84-019f. 12. reinhardt jw. side-effects: mercury contribution to body burden from dental amalgam. adv dent res 1992; 6:110–13. 13. trivedi sc, talim st. the response of human gingival to restorative materials. j prosthet dent 1973; 29:73–80. 14. turgeon j, lemay l-p, cleroux r. periodontal effects of restoring proximal tooth surfaces with amalgam: a clinical evaluation in children. can dent assoc 1972; 37:255–26. 15. freden h, hellden l, milleding p. mercury content in gingival tissues adjacent to amalgam fillings. odontol revy 1974; 25:207–10. 16. siblerud rl. the relationship between mercury from dental amalgam and oral cavity health. ann dent 1990; 49:6–10. 17. ngim ch, devathasan g. epidemiologic study on the association between body burden mercury level and idiopathic parkinson’s disease. neuroepidemiology 1989; 8:128–41. 18. arenholt, bindslev d. environmental aspects of dental filling materials. eur j oral sci 1998; 106:713–20. 19. pinto of, huggins ha. mercury poisoning in america. j int acad prevent med 1976; 3(2):42–58. 20. bauer jg, first ha. the toxicity of mercury in dental amalgams. can dent assoc j 1982; 10(6):47–61. 21. gay dd, cox rd, reinhardt jw. chewing releases mercury from fillings. lancet 1979; 1:985–6. 22. langan dc, fan pl, hoos aa. the use of mercury in dentistry: a critical review of the recent literature. j am dent assoc 1987; 115:867–80. 23. mchugh wd. statement: effects and side effects of dental restorative materials. adv dent res 1992; 6:139–44. 24. who. mercury, environmental health criteria 1. geneva: world health organization, 1976. p. 21–25. 25. arenholt, bindslev d. dental amalgamenvironmental aspects. adv dental amalgamenvironmental aspects. adv dent res 1992; 6:125–30. 190 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 190–194 original article the effect of persea americana mill. seed extract on inflammatory cells and fibroblast formation in tooth extraction socket healing yessy ariesanti, irvan septrian syah putra rasad, maylan nimas and nadira syabilla department of oral and maxillofacial surgery, faculty of dentistry, universitas trisakti, jakarta, indonesia abstract background: inflammatory cells and fibroblasts have an essential role in the wound healing process. persea americana mill. seed categorises as a waste; it contains rich nutrients that can accelerate wound healing activity. purpose: this study aims to determine the effect of persea americana mill. seed against inflammatory cells and fibroblast formation in tooth extraction socket healing. methods: ninety-six sprague dawley rats had their lower left molars removed. forty-eight rats tested for inflammatory cells were divided into four groups: negative control group (ic1), positive control group (ic2), persea americana mill. seed extract concentrations of 50% (ie1) and 90% (ie2). another 48 rats used for fibroblast were divided into three groups: the control group (fc1), persea americana mill. seed concentrations of 50% (fe1) and 90% (fe2). the gel was applied to the socket under general anaesthesia. four rats from each group were decapitated for histopathological tissue preparations with haematoxylin eosin (he) staining on the 3rd, 5th and 7th days for inflammatory cells and the 3rd, 5th, 7th and 14th days for fibroblast formation. the preparations for each research were scored under the microscope at 40x magnification. the obtained data was analysed using the kruska—wallis and the mann—whitney test. results: a significant decrease (p<0.05) of inflammatory cells in ie2 on the 5th and 7th day. a significant increase (p<0.05) of fibroblast formation between treatment and control groups and no significant difference (p>0.05) between fe1 and fe2 was based on the interval days. conclusion: persea americana mill. seed extract can decrease the inflammatory cells and accelerate the fibroblast formation in tooth extraction socket healing. keywords: fibroblast; inflammatory cells; persea americana mill.; tooth extraction socket healing correspondence: yessy ariesanti, department of oral and maxillofacial surgery, faculty of dentistry, universitas trisakti. jl. kyai tapa no. 260 grogol, jakarta, 11440, indonesia. email: yessy.ariesanti@trisakti.ac.id introduction tooth extraction is one of the most frequent oral surgical procedures and can lead to post-extraction complications such as pain, swelling, infection and mastication problems.1,2 the tooth extraction socket heals when the surface closes and when the tissue strength is normal.3 the tooth extraction process will damage the hard and soft tissue, and physiologically, the body will heal the wound. wound healing in post-dental extraction is a complex process to restore the epithelial regeneration and the formation of connective tissue in the general principle of wound healing.4 wound healing is divided into four phases: the haemostasis phase, the inflammation phase, the proliferation phase and the maturation phase.4,5 in the early stage of the inflammation phase, the first inflammatory cell chemically attracted is the neutrophil in polymorphonuclear (pmn) cells. neutrophils have the function of attacking and destroying invading microorganisms during blood circulation in the early stage of the inflammatory process.6,7 neutrophils appear in large numbers during the first days of inflammation. many neutrophils are present due to the infiltration and accumulation of leukocytes from the blood vessels into the injury site.7 after 24–48 hours, the neutrophil cells transition into macrophages.8 macrophages can phagocytise microorganisms and also secrete growth factors such as fibroblast growth factors (fgf).8,9 the formation of fibroblasts plays an essential role in the wound healing process, and fibroblasts can fuse the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p190–194 mailto:yessy.ariesanti@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p190-194 191ariesanti et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 190–194 wound edges together, bringing them closer and attached.10 fibroblasts migrate to the wound site and proliferate, resulting in a predominance of their numbers at the wound site.11 fibroblasts first significantly create on day three and reach their peak on day seven.12 another study stated that the fibroblast formation starts to decrease to normal levels by around day fourteen. fibroblasts infiltrate and degrade the fibrin clot by producing extracellular matrix components. this matrix complex supports and regulates fibroblast migration and activity, as well as granulation tissue generation and epithelialisation.13 most of the world’s population began to reintroduce the concept of natural life, where natural ingredients are used again, including treatment with medicinal plants. based on data from world health organization, about 80% of the world’s population uses medicinal plants as alternative medicine.14 persea americana mill. is a plant widely grown in a tropical country and is one of the medicinal plants in various health fields because it contains essential bioactive ingredients.15 in addition, it is also known for its fruit, leaves and seeds having multiple benefits and high nutritional content.16,17 the seeds of the fruit are non-edible and categorised as wastes.18 the seeds contain several secondary metabolite compounds based on phytochemical screening, namely alkaloids, triterpenoids, tannins, flavonoids and saponins. these secondary metabolite compounds, such as tannins, flavonoids and saponins, can stimulate the migration and formation of fibroblasts in the wound area.12,18 other studies have also shown that persea americana mill. seed extract is a rich source of oleic acid and contains essential fatty acids. when used in pharmaceutical formulations for topical use, it can decrease inflammatory cells during wound healing. thus, it can be considered an option to treat a wound.16,19,20 this study aims to determine the effect of persea americana mill. seed extract against inflammatory cells and fibroblast formation in tooth extraction healing in sprague dawley rats. materials and methods the research is an in vivo experimental laboratory with a randomised controlled and post-test control group design. the ethical clearance was approved by the dentistry ethics committee in universitas trisakti (letters no. 347/ke/ fkg/8/2016 and 385/ke/fkg/11/2016). the research was conducted from october 2016–january 2017. the material extraction of persea americana mill. seed was conducted at balai penelitian tanaman rempah dan obat (balittro), cimanggu, bogor, v-stem laboratory, bogor district, indonesia, and the research with sprague dawley rats was conducted at pertanian bogor institute (ipb), babakan, bogor, indonesia. fresh persea americana mill. seeds were obtained from plantation cultivation in bangka belitung, indonesia, then were washed thoroughly and dried in an oven for 24 hours. after drying, the persea americana mill. seeds were cut into small pieces and made into powder, then extracted with ethanol 96% concentrations for 30 minutes. the extracts continued with the maceration process for 24 hours and then were filtered with a buchner funnel (fisherbrand, new hampshire, usa). the obtained filtrate was evaporated with a rotary evaporator (buchi r-110, flawil, switzerland) at a temperature of 40˚c and given vacuum pressure to obtain a thick extract until it did not drip. persea americana mill. seed gel extract was made by mixing the thick extract of persea americana mill. seeds into 1% sodium carboxymethyl cellulose (cmc-na) solution (wealthy, jiangsu, china) as a gelling agent. the research samples used were 96 white male sprague dawley rats, age 2–3 months. the condition of the rats was always monitored so that their food and drinking needs were filled. the samples were divided equally for inflammatory cells and fibroblast formation. there were 48 rats used for inflammatory cell research that were randomly divided into four groups (n=12). group i was the negative control group (ic1), which did not get any treatment on the socket. group ii was the positive control group (ic2), which was treated with povidone-iodine. group iii was the treatment group administered with persea americana mill. seed extract concentration of 50% (ie1). group iv was the treatment group administered with persea americana mill. seed extract concentration of 90% (ie2). another 48 rats used for fibroblast formation research were divided into three groups (n=16). group i was the control group (fc1), which did not get any treatment on the socket. group ii was the treatment group with persea americana mill. seed extract concentration of 50% (fe1). group iii was the treatment group with persea americana mill. seed extract concentration of 90% (fe2). the lower left molars of the samples were extracted with forceps under general anaesthesia with ketamine 50–80mg/ kg (kepro b.v., deventer, netherlands) and xylazine 20mg/ kg (agrovet market, lima, peru) infiltration followed with the topical gel application of the persea americana mill. seed extract on the socket for one minute using a plastic instrument. the ie1 and ie2 were given the extracted gel every day, while the ic2 was given povidone-iodine. the same tooth extraction procedure and topical gel application of the persea americana mill. seed extract were applied every day for fe1 and fe2. tissue preparations were made with decapitating four rats from each group under general anaesthesia with ketamine-xylazine infiltration on the 3rd, 5th, and 7th days for inflammatory cells and the 3rd, 5th, 7th and 14th days for fibroblast formation. in each day interval, the left mandible was cut to the size of a tooth socket fixated with 10% formalin and decalcified to be used as a tissue sample for haematoxylin eosin (he) staining. the sample slides were read and assessed under a light microscope (nikon e-100, tokyo, japan) per five-micrometre fields of view on a binocular light microscope with 40x magnification. each preparation was assessed using a scoring system by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p190–194 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p190-194 192 ariesanti et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 190–194 two pathologists. the data obtained was analysed using the statistical package for the social sciences (spss version 20.0, ibm, new york, usa) program with kruskal–wallis and mann–whitney testing. results the histopathological examination showed inflammatory cells on the 3rd, 5th and 7th days in ic1, ic2, ie1 and ie2. the inflammatory cells were seen the most on day three in all groups; they decreased on day five and were at the lowest level on day seven. based on the interval of days in the research, the ie2 preparation showed a notable decrease in inflammatory cells on day three and day seven. figure 1 shows that inflammatory cells in ie2 (yellow arrows) were widely spread on day three and decreased on day seven. the groups with the highest average score on the 3rd day are ic2 and ie2, while the lowest average scores are ic1 and ie1. on the 5th day, ie2 started to decrease, and the other groups remained the same. on the 7th day, every group had decreased with ic1 having the highest average score while ie1 and ie2 had the lowest average score in inflammatory cells (figure 2). figure 1. histopathological appearance shows the inflammatory cells (yellow arrow) in ie2 were widely spread on day three (a) and decreased on day seven (b). 2.6 2.6 2.3 3 3 2 2.6 2.6 1 3 2.6 1 0 0.5 1 1.5 2 2.5 3 3.5 day 3 day 5 day 7 av er ag e interval of days (ic1) negative control group (ic2) positive control group (ie1) persea americana mill. 50% (ie2) persea americana mill. 90% figure 2. the average number of inflammatory cells on the 3rd, 5th and 7th day in each experimental group. figure 3. histopathological appearance shows a significant increase in fibroblast formation (red arrow) of fe2 on the 14th day. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p190–194 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p190-194 193ariesanti et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 190–194 the kruskal–wallis test concluded there was no significant difference (p>0.05) for inflammatory cell number in all four groups (ic1, ic2, ie1 and ie2). kruskal–wallis test results in ie2 showed a significant difference (p<0.05) in every interval of days (3rd, 5th and 7th day), with the highest average of inflammatory cells on the 3rd day. using the mann–whitney test, the data showed a significant difference (p<0.05) in inflammatory cell decrease on the 5th and 7th day in ie2. the histopathology showed fibroblast formation was not visible in fc1 and fe1 on the 3rd day, while the fibroblast formation had been formed from the 3rd day until the 14th day in fe2; the fibroblast formation was seen the most widely spread on the 14th day. fibroblast formation in fc1 was less noticeable than fe1 and fe2, while the comparison of fibroblast formation between fe1 and fe2 in the interval of the day was not much different. the highest increase in fibroblast formation, pointed out by the red arrows, in fe2 was seen on the 14th day (figure 3). the fibroblast formation average score on the 3rd day was only seen in fe2, while fc1 and fe1 were not formed yet. fibroblasts have formed in all groups on the 5th day with the highest fe2. fibroblast formation rapidly increased on the 7th day in every group, and on the 14th day, fibroblast formation still increased with fe2 as the highest, followed by fe1 and fc1 as the lowest (figure 4). the kruskal–wallis test concluded that there was a significant difference (p<0.05) in fibroblast formation between the control group (fc1) and the treatment groups (fe1 and fe2). the data continued with the mann–whitney test; no significant difference (p>0.05) in fibroblast formation in fe1 and fe2 based on day interval. discussion this research showed the highest number of inflammatory cells in the control group, and the treatment group was on the 3rd day. the inflammatory cells in ic1 and ic2 did not decrease on day five. the decrease occurred on day seven but was not significant. there was no decrease in ie1 on day five but a significant decrease on day seven, while in the ie2 group, there was a significant decrease on day five and day seven. it shows that persea americana mill. seed is rich in nutrients, and secondary polyphenol compounds are beneficial for antioxidants, are anti-inflammatory and promote wound healing activities.18 the previous research also found the use of oleic acid in persea americana mill. can promote a reduction in the number of inflammatory cells in the injured tissue.21 this research proves that ie2 is more effective at decreasing inflammatory cells than ie1, as the content in ie1 was not adequate for reducing inflammatory cells. this proves that the greater the concentration of persea americana mill. extract, the higher its effectiveness in reducing inflammatory cells. this analysis is in line with the previous research regarding the effects of foeniculum vulgare mill. extract on the healing of labial gingiva mucosal wounds, as it was stated that the concentration of plant extracts is too low to contain chemically active compounds, so the biological function is not optimal.22 the time interval in this research began on day three because it is based on the theory that states that the proliferative phase postoperatively begins on day three to week three. histopathology examination showed that fibroblast formation had formed on day three of fe2. however, histopathology showed fibroblast formation was not visible in fc1 and fe1 on day three. this condition is related to the incomplete or nonoptimal number of fibroblast cell mediators in the wound healing process. the highest fibroblast formation was seen on day fourteen in all groups. it proves that even without therapy, physiologically, the number of fibroblasts will increase from day three to day seven, with the number remaining high on day fourteen. however, in the treatment groups, fibroblasts physiologically increased faster with the support of persea americana mill. seed extract. the results align with the previous study, and fibroblasts will appear in the wound area after three days, with the number of fibroblast formations peaking on the seventh day after trauma. however, although the fibroblasts were already high on day seven, they continued to increase until day fourteen. fibroblast formation occurred faster in the treatment group (fe1 and fe2) than in the control group. the increase 0 1 1.3 2.3 0 1.3 2.3 2.6 1 2.3 2.6 3 0 0.5 1 1.5 2 2.5 3 3.5 day 3 day 5 day 7 day 14 av er ag e interval of days (fc1) control group (fe1 )persea americana mill. 50% (fe2) persea americana mill. 90% figure 4. the average number of fibroblast formation on the 3rd, 5th, 7th and 14th day in each experimental group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p190–194 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p190-194 194 ariesanti et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 190–194 in the average number of fibroblasts in the treatment group was due to the effect of the persea americana mill. seed extract. these results are in line with the theory that the increase in the number of fibroblast cells in the treatment group was influenced by the provision of nutrients obtained from the persea americana mill. seed extract that is useful in accelerating the wound healing process by increasing the process of fibroblast formation.19 statistically, there was no significant difference in fibroblast formation in fe1 and fe2 based on day intervals. however, the average fibroblast formation in fe2 was higher than in fe1 based on the day interval. this result is because fe2 contains more persea americana mill. seed extract than fe1. the increase of fibroblast formation in the extract group was influenced by the provision of nutrients such as flavonoids, tannins and saponin obtained from persea americana mill. seed extract.18 flavonoids help reduce the duration of the inflammatory reaction, enhance growth factor proliferation and result in fibroblast formation. tannins can promote the formation of fibroblasts, capillaries and produce growth factors to stimulate the proliferation growth of fibroblasts. saponins can increase the proliferation of monocytes and macrophages, which secrete growth factors and stimulate the migration and proliferation of fibroblasts in the wound area, accelerating wound healing by increasing the process of fibroblast formation.12,18 this research concluded that persea americana mill. seed extract can decrease the inflammatory cells and accelerate the fibroblast formation. persea americana mill. seed extract concentration of 90% is more effective than 50% for decreasing inflammatory cells and accelerating fibroblast formation in tooth extraction socket healing in sprague dawley rats. acknowledgement the authors are thankful to the faculty of dentistry, universitas trisakti, jakarta, indonesia for facilitating the research. references 1. wang cw, yu sh, fretwurst t, larsson l, sugai j v, oh j, lehner k, jin q, giannobile w v. maresin 1 promotes wound healing and socket bone regeneration for alveolar ridge preservation. j dent res. 2020; 99(8): 930–7. 2. goswami a, ghorui t, bandyopadhyay r, sarkar a, ray a. a general overview of post extraction complications-prevention, management and importance of post extraction advices. fortune j heal sci. 2020; 03(03): 135–47. 3. nignsih jr, haniastuti t, handajani j. re-epitelisasi luka soket pasca pencabutan gigi setelah pemberian gel getah pisang raja (musa sapientum l) kajian histologis pada marmut (cavia cobaya). jikg (jurnal ilmu kedokt gigi). 2019; 2(1): 1–6. 4. luthfi m, juliastuti ws, risky ya, wijayanti eh, rachmawati ae, asyhari npo. expression of fibroblast cells after extraction of wistar rat teeth after topical application of okra fruit (abelmoschus esculentus) gel. infect dis rep. 2020; 12(suppl 1): 8726. 5. dwivedi d, dwivedi m, malviya s, singh v. evaluation of wound healing, anti-microbial and antioxidant potential of pongamia pinnata in wistar rats. j tradit complement med. 2017; 7(1): 79–85. 6. rosales c. neutrophil: a cell with many roles in inflammation or several cell types? front physiol. 2018; 9: 113. 7. li jj, zreiqat h. tissue response to biomaterials. in: encyclopedia of biomedical engineering. elsevier; 2019. p. 270–7. 8. hirayama d, iida t, nakase h. the phagocytic function of macrophage-enforcing innate immunity and tissue homeostasis. int j mol sci. 2017; 19(1): 92. 9. fitriani d, prasetyaningrum n, sinta gitaning r. the effect of avocado’s (persea americana) ethanolic extract for increasing macrophages cells in rattus norvegicus post extraction socket. in: proceedings of the international dental conference of sumatera utara 2017 (idcsu 2017). paris, france: atlantis press; 2018. p. 62–5. 10. ariesanti y, sandra f, claresta b, alvita l. coffea canephora bean extract induces nih3t3 cell migration. indones biomed j. 2021; 13(2): 216–20. 11. rinaldi dh, kamadjaja db, sumarta npm. the effects of breadfruit leaf (artocarpus altilis) extract on fibroblast proliferation in the tooth extraction sockets of wistar rat. dent j (majalah kedokt gigi). 2018; 51(3): 143–6. 12. khoswanto c, juliastuti ws, adla ka. the effect of avocado leaf extract (persea americana mill.) on the fibroblast cells of postextraction dental sockets in wistar rats. dent j (majalah kedokt gigi). 2018; 51(3): 129–32. 13. bainbridge p. wound healing and the role of fibroblasts. j wound care. 2013; 22(8): 407–12. 14. world health organization. who global report on traditional and complementary medicine 2019. world health organization. geneva: world health organization; 2019. p. 18–9, 45. 15. rauf a, pato u, ayu df. aktivitas antioksidan dan penerimaan panelis teh bubuk daun alpukat (persea americana mill.) berdasarkan letak daun pada ranting. jom faperta. 2017; 4(2): 1–12. 16. ramos-aguilar al, ornelas-paz j, tapia-vargas lm, ruiz-cruz s, gardea-béjar aa, yahia em, ornelas-paz j de j, pérez-martínez jd, rios-velasco c, ibarra-junquera v. the importance of the bioactive compounds of avocado fruit (persea americana mill) on human health. biotecnia. 2019; 21(3): 154–62. 17. amado dav, helmann gab, detoni am, carvalho slc de, aguiar cm de, martin ca, tiuman ts, cottica sm. antioxidant and antibacterial activity and preliminary toxicity analysis of four varieties of avocado (persea americana mill.). brazilian j food technol. 2019; 22: e2018044. 18. setyawan hy, sukardi s, puriwangi ca. phytochemicals properties of avocado seed: a review. iop conf ser earth environ sci. 2021; 733: 12090. 19. bhuyan dj, alsherbiny ma, perera s, low m, basu a, devi oa, barooah ms, li cg, papoutsis k. the odyssey of bioactive compounds in avocado (persea americana) and their health benefits. antioxidants (basel, switzerland). 2019; 8(10): 426. 20. nicolella hd, neto fr, corrêa mb, lopes dh, rondon en, dos santos lfr, de oliveira pf, damasceno jl, acésio no, turatti icc, tozatti mg, cunha wr, furtado ra, tavares dc. toxicogenetic study of persea americana fruit pulp oil and its effect on genomic instability. food chem toxicol. 2017; 101: 114–20. 21. de oliveira ap, franco e de s, rodrigues barreto r, cordeiro dp, de melo rg, de aquino cmf, e silva aar, de medeiros pl, da silva tg, góes aj da s, maia mb de s. effect of semisolid formulation of persea americana mill (avocado) oil on wound healing in rats. evid based complement alternat med. 2013; 2013: 472382. 22. indraswary r. efek konsentrasi ekstrak buah adas (foeniculum vulgare mill.) topikal pada epitelisasi penyembuhan luka gingiva labial tikus sprague dawley in vivo. maj ilm sultan agung. 2011; 49: 124. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p190–194 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p190-194 113113 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 113–118 serum c-reactive protein and c-reactive gene (-717c>t) polymorphism are not associated with periodontitis in indonesian male patients antonius winoto suhartono,1 benso sulijaya,2 niniarty zeiroeddin djamal,1 sri lelyati chaidar masulili,2 christopher talbot,3 and elza ibrahim auerkari1 1department of oral biology, faculty of dentistry, universitas indonesia, jakarta indonesia 2department of periodontic, faculty of dentistry, universitas indonesia, jakarta indonesia 3department of human genetics, leicester university uk abstract background: periodontitis is an inflammatory disease caused by periodontal pathogens and influenced by multiple risk factors such as genetics, smoking habit, age and systemic diseases. the inflammatory cascade is characterized by the release of c-reactive protein (crp). periodontitis has been reported to have plausible links to increased level of crp, which in turn has been associated to elevated risk of cardiovascular disease (cvd). purpose: the purpose of this study was t o investigate the relationship amongst the severity of periodontitis, crp level in blood and crp (-717 c>t) gene polymorphism in male indonesian smokers and non-smokers. method: the severity of periodontitis was assessed for 97 consenting male indonesian smokers and non-smokers. the crp level of the subjects was determined by using immuno-turbidimetric assay performed in parahita diagnostic center laboratory iso 9001: 2000 cert no. 15225/2. the rate of crp (-717c>t) gene polymorphism was determined by using pcr-rflp in oral biology laboratory, faculty of dentistry, universitas indonesia. result: the results suggest that the crp protein level is not significantly associated with the tested crp gene polymorphism (p>0.05). also, while the severity of periodontitis increased significantly with subject age, the crp level in blood serum was not significantly related to the severity of periodontitis. the genotypes of the tested polymorphism did not show significant association with the severity of periodontitis either in smokers or in the combined population including smokers and non-smokers. the results naturally do not exclude such associations, but suggest that to discern the differences the sample size must be considerably increased. conclusion: the crp (-717c>t) gene polymorphism and crp level in blood serum were not found to be associated with the severity of periodontitis in male smokers or in the combined population of smokers and non-smokers. keywords: periodontitis; smoking; crp; polymorphism; cvd correspondence: elza ibrahim auerkari, c/o: departemen biologi oral, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat 10430, indonesia. e-mail: eauerkari@yahoo.com introduction in a worldwide population, the national health and nutrition examination survey iii (nhanes 1988-1994) found the prevalence of gingivitis in 50% adult and 40-60% in schoolchild.1 world health organization (who) stated that severity level of periodontal disease that being marked by the depth of periodontal pocket (≥ 6.0 mm) was found in 10-15% adult people all over the world2 and 13.1% in south east asia.3 particularly in indonesia, periodontal disease took as the first place (61%) in top ten most complained diseases according to the indonesian health profile (profil kesehatan indonesia) in 2001.4 unfortunately only 29.6% of them had received dental treatment (according to national basic health research/ riset dasar kesehatan nasional riskesdas) in 2007.5 this epidemiologic data reveals that the prevalence of periodontal disease still high and the ability of patient to be treated is low. previous study has proved that individual with periodontal disease has an increasing of c-reactive protein (crp) level in its blood.6,7 this protein is an acute protein phase that being controlled as the sign of inflammatory status, and had been identified as main risk factor of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 114 suhartono, et al./dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 113–118 atherosclerosis complication.7 another study found relationship between loss of teeth or periodontitis with the risk of atherosclerosis. this sign was immune system fast response toward persistent inflammation.8 there was also significant increase in adjusted mean levels of crp in subject with high attachment loss when compared to subjects with healthy periodontium.9 it could be proposed that patients with periodontitis may have elevated circulating levels of these inflammatory markers like c-reactive protein and hence increase the risk for atherosclerosis.10 periodontitis and atherosclerosis have mutual complex etiologic factors and shared mutual potential bacterial mechanism.11 the pathway is that periodontal pathogen bacteria and its products will leads to endothelial vascular destruction. its destruction will indirectly activate the production of platelet. the aggregation of platelets will cause thrombus-embolic formation.12,13 various studies concerning assessment of etiologic factor of periodontal disease, implied that periodontal disease was multifactorial disease with interacted manifestation between three elements which are bacterial, host and environmental factor. host immune system is influenced by genetic and epigenetic factor. this study was focused to analyze one of the genetic factors of the periodontal disease’s severity which is genetic polymorphism in each individual based on structure difference, diversity of gene expression and function.14 this study i s aimed to investigate the relationship among the severity of periodontitis, crp level in blood s e r u m and crp (-717 c>t) gene polymorphism in indonesian male smokers and non-smokers. materials and methods this study is analytic study with laboratory approach. ninety-seventh male subjects were randomly selected from all patients in department of periodontology, dental hospital faculty of dentistry universitas indonesia. intra oral examination and periodontal health status, attachment loss (al), probing depth were assessed for all subjects using a standardized procedure at six locations on each tooth. the sample is stored samples in -20°c (dna and blood samples) archived in oral biology laboratory, faculty of dentistry and biology laboratory faculty of medicine, universitas indonesia. the ethical clearance was approved by the ethical committee of the faculty of dentistry, universitas indonesia and all patients signed written informed consent. ninety-seventh samples (25-60 years old) were analyzed for its severity of periodontal disease, smoking status (smoker or non-smoker), and crp level. systemic disease is excluded for this study. the samples known divided into 59 smokers and 38 non-smokers based on its cigarette consumption habit characteristic. severity of periodontal disease were divided into mild (cal<2.0 mm), moderate (cal 2.0 ≤ 4.0 mm), and severe (>4.0 mm). levels of crp were measured from peripheral blood samples by using immuno-turbidimetric technique assay performed in parahita diagnostic center laboratory iso 9001: 2000 cert no. 15225/2. the rate of crp (-717c>t) gene polymorphism was determined by using pcr-rflp in oral biology laboratory, faculty of dentistry, universitas indonesia. patient who under medication such as antibiotics, corticosteroids, anti-inflammatory drugs and who had a history of periodontal treatment in the past 6 months were excluded from this study. to survey the genotype-phenotype variations related to the gene locus polymorphisms of crp and crp (-717c>t) gene polymorphism, the polymorphism status of these genes was determined from samples of peripheral blood. dna fragments amplifications were done using polymerase chain reaction (pcr) method. 12.5 ml top tag master mix 2x (1.25 u top tag dna polymerase, 1x pr buffer 1.5 mm mgcl2, 200 mm dntp), 2.5 µl coral load, and 4 ml ddh2o were used for every 25 ml reactan amplification. else 5 µl primer forward, 1.5 µl primer reverse, and 5 µl dna template were used. solution was mixed with vortex before went into pcr machine. primer forward 5’-actggacttttactgtcagggc-3’ and primer reverse 5’-atcccatctat gagtgagaacc-3’ were used for crp-717 c>t dna samples were amplified fp tu 35 cycles with early denaturation in 94° for 5 minutes, then went into cycle consists of annealing and elongation. time elongation extension 72°c for 7 minutes 10 figure 1. crp polymorphism. crp polymorphism could take place in promoter (left), utr (right), exon (right-center), and intron (left-center). 15 figure 2. enzyme sacii digest electrophoresis based on molecules weight. note: ss = ct, ss = ct, ss = tt. kd figure 1. crp polymorphism. crp polymorphism could take place in promoter (left), utr (right), exon (right-center), and intron (left-center).15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 115115suhartono, et al./dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 113–118 was added at the end of cycle. after dna amplification process, amplicons were stored at 4°c. amplification outcomes (pcr) were detached with electrophoresis in agarose gel 2% (promega) contains 0.1 µl etidium bromide (0.5 mg/ml) inside dapar solution tae 1x (0.04) m trisacetate, 0.002 m edta ph 8.0). 5 µl dna pcr yield were mixed with 2 µl tracking dye (0.25% bromopgenol blue, 0.25% xylene xyanole, 25% sucrose), afterwards went into electrophoresis well. the dna fragment bands were separated with electrophoresis in 80 voltages for 60 minutes. 100 pb dna ladder were used as marker. the dna fragment bands outcome of electrophoresis were observed with uv illuminator and photographed using digital camera. there are crp (-717c>t) polymorphism at various location, it could take place in promoter region, first exon, intron, second exon or in utr (un-translated regions).20 restriction fragment length polymorphism (rflp) technique was used to detect the presence of crp717 c>t polymorphism. the cutting of pcr yields were done with 20 µl 65°c sac ii enzyme for 4 hours. afterwards, it transferred to 85°c of temperature to inactivation. the outcomes of the cutting were examined using electrophoresis with agarose 3% and buffer tae. visualization of the outcome was done using electrophoresis with agarose gel 3% stained by 0.5 ml 0.1% etidium bromide, and poured into dapar tae 1x solution. electrophoresis was set in 80 voltages, 400 ma for 60 minutes. for dna visualization, gel was placed in gel doc. chi-square testing with spss 18.0 was mainly used in the statistical analysis, both for comparing results in the test groups and for assessing the allele and genotype frequencies with predictions with respect to the hardyweinberg equilibrium. statistical significance was assumed with p<0.05. 10 figure 1. crp polymorphism. crp polymorphism could take place in promoter (left), utr (right), exon (right-center), and intron (left-center). 15 figure 2. enzyme sacii digest electrophoresis based on molecules weight. note: ss = ct, ss = ct, ss = tt. kd figure 2. enzyme sacii digest electrophoresis based on molecules weight. note: ss = ct, ss = ct, ss = tt. 0 5 10 15 20 25 30 35 40 g en ot yp e fr eq ue nc y gene crp 717 cc ct tt figure 3. genotype frequency in smoker and non-smoker group 0 5 10 15 20 25 30 35 40 g en ot yp e fr eq ue nc y gene crp 717 cc ct tt figure 3. genotype frequency in smoker and non-smoker group figure 3. genotype frequency distribution in smoker and nonsmoker group. table 1. genotype crp-717 c>t (n, %) distribution in smoker and non-smoker genotype cc ct tt total smoker 33 (55.93%) 24 (40.68%) 2 (3.39%) 59 (100%) non-smoker 24 (63.15%) 8 (21.05%) 6 (15.80%) 38 (100%) total 57 (58.76%) 32 (32.99%) 8 (8.25%) 97 (100%) table 2. relationship between the severity of periodontal disease (pp) with crp level (mg/l) pp mild(11) moderate(47) severe(39) p value crp (mg/l) median (r) 0.09 (0.01-1.35) 0.10 (0.01-0.94) 0.11 (0.02-2.08) 0.740* mean±sd 0.21±0.39 0.20±0.23 0.22±0.36 * kruskal-wallis test, posthoc analysis was using mann-whitney to compare mild, moderate, and severity group p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 116 suhartono, et al./dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 113–118 results there were three genotypes from genotype and alotype distribution enzyme sacii cutting result, that is cc at 138bp band, ct at 138bp, 112bp, 40 bp, and tt at 112bp and 40bp (figure 2). all of examined genotype subjects were divided based on wild type or dominant homozygote (cc), mutant heterozygote (ct), and recessive homozygote (tt). frequency of smoker and non-smoker with those three genotypes were showed in table 1. there is a significant difference on severe group, so that it could be concluded that there is a differences between the mild-moderate and severe group (table 5). we could assumed that non-smoker group (left) and smoker (right) group have much the same of genotype, most of it was cc, the ct and tt was the least (figure 3). discussion from all subject population consists of smoker and non-smoker group like being showed in table 2-3, could be grouped based on the degree of periodontal disease severity, mild, moderate and severe. there were 3 people (5%) with mild periodontal disease, while 27 people (46%) with moderate periodontal disease, and 29 people (49%) with severe periodontal disease in smoker group. while table 3. relationship between severity of periodontal disease (pp) with age and crp with genotype in all subject population either smoker or non-smoker; r = data interval pp mild(11) moderate(47) severe(39) p value age (years) median (r) 34 (25-51) 38 (25-56) 45 (25-60) 0.002* mean±sd 35.0±9.6 37.3±9.4 44.2±9.6 genotype cc 10(17.5%) 25(43.9%) 22(38.6%) 0.173** ct 0(0.0%) 18(56.3%) 14(43.8%) tt 1(12.5%) 4(50.0%) 3(37.5%) smoking no 8(21.1%) 20(52.6%) 10(26.3%) 0.005** yes 3(5.15) 27(45.8%) 29(49.2%) * kruskal-wallis test, posthoc analysis was using mann-whitney to compare mild, moderate, and severity group ** chi-square test p<0.05 table 4. relationship between genotype with crp level (mg/l) genotype cc (57) ct (32) tt (8) p value crp (mg/l) median (r) 0.09 (0.01-2.08) 0.13 (0.01-0.78) 0.20 (0.03-0.94) 0.181* mean± sd 0.20 ± 0.35 0.20 ± 0.20 0.30 ± 0.31 * kruskal-wallis test, posthoc analysis was using mann-whitney to compare mild, moderate, and severity group p<0.05 table 5. relationship between genotype with age and crp and severity of periodontal disease (pp) in all subject population either smoker or non-smoker; r = data interval. genotype cc (57) ct (32) tt (8) p value age (years) median (r) 38.0 (25-60) 40.5 (25-56) 49.5 (26-55) 0.038* mean± sd 38.4 ± 9.8 40.5 ± 10.2 47.3 ± 9.0 pp mild 10 (90.91%) 0 (0.0%) 1 (9.09%) 0.173** moderate 25 (53.19%) 18 (38.30%) 4 (8.51%) severe 22 (56.41%) 14 (35.90%) 3 (7.69%) smoking no 24 (63.15%) 8 (21.05%) 6 (15.80%) 0.027** yes 33 (55.93%) 24 (40.68%) 2 (3.39%) * kruskal-wallis test, posthoc analysis was using mann-whitney to compare mild, moderate, and severity group ** chi-square test p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 117117suhartono, et al./dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 113–118 in non-smoker group, there were 8 people (21%) with mild periodontal disease, 20 people (53%) with moderate periodontal disease and 10 people (26%) with severe periodontal disease. from various studies reported that smoking could affect systemic or local disease especially periodontal disease. individual with poor oral hygiene would increase the periodontal disease susceptibility, and decline the immune response.15 smoking habit as a part of environment factor along with genetic factor that could affect the patient susceptibility toward periodontal disease. in conclusion, from statistical analytic as shown in table 3, there is a significant difference between the two group, smoker and non-smoker with p=0.005. in this study, the polymorphisms of crp (genotype cc, ct, tt) and crp (-717c>t) genes have been compared to the periodontal disease degree and serum crp level of 97 indonesian male subjects. these studies also compare the relationship between the degree of periodontal disease and the smoking status. all samples included relatively few mild periodontitis subjects in comparison with the number of those with moderate and severe groups; this portion seems relevant with the plausible theoretical revealed that oral infection in severe attachment loss of periodontal tissue could spread its toxin systemically through capillary blood vessel and expressed in peripheral blood. the results prove a clear relationship between the severity of periodontitis and smoking habit, so that severe periodontitis was significantly associated with higher lifetime smoking exposure (p=0.005). moreover there also a confirmation result shown in table 3, that the severity of periodontitis is related with increasing of age (p=0.002). the genotype of crp (ct, ct, tt) found significantly correlated with age (p=0.038) and smoking status (p=0.027) as seen in table 4. in contrast, the genotype of crp is not related with periodontitis severity. periodontal bacteria such as porphyromonas gingivalis, aggregatibacter actiomycetemcomitans, prevotella intermedia, treponema denticola, and eikenella corrodens, was found in atherosclerosis plaque.15 indeed, p. gingivalis has the highest risk because of its ability to regulate adhesion molecules like icam-1, vcam-1, selectins (p and e). these molecules are necessary to bind leucocytes in the endothelium layer at the early stage of atherosclerosis.16 these bacteria will produce a leucotoxin that induces degranulation and lysis in human neutrophils, caspase-1 activation, and abundant secretion of cytokine from human macrophages, promoting a tissue destruction including loss of alveolar bone.17 therefore, theoretical based revealed that the level of crp by the induction of bacterial leucotoxin are supposed largely expressed in severe periodontitis group. contrast with the result of our study that shown insignificant relation between the polymorphisms of crp (genotype cc, ct, tt) and crp (-717c>t) genes and periodontitis severity (table 2-3 and table 3-4). chronic periodontitis patients either with or without atherosclerosis symptoms had increase of crp level.18 chronic periodontitis patients with atherosclerosis symptoms had twice crp level than patients without a t h e r o s c l e r o s i s s ym p t o m s . ot h e r s t u d y b y d e freitas et al.19 stated that non-surgical periodontal therapy could reduce crp level significantly. blum20 did research to post-therapy periodontal disease patients and after three months, he found a decreasing of crp level and a reducing the risk of cardiovascular disease. however, the reported association between periodontitis, cvd, and individual polymorphisms of crp (-717c>t) genes is tend to be correlated. if the links between individual genes and their polymorphisms are relatively weak, this means that the individual polymorphisms itself are not predominantly work as influential risk indicators to the disease. economically the conventional indicators for periodontitis examination such as measurement of pocket depth and alveolar bone loss may remain more useful and effective. while for cvd indicator, the high blood pressure, smoking, obesity, and crp level is suited. an appropriate treatment of peridontitis can reduce inflammation and circulating crp levels, which directly reduced the risk of cvd.17 furthermore, the potential indicators like crp (-717c>t) genes polymorphism status did not serve as a complementary factor to confirm the conventional indicators and other research should be made to tested it accurately. it can be concluded that no relation between polymorphism promoter gene crp-717c>t with the degree of periodontal disease severity in either smoker or non-smoker male, except the difference between moderate and severe group of the severity degree. there’s no association between crp level in serum with the severity of periodontal disease either in smoker or non-smoker male. acknowledgement this study was supported by drpm-ui. authors wish to gratefully acknowledge financial support from drpmui for this work. references 1. academy repor t. epidem iolog y of per iodonta l diseases. j periodontol 2005; 76: 1406-19. 2. petersen pe, ogawa h. strengthening the prevention of periodontal disease: the who approach. j periodontol 2005; 76(12): 2187-93. table 6. the significant differences among the severity of periodontitis severity p value mild-moderate 0.409 mild-severe 0.011* moderate-severe 0.002* * p<0.05 mann-whitney test dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 118 suhartono, et al./dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 113–118 3. kasebaum nj, bernabe e, dahiya m, bhandari b, murray cjl, marcenes w. global burden of severe periodontitis in 1990-2010: a systemic review and meta-regression. j dent res 2014; 93(11): 1045-53. 4. departemen kesehatan r i. profil kesehatan 20 01 menuju indonesia sehat 2010 jakarta: pusat data departemen kesehatan; 2002; p. 40. 5. badan penelitian dan pengembangan kesehatan departemen kesehatan ri. riset kesehatan dasar 2007 (riskesdas). desember 2008; p. 97-112 6. d’aiuto f, ready d, tonetti ms. periodontal disease and c-reactive proteinassociated cardiovascular risk. j periodontal res 2004; 39(4): 236-41. 7. dave s, batista jr el, van dyke te. cardiovascular disease and periodontal diseases: commonality and causation. compendium of continuing education in dentistry 2004; 25(7 suppl 1): 26-37. 8. glu r ich i, g rossi s, a lbi n i b. syst em ic i n f la m mat ion i n cardiovascular and periodontal disease: a comparative study. clin diagn lab immunol 2002; 9: 425–32. 9. smith gd, ebrahim s. ‘mendelian randomization’: can genetic epidem iolog y cont r ibut e to u nder st a nd i ng envi ron ment a l determinants of disease?. int j epidemiology 2003; 32: 1-22. 10. mendis s, puska p, norrving b. world health organization. global atlas on cardiovascular disease prevention and control. geneva: world health organization in collaboration with the world heart federation and the world stroke organization; 2011; p. 8-13. 11. ryan td, moïse d. periodontal infections and cardiovascular disease: the heart of the matter. j am dent associ 2006. 137; 1420. 12. djais a. periodontitis sebagai faktor resiko jantung koroner aterosklerosis. jurnal kedokteran gigi pdgi 2006; 56: 53-9. 13. loos bg. systemic effects of periodontitis. int j dent hyg 2006; 4 (suppl 1): 34-8. 14. ramírez jh, arce rm, contreras a. periodontal treatment effects on endothelial function and cardiovascular disease biomarkers in subjects with chronic periodontitis: protocol for a randomized clinical trial. trial 2011; 12(46): 1-10. 15. chun y-hp, chun k-rj, olguin d’a, wang h-l. biological fou ndat ion for per iodont it is as a potent ia l r isk factor for atherosclerosis. j periodontal res 2005; 40(1): 8795. 16. auerkari ei, suhartono aw, djamal nz, verisqa f, suryandari da, kusdhany ls, masulili slc, talbot c. crp and il-1b gene polymorphisms and crp in blood in periodontal disease. open dent j 2013; 7: 88-93. 17. thakare ks, deo v, bhongade ml. evaluation of the c-reactive protein serum levels in periodontitis patients with or without atherosclerosis. indian j dent res 2010; 21(3): 326-9. 18. de freitas cot, gomes-filho is, naves rc, da cruz ss, santos cast, barbosa mds. effect of non-surgical periodontal therapy on the levels of c-reactive protein: a pilot study. rev odonto cienc 2011; 26(1): 16-21. 19. blum a, front e, peleg a. periodontal may care improves systemic inflammation. clin invest med 2007; 30 (3): 114-7. 20. smith gd, ebrahim s. ‘mendelian randomization’: can genetic epidem iolog y cont r ibut e to u nder st a nd i ng envi ron ment a l determinants of disease?. int j epidemiology 2003; 32: 1 22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p113-118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p113-118 vol 50 no 4 desember 2017.indd 178 research report composite resin shear bond strength on bleached dentin increased by 35% sodium ascorbate application tunjung nugraheni,1 n. nuryono,2 siti sunarintyas,3 and ema mulyawati1 1department of conservative dentistry 2department of chemistry 3department of biomaterial faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: restoration of the teeth immediately after bleaching with 35% hydrogen peroxide (h2o2) is contraindicated due to the remnant of free radicals that will stay inside enamel and dentin for 1-3 weeks and reduce the adhesion of composite resin. sodium ascorbate is an antioxidant substance known to bind free radical residues, thereby shortening the delay in restoration. purpose: the purpose of this study was to examine the resin bond strength of bleached dentin influenced by the application of 35% sodium ascorbate. methods: nine premolars were divided into their crown and root sections, with the crown subsequently being cut into four equal parts to obtain 36 samples. these were then divided into four groups, each containing nine samples. group a (control): samples were bleached using35% hydrogen peroxide, immersed in an artificial saliva, stored in an incubator at 37°c for seven days and then filled with a composite resin. group b:samples were also bleached by means of 35% h2o2 followed by one application of 0.025 ml 35% sodium ascorbate for 5 minutes and restored with composite resin. group c: samples were bleached with 35% h2o2, followed by two applications of 0.025 ml 35% sodium ascorbate for 5 minutes, and restored with a composite resin. group d: dentin was bleached with 35% h2o2 followed by three applications of 0.025 ml sodium ascorbate 35% for 5 minutes and restored with a composite resin. the shear bond strength of the composite resin was measured by a universal testing instrument (zwick, usa). data was analyzed by means of one-way anova and lsd. results: the highest mean shear bond strength of composite resin was in group c, while the lowest was in group b. the result of one-way anova indicated a difference in the shear bond strength of composite resin in the four treatment groups (p < 0.05). an lsd test showed there to be a difference in shear bond strength of composite resin between group a and groups c and d or between group b and groups c and d. there was no difference in shear bond strength of composite resin between group a and group b or between group c and group d. conclusion: application frequency of 35% sodium ascorbate affect on shear bond strength of composite resin restoration in bleached dentin by 35% h2o2. keywords: 35% sodium ascorbate; bleached dentin; 35% hydrogen peroxide; shear bond strength correspondence: tunjung nugraheni, department of conservative dentistry, faculty of dentistry universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: tunjungnugraheni22@gmail.com. introduction there are two kinds of bleaching procedure: extracoronal and intracoronal. the materials used for bleaching are sodium perborate, 30–38% hydrogen peroxide (h2o2) and 10–35% carbamide peroxide. the bleaching materials release free radicals perhydroxyl (hoo•) and oxygen nascen (on/o•). the free radicals react with the double bonds of chromogenic molecules in enamel and dentine, dividing into simpler molecules that are less reflective and cause the color of teeth to become brighter. posttreatment of intracoronal bleaching requires composite resin restoration to fill cavity that serves to prevent toothrecontamination by bacteria, thereby minimizing root canal failure. restoration of the tooth immediately after bleaching is contraindicated since postbleaching procedures often dental journal (majalah kedokteran gigi) 2017 december; 50(4): 178–182 doi: 10.20473/j.djmkg.v50.i4.p178–182 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i4.p178-182 179179nugraheni, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 178–182 have peroxide and residues inside the dentin tubule, between the collagen matrix and in the inteprismatic enamel, which remain active for a period of time. the trapped ion peroxide and residues can survive until eliminated by pulp microcirculation and then diffuse to external tooth surfaces.1 the residue of peroxide and oxygen nascen are free radicals which can cause a decrease in adhesion of composite resin and an increase in microleakage.2 these molecules will interfere with the propagation of vinyl radical composite resin, causing the early termination of the polymer chain, and will also form polymers with such limited mechanical power that the strength of the bonding declines, resulting in weak adhesion and microleakage.3–5 microleakage can lead to discoloration of marginal restoration and, ultimately, root canal treatment failure. h2o2 can alter the surface of the tag resin on composite resins. resin tags on bleached teeth are fewer in number and shorter than on those that are not bleached. this is probably due to bleaching materials producing mechanical modifications to the inter tubular and peritubular dentine that can lead to biomechanical changes.6 composite resin restoration must be undertaken 1–2 weeks after bleaching is completed because it is expected that free radical residues on the bleached teeth will gradually reduce, resulted in the need for longer treatment. one way to shorten the delay time is through the application of antioxidants.7,8 antioxidants are substances that can bind free radicals,one such recommended antioxidant being sodium ascorbate.8 sodium ascorbate is a sodium salt of l-ascorbic acid, consisting of a white or yellowish, odorless, non-toxic, crystalline powder or solid, that is soluble in water and partially soluble in ethanol. sodium ascorbate in liquid form has a ph of 6.5–8 and constitutes a biocompatible antioxidant.9 sodium ascorbate will be oxidized to dehydroascorbic acid after contact with on.10 controversy persists regarding the concentration, frequency and duration of sodium ascorbate applications necessary to reduce the residual bleaching material. the most widely used concentration in various studies is 10% sodium ascorbate.11 previous research has shown that up to ten minutes’ application of 10% sodium ascorbate is effective in increasing the shear bond of composite resin in bleached teeth by 15–35% carbamide peroxide.12– 14carbamide peroxide is the material commonly used for extra coronal bleaching on vital teeth. in intracoronal bleaching, the most commonly used material is 35% h2o2. briso et al.6 showed that 10% sodium ascorbate increases the tensile strength of composite resin attachment in enamel bleached using10% carbamide peroxide, but proves ineffective on enamel and dentine bleached by means of 35% hydrogen peroxide. similar results were demonstrated by turkmen et al.,15 10% sodium ascorbate was ineffective in raising the shear bond strength of composite resins on teeth bleached using 35% and 38% h2o2. the higher concentrations of h2o2 would leave more free radical residue and, therefore, require antioxidants at higher concentrations. freire et al.16confirm the amount of sodium ascorbate required to reduce h2o2 depends on the concentration of hydrogen peroxide used. freire et al.17 and murad et al.18 also argued that longer time applications do not prove effective. according to freire et al.17 sodium ascorbate can rapidly lose its antioxidant power. consequently, repeated applications are more effective than extending the application time. the aim of this study was to investigate the effectiveness of 35% sodium ascorbate to improve the shear bond strength of composite resin on dentin that bleached by 35% h2o2. materials and methods nine premolars free of caries and cracks were stored in a 10% saline buffer for a period of two months prior to the study. nine premolars were separated into their crown and root sections, with the crown subsequently being cut into four equal parts to obtain 36 samples. the samples were divided into four groups consisting of nine parts each was embedded in 2 × 2 × 2 cm self-cured acrylic. the samples were bleached using 0.025 ml of 35% h2o2 (opalescene endo, ultradent products, south jordan, usa). each sample was subsequently placed in a sterile plastic box and stored for 120 hours in an incubator at 37°c. the samples were washed with 5ml of distilled water before being dried for 10 seconds using a water syringe. bleaching was repeated twice. all of the 36 samples were divided into four groups: a, b, c, and d. group a (control) was soaked in artificial saliva and stored in an incubator at 37°c for seven days, before a restoration procedure was undertaken. group b was administered with 0.025 ml of 35% sodium ascorbate (sigma eldrich, singapura) for five minutes washed with 5 ml of distilled water by means of a syringe and dried for ten seconds, before undergoing a restoration procedure. 0.025 ml of 35% sodium ascorbate was applied by group c with the same procedure being repeated twice, prior to a restoration procedure. group d was administered with 0.025 ml of 35% sodium ascorbate with the same procedure being repeated three times, followed by a restoration procedure. the restoration procedures applied to each sample were completed with a composite resin restoration (z 350, 3m espe, usa). a bonding agent (tetric n-bond universal, ivoclar vivadent) was applied to each dentin surface of the sample and cured for ten seconds. a mould was mounted in the sample and filled with composite resin before being cured for 20 seconds. the samples were immersed in artificial saliva (faculty of mathematics and science, department of chemistry universitas gadjah mada) and stored in an incubator at 37°c for 24 hours. a shear bond strength test was performed on each sample using a universal testing instrument (zwick, usa) at the instrument laboratory of the faculty of engineering, universitas gadjah mada data was analyzed by means of one-way anova and lsd. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p178–182 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p178-182 180 nugraheni, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 178–182 results the highest mean shear bond strength of composite resin in dentin bleached by 35% h2o2 was that of group c, while the lowest was in group b (table 1). a one-way anova test showed there to be difference in the shear bond strength of the composite resin in the four treatment groups (table 2). an lsd test was subsequently conducted to quantify the difference between each group, the results of which can be seen in table 3. an lsd test showed that there was a difference in shear bond strength of composite resin between group a (control) and groups c and d, and between group b and groups c and d. in contrast, no difference in shear bond strength of composite resin between group a (control) and group b, or between groups c and d existed. discussion composite resin restoration is contra indication performed immediately after intracoronal bleaching, due to the presence of free radical residues that will affect the bond strength of the composite resin.19 the present study used 35% h2o2 because this material is often used in intracoronal bleaching techniques.20 sodium ascorbate is an antioxidant reputed to capture free radicals. the frequency of 35% sodium ascorbate application that was performed (once, twice and three times) refers to the study by freire which indicates that to remove all remaining free radicals, sodium ascorbate must be applied more than once.17 as an antioxidant, sodium ascorbate will provide one electron to bind to free radicals, becoming another more stable form of water and ascorbyl free radicals (afr). the afr pair produces one molecule of dehydroascorbic acid and one ascorbate. dehydroascorbic acid reacts with oxidants of reactive oxygen compounds such as hydroxyl radicals.5,8 a one-way anova test confirmed that there was a significant difference (p < 0.05) of shear bond strength of composite resin on dentine bleached with 35% h2o2 which was applied at different frequencies with 35% sodium ascorbate. this is in accordance with the research of freire et al.16 that showed the amount of sodium ascorbate required is directly proportional to the number of free radicals released by the bleaching materials. this study used 35% h2o2, a compound which it could be assumed released numerous free radicals. the higher the numbers of free radicals are released by bleaching materials, the more likely a greater residual post-bleaching. to capture the remaining free radicals requires more sodium ascorbate.16 the free radicals resulting from the breakdown of h2o2 are highly electrophilic and unstable because they lack electron pairs. as a result, the free radicals will try to locate pairs of electrons in order to become more stable.9 if, in bleached teeth, free radical residue still exists, then the restoration with composite is applied to the surface enabling free radicals to react with the composite resin monomer. the polymerization of the composite resin at the propagation stage will be disrupted resulting in early polymerization. inadequate polymerization produces polymers with low mechanical strength which, in turn, will result in low bonding to the tooth structure.8 the occurrence of shear bond strength may also be due to a loss of calcium or changes in the organic structure affecting tooth structure and leading to a decrease in bonding strength.21 the magnitude of changes that occur in tooth structure is related to the amount of free radicals released by bleaching materials. examination using an electron microscope on the post-bleaching teeth showed that the resin tags are short and irregular while, on some surfaces, they are not even present at all.1,16 freire et al. also highlighted granular and porous features with bubbles on the surface between the resin and bleached enamel. these bubbles can block the infiltration of the bonding material into the tooth structure.16 an lsd test showed that there was a significant difference in shear bond strength of composite resin between group a (control) and groups c and d, as well as between group b and groups c and d. the shear bond strength of composite resin in groups a and b is lower than in groups c and d. these results could probably be attributed to the fact that in groups a and b the remaining free radical residue was greater than in groups c and d. table 2. one-way anova of shear bond strength in dentin bleached with 35% h2o2 and with 35% sodium ascorbate applied variable sum of squares df mean square f sig between groups 119.913 3 39.971 6.884 0,001* within groups 185.807 32 5.806 total 305.720 35 *< 0,05 = significance. table 1. the mean shear bond strength of composite resin (n/ mm2) in dentin bleached by 35% h2o2 and with 35% sodium ascorbate applied group mean ± sd samples a 10.68 ± 2.63 9 b 9.11 ± 2.14 9 c 13.40 ± 1.54 9 d 13.34 ± 3.05 9 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p178–182 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p178-182 181181nugraheni, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 178–182 this finding is in accordance with the study of freire et al. which shows that to remove all residual free radicals after bleaching with 35% h2o2, the application of 35% sodium ascorbate should be carried out more than once.17 the remaining free radicals will result in early termination. consequently, the mechanical properties of the composite resin in the group soaked in saliva for one week and receiving a one-off application of 35% sodium ascorbate are lower when compared to the mechanical properties of the composite resin in the group receiving two or three applications of 35% sodium ascorbate. more free radical residue will also result in greater loss of calcium and changes in the structure of more organic tissues, including collagen. to achieve a strong bond with dentin, collagen is required to create a dentin hybrid layer on a resin composite.19 hydrogen peroxide can lead to denaturation of collagen so that no effective hybrid layer is formed resulting in weakly-formed bonds. the lower mechanical properties of the composite resin and the greater denaturation of collagen will result in lower shear strength.22 an lsd test showed no significant difference in shear bond strength between groups c and d. this result was probably due to free radical residue being attached when 35% sodium ascorbate is applied twice to dentin. when the application is propagated three times, it proves unsuccessful. the application of 35% sodium ascorbate more than once leaves no free radical. therefore, complete composite resin polymerization can take place.17 this study showed that the application of 35% sodium ascorbate can improve the shear bond strength of composite resin on teeth bleached with 35% h2o2. therefore, with composite resin restoration after bleaching a one-week delay is unnecessary. the shear bond strength of composite resin is higher if teeth bleached with 35% h2o2 had 35% sodium ascorbate applied to them more than once. this study used sodium ascorbate in gel form due to the consideration greater ease and control of application. the selection of gel form was also based on the research of awdah et al. which states that bleached teeth treated with sodium ascorbate gel form or solution showed the same shear bond strength. the gel form has a high viscosity, is easier to apply and can be applied to the tooth surface for a greater duration compared to a solution form.23 in conclusion application frequency of 35% sodium ascorbate affects on shear bond strength of composite resin restoration on bleached dentin by 35% h2o2. acknowledgement the authors would like to thank the indonesian government (bpdn) for the scholarship funding this article. this topic have been orally presented at the ichs forum on august 18, 2017. the authors deny any conflicts of interest related to this study. references 1. torres crg, koga af, borges ab. the effects of anti-oxidant agents as neutralizers of bleaching agents on enamel bond strength. braz j oral sci. 2006; 5: 971–6. 2. briso alf, toseto rm, rahal v, dos santos ph, ambrosano gmb. effect of sodium ascorbate on tag formation in bleached enamel. j adhes dent. 2012; 14(1): 19–23. 3. feiz a, khoroushi m, gheisarifar m. bond strength of composite resin to bleached dentin: effect of using antioxidant versus buffering agent. j dent (tehran). 2011; 8(2): 60–6. 4. kunt ge, yılmaz n, şen s, ömür dede d. effect of antioxidant treatment on the shear bond strength of composite resin to bleached enamel. acta odontol scand. 2011; 69: 287–91. 5. lima af, fonseca fm, freitas ms, palialol ar, aguiar fh, marchi gm. effect of bleaching treatment and reduced application time of an antioxidant on bond strength to bleached enamel and subjacent dentin. j adhes dent. 2011; 13(6): 537–42. 6. briso alf, rahal v, sundfeld rh, dos santos ph, alexandre rs. effect of sodium ascorbate on dentin bonding after two bleaching techniques. oper dent. 2014; 39(2): 195–203. 7. da silva jmg, botta ac, barcellos dc, pagani c, torres crg. effect of antioxidant agents on bond strength of composite to bleached enamel with 38% hydrogen peroxide. mater res. 2011; 14(2): 235–8. 8. park j, kwon t, kim y. effective application duration of sodium ascorbate antioxidant in reducing microleakage of bonded composite restoration in intracoronally-bleached teeth. restor dent endod. 2013; 38(1): 43–7. 9. uysal t, ertas h, sagsen b, bulut h, er o, ustdal a. can intracoronally bleached teeth be bonded safely after antioxidant treatment?. dent mater j. 2010; 29: 47–52. 10. oskoee ss, oskoee pa, soroush mh, ajami aa, beheshtirouy m,pour naghi-azha r f. effect of 10% sodium ascorbate on streptococcus mutans adherence to bleached bovine enamel surface. african j biotechnol. 2010; 9(33): 5419–22. 11. t hapa a, viveka na nda pa r, t homas ms. eva luation a nd comparison of bond strength to 10% carbamide peroxide bleached enamel following the application of 10% and 25% sodium ascorbate and alpha-tocopherol solutions: an in vitro study. j conserv dent. 2013; 16(2): 111–5. 12. güler e, gönülol n, özyilmaz öy, yücel aç. effect of sodium ascorbate on the bond strength of silorane and methacrylate composites after vital bleaching. braz oral res. 2013; 27(4): 299–304. 13. whang h, shin d. effects of applying antioxidants on bond strength of bleached bovine dentin. restor dent endod. 2015; 40(1): 37–43. 14. kadiyala a, saladi hk, bollu ip, burla d, ballullaya sv, devalla s, maroli s, jayaprakash t. effect of different anti-oxidants on shear bond strength of composite resins to bleached human enamel. j clin diagn res. 2015; 9(11): zc40–3. 15. türkmen c, güleryüz n, atali py. effect of sodium ascorbate and delayed treatment on the shear bond strength of composite resin to enamel following bleaching. niger j clin pract. 2016; 19: 91–8. table 3. lsd test of shear bond strength in dentin bleached by 35% h2o2 and with 35% sodium ascorbate applied group a b c d a – 0.177 0.023* 0.025* b – – 0.001* 0.001* c – – – 0.961 d – – – – note: *< 0,05 = significance; a (control) = soaked in saliva for 1 week; b = one application of 35% sodium ascorbate; c = two s application of 35% sodium ascorbate; d = three applications of 35% sodium ascorbate. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p178–182 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p178-182 182 nugraheni, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 178–182 16. freire a, souza em, caldas dbm, rosa ear, bordin cfw, de carvalho rm, vieira s. reaction kinetics of sodium ascorbate and dental bleaching gel. j dent. 2009; 37: 932–6. 17. freire a, durski mt, ingberman m, nakao ls, souza em, vieira s. assessing the use of 35 percent sodium ascorbate for removal of residual hydrogen peroxide after in-office tooth bleaching. jada. 2011; 142: 836–41. 18. murad cg, de andrade sn, disconzi lr, munchow ea, piva e, pascotto rc, moura sk. influence of 10% sodium ascorbate gel application time on composite bond strength to bleached enamel. acta biomater odontol scand. 2016; 2(1): 49–54. 19. roberson tm, heymann ho, swift ej. sturdevant’s art and science of operative dentistry. 4th ed. st. louis: mosby; 2002. p. 593–648. 20. ingle ji, bakland lk, baumgartner jc. ingle’s endodontic. 6th ed. ontario: bc decker; 2008. p. 1383-99. 21. cavalli v, shinohara ms, ambrose w, malafaia fm, pereira pnr, giannini m. influence of intracoronal bleaching agents on the ultimate strength and ultrastructure morphology of dentine. int endod j. 2009; 42(7): 568–75. 22. van noort v. introduction to dental materials. 3rd ed. london: mosby; 2007. p. 99-126. 23. al awdah as, al habdan aha, al muhaisen n, al khalifah r. the effect of different forms of antioxidant surface treatment on the shear bond strength of composite restorations to bonded to office bleached enamel. res rev j dent sci. 2015; 4: 5–11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p178–182 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p178-182 76 nanotechnology in dentistry melanie sadono djamil biochemistry department faculty of dentistry trisakti university jakarta indonesia abstract technology has continuously improved along with the complexity of devices. nowadays, it is widely accepted that micro-technology, which is defined as a further reduction in the size of interconnections and components, is achieved by a conventional “top-down” method. we have now moved to a new concept and approach for fabrication from small to bigger building-block elements, which is called nanotechnology. nanotechnology is the fabrication technology of tiny parts that is achieved by a “bottom-up” method. nanotechnology has been developed in many areas of life sciences, such as in dentistry. this presentation provides some examples that illustrate the progress in technological growth, especially in the nanoscale. in the developments of nanotechnology, we are also concerned in many ways about its ethics and the laws of physics. the expansion in nanotechnology shows that much multidisciplinary research is being done in the nanoscale area. in dentistry, one of the examples is research in dental materials such as nanoleakage types in the use of various adhesives with resin composition. nanodiagnostics are nanotechnology in applied molecular diagnostics. all these fields have applications in diagnostics and in point-of-care hand-held devices. key words: nanotechnology, nanoscale, nanodiagnostics, nanoleakage correspondence: melanie sadono djamil, c/o: bagian biokimia, fakultas kedokteran gigi universitas trisakti. jln. kyai tapa, grogol jakarta, indonesia. e-mail: melaniehendriaty@trisakti.ac.id introduction in the middle of the 20th century, a new technology was developed in various fields of science called microtechnology. in the ensuing progress at the beginning of the 21st century, a technological revolution occurred that began with new developments in the technology of computers and the internet, namely nanotechnology. nanotechnology has since colored research and technological developments in engineering, basic sciences, medicine, dentistry, and even social sciences. the methods of fabrication of materials and devices are today known through the molecular level: casting, grinding, milling up to the lithographic movement of a group of atoms. as if we wanted to make a lego construction with our hands in boxing gloves: you cannot really snap the pieces together the way you like.1 in the future, nanotechnology will allow us to be free of those boxing gloves so that we can readily build from the base the construction that we want, which is strong and easy in terms of the laws of physics that indeed enable this to be possible. similarly with the developments in computer science in coming decades, it may be possible to make a new generation of computers that have a clearer resolution, are stronger, lighter and more precise.1 the trends in nano-scale research are getting brighter and expanding to such an extent that discussions in all branches of knowledge are heading towards nano-science and nanotechnology. what follows in this paper is a discussion of the formation of organic and inorganic materials, as well as hybrid materials, on a nano-scale through methods of chemistry, physics and biology, as well as involving nanostructures and nano-devices.2 the position of nanotechnology and its affect on all branches of science and life are very significant, particularly where the aim of such science is to change a situation that is not good or beneficial into something better. an example of such assistance from nanotechnology in the community is water filtering systems using nanotechnology that produce better sources of water and better sanitation.2 in the basic sciences, particularly molecular biology, there have been 50 years of developments in dna structure, which was introduced by watson and crick in 1954. it was 20 years later when their work got acknowledged with the nobel prize. it began with a central dogmatic theory that was followed later by scientific advances in genetics, which originated in biological phenomena such as theories of recombination and growth.2 initially, there was an understanding of the formation of a product made from atomic elements, and how those atoms were structured. if we recombine the atoms of coal, we can create diamonds; if we recombine the atoms of sand (and adding other trace elements), we get computer chips. if we recombine the atoms of feces, water and air, we get potatoes.1 77djamil: nanotechnology in dentistry nanotechnology of dna structure dna is more than merely the secret of life because it is a versatile component in the formation of structures or devices. in 2003, we had reached 50 years since the double-chain structure of dna was discovered. their discovery is the basis of genetical chemistry and biology. now, many researchers have proven that genes can affect the development and growth of organisms. in fact, dna has functions other than in biochemistry, such as in modern biotechnology where the long dna chains can be used as computer materials. this is called a non-biological use of dna.3 figure 1. an example of a nano-scale dna structure, comprising of a pair of phosphate ions and sugar molecules that are paired with complementary base a & t and c & g, by a weak bond. figure 2. nanotechnology of basic dna structure. dna is an ideal molecule with a structure of a nanometer scale; the chains can form themselves into a complex form of double chains each with its own complement. the main aim of dna-based nanotechnology is a continuation of two-dimensional success to be threedimensional. basically, nanotechnology of dna structure is a concept of combining the stable dna branches with the ends adhering cohesively (figure 2).2 nanomachines the centre or core of nanotechnology is a molecularscale machine. dna has proven to play a decisive role in the formation of such a machine. several machines/devices have been made originating from dna. the mechanism of their formation is based on a transition of the structure of dna molecules, such as a change of the conformation from a double-helix form into another shape.4 removal of co-hexamine deposition of co-hexamine z-dna b-dna coloring molecules figure 3. nanomechanical b-z device. shows controlled movement from the two double helixes (blue and orange) that are connected by a hand comprising 20 alkali pairs (purple). figure 4. semi-conductor solutions of same material (cdse), where different colors are given off due to the size of the particles. figure 5. surgical knife made from microstructured-silicon with a diamond-layered tip. conventional dna is a helix that twists to the right (right-handed dna structure), which we can imagine as if we were climbing a spiral staircase with our left hand 78 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 76–80 holding the inside handrail and our right hand holding the outside handrail. the conventional dna is called b-dna, is soluble in water and has high energy.2 figure 6. teeth filled with the latest generation fillings (nanofillers and nano-hybrids). in addition to that, since 1979, alexander rich and colleagues at mit1 have known of z-dna that spirals to the left (left-handed dna structure). one of the dna structures of the spine contains negative phosphate groups that approach each other in the z-dna structure area. they contain a lot of cytosine and guanine alkalis.2 nanomedicine in years to come, the development of nanotechnology applications is going to be quite intensive, including in the fields of health. nanotechnology has not only attracted scientists but also governments, academics and industry, even though it is difficult to give an exact definition of ‘nanotechnology’, and where it differs from microtechnology. it is most likely that the defining point is the difference in their formation, namely micro-structures are formed by a “top down” process while nano-structures are formed through a “bottom up” process.5 micro-technology can be defined as the formation of structures through a “bottom up” process of the bulk deposition and removal of a material, as occurs in a microelectronic industry. this is seen as a “top down” approach resulting in a final structure, which begins with materials of a large physical size.6 conversely, nanotechnology is the formation of a structure through a “bottom up” process, starting at the molecular level and using atoms or molecularscale materials, which results in the desired structure. nanotechnology is not merely a ‘laboratory curiosity technology’. such a view has circulated in the markets, such as with sunscreens that have nano-particles added, window panes made with nano-engineering so that dirt does not easily stick to them, and wall paint that changes its color when viewed from different angles. since 2003 in america, research into nanotechnology itself has consumed more than usd700 million. health sciences and biomedicine are the prime targets.6 evaluation of nano-particle biology has been around long, and so will be soon be familiar and cleaned of its macrophage pharmaco-kinetic medicine is layered with nano-particles and given intravenously so that the length of time of the medicine undergoes a change. the pharmacokinetics of medications and the biological characteristics of the particles are therefore estimated to remain long in the blood circulation, whether they need to be administered once or repeatedly, needs to be designed precisely so that the optimal effect of the medicine, for both therapy and diagnostics, is achievable.7 phosphorus and biolabels nano-particles have physical characteristics that are generally very different from the bulk material. an example is quantum dots whose electronics is set by the theories of quantum physics: if they are hit by light of a specific wave length, they will emit light at a different wave length, which is related to the size of the particles. the picture below shows solutions containing the same semi-conductor material (cdse), but produce different colors; this is caused by the size of the particles.8 oxonica (oxford, uk) has developed a phosphorous material that emits color of a narrow wave length. this can be used as a replacement material/solution for use in diagnosis (where luminescent organic dyes are normally used) with high stability, nontoxicity, all colors readable at a single wave length, colors produced being stable at specific times, the screening process can be automated – because the output spectrum of nano-particles have sharp biolabels and high quality results.9 healing of wounds nucryst (wakefield, usa) has made a wound-healing material (dressing) that is generally used in specialist burn-treatment hospitals in america. the dressing contains nanocrystalline silver that stops 150 types of fungus and bacteria, including several bacteria that are resistant to antibiotics. meanwhile, another company has placed nanoparticles into a plastic material to make it biocidal. this can be useful for devices that are placed inside the body.10 batteries for portable devices batteries can be modified by nanotechnology. ntera (dublin, ireland) has developed a nano-scale low-cost refillable battery material of ion lithium. the metal oxide in the new nano-structure will replace the traditional type of electrode material in ion lithium batteries, which can be refilled several times.4 surgical devices the characteristics of materials change drastically according to the thickness of some molecules. gfd (ulm, germany) has produced a surgical knife from microstructured-silicon with a diamond-layered tip (see picture below). diamond is a material that is chemically rigid, and silicon is non-magnetic and biocompatible. the knife therefore makes sharper incisions and with a lower penetration pressure.11 79djamil: nanotechnology in dentistry implants nanotechnology has enabled the creation of new surfaces, visual effects and overall changes that are beneficial. sus tech gmbh (darmstadt, germany) has worked on nano-apatite and other biocomposites for the surface of tooth and bone prostheses. generally, the use of artificial spine implants leads to irritation for the user because the surface is not hard enough, so by making the surface tougher, the life of the implant is extended.6 nano-capsules nanotechnology can assist in the precise delivery of medicine to the target organ. layered with nano-scale polymer particles, materials become hydrophilic and immune to natural bodily mechanisms. during the past five years, hundreds of fabricated protein medicines have reached the market, for example, insulin, growth factor supplements, cytocines, and monoclonal antibodies. protein itself cannot be taken orally because the digestive system will break it down into amino acid and the protein molecules are too large to be given transdermally. consequently, the administration of such medicines by inhalation is very necessary.10 nano-crystals (king of prussia, pennsylvania, usa) allow the provision of medicines in various forms. medicines in nano-crystal form are more readily absorbed. this new technique stabilizes the medicine particles with polymer on its surface so that it changes clinical patches and medication that has a bad consistency.4 ferex (san diego, california, usa) has been increasingly developed and marketed in the technological form of magnetic target carriers (mtc) to specifically reach a target site and to enable the release of the pharmacological materials that can be absorbed by the mtcs.4 discussion in dentistry nanotechnology have been used, one of the examples in dental materials. resin composites that are combined with particles of nano-fill resin composites (supreme) and nanohybrid (grandio) plus an ormocerbased tooth-colored restorative material (admira) after the finishing and polishing procedure will improve the rough surface. these days, patients can choose the treatment that is going to be done on their teeth, especially from an esthetic point of view, as well as biocompatibility, durability, length of use, and safety. posterior esthetic fillings are generally not so good from a biological and physio-chemical point of view compared to gold and amalgam. first generation composite resins were limited in their resistance to the use abrasions, and their color varied; they shrank after polymerization, had low strength and elasticity, and were easily fractured. new formulas—types of nano-fillers—reduce the shrinkage after polymerization, increase the strength and elasticity, reduce the fractures, and can withstand abrasion and congestive discoloration. in addition, various combinations of light, heat, pressure, vacuums and nitrogen are used to increase the degree of strength after the post-curing period and repair the physical characteristics and mechanics of the resin. other additional results of the latest generation fillings are their optical characteristics that are more translucent, fluorescent and opalescent so that they resemble the structure of real teeth.12 esthetic dentistry has been expanding through innovations in bonding systems, restorative materials, function-based treatments, and the design of conservation preparations. the latest generations of direct resin composites for posterior teeth are very beneficial for certain cavities, particularly those that are not too wide, or just small cavities, keeping in mind those shrinkages still occur.13 i n d i r e c t r e s i n c o m p o s i t e s y s t e m s a r e m o r e recommendable for large cavities. these indirect systems can satisfy the mechanical and biological requirements, while also achieving esthetic results, and shrinkage is less and more easily manageable. the materials used, which are called ceramic optimized polymers, can maintain a high density of inorganic ceramic micro-fillers compared to previous generations. these second-generation indirect systems are known as resin or a porcelain composite whose limitations cannot be overlooked but the value of their use is high.13 the materials can be classified as ‘micro-hybrids’ that include combinations of inorganic particles (as fillers) and organic polymers (matrix) with a ratio of 2:1. the fillers are a main determining material of the clinical and physiochemical characteristics of a resin composite. the particles of the sub-micron fillers can determine the characteristics of the surface, such as the polishing capacity and wear resistance. the use is affected by the size of the fillers, and the shape, burden and bonding of the matrix.12 as additions, there are diverse combinations of light, heat, pressure and vacuums, as in the use of nitrogen, for speeding up the process after curing in order to improve the physical characteristics of the second generation indirect resin systems. the curing process removes the monomer remnants, and enables the optimal polymerization to be achieved. oxygen is removed through a vacuum process, and pressure or nitrogen can remove trapped air, so that the opacity of the restorative material can be preserved. precise and good manipulation can result in maximum strength and homogeneity, esthetic qualities, color stability, and extend the strength of the material in use.12 based on the results of research done by suzuki14 by comparing several denture materials using nano-filled filling material (veracia) with micro-filled dentures (srorthosit, endura, duardent, surpass), cross-linked acrylic (sr-postaris, genios-p, creapearl, vitapan physiodens, premium 8, integral), it was proven that dentures with basic materials of nano-composites were stronger and more wear resistant compared to using acrylic, but almost the same as micro-filled dentures.14 80 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 76–80 the in-vitro studied use of the developments of nano-filler and nano-composite filling materials to other composites such as hybrids, micro-hybrids and microfillers. the researches concluded that nano-fillers and nano-composites were stronger and esthetically better with the translucent characteristics of the teeth, and are usable for both anterior and posterior teeth. with the increasing developments of bionanomaterial science, we are forced to go into it more deeply both theoretically and through research so that we can understand the structures and functions of bionanomaterials. we also need to look into the use of dna, rna and peptides as the basis of the developments in biology and nanomaterials. particularly deserving of our attention are biochemistry, biophysics, thermodynamics, and the electronic content of dna, rna and peptides because they represent important matters in interdisciplinary life sciences and material sciences. t h e r e a r e m a n y b e n e f i t s f r o m s t u d y i n g a n d using nanobiochip materials, interface materials and nanobiosensors, as well as systems of nano-drug-delivery, in industry, resistance, and applications in health sciences. we therefore need to always review our current uses so that the developments of bionanomaterial technology can be beneficial for the whole world. references 1. doty rc, fernig dg, levy r. nanoscale science: a big step towards the holy grail of single molecule biochemistry and molecular biology. cell mol life sci 2004 augustus; 61(15):1843–9. 2. seeman nc. biochemistry and structural dna nanotechnology: an evolving symbiotic relationship. j biochemistry 2003 june 24; 42(24):7259–69. 3. minguez n, ellacuria j, soler ji, triana r, ibaseta g. advances in the history of composite resins. j hist dent 2003 november; 51(3):103–5. 4. allen kl, schenkel ab, estafan d. 2004. an overview of the cerec 3d cad/cam system. gen dent. may-jun; 52(3):234–5. 5. carella m, volinia s, gasparini p. nanotechnologies and microchips in genetic diseases. j nephrol 2003 july-augustus; 16(4):597–602. 6. silva ga. introduction to nanotechnology and its applications to medicine. surg neurol 2004 march; 61(3):216–20. 7. williams d. nanocrystalline metals: another opportunity for medical devices? med device technol 2003 november; 14(9):12, 16–7. 8. vo-dinh t. nanobiosensors: probing the sanctuary of individual living cells. j cell biochem 2002; 39(suppl):154-61. 9. jain kk. nanodiagnostics: application of nanotechnology in molecular diagnostics. expert rev mol diagn 2003 march; 3(2):153–61. 10. huikko k, kostiainen r, kotiaho t. introduction to micro-analytical systems: bioanalytical and pharmaceutical applications. eur j pharm sci 2003 october; 20(2):149–71. 11. wilkinson jm. microand nanotechnology fabrication processes for metals. med device technol 2004 june; 15(5):21–3. 12. burgess jo, gallo jr, ripps ah, walker rs, ireland ej. clinical evaluation of four class 5 restorative materials: 3-year recall. am j dent 2004 june; 17(3):147–50. 13. tay fr, pashley dh, yiu c, cheong c, hashimoto m, itou k, yoshiyama m, king nm. nanoleakage types and potential implications: evidence from unfilled and filled adhesives with the same resin composition. am j dent 2004 june; 17(3):182–90. 14. terry da, leinfelder kf. an integration of composite resin with natural tooth structure: the class iv restoration. pract proced aesthet dent 2004 april; 16(3):235–42. quiz 244. 15. mitra sb, wu d, holmes bn. an application of nanotechnology in advanced dental materials. j am dent assoc 2003 october; 134(10):1382–90. 102 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 102–105 research report differences in surface roughness of nanohybrid composites immersed in varying concentrations of citric acid gabriela kevina alifen, adioro soetojo, and widya saraswati department of conservative dentistry, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: the surface roughness of restoration is important in predicting the length of time it might remain in the mouth. conditions within the oral cavity can affect the surface roughness of a restoration. nanohybrid composite is widely used in dentistry because it can be applied to restore anterior and posterior teeth. athletes routinely consume isotonic drinks which are acidic and even more erosive than the carbonated variety because they contain a range of acids; the highest content of which being citric acid. purpose: the aim of the study was to analyze the surface roughness of nanohybrid composite after having been subjected to immersion in varying concentrations of citric acid. methods: two isotonic drinks (pocari sweat and mizone) were analyzed using high performance liquid chromatography (hplc) to quantify the respective concentrations of citric acid which they contained. a total of 27 samples of cylindrical nanohybrid composite were prepared before being divided into three groups. in group 1, samples were immersed in citric acid solution derived from pocari sweat. those of group 2 were immersed in citric acid solution derived from mizone; while group 3, samples were immersed in distilled water as a control. all samples were immersed for 7 days, before their surface roughness was tested by means of a surface roughness tester (mitutoyo sj-201). data was analyzed using a one-way anova test. results: the results showed that there was no significant difference in surface roughness between groups 1, 2 and 3 (p=0.985). conclusion: no difference in surface roughness of nanohybrid composites results from prolonged immersion in varying concentrations of citric acid keywords: composite; nanohybrid; citric acid; surface roughness; ph correspondence: adioro soetojo, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: adioros@yahoo.com introduction nanohybrid composite consists of both micrometersized and nanometer-sized particles1 and represents a universal composite for both anterior and posterior restoration. it can also be used for esthetic purposes, being suitable for filling both posterior teeth requiring great pressure and anterior teeth.2 if the composite resin is eroded, teeth with fillings formed from this substance can suffer loss of anatomy and secondary caries, in addition to experiencing increased surface roughness of the restoration. this can, in turn, lead to the formation of plaque and deposits staining the restoration. this condition leads to irritated soft tissues which can develop into gingivitis, as well as a decrease in restorative resilience.3 the surface roughness of composite resin is affected by filler content, volume, matrix type, and coupling agent disintegration within the surfaces of the composite resin fillers.4 athletes consume isotonic drinks before, during, and after exercise to minimize dehydration and excessive changes in electrolyte balance.5 in a number of countries, researchers have published data on the relationship between athletes and dental erosion. in the united states, some have reported that more than 35% of athletes experience tooth erosion.6,7 a similar prevalence occurred in the united kingdom, where 36-85% of athletes surveyed suffered from the condition.8 it was also found to afflict 45% of athletes who participated in the london olympic games in 2012.9 dental erosion affected 25% of the 12-17 year old swimmers in lithuania and 50% of 18-25 year old swimmers in lithuania.10 similarly, an australian study dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p102–105 mailto:adioros@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p102-105 103103alifen, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 102–105 reported that between 20-30% of athletes surveyed suffered from dental erosion.11 one of the factors affecting dental health is the consumption of isotonic drinks with low ph, i.e. approximately ph 2.4 to 4.5, which is below the critical ph level. the majority of soft drinks, including the isotonic variety, contain several types of acids, such as phosphoric, citric, malic, and tartaric.12,13 previous research also reported that energy drinks consumed during exercise contain citric acid which damages the organic fillers of composite.14 in fact, citric acid is a weak acid often used as an additive in food and drinks. furthermore, there is no clear-cut critical ph concentration below which erosion will occur.15 therefore, this study aimed to analyze the surface roughness of nanohybrid composites immersed in varying concentrations of citric acid. materials and methods this laboratory-based experimental research employed a post test-only control group design. the concentrations of citric acid solution used were adjusted to those contained in some isotonic drinks available in indonesia. isotonic drinks analysed in this research were pocari sweat and mizone, widely consumed by the public in indonesia based on the top brand index in the year 2011 and 2015. a high performance liquid chromatography (hplc) test was performed in advance to determine the concentration of citric acid contained in each of the isotonic drinks. based on hplc test, the concentration in pocari sweat was 2509.2 ppm, while that of mizone was 1897.6 ppm. pure citriid acid was made for each test results in accordance to its concentration: pure citric acid with concentration of 2509.2 ppm has a ph of 2, while the one with a concentration of 1897.6 ppm has a ph of 3.. the research sample used nanohybrid composite resin (filltek z250 xt, 3m espe, mn, usa) in a cylindrical shape with a diameter of 5 mm and a thickness of 2 mm, activated by light curing method.16 the number of samples was 27, divided into three groups. group 1 used nanohybrid composites immersed in citric acid at a concentration of 2509.2 ppm. group 2 used nanohybrid composites immersed in citric acid at a concentration of 1897.6 ppm. meanwhile, group 3 used nanohybrid composites immersed in distilled water as a control. a cast for the samples was made of 2 mm thick acrylic plates and a hole diameter of 5 mm.16,17 the cast was smeared with vaseline and subsequently placed on a glass plate with a celluloid strip attached. nanohybrid composite resin was then introduced into the cast on the glass plate covered with the celluloid strip and subjected to a weight load for 30 seconds to produce a flat and smooth surface.18 the scales and glass plate were then lifted. thereafter, polymerization was carried out using light curing units (cure rite, caulk, dentsply, canada) at a wavelength of 400-500 nm and an average light intensity of 637 mw/ cm2 for 20 seconds (in accordance with the manufacturer’s instructions). sample preparation was conducted to obtain 27 samples with flat, smooth, shiny surfaces. the entire sample was randomly divided into three groups and then subjected to prolonged immersion in the solutions. the samples were immersed for 7 days in each of the test solutions which were replaced daily in order to maintain their stability. the immersion time was determined based on the assumptions that, with each instance of drinking, residual beverage may remain in the mouth for about 15 minutes, and that the 7-day immersion is equivalent to a period of 672 days (7 x 24 hours x 60 minutes divided by 15 minutes per day) or about 2 years of isotonic drink exposure to restorative material in the oral cavity.17 immersion was conducted within sealed bottles placed in a sealed box at room temperature to avoid sunlight possibly negatively affecting the stability of the solutions. after 7 days of immersion, samples were taken from each test group and then dried with blotting paper. the surface roughness of the samples was then investigated using a surface roughness tester (mitutoyo sj-201, california, america) with an accuracy of 0.01-100 μm. the parameter used in the surface roughness test was that of roughness average (ra) which shows an average value for the surface roughness of the whole formation of the peaks and valleys recorded by the tool.18,19 data was collected twice from the intersection at the mid point of the samples. the data obtained was tabulated for each group and analyzed for normality with a kolmogorov smirnov test. a levene test was used to measure the homogeneity of the data. normally distributed and homogeneous data would be examined using a one-way anova test to reveal any significant differences between the sample groups. table 1. results of the difference test on the surface roughness of nanohybrid composites immersed in citric acid solution at different concentrations for seven days group n mean (μm) standard deviation (μm) p 1 9 0.2211 0.05413 0.985 2 9 0.2183 0.04465 3 9 0.2178 0.02682 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p102–105 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p102-105 104 alifen, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 102–105 results this research focused on the surface roughness of nanohybrid composites immersed in citric acid solution at different concentrations. the results are shown in table 1 and figure 1. from the table and figure above, group 1 composites immersed in citric acid at a concentration of 2509.2 ppm, had a similar mean surface roughness to that of group 2 composites immersed in citric acid at a concentration of 1897.6 ppm. the mean surface roughness in group 1 and group 2 was 0.2211 and 0.2183 μm, respectively; while in group 3, composites immersed in distilled water as a control produced results of 0.2178 μm. these three findings were compared and tested statistically using one-way anova, producing the result p = 0.985 (p>0.05). the result confirmed that there was no significant difference in the surface roughness of nanohybrid composites immersed in citric acid solutions of different concentrations. discussion based on the results of this research, no differences existed in the surface roughness of the composites after immersion in pure citric acid solutions at certain concentrations equivalent to those of specific isotonic drink products in indonesia, namely; pocari sweat and mizone. the results of the hplc test also indicated that the highest level of acid contained in those isotonic drinks was citric acid. previous research had similarly found that citric acid is the most acidic ingredient in energy drinks and sport drinks.14 such research had also used the same detector, a surface roughness tester, to detect the surface roughness of composites with the same accuracy value.3,17 nevertheless, this surface roughness tester (mitutoyo sj-201, california, america) has some limitations when used to examine the surface roughness of materials with nanometer-sized particles since it can only detect the loss of micrometersized composite particles (0.01-100 μm). consequently, if the size of composite particles released is smaller, they will remain undetected. the complex structure of a surface cannot be fully characterized by means of surface-only roughness measurements.20 more valid predictions of clinical performance can be made when the surface roughness measurements are combined with an sem analysis that permits evaluation.20 a study by ergücü et al., used roughness measurement and sem to reveal the damage to the surface of all the resin composite tested. it was observed that roughness measurements were largely confirmed by sem analysis.20 in this research, the results showed that citric acid alone did not cause the surface roughness of the nanohybrid composites. citric acid is a weak organic acid, commonly used as a food additive. therefore, the strength of the acid is insufficient to damage the bonds contained in the composites that have been perfectly polymerized.15 the composites used in this research were nanohybrid in nature, which featured a combination of filler particle sizes, thus causing the bonding between fillers and matrix to be stronger. nanohybrid composites also have high filler contents, so they have a higher resistance to acid conditions.21 the strength of the acid that can cause erosion is not only dependent on the concentration or the ph of the solution, but also on the amount of acid that is available (titratable acidity), the degree of dissociation (pka), and the function of acid as a chelating agent.22 there are some researchers who claim that soft drinks have a low ph and high titratable acidity (ta).7,23 however ta is more important in determining the erosive potential of a drink rather than its degree of acidity (ph).24 the degree of acidity (ph) merely represents the hydrogen ion concentration of a drink, while ta measures the total acid concentration of a solution.25 although the types of beverages are acidic, their ph values and their ta are different.23 based on the results of this research, there was no difference in the surface roughness of the composites between the group using citric acid solution with an acidity degree of 2 and the control group. in contrast to this research, a previous investigation using a drink with an acidity degree of 2.97 showed a difference in the surface roughness of nanohybrid composites.3 another previous piece of research showed that citric acid can dissolve or erode the surface of a tooth15 and that citric acid has the capacity to chelate (chelating agent) so that it can interact with saliva as well as instantly soften and dissolve tooth mineral.26 in this research, however, the test of the citric acid exposure was only conducted on the composite filling materials, which did not contain calcium ions. moreover, it did not use saliva that contains calcium minerals, but only used distilled water. as a result, the role of citric acid as a chelating agent, which was thought to affect the potential for erosion, could not be detected. it can be concluded that there is no significant difference in the surface roughness 0.3 0.25 0.2 0.15 0.1 0.05 µm 0. group 2group 1 group 3 figure 1. mean surface roughness of nanohybrid composites immersed in citric acid solution at different concentrations for seven days. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p102–105 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p102-105 105105alifen, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 102–105 of nanohybrid composites immersed in citric acid solution at different concentrations. nevertheless, further studies into the surface roughness are expected to use another additional tool to detect the surface topography such as ta as a parameter substituting ph. references 1. sakaguchi r, powers j. craig’s restorative dental materials. 13th ed. saint louis: elsevier; 2012. p. 161-75. 2. jain a, deepti d, tavane pn, singh a, gupta p, gupta a, sonkusre s. evaluation of microleakage of recent nano-hybrid composites in class v restorations : an in vitro study. int j adv heal sci. 2015; 2(1): 8–12. 3. tantanuch s, kukiattrakoon b, peerasukprasert t, chanmanee n, chaisomboonphun p, rodklai a. surface roughness and erosion of nanohybrid and nanofilled resin composites after immersion in red and white wine. j conserv dent. 2016; 19(1): 51–5. 4. rajavardhan k, sankar a, kumar m, kumar k, pranitha k, kishore k. erosive potential of cola and orange fruit juice on tooth colored restorative materials. ann med health sci res. 2014; 4(suppl 3): s208-12. 5. sawka mn, burke lm, eichner er, maughan rj, montain sj, stachenfeld ns. american college of sports medicine position stand. exercise and fluid replacement. med sci sports exerc. 2007; 39(2): 377–90. 6. deery c, wagner ml, longbottom c, simon r, nugent zj. the prevalence of dental erosion in a united states and a united kingdom sample of adolescents. pediatr dent. 22(6): 505–10. 7. mathew t, casamassimo ps, hayes jr. relationship between sports drinks and dental erosion in 304 university athletes in columbus, ohio, usa. caries res. 2002; 36(4): 281–7. 8. ashley p, di iorio a, cole e, tanday a, needleman i. oral health of elite athletes and association with performance: a systematic review. br j sports med. 2015; 49(1): 14–9. 9. needleman i, ashley p, petrie a, fortune f, turner w, jones j, niggli j, engebretsen l, budgett r, donos n, clough t, porter s. oral health and impact on performance of athletes participating in the london 2012 olympic games: a cross-sectional study. br j sports med. 2013; 47(16): 1054–8. 10. zebrauskas a, birskute r, maciulskiene v. prevalence of dental erosion among the young regular swimmers in kaunas, lithuania. j oral maxillofac res. 2014; 5(2): 1–7. 11. seow wk, thong km. erosive effects of common beverages on extracted premolar teeth. aust dent j. 2005; 50(3): 173–8. 12. panigoro s, pangemanan dhc, juliatri. kadar kalsium gigi yang terlarut pada perendaman minuman isotonik. e-gigi. 2015; 3(2): 356–60. 13. kumar s, sabikhi l. innovations in ready-to-drink (rtd) nonalcoholic beverages. in: innovative trends in dairy and food products formulation. karnal: national dairy research institute; 2012. p. 45–6. 14. erdemir u, yildiz e, eren mm, ozel s. surface hardness evaluation of different composite resin materials: influence of sports and energy drinks immersion after a short-term period. j appl oral sci. 2013; 21(2): 124–31. 15. lussi a. dental erosion: from diagnosis to therapy. withford gm, editor. vol. 20, monographs in oral science. basel: karger; 2006. p. 233. 16. paula ab de, alonso rcb, araújo gas de, rontani jp, corrersobrinho l, puppin-rontani rm. influence of chemical degradation and abrasion on surface properties of nanorestorative materials. brazilian j oral sci. 2015; 14(2): 100–5. 17. nurmalasari a. perbedan kekasaran permukaan komposit nano pada perendaman teh hitam dan kopi. j wiyata. 2015; 2(1): 48–53. 18. carvalho f, sampaio c, fucio s, carlo h, sobrinho lc, rontani rp. effect of chemical and mechanical degradation on surface roughness of three glass ionomers and a nanofilled resin composite. oper dent. 2012; 37(5): 509–17. 19. bala o, arisu hd, yikilgan i, arslan s, gullu a. evaluation of surface roughness and hardness of different glass ionomer cements. eur j dent. 2012; 6(1): 79–86. 20. ergücü z, türkün ls. surface roughness of novel resin composites polished with one-step systems. oper dent. 2007; 32(2): 185–92. 21. han l, okamoto a, fukushima m, okiji t. evaluation of flowable resin composite surfaces eroded by acidic and alcoholic drinks. dent mater j. 2008; 27(3): 455–65. 22. wan bakar wz, abdullah a, hussien a. erosion effect of acidic drinks on two types of glass ionomer cement. malays dent j. 2011; 33(2): 27–32. 23. tenuta lma, fernández ce, brandão acs, cury ja. titratable acidity of beverages influences salivary ph recovery. braz oral res. 2015; 29(1): 1–6. 24. rai vr, bai ja. benefecial microbes in fermented and functional foods. boca raton: crc press; 2014. p. 600. 25. bamise ct, esan ta, oziegbe eo, alo if. ph and titratable acidity of different cough syrups in nigeria. tanzania dent j. 2014; 18(2): 49–55. 26. bamise c, kolawol k, oloyede e. the determinants and control of soft dr inks-incited dental erosion. rev clínica e pesqui odontológica. 2009; 5(2): 141–54. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p102–105 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p102-105 196 volume 46, number 4, december 2013 efek ekstrak daun singkong (manihot utilissima) terhadap ekspresi cox-2 pada monosit yang dipapar lps e.coli (the effect of manihot utilissima extracts on cox-2 expression of monocytes induced by lps e. coli) zahara meilawaty bagian biomedik fakultas kedokteran gigi universitas jember jember indonesia abstract background: periodontal disease is a common and widespread disease in the community. gram negative bacteria have a role in periodontitis. these bacteria secrete a variety of products such as endotoxin lipopolysaccharide (lps), which causes the occurrence of inflammation or infection. the body defense responses are neutrophils and mononuclear cells (monocytes and macrophages). in response to defense mechanism, the body will be expressed enzyme cyclooxygenase (cox) which functions convert arachidonic acid to prostaglandins. cassava leaf cells known to play a role in reducing inflammation, but the mechanism for inhibiting cox-2, is not known. purpose: the study was aimed to determine the effect of cassava leaf extract (manihot utilissima) on expression of enzyme cox2 in monocytes which were exposed by lps e. coli. methods: this study was in vitro experimental studies with the design of posttest only control group design. the sample was the cassava leaves extract (manihot utilissima) at concentration of 12.5 % and 25 %. the expression of cox-2 was determined by immunocytochemistry method. isolated monocytes were incubated in cassava leaf extract, and then exposed to lps, after washing imunostaning procedure was performed using a monoclonal antibody (mab) anti-human cox-2. the research data was the number of monocytes that express cox-2. results: expression of cox-2 in the group cassava leaf extract was higher than the group that induced by lps e. coli only. conclusion: cassava leaf extract did not inhibit the expression of cox-2 in monocytes which were exposed by lps e. coli. key words: lps, monocytes, cox-2, cassava leaves, manihot utilissima abstrak latar belakang: penyakit periodontal merupakan penyakit umum dan tersebar luas di masyarakat. bakteri yang banyak berperan pada periodontitis adalah gram negatif. bakteri ini mengeluarkan berbagai produk antara lain endotoksin lipopolisakarida (lps) yang menyebabkan inflamasi atau infeksi. respon pertahanan tubuh pertama adalah netrofil dan sel mononuklear (monosit dan makrofag). pada respon pertahanan tubuh akan diekspresikan enzim siklooksigenase (cox) yang berfungsi mengubah asam arakidonat menjadi prostaglandin. daun singkong diketahui berperan dalam menurunkan sel radang, tetapi mekanisme dalam menghambat cox-2, belum diketahui. tujuan: penelitian ini bertujuan untuk meneliti ekstrak daun singkong terhadap ekspresi enzim cox-2 pada monosit yang dipapar lps e. coli. metode: penelitian ini merupakan studi eksperimental in vitro dengan rancangan the posttest only control group design. sampel adalah ekstrak daun singkong (manihot utilissima) dengan dosis 12,5% dan 25%. ekspresi cox-2 diteliti dengan metode imunositokimia. isolat monosit diinkubasi ekstrak daun singkong, kemudian dipapar lps, setelah pencucian kemudian dilakukan prosedur imunostaning menggunakan antibodi monoklonal (mab) anti human cox-2. data penelitian adalah jumlah monosit yang research report 197meilawaty: efek ekstrak daun singkong (manihot utilissima) terhadap ekspresi cox-2 mengekspresikan cox-2.hasil: ekspresi cox-2 pada kelompok ekstrak daun singkong lebih tinggi dibandingkan kelompok yang hanya diinduksi lps e.coli. simpulan: ekstrak daun singkong tidak menghambat ekspresi cox-2 pada monosit yang dipapar lps e. coli. kata kunci: lps, monosit, cox-2, daun singkong, manihot utilissima korespondensi (correspondence): zahara meilawaty, bagian biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: zhr_mel@yahoo.com pendahuluan penyakit periodontal merupakan penyakit umum dan tersebar luas di masyarakat, bisa menyerang anak-anak, orang dewasa maupun orang tua. salah satu bentuk penyakit periodontal adalah keradangan yang menyerang jaringan periodontal, dapat hanya mengenai gingiva yang disebut dengan gingivitis atau mengenai jaringan periodontal yang lebih luas yaitu ligamen periodontal, sementum dan tulang alveolar.1 bakteri yang paling banyak berperan tehadap timbulnya periodontitis adalah bakteri gram negatif, diantaranya yaitu porphyromonas gingivalis, actinobacillus actinomycetemcomitans, prevotella intermedia, dan bacteriodes forsythus. bakteri gram negatif anaerob ini, mengeluarkan berbagai produk antara lain endotoksin biologi aktif atau lipopolisakarida (lps) yang menyebabkan aktivitas biologis sehingga terjadinya kerandangan.2,3 lipopolisakarida adalah salah satu penyebab kelainan periodontal. bahan ini merupakan struktur utama dinding sel bakteri gram negatif anaerob yang berfungsi untuk integritas struktur bakteri dan melindungi bakteri dari sistem pertahanan imun hospes.4 lipopolisakarida mampu menimbulkan stimulasi pada berbagai sel imun, baik in vitro maupun in vivo, substansi ini mempunyai relevansi klinis yang penting karena berperan langsung dalam patogenesis infeksi bakteri gram negatif. infeksi yang diakibatkan aktivitas bakteri dapat menimbulkan respon pertahanan didalam tubuh berupa respon imun spesifik maupun non spesifik. respon imun yang berperan sebagai garis pertahanan pertama terhadap invasi bakteri adalah netrofil dan sel mononuklear (monosit dan makrofag).5 sel darah yang berfungsi sebagai sistem kekebalan bagi tubuh adalah leukosit. leukosit bergerak bebas dalam darah sebagai organisme selular bebas pada sistem kekebalan tubuh. leukosit terdiri dari fagosit makrofag, neutrofil, sel dendritik, sel mast, eosinofil, basofil dan sel pembunuh alami yang merupakan mediator penting pada sistem kekebalan adaptif. monosit merupakan jenis leukosit yang membentuk makrofag. peran monosit ketika terjadi infeksi adalah meninggalkan aliran darah dan bergerak ke dalam jaringan untuk mengidentifikasi dan membunuh patogen dengan menyerang patogen yang lebih besar melalui kontak langsung kemudian membunuh mikroorganisme. monosit bertindak sebagai fagosit yang berperan dalam merespon adanya bakteri patogen, sehingga viabilitas monosit menjadi faktor penting pada sistem kekebalan tubuh.6 enzim siklooksigenase (cox) merupakan target utama obat antiinflamasi nonsteroid, cox merupakan enzim yang berperan dalam merubah asam arakidonat menjadi prostaglandin yang bertanggungjawab terhadap inflamasi, rasa sakit, proliferasi sel dan respon biologis lainnya. cox2 dapat diinduksi oleh sitokin, growth factor, dan stimulus lainnya berdasarkan respon inflamasi. cox-2 biasanya diekspresikan bila terjadi inflamasi atau pada keadaan patologis lainnya, cox-2 juga diekspresikan di saraf otak dan ginjal. secara farmakologi, penghambatan cox dapat digunakan sebagai relief of pain dari gejala inflamasi. semua obat ains, salah satunya ibuprofen bekerja sebagai antiinflamasi dengan menghambat sintesis prostaglandin dengan cara menghambat enzim cox yang mengkatalis reaksi asam arakidonat menjadi senyawa endoperoksidase. obat ini diindikasikan untuk luka pada jaringan lunak, fraktur, ekstraksi gigi, vasektomi, pasca melahirkan, pasca operasi; dapat menekan terjadinya inflamasi. tetapi, penggunaan obat ains dapat menimbulkan efek samping, diantaranya dapat menyebabkan terjadinya perdarahan gastrointestinal, memperlama waktu perdarahan, serta dapat merusak fungsi ginjal.7,8 selama ini, masyarakat hanya mengenal daun singkong sebagai sayuran dan bahan makanan. masyarakat kurang mengetahui bahwa daun singkong memiliki banyak manfaat di dunia kesehatan karena memiliki kandungan vitamin c yang cukup tinggi (sekitar 27,5%), senyawa organik flavonoid, triterpenoid, tanin serta saponin. konsumsi vitamin c sangat bermanfaat dalam proses penyembuhan luka karena dapat mempengaruhi tingkat keparahan respon inflamasi dan kualitas penyembuhan.6,9 penelitian lain juga telah membuktikan bahwa vitamin c dapat menurunkan jumlah neutrofil pada proses penyembuhan luka tikus wistar jantan.10 flavonoid dan saponin sejak lama diketahui memiliki aktivitas antimikroba dan antivirus. demikian juga triterpenoid yang sering ditemukan pada banyak tanaman obat dan diketahui memiliki aktivitas antivirus dan antibakteri, serta dapat mengobati kerusakan pada kulit.11 flavonoid yang diisolasi dari daun singkong sebesar 100-200 µg/ml dapat mengurangi degranulasi sel mast yang diinduksi senyawa 48,80 albumin pada sebuah penelitian in vitro. flavonoid diyakini dapat menghambat prostaglandin.12 ekstrak daun singkong juga diketahui berpotensi dalam menurunkan jumlah neutrofil pada proses penyembuhan luka tikus wistar jantan.13 tetapi mekanisme kerja ekstrak daun singkong itu sendiri terhadap ekspresi 198 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 196–201 enzim cox-2, yaitu enzim yang berperan dalam merubah asam arakidonat menjadi prostaglandin pada inflamasi sampai saat ini belum diketahui. berdasarkan uraian di atas, timbul suatu permasalahan bagaimana potensi ekstrak daun singkong terhadap ekspresi cox-2 pada model inflamasi. tujuan penelitian ini adalah meneliti ekstrak daun singkong (manihot utilissima) terhadap ekspresi enzim cox-2 pada monosit yang dipapar lps e. coli. bahan dan metode penelitian ini merupakan studi eksperimental in vitro dengan rancangan the posttest only control group design. variabel bebas adalah ekstrak daun singkong (manihot utilissima) dengan dosis 12,5% dan 25%. variabel tergantung adalah ekspresi cox-2 pada monosit. variabel terkendali adalah jenis dan konsentrasi monosit dan lps e. coli serta prosedur penelitian. daun singkong (manihot utilissima) yang digunakan didapatkan dari daerah tempurejo, kecamatan tempurejo jember. daun yang diambil adalah daun yang masih hijau, utuh dan berada di bagian tengah pohon untuk menghindari kandungan sianida yang berlebihan pada daun yang terlalu muda. daun singkong terlebih dahulu diidentifikasi di herbarium jemberiense, laboratorium botani dan kultur jaringan, jurusan biologi, fakultas mipa universitas jember. untuk pembuatan ekstrak, daun singkong dicuci bersih, dipotong kecil-kecil dan dikeringkan dengan cara diangin-anginkan selama 24 jam di dalam ruangan dengan suhu ruang, yang tidak terkena sinar matahari secara langsung, kemudian dioven selama 3 jam dalam suhu 45 0c. setelah itu, daun yang kering tersebut digiling menggunakan blender, diayak dengan ayakan 50 maze sehingga didapatkan serbuk halus sebanyak total 400 gram serbuk daun. setelah itu, serbuk daun dimaserasi dengan etanol 95% selama 2 hari dan dilakukan pengadukan setiap hari. selanjutnya, larutan tersebut dipekatkan dengan rotavapor (rotary evaporator) dengan suhu 50°c dan putaran 90 rpm menjadi ekstrak daun singkong dengan konsentrasi 100%. penyimpanan ekstrak 100% ini diletakkan dalam kulkas. isolasi monosit dilakukan dengan metode ficoll hypaque centrifugation.14 sebanyak 12 cc darah (heparinized whole blood) dibagi menjadi dua, sentrifuse 600 rpm selama 10 menit pada suhu ruang. serum yang mengandung platelet dipisahkan, sisa darah diencerkan dengan hbss sehingga menjadi 9 cc. setelah itu menyiapkan dua tabung falcon, masing-masing diisi dengan 3 cc ficol. selanjutnya melapiskan darah (secara berhati-hati) di atas lapisan ficol dengan mikropipet. darah yang telah dilapiskan disentrifuse selama 30 menit, 1400 rpm, sehingga terbentuk 4 lapisan dari atas ke bawah adalah (plasma, mononuklear, ficol dan polinuklear+rbc). lapisan mononuklear (interface plasma-ficol) yang mengandung limfosit dan monosit dipisahkan dan dimasukkan dalam tabung falcon. kemudian dicuci dengan hbss dan disentrifuse 600 rpm, 10 menit, sebanyak 2 kali untuk menghilangkan kontaminan platelet. hasil pencucian diresuspensi dalam hbss sebanyak 2500 µl, kemudian dilakukan pipeting. suspensi sel mononuklear kemudian dilapiskan pada plastic microplate (24 well) yang didasarnya telah diberi cover slip sebanyak 100 µl tiap well, kemudian diinkubasi selama 1 jam, 37 °c. medium inkubasi yang mengandung limfosit dibuang, sisanya yang mengandung monosit dicuci 3 x dengan hbss. hasil pencucian pelet monosit diresuspensi dengan rpmi sebanyak 1000 µl tiap well. kemudian tambahkan penstrep (5 µl dan fungison 5 µl) pada tiap well, pipeting medium secara hati-hati, monosit siap untuk diinkubasi dengan daun singkong. suspensi isolat monosit, masing-masing dibagi menjadi 4 kelompok uji (masing-masing terdiri dari 3 well) yaitu: (1) k = kontrol, tidak diinkubasi eds, tetapi ditambahkan rpmi 1000 µl; (2) p1= tidak diinkubasi eds, tetapi ditambahkan rpmi 1000 µl; (3) p2= diinkubasi eds 12,5% (sebanyak 200 µl); (4) p3= diinkubasi eds 25% (sebanyak 200 µl). inkubasi dilakukan dalam inkubator shaker dengan 5% co2, 37 °c selama 18 jam. setelah 18 jam, isolat monosit pada kelompok 2, 3 dan 4 kemudian dipapar dengan lps e.coli sebanyak 5 µl tiap well kemudian diinkubasi selama 1 jam pada suhu 370c dan 5% co2. setelah inkubasi pemaparan lps selama 1 jam, dilakukan pencucian. kemudian dilakukan prosedur imunostaning menggunakan antibodi monoklonal (mab) anti human cox-2. ekspresi cox-2 dianalisis dengan metode imunositokimia. ekspresi cox-2 ditunjukkan oleh monosit yang membran selnya berwarna coklat, pengamatan dilakukan di bawah mikroskop dengan pembesaran 400 kali. data penelitian adalah jumlah rata-rata monosit yang mengekspresikan cox-2 dihitung per 100 sel. data penelitian diuji normalitasnya menggunakan uji shapiro-wilk dan homogenitasnya menggunakan uji levene. selanjutnya dilakukan uji parametrik menggunakan uji one way anova untuk mengetahui perbedaan ekspresi cox2 dan dilanjutkan dengan uji lsd untuk membandingkan ekspresi cox-2 monosit antar kelompok percobaan. hasil ekspresi cox-2 dilihat menggunakan mikroskop dengan pembesaran 400 kali. data penelitian adalah jumlah rerata monosit yang mengekspresikan cox-2 dihitung per 100 sel. rerata dan simpangan baku ekspresi cox-2 pada masing-masing kelompok dapat dilihat pada gambar 1. rerata ekspresi cox-2 tertinggi terdapat pada kelompok ekstrak daun singkong 12,5% sebesar 55,67, kemudian kelompok ekstrak daun singkong 25% sebesar 47,33; setelah itu kelompok yang hanya diberi lps e. coli sebesar 30,67; dan ekspresi cox-2 terendah terdapat pada kelompok kontrol sebesar 13,67 (gambar 1). data yang didapat diuji normalitasnya terlebih dahulu menggunakan uji shapiro-wilk sebelum dianalisis menggunakan uji parametrik. hasil uji normalitas 199meilawaty: efek ekstrak daun singkong (manihot utilissima) terhadap ekspresi cox-2 menunjukkan bahwa data yang diuji mempunyai nilai sig 0,344 (p>0,05), ini berarti data terdistribusi secara normal sehingga memenuhi syarat untuk dilakukan uji parametrik. oleh karena itu, selanjutnya diuji dengan menggunakan uji parametrik one-way anova yang rangkumannya dapat dilihat pada tabel 1. ekspresi cox-2 pada masing-masing perlakuan mempunyai perbedaan yang bermakna (p<0,05) dapat dilihat pada tabel 1, selanjutnya untuk mengetahui perbedaan ekspresi cox-2 antara masing-masing kelompok dilakukan uji lsd. terdapat perbedaan yang bermakna (p<0.05) antara kelompok kontrol dengan kelompok ekstrak daun singkong 12,5%; kelompok kontrol dengan kelompok ekstrak daun singkong 25%; kelompok lps e. coli dengan kelompok ekstrak daun singkong 12,5% (tabel 2). gambaran mikroskopis ekspresi cox-2 dengan pembesaran 1000x dapat dilihat pada gambar 2. pembahasan penelitian ini adalah penelitian in vitro yang menggunakan sel monosit. hasil penelitian yang terlihat pada gambar 1 menunjukkan bahwa rerata ekspresi cox2 pada kelompok yang diinduksi lps e. coli dan ekstrak daun singkong menunjukkan kecendrungan yang lebih banyak dibandingkan kelompok yang hanya diberi lps e. coli ataupun kelompok kontrol. hal ini juga menunjukkan bahwa pemberian ekstrak daun singkong 12,5% dan 25% mempunyai pengaruh yang bermakna terhadap peningkatan ekspresi cox-2, bahwa ekstrak daun singkong 12,5% dan 25% tidak menghambat ekspresi cox-2 pada sel monosit yang diinduksi lps e. coli. hasil ini tidak sesuai dengan hipotesis, bahwa pemberian ekstrak daun singkong dapat menghambat ekspresi cox-2 pada sel monosit yang diinduksi lps e. coli. pada penelitian ini, didapatkan ekspresi cox-2 pada kelompok kontrol sangat sedikit atau paling rendah. hal ini disebabkan pada kelompok kontrol tidak diinduksi lps e. coli sehingga seharusnya sel monosit tidak mengekspresikan cox-2. cox-2 secara normal ditemukan dalam jumlah yang tidak signifikan tetapi dapat diinduksi oleh sitokin atau growth factor, tetapi akan lebih banyak diekspresikan pada keadaan inflamasi atau keadaan patologis lainnya.8 lipopolisakarida bersifat endotoksik karena lps mengikat reseptor cd14/ toll-like receptor-4 (tlr4) yang mengakibatkan sekresi sitokin proinflamasi dari beberapa tipe sel. cd14 merupakan reseptor permukaan sel pada makrofag dan monosit untuk karbohidrat. makrofag yang berikatan dengan bakteri oleh karena adanya cd14, akan mensekresi sitokin [interleukin-1α (il-1α), il-1β, il16, tumor necrosis factor-α (tnf-α) dan mediator lipid inflamation yaitu prostaglandin (pge2)]. 4 keadaan inflamasi membuat ekspresi cox akan meningkat, baik gambar 1. grafik batang rerata ekspresi cox-2 berdasarkan kelompok perlakuan. gambar 2. gambaran mikroskopis ekspresi cox-2, pembesaran 1000x. tanda panah warna hitam menunjukkan monosit yang mengekspresikan cox-2, tanda panah merah menunjukkan monosit yang tidak mengekspresikan cox-2. sel yang mengekspresikan cox-2 terlihat berwarna coklat, dan yang tidak mengekspresikan cox-2 berwarna biru. counterstain menggunakan mayer’s hematoxylin. tabel 1. rangkuman hasil uji one way anova potensi ekstrak daun singkong (manihot utilissima) dalam memodulasi cox-2 pada monosit yang dipapar lps e. coli f sig. between group 12,255 0,002 tabel 2. rangkuman hasil uji lsd potensi ekstrak daun singkong (manihot utilissima) dalam memodulasi cox-2 pada monosit yang dipapar lps e. coli kontrol lps eds 12,5% eds 25% kontrol .054 .001* .002* lps .010* .058 eds 12,5% .300 eds 25% 13.67 55.67 47.33 30.67 200 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 196–201 cox 1 maupun cox-2. cox 1 merupakan enzim yang ditemukan di banyak sel dan jaringan normal, berperan pada fungsi fisiologis seperti sekresi mukus untuk melindungi mukosa pencernaan, hemostasis, penyembuhan luka, ovulasi, dan untuk memelihara fungsi ginjal. cox-2 baru akan terbentuk setelah diinduksi oleh sitokin dan mediator inflamasi lainnya di daerah inflamasi atau pada keadaan patologis lainnya.8,15,16 pada kelompok yang diinduksi lps e. coli seharusnya paling banyak mengekspresikan cox-2. lps e. coli bisa menginduksi inflamasi atau peradangan, kondisi inflamasi ini bisa menginduksikan ekspresi cox-2. tetapi pada hasil penelitian ini, ekspresi cox-2 hanya sedikit, secara statistik tidak berbeda bermakna dengan kelompok kontrol. hal ini diduga karena lps e. coli akan menstimulasi monosit untuk menghasilkan mediator inflamasi, apabila mediator inflamasi tersebut dikeluarkan dalam jumlah berlebihan akan menyebabkan kerusakan jaringan dan akhirnya dapat membuat monosit lisis.17 hasil ini diperkuat dengan hasil uji viabilitas yang sudah dilakukan penulis sebelumnya yang menunjukkan nilai viabilitas 24,56 %; berarti hanya ada sekitar 25 sel monosit yang hidup dari 100 sel monosit yang diamati pada kelompok yang diberi lps. sel yang lisis atau mati tidak dapat merespon antibodi cox-2 dan tidak dapat mengekspresikan cox-2, sehingga pada penelitian ini hasil ekspresi cox-2 pada kelompok yang hanya diinduksi lps e.coli lebih rendah daripada kelompok yang diberi ekstrak daun singkong. potensi flavonoid dalam menekan inflamasi adalah dengan jalan memblokir siklus siklooksigenase (cox) dan lipoksigenase, sehingga sel radang yang bermigrasi terbatas dan tanda-tanda klinis peradangan berkurang. flavonoid juga dapat bertindak melindungi lipid membran terhadap agen yang merusak.11 diduga aksi ini yang menjaga membran sel tidak mudah dirusak bakteri dan tetap berfungsi dengan baik. saponin selama ini diketahui dapat bekerja sebagai antibakteri. ketika berinteraksi dengan sel bakteri, saponin dapat meningkatkan permeabilitas membran sel bakteri sehingga terjadi hemolisis sel bakteri. saponin juga memiliki efek antiinflamasi yang hampir sama dengan flavonoid, memblokir jalur prostaglandin sebagai penghambat aktifasinya, namun tidak berpengaruh terhadap sintesisnya. dengan dihambatnya pelepasan prostaglandin maka keluarnya sel radang dapat ditekan.11 adanya saponin dalam ekstrak daun singkong diduga dapat mendukung proses penyembuhan luka lebih cepat dengan meminimalisir kontaminasi bakteri sehingga epitel dapat bermitosis dan berproliferasi dengan baik. tannin dan triterpenoid diketahui memiliki aktivitas antioksidan pada beberapa tanaman obat.12 antioksidan berperan menangkap radikal bebas yang dapat menyebabkan kerusakan membran sel. cedera pada membran sel tersebut kemudian mengaktifkan histamin yang nantinya menjadi mediator sel radang.18 antioksidan di dalam tannin dan triterpenoid diduga dapat mengurangi adanya radikal bebas yang dapat merusak membran sel dan mengurangi pelepasan mediator sel radang. pada penelitian ini, ekspresi cox-2 pada kelompok yang diberi ekstrak daun singkong lebih tinggi dibandingkan kelompok kontrol dan kelompok yang diinduksi lps e. coli. hal ini diduga karena sel monosit yang telah diberi ekstrak daun singkong lebih tahan terhadap induksi lps e. coli sehingga banyak sel monosit yang hidup. ekspresi cox-2 paling tinggi terdapat pada kelompok yang diberi ekstrak daun singkong 12,5% dibanding kelompok yang diberi ekstrak daun singkong 25%. hal ini kemungkinan karena ekstrak daun singkong 25% mempunyai toksisitas yang lebih tinggi. konsentrasi obat atau bahan alami yang besar dapat menyebabkan toksisitas yang besar pula, sehingga dapat melisiskan sel monosit. semakin tinggi dosis saponin pada colocynth (citrullus colocynthis) yang diberikan pada tikus, maka kematian tikus semakin tinggi, dosis saponin 100 mg/kg berat badan bersifat toksik.19 hal tersebut juga mungkin yang terjadi pada hasil penelitian ini, pemberian ekstrak dengan konsentrasi lebih tinggi menyebabkan turunnya viabilitas. viabilitas monosit yang diinkubasi ekstrak daun singkong 25% lebih kecil jika dibandingkan dengan viabilitas monosit yang diinkubasi dengan ekstrak daun singkong 12,5%. viabilitas sel monosit pada kelompok yang diberi ekstrak daun singkong 12,5% yaitu sebesar 62,65%, sedangkan viabilitas sel monosit yang diberi ekstrak daun singkong 25% hanya sebesar 43,44%. pada penelitian in vitro ini sel monosit yang sudah lisis tidak dapat meregenerasi selnya kembali, dan juga karena waktu hidup monosit yang pendek disirkulasi darah hanya 8 jam, sehingga sel monosit yang lisis tersebut tidak dapat mengekspresikan cox-2. hasil penelitian ini menunjukkan bahwa ekstrak daun singkong (manihot utilissima) tidak menghambat ekspresi enzim cox-2 pada monosit yang dipapar lps e. coli. ucapan terima kasih terimakasih kepada rektor dan ketua lembaga penelitian universitas jember atas dana dipa universitas jember tahun 2013 sehingga penelitian ini terlaksana. daftar pustaka 1. kurniawati a. hubungan kehamilan dan kesehatan periodontal. j biomed unej 2005; ii(2): 43-51. 2. djais ai. periodontitis sebagai faktor resiko jantung koroner aterosklorosis. j pdgi. 2006; 56(2): 53-9. 3. fitria e. kadar il-1b dan il-8 sebagai penanda periodontitis, faktor resiko kelahiran prematur. j pdgi 2006; 56(2): 60-4. 4. i n d a hya n i de , sa nt o s o a s, ut o r o t. pe nga r u h i n d u k si lipopolisakarida (lps) terhadap osteopontin tulang alveolaris tikus pada masa erupsi gigi. ind j dent 2007; 14(1): 2-7. 5. susilowati h, haniastuti t, santoso as. produksi nitrat oksida dan aktivitas fagositosis makrofag mencit setelah stimulasi dengan lipopolisakarida. maj ked gigi 2009; 16(1): 19-24. 6. robbins sl, cotran rs, kumar v. basic pathology. 7th ed. philadelphia: wb. saunders company; 2003. p. 33-78. 7. tripathi kd. essentials of medical pharmacology. 5th ed. new delhi: jaypee brothers; 2003. p. 156-84. 201meilawaty: efek ekstrak daun singkong (manihot utilissima) terhadap ekspresi cox-2 8. cicconetti a, bartoli a, ripari f, ripari a. cox-2 selective inhibitors: a literature review of analgesic efficacy and safety in oral-maxillofacial surgery. j oral surg oral med oral pathol oral radiol endod 2004; 97(2): 139-46. 9. yendriwati. kebutuhan vitamin c dan pengaruhnya terhadap kesehatan tubuh dan rongga mulut. dentika dental journal 2006; ii(1): 78-83. 10. isgianto wa. pengaruh vitamin c terhadap jumlah neutrofil pmn pada proses penyembuhan luka pada gingiva tikus (rattus norvegiccus). skripsi. jember: fakultas kedokteran gigi universitas jember; 2005. 11. robinson t. 1991. kandungan organik tumbuhan tinggi. edisi 6. padmawinata k, editor. bandung: itb; 1995. h. 154, 191-3. 12. adi lt. tanaman obat dan jus untuk asam urat dan rematik. jakarta: agromedia pustaka; 2006. h. 30-3. 13. nurdiana ar. potensi ekstrak daun singkong (manihot esculanta) terhadap jumlah neutrofil pada proses penyembuhan luka tikus wistar (rattus norvegiccus). skripsi. jember: fakultas kedokteran gigi universitas jember; 2013. 14. purwanto. peran streptococcus mutans dan monosit pada degradasi kolagen tipe iv dan agregasi platelet. disertasi. malang: universitas brawijaya; 2010. 15. isbagio h. peranan obat antiinflamasi non steroid terhadap nyeri dan inflamasi pada penyakit reumatik. cermin dunia kedokteran 1992; no 78: 32-35 16. goodman, gilman. goodman & gilman’s the pharmacological basis of therapeutics. 10th ed. toronto: mc graw hill; 2001. p. 687-71. 17. newman mg, carranza fa, takei hh, klokkevold pr. carranza’s clinical periodontology. 10th ed. philadelphia: saunders; 2006. p. 133-47. 18. price sa, wilson lm. 1995. patofisiologi: konsep klinis prosesproses penyakit. edisi 6. pendit bu, editor. jakarta: egc; 2005. h. 35-46. 19. diwan fh, abdel-hasan ia, mohammed st. effect of saponin on mortality and histopathological changes in mice. eastern mediterranean health j 2000; 6(2-3): 345-51. . �� vol. 45. no. 1 march 2012 research report relationship between salivary fluor concentration and caries index in ��–�� years old children vidyana pratiwi1, dudi aripin2, and ame suciati setiawan3 1 internship 2 department of conservative dentistry 3 department of oral biology faculty of dentistry, padjadjaran university bandung indonesia abstract background: dental caries is a bacterial infection leading to dissolution and localized damage of hard tissues. the assessment of caries risk is based on several caries indicators including clinical conditions (dmf-t index), environment (fluor), and general health. purpose: the objective of this study was to assess the relationship between salivary fluor concentration and caries index in children aging 12–15 years old at smp negeri 2 ptpn viii pangalengan. methods: this study is an observational analytical study using crosssectional approach and is conducted in a field trial manner. the study sample consists of 80 students in the age of 12 to 15 years old at smp negeri 2 ptpn viii selected through probability sampling manner using simple random sampling method. results: the result of this study shows a dmf-t index of 4.32 and salivary fluor concentration mean of 0.018. pearson product moment correlation test shows that there is a weak correlation between salivary fluor concentration and dmf-t index. conclusion: it is concluded that the salivary fluor concentration has an insignificant correlation with the dmf-t index since the fluor concentration in saliva is very low. key words: fluor concentration, saliva, dmf-t index abstrak latar belakang: karies gigi adalah penyakit infeksi bakteri yang berakibat pada disolusi dan kerusakan terlokalisasi jaringan keras. penilaian risiko karies berdasarkan atas beberapa indikator karies yaitu kondisi klinis (indeks dmf-t), lingkungan (fluor), dan kesehatan umum. tujuan: penelitian ini bertujuan untuk mengetahui hubungan konsentrasi fluor yang terdapat dalam saliva dengan indeks karies pada anak usia 12-15 tahun di smp negeri 2 ptpn viii pangalengan. metode: jenis penelitian ini adalah penelitian analitik observasional dengan pendekatan cross sectional yang dilakukan di lapangan. sampel penelitian sebanyak 80 orang siswasiswi usia 12–15 tahun di smp negeri 2 ptpn viii pangalengan yang dipilih secara probability sampling dengan metode simple random sampling. hasil: hasil penelitian menunjukkan indeks dmf-t 4,32 dan rerata konsentrasi fluor dalam saliva sebesar 0,018. tes korelasi pearson product moment menunjukkan hubungan yang tidak kuat antara konsentrasi fluor dalam saliva dengan indeks dmf-t. kesimpulan: dapat disimpulkan konsentrasi fluor dalam saliva mempunyai hubungan dengan indeks dmf-t tetapi tidak signifikan dikarenakan nilai konsentrasi fluor dalam saliva yang sangat rendah. kata kunci: konsentrasi fluor, saliva, indeks dmf-t correspondence: ame suciati setiawan, c/o: bagian biologi oral, fakultas kedokteran gigi universitas padjadjaran bandung. jl. sekeloa selatan i bandung, indonesia. e-mail: amesuciati@gmail.com �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 35–38 introduction dental caries is a multifactorial disease. caries can be rooted from 4 factors, i.e. host, microorganism, substrate or diet, and time. the integration of the caries etiological factors can be described as four overlapped circles.1 until now, the incidence of dental caries in developing countries, especially in indonesia is still high in various age and socio-economic levels. this is in line with the findings in 2007 indonesian basic health research (riskesdas) which described the national dmf-t index as 4.85. this means that the average dental decay among indonesians is 5 teeth per individual. the biggest component is the extracted teeth/m-t of 3.86; meaning that average indonesian has 4 extracted teeth or 4 teeth that have been indicated for extraction. the dmf-t index of the west java province is 4.03. it means that the average dental decay among west java people is 4 teeth per person. the biggest component is the extracted teeth, m-t, of 3.71 or, in other words, most west java people have four extracted teeth or four teeth that are indicated to be extracted. the active dental caries prevalence is 39.0 and a caries experience of 58.4.2 based on the age, the prevalence of active dental caries in 12 year old group is 29.8 and in 15 year old group the prevalence is 36.1. meanwhile the caries experience in 12 year old group is 26.1 and the experience for the 15 year old group is 43.6. the number of decayed teeth increases along with age based on the dmf-t index. in the 12 year old group, the dmf-t index is 0.91 and in the 15 year old group, the dmf-t index increases to 1.14 and the highest dmf-t index is found in the age of more than 65 year old, i.e. 18.33.2 according to who, the 12 year old group is a critical indicator because about 76.97% of caries diseases occur in that age period. this age group is important because in general, children leave their elementary school at the age of 12 years old. in addition, all permanent teeth are assumed to have been erupted except for the third molar. the age of 12 years old is used for global monitoring age for caries while the age of 15 years old is considered as the age that the permanent teeth have undergone adaptation with oral environment for 3–9 years.3 caries risk assessment is a complex discussion. there are a lot of factors to be considered including social status, medical history, diet pattern, fluor use, plaque control, saliva and clinical status. the clinical status can be observed from the dmf-t index. the dmf-t index is an irreversible index used for permanent teeth where d (decayed) refers to a tooth that has one or more signs of caries attack that is not filled but still eligible for filling; m (missing) refers to the tooth that has been pulled out (self-destruct) due to caries or has to be extracted because of caries; and f (filling) that refers to a tooth that has one or more good fillings.4 in addition to the clinical status, the caries risk assessment can be done from the environmental condition aspects, i.e. fluor use, social status, diet habit and general health condition that includes diseases experienced and treatment as well as consumed medication.5-7 fluor can be found in drinking water, supplement and toothpaste. fluor is important in dental structure growth and development to achieved dental structures that have high resistance towards bacteria. one of the roles of fluor in reducing dental caries is the ability fluor in reducing acid production of plaque microorganism and in affecting cariogenic microorganism colonization on the dental surface.8 the results of a study by dean in united states in 1942 stated that there is a correlation between caries condition with fluor content in drinking water among children in the age group of 12-14 years old. good drinking fluoridation means that the water has 1 ppm fluor level. however, in warmer areas, the fluor concentration in drinking water is lower, i.e. 0.7 ppm.7 a study on the relationship between salivary fluor concentration and caries index in 12-15 years old children was conducted in smp negeri perseroan terbatas perkebunan nusantara (ptpn) viii pangalengan, to get homogenous sample with similar socio-economic and education background levels. materials and methods the population of this study was students of smp negeri 2 in malabar ptpn viii plantation, pangalengan which consisted of 243 children. the size of the sample was 80 chitosan 12–15 year old. the inclusion criteria were children of 12–15 year old, do not smoke, do not have systemic abnormalities, not under a long-term antibiotic therapy and willing to participate in the study. this study was analytical observational study with cross sectional approach (field trial). the procedure consisted of 2 examinations: clinical and laboratory examination. during the clinical examination, the subjects received questionnaire and filled in informed consent form. the subjects were selected according to the population criteria. he/she was asked to rinse his/her mouth before the operator examined the condition of the subject’s dentition, then dmf and dmf-t index was calculated. after the clinical examination, the subject was instructed to chew a paraffin gum and spitted the saliva out into a 2 ml sterile plastic tube using the spitting technique. the tube was kept in chiller. after clinical examination, saliva examination was done at laboratorium pengendalian kualitas lingkungan (lpkl). the saliva was moved into a measuring cup to be diluted using aquadest up to 5 times dilution and until reached 10 ml. the saliva was poured into a reaction tube, added with 2 ml of spadns fluor reagent and moved to a special tube for spectrophotometer dr 2400 to measure the fluor level. data were collected and presented in a table and analyzed using a statistical test (pearson product moment correlation) to assess whether there is a correlation between salivary fluor concentration and caries index. ��pratiwi, et al.: relationship between salivary fluor concentration and caries index results the subject characteristic data include: gender, age, dmf-t index and salivary fluor concentration. based on gender, out of 80 subjects, 48 of them are girls (60%) and 32 are boys (40%). in terms of the age, there are 4 categories: 16 subjects were in the 12 years old category (20%), 43 subjects (53.7%) were in the 13 years old category, 20 subjects (25%) were in the 14 years old category and one subject (1.25%) was in the 15 years old category. based on the dmf-t index, it was revealed that 36 subjects (45%) has a dmf-t index that is less than or equals 3, 25 subjects (31.25%) and have dmf-t index in the range of 4 to 6, 16 subjects (20%) have a dmf-t index in the range of 7 to 9 and 3 (3.75%) have a dmf-t index of 10 or more. the average dmf-t index of the 80 subjects is 4.23. the results of the salivary fluor level assessment show that 32 subjects (40.0%) have a salivary fluor concentration of less than or equals 0.0126, 21 subjects (26.25%) have a fluor concentration in the range of 0.026 to 0.0375, 20 subjects (25.0%) have a fluor concentration in the range of 0.0126 to 0.025, and the remaining 7 subjects (8.75%) have the highest salivary fluor concentration, i.e. above 0.0375. the average salivary fluor concentration value of the 80 subjects is 0.018. the results from the pearson product moment correlation analysis on the correlation between salivary fluor concentration and caries index show an r of -0.168 and tcalc. of 1.507, which is smaller than the ttable (1.991). this shows an inverted correlation between dmf-t index and salivary fluor concentration so that the higher the dmf-t, the lower the salivary fluor concentration and vice versa. however, the correlation is not significant. discussion the results of the study on dmf-t index in children in the age group of 12–15 years old at smp negeri 2 ptpn viii pangalengan show a value of 4.23. according to who, this rate shows that the subjects experience 4 decayed teeth and are included into the moderate caries severity level. the low salivary fluor concentration is due to the fact that the consumed fluor is not accumulated so that the amount of excreted fluor in the saliva is low. this low fluor excretion in saliva is caused by the fact that fluor consumed by the body is also deposited in the bone and teeth.9 fluor excretion can also be found in faeces, sweat and urine. the percentage of excretion is 80-90% and around 10% for urine and faeces, respectively.10,11 the concentration of fluor excreted by the salivary gland in normal condition is 0,007 to 0.05 ppm.10 the concentration can increase, especially after using fluorcontaining toothpaste or mouthwash but the concentration rebounds to normal immediately.4 however, the low salivary fluor concentration still has cariostatic function because fluor is a micromolecule that can be easily absorbed by the enamel and able to remineralize the enamel.10 however, the increased fluor concentration in the saliva will be better for the post eruption maturation. the fluor concentration value needed to reduce dental caries occurrence is around 1 ppm in saliva.9 the inverted correlation between salivary fluor concentration and dmf-t index is shown by the existance of cariostatic nature of fluor. the mechanism of caries prevention with fluor is that fluor binds apatite to produce fluoroapatite bound in dental enamel and increases the tooth resistance towards acid attacks produced by the cariogenic bacteria.12 fluor is able to reduce acid production by inhibiting the enzyme that metabolizes carbohydrates.13 fluor can also prevent mineral release from the crystal surface and improve remineralization with the presence of calcium and phosphate ions. fluor excreted in the saliva can inhibit several enzyme processes that reduce the amount of acid produced by the bacteria in the saliva and plaque. 14 the enzyme is enolase, an enzyme that is needed by the bacteria to metabolize sugar. the fluor in saliva will bind the magnesium ion to form magnesium fluor. magnesium is the ion that is also needed by enolase. as an effect of this inhibition by fluor, glycolysis in bacterial cell is inhibited that can not produce enough energy and the bacterial growth is restrained.15 the fluor concentration in saliva can prevent caries but fluor concentration is not the only factor involved in caries prevention process. there are other things to be considered such as the period of exposure, time of exposure, frequency and other factors related to systemic caries prevention mechanism using fluor.16 this study shows an insignificant correlation between dmf-t index and salivary fluor concentration due to confounding factors in saliva detected by spectrophotometer dr 2400 that include salivary inorganic components. those components are sodium, potassium, calcium, magnesium, chloride and phosphate. fluor concentration is smaller than the concentration of other inorganic components with sodium and potassium as the components with the highest concentration. the fluor testing using spectrophotometer dr 2400 is very sensitive to some small number of confounding factors. another factor that affects the insignificancy of the result is that the special tube for spectrophotometer dr 2400 that is not sterile and clean enough. the test should be repeated for each saliva samples to make sure that the result of the test is accurate. however, it is impossible to repeat the test due to limited amount of saliva. in addition to limited instruments and saliva, the insignificant result may occur due to the insufficient knowledge and skills of the researcher for conducting this type of study. based on the results of this study on the relationship of salivary fluor concentration and caries index in 12-15 year old children at smp negeri 2 ptpn viii pangalengan, it can be concluded that salivary fluor concentration has an �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 35–38 insignificant correlation with dmf-t index due to the very low concentration of fluor in saliva. references 1. fejerskov o, kidd eam. dental caries the disease and its clinical management. uk: blackwell munksgaard; 2003. p. 4 –5, 71, 101–2. 2. indonesia. litbang departemen kesehatan republik indonesia. hasil riset kesehatan dasar. penerbit depkes ri; 2007. p. 104–7. 3. pintauli s, hamada t. menuju gigi and mulut sehat, pencegahan and pemeliharaan. medan: usu press; 2008. p. 4–6, 1–18. 4. burt ba, eklund sa. dentistry, dental practice, and the community. 5th ed. united states of america: wb saunders company; 1999. p. 178, 291, 297–8. 5. angela a. pencegahan primer pada anak yang berisiko karies tinggi. majalah kedokteran gigi medan 2005; 38(3): 130–4. 6. sumawinata n. evaluasi and pengendalian faktor risiko karies. edisi 7. jurnal kedokteran gigi indonesia 2000; p. 417–24. 7. kidd eam. essential of dental caries the disease and its management. 3rd ed. new york: oxford university press; 2005. p. 2, 7–8, 10, 18, 60–4, 110–2. 8. erawanto bb, pudyani ps. pengaruh pelepasan fluor dari elastomeric ligature terhadap jumlah streptococcus mutans dalam saliva. majalah ilmu kedokteran gigi (mikgi) 2003; 5(10): 225–58. 9. supit jh, suwelo is, sunawan h. hubungan kandungan fluor saliva dengan karies gigi anak. jurnal kedokteran gigi pdgi 1995; 44(2): 63–6. 10. mellberg jr, ripa lw, leske gs. fluoride in preventive dentistry theory and clinical applications. chicago: quintessence publishing co, inc; 1983. p. 28, 41–42, 58–61, 81–92. 11. fejerskov o, ekstrand j, burt ba. fluoride in dentistry. copanhagen: munksgaard; 1996. p. 55-64, 216–20. 12. nisengard rj, newman mg. oral microbiology and immunology. 2nd ed. united states of america: wb saunders co; 1994. p. 343, 355–6. 13. cameron ac, widmer rp. handbook of pediatric dentistry. china: mosby elsevier; p. 28–9. 14. agtini md. fluor sistemik and kesehatan gigi. cermin dunia kedokteran 1988; 52: 45–7. 15. djamil ms. mekanisme fluor menghambat kerja enzim air liur. jurnal kedokteran gigi universitas indonesia 2000; (7): 1–6. 16. sugito fs. peranan teh dalam mencegah terjadinya karies gigi. edisi 7. jurnal kedokteran gigi indonesia 2000; 375–9. vol 49 no 1 jan-mrt 2016.indd 2727 research report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 28–32 effects of anadara granosa shell combined with sardinella longiceps oil on oesteoblast proliferation in bone defect healing process rima parwati sari,1 eddy hermanto,2 dinda divilia,1 indira candra,1 wisnu kuncoro,1 and tantri liswanti1 1department of oral biology 2department of oral surgery faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: alveolar bone damage is the most common case in dentistry. one way to fix the bone damage is by using bone graft. anadara granosa shell is a potential bone substitute since it is rich in calcium which can be processed into hydroxyapatite. the addition of sardinella longiceps oil rich in omega-3 can modulate inflammation, thus accelerating the healing process. purpose: this study aimed to determine effects of application of anadara granosa shell combined with sardinella longiceps oil on osteoblast proliferation in the healing process of bone defects. method: the subjects were 32 male rats type wistar divided into 4 groups (n = 8). making defect was performed on the right bone of the femurs with a half of the diameter of round mcisinger® germany bur sized 18. the first group (k) is a negative control group that was not given anything. the second group (ag) was given anadara granosa pasta. the third group (am10) was given anadara granosa pasta combined with 10% sardinella longiceps oil. and, the fourth group (am30) was given anadara granosa pasta combined with 30% sardinella longiceps oil. next, preparations and animal euthanasia were performed on the 7th day after the treatment. the number of osteoblasts then was measured after making preparations for hpa with hematoxylin eosin staining (he). afterward, tabulation of data followed by statistical analysis of anova and hsd tukey was carried out. result: the average number of osteoblasts in groups k, ag, am10, and am30 was 19.00, 34.63, 33.50, and 38.50. the results of anova test showed a significant difference (p<0.05). similarly, the results of tukey-hsd test also showed significant differences (p <0.05) between group k and all other groups (ag, am10, and am30). nevertheless, there were no significant differences between group ag and groups am10 and am30, as well as between group am10 and group am30. conclusion: the application of the combination of anadara granosa shell and sardinella longiceps oil can not increase the proliferation of osteoblasts in the healing process of bone defects. keywords: anadara granosa; sardinella longiceps oil; bone graft; osteoblasts; bone healing correspondence: rima parvati sari, department of oral biology, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: rimaparwatisari@gmail.com introduction bone damage usually can occur because of trauma, tumor, congenital abnormalities, infection, inadequate prosthesis, and systemic disease.1 in dentistry, alveolar bone damage as the highest prevalence of bone damage is commonly caused by periodontal disease. periodontal disease is an oral and dental problem with the prevalence of the disease reached 96.58% in indonesia.2 in addition, complications after tooth extraction due to sizeable trauma can cause damage to the alveolar bone.3 naturally, abnormal condition can occur in the body. for instance, bone defects can be cured by mechanical balance in the body. this process is known as bone healing. the process of bone healing plays an important role after the treatment in dentistry.4 generally, bone healing requires a long period to get back on the normal state.5 the bone tissue healing process is generally the same as wound dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p27-31 28 sari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 27–31 healing process in soft tissue, but the difference is related to physiological ability to repair the hard tissues during the healing process.6 the bone healing process begins with the formation of hematoma and the response of inflammatory cells lasting for 24-48 hours followed by reparative phase occurred in the first few days before the inflammation is reduced, and then last for several weeks, resulting in the development of tissue repair. in this phase, pluripotential mesenchymal cells start to form other cells, such as fibroblasts, chondroblasts, and osteoblasts.5 osteoblast activity appears first at reparative phase. osteoblasts will be seen in the cortex of the bone, a few millimeters from the defect area.7 osteoblasts function as the main cells in the bone healing process. osteoblasts then will produce, secrete, depose, and mineralize bone matrix.8 based on these functions, it is necessary to increase the activity of osteoblasts to accelerate the bone healing process. a small number of osteoblastic activities occur continuously in all the living bone tissue (about 4 percent of all the bone surfaces in adults at various times), resulting in the least number of new bone formed constantly.9 bone formation, moreover, can occur due to several factors affecting the acceleration of healing process, such as use of bone graft. bone graft has been widely used as a substituting bone material, especially in periodontal therapy.10 graft tissue including bone, has been widely used until now. in general, there are two main functions of bone graft for recipients’ bone, namely to encourage osteogenesis (bone formation) and to provide mechanical support in the framework of the recipients.11 bone graft, furthermore, is used to stimulate bone healing and stabilize the dimensions of the alveolar bone in the field of dentistry.12 bone graft is also considered as an appropriate action to increase the height of the alveolar crest, remodel jawbone, transfer microvascular free tissue, and reshape the alveolar crest.13 in addition, xenograft is one form of bone grafts that has biocompatible properties in humans and has higher osteoconductive properties than alloplast. additionally, xenograft can provide a suitable medium for osteogenesis by bone marrow cells.14 currently, xenograft materials widely used are derived from bone bovine. the main compound used in bone graft is calcium phosphate as the major component of bone mineral constituent. the calcium phosphate compound is an inorganic material that has bioactive and biocompatible properties. calcium phosphate compound used as bone graft can be in a crystalline phase or in an amorphous phase, namely tricalcium phosphate (ca3 (po4) 2) and hydroxyapatite (ca10 (po4) 6oh2).15 the form of the most stable calcium phosphate is hydroxyapatite (ha). ha is not only considered as a bioactive ceramic material with high bioaffinity as well as non-corrosive, non-toxic and biocompatible properties to the human body, but also as one of the calcium phosphate crystals that give the tough nature in bone.16 one of the natural ingredients found as a valuable economic resource, which generally has not been utilized is anadara granosa. anadara granosa is mostly used as food rich in protein. meanwhile, the shell is discarded into the waste. several researches actually have been conducted to increase the additional value of anadara granosa. for instance, anadara granosa shells containing a lot of calcium are used for bone healing process. anadara granosa shells also contain other minerals, such as cac (98.7%), mg (0.05%), na (0.9%), p (0.02%) and other elements (0.2%).1 similarly, a research conducted by hafisko et al.18 showed that anadara granosa shells contains caco3 that can be converted into more biocompatible hydroxyapatite compounds than calcium carbonate alone, so the process is more osteoconductive conditioned by inducing bmp-2. bmp-2 can increase the differentiation of periosteal cells, derivatives of mesenchymal stem cell (msc) in the formation of chondroblasts and osteoblas.19 thus, the use of ha bone graft can effectively improve bone healing. another stimulus to accelerate the bone healing process is omega-3. some studies suggest that omega-3 can reduce the production of proinflammatory cytokines and eicosanoids by inhibiting the metabolism of arachidonic acid (aa), the substrates of eicosanoids (prostaglandin). omega-3 may also alter the inflammatory gene expression through transcription factor activity, therefore, omega-3 can be potentially considered as anti-inflammatory.20 high omega-3 diet, lead to an increase in both activity of serum isoenzyme of alkaline phosphatase (alp), an enzyme marker of osteoblast activity, and activity of osteoblasts.21 high omega-3 diet can also lower the production of prostaglandin e2 (pge2).22 pge2 and proinflammatory cytokines are mediators which play an important role for the occurrence of bone resorption. high omega-3 diet, consequently, can result in a decrease in the formation and activity of osteoclasts, which serve to bone resorption.23 fish species in indonesia mostly consisted of fish oil is sardinella longiceps. fish oil derived from sardinella longiceps contains a lot of omega-3, namely 13.70% eicosapentaenoic acid (epa) and 8.91% docosahexaenoic acid (dha). sardinella longiceps is a type of pelagic fish (containing oil) distributed in all waters in indonesia. the largest number of sardinella longiceps is found in the strait of bali, around muncar near banyuwangi (east java).24 the use of sardinella longiceps oil as a therapy in wound healing has already been proven by wijaya25 who observed the healing of cut wound in wistar rats using ointment of sardinella longiceps oil as the test material. the research also shows that at concentrations of 10%, sardinella longiceps oil can make cut wound cover 100% within 7 days. finally, based on the description above, this research aimed to to know effects of application of anadara granosa shell combined with sardinella longiceps oil on osteoblast proliferation in the healing process of bone defects. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p27-31 2929sari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 27–31 materials and method this research was a true experimental research with completely randomized design. it means that the control group and the selection of experimental animals in the treatment groups were randomly conducted. experimental animals used in this research were 32 male rats aged 5 months old and weighed 200-250 grams. tools used in this research, moreover, were animal cages, storage tubes for femurs’ right bones, scissors, scalpels, anatomical tweezers, resparatorium, scales for animals, food and beverage, microtome for cutting, light microscopy, glass objects, glass cover, bottles, shelves for painting, syringes 3cc, round bur (straight and piece) of mcisinger® germany sized 18, micromotor 1200 rpm, fur shaver, probe for eating, dappen glass, as well as separating disc. meanwhile, materials used were anadara granosa powder processed by hafisko et al. with nano size.18 sardinella longiceps oil processing in banyuwangi, membranes, sewing silk thread, needles, cotton or tissue, rat food, distilled water to drink those rats replaced every today, 10% buffered formalin, ketamine hydrochloride, xylazine hydrochloride, 10% povidine iodine, 30%, 50%, 70%, 80%, and 96% alcohol, novalgin, as well as 10% edta. before conducting the research, anadara granosa shell pasta (ag), a combination of anadara granosa and 10% sardinella longiceps oil (am10), and a combination of anadara granosa and 30% sardinella longiceps oil (am30) were made. ag shell pasta was made from 5 grams of anadara granosa shell powder mixed with glycerin to obtain 1 ml of pasta. the pasta was mixed and condensed until well blended and homogeneous, and viscosity values obtained were quite thick, so easy to apply. preparations of am10 and am30 then were made by mixing 5 grams of anadara granosa powder, 0.1 ml and 0.3 ml of sardinella longiceps oil, and glycerin to obtain 1 ml of pasta. all those rats were acclimatized for one week. they were kept in cages with a size of 40 cm (l) x 30 cm (l) x 14cm (t). afterwards, they were divided into 4 groups, namely the negative control group (c), the group ag using anadara granosa pasta, the group am10 using anadara granosa pasta combined with sardinella longiceps oil 10%, and the group am30 using anadara granosa pasta combined with sardinella longiceps oil 30%. each group consisted of 3-4 rats. then those were given food in a small container every morning, afternoon, and evening, while drink was supplied in bottles of 300 ml equipped with a small pipe filled with cooked water (ad libitum). bone defects then were performed on the femurs’ right bone with the following procedure. first, anesthetics was performed using 1 ml of ketamine mixed with 0.5 ml of xylazine, then injected with a dose of 1.5 ml / 100 g bm on the femurs’ right bone intramuscularly.26 second, after they began to be unconscious, fur in part to be done the defect was sheared. third, 10% povidine iodine was applied on the area around the defect for five minutes.27 fourth, the soft tissue (skin and muscle) around the defect area was incised about 2 cm using a scalpel and removed using a periosteal elevator. after finding femurs’ right bone, a hole defect then was made using bone bur (straight and piece) sized half of the diameter of round mcisinger® germany bur sized 18. in the first group, the holes then were not given any materials because it was used as a negative control (c). in the second group, the holes were given anadara granosa pasta (ag). in the third group, the holes were given am10, while in the fourth group, the holes were given am30. after filling, the holes were covered with membrane materials and sewed to cover the soft tissue and the skin on the femurs’ right bone, so the graft materials would not be wasted (out of the defect areas). 28 analgesics then was given one hour after the surgery, ie novalgin® at a dose of 175 mg / kg dissolved in saline to control swelling and pain.29 afterward, making preparations and animal euthanasia were performed on the 7th day after the treatment. those rats were sacrificed first with ether, and the tissues of the femurs’ right bone were removed. the tissues then were peeled to take the bone part after bone grafting by cutting with a separating disc, and put in 10% buffered formalin solution so that the tissues would not be rot and hard, and the affinity values of tissue against staining material increased.27 after tissue fixation process, decalcification process was conducted using edta for 2 months. specimens of the femurs were made in the form of a sagittal slice preparation with he staining. the number of osteoblasts in the defect areas then was observed using light microscope with a magnification of 400x. next, the acquired data were tabulated. and, several statistical tests were performed using a parametric test, oneway anova test, followed by tukey-hsd test. results the results of the histological examination showed that there were osteoblasts in the defect areas of all the research groups (figure 1). the calculation results, moreover, showed that the average number of osteoblasts in group k was 19.00 ± 1.74, in group ag was 34.63 ± 1.94, in group am10 was 33.50 ± 2.94, while in group am30 was 38.50 ± 2.28. figure 2 shows that the lowest average of osteoblast count was found in the control group, while the highest average of osteoblast count was found in group am30. the results of the statistical test using spss 18.0, moreover, indicated that the data were normally distributed with (p> 0.05) and homogeneous (p = 0.324). the results of the statistical test using one way anova test, furthermore, showed that there was a significant difference. another statistical test, tukey-hsd test, was then performed to compare one group to another group. in addition, the results of tukey hsd test showed that there was a significant difference between group k and the treatment groups using bone graft. there was no significant difference between group ag and both group dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p27-31 30 sari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 27–31 am10 and group am30 as well as between group am 10 and group am30. discussion animals used in his research were male wistar rats since there is no hormonal influence that can affect healing mechanisms. decalcification process in wistar rats is faster than in other experimental animals. a rat femur also has certain characteristics that are relatively similar to humans, for instance, histologically epicondyle of the femurs has trabecular bone growing well below the cortical bone, resulting in providing a substantial model for dentistry.30, 31 moreover, improvements in bone defect healing process involve a complex physiological sequence. once damage occurs, hematoma will appear around the site of trauma triggering fibrous tissues to form a lesion area and to develop into callus.32 in some ways, the bone will activate all intraosseous and periosteum osteoblasts maximally in the defect areas. number of new osteoblasts are formed from progenitor cells as bone stem cells in the surface of the tissue covering the bones, called as bone membrane.9 bone graft as a substituting bone material actually has been widely used because of its osteoconductive properties.10 contribution of the graft begins during the osteoconductive process, namely to make framework as the bone matrix in the recipient tissue. this is followed by stimulation of bone formation during bone healing.11 hydroxyapatite possess good biocompatibility and bioactivity. continually, ha will improve osteoconductive process. the use of ha bone graft can effectively improve bone healing caused by bone defect. the addition of ha bone graft can also attach bioactivity and osteoconductivity in making improvements during the mineral phase.33 based on crystallography and chemical properties, ha approaches bone structure, thus providing an inorganic component in the form of crystalline hydroxyapatite together with sodium, magnesium, carbonate, and structural skeleton.34 similarly, the results of this research showed a significant difference between the negative control and the treatment groups ag, am10, and am30. omega-3 is one of eicosanoids rich in eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). epa and dha play a role in generating resolvins and related compounds, such as protectins through pathways that involve cyclooxygenase and lipoxygenase enzymes. resolvin e1, resolvin d1, and d1 protectin inhibit transendothelial migration of neutrophils, thereby preventing the infiltration of neutrophils at sites of inflammation. resolvin d1 inhibits the production of il-1β, while protectin d1 inhibit the production of tnf and il-1β. therefore, resolvins and related compounds play a very important role in stopping the ongoing inflammatory process and in limiting tissue damage. in addition, epa and dha in sardinella longiceps oil can also inhibit inflammatory mediators, such as il-6, il-8, and tnf-α. sardinella longiceps oil actually can decrease the activation of nfκb more than corn oil.35 decreasing in inflammation products can reduce the ratio of rankl/opg. opg protects bone skeleton by preventing bond between rankl and rank as pre osteoclast receptors, thereby suppressing the formation of osteoclasts figure 1. histological results of osteoblasts in each group. group c (negative control), group ag using anadara granosa pasta, group am10 using anadara granosa pasta combined with sardinella longiceps oil 10%, and group am30 using anadara granosa pasta combined with sardinella longiceps oil 30%. the presence of osteoblasts was indicated by green arrows. c figure 2. the average number of osteoblasts in each group table 2. the results of tukey-hsd test group ag am10 am30 c .030* .049* .005* ag 0.997 .883 am10 .781 control 34.625 33.5 38.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p27-31 3131sari, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 27–31 and activating more osteoblasts then leading to new bone formation.36 although there was statistically no significant difference, the average number of osteoblasts in the groups using the combination of anadara granosa shell pasta and sardinella longiceps oil was higher than in the group using anadara granosa shell powder only. this may be due to the non-optimal physicochemical and biomaterial properties of the preparation materials used. thus, further researches need to be improve bone graft preparations. the tendency of increase in the number of osteoblasts with higher concentration may be the reason for increasing the concentration of sardinella longiceps oil added to anadara granosa shell powder. finally, it can be concluded that the application of a combination of anadara granosa shell and sardinella longiceps oil can not increase the proliferation of osteoblasts in the healing process of bone defects. references 1. ferdiansyah, rushadi d, rantam fa, aulani'am. regenerasi pada massive bone defect dengan bovine hydroxyapatite sebagai scaffold mesenchymal stem cel. jbp 2011; 13(3): 179-95. 2. situmorang n. profil penyakit periodontal penduduk di dua kecamatan kota medan tahun 2004 dibandingkan dengan kesehatan mulut tahun 2010. dentika dental jurnal 2005; 9(2): 71-7. 3. matoses vf, vinarta mm, domingo ta, matoses if, liacer vjf. treatment of multiple traumatized anterior teeth associated with an alveolar bone fracture in a 20-year-old patient: a 3-year follow up. j clin exp dent 2014; 6(4): e425-9. 4. duvall cl, taylor wr, weiss d, wojtowicz am, guldberg re. impaired angiogenesis, early callus formation, and late stage remodelling in fracture healing of osteopontin-deficient mice. j bone miner res 2007; 22(2): 286-97. 5. sfeir c, lawrence ho, bruce a, azari k, jeffrey oh. bone regeneration and repair: biology and clinical applications. totowa, nj: humana press inc; 2005. p. 21-44. 6. oryan a, alidadi s, moshiri a. current concern regarding healing of bone defects. hard tissue 2013; 2(2): 1-12. 7. sfeir c, ho l, doll ba, azari k, hollinger jo. fractur repair. totowa, nj: humana press inc; 2005. p. 21-44. 8. pramusita a, prahasanti c, augustina ef. aplikasi serbuk natural hidroksiapatit-kitosan dengan kadar 70:30 dan 30:70 terhadap proliferasi osteoblas. periodontic journal 2013; 5(1): 1-2. 9. guyton ac, hall je. buku ajar fisiologi kedokteran. edisi 11. jakarta: egc; 2008. p. 305-308 10. sukumar s, drizhal i. bone grafts in periodontal therapy. acta medica (hradec králové. 2008; 51(4): 203-7. 11. mao t, kamakshi v. bone grafts and bone substitutes. int j pharm pharm sci 2014; 6(suppl 2): 88-91. 12. drismayanti i, sariatun t, a’la ub, ruslin m, jubhari eh. perbandingan tingkat keberhasilan implan antara osteogenesis autogeneous bone graft. dentofasial 2012; 11(3): 180-6. 13. keskin d, gundogdu c, atac ac. experimental comparison of bovinederived xenograft, xenograft-autologous bone marrow and autogenous bone graft for the treatment of bony defects in the rabbit ulna. med princ pract 2007; 16: 299–305. 14. barrère f, van blitterswijk ca, de groot k. bone regeneration: molecular and cellular interactions with calcium phosphate ceramics. int j nanomedicine 2006; 1(3): 317–32. 15. leukers b, gülkan h, irsen sh, milz s, tille c, schieker m, seitz h. hydroxyapatite scaffolds for bone tissue engineering made by 3d printing. j mater sci mater med 2005; 16(12): 1121-4. 16. awang-hazmi aj, zuki abz, noordin mm, jalila a, norimah y. mineral composition of the cockle (anadara granosa) shells of west coast of peninsular malaysia and it's potential as biomaterial for use in bone repair. journal of animal and veterinary advances 2007; 6(5): 591-4. 17. verwilghen c, chkir m, rio s, nzihou a, sharrock p, depelsenaire g. convenient conversion of calcium carbonate to hydroxyapatite at ambient pressure. materials science and engineering: c 2009, 29 (3) : 771–773 18. mi m, jin h, wang b, yukata k, sheu tj, ke qh, tong p, im hj, xiao g, chen d. chondrocyte bmp2 signaling plays an essential role in bone fracture healing. gene 2013; 512(2): 211-8. 19. calder pc. omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology. british journal of clinical pharmacology 2012; 75(3): 645-62. 20. dana m, grigorie d, stefanovici g, vladoiu s, busu c, calarasu r, dum itrache c. alkaline phosphatase biochem ical and histochemical marker for primary osteoblast in proliferative phase. rom j endocrinol 2004; 42 :1-4 21. watkins ba, li y, lippman he, feng s. modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism. pubmed journal 2003; 68(6): 387-98. 22. indahyani de, santoso als, utoro t, soesatyo mhne, sosroseno w. effect of fish oil on lipopolysaccharide-induced hydroxyapatite loss in rat alveolar bone: a preliminary study online. j health allied scs 2008; 7(4): 7. 23. pusat penyuluhan kelautan dan perikanan. pengelolahan ikan lemuru. materi penyuluhan terkait pengelolahan ikan lemuru. jakarta. 2012. p. 4-6 24. tanideh n, abdordideh e, yousefabad sla, daneshi s, hosseinabadi ok, samani sm. a comparison of the effects of honey, fish oil and their combination on wound healing in rat. journal of coastal life medicine 2016; 4(9): 683-8. 25. fleckhell p. laboratory animal anastesia. third edition. newcastleupon-tyne, uk: elsevier; 2009. p. 187. 26. rokn ar, khodadoostan ma, ghahroudi aarr, motahhary p, fard mjk, bruyn hd, afzalifar r, soolar e, soolari a. bone formation with two types of grafting materials: a histologic and histomorphometric study. open dent journal 2011; 5: 96104. 27. kopschina mi, marinowic dr, klein cp, araujo ca, freitas ta, hoff g, silva jb. effect of bone marrow mononuclear cells plus platelet-rich plasma in femoral bone repair in rats. braz j vet res anim sci 2012; 49(3): 179-84. 28. dorsch mm, otto k, hedrich hj. does preoperative administration of metamizol (novalgin®) affect postoperative body weight and duration of recovery from ketamine– xylazine anaesthesia in mice undergoing embryo transfer: a preliminary report. laboratory animals 2004; 38: 44–9. 29. asakura t. bone regeneration on the epicondyle of the femur supported by silk fibroin-based scaffold : a model system for dental surgery. journal of insect biotechnology and sericoloy 2011; 80: 25-30. 30. ridwan e. etika pemanfaatan hewan percobaan dalam penelitian kesehatan. j indon med assoc 2013; 63(3): 114. 31. berendsen ad, pinnow el, maeda a, brown ac, mccartneyfrancis n, kram v, owens rt, robey pg, holmbeck k, de castro lf, kilts tm, young mf. biglycan modulated angiogenesis and bone formation during fracture healing. matrix biol 2014; 35: 223-31. 32. de guzman rc, saul jm, ellenburg md, merrill mr, coan hb, smith tl, van dyke me. bone regeneration with bmp-2 delivered from keratose scaffolds. biomaterials 2013; 34(6): 1644-56. 33. murray rk, granner dk, mayes pa, rodwell vw. biokimia harper. edisi 27. jakarta: egc; 2006. p. 575-578 34. calder pc. omega-3 fatty acids and inf lammatory processes. nutrients 2010; 2(3): 355-74. 35. boyce bf, xing l. biology of rank, rankl and osteoprotegerin. arthritis res ther 2007; 9 suppl 1: s1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p27-31 dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  results, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st. louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1):[24 screens]. available from: ��l:h���://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. h. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. h. 8–21. ii. literature reviews preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references.  photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. notes to authors the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. editor 183183 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 183–186 research report proliferation of odontoblast-like cells following application of a combination of calcium hydroxide and propolis ira widjiastuti, sri kunarti, fauziah diajeng retnaningsih, evri kusumah ningtyas, debby fauziah suryani and andrie handy kusuma department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: one purpose of operative dentistry is the maintenance of healthy pulp by reducing the need for root canal treatment and the possibility of undesirable scenarios such as tooth loss. propolis is a plant-derived substance that contains a resin produced by honeybees belonging to the apis mellifera species. purpose: this study aimed to investigate the effect of a combination of calcium hydroxide (ca(oh)2) and propolis extract on odontoblast-like cell proliferation in wistar rats (rattus norvegicus). methods: this research constituted a true experimental laboratory-based investigation with post-test control group design. thirty wistar rats were randomly divided into six groups. the first molar pulp of each sample was perforated on occlusal surfaces using a low speed round bur. on day 3, the samples were divided into six groups (n=10): group i: control; group ii: ca(oh)2 + 11%; propolis extract; group iii: ca(oh)2 + aquadest, and on day 7: group iv: control; group v: ca(oh)2 + 11% propolis extract; group vi: ca(oh)2 + aquadest. all samples were filled with restorative material. they were subsequently sacrificed after 3 and 7 days post-pulp capping administration and the afflicted tooth extracted for hematoxylin and eosin (h&e) staining. the resulting data was subjected to statistical analysis to ascertain the proliferation of odontoblast-like cells. the significance of differences between the groups was determined by a one-way anova test followed by a post hoc tuckey hsd. a p-value <0.05 was considered to be significant. results: on day 3, a significant difference existed between group ii (ca(oh)2–propolis) and group i (control group) and group iii (ca(oh)2–aquades), whereas ca(oh)2–propolis revealed that the proliferation of odontoblast-like cells was higher. meanwhile, on day 7, there was a significant difference between all groups whereas, with regard to ca(oh)2–propolis, the proliferation of odontoblast-like cells in group v was higher. conclusion: application of combination of ca(oh)2-propolis extract can increase the proliferation of odontoblast-like cells in pulp tissue on days 3 and 7. keywords: calcium hydroxide; odontoblast like cell; propolis extract correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ira-w@fkg.unair.ac.id introduction one objective of operative dentistry is the maintenance of healthy pulp which reduces the need for root canal treatment.1 direct pulp capping is a procedure that introduces biocompatible materials and bio-inductors into exposed pulp tissue in order to maintain its vitality,2 and induce the differentiation of odontoblast-like cells, in addition to repairing exposed dentin tissue with the formation of reparative dentin.1 the purpose of this treatment is to seal the pulp, thereby protecting it from bacterial penetration, in order to induce it to initiate dentin bridge formation and maintain healthy pulp tissue.1,2 dentin bridge is often described as reparative dentin. in fact, the former can be defined as a new matrix deposition located in close proximity to certain material such as that used for pulp capping.3 the success of direct pulp capping depends on biocompatibility with the tissue and an effective physicochemical composition. the contemporary gold standard for pulp capping material is calcium hydroxide (ca(oh)2 ) which has been employed in a range of therapies, including; direct pulp capping, indirect pulp capping, apexogenesis, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p183–186 http://dx.doi.org/10.20473/j.djmkg.v52.i4.p183-186 http://e-journal.unair.ac.id/index.php/mkg 184 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 183–186 apexification, root resorption, iatrogenic root perforation, root fracture, tooth replantation, and intracanal dressing.4 nevertheless, it also suffers from certain disadvantages. for example, it can induce pulp inflammation for a period of up to three months, render the pulp tissue response unpredictable and cause irregular reparative dentin formation potentially leading to tunnel defects.5 recently, a range of studies have investigated the application of propolis to dentistry because of its numerous positive properties such as; anti-inflammatory, antibacterial, and the ability to induce the reorganization of pulp tissue.6 propolis is a substance that contains a plant-based resin produced by honeybees of the apis mellifera species which consists of more than 200 elements, including; phenolic acids, flavonoids, esters, aromatic aldehydes, alcohol, amino acids, fatty acids, vitamins and minerals.3,7 in dentistry, propolis has been widely employed because of the protection against caries that it provides to teeth. moreover, 30% propolis has also been recommended for use in irrigation during root canal treatment, while the resin formation is employed as pulp capping material to protect vital pulps derived from one of the flavonoids with the highest propolis composition.6 it can also induce the formation of reparative dentin by stimulating the release of transforming growth factor-β1 (tgf-β1) capable of inhibiting pulp inflammation and accelerating collagen synthesis by pulp cells.8 the numerous benefits of propolis underlies the authors’ support for the integration of natural remedies with modern medicine by combining calcium hydroxide with propolis as a pulp capping material in the hope that the efficacy of each ingredient can compensate for deficiencies in the others. studies showed that a combination of ca(oh)2–propolis used as a pulp capping material produces no toxic reactions and is capable of significantly reducing inflammation.6 therefore, the purpose of this study was to investigate the effect of a combination of ca(oh)2 and propolis extract as pulp capping material on odontoblast-like cell proliferation in wistar rats (rattus norvegicus). materials and methods this research constituted an experimental laboratory study incorporating a post-test control group design. all procedures and treatments which the animal subjects of this research underwent were approved by the ethical committee of the faculty of dentistry, universitas airlangga (document no. 277/hrecc/fodm/x/2018). propolis extract was produced by maceration using 96% ethanol. the research subjects comprised 30 healthy male wistar rats (aged 6-18 weeks, weighing 200-300 grams) which were anaesthetized using 100mg of ketamine (ketalar®, warner lambert, irlandia) and 10mg/kg of xylazine hcl (rompun®, bayer, leverkusen, jerman) in sterile phosphate buffered saline (pbs). the pbs was then placed on a fixation board. first, a cavity was created on the occlucal of right maxillary first molar using a low speed handpiece with a round tapered diamond bur (diameter 0.84) until it reached the pulp chamber. the pulp was perforated (diameter 0.46 mm) with a low speed handpiece featuring a round diamond bur. the wistar rats were divided randomly into six groups, namely: group i (not treated with any pulp capping, observed on the third day), group ii (treated with a combination of ca(oh)2 (emsure acs, reag. ph eur, germany) and 11% propolis extract, observed on the third day), group iii (treated with a combination of ca(oh)2 and aquadest observed on the third day), group iv (not treated with any pulp capping material, observed on the seventh day), group v (treated with a combination of ca(oh)2 and 11% propolis extract, observed on the seventh day), and group vi (treated with a combination of ca(oh)2 and aquadest, observed on the seventh day).9 for all groups, a micro applicator was employed to apply the pulp capping materials. all of the cavities were filled with restoration material (cention n, ivoclar vivadent). a necropsy was performed on the third and seventh days, followed by decapitation and separation of the maxilla. cutting was performed using a rotary microtome at a thickness of 5 µm, the resulting tissue being placed on a glass object and stained using haematoxilin and eosin (h&e) and observed through a microscope (olympus cx 23 binocular led, japan) to enable calculation of the odontoblast-like cells. the data obtained was analyzed statistically to examine the proliferation of odontoblast-like cells. the significance of differences between groups was determined by means of a one-way anova test followed by a post hoc tukey hsd. a p-value < 0.05 was considered to be significant. results figure 1 shows the histological examination of odontoblastlike cells on days 3 and 7. the mean and standard deviation of each sample group used to quantify the proliferation of odontoblast-like cells on days 3 and 7is shown in the table 1. table 1. mean and standard deviation of odontoblast-like cells on days 3 and 7 group mean ± sd i 6 ± 1.58 ii 12.8 ± 1.30 iii 6.2 ± 1.30 iv 7 ± 1.58 v 16.8 ± 1.30 vi 10.8 ± 0.83 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p183–186 http://dx.doi.org/10.20473/j.djmkg.v52.i4.p183-186 http://e-journal.unair.ac.id/index.php/mkg 185widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 183–186 a b c d e f figure 1. histological examination of odontoblast-like cells on days 3 and 7. the red arrow indicates the odontoblast-like cells (magnification 1000x). day 3 (a) group i: control (b) group ii: ca(oh)2+ 11% propolis (c) group iii: ca(oh)2 + aquadest. day 7 (d) group iv: control (e) group v: ca(oh)2+ 11% propolis (f) group vi: ca(oh)2 + aquadest. table 2. tuckey hsd on day 3 and 7 * p<0.05 = significant difference existed day 3 day 7 group i group ii group iii group iv group v group vi day 3 group i group ii .000* group iii 1.000 .000* day 7 group iv .843 .000* .931 group v .000* .001* .000* .000* group vi .000* .211 .000* .002* .000* the conducting of a tukey hsd indicated a significant difference in the proliferation of odontoblast-like cells between all groups (p<0.05). the contents of table 2 show that on day 3 there was a significant difference between group ii (ca(oh)2–propolis) and group i (control groups) and group iii (ca(oh)2–aquades), whereas group ii (ca(oh)2–propolis) presented a proliferation of odontoblast-like cells higher than that in group i (control) and group iii (ca(oh)2–aquades). meanwhile, on day 7 a significant difference existed between group iv (control) and group v (ca(oh)2–propolis); group iv (control) and group vi (ca(oh)2 + aquadest); and group v (ca(oh)2– propolis) and group vi (ca(oh)2 + aquades, whereas group v (ca(oh)2–propolis) experienced a proliferation of odontoblast-like cells higher than that in group iv (control) and group vi (ca(oh)2–aquades). discussion preparation of deep cavity caries can culminate in pulp perforation. the basic principle of operative dentistry is to maintain the health and function of the dentin-pulp complex, especially in cases of exposed pulp.10 pulp tissue also possesses the ability to repair itself in the manner of other connective tissue.11 the healing characteristics of exposed pulp tissue include; reorganization of damaged soft tissue, differentiation between sub-odontoblast and odontoblast-like cell and formation of reparative dentin.10 pulp tissue damage leads to inflammation. fibroblasts migrate immediately to the site of destruction, proliferation, and differentiation into odontoblast-like cells, in addition to producing the collagen matrix that subsequently becomes a hard tissue barrier protecting the remaining pulp tissue from irritants.12 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p183–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p183-186 186 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 183–186 in this study, enhancement of the significant difference in odontoblast-like cells occurred after application of ca(oh)2–11% propolis extract because propolis promotes antibacterial, anti-inflammatory, antioxidant, and immunomodulatory activity in order to prevent infection and increase cell regeneration.11,13 it is also known that propolis extract can stimulate tgf-β1 production, while tgf-β1 can induce proliferation of fibroblasts.11 jahromi et al. (2014) stated that ca(oh)2 application promotes less cell viability than propolis application.14 shaher et al. (2004), also reported that ca(oh)2 was almost ten times more cytotoxic than the propolis in pulp cells.13,14 in another study, the combination of ca(oh)2 with propolis produced no toxic reactions and reduced inflammation significantly in the connective tissue of rats.14 in this study, groups with a ca(oh)2 + 11% propolis combination (group ii and group v) experienced a proliferation of odontoblast-like cells higher than that of the control group and ca(oh)2-aquades group. this contrast could be caused by the properties of propolis which are known to have numerous advantages, one of them being immunomodulatory activity which promotes the healing process commencing with the formation of collagen fiber.10 two active components of propolis are flavonoids and caffeic acid whose function is to inhibit the arachidonic acid lipoxygenase pathway causing a reduction in the inflammatory response.15 however, proliferation of odontoblast-like cells on day 3 was less pronounced than that on day 7. this phenomenon was caused by acute inflammation that presented between day 0 and day 3 on which the proliferation of odontoblast-like cells commences, reaching its peak on day 7. this is because on day 7 there was a transition from acute to chronic inflammation in the pulp which involved odontoblastlike cells. chronic inflammation histologically marked by a form of granulated tissue consists of infiltration of chronic inflammatory cells, proliferation of capillary vessels, and proliferation of fibroblasts.10 in conclusion, the combination of ca(oh)2-propolis as a pulp capping material can increase the proliferation of odontoblastlike cell in pulp tissue on days 3 and 7. references 1. tziafas d, smith aj, lesot h. designing new treatment strategies in vital pulp therapy. j dent. 2000; 28(2): 77–92. 2. parolia a, kundabala m, rao nn, acharya sr, agrawal p, mohan m, thomas m. a comparative histological analysis of human pulp following direct pulp capping with propolis, mineral trioxide aggregate and dycal. aust dent j. 2010; 55(1): 59–64. 3. nanci a. ten cate’s oral histology : development, structure, and function. 9th ed. st. louis: elsevier; 2017. p. 326–7. 4. farhad a, mohammadi z. calcium hydroxide: a review. vol. 55, international dental journal. fdi world dental press ltd; 2005. p. 293–301. 5. dwiandhono i, effendy r, kunarti s. the thickness of odontoblastlike cell layer after induced by propolis extract and calcium hydroxide. dent j (majalah kedokt gigi). 2016; 49(1): 17–21. 6. montero jc, mori gg. assessment of ion diffusion from a calcium hydroxide-propolis paste through dentin. braz oral res. 2012; 26(4): 318–22. 7. mendonça icg, medeiros mlbb, penteado rapm, parolia a, porto iccm. an overview of the toxic effects and allergic reactions caused by propolis. pharmacologyonline. 2013; 2: 96–105. 8. ansorge s, reinhold d, lendeckel u. propolis and some of its constituents down-regulate dna synthesis and inf lammatory cytokine production but induce tgf-β1 production of human immune cells. zeitschrift fur naturforsch sect c j biosci. 2003; 58(7–8): 580–9. 9. dharsono va. aplikasi kombinasi kalsium hidroksida dan ekstrak propolis terhadap ekspresi dmp1 dan dsp pada odontoblas gigi tikus. thesis. surabaya: universitas airlangga; 2018. p. 26–36. 10. yu c, abbott p. an overview of the dental pulp: its functions and responses to injury. aust dent j. 2007; 52: s4–6. 11. karube h, nishitai g, inageda k, kurosu h, matsuoka m. naf activates mapks and induces apoptosis in odontoblast-like cells. j dent res. 2009; 88(5): 461–5. 12. sabir a, tabbu cr, agustiono p, sosroseno w. histological analysis of rat dental pulp tissue capped with propolis. j oral sci. 2005; 47(3): 135–8. 13. al-shaher a, wallace j, agarwal s, bretz w, baugh d. effect of propolis on human fibroblasts from the pulp and periodontal ligament. j endod. 2004; 30(5): 359–61. 14. zare jahromi m, ranjbarian p, shiravi s. cytotoxicity evaluation of iranian propolis and calcium hydroxide on dental pulp fibroblasts. j dent res dent clin dent prospects. 2014; 8(3): 130–3. 15. sabir a. respons inflamasi pada pulpa gigi tikus setelah aplikasi ekstrak etanol propolis (eep) (the inflammatory response on rat dental pulp following ethanolic extract of propolis (eep) application). dent j (majalah kedokt gigi). 2005; 38(2): 77–83. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p183–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p183-186 contents page printed by: airlangga university press. (139/09.10/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 43 number 2 april 2010 issn 1978 3728 dental journal majalah kedokteran gigi 1. erythema multiforme as the result of taking carbamazepine maharani laillyza apriasari and m. jusri .................................................................................... 49–53 2. tensile bond strength of hydroxyethyl methacrylate dentin bonding agent on dentin surface at various drying techniques kun ismiyatin ................................................................................................................................... 54–57 3. the efficacy of honey solution as plaque reducing agent dewi nurul m, indria rizki s, indriani s, masyitoh, and auerkari ei ..................................... 58–61 4. optimum dose of 2-hydroxyethyl methacrylate based bonding material on pulp cells toxicity widya saraswati .............................................................................................................................. 62–66 5. ankylosis of the temporomandibular joint and mandibular growth disturbance caused by neglected condylar fracture in childhood endrajana ........................................................................................................................................ 67–71 6. the effect of monofluorophosphate implant in white rat mothers towards the level of fluor in the incisors of the young babies (rattus-rattus) widjijono .......................................................................................................................................... 72–75 7. early removal of odontoma resulting in spontaneous eruption of the impacted teeth achmad harijadi ............................................................................................................................. 76–80 8. dental modifications: a perspective of indonesian chronology and the current applications rusyad adi suriyanto and toetik koesbardiati ............................................................................ 81–90 9. enamel defect of deciduous teeth in small gestational age children willyanti s syarif, roosje r. oewen, sjarif h. effendi and bambang sutrisna ....................... 91–96 10. integrated orofacial therapy in chronic rhinosinusitis management for children with sleep bruxism haryono utomo ................................................................................................................................ 97–101 11. the apical leakage of mineral trioxide agregate as the retrograde filling material with various mixing agents ema mulyawati ................................................................................................................................ 102–106 daftar isi 195195 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 195–199 original article the effects of mixing slurry water with type iii gypsum on setting time, compressive strength and dimensional stability chindy fransiska br nainggolan and dwi tjahyaning putranti department of prosthodontics, faculty of dentistry, university of sumatera utara, medan, indonesia abstract background: type iii gypsum is a material used to make dental master casts. it may be added to an accelerator, such as slurry water, to shorten setting time. calcium sulphate in slurry water may affect setting time, compressive strength and dimensional stability. purpose: the study evaluated the effect of slurry water on the setting time, compressive strength and dimensional stability of type iii gypsum. methods: eighty-one samples were made of type iii gypsum, divided into three groups: group a was gypsum mixed with 1% slurry water, group b, gypsum mixed with 2% slurry water and group c, gypsum mixed with distilled water. each sample was formed using a standardised master mould. for testing setting time, a cylindrical mould 25 mm in diameter and height was used; for compressive strength testing, the cylindrical mould was 20 mm in diameter and 40 mm in height; and for dimensional stability testing, a pair of cylindrical, ruled block and mould were used. setting time was tested using vicat’s apparatus; compressive strength was tested using a universal testing machine; and dimensional stability was tested using digital callipers. the data were analysed by one-way analysis of variance (anova) and least significant difference (lsd) tests. results: one-way anova and lsd tests showed significant differences in the effect of slurry water on the setting time, compressive strength and dimensional stability of type iii gypsum (p<0.05). conclusion: the use of slurry water can shorten setting time, decrease compressive strength and increase dimensional change of type iii gypsum. keywords: compressive strength; dimensional stability; setting time; slurry water; type iii gypsum correspondence: chindy fransiska br nainggolan, department of prosthodontics, faculty of dentistry, university of sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: nainggolanchindy@gmail.com introduction gypsum is a mineral widely found in nature, categorised as calcium sulphate dihydrate (caso4.2h2o). it is usually white to yellowish white and is found as a solid mass.1–3 gypsum products are widely used in dentistry for the production of study and master casts. to form a powder from mineral gypsum, a manufacturer heats the dihydrate, which cause it to lose water. it is then ground to produce a powdered hemihydrate (caso4.½h2o). when the hemihydrate is again mixed with water, it is converted back to a dihydrate and again becomes a solid mass.3 according to the specification no. 25 of the american dental association (ada), dental gypsum products are classified into five types: type i, impression plaster; type ii, model plaster; type iii, dental stone; type iv, high strength dental stone; and type v, high strength, high expansion dental stone.1–4 type iii gypsum, known as dental stone, is often used to produce a working cast (master cast) due to its high strength against fracture and abrasion. dental stone is denser and stronger than plaster, because its particle characteristics require less water.3 the properties of type iii gypsum are setting time, compressive strength, hardness and abrasion resistance, setting expansion, dimensional stability and detail reproduction.1–4 the working cast must have a specific setting time, strength, and dimensional stability over time, so as to guarantee the accuracy of the model – properties that are very important during the manufacturing process. based on the ada specification no. 25, type iii gypsum has a setting time of 8–16 minutes, compressive strength of 20.7–34.5 mpa and dimensional stability of 0–0.20%.3 setting time, compressive strength, and dimensional stability values are affected by the addition of an accelerator.1 the use of different types of water, such as slurry water and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p195–199 mailto:nainggolanchindy@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p195-199 196 nainggolan and putranti/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 195–199 distilled water, can be used in the gypsum manipulation process. several studies have identified that the addition of an accelerator can change gypsum’s properties, due to differences in mineral content of different solutions. slurry water is an accelerator that contains calcium sulphate and is obtained by placing clean, completely set dental stone pieces in a plastic container of distilled water and allowing them to soak for 48 hours.5 use of slurry water is a must.6 the calcium sulphate contained in slurry water acts as a catalyst that causes calcium sulphate dihydrate particles to form faster, thereby reducing the setting time when making a master cast. thus, the aim of this study is to evaluate the effects of 1% and 2% slurry water on the setting time, compressive strength and dimensional stability of type iii gypsum. materials and methods this experimental research was carried out in a laboratory setting using a post-test-only control group design. the samples used were three different mixtures of type iii gypsum, produced and divided into three groups: group a was type iii gypsum mixed with 1% slurry water, group b, type iii gypsum mixed with 2% slurry water and group c, type iii gypsum mixed with distilled water. each sample was formed using a standardised master mould according to ada specifications: the master mould for setting time testing (ada no. 25) was cylindrical and 25 mm in both diameter and height; for compressive strength testing (ada no. 25) the cylindrical mould was 20 mm in diameter and 40 mm in height;7 and for stability dimension testing (ada no. 19) a pair of cylindrical ruled block and gypsum mould with a 38 mm outer diameter, 30 mm inner diameter and 20 mm height.8 the number of samples in this study was calculated using the federer formula, with nine samples for each of the three studied groups, totalling 27 samples. each sample was then tested for setting time, compressive strength and dimensional stability – an overall total of 81 samples. the study was conducted in december 2020 at the prosthodontics laboratory, faculty of dentistry, university of sumatera utara and the impact and fracture research center, laboratory of mechanical engineering, university of sumatera utara. the research ethics commission at the university of sumatera utara approved the study (no: 832/ kep/usu/2020), according to the declaration of helsinki on medical protocols and ethics. the manufacture of slurry water began by mixing 100 g of type iii gypsum powder with 30 ml of distilled water in a rubber bowl using a spatula for 60 seconds at a speed of about two revolutions per second until homogenous. the dough was then poured into a container and allowed to stand for 48 hours. gypsum that had hardened was then crushed to form pieces. to make 1% slurry water, 1 g of gypsum pieces were soaked in 100 ml of distilled water for 48 hours (group a), and 2 g of gypsum pieces were soaked in 100 ml of distilled water for 48 hours, to make 2% slurry water (group b). slurry water was stored at room temperature (figure 1).9 to make the research sample, each master mould was first smeared with vaseline, as evenly and as thinly as possible. the slurry water was shaken well before use, after which 30 ml of each solution (1%, 2% slurry water and distilled water) was weighed out using digital scales and placed in separate rubber bowls. next, 100 g of type iii gypsum powder, which had been weighed, was added gradually to each rubber bowl and stirred using a spatula for 60 seconds at a speed of approximately two revolutions per second until homogeneous. once homogeneous, the gypsum mixture was poured slowly, with the help of a spatula, into the master mould, positioned on a glass slab that was vibrated for a few seconds until the mould was full. the excess dough was flattened using a glass slab placed on top of the master mould and pressed firmly until it touched the top surface of the master model. setting time was tested using vicat’s apparatus. the mould containing type iii gypsum dough was placed under the measuring needle, which was 1 ± 0.005 mm in diameter and a plunger weighing 2.942 ± 0.005 n (300 ± 0.5 grams). the setting time was determined by bringing the tip of the needle into contact with the surface of the material. the needle was released and allowed to penetrate the sample at 15-second intervals. the needle was wiped clean after each penetration and the master mould moved to permit the next penetration in a new area. the setting time of the type iii gypsum was calculated from the start of the mixing process until the time when the needle could no longer penetrate.1,7 compressive strength was tested after the samples had hardened completely for 24 hours, by applying a compressive load to each sample until it broke, using a universal testing machine. the data obtained at the failure point of each sample were recorded in kilogram-force (kgf), and the results of the type iii gypsum compressive strength calculated in megapascals (mpa) by using the formula:7 compressive strength = f a = p x 9.8 πr�figure 1. 1% and 2% slurry water. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p195–199 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p195-199 197nainggolan and putranti/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 195–199 where: f = the force or load at point of failure (n) a = cross-sectional surface area (mm2) p = compressive load (kgf) dimensional stability was evaluated 24 hours after making the samples, by measuring the length of the lines using digital callipers. the distance between the crosslines (cd – c’d’) on lines a, b and c of each sample was measured. the measurements were then totalled, and the mean value obtained. next, the data from the measurement of dimensional changes were converted into a percentage by using the formula:9 𝐿� − 𝐿� 𝐿� 𝑥 100% where: l1 = the mean of line length obtained on the sample (mm) l0 = the line length on the master mould (mm) all the data were analysed using statistical tests run on the spss software program. the mean and standard deviation of the data were obtained using descriptive analysis. the effect of mixing slurry water with type iii gypsum on setting time, compressive strength and dimensional stability were evaluated using a one-way analysis of variance (anova). the differences between the studied groups were compared using the least significant difference (lsd) post hoc test. to use one-way anova, the data obtained from the study must be normally distributed. to establish whether the data were normally distributed or not, a normality test was carried out using shapiro–wilk test (p>0.05). results table 1 shows the mean and standard deviation of setting time, compressive strength and dimensional stability of type iii gypsum mixed with each of the three different solutions. statistical analysis using the shapiro–wilk test established that the data were normally distributed (p>0.05), following which a one-way anova was used to evaluate the effects of mixing slurry water with type iii gypsum on setting time, compressive strength and dimensional stability. the anova test showed a statistically significant difference (p<0.05) in the effect of mixing slurry water with type iii gypsum on setting time, compressive strength and dimensional stability (table 2). the lsd multiple comparison test showed a significant difference (p<0.05) between all the studied groups (table 3). discussion in this study, type iii gypsum powder was mixed with three different types of solutions, namely 1% slurry water, 2% slurry water and distilled water as a control. slurry water contains minerals, unlike distilled water. because the solutions used have different mineral compositions, this can affect the setting time, compressive strength and dimensional stability of type iii gypsum. the mean and standard deviation of setting time in group c was the longest relative to groups a and b. table 1. mean and standard deviation of setting time, compressive strength and dimensional stability of type iii gypsum mixed with 1% slurry water (a), 2% slurry water (b) and distilled water (c) group setting time (s) compressive strength (mpa) dimensional stability (%) mean sd mean sd mean sd a 421.67 31.25 22.37 0.59 0.088 0.028 b 346.89 22.61 20.07 0.57 0.130 0.032 c 596.22 57.82 25.87 2.00 0.053 0.028 table 2. one-way anova results of setting time, compressive strength and dimensional stability of type iii gypsum mixed with 1% slurry water, 2% slurry water and distilled water variable n f-value p-value setting time 27 91.50 0.0001 compressive strength 27 49.33 0.0001 dimensional stability 27 15.35 0.0001 table 3. least significant difference results of setting time, compressive strength and dimensional stability of type iii gypsum mixed with 1% slurry water (a), 2% slurry water (b) and distilled water (c) group p-value setting time compressive strength dimensional stability a b 0.001 0.001 0.006 a c 0.000 0.000 0.020 b c 0.000 0.000 0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p195–199 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p195-199 198 nainggolan and putranti/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 195–199 the type iii gypsum, when mixed with 1% and 2% slurry water, had shorter setting times than when mixed with distilled water, due to the presence of calcium sulphate. the results of this study also showed that the type iii gypsum mixed with 2% slurry water group had a shorter setting time than the type iii gypsum group mixed with 1% slurry water, due to the respective differences in the amount of calcium sulphate contained in 1% and 2% slurry water. calcium sulphate dihydrate particles act as the core of the crystallisation process. the greater the number of calcium sulphate dihydrate particles, the greater the crystallisation formation.3,10 the presence of a sufficient number of crystallisation nuclei in the slurry water can accelerate the crystallisation process and the solubility of calcium sulphate hemihydrate to become dihydrate, with the result that the setting time of type iii gypsum becomes faster. the results of this study are in line with previous research conducted by denizoglu et al.,11 which compared the setting times of type iii gypsum mixed with distilled water, tap water, 2% and 16% slurry water and found that the use of slurry water shortened type iii gypsum setting time. the mean setting time of type iii gypsum mixed with 16% slurry water was shorter than that of the type iii gypsum mixed with 2% slurry water.11 the results of the study for the compressive strength variable showed that the type iii gypsum mixed with distilled water group had the highest compressive strength value compared to the gypsum type iii groups mixed with 1% and 2% slurry water. the results of this study are in line with research conducted by dewi,9 which found that use of distilled water had the highest compressive strength value when compared to the use of tap water and slurry water. the addition of slurry water, which acts as an accelerator, can reduce the compressive strength of gypsum, due to the crystal shape becoming irregular, which reduces intracrystalline cohesion.9 furthermore, the compressive strength of gypsum is closely related to its surface hardness. the higher the surface hardness value of gypsum, the higher its compressive strength value. ayoub et al.,12 found that the greatest type iv gypsum surface hardness was obtained in samples mixed with distilled water, while the least gypsum surface hardness was obtained in samples mixed with slurry water. different mineral content between slurry water, both 1% and 2%, and distilled water affects the value of the compressive strength of type iii gypsum. musa et al.,13 showed that use of distilled water produced the greatest compressive strength value compared to five other treatment groups, namely tap water, tap water mixed with gypsum powder, slurry water made with tap water, distilled water mixed with powder gypsum and slurry water made with distilled water. the gypsum group mixed with slurry water had a lower compressive strength than the group using distilled water. this may be related to the absence of any mineral content in the distilled water, such that the crystal form is regular, relatively non-porous and denser. the mineral contained in the slurry water, namely calcium sulphate, may reduce intracrystalline cohesion, resulting in a reduction in the gypsum compressive strength. mixing slurry water with type iii gypsum reduces the compressive strength, because calcium sulphate particles cause the crystals to become irregular in shape, which affects the ability of gypsum crystals to grow and causes pores, resulting in a more brittle gypsum product.13 vyas et al.,14 found that, generally, the gypsum group with sulphate additives had a lower resistance to compressive strength than the control group without additives. incorporating additives may cause an increase in the concentration of additives in the gypsum dough, such that the number of gypsum crystals formed from the overall volume decreases, with a corresponding decrease in intracrystalline cohesion, resulting in gypsum products with a low compressive strength. the function of additives is to increase the reaction rate, so it is possible that the reaction occurs so fast that some hemihydrate crystals are not completely formed into dihydrates, which causes an increase in hemihydrate crystals, producing a weak gypsum product.14 the results of the study for the dimensional stability variable found that the type iii gypsum mixed with distilled water group had the lowest dimensional stability value compared to the groups of type iii gypsum mixed with 1% and 2% slurry water. this is related to the different mineral content between distilled water and slurry water. the results of this study are in line with research conducted by dewi,9 who found that the use of distilled water had the smallest dimensional change value when compared to the use of tap water or slurry water. the dimensional stability of gypsum is generally closely related to its setting expansion. the higher the gypsum expansion value, the higher the gypsum dimension change value. denizoglu et al.,11 found that mixing gypsum with 2% and 16% slurry water increased the expansion value in the first 24 hours. 16% slurry water had the greatest expansion value when compared to distilled water and 2% slurry water. this may be caused by the presence of calcium sulphate in the slurry water, which acts as a nucleation core for the growth of calcium sulphate dihydrate crystals, resulting in an increased number of dihydrate crystals, which causes greater overlapping of crystals and a greater setting expansion.11 the value of dimensional stability of gypsum mixed with slurry water is greater than that of the group of gypsum mixed with distilled water. this may be due to the relatively lower water content of slurry water, which causes a greater setting expansion. this reduced water content results from an increase in calcium sulphate particles, which attract water particles during the gypsum setting process, with a resulting lower water content.10 alberto et al.,15 noted that water content may affect the setting expansion of gypsum. the lower the water content at the time of mixing, the greater the setting expansion of gypsum. the water content in gypsum affects both the internal growth of the dihydrate crystals and the protusion of the dihydrate crystals.15 from the data analysis and discussion, the study concludes that dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p195–199 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p195-199 199nainggolan and putranti/dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 195–199 the use of slurry water, both 1% and 2%, may shorten setting time, decrease compressive strength and increase dimensional change of type iii gypsum. further analysis of the effects of slurry water on other properties of type iii gypsum is needed. references 1. sakaguchi r, ferracane j, powers j. craig’s restorative dental materials. 14th ed. st. louis: mosby elsevier; 2016. p. 252–8. 2. powers j, wataha j. dental materials: foundations and applications. 11th ed. st. louis: mosby; 2014. p. 118–28. 3. hatrick cd, eakle ws, bird wf. dental materials: clinical applications for dental assistants and dental hygienists. 2nd ed. st. louis: elsevier; 2011. p. 203–6. 4. mccabe jf, walls awg. bahan kedokteran gigi. 9th ed. jakarta: egc; 2014. p. 45–56. 5. kumar rn, reddy sm, karthigeyan s, punithavathy r, karthik ks, manikandan r. the effect of repeated immersion of gypsum cast in sodium hypochlorite and glutaraldehyde on its physical properties: an in vitro study. j pharm bioallied sci. 2012; 4(suppl 2): s353-7. 6. choudhary s, banerjee a, giri tk, rohilla ak. study of surface hardness of gypsum casts made with slurry water : an in vitro study. iosr j dent med sci. 2016; 15(12): 23–6. 7. hooman z, nafiseh k, seyfollah s, amir f. comparison of setting time, setting expansion and compressive strength of gypsum cast produced by mixing of gypsum powder with distilled water or 0.05% sodium hypochlorite. j dent sch shahid beheshti univ med sci. 2013; 31(3): 162–9. 8. raipure p, kharsan v. comparative evaluation of dimensional accuracy and surface detail reproduction of elastomeric impression material when treated with three retraction cord medicaments : an in vitro study. int j dent med res. 2014; 1(2): 12–20. 9. dewi ls. pengaruh pemakaian slurry water dan air bersih terhadap kekuatan kompresi dan perubahan dimensi gipsum tipe iii pada pembuatan model kerja gigi tiruan. thesis. medan: universitas sumatera utara; 2014. p. 35, 52. 10. taqa a, mohammed n, alomari a. the effect of different water types on the water powder ratio of dental gypsum products. alrafidain dent j. 2012; 12(1): 142–7. 11. denizoglu s, yanikoglu n, baydas b. the linear setting expansions of the dental stone and whose initial setting times. dentistry. 2015; 5(6): 1000308. 12. ayoub w ul, magray ia, jan t, bashir a. comparison of tap water, distilled water and slurry water on surface hardness of gypsum die an in vitro study. int j appl dent sci. 2019; 5(2): 281–3. 13. musa l, nafea i, hasan n. evaluation the effect of mixed different types of water with dental stone on the compressive strength . alkufa univ j biol. 2010; 2(1): 1–7. 14. vyas r, idris ba, al-sayyid ma, al-getlawi mh. compressive strength of gypsum product with various sulfates. cairo dent j. 2008; 24(2): 199–203. 15. alberto n, carvalho l, lima h, antunes p, nogueira r, pinto jl. characterization of different water/powder ratios of dental gypsum using fiber bragg grating sensors. dent mater j. 2011; 30(5): 700–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p195–199 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p195-199 6767 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 67–70 research report nickel release and the microstructure of stainless steel orthodontic archwire surfaces after immersion in detergent and non-detergent toothpaste: an in vitro study hilda fitria lubis,1 kholidina imanda harahap2 and dina hudiya nadana lubis1 1department of orthodontics, 2department of dental material and technology, faculty of dentistry, universitas sumatera utara medan – indonesia abstract background: stainless steel is a material that can be used in orthodontics for components of dental braces, such as brackets, archwires and molar bands. orthodontic archwires exposed to toothpaste can release nickel ions that cause hypersensitivity. the excessive use of sodium lauryl sulphate in detergent toothpaste can cause mouth irritation, severe ulceration, decreased salivary solubility and taste sensitivity changes. purpose: the aim of this study is to compare the nickel ion released by stainless steel archwires after immersion in detergent and non-detergent toothpaste. methods: forty stainless steel archwires from ortho organizer (0.016 x 0.022in) were divided into two groups (n=20). group 1 comprised stainless steel archwires immersed in detergent toothpaste. group 2 consisted of stainless steel archwires immersed in non-detergent toothpaste. these archwires were immersed in 1.5g toothpaste then kept in an incubator at 37°c for around 24 hours. after that, the archwires were removed from the toothpaste, and the toothpaste was dissolved in 25ml of aquadest. the amount of nickel ion released was examined by using inductively coupled plasma optical emission spectrometry (icp-oes). after that, the structure of the sample surface was examined with a scanning electron microscope (sem). a statistical analysis was done using the shapiro–wilk normality test (p>0.05). an independent t-test was carried out to compare the two groups (p<0.05). results: the mean of nickel ion release in group 1 was 0.214±0.319mg/l, and in group 2 it was 0.168±0.107 mg/l. there was no significance between the groups (p=0.323; p>0.05). the sem images of the archwire surfaces showed that there were more corrosive contour changes in the archwire surface in group 1 than in group 2. conclusion: there was no difference between the nickel ion released from stainless steel orthodontic archwires after immersion in detergent and non-detergent toothpaste. after immersion in detergent toothpaste, stainless steel archwire surfaces showed more corrosive contour changes than those immersed in non-detergent toothpaste. keywords: nickel ion; orthodontic archwires; sodium lauryl sulphate; stainless steel; tooth paste correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no 2, medan 20155, indonesia. email: hildadrgusu@gmail.com introduction fixed orthodontic appliances basically consist of brackets attached to the teeth, rings embracing molar teeth and arches connecting the individual parts of the appliance. orthodontic archwires are elements generating forces that allow for the movement of teeth as well as providing the base along which they move.1 orthodontic archwires are made from a cobalt-chromium-nickel alloy (cocrni), containing about 40% cobalt, 20% chromium, 15% nickel, 16% iron and an addition of molybdenum and manganese.2 stainless steel has been the most commonly used material in fixed and removable orthodontic treatment since 1932.3 stainless steel that has a composition of 8%–12% nickel, 17%–22% chromium, 71% iron and 0.2% carbon is widely used on bracket, molar band and archwire. the nickel and chromium give stainless steel its ductility and corrosion resistance.4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p67–70 mailto:hildadrgusu@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p67-70 68 lubis et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 67–70 oral hygiene is an important factor that must be controlled during orthodontic treatment since it can affect the quality and duration of the therapy.5 during orthodontic treatment, patients must brush their teeth daily, but the toothpaste that they use can cause the corrosion of the archwire in the oral environment. a previous study into the corrosive behaviour of dental applications with the presence of vicco® toothpaste showed that corrosion resistance is highest when artificial saliva is mixed with toothpaste, followed by toothpaste alone and, lastly, artificial saliva alone.6 homogenous corrosion resistance is the most important characteristic of stainless steel. however, it is not an intrinsic property, but results from the behaviour of the material surface in interaction with its environment. indeed, corrosion resistance in stainless steel is developed by the formation of a passive surface film, which acts as a blockade between the surface and the surrounding environment.7 the corrosion of orthodontic appliances in the oral environment has concerned clinicians for some time, and this concern is focused on two principal issues: whether corrosion products, if produced, are absorbed into the body and cause either localized or systemic effects and what the effects of corrosion are on the physical properties and the clinical performance of orthodontic appliances.8 in general, orthodontic materials are considered biocompatible, but there are side effects that have been reported in the literature, including allergic, inflammation, cytotoxicity, and mutagenicity.9 the estimated incidence of an allergic reaction in orthodontic patients is 1:100, with 85% of these being contact dermatitis.10 with a view to furthering the understanding of such problems, we are studying the amount of nickel ion released in orthodontic archwires after immersion in detergent and non-detergent toothpaste, and the change in the morphology of the orthodontic archwire surface. the release of nickel ion was analysed using inductively coupled plasma optical emission spectrometry (icp-oes) and the change in the morphology of the archwire surfaces was analysed using scanning electron microscope (sem). materials and methods the research type was an experimental laboratory posttestonly control group design using a comparison group. the sample used in this research was composed of 40 pieces of stainless steel archwire (ortho organizer, langenhagen, germany), with a diameter of 0.016 x 0.022in, which were divided into two groups. group 1 were immersed in the detergent toothpaste (pepsodent, tangerang, indonesia) and group 2 in the non-detergent toothpaste (enzim fresh mint, depok, indonesia). each piece was cut into 2cm strips and immersed in 1.5g of either detergent or nondetergent toothpaste with 2.5ml of aquadest and stored in an incubator for 24 hours at 37oc. after that, the archwire pieces were removed from the toothpaste, washed with distilled water and dried. the samples were also tested with a scanning electron microscope (sem) (hitachi tm3000 tabletop microscope, japan) in the physics laboratory unimed to observe the structure of the archwire surface after an immersion. the toothpaste was dissolved in 25ml aquadest and stirred until homogenous, and then a measurement was taken of the amount of nickel ion released with inductively coupled plasma optical emission spectrometry (icp-oes) (varian liberty series ii, united state) in btklpp medan. the results of the examination gave a figure in mg/l. using the statistical package for social science (spss) 17.0 edition (chicago, us) a statistical analysis was performed with a shapiro–wilk normality test (p>0.05). the data obtained was analysed statistically by using an independent t-test to compare the difference between the two groups (p<0.05). a d c b figure 1. ss group 1 magnification 1500x (a); ss group 1 magnification 2000x (b); ss group 2 magnification 1500x (c); ss group 2 magnification 2000x (d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p67-70 69lubis et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 67–70 results the average amount of nickel ion released in group 1 was 0.252 mg/l, and in group 2 was 0.197 mg/l. the results of the normality test showed that the data was normally distributed in both group 1 (p=0.884; p>0.05) and group 2 (p=0.884; p>0.05). an independent t-test was carried out and the results showed no significant difference between the amount of nickel ion released after immersion in the detergent and non-detergent toothpaste (p=0.213; p>0.05) (table 1). the sem images of the archwire surfaces showed that those belonging to group 1 were rougher than those from group 2. the results also revealed that the archwire surfaces in group 1 had more corrosive contour changes than those in group 2 (figure 1). discussion this study measured the number of nickel ions released by stainless steel archwire after immersion in detergent and non-detergent toothpaste. the results of this study were in line with the study of brandao et al.11 which measured the release of nickel ions from a stainless steel bracket that was immersed in various toothpastes that contained detergent. nickel ions can be released because of the substance contained in toothpaste.11 according to schmalz et al.12 nickel ions have soluble properties in liquids, so the length of time of the exposure of archwire to a liquid can affect the release of metal ions. nickel ions have a high tendency to be released because the nickel atom is not strongly attached to the intermetallic compound.12 several components of toothpaste can cause changes in metal. the changes of orthodontic material properties can have a negative effect on orthodontic treatment.13 inorganic components, such as phosphate, sodium and potassium, contained in toothpaste act as electrolyte media that can trigger electrochemical reactions. electrochemical reactions are reactions that occur at the anode, where oxidisation takes place, and cathode, where reduction occurs. metal ions act as anodes and h+ from electrolyte media act as cathodes.14 detergent toothpaste contains sodium lauryl sulphate, which can cause the release of nickel ions, due to the presence of sodium ions that can trigger electrochemical reactions. sodium ions will react with water to form hydroxide bonds that occur at the cathode (reduction reaction).15,16 in addition, sodium lauryl sulphate also contains sulphate (so4) that can affect the release of metal ions.15 sulphate ions will react with water to form sulphuric acid, which occurs at the cathode (reduction reaction).16 the presence of these ions results in the release of a protective layer (cr2o3), which is the outermost layer of stainless-steel wire. the release of the protective layer can cause nickel ions to detach. this research is in accordance with the research conducted by minanga et al.17 on the release of nickel ions and the immersion of chromium orthodontic stainless steel brackets in mouthwash containing sodium. the results of the study showed the composition of the material and the reactions of the solution where the metal was immersed. there were various types of sodium, namely sodium fluoride, sodium citrate, sodium benzoate, sodium lauryl sulphate, and sodium saccharin. the study showed that the acid contained in mouthwash, namely citric acid and benzoic acid, canresult in the release of metal ions.17 another possible cause of the release of nickel ions in this study is the presence of fluoride ions found in detergent and non-detergent toothpaste. according to alavi et al.18 the release of fluoride ions will combine with hydrogen to produce hydrofluoric acid (hf), which can damage the oxide layer in orthodontic wires, resulting in the release of metal ions, such as nickel and chromium. fatimah et al.19 states that fluoride can reduce the resistance of stainless steel orthodontic wire to corrosion. this can occur because the effects of fluoride can damage the protective layer of the wire. in this study, the non-detergent toothpaste contains citric acid (c6h8o7), which can trigger the release of nickel ions. according to fontana, citric acid (c6h8o7) has high enough h+ particles to lead to faster corrosion rates.20 nickel ions released from archwires can be carcinogenic, mutagenic, cytotoxic and allergic. the release of nickel ions from archwires can result in gingival hyperplasia, labial desquamation, angular cheilitis, swelling, and burning in the oral mucosa.5 the average intake of nickel per day in food is 300–500µg.7 the concentration of nickel in drinking water is generally below 20µg / l.7,21 a nickel content of more than 50% can cause manifestations of allergic reactions.21 the biologic effect of the corrosion product was measured by gingival fibroblast cell viability in a released metal ion solution. the physical effect of the released metal ions was evaluated by the morphology of the appliance surface and the surface roughness of the brackets and archwires. 22 it can be concluded that both the detergent and non-detergent tooth paste can cause the release of nickel ion and surface roughness in stainless steel orthodontic archwire. further research needs to be conducted with a variety of orthodontic wire types, and variations in the immersion and stirring times. in addition, clinical research is needed to see the effects of nickel ion release. table 1. the differences in the nickel ions released after immersion in the detergent and non-detergent toothpaste. groups mean(mg/l) ± sd p detergent toothpaste 0.252 ± 0.139 0.213 non-detergent toothpaste 0.197 ± 0.107 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p67-70 70 lubis et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 67–70 references 1. małkiewicz k, sztogryn m, mikulewicz m, wielgus a, kamiński j, wierzchoń t. comparative assessment of the corrosion process of orthodontic archwires made of stainless steel, titanium–molybdenum and nickel–titanium alloys. arch civ mech eng. 2018; 18(3): 941–7. 2. kotha rs, alla rk, shammas m, ravi rk. an overview of orthodontic wires. trends biomater artif organs. 2014; 28(1): 32–6. 3. gajapurada j, ashtekar s, shetty p, biradar a, chougule a, bhalkeshwar, bansal a, zubair w. ion release from orthodontic brackets in three different mouthwashes and artificial saliva: an in-vitro study. iosr j dent med sci. 2016; 15(4): 76–85. 4. hussain hd, ajith sd, goel p. nickel release from stainless steel and nickel titanium archwires an in vitro study. j oral biol craniofacial res. 2016; 6(3): 213–8. 5. cozzani m, ragazzini g, delucchi a, mutinelli s, barreca c, rinchuse dj, servetto r, piras v. oral hygiene compliance in orthodontic patients: a randomized controlled study on the effects of a post-treatment communication. prog orthod. 2016; 17: 1–6. 6. souza rd, chattree a, rajendran s, souza rd, chemica dp. stainless steel alloys for dental application: corrosion behaviour in the presence of toothpaste vicco. der pharma chem. 2017; 9(8): 25–31. 7. patnaik l, ranjan maity s, kumar s. status of nickel free stainless steel in biomedical field: a review of last 10 years and what else can be done. mater today proc. 2019; : 1–6. 8. house k, sernetz f, dymock d, sandy jr, ireland aj. corrosion of orthodontic appliances—should we care? am j orthod dentofac orthop. 2008; 133(4): 584–92. 9. pazzini ca, pereira lj, marques ls, ramos-jorge j, aparecida da silva t, paiva sm. nickel-free vs conventional braces for patients allergic to nickel: gingival and blood parameters during and after treatment. am j orthod dentofac orthop. 2016; 150(6): 1014–9. 10. maheshwari s, verma sk, dhiman s. metal hypersensitivity in orthodontic patients. j dent mater tech. 2015; 4(2): 111–4. 11. brandão gam, simas rm, almeida lm de, silva jm da, meneghim m de c, pereira ac, almeida ha de, brandão amm. evaluation of ionic degradation and slot corrosion of metallic brackets by the action of different dentifrices. dental press j orthod. 2013; 18(1): 86–93. 12. schmalz g, arenholt-bindslev d. biocompatibility of dental materials. springer; 2009. p. 379. 13. hosseinzadeh nik t, hooshmand t, farhadifard h. effect of different types of toothpaste on the frictional resistance between orthodontic stainless steel brackets and wires. j dent (tehran). 2017; 14(5): 275–81. 14. bardal e. corrosion and protection. springer; 2004. p. 315. 15. sidik f. analisa korosi dan pengendaliannya. j foundry. 2013; 3(1): 25–30. 16. chang r. kimia dasar: konsep-konsep inti. achmadi ss, simarma l, editors. jakarta: erlangga; 2005. p. 105. 17. minanga ma, anindita ps, juliatri. pelepasan ion nikel dan kromium braket ortodontik stainless steel yang direndam dalam obat kumur. pharmacon j ilm farm – unsrat. 2016; 5(1): 135–41. 18. alavi s, farahi a. effect of fluoride on friction between bracket and wire. dent res j (isfahan). 2011; 8(suppl 1): s37-42. 19. fatimah di, anggani hs, ismah n. effect of fluoride mouthwash on tensile strength of stainless steel orthodontic archwires. iop j phys conf ser. 2017; 884: 1–5. 20. fontana mg. corrosion engineering. 3rd ed. siangapore: mcgrawhill; 1986. 21. noble j, ahing si, karaiskos ne, wiltshire wa. nickel allergy and orthodontics, a review and report of two cases. br dent j. 2008; 204(6): 297–300. 22. yanisarapan t, thunyakitpisal p, chantarawaratit p on. corrosion of metal orthodontic brackets and archwires caused by fluoridecontaining products: cytotoxicity, metal ion release and surface roughness. orthod waves. 2018; 77(2): 79–89. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p67-70 dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed 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reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. vol 38 no 2-2005 68 viabilitas sel fibroblas bhk-21 pada permukaan resin akrilik rapid heat cured (viability of fibroblast bhk-21 cells to the surface of rapid heat cured acrylic resins) anita yuliati bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract acrylic resins are widely used in the fabrication of denture bases and have been shown to be cytotoxic as a result of substances that leach from the resin. numerous reports suggest that residual monomer may be responsible for mucosal irritation and sensitization of tissues. this information is important in eddition to the information of the biologiced effect of such materials. the purpose of this study was to know the viability of fibroblast bhk-21cells to the surface of rapid heat cured acrylic resins. the sample of 5 mm in diameter and 1 mm thickness was cured in water bath for 20, 30, and 40 minutes at 100° c. bhk-21 cells were grown in medium eagle to be 2 × 105 cell/ml in 96 well micro titer plates as the added sample and incubated at 37° c for 24 hour. five hours before the end of the incubation mtt solutionwas added from step one to each well containing cells. viability cells were measured by spectrophotometer at 550 nm. the data were statistically analyzed by using one-way analysis of variance followed by lsd test. the result indicated that viability of fibroblast bhk-21 cells did not decrease to the surface of resin acrylic rapid heat cured. key words: viability bhk-21 cells, rapid heat cured acrylic resins korespondensi (correspondence): anita yuliati, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132. pendahuluan polimetil metakrilat yang merupakan material dasar dari resin akrilik di bidang kedokteran gigi digunakan sebagai material pembuatan basis gigi tiruan lepasan semenjak mulai diperkenalkan pada tahun 1937.1 material ini mempunyai beberapa keunggulan antara lain estetik yang baik, kekuatan tinggi, menyerap air rendah, daya larut rendah, mudah dilakukan reparasi, proses manipulasi mudah karena tidak memerlukan peralatan rumit.2 oleh karena itu resin akrilik masih menjadi pilihan utama dokter gigi sebagai pembuatan basis gigi tiruan lepasan, meskipun saat ini telah banyak digunakan material logam campur sebagai basis gigi tiruan lepasan. perkembangan material untuk pembuatan basis gigi tiruan telah dirasakan pada saat ini dengan dipasarkan resin akrilik jenis rapid heat cured. pabrik pembuat material tersebut menyebutkan bahwa resin akrilik ini mempunyai fitting yang baik, komfortabel, free bubble, kuat, cadmiumfree. keunggulan jenis resin akrilik ini tidak memerlukan waktu yang lama untuk proses polimerisasi. menggunakan perbandingan antara bubuk dan cairan resin akrilik yang tepat berdasarkan petunjuk pabrik dan jenis resin akrilik ini hanya memerlukan waktu selama 20 menit untuk proses polimerisasi. hal ini berbeda dengan resin akrilik yang sebelumnya, memerlukan waktu sekitar 120 menit untuk proses polimerisasi. apabila proses polimerisasi dari resin akrilik berjalan singkat, akan menyebabkan kandungan monomer yang belum bereaksi menjadi polimer masih tetap tinggi.3,4 hal ini telah terbukti bahwa resin akrilik jenis rapid heat cured bila proses polimerisasi selama 20 menit, kandungan monomer sisa yang terdeteksi dengan kromatografi gas sebesar 1,9%. kandungan monomer sisa tersebut cukup tinggi bila dibandingkan dengan resin akrilik yang diproses dengan polimerisasi waktu yang lama.5 kandungan monomer sisa dalam resin akrilik yang tinggi perlu mendapatkan perhatian. bila material tersebut digunakan di dalam rongga mulut dapat mengakibatkan terjadi iritasi pada mukosa rongga mulut yang manifestasinya berupa kemerahan, rasa sakit dan pembengkakan.6 peneliti lain juga melaporkan terjadi iritasi mukosa yang disebabkan pelepasan monomer sisa dari resin akrilik yang telah mengeras.7 syarat material di bidang kedokteran gigi terutama yang digunakan di dalam mulut harus bersifat biokompatibel, artinya dapat diterima oleh inang, tidak toksik, tidak iritan, tidak bersifat karsinogenik dan tidak menimbulkan reaksi alergi.8 kekurangan material resin akrilik adalah bersifat toksik.2 metode kultur sel sering digunakan untuk pengujian efek biologi pada tingkat awal dari suatu material yang digunakan pada kedokteran gigi untuk mengetahui efek toksisitas.8,9 sel fibroblast bhk-21 adalah sel yang paling 69yuliati: viabilitas sel fibroblas bhk-21 banyak digunakan para peneliti untuk uji toksisitas material di bidang kedokteran gigi. toksisitas dari material resin akrilik jenis rapid heat cured ini tidak dilaporkan oleh pabrik. toksisitas material yang digunakan di kedokteran gigi berhubungan dengan viabilitas sel yang hidup. pada penelitian ini viabilitas sel ditentukan dengan mtt colorimetric bioassays untuk mendeteksi secara kuantitatif sel hidup.10 oleh karena itu perlu diteliti bagaimana viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik jenis rapid heat cured bila proses polimerisasi diperpanjang. penelitian ini bertujuan untuk mengetahui viabilitas sel fibroblas bhk-21 pada permukaan resin akrilik jenis rapid heat cured yang proses polimerisasinya diperpanjang. manfaat penelitian ini dapat digunakan sebagai pertimbangan yang tepat dalam menentukan lama proses polimerasasi resin akrilik jenis rapid heat cured untuk menghasilkan material yang biokompatibel. bahan dan metode jenis penelitian ini adalah eksperimental laboratoris. bahan yang digunakan untuk penelitian adalah resin akrilik rapid heat polymerized (biocryl-2, altripon co. inc box 3526 paranaque, philippines), gips keras (moldano), sel bhk-21 diperoleh dari stok (tdc unair), mtt (sigma cat.no.m-5655), media eagle's, bovine serum albumin, fosfat bufer salin, lempeng kultur sel 96 sumuran beralas datar, larutan tripsin versene. alat yang digunakan adalah model master kuningan diameter 5 mm tebal 1 mm, kuvet logam, timbangan digital, termometer, pipet mikro, laminar flow cabinet, inkubator, spektofotometer. penelitian dilakukan di bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi dan tropical disease center universitas airlangga. waktu penelitian juli sampai agustus 2003. pembuatan mould dilakukan dengan mengaduk gips keras dan air dengan perbandingan 100 gram gips dan 24 ml air (aturan pabrik) diaduk menggunakan spatel di atas vibrator. adonan dimasukkan ke dalam kuvet yang telah disediakan di atas vibrator. kemudian master kuningan diameter 5 mm dan tebal 1 mm diletakan ditengah kuvet, gips dibiarkan sampai mengeras. setelah gips mengeras, permukaan gips diulasi vaselin dan kuvet atas dipasang, selanjutnya adonan gips diisi di atas vibrator dan dipres, dibiarkan sampai gips mengeras. selanjutnya kuvet dibuka, master model diambil sehingga diperoleh suatu cetakan (mould). pembuatan sampel dengan cara, mould yang diperoleh diulasi dengan bahan separator dan dibiarkan sampai kering. kemudian mencampur resin akrilik jenis rapid heat cured dengan perbandingan 4 gram polimer: 2 ml monomer dalam mangkuk porselen. setelah 5 menit adonan mencapai dough stage, adonan dimasukkan ke dalam mould sampai penuh dan ditutup dengan kertas selopan, selanjutnya kuvet lawan dipasang dan dipres dengan pres hidrolik bertekanan 50 kg/cm2. kemudian kuvet dibuka kelebihan resin akrilik dipotong. prosedur ini diulangi sampai tiga kali. resin akrilik dibiarkan selama 15 menit (aturan pabrik). selanjutnya, dilakukan penggodokan dalam air selama 20, 30 dan 40 menit pada suhu 100° c. setelah itu kuvet diambil dari pengodokan dan dibiarkan sampai dingin pada suhu kamar. selanjutnya sampel dikeluarkan dari kuvet, sisa gips yang menempel pada sampel akrilik dibersihkan.11 sampel yang didapat pada setiap kelompok dipotong dengan ukuran 5 × 5 m. jumlah sampel pada setiap kelompok perlakuan berjumlah 6 buah. kultur sel bhk-21 dalam bentuk cell-line ditanam dalam botol. setelah confluent, kultur dipanen dengan menggunakan larutan trypsine versene. hasil panen diambil sedikit dan ditanam kembali dalam media eagle yang mengandung 10% bovine serum albumin diinkubasi selama 24 jam. kemudian sel dipindahkan dalam botol kecil dan dibuat dengan kepadatan 2 × 105 sel/ml, sel tersebut siap digunakan untuk pengujian sampel. pengujian viabilitas sel pada resin akrilik menggunakan lempeng kultur sel 96 sumuran beralas datar. pengujian ini dilakukan sesuai standar protocol yang dianjurkan untuk esei mmt.12 pada setiap sumuran dimasukkan 200 μl media yang berisi sel dengan kepadatan 2 x 105 sel/ml. sebelum diuji sampel disterilkan terlebih dahulu dengan ultra violet selama 15 menit, selanjutnya sampel dimasukkan dalam lempeng sumuran. pada penelitian ini pengujian dilakukan secara duplo. kemudian lempeng sumuran diinkubasi selama 20 jam pada suhu 37° c. setelah itu, pada setiap sumuran dimasukkan 20 μl mtt yang telah dilarutkan dalam pbs, diinkubasi kembali selama 5 jam suhu 37° c. selanjutnya sampel diambil dari setiap sumuran, dan pada setiap sumuran ditambahkan 200 μl dmso dan dipipet naik turun untuk melarutkan kristal yang terbentuk. lempeng sumuran diinkubasi kembali selama 5 menit pada suhu 37° c. selanjutnya, lempeng sumuran dibaca pada spektofotometer dengan panjang gelombang 550 nm. hasil yang diperoleh dinyatakan dalam optical density (absorben). besar absorben setiap sumuran menunjukkan jumlah viabilitas sel dalam kultur media. 13, 14 hasil hasil penelitian tentang viabilitas sel fibroblast bhk21 pada permukaan resin akrilik jenis rapid heat cured terlihat pada tabel 1. pada tabel 1 terlihat bahwa resin akrilik dengan penggodokan selama 40 menit paling tinggi rerata jumlah viabilitas selnya, sedangkan resin akrilik dengan penggodokan selama 20 menit paling rendah rerata viabilitas selnya. data yang diperoleh homogen dan berdistribusi normal, diuji dengan one sample kolmogorov-smirnov test. untuk mengetahui perbedaan viabilitas sel fibroblast bhk-21 terhadap perlakuan resin akrilik rapid heat cured dilakukan perhitungan statistik 70 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 68–72 dengan menggunakan anova satu arah, didapatkan nilai p < 0,05. hasil analisis ini menunjukkan bahwa viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik rapid heat cured terdapat perbedaan yang bermakna. untuk menentukan ada perbedaan antar perlakuan dilakukan uji lsd, seperti terlihat pada tabel 2. tabel 1. rata-rata dan simpang baku viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik rapid heat cured (optical density) kelompok perlakuan jumlah sampel rata-rata ± simpang baku kontrol positif 6 0,881 ± 0,036 penggodokan selama 20 menit 6 0,612 ± 0,092 penggodokan selama 30 menit 6 0,626 ± 0,112 penggodokan selama 40 menit 6 0,694 ± 0,103 pada tabel 2, terlihat viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik jenis rapid heat cured yang diperpanjang lama polimerisasinya dibandingkan kelompok kontrol positif (sel dan media tanpa sampel) ada perbedaan yang bermakna, berarti terjadi penurunan viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik jenis rapid heat cured dibandingkan kelompok kontrol. bila polimerisasi resin akrilik jenis rapid heat cured diperpanjang 20, 30 dan 40 menit tidak ada perbedaan yang bermakna, berarti tidak terjadi penurunan viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik jenis rapid heat cured. pembahasan basis gigi tiruan dari resin akrilik yang berpolimerisasi dengan sistem pemanasan dan kimia merupakan pilihan untuk pembuatan gigi tiruan lengkap dan sebagian lepasan. dalam aplikasinya resin akrilik yang digunakan sebagai basis gigi tiruan akan kontak dan berada di dalam lingkungan rongga mulut, sehingga memerlukan penelitian tentang biokompatibilitas yang berhubungan dengan material tersebut. 13 uji viabilitas sel merupakan bagian dari uji toksisitas yang digunakan untuk mengevaluasi secara biologi efek suatu material kedokteran gigi yang diperlukan secara langsung terhadap jaringan dalam kultur sel untuk prosuder skrining standar yang direkomendasikan yang perhatian utama pada sifat iritasi lokal.8 bila dilihat dari tabel 1, hasil ini sama dengan yang dilaporkan oleh peneliti sebelumnya, meskipun metode yang digunakan untuk mendeteksi viabilitas sel berbeda.11 penelitian ini menggunakan uji enzimatik untuk menentukan viabilitas sel atau toksisitas suatu material dengan esei mtt {3-(4,5-dimethylthiaziol-2yl)-2,5-diphenyl-tetrazolium bromide}. esei ini banyak digunakan untuk mengukur proliferasi seluler secara kuantitatif atau untuk mengukur jumlah sel yang hidup. keuntungan esei ini adalah pengukuran akurat dan sensitif karena menggunakan alat spektrofotometer yang dapat mendeteksi perubahan metabolisme sel secara jelas, manipulasi mudah, peralatan yang digunakan biasa tersedia di laboratorium, menghemat waktu, tenaga, tidak menggunakan isotop radioaktif. 12 resin akrilik diproses dengan penggodokan selama 20 menit paling rendah jumlah viabilitas selnya. hal ini kemungkinan disebabkan pada pemanasan 20 menit masih tinggi kandungan monomer sisa yang ada dalam massa resin akrilik tersebut yang belum membentuk rantai polimer. metode polimerisasi resin akrilik yang digunakan di kedokteran gigi ada beberapa cara, misal cara konvensional (penggodokan dalam air), radiasi gelombang mikro dan sinar tampak. proses polimerisasi resin akrilik menggunakan cara konvensional sebaiknya penggodokan resin akrilik dalam air selama 90 menit pada suhu 70° c dilanjutkan 30 menit pada suhu 100° c.15 bila proses polimerisasi dilakukan dalam waktu singkat dan suhu rendah, proses polimerisasi tidak berjalan sempurna, kandungan monomer sisa masih tinggi.3 terjadinya monomer sisa dipengaruhi oleh perbandingan pencampuran antara bubuk dan cairan resin akrilik dari setiap jenis bahan dan prosedur prosesing resin akrilik.14 hasil suatu penelitian, menyimpulkan kandungan monomer sisa dari resin akrilik jenis rapid heat cured paling tinggi pada penggodokan 20 menit dan paling rendah pada penggodokan selama 40 menit.5 hal yang sama juga telah dilaporkan oleh peneliti lain, kandungan monomer sisa dari resin akrilik yang di proses selama 20 menit pada suhu 100° c paling tinggi dibandingkan yang diproses pada suhu tinggi dan waktu lama.14 tabel 2. uji lsd viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik rapid heat cured kelompok perlakuaan kontrol positif penggodokan 20 menit penggodokan 30 menit penggodokan 40 menit kontrol positif − s s s penggodokan 20 menit − ns ns penggodokan 30 menit − ns penggodokan 40 menit − keterangan: ns = tidak bermakna; s = bermakna 71yuliati: viabilitas sel fibroblas bhk-21 resin akrilik yang digodok selama 40 menit paling tinggi viabilitas sel dibandingkan dengan perlakuan yang lain. hal ini kemungkinan disebabkan kandungan monomer sisa menurun. kandungan monomer sisa resin akrilik akan menurun bila proses polimerisasi lama dan suhu penggodokan ditingkatkan.16 viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik jenis rapid heat cured dari penelitian ini berbeda bermakna dibandingkan kelompok kontrol (tabel 2). keadaan ini membuktikan bahwa viabilitas sel dipengaruhi oleh sesuatu komponen yang terlepas dari resin akrilik. komponen yang terlepas tersebut kemungkinan adalah monomer metil metakrilat yang tidak bisa terpolimerisasi secara sempurna. hal ini sesuai yang dikatakan oleh peneliti sebelumnya, bahwa proses polimerisasi tidak pernah berjalan sempurna. tidak terpolimerisasinya monomer akan tertinggal dalam polimer sesudah proses kuring.17 komponen dari resin akrilik misal: dimetakrilat, metil metakrilat, asam benzonat, dan formaldehid merupakan komponen toksik yang dapat terlepas dari resin komposit menyebabkan efek toksisitas.13 metil metakrilat merupakan komponen utama monomer resin akrilik kemungkinan mempengaruhi viabilitas sel pada penelitian ini. monomer metil metakrilat memperlihatkan tanda toksik pada monosit, granulosit dan sel endotel pada penelitian in vitro setelah diinkubasi satu menit pada dosis 10 mg ml-1.18 evalusi dari berbagai merek dagang basis gigi tiruan, eluates dari resin akrilik mempunyai efek toksisitas terhadap sel epitel rongga mulut, gingival fibroblast manusia. efek ini dapat menghambat pertumbuhan sel, replikasi dna, sentesis rna, dan proses metabolik sel.13 evaluasi mikroskop pada uji mtt esei terlihat, sel gingival dan periodontal ligamen fibroblas yang terpapar methyl methacrylate (mma), isobutyl methacrylate (ibma) dan 1,6-hexanediol dimethacrylate (1,6-hdma) setelah inkubasi 24 jam memperlihatkan karakteristik morfologi sel menjadi tidak normal. sel terlihat pyknosis, membulat, membengkak, batas membran sel tidak teratur dan kehilangan perlekatan pada lempeng mikrotiter. mtt esei dapat menentukan aktivitas dehidrogenase mitokondria sel yang digunakan sebagai indikator dari viabiltas sel.19 bila ditinjau dari hubungan antara struktur monomer dan toksisitas dapat disimpulkan bahwa kelompok hidroksil dan metakrilat nampaknya akan meningkatkan toksisitas, dimetakrilat dengan 23 rantai oxyethylene memperlihatkan tingkat toksisitas yang tinggi daripada dimetakrilat dengan 14 rantai oxyethylene, efek toksisitas dari monomer berkorelasi dengan logarithm dari koefisient oktanol/air (log p).20 resin akrilik jenis rapid heat cured yang proses polimerisasi diperpanjang dari anjuran pabrik, viabilitas sel yang terdeteksi tidak ada perbedaan yang bermakna. tidak terjadi penurunan viabilitas sel fibroblast bhk 21 pada permukaan resin akrilik. toksisitas dapat dihubungkan dengan viabilitas sel yang merupakan faktor utama biokompatibilitas suatu material di kedokteran gigi yang secara umum ditentukan dengan uji sel kultur secara in vitro. dibandingkan dengan penelitian secara in vivo, pada penelitian secara in vitro lebih mudah dikontrol, sistem parameter penilaian yang digunakan lebih sederhana, meminimalkan variabel confounding, dan penentuan mekanisme toksisitas lebih spesifik. meskipun hasil penelitian secara in vitro secara kuantitatif tidak dapat dikorelasikan dengan hasil in vivo. beberapa klinikus melaporkan ada toksisitas jaringan ketika jaringan tersebut terpapar komponen yang terlepas dari resin akrilik yang terpolimerisasi. jaringan rongga mulut secara langsung akan kontak secara in situ oleh resin akrilik yang terpolimerisasi sehingga mendapatkan konsentrasi bahan kimia tertinggi. hal ini akan berpengaruh terhadap kerusakan jaringan yang lebih besar.19 oleh karena itu metode yang digunakan untuk mengurangi substansi yang terlepas dari basis gigi tiruan yang baru diproses sebelum digunakan dalam rongga mulut, sebaiknya gigi tiruan direndam dalam air paling sedikit satu hari atau direndam dalam air panas suhu 50° c.14, 21 hasil penelitian ini dapat disimpulkan tidak terjadi penurunan viabilitas sel fibroblast bhk-21 pada resin akrilik jenis rapid heat cured bila lama polimerisasi diperpanjang dari 20 menit. daftar pustaka 1. craig rg, powers jm. restorative dental materials. 11st ed. st louis, mo, mosby; 2002. p. 636–89. 2. parvizi a, linquist t, schneider r, williamson d, boyer d, dawson dv. comparasion of the dimensional accuracy of injectionmolded denture base materials to that of conventional pressurepack acrylic resin. j prosthodont 2004; 13 (2): 83–9. 3. combe ec. notes on dental materials. 6th ed. new york: churchil livingstone; 1992. p. 158–60. 4. harrison a, huggett r. the effect of curing cycle on residual monomer level of acrylic resin denture base polymer. j prosthet dent 1992; 20: 370–74. 5. intan nirwana. kandungan monomer sisa pada resin akrilik rapid heat cured dengan proses kuring berbeda. majalah kedokteran gigi 2001; 34(3): 119–21. 6. hensten, petterson a, yacobson n. preceived side effect of biomaterials in prosthetic dentistry. j prosthet dent 1991; 65:138– 44. 7. taira m, nakao h, matsumoto t, takahashi j. cytotoxic effect of methyl methacrylate on 4 cultured fibroblasts. int j prosthodont 2000; 13: 311–15. 8. anussavice kj. phillips' science of dental materials. 11st ed. elsevier science (usa) saunders; 2003. p. 172–94. 9. schmalz g. the biocompatibility of non-amalgam dental filling materials. eur j oral sci 1998; 106: 696–706. 10. rose ec, bumann j, jonas ie, kappert hf. contribution to the biological assessment of orthodontic acrylic materials. measurement of their residual monomer output and cytotoxicity. j orofac orthop 2000; 61(4): 246–57. 11. intan nirwana. sitotoksisitas resin akrilik rapid heat polymerized terhadap kultur sel bhk. majalah kedokteran gigi 2004; 37(1): 15–8. 72 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 68–72 12. dash p. standard protocols mtt assay. 2002. avalaible http:// web.bham.ac.uk/can4psd4/brum/mtt.html. accessed 8/1/2002. 13. sheridan pj, koka a, ewoldsen no, lefebre ca, lavin mt. cytotoxicity of denture base resins. int j prosthodont 1997; 10: 73–7. 14. kedjarune u, charoenworaluk n, kootongkaew s. release of methyl methacrylate from heat-cured and autopolymerized resins: cytotoxicity testing releated to residual monomer. australian dental journal. 1999; 44: (1) 25–30. 15. de clerek jp. microwave polymerization of acrylic resin used in dental prostheses. j prosthet dent 1987; 57: 650–8. 16. dogan a, bek b, cevik nn, usanmaz a. the effect of preparation conditions of acrylic denture base materials on the level of residual monomer, mechanical properties and water absorption. j.dent 1995; 23: 313–8. 17. kostoryz el, eick jd, glaros ag, judy bm, welshons wv, busmater s and yourtee dm. biocompatibility of hydroxylated metabolisme of bisgma and bfdge. j dent res 2003; 82: (5) 367–71. 18. dahl oe, garvik lj, lyberg t. toxic effects of methylmethacrylate monomer on leukocytes and endothelial cells in vitro. acta orthop scan 1994; 65: 147–53. 19. lai y, chen yt, lee sy, shienh tm, hung sl. cytotoxic effects ofdental resin liquids on primary gingival fibroblasts and periodontal ligament cells in vitro. j oral rehab 2004; 31 (12): 1165–72. 20. yoshi e. cytotoxic effects of acrylates and methacrylates : relationships of monomer structures and cytotoxicity. j biomed mater res 1997; 37: 517–24. 21. tsuchiya h, hoshino y, tajima k and takagi n. leaching and cytotoxicity offormaldehyde and methyl maethacrylate from acrylic resin denture base materials. j prosthet dent 1994; 71(6): 618–24. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no4-2005-isi.pmd 201 the graptophyllum pictum extract effect on acrylic resin complete denture plaque growth endang wahyuningtyas department of prosthodontics faculty of dentistry gadjah mada university yogyakarta indonesia abstract graptophyllum pictum, in indonesian is named ‘daun ungu’, is one of the traditional plants usually used as haemorrhoids medicament in indonesia; it is composed from alkaloid, pectin, saponin, tannin, flavonoid and alcohol. graptophyllum pictum is able to prevent the growth of streptococcus mutans. the purpose of this investigation is to study the effect of graptophyllum pictum extract toward the growth of plaque on acrylic resin complete denture. for this plaque growth research, 40 samples of acrylic resin complete denture were used, which divided into four groups. those samples were first treated by cleansing each denture from saliva with running water. the existence of plaque on the complete denture was known by means of disclosing agent. the plaque was then scored by using modified quigley hein index, in this case until the plaque was scored zero. the second treatment was soaking the group i to iv denture samples in graptophyllum pictum extract with various concentration 5%, 10%, 20% and 40%, each for 15 minutes. following this, the dentures were worn by the patients again for four hours, and then removed and washed with water. then the disclosing agent were rubbed on the dentures and subsequently washed with water. the obtained data were analyzed by one-way variant analysis and t-test. the study result showed there were significant differences among the growth of plaque on acrylic resin complete denture which was soaked in different concentration of graptophyllum pictum extract (p < 0.05). regarding this result it can be concluded that graptophyllum pictum extract could inhibit the plaque growth on acrylic resin complete denture. and it seems also that the highest plaque growth inhibition on acrylic resin complete denture is caused by the 40% concentration extract of graptophyllum pictum. key words: graptophyllum pictum extract, plaque, acrylic resin complete denture correspondence: endang wahyuningtyas, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii sekip utara, yogyakarta 55281, indonesia. introduction metacrilate polymethyl acrylic resin is a common material widely-used in the construction of complete denture bases. acrylic resin has a good aesthetic property, could match the colour of the substituted mouth tissue naturally, stable form, besides would not cause irritation, non-toxic, and is easy to fabricate and manipulate, as well as to repair if broken.1 most of the acrylic resin’s defect are caused by the remaining monomer, water absorbent as well as porosity.2 while wearing an acrylic resin complete denture, the mucosa underneath will be covered by the device for a long time, this matter will hinder the cleansing of the denture surface facing the mucosa, by tongue and saliva.3 the supporting mucosa tissue of the acrylic resin complete denture, will alter. and this matter related very much to the amount of plaque formation at the fitting surface. plaque is a soft non mineralized deposit, originated from bacteria attached to an adhesive matrix which is form-by saliva-glycoprotein and extra cellular bacteria on teeth as well as dentures surfaces. this plaque’s matrix is comprised of 80% water and 20% solid substances.4 the denture’s plaque is formed due the long wearing within a certain period of time. the plaque structure on a denture has the same basic composition as those found at an natural teeth.5 denture plaque is the source of periodontal diseases, bad breath odour, changing the denture colour and cause denture stomatitis as well, an infection of the mucosa tissue especially underneath the denture.6 streptococcus mutans is the greatest amount of bacteria found in the dental plaque, because this is their main habitat. they multiply and colonized on the teeth surfaces forming dental plaque.7 because that matter acrylic resin complete denture wearers should attend the hygienic condition by keeping the oral and dentures clean. the purpose of routine denture cleansing is to avoid plaque growth and prevent their accumulation as well as mucin and calculus deposits.5 generally denture cleansing can be done by mechanical or chemical method. mechanical cleansing is done by brushing the denture with a soft tooth brush and chemical cleansing is by soaking it into a denture cleansing or disinfectant solution especially for that purpose.8, 9 graptophyllum pictum, indonesian named ‘daun ungu (violet leave)’ is one of the traditional herbal plants 202 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 201–204 commonly grow in indonesia as shrubs. graptophyllum pictum has pharmacological specialty for various health problems, such as: to release anuria, constipation, haemorrhoids, maturing boil process, weak laxative for purgation, skin softener and to enhance menstrual blood flow.10 graptophyllum pictum consist of chemical content: alkaloid, pectin, and formic acid. it contains also steroid, saponin, tannin, flavonoid and alcohol.11 tannin can be locally applied on wounds at the throat and oral cavity, the later especially in stomatitis. tannin has a physiological action against bacteria growth.12 as a detoxification agent, tannin can precipitate protein and form a specific compound interacting with protein and saliva pellicle13 to inhibit the attachment of streptococcus mutans as well as reducing it.7 alkaloid has a physiological action against bacteria’s growth.12, 14 flavonoid is a phenol compound, that can dissolve in water, and can be extracted by 70% ethanol. flavonoid has an antiviral, antibacterial and anti-inflammation character as well. the general characteristic of phenolic compound is able to increase cell permeability to form a complex compound with protein by hydrogen bond.15 graptophyllum pictum extract can inhibit the growth of streptococcus mutans bacteria.16 based on those backgrounds, arouse problem questions, whether graptophyllum pictum influencing the plaque formation on acrylic resin complete denture or not. this is stated in the purpose of this study. hopefully this study may give the information whether graptophyllum pictum extract is suitable as a cleansing agent for acrylic resin complete denture. materials and method in this study, extract of graptophyllum pictum was made by means of: 100 grams dried old leaves of graptophyllum pictum, grounded to powder form, and then extracted in 70% ethanol with sohlet instrument for 2–3 hours until the process of extraction finished. the extract solution was concentrated with a vacuum rotary evaporator to obtain a dry extract form. the graptophyllum pictum extract tester solution was made of 5 grams main extract added with distillate aqua until 100 ml volume was reached, and then shook until homogenously mixed, and a 5% tester solution was obtained. to obtain 10%, 20% and 40% tester solution, the procedure was the same, except the quantity of graptophyllum pictum extract was increased respectively 10, 20 and 40 grams. the plaque accumulations on the surface of acrylic resin complete dentures were observed by clinical research method. the subjects observed consisted of complete upper dentures (cud) wore by patients at the prosthodontic clinic, faculty of dentistry, gajah mada university with the following criterion: minimally the cud had to be worn for 1 month. the patient’s age were about 40 years old or more, and did not suffer of any systemic diseases. in this case forty research subjects were examined. the first treatment of the study was the plaque examination, which attached on the complete denture by rubbing disclosing agent. each cud was taken out from the patient’s mouth and washed with running water until the saliva disappeared. the disclosing agent was rubbed to the cud surface evenly by means of cotton buds which was soaked in before, and then washed under running water and subsequently scored by modified quigley hein index. the cud should be cleansed from the disclosing agent solution, and the plaque was scored until reaching zero. the 40 research subjects in the second treatment were divided into four groups, where each consisted of 10 study subjects. the complete dentures in the first group were soaked in 5% graptophyllum pictum extract solution, the second group soaked in 10% solution, the third soaked in 20% and the last group soaked in 40% solution. each complete denture was soaked for 15 minutes. then the complete denture was removed out of the extract solution, brushed with a toothbrush using the soaking liquid. the patient wore back the denture for 4 hours. after 4 hours, the cud was taken out, and washed with water until the saliva disappeared. the disclosing agent was rubbed on its surface, then washed off with water and scored by modified quigley hein index. the plaque accumulation on the cud surfaces were divided into 8 location parts, namely: 4 location on the buccal surface of cud as follows: a= right posterior buccal surface; b= right anterior buccal surface; c= left anterior buccal surface and d= left posterior buccal surface. and the other 4 location parts were on the fitting surface of the cud (fit-surface facing to the palatum mucosa) as follows: e= right posterior fitting surface; f = left posterior fitting surface; g= right anterior fitting surface, and h= left anterior fitting surface. the counting of the plaque was scored according to modified quigley hein index: 0 score = no plaque; 1 score = light plaque, denoting that 1%-25% of the area was covered with plaque; 2 score = moderate plaque, 26%-50% of the area was covered with plaque; 3 score = heavy plaque: 51%-75% of the area was covered with plaque; 4 score = very heavy plaque: 76%-100% of the area was covered with plaque. cud plaque score the total plaque score at 8 scored areas (a–h) 8 = the obtained data was analyzed by one-way variant-analysis and t-test. 203sadamori: comparison of recognition about denture adhesive figure 1. graptophyllum pictum (daun ungu).plant results the average counting result of the plaque growth at the acrylic resin complete denture in each graptophyllum pictum extract soaking group can be seen in table 1. table 1. the average and standard deviation result of plaque formation after soaking in different graptophyllum pictum extract concentration concentration n average standard deviation 5% 10% 20% 40% 10 10 10 10 2.729 1.925 1.166 0.425 0.002 0.008 0.007 0.005 it was showed in table 1 that the highest plaque growth was in 5% graptophyllum pictum extract, namely 2.729 ± 0.002, while the lowest plaque growth average was in 40% concentration, namely 0.425 ± 0.005. to know the result differences between each study group, a one-way variant analysis was performed, which will show the significant difference of each graptophyllum pictum extract concentration to the plaque growth on the acrylic resin complete denture (p < 0.05). a t-test was conducted to know the inter group differences, as seen in table 2. the t-test result in table 2 showed a significant difference among each different graptophyllum pictum extract concentration soaking group, namely of 5% and 10% concentration, 5% and 20%, 5% and 40%, 10% and 20%, 10% and 40%, as well as 20% and 40% to the plaque growth existence on the acrylic resin complete denture (p < 0.05). table 2. the t-test result of each inter group which cud were soaked in different concentration of graptophyllum pictum extract concentration 5% 10% 20% 40% 5% 10% 20% 40% 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* discussion the result of the study showed the highest plaque growth inhibition were found in those soaked in 40% graptophyllum pictum extract solution, and the lowest inhibition were those soaked in 5% concentration solution. the active composition content of graptophyllum pictum were tannin and flavonoid.11 tannin had the antibacterial power to inhibit glucose growth from streptococcus mutans, so that it was able to get hold of the plaque growth.7 based on those findings, presumably the low inhibiting effect result of the 5% graptophyllum pictum extract solution, was caused by the low content of tannin and flavonoid, so that the solution was not effectively enough to inhibit. the 40% graptophyllum pictum extract solution content higher tannin and flavonoid, so that it could function more effectively in the inhibition of the cud‘s plaque growth. the outcome of this study was matched with kozai et al finding,17 who explained that tannin could inhibit the formation of insoluble glucan from sucrose by glucotransferase which had an important role in the plaque growth. the one-way anova test result showed there was a significant difference in the graptophyllum pictum extracts various concentrations to the plaque growth at acrylic resin complete denture (p < 0.05). the t-test result showed also a significant difference among acrylic resin complete dentures soaking in various graptophyllum pictum extract solution groups to the existence of plaque growth (p < 0.05). result of the study showed that graptophyllum pictum which content tannin and flavonoid has the ability to disrupt protein forming plaque. these study results was cause by tannin and flavonoid which was able to destroy protein, so that it can inhibit the plaque growth. the higher concentration of graptophyllum pictum extracts solution, the more will be the amount of tannin and flavonoid, and consequently the inhibiting power will be higher to the plaque growth. these findings matching to pelczar and chan13 opinion that tannin could interact with protein and saliva pellicle to cause protein precipitation. following wu yuan et al.7 tannin as a detoxification agent could precipitate protein and form a certain compound, that interact with protein and saliva pellicle to avoid attachment of streptococcus mutans and lessen the bacteria’s as well. 204 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 201–204 the statement support the findings that graptophyllum pictum extract could inhibit the growth of streptococcus mutans.16 harborne15 wrote that flavonoid was able to form a complex compound with protein by hydrogen bond, so that this protein precipitation could inhibit plaque growth. based on the study findings of graptophyllum pictum extract solution on the plaque growth at the surface of cud, a conclusion could be drawn as follows: the 40% concentration of graptophyllum pictum extract had the highest plaque growth inhibition action on the denture. so graptophyllum pictum extract solution can be suggested to be used as an alternative material for cleansing acrylic resin dentures. references 1. phillips rw. skinner’s science of dental materials. 9th ed. philladelphia: wb saunder co; 1991. p. 199-204. 2. combe ec. notes on dental materials. 6th ed. edinburg: churchill livingstone. 1992; p. 79-120, 157-61. 3. basker rm, davenport jc, tomlin, hr. prosthodontics treatment for edontulous patient. 1 st ed. london: the macmillan press; 1976. p. 163-5. 4. roth gi, calmes r. oral biology. st louis: the cv mosby co; 1981. p. 329-32, 369-71. 5. jorgensen eb. materials and methods for cleaning dentures. j prosthet dent 1979; 42(6):619-22. 6. abelson dg. denture plaque and denture cleanser. j prosthet dent 1981; 42:376-9. 7. wu yuan cd, chen cy, wu rt. gallotanins inhibit growth, water insoluble glucan synthesis, and aggregation of mutans sreptococcoci. j dent res 1988; 1:51-5. 8. dills ss, olshan am, goldner s, brogdon s. comparison of the antimicrobial capability of an abrasive paste and chemical soak denture cleanser. j prosthet dent 1988; 60:467-70. 9. woodall jr. comprehensive dental hygiene care. 2nd ed. st louis: cv mosby co; 1985. p. 204-9. 10. setiawan d. atlas tumbuhan obat indonesia. jilid 1. yogyakarta: trubus agriwidya; 1999. h.16-7. 11. thomas ans. tanaman obat tradisional ii. yogyakarta: kanisius; 1992. h. 9-10. 12. martin ew, cook ef. remingtons practice on pharmacy. 12th ed. new york: mack publishing co; 1961. p. 67-9. 13. pelczar mj, chan ecs. 1988. dasar-dasar mikrobiology. hadioetomo rs, dkk. jakarta: penerbit universitas indonesia; 1988. h. 456-8. 14. jawetz em. review of medical microbiology. 16th ed. san francisco: longo medical pub; 1986. p. 143-8, 297-9. 15. harbone. denture plaque distribution and the effectiveness of denture cleaner. quintessence int 1987; 27:341-5. 16. wahyuningtyas e, indrastuti m. pengaruh ekstrak graptophylum pictum terhadap pertumbuhan bakteri streptococcus mutans pada resin akrilik. maj ked gigi (dent j); edisi khusus temu ilmiah nasional iv 2005:298-301. 17. kozai k, shoto m, yamaguchi m, nagasaka n, pradopo s. potential of gambir as an inhibitor of dental plaque formation. maj ked gigi (dent j)1995; 28:(5):95-6. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 74 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 74–77 original article the severity and direction prevalence rate of patients with a mandible deviation compared to cobb’s angle kimberly clarissa oetomo1, i gusti aju wahju ardani1, thalca hamid1 and komang agung irianto2 1department of orthodontics, faculty of dental medicine, universitas airlangga 2department of orthopaedics and traumatology, faculty of medicine, universitas airlangga surabaya – indonesia abstract background: patients with mandible deviation often have idiopathic scoliosis, which might affect the result of orthodontic and orthopaedic treatment. orthodontic treatment not only focuses on aesthetic and functional but also orthopaedic stability. a thorough examination is needed to obtain orthopaedic stability by evaluating the occlusion and posture to establish the best strategy of treatment and interdisciplinary approach. purpose: this study was conducted to assess the correlation between mandible deviation and idiopathic scoliosis. methods: this is a descriptive-analytic study with a cross-sectional approach. from 60 samples, 35 patients were chosen based on the inclusion criteria of the total sampling technique. patients were referred to have skull posteroanterior (pa) and a thoracolumbar pa radiograph taken. skull pa radiographs were analysed with grummon’s method using the orthovision program. cobb’s angle analysis was used by the radiologist to analyse the thoracolumbar pa radiographs. the data gathered was then further analysed using the spearman test and the crosstabs test, using spss 23.0. results: correlation between mandible deviation and the severity of idiopathic scoliosis is not significant (p=0.866). the direction prevalence of mandible deviation towards cobb’s angle is 54.3% to the right and 45.7% to the left. all patients with mandible deviation have cobb’s angle. conclusion: there is no correlation between mandible deviation and the severity of idiopathic scoliosis. however, many cases showed that the direction of mandible deviation and of idiopathic scoliosis is the same. keywords: cobb’s angle; idiopathic scoliosis; mandible deviation correspondence: kimberly clarissa oetomo, department of orthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: kimi_ko7@yahoo.com introduction mandibular deviation is a craniofacial deformity with a lateral shift of its midline.1 mandibular deviation with a lateral shift may affect the oral function and body appearance. individuals with lateral mandible deviation often have a temporomandibular joint disorder (tmd) because of the asymmetrical occlusion.2 if this prolonged, more severe tmd, facial asymmetry, and an imbalance of musculature might occur, affecting the body’s symmetrical coordination and balance. previous studies have shown that patients with mandible deviation often have abnormal cervical vertebrae morphology.3–5 a high prevalence of orthopaedic pathological findings is also reported in patients who need an orthodontic treatment because of the occurrence of mandible deviation; for example, 91% of the group examined by hirschfelder and hirschfelder, and 83% of patients of 420 patients investigated by muller-wachendorf.6–8 scoliosis is the orthopaedic pathology that defines a three-dimensional deviation of the spinal axis. this might be linked indirectly to some mild forms of mandible deviation in the transversal dimension.9 idiopathic scoliosis is an orthopaedic condition characterised by inadequate posture, which is progressive and often revealed in childhood, common in children ten years or older.10 worsened scoliosis might occur during growth. scoliosis management is often delayed by a lack of awareness among patients and parents.11 the prevalence of idiopathic scoliosis is 0.47% to 5.20% around the world.12 the prevalence rate of idiopathic scoliosis in surabaya, indonesia, is 2.94%.10 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p74–77 mailto:kimi_ko7@yahoo.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p74-77 75oetomo et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 74–77 the possibility of mandible deviation and scoliosis being related offers a new perspective to the orthodontist and orthopaedist in examining patients. this study investigated the relationship between mandible deviation and idiopathic scoliosis. materials and methods a total of 35 patients were chosen from 60 subjects from a private clinic and the orthopaedic and traumatology hospital surabaya, which fulfilled the inclusion criteria. the inclusion criteria were: 1) patient with mandible deviation, 2) no orthopaedic surgery procedures, 3) no orthodontic treatment, 4) age 9–35 years old, 5) no gender limitation. the exclusion criteria were: 1) have a history of facial trauma, 2) have a spinal trauma history, 3) had had orthognathic surgery. this study was ethically approved by universitas airlangga faculty of dental medicine health research ethical clearance commission (680/ hrecc.fodm/x/2019), and written informed consent was obtained from the patients. patients were asked to sign an informed consent form that included an agreement to allow their data to be used for research. they also agreed to have their thoracolumbar posteroanterior (pa) and skull pa x-rayed. tracing was performed using the orthovision program’s version of grummon’s (linear asymmetry) analysis (figure 1).13 the horizontal reference line (hrl) is a line connecting the right and left lateral orbital (lo). the vertical reference line (vrl) is defined as the perpendicular bisector of the right and left lateral orbital. menton (me) is the point of the chin. the direction of the mandible deviation can be seen from the me’s position towards the vrl. cobb’s angle is formed between the upper-end plate on the superior end and the lower endplate on the spinal curve’s inferior-end vertebrae. the curve’s direction was also determined as to whether the endpoint of the curve’s position is on the right or left of the midline of the vertebrae (figure 2). when cobb’s angle is more than 10°, it can be concluded that the subject has scoliosis. 9,14 all data were evaluated with a normality test. the spearman test was performed to investigate the correlation between the severity of mandible deviation and cobb’s angle (scoliosis). the crosstabs test was performed to investigate the prevalence of mandible deviation direction against cobb’s angle. the statistical package for social sciences (spss) (ibm, new york, usa) version 23.0 for windows was used to conduct the statistical analysis. results this study was performed on a total of 35 patients chosen from 60 subjects (6 males and 29 females) aged 9–35 years old. a total of 14 subjects out of 35 have a mandible deviation to the right. the other 21 patients have a mandible deviation to the left. of these, 19 patients have scoliosis to the right and 16 to the left. a one-sample kolmogorov– smirnov test was performed on the me deviation degree (p=0.531; normal) and cobb’s angle (p=0.035; abnormal), to discover any correlation between the severity of the two variables. from the spearman test, the correlation between me deviation and cobb’s angle is not significant (p=0.866), table 1. cobb’s angle direction totalright percentage left percentage me deviation towards vrl direction right 14 100% 0 0% 100% left 5 23.8% 6 76.2% 100% total 19 54.3% 16 45.7% 100% figure 1. skull pa analysis using orthovision program (3.07° deviating to the right). figure 2. thoracolumbar pa analysis with cobb’s angle method (left).14 subject’s thoracolumbar pa (right) (10° cobb’s angle to the right). the direction prevalence rate of patients with me deviation towards cobb’s angle dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p74–77 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p74-77 76 oetomo et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 74–77 showing no correlation. table 1 shows that 100% of the subjects with mandible deviation to the right have a right scoliosis curve direction. 23.8% of the patients with mandible deviation to the left have a right curve direction, and 76.2% of the subjects with mandible deviation to the left have a left curve direction. discussion analysis of mandible deviation can be conducted using a skull pa. mandible deviation may lead to temporomandibular joint disorders, imbalanced strength of craniofacial muscles, and the symmetry, coordination, and balance of other muscles.15 a previous study reported by sambataro et al.16 revealed that patients with mandible deviation have abnormal cervical vertebrae and spine, as it interferes with upright posture stability on an unstable platform. this suggested that changes in the stomatognathic system affect posture and balance. vertebrae and muscles work together to make the head posture stable and play a role in head movement.16 more female patients were considered as females are more prone to idiopathic scoliosis.17,18 this phenomenon is caused by the fact that more of the oestrogen hormone can be found in females. oestrogen significantly increases the incidence of the scoliotic curve and the curve severity, showing that oestrogen is a factor that contributes to idiopathic scoliosis.17 idiopathic scoliosis needs to be treated as soon as possible, as it progresses and becomes more severe.12,19,20 this study showed no significant correlation between mandible deviation and scoliosis (cobb’s angle). the correlation in this research could be positive between scoliosis and mandible deviation. another possibility is that scoliosis is associated with the severity of mandibular deviation. some patients had mild-to-moderate scoliosis, which then shows no apparent correlation between scoliosis and mandible deviation. it needs to be noted that in approximately 10% of subjects, cobb’s angle will get more severe as the individual grows and may reach 58–100%.14,17 scoliosis is a three-dimensional deformity, whereas the thoracolumbar pa that was used is a two-dimensional presentation. thus, the spinal curve might be 20% more severe than the radiograph. a distortion of 2°–7° can be found.21 to prevent this, elimination of the aetiologies needs to be conducted, as otherwise this progress cannot be avoided this study also showed the direction prevalence where 100% of the patients with mandible deviation to the right have cobb’s angle to the right (curve direction). many factors might affect this result. there is a possibility that the patient might have unilateral chewing habits where the masticatory muscles are often asymmetrical, producing asymmetrical stress distribution in the mandible. evidence of this can be seen from the depth of the mandibular notch from the skull pa. this condition produces an asymmetrical stress distribution on the cervical spine and leads to the spine deformation.22 the presence of a chewing side preference increases muscle misfit.5 cervical muscles, the sternocleidomastoid and the trapezius work together, connecting the spine, cervical spine, and mandible. patients with right side dominance are more prevalent; they tend to chew on the right side and use the right limbs for activities, which result in the shortening of the muscles on the right side.3,5,22 this condition produces cobb’s angle to the right. the occurrence of an asymmetrical condyle may also play a role in mandible deviation. imbalanced occlusion in patients with mandible asymmetry causes abnormal load distribution on articular surfaces and the remodelling of the condyle. 23 thus, internal derangement and temporomandibular joint disorders might occur. condyles play a significant role as the centre of the mandible’s growth and as a pivot for mandible rotation. as the centre of growth, the condyle remodelling process occurs to respond to continuous stimulation during the mandible movement. surface morphology and bone density are related to mandible asymmetry pathogenesis and imbalanced force distribution on both occlusal surfaces.24 a study by oh et al.25 suggested a correlation between mandible deviation and condyles’ asymmetry. the possibility of occlusal cant occurrence in mandible deviation patients also leads to idiopathic scoliosis. occlusal cant may also lead to mandible deviation, as asymmetrical occlusal guidance takes place. occlusal cant causes a unilateral chewing habit. as a reaction, the side that is used more for chewing will receive higher occlusal force, preventing teeth eruption on that side.25,26 this situation then causes cranial strain that results from unilateral muscle hypertrophy where compression on c1 (atlas) and c2 (axis) can be found. formation of cobb’s angle or idiopathic scoliosis can be expected.5,6 most of the patients have a mandible deviation in the same direction as cobb’s angle. this can be caused by damaging habits such as unilateral mastication during the growth period; thus, the mandible’s side that received the greater load grows more and deviates to the opposite side. this corresponds to previous studies showing that abnormal morphology of vertebrae is related to mandible deviation.3,22 more patients have cobb’s angle formation to the right (54.3%). this corresponds to previous studies by grauers et al.,12 choudhry et al.,17 and blom et al.20, which suggested that the most common deformity is cobb’s angle to the right. side dominance, a preference or hand-differences in task performance and represents an expression of the brain’s motor cortex asymmetry, might play a role as righthanded patients are more prevalent. stronger superficial extrinsic back muscles can be found on the dominant side. here, right side dominance is more frequent and leads to a spinal curve to the right.5 this study has limitations, such as the screening procedures used, which only use a radiographic approach, and only a small sample of patients dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p74–77 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p74-77 77oetomo et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 74–77 was involved. further research involving muscle activity, sample grouping based on the severity of scoliosis, age, gender, type of scoliosis (using lenke’s classification), cone-beam computed tomography (cbct) usage, and a larger sample size is suggested. in conclusion, this study showed a positive correlation between the direction of mandible deviation and that of idiopathic scoliosis. the same direction between mandible deviation and cobb’s angle formation can be found. the more frequent direction of scoliosis and mandible deviation is to the right. there is no correlation between the severity of mandible deviation and idiopathic scoliosis. references 1. lin h, zhu p, lin y, wan s, shu x, xu y, zheng y. mandibular asymmetry: a three-dimensional quantification of bilateral condyles. head face med. 2013; 9(1): 42. 2. thiesen g, gr ibel bf, freitas mpm, oliver dr, k im k b. mandibular asymmetries and associated factors in orthodontic and orthognathic surgery patients. angle orthod. 2018; 88(5): 545–51. 3. de la madrid fajardo v, morales garfias f, ondarza rovira r, justus doczi r, garcía-lópez s. influence of an occlusal imbalance in the deviation and alignment of the vertebral spine in rats: a controlled trial. rev mex ortod. 2016; 4(1): e23–9. 4. zhou s, yan j, da h, yang y, wang n, wang w, ding y, sun s. a correlational study of scoliosis and trunk balance in adult patients with mandibular deviation. plos one. 2013; 8(3): e59929. 5. arienti c, buraschi r, donzelli s, zaina f, pollet j, negrini s. trunk asymmetry is associated with dominance preference: results from a cross-sectional study of 1029 children. brazilian j phys ther. 2019; 23(4): 324–8. 6. pacel la e , da r i m, g iova n non i d, cat er i n i l , mez io m. the relationship between temporomandibular disorders and posture: a systematic review. webmed cent orthod. 2017; 8(11): wmc005374. 7. hirschfelder u, hirschfelder h. effects of scoliosis on the facial bones. fortschr kieferorthop. 1983; 44(6): 457–67. 8. müller-wachendorff r. untersuchungen über die häufigkeit d e s au f t r e t e n s vo n g e b i ß a n o m a l ie n i n ve r b i n d u ng m it skelettdeformierungen mit besonderer berücksichtigung der skoliosen. fortschr kieferorthop. 1961; 22(4): 399–408. 9. cheng jc, castelein rm, chu wc, danielsson aj, dobbs mb, grivas tb, gurnett ca, luk kd, moreau a, newton po, stokes ia, weinstein sl, burwell rg. adolescent idiopathic scoliosis. nat rev dis prim. 2015; 1: 15030. 10. komang-agung is, dwi-purnomo sb, susilowati a. prevalence rate of adolescent idiopathic scoliosis: results of school-based screening in surabaya, indonesia. malaysian orthop j. 2017; 11(3): 17–22. 11. peng y, wang s-r, qiu g-x, zhang j-g, zhuang q-y. research progress on the etiology and pathogenesis of adolescent idiopathic scoliosis. chin med j (engl). 2020; 133(4): 483–93. 12. grauers a, einarsdottir e, gerdhem p. genetics and pathogenesis of idiopathic scoliosis. scoliosis spinal disord. 2016; 11: 45. 13. gr ummons dc, kappeyne van de coppello ma. a frontal asymmetry analysis. j clin orthod. 1987; 21(7): 448–65. 14. kim t-h, kim j-h, kim y-j, cho i-s, lim y-k, lee d-y. the relation between idiopathic scoliosis and the frontal and lateral facial form. korean j orthod. 2014; 44(5): 254–62. 15. hwang s-a, lee j-s, hwang h-s, lee k-m. benefits of lateral cephalogram during landmark identification on posteroanterior cephalograms. korean j orthod. 2019; 49(1): 32–40. 16. sambataro s, bocchieri s, cervino g, la bruna r, cicciù a, innorta m, torrisi b, cicciù m. correlations between malocclusion and postural anomalies in children with mixed dentition. j funct morphol kinesiol. 2019; 4: 45. 17. choudhry mn, ahmad z, verma r. adolescent idiopathic scoliosis. open orthop j. 2016; 10: 143–54. 18. jada a, mackel ce, hwang sw, samdani af, stephen jh, bennett jt, baaj aa. evaluation and management of adolescent idiopathic scoliosis: a review. neurosurg focus. 2017; 43(4): e2. 19. zheng s, zhou h, gao b, li y, liao z, zhou t, lian c, wu z, su d, wang t, su p, xu c. estrogen promotes the onset and development of idiopathic scoliosis via disproportionate endochondral ossification of the anterior and posterior column in a bipedal rat model. exp mol med. 2018; 50(11): 1–11. 20. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. uk: crc press; 2018. p. 455–89. 21. kim h, kim hs, moon es, yoon c-s, chung t-s, song h-t, suh j-s, lee yh, kim s. scoliosis imaging: what radiologists should know. radiographics. 2010; 30(7): 1823–42. 22. shimazaki t, motoyoshi m, hosoi k, namura s. the effect of occlusal alteration and masticatory imbalance on the cervical spine. eur j orthod. 2003; 25(5): 457–63. 23. moraes kjr de, cunha da da, albuquerque lca, carvalho cc de, silva hj da. chewing preference and its relationship with postural muscular electric potential. rev cefac. 2018; 20(5): 648–56. 24. okeson jp. management of temporomandibular disorders and occlusion. 7th ed. st. louis: elsevier mosby; 2012. p. 234–7. 25. oh m-h, kang s-j, cho j-h. comparison of the three-dimensional structures of mandibular condyles between adults with and without facial asymmetry: a retrospective study. korean j orthod. 2018; 48(2): 73–80. 26. velásquez rl, coro jc, londoño a, mcgorray sp, wheeler tt, sato s. three-dimensional morphological characterization of malocclusions with mandibular lateral displacement using conebeam computed tomography. cranio. 2018; 36(3): 143–55. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p74–77 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p74-77 vol 52 no 1 jan-mar 2019_new.indd 4545 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 45–50 research report the role of kuniran (u. moluccensis) and gurami (o. goramy) fish thorns and scales in increasing salivary leukocyte and monocyte cells viability against streptococcus mutans i dewa ayu ratna dewanti,1 i dewa ayu susilawati,1 p. purwanto,1 pujiana endah lestari,1 roedy budirahardjo,2 dyah setyorini,2 ristya widi endah yani,3 erawati wulandari,4 and melok aris wahyukundari5 1department of biomedical science 2department of pedodontics 3department of dental public health 4department of conservative dentistry 5department of periodontics faculty of dentistry, universitas jember, jember – indonesia abstract background: kuniran thorns and gurami fi sh scales are rich in protein and minerals such as dentin believed to increase cell viability against streptococcus mutans (s. mutans) that causes dental caries. these, in turn, can cause systemic diseases if left untreated. purpose: this study aims to analyze the infl uence of kuniran thorns and gurami fi shes scales on the viability of monocytes and salivary leukocytes against s. mutans. methods: monocytes and leukocytes salivary cells were placed on a microtiter plate and treated according to the nature of each group. this study comprised the following groups: control group: untreated; s. mutans group: induced by s. mutans; gurami thorn group: thorns + s. mutans; gurami scales group: scales + s. mutans; kuniran thorn group: thorns + s. mutans; kuniran scales group: scales + s. mutans. viability analysis involved staining with tripan blue. furthermore, the number of viable cells (white) was calculated under an inverted microscope at 200 times magnifi cation from fi ve fi elds of view. data was analyzed by means of an anova test followed by lsd test. results: the anova and lsd tests confi rmed signifi cant diff erences (0.01<p). kuniran thorns and gurami fi sh scales increased the viability of monocytes and salivary leukocytes, but not signifi cantly. the content of fl avonoids, amino acids, omega 3, omega 6 and antioxidants increased leukocyte metabolism, thereby increasing cell resistance to s. mutans infection. conclusion: kuniran thorns (u. moluccensis) and gurami (o. goramy) fi sh scales increase the viability of salivary leukocyte and monocyte cells against streptococcus mutans. keywords: osphronemus goramy; scales; streptococcus mutans; thorns; upeneus moluccensis; viability correspondence: i dewa ayu ratna dewanti, faculty of dentistry, universitas jember. jl. kalimantan 37, jember, 68121, indonesia. e-mail: idewadewanti@yahoo.com introduction in 2018, the national basic health research department (riskesdas) stated that the rate of dental and oral problems stood at 57.6%. dental caries remain an intractable national problem in indonesia which, according to riskesdas figures for 2013 and 2018, had reached the level of 4-5 teeth per person, while the international standard stood at 2.5 teeth.1 streptococcus mutans (s. mutans) is a bacterium constituting the main cause of dental caries whose spread can lead to systemic diseases. consequently, researchers are increasing their understanding of its presence and studying the pathogenesis of dental caries together with their systemic expansion. there is evidence that bacteria (s. mutans) originating in dental caries play a role in the occurrence of endocarditis 2 since they are found in the coronary atherosclerotic plaque of patients who suffer fatal heart attacks.3 since monocytes and salivary leukocytes play a role in the spread of s. mutans from the mouth throughout the systems of the body, the importance of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 mailto:idewadewanti@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 46 dewanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 45–50 the role of leukocytes in saliva and monocytes in blood in the spread of s. mutans is evident. on the other hand, prevention and treatment of dental caries can be achieved by employing natural resources readily available in indonesia. two such resources are fish thorns and scales which, since they are regarded as waste, remain underutilised. however, such materials must be compatible.4 in jember, kuniran (upeneus moluccensis) and gurami (osphronemus goramy) are two of the five most common fish species5 with the latter representing an important national aquaculture commodity.6 therefore, this study seeks to analyze the viability of kuniran thorns and gurami fish scales in monocytes and salivary leukocytes acting against s. mutans. researchers have previously proved that fish thorns and scales consist of proteins that supposedly act as immunomodulators because they contain omega 3, omega 6, flavonoid, various amino acids (alanine, leucine-isoleucine, valine, arginine, methionine, proline, glutamic acid, histidine, lysine, glycine, serine and tyrosine) which are believed to prevent dental caries. a number of researchers have confirmed that fish waste such as scales, thorns and skin represent a potential source of collagen that is crucial to tissue formation and organ function, while also being involved in various functional expressions of cells.4,7–9 scientists from nanyang technological university (ntu) in singapore have demonstrated that collagen derived from fish scales is highly effective across a variety of biomedical applications such as wound healing.10 research conducted by zhou et al. (2013), strongly suggests that fish scales containing collagen can increase blood vessel and lymph formation, thereby promoting tissue repair and regeneration.11 however, the findings of these studies suggest that the viability of the scales and thorns of gurami and kuniran fish remains unproven. it is known that gurami (fresh water) and kuniran (salt water) are the types of fish most frequently consumed by the population of jember, east java, indonesia. cell viability testing is central to drug discovery studies.12 determination of cell viability is the most commonly employed method of assessing the impact of various types of stressors in both toxicity and industrial microbiology studies. viability is defined as the percentage of living cells within the entire population.13 therefore, this study was intended to analyze the effects of thorns and scales from gurami and kuniran fish on the viability of salivary leukocyte and monocyte cells after induction of s. mutans. materials and methods the research, approved by the ethical committee, faculty of dentistry, university of jember (079/un.25.8/kepk/ dl/2018), was conducted between april and september 2018. the materials comprised the following: peripheral blood, s. mutans from the microbiology laboratory, faculty of dentistry, university of jember; ficoll-hypaque (sigma); hbss/hank’s balanced salt solution (gibco); rpmi medium (roswell park memorial institute/gibco); immunostaining kit (daco); pbs/phosphate bufer saline (sigma); dab/diaminobenzidine (daco); hrp/horseradish peroxidase (daco) and fungizone (gibco). the fish thorns/scales were placed in an alumunium pan once the water temperature had reached 80°c and boiled for 60 minutes, before being dehydrated in a drying oven for 48 hours at 65°c, mashed using a grinder mill and sieved through a 100 mesh filter. ±1g of the dry sample was subsequently weighed and dissolved using pbs at a ratio of 1:1.14 blood and saliva were collected from healthy individuals (males deemed to be free of systemic disease and caries who were teetotal non-smokers). saliva was collected by means of the subject spitting onto a dish placed on ice. the blood and saliva was then carefully layered with histopaque and ficollpaque to avoid their mixing, before being centrifugated at 1,900 rpm for 30 minutes at 26oc. the monocyte and salivary leukocyte layers were added to hbss (1:1) and pipetted. monocytes and salivary leukocytes were filtered on each coverslip (@ 100 μl), placed in a microplate and incubated for 15 minutes. 1 cc of rpmi medium, 20 μl of stain and 5 μl of fungizone were added to each well and incubated for 20 minutes, washed twice to eradicate any contamination and, finally, treated according to the group.15 the monocyte group consisted of six groups: a control group; a monocyte isolates untreated s. mutans group; a monocyte isolate induced by s. mutans group; a gurami thorn group: @ 100 μl of monocyte isolates were added to 200 μl of gurami thorns and 100 μl of s. mutans; a gurami scales group: @ 100 μl of monocyte isolates were added to 200 μl of gurami scales and 100 μl of s. mutans. the salivary leukocytes group consisted of six groups: a control group: untreated salivary leukocytes isolates; s. mutans group: salivary leukocytes isolates induced by s. mutans; gurami thorns group: @ 100 μl of salivary leukocytes isolates were added to 200 μl thorns of gurami and 100 μl of s. mutans; gurami scale group: @ 100 μl of salivary leukocytes isolates were added to 200 μl of scales gurami and 100 μl of s. mutans; kuniran thorn group: @ 100 μl of salivary leukocytes isolates were added to 200 μl of kuniran thorns and 100 μl of s. mutans; kuniran scales group: @ 100 μl of salivary leukocytes isolates were added to 200 μl of kuniran thorns and 100 μl of s. mutans. all groups were painted with tripan blue to facilitate analysis of cell viability. treatment ensued with s. mutans (@ 100 μl and thorns/scales @ 200 μl. cell viability was analyzed by counting the number of living cells (white) under the inverted microscope at 200 times magnification from five fields of view. the data obtained was subsequently analyzed by means of anova and lsd tests. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 47dewanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 45–50 results the results of the viability of salivary monocyte and leukocyte cells exposed by thorns and scales of kuniran and gurami fish after being induced by s. mutans were as follows. the anova analysis results were p=0.000 (p<0.05), while the lsd test produced a significant difference between the control with gurami thorns and scales and the control with kuniran thorns and scales. however, there were no significant differences between gurami thorns and gurami scales p=0.86 (p>0.05), gurami thorns with kuniran scales p=0.14 (p> 0.05), gurami thorns with kuniran scales p=0.147 (p>0.05), gurami scales with kuniran scales p=0.765 (p>0.05). the anova analysis results were p=0.000 (p<0.05), while the lsd test indicated a significant difference between the control group with gurami thorns and scales and the control group with kuniran thorns and scales. however, there were no significant differences between gurami thorns with gurami scales p=0.86 (p>0.05), gurami thorns with kuniran thorns p=0.14 (p>0.05), gurami thorns with kuniran scales p=0.147 (p>0.05) and gurami scales with kuniran scales p=0.765 (p> 0.05). these results illustrated that kuniran thorns and scales possess the same ability to increase the viability of salivary leukocytes, whereas gurami scales increased the viability of salivary leukocytes compared to their scales. it could be said that the thorns and scales of kuniran and gurami fish usually increased both the viability of monocytes and salivary leukocytes (tables 1-6 and figures 1-4). table 1. normality test of monocyte cells viability one-sample kolmogorov-smirnov test treatment viability of monocyte cells 2424n normal parametersa,b 92.41673.5000mean 59.960071.74456std. deviation most extreme differences .401.138absolute .235.138positive -.401-.138negative 1.966.678kolmogorov-smirnov z .001.748asymp. sig. (2-tailed) a. test distribution is normal; b. calculated from data. table 2. test of homogeneity of monocyte cells viability levene statistic sig.df2df1 5 18 .796.467 table 3. one-way anova of monocyte cell viability sum of squares df mean square sig.f between groups .00012146.74316533.067582665.333 within groups 1.3611824.500 total 82689.833 23 table 5. test of salivary leucocyte homogeneity sig.df2df1levene statistic .692185.612 table 4. normality test of salivary leucocyte viability one-sample kolmogorov-smirnov test treatment viability of monocyte cells 2424n normal parametersa,,b 44.83333.5000mean std. deviation 27.641601.74456 most extreme differences .375.138absolute .222.138positive -.375-.138negative 1.837.678kolmogorov-smirnov z asymp. sig. (2-tailed) .748 .002 a. test distribution is normal; b. calculated from data. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 48 dewanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 45–50 table 6. one-way anova of salivary leucocytes viability sig.fmean squaredfsum of squares .0002938.9123510.367517551.833between groups 1.1941821.500within groups 2317573.333total 10.5 3 63.75 65.25 61.5 65 0 10 20 30 40 50 60 70 80 control s. mutans gurami thorns gurami scales kuniran thorns kuniran scales th e nu m be r of c el ls figure 3. diagram of salivary leukocytes viability 13.5 5.5 133.75 135.25 131.5 135 0 20 40 60 80 100 120 140 160 control s. mutans gurami thorns gurami scales kuniran thorns kuniran scales th e nu m be r of c el ls figure 1. diagram of monocytes viability control s. mutans kuniran scales kuniran thorns gurami scales gurami thorns figure 2 microscopic description of monocytes viability of kuniran and gurami fish scales and thorns (arrow). observations. conducted with an inverted microscope at 200x magnification. control s. mutans gurami thorns kuniran scales kuniran thorns gurami scales figure 4. microscopic description of salivary leucocyte viability of kuniran and gurami fish scales, thorns (arrow). observations using a inverted microscope at 200x magnification. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 49dewanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 45–50 discussion as caries-related bacteria penetrate deeply into dentin, coming into close proximity to the pulp, inflammatory cells (such as lymphocytes, macrophages and neutrophils) infiltrate the bacterium-invaded area resulting in the development of pulpitis. many types of cytokines and adhesion molecules are responsible for the initiation and progression of pulpitis.16 bacteria (s. mutans) then spread systemically through the blood vessels in the oral cavity to other parts of the body. at this point, the role of immunocompetent cells such as monocytes, macrophages, neutrophils, lymphocytes is crucial in preventing the spread of s. mutans from the oral cavity to other parts of the body’s systems. immunocompetent dental pulp cells have an important function in maintaining the structural integrity of connective tissue. these cells include odontoblasts which produce proinflammatory cytokines and express adhesion molecules in response to pathogens such as pathogen-associated molecular patterns (pamp) which are structures expressed by microorganisms. generally, the initial recognition of microbial pathogens is mediated by pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the nucleotide-binding oligomerization domain-like receptors (nlrs).17–19after recognition by receptors, the cells will initiate proinflammatory cytokines and oxidant-producing phagocytic processes, both of which cause damage to cells and tissues. therefore, a material is required that can increase cell resistance to damage caused by bacterial infections, especially s. mutans. the research reported here has proved that the thorns and scales of kuniran and gurami fish can inhibit the growth of s. mutans by increasing the viability of monocytes and salivary leukocytes. kuniran and gurami fish thorns and scales increased the viability of monocytes and salivary leukocytes due to their content, including: amino acids, flavonoids and unsaturated fatty acids (omega 3, omega 6). amino acids play a role in the vitamin b process (especially that of b5 and b6), produce leucine and isoleucine in protein synthesis, form antibodies, activate various types of hormones, energy providers, ketone and glucose makers. arginine can strengthen the immune system, while methionine nourishes blood vessels, reduces inflammation and treats allergies. glycine can be employed for wound healing and arginine for various metabolic urea synthesis, lymphocyte proliferation and wound healing. glutamine is one of the three amino acids present in glutathione which are antioxidant compounds used as ingredients in leukocytes metabolism. glutathione is one of the antioxidants with a role in protecting cells from damage caused by reactive oxygen. on the other hand, the main components of cell membranes are phospholipids, glycolipids and cholesterol. these components contain polyunsaturated fatty acids which are highly susceptible to oxidation that causes free radicals.20,21 antioxidants can prevent cell damage caused by donating hydrogen electrons to free radicals. antioxidants can give hydrogen atoms to lipid radicals (r •, roo •) and transform them into more stable forms. in addition, antioxidants can also slow the rate of auto-oxidation resulting in the presence of amino acids, thought to positively affect body cell metabolism, thereby enhancing cell resistance to infection. the flavonoid content is thought to work through its inhibiting of the production of nitric oxide (no) through the mechanism of the cytokine-induced no synthase (inos) enzyme. it may also inhibit arginine transport through the mechanism of cationic amino acid transporter-2 mrna (cat-2 mrna). it is said that flavonoids are potential cancer-reducing compounds which can inhibit oxidation reactions induced by enzymes or non-enzymes and act as a good source of hydroxyl and superoxide radicals that protect membrane lipids from reactions that can damage cells (monocytes and salivary leukocytes).22 unsaturated fatty acids constitute the main component of phospholipids which act as a constituent of cell membranes, dna and proteins. dna represents a cell’s genetic device, while proteins play an important role as enzymes, receptors, antibodies, matrix formers and cytoskeleton. research has demonstrated that omega-3 polyunsaturated fatty acid fish oil reduces stress-induced oxidative dna in vascular endothelial cells.23 therefore, all bioactive components of kuniran and gurami fish thorns and scales increase cell viability. the conclusion of this research is that the thorns and scales of kuniran (u. moluccensis) and gurami (o. goramy) fish can increase the viability cells of salivary leukocytes and monocytes against s. mutans. acknowledgments the authors express their gratitude to the indonesian ministry of research, technology and higher education (ristekdikti) for its provision of funding for this research and to the chairperson of the institute for research and community service (lp2m), universitas jember, and dean of the faculty of dentistry, universitas jember for their recommendations relating to its conduct. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. 2. lockhart pb, brennan mt, thornhill m, michalowicz bs, noll j, bahrani-mougeot fk, sasser hc. poor oral hygiene as a risk factor for infective endocarditis–related bacteremia. j am dent assoc. 2009; 140(10): 1238–44. 3. nomura r, nakano k, nemoto h, fujita k, inagaki s, takahashi t, taniguchi k, takeda m, yoshioka h, amano a, ooshima t. isolation and characterization of streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis. j med microbiol. 2006; 55(8): 1135–40. 4. yamamoto k, igawa k, sugimoto k, yoshizawa y, yanagiguchi k, ikeda t, yamada s, hayashi y. biological safety of fish (tilapia) collagen. biomed res int. 2014; 2014: 1–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 50 dewanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 45–50 5. abdullah fn, solichin a, saputra sw. aspek biologi dan tingkat pemanfaatan ikan kuniran (upeneus moluccensis) yang didaratkan di tempat pelelangan ikan (tpi) tawang kabupaten kendal provinsi jawa tengah. manag aquat resour j. 2015; 4(2): 28–37. 6. budi ds, alimuddin, suprayudi ma. growth response and feed utilization of giant gourami (osphronemus goramy) juvenile feeding different protein levels of the diets supplemented with recombinant growth hormone. hayati j biosci. 2015; 22: 12–9. 7. jeevithan e, qingbo z, bao b, wu w. biomedical and pharmaceutical application of fish collagen and gelatin: a review. j nutr ther. 2013; 2(4): 218–27. 8. duan r, zhang j, du x, yao x, konno k. properties of collagen from skin, scale and bone of carp (cyprinus carpio). food chem. 2009; 112(3): 702–6. 9. herpandi, huda n, adzitey f. fish bone and scale as a potential source of halal gelatin. j fish aquat sci. 2011; 6(4): 379–89. 10. na nya n g te c h n o l o g i c a l un ive r s it y. s c i e n t i s t s d i s c ove r f ish sca le der ive d col lagen ef fe ct ive for hea l i ng wou nds. sciencedaily. 2018. available from: www.sciencedaily.com / releases/2018/03/180312091405.htm. accessed 2018 sep 19. 11. zhou g, zhang g, wu z, hou y, yan m, liu h, niu x, ruhan a, fan y. research on the structure of fish collagen nanofibers influenced cell growth. j nanomater. 2013; 2013: 1–6. 12. single a, beetham h, telford bj, guilford p, chen a. a comparison of real-time and endpoint cell viability assays for improved synthetic lethal drug validation. j biomol screen. 2015; 20(10): 1286–93. 13. kwolek-mirek m, zadrag-tecza r. comparison of methods used for assessing the viability and vitality of yeast cells. fems yeast res. 2014; 14(7): 1068–79. 14. ha nura a b, tr ila ksa n i w, suptija h p. cha racter ization of nanohidroxyapatite from tuna’s thunnus sp bone as biomaterials substance. j ilmu dan teknol kelaut trop. 2017; 9(2): 619–29. 15. h ist opa que ® -1119 st e r i le -f i lt e r e d , d en sit y: 1.119 g /m l | sig m a -a ld r ich . p r ot o c ol s & a r t icle s s e c t io n. ava i l a ble f r o m : h t t p s : // w w w. s i g m a a l d r i c h . c o m /c a t a l o g / p r o d u c t / sigma/11191?lang=en&region=id. accessed 2018 sep 19. 16. nakanishi t, takegawa d, hirao k, takahashi k, yumoto h, matsuo t. roles of dental pulp fibroblasts in the recognition of bacteriumrelated factors and subsequent development of pulpitis. jpn dent sci rev. 2011; 47(2): 161–6. 17. jiang w, lv h, wang h, wang d, sun s, jia q, wang p, song b, ni l. activation of the nlrp3/caspase-1 inflammasome in human dental pulp tissue and human dental pulp fibroblasts. cell tissue res. 2015; 361(2): 541–55. 18. farges jc, alliot-licht b, renard e, ducret m, gaudin a, smith aj, cooper pr. dental pulp defence and repair mechanisms in dental caries. mediators inflamm. 2015; 2015: 1–16. 19. yumoto h, hirao k, hosokawa y, kuramoto h, takegawa d, nakanishi t, matsuo t. the roles of odontoblasts in dental pulp innate immunity. jpn dent sci rev. 2018; 54(3): 105–17. 20. faidah n, hernawati t, lamid m, ismudiono i, suprayogi tw, mulyati s. increased integrity of plasma membrane and acrosome cap spermatozoa limousin cattle at post thawing in frozen media by adding seawater extract. kne life sci. 2017; 3(6): 633–41. 21. rizal m, herdis. peranan antioksidan dalam meningkatkan kualitas semen beku. wartozoa. 2010; 2(3): 139–45. 22. duarte j, francisco v, perez-vizcaino f. modulation of nitric oxide by flavonoids. food funct. 2014; 5(8): 1653–68. 23. sakai c, ishida m, ohba h, yamashita h, uchida h, yoshizumi m, ishida t. fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced dna damage in vascular endothelial cells. minamino t, editor. plos one. 2017; 12(11): 1–13. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p45–50 http://www.sciencedaily.com/ https://www.sigmaaldr http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p45-50 219219 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 219–223 research report the potency of andrographis paniculata nees extract to increase the viability of monocytes following exposure to porphyromonas gingivalis yani corvianindya rahayu,1 didin erma indahyani,2 sheila dian pradipta3 and anis irmawati4 1department of oral biology, faculty of dentistry, universitas jember, jember – indonesia 2department of biomedical, faculty of dentistry, universitas jember, jember – indonesia 3undergraduate student, faculty of dentistry, universitas jember, jember – indonesia 4department of oral biology, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: periodontitis is a chronic infectious disease affecting the global population. in indonesia, the prevalence of periodontal disease has reached 57.6% across all age groups. the bacterium considered as the orginator factor of periodontitis is porphyromonas gingivalis (p. gingivalis). herbal ingredients are currently being promoted as a form of treatment because of the minimal side effects they induce. andrographis paniculata nees (apn) extract produces pharmacological effects, including ones immunomodulatory in character, rendering possible its application as a preparation for treating periodontitis. purpose: the purpose of the study was to prove the potency of andrographis paniculata nees extract in increasing the viability of monocytes following exposure to p. gingivalis. methods: the sample was divided into four groups, namely; control negative (c-): monocytes in the medium, not exposed to p. gingivalis; control positive (c+): monocytes in the medium, exposed to p. gingivalis; treatment i (ap25): monocytes with 25% apn extract, exposed to p. gingivalis; treatment ii (ap50): monocytes with 50% apn extract, exposed with p. gingivalis. the monocytes were exposed to 100 ul p. gingivalis for 4.5 hours and stained with trypan blue. observations were conducted using an inverted microscope at 200x magnification. the percentage of viable monocytes was calculated based on the ratio of the number of the cells which absorbed trypan blue staining to that which did not. data was tested using a one-way anova followed by an lsd test. results: there were significant differences between the treatment groups in the number of viable monocytes (p=0.001) they contained. monocyte viability was higher in the 25% apn extract group than that exposed to 50% p. gingivalis. conclusion: andrographis paniculata nees extract demonstrates the potency to increase monocyte viability following exposure to p. gingivalis. keywords: andrographis paniculata nees extract; monocytes viability; porphyromonas gingivalis correspondence: anis irmawati, departement of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: anis-m@fkg.unair.ac.id introduction periodontitis, a disease potentially causing damage to supporting tissues, is triggered by porphyromonas gingivalis (p. gingivalis), a gram-negative anaerobic bacterium which can invade periodontal tissues, while evading the host’s defences.1,2 during chronic inflammation, p. gingivalismediated periodontal disease constitutes a risk factor in several systemic diseases among others; diabetes, pre-term birth, strokes, and atherosclerotic cardiovascular disease.3 it has major virulence factors, namely; lipopolysaccharide, capsule, gingipains and fimbriae.4 during bacterial attack, lipopolysaccharide (lps) of p. gingivalis stimulates an increase in monocyte levels in the periodontal tissues and activates monocytes in peripheral blood vessels. monocytes which are not activated will produce free radicals through a metabolic process .5 free radical production in activated monocytes increases as the result of phagocytosis against infection. free radical production from various biological and environmental dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p219–223 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p219-223 220 rahayu, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 219–223 table 1. the different monocyte viability test results for all groups produced by a one-way anova test groups mean ± sd one-way anova (p) c73.5 ± 2.38 0.0001 c+ 64.25 ± 2.36 ap25 82.75 ± 2.62 ap50 96 ± 2.16 table 2. the different monocyte viability results for groups produced by a lsd test treatment groups cc+ ap25 ap50 c.004 .003 .000 c+ .000 .000 ap25 .000 ap50 sources is caused by an imbalance of natural antioxidants which leads to various disease-associated inflammation.6 the effect of oxidative stress and its associated factors are an important problem within human health. endogenous enzymatic and non-enzymatic antioxidant substances are incapable of coping with the overload of reactive oxygen species (ros) which leads to imbalances within the process, cell damage, and health problems.7 therefore, in this case, the body requires external antioxidants, referred to as exogenous antioxidants, to increase monocytes viability.8 andrographis paniculata nees (apn), commonly known as the “king of bitters,” is an herbaceous plant which includes the acanthaceae family found in all regions of tropical and subtropical asia and southeast asia. in indonesia, most people refer to it as sambiloto (java) to. the andrographolide contained in apn exhibits immunostimulatory, antiviral, and antibacterial pharmacological properties.9,10 the purpose of this research was to prove the potency of apn extract to increase monocytes viability after exposure to p. gingivalis. materials and methods this study was approved by the ethical committee, university of jember (permit number: 000651/kkep/ fkg-ugm/ec/2016). p. gingivalis strain atcc 33277 was supplied by the department of microbiology, faculty of dental medicine, universitas jember (identification number: 0970mikro/s. ket/2016). apn powder was obtained from materia medica, batu, east java, indonesia which had utilized the following extraction process. first, n 400 g of apn leaf powder was moistened with 96% ethanol solvent until 2,000 ml of solution was obtained. the jar containing it was closed for 48 hours and agitated in a digital shaker at 50 rpm. the liquid extract was subsequently filtered and placed in an erlenmeyer flask. remaseration was performed twice with 1,000 ml of 96% ethanol before being soaked overnight in a shaker. finally, the liquid extract was evaporated in a rotary evaporator for 2.5 hours. monocytes were isolated by collecting 9cc of blood using 1 mm ethylene diamine tetraacetic acid (edta) as an anticoagulant. the blood, all solutions, and equipment were conditioned at 4°c prior to use and agitated gently before extraction of an aliquot. iodixanol working solution (ws) at 40% (w/v) concentration was prepared with optiprep 4 vol of diluted with 2 vol of diluent (axis-shield density gradient media, usa). this solution had a density of approximately 1.217 g/ml. 1.072 g/ml-1.074 g/ml density barrier solution was prepared by ws diluted with 2.14 ml + 5 ml and 2.27 + 5 ml diluent, respectively. 4.24 ml of ws was mixed with 10 ml of whole blood (wb) in a 15 ml centrifuge tube; 5 ml of one of the density barrier solutions over 5 ml of the blood, and then layered with 0.5 ml of diluents (ws mix wb) on top. the solution was centrifuged at 700g in a swinging-bucket rotator for 30 minutes at 4°c. 100 µl of mononuclear cells was pipetted onto the microplate, suspended again with 1000 µl rpmi media, and then added to 20 µl fungizon.11 the p. gingivalis used was p. gingivalis atcc 33277 at a concentration of 106 which was measured by densicheck until a density of 0.5 mcfarland was achieved. in the subsequent stage, monocytes were divided into four groups. control negative (c-): monocytes not exposed to p. gingivalis; control positive (c+): monocytes exposed to p. gingivalis; treatment i (ap25): monocytes with 25% apn extract and exposed to p. gingivalis; and, treatment ii (ap50): monocytes with 50% apn extract and exposed to p. gingivalis. a 0.4% trypan blue exclusion test was conducted to determine monocyte viability. monocytes that did not absorb trypan blue staining were counted as viable. the percentage of monocytes viability was calculated by dividing the number of viable monocyte cells by the total number of monocyte cells and multiplying the result by 100%. the data produced was subsequently tested statistically using ibm with spss 20.0 software. a oneway analysis of variance (anova) and a lsd test were administered for pairwise comparison with a significance of 0.05. results the results of data analysis can be observed in table 1 indicated that significant differences existed in all experimental groups. the results of data analysis contained in table 2 revealed that 25% of the apn extract group (ap25) had a significant difference compared to the negative control group (c-), positive control group (c+), dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p219–223 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p219-223 221rahayu, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 219–223 a b c d figure 1. monocytes viability counted using an inverted microscope (200x) and trypan blue staining. viable monocytes are light-colored (red arrows), while unviable monocytes are dark-colored (purple arrows). monocytes viability in cgroup (a), c+ group (b), ap25 group (c), and ap50 group (d). in the ap50 group, monocyte viability was higher than in the other groups. and 50% apn extract group (ap50). the 50% apn extract group (ap50) had a significant difference compared to the negative control group (c-), positive control group (c+), and 25% apn extract (ap25) group. figure 1 contains images of monocyte viability counted by means of an inverted microscope (200x) and trypan blue staining. viable monocytes were light-colored (red arrows), while unviable monocytes were dark-colored (purple arrows). monocyte viability was highest in the ap50 group compared to the other groups (light-colored). discussion the overall results of this research indicated that the presence of apn leaf extract induced a higher increase in monocyte viability in the treatment group compared to the control group. based on the contents of table 1, the results of this study showed that there were significant differences in all experimental groups. apn is an effective antiinflammatory and a correlation existed between increasing the extract dose and a more potent anti-inflammatory effect. the positive control group had the lowest average monocyte viability of all the groups due to the virulence factors of p. gingivalis, i.e. lps, fimbria, and gingipain.4 the lps in p. gingivalis induced an increase in the superoxide and nitric oxides contained in monocytes. nitric oxide in the form of gas molecules constitutes another reactive species of ros which is toxic.12 the imbalance between free radical production and antioxidant defenses leads to an oxidative stress causing lipid peroxidation of cell membranes. the presence of excessive lipid peroxidation causes damage to cell membranes precipitating lysis in the monocytes. lps acts as protypical endotoxin which binds cd14/tlr-4, a complex receptor in different types of cells, especially monocytes. binding of cd14 by lps results in the release of proinflammatory cytokines, namely; il-1α, il-1β, il-16, tnf-α and lipid inflammatory prostalglandins e2 (pge2) mediators. 13 during the inflamatory process, inflammatory cells produce inflammatory mediators such as arachidonic acid and chemokines that demonstrate higher solubility. both of these inflammatory mediators will work by activating inflammatory cells in the infection site and releasing more reactive species. certain markers, in addition to being capable of stimulating signal transduction cascade, can also cause changes in transcription factors, including; nuclear factor kappa b (nf-κb), signal transducers and activators in transcription 3, nf-e2 related factor-2, nuclear factor in activated t cells (nfat), and hypoxia-inducible factor1α (hif1-α), which mediate cellular stress reactions. the subsequent stage is the initiation of cyclooxygenase-2 (cox-2), inducing of nitric oxide synthase (inos), and high expression of inflammatory cytokines, including tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), il-6, and chemokines.14–16 however, if the number of inflammatory mediators in monocytes is excessive, tissue damage may ensue commencing with cell lysis.17 gingipain in p. gingivalis degrades cd14 receptors which causes the hyper-responsiveness of monocytes to bacterial infection.18 gingipain in p. gingivalis manipulates host molecules proactively by intervening in cross-talk dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p219–223 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p219-223 222 rahayu, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 219–223 between c5a receptors and tlr signaling to avoid bacterial clearance. in-vitro studies have indicated that gingipain plays a role in the regulation of inflammatory mediators in a number host cells, including il-1α, il-1β, and il-18.19 the excessive amount of inflammatory mediators in monocytes causes tissue damage which leads to lysis.20 anti-inflammatory activity of andrographolide has been studied using a number of in vivo and in vitro experimental paradigms, including human whole genome dna microarrays. the most common anti-inflammatory and immunomodulatoy activities of andrographolide are barriers to mitogen-activated protein kinase/extracellular signal (mapk/erk) or regulated kinase signalling (specifically p38 mapk/erk1/2) and final transcription factors such as nuclear factor kappa b (nf-κb) and nuclear factor of activated t cells (nfat).21 inflammation is a complex interaction between organisms and pathogens which induces macrophage activation and secretion of proinflammatory cytokines, such as tnf-α, il-1β, and il-6, as the body’s immune system response. research previously conducted confirmed that lps is a powerful inflammatory trigger capable of stimulating macrophages to synthesize tnf-α, il-1β, and il-6.22 as indicated by the contents of table 2, this study showed that the ap25 and ap50 groups had a significant difference to cand c+ group, while ap25 also demonstrated a significant difference to ap50. it was suspected that the active ingredients in apn leaves are andrographolide and flavonoids capable of suppressing the inflammatory process. flavonoids contained in the apn leaves extract act as antioxidants which inhibit the formation of free radical reactions (peroxide) during lipid oxidation by donating one or more electrons to free radicals causing them to become muted. antioxidants delay or prevent the formation of free radical reactions (peroxide) in the lipid oxidation.23 in vitro studies have confirmed that flavonoids are strong inhibitors of lipid peroxidation, as traps of ros or reactive nitrogen, and are also able to inhibit cyclooxygenase and lipooxygenase enzyme activity.24 flavonoids derived from polyphenols can inhibit the oxidation reaction through a radical arrest mechanism (radical scavenging) by donating an electron to the unpaired electrons in free radicals causing a reduction in their number.23 flavonoids significantly inhibit the protease activity of porphyromonas gingivalis gingipain depending on the size of the dose administered.24 monocyte viability of the ap25 group was higher compared to the cand c+ groups. the ap50 group demonstrated the highest level of monocyte viability. the results of this study are compatible with those of the research conducted by chandrasekaran (2011) which found that andrographolide is the main active component of andrographis paniculate which provides anti-inflammatory effects by inhibiting the activation of the nf-κb/mapk signaling pathway and inducing pro-inflammatory cytokines. andrographolide can inhibit the expression of inos through lps, which is activated by macrophages and prostaglandin e2 (pge2) production.25 apn can suppress isoform inos and cox-2 and decrease nitric oxide and the production of pge2.14 andrographolide reduces the expression and production of pro-inflammatory cytokines (il-1α, il-6, tnf-α) and proinflammatory mediators pge2-stimulated by lps, while also reducing the production of superoxide anion radicals and hydrogen peroxide.13 it was concluded that apn extract is highly effective in increasing monocyte viability after exposure to p. gingivalis. references 1. nazir ma. prevalence of periodontal disease, its association with systemic diseases and prevention. int j health sci (qassim). 2017; 11(2): 72–80. 2. varghese ss, thomas h, jayakumar nd, sankari m, lakshmanan r. estimation of salivary tumor necrosis factor-alpha in chronic and aggressive periodontitis patients. contemp clin dent. 2015; 6: s152–6. 3. hayashi c, gudino c v., gibson fc, genco ca. pathogen-induced inflammation at sites distant from oral infection: bacterial persistence and induction of cell-specific innate immune inf lammatory pathways. mol oral microbiol. 2010; 25(5): 305–16. 4. bostanci n, belibasakis gn. porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen. fems microbiol lett. 2012; 333(1): 1–9. 5. duerden bi, collier l, balows a, sussman m. topley & wilson’s microbiology and microbial infection: systematic bacteriology. 9th ed. new york: hodder education publishers; 1998. p. 1501. 6. ishikawa t, ohara-nemoto y, tajika s, sasaki m, kimura s. the production of secretory leukocyte protease inhibitor from gingival epithelial cells in response to porphyromonas gingivalis lipopolysaccharides. in: interface oral health science 2009. springer japan; 2010. p. 275–6. 7. krishnaiah d, sarbatly r, nithyanandam r. a review of the antioxidant potential of medicinal plant species. food bioprod process. 2011; 89(3): 217–33. 8. pendyala g, thomas b, kumari s. the challenge of antioxidants to free radicals in periodontitis. j indian soc periodontol. 2008; 12(3): 79–83. 9. ajaya kumar r, sridevi k, vijaya kumar n, nanduri s, rajagopal s. anticancer and immunostimulatory compounds from andrographis paniculata. j ethnopharmacol. 2004; 92(2–3): 291–5. 10. churiyah, pongtuluran ob, rofaani e, tarwaadi. antiviral and immunostimulant activities of andrographis paniculata. hayati j biosci. 2015; 22(2): 67–72. 11. graham jm. separation of monocytes from whole human blood. scientificworldjournal. 2002; 2: 1540–3. 12. hussain sp, hofseth lj, harris cc. radical causes of cancer. nat rev cancer. 2003; 3(4): 276–85. 13. gabrielian es, shukarian ak, goukasova gi, chandanian gl, panossian ag, wikman g, wagner h. a double blind, placebocontrolled study of andrographis paniculata fixed combination kan jang in the treatment of acute upper respiratory tract infections including sinusitis. phytomedicine. 2002; 9(7): 589–97. 14. federico a, morgillo f, tuccillo c, ciardiello f, loguercio c. chronic inflammation and oxidative stress in human carcinogenesis. int j cancer. 2007; 121(11): 2381–6. 15. kurniasih r, wijaya a. peran radikal bebas pada reperfusi cerebral atau miokardium. in: forum diacnosticum prodia diacnostics educational services. jakarta: prodia; 2002. p. 1–24. 16. said ea, dupuy fp, trautmann l, zhang y, shi y, el-far m, hill bj, noto a, ancuta p, peretz y, fonseca sg, van grevenynghe j, boulassel mr, bruneau j, shoukry nh, routy jp, douek dc, haddad ek, sekaly rp. programmed death-1-induced interleukin-10 production by monocytes impairs cd4 + t cell activation during hiv infection. nat med. 2010; 16(4): 452–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p219–223 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p219-223 223rahayu, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 219–223 17. saranya p, geetha a, selvamathy smk. a biochemical study on the gastroprotective effect of andrographolide in rats induced with gastric ulcer. indian j pharm sci. 2011; 73(5): 550–7. 18. maekawa t, k rauss jl, abe t, jotwani r, triantafilou m, tr iantafilou k, hashim a, hoch s, cur tis ma, nussbaum g, lambris jd, hajishengallis g. porphyromonas gingivalis manipulates complement and tlr signaling to uncouple bacterial clearance from inflammation and promote dysbiosis. cell host microbe. 2014; 15(6): 768–78. 19. hamedi m, belibasakis gn, cruchley at, rangarajan m, curtis ma, bostanci n. porphyromonas gingivalis culture supernatants differentially regulate interleukin-1β and interleukin-18 in human monocytic cells. cytokine. 2009; 45(2): 99–104. 20. goodman ls, hardman jg, limbird le, gilman ag. goodman & gilman’s the pharmacological basis of therapeutics. 10th ed. toronto: mcgraw-hill; 2001. p. 2148. 21. chao ww, kuo yh, lin bif. anti-inflammatory activity of new compounds from andrographis paniculata by nf-κb transactivation inhibition. j agric food chem. 2010; 58(4): 2505–12. 22. nester ew, anderson dg, roberts jr. ce, nester mt. microbiology: a human perspective. 5th ed. new york: mcgraw-hill; 2007. 23. chandrasekaran c v., thiyagarajan p, deepak hb, agarwal a. in vitro modulation of lps/calcimycin induced inflammatory and allergic mediators by pure compounds of andrographis paniculata (king of bitters) extract. int immunopharmacol. 2011; 11(1): 79–84. 24. dong hj, zhang zj, yu j, liu y, xu fg. chemical fingerprinting of andrographis paniculata (burm. f.) nees by hplc and hierarchical clustering analysis. j chromatogr sci. 2009; 47(10): 931–5. 25. chiou wf, chen cf, lin jj. mechanisms of suppression of inducible nitric oxide synthase (inos) expression in raw 264.7 cells by andrographolide. br j pharmacol. 2000; 129(8): 1553–60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p219–223 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p219-223 �� pulp tissue vacuolization and necrosis after direct pulp capping with calcium hydroxide and transforming growth factor-β� sri kunarti department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract mechanical pulp exposure by a rotary cutting instrument or a hand-cutting instrument often happens in deep caries. application of protective dressing can protect the pulp from additional injury by facilitating healing and repair. pulp capping has been suggested as one treatment of choice after pulp exposure to maintain pulp vitality. tgf-b1 is growth factor that has important rule in wound healing. the application of ca(oh)2 and exogenous tgf-b1 as direct pulp capping tr4eatment must be experimented in-vivo to see the vacuolization and necrosis in 7, 14, and 21 days after application. this research was done in vivo experiment from orthodontic patients indicated for premolar extraction, between ages 10–15 years. a class v cavity preparation was created in the buccal aspect 1 mm above gingival margin until pulp exposure. cavity was irrigated slowly with saline solution and dried with a sterile small cotton pellet. group 1 calcium hydroxide was applied as manufacture procedure. group 2, the sterile absorbable collagen membrane used, as inert carrier of tgf-b1 was soaked with 5 ml. all groups were covered by a teflon pledge to separate pulp capping agent from glass ionomer cement restoration. teeth extracted in 7, 14 and 21 days after treatment. all samples were hystopathologically examined. there were significant difference of tgf-b1 (p < 0.05) in the vacuolization day 14th and 21th compared with 7th. there were not significant difference in necrosis for all variables. vacuolization and necrosis decreased in the application of tgf-b1. key words: direct pulp capping, ca(oh)2 , tgf-b1, vacuolization, necrosis correspondence: sri kunarti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction the treatment maintaining pulp vitality could be done in pulp inflammation with diagnosis of reversible pulpitis. pulp inflammation is caused by the opening of pulp space/perforation due to appliance wound or caries. pulp protective dressing is needed to achieve speedy recovery and tissue repair. calcium hydroxide [ca(oh)2] has been used for more than seventy years and the main advantage is the capability to stimulate pulp tissue to form dentin bridge. however, calcium hydroxide also has some disadvantages such as: high ph (ph = 12,5) could cause liquivactive necrosis in most superficial pulp layer.1 caustic effect is caused by high ph, easily solved and triggering coagulation necrosis at the area between necrotic and vital pulp layer.2 alkali ph of calcium hydroxide induces local necrosis in contact with pulp and neutralize acid produced during inflammation response. the alkali properties could increase the risk of pulp disorder and apical lesion.3 multiple tunnel defect occurs during the formation of dentin bridge in which it would allow the entrance of bacteria and toxin into dentin resulting in treatment failure.4 multiple tunnel defect is not only morphologic disorder in pulp permanent protection but also pulp biologic protective failure against bacteria.5 at the present the tendency of application development of dental material is tissue engineering and growth factor which is a new approach based on cellular and molecular mechanism on dentinogenesis regulation during dental tissue repair and the capability of clinical exploitation,6 especially, either in vivo or in vitro study using tgf-β1. the study has been done on the application of tgf-β1 in rat molar using direct pulp capping.3 this material is multipotent physiologic molecule, regulating growth and development, inducing trauma recovery as well as regenerating tissue. through receptor binding mechanism, signal transduction and gene activation would cause cell proliferation and secretion of extra cellular matrix to be reparative dentin.6 transforming growth factor-β1 would increase the formation of reparative dentin compared with other growth factor such as: egf, fgf, pdgf.3 inflammation response is protective mechanism of pulp tissue which has injury such as trauma (mechanical, chemical) or due to bacterial invasion, then, followed by tissue repair or recovery. inflammation response is an accumulative cellular and vascular incidence such as vacuolization and necrosis. vacuolization is cell inflammation due to reversible hydropic change. if recovery process occurs, vacuolization will reduce, in contrast if inflammation increases so vacuolization will be higher, and �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 35-38 also necrotic tissue will increase in severe inflammation. the purpose of this study is to know the effect of tgf-β1 application against vacuolization and nicrotic pulp tissue. material and method this study was done on orthodontic patient’s premolar which required extraction. anesthesia was done in buccal fold using 6 ml xylestesin f. rubberdam and saliva ejector were used and 70% alcohol as disinfectant was applied in preparation on buccal side 1 mm above gingival margin. light pressure intermittent drilling was also used. preparation was done in a slanting direction toward the apical using no. 3 round drilling 1.5 mm in diameter approaching the pulp, then, using no.1 round drilling with, 0.5 mm in diameter penetrating thin dentin layer perforating pulp space. slowly cavity was irrigated using 0.5 ml saline solution, then, it was dried cotton pellet sterile. ca(oh)2 visible light in 1 mm tube package (calcimol) applied on pulp and illuminated for 40 seconds. in another group, 20 ng/ml tgf-β1 was used with by filling material of type ii glass ionomer cement. teflon pledged was applied to avoid the possibility of reaction between type ii glass ionomer cement and pulp capping materials. hystophatologic preparation was made since tooth extraction was done. fixation using 10% formaline buffer for 48 hours decalcification applying solution consist of alcl, formic acid, 37% hcl and aquadest. the next process would be dehydration with the purpose to extract water from tissue and replaced by medium which could be hardened (parafine), the next step clearing with xylene and infiltration hardening, tissue could be cut using microtom with 4 µm thickness. the available preparation was stained using hemotocycline and eosin. evaluation of vacuolization was analyzed quantitatively (figure 1). while necrosis evaluation was done based on criteria of score 1–4,3,8,9 and it was read under microscope with graticule. necrosis score 1 = no necrotic tissue, 2 = necrotic tissue is less or equal to1/3 of perforation width, 3 = necrotic tissue is more than 1/3 until 2/3 of preparation width and 4 = necrotic tissue more than 2/3 of perforation width (figure 2). result statistical parametric test was done to analyze rational scale of vacuolization number. the mean vacuolization of 7, 14, 12 day observation, after treatment tgf-β1. the result of one way anova test ca(oh)2 group has shown insignificant difference (p > 0.05) lower then control group tgf-β1 group has shown significant difference (p > 0.05) between treatment variables. analysis result mean and standard deviation of vacuolization in ca(oh)2 and tgf-β1 group on 7, 14, and 21 day observation could be seen on the table 1. table 1. mean and standard deviation of vacuolization in ca(oh)2 group and tgf-β1 in 7, 14, and 21 days after treatment variable material 7 days 14 days 21 days mean sd mean sd mmean sd vacuolization ca(oh)2 tgf-β1 13.1250 12.7500 2.6424 2.1213 9.8750 8.6250 3.0909 2.9246 12.2500 9.5000 3.9551 2.4495 sd: standard deviation figure 2. necrotic area. figure 1. vacuolization ( ), the core directed to the edge pushed by intracellular liquid. ��kunarti: pulp tissue vacuolization and necrosis after direct pulp capping through normality homogeneity test, unhomogenesis vacuolization data, dunnet t3 test was done to analyze significant level. kruskal walis was done to analyze necrosis data, statistical analysis has shown significant decrease of tgf-β1 comparison between 7–14 days (p = 0.004) and 7–21 days (p = 0.017). no significant difference on necrosis data analysis in both groups (table 2). table 2. lsd test of vacuolization and necrosis comparison between ca(oh) 2 group and tgf-β1 group in 7–14 days, 14–21 days and 7–21 days variable observation time p value ca(oh)2 tgf-β1 vacuolization necrosis 7–14 days 7–14 days 14–21 days 7–14 days 7–14 days 14–21 days 0.06 0.599 0.162 0.054 0.813 0.124 0.004 0.017 0.495 0.063 0.134 0.264 necrosis data taken from t-test comparison between tgf-β1 and ca(oh)2 in 21 days after treatment shows significant difference (table 3). table 3. t test of vacuolization and necrosis comparison between ca(oh)2 with tgf-β1 in group 7, 14, and 12 day variable p value ca(oh)2 tgf-β1 7 days 14 days 21 days vacuolization necrosis 0.759 0.143 0.420 0.143 0.117 0.038 discussion significant difference and decreasing of mean value are found in comparison of vacuolization tgf-β1 in 7, 14, and 21 days it shows that healing process due to the present of reversible degenerative vacuolar. vacuole is inflammation cell which is also called hydropic change. cytoplasm consists of cell containing fluid from invagination of cell membrane, endoplasmic dilatation of rough recticulum, and inflammation of mitochondria due to cell reaction on physical and pathological stimulation. injury contributes loss of cell volume control. usually, cell must secrete metabolic energy to pump out the secretion of natrium ion in order to maintain cell’s internal environmental stability in which it occurs in cell membrane level. anything disturbs cell metabolic energy or a little cell membrane injury could result cell disability to pump out adequate natrium, consequently, osmotic pressure change would occur in which natrium concentration in cell would increase or water would penetrate into cell resulting in morphologic change in the form of cell inflammation.10 this kind of change frequently happened in intra cell lipid accumulation. microscopically, cytoplasm cell seemingly vacuolated in condition which is really similar to hydropic change. in this he staining, lipid would dissolve during the process of preparation so it would appear as an empty space in which previously containing lipid ball. this degeneration process is reversible excepts the presence of continues possibly due to effect of ca(oh)2 on day 7, 14, and 21, the increase has been shown from day 14 until day 21 meaning that degenerative process still continues possibly due to the effect of ca(oh)2 stimulation which contributes the imbalance of intercellular ion change caused by metabolic disturbance due to alkali ph. significant difference (p = 0.038) is found in necrosis comparison between tgf-β1 and ca(oh)2 on day 21 and lowering median value. necrosis occurs because of disintegration of cell or autolysis and entrance of enzyme into tissue. necrotic tissue would be cleaned by phagocytosis. necrosis in ca(oh)2 is possible due to alkali ph resulting in caustic effect on the underlying tissue. morphologic appearance of necrotic tissue is different depends on the result of lytic activity on dead tissue. if the enzyme activity is inhibited by local condition, so necrotic cells would defend the form and tissue would defend the character for some time. this type of necrosis is called coagulation necrosis which is frequently found if necrosis is due to the loss of blood supply.10 in this study no significant difference in necrotic pulp tissue in 7, 14, 21 days, due to coagulated necrosis layer and it could happen for several days until several weeks. the region consists of debris, fragment of dentin, blood, material particle and inflammation cell and this region is also plasma protein which is denatured in obligation zone as caustic effect of pulp capping material. demarcation line found between zone of coagulation necrosis and vital pulp tissue is resulting from material reaction with tissue protein forming globular protein layer for recovery purpose.11 necrotic cell is shown by specific core change or the loss of the cell integrity resulting from damaging cell membrane. necrosis median value in tgf-β1 permanently in 7 days until 21 days after treatment, while median value in ca(oh)2 is higher in 7 days until 14 days, then until 21 days. the above description concludes that the use of tgf-β1 would lower vacuolization and more necrotic tissue occurs in ca(oh)2. references 1. trope m, chivian n, sigurdsson a. traumatic injuries. in: cohen s, burns rc, eds. pathways of the pulp. 8th ed. st louis: mosby inc; 2002. p. 560–72. 2. craig rg, power jm, wataha jc. cement: dental materials properties and manipulation. 7th ed. st louis: mosby inc; 2000. p. 129–31. �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 35-38 3. hu cc, zhang c, qian q, tatum nb. reparative dentin formation in rat molars after direct pulp capping with growth factors. j endod 1998; 24:744–51. 4. gorecka v, suliborski s, biskupski t. direct pulp capping with a dentin adhesive resin system in children’s permanent teeth after traumatic injuries: case reports. quintessence int 2000; 31:241–48. 5. cox cf, subay rk, ostro e, suzuki s, suzuki sh. tunnel defects in dentin bridges: formation following direct pulp capping. op dent 1996; 21:4–11. 6. goldberg m. cell and extra cellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. crit rev oral biol med 2004; 15:13–27. 7. holland r, de souza v, de mello w, nery mj, bernabe pfe, filho jao. permeability of the hard tissue bridge formed after pulpotomypermeability of the hard tissue bridge formed after pulpotomy with calcium hydroxide: a histologic study. j am dent assoc 1979; 99:472–5 8. tsuneda y, hayakawa t, yamamoto h, ikemi t, nemoto k. a histopathological study of direct pulp capping with adhesive resins. oper dent 1995; 20:223–9. 9. kitasako y, shibata s, arakawa m, cox cf, tagami j. a light and transmission microscopic study of mechanically exposed monkey pulps. oral surg oral med oral pathol oral radiol endod 2000; 89:224–30. 10. abrams gd. cedera dan kematian sel. in: price sa, wilson lm, eds. patofisologi konsep klinik proses-proses penyakit. cetakan ke-4. indonesia. jakarta: egc; 1995. p. 22–33. 11. stanley hr. pulp capping: conserving the dental pulp-can it be done? is it worth it? oral surg oral med oral pathol 1989; 68:628–39. 210 vol. 44. no. 4 december 2011 literature review volatile sulphur compounds elimination: a new insight in periodontal treatment ernie maduratna setiawatie1 and rikko hudyono2 1 department of periodontics/institute of tropical disease, airlangga university 2 resident of department of periodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: recent evidences had demonstrated a link between halitosis and apoptosis in periodontitis. periodontal pathogenic micro-organisms produce volatile sulphur compounds (vscs). vscs are toxic to periodontal tissue. purpose: the purpose of this paper was to reveal the mechanism of vscs in periodontal breakdown according to the most recent knowledges. reviews: halitosis is mainly attributed to vscs such as hydrogen sulfide, methyl mercaptan and dimethyl sulfide. several studies demonstrated a strong relationship between vscs and periodontal disease progression. vscs are released from amino acid breakdown from food, protein, cells, blood and saliva. in prone subjects, the vscs may cause alteration in tissue integrity by increasing its permeability and facilitate the endotoxin to penetrate the tissue barrier. they may also causing apoptotic in gingival and periodontal tissue, which are considered the main pathogenesis in aggravating the periodontitis. vscs may also initiate the increase of proinflammatory cytokines which is considered to have negative effects in host response. conclusion: vscs had been shown to have detrimental effects in gingival and periodontal ligament cells. the use of chlorine dioxine agent and topical antioxidant is beneficial in controlling the periodontal disease severity. key words: periodontitis, volatile sulphur compounds (vscs), apoptosis, chlorine dioxine abstrak latar belakang: penelitian terakhir menunjukkan adanya hubungan antara halitosis dengan terjadinya apoptosis pada periodontitis. mikroorganisme penyebab periodontitis memproduksi volatile sulphur compounds (vscs) yang bersifat toksik terhadap jaringan periodontal. tujuan: tujuan penulisan ini adalah membahas mekanisme vscs dalam menyebabkan kerusakan periodontal berdasarkan penelitian terakhir yang ada. tinjauan pustaka: halitosis seringkali dikaitkan dengan timbulnya vscs seperti hidrogen sulfida, metil merkaptan, dan dimetil sulfida. penelitian terakhir menunjukkan bahwa vscs yang dilepaskan dari pemecahan asam amino makanan ternyata memiliki korelasi dengan kerusakan jaringan periodontal. pada subjek yang peka, vscs dapat menyebabkan terjadinya disintegritas epitel dengan meningkatkan permeabilitasnya sehingga endotoksin dapat masuk melewati epitel. adanya vscs tersebut memicu terjadinya apoptosis pada jaringan gingiva dan ligamen periodontal, dimana proses ini menyebabkan keparahan penyakit periodontal. vscs juga dapat mencetuskan peningkatan sitokin proinflamasi yang dapat menyebabkan kerusakan jaringan periodontal. kesimpulan: vscs dapat menyebabkan kerusakan pada gingiva dan ligamen periodontal. penggunaan bahan chlorine dioxine untuk dan antioksidan topikal sangat berguna dalam mengontrol keparahan penyakit periodontal. kata kunci: periodontitis, volatile sulphur compounds (vscs), apoptosis, chlorine dioxine correspondence: ernie maduratna setiawatie, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: setiowati_ernie@yahoo.co.id 211setiawatie and hudyono: volatile sulphur compounds elimination introduction halitosis originated from ‘halitus’, the latin word for breath, means a complaint analogous to body odor and is used to refer the offensive odor emanating from the oral cavity. with up to 50% of people worldwide assessing themselves as having frequent or constant incidents of malodor, it is a common complaint of many adults.1 halitosis usually affects interpersonal social communication and has also become an important market for the pharmacological and cosmetic industries. it is estimated that 20% of young male in switzerland suffer from halitosis.2 another study had shown the percentage of halitosis ranged vary from 22–50%. according to the american dental association, 50% of the adult population had suffered from an occasional oral malodor disorder, while 25% seemed to have a chronic problem. consequently, halitosis become a major sources of multimillion-dollar industry. in the usa alone, over us$500 million are spent annually on mouthwashes, sprays, and related over-the-counter products toward to management of this common problem.3,4 there are various compounds that produce unpleasant smelling in human oral environment, such as hydrogen sulfide, methyl mercaptan, dimethylsulfide, butyrate, isovalerate, skatole, trymethylamine, and putrescine. the hydrogen sulfide, methyl mercaptan and dimethyl sulfide, that arise from bacterial metabolism of aminoacids, mainly contribute to oral malodor. they comprise up to 90% the volatile sulphur compound (vscs) content of mouth air and have been shown to increase with periodontal disease.5 it has been demonstrated that the intensity of clinical bad breath is significantly associated with amount of intra-oral vscs level. the periodontal pocket is an ideal environment for vscs production with respect to the bacterial profile and sulfur source. in addition, vscs also accelerate in periodontal tissue destruction.6 this may explain why patients with periodontal diseases often complain of oral malodor.the purposes of this paper is to explain the mechanism of vscs in aggravating periodontal disease. periodontal disease periodontal disease is one of the two major dental diseases that affect human populations worldwide at high prevalence rates. an advanced periodontal disease with deep periodontal pockets (6 mm or more) affects 10% to 15% of adults worldwide. the available evidence shows that important risk factors for periodontal disease relate to poor oral hygiene, tobacco use, excessive alcohol consumption, stress, and diabetes mellitus. periodontitis is defined as an inflammatory disease of the supporting tissues of teeth caused by specific microorganism or group of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, recession or both. periodontitis results in the formation of soft tissue pockets. severe periodontitis can result in loosening of teeth, occasional pain and discomfort, impaired mastication, and eventual tooth loss.the microflora of the mouth contains three hundreds of species of aerobic and anaerobic bacteria. these organisms grow on tooth surfaces as complex, mixed, interdependent colonies in biofilms, and are attached and densely packed against the tooth in the deeper layers, with more motile forms in the superficial layers.7 the pathogenesis of periodontal tissue commonly thought as a response to bacterial challenge. infection of periodontal tissues with these and other organisms is accompanied by the release of bacterial endotoxins such as leucotoxins, collagenases, fibrinolysins, and other proteases. although bacteria are necessary for periodontal disease to take place, a susceptible host is also needed. the host response is essentially protective. however, either hyporesponsiveness or hyper-responsiveness of certain pathways can result in enhanced tissue destruction. either the host factors or the activity of proteolytic enzymes from bacterial challenges may result in tissue damage. the continuity of periodontal tissues is maintained by homeostasis between regeneration and apoptosis. apoptosis plays an important role in this disease severity.8 volatile sulphur compounds (vscs) periodontal diseases, in particular, necrotizing ulcerative gingivitis and severe periodontitis, can give rise to malodor. pericoronitis, dry socket, other oral infections or ulcers can also cause malodor as can any causes of bleeding in the mouth.the large surface area of the tongue and its papillary structure allows it to retain considerable quantities of food and debris which support a large microbial population, giving rise to malodor predominantly by the generation of vscs such as hydrogen sulphide (h2s) and methyl mercaptan (ch3sh). 9,10 gram-negative anaerobes are probably the main organisms capable of producing sulphur compounds which they release by putrefaction of oral debris, blood and serum products.8 periodontal organisms including treponema denticola, porphyromonas gingivalis, prevotella intermedia, bacteroides forsythus and fusobacteria can produce both sulphides and mercaptans, the main vscs.9 vscs are found in the gingival crevice and are released particularly from deep periodontal pockets and where there is attachment loss.12 vscs are likely to arise mainly from the breakdown of cysteine, cystine, and methionine, peptides and amino acids found free in gingival crevicular fluid, in saliva or produced as a result of proteolysis of protein substrates.13 mass spectrometric (ms) and gas chromatographic (gc) analyses have identified h2s, methyl mercaptan, dimethyl sulphide, (ch3)2s, and dimethyl disulphide, (ch3)2s2, as the principal malodorous products of salivary putrefaction.14–16 hydrogen sulfide has been shown previously to exert proapoptotic activity. h2s may cause micronuclei formation (indicating dna damage) and cell cycle arrest (g1 phase). it may also resulted in stabilization of p53 coupled with induction of downstream proteins such as p21, bax, and cytochrome c, as well as translocation of bax from the cytosol to the mitochondria and release of cytochrome c from mitonchondria. it was 212 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 210–214 shown that h2s did not up-regulate cell levels of the antiapoptotic protein, bcl-2. mounting evidence indicates that, on dna damage, p53 promotes apoptosis by activating bax, which enhances apoptosis by stimulating the release of cytochrome c and the formation of apoptosomes. however, the precise molecular mechanisms involved in h2s-induced apoptosis are, as yet, unknown. it is likely that dna damage caused by h2s induces p53, which then activates bax. induction of bax might then promote apoptosis by targeting the mitochondria and releasing cytochrome c to cytosol. h2s causes dna lesions and up-regulates the genome guardian p53, which leads, in turn, to translocation of proapoptotic bax and the release of cytochrome c and ultimately results in apoptotic cell death.17 h2s were reported to induce cell apoptosis and to suppress expression of some leukocyte and endothelial adhesion molecules. the proapoptotic effect of h2s in cells via activation of mitogen-activated protein kinase pathways has been suggested to be an important endogenous modulator of cellular apoptosis. several recent reports provide evidence suggesting a role for h2s in inflammation. h2s can scavenge peroxynitrite and can interfere with the ability of neutrophils, through hypochlorous acid, to kill microbes and other cells (figure 1). h2s inhibited the opening of mitochondrial mptp. the opening of the mptp plays a pivotal role in the induction of apoptosis in cardiomyocytes. h2s-mediated inhibition of the mptp is regulated via the phosphorylation of gsk-3b (ser9) (figure 2). the inhibitory effects of h2s on mptp opening are mediated by signaling elements such as gsk-3b, and h2s does not directly act on mitochondrial mptps. 16–20 acute exposure to h2s was also associated with increased expression of toll-like receptor 4 (tlr-4),20 that activate intracellular signaling, resulting in the induction of a variety of effector genes and the production of inflammatory cytokines. this response was coupled with an increase in expression of genes involved in matrix remodeling (e.g., laminin gamma 2, microtubule-associated protein 6, fibrinogen-like 2). figure 2. h2s-mediated inhibition of the mptp is regulated via the phosphorylation of gsk-3b.22 discussion gingival epithelial tissues play a key role in periodontal pathogenesis by forming a barrier against penetration by periodontally pathogenic microorganisms and the detrimental products of microorganisms. in healthy gingiva, oral epithelium plays a key role as a barrier against pathogens or toxic compounds.20 the maintenance of this epithelial barrier is therefore extremely important for the preservation of normal gingival structure and function. however, during the progression of periodontal disease, this barrier can be affected. several studies have focused on the relationship between oral malodorous compounds and this epithelial barrier. vscs have been reported to increase the permeability of the tissue and the penetration of lipopolysaccharide and prostaglandin in a crevicular epithelial model. a study used a porcine model for gingival crevicular epithelia to show that exposure to concentrations of vscs much lower than those found in periodontal pockets caused increased permeability of sublingual nonkeratinized mucosa. another study demonstrated that protein content of ch3sh-exposed epithelial cell cultures was decreased by approximately 25% and seemed to be irreversible although it was incubated for next-24 hours in a mercaptan-free environment. furthermore, vscs induced an important decrease in figure 1. hydrogen sulfide modulates inflammatory processes at the leukocyte-endothelial interface. a) under normal conditions, h2s is synthesized in blood vessels primarily via cystathionine-g-lyase (cse), which is expressed in endothelial cells and smooth muscle cells. h2s tonically down-regulates leukocyte adherence via activation of atp-activated potassium channels (katp) on leukocytes and the endothelium. b) when endogenous h2s synthesis is inhibited, leukocyte rolling and adherence to the vascular endothelium increase, likely due in part to elevated expression of adhesion molecules on leukocytes (cd11/cd18) and endothelial cells (p-selectin).20 213setiawatie and hudyono: volatile sulphur compounds elimination the collagen content of the vsc-exposed cell cultures. another study also demonstrated the capability of vscs in suppressing collagen synthesis by 39% and increasing the intracellular degradation of newly synthesized collagen from 26% to 42%. human gingival fibroblasts were exposed to hydrogen sulfide and methyl mercaptan, total protein synthesis was reduced by 18% and 35%, respectively.21–23 it seems that increased concentrations of ch3sh have an inhibitory effect on both cell growth and proliferation in human oral epithelial cell lines.the changes in total protein were accompanied by a corresponding decrease in collagenous protein, which resulted from increased degradation and suppressed synthesis. methyl mercaptan suppressed dna synthesis by 44% and altered collagen metabolism in fibroblast cultures. methyl mercaptan reduces collagen synthesis by 39%, while increases intracellular degradation of newly synthesized collagen by 62%. hydrogen sulfide was also shown to induce cell cycle arrest in oral epithelial cells by the expression of p21(cip1) in ca9-22 cells, which may contribute to delayed epithelial repair. exposure to 5 and 10 ng/ml of h2s significantly decreased dna synthesis. cell cycle analysis also showed that exposure to both concentrations of h2s significantly increased the proportion of cells in g1 phase and significantly decreased the proportion of cells in s phase.24 volatile sulfur compounds were also reported to increase the production of interleukin-1 and prostaglandin e2 (pge2), activate matrix metalloproteinase, then increase collagen degradation and reduce collagen synthesis inhuman gingival fibroblasts.25 vsc may also induce osteoclasts activation which may deleterious towards alveolar bone and periodontium. in the murine cell culture, ii et al demonstrated the capability of h2s to induce pathologic changes in rat alveolar bone. cathepsin k protein, a specific marker for osteoclasts, was expressed in the h2s-induced multinuclear cells. apoptosis plays an important role in the onset and progress of periodontal conditions. activation of caspase-3, p53 or bcl-2 was found in human gingival tissues with periodontitis, and periodontal pathogenic microorganisms have been reported to cause apoptosis in periodontal tissues. the initial apical migration of junctional epithelium in lipopolysaccharideinduced experimental periodontitis appeared to occur simultaneously with the apoptosis of periodontal ligament fibroblasts.25-27 it was therefore suggested that the apoptosisrelated detachment of connective tissue may cause the migration of junctional epithelium. callenic and others found there were two main mechanisms in the apoptotic process involve activation of an intrinsic pathway, in which the mitochondrion plays a central role, and activation of an extrinsic pathway, involving a receptor–ligand-mediated mechanism.28 to distinguish between the two pathways, they analyzed mitochondrial changes. increased production of ros in mitochondria causes disruption of the electrochemical gradient across the inner mitochondrial membrane, which then activates the apoptotic process.29 they also found that h2s increased ros and caused a significant loss of the mitochondrial inner transmembrane potential. collapse of this potential is associated with early stages of apoptosis; moreover, it leads to a key event in the mitochondrial pathway of apoptosis, that is, the release of cytochrome c from the mitochondria intermembrane into cytosol. in response to h2s, the release of cytochrome c was significantly increased especially in 48 hours.19,30 h2s was shown to induce genomic dna damage in human gingival fibroblasts.30,31 callenic and others also observed an increment in the number of dna strand breaks at the genomic level, proving the genotoxic effect of h2s by using single-cell electrophoresis gel.25 genomic dna damage suggests that other molecular pathways, such as the p53 pathway, might be involved in the apoptotic process and that h2s may have pathological effects on human gingiva at the genomic level. it is concluded that the vscs, the main source of oral malodor may be considered periodontally-toxic. the vscs may increase epithelial permeability, trigger the pro-inflammatory cytokines, and induce apoptotic process in periodontal ligament. these may lead to peridontal destruction in susceptible host. it is suggested the vscs elimination as an integral part of periodontal treatment in any phase. the use of potent vscs eliminator, such as chlorine dioxine shall be considered. the use of chlorine dioxine and topical antioxidant may be beneficial especially in preliminary and maintenance phase to control the disease progression, and in surgical phase to reduce the deleterious effect of vscs towards the periodontal regeneration. references 1. armstrong bl, sensat ml, stoltenberg jl. halitosis: a review of current literature. j of dent hyg 2010; 84(2): 65–74. 2. bornstein mm, stocker bl, seemann r, bürgin wb, lussi a. prevalence of halitosis in young male adults: a study in swiss army recruits comparing self-reported and clinical data. j periodontol 2009; 80(1): 24–31. 3. pratibha pk, bhat gs. oral malodor: a review of the literature. j of dent hyg 2006; 80(3): 1–9. 4. nachnani s. oral malodor: causes, assessment, and treatment. compendium 2011; 32(1): 22–31. 5. porter sr, scully c. oral malodour (halitosis). bmj 2006; 333: 632–5. 6. johansson b. bad breath prevalence, periodontal disease and inflammatory markers. thesis. stockholm: kongl carolinska medico chirurgiska intitutet. 2005. 7. kampoo k. bacteriological investigations of the human oral microbiota in health and disease. dissertation. manchester: university of manchester; 2011. 8. ryan me, gu y. host modulation in: newman mg, takei hh, klokkevold pr, carranza fa, eds. clinical periodontology. 11periodontology. 11th ed. st louis: saunders; 2012. p. 492–500. louis: saunders; 2012. p. 492–500. 9. laurina z, pilmane m, care r. growth factors/cytokines/defensins and apoptosis in periodontal pathologies. stomatol baltic dent maxillofac j 2009; 11: 48–54. 10. tsai cc, chou hh, wu tl, yang yh, ho ky, wu ym, ho yp. the levels of volatile sulfur compounds in mouth air from patients with chronic periodontitis. j periodont res 2008; 43: 186–93. 214 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 210–214 11. washio j, sato t, koseki t, takahashi n. hydrogen sulfide-producing bacteria in tongue biofilm and their relationship with oral malodour. j med microbiol 2005; 54(9): 889–95. 12. rioldan s, herrera d, sanz m. biofilms and the tongue: therapeutical approaches to control halitosis. clin oral invest 2003; 7: 189–97. 13. van den velde s, van steenberghe d, van hee p, quirynen m. detection of odorous compounds in breath. j dent res 2009; 88(3): 258–9. 14. john m, vandana kl. detection and measurement of oral malodor in periodontitis patients. indian j dent res 2006; 17: 2–6. 15. quirynen m, van den velde s, vandekerckhove b, dadamio j. oral malodor. in: newman mg, takei hh, klokkevold pr, carranza fa, eds. clinical periodontology. 11periodontology. 11th ed. st. louis: saunders; 2012.; 2012. p. 331–8. 16. nakano y, yoshimura m, koga t. correlation between oral malodor and periodontal bacteria. microbes infect 2002; 4: 679–83. 17. cortelli jr, barbosa md, westphal ma. halitosis: a review of associated factors and therapeutic approach. braz oral res 2008; 22(s1): 44–54. 18. makino y, yamaga t, yoshihara a, nohno k, miyazaki h. association between volatile sulphur compounds and periodontal disease progression in elderly non-smokers. j periodontol 2011 aug 23. 19. baskar r, li l, moore pk. hydrogen sulfide-induces dna damage and changes in apoptotic gene expression in human lung fibroblast cells. faseb j 2007; 21(1): 247–55. 20. yang g, wu l, wang r. pro-apoptotic effect of endogenous h2s on human aorta smooth muscle cells. faseb j 2006; 20(3): 553–5. 21. yao l, huang x, wang yg, cao yx, zhang cc, zhu yc. hydrogen sulfide protects cardiomyocytes from hypoxia/ reoxygenation-induced apoptosis by preventing gsk-3b-dependent opening of mptp. am j physiol heart circ physiol 2010; 298: 1310–9. 22. zanardo rc, brancaleone v, distrutti e, fiorucci s, cirino g, wallace jl. hydrogen sulfide is an endogenous modulator of leukocytemediated inflammation. faseb j 2006; 20(12): 2118–20. 23. novak mj, novak kf. chronic periodontitis. in: newman mg, takei hh, klokkevold pr, carranza fa, eds. clinical periodontology. 11, eds. clinical periodontology. 11periodontology. 11th ed. st louis: saunders; 2012. p. 160–5. louis: saunders; 2012. p. 160–5. 24. takehara s, yanagishita m, podyma-inoue pa, ueno m, shinadak, kawaguchiy. relationship between oral malodor and glycosylated salivary proteins. j med dent sci 2010; 57: 25–33. 25. yasukawa t, ohmori m, sato s. the relationship between physiologic halitosis and periodontopathic bacteria of the tongue and gingival sulcus. odontology 2010; 98(1): 44–51. 26. kurata h, awano s, yoshida a, ansai t, takehara t. the prevalence of periodontopathogenic bacteria in saliva is linked to periodontal health status and oral malodour. j med microbiol 2008; 57(5): 636–42. 27. calenic b, yaegaki k, murata t, imai t, aoyama i, sato t, ii h. oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic dna damage in human gingival epithelial cells. j periodont res 2010; 45: 31–7. 28. takeuchi h, setoguchi t, machigashira m, kanbara k, izumi y. hydrogen sulfide inhibits cell proliferation and induces cell cycle arrest via an elevated p21 cip1 level in ca9-22 cells. j periodontal res 2008; 43: 90–5. 29. nussbaum g, shapira l. how has neutrophil research improved our understanding of periodontal pathogenesis?. j clin periodontol 2011; 38(suppl. 11): 49–59. 30. yaegaki k, qian w, murata t, imai t, sato t, tanaka t, kamoda t. oral malodorous compound causes apoptosis and genomic dna damage in human gingival fibroblasts. j periodont res 2008; 43: 391–9. 31. fujimura m, calenic b, yaegaki k, murata t, ii h, imai t, sato t, izumi y. oral malodorous compound activates mitochondrial pathway inducing apoptosis in human gingival fibroblasts. clin oral invest 2010; 14: 367–73. � dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg � trans-adapted, reliability, and validity of children fear survey schedule-dental subscale in bahasa indonesia arlette suzy,1 julian amriwijaya2 and efi fitriana2 1department of pediatric dentistry, faculty of dentistry, universitas padjadjaran 2faculty of psychology, universitas padjadjaran bandung indonesia abstract background: the most frequently used measuring instrument for determination of dental fear in children nowadays is the children’s fear survey schedule-dental scale (cfss-ds). purpose: the purpose of this study was to explore the reliability and validity of the scale with indonesian trans adapted version of the scale, thus the scale can be reliable to be used in other similar research in indonesia. methods: total of 113 participants, who were parent’s 3 to 12 years old children. children were divided into two age groups, group i 3-6 year old (83 children and group ii 7-12 year old (30 children). eighty three children from the first group were divided into first dental visit group (30 children) and non first dental visit group (53 children). test-retest approach was applied to 30 first dental visit children aged 3-6 year old. original scale was translated to indonesian language. result: the result showed the high value of the cronbach’s coefficient of internal consistency α=0.956. three factors were extracted by screen test method with eigen values higher than 1, which explained 93.05% variance of results. conclusion: cfss-ds scale is reliable and valid psychometric instrument for dental fear evaluation in children in bahasa indonesia. the differences between this study and those of others may appear due to many factors. keywords: reliability; validity; cfss-ds; children; bahasa indonesia correspondence: arlette suzy, c/o: departemen ilmu kesehatan gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. raya bandung sumedang km 21, jatinangor bandung 45363. e-mail: a.suzy@unpad.ac.id introduction recently, there have been many advancements in dental technology regarding pain management and many strategies are established to make patients feel comfortable during dental treatment. however, these have not changed the individual perception that makes them see dental treatment as something that creates fear. the international classification has included this anxiousness and fear toward dental procedures into “specific phobia”.1 fear toward dental treatment or dental fear (df) is a major problem to an individual, especially in children and adolescent. the df prevalence in children and adolescent is about 5-20% in some countries,2 depending on the measuring method, the children’s age, and culture.3 several measuring methods have been used to measure df in children, including behavioral rating such as frankl scale;4,5 physiological measurement such as heart rate, skin galvanic reflect and nasal skin temperature and also questionnaire.6 other community-based study and largescale study performed in school or clinic usually depends on questionnaire data to assess the df prevalence.3 currently, there is only little information related to df in children in a long-term perspective. the model theory of berggren in 1984 predicts that individual with df tends to postpone dental treatment and eventually leads to poor dental and oral health.7 in children, this delay can be noticed through the postponing of first dental visit that should be established when the child is already one year old or, at the latest, 3 years old.8,9 the postponing of dental treatment may persist in adult individuals.7,10 df in adult can be complex in terms of research report dental journal (majalah kedokteran gigi) 20�5 march; 48(�): �–6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 2 suzy, et al/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 1–6 various psychological factors frequently found in adults. study in woman population shows that df is related to several psychological factors, such as depression.11 the result of some studies shows that df is related to various long-term medical effects.12-13 therefore, a df assessment that is suitable for children, adolescence, and adults is very valuable. one of the questionnaires that is usually used to assess df is the children’s fear survey schedule-dental subscale (cfss-ds).14 sherer, as stated by beena, then developed the cfss-ds as a tool to assess df.15 cuthbert used this measuring instrument in their study and modified it to assess df. currently, the developed instrument has been translated and adapted into several languages and countries,3 but not in indonesian. the cronhbach coefficient for internal reliability for this instrument is about α=0.85 to 0.92.3, 16-20 in addition to be used worldwide with high reliability level, cfss-ds must have a simple and fast application for evaluating df. on the contrary, even though it has good results, several studies consider that df self-reporting measurement cannot be used to differentiate common fear and df.21 a self-report measurement tool should demonstrate reliability by being both repeatable, yielding consistent results in a group of stable patients and internally consistent which demonstrating that the items on the questionnaire are strongly related to each other.22 most scientific questionnaires used in dentistry have been developed in english-speaking areas. international multicenter-based studies including populations with different cultural backgrounds and other languages are growing. the process of cross-cultural adaptation tries to produce equivalency between source and target based on content.23 there is a great need for cross-cultural adapted questionnaires as they allow comparison of data across different countries. until now, studies on df in indonesia using cfss-ds are very rare. considering the high reliability and validity of this instrument in other countries, trans-adaptation and cfss-ds psychometric analysis, which is applicable in indonesia, needs to be performed. hence this study was aimed to translate cfss-ds from english to indonesian; perform a transcultural adaptation; and test the indonesian version (cfss-ds) psychometrically with regard to reliability and validity in a cross-sectional study. material and methods the process of questionnaire development followed a standard procedure in six phases, known as ‘stages’, according to the established guidelines for self-assessment instruments this procedure is internationally recognized and has been well documented in numerous applications.23 the study has been approved by the ethical committee for research from faculty of dentistry universitas padjadjaran. cfss-ds consists of 15 questions related to dental treatment using the likert scale optional answers of 1-5 (1-not afraid, 5-very afraid). the total score that indicates df is minimum of 38.15 a single translator, and then being re-translated into english by other translator by observing the comparability with the original version translate cfss-ds from english into bahasa indonesia (table 1). the indonesian version is applied on a small number of participants and then a modification on the translation was performed for better comparison. the study participants were parents who take their 3-12 years old children to the pediatric dentistry department of bandung dental hospital, indonesia. pediatric patients with acute dental symptoms or other dental emergencies were not included in the study. the participants were parents of 3-6 years old children who never visited dentist before (n=30); parents with 3-6 years children old who had visited dentist in the past (n=53); and parents with 7-12 years old children who had visited dentist in the past (n=30). total participants were 113 parents. demographic data is shown in table 2. the purpose of this study was explained to the parents and a written informed consent was also provided. the cfss-ds questionnaire was given and completed by the parents before dental treatment. a test-retest approach was performed for reliability verification during within 10 days period between the tests to explore the consistency of participants in answering the questionnaire.3 psychometric is a set of statistical models and methods developed especially to summarize, describe, and make a conclusion from data collected in psychological studies.24 psychometric is needed to explore the psychometric characteristics of each questionnaire before being used. some of the basic psychometric characteristics from a questionnaire are reliability and validity.25, 26 table 1. translated version of the scale from english to bahasa indonesia no items apakah anak anda merasa takut: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15 pada dokter gigi? pada dokter? disuntik? bila seseorang memeriksa mulutnya? bila diminta membuka mulut? bila ada orang yang tidak dikenal menyentuhnya? bila diperhatikan oleh orang lain? bila giginya di-bor oleh dokter gigi? bila membayangkan giginya dibor dokter gigi? pada suara bor dokter gigi? bila seseorang memasukkan alat-alat dokter gigi ke dalam mulutnya? tersedak oleh alat-alat dokter gigi? bila harus pergi ke rumah sakit? melihat orang berpakaian putih? bila perawat gigi membersihkan gigi dan mulutnya? � dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �suzy, et al/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 1–6 the intraclass correlation coefficient (icc 2,1; two observation time points of one item) was calculated within 10 days for two surveys in order to evaluate the reproducibility of the results (under constant conditions).27 a random sampling strategy was used to select the patients, who were asked to complete the questionnaire at test-retest approach to maximize the probability that the patients who received the questionnaire were representative of the entire population.28 an icc value of 0.00 indicated ‘no reliability’, >0.75 was defined as ‘good’ and 1.00 as ‘perfect’.27 chronbach’s alpha quantified the level of the relationship between different items within the questionnaire (internal consistency, homogeneity, item to total correlation), and thus determined how well the instrument, as a single entity, measured the individual properties (0.00 = none, 1.00 = perfect homogeneity).27-29 chronbach’s alpha of the single items represents the homogeneity between the test–retest results. a bland–altman plot and analysis for assessing the agreement between the test and retest measurements was performed.28 the measuring instrument validity is defined as precision and accuracy of a measuring instrument related to its measuring function. one of the validity instruments is construct validity.30 in general; the validity of an individual item is quantified by bivariate correlations between the item and a comparable instrument with the same construct. the construct validity was rated as follows: spearman’s rank correlation r ≥ 0.81–1.0 excellent, 0.61–0.80 very good, 0.41–0.60 good, 0.21–0.40 sufficient, and 0.00–0.20.19 the statistical method used was descriptive statistic for age and sex/gender distributions. the cfss-ds scale reliability was defined using the cronbach coefficient for internal consistency. the test-retest reliability approach was quantified using the intraclass correlation coefficient icc, resulting from analysis of variance. for every icc coefficient, the corresponding 95% confidence interval is also stated as a measure of precision. the cfss-ds validity was defined by construct validity exploration using factor analysis through varimax rotation. the discriminant validity was analyzed using t-test based on the age group (3-6 years old and 7-12 years old) and also based on the information of previous visit/ never visit to the dentist. all statistical analysis was performed using statistical package version 21.0 (spss inc., chicago, il, usa) for mac osx. results the survey took place from december 2013 to the end of february 2014. one hundred and thirteen subjects out of 150 responded (75%), which consist of parents of 3-12 years old children (mean 5.56 + 2.16). of these, 30 were selected at random to create a subsample for the testretest reliability testing. all the participants completed the questionnaire in the clinic. the whole process (stages 1-6) of transcultural adaptation and translation was implemented according to standardized guidelines.27 the process of forward and backward translation ran without any major obstacle. definitive adaptation took place at the consensus meeting and consisted mainly of simplification of the content of various questions. all transcultural adaptations correspond to colloquial speech in indonesian-speaking areas. in the pilot phase, the cfss-ds was tested on parents of patients (n=50). there were no difficulties with the contextual interpretations of different points or with the grading of the response options. a sentences ‘are your child afraid of…’ as a heading sentence was added before the following items in order to increase ease of reading. testing the scoring system consist of item analysis, cfssds reliability, cfss-ds factor analysis, discriminant validity. descriptive data of cfss-ds distribution showed cfss-ds score range from 15-69 (mean 27.62+11.77) with the skew 1.519, and se-skew .227. the distribution is asymmetric because the skewness value is more than two times higher than se-skew value. arithmetic means and deviation standards of the component as a result of cfss-ds subject (table 3). the highest mean on subjects is found in the following cfssds components: 3) injections, 8) the dentist drilling, 4) having somebody examine the mouth, 5) having to open your mouth, and 1) dentists. the icc values for the items ranged from 0.501 (item 9) to 0.901 (item 1 and 2). the use of internal consistency determination is consistent for result validity exploration obtained from cfss-ds scale. the highest score of cronbach coefficient for internal consistency is α=0.966. the corrected value of total-items correlation (table 5). the value for all scale items is indicated higher than the r value for an alpha of 0.05 and a degree of freedom of df=n-2=0.16, showing 15 valid scale items. the pearson correlation value for the test-retest approach reliability shows a high correlation r=0.853 (p=0.000). the cfss-ds component factor analysis uses varimax rotation to explore psychometric instrument validity. two factors are extracted using screen-test method with an eigen value that is higher than 1, which explains 78.744% of variants. the cfss-ds component analysis result is shown in table 3. table 2. demographic and descriptive data age (years) (mean+sd) 5.56 + 2.16 gender distribution % n girls 63.7 72 boys 36.3 41 dental visit % n first 83 73.5 recall 30 26.5 sd: standard deviation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 4 suzy, et al/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 1–6 the first factor explains 67.944% variants and shows cfss-ds that illustrates a situation that is not related directly to dentist and varied. therefore, for factor 1, the analysis factor was performed again (table 3). factor two explains 10.800% variants and shows cfss-ds component that illustrates dentist directly (having to open your mouth). the component factor analysis that is included in factor 1. two factors are extracted using screen-test method with an eigen value that is higher than 1, which explains 82.250% variants. factor 1a (73.690%) explains the fear related to less-invasive relationship with dentist (doctors, having a stranger touch you, having somebody look at you). factor 1b (8.560%) shows fear that is related to the invasive procedure (injections, the dentist drilling, the sight of the dentist drilling). scatter diagram of the differences plotted against the averages of test and retest measurements (figure 1). the mean difference was 4.4 points, sd 7.3 (cfss-ds total score test mean = 40.83, sd 14.06; cfss-ds total score retest mean = 36.43, sd 12.34). the pediatric patients in this study are differentiated based on age group and whether they have visited/never visited (recall/first visit) a dentist. the t-test result shows a significant difference of cfss-ds value mean between children who have/have never visited dentist (p=.000), and also between 3-6 years old children and 7-12 years old children (p=.010). discussion many studies on df in children have been performed in several countries. as a culture and social norm, behavior can affect expression and fear development in children and, because of variations of dental treatment system in various cultures; normative data from every culture is needed. in this study, the indonesian version of cfss-ds showed good internal consistency, test-retest reliability, construct validity and discriminant. the indonesian version of scale is longer in sentences than the english version in some items due to be more understandable. such as the word “choking” is added by the words “by dentist instrument”, so it is more adaptable for participant to understand what it is meaning for without changing the purpose of the original question. table 3. descriptive (n=113) and reliability (n=30) data item content mean sd icc ca ritemtotal factors factors 1 2 1a 1b 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. dentists doctors injections having somebody examine the mouth having to open your mouth having a stranger touch you having somebody look at you the dentist drilling the sight of the dentist drilling the noise of the dentist drilling having someone put instruments in your mouth choking having to go to the hospital people in white uniforms having the nurse clean your teeth 2.00 1.59 2.81 2.09 2.09 1.65 1.64 2.14 1.60 1.80 1.74 1.65 1.69 1.57 1.56 .945 .903 1.292 1.057 1.023 .906 .0887 1.209 .762 1.045 1.007 .924 9.83 .865 .865 .901 .901 .720 .804 .788 .832 .837 .818 .501 .818 .753 .770 .787 .900 .900 .960 .955 .966 .956 .956 .953 .953 .959 .959 .952 .952 .953 .952 .954 .954 .548 .827 .395 .735 .739 .898 .891 .659 .598 .915 .918 .895 .942 .869 .868 .081 .707 .719 .322 .324 .812 .812 .731 .660 .809 .815 .821 .832 .720 .716 .909 .479 -.254 .865 .876 .465 .455 .153 .167 .456 .473 .444 .498 .541 .544 .793 .104 .908 .901 .392 .412 .734 .836 .880 .918 .902 .899 .335 .843 .302 .305 .747 .603 .575 .433 .436 .347 .232 .233 % of explained variance eigen value 67.944 10.192 10.800 1.620 73.690 8.843 8.560 1.027 sd: standard deviation; ca: chronbach’s alpha; icc: intraclass correlation coefficient figure 1. bland-altman plot of the difference against the mean for test-retest data. 5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 5suzy, et al/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 1–6 in the process of transcultural adaptation of the questionnaire, all the questions were worded precisely and comprehensibly. the question using word ‘having’ (item 47, 11, 13, and 15) was made more precise in the indonesian version by use the phrase ‘being…by….’ (example: item 6: ‘having a stranger touch you’ transformed in to ‘being touched by a stranger’). studies in several countries support cfss-ds as a psychometric instrument with high reliability to measure df in children. the coefficient values of α cronbach cfssds in several studies are α=0.85 in greek sample,17 α=0.89 in japanese sample,3 α=0.861 in bosnia herzegovina children.16 a value of α = 0.85is also resulted in a test in dutch and finland. the α cronbach coefficient for internal consistency in this study is 0.956, which is similar to that of a study in taiwan, α=0.90,31 and in india, α=0.92.32 based on the result of other studies, it is predicted that this study result is based on the item description in the cfss-ds scale that is quite distinct and easily understood by the subject. based on the cfss-ds scale structure factor, there are three factors indicated, i.e. factor i (explains 73.69% variants), which is characterized by fear due to less-invasive procedure; factor ii (explains 8.56% variants), which is characterized by dental invasive procedure characteristic.; and factor iii, which is marked by direct fear of dentist (explains 10.80% variants). nakai et al.3 also identified three factors while the study in bosnia herzegovina children identified four factors.16 the discriminant validity with t-test is applied to find the difference in df level based on children groups (3-6 years old and 7-12 years old) and also based on dental visit experiences (previous visit or never visited). the test result shows a significant difference on df score (p value = .000) in children who have visited/never-visited dentist. children who have never visited a dentist show higher df score (31.87+14.03) compared to the children who had visited dentist in the past (22.81+5.571). this result is relevant with the result from yoshida’s study showing that the cfss-ds score mean for the first visit is 38.1+13.2 in the first visit and 23.8+7.1 in children who had visited dentist in the past.33 the t-test analysis shows a significant difference (pvalue .002) between the scores of 3-6 years old group (29.33+12.63) and 7-12 years group (22.90+6.042). this is in compliance with raj’s study that concludes that df is reduced along with the increased age (cfss-df score of 4-6 years old group is 28.78+ 5.742 and for 10-14 years old group, it is 25.93+ 5.586). in conclusion cfss-ds scale is reliable and valid psychometric instrument for dental fear evaluation in children in bahasa indonesia. the differences between this study and those of others may appear due to many factors, such as socioeconomic, parental age, and education background that was not being tested in this study. references 1. nicolas e, bessadet m, collado v, carrasco p, rogerleroi v, hennequin m. factors affecting dental fear in french children aged 5-12 years. int j paediatr dent 2010; 20(5): 366–73. 2. gao x, hamzah sh, yiu yck, mcgrath c, king mn. dental fear and anxiety in children and adolescents: qualitative study using youtube. j med internet res 2013; 15(2): e29. 3. nakai y, hirakawa t, milgrom p, coolidge t, heima m, mori y. the children’s fear survey schedule-dental subscale in japan. community dent oral epidemiol 2005; 33(3): 196–204. 4. sharma a, tyagi r. behavior assessment of children in dental settings: a retrospective study. das um, editor. int j clin ped dent 2011; 4(1): 35–9. 5. shinohara s, nomura y, shingyouchi k, takase a, ide m, moriyasu k. structural relationship of child behavior and its evaluation during dental treatment. j oral sci 2005; 47(2): 91–6. 6. farhat-mchayleh n, harfouche a, souaid p. techniques for managing behaviour in pediatric dentistry. j can dent assoc 2009; 75(4): 283a-f. 7. de jongh a, schutjes m, aartman iha. a test of berggren’s model of dental fear and anxiety. eur j oral sci 2011; 119(5): 361–5. 8. kuthy ra, pendharkar b, momany et, jones mp, askelson nm, chi dl. factors affecting age at first dental exam for medicaid-enrolled children seen at federally qualified health centers. pediatr dent 2013; 35(3): e100–6. 9. saraiva m, bettiol h, barbieri ma, holanda l. p1-173 delay in the first dental visit in a brazilian cohort study. j epid comm health 2011; 65(suppl 1): a114–4. 10. pohjola v, mattila ak, joukamaa m, lahti s. anxiety and depressive disorders and dental fear among adults in finland. eur j oral sci 2011; 119(1): 55–60. 11. hägglin c, carlsson sg, hakeberg m. on the dynamics of dental fear: dental or mental? eur j oral sci 2013; 121(3pt2): 235–9. 12. carter ae, carter g, boschen m, alshwaimi e, george r. pathways of fear and anxiety in dentistry: a review. world j clin cases 2014; 2(11): 642-53. 13. moore r. psychosocial aspects of dental anxiety and clinical pain phenomena. aarhus universitet; 2000. p. 1–1. 14. javadinejad s, farajzadegan z, madahain m. iranian version of a face version of the modified child dental anxiety scale: transcultural adaptation and reliability analysis. j res med sci 2011; 16(7): 8727. 15. beena jp. dental subscale of children’s fear survey schedule and dental caries prevalence. eur j dent 2013; 7(2): 181-5. 16. bajric e, kobaslija s, juric h. reliability and validity of dental subscale of the children’s fear survey schedule (cfss-ds) in children in bosnia and herzegovina. basic med sci 2011; 11(4): 214–8. 17. arapostathis kn, coolidge t, emmanouil d, kotsanos n. reliability and validity of the greek version of the children’s fear survey schedule-dental subscale. int j paed dent 2008; 18(5): 374–9. 18. krikken jb, van wijk aj, cate ten jm, veerkamp js. measuring dental fear using the cfss-ds. do children and parents agree?. int j paediatr dent 2012; 23(2): 94–100. 19. marginean i, filimon l. dental fear survey: a validation study on the romanian population. j psych educ res 2011; 19(2): 124–38. 20. singh p, pandey rk, nagar a, dutt k. reliability and factor analysis of children′s fear survey schedule-dental subscale in indian subjects. j ind soc pedod prev dent 2010; 28(3): 151. 21. el-housseiny aa, alamoudi nm, farsi nm, el derwi da. characteristics of dental fear among arabic-speaking children: a descriptive study. bmc oral health 2014; 14: 118. 22. kruse s, schneeberg a, brussoni m construct validity and impact of mode of administration of the pedsql among a pediatric injury population. health qual life outcomes 2014; 12(1): 168. 23. beaton de, bombardier c, guillemin f, ferraz mb. guidelines for the process of cross-cultural adaptation of self-report measures. spine (phila pa 1976) 2000; 25(24): 3186–91. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 6 suzy, et al/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 1–6 24. rao cr, sinharay s. psychometrics. elsevier; 2007. p. 34-57. 25. lu y, fang jq. advanced medical statistics. world scientific; 2003. p. 118. 26. matthews de, farewell vt. using and understanding medical statistics. karger; 2007. p. 45-87. 27. portney lg, watkins mp. foundations of clinical research: applications to practice. pearson/ prentice hall; 2009. p. 67-99. 28. devon ha, block me, moyle-wright p, ernst dm, hayden sj, lazzara dj, savoy sm, kostas-polston e. a psychometric toolbox for testing validity and reliability. j nurs scholars 2007; 39: 155–64. 29. everitt bs. medical statistics from a to z: a guide for clinicians and medical students. cambridge university press; 2006. p.24-89. 30. feinstein ar. principles of medical statistics. taylor & francis; 2001. p. 45-51. 31. lee c-y, chang y-y, huang s-t. higher-order exploratory factor analysis of the dental subscale of children’s fear survey schedule in a taiwanese population. community dent health 2009; 26(3): 183–7. 32. singh-manoux a, hillsdon m, brunner e, marmot m. effects of physical activity on cognitive functioning in middle age: evidence from the whitehall ii prospective cohort study. am j pub health 2005; 95(12): 2252–8. 33. yoshida t, nakai y, matsumi y, murakami. does regular attendance decrease children’s dental fear? a longitudinal study. washington: proceeding iadr conference; 2013. p. 56-61. 34. raj s, agarwal m, aradhya k, konde s, nagakishore v. evaluation of dental fear children during dental visit using children’s fear survey schedule-dental subscale. int j clin ped dent 2013; 6(1): 12-5. 82 volume 47, number 2, june 2014 penurunan jumlah streptococcus mutans pada saliva anak dengan ortodonti cekat setelah konsumsi yoghurt (reduction of salivary mutans streptococci in children with fixed orthodontic appliance after yoghurt consumption) dewi anggreani bibi, udijanto tedjosasongko, dan irmawati departemen ilmu kedokteran gigi anak universitas airlangga surabaya indonesia abstract background: orthodontic treatment using fixed appliances in children increases with the case of malocclusion in indonesia. the patients with fixed orthodontics have higher risks of caries. purpose: the study was aimed to examine the influences short term daily consumption fruit-flavored yoghurt on salivary mutans streptococci in pediatric patients during orthodontic treatment with fixed appliances. methods: this was an experimental laboratory study with a double-blind randomized crossover design. the subjects were 26 children in range of age 11 to 15 years old who were under orthodontic treatment using fixed appliances. subjects were divided into 2 (two) groups which consist of 13 children each. group a were asked to consumed a 150 ml of yoghurt bifidobacterium bifidum and lactobacillus acidophilus once a day for 2 weeks, while group b were asked to consumed milk once a day for the same period of time. after “washout” period for 2 weeks, the subjects of group a and b crossed over the drink, group a was asked to consumed milk and group b was asked to consumed yoghurt for another 2 weeks. before and after consuming yoghurt or milk, the subject’s saliva samples were taken and the colonies of mutans streptococci were counted on tyc media. results: statistical analysis showed that on subjects who consumed yoghurt the colony number of mutans streptococci reduced significantly (p < 0.05). conclusion: short-term daily consumption of the probiotic yoghurt containing bifidobacterium bifidum bacteria and lactobacillus acidophilus could reduce the number of salivary mutans streptococci in pediatric patient during orthodontic treatment with fixed appliances. key words: probiotic yoghurt, children, mutans streptococci, bifidobacterium bifidum, lactobacillus acidophillus abstrak latar belakang: kebutuhan perawatan ortodonti menggunakan alat cekat pada anak-anak meningkat seiring bertambahnya jumlah kasus maloklusi di indonesia. namun faktanya penggunaan piranti ortodonti cekat berisiko terjadinya karies disekitar bracket. tujuan: penelitian ini bertujuan meneliti pengaruh konsumsi yoghurt buah dalam jangka pendek terhadap jumlah streptococcus mutans pada saliva anak pemakai ortodonti cekat. metode: jenis penelitian ini eksperimental laboratoris dengan desain penelitian double blind randomized crossover design. subjek penelitian adalah 26 anak berusia 11-15 tahun yang sedang menjalani perawatan ortodonti dengan piranti cekat. subjek dibagi dalam 2 kelompok dengan masing-masing 13 anak. kelompok a diminta untuk mengkonsumsi 150 ml yoghurt bifidobacterium bifidum dan lactobachillus acidophillus sekali sehari selama 2 minggu, sedang kelompok b diminta untuk mengkonsumsi susu sekali sehari selama 2 minggu. setelah periode “washout” selama 2 minggu, kedua kelompok bertukar minuman, kelompok a mengkonsumsi susu sedang kelompok b mengkonsumsi yoghurt selama 2 minggu. setiap sebelum dan sesudah mengkonsumsi yoghurt dan susu, sampel saliva subjek diambil dan dilakukan penghitungan jumlah koloni streptococcus mutans pada media tyc. hasil: analisa statistik menunjukkan bahwa setelah mengkonsumsi yoghurt jumlah koloni streptococcus mutans dalam saliva subjek berkurang secara signifikan (p < 0,05). simpulan: konsumsi probiotik yoghurt yang mengandung bifidobacterium research report 83bibi, at al.: penurunan jumlah streptococcus mutans pada saliva anak bifidum dan lactobachillus acidophillus dapat mengurangi jumlah koloni streptococcus mutans dalam saliva anak selama perawatan ortodontik cekat. kata kunci: probiotik yoghurt, anak, streptococcus mutans, bifidobacterium bifidum, lactobachillus acidophillus korespondensi (correspondence): dewi anggreani bibi, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dewi_bibi@yahoo.com pendahuluan pada anak dengan ortodonti cekat, karies disekitar bracket merupakan salah satu resiko yang sering terjadi.1 prevalensi karies yang tinggi 90% terjadi pada anak-anak dan terutama anak pemakai ortodonti cekat dimana terjadi aktifitas mikroorganisme yang menyebabkan enamel demineralisasi yang secara klinis terlihat sebagai white spot lesion disekitar brackets.2,3 berbagai mekanisme dapat digunakan dalam mencegah karies, antara lain dengan mengubah kebiasaan pasien dalam mengkonsumsi makanan yang berkarbohidrat tinggi, meningkatkan kebersihan mulut dan menggunakan obat kumur antiseptik, namun obat kumur tidak direkomendasikan untuk anak karena mempunyai efek samping jika digunakan dalam jangka panjang dan pada kenyataannya rasa obat kumur tidak disukai sebagian besar anak-anak. probiotik adalah nama untuk mikroorganisme yang bila diberikan dalam jumlah yang cukup dapat memberikan manfaat kesehatan pada hostnya dan akan berpean sebagai bantuan untuk melawan infeksi.4,5 keuntungan probiotik yang berhubungan terhadap faktor kesehatan secara umum telah banyak diteliti, yaitu dapat mengurangi kerentanan terhadap infeksi, mengurangi resiko alergi dan ketidaktoleran terhadap laktosa, menurunkan tekanan darah dan nilai serum kolesterol.6 namun pengaruh probiotik terhadap kesehatan mulut, sangat sedikit yang dieksplorasi. bifidobacteriumdan lactobachillus acidophillus yang sering digunakan dalam yoghurt adalah bakteri non patogen yang dapat menghambat pertumbuhan bakteri patogen menghasilkan bakteriosin serta mempunyai sifat bakteriostatik dan bakteriosid dengan target melindungi membran plasma akibat kerusakan yang disebabkan infeksi bakteri.7 penelitian yang dilakukan cildir et al.8 membuktikan bahwa probiotik yoghurt yang mengandung lactobachillus dan bifidobacterium dn-173 010 secara konsisten menunjukkan penurunan jumlah streptococcus mutans pada saliva.9 tujuan penelitian ini adalah untuk meneliti pengaruh konsumsi jangka pendek yoghurt buah yang mengandung probiotik bifidobacterium bifidum dan lactobacillus acidophillus terhadap jumlah streptococcus mutans pada saliva anak pemakai ortodonti cekat. bahan dan metode penelitian ini dilakukan di smp muhammadiyah 1 sidoarjo. jenis penelitian ini eksperimental laboratoris dengan desain penelitian double blind randomized cross over design dengan teknik pengambilan sampel yang digunakan adalah simple random sampling. subjek penelitian ini 26 anak sehat (18 perempuan dan 8 lakilaki), berusia11-15 tahun yang telah memakai ortodontik cekat pada dua sisi rahang selama 6 bulan. kriteria subjek yang ditentukan yaitu tidak mempunyai kebiasaan mengkonsumsi xylitol, tidak sedang menjalani terapi antibiotik sistemik, tidak sedang perawatan topikal aplikasi fluoride dalam kurun waktu 4 minggu sebelum penelitian dimulai, tidak menggunakan obat kumur antiseptik selama penelitian, mempunyai kesehatan gigi dan mulut yang baik (skor indeks dmf-t = 0-2) dan karies sudah dirawat, serta kebiasaan menyikat gigi teratur 2 kali sehari menggunakan pasta gigi berfluoride. subjek diberi penjelasan secara verbal dan tertulis, kemudian diberikan informed consent yang diisi oleh orangtuanya. subjek dihimbau untuk menjaga kebersihan mulutnya dengan kebiasaan menyikat gigi 2 kali sehari. subjek dalam penelitian ini dibagi dalam 2 kelompok dengan masing-masing kelompok terdiri dari 13 anak. kelompok a diminta untuk mengkonsumsi 150 ml yoghurt rasa strawberry yang mengandung bifidobacterium bifidum, bb-12: 5.2 x 107 kol dan lactobacillus acidophillus, la5: 3.2 x 108 kol (bio kul diamondtm), sedang kelompok b diminta untuk mengkonsumsi susu strawberry 140 ml (milkuat danonetm). subjek tidak diperbolehkan menggosok gigi selama satu jam setelah mengkonsumsi yoghurt. yoghurt dan susu diberikan secara acak dimana kedua sediaan yang diberikan memilki cita rasa dan konsistensi yang sama, kemudian diletakkan pada botol yang sudah ditandai ‘a’ atau ‘b’. kadarnya tidak diketahui, baik oleh subjek yang diuji atau peneliti dan kodenya tidak akan diungkapkan sampai setelah uji statistik (double blind dengan rancangan penelitian randomized crossover design). pada seminggu pertama kedua kelompok tidak diperkenankan mengkonsumsi yoghurt dan susu untuk menyamakan kondisi awal. kemudian kedua kelompok diminta mengkonsumsi yoghurt atau susu sekali sehari setelah makan malam selama 2 minggu. setelah 2 minggu kedua kelompok tidak diperbolehkan mengkonsumsi yoghurt dan susu selama 6 minggu, hal ini merupakan upaya menghilangkan efek kedua minuman tersebut (washout). setelah periode washout, kedua kelompok bertukar minuman, kelompok a mengkonsumsi susu dang kelompok b mengkonsumsi yoghurt selama 2 minggu lagi. 84 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 82–86 setiap sebelum dan sesudah mengkonsumsi yoghurt dan susu, sampel saliva subjek diambil tanpa stimulasi pada pagi hari pukul 6.30 wib sebelum menggosok gigi dan saliva segera ditampung dalam tabung reaksi. saliva diambil 0,1 ml menggunakan mikropipet. pengambilan saliva ini dilakukan 4 kali, yaitu pada hari ke-8, ke-22, ke-65, dan hari ke-77. sampel saliva segera dibawa ke laboratorium mikrobiologi fakultas kedokteran gigi universitas airlangga untuk dilakukan pembiakan bakteri streptococcus mutans yang dikultur pada media brain heart infusion (bhi) yang diinkubasi pada suhu 37o c selama 24 jam dan masing-masing diambil sebanyak 0,5 ml untuk dilakukan pengenceran sebanyak 4 kali. pada pengenceran keempat, sampel saliva diambil mengunakan mikropipet sebanyak 0,1 ml dari media bhi dan ditanam pada petridish dengan media agar tryptone yeast cystine (tyc) menggunakan metode spreading dan diinkubasi pada suhu 37o c selama 48 jam. dilakukan penghitungan jumlah koloni streptococcus mutans (cfu) dan morfologinya diidentifikasi melalui bantuan mikroskop binokuler cahaya dengan pembesaran 1000x. hasil data penelitian ini dianalisa dengan program statistik (spss inc., versi 15.0, chicago, illinois, usa). hasil dari hasil yang diperoleh menunjukkan adanya penurunan jumlah koloni streptococcus mutans pada subjek setelah mengkonsumsi yoghurt yang mengandung bifidobacterium bifidum, bb-12: 5,2 x 107 kol dan lactobachillus acidophillus, la-5: 3,2 x 108 kol (bio kul diamondtm) (gambar 1). hasil perhitungan yang diuji melalui analisa uji mann-whitney test didapatkan hasil perhitungan rerata tertinggi penurunan jumlah koloni streptococcus mutans adalah pada kelompok yang mengkonsumsi yoghurt. hal ini menunjukkan bahwa probiotik yoghurt memiliki efektifitas yang lebih baik dalam menurunkan jumlah koloni streptococcus mutans (tabel 1). pembahasan pada penelitian ini jumlah streptococcus mutans dalam saliva dihitung dengan menggunakan uji hitung koloni dan hasil uji statistiknya menunjukkan koefisien korelasi yang kuat (r = 0,43). penelitian sebelumnya menggunakan caries-risk-test (crt) untukmendeteksi streptocoocus mutans dan hasil hitung streptococcus mutans dalam saliva juga menunjukkan koefisien korelasi yang kuat dengan menggunakan metode konvensional laboratoris (r = 0,76).10-12 yoghurt rasa buah dipilih sebagai bahan yang digunakan dalam penelitian ini yang merupakan suplemen probiotik karena bahan ini memiliki kapasitas buffer yang tinggi dan non erosive serta potensi terhadap kariesnya rendah.13 selain itu, yoghurt sangat digemari oleh anakanak dan tidak perlu penyesuaian untuk dikonsumsi. tidak ada efek samping yang ditemukan setelah mengkonsumsi yoghurt selama periode trial. hasil penelitian ini menunjukkan bahwa subjek yang mengkonsumsi yoghurt secara rutin selama 2 minggu terjadi penurunan jumlah streptococcus mutans pada salivanya dan hasil ini diperkuat dengan penelitian sebelumnya yang tabel 1. prosentase penurunan koloni streptococcus mutanssetelah konsumsi yoghurt dan susu selama 2 minggu sediaan jumlah subjek mutans streptococci menurun tetap meningkat prosentase penurunan s. mutans setelah konsumsi probiotik yoghurt strawberry selama 2 minggu 26 20 subjek 5 subjek 1 subjek 76.9 % setelah konsumsi susu rasa strawberry selama 2 minggu 26 5 subjek 21 subjek 0 19,23 % 1 grafik 1. perbandingan selisih rata-rata jumlah koloni streptococcus mutans grup a dan grup b setelah pemberian yoghurt dan susu pada periode 2 dan periode 4 -40 -20 0 20 40 60 80 1 2 3 4 m ea n before after difference susu grup a yoghurt grup a yoghurt grup b susu grup b gambar 1. perbandingan selisih rata-rata jumlah koloni streptococcus mutans grup a dan grup b pada pemberian yoghurt dan susu pada periode 2 dan periode 4. 85bibi, at al.: penurunan jumlah streptococcus mutans pada saliva anak menggunakan strain bakteri bifidobacterium animalissub sp. lactis dn-173010 (2 x 108) cfu/g dan lactobacillus sebagai derivate probiotik.14,15 pertumbuhan bakteri patogen dapat dihambat oleh bakteri probiotik ini dijelaskan melalui kombinasi sistem imun sistemik dan lokal serta mekanisme pertahanan tubuh.16 prinsip imunologi mengatakan bahwa semakin meningkatnya pertahanan imun mukosa dan aktivitas makrofag akan meningkatkan sel killer, sel-t dan interferon.17 bakteri probiotik dapat mempengaruhi sistem imun, baik lokal maupun humoral. pada respon imun seluler dikatakan bahwa probiotikakan meningkatkan proliferasi splenosit sebagai akibat mitogen untuk sel-t dan sel-b.18 probiotik juga berperan terhadap peningkatan produksi sitokin, sebagai contoh strain streptococcus thermophillus akan meningkatkan produksi sitokin tnf dan il-6 melalui sel makrofag. strain lactobacillusbulgaricus, bifidobacterium culolescenti dan bifidobacterium bifidum akan meningkatkan produksi il-6 melalui sel t-helper.19 bakteriosin yang dihasilkan bakteri probiotik merupakan senyawa peptida antimikroba, yaitu senyawa dengan berat molekul rendah baik berupa protein peptida pendek yang memiliki aktivitas menghambat atau membunuh mikroba (antimikroba), sebagai akibat dari lemahnya proton motive force dengan cara merusak struktur dan komponen membran sel bakteri streptococcus mutans dan lactobacillus.20 bakteriosin yang dihasilkan akan menetap dalam rongga mulut, dapat beradhesi dengan berbagai sel dalam mulut dan menghasilkan bakteriosin tipe lanbiotik yang poten terhadap bakteri gram positif sehingga terjadi hambatan pertumbuhan bakteri streptococcus mutans dan lactobacillus.21 bifidobacterium bifidum dan lactobacillus acidophillus berperan sebagai immunomodulator terutama akan meningkatkan jumlah sel penghasil iga sekretori (siga) yang spesifik dalam saliva dan sel penghasil immunoglobulin lainnya, serta merangsang pelepasan interferon lokal yang memfasilitasi transport antigen. siga merupakan immunoglobulin yang dominan dalam saliva dan berperan dalam proteksi terhadap mikroorganisme kariogenik. siga dalam saliva dapat menghambat permulaan perlekatan streptococcus mutans pada pelikel saliva di permukaan gigi. aktivitas siga juga dapat menghambat fungsi enzim glukosiltransferase dari streptococcus mutans.22 lactobachillus acidophillus mampu berkompetisi dengan bakteri lain yang bersifat patogen, dengan mempertahankan keadaan homeostasis dari lingkungannya, hal ini disebut sebagai produksi dl-lactic acid, yaitu pembentukan asam laktat dan amilase yang dapat membantu pertumbuhan bakteri probiotik lain menghasilkan asam laktat dan amilase yang dapat membantu pertumbuhan bakteri probiotik lain menghasilkan asam laktat.23 mikroba yang berada berbatasan dengan breket ortodontik dapat menjadi jalan masuk langsung dan ditempati oleh spesies streptococcus mutans yang nantinya diganti oleh bifidobacteria yang tidak berbahaya. teori ini dijelaskan secara in vitro dan menunjukkan bahwa bifidobacteria dapat bertahan hidup dalam saliva dan berikatan dengan fusobacterium nucleatum pada hidroksiapatit yang tertutupi.24 probiotik juga pernah diteliti dalam hubungannya dengan kesehatan mulut. di bidang kedokteran gigi, penelitian dengan bifidobacterium dan lactobacillus menunjukkan kemampuan kedua bakteri tersebut dalam berinteraksi dengan streptococcus mutans dan menurunkan jumlah bakteri patogen tersebut.25 penelitian cildir et al.8 menyatakan bahwa bifidobacterium dan lactobacillus dalam yoghurt mengurangi kemampuan perlekatan dari streptococcus mutans dan kedua bakteri ini dapat berkolonisasi dan melekat pada permukaan email. hal ini menunjukkan peran probiotik dalam profilaksis karies. perbedaan penelitian ini dengan penelitian sebelumnya cildir et al.8 adalah perbedaan probiotic strain dan dosis probiotik yang dikonsumsi dalam sehari. pada penelitian ini menggunakan probiotik yoghurt yang mengandung strain bakteri bifidobacterium bifidum, bb-12: 5,2 x 107 kol dan lactobachillus acidophillus, la-5: 3,2 x 108 kol (bio kul diamondtm) dengan dosis 150 ml/hari dan kontrol menggunakan susu sapi (milkuat danone tm), sedangkan penelitian terdahulu menggunakan probiotik yoghurt yang mengandung strain bakteri bifidobacterium animalissub sp. lactis dn-173010 (2 x 108) cfu/g dan lactobacillus (activia cilekli meyveli, danone, istanbul, turkey) dan kontrol menggunakan yoghurt (cilek meyveli) dengan dosis 200 gram/hari. tujuan dilakukannya perbedaan ini adalah melihat efek probiotik dengan strain yang berbeda dan dosis yang lebih rendah sudah berpengaruh terhadap penurunan jumlah koloni streptococcus mutans. penentuan subjek pada penelitian ini adalah anak dan remaja dimana merupakan subjek yang tepat karena berhubungan dengan faktor resiko terjadinya demineralisasi enamel. penelitian ini menunjukkan bahwa mengkonsumsi probiotik yoghurt bifidobacterium bifidum, bb-12: 5,2 x 107 kol dan lactobachillus acidophillus, la-5: 3,2 x 108 kol setiap hari secara rutin efektif menurunkan jumlah koloni streptococcus mutans pada saliva anak pemakai ortodontik cekat. daftar pustaka 1. kupietzky a, majumdar ak, shey z, binder r, matheson pb. colony forming unit levels of salivary lactobacilli and streptococcus mutans in orthodontic patiens. j clin pediatr dent 2005; 30(1): 514. 2. mitchell l. decalcifiction during orthodontic treatment with fixed appliance an overview. br j orthod 1992; 19(3): 199-205. 3. ahn sj, lim bs, lee sj. prevalence of cariogenic streptococci on incisor brackets detected by polymerase chain reaction. am j orthod dentofacial orthop 2007; 131(6): 736-41. 4. rasic jl. the role of dairy foods containing bifido and acidophillus bacteria in nutrition and health. north european dairy j 1983; 4: 80-8. 5. doron s, gorbach sl. probiotics: their role in the treatment and prevention of disease. expert rev anti infect ther 2006; 4(2): 26175. 86 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 82–86 6. reid g, jass j, sebulsky mt, mccormick jk. potential uses of probiotics in clinical practise. clin microbiol rev 2003; 16(4): 65872. 7. verschuere l, rombaut g, sorgeloos p, verstraete w. probiotic bacteria as biological control agents in aquaculture. microbiol mol biol rev 2000; 64(4): 655-71. 8. cildir sk, germec d, sandalli n, ozdemir f.i, arun t, twetman s, caglar e. reduction of salivary mutans streptococci in orthodontic patiens during daily consumption of yoghurt containing probiotic bacteria. eur j orthod 2009; 31: 407-11. 9. tahmourespour a, kermanshahi rk. the effect of a probiotic strain (lactobacillus acidophillus) on the plaque formation of oral streptococci. bosn j basic med sci 2011; 11(1): 37-40. 10. scheie a a, arneberg p, krogstad o. effect of orthodontic treatment on prevalence ofstreptococcus mutans in plaque and saliva. scand j dental res 1984; 92: 212-7. 11. twettman s, stahl b, petersson lg. caries risk assesment in schoolchildren with two different chair-side-tests. prophylaxe impuls 2000; 4: 66-70. 12. karjalainen s, söderling e, pienihäkkinen k. validation and interexaminer agreement of mutans streptococci levels in plaque and saliva of 10-year-old children using simple chair-side tests. acta odontol scand 2004; 62(3): 153-7. 13. caglar e, lussi a, kargul b, ugur k. fruit yoghurt: any errosive potential regarding teeth?. quintessence int 2006; 37(8): 647-51. 14. kargul b, caglar e, lussi a. errosive and buffering capacities of yogurt. quintessence int 2007; 38(5): 381-5. 15. caglar e, sandalli n, twetman s, kavaloglu s, ergeneli s, selvi s. effect of yogurt with bifidobacterium dn-173 010 on salivary mutans streptococci and lactobacilli in young adults. acta odontol scand 2005; 63(6): 317-20. 16. meurman jh. probiotics. do they have a role in oral medicine and dentistry?. eur j oral sci 2005; 113: 188-96. 17. fuller r, gibson gr. modification of the intestinal microflora using probiotics. scand j gastroenterol 1997; 222: 28-31. 18. simone cdr. the role of probiotics in modulation of immune system in man and in animal. int j immmunother 1993; 9: 23-8. 19. nicaise p, gleizes a, forestier f, quéro am, labarre c. influence and intestinal bacterial flora on cytokine (il-1, il-6 and tnf-alpha production by mouse peritoneal macrophages. eur cytokine netw 1993; 4(2): 133-8. 20. marshall sh. antimicrobial peptides: as natural alternative to chemical antibiotics and a potential for applied biotechnology. electron j biotech 2003; 3-6. 21. burton jp, chilcott cn, tagg jr. the rationale and potential for the reduction of oral maladour using streptococcus salivarius probiotics. oral dis 2005; 11(suppl 1): 29-31. 22. devijanti r. antibodi monoklonal streptococcus mutans 1 (c) 67 kda dalam pasta gigi untuk menghambat pertumbuhan streptococcus mutans. adln lib universitas airlangga; 2004. h. 34-6. 23. fernandez antonio jf, domingo teresa a, oltra david p, diago minguel p. probiotic treatment in oral cavity: an update. j oral medicine and pathology valencia university medical and dental school 2007; 1-3. 24. haukioja a, yli-knuuttila h, loimaranta v, kari k, ouwehand ac, meurman jh, tenovuo j. oral adhesion and survival of probiotic and other lactobacilli and bifidobacteria in vitro. oral microbiol and immunol 2006; 21(5): 326-32. 25. agarwal e. probiotics: a novel step towards oral health. archives of oral science & research 2011; 1(2): 108-15. 150 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 150–154 original article ethanol extract of imperata cylindrica leaves inhibits proliferation and migration of hsc-3 cell lines moehamad orliando roeslan1 and gabriella tasha2 1department of oral biology, faculty of dentistry, trisakti university, jakarta, indonesia 2undergraduate student, faculty of dentistry, trisakti university, jakarta, indonesia abstract background: squamous cell carcinoma (scc) is classified as the most common type of oral cancer up to 90% of all malignant neoplasm in the oral cavity. currently, the only treatments for scc are surgery and/or radiation or chemotherapy, which can cause various side effects. cogon grass leaves (imperata cylindrica) have been considered an alternative cancer treatment that may reduce side effects. imperata cylindrica (i. cylindrica) leaf extract can inhibit cancer cell proliferation and migration by withholding the cell cycle in the gap 1/synthesis (g1/s) and gap 2/mitosis (g2/m) phases. therefore, the levels of matrix metalloproteinase-2 (mmp-2) and matrix metalloproteinase-9 (mmp-9) is decreased and cancer is not progressing. purpose: the study aims to determine the effect of i. cylindrica leaf extract on the proliferation and migration of human oral squamous carcinoma-3 (hsc-3) cell lines. methods: this in vitro experimental study was conducted with nine study groups. the treatment group was divided into seven concentrations—640 ppm, 320 ppm, 160 ppm, 80 ppm, 40 ppm, 20 ppm and 10 ppm. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium-bromide (mtt) assay and scratch assay were carried out to assess the effect of i. cylindrica leaf extract on hsc-3 cell proliferation and migration. results: ethanol extract of i. cylindrica has a significant effect compared to the negative control towards the proliferation and migration of hsc-3 cells. conclusion: this study shows that i. cylindrica ethanol leaf extract can inhibit proliferation and migration of hsc-3 cells. keywords: hsc-3 cell; imperata cylindrica; mtt assay; oral cancer; scratch assay correspondence: moehamad orliando roeslan, department of oral biology, faculty of dentistry, trisakti university. jl. kyai tapa no. 1, jakarta barat 11440, jakarta, indonesia. email: orliando.roeslan@trisakti.ac.id introduction oral cavity scc is a type of cancer in humans that has a high prevalence in comparison to all malignant oral cancers.1 the incidence of scc mainly occurs in people over 40 years of age. its prevalence in men is greater than in women. the aetiology of oral scc is multi-factorial—a combination of genetic and predisposing factors.2 the lateral side of the tongue, gingival mucosa, tongue ventral and the floor of the mouth are the predilections of scc.3 referring to world health organization (who) data in 2018, the death rate for oral cancer was ranked at 15 from those of 33 types of cancer, namely 177,384 deaths.4 currently, the treatment for scc is surgery alone, or it can be combined with radiation or chemotherapy.5 in the world of modern medicine, traditional medicine is starting to be developed again because of the high incidence of oral cancer.6,7 one of the natural ingredients that has anticancer activity is cogon grass leaves (i. cylindrica).8 previous research showed that the leaf extract of i. cylindrica has an effect on the scc-9 cell line by inhibiting cell proliferation.9 cell migration can be reduced by the presence of flavonoids and alkaloids in the leaves of i. cylindrica. they achieve this by decreasing the expression of mmp-2 and mmp-9, which are proenzyme members of zinc-endopeptidases and play a role in cancer development.10 however, there was no similar study that used leaf extract of i. cylindrica on the hsc-3 cell line. cancer cell line hsc-3 cells are a type of the human oral scc cell culture that is suitable for use as a study model. this cell line has the highest metastatic ability among cells other cultures, such as hsc-2, hsc-4 and hsc-7.11 the aim of this study is to determine effect of i. cylindrica leaf extract on the proliferation and migration of hsc-3 cell lines. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p150–154 mailto:orliando.roeslan@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p150-154 151roeslan and tasha/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 150–154 materials and methods the leaves of i. cylindrica were grown and taken from bogor, west java, indonesia. the leaves, stem and root of the i. cylindrica were sent to the indonesian institute of science for authentication. after the leaves dried under indirect sunlight, they were ground to powder. five hundred millilitres of ethanol was used to extract 50 g of leaf powder into a soxhlet extractor at 79°c for 70 hours. after that, the liquid extract was evaporated in a rotary evaporator at 50°c under vacuum. the leaf extract of i. cylindrica was stored at 4°c in the refrigerator until it was used.12 qualitative phytochemical screening was done to assess the constituents in ethanol extract of the i. cylindrica leaves. five percent of the sodium hydroxide (naoh) solution was mixed to 1 ml of crude extract, which generated a yellow colour. the presence of flavonoid was confirmed after hydrogen chloride (hcl) was added and the yellow colour disappeared.13 one milligram of extract was dissolved with 10 ml of chloroform, then an equal volume of sulphuric acid (h2so4) was added. the presence of steroid was confirmed when the h2so4 layer turned yellow with green fluorescence and the layer above it turned red.14 the crude extracts were mixed with 1 ml of chloroform and h2so4. the presence of terpenoid was confirmed after it generated a red-brown colour.15 fifty grams of crude extract was mixed with 5% ferric chloride (fecl3.)the presence of tannin was confirmed after the development of a bluish black colour.13 dragendorff’s solution was added to 0.1 g of extract. the development of an orange-red precipitate confirmed the presence of alkaloid.13 the hsc-3 cell line was cultured in dulbecco’s modified eagle’s medium (dmem; gibco, usa) with an addition of 10% fetal bovine serum (fbs; gibco, usa) and 1% penicillin-streptomycin (invitrogen, usa). the cells were incubated under humidified conditions at 37°c and in atmospheric air supplemented with 5% co2 in an incubator (memmert, germany). the medium was changed regularly and the cells were passaged every 3-4 days, passaged 3 were used for experiments.12 a viable cell number was determined by the mitochondrial-dependent reduction of the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2h-tetrazolium-bromide (mtt; sigma aldrich, usas) method as previously described.16 the number of cells being cultured in 96-well plates was 1 ×x 105 cells/well, then were incubated for 24 hours. the cells were then treated with various concentrations (10, 20, 40, 80, 160, 320 and 640 ppm) of i. cylindrica extract for 24 hours. positive control was 3 µm of doxorubicin. after mtt was added, the final concentration was 0.5 mg/ml. the cells were then incubated for four hours at 37°c and 5% co2. then dimethyl sulfoxide (sigma aldrich, usa) was added and a microplate reader (tecan, salzburg, austria) was used to measure the absorbance at 570 nm. the formula to calculate cell viability (%) is: table 1. qualitative phytochemical assay of ethanol extracts from i. cylindrica assay results flavonoid + steroid + terpenoid + tannin + alkaloid + [a] test / [a] control x 100 where [a] test is the absorbance of the test sample and [a] control is the absorbance of the control. the in vitro scratch assay was used to test the effects of the i. cylindrica leaf extract on the migration of hsc-3.17 the cells (1 ×x 106 cells/well) were cultured in 6-well plates containing dmem with an addition of 10% fbs and 1% penicillin-streptomycin. after it reached 90% confluence, the scratches were created vertically and horizontally in the centre of the well and monolayers of each well. phosphatebuffered saline was used to wash cellular debris from the scratch. the cells in the well were then treated with various concentrations (10, 20, 40, 80, 160 and 640 ppm) of i. cylindrica leaf extract. the scratches were observed under an inverted microscope periodically (0, 6, 12 and 24 hours). the negative control was dmem without extracts. three µm of doxorubicin was used as the positive control. image j software (national institutes of health, bethesda, md) was used to calculated the gap area in the monolayers after pictures were taken. the formula to calculate gap closure is: percentage gap closure = [(at=0h–at=24h)] x 100 at=0h where, at=0h is the area of the wound measured immediately after scratching and at=24h is area of the wound measured 24 hours after scratching. the shapiro-wilk test was used to asses normality. the differences between experimental groups were analysed using one-way and two-way analysis of variance (anova) with the post-hoc test. a p-value (p<0.05) was considered statistically significant. results qualitative phytochemical assay showed that i. cylindrica ethanol leaf extract contains flavonoid, steroid, terpenoid, tannin and alkaloid (table 1). the results of the cell proliferation assay indicated that the i. cylindrica ethanol leaf extract at all concentrations was significantly different compared to the negative control (figure 1). while compared to the positive control, concentrations 640, 320, 160, 80 and 40 ppm had no significant difference. the results of the scratch assay showed that a small percentage means that the gap is closing. the negative control group showed that at six hours, the gap started to close by 61.82% and at 12 hours the whole gap was dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p150–154 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p150-154 152 roeslan and tasha/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 150–154 already closed by the cells. on the contrary, the positive control and concentration 640 ppm groups showed that at six hours, cells had already undergone apoptosis. while at concentration 320 ppm, from six to 24 hours, the gap was more open (114% to 145%). this means that after a longer time, cells started to undergo apoptosis. at concentration 10 to 160 ppm, it showed that after a longer time, the gap was closing. it means that concentration 10 to 160 ppm could not inhibit hsc-3 migration (table 2). discussion in previous studies, i. cylindrica leaf extract which contained flavonoid and alkaloid showed anti-cancer activity by inhibiting proliferation, inflammation, angiogenesis, invasion and metastasis.18,19 flavonoid and alkaloid work by inducing the inhibition of the cell cycle in the gap 1/synthesis (g1/s) and gap 2/mitosis (g2/m) phases and cell apoptosis. this ultimately leads to inhibition of cancer cell proliferation.20 the results of the cytotoxicity assay showed that concentrations 40 to 640 ppm had no significant difference compared to the positive control. this showed that as low as at concentration 40 ppm, i. cylindrica leaf extract could inhibit proliferation of hsc-3. this result is in line with previous research which stated that i. cylindrica leaf extract can inhibit proliferation and induce apoptosis of scc-9.21 however, in that research the highest inhibition was approximately 50%, while in this research the highest inhibition was 74.5%. this could be because of cell difference and solvent difference for extracting from the plant. previous research also showed that i. cylindrica leaf extract could decrease proliferation (inhibition up to 50%) of the colorectal cancer cell line (ht-29).22 the benefit of this scratch assay is that it can study the regulation of cell migration through cell–extracellular matrix (ecm) interactions and cell–cell interactions.23 in this study, the concentration of 640 ppm at 6, 12 and 24 hours could induce hsc-3 to undergo apoptosis, the same as the positive control. at concentration 320 ppm, some of the cells already started to undergo apoptosis at table 2. scratch assay of hsc-3 under various concentrations. the data is expressed as mean ± sd (n = 3) concentration hour 0 6 12 24 control (-) 100 61.82 ± 49.25 cell already closed the gap cell already closed the gap control (+) 100 apoptosis apoptosis apoptosis 640 ppm 100 apoptosis apoptosis apoptosis 320 ppm 100 114.64 ± 19.64 125.53 ± 19.64 145.55 ± 19.64 160 ppm 100 93.69 ± 6.26 89.5 ± 6.26 85.31 ± 6.26 80 ppm 100 87.62 ± 9.48 87.19 ± 9.48 76.81 ± 9.48 40 ppm 100 75.099 ± 29.3 69.13 ± 29.3 29.2 ± 29.3 20 ppm 100 73.05 ± 43.99 33.13 ± 43.99 cell already closed the gap 10 ppm 100 77.01 ± 43.22 46.48 ± 43.22 cell already closed the gap figure 1. cell proliferation after treated with ethanol extracts of i. cylindrica leaves with various concentration. negative control was without treatment. positive control was with 3 µm of doxorubicin. the data was expressed as mean ± sd (n = 3). * indicates a significant difference versus the negative control group (p<0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p150–154 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p150-154 153roeslan and tasha/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 150–154 6 hours. at 12 and 24 hours more cells had undergone apoptosis. meanwhile, at concentration 10 to 160 ppm, i. cylindrica leaf extract could not induce hsc-3 to undergo apoptosis. vimentin, n-cadherin and β-catenin are proteins that play a role in hsc-3 becoming less invasive. these proteins affect cancer invasion and tumour metastasis. the alteration of cell state in hsc-3 cells was also caused by epithelial-mesenchymal transition (emt) programs that are characterized by a mesenchymal marker.24 a previous study showed that inhibition of emt promoters, the epithelial cell adhesion molecule (epcam) and survivin-1 also inhibit cancer cell migration.25 based on the qualitative phytochemical assay, the i. cylindrica leaf extract that is used in this research contains flavonoid, steroid, terpenoid, tannin and alkaloid. in carcinogenesis, flavonoid are secondary plant metabolites that have the ability to decrease proliferation, metastasis and angiogenesis and to increase apoptosis by interfering in multiple signal transducing pathways.26 flavonoid that are distributed mainly in leaves are flavones.27 apigenin, one of the flavones, induced the scc25 cell to apoptosis by leading to halting the cell at the g2/m phase via an increasement level of reactive oxygen species (ros).28 in a previous study, apigenin induced achn (kidney-cancerderived cell line), 786-0 cell lines to undergo apoptosis via cell arrest at the g2/m phase, dna damage and p53 upregulation.29 luteolin, another flavones, induced hela cells to undergo apoptosis by the escalation of caspase-3 and -8, receptor (fas/fasl, dr5/trail, and fadd) upregulation and inhibition of b-cell lymphoma 2 (bcl-2) and b-cell lymphoma-extra large (bcl-xl) expression.30 terpenoids consist of 4 large groups—steroids, terpenes, saponins and cardiac glycosides. every year new terpenoid are being discovered. there are 40,000 compounds that are included in terpenoids.31 a previous study showed annona cherimola, with a high content of terpenoid, induced apoptosis of acute myeloid leukemia cell lines through a bax/bcl-2 dependent mechanism.32 the shifting of bax in cytosol to mitochondria through the bax pores caused apoptosis via dna damage.33 a previous study showed that steroid induced apoptosis via enhancement of caspase-3.34 alkaloid isolated from zanthoxylum nitidum possesses the ability to induce apoptosis on hsc-3 and hsc-4 cell lines via blockage expression of signal transducer and activator of transcription 3 (stat3).35 the stat3 signalling pathway showed a contribution to the progression of a tumor.36 another previous study on alkaloid from a medicinal plant, showed apoptosis activity on jurkat t cells by upregulating caspase-8 and -9 and effector caspases-3 and -6. it also enhanced expression of bax and p53.36 flavonoid, steroid, terpenoid, and alkaloid from medicinal plants have many mechanisms to induce apoptosis on various cancer cell lines. these compounds are found in i. cylindrica leaf extract and promising as anti-cancer agents. in the future, we need to investigate the predominant compound in i. cylindrica that possesses activity to induce apoptosis in cancer cells. in conclusion, i. cylindrica leaf ethanol extract could inhibit hsc-3 cell proliferation at concentration 10 ppm and higher. this extract also could inhibit hsc-3 migration at concentration 320 ppm at 6 hours and higher. acknowledgement the authors would like to thank dr. indra kusuma, m. biomed. from faculty of medicine, yarsi university, jakarta, indonesia, who provided the hsc-3 cells. references 1. johnson nw, jayasekara p, amarasinghe aa. squamous cell carcinoma and precursor lesions of the oral cavity: epidemiology and aetiology. periodontol 2000. 2011; 57(1): 19–37. 2. bilgic o, duda l, sánchez md, lewis jr. feline oral squamous cell carcinoma: clinical manifestations and literature review. j vet dent. 2015; 32(1): 30–40. 3. pires fr, ramos ab, oliveira jb, tavares as, luz ps, santos tc. oral squamous cell carcinoma: clinicopathological features from 346 cases from a single oral pathology service during an 8-year period. j appl oral sci. 2013; 21(5): 460–7. 4. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2018; 68(6): 394–424. 5. huang sh, o’sullivan b. oral cancer: current role of radiotherapy and chemotherapy. med oral patol oral cir bucal. 2013; 18(2): e233–40. 6. al-koshab m, alabsi am, mohd bakri m, ali-saeed r, selvi naicker m. antitumor activity of ficus deltoidea extract on oral cancer: an in vivo study. j oncol. 2020; 2020: 5490468. 7. hassabou nf, farag af. anticancer effects induced by artichoke extract in oral squamous carcinoma cell lines. j egypt natl canc inst. 2020; 32: 17. 8. fatmawati s. bioaktivitas dan konstituen kimia tanaman obat indonesia. yogyakarta: deepublish; 2019. p. 181. 9. mbaveng at, kuete v, efferth t. potential of central, eastern and western africa medicinal plants for cancer therapy: spotlight on resistant cells and molecular targets. front pharmacol. 2017; 8: 343. 10. quintero-fabián s, arreola r, becerril-villanueva e, torresromero jc, arana-argáez v, lara-riegos j, ramírez-camacho ma, alvarez-sánchez me. role of matrix metalloproteinases in angiogenesis and cancer. front oncol. 2019; 9: 1370. 11. ma d, fang j, liu y, song jj, wang yq, xia j, cheng b, wang z. high level of calpain1 promotes cancer cell invasion and migration in oral squamous cell carcinoma. oncol lett. 2017; 13(6): 4017–26. 12. tessalonica s, roeslan mo. effect of ethanol extracts from persea americana leaves on hsc-3 proliferation. j indones dent assoc. 2020; 3(2): 65–70. 13. roghini r, vijayalakshmi k. phytochemical screening, quantitative analysis of flavonoids and minerals in ethanolic extract of citrus paradisi. int j pharm sci res. 2018; 9(11): 4859–64. 14. hossain ma, al-raqmi ka, al-mijizy zh, weli am, al-riyami q. study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown thymus vulgaris. asian pac j trop biomed. 2013; 3(9): 705–10. 15. das bk, al-amin mm, russel sm, kabir s, bhattacherjee r, hannan jma. phytochemical screening and evaluation of analgesic activity of oroxylum indicum. indian j pharm sci. 2014; 76(6): 571–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p150–154 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p150-154 154 roeslan and tasha/dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 150–154 16. riss tl, moravec ra, niles al, duellman s, benink ha, worzella tj, minor l. cell viability assays. in: markossian s, grossman a, brimacombe k, arkin m, auld d, austin cp, baell j, chung tdy, coussens np, dahlin jl, devanarayan v, foley tl, glicksman m, hall md, haas j v, hoare srj, inglese j, iversen pw, kales sc, lal-nag m, li z, mcgee j, mcmanus o, riss t, xu x, editors. assay guidance manual. bethesda (md): eli lilly & company and the national center for advancing translational sciences; 2004-; 2016. p. 1–25. 17. roeslan mo, ayudhya tdn, yingyongnarongkul be, koontongkaew s. anti-biofilm, nitric oxide inhibition and wound healing potential of purpurin-18 phytyl ester isolated from clinacanthus nutans leaves. biomed pharmacother. 2019; 113: 108724. 18. isah t. anticancer alkaloids from trees: development into drugs. pharmacogn rev. 2016; 10(20): 90–9. 19. romagnolo df, selmin oi. flavonoids and cancer prevention: a review of the evidence. j nutr gerontol geriatr. 2012; 31(3): 206–38. 20. stoddart mj. cell viability assays: introduction. in: stoddart m, editor. mammalian cell viability methods in molecular biology (methods and protocols), vol 740. humana press; 2011. p. 1–6. 21. keshava r, muniyappa n, gope r, ramaswamaiah as. anti-cancer effects of imperata cylindrica leaf extract on human oral squamous carcinoma cell line scc-9 in vitro. asian pacific j cancer prev. 2016; 17(4): 1891–8. 22. kwok ah, wang y, ho ws. cytotoxic and pro-oxidative effects of imperata cylindrica aerial part ethyl acetate extract in colorectal cancer in vitro. phytomedicine. 2016; 23(5): 558–65. 23. cory g. scratch-wound assay. in: wells cm, parsons m, editors. cell migration methods in molecular biology (methods and protocols), vol 769. humana press; 2011. p. 25–30. 24. kim dh, xing t, yang z, dudek r, lu q, chen yh. epithelial mesenchymal transition in embryonic development, tissue repair and cancer: a comprehensive overview. j clin med. 2017; 7(1): 1–25. 25. yang f, ma j, wan j, ha w, fang c, lu h, zhang w. epithelialmesenchymal transition of circulating tumor cells in prostate cancer is promoted by survivin. j int med res. 2020; 48(1): 300060519892395. 26. ravishankar d, rajora ak, greco f, osborn hm. flavonoids as prospective compounds for anti-cancer therapy. int j biochem cell biol. 2013; 45(12): 2821–31. 27. panche an, diwan ad, chandra sr. flavonoids: an overview. j nutr sci. 2016; 5: e47. 28. chan lp, chou th, ding hy, chen pr, chiang fy, kuo pl, liang ch. apigenin induces apoptosis via tumor necrosis factor receptor and bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin. biochim biophys acta gen subj. 2012; 1820(7): 1081–91. 29. meng s, zhu y, li jf, wang x, liang z, li sq, xu x, chen h, liu b, zheng xy, xie lp. apigenin inhibits renal cell carcinoma cell proliferation. oncotarget. 2017; 8(12): 19834–42. 30. ham s, kim kh, kwon th, bak y, lee dh, song ys, park sh, park ys, kim ms, kang jw, hong jt, yoon dy. luteolin induces intrinsic apoptosis via inhibition of e6/e7 oncogenes and activation of extrinsic and intrinsic signaling pathways in hpv-18-associated cells. oncol rep. 2014; 31(6): 2683–91. 31. stephane ffy, jules bkj. terpenoids as important bioactive constituents of essential oils. in: essential oils bioactive compounds, new perspectives and applications. intechopen; 2020. p. 1–32. 32. ammoury c, younes m, el khoury m, hodroj mh, haykal t, nasr p, sily m, taleb ri, sarkis r, khalife r, rizk s. the pro-apoptotic effect of a terpene-rich annona cherimola leaf extract on leukemic cell lines. bmc complement altern med. 2019; 19: 365. 33. wang p, wang p, liu b, zhao j, pang q, agrawal sg, jia l, liu ft. dynamin-related protein drp1 is required for bax translocation to mitochondria in response to irradiation-induced apoptosis. oncotarget. 2015; 6(26): 22598–612. 34. gr uver-yates a l, cidlowsk i ja. tissue-specific actions of glucocorticoids on apoptosis: a double-edged sword. cells. 2013; 2(2): 202–23. 35. kim lh, khadka s, shin ja, jung jy, ryu mh, yu hj, lee hn, jang b, yang ih, won dh, kwon hj, jeong jh, hong sd, cho np, cho sd. nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of stat3. oncotarget. 2017; 8(53): 91306–15. 36. xu w, wang x, tu y, masaki h, tanaka s, onda k, sugiyama k, yamada h, hirano t. tetrandrine and cepharanthine induce apoptosis through caspase cascade regulation, cell cycle arrest, mapk activation and pi3k/akt/mtor signal modification in glucocorticoid resistant human leukemia jurkat t cells. chem biol interact. 2019; 310: 108726. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p150–154 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p150-154 187187 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 187–191 research report the correlation between exposure to cigarette smoke and the degree of mucosal epithelium-based dysplasia in rattus norvegicus tongues dorisna prijaryanti,1 diah savitri ernawati,1 desiana radithia,1 hening tuti hendarti1 and rosnah binti zain2 1department of oral medicine, faculty of dental medicine, universitas airlangga. surabaya – indonesia 2department of oral pathology and oral medicine, faculty of dentistry, mahsa university, bandar saujana putra – malaysia abstract background: cigarette smoke contains various carcinogenic substances such as polycyclic aromatic hydrocarbons and nitrosamines. these chemicals not only have the potential to damage dna, but can also induce genetic mutations and activate genes that function during apoptosis. thus, if the gene is dysregulated, it will cause cells to survive, proliferate and subsequently lead to the development of cancerous ones. histologically, the carcinogenic process affecting the oral cavity starts with hyperplasia and dysplasia, followed by severe dysplasia then leading to invasive cancer and metastatic processes in other bodies. purpose: this study aims to reveal the correlation between exposure to cigarette smoke and the degree of epithelial dysplasia evident in research subjects. methods: this study used 27 samples of rattus norvegicus tongue, divided into three groups, namely; a control group, a treatment group subjected to four weeks’ exposure to cigarette smoke, and a treatment group subjected to exposure lasting eight weeks. each rat was placed in an individual chamber and exposed to smoke from 20 cigarettes introduced by a pump via a pipe for 7.5 minutes. the degree of epithelial dysplasia in each case was subsequently observed microscopically using he staining technique. results: mild epithelial dysplasia increased by 0.82%, during the fourth week of exposure to cigarette smoke and by 2.99% during the eighth week. similarly, moderate epithelial dysplasia rose by 5.29% during the fourth week of exposure and 5.99% during the eighth week. severe epithelial dysplasia also increased by 2.2% during the fourth week of exposure and by 2.66% during the eighth week. conclusion: the longer the exposure to cigarette smoke, the higher the degree of ensuing dysplasia. keywords: epithelial dysplasia, exposure to cigarette smoke, oral cancer, rattus norvegicus correspondence: desiana radithia, department of oral medicine, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: deisy.radithia@fkg.unair.ac.id introduction smoking is currently regarded as widespread across indonesia, indulged in on a daily basis by significant numbers of individuals.1 worryingly, the population of passive smokers is reportedly outstripping that of active ones.2 in fact, both categories of smoker run the risk of developing cancer since cigarettes contain various carcinogenic substances, such as polycyclic aromatic hydrocarbons (pahs) 3, and nitrosamines consisting of 4 (methylnitrosamino) -1(3-pyridyl) -1-butanone (nnk) and n’-nitrosonornicotine (nnn). each substance is known to have carcinogenic properties in relation to human beings.4 genetic apoptosis is a process of programmed cell death which spontaneously promotes soft tissue growth and plays a regulatory role in the physiological development of cells. dysregulation in the apoptotic pathway will promote the longevity of cells, while the resulting cell proliferation induces malignancy. consequently, apoptosis is considered a marker of oral epithelial dysplasia (oed) the presence of which is employed to determine the prognosis of a cancer. in an oral squamous cell carcinoma (oscc), a lack of apoptosis and an increased degree of dysplasia will usually be detected.5 oral epithelial dysplasia can increase in individuals whose daily consumption of cigarettes can be as high dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p187–191 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p187-191 188 prijaryanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 187–191 as 20.6 a previous study conducted by trombitas et al. (2016),7 posited that exposure to cigarette smoke can affect the severity of wounds in rats since epithelial dysplasia was detected in the nasal septum mucosa of those injured and exposed to cigarette smoke for a period of five days. in contrast, orthokeratotic keratinization was diagnosed in those injured but not subsequently exposed to cigarette smoke.7 hence, this study aims to examine the correlation between the duration of exposure to cigarette smoke and the degree of dysplasia in the lingual mucosal epithelium of rattus norvegicus. materials and methods this research was approved by the universitas airlangga faculty of dental medicine health research ethical clearance commission (eligibility number 067/hrecc. fodm/iii/2019). the subjects of this study comprised 27 three-month old, male rattus norvegicus, 170g (± 10%) in weight, which were divided into three groups, namely; a control group and two treatment groups. their respective members were initially required to undergo a one-week period of adaptation, demonstrate excellent health, have clear vision, shiny fur, agility, and excrete solid stools. the research subjects were housed in plastic box cages equipped with effective ventilation and lighting at room temperature. the members of all the control and treatment groups were placed on the same diet consisting of rice husks and water. treatment group 1 (p1) was exposed for four weeks to cigarette smoke delivered by means of a pump, while treatment group 2 (p2) was exposed for a period of eight weeks. the volume of smoke to which the subjects were exposed was maintained at a consistent daily level equivalent to that of 20 cigarettes. the smoke contained nicotine (1.2-4.5 mg), tar (46.8 mg), and carbon monoxide (28.3 mg).8 the group control (k1) members were sacrificed on the 29th day before surface tissue was removed from their tongues. meanwhile, the treatment group subjects were euthanised at weeks four (pi) and eight (p2) by placing them in glass boxes into which a lethal dose of ether was gradually introduced. the tongue mucosa was subsequently removed by means of a 2 cm scalpel incision before being placed in 10% formalin. tissue preparation was then completed. the degree of dysplasia was examined using he staining and a nikon e100 light microscope at 400x magnification. the data obtained was analysed with a statistical package for the social sciences (spss) 19 (ibm, new york, us). a manova test was conducted to determine the respective effects of 4and 8-week exposure to cigarette smoke on each degree of dysplasia in the mucosal epithelium of the research subjects’ tongues. however, prerequisite tests were initially performed, namely; variant homogeneity tests using levene’s test for equivalence of error variances and box’s test for equivalence of covariance matrices. the manova test results were then subjected to a range of analyses including; pillai’s trace, wilks’ lambda distribution, hotelling’s trace, and roy’s largest root. finally, in order to determine the correlation between exposure to cigarette smoke and each degree of dysplasia, a between subject effect analysis was carried out. meanwhile, a post hoc bonferroni test was conducted to identify differences in the degree of dysplasia demonstrated by the group exposed to cigarette smoke. results in the control group (k1) not exposed to cigarette smoke (figure 1), the condition of the squamous epithelial cells was found to be normal. meanwhile, in the four-week exposure to smoke group (p1), mild, moderate and severe epithelial dysplasia (figure 2) was detected, as was the case with the group exposed to smoke for eight weeks (p2), (figure 3). at this point, a number of pre-tests for homogeneity of variance and homogeneity of variance-covariance matrices were conducted, the results of which indicated significance values > 0.05. therefore, a manova test was performed whose results, as illustrated by the contents of table 1, showed that f values for pillai’s trace, wilks’ lambda distribution, hotelling’s trace, and roy’s largest root had a significance value of 0.000 <0.05. these findings confirmed the significant effect of 4and 8-week exposure to cigarette smoke on each degree of epithelial dysplasia in groups p1 and p2. moreover, based on the between subject effect test results contained in table 2, a correlation existed between exposure to cigarette smoke and the degree of dysplasia, figure 1. a histopathological image of the control group. normal squamous epithelial cells (he 400x). table 1. the effects of 4and 8-week exposure to cigarette smoke on each degree of dysplasia in the mucosal epithelium of rattus norvegicus tongue cigarette smoke exposure degree of dysplasia f significance pillai’s trace 15.284 0.000 wilks’ lambda 31.589 0.000 hotelling’s trace 58.874 0.000 roy’s largest root 124.058 0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p187–191 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p187-191 189prijaryanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 187–191 a c b d figure 2. pictures of epithelial dysplasia in group p1. a) mild dysplasia, b) moderate dysplasia, c) severe dysplasia, d) hyperplasia (microscope h&e 400x light). a b c d e figure 3. histopathological pictures in group p2. a) mild dysplasia, b) moderate dysplasia, c) severe dysplasia, d) hyperplasia, e) carcinoma in situ. (microscope h&e 400x light). table 2. the correlation between exposure cigarette smoke and each dysplasia degree in the mucosal epithelium of rattus norvegicus tongue degrees of dysplasia f significance m mild 47.073 0.000 0.780 moderate 43.317 0.000 0.765 severe 20.574 0.000 0.601 table 3. the results of a post hoc bonferroni test on the degrees of dysplasia in group k1, group p2, and group p3 degrees of dysplasia smoke exposure mean significance mild control – 4 weeks -3.11 0.050 control – 8 weeks -11.33 0.000 4 weeks – 8 weeks -8.22 0.000 moderate control – 4 weeks -10.00 0.000 control – 8 weeks -11.33 0.000 4 weeks – 8 weeks -1.33 0.979 severe control – 4 weeks -5.89 0.000 control – 8 weeks -7.11 0.000 4 weeks – 8 weeks -1.22 0.938 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p187–191 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p187-191 190 prijaryanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 187–191 such as mild, moderate, and severe with a significance value of 0.00 <0.05. this indicates that each degree of dysplasia was caused by an increase in the duration of exposure to cigarette smoke. the mean differences in the degree of dysplasia in group k1, group p1, and group p2 indicated an increase in the average degree of dysplasia in the mucosal epithelium of rattus norvegicus tongue along with the greater duration of exposure to cigarette smoke as can be seen from the contents of table 3. the degree of mild epithelial dysplasia increased in group p1 by 0.82% and by 2.99% in group p2. similarly, the degree of moderate epithelial dysplasia increased by up to 5.29% in group p1 and 5.99% in group p2. the degree of severe epithelial dysplasia also increased by 2.2% in group p1 and 2.66% in group p2. discussion in this study, the subjects were exposed to cigarette smoke for either 29 days or 59 days with the objective of rendering them more likely to become passive smokers, also referred to as second-hand smokers (shs) or environmental tobacco smokers (ets).9 actually, both active and passive smokers are suspected of possessing one of the predisposing factors for oral squamous cell carcinoma.10 according to the results of this study, those subjects exposed to cigarette smoke for 29 days presented mild dysplasia up to a level of 0.82%. 5.29%, also presented moderate dysplasia, while 2.2% suffered from severe dysplasia. on the other hand, 2.99% of those subjects exposed to cigarette smoke for 59 days presented mild dysplasia, while 5.99% had moderate dysplasia and 2.66% severe dysplasia. there was even a subject in group p2 which presented carcinoma in situ. a previous study conducted by martins et al. (2012)11 produced similar findings to those of this analysis, namely; that 60% of subjects exposed to cigarette smoke for 260 days presented lingual dysplasia, while a further 20% suffered from dysplasia of the pharynx and one subject had severe dysplasia or carcinoma in situ. similarly, another previous study conducted by martins et al. (2012) indicated that exposure to cigarette smoke carries the potential risk of carcinoma, although the treatment applied differed from that employed in this study.11 in the research conducted by martins et al. referred to above, the subjects were exposed to the smoke produced by ten cigarettes per day, while being provided with food and drink ad libitium. unlike martins’ study, this research involved the burning of 20 clove cigarettes (kreteks) per day which contained higher levels of nicotine (1.2-4.5 mg), tar (46.8 mg) and carbon monoxide (28.3 mg). moreover, the subjects were provided with food and drink devoid of additional substances. when a kretek is burned the temperature of a mixture of tobacco and cloves is raised with the result that the level of carbon dioxide and nicotine can be as much as three times as high, while that of tar may increase fivefold.12 moreover, another previous study performed by radwan et al. (2016),13 produced similar results. this previous study asserted that male albino rats passively exposed three times a day to the smoke produced by four cigarettes during a period of 40 days (group 2) or 60 days (group 3) would present symptoms of epithelial dysplasia. these results indicated that a lingual dorsum epithelium developed under normal conditions in the control group that had not been exposed to cigarette smoke (group 1), while the subjects in group 2 experienced a change in their epithelial cell architecture accompanied by desquamation and hyperkeratosis. in addition, the group 3 subjects presented symptoms of hyperkeratosis accompanied by macrophages and lymphocytes. according to xue et al. (2014), second hand smoke (shs)4 can increase the risk of nasopharyngeal cancer, nasal cavity cancer, breast cancer, leukemia, lymphoma, and brain tumors in children. shs contains a variety of toxic chemicals such as formaldehyde which can irritate the eyes, nose and throat, in addition to hydrogen cyanide, carbon monoxide, and ammonia which can weaken the natural airbourne cleaning mechanism which eradicates toxins. moreover, shs contains polycyclic aromatic hydrocarbons and nitrosamines potentially damaging to dna. arsenic, benzene, cadmium and tar constitute other chemicals also contained in tobacco.9 nitrosamines consist of 4(methylnitrosamino) -1 (3-pyridyil) -1-butanone (nnk), and n ‘nitrosonornicotine (nnn) which contain carbon monoxide, hydrogen cyanide, and oxygen radicals that induce an increase in ros (reactive oxygen species). as a result, the activation of ros has a pathological effect culminating in dna damage4,9 which will, in turn, stimulate p53 to activate the bcl-2 family consisting of anti-apoptosis proteins (bcl-2, bcl-xl and mcl1), pro-apoptotic protein (bax, bak), and the third protein (bh3), usually referred to as bh3only protein (bad, bid). bax is a pro-apoptotic protein which, if activated or increased, will induce porosity in the mitochondria culminating in the release of cytochrome c. this chain of events activates not only caspase 3 as the executor, but also caspase 9, while also causing apoptosis.14 in other words, if the anti-apoptotic protein is activated, the cytochrome release can activate caspase 9, resulting in the absence of apoptosis, thereby enabling cells to survive. nevertheless, if this condition occurs continuously, then abnormal cell proliferation will ensue. such abnormal cell proliferation can lead to hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia, and carcinoma in situ. similarly, this condition was found to have occurred by the research reported here following controlled exposure to cigarette smoke for 29 days and 59 days. the incidence of epithelial dysplasia was detected since oral epithelial dysplasia (oed) is histopathologically associated with an increased risk of oral cancer6 and can be considered one of the markers employed to detect early oral squamous cell carcinoma (oscc).15 jain (2019)16 even argues that, histologically, the process of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p187–191 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p187-191 191prijaryanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 187–191 oral carcinogenesis commences with hyperplasia, moving through dysplasia to severe dysplasia before, finally, culminating in invasive and metastatic conditions which reflect the combined changes transforming normal cells into cancerous ones. dysplasia in the epithelium is even considered to be the initial stage of cellular morphological change which precedes malignancy.15 in addition, epithelial dysplasia constitutes a developmental abnormality which indicates histological cellular and architectural changes in the epithelium. characteristic features of cell architecture in epithelial dysplasia can be manifested by irregular layers of epithelium, loss of basal cell polarity, proliferation of mitotic features, abnormalities of mitotic and keratinous surfaces without the presence of a rete peg.17 however, in this study, the different degrees of dysplasia in each treatment group can be due to gene variations resulting in cell changes in a single group. according to ge et al. (2016),18 approximately 600 genes influence the process of normal cell changes culminating in malignancy, including transcription factors, oncogenes, differentiation markers, tumour suppressors, and metastatic proteins. genetically, changes that occur during carcinogenesis can be due to mutations, amplifications, rearrangements, and deletions.16 this carcinogenic stage may be preceded by initiation, promotion, progression, and metastasis. the initiation stage involves structural changes, gene mutations occurring either spontaneously or because of stimulation resulting from exposure to carcinogens. such genetic changes subsequently cause dysregulation of cell proliferation pathways, survival, and differentiation which can be influenced by several factors, including the size and type of carcinogenic metabolism, as well as the response of dna repair functions. the subsequent promotion stage is considered a relatively protracted and reversible process in which active cells undergoing preneoplastic proliferation can accumulate. this period can be changed by chemo-preventive materials which will affect the growth rate. the subsequent progression stage is considered to be that between premalignant lesions and progression to invasive cancer. progression is also considered the final stage in neoplastic transformation, where genetic changes, phenotypes, and proliferation occur. it involves the rapid growth in tumor size in which cells undergo further mutations that will potentially become invasive and metastatic. furthermore, the metastastic stage is the one in which cancer cells spread from their original location to other sites around the body through the bloodstream or lymphatic system.19 finally, the different degrees of dysplasia in each treatment group in this study could also be caused by the nicotine and other substances passing into the subjects’ body differing from each other, despite the equal duration of exposure to cigarette smoke. in other words, the degree of dysplasia could be influenced by several factors, such as genetic and carcinogenic, which affect the body. in conclusion, the more protracted the exposure of rattus norvegicus to cigarette smoke, the higher the degree of epithelial dysplasia, indicating that such exposure plays an active role in the carcinogenic process affecting the oral mucosa. references 1. martini s. makna merokok pada remaja putri perokok (the significance of smoking in young female smokers). j psikol pendidik san perkemb. 2014; 3(2): 119–27. 2. kosen s, thabrany h, kusumawardani n, martini s. review of evidence series: health andeconomic costs of tobacco in indonesia. jakarta: kementerian kesehatan republik indonesia; 2017. p. 116. 3. kushihashi y, kadokura y, takiguchi s, kyo y, yamada y, shino m, kano m, suzaki h. association between head-and-neck cancers and active and passive cigarette smoking. health (irvine calif). 2012; 04(09): 619–24. 4. xue j, yang s, seng s. mechanisms of cancer induction by tobaccospecific nnk and nnn. cancers (basel). 2014; 6(2): 1138–56. 5. kesarwani p, choudhary a, gupta r. apoptosis: a prognostic marker in oral epithelial dysplasia and oral squamous cell carcinoma. int j dent heal concerns. 2016; 1(2): 1–4. 6. jaber ma. oral epithelial dysplasia in non-users of tobacco and alcohol: an analysis of clinicopathologic characteristics and treatment outcome. j oral sci. 2010; 52(1): 13–21. 7. trombitas v, nagy a, berce c, tabaran f, albu s. effect of cigarette smoke on wound healing of the septal mucosa of the rat. biomed res int. 2016; 2016: 1–11. 8. teague s v., pinkerton ke, goldsmith m, gebremichael a, chang s, jenkins ra, moneyhun jh. sidestream cigarette smoke generation and exposure system for environmental tobacco smoke studies. inhal toxicol. 1994; 6(1): 79–93. 9. beegom aa. passive smoking and oral cancer risk: a case report. kerala med j. 2014; 7(3): 74–8. 10. glick m, feagans wm. burket’s oral medicine. 12th ed. shelton: people’s medical publishing house; 2015. p. 556. 11. martins rhg, marques madeira sl, fabro at, rocha nds, de oliveira semenzati g, alves kf. effects to exposure of tobacco smoke and alcohol on the tongue and pharynx of rats. inhal toxicol. 2012; 24(3): 153–60. 12. amtha r, razak ia, basuki b, roeslan bo, gautama w, puwanto dj, ghani wmn, zain rb. tobacco (kretek) smoking, betel quid chewing and risk of oral cancer in a selected jakarta population. asian pacific j cancer prev. 2014; 15(20): 8673–8. 13. radwan lrs, grawish me, elmadawy sh, el-hawary ym. ultrasurface morphological changes in the rat tongue posterior onethird exposed to passive smoke. ec dent sci. 2016; 4(4): 846–53. 14. kumar v, abbas ak, aster jc. robbins and cotran pathologic basis of disease. 9th ed. philadelphia: elsevier saunders; 2015. p. 301–3. 15. nikitakis ng, pentenero m, georgaki m, poh cf, peterson de, edwards p, lingen m, sauk jj. molecular markers associated with development and progression of potentially premalignant oral epithelial lesions: current knowledge and future implications. oral surg oral med oral pathol oral radiol. 2018; 125(6): 650–69. 16. jain a. molecular pathogenesis of oral squamous cell carcinoma. in: squamous cell carcinoma hallmark and treatment modalities. intechopen; 2019. p. 1–15. 17. sadiq h, gupta p, singh n, thakar s, prabhakar i, thakral j. various grading systems of the oral epithelial dysplasia: a review. int j adv heal sci. 2015; 1(11): 20–6. 18. ge s, zhang j, du y, hu b, zhou z, lou j. dynamic changes in the gene expression profile during rat oral carcinogenesis induced by 4-nitroquinoline 1-oxide. mol med rep. 2016; 13(3): 2561–9. 19. siddiqui ia, sanna v, ahmad n, sechi m, mukhtar h. resveratrol nanoformulation for cancer prevention and therapy. ann n y acad sci. 2015; 1348(1): 20–31. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p187–191 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p187-191 201201 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 201–205 correlation between working positions and lactic acid levels with musculoskeletal complaints among dentists fiory dioptis putriwijaya,1 titiek berniyanti,2 and indeswati diyatri3 1department of dental public health, institut ilmu kesehatan bhakti wiyata, kediri indonesia 2department of dental public health, faculty of dental medicne, universitas airlangga, surabaya-indonesia 3department of oral biology, faculty of dental medicne, universitas airlangga, surabaya-indonesia abstract background: musculoskeletal complaints have been common for dentists since their body is unknowingly often in inappropriate positions when caring for patients. for example, they bend towards patients, suddenly move, and then rotate from one side to another. the repetitive movements are done in long term. high activities and sufficient recovery time can cause a buildup of lactic acid in their blood leading to obstruction of the energy intake from the aerobic system in their muscle cells, resulting in fatigue. as a result, such conditions trigger decreased muscle performances. purpose: this study aimed to determine the correlation between working positions and lactic acid levels with the risk of musculoskeletal disorders among dentists at public health centers in surabaya. method: this research was an analytical observational research using cross sectional approach. sampling technique used in this research was cluster random sampling with nineteen samples. to evaluate the working positions of those samples, a rapid entire body assessment (reba) method was used. meanwhile, to observe the musculoskeletal disorders of those samples, a nordic body map was used. data obtained then were analyzed using pearson correlation test with a significance level (p<0.05). result: results of data analysis using the pearson correlation test showed that the significance value obtained was 0.036. it indicates that there was a correlation between the working positions and the lactic acid levels with the musculoskeletal disorders in those dentists. the results of the pearson correlation test also revealed that there was a correlation between the working positions and the lactic acid levels among those dentists with a significance value of 0.025. conclusion: it may be concluded that the wrong body positions during working can increase lactic acid level in the body of dentists. the increased level of lactic acid then can affect their muscles, leading to the high risk of musculoskeletal disorders. keywords: lactic acid; musculoskeletal complaints; working positions; reba; nordic body map correspondence: titiek berniyanti, department of dental public health, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: berniyanti@gmail.com introduction statistically, musculoskeletal disorders have become common for dentists. hayes et al.1 conducted a review of 95 reports revealing that musculoskeletal risks are experienced by 64-93% of the respondents. body regions most commonly involved are back (36.3-60.1%), neck (19.8-85%), and hands (60-69.5%). the prevalence of musculoskeletal disorders in dentists in saudi arabia is 82.9%, australia 87.2%, india 78%, lithuania 86.5%, and turkey reached 94%.2 from these data, it can be said that the prevalences of musculoskeletal disorders are high among dentists in some countries. in indonesia, the prevalence of musculoskeletal disorders is still unknown since there have been no data on the prevalence of musculoskeletal disorders among dentists in indonesia, especially in the city of surabaya. however, based on screening results conducted in faculty of dentistry, universitas indonesia using discomfort body map and brief survey instruments, there are 80% dentists working in clinics suffering from msd mainly on neck, shoulders, forearms, hands, and back.2 one of factors causing musculoskeletal syndrome suffered by dentists is that they care more with the comfort for patients treated, but less attention to their comfort when caring for patients. dentists consider more on the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p201-205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p201-205 202 putriwijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 201–205 need to move toward their patients rather than to adjust the seating position of their patients on dental chair. thus, most musculoskeletal disorders occur because their body is unknowingly often in inappropriate positions when caring for patients. when performing a tooth preparation or tooth extraction, for example, sometimes dentists bend towards patients, suddenly move, and then rotate from one side to another. the entire movement is done many times in long term. as a result, it triggers musculoskeletal syndrome.3 in general, factors triggering dentists to suffer musculoskeletal syndrome can be categorized into technical factors and non-technical factors. technical factors are dental unit, work lights, and other equipments that are not ergonomic. meanwhile, non-technical factors are their working positions when caring for patients as well as their life style.3 consequently, dentists have a high risk of musculoskeletal disorders, such as pain or discomfort in their neck, shoulders, wrist, and back.4 strong muscle contractions in the long term, furthermore, can trigger a condition, known as muscle fatigue. fatigue is the result of inability of both contraction and metabolic processes of muscle fibers to continually supply the same work output. at first, nerves work well, and nerve impulse runs normally through the muscle-nerve correlation moving into muscle fibers, but the contractions are increasingly getting weaker later because of atp deficiency in the muscle fibers. this condition is caused by barriers in flow of blood to the muscles contracting, then leading to muscle fatigue in one minute or more due to the loss of the nutrient supply.5 thus, feeling tense or tired in the body can be considered as an indication of accumulated lactic acid in the muscles. lactic acid occurs due to combustion process in the active muscle. during the combustion process, according to fox cit. sitepu,6 in addition to energy, lactic acid is also generated as combustion residue. the longer the activity is executed, the smaller the energy is generated and the greater the combustion residue in the form of lactic acid is deposited. the accumulation of lactic acid then can trigger tiredness or fatigue. therefore, lactic acid is closely related to the ability of muscles to contract. unfortunately, body has a number of limitations to tolerate lactic acid, and each individual has a different lactic acid threshold. lactic acid levels will increase during moving which source of energy is from anaerobic glycolysis system.7 for these reasons, this research aimed to determine the correlation between working positions and lactic acid levels with musculoskeletal disorders among dentists. as a result, this research can be expected to give several alternative solutions for the problem. materials and methods this research was an analytic observational research using cross sectional approach. sampling technique used in this research was cluster random sampling with nineteen samples. criteria used in sampling were a maximum age of 50 years, premenopausal condition, having no menstruation (during the time of blood sampling), a minimum term of 5 years, physically and mentally healthy condition, and willing to be sampled in this research. moreover, to assess the working positions of dentists, reba method was used. their working positions were assessed by giving a risk score from one to fifteen. the highest score indicated a high level of risk rising in the working positions, while the lowest score assured that the working positions was free of ergonomic hazard. to reveal the musculoskeletal complaints, furthermore, nordic body map questionnaire technique was used. this questionnaire uses images of the human body that has been divided into nine main sections, namely: neck; shoulder; upper back; elbow; lower back; wrist/ hand; waist/ buttocks; knee; heel/ feet.8 meanwhile, assessment of this nordic body map questionnaire can be categorized into four categories, namely low with a score of 0-20, middle with a score of 21-41, high with a score of 42-62, and very high with a score of 63-84.10 in addition, blood samples were carried out to examine lactic acid levels of those dentists. their blood was taken in their vein as much as 2cc syringe. after that the blood samples were stored in red blood sample tubes, and then centrifuged in the laboratory to take the serum. their blood serum was dropped on test strips, and then inserted into accutrend plus instrument to evaluate lactic acid levels. the working positions of those dentists were evaluated when they were performing tooth filling. blood samples were conducted after they had finished the treatment. the data obtained then were analyzed using pearson correlation test with a significance level (p)<0.05. results in this research, the working positions of those samples were evaluated using reba method. table 1 shows that the highest frequency was found in 11 respondents (57.9%) with a score of 2-3 indicating the first risk level, low risk category. nevertheless, this indicates that further treatment was necessary. table 2, moreover, shows the descriptive overview of the mean and standard deviations of the working positions and lactic acid levels using reba method. the mean and standard deviations of the working positions were 3.16 ± 1.259, while the mean and standard deviations of the lactic acid levels were 3.28 ± 1.853 mm/ l. in other words, the physical activities of those dentists led to increase lactic acid levels as much as 3.28 mm/ l. table 3 shows that of the 19 respondents, there were only low, moderate, and high-risk categories. 11 respondents (57.9%) had low complaint level, 5 respondents (26.3%) had moderate complaint level, and 3 respondents (15.8%) had high complaint level. the mean and standard dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p201-205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p201-205 203203putriwijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 201–205 deviations of musculoskeletal complaints using nordic body map method amounted to 19.63 ± 15.148 as seen in table 4. based on table 5, the highest frequency of the musculoskeletal disorders was found on the right shoulder (78.9%), followed with on the back (68.4%) and on the upper neck (63.2%). this indicates that the majority of those dentists performed posterior maxillary tooth fillings with the wrong position, resulting in pain on the shoulder area, back, and neck. in addition, based on tables 6, 7 and 8, results of the pearson correlation test showed that there was a significant correlation between the working positions and the lactic acid levels with the musculoskeletal complaints with a table 1. frequency distribution of the working positions among those dentists at some public health centers in surabaya in 2016 no score of working positions (reba) risk levels total percentage (%) 1 1 0 0 0 2 2-3 1 11 57.9 3 4-7 2 8 42.1 4 8-10 3 0 0 5 11-15 4 0 0 total 19 100 table 2. mean values of the working positions and the lactic acid levels among those dentists at some public health centers in surabaya in 2016 working positions (reba) lactic acid levels (mm/l) n 19 19 mean ± sd 3.16 ± 1.259 3.28 ± 1.853 table 3. frequency distribution of the musculoskeletal complaints among those dentists at some public health centers in surabaya in 2016 no. musculoskeletal complaints total percentage (%) 1 2 3 low moderate high 11 5 3 57.9 26.3 15.8 total 19 100 table 4. mean values of the musculoskeletal complaints among those dentists at some public health centers in surabaya in 2016 musculoskeletal complaints (nordic body map) n 19 mean ± sd 19.63 ± 15.148 table 5. frequency distribution of the location of the musculoskeletal complaints among those dentists at some public health centers in surabaya in 2016 no the location of musculoskeletal complaints total percentage (%) 1 upper neck 12 63.2 2 lower neck 10 52.6 3 left shoulder 11 57.9 4 right shoulder 15 78.9 5 left upper arm 11 57.9 6 back 13 68.4 7 right upper arm 10 52.6 8 waist 9 47.4 9 buttock 6 31.6 10 bottom 1 5.3 11 left elbow 2 10.5 12 right elbow 1 5.3 13 left forearm 3 15.8 14 right forearm 5 26.3 15 left wrist 4 21.1 16 right wrist 9 47.4 17 left hand 8 42.1 18 right hand 7 36.8 19 left thigh 2 10.5 20 right thigh 2 10.5 21 left knee 4 21.1 22 right knee 2 10.5 23 left calf 7 36.8 24 right calf 6 31.6 25 left ankle 4 21.1 26 right ankle 5 26.3 27 left leg 5 26.3 28 right leg 5 26.3 significance value of 0036. similarly, there was also a significant correlation between the working positions and the lactic acid levels with a significance value of 0.025. each of the correlation coefficients showed positive values. it means that the correlation between the working position, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p201-205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p201-205 204 putriwijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 201–205 the lactic acid levels, and the musculoskeletal complaints was directly proportional. in other words, the working positions might trigger an increase in the lactic acid levels. the higher the lactic acid levels are, the greater the levels of the musculoskeletal disorder risk are. discussion based on results of the pearson correlation test, there was a significant correlation between the working positions and the musculoskeletal complaints with a significance value of 0.036. musculoskeletal disorders in those dentists in this research were mostly found on right shoulder, back, and upper neck. this indicates that during caring for their patients the majority of those dentists took standing and bending positions in a long time repeatedly with the neck position likely to come forward. dentists are generally characterized by their static and rigid posture in taking care of their patients. that their patients are treated at top of the dental chair makes them sit, stand or bend for a long time. as results, these positions make them experience pain or discomfort on neck area, shoulder, and spine, leading to musculoskeletal disorders, such as low back pain.7 human body, actually can tolerate with standing position in one posture only for 20 minutes. if more than this limit, tissue elasticity will slowly be reduced, and eventually muscle pressure increases as well as discomfort is felt in the hip area. if the back muscles receive static loads when table 6. correlation between the working positions and the musculoskeletal complaints in those dentists at some public health centers in surabaya in 2016 independent variable dependent variable p r working positions (reba) nordic body map 0.036 0.484 table 7. correlation between the lactic acid levels and the musculoskeletal complaints in those dentists at some public health centers in surabaya in 2016 independent variable dependent variable p r lactic acid levels nordic body map 0.036 0.484 table 8. correlation between the lactic acid levels and the working positions in those dentists at some public health centers in surabaya in 2016 independent variable dependent variable p r working positions (reba) lactic acid levels 0.025 0.513 standing for a long time, it can lead to complaints of damage to the joint, ligaments, and tendons. complaints to the damages are usually known as musculoskeletal complaints or injuries on the musculoskeletal system.11 dentists during their working often perform a variety of extreme static postures, such as lowered head, bow, lean over, raised hands, and elevated shoulder. the extreme postures, consequently, trigger muscle fatigue and mechanical pain in the neck, shoulder, and lower back.12 during working, the need for blood circulation, can increase ten to twenty times. the increased blood circulation in the muscles during working then can force the heart to pump more blood. standing for long time can make muscles tend to work static. static muscular work is characterized by long muscle contractions, usually corresponding to the posture of the body. however, the static muscle contractions in the long term are not recommended since they will cause pain. one of the causes of back pain is sitting or standing for long time or having similar motion performed continuously, resulting in muscle stiffness (spasms).11 sitting posture during working, actually requires less energy than standing since this position reduces the amount of static load on leg muscles.13 however, the wrong sitting posture will lead to several problems, such as back pain, neck pain, and waist pain since pressure on the back of the spine will increase in the sitting posture than in standing or lying down position. tense sitting posture even requires more muscle or spinal cord activities. thus, the sitting posture can affect the performance of a worker. all the activities and movements of the human body actually require contraction of muscles, including respiration processes involving the muscles of inspiration and expiration to contract and relax on an ongoing basis. the process of muscle contraction requires muscle energy sources that are available in the body’s metabolism, namely atp formation, breaking down muscle glycogen into glucose. in aerobic metabolism, this process also requires o₂. this energy-making process is necessary to maintain the quality of contraction and the force of muscle contraction continuously. nevertheless, this process also generates metabolic wastes. the accumulation of metabolic wastes, such as lactic acid, can trigger fatigue and muscle glycogen reduction. the increased co₂ during the process also leads to an inability of the muscle to maintain the same work output or a decrease in the muscular endurance.14 muscles get their energy from aerobic and anaerobic metabolisms in the form of adenosine triphosphate (atp). if the muscle contraction continuously occurs, the need for atp will not be met only through the aerobic metabolism, as a result, the anaerobic metabolism will become a viable alternative to meet the body’s needs. however, the anaerobic metabolism can give disadvantages. for instance, the anaerobic metabolism can generate lactic acid, triggering pain and fatigue sensations.15 in addition, lactic acid in the blood is always derived from anaerobic metabolism in erythrocytes.7 nevertheless, the total amount of lactic acid in the body is relatively fixed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p201-205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p201-205 205205putriwijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 201–205 in a healthy person having a rest, the amount of lactic acid is about 1-2 mm/ l or 1-1.8 mm/ l. therefore, the blood lactic acid level that exceeds 6 mm/ l, according to jansen can interfere with the working muscle cells, leading to disorientation of the muscle movement coordination. in this research, the mean level of lactic acid in the nineteen respondents was 3.28±1.853 mm/l with the highest mean level of 7.3 mm/ l and the lowest mean level of 0.2 mm/ l. tolerance limit to the level of lactic acid concentration in the muscles and blood during physical exercise actually still has not been known. nevertheless, the tolerance limit to the level of lactic acid in humans is estimated at above 20 mm/ l blood or 25 mm/ kg/ wet muscle weight, and even can reach above 30 mm/ l in the dynamic exercise with high intensity.7 consequently, the lactic acid will lower the ph in the muscles and blood. the decreased ph will inhibit the glycolytic enzyme, and then will interfere with chemical reactions in the muscle cells. this condition, as a result, will lead to weak muscle contraction, and ultimately will make the muscles fatigue.7 it may be concluded that the wrong body positions during working can increase lactic acid level in the body of dentists. the increased level of lactic acid then can affect their muscles, leading to the high risk of musculoskeletal disorders. references 1. widinugroho. bp. 2011. evaluasi postur kerja mahasiswa /i tingkat profesi fkg-ui pada tindakan pembersihan karang gigi dengan posisi duduk dalam virtual environment. skripsi. jakarta: universitas indonesia. 2. wijaya at, darwita rr, bahar a. the relation between risk factors and musculoskeletal impairment in dental students: a preliminary study. journal of dentistry indonesia 2011; 18(2): 33-7. 3. andayasari l, anorital. gangguan muskulosskeletal pada praktik dokter gigi dan upaya pencegahannya. media litbang kesehatan 2012; 22(2). 4. alexopoulos ec, stathi ic, charizani f. prevalence of musculoskeletal disorders in dentists. bmc musculoskelet disord. 2004; 5: 16. 5. arthur cg. buku ajar fisiologi kedokteran. edisi 3. jakarta: egc; 1995. 6. sitepu id. efektifitas massage terhadap penurunan kelelahan otot tangan operator komputer puskom unimed tahun 2007. tesis. medan: universitas sumatera utara; 2007. 7. dyah wa. perbedaan pengaruh circulo massage dan contrasbath terhadap kadar asam laktat pada latihan beban ditinjau dari vo2max. tesis. surakarta: universitas sebelas maret; 2016. 8. dewayana ts, azmi n, riviana. identifikasi resiko ergonomi pada pekerja di pt. asaba industry. bandung: jati undip; 2008. 9. sutrio, firdaus o. analisis pengukuran rula dan reba petugas pada pengangkatan barang di gudang dengan menggunakan software ergolntelligence (studi kasus: petugas pembawa barang di toko dewi bandung). prosiding seminar nasional ritektra 2011, program studi teknik industri, fakultas teknik, bandung: universitas widyatama; 2011. 10. tarwaka. ergonomi industri: dasar-dasar pengetahuan ergonomi dan aplikasi di tempat kerja. surakarta: harapan press; 2015. 11. susanti n, hartiyah, kuntowato d. hubungan berdiri lama dengan keluhan nyeri punggung bawah miogenik pada pekerja kasir di surakarta. jurnal pena medika 2015; 5(1): 60–70. 12. anggraini w. kelelahan kerja tulang belakang akibat penyimpangan prinsip ergonomik dalam praktek dokter gigi. jurnal kedokteran gigi universitas indonesia 2000; 7(edisi khusus): 14-20. 13. bayu, darmadi, mahayana. hubungan faktor waktu kerja. waktu istirahat dan sikap kerja terhadap keluhan nyeri tengkuk pada pengerajin ukiran kayu. jurnal kesehatan lingkungan 2014; 4(1): 6-15. 14. arthur cg, john e. buku ajar fisiologi kedokteran. edisi 11. jakarta: egc; 2008. 15. sukedana p, made l. prevalensi keluhan muskuloskeletal dan keluhan kesehatan lainnya pada pekerja pura batu padas di desa tamblang dalam konsep health ergonomic. jurnal ergonomi indonesia 2016; 2(1): 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p201-205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p201-205 220 volume 47, number 4, december 2014 hubungan tweed triangle dan posisi bibir terhadap garis estetik (relationship between tweed triangle and the lips position to esthetic line) intan oktaviona, i.g.a. wahju ardani dan achmad sjafei departemen ortodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: the aimed of almost all of patient came for orthodontics treatment, were to recover face esthetic or their profile. the facial profile not only determined by the hard tissue, but will also be influenced by the soft tissue such as nose, lip, and chin. tweed has done face analysis with cephalometric by using three angles which are fma, fmia, and impa. there are many facial profile analysis that usually use in lateral cephalometric one of them is ricketts analysis that draw the line from pogonion to the tip of nose. the biggest tribe in indonesia is javanese, the javanese have a specific characteristic. javanese people do not have a long nose and the chin not really uppermost, thick lip, convex profile and female’s profile more convex than male. purpose: the study was aimed to examine the relation between tweed triangle, upper lip and lower lip to esthetic line according to ricketts. methods: the facial profile pictures of subjects which were taken base on sample criteria changed into silhouette black and white. the photo selected by orthodontist and lay persons. then the lateral cephalometric radiograph was taken and measured the angle of tweed triangle and the lip position to esthetic line. results: the fma were 28.830, fmia were 56.740 and impa were 94.430. the upper lip distance among males were closer to the esthetic line than female. the lower lip of males located in front of esthetic line and female lower lip located rare of esthetic line. conclusion: there was corelation between fma and fmia with lower lip. there was no corelation between impa towards upper lip and also lower lip. key words: tweed triangle, upper lip, lower lip, esthetic line abstrak latar belakang: tujuan dari hampir seluruh pasien yang datang untuk perawatan ortodonti adalah untuk memperbaiki estetis wajah atau profilnya. profil fasial tidak hanya ditentukan oleh jaringan keras, akan tetapi juga akan sangat dipengaruhi oleh jaringan lunak hidung, bibir, dan dagu. tweed melakukan analisis wajah pada sefalometri menggunakan 3 sudut dalam segitiga yaitu fma, fmia, dan impa. beberapa analisis profil facial yang sering digunakan pada sefalometri lateral antara lain analisis ricketts yang merupakan garis yang ditarik dari pogonion ke ujung hidung. suku terbesar di indonesia adalah suku jawa, suku jawa memiliki ciri ragawi tertentu antara lain: hidung orang jawa tidak terlalu mancung dan dagu tidak begitu menonjol, bibir tebal, proporsi jaringan lunak yang cembung dan perempuan lebih cembung daripada laki–laki. tujuan: meneliti hubungan antara tweed triangle terhadap posisi bibir atas dan bibir bawah terhadap garis estetik. metode: foto profil wajah berdasarkan kriteria sampel, yang dibuat siluet hitam putih, dan selanjutnya dipilih oleh dokter gigi spesialis ortodonti dan orang awam. selanjutnya sampel terpilih akan difoto sefalometri lateral dan akan dihitung besarnya sudut tweed triangle dan posisi bibir terhadap garis estetik. hasil: besarnya nilai fma 28.830, fmia 56.740, dan impa 94.430. jarak bibir atas laki-laki lebih mendekati garis estetik dibandingkan dengan perempuan, sedangkan bibir bawah laki – laki terletak di depan garis estetik dan bibir bawah perempuan mendekati garis estetik. simpulan: research report 221oktaviona, et al.: hubungan tweed triangle dan posisi bibir terhadap garis estetik terdapat hubungan antara fma dan fmia terhadap bibir bawah. tidak terdapat hubungan antara impa terhadap bibir atas maupun bibir bawah. kata kunci: tweed triangle, bibir atas, bibir bawah, garis estetik korespondensi (correspondence): intan oktaviona, departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no.47 surabaya 60132, indonesia. e-mail: intanov59@gmail.com pendahuluan perawatan ortodonti merupakan perawatan kedokteran gigi yang memperbaiki susunan gigi sehingga dapat meningkatkan kemampuan mastikasi, fonetik, serta estetik.1 memperbaiki estetika wajah dan gigi merupakan motivasi utama dalam mencari perawatan ortodonti. sarver dan ackerman2 menyatakan bahwa para artis dan dokter telah berusaha selama berabad-abad untuk menentukan proporsi ideal wajah. penelitian masih berlangsung hingga kini dan sangat menarik bagi para ahli ortodonti karena dapat dijadikan pedoman untuk penilaian estetika. penampilan profil wajah tidak hanya ditentukan oleh jaringan keras, akan tetapi dipengaruhi oleh jaringan lunak hidung, bibir, dan dagu. tweed melakukan analisis wajah pada sefalometri menggunakan 3 sudut dalam segitiga yaitu frankfort mandibular angle (fma), frankfort mandibular incisor angle (fmia), dan incisor mandibular plane angle (impa). hubungan dari ketiga sudut sefalometri tersebut memberikan informasi diagnosa tentang pola vertikal skeletal pasien, hubungan insisivus mandibula dengan tulang basal, dan jumlah relatif protrusi, atau berkurangnya ukuran wajah. ukuran rerata untuk fma, fmia, dan impa berturut–turut 25° , 68°dan 87°. beberapa analisis profil wajah yang sering digunakan pada sefalometri lateral antara lain analisis ricketts.4 analisa ricketts menggunakan garis estetika (garis e) yang merupakan garis yang ditarik dari pogonion ke ujung hidung. standar normal (menurut ras kaukasoid): bibir atas terleltak 2–3 mm di belakang garis tersebut dan bibir bawah 1–2 mm di belakang garis tersebut.5 berbagai suku terdapat di indonesia, suku terbesar di indonesia adalah suku jawa, suku jawa adalah suku yang terdapat di jawa tengah, jawa timur, dan yogyakarta. menurut heryumani6 suku jawa memiliki ciri ragawi tertentu antara lain: hidung orang jawa tidak terlalu mancung dan dagu tidak begitu menonjol, bibir tebal, proporsi jaringan lunak yang cembung (proporsi hidung, bibir dan dagu) dan perempuan lebih cembung dari laki–laki. tujuan dari penelitian ini adalah untuk meneliti hubungan antara tweed triangle dan posisi bibir atas dan bibir bawah terhadap garis estetik menurut ricketts pada orang jawa. bahan dan metode penelitian ini menggunakan subjek mahasiswa dari suku jawa fakultas ilmu sosial dan ilmu politik, fakultas kedokteran gigi dan fakultas hukum universitas airlangga. subyek yang telah terpilih kemudian difoto profil wajah, lalu hasilnya disiluet hitam putih. selanjutnya foto profil hitam putih akan di seleksi oleh 7 ortodontis dan 7 orang awam. hanya foto yang dipilih paling sedikit oleh 4 ortodontis dan 4 orang awam yang akan difoto sefalometri lateral. setelah difoto sefalometri, hasilnya akan ditelusuri satu kali dengan dua kali pengukuran. titik-titik referensi sefalometri dan cara mengukurnya: (a) orbita: tangen satu ujung penggaris ke tepi paling atas dari ear rods dan bergerak ke ujung atas lainnya sampai menyentuh rim infraorbital dari orbita; (b) porion: design yang paling luar dan paling superior dari ear rods; (c) pogonion: garis yang tegak lurus dengan fh ke depan lalu ke belakang dimana sentuhan pertama pada dagu merupakan pogonion; (d) pronasale: garis yang tegak lurus dengan fh ke depan lalu ke belakang dimana sentuhan pertama pada hidung merupakan pronasale. sefalogram ditelusuri dengan kertas asetat dan pensil 4h di atas pencahayaan tracing box untuk mencari titik pronasale dan titik pogonion, kemudian ditarik garis dari kedua titik tersebut sehingga didapat garis estetik, kemudian diukur jarak antara bibir atas dan bibir bawah terhadap e–line menggunakan penggaris dengan ketelitian 0,5 mm. sudut–sudut tweed triangle dibuat bidang frankfort horizontal yaitu garis yang ditarik dari ujung atas porion ke orbita, selain itu juga dibuat bidang mandibular plane, yaitu garis yang menyinggung bidang mandibular, dan yang terakhir dibuat garis yang ditarik dari ujung apeks insisivus bawah ke ujung insisivus bawah. sudut fma diukur dengan menggunakan busur pada perpotongan antara bidang frankfort horizontal dengan bidang mandibular. sudut fmia diukur dengan menggunakan busur pada perpotongan antara frankfort horizontal dengan garis yang ditarik dari ujung apeks insisivus bawah ke ujung insisivus bawah. sudut impa diukur dengan menggunakan busur pada perpotongan antara bidang mandibular dengan garis yang ditarik dari ujung apeks insisivus bawah ke ujung insisivus bawah. analisis data secara deskriptif 222 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 220–225 dan uji korelasi dengan menggunakan spss 15.0 untuk windows. hasil sampel penelitian adalah 64 foto sefalometri mahasiswa dari suku jawa yang terdiri dari laki-laki dan perempuan dengan rata–rata usia 18–25 tahun yang telah dipilih berdasarkan kriteria sampel dan pemilihan oleh tujuh dokter gigi spesialis ortodonti dan tujuh orang awam. analisa hasil penelitian berupa uji normalitas, uji–t dan uji korelasi. uji normalitas digunakan untuk mengetahui apakah data berdistribusi normal atau tidak. uji normalitas yang digunakan adalah kolmogorov–smirnov. pada tabel 1 semua nilai p > 0.05 sehingga dapat diketahui bahwa semua data dalam penelitian ini berdistribusi normal. selanjutnya dilakukan analisis dengan independent t–test untuk mengetahui ada tidaknya perbedaan yang signifikan usia antara laki–laki dan perempuan. tabel 2 menunjukkan antara usia laki–laki dan perempuan tidak berbeda yang artinya data untuk usia subjek laki-laki dan perempuan adalah homogen. uji selanjutnya menggunakan paired t–test untuk mengetahui ada tidaknya perbedaan yang signifikan antara ukuran sefalometri laki-laki dan perempuan (tabel 3). pada tabel 3 tidak terdapat perbedaan bermakna untuk ukuran sefalometri fma, fmia, dan impa dengan masing–masing nilai p>0.05 menunjukkan bahwa tidak ada perbedaan bermakna antara ketiga ukuran tersebut. sedangkan untuk ukuran sefalometri ls e–line dan li e–line terdapat berbeda yang bermakna antara laki–laki dan perempuan dengan nilai p<0.05. jarak bibir atas pada laki–laki lebih dekat di belakang garis estetik dibandingkan dengan perempuan, sedangkan jarak bibir bawah pada laki–laki terletak di depan garis estetik dan pada perempuan terletak di belakang garis estetik. selain itu uji paired t–test digunakan untuk menguji perbedaan antara pengukuran sefalometri pertama dan pengukuran kedua pada subjek laki–laki dan perempuan. pengukuran sefalometri pertama dan kedua dengan selang waktu 2 minggu pada tabel 4 masing–masing nilai p > 0.05, hal ini menunjukkan bahwa tidak ada perbedaan bermakna yang artinya pengukuran yang dilakukan dua kali dengan selang waktu 2 minggu tetap memberikan hasil yang sama pada subjek laki–laki untuk semua ukuran sefalometri. pengukuran sefalometri pertama dan kedua pada tabel 5 masing – masing nilai p > 0.05, hal ini berarti tidak berbeda bermakna yang artinya pengukuran yang dilakukan dua kali dengan selang waktu 2 minggu tetap memberikan hasil tabel 1. hasil uji kolmogorov smirnov variabel mean sd p fma 28.83 7.09 0.71 fmia 56.74 5.87 0.99 impa 94.43 6.49 0.68 ls e – line -1.63 2.03 0.15 li e – line -0.68 2.11 0.53 nilai p < 0.05: berbeda bermakna tabel 2. perbedaan usia pada subjek laki–laki dan perempuan dengan independent t–test variabel n rerata sd p laki – laki 22 20.05 1.53 0.48 perempuan 42 19.71 1.31 nilai p<0.05: berbeda bermakna tabel 3. perbedaan ukuran sefalometri pada subjek laki – laki dan perempuan dengan uji paired t – test variabel laki – laki perempuan p fma 28.25 29.14 0.64 fmia 58.68 55.73 0.05 impa 93.07 95.14 0.23 ls e – line -0.60 -2.16 0.003 li e – line 0.21 -1.14 0.01 nilai p<0.0 : berbeda bermakna tabel 4. perbedaan pengukuran 1 dan pengukuran 2 pada tracing sefalometri subjek laki–laki dengan uji paired t – test variabel pengukuran 1 pengukuran 2 p fma 28.25 34.36 0.15 fmia 58.68 57.02 0.15 impa 93.07 92.70 0.65 ls e – line -0.60 -0.68 0.36 li e – line 0.21 0.10 0.46 nilai p<0.05: berbeda bermakna tabel 5 perbedaan pengukuran 1 dan pengukuran 2 pada tracing sefalometri subjek perempuan dengan uji paired t – test variabel pengukuran 1 pengukuran 2 p fma 29.14 29.13 0.99 fmia 55.73 55.26 0.47 impa 95.14 95.61 0.47 ls e – line -2.16 -2.15 0.91 li e – line -1.14 -1.08 0.47 nilai p < 0.05 : berbeda bermakna 223oktaviona, et al.: hubungan tweed triangle dan posisi bibir terhadap garis estetik yang sama pada subjek perempuan untuk semua ukuran sefalometri. uji statstik selanjutnya adalah uji korelasi pearson untuk mengetahui ada tidaknya hubungan antara tweed triangle dan bibir atas dan bibir bawah. dari hasil pengamatan antara fma dengan fmia dan fma dengan impa (p<0.05) hal ini menunjukkan bahwa terdapat hubungan atau korelasi, dan nilai r yang negatif (tabel 6). apabila nilai fma semakin besar maka nilai fmia akan semakin kecil dan sebaliknya. apabila nilai fma semakin besar maka nilai impa akan semakin kecil dan sebaliknya. antara fma dengan ls e–line (p > 0.05) tidak terdapat hubungan atau korelasi, sedangkan antara fma dengan li e–line (p<0.05) terdapat hubungan atau korelasi. hubungan antara kedua variabel ini searah dimana apabila nilai fma semakin besar maka nilai li e–line juga akan semakin besar dan sebaliknya. antara fmia dengan impa (p<0.05) terdapat hubungan atau korelasi, dan hubungan antara kedua variabel ini berkebalikan dimana apabila nilai fmia semakin besar maka nilai impa akan semakin kecil dan sebaliknya. antara fmia dengan ls e–line (p>0.05) tidak terdapat hubungan atau korelasi, sedangkan antara fmia dengan li e–line (p<0.05) terdapat hubungan atau korelasi. hubungan antara kedua variabel ini berkebalikan dimana apabila nilai fmia semakin besar maka nilai li e–line akan semakin kecil dan sebaliknya. antara impa dengan ls e–line (p>0.05) dan fmia dengan li e–line (p >0.05) tidak terdapat hubungan atau korelasi. antara ls e–line dengan li e–line (p<0.05) terdapat hubungan atau korelasi. hubungan antara kedua variabel ini searah yaitu apabila nilai ls e–line semakin besar maka nila li e–line juga akan semakin besar dan sebaliknya. pembahasan subyek adalah mahasiswa suku jawa yang berada di surabaya karena populasi jawa adalah kelompok populasi terbesar di indonesia dengan jumlah mencapai 41% dari total populasi. subyek yang dipilih adalah mahasiswa yang berusia 18–25 tahun karena profil jaringan lunak dipengaruhi oleh umur. usia minimal pada sampel ini adalah 18 tahun dikarenakan pada usia ini telah melewati pubertas dan fase tumbuh kembangnya telah selesai.7 penyeleksian oleh orang awam dari populasi jawa untuk tabel 6. hubungan antara fma, fmia, impa terhadap ls e line dan li e line dengan uji korelasi pearson total variabel fmia impa ls e – line li e – line r p r p r p r p fma -0.51 0.00 -0.63 0.00 0.24 0.06 0.26 0.03 fmia -0.35 0.00 -0.17 0.19 -0.27 0.03 impa -0.11 0.38 -0.05 0.72 ls e-line 0.82 0.00 nilai p < 0.05 adalah berbeda bermakna (signifikan) mendapatkan sampel yang dapat mewakili kriteria wajah yang disukai dan diterima menurut populasi jawa. penilaian estetika bersifat self preference. pemahaman estetika setiap orang dipengaruhi pengalaman personal dan lingkungan sosial. pendapat ortodontis belum tentu diterima oleh oleh persepsi orang awam.8 pengukuran tweed triangle pada sampel menunjukkan bahwa besar sudut fma, fmia, dan impa untuk laki – laki dan perempuan mahasiswa universitas airlangga populasi jawa adalah 28.830, 56.740, dan 94.430. pada subyek laki-laki nilai rerata fmia (57.850) lebih besar dibandingkan dengan subyek perempuan (55.490), perbedaan ini menunjukkan bahwa subyek perempuan memiliki proklinasi insisivus bawah lebih besar dibandingkan dengan laki – laki. jika pengukuran penelitian ini dibandingkan dengan nilai baku dari tweed yaitu fma 250, fmia 650 dan impa 900, nilai fma penelitian ini 28.830 lebih besar 3 derajat lebih dibanding nilai baku tweed. hal ini menunjukkan bahwa sampel populasi jawa yang merupakan ras deutro melayu memiliki pertumbuhan vertikal dari mandibular yang lebih besar dibandingkan dengan ras kaukasia. nilai fma yang besar menunjukkan divergen wajah, sehingga profil terlihat lebih cembung. jika fmia penelitian ini dibandingkan dengan nilai baku tweed yaitu 650, hasilnya lebih kecil 8 derajat lebih dibanding nilai baku tweed. hal ini menunjukkan bahwa sampel penelitian dari populasi jawa memiliki proklinasi insisivus bawah lebih besar. sedangkan jika impa penelitian ini dibandingkan dengan nilai baku tweed yaitu 900, hasilnya lebih besar 4 derajat dibanding nilai baku tweed. hal ini menunjukkan bahwa sampel penelitian ini memiliki proklinasi insisivus bawah yang lebih besar dibandingkan dengan ras kaukasia. penelitian tweed triangle dan jaringan lunak dari populasi jepang dengan oklusi normal dan profil facial yang baik didapatkan hasil fma 27.280, fmia 57.220, dan impa 95.500.9 dari hasil studi ini dan laporan dari peneliti jepang lainnya, mereka menyarankan bahwa besarnya nilai fmia untuk mendapatkan profil facial yang baik pada populasi jepang yaitu sebesar 570, sedangkan pada penelitian ini, nilai fmia adalah 57.2050. nilai fmia yang besar menunjukkan adanya retroklinasi insisivus sedangkan fmia yang kecil menunjukkan adanya proklinasi insisivus. uji korelasi statistik ditemukan bahwa antara fma dan fmia keduanya memiliki hubungan korelasi negatif. hal ini sesuai dengan hasil penelitian, dimana nilai fma yang 224 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 220–225 besar mengakibatkan berkurangnya nilai fmia. nilai fmia yang kecil menunjukkan adanya proklinasi insisivus bawah yang memberikan dampak pada bibir yaitu bibir bawah akan terletak pada atau di depan garis estetik sehingga membentuk profil wajah yang cembung. posisi optimal geligi pada rahang dan wajah lebih ditentukan oleh posisi gigi insisivus atas daripada posisi gigi insisivus bawah, dan gigi insisivus atas memegang peranan penting sebagai petunjuk anterior dari gerakan protrusi mandibula.10 penelitian tentang sudut interinsisial terhadap jaringan lunak terdapat hubungan antara profil jaringan lunak wajah dengan sudut interinsisal, dimana korelasi hasil penelitian menuju ke arah negatif yang berarti semakin besar sudut interinsisal maka semakin kecil jarak bibir atas (ls) dan bibir bawah (li) terhadap garis estetik. hal ini dibuktikan dalam penelitian dimana nilai fmia yang semakin besar akan mengakibatkan nilai ls e – line yang semakin kecil.7 pada penelitian tweed triangle pada populasi brazil, didapatkan nilai rata – rata fma fmia dan impa adalah 29,1250, 50,9060, dan 99,8750. berdasarkan hasil dari studi sampel didapatkan kesimpulan yaitu populasi kulit hitam brazil memiliki proklinasi insisivus bawah dan profil wajah yang relatif lebih cembung yang dibuktikan dengan labial tipping yang berlebihan dan proklinasi gigi insisivus bawah dibandingkan dengan populasi kulit putih. adalah penting pada rencana perawatan ortodonti mengacu pada faktor etnik,11 sedangkan pada penelitian tentang tweed triangle pada populasi nepal, didapatkan hasil rata – rata fma, fmia, dan impa sebesar 280, 560, dan 960. dari penelitian ini juga diketahui bahwa tidak ada perbedaan yang signifikan antara populasi laki– laki dan perempuan. antara laki–laki dan perempuan dari populasi nepal memiliki pertumbuhan horizontal yang besarnya sama yaitu 280. signifikansi akan terjadi ketika hasil rata–rata tweed triangle pada populasi nepal dibandingkan dengan ras kaukasia dalam nilai baku tweed yaitu fma 250, fmia 650, dan impa 900.12 sudut tweed triangle pada orang jawa dari hasil penelitian memiliki nilai yang mendekati besarnya sudut tweed triangle pada populasi jepang dan nepal. selain dipengaruhi oleh umur dan jenis kelamin, profil jaringan lunak juga dipengaruhi oleh etnik dan ras.13 jepang dan nepal termasuk dalam ras mongoloid sedangkan populasi jawa termasuk dalam ras deutro melayu. orang jawa merupakan sub – ras sekunder mongoloid yaitu berasal dari subras deutro melayu.14 garis estetik pada hasil penelitian terlihat bahwa garis estetik pada laki – laki dan perempuan mahasiswa universitas airlangga tahun 2009 – 2011 orang jawa menunjukkan bibir atas dan bibir bawah yang terletak di belakang garis estetik berdasarkan garis referensi untuk garis estetik menurut ricketts yaitu garis yang menyinggung ujung hidung dan ujung dagu. populasi jawa memiliki ciri bibir tebal, hidung yang tidak terlalu mancung, dan dagu tidak terlalu menonjol, sehingga profil wajah cenderung cembung. profil wajah seseorang dikatakan cembung apabila dua garis acuan yaitu garis dari dahi ke bibir atas dan garis dari bibir atas ke dagu membentuk sudut yang menunjukkan dagu terletak relatif di belakang. dari hasil penelitan, rata – rata sampel menunjukkan dagu yang relatif di belakang, sehingga profil orang jawa pada sampel ini memiliki profil wajah yang cembung menurut rakosi. tetapi apabila penelitian ini menggunakan analisis ricketts yang mengatakan bahwa profil wajah yang cembung apabila berada di depan atau sedikit menyentuh garis estetik dan besarnya rata–rata bibir atas dan bibir bawah yaitu 4 mm dan 2 mm, hasil penelitian ini bukan merupakan profil cembung. hal ini mungkin disebabkan karena analisis ricketts menggunakan standar ras kaukasoid, sehingga akan berbeda hasilnya bila dibandingkan dengan penelitian ini yang merupakan ras deutromelayu. dari hasil penelitian juga terlihat bahwa jarak antara bibir atas maupun bibir bawah laki–laki terhadap e–line lebih mendekati garis estetik dibanding perempuan. pada usia ini, pertumbuhan mandibula pada laki–laki lebih lambat 2 tahun dibandingkan perempuan, sehingga mandibula pada laki–laki nampak lebih retruded dan bibir atas lebih maju dibanding perempuan. penelitian pada populasi korea ditemukan bahwa ls e–line -1.66 mm dan li e – line -0.04 mm pada perempuan, sedangkan untuk laki – laki ls e – line ls e – line -1.66 mm dan li e – 0.09 mm. sedangkan pada populasi mongolia ditemukan ls e – line -0.8 mm dan li e – line -0.11 mm untuk perempuan dan ls e – line -0.83 mm dan li e – line -0.21 mm.15 dari hasil beberapa penelitian menunjukkan kesamaan antara populasi jawa dengan populasi korea dan mongolia. hal ini dipengaruhi oleh ras yang sama pada populasi penelitian. selain itu dari hasil penelitian didapatkan bahwa terdapat korelasi positif antara bibir atas dan bibir bawah. pertumbuhan jaringan lunak dan keras tidak berlangsung sendiri–sendiri, melainkan saling berpengaruh dan saling bergantung. pertumbuhan otot dipengaruhi oleh pertumbuhan tulang di bawahnya. penelitian mengenai profil wajah mengemukakan adanya korelasi positif antara bagian jaringan keras dan bentuk profil muka jaringan lunak. jarak bibir atas dan bawah terhadap e – line dipengaruhi oleh pertumbuhan hidung, posisi bibir, dan pertumbuhan dagu.16 penelitian tentang profil dentofacial pada maloklusi skeletal ditemukan bahwa terdapat hubungan antara sudut impa dengan li e–line. impa digunakan sebagai pemandu agar meletakkan insisiv bawah pada tulang basal. posisi insisiv bawah terhadap mandibula agar memberikan perawatan yang stabil yaitu 87–92 derajat.17 hal ini sesuai dengan teori bahwa posisi insisiv bawah berpengaruh pada profil wajah sehubungan dengan letak bibir bawah terhadap e – line. dari hasil penelitian, tidak ditemukan adanya hubungan antara impa terhadap posisi bibir atas maupun bibir bawah. hal ini berbeda dengan beberapa penelitian sebelumnya, hal ini mungkin dikarenakan karena subyek yang digunakan merupakan subyek dengan keadaan profil 225oktaviona, et al.: hubungan tweed triangle dan posisi bibir terhadap garis estetik wajah dan keadaan gigi yang normal, sedangkan pada penelitian sebelumnya sampel yang digunakan merupakan subyek dengan keadaan gigi yang maloklusi, selain itu juga subyek menjalani perawatan ortodonti cekat serta pencabutan gigi. hasil penelitian ini menunjukkan bahwa besar nilai tweed triangle pada orang jawa pada mahasiswa universitas airlangga (fakultas ilmu sosial dan ilmu politik, fakultas kedokteran gigi dan fakultas hukum) adalah fma 28.830, fmia 56.740, dan impa 94.430. hubungan antara fma dengan bibir bawah merupakan hubungan atau korelasi yang positif, sedangkan fma dengan bibir atas tidak ditemukan hubungan atau korelasi. hubungan antara fmia dengan bibir bawah merupakan hubungan atau korelasi yang negatif, sedangkan fmia dengan bibir atas tidak ditemukan hubungan. hubungan antara impa dengan bibir atas dan bibir bawah tidak ditemukan hubungan. hubungan antara bibir atas dan bibir bawah merupakan hubungan atau korelasi yang positif. nilai rerata fmia pada laki–laki lebih besar dibandingkan dengan perempuan. posisi bibir pada laki–laki lebih mendekati garis estetik dibandingkan dengan perempuan. ucapan terima kasih penelitian ini terlaksana atas dana boptn kolaborasi dosen mahasiswa tahun 2013 dengan tim: dr. i.g.a. wahju ardani, drg., m.kes., sp.ort, jusuf sjamsudin, drg., sp.ort.(k) dan achmad sjafei, drg., ms., sp.ort.(k). daftar pustaka 1. lau pyw, wong rwk. risk and complications in orthodontic treatment. hong kong dental j 2006. available from: url: http:// orthofree.com/cms/assets/22.pdf. accesssed november 30, 2011. 2. sarver dm, ackerman jl. orthodontics about face: the re–emergence of the esthetic paradigm. am j orthod dentofacial orthop 2000; 117(5): 575-6. 3. pereira cb, galvao ca, evans wg, preston cb. a cephalometric evaluation of brazillian prehistoric man. j dent assoc s afr 1983; 38(10): 627-31. 4. jacobson a. radiographic cephalometry from basics to 3–d imaging. 2nd ed. canada: quintessence publishing; 2006. p. 71–98. 5. graber tm. dentofacial orthopedics with fungtional appliances. amerika: mosby; 1997. p. 11, 53–4. 6. heryumani. proporsi sagital wajah laki–laki dan perempuan dewasa etnik jawa (studi pada mahasiswa fakultas kedokteran gigi universitas gadjah mada). majalah ilmiah kedokteran gigi (scientific journal in dentistry) 2007; 22. 7. nurbayati s. hubungan sudut interinsisal terhadap profil jaringan lunak pasien rsgmp fakultas kedokteran gigi universitas sumatera utara. sk ripsi. medan: fakultas kedokteran gigi universitas sumatera utara; 2011. 8. flores-mir c, silva e, barriga mi, lagravere mo, major pw.. lay person’s perception of smile aesthetics in dental and facial views. j orthod 2004; 31(3): 204-9. 9. iwasawa t, moro t, nakamura k. tweed triangle and soft – tissue consideration of japanese with normal occlusion and good facial profile. am j orthod 1997; 72(20): 119–27. 10. zen y. pola hubungan antara konveksitas, posisi gigi insisivus, dan posisi bibir dalam analisa ricketts. majalah ilmiah kedokteran gigi 2005; 20(63): 160–8. 11. kuramae mayury, maria beatriz borges de araújo magnani, darcy flávio nouer, gláucia maria bovi ambrosano, roger cristiano inoue. analysis of tweed’s facial triangle in black brazilian youngsters with normal occlusion. braz j oral sci 2004; 3(8). 12. bhattarai p, shrestha rm. tweeds analysis of nepalese people. nepal med coll j 2011; 13(2): 103-6. 13. hazar s, sercan a, hayal b. soft tissue profile changes in anatolian turkish girls and boys following orthodontic treatment with and without extraction. turk j med sci 2003; 34: 171-8. 14. mahyastuti rd, christnawati. perbandingan posisi bibir dan dagu antara laki-laki dan perempuan jawa berdasarkan analisa estetik profil muka menurut bass. majalah ilmiah kedokteran gigi 2008; 23(1): 1-7. 15. kim jh, odontuya g, bazar a, shin jl, tae wk. comparison of cephalometric norms between mongolian and korean adults with normal occlusion and well balances profile. korean j orthod 2011; 41(1): 42–50. 16. suhardono d. korelasi biometrik antar organ profil muka orang indonesia. tesis. surabaya: universitas airlangga; 1983. 17. wa hju a. p rofil dentofacial pada malok lusi skeletal (studi sefalometri). karya tulis akhir. surabaya: universitas airlangga; 2010. 48 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 48–52 original article wound healing induces vegf expression stimulated by forest honey in palatoplasty sprague dawley reine zhafirah1, alifah nur aida1, helmi hirawan2, tirta wardana3,4 1undergraduate student, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 2department of oral surgery, dental medicine study programme, faculty of medicine, jenderal soedirman university purwokerto, indonesia 3department of biomedicine, dental medicine study programme, faculty of medicine, jenderal soedirman university purwokerto, indonesia 4research integrity research laboratory, faculty of medicine, jenderal soedirman university, purwokerto, indonesia abstract background: cleft palate is a craniofacial disorder with definitive therapy using the v–y pushback technique palatoplasty, which has the impact of leaving the bone exposed on the palate with long wound healing and a high risk of infection. forest honey has high antioxidants and the ability to accelerate wound healing. purpose: this study aims to determine the effect of forest honey on vascular endothelial growth factor (vegf) expression to accelerate the wound healing process after palatoplasty biopsy. methods: posttest only control group design using sprague dawley palatoplasty was performed on 15 rats which were divided into three groups, namely the honey treatment (kp), aloclair as a positive control (kpp), and aquadest as a negative control (kkn). as much as 25 mg of honey was given therapeutically, and vegf expression analysis post-biopsy palatoplasty was measured using the elisa test. anova analysis was carried out to determine the significant differences between each treatment, and in silico analysis was conducted to determine the compounds’ role in honey on the mechanism of vegf expression. results: statistical analysis of vegf expression in the kp group was 41.10 ng/ml ± 0.26, the kkp was 39.57 ± 0.27, while the kkn was 33.26 ± 0.62 (p≤ 0.01). in silico study, genistein (c15h10o5) targets several signaling pathways such as pi3k-akt, ampk, and mtor, affecting accelerated proliferation and angiogenesis. conclusion: in wound healing acceleration, forest honey induced vegf expression through the genistein mechanism of angiogenesis and cell proliferation. keywords: forest honey; vegf; palatoplasty; angiogenesis; in silico article history: received 24 may 2022, revised 2 august 2022, accepted 28 september 2022 correspondence: tirta wardana, department of biomedicine, school of medicine, faculty of medicine, jenderal soedirman university, purwokerto, indonesia. email: tirta.wardana@unsoed.ac.id introduction the cleft palate’s abnormal formation causes an abnormal palate cleft, which is usually congenital. the cleft palate is a craniofacial abnormality that occurs in 1:1000 births.1 the highest incidence occurs in asians and is dominant in boys compared to girls with a 2.5:1 ratio.2 one of the therapies used to treat cleft palate is the use of definitive cleft palate therapy. the definitive therapy for cleft palate is reconstructive surgery to close the cleft by connecting existing tissue and is called palatoplasty.3,4 palatoplasty is a surgical procedure to repair the cleft palate in the mouth aimed at improving facial appearance, improving swallowing function, helping improve speech function, reducing hearing loss, and reducing the psychological impact on the patient.5,6 palatoplasty will cause injury, which will occur in the wound healing process through four phases: hemostasis, inflammation, proliferation, and remodeling. an essential factor in wound healing is the positive regulator and stimulation of the angiogenesis mechanism, which considerably impacts epidermal repair, granulation tissue, and quality repair formation.7–10 vascular endothelial growth factor (vegf) is an angiogenesis regulatory protein that plays a role in the angiogenic response stage by stimulating the degradation of the extracellular matrix around endothelial cells, increasing the proliferation and migration of endothelial cells, and helping the formation of blood vessel structures.7,11,12 in addition to its role in angiogenesis, vegf is also unique copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p48–52 mailto:tirta.wardana@unsoed.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p48-52 49zhafirah et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 48–52 in its effects on multiple components in the wound healing cascade, epithelization, and collagen deposition. therefore, vegf is one of the potential targets for the surgical wound healing response. after a cleft palate biopsy, postoperative wound care is critical to speed up the healing process. alternative treatments, especially bee honey, can develop for wound healing along with topical antimicrobial agents obtained from natural ingredients, which have no side effects.13 honey contains energy, carbohydrates, amino acids, proteins, and various active compounds such as alkaloids, flavonoids, steroids, triterpenoids, quinones, and hydrogen peroxide (h2o2), which have antiinflammatory, antibacterial, anti-allergic, antioxidant, and anti-carcinogenic functions.14 in addition, the contents of honey can stimulate vegf expression to affect the angiogenesis process. as previously reported, flavonoids could increase vegf expression in oral aphthous ulcers, new blood cell formation, and pseudotubule formation in the aortic ring test.8,9,12,15–18 therefore, we aimed to determine the role of forest honey administration on vegf expression for wound healing in a palatoplasty model in sprague dawley. materials and methods true experimental research laboratories in vivo were conducted with the sprague dawley animal model with posttest only control group design. fifteen samples from each group, namely the treatment group, were given black honey (kp), the positive control was given aloclair, and the negative control was given aquadest. the analysis showed the difference in vegf level against the treatment given. after obtaining the feasibility of all the methods carried out, the study was performed. the ethical approval was issued by the ethics committee faculty of medicine jenderal soedirman university on july 22, 2020, with reference number 127/kepk/vii/202. making animal models of palatoplasty in sprague dawley rats used a punch biopsy with asepsis on the hard palate using povidone with cotton pellets. anesthetic ketamine was given at a dose of 1–2 mg/kg intravenously or 5–10 mg/kg intramuscularly. punch biopsy was performed using a seamless premier® uni-punch width of 3 mm on the hard palate to the exposed periosteum. the bone was seen without any bleeding again, resembling the v–y pushback technique. maintenance was carried out in 25 cm x 12 cm x 15 cm / 0.0045 m3 with sufficient light and air conditions and far from noisy conditions. the environment for rearing experimental animals has a temperature of around 16–27ºc with a humidity of 40%–70%. animal models are standard fed ad ii and drink aquadest ad libitum, with the amount of feed every morning being about 20 g/head/day. after punch biopsy, applying forest honey treatment to the wound covered the entire area with a laboratory spatula topically. according to previous research, the dosage was done with 25 mg of honey, which is the maximum concentration.19 the positive control group (kkp) was treated with aloclair and the negative control group (kkn) with aqueduct at the same dose. treatment was given routinely in the morning until the fourth day. the tissue around the punch biopsy wound was taken using blade no. 15, as much as 15 mg. vegf level analysis was carried out to determine changes in expression using elisa. expression level testing was carried out using the indirect sandwich technique using the bt lab kit (cat no. e0659ra). a standard dilution buffer was constructed with serial concentration as the horizontal axis, and the optical density (od) value was taken as a vertical axis. the od was detected at a wavelength of 450 nm.20,21 all procedures followed all advice from the company. statistical data analysis was performed using spss version 25 (ibm inc, chicago, il, usa) for windows. expression data analysis was carried out based on mean ±sd. one-way anova was conducted to analyze the differences between the treatment groups. a confidence interval (ci) of 95% with p<0.05 shows statistical significance, graph and curve performed using graphpad prism software version 9.0 for windows (graphpad software, san diego, ca, usa). in silico analysis was carried out using web server-based online database software and pubmed to analyze the content of compounds contained in forest honey.22 analysis of compounds and chemical bond structures used http://www. pubchem.ncbi.nlm.nih.gov/ using canonical smiles.23 the analysis of the compound and target protein mechanism used http://stitch.embl.de/,24 while the interaction mechanism and the mechanism that was influenced by chemical compounds found in forest honey were analyzed using http:///string.db.org/ in the kyoto encyclopedia of genes and genomes (kegg) section pathways.25 results animal models of palatoplasty were performed using punch biopsy methods with a seamless premier® unipunch 3 mm on the palates of the rats. more minor diameter biopsy wounds can heal independently, but a biopsy diameter greater than 3 mm can cause scarring, requiring 1-2 stitches to be sutured. the wound was characterized by exposure to the palate and normal bleeding from the wound and surface. the effect of giving forest honey on vegf levels in wounds was measured using elisa by reading the od value with a wavelength of 450 mm. the absorbance value used is the average of the two repetitions. vegf levels in the treatment group given forest honey were 41.10 ± 0.26 ng/ml, in the positive control group given aloclair they were 39.57 ± 0.27 ng/ml, and in the negative control group they were negative 33.26 ± 0.62 ng/ml (figure 1). copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p48–52 http://www http://stitch.embl.de/ http:///string.db.org/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p48-52 50 zhafirah et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 48–52 the post hoc lsd test conducted expression change analysis to determine significant differences between the groups. the results showed that there was a very significant difference in each group. the negative control group, the positive control group, and the treatment group obtained a significant difference (p≤0.01). statistical results analyzed of vegf level expression were kp (treatment group) = 41.10 ng/ml 0.26, kkp = 39.57 0.27, and kkn = 33.26 0.62 (p≤ 0.01). in a silico study, we analyzed genistein (c15h10o5), as one of the honey’s ingredients which has been promising, by targeting several signaling pathways such as pi3k-akt, ampk mtor, prostate cancer, and insulin resistance. changes in the mechanism can trigger accelerated proliferation and angiogenesis (figure 2). discussion cleft palate craniofacial deformity or palatoschisis affects the sufferer because it will cause eating disorders, speech disorders, psychological disorders, and impaired tooth growth. the cause of cleft palate is due to the interaction between genetic and non-genetic (environmental) factors. surgical treatment (palatoplasty) is performed to treat palatoschisis. one of the techniques is called a punch biopsy, which is simple and easy, and the diameter of the biopsy is small, so there is no need for suturing the wound.26 however, palatoschisis needs to be treated as early as possible and antibacterial properties used to speed up wound healing. the increased expression of vegf in the administration of honey indicated an increasing amount of neovascularization compared to the control group. honey is considered one of the candidates among natural ingredients, which the data shows accelerates wound healing after palatoplasty surgery due to its high content of flavonoids and h2o2. 27–29 the content of flavonoids in wild honey shows a rate of 20.43%. flavonoids are known to have anti-inflammatory and antioxidant properties and induce vegf. the dynamic content of flavonoids in honey, such as quercetin,30 apigenin,31 genistein,32 and kaempferol,33 can activate hypoxia-inducible factor-1 (hif-1), which then induces vegf.34 the content of flavonoids in honey such as quercetin, apigenin, genistein, and kaempferol has been widely reported to have a role in wound healing. quercetin is often recommended as a drug for oral ulcers to increase collagen in wounds and accelerate angiogenesis.32,35 quercetin is known to increase vegf by activating hif-1. in addition, apigenin and genistein pass through the phosphatidylinositol-3-kinase (pi3k) pathway by binding to protein kinase 1 (akt1), and increasing endothelial nitric oxide figure 1. statistical analysis of vegf expression. kp (group treatment with honey), kkp (aloclair/ positive control), kkn (aquadest/negative control) (p≤ 0.01). figure 2. in silico analysis of the role of genistein, which is one of the ingredients in forest honey and plays an essential role in the mechanism of angiogenesis and cell proliferation. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p48–52 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p48-52 51zhafirah et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 48–52 synthase (enos) to increase vegf expression.36–40 this plays an essential role in the mechanism of angiogenesis and cell proliferation. in a previous study, hu reported that the kaempferol content in honey could increase the activation of vegf receptor-2 (vegfr-2) receptors to increase vegf protein expression and keratinocyte cell migration so that re-epithelialization occurs.33 in this study, to determine the role of honey on vegf expression through in vivo and in silico analysis, we found that genistein compounds directly target pi3k and akt, thereby influencing the angiogenesis pathway. the process of the angiogenesis mechanism is mainly mediated through the interaction of vegf-a with vegfr-2. other vegf ligand and receptor variants play a secondary role in this process.7 one of the wound healing process effects is hypoxia due to the lack of oxygen in the cells around the wound. hypoxia stimulates the vegf flt-1 receptor, and then hypoxia can regulate the expression of vegf and its receptors so that angiogenesis occurs through hif-1 by inducing vegf.41 this study proved that forest honey had increased vegf expression in palatoplasty of wound healing in animal models. the 25 mg of forest honey increased vegf expression higher than the positive and negative controls with a fold change of 1.038 times higher (41.10 ng/ml) than the positive control given aloclair (39.57 ng/ml), and the control negative in the form of aquadest (33.26 ng/ml). thus, we recommend alternative natural treatment using forest honey to support the acceleration of postbiopsy wound healing. forest honey affects the increased expression of vegf protein in punch biopsy wounds of the palate in sprague dawley rats. the use of 25 mg of honey affected vegf protein expression with a fold change of 1.038 times compared to the positive control and 1.235 times that of the negative control group. in conclusion, we have confirmed the role of forest honey in accelerating wound healing in a palatoplasty animal model—the ability of forest honey to increase vegf protein expression. analysis in silico indicated the role of genistein in forest honey that has affected the cell’s angiogenesis and proliferation mechanism. references 1. bennun rd, harfin jf, sándor gkb, genecov d. cleft lip and palate management. bennun rd, harfin jf, sándor gkb, genecov d, editors. cleft lip and palate management: a comprehensive atlas. hoboken, nj, usa: joh n wiley & sons, i nc; 2015. p. 1–267. 2. vyas t, gupta p, kumar s, gupta r, gupta t, singh h. cleft of lip and palate: a review. j fam med prim care. 2020; 9(6): 2621. 3. raj n, raj v, aeran h. interim palatal lift prosthesis as a constituent of multidisciplinary approach in the treatment of velopharyngeal incompetence. j adv prosthodont. 2012; 4(4): 243. 4. rossell-per r y p, cotr ina-rabanal o, ba r renechea-ta razona l, vargas-chanduvi r, paredes-aponte l, romero-narvaez c. mucoperiosteal f lap necrosis after primar y palatoplasty in patients with cleft palate. arch plast surg. 2017; 44(03): 217–22. 5. oliveira mhm de f, rezende al de f, ibiapina c da c, godinho rn. hearing impairment in children with cleft lip and cleft palate. rev médica minas gerais. 2015; 25(3): 400–5. 6. gongorjav na, luvsandorj d, nyanrag p, garidhuu a, sarah eg. cleft palate repair in mongolia: modified palatoplasty vs. conventional technique. ann maxillofac surg. 2012; 2(2): 131. 7. johnson ke, wilgus ta. vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. adv wound care. 2014; 3(10): 647–61. 8. dipietro la. angiogenesis and wound repair: when enough is enough. j leukoc biol. 2016; 100(5): 979–84. 9. g reaves ns, ashcrof t k j, bag uneid m, bayat a. cu r rent understanding of molecular and cellular mechanisms in fibroplasia and angiogenesis during acute wound healing. j dermatol sci. 2013; 72(3): 206–17. 10. gonzalez ac de o, costa tf, andrade z de a, medrado arap. wound healing a literature review. an bras dermatol. 2016; 91(5): 614–20. 11. kumar p, kumar s, udupa ep, kumar u, rao p, honnegowda t. role of angiogenesis and angiogenic factors in acute and chronic wound healing. plast aesthetic res. 2015; 2(5): 243. 12. neve a, ca ntatore f p, ma r uotti n, cor rado a, r ibatti d. extracellular matrix modulates angiogenesis in physiological and pathological conditions. biomed res int. 2014; 2014: 756078. 13. eteraf-oskouei t, najafi m. traditional and modern uses of natural honey in human diseases: a review. iran j basic med sci. 2013; 16(6): 731–42. 14. miguel m, antunes m, faleiro m. honey as a complementary medicine. integr med insights. 2017; 12(2): 117863371770286. 15. guo d, wang q, li c, wang y, chen x. vegf stimulated the angiogenesis by promoting the mitochondrial functions. oncotarget. 2017; 8(44): 77020–7. 16. daaboul he, dagher c, taleb ri, bodman-smith k, shebaby wn, el-sibai m, mroueh ma, daher cf. β-2-himachalen-6-ol inhibits 4t1 cells-induced metastatic triple negative breast carcinoma in murine model. chem biol interact. 2019; 309: 108703. 17. varoni em, lodi g, sardella a, car rassi a, iriti m. plant polyphenols and oral health: old phytochemicals for new fields. curr med chem. 2012; 19(11): 1706–20. 18. ma j, zhou x. pro-angiogenic and anti-angiogenic effects of small molecules from natural products. in: nutraceuticals and natural product derivatives. hoboken, nj, usa: john wiley & sons, inc.; 2018. p. 81–109. 19. ibnu ys. potensi madu sebagai terapi topikal otitis eksterna. j ilm kedokt wijaya kusuma. 2019; 8(2): 7–22. 20. zhu l, he j, cao x, huang k, luo y, xu w. development of a double-antibody sandwich elisa for rapid detection of bacillus cereus in food. sci rep. 2016; 6(1): 16092. 21. wang s, liu j, yong w, chen q, zhang l, dong y, su h, tan t. a direct competitive assay-based aptasensor for sensitive determination of tetracycline residue in honey. talanta. 2015; 131: 562–9. 22. national library of medicine. ncbi pubmed overview. national center for biotechnology information. 2021. 23. national center for biotechnology information. pubchem database ncbi. 2021. available from: https://pubchem.ncbi.nlm.nih.gov/ docs/about/. 24. szklarczyk d, santos a, von mering c, jensen lj, bork p, kuhn m. stitch 5: augmenting protein-chemical interaction networks with tissue and affinity data. nucleic acids res. 2016; 44(d1): d380-4. 25. hermawan a, putri h. bioinformatics studies provide insight into possible target and mechanisms of action of nobiletin against cancer stem cells. asian pacific j cancer prev. 2020; 21(3): 611–20. 26. levitt j, bernardo s, whang t. how to perform a punch biopsy of the skin. n engl j med. 2013; 369(11): e13. 27. almaz ai, purnawati rd, istiadi h, susilaningsih n. the effect of honey in second degree burn healing on wistar rats (overview of angiogenesis and the number of fibroblasts). sains med. 2020; 11(1): 27–32. 28. martinotti s, ranzato e. honey, wound repair and regenerative medicine. j funct biomater. 2018; 9(2): 34–40. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p48–52 https://pubchem.ncbi.nlm.nih.gov/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p48-52 52 zhafirah et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 48–52 29. oryan a, alemzadeh e, moshiri a. biological properties and therapeutic activities of honey in wound healing: a narrative review and meta-analysis. j tissue viability. 2016; 25(2): 98–118. 30. gopalakrishnan a, ram m, kumawat s, tandan s, kumar d. quercetin accelerated cutaneous wound healing in rats by increasing levels of vegf and tgf-β1. indian j exp biol. 2016; 54(3): 187–95. 31. tu f, pang q, chen x, huang t, liu m, zhai q. angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway. int j mol med. 2017; 40(6): 1639–48. 32. sergiel i, pohl p, biesaga m. characterisation of honeys according to their content of phenolic compounds using high performance liquid chromatography/tandem mass spectrometry. food chem. 2014; 145: 404–8. 33. hu w-h, wang h-y, xia y-t, dai dk, xiong q-p, dong tt-x, duan r, chan gk-l, qin q-w, tsim kw-k. kaempferol, a major flavonoid in ginkgo folium, potentiates angiogenic functions in cultured endothelial cells by binding to vascular endothelial growth factor. front pharmacol. 2020; 11: 526–38. 34. nugroho am, elfiah u, normasari r. pengaruh gel ekstrak dan serbuk mentimun (cucumis sativus) terhadap angiogenesis pada penyembuhan luka bakar derajat iib pada tikus wistar. e-jurnal pustaka kesehat. 2016; 4(3): 443–8. 35. fu j, huang j, lin m, xie t, you t. quercetin promotes diabetic wound healing via switching macrophages from m1 to m2 polarization. j surg res. 2020; 246: 213–23. 36. favaro e, granata r, miceli i, baragli a, settanni f, cavallo perin p, ghigo e, camussi g, zanone mm. the ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/akt, extracellular signal-related kinase (erk)1/2 and camp/protein kinase a (pka) signalli. diabetologia. 2012; 55(4): 1058–70. 37. eller-borges r, batista wl, da costa pe, tokikawa r, curcio mf, str umillo st, sar tori a, moraes ms, de oliveira ga, taha mo, fonseca f v., stern a, monteiro hp. ras, rac1, and phosphatidylinositol-3-kinase (pi3k) signaling in nitric oxide induced endothelial cell migration. nitric oxide biol chem. 2015; 47: 40–51. 38. abhinand cs, raju r, soumya sj, arya ps, sudhakaran pr. v egf-a / v egf r 2 sig na l i ng net work i n endot hel ia l cel ls relevant to angiogenesis. j cell commun signal. 2016; 10(4): 347–54. 39. alzoubi a, ghazwi r, alzoubi k, alqudah m, kheirallah k, khabour o, allouh m. vascular endothelial growth factor receptor inhibition enhances chronic obstructive pulmonary disease picture in mice exposed to waterpipe smoke. folia morphol (warsz). 2018; 77(3): 447–55. 40. salehi b, machin l, monzote l, sharifi-rad j, ezzat sm, salem ma, merghany rm, el mahdy nm, kılıç cs, sytar o, sharifi-rad m, sharopov f, martins n, martorell m, cho wc. therapeutic potential of quercetin: new insights and perspectives for human health. acs omega. 2020; 5(20): 11849–72. 41. lugano r, ramachandran m, dimberg a. tumor angiogenesis: causes, consequences, challenges and opportunities. cell mol life sci. 2020; 77(9): 1745–70. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p48–52 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p48-52 � volume 47, number 1, march 2014 research report combination of aloe vera and xenograft induction on decreasing of nf-kb of tooth extraction socket preservation in cavia cobaya utari kresnoadi1 and retno pudji rahayu2 1 department of prosthodontics 2 department of oral and maxillofacial pathology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: tooth extraction can naturally cause inflammation triggering osteoclast proliferation and alveolar bone resorption. preservation of the tooth extraction sockets is needed for patients in order to reduce alveolar bone resorption risks. aloe vera is known to have anthraquinones components, namely aloin, aloe emedin, and barbaloin, considered as anti-inflammation. therefore, to overcome the inflammation, the role of nf-kb is very significant to decrease nuclear factor kappa b (nf-kb). as a result, inflammation risks will be decreased. purpose: the study was aimed to determine the induction effect of combination of aloe vera and xcb into tooth extraction sockets to reduce inflammation by reducing nf-kb expression, osteoclasts and osteoblasts. methods: forty-eight cavia cobaya were divided into eight groups, each group consisted of six animals. the mandibular incisors of those cavia cobaya were extracted and induced with either peg, xcb, aloe vera, or the combination of aloe vera + xcb. those animals were sacrificed on day 7 and day 30 after the extraction. then immunohistochemical and histopathology examinations were conducted to observe nf-kb expression, osteoblasts and osteoclasts. results: it was known that in group induced with the combination of aloe vera and xenograft concelous bovine, the growth of osteoblasts was high, while nf-kb expression and osteoclasts reduced. conclusion: it can be concluded that the induction of the combination of aloe vera and xcb into the tooth extraction sockets can reduce nf-kb expression and osteoclast, as a result, alveolar bone resorption risks decrease, and osteoblast increase. key words: aloe vera, nf-kb, tooth extraction socket, xenograft abstrak latar belakang: trauma mekanis akibat pencabutan gigi asli menyebabkan keradangan. keradangan memicu proliferasi osteoklas sehingga menyebabkan resorpsi tulang alveolararis. pada pembuatan gigi tiruan, resorpsi tulang alveolar yang terjadi, sangat tidak diinginkan, sebab resorpsi tulang alveolar mengurangi keberhasilan pembuatan gigitiruan. diperlukan preservasi soket pencabutan gigi asli pada penderita untuk mencegah terjadinya resorpsi tulang alveolar. aloe vera mempunyai komponen anthraquinon yaitu aloin, aloe emodin, barbaloin yang merupakan anti inflamasi yang dapat secara cepat menyembuhkan luka, sehingga berpotensi untuk digunakan pada preservasi soket. didalam mengatasi keradangan peran nf-kb sangat berarti, sebab penurunan nf-kb akan mengurangi terjadinya inflamasi. tujuan: penelitian ini bertujuan untuk menguji apakah induksi kombinasi lidah buaya dan xcb ke soket pencabutan gigi dapat mengurangi peradangan dengan mengurangi ekspresi nf kb , osteoklas dan osteoblast. metode: empat puluh delapan ekor cavia cabaya yang terdiri dari 8 kelompok, tiap kelompok 6 ekor, kelompok pengisian peg (kontrol), kelompok pengisian xcb, kelompok pengisian aloe vera dan kelompok pengisian kombinasi aloe vera dan xcb, kelompok ini terdiri dari kelompok 7 dan 30 hari, kemudian diperiksa dengan imunohistokimia ekspresi nf-kb dan pemeriksaan histologi untuk osteoblas dan osteoklas. hasil: kelompok yang diisi kombinasi alo vera dan xenograft concelous bovine pada soket pencabutan gigi, menunjukan nilai tertinggi dalam pertumbuhan osteoblas dan penurunan pada ekspresi nf-kb dan osteoklas. simpulan: induksi kombinasi aloe vera � dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 1–6 dan xenograft concelous bovine pada preservasi soket pencabutan gigi dapat menurunkan ekspresi nf-kb dan osteoklas, menurunkan resiko resorpsi tulang alveolar dan meningkatkan osteoblas. kata kunci: aloe vera, nf-kb, soket pencabutan gigi, xenograft ccorrespondence: utari kresnoadi, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: ut.kres@yahoo.com introduction original tooth extraction may cause trauma, triggering inflammation and later stimulating the growth of osteoclasts and the resorption of alveolarar bone. thus, it is necessary to preserve the extraction sockets in order to reduce the risks of alveolar bone resorption. alveolar bone resorption can also cause dentures making become unsuccessful. in general, the success of dentures actually depends on retention factor, stabilization factor, and convenience factor of denture use, which can be achieved by supporting anatomical condition related to prominent ridge. therefore, the prevention of alveolar bone resorption should be conducted during tooth extraction by reserving the extraction socket. in general surgery, especially oral surgery, and periodontics, graft has been used to repair bone defects and augmentation.1-6 the use of graft in long period, according to pachene et al. cit. lanza et al., 7 is not stable even though the design and development of tissue engineering products have benefited the clinical use of various biodegradable polymers for many years. thus, it takes an effort to tissue engineering for reserving tooth extraction socket by combining aloe vera and graft materials in order to reduce alveolar bone resorption risks and get a prominent ridge. aloe vera is considered to have both a function as a biogenic stimulator and hormones stimulating wound healing activity. aloe vera liquid even can prevent scartissue incision, and when the gel is used after surgery, the incision will be healed faster.8 similarly, some studies also suggest that there is anthraquinones components in aloe vera, namely aloin, aloe emodin, and barbaloin, which have important role as anti-inflammatory, anti-bacteria, and anti-virus so that aloe vera can reduce inflammation caused by extraction trauma and also induce extraction wound healing process.9-14 to reduce inflammation risk, the physiological role of nuclear factor kappa b (nf-kb) is very meaningful in immune system. for instance, nuclear factor kappa b can control transcription, cytokines, antimicrobial effector as well as genes that regulate cellular differentiation, life survival and cell proliferation, consequently, it then can regulate various aspects of innate and adaptive immune responses.15 lorenzo et al.,16 moreover, said that tumor necrosis factor α (tnf-α) and interlukin 1 (il-1) can stimulate osteoclast formation. interlukin-1 can stimulate activities of receptor activator of nuclear factor kb ligand (rankl) to induce osteoclastogenesis, and can also be considered as a strong resorption stimuli for prostaglandin synthesis in bones, whereas tnf-α is a potent stimulator of bone resorption. therefore, the increasing of nf-kb expression can cause inflammation triggered by the increasing of tnf-α cytokines. as a result, it takes an effort to reduce the inflammation caused by the tooth extraction trauma. therefore, this research was aimed to determine whether the induction of the combination of aloe vera and xcb into tooth extraction sockets can reduce inflammation by reducing nf-kb expression, osteoclasts and osteoblasts. materials and methods this research can be considered as an experimental study with a randomized post test control group design. animals used in this research were male cavia cobaya (guinea pig) with body weight of 300-350 g at the age of 3-3.5 months. in addition, materials used in this research were aloe vera extracts, sterile distilled water, xcb from dr. soetomo hospital tissue bank, absolute alcohol, 70% alcohol, anti nf-kb p65 monoclonal antibodies (santa cruz), reagents for immunostaining kit (biocare) used in immunohistochemical examination, and reagents for hematoxilen-eosin staining (he). thus, indirect immunohistochemical examination could be conducted by using primary antibodies and also secondary antibodies. furthermore, tools used in this research were a set of tools for immunohistochemical examination techniques, a set of tools for making preparations, micropipette, tip (yellow, white, blue), light microscopy, object glass, and cover glass. those experimental animals had been taken care in biochemistry laboratory of faculty of medicine universitas airlangga. tissue preparations used in this research was conducted in anatomical pathology of dr. soetomo hospital. aloe vera extracts was prepared in physical chemistry laboratory of faculty of pharmacy, universitas airlangga, while the making of freeze dried aloe vera was conducted in faculty of biology laboratory of science and technology, universitas airlangga. the processes of immunohistochemical staining and he staining for immunohistochemical examination were conducted in biochemistry and biomolecular engineering laboratory of faculty of medicine, universitas brawijaya. �kresnoadi and rahayu: the induction of combination of aloe vera and xenograft the procedures of this research, furthermore, consisted of several steps. forty-eight cavia cabaya animals were divided into eight groups, each of which consisted of six animals. before their lower right incisors were extracted by using a special pliers (needle holder), they had intravenously been anaesthetized with ketamine 0.2 cc or 300 g bm.17 then, their extraction sockets were induced with either peg, peg + xcb, aloe vera + peg, and a combination of aloe vera + xcb + peg as much as 0.1 cc of the appropriate volume of the extraction socket, and then stitched. the sockets of those animals in group 1 and group 2 were induced with polyethylene glycol (peg), and then examined on day 7 and day 30 after the treatment. meanwhile, the sockets of those animals in group 3 and group 4 were induced with xcb + peg, and then examined on day 7th and day 30th after the treatment. the sockets of those animals in group 5 and group 6 were induced with aloe vera and peg, and then examined on day 7th and day 30th after the treatment. and, the sockets of those animals in group 7 and group 8 were induced with the mixture of aloe vera 500 mg + xcb 500 mg + peg 24 g, and then examined on day 7th and day 30th after the treatment.18 after 7 days and 30 days, those animals were killed, and their jaws were cut off. the preparation consisted of hard materials was decalcified first with 2% nitric acid for approximately 14 days, and then paraffin block preparations were made. those paraffin blocks were cut with a rotary microtome with a thickness of about 4 microns, and placed on a glass object. deparaffinization was conducted by dissolving in xylol for 2 x 3 minutes. the rest xylol was washed with absolute alcohol 99%, 95%, 90%, 80%, and 70% for 2 x 1 minutes. next, the residual alcohol was washed with running water. nf-kb expression, osteoblasts and osteoclasts were examined by using a light microscope after immunohistochemical staining and he staining were then conducted. for the purposes of calculating, moreover, the code of the already coded slides was closed, and a new number was given randomly for each slide. each slide was then observed with 1000x magnification and 10 fields of view. afterwards, the results of the observation were written on the worksheet and calculated for their average value per field of view. the results of the calculation were then tabulated and tested with kolmogorov-smirnov test and analysis of variants (anova) test. finally, to compare the results of the groups, tuckey hsd multiple comparison test was conducted. results the mean (m) and standard deviations (sd) of nf-kb expression, osteoblasts, and osteoclasts in each treatment groups 7 days and 30 days after the treatment can be seen in figure 1. based on figure 1, it can be said that on day 7th and day 30th , nf-kb expression was decreased in either group control or all treatment groups. but, the biggest decreasing was in the group induced with the combination of aloe vera and xcb. on day 7th , the number of osteoblasts was increased in all of the groups. the biggest increasing was in the group induced with the combination of aloe vera and xcb. on day 30th , the number of osteoblasts in group control and that in the group induced with xcb were significantly increased, while that in the group induced with aloe vera was slightly decreased. the highest number of osteoblasts was found in the group induced with the combination of aloe vera and xcb. on the other hand, the number of osteoclasts was decreased in all of the groups. but, the smallest decreasing was in the group induced with the combination of aloe vera and xcb. then nf-kb expression can be seen in figure 2. the results of anova test on nf-kb expressions on day 7th and day 30th after the examination can be seen in table 1. the results of anova test on nf-kb expression on day 7 and day 30 after the examination show that there was significant difference between all of the treatment groups induced with either xcb, aloe vera, or the combination of aloe vera and xcb and group control with p = 0.001<0.05. after anova test, tukey hsd multiple comparison test was conducted with the following results as seen in table 2. in table 2, it is known that there were significant differences among the treatment groups, especially with group control. however, there was no significant difference table 1. nf-kb expressions on day 7 and day 30 after the examination variation df f p among the groups 7 148.570 0.001 within the groups 40 total 47 note: df = degrees of freedom; f = frequency; p = probability figure 1. the graphs of nf-kb expression, osteoblasts, and osteoclasts in group control, group induced with xcb, group induced with aloe vera, group induced with aloe vera + xcb on day 7 and day 30 after the examination. 7 days 30 days osteoblast osteoclast osteoblast osteoclast control � dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 1–6 figure 2. the pictures of nf-kb expression after immunohistochemical staining. note: a+b : nf-kb expression in group control after immunohistochemical examination on day 30; c+ d : nf-kb expression in the group induced with xcb after immunohistochemical examination on day 30; e + f : nf-kb expression in the group induced with aloe vera after immunohistochemical examination on day 30; g + h : nf-kb expression in the group induced with the combination of aloe vera + xcb after immunohistochemical examination on day 30 blue arrow points to nf-kb expression between group xcb on day 30 and group aloe vera on day 7 with p=0.141>0.05. similarly, there was no significant difference between the group aloe vera on day 7 and the group aloe vera and xcb on day 7 with p = 0.954>0.05. there was also no significant difference between group aloe vera on day 30 and group aloe vera + xcb on day 7 with p=0.203>0.05. finally, it is also known that there was also no significant difference between group aloe vera on day 30 and group aloe vera + xcb on day 30 with with p=0.983>0.05. discussion this study was aimed to measure nf-kb expression since it has a main physiological role in immune system as well as in inflammation process.15 tooth extraction always causes mechanical trauma triggering inflammation. when exposed to trauma, incompetent macrophage cells and mast cells will increase tnf-α. most of the immune response is actually regulated by nf-kb in sitosol and bound into ikb. consequently, phosphorylation of ikb will occur and then trigger proteasome degradation and nf-kb releasing to �kresnoadi and rahayu: the induction of combination of aloe vera and xenograft translocate to nucleus.19, 20 inflammation makes osteoclasts increased, and then proinflammatory cytokines, tnf-α, will also be increased. consequently, the receptor activator of nuclear factor kb ligand (rankl) and the receptor activator of nuclear kb (rank) will then be increased. rank and rankl can be considered as a mediator of osteoclast differentiation signals. therefore, when osteoclasts increases, bone resorption will occur.21, 22 based on the results of the examination, it is known that nf-kb expression was decreased in all of the groups. but, the lowest one was in the group induced with the combination of aloe vera and xcb. the results indicate that the decreasing of nf-kb can reduce inflammation risks. similarly, a research conducted by hayden et al.15 also showed that nf-kb can be considered as an important mediator of local and systemic inflammation, and also can affect on tnf-α as proinflammatory cytokine. therefore, nf-kb is essential for the propagation and elaboration of cytokine responses.15 in figure 1, furthermore, it is known that the number of osteoblasts was increased in all of the groups. however, the highest occurred in the group induced with the combination of aloe vera and xcb. it is also known that the number of osteoclasts was decreased. the lowest one was in the group induced with the combination of aloe vera and xcb. thus, it can indicate that the decreasing of nf-kb can lead to the decreasing of osteoclast growth. thereby, bone resorption will be decreased, and the number of osteoblasts will be increased. the occurrence of bone resorption is actually affected by osteoclast activating factor (oaf).23-25 nf-kb (nuclear factor kb) as a transcription factor involves in various activities, including the regulation of the immune response, the maturation of immune cells, the development of secondary lymphoid organs, and osteoclast genesis.26 finally, the study suggested that the induction of the combination of aloe vera and xenograft concelous bovine into tooth extraction sockets could reduce nf-kb expression. as a result, osteoclasts will decreased, while osteoblasts will increased. thus, alveolar bone growth then will be improved. tabel 2. the results of multiple comparison test on bmp2 expression groups group control on day 7 group control on day 30 group xcb on day 7 group xcb on day 30 group aloe vera on day 7 group aloe vera on day 30 group aloe vera+xcb on day 7 group aloe vera+xcb on day 30 group control on day 7 -s s s s s s s group control on day 30 ns -ns s s s s s group xcb on day 7 s s -ns ns s s s group xcb on day 30 s s ns -ns ns s s group aloe vera on day 7 s s ns ns -ns s s group aloe vera on day 30 s s s ns ns -ns ns group aloe vera+xcb on day 7 s s s s s ns -ns group aloe vera+xcb on day 30 s s s s s s ns -note: s : significant; ns: no significant � dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 1–6 references 1. lieberman jr, friedlaender ge. bone regeneration and repair. 1th ed. totowa, new jersey: humana press; 2005. p. 22-32. 2. boix d, weiss p, gauthier o, guicheux j, bouler jm, pilet p, daculsi g, grimandi g. injectable bone substitute to preserve alveolarar ridge resorption after tooth extraction: a study in dog. j mater sci mater med 2006; 17(11): 1145-52. 3. steigmann m. a bovine-bone mineral block for the treatment of severe ridge deficiencies in the anterior region: a clinical case report. int j oral maxillofac implants 2008; 23(1): 123-8. 4. barone a, aldini nn, fini m, giardino r, calvo guirado jl, covani u. xenograft versus extraction alone for ridge preservation after tooth removal: a clinical and histomorphometric study. j periodontol 2008; 79(8): 1370-7. 5. mardas n, chadha v, donos n. alveolarar ridge preservation with guided bone regeneration and a synthetic bone substitute or a bovine-derived xenograft: a randomized, controlled clinical trial. clin oral implants res 2010; 21(7): 688-98. 6. pelegrine aa, sorgi da costa ce, pizzigati correa me, marques jr jfc. clinical and histomorphometric evaluation of extraction socket treted withan autologous bone marrow graft. clinical oral implant rest 2010; 21: 535-42. 7. lanza r, langer r, vacanti j. principle of tissue enginering. 3rd ed. china: elsevier inc; 2007. p. 324. 8. roostita & tim redaksi qanita. lidah buaya. cetakan i. bandung: pt. mizan pustaka; 2008. h. 19-38. 9. kurniawati r, marminah mt. efek anti inf lamasi gel lidah buaya (aloe vera linn) terhadap tikus putih. prosiding seminar nasional tumbuhan obat indonesia xxix, solo, 2006; h. 82-9. 10. yu cs1, yu fs, chan jk, li tm, lin ss, chen sc, hsia tc, chang yh, chung jg. aloe-emodin affects the levels of cytokines and functions of leukocytes from sprague-dawley rats. in vivo 2006; 20(4): 505-9. 11. hamman jh. composition and applications of aloe vera leaf gel. molecules 2008; 13(8): 1599-616. 12. park my, kwon hj, sung mk. evaluation of aloin and aloe-emodin as anti-inf lammatory agents in aloe by using murine macrophages. biosci biotechnol biochem 2009; 73(4): 828-32. 13. lawrence r, tripathi p, jeyakumar e. isolation, purification and evaluation of antibacterial agents from aloe vera. braz j microbiol 2009; 40(4): 906-15. 14. moghadasi sm, verma sk. aloe vera their chemicals composition and applications: a review. int j biol med res 2011; 2(1): 46671. 15. hayden ms, west ap, ghosh s. nf-kappab and the immune response. oncogene 2006; 25(51): 6758-80. 16. lorenzo j, horowitz m, choi y. osteoimmunology: interactions of the bone and immune system. endocr rev 2008; 29(4): 403-40. 17. kusumawati d. bersahabat dengan hewan coba. cetakan pertama. jogjakarta: gajah mada university press; 2004. h. 67. 18. kresnoadi u. tall like receptor 2 sebagai signaling pathway osteogenesis tulang alveolar yang diinduksi kombinasi aloe vera dan graft. disertasi. surabaya: program doktor ilmu kedokteran, universitas airlangga; 2012. 19. tergaonkar v, correa rg, ikawa m, verma im. distinct roles of ikappab proteins in regulating constitutive nf-kappab activity. nat cell biol 2005; 7(9): 921-3. 20. yenari ma, liu j, zheng z, vexler zs, lee je, giffard rg. antiapoptotic and anti-inf lammatory mechanisms of heat-shock protein protection. ann n y acad sci 2005; 1053: 74-83. 21. lundy ft, linden gj. neuropeptides and neurogenic mechanisms in oral and periodontal inf lammation. crit rev oral biol med 2004; 15(2): 82-98. 22. kulka m, sheen ch,tancowny bp, grammer lc, schleimer rp. neuropeptide active human mast cell degranulation and chemokin production. immunology 2007; 123: 398-410. 23. lorenzo j. interaction beetwen imunno and bone cell: new insight with many remaining question. j clinical investigation 2000; 106(6): 749-52. 24. winkler s. implant site development and alveolarar bone resorption patterns. j oral implantol 2002; 28(5): 226-9. 25. fickl s, zuhr o, wachtel h, kebschull m, hürzeler mb. hard tissue alterations after socket preservation with additional buccal overbuilding: a study in the beagle dog. j clin periodontol 2009; 36(10): 898-904. 26. wong et, tergaonkar v. roles of nfkb in health and disease: mechanisms anda therapheutic potential. clin sci 2009; 116: 45165. 124 volume 46, number 3, september 2013 research report determination of fluoride content in toothpaste using spectrophotometry susanti pudji hastuti, devinta lestari and yohanes martono department of chemistry faculty of science and mathematics satya wacana christian university salatiga indonesia abstract background: intake excessive fluoride in children’s teeth are generally marked with white and brown patches. excessive fluoride of more than 4.0 mg/l can cause a person suffering from poisoning, fragility of the bones (osteoporosis), liver and kidney damage. knowledge about the spectrophotometry for determination method of fluoride content in commercially available toothpaste is very few. purpose: the purposes of study were to examine the suitable method for fluoride extraction and to determine out the accuracy, precision, linearity, and stability of the measurement method of fluoride content in toothpaste. methods: the suitable f extraction method was determined by the comparison among 3 methods of extraction; e.g. the dried samples were immersed in (1) distilled water, (2) 96% hcl, and (3) 96% hno3; and the validation methods of measurement were the maximum wavelength, standart curve, accuracy test, precision test, and stability test. results: result showed that the fluoride extraction by using the concentrated hno3 was found to have the highest levels of fluoride, followed by hydrochloric acid dissolution (hcl) and distilled water, while the method of validation showed that spadns revealed the acceptable accuracy. precision has the rsd ≤ 2.00%. furthermore the stability test result showed that the measurement of fluoride less than 2 hours was still reliable. conclusion: the study suggested that the best result of fluoride extraction from toothpaste could be gained by using concentrate hno3, and the spectrophotometer (uv-vis mini shimadzu u-1240) and spadns have the acceptable accuracy. key words: spectrophotometry, fluoride content, toothpaste abstrak latar belakang: pemasukan fluoride yang berlebihan pada gigi anak ditandai dengan bercak putih dan coklat. fluoride lebih dari 4.0 mg / l dapat menyebabkan seseorang menderita keracunan , kerapuhan tulang (osteoporosis), kerusakan hati dan ginjal. pengetahuan tentang spektrofotometri untuk metode penentuan kadar fluoride dalam pasta gigi yang tersedia secara komersial sangat sedikit . tujuan: penelitian ini bertujuan meneliti metode yang tepat untuk mengektrak kandungan fluoride dan mengukur akurasi, presisi, linearitas dan stabilitas pengukuran kandungan fluoride pada pasta gigi. metode: metode ekstrak yang tepat ditentukan dengan membandingkan 3 metode, yaitu dengan perendamam sampel kering dalam (1) air destilasi, (2) hcl 96%, dan (3) hno3 96%; dan validasi metode yang memperhitungkan panjang gelombang, kurva standar, tes akurasi, presisi, dan stabilitas. hasil: hasil menunjukkan bahwa metode ekstrasi fluoride pada pasta gigi dengan menggunakan metode hno3 didapatkan level fluoride tertinggi, diikuti dengan metode hcl dan air destilasi. hasil validasi metoda menunjukkan bahwa penggunaan spadns akurasinya dapat diterima. presisi mempunyai rsd ≤2,00%. pada tes stabilitas didapatkan hasil bahwa pengukuran kadar fluorida dalam waktu 125hastuti, et al.,: determination of fluoride content in toothpaste using spectrophotometry tidak lebih dari 2 jam masih dapat dilakukan. simpulan: penelitian ini menunjukkan bahwa hasil terbaik ekstraksi fluoride dari pasta gigi dapat diperoleh dengan menggunakan hno3, dan spectrophotometer (uv-vis mini shimadzu u-1240) dan spadns memiliki pengukuran yang akurat. kata kunci: metode spektrofotometrik, kandungan fluorida, pasta gigi correspondence: susanti pudji hastuti, c/o: departemen kimia, fakultas matematika dan ilmu pengetahuan alam, universitas kristen satya wacana. jl. diponegoro 52-60 salatiga 50711, indonesia. e-mail: susanti012@yahoo.com introduction the use of toothpaste is a part of tooth brushing which is needed for healthy teeth. toothpaste makes the teeth cleaner and reduce oral microorganism.1 fluoride in toothpaste is one of the substances that needed for healthy teeth. the mechanisms of fluoride in dental caries prevention are; reducing the enamel solubility caused by acid, lowering the enamel surface permeability and inhibiting the fermentation of carbohydrates by microorganisms of the oral cavity.2 the needs of fluoride is between 0.7 to 0.9 mg/l (parts per million).3 therefore, despite the growing controversy, the provision of fluoride in toothpaste should not be exaggerated. this is due to the excess fluoride (fluorosis) can cause cells die and the teeth become brittle. the degree of dental fluorosis depends on the amount of fluoride exposure up to age of 8 to10 years old. the f a c t t h a t a n a d u l t s h o w s n o s i g n s o f d e n t a l flu o r o s i s d o e s n o t m e a n t h a t h i s o r h e r flu o r i d e intake is within the safety limit.4 excessive fluoride in children’s teeth are generally marked with white and brown patches. excessive fluoride of more than 4.0 mg/l can cause a person suffering from poisoning, the fragility of the bones (osteoporosis), liver and kidney damage.3-7 to ensure accuracy of fluoride content measurement in toothpaste, the validation of method need to be done. validation of the method according to united states pharmacopeia (usp) in martono8 is aimed to determine that the analysis method is accurate, specific, reproducible, and hold in the range of analytes to be analyzed. some parameters according to the usp are accuracy, precision, linearity, and stability. accuracy is the closeness between the measured values, which acceptable to the convention, the true value, or value of referrals. accuracy is measured as the amount of analyte recovered in a measurement by performing spiking on a sample. accuracy can be obtained by comparing the results of measurements with standard reference materials. international conference on harmonisation (ich) recommends the collection of data from nine times the assay with 3 different concentrations (e.g. 3 concentrations with 3 times replication). data are reported as percent recovery (% recovery). precision is a measurement of repeatability analysis method, and is usually expressed as relative standard deviation of statistically different samples. there are three levels of precision, i.e. repeatability, between the precision (intermediate precision), and reproducibility. the precision are include: standard deviation, relative standard deviation (rsd) or coefficient of variation (cv), and the range of beliefs. data for precision test is often collected as part of other studies related to precision, linearity or accuracy. usually 6 to 15 replications are done for a single sample of each concentration. in testing, the value of rsd is 1-2% for the active compounds in large quantities, while for compounds with a little amount, rsd ranged between 1-5%.8-10 linearity is the ability to obtain test result which is proportional to the concentration of analyte in a given range. linearity is measurement of how well the calibration curve method connects between the response (y) with concentration (x). linearity can be measured by performing a single measurement at different concentrations. the data obtained is processed by the method of least squares, and then determined the value of the slope (slope), intercept, and correlation coefficient (r).10 to obtain the reproducible and reliable test results, the sample, reagents and raw materials used should be stable at a certain time. the stability of all solutions and reagents is very important, either in relation to temperature or in respect of time. if the solution is not stable at room temperature, the temperature should be decreased 2-8°c to increase the stability of samples and standards. the purposes of study were to examine the suitable method for the fluoride extraction and to determine the accuracy, precision, linierity, and stability of the method concerning to the measurement of fluoride content in toothpaste. materials and methods determination of maximum wavelength was done by mixing 5 ml of distilled water with 1 ml of spadns reagent, then performed using a scanning spectrophotometer (uv-vis mini shimadzu u-1240). measurements were taken at region of 350-700 nm wavelength. ten series of fluoride standard solutions levels are: 0.2 mg/l; 0.4 mg/l; 0.6 mg/l; 0.8 mg/l; 1 mg/l; 1.2 mg/l; 1.4 mg/l; 1.6 mg/l; 1.8 mg/l and 2 mg/l respectively (each made in a volume of 10 ml) was prepared for configuration of standard curve. from each level taken 5 ml of solution 126 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 124–129 table 2. the accuracy of fluoride standard solution based on the measurement of absorbance at λ 550 nm concentration of fluoride standard solution (mg/l) a550 (x 100) average a550 measurable levels of fluoride (mg/l) recovery (%) average of recovery (%)i ii iii 0.4 181.9 181.9 181.9 181.9 0.4286 107.15 99.84 1.0 181.2 181.2 181.2 181.2 0.8286 82.86 1.8 179.2 179.2 179.2 179.2 1.9714 109.52 table 1. the comparative analysis of fluoride method fluoride level (mg/l) sample code method 1 method 2 method 3 1 2 3 1 2 3 1 2 3 x 0.85 0.853 0.85 0.803 0.80 0.80 0.91 0.913 0.91 y 0.713 0.71 0.716 0.807 0.807 0.81 0.857 0.85 0.85 z 0.81 0.81 0.813 0.87 0.87 0.87 0.907 0.903 0.893 was then added 1ml of reagent spadns into each test tube, stirred until homogeneous and then incubated for 5 min at room temperature. the absorbance of each solution was measured with a spectrophotometer (uv-vis mini shimadzu u-1240) at a wavelength of 550 nm (maximum wavelength after scanning).8,10 accuracy and precision was obtained with standard solutions of fluoride levels of 0.4 mg/l, 1 mg/l and 1.8 mg/ l respectively which is made from a standard solution of fluoride of 2 mg/l. from each level taken 5 ml of solution was then added 1 ml of reagent spadns into each test tube, stirred until homogeneous and then incubated for 5 min at room temperature. the absorbance of each solution was measured with a spectrophotometer (shimadzu uvvis mini u-1240) at a wavelength of 550 nm (maximum wavelength after scanning). assay performed nine times, including three kinds of levels, each of 3 replications.8,10 stability of reagent spadns was done by comparing volume of reagent at 0.5 ml and 1 ml within 5 ml of standard fluoride solution in three different levels of 0.4 mg/l, 1 mg/l, and 1.8 mg/l respectively and then measured with a spectrophotometer (uv-vis mini shimadzu u-1240) at a wavelength of 550 nm. spadns reagent stability is determined by mixing 5 ml of distilled water with 1 ml of reagent spadns. determination of wavelength and absorbance of the mixture was done by using a spectrophotometer (uv-vis mini shimadzu u-1240). measurements were made every 5 minutes in a span of 120 minutes and on the wavelength region of 350-700 nm.11 six adults toothpastes and 3 children toothpastes which commercially available, were dried in order to make comparison between the method used in the comparison of fluoride extraction by putting the samples in an oven at ±105o c temperature until constant mass was obtained while cooling in the desicator. furthermore as a first step, the optimization method performed by a comparison of three methods of sample dissolution.12 there were three methods used, in method 1: the dried samples (1 g) was immersed in 50 ml of distilled water for 24 hours in a porcelain dish. the mixture was filtered and put in a 100 ml volumetric flask, then add distilled water until the calibration line (tera-line fulfillment to be done by washing the residue). prior to the measurement of fluoride levels, first centrifuge solution at a speed of 3000 rpm for 10 minutes. method 2: the dried samples (1 g) was included in a porcelain dish and immersed in 5 ml of concentrated hcl (96%) for 24 hours in the fumehood. after that, heated ± 2 hours at a temperature of 50ºc using a hot plate in the fumehood. once heated, add another 5 ml of concentrated hcl and heating was continued until no white gas formed. the solution was then cooled, then filtered and put in a 100 ml volumetric flask, then add distilled water until the calibration line. method 3: the dried samples (1 g) was included in a porcelain dish and immersed in 5 ml of concentrated hno3 (96%) for 24 hours in the fumehood. after that, heated ± 2 hours at a temperature of 50ºc using a hot plate in the fumehood. once heated, add another 5 ml of concentrated hno3 and a few drops of h2o2. heating is continued until no more gas to form nitrogen oxides and the resulting brown solution nodes. the solution was cooled, then filtered and the filtrate was added in 100 ml flask, then add distilled water until the calibration line. 127hastuti, et al.,: determination of fluoride content in toothpaste using spectrophotometry table 4. stability of spadns reagent t (minutes) λ (nm) absorbance t (minutes) λ (nm) absorbance 0 5 10 15 20 25 30 35 40 45 50 55 60 549 549 547 548 551 549 548 548 550 549 550 547 548 1.843 1.834 1.837 1.840 1.839 1.842 1.832 1.831 1.836 1.832 1.836 1.825 1.838 65 70 75 80 85 90 95 100 105 110 115 120 548 549 549 548 550 550 548 550 548 549 548 550 1.830 1.827 1.827 1.836 1.832 1.839 1.828 1.830 1.836 1.833 1.834 1.837 table 3. repeatability of fluoride standard solution based on the measurement of absorbance at λ 550 nm standard levels of fluoride (mg/l) repeatation a550 (x 100) measurable levels of fluoride (mg/l) 0.4 1 181.9 0.4286 2 181.9 0.4286 3 181.9 0.4286 average 181.9 0.4286 sd 0 0 rsd (%) 0 0 1 1 181.2 0.8286 2 181.2 0.8286 3 181.2 0.8286 average 181.2 0.8286 sd 0 0 rsd (%) 0 0 1.8 1 179.2 1.9714 2 179.2 1.9714 3 179.2 1.9714 average 179.2 1.9714 sd 0 0 rsd (%) 0 0 results the comparison of three extraction methods of samples could be seen in table 1. accuracy was expressed as percent recovery of analyte added.13 (table 2). precision was determined by measuring the spread of individual results from the average, if the procedure was applied repeatedly in samples which were taken from a homogeneous mixture.13 (table 3). the stability of the volume of reagents was very important thing (table 4). disccusion all three methods of sample extraction were compared, and the result showed extraction by using concentrated hno3 was found to have the highest levels of fluoride, followed by hydrochloric acid dissolution (hcl) and distilled water. this result is supported by oyewale12 which states that the use of hno3 extraction gave the optimum extraction for all inorganic parameters or components of all different toothpastes. 128 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 124–129 the component of toothpastes are combined of calcium carbonate and phosphates, which have low solubility in water. acidic condition is often required to release some of components into water soluble form. this may partly account for the acidic nature of some of the toothpaste samples. furthermore oyewale12 stated that the fluoride content is all essentially available even in aqueous medium since it is often incorporated in toothpastes as sodium salts, which are generally soluble in water. based on this experiment, the f extraction from toothpaste can be determined using concentrated hno3. an important point related to validation method is selection of the maximum wavelength which in turn will be used for the manufacture of standard curves. the results showed that the wavelength of maximum absorption was at a wavelength of 550 nm. validation method was done using reagents spadns and an instrument of spectrophotometer (uv-vis mini shimadzu u-1240). spadns reagent is a mixture of solution spadns [sodium 2 (para-sulfophenylazo) -1, 8-dihydroxy-3, 6naphtalene disulfonate] with a solution of zirconyl chloride octahydrate, zrocl2.8h2o, in acidic conditions. 13,14 determination of fluoride was done by using spectrophotometry method and it based on metal displacement from a colored complex or the formation of a mixed-ligand complex, zr(iv)-f-spadns. fluoride addition will bleach spadns-zirconyl chloride and degrade the red colored complex. the degree of bleaching was determined with a spectrophotometer, and the concentration of fluoride ions was assessed by comparison with standard solutions. the color loss was measured at wavelength on maximum absorbtion of mixed-ligand complex.14 the reaction is as follows : 6 disccusion all three methods of sample extraction were compared, and the result showed extraction by using concentrated hno3 was found to have the highest levels of fluoride, followed by hydrochloric acid dissolution (hcl) and distilled water. this result is supported by oyewale12 which states that the use of hno3 extraction gave the optimum extraction for all inorganic parameters or components of all different toothpastes. the component of toothpastes are combined of calcium carbonate and phosphates, which have low solubility in water. acidic condition is often required to release some of components into water soluble form. this may partly account for the acidic nature of some of the toothpaste samples. furthermore oyewale12 stated that the fluoride content is all essentially available even in aqueous medium since it is often incorporated in toothpastes as sodium salts, which are generally soluble in water. based on this experiment, the f extraction from toothpaste can be determined using concentrated hno3. an important point related to validation method is selection of the maximum wavelength which in turn will be used for the manufacture of standard curves. the results showed that the wavelength of maximum absorption was at a wavelength of 550 nm. validation method was done using reagents spadns and an instrument of spectrophotometer (uv-vis mini shimadzu u-1240). spadns reagent is a mixture of solution spadns [sodium 2 (para-sulfophenylazo) -1, 8-dihydroxy-3, 6-naphtalene disulfonate] with a solution of zirconyl chloride octahydrate, zrocl2.8h2o, in acidic conditions.13,14 determination of fluoride was done by using spectrophotometry method and it based on metal displacement from a colored complex or the formation of a mixed-ligand complex, zr(iv)f-spadns. fluoride addition will bleach spadns-zirconyl chloride and degrade the red colored complex. the degree of bleaching was determined with a spectrophotometer, and the concentration of fluoride ions was assessed by comparison with standard solutions. the color loss was measured at wavelength on maximum absorbtion of mixed-ligand complex.14 the reaction is as follows : zr-spadns + spadns + zrf62+ nh2o6fh+ (red) (colorless) this is consistent with the theory spadns method that the use of wavelength will be in the region of 550-580 nm wavelength. in addition, the use of 550 nm wavelength on a spectrophotometer supported by theory which stated that the red light located on the complementary wavelength region of 490-560 nm.14 the red color observed (as seen by the eye) is the color of the reagent spadns. as an indication of the content of fluoride, the red color of the reagent spadns will be degraded, and if the higher fluoride content, the red color will fade (degradation increases) so that the smaller absorbance.15 the results of absorbance measurements of fluoride standard solution at various levels in the manufacturing of standard curves. the results of the linear regression relationship is created from “y = bx + a”, where “y” is the response (absorbance), “b” is the slope and “a” is the intercept. equation of the regression line (content vs absorbance) and then “y = 1.75 x + 182.6” with r = 1. standard curve was made to give a perfect correlation coefficient (r = 1). to examine the accuracy of the developed methods from the experiments, standard addition method was carried out, then the percentage recovery was calcutated.16 accuracy can be determined by the assay minimum of 9 times, covering a certain range e.g. 3 different levels, each of 3 replications. the average percent recovery were in the range of 98-102% 10 as seen in table 2. the study showed that the spectrophotometer instrument (uv-vis mini shimadzu u-1240) and methods developed spadns have acceptable accuracy.8,14 the test precision was conducted on three kinds of different levels with 3 repetitions for each target compound. the low relative standard deviation (rsd) values indicates precision of the method. in majority, the determinations were below 2%, indicating high degree of agreement (repeatability) between experimental values.17 determination of repeatability can be done with a minimum of 6 times determination levels of 100%, or 9 times in the range of levels with 3 different levels, each repeated a number of 3 times. according to ermer and miller 10 it may be accepted if the repeatability rsd ≤ 2.00% of the test. the repeatability of fluoride standard solution based on the measurement of absorbance at λ 550 nm, the three standard levels of fluoride (mg/l) and measurable levels of fluoride (mg/l) gave the rsd of 0%. this shows that the repeatability with rsd ≤ 2.00% of the test is acceptable.17 the stability of all solutions and reagents is very important, whether in relation to temperature or in respect of time.8 comparison the stability of spadns reagent volume based on absorbance at λ 550 nm showed that the absorbance was stable, when the volume reached spadns reagent was added to the standard solution of 1 ml. in contrast the use of 0.5 ml of reagent gave absorbance measurement results of an unstable (fluctuating). the possibility of this result was because the amount of spadns reagent still not enough to indicate the amount of fluoride (levels) contained in the standard solution. therefore, the determination of fluoride levels in this study was carried out by applying the ratio of the volume of the sample with a spadns reagent by 5:1 (v: v). the results showed that up to 120 minutes, a mixture of sample and spadns reagent continued to show stable and unaffected by environmental factors, especially from the air. this is according to us environmental protection agency (usepa)19 which also states that during 2 hours of color formed from the sample and spadns reagent will remain stable. this means that measuring the levels of fluoride in the range of no more than 2 hours is still reliable. the study suggested that the best result of fluoride extraction from toothpaste could be gained by using concentrate hno3, and the validation test results showed that the spectrophotometer (uv-vis mini shimadzu u-1240) and spadns have the acceptable accuracy. precision has rsd ≤ 2.00%. the stability test results revealed that the measurement of fluoride in the range of less than 2 hours is still reliable. 129hastuti, et al.,: determination of fluoride content in toothpaste using spectrophotometry references 1. andriewongso. pasta gigi. (on line) http://www.andriewongso. com/awartikel-1984-tahukah_anda-pasta_gigi. 2008. accessed march 20, 2009. 2. arnold w, dorow a, langenhorst s, gintner z, bánóczy j, gaengler p. effect of fluoride toothpastes on enamel demineralization. bmc oral health 2006; 6: 8. 3. meenakshi, maheshwari rc. fluoride in drinking water and its removal. j hazard mater 2006; 137(1): 456-63. 4. oxford instruments molecular biotools limited. determination of fluoride content in toothpaste. usa: http://www.oxford-instruments. com /oxfordinstr uments/media /industrial-analysis/magneticresonance-pdfs/determination-of-fluoride-content-in-toothpaste. pdf 2008. p. 1-2. 5. darmawan l. cara cepat membuat gigi sehat dan cantik dengan dental cosmetics. jakarta: gramedia pustaka utama; 2007. p.144. 6. moghaddam aa, fijani e. distribution of fluoride in groundwater of maku area, northwest of iran. environmental geology 2008; 56(2): 281-7. 7. msonda kwm, masamba wrl, fabiano e. a study of fluoride groundwater occurrence in nathenje, lilongwe, malawi. physics and chemistry of the earth 2007; 32: 1178-84. 8. martono y. validasi metoda kromatografi cair kinerja tinggi isokratik untuk penetapan kadar asam galat, kafein dan epigalokatekin galat pada berbagai produk teh celup. thesis. yogyakarta: program studi ilmu farmasi, fakultas farmasi universitas gadjah mada; 2009. 9. gujarathi sc, shah ar, jagdale sc, datar pa, choudari vp, bhanudas sk. spectrophotometric simultaneous determination of aspirin and ticlopidine in combined tablet dosage form by first order derivative spectroscopy, auc and ratio derivative spectrophotometric methods. int j pharm sci review and research 2010; 3(1): 115-9. 10. ermer j, miller jh. method validation in pharmaceutical analysis. a guide to best practice. weinheim: wiley-vch verlag gmbh & co.kgaa; 2005. p. 4-22. 11. gandjar ig, rohman a. kimia farmasi analisis. yogyakarta: pustaka pelajar; 2007. 12. oyewale ao. estimation of the essential inorganic consituents of commercial toothpaste. scientific & industrial research 2005; 64: 101-7. 13. ha r m it a. pet u nju k p ela k sa na a n va l id asi metod a d a n ca ra perhitungannya. majalah ilmu kefarmasian 2004; 1(3): 117-35. 14. battaleb-looie s, moore f. a study of f luoride groundwater occurrence in posht-e-kooh-e-dashtestan, south of iran. world applied sci j 2010; 8(11): 1317-21. 15. hach company. 2006. fluoride for water and seawater spadns, spadns 2 and ion-selective electrode methods. (on line) http://www. hach.com /fmmimghach?/code%3aex_fluoride15801/1. accessed september 15, 2013. 16. sharma mc, sharma s. validated simultaneous spectrophotometric estimation of paroxetine hcl bulk and tablet dosage form using ferric chloride. j opteoelectronics and biomedical materials 2010; 2(4): 185-9. 17. garcia pl, santoro mirm, singh ak, kedor-hackmann erm. determination of optimum wavelength and derivative order in spectrophotometry for quantitation of hydroquinone in creams. brazilian j pharm sci 2007; 43: 397404. 18. sharma r, pervez s. study of dental fluorosis in subjects related to a phosphatic fertlizer plant environment in chhattisgarh state. scientific & industrial research 2004; 63: 985-88. 19. us environmental protection agency. method 13a determination of total f luoride emissions from stationary sources. spadns zirconium lake method. (on line) http://www.epa.gov/ttn/ emc/promgate/m-13a. accessed september 16,2013. 81 effect of pressure and polishing technique on surface roughness of cold cured acrylic resin sianiwati goenharto orthodontic department faculty of dentistry airlangga university surabaya indonesia abstract the smoothness of acrylic surface plays an important role in producing removable orthodontic appliances. int this study, we examine the effect of pressure and polishing technique on surface roughness of cold cured acrylic resin. forty eight samples were prepared and classified into two groups: acrylic resin polymerization with and without pressure. each group was classified into four subgroups: being polished with abrasive stone, bur for acrylic, silicone polisher and without being polished as control group. surface roughness was measured using surface roughness tester. the surface roughness of polymerized acrylic with and without pressure and polished with different technique was analyzed using one-way anova, continued by dunnet test. t-test was done to know whether there was the effect of pressure on surface roughness after being polished using certain technique. the result showed that pressure and polishing technique affected surface roughness significantly (p = 0.001). on the group of polymerization with pressure, surface roughness resulted from polishing with bur of acrylic showed significant difference with silicone polisher, whereas on the group without pressure, polishing with bur of acrylic showed significant difference with abrasive stone. of the three polishing techniques, there was significant difference of surface roughness of cold cured acrylic resin (t = 0.002). it is concluded that pressure and polishing technique affected the surface roughness of cold cured acrylic resin. polishing technique using bur of acrylic, followed by abrasive paper, rotating felt cone and soft brush showed less surface roughness on the group of polymerization with or without pressure. key words: polymerization, pressure and polishing technique, cold cured acrylic correspondence: sianiwati goenharto, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the smoothness of acrylic resin surface in removable orthodontic appliances is an important thing. a smooth exposed surface that is highly is good in aesthetic, give comfort, make oral health better, and the risk of plaque and debris attachment is smaller. nowadays, the role of heat cured acrylic in the process of making removable orthodontic appliance has been replaced by cold cured acrylic,1 including which polymerized with visible light cured.2 the base plate produced by heat cured acrylic is more solid, harder, no porosity with more stable color. however, the process is more complicated because wax model should be made and the acrylic processing will take long time. the instrument and the material which are needed are quite a lot, because, wax model should be flasked before polymerization process which need heat and pressure.1 the use of cold cured acrylic material will give advantage because it is cheaper, the working method is easier, time saving and the dimensional changing is small.3,4 the particle size of cold cured acrylic powder is smaller than heat cured, so it is easier to be absorbed by monomer liquid.5 changing the design of removable orthodontic appliance or reparation process can be done easier with this material. the main disadvantage of this material is the porosity is high so the debris will be easily attached and difficult to clean. giving the pressure during polymerization process will enable to reduce acrylic porosity. the pressure over 4 atmosphere will not give any affect while 2–3 atmosphere will give positive effect.6 to achieve smooth surface, acrylic resin needs to perform polishing stage. there are various kinds of acrylic polishing techniques. the previous study showed that the use of abrasive stone followed consecutively by coarse abrasive disc, medium abrasive disc, fine abrasive disc, rotating felt cone with pumice slurry, rotating soft brush with chalk powder, will give the best polishing out come.7 at the diploma iii laboratory of dental health technique program of faculty of dentistry airlangga university, bur of acrylic is usually used to polish acrylic, followed by abrasive paper, rotating felt cone with pumice slurry and also, rotating soft brush with chalk powder. silicone polisher which can be used to smoothen the acrylic is available on the market. the use of silicone polisher is more practical and more advantageous because it will not defect the wire or the alloy which is possible on the base palate. the purpose of this study is to examine the effect of pressure and polishing technique toward surface roughness of cold cured acrylic resin, so it will be useful to increase the quality of removable orthodontic appliances. 82 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 81–84 material and method this study was experimental laboratory with factorial design. to make the sample, brass plate master model, 60 × 10 × 2 mm in size was prepared, then hard gypsum mold was made according to the master model. acrylic plate was made in gypsum mold using layering technique by spreading a layer of cold cured acrylic power (vertex, dentaurum) and followed by adding monomer liquid until the whole monomer infiltrate into the polymer powder. the process was repeated until the required form according to the criteria was achieved. forty eight samples were prepared and classified into two groups i.e. acrylic polymerization groups with and without pressure. immediately after acrylic processing was completed, samples of the first group were put into polyclav containing water in 50 °c and the pressure given was 2,2 atmosphere for 10 minutes. the second group samples were done without pressure (without polyclav). polymerization groups with (group 1) or without (group 2) pressure was classified into four subgroups, consisted of six specimens in which would be polished in different technique. technique i (subgroup) using rough abrasive stone for 40 seconds, followed by fine abrasive stone for 20 seconds, rotating felt cone with pumice slurry for 45 seconds, and polished by soft brush with chalk powder for 15 seconds. technique ii (subgroup 2) using bur of acrylic for 30 seconds, followed by abrasive paper no 0 for 15 seconds, rotating felt cone with pumice slurry for 45 seconds, and polished by soft brush with chalk powder for 15 seconds. technique iii (subgroup 3) using rough silicone polisher for 60 seconds, continued by smooth silicone polisher for 30 seconds, rotating felt cone with pumice slurry for 45 seconds, and polished by soft brush with chalk powder for 15 seconds. subgroup 4 was not polished (as control group). the acrylic plate should be cleansed with running water when the instrument changed. surface roughness of the acrylic resins was examined by surface roughness tester. the specimen was placed on the table so that the stylus of the instrument could freely move on specimen surface. the measurement distance was 5 mm and the instrument moved in 0,75 m/second. surface roughness was counted using pertograph i.e arithmetic mean of five differences peak to valley height. rz showed the height and the depth of surface roughness in micron. the data of acrylic resin surface roughness which polymerized with and without pressure and polished by different technique was tabulated and analyzed using one-way anova continued by dunnett test with 0,05 significant grade. t-test was performed in order to know whether there was the effect of pressure on the surface roughness after being polished using certain technique. result the data of average surface roughness (rz) of cold cured acrylic resin which was polymerized with and without pressure and polished by using different technique can be seen on table 1. table 1 shows that the value of rz in group without pressure with all technique relatively higher than the group with pressure. before one-way anova test was done, kolmogorov-smironov test showed p > 0.05 which means the data of the whole samples groups had normal distribution. the value of homogeneity variant test: p = 0.028 (p < 0.05) means the data was homogeneous. in order to know whether there is the effect of polishing technique in surface roughness of cold cured acrylic which polymerized with pressure, one-way anova test was done and p = 0.001 (p < 0.05) was found, means there was significant difference on surface roughness of cold cured acrylic resins which polished in different technique. in order to know whether there is significant difference of the three polishing techniques on the surface roughness, dunnet test was done (table 2). tabel 2 showed that all techniques resulting significant difference of surface roughness with controlled group, as well as between technique ii and iii. to know whether there is the effect of polishing technique in surface roughness of polymerized acrylic without pressure, one-way anova was done and table 1. the mean and the standard deviation of surface roughness (rz) of cold cured acrylic resin on various groups group/subgroup number of samples mean rz (mm) standard deviation polymerization with pressure, polishing technique i polymerization with pressure, polishing technique ii polymerization with pressure, polishing technique iii polymerization with pressure, without polishing polymerization without pressure, polishing technique i polymerization without pressure, polishing technique ii polymerization without pressure, polishing technique iii polymerization without pressure, without polishing 6 6 6 6 6 6 6 6 2.47 2.02 2.98 43.60 6.97 2.45 10.02 67.33 0.46 0.17 0.49 15.95 1.59 1.00 6.51 17.57 83goenharto: effect of pressure and polishing p = 0.001 (p < 0.005) was found, means polishing technique affect the surface roughness of polymerized acrylic without pressure. on homogeneity test, p = 0.001 (p < 0.005) was found so in order to know whether there is significant difference of the three polishing technique towards the surface roughness, dunnet test was done (table 3). table 3 showed that all techniques giving significant difference of surface roughness with controlled group as well as technique i and ii. t-test was done in order to know whether there is difference of surface roughness of polymerized cold cured acrylic with and without pressure, and in the three technique t = 0.002 (t < 0.005) was found, which means there is effect of pressure in surface roughness of cold cured acrylic after being polished with a certain technique. t value in controlled group was 0.537 (t > 0.05) means that there was no difference of surface roughness of polymerized cold cured acrylic with and without pressure. discussion the smoothness surface of acrylic resin on orthodontic treatment with removable appliance is considerably important factor to support the success of treatment. a rough surface may be uncomfortable and food debris and plaque can adhere easily to it.8 polishing is done to inhibit adhesion, to make the surface feel smooth and to increase aesthetic.9 resin which is smoothly polished and shiny, will be more comfortable for the patient, debris will not be easily attached, and also it will reduce the risk of decreasing hygiene and oral health, in addition, the possibility of having bad smell after being worn for some times will be prevented. this study showed that polishing technique will affect the surface roughness of cold cured acrylic resin which was polymerized with pressure (p = 0.02). dunnet test shows (table 2) that polishing with the three techniques can result significant surface roughness which was lower than unpolished group. the result can be understood because the purpose of polishing is to achieve smoother surface. the three techniques which are applied in this study are based on polishing principle i.e by using rougher to smoother instrument with acrylic resin cleansing in running water every time prior to instrument change,8 because particle material that left on the surface can be scratched during polishing period.10 grinding and polishing phases should be done step by step without neglecting any step. polishing technique with silicone polisher (technique iii), had significantly higher surface roughness compared with bur for acrylic (technique ii). in this case, it is due to sharpness factor and the hardness of instrument. bur for acrylic is sharper and harder compared with silicone polisher that made from such a rubber, and also abrasive paper is sharper than fine silicone polisher. sharp instrument is more capable to cut and eliminate the rough part. silicone polisher is more blunt but advantageous because it does not defect either the wire or the alloy on the design of removable orthodontic appliance. careless use of sharp instrument can contribute defect on the other component of removable orthodontic appliances. this study also showed that polishing technique might affect the average roughness of cold cured acrylic resin with which is polymerized without pressure (p = 0.001). table 2. significant difference of surface roughness (rz) among the groups of different polishing technique in polymerization with pressure using dunnet test. subgroup technique i technique ii technique iii control technique i technique ii technique iii control – 0.277 – 0.385 0.019* – 0.007* 0.007* 0.007* note: * = significant difference (p < 0.05) table 3. the significant difference of the surface roughness (rz) between groups with different polishing technique in polymerization without pressure using dunnet test subgroup technique i technique ii technique ii control technique i technique ii technique iii control – 0.002* – 0.826 0.157 – 0.002* 0.001* 0.001* – notes: * = significant difference (p < 0.05) 84 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 81–84 dunnet test (table 3) showed that polishing with the three techniques resulting significantly lower surface roughness than in the unpolished group. in polymerization group without pressure, polishing technique with abrasive stone (technique i) gave significant higher surface roughness compared with bur for acrylic (technique ii). in this case, it is possible due to the characteristic of bur of acrylic made of metal (tungsten carbide) relatively harder compared with abrasive stone made of silicone carbide. the harder material will be more capable to dispose the rough part. this result differs from polymerization group with pressure. this is possibly because the porosity which affect the smoothness of acrylic resin surface. giving the pressure during the polymerization process of cold cured acrylic can decrease the amount of porosity. porosity is caused by the residual monomer. polymerization of cold cured acrylic resin is never as complete as heat cured acrylic resin. in this case, it will result more residual monomer on cold cured acrylic resin i.e. 3–5% compared with the amount of residual monomer on heat cure i.e. only 0,2–0,5%.11 one of the techniques to reduce the amount of residual monomer recommended is auto polymerization process in the water with pressure. the process of cold cured acrylic polymerization in a pressure pot might result stronger acrylic with less porosity and shrinkage. the use of polyclav also make acrylic resin immersed in water. the water immersion might also be able to reduce residual monomer due to the occurrence of releasing residual monomer.13 polishing can also dispose the excessive material, smoothen the rough surface,9 and also decrease the amount of residual monomer.14 therefore, on polymerization process with pressure, due to the less amount of porosity, there was no significant different of surface roughness resulted from polishing technique i and ii. although abrasive stone and bur of acrylic are sharper than silicone polisher but there was no significant different on surface roughness of polymerization group without pressure. this is possibly caused by the solidity of acrylic that polymerized without pressure is lower than polymerization with pressure, so even though silicone polisher is more blunt but capable to dispose the rough part similar with the other two techniques. in addition the value of standard deviation on polishing group with silicone polisher was high. in this study, it was difficult to obtain polishing with true uniformity. in fact the speed of movement and pressure performed during polishing might influence the polishing result.3 this study also showed that the three techniques of polishing, pressure will affect the surface roughness of cold cured acrylic resin (t = 0,002). on unpolished group, significant difference was not found. polishing is very important because without it, a rough acrylic will be produced either in the group of polymerization with or without pressure. so, polishing factor plays more important role in surface roughness decreasing than pressure factor. it is concluded that pressure and polishing technique affected the surface roughness of cold cured acrylic resin. polishing technique using bur of acrylic, followed by abrasive paper, rotating felt cone and soft brush showed less surface roughness on the group of polymerization with or without pressure. acknowledgement the author would like to thank to lembaga penelitian universitas airlangga which had given the sponsorship to do this study. refferences 1. adams cp. the design and construction of removable orthodontic appliances. 4th ed. bristol: john wright & sons ltd; 1990. p. 186–8. 2. brown k. light-curing acrylic resin as an orthodontic baseplate materials. quint int 1998; 29:508–12. 3. combe ec. notes on dental materials. 7th ed. edinburg, london, melbourne, new york: churchill livingstone; 1992. p. 258–60, 332–4. 4. isaacson kg, muir jd, reed rt. removable orthodontic appliances. oxford, auckland, boston: wright; 2002. p. 35. 5. van noort r. introduction to dental materials. 2nd ed. edinburg: mosby; 2002. p. 228–9. 6. furnish gm, o’toole tj, fraunhofer ja. the polymerization of acrylic resin orthodontics. j prost dent 1983; 49:276–8. 7. ulusoy m, ulusoy n, aydin ak. an evaluation of polishing techniques on surface roughness of acrylic resins. j prost dent 1986; 56:107–12. 8. craig rg, powers jm, wataha jc. dental materials: properties and manipulation. 8th ed. st. louis: mosby; 2004. p. 110–2, 270–89. 9. gladwin m, bagby m. clinical aspect of dental materials: theory, practice and cases. 2nd ed. philadelphia, baltimore: lippincott williams & wilkins; 2004. p. 153, 205–8. 10. craig rg. restorative dental materials. 10th ed. st. louis, baltimore, boston: mosby; 2002. p. 521. 11. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia: wb saunders co; 2003. p. 663–79. 12. hatrick cd, eakle ws, bird wf. dental materials: clinical applications for dental assistants and dental hygienists. philadelphia, london, new york: saunders; 2003. p. 263. 13. retno a, intan n, anita y. jumlah pelepasan monomer sisa resin akrilik jenis heat cured dalam air. maj ked gigi (dent j) 2005; ed khusus: 43–6. 14. kejarune u. release of methyl methacrylate from heat cured and autopolymerized resin; cytotoxicity testing related to residual monomer. australian dent j 1999; 44(1):25–30. editorial board of dental journal (majalah kedokteran gigi) sk: 118/j03.1.21/kp/2008 january 2nd, 2008 january 2nd, 2010 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: dr. elly munadziroh, drg, m.s. (dental material – airlangga university) editorial boards: prof. dr. m rubianto, drg, m.s., sp.perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d., fds. (conservative dentistry – university of guy’s dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery – university of saarland, homburg, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery – university science malaysia, malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic – university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry – university of hiroshima, japan); prof. kozai katsuyuki, d.d.s., ph.d. (pediatric – university of hiroshima, japan); dr. nugrohowati, drg, m.kes. (conservative dentistry – prof. dr. moestopo university); dr. m. suharsini, drg, m.s., sp.kga. (pediatric dentistry – indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic – jember university); widowati witjaksono, drg., ph.d. (periodontic – university science malaysia, malaysia); prof. dr. a.g.m. tielens (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands); kok van kessel (medical microbiology – university medical centre, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. lakshman samaranayake (oral microbiology – the university of hongkong). managing editors: dr. r. darmawan setijanto, drg, m.kes. (department of dental public health – airlangga university); prof. dr. arifzan razak, drg, msc, sp. pros. (prostodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp. kg. (conservative dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d.,sp.ort. (ortodontic – airlangga university); prof. dr. mieke sylvia m. a. r., drg, m.s.,sp.ort. (ortodontic – airlangga university); prof. dr. istiati soehardjo, drg, m.s. (oral biology – airlangga university); dr. anita yuliati, drg, m.kes. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); seno pradopo, drg, s.u., ph.d. sp. kga. (pediatric dentistry – airlangga university); udijanto tedjosasongko, drg, ph.d.,sp.kga. (pediatric dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg.,su.,sp.bm. (oral maxillofacial surgery – airlangga university); markus budi rahardjo, drg, m.kes. (oral biology – airlangga university); endang pudjirochani, drg, m.s., sp. pros. (prostodontic – airlangga university); ira widjiastuti, drg, m.kes.sp.kg. (consevative dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); bagus soebadi, drg, mhped. sp.pm. (oral medicine – airlangga university); ketut suardita, drg.,ph.d. (conservative dentistry – airlangga university); sianiwati goenharto, drg., m.s. (ortodontic – airlangga university); devi rianti, drg., m.kes. (dental material – airlangga university); chiquita prahasanti, drg.,sp.perio. (periodontic – airlangga university); dr. eha renwi astuti, drg., m.kes. (roentgen – airlangga university); dr. diah savitri ernawati, drg.,msi. (oral medicine – airlangga university); rostiny, drg., m.kes.,sp.pros. (prostodontic – airlangga university). administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 41 no. 2 april–june 2008: prof. nairn hutchinson fulton wilson, msc. ph.d., fds. (conservative dentistry – university of guy’s dental school, london) prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery – university of saarland, homburg, germany) prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a) endrajana, drg., ms., sp.bm. (oral maxillofacial surgery – airlangga university) achmad harijadi, drg., ms., sp.bm. (oral maxillofacial surgery – airlangga university) sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478 / 5030255. fax. (031) 5039478 / 5020256 e-mail: dental_journal@yahoo.com website: www.dentj.fkg.unair.ac.id www.journal.unair.ac.id accredited no. 48/dikti/kep/2006 volume 41 number 2 april-june 2008 issn 1978 3728 dental journal majalah kedokteran gigi contents page printed by: airlangga university press. (126/07.08/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail:aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 41 number 2 april-june 2008 issn 1978 3728 dental journal majalah kedokteran gigi 1. the level of streptococcus mutants and lactobacillus in saliva of dental caries and free caries children seno pradopo ................................................................................................................................... 53–55 2. combination of natural teeth and osseointegrated implants as prosthesis abutments in a posterior cantilever bridge michael josef kridanto kamadjaja ............................................................................................... 56–61 3. students’ evaluation of preclinical simulation for all ceramic preparation (in faculty of dentistry universiti kebangsaan malaysia) natasya ahmad tarib and marlynda ahmad ............................................................................... 62–66 4. differences in cytotoxicity between 5% tetracycline hydrochloride and 15% edta as root canal irrigant devi eka juniarti, karlina samadi, and achmad sudirman ....................................................... 67–69 5. the role of partial denture in management of hypohidrotic ectodermal dysplasia tania saskianti, seno pradopo, prawati nuraini, and michael josef kridanto kamadjaja ........................................................................................................................................ 70–73 6. the use of 90% aloe vera freeze drying as the modulator of collagen density in extraction socket of incicivus cavia cobaya ester arijani and christian khoswanto ........................................................................................ 74–76 7. management of zygomatic-maxillary fracture (the principles of diagnosis and surgical treatment with a case illustration) david b. kamadjaja and coen pramono d ................................................................................... 77–83 8. the transversal strength of acrylic resin plate after being immersed soaking in noni fruit (morinda citrifolia linn.) juice sri redjeki indiani ........................................................................................................................... 84–87 9. pulp tissue inflammation and angiogenesis after pulp capping with transforming growth factor b1 sri kunarti ........................................................................................................................................ 88–90 10. the effect of exposure duration of self etch dentin bonding on the toxicity of human gingival fibroblast of cell culture sri lestari ......................................................................................................................................... 91–94 11. effect of il-1 and gustducin expression change on bitter taste during fever jenny sunariani ............................................................................................................................... 95–99 12. potency of probiotic therapy for dental caries prevention indah listiana kriswandini ............................................................................................................ 100–102 109 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 109–113 case report interdisciplinary approach for pathologic tooth migration in advanced periodontal disease patient marie louisa department of periodontics, faculty of dentistry, universitas trisakti, jakarta, indonesia abstract background: recent treatment trends have included an interdisciplinary approach to cases and have stressed the importance of orthodontic treatment in optimising the prognosis for patients with periodontal disease. orthodontic intrusion is a reliable method to improve periodontal support as research has previously documented. purpose: this case study demonstrates an interdisciplinary approach to treating a patient with moderately advanced periodontitis disease and pathologic tooth migration (ptm) of the upper left central incisor to enhance structure, function and aesthetics. case: a 46-year-old systemically healthy male patient came with a chief complaint of a protruding and elongated tooth. clinical examination showed a mobile, extruded incisor along with bleeding on probing and suppuration. periodontal and radiographic examinations showed generalised horizontal bone loss combined with infrabony defects at the pathologically migrated upper left central incisor. case management: periodontal inflammation was treated with a combination of nonsurgical and surgical therapy. afterward, orthodontic treatment was done using a self-ligating system. the intrusion of a pathologically extruded tooth improved infrabony defects, creating a favourable bone level and probing depth. conclusion: the combination of periodontal–orthodontic therapy achieves satisfactory outcomes if periodontal inflammation is controlled, physiologic forces are used, and oral hygiene is maintained throughout therapy. keywords: interdisciplinary; orthodontic; intrusion; periodontitis; pathologic tooth migration correspondence: marie louisa, department of periodontics, faculty of dentistry, universitas trisakti. jl. kyai tapa, jakarta, 11440, indonesia, e-mail: marielouisa@trisakti.ac.id introduction pathologic tooth migration (ptm) is a tooth position change due to disruption of the forces that hold it in the normal position. the etiologic factors of ptm are varied; however, periodontal bone loss appears to be a primary factor. ptm may damage patients’ smile aesthetics and affect their confidence. to resolve this problem, an interdisciplinary approach is frequently required; it includes periodontal, orthodontic and restorative treatment.1,2 the decision to keep the periodontally involved tooth with ptm necessitates a properly defined treatment plan. initial periodontal treatment to resolve inflammation and reduce probing depths has to precede orthodontic therapy.3,4 studies imply that a new attachment is possible in association with orthodontic tooth intrusion. moreover, the procedure has the potential to re-establish a wholesome and well-functioning periodontium with a favourable aesthetic result.2 this case study demonstrates periodontal and orthodontic intervention in a patient with moderately advanced periodontitis combined with ptm of an anterior tooth followed by prosthetic rehabilitation to enhance structure, function and aesthetics, while emphasising the role of an interdisciplinary approach. case a 46-year-old male with an acute periodontal lesion, generalised periodontitis, along with functional and aesthetic issues was referred to a periodontist. his upper left central incisor had migrated and was showing mobility with a history of intermittent swelling and suppuration (figure 1a and 1b). the patient’s chief concern was his appearance and impaired function. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p109–113 mailto:marielouisa@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p109-113 110marie louisa/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 109–113 he was a non-smoker and in good general health. clinical examination revealed probing depth around 4–10 mm, recession 1–5 mm and teeth mobility grade i and ii on anterior teeth and molars. the most severe attachment loss was found on the extruded and protruded upper left central incisor with miller mobility grade ii and recession class iii. initial probing depths were around 9–10 mm with bleeding on probing and suppuration. the vitality test on the severely affected tooth was negative. a dental radiograph revealed severe bone loss, widened periodontal space and loss of lamina dura (figure 1c). patient was diagnosed with generalised moderate to advanced periodontitis with an acute periodontal lesion on the upper left central incisor. the prognosis for the upper left central incisor was deemed hopeless.5 treatment options included tooth extraction and prosthetic replacement. the best options for retaining the tooth were regenerative periodontal surgery followed by orthodontic treatment. after a thorough explanation, the patient preferred the regenerative–orthodontic approach and signed the informed consent form. case management the treatment started with cause-related non-surgical periodontal therapy, which included plaque control, scaling, root planing and temporary periodontal splint placement using wire, followed by subgingival administration of metronidazole (ti–es). since the tooth vitality tested negative, root canal treatment was carried out. four weeks later, there was no bleeding on probing and suppuration although periodontal pockets remained deep. guided tissue regeneration was done using demineralised freeze-dried bone allograft and collagen membrane to reduce vertical bone loss in the upper anterior region (figure 2). six months after surgery, a re-evaluation showed a stable periodontal condition with reduced probing depth (2–5 mm), no sign of inflammation and good oral hygiene. after removal of the temporary splinting, orthodontic treatment was started. the pre-orthodontic examination revealed skeletal class ii malocclusion with overjet 7 mm, overbite 3 mm and a point – nasion – b point angle 7° (figure 3a). canine relationship class i was on the left side and class ii — on the right side along with bilateral first molar relationship class ii (figure 3b and c). during centric occlusion, there was interference between the upper left central incisor and lower lip (seen as a red x in figure 3a). the patient had refused to undergo more invasive procedures such as teeth extraction or surgical correction. the orthodontic treatment goals were to align the teeth, achieve a stable occlusion and improve the smile aesthetics. the treatment started using roth self-ligating brackets with a 0.022 x 0.028 in the slot. bonded orthodontic molar tubes were used because poorly adapted molar bands could harm the periodontal tissue. the treatment began with a b c figure 1. pre-treatment intraoral facial view (a), upper occlusal view (b) and dental radiograph showed extruded left central incisor (blue lines) as compared with the neighbouring tooth (orange lines) (c). figure 2. surgical treatment. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p109–113 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p109-113 111 marie louisa/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 109–113 a b c figure 3. pre-orthodontic lateral cephalometry with x sign indicating left central incisor interference with lower lip during centric occlusion (a), left view of occlusion (b) and right view of occlusion (c). a b c figure 4. orthodontic treatment intraoral facial view (a), aligned teeth after debonding (b), and preand post-orthodontic radiograph comparison showing intruded left central incisor (blue line on incisal plane was levelled with orange line) and increased density of surrounding alveolar bone (inside the blue circles) (c). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p109–113 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p109-113 112marie louisa/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 109–113 0.012-in, 0.014-in, 0.016-in and 0.016 × 0.022 in niti to align and level the teeth with light force for nine months. gradually, the left central incisor was intruded and brought to the occlusal plane (figure 4a). retraction of the upper front teeth was performed with 0.017 x 0.025-in stainless steel arch wire with cuspid hooks supported by class ii elastics for 6 months. the patient was then kept in a retentive phase to stabilise the teeth in a new position for 5 months. the total treatment time was 20 months. before debonding (figure 4b), a dental radiograph was taken. a comparison of preand post-orthodontic radiographs revealed intruded upper left central incisor (seen as blue lines compared with red lines in figure 4c) with no evidence of root resorption, normal periodontal space, intact lamina dura and increased density of alveolar bone (seen inside blue circles in figure 4c). after debonding, the patient had an improved smile with no interference between the upper left central incisor and lower lip during centric occlusion. no mobility observed along with normal overjet and overbite. removable hawley-type retainers were chosen to facilitate patient’s oral hygiene care. the significant deficiency in gingival scallop since the beginning of treatment was not improved to a satisfactory level, even after intrusive orthodontic movement. afterwards, the patient was referred for restorative treatment using dental veneers to close black triangles and improve his smile (figure 5a). at the end of treatment, the patient was pleased with the results provided by comprehensive dental treatments. periodic follow-ups were scheduled along with continuous reinforcement of oral hygiene instruction. four months after restorative treatment, re-evaluation revealed normal probing depth within 2 mm (figure 5b) without any sign of inflammation. a b figure 5. post-treatment extra oral-facial view (a) and normal probing depth (2 mm) on left central incisor without any sign of inflammation during four-month control (b). discussion pathologic tooth migration is a common complication of moderate to severe periodontitis and often becomes the motivation to seek treatment. occlusal trauma and periodontitis are mutually aggravated due to ptm. common consequences of ptm are greater loss of attachment, extrusion and mobility of the displaced tooth. periodontitis patients often suffer from extrusion and spacing of anterior teeth, resulting in functional and aesthetic issues. orthodontic correction in these cases can relieve occlusal trauma, stabilise the dentition and enhance the periodontal support.1,2 several studies have shown that orthodontic treatment can be done in stabilised periodontal patients.1 in this case, both nonsurgical and surgical periodontal therapies were completed prior to orthodontic treatment in order to eliminate inflammation. re-evaluation six months later revealed no inflammation, reduced pocket depth, and reduced tooth mobility. the orthodontic treatment was initiated along with professional periodontal maintenance and meticulous plaque control procedures to ensure the stability of long-term treatment.3,4 at the beginning of treatment, niti wires were chosen in order to exert light force during aligning and levelling.5 bite correction and finishing stages were then completed using stainless steel wires. bonded orthodontic molar tubes were used because poorly adapted molar bands would damage the subgingival supporting tissues, leading to infection and subsequent alveolar bone loss.6 sabatoski et al.7 suggest that orthodontic tooth movement can also enhance the periodontal ligament cells mitotic activity. intrusion and retraction towards the bone have a potential osteogenicity. these movements dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p109–113 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p109-113 113 marie louisa/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 109–113 encourage bone apposition and sometimes improve bony defect.7 kumar et al.1 claim that intrusive movement promotes new attachment. this movement also appears to be more effective and less invasive in realigning an extruded tooth.1 after debonding, periodontally compromised patients may have troubles such as a relapse. therefore, they require permanent retention that can be achieved by fixed or removable retainers.8 in this case, removable hawley retainers were used in both arches to facilitate the patient’s plaque control procedure.9 upon completion of orthodontic treatment, the patient was scheduled for follow-up every 4 months to maintain his periodontal condition. a combined periodontal–orthodontic approach can help modify the papillae height. the goal is to reduce the distance between the alveolar bone crest and the interproximal contact point. if this distance is 5 mm, 98% of the embrasure papilla space can be completely filled; when the distance is 6 mm, only 56% can be completely filled; and when it is near 7 mm, the percentage goes down to 27%. when the distance is about 10 mm, the space cannot be reduced.10 in this case, due to the severe bone loss from previous inflammation, the distance was around 11 mm, even after intrusive movement. hence, restorative treatment using dental veneers was finally chosen to close the black triangles and improve patient’s smile. treatment for periodontitis patient with ptm was complex due to the interdisciplinary treatments needed. the keys to achieve satisfactory outcomes are complete elimination of inflammation, controlled orthodontic force and adequate oral hygiene maintenance. references 1. kumar n, jhingta p, negi ks, bhardwaj vk, sharma d, thakur as. combined periodontal-orthodontic treatment of pathologic tooth migration: a case study with 10-year follow-up. contemp clin dent. 2018; 9(suppl 2): s377–81. 2. vinod k, reddy yg, reddy vp, nandan h, sharma m. orthodonticperiodontics interdisciplinary approach. j indian soc periodontol. 2012; 16(1): 11–5. 3. jepsen k, jaeger a, jepsen s. esthetic and functional rehabilitation of a severely compromised central incisor: an interdisciplinary approach. int j periodontics restorative dent. 2015; 35(3): e35-43. 4. ramachandra cs, shetty pc, rege s, shah c. ortho-perio integrated approach in periodontally compromised patients. j indian soc periodontol. 2011; 15(4): 414–7. 5. ioannou al, kotsakis ga, hinrichs je. prognostic factors in periodontal therapy and their association with treatment outcomes. world j clin cases. 2014; 2(12): 822–7. 6. rath s, datan s, gupta a. ortho-perio management of malocclusion in an adult patient. j interdiscip dent. 2017; 7(1): 41–4. 7. sabatoski cv, bueno rc, reyes pacheco aa, pithon mm, tanaka om. combined periodontal, orthodontic, and prosthetic treatment in an adult patient. case rep dent. 2015; 2015: 716462. 8. littlewood sj, kandasamy s, huang g. retention and relapse in clinical practice. aust dent j. 2017; 62(suppl 1): 51–7. 9. li b, xu y, lu c, wei z, li y, zhang j. assessment of the effect of vacuum-formed retainers and hawley retainers on periodontal health: a systematic review and meta-analysis. plos one. 2021; 16(7): e0253968. 10. jamwal d, kanade k, singh tanwar v, waghmare p, landge n, student p, professor a, author c. treatment of interdental papilla: a review. galore int j heal sci res. 2019; 4(2): 1–12. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p109–113 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p109-113 �� vol. 45. no. 1 march 2012 research report crude toxin of aggregatibacter actinomycetemcomitans serotype-b increase parp-� expression in gingival epithelium ernie maduratna setiawatie department of periodontics/institute of tropical disease, airlangga university faculty of dentistry, airlangga university surabaya indonesia abstract background: aggregatibacter actinomycetemcomitans (a. actinomycetemcomitants) serotype-b has long been associated with aggressive periodontitis. gingival epithelial cell is exquisitely sensitive to the toxin so that may lead to disruption of the epithelial protective barrier, facilitating invasion and perturbation of the underlying connective tissue. currently suggested that aa serotypeb produce protein toxin that caused dna strand breaks. parp-1 is an abundant nuclear protein functioning as a dna nick-sensor enzyme. parp-1 was one of the first identified substrates of caspases, the main executioners of apoptosis. therefore, a role for parp-1 in the regulation of apoptosis has been suggested. purpose: the purpose of this study was to prove parp-1 expression in gingival epithelium caused by toxin exposure of a. actinomycetemcomitant serotype-b. methods: this is an experimental study involving twenty adult mice strain swiss webster (balb c) divided randomly into two groups: control group (group a) and toxin group (group b). both group were acclimated for one week before treatment. group a was applied topically with sterile distillated water every 12 hours. group b was applied topically by 100μg/ml of crude toxin a. actinomycetemcomitant serotype b at the buccal area of mandibular anterior teeth using hamilton syringe. the mice were sacrificed at 24 hours after toxin application, and then the tissue sections of gingival epithelium were stained with immunohistochemistry to reveal the parp-1 expression. the data were analyzed with t-test. results: the parp-1 expression exhibited an increase with the toxin group (mean= 48.9; sd= 2.01) compared with the control group (mean= 25.21; sd= 1.72). dna fragmentation appeared from the agarose gel examination, marked as dna laddering, indicate the cell apoptosis. conclusion: in conclusion the crude toxin exposure of a. actinomycetemcomitant serotype-b leads to dna fragmentation and increase parp-1 expression. key words: crude toxin of aggregatibacter actinomycetemcomitans serotype-b, parp-1, dna fragmentation abstrak latar belakang: aggregatibacteractinomycetemcomitans (a. actinomycetemcomitant) serotype-b merupakan etiologi utama periodontitis agresif. sel epitel gingiva sangat sensitif terhadap toksin sehingga dapat mengganggu epitel sebagai pertahanan awal gingiva, membantu invasi toksin dan mengganggu jaringan ikat dibawahnya. saat ini diketahui bahwa toksin bakteri aa serotype-b menyebabkan putusnya rantai dna. parp-1 merupakan protein dalam intisel yang berfungsi sebagai dna nicksensor enzyme. parp-1 merupakan penanda awal apoptosis, sehingga peran parp-1 dalam pengaturan apoptosis perlu diteliti tujuan: tujuan dari penelitian ini adalah untuk meneliti ekspresi parp-1 pada epitel gingiva yang dipapar toksin bakteri a. actinomycetemcomitant serotype-b. metode: penelitian eksperimen pada 20 mencit strain swiss webster (balb c) dibagi secara random dalam 2 kelompok, kelompok kontrol (group a) dan kelompok perlakuan (group b). kedua kelompok diaklimasi sebelumnya selama 1 minggu. kelompok diaplikasi secara topikal dengan air destilasi steril setiap 12 jam. kelompok b diaplikasi 100 μg/ml toksin a. actinomycetemcomitant serotype-b secara topikal dengan menggunakan hamilton syringe. mencit dimatikan 24 jam setelah aplikasi toksin kemudian potongan epitel gingiva dilakukan pemeriksaan secara imunohistokimia untuk melihat ekspresi parp-1. data dianalisis dengan uji-t hasil: ekspresi parp-1 menunjukkan penigkatan pada kelompok perlakuan (mean = 48,9; sd = 2,01) bila dibanding kelopok kontrol (mean= 25,21; sd= 1,72). tampak adanya gambaran dna fragmentasi pada pemeriksaan gel elektroforesis yang menunjukkan adanya apoptosis. kesimpulan: dapat disimpulkan bahwa paparan toksin a. actinomycetemcomitant serotype-b menyebabkan dna fragmentasi, dan meningkatkan ekspresi parp-1. �0 dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 39–42 introduction aggressive periodontitis is a form of periodontal disease, which has a sign of aggressive destruction of periodontal ligament and alveolar bone. the loss of attachment is three times higher than chronic periodontitis causing premature tooth loss. the prevalence of aggressive periodontitis can reach a significant number of 10 to 15 percent.1 the trigger of aggressive periodontitis is the predominant specific bacterium of aggregatibacter actinomycetemcomitans (a. actinomycetemcomitant) serotype-b.2 the bacterium and its end-products can interact with gingival epithelium during the chronic infection, furthermore will penetrate into fibroblast, periodontal ligament and alveolar bone. a. actinomycetemcomitans inhibits the proliferation and supporting the apoptosis of gingival cell, fibroblast, osteoblast, macrophage, lymphocyte b and lymphocyte t.3,4 apoptosis plays an important role in the homeostasis of cells and tissue in response to damage. parp-1-mediated poly (adp-ribosylation) of nuclear proteins is required for apoptosis. during apoptosis, caspase-7 and caspase-3 cleave parp-1 into two fragments: p89 and p24.5 parp-1 also plays a central role in a caspase-independent apoptosis pathway mediated by apoptosis-inducing factor (aif). the induction of apoptosis at the host cell triggered by specific pathogenic bacterium is a new phenomenon in the pathogenesis of periodontal diseases.6 the incidence of apoptosis at the underlying gingival pocket epithelium can be continuously occur and can caused further destruction by decreased fibroblasts, diminished lymphocytes functions and declined osteoblast. if it occurs, tooth will lose its attachment to periodontium and will cause premature tooth detachment and loss.7 the gingival cell acts as the port d’entrée of a. actinomycetemcomitans into the periodontium, thus taking part in the development of inflammation and the progressivity of periodontal disease. this research is aimed to examine of parp-i expression in gingival epithelium that exposed to a. actinomycetemcomitant serotype-b toxin. materials and methods this experimental research was using male rat (musmusculus strain swiss webster balb c). the methodology was using post test only group design. the analysis unit was gingival epithelial cell of the buccoanterior rat mandible, physically fit, aged at 2.5 months with body weight of 25–35 gram obtained from pharma veterinary centre surabaya. first treatment (control group): ten rats were given sterile distillated water topically at the bucco-anterior mandibular gingiva every twelve hours with aid from disposable oral sponge swab (rynell inc, usa) immersed into sterile distillated water until thoroughly wet, then applied with double lateral strokes. second treatment (treatment group): ten rats were applied topically with a. actinomycetemcomitans serotype-b toxin at 100µg/ml at the bucco-anterior mandibular gingiva using hamilton syringe (reno, nevada usa). after twenty-four hours, the rats were eliminated and the mandibular gingiva were incised as biopsy specimen. the immunohistochemistry examination with the method of streptavidin –biotin-complex were used to get the expression of parp-1. the counting of gingival epithelial cells which expressed parp-1 was done under light microscope with 400 times magnification. cells which proved to be positive gave brownish color between bluish/greenish epithelial cells. every reserves were examined at 4 different places clockwise 3, 6, 9, and 12. each field of vision are examined and counted at two places according to 6 and 12 needle using counting room and counter. this research used the t-test statistical analysis with 95% sensitivity (p < 0.05). to strengthen the image of early apoptosis using parp-1 expression, the dna fragmentation test was performed with electrophoresis gel. dna was purified from epithelial gingiva cultured cells using a dna extraction kit (stratagene #200600; la jolla, ca). the purified dna was electrophoresed using a 2% agarose gel with ethidium bromide staining. results the results of immunohistochemical examination shows that the number of cells expressing parp-1 is shown in table 1. on the induction of aa serotype-b crude toxin shows an increasing number of cells expressing parp-1 with an average rating of 48.9, whereas in the control group obtained the results of 25.21. normality test is done using kolmogorov-smirnov test. normal distribution group continued by parametric test (t-test) to know the difference in the group with 5% significant rate. table 1 shows that significant difference in number of cells which express parp-1 in gingival kata kunci: toksin aggregatibacteractinomycetemcomitans serotype-b, parp-1, fragmentasi dna correspondence: ernie maduratna setiawatie, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: setiowati_ernie@yahoo.co.id table 1. mean, standard deviation, the significance of number of cells which express parp-1 in gingival epithelial cells that exposed to a. actinomycetemcomitant serotype-b bacterial toxin group n mean sd significance control 10 25.21 2.01 0.01 toxin 10 48.9 1.72 0.01 ��setiawati: crude toxin of aggregatibacter actinomycetemcomitans serotype-b epithelial cell that exposed aa serotype b toxin. in figure 1 can shown that the results of immunohistochemical examination, the gingival epithelium biopsy of mice to detect the expression of parp-1 with a magnification of 400√. at figure 2, there are many dna laddering which indicates the dna has been fragmented at the intoxicated cells. apoptosis indicated by the fragmentation at 180–200 basepairs and its multiples. on the control group, there was no dna fragmentation. discussion periodontitis, one of the most common human infectious diseases, is an acute or chronic infectious condition that can result in the inflammatory destruction of periodontal tissues such as periodontal ligaments and alveolar bone. among more than 300 species of bacteria in the oral cavity, porphyromonas gingivalis and a. actinomycetemcomitans (aa) are the major periodontopathic bacteria, and several virulence factors related to the pathogenesis of periodontitis. therefore, identification of certain virulence factors of aa serotype-b bacteria would aid in the development of preventive strategies against periodontal diseases, especially agressive periodontitis. apoptosis plays an important role in the homeostasis of cells and tissue in response to damage. the process of apoptosis/programmed death of cells shows changes in cell morphology include chromatin condensation, cell membrane shrinkage, the outbreak of nucleus and apoptotic bodies was a sign of dna fragmentation in nucleosom, releasing parp1. based on these results, it seems that the exposure of aa serotype-b crude toxins can increase the number of parp1 in the cells of the gingival epithelium. this is caused by the aa serotype-b bacterial toxin, both endotoxin and exotoxin can cause apoptosis via different paths. endotoxin (lipopolysacharida) through lps binding protein binds the cd14 in membrane cells will activate ip3 receptors that activate calcium release from the endoplasmic reticulum. lps also activates tnf alpha which will trigger apoptosis in an external way. lps induces apoptosis signal in fibroblast through tumor necrosis factor receptor 1 (tnfr1), thus a b figure 1. overview of parp-1 expression in rat gingival epithelial cells using monoclonal antibody against parp-1 with a magnification of 400√. a) control, b) the provision of a. actinomycetemcomitant serotype-b bacterial toxin. red arrow shows the positive results of brown spots on the nucleus of gingival epithelial cells, whereas blue arrows indicate a negative result of no brown spots on gingival epithelial cell nucleus. figure 2. dna fragmentation from electrophoresis gel examination. by using marker (100 bp dna ladder promega) shows dna laddering at 200 bp and its multiples at the first lane. �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 39–42 increasing the activity of caspase 3 and caspase 8. cylolethal descending toxin (cdt) from actinomycetemcomitans bacteria inhibit epithelial cell growth, cdt bind to gm3 ganglioside specific receptor in nucleus using the enzyme dna-like a nuclease that will lead to dna fragmentation thus inhibiting cell cycle g0/g1 and g2/m and inhibit cell to enter the mitotic phase. the breakdown of dna internucleosome chain is an early sign of apoptosis. the breakdown of dna chain will activate parp-1 enzyme, which is an enzyme that activate dna repair in the nucleus. but if there is excessive activation, parp-1 will result in the loss of nad+, causing apoptosis.8,9 parp-1 is an enzyme in the nucleus which always present in eukariota. it consists of proteins with a molecular weight of 116 kda. parp-1 is the most dominant member of the poly adp-ribose polymerases (parps) protein in the nucleus, representing more than 85%. parp-1 plays a role in dna repair that suffers injury. in every 1000 bp of dna, there is 1 parp molecule. if the dna is damaged, there is an increase of 500 times of parp to bind to the broken dna chain. parp-1 produces 50–200 poly adpribose (par) to be given to the histones protein, dnapolymerases, topoisomerase, dna ligase, and transcription factors. parp-1 catalyzes the breakdown of nicotinamide adenine dinucleotide (nad+) into nicotinamide and adp ribose, subsequently polymerized forming a branch of poly adp-ribose nucleic acids. in basal state, parp-1 activity is very low, but if there are damages to dna, parp-1 will hyperactively break nad+ to create poly adp-ribose branch, in turn, will generate the histone proteins and enzymes that help the dna repair. at low levels of dna damage, parp-1 has the function of repairing the dna. but at medium or high level of dna damage, parp-1 may lead to cell’s death through apoptosis or necrosis. parp-1 requires nad+ to be hydrolyzed and adp-ribose units. parp-1 that is too active will cause nad+ depletion, which continues to deplete the atp.10 in the present study, we demonstrated that crude toxin periodontopathic bacteria aa serotype b increase parp-1 expression which early marker of cell death. this cell death is due to apoptosis, since of the crude toxin induced dna ladder formation. a. actinomycetemcomitans serotype-b have three cytotoxic factors such as leukotoxin, cytolethal distending toxin (cdt), and endotoxin lps which induces both cell cycle arrest and apoptosis. leukotoxin induced apoptosis in hl-60 cells, which was consistent with the present.11 furthermore, it has been reported that both cell cycle arrest and apoptosis were induced by partially purified a. actinomycetemcomitans serotype-b toxin in mouse hybridoma cell line hs-72 cells. it is likely that a. actinomycetemcomitans serotype-b produces several types of toxins which induce different cytotoxic effects against mammalian cells. treatment with the extract from the a. actinomycetemcomitans serotype-b induced pore formation on the cell membrane, nucleosomal dna ladder formation, and caspase-3 activation.12-14 the dna breakage resulted in the rapid activation of parp-1. at the same time, atp and nad+ concentrations decreased and nicotinamide accumulated extracellularly. these findings collectively indicated the rapid activation and central role of parp in the pathogenesis of periodontitis.15 this research showed that the a. actinomycetemcomitant serotype-b crude toxin cause dna fragmentation and increased parp-1 expression in gingival epithelial cells. the author recommends providing cytoprotection materials on gingival epithelium to promote epithelial cell survival against aa serotype-b crude toxin. inhibition of parp-1 may represent a novel host response modulatory approach for the therapy of periodontitis. in conclusion, the crude toxin exposure of a. actinomycetemcomitant serotype-b leads to dna fragmentation and increase parp-1 expression. references 1. newman mg, takei n, klokkevold p, carranza f. carranza’ clinical periodontology. 10th ed. philadelpia, wb saunder; 2006. 168–81, 409. 2. daniel hf, kenneth m, david f, karen f, javier f, cebile n, marie m, john g. a. actinomycetemcomitans and its relationship to initiation of localized aggressive periodontitis. j clin microbiol 2007; 45: 3859–69. 3. suzuki t, kobayashi m, isatsu k, nisihara t. mechanisms involved in apoptosis of human macrophages induced by lipopolysaccaride from actinobacillus actinomycetemcomitans in the presence of cycloheximide. infect immune 2004; 72: 1856–65. 4. k ato s, na k a sh i m a , sug i mu r a , nish i ha r a t, kowa sh i y. actinobacillus actinomycetemcomitans induces apoptosis in human monocytic thp-1 cells. med microbiol 2005; 54: 293–8. 5. lohinai zjg, mabley e, fehér, marton a, komjáti k, szabó c. role of the activation of the nuclear enzyme poly (adp-ribose) polymerase in the pathogenesis of periodontitis. j dent res 2003; 82: 987–92. 6. gamonal aj, gomez m, silvà a, gonzalez ma. new knowledge of the pathogenesis of periodontal disease. quintessence int 2004; 35: 706–16. 7. bosshardt dd, lang npthe junctional epithelium: from health to disease. j dent res 2005; 84: 9–20. 8. bascones a, gamonal j, gomez m, silvà a, gonzalez m a. new knowledge of the pathogenesis of periodontal disease. quintessence int 2004; 35: 706–16. 9. alikhani m, alikhani z, graves dt. apoptotic effects of lps on fibroblasts are indirectly mediated through tnfr1. j dent res 2004; 83(9): 671-6. 10. lászló v, csaba s. the therapeutic potential of poly (adp-ribose) polymerase inhibitors. pharmacol rev 2002; 54: 375. 11. shenker b, hoffmaster r, zekavat a, yamaguchi n. induction of apoptosis in human t cells by actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of g2 arrest of the cell cycle. j immune 2001; 167: 435–41. 12. yamaguchi n, kubo c, masuhiro y, lally e, koga t. tumor necrosis factor alfa enhances actinobacillus actinomycetemcomitans leukotoxin–induced hl-60 cell apoptosis by stimulating lymphocyte function associated antigen 1 expression. infect immune 2004; 72: 269–72. 13. zhang l, pelech s, uitto v. long-term effect of heat shock protein 60 from actinobacillus actinomycetemcomitans on epithelial cell viability and mitogen-activated protein kinases. infect and immun 2004; 72: 38–45. 14. dirienzo jm, song m, wan ls, ellen rp. kinetics of kb and hep-2 cell responses to an invasive, cytolethal descending toxin–producing strain a. actinomycetemcomitans. oral micrbiol immunol 2002; 17: 245–51. 15. lucas h, bartold pm, dharmapatni, holding ca, haynes dr. inhibition of apoptosis in periodontitis. j dent res 2010; 89: 29–33. 76 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 76–80 correlation between estrogen and alkaline phosphatase expression in osteoporotic rat model sherman salim department of prosthodontics faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: osteoporosis is a systemic disease that can decrease bone density as a result of imbalance bone remodeling and bone resorption. estrogen reduction due to menopause can increase osteoclast activity and furthermore decrease bone density. estrogen can stimulate alkaline phosphatase (alp) expression, collagen type i and osteocalcin in bone remodelling process. ovariectomized rat is a common animal for studying patofisiology, diagnosis and treatment osteoporosis patient. purpose: to evaluate correlation between estrogen and alp expression in osteoporotic rat model mandible. methode: 18 female wistar rats, 2 months old, 200 grams were divided into 2 groups, ovariectomized group and sham surgery as control group. surgery was done under intra muskular anesthesia using combination 2% xylazine 1cc and 10% ketamine 1cc. after 12 weeks, mandible was taken for alp examination and blood from heart was taken to evaluate the amount of estrogen. result: there was significant correlation between estrogen and alp expression in osteoporotic rat model mandible. conclusion: the amount of estrogen can influence alp expression activity. keywords: osteoporosis; estrogen; alkaline phosphatase; ovariectomy correspondence: sherman salim, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: sherman.salim@yahoo.com introduction oral health condition of elderly patients has an important role equal to quality of life, in addition to several other life factors, such as socio-economical, physical, and psychosocial reasons. consequently, demand to get better dental and oral cavity treatment increases. based on global epidemiological data report, 1 out of 3 women and 1 out of every 50 men over the age of 50 years suffered from osteoporosis. several researches even have reported that there is a correlation between systemic osteoporosis and bone resorption in oral cavity.1 osteoporosis is a systemic disease that can decrease bone density as a result of imbalance between bone formation with bone resorption. postmenopausal osteoporosis is the most common form of osteoporosis, leading to increased risk of fractures after menopause manifestations such as wrist fractures, spine fractures, and hip fractures with a mean age over 60 years.2 bone density is a specific factor influencing the risk of bone fractures. according to who, a state of osteoporosis if bone density more than 2.5 standard deviation, below the average for normal young people (t<-2.5) in dual energy x-ray absorptiometry (dexa) measurement.3 moreover, an estimated 40% of women and 13% of men over at the age of 50 will experience a fracture due to osteoporosis.4 osteoporosis is classified into two categories, namely primary osteoporosis and secondary osteoporosis. primary osteoporosis is associated with menopause and aging conditions. meanwhile, secondary osteoporosis is due to certain health conditions, along with drug therapies that influence bone density or increasing of bone density loss. the condition is generally associated with endocrine disorders (cushing syndrome, hyperparathyroidism, insulin-dependent diabetes mellitus (iddm), adrenal insufficiency), rheumatoid arthritis, hematological disorders, and malignancies (leukemia, lymphoma).5 hormonal changes that occur as the condition of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p76-80 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p76-80 7777salim/dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 76–80 menopause are important factors cause decrease women bone density.6 the decline in estrogen levels associated with post-menopausal osteoporosis condition then can increase osteoclasts activity, and subsequently can lead to osteoporosis decreasing the attachment of the bone into the teeth in the oral cavity.7 there are several methods to measure bone density, such as inspection dexa, which has good accuracy in diagnosis. this examination is very important to improve successful rate of dental treatment, expecially prosthodontics such as complete dentures, partial dentures, as well as dental implants. however, the use of this measuring tool has limitations in its implementation because it is conducted on a particular health facility and the cost is relative high. therefore, simple examination is needed for early identification of osteoporosis during dental care, such as a examination of estrogen levels and alp expression. the examination may be an option for an early indicator of osteoporosis sufferers, which is more efficient with a good degree of diagnostic accuracy. ovx model rats are animals mostly used in a study of osteoporosis pathophysiology, focused on its diagnosis and treatment. these animals have also been validated as a clinically relevant model of post-menopausal bone density loss.4 in this research, immunohistochemical examination (ihc) was conducted in the osteoporotic rats model mandible. this research was conducted to evaluate estrogen levels on mandibular bone alp expressions in both model rats suffering from osteoporosis and normal rats. thus, the results of this research are expected to provide information about evaluation of estrogen levels and immunohistochemical examination of alp in both normal rats and model rats suffering from osteoporosis. materials and method this research was a laboratory experimental research using 18 female wistar rats, 3 months old. those animals were not in a state of pregnancy, did not suffer from any disease, as well as did not undergo hormone therapy. this research was approved by health research ethics committee (kkepk) from faculty of dental medicine, universitas airlangga no. 15/ kkepk.fkg/ i/2016. wistar rats then were divided into two groups, group of shs (sham surgery) rats as the control group and the group of ovx rats. model rats suffering from osteoporosis were classified into the treatment group. they were weighed and had intramuscular anesthesia with a combination of 2% xylazine (1 cc) and 10% ketamine (1 cc). ovariectomy was performed by making a ventral incision from the umbilicus to the pubis. the blood vessels of the ovary and the fallopian tubes were ligated separately. the ovarium was taken bilaterally, and then the peritoneum incision was closed using a simple interrupted suture technique. shs was performed in the control group by making a ventral incision from the umbilicus to the pubis, and then returned to its previous position and closed the peritoneum incision by using a simple interrupted suture technique.9 those ovx female wistar rats were maintained for 12 weeks in a cage with simultaneous treatment, in which they got 12 hours of light and 12 hours without light. they were also fed with comfeed concentrate starter (calf starter) containing yellow corn, wheat bran, sbm, drops, palm olien essential amino acids, essential minerals, premix, and vitamin. they also got drink at the same time. blood samples were obtained after those rats were anaesthetized using ketamine before termination. blood samples were taken directly from the heart through the apex as much as 3 ml using 5 ml of disposable syringe. blood samples taken from the rat heart were then inserted into a test tube and waited for 3 hours to generate serum. the serum was centrifuged at 3000 rpm at room temperature for thirty minutes with microliter centrifuge (micro 200, hettich gmbh & co.kg). centrifugation process was performed to obtain pure serum. the serum then was added into eppendrof tubes, and stored in a refrigerator at a temperature of 40 c. to determine estrogen levels in those rats, an examination with indirect elisa method was performed. antigens were derived from the serum of blood samples taken from the heart. the blood samples were taken from all the experimental animals treated. indirect elisa testing was performed using 100μl of antigens in coating buffer with a ratio of 1: 9, and then put in wells of elisa plates and incubated overnight at a temperature of 40ºc. they were washed with pbs-tween 3 times. they were added with 50 ml of blocking buffer (1% bsa in pbs). they were washed with pbs-tween 3 times. they were added with 100 ml of primary antibody in 1% pbs-bsa solution with a ratio of 1: 500, and then incubated for 2 hours. they were washed with pbs-tween 3 times, added with 100 ml of the secondary antibody in tris buffer saline at a ratio of 1: 2500, and then incubated for 1.5 hours. they then were washed with pbs-tween 2 times. they were added with 50 ml of pnpp substrate, incubated for 30 minutes, and added with 50 ml of 1n naoh as the reaction stopper. the absorbance was read using elisa reader at a wavelength of 405 nm.10 after blood sample was taken, rats were terminated and mandible was taken for immunohistochemistry examination. direct method with a monoclonal antibody was used to detect a marker of cells, directly labeled with alp enzyme. to eliminate the activity of endogenous peroxidase, 3% h2o2 was used. to clean protein debris that might cover epitope with materials that would be used to detect, 0.025% trypsin in pbs was used. ihc examination then was conducted by deparaffinization, integrating successive tissue incision into xylitol, absolute ethanol, 95% ethanol, 80% ethanol, and 70% ethanol, and then watered under running water for 10-15 minutes. it was put into a solution of 3% h2o2 for 30 minutes, washed with pbs, put in 0.025% trypsin for 6 minutes at 370 c, and then washed dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p76-80 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p76-80 78 salim/dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 76–80 again using pbs. afterwards, it was put into monoclonal antibody labeled with alp enzyme, washed with pbs, put into chromogen substrates, washed with pbs, washed using aquadestilata, and then put into mayer’s haematoksilin. it was washed using running water, and then dehidrationclearing-mounting was performed.11 variables studied in this research included changes in both estrogen levels using elisa and alp expression using ihc examination. ihc examination was conducted using qualitative observation supported by quantitative data in one field of view. quantitative data obtained were tabulated using microsoft office excel, and then analyzed using spss 20.0 for windows with shapiro-wilk analysis to determine whether the distribution of the data was normal or not. if the distribution of data was normal, the analysis would be continued using parametric analysis of independent t-test. results this research focused on the correlation between estrogen levels and alp expression in all groups, used nine samples in each group, namely the group of normal rats (as the control group) and the group of ovx rats (osteoporosis). normality test was conducted to determine the normal distribution in all the variables studied. ekspresi alp table 1. mean and standard deviation of estrogen levels and alp expression in the group of mandibular bone osteoporosis rats and the group of normal rats parameter group mean standard deviation significance of difference estrogen normal 86.5091 23.19584 0.075 osteoporosis 67.3417 25.65043 alp normal 1.0000 0.75366 0.001* osteoporosis 4.8000 2.78959 10 table 1. mean and standard deviation of estrogen levels and alp expression in the group of mandibular bone osteoporosis rats and the group of normal rats parameter group mean standard deviation significance of difference estrogen normal 86.5091 23.19584 0.075 osteoporosis 67.3417 25.65043 alp normal 1.0000 0.75366 0.001* osteoporosis 4.8000 2.78959 figure 1. expressions of alp on immunereactive osteogenic cells (arrows) among the control groups of shs (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). figure 2. expressions of alp on immunereactive osteogenic cells (arrows) among the treatment groups of ovx (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). table 2. correlation test on estrogen levels and alp expression among the research groups group parameter coefficient significance of correlation normal estrogen 0.162 0.635 alp osteoporosis estrogen -0.014 0.965 alp ovx shs figure 1. expressions of alp on immunereactive osteogenic cells (arrows) among the control groups of shs (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). 10 table 1. mean and standard deviation of estrogen levels and alp expression in the group of mandibular bone osteoporosis rats and the group of normal rats parameter group mean standard deviation significance of difference estrogen normal 86.5091 23.19584 0.075 osteoporosis 67.3417 25.65043 alp normal 1.0000 0.75366 0.001* osteoporosis 4.8000 2.78959 figure 1. expressions of alp on immunereactive osteogenic cells (arrows) among the control groups of shs (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). figure 2. expressions of alp on immunereactive osteogenic cells (arrows) among the treatment groups of ovx (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). table 2. correlation test on estrogen levels and alp expression among the research groups group parameter coefficient significance of correlation normal estrogen 0.162 0.635 alp osteoporosis estrogen -0.014 0.965 alp ovx shs figure 2. expressions of alp on immunereactive osteogenic cells (arrows) among the treatment groups of ovx (ihc staining, magnification 400x; h600l nikon microscope; fi2 300 megapixel camera ds). table 2. correlation test on estrogen levels and alp expression among the research groups group parameter coefficient significance of correlation normal estrogen 0.162 0.635 alp osteoporosis estrogen -0.014 0.965 alp table 1 shows that the normal group had a higher mean value of estrogen than the osteoporosis group. in contrary, the osteoporosis group had a higher mean value of alp than the normal group. based on the results of the data distribution test, the distribution of all data was normal, so the analysis was continued using parametric analysis of independent t-test. based on the results of independent t-test, furthermore, there was no significant difference in estrogen levels between the normal group and the osteoporosis group. however, there were significant differences in alp expression between the normal group and the osteoporosis group. the results of pearson correlation test showed that there was no significant correlation between estrogen levels and alp expression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p76-80 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p76-80 7979salim/dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 76–80 discussion osteoporosis in the elderly will trigger risk of fracture due to a decrease in bone density. osteoporosis is a disease with some distinctive properties, such as low bone mass, accompanied by changes in bone microarchitecture and deterioration of bone tissue quality, increasing risk of bone fragility fractures.12 bone tissue continuously experiences formation and resorption (bone remodeling). at the age of 20 years old, bone formation process is highly active, far beyond bone resorption process. at the age of 20-40 years old, both processes then are nearly active, while at the age of 40 years old, bone resorption process is more active than bone formation process, leading to decrease in bone density. osteoporosis, furthermore, can occur in the elderly population, especially postmenopausal women because of several factors, especially estrogen levels in bone remodeling process. estrogen plays an important role in bone metabolism contributing to an increase in the number of osteoblasts as well as their function, and also to reduce bone resorption. there are estrogen receptors in osteoblasts, osteocytes, and osteoclasts. estrogen also has been reported to have ability in increasing levels of osteoprotegerin (opg), a protein produced by osteoblasts, which can inhibit bone resorption by preventing rankl from binding to rank receptors. therefore, estrogen plays an important role in osteoclastogenesis process. estrogen inhibits local factors that hinder bone formation process or increase local factors that stimulate bone formation process. for this reason, a decrease in estrogen is an important pathogenic factor in bone loss associated with osteoporosis.13 examination with indirect elisa method, moreover, was conducted using blood samples to determine levels of estrogen from their heart. the results of independent t-test showed that there was a significant difference (p=0.075) between the normal group (86.5091) and the osteoporosis group (67.3417). this may happen because there are other sources of synthesis that can replace the main source of estrogen in their ovaries during osteoporosis condition. other sources of estrogen synthesis can be found in mesenchymal cells of adipose tissue, skin, osteoblasts, osteoclasts in bone, vascular endothelial as well as some places in the brain, such as preoptic medial/anterior hypothalamus, medial basal hypothalamus, and amygdala, which are only active at the level of the local tissue with a high concentration. at the beginning of osteoporosis, those sources of synthetic estrogen systemically controlled by the ovaries begin to decline. as a result, it will trigger the reactive properties of estrogen synthesized in the local tissue to get into the systemic circulation so that it can act as a substitute for the primary synthesis in maintaining levels of estrogen.14 nevertheless, the reactive properties of estrogen derived from the local tissue are only temporary and occurs only in the early stage of osteoporosis. osteoporosis at the advanced stage then would affect the local tissue in synthesizing estrogen so that estrogen levels in the systemic circulation reached its lowest level.15 similarly, a research on model animals conducted by gao16 shows that changes in hormones during menopause contribute to changes in body composition and fat distribution. increased adipose tissue in a group of menopause rats triggers increased production of estrogen by the local tissue. decreased ovarian function in the early stage of osteoporosis can also lead to an increase in adipose tissue mass not influenced by dietary adjustments and pathological metabolic conditions. consequently, it is important to recognize clinically since in those ovariectomized rats there was an increase in fat accumulation due to estrogen deficiency and decreased physical activity. in this research, alp obtained showed a significant difference (p=0.001) between the normal group (1.0000) and the osteoporosis group (4.8000). bone remodeling in osteoporosis is an ongoing process. however, bone resorption and bone formation cannot be balanced due to estrogen deficiency. this is indicated by a decline in the capacity of osteoblasts in the form of new bone matrix. in a research conducted by lim and kim,17 there are significant differences in trabecular bone mass after 12 weeks of post-ovariectomy in wistar rats. similarly, a research on ovariectomized wistar rats conducted by lasota18 also shows that after 12 weeks of post-ovariectomy there are changes in bone density and bone mineral structure. therefore, this research used a 12-week evaluation period after ovariectomy because there has been a reduction in bone density and bone structure representing the loss of bone density after menopause. in addition, the markers of bone remodeling are a biochemical product measured in blood or urine reflecting bone metabolism activity, but not having a function as a controller of skeletal metabolism. markers of bone remodeling are simply categorized as markers of bone formation. among the markers of bone formation, alp is secreted by osteoblasts towards the extracellular fluid that can be measured in serum.19 besides serum activity of osteoblasts in bone formation process, type i collagen and proteoglycan can also be considered as bone matrix through a process, called ossification, which in the active condition then will produce osteoid tissue. osteoblasts secrete a large amount of alp in the process of bone formation. alp plays a role in mineralization process, which is to prepare alkaline atmosphere (basic) in osteoid tissue formed so that calcium can be deposited on the tissue.20 increased alp expression then indicates osteogenic differentiation. during the differentiation process from pre osteoblast to osteoblasts, alp activity will increase. meanwhile, at the beginning of osteoblasts formation, alp activity decrease, but during maturation process of osteoblasts, alp activity will increase again.21 based on the results of pearson correlation test, there was no significant correlation between estrogen and alp in both of the normal group and the group of ovariectomized rats. this is due to estrogen levels in the systemic circulation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p76-80 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p76-80 80 salim/dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 76–80 affecting osteoblasts in the process of bone formation. it did not directly affect the activity of alp expression in osteoblasts during the differentiation process from pre osteoblast to maturation. similarly, gao16 mentions that the expression of alp activity changes in accordance with the phases of bone formation in osteoblasts, but is not affected by estrogen levels in the systemic circulation. in this research, estrogen levels also were not directly related to the activity of alp expression caused by alp expression changes according to the phases of bone formation. it can be concluded that estrogen levels in model rats suffering from osteoporosis are lower than normal ones. meanwhile, mandibular bone alp expression in model rats suffering from osteoporosis is higher than normal one. it indicates that there is no significant correlation between estrogen levels and mandibular bone alp expression in model rats suffering from osteoporosis and normal ones. references 1. amadei su, souza dm, brandão aah, rocha rf. influence of different durations of estrogen deficiency on alveolar bone loss in rats. braz oral res 2011; 25(6): 538-43. 2. dodd dz, rowe dj. the relationship between postmenopausal osteoporosis and periodontal disease. j dent hyg 2013; 87(6): 33644. 3. kanis ja. world health organization scientific group assessment of osteoporosis at the primary health-care level. technical report. world health organization collaborating centre for metabolic bone diseases1, uk: university of sheffield. 2007. 4. durão. bone regeneration in osteoporotic conditions: healing of subcritical-size calvarial defects in the ovariectomized rat. int j oral maxillofac impl 2012; 27(6): 1400-8. 5. chhina k, rakhi s. osteoporosis: a risk factor in periodontal disease. indian j dent sci 2010; 30-5. 6. khojastehpour. comparison of the mandibular bone densitometry measurement between nor mal, osteopenic and osteoporotic postmenopausal women. j dent (tehran) 2013; 10(3): 203-9. 7. nebel d, bratthall g, warfvinge g, nilsson bo. effects of ovariectomy and aging on tooth attachment in female mice assessed by morphometric analysis. acta odontol scand 2009; 67(1): 8-12. 8. rahnama m, światkowski w. effect of ovariectomy on biochemical markersof bone turnover (alp, acp) and calcium content in rat mandible and teeth. bull vet inst pulawy 2002; 46: 281-7. 9. tabata y. tissue regeneration based on drug delivery technology. topics in tissue engineering, finland: university of oulu. 2003. p. 1-32. 10. albert b. molecular biology of the cell. 3rd ed. new york and london, usa: garland publishing, inc; 1998. p. 125-30. 11. schacht v, kern js. basics of immunohistochemistry. j invest derm 2015; 135(3): e30. 12. viguet-carrin s, garnero p, delmas pd. the role of collagen in bone strength. osteoporos int 2006; 17(3): 319-36. 13. lenora j. bone turnover markers and prediction of bone loss in elderly women. dissertation. lund university 2009. p. 87-8. 14. kini u, nandeesh bn. physiology of bone formation, remodeling, a nd metabolism. spr inger-verlag berlin heidelberg; 2012. p. 29-59. 15. duarte pm, gonçalves p, casati mz, de toledo s, sallum ea, nociti fh jr. estrogen and alendronate therapies may prevent the influence of estrogen deficiency on the toothsupporting alveolar bone: a histometric study in rats. j periodont res 2006; 41(6): 541-6. 16. gao x. establishing a rapid animal model of osteoporosis with ovarectomy plus low calcium diet in rats. int j clin exp pathol 2014; 7(8): 5123-8. 17. lim dw, kim yt. anti-osteoporotic effects of angelica sinensis (oliv.) diels extract on ovariectomized rats and its oral toxicity in rats. nutrients 2014; 6(10): 4362-72. 18. lasota a. experimental osteoporosisdifferent methods of ovariectomy in female white rats. proceedings annales academiae medicae bialostocensis 2004; p. 49. 19. vasika ra n s, eastell r, br uyère o, foldes a j, ga r nero p, griesmacher a, mcclung m, morris ha, silverman s, trenti t, wahl da, cooper c, kanis ja. markers of bone turnover for the prediction of fracture risk and monitoring of osteoporosis treatment: a need for international reference standards. osteoporosis int 2011; 22(2): 391-420. 20. yudaniayanti is. alkaline phosphatase activity to femoral fractures healing with excessive caco 3 treatment in male rat (sprague dawley). media kedokteran hewan 2005; 21(1): 15-26. 21. hendrijantini n, kresnoadi u, salim s, agustono b, retnowati e, syahrial i. study biocompatibility and osteogenic differentiation potential of human umbilical cord mesenchymal stem cells (hucmscs) with gelatin solvent. j biomed sci engineer 2015; 8(7): 420-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p76-80 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p76-80 p-issn: 1978-3728 e-issn: 2442-9740 volume 52, number 2, june 2019 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2019 january 2nd – december 31st, 2019 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (department of dental health techniques, faculty of vocational studies, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); al. supartinah santoso (department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, indonesia); trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); irna sufiawati (department of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); adioro soetojo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); latief mooduto (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontology, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); ernie maduratna setiawati (department of periodontology, faculty of dental medicine, universitas airlangga, indonesia); i. b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); priyawan rachmadi (department of dental material science and technology, faculty of dental medicine, universitas airlangga, indonesia); ari triwardhani (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); david kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); ira widjiastuti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); nurina febriyanti ayuningtyas (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (+6231) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg/index accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (+6231) 5992246, 5992247, telp./fax. (+6231) 5992248. e-mail: aup.unair@gmail.com volume 52, number 2, june 2019 p-issn: 1978-3728 e-issn: 2442-9740 1. the assistance of er,cr:ysgg laser in pulp injury related to anterior teeth trauma gülşah balan, harry huiz peeters, and serap akyüz .................................................................. 57–60 2. topical application of snail mucin gel enhances the number of osteoblasts in periodontitis rat model h. hendrawati, hanindya noor agustha, and rezmelia sari ...................................................... 61–65 3. assessment of behavioral factors associated with dental caries in pre-school children of high socioeconomic status families bushra rashid noaman ................................................................................................................... 66–70 4. the potential effect of moringa oleifera leaves extract on vascular endothelial growth factor expression in wistar rat oral cancer cells dwicha rahma nuriska hartono, theresia indah budhy sulisetyawati, and edhi jularso .... 71–75 5. the management of herpes labialis, oral thrush and angular cheilitis in cases of oral diabetes maharani laillyza apriasari .......................................................................................................... 76–80 6. the activity of polyclonal igy derived from aggregatibacter actinomycetemcomitans and porphyromonas gingivalis in inhibiting colonization of fusobacterium nucleatum and streptococcus sanguinis oktaviani suci lestari, rini devijanti ridwan, tuti kusumaningsih, and s. sidarningsih .... 81–85 7. density of streptococcus mutans biofilm protein induced by glucose, lactose, soy protein and iron indah listiana kriswandini, indeswati diyatri, and intan amalia putri ................................... 86–89 8. the effects of topical application of red pomegranate (punica granatum linn) extract gel on the healing process of traumatic ulcers in wistar rats sri hernawati, yonanda az zikra, and dwi warna aju fatmawati .......................................... 90–94 9. effects of glycerin application on the hardness of nanofilled composite immersed in tamarind soft drinks titis mustikaningsih handayani, raditya nugroho, lusi hidayati, dwi warna aju fatmawati, and agus sumono ......................................................................................................... 95–99 10. permanent tooth eruption based on chronological age and gender in 6-12-year old children on madura agus marjianto, mieke sylvia, and soegeng wahluyo ................................................................. 100–104 11. the role of cox-2, caspase-1 and il-17 in pericoronitis-related inflammation due to lower third molar impaction adi prayitno ..................................................................................................................................... 105–109 66 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 66–70 research report the effects of audio-video instruction in brushing teeth on the knowledge and attitude of young slow learners in cirebon regency yayah sopianah,1 muhammad fiqih sabilillah,2 and oedijani3 1,2department of dental nursing, poltekkes kemenkes, tasikmalaya indonesia 3department of dental and oral diseases, faculty of medicine, universitas diponegoro, semarang indonesia abstract background: young slow learners are children with special needs who require special attention to satisfy their personal hygiene needs, especially those of dental hygiene since they are particularly susceptible to tooth decay. changing the knowledge and attitudes of those slow learners can be achieved by a proper method purpose: this study aimed to analyze the effects of teaching effective methods of brushing teeth by means of an audio video approach on the knowledge and attitude of young slow learners in cirebon regency. methods: this study was quasi-experimental in nature using pre test-post test methods within a two-group design. the sampling technique employed was purposive in nature. the number of young slow learners as respondents in this research totalled 31 individuals. those children were, subsequently, divided into two groups; group i containing 16 children, instructed in tooth brushing techniques by means of an audio video method and group ii composed of 15 children who were taught tooth brushing techniques manually. the variables measured consisted of subjects’ knowledge of and attitude towards how to brush teeth most effectively identified by means of a questionnaire, in order that the interval data could be collected. results: the results of an independent t-test showed there to be significant differences in the mean scores of the knowledge variable and the attitude variable (p = 0.003 and p = 0.000 respectively) between groups i and ii, at that stage of the investigation. conclusion: it can be concluded that instructional audio-videos on how to brush teeth most effectively can improve both the knowledge and attitude of children with slow learning problems. keywords: audio video; knowledge; attitude; young slow learners correspondence: yayah sopianah, dental nursing department of poltekkes kemenkes, tasikmalaya. jl. tamansari no. 210 tasikmalaya, indonesia. e-mail: yayahsopianah@gmail.com. introduction young slow learners are children with special needs, who are physically, mentally, intellectually, socially, and emotionally disadvantaged, a condition significantly influencing their growth and development processes compared to those of normal children.1 the world health organization (who) estimates that individuals with such disabilities to represent about 7-10% of the total number of children in indonesia. however, according to national socio-economic survey data, in early 2011 there were 679,048 school-age children with special needs, constituting approximately 21.42% of the total number of children with special needs.2 moreover, based on the national socio-economic survey of march 1, 2011, out of a total population of indonesian children amounting to 82,980,000, no fewer than 9,957,600 demonstrated some form of special needs, being categorized as individuals with disabilities. meanwhile, the proportion of gifted children demonstrating special and exceptional intelligence stood at 2.2% of the school-age population (4-18 years old) or about 1,185,560 children.1 actually, it is essential to identify individuals with special needs within groups of children since they require particular attention, such as medical services, tailored education, or specific exercises aimed at reducing their limitations in order to maximize their independence when living as part of a community.2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p66-70 mailto:yayahsopianah@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p66-70 6767sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 66–70 one of the groups of children with special needs requiring special provision is that of young slow learners. according to the national institute of health, the united states of america (usa),3 slow learning constitutes a learning disturbance in children and adolescents characterized by a significant gap between the expected level of intelligence and academic ability and that actually achieved. it is further explained that learning difficulties are probably caused by disturbances within the central nervous system (brain neurobiological disorders) resulting in developmental disorders, such as those of speech development, reading, writing, understanding, and numeracy. slow learners tend to demonstrate limited ability with an intelligence quotient (iq) score of between 70 and 89 or slightly below what is generally considered to be normal. however, individuals falling within this category are not regarded as being mental retardation.4 the phenomenon of slow learning has recently become a significant problem since the number of children presenting this type of problem is outstripping that of those suffering from other disabilities, such as mental retardation, learning disabilities, visual/hearing disorders, and brain/ head trauma.5 the proportion of slow learners has even reached 14% of the total population of children with special needs. once this percentage exceeds 10%, it is considered to be relatively large. moreover, the physical condition of slow learners is similar to that of normal children, causing them to be neglected although they actually need relatively special guidance. in other words, slow learners are children with disorders, but who do not present physical symptoms which highlight their developmental condition.6 slow learners are also characterized by a slightly lower than average intellectual potential, but are excluded from the various categories of mental disorder. such individuals take a long time and require repetitive practice in order to be able to complete tasks, both academic and non-academic in nature.1 child health care for slow learners, for instance, requires more attention since their condition can trigger oral health problems due to their not being prioritised within individual health services.7 based on the results of preliminary research, the majority of young slow learners (80.64%) suffer from poor oral hygiene and unattended dental needs. these problems will worsen such children’s general health, while also undermining their overall feelings of well-being, thus increasing the difficulties in satisfying their nutritional requirements. consequently, dental hygiene in young slow learners should attract more concern since their oral hygiene and dental health status remain poor. according to the who global oral health indicator on the dental and oral health status of indonesians, the government has succeeded in maintaining that of children, adolescents, adults, and elderly people. in addition the ministry of health’s target of keeping every child free from caries and able to maintain healthy teeth and mouth has reached 90%.8 changing the knowledge and attitudes of those slow learners can actually only be achieved through a deliberate policy with a grand design, involving certain processes.9 a method used to implement the master plan must be supported by an appropriate strategy.10 thus, a proper method is very helpful for the effecting of behavioral changes in target populations. changes in behavior or adoption of behaviors based on knowledge and attitude can be enduring in nature. there are actually various factors affecting one’s knowledge and attitude, one of which is the media.11 the media can support participants in acquiring a better understanding of health education. indeed, health workers use its various channels as a means of delivering health-related materials. the health education media most commomly used for demonstrations are models of human body (dummies) and audio video.12 the latter, is one form of audio-visual aid that can display a moving object together with natural sound. it may hold learners’ attention and concentration during the teaching process as well as enabling them to focus on those explanations of material that need to be emphasised.13 video has several advantages. for instance, it is dynamic, impressive and stimulating, thus accelerating the development of one’s understanding.14 however, this particular audio-visual aid also has several drawbacks. for example, an unclear image can lead to a lack of certainty on the part of the audience. video also requires some means of projection equipment to display the image, thereby rendering it comparatively expensive. nevertheless, previous research shows that video could positively influence the dental and oral health care behavior of those slow learners with special needs.15 consequently, it is necessary to adopt an appropriate approach to providing dental and oral health services for young slow learners in order to increase their self-reliance in maintaining healthy teeth and achieve optimal dental and oral health. the approach must be capable of stimulating their health as well as prioritizing and promoting preventive efforts at an early age in order to improve their behavior in maintaining oral health. this study aimed to analyze the effects of teaching effective brushing by means of an audio video-based methodology on the knowledge and attitudes of young slow learners. therefore, the results were expected to influence the behavior of those young slow learners in maintaining their oral health as well as in improving their dental and oral hygiene by means of an audio video method. materials and methods this study was a quasi-experimental research incorporating the use of pre test-post test methods within a two-group design.16 the sampling technique used was purposive in nature. the number of respondents with slow dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p66-70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p66-70 68 sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 66–70 learning problems participating in this research totalled 31 divided into two groups. group i consisted of 16 children instructed in brushing technique using an audio video method, while group ii contained 15 individuals who were taught by means of demonstration. before performing intervention, groups i and ii had to complete questionnaires. this research was conducted at sekolah dasar negeri 1 astana (model inclusion school) gunung jati subdistrict cirebon regency west java province. both groups were evaluated at day 2117 with the variables measured at this stage of the research being knowledge of and attitude to tooth brushing collected from subjects by means of questionnaires. data collected was then subjected to an independent t-test. the knowledge variable in this research was interpreted as the slow learners’ level of understanding and awareness about dental and oral hygiene maintenance. the attitude variable in this research was interpreted as their attitude to the maintenance of oral hygiene. results characteristics of the research respondents based on gender can be seen in table 1. based on table 1, the majority of respondents in groups i and ii were male at 56.25% and 60% respectively. another characteristic of the research respondents, namely, that based on age is shown table 1. characteristics of the research respondents based on gender no gender group i group ii n % n % 1 males 9 56.25 9 60 2 females 7 43.75 6 40 total 16 100 15 100 in table 2. based on the contents of table 2 most of the respondents in groups i and ii were aged ten years old. in group i, they numbered five (31.25%), while in group ii the total was four (26.67%). the independent sample t-test results illustrating the mean knowledge of the respondents in both groups based on the pre test and the post test can be seen in the table 3. based on the results of the pre test and post test in table 3, a difference in the mean scores of the knowledge variable between groups i and ii existed. the mean score of the knowledge variable of group i young slow learners during the pre test was 43.75, higher than that of group ii at 35.11. however, there was no significant difference in the mean scores of the knowledge variable between groups i and ii during the pre test as indicated by a p value of 0.81 (p>0.05). similarly, the mean scores of the knowledge variable of the group i young slow learners during the post test was table 2. characteristics of the research respondents based on age no. age (years) group i group ii n % n % 1 8 2 12.5 2 13.33 2 9 4 25 3 20 3 10 5 31.25 4 26.67 4 11 1 6.25 2 13.33 5 12 2 12.5 2 13.33 6 13 2 12.5 2 13.34 total 16 100 15 100 table 3. analysis of the mean score for the knowledge variable in both groups based on pre test and post test results no. knowledge variables pre test post test n mean p n mean p 1 group i 16 43.75 0.81 16 55.00 0.003 2 group ii 15 35.11 15 40.45 table 4. the analysis results of the mean score of the attitude variable in groups i and ii based on pre test and post test results no. knowledge variables pre test post test n mean t p n mean t p 1 group i 16 40.56 1.66 0.81 16 47.43 5.083 0.000 2 group ii 15 38.13 15 41.26 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p66-70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p66-70 6969sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 66–70 55.00, a level higher than that of group ii (40.45). there was a significant difference between groups i and ii’s knowledge variable mean scores during the post test as indicated by a p value of 0.003 (p>0.05). the independent sample t-test results indicating the mean scores of the attitude variable for both groups based on the pre test and post test results can be seen in table 4. based on the pre test and post test results, there was no difference in the mean scores of the attitude variable between groups i and ii (p=0.253) during the pre test. in contrast, there was a significant difference in the mean scores of the attitude variable between groups i and ii (p=0.000) during the post test (table 4). in addition, the mean score of the knowledge variable of those young slow learners in group i during the post test was 47.43, higher than for group ii at 41.26. there was a significant difference in the mean scores of the attitude variable during the post test between groups i and ii as indicated with a t value of 5.083 and a p value of 0.000 (p>0.05). discussion children with a slow learning condition are also known as slow learners and include individuals with special needs who require specific forms of support. slow-learning constitutes a learning disorder in children and adolescents characterized by a significant gap between the level of intelligence and academic ability. it is most probably caused by disturbances to the central nervous system, especially the brain (neurobiological disorders) that can cause developmental problems, such as ones affecting speech development, reading, writing, understanding, and numeracy.8 slow learners typically demonstrate restricted iq levels between 70 and 89 which, although slightly below normal, are not necessarily indicative of mental retardation.4 as a result, they tend to require considerable time and repeated attempts to complete both academic and nonacademic tasks.1 based on an analysis of the respondent characteristics (gender and age) in the research described here, there was a balance in the frequency and percentage of data distribution in both groups i and ii consisting of 16 and 15 children respectively, given intervention in the form of audiovisual teaching about brushing technique, see tables 1 and 2. the results of the analysis confirmed no significant difference between the groups i and ii pre test mean scores. however, the mean score of the pre test for group i was higher than that for group ii. this is because these respondents’ knowledge about tooth brushing remained low due to an uninteresting and non-repetitive teaching methodology. this result is in line with that of a research project conducted by fitrika.8 the study also indicates that there was a significant difference in the mean scores of the knowledge variable between groups i and ii during the post test. group i was significantly higher than group ii (table 3) because the material of the two groups was delivered using different methods. the slow learners in group i acquired the material by means of an audio video presentation method, while those in group ii acquired the material on a face-to-face basis. the former employed displays such attractive images that could attract the attention and hold the concentration of those slow learners during the learning process as well as focusing on those explanations of the materials requiring considerable emphasis. according to the statistical analysis, there was no significant difference in the mean scores of the attitude variable between groups i and ii during the pre test. nevertheless, the mean score of the attitude variable in group i during the pre test was higher than of group ii (table 4). besides, it was established that after the post test, the mean score of the attitude variable of group i was higher than of group ii. this result was supported by statistical analysis, confirming there to be a significant difference in the mean scores of the attitude variable result between groups i and ii (table 4) during the post test. this is because the teaching method used with group i was different from that with group ii and affected the mean result for attitudes. based on the description above related to behavior influenced by knowledge and attitude variables, a dynamic balance between such variables promoting both positive dental and oral hygiene habits and results was to be expected. factors potentially affecting the behavior of young slow learners with regard to dental and oral health care can be assigned to one of three categories, namely; predisposing factors, supporting factors and driving factors. predisposing factors consist of triggering behavioral changes motivating the formation of particular behavior, including; knowledge, attitudes, beliefs, and values. supporting factors are ones facilitating individual or group behavior, encompassing three main aspects. these are firstly, the availability, affordability and effectiveness of health care resources, secondly, community priorities and commitments, and thirdly, government and health-related measures. driving factors constitute those which encourage the strengthening of the behavior of community leaders, health workers, teachers, and families.8,18,19 the use of audio video depicting a moving object accompanied by natural or appropriate sound can promote the concentration of the young slow learners during the teaching process. in addition, it can focus their attention on the explanation of material deserving of special emphasis. an audio video method also can stimulate the brain cells of those young slow learners since audio-visualization can attract attention and focus it on explanations, thus resulting in a better understanding of the concept compared dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p66-70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p66-70 70 sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 66–70 to that resulting from a manual method. in conclusion, the provision of awareness-raising material regarding effective forms of tooth brushing to young slow learners via audio video methods can change their knowledge and attitude related to their oral hygiene activity. references 1. deputi bidang perlindungan anak. panduan penanganan anak berkebutuhan khusus bagi pendamping (orang tua, keluarga, dan masyarakat). jakarta: kementerian pemberdayaan perempuan dan perlindungan anak republik indonesia; 2013. p. 1-17. 2. direktorat jenderal bina gizi dan kesehatan ibu anak. pedoman pelayanan kesehatan anak di sekolah luar biasa (slb) : bagi petugas kesehatan. jakarta: kementerian kesehatan republik indonesia; 2011. p. 1-44. 3. idris r. mengatasi kesulitan belajar dengan pendekatan psikologi kognitif. lentera pendidikan. 2009; 12(2): 152–72. 4. sugiarti r, pribadi as. analisis faktor yang mempengaruhi keberhasilan belajar siswa slow learner di sekolah luar biasa (slb) negeri semarang. wacana. 2013; 5(9): 1–17. 5. shaw s, grimes d, bulman j. educating slow learners: are charter schools the last, best hope for their educational success? chart sch resour j. 2005; 1(1): 10–9. 6. khaliq f, anjana y, vaney n. visual evoked potential study in slow learners. indian j physiol pharmacol. 2009; 53(4): 341–6. 7. bernal c. maintenance of oral health in people with learning disabilities. nurs times. 2005; 101(6): 40–2. 8. sabilillah mf, kristiani ak. hubungan oral hygiene dengan keterampilan menggosok gigi pada anak tunanetra. actual res sci acad. 2017; 2(2): 23–8. 9. mubarak wi. promosi kesehatan untuk kebidanan. jakarta: salemba medika; 2011. p. 80-4. 10. heikkilä a, lonka k. studying in higher education: students’ approaches to learning, self-regulation, and cognitive strategies. stud high educ. 2006; 31(1): 99–117. 11. notoatmodjo s. pendidikan dan perilaku kesehatan. jakarta: rineka cipta; 2003. p. 15-49. 12. golden sd, earp jal. social ecological approaches to individuals and their contexts: twenty years of health education & behavior health promotion interventions. heal educ behav. 2012; 39(3): 364–72. 13. reiser ra. a history of instructional design and technology: part i: a history of instructional media. educ technol res dev. 2001; 49(1): 53–64. 14. farrell eh, whistance rn, phillips k, morgan b, savage k, lewis v, kelly m, blazeby jm, kinnersley p, edwards a. systematic review and meta-analysis of audio-visual information aids for informed consent for invasive healthcare procedures in clinical practice. patient educ couns. 2014; 94(1): 20–32. 15. sallam am, badr sby, rashed ma. effectiveness of audiovisual modeling on the behavioral change toward oral and dental care in children with autism. indian j dent. 2013; 4(4): 184–90. 16. sandelowski m. whatever happened to qualitative description? res nurs health. 2000; 23(4): 334–40. 17. maher ca, lewis lk, ferrar k, marshall s, de bourdeaudhuij i, vandelanotte c. are health behavior change interventions that use online social networks effective? a systematic review. j med internet res. 2014; 16(2): e40. 18. sabilillah mf, taftazani rz, sopianah y, fatmasari d. pengaruh dental braille education (dbe) terhadap oral hygiene pada anak tunanetra. j kesehatan gigi. 2016; 3(2): 7–13. 19. sopianah y. hubungan mengunyah unilateral dengan status kebersihan gigi dan mulut pada mahasiswa tingkat i jurusan keperawatan gigi. j kesehatan bakti tunas husada. 2017; 17(1): 176–82. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p66-70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p66-70 5151 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 51–56 research report protection against periodontal destruction in diabetic condition with sardinella longiceps fish oil: expression of matrixmetalloproteinase 8 and tissue inhibitor of metalloproteinase 1 dian widya damaiyanti, dian mulawarmanti, and kristanti parisihni department of oral biology faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: there is strong evidence to support the claim that periodontitis may be more prevalent among diabetic individuals. collagen degradation represents one of the key events in periodontal destructive lesions. the level of matrix metalloproteinase 8 (mmp-8) and tissue inhibitor of metalloproteinase 1 (timp-1) are key to periodontal collagenolysis and associated with the severity of periodontal inflammation and disease. host modulatory therapy has been proposed as a treatment for periodontal diseases. sardinella longiceps (lemuru) fish oil containing polyunsaturated fatty acids (pufas), including omega 3 and 6, has been shown to possess therapeutic anti-inflammatory and protective properties effective against inflammatory diseases, including periodontitis. purpose: the study aimed to examine the effect of dietary supplementation of sardinella longiceps fish oil on protection against periodontal destruction resulting from the expression of mmp-8 and timp-1. methods: wistar rat samples are divided into four groups: a negative control group and three groups receiving sardinella longiceps fish oil treatment (4 ml/ weight (kg), 8 ml/ weight (kg) and 16 ml/ weight (kg). one week before treatment, all groups were administered with streptozotocin (stz) 65 ml/ weight (kg) and nicotinamide 110 ml/ weight (kg) to induce diabetic conditions. immunohistochemistry slides of periodontal tissues were prepared after three weeks of treatment. the expression of mmp-8 and timp-1 was counted using the hscore index, data was analyzed by means of non-parametric methods using kruskal-wallis, and mann-whitney tests. results: statistical analyses confirmed a significant increase in mmp-8 expression and a reduction in timp-1 expression in the negative control group compared to the treatment group (p<0.05). meanwhile, the treatment group showed a significant reduction in mmp-8 expression and a marked increase in timp-1 expression, with the best result produced by the administering of 16 ml/ weight (kg) sardinella longiceps fish oil to the treatment group (p<0.05). conclusion: dietary supplementation of sardinella longiceps fish oil can protect against periodontal destruction under diabetic conditions, by decreasing mmp-8 expression and increasing timp-1 expression. keywords: diabetes; lemuru; mmp-8 expression; periodontitis; timp-1 expression correspondence: dian widya damaiyanti, departement of oral biology, faculty of dentistry universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: damaiyanti@hangtuah.ac.id introduction diabetes mellitus (dm) is a metabolic disorder characterized by hyperglycemia, a high blood glucose level. from an epidemiological perspective, it is estimated that the prevalence of dm in indonesia will reach 21.3 million people by 2030.1 hyperglycemia will trigger an increase in prostaglandin e2 (pge2) and cytokine expression through activation of diacyl glycerol (dag)-proteinkinase c (pkc). this increase in inflammatory mediators will, in turn, initiate a process of periodontal tissue destruction.2 clinical studies have confirmed greater prevalence of periodontitis in patients with diabetes mellitus. fibroblast cells do not function properly in environments featuring a high concentration of glucose. in addition, collagen produced by fibroblasts is susceptible to rapid degradation by matrix metalloproteinase (mmp) enzymes, the production of which is increased in diabetics rendering them susceptible dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 52 damaiyanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 51–56 to more rapid loss of attachment and bone damage.3 the main mmps found in diabetics include mmp-8 and mmp9.4 mmp-8 (collagenase 2) is one of the main biomarkers of connective tissue destruction in periodontitis.4,5 the release of mmp-8 will be offset by that of the tissue inhibitor of metalloproteinase (timp). periodontal damage will be affected by the ratio of mmp-8 to timp-1, the latter of which is produced by endothelial cells, and macrophages as protection against mmps. an imbalance between mmps and timp will lead to destruction during periodontitis pathology.5,6 an in vitro study shows that the monocyte cells of diabetics experience an excessive response with increased expression of inflammatory mediators such as il-1, tnfα and pge2.7 inflammatory mediators such as il-1β, and tnfα increased regulation of mmps expression resulting in periodontal collagen degradation.8 sardinella longiceps fish oil, obtained from fish canning industry waste, contains polyunsaturated fatty acids (pufas) such as eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). the epa and dha content of sardinella longiceps fish oil is one of the highest.9 omega 3 contains several metabolites, resolvin and two-component protectins among others, that demonstrate the ability to reduce the production of pro-inflammatory enzymes and cytokine including cox2, tnfα and il-β.10 administering omega-3 and omega-6 fatty acids together to research models whose feet had sustained injuries after diabetes induction resulted in more rapid wound healing. it seems that a combination of omega-3 and omega-6 can control cell diffusion and inflammation under diabetic conditions, the mechanism probably one of altering the fibroplastic or maturational phases of the healing response.11 based on the findings of research into omega 3 (epa & dha) diets in cases of periodontitis, it is known that omega 3 can reduce mmp, thereby inhibiting osteoclast activity.12 this result was also produced by research into the apoptosis of osteoblasts in alveolar bone. the research involving the administering of a 1 ml/300 gram dose of sardinella longiceps fish oil to rats indicated significantly smaller apoptosis osteoblasts in alveolar bone than those induced by lipopolysaccharide (lps).13 provision of omega 3 for two weeks in the periodontitis model produced an increase in timp-1 levels.14 research involving the administering of omega 3 and omega 6 derived from toman fish oil at doses of 4 ml, 8 ml and 16 ml/ weight (kg) once a day for seven days produced significant wound healing results. this was due to the ability of omega 3 and omega 6 to act as immunomodulators in controlling inflammation. based on the foregoing information, the researchers wanted to identify the effect of a diet containing sardinella longiceps fish oil extract at doses of 4 ml, 8 ml, and 16 ml/ weight on the expression of mmp-8 and timp-1 in the periodontal tissue of wistar rats as markers of periodontal destruction induced by diabetes mellitus. materials and methods all experiments were approved by the ethics committee faculty of dentistry, universitas hang tuah with number certificate ec/013/kepk-fkguht/viii/2019 and performed following the guidelines. experimental diabetes mellitus is produced by injecting a single dose of streptozotocin (sigma aldrich, singapore). forty male wistar rats were injected with approximately 110 ml/ weight (kg) of intra peritoneal nicotinamide (merck, germany) dissolved in phosphate buffer saline (pbs) (merck certipur, germany). 15 minutes later a single intraperitoneal dose 65 mg/ weight (kg) of stz was injected into wistar rats that had fasted for between 8 and 12 hours overnight. after seven days of induction with stz, the rats that presented an increased blood glucose level in excess of 126 mg/ ml were considered to be diabetic.15,16 after the induction of diabetes, the wistar rats were divided into four groups, namely: k0 diabetes induction, administered with sardinella longiceps fish oil treatment; k1 diabetes induction, administered with sardinella longiceps fish oil treatment 4 ml/ weight (kg); k2 induction of diabetes, administered with sardinella longiceps fish oil treatment 8 ml/ weight (kg) and k3 diabetes induction, administered with sardinella longiceps fish oil treatment 16 ml/ weight (kg). treatment with sardinella longiceps fish oil involved oral administration once a day for a period of three weeks. both the control rats and their medicated counterparts were sacrificed after three weeks’ administration of sardinella longiceps fish oil or one month post-diabetes induction with stz. the rats were inserted in a glass tube and administered a lethal dose of ether for three minutes. following removal of their jaws, which were subsequently immersed in a fixing solution, the rats were interred. the specimen processing technique was continued using a paraffin method, immunohistochemistry staining techniques with monoclonal primary antibodies, mmp-8 and timp-1 and secondary antibodies that had been biotinated.17 calculation of mmp-8 and timp-1 expressions using a modification of hscore technique was conducted with an olympus cx-21 microscope using an optilab program at 400x magnification through three visual fields. the intensity of scores was assessed according to category: 0, absent; 1, weak; 2, moderate and 3, intense before being included in the formula hscore = pi (i +1) where i is the intensity score, pi is the percentage of the number of cells stained and 1 represents a correction factor.18 the data was analyzed by means of a nonparametric kruskal-wallis test the data from which was in ordinal form. in cases of difference, a mann-whitney test was subsequently conducted to identify the significance difference. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 53damaiyanti, et al./ dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 51–56 table 1. mmp-8 significance test results between groups group k1 group k2 group k3 mmp-8 timp-1 mmp-8 timp-1 mmp timp-1 negative control group 0.019 0.037 0.02 0.02 0.019 0.02 group k1 0.078 0.02 0.019 0.02 group k2 0.019 0.021 figure 1. mmp-8 expression after a 3-week period of treatment with sardinella longiceps fish oil. figure 2. expression of timp-1 after 3 weeks treatment with sardinella longiceps fish oil. 9.58 5.92 4.67 2.50 0.00 2.00 4.00 6.00 8.00 10.00 12.00 k0 k1 k3 h sc or e experiment group 1.08 2.00 5.75 8.83 0.00 2.00 4.00 6.00 8.00 10.00 12.00 k0 k1 k2 k3 h sc or e experiment group k2 figure 1. mmp-8 expression after a 3-week period of treatment with sardinella longiceps fish oil. figure 1. mmp-8 expression after a 3-week period of treatment with sardinella longiceps fish oil. figure 2. expression of timp-1 after 3 weeks treatment with sardinella longiceps fish oil. 9.58 5.92 4.67 2.50 0.00 2.00 4.00 6.00 8.00 10.00 12.00 k0 k1 k3 h sc or e experiment group 1.08 2.00 5.75 8.83 0.00 2.00 4.00 6.00 8.00 10.00 12.00 k0 k1 k2 k3 h sc or e experiment group k2 figure 2. expression of timp-1 after 3 weeks treatment with sardinella longiceps fish oil. results observation and calculation of mmp-8 and timp-1 expression in the periodontal tissue area were conducted one month after diabetes induction and treatment. the results indicated that the highest median value of mmp-8 occurred in the k0 group compared to the treatment group, while the highest median of timp-1 was observed in the k3 groups compared to the control group. it can be seen that the highest mmp-8 score was recorded in the k0 group and the lowest score in the k3 group (figure 1). the results of the timp-1 calculation show that the highest score occurred in the group k3 (figure 2). based on the contents of table 1, there were significant differences between the control group and groups k1, k2 and k3 as indicated by the value p<0.05 for both the mmp-8 and timp-1 scores. in contrast, with regard to the mannwhitney test, there were no significant differences between mmp-8 scores of the control, k1 and k2 groups where the value of p> 0.05. these results showed that k3 group registered the lowest mmp-8 score expression and the highest timp-1 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 54 damaiyanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 51–56 score compared to the other groups, while the k0 group had the highest mmp-8 score and the lowest timp-1 score (table 1). the expression of mmp-8 and timp-1 was calculated using the color intensity and proportion scores, which were then calculated using the formula. the dark brown ihc staining visible in the picture indicates a positive expression (figure 3 dan 4). discussion systemic conditions have been identified as the cause of an increase in periodontal disease. biological mechanisms known to occur under diabetic conditions include: increased production of advanced glycation end products (ages), hyperinflammatory reactions, poor quality collagen, figure 3. mmp-8 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml/ weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). figure 4. timp-1 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml /weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). figure 3. mmp-8 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml/ weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). figure 3. mmp-8 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml/ weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). figure 4. timp-1 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml /weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). figure 4. timp-1 expression (dark brown, blue arrow) in periodontal ligament at 400x magnification. (a) group k0 (diabetes induction without treatment); (b) group k1 (diabetes induction, sardinella longiceps oil treatment 4 ml/ weight (kg)); (c) group k2 (diabetes induction, sardinella longiceps fish oil treatment 8 ml /weight (kg)); (d) group k3 (diabetes induction, sardinella longiceps fish oil treatment 16 ml/ weight (kg)). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 55damaiyanti, et al./ dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 51–56 changes in microcirculation, host defense dysfunction and increased matrix metalloproteinases. the formation of ages plays an important role in increasing the sensitivity of the formation of inflammatory mediators by endothelial cells and monocytes. the accumulation of ages in the plasma and tissues in diabetic patients is associated with the occurrence of periodontal disease.19,20 the pathology of activation or increase in mmps and the production of timp protection systems by bacteria or by inflammatory mediators is characteristic of chronic periodontitis.5 this characteristic was shown by the stzinduced negative control group for diabetes after one month and subsequently examined using ihc, which resulted in a small increase in mmp-8 and timp-1 production (table 1), collagen degeneration and characteristic of chronic periodontitis. levels of mmps and timp-1 are very important because they can indicate the progression of periodontitis. increased mmp and production of timps are less able to direct periodontal attachment loss.5,14 the administration of sardinella longiceps fish oil was conducted one week post-induction for three weeks, while the research subjects were euthanised one month post-stz induction. the results of this study indicated the smallest mmp-8 expression to have occurred in the 16 ml/ weight (kg) sardinella longiceps fish oil group and demonstrated a significant difference compared to the control group, 4 ml/ weight (kg) sardinella longiceps fish oil group and 8 ml/ weight (kg) sardinella longiceps fish oil group. the decrease in mmp-8 expression was in accordance with the results of timp-1 expression in the 16 ml/ weight (kg) sardinella longiceps fish oil group which experienced a significant increase compared to the control group, 4 ml/ weight (kg) sardinella longiceps fish oil group and 8ml weight (kg) sardinella longiceps oil group. it was concluded that the 16 ml/ weight (kg) lemuru fish oil group had the lowest mmp-8 expression and the highest timp-1 expression. the control group results indicated an increase in mmp-8 without being followed by a rise in timp-1. in theory, this could lead to the degradation of collagen and destruction of periodontal tissue. this study proves the expression of mmp-8 in the negative control group, diabetic rats increased without being offset by the production of mmp-8 inhibitors, namely timp-1, which is limited in scale. increased mmp-8 has been reported to be associated with the development of gingivitis into periodontitis.5 the treatment group showed results that support the theory that a decrease in mmp-8 and an increase in timp-1, due to the administration of sardinella longiceps fish oil containing omega 3, can reduce the production of inflammatory mediators such as pge2. prostaglandin e2 (pge2) causes stimulation of inflammatory mediators and mmps, including osteoclast formation.8 inhibition of pge2 production can, in turn, lead to restrictions on mmp production, while sardinella longiceps oil with polyunsaturated acid (pufa) content can reduce pge2 production.10 the positive effects of omega-6 have also been reported in terms of their ability to affect the production of eicosanoids, pge2, leukotrienes and lipoxins. they also inhibite cyclooxygenase, promote growth hormone secretion from the anterior lobe of the pituitary gland by derived eicosanoids from omega-6 fatty acids. moreover, they prevent a reduction in arachidonic acids in plasma, as well as membrane phospholipids in diabetes, mitogenic effects and an increase in collagen formation.11 sardinella longiceps fish oil contains pufa consisting of omega 3 and 6. omega 3 is proven to regulate various proteins in periodontal tissues such as mmp-8, mmp-13, mmp-14 and timp-1 in the periodontitis-induced lps model.14 in experimental studies of periodontitis in rats, the use of selective cox-2 and prophylactic omega 3 fatty acids, both singly and in combination, caused inhibition of mmp-8 expression in gingival tissue.8 this finding is consistent with the results of the study of the sardinella longiceps oil treatment group which experienced a decrease in mmp-8 expression compared to the negative control group. the largest reduction occurred in the 16 ml/ weight (kg) sardinella longiceps fish oil treatment group. timps that control mmp activity and act as regulators of extracellular matrix destruction by mmp have an important role to play in tissue remodeling and pathology from periodontal tissue destruction. timp levels are generally more elevated in healthy periodontal tissues than those affected by periodontal inflammation, which leads to the excessive production of mmps.8,5 this finding is in accordance with the study of the control group which showed high mmp-8 expression, while that of timp-1 was low. contrastingly, in the treatment group mmp-8 expression decreased and timp expression increased. in conclusion, administering sardinella longiceps fish oil to diabetic rats was found to reduce mmp-8 expression and increase timp-1 expression. the most impressive results occurred in the group which received a 16 ml/ weight (kg) dose of sardinella longiceps fish oil. references 1. budiman, fitriana a. the increase of periodontal tissue in type 2 diabetes mellitus patients based on index cpitn. j kesehat masy. 2016; 12(1): 18–24. 2. santoso o, waspadji s. the effect of non-surgical periodontal therapy on systemic immune response and blood glucose level of niddm patients. med j indones. 2008; 17(1): 20–4. 3. mirza baq, syed a, izhar f, ali khan a. bidirectional relationship between diabetes and periodontal disease: review of evidence. j pak med assoc. 2010; 60(9): 766–8. 4. collin hl, sorsa t, meurman jh, niskanen l, salo t, rönkä h, konttinen yt, koivisto am, uusitupa m. salivary matrix metalloproteinase (mmp-8) levels and gelatinase (mmp-9) activities in patients with type 2 diabetes mellitus. j periodontal res. 2000; 35(5): 259–65. 5. rios mh. identification of collagenolytic mmp networks as potential biomarkers in progressive chronic periodontitis subjects and mmp-8 null allele model. dissertation. helsinki: univerity of helsinki; 2012. p. 9–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 56 damaiyanti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 51–56 6. bıyıkoğlu b, buduneli n, kardeşler l, aksu k, pitkala m, sorsa t. gingival crevicular fluid mmp-8 and -13 and timp-1 levels in patients with rheumatoid arthritis and inflammatory periodontal disease. j periodontol. 2009; 80(8): 1307–14. 7. woodward-lopez g, ritchie ld, gerstein de, crawford pb. obesity: dietary and developmental influences. crc press taylor & francis; 2006. p. 5–31. 8. yucel-lindberg t, båge t. inflammatory mediators in the pathogenesis of periodontitis. expert rev mol med. 2013; 15: 1–22. 9. khoddami a, ariffin aa, bakar j, ghazali hm. quality and fatty acid profile of the oil extracted from fish waste (head, intestine and liver)(euthynnus affinis). african j biotechnol. 2012; 11(7): 1683–9. 10. peeran sw, al-taher ob, el mugrabi mh, grain a, alsaid fm, naveen kumar pg. therapeutic role of dietary omega-3 fatty acids in periodontal disease. univers res j dent. 2014; 4(2): 82–6. 11. jafari naveh hr, taghavi mm, shariati m, vazeirnejad r, rezvani me. both omega-3 and omega-6 polyunsaturated fatty acids stimulate foot wound healing in chronic diabetic rat. african j pharm pharmacol. 2011; 5(14): 1713–7. 12. boeyens jca, deepak v, chua wh, kruger mc, joubert am, coetzee m. effects of ω3and ω6-polyunsaturated fatty acids on rankl-induced osteoclast differentiation of raw264.7 cells: a comparative in vitro study. nutrients. 2014; 6(7): 2584–601. 13. indahyani de. minyak ikan lemuru (sardinella longicep) menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (fish oil of lemuru (sardinella longicep) reduced the osteoblast apoptosis in wistar rat alveolar bone). dent j (majalah kedokt gigi). 2013; 46(4): 185–8. 14. vardar-sengul s, buduneli e, turkoglu o, buduneli n, atilla g, wahlgren j, sorsa t, baylas h. the effects of selective cox-2 inhibitor/celecoxib and omega-3 fatty acid on matrix metalloproteinases, timp-1, and laminin-5γ2chain immunolocalization in experimental periodontitis. j periodontol. 2008; 79(10): 1934–41. 15. srinivasan k, ramarao p. animal models in type 2 research: an overview. indian j med res. 2007; 125(3): 451–72. 16. rosandria dea. pengaruh penggunaan matras elektromagnetik terhadap toleransi glukosa darah tikus putih (rattus novergicus) diabetik tipe ii. thesis. surabaya: universitas airlangga; 2012. p. 17–34. 17. sudiana ik. teknologi ilmu jaringan dan imunohistokimia. sagung seto. jakarta. surabaya: sagung seto; 2004. p. 36–44. 18. balli u, keles gc, cetinkaya bo, mercan u, ayas b, erdogan d. assessment of vascular endothelial growth factor and matrix metalloproteinase-9 in the periodontium of rats treated with atorvastatin. j periodontol. 2013; 85(1): 178–87. 19. botero je, yepes fl, roldán n, castrillón ca, hincapie jp, ochoa sp, ospina ca, becerra ma, jaramillo a, gutierrez sj, contreras a. tooth and periodontal clinical attachment loss are associated with hyperglycemia in patients with diabetes. j periodontol. 2012; 83(10): 1245–50. 20. matthews dc. the relationship between diabetes and periodontal disease. j can dent assoc. 2002; 68(3): 161–4. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p51–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p51-56 204 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 204–208 case report peripheral ossifying fibroma of the anterior maxillary gingiva ganendra anugraha and ni putu mira sumarta department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: peripheral ossifying fibroma is a rejuvenation of the reactive gingiva, usually occurring in the anterior maxillary gingiva. the condition is often clinically ambiguous when diagnosed on the basis of gingival hyperplastic lesions such as focal fibrous hyperplasia, peripheral giant cell granuloma, peripheral fibroma and pyogenic granuloma because peripheral ossifying fibroma has a tendency to recur with a ratio of around 20%. the literature on the subject predominantly classifies peripheral osifying fibroma as an epulis type, but it has also been identified as a peripheral mesenchymal tumor presenting similar clinical symptoms to ossified fibrous epulis. purpose: the purpose of this article is to explain the rare case of peripheral ossifying fibroma in the anterior maxillary gingiva which can be clinically misdiagnosed as reactive gingival hyperplastic lesions. case: a case report of peripheral ossifying fibroma in the left lateral incisor and canine of the maxillary gingiva in a 26 year-old male. the patient chiefly complained of a painless, slow growing gingival enlargement on the upper left jaw during the previous five years. clinical examination confirmed it to be a single, hard swelling in the 21-24 region, pale in color and with a rough surface. case management: the procedure constituted a complete surgical excision of the lesion together with the underlying periosteum curettage intended to prevent recurrence. the histopathologic examination results indicated tissue with squamous epithelial lining, stroma consisting of fibroblasts, and immature trabecula with osteoblastic rimming between collagen tissue without signs of malignancy. osteoblastic rimming has specific features in histopathologic examination of ossifying fibroma. conclusion: peripheral ossifying fibroma is a rare solitary enlargement in the oral cavity frequently misdiagnosed as ossified fibrous epulis. a definitive diagnosis is made by means of histopathologic examination. the condition has a low reccurance rate. keywords: gingival enlargement; gingival overgrowth; peripheral ossifying fibroma correspondence: ganendra anugraha, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: ganendra@fkg.unair.ac.id introduction peripheral ossifying fibroma is a non-encapsulated lesion consisting of fibrous tissue that contains a number of mineral-based materials that transform bones into ossifying fibroma.1 the etiology and pathogenesis of ossifying fibroma is uncertain. a number of researchers assert that ossifying fibroma constitutes a neoplastic process, whereas others argue that this lesion represents a reactive process involving growth from periodontal ligament cells.2 peripheral ossifying fibroma has various names such as peripheral fibroma, cemento ossifying fibroma, peripheral odontogenic fibroma, and calcifying fibroblastic granuloma. it is one of the lesions that presents as a gingival mass, usually emerging from interdental gingiva and, apparently, from the periodontal ligament.3 the pathogenesis of this lesion is uncertain. peripheral ossifying fibroma is considered a reactive process, originating in the periosteal or periodontal membrane. it is also reported that this process constitutes a maturation of pyogenic granuloma or a peripheral giant cell granuloma.4 localized gingival enlargement generally leads to a reactive proliferation lesion rather than a neoplasm.5 reactive or inflammatory lesions represent more than 90% of histopathologically-analyzed gingival biopsies 6 and are generally included in the diagnosis of pyogenic granulomas, fibrous hyperplasia, peripheral ossifying fibromas and peripheral giant cell granulomas. however, certain authors dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p204–208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p204-208 205anugraha, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 204–208 have stated that peripheral ossifying fibroma is a variant of central ossifying fibroma and, therefore, a neoplastic process.2,7 clinically, the peripheral ossifying fibroma appears as a sessile or pedunculated nodular mass, usually less than 2 cm in size but which can occasionally reach 6 cm, reddish to pink in colour and, frequently, with an ulcerated surface.8 in general, this is often found to occur at some point between birth and 20 years of age and predominantly in women. low predilection of the maxilla and the incisive cusp region is also evident. according to the literature, peripheral ossifying fibroma constitutes a rare example in distribution cases of reactive hyperplasia depending on the type of lesion, with only two cases (1%) out of 197 reported in tabriz university’s journal of medical sciences. normal gingival growth is typically observed in the interdental papillae and consists of 9% of gingival growth. this case is more prevalent in adult females than males with the anterior maxillary region being the most frequently affected.9 in most cases, a radiograph confirms no involvement of bone.3 however, in some cases, superficial bone erosion may occur. the lesion can be excised surgically and microscopic examination subsequently performed to confirm the diagnosis. histopathological examination reveals stratified squamous epithelium and connective tissue in large numbers accompanied by fibroblast cell proliferation.6 calcification can be observed in connective tissue in the form of interconnected bone and osteoid trabeculae, that part of soft tissue in central ossifying fibroma.10 this report describes a case of peripheral ossifying fibroma in the maxillary gingiva of a 26-year-old male patient. case a 26 year old male patient consulted the oral and maxillofacial surgery deparment at the dental hospital of universitas airlangga, complaining of having experienced gingival swelling in the upper anterior region. the growth of this lesion was initially limited and it had grown gradually to its present size over the course of 2-3 years. it constituted a hard solitary sessile gingival and non-ulcerated swelling with a rough surface normal in color for a gingiva and nontender on palpation. the swelling, approximately 3x2x1 cm in size, extending from distal 21 tooth to distal 23 tooth, involving attached gingiva and marginal gingiva (figure 1). the teeth in question were free of caries and calculus, while the panoramic radiograph indicated the absence of bone loss around the tooth (figure 2). an expanded periodontal gap between the left central incisor and the lateral incisor was evident from the intraoral periapical radiographic view (figure 3). the patient’s medical and family history was non-contributory and his lifestyle was healthy. figure 1. clinical appearance of gingival swelling with defined border from the 21-23 region with a rough surface and normal color of gingiva. figure 2. panoramic view indicating normal appearance. figure 3. periapical radiograph illustrating a widenend periodontal space between teeth 21 and 22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p204–208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p204-208 206 anugraha, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 204–208 case management following administration of a local anaesthesia (2 ml pehacain), a mucoperisteal flap was created with a blade scalpel no.15 in the gingival margin of teeth 2123. on excision of the bony lesion, including the rough and hardened gingiva, the lesion was found to be well demarcated from healthy bone (figure 4). both the bony and gingival lesions were sent for histopathologic examination. on wound closure, care was taken to maintain the normal gingival architecture of the cervical margin of 21-23 teeth by undermining and recontouring the flap margin which was to be attached to the cervical margin of 21-23 teeth and sutured appropriately with silk 3.0. the diagnosis of peripheral ossifying fibroma was confirmed through histopathologic examination which indicated the presence of parakeratinized stratified squamous epithelium with stroma consisting of fibroblast and immature bone trabeculae. osteoblastic rimming within collagen tissue was also observed which was a distinctive finding in ossifying fibroma (figure 5). no malignancy was evident in the course of this examination. based on these findings, a final diagnosis of peripheral ossifying fibroma was arrived at. the wound healed and an evaluation of the gingiva conducted six months after excision confirmed the presence of normal contours without gingival recession that showed the marginal flap to be perfectly adapted to the cervical margin of 21-23 teeth (figure 6). no recurrence occurred. discussion ossifying fibroma is a benign bone neoplasm with the potential for excessive growth, bone destruction, and recurrence. the etiology and pathogenesis of peripheral ossifying fibroma remains undefined. several researchers claim peripheral ossifying fibroma to be a neoplastic process, while others argue that it is a reactive process. this lesion results from damage to periodontal ligament cells.11,12 the main causative factors in pof consist of chronic irritation and trauma, possibly originating in the subgingival plaque and calculus. in addition, the potential for this lesion to occur can also be related to the use of orthodontic devices as has been shown to be true of 3.8% of adult cases and 7% of pediatic cases.13 inflammatory hyperplasia of the periodontal ligament is a factor causing histogenesis in peripheral ossifying fibroma.8 these clinical results include swelling of the gingiva, the proximity of the gingiva to the parodontal ligament, and the correlation of age distribution of lesions with the number of the lost teeth and their parodontal ligament. it is similar, if not identical, to cementifying fibroma both clinically and microscopically. composed of a stroma of fibrous connective tissue in which new bone is formed, it is known as one of the benign fibroosseous lesions affecting the jaw.14 ossifying fibroma consists of craniofacial bone and is typically divided into the two groups of central and peripheral ossifying fibroma. the central group is formed from the endosteum or periodontal ligament which is associated with the apex and extends from the bone medulla, whereas the peripheral type indicates a relationship with periodontal ligaments which occur in soft tissue. peripheral ossifying fibroma is not a peripheral part of central ossifying fibroma.12 figure 4. intraoperative photograph confirming the lesion to be well demarcated from the healthy bone. figure 5. histopathologic examination (black arrow indicating osteoblastic rimming within collagen tissue). figure 6. postoperative photograph six months after excision showing normal contours of the gingiva without recession indicating that the marginal flap was perfectly adapted to the cervical margin of teeth 21-23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p204–208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p204-208 207anugraha, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 204–208 ossifying fibroma is thought to originate in periodontal membrane tumors resulting from mesenchymal cell blasts, present in the periodontal membrane, which have the ability to produce cementum, alveolar bone and fibrous tissue. based on its character, ossifying fibroma is also classified as falling within the neoplastic disease group with a low recurrence rate in general.15 fibrous connective tissue in the periodontal membrane is composed of collagen fibers, mucopolysaccharides and oxytalan fibers. ossifying fibromas occur in both central and peripheral locations of the jaw bones. histologically, the lesion is composed of varying amounts of mature and immature bony trabeculae, cementum-like tissue, dystrophic calcifications, all in different configurations with varied stromal collagen content and cellularity.11 peripheral ossifying fibroma is a solid, slow-growing mass which can be either sessile or pedunculated. it is most often located in the gingival papilla between the teeth and maxilla, rather than the lower jaw within the anterior region or posteriorly, can occur at any age but particularly during the second decade of life, and predominantly affects women rather than men at a ratio of 3:2. the surface of this ossifying fibroma lesion is either smooth or ulcerated and reddish pink and measures approximately 1.5 cm in diameter, although lesions with a diameter of between 6 and 9 cm have also been reported. the pathogenesis of peripheral ossifying fibroma remains unclear, possibly originating from the periodontal ligament. the majority of peripheral ossifying fibromas occur in the gingiva (interdental papilla, proximal portion of the gingiva to the periodontal ligament, oxytalan fiber with mineralized matrix in certain lesions, age distribution related to the number of permanent teeth lost, and fibrocellular response in the periodontal ligament).3 ossifying fibroma can be caused by local trauma or irritation such as dental plaque, calculus, microorganisms, masticatory force, ill-adjusted dentures or inadequate restoration. the influence of hormones is also likely to be a causative factor in women.2,6 the prevalence of peripheral ossifying fibroma recurrence is between 8 and 20 percent. surgical excision may be the treatment of choice after removing local causative factors such as plaque, calculus, ill-fitting dentures and poor reconstruction. excision involves removing the ligament and periosteum at the base of the lesion in order to reduce the likelihood of recurrence.16 as in this case report, gingival mass suffered by the patient involves the marginal gingiva of 21-21 teeth without any local irritant from dental calculus or remaining sharp tooth root. the peripheral ossifying fibroma has numerous radiographic features. it has been observed that the radiographic calcification foci are dispersed in the central area of the class, but not all lesions exhibit radiographic calcifications. radiographs indicate no presence of bone. superficial bone degradation is found in rare cases. histopathological evaluation of biopsy specimens results in a definite diagnosis of peripheral ossifying fibroma. during microscopic examination, the following characteristics are usually observed: intact or ulcerated stratified squamous surface epithelium; potentially mature mineralized material; epithelial proliferation ranging from sparse to abundant; benign fibrous connective tissue with varying fibroblast content; myofibroblasts and collagen; lamellar or woven osteoid; and cement-like material or dystrophic calcifications; while acute and chronic leak cells are also identified. the results of histopathologic examination in this case produced consistent findings with peripheral ossifying fibroma in the form of parakeratinized stratified squamous epithelium with stroma consisting of fibroblast and immature bone trabeculae. osteoblastic rimming within collagen tissue was also observed. no malignancy was evident during this examination. the characteristic of this pattern occurs only in large lesions that form fibromic irritations or pyogenic granulomas. in most cases, radiology indicates no effect on bones, although their superficial erosion can be detected. the migration and mobility of adjacent teeth can also be observed.17 it can be concluded that peripheral ossifying fibroma in the oral cavity is a rare, discrete swelling that has frequently been clinically diagnosed as ossified fibrous epulis. histopathological research is essential for such lesions to be definitively diagnosed and treated. although it has a low rate of recurrence, peripheral ossifying fibroma will require complete surgical excision, including the underlying periosteum, to prevent recurrence. references 1. shekhar mg, bokhari k. juvenile aggressive ossifying fibroma of the maxilla. j indian soc pedod prev dent. 2009; 27(3): 170–4. 2. liu y, you m, wang h, yang z, miao j, shimizutani k, koseki t. ossifying fibromas of the jaw bone: 20 cases. dentomaxillofacial radiol. 2010; 39(1): 57–63. 3. shah j, sharma s. peripheral ossifying fibroma: an unusual presentation. int j oral heal sci. 2018; 8: 47–50. 4. hosseini fa, moslemi e. central ossifying fibroma, periapical cemento-osseous dysplasia and complex odontoma occurring in the same jaw. clin pract. 2011; 1(2): 67–9. 5. hegde u, nagpal b, archana s, shetty sk, guledgud m v. peripheral ossifying fibroma : reactive or neoplastic lesion ??? int j adv res. 2015; 3(11): 1566–70. 6. chaturvedy v, gupta ak, gupta hl, chaturvedy s. peripheral ossifying fibroma, some rare findings. j indian soc periodontol. 2014; 18(1): 88–91. 7. mishra mb, bhishen ka, mishra s. peripheral ossifying fibroma. j oral maxillofac pathol. 2011; 15(1): 65–8. 8. amberkar vs, mohankumar kp, chawla sk, madhushankari gs. peripheral ossifying fibroma: revisited. int j oral heal sci. 2017; 7: 35–40. 9. kashyap rr, nair gr, gogineni sb. asymptomatic presentation of aggressive ossifying fibroma: a case report. case rep dent. 2011; 2011: 1–4. 10. john rr, kandasamy s, achuthan n. unusually large-sized peripheral ossifying fibroma. ann maxillofac surg. 2016; 6(2): 300–3. 11. khan sa, raj v, sharma nk, sethi t. ossifying fibroma of maxilla in a male child: report of a case and review of the literature. natl j maxillofac surg. 2011; 2: 73–9. 12. sathyabama v, saravanan c, sharma ss, kamal kanthan r. ossifying fibroma of maxilla: a case report with review of literature. indian j multidiscip dent. 2011; 7(1): 293–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p204–208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p204-208 208 anugraha, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 204–208 13. arunachalam m, saravanan t, shakila kr, anisa n. peripheral ossifying fibroma: a case report and brief review. j res dent sci. 2017; 8: 41–5. 14. worawongvasu r, songkampol k. fibro-osseous lesions of the jaws: an analysis of 122 cases in thailand. j oral pathol med. 2010; 39(9): 703–8. 15. lawson sla, medji s, atigossou d, bio-tchane i, kpemissi e, amaglo ks. ossifying fibroma of the maxillary sinus at the kara (togo) teaching hospital. eur ann otorhinolaryngol head neck dis. 2010; 127(6): 217–20. 16. suramya s, gujjari sk, sreeshyla hs. peripheral ossifying consequence fibroma in pregnancy: case report multifactorial. int j med dent sci. 2014; 3(2): 518–23. 17. raffi rm, shubha c, sujatha gp, ashok l. peripheral ossifying fibroma – a case report. int dent j student’s res. 2019; 7(2): 43–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p204–208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p204-208 �� volume 47, number 1, march 2014 uji sensitivitas dan spesifisitas perangkat lunak “prediktor karies anak” (the sensitivity and specificity test of software for dental caries prediction in children) quroti a’yun,1 julita hendrartini,2 al. supartinah santoso,3 dan lukito edi nugroho4 1poltekkes negeri kemenkeskemenkes 2departemen ilmu kedokteran gigi masyarakat, fakultas kedokteran gigi universitas gadjah mada 3departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas gadjah mada 4 jurusan teknik elektro dan teknologi informasi, fakultas teknik universitas gadjah mada yogyakarta indonesiata indonesia abstract background: the prevalence of dental caries in children is high, therefore preventive actions is needed. so far the computertherefore preventive actions is needed. so far the computerthe computer software that have been used for caries predictor is cariogram, which determine the condition of teeth and oral mouth. recently cariogram, which determine the condition of teeth and oral mouth. recentlycariogram, which determine the condition of teeth and oral mouth. recentlyteeth and oral mouth. recentlyoral mouth. recently “prediktor karies anak” (pediatric caries predictor) software have been developed not only determine the condition of teeth and software have been developed not only determine the condition of teeth andve been developed not only determine the condition of teeth andteeth and oral mouth but also child’s behavior, maternal behavior, and the environment.child’s behavior, maternal behavior, and the environment. purpose: the objective of this study was to examinehe objective of this study was to examine the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of �prediktor karies anak� a softwareof �prediktor karies anak� a software for dental caries prediction in children. methods: this study study was an observational study with cross-sectional plan, carried out on 67 primary school children aged 10-12 years. the research instrument was software of “prediktor karies anak” (pediatric cariessoftware of “prediktor karies anak” (pediatric caries predictor) and cariogram. the data of this research was the percentage of new caries occurrence and caries risk categorized intowas the percentage of new caries occurrence and caries risk categorized intointo high and low, and analyzed with a 2 x 2 table. results:s: the data of 67 children was analyzed using “prediktor karies anak”softwaresoftware and revealed 38 children had low caries risk and 29 children had high caries. the data then re-analyzed using cariogram software had low caries risk and 29 children had high caries. the data then re-analyzed using cariogram softwaredata then re-analyzed using cariogram software showed that 37 children had low caries risk, and 30 children had high caries risk. sensitivity of “prediktor karies anak” software wased that 37 children had low caries risk, and 30 children had high caries risk. sensitivity of “prediktor karies anak” software was“prediktor karies anak” software waswas 87%, specificity was 92%, the ppv was 90%, and npv was 89%. conclusion: “prediktor karies anak”software had high sensitivity,“prediktor karies anak”software had high sensitivity,had high sensitivity, specificity, ppv, and npv and could be used as an instrument to predict new caries on children.could be used as an instrument to predict new caries on children.new caries on children. key words: words: sensitivity, specificity, pediatric caries predicator, cariogram, computer software abstrak latar belakang:belakang:elakang: prevalensi karies pada anak tergolong tinggi, sehingga perlu dilakukan tindakan pencegahan. selama inirevalensi karies pada anak tergolong tinggi, sehingga perlu dilakukan tindakan pencegahan. selama ini perangkat lunak komputer yang digunakan untuk memprediksi karies gigi adalah cariogram, yang mengukur faktor keadaan gigicariogram, yang mengukur faktor keadaan gigi dan mulut. baru-baru ini telah dikembangkan perangkat lunak �prediktor karies anak� yang tidak hanya mengukur kondisi gigi danbaru-baru ini telah dikembangkan perangkat lunak �prediktor karies anak� yang tidak hanya mengukur kondisi gigi dan rongga mulut tetapi juga perilaku anak, perilaku ibu, dan lingkungan. tujuan: tujuan penelitian ini adalah untuk menguji sensitivitas,menguji sensitivitas, spesifisitas, nilai duga positif (ndp) dan nilai duga negatif (ndn) perangkat lunak �prediktor karies anak� suatu perangkat lunaknilai duga positif (ndp) dan nilai duga negatif (ndn) perangkat lunak �prediktor karies anak� suatu perangkat lunak duga negatif (ndn) perangkat lunak �prediktor karies anak� suatu perangkat lunak (ndn) perangkat lunak �prediktor karies anak� suatu perangkat lunak�prediktor karies anak� suatu perangkat lunak untuk memprediksi karies gigi pada anak. metode: jenis penelitian ini adalah observasional dengan rancangan cross sectional, yang dilakukan pada 67 anak sekolah dasar usia 10-12 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� dan67 anak sekolah dasar usia 10-12 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� dan anak sekolah dasar usia 10-12 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� dansekolah dasar usia 10-12 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� danusia 10-12 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� dan2 tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� dan tahun. instrumen penelitian adalah perangkat lunak �prediktor karies anak� danperangkat lunak �prediktor karies anak� dan�prediktor karies anak� dan cariogram. data penelitian ini berupa persentase terjadinya karies baru yang dikategorikan resiko karies tinggi dan rendah, danpersentase terjadinya karies baru yang dikategorikan resiko karies tinggi dan rendah, dan yang dikategorikan resiko karies tinggi dan rendah, dandikategorikan resiko karies tinggi dan rendah, danegorikan resiko karies tinggi dan rendah, dangorikan resiko karies tinggi dan rendah, danresiko karies tinggi dan rendah, dan dianalisis dengan tabel 2 x 2. hasil: data dari 67 anak yang dianalisis dengan menggunakan perangkat lunak “prediktor karies anak” menunjukkan bahwa 38 anak memiliki risiko karies rendah dan 29 anak-anak memiliki resiko karies tinggi. data tersebut dianalisa kembali dengan perangkat lunak cariogram, dan hasilnya menunjukkan bahwa 37 anak mempunyai resiko karies rendah, dan 30 anak37 anak mempunyai resiko karies rendah, dan 30 anak research report �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 45–51 mempunyai resiko karies tinggi. sensitivitas “prediktor karies anak” 87%, spesifisitas 92%, ndp 90% dan ndn 89%.ensitivitas “prediktor karies anak” 87%, spesifisitas 92%, ndp 90% dan ndn 89%.7%, spesifisitas 92%, ndp 90% dan ndn 89%.%, spesifisitas 92%, ndp 90% dan ndn 89%.2%, ndp 90% dan ndn 89%.%, ndp 90% dan ndn 89%.0% dan ndn 89%.% dan ndn 89%.9%.%. simpulan: “prediktor karies anak” mempunyai sensitivitas, spesifitas, ndp dan ndn yang tinggi dan dapat dipakai sebagai instrumen untuk memprediksi terjadinya karies baru pada anak.terjadinya karies baru pada anak.karies baru pada anak. baru pada anak.anak. kata kunci: sensitivitas, spesifisitas, prediktor karies anak, cariogram, perangkat lunak komputer korespondensi (correspondence): quroti a’yun, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara, yogyakarta 55281, indonesia. e-mail: ayunquroti@yahoo.com pendahuluan karies gigi banyak terjadi pada anak-anak dan kebanyakan tidak dilakukan perawatan sehingga berdampak terjadinya gangguan pengunyahan dan mempengaruhidan mempengaruhi pertumbuhan dan perkembangan anak.1 karies gigi merupakan penyakit multi faktorial yang disebabkan oleh faktor langsung dan tidak langsung. faktor langsung, yaitu keadaan gigi dan mulut, antara lain; bakteri, turunnya resistensi gigi dan lingkungan, diet karbohidrat, dan memerlukan waktu untuk dapat terjadinya karies.2,3 faktor tidak langsung yang berperan terjadinya karies pada anak adalah faktor anak, keluarga dan pengaruh lingkungan. faktor anak antara lain meliputi perilaku dan pemanfaatan pelayanan kesehatan gigi dan mulut.4 pengalaman karies pada masa lampau merupakan salah satu faktor karies dan diukur berdasarkan indeks dmf-t/dmf-t.2 faktor keluarga antara lain perilaku ibu dalam memelihara kesehatan gigi mulut anak dan menyediakan makanan untuk anaknya.5 salah satu faktor lingkungan anak adalah sekolah, oleh karena itu program usaha kesehatan gigi sekolah (ukgs)kesehatan gigi sekolah (ukgs)gigi sekolah (ukgs) berperan penting dalam meningkatkan kesehatan gigi dan mulut anak.6,7 pengukuran resiko terjadinya karies perlu dilakukan untuk menurunkan prevalensi karies gigi pada anak yang tinggi untuk program perencanaan kesehatan dan pengawasan penyakit gigi dan mulut.8 pengukuran resiko karies ditujukan untuk pencegahan keparahan dan terjadinya karies baru.9,10 seiring dengan perkembangan teknologi yang semakin pesat, maka untuk memprediksi resiko terjadinya karies pada masa mendatang memerlukan alat bantu dengan program berbasis komputer.rogram berbasis komputer. cariogram adalah salah satu instrumen untuk memprediksi resikosalah satu instrumen untuk memprediksi resiko terjadinya karies baru yang mengukur 9 faktor resiko yaitu; pengalaman karies, penyakit yang berpengaruh, kandungan makanan, frekuensi makan, banyaknya plak, program fluor, sekresi saliva, kapasitas buffer dan penilaian klinik.2 faktor perilaku anak, orang tua dan lingkungan sekolah anak, orang tua dan lingkungan sekolah diperkirakan memiliki peran penting dalam memperkirakan terjadinya karies pada anak. cariogram belum memasukkan faktor-faktor tersebut sehingga dilakukan penyusunan alatyusunan alat baru yang dapat memprediksi terjadinya karies pada anak dengan melibatkan faktor tersebut. studi sebelumnya telah berhasil menyusun perangkaterangkat lunak “prediktor karies anak” berdasarkan hasil penelitian“prediktor karies anak” berdasarkan hasil penelitianberdasarkan hasil penelitian pada 430 anak sd usia 10-12 tahun di wilayah daerah istimewa yogyakarta. hasil penelitian sebelumnyasebelumnya menunjukkan bahwa dari 11 faktor resiko karies anak diperoleh 9 faktor resiko yang bermakna, yaitu: ph saliva; banyaknya plak; pengalaman karies; pemanfaatan; banyaknya plak; pengalaman karies; pemanfaatan pelayanan kesehatan; perilaku ibu dalam memilih makanan anak; pengetahuan anak tentang kesehatan gigi; perilaku anak dalam pemeliharaan kesehatan gigi; perilaku anak dalam kebiasaan makan anak dan usaha kesehatan gigi sekolah (ukgs).11 sebelum dipergunakan sebagai salah satu alat untuk memprediksi resiko karies baru, prediktor baru, prediktor karies anak perlu dilakukan uji diagnostik yang meliputi; sensitivitas, spesifisitas, nilai duga positif (ndp) dan nilai duga negatif (ndn). uji ini menggunakan perangkat lunak cariogramariogram sebagai gold standar. sensitivitas adalah kemampuan alat mendeteksi subjek yang sakit dan spesifisitas adalah kemampuan alat untuk mendeteksifisitas adalah kemampuan alat untuk mendeteksi subyek yang tidak sakit. ndp adalah probabilitas seseorang menderita penyakit bila hasil uji diagnostinya positif dan ndn adalah probabilitas seseorang tidak menderita penyakit bila hasil uji diagnostiknya negatif.12,132,13 penelitianenelitian ini bertujuan menguji sensitivitas, spesifisitas, nilai dugabertujuan menguji sensitivitas, spesifisitas, nilai duganilai duga positif (ndp) dan nilai duga negatif (ndn) perangkat lunak "prediktor karies anak". bahan dan metode desain penelitian yang digunakan adalah observasional dengan rancangan cross sectional. populasi penelitian adalah anak sekolah dasar dan orangtuanya di sd negeri sekolah dasar dan orangtuanya di sd negeri godean i, di wilayah kabupaten sleman, propinsi daerah istimewa yogyakarta. sampel sebanyak 67 anak yang diambil secara purposive sampling, yaitu anak usia 10-12 tahun setelah mendapat ethical cleareance dan informed consent dari orang tuanya. setiap anak mendapatkan 2 kali pengukuran resiko terjadinya karies, yaitu pengukuran pertama denganpertama dengan dengan perangkat lunak “prediktor karies anak” yang disusun“prediktor karies anak” yang disusunyang disusun oleh peneliti, dan pengukuran kedua dengan perangkatkedua dengan perangkat dengan perangkat lunak cariogram.ariogram. variabel bebas adalah resiko terjadinya karies, variabel tak terkendali adalah struktur dan posisi gigi geligi, kandungan fluor air minum, tingkat pendidikan ibu dan tingkat sosial ekonomi keluarga. variabel terkendali adalah status gizi baik, tidak memiliki penyakit sistemik, dan telah menerima program ukgs tahap i. faktor resiko karies yang diukur dalam perangkat lunak “prediktor karies anak” terdiri atas 9 faktor yaitu:“prediktor karies anak” terdiri atas 9 faktor yaitu: ph saliva diukur dengan ph meter; banyaknya plak; banyaknya plak banyaknya plak ��a’yun, et al.: uji sensitivitas dan spesifisitas perangkat lunak “prediktor karies anak” diukur berdasarkan indeks phpm (marten & meskin); meskin);meskin);; pengalaman karies, diukur berdasarkan indeks def-t/ dmf-t; pemanfaatan pelayanan kesehatan; perilaku; pemanfaatan pelayanan kesehatan; perilaku pemanfaatan pelayanan kesehatan; perilaku; perilaku perilaku orang tua dalam memilih makanan untuk anaknya dengan;; pengetahuan anak tentang kesehatan gigi dan mulut;; perilaku anak dalam memelihara kesehatan gigi; perilakuperilaku anak dalam kebiasaan makan dan pelaksanaan ukgs oleh guru. ada 9 faktor resiko karies yang diukur dalam cariogram, yaitu: pengalaman karies berdasarkan: pengalaman karies berdasarkanpengalaman karies berdasarkan indeks def-t/ dmf-t; penyakit umum yang berhubungan; penyakit umum yang berhubunganpenyakit umum yang berhubungan dengan penyakit gigi; frekuensi makan makanan manis;; frekuensi makan makanan manis; banyaknya plak menggunakan indeks silness-loe; jumlah; jumlahjumlah streptococcus mutans menggunakan mucount; program; program fluor; sekresi saliva; kapasitas salivasekresi saliva; kapasitas saliva; kapasitas saliva buffer dan penilaianpenilaian klinik (gambar 1). hasil yang diperoleh berupa persentase prediksi terjadinya karies baru, dan dikelompokkan dalam resiko karies tinggi dan rendah. hasil pengukuran dengan “prediktor karies anak” dikatakan resiko karies tinggi jikaprediktor karies anak” dikatakan resiko karies tinggi jika” dikatakan resiko karies tinggi jika dikatakan resiko karies tinggi jika persentase terjadinya karies baru 55-98,9%, dan rendah, jika persentase terjadinya karies baru 8,8254%.11 hasil pengukuran dengan cariogram dikatakan resiko karies tinggi jika peluang gigi sehat 0-20% dan resiko karies rendah jika peluang gigi sehat 21-100%.14 sensitivitas adalah hasil perbandingan positif benar terhadap positif benar + negatif semu. spesifisitas hasil perbandingan antara negatif benar terhadap positif semu+ negatif benar. ndp adalah perbandingan antara positif benar terhadap posif benar + positif semu dan ndn adalah perbandingan antara negatif benar terhadap negatif benar+ negatif semu.12,13 penghitungan menggunakan tabel 2 x 2 seperti ditunjukkan pada tabel 1. hasil distribusi sebagian besar responden adalah anakistribusi sebagian besar responden adalah anak perempuan (53,73%), tingkat pendidikan ibu adalah smp (52,24%), dan keluarga mempunyai pendapatan yang rendah (52,24) (tabel 2). hasil penelitian dengan menggunakan (tabel 2). hasil penelitian dengan menggunakan. hasil penelitian dengan menggunakan perangkat lunak prediktor karies anak menunjukkanmenunjukkan bahwa, 100% anak mempunyai ph saliva tinggi, 73%tinggi, 73% anak menunjukkan banyaknya plak yang rendah, dan 57% anak mempunyai pengalaman karies yang rendah (tabeltabel 3). sebanyak 84% ibu dalam memanfaatkan pelayanan4% ibu dalam memanfaatkan pelayanan kesehatan gigi untuk anaknya tergolong rendah dan 87% ibu dalam memilih makanan untuk anaknya tergolong buruk. sebanyak 70% anak mempunyai pengetahuan tentang kesehatan gigi dan mulut yang baik dan perilaku anak dalam pemeliharaan kesehatan gigi dan kebiasaan makan tergolong baik, yaitu 70%, dan 87%. semua anak berpendapat bahwa ukgs yang dilaksanakan oleh guru masih tergolong kurang. hasil pengukuran resiko karies dengan perangkat lunak prediktor karies anak, diperoleh 38 anak mempunyai resiko karies rendah dan 29 mempunyai resiko karies tinggi (tabel 4). hasil pengukuran dengan perangkat lunak cariogram menunjukkan 37 anak mempunyai resiko karies rendah, dan 37 mempunyai resiko karies tinggi. sensitivitas perangkat lunak prediktor karies anak yaitu 26/30 x 100% = 87%, sedangkan spesifisitas diperoleh 34/37 x 100% = 92%. hasil pengukuran ndp adalah 26/29 x 100% = 90%, dan ndn diperoleh 34/38 x 100% = 89%. pembahasan hasil pengukuran resiko terjadinya karies pada anak menggunakan prediktor karies anak memperoleh resikomemperoleh resiko karies yang rendah, dikarenakan sebagian besar faktor resiko hasil pengukuran dengan prediktor karies anak tabel 11. rumus penghitungan sensitivitas, spesifisitas, ndp dan ndn hasih hasil teshas hasil uji cariogram resiko tinggi resiko rendah jumlah prediktor karies anak resiko tinggi a b a + b resiko rendah c d c + d jumlah a + c b + d a+b+c+d keterangan: “ a” adalah subjek yang diprediksi resiko karies tinggi oleh prediktor karies anak dan cariogram (positif benar); “b” adalah subjek yang diprediksi resiko karies tinggi oleh prediktor karies anak dan resiko karies rendah oleh cariogram (positif semu); “c” adalah subjek yang diprediksi resiko karies rendah oleh prediktor karies anak dan resiko karies tinggi oleh cariogram (negatif semu); “d” adalah subjek yang diprediksi resiko karies rendah oleh prediktor karies anak dan cariogram (negatif benar). tabel 22. deskripsi karakteristik anak dan ibu karakteristik kriteria frekuensi n (%) jenis kelamin anak pendidikan ibu tingkat ekonomi laki-laki perempuan sd smp smu pt rendah menengah tinggi 31 35 10 35 13 10 35 19 13 46,27 53,73 14,93 52,24 17,90 14,93 52,24 28,36 19,4019,40 �8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 45–51 tabel 3. distribusi faktor resiko karies hasil pengukuran dengan prediktor karies anak faktor resiko batas katagori n % ph saliva banyaknya plak (phpm) pengalaman karies pemanfaatan pelayanan kesehatan perilaku ibu dalam memilih makanan pengetahuan anakk perilaku anak dalam pemeliharaan kesehatan gigi dan mulut gigi dan mulutgigi dan mulut perilaku anak dalam kebiasaan makan anak ukgs 0: > 6,5 1: < 6,5< 6,55 0: 0-15 1:16-3 0: 01: >3 0: >2 1: 0-1 0: 16-21 1: 0-15 0: 6 1: 1-5 0: 7-12 1: 0-6 0 : 9-15 1: 0-8 0: 3 1:0-2 67 0 49 18 38 29 11 56 9 58 47 20 47 20 58 9 0 67 100 0 73 17 57 43 17 84 13 87 70 30 70 30 87 13 0 100 keterangan : 0 : resiko rendah;1 : risko tinggi tabel 44. penghitungan sensitivitas, spesifisitas, ndp dan ndn hasih hasil teshas hasil uji cariogram resiko tinggi resiko rendah jumlah prediktor karies anak resiko tinggi 26 3 29 resiko rendah 4 34 38 jumlah 30 37 67 gambar 1. contoh hasil pengukuran prediksi karies baru dengan cariogram.2 tergolong rendah. hasil pengukuran tersebut menunjukkan semua anak mempunyai ph saliva yang tinggi, sebagian besar banyaknya plak dan pengalaman karies tergolong rendah. derajat keasaman (ph) saliva pada anak bersifatt keasaman (ph) saliva pada anak bersifat keasaman (ph) saliva pada anak bersifat basa, dikarenakan sekresi saliva pada anak yang cukup tinggi, sehingga berdampak pada volume saliva yang tinggi pula.3 karies terjadi jika terdapat plak pada permukaan gigi dan apabila tidak segera dibersihkan dan berkontak dengan bakteri dapat mengakibatkan penurunan ph plak di bawah 5,5. penurunan ph yang berulangkali memungkinkan ��a’yun, et al.: uji sensitivitas dan spesifisitas perangkat lunak “prediktor karies anak” terjadinya demineralisasi lapisan gigi, yang merupakan awal terjadinya karies.8,15 anak yang mempunyai pengalaman karies yang tinggi, pada usia berikutnya akan menderita karies yang tinggi pula, jika perilaku terhadap kesehatan giginya tidak, jika perilaku terhadap kesehatan giginya tidak dilakukan perubahan.16 keadaan tersebut dikarenakan gigi yang mengalami karies terdapat lebih banyak bakteri, sehingga akan menghasilkan asam lebih banyak. penurunan ph plak terjadi lebih besar dibanding dengan gigi yang tidak mengalami karies.17 pada penelitian ini pengalaman karies anak tergolong rendah, sehingga pengukuran dengan prediktor karies anak menghasilkan prediksi terjadinya karies adalah rendah. faktor resiko pengalaman karies padafaktor resiko pengalaman karies padaaktor resiko pengalaman karies pada prediktor karies anak mempunyaianak mempunyaimempunyai prevalence odds ratio (por) yang tinggi, yaitu 4,048, sehingga dapat dikatakan bahwa anak yang mempunyai pengalaman karies yang tinggi, akan mempunyai resiko 4 kali lebih tinggi terjadi karies, dibanding anak dengan pengalaman karies yang rendah.11 perilaku ibu dalam memanfaatkan pelayanan kesehatan gigi dan memilih makanan untuk anaknya termasuk dalam memilih makanan untuk anaknya termasuk dalam kriteria buruk. pada penelitian ini, pemanfaatan pelayanan kesehatan gigi masih buruk (<2 kali/tahun), dikarenakan sebagian besar anak mempunyai pengalaman karies yang rendah dan tidak ada keluhan sakit gigi.11 anak yang mempunyai pengalaman karies yang rendah jarang mengeluh sakit gigi.18 anak dan orang tua akan termotivasi untuk memeriksakan kesehatan gigi, jika anak merasa terganggu dengan kesehatan gigi dan mulutnya.19 dokter gigi selalu menekankan pentingnya memelihara kesehatan gigi pada anak yang sering melakukan kontrol ke dokter gigi, sehingga akan berdampak pada oral hygiene anak yang baik pula.20 selain hal tersebut, pemanfaatan pelayanan kesehatan gigi, juga dipengaruhi oleh tingkat pendidikan dan keadaan sosial ekonomi.17 pada penelitian ini sebagian besar ibu, mempunyaiagian besar ibu, mempunyai pendidikan tergolong rendah, yaitu sd dan smp. pendidikan ibu yang rendah akan mempengaruhi kemampuan orangtua dalam mengakses informasi kesehatan dan pemahaman terhadap manfaat perawatan kesehatan gigi dan mulut bagi anak. hal tersebut akan menimbulkan motivasi dan sikap yang kurang baik terhadap perawatan kesehatan gigi dan mulut anak.21 ibu yang tidak peduli terhadap kesehatan gigi, tidak akan memperhatikan kesehatan gigi anaknya,tidak akan memperhatikan kesehatan gigi anaknya, sehingga status kesehatan gigi dan mulut anak akan buruk pula.22 tingkat pemanfaatan pelayanan kesehatan gigi anak usia dipengaruhi juga oleh tingkat pendapatan orangtua.. pada penelitian ini lebih dari separuh jumlah orangtua mempunyai tingkat pendapatan yang rendah, sehingga kemampuan dalam memanfaatkan pelayanan kesehatan gigi juga rendah.23 semakin tinggi tingkat pendapatan orang tua, semakin baik pula pemanfataan pelayanan kesehatan gigi.11 sebagian ibu mempunyai perilaku dalam memilih makanan untuk anaknya masih tergolong buruk. hal tersebut dikarenakan, dalam memberikan makanan pada anak cenderung makanan yang bersifat kariogenik.12 tingkat pendidikan sebagaian besar ibu adalah rendah sehingga berdampak terhadap pengetahuan dan perilaku dalam memilih makanan untuk anaknya. ibu dengan pendidikan yang rendah cenderung memberikan makanan kesukaan anaknya, tanpa memperhatikan dampaknya bagi kesehatan gigi anaknya.24 pengetahuan anak tentang kesehatan gigi dan mulut sebagian besar baik, sehingga perilaku anak anak dalam pemeliharaan kesehatan gigi dan kebiasaan makan yang baik pula, yaitu. orang akan berperilaku terhadap kesehatan gigi yang baik, jika mempunyai dasar pengetahuan tentang kesehatan gigi yang baik pula.25 pengetahuan dipengaruhi oleh faktor predisposisi, yaitu status ekonomi, umur, jenis kelamin, dan susunan dalam keluarga. umur mempengaruhi daya tangkap dan pola pikir seseorang. semakin bertambah umur akan semakin berkembang pula daya tangkap dan pola pikirnya, sehingga pengetahuan yang diperolehnya semakin membaik. pada anak usia 10-12 tahun tahap perkembangan sudah memasuki tahapan cara berfikir secara logis, masuk akal dan semakin tersosialisasi.26 begitu juga dalam hal kebiasaan makan, anak sudah bisa memilih makanan yang sehat, bisa memahami cara pencegahan dan pengetahuan tentang proses terjadinya karies.27 pengetahuan tentang kesehatan gigi yang baik akan memotivasi seseorang untuk berperilaku yang baik dalam memelihara kesehatan gigi dan mulut.6 anak usia sekolah sebenarnya sudah mulai terampil gambar 2. contoh hasil pengukuran prediksi karies baru dengan prediktor karies anak. �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 45–51 menyikat gigi, karena perkembangan motorik halus dan kasar yang semakin baik. perubahan biologis pada anak usia sekolah dimulai dengan menguasai keterampilanketerampilan motoriknya. perkembangan kemampuan fisik pada anak tampak pada kekuatan koordinasi, fleksibilitas dan keseimbangan, kelancaran, kemampuan melakukan kontrol dan variasi gerakan. berbagai gerakan dasar dan variasinya yang telah bisa dilakukan sebelumnya akan mengalami peningkatan kualitas atau mengalami penyempurnaan.29 di dalam pelaksaanan ukgs oleh guru, semua anak menganggap masih kurang. usaha pencegahan karies gigi pada anak, diperlukan adanya kerjasama yang baik antara pihak sekolah, dalam hal ini guru, orang tua dan anak didik.6,7 usaha kesehatan gigi sekolah menitikberatkan pada upaya penyuluhan, kegiatan menyikat gigi bersama, dan pemeriksaan gigi dan mulut secara rutin. oleh karena itu, guru di sekolah perlu mengingatkan metode menyikat gigi yang benar, frekuensi menyikat gigi paling sedikt 2 kali sehari, yaitu sebelum tidur dan sesudah sarapan, dan setiap menyikat gigi selalu mempergunakan pasta gigi berfluor.23 pelaksanaan ukgs diharapakan mampu mempengaruhi dan memotivasi anak usia sekolah dalam memelihara kesehatan giginya, sehingga diharapkan resiko karies akan turun. informasi tentang kesehatan gigi dan mulut pada anak diperoleh diantaranya dari guru di sekolah.6 peran dan dukungan dari komponen sekolah terutama guru sangat menentukan keberhasilan program kesehatan sekolah, karena informasi dari guru dapat terjadi secara langsung pada anak maupun tidak langsung melalui orangtuanya.30 pengukuran resiko karies yang dilaksanakan pada 67 anak, didapatkan 30 anak diprediksi resiko karies tinggi oleh cariogram, 26 anak diprediksi resiko karies tinggi26 anak diprediksi resiko karies tinggi oleh prediktor karies anak, sehingga dapat dikatakan sensitifitas prediktor karies anak sebesar 87%. spefisifisitas prediktor karies anak diperoleh 92%, artinya pada 37 anak yang diprediksi karies rendah oleh cariogram, terdapat 3434 anak mempunyai resiko karies rendah yang diukur dengan prediktor karies anak. hasil ini menunjukkan bahwa perangkat lunak prediktor karies anak dapat mengukur 87% anak yang mempunyai resiko karies tinggi, dan dapat mengukur 92% anak yang mempunyai resiko karies rendah. dari 29 anak yang diprediksi resiko karies tinggi oleh prediktor karies anak, ternyata 26 anak benar-benar mempunyai resiko karies tinggi, sehingga diperoleh ndp adalah 90%. hasil ini menunjukkan bahwa jika pemeriksaaan ini memberikan hasil yang positif, maka 90% dari uji ini mendeteksi adanya resiko karies tinggi, dan 10% mendeteksi risisko karies rendah. hasil pemeriksaan pada 38 anak yang mempunyai resiko karies rendah, ternyata 34 anak benar-benar mempunyai resiko karies rendah, sehingga dapat dikatakan bahwa ndn adalah 89%. hasil ini menunjukkan bahwa, bila uji ini memberikan hasil negatif, maka 89% dari uji ini mendeteksi anak yang mempunyai karies rendah, adapun sebesar 11% memdeteksi anak yang mempunyai resiko karies tinggi. model prediksi karies pada umumnya mempunyai sensitivitas antara 29-70% dan spesifisitas 65-80%.9 hasil sensitivitas, spesitivitas, ndp dan ndn prediktor karies anak pada penelitian ini, tidak berbeda jauh dengan hasil penelitian resiko karies pada anak dengan mempergunakan cariogram, yaitu diperoleh sensitivitas adalah 93%, spesifitas 63%, ndp sebesar 87% dan ndn sebesar 63%.30 hasil pengujian sensitivitas dan spesifisitas simulator resiko karies pada anak prasekolah dengan menggunakan irene’s donut, diperoleh sensitivitas sebesar 86% dan spesifisitas 69%.31 alat prediksi karies yang mempunya nilai sensitivitas, spesifisitas, ndp dan ndn 70-100% termasuk dalam katagori bagus sampai sangat bagus, sehingga dapat dikatakan mempunyai validitas yang tinggi.32 perlu dilakukan penelitian lebih lanjut dengan menggunakan populasi yang lebih luas. selain itu perlu melakukan penelitian tentang ada atau tidaknya perubahan perilaku anak maupun orangtua setelah dilakukan pengukuran prediksi resiko terjadinya karies. dari hasil penelitian tersebut di atas, dapat ditarik kesimpulan bahwa "prediktor karies anak" mempunyai sensitivitas, spesifitas, ndp dan ndn yang tinggi dan dapat dipakai sebagai instrumen untuk memprediksi terjadinya karies baru pada anak. ucapan terima kasih ucapan terima kasih ditujukan kepada orangtua/wali murid dan siswa kelas va dan b sekolah dasar negeri dan b sekolah dasar negeri godean i, kabupaten sleman, yogyakarta. daftar pustaka 1. benzian h, monse b, heinrich-weltzien r, hobdell m, mulder j, van palenstein helderman w. untreated severe dental decay: a neglected determinant of low body mass index in 12-year-old filipino children. bmc public health 2011;11: 558. 2. bratthall d, petersson gh, stjernsward jr. cariogram manual. internet version 2.01, http:/ www. db.od.mah.se/car/cariogram /cariograminf and cheo. html., 2004 accessed march 23, 2011 . 3. cameron ac, widmer rp. handbook of pediatric dentistry. 3rd ed. mosby elsevier limited; 2008. p. 39-41. 4. fisher-owen, sa, gansky sa, platt lj, weintraub j, soobader m, bramlett md, newacheck pw. influences on children’s oral health: conceptual model. american academy of pediatrics, p. 510-520,academy of pediatrics, p. 510-520, 510-520, htpp: //pediatrics. aapublication.org/conten/120/3/e510.full.html, 2011. accessed january 10, 2012 5. qiu, rm, wong, mcm, lo, ecm, lin, hc, relationship between children’s oral health-related behaviors and their caregiver’s sense of coherence, bmc public health. 2013, 12(239): 1-7. 6. darwita, rr, novrida h, budiharto, pratiwi pd, amalia r, asri sr. improving oral health awareness in primary school student. j indon med assoc 2011; 61(5): 204-9. 7. bhardwaj vk, sharma, kr, luthra, rp, jhingta p, sharma d, justa a. impact of school-based oral health education program oral health of 12 and 15 years old school children. j education and health promotion 2013; 2: 1-4. 8. hunstad mn, antonsen gm. masteroppgave: caries risk assessment. universittet, det helsevitenskapelige fakultet, institutt for kliniks odontologi; 2011. p. 1-16. ��a’yun, et al.: uji sensitivitas dan spesifisitas perangkat lunak “prediktor karies anak” 9. aleksejuniene j, holst d, brukiene v. dental caries risk studies revisited: causal approaches needed for future inquiries. int j environt res public health 2009; 6(12): 2992-3009. 10. giacaman ra, reyes pm, leon vb. caries risk assessment in chileon adolescent and adult and its association with caries experience. braz oral res 2013; 27(1): 7-13. 11. a’yun q, hendrartini j, santoso als, nugroho le, prediction about the incidence of caries in children base on children’s bahavior, parent’s and environment. sciences the indonesian journal of dental research, proceeding of the international symposium on oral and dental, 2013; p. 149-55. 12. pusponagoro, sastroasmoro s, ismail s. dasar-dasar metodologis penelitian klinis. edisi ke 3.. jakarta: sagung seto; 2010. h. 193216. 13. petrie a, sabin c. medical statistic and a glance. 3rd ed. singapore: ho printing singapore; 2010. p. 115-6. 14. petersson gh, fure s, bratthall d. evaluation of a computer-based caries risk assessment program in aldery of group indivual. acta odontol scan 2003; 61: 164-71. 15. kidd eam, essential of dental caries. 3rd edition. new york, usa: oxford university press; 2005. p. 1-5. 16. lian cw, phing ts, chat cs, shin bc, baharuddin lh, che’jalil zbj. oral health knowledge, attitude and practice among secondary school student in kucing sarawak. archives of oraofacial sciences 2010; 5(1): 9-16. 17. jamieson lm, mejia gc, slade gd, robert-thomson kf. predictor of untreated dental decay among 15-34-year-old australian. community dent oral epidemiol 2011; 37: 24-7. 18. maharani da, anton r. mother’s dental health behaviors and mother-child dental caries experience: study of a suburb area in indonesia makara kesehatan 2012; 16(2): 72-6. 19. zhu l, petersen pe, hong yw, jin yb, bo xz. oral health knowledge, attitudes and behavior of adult in china. int dent j 2005; 55: 231-41. 20. medina-solis ce, maupome g, herrera ms, perez-nunez r, avilaburgos l, lamadrid-figueroa h. dental health service utilization and association factors in children 6 to 12 years old in low-income country. american association of public health dentistry 2008; 68: 1. 21. sumanti v, widarsa t, duarsa dp. faktor yang berhubungan dengan partisipasi orangtua dalam perawatan gigi anak di puskesmas tegalalang i. public health and preventive medicine archive 2013; 1: 1. 22. amin m, nyachhon p, elyasi m, al-nuaimi m. impact of oral health education workshop on parent’s oral health knowledge, attitude, and perceived behavioral control among african immigrants. j oral disease 2014; 1-7. 23. guiney h, woods n, whelton h, morgan k. predictors of utilitation of dental care servicees in a nationally representative sample of adult. community dental health 2011; xx: 1-5. 24. dye ba, vargas cm, lee jj, magder l, tinanoff n. assessing the relation between children’s oral health status and that of their mothers. j am dent assoc 2011; 142: 173-83. 25. budiharto. pengantar ilmu perilaku kesehatan dan pendidikan kesehatan gigi. edisi ke-7. jakarta: egc; 2010. p. 1-5. 26. notoatmojo s. ilmu perilaku kesehatan. 1st ed. jakarta: pt. rineke cipta; 2010. h. 20-33. 27. pinkham jr, cassamassiomo ps, field hw, tigue dj, nowak aj. pediatric dentistry. 4th ed. st. louis: elsevier saunders; 2005. p. 469. 28. santrock jw. masa perkembangan anak. edisi ke-11. jakarta: penerbit salemba humanika; 2011. p. 143-6. 29. kemenkes. pedoman usaha kesehatan gigi sekolah (ukgs). jakarta: kemenkes; 2012. h. 11-20. 30. suzuki pbe, calvo ljc, reyes bia, jau rag, ramirez oi, alvarez ra, rodliquez ljp. predicting risk of caries in schoolchildren from northwestern mexico (longitudinal study). rev invest clin 2013; 65(10): 24-9. 31. adyatmaka i. model simulator resiko karies gigi pada anak prasekolah. disertasi. jakarta: pascasarjana universitas indonesia; 2008. p. 131-2. 32. ditmyer mm, dounis g, howard km, mobley c, cappelli d. validation of amultifactorial risk factor model used for predicting future caries risk with nevada adolescents. bmc oral health 2011; 11(18): 1-8. 140 volume 46, number 3, september 2013 dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring (epstein-barr virus (ebv) dna as biomaker of nasopharyngeal carcinoma diagnosis) janti sudiono1 dan irma hassan2 1bagian patologi oral, fakultas kedokteran gigi universitas trisakti 2bagian patologi anatomi, fakultas kedokteran universitas tarumanagara jakarta – indonesia abstract background: nasopharyngeal carcinoma (npc) is a malignant neoplasm arising from the mucosal epithelium of the nasopharynx with various cells differentiation. nasopharyngeal carcinoma is vastly more common in certain regions of east asia, south asia and africa with viral, dietary which is typically includes consumption of salted vegetables, fish, meat and genetic factors that implicated in its causation. the undifferentiated is the most common type of npc and strongly associated with epstein-barr virus (ebv) infection. purpose: this paper was aimed to review about molecular biomarker as non invasive diagnosis of npc especially in related to ebv infection in nasopharyngeal epithelial cells. reviews: the pathogenesis of npc particularly the endemic type seems to follow a multi-step process, in which ebv, ethnic background, and environmental carcinogens all seem to play important role. ebv dna plasm level is used continuously in clinic as a promise, sensitive and specific molecular marker diagnostic that reflected the stage, treatment response and prognosis of npc. detection of nuclear antigen associated with epstein-barr virus (ebna) and viral dna has revealed that ebv can infect epithelial cells and associated with their transformation in carcinogenesis. latent membrane protein (lmp-1 and lmp-2) oncogenes ebv encoded related to proliferative gene expression indicated invasive and progressive growth of npc. conclusion: the new biomarkers for npc, including ebv dna in serum; ebv dna and bamh1-a reading frame-1 (barf1) mrna in npc brushings have been developed for the molecular non invasive diagnosis of this tumour. key words: nasopharyngeal carcinoma, epstein-barr virus, environmental carcinogen, molecular biomarker abstrak latar belakang: nasopharyngeal carcinoma (npc), sering dikenal sebagai kanker nasofaring merupakan tumor ganas yang berasal dari epitel mukosa nasofaring dengan derajat diferensiasi sel yang bervariasi. paling banyak ditemukan di asia selatan, asia timur, dan afrika. virus, pola diet tipikal seperti konsumsi sayuran, ikan dan daging yang diasinkan, dan faktor genetik merupakan faktor kausatif. tipe undifferentiated paling banyak ditemukan dan sangat berkaitan dengan infeksi virus epstein barr (ebv). tujuan: tujuan penulisan ini akan meninjau pustaka mengenai biomarker molekular sebagai alat diagnostik yang non invasif untuk npc terutama dalam kaitannya dengan infeksi ebv pada sel epitel nasofaring. tinjauan pustaka: patogenesis npc terutama pada tipe endemik, merupakan proses multi tahap, dan semua faktor seperti ebv, latar belakang etnik, dan karsinogen lingkungan berperan penting. level plasma dna ebv digunakan secara rutin di klinik sebagai suatu marker diagnostik molekular yang menjanjikan, sensitif, dan spesifik sebagai cerminan stadium, respon terhadap pengobatan dan prognosis npc. terdeteksinya antigen inti yang berkaitan dengan ebv (ebna) dan dna virus menyatakan bahwa ebv menginfeksi sel epitel dan terkait dengan transformasi sel dalam karsinogenesis. protein membran laten-1 dan 2 onkogen (lmp1 dan lmp2) mengkode ebv berkaitan dengan ekspresi gen pertumbuhan sel yang mengindikasikan pertumbuhan yang sangat invasif dan progesif dari npc. simpulan: biomarker npc terkini literature reviews 141sudiono dan hassan: dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring seperti pengukuran ebv dna dalam serum; ebv dna dan barf1 (bamh1-a reading frame-1) mrna pada sitologi npc telah dikembangkan untuk diagnosis molekular yang non invasif. kata kunci: nasopharyngeal carcinoma, virus epstein barr, karsinogen lingkungan, biomarker molekular korespondensi (correspondence): janti sudiono, bagian patologi oral, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa, grogol, jakarta 11440, indonesia. e-mail: jantish@hotmail.com pendahuluan the american cancer society menyatakan bahwa pada tahun 2009 ditemukan sekitar 35.720 kasus baru dari kanker mulut dan orofaring, meliputi 25.240 pada pria dan 10.480 pada wanita, dengan 7.600 kematian. kasus baru dan kematian karena kanker rongga mulut dan orofaring semenjak 20 tahun terakhir nampak berkurang. hal ini kemungkinan karena berkurangnya pemakaian tembakau di amerika. kanker nasofaring (nasopharyngeal carcinoma/ npc) sangat jarang ditemukan di inggris. lebih sering ditemukan pada kelompok etnik tertentu yang tinggal di inggris, contoh populasi cina. sekitar 240 kasus didiagnosis pertahun.1 di indonesia, di antara kanker tubuh lain, angka kematian kelima tertinggi ditempati oleh npc yang menempati peringkat ke tiga pada pria, sedangkan pada wanita menempati peringkat tertinggi ke lima.2 rata-rata prevalensi npc di indonesia adalah 6,2/100.000 dengan 13.000 kasus npc baru setiap tahun, namun demikian data npc di indonesia sedikit sekali. pada periode tahun 19962005 di rscm terdata kasus npc sebanyak 1.121.3 pendapat umum mengakui bahwa npc berasal dari sel epitel gepeng yang berasal dari mukosa saluran pernapasan dan stroma sub mukosa yang mengandung jaringan limfoid serta kelenjar. klasifikasi npc berdasarkan pada topografi dan morfologi sel dominan yang dilihat berdasarkan tingkat diferensiasi dan keratinisasi sel, terbagi dalam tipe berdiferensiasi berkeratin, berdiferensiasi tak berkeratin dan tipe undifferentiated tak berkeratin. faktor yang berperan dalam patogenesis npc termasuk virus epstein bar, kerentanan genetik dan faktor risiko dari lingkungan. beberapa faktor lingkungan yang diduga berkaitan dengan npc adalah diet, paparan bahan kimia di tempat pekerjaan dan tembakau. banyak penelitian tentang bagaimana infeksi epstein-barr virus (ebv) dan faktor risiko lainnya menyebabkan sel nasofaring menjadi kanker. para peneliti berharap bahwa penelitian pada akhirnya akan dapat berhasil mendapatkan suatu vaksin untuk mencegah terjadinya npc dengan menghindari terjadinya infeksi ebv. penemuan terakhir tentang ebv dan interaksinya terhadap sel nasofaring serta reaksi sistem imun terhadap ebv telah berhasil menemukan suatu tes darah untuk mendapatkan biomarker molekular yang dapat membantu mendeteksi npc stadium awal dan dapat memprediksi respon terhadap pengobatan dengan lebih baik. tes ini sedang diteliti di daerah belahan dunia di mana kanker ini sering dijumpai.4 tulisan ini bertujuan meninjau pustaka mengenai infeksi ebv pada sel nasofaring sebagai biomaker molekular untuk alat diagnostik non invasif. epidemiologi nasopharyngeal carcinoma merupakan kanker yang umum terjadi di cina selatan dan asia tenggara.5 npc merupakan tumor endemis di asia selatan terutama pada pria dan sangat jarang ditemui di bagian lain dunia. predileksi umur dekade 5, 6, dan 7 meskipun dapat pula terjadi pada dekade 2 dan 3.6 angka kematian penderita npc etnik cina yang lahir di amerika meningkat 20x dibanding pada populasi etnik lainnya. juga angka insidensi npc ditemukan tinggi di tunisia dan afrika timur.7 angka kejadian npc di inggris per tahun sekitar 0,3/1.000.000 penduduk pada usia sampai 14 tahun dan 1-2/1.000.000 untuk usia 15-19 tahun. insidensi lebih tinggi ditemukan pada populasi cina dan tunisia yang tinggal di inggris. sekitar 1/3 neoplasma nasofaring ditemukan pada usia anak. di cina bagian selatan, asia selatan, mediterania dan alaska, insidensi npc agak meningkat. angka kejadian di cina sekitar 2/1000.000. di negara lain seperti india, insidensi seimbang dengan di inggris yaitu sekitar 0,9/1000.000, dengan puncak usia sama seperti di inggris, pada usia lebih muda yaitu pada dekade kedua.8 di indonesia, data terbaru tahun 2008 menunjukkan bahwa insidensi dan mortalitas tertinggi ke lima di antara kanker tubuh ditempati oleh npc. pada pria insidensi sebanyak 9,4 kasus baru/100.000 orang per tahun dan pada wanita sebanyak 3,8. angka mortalitas pada pria 6,0 kematian/100.000 orang per tahun sedangkan pada wanita angka mortalitas sebesar 2,4 (tabel 1).2 etiologi meskipun penyebab pasti npc belum diketahui, namun diduga ebv berperan penting di samping faktor lingkungan.5,6 terpapar banyak karsinogen lingkungan berperan dalam kecenderungan peningkatan insiden kanker.9 penelitian patogenesis npc yang telah dilakukan akhir-akhir ini bertujuan untuk mengetahui peran kombinasi faktor lingkungan dan genetik dan dinyatakan bahwa ada hubungan antara profil human leukocyte antigen (hla) pada populasi cina yang mengindikasikan kerentanan genetik.7 pendapat ini juga didukung oleh peneliti lain yang menyatakan bahwa npc merupakan hasil dari kerentanan genetik dan interaksi dari karsinogen lingkungan dengan ebv yang menginduksi terjadinya tumor. peran ebv, sayuran dan ikan yang diawetkan serta peningkatan kerentanan genetik ditemukan pada populasi dengan tipe 142 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 140–147 hla tertentu.10 epstein-barr virus, latar belakang etnik dan karsinogen lingkungan semuanya berperan penting dalam etiologi npc terutama pada tipe endemik.8 interaksi antara faktor host (hla-a2, hla-bsin2 loci dan tempat lahir), infeksi oleh ebv dan faktor lingkungan merupakan faktor risiko yang tinggi di antara penduduk cina selatan.3,8 beberapa faktor lingkungan yang diduga berkaitan dengan terjadinya npc adalah tembakau, paparan bahan kimiawi, obat tradisional cina dan diet. di antara makanan, penelitian mengenai makanan yang diawetkan telah banyak dilakukan dengan hasil menyatakan bahwa terjadi peningkatan risiko terkena npc berkaitan dengan konsumsi ikan yang diasinkan. makanan lain yang diawetkan yang juga berperan adalah saus ikan yang difermentasi, pasta udang yang diasinkan, kacang kedele yang diasinkan, kacang yang difermentasi, buah plum yang diawetkan, dan telur bebek yang diasinkan, sayuran kalengan. terpapar oleh bahan aditif pada makanan seperti pengawet nitrit dan zat warna azo berkaitan dengan induksi proses karsinogenesis.1113 nitrit dan nitrat yang digunakan dalam daging akan berikatan dengan myoglobin dan menghambat pembentukan eksotoksin botulinum, namun sangat karsinogenik.14 studi di cina dan hongkong menunjukkan bahwa bayi dan anak muda yang mengkonsumsi ikan yang diasinkan akan mendapatkan risiko tinggi npc di kemudian hari. populasi etnik cina yang tinggal di inggris mempunyai angka ratarata npc lebih tinggi dibanding etnik lainnya dan hal ini mungkin disebabkan oleh faktor kebiasaan makannya. beberapa penelitian membuktikan peningkatan risiko npc di antara populasi yang minum teh yang dibuat dari tanaman obat cina.1 diet yang buruk meningkatkan risiko akan npc, hal ini disebabkan karena kurangnya vitamin dan mineral. konsumsi banyak sayuran, buah buahan segar dan sumber lain dari vitamin c mempunyai risiko lebih rendah terhadap terjadinya npc. hal ini penting dilakukan selama kehidupan terutama pada usia anak.15 terpapar asap kayu bakar untuk memasak di rumah selama bertahun-tahun dan penggunaan larutan toksik tertentu di tempat pekerjaan berkaitan dengan risiko terjadinya npc. studi di afrika menunjukkan bahwa pemaparan dengan asap batu bara selama masa anak dapat meningkatkan risiko npc dan ini tidak terjadi bila terpapar pada usia dewasa. juga ada penelitian yang menyatakan bahwa risiko terkena npc meningkat pada paparan klorofenol yang digunakan dalam pestisida dan pengawet kayu.1 merokok menyebabkan peningkatan risiko terkena npc sebesar 3x lipat bila merokok sudah dilakukan dalam jangka waktu lama (25 tahun atau lebih). tidak seperti kanker kepala dan leher lainnya, alkohol tampaknya tak meningkatkan risiko terkena npc. di amerika, pemakaian tembakau dan alkohol terutama berkaitan dengan terjadinya npc berdiferensiasi baik berkeratin.1 pendapat mengenai pengaruh alkohol terhadap risiko terjadinya npc masih belum diakui sepenuhnya, namun ada yang menyatakan bahwa konsumsi alkohol berat berkaitan dengan peningkatan risiko terhadap terjadinya npc.16 risiko terkena npc lebih tinggi pada seseorang yang mempunyai anggota keluarga yang mengidap penyakit ini. risiko anggota keluarga terkena tampaknya lebih tinggi bila ada keluarganya yang terdiagnosis sebelum usia 40 tahun dan hubungan keluarga yang dimaksud adalah yang merupakan satu keluarga inti seperti orang tua, kakak atau adik, anak laki-laki atau perempuan. penderita penyakit kronis pada telinga, hidung, dan tenggorokan di masa lalu seperti rhinitis, hidung tersumbat kronis, infeksi telinga tengah (otitis media) dan polip, mempunyai risiko lebih tinggi terkena npc.1 penelitian menyatakan ada hubungan antara iga dengan antigen kapsid ebv (vca). hasil analisis statistik menyatakan bahwa iga/vca merupakan faktor prediktor utama bagi risiko terjadinya npc dan ikan yang diawetkan merupakan faktor prediktor kuat kedua. pada tipe npc undifferentiated terlihat peningkatan titer antibodi igg dan iga melawan vca dan antigen awal.15 ada pendapat yang mengemukakan bahwa tanpa ebv dan faktor genetik merupakan hal yang berlebihan bila menyatakan bahwa ikan yang diasinkan bukan merupakan faktor etiologi penting bagi npc.15 adham dkk.3 menemukan pada penelitian terhadap 1121 kasus npc di rscm, infeksi ebv pada usia muda dikombinasi dengan seringnya terpapar karsinogen dan co-carcinogen lingkungan tampaknya merupakan penyebab terjadinya npc. patogenesis ebv dalam terjadinya npc epstein-barr virus merupakan virus herpes yang berada di mana mana dan yang menginfeksi lebih dari 90% populasi dewasa di dunia. infeksi primer ebv umumnya terjadi pada awal kehidupan dan asimtomatik. infeksi primer ebv yang berlanjut bermanifestasi sebagai infeksi mononukleosis yang merupakan infeksi oleh virus, bersifat self limiting namun sangat menular dengan karakteristik tabel 1. angka kejadian kanker mulut di indonesia kanker insidensi kematian jumlah jumlah pria asr* wanita asr* pria asr* wanita asr* bibir, rongga mulut 2.693 2,8 2.310 2,1 1.153 1,3 990 0,9 nasofaring 10.035 9,4 4.230 3,8 6.084 6,0 2.564 2,4 lainnya 1.317 1,5 619 0,6 1.077 1,2 506 0,5 *asr= jumlah kasus baru atau kematian tiap 100.000 orang per tahun.2 143sudiono dan hassan: dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring demam, sakit pada tenggorok dan kondisi tubuh yang lemah. pada setiap kasus, infeksi primer diikuti oleh keberadaan virus sepanjang hidup yang pada sebagian besar kasus bersifat asimtomatik.17 epstein-barr virus menetap sepanjang kehidupan pada >95% populasi manusia dewasa yang terinfeksi. epstein-barr virus dikontrol dengan sempurna oleh sistem imun namun sebagian kecil berkembang menjadi penyakit terkait, pada sebagian besar individu terutama dalam bentuk keganasan primer dari sel b dan sel epitel.18 selain infeksi ebv, infeksi oleh beberapa virus onkogen lainnya dapat pula berperan dalam terjadinya kanker. dari total perkiraan kanker terkait infeksi pada tahun 2002, agen utama adalah helicobacter pylori (5,5%), human papilloma virus (5,2%), virus hepatitis b dan c (4,9%), ebv (1%), human immunodeficiency virus (hiv) bersama human herpes virus 8 (0,9%). agen penyebab yang paling kecil sebagai penyebab kanker adalah schistosoma (0,1%), virus human t-cell lymphotropic tipe i (0,03%) dan parasit pada liver (0,02%). bila infeksi ini dicegah maka akan berdampak terhadap angka penurunan kanker, yaitu sebesar 26,3% di negara berkembang (1,5 juta kasus/tahun) dan sebesar 7,7% di negara maju (390,000 kasus).19 epstein-barr virus dikategori sebagai kelompok karsinogen pertama oleh international agency for research on cancer (iarc) kerena keterkaitannya dengan npc.17 diduga ebv berperan dalam npc dengan terdeteksinya ebv pada sel tumor dan limfosit b meskipun hal ini belum terbukti secara luas.7 virus ini berkaitan dengan sejumlah kanker termasuk limfoma hodgkin dan limfoma burkit di samping npc.1 epstein-barr virus terdeteksi pada semua sampel npc dengan beberapa teknik pemeriksaan seperti sikatan sel epitel nasofaring (brushing), pcr, hibridisasi in situ, dan metode imunohistokimia.20,21 hubungan npc dengan ebv telah diakui sejak beberapa dekade yang lalu meskipun peran virus ini dalam patogenesis npc masih kontroversi. nasopharyngeal carcinoma sangat berkaitan dengan ebv terutama pada tipe yang undifferentiated. namun demikian, ebv bukanlah faktor penting satusatunya dalam patogenesis npc. bahan etiologi lain seperti lingkungan dan genetik, merupakan hal penting lainnya yang berperan dalam perkembangan multi tahap dari keganasan. kerentanan genetik seperti hla-a2 dan hla-bsin2 loci berperan sebagai faktor predisposisi.3 semua kasus tumor tipe undifferentiated menunjukkan ebv positif, terlepas dari asal geografi tumor, virus ditemukan pada semua sel tumor. infeksi ebv menginduksi ketahanan sel terhadap kematian (immortality) melalui mekanisme aktivasi telomerase yang merupakan suatu ensim yang secara normal tertekan namun teraktivasi selama perkembangan kanker. akhir akhir ini terbukti bahwa untuk mengaktivasi atau menghambat gen reverse transcriptase telomerase manusia adalah melalui modulasi jalur signal intra selular.5 pada npc tipe undifferentiated gambar 1. n p c t i p e u n d i f f e r e n t i a t e d. t e r l i h a t s e p e r t i limfoepitelioma, sel tumor tampak tipikal dengan inti besar, nukleoli menonjol dan eosinofilik.6 gambar 2. npc tipe undifferentiated. terlihat sel dengan sitokeratin positif mengindikasikan suatu proliferasi dari sel epitel.6 144 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 140–147 dengan ebv positif pada populasi kulit putih, diduga hpv sebagai faktor etiologi. 22 penelitian in vitro mendapatkan infeksi ebv yang menetap pada sel epitel menyebabkan sel epitel menjadi rentan terhadap paparan karsinogen lingkungan, contoh bentuk karsinogen lingkungan adalah faktor diet seperti ikan yang diasinkan, mekanisme ebv memasuki sel epitel masih belum terpecahkan.17 selain potensi perubahan genetik dari infeksi ebv, adanya infeksi laten pada sel epitel tampaknya berperan besar dalam perkembangan npc.23 biomarker molekular terkait ebv pada npc penelitian menggunakan pcr membuktikan adanya ebv dna dalam sel darah tepi dapat digunakan sebagai indikator prognostik dari npc.24 penelitian lain menyatakan bahwa plasma ebv dna merupakan marker molekular yang sensitif dan spesifik yang dapat merefleksikan stadium, respon terhadap pengobatan dan prognosis npc. konsentrasi ebv dalam plasma sebelum dan sesudah pengobatan dapat digunakan untuk prediksi terhadap kemungkinan terjadinya metastasis jauh, dan dapat menseleksi pasien yang mempunyai risiko tinggi serta dapat digunakan dalam menentukan kombinasi metoda perawatan. 25 epstein-barr virus mengkode dua onkogen, latent membrane protein-1 (lmp1) dan bamh1-a reading frame-1 (barf1). lmp1 termasuk famili gen laten dan barf1 ditetapkan sebagai satu dari famili gen awal. namun, onkogen barf1 diekspresikan dalam jumlah tinggi pada npc dan kanker lambung sebagai suatu keadaan laten tipe ii dan pada infeksi primer sel epitel oleh ebv secara in vitro sebagai keadaan laten tipe i. ekspresi barf1 juga ditemukan pada limfoma burkit. sekresi masif dari protein barf1 juga ditemukan pada serum dan saliva penderita npc. barf1 diekspresikan pada stadium laten dan meningkat ekspresinya selama stadium litik.26 infeksi ebv laten pada sel b diklasifikasi dalam 3 tipe, tipe i, ii dan iii. tipe i umum pada limfoma dan mengekspresikan dengan sangat terbatas protein virus, terutama ebvencoded nuclear antigens (ebna1), ebv-encoded nonpolyadenylated rnas (ebers) dan barf0. tipe ii mengekspresikan ebna1, lmp1, ebers, barf0 dan lmp2. tipe iii mengekspresikan beberapa protein virus seperti ebna1, ebna2, ebna3a, 3b dan 3c, lmp1, lmp2a, lmp2b, barf0 dan ebers.27 nasopharyngeal carcinoma termasuk tipe ii. pada percobaan in vitro, sel epitel primer tidak mengalami kematian oleh ebv yang mengekspresikan ebna1, ebers, lmp2a dan barf1,28,29sehingga termasuk infeksi tipe ii kecuali bila tak ada ekspresi lmp1. barf1 secara masif disekresikan dalam serum penderita npc. barf1 murni dalam serum menunjukkan aktivitas mitogenik yang sangat kuat. 30 epstein-barr virus menginduksi dan mengontrol proliferasi sel melalui ekspresi 10 gen viral yang mengkode 6 protein inti (ebna1, 2, 3a, 3b, 3c dan lp), 3 protein membran (lmp1, lmp2a, lmp2b) dan 2 rna kecil. keterbatasan ekspresi gen viral lmp2a in situ menyatakan bahwa protein ini mungkin mempunyai peran kunci terhadap terjadinya dan menetapnya keadaan laten dan atau reaktivasi dari keadaan laten dan atau reaktivasi infeksi dari keadaan laten. lmp2b terlokalisasi pada daerah perinuklear di sel yang terinfeksi sementara dan terletak bersama dengan lmp2a. lmp2b dapat berfungsi sebagai regulator negatif dari lmp2a karena strukturnya yang hampir identik dengan lmp2a dan ketiadaan dari signaling.31 lmp1 mengkode ebv onkogen merupakan suatu efektor kunci dari transformasi neoplastik sel b dimediasi ebv. lmp1 mengkode ebv onkogen menunjukkan potensi onkogenik pada fibroblas hewan pengerat dan menginduksi proses menuju transformasi neoplastik dalam sel b dan sel epitel. lmp1 mengkode ebv onkogen memungkinkan virus untuk bertahan dalam jangka waktu yang lama dalam sel sistem imun. lmp1 berfungsi aktif pada reseptor famili tumor necrosis factor receptor (tnfr); berikatan dengan membran plasma; mengawali aktivasi jalur signaling seperti nf-kb, mitogen-activated protein kinases (mapks), c-jun n-terminal kinases (jnk), p38, jalur janus kinase atau transducer and activator of transcription (jak/stat) oleh protein adaptor termasuk tnf, tumor necrosis factor receptor associated factors (trafs) dan tnf receptor associated death domain protein (tradd); meningkatkan ekspresi molekul adhesi lymphocyte function-associated antigen1 (lfa-1), intercellular adhesion molecule 1 (icam-1), dan molekul kostimulator b7-1 dari sel b; serta meregulasi sekresi antibodi dan sitokin oleh sel b. ikatan dari sinyal dan karakteristik interaksi molekular yang meregulasinya merupakan dasar dari kemampuan protein kunci ebv ini untuk melakukan transformasi neoplastik.32 lmp1 dan cd40 mempunyai banyak instrumentasi dalam transduksi sinyal. aktivasi nf-kb oleh induksi ebv lmp1 penting dalam ketahanan sel b terhadap transformasi neoplastik oleh ebv.33 ebv lmp1 terekspresi pada semua keganasan terkait ebv. ekspresi lmp1 pada sel b manusia menginduksi aktivasi dan ekspresi molekul adhesi. interaksi lmp1traf memediasi aktivasi jalur signaling penting untuk mengendalikan ketahanan dan pertumbuhan sel yang terinfeksi ebv.34 gen lmp2 diekspresikan pada sel b yang terinfeksi laten dan mengkode dua bentuk protein yang sama, lmp2a dan lmp2b identik, kecuali pada tambahan ujung sitoplasma n-119 aa pada lmp2a. ebv lmp2a meregulasi reaktivasi virus laten. ebv lmp2b memodulasi aktivitas ebv lmp2a. ebv lmp2a merupakan protein transmembran yang memblok sinyal tyrosine kinase dan dipercaya dapat mengatur aktivitas replikasi virus pada sel b yang terinfeksi laten. tidak diketahui apakah lmp2b diperlukan untuk transformasi pertumbuhan neoplastik yang dimediasi ebv. ada yang melaporkan bahwa lmp2a tidak diperlukan untuk transformasi neoplastik.31 terdeteksinya ebna1 mrna dalam sirkulasi darah merefleksikan peningkatan jumlah sel b positif ebv seperti yang telah dinyatakan oleh 145sudiono dan hassan: dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring penelitian sebelumnya bahwa peningkatan ebv dna dalam sel mononuklear darah tepi ditemukan pada penderita npc populasi taiwan.35 fenomena ini berkaitan dengan infeksi sel b oleh ebv yang menyebabkan lisis lokal sel b akibat replikasi ebv pada kasus npc,36 serta merupakan suatu refleksi reaktivitas igg and iga terhadap lytic ebv antigens yang mengindikasikan adanya replikasi virus.37 frekuensi replikasi sel b positif ebv yang meningkat pada sirkulasi menyebabkan peningkatan kadar ekspresi ebna1 mrna.38 selanjutnya, transkripsi ebna1 dalam darah mengindikasikan bahwa tidak semua ebv dna mengalami fragmentasi namun sebagian virus dna yang bersirkulasi merupakan sel yang utuh dan aktif melakukan transkripsi genom ebv38 sesuai dengan yang ditemukan oleh lin dkk.35 dan shotelersuk dkk.39 kadar ebv dna dalam plasma darah tampaknya berkorelasi dengan respon terapi radiasi dan kemo serta merupakan indikator prognosis yang bebas dan tidak dipengaruhi oleh indikator lainnya.40 analisis kinetik secara in vivo terhadap dna tumor dalam sirkulasi selama perawatan merupakan instrumen yang bermanfaat untuk mengevaluasi respon pengobatan npc. 41 kadar ebv dna terbukti lebih kuat dibanding sistem stadium klinis menggunakan pemeriksaan endoskopi, computed tomography (ct), dan magnetic resonance imaging (mri) untuk memprediksi penyakit dan juga untuk memperkirakan muncul kembalinya penyakit secara klinis. oleh karenanya, ini dapat diharapkan sebagai marker tumor yang menjanjikan yang dapat digunakan di klinik secara rutin.42,43 pembahasan hampir setengah kasus npc bermanifestasi awal atau terdeteksi sebagai pembesaran kelenjar limfe servikal.3,6,7 sejauh ini, di antara semua tumor ganas pada suku cina, npc merupakan kanker yang paling tinggi frekuensinya. demikian pula dengan di indonesia, npc merupakan kanker dengan angka mortalitas tertinggi di antara kanker tubuh lainnya.2 npc tipe undifferentiated merupakan tipe yang terbanyak, terutama banyak ditemukan di daerah endemis seperti asia tenggara dan bagian dari afrika. di hongkong, npc tipe undifferentiated berjumlah 18% dari semua kanker, dibandingkan dengan di seluruh dunia yang angka prevalensinya hanya sekitar 0,25%.7 tipe undifferentiated (gambar 1 dan 2) merupakan tipe npc yang paling sering ditemukan dan merupakan endemi di daerah tertentu, terutama di asia tenggara.44 tipe berdiferensiasi berkeratin terjadi pada populasi dengan usia lebih tua dan korelasinya terhadap ebv tidak sama seperti pada tipe undifferentiated dan tipe undifferentiated sangat berkaitan erat dengan ebv.7,17 bermacam-macam faktor risiko dari lingkungan (diet, paparan substansi kimiawi melalui pernapasan, kebiasaan etnik) berperan terhadap terjadinya npc namun belum ada pembuktian secara positif mengenai hubungan antar faktor. penelitian akhir-akhir ini ditujukan pada peran kombinasi faktor lingkungan dan genetik dalam patogenesis npc misal profil hla pada populasi cina mengindikasikan kerentanan genetik terhadap faktor karsinogenik lingkungan.7 patogenesis npc terutama pada tipe endemik agaknya mengikuti proses multi tahap di mana ebv, latar belakang etnik dan karsinogen lingkungan semuanya berperan penting dan saling terkait.8 diet daging dan ikan dengan kadar garam tinggi atau makanan yang diawetkan banyak ditemukan di daerah dengan angka kejadian npc yang tinggi seperti di bagian asia, afrika utara dan artic. makanan ini mengandung nitrat dan nitrit dengan kadar tinggi yang bereaksi dengan protein, membentuk nitrosamin yang merupakan bahan kimiawi yang dapat merusak dna. di samping itu zat aditif yang ditujukan sebagai pengawet dengan menghambat pembentukan eksotoksin botulinum akan berikatan dengan myoglobin dan membentuk senyawa yang sangat karsinogenik.14 dapat dikatakan bahwa yang merupakan faktor risiko tinggi di daerah endemis npc adalah interaksi antara faktor host (hla-a2, hla-bsin2 loci dan tempat lahir), infeksi oleh ebv dan faktor lingkungan.3,8 ada pula peneliti yang menemukan bahwa infeksi ebv pada usia muda dikombinasi dengan seringnya terpapar oleh karsinogen dan ko-karsinogen lingkungan tampaknya merupakan penyebab terjadinya npc.3 penelitian kontrol-kasus yang telah dilakukan di cina membuktikan adanya hubungan antara kebiasaan makan, paparan dalam pekerjaan, penggunaan tembakau dan alkohol, riwayat keluarga yang menderita npc dan iga dengan antigen kapsid ebv (iga/vca). iga/vca positif dan konsumsi ikan yang diasinkan berhubungan dengan risiko tinggi npc. 15 pendapat yang sama menyatakan bahwa 85% pasien npc menunjukkan antibodi terhadap ebv dan juga mengandung ebv iga dalam serum. ebv genom terdeteksi pada sekitar 75-100% kasus npc tipe undifferentiated. ebv dapat dideteksi dengan beberapa teknik seperti sitologi dengan sikat (brush biopsy), pemeriksaan pcr, hibridisasi in situ, dan metode imunohistokimia.20,21 deteksi ebv pada tipe berdiferensiasi berkeratin bervariasi dan umumnya tersebar pada sel intraepitelial yang displastik,7 namun demikian, ada pendapat yang menyatakan bahwa untuk memonitor prognosis pasien npc, plasma yang bebas dari ebv-dna lebih sensitif dan dapat diandalkan dibanding dengan vca/ iga dan ebv/iga.45 ebv secara konsisten terdeteksi pada npc baik di daerah yang insidensinya tinggi maupun rendah.23 tak ada tumor pada manusia yang mempunyai hubungan sekonsisten hubungan ebv dengan npc tipe undifferentiated.17 ebv dikategorikan oleh iarc sebagai karsinogen utama dalam keterkaitannya dengan npc. 17 gen spesifik ebv secara konsisten terekspresi pada npc dan pada lesi displastik sebagai stadium awal npc. terdeteksinya bentuk tunggal dari dna virus menyatakan bahwa tumor terjadi dari proliferasi klonal sel tunggal yang awalnya terinfeksi oleh ebv.22 pada hampir semua sel npc, ebv menggunakan beberapa mekanisme intraselular 146 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 140–147 untuk menyebabkan evolusi onkogenik dari sel yang terinfeksi virus ini. penelitian membuktikan bahwa >85% npc primer menunjukkan aktivitas telomerase yang tinggi melalui mekanisme yang terlibat dalam infeksi ebv yang menyebabkan immortalitas sel.5 infeksi ebv ditemukan pada karsinoma in situ nasofaring yang diduga merupakan lesi prekursor dari npc. penemuan ini menyatakan bahwa infeksi ebv terjadi sebelum pertumbuhan invasif dimulai.17 meskipun penderita npc menunjukkan respon imun akibat terinfeksi ebv namun tidak cukup untuk membunuh sel kanker. peneliti berusaha untuk menggunakan jalur lain untuk meningkatkan sistem imun sebagai target yang lebih tahan terhadap infeksi ebv melalui jalur imunoterapi. salah satu cara yang dapat digunakan adalah mengekstrak sel limfosit t cd4+ dan t cd8+ dari darah penderita npc dan mengubahnya di laboratorium untuk meningkatkan jumlah dan ketahanannya untuk membunuh ebv. sel ini kemudian disuntikkan kembali pada penderita. hasil awal dengan sejumlah kecil penderita menunjukkan hasil yang menjanjikan dan saat ini studi yang lebih besar sedang dilakukan. ilmuwan akhir-akhir ini menemukan bagaimana mutasi gen tertentu terjadi pada sel nasofaring yang menyebabkannya menjadi kanker. uji klinik menggunakan virus untuk menggantikan gen supresor tumor p53 yang rusak pada sel kanker memberikan hasil yang menjanjikan. pendekatan terapi gen ini sedang diteliti.4 antibodi terhadap antigen kapsid dari ebv dan antibodi yang menetralisir ebv dnase merupakan faktor prediktor adanya npc.46 protein virus, lmp1 dan lmp2 mempunyai efek yang sangat nyata terhadap ekspresi gen pertumbuhan sel dalam bentuk pertumbuhan yang sangat invasif dan ganas dari npc. 23 lmp1 merupakan suatu molekul membran integral yang diekspresikan oleh ebv selama masa laten virus dan mununjukkan kemampuan mengaktivasi reseptor famili tnf. lmp1 dibutuhkan untuk mematikan sel b atau monosit dengan diinduksi oleh ebv. potensi transformasi lmp1 dimediasi oleh sitoplasma terminal c yang mengaktivasi bermacam-macam jalur signal selular termasuk nfkb dan jnk. lmp-ct (= lmp mutan) tidak menunjukkan kemampuan sitostatika pada sel yang tak terinfeksi. lmp1-ct menginhibisi ketahanan t-cell line yang mengalami transformasi oleh induksi lmp1 ebv, mengekspresikan keadaan laten virus yang umum dijumpai pada mayoritas tumor terkait ebv pada manusia. 47 lmp1 dapat secara simultan menginduksi dan menginhibisi apoptosis sel b. induksi apoptosis dilakukan oleh transmembrane domain lmp1. inhibisi apoptosis sel b dilakukan oleh terminal karboksi dari lmp1 yang membutuhkan unfolded protein response (upr). ekspresi dari mrna dari bcl-2a1, mengkode apoptotic homolog bcl2, berkorelasi secara langsung dengan ekspresi lmp1 pada sel b positif ebv, dan ekspresinya menginhibisi apoptosis yang diinduksi oleh transmembrane domains lmp1.48 seringnya dijumpai ekspresi dari lmp1 pada npc tipe undifferentiated menunjukkan peran protein virus onkogen ini sebagai molekul efektor kunci dalam patogenesis npc. gen lmp2 diekspresikan pada sel b yang terinfeksi laten. tidak diketahui apakah lmp-2 ikut berperan untuk transformasi pertumbuhan yang dimediasi ebv.31 ekspresi lmp-1 dan -2 onkogen yang mengkode ebv pada jaringan biopsi npc ditemukan lebih tinggi pada pasien usia muda (<30 tahun) yang mengindikasikan replikasi virus yang tinggi dalam sel. keadaan ini juga berkaitan dengan progresifitas lokal npc.3 sensitivitas dan spesifitas penggunaan ebv dna sebagai marker diagnostik dalam sirkulasi untuk mendeteksi adanya npc dengan analisis real-time pcr sangat tinggi.42 deteksi ebv-dna dalam plasma akan meningkatkan aplikasi klinis dari sistem klasifikasi tnm dari npc di dalam praktek berdasarkan tingkatan molekular.49 adanya ebna1 dan dna virus dalam sirkulasi darah tepi merupakan faktor penting yang mengindikasikan risiko tinggi yang signifikan untuk berkembangnya metastasis, juga terhadap survival rate (angka kesintasan hidup) yang rendah.24 kesimpulan dari penulisan ini adalah gen spesifik ebv secara konsisten terekspresi pada npc terutama pada tipe undifferentiated yang merupakan tipe yang paling sering ditemukan, terutama di daerah endemis seperti cina bagian selatan dan pada ras cina dan afrika termasuk mereka yang sudah bermigrasi. biomarker npc terkini seperti pengukuran ebv dna dalam serum; ebv dna dan barf1 (bamh1-a reading frame-1) mrna pada sitologi npc telah dikembangkan untuk diagnosis molekular yang non invasif. daftar pustaka 1. cancer research uk. http://www.cancerresearchuk.org/cancerhelp/type/nasopharyngeal-cancer/about/nasopharyngeal-cancerrisks-and-causes. accessed 2013. 2. globocan (iarc) 2008. section of cancer information. http://www. mdguidelines.com/cancer-oral-cavity-and-oropharynx/definition htttp://globocan.iarc.fr/factsheets/populations/facsheet.asp?u. accessed october 20, 2011. 3. adham m, kurniawan an, muhtadi ai, roezin a, hermani b, gondhowiardjo s, tan ib, middeldorp jm. nasopharyngeal carcinoma in indonesia: epidemiology, incidence, signs, and symptoms at presentation. chin j cancer 2012; 31(4): 185-96. 4. http://www.cancer.org/cancer/nasopharyngealcancer/detailedguide/ n a s o p h a r y n g e a l c a n c e r-w h a ti s n a s o p h a r y n g e a l c a n c e r, 08/08/2012. 5. liu jp, cassar l, pinto a, li h. mechanisms of cell immortalization mediated by eb viral activation of telomerase in nasopharyngeal carcinoma. cell res. 2006; 16(10): 809–17. 6. neville bw, damm dd, white dh. color atlas of clinical oral pathology. 2nd ed. london: deker, hamilton; 2003. p. 230. 7. rubin e, gorstein f, rubin r, schwarting r, strayer d. rubin’s pathology: clinicopathologic foundations of medicine. 4th ed. in: the head and neck: tumors of the nasopharynx. baltimore: lippincott; 2005. p. 1294–5. 8. brennan b. nasopharyngeal carcinoma. orphanet j rare dis 2006; 1: 23. 9. belpomme d, irigaray p, sasco aj, newby ja, howard v, clapp r, hardell l. the growing incidence of cancer: role of lifestyle and screening detection (review). int j oncol 2007; 30(5): 1037–49. 10. leung sf, zee b, ma bb, hui ep, mo f, lai m, chan kc, chan ly, kwan wh, lo ym, chan at. plasma epstein-barr viral deoxyribonucleic acid quantitation complements tumor-nodemetastasis staging prognostication in nasopharyngeal carcinoma. j clin oncol 2006; 24(34): 5414–8. 147sudiono dan hassan: dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring 11. sasaki yf, kawaguchi s, kamaya a, ohshita m, kabasawa k, iwama k, taniguchi k, tsuda s. the comet assay with 8 mouse organs: results with 39 currently used food additives. mutat. res 2002; 519: 103–19. 12. belpomme d, irigaray p, hardell l, clapp r, montagnier l, epstein s, sasco aj. the multitude and diversity of environmental carcinogens. environ res 2007; 105(3): 414–29. 13. irigaray p, newby ja, clapp r, hardell l, howard v, montagnier l, epstein s, belpomme d. lifestyle-related factors and environmental agents causing cancer: an overview. biomed pharmacother 2007; 61(10): 640–58. 14. divisi s, tommaso d, salvemini s, garramone m, crisci r. diet and cancer. acta biomed 2006; 77: 118–23. 15. yuan jm, wang xl, xiang yb, gao yt, ross , yu mc. preserved foods in relation to risk of nasopharyngeal carcinoma in shanghai, china. int j cancer 2000; 85: 358–63. 16. chen l, gallicchio l, boyd-lindsley k. alcohol consumption and the risk of nasopharyngeal carcinoma: a systematic review. nutr cancer 2009; 61(1): 1–15. 17. niedobitek g. epstein-barr virus infection in the pathogenesis of nasopharyngeal carcinoma. mol pathol 2000; 53(5): 248–54. 18. chijioke o, azzi t, nadal d, münz c. innate immune responses against epstein barr virus infection. j leukoc biol 2013; 94(6): 1185–90. 19. parkin dm. the global health burden of infection-associated cancers in the year 2002. cancer 2006; 118(12): 3030–44. 20. bu rgos js. i nvolvement of t he epst ei n-ba r r v i r us i n t he nasopharyngeal carcinoma pathogenesis. med oncol 2005; 22(2): 113–21. 21. stevens sj, verkuijlen sa, hariwiyanto b, harijadi, paramita dk, fachiroh j, adham m, tan ib, haryana sm, middeldorp j m. non i nvasive d iagnosis of nasopha r yngea l ca rci noma: nasopharyngeal brushings reveal high epstein-barr virus dna load and carcinoma-specific viral barf1 mrna. int j cancer 2006; 119(3): 608–14. 22. maxwell jh, kumar b, feng fy, mchugh jb, cordell kg, eisbruch a, worden fp, wolf gt, prince me, moyer js, teknos tn, chepeha db, stoerker j, walline h, carey te, bradford cr. hpv-positive/ p16-positive/ebv-negative nasopharyngeal carcinoma in white north americans. head neck 2010; 32(5): 562–7. 23. raab-traub n. epstein-barr virus in the pathogenesis of npc. semin cancer biol 2002; ; 12(6): 431–41. 24. lin jc, chen ky, wang wy, jan js, liang wm, tsai cs, wei yh. detection of epstein-barr virus dna the peripheral-blood cells of patients with nasopharyngeal carcinoma: relationship to distant metastasis and survival. j clin oncol 2001; 19(10): 2607–15. 25. hou x, zhang l, zhao c, li s, lu lx, han f, shao jy, huang py. prognostic impact of plasma epstein-barr virus dna concentration on distant metastasis in nasopharyngeal carcinoma. ai zheng 2006; 25(7): 785–92. 26. fiorini s, ooka t. secretion of epstein-barr virus-encoded barf1 oncoprotein from latently infected b cells. virol j 2008; 5: 70. 27. rickinson ab, kieff e. epstein-barr virus. in: fields bn, knipe dm, howley, editors. fields virology. 5th ed. philadelphia: lippincottwilliams & wilkins publishers; 2007. p. 2655–700. 28. danve c, decaussin g, busson p, ooka t. growth transformation of primary epithelial cells with a npc-derived epstein-barr virus strain. virology 2001; 288: 223–35. 29. feederle r, neuhierl b, bannert h, geletneky k, shannon-lowe c, delecluse hj. epstein-barr virus b95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection. intl j cancer 2007; 121: 588–94. 30. houali k, wang xh, shimizu y, djennaoui d, nicholls j, fiorini s, bouguermouh a, ooka t. a new diagnostic marker for secreted epstein-barr virus-encoded lmp1 and barf1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma. clin cancer res 2007; 13: 4993–5000. 31. longnecker r. epstein–barr virus latency: lmp2, a regulator or means for epstein–barr virus persistence?. advances in cancer research 2000; 79: 175–200. 32. eliopoulos ag, young ls. lmp1 structure and signal transduction. semin cancer biol. 2001;; 11(6): 435–44. 33. tao y, zhang xg. signal transduction and biological characteristics of eb virus-encoded latent membrane protein 1 and its correlation with cd40. zhongguo yi xue ke xue yuan xue bao. 2004; 26(5): 585–90. 34. soni v, cahir-mcfarland e, kieff e. lmp1 trafficking activates growth and survival pathways. adv exp med biol 2007; 597: 173–87. 35. lin jc, chen ky, wang wy, jan js, liang wm, tsai cs, wei yh. detection of epstein-barr virus dna in the peripheral blood cells of patients with nasopharyngeal carcinoma: relationship to distant metastasis survival. j clin oncol 2001; 19: 2607–15. 36. feng p, ren ec, liu d, chan sh, hu h. expression of epstein-barr virus lytic gene brlf1 in nasopharyngeal carcinoma: potential use in diagnosis. j gen virol 2000; 81: 2417–23. 37. fachiroh j, schouten t, hariwiyanto b, paramita dk, harijadi a, haryana sm, middeldorp jm. molecular diversity of epstein-barr virus igg iga antibody responses in nasopharyngeal carcinoma: a comparison of chinese, indonesian and european cases. j infect. dis 2004; 190: 53–62. 38. hochberg d, middeldorp jm, catalina m, sullivan jl, luzuriaga k, thorley-lawson da. demonstration of the burkitt’s lymphoma epstein-barr virus phenotype in dividing latently infected memory cells in vivo. proc natl acad sci 2004; 101: 239–44. 39. shotelersuk k, khorprasert c, sakdikul s, pornthanakasem w, voravud n, mutirangura a. epstein-barr virus dna in serum/ plasma as a tumor marker for nasopharyngeal cancer. clin cancer res. 2000; 6(3): 1046–51. 40. lin jc, wang wy, chen ky, wei yh, liang wm, jan js, jiang rs. quantification of plasma epstein-barr virus dna in patients with advanced nasopharyngeal carcinoma. n engl j med. 2004; 350: 2461–70. 41. lo ym, leung sf, chan ly, chan at, lo kw, johnson pj, huang dp. kinetics of plasma epstein-barr virus dna during radiation therapy for nasopharyngeal carcinoma. cancer res 2000; 60(9): 2351–5. 42. chan kc, lo ym. circulating ebv dna as a tumor marker for nasopharyngeal carcinoma. semin cancer biol 2002; 12(6): 489–96. 43. chu ea, wu j m, tun kel de, ishma n sl. nasopha r yngeal carcinoma: the role of the epstein-barr virus. medscape j med 2008; 10(7): 165. 44. spano jp, busson p, atlan d, bourhis j, pignon jp, esteban c, armand jp. nasopharyngeal carcinomas: an update. eur j cancer. 2003; 39(15): 2121–35. 45. cao sm, min hq, gao js, hong mh, xiao xb, zhang cq, liu xd, zhang al, guo x. significance of cell-free epstein-barr virus dna in monitoring prognosis of nasopharyngeal carcinoma. ai zheng mar 2003; 22(3):302–6. 46. chien yc, chen jy, liu my, et al. serologic markers of epsteinbarr virus infection and nasopharyngeal carcinoma in taiwanese men. n engl j med 2001; 345 (26): 1877–82. 47. adriaenssens e, mougel a, goormachtigh g, loing e, fafeur v, auriault c, coll j. a novel dominant-negative mutant form of epstein-barr virus latent membrane protein-1 (lmp1) selectively and differentially impairs lmp1 and tnf signaling pathways. oncogene. 2004; 23(15): 2681–93. 48. pratt zl, zhang j, sugden b. the latent membrane protein 1 (lmp1) oncogene of epstein-barr virus can simultaneously induce and inhibit apoptosis in b cells. j virol. 2012; 86(8): 4380–93. 49. zhang y, gao hy, feng hx, deng l, huang my, hu b, cheng g, wu ql, cui nj, shao jy. quantitative analysis of epstein-barr virus dna in plasma and peripheral blood cells in patients with nasopharyngeal carcinoma. zhonghua yi xue za zhi. 2004; 84(12): 982–6. mkgs vol 45 no 2 april-juni 2012.indd issn 1978 3728volume 45 number 2 june 2012 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm (oral and maxillofacial surgery – airlangga university); prof. dr. m. rubianto, drg., ms., sp.perio (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic university of hiroshima, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontica – airlangga university); prof. dr. latief mooduto, drg., m.s., sp.kg (conservative dentistry – airlangga university); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort (orthodontic – airlangga university); prof. dr. istiati soehardjo, drg., ms (oral biology – airlangga university); prof. dr. anita yuliati, drg., m.kes (dental material – airlangga university); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – airlangga university); prof. dr. diah savitri ernawati, drg., m.si (oral medicine – airlangga university); thalca i. agusni, drg., mhped., ph.d., sp.ort (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes (dental public health – airlangga university); dr. elly munadziroh, drg., ms (dental material – airlangga university); priyawan rachmadi, drg., ph.d (dental material – airlangga university); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes (dental radiology – airlangga university); bagus soebadi, drg., mhped (oral medicine – airlangga university); endang pudjirochani, drg., ms., sp.pros (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes (oral biology – airlangga university); susy kristiani, drg., m.kes (oral biology – airlangga university); ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms (orthodontic – airlangga university); devi rianti, drg., m.kes (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros (prosthodontic – airlangga university); an’nissa chusida, drg., m.kes (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg (conservative dentistry – airlangga university); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university); hendrik setiabudi, drg., m.kes (oral biology – airlangga university); otty ratna wahyuni, drg., m.kes (dental radiology – airlangga university); anis irmawati, drg., m.kes (oral biology – airlangga university); yuliati, drg., m.kes (oral biology – airlangga university); retno palupi, drg., m.kes (dental public health – airlangga university); eka augustina, drg., sp.perio (periodontica – airlangga university); febriastuti, drg., sp.kg (conservative dentistry – airlangga university); mega m. puteri, drg., sp.kga (pediatric dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 45 number 2 june 2012: prof. dr. regina titi christinawati, drg., m.sc. (oral biology – gadjah mada university) endrajana, drg., ms., sp.bm (oral and maxillofacial surgery – airlangga university) kus harijanti, drg., ms., sp.pm (oral medicine – airlangga university) dr. retno indrawati, drg., m.si (oral biology – airlangga university) dr. theresia indah budhy, drg., m.kes. (oral biology – airlangga university) dr. indah listiana kriswandini, drg., m.kes. (oral biology – airlangga university) david buntoro kamadjaja, drg., mds., sp.bm. (oral and maxillofacial surgery – airlangga university) agung krismariono, drg., m.kes., sp.perio (periodontic – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 design cover photo by setyabudi, drg., mars., sp.kg contents page printed by: airlangga university press. (151/11.12/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 45 number 2 june 2012 issn 1978 3728 1. the importance of masticatory functional analysis in the diagnostic finding and treatment planning for prosthodontic rehabilitation harry laksono, agus dahlan, and sonya harwasih .................................................................... 59–67 2. the management of chronic traumatic ulcer in oral cavit maharani laillyza apriasari .......................................................................................................... 68–72 3. simplified digital infra red photography: an alternative tool in bite mark forensic investigation haryono utomo and mieke sylvia ................................................................................................. 73–78 4. the relation between salivary siga level and caries incidence in down syndrome children rosdiana and mochammad fahlevi rizal ...................................................................................... 79–83 5. inhibition of 10% alpinia galanga and alpinia purpurata rhizome extract on candida albicans growth fakhrurrazi, rachmi fanani hakim, and cut cahya .................................................................. 84–88 6. pulpal inflammation after vital tooth bleaching with 38% hydrogen peroxide ardiny andriani, juni handajani, and tetiana haniastuti ......................................................... 89–92 7. the increasing of enamel calcium level after casein phosphopeptide-amorphous calcium phosphate covering widyasri prananingrum and puguh bayu prabowo .................................................................... 93–96 8. cytotoxicity of betel leaf (piper betel l.) against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells suprapto ma’at ................................................................................................................................ 97–101 9. deoxypyridinoline level in gingival crevicular fluid as alveolar bone loss biomarker in periodontal disease agustin wulan suci dharmayanti ................................................................................................. 102–106 10. craniofacial morphology of children with complete unilateral cleft lip and palate following labioplasty and palatoplasty sigit handoko utomo, krisnawati, and benny m. soegiharto ................................................... 107–113 11. seroprevalence of herpes simplex virus types 1 and 2 and their association with cd4 count among hiv-positive patients irna sufiawati, sunardhi widyaputra, and tony s. djajakusumah ........................................... 114–120 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 116 automation of gender determination in human canines using artificial intelligence f. fidya1 and bayu priyambadha2 1department of oral biology, faculty of dentistry, universitas brawijaya 2department of software engineering, faculty of computer science, universitas brawijaya malang indonesia abstract background: gender determination is an important aspect of the identification process. the tooth represents a part of the human body that indicates the nature of sexual dimorphism. artificial intelligence enables computers to perform to the same standard the same tasks as those carried out by humans. several methods of classification exist within an artificial intelligence approach to identifying sexual dimorphism in canines. purpose: this study aimed to quantify the respective accuracy of the naive bayes, decision tree, and multi-layer perceptron (mlp) methods in identifying sexual dimorphism in canines. methods: a sample of results derived from 100 measurements of the diameter of mesiodistal, buccolingual, and diagonal upper and lower canine jaw models of both genders were entered into an application computer program that implements the algorithm (mlp). the analytical process was conducted by the program to obtain a classification model with testing being subsequently carried out in order to obtain 50 new measurement results, 25 each for males and females. a comparative analysis was conducted on the program-generated information. results: the accuracy rate of the naive bayes method was 82%, while that of the decision tree and mlp amounted to 84%. the mlp method had an absolute error value lower than that of its decision tree counterpart. conclusion: the use of artificial intelligence methods produced a highly accurate identification process relating to the gender determination of canine teeth. the most appropriate method was the mlp with an accuracy rate of 84%. keywords: sexual dimorphism; canines;artificial intelligence; automation correspondence: fidya, department of oral biology, faculty of dentistry, universitas brawijaya. jl. veteran malang 65145, indonesia. e-mail: fidya.fk@ub.ac.id introduction determining the gender of an individual constitutes an important element within the human identification process which can be accomplished by measuring the skeleton. this measurement is obtained through a comparison of males and females.1 teeth, being one part of the human skeleton, can be measured in both living and deceased humans.2 the measuring of teeth, a method known as odontometry and considered easily applicable, inexpensive, and reliable, provides useful information for the purposes of determining gender within a defined population.3 teeth constitute the most durable part of human body, able to withstand various external irritants; biological, chemical, mechanical, or temperature-related. tooth morphology executes an important role, not only in indicating differences between the activities associated with occlusion and determining the frequency of dental tissue and skeletal anomalies in orthodontic treatment, but also in sex determination.4 the gender difference is evident from the permanent dentition due to hormonal variations specifically affecting the respective size and shape of the two genders prior to adulthood.5 among human teeth, canines demonstrate the highest degree of sexual dimorphism, as demonstrated by studies conducted on multiple populations. canines are retained longer compared to other teeth due to their rarely suffering from caries and periodontal tissue damage. the ability of primates to survive is also shown in the course of the gender identification process by means of odontometric analysis to research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 116–120 doi: 10.20473/j.djmkg.v50.i3.p116-120 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p116-120 http://e-journal.unair.ac.id/index.php/mkg mailto:fidya.fk@ub.ac.id 117117fidya and priyambadha/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 116–120 be dependent upon canine teeth due to their high durability and utility.6 in humans, canine size has been shown to constitute a significant difference between males and females, but the process of manual measurement is of greater duration and requires an expert operator to arrive at a determination of gender. the use of a computer-based gender classification model can significantly accelerate this process since it consists of a mathematical formula implemented by an algorithm that allocates data to certain categories or classes.7 the classification model emerged from data pattern extracts by using the algorithm of an artificial neural network (ann) which constitutes a method of artificial intelligent (ai).8 there are several classification methods potentially employable in this case. however, the most appropriate process for the identification of sexual dimorphism based on canine size should be determined by means of several factors, including; accuracy, error rate, and level of agreement between experts as to the particular classification model to be applied.9 the central focus of ai is the feasibility of developing an intelligence system approximating human ability with the intention of combining software and hardware. within this system, one particular area of observation is referred to as an expert system which stores the ability of an expert in a particular domain within a computer program enabling the machine to make decisions or find solutions. these systems are applied in various fields such as medical diagnostics, exchange markets, robotics, law, science, and entertainment.10 the implementation of this system is expected to accelerate the identification of gender and respond to present day challenges requiring scientific analysis of large amounts of data.11 material and methods the research subjects consisted of 150 student dental cast models, equally divided between male and female, from the dental laboratory collections of universitas brawijaya and universitas airlangga. the model employed was caries-free, with no abnormalities in the canine maxilla or mandibula. measurement of the mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) diameter of maxillary and mandibular canines for all models was effected by means of a tricle brand vernier caliper no.3965-006 prohex technology germany using a millimeter (mm) scale. the method of measuring the canine diameter is shown in figure 1. the diameters of the mesiodistal, buccolingual and diagonal of maxillary and mandibular canines of both genders were inputted into an application computer program implementing the algorithm multi-layer perceptron (mlp). one hundred cast models that equally divided in number between male and female were then used as training data calculated by means of the algorithm naive bayes, decision tree, and mlp in a 3.91 weka program on a computer with an intel core i3 processor. analysis was conducted by the program in order to obtain data patterns. further testing was carried out using 50 new measurement results from 25 members of either gender. measurement data from another 50 cast models that divided equally between male and female was subjected to testing in order to measure the accuracy of gender identification using ai methods. the statistical analysis applied consisted of cohen’s kappa coefficient with a value range of 0-1. the value match between the training data and testing data was calculated using cohen’s kappa coefficient which is a method of measuring the validity and reliability of the data. results the data relating to mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) maxillary and mandibular canines was entered into the weka program 3.9.1. three methods of artificial intelligence were employed that demonstrate the extent of matched value between experts and the results of the system. the implementation results for the three models of classification; naive bayes, decision tree, and mlp are contained in tables 1, 2 and 3. table 1 features simulation method results relating to a research population of 50 canines. application of the naive bayes method classified the correct data as 0.82, the error rate as 0.18, the level of agreement with the expert as 0.64 and the mean absolute error as 0.2288. the mean absolute error is a value that indicates the average absolute error between the system 3 of gender and respond to present day challenges requiring scientific analysis of large amounts of data.11 material and methods the research subjects consisted of 150 student dental cast models, equally divided between male and female, from the dental laboratory collections of universitas brawijaya and universitas airlangga. the model employed was caries-free, with no abnormalities in the canine maxilla or mandibula. measurement of the mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) diameter of maxillary and mandibular canines for all models was effected by means of a tricle brand vernier caliper no.3965-006 prohex technology germany using a millimeter (mm) scale. the method of measuring the canine diameter is shown in figure 1. figure 1. measuring the canine diameter (mm). (note: a= mesiodistal, b= buccolingual, c= mesiobuccal distolingual, d= distobuccal mesiolingual). the diameters of the mesiodistal, buccolingual and diagonal of maxillary and mandibular canines of both genders were inputted into an application computer program implementing the algorithm mlp. 100 cast models (equally divided in number between male and female) were then used as training data calculated by means of the algorithm naive bayes, decision tree, and multilayer perceptron (mlp) in a 3.91 weka program on a computer with an intel core i3 processor. analysis was conducted by the program in order to obtain data patterns. further testing was carried out using 50 new measurement results from 25 members of either gender. measurement data from another 50 cast models (again divided equally between male and female) was subjected to testing in order to measure the accuracy of gender identification using artificial intelligence methods. 3 of gender and respond to present day challenges requiring scientific analysis of large amounts of data.11 material and methods the research subjects consisted of 150 student dental cast models, equally divided between male and female, from the dental laboratory collections of universitas brawijaya and universitas airlangga. the model employed was caries-free, with no abnormalities in the canine maxilla or mandibula. measurement of the mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) diameter of maxillary and mandibular canines for all models was effected by means of a tricle brand vernier caliper no.3965-006 prohex technology germany using a millimeter (mm) scale. the method of measuring the canine diameter is shown in figure 1. figure 1. measuring the canine diameter (mm). (note: a= mesiodistal, b= buccolingual, c= mesiobuccal distolingual, d= distobuccal mesiolingual). the diameters of the mesiodistal, buccolingual and diagonal of maxillary and mandibular canines of both genders were inputted into an application computer program implementing the algorithm mlp. 100 cast models (equally divided in number between male and female) were then used as training data calculated by means of the algorithm naive bayes, decision tree, and multilayer perceptron (mlp) in a 3.91 weka program on a computer with an intel core i3 processor. analysis was conducted by the program in order to obtain data patterns. further testing was carried out using 50 new measurement results from 25 members of either gender. measurement data from another 50 cast models (again divided equally between male and female) was subjected to testing in order to measure the accuracy of gender identification using artificial intelligence methods. 3 of gender and respond to present day challenges requiring scientific analysis of large amounts of data.11 material and methods the research subjects consisted of 150 student dental cast models, equally divided between male and female, from the dental laboratory collections of universitas brawijaya and universitas airlangga. the model employed was caries-free, with no abnormalities in the canine maxilla or mandibula. measurement of the mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) diameter of maxillary and mandibular canines for all models was effected by means of a tricle brand vernier caliper no.3965-006 prohex technology germany using a millimeter (mm) scale. the method of measuring the canine diameter is shown in figure 1. figure 1. measuring the canine diameter (mm). (note: a= mesiodistal, b= buccolingual, c= mesiobuccal distolingual, d= distobuccal mesiolingual). the diameters of the mesiodistal, buccolingual and diagonal of maxillary and mandibular canines of both genders were inputted into an application computer program implementing the algorithm mlp. 100 cast models (equally divided in number between male and female) were then used as training data calculated by means of the algorithm naive bayes, decision tree, and multilayer perceptron (mlp) in a 3.91 weka program on a computer with an intel core i3 processor. analysis was conducted by the program in order to obtain data patterns. further testing was carried out using 50 new measurement results from 25 members of either gender. measurement data from another 50 cast models (again divided equally between male and female) was subjected to testing in order to measure the accuracy of gender identification using artificial intelligence methods. 3 of gender and respond to present day challenges requiring scientific analysis of large amounts of data.11 material and methods the research subjects consisted of 150 student dental cast models, equally divided between male and female, from the dental laboratory collections of universitas brawijaya and universitas airlangga. the model employed was caries-free, with no abnormalities in the canine maxilla or mandibula. measurement of the mesiodistal, buccolingual, and diagonal (mesiobuccal distolingual and distobuccal mesiolingual) diameter of maxillary and mandibular canines for all models was effected by means of a tricle brand vernier caliper no.3965-006 prohex technology germany using a millimeter (mm) scale. the method of measuring the canine diameter is shown in figure 1. figure 1. measuring the canine diameter (mm). (note: a= mesiodistal, b= buccolingual, c= mesiobuccal distolingual, d= distobuccal mesiolingual). the diameters of the mesiodistal, buccolingual and diagonal of maxillary and mandibular canines of both genders were inputted into an application computer program implementing the algorithm mlp. 100 cast models (equally divided in number between male and female) were then used as training data calculated by means of the algorithm naive bayes, decision tree, and multilayer perceptron (mlp) in a 3.91 weka program on a computer with an intel core i3 processor. analysis was conducted by the program in order to obtain data patterns. further testing was carried out using 50 new measurement results from 25 members of either gender. measurement data from another 50 cast models (again divided equally between male and female) was subjected to testing in order to measure the accuracy of gender identification using artificial intelligence methods. a b c d figure 1. measuring the canine diameter; a) mesiodistal; b) buccolingual; c) mesiobuccal distolingual; d) distobuccal mesiolingual. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p116-120 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p116-120 118 fidya and priyambadha/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 116–120 outcomes and the actual results.12 table 2 describes the implementation results of the decision tree method. the correct data was 0.84, the error rate 0.16, the level of agreement with the expert 0.68 and the mean absolute error 0.2528. table 3 contains the results of the mlp method. the correct data was 0.84, the error rate 0.16, the level of agreement with the expert 0.68 and the mean absolute error 0.2049. the three methods of classification demonstrate almost the same performance if implemented on a case identification of canine sexual dimorphism. based on the comparison of results graph in figure 2, the naive bayes method provides lower classification accuracy than either of the other two methods. the correct data was 0.82, the error rate 0.18, the level of agreement with the expert 0.64 and the mean absolute error 0.2049. the decision tree and mlp methods have the same level of accuracy with regard to true/false classification and its kappa value. concerning accuracy, the decision tree and mlp methods constitute appropriate choices. however, one more indicator should be considered in the classification process, namely; the mean absolute error. mlp has a mean absolute error lower than that of decision tree. this value indicates that the average error occurring during the classification process recorded by mlp was lower than that of decision tree. therefore, as far as canine sexual dimorphism is concerned, the most appropriate method delivering optimum performance was that of mlp. discussion the term ‘sexual difference’ refers to the contrasts in the size, height, and appearance of males and females.4 the analysis of teeth provides reliable information with a low incidence of observer error. however, it also needs to provide a high level of measurement accuracy due to the relatively small dimensions involved.13 tooth-based sexual determination relates to the size and shape of the teeth since male teeth are usually larger than those of females.14 certain ancient non-human primates and extinct hominid species exhibit dental dimensions of sexual dimorphism, especially in the case of canine teeth. this dimorphism is most probably the result of intra-species evolutionary selection and rivalry of a sexual, territorial or other resource-based nature.15 certain studies show significantly different results with regard to the diameter of both maxillary and mandibular canines in each of the two genders.16,17 table 1. results of naive bayes method no. indicator value 1. correctly classified instances 0.82 2. incorrectly classified instances 0.18 3. kappa statistic 0.64 4. mean absolute error 0.228 table 2. results of decision tree method no. indicator value 1. correctly classified instances 0.84 2. incorrectly classified instances 0.16 3. kappa statistic 0.68 4. mean absolute error 0.2528 table 3. results of mlp method no. indicator value 1. correctly classified instances 0.84 2. incorrectly classified instances 0.16 3. kappa statistic 0.68 4. mean absolute error 0.2049 5 4. mean absolute error 0.2528 table 5 contains the results of the multi-layer perceptron method. the correct data was 0.84, the error rate 0.16, the level of agreement with the expert 0.68 and the mean absolute error 0.2049. table 5. results of multi-layer perceptron method no. indicator value 1. correctly classified instances 0.84 2. incorrectly classified instances 0.16 3. kappa statistic 0.68 4. mean absolute error 0.2049 figure 2. comparison of correctly classified, incorrectly classified, kappa coefficient and mae of classification methods. the three methods of classification demonstrate almost the same performance if implemented on a case identification of canine sexual dimorphism. based on the comparison of results graph in figure 2, the naive bayes method provides lower classification accuracy 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 correctly classified instances (%) incorrectly classified instances (%) kappa coefficient (k) mean absolute error (mse) comparison of the characteristics of the algorithms multi-layer perceptron naïve bayes decission tree correctly classified instances (%) mlp decision tree incorrectly classified instances (%) kappa coefficient (k) mean absolute error (mse) figure 2. comparison of correctly classified, incorrectly classified, kappa coefficient and mae of classification methods. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p116-120 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p116-120 119119fidya and priyambadha/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 116–120 among the range of human teeth, the canines can be regarded as key to understanding the jaw due to their size, sturdiness, and morphological resistance to caries and periodontal disease because they survive relatively longer than other teeth. their length and sturdy shape also enable them to withstand severe post-mortem conditions such as explosions or air disasters.18 different mean values between males and females are found in all dimensions of canine teeth, mesiobuccal and distolingual. statistical results of the study indicate significant differences between male and female.19 the results presented here are in accordance with those of other odontometric studies found not only in humans, but also in anthropoid apes and certain species of monkey. the canines of the two jaws of the species are more dimorphic than those of others, while the upper teeth of males all possess a much larger buccolingual dimension. another study also confirmed that male tooth size is larger than that of females and canine teeth represent the largest sexually dimorphic teeth.20,21 the gender-determined dimorphism of teeth has been analyzed from a genetic perspective within which it is reported that the y chromosome can affect tooth growth by increasing mitotic activity through amelogenesis and dentinogenesis, resulting in dentine thickening in males. the amelogenin gene, found in both x and y chromosomes, implies dimorphism with regard to tooth size. no significant differences between the sexes with regard to thickness of the enamel were detected, while the deposition of dentine was more prominent in males than females. these findings were in keeping with data from the results of the research conducted. mitosis occurred in the y chromosomes, permitting the deposition of enamel and dentin, while in the x chromosomes deposition was limited to enamel alone. this may explain the difference in size between male and female teeth (especially linear measurements) noted in the literature.14 in contrast, sex hormone modification is of more limited interest.5 a statistically significant difference was also evident in relation to tooth volume. however, due to the effects of aging on the pulp chamber, its volume-related results and ratio to less gender-related dental volume dimorphism are more appropriately studied by age rather than sex.14 gender determination by means of artificial intelligent was performed in several stages. firstly, tooth size data was collected and labelled “canines”. secondly, an mlp learning process was conducted. thirdly, an mlp testing process and the analysis of results were completed. the labeled data was that which had already been categorized as male or female. data relating to canine tooth size was obtained from measurements of dental cast impressions. the next step consisted of the learning process using mlp models, the objective of which was to explore the pattern of existing data on the size of the dataset of labelled canines. the central focus of this learning process was the search for weight value (w) that was appropriate to the classification model. the weighing process was repeated until the optimal weight values for the classification process had been identified. the testing process or trial constituted a procedure to assess the classification model against a set of labelled data. the classification model developed during the learning process would be used to identify gender. the data used in this testing process consisted of labeled data, whose results would be compared with the data system of testing so that the performance of the naive bayes, decision tree, and mlp model could be examined. analysis of the results was affected by comparing the system results and those of the data testing. the process of extracting data was completed by running a learning process on the classification model using labeled canine size data. after obtaining the pattern and applying a classification model, the determination of gender on the basis of canine size could be completed. test results based on a statistical approach were used to arrive at a conclusion. the characteristic of every method was that point at which the model was most relevant to this case. in conclusion, the use of ai methods produced a highly accurate identification process relating to the gender determination of canine teeth. the most appropriate method was the mlp with an accuracy rate of 84%. references 1. srivastava rk, kumar a, ali i, wadhwani p, awasthi p, parveen g. determination of age and sex and identification of deceased person by forensic procedures. univers res j dent. 2014; 4(3): 153–7. 2. artaria md. antropologi dental. 1st ed. yogyakarta: graha ilmu; 2009. p. 102. 3. khangura rk, sircar k, singh s, rastogi v. sex determination using mesiodistal dimension of permanent maxillary incisors and canines. j forensic dent sci. 2011; 3(2): 81–5. 4. duraiswamy p, tibdewal h, patel k, kumar s, dhanni c, kulkarni s. sex determination using mandibular canine index in optimalfluoride and high-fluoride areas. j forensic dent sci. 2009; 1(2): 99–103. 5. guatelli-steinberg d, sciulli pw, betsinger tk. dental crown size and sex hormone concentrations: another look at the development of sexual dimorphism. am j phys anthropol. 2008; 137(3): 324–33. 6. reddy vm, saxena s, bansal p. mandibular canine index as a sex determinant: a study on the population of western uttar pradesh. j oral maxillofac pathol. 2008; 12(2): 56–9. 7. patel k, shah v. implementation of classification using association rule mining. int j emerg technol comput appl sci. 2013; 2(4): 166–9. 8. russell sj, norvig p. artificial intelligence: a modern approach. 3rd ed. new jersey: pearson education; 2010. p. 1132. 9. zhang c, liu c, zhang x, almpanidis g. an up-to-date comparison of state-of-the-art classification algorithms. expert syst appl. 2017; 82: 128–50. 10. silitonga dv, budiharto w. an expert system of measurement of individual knowledge for teeth treatment. int j softw eng its appl. 2015; 9(4): 11–8. 11. miladinovic m, mihailovic b, jankovic a, tosic g, mladenovic d, zivkovic d, duka m, vujicic b. reasons for extraction obtained by artificial intelligence. sci j fac med niš. 2010; 27(3): 143–58. 12. de myttenaere a, golden b, le grand b, rossi f. mean absolute percentage error for regression models. neurocomputing. 2016; 192: 38–48. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p116-120 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p116-120 120 fidya and priyambadha/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 116–120 13. pilloud ma, hefner jt. biological distance analysis : forensic and bioarchaeological perspectives. 1st ed. london: elsevier; 2016. p. 520. 14. de angelis d, gibelli d, gaudio d, noce fc, guercini n, varvara g, sguazza e, sforza c, cattaneo c. sexual dimorphism of canine volume: a pilot study. leg med. 2015; 17(3): 163–6. 15. plavcan jm, ruff cb. canine size, shape, and bending strength in primates and carnivores. am j phys anthropol. 2008; 136(1): 65–84. 16. sharma m, gorea rk. importance of mandibular and maxillary canines in sex determination. j punjab acad forensic med toxicol. 2010; 10: 27–30. 17. da costa ytf, lima lnc, rabello pm. analysis of canine dimorphism in the estimation of sex. brazilian j oral sci. 2012; 11(3): 406–10. 18. kakkar t, sandhu js, sandhu s v., sekhon ak, singla k, bector k. study of mandibular canine index as a sex predictor in a punjabi population. indian j oral sci. 2013; 4(1): 23–6. 19. davoudmanesh z, shariati m, azizi n, yekaninejad s, hozhabr h, oliadarani kf. sexual dimorphism in permanent canine teeth and formulas for sex determination. biomed res. 2017; 28(6): 2773–7. 20. acharya ab, mainali s. univariate sex dimorphism in the nepalese dentition and the use of discriminant functions in gender assessment. forensic sci int. 2007; 173(1): 47–56. 21. angadi p v., hemani s, prabhu s, acharya ab. analyses of odontometric sexual dimorphism and sex assessment accuracy on a large sample. j forensic leg med. 2013; 20(6): 673–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p116-120 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p116-120 issn 1978 3728volume 45 number 1 march 2012 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm (oral and maxillofacial surgery – airlangga university); prof. dr. m. rubianto, drg., ms., sp.perio (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic university of hiroshima, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontica – airlangga university); prof. dr. latief mooduto, drg., m.s., sp.kg (conservative dentistry – airlangga university); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort (orthodontic – airlangga university); prof. dr. istiati soehardjo, drg., ms (oral biology – airlangga university); prof. dr. anita yuliati, drg., m.kes (dental material – airlangga university); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – airlangga university); prof. dr. diah savitri ernawati, drg., m.si (oral medicine – airlangga university); thalca i. agusni, drg., mhped., ph.d., sp.ort (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes (dental public health – airlangga university); dr. elly munadziroh, drg., ms (dental material – airlangga university); priyawan rachmadi, drg., ph.d (dental material – airlangga university); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes (dental radiology – airlangga university); bagus soebadi, drg., mhped (oral medicine – airlangga university); endang pudjirochani, drg., ms., sp.pros (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes (oral biology – airlangga university); susy kristiani, drg., m.kes (oral biology – airlangga university); ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms (orthodontic – airlangga university); devi rianti, drg., m.kes (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros (prosthodontic – airlangga university); an’nissa chusida, drg., m.kes (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg (conservative dentistry – airlangga university); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university); hendrik setiabudi, drg., m.kes (oral biology – airlangga university); otty ratna wahyuni, drg., m.kes (dental radiology – airlangga university); anis irmawati, drg., m.kes (oral biology – airlangga university); yuliati, drg., m.kes (oral biology – airlangga university); retno palupi, drg., m.kes (dental public health – airlangga university); eka augustina, drg., sp.perio (periodontica – airlangga university); febriastuti, drg., sp.kg (conservative dentistry – airlangga university); mega m. puteri, drg., sp.kga (pediatric dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 45 no. 1 march 2012: sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 83/dikti/kep/2009 design cover photo by setyabudi, drg., mars., sp.kg contents page printed by: airlangga university press. (048/03.12/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 45 number 1 march 2012 issn 1978 3728 1. immediate overdenture for improving aesthetic of anterior teeth with periodontal problem fx. ady soesetijo ............................................................................................................................. 1–5 2. orthodontic treatment considerations in down syndrome patients sianiwati goenharto ........................................................................................................................ 6–11 3. the effective concentration of red betel leaf (piper crocatum) infusion as root canal irrigant solution fani pangabdian, slamet soetanto, and ketut suardita .............................................................. 12–16 4. degrees of chitosan deacetylation from white shrimp shell waste as dental biomaterials sularsih, anita yuliati, and coen pramono d ............................................................................... 17–21 5. antimicrobial effect of chlorine dioxide on actinobacillus actinomycetemcomitans in diabetes mellitus rats treated with insulin tantin ermawati and kwartarini murdiastuti ............................................................................. 22–27 6. genetic variability of candida albicans in hiv/aids patient with and without arv therapy and non hiv/aids retno puji rahayu, widiyanti p, and arfijanto m ....................................................................... 28–34 7. relationship between salivary fluor concentration and caries index in 12–15 years old children vidyana pratiwi, dudi aripin, and ame suciati setiawan ........................................................... 35–38 8. crude toxin of aggregatibacter actinomycetemcomitans serotype-b increase parp-1 expression in gingival epithelium ernie maduratna setiawatie ........................................................................................................... 39–42 9. effectiveness of bleaching agent on composite resin discoloration galih sampoerno ............................................................................................................................ 43–47 10. analysis of importance level and quality achievement aspect in dental health service (a case study on waru sidoarjo community dental health service) taufan bramantoro and retno palupi .......................................................................................... 48–51 11. effect of robusta coffee beans ointment on full thickness wound healing yorinta putri kenisa, istiati, and wisnu setyari j ....................................................................... 52–57 202 volume 46, number 4, december 2013 the effect of cpp-acp containing fluoride on streptococcus mutans adhesion and enamel roughness yulita kristanti,1 widya asmara,2 siti sunarintyas,3 and juni handajani4 1 department of conservative dentistry, faculty of dentistry, universitas gadjah mada 2 department of microbiology, faculty of veterinary, universitas gadjah mada 3 department of dental biomaterials, faculty of dentistry, universitas gadjah mada 4 department of oral biology, faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: direct contact between the bleaching agent and the enamel surface results in demineralization, alteration in surface roughness and bacterial adhesion. many studies try to minimize this side effect through different way. purpose: the aim of this study was to determined the effect of calcium phospho peptide-amorphous calcium phosphate (cpp-acp) containing fluoride application before and after bleaching procedure on the adhesion of s. mutans and enamel roughness. methods: the samples were 6 teeth which were divided into 4 groups, and each tooth was cut into four pieces. group a and c were treated with cpp-acp after bleaching, while group b and d were treated with cpp-acp before and after bleaching. cpp-acp used in group c and d was the one that contain fluoride. after treatment, all samples were sterilized, immersed in steril human saliva for one hour, then immersed into s. mutans suspension of 108 cfu. samples were incubated overnight. on the next day the samples were put into steril bhi and vortexed for one minute to detach the bacteria. fifteen ml bhi containing bacteria was poured into tys agar then incubated 37°cfor 48 hours. bacterial colony was counted with colony counter. the sem examination was done on all samples. results: application of desensitizing agent reduced the s.mutans adhesion significantly among groups (p<0.05) except between group a and c. sem evaluation revealed significant differences among groups. conclusion: the application of cpp-acp containing fluoride before and after bleaching was effective to reduce the accumulation of s.mutans colony and enamel roughness. key words: cpp-acp, fluoride, adhesion, streptococcus mutans abstrak latar belakang: kontak langsung antara bahan bleaching dan permukaan enamel menyebabkan demineralisasi, perubahan kekasaran permukaan dan berpengaruh terhadap banyaknya bakteri streptococcus mutans (s. mutans) yang melekat. banyak peneliti mencoba meminimalkan efek samping ini dengan cara yang beragam. tujuan: penelitian ini bertujuan untuk meneliti efek aplikasi cpp-acp mengandung fluor sebelum dan setelah bleaching terhadap adhesi s.mutans dan kekasaran enamel. metode: sampel penelitian adalah 6 buah gigi yang dibagi dalam 4 kelompok, kemudian masing-masing gigi dibelah menjadi 4 bagian. kelompok a dan c diaplikasi dengan cpp-acp setelah bleaching, sedang kelompok b dan d diaplikasi cpp-acp sebelum dan setelah bleaching. cpp-acp yang digunakan pada kelompok c dan d adalah yang mengandung fluor. setelah perlakuan, semua sampel disterilkan dan direndam dalam saliva steril, lalu direndam dalam suspensi s. mutans 108 cfu dan diinkubasi 24 jam. hari berikutnya sampel dimasukkan dalam bhi steril, divortex 1 menit untuk melepaskan bakteri. lima belas ml bhi yang berisi s. mutans tersebut diambil untuk dikultur dalam agar tys dan diinkubasi 37°c selama 48 jam. bakteri yang tumbuh dihitung dengan colony counter. pemeriksaan sem dilakukan untuk meneliti permukaan enamel. hasil: aplikasi cpp-acp(f) menurunkan jumlah bakteri yang melekat pada enamel secara signifikan (p<0,05) pada semua kelompok, kecuali antara kelompok a dan c. simpulan: aplikasi cpp-acp mengandung fluor sebelum dan sesudah bleaching efektif mengurangi akumulasi s. mutans dan kekasaran pada permukaan enamel. kata kunci: cpp-acp, fluor, adhesi, streptococcus mutans research report 203kristanti, et al.: the effect of cpp-acp containing fluoride correspondence: yulita kristanti, c/o bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara yogyakarta 55281, indonesia. e-mail: litaugm11@gmail.com introduction information concerning esthetic dentistry that can be accessed easily make people more concerned to improve their performance. brighter teeth are something that encourages people to see a dentist to bleach their teeth. bleaching can be categorized into 2 groups: intracoronal bleaching and extracoronal bleaching. intracoronal bleaching is performed to bleach non vital teeth, while extracoronal bleaching is indicated for vital teeth. bleaching for vital teeth can be classified into at home bleaching (professionally dispensed) and in office bleaching (professionally administered).1 people prefer their teeth to be bleached by a dentist (in office bleaching technique) than to do it by themselves because it does not take a long time to see the bleaching result. beside that, they also feel much secured if the bleaching process is done by the dentist because of better gingival protection and better sensitivity control. this procedure is suitable for patient with bleaching tray intolerance although it result in higher tooth sensitivity than at home bleaching because of higher concentration used in this technique.2 the mechanism of bleaching discolored teeth still unclear. it differs according to the type of discoloration involved, the chemical and physical condition at the time of the reaction. bleaching agent mainly oxidizers, slowly degrading organic structure into chemical product such as carbon dioxide that are lighter in color. the oxidation reduction reaction that occurs during bleaching is known as a redox reaction. the mechanism of bleaching involve the degradation of the extracellular matrix and oxidation of chromosphores located within enamel and dentin.3,4 beside tooth sensitivity, there are several bleaching side effect that must be anticipated such as mineral loss, surface roughness and increasing bacterial adhesion such as streptococcus mutans (s. mutans). s. mutans has ability to metabolize many sugars and produces various organic acid. s. mutans is strong acid producer hence cause an acidic environment. consequently it lowers the ph of the surrounding environment that will lead to tooth demineralization.5,6 according to mithra and moeny remineralization using desensitizing agent calcium phospho peptide-amorphous calcium phosphate (cpp-acp) 3 minutes twice a day was achieved after 35 days.7 meanwhile the bleaching effect results in enamel porousity which enhances trans–enamel diffusion to reach deep area of dentin and pulp chamber.8 in vitro studies have demonstrated that a high concentration of toxic components released from hidrogen peroxide 35% bleaching gels used for in office treatment are capable of diffuse through enamel and dentin and they significantly decrease the metabolism of pulp cells.9,10 several studies have reported the effect of hydrogen peroxide on the structure of dental tissue such as pulp sensitivity, cervical resorption, release of selected components of dental materials and alteration of enamel surface.4,8-10 however little has been reported about the adhesion of s. mutans and its relationship with the difference morphological alteration of the outer enamel surface. the aim of this study was to determine the effect of fluoride and non fluoride desensitizing agent that applied prior to and after bleaching procedure on the adhesion of s. mutans to enamel and enamel roughness. materials and methods six maxillary extracted premolar were used as sample of this study. samples were cut into 4 pieces. furthermore, the 24 pieces classified into 4 groups, each group contains 6 specimen. group a was treated using non fluoride cppacp after bleaching procedure was performed. group b was treated using non fluoride cpp-acp before and after in office bleaching was performed. group c was treated using cpp-acp containing fluoride after bleaching was performed, and group d was treated using cppacp containing fluoride before and after bleaching was performed. group a was bleached using hydrogen peroxide 40% for one hour, washed, dried, followed by the application of non fluoride cpp-acp for 30 minutes, immersed into human saliva for one hour then immersed into s. mutans suspension of 108 cfu, incubated 37°c (24 hours). the day after the teeth were put into 3 ml sterilized brain heart infusion (bhi), vortex for 1 minute.11 after diluting 10,3 0.1 ml bhi containing s. mutans were cultured in trypticase-soy-sucrose-bacitracin (tys 20b) followed by incubating at 37°c for 48 hours. s. mutans colony were counted using colony counter. in group b, before in office bleaching procedure was performed, the non fluoride cppacp was applied for 30 minutes then the samples were washed and dried. after bleaching, the non fluoride cppacp was aplied again, followed by washing and immersed into human saliva for one hour, immersed into s. mutans suspension 108 cfu, incubated 37°c (24 hours). the day after the teeth were put into 3 ml sterilized bhi, vortex for 1 minute.12 after diluting 10,3 0.1 bhi containing s. mutans were cultured in tys 20b followed by incubating 37 °c for 48 hours.12 s. mutans colony were counted using colony counter. group c was treated with the same procedure as group a, but in group c the cpp-acp used was the one that containing fluoride. cpp-acp (f) was also used in group d, it was applied before and after bleaching procedure. 204 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 202–206 results the average of s. mutans colony accumulation was shown in figure 1. the result showed that the the accumulation colony of s. mutans decrease when the cppacp was used before and after bleaching. cpp-acp (f) also reduced the amount colony of s. mutans (figure 1). the one way anova showed there was a significant difference (p<0.005) of the accumulation colony of s. mutans among groups with once and twice cpp-acp application, with and without fluoride (table 1). the result of this research showed there was a significant difference between groups (p<0.005), except between group a and c (p>0.005). this may be caused by low fluoride dosage when the da applied only once. there were scanning electron microscope (sem) evaluation for detecting enamel surface roughness with 2000 magnification. in group a (cpp acp after bleaching only): the dissolved periphery of enamel prism and the porosities could be seen. areas of remineralization noted although at some places dissolved prism core and dissolved interprismatic substance are still evident (figure 2). in group b (cpp acp before and after bleaching) the configuration of the enamel was apparent with few porous defect. areas of remineralization are evident clearly (figure 3). in group c (cpp-acp (f) after bleaching only) the porosities still could be seen clearly, but not as much as in group a. certain areas of remineralization were evident (figure 4). in group d (cpp acfp before and after bleaching): the areas of mineralized deposits were more evident. the different pattern among group a, b, c and d maybe due to variation in crystallite orientation in the enamel prism (figure 5). from this different morphological alteration point of view, sem evaluation support the result figure 1. average of s. mutans accumulation after once and twice da application, with and without fluoride note: a. bleaching → cpp-acp b. cpp-acp → bleaching → cpp-acp c. bleaching → cpp-acp (f) d. cpp-acp (f) → bleaching → cpp-acp(f) 6 figure 2. group a. figure 3. group b. figure 4. group c. figure 5. group d. in group a (cpp acp after bleaching only): the dissolved periphery of enamel prism and the porosities can be seen. areas of remineralization noted although at some places dissolved prism core and dissolved interprismatic substance are still evident (figure 2). in group b (cpp acp before and after bleaching) the configuration of the enamel is apparent with few porous defect. areas of remineralization are evident clearly (figure 3). in group c (cpp acfp after bleaching only) the porosities still can be seen clearly, but not as much as in group a. certain areas of remineralization are evident (figure 4). in group d (cpp acfp) before and after bleaching): the areas of mineralized deposits are more evident. the different pattern among group a, b, c and d maybe due to variation in crystallite orientation in the enamel prism (figure 5). from this different morphological alteration point of view, sem evaluation support the result of this research. the higher the topography irregularities (roughness) of a material, the more the s. mutans colony could be counted. figure 2. group a (bleaching→cpp-acp) the dissolve peripheral enamel prism and porosites apreared 6 figure 2. group a. figure 3. group b. figure 4. group c. figure 5. group d. in group a (cpp acp after bleaching only): the dissolved periphery of enamel prism and the porosities can be seen. areas of remineralization noted although at some places dissolved prism core and dissolved interprismatic substance are still evident (figure 2). in group b (cpp acp before and after bleaching) the configuration of the enamel is apparent with few porous defect. areas of remineralization are evident clearly (figure 3). in group c (cpp acfp after bleaching only) the porosities still can be seen clearly, but not as much as in group a. certain areas of remineralization are evident (figure 4). in group d (cpp acfp) before and after bleaching): the areas of mineralized deposits are more evident. the different pattern among group a, b, c and d maybe due to variation in crystallite orientation in the enamel prism (figure 5). from this different morphological alteration point of view, sem evaluation support the result of this research. the higher the topography irregularities (roughness) of a material, the more the s. mutans colony could be counted. figure 3. group b (cpp-acp→bleaching→cpp-acp) the configuration of enamel revealed few porous defect 6 figure 2. group a. figure 3. group b. figure 4. group c. figure 5. group d. in group a (cpp acp after bleaching only): the dissolved periphery of enamel prism and the porosities can be seen. areas of remineralization noted although at some places dissolved prism core and dissolved interprismatic substance are still evident (figure 2). in group b (cpp acp before and after bleaching) the configuration of the enamel is apparent with few porous defect. areas of remineralization are evident clearly (figure 3). in group c (cpp acfp after bleaching only) the porosities still can be seen clearly, but not as much as in group a. certain areas of remineralization are evident (figure 4). in group d (cpp acfp) before and after bleaching): the areas of mineralized deposits are more evident. the different pattern among group a, b, c and d maybe due to variation in crystallite orientation in the enamel prism (figure 5). from this different morphological alteration point of view, sem evaluation support the result of this research. the higher the topography irregularities (roughness) of a material, the more the s. mutans colony could be counted. figure 4. group c (bleaching→cpp-acp (f)) the porosities could be seen and certain area of remineralization were evident groups c ol on y n um be r of m s 205kristanti, et al.: the effect of cpp-acp containing fluoride 6 figure 2. group a. figure 3. group b. figure 4. group c. figure 5. group d. in group a (cpp acp after bleaching only): the dissolved periphery of enamel prism and the porosities can be seen. areas of remineralization noted although at some places dissolved prism core and dissolved interprismatic substance are still evident (figure 2). in group b (cpp acp before and after bleaching) the configuration of the enamel is apparent with few porous defect. areas of remineralization are evident clearly (figure 3). in group c (cpp acfp after bleaching only) the porosities still can be seen clearly, but not as much as in group a. certain areas of remineralization are evident (figure 4). in group d (cpp acfp) before and after bleaching): the areas of mineralized deposits are more evident. the different pattern among group a, b, c and d maybe due to variation in crystallite orientation in the enamel prism (figure 5). from this different morphological alteration point of view, sem evaluation support the result of this research. the higher the topography irregularities (roughness) of a material, the more the s. mutans colony could be counted. figure 5. group d (cpp-acp (f)→bleaching→cpp-acp (f) the areas of mineralized deposit were more evident of this research. the higher the topography irregularities (roughness) of a material, the more the s. mutans colony could be counted. discussion the result showed there were significant difference occured on the accumulation of s. mutans colony among group a and b, group a and d, group b and c. this fenomena has suggested that there is a relationship between the accumulation of s. mutans with different surface roughness among group a and b, group a and d, group b and c as a result of bleaching treatment with tooth bleaching containing 40% h2o2 (figure 2-5). tooth bleaching releases reactive free radicals that will influence the reducing agent, so the yellowish pigment (xanthopterin) will be oxidated and become a whitening pigmen (leucopterin).13 bleaching agent can produce undesirable effect such as hypersensitivity, gingival irritation, micromorphological defect due to demineralization and effect on restorative material.1 the result of this research shown that highest mean value of s. mutans colony can be noticed in group a, followed by group c, group b and group d. this means that application cpp-acp (either with or without fluor) before and after bleaching were effective in reducing s. mutans colony. mean value of s. mutans colony in group c below the mean value of s. mutans in group a. this means that fluoride containing da showed better result in reducing s. mutans colony. sem evaluation showed that group a display the highest roughness value, followed by group c, b and d. surface roughness can be measured by qualitative method such as sem or quantitative method such as profilometry. sem is a powerful magnification tool that utilizes focused beams of electron to obtain information while profilometer is a simple tool to measure surface’s profile in order to quantify its roughness. it determines line roughness, in either vertical or horizontal direction, but it can not penetrate certain micro irregularities. this tool is easy to operate and rapid to obtain the measuring result. sem and profilometry have limitation in defining surface topography. the electron beam techniques in sem does not allow visualization of three dimensional surface texture.14,15 according to katsikogianni, the first stage of bacterial adhesion consist of the initial attraction of the cells to the surface followed by absorption and attachment. at the second stage, molecular-specific reaction between bacterial surface structure and substratum surface become predominant.16 there was no significant difference occured between group a and c. this fact can be studied from three perspectives. first, it was suggested that s. mutans can adapt to fluoride because of either widespread or longterm use of fluoride. there are few alteration could be detected in fluoride resistant s. mutans, one of them is the fatty acid membrane. membrane of fatty acid plays an important role in maintaining normal physiological cell function, tolerance of physiological stressor including oxidative stress.17,18 cell membranes, which are structurally made of large amount of polyunsaturated fatty acid are highly susceptible to oxidative attacks. oxidative attack will result in oxidative stress when the balance between the existace of reactive oxygen species and antioxidant defence is lost. the free radical mediated oxidative stress result in oxidation of membrane of lipoprotein, glycoxidation, oxidation of dna, subsequently cell death result.19 in fluoride resistance s. mutans, the unsaturated/ saturated ratio during the stationary phase was higher than the wild-type strain. a significant difference in the amount of long chain mono saturated fatty acids between fluoride resistant strain and wild-type strain was detected in acidic condition. previous study conducted by zhu et al.17 showed that the level of gene that is responsible for biosynthesis fatty acid (fabm) rna in the fluoride resistant-strain was significantly higher than that of the wildtype strain in the acidic condition as well although the sequence of the fabm gene was the same in the fluoride resistant strain as table 1. one way anova of the accumulation colony of s. mutans after once and twice desensitizing agent application, with and without fluoride sum of squares df mean square f sig between groups 104163.792 3 34721.264 103.028 .000 within groups 6740.167 20 337.008 total 110903.958 23 206 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 202–206 the one reported for the wild-type strain. the fabm gene sequence from the fluoride resistant strain was 100% homologous to the wild-type strain. a single gene product of fabm in s. mutans is responsible for the synthesis of monounsaturated fatty acid and is necessary for survival in acidic environment. alteration in the lipid content of membranes of an organisme is the major importance in response to environmental stress. these findings are consistent with zhu et al.17 if that fluoride-resistant s. mutans exhibit greater ability to resist acid stress compare to wild-type s. mutans. another possibilities that influence this result is the concentration of the cpp-acp containing fluoride in group c is to low (900 ppm). if the cpp-acp only applied once after bleaching it means the fluoride delivered still below the traditional treatment as in mouth rinse or in tooth paste (1500 ppm). many researcher still confuse to formulate the precision consentration of fluoride, so debate around fluoride consentration still persist. treatment with 5000 ppm fluoride significantly enhanced remineralization and inhibit demineralization when compared with treatment with 1500 ppm.19 in group d, cpp-acp containing fluoride was delivered twice before and after bleaching. it means the total fluoride delivered was 1800 ppm. present findings showed lower surface roughness value and lower s. mutans colony accumulation (data not shown). our results indicated that fluoride combinated with cpp acfp that was delivered before and after bleaching (group d) showed lower s. mutans colony accumulation than cpp acp without fluoride that delivered in the same manner (group b) this is because fluoride can inhibit glucocyltranferase produced by s. mutans by inhibiting enolase that has an important role in glycolysis. beside that, fluoride was involved in developing complex metalfluor, usually aluminium floride (alf4) that account for inhibiting glucan formation from glucose 6–phosphatase that result in inhibiting glucosyltransferase activity.20 on the colony counting method, the difference between alive and death s. mutans could not be seen, so it is possible to have false positive. the surface roughness value of group a seems to be the same as in group c. this condition could be as a result of contaminant such as residual of the desensitizing agent used that can not be rinsed because of the certain retentive topography on the enamel surface. the variation of enamel topography it self has wide diversity. every part of enamel surface has diferent surface roughness value that can influence its agent retention capability. in this study the surface roughness value of human enamel before treatment are almost above 1 µm. it means that the starting point of surface roughness value was above critical value for bacterial colonization (0.2 µm).21 the study suggested that the application of cpp-acp containing fluoride before and after bleaching was effective in reducing s. mutans accumulation and enamel roughness. references 1. greenwall l, dunitz m. bleaching techniques in restorative dentistry. london: the livery house; 2001. p. 1-60. 2. gurgan s, cakir fy, yazici e. different light-activated in office bleaching system: a clinical evaluation. lasers med sci 2010; 25(6): 817-22. 3. ingle ji, backland lk. endodontics. 4 th ed. london: hamilton; 2002. p. 868-75. 4. goldberg m, grootveld m, lynch e. undisreable and adverse effect of tooth-whitening products: a review. clin oral investig 2010; 14(1): 1-10. 5. fozo em, quivey rg jr. shift in membrane fatty acid profile of streptococcus mutans enhace survival in acidic environments. appl environ microbiol 2004; (70)2: 929-6. 6. forssten sd, bjorklund m, ouwehand ac. streptococcus mutans, caries and simulation models. nutrients 2010; 2(3): 290-8. 7. hegde mn, moeny. remineralization of enamel subsurface lesions with casein phosphopeptide-amorphous calcium phosphat: a quantitative energy dispersive x ray analysis using scanning electron microscopy: an in vitro study. j conserv dent 2012; 15(1): 61-7. 8. gökay o, müjdeci a, algin e. in vitro peroxide penetration into the pulp chamber from newer bleaching product. int endod j 2005; 38(8): 516-20. 9. coldebella cr, riberio ap, sacono nt, trindade fz, hebling j, costa ca. indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblastlike cell. braz dent j 2009; 20(4): 267-4. 10. trindade fz, ribeiro ap, sacono nt, oliveira cf, lessa fc, hebling j, costa ca. trans-enamel and trans-dentinal cytotoxic effects of a 35% hp bleaching gel on cultured odontoblast cell lines after constitutive applications. int endod j 2009; 42(6): 516-524. 11. elsayed me, sultan ko, el-hameed hma, elsayed ae. detection of bacterial colonization around cobalt chromium versus zirconium copings on natural teeth supporting overdenture. two different in vitro studies. j am sci 2012; 8(7): 799-803. 12. anggraeni a, yuliati a, nirwana i, perlekatan koloni streptococcus mutans pada permukaan resin komposit sinar tampak. maj ked gigi (dent j) 2005; 38(1): 8-11. 13. mithra nh, krishna s, shishir s. overview of in-office bleaching of vital teeth. irjp 2012; 3(11): 12-6. 14. giacomelli l, derchi g, frustaci a, bruno o, covani u, barone a, de santis d, chiapelli f. surface roughness of commercial composites after different polishing protocols: an analysis with atomic force microscopy. the open dent j 2010; 4: 191-4. 15. kakaboura a, fragouli m, rahiotis c, silikas n. evaluation of surface characteristics of dental composites using profilometry, scanning electron, atomic force microscopy and gloss-meter. j mater sci mater med 2007; 18: 155-63. 16. katsikogianni m, missirlis yf, concise review of mechanism of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interaction, european cell and matrials 2004, 8:37-57. 17. zhu l, zhang z, liang j. fatty-acid profiles and expression of the fabm gene in a fluoride-resistant strain of streptococcus mutans. arch oral biol 2012; 57(1): 10-4. 18. singh, sc, sinha rp, hader dp. role of lipids and fatty acids in stress tolerance in cyanobacteria 2002; 41: 297-308. 19. ten cate jm, buis mj, miller cc, exterkate ra. elevated fluoride product enhance remineralization of advanced enamel lesions. j dent res 2008; 87(10): 943-7. 20. al-jumaily efa, al-mudhalal nha, muhimen naa. the effects of inhibitors and anti gtf-1b antibody on growth of mutans streptococci streptococcus sobrinus (serotype g) n10 strain j pharmacy 2013; 3(4): 5-9. 21. botta ac, mollica fb, riberio cf, araujo mam, nicolo rd, balducci i. influence of topical acidulated phosphate fluoride on surface roughness of human enamel and different restorative materials; rev odonto cienc 2010; 25 (1): 83-7. 124 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 124–127 original article the possibility of polymorphonuclear leukocyte activation in dental socket healing by freeze-dried aloe vera induction pratiwi soesilawati, ester arijani rachmat, ira arundina and nita naomi department of oral biology, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: research has proved that aloe vera has anti-inflammatory, anti-bacterial, and immunomodulator properties that can accelerate the healing process. these properties could be beneficial in the tooth extraction wound-healing process. neutrophils are the first inflammatory cells to invade and are the predominant cell markers in the wound for 24 hours after injury. polymorphonuclear leukocytes (pmn) provide initial protection against micro-organisms, as they engulf and digest foreign bodies. purpose: the aim of this study was to prove the effect of aloe vera 90% gel application on the pmn count in the healing process of cavia cobaya following tooth extraction. methods: this laboratory experimental research was done using post-test only control group design. the lower incisive of twelve male cavia cobayas were extracted then divided into four groups. group p1 and p3 had 90% aloe vera gel applied into the socket and sutured; group k1 and k3, received no 90% aloe vera gel application and acted as controls. samples from group p1 and k1 were terminated and had their mandibula taken one day after tooth extraction, and those from group p3 and k3 were terminated after three days. tissues were then processed into histology slides, and pmn cells were counted. results: there were significant differences in the pmn count between groups k1, p1, and k3 compared with p3 (p < 0.05). higher pmn count was shown on the group containing aloe vera 90% gel compared with control group, both on day one and three after tooth extraction. conclusion: aloe vera 90% gel can increase the pmn count in the inflammation phase of the tooth extraction wound-healing process. keywords: aloe vera 90% gel; pmn count; tooth extraction; wound healing correspondence: pratiwi soesilawati, department of oral biology, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr moestopo 47 surabaya, 60132 indonesia. email: pratiwi-s@fkg.unair.ac.id introduction tooth extraction is the removal of a tooth, which creates a wound in its socket. post-extraction wounds normally go through a healing process, but sometimes complications can slow down this process. some research stated that post-extraction wounds treated by the use of various natural products, such as aloe vera, can minimise the risk of complications and promote wound healing because of their therapeutic properties.1 wound healing is a complex phenomenon. immediately after injury, extravasated blood constituents form a hemostatic plug. the inflammatory stage is the first attempt of the repair process, and corresponds to the proliferation and remodelling stage that constitutes wound healing. the process begins with the infiltration of leukocyte, as a primary cellular component of inflammation response in the wound area.2 neutrophils are the first type of white blood cell to be attracted into the wound within a few hours and are the predominant cell marker in the injury site for 24 hours after injury. polymorphonuclear leukocyte (pmn) provides initial protection against micro-organisms, as they engulf and digest foreign bodies. after two to three days, mature and senescent pmns undergo apoptosis and phagocytosis, which is then cleared locally by tissue phagocytes, such as macrophage.3–5 additional pmn cell functions that contribute to tissue restoration by promoting angiogenesis and cell proliferation are important in the wound-healing process. these functions dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p124–127 mailto:pratiwi-s@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p124-127 125soesilawati et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 124–127 have also been uncovered in tumours, sterile inflammation and ischemic injury. pmn cell functions are impaired in patients with chronic diseases ssociated with compromised healing, such as diabetes. the use of a substance that can improve pmn cells will significantly help the woundhealing process for people with this condition.6 the aloe plant consists of more than 360 species, with the most commonly used being the aloe vera species (aloe barbadensis miller). research involving cytotoxic tests of variable concentrations of aloe vera against fibroblast cell culture shows that a concentration of aloe vera 90% is nontoxic against fibroblast cell culture.7 the research result states that aloe vera promotes wound healing by improving cell regeneration and increasing the number of phagocytic cells, that it has a hypoglycemic effect, is antiinflammatory, anti-bacterial, anti-viral, anti-fungal, and is an immunomodulator.8,9 aloe vera contains mannose-6-phospates, chromones, β-sitosterol, and bradykinase as an anti-inflammation agent. acemannan, which is derived from aloe vera, is an immunomodulator that stimulates various cytokines and growth factors in the wound-healing process. this property of aloe vera might impact on the pmn count through a vascular (vasodilatation/vasoconstriction), immunomodulator, and anti-inflammation effect during the inflammation phase in the wound-healing process following tooth extraction.9,10 the purpose of this research is to prove the effect of aloe vera 90% gel application on the pmn count in the wound-healing process following tooth extraction of cavia cobaya. materials and methods this was a laboratory experimental research with posttest only control group design. twelve healthy male samples of cavia cobaya, weight 300–500-gram, age 2–3 months old, were used for right mandibular incisive extraction. samples were divided into four groups (p1, p3, k1, k3). groups p1 and p3 were the experimental groups, in which aloe vera 90% gel was applied and sutured in the post-extraction socket. groups k1 and k3 were the control groups in which aloe vera 90% gel was not applied in the post-extraction socket. samples from groups p1 and k1 were terminated one day after tooth extraction; those in groups p3 and k3 were terminated three days after the extraction. aloe vera (l.) webb species were used in this research. they underwent freeze drying and were dissolved with natrium carboxymethyl cellulose (cmc-na) to form a gel with 90% concentration. the right mandibular incisive was carefully extracted using a needle holder and elevator, and the socket was irrigated with sterile aquadest. cavia cobaya was then euthanised with a lethal dose of 10% ether. the mandibula was separated from its joint and, for the fixation process, was first put into formalin buffer 10% solution for 24 hours at -80º c, then underwent decalcification using a nitric acid solution for 48 hours. after the mandibular bone had softened, the lower right incisor region was cut into rectangular pieces and put into formalin buffer 10% solution for 24 hours at -80º c. the remaining tissue was thinly sliced into 1x1x1/2 cm, then was dehydrated, cleared, embedded, blocked, sectioned into 5–7 micron width, and stained with haematoxylin and eosin solution so the specimen could be analysed histopathologically.11 the histology slide was then put under an olympus microscope bx53 and olympus camera dp73, which had a sensor that was connected to the computer with cell sens imaging system software. the magnification was set to 40x, and the resulting image were shown on a computer screen with live mode action, operating the microscope to get a clear field of pmn and its features. the snapshot feature was then used to get an exact photo of recognised pmn, then the cell d imaging system by olympus was used to count the pmn automatically, and the results were ready to be analysed.12 the data was statistically processed using the kruskal-wallis and post hoc (tukey hsd) test (p<0.05). results there is a pmn count difference between the experimental and control group, with a higher pmn count in the experimental group on day one (p1) and day three (p3), as shown in figure 1. table 1 shows the pmn count of the control group on day one (k1), the experimental group on day one (p1), the control group on day three (k3), and the experimental group on day three (p3). aloe vera’s ability to inhibit pmn infiltration on day three is discovered by counting the pmn mean percentage between groups shown by the following equation: a b dc figure 1. histopathology specimen a) control group day one (k1), b) experimental group day one (p1), c) control group day three (k3), and d) experimental group day three (p3). black arrows show whole and rounded pmn cells, while blue arrows show pmn shrinkage with cytoplasmic projections. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p124–127 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p124-127 126 soesilawati et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 124–127 table 2. increased percentage of mean pmn cell number between control and experimental group control group = 358.25% experimental group = 290.18% table 3. tukey hsd test result between control group and experimental group sig. k1 p1 k3 p3 k1 0.547 0.472 0.001* p1 0.999 0.004* k3 0.004* p3 table 1. pmn count by histopathological examination group cell count mean standard deviation k1 6 4 2 ± 0.1754 2 p1 17 17 1 ± 0.17518 16 k3 15 18.33 3.05 ± 0.25319 21 p3 40 66.33 22.85 ± 0.36278 81 table 2 shows that the increase in percentage of mean pmn count in the experimental group is less than in the control group, so the experimental group has a stronger ability to inhibit pmn infiltration compared with control group. the post hoc (tukey hsd) test was then used to compare the 4 groups of variables, and the results can be seen in table 3. group k1 and p3 (p = 0.001), p1 and p3 (p = 0.004), k3 and p3 (p = 0.004) have a p score < 0.05, showing there is a significant difference in the pmn count. group k1 and p1 (p = 0.547), k1 and k3 (p = 0.472), p1 and k3 (p = 0.999) show no significancy of pmn count difference. discussion post extraction-socket wound healing starts with thrombus formation that stimulates pmn infiltration to the wound site.13 pmn then undergo apoptosis and phagocytic clearance by macrophage, and are replaced with macrophage and other mononuclear leukocytes on day two or day three.2,5 the pmn count of the control (k1) and experimental group (p1) on day one was less than on day three (table 1), while the theory stated that extravasated neutrophils influx after wounding increased most rapidly over the initial 12 hours and reached a maximum value between day one and two, with the level plateauing up to day three, and decreasing precipitously on day five.14 this might be caused by the pmn count on day three having included apoptotic pmn, which had not undergone phagocytic clearance by macrophages. in figure 1, on day three, both group k3 and p3 showed some pmn cells still in an apoptotic state (blue arrows show pmn with cytoplasmic projections). another cause for the higher pmn count on day three is the upregulated anti-apoptotic genes, immediate early response gene x-1 (iex1) also known as immediate early response 3 (ier3) and bcl2 related protein a1 (bcl2a1) defined as target genes of nuclear factor κb (nf-κb), a transcription factor that is activated through interleukin 1 beta (il-1β) and tumour necrosis factor alpha (tnf-α) signalling. because macrophages and pmns both produce il-1β and tnf-α at sites of infection, it might be responsible for the inhibition of normal cell apoptosis.15 the normal process of engulfment of apoptotic neutrophils by tissue phagocytes triggers anti-inflammatory signals, which decrease their production of interleukin 23 (il-23), a key cytokine for the induction of interleukin 17 (il-17) by both innate and adaptive immune cells. the resulting inhibition of il-17 production, in turn, leads to decreased production of granulocyte-colony stimulating factor (g-csf) by cells such as fibroblasts, thereby limiting stimulus for neutrophils production to maintain steady-state neutrophil counts.5 another possibility is described by a research of full thickness skin burn on rats treated by aloe vera 95% gel, which shows there was a significant decrease of pmn infiltration in the granulation tissue that appeared at the base of the burnt area detected on day four.16 the pmn count on day one of the experimental group (p1) is higher than the control group (k1) (figure 1). pure acemannan isolated from aloe vera leaves have hematopoietic activity by stimulating macrophages to release gm-csf, thus increasing cell numbers and improving pmn function.9 gm-csf is a monomeric glycoprotein secreted by macrophage and other cells, which functions as a growth factor of white blood cells, and stimulates stem cells to produce granulocytes that include neutrophils.17 some research stated that polysaccharides (mannan, mannose, galactose, β-glucans) have hematopoietic activity linked with specific carbohydrate receptors that mediate the binding between hematopoietic progenitor stem cells and the stroma. study has shown that ppg-glucan improves hematopoietic cells’ mobilisation towards the peripheral area to release progenitor cells, which increase the amount of spleen gm-csf. another study, that uses pure polymannan (>99%) shows a significant expansion of progenitor cells in the spleen.10 other content of aloe vera is glycoprotein fraction (14kda) which synthesises less thromboxane a2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p124–127 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p124-127 127soesilawati et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 124–127 (txa2) in in vitro analysis.16 txa2 produced by active platelets has a vasoconstrictor effect and plays an important role in wound healing and the inflammation process. decreased txa2 synthesis causes vasodilatation and thus accelerates pmn extravasated to the wound site.18 the pmn count on day three of experimental group (p3) was higher than the control group (k3) (figure 1), but the percentage of increasing cells number was lower than the control group (table 2). on day three, pmn cells became apoptotic and were cleared by macrophages. this downward pattern of pmn cells confirmed that there was no prolonged inflammatory response.19 this clearance process was also marked by the release of the tissuerepairing cytokines, transforming growth factor-β (tgfβ) and interleukin-10 (il-10), which initiates tissue repair. thus, substances that promote pmn cells’ apoptosis have a therapeutic potential to accelerate wound healing and tissue repair.20 aloe vera contains amino acid (phenylalanine, tryptophane) and salicylic acid, which inhibit pge2 synthesis from arachidonic acid, which inhibits vasodilatation and inflammatory mediator effect (histamine, serotonin), and thus has the ability to inhibit pmn infiltration to the wound site.18 another research stated that chromones component (c-glucosyl chromone) isolated form aloe vera can inhibit the cyclooxygenase (cox) cycle and lower pge2 production.16 aloe vera contains bradykinase, which can break down the bradykinin compound. bradykinin is an inflammation mediator that causes vasodilatation. hydrolysis of bradykinin can reduce the vasodilatation effect, which causes an inhibition of leukocyte infiltration.21 gibberellins from aloe vera have an anti-inflammatory effect by inhibiting the cox cycle during the inflammation phase, which inhibits pmn infiltration. in vivo analysis of diabetes-induced rats show that aloe vera has an active component similar to gibberellin, which can lower the amount of pmn infiltration up to 60.1%.22 this research has proved a differential count of pmn in the inflammation phase. the experimental groups treated by aloe vera 90% gel show a significant increase in pmn count compared with control group in the tooth extraction wound-healing process. acknowledgements we thank lita rakhma yustinasari, drh and suryo kuncorojakti, drh for the preparation and processing supported by faculty of veterinary medicine, airlangga university. this study was also supported by dana riset kolaborasi dosen mahasiswa, faculty of dental medicine, airlangga university. the authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. references 1. nimma vl, talla hv, bairi jk, gopaldas m, bathula h, vangdoth s. holistic healing through herbs: effectiveness of aloe vera on post extraction socket healing. j clin diagnostic res. 2017; 11(3): zc83–6. 2. gonzalez ac de o, costa tf, andrade z de a, medrado arap. wound healing a literature review. an bras dermatol. 2016; 91(5): 614–20. 3. wang j. neutrophils in tissue injury and repair. cell tissue res. 2018; 371(3): 531–9. 4. nanci a. ten cate’s oral histology: development, structure, and function. 8th ed. st louis: mosby elsevier; 2012. p. 379–83, 388–9. 5. hajishengallis e, hajishengallis g. neutrophil homeostasis and periodontal health in children and adults. j dent res. 2014; 93(3): 231–7. 6. phillipson m, kubes p. the healing power of neutrophils. trends immunol. 2019; 40(7): 635–47. 7. sumarta npm, danudiningrat cp, rachmat ea, soesilawati p. cytotoxicity difference of 316l stainless steel and titanium reconstruction plate. dent j (majalah kedokt gigi). 2011; 44(1): 7–11. 8. hashemi sa, madani sa, abediankenari s. the review on properties of aloe vera in healing of cutaneous wounds. biomed res int. 2015; 2015: 714216. 9. cock ie. problems of reproducibility and efficacy of bioassays using crude extracts, with reference to aloe vera. pharmacogn commun. 2011; 1(1): 52–62. 10. sierra-garcía gd, castro-ríos r, gonzález-horta a, lara-arias j, chávez-montes a. acemannan, an extracted polysaccharide from aloe vera: a literature review. nat prod commun. 2014; 9(8): 1217–21. 11. yuliati, kusumaningsih t, rananda i, soesilawati p. increasing macrophages in tooth extraction wound healing after induction of freeze-drying gel aloe vera 90% on cavia cobaya. malaysian j med heal sci. 2020; 16(4): 87–91. 12. olympus corporation. user manual cellsens 1.18 life science imaging software. tokyo: olympus; 2017. p. 7–39, 139–75. 13. cross ss. underwood’s pathology: a clinical approach. 6th ed. china: churchill livingstone elsevier; 2013. p. 179–80. 14. reinke jm, sorg h. wound repair and regeneration. eur surg res. 2012; 49(1): 35–43. 15. wilkinson hn, hardman mj. wound healing: cellular mechanisms and pathological outcomes. open biol. 2020; 10(9): 200223. 16. abdel hamid aa, soliman mf. effect of topical aloe vera on the process of healing of full-thickness skin burn: a histological and immunohistochemical study. j histol histopathol. 2015; 2: 3. 17. francisco-cruz a, aguilar-santelises m, ramos-espinosa o, mataespinosa d, marquina-castillo b, barrios-payan j, hernandezpando r. granulocyte-macrophage colony-stimulating factor: not just another haematopoietic growth factor. med oncol. 2014; 31(1): 774. 18. jamil m, mansoor m, latif n, naz r, anwar f, arshad m, gul j, ullah s, saddam m. review: effect of aloe vera on wound healing. pakistan j sci ind res. 2020; 63(1): 48–61. 19. kamadjaja db, harijadi a, soesilawati p, wahyuni e, maulidah n, fauzi a, rah ayu f, simanjuntak r, soesanto r, asmara d, rizqiawan a, agus p, pramono c. demineralized freeze-dried bovine cortical bone: its potential for guided bone regeneration membrane. int j dent. 2017; 2017: 5149675. 20. haque n, widera d, govindasamy v, soesilawati p, abu kasim nh. extracellular vesicles from stem and progenitor cells for cell-free regenerative therapy. curr mol med. 2021; 21. 21. gupta vk, malhotra s. pharmacological attribute of aloe vera: revalidation through experimental and clinical studies. ayu. 2012; 33(2): 193–6. 22. choche t, shende s, kadu p. extraction and identification of bioactive components from aloe barbadensis. j pharmacogn phytochem. 2014; 2(1): 14–23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p124–127 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p124-127 119119 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 119–125 potential of jatropha multifida sap against traumatic ulcer basri a. gani,1 abdillah imron nasution,1 nazaruddin,2 lidya sartika,1 and rahmat kurniawan alam1 1department of oral biology, faculty of dentistry, universitas syiah kuala, banda aceh-indonesia 2department of pathology, faculty of veterinary, universitas syiah kuala, banda aceh-indonesia abstract background: traumatic ulcer is a lesion in oral mucosa as a result of physical and mechanical trauma, as well as changes in salivary ph. jatropha multifida sap can act as antimicrobial, anti-inflammatory and re-epithelialization, and can also trigger the healing process of ulcers. purpose: research was aimed to determine the potential of jatropha multifida sap against traumatic ulcer base on clinical and histopathological healing process. method: this research was conducted laboratory experimental model, with rats (rattus norvegicus) as the subject as well as jatropha multifida sap for ulcer healing. those subjects were divided into four groups: two treatment groups administrated with pellet and jatropha multifida sap, one group as the positive control group administrated with 0.1% triamcinolone acetonide, and one group as the negative control group administrated with 0.9% nacl. ulcer manipulation was used 30% h2o2, and evaluation of ulcer healing was used clinical and histopathological approach. result: clinically, the healing process of ulcers in the treatment group with jatropha multifida sap was faster than that in the positive control group with 0.1% triamcinolone acetonide, indicated with the reduction of the ulcer size until the missing of the ulcers started from the third day to the seventh one (p≤0.05). histopathologically inflammatory cells (lymphocytes, and plasma cells) declined started from the third day, and the formation of collagen and re-epithelialization then occurred. on the seventh day, the epithelial cells thickened, and the inflammatory cells infiltrated. statistically, those groups were significant (p≤0.05). conclusion: jatropha multifida sap has a significant potential to cure traumatic ulcers on oral mucosa clinically and histopathologically. keywords: traumatic ulcer; jatropha multifidi; oral mucosa correspondence: basri a. gani, c/o: departemen biologi oral, fakultas kedokteran gigi universitas syiah kuala. jln. teuku nyak arief, darussalam, banda aceh, aceh, 23111, indonesia. e-mail: basriunoe@gmail.com introduction ulcer is a lesion on soft tissues of oral mucosa caused by physical trauma, thermal, chemical and also trigerred by infectious agents (bacteria, viruses, and fungi), systemic diseases, cytotoxic drugs , immunological system disorders, neoplasm, radiotherapy, smoking, alcohol and allergy.1-3 ulcer on oral cavity can cause damage to oral mucosa epithelial cells, interfering the secretion of secretory immunoglobulin a (siga) and extracellular matrix proteins as mucosal defenses against the antigen. additionally, ulcer can reduce comfort and masticatory function, affecting nutritional intake, consequently, disrupting the healing process of ulcer.4 ulcer would be classified into two phases, acute and chronic phases.2 the acute phase is characterized by pain as a result of early trauma and will heal itself in 7-10 days. the chronic phase is characterized by irritation as the effect of dental friction such as the adaptation failure of restoration and prosthesis materials.5,6 some diseases manifested commonly in the oral cavity as chronic ulcers are hiv, syphilis, tuberculosis, squamous cell carcinoma, and deep fungal infection.7 healing ulcers in the acute phase is generally facilitated by saliva, secretory immunoglobulin a (siga) and growth factors. chronic phase is often become the trigger of infectious diseases in oral cavity.8-10 martin,11 reported that ulcer healing process in the chronic phase normally takes a long time (30-45 days) to go through several phases, including homeostasis, inflammatory, proliferative, and maturation phase considered as a refinement phase of new tissue formation into permanent tissue.12,13 generally, siga, collagen binding protein, igm, igg and polymorphonuclear (pmn) can accelerate the healing dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 120 gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 process of ulcer and also improve the tissue suffering from trauma.4,14 some of those antibody proteins have a working phase based on the development level of ulcers or wounds.12 in the chronic phase, potential of those antibodies will decrease their effectiveness in healing ulcers, thus, requiring a trigger or therapeutic agents to accelerate the healing process of ulcers and improve the function of the body’s defense system against ulcers.15,16 the 0.1% triamcinolone acetonide is a topical corticosteroid often used in ulcer treatment, and can trigger the intensity of immune system during ulcer healing process. 17 however, the use of this synthetic drug in long term could be triggered the resistance of immune system,18 epithelial cell atrophy, skin hypopigmentation, and adrenal suppression.19 in addition, it can lead to increasing blood glucose, osteoporosis, and hypertension.20,21 alternative medicine derived from herbal ingredients is needed as ulcer treatment option since it is also more economical. 22 jatropha multifida is one of the herbs that can be used as an alternative medicine for healing traumatic ulcers and skin wound.23 some researchers claim that it contains several chemical substances, namely alkaloids and saponins acting as antioxidant and anti-bacterial; tannin playing a role in granulation process, stopping bleeding, and acting as anti-inflammatory and anti-microbial; and flavonoids acting as anti-oxidant and contributing in collagen fiber formation by preventing elastin degradation and improving vascularization.24,25 thus, this research was aimed to test the potential of jatropha multifida against traumatic ulcers clinically and histopathologicaly. materials and methods this research was a laboratory experimental research conducted at biology laboratory, faculty of mathematics and natural sciences, as well as at pathology laboratory and laboratory of experimental animal model in veterinary faculty of universitas syiah kuala in 2013. the subjects of this research were forty-eight male wistar rats at the age of 2-3 months. those subjects were divided into four groups: two treatment groups administrated with pellet and jatropha multifida sap, one group as the positive control group administrated with 0.1% triamcinolone acetonide, and one group as the negative control group administrated with 0.9% nacl. this research passed ethical clearance for animal models from the ethics committee of the faculty of medicine, university of syiah kuala 175/ke/fk/2013 dated may 24, 2013. jatropha multifida sap was obtained from its stems. it was collected and put into 30 ml test tubes, and then centrifuged for 15 minutes at 3000 rpm at 4°c. pellet and supernatant obtained from the centrifugation were separated through decantation technique, and each fragment was then tested with phytochemical test for tannins, flavonoids, alkaloids and saponins contained.22,25 rats (rattus norvegicus) that had been acclimatized for 7 days were given light-dark cycle treatment for 12/12 hours with standard food and water ad libitum. modification of ulcer was then conducted by using 0.25 ml of 30% h2o2 smeared 1.5 cm on the right side of the lower jaw mucosa using a disposable micro-applicator sized 1.5 mm twice a day, morning and evening with a gap of 6 hours per day every 5 minutes during three days.21 observation was conducted from day 0, day 3 to day 7 to measure the size of the ulcer area. subjects were divided into four groups, each of which consisted of twelve rats. those rats in the negative control group were administrated with 0.25 ml of 0.9% nacl, while those in the positive control group were administrated with 0.25 mg of 0.1% triamcinolone acetonide. in addition, those in the treatment groups were administrated with 0.25 mg of jatropha multifida sap. after the size of the ulcer area was measured, the subjects were sacrificed. excision on mandibular labial mucosa was done and tissue was subsequently fixed in 10% formalin solution for making histophatological preparations.26 clinical assessment was then conducted by measuring the size of the ulcer area using a periodontal probe vertically, horizontally and diagonally on both sides. for getting an average diameter, a formula was used as follow:22,27 l = area of ulceration (mm2) π= 3.14 (provisions) d = average diameter (mm) a f t e r w a r d s , m u c o s a l h e a l i n g u l c e r s w e r e histopathologically measured with haematoxylin and eosin (he) technique. this technique involves several phases, ie. tissue fixation, dehydrating, clearing, embedding, sectionin, and mounting the tissue with cover glass, then observed under a microscope with 4x and 10x magnifications.24 confirmation of the ulcer healing process was assessed on the basis of scoring. score 1 for total healing process of epithelium with fibrosis occurred on the underlying connective tissue and no inflammatory cells; score 2 for total healing process of epithelium with fibrosis occurred on the underlying connective tissue and inflammatory cells, such as macrophages, plasma cells and lymphocytes; score 3 for ulcers with 2/3 of the width of the ulcer covered by epithelium, mild fibrosis occurred on the underlying connective tissue and inflammatory cells, such as macrophages, plasma cells and lymphocytes; score 4 for ulcers with 1/3 of the width of the ulcer covered by epithelium, moderate fibrosis occurred on the underlying connective tissue and inflammatory cells, such as macrophages, plasma cells and lymphocytes; and score 5 for ulcers with less than 1/3 of the width of the ulcer improved and high number of connective tissue and inflammatory cells.28 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 121121gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 finally, data obtained were analyzed by anova split plot test followed by least significant difference (lsd) test and the results of the histopathological observation were then analyzed with the kruskal wallis test followed by mann-whitney test. results based on the phytochemical test, the supernatant and pellet of the positive (+) jatropha multifida sap contains flavonoids, alkaloids, and tannins. thus, jatropha multifida sap was used as a material for healing traumatic ulcers in rattus norvegicus rats. the healing process of the ulcer was clinically assessed by measuring the ulcer area after the administration of jatropha multifida sap, whereas histopathologically assessed by observing the picture of cellular activity based on collagen tissue and epithelial cell repair, inflammatory response and infiltration with he staining. on day 0, there were similar histopathological features on the ulcers in those four groups. the ulcers were formed, therefore, the healing process still did not occur. the connective tissue and inflammatory cells even were still dominated with granulocytes, neutrophils, macrophages, plasma and lymphocytes (figure 1). on day 3, however, there were different histopathological features in those four groups. in the treatment groups with supernatant and pellet derived from jatropha multifida sap, there were two thirds of the ulcers covered by epithelial cells of connective tissues, containing the moderate number of fibrosis. in those two treatment groups, moreover, the number of macrophages, lymphocytes and plasma cells were lower than on day 0 as well as in the positive control group (figure 2). meanwhile, in the negative control group, one-third of the ulcers were covered by epithelial cells of connective tissues, containing the moderate number of fibrosis. in this group, the number of inflammatory cells was still the same as on day 0, ie neutrophils, macrophages, plasma cells and lymphocytes. nevertheless, on day 7, the ulcers in this group had different histopathological features from the treatment groups and the control positive group. in the treatment groups and the control positive group, the ulcers were totally covered by table 1. the assessment of the ulcer healing process based on histopathological description evaluated with traumatic ulcer healing score day treatments number of preparations score histopathological description of the traumatic ulcers 0 supernatant and pellet derived from jatropha multifida sap 3 5 less than a third of the traumatic ulcers were covered by epithelial cells of connective tissue containing the moderate number of fibrosis, and the number of inflammatory cells was high, such as neutrophil granulocytes, macrophages, plasma and lymphocytes. 0.1% triamcinolone acetonide 3 0.9% nacl 3 3 supernatant and pellet derived from jatropha multifida sap 3 3 two-thirds of the traumatic ulcers were covered by epithelial cells of connective tissue containing the moderate number of fibrosis, and the number of inflammatory cells was also moderate, such as macrophages, plasma cells and lymphocytes. 0.1% triamcinolone acetonide 3 0.9% nacl 3 4 one-third of the traumatic ulcers was covered by epithelial cells of connective tissue containing the moderate number of fibrosis, and the number of inflammatory cells was emerged, such as macrophages, plasma cells and lymphocytes 7 supernatant and pellet derived from jatropha multifida sap 3 1 the epithelial cells of connective tissue were totally improved based on the emergence of fibrosis and none or few of inflammatory cells.0.1% triamcinolone acetonide 3 0.9% nacl 1 3 one-third of the traumatic ulcers were covered by epithelial cells of connective tissue containing the moderate number of fibrosis, and the number of inflammatory cells emerged, such as macrophages, plasma cells and lymphocytes. 2 2 the epithelial cells of connective tissue were totally improved based on the emergence of fibrosis and the moderate number of inflammatory cells, such as macrophages, plasma cells and lymphocytes. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 122 gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 table 2. the average of the diameter size of the traumatic ulcers on day 0, day 3 and day 7 groups day 0 (mm) 3 (mm) 7 (mm) supernatant derived from jatropha multifida sap 11.1 5.20 0 pellet derived from jatropha multifida sap 12.3 6.32 0 possitive control (0.1 % triamcinolon acetonide) 10.12 6.32 0 negative control (0.9% nacl) 13.3 8.7 1.05 1 figure 1. figure 2. figure 1. histopathological description of the traumatic ulcers on the rats on day 0. (a) epithelium layer, getting destructed (black arrows); (b) infiltration of inflammatory cells in the connective tissue (black arrows); (c) inflammatory cells, namely a) neutrophils; b) macrophages; c) lymphocytes, and d) plasma cells. (d) neutrophils (blue arrows). he staining with an electric microscope magnification of 400x. 1 figure 1. figure 2. 1 figure 1. figure 2. figure 2. histopathological description of the traumatic ulcers on the rats on day 3. (a) supernatant: re– epithelialization, started to emerge (black arrows); (b) pellet: epithelial layer (black arrow), vasodilatation (red arrow); (c). infiltration of inflammatory cells and the formation of collagen fibers (black arrow); (d) the types of inflammatory cells; a) neutrophil, b) macrophages, c) lymphocytes, and d) plasma cells; (e) control (+): epithelial layer getting thicker (black arrow), vasodilatation (red arrow), infiltration of inflammatory cells (blue arrow); (f) control (-): thin epithelium (black arrow), infiltration of inflammatory cells (blue arrow). he staining with an electric microscope magnification 400x. epithelial cells of the connective tissues, and infiltration of inflammatory cells was occurred (figure 3). meanwhile, in the negative control group, the ulcers were still not totally covered by epithelial cells of connective tissue, and inflammatory cells, such as macrophages, plasma cells and lymphocytes were still found (table 1). clinically after three days, the size of the ulcers was various between 11 to 13 mm (table 2). there was also a reddish color on the mucosa, yellow-gray on the center of the ulcers, and mucosal erythema was formed. based on statistical analysis using anova split plot, the scoring of healing ulcers in the supernatant and pellet fractions derived from jatropha multifida sap clinically had a significant effect on ulcer healing process (p≤0.05) as well as on ulcer healing time on days 0, 3, and 7 (p≤0.05). based on the results of lsd test, furthermore, there was significant difference between the treatment groups, supernatant and pellet, and the negative control group (p≤0.05). but, there was no significant difference between the treatment groups, supernatant and pellet, and the positive control group (p≥0.05). finally, there was significant difference between the negative control group and the positive control group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 123123gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 discussion this research used 30% h2o2 for making artificial traumatic ulcers due to chemical trauma with the normal size of ulcer, 0.3-1 cm.29 besides less toxic to the body, h2o2 has strong oxidizing properties against tissues and leaves no residue as one of the requirements for mucosa irritant.30 the residue of h2o2 then can be broken down by catalase enzyme into water (h2o) and oxygen (o2) that are not dangerous.31 jatropha multifida sap can stimulate catalase enzyme on mucosa membranes characterized by the emergence of bubbles on the surface of the ulcers with clinical symptoms and redness on the mucosa with greyishyellow erythema. 32 the results in table 1 showed that jatropha multifida sap was able to reduce the diameter of the ulcers from day 3 to 7. on day 0, the clinical features of the edge of the ulcers showed reddish color, while histopathologically this group still showed inflammatory reaction (figure 1). this condition is as a result of increased blood flow to damaged tissue at the commencement of the inflammatory process, while a few hours after the formation of the ulcers, epithelial cells will be formed and grown from the wound edges, and then will migrate into the live connective tissue.9 consequently, thickening of the epidermis of the ulcers will occur within 24 hours, and perfect reepithelialization then will occur less than 48 hours after the formation of the ulcers.33,34 on day 3, histopathologically 2/3 of the ulcers were covered by epithelial cells (table 2), and cell proliferation (figure 2) occurred, facilitated by fibroblasts from mesenchyme cells. in this phase, fibroblasts will produce collagen fibers connecting the edges of the ulcer to provide strength and integrity, resulting in better healing process.35 the increasing of fibroblasts then can trigger the increasing of collagen fibers, as a result, the wound healing process and the first emergence of fibroblasts can significantly be accelerated on day 3, and reach the peak on day 7.36 jatropha multifida sap contains saponin, in addition to a role in epithelialization, also can activate the function of tgf-β by fibroblast receptors stimulating migration and proliferation of fibroblasts further.37 on day 3, neutrophils will be replaced by macrophages to activate the function of t cells and the differentiation of b cells as a specific defense system.11 clinically, the results of this research showed those three conditions, consequently, the size of the ulcers was reduced. based on the theory, the wound healing process of the ulcers consists of inflammatory phase, proliferative phase and maturation phase.38 inflammatory reaction is usually started from day 1 to day 3 characterized by the occurrence of blood vessel vasodilatation with infiltration of inflammatory cells into the ulcer area, such as neutrophils, macrophages, plasma cells and lymphocytes.39 after the trauma, inflammatory cells present in the injured tissue to destroy bacteria and remove debris from dead cells and broken matrix, so the healing process can be continued.40 the inflammatory phase is characterized by cell infiltration of neutrophils, macrophages and lymphocytes, while the proliferative phase occurs simultaneously with the inflammatory phase by showing epithelial proliferation, angiogenesis, collagen synthesis and extracellular matrix formation followed by tissue remodeling and scarring formation.9 this proliferation phase occurs from day 3 to day 14, which is characterized by formation of granulation tissue in the wound.41 granulation tissue is a combination of cellular elements, including fibroblasts and inflammatory cells, along with the growth of new capillaries from the matrix of collagen, fibronectin and hyaluronic acid.42 in the maturation phase, the number of fibroblasts decreases periodically, and then the re-formation of new collagen fibers and vascular maturation occur.43 on day seven, the ulcers had completely been cured significantly (p≤0.05), and epithelial cells of the connective tissue containing solid collagen were totally improved (figure 3 and table 2). the increasing of blood flow to the area of infection and the decreasing of inflammatory cells are actually in proportion to the reduction of the infection on the ulcer.44 consequently, collagen fibers quickly will become a major factor forming a matrix to support tissue healing process and increase the rigidity and strength of the wound area tension.45 based on clinical assessment, fragments of the supernatant and pellet from jatropha multifida sap have significant potential to cure ulcers. the existence of flavonoids, alkaloids, and tannins in the second fragment may accelerate the inflammatory phase and trigger the proliferation phase, as a result, the formation of collagen fibers can be accelerated to the granulation 2 figure 3. 2 figure 3. figure 3. histopathological description of the traumatic ulcers on the rats on day 7. (a) supernatant: epithelial layer, that had already thickened (black arrow) and connective tissue; (b) pellets: re-epithelialization (yellow arrow) and connective tissue; (c) control (+): epithelial layer, getting thicker (black arrow), connective tissue (yellow arrow); (d) control (-): thin epithelial layer (black arrow), infiltration of inflammatory cells (blue arrow). he staining with an electric microscope magnification 400x. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 124 gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 process on the edge of the ulcers.22 a study conducted by jin et al.,46 based on clinical observation and histopathological examination on traumatic ulcers up 10 days, the healing process was directly proportional to the length of day needed, meaning that the longer the day needed, the more decreasing the measure of the ulcer5 where, wound healing is influenced by connective tissue.46 the decreasing of the infection and the total healing process of the ulcers were triggered by active agents contained in jatropha multifida sap, named tannins and flavonoid. both active agents always play significant roles as astringents during epithelialization by increasing the activities of extracellular proteins in the epithelial cells to increase production of collagen fibers as the initial phase of wound healing process.47,48 jatropha glandulifera roxb still considered as a family of jatropha multifida was also containing tannins and flavonoids that has antibacterial effect for wound infections and ulcers.49 a research about bioactive compounds (tannin and flavonoid) that derived from arecha cathecu shows that it’s bioactive could be trigger re-epithelialization and accelerate the wound healing process. saponin contained in jatropha gossypifolia linn sap can also play a role in reepithelialization process and inhibit bacterial infection.50,51 revascularization of injuries can occur simultaneously with fibroplasias and blood capillary formation derived from blood vessels adjacent to wound.52 several investigators have reported that cytokines, acidic fibroblast growth factor (afgf), epidermal fibroblast growth factor (efgf), bfgf and tgf α β are components of the body’s immune playing significant roles as stimulators for re-epithelization and revascularization of wound healing.53,54 in conclusion, pellet and supernatant of jatropha multifida sap has been played a role on the healing process of traumatic ulcer, clinically and histopathologically which indicated by shrinking the diameter of the ulcers, decreasing the number of inflammatory cells and re-epithelialization and to step up the collagen tissues. acknowledgement the research was funded by ministry of education and cultural, indonesia, through universitas syiah kuala, contract number: 187/un11/s/lk-pnbp/2013. references 1. duarte cm, quirino mr, patrocinio mc, anbinder al. effects of chamomilla recutita (l.) on oral wound healing in rats. med oral patol oral cir bucal 2011; 16(6): e716-21. 2. turker sb, sener id, kocak a, yilmaz s, ozkan yk. factors triggering the oral mucosal lesions by complete dentures. arch gerontol geriatr 2010; 51(1): 100-4. 3. scully c, shotts r. mouth ulcers and other causes of orofacial soreness and pain. western journal of medicine 2001; 174(6): 42124. 4. scully c. challenges in predicting which oral mucosal potentially malignant disease will progress to neoplasia. oral dis 2014; 20(1):1-5. 5. cavalcante gm, sousa de paula rj, souza lp, et al. experimental model of traumatic ulcer in the cheek mucosa of rats. acta cir bras 2011; 26(3):227-34. 6. demidova-rice tn, hamblin mr, herman im. acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 1: normal and chronic wounds: biology, causes, and approaches to care. advances in skin & wound care 2012; 25(7): 304-14. 7. scully c, shotts r. mouth ulcers and other causes of orofacial soreness and pain. british medical journal 2000; 321(7254): 16265. 8. anura a. traumatic oral mucosal lesions: a mini review and clinical update. oral health dent manag 2014; 13(2): 254-9. 9. lim ys, kwon sk, park jh, et al. enhanced mucosal healing with curcumin in animal oral ulcer model. laryngoscope 2015. 10. zoller m, silinski s, ludwig c, et al. mucocutaneous candidiasis in a mandrill (mandrillus sphinx). j comp pathol 2012; 147(2-3): 381-5. 11. martin p, nunan r. cellular and molecular mechanisms of repair in acute and chronic wound healing. br j dermatol 2015; 173(2): 370-8. 12. guo s, dipietro la. factors affecting wound healing. journal of dental research 2010; 89(3): 219-29. 13. dryden sv, shoemaker wg, kim jh. wound management and nutrition for optimal wound healing. atlas oral maxillofac surg clin north am 2013; 21(1): 37-47. 14. hashemipour ma, ghasemi ar, dogaheh ma, torabi m. effects of locally and systemically applied n-3 fatty acid on oral ulcer recovery process in rats. wounds 2012;24(9):258-66. 15. koh tj, dipietro la. inflammation and wound healing: the role of the macrophage. expert reviews in molecular medicine 2011; 13: e23-e23. 16. geethalakshmi r, sakravarthi c, kritika t, arul kirubakaran m, sarada dvl. evaluation of antioxidant and wound healing potentials of sphaeranthus amaranthoides burm.f. biomed research international 2013; 2013: 607109. 17. deshmukh ra, bagewadi as. comparison of effectiveness of curcumin with triamcinolone acetonide in the gel form in treatment of minor recurrent aphthous stomatitis: a randomized clinical trial. international journal of pharmaceutical investigation 2014; 4(3): 138-41. 18. pakfetrat a, delavarian z, falaki f, khorashadizadeh m, saba m. the effect of pimecrolimus cream 1% compared with triamcinolone acetonide paste in treatment of atrophic-erosive oral lichen planus. iranian journal of otorhinolaryngology 2015; 27(79): 119-26. 19. coondoo a, phiske m, verma s, lahiri k. side-effects of topical steroids: a long overdue revisit. indian dermatology online journal 2014;5(4):416-25. 20. cellini m, pazzaglia a, zamparini e, leonetti p, campos ec. intravitreal vs. subtenon triamcinolone acetonide for the treatment of diabetic cystoid macular edema. bmc ophthalmology 2008; 8: 5-5. 21. pastar i, stojadinovic o, yin nc, et al. epithelialization in wound healing: a comprehensive review. advances in wound care 2014; 3(7): 445-64. 22. thakur r, jain n, pathak r, sandhu ss. practices in wound healing studies of plants. evidence-based complementary and alternative medicine : ecam 2011; 2011: 438056. 23. cazander g, jukema gn, nibbering ph. complement activation and inhibition in wound healing. clinical and developmental immunology 2012;2012:534291. 24. falodun a, imieje v, erharuyi o, et al. isolation of antileishmanial, antimalarial and antimicrobial metabolites from jatropha multifida. asian pacific journal of tropical biomedicine 2014; 4(5): 374-78. 25. r a mpa d a r a t h s, p ucho oa d, r a ng ho o sa n mu k h iya v m. antimicrobial, phytochemical and larvicidal properties of jatropha multifida linn. asian pac j trop med 2014; 7s1: s380-3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 125125gani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 119–125 26. suliman nm, åstrøm an, ali rw, salman h, johannessen ac. clinical and histological characterization of oral pemphigus lesions in patients with skin diseases: a cross sectional study from sudan. bmc oral health 2013; 13: 66-66. 27. wagner vp, meurer l, martins mat, et al. influence of different energy densities of laser phototherapy on oral wound healing. journal of biomedical optics 2013; 18(12): 128002. 28. bradbury s, walkley n, ivins n, harding k. clinical evaluation of a novel topical negative pressure device in promoting healing in chronic wounds. advances in wound care 2015; 4(6): 346-57. 29. pritchett s, green d, rossos p. accidental ingestion of 35% hydrogen peroxide. can j gastroenterol 2007;21(10):665-7. 30. ciechanowicz r, sein anand j, chodorowski z, kujawska-danecka h. acute intoxication with hydrogen peroxide with air emboli in central nervous system--a case report. przegl lek 2007; 64(4-5): 339-40. 31. french lk, horowitz bz, mckeown nj. hydrogen peroxide ingestion associated with portal venous gas and treatment with hyperbaric oxygen: a case series and review of the literature. clin toxicol (phila) 2010; 48(6): 533-8. 32. kanth bs, kumar as, shinde db, et al. new bioactive macrocyclic diterpenoids from jatropha multifida. bioorg med chem lett 2011; 21(22): 6808-10. 33. bharara m, schoess j, nouvong a, armstrong dg. wound inflammatory index: a “proof of concept” study to assess wound healing trajectory. j diabetes sci technol 2010; 4(4): 773-9. 34. robb wj. self-healing: a concept analysis. nurs forum 2006; 41(2): 60-77. 35. dogan a, ozdemir a, kubar a, oygur t. healing of artificial fenestration defects by seeding of fibroblast-like cells derived from regenerated periodontal ligament in a dog: a preliminary study. tissue eng 2003; 9(6): 1189-96. 36. jeon yk, jang yh, yoo dr, et al. mesenchymal stem cells’ interaction with skin: wound-healing effect on fibroblast cells and skin tissue. wound repair regen 2010; 18(6): 655-61. 37. lee ch, shah b, moioli ek, mao jj. ctgf directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. j clin invest 2010; 120(9): 3340-9. 38. mcelligott d. healing: the journey from concept to nursing practice. j holist nurs 2010; 28(4): 251-9. 39. benbow m. exploring the concept of moist wound healing and its application in practice. br j nurs 2008; 17(15): s4, s6, s8 passim. 40. pringuey-criou f. [healing garden: primary concept]. encephale 2015. 41. kwansang j, itthipanichpong c, limpanasithikul w. evaluation of wound healing activity of thunbergia laurifolia supercritical carbon dioxide extract in rats with second-degree burn wounds. j adv pharm technol res 2015; 6(3): 103-7. 42. shi gb, wang b, wu q, et al. evaluation of the wound-healing activity and anti-inflammatory activity of aqueous extracts from acorus calamus l. pak j pharm sci 2014; 27(1): 91-5. 43. jorgensen ln. collagen deposition in the subcutaneous tissue during wound healing in humans: a model evaluation. apmis suppl 2003; (115): 1-56. 44. seleit i, bakry oa, samaka rm, tawfik as. immunohistochemical evaluation of leptin expression in wound healing: a clue to exuberant scar formation. appl immunohistochem mol morphol 2015. 45. sharma av, ganguly k, paul s, maulik n, swarnakar s. curcumin heals indomethacin-induced gastric ulceration by stimulation of angiogenesis and restitution of collagen fibers via vegf and mmp-2 mediated signaling. antioxid redox signal 2012; 16(4): 351-62. 46. jin sg, kim ks, yousaf am, kim dw, jang sw, son mw, kim yh, yong cs, kim jo, choi hg. mechanical properties and in vivo healing evaluation of a novel centella asiatica-loaded hydrocolloid wound dressing. int j pharm 2015; 490(1-2): 240-7. 47. de jesus nz, de souza falcão h, gomes if, de almeida leite tj, de morais lima gr, barbosa-filho jm, tavares jf, da silva ms, de athayde-filho pf, batista lm. tannins, peptic ulcers and related mechanisms. int j mol sci 2012; 13(3): 3203-28. 48. vilar dde a, vilar ms, de lima e moura tf, raffin fn, de oliveira mr, franco cf, de athayde-filho pf, diniz mde f, barbosa-filho jm. traditional uses, chemical constituents, and biological activities of bixa orellana l.: a review. scientific world journal 2014; 2014: 857292. 49. mujumdar am, misar av. anti-inflammatory activity of jatropha curcas roots in mice and rats. j ethnopharmacol 2004; 90(1): 11-5. 50. modolo lv, de souza ax, horta lp, araujo dp, de fátima â. an overview on the potential of natural products as ureases inhibitors: a review. j adv res 2015; 6(1): 35-44. 51. nur sazwi n, nalina t, rahim zha. antioxidant and cytoprotective activities of piper betle, areca catechu, uncaria gambir and betel quid with and without calcium hydroxide. bmc complementary and alternative medicine 2013; 13: 351-51. 52. duraisamy y, slevin m, smith n, bailey j, zweit j, smith c, ahmed n, gaffney j. effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: possible relevance to wound healing in diabetes. angiogenesis 2001; 4(4): 277-88. 53. lee ch, shah b, moioli ek, mao jj. ctgf directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. j clin invest 2015; 125(10): 3992. 54. ching yh, sutton tl, pierpont yn, robson mc, payne wg. the use of growth factors and other humoral agents to accelerate and enhance burn wound healing. eplasty 2011; 11: e41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p119-125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p119-125 vol 38 no 2-2005 73 bahan pemutih gigi dengan sertifikat ada/iso (tooth bleaching material with ada/iso certificate) asti meizarini dan devi rianti bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract bleaching of teeth for cosmetic reasons is a popular aspect of cosmetic dentistry because patients realize the aesthetical benefits of these products. the dentist as a clinician's practitioner must be knowledgeable of the products and their application techniques. bleaching materials which are safe and effective are the ada accepted or manufactured by those which have already haved iso certificate. dentist must have enough knowledge about in-office bleaching prescribed for home-use bleaching including their contra indication and side effects, to advise the patients and provide effective bleaching services. key words: bleaching materials, ada accepted, in-office bleaching, home-use bleaching korespondensi (correspondence): asti meizarini, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132 surabaya, indonesia. pendahuluan keinginan penderita untuk mendapatkan senyum yang lebih cerah dan lebih putih menyebabkan kebutuhan pelayanan gigi kosmetik meningkat. salah satu bentuk pelayanan gigi kosmetik adalah memutihkan gigi. tidak ada alat atau material kedokteran gigi yang sepenuhnya aman, termasuk bahan pemutih gigi. pemilihan serta penggunaan alat atau material kedokteran gigi didasarkan asumsi bahwa keuntungan penggunaannya jauh melebihi risiko biologis yang diketahui. mutu dan sifat material kedokteran gigi harus mempunyai standar spesifikasi yang dapat diukur, perlu identifikasi persyaratan sifat fisik dan kimia material, sehingga dapat digunakan dengan hasil yang memuaskan atau tepat guna.1 american dental association (ada) pada tahun 1994 mulai memformulasikan panduan pengujian bahan pemutih gigi yang aman dan efektif. aman menurut definisi ada adalah aman secara biologis bukan klinis. tingkat keberhasilan dinilai sampai dengan 6 bulan setelah perawatan dimulai, dengan cara memantau perubahan histologis pulpa, iritasi terhadap jaringan gingiva serta efek samping penggunaannya untuk mendapatkan sertifikasi dari ada.2 council on scientific affairs (csa) dari ada merupakan badan yang bertanggung jawab terhadap pengembangan standar dan pemberian sertifikat pada produk yang memenuhi persyaratan spesifikasi. council on scientific affairs (csa) juga bertanggung jawab untuk mengevaluasi material pemutih gigi. label pada material yang telah diuji dan disetujui oleh csa, diberi ijin untuk ditandai dengan tulisan disetujui oleh ada dan merupakan standar nasional amerika. international organization for standardization (iso) adalah organisasi internasional, non pemerintah yang mengembangkan standar internasional, terdiri dari organisasi standar nasional lebih dari 80 negara. iso technical committee (tc) 106 tugasnya menstandarisasi terminologi, metode pengujian dan menentukan spesifikasi untuk material, peralatan kedokteran gigi pada tingkat internasional.1 adanya sertifikat ada/iso menunjang didapatkannya material yang aman dan efektif untuk perawatan gigi. kandungan utama pemutih gigi kandungan utama bahan pemutih gigi tergantung dari produsen pembuatnya, diantaranya hydrogen peroxide, carbamide peroxide atau urea peroxide atau sistim non hydrogen peroxide yang mengandung sodium chloride, oxygen dan natrium fluoride. beberapa produk mengandung bahan tambahan potasium nitrat dan fluoride untuk membantu mengurangi sensitifitas gigi.3,4 faktor penyebab perubahan warna gigi dapat dilihat pada tabel 1. warna alami enamel adalah putih translusen dan warna struktur gigi di bawah enamel cenderung tampak. dentin berada di bawah enamel, dengan warna normal kekuningan, tetapi oleh karena struktur porous dan adanya persyarafan gigi akan menembus warna dentin yang menyebabkan warna gigi menjadi lebih gelap sampai kearah kuning kecoklatan. hal ini seiring dengan pertambahan usia. perawatan saluran akar cenderung membuat gigi menjadi lebih gelap karena syaraf yang mati dapat terdorong saat perawatan saluran akar sehingga warna gigi berubah menjadi kecoklatan oleh karena syaraf tersebut dapat menembus tubuli dentin di sekitarnya.5 74 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 73–76 mekanisme kerja pemutih gigi mekanisme kerja bahan pemutih peroxide dan non peroxide yaitu dengan cara masuk melalui perantara enamel ke tubuli dentin dan mengoksidasi pigmen pada dentin, menyebabkan warna gigi menjadi lebih muda. proses ini dapat dipercepat menggunakan pemanasan dengan sinar berintensitas cahaya rendah atau sinar dengan intensitas cahaya tinggi, misalnya sinar kuring komposit konvensional, sinar laser, sinar plasma arc dengan intensitas tinggi. beberapa pabrik menyarankan penggunaan etsa asam sebelum aplikasi pemutih kimia untuk mempertinggi penetrasi dari material pemutih. berdasarkan hasil penelitian menunjukkan bahwa etsa asam tidak memperbaiki hasil pemutihan, bahkan gigi perlu dilakukan pemulasan akibat permukaannya menjadi kasar karena penggunaan etsa asam tersebut.3 prosedur untuk pemutihan gigi ada berbagai macam cara. pemutihan gigi dapat dikerjakan di klinik oleh dokter gigi secara langsung atau dilakukan di rumah dengan pantauan dokter gigi (lihat tabel 2). sebelum dilakukan prosedur pemutihan, warna gigi pasien harus di data terlebih dahulu, sehingga dapat tabel 1. perubahan warna gigi dan penyebabnya.4 penyebab perubahan warna gigi warna gigi kesehatan mulut jelek kuning, coklat, hijau, hitam kopi, teh, makanan coklat sampai hitam faktor dari luar gigi produk tembakau kuning kecoklatan sampai hitam obat-obatan selama pertumbuhan gigi tetracycline garis coklat, abu-abu, hitam fluoride bercak coklat, putih atau garis obat-obatan setelah pertumbuhan gigi minocycline coklat, abu-abu penyakit/kondisi selama pertumbuhan gigi kondisi kelainan darah merah, coklat, ungu trauma biru, hitam, coklat perubahan pada pulpa obliterasi saluran akar kuning nekrosis pulpa dengan perdarahan abu-abu, hitam nekrosis pulpa tanpa perdarahan kuning, abu-abu kecoklatan penyebab lain pada gigi nonvital trauma selama ekstirpasi pulpa abu-abu, hitam sisa jaringan dalam ruang pulpa coklat, abu-abu, hitam material restorasi gigi coklat, abu-abu, hitam faktor dari dalam gigi material perawatan saluran akar abu-abu, hitam fluorosis putih, coklat kombinasi proses ketuaan kuning tabel 2. pemilihan prosedur pemutihan gigi.6 pemutihan gigi di rumah pemutihan gigi di klinik pemutihan gigi dengan kombinasi kekuatan sinar pilihan penderita penderita dengan perubahan warna gigi ringan, ingin diputihkan satu atau dua tingkat dan punya waktu untuk pemakaian di rumah penderita dengan perubahan warna gigi ringan sampai akut, ingin efek pemutihan lebih nyata penderita dengan perubahan warna gigi ringan sampai berat, ingin hasil secara langsung bahan yang digunakan carbamide peroxide (10−22%) atau gel pemutih non peroxide carbamide peroxide (34−44%) hydrogen peroxide (30−50%) lokasi rumah, 2−4 jam perhari klinik gigi klinik gigi teknik buat sendok cetak di klinik. sendok cetak dan cairan pemutih dibawa pulang, kembali ke klinik periodik untuk kontrol perubahan carbamide peroxide diaplikasikan pada sendok cetak, dimasukkan mulut sedikitnya 30 menit tiap perawatan. aplikasi tambahan dilakukan penderita dirumah cairan diaplikasikan pada gigi dan diaktivasi dengan sumber panas atau sinar khusus hasil butuh 3−4 minggu untuk mengukur hasil yang terlihat beberapa hasil terlihat setelah 30 menit perawatan pada beberapa kasus tampak perubahan warna secara langsung rata-rata jumlah perawatan sekali sehari selama 2−3 jam untuk 4−6 minggu dapat digunakan sebagai perawatan pertama untuk perawatan harian dirumah satu visit. pemakaian dirumah disarankan tergantung noda gigi yang akan dihilangkan 75meizarini: bahan pemutih gigi dibedakan dengan hasil warna gigi setelah pemutihan menggunakan shade guide. semua produk yang telah disetujui ada untuk pemakaian di rumah biasanya menggunakan carbamide peroxide 10% yang diaplikasikan pada sendok cetak. produk dengan konsentrasi carbamide peroxide lebih dari 10% tidak disetujui sebagai bahan yang aman dan efektif oleh ada untuk pemakaian di luar klinik gigi.2 pemakaian bahan pemutih di klinik yang dikerjakan oleh dokter gigi dianjurkan untuk penderita yang ingin hasil secara singkat, tanpa penggunaan sendok cetak. penggunaan bahan pemutih tambahan di rumah dianjurkan untuk mempercepat hasil pemutihan.3 indikasi dan kontra indikasi penggunaan bahan pemutih gigi perawatan pemutihan gigi tidak dapat di indikasikan untuk semua orang. indikasi perawatannya untuk penderita dengan perubahan warna yang disebabkan proses penuaan, konsumsi makanan, minuman, obat antara lain tetrasiklin, serta fluorosis.7 kontra indikasi penggunaan bahan pemutih gigi, adalah penderita yang alergi terhadap komponen bahan pemutih gigi atau bahan sendok cetak, penderita dengan gigi sangat sensitif, penderita dengan gangguan temporomandibular joints (tmj), penderita hamil, penderita dengan restorasi geligi anterior yang berubah warna. penderita yang terlalu berharap akan hasil pemutihan gigi juga tidak dianjurkan melakukan hal ini, karena kemungkinan hasilnya akan mengecewakan secara psikis.3,7 efek samping bahan pemutih gigi penggunaan bahan pemutih gigi dapat menimbulkan efek samping berupa gigi yang sensitif, iritasi pada mukosa dan rasa sakit pada tmj. gigi sensitif yang timbul akibat proses pemutihan gigi, umumnya dalam waktu singkat, dapat ditanggulangi dengan memendekkan waktu proses pemutihan setiap harinya, pengulasan fluor, potasium nitrat atau bahan desentizing lain. iritasi pada mukosa gingiva dan tenggorokan biasanya disebabkan bahan pemutih yang berlebihan, keluar dari sendok cetak sehingga mengiritasi mukosa atau kemungkinan tertelan. sakit pada otot pengunyahan dan tmj untuk penderita yang menggunakan sendok cetak sepanjang malam, disebabkan karena adanya perubahan pada kondili.3,4 pembahasan dahulu metode pemutihan gigi vital, umumnya menggunakan cairan hydrogen peroxide dikombinasi dengan pemanasan. metode ini menyebabkan gigi menjadi sensitif, yang bervariasi selama proses pemutihan atau setelahnya. penggunaan hydrogen peroxide 35% dengan atau tanpa pemanasan, dapat menyebabkan perubahan pulpa secara histologis, meskipun umumnya akan sembuh dalam waktu 60 sampai 92 hari.8 hydrogen peroxide sendiri dapat menghambat aktivitas ensim pulpa meskipun masuk ke pulpa dengan jumlah sedikit. dari hasil penelitian seale and wilson cit. tam,8 di laporkan ada perubahan respon pulpa secara permanen pada anjing karena penggunaan hydrogen peroxide 35% in vitro. sampai sekarang belum ada laporan adanya perubahan pulpa permanen pada penggunaan carbamide peroxide untuk pemakaian di rumah.8 di amerika serikat, hanya formula yang mengandung carbamide peroxide 10% yang dapat disetujui ada oleh csa.9 bahan dasar carbamide peroxide 10% terdiri dari 3% hydrogen peroxide dan 7% urea. urea dalam carbamide peroxide berperan sebagai stabilisator untuk memperpanjang shelf life dan memperlambat pelepasan hydrogen peroxide.10 produk yang dianggap aman dan efektif untuk pemakaian di rumah dan telah disetujui ada pada maret 2003 adalah colgate platinum daytime professional whitening system 10%, nite white classic whitening gel 10%, opalescence whitening gel 10%, patterson brand tooth whitening gel 10%, rembrandt lighten bleaching gel 10%.2,3 penggunaan teknik pemutihan gigi di rumah menggunakan carbamide peroxide 10%, tidak menyebabkan terjadinya masalah kelainan jaringan lunak secara bermakna, bila sendok cetak di desain agar bahan pemutih tidak kontak langsung dengan jaringan lunak. pada konsentrasi 30%, hydrogen peroxide akan memutihkan (memucatkan) jaringan gingiva untuk sementara waktu. di canada carbamide peroxide atau hydrogen peroxide boleh digunakan sebagai bahan pemutih dengan konsentrasi terbatas, 10% dan 3%, dan harus diperhatikan agar penggunaan tidak lebih dari 14 hari, kecuali di bawah pantauan dokter gigi. bila perawatan pemutihan gigi perlu diperpanjang, dokter gigi harus memantau setiap 2 minggu, meskipun ada pendapat yang menyatakan bahwa efek samping yang ditimbulkan minimal, untuk pemakaian pemutih gigi di rumah sampai dengan 6 bulan.8 penelitian matis et al.10 membandingkan penggunaan bahan pemutih carbamide peroxide pada konsentrasi 10% dan 15%, mendapatkan hasil efek pemutihan carbamide peroxide 15% lebih cepat, tetapi hasil pemutihan setelah 6 minggu tidak menunjukkan perbedaan. hal ini berarti carbamide peroxide dengan konsentrasi yang lebih rendah memerlukan waktu yang lebih lama untuk memutihkan gigi, tetapi hasil akhir pemutihan gigi sama sesudah 6 minggu. penelitian zekonis et al.11 yang membandingkan melalui evaluasi klinik perawatan pemutihan gigi di klinik menggunakan hydrogen peroxide 35% selama 60 menit (2 kali kunjungan) dan perawatan di rumah menggunakan carbamide peroxide 10% selama 14 hari, menunjukkan bahwa 84% subyek melaporkan perawatan dirumah lebih efisien dan 16% melaporkan tidak ada perbedaan hasil perawatan pemutihan antara di klinik dan di rumah. pada kasus penderita yang tidak dapat menggunakan prosedur pemutihan di rumah karena berbagai alasan, 76 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 73–76 misalnya waktu perawatan lama, pemakaian sendok cetak yang tidak nyaman dan mengiritasi atau rasa tidak enak, iritasi gingiva atau perut karena bahan pemutih. penderita ini disarankan melakukan perawatan di klinik atau power bleaching untuk mendapatkan hasil pemutihan lebih cepat tanpa pemakaian sendok cetak yang lama. penderita cukup sekali dirawat di klinik dokter gigi untuk melakukan prosedur pemutihan.12 dokter gigi memegang peranan penting dalam penggunaan bahan pemutih yang aman, mendiagnosa kondisi gigi geligi dan etiologi perubahan warna, menentukan metode perawatan yang akan dikerjakan, menetapkan macam dan jumlah bahan pemutih yang dipakai, menuntun dan memantau penderita selama perawatan pemutihan di rumah, menilai efektifitas perawatan dan merawatnya bila ada efek samping.6 sebagai kesimpulan, untuk mendapatkan perawatan pemutihan gigi yang aman dan efektif, selain menggunakan bahan yang telah disetujui ada/iso, dokter gigi harus mempunyai pengetahuan mengenai pemutihan gigi di klinik, penggunaan pemutih gigi di rumah, termasuk kontra indikasi dan efek sampingnya agar dapat memberikan saran dan pelayanan pemutihan gigi yang efektif dan hasil memuaskan. daftar pustaka 1. anusavice. phillips' science of dental materials. 11st ed. saunders; 2003. p. 9–17. 2. matis ba. tray whitening: what the evidence shows. compendium of continuing education in dentistry 2003; 24(4a): 354–62. 3. matis ba. the question-at-home or in-office bleaching: evidence based concepts to empower dental professionals. available at: bmatis@iupui.edu. accesed august 27, 2004. 4. hatrick cd, eakle ws, bird wf. dental materials: clinical applications for dental assistants and dental hygienists. philadelphia: saunders; 2003. p. 101–6. 5. spiller. dental bleaching. available at: http://www.doctorspiller. com/bleaching.htm. accessed january 19, 2005. 6. o'brien wj. dental materials and their selection. 3rd ed. chicago: quintessence publ co; 2002. p. 162–3. 7. anonim. bleaching. oral health-dentistry. viahealth disease and wellness information. available at: www:/viahealthplan/ index.htm. accessed december 13, 2004. 8. tam l. the safety of home bleaching techniques. j can dent assoc 1999; 65: 453–5. 9. tam l. clinical trial of three 10% carbamide peroxide bleaching products. j can dent assoc 1999; 65: 201–5. 10. matis ba, mousa hn, cochran ma, eckert gj. clinical evaluation of bleaching agents of different concentrations. quintessence int 2000; 31: 303–10. 11. zekonis r, matis ba, cochran ma, al shetri se, eckert gj, carlson tj. clinical evaluation of in-office and at-home bleaching treatments. op dent 2003; 28(2): 114–21. 12. sun g. lasers and light amplification in dentistry. the role of laser in cosmetic dentistry. available at: htpp:/www.w3.org/tr/rechtml40. accessed february 24, 2005. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the purpos, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  results, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. h. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. h. 8–21. ii. literature reviews preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references.  photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. editor p-issn: 1978-3728 e-issn: 2442-9740 volume 54, number 1, march 2021 editorial team of dental journal (majalah kedokteran gigi) sk: 17/un3.1.2/2021 january 4 – december 31, 2021 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: muhammad dimas aditya ari, drg., m.kes (department of prosthodontics, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); aulia ramadhani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); nastiti faradilla ramadhani (department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers arlette suzy setiawan (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); irna sufiawati (deparment of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); sri oktawati (department of periodontics, faculty of dentistry, universitas hasanuddin, indonesia); diah savitri ernawati (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); tuti kusumaningsih (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); eha renwi astuti (department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); irma josefina savitri (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); dini setyowati (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); ratri maya sitalaksmi (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); tania saskianti (department of pediatric dentistry, faculty of dental medicine, universitas airlangga, indonesia); retno indrawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 preface dental journal (majalah kedokteran gigi) is proud to be one of certified scientific journals by the ministry of research and technology and national research and innovation agency. we have been publishing since 1968. we are supported by indonesian and international editors. quality is always maintained in order to provide our readers with update scientific information. with the spirit and vision to be an internationally indexed journal, dental journal (majalah kedokteran gigi) is published in english and now available both print and online. starting from 2021, we have made updates related to the classifying of scientific articles that we publish. we classify these into original articles, case report and review articles. the classification will be included in the table of contents for each edition according to the published article and become an option during the submission process as well as a template for updating the manuscript that we provide. as detailed further information can be accessed on our online journal portal. we would also like to acknowledge generous supports from the dean & vice dean of faculty of dental medicine universitas airlangga; the institute of innovation, journal development, publishing and intellectual property rights universitas airlangga; and national scientific journal reviewer board of the indonesian ministry of education. best regards, chief editor muhammad dimas aditya ari, drg., m.kes printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 54, number 1, march 2021 p-issn: 1978-3728 e-issn: 2442-9740 1. epidemiological survey for dental trauma among 12-year-old children in erbil city, iraq mohamed salim younus and karam ahmed ............................................................................... 1–4 2. effectiveness of reminder sticker books at increasing dental health knowledge and oral hygiene muhammad chair effendi, edina hartami, merlya balbeid and ghea dewi hapsari ............ 5–10 3. the comparative micro-ct analysis on trabecular bone density between hydroxyapatite gypsum puger scaffold application and bovine hydroxyapatite scaffold application amiyatun naini ................................................................................................................................ 11–15 4. the enhancement of type 1 collagen expression after 10% propolis-carbonated hydroxyapatite application in periodontitis-induced rabbits indi kusumawati, suryono and ahmad syaify ............................................................................. 16–20 5. adjunctive radiograph diagnostic in vertical mandibular asymmetry kirubanandan sathya moorthy, ervina sofyanti, trelia boel, jesslyn okto govanny and aditya rachmawati .................................................................................................................. 21–24 6. comparison between school and home-based dental health promotion in improving knowledge, parental attitude and dental health of children with mild disabilities putri raisah, rosa amalia and bambang priyono ....................................................................... 25–30 7. correlation between salivary zinc levels and salivary volume on taste disorders in elderly patients dewi kania intan permatasari, tenny setiani dewi and dewi marhaeni diah herawati ...... 31–34 8. silicone loop alternative for posterior bitewing radiography shinta amini prativi, shanty chairani and tyas hestingsih ...................................................... 35–38 9. cultivation and expansion of mesenchymal stem cells from human gingival tissue and periodontal ligament in different culture media banun kusumawardani, dwi merry christmarini robin, endah puspitasari, irma josefina savitri and dea ajeng pravita suendi ................................................................... 39–45 10. correlation between carbohydrate intake and dental caries in obese individuals ignatius setiawan, ananda sagita, ibnu suryatmojo, dewi marhaeni diah herawati, irna sufiawati and sunardhi widyaputra .................................................................................... 46–51 11. streptococcus mutans detection on mother-child pairs using matrix-assisted laser desorption ionization – time of flight mass spectrometry and polymerase chain reaction udijanto tedjosasongko, dwi mulia ramadhaniati and seno pradopo .................................... 52–56 contents page �� antimicrobial effect of calcium hydroxide as endo intracanal dressing on streptococcus viridans nanik zubaidah department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract calcium hydroxide had been used as the intra-canal dressing in endodontic treatment due to its high alkaline and antimicrobial capacity. it can also dissolve the necrotic tissue, prevent dental root resorbtion and regenerate a new hard tissue. the aim of this study was to determine the concentration of calcium hydroxide which had the highest antimicrobial effect on streptococcus viridans. samples were divided into 5 groups; each group consisted of 8 samples with different concentration of calcium hydroxide. group i: 50%, group ii: 55, group iii: 60%, group iv: 65%, group v: 70%. the antimicrobial testing was performed using diffusion method against streptococcus viridans. the result of susceptibility test was showed by the inhibition zone diameter which measured with caliper (in millimeter). we analyzed the data using one-way anova test with significant difference 0.05 and subsequently lsd test. the study showed that calcium hydroxide with concentration 60% has the highest antimicrobial effect. key words: calcium hydroxide, streptococcus viridans, antimicrobial effect correspondence: nanik zubaidah, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction since calcium hydroxide applied in dentistry, it has been used for many purposes such as cavity liner; direct and indirect pulp capping; apexification; root resorption prevention; iatrogenic perforation; improvement in cavity base and root canal wall; treatment of horizontal and vertical root fracture; main substance of root canal paste (sealer) and root canal (filler); and irrigation material.1–3 along with science development and modern technology, calcium hydroxide as intracanal dressing has been introduced in modern endodontic treatment to reach sterile root canal. it is due to the capability of calcium hydroxide to solve necrotic tissue; stimulate odontoblast activity and capable penetrate into accessories root canal and dentinal tubule so it would lower permeability of dentinal surface; capable to neutralize acid condition produced by osteoclast activity therefore it could prevent eliminated tissue from further damage; due to presence of calcium ion it could promote excessive exudates to dry and make permeability of capillary blood vessel decrease; and finally calcium hydroxide has very effective antimicrobial effect in endodontic treatment.1,4 grossman et al.,5 suggested that a medicine which is used as intracanal dressing must fulfill some requirements such as: germicide and fungicide; non irritated substance on periapical tissue; stable in solution; long antimicrobial effect; presence of blood, serum and derivate of tissue protein do not decrease the effect; low surface tension; does not change tooth color. tronstad et al.,6 suggested that placing calcium hydroxide into root canal would increase ph, contribute alkalis environment in the adjacent tissue by diffusing hydroxyl ion through dentinal tubule. increasing ph would make calcium hydroxide bactericidal and inhibit osteoclast activity. when calcium hydroxide is solved in sterile aquabidest, it would decompose in calcium ion (ca2+) and hydroxyl ion (oh--). the occurrence of hydroxyl ion in solution would make the environment alkalis (ph = 12.5 at 37° c) and destroy bacterial membrane. that condition will killed bacteria.3,7 according to siquira and lopes8 the antibacterial effect of calcium hydroxide used as intra-canal dressing is due to its ability to destroyed cytoplasmic membrane cell of bacterial, to denaturized protein and destroyed dna of bacteria. the most common bacteria found in infected root canal is gram positive bacteria such as: streptococcus viridans including streptococcus hemolyticus, lactobacillus and staphylococcus and followed by gram negative bacteria and some fungi group.8,9 grossman et al.5 found the domination of streptococcus α hemolitycus such as streptococcus viridans (63%), staphylococcus albus (17%), dipteroid bacilli (6.5%), staphylococcus aureus, bacillus proteus, bacillus coli. saifudin10 found streptococcus α hemolyticus (76.6%) and anaerobe obligate bacteria (23.4%) infected root canal. in early 1900, a theory was found on focus infection using intracanal dressing with strong antimicrobial effect for root canal treatment. it contributes sterile root canal and periapical tissue and prevented the possibility of �0 dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 39-42 spreading dangerous bacteria into the body.6 grossman et al.5 and suzuki et al.11 stated that intracanal dressing could destroy pathogenic bacteria and kill microbial flora of infected root canal. in general the clinical use of the 50% calcium hydroxide (50 gram calcium hydroxide powder in 100 ml sterile aquabidest) is based on factory procedure.12 hosoya et al.2 and estrela13 used 44% and 38% calcium hydroxide with ph = 11.24 in their study to examine calcium ion (ca2+) and hydroxyl ion (oh–) release in the adjacent apical of root canal for 3 days. sjogren et al.14 stated that calcium hydroxide paste is still effective as long as it is in root canal and shows antimicrobial force for seven days. grossman et al.5 reported that calcium hydroxide is strong disinfectant in root canal. in this study, pure calcium hydroxide was used not only because its cost is relatively cheap but also because it is availability. it is expected that pure calcium hydroxide would be able to be applied as intracanal dressing in endodontic treatment. the optimum concentration of calcium hydroxide as intra-canal dressing which has highest antimicrobial effect on streptococcus viridans is still need to be studied further. the purpose of this study was to determine the concentration of calcium hydroxide which has optimal antimicrobial effect against streptococcus viridans. the advantage of this study is to determine concentration of calcium hydroxide applied as intracanal dressing, so optimal endodontic treatment could also be reached. material and method the study used the post test only controlled group design. the study was done at dentistry and oral health department of dentistry faculty, airlangga university and antimicrobial test was done in microbiology laboratory of dr. soetomo general hospital, surabaya. the materials were pure calcium hydroxide powder (m2047, merck darmstad, germany), sterile aquabidest (kimia farma), media brain heart infusion (bhi), media chocolate agar, normal saline (naocl 0.85%), streptococcus viridans (isolated from the patient). calcium hydroxide paste preparation calcium hydroxide paste was made by mixing calcium hydroxide powder with sterile aquabidest with concentration of 50%, 55%, 60%, 65%, and 70% until paste was formed. the mixture was made in appendorf tube, after mixed using sterile spatel cement for one minute the mixture was homogenized with vortex for 30 seconds.13 isolation of streptococcus viridans streptococcus viridans was isolated from the patient’s maxillary anterior teeth with the diagnosis of necrotic pulpa and periapical lesion. the procedure was done as follows; the working region was isolated by rubber dam and 70% alcohol was applied at the surface’s tooth. cavity entrance was made using sterile round bur and removed the pulp wall. sterile paper point was inserted into root canal for one minute, and then paper point was put into brain heart infusion (bhi) and incubated in 37° c for 24 hours. the bacteria culture was re-inoculated by spreading it with ose into blood agar media. after 37° c incubation for 24 hours, the bacterial growth was examined using light microscope. the bacteria was re-cultured using chocolate agar plate and incubated 37° c for 24 hours to determine coccus gram positive bacterial growth (chain form, α hemolytic). the identification was done by gram staining. antibacterial examination suspension was done by taking colonies of streptococcus viridans and its culture media using ose, and then mixed by normal saline (nacl 0.85%) until turbidity equal to standard mc. farland 0.5. one ml of s. viridans suspension was taken and put into petridish containing muller hinton agar media and spread using sterile spreader. wells were made at the surface of agar media by placing platinum ring with diameter of 6 mm and 9 mm height. twenty five µl of calcium hydroxide with concentration of 50% (group i), 55% (group ii), 60% (group iii), 65% (group iv), 70% (group v) were put into the wells in muller hinton agar media using pippete and incubated at 37° c for 24 hours. the inhibition zone was measured using caliper (0.5 accuracy; in millimeter).16 result the mean and standard deviation of inhibition zone for 50%, 55%, 60%, 65%, and 70% calcium hydroxide against streptococcus viridans showed on table 1 and figure 1. the result showed that 60% of calcium hydroxide had the highest inhibition zone comparing to other groups. table1. the mean and standard deviation of inhibition zone calcium hydroxide in various concentration against streptococcus viridans (mm) concentration n x sd group i 50% group ii 55% group iii 60% group iv 65% group v 70% 8 8 8 8 8 14.6250 14.8750 15.7500 13.1250 11.350 1.1877 1.6421 1.4880 1.260 1.1877 note: n = number of samples, x = mean of inhibition zone, sd = standard deviation statistical analysis using one direction anova test with significance level p = 0.05 was done to determine the difference of inhibition zone of calcium hydroxide against streptococcus viridans. the statistical result showed that there was a significant difference of inhibition zone of calcium hydroxide in various concentration against streptococcus viridans (p < 0.05). least significant ��zubaidah: antimicrobial effect of calcium hydroxide as endo intracanal dressing difference (lsd) test was done to determine the difference of diameter of inhibition zone (table 2). discussion an intra-canal dressing is necessarily given prior to root canal preparation in endodontic treatment to sterilize root canal either from anaerobe or facultative anaerobe bacteria. the dominant bacterial found in root canal are streptococcus viridans which is facultative anaerobe. calcium hydroxide used as an intracanal dressing in endodontic treatment because it has an alkalis ph (12.5) and high anti microbial effect.5,17,22 the antimicrobial effect of material or drugs can be examined by sensitivity tests against bacteria, such as dilution or diffusion method. agar diffusion method was chosen based on several reasons. first, this method could be used for activity evaluation of antimicrobial medicine which the concentration had been determined. second, the time needed to analyze the inhibition zone was relatively short. third, the equipments were simple and easily obtained. fourth, the cost was relatively inexpensive, and the most important was the method could determine the bactericide effect of medicine by observing inhibition zone occurred in agar media. this method is generally used to examine anti microbial effect of materials including material of endodontic treatment.17,18 di fiore,19 siqueira and uzeda,18 gomes et al.20 stated that the result of anti microbial test using agar diffusion method depends on several aspects: the size of material molecule, solubility and diffusibility of material in agar media, medicine sensitivity, source of bacterial (strain of species colony), the number of inoculated bacteria, ph of substrate in plate, agar viscosity, condition of agar storage, incubation time and metabolic activity of bacteria. the higher the solubility and diffusibility of material against the media the bigger inhibition zone will be. fifty percent of calcium hydroxide is the lowest concentration. the result of previous study showed that the viscosity of calcium hydroxide mixed with sterile aquabidest similar to paste. in this case, it is similar with clinical use in which the ratio between calcium hydroxide powder and sterile aquabidest with 50% concentration. gomes et al.20 using 50% concentration of calcium hydroxide paste proved that gram negative anaerobe bacteria was more sensitive to calcium hydroxide than gram positive anaerobe facultative bacteria. sixty percent concentration of calcium hydroxide showed mean of inhibition zone was 15,7500 mm (table 1) which the highest antimicrobial effect comparing to the other groups. it might due to ion hydroxyl released (oh–) from calcium hydroxide. ion hydroxyl (oh–) is highly free radical oxidant shows strong reactivity against bacterial cell.21 effect of ion hydroxyl (oh–) is very reactive and quickly combines itself with lipid, protein and nucleate acid resulting lipid peroxides. it would increase membrane permeability of bacterial cells, and followed by protein denaturation, inactivating enzyme and dna destruction which kill the bacteria.1,12 in this concentration calcium hydroxide has not reached the saturation point so calcium hydroxide is still capable to diffuse into muller hinton agar culture media which has been exposed by streptococcus viridans. table 2 showed the antimicrobial test result of calcium hydroxide against streptococcus viridans. calcium hydroxide 55%, and 60% concentration compare to group 50%, 60% concentration compare to 55%, did not show significant difference. it might due to viscosity of the mixture and the number of released hydroxyl ion was the same. in 65% concentration compare to 50%, 55%, and 60% concentration of calcium hydroxide, and also 70% concentration comparing to 50%, 55%, 60%, 65% calcium table 2. the result of lsd test in diameter of inhibition zone of calcium hydroxide in various concentration against streptococcus viridans concentration group i group ii group iii group iv group v group i group ii group iii group iv group v --0.712 --0.102 0.201 --0.032* 0.013* 0.000* --0.000* 0.000* 0.000* 0.013* --*) significant difference (p < 0.05) figure 1. mean of inhibition zone for 50%, 55%, 60%, 65%, and 70% calcium hydroxide against streptococcus viridans. 14.625 14.875 15.75 13.12511.375 0 5 10 15 20 50% 55% 60% 65% 70% concentration ca (oh)2 �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 39-42 hydroxide showed significant difference in inhibition zone against streptococcus viridans. it might due to the viscosity of calcium hydroxide powder and sterile aquabidest has reached saturation point in 65% and 70% concentration, consequently, hydroxyl ion is difficult to release into agar media resulting the decrease of number of hydroxyl ion and small inhibition zone. the diffusibility and solubility of calcium hydroxide in agar media was very low due to high viscosity of material. safavi and nakayama7 suggested that the effect of water insoluble solvent would decrease the affectivity of calcium hydroxide. it is also proved by suzuki11 by mixing calcium hydroxide and pure glycerin or propylene glycol (including calcium hydroxide vehicle). both materials are non-polar therefore it does not show inhibition zone of bacteria. since the solution has reached saturation point, so, hydroxyl ion (oh–) can not diffuse into agar culture media. the number of hydroxyl (oh–) which has been released will be less and the anti microbial effect will decrease. there is other factor such as buffer capacity of culture media which lowering the ph. it makes the antimicrobial of calcium hydroxide reduce.23, 26 the study showed that calcium hydroxide with 60% concentration showed the highest antimicrobial effect compared to calcium hydroxide with 50%, 55%, 65%, and 70% concentration. references 1. estrela c. calcium hydroxide: study based on scientific evidences.1. estrela c. calcium hydroxide: study based on scientific evidences. j appt oral sci 2003; 11(4):269–82. 2. hosoya n, takahashi g, arai t, nakamura j. calcium concentration and ph of periapical environment after applying calcium hydroxide into root canals in vitro. j endod 2001; 27(5):343–6. 3. solak h, oztan md. the ph change of four different calcium3. solak h, oztan md. the ph change of four different calciumthe ph change of four different calcium hydroxide mixture used for intracanal medication. j oral rehab 2003;2003; 30:436–9. 4. leonardo mr, sielveira ff. calcium hydroxide root canal dressing. histopathological evaluation of periapical repair at different time periods. j braz dent 2002; 13:17–22. 5. grossman li, oliet s, del rio ce. ilmu endodontik dalam praktek. abyono r, editor. cetakan i. jakarta: penerbit buku kedokteran egc; 1995. p. 248–50, 256. 6. tronstad l, andreasen jo, hasselgren g, kristerson l, riis i. ph change in dental tissue alter root canal filling with calcium hydroxide. j endod 1981; 7:17. 7. safavi k and nakayama ta. influence of mixing vehicle oninfluence of mixing vehicle on dissociation of calcium hydroxide in solution. j endod 2000; 26:649–52. 8. siqueira jf, lopes hp. mechanisms of antimicrobial activity of calcium hydroxide: a critical review. j intern endod 1999; 32:361–9. 9. pitt ford tr. endodontic in clinical practice. london: united medical and dental schools, university of london, uk; 1997. p. 108–9. 10. saifuddin i. khasiat dan efek iritasi antiseptik poliantibiotika dengan metronodazol sebagai obat sterilisasi saluran akar. surabaya: penelitian laboratorium; 1986. p. 13–14. 11. suzuki k, higuchi n, horiba n, matsumoto t, nakamura h. antimicrobial effect of calcium hydroxide on bacteria isolated from infected root canals. j conservative dentistry in japan 1999; 35:43–47. 12. siqueira jf, uzeda m. influence of different vehicle on the antibacterial effect of calcium hydroxide. j endod 1998; 24(10):663. 13. estrela c, pimenta fc, ito yoko i, bammann l. in vitro determination of direct antimicrobial effect of calcium hydroxide. j endod 1998; 24(1):15–17. 14. sjogren u, fidgor d, spangberg l, sundqvist g. the antimicrobialthe antimicrobial effect of calcium hydroxide as a shortterm intracanal dressing. int j endod 1991; 24(1):19, 25.1991; 24(1):19, 25.24(1):19, 25. 15. baron ej, peterson lr, finegold sm. bailey and scott’s diagnostic microbiology. 9th ed. toronto: cv mosby; 1994. p. 342–5. 16. wistreich ga, lechtman md. laboratory exercises in microbiology. antibiotic sensitivity testing method. third edition. london: glencoe publishing co, inc; 1980. p. 234–7. 17. morrier jj, benay g, hortman c. antimicrobial activity of calcium hydroxide dental cements. an in vitro study. j endod 2003; 29(1):51–53. 18. siqueira jf, uzeda m. intracanal medicaments: evaluation of the antibacterial effect of chlorhexidine, metronidazole and calcium hydroxide associate with three vehicle. j endod 1997; 23(3):167–9. 19. di fiore pm, peter d, setterstrom ja. the antibacterial effects ofthe antibacterial effects of calcium hydroxide apexification pastas on streptococcus sanguis. j oral surg 1983; 55 (1):91–93. 20. gomes bp, ferraz cc, garrido fd. microbial susceptibility to calciummicrobial susceptibility to calcium hydroxide pastes and their vehicle. j endod 2003; 29(11):758–61. 21. oztan md, yilmaz s, kalayci a, zaimoglu l. a comparison of the in vitro cytotoxicity of two root canal sealer. j of oral rehab 2003; 30:426–9. 22. widodo t. paradigma baru pada perawatan endodontik. kumpulan naskah seminar sehari tema ramah perlakuan pada jaringan gigi, ikorgi; 2000. p. 2, 6–7. 23. barbosa cam, goncalves rb. evaluation of antibacterialevaluation of antibacterial activities of calcium hydroxide, chlorhexidine, and camphorated paramonochlorophenol as intracanal medicament. clinical and laboratories study. j endod 1997; 23(5):297–9. 24. walton, torabinejad. pembersihan dan pembentukan saluran akar. in: prinsip dan praktik ilmu endodontik. sumawinata n, nursasongko b, editor. jakarta: penerbit buku kedokteran egc; 1998. p. 295–8. 25. clement dj. medical center. available at: endodontics.mcendo@ flash net. accessed april 29, 2000. 26. spangberg lsw. instrumen, materials and devise. in: cohen s, burns re, editors. pathways of the pulp. 2nd ed. st louis: the cv mosby co; 1998. p. 476. references no 10 tidak ada, no 12 dobel ?? vol 49 no 1 jan-mrt 2016.indd 32 dental journal (majalah kedokteran gigi) 2016 march; 49(1): 33–37 salivary neutrophils isolation of severe early childhood caries patients with flow cytometry analysis using magnetic beads and cd177 marker muhammad luthfi and tuti kusumaningsih department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: neutrophils are the first line of defense, not only serving as he killer of microbes through phagocytosis process, in which reactive oxygen species (ros) and anti-microbial peptides were released, but also regulating activation of immune response. cd177 is a tidylinositol glycosylphosphate glycoprotein with a molecular weight of 5864-kda exclusively found on neutrophils, neutrophilic metamyelocytes, and mielosit. cd177 expression, a protein on the cell surface with an average size ranging from 45% to 65%, is only found on subpopulations of neutrophils. purpose: this study aims to analyze the effects of salivary neutrophil isolation using magnetic beads and cd177 marker on s-ecc patients. method: the study is an observational analytic research with cross sectional approach using flow cytometry analysis on the s-ecc patients and the caries-free children who were asked to use mouthwash, nacl 1.5%. for the isolation of neutrophils, magnetic beads labeled with fitc funds and cd177+ marker were used. result: there were 77.66% of salivary neutrophils expressing cd177+ markers, successfully isolated in the s-ecc patients, while in the caries-free children there were 63.67% of salivary neutrophils. conclusion: in the s-ecc patients, there were 77.66% of salivary neutrophils expressing cd177markers, successfully isolated, while in the caries-free children there were 63.67% of salivary neutrophils. keywords: magnetic beads; salivary neutrophils; s-ecc correspondence: muhammad luthfi, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: m.luthfi7@yahoo.com introduction dental caries in preschool children is a very serious health problem, requiring special attention since dental caries is a focal infection that causes a variety of systemic diseases, and it is not possible to recover the formation of tooth structure when a cavity/hole occurs. dental caries is a disease that is irreversible, consequently, it needs to be cured since its impact is huge in children. for instance, it can cause difficulty in chewing, malnutrition, gastrointestinal disorders, growth disorders, especially weight and height, articulation disorders of speech, and impaired social and cognitive development. dental caries can be considered as a continuous problem that burden children,1 such as affecting the physical and mental health of the children and increasing the risk of dental caries to become the permanent one.2 streptococcus mutans (s. mutans) are the primary etiologic agents of early childhood caries (s-ecc) since they have some mechanisms to colonize the tooth surface, and under certain conditions, they can alter into cariogenic species significantly higher in the oral biofilm environment. as a result, it indicates a causative relation between dental caries and the increasing of s. mutans. in other words, the increasing of the number of s. mutans in saliva can be an indication of the increasing of dental caries prevalence.3 in the s-ecc cases, the increasing of the number of s. mutans can cause the migration of neutrophils out of the bloodstream into oral mouth to perform phagocytosis against microbial pathogens in an effort of homeostasis. research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p32-36 3333luthfi and kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 32–36 in recent years, the perspective about neutrophils has changed dramatically. neutrophils are considered as a key component of the first line of defense against microbes.3 neutrophils not only act as the killer of microbes through phagocytosis process, in which reactive oxygen species (ros) and anti-microbial peptides were released, but also regulate the activation of immune response.5 neutrophils, moreover, can produce a variety of cytokines, chemokine, and growth factors. therefore, neutrophils can be considered as the major contributor to the production of proinflammatory cytokines on infection area.6 furthermore, neutrophils isolated from saliva by a nylon filter sequentially with 20 and 11μm nylon filter that is often used nowadays still have not been able to get the maximum results. meanwhile, the latest method using magnetic beads labeled with cd177 and analyzed using flow cytometry can be considered as one alternative method for the isolation of neutrophil cells. cd177 is a tidylinositol glycosylphosphate glycoprotein with a molecular weight of 58-64-kda contained exclusively in neutrophils, neutrophilic metamyelocytes, and mielosit.7,8 cd177 expression is only found on subpopulation of neutrophils, a protein on the cell surface with an average size ranging from 45% to 65%.9 based on the above information, the researcher wants to isolate and analyze salivary neutrophils with such method in order to obtain optimal neutrophil cells from saliva of severe early childhood caries (s-ecc) patients and caries-free children. materials and methods students of kindergartens selected as sampling sites in the area of surabaya were examined for dental caries by measuring the index of the decayexfoliation filling (def-t). the children were divided into two groups, namely caries-free group and severe caries group with def-t more than 6. all the subjects in the sample aged between 4 to 6 years. prior to the sampling process, questionnaire sheet and informed consent were distributed to the parents of those students. sampling process was conducted by researcher and trained personnel using a standard protocol. subjects should not eat, drink, chew gum, or brush teeth for 60 minutes prior to the sampling process. after collected, the samples were stored at -80° c for analysis.10 salivary neutrophils were obtained by asking the subjects to rinse their mouth with 10 ml of sterile 1.5% nacl solution for 30 seconds, and then expectorated in a sterile glass. this procedure was repeated four times. the samples were subsequently centrifuged at 450 g for 15 minutes at a temperature of 4° c. pellets obtained from the centrifugation result were mixed with 2 ml of rpmi medium. neutrophil cells then were identified using human neutrophils enrichment kit of easy sep brand in with the following methods: the cell suspension with a concentration of 5 x 107 cells/ml was placed in a polystyrene easysep® magnet tubes sized 5 ml (12 x 75 mm). falcon ™ 5 ml polystyrene tube (becton dickinson, catalog # 352 058) was then added with 50 ul/ ml of easysep® neutrophil cell cocktail (e.g. for 2 ml of cells, 100 ul of cocktail is added). it was stirred well and incubated at 4° c for 10 minutes. three mix easysep® nanoparticles were then used to ensure whether the cells were in a homogeneous suspension by conducting pipetting five times. afterward, nanoparticles were added into 100 ul of cells/ ml (2 ml of cells were added into 200 ul of nanoparticles). it was then stirred well and incubated at 4° c for 10 minutes. 2.5 ml of the cell suspension was then put on the tube (without cap) to the magnet for 5 minutes. the next stage, easysep® magnet was removed in a single motion sequence, i.e. reversing the magnet and the tube. cells that were not needed on magnetic beads already labeled were remained and bound in the tube. the tube was in the inverted position for 2-3 seconds, and then returned to the upright position. after that, the empty tube was taken from easysep® magnet and replaced with a new tube containing supernatant fraction placed on the magnet, and then left for 5 minutes. cells in the new tube was then ready for use. analysis of cell suspensions using flow cytometry was performed on a fluorescence activated by cell facscan analyzer (becton dickinson). at least 25,000 events were analyzed for each sample of salivary neutrophils. each sample of salivary neutrophils was identified to get profile of those neutrophils using fsc and ssc based on the size and granular respectively in the neutrophil suspension. cells that had been identified their profile were then analyzed, and positive staining for neutrophil marker was conducted to define events beyond the level of fluorescence. neutrophils that were more than 70% of the isotope and matched with staining control were studied. the percentage of neutrophil cells was determined by subtracting isotope positive staining cells-matched with positive staining antibody cells. meanwhile, the percentage of fluorescent neutrophil cells was determined by gating on both of the cells reacting negatively to propidium iodide which had been labeled. back gating for fsc was then compared to ssc plot to verify the morphology of cells stained positive. results figures 1 and 2 show the isolation results of salivary neutrophils cultured on hank's balanced salt solution (hbss) media observed using olympus inverted microscope with a magnification of 200x on days 1 and 3. they were prepared based on cell sorting method using human neutrophils enrichment kit of easy sept brand and analyzed with flow cytometry using cd177 marker. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p32-36 34 luthfi and kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 32–36 figures 3 and 4 show the isolation of neutrophils on day 1 and 3, using flow cytometry which had previously been prepared using beads magnitude followed by manipulation using human neutrophils enrichment kit of easy sep brand. the results of analysis using flow cytometry show that the number of salivary neutrophils isolated in the caries-free children was 63.67% (figure 3), while that in the s-ecc patients was 77.66% (figure 4). discussion neutrophils are important effector cells participating in innate immune response playing an important role in the first line of host defense against invading pathogens. neutrophils isolated from the oral cavities of both the caries-free children and the s-ecc patients using magnitude beads analyzed by flow cytometry labeled with cd177. based on the analysis, the percentage of neutrophil cells expressing cd177 in the caries-free children was 63.67%, while that in the s-ecc patients was 77.66%. this indicates that the isolation was accurate enough to get neutrophil cells of saliva since in addition to neutrophils, there are a lot of innate immunity cells in saliva, such as eosinophil, basophils, macrophage, and others. human body has cells that function to the defense collectively forming immune system.11 under normal condition, the number of neutrophils is very high in circulation, reached 60-70% of circulating leukocytes. the number of neutrophils in case of inflammation increases rapidly to more than 90%. under the influence of several factors associated with humoral and cellular signals, the neutrophils will migrate to the site of injury or infection that serves as members of anti-pathogens and damaged cells. after being at the site of infection, immune cells, such as neutrophils, monocytes, macrophages, dendritic cells, and mast cells are able to produce specific anti-microbial peptides, such as proteases and reactive oxygen radicals to figure 1. neutrophil cell culture on day 1 after incubation (arrows show the possibility of neutrophil cells, but must be confirmed by further tests). figure 2. neutrophil cell culture on day 3 after incubation (arrows show the possibility of neutrophil cells, but must be confirmed by further tests). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p32-36 3535luthfi and kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 32–36 (a) (b) (c) figure 3. the isolation of salivary neutrophils using labeled magnitude beads and cd177 marker analyzed using flow cytometry in the caries-free children was 63.67%. (a) getting neutrophils by ssc and fsc height; (b) histogram of neutrophils expressing cd177; (c) neutrophils expressing cd177. (a) (b) (c) figure 4. the isolation of salivary neutrophils using labeled magnitude beads and cd177 marker analyzed using flow cytometry in the s-ecc patients was 63.67%. (a) getting neutrophils by ssc and fsc height; (b) histogram of neutrophils expressing cd177; (c) neutrophils expressing cd177. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p32-36 36 luthfi and kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 32–36 facilitate the murder of microbes by disturbing bacterial membrane and metabolism.12 in the human oral cavity, there are actually about 300 to 500 species of microbes mostly consisted of commensal and opportunistic bacteria. the relation between bacteria in the oral cavity and the host dynamically configured is considered as the balance of bacterial virulence factors and host-immune system strength. in saliva, neutrophils are the first line of defense as the most prominent of immune cells for defense against pathogenic microbes. the importance of neutrophils in the host immune system of patients with neutropenia or defects in neutrophil function can lead to a tendency for the occurrence of serious infections.13 recruitment, internal migration, phagocytosis, and activation processes of neutrophil are highly coordinated to prevent or eliminate infection in humans. in the area of infection, neutrophils bind and immerse microbes through a process, known as phagocytosis. neutrophils recognize surface-bound or free molecules secreted by bacteria, including glycan peptide, lipoprotein, lipoteichoic acid (lta), lipopolysaccharide (lps), dna containing cpg, and flagellin. this pathogen molecule is known as pathogenassociated molecular pattern (pamps), interacting directly with a number of pathogen recognition receptors (prrs) expressed on the surface of cells, including toll like receptors (tlrs).14 in conclusion, the number of neutrophils in s-ecc patients successfully isolated was 77.66%, while that in the caries-free children was 63.67%. references 1. clarke m, locker d, berall g, pencharz p, kenny dj, judd p. malnourishment in a population of young children with severe early childhood caries. pediatr dent 2006; 28(3): 254-9. 2. wigen ti, wang nj. caries and background factors in norwegian and immigrant 5-year-old children. community dent oral epidemiol 2010; 38(1): 19–28. 3. corby pm, lyons-weiler j, bretz wa, hart tc, aas ja, boumenna t, goss j, corby al, junior hm, weyant rj, paster bj. microbial risk indicators of early childhood caries. j clin microbiol 2005; 43(11): 5753-9. 4. elbim c, katsikis pd, estaquier j. neutrophil apoptosis during viral infections. open virol j 2009; 3: 52-9. 5. nathan c. neutrophils and immunity: challenges and opportunities. nat rev immunol 2006; 6(3): 173-82. 6. mantovani a, cassatella ma, costantini c, jaillon s. neutrophils in the activation and regulation of innate and adaptive immunity. nat rev immunol 2011; 11(8): 519-31. 7. stroncek df. neutrophil-specific antigen hna-2a, nb1 glycoprotein, and cd177. curr opin hematol 2007; 14(6): 688-93. 8. sachs uj, andrei-selmer cl, maniar a, weiss t, paddock c, orlova vv, choi ey, newman pj, preissner kt, chavakis t, santoso s. the neutrophil-specific antigen cd177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (cd31). j biol chem 2007; 282(32): 23603-12. 9. meyerson hj, osei e, schweitzer k, blidaru g, edinger a, balog a. cd177 expression on neutrophils: in search of a clonal assay for myeloid neoplasia by flow cytometry. am j clin pathol 2013; 140(5): 658-69. 10. phattarataratip e. the role of salivary antimicrobial peptides in shaping streptococcus mutans ecology. iowa research online 2010. available from: http://ir.uiowa.edu/etd/724/ 11. parham p. element soft the immune system and their roles in defense. in: parham p, editors. the immune system. 3rd ed. new york: garland publishing; 2009. p. 2-3. 12. pasupuleti m, schmidtchen a, malmsten m. antimicrobial peptides: key components of the innate immune system. crit rev biotechnol 2012; 32(2): 143-71. 13. rosenzweig sd, holland sm. phagocyte immunodeficiencies and their infections. j allergy clin immunol 2004; 113(4): 620–6. 14. akira s, takeda k. toll-like receptor signalling. nat rev immunol 2004; 4(7): 499-511. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p32-36 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �2 the application of lesion sterilization and tissue repair �mixmp for treating rat's dental pulp tissue raditya nugroho,1 ananta tantri budi,2 and sri kunarti2 1 department of conservative dentistry, faculty of dentistry, universitas jember, jember-indonesia 2 department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: lesion sterilization and tissue repair (lstr) 3mix-mp are three broad-spectrum antibiotics, including metronidazole, ciprofloxacin and minocycline are mixed with propylene glycol or macrogol. there is the possibility of the healing process that marked proliferation of new blood vessels and proliferation of fibroblasts in the treatment of irreversible pulpitis by pulp capping lstr 3mixmp because of the principle of the method lstr 3mix-mp is to kill bacteria. purpose: the purpose of this study to prove the effect of lstr 3mix-mp on chronic inflammation and the healing process in rat dental pulp tissue in vivo. methods: rattus norvegicus anaesthetized by using ketamine and xylazine dissolved in sterile isotonic saline solution (0.2 ml/50gr mm) on the upper right thigh. cavity preparation class i to perforation by using a low speed tapered diamond round bur. in the treatment group, rats were treated 3mix-mp at a dose of 10 mg and then covered with glass ionomer cement for 7 days on the pulp that has been opened for 3 days. the control group treated with saline irrigation on the pulp that has been opened for 3 days. rats were killed after seven days, and then made preparations pulp tissue to count the number of lymphocytes, macrophages, plasma cells, blood vessels, and fibroblasts results: there is an increase in the average number of macrophage cells, plasma, and fibroblasts; and decreased lymphocytes and blood vessels in the treated group exposure lstr 3mix-mp. conclusion: lstr 3mix-mp can reduce chronic inflammation process and enhance the healing process in rat dental pulp tissue. keywords: lstr 3mix-mp; chronic inflammation; healing correspondence: raditya nugroho, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas jember. jl. kalimantan i no. 37 jember 68121. e-mail: ranugtab@gmail.com research report introduction lesion sterilization and tissue repair (lstr), also called non instrument endodontic treatment (niet), is a new treatment with or without pulp and periapical infections by using a mixture of three antibiotics as disinfection.1 the mixture of those three antibiotics is considered as quite powerful antibiotics in eradicating bacteria compared to a single antibiotic.2,3 thus, this disinfection treatment is expected to heal the lesion fast. this treatment actually was developed by cariology research unity of the niigata university school of dentistry in 1988. there was disagreement about lstr in the earlier studies showing that lstr therapy, using a mixture of metronidazole, ciprofloxacin, and minocycline (three materials) and a mixture of carrier materials, such as macrogol and propylene glycol (mp), has toxic effect on the cell culture of fibroblast.1,4 in addition, the use of antibiotic pasta may cause bacterial resistance.2 thus, the use of lstr is still questionable and requires further research. moreover, there are different opinions on the mixture ratio and the proportion of antibiotics used in lstr, namely metronidazole, ciprofloxacin and minocycline (3mix). some researchers use a mixture of 3: 1: 1, while others use 3: 1: 3 like what hoshino did. three antibiotics (3mix) were mixed with propylene glycol or macrogol as the carrier (mp) of 3mix into the dentinal tubules killing all the bacteria in lesion. 3mix is incorporated into mp using the following 1:5 (mp:3mix) or 1:7 (standard mix).5,6 dental journal (majalah kedokteran gigi) 20�5 march; 48(�): �2–�5 �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��nugroho, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 12–15 inflammation, furthermore, is a protective mechanism that is limited to trauma or microbial invasion by destroying, diluting, or restricting hazardous materials causing tissue damage. acute inflammation even can cause the elimination of harmful agents followed by decreasing the reaction and repairing damaged tissue. if the cause of the inflammation cannot be removed, there will be a chronic inflammation. chronic inflammation is characterized by mononuclear infiltration (lymphocytes, macrophages, and plasma cells), tissue destruction and repair marked with the proliferation of new blood vessels and fibroblasts. inflammatory healing reaction then will arise immediately after the injury while acute inflammatory reaction will be running. nevertheless, the healing process still can occur if the cause of the injury can be destroyed or neutralized. this process consists of the replacement of dead cells by live cell.7-9 direct pulp capping treatment have used pulp perforation technique due to mechanical bur trauma or deep caries cleaning with reversible pulpitis diagnosis. in the clinics of faculty of dental medicine, universitas airlangga, however, reversible pulpitis with pulp perforation treatment is rare. in general, people come up with superficial dental caries, or teeth that were perforated before with irreversible pulpitis diagnosis. irreversible pulpitis is an inflammation of the pulp that cannot recover normally even if the cause of inflammation is removed. damage that occurs in irreversible pulpitis can be caused by surgical procedures or blood flow interruption in the pulp due to trauma. in irreversible pulpitis, there is also a bacterial infection occurred in pulp.10 a treatment using lstr 3mix-mp is expected to heal irreversible pulpitis since a previous research conducted by takushige et al. shows that the perforation of pulp and pulpitis with spontaneous pain can cause positive clinical results.11 nevertheless, the mechanism of pulp therapy using lstr 3 mix-mp is still unclear. there is still a possibility of irreversible pulpitis healing process in pulp capping treatment using lstr 3mix-mp since its principle is to kill bacteria. it means that if bacteria can be removed, then inflammation can subside, thereby allowing dental pulp tissue repair.11 therefore, a further research is needed to know the effectiveness of the provision of lstr 3mix-mp on the healing process by calculating a chronic inflammatory cells (lymphocytes, macrophages, and plasma cells), fibroblasts, and blood capillaries. materials and methods in this research, experimental animals used were male wistar strain rattus norvegicus aged 8-16 weeks and weighed 200-250 grams. moreover, samples used for control group were treated with saline irrigation in their pulp that has been opened for 3 days. meanwhile, samples used for treatment group were similarly treated to a previous research conducted by sabir12 in which those animals were anaesthetized by using ketamine and xylazine dissolved in sterile isotonic saline solution (0.2 ml/ 50gr mm) on their upper right thigh. thus, the similar working principle of asepsis was conducted. all the tools were sterilized by dry heat of 160° c for 1 hour. the disinfection was conducted on the upper right thigh of those animals with betadine before intramuscular anesthesia was conducted. the first right molars of the lower jaw were disinfected with 70% alcohol. class i cavity with perforation was prepared by using low speed tapered round diamond bur no. 200s (intensive, switzerland) with a bur’s diameter of 0.84 mm closer to the pulp, and then the thin layer of dentin was penetrated to the perforation in the pulp chamber area. those animals were given 3mix-mp at a dose of 10 mg, and then the pulp that had been opened for 3 days filled with glass ionomer cement for 7 days. 3mix-mp used was a pasta combination of lstr, a mixture of ciprofloxacin, metronidazole, and minocycline, with a ratio of 1: 3: 3 (3mix) mixed with macrogol and propylene glycol to be formed. all samples in the control group and in the treatment group were sacrificed after seven days of the treatment. their jawbone in the interdental areas of mandibular molars then was cut and put into the fixation solution. this research using independent t-test with significance level of 95%(p<0.05) to see the difference in the number of lymphocytes, macrophages, plasma cells, blood vessels, and fibroblasts between control and treatment groups. the normality of the data was tested first by using kolmogorovsmirnov test, while the homogeneity of the data was tested by using levene test. results it can be seen that the average numbers of macrophages, plasma cells, and fibroblasts in the treatment group exposed with lstr 3 mix-mp on their dental pulp tissue had larger perforations than those in the control group without lstr 3 mix-mp exposure (figure 1). the average numbers of lymphocytes and blood vessels in the treatment group were lower than those in the control group. in short, it can be said that the numbers of lymphocytes, macrophages, blood vessels and fibroblasts in the treatment group were significantly different from those in the control group with the significance value less than 0.05 (p<0.05). meanwhile, the number of plasma cells in the treatment group was not significantly different from that in the control group with the significance value greater than 0.05 (p>0.05). discussion this research used wistar strain rattus norvegicus as experimental animals because those animals are easy to care, and also have a similar healing reaction to humans. in other words, their dental pulp has a similar reaction to humans dental pulp. thus, the lower right first molar tooth dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �4 nugroho, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 12–15 was selected not only because the structure and anatomy of their tooth are similar to human’s tooth, which can access to the pulp tissue, but also because the speed of attrition due to mastication on the occlusal surface of their molars is slower than their incisive.12 based on the result of histopathological examination on the pulp tissues in the treatment group, then it is known the average numbers of macrophages, plasma cells, and fibroblasts in the treatment group were increased. meanwhile, the average number of blood vessels in the treatment group was decreased compared to in the control group. the increasing average number of lymphocytes, macrophages, plasma cells, and fibroblasts and the decreasing average number of lymphocytes and blood vessels were associated with the healing process. in the healing process, there are actually several phases, namely inflammation, proliferation, and maturation. in inflammation phase, vascular and cellular responses respond to injury. both injury and thermal and mechanical exposure can usually cause interference with microvasculature, and subsequently can lead to bleeding. during this inflammation process, the vasoconstriction of blood vessels occurs with mediators, such as epinephrine, norepinephrine, prostaglandins, serotonin, and thromboxane. vasoconstriction actually plays a role in reducing the occurrence of bleeding at the wound with the addition of platelet aggregation factors-other healing factors. vasoconstriction is then followed by longer vasodilation period mediated by histamine, prostaglandins, kinins, and leukotrienes. vasodilation is characterized by erythema, edema, and heat that occurs after an injury. vasodilatation is an important phase in which blood flow to the injured area is increased, followed by inflammatory cells and important factors in fighting infections and cleaning tissue damaged by the injury.12 in proliferation phase, the formation of granulation tissue occurs. the formed granulation tissue consists of inflammatory cells, fibroblasts, new blood vessels in fibronectin matrix, collagen, glycosaminoglycans, and proteoglycans. this formation of granulation tissue occurs 3 to 5 days after the injury, and overlaps with the inflammation phase. in the proliferation phase, moreover, there are fibroplasia and angiogenesis processes. in the fibroplasia process, fibroblasts are the most important component. fibroblasts are responsible for the formation of collagen, elastin, fibronectin, and glycosaminoglycans. the growth of fibroblast proteases in the wound can be considered as a form of the reduction of inflammatory cells. the existence of demand for the inflammation process will make the production of chemotactic factors, called inflammatory cells decreased and disappeared. fibroplasia begins 3-5 days after the injury and can last for 14 days. fibroblasts migrate and proliferate in response to the presence of fibronectin, platelet-derived factor (pdgf), fibroblast growth factor (fgf), transforming growth factor (tgf), and c5a. in the angiogenesis process, the amount of blood supply is vital to maintain the shape of the newly formed tissue characterized by the presence of erythema at the newly formed scar. in the next process, the blood vessels will be lost because it is not needed anymore, so is in the formed scar. macrophages are something essential in stimulating angiogenesis and in producing macrophagederived angiogenesis factor in response to the deeper tissue oxygenation. the function of these factors are as chemoattractant in endothelial cells. basic fibroblast growth factors are actually secreted by macrophages. meanwhile, vascular growth factors are secreted by epidermal cells, considered as important factors in the occurrence of angiogenesis. angiogenesis produce large blood flow in the injured area, and consequently will increase the perfusion factors-healing factors. in the next process, the cessation of angiogenesis will occur as a termination request from new blood vessels. the new blood vessels become too important to be lost by the presence of apoptosis. figure 1. the average number of lymphocytes, macrophages, plasma cells, blood vessels, and fibroblasts. 1 figure 1. the average number of lymphocytes, macrophages, plasma cells, blood vessels, and fibroblasts. 152.5 35.623 14.375 31.625 136.875 29.375 43.75 17 11.125 311.25 0 50 100 150 200 250 300 350 lymphocytes macrophages plasma cells blood vessels fibroblasts th e av er ag e nu m be r o f c el ls control group treatment group �5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �5nugroho, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 12–15 in the maturation phase, moreover, collagen and cytokines play a role. collagens are an important component in all phases of wound healing. synthesized by fibroblasts, they impart integrity and strength to all tissues and play a key role, especially in the proliferative and remodelling phases of repair. collagens act as a foundation for the intracellular matrix formation within the wound. collagen remodeling during the maturation phase relies on the synthesis of collagen. collagenase and matrixmetalloproteinase in the wound actually helps to eliminate excess collagen during the synthesis of new collagen. tissue inhibitor of metalloproteinase will inhibit collagenolitic enzyme, so there is a balance between the formation of new collagen and the disposal of old collagen. in addition to collagen, cytokines also play a role as an important mediator in the wound healing process.9,13 based on the results in this research, it is known that the numbers of macrophages, plasma cells, and fibroblasts in the treatment group were bigger than those in the control group associated with the provision of lstr 3mix-mp. it is because in the control group, inflammation process was still on progress to become chronic inflammation although the process could also improve to healing process. the healing process that occurred in the control group, however, was slower than in the treatment group. it is because in the control group, substance or agent that can help the healing process go faster was not given. in addition, it is also known that the average number of blood vessels in the treatment group was about 11.125 smaller than in the control group, about 31.625. the low average number of blood vessels in the treatment group could be due to the faster healing process. it is because in the angiogenesis phase, as described earlier, there was a reduction or even elimination of the blood vessels that were not important. the loss of blood vessels actually has been programmed by the process of apoptosis. it can be seen that the average number of fibroblasts in the treatment group was significantly increased compared to those in the control group. this condition is because of fibroplasia process in the proliferative phase. fibroblasts play an important role in the healing process because fibroblasts are responsible for the formation of new tissue and the process of maturation in the next process. in the perforation area, the presence of fibroblasts is very important since they together with odontoblastoid synthesize collagen type i, which is instrumental in the formation of reparative dentin.14 in the treatment group, the healing process can be due to the provision of lstr 3mix-mp, which acts as an antibiotic. antibiotics in lstr 3mix-mp consist of metronidazole, ciprofloxacin, and minocycline. the use of metronidazole alone has a role in inhibiting amoeba, trichomonas, and anaerobic bacterial infections. metronidazole has a broad bactericidal spectrum. meanwhile, ciprofloxacin used in mixing antibiotics has a broadantibiotic spectrum that can fight gram-positive and gram-negative bacteria. minocycline, a group of tetracycline, has a broad antibacterial activity which includes gram-positivenegative, aerobic, and anaerobic bacteria. in short, it can be said that after the dental pulp of those rats was drilled, the dental pulp was left open to let bacterial colonization, including good bacteria from dental bacteria, bacteria in saliva, and bacteria found in periodontal tissues. inflammation process then occurred. but, inflammation process occurred in the control group lasted longer and became chronic inflammation process, while the inflammation process in the treatment group did not last long and got faster healing process. the existence of the healing process that occurs in the treatment group can be associated with a mixture of antibiotics, namely metronidazole, ciprofloxacin, and minocycline which has anti-bacterial power.11 in conclusion, lstr 3mix-mp can reduce chronic inflammation process and enhance the healing process in rat dental pulp tissue. references 1. hendrawan c. uji toksisitas lession sterillization and tissue repair (3mix-mp) terhadap kultur sel fibroblas. undergraduate theses airlangga university library; 2007. 2. huang gj. a paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. j dent 2008; 36(6): 379-86. 3. mohammadi z. antibiotics as intracanal medicaments: a review. cdaj 2009; 137: 99. 4. prabhakar ar, sridevi e, raju os, satish v. endodontic treatment of primary teeth using combination of antibacterial drugs: an in vivo study. j indian soc pedod prev dent 2008; 26 (suppl 1): s5-10. 5. parasurama v, muljibhai bs. 3mix mp in endodontics: an overview. jdms 2012; 3: 36-45. 6. handajani j, haniastuti t, ohshima h, hoshino e. survival of root canal pulp tissue after pulpitis. j lstr ther 2010; 9: 1-6. 7. kumar v, cotran rs, robbins sl. robbins basic pathology. michigan: saunders; 2003. p. 31-5. 8. baratawidjaja k. imunologi dasar. edisi-4. jakarta: balai penerbit fk ui; 2004. p. 153-70. 9. kumar v, cotran rs, robbins sl. buku ajar patologi. jakarta: egc; 2007. p. 35-65. 10. ingle ji, bakland lk. endodontics. fifth edition. london: bc decker inc; 2002. p. 31-40. 11. takushige t, cruz ev, moral aa, hoshino e. non surgical treatment of pulpitis, including those with history of spontaneous pain, using a combination of antibacterial drugs. j lstr ther 2008; 7: 1-5. 12. sabir a. respon inflamasi pada pulpa gigi tikus setelah aplikasi ekstrak etanol propolis (eep). oral sci j 2005; 47: 77-83. 13. velnar t, hainar t, smrkolj. the wound healing process: an overview of the cellular and molecular mechanism. j inmedres 2009; 37: 1528-42. 14. goldberg m, smith aj. cell and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. crit rev oral biol med 2004; 15(1): 13-27. 82 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 82–86 original article the antifungal susceptibility of candida albicans isolated from hiv/aids patients sri rezeki1, siti aliyah pradono2, gus permana subita2, yeva rosana3, sunnati4 and basri a. gani5 1 department of oral medicine, faculty of dentistry, universitas syiah kuala, banda aceh, indonesia 2 department of oral medicine, faculty of dentistry, universitas indonesia, jakarta, indonesia 3 department of microbiology, faculty of medicine, universitas indonesia, jakarta, indonesia 4 department of periodontics, faculty of dentistry, universitas syiah kuala, banda aceh, indonesia 5 department of oral biology, faculty of dentistry, universitas syiah kuala, banda aceh, indonesia abstract background: candida albicans was found to be dominant in patients with human immunodeficiency virus / acquired immunodeficiency syndrome (hiv/aids). the antifungals fluconazole, ketoconazole, and nystatin were used as oral candidiasis therapy for hiv/aids, each of which has differing susceptibility in oral candidiasis therapy. purpose: the present study aimed to evaluate the susceptibility and antifungal resistance to oral c. albicans in hiv/aids patients. methods: the subjects followed the universal precaution principles. oral candida species were isolated from the saliva of 98 hiv/aids subjects. identification of candida species was carried out by the mycobiotic agar of api 20 c aux system. susceptibility and resistance antifungal tests on the candida species were performed using a fungus atb kit. results: candida albicans was the most dominant species found from 98 subjects (95%). the rest were other candida species. there are 41 subjects (42%) with a history of oral candidiasis, and 57 subjects (58%) without. the history of those who used antifungals were: nystatin = 60 subjects (61%), fluconazole = 39 subjects (40%), and ketoconazole = two subjects (2%). these antifungals have a susceptibility above 80% against c. albicans, except the nystatin group (79%) (p>0.05; 0.628), but fluconazole has a strong correlation (r=0.820) to susceptibility, susceptibility-dependent dose, and resistance. conclusion: candida albicans was dominant in the saliva of hiv/aids patients. this fungus was effectively treated by fluconazole, ketoconazole and nystatin. these antifungals had a high susceptibility at ≤ 8 μg/ml to c. albicans. keywords: antifungal; candida albicans; hiv/aids; susceptibility and resistance correspondence: sri rezeki, department of oral medicine, faculty of dentistry, universitas syiah kuala. jl. teuku nyak arief darussalam, banda aceh, 23111 indonesia email: iy_99fkg@unsyiah.ac.id introduction in indonesia (data 2005–2019), the number of people living with human immunodeficiency virus (hiv) was 338,363. meanwhile, an acquired immunodeficiency syndrome (aids) was reported in 115,601 cases.1 one of the conditions experienced by people with hiv/aids is mucosal candidiasis in the oral, esophageal, or vaginal regions. the circumstances of oral candidiasis in hiv infections vary from 7% to 93%, depending on the patient mix, diagnostic criteria, and research methods.2 it is considered an important marker of immune decline as an early manifestation of hiv infection. in addition, there is a decrease in cd4 lymphocyte cells <200.3 oropharyngeal and esophageal candidiasis are common manifestation in hiv patients. the disease is caused by c. albicans and other oral candida species. antiretroviral therapy (art) can reduce the prevalence of oropharyngeal and esophageal candidiasis in refractory disease.4 the resistance of fluconazole or azole is influenced by repeated long-term exposure. in this permutation, the majority of cases relate to the acquisition of resistance to c. albicans.5 both local and systemic predisposing factors lead to candida’s commensal changes, developing into oral candidiasis. these infections of esophageal candidiasis, as well as morbidity, and even mortality, were reported as secondary complications.6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p82–86 mailto:iy_99fkg@unsyiah.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p82-86 83rezeki et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 82–86 the antifungal drugs fluconazole, ketoconazole, and nystatin effectively treat hiv oropharyngeal and esophageal candidiasis. therefore, their widespread use has the potential to increase c. albicans resistance. these antifungals, when observed in different groups of hiv patients, were resistant to c. albicans. one-third of c. albicans hiv isolates were resistant to several antifungals.7 mohamadi et al.8 revealed that c. albicans remains the most common species that rapidly developed into fluconazole, ketoconazole, and nystatin resistant. the resistance of these antifungals has an impact on dehydration, malnutrition, and increased hiv infection. it is often reported in cases of esophageal and oropharyngeal candidiasis. monroy-pérez et al.9 reported that fluconazole, ketoconazole, and nystatin have varying susceptibility to c. albicans. however, these three antifungals have the same principle action for inhibiting the development of c. albicans by damaging the cell walls and inhibiting synthesis protein.10 susceptibility of fluconazole, ketoconazole, and nystatin to c. albicans in hiv/aids patients can be used as a reference for determining relevant and appropriate doses for hiv/aids oral candidiasis, which can reduce the prevalence of resistance. this study evaluated the susceptibility of antifungals to oral c. albicans isolates from hiv/aids patients. materials and methods the research was approved ethical clearance by the faculty of dentistry, universitas syiah kuala, banda aceh, indonesia, no.187/ke/fkg/2020. saliva was collected from 98 people with hiv/aids at the aids center of cipto mangunkusumo hospital (rscm), jakarta, indonesia. the isolation and characterization of candida species from the samples were carried out at the microbiology laboratory, faculty of medicine, university of indonesia. criteria for inclusion were patients with hiv/aids who had not used antifungal drugs for the previous week, were communicative and were cooperative. the patients included male and female patients. meanwhile, the exclusion criteria included people living with hiv/aids who have refused to be research subjects, who have used antifungal drugs for the past week, were non-communicative and were non-cooperative. the subjects were asked to sign an informed consent, and questionnaire sheets were filled in with the subjects’ demographic data obtained from interviews. interviews were conducted before sampling, and the medical records of subjects were obtained from the rscm’s aids clinic, jakarta, indonesia. the researcher applied the sampling procedure to people with hiv/aids using personal protective equipment and precautions, such as gloves and masks, hand hygiene and sterility, disposing of medical waste contaminated with the saliva of research subjects, and applying laboratory work safety procedures. the following oral rinse technique was used to collect the saliva: the subjects were instructed to rinse their mouths in 10 ml of phosphate buffer saline (pbs) (sigmaaldrich, darmstadt, germany) for 15 sec. the saliva was then expectorated into sterile containers and stored at -25°c.11 next, the oral saliva specimen was cultured on mycobiotic® agar at 35°c for 48 h for identified a candida species. 12 the suspensions of c. albicans cultured on the mycobiotic® agar medium using 0.85% nacl and adjusted to mcfarland 2 (6 x 108 cfu/ml).13 then, 100 μl of candida suspension in 0.85% nacl was mixed to api 20 liquid medium. then homogenization was carried out. subsequently, 200 μl was taken and placed into a test vial. a fermentation assay was conducted on several carbohydrates in parallel. after 48 h of incubating at 30oc, a well-observed growth was taken randomly. a positive reaction was characterised by turbidity in each well observed after incubation at 30oc for 48 h.9 the fungus atb® commercial kit was used to evaluate the susceptibility test, with the maximal assay in 128 μg/ml. a 20 μl suspension of c. albicans in 0.85% nacl was added to the fungus atb® media. after homogenisation, each well was inoculated with 135 μl of homogeneous inoculums and incubated at 37°c for 24 h. the growth of c. albicans on the strip was read visually, according to the instructions kit, with baseline turbidity as the indicator of the growth of c. albicans in fluconazole, ketoconazole and nystatin in concentrations of <1, 2, 4, 64, and >128 μg/ml, which was an indicator of susceptibility (≤8 μg/ml), susceptibility-dose dependent (s-dd) (16–64 μg/ml) and resistance (>128 μg/ ml). the growth of c. albicans characterised by turbidity relates to the atb® kit: 0 (no growth), 1 (weak growth), 2 (reduction in growth), 3 (growth reduced slightly), and 4 (no reduction in growth).14 the kruskal–wallis test analysed the data of susceptibility, s-dd, and fluconazole-resistant against c. albicans. the significance limit of p<0.05 and the correlation coefficient (r = 1) was strongly correlated. results data from study subjects included in this study are shown in table 1, representing age group, sex, history of hiv/aids transmission, history of antifungal agents and oral candidiasis. these indicators could be the reference for this study, which also correlated with candida species susceptibility. c. albicans were found predominantly in hiv/aids subjects without a history of candidiasis (table 2). its antifungals were reported susceptible to c. albicans and other candida species. the data from table 2 refer to an in vitro assay related to antifungal susceptibility to c. albicans, which were selected to represent the dominant population of candida species from hiv/aids subjects. table 3 shows that the three antifungal drugs have a high susceptibility above 80%, but not significantly dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p82–86 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p82-86 84 rezeki et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 82–86 (p> 0.05; 0.628), which means that all drugs have a potential antifungal effect on c. albicans. three antifungal drug concentrations showed significant differences in fluconazole’s susceptibility against c. albicans (p<0.05; 0.024). it means that the concentration of the three drugs has a strong influence on fungicidal or fungistatic properties. the subject status was related to susceptibility, s-dd, and resistance of the three antifungal drugs against c. albicans (p> 0.05; 0.175). based on spearman’s rho correlation, drug status (susceptibility, s-dd, and resistance) has a strong relationship with the concentration of the three groups of antifungal drugs (r = 0.820). the standard susceptibility ≤ 8 μg/ml, s-dd 16-64 μg/ml, and resistant ≥ 128 μg/ml is based on the atb® fungus kit. table 1. distribution and frequency characteristics of hiv/aids subjects characteristics n % gender female 10 10 male 88 90 age (year) 20–29 47 48 30–39 40 41 40–49 10 10 50–59 1 1 risk factors for hiv transmission intravenous drug users (ivdu) 61 69 homosexual intercourse 1 1 heterosexual intercourse 22 22 ivdu and homosexual intercourse 1 1 ivdu and heterosexual intercourse 5 5 ivdu and tattoo/piercing 2 2 history of antifungal agents fluconazole no 59 60 yes 39 40 nystatin no 38 39 yes 60 61 ketoconazole no 96 98 yes 2 2 oral candidiasis no 57 58 yes 41 42 table 2. susceptibility and resistance antifungal on the candida species and oral candidiasis history in hiv/aids patients candida species n % fluconazole ketoconazole nystatin oral candidiasis history susceptible resistant susceptible resistant susceptible resistant yes no c. albicans 93 95 80 13 81 12 80 13 39 50 c. dubliniensis 2 2 2 0 1 1 2 0 1 2 c. guillermondii 1 1 1 0 1 0 1 0 1 2 c. famata 1 1 1 0 1 0 1 0 0 2 c. tropicalis 1 1 1 0 1 0 1 0 0 1 table 3. antifungal susceptibility on c. albicans in hiv/aids patients antifungals (μg/ml) fluconazole ketoconazole nystatin p-value n % status turbidity n % status turbidity n % status turbidity 1 74 80 susceptible 0 75 81 susceptible 0 73 79 susceptible 0 *p>0.05 (0.628) 2 3 3 susceptible 1 1 1 susceptible 1 1 1 susceptible 1 4 3 3 susceptible 1 1 1 susceptible 1 1 1 susceptible 1 64 1 1 s-dd 2 1 1 s-dd 2 1 1 s-dd 2 128 12 13 resistant 3 15 16 resistant 3 17 18 resistant 3 * kruskal–wallis test and spearman’s rho correlation; s-dd (susceptibility-dose dependent); susceptible ≤ 8 μg/ml, s-dd 16-64 μg/ml, resistant ≥ 128 μg/ml, and 250 μg/ml not recommended: turbidity indicator, 0 (no growth), 1 (weak growth), 2 (reduction in growth), 3 (growth reduced slightly), 4 (no reduction in growth). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p82–86 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p82-86 85rezeki et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 82–86 discussion this research reported that subjects without candidiasis are more dominant than with oral candidiasis. these subjects have susceptibility and resistance to fluconazole, ketoconazole, and nystatin in low. generally, these antifungals have a vulnerability to the oral candida species, mainly c. albicans. the presence of c. albicans in the oral candidiasis of hiv patients is confirmed by patil et al.15 who found 95.2% of c. albicans cases leading to oropharyngeal candidiasis in hiv/aids. this study indicates that the male population is higher than the female population with an average age of 20–29 years and ages 30–39 (table 1). the most dominant transmission is through intravenous and heterosexual injections. glick et al.16 reports that men who have sex with men (msm) have higher rates of hiv and sexually transmitted infections (stis) than women and other heterosexual men. this increased risk persists across all age groups and reflects various biological and behavioural factors, but there are some direct comparisons of sexual behaviour patterns between populations.16 in this research, we found the different effects of three antifungal types to treat oral candidiasis on hiv/aids subjects (table 3). they have a susceptibility to preventing the oral candidiasis treatment of subjects with hiv/aids. the oral candidiasis prophylaxis used fluconazole (200 mg/day) with a cd4 count <100 cells/μl also in patients with cd4 100–200 cells/μl. additionally, long-term oral candidiasis prophylaxis with fluconazole is likely to lead to resistance.17 based on the characteristics analyses of hiv/aids, 42% of the subjects had a history of candidiasis, 58% had no history of candidiasis. the candida species cultures from the 98 subjects showed 95% c. albicans, while the rest were other candida species. it is, therefore, explained that in hiv, oral candidiasis is related to the history of antifungal use (table 1). the subjects had a history of antifungal use with different percentages of antifungals. garcia-cuesta et al.18 explain that the oral antifungal of candidiasis of a patient with hiv status is often given several antifungal types to prevent oral candida species symptoms and infection. the use of an antifungal mixture to maintain the fungal population’s balance in the oral cavity can also help increase the mucosal oral defence system to prevent candida species’ adhesion.19 the development of oral candidiasis in hiv/aids patients is highly dependent on the history of antifungal treatment. it is reasonable to suspect that some subjects in this study have had proper oral candidiasis treatment. nevertheless, the prevalence of c. albicans in hiv is always associated with resistance to fluconazole.20 in general, the injection of narcotic drugs and heterosexual relationships tend to be experienced by other people living with hiv. these trends indicate that the susceptibility population is strongly related to the level of resistance to fluconazole or other antifungal drugs such as nystatin and ketoconazole. maheshwari et al.21 reported that in general hiv-positive patients with cd4+ cell counts between 200 and 400/μl had more colonisation of c. albicans and c. dubliniensis. table 3 shows that the lowest concentration of fluconazole has susceptibility to oral c. albicans. in contrast, the highest concentration of fluconazole is resistant to the antifungal of c. albicans essence of hiv/aids. thus, fluconazole toxicity to candida species at the lowest concentration is becoming higher. this antifungal has the ability to prevent candida infections by inhibiting dna synthesis and ergosterol biosynthesis in fungal cell membranes, changing cell surface hydrophobicity, and influencing the synthesis of triglycerides and phospholipids.22 based on this study, resistance occurs due to intense administration with varying doses. this finding concurs with previous research showing that resistance occurred, on average, in minimal inhibition concentration (mic) of fluconazole concentrations of up to three times due to longterm treatment.23 patil et al.24 reported that the subject’s decreased resistance could support oral candidiasis development. as many as 95% of c. albicans cases were reported as a trigger factor for oral candidiasis. the oral candida species’ resistance was reported to be less than sensitive, but it can threaten oral candidiasis if hiv/aids infection persists.24 this study found that susceptibility frequency is higher than that of resistance, so these antifungals are recommended to prevent oral candidiasis in hiv/aids. the antifungal drugs used in this research have different susceptibility and resistance properties. however, they have the same tendency to suppress the growth of c. albicans, as shown in tables 2 and 3. it is indicated that fluconazole, ketoconazole, and nystatin have a high susceptibility, with an average above 80%. these antifungals have a substantial effect on c. albicans, both fungicidal and fungistatic. other findings from this study are that the three drugs’ susceptibility and resistance. whaley et al.25 reported that in the treatment of hiv, the administration of azole groups could cause c. albicans resistance or trigger the development of non-c. albicans species, such as c. glabrata and c. krusei, which intrinsically cause resistance. the opposition to one of the azole antifungal drugs is often associated with other azole resistance.25 period exposure is a risk factor in fluconazole resistance. refractory oropharyngeal candidiasis of treatment by fluconazole is most frequently used in hiv.8 the tracing of the possible history of antifungal drug administration was obtained through periods of hiv infection and diagnosed hiv patients. the higher possibility of opportunistic infections impacts oral candidiasis with treatment by antifungals, which then affects susceptibility. however, these allegations still need further analysis regarding the relationship between the length of hiv infection with a history of antifungal use and susceptibility based on more accurate data. in this study, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p82–86 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p82-86 86 rezeki et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 82–86 the susceptibility test was not carried out on other candida species because the population was too small. there was also no antifungal combination to test the susceptibility to c. albicans. it can be concluded that candida albicans was dominant in the saliva of hiv/aids patients. this fungus was effectively treated by fluconazole, ketoconazole, and nystatin. these antifungals had high susceptibility at ≤ 8 μg/ml to c. albicans. references 1. rahmawati m. penangulangan hiv/aids di indonesia dalam ancaman rkuhp: proyeksi dampak kriminalisasi perilaku beresiko transmisi hiv/aids dalam rkuhp terhadap penanggulangan hiv/ aids di indonesia. jakarta: institute for criminal justice reform; 2019. p. 7–8. 2. anwar kp, malik a, subhan kh. profile of candidiasis in hiv infected patients. iran j microbiol. 2012; 4(4): 204–9. 3. wilson d. candida albicans. trends microbiol. 2019; 27(2): 188–9. 4. thompson gr, patel pk, kirkpatrick wr, westbrook sd, berg d, erlandsen j, redding sw, patterson tf. oropharyngeal candidiasis in the era of antiretroviral therapy. oral surg oral med oral pathol oral radiol endod. 2010; 109(4): 488–95. 5. mulu a, kassu a, anagaw b, moges b, gelaw a, alemayehu m, belyhun y, biadglegne f, hurissa z, moges f, isogai e. frequent detection of ‘azole’ resistant candida species among late presenting aids patients in northwest ethiopia. bmc infect dis. 2013; 13: 82. 6. rodrigues cf, rodrigues me, henriques m. candida sp. infections in patients with diabetes mellitus. j clin med. 2019; 8(1): 76. 7. katiraee f, teifoori f, soltani m. emergence of azole-resistant candida species in aids patients with oropharyngeal candidiasis in iran. curr med mycol. 2015; 1(3): 11–6. 8. mohamadi j, motaghi m, panahi j, havasian mr, delpisheh a, azizian m, pakzad i. anti-fungal resistance in candida isolated from oral and diaper rash candidiasis in neonates. bioinformation. 2014; 10(11): 667–70. 9. monroy-pérez e, paniagua-contreras gl, rodríguez-purata p, vacapaniagua f, vázquez-villaseñor m, díaz-velásquez c, uribe-garcía a, vaca s. high virulence and antifungal resistance in clinical strains of candida albicans. can j infect dis med microbiol. 2016; 2016: 5930489. 10. hasim s, coleman jj. targeting the fungal cell wall: current therapies and implications for development of alternative antifungal agents. future med chem. 2019; 11(8): 869–83. 11. tellier r, li y, cowling bj, tang jw. recognition of aerosol transmission of infectious agents: a commentary. bmc infect dis. 2019; 19: 101. 12. lozano moraga cp, rodríguez martínez ga, lefimil puente ca, morales bozo ic, urzúa orellana br. prevalence of candida albicans and carriage of candida non-albicans in the saliva of preschool children, according to their caries status. acta odontol scand. 2017; 75(1): 30–5. 13. arastehfar a, daneshnia f, kord m, roudbary m, zarrinfar h, fang w, hashemi sj, najafzadeh mj, khodavaisy s, pan w, liao w, badali h, rezaie s, zomorodian k, hagen f, boekhout t. comparison of 21-plex pcr and api 20c aux, maldi-tof ms, and rdna sequencing for a wide range of clinically isolated yeast species: improved identification by combining 21-plex pcr and api 20c aux as an alternative strategy for developing countries. front cell infect microbiol. 2019; 9: 21. 14. zhang l, wang h, xiao m, kudinha t, mao l-l, zhao h-r, kong f, xu y-c. the widely used atb fungus 3 automated readings in china and its misleading high mics of candida spp. to azoles: challenges for developing countries’ clinical microbiology labs. plos one. 2014; 9(12): e114004. 15. pat i l s, maju md a r b, sa ro de sc, sa ro de gs, awa n k h. oropharyngeal candidosis in hiv-infected patients-an update. front microbiol. 2018; 9: 980. 16. glick sn, morris m, foxman b, aral so, manhart le, holmes kk, golden mr. a comparison of sexual behavior patterns among men who have sex with men and heterosexual men and women. j acquir immune defic syndr. 2012; 60(1): 83–90. 17. berkow el, lockhart sr. fluconazole resistance in candida species: a current perspective. infect drug resist. 2017; 10: 237–45. 18. garcia-cuesta c, sarrion-pérez m-g, bagán j v. current treatment of oral candidiasis: a literature review. j clin exp dent. 2014; 6(5): e576-82. 19. williams d, lewis m. pathogenesis and treatment of oral candidosis. j oral microbiol. 2011; 3: 1–11. 20. gaitán-cepeda la, sánchez-vargas o, castillo n. prevalence of oral candidiasis in hiv/aids children in highly active antiretroviral therapy era. a literature analysis. int j std aids. 2015; 26(9): 625–32. 21. maheshwari m, kaur r, chadha s. candida species prevalence prof ile in h i v seropositive patients f rom a major ter tia r y ca re hospit a l i n new del h i, i nd ia . j pat hog. 2016; 2016: 6204804. 22. brito gnb, inocêncio ac, querido smr, jorge aoc, koga-ito cy. in vitro antifungal susceptibility of candida spp. oral isolates from hiv-positive patients and control individuals. braz oral res. 2011; 25(1): 28–33. 23. butts a, reitler p, nishimoto at, dejarnette c, estredge lr, peters tl, veve mp, rogers pd, palmer ge. a systematic screen reveals a diverse collection of medications that induce antifungal resistance in candida species. antimicrob agents chemother. 2019; 63(5): e00054-19. 24. patil s, rao rs, majumdar b, anil s. clinical appearance of oral candida infection and therapeutic strategies. front microbiol. 2015; 6: 1391. 25. whaley sg, berkow el, rybak jm, nishimoto at, barker ks, rogers pd. azole antifungal resistance in candida albicans and emerging non-albicans candida species. front microbiol. 2016; 7: 2173. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p82–86 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p82-86 153 volume 47, number 3, september 2014 efektifitas siwak (salvadora persica) dan pasta gigi siwak terhadap akumulasi plak gigi pada anak-anak (effectiveness of siwak (salvadora persica) and siwak toothpaste on dental plaque accumulation in children) indra bramanti, iwa sutardjo rs, navilatul ula, dan muhammmad isa departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas gadjah mada yogyakarta indonesia abstract background: siwak contain of salvadorine with an antiseptic effect. there were many reports about antibacterial effect of siwak on the cariogenic bacterial, pathogen periodontal, and dental plaque accumulation. purpose: the study was aimed to determine the effect of siwak and siwak toothpaste on accumulated dental plaque in children. methods: the subjects were 39 teenage children in range of age 12-15 years old, and were divided on 3 groups. each subject group was asked to brush their teeth 3 times a day using siwak; siwak toothpaste; and toothpaste with no additional substance as control, respectively. after brushing for a week, plaque scoring was performing using modified personal hygiene performance index (php-m). data were analysed using one way anova. results: the plaque score on siwak group lower significantly than control group, but there was no significant difference between siwak group and siwak toothpaste group. conclusion: the study suggested that siwak and siwak toothpaste had the same effect on decreasing plaque accumulation in children. key words: siwak, siwak toothpaste, dental plaque abstrak latar belakang: siwak mengandung salvadorine yang berefek sebagai antiseptik. siwak juga dilaporkan memiliki efek antibakteri terhadap bakteri kariogenik dan pathogen periodontal, dan menghambat pembentukan plak. tujuan: penelitian ini bertujuan untuk menguji efek siwak dan pasta gigi siwak terhadap akumulasi plak pada anak. metode: subyek penelitian adalah 39 anak remaja berusia 12-15 tahun yang dibagi dalam 3 kelompok. setiap subyek dalam kelompok yang sama diminta untuk menyikat gigi sehari 3 kali dengan menggunakan siwak; pasta gigi siwak; dan pasta gigi murni tanpa tambahan bahan sebagai control. setelah selama seminggu menyikat gigi, dilakukan pengukuran skor plak menggunakan indeks php-m. data yang diperoleh dianalisis menggunakan anova satu jalur. hasil: skor plak kelompok siwak lebih rendah secara signifikan dibanding kelompok pasta gigi murni, namun antara kelompok siwak dan pasta gigi siwak tidak terdapat perbedaan yang bermakna. simpulan: studi ini menunjukkan bahwa siwak dan pasta gigi siwak memiliki efek yang sama dalam menurunkan akumulasi plak pada anak. kata kunci: siwak, pasta gigi mengandung siwak, plak gigi korespondensi (correspondence): indra bramanti, departemen ilmu kedokteran gigi anak, universitas gadjah mada. jl. denta no. 1, sekip utara, yogyakarta, 55281, indonesia. e-mail: bramantikg@gmail.com research report 154 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 153–157 pendahuluan kesehatan gigi dan mulut mencerminkan kesehatan secara keseluruhan.1 di indonesia, kesehatan gigi dan mulut merupakan hal yang perlu mendapat perhatian serius dari tenaga kesehatan. hal ini terlihat dari tingginya angka penyakit gigi dan mulut penduduk indonesia yang mencapai 90% dengan prevalensi terbesar adalah penyakit jaringan penyangga gigi dan karies gigi. keduanya bersumber dari terabaikannya kebersihan gigi dan mulut sehingga berakibat pada terjadinya akumulasi plak. plak merupakan lapisan tipis yang melekat erat di permukaan gigi dan jaringan sekitar gigi yang mengandung kumpulan bakteri dan tidak dapat dibersihkanhanya dengan kumur.2 produk bakteri akan menurunkan ph plak sehingga akan terjadi demineralisasi email yang berlanjut menjadi karies gigi.3 usaha untuk meningkatkan kesehatan gigi dan mulut salah satunya dilakukan dengan cara menghilangkan plak secara teratur. hal tersebut dimaksudkan untuk mencegah agar plak tidak tertimbun, sehingga dapat menyebabkan kerusakan jaringan pada rongga mulut, baik gigi ataupun jaringan sekitar gigi. plak tidak dapat dibersihkan hanya dengan berkumur-kumur, tetapi juga harus dibersihkan dengan cara mekanis. sampai saat ini cara mekanis yang paling efektif untuk membersihkan plak adalah menyikat gigi.3 salah satu alternatif dalam menyikat gigi adalah menggunakan siwak. siwak merupakan tumbuhan berfamili salvadoraceae yang biasanya digunakan oleh orang islam untuk membersihkan gigi.4 beberapa peneliti melaporkan adanya efek antibakteri dari siwak terhadap bakteri kariogenik dan pathogen periodontal khususnya spesies bacterioides serta menghambat pembentukan plak.5,6 penelitian yang lain menyatakan bahwa ekstrak siwak memiliki daya antibakteri terhadap streptococcus mutans dan s.faecalis.7 aktifitas siwak dalam menurunkan pertumbuhan bakteri s.mutans disebabkan karena kandungan scnyang ketika bereaksi dengan kelompok sulfhidril dalam enzim bakteri akan menyebabkan kematian bakteri,8 sehingga produk asam tidak terbentuk dan ph plak tidak mengalami penurunan.9 saat ini sudah tersedia pasta gigi yang mengandung siwak sebagai bahan aktifnya.10 pasta yang mengandung siwak memiliki daya hambat yang besar terhadap pertumbuhan bakteri rongga mulut, khususnya s.mutans.11 hal yang dikhawatirkan sebagai efek samping dari pasta gigi adalah adanya sifat abrasif. kandungan yang terlalu abrasif dapat menjadi salah satu penyebab terjadinya trauma akibat gosokan pada gigi dan mengiritasi mukosa mulut. saat ini belum diketahui apakah pasta gigi yang menggunakan siwak sebagai bahan dasarnya lebih baik dari pada siwak alami dalam menjaga kesehatan gigi dan mulut, sehingga perlu penelitian untuk menguji efektivitas penggunaan siwak alami dengan pasta gigi berbahan dasar siwak. penelitian ini bertujuan untuk menguji efek siwak dan pasta gigi siwak terhadap akumulasi plak pada anak. bahan dan metode jenis penelitian ini adalah pre and postest control group design quasi experimental. penelitian dilakukan pada 39 siswa dan siswi pondok pesantren taruna al quran, lempongsari, yogyakarta. kriteria subyek adalah anak usia 12-15 tahun dengan periode gigi permanen, pria dan wanita tidak dibedakan, kooperatif, susunan gigi tidak berjejal, tidak karies pada daerah yang akan diskoring, serta tidak memakai protesa dan alat ortodonsi. penelitian mendapat persetujuan dari komisi etik (ethical clearance) fakultas kedokteran gigi universitas gadjah mada, dan tiap subyek diberi lembar persetujuan yang ditandatangani walinya sebagai tanda persetujuan (informed consent). pada semua subyek dilakukan skaling dengan tujuan menyamakan kondisi rongga mulut subyek sebelum perlakuan. selanjutnya, subyek diberi edukasi cara dan frekuensi menyikat gigi yaitu dengan metode secara vertikal (dilakukan 3 kali sehari yakni pada pagi hari saat bangun tidur, setelah makan pagi dan malam hari sebelum tidur) selama 7 hari dan dilakukan pemeriksaan plak yang pertama (sebelum perlakuan). plak diskor dengan metode phpmenurut martens dan meskin dengan gigi yang diperiksa berjumlah 6, yaitu gigi paling posterior kanan atas, kaninus kanan atas (jika tidak ada dipakai gigi anterior lainnya), premolar kiri atas, gigi paling posterior kiri bawah, kaninus kiri bawah (jika tidak ada dipakai gigi anterior lainnya) dan premolar kanan bawah. pemeriksaan plak dilakukan pada permukaan mahkota bagian fasial, bukal, lingual dan palatal dengan membagi tiap permukaan mahkota gigi menjadi lima area subdivisi, menggunakan bahan pewarna gigi (disclosing solution), dengan nilai 0 untuk bagian yang tidak terwarnai (tidak ada plak) dan nilai 1 untuk bagian yang terwarnai (ada plak). subyek dibagi menjadi 3 kelompok perlakuan yakni kelompok menyikat gigi menggunakan siwak, kelompok pasta gigi siwak, dan kelompok pasta gigi dasar (tanpa bahan aktif, sebagai kontrol), kemudian diinstruksikan untuk menyikat gigi sesuai kelompok perlakuan dengan menggunakan metode menyikat gigi yang sama selama 7 hari. pemeriksaan kembali dilakukan untuk melihat pengaruh menyikat gigi sebelum dan sesudah perlakuan serta perbedaan pengaruh menyikat gigi menggunakan siwak dibandingkan pasta gigi siwak terhadap pembentukan plak gigi. data yang diperoleh dari hasil pengukuran indeks php merupakan selisih hasil pengukuran sebelum dan sesudah menyikat gigi. data tersebut selanjutnya dihitung reratanya berdasarkan kelompok perlakuan dan dianalis menggunakan one way anova dengan tingkat kepercayaan 95%. hasil hasil penelitian menunjukkan adanya penurunan skor plak sebelum dan sesudah menyikat gigi pada tiap 155bramanti, et al.: efektifitas siwak (salvadora persica) dan pasta gigi siwak kelompok perlakuan (tabel 1). rerata skor plak sebelum dan sesudah menyikat gigi pada masing-masing kelompok perlakuan menunjukkan adanya penurunan (gambar 1). kelompok menyikat gigi menggunakan siwak memiliki rata-rata penurunan indeks plak paling tinggi, sedangkan penurunan skor plak terendah terdapat pada kelompok control (gambar 2). hasil uji normalitas dan homogenitas variannya menunjukkan bahwa data yang diuji mempunyai nilai sig p > 0,05, yang berarti data terdistribusi secara normal sehingga memenuhi syarat untuk dilakukan uji parametrik. uji hipotesis dilakukan dengan menganalisis data menggunakan uji one way anova yang menunjukkan adanya perbedaan yang bermakna indeks plak pada ketiga kelompok perlakuan (p = 0,01 yang berarti p < 0,05). selanjutnya untuk mengetahui perbedaan antara ketiga kelompok perlakuan tersebut dilakukan uji lsd (tabel 2). terdapat perbedaan bermakna antara kelompok kontrol dengan kelompok siwak (p < 0,05), sedang antara kelompok pasta gigi siwak dengan kelompok siwak tampak tidak ada perbedaan bermakna (p > 0,05) (tabel 2). hal tersebut berarti menyikat gigi menggunakan siwak dapat menurunkan skor plak lebih besar secara bermakna dibanding dengan menggunakan pasta dasar. adapun penurunan skor plak setelah menyikat gigi menggunakan tabel 1. rerata dan simpangan baku skor plak awal (sebelum perlakuan), skor plak akhir (setelah perlakuan), dan selisih skor plak perlakuan jumlah subyek (n) skor plak skor plak awal skor plak akhir selisih skor plak awal dan akhir selisih indeks plak awal dan akhir x ± sd x ± sd x ± sd x ± sd kontrol 13 27,15 ± 10.14 22,92 ± 10,37 4,23 ± 3,37 0,71 ± 0,56 pasta gigi siwak 13 28,08 ± 10.44 22,00 ± 11,35 6,08 ± 3,80 1,01 ± 0,63 siwak 13 26,15± 7.84 17,00 ± 7,52 9,15 ± 4,54 1,53 ± 0,76 gambar 1. rerata skor plak awal (sebelum menyikat gigi) dan skor plak akhir (sesudah menyikat gigi) pada ketiga kelompok perlakuan. gambar 2. rerata selisih indeks plak pada masing-masing kelompok perlakuan. tabel 2. hasil uji lsd indeks plak setelah meyikat gigi dengan siwak, pasta gigi siwak dan pasta gigi dasar perlakuan sig. (p) skor plak sig. (p) indeks plak kontrol pasta gigi siwak 0,239 0,239 siwak 0,003* 0,003* pasta gigi siwak kontrol 0,239 0,239 siwak 0,054 0,054 siwak kontrol 0,003* 0,003* pasta gigi siwak 0,054 0,054 keterangan: *= berbeda signifikan (p<0,05) tabel 3. hasil uji tukey test (anava) indeks plak untuk mengetahui kelompok yang sama dan yang berbeda perlakuan jumlah subyek (n) α = 0,05 1 2 kontrol 13 0,7050 pasta gigi siwak 13 1,0128 1,0128 siwak 13 1,5257 signifikansi 0,463 0,128 156 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 153–157 siwak dibandingkan dengan pasta gigi siwak tidak berbeda secara bermakna. kesimpulan dari hasil uji lsd dapat dilihat pada hasil uji menggunakan tukey test berikut ini (tabel 3). dua kelompok besar yakni kelompok i yang menunjukkan tidak adanya perbedaan bermakna antara kelompok kontrol dengan kelompok pasta gigi siwak dan kelompok ii yang menunjukkan tidak adanya perbedaan bermakna antara kelompok pasta gigi siwak dan siwak. adapun antara kelompok i dan ii terdapat perbedaan yang bermakna. pembahasan pada penelitian ini subyek penelitian merupakan anak usia 12-15 tahun siswa dan siswi pondok pesantren taruna al quran. penelitian dilakukan di satu pondok pesantren dengan maksud subyek memiliki pola makan yang sama karena pola makan dapat mempengaruhi pembentukan plak gigi, sehingga dalam hal ini diperlukan adanya pengendalian pola makan diantaranya menyangkut jenis atau bahan makanan.3,12 hal ini dibuktikan dengan penelitian oleh chemiawan yang menyimpulkan bahwa tingkat kebersihan gigi dan mulut anak vegetarian lebih baik dibandingkan dengan non vegetarian.13 hasil penelitian menunjukkan adanya perbedaan yang bermakna rerata selisih skor plak sebelum dan sesudah menyikat gigi menggunakan siwak dengan pasta gigi dasar. adapun antara kelompok siwak dengan pasta gigi siwak tidak menunjukkan perbedaan yang bermakna walaupun hasil dari rerata selisih skor plak sebelum dan sesudah menyikat gigi menggunakan siwak lebih besar dibandingkan dengan kelompok pasta gigi siwak. hal tersebut berarti bahwa menyikat gigi menggunakan siwak mampu menghambat pembentukan plak gigi, namun tidak ada perbedaan dengan menyikat gigi menggunakan pasta gigi siwak. kelebihan siwak dalam membersihkan gigi dan mulut disebabkan oleh efek mekanik dari seratserat batang serta juga disebabkan dari kemampuan siwak dalam melepaskan senyawa aktif yang bermanfaat.14 siwak (s. persica) mengandung lebih dari 20 zat (salvadourea dan salvadorine, saponin, tanin, vitamin c, silika, resin, cyanogenic glycoside dan benzylsothio-cyanate) yang dibutuhkan untuk meningkatkan kebersihan mulut, diantaranya salvadorineyang berefek antiseptik, asam tanat yang bersifat astringensia dan minyak atsiri meningkatkan air liur.15 daya antibakterial dan efek pembersih pada siwak berhubungan dengan tingginya kandungan sodium klorida dan potassium klorida.15 tidak adanya perbedaan menyikat gigi menggunakan siwak dibandingkan dengan pasta gigi siwak dalam menghambat pembentukan plak gigi disebabkan keduanya sama-sama berkhasiat dalam menghambat pembentukan plak. efek terapeutik dan profilaktik dari siwak disebabkan oleh adanya pembersihan mekanis, pelepasan zat kimia aktif yang terdapat didalamnya dan atau kombinasi keduanya. adanya substansi silica pada siwak juga membantu aksi mekanis siwak terhadap pembersihan plak.6 adapun pasta gigi siwak mengandung chloride yang berguna dalam mengangkat stain, silica yang merupakan bahan pembersih gigi serta trymetylamine yang berfungsi dalam mengurangi kalkulus dan stain,6 sehingga keduanya pasta gigi siwak dan siwak sama-sama memiliki kemampuan dalam menghambat pembentukan plak gigi. penelitian yang dilakukan oleh desire, dkk 16 menyimpulkan bahwa menyikat gigi menggunakan pasta gigi siwak dapat menurunkan pertumbuhan koloni s.mutans plak gigi anak setelah pemakaian selama 7 hari. hasil penelitian ini juga menunjukkan bahwa antara kelompok pasta gigi dasar dengan pasta gigi siwak tidak terdapat perbedaan yang bermakna. adanya perbedaan yang tidak bermakna rerata selisih skor plak antara kelompok kontrol dengan pasta gigi siwak, diantaranya dimungkinkan karena penelitian ini dilakukan pada anak usia remaja (12-15 tahun), dimana motorik anak sudah berkembang dengan baik sehingga dapat menyikat gigi berdasarkan metode yang disarankan dengan optimal. selain itu, baik pasta gigi dasar maupun pasta gigi siwak sama-sama mengandung detergen yang bersifat abrasif berfungsi dalam membersihkan plak. kelompok kontrol melakukan sikat gigi meggunakan pasta gigi dasar yang mengandung sodium lauryl sulfat yang merupakan detergen berfungsi sebagai bahan pembersih plak. hasil tersebut berbeda jika dibandingkan dengan siwak sebelum dikemas menjadi pasta gigi yang menunjukkan adanya perbedaan yang bermakna dalam menurunkan pembentukan skor plak gigi. walaupun tidak ada perbedaan yang bermakna antara kelompok pasta gigi siwak dengan siwak, namun dari sebagian besar subyek penelitian yang menggunakan siwak mengungkapkan bahwa menggunakan siwak dirasakan lebih praktis, karena tidak perlu memakai pasta gigi, tidak perlu berkumur, sehingga memudahkan dalam penggunaannya. hal tersebut sama dengan penelitian sebelumnya yang menunjukkan bahwa menyikat gigi menggunakan siwak lebih efektif jika dibandingkan dengan sikat gigi dalam menurunkan plak gigi dan gingivitis, serta meningkatkan kebersihan mulut pada bagian interproksimal dan fissure gigi.17 menurut world health organization report series, siwak dapat menghilangkan plak tanpa menyebabkan luka pada gigi.5 peneliti menyarankan untuk dilakukan penelitian lebih lanjut tentang pemakaian pasta gigi yang mengandung siwak dibandingkan pasta gigi siwak terhadap kesehatan rongga mulut dalam waktu yang lebih lama dan dengan metode yang lebih teliti. dapat disimpulkan bahwa siwak dan pasta gigi siwak memiliki efek yang sama dalam menurunkan akumulasi plak pada anak. ucapan terima kasih penelitian ini didanai oleh dana masyarakat fakultas kedokteran gigi universitas gadjah mada 2013. terima kasih dan penghargaan disampaikan kepada ponpes taruna al quran, yogyakarta dan prof. dr. drg. iwa sutardjo rs, s.u., sp.kga(k). 157bramanti, et al.: efektifitas siwak (salvadora persica) dan pasta gigi siwak daftar pustaka 1. glick g, ship. burket’s oral medicine.7th ed. india: bc dekker inc; 2008. 2. anitasari s, rahayu ne. hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran kotamadya samarinda provinsi kalimantan timur. dental journal 2005; 38(2): 88. 3. caranza fa. carranza’s clinical periodontal. 10th ed. st. louis: wb saunders co; 2006. p. 137-55, 728-37. 4. salehi p, momeni, danaie sh. comparison of the antibacterial effects of persica mouthwash with chlorhexidine on streptococcus mutans in orthodontic patients. daru 2006; 14: 178-82 5. zaenab m, anny hw, logawa b. uji antibakteri siwak (salvadora persic linn.) terhadap streptococccus mutans (atc3 1987) dan bacteroides melaninogenicus. makara kesehatan 2004; 8(2): 3740. 6. almas k, al-zeid z. the immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study. j contemp dent parct 2004; 5(1): 105-114 7. almas k, skaug n, ahmad i. an invitro antimicrobial comparison of miswak extract with commercially available non alcohol mouthrinses. int j dent hygiene 2005; 3(1): 18-24. 8. dorouth ia, christy aa, skaug n, egeberg pk. identification and quantification of some potentially antimicrobial anionic components in miswak extract. indian j pharmacol 2000; 32: 11-4. 9. el rahman hf, skaug n, francis gw. in vitro microbial effects of crude miswak extract on oral pathogens. saudi dent j 2002; 14: 26-32. 10. soesilowati p, devijanti r. perbedaan daya hambat terhadap streptococcus mutans dari beberapa pasta gigi yang mengandung herbal. maj ked gigi (dent j) 2005; 38(2): 64-7. 11. sutadi h, mangundjaya s. effect of salvadora persica indentifrice on streptococcus mutans of school children. 1st international conference of pediatric dentistry. kuala lumpur malaysia, 2006. 12. mcdonald re, avery dr. dentistry for the child and adolescent. 6th ed. st louis: mosby; 1994. p. 256-82. 13. chemiawan e, riyanti e, fransisca f. perbedaan tingkat kebersihan gigi dan mulut antara anak vegetarian dan non vegetarian di vihara maitreya pusat jakarta. jurnal pdgi 2007; edisi khusus pin ikga ii: 79-84. 14. dorouth ia, skaug n. comparative oral health status of an adult sudanese population using miswak and toothbrush regularly. saudi dent j 2004; 16(1): 29-38. 15. dorouth ia. miswak as an alternative to the modern toothbrush in preventing oral disease. thesis. bergen: norway; 2013. 16. desiree s, sutadi h, hayati r. peran pasta gigi yang mengandung siwak terhadap koloni streptococcus mutans dalam plak gigi anak. jurnal pdgi 2007; edisi khusus pin ikga ii: 90-4. 17. al-otaibi m, al harthy m, soder b, gustafsson a, angmarmansson b. comparative effect of chewing sticks and toothbrushing on plaque removal and gingival health. oral health prev dent 2003; 1: 301-7. 215 vol. 44. no. 4 december 2011 literature review piperin and piplartin as natural oral anticancer drug berlian bidarisugma1, mar'atus sholikhah2, sarah usman balbeid3, and anis irmawati4 1,2,3 dental student 4 department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: since the last few decades, oral cancer as pathology has become an attention in medicine and dentistry. the majority cases of oral cancer are affecting people with smoking habit and alcohol consumption. many herbs contain substances which can stop cancer cells proliferation, such as piper retrofractum/retrofracti fructus, an herb plant from piperaceae family which contains piperin and piplartin. purpose: the purpose of this study was to examine the mechanism of piperin and pilplartin as natural oral anticancer drug. reviews: piperin and piplartin has function as antioxidant that can protect body cell from damage caused by free radicals. piperin works synergistically with another bioactive substance like capsaicin and curcumin. piperin increase the number of serum and life time of serum from a few nutrition substance like co-enzyme q10 and beta-carotene. beta-carotene can catch reactive o2 and peroxil radicals. the activity of anticancer piplartin related with obstruction of proliferation cell rate, observe form ki67 reduction as antigen in nucleus that associated with g1, s, g2, and m phase in cell cycle. comparing with piplartin, piperin is more potential to inhibit proliferation rate of ki67, but piplartin’s antiproliferation mechanism will increase if supported by piperin. conclusion: piperin and piplartin contained in javanese chili are potential for natural oral anticancer, by directly or indirectly suppress tumor cell development by increasing the number of immunity cells (immunomodulator), and by inhibiting cell proliferation with reduction of ki67, nucleus antigen that associated with g1,s,g2, dan m phase of cell cycle. key words: oral cancer, piperin, piplartin abstrak latar belakang: sejak beberapa dekade terakhir, patologi kanker rongga mulut telah banyak menjadi perhatian di bidang kedokteran dan kedokteran gigi. risiko paling tinggi ditemukan pada penderita perokok dan peminum alkohol. banyak tanaman herbal yang memiliki kandungan untuk menghambat pertumbuhan sel kanker atau antiproliferasi sel, seperti tanaman herbal yang berasal dari suku piperaceae, salah satunya adalah cabe jawa (piper retrofractum) yang mengandung piperin dan piplartin. tujuan: artikel ini bertujuan untuk mengetahui mekanisme kerja piperin dan pilplartin sebagai antikanker alami rongga mulut. tinjauan pustaka: piperin dan piplartin berfungsi sebagai antioksidan yang dapat melindungi sel tubuh dari kerusakan akibat radikal bebas. piperin bekerja secara sinergis dengan zat-zat bioaktif lainnya seperti capsaicin dan curcumin. piperin meningkatkan jumlah serum dan umur serum dari beberapa substansi nutrisi seperti koenzim q10 dan betakaroten. betakaroten mampu menangkap oksigen reaktif dan radikal peroksil. aktivitas antitumor piplartin berhubungan dengan penghambatan laju proliferasi sel, ditinjau dari reduksi ki67 yaitu antigen pada inti sel yang berasosiasi dengan g1, s, g2, dan m pada siklus sintesa sel. �alam mekanisme kerjanya piplartin akan lebih meningkat aktivitas antiproliferasinya jika disinergiskan dengan piperin. kesimpulan: piperin dan piplartin yang terkandung dalam cabe jawa berpotensi sebagai antikanker rongga mulut alami, dengan menekan perkembangan sel tumor baik secara langsung 216 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 20111: 215–219 introduction since the last few decades, oral cancer as pathology has become an attention in medicine and dentistry among health professionals and the public because there is increased death rate and number of cancer patients. according to world health organization (who), each year the number of cancer patients in the world increased to 6.25 million people. in the next 10 years an estimated of 9 million people will die each year by cancer. two-thirds of cancer patients in the world will be in developing countries.1 highest risk was found in tobacco smokers and alcoholic dringkers. expenses incurred for treatment of cancers in developed countries is still quite high. it is also felt by the developing countries. the spread of cancer cases is due to the low level of public awareness on the danger of cancer, the high cost of treatment and care, leads to only certain people who can cover medical cost, even though cancer treatment can only slow down the spread of cancer cells.2 many curative measures has been taken to cope the oral cancer include radiation therapy, chemotherapy, surgery, and combination therapy. radiation therapy is a treatment that uses an ion light that can destroy the dna cell in cancer cells, so cancer cells can not grow. radiation therapy has side effects depend on the area treated, dose, and distance to the cancerous tissue lesions that rapidly divide. long-term effects of this radiation therapy include infertility, fibrosis, permanent hair loss, osteoradionecrosis, and others. chemotherapy is one form of palliative therapy, used when the cancer recurs or metastasis occurred. chemotherapy uses chemicals that destroy cancer cells. the side effects of this therapy are fatigue, nausea and vomiting, digestive disorder, hair loss, weakness in the muscles and nerves, disorders of blood formation, and others. surgery is often performed when the lesion involves throat, but can also be done in the oral cavity. surgery is performed to remove the entire lesion to prevent the spread of cancer cells in the lymph nodes, blood vessels, and nerves. combination therapy is a combination of several therapies. 3 on the whole oral cancer therapies that exist today still have an adverse effect on patients. to cope with an increasing number of patients with oral cancer cases, especially in developing countries, need effective and efficient efforts. one of the efforts is the prevention of oral cancer. in addition, prevention efforts should be affordable by all levels of society to reduce the number of cancer patients. empirically herbs are believed to have optimal efficacy in curing various diseases. many herbal plants that contain substances to inhibit cancer cell growth or antiproliferrative cell, such as herbal plants from the family of piperaceae. javanese chili (piper retrofractum) with the content of piperine and piplartin can act as anti-proliferative cells, where both substances are also found in black pepper. piperine and piplartin also serves as an antioxidant that may protect body cells from free radical damage. these herbs can be used as an alternative to oral cancer prevention (chemo-prevention).4–6 piperine pharmacological effects are antioxidant, antipyretic, analgesic, anti-inflammatory, and central nervous system suppressor. pharmacological effects as anti-proliferative piplartin is a substance that can inhibit cell growth and reduce cells number.7 javanese chili is a plant easily found in indonesia compared to black pepper, cayenne and the market price is relatively cheaper to black pepper. javanese chili contains about 2.03 to 3.65% piperine, while the level of black pepper piperine is around the 3–5%. 8,9 although the levels of piperine in black pepper is higher, it is still possible to continue using javanese chili as a natural oral anticancer. this review describe the mechanism of piperin and piplartin as active ingredients in javanese chili (fructus retrofracti) as an alternative natural oral anticancer drug (oral cancer chemo-prevention). oral cancer cancer is a disease caused by abnormal and uncontrolled cells mutations. if cell growth is not stopped then the growth will continue slowly. if the cancer has invaded a cell or group of cells, the progress will be faster, doubled on an ongoing basis. growth of benign (non-cancerous, benign) and malignant growth (cancer, malignant) can be derived from various tissues in and around the mouth, including the bones, muscles and nerves.10 oral cavity cancer has a multifactorial cause and a process that consists of several stages, namely initiation, promotion and development of progressive oral cancer etiology.8 etiology of cancer can be grouped on local factors, environmental factors and host factors. local factors include poor oral hygiene, chronic irritation of the restorations, dental caries, denture. environmental factors, including chemical carcinogens and their use of cigarettes, tobacco, ionizing radiation, viruses, sunlight. host factors, including age, gender, nutrition, immune response and maupun tidak langsung melalui peningkatan sel imun (immunomodulator), dengan penghambatan laju proliferasi sel, ditinjau dari reduksi ki67, yaitu antigen pada inti sel yang berasosiasi dengan g1, s, g2, dan m pada siklus sintesis sel. kata kunci: kanker rongga mulut, piperin, piplartin correspondence: anis irmawati, c/o: departemen biologi oral, fakultas kedokteran gigi, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. email: irmaamky@yahoo.com 217bidarisugma, et al.,: piperin and piplartin as natural oral anticancer drug genetic. etiologic factors are combination of three factors. in the last decades, the molecular pathogenesis of neoplasm suggests that the neoplasm is a genetic disease. tumor formation as a result of genetic drift is caused by etiologic factors resulting in excessive and uncontrolled cell division. genes that were target genetic changes are oncogenes (genes that promote growth), antioncogenes (genes that inhibit growth) and genes that regulate apoptosis.11 signs to consider on the possibility of early oral cancer in advanced stages are the white patches, scaly, persistent, pigment spots which suddenly increase in size, this non-healing ulcer, swelling and bleeding gums, which eventually forming progressive facial asymmetry.12 early stages of oral cancer does not cause pain and are usually found on routine dental examination. cancer on the floor of mouth is usually a squamous cell carcinoma, which looks like an open wound and tend to grow into the underlying structures. 13 javanese chili (piper retrofractum) javanese chili plant height is between 10–12 meters long, with round trunk and woody, branching with soft consistency. the fruit is oval with light green color when young and red when matured. small fruit size arranged into one shaped like a chili and a length of 2–7 cm. spicy fruit flavors and smells fresh. javanese chili fruit contains piperine, palmitic acids, tetrahydropiperic acids, 1-undercyclnyl-3, 4-methylenedioxy benzene, piperidine, essential oils, n-isobutildecatrans-2-trans-4dienamide and sesamin. meanwhile, the root contains piperine, piplartin and piperlongumin. substances in its fruit essential oil contains in an amount of about 1% of dry weight. essential oil contains 6% piperine. researches in several countries stated that the average amount of javanese chili essential oil almost the same as black pepper, about 0.9% consisting of 0.19% piperine alkaloids. javanese chili fruit is used to cleanse the mouth, reducing mouth odor, reduce tooth pain, and treat gingivitis. safety of javanese chili fruit as a raw material have been studied in medicine and obtained the conclusion that use empirical form of javanese pepper fruit infusion which is safe and classified as relatively harmless materials. javanese chili fruit has androgenic, anabolic and anti-proliferative effect.14 piperine and piplartin piperine is an alkaloid contained in the piperaceae plant family. piperine is potential as antioxidant, sedative, anti-inflammatory, antiproliferative and analgesic. piperine also serves as an antioxidant that may protect body cells from free radicals.15,16 piperine is the solid substance and not soluble in water. the molecular structure of piperine is c17h19no3, molecule weight 285.34 daltons. piperine is a trans-trans stereoisomer of 1-piperoylpiperidine or known as (e, e)-1-[5-(1,3-benzodioxol-5-y1)-1-oxo-2,4pentdienyl] piperidine.17 piperine may increase the activity of some nutritional substances and drug.18 this drug have anti-inflammatory activity, analgesic, and support the metabolic process of digestion.19,20 piperine was found to be non-specific inhibitors on the metabolism of drugs and xenobiotics. piperine inhibits the cytochrome p450 as well as hepatic udp-glucuronyltransferase and arylhydrocarbon hydroxylase and other enzymes contained in the drug and xenobiotic.21,22 many studies have shown that piperine can inhibit lipid peroxidation. piperine has been shown to stimulate the secretion of digestive enzymes in pancreas such as amylase, trypsin, chymotrypsin and lipase in rats. piperine work synergistically with other bioactive substances such as capsaicin and curcumin.23,24 piperine may increase the number and age of coenzyme q10 and beta carotene. beta carotene is able to capture reactive oxygen and peroxyl radicals that play a role in the process of cancer. analgesic and antiinflammatory effects of beta-carotenerelated activities as antioxidant.25-27 there is evidence that piperine-containing antioxidant, anticonvulsant, anticarcinogenic and anti-inflammatory.28 piplartin is an alkaloid contained in piperaceae plant family. piplartin is potential as anti-proliverative agents. the chemical structure: {5,6dihydro-1-[l-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]2(1h) pyridinone}. piplartin have antidepressant, cytotoxic, and anti-proliferrative effects.30 discussion oral cancers in developing countries require effective prevention efforts. curative attempts to cope with cancer of the oral cavity still cause adverse side effects and high cost. use of medicinal herbs is increasingly popular among modern medicine and dentistry, including the use of herbs as natural anti-cancer drugs. one herb that potential as a natural anticancer is javanese chili in piperaceae plant family. javanese chili containing active substance of piperine and piplartin possess anti-proliferative cells. piperine and piplartin also serves as an antioxidant that may protect body cells from free radical damage. piperine and piplartin can suppress tumor cell growth both directly and indirectly through increased activity of the immune system in advance (immunomodulators).28 circumstances that indicate the role of immune system in cancer is that a tumor can be cured. in patients with immune deficiency and received immunosuppression therapy, malignancy can be doubled to 200 times. malignant transformation causes a change in the phenotype of normal cells, the loss component of the surface antigen, a neo-antigen, and other changes in the cell membrane. this will affect the immune response of antigen that stimulates the body to produce antibodies. distribution of tumor antigens found is divided into 3 classes. class 1 antigen is found only in certain tumors, are not found in normal cells as well as other malignancies. class 2 antigens are found in tumors, and class 3 antigen is found in normal cells and malignant cells.31 piplartin work synergistic with piperine. khajuria et al., noted that piperine and piplartin showed positive activity in inhibiting some tumor cells. in research conducted in218 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 20111: 215–219 figure 1. the role and mechanism of piperine and piplartin as anticancer drug. vivo testing of antitumor activity of piperine and piplartin. in-vivo test involved 60 female mice in which each of 10 groups of mice transplanted with sarcoma 180.28 analysis of histopathology and morphology of tumor cells with multiple organs such as liver, kidney, and lung, demonstrated piperine and piplartin as a treatment against the tumor cells. piperine and piplartin with a dose of 50 or 100 mg/day continuously in 7 days can inhibit tumor cell growth in mice that had been transplanted with sarcoma 180. the average resistance was 28.7–52.3% for piperine and 55.1-56.8% for piplartin. piplartin antitumor activity associated with inhibition of cell proliferation rate, in terms of reduction of ki67, an antigen on the cell nucleus that is associated with the g1, s, g2, and m on the synthesis of the cell cycle. piplartin as an anticancer role in the initiation phase of the cell by inhibiting the rate of cell proliferation. compared with piplartin, piperine is still not enough potential in inhibiting the proliferation rate of ki67 antigen. however, the mechanism of action, will increase anti-proliferative activity of piplartin if synergized with piperin.28 piperine and piplartin also serves as an antioxidant that may protect body cells from free radicals damage. this herb can be used as an alternative to oral cancer prevention (oral cavity cancer chemo-prevention).4-6 the pharmacological effects of piperine as an antioxidant, antipyretic, analgesic, anti-inflammatory, and suppress the central nervous system. the pharmacological effects of piplartin as antiproliferative agent, a substance that can inhibit cell growth and reduce the number of cells.7 anti-cancer mechanism can be explained as follows (figure 1): the first mechanism is an inhibition of carcinogens. carcinogen inhibitor works as a barrier to the formation of procarcinogenic to be carcinogenic. the second mechanism is inhibition on the formation of cells initiation from normal cell. cells initiation can lead to tumor cell if the cell promoter happens continuously. this mechanism plays a role in the formation of dna repair or repair of the mutated dna structure. the last mechanism is by inhibiting the formation of primary tumor cells into secondary tumor cells that more malignant or cancerous. inhibiting agents that play a role in this mechanism is an immunomodulator. immunomodulator will increase or decrease the body's immune cells in accordance with necessary needs.32 piplartin as an anticancer role in the initiation phase of the cell by inhibiting the rate of cell proliferation. piperine and piplartin also play a role in the stage of inhibition of primary tumor cells into secondary tumor cells by enhancing immune cell first (immunomodulator).28 the content of active piperine and piplartin ingredient have shown that javanese chili can be used as a natural alternative to oral anticancer drug. however, its use has not been optimal. a breakthrough is needed to process javanese chili fruit as a natural oral anticancer drug that is easy to consume and apply in everyday use. alternative processing of javanese chili fruit as a natural oral anticancer may include food products such as candy, and product of dental care such as toothpaste and moutwash. javanese chili fruit extracts can be added as instant 219bidarisugma, et al.,: piperin and piplartin as natural oral anticancer drug beverage to be consumed every day with the right dose. it can be concluded that piperine and piplartin contained in javanese chili are potential as natural oral anticancer drug, by suppressing tumor cell growth either directly or indirectly through increasing immune cells (immunomodulator). references 1. siswono. setiap tahun 190 ribu penderita kanker baru. 2005. available at: http://www.gizi.net/. accessed january 4, 2007. 2. bernadus c. kanker rongga mulut kenali gejalanya. 2008. available at www.summarizehuman knowledge.com. accessed october 11, 2008. 3. bayani rnb. kanker rongga mulut disebabkan oleh kebiasaan menyirih (laporan kasus). skripsi. medan: fakultas kedokteran gigi universitas sumatra utara; 2009. 4. khajuria a, zutshi u, bedi kl. permeability characteristics of piperine on oral absorption-an active alkaloid from peppers and a bioavailability enhancer. ind j exp biol 1998; 36(1): 46–50. 5. null g. beta carotene. new england journal medicine 2001; available at: www.garynull.com/document/beta carotene/htm. accessed november 26, 2005. 6. vogel hg. drug discovery & evaluation: pharmalogical assays. 2nd ed. new york: springer; 2002. p. 304–5. 7. winarto wp. cabe jawa. si pedas berkhasiat obat. jakarta: penerbit agromedia pustaka; 2003. p. 35-42 8. melati m, aziz sa, ghulamahdi m. studi cabe jawa biasa (piper retrofractum vahl.) dan cabe jawa perdu dari tiga sentra produksi dengan keragaman intensitas cahaya dan pemupukan. available at http://repository.ipb.ac.id/bitstream/handle/123456789/41764/ maya%20melati-studi_cabe%20jawa_hb.pdf?sequence=3. accessed october 11, 2008. 9. wood ab, maureen l, barrow, james dj. piperine determination in pepper (pipper nigrum l.) and its oleoresins a reserved-phase high performance liquid chromatogrphic method. flovour and fragrance j 1988; 3: 55–64. 10. hasibuan s. prosedur deteksi dini dan diagnosis kanker rongga mulut. usu digital library 2004; available at: repository.usu. ac.id/bitstream/123456789/1159/1/fkg-sayuti2.pdf. accessed at october 11, 2008. 11. tambunan gw. diagnosis dan tatalaksana sepuluh jenis kanker terbanyak di indonesia. handoyo m, editor. edisi ke-2. jakarta: penerbit buku kedokteran egg; 1993. p. 185–98. 12. scully c. oncogen, onco-supressor, carcinogenesis and oral cancer. br dent j 1992; 173(2): 53. 13. bolden te. the prevention and detection of oral cancer. in: stallard re. a textbook of preventif dentistry. 2nd ed. philadelphia: wb saunders company; 1982. p. 277–306. 14. winarto wp. cabe jawa. si pedas berkhasiat obat. jakarta: penerbit agromedia pustaka. 2003. 15. raguso ra, pichersky e. a day in the life of a linalool molecule: chemical communication in a plant-pollinator system. part 1: linalool biosynthesis in flowering plants. plant species biol 1999; 14(7): 95–120. 16. shoba g, joy d, joseph t, majeed m, rajendran r, srinivas ps. influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. planta med 1998; 64(4): 353–6. 17. d'hooge r, pei yq, raes a, lebrun p, bogaert pp, deyn pp. anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonists. arzneimittelforschung 1996; 46(6): 557–60. 18. vogel hg. drug discovery & evaluation: pharmalogical assays. 2nd ed. new york: springer; 2002. 19. badmaev v, majeed m, norkus ep. piperine an alkaloid derived from black pepper increases serum response of beta-carotene during 14-days of oral beta-carotene supplementation. nutr res 1999; 19: 381–8. 20. badmaev v. majeed m, prakash l. piperine derived from black pepper increases the plasma levels of coenzyme q10 following oral supplementation. j nutr biochem 2000; 11(2): 109–13. 21. atal ck, dubey rk, singh j. biochemical basis of enhanced drug bioavailability by piperine: evidence that piperine is a potent inhibitor of drug metabolism. j pharmacol exp ther 1985; 232(1): 258–62. 22. bezerra dp, castro fo, alves apnn, pessoa c, silveira er, lima mas, elmiro fjm. effect of the association of alkaloids from peppers and 5-un in the treatment of sarcoma. j braz and chemical society 2005; 180 (s180). 23. mutschler e. arzneimittelwirkungen. widianto mb, ranti as, editors. dinamika obat. edisi v. bandung: penerbit itb; 1986. p. 177-8. 24. williamson em, okpako dt, evans fj. selection, preparation, and pharmacologycaly evaluation of plant material. volume i. new york: john willey and sons; 1996. p. 131–7. 25. malini t, manimaran rr, arunakaran j, aruldhas mm, govindarajulu p. effects of piperine on testis of albino rats. j ethnopharmacol 1999; 64: 219–25. 26. paiva sar, russel rm. b-carotene and other carotenoids as antioxidants. journal of the american college of nutrition 2000; 18(5): 426–33. 27. unchern s, nagata k, saito h, fukuda j. piperine, a pungent alkaloid, is cytotoxic to cultured neutrons from the embryonic rat brain. biol pharm bull 1994; 17: 403–6. 28. khajuria a, thusu n, zutshi u, bedi kl. piperine modulation of carcinogen induced oxidative stress in intestinal mucosa. mol cell biochem 1998; 189: 113-8. 29. lieber cs, leo ma. alcohol, vitamin a, and b-carotene: adverse interactions including hepatotoxicity and carcinogenicity. am j clin nut 1999; 69(6): 1071–85. 30. bano g, raina rk, zutshi u, bedi kl, johri rk, sharma sc. effect of piperine on bioavailability and pharmacokinetics of propanolol and theophylline in healthy volunteers. eur j clin pharmacol 1991; 41: 615–7. 31. katzung bg. 1987. basic and clinical pharmacology. 3rd ed. bagian farmakologi, fakultas kedokteran, iniversitas airlangga, farmakologi dan klinik. jakarta: salemba medika; 2001. p. 126–9. 32. gunawan g, sulistia. farmakologi dan terapi. edisi 5. jakarta: gaya baru; 2007. 130 volume 46, number 3, september 2013 research report analisis heteroplasmy dna mitokondria pulpa gigi pada identifikasi personal forensik (heteroplasmy analysis of dental pulp mitochondrial dna in forensic personal identification) ardyni febri k,1 retno pudji rahayu1 dan agung sosiawan2 1 departemen patologi mulut dan maksilofasial 2 departemen biologi oral fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: mitochondrial dna (mtdna) sequence analysis of the hypervariable control region has been shown to be an effective tool for personal identification. the high copy and maternal mode of inheritance make mtdna analysis particularly useful when old samples or degradation of biological samples prohibits the detection of nuclear dna analysis. dental pulp is covered with hard tissue such as dentin and enamel. it is highly capable of protecting the dna and thus is extremely useful. one of the diasadvantages of mitochondrial dna is heteroplasmy. heteroplasmy is the presence of a mixture of more than one type of an organellar genome within a cell or individual. it can lead to ambiguity in forensic personal identification. due to that, the evidence of heteroplasmy in dental pulp is needed. purpose: the study was aimed to determine the heteroplasmy occurance of mitocondrial dna in dental pulp. methods: blood and teeth samples were taken from 6 persons, each samples was extracted with dnazol. dna samples were amplified with pcr and sequencing to analyze the nucleotide sequences polymorphism of the hypervariable region 1 in mtdna and compared with revised cambridge reference sequence (rcrs). results: the dental pulp and blood nucleotide sequence of hypervariable region 1 mitochondrial dna showed polymorphism when compared with rcrs and heteroplasmy when compared between dental pulp with blood. conclusion: the study showed that heteroplasmy was found in mithocondrial dna from dental pulp. key words: personal identification, mtdna, dental pulp, heteroplasmy abstrak latar belakang: analisis sekuens dna mitokondria (mtdna) regio kontrol hypervariable telah terbukti menjadi alat efektif untuk identifikasi personal. kopi dna yang banyak dan pewarisan maternal membuat analisis mtdna sangat berguna ketika sampel lama atau sampel biologis yang terdegradasi menghambat deteksi analisis dna inti. pulpa gigi terlindung jaringan keras seperti dentin dan enamel. hal ini membuat pulpa mampu melindungi dna dan dengan demikian sangat berguna untuk identifikasi. salah satu kekurangan dna mitokondria adalah heteroplasmy. heteroplasmy adalah adanya campuran lebih dari satu jenis genom dalam sel atau individua. hal ini dapat menyebabkan ambiguitas pada identifikasi pribadi forensik. oleh sebab itu, identifikasi personal menggunakan pulpa gigi harus memperhatikan kejadian heteroplasmy. tujuan: penelitian ini bertujuan untuk meneliti kejadian heteroplamy dna mitokondria pada pulpa gigi. metode: sampel darah dan gigi diambil dari 6 orang, masing-masing sampel diekstraksi dengan metode dnazol. sampel dna diamplifikasi dengan pcr dan sequencing untuk menganalisis polimorfisme urutan nukleotida di hypervariable region 1 mtdna dan dibandingkan dengan revised cambridge reference sequence (rcrs). hasil: sekuens nukleotida pulpa gigi dan darah daerah pada hypervariable region 1 dna mitokondria menunjukkan polimorfisme bila dibandingkan dengan rcrs dan heteroplasmy 131febri, dkk.: analisis heteroplasmy dna mitokondria pulpa gigi bila dibandingkan antara pulpa gigi dengan darah. simpulan: penelitian ini menunjukkan bahwa heteroplasmy dapat ditemukan pada dna mitokrondia pulpa gigi. kata kunci: identifikasi personal, mtdna, pulpa gigi, heteroplasmy korespondensi (correspondence): ardyni febri k, departemen patologi mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga, surabaya, indonesia. email: rumahnya.ve@gmail.com pendahuluan identifikasi personal menggunakan gigi merupakan hal yang sudah lama dilakukan. menurut heinemann, sebagaimana dikutip oleh svensson,1 penggunaan sampel gigi ini ditemukan pertama kali sejak permulaan abad 49 sebelum masehi yaitu agrippina dari romawi dapat diidentifikasi berdasarkan karakteristik dari gigi. darah merupakan spesimen identifikasi forensik yang sering digunakan dan merupakan gold standard akan tetapi ketika terjadi suatu peristiwa yang menyebabkan tubuh seseorang hancur, maka diperlukan spesimen lain yang digunakan sebagai sampel yaitu gigi.2 apabila kecelakaan terjadi di mana anggota tubuh yang lain telah hancur atau rusak akan tetapi kondisi gigi masih relatif baik maka akan didapatkan dna mitokondria pulpa gigi.3 menurut tsutsumi2 hal tersebut disebabkan dentin dan enamel memberikan semacam perlindungan bagi dna gigi. perlindungan tersebut menyebabkan dna gigi 100% secara tepat masih dapat digunakan untuk analisis gender dengan analisa polymerase chain reaction (pcr) setelah gigi dipanaskan pada suhu 100° c selama 15 menit.4 mitochondrial dna (mtdna) sebagai sampel identifikasi personal dalam bidang forensik banyak digunakan karena mempunyai struktur molekul berbentuk sirkuler yang stabil dan kemampuan menggandakan diri yang banyak pada tiap sel sehingga mtdna lebih efektif daripada dna inti.5 rangkaian analisa (sequence analysis) pada mtdna manusia telah digunakan secara luas untuk menggambarkan spesimen forensik secara biologis terutama ketika tidak ada sampel dna inti yang mencukupi untuk diambil. terdapat permasalahan pada mtdna yang perlu diketahui antara lain pewarisan maternal mtdna, heteroplasmy dan diperlukan sensitifitas peralatan yang tinggi untuk deteksi mtdna. mtdna terdiri dari 13 polipeptida untuk protein kompleks rantai respirasi, 22 trna dan 2rrna yang berfungsi dalam proses sintesis protein mitokondria, serta daerah yang tidak terkode (non coding region) yang disebut displacement loop (d-loop). daerah ini memiliki makna sangat besar bagi pemeriksaan forensik karena sekuens yang terdapat pada d-loop ini cenderung bervariasi (polymorphism) pada masing-masing individu, yaitu pada daerah hvr1 (nt16024-16383) dan hvr2 (nt 57-372).6 variasi nukleotida (transisi, transversi dan insersi) lebih banyak muncul pada hvr 1 yaitu sebanyak 77 posisi (24,8%) daripada hvr 2 sebanyak 56 posisi (19,9%).2 heteroplasmy adalah suatu keadaan di mana terdapat dua atau lebih tipe mtdna dalam mitokondrion tunggal, pada sel atau individu.7 heteroplasmy yang terjadi pada dna mitokondria terjadi karena mitokondria berhubungan erat dengan sistem transportasi elektron sehingga hal ini menyebabkan dna mitokondria rentan terjadi mutasi.8 berdasarkan uraian di atas maka identifikasi forensik yang menggunakan dna mitokondria pulpa gigi harus memperhatikan kemungkinan terjadi heteroplasmy untuk menghindari ambiguitas identitas seseorang. penelitian ini bertujuan untuk meneliti kejadian heteroplasmy dna mitokondria pada pulpa gigi. bahan dan metode jenis penelitian yang digunakan adalah analitic observational cross-sectional study. ethical clearance penelitian diperoleh dari komisi kelaikan etik penelitian kesehatan fakultas kedokteran gigi universitas airlangga. sampel yang digunakan ialah gigi premolar utuh dan sehat yang didapatkan dari pasien yang akan dilakukan perawatan ortodontik pada bpg puskesmas pegirian dan praktek pribadi peneliti dengan usia 20–30 tahun dan jenis kelamin laki-laki maupun perempuan. jumlah sampel pada penelitian ini ditetapkan sebanyak 6 sampel pada kelompok darah dan 6 sampel pada kelompok pulpa gigi. bahan untuk ekstraksi dna: dnazol reagent, larutan 100% dan 70% ethanol serta destilated water (sigma). bahan untuk amplifikasi dna: a) dna target: dna sasaran yang berasal dari dna mitokondria gigi premolar dan darah sebagai gold standard; b) primer yang digunakan: hypervariable region (hvr) 1 (nt34-159) primer mtdna amplicon size 126 bp. forward: 5’ggg agg tct cca tgc att tgg ta 3’, reverse: 5’aaa taa tag gat agg cag gtc 3’; pcr mix 25 μl yang terdiri : 2.5 μl 10x buffer, 1 μl dntp (atp, ctp, ttp, gtp), 1 μl primer f, 1 μl primer r, 0.6 μl mgcl2 , 0.3 μl taq polymerase, 16.6 μ l dh2o dan 2 μl sampel mtdna (promega corp). gigi premolar yang sehat diambil jaringan pulpanya dengan cara mencari orifice pulpa menggunakan mikromotor lowspeed, kemudian pulpa diambil menggunakan jarum ekstirpasi dan pulpa yang didapat dimasukkan ke dalam tabung eppendorf dan disonikasi selama 15 menit.2 pengambilan darah sampel sebanyak 5 ml dari vena cubiti dilakukan sebagai kontrol. 132 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 130–134 isolasi dna gigi dengan menggunakan dnazol (invitrogen tech-linesm): pellet gigi yang sudah dicuci yang berasal dari pulpa gigi yang telah diambil dicampur dengan 1 ml dnazol dengan cara divorteks kemudian diinkubasi selama 5 menit pada suhu kamar. dilakukan sentrifuse 10.000 rpm selama 10 menit pada suhu 4° c kemudian supernatan diambil dan dimasukkan ke dalam tabung baru. selanjutnya ditambahkan 0.5 ml ethanol absolute (100%), dicampur perlahan, kemudian diinkubasi selama 3 menit. dilakukan sentrifuse 4.000 rpm selama 2 menit pada suhu 4° c, kemudian supernatan dibuang secara hati-hati agar dna tidak ikut terbuang. dilakukan pencucian pellet dengan ethanol 75% 1 ml dan setiap kali dicuci dengan ethanol 75%, diulang selama 6 kali. selanjutnya disentrifuse 4000 rpm selama 2 menit. tabung diletakkan dengan posisi tegak selama 1 menit setelah itu buang ethanol 75% dengan cara pippeting atau decanting. pencucian dengan ethanol 75% sebanyak 2 kali. pellet dikeringkan dengan cara membiarkan tabung terbuka selama 15 detik sesudah ethanol 75% dibuang. pellet ditambah dengan larutan naoh 8mm sebanyak 0,3 ml sebagai pelarut dna, divorteks kemudian di spindown dan disimpan pada suhu -20° c.10 pengukuran kadar dan kemurnian dna melalui uv-spectrophotometer. pada larutan dna hasil uv spektrofotometer pada panjang gelombang 260 dan 280 nm, dihitung sebagai berikut: kadar dna = (bacaan pada λ 260) x fp (faktor pengenceran) x 1 od, dimana 1 optical density (od) setara 50 ng/μl atau 50 μg/ml untuk dna untai ganda. kemurnian dna dihitung dengan rasio perbandingan absorpsi panjang gelombang 260 nm dan 280 nm (a260/a280). hasil ekstraksi dna dari gigi dilakukan elektroforesis dengan menggunakan agarose gel 2% untuk mengetahui band/pita pada tiap sampel.10 kemudian dilakukan amplifikasi pcr mtdna 126 bp (nt34-159) dengan tahapan: seluruh reagen pcr serta dna sampel yang telah dimasukkan pada tabung eppendorf divorteks dengan di spin down selama 10 detik kemudian diamplifikasi sesuai dengan primer yang digunakan sesuai petunjuk referensi. primer: mtdna 126 bp, initial denaturation: 95° c 3 menit, denaturation: 94° c 1 menit, annealing: 56° c 1 menit, ekstension: 72° c 1 menit, final extention: 72° c 3 menit, cycle: 30x, elongation: 72° c–5 menit. hasil pcr yang positif dimurnikan (purification), dna quantity, labelling, purifying extention product/precipitation kemudian dilakukan proses sequencing. hasil berdasarkan hasil pcr dna dari gigi premolar dan darah sampel, didapatkan gambaran elektroforesis mtdna sebagai berikut (gambar 1). keenam sampel mtdna darah dan gigi yang telah dipurifikasi kemudian dilakukan proses sequencing dan analisa mtdna untuk mengetahui polimorfisme dan heteroplasmy yang mungkin terjadi pada basa nukleotidanya. polimorfisme yang terjadi pada sampel darah jika dibandingkan dengan cambridge reference sequence r a t a r a t a s e b e s a r 0 , 8 0 % d a r i 1 2 6 b p ( t a b e l 1 ) . gambar 1. hasil elektroforesis dna gigi dan darah. keterangan: d1-6: sampel darah; g1-6: sampel gigi; k (-): kontrol negatif; m: marker 100 bp. d1 d2 d3 d4 d5 d6 k (-) m k (-) g1 g2 g3 g4 g5 g6 k(-) m tabel 1. polimorfisme pada sampel darah sampel deteksi polimorfisme jumlah % 1 terdeteksi 1 0,80 2 terdeteksi 1 0,80 3 terdeteksi 1 0,80 4 terdeteksi 1 0,80 5 terdeteksi 1 0,80 6 terdeteksi 1 0,80 rata-rata 0,80 133febri, dkk.: analisis heteroplasmy dna mitokondria pulpa gigi penjumlahan total persentase keenam sampel dibagi enam. rata-rata terjadi heteroplasmy sebesar 20,90% dari 126 bp (tabel 3). persentase heteroplasmy tiap sampel didapatkan berdasar perhitungan jumlah titik yang terjadi heteroplasmy dibagi 126 bp. kemudian untuk persentase rata-rata didapatkan dari penjumlahan total persentase keenam sampel dibagi 6. berdasarkan hasil pengamatan keenam sampel antara sampel darah dan gigi didapatkan rata-rata transisi sebanyak 1.650% dan rata-rata transversi sebanyak 1.852% dari penjumlahan persentase rata-rata tiap kelompok (tabel 4). pembahasan heteroplasmy merupakan suatu kejadian di mana terdapat dua atau lebih mtdna dalam satu mitokondrion. pada banyak penelitian dikatakan bahwa heteroplasmy merupakan suatu situasi yang umum terjadi (1-2% dari genom mitokondria) namun keberadaan heteroplasmy bisa jadi akan mengubah penilaian investigasi identitas individu di bidang forensik. sampel darah sukarelawan digunakan sebagai kontrol (pembanding) karena darah merupakan sumber dna yang handal untuk digunakan secara identifikasi dan menjadi gold standard dalam pemeriksaan forensik. 10 berdasar hasil penelitian (tabel 1 dan 2) nampak perbedaan antara sampel darah dengan pulpa gigi di mana titik-titik terjadi polimorfisme pada mtdna darah lebih sedikit yaitu dengan rerata sebesar 0.80% dari mtdna pulpa gigi sebesar 19,31% jika dibandingkan dengan cambridge reference sequence. begitu pula hasil sequencing mtdna pulpa gigi (tabel 3) banyak menunjukkan titik basa nukleotida yang mengalami heteroplasmy dibandingkan dengan mtdna darah dengan rerata persentase heteroplasmy sebesar 20.9%. level mutasi mtdna yang tinggi biasa ditemukan pada jaringan postmitotic seperti tulang dan otak sedangkan level mutasi yang rendah ditemukan pada jaringan dengan tingkat pembelahan (mitosis) yang cepat seperti darah.11 jaringan post-mitotic ialah jaringan yang tidak memiliki kemampuan untuk bermitosis. sel-sel pada darah akan mengalami mitosis terus menerus yang diproduksi dari tulang belakang. selama siklus sel, replikasi dna terjadi pada fase s (synthesis) dan berlangsung selama 10 jam. keuntungan mengetahui pola sekuens, tidak hanya membantu proses identifikasi forensik tetapi juga dalam bidang antropologi dan arkeologi oleh karena perbedaan posisi heteroplasmy pada tiap suku bangsa disebabkan oleh perbedaan karakteristik haplotypes mtdna yang terjadi sejak adanya migrasi manusia dari afrika sehingga perbedaan posisi mutasi dan heteroplasmy antara darah dan gigi dengan rcrs disebabkan karena pada rcrs menggunakan haplogroup ras caucasian (eropa) yaitu h2a2a sedangkan pada orang indonesia (asia tenggara) termasuk pada haplogroup n9a.12 mutasi adalah perubahan tabel 2. polimorfisme pada sampel gigi sampel deteksi polimorfisme jumlah % 1 terdeteksi 15 11,90 2 terdeteksi 29 23,02 3 terdeteksi 21 16,67 4 terdeteksi 28 22,22 5 terdeteksi 20 15,87 6 terdeteksi 33 26,19 rata-rata 19,31 tabel 3. analisis heteroplasmy antara sampel darah dengan gigi sampel deteksi heteroplasmy jumlah % 1 terdeteksi 18 14,29 2 terdeteksi 29 23,02 3 terdeteksi 28 22,22 4 terdeteksi 28 22,22 5 terdeteksi 22 17,46 6 terdeteksi 33 26,19 rata-rata 20,90 tabel 4. karakteristik polimorfisme pada keenam sampel antara gigi dan darah tipe jumlah % rata-rata tiap kelompok ag 11 1,455 ga 8 1,058 tc 18 2,380 ct 13 1,720 rata-rata% transisi 1,650 at 10 1,323 ac 11 1,455 gt 23 3,042 gc 22 2,910 ca 8 1,058 cg 6 0,794 ta 17 2,249 tg 15 1,984 rata-rata transversi 1,852 insersi delesi persentase rata-rata didapatkan dari penjumlahan total persentase keenam sampel dibagi enam. polimorfisme yang terjadi pada sampel gigi jika dibandingkan dengan cambridge reference sequence rata-rata sebesar 19,31% dari 126 bp (tabel 2). persentase rata-rata didapatkan dari 134 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 130–134 pada materi genetik yang terjadi secara tiba-tiba, acak, dan merupakan dasar bagi sumber variasi organisme hidup yang bersifat terwariskan (heritable). mutasi terdiri atas dua jenis yaitu mutasi kromosom dan mutasi gen. mutasi kromosom adalah perubahan yang terjadi pada kromosom, meliputi: duplikasi (penambahan), delesi (lenyapnya gen dari suatu kromosom), inversi (perubahan letak segmen kromosom), pindah silang (pertukaran gen dari kromosom homolog), translokasi (pertukaran dari kromosom (nonhomolog). mutasi gen secara umum adalah perubahan apapun dalam bahan genetik yang disebabkan oleh lima hal, yaitu: mutasi noktah yang meliputi perubahan pada kodon tunggal, transisi, transversi, insersi dan delesi namun secara lebih terbatas mutasi lebih menunjuk ke perubahan dalam gen (intragenik). tabel 4 menunjukkan karakteristik polimorfisme antara sampel darah dan gigi dari keenam sampel yaitu rata-rata persentase transisi sebesar 1.65% dan transversi sebesar 1.85% sedangkan 0% untuk delesi dan insersi. mutasi transisi adalah penggantian purin yang satu dengan purin yang lain atau pirimidin yang satu dengan pirimidin yang lain, dan umumnya terjadi selama replikasi dna. pada peristiwa ini terjadi pergeseran elektron yang menyebabkan bentuk molekul menjadi sedikit berubah. pergeseran tautomer pada basa dna mengubah sifat pasangan basa sehingga a dapat berpasangan dengan c, dan t dengan g. mutasi transversi yaitu penggantian basa purin dengan pirimidin atau sebaliknya.13 penyebab transversi berbeda dengan penyebab transisi karena penyebab transversi terkait dengan sistem reparasi dna yang rentan terhadap kesalahan.14 secara teori faktor yang mempengaruhi level heteroplasmy mtdna seseorang antara lain kecepatan mutasi tiap individu, banyaknya mutasi yang dibawa ibu, jumlah efektif molekul mtdna per sel, jumlah pembelahan sel tiap generasi.15 teori bottleneck mechanism yang terjadi pada segregasi mtdna ditengarai juga menjadi penyebab variasi level heteroplasmy. penelitian ini menunjukkan bahwa heteroplasmy dapat ditemukan pada dna mitokondria pulpa gigi daftar pustaka 1. svensson k. a proposal of an investigation of the reliability of mitochondrial dna as a tool in forensic dentistry. huddings. sweden: institute of odontology. karolinska institute. 2002. p. 345–39. 2. tsutsumi h, komuro t, mukoyama r, nogami h. hypervariable region structure and polymorphism of mtdna from dental pulp and a family analysis. j oral sci 2006 sep;48(3):145–52. 3. singh d, bastian t.s, anil s, rohit j. use of mitochondrial dna in forensic odontology: a review. indian j forensic odontology 2008; 1(2): 35–40. 4. urbani c, lastrucci rd, kramer b. the effect of temperature on sex determination using dna-pcr analysis of dental pulp. j forensic odontostomatology 1999; 17(2): 35–39. 5. butler jm. advanced topics in forensic dna typing: methodology. san diego: elsevier academic press; 2012. p. 112. 6. soekry ek, sosiawan a. efek temperatur ekstrim terhadap kualitas dna inti dan dna mitokondria sebagai bahan identifikasi forensik. surabaya: laporan penelitian dik rutin unair; 2006. h. 1–30. 7. salas a, mv, lareu. a. carracedo. heteroplasmy in mtdna and the weight of evidence in forensic mtdna analysis: a case report. int j legal med 2001; 114: 186–90. 8. taylor rw, doug m.t. mitochondrial dna mutations in human disease. nat rev genet 2005; 6(5): 389–402. 9. yudianto a. analysis of nucleotide damage on str codis, mini str codis, y-str, & mtdna due to high temperature exposure in molecular forensic identification. disertasi. surabaya: universitas airlangga; 2010. h. 57–85. 10. lindstrom jd. a more efficient means to collect and process reference dna samples. nij cooperative agreement 2009-dnbx-k160. 2012; p. 8. 11. kujoth gc, hiona a, pugh td, someya s, panzer k, wohlgemuth se, hofer t, seo ay, sullivan r, jobling wa, morrow jd, van remmen h, sedivy jm, yamasoba t, tanokura m, weindruch r, leeuwenburgh c, prolla ta.. mitochondrial dna mutations, oxidative stress and apoptosis in mammalian aging. science 2005; 309(5733): 481–4. 12. van oven m, kayser m. updated comprehensive phylogenetic tree of global human mitochondrial dna variation. hum mutat 2009; 30(2): e386–94. 13. bandelt hj, quintana-mu rci l, salas a, macaulay v. the fingerprint of phantom mutations in mitochondrial dna data. am j hum genet . 2002; 71(5): 1150–60. 14. brown ta. genomes. 2nd ed. england: garland science; 2002. p. 14. 15. goto h, dickins b, afgan e, paul im, taylor j, makova kd, nekrutenko a. dynamics of mitochondrial heteroplasmy in three families investigated via a repeatable re-sequencing study. genome biol. 2011; 12(6): r59. 87 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 87–91 original article using the demirjian method for estimating the dental age of children in surabaya, indonesia beshlina fitri widayanti roosyanto prakoeswa1, arofi kurniawan1, an’nisaa chusida1, beta novia rizky1, anugerah i’zaaz darmawan2, andini kamilia nur aisyah2, aspalilah alias3 1department of forensic odontology, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2undergraduate student of dental medicine education study program, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3department of basic sciences and oral biology, faculty of dentistry, universiti sains islam malaysia, negeri sembilan, malaysia abstract background: the demirjian method is used in assessing the stages of growth and development of teeth to calculate a person’s estimated age. in 1973, demirjian identified the eight stages of tooth growth and development and their respective criteria. purpose: to analyze the validity of demirjian’s method for estimating dental age among children aged 6–17 years old in surabaya, indonesia. methods: from august–october 2020, 162 panoramic radiographs of patients aged 6–17 years were taken at the radiology department of airlangga dental hospital. data analysis was conducted using spss software for different tests, including a paired t test. results: using the demirjian method, there was no significant dissimilarity between chronological age (ca) and estimated dental age (eda) in the male group. however, a significant dissimilarity was found between ca and eda in the female group. conclusion: demirjian’s method can be used as a tool for estimating the dental age of males age 6-17 years old in surabaya. keywords: children; demirjian method; dental age estimation; forensic odontology; panoramic article history: received 6 june 2022; revised 31 august 2022; accepted 9 september 2022 correspondence: arofi kurniawan, department of forensic odontology, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya 60132, indonesia. email: arofi.kurniawan@fkg.unair.ac.id introduction indonesia is an archipelago of thousands of islands connected by straits and seas located between the asian and australian continents and the indian and pacific oceans.1 indonesia is prone to natural disasters caused by geographical conditions, climate and geology.2 in the case of a mass disaster, age estimation can simplify the identification of victims and enable grouping by ages.3 an estimate of age can also be necessary for living individuals involved in criminal or civil law cases, including falsification of employment age, marriage, athletes, child guardianship, immigration, or rape.4 legally valid evidence of age is important to determine whether an individual is legally a child or an adult, and there are differences in legal and judicial processes for children and adults.5 age estimation is also valuable evidence when a birth certificate does not exist or is in doubt. the body parts that are generally used for age estimation are skeletal and dental.6 skeletal maturation as a tool for age estimation has limitations because age can only be estimated within a certain range and with a large age standard deviation. in comparison, teeth have several advantages as an age estimation medium, including the ability to estimate the age of an individual from prenatal through to adulthood.7 assessment of tooth growth and development can be performed clinically or radiographically. the radiographic method has advantages because it is easier than other methods, is non-invasive, and can be performed on living or dead humans. one of the radiographic methods used was developed by demirjian et al.8 and groups tooth growth and development into eight stages with their respective criteria. the demirjian method is used in assessing the stages of growth and development of teeth to estimate chronological age, but it can also be used to see how much tooth development and growth has occured.9 in this study, panoramic radiograph images were used to assess tooth growth and development via the demirjian method. panoramic photos are easy to obtain and simple copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p87–91 mailto:arofi.kurniawan@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p87-91 88prakoeswa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 87–91 to perform, the dose of x-rays the patient is exposed to is relatively low, and the photos can be used in patients with trismus, are easier to apply to children, are relatively fast and convenient, have minimal distortion, and provide an overall picture of teeth and surrounding tissue.10,11 previous research on estimating dental age has found that the demirjian approach performs well in a variety of populations. as indonesia’s population has millions of people from different cultural and religious backgrounds, there is an urgent need for a reliable mechanism to identify a range of victims in the event of a large-scale tragedy.11 this study sought to evaluate the extent of applicability of the demirjian method for estimating the age of indonesian children and adolescents in surabaya. prior to this research, there was no dental age estimation method specifically for the surabaya population and no specific research on applying the demirjian method in surabaya. the author carried out this research by adapting an existing dental age estimation method. this study aimed to analyze the accuracy of demirjian’s dental age estimation method for children aged 6–17 in surabaya, indonesia. materials and methods this study was an observational analysis based on the panoramic radiographs of a population of 162 airlangga dental hospital patients aged 6–17 years (80 males and 82 females) from the radiology department, surabaya, in august –october 2020. the sample for this study was selected based on a purposive sampling technique with key inclusion criteria, including the panoramic radiograph used was not opaque, all parts of the studied teeth are visible on the panoramic radiograph, and there are no missing teeth in the studied region. the key exclusion criteria were radiographs with pathological features on teeth and surrounding tissues, patients using orthodontics or denture appliances, and developmental anomalies.12 ethics approval for this study was obtained from the ethics committee of the indonesian dental hospital airlangga university health research (001/un3.9.3/etik/pt/2021). all sample measurements were checked three times by a single observer at one-week intervals. all digital panoramic radiographs were scored using the demirjian method, and the calcification stage of the seven left mandibular teeth was assessed. the radiographic images were used to categorize tooth development into the eight stages (a to h), and figure 1 illustrates the particular parameters required for each stage for single-rooted and multi-rooted teeth.13 the score for every tooth was converted into a table based on gender (table 1),13 and the scores for the seven teeth were added up by the observer to identify the estimated age. figure 1. dental development stages a to h for single and multi-rooted teeth.13 table 1. maturity score for each stage by demirjian et al.13 sex tooth stagea b c d e f g h male m2 2.1 3.5 5.9 10.1 12.5 13.2 13.6 15.4 m1 8 9.6 12.3 17 19.3 p2 1.7 3.1 5.4 9.7 12 12.8 13.2 14.4 p1 7 11 12.3 12.7 13.5 c 3.5 7.9 10 11 11.9 i2 3.2 5.2 7.8 11.7 13.7 i1 1.9 4.1 8.2 11.8 female m2 2.7 3.9 6.9 11.1 13.5 14.2 14.5 15.6 m1 4.5 6.2 9 14 16.2 p2 1.8 3.4 6.5 10.6 12.7 13.5 13.8 14.6 p1 3.7 7.5 11.8 13.1 13.4 14.1 c 3.8 7.3 10.3 11.6 12.4 i2 3.2 5.6 8 12.2 14.2 i1 2.4 5.1 9.3 12.9 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p87–91 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p87-91 89 prakoeswa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 87–91 chronological age (ca) was defined as an individual’s date, month, and year of birth, and was calculated by subtracting the patient’s recorded date of birth from the date the panorama photo was taken. the estimated dental age (eda) was the age determined using demirjian’s method and applied on an orthopantomograph with each gender calculated independently.13 to extinguish bias, data calculations were performed three times at weekly intervals by a single observer. samples were analyzed by statistical tests using ibm® spss® statistics version 26.0 (ibm, armonk, ny, usa). the reliability of each variable was tested using cronbach’s alpha, the normality test was done using the kolomogorovsmirnov test, and the homogeneity test used levene’s test. variables showing p > .05 are detailed using the paired t test for the comparative test. results cronbach’s alpha was used to measure inter-examiner agreement in the grading stages of tooth development (α = .975). with a p value greater than .05, the kolmogorovsmirnov test result indicated a normal distribution of data which was suitable for further statistical analysis. the paired t test was employed to examine the significance of differences between ca and eda. table 2 provides a summary of the statistical analysis of ca and eda by subject totals. the general mean difference between ca and eda was -0.05 ± 1.31 for males and -0.72 ± 1.15 for females. the paired t test for demirjian’s method showed no significant dissimilarity amongst the ca and eda for males (p > .05). the opposite result was found for the female group, with the paired t test showing a significant dissimilarity between the ca and eda (p < .05). table 3 shows the statistical analysis of ca and eda by age group. for the 6–11-year-old group, the mean difference between ca and eda was -0.17 ± 1.00 and -0.46 ± 0.76 for males and females, respectively. in the 12–17-year-old age group, the mean difference between ca and eda was 0.08 ± 1.60 and -0.96 ± 1.39 for males and females, respectively. the p value for males in the 6–11-year-old group and 12–17-year-old group showed that there were no significant differences amongst dental age estimation and chronological age (p > .05). in contrast, the p value for females aged 6–11 years and 12–17 years showed that there was a significant dissimilarity between eda and ca (p < .05) discussion the demirjian approach assesses chronological age by using the calcification sequence of a person’s teeth as an indicator of age. there is disagreement amongst researchers about whether demirjian and dental age estimation methods can be applied to all types of populations, as different populations can yield different results between investigators.13 the comparative test conducted in this study showed no significant dissimilarity between the ca and the eda of males across all age groups using the demirjian method. this agrees with previous research conducted by sinha et al.,14 zhai et al.,15 and bagherian and sadeghi16 that there is no significant dissimilarity between chronological age and estimated age found using the demirjian method. in a study conducted by sinha et al.,14 the mean difference between ca and eda was 0.02 ± 0.31 with p = .245, which indicated that demirjian’s method could be used for age estimation among the population of northern india. this is also in line with research conducted by zhai et al.15 about the population of northern china. in the current study, the mean dissimilarity between ca and eda was 0.47 ± 1.21 with a p value of .072 in the male group. research by bagherian16 on the population in iran also showed similar results, with the mean dissimilarity between ca and eda equal to 0.15 ± 0.51 with a p value of .075 for the male group. table 2. statistical analysis of ca and eda by subject totals gender n ca eda age difference mean sd mean sd mean sd p value remarks male 80 11.41 3.43 11.35 3.45 -0.05 1.31 0.700 underestimated female 82 11.79 3.39 11.07 3.26 -0.72 1.15 0.000* underestimated total 162 11.60 3.41 11.21 2.35 -0.39 1.27 0.000 underestimated paired t test (*p < .05) table 3. statistical analysis of ca and eda of the age group gender age group ca eda age difference mean sd mean sd mean sd p value remarks male 6–11 8.72 1.79 8.54 1.41 -0.17 1.00 0.250 underestimated 12–17 14.54 1.86 14.62 1.86 0.08 1.60 0.749 overestimated female 6–11 8.75 1.44 8.28 1.07 -0.46 0.76 0.000* underestimated 12–17 14.69 1.78 13.72 2.27 -0.96 1.39 0.000* underestimated paired t test (*p < .05) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p87–91 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p87-91 90prakoeswa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 87–91 these three studies had a p value greater than .05 which indicates no significant dissimilarity between ca and eda using demirjian’s method.11 these results are consistent with the findings of this study that the demirjian method can be applied to the population of surabaya in indonesia as the method showed no significant dissimilarity between ca and eda. in comparison, research by kurniawan et al.17 and agitha et al.4 showed that willems’ dental age estimation method was applicable to the surabaya population, with no significant dissimilarity found between ca and eda. differences in the determination of age estimates can reflect a child’s general developmental shift and various factors that contribute to changes in dental development.18 an individual’s growth and development depend on intrinsic and extrinsic factors. for example, variations in nutritional selection can significantly alter individual growth.5 few studies have considered the timing of relationships of tooth formation which can vary widely among population groups. previous studies have consistently overestimated age-related changes, and this suggests that genetic and environmental factors may influence variation in the timing of tooth development.19 the age difference found between the male and female samples in the current study may be due to gender-specific factors, and adjustments made for other maturation parameters in female developmental stages, such as sexual maturation, skeletal development and height.20 the growth spurt process causes an acceleration of tooth maturation and is often associated with a spike in tooth age within one age group. growth spurts occur early after birth and again at the age of about 6–7 years, and last for approximately 3–4 months. however, there are differences in the later growth spurts of males and females. accelerated growth occurs in females at approximately 12 years of age and at age 14 years in males. there is also a large variation in growth acceleration, with a standard deviation of one year, and sometimes growth spurts occur in males over the age of 16 years.21 estimates of dental age must be as detailed as possible to undertake forensic examinations.8 based on the results of this study, demirjian’s method showed no significant dissimilarity in the eda and the ca of males therefore, it could be used in surabaya as a method for estimating the age of children and adolescents aged 6–17 years. if the result of the difference between ca and eda is closer to zero, the higher the precision of age estimate for that method when applied to certain populations. in addition, the use of the mean error prediction, which shows a maximum result of 1, can be considered accurate.22 the process of age identification can be done using a combination of several methods, such as teeth and bones. this can increase the reliability of age identification for more accurate results than using only one method.22 maber et al.23 and liversidge11 highlight that research results can differ based on variations between the sample population and general population standards, such as age, sample size, sample bias, sample population biological variations, environment, eating habits, and accuracy in evaluating the method used. although there are differences in the results of this study, the difference between chronological age and dental age in each age group is still within the limits determined by forensic anthropology, which is between ± 0.5 years to ± 1 year in adult and child populations.24 in conclusion, the demirjian method can be utilized to calculate the estimated dental age of children from surabaya, indonesia; however, further research is needed for females in this population. the results obtained in this study may differ when applied to other populations due to various factors. further explorations with larger sample sizes will strengthen the reliability of using the demirjian approach in indonesia. references 1. badan pusat statistik. statistik indonesia 2018. subdirektorat publikasi dan kompilasi statistik, editor. jakarta: bps-statistics indonesia; 2018. p. 1–762. 2. p usat pena ngg ula nga n k r isis kesehat a n. pedoma n tek n is penanggulangan k risis kesehatan akibat bencana: technical guidelines for health cr isis responses on disaster. ja ka r ta: kementerian kesehatan republik indonesia; 2010. p. 1–170. 3. prakoeswa bfwr, kurniawan a, chusida a, marini mi, rizky bn, margaretha ms, utomo h, i’zaaz darmawan a, aisyah akn, alias a, wahjuni or, marya a. children and adolescent dental age estimation by the willems and al qahtani methods in surabaya, indonesia. dias fj, editor. biomed res int. 2022; 2022: 1–4. 4. agitha sra, sylvia m.a.r m, utomo h. estimasi usia anak etnis tionghoa di indonesia dengan menggunakan metode willems. j biosains pascasarj. 2016; 18(1): 35–9. 5. swastirani a, utomo h, sylvia mar m. estimsasi usia dengan orthopantomogram pada pasien rumah sakit gigi dan mulut pendidikan universitas airlangga. e-prodenta j dent. 2018; 2(1): 124–9. 6. putri as, nehemia b, soedarsono n. prakiraan usia individu melalui pemeriksaan gigi untuk kepentingan forensik kedokteran gigi (age estimation through dental examination in forensic denstistry). j pdgi. 2013; 62(3): 55–63. 7. kurniawan a, chusida a, atika n, gianosa tk, solikhin md, margaretha ms, utomo h, marini mi, rizky bn, prakoeswa bfwr, alias a, marya a. the applicable dental age estimation methods for children and adolescents in indonesia. pucci cr, editor. int j dent. 2022; 2022: 6761476. 8. demirjian a, goldstein h, tanner jm. a new system of dental age assessment. hum biol. 1973; 45(2): 211–27. 9. boel t, bahri ta. age estimation using schour-massler method compared to the demirjian method. dentika dent j. 2019; 22(1): 15–9. 10. larasati aw, irianto mg, bustomi ec. peran pemeriksaan odontologi forensik dalam mengidentifikasi identitas korban bencana masal. major med j lampung univ. 2018; 7(3): 228–33. 11. liversidge hm. the assessment and interpretation of demirjian, goldstein and tanner’s dental maturity. ann hum biol. 2012; 39(5): 412–31. 12. apriyono dk. metode penentuan usia melalui gigi dalam proses identifikasi korban. cermin dunia kedokt. 2016; 43(1): 71–4. 13. demirjian a, goldstein h. new systems for dental maturity based on seven and four teeth. ann hum biol. 1976; 3(5): 411–21. 14. sinha s, umapathy d, shashikanth m, misra n, mehra a, singh a. dental age estimation by demirjian′s and nolla′s method: a comparative study among children attending a dental college in lucknow (up). j indian acad oral med radiol. 2014; 26(3): 279. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p87–91 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p87-91 91 prakoeswa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 87–91 15. zhai y, park h, han j, wang h, ji f, tao j. dental age assessment in a northern chinese population. j forensic leg med. 2016; 38: 43–9. 16. bagherian a, sadeghi m. assessment of dental maturity of children aged 3.5 to 13.5 years using the demirjian method in an iranian population. j oral sci. 2011; 53(1): 37–42. 17. kurniawan a, agitha sra, margaretha ms, utomo h, chusida a, sosiawan a, marini mi, rizky bn. the applicability of willems dental age estimation method for indonesian children population in surabaya. egypt j forensic sci. 2020; 10(1): 0–3. 18. sykes l, bhayat a, bernitz h. the effects of the refugee crisis on age estimation analysis over the past 10 years: a 16-country survey. int j environ res public health. 2017; 14(6): 630. 19. kumaresan r, cugati n, chandrasekaran b, karthikeyan p. reliability and validity of five radiographic dental-age estimation methods in a population of malaysian children. j investig clin dent. 2016; 7(1): 102–9. 20. nik-hussein nn, kee km, gan p. validity of demirjian and willems methods for dental age estimation for malaysian children aged 5-15 years old. forensic sci int. 2011; 204(1–3): 208.e1-6. 21. rahardjo p. diagnosis ortodontik. surabaya: airlangga university press; 2008. p. 1–100. 22. koç a, özlek e, öner talmaç ag. accuracy of the london atlas, willems, and nolla methods for dental age estimation: a crosssectional study on eastern turkish children. clin oral investig. 2021; 25(8): 4833–40. 23. maber m, liversidge hm, hector mp. accuracy of age estimation of radiographic methods using developing teeth. forensic sci int. 2006; 159: s68–73. 24. a mba rkova v, galić i, voda nović m, biočina-lukenda d, brkić h. dental age estimation using demirjian and willems methods: cross sectional study on children from the former yugoslav republic of macedonia. forensic sci int. 2014; 234: 187.e1-187.e7. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p87–91 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p87-91 126 dental journal (majalah kedokteran gigi) 2019 september; 52(3): 126–132 research report the effect of a combination of propolis extract and bovine bone graft on the quantity of fibroblasts, osteoblasts and osteoclasts in tooth extraction sockets louisa christy lunardhi, utari kresnoadi and bambang agustono department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: tooth extraction is a procedure frequently performed in the field of dentistry that can cause alveolar bone resorption during the healing process. therefore, preservation of sockets is necessary to maintain alveolar bone which represents one of the important factors in the successful manufacture of dentures. a combination of propolis extract and bovine bone graft (bbg) can accelerate bone regeneration. purpose: the purpose of this study was to determine the effect of a combination of propolis extract and bbg on the quantity of fibroblasts, osteoblasts, and osteoclasts in the tooth extraction socket. methods: 56 cavia cobaya were divided into eight groups. the lower left incisor of each subject was extracted and induced with polyethylene glycol (peg), propolis extract+ peg, bbg + peg, combination of propolis extract + bbg + peg at a concentration of 2% active substance. experimental subjects were sacrificed on days 3 and 7. histopathological examination with a microscope at 400x magnification was conducted to calculate the quantity of fibroblasts, osteoblasts, and osteoclasts. statistical analysis was performed by one-way anova and tukey hsd tests. results: the highest average quantity of fibroblasts and osteoblasts and the lowest average quantity of osteoclasts occurred in the group to which a combination of propolis extract and bbg had been administered on both days 3 and 7. according to the statistical analysis results, all the treatment groups recorded a significant difference in the quantity of fibroblasts, osteoblasts, and osteoclasts with a p value: 0.000 (p<0.05). conclusion: a combination of propolis extract and bbg can increase the quantity of fibroblast and osteoblast cells, while reducing the number of osteoclast cells in tooth extraction sockets treated with 2% concentration of the active substance. keywords: bovine bone graft (bbg); fibroblasts; osteoblasts; osteoclasts; propolis extract; socket preservation correspondence: utari kresnoadi, department of prosthodontics, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia, e-mail: utari-k@fkg.unair.ac.id introduction tooth extraction constitutes a commonly performed dental procedure that invariably precedes bone resorption and regeneration. tooth extraction followed by the socket healing process usually leads to alveolar bone deformities, including a reduction in residual ridge height and width.1 after extraction, bleeding in the tooth socket precedes initiation of the inflammatory mediator and produces a blood clot that seals the exposed socket. inflammation causes an increase in pro-inflammatory cytokines, such as tumor necrosis factor alpha (tnf-α) and interleukin-1β (il1β) and osteoclasts, with the result that receptor activators of both nuclear factor kappa-b (rank) and nuclear factor kappa-b ligand (rankl) proliferate. if osteoclasts increase in number, alveolar bone resorption ensues.1 three to five days post-extraction, fibrin degeneration will occur together with the formation of granulation tissue containing blood vessels, fibroblasts, and chronic inflammatory cells. during the process of inflammation, fibroblasts stimulate osteoprogenitor (opg) to inhibit rankl binding to rank and trigger an increase in fibroblasts growth factor-2 (fgf-2) which plays a role in the process of osteoblast cells proliferation and differentiation. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 127lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 within a period of seven days post-extraction, immature bone has formed which differs from osteoblasts and bone matrix.2 alveolar bone resorption is known to occur rapidly for one year after tooth extraction and will continue during the subsequent 12 months. a 25% reduction in bone width in the cervical region and a 40% reduction of width in the labial plate of the bone occurs in the first and third years after extraction.3 large resorption which is not offset by rapid bone regeneration can change the structure of the maxilla and mandible bones, thereby rendering the use of dentures problematic.3 consequently, it is important to perform socket preservation in order to maintain the alveolar bone after tooth extraction and minimize alveolar bone resorption.4 a biomaterial often used in socket preservation procedures is bone graft, one form of which is bovine bone graft (bbg). bbg, produced from cow bone which has been demineralized to remove any organic minerals present, has an osteoconductive effect in forming scaffold for bone regeneration.5 natural ingredients, one of which is propolis (a resin material found in beehives), are extensively employed in traditional medicine. propolis, which had previously been obtained from lawang, east java, possesses certain bioactive components such as artepyline, apigenin, flavonoid, cinnamic acid, saponin, quercetin, terpenoid and caffeic acid-phenetyl ester (cape) which have many beneficial effects, including; antibacterial, antifungal, antiinflammatory, antiviral, antioxidant, immunostimulative, and anticancer. propolis extract is also known to contain polyphenolic compounds such as flavonoids and cape which can increase the number of osteoblasts and are anti-inflammatory.6 therefore, this study aims to prove the effects of the combination of propolis extract and bbg on the quantity of fibroblasts, osteoblasts and osteoclasts in the tooth extraction socket on days 3 and 7. materials and methods this investigation constituted experimental laboratory research which received ethical approval from the ethics committee, faculty of dental medicine, universitas airlangga (number 316/hrecc.fodm/xii.2018). the research population of 56 male cavia cobaya (c. cobaya) aged 3-3.5 months and 300-350mg in weight were divided into eight groups of seven subjects. these were subsequently designated either 3-day groups or 7-day groups each of which received four treatments. the c. cobaya were taken from their treatment facility and anesthetized intravenously with 0.2cc/300 g ketamine. extraction of the lower left incisor was effected using needle holder pliers. the extraction socket was then filled with polyethylene glycol (peg) suspension, propolis extract + peg, bovine bone graft (bbg) + peg, and a combination of propolis extract + bbg + peg at an active substance concentration of 2%. up to a maximum of 0.1cc of each mixture was administered to the socket which was then sutured with polyamide monofilament.7 the groups were classified as follows. groups i and ii: tooth extraction sockets were filled with 25g of peg only. groups iii and iv: tooth extraction sockets were filled with 0.5g of propolis extract and 24.5g of peg. groups v and vi: tooth extraction sockets were filled with 0.5g of bbg and 24.5g of peg. groups vii and viii: tooth extraction sockets were filled with a combination of 0.5g of propolis extract, 0.5g of bbg and 24g of peg. groups i, iii, v and vii were examined after three days and groups ii, iv, vi and viii after seven days. in preparation for histopathologic (hpa) examination, after three days and seven days of the treatment the c. cobaya were euthanized and their jaws removed by means of a surgical incision. their mandibles were cut to be decalcified with ethylene diamine tetra acetate (edta) for a period of three months. paraffin blocks were manufactured and cut to a thickness of 4µ with a rotary microtome before being deparaffinized by being dissolved in xylol for 2x3 minutes. the residual xylol was respectively washed with 99%, 95%, 90%, 80%, and 70% absolute alcohol for 2x1 minutes. any residual alcohol was removed with running water. at that point, hematoxylin eosin (he) staining was subsequently performed for 30 seconds before rinsing with water. staining with he was subsequently conducted for 1-2 minutes prior to washing with 70%, 80%, 90%, 95%, 99% absolute alcohol for 2x1 minutes. fibroblast, osteoblast, and osteoclast cells were washed out with 99%, 95%, 90%, 80%, 70% absolute alcohol for 2x1 minutes. observation was subsequently carried out under a light microscope, each slide being examined at 400x magnification and a maximum of 8 fields of view (fov). the calculation results were recorded in a worksheet with a mean value per field of view. at this point, the quantity of fibroblasts, osteoblasts, and osteoclasts was calculated.7 for statistical analysis, a kolmogorov smirnov statistical test was performed on the data obtained. in order to identify differences between the groups, a one-way anova test was conducted, followed by a multifactorial comparison test involving a tukey honest significant difference (hsd) test. a levene’s homogeneity statistical test was also performed. results a hpa examination identified the presence of fibroblast, osteoblast and osteoclast cells. fibroblast cells appeared as large cells with slim and spindle-shaped branches, an oval or elongated core, and fine chromatin (figure 1). osteoblast cells could be observed as circular cells located on the edge of the trabecular bone (figure 2), while osteoclast cells appeared as large cells with multiple nuclei (figure 3). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 128 lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 a b c d figure 1. black arrows indicate histopathological staining identifying fibroblasts on day 7 (he staining observed through a light microscope at a magnification of 400x). (a) control group (peg); (b) propolis extract + peg; (c) bbg + peg; (d) a combination of propolis extract +bbg+ peg. a b d c figure 2. black arrows indicate histopathological staining identifying osteoblasts on day 7 (he staining observed through a light microscope at a magnification of 400x). (a) control group (peg); (b) propolis extract + peg; (c) bbg + peg; (d) combination of propolis extract +bbg+ peg. a b c d figure 3. black arrows indicate histopathological staining identifying osteoclasts on day 7 (he staining observed through a light microscope at a magnification of 400x). (a) control group (peg); (b) propolis extract + peg; (c) bbg + peg; (d) combination of propolis extract +bbg+ peg. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 129lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 figure 4. the bar chart displays the average and standard deviations of the quantity of fibroblasts after 3 and 7 days of treatment. the y-axis shows the average quantity of fibroblasts. the x-axis shows treatment groups which the socket filled with peg, propolis+peg, bbg+peg, propolis+bbg+peg. figure 5. the bar chart displays the average and standard deviations of the quantity of osteoblasts after 3 and 7 days of treatment. the y-axis shows the average quantity of osteoblasts. the x-axis shows treatment groups which the socket filled with peg, propolis+peg, bbg+peg, propolis+bbg+peg. figure 6. the bar chart displays the average and standard deviations of the quantity of osteoclasts after 3 and 7 days of treatment. the y-axis shows the average quantity of osteoclasts. the x-axis shows treatment groups which the socket filled with peg, propolis+peg, bbg+peg, propolis+bbg+peg. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 130 lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 table 1. statistical analysis data relating to the quantity of fibroblasts, osteoblasts, osteoclasts in each treatment group on day 3 group mean ± sd normality test homogenity test one-way anova test tuckey hsd test i iii v vii fibroblasts i 12.86 ± 1.77 0.789 0.546 0.000 * * iii 15.29 ± 2.63 0.494 * v 18.57 ± 2.23 0.595 * * vii 22.29 ± 2.29 0.999 * * * osteoblasts i 10.29 ± 1.11 0.985 0,219 0.000 * * iii 12.00 ± 1.41 0.964 * v 13.00 ± 1.15 0.833 * * vii 18.86 ± 2.61 0.858 * * * osteoclasts i 15.71 ± 1.80 0.960 0.492 0.000 * * iii 13.57 ± 1.72 0.988 * v 11.86 ± 1.35 0.986 * vii 9.71 ± 1.11 0.985 * * *= there is a significant difference (p<0.05) note: a normality test score of p>0.05 means the data follows normal distribution; a homogeneity test score of p>0.05 means the data demonstrates homogenous distribution; a one-way anova test score of p<0.05 means that significant difference exists; group i: 25 grams of peg on day 3; group iii: propolis extract+peg on day 3; group v: bbg+peg on day 3; group vii: propolis extract+bbg+peg at an active substance concentration of 2% on day 3. table 2. statistical analysis data on the quantity of fibroblasts, osteoblasts, osteoclasts in each treatment group on day 7 group mean ± sd normality test homogenity test one-way anova test tuckey hsd test ii iv vi viii fibroblasts ii 15.71 ±2.75 0.985 0.942 0.000 * * iv 17.86 ±3.13 0.997 * * vi 23.00 ±3.42 1.000 * * * viii 27.57 ±2.88 0.997 * * * osteoblasts ii 11.86 ±1.35 0.986 0.829 0.000 * * * iv 14.71 ±1.25 0.701 * * * vi 19.00 ±2.38 0.905 * * * viii 27.86 ±1.35 0.986 * * * osteoclasts ii 12.86 ±1.46 0.761 0.602 0.000 * * * iv 10.29 ±0.95 0.422 * * * vi 8.00 ±0.82 0.905 * * viii 5.71 ±1.25 0.701 * * * *= there is a significant difference (p<0.05) note: a normality test score of p>0.05 signifies that the data follows normal distribution; a homogeneity test result of p>0.05 indicates that the data demonstrates homogenous distribution; a one-way anova test score of p<0.05 means there is significant difference; group ii: 25 grams of peg on day 7; group iv: propolis extract+peg on day 7; group vi: bbg+peg on day 7; group viii: propolis extract+bbg+peg at the active substance concentration of 2% on day 7. the calculated averages and standard deviations of the fibroblasts, osteoblasts, and osteoclasts in each treatment group and the control group are contained in figures 4-6. a histopathologic (hpa) examination indicated that the quantity of fibroblasts and osteoblasts was higher in group viii whose members had been administered a combination of propolis extract, bbg, and peg on day 7, than in the group control which only received peg on day 3. the quantity of fibroblasts and osteoblasts on day 7 was higher than that on day 3. on the other hand, the highest concentration of osteoclasts in the tooth extraction socket was found in the control group (group i) on day 3, while the lowest was in group viii which had received a combination of propolis extract, bbg, and peg on day 7. the quantity of osteoclasts on day 3 was higher than that on day 7. prior to an analysis of the test results for each group, a kolmogorov-smirnov normality test was conducted. in this research, all the research groups had a p value greater than 0.05 signifying that data derived from all dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 131lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 of them demonstrated normal distribution. based on the homogeneity test results, all research groups had a significance value greater than 0.05. this indicates that all groups had homogeneous variance. given the prerequisite normal and homogeneous distribution, a one-way anova test was conducted to quantify the significance among the various groups. based on the results of the one-way anova test, a significant value of 0.000 was obtained, which was lower than 0.05. this indicates that there was a significant difference in the quantity of fibroblasts, osteoblasts, and osteoclasts between the control and treatment groups. the results of a tukey hsd test confirmed a significant difference in the quantity of fibroblasts, osteoblasts, and osteoclasts between the control group (peg) and the treatment group (propolis extract, bbg, and peg) on both day 3 and day 7, with a significance of p = <0.05 (see table 1 and 2). discussion based on the results outlined above, it is evident that the quantity of fibroblasts increased between days 3 and 7 in both the control and treatment groups. the quantity of fibroblasts on day 7 was higher than that on day 3. this may have occurred because the inflammatory phase enters the healing stage on day 7 and the combination of propolis extract and bbg affects the ros produced by macrophages. propolis was obtained from the research and consultation laboratory lawang, east java, indonesia. it contained cinnamic acid (2.56%), apigenin (1.05%), flavonoid (1.28%), saponin (0.82%), quercetin (1.03%) and terpenoid (1.15%) which produced several effects, including; anti-inflammatory, antibacterial, antiviral, immunostimulatory, antifungal, and anticancer. the flavonoids present in propolis promote wound healing by increasing the formation of fibroblast growth factor-2 (fgf-2) and vascular endothelial growth factor a (vegfa). fgf2 constitutes a pleiotropic growth factor capable of stimulating fibroblast cells and progenitor osteoblasts.8 this assertion concurs with the findings of research conducted by puspasari et. al.9 which showed that the expression of fgf2 in the group whose members received an application of propolis extract was significantly higher than that of the control group. caffeic acid phenethyl ester (cape) represents a group of flavonoids in propolis that has an antioxidant effect which can include excessive oxidative reactions as a result of inflammatory reactions and metabolic processes followed by cell injury. as an anti-inflammatory, cape acts to inhibit phospholipase in the arachidonic acid cascade. consequently, it does not release prostaglandins and leukotreins. cape can also inhibit lipoxygenase (lox) and cylooxygenase (cox) which play a role in the metabolic pathway. cox is inhibited by flavonoids which suppress the stimulation and synthesis of prostaglandins and thromboxanes. lox is inhibited by a propolis component, quercetin, which impede leukotrin and lipoxin stimulation. cape is lipophilic and facilitates cell infiltration, releases anti-inflammatory cytokines (tgf-β, il-10, il-4), inhibits both pro-inflammatory cytokine (tnf-α, il-6, il-1β) and the activity of nuclear factor-kb (nf-kb), while increasing fgf-2 proliferation.9 in this research, the anti-inflammatory effect of propolis extract was employed to reduce inflammation resulting from tooth extraction trauma by stimulating fibroblast growth. bbg produces an osteoconductive effect that can promote the wound healing process and enhance bone formation. based on the foregoing results, it is evident that the osteoblasts increased in number between days 3 and 7, while the quantity of osteoclasts in both the control and treatment groups declined significantly. the quantity of osteoblasts was higher on day 7 than on day 3. the quantity of osteoclasts was initially high in the control group, but decreased on day 7. this could be due to propolis extract and bbg impeding the inflammatory process with the result that both the wound healing and bone regeneration processes were accelerated. research conducted by yuanita, et.al.10 shows that the administration of propolis extract can inhibit rankl binding on rank receptor, resulting in inhibited activation of nf-kb and a subsequent decrease in the number of osteoclasts.10 propolis has an antioxidant effect and will inhibit reactive oxygen species (ros), a free radical that plays a role in bone resorption, and the process of osteoclastogenesis by activating osteoclast cells. ros can stimulate the phosphorylation process involving kappa β (ikβ) inhibitors which function to bind nf-kb, causing it to remain inactive in the cytoplasm. when ikβ is phosphorylated, ikβ and nf-kb bonds are released and nf-kb becomes active in moving to the cell nucleus. this process is called nf-kb activation. the presence of flavonoid in propolis acts against the formation of ros thereby inhibiting the activation of nf-kb. this may induce a reduction in bone resorption.11 propolis extract can accelerate osteoblast cell maturation and bone remodelling activity. osteoblast cells are regulated by several growth factors, such as bone morphogenic protein (bmp), runt-related transcription factor 2(runx2) and osterix. propolis extract containing flavonoid plays a role in increasing osterix and runx2 with the result that osteoblasts mature more rapidly, thereby promoting bone remodelling. propolis extract containing flavonoid can reduce bone resorption and increase bone remodelling.11 saponins in propolis play an active role in intensifying alkaline phosphate (alp) activity, increasing mineralization, and promoting expression of the osteogenic alp, runx2 gene. runx2 is a transcription factor involved in osteoblast differentiation and bone formation. saponins also increase dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 132 lunardhi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 126–132 the activity of bone morphogenic protein (bmp) which serves as a signal of bone formation, while bmp-2 promotes runx2 gene expression.12 in addition, quercetin in propolis promotes an increase in the formation of osterix and runx2 which plays a role in stimulating osteoblast differentiation and bone formation by suppressing lipopolysaccharide (lps).13 cinnamic acid in propolis acts as an immunodulator. it can also increase alp activity and calcium that can stimulate bone formation, while also inhibiting the production of nf-kb and tnf-α.14 consequently, since propolis extract reduces proinflammatory cytokines (il-1, tnf-α), inhibits nf-kb and increases certain osteoblast progenitors (bmp-2, osterix, runx2), it can be argued an inverse relationship exists between osteoblasts and osteoclasts. the reduction in the number of osteoclast cells will occur when their growth is stimulated by homeostasis in bone cells.15 similarly, the results of this research indicate that a combination of propolis extract and bbg increases the quantity of osteoblasts, but reduces that of osteoclasts. another cause influencing the increase in osteoblast cells in this research is the administration of graft material (bbg) combined with propolis extract. bbg constitutes a graft material in the form of xenograft employed in this study. xenograft is used to stimulate the proliferation of fibroblasts, osteoblasts, and endothelial cells. bbg is the most commonly used graft because it contains hydroxyapatite which is almost identical to human bone and enables the graft to revascularize and be replaced with new bone. inorganic material from bbg is capable of supporting the attachment and proliferation of osteoblast cells which represents the first step in the process of osteogenesis. the material supports the bone matrix for regulation through three mechanisms, namely; formation of a strong filling space, establishment of osteoblast adhesion and proliferation and stimulation of bone formation.16 bbg produces an osteoconductive effect which acts as a scaffold in a medium for stem cells and osteoblast cells to attach to, live within, and develop properly in bone defects. in addition, scaffold promotes the development of blood vessels during the formation of new bone. the osteoconductive effect in graft can stimulate bone growth and cause bone apposition in existing bone.16 it can be concluded, therefore, that a combination of propolis extract and bbg with an active substance concentration of 2% can increase the quantity of both fibroblasts and osteoblasts, while decreasing the quantity of osteoclasts on days 3 and 7 after tooth extraction. this, in turn, can accelerate wound healing and bone regeneration. references 1. araújo mg, silva co, misawa m, sukekava f. alveolar socket healing: what can we learn? periodontol 2000. 2015; 68(1): 122– 34. 2. cassini-vieira p, araújo fa, da costa dias fl, russo rc, andrade sp, teixeira mm, barcelos ls. inos activity modulates inf lammation, angiogenesis, and tissue fibrosis in polyetherpolyurethane synthetic implants. mediators inflamm. 2015; 2015: 1–9. 3. mittal y, jindal g, garg s. bone manipulation procedures in dental implants. indian j dent. 2016; 7(2): 86–94. 4. fee l. socket preservation. br dent j. 2017; 222(8): 579–82. 5. singh j, takhar rk, bhatia a, goel a. bone graft materials: dental aspects. j nov res healthc nurs. 2016; 3(1): 99–103. 6. khurshid z, naseem m, zafar ms, najeeb s, zohaib s. propolis: a natural biomaterial for dental and oral healthcare. j dent res dent clin dent prospects. 2017; 11(4): 265–74. 7. kresnoadi u, ariani md, djulaeha e, hendrijantini n. the potential of mangosteen (garcinia mangostana) peel extract, combined with demineralized freeze-dried bovine bone xenograft, to reduce ridge resorption and alveolar bone regeneration in preserving the tooth extraction socket. j indian prosthodont soc. 2017; 17(3): 282–8. 8. jacob a, parolia a, pau a, davamani amalraj f. the effects of malaysian propolis and brazilian red propolis on connective tissue fibroblasts in the wound healing process. bmc complement altern med. 2015; 15: 294. 9. puspasari a, harijanti k, soebadi b, hendarti ht, radithia d, ernawati ds. effects of topical application of propolis extract on fibroblast growth factor-2 and fibroblast expression in the traumatic ulcers of diabetic rattus norvegicus. j oral maxillofac pathol. 2018; 22(1): 54–8. 10. yuanita t, zubaidah n, kunarti s. expression of osteoprotegrin and osteoclast level in chronic apical periodontitis induced with east java propolis extract. iran endod j. 2018; 13(1): 42–6. 11. perkasa mia, yogyarti s, harijanto e. propolis extract gel application to the socket of cavia cobaya after tooth extraction to the quantity of osteoclast. mater dent j. 2017; 8(1): 23–8. 12. ke k, li q, yang x, xie z, wang y, shi j, chi l, xu w, hu l, shi h. asperosaponin vi promotes bone marrow stromal cell osteogenic differentiation through the pi3k/akt signaling pathway in an osteoporosis model. sci rep. 2016; 6: 35233. 13. wang xc, zhao nj, guo c, chen jt, song jl, gao l. quercetin reversed lipopolysaccharide-induced inhibition of osteoblast differentiation through the mitogen-activated protein kinase pathway in mc3t3-e1 cells. mol med rep. 2014; 10(6): 3320–6. 14. conti bj, búfalo mc, golim mda, bankova v, sforcin jm. cinnamic acid is partially involved in propolis immunomodulatory action on human monocytes. evidence-based complement altern med. 2013; 2013: 1–7. 15. kresnoadi u, hadisoesanto y, prabowo h. effect of mangosteen peel extract combined with demineralized freezed-dried bovine bone xenograft on osteoblast and osteoclast formation in post tooth extraction socket. dent j (majalah kedokt gigi). 2016; 49(1): 43–8. 16. kumar p, fathima g, vinitha b. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(5): 125–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p126-132 vol 38 no 3 2005 103 identifikasi epitop dari streptococcus mutans terhadap sekretori imunoglobulin a saliva (the identification of streptococcus mutans epitopes to secretory immunoglobulin a saliva) anita yuliati bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract s. mutans is one of the etiology agent of dental caries, these bacteria have a surface protein of about 185 kda named ag i/ii. the secretory of siga saliva to ag i/ii of s.mutans has shown to be able to prevent colonization in human oral cavity. peptides derived from the 824 to 853 residues of the p region of antigen i/ii s. mutans related to the pathogenesis of dental caries. the aim of this study was to identify the overlapping sequence of amino acids (epitope) derived from the 624 to 853 residues of p of antigen i/ii s. mutans to siga saliva on caries and caries-free subject in a observational cross sectional study. the p region of antigen i/ii s.mutans was cut into 22 peptides of 9 mer sequences with an overlapping of 8 mer and an offset of 1 mer, synthesized on polyethylene pins and tested for the reactivity with an elisa indirect method to siga saliva on caries and caries-free subject. the results of this study showed that amino acid sequences with tppvkp (832–837) and taptkpty (838–845) were reactive to siga saliva on caries and caries-free subject. the conclusion of this study was that the overlapping common sequence of amino acid (epitopes) corresponding to tppvkp (832–837) and taptkpty (838–845) was identified as caries marker epitopes in human. key words: siga saliva, caries and caries-free subject, caries marker epitopes, antigen i/ii s.mutans korespondensi (correspondence): anita yuliati, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan karies gigi pada manusia merupakan penyakit infeksi menyerang jaringan keras rongga mulut yaitu gigi, yang melibatkan bakteri. streptococcus mutans (s. mutans) adalah agen etiologi sebagai penyebab utama. secara umum bakteri s. mutans dan sterptoccocus sobrinus (s. sobrinus) sering diisolasi dari rongga mulut manusia.1 s. mutans telah disepakati sebagai agen utama etiologi karies gigi pada manusia dan binatang coba, karena bakteri ini ditemukan pada populasi yang mempunyai prevalensi karies tinggi, rendah dan paling rendah.2 di antara serotipe s. mutans yang ada, serotipe c predominan ditemukan pada plak dan saliva. bakteri ini secara struktural dan antigenetikal mengekpresikan protein permukaan yang disebut antigen i/ii, b, sr dan pac yang mempunyai berat molekul 185 kda. antigen ini oleh para peneliti ditetapkan berperan dalam patogenesis karies gigi, dan efektif sebagai vaksin dalam pencegahan karies gigi.3,4 antigen i/ii s. mutans ini mempunyai sifat adesif, pada saat bakteri tersebut melekat pada komponen inangnya selama berkolonisasi dan infeksi sehingga menjadi fokus sejumlah peneliti.1 antigen protein permukaan ini berpengaruh dalam perlekatan s. mutans dengan acquired pellicles pada permukaan gigi.5 antibodi dalam sistem imun lokal maupun sistemik terhadap s. mutans ikut berperan dalam proteksi terhadap karies gigi. sistem imun saliva lokal diperankan oleh sekretori iga (siga). sekretori iga (siga) saliva dapat berikatan secara spesifik dengan adesin atau epitop dari bagian antigen i/ii s. mutans, sehingga adesin ini tidak dapat berikatan lagi dengan pelikel saliva dan s. mutans tidak akan berkolonisasi pada permukaan gigi.4 urutan lengkap nukleotida dari gen protein antigen i/ ii s. mutans serotipe c telah berhasil ditentukan. gen ini terdiri dari 4,695 bp, mengkode protein dengan berat molekul 170.773 da. sekuen nukleotida gen s. mutans berperan dalam identifikasi terpenting bagian fungsional dan antigenisitas epitop molekul antigen i/ii. 6,7 karakteristik molekul gen dari antigen i/ii s. mutans serotipe c yang terlibat dalam karies gigi berada pada residu asam amino 824–853. residu asam amino tersebut masih mengandung banyak epitop yang antigenik.8 dalam urutan asam amino yang begitu panjang ada satu atau dua (sikuen) epitop dengan sikuen asam amino tertentu yang paling dominan dalam proses karies gigi. metode mutakhir yang digunakan untuk mengidentifikasi suatu epitop dari protein yang telah diketahui urutan asam amino secara linier, adalah epitope scanning. metode ini telah banyak digunakan oleh peneliti di 104 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 103–107 mancanegara. metode epitope scanning ini membutuhkan pengetahuan tentang sikuen protein untuk mensintesis sejumlah peptida kecil, yang pada umumnya tumpangtindih dalam sikuen asam amino (overlapping common sequence). peptida kecil ini kemudian diuji dengan sistem asai sehingga epitop yang spesifik dapat ditentukan.9 oleh karena urutan asam amino pada regio p (824–853) antigen i/ii s. mutans telah diketahui, maka residu urutan asam amino tersebut dapat dibuat secara sintetik dan dapat mengidentifikasi epitop dari antigen tersebut terhadap respons imun saliva lokal siga dengan metode epitope scanning. permasalahan penelitian ini adalah terletak pada urutan residu asam amino atau overlapping common sequence asam amino (epitop) manakah dari regio p (824–853) antigen i/ii s. mutans yang reaktif terhadap siga saliva pada subyek karies dan bebas karies. tujuan dari penelitian ini adalah untuk mengidentifikasi urutan residu asam amino atau overlapping common sequence asam amino (epitop) yang reaktif terhadap siga saliva pada subyek karies dan bebas karies. manfaat penelitian ini adalah dengan teridentifikasinya epitop terhadap siga saliva dapat digunakan sebagai petanda (marker) untuk sarana diagnostik karies gigi. bahan dan metode jenis penelitian ini adalah observasional. penelitian dilakukan di laboratorium ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya dan laboratorium a. 470 institute of postgraduate studies and reseacrh (ipsp) university malaya kuala lumpur. bahan yang digunakan untuk penelitian dibuat terlebih dahulu dalam bentuk kit dengan cara, residu asam amino pada regio p (824–853) antigen i/ii s. mutans, dimasukkan dalam komputer dengan software antibody epitope scanning kit dengan program gnet, dipotong menjadi 9 mer dengan tumpang-tindih 8 dan offset 1. hasil dari pemotongan residu asam amino dibuat secara sintetik dalam bentuk potongan peptida. kemudian potongan peptida tersebut dikonjugasikan pada permukaan pin yang terbuat dari polyethylene. prosedur tersebut merupakan metode pembuatan epitope scanning kit yang dikerjakan oleh chiron technologies australia. sodium dodecyl sulphate (merck), 2-mercaptoetethanol (merck), methanol (bdh), fosfat bufer salin, konjugat anti human iga berlabel hrp, abts (sigma, a-1888), hidrogen peroksidase (merck). alat yang digunakan dalam penelitian, sonication bath, waterbath, shaker, lempeng micro-elisa dengan dasar datar (maxisorp, nuc), mikro pipet, micro-elisa reader (dynex technologies). sampel penelitian subyek karies dan bebas karies diambil dari mahasiswa fakultas kedokteran gigi universitas airlangga dengan kriteria penerimaan subyek karies adalah umur 17–25 tahun, lahir di surabaya, dmfs subyek karies 8–12, keadaan umum sehat, detro-malay, etnik jawa, pola makan sama, kesehatan rongga mulut baik. kriteria penerimaan subyek bebas karies sama dengan subyek karies, hanya dmfs subyek karies 0 atau 1.10 kriteria penolakan subyek karies dan bebas karies adalah; subyek penelitian sedang menjalani pengobatan dengan kortikosteroid atau imunosupresif yang lain dalam satu bulan terakhir, malnutri berat, penyakit sistemik yang dapat mengganggu sistem imunologis humoral dan mukosal, kesehatan rongga mulut jelek. jumlah sampel untuk subyek karies dan bebas karies masing-masing 15 orang. pengambilan sampel saliva pada subyek karies dan bebas karies dilakukan dengan cara, subyek diinstruksikan untuk menggosok gigi dan makan pagi, selanjutnya tidak boleh makan dan minum selama 2 jam. subyek disuruh meludah pada wadah yang steril sebanyak 5 ml. pelaksanaan pengambilan sampel dilakukan pada pagi hari antara jam 9.00–10.00. saliva yang diperoleh disentrifus dengan kecepatan 12.000 γ selama 10 menit pada suhu 4° c. supernatan diambil, dan dibagi dalam beberapa aliquot dan disimpan dalam –20° c sampai dilakukan pengujian. pungujian sampel dari subyek bebas karies dan karies dilakukan sebagai berikut, kit yang telah dipesan dimasukkan dalam larutan pre-coat buffer sebanyak 30 ml dalam kotak kecil dan digoyang di atas shaker selama 30 menit. pin dicuci sebanyak 3 kali dengan 0,01m pbs. kemudian ke dalam larutan pre-coat buffer tersebut dimasukkan sampel saliva dengan pengenceran 1:10, digoyangkan di atas shaker selama 30 menit. pin dicuci kembali sebanyak 3 kali dengan 0,01m pbs. tahap berikutnya, dimasukkan konjugat goat anti-human iga dalam dengan pengenceran 1 : 3.000 dalam 30 ml pelarut konjugat, dan diinkubasi di atas shaker selama 30 menit. pin dicuci kembali seperti cara di atas. pada sumuran lempeng micro elisa dimasukkan 100 μl larutan substrat yang telah ditambahkan h2o2 30%, kemudian pin dimasukkan dalam sumuran dan diinkubasi di atas shaker selama 20 menit sampai terjadi perubahan warna hijau. bila sudah terjadi perubahan warna pin diambil, adanya perubahan warna dibaca dengan micro elisa reader pada λ 405 nm. peptida yang reaktif dengan sampel siga subyek bebas karies dan karies dinyatakan dengan absorben. nilai absorben menandakan kereaktifan peptida yang diuji terhadap siga. pin yang telah digunakan untuk pengujian tidak boleh langsung digunakan lagi, apabila akan digunakan lagi untuk pengujian berikutnya maka harus dilakukan pembersihan atau distruption dan sonication (pencucian) terlebih dahulu. penentuan interpretasi hasil elisa menggunakan cara plot algorithm, yaitu rerata dari setengah absorben terendah ditambah tiga kali standar deviasi.11 nilai tersebut sebagai ambang atas nilai rujukan (cut-off value). nilai absorben di atas nilai rujukan dinyatakan signifikan.12,13 105yuliati: identifikasi epitop dari streptococcus mutans hasil hasil pemotongan residu asam amino (824–853) antigen i/ii s. mutans dengan program komputer gnet diperoleh dua puluh dua peptida dengan sikuen asam amino yang saling tumpang-tindih, seperti yang terlihat pada tabel 1. selanjutnya, ke dua puluh dua peptida tersebut yang telah dikonjugasikan pada permukaan pin diuji reaktivitasnya terhadap siga saliva subyek karies dan bebas karies. hasil reaktivitas setiap peptida ditentukan nilai rujukannya. peptida yang mempunyai rerata dari setengah absorben terendah ditambah tiga kali standar deviasi dinyatakan di atas nilai rujukan. hasil nilai rujukan dari setiap sampel subyek karies dan bebas karies terhadap kedua puluh dua peptida yang diuji dapat dilihat pada tabel 2. penentuan epitop dilakukan dengan cara, urutan asam amino setiap peptida dijabarkan dan dikelompokkan dengan nomer peptida yang berdekatan. pengelompokkan berdasarkan pada setiap overlapping common sequence asam amino (epitop) minimal 6 mer dengan rentangan paling banyak offset 4. penentuan cara epitop ini mengacu pada peneliti sebelumnya.14 presentase atau frekuensi dari setiap kelompok sampel yang diuji dilakukan uji statistik uji z dengan α = 0,05 dan z tabel = 1,96. diperoleh perbedaan yang bermakna antara siga saliva subyek bebas karies dan karies terhadap epitop urutan asam amino tppvkpt (832–837) dan taptkpty (838–845). pembahasan walaupun diketahui bahwa suatu molekul besar merupakan imunogen, tetapi hanya bagian tertentu dari molekul saja yang terlibat dalam ikatan dengan antibodi yang ditimbulkan. bagian tersebut selain menentukan spesifitas reaksi antigen-antibodi juga sebagai penentu timbulnya respons imun. bagian ini dapat diikat dengan tabel 1. dua puluh dua urutan asam amino antigen i/ii s. mutans regio p (824–853) setelah dipotong dengan program komputer gnet no. nomer peptida urutan asam amino no. nomer peptida urutan asam amino 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. peptida 1 peptida 2 peptida 3 peptida 4 peptida 5 peptida 6 peptida 7 peptida 8 peptida 9 peptida 10 peptide 11 pkvtkekpt (824−832) kvtkekptp (825−833) vtkekptpp (826−834) tkekptppv (827−835) kekptppvk (828−836) ekptppvkp (829−837) kptppvkpt (830−838) ptppvkpta (831−839) tppvkptap (832−840) ppvkptapt (833−841) pvkptaptk (834−842) 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22 peptida 12 peptida 13 peptida 14 peptida 15 peptida 16 peptida 17 peptida 18 peptida 19 peptida 20 peptida 21 peptide 22 vkptaptkp (835−843) kptaptkpt (836−844) ptaptkpty (837−845) taptkptye (838−846) aptkptyet (839−847) ptaptkpte (840−848) taptkptek (841−849) aptkptekp (842−850) ptkptekpl (843−851) tkptekplk (844−852) kptekplkp (845−853) tabel 2. reaktivitas ke dua puluh dua peptida yang memberikan absorben di atas cut-off value terhadap siga saliva subyek karies dan bebas karies subyek bebas karies subyek karies no. sampel cut-off value nomer peptida di atas cut-off value no. sampel cut-off value nomer peptida di atas cut-off value 1. 0,093 8,10 1. 0,129 2. 0,192 20 2. 0,115 3,7,14,15 3. 0,182 2,3 3. 0,120 3,10,14 4. 0,067 2,7,10 4. 0,109 3,6,7,8,9,14,15 5. 0,20 2,9,15,21 5. 0,111 3,7,14,15 6. 0,185 3 6. 0,118 7,10,13,14 7. 0,073 6,7,8,9,14 7. 0,114 3,8,9,15 8. 0,063 14 8. 0,117 7,9,20,21 9. 0,183 20 9. 0,091 2,5,7,9,10,11,13,15 10. 0,224 9,21 10. 0,129 7,9,14,20,21 11. 0,087 7,9,11,12,13,20,21 11. 0,154 14,20,21,22 12. 0,187 20 12. 0,195 7,9,10,20,21,22 13. 0,090 7,9,12,13,14,20 13. 0,10 7,9,10,11,12,14 14. 0,063 3,6.7,8,9,10 14. 0,204 7,9,20,21,22 15. 0,062 10,14 15. 0,160 14,20,21,22 106 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 103–107 spesifik oleh reseptor pada limfosit. bagian dari molekul yang terdapat pada permukaan ini disebut determinan antigenik atau epitop.15,16 epitop merupakan bagian dari molekul yang secara spesifik dikenali oleh paratop atau binding sites dari molekul antibodi. pada antigen protein, epitop ditentukan oleh beberapa residu asam amino yang akan dikenali oleh antibodi yang disebut kontak residu. kontak residu harus terbuka dan bebas tanpa ditutupi oleh rangkaian asam amino lainnya atau lipatan asam amino.17 pada penelitian ini, prasyarat tersebut telah terpenuhi karena menggunakan protein yang telah diketahui struktur primernya untuk menentukan suatu epitop. konsep lain yang ditetapkan oleh beberapa peneliti menyebutkan bahwa suatu epitop membutuhkan dua sifat yaitu reaktivitas dan imunogenesitas. 18 hasil keseluruhan dari penelitian ini, seperti yang ditampilkan pada tabel 3 menunjukkan bahwa ada epitop yang hanya reaktif terhadap antibodi dalam saliva subyek karies, dan saliva subyek bebas karies, tetapi frekuensi antibodi antara kedua kelompok tersebut berbeda. pada perhitungan uji statistik proporsional didapatkan perbedaan yang bermakna dengan z hitung > z tabel. dengan demikian, ada 2 epitop yaitu: overlapping common sequence asam amino (epitop) tppvkpt (832–837) yang reaktif terhadap antibodi yang terdapat pada 78,6% saliva subyek karies dan hanya 40% saliva subyek bebas karies. overlapping common sequence asam amino (epitop) taptkpty (838–845) yang reaktif terhadap antibodi yang terdapat pada 71,4% saliva subyek karies dan hanya 26,6% saliva subyek bebas karies. kedua epitop tersebut disebut epitop marka karies. istilah epitop marka karies tidak pernah ditemukan dalam literatur. peneliti sendiri yang memperkenalkan istilah epitop marka karies dengan alasan, epitop ini hanya reaktif terhadap antibodi dalam hal ini siga saliva yang terdapat pada sebagian besar sampel subyek karies dan sebagain kecil pada subyek bebas karies. kemungkinan keadaan tersebut disebabkan oleh karena, antibodi dalam saliva subyek bebas karies maupun subyek karies mempuyai kadar siga yang sama tinggi, sehingga kedua kelompok tersebut derajat kereaktivannya sama terhadap 22 peptida yang diuji. hal ini tidak sesuai dengan suatu hipotesis yang menyatakan bahwa respons antibodi saliva meningkat terhadap bakteri kariogenik pada individu yang resisten karies, dan observasi ini tidak menambah atau membedakan secara kualitatif dari antibodi spesifik dalam subyek yang resisten karies dibandingkan dengan subyek yang peka karies. lebih lanjut dikatakan bahwa, korelasi antara karies positif dan level antibodi siga terhadap s. mutans masih menjadi perdebatan yang besar.19 antibodi dalam saliva pada subyek bebas karies masih mampu mengikat epitop dari antigen i/ii s. mutans, khususnya pada regio asam amino 824–853, sehingga s. mutans masih tetap berakumulasi pada permukaan gigi. perlu diperhatikan, saliva yang digunakan dalam penelitian ini adalah total saliva, yang dapat berasal dari saliva dan cairan ginggiva, meskipun dikatakan konsentrasi iga tertinggi di saliva. konjugat yang digunakan dalam penelitian ini adalah anti human iga bukan anti human siga, karena tidak ada perusahan yang memproduksi konjugat anti human siga yang berlebel enzim. keterangan perusahaan mengatakan kesukaran dalam pemurnian siga dan bila ada konjugat dalam bentuk siga mudah terjadi kerusakan karena sifatnya yang tidak stabil selama penyimpanan. oleh karena itu dalam penelitian ini digunakan siga bukan iga saliva, karena iga total dalam saliva 90–95% berbentuk siga,20 atau mokelul yang diproduksi dalam kelenjar ludah 98% berbentuk siga.21 penggunaan saliva secara keseluruhan pada penelitian ini lebih mencerminkan keadaan rongga mulut sebenarnya. tampaknya kedua epitop marka karies tppvkpt dan taptkpty tersebut dapat membentuk ikatan terhadap antibodi spesifik yang tidak protektif. epitop tersebut dapat berinteraksi dengan salah satu reseptor saliva yang berada dalam rongga mulut terutama pada permukaan gigi. dengan demikian s. mutans akan tetap adherence dan berkolonisasi di permukaan gigi. adanya bakteri yang adherence pada permukaan gigi terutama pada permukaan halus dan fisura gigi atau bagian gigi lainnya merupakan proses awal karies gigi. residu asam amino 832–845 dari antigen i/ii s. mutans merupakan daerah fungsional untuk perlekatan dengan salah satu komponen iga sekretori. hasil ini sesuai dengan yang dilaporkan oleh peneliti sebelumnya, residu asam amino 39–864 dari molekul pac berperan penting dalam perlekatan protein terhadap komponen saliva. lebih lanjut dikatakan pemotongan yang pendek pada molekul pac yaitu fragmen pac-5, pada residu asam amino 828–1000 (regio p) dari s. mutans yang diuji dengan sandwich elisa memperlihatkan bahwa fragmen tersebut melekat pada komponen saliva oleh karena pada regio tersebut juga mempunyai daerah fungsional untuk perlekatan saliva.22 bila dilihat dari segi susunan atau urutan residu asam aminonya, kedua epitop yang terdeteksi merupakan epitop continous epitope dengan rentangan pendek kurang dari 10 residu asam amino dari sikuen yang sesuai dengan fragmen peptida dari protein yang secara antigenik berhubungan dengan protein induknya. continous epitope mungkin hanya merupakan salah satu bagian dari suatu epitop discontinous yang lebih luas.23,24 bila tidak ada penjelasan yang lebih lanjut, istilah epitop biasanya dipakai untuk epitop sel b, bagian antigen yang dikenal oleh reseptor sel b dan antibodi. tak ada suatu tolok ukur kimia fisik yang unik sebagai sifat yang khas dari epitop sel b. suatu sifat yang penting dari epitop adalah asesibilitas yang diperlukan untuk dikenali oleh reseptor sel b dan oleh antibodi, adalah hal yang amat penting bagi para peneliti untuk menentukan sikuen yang spesifik pada protein (epitop) yang dikenali oleh antibodi atau oleh sel t. 24 epitop marka karies yang terdeteksi pada penelitian ini terdiri dari enam dan tujuh mer urutan asam amino. hasil ini sesuai dengan pernyataan peneliti sebelumnya, oktamer adalah panjang minimal yang dibutuhkan untuk 107yuliati: identifikasi epitop dari streptococcus mutans mendeteksi seluruh epitop antibodi sel b linier.25 epitop marka karies hanya mempunyai arti sebagai marka adanya karies gigi dan tidak mempunyai arti dalam mencegahan karies, sebab antibodi terhadap epitop ini tidak protektif. epitop ini mungkin hanya mempunyai nilai sebagai salah satu sarana diagnostik dari karies, atau sebagai landasan untuk pembuatan kit uji serologis guna mendeteksi adanya karies gigi pada stadium dini. kesimpulan dari hasil penelitian ini adalah overlapping common sequence asam amino (epitop) tppvkpt (832–837) dan taptkpty (838–845) dari antigen i/ii s. mutans merupakan epitop marka karies terhadap siga saliva. daftar pustaka 1. chia js, you cm, hu cy, chiang bl, chen jy. human t-cell responses to the glucosyltransferases of streptococcus mutans. clin diagn lab immunol 2001; 8(2):441–5. 2. ethesis. review of literature. general bacteriological aspects of mutans streptococci. 2004. available:http//ethesis.helsinki.fi/ julkaisut/laa/hamma/vk/gronross/ch2.htm. accessed march 28, 2004. 3. brady lj, van tilburg mlja, alford ce, mc arthur wp. monoclonal antibody-mediated modulation of humoral immune response againt mucosally applied streptococcus mutans. infec immun 2000; 68:1796–05. 4. lehner t, russell mw, caldwell j. immunisation with a purifed protein from streptococcus mutans against dental caries in rhesus monkeys. lancet 1980; 10:995–6. 5. peterson fc, assey s, van der mei hc, busscher hj, scheice aa. funtional variation of the antigen i/ii surface protein in streptococcus mutans and streptococcus intermedius. infect immun 2002; 70(1):249–56. 6. kelly c, evans p, bergmeier l, lee sf, proguiske-fox a, harris ac, aitken a, bleiweis as, lehner t. sequence analysis of the cloned streptococcal cell surface antigen i/ii. febs letter 1989; 127–32. 7. van dolleweerd cj, chargelegue d, ma jkc. characterization of the conformational epitope of guy’13, a monoclonal antibody that prevents streptococcus mutans colonization in humans. infect immun 2003; 71(2):754–65. 8. okahashi n, sasakawa c, yoshikawa m, hamada t, koga t. mocelucar characterization of a surface protein antigen gene from serotype c streptococcus mutans, implicated in dental caries. mol microbiol 1989; 3:673–8. 9. worthington j, morgan k. epitope mapping using synthetic peptides. peptide antigen. a pratical approach. oxford, new york, tokyo: oxford university press; 1994. p. 181–217. 10. perrone m, gfell le, fontana m, gregory rl. antigenic characterization of fimbria preparations from streptococcus mutans isolates from caries-free and caries-susceptible subjects. clin diagn lab immunol 1997; 4:291–6. 11. stren ps. predicting antigenic sites on proteins.tibtech 1991; 9:163–9. 12. lachumanan r, devi s, cheong ym, rodda sj, pang t. epitope mapping of the sta58 major outer membrane protein of rickettsia tsutsugamushi. infect immun 1993; 61:4527–31. 13. panchanathan v, naidu br, devi s, dipasquale a, mason t, pang t. immunogenic epitopes of salmonella typhi groel heat schock protein reactive with both monoclonal antibody and patient sera. immunology letter 1998; 62:105–9. 14. tam jp. immunization with peptide-carrier complexes: traditional and multiple-antigen peptide systems. peptide antigen. a practical approach. oxford, new york: wisdom gb irl preess; 1994. p. 84–115. 15. goer j. immunochemical techniques laboratory manual. san diego, new york, boston, london, sydney, tokyo, toronto: academic press. harcourt brace jovanovich, publisher; 1993. p. 126–33. 16. baratawidjaya kg. imunologi dasar. edisi ke-4. jakarta: fakultas kedokteran universitas indonsesia; 2000. p. 22–32. 17. sosroseno w. pedoman kuliah. dasar-dasar imunologi untuk kedokteran gigi. yogyakarta: universitas gajah mada; 1993. h. 3–5. 18. van regenmortel mhv. the concept and operational definition of protein epitopes. phil trans r scot lond 1989; 323:451–66. 19. hocini h, iscaki s, bouvet jp, pillot j. unexpectedly high level of some presumably protective secretory immunoglobulin a antibodies to dental plaque bacteria in salivas of both caries-resistent and cariessusceptible subjects. infect immun 1993; 61:3597–604. 20. challacombe sj, shirlaw pj. immunology of diseases of the oral cavity. handbook of mucosal immunology. san diego, new york, boston, london, sydney, tokyo, toronto: academic press inc; 1994. p. 607–24. 21. amerongen an. ludah dan kelenjar ludah arti bagi kesehatan gigi. yogyakarta: gajah mada university press; 1992; h. 43–124. 22. nakai m, okahashi n, ohta h, koga t. saliva-binding region of streptococcus mutans surface protein antigen. infect immun 1993; 61:4344–9. 23. lane dp, stephen cw. epitope mapping using bacteriophage peptide libaries. current opinion in immunology 1993; 5:268–71. 24. pellequer jl, westhof e,van regenmortel hv. epitope predictions from the primary structure of proteins. peptide antigen. a pratical approach. oxford new york tokyo: wisdom gbirl press oxford university press; 1994. p. 7–25. 25. tribbick g. antibody epitope mapping with multipintm peptides. immunology method manual. clayton, victoria, australia: chiron mimotopes pty ltd; 1997. p. 818–25. tabel 3. frekuensi, persentasi dan nilai z dari epitop yang ditemukan dengan siga saliva terhadap subyek bebas karies dan karies saliva subyek penelitian bebas karies karies epitop fre % n fre % n kemaknaan vtkekptp 5 33,3 15 6 42,8 15 zhit = 0,53 < 1,98 tppvkpt 6 40 15 11 78,6 15 zhit = 2,11 > 1,98 * pvkptap 10 66,6 15 8 57,1 15 zhit = 1,41 < 1,98 taptkpty 4 26,6 15 10 78,4 15 zhit = 2,41 > 1,98 * tyetekpl 7 46,6 15 6 42,8 15 zhit = 0,21 < 1,98 keterangan: fre = frekwensi; n = jumlah sampel tiap kelompok; * = berbeda bermakna << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 121 volume 47, number 3, september 2014 research report pengaruh chitosan belangkas (tachypleus gigas) nanopartikel terhadap celah antara berbagai jenis semen ionomer kaca dengan dentin (the effect of horseshoe crab (tachypleus gigas) dderived nanoparticle chitosan on interface between various glass ionomers and dentin) henny sutrisman,1 trimurni abidin1 dan harry agusnar2 1 departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas sumatera utara 2 departemen ilmu kimia, fakultas matematika dan ilmu pengetahuan alam universitas sumatera utara medan indonesia abstract background: the development of dental material restoration is regarded to be relevant to obtain a better bonding between dental structure and restorative materials. glass ionomer cement (gic) is a bioactive material. resin-modified gic (rmgic) is an alternative to the conventional glass ionomer. nowadays with nano technology, this material is available in nano particle glass ionomer form in order to enhance the bond strength between tooth structure and restoration. the use of the natural product in dentistry such as chitosan has increased widely. chitosan is one of the natural materials that used to improve the bioactivity of the glass ionomer. studies showed that addition of chitosan high molecule to gic can increase mechanical performance and capability and also as a catalyst to release fluoride ions. purpose: this study was aimed to examine the effect of the addition of high molecular nanoparticle chitosan derived from horseshoe crab (tachypleus gigas) on interface of rmgic, nano rmgic and the dentin surface. methods: nano particle chitosan was added to the restorative materials and then applied to the class i cavity of premolar and then the tooth was sectioned with diamond disc. specimens were prepared for sem examination. results: the result showed that the addition of chitosan increases adhesion between restoration and dentin structure. conclusion: the addition of nanoparticle chitosan with a high molecular weight of 0.015% into rmgic and nanoparticle rmgic can improve the adhesion of restorative material to dentin structure. key words: chitosan high molecule, glass ionomer cements, adhesive interface, scanning electron microscopy abstrak latar belakang: perkembangan restorasi bahan gigi untuk mendapatkan ikatan yang lebih baik antara struktur gigi dan bahan restoratif. semen ionomer kaca (sik) adalah bahan bioaktif. semen ionomer kaca modifikasi resin (sikmr) adalah sebuah alternatif untuk ionomer kaca konvensional. saat ini dengan teknologi nano, bahan ini tersedia dalam bentuk partikel nano ionomer kaca untuk meningkatkan kekuatan ikatan antara struktur gigi dan restorasi. penggunaan produk alami dalam kedokteran gigi seperti kitosan telah meningkat banyak. kitosan merupakan salah satu bahan alami yang digunakan untuk meningkatkan bioaktivitas dari ionomer kaca. studi menunjukkan bahwa penambahan molekul tinggi kitosan untuk gic dapat meningkatkan kinerja mekanik dan kemampuan dan juga sebagai katalis untuk melepaskan ion fluoride. tujuan: penelitian ini bertujuan untuk menguji pengaruh penambahan kitosan nanopartikel molekul yang berasal dari kepiting tapal kuda (tachypleus gigas) terhadap permukaan antara rmgic, nano rmgic dan permukaan dentin. metode: nano partikel kitosan ditambahkan pada bahan restoratif dan kemudian diterapkan pada kavitas kelas i premolar dan kemudian gigi tersebut dipotong dengan disc berlian. sampel disiapkan untuk pemeriksaan sem. hasil: hasil penelitian menunjukkan bahwa penambahan kitosan meningkatkan adhesi antara restorasi dan struktur dentin. simpulan: 122 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 121–125 penambahan nanopartikel kitosan dengan berat molekul tinggi 0,015% pada sikmr dan sikmr nano dapat meningkatkan adhesi bahan restorasi struktur dentin. kata kunci: molekul kitosan, semen ionomer kaca, kekuatan adhesi, scanning electron microscopy korespondensi (correspondence): henny sutrisman, departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas sumatera utara. jl. alumni no. 2 kampus usu, medan 20155, indonesia. e-mail: hunny_soe@yahoo.com pendahuluan perkembangan bahan dan teknik restorasi memiliki peranan penting untuk mencapai ikatan yang baik antara bahan restorasi dengan struktur gigi. penelitian-penelitian selama beberapa periode memungkinkan berkembangnya beragam teknik dan bahan restorasi sehingga dapat meningkatkan ikatan terhadap permukaan gigi dan meminimalkan celah antara gigi dan restorasi.1-3 di antara material kedokteran gigi yang dikembangkan akhir-akhir ini adalah semen ionomer kaca (sik). semen ionomer kaca modifikasi resin (sikmr) dan semen ionomer kaca nanopartikel (sikmrn) digunakan sebagai alternatif untuk memperbaiki sifat-sifat fisis sik seperti estetis, translusensi, dan kekuatan yang lebih baik.4-6 salah satu masalah utama dalam kedokteran gigi restoratif adalah sulitnya memperoleh penyatuan yang baik antara bahan restorasi dengan permukaan gigi.1 kebocoran mikro merupakan salah satu masalah yang berperan dalam kegagalan dari bahan restorasi yang digunakan terhadap perembesan bakteri, ion molekul, bahan khemis dan cairan. kebocoran mikro telah dihubungkan pada berbagai kondisi termasuk respon pulpa, sensitivitas pasca perawatan, karies sekunder dan kerusakan beberapa bahan restorasi yang memicu kegagalan dari restorasi tersebut.7 ikatan sik ke dentin terdiri dari ikatan khemis dan mekanis. kekuatan ikatan pada dentin tidak sebaik kekuatan ikatan pada enamel. walaupun terjadi interaksi khemis antara sik dengan dentin, nilai kekuatan ikatan bahan ini masih lebih rendah dibandingkan dengan kekuatan ikatan sistem adhesif terhadap dentin dan enamel.2,3,7 pada kavitaskavitas dalam, perlu diperhatikan ikatan yang terjadi antara bahan restorasi dengan dentin. dentin merupakan jaringan vital yang terhubung langsung ke pulpa melalui tubulus dentin yang berisi cairan sehingga adanya pergerakan cairan pada tubulus dentin dapat mengganggu perlekatan antara bahan restorasi dengan struktur gigi. selain itu, diameter tubulus dentin yang mengarah ke pulpa semakin besar sehingga dibutuhkan suatu bahan restorasi yang memiliki biokompatibilitas baik dan mampu melindungi daerah pulpodentinokompleks serta memiliki perlekatan yang baik terhadap dentin untuk menghindari terjadinya kebocoran mikro.3,8 produk-produk alam yang dapat dihubungkan sebagai biomaterial di bidang kedokteran gigi saat ini semakin berkembang pesat penggunaannya, salah satunya adalah pemakaian kitosan molekul tinggi. kitosan merupakan biomaterial yang terus dikembangkan karena memiliki berbagai manfaat medikal dan terbukti aman untuk manusia. beberapa penelitian menunjukkan penggunaan kitosan molekul tinggi mampu menstimulasi pembentukan dentin reparatif, meningkatkan performa mekanis dan pelepasan ion fluor.9,10 kitosan dalam bentuk terprotonisasi menunjukkan kerapatan muatan yang tinggi dan bersifat sebagai polielektrolit kationik dan sangat efektif berinteraksi dengan biomolekul bermuatan negatif dan biomolekul permukaan. penambahan kitosan ke dalam sik komersil dapat meningkatkan performa mekanis dan mampu sebagai katalisator dalam pelepasan ion fluor. efek ini dijelaskan berdasarkan penemuan jaringan polimerik yang berikatan kuat di sekitar pengisi anorganik, sehingga terjadi ikatan yang lebih baik.10,11 tujuan penelitian ini adalah meneliti efek penambahan kitosan molekul tinggi nanopartikel yang berasal dari kepiting tapal kuda (tachypleus gigas) pada varian semen ionomer kaca terhadap daya adhesif material tersebut pada permukaan dentin. bahan dan metode penelitian ini merupakan penelitian eksperimental laboratorium dengan menggunakan gigi premolar yang sudah diekstraksi untuk kebutuhan ortodonti yang telah disetujui oleh komite etik fakultas kedokteran universitas sumatera utara dengan no. 362. beberapa penelitian menyiapkan kitosan nanopartikel dengan melarutkan kitosan dalam larutan asam lemah ditambahkan larutan yang bersifat basa, seperti amoniak, dan distirer dengan kecepatan 200 rpm kemudian ditempatkan dalam ultrasonic bath untuk memecah partikel-partikel gel kitosan menjadi lebih kecil.12 satu gram kitosan (tachypleus gigas) yang diperoleh dari laboratorium fakultas matematika dan ilmu pengetahuan alam universitas sumatera utara dilarutkan dalam 50 ml larutan asam lemah (asam asetat 1%) dan diaduk pada kecepatan 200 rpm. larutan kitosan ditetesi dengan larutan amoniak dan diaduk kembali. larutan yang telah membentuk pasta tersebut dimasukkan ke dalam ultrasonic bath untuk memecah kan partikel kitosan menjadi nanopartikel. selanjutnya disaring dan residunya dicuci dengan aquadest untuk menghilangkan bau amoniak. 123sutrisman, et al.: pengaruh chitosan belangkas (tachypleus gigas) nanopartikel sampel dibuat dari gigi premolar rahang bawah yang tidak karies dan sudah diekstraksi untuk kebutuhan ortodonti, ditandai dari cemento-enamel junction kearah koronal untuk menentukan kedalaman preparasi kavitas. preparasi kavitas klas i dilakukan pada masing-masing gigi sampel dipersiapkan dengan mengaduk pasta sikmr dan sikmrn dengan menambahkan kitosan nano masingmasing sebanyak 0,015% b/v. penambahan berat kitosan nanopartikel pada penelitian ini didasarkan penelitian sebelumnya yang menunjukkan penambahan berat kitosan nano sebanyak 0,015% b/v pada sikmr dan sikmrn dapat meningkatkan proliferasi sel. 13 perlakuan pada sampel yaitu sikmr dan sikmrn yang telah ditambahkan kitosan nano diaplikasikan pada kavitas gigi premolar dan dilakukan penyinaran. gigi dibagi dua dengan menggunakan bur cakram dan semprotan air pada arah bukal dan lingual kemudian akar gigi dipotong. bagian mahkota gigi yang sudah dibagi dua ditanam dalam mould spuit yang berisi dental stone dengan diameter 5 mm dan tinggi 3 mm. setelah cetakan mengeras, cetakan dikeluarkan dari mould (gambar 1a). pemeriksaan sem bertujuan untuk mendapatkan gambaran mikrostruktur sampel dan permukaan antara bahan restorasi dengan dentin dapat dianalisis secara kualitatif. setelah pengaplikasian bahan uji, sampel dilapisi (coating) dengan cairan emas (gambar 1b). sampel diletakkan pada chamber yang vakum dan berada tepat ditengah-tengah chamber. kemudian alat dihidupkan dengan daya 20 kv. sampel digeser secara perlahan untuk mendapatkan daerah yang akan difoto pada layar sem. brightness, kontras dan fokus disesuaikan sampai didapatkan gambaran yang baik. pengambilan foto dilakukan dengan pembesaran 500, 1000 dan 2000. hasil ukuran butir partikel kaca dengan kerapatan antar partikel lebih kecil terlihat pada sikmrn sedangkan pada sikmr terlihat butiran partikel kaca dan kerapatan antara partikel kaca yang lebih besar. pada sikmr dan sikmrn yang ditambahkan kitosan nanopartikel terlihat kerapatan gambar 1. (a) cetakan yang telah dikeluarkan dari mould; (b) sampel yang telah dicoating. gambar 2. gambaran sem permukaan mikrostruktur. (a) sikmr tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (b) sikmrn tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (c) sikmr yang ditambahkan kitosan nanopartikel (pembesaran 2000x); (d) sikmrn yang ditambahkan kitosan nanopartikel (pembesaran 500x). a b c d 5 gambar 1. (a) cetakan yang telah dikeluarkan dari mould; (b) sampel yang telah dicoating. hasil ukuran butir partikel kaca dengan kerapatan antar partikel lebih kecil terlihat pada sikmrn sedangkan pada sikmr terlihat butiran partikel kaca dan kerapatan antara partikel kaca yang lebih besar. pada sikmr dan sikmrn yang ditambahkan kitosan nanopartikel terlihat kerapatan antar partikel lebih kecil bila dibandingkan sikmr dan sikmrn yang tidak ditambahkan kitosan nanopartikel (gambar 2). (a) (b) (c) (d) gambar 2. gambaran sem permukaan mikrostruktur. (a) sikmr tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (b) sikmrn tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (c) sikmr yang ditambahkan kitosan nanopartikel (pembesaran 2000x); (d) sikmrn yang ditambahkan kitosan nanopartikel (pembesaran 500x). 5 gambar 1. (a) cetakan yang telah dikeluarkan dari mould; (b) sampel yang telah dicoating. hasil ukuran butir partikel kaca dengan kerapatan antar partikel lebih kecil terlihat pada sikmrn sedangkan pada sikmr terlihat butiran partikel kaca dan kerapatan antara partikel kaca yang lebih besar. pada sikmr dan sikmrn yang ditambahkan kitosan nanopartikel terlihat kerapatan antar partikel lebih kecil bila dibandingkan sikmr dan sikmrn yang tidak ditambahkan kitosan nanopartikel (gambar 2). (a) (b) (c) (d) gambar 2. gambaran sem permukaan mikrostruktur. (a) sikmr tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (b) sikmrn tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (c) sikmr yang ditambahkan kitosan nanopartikel (pembesaran 2000x); (d) sikmrn yang ditambahkan kitosan nanopartikel (pembesaran 500x). 5 gambar 1. (a) cetakan yang telah dikeluarkan dari mould; (b) sampel yang telah dicoating. hasil ukuran butir partikel kaca dengan kerapatan antar partikel lebih kecil terlihat pada sikmrn sedangkan pada sikmr terlihat butiran partikel kaca dan kerapatan antara partikel kaca yang lebih besar. pada sikmr dan sikmrn yang ditambahkan kitosan nanopartikel terlihat kerapatan antar partikel lebih kecil bila dibandingkan sikmr dan sikmrn yang tidak ditambahkan kitosan nanopartikel (gambar 2). (a) (b) (c) (d) gambar 2. gambaran sem permukaan mikrostruktur. (a) sikmr tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (b) sikmrn tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (c) sikmr yang ditambahkan kitosan nanopartikel (pembesaran 2000x); (d) sikmrn yang ditambahkan kitosan nanopartikel (pembesaran 500x). 5 gambar 1. (a) cetakan yang telah dikeluarkan dari mould; (b) sampel yang telah dicoating. hasil ukuran butir partikel kaca dengan kerapatan antar partikel lebih kecil terlihat pada sikmrn sedangkan pada sikmr terlihat butiran partikel kaca dan kerapatan antara partikel kaca yang lebih besar. pada sikmr dan sikmrn yang ditambahkan kitosan nanopartikel terlihat kerapatan antar partikel lebih kecil bila dibandingkan sikmr dan sikmrn yang tidak ditambahkan kitosan nanopartikel (gambar 2). (a) (b) (c) (d) gambar 2. gambaran sem permukaan mikrostruktur. (a) sikmr tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (b) sikmrn tanpa penambahan kitosan nanopartikel (pembesaran 2000x); (c) sikmr yang ditambahkan kitosan nanopartikel (pembesaran 2000x); (d) sikmrn yang ditambahkan kitosan nanopartikel (pembesaran 500x). 124 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 121–125 antar partikel lebih kecil bila dibandingkan sikmr dan sikmrn yang tidak ditambahkan kitosan nanopartikel (gambar 2). perlekatan sikmr dan sikmrn yang ditambahkan kitosan nanopartikel dengan dentin terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin. meskipun tidak terdapat celah antara dentin dengan sikmrn, tetapi butiran partikel pada sikmrn yang ditambahkan kitosan nanopartikel lebih kecil dibandingkan sikmrn tanpa penambahan kitosan nanopartikel. selain itu, kerapatan antar partikel lebih baik pada sikmrn yang ditambahkan kitosan nanopartikel (gambar 3). pembahasan permukaan kontak restorasi dengan dentin dapat mengindikasikan kemampuan beberapa bahan dalam mencegah perkembangan karies rekuren dan sensitifitas pasca perawatan sebagai akibat dari kebocoran mikro pada permukaan tersebut. penggunaan bahan restoratif adhesif yang memiliki kemampuan penutupan baik disertai pelepasan fluor dapat menurunkan dan mencegah terbentuknya celah pada daerah marginal. semen ionomer merupakan bahan potensial yang diletakkan pada daerah kritis untuk memperoleh adhesi dari interaksi khemis gambar 3. gambaran permukaan antara bahan restorasi dengan dentin (pembesaran 500x). (a) sikmr tanpa penambahan kitosan nanopartikel; (b) sikmrn tanpa penambahan kitosan nanopartikel; (c) sikmr yang ditambahkan kitosan nanopartikel; (d) sikmrn yang ditambahkan kitosan nanopartikel dengan dentin. dengan dentin. bahan restorasi semen ionomer kaca dapat memberikan penutupan yang optimal dan melindungi restorasi dari kebocoran marginal. kemampuan pelepasan fluor semen ionomer kaca dapat membantu mengendalikan perkembangan karies rekuren dan patologi pulpa yang dapat menggagalkan perawatan restoratif dalam waktu yang singkat.2,3 s i k m r d a n s i k m r n d i k e m b a n g k a n u n t u k meningkatkan sifat-sifat mekanikal yang lebih baik dibandingkan sik. kedua bahan ini memiliki estetis, translusensi, dan kekuatan yang lebih baik dibandingkan sik. sikmr dan sikmrn dipilih sebagai material uji pada penelitian ini karena jenis semen ini dapat berikatan secara khemis dengan jaringan keras gigi tanpa diikuti kontraksi. kondisi ini dapat mencegah terjadinya kebocoran marginal yang merupakan penyebab utama kegagalan restorasi karena memudahkan masuknya bakteri ke dalam pulpa.6,14,15 pada gambaran mikrostruktur permukaan sikmr dan sikmrn tampak ukuran partikel kaca terkecil terdapat pada sikmrn dengan kepadatan yang merata, dan pada sikmr tampak kepadatan merata dengan ukuran partikel kaca yang lebih besar. hal ini berkaitan dengan ukuran partikel nano yang dimiliki sikmrn sehingga penghantaran molekul yang terjadi pada jaringan lebih optimal dan kemampuan dalam skala yang cukup kecil a b c d 6 perlekatan sikmr dan sikmrn yang ditambahkan kitosan nanopartikel dengan dentin terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin. meskipun tidak terdapat celah antara dentin dengan sikmrn, tetapi butiran partikel pada sikmrn yang ditambahkan kitosan nanopartikel lebih kecil dibandingkan sikmrn tanpa penambahan kitosan nanopartikel. selain itu, kerapatan antar partikel lebih baik pada sikmrn yang ditambahkan kitosan nanopartikel (gambar 3). (a) (b) (c) (d) gambar 3. gambaran permukaan antara bahan restorasi dengan dentin (pembesaran 500x). (a) sikmr tanpa penambahan kitosan nanopartikel; (b) sikmrn tanpa penambahan kitosan nanopartikel; (c) sikmr yang ditambahkan kitosan nanopartikel; (d) sikmrn yang ditambahkan kitosan nanopartikel dengan dentin. pembahasan permukaan kontak restorasi dengan dentin dapat mengindikasikan kemampuan beberapa bahan dalam mencegah perkembangan karies rekuren dan sensitifitas pasca perawatan sebagai akibat dari kebocoran mikro pada permukaan tersebut. penggunaan bahan 6 perlekatan sikmr dan sikmrn yang ditambahkan kitosan nanopartikel dengan dentin terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin. meskipun tidak terdapat celah antara dentin dengan sikmrn, tetapi butiran partikel pada sikmrn yang ditambahkan kitosan nanopartikel lebih kecil dibandingkan sikmrn tanpa penambahan kitosan nanopartikel. selain itu, kerapatan antar partikel lebih baik pada sikmrn yang ditambahkan kitosan nanopartikel (gambar 3). (a) (b) (c) (d) gambar 3. gambaran permukaan antara bahan restorasi dengan dentin (pembesaran 500x). (a) sikmr tanpa penambahan kitosan nanopartikel; (b) sikmrn tanpa penambahan kitosan nanopartikel; (c) sikmr yang ditambahkan kitosan nanopartikel; (d) sikmrn yang ditambahkan kitosan nanopartikel dengan dentin. pembahasan permukaan kontak restorasi dengan dentin dapat mengindikasikan kemampuan beberapa bahan dalam mencegah perkembangan karies rekuren dan sensitifitas pasca perawatan sebagai akibat dari kebocoran mikro pada permukaan tersebut. penggunaan bahan 6 perlekatan sikmr dan sikmrn yang ditambahkan kitosan nanopartikel dengan dentin terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin. meskipun tidak terdapat celah antara dentin dengan sikmrn, tetapi butiran partikel pada sikmrn yang ditambahkan kitosan nanopartikel lebih kecil dibandingkan sikmrn tanpa penambahan kitosan nanopartikel. selain itu, kerapatan antar partikel lebih baik pada sikmrn yang ditambahkan kitosan nanopartikel (gambar 3). (a) (b) (c) (d) gambar 3. gambaran permukaan antara bahan restorasi dengan dentin (pembesaran 500x). (a) sikmr tanpa penambahan kitosan nanopartikel; (b) sikmrn tanpa penambahan kitosan nanopartikel; (c) sikmr yang ditambahkan kitosan nanopartikel; (d) sikmrn yang ditambahkan kitosan nanopartikel dengan dentin. pembahasan permukaan kontak restorasi dengan dentin dapat mengindikasikan kemampuan beberapa bahan dalam mencegah perkembangan karies rekuren dan sensitifitas pasca perawatan sebagai akibat dari kebocoran mikro pada permukaan tersebut. penggunaan bahan 6 perlekatan sikmr dan sikmrn yang ditambahkan kitosan nanopartikel dengan dentin terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin. meskipun tidak terdapat celah antara dentin dengan sikmrn, tetapi butiran partikel pada sikmrn yang ditambahkan kitosan nanopartikel lebih kecil dibandingkan sikmrn tanpa penambahan kitosan nanopartikel. selain itu, kerapatan antar partikel lebih baik pada sikmrn yang ditambahkan kitosan nanopartikel (gambar 3). (a) (b) (c) (d) gambar 3. gambaran permukaan antara bahan restorasi dengan dentin (pembesaran 500x). (a) sikmr tanpa penambahan kitosan nanopartikel; (b) sikmrn tanpa penambahan kitosan nanopartikel; (c) sikmr yang ditambahkan kitosan nanopartikel; (d) sikmrn yang ditambahkan kitosan nanopartikel dengan dentin. pembahasan permukaan kontak restorasi dengan dentin dapat mengindikasikan kemampuan beberapa bahan dalam mencegah perkembangan karies rekuren dan sensitifitas pasca perawatan sebagai akibat dari kebocoran mikro pada permukaan tersebut. penggunaan bahan 125sutrisman, et al.: pengaruh chitosan belangkas (tachypleus gigas) nanopartikel untuk berinteraksi dengan komponen intraselular termasuk dna. selain itu, pada prinsip rekayasa jaringan, ukuran partikel material dapat mempengaruhi efek biologi, yaitu makin kecil ukuran partikel, makin luas permukaannya, sehingga makin meningkat pula interaksi material dan jaringan sekitarnya.6 suatu restorasi harus mampu melindungi pulpodentinal kompleks untuk mencegah rangsangan tambahan bagi jaringan pulpa akibat prosedur operatif, toksisitas bahan restorasi serta penetrasi bakteri akibat terjadinya kebocoran mikro. proteksi pulpodentinal kompleks juga berguna untuk memulihkan vitalitas pulpa. oleh karena itu dbutuhkan suatu restorasi yang dapat berikatan baik dengan struktur gigi dan mampu memperbaiki kerusakan pada daerah pulpodentinokompleks.2,3,16,17 penggunaan produk-produk alam di bidang kedokteran gigi saat ini semakin berkembang pesat, maka dengan banyaknya belangkas (tachypleus gigas) di sumatera utara, memungkinkan untuk dikembangkan bahan baru yaitu kitosan dan derivatnya di bidang kedokteran gigi. ukuran partikel kitosan berskala nanometer akan meningkatkan luas permukaan sampai ratusan kali dibandingkan dengan partikel yang berukuran mikrometer, sehingga dapat meningkatkan efektifitas kitosan dalam mengikat gugus kimia lainnya.18 hal ini akan meningkatkan efisiensi proses fisika-kimia pada permukaan kitosan tersebut, karena memungkinkan interaksi pada permukaan yang lebih besar.18 butiran partikel kaca dengan ukuran kecil juga terlihat pada sikmr dan sikmrn yang ditambahkan kitosan belangkas dengan kerapatan antar butir partikel lebih padat. ukuran butir partikel yang semakin kecil akan menghasilkan perlekatan yang lebih baik. perlekatan antara dentin dengan sikmr dan sikmrn yang ditambahkan kitosan belangkas nanopartikel terlihat lebih baik dan tidak terdapat celah antara bahan restorasi dengan dentin apabila dibandingkan dengan sikmr dan sikmrn yang tidak ditambahkan kitosan belangkas nanopartikel. hal ini karena pada pasta sikmr dan sikmrn terdapat gugus fas (fluoro alumino silikat) sedangkan pada pasta pelarutnya terdapat asam poli akrilat (polyacrilic acid/ paa). di sisi lain, gugus kitosan mempunyai gugus amin yang mampu mengikat partikel hidroksil dan gugus karboksilat dari paa oleh ikatan hidrogen. ikatan yang dibentuk oleh kitosan dan paa di sekitar partikel anorganik dapat mengurangi tegangan pada permukaan antar komponen sik modifikasi resin, sehingga meningkatkan sifat mekanik bahan tersebut.10 hipotesa penggunaan partikel berukuran nano yang dianut sekarang adalah berupa penyebaran partikel pengisi yang lebih merata serta peningkatan luas daerah interfasial antara matriks dengan filler sehingga menghasilkan restorasi yang lebih fleksibel, menurunnya kekasaran permukaan restorasi serta perlekatan yang lebih baik.19 dapat disimpulkan bahwa penambahan kitosan molekul tinggi nanopartikel dengan berat 0,015% b/v pada varian semen ionomer kaca (sikmr dan sikmrn) mampu meningkatkan perlekatan antara material terhadap dentin. daftar pustaka 1. souza-gabriel ae, chimello-sousa dt, palma-dibb rg, pecora jd, corona sam. morphologic assessment of dental surface/ glass ionomer cement interface: influence of er: yag laser pretreatment. rsbo 2012; 9(4): 382-7. 2. mauro sj, sundfeld rh, bedran-russo akb, fraga briso alf. bond strength of resin-modified glass ionomer to dentin: the effect of dentin surface treatment. j minim interv 2009; 2(1): 45-53. 3. sikri vk. textbook of operative dentistry. second edition. india: cbs; 2008. p. 358. 4. lohbauer u. dental glass ionomer cements as permanent filling materials? properties, limitations and future trends. materials 2010; 3: 76-96. 5. ghavamnasiri m, mousavinasab m, mohtahsam m. a histopathologic study on pulp response to glass ionomer cements in human teeth. j dent 2005; 2(4): 135-41. 6. suprastiwi e. bioaktivitas semen ionomer kaca dalam menginduksi peningkatan kadar alp, dmp-1, dan pembentukan dentin reparatif (penelitian in vivo pada macaca nemestrina). disertasi. jakarta: fakultas kedokteran gigi universitas indonesia; 2011. 7. patel ag. microleakage in new resin-modified glass ionomer cements using new no-rinse conditioners: an in-vitro study. thesis. indiana: master of science in dentistry, indiana university school of dentistry; 2011. p. 5-9. 8. bouillaguet s. biological risks of resin-based materials to the dentinpulp complex. crobm 2004; 15(1): 47-60. 9. trimurni a, harry a, wandania f. efek dentinogenesis kitosan dan derivatnya terhadap inflamasi jaringan pulpa gigi reversible. laporan akhir penelitian riset pembinaan iptek kedokteran. medan: fakultas kedokteran gigi universitas sumatera utara; 2006. h. 16-8, 27-30, 39-41. 10. petri dfs, donega j, benassi am, bocangel ja. preliminary study on chitosan modified glass ionomer restoratives. dent mater 2007; 23(8): 1004-10. 11. sugita p, wukisari t, sjahriza a, wahyono a. kitosan sumber biomaterial masa depan. bogor: ipb press; 2009. h. 27, 125. 12. siregar m. pengaruh berat molekul kitosan nanopartikel untuk menurunkan kadar logam besi (fe) dan zat warna pada limbah industri tekstil jeans. tesis. medan: pascasarjana universitas sumatera utara; 2009. h. 514. 13. sutrisman h. efek penambahan kitosan molekul tinggi nanopartikel pada semen ionomer kaca nanopartikel terhadap viabilitas sel pulpa (in vitro).tesis. medan: fakultas kedokteran gigi universitas sumatera utara; 2013. 14. bonifacio cc, kleverlaan cj, raggio dp, werner a, rcr de carvalho, we van amerongen. physical-mechanical properties of glass ionomer cements indicated for atraumatic restorative treatment. australian dent j 2009; 54: 233-7. 15. croll tp, berg jh. nano-ionomer restorative cement: observations after 2 years of use. inside dentistry 2009; 5(1): 1-6. 16. goldberg m, smith aj. cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. crit rev oral biol med 2004; 15(1): 13-27. 17. ferracane jl, cooper pr, smith aj. can interaction of materials with the dentin–pulp complex contribute to dentin regeneration?. odontology 2010; 98(1): 2–14. 18. ningsih w. pengaruh viskositas larutan kitosan nanopartikel sebagai penyalut asam askorbat untuk menyerap asam lemak bebas (alb) dalam minyak goreng curah. tesis. medan: universitas sumatera utara; 2010. h. 1-21. 19. saunders sa. current practicality of nanotechnology in dentistry. part 1: focus on nanocomposite restoratives and biomimetics. clinical. cosmetic and investigational dentistry 2009; 1: 47–61. 87 volume 47, number 2, june 2014 aplikasi teori belajar sosial dalam penatalaksanaan rasa takut dan cemasan anak pada perawatan gigi (application of social learning theory in the management of children dental fear and anxiety) arlette suzy setiawan departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas padjadjaran bandung indonesia abstract background: dental anxiety is a prevalent problem faced by dentists till nowadays, especially in treating child patients. several methods in managing anxiety are available, but there is no single method can be applied extensively. purpose: this article was aimed to describe the application of social learning theory in preventing or reducing dental anxiety in children. literature review: anxiety and fear are psych feelings that is experienced by a person. a child developed these feelings by learning from their own past experienced or by information obtained from their environment. if fear and anxiety can be learned by a child, thus the opposite term which is preventing the feelings is assumed can be also offered to learned. this application actually being applied in the field of dentistry as modeling, but the exploration of how this process is due is often being ignored. conclusion: social learning theory provides an easy preventive approach and effective intervention that can be applied to children in 4-9 years old to reduce dental anxiety. key words: anxiety, children, dental treatment, social learning abstrak latar belakang: kecemasan pada perawatan gigi merupakan hal yang paling sering dijumpai dan merupakan masalah yang dihadapi oleh dokter gigi sampai saat ini, terutama pada pasien anak. berbagai metode penatalaksanaan kecemasan banyak tersedia, namun tidak satu pun metode yang dapat diterapkan secara luas. tujuan: makalah ini disusun untuk membahas mengenai aplikasi teori belajar sosial dalam mencegah kecemasan pada anak saat perawatan gigi. tinjauan pustaka: rasa cemas dan takut merupakan perasaan psikis yang dialami seorang individu. perasaan ini pada seorang anak lebih banyak didapat dari proses belajar dalam menyerap informasi berdasarkan pengalaman pribadi ataupun informasi dari lingkungan sekitar. bila rasa cemas dan takut dapat dipelajari oleh seorang anak, maka diasumsikan bahwa menghindari timbulnya perasaan ini dapat pula diajarkan pada anak. aplikasi hal tersebut sebenarnya telah diterapkan di bidang kedokteran gigi melalui modeling, namun eksplorasi bagaimana proses pembelajaran ini berlangsung sering kali terabaikan. simpulan: teori belajar sosial memberikan pendekatan preventif yang mudah dan intervensi yang efektif yang dapat digunakan pada anak usia 4-9 tahun untuk mengurangi kecemasan anak saat perawatan gigi. kata kunci: kecemasan, anak, perawatan gigi, belajar social korespondensi (correspondence): arlette suzy setiawan, departemen kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung indonesia. e-mail: a.suzy@unpad.ac.id literature reviews 88 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 87–91 pendahuluan saat ini telah banyak dicapai kemajuan dalam teknologi kedokteran gigi dalam tatalaksana nyeri dan berbagai strategi yang dapat membuat pasien nyaman dalam menjalani perawatan gigi, namun hal tersebut tetap tidak merubah persepsi sebagian individu yang membuat kedokteran gigi ditakuti. klasifikasi internasional telah memasukkan kecemasan dan rasa takut terhadap kedokteran gigi ini di bawah seksi “fobia spesifik”. 1 dalam praktek dokter gigi, sering dialami bahwa sebagian besar anak tidak bekerja sama selama perawatan sehingga kadang-kadang menjadi sangat sulit untuk mengendalikannya. kesulitan tersebut tidak hanya terkait dengan prosedur teknis perawatan, tetapi juga dengan gangguan emosional yang berbeda dari anak. gangguan emosional yang paling umum ditunjukkan selama perawatan gigi adalah rasa takut dan cemas.2 menurut gao et al.,3 rasa takut dan cemas terhadap perawatan gigi (dental fear and anxiety, dfa) merupakan masalah besar bagi sebagian individu, terutama anak dan remaja. prevalensi dfa pada anak dan remaja berkisar antara 5-20% di berbagai negara, dengan beberapa kasus menunjukkan kasus yang mengarah pada dental phobia (dfa berat). perilaku anak dan remaja dengan dfa dapat mempengaruhi hasil perawatan, menciptakan stres kerja pada dokter gigi dan stafnya,serta tidak jarang menjadi penyebab perselisihan antara dokter gigi dengan pasien atau orang tua mereka. anak akan mencoba segala cara untuk menghindari atau menunda pengobatan, sehingga kesehatan rongga mulut tidak terjaga.4,5 selain dampaknya terhadap perawatan gigi, dfa juga dapat menyebabkan gangguan tidur, mempengaruhi kehidupan sehari-hari dan memiliki dampak negatif pada seseorang fungsi psikososial. dfa diperoleh di masa kanak-kanak dapat bertahan hingga dewasa dan merupakan prediktor signifikan untuk menghindari kunjungan ke dokter gigi pada usia dewasa. hal tersebut merupakan tahap penting untuk mencegah dfa sehingga kesehatan mulut anak dapat dicapai secara optimal.3 makalah ini membahas konsep mengurangi dfa pada anak menggunakan teori belajar sosial..6 konsep takut atau kecemasan merupakan suatu konsep yang dikembangkan di bidang psikologi, namun bila konsep ini diaplikasikan pada bidang kedokteran gigi, maka kedua bidang ini harus berkerja sama dalam membantu pasien dan dokter gigi mencapai penatalaksanaan terbaik terhadap anak dengan dfa. rasa takut dan cemas pada perawatan gigi takut (fear) adalah respon emosional terhadap ancaman atau bahaya. hal tersebut terdiri dari perubahan fisiologis, perasaan dari dalam diri, suatu tindakan perilaku luar.7 rasa takut dapat menyebabkan berbagai perubahan fisiologis, seperti pucat, dilatasi pupil, takikardia, spasme jantung, hiperperistaltik, hiper/hiposekresi gastrointestinal, dan peningkatan aliran adrenalin. selain itu dapat menyebabkan sejumlah perasaan tidak menyenangkan seperti perasaan akan teror, pucat, jantung berdebar, ketegangan otot, kekeringan pada tenggorokan dan mulut, perasaan tenggelam di perut, mual, muntah, diare, iritabilitas, kesulitan bernafas, kehilangan nafsu makan, insomnia, dan dorongan untuk lari dan bersembunyi. perubahan perilaku eksternal dapat tercermin sebagai pola mengagetkan, penarikan atau penghindaran, atau melarikan diri. hal ini dapat menyebabkan individu untuk tetap terdiam atau bergerak.8,9 kecemasan (anxiety) adalah salah satu yang paling umum dari semua emosi manusia.3,10 hal ini termasuk: (1) kesadaran fisik dan mental terhadap ketidakberdayaan; (2) adanya ancaman yang akan dating; (3) perasaan bahaya yang berasal dari dalam, hasil penilaian kognitif; dan (4) sebuah keraguan yang tidak dapat terpecahkan tentang sifat ancaman, cara terbaik untuk menguranginya, dan kapasitas subjektif seseorang untuk secara efektif memanfaatkan sarana tersebut. bagaimana seseorang menilai situasi tersebut tergantung pada dua faktor: (1) faktor-faktor tersebut berasal dalam objek stimulus atau peristiwa itu sendiri; dan (2) variabel interpersonal. berkenaan dengan yang pertama, beberapa individu dikondisikan untuk bereaksi secara negatif pada perawatan gigi dan banyak aspek yang terkait dengannya. kedua, kemampuan seseorang untuk mengatasi atau mengelola situasi yang mengancam mengatur respon yang akan menyertainya.8 kedua faktor di atas dipengaruhi oleh pengalaman masa lalu individu, kepribadian, dan kemampuan untuk menghadapi kejadian yang menyebabkan rasa tersebut. ketakutan dibedakan dari kecemasan atas dasar kemampuan seseorang untuk mengidentifikasi objek eksternal yang mengancam dan untuk mengenali adanya perilaku yang akan mengurangi atau memperbaiki bahaya yang dirasakan. kecemasan juga dapat dipertimbangkan sebagai keadaan emosional di mana seseorang merasa tidak nyaman, gelisah, atau takut. seseorang biasanya akan mengalami kecemasan bila menghadapi peristiwa yang mereka tidak dapat mengendalikan atau memprediksi, atau tentang peristiwa atau situasi yang mereka dapat mempertimbangkan mengancam dan berbahaya. ada perasaan kerentanan, dan kecemasan yang parah dapat bertahan dan akhirnya bahkan mengarah pada ketidakberdayaan.8,11 bidang psikologi mendokumentasikan dfa pada tahun 1947 dengan diterbikannya buku “dental anxiety: fear of going to the dentist” oleh coriat. buku tersebut menuangkah bahwa rasa takut pergi ke dokter gigi adalah salah satu bentuk yang paling umum dari ketakutan dalam kehidupan sehari-hari. pemahaman psikologis tentang rasa takut ini dapat bermanfaat dalam menangani pasien12 secara garis besar bidang psikologi memahami rasa takut dipelajari oleh seorang anak. jika rasa takut dapat dipelajari, maka secara logika dapat pula untuk tidak dipelajari.13 rachman pada akhir tahun 1990-an mengembangkan suatu model yang menggambarkan bagaimana rasa takut dipelajari seorang individu. model yang dikembangkan rachman terdiri dari tiga jalur utama dalam mempelajari rasa takut, yaitu pengkondisian, jalur 89setiawan: aplikasi teori belajar sosial dalam penatalaksanaan rasa takut dan cemasan anak informasi, dan pembelajaran modeling.14 makalah ini hanya membahas mengenai pembelajaran modeling karena keterkaitannya dengan teori pembelajaran sosial dari albert bandura. teori belajar sosial teori yang dikembangkan oleh albert bandura pada akhir tahun 1960-an mengenai proses belajar sosial pada awalnya dimaksudkan untuk mengembangkan sifat agresi anak dari usiah 3-5 tahun melalui percobaan boneka bobo.6 kemudian teori tersebut lebih dikembangkan secara luas dalam menggambarkan bagaimana anak mendapatkan rasa takut dan juga bagaimana agar anak dapat mengatasi rasa takut ini.13 teori belajar sosial menyatakan bahwa anak dapat mempelajari rasa takut melalui observasi dari anak lain atau dari pengalaman masa lalu. pada intinya, teori tersebut memahami bahwa seorang inidividu dapat belajar dari menonton pengalaman individu lainnya mengenai situasi yang serupa. melalui pengamatan terhadap pengalaman orang lain, maka bagian dari pengalaman tersebut juga akan menjadi pengalaman anak. bandura menyatakan bahwa “many intractable fears arise not from personally injurious experiences, but seeing others respond fearfully toward or be hurt by threatening objects. similarly, evaluations of places, persons, or things often originate from exposure to modeled attitudes.”6 seorang anak yang melihat anak lain menunjukkan reaksi tantrum saat perawatan gigi dapat menjadikan anak tersebut mengalami dfa yang berkaitan dengan teori belajar sosial. beberapa penelitian yang dirangkum oleh do menunjukkan bahwa observasi dan pengalaman mengamati anak lain mempengaruhi dfa, antara lain hasil penelitian ost dan hughdahl mengenai dental phobia membuktikan bahwa 68% subjek mendapatkan rasa takut melalui pengkondisian, tetapi hasil lain yang lebih penting dari penelitian ini adalah bahwa penelusuran dfa pada orang dewasa menunjukkan 12% mendapatkan dfa dari pengalaman dalam mengamati pengalaman anak lain.15 teori belajar sosial tidak hanya menempatkan anak sebaya lain sebagai pelaku transfer rasa takut pada anak. orang tua berpotensi menurunkan rasa takut pada anaknya melalui modeling.16-18 penelitian townsend et al. seperti yang dikutip oleh do15 melaporkan bahwa ibu dari anak yang mengalami dfa lebih cemas dibandingkan dengan ibu dari anak yang non dfa.19 lara20 juga melaporkan bahwa ayah juga berperan penting dalam transfer rasa takut kepada anaknya. proses belajar sosial bekerja melalui lima elemen berbeda, yaitu efikasi diri, pencapaian kinerja, pengalaman yang mewakili, persuasi verbal, dan gairah emosional.6 efikasi diri adalah konsep lain dalam teori belajar sosial. tujuan teori tersebut adalah membangun efikasi diri atau membentuk persepsi individu mengenai kemampuan kinerjanya.15,21 bandura menyatakan, “perceived selfefficacy not only reduces anticipatory fear and inhibitions but, through expectations of eventual success, it affects coping effort once they are initiated.”6 terbentuknya efikasi diri terjadi melalui pencapaian kinerja, pengalaman yang serupa, persuasi verbal, dan gairah emosional. pencapaian kinerja dipertimbangkan sebagai sumber terkuat dari efikasi diri. pengalaman pribadi yang berhasil dapat meningkatkan ekspektasi keberhasilan selanjutnya; keberhasilan pribadi yang berulang mengarah pada terbentuknya efikasi diri yang kuat.6,15 pengalaman yang serupa membentuk efikasi diri melalui “seeing others perform threatening activities without adverse consequences can create expectations in observers they too will eventually succeed if they intensify and persist in their efforts.”6 persuasi verbal terdiri dari rasa seseorang dipimpin orang lain melalui sugesti persuasive, menjadikan seseorang percaya bahwa mereka dapat mengatasi apa yang menghambatnya di masa lalu. persuasi verbal merupakan kemungkinan terlemah dalam membentuk efikasi diri yang dapat bertahan lama. gairah emosional berkaitan dengan efikasi diri dalam persepsi situasi yang mengancam. meningkatnya gairah emosional secara tipikal menurunkan kinerja, sehingga individu cenderung lebih berhasil jika mereka tidak dalam keadaan emosi tinggi atau tidak merasa sensasi fisik atau emosi yang berhubungan dengan kecemasan dan takut.6 aplikasi teori belajar sosial pada anak dengan dfa saat ini dokter gigi menggunakan berbagai teknik dalam mengendalikan perilaku negative anak seperti teknik tell-show-do, voice control, hand over mouth, dan sedasi n2o.22,23 beberapa anak dapat menunjukkan perubahan perilaku dengan teknik tersebut di atas, namun pada beberapa kasus tertentu teknik tersebut tidak efektif, terutama bila anak mengalami dfa. aplikasi teori belajar sosial di kedokteran gigi dimaksudkan untuk mengurangi atau mencegah timbulnya dfa pada anak sebelum anak tersebut pergi ke dokter gigi. atau dengan kata lain, teori belajar sosial ini diaplikasikan pada tingkat preventif. anak diharapkan mendapatkan aspek teori belajar sosial sebelum perawatan gigi pertamanya. intervensi preventif dapat berbentuk modeling film/ in vivo dan modeling partisipan.15 teori belajar sosial dimaksudkan bandura untuk anak usia antara 3 sampai 5 tahun,6 namun penelitian yang ditujukan untuk mengetahui efek teori belajar sosial dalam mengurangi dfa terbatas pada anak usia 4 hingga 9 tahun. dengan kata lain, aplikasi teori belajar sosial efektif pada anak 4-9 tahun dan berpotensi efektif pada anak kurang dari 4 tahun.15 modeling in-vivo/film modeling in vivo adalah teknik yang menempatkan anak menonton individu lain (model) baik dalam bentuk film atau in vivo (kehidupan nyata) menjalani perawatan gigi. selama sesi modeling, anak menonton model menjalani setiap tahap perawatan gigi. hal tersebut menunjukkan dua komponen kunci dari teori belajar sosial, yaitu pembelajaran melalui situasi yang sama dan pencapaian kinerja. anak akan mengobservasi model menunjukkan keterampilan dalam mengatasi situasi yang kurang menyenangkan melalui bernapas dalam, relaksasi, selama perawatan gigi.15 90 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 87–91 proses ini mengajarkan anak berbagai alternatif dalam mengatasi dfa, bukan dengan tantrum atau penolakan. hal penting dalam modeling in vivo/ film adalah pencapaian kinerja. anak harus melihat model secara sukses menyelesaikan perawatan gigi yang disebabkan oleh perilaku model dalam mengembangkan keterampilan coping yang positif. keberhasilan bisa ditunjukkan dalam bentuk selesainya perawatan gigi atau pemberian positive reinforcement.15 modeling partisipan proses ini melibatkan partisipasi aktif dari observer. secara tipikal observer diminta untuk menonton model yang serupa dengan yang ada pada modeling in vivo atau film. selain itu, anak diminta untuk berlatih keterampilan yang ditunjukkan oleh model selama proses modeling. penggunaan pencapaian kinerja dan pengalaman yang serupa sama pentingnya seperti dalam modeling in vivo atau film. anak harus melihat model berhasil dalam pengalamannya mengatasi rasa cemas mereka dalam perawatan gigi.15 pembahasan dental fear and anxiety pada anak seringkali merupakan penyebab utama masalah penatalaksanaan tingkah laku yang mengganggu perawatan gigi reguler. efek negatif lain dari dfa adalah terbentuknya internalisasi secara alami reaksi penolakan secara psikologis maupun perilaku hingga menghindari perawatan gigi. hal tersebut akan berdampak timbulnya konsekuensi negatif bagi kesehatan gigi anak yang tentu berpotensi menciptakan masalah lain yang saling berkaitan, seperti stigma sosial atau berkembangnya rasa rendah diri pada anak.24,25 berbagai penelitian telah mengembangkan dalam tatalaksana dfa. intervensi musik,26,27 modifikasi lingkungan kerja,28,29 biofeedback,30,31 adalah contoh beberapa penelitian yang ditujukan dalam mengurangi dfa saat perawatan gigi. peneliti bidang psikologi sendiri telah banyak melakukan penelitian mengenai dfa sejak tahun 1980-an.32-36 intervensi psikolog dalam mengurangi anxiety disorder melalui cognitive behavior therapy telah memasukkan poin khusus mengenai dfa.37,38 hal tersebut menunjukkan betapa pentingnya masalah dfa untuk diatasi. faktor yang dapat memberikan kontribusi bagi timbulnya dfa penting untuk diketahui agar tatalaksana memberikan hasil yang memuaskan.1,39 bila dikaitkan dengan teori belajar sosial yang menyatakan bahwa rasa takut dipelajari oleh anak, maka faktor yang dapat membentuk perilaku tersebut dapat dimodifikasi. walaupun hasil penelitian brown pada pertengahan thun 1980-an menunjukkan bahwa dfa lebih merefleksikan kecemasan secara umum dibendingkan dengan takut yang dipelajari sebagai respon terhadap situasi spesifik,40 namun penelitian mengenai hubungan pola pengasuhan dengan dfa menunjukkan adanya hubungan yang positif.41 inti dari pengembangan teori belajar sosial itu sendiri berada dalam tingkat preventif, yaitu mencegah terjadinya dfa, yang dapat merupakan masukan berharga dalam bidang kedokteran gigi. o l l e n d i c k d a n k i n g m e l a l u i p e d o m a n y a n g dikembangkan oleh american psychological association divisi 12, yaitu kriteria chambless menemukan bahwa modeling film efektif dalam perawatan fobia. penelitian lain pada anak-anak antara usia 5-11 tahun yang belum pernah ke dokter gigi diperlihatkan film yang menunjukkan anak usia 4 tahun menjalani perawatan gigi. anak yang menonton film tersebut memiliki skor yang lebih rendah secara signifikan pada behavioral profile rating scale (bprs) dibandingkan dengan kelompok kontrol yang menonton film yang tidak berkaitan dangan modeling.15 penelitian yang menelaah modeling in vivo menemukan bahwa bila anak mengamati perawatan gigi anak lain sebelum jadwal perawatan giginya menujukkan bahwa anak yang mengamati memiliki reduksi signifikan dalam perilaku negatif. williams et al. juga menemukan bahwa anak yang diobservasi anak lain juga menunjukkan penurunan signifikan dalam perilaku negatifnya.15 seiring dengan hasil penelitian tersebut, farhat-mchayleh42 melaporkan bahwa model in vivo lebih efektif dibandingkan dengan metode tell-show-do. modeling partisipan telah terbukti sebagai perawatan untuk fobia. ollendick dan king menemukan bahwa modeling partisipan lebih efektif dibandingkan baik modeling film/ in-vivo dan perawatan pengkondisian klasik dari desensitisasi sistematik dalam perawatan fobia. bukti lain yang mendukung superioritas modeling partisipan adalah hasil penelitian klingman et al. yang menyatakan bahwa anak dari kelompok modeling lebih kooperatif dan kurang cemas dibandingkan kelompok modeling film.15 beberapa cara meningkatkan keefektifan modeling secara umum, baik secara film ataupun partisipan. karakteristik model itu sendiri dapat mengganggu keefektifan intervensi. model yang baik harus serupa dengan pengamat. model untuk anak dengan dfa harus anak yang serupa karakteristik secara demografi. model dapat menjadi sangat efektif bila pengamat melihat model tersebut dalam pandangan yang tinggi. contoh yang baik dari model yang dipandang tinggi oleh anak dapat berupa karakter kartun popular saat ini, misalnya karakter seperti sponge bob atau barney.15 keefektifan model dapat ditingkatkan dengan menggunakan model “coping”. untuk anak dengan dfa, model coping lebih efektif dibandingkan dengan model mastery. model coping akan mengekspresikan rasa takut dan kesulitan dalam situasi modeling sementara model mastery akan menunjukkan keahliannya dalam situasi modeling. namun utnuk anak yang baru ke dokter gigi dan pernah menonton model coping, menunjukkan penurunan perilaku tidak kooperatif dibandingkan kelompok yang menonton model mastery.15 terakhir, anak harus memiliki pencapaian kinerja. anak harus terdorong memperlihatkan keterampilan coping yang 91setiawan: aplikasi teori belajar sosial dalam penatalaksanaan rasa takut dan cemasan anak positif. setelah perawatan selesai, anak akan merasi efikasi dirinya meningkat dalam perawatan gigi.15 dapat disimpulkan bahwa dfa merupakan masalah yang cukup serius dan membutuhkan penatalaksanaan yang komprehensif antara bidang kedokteran gigi dan psikologi. teori belajar sosial memberikan pendekatan preventif yang mudah dan intervensi yang efektif yang dapat digunakan pada anak usia 4-9 tahun. dokter gigi dapat menyediakan film, atau pun kesempatan observasi in vivo pada ruang prakteknya. setelahnya dokter gigi dapat menurunkan perilaku disrupsi dengan meminta anak mempraktekkan dengan model. modeling in vivo dapat merupakan teknik yang lebih aplikatif bagi dokter gigi. model yang digunakan dapat ibu atau ayah, ataupun saudara pasien anak. namun hal penting yang tidak bisa dilupakan adalah usia anak. daftar pustaka 1. nicolas e, bessadet m, collado v, carrasco p, rogerleroi v, hennequin m. factors affecting dental fear in french children aged 5-12 years. int j paediatr dent 2010; 20(5): 366–73. 2. sharma m, mittal r. assessment of psychological effects of dental treatment on children. contemp clin dent 2012; 3(5): 2. 3. gao x, hamzah sh, yiu yck, mcgrath c, king mn. dental fear and anxiety in children and adolescents: qualitative study using youtube. j med internet res 2013; 15(2): e29. 4. sartory g, heinen r, pundt i, jöhren p. predictors of behavioral avoidance in dental phobia: the role of gender, dysfunctional cognitions and the need for control. anxiety, stress & coping 2006; 19(3): 279–91. 5. watson rj. an exploration of children’s dental anxiety. in: jones l, editor. new zealand: massey university; 2010. p. 1–134. 6. bandura a. social learning theory. oxford, england: prentice-hall; 1977. p. 61, 80-1. 7. nolen-hoeksema s, fredrickson bl, loftus gr, wagenaar wa. atkinson and hildegard’s introduction to psychology. 15th ed. farmington hill: wadsworth/cengage learning; 2009. 8. weiner aa. the fearful dental patient. 1st ed. iowa: wiley-blackwell; 2011. p. 1–311. 9. mostofsky di, fortune f. behavioral dentistry. 2nd ed. oxford: john wiley & sons, inc; 2014. 10. kennedy b. anxiety disorders. farmington hill: wadsworth/ cengage learning; 2010. p. 1–113. 11. shiota mn, kalat jw. emotion. 2nd ed. belmont: wadsworth cancage learning; 2012. p. 1–484. 12. capps d, carlin n. sublimation and symbolization: the case of dental anxiety and the symbolic meaning of teeth. pastoral psychol 2011; 60(6): 773–89. 13. craske mg, hermans d, vansteenwegen d. fear and learning. washington: american psychological association; 2006. p. 1–313. 14. rachman sj. fear and courage. 2nd ed. new :york: wh freeman and company; 1990. 15. do c. applying social learning theory to children with dental anxiety. j contemp dent pract 2004; 5(1): 126–35. 16. salem k, kousha m, anissian a, shahabi a. dental fear and concomitant factors in 3-6 year-old children. j dent res dent clin dent prospects 2012; 6(2): 70–4. 17. goettems ml, ardenghi tm, romano ar, demarco ff, torriani dd. influence of maternal dental anxiety on oral health–related quality of life of preschool children. qual life res. 2010; 20(6): 951–9. 18. kulkarni s, jain m, mathur a, mehta p, gupta r, goutham b, et al. a relation between dental anxiety, the parental family and regularity of dental attendance in india. johcd 2009; 3(2): 29–35. 19. walton jw, johnson sb, algina j. mother and child perceptions of child anxiety: effects of race, health status, and stress. j ped psych 2009; 24(1): 29–39. 20. lara a, crego a, romero-maroto m. emotional contagion of dental fear to children: the fathers’ mediating role in parental transfer of fear. int j paediatr dent. 2012; 22(5): 324–30. 21. pajares f, urdan tc. self-efficacy beliefs of adolescents. illustrated. new york: iap information age pub., incorporated; 2006. 22. widmer rp, mcneil dw, mcneil cb, hayes-cameron l. child development, relationships and behaviour management. in: cameron ac, widmer rp, editors. handbook of pediatric dentistry (fourth edition). fourth edition. sydney: mosby; 2013. p. 9–24. 23. yeung y. distraction techniques for anxious dental patients. saad digest 2013; 29: 82–7. 24. berge m. dental fear in children: clinical consequences suggested behaviour management strategies in treating children with dental fear. eur arch paediatr dent 2008; 9(1): 41–6. 25. moore r, brødsgaa rd i, rosenberg n. the contr ibution of embarrassment to phobic dental anxiety. bmc psychiatr 2004; 4(1): 10. 26. setiawan a, sasmita is. the mozart effect towards dental anxiety in–year old children. dent j (maj ked gigi) 2010; 43(1): 1–4. 27. moola s. effectiveness of music interventions in reducing dental anxiety in paediatric and adult patients. in: pearson a, jordan z, hagger c, editors. adelaide: the university of adelaide; 2011. p. 1–76. 28. saphiro m, melmed rn, sgan-cohen hd, eli l, parush s. behavioural and physiological effect of dentl environment sensory adaptation on children’s dental anxiety. eur j oral sci 2007; 115(6): 479–83. 29. menezes abreu dm, leal sc, mulder j, frencken je. patterns of dental anxiety in children after sequential dental visits. eur arch paediatr dent 2011; 12(6): 298–302. 30. morarend q, spector m, dawson d, clark s, holmes d. the use of a respiratory rate biofeedback device to reduce dental anxiety: an exploratory investigation. appl psychophysiol biofeedback 2011; 36(2): 63–70. 31. dedeepya p, nuvvula s, kamatham r, nirmala sv.. behavioural and physiological outcomes of biofeedback therapy on dental anxiety of children undergoing restorations: a randomised controlled trial. eur arch paediatr dent 2014; 15(2): 97-103. 32. nocella j, kaplan rm. training children to cope with dental treatment. j ped psych 2005; 7(2): 175–8. 33. dahlquist lm, gil km, hodges j, kalfus gr, ginsberg a, holborn s. the effects of behavioral intervention on dental flossing skills in children. j ped psych 2005; 10(4): 403–12. 34. knapp lg. effects of type of value appealed to and valence of appeal on children’s dental health behavior. j ped psych 2004; 16(6): 675–86. 35. christiano b, russ sw. matching preparatory intervention to coping style: the effects on children’s distress in the dental setting. j ped psych 2005; 23(1): 17–27. 36. bernard rs. pediatric procedural approach-avoidance coping and distress: a multitrait-multimethod analysis. j ped psych 2004; 29(2): 131–41. 37. prangnell sj, green k. a cognitive behavioural intervention for dental anxiety for people with learning disabilities: a case study. british j learning disabilities 2008; 36(4): 242–8. 38. nash m. dental anxiety: how cbt might help support anxious patients. dent nurs 2013; 9(2): 85–7. 39. klingberg g. dental anxiety and behaviour management problems in paediatric dentistry — a review of background factors and diagnostics. eur arch paediatr dent 2008; 9(1): 11–5. 40. brown d, clive wright fa, mcmurray n. psychological and behavioral factors associated with dental anxiety in children. j behav med 1986; 9(2): 213–8. 41. krikken jb, veerkamp jsj. child rearing styles, dental anxiety and disruptive behaviour; an exploratory study. eur arch paediatr dent 2008; 9(1): 23–8. 42. farhat-mchayleh n, harfouche a, souaid p. techniques for managing behaviour in pediatric dentistry. j can dent assoc 2009; 75(4): 283–283f. 160 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 160–164 case report introduction according to indonesian basic health research 2018, 57.6 per cent of dental and oral problems that happen in indonesia often lead to tooth extraction.1 after the procedure, the alveolar bone will undergo the healing process, and, during that time, vertical and horizontal resorption of the bone will occur.2 as much as 25 per cent of bone width will be lost in the first year after tooth extraction, and this will reach 40 per cent in the third year. after the first year of healing, the resorption rate will significantly drop but will still continue perpetually. after tooth extraction, the patient will need tooth replacement to rehabilitate the oral function, aesthetic and equilibrium. there are a lot of ways to replace missing teeth. one of the treatments of choice is dental implant.3 implant placement can be done immediately after tooth extraction or after the post-extraction wound has healed, whether it is early implant placement or delayed/ conventional implant placement. early implant placement is done within four to eight weeks after extraction, where soft tissue healing has taken place; whereas, delayed implant placement is done within 12 to 16 weeks after tooth extraction, where there has been partial bone healing.4 immediate implant placement has some advantages, such as preserving the alveolar bone dimension, reducing the amount of surgery and shortening the treatment time. additionally, by performing immediate implant placement, flap incision may be avoided. however, immediate implant placement also has some disadvantages, such as increased risk of infection and the presence of a gap between the implant surface and the socket wall. although there are some disadvantages, immediate implant placement is sometimes needed to avoid resorption of the ridge and to avoid a future bone augmentation procedure.5,6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p160–164 oral rehabilitation using immediate implant placement in mandibular lateral incisors – a case report nila sari1, abil kurdi2, bambang agustono satmoko tumali2 and muhammad dimas aditya ari2 1resident in prosthodontics, 2department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: dental problems often lead to missing teeth. after tooth extraction, the alveolar bone will undergo a healing phase, and this will cause some vertical and horizontal resorption. immediate implant placement can shorten treatment time and preserve the rest of the alveolar bone. purpose: the purpose of this study is to present a case of oral function, equilibrium and aesthetic rehabilitation using immediate implant placement. case: a male patient aged 31 came to dental hospital universitas airlangga with a fractured anterior tooth. the tooth had fractured two weeks before he came to the hospital, and he wanted to improve his appearance. case management: the mandibular incisor was fractured, and its residual root remained. the treatment plan was to undertake an immediate implant placement. the type of implant chosen was a bone level tapered implant sc roxolid® sla ø 2.9 mm and 10 mm long. surgery was performed in two stages. the first stage was to extract the residual root, position the implant and apply the bone graft. the second stage was to position the healing abutment. a crown impression was made using the closed tray technique. the crown was cemented to the abutment. conclusion: immediate implant placement is an aesthetic means of rehabilitating a missing tooth, such as an anterior mandibular tooth. keywords: aesthetic zone; dental implant; edentulous; human & health; immediate placement correspondence: bambang agustono satmoko tumali, department of prosthodontics, faculty of dental medicine, universitas airlangga; jl. mayjen prof. dr moestopo 47 surabaya, 60132 indonesia. email: bambang-a-s-t@fkg.unair.ac.id; khrisna@indo.net.id mailto:bambang-a-s-t@fkg.unair.ac.id mailto:khrisna@indo.net.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p160-164 161sari et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 160–164 implant loading can be divided into three types. immediate implant loading is performed within one week of implant placement. early implant loading is carried out within one week to two months after implant placement, and conventional loading is performed more than two months after implant placement. from some studies, the healing time to achieve osseointegration of the implant is at least three to four months without loading. the presence of micromotion on implants can interfere with the healing process causing fibrous tissue to form. this fibrous tissue will separate bone and implant. when micromotion reaches a certain threshold, it will cause damage to the implant. however, the development of recent implant technology can shorten waiting time for loading.4,7 a thorough examination and accurate diagnosis is the key to success for an immediate placement implant. radiography testing is also needed, such as panoramic and cone beam computerised tomography (cbct). one of the most important things to note is the teeth prognosis. it is also important to note the patient’s general health. a patient with uncontrolled systemic disease, a smoking habit, unhealthy adjacent teeth or inadequate buccal, lingual bone and septum is not indicated for immediate implant placement.8,9 the purpose of this study is to show that immediate implant placement is one of the treatment plans that can be considered in such cases. it can help rehabilitate oral function and equilibrium as well as the facial aesthetic. case a male patient aged 31 years old came to dental hospital universitas airlangga because he wanted to have a denture to improve his appearance. the anterior mandibular tooth had been fractured two weeks before he came. the patient claimed that he was not suffering from any systemic disease. extraoral examination showed that the temporomandibular joint, eyes, nose and lips were normal. the patient had an oval-shaped face. intraoral examination showed that there was a gangrene radix of tooth 31 (figure 1). a cbct x-ray was taken to help determine the width and height of the alveolar bone (figure 2). the cbct image showed that the radix was 9.29 mm long and its bucco-lingual width was 5.4 mm. the narrowest part of the bone was 4.31 mm wide. the distance between the crown of tooth 41 and tooth 32 was 5.83 mm, and the distance between their roots was 7.65 mm. the distance from the apical tip of tooth 31 to the edge of the mandibular cortex was 20.4 mm. the patient was informed of the treatment options, and he agreed with the treatment. the patient then signed an informed consent. case management the treatment chosen for this case was an implantsupported solitary crown for tooth 31. the implant would be immediately placed after tooth extraction. before the procedure, tooth scaling was carried out to manage the patient’s oral hygiene. the first surgery was performed to place the implant fixture. the first step was to anesthetise the buccal and lingual area using articaine hydrochloride four per cent. a full thickness flap was made on the top of tooth 31’s ridge towards the gingival margin of tooth 32, and then a vertical incision was made on the distal part of tooth 32. atraumatic extraction was carried out (figure 3) by dividing the root mesio-distally into two parts, then it was extracted using an elevator and forceps. the post-extraction socket was thoroughly debrided to remove any granulation tissue. figure 1. frontal intraoral view of the patient showing a missing anterior mandibular tooth. sagittal (a) axial (b) coronal (c) figure 2. cbct image containing the available space for implant placement from sagittal view (a), coronal view (b) and axial view (c). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p160–164 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p160-164 162 sari et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 160–164 a b a figure 3. atraumatic tooth extraction by dividing the left root (a) and extracting the tooth from the socket (b). a b c figure 4. procedure of manual implant fixture placement using implant ratchet (a) followed by bone graft and membrane application (b) and sutured surgery site (c). the osteotomy site was prepared with drills in sequential order starting with a needle drill (ø 1.6 mm) followed by a pilot drill (ø 2.2 mm). drilling maximal speed was 800 rpm, until 10 mm deep and 4 mm below the adjacent tooth’s cemento-enamel junction (cej), then after every drilling, the depth was checked with a depth gauge. profile drilling and tapping was done manually, followed by implant placement with simultaneous guided bone regeneration (gbr). the implant fixture using a bone level tapered implant staumann sc roxolid® sla ø 2.9 mm and 10 mm long was inserted into the osteotomy site until 4 mm below the cej of the adjacent teeth (figure 4a). a xenograft (biooss small granule) was applied to the exposed implant part on the buccal side and the gap between implant and socket wall. the graft was then covered by a resorbable membrane (bio-gide) and sutured (figure 4b and 4c). an antibiotic (lincomycin 500 mg) and antiinflammatory drug (cataflam 50 mg) were prescribed for five days. the patient was asked to come back to the dental practice one week after surgery to have the sutures removed. on the first day post-surgery control, the patient reported no complaint. clinical examination showed redness and swelling on the gingival margin around the implant. a second review took place a week after surgery. the patient had no complaint, and there was no sign of inflammation on the gingiva surrounding the implant. the sutures were removed, then an adhesive bridge was placed as a temporary restoration (figure 5a), and the patient was asked to come back to the dental practice after six months or if there were any problems. before the second surgery was performed, the temporary adhesive bridge was taken off, and the surrounding gingiva was evaluated. the surgery was carried out to insert the healing abutment (figure 5b), and one week after surgery, the patient was asked to come back to the dental practice to take a final impression. a week after healing abutment placement, the implant and surrounding gingival condition were evaluated: there was no pain and abnormalities in percussion and palpation; no implant mobility; no pain and redness in the soft tissue around the implant. the implant was irrigated with saline a b figure 5. the temporary adhesive bridge six months after placement (a) and the attachment of healing abutment (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p160–164 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p160-164 163sari et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 160–164 and dried after the healing abutment was removed. then the clinician inserted an impression post into the implant and secured it with a screwdriver (figure 6a). the impression cap was then applied on top of the fixed impression post. medium body silicone impression material was injected around the impression post, then the impression tray, filled with impression material, was placed in the mouth. after the impression material had set, the tray was taken out of the mouth. the impression post then was unscrewed and taken out of the mouth. the impression post was fixed to the analogue using a guide screw and the colour code of the polymer impression cap checked for suitability. the impression post was positioned and pushed into place in the impression tray. an impression of the upper jaw was taken with alginate. tooth colour was determined using a shade guide, then the laboratory was informed for manufacturing the crown. the healing abutment then was reinserted to the implant. the impression was performed with a closed tray technique (figure 6b). the last step was to insert the solitary crown onto the implant. after taking off the healing abutment, the gingival condition surrounding the implant was examined. there was no redness or swelling; the implant showed no response on percussion and no pain during palpation. an abutment (variobase) was then inserted manually using an implant ratchet with insertion torque of 35 n. occlusion, retention, stability and comfort had been checked before the zirconia crown was cemented. articulating paper was used to check whether there was any premature contact on the crown, then the crown was cemented using temporary cement. a week after temporary insertion, the patient had no complaint, and the clinical examination showed no redness or swelling around the crown. percussion and palpation produced no pain response. the crown was then cemented permanently using fuji i luting cement (figure 7). discussion post-extraction alveolar resorption is a phenomenon that cannot be avoided. when a tooth is extracted, bone loss will occur mostly over the first six months after extraction (40 per cent of bone height and 60 per cent of bone width). this is a continuous process with the rate of 0.25–0.5 per cent of bone loss per year. implant treatment needs around two to three months for socket remodelling and three to six months for osseointegration.10 immediate implant placement should be considered as a treatment plan to shorten treatment time. the major benefit of doing immediate implant placement is that less time is needed for healing, and it can preserve the dimension of bone tissue around the extracted tooth area. earlier implant placement can result in a better crown/ implant ratio, thus giving a better aesthetic and inter-jaw relation, also preserving alveolar bone.11,12 immediate implant placement is usually indicated for a tooth that needs to be extracted because of external trauma, endodontic lesion, root fracture, root resorption or root perforation. contraindicated conditions are active infection, inadequate bone below the tooth apex (cannot give primary stability) and severe gingival recession.12 immediate implant placement is divided into three categories: (1) immediate placement with immediate loading; (2) immediate placement with early loading; and (3) immediate placement with conventional loading. in this case, immediate implant placement was performed to shorten the treatment period and to prevent major bone resorption. conventional loading was chosen because there was considerable bone loss on the facial side (aesthetic zone) so that it needed a gbr procedure. the success rate of immediate implant placement with conventional loading is 96 per cent.13–15 guided bone regeneration is a reconstructive procedure for the alveolar ridge using a bone graft and membrane. this procedure is to improve inadequate bone dimension usually found because of trauma, infection or periodontal disease. inadequate bone dimension can affect the aesthetic and long-term prognosis of a dental implant and a b figure 6. fixing the impression post (a) followed by the closed tray technique impression, and the implant analogue was placed on the final impression (b). figure 7. frontal intraoral view post solitary crown insertion. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p160–164 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p160-164 164 sari et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 160–164 its superstructure. in this case, bone defect was found on the facial side so that gbr procedure was needed after immediate implant placement.16,17 bio-oss was used for the gbr procedure. bio-oss is derived from bovine bone that is processed to completely remove the organic component. according to kim et al.18 (2020), there is no difference between bone augmentation using bio-oss or autogenous bone graft. in a gbr procedure, the barrier membrane plays a significant role in bone regeneration. membranes can prevent soft tissue growth into the defect and maintain the defect cavity throughout the bone regeneration process. bio-gide, the membrane used in this case, is an absorbable collagen membrane.19 dental implants are very variable in size and shape. these variations are based on the condition of the alveolar bone: height, width, length and angulation.20 in this case, a bone-level implant was chosen because it would be placed in the anterior region of the jaw. the implant diameter was determined by the space available between two adjacent teeth, and the length of the implant was determined by the length of bone available. the diameter should leave a 1.5 mm bone surrounding the implant.20 space available in this case was 6 mm so that a blt implant ø 2.9 mm and 10 mm long was chosen. a solitary zirconia layered crown was used as the restoration because it has good mechanical characteristics and aesthetic.21 the patient was taught how to maintain his oral hygiene and, most importantly, the area surrounding the implant. he was instructed to use dental floss and come back to the dental practice every six months to have a routine dental check-up. in conclusion, immediate implant placement with conventional loading is one of the implant placement techniques that can be performed in such cases. it can help rehabilitate oral function and shorten treatment time. success rate can be increased with accurate diagnosis and thorough examination. references 1. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 180. 2. samyukta, abirami g. residual ridge resorption in complete denture wearers. j pharm sci res. 2016; 8(6): 565–9. 3. mittal y, jindal g, garg s. bone manipulation procedures in dental implants. indian j dent. 2016; 7(2): 86–94. 4. gallucci go, hamilton a, zhou w, buser d, chen s. implant placement and loading protocols in partially edentulous patients: a systematic review. clin oral implants res. 2018; 29(suppl 1): 106–34. 5. annibali s, bignozzi i, iacovazzi l, la monaca g, cristalli mp. immediate, early, and late implant placement in first-molar sites: a retrospective case series. int j oral maxillofac implants. 2011; 26(5): 1108–22. 6. lanza a, scognamiglio f, femiano f, lanza m. immediate, early, and conventional implant placement in a patient with history of periodontitis. case rep dent. 2015; 2015: 217895. 7. zhu y, zheng x, zeng g, xu y, qu x, zhu m, lu e. clinical efficacy of early loading versus conventional loading of dental implants. sci rep. 2015; 5. 8. ebenezer v, balakrishnan k, asir r v, sragunar b. immediate placement of endosseous implants into the extraction sockets. j pharm bioallied sci. 2015; 7(suppl 1): s234–7. 9. swathi k v. immediate implants placement-a review. j pharm sci res. 2016; 8(11): 1315–7. 10. soni r, singh a, vivek r, baranwal h, chaturvedi t, srivastava a. immediate implant placement in mandibular anterior region with dehiscence. j dent implant. 2013; 3(2): 177. 11. sabir m, alam mn. survival of implants in immediate extraction sockets of anterior teeth: early clinical results. j clin diagn res. 2015; 9(6): zc58–61. 12. singh a, gupta a, yadav a, chaturvedi tp, bhatnagar a, singh bp. immediate placement of implant in fresh extraction socket with early loading. contemp clin dent. 2012; 3(suppl 2): 219–22. 13. chen st, hamilton a. immediate implant placement: indications, surgical and prosthetic procedures and outcomes. forum implantol. 2019; 15: 22–39. 14. enkling n. immediate dental implant placement, immediate restorative treatment and immediate loading: treatment options in dental practice? dtsch zahnärztliche zeitschrift int. 2021; 3(3): 91–7. 15. ghouraba r f, al-hessy a a, el-sheik h m m. compa rative radiographical evaluation of immediate and immediate -delayed implant placement on the facial bone thickness. ec dent sci. 2019; 18(10): 2363–74. 16. saad m, assaf a, maghaireh h. guided bone regeneration: evidence & limits. smile dent j. 2012; 7(1): 8–16. 17. farzad m, mohammadi m. guided bone regeneration: a literature review. j oral heal oral epidemiol. 2012; 1(1): 3–18. 18. kim yj, saiki cet, silva k, massuda ckm, de souza faloni ap, braz-silva ph, pallos d, sendyk wr. bone formation in grafts with bio-oss and autogenous bone at different proportions in rabbit calvaria. int j dent. 2020; 2020: 2494128. 19. lee s-w, kim s-g. membranes for the guided bone regeneration. maxillofac plast reconstr surg. 2014; 36(6): 239. 20. resnik r. misch’s contemporary implant dentistry. 4th ed. canada: mosby elsevier; 2020. p. 331–40. 21. zarone f, russo s, sorrentino r. from porcelain-fused-to-metal to zirconia: clinical and experimental considerations. dent mater. 2011; 27(1): 83–96. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p160–164 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p160-164 85 lipopolysaccharide (lps) introduction during growth and development period of rat’s tooth toward the occurrence of enamel hypoplasia didin erma indahyani*, al-supartinah santoso**, totok utoro***, and marsetyawan hne soesatyo**** **** department of oral biology, faculty of dentistry jember university, jember-indonesia **** department of pediatric dentistry, faculty of dentistry gadjah mada university, **** department of anatomy and pathology, faculty of medicine gadjah mada university, yogyakarta-indonesia **** department of histology, faculty of medicine gadjah mada university, yogyakarta-indonesia abstract the aim of this study is to know the effect of lipopoly saccharide (lps) induction during growth and development period specifically the occurrence of hypoplasia on tooth enamel. 5 day old male wistar rats divided into two groups. group 1 (control) under went no treatment. group 2 (treatment) under went lps induction every 24 hour for 8 days on buccal fold right maxillary first molar. after 21 days old the rats were sacrificed and the tooth was resected. hypoplasia hypo calcification index (hhi) was used to determine the degree of hypoplasia by clinical examination. radiograph of maxilla was also taken to analyze the apacities of enamel by using corel draw version 11. the result showed that group under went lps induction hypoplasia occurred on its molar tooth and more radiolucent than control groups. the conclusion is lps induction during growth and development period of rats tooth causing enamel hypoplasia. key words: lipopolysaccharide, growth and development tooth, hypoplasia, rat correspondence: didin erma indahyani, c/o: bagian biologi oral, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. introduction growth and development of tooth consist of several stages; complex process and involve coordination as well as interaction between neural crest and epitel.1 the stage and process consist of initiation (bud stage), proliferation (cap stage) histodifferentiation and morfodifferentiation (bell stage), apposition as well as calcification (mineralization). the change occurring during the period of growth and development would contribute tooth abnormality. the change might be resulted from various kind of local and systemic factors.2 one of local factors inducing tooth abnormality is bacterial infection during the period of tooth growth and development. periapical lesion and alveolar bone impairment affected by either bacteria or product of other bacteries, one of them is lipopolysaccharide (lps). lps is the main structure of cell wall of gram negative bacteria to maintain the integrity of bacterial structure and to protect the bacteria from host immunity. in addition, lps is endotoxin contributing the occurrence of cytokine proinflammatory secretion from several types of cell.3 it could be suggested that lps has an important role as the cause of periapical lesion.4 this condition would induce local factor cytokine proinflammatory such as: interleukin -1 (il-1), il–1, il–6, tumor necrosis factor(tnf-) and eikosanoid i.e. prostaglandin (pge2). 5 prostaglandine and cytokine proinflamatory might result distruction of periodontal tissue by stimulating the formation and the increase of osteoclast activities.6 enamel hypolasia is one of abnormalities of tooth enamel which frequently found in children’s teeth caused by the disturbance during the period of tooth growth and development. in general, clinical feature of enamel hypoplasia is shown by enamel agenesis which looks like a small well, horizontally arranged found on buccal region. if severe hypoplasia occurs, most parts of tooth enamel will disappear.7 hypoplasia is called as quantitative abnormality, usually followed by hypocalcification which is called qualitative abnormality.8 hypocalcification (demarcated opacities) resulted from imperfect mineralization on enamel and manifest in the form of white spot.7 li et al.9 reported that enamel hypoplasia of children’s permanent teeth in rural area in china suspected caused by continuous caries on deciduous teeth which initially happened at the age of 3–5 years. study condacted by lo et al.,10 nicolau et al.,11 and broad bent et al.12 suggested that demarcated opacities and hypoplasia which happened in permanent tooth due to severe caries on the previous deciduous teeth even though the mechanism was still unknown. the purpose of this study is to know the effect of lps induction enamel hypoplasia. the outcome of the study is expected to be able to contribute scientific in formation on the mechanism of hypoplasia, so that local infection which could disturb the growth and development could be avoided. in addition, it is also expected as the basic guidance of further study in the future. 86 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 85–88 material and method this study was performed on 10 male wistar rats of 5 days old, the rats were classified into 2 groups (5 rats each): groups 1: was controlled group which didn’t undergo any treatment, groups 2: the rats were inducted using escherichia coli lps (sigma) with the dosage: 5 gr/0,005 ml saline on buccal fold of right maxillary first molar. lps induction was done by intra muscular injection once in 24 hours for 8 days.13 on the 21st day the rats were decapitated. the maxilla was observed to find whether hypoplasia occurred or not enamel of right molar. the degree of hypoplasia was assessed by hypoplasia hypocalcification index (hhi)14 occurrence or hypocalcification, as follows 0 = no hypoplasia occurrence or hypo calcification, 1 = hypocalcification on half of incisal or occlusal crown, 2 = hypocalcification on half of cervical crown, 3 = hypoplasia on half incisal or occlusal crown, 4 = hypoplasia on half of cervical crown, 5 = hypocalcification less than half incisal occlusal crown, 6 = hypocalcification more than half of incisal occlusal crown or affected more than one crown, 7 = hypoplasia less than half of incisal occlusal crown, 8 = hypoplasia more than half insisal occlusal or affected more than one occlusal, and 9 = hypocalcification/hypoplasia excluded the above criteria (disfusive hypoplasia, limited one occlusal expected incisal). the result of radiopaque was put on x-ray viewer, then, recorded by digital camera and the result was transferred into computer. radiopaque and radiolucent feature of tooth enamel was observed using corel draw version 11 (corel, ottawa canada) assessing the size of red blue green (rgb) to see the difference of radiopaque of every radiopaque.15 the combination of rgb would produce white color, so, it is called additive color. the computer monitor would produce color shining ray through rgb. the mean of the total component of rgb would come maximal score = 255, and it would show pure white while the mean of total rgb would come: 0, it would show pure black. statistic analysis would be done using t-test in order to know whether there is or there is no statistical difference between the two groups either based on hhi radiopaque of radiopaque. result there was no evidence of hypoplasia (figure 1a) on the rats of groups 1 with hhi index = 0 (either hypoplasia or hypocalcification undetected), meanwhile on the rats of group 2 hypoplasia was found in upper right molar (figure 1-b). on radiopaque examinations showed that, group 1 had mean rgb higher than group 2, therefore, it could be suggested that the enamel showed more radiopaque companing to group 2 (figure 2). the result of hhi examination and radiopaque feature of radiopaque could be seen on table 1. statistical analysis using t-test showed significant difference (p < 0,005) between group 1 and 2 on hhi, radiopaque feature of radiopaque showed insignificant difference (p > 0,005) between group 1 and 2 (table 2). figure 1. rats molar enamel with and without enamel hypoplasia. note: a) rat’s molar in control group (without any treatment and enamel hypoplasia, b) rat’s molar in treated group, hypoplasia was found (lps induction was done) figure 2. radiopaque of rat’s molar with and without enamel hypoplasia. note: a) control group without treatment, b) treated group with lps induction. on figure b is more radiolucent compared to figure a  radiolucent enamel tooth table 1. the result of hypoplasia observation using hypoplasia hypocalcification index (hhi) and radiopaque observation using corel draw version 11 n groups hhi radiophaque 1 2 3 4 5 6 7 8 9 10 i i i i i ii ii ii ii ii 0 0 0 0 0 8 8 8 1 3 196,6 198,4 195,73 196,2 198,2 180,5 88,4 170,87 179,6 102,1 note: n = sample; hhi = hypoplasia hypocalcification index 87indayani: lipopolysaccharide (lps) discussion the outcome of this study identified that lps induction during the period of tooth growth and development resulting the presence of enamel hypoplasia. lps consist of 3 parts: o-polysaccharide chain, core of polysaccharide and lipid-a. lipid-a is a part of endotoxic lps due to receptor cd14 bind / toll-like receptor-4 (tlr4).3 cd14 is receptor of cell surface on macrophage and monocyte.16,17 macrophage and monocyte cell which make bacterial binding due to the presence of cd14 would secrete cytokine and lipid inflammation mediator.18 the study done by yoshimura et al.19 suggested that lps a. actinomycetemcomitans and e. coli are capable to induct pmn secreting il-1b, il-8, tnf-a and interleukine receptor antagonist (il-1ra) in large number, while pmn which inducted to lps p. gingivalis, il-1b is not detected. monocyte which inducted by lps p. gingivalis and e. coli would secret higher il-1b than pmn. il-1b, il-8 and tnf-a stimulate the elevation of osteoclast amount and activities. oseteoclast is kind of cell which functions in degradation of bone matrix.6 induction lps e. coli contributes the elevation of osteoclast number and activities and alveolaris bone resorption.13 either chronic or acute local infection in periodontal tissue which are presented during the period of tooth growth might contribute the failure of dental germ to perform the stage growth i.e. initiation (bud stage) proliferation (cup stage), histodifferentiation and morfodifferentiation (bell stage), aposition as well as calcification resulting the presence of abnormality of the form, number, quality and the quantity of eruptive teeth.20 in enamel hypoplasia, failure is usually presented during the process of amelogenesis.2 according to mc donald & avery2 and wikipedia:20 amelogenesis (enamel formation) is apart of the whole process of tooth growth. amelogenesis occurs after perfect dentin formation done by ameloblast. amelogenesis consist of two stages i.e. secretory stage (aposition) involving organic matrix formation and it is called enamel matrix, this stage functions during the process of enamel mineralization. the second stage is maturation i.e. enamel matrix has perfect calcification.2,20 infection which is presented during the period of enamel matrix formation would contribute the occurrence of hypolasia and if happens during the period of calcification would contribute enamel hypocalcification.2 the stage of matrix aposition and deposition done by formative cell would be the most sensitive stage during the period of growth.21 lps induction resulting stimulation of osteoclast cell which could perform tissue resorption. enamel prisms which has been deposited during aposition and deposition stage would be destroid.22 this case is due to stimulated osteoclast by lps activity resulting the presence of resorption. in addition, ameloblast cell is the most sensitive cell environmental change.23 physiologic change would affect ameloblast and contribute enamel structural change. in general the change could not be seen clinically but it would appear through the microscope. disturbance or infection might result the disturbance of enamel matrix secretion by ameloblast or death of ameloblast. the presented abnormality usually would appear clinically.1 basically, the growth and the development of rats and human molar indicate the same stage during embrional to eruption stage.24 molar amelogenesis on rats has initiated since the age of 20–21 days intrauterine and perfectly formed at the age of 11 days post uterine.21 lps induction done on 5 day-old rats resulting the disturbance of amelogenesis or going process. lps contributes the presence of elevation il-1a, il-1b, il-6, tumor necrosis factor (tnf-a) and pge2, consequently, increasing the number and the activity of osteoclast. this case would cause obstruction of alveolaris bone and the body would fail to stimulate the formation of fibrous wall which might localize the infection, as a result infection would disseminate on dental germ about 20–30%.25 the number and the activity of osteoclast and adontoclast would continually degrade either base matrix or enamel matrix which is newly formed dental germ and disturb ameloblast cell. on 5 day-old rats ameloblast cell is forming enamel matrix and deposition process of organic and inorganic material is occurring. lps induction would cause the death of ameloblast cell therefore enamel matrix with would fail to be formed. in normal condition ameloblast cell secretes enamel matrix with certain immunity.1 due to failure of enamel matrix formation, so there is some enamel which failed to be formed until the period of tooth eruption. as a result enamel becomes thin and either some holes would appear on the surface. table 2. the result of t-test on the presentence of hypoplasia and radiopaque score of rats tooth enamel t-test for equality of means t df sig. (2-tailed) mean difference std. error difference 95% confidence interval of the difference lower upper hhi radiopak –3.725 2.608 4.000 4.006 .020 .059 –5.60000 52.73200 1.50333 20.21627 –9.77391 –3.36594 –1.42609 108.82994 note: t= t count; df = degree of freedom; sig = significant; hhi = hypoplasia hypocalcification index 88 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 85–88 after the thickness of enamel matrix is considered adequate by ameloblast cell, the function of ameloblast cell would change and have important role in the process of enamel maturation. during the occurrence of maturation process, the change of enamel organic component is presented either qualitatively or quantitatively. other changes also happen in enamel organic component i.e. calcium and phosphate influx presented in short time. this condition can cause the presence of crystal growth located in loophole which formed due to disappearance of organic material and water. striated border and alkaline phosphates in ameloblast have importance role on organic ion transport through cell membrane enamel maturation.1 the decrease and the presence of change on ameloblast cell due to lps induction deposition process of either organic and inorganic material would be disturbed, so, the process of enamel mineralization is not perfect. thin enamels due to mineralization disturbance which make the teeth look brownish yellow. brownish yellow color due to dentin appearance look transparent and several part of dentin opened. dentin was looked yellower because the organic content is lower comparing to enamel. the radiophaque shows that rats with hypoplasia, the enamel is more radiolucent compared with control group (figure 2). solid material would adsorb x-ray stronger which causes the radiophaque clear (white) which is called radiophaque, while material with low density, the adsorption would be also weak, which gives dark region or it is called radiolucent.26,27 in normal condition enamel would be more radiograph compared with other body tissue, because enamel consist of 90% mineral so enamel would be more solid.26 on enamel hypoplasia due to the disturbance in matrix deposition either organic or inorganic the density of enamel is lower, as it is reported by mahoney et al.28 that tooth with hypoplasia would get lowering density 10% or between: 2,30–2,50 gr/cm3. the conclusion is lps induction during tooth growth and development causing enamel hypoplasia. references 1. nancy a. ten cate’s: oral histology (development, structure, and function). 6th ed. st louis: mosby inc; 2003. p. 141–91. 2. mcdonald re, avery dr, hartsfield jrjk. acquired and developmental disturbances of the teeth and associated oral structures. in: mcdonald re, avery dr, editor. dentistry for the child and adolescent. 7th ed. missouri: mosby inc; 2000. p. 115–7. 3. wikipedia. lipopolisaccharide. available from: http://en.wikipedia. org/w/index.php. accessed february 23, 2006. 4. sundqvist g. taxonomy, ecology and pathogenicity of the root canal flora. oral surg oral med oral pathol oral radiol endod 1994; 78:522–30. 5. stashenko p. interrelationship of dental pulp and apical periodontitis. in: hargreaves km, goodis, editor. dental pulp. chicago: quintessence publishing co inc; 2002. p. 389–409. 6. schwartz z, goultschin j, dean dd, byan bd. mechanisms of alveolar bone destruction periodontitis. in: page rc, kornman ks, editor. the pathogenesis of perodontitis. periodontology 2000; 1997. 14:158–72. 7. schuurs ahb, moorer wr, prahl-andersen b, van velzen skt, visser jb. patologi gigi-geligi: kelainan-kelainan jaringan keras gigi. sutatmi suryo, editor. yogyakarta: gadjah mada university press; 1992. p. 61–3. 8. w e e r h e i j m k l , j a l e v i k b , a l a l u u s u a s , n o n f l u o r i d e hypomineralizations in the permanen first molars and their impact on the treatment need. caries res 2001; 35:36–40. 9. li y, navia jm, bian jy. caries experience in deciduous dentition of rural chinese children 3–5 years old in relation to the presence or absence of enamel hypoplasia. caries res 1996; 30(1):8–15. 10. lo ec, zheng cg, king nm. relationship between the presences of demarcated opacities & hypoplasia in permanent teeth & caries in their primary predecessors. caries res 2003; 37(6): 456–61. 11. nicolau b, marcenes w, bartley m, sheiham a. a life course approach to assessing causes of dental caries experience: the relationship between biological, behavioral, socio-economic and psychological conditions and caries in adolescents. caries res 2003; 37:319–26. 12. broadbent jm, thomson wm, williams sm. does caries in primary teeth predict enamel defects in permanent teeth? a longitudinal study. j dent res 2005; 84(3):260–4. 13. umezu a, kaneko n, toyama y, wanatabe y, itoh h. appearance of osteoclast by injections of lipopolysaccharides in rat periodontal tissue. j periodont res 1989; 24:378–83. 14. hargreaves ja, cleaton-jones pe, williams sdl. hypocalsification and hypoplasia in permanent teeth of children from different ethnic groups in south africa assessed with a new index. adv dent rest 1989; 3(2):126–31. 15. jamani kd, aqrabawi j, fayyad am. a radiographic study of the relationship between technical qualiyty of coronoradicular post and periapical status in a jordanian population. j oral sci 2005; 47:123–28. 16. akashi s, shimazu r, ogata h, nagai y, takeda k, kimoto m, miyake k. cutting edge: cell surface expression and lipopolysaccharide signaling via the tool-like receptor 4-md-2 complex on mouse peritoneal macrophages. j immunol 2000; 164:3471–5. 17. ziegle-heitbrock hwl, ulevitch rj. cd14: cell surface receptor and differentiation marker. immunol today. 1993; 14:121–5. 18. janeway ca, tarvers p, walport m, shlomchik m. immuno biology. 5th ed. new york: garland publishing; 2001. p. 67–8. 19. yoshimura a, hara y, kaneko t, kato i. secretion of il-1b, tnf-a, il-8 and il-1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. j periodont res 1997; 32:279–86. 20. w i k i p e d i a . t o o t h e n a m e l . a v a i l a b l e f r o m : h t t p : / / en.wikipediafoundation.org/w/index.php. accessed november 28, 2006. 21. farris, griffith. the rat in laboratory investigation. new york: hafner publishing co; 1971. p. 124. 22. heiserman dl. oral and maxillofacial pathology. available from: http://www.waybuilder.net/free.ed/qlinks03/contct.asp?usr. accessed november 28, 2006. 23. navarro lf, garcia aa, marco jm, llena-puy mc. a study of the clinical, histopathologic and ultrastructural aspects of enamel agenesis: report of case. j dent for child 1999; 208–12. 24. gaete m, lobos n, torres-quintana ma. mouse tooth development time sequence determination for the icr/jcl strain. j oral sci 2004; 46:135–41. 25. mcdonnell st, liversidge h, kinirons m. temporary arrest of root development in premolar of a child with hypodontia and extensive caries. int j paediatr dent 2004; 14(6):455–60. 26. white sc, pharooh mj. oral radiology: principles and interpretation. 5th ed. st louis: mosby co; 2004. p. 166–90. 27. wikipedia. x-ray. available from: http://en.wikipedia.org/wiki/ nuclear_power. accessed november 28, 2006. 28. mahoney ek, ismail fsm, kilpatrick n, swain m. mecahnical properties a cross hypomineralized/hypoplastic enamel of first permanent molar teeth. eur j oral sci 2004; 112:197–502. 148 volume 46, number 3, september 2013 research report efek ekstrak buah delima (punica granatum l) terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster (the pomegranate extracts (punica granatum l) effect on the wild p53 expression in oral mouth malignant cell of swiss webster strain mice) sri hernawati,1 fedik abdul rantam,2 i ketut sudiana3 dan retno pudji rahayu4 1bagian penyakit mulut, fakultas kedokteran gigi, universitas jember, jember-indonesia 2departemen mikrobiologi veteriner, fakultas kedokteran hewan, universitas airlangga, surabaya indonesia 3 departemen biomedik, fakultas kedokteran, universitas airlangga, surabaya-indonesia 4departemen patologi mulut dan maksilofasial, fakultas kedokteran gigi, universitas airlangga, surabaya-indonesia abstract background: squamous cell carcinoma is the most common cancer in the oral cavity. dna tests showed that almost 90% of cases revealed wild p53 gene mutations. wild p53 gene mutations cause p53 inactivation so the cell cycle does not stop in g1 phase but continues to s phase and g2 and m, it makes the mutated dna remains multiplied and apoptosis does not occur. one candidate of the cancer treatment alternatives is pomegranate extract (punica granatum l – pgl). purpose: the purpose of study was to examine the effect of pgl on wild p53 expression in oral cavity malignant cell of swiss webster strain mice. methods: thirtytwo swiss webster strain mice (balb/c) 5 months old were randomly divided into four groups. two control groups (k0: no benzopirene exposed and untreated; k1: benzopirene exposed and untreated); and 2 treatment groups (p1: benzopirene exposed and given ea; p2: benzopirene exposed and given pgl extract). the expression of wild p53 was determined by immunohistochemical techniques. results: the results showed that administration of pgl could increase the expression of wild p53 in malignant epithelial cells in the oral mucosa of mice, and the expression was higher than ea. conclusion: this study suggested that the pgl extract could express wild p53 in the oral cavity malignant cells of swiss webster strains mice. key words: punica granatum l (pgl), ellagic acid, malignant cells, wild p53 abstrak latar belakang: karsinoma sel skuamosa merupakan kanker yang sering terjadi pada rongga mulut. pemeriksaan dna menunjukkan hampir 90% kasus dijumpai adanya mutasi gen wild p53. mutasi gen wild p53 menyebabkan inaktivasi wild p53 sehingga siklus sel tidak berhenti pada fase g1 tetapi berlanjut ke fase s dan g2 dan m, sehingga dna yang mengalami mutasi tetap dilipatgandakan dan apoptosis tidak terjadi. salah satu kandidat obat kanker adalah ekstrak buah delima (punica granatum l pgl). tujuan: penelitian ini bertujuan untuk meneliti efek ekstrak pgl terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster. metode: tiga puluh dua ekor mencit (balb/c) strain swiss webster jantan berumur 5 bulan dibagi secara random menjadi 4 kelompok, yaitu 2 kelompok kontrol (k0: tidak dipapar benzopirene dan tidak diberi perlakuan; k1: dipapar benzopirene dan tidak diberi perlakuan); serta 2 kelompok perlakuan (p1: dipapar benzopirene dan diberi ea; p2: dipapar benzopirene dan diberi ekstrak pgl). pemeriksaan ekspresi wild p53 dilakukan dengan teknik imunohistokimia. hasil: hasil penelitian menunjukkan bahwa pemberian ekstrak pgl dapat meningkatkan ekspresi wild p53 pada sel epitel ganas pada mukosa rongga mulut mencit, dan lebih 149hernawati, dkk.: efek ekstrak buah delima (punica granatum l) terhadap ekspresi wild p53 timggi dibanding dengan pemberian ea. simpulan: penelitian ini menunjukkan bahwa ekstrak pgl dapat meningkatkan ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster kata kunci: punica granatum l (pgl), ellagic acid, sel ganas, wild p53 korespondensi (correspondence): sri hernawati, bagian penyakit mulut, fakultas kedokteran gigi universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: srihernawati.drg5@yahoo.com pendahuluan angka kejadian karsinoma sel skuamosa rongga mulut menempati urutan ke enam di dunia, di india dilaporkan setiap tahun 75.000-80.000 kasus baru, di singapura dan berbagai negara asia lainnya juga dilaporkan tinggi angka kejadiannya.1,2 beberapa penelitian di asia tenggara, diketahui bahwa mukosa bukal merupakan daerah karsinoma sel skuamosa yang paling umum yaitu sebesar 50-72%.2 penanganan yang kurang optimal menyebabkan karsinoma sel skuamosa rongga mulut bermetastasis. hal ini menyebabkan 30-65% penderita meninggal dunia dalam kurun waktu 5 tahun. pemeriksaan dna kasus karsinoma sel skuamosa rongga mulut menunjukkan hampir 90% kasus dijumpai adanya mutasi gen wild p53.3 mutasi gen menyebabkan inaktivasi wild p53 sehingga siklus sel tidak berhenti pada fase g1 tetapi berlanjut ke fase s, g2 dan m. pada proses apoptosis wild p53 berperan memicu faktor transkripsi p21 dan memicu aktivasi protein bax (protooncogene yang bersifat pro–apoptosis), menekan protein bcl-2 (protooncogene yang bersifat inhibitor apoptosis). wild p53 yang mengalami mutasi tidak dapat menginduksi apoptosis.4 apoptosis fisiologis merupakan proses kematian sel dalam rangka mempertahankan integritas tubuh secara keseluruhan dan berperan penting menjaga homeostatis. apoptosis patologis adalah membatasi proliferasi sel yang tidak diperlukan termasuk sel ganas. apoptosis patologis merupakan mekanisme yang efesien untuk mengeliminasi sel yang tidak diperlukan dan berbahaya. pada sel ganas, apoptosis ini mengalami gangguan maupun hambatan.5 wild p53 merupakan tumor supresor gen yang akan mengaktifasi pembentukan p-21. peningkatan p-21 yang disintesisakan menekan semua cyclin dependent protein kinase (cdk). terjadinya siklus pembelahan sel sangat tergantung pada ikatan kompleks antara cdk dengan cyclin.5 dengan terjadinya penekanan semua cdk, maka siklus sel akan berhenti. saat berhenti,wild p53 akan memicu aktivitas protein bax, dimana protein bax akan menekan gen bcl-2 (protein yang berperan sebagai anti apoptosis) pada membrane mitokondria, sehingga terjadi perubahan permeabilitas membrane mitokondria, kemudian terjadi pelepasan cytokrom-c kesitosol. cytokrom-c akan mengaktivasi apaf-1. apaf-1 mengaktivasi kaskadekaspase, dan kaspase yang aktif akan mengaktivasi dna-se sehingga terjadi apoptosis.6 berbagai upaya penatalaksanaan penyakit kanker masih banyak menemui kendala, yang mengakibatkan kurangnya keberhasilan dalam mencegah dan mengobati keganasan. salah satu upaya pengobatan yang sudah dirintis sejak zaman dulu adalah pemanfaatan fitofarmaka, menggali kandungan unsur kimiawi dalam tumbuh–tumbuhan yang potensial dapat dipakai sebagai obat. salah satu tanaman obat adalah buah delima (punica granatum l pgl). kelompok utama fitokimia buah delima adalah polyphenol, polyphenol buah delima terdiri dari flavonoids (flavonols, flavonols dan anthocyanins), hydrolyzable tannins (ellagitannins dan gallotannins) dan condensed tannins (proanthocyanidins). buah delima memiliki khasiat terapeutik antara lain; anti bakteri, anti virus, anti kanker,anti inflamas.6,7 whole ekstrak buah delima (pgl) dengan standarisasi 40% ellagic acid (ea) dapat menghambat perkembangan sel kanker, anti poliferasi, menginduksi apoptosis dan anti oksidan secara invitro.7,8 ekstrak buah delima dapat meningkatkan wild p53 secara in vitro pada biakan sel karsinoma skuamosa lidah manusia dengan dosis 250 ug/ml.9 standarisasi 40% ea dengan tujuan 40% dapat menggambarkan kekuatan delima yang bertanggung jawab terhadap aktivitas farmakologi.10 ellagic acid merupakan salah satu bahan aktif dari whole ekstrak buah delima (pgl) berada dalam buah delima dalam bentuk bebas sebagai ellagic acid-glycosides atau terikat dalam bentuk ellagitannins.11ellagic acid secara in vitro berfungsi sebagai anti kanker tapi masih jarang diteliti secara in vivo. aktivitas dan konsentrasi ea dalam plasma rendah disebabkan kelarutan dalam air rendah, disamping itu ea mudah mengalami transformasi dan degradasi sebelum diabsorbsi.12,13 whole ekstrak buah delima (pgl) mempunyai beberapa bahan aktif yang kemungkinan bekerja sinergis, diantaranya polyphenol dalam buah delima dapat meningkatkan kelarutan dan absorbsi ea sehingga whole ekstrak buah delima (pgl) mempunyai efek anti kanker yang lebih poten.14,15 apabila efek whole ekstrak buah delima (pgl) pada mencit strain swisswebster (balb/c) dapat terungkap, maka whole ekstrak buah delima (pgl) dapat dijadikan sebagai salah satu alternatif pengobatan terhadap karsinoma sel skuamosa rongga mulut. penelitian ini bertujuan untuk meneliti efek ekstrak pgl terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster. bahan dan metode jenis penelitian ini adalah penelitian eksperimental laboratorium. mencit yang digunakan adalah strain swiss 150 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 148–151 tabel 1. rerata dan standar deviasi sel yang mengekspresikan wild p53 kelompok (n=6) jumlah sel yang mengekspresikan wild p53 (rerata ± sd) kontrol (-)/k0 kontrol (+)/k1 (benzopirene + cmc) p1/(benzopirene + ea) p2/(benzopirene+pgl) 0,383± 0,194a 0,133 ± 0,216c 0,333 ± 0,160ab 0,357± 0,164ab gambar 1. h a s i l p e m e r i k s a a n p r e p a r a t d e n g a n t e k n i k imunohistokimia, sel mengekspresikan wild p53 (berwarna coklat) dengan pembesaran 400x. webster (balb/c), berat badan berkisar 30-50 gram, jenis kelamin jantan, berumur 5 bulan, yang diperoleh dari unit hewan coba universitas gajah mada, yogyakarta. mencit dibagi 4 kelompok, yaitu 2 kelompok kontrol; k0 (tidak dipapar benzopirene dan tidak diberi perlakuan), k1 (dipapar benzopirene dan tidak diberi perlakuan), dan 2 kelompok perlakuan; p1 (dipapar benzopirene dan diberi ea), p2 (dipapar benzopirene dan diberi whole ekstrak pgl), tiap kelompok ada 8 ekor mencit. whole ekstrak buah delima (pgl) adalah ekstraksi seluruh bagian buah delima dalam bentuk serbuk dan telah terstandarisasi mengandung 40% ea (produksi xian biof biotechnology co. ltd people republic of china). ellagic acid adalah kristal putih ea merupakan salah satu komponen bahan aktif whole ekstrak buah delima (pgl) yang diproduksi xian biof biotechnology co. ltd dengan whole ekstrak buah delima (pg). mencit dipapar benzopirene 0,04 mg/0,04 ml olium olivarum secara per oral seminggu 3 kali selama 4 minggu pada mukosa bukal sebelah kanan rongga mulut mencit. pada akhir minggu ke-9 jaringan mukosa rongga mulut mencit dibiopsi, kemudian dikorbankan. hewan model karsinoma sel skuamosa adalah mencit (balb/c) yang telah mengalami keganasan pada epitelnya akibat paparan benzopirene dengan gambaran mikroskopis; menunjukkan proliferasi sel–sel epitel skuamous, sel-sel atipia disertai perubahan bentuk rete peg processus, pembentukan keratin yang abnormal, susunan sel tidak teratur. whole ekstrak buah delima (pgl), ea diberikan per oral setiap hari selama 4 minggu. dosis whole ekstrak buah delima (pgl) ea adalah 75 mg/ kg/bb/hari dilarutkan dalam cmc-na 0,3 %. pemeriksaan laboratorium yang digunakan untuk ekspresi bcl-2 dengan menggunakan imunohistokimia. prosedur pemeriksaan ekspresi wild p53 meliputi: 1) persiapan reagen: tahap fiksasi. 2) pewarnaan; 3) pencucian; 4) pelebelan; dan 5) pembacaan. bahan untuk pemeriksaan imunohistokimia: h202 3%, tripsin 0.025%, pbs, aquadestilata, buffer substrat, xylol, etanol absolut, metanol, air, anti wild p53 (mouse antirat) antibodi, kaca obyek polil-lisin, buffer, formalin, labeled, antiglobulin, sekunder, antibody, streptavidin. gambaran imunohistokimia, sel yang mengekspresikan protein wildp53 berwarna coklat. penghitungan sel dilakukan pada 10 lapangan pandang dengan mikroskop menggunakan pembesaran 400x dan disajikan reratanya (gambar 1). analisis data penelitian menggunakan uji normalitas, uji homogenitas, uji anova, uji lsd. hasil analisis antar kelompok perlakuan menggunakan lsd. hasil h a s i l p e m e r i k s a a n p r e p a r a t d e n g a n t e k n i k imunohistokimia,ekspresi wild p53 terlihat pada tabel 1. hasil pemeriksaan preparat dengan teknik imunohistokimia menunjukkan bahwapemberian wholeekstrak buah delima terstandar(p2/benzopirene+pgl) menunjukkan peningkatan ekspresiwild p53 paling tinggi di bandingka ndengankelompokperlakuan yang lain. kenaikan ekspresi wild p53 pada kelompok p2 (benzopirene + pgl) (0,357 ± 0,164) tidak berbeda secara signifikan bila dibandingka nkelompokkontrol (ko/ mencit normal) ((0,383 ± 0,194) dan p1(benzopirene + ea) (0,333 ± 0,160) . kelompok p2, p1 dan k0 berbeda secara signifikan demgan kelompok k1 (0,133 ± 0,216). pembahasan wild p53 merupakan protein kelompok tumor supressor gene yaitu suatu protein yang berperan sebagai faktor pengendalian pertumbuhan sel, bekerja didalam inti sel, khususnya pada proses pengendalian siklus pembelahan sel. wild p53 merupakan faktor transkripsi terhadap pembentukan p21, peningkatan p21 yang disentesis akan menekan semua cdk, terjadinya siklus pembelahan sel sangat tergantung pada cdk. cdk ditekan dan tidak berfungsi sehingga siklus pembelahan sel akan berhenti. wild p53 akan memicu aktivitas bax (pro apoptosis), aktivitas bax akan menekan bcl (antiapoptosis), sehingga terjadi pelepasan cytochrome-c, mengaktifkan apaf-1 selanjutnya mengaktivasi kaskade-kaspase. kaspase yang aktif mengaktifkan dna-se, dna-se menembus membran inti dan merusak dna yang mutasi sehingga dna sel yang bersangkutan rusak (fragmentasi) dan akhirnya sel mengalami kematian (apoptosis).11 151hernawati, dkk.: efek ekstrak buah delima (punica granatum l) terhadap ekspresi wild p53 wild p53 memiliki paruh umur yang sangat singkat (4 sampai 5 menit) sedangkan bentuk p53 mutan dari protein ini lebih stabil, dengan paruh umur 6 jam. gen p53 diketahui bermutasi pada sekitar 70% kejadian kanker.12 pada karsinoma sel skuamosa rongga mulut, pemeriksaan dna menunjukkan mutasi wild p53, dijumpai hingga 90% kasus.3 mutasi wild p53 sebagai pointmutation, menghasilkan protein dengan struktur berubah yang mengisolasi protein wild p53, sehingga menginaktivasi aktivitas fungsi wild p53.12 ellagic acid dapat meningkatkan ekspresi reseptor kematian (apoptosis), antara lain peningkatan reseptor trail r2/dr2. ekspresi dr5 diatur oleh wild p53 sehingga bisa dihubungkan dengan wild p53, peningkatan wild p53 akan meningkatka reseptor dr5. dr5 mengikat stimulasi kematian dan menyebabkan aktivasi pro-caspase 8.13 ellagic acid dapat menginduksi pada g0/g1 melalui peningkatan level wild p53 dan aktivasi caspase-3 dalam sel t24 kanker bladder, melalui pemeriksaan flowcytometry dan pcr (poly chain reaction).14 ellagic acid telah terbukti memiliki aktivitas antioksidan, antiinflamasi dan dapat mencegah destruksi gen p53 oleh kanker. selain itu ellagic acid juga dapat berikatan dengan sel kanker dan membentuk suatu molekul kompleks, sehingga sel kanker menjadi inaktif.15 whole ektrak buah delima (pgl) terstandar lebih kuat efeknya terhadap peningkatan ekspresi wild p53 dibandingkan ea, hal ini membuktikan whole ekstrak buah delima (pgl) lebih efektif untuk meningkatkan wild p53. penelitian ini menginformasikan bahwa whole ekstrak buah delima (pgl) yang mengandung beberapa bahan aktif, memiliki efek unggul dan bekerja secara sinergis. empat kandungan bahan aktif dari buah delima (pgl), yaitu ea, caffeic acid, luteolin, punicid acid secara individual menunjukkan aktivitas antikanker pada sel kanker prostat. namun bila dikombinasikan, keempatnya menunjukkan aktivitas yang berlipat ganda8 dibanding ea lebih rendah karena ea mudah mengalami transformasi dan degradasi sebelum diabsorbsi, kelarutannya rendah dalam air, metabolisme ea tidak larut dalam intestinal.11 penelitian ini menunjukkan bahwa ekstrak pgl dapat mengekspresi wild p53 pada pada sel ganas rongga mulut mencit strain swiss webster daftar pustaka 1. epstein w. oral cancer.texbook of oral medicine. burket eleventh edition. hamilton: bc decker inc press; 2008 .p. 153-67. 2. solomon mc, carnelio s, gudattu v. molecular analysis of oral squamous cell carcinoma:a tissue microarray study. kamataka india: department of oral pathology, manipal college of dental science, manipal university manipal; 2010. p. 104-576. 3. safriadi m. patologi muluttumor neoplastik dan nonneoplastik rongga mulut. jogyakarta: cv andi; 2008. p. 73-83. 4. sudiana ik. patobiologi molekuler kanker. jakarta: penerbit salemba medika; 2008. p. 27-90. 5. salido gm, rosado ja. apoptosis; involvement of oxidative stress and intracellular ca2 homeostasis spain. dept of physiology university of ektremadure; 2009. p. 35-229. 6. lansky ep, newman ra. punica granatum (pomegranate) and its potential for preventif and treatment of inflammation and cancer. j ethnopharmaceol 2007; 109(2): 177-206. 7. jurenka j. therapeutic applications of pomegranate (punica granatum l): areview. altern med rev 2008; 13(2): 128-44. 8. seeram np, schulman rn, heber d. pomegranate ancient roots to modern medicine. 1st ed. new york: taylor and francis group; 2006. p. 2-99. 9. kholifa m. pengaruh konsentrasi ekstrak etanol buah delima (punica granatum linn) terhadap peningkatan apoptosis sel kanker lidahmanusia sp-c1 in vitro. biomedika 2010; 2(2): 72-80. 10. saifudin a, rahayu v, teruna hy. textbook standardisasi bahan obat alam. edisi pertama. yogyakarta: press universitas gajah mada 2011; l(1): 1-26. 11. kresno sb. textbook ilmu dasar onkologi. edisi kedua. jakarta: badan penerbit fakultas kedokteran universitas indonesi; 2011. h. 66-129. 12. william. molecular pathogenesis of oral squmous carcinoma. j clin oral pathology birminghan dental hospital and school; 53: 72-165. 13. mohammad hf. ellagic acid mediated ck2 inhibition. anatural multifunction strategy to trigger carvical cancer cell death invitrio and in vivo. dissertation. department of chemistry, cleve land state university; 2002. p. 1–24. 14. lansky ep, newman ra. punica granatum (pomegranate) and its potential for preventif and treatment of inflammation and cancer. j ethnopharmacol 2007; 109(2): 128-44. 15. seeram np, adam ls, henning sm, niu y, zhang y, nair mg, heber d. in vitro antiproliferative, apoptosis and antioxidant activitics of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenol as found in pomegranate juice. j nutr biochem 2005; 16(6): 360-7. � volume 47, number 1, march 2014 research report the role of hsp�0, cd-8 and ifn-γ in immunopathobiogenesis of periapical granuloma in dental caries risya cilmiaty1 and mandojo rukmo2 1 department of dental and oral, faculty of medicine, university of sebelas maret, surakarta-indonesia 2 department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: the incidence of dental caries with periapical granulomas in indonesia is quite high. however, the mechanism of the formation of periapical granulomas in dental caries caused by bacterial infection in immunopathobiogenesis cannot be explained completely. thus, this explanation is necessary in order to be used as a basis for diagnostic, preventive and therapeutic measures. purpose: this research was aimed to determine the role of hsp60, cd-8 and ifn-γ in immunopatobiogenesis of periapical granuloma in dental caries. methods: this research was an analytic observational study with cross sectional approach. samples of this research were 36 teeth of patients with dental caries, consisting of 18 caries teeth with periapical granulomas and 18 caries teeth without periapical granulomas. the variables observed in this research were hsp60, cd-8 and ifn-γ. measurements were conducted by using immunohistochemical methods on periapical tissue. results: the mean of hsp60, cd-8 and ifn-γ in granuloma group was significantly higher than those in non granuloma group (p<0.05). the positive role of ifn-γ on the incidence of granulomas appeared to be more prominent. conclusion: the study suggested that in immunopathobiogenesis of periapical granuloma in dental caries, hsp60, cd-8 and ifn-γ played important roles, but the role of ifn-γ was found to be more prominent. key words: dental caries, immunopathobiogenesis, periapical granuloma, hsp60, cd-8, ifn-γ abstrak latar belakang: angka kejadian gigi karies dengan granuloma periapikal di indonesia cukup tinggi, namun mekanisme terbentuknya granuloma periapikal pada gigi karies yang disebabkan oleh infeksi bakteri secara imunopatobiogenesis belum dapat dijelaskan secara tuntas. adanya penjelasan ini diperlukan agar dapat digunakan sebagai dasar pengembangan diagnosis, langkah preventif dan terapinya. tujuan: penelitian ini bertujuan untuk mengetahui peran hsp60, cd-8 dan ifn-γ dalam immunopatobiogenesis dari granuloma periapikal karies gigi. metode: penelitian ini merupakan penelitian observasional analitik dengan pendekatan cross sectional. sampel dari penelitian ini adalah 36 gigi pasien dengan karies, yang terdiri dari 18 karies gigi dengan granuloma periapikal dan 18 karies gigi tanpa granuloma periapikal. variabel yang diamati adalah hsp60, cd-8 dan ifn-γ. pengukuran dilakukan dengan menggunakan metode imunohistokimia pada jaringan periapikal. results: rerata hsp60, cd-8 dan ifn-γ pada kelompok granuloma secara signifikan lebih tinggi dibanding kelompok non granuloma (p <0,05). peran positif dari ifn-γ terhadap kejadian granuloma tampaknya lebih menonjol. simpulan: studi ini menunjukkan bahwa dalam imunopatobiogenesis dari granuloma periapikal karies gigi, hsp60, cd-8 dan ifn-γ memainkan peran penting, tetapi peran ifn-γ ditemukan lebih menonjol. kata kunci: gigi karies, imunopatobiogenesis, granuloma periapikal, hsp60, cd-8, ifn-γ correspondence: risya cilmiaty, c/o: departemen gigi dan mulut, fakultas kedokteran universitas sebelas maret. jl. ir. sutami 36 a kentingan, surakarta 57126, indonesia. e-mail: h_risya@yahoo.co.id 8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 7–12 introduction dental caries with chronic periapical can be considered as a multifactorial infectious disease caused by irritants in necrotic pulp moving into periapical tissues. cases involving chronic periapical disease was necrotic pulp can be treated with root canal treatment. the continuous exposure of those irritants to the periapical tissues will stimulate host defense in the form of periapical granulomas. the healing process of periapical granulomas is actually associated with the body’s immune response. thus, it is possible for recurrence despite the application of root canal treatment, or it may even develop into a radicular cyst, which is more difficult to treat. therefore, if the process to being granuloma formation can be prevented, many difficulties in healing process of periapical granulomas can be overcome. unfortunately, the mechanism of immunopathobiogenesis of periapical granulomas caused by chronic periapical in dental caries still cannot be explained. many researches only focus on the causes of dental pulp necrosis, such as tumor necrosis factor α (tnf α) and interleukin-6 (il-6) in periapical lesions.1 the mechanism of periapical granuloma is necessary to be studied since the number of cases of dental pulp necrosis is more commonly found in patients with dental caries. histological changes in the periapical tissues caused by bacterial invasion, moreover, will be indicated by the presence of granulation tissue containing with macrophages, lymphocytes, plasma cells, neutrophils, and fibrovascular elements in varying numbers. at the same time, there will also be damage to the periapical tissues and bone resorption.3 periapical granuloma is actually composed of granulation tissue surrounded by a cell wall of fibrous connective tissue. in histopathological examination of the chronic lesions, it can be known the existence of lymphocytes, plasma cells, neutrophils, histiocytes, eusinophils, and epithelial cell rests of mallesse.4 lymphocytes are the predominant cell type (50%), which number is closely related to the total number of the following cells, namely cds -4 t cells and cd-8 t cells. therefore, in chronic lesions the number of cd-8 t cells is increased. all of their structures are surrounded by a capsule of fibrous connective tissues consisting of cd-8 t lymphocytes.3 therefore, it can be said that pathological periapical granuloma is frequently caused by dental caries.1 dental caries is considered as a chronic pathological process caused by microorganisms found in dental caries tissue as a potential immunogen leading to changes in the pulp tissue related to immune response.5, 6 in other words, bacteria can be considered as an important factor in the development and growth of dental caries. the presence of anaerobic bacteria considered as pathogenic can trigger macrophages to form periapical granulomas. in the process of periapical granuloma formation, various components have important roles, namely hsp60 as chaperone playing a role in the fraction of proteins involved in apc. it is because hsp60 synthesized in the exosome is used to assist the synthesis and maturation of proteins to become functional ones. consequently, the processing of epitope can be run, and hsp60 will be recognized by ctl/cd-8, which then will secrete ifn-γ. 7, 8 furthermore, ifn-γ released by both th-1 and ctl/cd-8 will induce macrophage activities. thus, those macrophages will migrate around those containing intracellular bacteria, and then create granuloma.9 it is because the presence of pathogenic bacteria will trigger some histiocytes to develop into macrophages and apc leading to the formation of granulomas, while some others will develop into phagocytic causing no granulomas.the study was aimed to determine the role of hsp60, cd-8 and ifn-γ in immunopaobiogenesis of periapical granuloma in dental caries. materials and methods this research was an analytic observational research with cross sectional approach. the samples of this research were selected from the population based on inclusion criteria (purposive sampling). the samples were divided into two groups, consist of group with granulomas and group with no-granulomas. the subjects were 36 patients who were indicated for tooth extraction and to accept informed consent about the purposes and objectives of this research. those patients classified into group with granulomas must have a diagnosis of dental pulp necrosis with periapical granulomas. diagnose was determined through clinical and radiographic examinations. clinically the depth of cavity reached the pulp and radiographic figure showed that there was radiolucent area with distinct border with 0.5 mm-2 of diameter. in the other hand, those patients who were classified into non-granulomas group must have a diagnosis of dental pulp necrosis without periapical granulomas indicated clinically by the depth of pulp cavity and the radiographic figure showed no periapical radiolucency. in the group with granulomas, periapical tissues around tooth extraction were taken by using a pair of tweezers. meanwhile, in the group with non-granulomas, periapical tissues taken were tissues attached to the extracted tooth. those periapical tissues were partially fixed in 10% buffered formalin, then histopathologically examined by using hematoxylin eusin (he) staining, and observed by using a microscope with a magnification of 400 times. afterwards, the number of cells producing hsp60, cd-8 and ifn-γ contained in those periapical tissues was calculated by using immunohistochemical method. in the immunohistochemical method, staining process was conducted by using monoclonal antibodies against hsp60, cd-8 and ifn-γ. then those were observed by using a light microscope magnification of 400 times, and also carried out by means of counters in five visual fields. next, the results �cilmiaty and rukmo: the role of hsp60, cd-8 and ifn-γ in immunopathobiogenesis figure 1. the incision of apex tissue: (a) granuloma using cd-8; (b) non-granuloma using cd-8; (c) granuloma using hsp60; (d) non-granuloma using hsp60; (e) granuloma using ifn-γ; (f) non-granuloma using ifn-γ. brownish color for marking positive reaction to hsp60 in macrophages, and brown color for marking positive reaction to cd-8 and ifn-γ in lymphocytes. magnification 400 x. 5 results the results of immunohistochemical examination using monoclonal antibody, anti-hsp60, cd-8 lymphocytes and ifn induced into granuloma and non-granuloma tissues can be seen in figure 1. (a) (b) (c ) (d) (e) (f) figure 1. the incision of apex tissue: dental granulomas (a) and non-granulomas (b), using abm cd-8 in dental granulomas (c), using abm cd-8 in non-granulomas (d) using abm hsp60 in dental granuloma (e) using abm hsp60 in non-granulomas (f ) using ifnabm. brownish color for marking positive reaction to hsp60 in macrophages, and brown color for marking positive reaction to cd-8 and ifn in lymphocytes. magnification 400 x. of the examination showed the number of cells producing hsp60, cd-8 and ifn-γ that would give a positive reaction to monoclonal antibodies, divided by the total (number of cells) and multiplied by 100%.21 finally, multivariation statistical analysis was used to measure the differences of the average number of cells producing hsp60, cd-8 and ifn-γ among the groups. besides that, discriminant analysis was also conducted to obtain the mechanism of periapical granulomas and the patterns of variable contribution between in the group with granulomas and in the group with non-granulomas, so the dominant variable used to distinguish them could be determined. results the results of immunohistochemical examination using monoclonal antibody, anti-hsp60, cd-8 lymphocytes and ifn-γ induced into granuloma and non-granuloma tissues can be seen in figure 1. the calculation results of cells producing hsp60, lymphocytes cd-8, and ifn-γ in the group with granulomas and in the group with nongranulomas were obtained by using immunehistochemical method. the mean and standard deviation of those three variables on each group can be seen in table 1. based on the results, it can be said that the mean of hsp60, cd-8 lymphocytes and ifn-γ in the group with �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 7–12 table 1. the mean and standard deviation of immunity components, namely hsp60, cd-8 lymphocytes and ifn-γ immunity components (%/) granuloma (n = 18) non-granuloma (n = 18) mean standard deviation mean standard deviation hsp60 5.68 7.47 0.95 0.01 cd-8 3.71 4.56 0.19 0.00 ifn-γ 5.61 2.73 0.42 0.00 wilks’ lambda p = 0.000 table 2. fisher’s linear discriminant function with variable ifn-γ immunity components group granulomas non-granulomas ifn – γ 146.604 11.052 (constant) -4.808 -0.717 granulomas was larger than that in the group with nongranulomas (table 1). the results of multivariate analysis using wilks ‘lambda test shows p<.001. this means that there was a significant difference between the mean of hsp60, cd-8 lymphocytes and ifn-γ in the group with granulomas and that in the group with non-granulomas. moreover, based on the results of a statistical test with stepwise method, it is known that ifn-γ was the only one dominant variable in distinguishing the group with granulomas and the group with non-granuloma. the minimum of f value of ifn-γ was about 0.1, while the f values of hsp60 and cd-8 lymphocytes were 0.998 and 0.087. since the f values of hsp60 and cd-8 lymphocytes were higher than the maximum limit of 0.05 for inclusion in the analysis, hsp60 and cd-8 lymphocytes did not become discriminative with the grouping of granulomas/ non-granulomas. however, the discriminant analysis resulted in fisher’s linear discriminant function as shown in table 2. cross tabulation result from discriminant pattern can bee seen on the table 3. based on fisher’s linear discriminant function above, it was found that there were two groups of samples with granulomas incorrectly classified as the group with non-granulomas, while for the sample of non granulomas there was not any incorrect classification. the means of difference among those two groups was 94.4%, namely 88.9% for the group with granulomas and 100% for the group with non-granulomas. the results of cross-tabulations of the discriminant pattern can be seen in table 3. furthermore, the positive role of ifn-γ on the incidence of granulomas appeared more clearly as seen in figure 2, where the mean and standard deviations of both variables distinguishing the group with granulomas and the group with non-granulomas granuloma were calculated based on fisher’s function. discussion based on table 1, it is known that the average number of cells producing hsp60 in the group suffering from granulomas (5.68) was significantly different that in the group suffering from non-granulomas (0.95). similarly, a research conducted by suzuki et al.10 also showed that there was an increase in the local expression of hsp60 in periapical inflammatory lesions. this increasing of hsp60 expressions in patients with periapical granulomas is caused by immune response to the inflammatory later leading to the expression of mhc class i.7,9 the producing of the expression of mhc class i then will trigger a series of processes to produce several cytokines, including ifn-γ, tnf-α and tnf-b. on the other hand, the presence of mhc class i expression on the cell surface will also be recognized by cd-8 lymphocytes, thereby increasing the expression of cd-8. however, in the cases of periapical granulomas, the increasing of cd-8 expression does not occur since the release of ifn-γ activates monocytes (macrophages) which will surround intracellular bacteria. it happened because there is stimulation of ifn-γ , especially maf, which then will lead to the formation of granuloma.8,11 table 3. cross tabulation of the original data classification and the sample data classification based on discriminant pattern with ifn-γ group prediction of the sample datathe sample data classification based on based on discriminant pattern total granulomas non granulomas granulomas 16 (88.9%) 2 (11.1%) 18 (100%) nongranulomas 0 (0%) 18 (100%) 18 (100%) figure 2. discrimination pattern of ifn-ifn-γ. granulomas non granulomas ��cilmiaty and rukmo: the role of hsp60, cd-8 and ifn-γ in immunopathobiogenesis the average number of cells producing cd-8 in the group with granulomas (3.71) was significantly different that in the group with non-granulomas (0.19). the difference is caused by high immunity possessed by those patients with granulomas. as a result, bacteria entering to the body will be phagocytized by monocytes, so they will get phagolysosome and be neutralized by enzyme system (lysozyme). bacteria that are resistant to lysozyme will be destroyed by monocytes with reactive oxygen species (ros) system, especially super oxidant radicals, so the bacteria will get oxidative stress. to protect proteins contained in the body of bacteria from damage, the bacteria then will release stress proteins (hsp). when the attacks of intracellular bacteria occur continuously, the host cell will release excessive ros. as a result, anti-ros produced by the host will become unbalanced. in other words, ros is greater than anti-ros (scavenger enzyme), so the host cells will get oxidative stress. the host cells that distress then will lead to the damage of proteins in the host cells. thus, to prevent the damage, the host cell releases stress protein, hsp60. hsp60 is synthesized in the exosome and serves as chaperones, which will help folding and can be used to assist the synthesis and maturation of proteins to become functional ones. consequently, the processing of epitope can be run, and hsp60 will be recognized by ctl / cd-8, which then will secrete ifn-γ.7, 8 cd-8 lymphocyte can be considered as a specific phenotype of mononuclear cells used to differentiate asymptomatic lesions and symptomatic lesions in patients with periapical lesions. according to colic et al.,12 the percentage of cd-8 cells in symptomatic lesions is higher than that in asymptomatic lesions. in other words, the research proves the existence of cd-8 lymphocytes in asymptomatic lesions although it does not distinguish further the percentage of cd-8 cells in granuloma tissue and that in non-granuloma tissue in patients with asymptomatic periapical lesions. the average number of cells producing ifn-γ in the group with granulomas was 5.61 and that in the group with non-granulomas was 0.42 (table 1). it means that the increasing of immunity indicates the increasing of the ability of cells to eliminate bacteria. besides that, it also means that intracellular signals will be increased, so cytokines playing a role in delivering extracellular signal, namely il-12, will be improved. the cytokines also play a role in inducing t lymphocytes to secrete ifn-γ. the secretion of ifn-γ then will stimulate the activation of macrophages playing a role to prevent the diffusion of bacteria, thus, the cells containing intracellular bacteria will be surrounded by macrophages, and then granuloma will be formed.8,11 similarly, a research conducted by breloer et al.,13 also showed that the in vitro release of ifn-γ can dramatically be increased as the increasing of hsp60 moving to t cells and macrophages, but either the production of il-2 or the proliferation of t cells will not be increased. it means that the induction of ifn-γ is dependent entirely on the ability of macrophages to produce il-12. in patients suffering from dental caries with periapical granuloma, immunity components, namely hsp60, cd-8 lymphocytes and ifn-γ, have an important contribution as indicated with the increasing of cells producing cd-8, ifn-γ and hsp60. immunopathobiogenesis of periapical granulomas can show a relationship between those three components to the pattern of cells producing high cd-8, and ifn-γ, and hsp60. it means that those three immunity components have positive contribution in the immunopathobiogenesis of periapical granuloma. for instance, cd-8 lymphocyte expression will directly trigger the apoptosis of cd-8 lymphocytes, and then serve as a proapoptotic to inhibit cd-4 (th2 cells). these barriers will affect the balance resulting in the increasing of th1.14 on the other hand, hsp60 will also stimulate th1 through il-12, so cytokines (ifn-γ) will be secreted. cells producing ifn-γ will trigger the proliferation of cd-8, so cd-8 lymphocytes will be increased and ifn-γ produced will also be high. hsp60 is a mediator of immune stimulation with different mechanisms, and can be affected by lps. hsp60 and lps, according to osterloh et al.,15 can increases cytokine (ifn-γ) produced by th1 cells. the presence of hsp60 will induce the production of ifn-α, so that hsp60 functions as a classic signal that induces tissue damage to the innate immune system. if there is a bacterial infection, then hsp60 binds to lps and facilitates the detection of microbes by recognizing pathogen-associated molecular pattern (pamp) early and increasing tlr signaling. the result of a research conducted by suzuki et al.,10 showed that the epithelial residue of malassez had a negative reaction to hsp60 indicating no protective function of hsp60 against inflammation. this finding explains another function of hsp60 in immunepathobiogenesis of periapical granulomas since hsp60 is previously more often associated with epithelial proliferation and migration process into periapical lesions. similarly, the results of an in vitro research conducted by breloer et al.13 showed that hsp60 led to a pro-inflammatory response of cells in the adaptive immune system. it is also known that hsp60 triggers specific ifn-γ secretion with a high number of t cells in peritoneal exudates cells (pec) as the cells displaying the antigen (apc). in short, these results indicate that hsp60 as endogenous molecules can cause inflammatory response. these also indicate that the activation of cells of the innate and adaptive immune system by hsp60 is strongly influenced by the type of apc. the formation of granuloma can be considered as a chronic inflammatory process due to the failure of the acute inflammatory process, causing activation of persistent antigens and accumulation of macrophages continuously. it is because ifn-γ released by t cells causes the transformation of macrophages into epitheloid cells and datia cells.11 similarly, although the ability of macrophages to neutralize the bacteria that lead to a very high majority of bacteria will be destroyed by lysozyme system, then �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 7–12 causing no inflammatory reaction excessive, there is still humoral immunologic reaction. in this immunological reaction, macrophages will secrete il-12, which will induce lymphocytes (th1), so ifn-γ will be produced. then the expression of ifn-γ will trigger proliferation of cd-8 lymphocytes. consequently, it will secrete ifn-γ and activate macrophages. therefore, in patients suffering from dental caries with periapical granulomas, there will be no destruction of macrophages containing intracellular bacteria, but the activity of macrophages to surround those macrophages containing intracellular bacteria will be increased.8 the study suggested that in the immunopathobiogenesis of periapical granuloma in dental caries, hsp60, cd-8 and ifn-γ played important rules, but the role of ifn-γ was found to be more prominent. references 1. graves d. cytokines that promote periodontal tissue destruction. j periodontol 2008; 79(8 suppl): 1585-91. 2. badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia. laporan survei kesehatan rumah tangga (skrt). jakarta: studi morbiditas dan disabilitas dalam surkesnas; 2004. 3. radics t. the role of inflammatory and immunological processes in the development of chronic apical periodontitis. universitas of debrecen medical and health science center faculty of dentistry. 2004. 4. garcía cc, sempere fv, diago mp, bowen em. the post-endodontic periapical lesion: histologic and etiopathogenic aspects. med oral patol oral cir bucal 2007; 12(8): e585-90. 5. leprince jg, zeitlin bd, tolar m, peters oa. interactions between immune system and mesenchymal stem cells in dental pulp and periapical tissues. int endod j 2012; 45(8): 689-701. 6. trowbridge ho. immunological aspects of chronic inflammation and repair. j endod 1990; 16(2): 54-61. 7. clancy j. basic concepts in immunology: a student’s survival guide. the mcgraw-hill companies inc; 1998. 8. abbas ak, lichtman ah, pillai s. cellular and molecular immunology. 6th ed. usa: wb. saunders company; 2007. 9. goldsby ra, kindt tj, osborne ba. immunology. 4th ed. new york: wh freeman and company; 2000. p. 432-4. 10. matsumoto ma, ribeiro da. inducible nitric oxide expression correlates with the level of inflammation in periapical cysts. eur j dent 2007; 1(4): 212-5. 11. bratawidjaja kg, rengganis i. imunologi dasar. edisi ketujuh. jakarta: balai penerbit fakultas kedokteran indonesia; 2009. 12. colic m, lukic a, vucˇevic´ d, milosavljevic p, majstorovi i, marjanovic m, dimitrijevic j. correlation between phenotypic characteristics of mononuclear cells isolated from human periapical lesions and their in vitro production of th1 and th2 cytokines. archives of oral biology 2006; 51: 1120-30. 13. vercoulen y, van teijlingen nh, de kleer im, kamphuis s, albani s, prakken bj. heat shock protein 60 reactive t cells in juvenile idiopathic arthritis: what is new?. arthritis res ther 2009; 11(3): 231. 14. stolzing a, sethe s, scutt am. stressed stem cells: temperature response in aged mesenchymal stem cells. stem cells dev 2006; 15(4): 478-87. 15. osterloh a, kalinke u, weiss s, fleischer b, breloer m. synergistic and differential modulation of immune responses by hsp60 and lipopolysaccharide. j biol chem 2007; 282(7): 4669-80. 16. braakman i, hebert dn. protein folding in the endoplasmic reticulum. cold spring harb perspect biol 2013; 5(5): a013201. 17. z i m mer ma n n r, ey r isch s, a h mad m, hel ms v. p rotei n translocation across the er membrane. biochim biophys acta 2011; 1808(3): 912-24. 18. mori k, kawahara t, yoshida h, yanagi h, yura t. signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for unfolded protein-response pathway. genes cells 1996; i: 803-17. 19. cox js, chapman re, walter p. the unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane. mol biol cell 1997; 8(9): 180514. 20. sasaki h, balto k, kawashima n, eastcott j, hoshino k, akira s, stashenko p. gamma interferon (ifn-gamma) and ifn-gammainducing cytokines interleukin-12 (il-12) and il-18 do not augment infection-stimulated bone resorption in vivo. clin diagn lab immunol 2004; 11(1): 106-10. 21. sudiana ik. perbedaan produktivitas limfosit b yang diambil dari kelenjar getah bening dan limfa pada pembuatan antibody monok lonal toxoca racati. diser tasi. surabaya: universitas airlangga; 1999. dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation 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be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusionocclusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. editorial board of dental journal (majalah kedokteran gigi) sk: 118/j03.1.21/kp/2008 january 2, 2008-january 2, 2010 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: dr. elly munadziroh, drg, m.s. (dental material – airlangga university) editorial boards: prof. dr. m rubianto, drg, m.s., sp.perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d., fds. (conservative dentistry – university of guy’s dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery – university of saarland, homburg, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery – university science malaysia, malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic – university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry – university of hiroshima, japan); prof. kozai katsuyuki, d.d.s., ph.d. (pediatric – university of hiroshima, japan); dr. nugrohowati, drg, m.kes. (conservative dentistry – prof. dr. moestopo university); dr. m. suharsini, drg, m.s., sp.kga. (pediatric dentistry – indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic – jember university); widowati witjaksono, drg., ph.d. (periodontic – university science malaysia, malaysia); prof. dr. a.g.m. tielens (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands); kok van kessel (medical microbiology – university medical centre, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. lakshman samaranayake (oral microbiology – the university of hongkong). managing editors: dr. r. darmawan setijanto, drg, m.kes. (department of dental public health – airlangga university); prof. dr. arifzan razak, drg, msc, sp. pros. (prostodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp. kg. (conservative dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d.,sp.ort. (ortodontic – airlangga university); prof. dr. mieke sylvia m. a. r., drg, m.s.,sp.ort. (ortodontic – airlangga university); prof. dr. istiati soehardjo, drg, m.s. (oral biology – airlangga university); dr. anita yuliati, drg, m.kes. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); seno pradopo, drg, s.u., ph.d. sp. kga. (pediatric dentistry – airlangga university); udijanto tedjosasongko, drg, ph.d.,sp.kga. (pediatric dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg.,su.,sp.bm. (oral maxillofacial surgery – airlangga university); markus budi rahardjo, drg, m.kes. (oral biology – airlangga university); endang pudjirochani, drg, m.s., sp. pros. (prostodontic – airlangga university); ira widjiastuti, drg, m.kes.sp.kg. (consevative dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); bagus soebadi, drg, mhped. (oral medicine – airlangga university); ketut suardita, drg.,ph.d. (conservative dentistry – airlangga university); sianiwati goenharto, drg., m.s. (ortodontic – airlangga university); devi rianti, drg., m.kes. (dental material – airlangga university); chiquita prahasanti, drg.,sp.perio. (periodontic – airlangga university); dr. eha renwi astuti, drg., m.kes. (roentgen– airlangga university); dr. diah savitri ernawati, drg.,msi. (oral medicine – airlangga university); rostiny, drg., m.kes.,sp.pros. (prostodontic – airlangga university). administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478 / 5030255. fax. (031) 5039478 / 5020256 e-mail: dental_journal@yahoo.com website: www.dentj.fkg.unair.ac.id www.journal.unair.ac.id accredited no. 48/dikti/kep/2006 volume 41 number 1 january-march 2008 thanks to editors in duty of dental journal (majalah kedokteran gigi) volume 40 number 3 july – september 2007 : prof. edward c. combe, m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a) prof. h. ab. rani samsudin, d.d.s. fdsrc., am. (oral and maxillofacial – university science malaysia, malaysia) endrajana, drg., m.s.,sp.bm. (oral maxillofacial surgery – airlangga university) sudarjani gunawan, drg., m.s., sp.kg. (conservative dentistry – airlangga university) mintarsih djamhari, drg., m.s., sp.pm. (oral medicine – airlangga university) issn 1978 3728 dental journal majalah kedokteran gigi contents page printed by: airlangga university press. (068/04.08/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail:aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 41 number 1 january–march 2008 issn 1978 3728 dental journal majalah kedokteran gigi 1. three dimensional changes in maxillary complete dentures immersed in water for seven days after polymerization shinsuke sadamori, toshiya ishii, taizo hamada, and arifzan razak ...................................... 1–4 2. the in vitro assessment of anti proliferation activity of crude diethyl ether extract of dendrophthoe species against to myeloma culture cell mochamad lazuardi ......................................................................................................................... 5–9 3. coen’s ascending ramus fixator use for repositioning the ascending ramus during mandible reconstruction coen pramono d ................................................................................................................................ 10–14 4. the role of transforming growth factor beta in tertiary dentinogenesis tetiana haniastuti, phides nunez, and ariadna a. djais .............................................................. 15–20 5. morphological changes of alveolar bone due to orthodontic movement of maxillary and mandibulary incisors pinandi sri pudyani, darmawan sutantyo, and sri suparwitri .................................................... 21–24 6. the effect of psidium guajava linn leaf extract on candida albicans adherence and the transversal strength of acrylic resin amiyatun naini and sherman salim ................................................................................................ 25–29 7. side effects of mercury in dental amalgam titiek berniyanti and ninuk hariyani ............................................................................................ 30–34 8. pulp tissue vacuolization and necrosis after direct pulp capping with calcium hydroxide and transforming growth factor-β1 sri kunarti .......................................................................................................................................... 35–38 9. antimicrobial effect of calcium hydroxide as endo intracanal dressing on streptococcus viridans nanik zubaidah ................................................................................................................................. 39–42 10. gingival immunologic defense index: a new indicator for evaluating dental plaque infection risk in allergic children seno pradopo and haryono utomo .................................................................................................. 43–46 11. expression of toll-like receptors in the oral mucosa of patients with recurrent aphthous stomatitis diah savitri ernawati ........................................................................................................................ 47–51 preface we are now in the age of 38. in the period of 1968 to 2006, by the support and commitment of faculty of dentistry airlangga university and the customers, we have already made such significant progress on the quality as define below. at first, our journal served our local researchers to publish their articles, and then we served also national researchers in the field of dentistry. we try to serve international researchers to do scientific communication with local researchers by publishing their articles. due to the improvement toward international journal, we have already tried to increase the number of english articles in the period of 2002–2005. recently, at the end of 2005, we succeed to publish all england articles in volume 38 number 4 october–december 2005. in addition, for the reason of international communication demand, we already have web site (www.fkg.unair.ac.id/web/mkg.htm). to make the sign of our progression's milestone and due to the aesthetic reason, we change our cover. our new cover reflected our proceed spirits to serve our customers. best regard, chief editor, �� volume 47, number 1, march 2014 research report pengaruh posisi dan fraksi volumetrik fiber polyethylene terhadap kekuatan fleksural fiber reinforced composite (the effect of position and volumetric fraction polyethylene fiber on the flexural strength of fiber reinforced composite) catur septommy,1 widjijono2 dan rini dharmastiti3 1 bagian prostodonsia, fakultas kedokteran gigi institut ilmu kesehatan bhakti wiyata kediri, kediri indonesia 2 bagian biomaterial, fakultas kedokteran gigi universitas gadjah mada, yogyakarta – indonesia 3 jurusan teknik mesin dan industri, fakultas teknik universitas gadjah mada, yogyakarta indonesia abstract background: composite resin is a combination of filler and matrix. the additional of fiber in the composite resin has a function as load-bearing in mastication. polyethylene fiber has been used as a reinforced to receive the forces on the fixed denture fiber reinforced composite (frc). purpose: the purpose of this study was to determine effect of position and olumetric fraction of fibers on the flexural strength and modulus polyethylene frc. methods: this study used 7 groups with variations in the position and the volume of fiber. group i, position compression volume 1 sheet; group ii, 2 volume compression sheet position; group iii, volume 1 sheet neutral position; group iv, neutral position volume 2 sheets; group v, position tension volume 1 sheet; group vi position tension volume 2 sheets; and group vii without fiber. each group consisted of 6 samples and frc rod-shaped samples with size (25 x2 x 2) mm. samples were tested by three-point bending test with a universal testing machine. the data were analyzed by two-way anova and lsd test continued (α = 0,05). results: group vi had the highest mean flexural strength than others (360.74 mpa) and group iv had the highest flexural modulus than others (3.56 gpa). the flexural strength and modulus with the variation of position or volume showed a significant differences (p<0.05), while the interaction between position and volume showed no significant difference (p>0.05). conclusions: the position fiber on tension and additional two strips on volumetric fiber affected the increasing flexural strength and modulus of frc. key words: fiber reinfoced composite, polyethylene, position, volumetric, flexural abstrak latar belakang: komposit merupakan gabungan filler dan matriks. penambahan fiber pada komposit berfungsi sebagai penahan beban pengunyahan. polyethylene fiber telah digunakan sebagai penguat dalam menerima gaya-gaya pada gigi tiruan cekat fiber reinforced composite (frc). tujuan: penelitian ini bertujuan untuk mengetahui pengaruh posisi dan fraksi volumetrik fiber pada kekuatan fleksural polyethylene frc. metode: penelitian ini membuat 7 kelompok sampel polyethylene frc dengan variasi posisi dan volume fiber. kelompok i, posisi compression volume 1 lembar; kelompok ii, posisi compression volume 2 lembar; kelompok iii, posisi netral volume 1 lembar; kelompok iv, posisi netral volume 2 lembar; kelompok v, posisi tension volume 1 lembar; kelompok vi, posisi tension volume 2 lembar; dan kelompok vii, tanpa fiber. setiap kelompok terdiri atas 6 sampel dan sampel berbentuk batang frc dengan ukuran (25x2x2) mm. sampel diuji dengan three-point bending test dengan universal testing machine. data dianalisis dengan two-way anova dan dilanjutkan uji lsd (α=0.05). hasil: rerata kekuatan fleksural kelompok vi paling tinggi (360.74 mpa) dan kelompok iv memiliki modulus fleksural tertinggi (3.56 gpa). kekuatan dan modulus fleksural dengan variasi posisi atau volume menunjukkan perbedaan bermakna (p<0.05) sedangkan interaksi antara posisi dan volume menunjukkan perbedaan tidak bermakna ��septommy, et al.: pengaruh posisi dan fraksi volumetrik fiber polyethylene (p>0.05). simpulan: posisi fiber pada sisi tension frc dan penambahan volume 2 lembar fiber akan meningkatkan kekuatan fleksural frc. kata kunci: fiber reinfoced composite, polyethylene, posisi, volume, fleksural korespondensi (correspondence): catur septommy, bagian prostodonsia, fakultas kedokteran gigi institut ilmu kesehatan bhakti wiyata kediri. jl. kh wahid hasyim 65 kediri 64114, indonesia. e-mail: tommy.wiens@gmail.com pendahuluan material resin komposit adalah gabungan dari filler dan matrik, sebagian besar resin komposit untuk kedokteran gigi berupa resin komposit dengan filler partikulat.1 resin komposit partikulat merupakan resin komposit yang mengandung reinforce berupa partikulat dan memiliki sifat isotropis yaitu tidak mempunyai arah filler khusus dalam strukturnya sehingga sifatnya sama untuk semua arah filler, sedangkan resin komposit yang diberi penguat fiber memiliki sifat anisotropis yaitu sifatnya tidak sama dalam semua arah.2 resin komposit yang digunakan untuk gigi tiruan adalah jenis resin komposit yang diberi penguat fiber sebagai pengganti kerangka logam yang berfungsi sebagai penahan beban pengunyahan.3 penggunaan fiber pada bahan kedokteran gigi memiliki beberapa fungsi diantaranya meningkatkan kekuatan dan kekakuan, meningkatkan ketahanan bahan terhadap fraktur, serta menurunkan shrinkage.4 gigi tiruan cekat (gtc) logam membutuhkan waktu yang lama dalam proses pembuatannya karena tidak bisa dibuat langsung dan harus melewati prosedur laboratorium, sedangkan pada gtc fiber reinforced composite (frc) dapat dibuat secara langsung tanpa melalui prosedur laboratorium.5 kelebihan material resin komposit jika dibandingkan dengan logam adalah tahan terhadap korosi dan pada aplikasinya tidak perlu menghilangkan jaringan yang sehat selama preparasi. pada gtc anterior berbahan fiber reinforced composite lebih estetik dibandingkan dengan bahan logam yang berlapiskan porselin atau yang sering disebut pfm/porcelain fused to metal.6 frc merupakan kombinasi antara filler resin partikulat dengan fiber yang akan menghasilkan estetik dan biomekanik yang sama dengan jaringan gigi.3 hasil penelitian menunjukkan bahwa non-impregnated polyethylene fiber mempunyai kekuatan fleksural lebih baik daripada pre-impregnated glass fiber.5 pada preimpregnated fiber sudah terdapat penambahan polimer pmma (polymethyl methacrylate) atau monomer (acrylate atau methacrylate). 7 impregansi merupakan upaya peningkatan keefektifan pembasahan fiber dengan matrik polimer.8 walaupun polyethylene fiber tidak efektif dalam pembasahan fiber dengan matrik polimer, polyethylene fiber telah digunakan sebagai penguat dalam menerima gaya-gaya pada gigi tiruan cekat anterior dan memenuhi karakter estetika karena polyethylene fiber telah ada penambahan glass plasma dingin yang akan menghasilkan permukaan untuk siap berekasi dengan substrat.6,9 fiber reinforced composite merupakan kombinasi antara resin partikulat dengan fiber sehingga sifat mekanik dari kontruksi frc dipengaruhi oleh fraksi volumetrik, lokasi, dan arah fiber.10 hasil penelitian menunjukkan volume fiber uhmwpe antara 18,6% dan 75,8% tidak ada perbedaan dalam nilai modulus elastisitasnya dan begitu juga posisi fiber uhmwpe yang berbeda dengan fraksi volumetrik fiber yang sama didapatkan nilai modulus elastisitas yang berbeda.11 hal ini menunjukkan bahwa belum ada posisi dan volume polyethylene fiber yang optimal untuk meningkatkan sifat mekanik frc. penelitian ini bertujuan untuk mengetahui pengaruh posisi dan fraksi volumetrik polyethylene fiber pada kekuatan fleksural frc. gambar 1 skema area sisi tarikan (tension side) dan sisi tekanan (compression side). (a) sumbu netral berada pada bagian tengah sampel. (b) fiber terletak pada sisi tarikan, (c) fiber terletak pada sisi tekanan.12 �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 52–56 bahan dan metode penelitian ini menggunakan polyethylene fiber lebar 2 mm (ribbond-thm, ribbond inc. usa) yang telah dipotong sepanjang 25 mm; packable composite (filtek 3m espe, usa) yang terdiri atas matrik bis-gma (bisphenol a diglycidiyl ether dimethacrylate); matrik tegdma (tri{ethylene glycol}dimethacrylate), dan bahan pengisi 66% (volume) silica/zirconia dengan rata-rata ukuran partikel 0,6 µm; silane coupling agent (3m espe sil) yang memiliki komposisi 3-mps silane (3-methacryloyl oxypropyltrimethoxysilane) dan ethanol. fiber dipotong sepanjang 25 mm dengan menggunakan blade, kemudian setiap potongan fiber diberi silane sebanyak 1 tetes tiap sisinya kemudian dengan microbrush diulas sebanyak tiga kali tiap sisinya dan ditunggu selama 60 detik, lembaran fiber disiapkan sebanyak 1 lembar dan 2 lembar. untuk mempersiapkan sampel frc dengan posisi fiber pada compression side. cetakan logam dengan ukuran 2 x 2 x 25 mm diletakkan di atas glass slide diisi dengan resin komposit packable terlebih dahulu, lembaran fiber tersebut dipegang dengan pinset dan kemudian diletakkan ke cetakan dengan menyisakan ketebalan 0,5 mm dari permukaan atas cetakan sampel. ketebalan 0,5 mm diukur dengan bantuan probe who. setelah itu bagian atas diaplikasikan resin komposit sampai cetakan penuh, kemudian ditutup dengan glass slide dan diikat dengan rubber band lalu disinar dengan qth light cure dengan penyinaran dibagi menjadi 3 bagian sepanjang spesimen, masing-masing selama 40 detik. plat frc dilepas dari cetakan, kemudian dipoles dengan polishing disc dan diukur dengan jangka sorong. untuk mempersiapkan sampel frc dengan posisi fiber pada neutral, caranya sama seperti persiapan sampel dengan posisi fiber pada compression side. pada posisi neutral side, posisi fiber diletakkan setinggi 1 mm dari dasar cetakan. pada fiber dengan tension side, posisi fiber diletakkan setinggi 0,5 mm dari dasar cetakan sampel frc. skema posisi fiber pada sisi tension (sisi tarikan), sisi netral, dan sisi compression (sisi tekanan) dapat dilihat pada gambar 1. dilakukan 7 pengelompokan sampel, yaitu kelompok i dengan posisi compression volume 1 lembar; kelompok ii dengan posisi compression volume 2 lembar; kelompok iii dengan posisi netral volume 1 lembar; kelompok iv dengan posisi netral volume 2 lembar; kelompok v dengan posisi tension volume 1 lembar; kelompok vi dengan posisi tension volume 2 lembar; dan kelompok vii dengan tanpa fiber. sampel yang sudah dikelompokkan, kemudian disimpan di dalam inkubator pada temperatur 37° c selama 24 jam sebelum pengujian. perlakuan three–point bending test dilakukan dengan alat universal testing machine. uji ini dilakukan dengan meletakkan subjek penelitian pada papan penyangga dengan jarak tumpuan sejauh 20 mm (l) dan titik tengah sampel frc terkena tekanan yang berasal dari alat uji. setelah sampel mengalami patah, pada layar monitor akan menunjukkan suatu angka (f) yang merupakan tekanan maksimal yang dapat diterima oleh saat fraktur. selanjutnya data pengukuran yang diperoleh dimasukkan ke dalam rumus kekuatan fleksural σ=3fl/ 2bh2, h= kedalaman atau ketebalan bahan (mm); b = lebar bahan (mm). rerata dan standar deviasi dari tiap kelompok dibandingkan dan dianalisa dengan menggunakan two way anova kemudian dilanjutkan uji lsd (α=o,o5). hasil rerata kekuatan fleksural tertinggi pada polyethylene frc dengan posisi tension dan volume 2 lembar sebesar 360,74 mpa dan rerata kekuatan fleksural terendah pada kelompk tanpa fiber sebesar 68,03 mpa (tabel 1). hasil analisa two way anova terdapat perbedaan bermakna (p<0,05) antar kelompok dengan variasi posisi dan variasi volume (tabel 2). interaksi kelompok antara variabel posisi tabel 1. rerata dan standar deviasi kekuatan fleksural batang polyethylene frc dengan posisi dan volume fiber yang berbeda (mpa) kelompok frc n rerata standar deviasi posisi compression volume 1 lembar 6 71,99 11,51 posisi compression volume 2 lembar 6 87,09 18,48 posisi netral volume 1 lembar 6 169,34 90,14 posisi netral volume 2 lembar 6 252,03 81,32 posisi tension volume 1 lembar 6 256,96 66,07 posisi tension volume 2 lembar 6 360,74 100,71 tanpa fiber 6 68,03 4,57 tabel 2. rangkuman uji lsd kekuatan fleksural pada polyethylene frc dengan variabel posisi posisi fiber compression (i) netral (i) tension (i) tanpa fiber (i) compression (j) – 131,133* 229,3* -11,514 netral (j) – 98,167* -142,646* tension (j) – -240,814* tanpa fiber (j) – ��septommy, et al.: pengaruh posisi dan fraksi volumetrik fiber polyethylene dengan variasi volume menunjukkan perbedaan tidak bermakna (p>0,05). hasil analisis lsd untuk variasi posisi menunjukkan terdapat perbedaan bermakna antar rerata kekuatan fleksural dalam seluruh kelompok perlakuan (p<0,05), kecuali antara posisi compression dengan tanpa fiber tidak terdapat perbedaan bermakna (p>0,05). hasil analisis lsd untuk variasi volumetrik fiber menunjukkan terdapat perbedaan bermakna antar rerata kekuatan fleksural pada semua kelompok (p<0,05) (tabel 3). pembahasan tes 3-point bending merupakan tes simulasi standar untuk kontruksi jembatan, sifat yang dapat diketahui pada tes ini adalah kekuatan fleksural dan elastisitas. kekuatan fleksural merupakan kemampuan suatu restorasi untuk menahan gaya fleksural, yaitu kombinasi dari gaya tarik dan kompresi, saat sedang berfungsi di dalam mulut baik sebagai restorasi di daerah anterior maupun posterior. kekuatan fleksural suatu material penting untuk diketahui oleh para klinisi sebagai bahan pertimbangan dalam pemilihan material untuk restorasi.13 sebuah benda batang diberi beban akan terjadi pendistribusian tekanan, maka tekanan pada benda homogen akan didistribusikan merata pada semua bagian bahan.14 frc dapat dianggap sebagai bahan homogen walaupun kandungan bahannya tidak sama dalam semua bagian. frc yang memiliki sifat homogen akan memiliki kekutan fleksural yang berbeda apabila posisi fiber juga berbeda. fraktur pada frc mudah terjadi apabila bagian bawah frc didukung dengan matriks komposit lebih besar. fiber memiliki peranan penting dalam mendistribusikan tekanan pada frc. fiber yang digunakan pada penelitian ini memiliki struktur pola mata rantai silang yang istimewa dengan desain anyaman (braided) yang mengunci sehingga dapat meningkatkan ketahanan, stabilisasi, dan kekuatan geser antar serat untuk mencegah dari keretakan. desain anyaman yang mengunci tersebut juga efektif menghantarkan tekanan sepanjang anyaman fiber tanpa tekanan dihantar kembali menuju resin.15 pada benda homogen, penambahan volume fiber akan menambah kemampuan dalam menyerap energi dan meningkatkan ketahanan terhadap fraktur oleh karena resultan gaya yang diteruskan fiber dengan desain anyaman akan menjadi kecil.4 penguat fiber akan tepat apabila diletakkan pada area gigi tiruan yang lemah. area gigi tiruan yang lemah berada pada sisi yang mengalami tekanan tarik, tekanan tarik akan berdampak pada pemanjangan dimensi gigi tiruan.16 sebagian besar fraktur terjadi oleh karena komponen tekanan tarik.17 aplikasi tekanan tarik akan menyebabkan molekul-molekul berupaya bertahan terhadap tarikan yang berakibat terpisahnya ikatan antar molekul yang memungkinkan terjadinya fraktur. ketahanan terhadap tarikan dapat ditingkatkan melalui penambahan fiber. fiber yang diletakkan di dasar spesimen tepat pada sisi yang menerima beban tarik menunjukkan kekuatan fleksural tertinggi.18 penempatan fiber pada posisi tension tidak ada hubungan antara kekuatan fleksural dengan jumlah fiber oleh karena jumlah fiber memiliki pengaruh kecil terhadap kekuatan fleksural dan juga didapatkan tidak ada interaksi antara posisi dan jumlah fiber pada modulus fleksural.19 pengaturan desain fiber seperti penempatan fiber lebih penting pengaruhnya dibandingkan jenis fiber.10 pengaruh posisi fiber lebih dominan dalam mempengaruhi sifat mekanik frc dan untuk mengoptimalkan pengaruh fiber maka penambahan fiber harus diletakkan di sisi tarikan.20 ketahanan terhadap perubahan bentuk juga akan didapatkan apabila fiber diletakkan pada sisi tarikan karena pada sisi tarikan akan terjadi tekanan tarik maksimal. pada sisi tension spesimen akan terdapat gaya tarik yang maksimum, sedangkan sisi tengah/netral spesimen akan terdapat gaya geser yang maksimum.21 ikatan antar permukaan dapat terbebani oleh gaya tarik atau gaya geser, kualitas elastisitas suatu bahan dapat dipengaruhi oleh kekuatan antar atom atau antar molekul suatu bahan.22 fiber pada sisi kompresi dan kelompok tanpa fiber tidak menunjukkan perbedaan rerata kekuatan fleksural. pada sisi kompresi, gaya tarik dan gaya geser tidak langsung dialihkan ke penguat fiber, gaya geser dan gaya tarik akan didistribusi lebih dulu ke resin komposit. glass fiber pada sisi kompresi tidak akan memberi efek kekuatan fleksural dibandingkan glass fiber yang diletakkan pada sisi netral atau tension.19 penambahan fiber jenis anyaman secara signifikan dapat meningkatkan keuletan frc sebesar 9 kali dari resin komposit tanpa fiber.4 distribusi tekanan memberi pengaruh yang signifikan terhadap kekuatan frc, apabila distribusi tekanan lebih merata maka fraktur tidak mudah terjadi.16 penambahan jumlah fiber sebanyak 2 lembar akan meningkatkan kekuatan fleksural frc dibandingkan tabel 3. rangkuman uji lsd kekuatan fleksural pada polyethylene frc dengan variabel volumetrik volume fiber 1 lembar (i) 2 lembar (i) tanpa fiber (i) 1 lembar (j) – 67,188* -98,064* 2 lembar (j) – -165,252* tanpa fiber (j) – *= berbeda bermakna (p<0,05) �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 52–56 frc dengan jumlah 1 lembar fiber dan tanpa fiber. desain jembatan dengan 2 lembar fiber mempunyai kekakuan yang tertinggi dibandingkan desain jembatan dengan 1 lembar dan tanpa fiber.23 berdasarkan hasil penelitian untuk mengoptimalkan posisi dan volume fiber pada penggunaan gigi tiruan cekat maka penempatan posisi fiber pada sisi tension dengan volume 1 lembar fiber polyethylene sudah optimal untuk digunakan sebagai gigi tiruan cekat, hasil rerata kekuatan fleksural pada sisi tension dengan volume 1 lembar sebesar 256,96 mpa sedangkan kekuatan rerata tekanan kompresif pada gigitan gigi posterior sebesar 193 mpa.17 hasil penelitian ini menunjukkan bahwa penempatan posisi fiber polyethylene pada sisi tension frc akan meningkatkan kekuatan fleksural dan modulus fleksural frc. penambahan volume fiber polyethylene sebanyak dua lembar pada frc juga akan meningkatkan kekuatan fleksural frc. daftar pustaka 1. shalaby ws, salz u. polymers for dental and orthopedic applications. new york, usa: crc press taylor & francis group; 2007. p. 26. 2. tezvergil a, lassila lvj, vallittu pk. the effect of fiber orientation on the polymer ization sh r in kage st ra in of f iber-reinforced composites. j dent material 2006; 22(7): 610-6. 3. polacek p, jancar j. effect of filler content on the adhesion strength between ud fiber reinforced and particulate filled composites. composites science and technology 2008; 68: 251-9. 4. karbhari vm, wang q. influence of triaxial braid denier on ribbonbased fiber reinforced dental composite. j dent material 2007; 23(8): 969-76. 5. gaspa r junior ade a, lopes m w, gaspa r gda s, braz r. comparative study of flexural strength and elasticity modulus in two types of direct fiber-reinforced systems. braz oral res 2009; 23(3): 236-40. 6. turker sb, sener id. replacement of a maxillary central incisor using a polyethylene fiber reinforced composite resin fixed partial denture: a clinical report. j prosthet dent 2008; 100: 254-58. 7. latsumaki tm, lassila lvj, vallittu pk. the semi-interpenetrating polymer network matrix of fiber-reinforced composite and its effect on the surface adhesive properties. j mater sci: materials in medicine 2003; 14: 803-9. 8. abdulmajeed aa, narhi to, vallittu pk, lassila lvj. the effect of high fiber fraction on some mechanical properties of unidirectional glass fiber-reinforced composite. j dent material 2011; 27: 31321. 9. belli s, eskitascioglu g. biomechanical properties and clinical use of a polyethylene fibre post-core. material international dentistry south africa 2006; 8(3): 20-6. 10. van heumen cc, kreulen cm, bronkhorst em, lesaffre e, creugers nh. fiber-reinforced dental composites in beam testing. j dent material 2008; 24(11): 1435-43. 11. dyer sr, lassila lv, jokinen m, vallittu pk. effect of crosssectional design on the modulus of elasticity and toughness of fiber-reinforced composites materials. j prosthet dent 2005; 94(3): 219-26. 12. narva kk, lassila lvj, vallittu pk. the static strength and modulus of fiber reinforced denture base polymer. j dent mat 2005; 21: 4218. 13. mozartha m, herda e, soufyan a. pemilihan resin komposit dan fiber untuk meningkatkan kekuatan f leksural fiber reinforced composite (frc). jurnal pdgi 2010; 59: 29-34. 14. yusof a. mekanika bahan dan struktur. johor: universitas teknologi malaysia; 2001. h. 30. 15. yanti d, amalia h, sugiatno e. perbedaan kekuatan fleksural fiber reinforced composite dengan struktur leno weave dan long longitudinal polyethylene pada gigi tiruan cekat adhesif. j ked gigi 2011; 2(4): 230-5 16. ellakwa ae, shortall ac, shehata mk, marquis pm. the influence of fibre placement and position on the efficiency of reinforced composite bridgework. j oral rehabilitation 2001; 28(8): 785-91. 17. a nusavice k j. ph ilips science of denta l mater ia l. 11t h ed. philadhelphia: wb. saunders company; 2003. h. 36. 18. ellakwa ae, shortall ac, marquis pm. influence of fiber position on the flexural properties and strain energy of a fibre-reinforced composite. j oral rehabilitation 2003; 30(7): 679-82. 19. kanie t, arikawa h, fuji k, ban s. mechanical properties of reinforced denture base resin: the effect of position and the number of woven glass fibers. dental materials j 2002; 21(3): 261-9. 20. vakiparta m, yli-urpo a, vallittu pk. flexural properties of glass fiber reinforced composite with multiphase biopolymer matrix. j mater sci mater med 2004; 15(1): 7-11. 21. lassila lvj, va llitt u pk. t he effect of f iber position a nd polymerization condition on the f lexural properties of fiberreinforced composite. j contemp dent prac 2004; 5(2): 14-26. 22. sakaguchi rl, powers jm, craig s. restorative dental materials. 12th ed. st louis, missouri: mosby inc; 2006. p. 60. 23. li w, swain mv, li q, ironside j, steven gp. fibre reinforced composite dental br idge.pa r t i: exper imental investigation. biomaterials 2004; 25(20): 4987:93. vol 51 no 3 jul sep 2018_pus.indd 153 case report herbal-induced stevens-johnson syndrome with oral involvement and management in an hiv patient s. suniti and irna sufiawati departement of oral medicine faculty of dentistry universitas padjadjaran bandung indonesia abstract background: stevens-johnson syndrome (sjs) is an immune complex-mediated hypersensitivity reaction affecting the skin and mucous membranes. patients infected with human immunodeficiency virus (hiv) are at increased risk of developing sjs which is predominantly caused by an adverse reaction to medications, including herbal varieties. in recent years, the consumption of herbal medicines has increased, while their safety remains a matter for investigation. purpose: the purpose of this case report is to explain the occurrence of sjs caused by herbal medicine. case: a 43-year-old male patient with body-wide skin erosion was referred to the department of oral medicine and subsequently diagnosed with stevens-johnson syndrome due to his consumption of a herbal medicine containing zingiber rhizoma, coboti rhizoma, asari herbal and epimedi. the patient’s chief complaints included difficulty when opening the mouth, dysphagia and excessive production of saliva continuously contaminated with blood and sputum. extraoral examination showed a sanguinolenta crust on the lips. intra oral examination of oral mucous showed erosive lesions with bleeding and pain. a hiv test performed at a clinical pathology laboratory was positive for antibodies against hiv with a cd4 cell count of 11 cells/ml. case management: treatment consisted of the administering of nacl 0.9 %, hydrocortisone 0.1% and chlorhexidine digluconate 0.12% for 12 days. conclusion: sjs can be caused by herbal medicine and it is essential to be aware of the latter’s potential adverse effects, especially in immunocompromised patients. symptomatic management of oral lesions should be planned as an early intervention in order to decrease morbidity and mortality in sjs patients. keywords: herbal medicine; hiv management; stevens-johnson syndrome correspondence: irna sufiawati, department of oral medicine, faculty of dentistry universitas padjadjaran, jl. sekeloa selatan no.1 bandung 40132, indonesia. e-mail:irna.sufiawati@fkg.unpad.ac.id introduction stevens-johnson syndrome (sjs) is an immunecomplex-mediated hypersensitivity reaction affecting the skin and mucous membranes first described by dr. stevens and dr. johnson in 1922 as an acute mucocutaneous syndrome suffered by two young boys. the condition is characterized by mucocutaneous tenderness, hemorrhagic erosions, erythema and severe epidermal detachment presenting as blisters in areas of denuded skin.1–5 sjs features blister-sores often referred to as toxic epidermal necrolysis (ten) which is categorized according to the surface area of the body affected by epidermolysis, i.e. sjs (affected body surface area <10%), sjs/ten overlap (10–30%) and ten (>30%). the sjs ratio that occurs between males and females is 2:1 with a mortality rate of 5.4%.6 the incidence rate of herbal medicine-induced sjs in china is 2.5%, in malaysia it is 7.5%, while in singapore and the philippines it stands at 3.5%.6 other studies have shown that the majority of herbal medicine-induced sjs in hiv patients occurred in females (64.7%).7 mortality resulting from sjs depends on the extent of the body area affected and other accompanying conditions such as secondary infection and sepsis. hiv infection, increasing age, chronic conditions, hematological malignancy (non-hodgkin’s lymphoma and leukemia) and renal failure were also associated with sjs/ten and mortality.8,9 patients infected with human immunodeficiency virus (hiv) are at an increased risk of developing sjs which is dental journal (majalah kedokteran gigi) 2018 september; 51(3): 153–157 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p153–157 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p153-157 154suniti and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 153–157 reported to occur 100 times more frequently than in nonhiv patients. the incidence rate of sjs patients infected with hiv ranges from 40% to 69%.10–13 the mortality rate of sjs in individuals infected with hiv is 1:100.000, while sjs mortality rates reported in the literature range between 10% and 75%.1,12 hiv patients suffer dysregulation of t and b lymphocytes within the immune system. certain multifactorial changes including drug metabolism, oxidative stress, cytokine profile, hyperactivation of the immune system and genetic factors are suspected of playing a role in this mechanism.6 the etiology of sjs in hiv patients is predominantly a drug-induced reaction including antituberculosis drugs, sulphonamides, anticonvulsants and antiretrovirals ( n e v i r a p i n e ) . 1 , 1 0 , 1 3 t h e p a t h o p h y s i o l o g y o f d r u g hypersensitivity in hiv is multifactorial and related to changes in drug metabolism, dysregulation of the immune systems (immune hyperactivation, patient cytokine profile), oxidative stress, genetic predisposition, nsaids and viral factors such as epstein-barr virus and cytomegalovirus infections.13 herbal medicines are drugs and may, therefore, cause severe adverse drug reactions.6 sjs in hiv patients occurs most often due to several reactions to drugs, including herbal medicines.14 herbal medicine consumption has recently been increasing. however, the non-observing of regulations is still being investigated. herbal medicinal products containing mixed herbs (36.0%) as well as those administered orally (63.2%) predominate. the most frequent reactions were urticaria and rash (49.2%), urticaria (15%) and rash erythematous (13.4%), while anaphylactic reactions accounted for 9.5 %.15,16. many patients believe that herbal medicines produce fewer side-effects and, therefore, often believe them to be safe. in sjs patients suspected of suffering from herbal medicine-induced etiology, it is very difficult to identify the specific medicine causing the hypersensitivity because most patients consume a mixture of herbal medicines.11,17–21 a diagnosis of herbal medicine-induced sjs in hivpatients is reported here in addition to a literature-based description of sjs related to the characteristics and criteria used in both its diagnosis and oral treatment. case a 43-year-old man was diagnosed by the department of dermatology with multiple druginduced sjs (herbal medicine, doxycycline, paracetamol, amoxycillin clavulanic acid). the patient was also diagnosed as suffering from hiv infection. the patient was referred to the department of oral medicine with painful oral sensations. his chief complaints were difficulty in opening the mouth, dysphagia and excessive continuous production of blood and sputum-contaminated saliva. the departments of dermatology and internist medicine provided systemic therapy involving piracetam, amlodipine, doneperazil, metoclorpiramide, vitamin c, levofloxacin, dexamethasone, in addition to antiretrovirals such as lenofavir, lamivudin and efavirenz respectively. a general examination found the patient to be alert and welloriented. on admission, he exhibited maculopapular cutaneous eruptions on the neck, face and trunk. extra oral examination detected the presence of sanguinolenta crusts and excessive and continuous saliva production with blood and sputum on both the upper and the lower lips. an examination for non-anemic conjunctiva and lymph node indicated no abnormalities, while ophthalmic examination revealed conjunctivitis and diffuse erythema on the upper eyelids. intra oral examination could not be completely performed because the patient was unable to open his mouth sufficiently wide due to the pain (see figure 1). a laboratory examination, including a serology blood count, revealed a decline in the number of haemoglobin 13.3 mg/dl (normal: 14-17.6), haematocrit 40.1% (normal:41.550), mchc 33.2% (normal:33.4-35.5), albumin 2.895 gr/dl (normal:3.4-5.0), calcium ion 5.65 mg/dl (normal:4.7-5.2), cd4 cell count: 11 cells/μl (normal:410-1590) and cd 4%: table 1. the results of antimicrobial suspectibility testing microbiological examination cultures microbiology : sputumspecimen identification :isolate i streptococcus qordonii suspectibility ampicillin : intermediate clindamicin : resistent tertracyclin : resistent cefritriaxone : suspectible cefotaxime : suspectible levofloxacin : suspectible linezolid : suspectible figure 1. the first visit. a. multiple erythematous papules and plaques were visible on the face b. excessive and continuous production of saliva with blood and sputum was visible on the lips. c. multiple erythematous papules and plaques were present on the lower extremities d. multiple erythematous papules and plaques were present on the upper extremities. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p153–157 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p153-157 155 suniti and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 153–157 3.7% (normal: 31-60). a serology test for hiv-1 by elisa was positive. an antibiotic susceptibility test was also performed (see table 1) because hiv patients were often treated with antibiotics for chemoprevention of opportunistic disease and treatment of acute infection. based on clinical symptom and laboratory evaluation, a diagnosis of oral lesions associated with multiple drug-induced sjs was made and the treatment started. case management the departments of dermatology and internist medicine provide systemic therapy involving piracetam, amlodipine, donepezil, metoclorpiramide, vitamin c, levofloxacin, per oral dexamethasone and antiviral drug administration using lenofavir, lamivudin and evafirenz. patients were diagnosed with herbal medicine-induced sjs due to the occurrence of sjs after they had consumed herbal medicines composed of zingeber rhizoma, coboti rhizoma and asari epimedii herb extract. the patient was diagnosed with sjs due to having experienced myalgias, arthralgias and other flulike symptoms after three days’ consumption of the herbal medicine. the patient’s symptoms worsened resulting in difficulty swallowing solid food. on his previous visits to three different hospitals he was prescribed antibiotic doxycycline, amoxicillin-clavulanic acid and paracetamol, but without any subsequent improvement. the patient was subsequently referred to the department of oral medicine and diagnosed with multiple drug-induced sjs. on his first visit to the department of oral medicine, a compress with nacl 0.9% was applied to the patient’s oral cavity daily four times a day to maintain the area around the wound moist and promote the healing process. the patient was instructed to clean his teeth with a gauze moistened with nacl 0.9% at least three times a day. four days later, the patient still complained of a high temperature, pain when opening his mouth, pain on swallowing, bleeding lips, sputtered saliva and spontaneous blood flow. to treat the lesion present on the lip, topical corticosteroid hydrocortisone 1% cream was applied to the affected area three times a day. after the fourth day of treatment, the patient still complained of pain in the oral cavity and lower lip on opening his mouth. a closed compress containing gauze soaked in 0.9% nacl was applied to maintain oral hygiene, while the use of hydrocortisone 1% cream was discontinued. chlorhexidine digluconate 0.12% spray was expected to be capable of applying the drug to the soft palate. after n ine days’ t reatment with chlorhexidine digluconate 0.12% the oral lesions gradually improved as shown in figure 2. sanguinolenta crusts on the upper and lower lips began to reduce in size and the patient, although still experiencing pain, proved able to open his mouth. after 18 days of treatment, the oral lesions improved as shown in figure 3. chlorhexidinedigluconate 0.12% spray therapy was discontinued and the patient was prescribed 5 mg of prednisone in powder form in addition to 10ml of aguadest for daily rinses three times a day for ten days and 1% hydrocortisone cream for the upper and lower lip. saliva was diluted and the intensity of saliva secretion through figure 2. oral lesions after nine days of treatment. a. decrease in saliva around the lips. b. hemorrhagic crusting of the vermillion zone of the lips was noted. c. erythema of palatal mucous. figure 3. after 18 days of treatment lesions have improved. a. absence of erythema on the face. b. crusts on the lips have disappeared. c. the palatal mucous still featured an erythema. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p153–157 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p153-157 156suniti and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 153–157 the lips decreased, although the patient still complained of coughing and was instructed to start a soft diet. subsequent follow-up proved impossible as the patient died as a result of a decrease in his immunosuppressive capacity. discussion the patient took a mixed herbal medicine containing zingiber rhizoma, coboti rhizoma and asari epimedii herb extract. it was difficult to conclusively identify the causative ingredient that led to an allergic reaction to the patient. epimedii extract is usually employed in kidney and asthma therapy when combined with budasonide.22 many patients believe herbal medicines to produce fewer side-effects and believe them to be safe. however, herbal medicines are considered to be a drug and have the potential to cause sjs. identifying sjs patients who have consumed suspect herbal medicine is very difficult because they will usually have injested medicine of mixed herbal composition. the subject of this research was diagnosed as suffering from sjs because he developed the condition after consuming a herbal medicine, a condition exacerbated after his being given paracetamol and amoxicillin. the precise herbal medicines that induced sjs in this patient were difficult to identify due to his having consumed a mixture containing several such medicines. herbal medicines are often considered safe because of their natural content, whereas remedies such as guggul herbal medicine, svarnabhasma, race maniknya, ginger (zingeber officinale), gingko biloba (gingko), ginseng, st. johns wort, godanti, echinacea purpurea, lavanabhaskar, parad preparations, timothy grass and andrographis paniculata, among others, can cause hypersensitive reactions.18,23,24 studies have shown that such reactions to harmful herbal medicine often occur between the ages of 18 and 44 years. in this particular case, the patient was 43 years old. females report a higher rate of adverse drug reactions compared to males.18 it has been reported that the use of orally-administered herbal medicine constitute the common means of administration leading to an allergic drug reaction (36.0%). the most common allergic reactions include rash (16.2%), urticaria (15.3%), erythematosus rash (13.4%), rash (49.2%) and anaphylactic reactions (9.5%).5 the pathogenesis of drug hypersensitivity is not welldefined, although it is known to occur in a susceptible individual if there is exposure to a causative agent. herbal medicines are often considered to be safer to use than chemical drugs with the result that members of the public do not take herbal drug reactions sufficiently into account. herbal remedies contain various ingredients making it difficult to identify possible causes of the hypersensitive reaction. the sjs pathogenesis of the drug triggers an increase in the regulation of fasl produced by peripheral blood mononuclear cells. fasl will pair up with the fas receptors in keratinocyte cells. drug receptors trigger cells expressing mhc class 1 to produce cytotoxic cds, natural killer (nk) cells and nkt cells that accumulate in epidermal blisters before secreting perforin and granzym b result in keratinocyte apoptosis.1,6,10 the patient had no previous history of drug allergy. a tendency to experience allergic reactions begins to occur with the appearance of symptoms of immunodeficiency disease. hiv itself can be a dangerous condition that tends to cause an immune response rather than immune tolerance.13 adverse cutaneous drug reactions increased as the immune system deteriorated with an apparent decrease in cd4+ t-cell count. cd4+ lymphocites constitute central regulators of the immune system that th-1 and th-2, which are differentiated by cytokines, release. th-1 cells produce inf� and il-2 which are important mediators within the humoral immune response, while th-2 produces il-4, il-5, il-6 and il-10 that help b lymphocytes to produce antibodies. hiv infection not only causes immunodeficiency, but also leads to dysregulation of the immune system. once infected by hiv, changes in cytokines profiles appear due to an increase in production of il-4 which is always accompanied by greater production of il-5 and reduced production of ifn-�. in the early phase of hiv infection of the cytokines a normal balance persists. subsequently th-2 (il-4) increases, while th-1 (il-2) decreases. elevated serum ige levels in hiv patients are also associated with a reduction in cd4+ cells (less than 200/mm2). hiv, itself, causes the stimulation of b lympocytes poyclonally which, combined, finally cause an inappropriate immune response.6,25 drug hypersensitivity occurs in a susceptible individual in cases of exposure to a causative agent. a delay of 4-28 days between the initiation of drug use and the onset of the adverse reaction is that regarded as most likely to support drug causality in sjs. the pathogenesis of drug hypersensitivity reactions in hiv infection is not well defined. herbal medicines are often considered to be safer than chemical drugs with the result that herbal drug reactions are not taken into consideration by the public. symptomatic management of the oral lesions is necessary in order to enable the patient to have oral feeds which maintain nutritional balance. sjs is a life-threatening condition and, therefore, supportive care is an essential element of the therapeutic approach. the departments of dermatology and internal medicine provide systemic therapy involving the use of piracetam, amlodipine, donepezil, metoclorpiramide, vitamin c, levofloxacin, dexamethasone, antiviral lenofavir, lamivudin and evafirenz. piracetam has neuroprotective and antithrombotic effects which may help to reduce death and disability.26 amlodipine is a first-line agent effective in improving blood pressure and patient recovery outcomes.27 donepezil is used to arrest the decline in cerebral blood flow, promoting preservation of functional brain activity.28 metoclorpiramide is used to treat the nausea and vomiting often accompanying acute migraines.29 vitamin c is used in the treatment of hyperpigmentation.30 levofloxacin produces a broad spectrum of activity against several causative bacterial pathogens of community-acquired pneumonia (cap).31 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p153–157 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p153-157 157 suniti and sufiawati/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 153–157 corticosteroids may contribute to a reduced mortality rate in sjs and/or ten without increasing secondary infection.32 lenofavir, lamivudin and evafirenz are used in antiretroviral therapy.33 steven–johnson syndrome can be induced by herbal drugs. it is essential to be aware of their potential adverse effects, especially in immunocompromised patients. symptomatic management of oral lesions as an early intervention should be planned to decrease morbidity and mortality in sjs patients. hiv will further cause a decrease in the number of cd4+ lymphocytes and an increased ige level. this condition can lead to herbal medicine hypersensitivity. herbal medicines are considered safe because their natural origin is assumed to carry no risk. certain herbal medicines can induce hypersensitive reactions producing the same effects as chemical drugs. patients should become aware of these risks and report any serious adverse effects for the safety of others.when health care professionals construct drug histories they should actively ask their patients about all self-administered herbal medicines. references scully c. oral and maxillofacial medicine: the basis of diagnosis and1. treatment. 3rd ed. london: churchill livingstone; 2013. p. 277-80. deore ss, dandekar rc, mahajan am, shiledar v v. drug induced2. stevens johnson syndrome : a case report. int j sci study. 2014; 2(4): 84–7. vanker a, rhode d. human immunodeficiency virus and allergic3. disease. curr allergy clin immunol. 2009; 22(4): 168–72. mittmann n, knowles sr, koo m, shear nh, rachlis a, rourke4. sb. incidence of toxic epidermal necrolysis and stevens-johnson syndrome in an hiv cohort: an observational, retrospective case series study. am j clin dermatol. 2012; 13: 49–54. gerull r, nelle m, schaible t. toxic epidermal necrolysis and5. stevens-johnson syndrome: a review. crit care med. 2011; 39(6): 1521–32. yang s-c, hu s, zhang s-z, huang j, zhang j, ji c, cheng b. the6. epidemiology of stevens-johnson syndrome and toxic epidermal necrolysis in china. j immunol res. 2018; 2018: 1–10. mudzviti t, maponga cc, khoza s, ma q, morse gd. the impact7. of herbal drug use on adverse drug reaction profiles of patients on antiretroviral therapy in zimbabwe. aids res treat. 2012; 2012: 1–4. hsu dy, brieva j, silverberg nb, silverberg ji. morbidity and mor-8. tality of stevens-johnson syndrome and toxic epidermal necrolysis in united states adults. j invest dermatol. 2016; 136(7): 1387–97. knight l, muloiwa r, dlamini s, lehloenya rj. factors associated9. with increased mortality in a predominantly hiv-infected population with stevens johnson syndrome and toxic epidermal necrolysis. plos one. 2014; 9(4): 8–12. 10. yunihastuti e, widhani a, karjadi th. drug hypersensitivity in human immunodeficiency virus-infected patient: challenging diagnosis and management. asia pac allergy. 2013; 4: 54–67. 11. minhajat r, djaharuddin i, halim r, benyamin af, bakri s. drugs hypersensitivity reaction in patient with human immunodeficiency virus (hiv) infection. j allergy ther. 2017; 8: 1–4. 12. harr t, french le. toxic epidermal necrolysis and stevens-johnson syndrome. orphanet j rare dis. 2010; 5(39): 1–11. 13. virot e, duclos a, adelaide l, miailhes p, hot a, ferry t, seve p. autoimmune diseases and hiv infection: a cross-sectional study. medicine (baltimore). 2017; 96(4): 1–11. 14. blumenthal kg, ziegler jb. hypersensitivity reactions, dietary supplements, and the importance of the case report. j allergy clin immunol pract. 2016; 4: 177–8. 15. meincke r, pokladnikova j, straznicka j, meyboom rhb, niedrig d, russmann s, jahodar l. allergy-like immediate reactions with herbal medicines in children: a retrospective study using data from vigibase®. pediatr allergy immunol. 2017; 28(7): 668–74. 16. pokladnikova j, meyboom rhb, meincke r, niedrig d, russmann s. allergy-like immediate reactions with herbal medicines: a retrospective study using data from vigibase®. drug saf. 2016; 39(5): 455–64. 17. alostad ah, steinke dt, schafheutle ei. international comparison of five herbal medicine registration systems to inform regulation development: united kingdom, germany, united states of america, united arab emirates and kingdom of bahrain. pharmaceut med. 2018; 32: 39–49. 18. dookeeram d, bidaisee s, paul jf, nunes p, robertson p, maharaj vr, sammy i. polypharmacy and potential drug–drug interactions in emergency department patients in the caribbean. int j clin pharm. 2017; 39(5): 1119–27. 19. asher gn, corbett ah, hawke rl. common herbal dietary supplement-drug interactions. am fam physician. 2017; 96(2): 101–7. 20. mazzari alda, prieto jm. herbal medicines in brazil: pharmacokinetic profile and potential herb-drug interactions. front pharmacol. 2014; 5: 1–12. 21. rice jo. stockley’s herbal medicines interactions: a guide to the interactions of herbal medicines. second edition. j med libr assoc. 2014; 102(3): 221–2. 22. tang x, nian h, li x, yang y, wang x, xu l, shi h, yang x, liu r. effects of the combined extracts of herba epimedii and fructus ligustrilucidi on airway remodeling in the asthmatic rats with the treatment of budesonide. bmc complement altern med. 2017; 17: 1–12. 23. zhang p, ye y, yang x, jiao y. systematic review on chinese herbal medicine induced liver injury. evidence-based complement altern med. 2016; 2016: 1–15. 24. shi s, klotz u. drug interactions with herbal medicines. clin pharmacokinet. 2012; 51(2): 77–104. 25. sonaimuthu b, baghyanathan v. study on the functional role of immunoglobulin e as surrogate marker for hiv/aids infection. retrovirology. 2012; 9(suppl 2): p96. 26. ricci s, celani mg, cantisani ta, righetti e. piracetam for acute ischaemic stroke. cochrane database syst rev. 2012; (9): 1–16. 27. fares h, dinicolantonio jj, o’keefe jh, lavie cj. amlodipine in hypertension: a first-line agent with efficacy for improving blood pressure and patient outcomes. open hear. 2016; 3(2): 1–7. 28. mehta s, chandersekhar k, dutt l, nagpal rd, gupta m, kushwaha s, nag j, langade d, prasadrao g, patkar s, raju gsp, praveen kk, prasad bsv, roy t, pawar d. safety and efficacy of donepezil hydrochloride in patients with mild to moderate alzheimer�s disease: findings of an observational study. indian j psychiatry. 2012; 54(4): 337–43. 29. valkova m, stamenov b, peychinska d, veleva i, dimitrova p, radeva p. metoclopramide – induced extrapyramidal signs and symptoms – brief review of literature and case report. j imab. 2014; 20(6): 539–41. 30. telang ps. vitamin c in dermatology. indian dermatol online j. 2013; 4(2): 143–6. 31. blyth dm, markelz e, okulicz jf. cutaneous leukocytoclastic vasculitis associated with levofloxacin therapy. infect dis rep. 2012; 4: 35–7. 32. roongpisuthipong w, prompongsa s, klangjareonchai t. retrospective analysis of corticosteroid treatment in stevens-johnson syndrome and/or toxic epidermal necrolysis over a period of 10 years in vajira hospital, navamindradhiraj university, bangkok. dermatol res pract. 2014; 2014: 1–5. 33. menteri kesehatan republik indonesia. peraturan menteri kesehatan republik indonesia no 87 tahun 2014 tentang pedoman pengobatan antiretrovial. jakarta: kementerian kesehatan republik indonesia; 2014. p. 1–121. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p153–157 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p153-157 20 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 20–23 research report the potency of immunoglobulin y anti porphyromonas gingivalis to inhibit the adherence ability of porphyromonas gingivalis on enterocytes nova andriani hepitaria, indeswati diyatri, markus budi rahardjo and rini devijanti ridwan department of oral biology, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: pophyromonas gingivalis (p. gingivalis) bacteria are the main type of bacterium that cause chronic periodontitis. immunoglobulin y (igy) is a type of immunoglobulin found in poultry, such as chickens and birds. igy can be used as an alternative method of preventing the accumulation of plaque that causes chronic periodontitis. purpose: to determine the ability of igy anti p. gingivalis to inhibit adherence of p. gingivalis. methods: the samples were divided into eight groups, each group containing 10 ml of igy anti p. gingivalis and 50 ml of enterocyte cells. the control group contained 50 ml of igy anti p. gingivalis, and 50 ml of enterocyte cells. serial dilution was carried out to the first seven groups, with the first group containing 90 ml phosphate-buffered saline (pbs) and 10 ml igy anti p. gingivalis, and the second to seventh groups containing 50 ml pbs before adding 50 ml of enterocyte cells and 50 ml of bacterial suspension per group. the number of bacteria was calculated as an adherence index value using a light microscope. results: this study shows that igy anti p. gingivalis significantly reduces the adherence index value of p. gingivalis. conclusion: igy anti p. gingivalis has potency to inhibit the adherence of p. gingivalis. keywords: adherence; egg yolk; igy; pophyromonas gingivalis correspondence: rini devijanti ridwan, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo 47 surabaya 60132, indonesia. email: rini-d-r@fkg.unair.ac.id introduction periodontal disease or periodontitis is a bacterial infectious disease characterised by continuous inflammation, connective tissue damage, and alveolar bone destruction.1 severe periodontitis can lead to tooth loss. this disease can be found in approximately 5–20% of adults worldwide. periodontal disease is divided into three types: aggressive periodontitis; chronic periodontitis; and periodontitis caused by systemic disease manifestation.2 the bacterium that causes chronic periodontitis is pophyromonas gingivalis (p.gingivalis).3 this bacterium is a rod-shaped anaerobic gram-negative bacterium. p. gingivalis bacteria inhabit the subgingival area. some virulence factors of p. gingivalis bacteria include adhesives, capsules, lipopolysaccharides (lps), proteases, and outer membrane proteins. capsules can reduce the phagocytic activity for invasion; lpss, protease enzymes and membrane proteins can aid bacterial aggregation on the cell surface and fimbriae.4 pulling activity between the surface of the bacteria and the surface of the host cell is called bacterial adherence activity. there are three stages of bacterial adherence to the surface, namely transport, initial adherence (usually called bioattachment) and colonisation.5 antibiotics can be used to kill bacteria, but antibiotics also have a negative effect, including the resistance or increased ability of bacteria to stay alive in the presence of antibiotics.6 immunoglobulin y (igy) technology is an innovative technology that involves the non-invasive production of polyclonal antibodies from egg yolk, and due to its non-invasive nature, igy technology has provided new opportunities in both therapeutic and prophylactic applications in human and veterinary medicine.7 igy in the form of polyclonal antibodies is used as passive dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p20–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23 21hepitaria, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 20–23 immunisation, and these antibodies derive from egg yolk, colostrum, or concentrated cow’s milk.8 igy has the same biological role as igg in mammals, as a major type of immunoglobulin that provides defence against infectious agents.9 igy can be used to prevent periodontitis by inhibiting bacterial adherence to the cell surface, inhibiting enzyme activity, and neutralising the toxins produced by the periontopathogen.10 the aim of this research was to determine the ability of anti igy of p. gingivalis in egg yolk to inhibit the adherence of p. gingivalis bacteria. materials and methods this study was a laboratory in vitro experimental study with a post-test-controlled group design. igy specific p. gingivalis was obtained from chicken eggs that had been injected with p. gingivalis (atcc 33277) by as much as 1.5 x 109 bacterial colony, three times a week for three weeks. in the fourth week, the egg yolks were taken. the sample used was a mixture of enterocyte cells and igy that had been induced with p. gingivalis (atcc 33277) bacteria, and the control was a mixture of enterocyte cells with p. gingivalis (atcc 33277) bacteria with three times replication. this research required the following research tools: a centrifuge, an anaerobic jar, a shaking incubator, a measuring cup, a petri dish, a light microscope with 1000x magnification, a glass slide, a falcon tube, a microcentrifuge tube, a micropipette, a research subject, i.e. mice with body weight 135 g, specific p. gingivalis (atcc 33277) igy serum, a culture of p. gingivalis (atcc 33277) bacteria, phospate buffer saline (pbs), mueller-hinton broth (mhb), a solution containing pbs ph 7.4 + 1 mm dithiothreitol (dtt), a solution containing pbs ph 7.3, a solution containing pbs ph 7.4 + 1.5 mg ethylenediaminetetraactic acid (edta) + 0.771 mg dtt, methanol, violet crystals, safranin, lugol, and alcohol. one colony of p. gingivalis (atcc 33277) that had been grown in blood agar, was put into a test tube containing brain heart infusion (bhi) media using an oese and incubated in an anaerobic atmosphere using a gas generating kit for 24 hours at 37°c. after the incubation period, an equal concentration of bacteria was processed in another test tube containing bhi media, so that it was to the same mcfarland standard 0.5 (1.5 x 108 cfu/ml).11 isolation of enterocytes was performed using the weisler method.12 enterocytes were taken from the small intestine of 6–8-week-old mice weighing 135 g. the mice were humanely destroyed and then dissected to remove a part of the small intestine. the small intestine that had been taken from the body of the mice was then cut across and minced into small pieces, then washed using a solution containing pbs ph 7.4 + 1 mm dtt, to remove dirt and mucus. after the intestinal tissue was clean, it was put into a falcon tube, then as much as 20 ml of the solution containing pbs ph 7.3 was added. it was then put into a water heater at 37oc and shaken with a shaker for 30 minutes. the discarded supernatant was then replaced with as much as 30 ml of a solution containing pbs ph 7.4 + 1.5 mg edta + 0.771 mg dtt, and was shaken using a water bath for 30 minutes at 37°c. after being shaken, the supernatant was removed. falcon tubes containing enterocyte cells were washed using pbs. next, they were left until the enterocytes had all settled at the bottom of the tube. after the enterocytes had settled, the supernatant was removed, then 20 ml of pbs was added, and the tubes were inserted into the microcentrifuge tube and centrifuged at 1500 rpm for three minutes. separation of the p. gingivalis igy concentration was carried out using the dilution series method on the microcentrifuge tubes. the concentrations made were 1/10, 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, and these were put into eppendorf tubes. each tube was added 50 ml pbs solution except in a tube with a concentration of 1/10, in other six samples pbs solution was added as much as 90 µl and 10 µl igy p. gingivalis, then all of the samples homogenised using vortex. after that, 50 µl of the homogeneous solution was taken from the tube with a concentration of 1/10 using a micropipette and put into the tube with a concentration of 1/20, after which it was homogenised using a vortex. the same procedure was carried out until the concentration reached 1/640. in the tubes with a concentration of 1/640, as much as 50µl of the solution was removed.13 for adherence to the test procedure, cultures of bacteria were centrifuged at 6000 rpm and 4oc for 15 minutes. the precipitate was suspended in pbs containing 1% of bovine serum albumin (bsa). the bacterial content used was 108/ ml. then 50 µl of the enterocyte suspension was added to each concentration and shaken using a shaker in the water bath at 37oc for 30 minutes. as much as 50 µl of the mixture was then added to the bacterial suspension (108/ml) the mixture was incubated in the shaking incubator for 30 minutes at 37oc. it was then centrifuged at 1500 rpm and 4oc for 3 minutes, then as much as 100 µl of the liquid was disposed of. each precipitate was taken and smeared on a glass slide and painted with gram stain. the glass slides were observed under a light microscope with 1000x magnification, and the number of bacteria attached to enterocytes were counted, being calculated for each observation of 100 enterocytes.14 the kruskal wallis difference test was carried out in the control group and the treatment group to determine if the table 1. mean and standard deviation of the power of adherence of sample groups to p. gingivalis adherence pg n mean std. deviation control 6 12.7583 6.44508 a-1/10 6 4.0733 .53638 b-1/20 6 5.3417 .85873 c-1/40 6 5.4517 1.75848 d-1/80 6 4.8500 1.77213 e-1/160 6 6.2950 1.89505 f-1/320 6 4.1700 1.58865 g-1/640 6 9.1000 4.19729 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p20–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23 22 hepitaria, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 20–23 significance value was below 0.05 (p<0.05), i.e. showed significant differences between groups. then the bonferroni test was carried out. results the treatment group with p. gingivalis (atcc 33277) of concentration 1/640 had the largest mean of 9.1, whereas the treatment group with p. gingivalis (atcc 33277) of concentration 1/10 had the smallest mean of 4.07. the mean and standard deviation of the power of adherence in each treatment group of the p. gingivalis bacteria can be seen in table 1. the research data were analysed using the kruskall wallis test. the results of the kruskall wallis test in this study were p = 0.000 (p <0.05), so it could be interpreted that there were significant differences between the data of the entire group.the bonferroni test results, shown in table 2, obtained a value of 0.00 between the control group and the sample groups, except in the group 1/320. this shows that there was a significant difference between the control group and the sample groups. comparison between the sample groups produces a value of 1.000. this can be interpreted as there being no significant difference between the sample groups. figure 1 shows the result of the experiment in adherence of bacteria onto the enterocyte cells. discussion igy has several advantages compared to antibiotics, vaccines, and immunotherapy. the advantages of igy compared to antibiotics are: 1) it is natural; 2) it is safe and is not absorbed into the body circulation (no toxic tissue residues); 3) it avoids environmental contamination with synthetic chemical drugs; 4) it does not induce specific pathogenic resistance of microorganisms since it is directed to multi epitopic antigenic targets that need multiple genes for their synthesis; 5) it is highly specific in its reactivity and controls only targeted pathogens without affecting normal bacterial flora; 6) it has a potentially broad spectrum of specificity when customised against viruses, bacteria or fungi; and 7) it does not induce adverse side effects as synthetic drugs do.10 the advantage of igy as passive immunotherapy is that igy has rapid action and high specific activity; it can be given to all ages ranging from infants to adults, including babies with low birth weight (lbw), patients with immunodeficiency, and pregnant women. it is not toxic and can be stored for a long time. igy is attractive for oral immunotherapy because some of its properties can be taken from animals without hurting them, the binding to antigens is stronger than in mammalian igg, and it reacts more to the same antigen. igy is also a natural ingredient that does not cause side effects when taken orally unless the recipient has an allergy to eggs.15 based on the results and analysis of the data, it was found that p. gingivalis igy can inhibit the adherence of p. gingivalis bacteria. this is likely because igy is a polyclonal antibody that can capture various epitopes on the cell surface of the bacterium. antibodies are host proteins found in plasma and extracellular fluids that serve as the first response and comprise one of the principal effectors of the adaptive immune system. they are produced in response to molecules and organisms, which they ultimately neutralise and/or eliminate. the ability of antibodies to bind to an antigen with a high degree of affinity and specificity has led to their ubiquitous use in a variety of scientific and medical disciplines. the formation of antibodies is not only due to the binding of the epitope with the antibodies, b a figure 1. bacterial adherence in the sample group to p. gingivalis (a) in enterocyte cell. (b) control group. arrow indicates p. gingivalis adherence to the enterocyte cell. table 2. results of bonferroni test to p. gingivalis groups 1 2 3 4 5 6 7 8 1 2 .000* 3 .003* 1.000 4 .004* 1.000 1.000 5 .001* 1.000 1.000 1.000 6 .017* 1.000 1.000 1.000 1.000 7 .000* 1.000 1.000 1.000 1.000 1.000 8 1.000 .173 1.000 1.000 .532 1.000 .200 note: 1) control group; 2) treatment group 1/10; 3) treatment group 1/20; 4) treatment group 1/40; 5) treatment group 1/80; 6) treatment group 1/60; 7) treatment group 1/320; 8) treatment group 1/640. *there are significant differences between sample groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p20–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23 23hepitaria, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 20–23 but because bacteria are one type of antigen that has virulence factors that can stimulate antibody formation.16 the main mechanism of igy is binding to components on the surface of bacteria, such as outer membrane proteins, lipopolysaccharides,as well as colonisation tools such as vesicles and fimbriae which are virulence factors of the p. gingivalis bacteria.17 the adherence value of p. gingivalis bacteria is significantly different between the treatment groups and the control group because of the possibility that this bacterium has expressed its virulence factor, particularly adhesion.18 fragments of antigen binding (fab) possessed by igy anti p. gingivalis can bind and recognise proteins on the cell surface of the bacterium. fab from igy anti p. gingivalis can also bind to fimbriae, which are bacterial movement tools, to attach to periodontal tissue and carry out colonisation activities. as shown in this study, igy binds to enterocytes.19 the presence of fimbriae is a virulence factor that plays a major role in the adherence activity of the p.gingivalis.20 on periodontal tissue, periodontitis carried out by imbriae from this bacterium are bound by igy p.gingivalis,21 whereas to carry out pathogenic activities, the process that must be carried on is that bacteria must be attached to the periodontal tissue and then carry out colonisation activities with similar bacteria.21 comparing concentration groups, based on the results of the data analysis, there were significant differences in the comparison of the control group with the concentration of the p. gingivalis, while in the 1/640 dilution group no significant difference could be seen, indicating igy’s ability to inhibit bacterial adherence on enterocytes at a minimum concentration. however, comparing between fellow treatment groups, there was no significant difference. this shows that, to inhibit p. gingivalis bacteria, it was sufficient to use a minimum concentration of igy anti p. gingivalis in this study (1/320). the conclusion of this study is that igy anti p. gingivalis has the potency to inhibit the adherence of p. gingivalis on enterocytes, and this potency is not dependent on the concentration. references 1. vargas segura ai, ilyina a, segura ceniceros ep, silva belmares y, méndez gonzález l. etiology and microbiology of periodontal diseases: a review. african j microbiol res. 2015; 9(48): 2300–6. 2. aljehani ya. risk factors of periodontal disease: review of the literature. int j dent. 2014; 2014: 1–9. 3. ismail ad. oral bacterial interactions in periodontal health and disease. j dent oral hyg. 2014; 6(5): 51–7. 4. how ky, song kp, chan kg. porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. front microbiol. 2016; 7: 1–14. 5. sandle t. bacterial adhesion: an introduction. ivt nerwork. 2013; : 1–8. 6. nami y, haghshenas b, abdullah n, barzegari a, radiah d, rosli r, khosroushahi ay. probiotics or antibiotics: future challenges in medicine. j med microbiol. 2015; 64(2): 137–46. 7. baloch ar, zhang xy, schade r. igy technology in aquaculture a review. rev aquac. 2015; 7(3): 153–60. 8. ba cht ia r e w, a fd ha l a , meidyawat i r , so ejo e dono r d, poerwaningsih e. effect of topical anti-streptococcus mutans igy gel on quantity of s. mutans on rats’ tooth surface. acta microbiol immunol hung. 2016; 63(2): 159–69. 9. munhoz ls, vargas gdá, fischer g, lima m de, esteves pa, ḧbner s de o. avian igy antibodies: characteristics and applications in immunodiagnostic. cienc rural st maria. 2014; 44(1): 153–60. 10. rahman s, van nguyen s, icatlo fc, umeda k, kodama y. oral passive igy-based immunotherapeutics: a novel solution for prevention and treatment of alimentary tract diseases. hum vaccines immunother. 2013; 9(5): 1039–48. 11. wicaksono aw, setiawatie em, rubianto m. daya hambat ekstrak buah mengkudu (morinda citrifolia l.) terhadap pertumbuhan bakteri porphyromonas gingivalis. thesis. universitas airlangga: surabaya; 2013. p. 3. 12. hidayati dyn, santosaningsih d, khotimah h. characterization of pili protein as cell adhesion molecules of mycobacterium tuberculosis h37rv to mouse enterocytes: in vitro study. int j res rev. 2018; 5(4): 66–72. 13. reynolds j. serial dilution protocols. am soc microbiol. 2005; (september 2005): 1–7. 14. ridwan rd. the role of actinobacillus actinomycetemcomitans fimbrial adhesin on mmp-8 activity in aggressive periodontitis pathogenesis. dent j (majalah kedokt gigi). 2012; 45(4): 181–6. 15. thu hm, myat tw, win mm, thant kz, rahman s, umeda k, van nguyen s, icatlo fc, higo-moriguchi k, taniguchi k, tsuji t, oguma k, kim sj, bae hs, choi hj. chicken egg yolk antibodies (igy) for prophylaxis and treatment of rotavirus diarrhea in human and animal neonates: a concise review. korean j food sci anim resour. 2017; 37(1): 1–9. 16. lipman ns, jackson lr, trudel lj, weis-garcia f. monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ilar j. 2005; 46(3): 258–68. 17. chalghoumi r, beckers y, portetelle d, théwis a. hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infections in chicken: a review. biotechnol agron soc env. 2009; 13(2): 295–308. 18. pandit n, changela r, bali d, tikoo p, gugnani s. porphyromonas gingivalis: its virulence and vaccine. j int clin dent res organ. 2015; 7(1): 51. 19. gani ba, chismirina s, hayati z, b ew, bachtiar bm, wibawan i w t. t he abi l it y of igy to recog n ize su r face protei ns of streptococcus mutans. dent j (majalah kedokt gigi). 2009; 42(4): 189–93. 20. garrett tr, bhakoo m, zhang z. bacterial adhesion and biofilms on surfaces. prog nat sci. 2008; 18(9): 1049–56. 21. hasan a, palmer rm. a clinical guide to periodontology: pathology of periodontal disease. vol. 216, british dental journal. nature publishing group; 2014. p. 457–61. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p20–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p20-23 4949 correlation of cost, time, need, access, and competence with the public interest in installing dentures at non-professional dentist yayah sopianah,1 muhammad fiqih sabilillah,2 and ayyu fadilah3 1,2 lecturers dental nursing programme, poltekkes kemenkes tasikmalaya 3 dental nursing, poltekkes kemenkes tasikmalaya, tasikmalaya – indonesia abstract background: tooth loss can be caused by trauma, caries, and periodontal disease. it then can trigger an emotional impact on each individual, such as having a lack of confidence, feeling ashamed of the appearance, and trying to hide the missing teeth. as a result, people experiencing tooth loss want to put or replace their missing teeth with artificial ones. unfortunately, many of them prefer to visit non-professional dentists rather than professional ones providing the same services because of both internal and external factors. purpose: this research aimed to analyze factors affecting the public interest in losari district of brebes regency in 2016 to install dentures in non-professional dentists. method: this research focused on a correlation of cost, time, need, access, and competence with denture-making care interest using survey method. sampling was conducted with purposive sampling technique. the number of samples was thirty-six people. result: there was a significant correlation between public interest in installing dentures at non-professional dentists and cost factor (a p value of 0.010, <a 0.05). similarly, there was a significant correlation between public interest in installing dentures at non-professional dentists and time factor (a p value of 0.000, <a 0.05). there were significant correlations between the public interest in installing dentures at non-professional dentists and need (a p value of 0.004, <a 0.05), as well as between public interest in installing dentures at non-professional dentists and access factor (a p value of 0.000 <a 0.05). nevertheless, there was no correlation between public interest in installing dentures at non-professional dentists and competence factor (a p value of 0.482> a 0.05). conclusion: it can be concluded that there is a correlation between cost, time, need, and access with the public interest in installing dentures at non-professional dentists. community competence has no correlation with public interest in installing dentures at non-professional dentists. keywords: public interest; denture installation; non-professional dentists correspondence: yayah sopianah, dental nursing programme, poltekkes kemenkes tasikmalaya. jl. tamansari no. 210 tasikmalaya, indonesia. e-mail: sabilillah.fiqih@gmail.com research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 49–53 introduction teeth have an important role in human body. teeth play a role in chewing, improving performance aesthetically, and talking. every individual ideally will maintain their permanent teeth naturally throughout their life. however, teeth sometimes can be loose or revoked for various reasons. tooth loss also can be caused by trauma, caries, and periodontal disease. consequently, tooth loss can trigger an emotional impact on each individual, such as having a lack of confidence, feeling ashamed of the appearance, and trying to hide the missing teeth.1 the number of teeth revoked in people aged 65 years and above is sixteen. the prevalence of dental and oral problems, based on riskesdas data in 2013, was 25.9%, higher than the prevalence in 2007, only about 23.4%. the prevalence of denture installation was only about 4.5% of the population of indonesia. in detail, the prevalence of complete denture installation (gtl) was 30.43%, 39.13% for removable partial denture installation (gtsl), and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p49-53 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p49-53 50 sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 49–53 40.43% for permanent denture installation. unfortunately, tooth loss case is still often found anywhere, consequently, denture installation is getting higher to replace the missing teeth as well as to improve and maintain the function of the teeth.2 people experiencing tooth loss usually want to put or replace their missing teeth with artificial ones. nevertheless, many of them prefer to visit non-professional dentists rather than professional ones offering the same services. non-professional dental practice can easily be found almost throughout indonesia. initially, the non-professional dentists only offer denture making service. nowadays they also offer denture’s crown making service and dental filling service regardless of the medical rules since they have never studied about dentistry.3 in the manufacture of dentures, the non-professional dentists do not pay attention to the health of the tissue around the dentures. they also make dentures carelessly. the remaining roots even are still found, not removed during the installation of dentures, resulting in inflamed gingival tissue, swelling, poor oral hygiene, or denture stomatitis due to poor denture adaptation.4 the negative effects of the installation commonly found in their users are severe infections, swollen face, as well as red and swollen gingival area with trismus (unable to open the mouth) condition, besides of great halitosis (bad breath).5 factors that affect the public interest in installing dentures at non-professional dentists are internal and external factors. interest is actually something personal and closely related to attitude. interests and attitudes are fundamental to prejudice, affecting decision making. interest may also trigger a person to undertake activities actively, leading to something interested.1 based on a preliminary research conducted on fifteen respondents in losari district of brebes regency in february, there were 53% of them installing their dentures at non-professional dentists for various reasons. similarly, based on interview results, some non-professional dentists also claimed that there were still many people who preferred to install their dentures with them. however, there had not been any statistical data about this situation. this research aimed to examine factors affecting the public interest in installing dentures at non-professional dentists in losari district of brebes regency in 2016. materials and method this research focused on a correlation of cost, time, need, access, and competence with denture-making care interest using survey method. this research was conducted in losari district of brebes regency. samples in this research were people in losari district, installing dentures at non-professional dentists. sampling was performed with purposive sampling technique. it means that the samples were taken based on the consideration of the researcher, in accordance with inclusion and exclusion criteria that had been determined. inclusion criteria in this research were living in losari district, installing dentures at nonprofessional dentists, aged >17 years, and willing to be cooperative during collecting data. meanwhile, exclusion criteria in this research were installing dentures at professional dentists and unwilling to be cooperative during data collection. the number of samples was thirty-six people. variables measured in this research were internal factors (cost, time, and need) and external factors (access and competence) correlated with public interest in installing dentures at non-professional dentists. next, primary data collection was conducted by using questionnaire. in this research, people considered as samples had to sign informed consent first. they had to fill out a questionnaire about what factors affecting their interest in installing dentures at the non-professional dentist. this study uses statistical tests of crosstabs correlations. analysis of the data in this study using a computer program to test the hypothesis based on a significance level of <0.05 with 95% confidence level. results the frequency distribution of the respondents’ sex in table 1 shows that there were sixteen male respondents (44.44%) and twenty female respondents (55.55%). meanwhile, characteristics of the respondents’ age can be seen in table 2. the frequency distribution of the respondents’ age in table 2 demonstrates that most respondents were at the age of 51-66 years old, i.e. thirteen people (36.11%), and at least three respondents (8.33%) aged 31-40 years old. characteristics of the respondents’ education level can be seen in table 3. the frequency distribution of the respondents’ education level in table 3 depicts that the highest number of the respondents was found in high school education level with a percentage of 33.33%. meanwhile, the least number of the table 1. frequency distribution of the respondents’ sex no. sex frequency percentage (%) 1 males 16 44.44 2 females 20 55.55 total 36 100 table 2. frequency distribution of the respondents’ age no. age frequency percentage (%) 1 25-30 9 25 2 31-40 3 8.33 3 41-50 11 30.55 4 51-66 13 36.11 total 36 100 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p49-53 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p49-53 5151sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 49–53 status. furthermore, bivariate analysis was performed by correlating the independent variables (cost, time, need, access and competence) with the dependent variable (public interest) as seen in table 5. based on table 5, there was a significant correlation between cost, time, need and access with the public interest (p<0.05). meanwhile, there was no correlation between competence and public interest (p>0.05). discussion this research was conducted on people living in losari district of brebes regency. public interest in installing dentures at non-professional dentists in losari district of brebes regency was influenced by several factors, including internal factors and external factors. internal factors in this research were cost, time, and need. meanwhile, external factors in this research were access and competence. results of the bivariate analysis using correlation method with crosstab statistics show that there was a significant correlation between cost factor and public interest in installing dentures at the non-professional dentists in the losari district of brebes regency. this is because installing dentures at professional dentists requires a lot of money. if installing dentures in professional dentists will be charged service fees, the cost of making dentures will be increased. meanwhile, installing dentures at non-professional dentists is only charged for denture manufacturing cost. the cost of dental care of the professional dentists is more expensive than that at the non-professional ones because of the time and expenses incurred to pursue an education in order to gain their competence as professional dentists. on the other hand, the non-professional dentists pursue their dental care skills only from their predecessors or from their education as dental technicians who should deal with laboratory, not directly with the community.6 data on the socio-economic condition of the people of losari district of brebes regency, indicated that most of them work as farmers and small-scale traders. as a result, it can be understood if they prefer to seek cheaper health services. similarly, a research conducted by teo’filo and leles shows that 88.8% of the samples studied have financial constraints, thus, most of them prefer not to pursue denture treatment.7 economic status is the most important predictor for a person in making decisions to choose treatment. socio-economic condition of a family can be measured by family income.8 in addition, a research conducted by meutuah in medan shows that 92.5% of the research samples prefer to install dentures at nonprofessional dentists due to relatively cheaper cost than at professional dentists.9 the bivariate analysis using correlation method with crosstab statistic shows that there was a significant correlation between time factor and public interest in installing dentures at the non-profesional dentists in losari table 3. frequency distribution of the respondents’ education level no. education level frequency percentage (%) 1 non-school education level 1 2.78 2 elementary level 8 22.22 3 junior high level 10 27.78 4 senior high level 12 33.33 5 undergraduate level 5 13.89 total 36 100 table 4. frequency distribution of the respondents’ job status no. job status frequency percentage (%) 1 not working 23 63.88 2 working 13 36.11 total 36 100 table 5. results of the bivariate analysis on the correlation of cost, time, need, access and competence with interest cost sig. good sufficient interest high 100% .010 low 38.7% 61.3% time sig. good sufficient interest high 100% .000 low 19.4% 80.6% need sig. good sufficient interest high 60% 40% .004 low 9.7% 90.3% access sig. good sufficient interest high 100% .000 low 22.6% 77.4% competence sig. sufficient poor interest high 100% .482 low 90.3% 9.7% respondents was found in non-school education level with a percentage of 2.77%. characteristics of the respondents’ job status can be seen in table 4. the frequency distribution of the respondents’ job status in table 4 indicates that there were twenty-three respondents (63.88%) with unemployment status, while there were thirteen respondents (36.11%) with employment dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p49-53 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p49-53 52 sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 49–53 district of brebes regency. according to the respondents, the non-professional dentists work faster. those dentists even can be called to their homes of residents to make dentures without taking a long time. besides, treatment procedures performed by the non-professional dentists are very different from the procedures performed by professional ones requiring the respondents to visit them more than once to having denture treatment. non-professional dentists in makasar’s kodingareng island work quickly so that their patients do not have to wait long. most of those patients just need to cast their teeth before denture installation without examining the condition of their oral cavity first. consequently, there is a root residue under the dentures, resulting in inflammation of the gingival tissue. unlike at the non-prefessional dentists, professional dentists require a longer process since they have to undergo several stages of the treatment, such as diagnosis, jaw printing, preparatory treatment, laboratory processes, and denture installation.10 the bivariate analysis using correlation method with crosstab statistic indicates that there was a significant correlation between the need factor and the public interest in installing dentures at the non-professional dentist in the losari district of brebes regency since most of the people experiencing anterior tooth loss require tooth replacements for aesthetic reasons, while those experiencing posterior tooth loss require tooth replacements for mastication. tooth loss can also have an emotional impact, leading to stress. a research conducted by davis et al.,11 in london even shows that 45% of people experiencing tooth loss are difficult to accept the situation and feel less confident, as a result, they do not want to be seen by others if not using dentures. it indicates that dentures used to replace missing teeth can restore the confidence of the users. need factor is a factor often experienced by the elderly. the older the age is, the worse the nutritional status is.12 in other words, tooth loss at the old age can affect mastication. although tooth loss does not have a direct impact, the mastication system will decrease, affecting nutrient intake. alveolar bone generally will change, especially due to bone mineral loss since aging can affect resorption process of bone matrix. this process, unfortunately, can be accelerated by tooth loss. therefore, most of elderly people need for dentures.13 like the previous researchers, data obtained in this research also indicate that most respondents have understood the need of dental care and treatment for themselves. thus, they have a great interest in installing dentures at the non-professional dentists in order to replace the function of their missing teeth. dentures made by those dentists can immediately fulfill the needs of people who lost their teeth, in addition to quickly recover their lost confidence. the bivariate analysis using correlation method with crosstab statistic reveals that there was a significant correlation between access factor and public interest in installing dentures at the non-professional dentists in losari district of brebes regency since those dentists also live in the district of losari, so their access to the community is easier and closer. the presence of professional dentists is still very minimal and uneven in the district of losari. the nearest public health center even does not serve the manufacture of dentures. consequently, people prefer to go to the non-professional ones. the bivariate analysis using correlation method with crosstab statistic finds that there was no significant correlation between community competence factor and public interest in installing dentures at the non-professional dentists in losari district of brebes regency. this is because they perform their dental care and treatment without permission. it means that they perform their dental care and treatment only based on limited knowledge. they just concern with the satisfaction of the community without considering the impacts that will be experienced by the users of their services. based on the regulation of the minister of health no.39 of 2014 article 6 paragraph (2), the authority of non-professional dentists as clearly written is that they are indeed capable of making dentures, but the manufacture and installation of dentures must be conducted by those who have scientific knowledge about oral cavity health in order to prevent the users from infection, swollen face, as well as red and swollen gingival area. those are not concerned by the non-professional dentists.14 the competence of the non-professional dentists is clearly different from the competence of the professional ones. according to the regulation of the minister of health no. 512/ menkes/ per/ iv/ 2007 concerning the practice license and the implementation of medical practice in chapter 1 article 1 point 7, medical services are health services provided by professional doctors or dentists in accordance with their competence and authority, including promotive, preventive, diagnostic, consultative, curative and rehabilitative services.15 it can be concluded that there is a correlation between cost, time, need, and access with the public interest in installing dentures at non-professional dentists. community competence has no correlation with public interest in installing dentures at non-professional dentists. references 1. gunarsa ds. psikologi perawatan. jakarta: gunung mulia; 2008; 193-203. 2. tampubolon ns. dampak karies gigi dan penyakit periodontal terhadap kualitas hidup. 2010; 1-3. 3. rahmayani l, ifwandi, hasanah i. analisis pemakaian jasa pemasangan gigi tiruan sebagian lepasan akrilik pada dokter gigi dan tukang gigi di desa peuniti banda aceh. jurnal pdgi 2012; 61(2): 74-9. 4. angraeni a. persepsi masyarakat terhadap pembuatan gigi tiruan oleh tukang gigi di desa treman kecamatan kauditan. e-gigi. 2013 dec 11;1(2). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p49-53 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p49-53 5353sopianah, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 49–53 5. wahab sa. perbandingan karakteristik pengguna gigi tiruan yang dibuat di dokter gigi dengan tukang gigi di banjarmasin (tinjauan terhadap pengetahuan dan biaya pembuatan gigi tiruan). dentin. 2017 may 3;1(1). 6. lumunon to. gambaran determinan perilaku masyarakat dalam pemanfaatan jasa tukang gigi pada pembuatan gigi tiruan lepasan di desa treman kecamatan kauditan. e-gigi. 2014 jun 3;2(1). 7. teófilo lt, leles cr. patient self-perceived impacts and prostodontic need at the time and after tooth loss. braz dent journal 2007; 18(2): 91-6. 8. mcgrath c, bedi r. severe tooth loss among uk adults – who goes for oral rehabilitation?. j oral rehabil 2002; 29: 240–4. 9. meutuah s. hubungan karakteristik pengguna gigi palsu dengan pemanfaatan jasa tukang gigi di kota medan tahun 2008. 2009; 1-5. 10. putra mt. tingkat kepuasan masyarakat pulau kodingareng terhadap pelayanan kesehatan gigi dan mulut. 2012; 1-5. 11. davis dm, fiske j, scott b, radford dr. the emotional effects of tooth loss: a preliminary quantitative study. br dent j 2000; 188(9): 503. 12. forster s, gariballa s. age as a determinant of nutritional status: a cross sectional study. nutrition journal 2005; 4: 28. 13. damayanti dl, pros s. respon jaringan terhadap gigi tiruan lengkap pada pasien usia lanjut. abstrak. 2009; 1. 14. peraturan menteri kesehatan r.i nomor 39 tahun 2014. pembinaan, pengawasan dan perizinan, pekerjaan tukang gigi. jakarta. 2014; 7-8. 15. peraturan menteri kesehatan nomor 512/menkes/per/iv/2007. izin praktek dan pelaksanaan praktek kedokteran. jakarta. 2007; 1-14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p49-53 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p49-53 119119 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 119–123 original article evaluation of osteogenic properties after application of hydroxyapatite-based shells of portunus pelagicus michael josef kridanto kamadjaja, alya nisrina sajidah gatia, agtadilla novitananda, lintang maudina, harry laksono, agus dahlan, bambang agustono satmoko tumali and muhammad dimas aditya ari department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: after tooth extraction, the socket leaves a defect on the alveolar bone. the administration of shell crab-derived hydroxyapatite maintains bone dimensions that are important for achieving successful prosthodontic treatment. purpose: the aim of the study was to determine the osteogenic properties, such as the number of osteoclasts, osteoblasts and osteocytes, after the application of hydroxyapatite-based shell crab in the post-extraction sockets of wistar rats. methods: there were two groups: the control group (k) and the treatment group (t). wistar rats were randomly divided into control and treatment groups. after tooth extraction, hydroxyapatite gel derived from portunus pelagicus shells was applied to the tooth sockets of wistar rats. observations and calculations of osteoclasts, osteoblasts and osteocytes were carried out on the 14th and 28th days under a light microscope with 400 times magnification. statistical analysis was performed using one-way anova. results: there was a significant difference (p<0.05) between the k14 and p14 groups, k28 and p28 groups, k14 and k28 groups, and p14 and p28 groups. the results indicated that there were significant differences between groups of variables. conclusion: the application of shell crab-derived hydroxyapatite (portunus pelagicus) was able to decrease the number of osteoclasts and increase the number of osteoblasts and osteocytes. keywords: hydroxyapatite; portunus pelagicus; osteoblasts; osteoclasts; osteocytes correspondence: michael josef kridanto kamadjaja, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47, surabaya 60132 indonesia. email: michael-j-k-k@fkg.unair.ac.id. introduction tooth extraction is the process of removing the tooth from the alveolar bone when teeth are unable to withstand further treatment.1 the post-extraction socket healing process leaves an alveolar defect. along with the growth of bone in post-extraction sockets, there is also a process of resorption on the alveolar ridge.2 there is a decrease in the buccolingual dimensions, as well as a decrease in the apicoronal dimensions of the alveolar ridge, as is often found after tooth extraction.3 reduction of the alveolar ridge can interfere with prosthodontic treatment. resorption can lead to a loss of aesthetic and function, which can be harmful when paired with dental implants, especially in the anterior maxilla.4 bone resorption after tooth extraction would cause difficulties for implant placement. this can be overcome by preserving the socket. there are several methods that can be used to minimise the occurrence of bone resorption. among them is the use of demineralised freeze-dried bone allograft (dfdba), bioglass and hydroxyapatite (ha), which has been used in the form of both a resorbable and non-resorbable membrane.5 calcium phosphate bioceramics, such as ha, are popular materials for bone reconstruction.6 ha bioceramic materials form up to 70% of bone structure. ha can be produced synthetically from chemical reagents or can be synthesised from natural resources through hydrothermal transformation and the high-temperature calcination of bones.7 raw ha biomaterials are easily available and are abundant in indonesia. among the abundant raw materials is shell crab, which is one of indonesia’s export commodities. indonesia exports 604,215–625,000 tons of crab without shells per year.8 flower crabs (portunus pelagicus) has been a mainstay of indonesia’s export commodities to various countries around the world.8 crab shells contain calcium dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p119–123 mailto:michael-j-k-k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p119-123 120 kamadjaja et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 119–123 carbonate (caco3), which can be processed further into ha [ca5(po4)3(oh)]. ha’s structure is identical to that of human bone, which renders it a potential source of synthetic bone for bone grafts. in the field of dentistry, bone grafts are used to increase the alveolar ridge height, remodel the jawbone, transfer tissue free of microvascular problems and re-establish the alveolar crest.8 ha has osteoconductive properties and can stimulate mesenchymal cells to proliferate and differentiate in the bone regeneration process. porous ha forms a strong bond between the bones, accelerating the process of vascularisation. the porosity of the bone graft increases the osteoconductive properties and the colonisation of osteoblasts, facilitates the penetration of osteoblast cells and provides a medium for osteoblasts to attach to.9 osteoblasts and osteocytes secrete osteoprotegerin (opg), which acts as a binder for the rankl receptor and decreases the differentiation of osteoclasts.10 opg has been shown to function as an inhibiting factor for osteoclastogenesis in vivo and in vitro.11 this study aimed to determine the effect of ha crab shell on the number of osteoclasts, osteoblasts and osteocytes in the tooth sockets of wistar rats. materials and methods this study was approved by the institutional health research ethical clearance commission with certificate number 177/hrecc.fodm/vii/2018. an ha powder was made from crab shell by soaking the shell in water (ratio 3:20) for 15 minutes. the powder was immersed in chlorine and dissolved in water (10 ml of chlorine was used for 20 litres of water). it was soaked for 5 minutes. the calcination process was carried out by heating the material in a furnace at an initial temperature of ± 50°c, which slowly increased by 5°c/minute until the temperature reached 1000°c. it was maintained at this temperature for two hours. the ha powder was made into a gel by adding carrageenan powder and water with a ratio of 6:3:2, then mixed and heated slowly at 70°c for 10 minutes to form a gel compound. the experimental subjects were 36 wistar rats divided into four subgroups. each group consisted of nine wistar rats: control group at 14 days (k14), control group at 28 days (k28), treatment group at 14 days (p14) and treatment group at 28 days (p28). the rats were sedated with 10% ether. each had its mandibula left incisor extracted using sterile forceps. for the treatment groups, ha gel was applied to the sockets, which were then sutured with silk thread 3/0. meanwhile, the sockets of the control groups were sutured without the application of ha gel. all the wistar rats were sacrificed on the 14th and 28th days. rats were euthanised using ketamine at a lethal dose (66–88 mg/kg of body weight). the mandible was cut and immersed in a 10% formaldehyde solution for at least 24 hours. decalcification was performed using ethylenediaminetetraacetic acid. the tissue then underwent dehydration and was stored at 60°c for some time before being submerged in liquid paraffin. the paraffin blocks containing tissue were then cut using a microtome machine (3–4µm). the slices of tissue were then inserted into a water bath (30–40°c). the tissue pieces were carefully attached to a glass object and 1–2 drops of albumin were added to the tops of the tissue pieces. after that, the glass object was heated with a hot plate at a temperature of 30–40°c. haematoxylin and eosin staining was then used to measure the number of osteoclasts, osteoblasts and osteocytes. this observation was carried out under a light microscope with 400 times magnification. statistical analysis was performed using one-way anova with a p value of <0.05. results the measurements of the osteoblasts, osteoclasts and osteocytes on the 14th and 28th days from all groups can be seen in figure 1. histological imaging of the osteoblasts, osteoclasts and osteocytes can be seen in figures 2, 3 and 4. the data was analysed using the kolmogorov-smirnov test and levene’s test, and the results showed that the data were normally distributed (p>0.05) and homogenous (p>0.05). 0 2 4 6 8 10 12 14 16 18 control 14 day treatment 14 day control 28 day treatment 28 day m ea n osteoclasts osteoblasts osteocytes number of cells figure 1. diagram of the mean number of osteoclasts, osteoblasts and osteocytes from the control and treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p119–123 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p119-123 121kamadjaja et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 119–123 a b c d figure 2. a histological view of the osteoclasts on the 14th (a) and 28th (b) days of the control group and the 14th (c) and 28th (d) days of the treatment group. a b c d figure 3. a histological view of the osteoblasts on the 14th (a) and 28th (b) days of the control group and the 14th (c) and 28th (d) days of the treatment group. a b c d figure 4. a histological view of the osteocytes on the 14th (a) and 28th (b) days of the control group and the 14th (c) and 28th (d) days of the treatment group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p119–123 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p119-123 122 kamadjaja et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 119–123 after the homogeneity test was carried out, a significance test was conducted using the one-way anova test. the results showed that there were significant differences between groups of variables (p<0.05). a post hoc tukey test was also conducted to determine the significance of the number of osteoclasts, osteoblasts and osteocytes in each study group. the significant differences between each group can be seen in table 1. discussion tooth extraction is the most common procedure in the field of dentistry. the response to the body’s normal healing process after tooth extraction often causes significant bone resorption.12 after tooth extraction, the alveolar bone is gradually absorbed by the body. then, a remodelling process occurs, which results in a decrease in the dimensions of the alveolar bone. the vertical plane decreases and tends to be more palatal than its original position.13 the bone remodelling process consists of several phases, beginning with the activation phase. the activation phase involves the recruitment and activation of osteoclast monocyte-macrophage precursors from the circulation, resulting in the interaction of osteoclast precursor cells and osteoblasts. then, during the resorption phase, osteoclasts begin to dissolve the mineral matrix and decompose the osteoid matrix.12 the resorption phase is dominated by osteoclasts. next comes the recovery phase, in which the transition from bone resorption to bone formation occurs. bone absorbed in the resorption phase contains various mononuclear cells, including monocytes, osteocytes released from the bone matrix and preosteoblasts, which function to begin the process of new bone formation. in the formation phase, osteoblast cells are released on the surface to begin bone formation.13 the process is completed by the mineralisation phase, which begins 30 days after osteoid deposition.14 to maximise bone regeneration after tooth extraction and minimise the occurrence of bone resorption, the socket is filled with bone graft material. when filling the socket, actions that could cause trauma to the bone should be avoided, thereby reducing the occurrence of buccal, lingual and ridge alveolar resorption.12 calcium phosphate bioceramics, such as ha, are popular materials for bone reconstruction. bioceramic ha material forms up to 70% of the bone structure. ha is effectively used to replace part or all of the bone tissue. it can be used as a bone filling material. ha can produce a physicochemical interaction between ceramics and bone tissue, thus encouraging the binding and growth of new tissue.15 the ha in this study was made from crab shell, which was first made into a ha powder using a furnace, then converted into a crab shell-based ha gel. the crab shellbased ha gel used in this study contained 87.11% ha. the results showed a significant difference in the number of osteoclasts on the 14th and 28th days. this was because, on the 14th day, the resorption phase was dominated by osteoclasts. osteoclasts need 2–4 weeks for the remodelling cycle to complete bone resorption. meanwhile, on the 28th day, there was a decrease in the number of osteoclasts due to the commencement of the initial stage of the recovery phase. it was found that the number of osteoclasts on day 14 was higher than the number of osteoclasts on day 28 in both the control groups and the treatment groups. the results also showed that there was a decrease in the number of osteoclasts in the treatment group when compared with the number of osteoclasts in the control group on the 14th and 28th days. this indicates that the administration of crab shell-based ha can reduce the number of osteoclasts in sockets after extraction. the number of osteoblasts in p14 and p28 was higher than the number of osteoblasts in k14 and k28. no significant differences were found between p14 and p28. this is because, on the 28th day, an insignificant number of osteoblasts were formed due to the continuation of osteoblast cells in the maturation phase forming osteocytes for apoptosis.16 figure 1 also shows that there were significant differences between k14 and p14 and k28 and p28. this is because ha gel can trigger osteocytes to differentiate, so there is an increase in the number of osteocytes. however, the differences were only significant between the k14 and p14; the differences between k28 and p28 were not significant. this was due to osteocyte apoptosis occurring after a period of 10 to 14 days. osteocyte apoptosis plays a key role in activating the bone remodelling mechanism.17 although the p28 showed the highest number of osteocytes, maximum cell growth actually occurred before the 28th day. thus, the number of osteocytes did not increase much between days 14 and 28. this is because crab shell-based ha has osteoconductive and osteoinductive properties, facilitating the growth of new bone tissue in the gap between mineral particles in ha. adding crab shell-based ha particles can significantly reduce the number of osteoclasts. the formation of an apatite layer on the surface of a biomaterial has the ability to bind living bones. the potential for the osteoinductive properties of ha has been confirmed in previous studies. furthermore, the administration of ha is found to deposit a higher number of collagen fibres around the ha particles.18 ha can bind to bone tissue and provide a specific biological response that can stimulate osteoblast cells to form table 1. this data represents the p value between each group of all-day treatment. all groups showed significant differences (p<0.05) k14 k28 p14 p28 k14 0.044 0.000 0.000 k28 0.024 0.000 p14 0.024 p28 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p119–123 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p119-123 123kamadjaja et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 119–123 new bone tissue and help the bone regeneration process.18 combined with osteoconduction, it can increase osteoblast attachment. the activation of osteoblasts and osteocytes can produce opg.19 opg is one of the main factors in regulating osteoclast differentiation. opg is found to inhibit the spontaneous induction of bone absorption.20 opg is a feed receptor for rankl and competes with rank to bind rankl. as a result, opg can be an effective inhibitor for osteoclast cell maturation and osteoclast cell activation. when the bone resorption phase by osteoclasts is complete, the resorbed bone cavity contains various mononuclear cells, including monocytes, osteocytes released from the bone matrix and preosteoblasts, which function to initiate new bone formation. in conclusion, the administration of ha-based shell crab to wistar rats after tooth extraction can reduce the number of osteoclasts and increase the number of osteoblasts and osteocytes. references 1. pedersen gw. buku ajar praktis bedah mulut. 4th ed. jakarta: egc; 2013. p. 36. 2. alani afi. multiple techniques have been proposed to preserve alveolar bone after tooth loss. ann med health sci res. 2018; 8(1): 65–8. 3. hansson s, halldin a. alveolar ridge resorption after tooth extraction: a consequence of a fundamental principle of bone physiology. j dent biomech. 2012; 3: 1–8. 4. van heerden p. treatment concepts for socket grafting. int dent – african ed. 2012; 2(1): 70–4. 5. kim y-k, yun p-y, um i-w, lee h-j, yi y-j, bae j-h, lee j. alveolar ridge preservation of an extraction socket using autogenous tooth bone graft material for implant site development: prospective case series. j adv prosthodont. 2014; 6(6): 521–7. 6. ari mda, yuliati a, rahayu rp, saraswati d. the differences scaffold composition in pore size and hydrophobicity properties as bone regeneration biomaterial. j int dent med res. 2018; 11(1): 318–22. 7. komur b, altun e, aydogdu mo, bilgiç d, gokce h, ekren n, salman s, inan at, oktar fn, gunduz o. hydroxyapatite synthesis from fish bones: atlantic salmon (salmon salar). acta phys pol a. 2017; 131(3): 400–2. 8. raya i, mayasari e, yahya a, syahrul m, latunra ai. shynthesis and characterizations of calcium hydroxyapatite derived from crabs shells (portunus pelagicus) and its potency in safeguard against to dental demineralizations. int j biomater. 2015; 2015: 469176. 9. ardhiyanto hb. stimulasi osteoblas oleh hidroksiapatit sebagai material bone graft pada proses penyembuhan tulang. stomatognatic (j k g unej). 2012; 9(3): 162–4. 10. smith sy, varela a, samadfam r. bone toxicology. new york: spinger; 2017. p. 27–93. 11. jana s, shah r, thomas r, kumar abt, mehta ds. techniques for preservation of post-extraction alveolar bone loss: a literature review. j adv med med res. 2021; 33(10): 33–42. 12. udeabor s, halwani m, alqahtani s, alshaiki s, alqahtani a, alqahtani s. effects of altitude and relative hypoxia on postextraction socket wound healing: a clinical pilot study. int j trop dis heal. 2017; 25(3): 1–7. 13. pagni g, pellegrini g, giannobile w v., rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 1–13. 14. fogelman i, gnanasegaran g, van der wall h. radionuclide and hybrid bone imaging. heidelberg: springer; 2012. p. 44–6. 15. anchana devi c, perumal p. synthesis & application of hydroxyapatite bioceramics from different marine sources. j res environ earth sci. 2016; 2(11): 7–15. 16. bellido t. osteocyte-driven bone remodeling. calcif tissue int. 2014; 94(1): 25–34. 17. ca rdoso l , her ma n bc, verborgt o, laud ier d, majeska rj, schaff ler mb. osteocyte apoptosis controls activation of intracortical resorption in response to bone fatigue. j bone miner res. 2009; 24(4): 597–605. 18. sotto-maior bs, senna pm, aarestrup bj v, ribeiro ra, assis nm de sp, cury aadb. effect of bovine hydroxyapatite on early stages of bone formation. rev odonto ciência. 2011; 26(3): 198– 292. 19. supangat d, cahyaningrum se. synthesis and characterization of crab shell hydroxyapatite (scylla serrata) by wet application method. unesa j chem. 2017; 6(3): 143–9. 20. bonucci e, ballanti p. osteoporosis-bone remodeling and animal models. toxicol pathol. 2014; 42(6): 957–69. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p119–123 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p119-123 188 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 188–192 antioxidant activity test on ambonese banana stem sap (musa parasidiaca var. sapientum) hendrik setia budi, indah listiana kriswandini, and aditya dana iswara department of oral biology faculty of dentistry, universitas airlangga surabaya-indonesia abstract background: polymorphonuclear cells (pmn) release oxygen free radicals or reactive oxygen species (ros) during inflammation. as a result, ros level is higher than antioxidant level in our body during oxidative stress leading to prolong inflammation or continuous tissue damage. indonesia, on the other hand, is a country with various herbal medicines. for instance, ambonese banana (musa parasidiaca var. sapientum) is often used as herbal medicine. ambonese banana, moreover, has flavonoid, polyphenol, tannin, and saponin as antioxidants to reduce free radicals by transferring their hydrogen atom. medicine used to reduce the impact of free radicals is known as antioxidant. antioxidant is proved to accelerate wound healing. purpose: this research aims to analyze the effects of the antioxidant activity of ambonese banana stem sap extract. method: antioxidant activities in this research were examined with 1,1-diphenyl-2-picryl-hidrazyl (dpph) method by reacting with stable radical compounds. spectrophotometry with a wavelength of 517 nm was used to measure absorption results shown in purple. the absorption results then were calculated by ic50 reduction activity. result: there were significant differences of ambonese banana stem sap antioxidant activity (p<0.05) at the concentrations of 15%, 30%, and 60 %. all concentrations have greater absorbance scores than ic50 (>50%). conclusion: ambonese banana stem sap extract has antioxidant activities. keywords: herbal medicine, antioxidant, wound healing, inflammation correspondence: hendrik setia budi, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya, indonesia. e-mail: hendrik-s-b@fkg.unair.ac.id introduction wound is tissue damage, caused by physical factors and accompanied by a disturbance of normal tissue continuity structure. based on the damage of the tissue, wound can be divided into two, namely open wound and closed wound. however, healing process of those wounds is basically similar, but the speed of the process depend on infection suffered, surgical intervention obtained, and medicines used. in tooth extraction process, the healing process involves several stages, namely hemostasis stage (the formation of blood clots), inflammation process (leukocyte infiltration), proliferation stage (the formation of connective tissue), granulation and epithelialization stages, and remodeling stage. thus, local therapy can be conducted to reduce the systemic effects and stop excessive bleeding, so the healing process will not be disturbed.1,2 inflammation, moreover, is a complex reaction to the causative agents of injury, such as microbial and cell damage. the inflammatory response is closely related to the healing process since it can destroy the causative agents of inflammatory lesion and cause a chain of events aimed to heal or repair damaged tissue.3 in tooth extraction, reactive oxygen species (ros) is increased by phagocytic cells, namely monocytes, macrophages, and neutrophils (pmn). reactive oxygen compounds will involve oxidants in various pathological processes in the body.4,5 in the medical field, oxidants and free radicals are often confounded since both have similar properties. activities of both compounds often cause the same effects. medicines dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p188-192 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p188-192 189189budi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 188–192 that can reduce the activity of free radicals are known as antioxidants. free radicals or ros at their “physiological concentrations” can serve as a regulator of cell growth, differentiation, adhesion between cells, cell senescence, and apoptosis. nevertheless, if ros with a high concentration or more than antioxidants in the body is obtained, ros will be destructive. consequently, ros can oxidize fat and protein, as well as damage dna by mediating dna fragmentation. in addition, prolonged exposure of ros is suspected as the cause of chronic inflammation and tissue damage. increased ros on inflamed tissue even can cause endothelial dysfunction and tissue damage. increasingly with age, the levels of antiox idants in the body will be reduced, so the healing response becomes slower. ros can be neutralized by antioxidants, such as catalase, superoxide dismutase, glutathione and non-enzymes (vitamins c, e, a, and pyruvate).6 antioxidant activity, furthermore, can be examined with dpph (1,1-diphenyl-2-picrylhydrazyl) method using a spectrophotometer with a wavelength λ = 517 nm. a material is considered to have antioxidant activity when the percentage of the antioxidant activity was greater than or equal to 50%7,8 fresh banana stem extract, moreover, can shorten bleeding and clotting time due to the activation of clotting factors and response of endothelial glycoprotein-ib (gpib). glycoprotein plays a role in platelet adhesion to the endothelium so that activated platelets will release the contents of granules during healing process.9,10 banana stem sap contains polyphenols, flavonoids, saponins, anthraquinone, and tannin, which can capture free radicals to inhibit cell damage. finally, the results of the previous researches showed that the effective concentration of banana stem sap in healing wounds is 15%, 30%, and 60%. moreover, the results of biocompatibility, anti-inflammatory, and analgesic tests showed that banana stem sap at concentration up to 100% is relatively not toxic to fibroblasts, and has properties as anti-inflammatory and analgesics. 11, 12 thus, this research aims to analyze the effects of the antioxidant activity of ambonese banana stem sap extract, so it can be developed into medical herbs that have medicinal properties. materials and method this research is experimental laboratory research with post-test only control group design. the treatment group was banana stem sap extract with the concentrations of 15%, 30%, and 60%. making banana stem sap extract was conducted through several stages. the central part of banana stems was cut into small pieces, weighing 200 grams. those pieces were put into a blender and added with 200 cc of sterile distilled water. those pieces were blended for five minutes until smooth. it was filtrated using whatman filter paper no.1. since the result obtained was at a concentration of 100%, it then was diluted to obtain concentrations of 15%, 30%, and 60%. to make it at the concentration of 15%, 15 ml of 100% banana stem extract was dissolved in 100 ml of water. meanwhile, to make it at the concentration of 30%, 30 ml of 100% banana stem extract was dissolved in 100 ml of water. and, for the concentration of 60%, 60 ml of 100% banana stem extract was dissolved in 100 ml of water. preparations for positive control group were prepared using 200 mg of powdered vitamin c (l-ascorbic acid) 200 mg dissolved in 200 ml of distilled water. dpph reagent then was prepared by mixing 4 mg of powdered dpph with ethanol as much as 100 ml. for the blank solution, dpph reagent was mixed with distilled water at a ratio of 2: 1. finally, each group (concentrations of 15%, 30%, and 60%, positive control, and blank) was replicated seven times. thus, the total of samples was 35 samples. to examine antioxidant activity, dpph method was used. for the treatment groups (concentrations of 15%, 30%, and 60%), 2 ml of each group was taken and mixed with 1 ml of dpph reagent in each test tube. meanwhile, for the positive control group, 2 ml of vitamin c solution was mixed with 1 ml of dpph reagent in each test tube. and, for a blank solution, 2 ml of distilled water was mixed with 1 ml of dpph reagent. after all samples were homogeneous, those samples were put into cuvette, and then measured, using a spectrophotometer with a wavelength λ = 517 nm. absorbance score derived from the results of the spectrophotometer measurement was calculated, using the following antioxidant activity formula: 7 abs blanko abs sampel × 100% abs blangko the absorbance score of each sample was measured using a uv-vis spectrophotometry. absorbance scores obtained were then calculated using the percentage formula for antioxidant activity. a material can be indicated to be active as an antioxidants if the percentage of its antioxidant activity is more or equal to 50% or so-called inhibitor concentration 50 (ic50). ic50 is used to determine which concentration can reduce 50% of free radicals. ic50 is the standard for determining antioxidant activity.8 finally, one way anova was was conducted, followed by tukey’s post hoc test to determine the difference of antioxidant activity in those groups. results examination of banana stem sap extract was conducted using spectrophotometry. the results showed chemical compounds contained in the banana stem sap have antioxidant activities and play a role in the healing process (table 1). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p188-192 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p188-192 190 budi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 188–192 the results of one way anova test showed p<0.05, which means that ho was accepted. thus, it indicates that there were no statistically significant differences among the research groups. therefore, post-hoc tukey test was performed to determine significant difference in each group (table 4). the results of post hoc tukey test were used to determine which group pair has significant difference with a significance of p<0.05. in table 4 shows that among all groups, a significant difference was marked with an asterisk. discussion wound caused by medical intervention will lead to an inflammatory response as wound healing process. the release of inflammatory mediators, such as bradykinin, histamine, and free radicals from leukocytes, can increase vascular permeability. a high number of free radicals then can cause damage to cell damage, reduce the cell’s ability to adapt to the environment that would cause cell death, and inhibit the wound healing process. ascorbic acid or vitamin c is a sixlactone carbon atom synthesized from glucose contained in the liver. the chemical name of ascorbic acid is 2-oxo-l-threo-hexono1.4-lactone-2.3-enediol. the main form of ascorbic acid is called l-ascorbic acid and dehydroascorbic. vitamin c atom donates h + or h oxidized by ros that produces neutral tricarbonyl ascorbate free radicals. the hydrogen atom donor then can reduce free radicals • oh and roo •. in addition, vitamin c is a compound that has a very active antioxidant activity compared with other oligoresveratrol compounds. oligoresveratrol compound is derived polyphenolic compounds that have antioxidant activity.12 in this research, vitamin c has antioxidant activity amounted to 55.20%, so according to the standard ic50, vitamin c has an ability as an antioxidant. based on the results, the average of antioxidant activity in banana stem sap extract at the concentration of 15% was 64.04%, 69.63% at the concentration of 30%, and 73.17% at the concentration of 60%. it means that those scores are in accordance with the appropriate standard of ic50. therefore, it can be concluded that the concentrations of the banana stem sap extract have antioxidant activity. antioxidant activity, moreover, can be caused by flavonoids contained in green banana stem sap. these compounds act as free radical catchers because of containing hydroxyl group. thus, as reducing agents, flavonoids can act as hydrogen donors against free radicals. donor hydrogen released by flavonoids then will bind with • oh to produce neutral h2o. atomic hydrogen can also neutralize roo• which is the result of the reaction r• with o2.13 next, this group will neutralize free radical characters from dpph. if all the electrons in the free radical of dpph become in pairs, table 1. the test results of chemical compound compounds results polyphenols + tanin + saponins + flavonoids + table 2. the average of absorbance score with uv-vis spectrophotometry treatment ∑ ± sd control + 0.441 ± 0.03 concentration of 15 % 0.355 ± 0.35 concentration of 30% 0.299 ± 0.01 concentration of 60 % 0.265 ± 0.01 blank 0.987 ± 0 table 3. the average of the percentage of reduction activity of ic50 as antioxidant treatment ∑ ± sd control + 55.20 % ± 3.06 concentration of 15 % 64.04 % ± 4.16 concentration of 30% 69.63 % ± 0.58 concentration of 60 % 73.17% ± 0.43 table 4. the results of post hoc tukey test c(+) k15% k30% k60% c(+) 0.000* 0.000* 0.000* k15% 0.000* 0.000* k30% 0.000* k60% note: (c (+): positive control; k15%: at the concentration of 15%; k30%: at the concentration of 30%; and k60%: at the concentration of 60%). based on the observation and calculation results of the reduction activities conducted on 35 samples, the absorbance scores obtained in all research groups are as follow (table 2). the calculation of the absorbance scores in each study group was conducted. the percentage of the reduction activity was measured to analyze the ability of antioxidant activity (table 3). the highest percentage of the reduction activity was obtained at the concentration of 60% for 73.17%, while the percentage of the reduction activity was found at the concentration of 15% for 64.04%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p188-192 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p188-192 191191budi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 188–192 then the color of the solution will change from dark purple to yellow light, and the absorbance at 517nm wavelength will be lost. flavonoids can actually increase the production of sod, gpx, and cat. these enzymes play a role in reducing free radicals in the body. flavonoids can also bind cu 2+, which also plays a role in the formation of free radicals, • oh.14 phenol compound can chemically be defined as the presence of at least one aromatic ring carrying one (phenol) or more (polyphenols) hydroxyl groups. polyphenols are a group of chemical substances found in plants. this substance has a distinctive sign that has a lot of phenol group in the molecule. derivative polyphenols as antioxidants can stabilize free radicals to complete the lack of electron free radicals and inhibit the chain reaction of free radical formation. the mechanism of polyphenol compounds as antioxidants is to donate the hydrogen of the hydroxyl groups. polyphenols are components that contribute to the activity of antioxidants in fruits and sayuran.15 fenton reaction occurs when h2o2 binds to fe2+, which will generate • oh. polyphenols have the ability to bind fe2+ so that free radicals • oh will be reduced. donor hydrogen atoms of polyphenols can also neutralize • oh into h2o, and can neutralize roo • which is the result of the reaction r • with o2. 16 tannins are secondary metabolites of active compounds, which are known to have some of the properties, such as astringent, anti-diarrhea, anti-bacterial and antioxidant. tannins are components of organic substances that are very complex, consisting of phenolic compounds which are difficult to separate and crystallize, but precipitate proteins out of the solution and fuse with the proteins.17 tannins are divided into two groups: the hydrolyzed tannins and condensed tannins. tannins have complex biological roles ranging from precipitating proteins to chelating metals. tannins can also serve as a biological antioxidant with the ability to bind metal. metal compounds, such as fe2+ and cu2+ can react with h2o2 through fenton reaction that produces reactive • oh, as a result, by binding with the metals, • oh levels in the body can be in reduced.18,19 in general, compounds classified as a class of polyphenols and carotenoids have antioxidant properties. some researches show that saponin also acts as an antioxidant. saponins have antioxidant properties that reduce superoxide to form intermediate hydroperoxide cells and prevent biomolecular damage from free radicals. 20 saponins increase the production of sod that plays a role in reducing ros and h2o2, and has an ability to bind so that inhibits the fenton reaction from generating • oh. saponins also bind o2-•. as a result, ros binding with nitric oxide (no) can generate more reactive onoo-.21, 22 from the results shown in table 5.2, a solution of vitamin c as the positive control (+) has antioxidant activity smaller than the extract of banana stem sap used due to the crude extract. the crude extract actually contains some compounds that can be collected, such as flavonoids, saponins and polyphenols, and tannins that have antioxidant properties. meanwhile, vitamin c is a single compound. some of the compounds contained in the crude extract of banana stem sap even have a different damping mechanism of ros, thereby reducing a wide range of ros, such as ·oh, roo·, o2 −•, and onoo-. on the other hand, vitamin c just donates a hydrogen atom, and can only reduce some kinds of ros, such as ·oh, roo·.23, 24 in conclusion, antioxidant activities found in all of the banana stem sap concentrations indicate that the banana stem sap has the potential to be developed as a biomaterial medicine for wound healing. the optimum result obtained in this research was at the concentration of 60% with the antioxidant activity of 73.17%. references 1. petersen jk, krogsgaarda j, nielsena km, norgaarda eb. a comparison between 2 absorbable hemostatic agents: gelatin sponge (spongostan®) and oxidized regenerated cellulose (surgicel®). int j oral surgery 2008; 13(5): 406-10. 2. sarabahi s. recent advances in topical wound care. indian j plas surg 2012; 45(2): 379-87. 3. kumar v, abbas ak, fausto n. robbin and cohran in pathologic basic and disease. 7th ed. philadelphia, pennsylvania: elsevier; 2005. p. 47-119. 4. mittal m, siddiqui mr, tran k, reddy sp, malik ab. reactive oxidative species in inflamatory and tissue injury. antioxid redox signal 2014; 20(7): 1126-67. 5. mazen m. jamil al-obaidi, fouad hussain al-bayaty, rami al batran, jamal hussaini, goot heah kho. impact of ellagic acid in bone formation after tooth extraction: an experimental study on diabetic rats. the scientific world journal 2014; 2014: article id 908098. 6. nicholas b, helen a, neil s, yves b, stephen w, john h. reactive oxygen species (ros)-a family of fate deciding molecules pivotal in constructive inflammation and wound healing. european cells and materials 2012; 24: 249-65. 7. e r y a r . uji a k t iv it a s a nt iok sid a n ek st r a k met a nol bu a h lakum (cayrania trifolia) dengan metode dpph (2,2-difenil-1pikrilhidrazil). pontianak: universitas tanjungpura; 2013. 8. parwata imoa, wiwik sr, raditya y. isolasi dan uji antiradikal bebas minyak atsiri pada daun sirih (piper betle, linn) secara spektroskopi ultra violet-tampak. jurnal kimia 2009; 3(1): 7-13. 9. watson sp. platelet activation by extracellular matrix proteins in haemostasis and thrombosis. curr pharm des 2009; 15(12): 135872. 10. weremfo a, adinortey mb, pappoe anm. haemostatic effect of the stem juice of musa paradisiaca l. (musaceae). advance in biological research 2011; 5(4): 190-2. 11. budi hs, yuliastutik ws, arundina i, ariani w. uji sitotoksisitas getah pisang ambon (musa paradisiaca var. sapientum l.) terhadap sel fibroblas. oral biology dental journal 2011; 3(1): 17-25 . 12. budi hs, yuliastutik ws, yuliati, mulyaningtyas r. efek analgesik getah pisang ambon (musa paradisiaca var. sapientum l.) pada mencit (mus musculus). oral biology dental journal 2010; 2(2): 9-19. 13. purwaningsih s. aktivitas antioksidan dan komposisi kimia keong matah merah (cerithidea obtusa). ilmu kelautan 2012; 17(1): 3948. 14. li x, lin j, gao y, han w, chen d. antioxidant activity and mechanism of rhizoma cimicifugae. chem cent j 2012; 6(1): 140. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p188-192 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p188-192 192 budi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 188–192 15. waji r a, sugrani a. f lavonoid (quersetin)[ma kala h k im ia organik bahan alam. makassar: program pascasarjana,universitas hasanuddin matthews je, dr. denture cleansers – potential allergic – recommendations. media release ada. 2008; 1-2 16. larrain re. dietary high-tannin sorghum and oxidative stability of muscle and muscle foods. university of winconsin; 2007. p. 10-2. 17. hättenschwiler s, vitousek pm. the role of polyphenols in terrestrial ecosystem nutrient cycling. trends ecol evol 2000; 15(6): 238-43. 18. ilhami g, zubeyr h, mahfuz e. radical scavenging and antioxidant activity of tannic acid. arabian journal of chemistry 2010; 3(1): 43-53. 19. desmiaty y, ratih h, dewi ma, agustin r. penentuan jumlah tanin total pada daun jati belanda (guazuma ulmifolia lamk) dan daun sambang darah (excoecaria bicolor hassk.) secara kolorimetri dengan pereaksi biru prusia. ortocarpus 2008; 8: 106-9. 20. hagerman ae. tannin handbook. department of chemistry and biochemistry, miami university; 2011. 21. khan aa, naqvi ts, naqvi ms. identification of phytosaponins as novel biodynamic agents: an updated overview. asian j exp biol sci 2012; 3(3): 459-67. 22. widyastuti n. pengukuran aktivitas antioksidan dengan metode cuprac, dpph, dan frap serta korelasinya dengan fenol dan flavonoid pada enam tanaman. bogor: institut pertanian bogor; 2010. p. 6. 23. pande ps, mane vd, mishra mn. evaluation of antioxidant activity of saponin and tannin fractions isolated from thr leaves of tridax procumbens. int j pharm bio sci 2014; 5(1): 396-400. 24. elekofehinti oo, adanlawo ig, komolafe k, ejelonu oc. saponins from solanum anguivi fruits exhibit antioxidant potential in wistar rats. annals of biological research 2012; 3 (7): 3212-7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p188-192 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p188-192 9393 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 93–98 antibacterial effects of pluchea indica less leaf extract on e. faecalis and fusobacterium nucleatum (in vitro) agni febrina pargaputri,1 elly munadziroh,2 and retno indrawati3 1department of oral biology, faculty of dentistry, universitas hang tuah 2department of dental material, faculty of dental medicine, universitas airlangga 3department of oral biology, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: enterococcus. faecalis (e. faecalis) and fusobacterium nucleatum (f. nucleatum) are the most common bacteria found in infected tooth root canal. most of these bacteria often cause failure in endodontic treatments. pluchea indica less leaf is a species of plants that has several chemical properties. it consists of flavonoids, tannins, polyphenols, and essensial oils which have been reported as antibacterial agents. because of its benefits, the extract of pluchea indica less leaves may be potentially developed as one of root canal sterilization dressing. purpose: this study aimed to determine antibacterial activity of pluchea indica less leaves extract against e. faecalis and f. nucleatum bacteria. method: dilution method was conducted first to show minimum inhibitory concentration (mic) of the extract against e. faecalis and f. nucleatum. the antibacterial activity test on pluchea indica less leaves extract was performed on e. faecalis and f. nucleatum bacteria using agar diffusion method. the pluchea indica less leaves extract used for antibacterial activity test was at a concentrations of 100%, 50%, 25%, 12.5%, and 6.25%. thirty-five petridiscs were used and divided into five groups based on the extract concentration. result: the results showed strong and moderate antibacterial effects of the pluchea indica less leaves extract on e. faecalis at the concentrations of 100% and 50%, while on f. nucleatum only at the concentration of 100% with moderate effect. conclusion: pluchea indica less leaves extract has antibacterial activity against e. faecalis and f. nucleatum bacteria with strong-moderate effect. keywords: pluchea indica less leaves extract; enterococcus faecalis; fusobacterium nucleatum; antibacterial correspondence: elly munadziroh, departement of dental material, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: emunadziroh@yahoo.com introduction microorganisms play an important role in causing inflammation of pulp and periapical tissues. several researches have reported the existence of microorganisms in infected pulp and root canals, 90% of which are facultative anaerobic and gram-positive bacteria, followed by gramnegative bacteria and some fungal groups.1,2 teeth with necrotic pulp tissues usually show a large number of combinations bacteria. some species of bacteria, such as peptostreptococcus provetii, actinomyces odontolycus, porphyromonas endodontalis, and streptococcus salivarius, also are commonly found in infected root canals. in failed treatment of root canals, enterococcus faecalis (e. faecalis) bacteria are mostly found with a prevalences of 67% to 77% in persistent endodontic infection cases.3 in addition to e. faecalis bacteria, fusobacterium nucleatum (f. nucleatum) bacteria also are mostly found in root canals, as many as 60% to 70% of periodontal lesion cases.1 good root canal treatment can be achieved by removing all sources of infection through preparation, sterilization, and filling of the root canal. however, there is still a failure in root canal treatment. one of the main causes of root canal treatment failure is the presence of microorganisms that can survive both in root canal and in apical region of the teeth. it may be because the microorganisms still have an ability of resistance to medicaments used during root canal treatment.2-4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p93-98 94 pargaputri, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 93–98 giving intra-canal sterilization medicine as one of antimicrobial agents, therefore, becomes an important stage in root canal treatment, because it can kill microorganisms in root canal teeth.5,6 root canal sterilization drugs that have been used in dentistry since the past, nevertheless, are known to cause irritation in the periapical region and to be cytotoxic, because these contain active ingredients and toxic chemicals. these root canal sterilization drugs are largely classified into phenols, including formokresol, camphorated parachlorophenol, thymol, metakresilasetat, and halides (iodine-potassium iodide).7 in addition to phenols, compounds that also used as root canal medicament are calcium hydroxide, n2, halogen, such as sodium hypochlorite and iodide, and quaternary ammonium (quats). 8 however, a research conducted by grossman et al 8 shows that the use of formokresol can generate a high degree of irritation and can cause necrosis for 2-3 months. hydrogen peroxide and sodium hypochlorite also are known to generate less irritation than most intra-canal medicaments. meanwhile, cresatin is known to generate little inflammation. pluchea indica less (l.) is a plant that has long been known by the people of indonesia for its benefits. these plants are often used as hedge plants, has special smell and bitter taste. part of these plants that always used are its leaves and roots, which is traditionally function as efficacious fever, appetite enhancer, and sweat bullets. pluchea indica l. leaves contain chemical properties such as tannins, flavonoids, polifenolat, and essential oils that are known to have antibacterial effect.9,10 antibacterial effects of pluchea indica l. leaves has been reported by purnomo,11 which is said that pluchea indica l. leaves has antibacterial effect against staphylococcus sp, propinobacterium sp, dan corynobacterium. antibacterial test results on pluchea indica l. leaves extract against methicillin resistant staphylococcus aureus indicate a minimum inhibitory concentration (mic) of 20%,12 while antibacterial test results against streptococcus mutans showed a mic at concentration of 25%.13 alternative materials from plant extracts currently have been considered as antimicrobial agents since these alternative materials have natural effects, so that the side effects are expected to be lower than from chemical drugs. one of those herbs that has antibacterial properties is pluchea indica l. leaf.9 for those reasons, in this research we aimed to determine the antibacterial activity of pluchea indica l. leaves extract against e. faecalis and f. nucleatum bacteria, which are commonly found as the cause of infection in the pulp and periapical tissues as well as failure in root canal treatment. consequently, the results of this research are expected to be developed in dentistry as one of alternative to root canal sterilization drug. materials and method this research was a true experiment research with post test only-control-group design. this research used pluchea indica l. leaves at a concentration of 100%, 50%, 25%, 12.5%, and 6.25%. the number of samples in each group was seven. the tools used in this research were blender, funnel cups, stirrers, measuring cups 500 ml, erlenmeyer tube 500 ml, beaker glass 600 ml, rotary evaporator, filter paper, analytical balance (analytical balance cpa 423s sartorius), oese, spiritus burner, test tubes (bd falcon), micropipette (eppendorf), petridiscs, paper disc 5mm, and incubator (500 memmer). the materials used in this research include pluchea indica l. leaves fresh obtained from upt materia medika batu, ethanol 80%, sterile distilled water, brain heart infusion (bhi) medium, stock of e. faecalis bacteria, stock of f. nucleatum bacteria, mc farland 0.5, mueller hinton agar (mha) media, and pluchea indica l. leaves extract with concentration of 100%, 50%, 25%, 12.5%, 6.25%. pluchea indica less leaves were washed under running water. second, they were dried, aerated, and protected from the sun for 14 days until the leaves were dried and easily crushed. third, they were crushed in a blender and sieved to obtain the leaves powder. fourth, 500 grams of the powder was weighed using an analytical balance, and then macerated with 80% ethanol as much as 2 liters shielded from sunlight. the maceration was performed for 3 x 24 hours. but, every 1 x 24 hours, each extract was filtered and macerated back with 800 ml of new ethanol. finally, the results of the filtrate were then combined and evaporated with a rotary evaporator at temperature 60°c for two hours.14 pluchea indica l. leaves extracts were made in five concentration levels, namely 100%, 50%, 25%, 12.5%, and 6.25% w/ v (g/ ml) by weighing each extract as much as 1 g, 0.5gr, 0.25gr, 0.125gr, and 0.0625gr and then diluted with sterile distilled water as much as 1 ml. phytochemical screening was conducted on pluchea indica l. leaves extract. the screening procedure was performed flavonoid test, tannin test, poliphenols test, and phenols test. flavonoid test was done by mixing 1 mg sample of extracts with 0.5 mg magnesium powder and 5 drops hydrochloric acid (hcl) 2 n. the mixture then was heated over a water bath at temperature 60° c for five minutes in test tube, and then filtered. next, the filtrate in a test tube was added with three drops of amyl alcohol and then shaken vigorously. the presence of flavonoids was characterized by the formation of yellow to red colour that could be drawn by amyl alcohol. tannin test was done by mixing 1 mg sample of the extract with five drops solution of 1% gelatin in test tube. the presence of tannin then was marked with a white precipitate. poliphenols test was done by mixing 1 mg sample of extract with three drops solution of reagent fecl3 1% in test tube. the presence of poliphenols compound was marked with blue-black color. phenols test was done by mixing 1mg sample of extract with 3 drops solution of fecl3 1% and three (tetes) of k4fe(cn)6. the presence of phenol compound was marked with purple, blue, or green color.12 bacteria used in this research were derived from special infection hospital (rski) airlangga university dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p93-98 9595pargaputri, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 93–98 surabaya. the use of bacteria in this research was to create a suspension of e. faecalis and f. nucleatum bacterial colonies with bhib media in test tubes, then incubated at 37°c for 48 hours anaerobically. turbidity of the suspension of e. faecalis and f. nucleatum bacteria was equated with mc farland 0.5, wich is equivalent to the number of bacteria of 1.5 x 108 cfu/ ml. after obtaining the same turbidity, the suspension was diluted to reach a bacterial infectious dose of 1 x 106 cfu/ ml (for e. faecalis) and 1 x 108 cfu/ ml (for f. nucleatum).15,16 the antibacterial activity test on pluchea indica l. leaves extract against the growth of e. faecalis and f. nucleatum bacteria was performed using agar diffusion method and paper discs with a diameter of 5 mm.16 the paper discs were dipped in pluchea indica l. leaves extract with a concentration of 100%, 50%, 25%, 12, 5%, and 6.25% as much as 50 μl. for the control group, sterile distilled water as much as 50μl was used. each paper disc then was implanted in petridisc containing muller hinton agar (mha) solid media with e. faecalis bacterial colonies as much as 1 x 106 cfu/ ml and f. nucleatum bacterial colonies as much as 1 x 108 cfu/ ml. after that, each petridisc was incubated in incubator at 37° c for 24 hours anaerobically. inhibition zones formed around the paper discs then were measured using a caliper with a precision of 0.05mm. this procedure was repeated seven times for each group of e. faecalis and f. nucleatum bacteria.15,16 the inhibition zones formed around the paper discs on a group of e. faecalis and f. nucleatum bacteria are classified based on the response of bacterial growth inhibition (table 1).17 analysis of the data used kolmogorov-smirnov test to determine whether the distribution of the data was normal. next, one way anova test was performed to determine the significance of differences between the groups. results the test results showed that pluchea indica l. leaves extract contained several metabolites, such as flavonoids, tannins, polifenolat, and phenol. the metabolites contained at most are tannins (table 2). the antibacterial activity test on the pluchea indica l. leaves extract against e. faecalis and f. nucleatum bacteria was performed using agar diffusion method. previously, dilution method or serial thinning method was conducted to determine the mic of the extract. based on the results, it is known that mic value of the pluchea indica l. leaves extract against e. faecalis bacteria was at a concentration of 12.5%, while against f. nucleatum bacteria was at a concentration of 50%. then we made concentration of the extract above and below the mic, that was 100%, 50%, 25%, 12,5%, dan 6,25%. the results of antibacterial activity test using agar diffusion method can be known based on the inhibition zones formed around the paper discs on e. faecalis and f. nucleatum bacteria. the mean diameter of inhibition zones formed around the paper disc then was classified in response to bacterial growth inhibition as shown in table 3. table 3 shows the mean diameter of inhibition zones formed around the paper discs in the group of e. faecalis bacteria at the extract concentrations of 100% and 50% with medium to strong inhibition responses. meanwhile, at the concentrations below 50%, there was no inhibition zone. on the other hand, in the group of f. nucleatum bacteria, the diameter of the inhibition zone was seen only at the concentration of 100% with medium inhibition response, table 1. classification of inhibition response to the bacterial growth diameters of inhibition zones inhibition response >20 mm very strong 11 – 19 mm strong 5 – 10 mm moderate < 5 mm weak table 2. metabolites in pluchea indica less leaves extract metabolites test results flavonoids ++ tannins +++ polifenolat ++ phenol ++ note: the sign (+) indicates the tested extracts containing metabolites table 3. the mean diameter of the inhibition zones of the pluchea indica less leaf extract on the growth of e. faecalis and fusobacterium nucleatum bacteria along with their resistance response bacterial isolates mean diameter of inhibition zone (mm) control at the concentration of 100% at the concentration of 50% at the concentration of 25% at the concentration of 12.5% at the concentration of 6.25% e. faecalis 0 16.2 mm (strong) 7.76 mm (medium) 0 0 0 fusobacterium nucleatum 0 5.73 mm (medium) 0 0 0 0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p93-98 96 pargaputri, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 93–98 while at concentration below 100%, there was no inhibition zone. the size of diameter of inhibition zones formed around the paper discs in the group of e. faecalis and f. nucleatum bacteria can be seen in figure 1. the highest diameter of inhibition zone in the group of e. faecalis and f. nucleatum bacteria was seen at a concentration of 100%. this was consistent with the statement of pelczar dan chan,18 which states that the higher concentration of the extract is, the greater antibacterial effect will be produced. the results of one way anova statistical test conducted on the group of e. faecalis bacteria, moreover, showed that there was a significant value (p) less than 0.05 at the concentration of 100% and 50% when compared with the extract concentrations of 25%, 12.5%, 6.25%, and without the extract (control). it means that there was a significant difference in the mean diameter of the inhibition zone at the extract concentrations of 100% and 50% when compared with the concentrations of 25%, 12.5%, 6.25%, and without the extract (control). similarly, there was also a significant value (p) less than 0.05 in the group of f. nucleatum bacteria at the extract concentration of 100% when compared with the extract concentrations of 50%, 25%, 12.5%, 6.25%, and without the extract (control). it indicates that there was a significant difference in the mean diameter of the inhibition zone at the concentration of 100% when compared with at the concentrations of 50%, 25%, 12.5%, 6.25%, and without the extract (control). discussion inhibition responses of the pluchea indica l. leaves extract on the growth of e. faecalis bacteria were moderate and strong responses. at the concentration of 50%, the mean diameter of inhibition zone generated the smallest inhibition response, while at the concentration of 100%, the mean diameter of inhibition zone generated the largest inhibition response, relatively strong (table 3). on the other hand, the inhibition response of the pluchea indica less leaf extract on the growth of f. nucleatum bacteria was only seemed at a concentration of 100% with moderate response (table 3). the pluchea indica l. leaves extract has ability to inhibit the growth of e. faecalis and f. nucleatum bacteria. this was because pluchea indica l. leaves extract contains some compounds that act as antibacterial, such as tannins, flavonoids, and essential oils. in the chemical structure of tannin contains gallo and pirogallo groups, which can react with the bacterial membrane protein. ester aromatic ring from the gallo and pirogallo groups will bind to protein transport cell envelope of the bacteria, which then will caused protein leakage so that causing damage to cell wall of bacteria and caused bacteria dead.19-21 in addition, nonspecific bonding also occurs through hydrogen bonding of the groups, which can caused damage to the cytoplasmic membrane of the bacteria, so the membrane functions as a selective permeability barrier, carrier active transport function, as well as control of the internal composition of the cell, will be disrupted. therefore, if the function of the cytoplasmic membrane integrity is damaged, macromolecules and ions will be out of the cell, and then the cell will be damaged and dead.22 activities of flavonoids, moreover, are related to their ability to form complexes with proteins from the cell wall, which will result in damage to the permeability of the bacterial cell wall. flavonoids have an antibacterial effect because of its ability to interact with dna of bacteria.23 each flavonoid compound has an ability to damage the hydrogen bridge bonding of the strands of the dna double chain, resulting in the disruption of the stability of the double chain structure of bacterial dna later influencing the whole process of bacterial growth and metabolism. flavonoids are also capable of producing energy transduction that will affect the bacterial cytoplasm and slowly motility of bacteria. it is known based on the presence of hydroxyl ions in flavonoids that can chemically alter organic compounds and nutrient transport that can cause toxic effects on the bacterial cells.23,25 essential oils in the pluchea indica l. leaves extract, furthermore, play a role in damaging cell membranes and bacterial protein denaturation. the main content of essential oils is sinamaldehida, benzyl alcohol, and eugenol compounds. benzyl alcohol has solvent properties of fat and protein denaturation, which can cause damage to the bacterial cell membrane. protein denaturation process involves changes in molecular protein stability and causes both changes in protein structure and protein coagulation. proteins that undergo denaturation will lose its physiological activity and ability to function properly. changes that occur in the protein in the cell wall will lead to increased cell permeability. damage and increased permeability of the cell then will damage the bacterial cells.24 based on this research, it required high concentration of the extract to be able to inhibit the growth of e. faecalis and f. nucleatum bacteria. f. nucleatum bacteria required higher extract concentration of 100% compared to e. faecalis bacteria (50%). the results of this research differ from previous research that used the same extract. research by fadhlia26 that used pluchea indica l. leaves extract 32 phenol ++ note: the sign (+) indicates the tested extracts containing metabolites table 3. the mean diameter of the inhibition zones of the pluchea indica less leaf extract on the growth of enterococcus faecalis and fusobacterium nucleatum bacteria along with their resistance response bacterial isolates mean diameter of inhibition zone (mm) control at the concentration of 100% at the concentration of 50% at the concentration of 25% at the concentration of 12.5% at the concentration of 6.25% enterococcus faecalis 0 16.2 mm (strong) 7.76 mm (medium) 0 0 0 fusobacterium nucleatum 0 5.73 mm (medium) 0 0 0 0 figure 1. diameter of inhibition zone that seen around the paper disc in agar diffusion method (a) enterococcus faecalis, (b) fusobacterium nucleatum. a b figure 1. diameter of inhibition zone that seen around the paper disc in agar diffusion method (a) e. faecalis, (b) fusobacterium nucleatum. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p93-98 9797pargaputri, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 93–98 against e. faecalis bacteria, showed mic at a concentration of 25%. the difference of mic that was obtained from this research with previous research was likely to be caused by difference in methods used. previous research by fadhlia26 used dilution method to show antibacterial activity of pluchea indica l. leaves extract. mic obtained through dilution method was different with mic obtained from agar diffusion method. this difference was likely to be caused by media used for bacterial seeding. in dilution methods or a serial number thinning methods, bhib media containing rich in nutrients are used, so the bacteria can grow rapidly and obtain maximum results.16 the difference of mic in this research with previous research also could be caused due to the amount of compounds contained in the extract, so that it could influence the ability of the extract in inhibit the growth of bacteria. in this research we used whole extract of pluchea indica l. leaves, so that we could not know certainty the mechanism of antibacterial activity of each of metabolite compounds with contain in the extract. however, it could be suspected that tannin, flavonoids, and essential oils work sinergistically in inhibiting the growth of e. faecalis and f. nucleatum bacteria. this research showed high concentration of pluchea indica l. leaves extract to inhibit the growth of e. faecalis and f. nucleatum bacteria. this can be caused by the type of bacteria used in the research. f. nucleatum bacteria is a gram-negative bacteria that have high phospholipids on its cell wall, making them more permeable than the gram-positive bacteria. gram-negative bacteria, on the other hand, have double membranes, and there is a unique periplasm space between them, that is not found in grampositive bacteria. in the periplasm space, there are enzymes that are capable of damaging foreign molecules that come from outside the bacterial cell.27 gram-negative bacteria also have a hydrophilic coating on the outer membrane rich in lipopolysaccharide molecules, which serve as a barrier against the entry of antimicrobial substances. meanwhile, e. faecalis is gram-positive bacteria, which have outer membrane structure and different cell wall of gram-negative bacteria. gram positive bacteria only have a single plasma membrane, and the majority of gram-positive bacteria are more sensitive to antimicrobial or antibacterial materials.27 because of the high concentration of pluchea indica l. leaves extract that was obtained from this reserach to inhibit the growth of e. faecalis and f. nucleatum bacteria, it needs to be examined further about the toxicity and biocompatibility of the extract. so, it can be developed in dentistry as one of alternative to root canal sterilization drug derived from plant extracts. besides that, with the high concentration of the extract obtained in this research, the minimum inhibitory concentration (mic) could not be determined, so more researches are needed with smaller concentration range to determine the exact minimum inhibitory concentration and minimum bactericidal concentration of pluchea indica l. leaves extract against e. faecalis and f. nucleatum bacteria. based on the results of this research, it can be concluded that pluchea indica l. leaves extract can inhibit the growth of e. faecalis and f. nucleatum bacteria with moderate to strong response of growth inhibition, but the mic of the extract against e. faecalis and f. nucleatum bacteria could not be determined. references 1. bolstad ai, jensen hb, bakken v. taxonomy, biology, and periodontal aspects of fusobacterium nucleatum. clin microbiol rev 1996; 9(1): 55-71. 2. e rca n e , da l l i m, yav u z i̇, öz ek i nci t. i nvest igat ion of microorganism in infected dental root canals. j biotechnol & biotechnol 2006; 20(2): 166-72. 3. pinheiro et, gomes bp, ferraz cc, teixeira fb, zaia aa, souza filho fj. evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. journal of oral microbiology and immunology 2003; 18(2): 100–3. 4. siqueira jf. aetiology of root canal treatment failure: why welltreated teeth can fail. international endodontic journal 2001; 34: 1-10. 5. estrela cra, decurcio da, hollanda acb, silva ja. antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals. international endodontic journal 2007; 40: 85-93. 6. kartika iy. obat-obat untuk menanggulangi infeksi saluran akar. majalah kedokteran gigi usakti 2000; 15: 153. 7. walton, torabinejad. prinsip dan praktik ilmu endodonsi. edisi kedua. sumawinata n. jakarta: penerbit buku kedokteran egc; 1994. p. 361-4. 8. grossman li, oliet s, del rio ce. ilmu endodontik dalam praktek. edisi kesebelas. abyono r, editor. jaka r ta: penerbit buku kedokteran egc; 1995. p. 248-49; 251; 255-57. 9. soesilowati p, devijanti r. perbedaan daya hambat terhadap streptococcus mutans dari beberapa pasta gigi yang mengandung herbal. majalah kedokteran gigi (denta j) 2005; 38(2). 10. andarwulan n, batari r, sandrasari da, bolling b, wijaya hanny. flavonoid content and antioxidant activity of vegetables from indonesia. journal of food chemistry 2010; 121: 1231-5. 11. purnomo m. isolasi flavonoid dari daun beluntas (pluchea indica less) yang mempunyai aktivitas antimikroba terhadap penyebab bau keringat secara bioautografi. thesis. surabaya: universitas airlangga; 2001. p. 47-9. 12. sulistiyaningsih. potensi daun beluntas (pluchea indica less.) sebagai inhibitor terhadap pseudomonas aeruginosa multi resistant dan methicillin resistant stapylococcus aureus. bandung: laporan penelitian mandiri fakultas farmasi universitas padjadjaran; 2009. p. 41. 13. nahak mm. ekstrak etanol daun beluntas (pluchea indica l.) dapat menghambat pertumbuhan bakteri streptococcus mutans. thesis. denpasar: program pascasarjana universitas udayana; 2012. p. 79-80. 14. depkes ri. ekstra farmakope indonesia. edisi iv. jakarta: lembaga farmasi nasional; 2003. p. 769-71. 15. bahador a, khaledi a, ghorbanzadeh r. evaluation of antibacterial properties of nano silver iranian mta against fusobacterium nucleatum. european journal of experimental biology. 2013; 3(6):88-94. 16. hudzicki j . kirby-bauer disk diffusion susceptibility test protocol. united states of america: university of kansas medical center; 2013. p. 24. 17. arbet a, khotimah s, yanti ah. aktivitas antibakteri ekstrak daun benalu jambu air (dendropthoe pentandra (l.) miq) terhadap pertumbuhan salmonella typhi. jurnal protobiont 2014; 3(2): 26872. 18. pelczar mj, chan es. dasar-dasar mikrobiologi. jilid 1. hadioetomo rs, editor. jakarta: ui press; 2005.p. 452-7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p93-98 98 pargaputri, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 93–98 19. machado tb, pinto av, pinto mcfr, leal icr, silvam mg, amaral acf, kuster rm, netto-dos santoz kr. in vitro activity of brazilian medicinal plants, naturally occuring naphtoquinones and their analogues againts methicilline resistant staphylococcus aureus. int j antimicrob agents 2003; 21(3): 279-84. 20. jawetz b. melnick and adelberg’s. mikrobiologi kedokteran. penerjemah: bagian mikrobiologi fakultas kedokteran universitas airlangga. jakarta: salemba medika; 2001. p. 327-35. 21. soleimanpour s, sedighinia fs, afshar as, zarif r, asili j, ghazvini k. synergistic antibacterial activity of capsella bursapastoris and glycyrrhiza glabra against oral pathogens. jundishapur journal microbiol 2012; 6(8): 7262. 22. mohamed ssh, hansi pd, thirumurugan k. antimicrobial activity and phytochemical analysis of selected indian folk medicinal plants. international journal of pharma sciences and research 2010; 1(10): 430-4. 23. sabir a. pemanfaatan flavonoid di bidang kedokteran gigi. majalah kedokteran gigi (dent j) 2003; edisi khusus temu ilmiah nasional iii: 81-7. 24. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). majalah kedokteran gigi (dent j) 2005; 38(3): 75-9. 25. braga lc, shupp jw, cummings c, jett m, takahashi ja, carmo ls, chartone-souza e, nasclmento ama. pomegranate extract inhibits staphylococcus aureus growth and subsequent enterotoxin production. journal of ethnopharmacology 2005; 96: 335-9. 26. fadhlia cc. aktivitas antibakteri ekstrak daun beluntas (pluchea indica l.) terhadap pertumbuhan e. faecalis secara in vitro. electronic thesis and dissertation banda aceh: university of syiah kuala; 2015. p. 47-9. 27. konate k, hilou a, mavoungou jf, lepengue an, souza a, barro n, datte jy, m’batchi b, nacoulma og. antimicrobial activity of polyphenol-rich fractions from sida alba l. (malvaceae) againts cotrimazol-resistant bacteria strains. annals of clinical microbiology and antimicrobials j 2012; 11(5): 1-6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p93-98 147147 closed reduction in the treatment of neglected mandibular fractures at the department of oral and maxillofacial surgery, universitas airlangga olivia jennifer gunardi, riska diana, david buntoro kamadjaja and ni putu mira sumarta department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: the mandible is one of the bones most affected by facial fractures commonly resulting from trauma to the face. the ultimate goal of treatment is to re-establish the pre-injury dental occlusion (bite), mandibular anatomy and jaw function of the patient. treatment approaches range from conservative non-invasive management by ’closed’ reduction and immobilization using intermaxillary fixation (imf) to the more invasive surgery-based ’open’ reduction incorporating an internal fixation approach. purpose: the purpose of this case series was to describe the close reduction method as a form of treatment in cases of neglected mandibular fracture. cases: four cases of single or multiple mandibular fracture were presented. case management: all of the cases were managed using a closed reduction method and imf. conclusion: a closed reduction method in this case series produced encouraging results and could be considered an alternative in the treatment of neglected mandibular fractures with displacement. keywords: closed reduction; mandibular fracture; neglected mandibular fracture correspondence: ni putu mira sumarta, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail address: niputu.mira@fkg.unair.ac.id dental journal (majalah kedokteran gigi) 2019 september; 52(3): 147–153 case report introduction the mandible, despite being the largest and strongest facial bone, is one of the most affected by fractures with an incidence rate of 36-70%. these occur most frequently to males in their thirties as a result of a facial trauma.1–3 assault constitutes the most common cause of mandibular (jaw) fractures at 48-65%, followed by motor vehicle accidents, falls and gunshot wounds.1,4 this high rate of fractures can be explained by the unique characteristics of the mandible such as its prominence, unprotected facial position, mobility and limited bone support when compared to other facial bones.3,5,6 the mandible is the only mobile facial bone and in cases of injury to the maxillofacial region it is more vulnerable than the mid-face to fractures.7 depending on the direction and force of the trauma, fractures of the mandible frequently occur at different sites.7 the most commonly fractured areas are the body (29%), followed by the condyle (26%), angle (25%), and symphysis (17%), while the ramus (4%) and coronoid process (1%) are rarely fractured. the most common causes of fractures to the condyle, symphysis and angle include car accidents, motorcycle accidents, and physical assault respectively.8 mandible fractures can be complete or incomplete, open or closed, single, double, or comminuted and the result of direct or indirect mechanisms.4 depending on the location of the fracture, the patient can present with pain exacerbated by jaw movement, trismus, dental malocclusion, swelling, bleeding, external and intraoral tenderness, dysphagia, and step deformity at the fracture site.2,8 damage to the inferior alveolar nerve may induce anesthesia in the lower lip.8 furthermore, mandibular fractures can cause a variety of impairments, including temporomandibular joint syndrome, poor mastication, malocclusion, and chronic pain.6 the ultimate goal of treatment is to re-establish the preinjury dental occlusion (bite), mandibular anatomy and jaw function of the patient.2,9 reduction techniques in mandibular fracture treatment may be classified as open dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 148 gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 or closed depending on the presence or absence of direct visual access to the fracture site. closed reduction allows manipulation of the fracture segments taking advantage of dental occlusion without direct visual access, whereas open reduction involves direct visual access to the fracture site through a surgical incision. closed reduction and maxillomandibular fixation may be performed using splints in the form of bonded orthodontic brackets, arch bars, direct wires or eyelet wires. open reduction and internal fixation involves the use of wires, plates and other hard-wares placed directly across the fractured site by means of surgical access.10 treatment of mandible fractures with a closed reduction method is referred to as non-surgical treatment, while the fracture treatment performed without surgical procedures by manual repositioning of the fragment, gradual repositioning of the teeth and immobilization of the jaw using intermaxillary fixation (imf), is commonly termed maxillomandibular fixation (mmf).11 in order to achieve optimum results, the management of neglected mandible fractures with large displacement is ideally performed using open reduction internal fixation (orif). however, if the patient refuses to be treated with the orif method due, for example, to socio-economic reasons, or limitations on general anesthesia facilities exist, then the close reduction method could represent an alternative treatment option. conservative treatment, when properly indicated, allows for appropriate patient recovery, while reducing both surgery-related morbidity and the cost of resources.3 this case series aims to provide information on successful treatment involving the use of the close reduction method in four cases of neglected mandibular fracture. cases case 1: a 13-year-old male attended the dental hospital of the faculty of dental medicine at universitas airlangga complaining chiefly of difficulty in chewing and closing his mouth as the result of a traffic accident 11 days earlier. the patient had been thrown off a moving motorcycle, his chin subsequently making hard contact with the asphalt. the patient was examined in hospital shortly after the accident without any treatment having been initiated. the individual in question had a history of fainting during the incident, although this was not accompanied by resulting nausea or vomiting. moreover, he had no history of diabetes or drug allergies. extraoral examination confirmed facial asymmetry and swelling of the mandibular region, right superior palpebral hematoma and the right maxillary region, mandibular retrusion, limited mouth opening, step-off deformities and tenderness on palpation in the right mandibular parasymphysis region (figure 1). intraoral examination confirmed ecchymosis in the anterior region of the mandible, malocclusion, anterior and posterior open bite, right posterior scissor bite, displacement in the 41 and 42 regions, 10 mm overlapping, tooth mobility in the 41 region and tenderness on palpation in the mandibular symphysis region (figure 2). a panoramic radiograph indicateded a vertical fracture line to the inferior border of the mandible between regions 41 and 42. symphysis appeared separate and there was overlap of teeth 41, 31 with teeth 42, 43 (figure 3). on the basis of the clinical and radiological examination, this case was diagnosed as a mandibular symphysis fracture with displacement. case 2: a 15-year-old male attended the dental hospital in the faculty of dental medicine of universitas airlangga complaining chiefly of difficulty in closing his mouth for the previous three days due to being hit on his left cheek in a school fight. no history of fainting, nausea or vomiting was reported. extraoral clinical examination showed facial asymmetry, minimal swelling in the left buccal region and crepitation and tenderness in the left mandibular condyle region (figure 4). intraoral clinical examination showed limited mouth opening, malocclusion, anterior open bite, redness and minimal swelling, instability in the posterior mandibular region and step off deformity in the angle of the mandible region. a panoramic radiograph showed a fracture line in the left posterior mandible region distally from tooth 38 and figure 1. extraoral clinical examination showing facial asymmetry. figure 2. intraoral clinical examination demonstrating anterior and posterior open bite, in addition to fracture with displacement in regions 41 and 42 and two overlapping mandibular incisors (blue arrow). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 149gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 figure 3. panoramic radiograph indicating vertical fracture line (blue arrow). figure 4. extraoral clinical examination showing minimal facial asymmetry and submental swelling. figure 5. intraoral clinical examination showed laceration on the anterior left lower gingival mucosa (white arrow), right edge-to-edge occlusion, laceration with displacement in the 31 and 33 regions and step off deformity ± 3 mm inferiorly (white arrow). figure 6. panoramic radiograph showed fracture line in the left parasymphyseal mandible region mesial to tooth 33, and in the ascending ramus of the left mandible region to the left of the mandibular coronoid process (blue arrow). in the left mandibular condyle region. following clinical and radiological examination, this case was diagnosed as fractures to the left mandibular angle and condyle. case 3: a 41-year-old male came to dental hospital of the faculty of dental medicine at universitas airlangga with the chief complaints of difficulty in closing the mouth and gingival injury due to being hit on his chin and cheek by a stranger since three days prior to attending the hospital. extraoral clinical examination showed facial asymmetry, swelling in the left parasymphysis and mandibular ramus region, deviation of the mandible to the right during mouth opening, as well as step off deformity in the parasymphysis and left mandibular ramus region with tenderness on palpation (figure 5). intraoral clinical examination confirmed limited mouth opening, laceration to the gingival mucosa of the anterior labial mandible, malocclusion, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 150 gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 figure 10. extraoral clinical examination on 86th day showed no facial asymmetry. figure 11. intraoral clinical examination on 86th day showed normal occlusion without anterior and posterior open bite. figure 7. extraoral clinical examination indicating minimal facial asymmetry, laceration on and swelling in the submental region. figure 8. intraoral clinical examination indicating laceration to the anterior lower gingival mucosa and ± 1 mm inferior displacement in the 32 region, malocclusion, anterior and posterior open bite. figure 9. panoramic radiograph showed oblique fracture line in the left parasymphysis mandible region to the distal tooth 31 region, and left condyle region (blue arrow). anterior and posterior open bite, fracture with displacement in the 31 and 33 regions, step off deformity of ±3 mm, and instability in the left posterior mandibular region. a panoramic radiograph showed a fracture line stretching mesially in the left parasymphysis mandible from tooth 33, and in the region of the ascending ramus of the left mandible to the left mandibular coronoid process (figure 6). following clinical and radiological examination, this case was diagnosed as left parasymphysis and ascending ramus of the mandible fracture. case 4: a 23-year-old male presented the chief complaint of difficulty in chewing and closing the mouth due to a motorcycle accident six days prior to attending the hospital. extraoral examination revealed laceration to the right anterior mandible region (figure 7). intraoral examination confirmed discontinuity in the symphysis region and malocclusion (figure 8). a panoramic radiograph indicated the presence of a fracture line in the symphysis region, an anterior comminuted mandible fracture, and a left subcondylar fracture (figure 9). on the basis of clinical dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 151gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 figure 12. panoramic radiograph after treatment showed union of symphysis mandible without fracture line. figure 13. intraoral clinical examination on 6th week showed normal occlusion without anterior and posterior open bite. figure 14. intraoral clinical examination on 12th week showed normal occlusion without anterior and posterior open bite, and no step off deformity in the 33 region. figure 15. panoramic radiograph after treatment showed no fracture line in the parasymphysis and left ascending ramus of the mandible region. and radiological examination, this case was diagnosed as one of mandibular symphysis fracture, comminuted anterior mandible fracture, and left subcondylar fracture. case management case 1: closed reduction was performed using an arch bar in the teeth 15 to 26, 35 to 41 and 42 to 45 regions, while elastic bands were employed to reposition bone fragment. during the first three days, elastic band application was performed laterally to reposition two separate bone fragments. during the subsequent three days, the traction direction was changed first to anterior and then to medial until the 20th day. the imf was subsequently changed using wire in the anterior and posterior region up to the 34th day, which was then replaced by elastic bands up to the 48th day. at that point, the imf was removed, and the patient instructed to perform mouth opening and closing exercises in addition to following a soft diet for the ensuing dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 152 gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 two weeks. on the 86th day, the subject presented no facial asymmetry (figure 10) and normal occlusion (figure 11 and 12). case 2: closed reduction was performed using an arch bar on the teeth 16 to 26 and 37 to 46. after direct repositioning, fracture fragment stabilization was performed with imf by means of elastic bands. facial asymmetry was corrected on the third day, there was no open bite on the tenth day, centric occlusion was achieved on the 17th day, and step off deformity or crepitation in the angle and condyle mandible was absent on the 28th day. following removal of the imf, the patient was instructed to perform mouth opening and closing exercises. on the 45th day, he did not report any pain and demonstrated a normal ability to close his mouth and masticate (figure 13). case 3: debridement and suturing of wound laceration were first performed followed by closed reduction using an arch bar in the teeth 17 to 27 region, and 37 to 47 region. after direct repositioning, fracture fragment stabilization was performed with imf using elastic bands. facial asymmetry was corrected during the 1st week, while no anterior or posterior open bite was evident during the 4th week. following removal of the imf during the 8th week, the patient was instructed to performed mouth opening and closing exercises for a period of four weeks. during the 12th week, he demonstrated the ability to open and close his mouth normally (figure 14). moreover, a panoramic radiograph failed to detect a fracture line (figure 15). case 4: closed reduction by means of an arch bar was performed for six weeks and imf for four weeks. a two-week regime of mandible movement exercises was prescribed for the patient. the aim of this treatment included pain alleviation, accepted occlusion, maximum intercuspation (35-40 mm), and facial symmetry. on the 40th day, no anterior or posterior open bite was observable, while the subject demonstrated the ability to open and close mouth his normally during the 8th week (figure 16). evaluations conducted over a period of three months confirmed no post-treatment complications. discussion regardless of age, the pre-injury skeletal and dentoalveolar anatomy and function have to be re-established by anatomic reduction of fractures due to occlusion.12 treatment approaches range from conservative non-invasive management by ’closed’ reduction and immobilization using intermaxillary fixation (imf), to the more invasive surgical ’open’ reduction with internal fixation approach.2 mandibular fracture without displacement and malocclusion are managed by close observation, a liquid to soft diet, avoidance of physical activities and analgesics.12 intermaxillary mandibular fixation (imf) reestablishes the patient’s pre-surgery occlusion and, in certain cases, can stabilize the bone sufficiently to enable healing to take place. this technique can be performed in cases such as those involving favorable fractures, stable occlusion with sufficient dentition, and multiple small comminuted fractures.13 this case series report discusses complex neglected mandible fractures. all four patients agreed to be treated with the closed reduction technique, although ideally an open reduction method using orif was adopted to obtain maximum results. in these cases, the closed method was suitable as an alternative treatment choice. in the first and second cases, the mandible fracture occurred in adolescence which constitutes an ongoing development period. the management of pediatric mandibular fractures differs from those occurring in adults because of the need to consider ongoing growth and developing dentition.13 in children, not every fracture needs an open reduction and internal fixation. moreover, the surgeon must contemplate the interplay between fracture location and both bony growth and dental development in order to chose an intervention that reduces the potential for long-term impairment and deformity.14 growth can support the objective of restoring form and function, especially in children. treatment should be designed to support, rather than interfere with, this biological process.13 children have greater osteogenic potential and demonstrate more rapid healing rates than adults. therefore, anatomic reduction must be accomplished earlier and immobilization time should be shorter (2 weeks versus 4–6 weeks in adults).12 this is consistent with the opinion of chrcanovic who said that, for many authors, conservative treatment of pediatric facial fractures has been the standard care due to the high osteogenic potential of facial bones in children. the early healing of fractures occurs with significant subsequent remodeling under the influence of the forces of mastication.15 according to goodday,13 because of the high elasticity of the pediatric mandible, there is typically minimal displacement of the fracture fragments, rendering the injury amenable to a closed reduction. in contrast to adults, many pediatric mandibular fractures can be treated with conservative measures such as a soft diet alone.14 figure 16. intraoral clinical examination on 8th week showed normal occlusion without anterior and posterior open bite. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 153gunardi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 147–153 according to goth,16 a period of two to three weeks of mmf in children younger than 12 years is sufficient. after the age of 10, the development of permanent teeth provides for safer wire anchors. however, because children develop at different rates, the strength of the teeth should be carefully evaluated before any type of wire placement is installed. however, according to der-martirosian,1 patients frequently reject the treatment recommended by the clinician, either because they do not view positively the benefits of the treatment or because the risk and potential harmful side effects are perceived to be more serious than anticipated by clinicians. occasionally, a residual facial scar was the most frequently expressed concern with regard to surgical treatment as observable in the third and fourth cases. standard treatments that were used to repair mandibular fractures in this cases were non-surgical and referred to as mmf, and in most adults mandibular fractures require 4 to 6 weeks of stabilization by means of jaw wiring.1,13 in general, patients with nondisplaced or minimally displaced fractures may be managed conservatively through a combination of close observation, soft diet, analgesics, and activity precautions.9,17 the adoption of a simple method would reduce complications related to the treating of mandible fractures because open reduction increases the risk of morbidity. patients who chose the closed reduction method treatment had to be capable of cooperating on the basis of a regular follow-up schedule and to evaluate previous treatment results to ensure that no unintended movement changes occurred. closed reduction method in this series of cases produced encouraging results and it could be considered as an alternative to the treatment of neglected mandibular fracture with displacement. references 1. der-martirosian c, gironda mw, black ee, belin tr, atchison ka. predictors of treatment preference for mandibular fracture. j public health dent. 2010; 70(1): 13–8. 2. nasser m, pandis n, fleming ps, fedorowicz z, ellis e, ali k. interventions for the management of mandibular fractures. cochrane database syst rev. 2013; 2013(7): cd006087. 3. munante-cardenas jl, nunes phf, passeri la. etiology, treatment, and complications of mandibular fractures. j craniofac surg. 2015; 26(3): 611–5. 4. niedzielska i, puszczewicz z, mertas a, niedzielski d, rózanowski b, baron s, konopka t, machorowska-pieniązek a, skucha-nowak m, tanasiewicz m, paluch j, markowski j, orzechowska-wylȩgała b, król w, morawiec t. the influence of ethanolic extract of brazilian rreen propolis gel on hygiene and oral microbiota in patients after mandible fractures. biomed res int. 2016; 2016: 1–11. 5. anyanechi ce, saheeb bd. mandibular sites prone to fracture: analysis of 174 cases in a nigerian tertiary hospital. ghana med j. 2011; 45(3): 111–4. 6. boffano p, kommers sc, karagozoglu kh, gallesio c, forouzanfar t. mandibular trauma: a two-centre study. int j oral maxillofac surg. 2015; 44(8): 998–1004. 7. namdev r, jindal a, bhargava s, dutta s, singhal p, grewal p. patterns of mandible fracture in children under 12 years in a district trauma center in india. dent traumatol. 2016; 32(1): 32–6. 8. yuen h-w, mazzoni t. mandible fracture. statpearls. 2019; 2019: 1–4. 9. pickrell bb, serebrakian at, maricevich rs. mandible fractures. semin plast surg. 2017; 31(2): 100–7. 10. omeje ku, rana m, adebola ar, efunkoya aa, olasoji ho, purcz n, gellrich nc, rana m. quality of life in treatment of mandibular fractures using closed reduction and maxillomandibular fixation in comparison with open reduction and internal fixation a randomized prospective study. j cranio-maxillofacial surg. 2014; 42(8): 1821–6. 11. fonseca r, barber hd, powers m, frost de. oral and maxillofacial trauma. 4th ed. philadelphia: saunders; 2013. p. 912. 12. glazer m, joshua bz, woldenberg y, bodner l. mandibular fractures in children: analysis of 61 cases and review of the literature. int j pediatr otorhinolaryngol. 2011; 75(1): 62–4. 13. goodday rhb. management of fractures of the mandibular body and symphysis. oral maxillofac surg clin north am. 2013; 25(4): 601–16. 14. boyette jr. facial fractures in children. otolaryngol clin north am. 2014; 47(5): 747–61. 15. chrcanovic br, abreu mhng, freire-maia b, souza ln. facial fractures in children and adolescents: a retrospective study of 3 years in a hospital in belo horizonte, brazil. dent traumatol. 2010; 26(3): 262–70. 16. goth s, sawatari y, peleg m. management of pediatric mandible fractures. j craniofac surg. 2012; 23(1): 47–56. 17. wolfswinkel em, weathers wm, wirthlin jo, monson la, hollier lh, khechoyan dy. management of pediatric mandible fractures. otolaryngol clin north am. 2013; 46(5): 791–806. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p147–153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p147-153 207 volume 46, number 4, december 2013 sifat fisik hidroksiapatit sintesis kalsit sebagai bahan pengisi pada sealer saluran akar resin epoxy (physical properties of calcite synthesized hydroxyapatite as the filler of epoxy-resin-based root canal sealer) ema mulyawati,1 marsetyawan hnes,2 siti sunarintyas,3 dan juni handajani4 1 departemen konservasi gigi, fakultas kedokteran gigi universitas gadjah mada 2 fakultas kedokteran universitas gadjah mada 3 departemen biomaterial, fakultas kedokteran gigi universitas gadjah mada 4 departemen biologi oral, fakultas kedokteran gigi universitas gadjah mada yogyakarta indonesia abstract background: the filler addition to resin based sealers will enhance the physical properties of the polymer. because of its biological properties, the synthetic hydroxyapatite (ha) has been proposed as filler for dental material such as composite resin. the calcite synthesized ha is the ha produced of calcite minerals that came from many indonesian mining. purpose: the aim of study was to determine the effect of different concentration of calcite synthesized ha as the filler of the epoxy-resin-based root canal sealer on the physical properties such as its contact angle, the film thickness and the microhardness. methods: the crystal of the calcite synthesized hydroxyapatite with the size between 77.721-88.710 nm and the ratio of ca/p 1.6886 were synthesized at ceramic laboratory, mechanical engineering, using wet method of hydrothermal microwave. the powders of the epoxyresin were prepared by added the synthesized hydroxyapatite crystal in 5 different weight ratios (e.g.: ha-10%, ha-20%, ha-30%, ha-40% and ha-50%). each of these was mixed with the paste of 3:1 ratio using spatula on a glass plate until homogen and then measuring the contact angle and the film thickness. microhardness test was conducted after the mixture of experimental sealer was stored for 24 hrs at 37 oc to reach perfect polymerization. results: all of contact angles were <90o and were not significantly different to each other (p= 0.510). all groups had a film thickness in accordance with iso 6876 (<50 um) and with no statistical difference (p= 0.858). in the ha of 10%, 20%, 30% seen that the microhardness were increased, while in the ha-50% was decreased and in the ha-40% has the same microhardness to the control groups (ha-0%). conclusion: calcite synthesized ha as the filler did not affect contact angle and film thickness of the sealer. microhardness of the epoxy-resin based sealer could be increased using maximum 30% of the calcite synthesized ha as the filler. key words: physical properties, contact angle, film thickness, micro hardness, calcite synthesized hydroxyapatite, root canal sealer abstrak latar belakang: penambahan bahan pengisi pada sealer berbahan dasar resin akan meningkatkan sifat fisik polimer. karena sifat biologis bagus, hidroksiapatit (ha) sintetis digunakan sebagai bahan pengisi material kedokteran gigi seperti resin komposit. hidroksiapatit sintesis kalsit merupakan ha yang hasilkan dari mineral kalsit berasal dari berbagai daerah pertambangan di indonesia. tujuan: penelitian ini bertujuan untuk meneliti pengaruh berbagai konsentrasi ha sintesis kalsit sebagai bahan pengisi sealer berbahan dasar resin epoksi terhadap sifat fisiknya yaitu sudut kontak, ketebalan film dan kekerasan mikronya. metode: kristal ha sintesis kalsit yang berukuran 7,721-88,710 nm dengan rasio ca/p 1,6886 diperoleh dari sintesis di laboratorium keramik, teknik mesin, universitas gadjah mada menggunakan wet method dengan microwave hidrotermal. serbuk resin epoksi dipersiapkan dengan menambahkan kristal ha sintesis kalsit dalam lima konsentrasi yang berbeda yaitu ha-10%, ha-20%, ha-30%, ha-40% dan ha-50% (dalam berat). masing-masing serbuk diaduk dengan pasta resin epoksi dengan perbandingan 3:1 menggunakan spatula di research report 208 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 207–212 atas glassplate hingga homogen, selanjutnya dilakukan pengukuran sudut kontak dan ketebalan film. pengukuran kekerasan mikro dilakukan setelah sealer disimpan dalam inkubator 37 oc selama 24 jam sehingga mencapai polimerisasi sempurna. hasil: semua kelompok menunjukkan bahwa sudut kontak <90o dan menunjukkan tidak ada perbedaan yang signifikan (p=0,510). semua kelompok menunjukkan bahwa ketebalan filmnya sesuai dengan iso 6876 (<50 um) dan menunjukkan tidak ada perbedaan yang signifikan (p=0,858). pada kelompok ha-10%, 20% dan 30% kekerasan mikronya meningkat, sedangkan pada ha-50% menurun dan ha-40% kekerasannya sama dengan kelompok kontrol (ha-0%). simpulan: penambahan hingga 50% ha sintesis kalsit sebagai bahan pengisi sealer resin epoksi tidak mempengaruhi sudut kontak dan ketebalan film. kekerasan sealer dapat ditingkatkan dengan menambahkan ha sintesis kalsit maksimum hingga 30%. kata kunci: sifat fisik, sudut kontak, ketebalan film, kekerasan mikro, hidroksiapatit sintesis kalsit, sealer saluran akar korespondensi (correspondence): ema mulyawati, departemen ilmu konservasi gigi, fakultas kedokteran gigi, universitas gadjah mada. jl. denta i, sekip utara, yogjakarta 55281. indonesia. e-mail: emamulyawati@gmail.com pendahuluan obturasi saluran akar (root canal obturation) bertujuan untuk mendapatkan penutupan hermetis sepanjang sistem saluran akar terutama di daerah apikal.1 kerapatan apikal menjadi parameter utama suatu bahan obturasi saluran akar yang akan mencegah terjadinya kebocoran apikal sebagai penyebab utama kegagalan perawatan saluran akar. bahan obturasi saluran akar yang digunakan hingga saat ini adalah guta perca yang dalam aplikasinya harus dikombinasikan dengan sealer saluran akar (root canal sealer). fungsi utama sealer adalah mengisi celah yang terjadi antara guta perca dan dinding saluran akar, sehingga bahan obturasi dapat menutup saluran akar baik ke arah apikal maupun lateral sehingga mencegah terjadinya kebocoran apikal.2,3 secara fisik sealer harus dapat melekat atau beradaptasi dengan baik pada dinding saluran akar. sifat fisik yang mempengaruhi pelekatan sealer antara lain daya pembasahan (wettability) dan ketebalan film (film thickness).4 selain itu sealer harus mudah diaplikasikan pada dinding saluran akar, tidak mengerut setelah dimasukkan, radiopak, tidak mewarnai gigi, dan mudah dikeluarkan bila perlu.3,4 pada saat ini sealer resin merupakan sealer yang paling banyak digunakan. resin merupakan suatu polimer sintetik yang mempunyai sifat adaptasi baik pada dinding saluran akar yang disebabkan daya adesinya tinggi dan perubahan dimensi kecil.5 sealer resin lambat mengeras dan dalam temperatur tubuh memerlukan waktu antara 9-15 jam, sehingga waktu kerja dan waktu setting cukup panjang.6 kekurangan sealer berbahan dasar resin adalah selalu mengalami pengerutan pada saat polimerisasi (polimerization shrinkage) sehingga mempengaruhi kerapatan apikalnya. jenis resin yang banyak digunakan sebagai siler adalah resin-epoxy yang berbasis bisphenol a-diglycidyl ether dengan kandungan hexamethylentetramine sebagai bahan katalisator dan bismuth oxide untuk sifat radiopaknya.5,6 sampai saat ini belum ditemukan bahan yang memenuhi semua persyaratan ideal suatu sealer saluran akar. hal ini yang mendorong selalu dicari usaha untuk meningkatkan mutu suatu sealer, antara lain dengan menambahkan suatu bahan pengisi (filler) pada bahan sealer yang digunakan. penambahan bahan pengisi pada sealer resin akan mengurangi pengerutan selama proses polimerisasi. salah satu bahan alternatif yang banyak dikembangkan saat ini adalah kalsium fosfat termasuk hidroksiapatit (ha).7-9 hidroksiapatit sintetis sudah banyak digunakan dalam bidang kedokteran gigi antara lain sebagai bahan pengisi untuk beberapa material kedokteran gigi seperti resin komposit dan sistem adhesif,10,11 bahan pengganti tulang alveolar yang memacu penyembuhan kerusakan tulang periodontal dan memacu osteointegrasi dari implan titanium.12-14 hidroksiapatit merupakan senyawa kalsium berbentuk kristal heksagonal, yang dapat diperoleh dari tulang dan struktur jaringan keras gigi yaitu email dan dentin baik pada manusia maupun binatang.15 hidroksiapatit sintesis kalsit merupakan ha yang disintesis dari serbuk kalsit alami yang berasal dari beberapa daerah pertambangan di indonesia.16 hidroksiapatit dapat disintesis dari kalsit dengan wet method menggunakan hydrothermal microwave melalui transformasi dengan hydrogen phosphat. hasil sintesis kalsit melalui transformasi menghasilkan ha dengan ukuran diameter kristal berkisar antara 77,721– 88,710 nm, sedangkan ha200 antara 57,538–77,534 nm yang artinya ukuran kristal ha sintesis kalsit lebih stabil dibandingkan ha sintetis (ha-200). pengujian karakteristik ha sintesis kalsit secara atomic absorption spectroscopy (aas) didapatkan hasil bahwa rasio ca/p ha sintesis kalsit 1,6886 sedangkan ca/p ha secara stokiometri adalah 1,6667. dari hasil tersebut disimpulkan bahwa kemurnian ha sintesis kalsit mendekati kemurnian ha stokiometri yang sudah terbukti biokompatibel terhadap jaringan.17 penelitian ini bertujuan untuk meneliti pengaruh penambahan ha sintesis kalsit sebagai bahan pengisi (filler) siler resin epoxy terhadap sifat fisik sealer yaitu sudut kontak (contact angle), ketebalan film (film thickness) dan kekerasan mikro (microhardness). 209mulyawati et al.,: sifat fisik hidroksiapatit sintesis kalsit bahan dan metode kristal ha sintesis kalsit yang berdiameter antara 77,72188,710 nm dengan ratio ca/p 1.6886 disintesis di laboratorium keramik, jurusan teknik mesin, universitas gadjah mada. sintesis dilakukan dengan wet method menggunakan hydrothermal microwave. kalsit yang digunakan merupakan hasil olahan pt. omya, sidoarjo, jawa timur yang bersumber dari kalsit alami beberapa daerah pertambangan di indonesia seperti pacitan, trenggalek tulungagung, kulon progo, gunung kidul, magelang, bima dan jeneponto.16 jenis sealer resin yang digunakan adalah siler resin epoxy (dentsply, germany) yang terdiri atas cairan (liquid) yang mengandung resin epoxy a-diglycidyl ether dan serbuk (powder) yang mengandung hexamethylen-tetramine 25% sebagai katalisator dan bismuth oxide 75% untuk sifat radiopaknya. dipersiapkan serbuk resin yang telah ditambahkan kristal ha sintesis kalsit dengan formula kandungan ha 10%, 20%, 30%, 40% dan 50% (dalam berat). pencampuran sealer dilakukan secara manual di atas glass plate menggunakan spatula dengan perbandingan 3 volume unit powder dicampur dengan 1 volume unit resin. pencampuran dilakukan hingga mendapatkan konsistensi yang homogen, yang akan putus bila diangkat ke atas 1,5 2,5 cm dari glass plate. seterusnya dilakukan pengujian sifat fisik (physical properties) kelima kelompok experimental sealer yaitu siler ha-10%, ha-20%, ha-30%, ha-40%, ha-50% dan kelompok kontrol yaitu siler resin epoxy tanpa ha sintesis kalsit (ha-0%). semua pengujian baik sudut kontak, film thickness maupun kekerasan mikro pada semua kelompok siler masing-masing dilakukan 5 kali (n=5). daya pembasahan (wettability) dievaluasi dengan mengukur sudut kontak yaitu sudut internal (θ) antara siler (bahan adhesif) dengan substrat.4 bahan coba sealer sebanyak 0,3 ml diletakkan di atas object glass ukuran 2,5 x 7,5 cm dengan ketebalan 1 mm yang diletakkan pada bidang datar. setelah 3 menit dari mulainya pencampuran siler, lalu diambil foto siler tersebut dari arah samping segaris dengan ketebalan object glass. pengukuran sudut kontak dilakukan pada foto tersebut dengan menggunakan komputer. sudut kontak merupakan resultan antara kekuatan adesif sealersubstrat dengan kekuatan kohesive siler. ketebalan film dievaluasi sesuai dengan pedoman dari iso 6876.18 dipersiapkan dua buah glass plate ukuran 4,5 cm x 4,5 cm dengan ketebalan 5 mm. kedua glass plate tersebut ditumpuk dan diukur ketebalan kombinasi keduanya menggunakan sliding caliper berskala micron (sylvac s-cal pro, swiss). setelah itu bahan coba siler (experimental sealer) yang telah diaduk sebanyak 0,5 ml ditempatkan di tengah-tengah salah satu glass plate tersebut kemudian sealer ditekan menggunakan glass plate yang kedua. setelah 3 menit dari mulainya pencampuran siler, diletakkan beban seberat 15 kg secara vertikal di atas glass plate kedua tersebut. setelah 10 menit dari mulainya pencampuran sealer, ketebalan kedua glass plate dengan sealer di antara keduanya diukur menggunakan sliding caliper. perbedaan ketebalan antara kedua glass plate tanpa dan dengan sealer merupakan ketebalan film dari sealer tersebut. pengukuran dilakukan empat kali pada masingmasing sisi glass plate dan diambil nilai reratanya. kekerasan mikro dievaluasi menggunakan vickers microhardness tester (hmv shimadzu corp, kyoto, japan). siler yang telah diaduk dimasukkan dalam cetakan logam bentuk diskus dengan ukuran 8 mm (diameter) x 2 mm (ketebalan) dan disimpan dalam inkubator 37°c selama 7x 24 jam hingga mencapai polimerisasi sempurna. permukaan siler diratakan menggunakan carborundum disc. uji kekerasan dilakukan dengan menekan siler menggunakan indentor yang terbuat dari batu intan berbentuk piramida selama 10 detik dengan beban sebesar 200 g. pada bagian yang terkena indentor akan terbentuk cekungan berbentuk piramida, diukur panjang diagonal hasil identasi tersebut pada dua sisi dan diambil reratanya.19 hasil uji normalitas data dievaluasi menggunakan uji shapirowilk, sedangkan homogenitas variansi menggunakan levene-test. pada pengujian sudut kontak dan ketebalan film didapatkan hasil data normal dan homogen selanjutnya dilakukan uji anova satu jalur. hasil pengukuran sudut kontak dan ketebalan film dapat dilihat pada tabel 1. pada kelima kelompok bahan coba sealer (experimental sealer) dari kandungan ha-10% hingga 50% dan kelompok kontrol (ha-0%) menunjukkan hasil bahwa besar sudut kontaknya berkisar antara 67,43° hingga 71,13°, yang berarti < dari 90°, dan tidak ada perbedaan antara semua kelompok tersebut (p=0,510). demikian juga ketebalan film pada semua kelompok menunjukkan bahwa ketebalan filmya <50,00 um, yang sesuai dengan standar iso 6876, dengan hasil berkisar antara 44.85 hingga 47.85 um dan menunjukkan tidak ada perbedaan pada semua kelompok perlakuan (p= 0,858). tabel 1. nilai rerata dan standard deviasi (sd) sudut kontak dan ketebalan film sealer resin berbahan pengisi ha sintesis kalsit dengan berbagai konsentrasi persentase ha kalsit sudut kontak (°) ketebalan film (um) rerata ± sd rerata ± sd ha0% ha10% ha20% ha30% ha40% ha-50% 71,13 ± 1,89 71,02 ± 1,75 68,98 ± 3,49 67,43 ± 2,57 68,72 ± 3,24 70,07 ± 4,54 45,92 ± 3,04 44,85 ± 2,51 46,69 ± 3,16 47,85 ± 2,39 46,15 ± 2,70 47,35 ± 2,27 pada uji kekerasan mikro, varian tidak homogen (p<0,05) sehingga data diuji menggunakan kruskal-wallis dan mann-whitney. hasil uji kekerasan dapat dilihat pada tabel 2. 210 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 207–212 tabel 2. nilai rerata dan standard deviasi (sd) kekerasan mikro siler resin berbahan pengisi ha sintesis kalsit dengan berbagai konsentrasi dalam vickers hardness number (vhn) persentase ha kalsit kekerasan mikro (vhn) rerata ± sd ha-0% ha-10% ha-20% ha-30% ha-40% ha-50% 64,28 ± 1,11 73,23 ± 2,55 77,31 ± 2,20 80,51 ± 0,74 65,37 ± 2,03 55,22 ± 1,59 tabel 3. hasil uji mann-whitney kekerasan mikro pada semua kelompok perlakuan siler resin berbahan pengisi ha sintesis kalsit dengan berbagai konsentrasi pasangan kelompok p ha0% ha-10% ha0% ha-20% ha0% ha-30% ha0% ha-40% ha0% ha-50% ha-10% ha-20% ha-10% ha-30% ha-10% ha-40% ha-10% ha-50% ha-20% ha-30% ha-20% ha-40% ha-20% ha-50% ha-30% ha-40% ha-30% ha-50% ha-40% ha-50% *0,009 *0,008 *0,009 0,347 *0,009 *0,045 *0,009 *0,009 *0,009 *0,026 *0,008 *0,008 *0,009 *0,009 *0,009 keterangan: * berbeda bermakna (p<0,05) dari uji kruskal-wallis didapatkan hasil bahwa ada perbedaan pada semua kelompok perlakuan maupun kontrol (p<0,05) sehingga dilanjutkan dengan uji mannwhitney. dari uji mann-whitney diketahui bahwa pada penambahan ha-10% kekerasannya lebih tinggi (p= 0,009) dibandingkan ha-0%. kekerasan siler ha-20% lebih tinggi dibandingkan dengan siler ha-0% (p=0,008) maupun ha10% (p=0,045). pada siler ha-30% kekerasannya lebih tinggi dibandingkan ha-0% (p=0,009), ha-10% (p=0,009) maupun ha-20% (p=0,026). kekerasan siler ha-40% menurun dibandingkan dengan ha-30% (p=0,009), 20% (p=0,008) maupun 10% (p=0,009), tetapi tidak berbeda dengan siler ha-0% (p=0,347 ). sealer ha-50% menunjukkan kekerasan mikro yang lebih kecil (p<0,05) dibandingkan semua kelompok bahan coba sealer maupun kelompok kontrol (tabel 3). pembahasan penambahan partikel bahan pengisi dapat meningkatkan viskositas bahan adesif.20 viskositas suatu bahan berhubungan langsung dengan daya pembasahan (wettability) bahan tersebut. wettability merupakan hasil interaksi molekuler antara adesif dengan aderen atau substrat yang merupakan perlawanan terhadap daya kohesi bahan adesif dan merupakan faktor yang sangat menentukan terjadinya adesi yang baik dengan aderen. agar bahan adesif dapat mengalir dan melekat pada permukaan substrat viskositas bahan tersebut harus rendah.4 wettability secara tidak langsung dapat diketahui dengan mengukur sudut kontak. secara umum dikatakan bahwa sudut kontak yang kecil didapatkan apabila tegangan permukaan bahan adesif rendah dan energi permukaan aderen atau substrat tinggi. pada penelitian ini ha sintesis kalsit yang digunakan sebagai bahan pengisi berukuran kristal 77,721–88,710 nm17 dan menunjukkan hasil bahwa viskositasnya tidak berbeda dengan yang tanpa penambahan bahan pengisi. hal ini dapat dilihat dari pengukuran sudut kontak yang menunjukkan tidak ada perbedaan antara kelompok kontrol (tanpa ha) maupun semua kelompok sealer dengan tambahan bahan pengisi ha sintesis kalsil (kelompok ha 10%, 20%, 30%, 40% dan 50%). pada semua kelompok menunjukkan bahwa sudut kontaknya <90o. ini menunjukkan bahwa penambahan ha sintesis kalsit tidak mengubah kemampuan pembasahan (wettability) dari siler epoxy tersebut. kemampuan pembasahan suatu adesif dikatakan baik apabila sudut kontaknya kurang dari 90o dan dikatakan ideal apabila adesif dapat menyebar di seluruh permukaan aderen dengan sudut kontak 0o.4 apabila bahan adesif sudut kontaknya <90o maka bahan adesif dapat mengalir dan melekat dengan mudah, artinya bahan sealer ini dapat mengalir, mengisi celah yang kosong antara guta perca dan dinding saluran akar, berpenetrasi pada permukaan dentin saluran akar tanpa menghasilkan porusitas pada antar permukaannya. pada akhirnya bahan sealer yang sudah berpenetrasi akan mengeras dan menghasilkan ikatan yang berpautan (interlocking bonding) dengan struktur mikro dari dentin saluran akar dan menghasilkan retensi mikromekanikal sehingga kerapatan apikalnya bagus. pemilihan bahan-bahan resin epoxy sebagai siler antara lain karena sifat flownya yang tinggi dan viskositasnya rendah. hasil penelitian ini menunjukkan bahwa penambahan ha sintesis kalsit pada sealer resin hingga 50% terbukti tidak meningkatkan viskositasnya. hal ini kemungkinan karena partikel ha kalsit yang digunakan sebagai bahan pengisi berukuran nano yaitu antara 77,721–88,710 nm, sehingga siler resin ini dapat digolongkan dalam klasifikasi nanofiller. pada uji ketebalan film (film thickness) semua kelompok siler baik kontrol maupun dengan penambahan ha sintesis kalsit menunjukkan bahwa ketebalan filmnya <50 um. menurut iso 6876 sesuai dengan cara pengujian yang dilakukan dikatakan bahwa ketebalan ideal suatu sealer tidak boleh melebihi 50 um.18 apabila ketebalan film bahan adesif melebihi 50 um maka bahan sealer akan sulit dimanipulasi sehingga mempengaruhi adaptasi sealer dengan dinding saluran akar. pada penelitian ini terlihat bahwa penambahan ha sintesis kalsit tidak menurunkan 211mulyawati et al.,: sifat fisik hidroksiapatit sintesis kalsit daya pembasahannya maupun meningkatkan ketebalan filmnya. semakin tipis film thikness maka sealer akan semakin mudah diaplikasikan pada dinding saluran akar dan semakin mudah mengalir serta mengisi celah antara guta perca dengan dinding saluran akar. hasil pengukuran kekerasan mikronya, walaupun kekerasan mikro tidak berhubungan langsung dengan pelekatan, akan tetapi dapat dijadikan tolak ukur untuk menentukan tingkat polimerisasi atau derajat konversi sealer resin tersebut.21 derajat konversi merupakan jumlah persentase ikatan ganda karbon yang telah menjadi ikatan tunggal membentuk polimer. derajat konversi diperoleh dengan mengurangi persentase ikatan rangkap karbon dari 100%.22,23 hasil uji statistik menunjukkan bahwa penambahan hidroksiapatit sebagai bahan pengisi dapat meningkatkan kekerasan bahan sealer. ini sesuai dengan hasil penelitian terdahulu yang menyatakan bahwa penambahan bahan pengisi dapat meningkatkan sifat fisik maupun mekanis suatu resin.24,25 selain meningkatkan kekerasan resin bahan pengisi dapat mengurangi koefisien termal ekspansi, pengerutan polimerisasi (polymerization shrinkage) dan memudahkan aplikasinya. hidroksiapatit sintesis kalsit sebagai bahan pengisi tidak bereaksi dengan bahan-bahan siler tetapi partikelnya akan penetrasi diantara partikelpartikel resin epoksi, hexamethylen-tetramine dan bismuth oxide. hidroksiapatit yang terdiri atas unsur kalsium dan fosfat, yang merupakan unsur utama jaringan keras gigi baik email maupun dentin, ternyata dapat meningkatkan kekerasan sealer. hasil penelitian didapatkan bahwa penambahan ha10%, 20% dan 30% dapat meningkatkan kekerasan sealer. akan tetapi pada penambahan ha-40% tidak menunjukkan peningkatan kekerasan walaupun apabila dibandingkan dengan ha-0% kekerasannya sama, sedangkan pada penambahan ha-50% terjadi penurunan kekerasan. hal ini menunjukkan bahwa ada batasan tertentu pada penambahan bahan pengisi agar dapat meningkatkan sifat sealer. hasil penelitian ini menunjukkan bahwa penambahan ha sintesis kalsit akan meningkatkan kekerasan apabila tidak lebih dari 30%, dan pada penambahan 30% menunjukkan kekerasan paling tinggi dibandingkan kelompok lainnya. penurunan kekerasan mikro pada kelompok ha-50% menunjukkan terjadi gangguan pada proses polimerisasi. proses polimerisasi resin epoxy dipicu dengan adanya hexamethylen-tetramine. penambahan hidroksiapatit yang melebihi volume hexamethylen-tetramine sebagai katalisator, jumlahnya menjadi tidak cukup untuk menghasilkan reaksi polimerisasi yang sempurna. polimerisasi yang tidak sempurna akan menyisakan lebih banyak monomer sisa yang tidak berikatan, akibatnya kekerasan sealer akan menurun. kekerasan sealer rendah menunjukkan bahwa polimerisasi tidak sempurna atau derajat konversinya rendah, secara tidak langsung akan mempengaruhi kerapatan apikalnya. sealer resin yang tidak terpolimerisasi sempurna akan meningkatkan sensitivitas sealer terhadap lingkungan. idealnya resin sealer yang terpolimerisasi dengan sempurna akan mempunyai sifat fisik dan kimiawi yang sempurna yang ditunjukkan dengan tidak larutnya sealer pada lingkungan lembab. pada penggunaan klinis sealer selalu akan berkontak dengan jaringan yang lembab yaitu jaringan periodontal karena adanya foramen apikal, kanalis lateralis maupun asesoris yang menghubungkan bahan obturasi saluran akar dengan jaringan periodontal. sealer dengan derajat konversi yang rendah atau yang tidak terpolimerisasi sempurna akan lebih mudah larut dalam jaringan periodontal. pada kondisi lembab monomer yang tidak terpolimerisasi akan lebih cepat terlepas dari resin polimer sehingga mempengaruhi sifat fisik sealer yaitu mengurangi adaptasi sealer dengan dinding saluran akar.26,27 hasil penelitian ini menunjukkan bahwa pada batasan tertentu penambahan ha sintesis kalsit sebagai bahan pengisi (filler) dapat meningkatkan sifat fisis bahan yang berarti meningkatkan kualitas bahan, sebaliknya pada batasan tertentu penambahan bahan pengisi ha justru menurunkan kualitas fisik bahan. penambahan bahan pengisi juga dipercaya akan mengurangi pengerutan polimerisasi sealer resin ini. pengerutan polimerisasi terjadi selama proses polimerisasi berlangsung, yaitu dengan memendeknya jarak antar monomer.28 bahan pengisi ha sintesis kalsit yang berpenetrasi di antara partikel monomer epoxy a-diglycidyl, hexamethylen-tetramine dan bismuth oxide akan mencegah terjadinya pemendekan jarak antar monomer sehingga pengerutan polimerisasinya juga berkurang. dari penelitian ini didapatkan hasil bahwa penambahan ha sintesis kalsit 10% hingga 50% sebagai bahan pengisi siler resin epoxy tidak mempengaruhi kemampuan pembasahan (wettability) dan ketebalan film (film thikness) tersebut, artinya secara fisik siler tersebut memenuhi syarat utama siler saluran akar. selain itu penambahan ha sintesis kalsit hingga 30% dapat meningkatkan kekerasannya, tetapi penambahan hingga 50% justru menurunkan kekerasannya. berdasarkan hasil penelitian dapat disimpulkan bahwa penambahan hingga 50% ha sintesis kalsit sebagai bahan pengisi siler resin epoksi tidak mempengaruhi sudut kontak dan ketebalan film sedangkan kekerasan sealer dapat ditingkatkan dengan menambahkan ha sintesis kalsit maksimum hingga 30%. daftar pustaka 1. glickman gn, walton re. obturation. in: torabinejad m, walton re, eds. endodontic principles and practice. 4th ed. china: saunders elseiver; 2009. p. 298-339. 2. johnson wt, kulild jc. obturation of the cleaned and shaped root canal system. in: hargreaves, cohen, eds. pathways of the pulp. 10th ed. china: mosby elseiver; 2011. p. 349-88. 3. gutmann jl, dumsha tc, lovdahl pe. problem solving in endododintics. 4th ed. st. louis: mosby elseiver; 2006. p. 197238. 4. sakaguchi rl, power jm. craigs restorative dental materials. 12th ed. philadelphia: mosby elsevier; 2012. p. 327-47. 212 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 207–212 5. roberts s, kim jr, gu ls, kim yk, mitchell qm, pashley dh, tay fr. the efficacy of different sealer removal protocols on bonding of self-etching adhesives to ah plus-contaminated dentin. j endod 2009; 35(4): 563-7. 6. huang th, yang jj, li h, kao ct. the biocompatibility evaluation of epoxy resin-based root canal sealers in vitro. biomaterials 2002; 23(1): 77-83. 7. markovic d, zivijinovic v, kokovic v, jokanovic v. hydroxyapatite as root canal system filling material: cytotoxicity testing. materials science forum 2004; 453: 555-60. 8. khashaba rm, moussa mm, chutkan nb, borke jl. the response of subcutaneous connective tissue to newly developed calcium phosphate-based root canal sealers. int endod j 2011; 44(4): 34252. 9. collares fm, leitune vc, rostirolla fv, trommer rm, bergmann cp, samuel sm. nanostructured hydroxyapatite as filler for methacrylate-based root canal sealers. int endod j 2012; 45(1): 637. 10. domingo c, arcís rw, osorio e, osorio r, fanovich ma, rodríguez-clemente r, toledano m. hydrolytic stability of experimental hydroxyapatite-filled dental composite materials. dent mater 2003; 19(6): 478-86. 11. shojai s. effect of ha nanofiber on the properties of dental adhesive. dental mater 2010; 26: 471-82. 12. hayash i y, i ma i m, ya nag ig uch i k , vi lor ia i l , i ke d a t. hydroxyapatite applied as direct pulp capping medicine substitutes for osteodentin. j endod 1999; 25(4): 225-9. 13. anusavice kj. phillips science of dental material. 11th ed. st louis: elseiver science; 2003. p. 752-53. 14. okamoto h, arai k, matsune k, hirukawa s, matsunaga s, kiba h. the usefulness of new hydroxyapatite as a pulp capping agent in rat molars. j oral med sci 2006; 5: 506. 15. hargreaves km, goodis he. seltzer and bender’s: dental pulp. 3rd ed. china: quintessence book pub co, inc; 2002. p. 309–24. 16. sukandarrumidi. bahan galian industri. yogyakarta: gadjah mada university press; 2004. h. 45-105. 16. syamsudin. analisis uji tekan dan porositas materials kompaksi sinter ha-zno sebagai material subtitusi tulang. tesis. yogyakarta: sekolah pasca sarjana universitas gadjah. mada; 2010. h. 43-72. 17. bhavan m, shah b, marg z. iso 6876: indian standard. dental root canal sealer materials. new delhi: bureu of indian standards; 2012. p. 1-5. 18. safarcherati h, alaghehmand h. hardness of composite resin polimeriuzed with different light-cure units. caspian j dent res 2012; 1: 32-5. 19. hanemann t. influence of particle properties on the viscosity of polymer alumina composit. ceramics international 2008; 34: 2099105. 20. thiab ss. influence of light curing method and curing time on the surface hardness and degree of cure in composite resins. j babylon univ 2012; 2: 778-95. 21. finan l, palin wm, moskwa n, mcginley el, fleming gj. the influence of irradiation potential on the degree of conversion and mechanical properties of two bulk-fill flowable rbc base materials. dent mater 2013; 29(8): 906-12. 22. salazar dc, dennison j, yaman p. inorganic and prepolymerized filler analysis of four resin composites. oper dent 2013; 38(6): e201-9. 23. hervás-garcía a, martínez-lozano ma, cabanes-vila j, barjauescribano a, fos-galve p. composite resins. a review of the materials and clinical indications. med oral patol oral cir bucal 2006; 11(2): e215-20. 24. lombardini m, chiesa m, scribante a, colombo m, poggio c. influence of polymerization time and depth of cure of resin composites determined by vickers hardness. dent res j (isfahan) 2012; 9(6): 735-40. 25. malacarne j1, carvalho rm, de goes mf, svizero n, pashley dh, tay fr, yiu ck, carrilho mr. water sorption/solubility of dental adhesive resins. dent mater 2006; 22(10): 973-80. 26. ghulman ma. effect of cavity configuration (c factor) on the marginal adaptation of low-shrinking composite: a comparative ex vivo study. int j dent 2011; 2011: 159749. 27. lee ib, cho bh, son hh, um cm. a new method to measure the polymerization shrinkage kinetics of light cured composites. j oral rehabil 2005; 32(4): 304-14. 4343 effects of citrus limon essential oil (citrus limon l.) on cytomorphometric changes of candida albicans rina prabajati,1 iwan hernawan,2 and hening tuti hendarti2 1dinas kesehatan kabupaten lumajang 2department of oral medicine, faculty of dential medicine, universitas airlangga surabaya indonesia abstract background: the most common fungal infection found in oral cavity is oral candidiasis, largely caused by candida species, particularly candida albicans (c. albicans). candida infection can get worse since it is difficult to be treated and resistant with antifungal drugs. therefore, new drugs and compounds as well as alternative therapies involving natural sources that have antifungal activities have continually been developed. limonene, β-pinene, and ɣ-terpinene contained in citrus limon essential oil have been known to have quite good antifungal activities against c. albicans. purpose: this research aimed to examine and analyze the effects of citrus limon essential oil on cytomorphometric changes of c. albicans. method: the research used post test only control group design. based on the results of the pre-elementary research on antifungal activities of citrus limon essential oil against c. albicans, citrus limon essential oil used in this research was on concentrations of 1.56%, 1.37%, 1.17%, 0.98%, and 0.78%. citrus limon essential oil by c. albicans inoculum and incubated for 24 hours and 48 hours. after the incubation, those c. albicans cells were fixed, dried, and then observed using a scanning electron microscopy. result: the most effective concentrations of citrus limon essential oil triggering cytomorphometric changes of candida albicans were at 1.37% and 1.56% with the incubation period of 48 hours. conclusion: c. albicans can undergo necrosis process through cytomorphometric changes after the administration of citrus limon essential oil at concentrations of 1.56% and 1.37% with the incubation period of 48 hours. keywords: citrus limon; candida albicans; necrosis; cytomorphometric changes correspondence: rina prabajati, department of oral medicine, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: rinapraba2@gmail.com. research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 43–48 introduction candida is an opportunistic organism in oral cavity, triggering no disease in healthy people, but leading to an infection in the body with low immune. candida albicans (c. albicans) is a commensal organism colonizing on the skin and mucosal tissue of gastrointestinal and genitourinary tracts. when there is an imbalance between c. albicans and other oral microbial components, c. albicans will proliferate, colonize, and invade mucosal tissues to trigger opportunistic infection.1 c. albicans infection in people with hiv/ aids is a type of infection most commonly found at around 70-80% and also considered as a major cause of oral candidiasis, followed by candida guilliermondii approximately at around 11.11%.2 in recent years, many reported cases of oral candidiasis infections are caused by candida glabrata.3 population of candida glabrata is almost half of the total population of non-c. albicans.4 however, c. albicans is still considered as the largest species of oral candidiasis in both hiv/ aids patients and other immunocompromised patients.2 antifungal compounds widely used in the medical field for the treatment of fungal infections are derived from the polyene group, such as nystatin, amphotericin, and natamisin, as well as from the azole group, such as imidazole and triazole. nevertheless, this conventional treatment of fungal infection is not considered to be beneficial in fungal infections treatment that have been resistance to antifungal compounds.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 44 prabajati, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 43–48 lemon rinds, contain essential oil, formed within the endoplasmic reticulum of the plant cells and then obtained from steam distillation or extraction process of the fruit, flowers, wood, roots, leaves, and seeds of the plant.7 the essential oil has anti bacterial, anti-oxidant, and anti-fungal functions. the essential oil contained on the outer part (pericarp) of lemon rinds is largely composed of limonene (90%), citral (5%), terpinol, linodylα-pinene, camphene, β-pinene, sabinene, myrcene, γ-terpinene, linalool, β-bisabolene, trans-α-bergamotene, and geranyl acetate.8 a previous research even reveals that limonene component found in the essential oil has good anti-fungal effects on trichophyton rubrum.9 in another previous research, lemon rinds, moreover, contain some anti-fungal compounds classified into terpenoids, namely limonene, β-pinene, and γ-terpinene, which have strong anti-fungal activities against c. albicans. terpenoids inhibit ergosterol synthesis that occurs in the cell membrane of c. albicans.10 consequently, the synthesis of nucleic acids is disrupted, resulting in increased cell membrane permeability.11 limonene, β-pinene, and γ-terpinene also can inhibit the metabolism of c. albicans, interfering organelles balance. the imbalance in organelles then makes intracellular components of the organelles disrupted as well as dna damaged, resulting in the death of c. albicans.12 another previous research even reveals that the extract of lemon rinds combined with 96% petroleum ether has inhibitory effects on the growth of c. albicans in vitro.10 cell necrosis is morphologically characterized by increased cell volume (oncosis), organelle swelling, and plasma membrane rupture, followed by intracellular component secretion. some ingredients even can trigger the death of fungal cells, such as h2o2, acetic acid, as well as some metals and materials/ anti-fungal drugs. anti-fungal ingredients at low concentrations can lead to apoptosis, but at high concentrations can cause necrosis as a result of radical damage to the cellular structure and integrity.13 a research conducted by kim et al on the death process of c. albicans given amphotericin b and flucytosine shows that the morphology of c. albicans cells undergo the process of death through cell membrane damage.14 similarly, a research conducted by dai et al.12 finds that the administration of the root extract of scutellaria baicaleinsis on c. albicans can trigger apoptosis in the cells of c. albicans, leading to death. hao et al.16 argues that caspofungin containing antifungal activities can trigger apoptosis and necrosis on the cells of c. albicans. equol as a soy isoflavone also has antifungal activities against c. albicans by triggering the ultrastructural changes of the cells. for those reasons, this research aimed to analyze the effects of citrus limon essential oil on cytomorphometric changes (necrosis process) of c. albicans using a scanning electron microscopy (sem). materials and method this research was a laboratory research with a purely experimental approach. this research was conducted to observe changes in cell size and morphology (cytomorphometrics) of c. albicans treated with the essential oil of lemon rinds by using a sem. based on results of the preliminary research, the essential oil of lemon rinds has inhibitory effect at a concentration of 0.78%, and fungicidal effect at a concentration of 1.56%. therefore, in this research the essential oil of lemon rinds used was at five different concentrations, namely 1.56%, 1.37%, 1.17%, 0.98%, and 0.78%. those five groups of different concentrations as well as a control group without any treatment were incubated for 24 hours. next, there were also five groups of different concentrations, namely 1.56%, 1.37%, 1.17%, 0.98%, and 0.78%, as well as a control group without any treatment, incubated for 48 hours. after that, fixation process and drying were performed, and then observation was conducted using a sem at a magnification of 3500x. the images resulted from the observation using a sem were calibrated to measure the cell size of c. albicans, six of which were taken from each treatment group. the data obtained then were processed using a statistical analysis, a one-way anova to determine differences between groups, followed by lsd test.17 results based on results of the measurement of the cell size of c. albicans, the mean and standard deviations of the cell size of c. albicans were obtained from each group. the normality of the data then was analyzed using kolmogorovsmirnov test. result of the kolmogorov-smirnov test on the groups with the incubation period of 24 hours showed a significance value of 0.05. this result indicated that the data in each group were distributed normally. meanwhile, result of the homogeneity test using levene’s test on the groups with the incubation period of 24 hours showed a significance value of 0.193. this result illustrated that the data were homogeneous. next, result of the one-way anova test on the groups with the incubation period of 24 hours showed a significance value of 0.583 (p>0.05). this result demonstrated that there was no significant difference between the control group and the five groups of different concentrations with the incubation period of 24 hours. result of the kolmogorov-smirnov test on the groups with the incubation period of 48 hours showed a significance value 0.05. this result indicated that the data in each group were distributed normally. result of the homogeneity test using levene’s test on the groups with the incubation period of 48 hours showed a significance value of 0.073. this result illustrated that the data were homogeneous. result of the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 4545prabajati, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 43–48 table 1. results of the lsd test on the five groups of different concentrations with the incubation period of 48 hours group (1.56%) (1.37%) (1.17%) (0.98%) (0.78%) control (1.56%) 0.855 0.097 0.045* 0.015* 0.014* (1.37%) 0.136 0.066 0.022* 0.021* (1.17%) 0.079 0.386 0.377 (0.98%) 0.619 0.606 (0.78%) 0.986 control note: * there was a significant difference 5 moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. f 1µm=7,45mm 1µm=5,78mm 1µm=13,56mm 1µm=6,23mm 5 moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. f 1µm=7,45mm 1µm=5,78mm 1µm=13,56mm 1µm=6,23mm 5 moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. f 1µm=7,45mm 1µm=5,78mm 1µm=13,56mm 1µm=6,23mm 5 moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. f 1µm=7,45mm 1µm=5,78mm 1µm=13,56mm 1µm=6,23mm 5 moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. f 1µm=7,45mm 1µm=5,78mm 1µm=13,56mm 1µm=6,23mm 6 figure 1. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 24 hours. (a) control: the surface of cells was smooth, round or oval, and colonizing; (b) the group with the concentration of 0.78%: the surface of some cells was rough and not round perfectly (). some cells were separated from colonies (); (c) the group with the concentration of 0.98%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of some cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of cells was rough and not round (); (f) the group with the concentration of 1.56%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (). 1µm=6,51mm 1µm=6,1mm 1µm=5,69mm 1µm=5,95mm a b c d e f figure 1. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 24 hours. (a) control: the surface of cells was smooth, round or oval, and colonizing; (b) the group with the concentration of 0.78%: the surface of some cells was rough and not round perfectly (). some cells were separated from colonies (); (c) the group with the concentration of 0.98%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of some cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of cells was rough and not round (); (f) the group with the concentration of 1.56%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (). 6 figure 1. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 24 hours. (a) control: the surface of cells was smooth, round or oval, and colonizing; (b) the group with the concentration of 0.78%: the surface of some cells was rough and not round perfectly (). some cells were separated from colonies (); (c) the group with the concentration of 0.98%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of some cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of cells was rough and not round (); (f) the group with the concentration of 1.56%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (). 1µm=6,51mm 1µm=6,1mm 1µm=5,69mm 1µm=5,95mm 6 figure 1. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 24 hours. (a) control: the surface of cells was smooth, round or oval, and colonizing; (b) the group with the concentration of 0.78%: the surface of some cells was rough and not round perfectly (). some cells were separated from colonies (); (c) the group with the concentration of 0.98%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of some cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of cells was rough and not round (); (f) the group with the concentration of 1.56%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (). 1µm=6,51mm 1µm=6,1mm 1µm=5,69mm 1µm=5,95mm 6 figure 1. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 24 hours. (a) control: the surface of cells was smooth, round or oval, and colonizing; (b) the group with the concentration of 0.78%: the surface of some cells was rough and not round perfectly (). some cells were separated from colonies (); (c) the group with the concentration of 0.98%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of some cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of cells was rough and not round (); (f) the group with the concentration of 1.56%: the surface of cells was rough and not round perfectly (). some cells were separated from colonies (). 1µm=6,51mm 1µm=6,1mm 1µm=5,69mm 1µm=5,95mm 7 figure 2. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 48 hours. (a) the control group: the surface of the cell was smooth and round in colonies; (b) the group with the concentration of 0.78%: the surface of few cells was rough, not round (), and not colonizing (); (c) the group with the concentration of 0.98%: the surface of the cells was rough and not round (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of the cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of the cells was rough, stood out, not round (), and not colonizing () (f) the group with the concentration of 1.56%: the surface of the cells was rough, stood out, not round (), and not colonizing (). discussion c. albicans fungi are often resistant to antifungal therapy, such as fluconazole and amphoterisin. thus, another more effective antifungal therapy needs to be developed as an alternative by considering the death process of c. albicans cells. consequently, there are so many researches focused on alternative materials containing better antifungal activities. hydrophobic molecules composing of essential oil are known to be able to attack ergosterol in fungal cell membranes. they will trigger changes in membrane permeability as well as damage to the membrane, and then ultimately the cells of the fungi will be secreted, resulting in cell death. essential oil molecules can also interfere with the enzymes bound to the fungal cell membranes, thereby disrupting the formation of cell membranes. in other words, the essential oil can kill and inhibit the growth of fungi.18 1µm=6,65mm 1µm=5,56mmm 1µm=5,95mm comment [hh11]: give reference 7 figure 2. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 48 hours. (a) the control group: the surface of the cell was smooth and round in colonies; (b) the group with the concentration of 0.78%: the surface of few cells was rough, not round (), and not colonizing (); (c) the group with the concentration of 0.98%: the surface of the cells was rough and not round (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of the cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of the cells was rough, stood out, not round (), and not colonizing () (f) the group with the concentration of 1.56%: the surface of the cells was rough, stood out, not round (), and not colonizing (). discussion c. albicans fungi are often resistant to antifungal therapy, such as fluconazole and amphoterisin. thus, another more effective antifungal therapy needs to be developed as an alternative by considering the death process of c. albicans cells. consequently, there are so many researches focused on alternative materials containing better antifungal activities. hydrophobic molecules composing of essential oil are known to be able to attack ergosterol in fungal cell membranes. they will trigger changes in membrane permeability as well as damage to the membrane, and then ultimately the cells of the fungi will be secreted, resulting in cell death. essential oil molecules can also interfere with the enzymes bound to the fungal cell membranes, thereby disrupting the formation of cell membranes. in other words, the essential oil can kill and inhibit the growth of fungi.18 1µm=6,65mm 1µm=5,56mmm 1µm=5,95mm comment [hh11]: give reference 7 figure 2. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 48 hours. (a) the control group: the surface of the cell was smooth and round in colonies; (b) the group with the concentration of 0.78%: the surface of few cells was rough, not round (), and not colonizing (); (c) the group with the concentration of 0.98%: the surface of the cells was rough and not round (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of the cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of the cells was rough, stood out, not round (), and not colonizing () (f) the group with the concentration of 1.56%: the surface of the cells was rough, stood out, not round (), and not colonizing (). discussion c. albicans fungi are often resistant to antifungal therapy, such as fluconazole and amphoterisin. thus, another more effective antifungal therapy needs to be developed as an alternative by considering the death process of c. albicans cells. consequently, there are so many researches focused on alternative materials containing better antifungal activities. hydrophobic molecules composing of essential oil are known to be able to attack ergosterol in fungal cell membranes. they will trigger changes in membrane permeability as well as damage to the membrane, and then ultimately the cells of the fungi will be secreted, resulting in cell death. essential oil molecules can also interfere with the enzymes bound to the fungal cell membranes, thereby disrupting the formation of cell membranes. in other words, the essential oil can kill and inhibit the growth of fungi.18 1µm=6,65mm 1µm=5,56mmm 1µm=5,95mm comment [hh11]: give reference a d e f cb figure 2. the cells of c. albicans were exposed to the essential oil of lemon rind with the incubation period of 48 hours. (a) the control group: the surface of the cell was smooth and round in colonies; (b) the group with the concentration of 0.78%: the surface of few cells was rough, not round (), and not colonizing (); (c) the group with the concentration of 0.98%: the surface of the cells was rough and not round (). some cells were separated from colonies (); (d) the group with the concentration of 1.17%: the surface of the cells was rough, not round (), and not colonizing (); (e) the group with the concentration of 1.37%: the surface of the cells was rough, stood out, not round (), and not colonizing () (f) the group with the concentration of 1.56%: the surface of the cells was rough, stood out, not round (), and not colonizing (). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 46 prabajati, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 43–48 one-way anova test on the groups with the incubation period of 48 hours showed a significance value of 0.038 (p <0.05). this result demonstrated that there was a significant difference between the control group and the five groups of different concentrations with the incubation period of 48 hours. results of the lsd test can be seen in table 1. based on table 1, there were significant differences between the group with the concentration of 0.98% and the group with 1.56%, between the group with the concentration of 0.78% and the group with the concentration of 1.56%, between the group with the concentration of 0.78% and the group with the concentration of 1.37%, between the control group and the group with the concentration of 1.56, as well as between the control group and the group with the concentration of 1.37%. consequently, it can be said that the essential oil of lemon rinds in the treatment groups with the concentrations of 1.56% and 1.37% had a significant change in the cell size of c. albicans compared to the control group and the other treatment groups with the concentrations of 1.17%, 0.98% and 0.78%. moreover, results of the observation using a sem on all the groups of different concentrations, 1.56%, 1.37%, 1.17%, 0.98%, 0.78%, and the control group with both of the incubation periods, 24 hours and 48 hours indicate the morphological changes in the cell wall. discussion c. albicans are often resistant to antifungal therapy, such as fluconazole and amphoterisin. thus, another more effective antifungal therapy needs to be developed as an alternative by considering the death process of c. albicans cells. consequently, there are so many researches focused on alternative materials containing better antifungal activities. hydrophobic molecules composing of essential oil are known to be able to attack ergosterol in fungal cell membranes. they will trigger changes in membrane permeability as well as damage to the membrane, and then ultimately the cells of the fungi will be secreted, resulting in cell death. essential oil molecules can also interfere with the enzymes bound to the fungal cell membranes, thereby disrupting the formation of cell membranes. in other words, the essential oil can kill and inhibit the growth of fungi.18 based on results of the gcms test, the essential oil of lemon rinds contained limonene (19.79%), β-pinene (1.06%), and γ-terpinene (0.45%). biological activities of the essential oil of lemon rinds are related to monoterpenes compounds characterized by high concentrations of limonene, β-pinene, γterpinene, and linalool contained in the components of the essential oil.19 limonene can trigger interference to the cell membrane of c. albicans, resulting in secretion of the cellular components. limonene can also change the structure of methylesterification from pectin, a major component of the fungal cell wall. such changes in the structure of pectin are associated with changes in cell adhesion and plasticity, ph and ionic content of the cell wall, as well as effects of c. albicans growth, membrane integrity, and permeability.20 β-pinene, can trigger both disruption of the cell membrane of c. albicans as well as interference to the functioning of mitochondria. b-pinene can also interfere with the movement of ions h + and k + in the cells of c. albicans, resulting in interference to the function of mitochondria in providing energy for cells in the form of atp. b-pinene as an oxidant can increase permeability of the mitochondria and atpase inhibitors that can block the formation process of energy by mitochondria.21 γ-terpinene, can interfere with the synthesis of ergosterol in c. albicans. ergosterol plays an important role in the cell growth of c. albicans. ergosterol, a predominant lipid molecule in fungal cells, serves to regulate fluidity of membrane as well as permeability and activity of membrane-bound enzyme. terpinene also can interfere with the synthesis of proteins that can interfere with metabolic processes in the nucleus of c. albicans cells.22 the essential oil of lemon rinds contains anti-fungal compounds that shows a variety of biological activities. the essential oil also can affect changes in the colony and morphology of c. albicans cells by lowering their enzymatic activities and reducing their ability to assimilate through the active components contained, such as pinene and γterpinene.23 the antifungal mechanism of the essential oil together with its monoterpen compounds can generate toxic effects on the membrane structure and functions of c. albicans.24,22 in fungal cells, α-pinene and β-pinene can disrupt the integrity of cells, inhibit respiration and ion transport process, and increase the permeability of membrane.22 apart from its cytotoxic effects, γterpinene activities through interactions with the cell membrane can lead to loss of atp synthesis capacity required for setting the cell functions. 25 based on results of the one-way anova test on the those five groups of different concentrations with the incubation period of 24 hours, there was no significant difference between those five groups of different concentrations and the control group. this indicates that there was no difference in the size of c. albicans cells between those five groups of different concentrations with the incubation period of 24 hours and the control group. there was no significant change in the size of c. albicans cells after treated by the administration of the lemon rind essential oil at the concentrations of 1.56%, 1.37%, 1.17%, 0.98%, and 0.78% incubated for 24 hours in liquid sabouroth dextrose media. based on results of the one-way anova test on those five groups of different concentrations with the incubation period of 48 hours, there were significant differences between those five groups of different concentrations and the control group. this illustrates that there were significant differences in the size of c. albicans cells between those five groups of concentration with the incubation period of 48 hours and the control group. there was a significant change in the size of c. albicans cells after treated by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 4747prabajati, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 43–48 the administration of the lemon rind essential oil at the concentrations of 1.56%, 1.37%, 1.17%, 0.98%, and 0.78% incubated for 48 hours in liquid sabouroth dextrose media. based on the results of the one-way anova test showed that there were some groups with the concentrations of 1.37% and 1.56% had higher significant values than the other groups. this means that the essential oil of lemon rinds at the concentrations of 1.37% and 1.56% can cause a significant change in the cell size of c. albicans compared to the control group. the mean cell size of c. albicans in the control group was 6.96 µm, while the mean cell size of c. albicans in the group with the concentration of 1.56% was 10.91 µm and 10.63 µm in the group with the concentration of 1.37%. all the treatment groups with the incubation period of 48 hour had greater changes in the cell size of c. albicans than the control group, especially in the groups with the concentrations of 1.37% and 1.56%. the results of the observation using a sem showed that the cell size of c. albicans in the groups with the essential oil of lemon rinds changed into the larger ones. the surface of those c. albicans cells was rough because the cell wall was wrinkled, swollen, and stood out, indicating of damage to the cell wall of c. albicans due to swelling of organelles inside the cells, resulting in an increase in volume suppressing the cell wall. as a result, the cell wall cracked. some of the fungal cells were also scattered and did not form colonies. it means that the essential oil could make permeability change, triggering osmotic imbalance. thus, there were curves on the damaged cell wall. changes in the cell size occur during the process of necrosis indicate the cell necrosis process occurs slowly and involves damage to the cell wall.26 the use of a sem in this research was to illustrate dimensional effects of the essential oil of lemon rinds against c. albicans cells. c. albicans cells experienced stiffness, and then clump together before they were totally destroyed by the essential oil. the cell walls became thick after the treatment due to the accumulation of membrane components surrounding the cell wall surface. this cell wall thickening can also be caused by increased leaks of amino acid transmembrane and other cytoplasmic components, while the peripheral cytoplasm becomes porous looked as indentations, so lowering the density of the cytoplasm, indicating necrosis.27 the use of incubation period variables, 24 hours and 48 hours, aimed to determine the effective time required by the essential oil of lemon rinds to affect c. albicans. incubation period can affect fungicidal activities of antifungal compounds against c. albicans. 28 based on results of the observations on the necrosis process of c. albicans using a sem, there was no significant difference in the size of c. albicans cells between the five groups of different concentrations (1.56%, 1.37%, 1.17%, 0.98%, and 0.78%) with the incubation period of 24 hours and the control group. this means that the essential oil of lemon rinds with the incubation period of 24 hours didnot demonstrate antifungal activities against c. albicans. the results of the observations on the necrosis process of c. albicans using a sem also showed that there were morphological differences between the five groups of different concentrations (1.56%, 1.37%, 1.17%, 0.98%, and 0.78%) with the incubation period of 24 hours and the control group. in the control group, the cells of c. albicans appeared round and smooth, as well as in the form of colonies. meanwhile, in the five groups of different concentrations (1.56%, 1.37%, 1.17%, 0.98%, and 0.78%), the cells of c. albicans did not appear in colonies or round, but seemed rough or shriveled, and dispersed without forming any colonies. such changes in the morphology of c. albicans cells that looked rough and shriveled were actually related to cellular physiological stress triggered by anti-fungal compounds, resulting in apoptosis process.14 there were differences in the size of c. albicans cells between the five groups of different concentrations (1.56%, 1.37%, 1.17%, 0.98%, and 0.78%) with the incubation period of 48 hours and the control group. results of the lsd test reveal that the significant differences in the size of c. albicans cells were found between the control group and the groups with the concentrations of 1.56% and 1.37% compared to the other groups with the concentrations of 1.17%, 0.98%, and 0.78%. this indicates that the essential oil of lemon rinds with the incubation period of 48 hours could trigger a significant necrosis process against c. albicans, mainly on the groups with the concentrations of 1.56% and 1.37%. it can be concluded that the essential oil of lemon rinds at concentrations of 1.56% and 1.37% with an incubation period of 48 hours can trigger cytomorphometric changes, especially changes in the morphology and size of c. albicans cells (characterized by necrosis). references 1. siar ch, ng kh, rasool s, ram s, abdul jalil a, ng kp. oral candidosis in non-hodgkin‟s lymphoma: a case report. j oral sci 2003; 45(3): 161-4. 2. okonkwo ec, alo mn, nworie o, orji jo, agah mv. prevalence of oral candida albicans infection in hiv sero-positive patients in abakaliki. american journal of life sciences 2013; 1(2): 72-6. 3. li l, redding s, bagtzoglou ad. candida glabrata, an emerging oral opportunistic pathogen. j dent res 2007; 86: 204-15. 4. meurman jh, siikala e, richardson md, rautemaa r. non-candida albicans, candida yeast of the oral cavity. in: mendez-villas a. communicating current research and educational topics and trends in applied microbiology. manchester: formatex publishing; 2007. p. 719-31. 5. leite mca, bezerra apd, sousa jp, guerra fqs, lima eo. evaluation of antifungal activity and mechanism os action of citral against candida albicans. hindawi pub. co; 2014. p. 1-9. 6. baktir a, masfufatun, hanum gr, karsono p. construction and characterization of the intestinal biofilm model of candida spp. research journal of pharmaceutical, biological and chemical sciences 2014; 5(1): 204-11. 7. ulfah, m. 2007,‘minyak esensial alternatif pengganti antibiotika’, (online), http://netfarm.blogsome.com /2007/10/01/antibiotikdanruminansia/. diakses tanggal6 april 2014. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 48 prabajati, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 43–48 8. katzer, g 2002,‘lime (citrus)’. online. http://wwwang.kfunigraz. ac.at/~katzer/engl/citr_aur.html. 4p. diakses 6 april 2014jam 09.00 wib. 9. chee hy, kim h, lee mh. antifungal activity of limonene against tricophytonrubrum. journal of microbiology 2009; 37(3): 243-6. 10. hamid aa, delvi f, rizqi pp. uji efek antifungi ekstrak kulit lemon (citrus limon l.) secara in vitro. jurnal penelitian fakultas kedokteran universitas brawijaya 2012; 1-15. 11. andres mt, monica vd, jose ff. human lactoferin induces apoptosis-like cell death in candida albicans: critical role of k+ -channel-mediated k+ eff luxv. journal antimicrob agents chemother 2008; 52(11): 4081-8. 12. dai bd, cao yy, huang s, xu yg, gao ph, wang y, jiang yy. baicelain induces programmed cell death in candida albicans. j microbiol biotechnol 2009; 19(8): 803-9. 13. eisenberg t, gutierrez dc, buttner s, tavernarakis n, madeo f. necrosis in yeast. usa: springer; 2010. p. 257-68. 14. kim ks, kim ys, han i, kim mh, jung mh, park hk. quantitative and qualitative analyses of cell death process in candida albicans treated by antifungal agents. plosone 2011; 6(12): 1-6. 15. hao b, cheng s, clancy cj, nguyen mh. caspofungin kills candida albicans by causing both cellular apoptosis and necrosis. antimicrob agents chemother 2013; 57(1): 326-32. 16. lee ja, chee hy. in vitro antifungal activity of equol against candida albicans. mycobiology 2010; 38(4): 328-30. 17. sujarweni vw. spss untuk paramedis. gava media; 2012. p. 31-44, 141-66. 18. ridawati, jenie bsl, djuwita i, sjamsuridzal w. aktivitas antifungal minyak atsiri jinten putih terhadap candida parapsilosis ss25, c. orthopsilosis nn14, c. metapsilosis mp27 dan c. etchellsi mp18. makara sains 2011; 15(1): 58-62. 19. zohra hf, rachida a, malika m, benalli s, samir aa, meriem b. chemical composition and antifungal activity of essential oils of algerian citrus. african journal of biotechnology 2015; 14(2): 1048-1055. 20. w i l k i n s m r , w id m e r w w, g r o h m a n k . si m u lt a n e o u s saccha r i f icat ion a nd fer ment at ion of cit r us pel l waste by saccharomyces cerevisiae to product ethanol. process biochemistry 2007; 42: 1614-9. 21. hafedh h, fethi ba, mejdi s, emira n, amina b. effect of mentha longifolia l. essential oil on the morphology visualized by atomic force miceroscopy. african journal of microbiology research 2010; 4(11): 1122-7. 22. parveen m, hasan k, takahashi j, murata y, kitagawa e, kodama o, iwahashi h. response sacharomyces cerevisiae to a monoterpene evaluation of antfungal potential by dna microarray analysis. journal of antimicrobial chemotherapy 2004; 54: 46-55. 23. rajkowska k, styczynska ak, maroszynska m, dabrowska m. the effect of thyme and tea tree oils on morphology and metabolism of candida albicans. acta biochimica polonica 2014; 61(2): 305-10. 24. cox sd, mann cm, ma rk ham j l, bell hc, gustafson j e, warmington jr, wyllie sg. the mode of antimicrobial action of the essential oils of melaleuca alternifolia. j appl microbiol 2000; 88: 170-5. 25. custodio jba, ribeiro mv, silva fsg, machado m, sousa mc. the essential oils component p-cymene induced proton leak through foatp synthase and uncoupling of mitochondrial respiration. j exp pharmacol 2011; 3: 69-76. 26. staniszewska m, bondaryk m, kopec es, siennicka k, sygitowicz g, kurzatkowski w. candida albicans morphologies revealed by scanning electron microscopy analysis. brazillian journal of microbiology 2013; 44(3): 813-21. 27. widodo gp, sukandar ey, adnyana ik, sukrasno. mechanism of action of coumarin against candida albicans by sem /tem analysis. itb j sci 2012; 44(2): 145-51. 28. clancy cj, huang h, cheng s, derendorf h, nguyen mh. characterizing the effects of caspofungin on candida albicans, candida parapsilosis, and candida glabrata isolates by simultaneous time-kill and postantifungal-effect experiments. j antimicrobial agents and chemotherapy 2006; 5(7): 2569-72. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p43-48 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p43-48 vol 50 no 4 desember 2017.indd 194 research report dental journal (majalah kedokteran gigi) 2017 december; 50(4): 194–198 the effects of anadara granosa shell-stichopus hermanni on bfgf expressions and blood vessel counts in the bone defect healing process of wistar rats rima parwati sari,1 sri agus sudjarwo,2 retno pudji rahayu,3 widyasri prananingrum,4 syamsulina revianti,1 hansen kurniawan,5 and aisah faiz bachmid1 1department of oral biology, faculty of dentistry, universitas hang tuah 2department of pharmacology, faculty of veterinary medicine, universitas airlangga 3department of oral pathology and maxilofacial, faculty of dental medicine, universitas airlangga 4department of dental engineering and materials science, faculty of dentistry, universitas hang tuah 5department of periodontics, faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: bone damage can be caused by various factors with treatment usually involving graft materials being applied to the defective area. moreover, in the bone defect healing process, blood vessels are also considered to be an important energy source for cell proliferation. one of the angiogenic factors playing an important role in blood vessel formation is basic fibroblast growth factor (bfgf). furthermore, synthesized hydroxyapatite derived from anadara granosa (ag) shells constitutes one of the potential materials for use in bone graft. the gold sea cucumber genus stichopus hermanni (sh) possesses the ability to stimulate endothelial progenitor cells inducing bfgf. purpose: this study aims to investigate the effects of ag shells and sh on bfgf expressions and blood vessel counts within the bone healing process. methods: twenty four male wistar rats were divided into three groups, namely: a control group (c), a treatment group was administered with blood cockle shell (ag), and a treatment group with blood cockle shell and golden sea cucumber (ag+sh). defects were made on their femurs measuring half the diameter of a circular, no. 018. bur these rats were subsequently sacrificed on day 7 after surgery. the expressions of bfgf were measured by means of ihc technique, while the number of blood vessels was quantified using he technique. the resulting data was subjected to statistical analysis using an anova test followed by an lsd test (p < 0.05). results: the one-way anova test results combined with those of an lsd test showed there to be significant differences in bfgf expressions and blood vessel counts between the control group (k) and the treatment group (ag) as well as between the treatment group (ag) and the treatment group (ag+sh). conclusions: a combination of anadara granosa shell and stichopus hermanni can increase the expression of bfgf and the number of blood vessels on day 7 during the bone healing process in wistar rats. keywords: anadara granosa shell; stichopus hermanni; basic fibroblast growth factor; blood vessel; bone healing correspondence: rima parwati sari, department of oral biology, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: rimaparwatisari@gmail.com. introduction bone damage or bone defects can be caused by congenital factors, trauma, infection, or jaw tumors.1 when bone damage occurs, a healing process consisting of three stages, namely an inflammatory phase, a reparation phase and a maturation phase2 will ensue. in the early stages, blood vessel vasoconstriction occurs followed by a hemostasis phase and one of inflammatory cell infiltration. a blood clot progressively filling the defective area will be replaced by granulated tissue. thereafter, the fiber remains will diffuse into the bone leading to a temporary matrix derived from the blood clot being formed. granulation tissue will also subsequently be formed. at the end of this dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p194–198 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p194-198 195195sari, et al./dent. j. (majalah kedokteran gigi) 2017 desember; 50(4): 194–198 process, a remodeling phase characterized by tissue and collagen remodeling, epidermal maturation and wound shrinkage occurs.3,4 another important factor accelerating the healing process of damaged bone significant in size and volume is new vascularization tissue formation on the bone.5 the formation of bone vascularization is triggered by an angiogenesis process together with several other factors. one which plays an important role in the formation of bone vascularization is basic fibroblast growth factor (bfgf).6 bfgf acts as an intermediary between the formation of vascularization in new bone and the occurrence of chondrocyte and osteoblast differentiations since it appears in the early phase of proliferation during the bone healing process.6,7 bfgf is also considered to be one of the growth factors possessing the ability to induce all stages required for angiogenesis and is involved in both wound repair and tissue development.8 in tissue healing, bfgf is regarded as a growth factor that plays a role in stimulating endothelial proliferation, migration and blood vessel formation. together with platelet-derived growth factor (pdgf) and vascular endothelial growth factor (vegf), bfgf synergizes to neutralize tissue.9 as medical science develops, therapeutic modalities for the reconstruction of bone defects become increasingly available. such modalities include local bone transport, bone elongation or reduction and bone graft.5 initially, the bone graft used was taken from the bones of the individual itself, but this can result in damage to other areas of the body.10 this situation then prompted efforts to develop bone graft from other natural sources, such as anadara granosa (ag) shells. ag shells have a calcium carbonate (caco3) content of more than 95%.11 in reality, caco3 also has the potential to act as bone substitute material. however, bone structure and composition consist of hydroxyapatite (hap). therefore, caco3 material must be converted into hap structure through a calcification process in the body. as a result, this research utilised hap synthesized from ag shells as bone graft material through a precipitation process to restore damaged bone.12 hap, an inorganic salt contained in bone, has a chemical synthesis form similar to that of bone, non-immunogenic properties and a stable crystal form.13 in addition, hap is non-degradable, a quality required by graft materials. during the healing process in bone, hydroxyapatite releases calcium phosphate, thus increasing the saturation of body fluids and precipitating the biological apatite of the body in the area of the defect. the biological apatite contains endogenous proteins and acts as a matrix for the attachment and growth of osteogenic cells.14 consequently, when a bone graft is carried out, the bone will undergo bone-repair phases involving hemostasis, inflammation, proliferation, revascularization, soft callus and hard callus formation and, finally, a remodeling phase.15 the use of hap in bone graft can also stimulate macrophages in the area of defects enabling the production of cytokines that stimulate growth factors, including fgf. in order to promote more rapid bone repair significantly greater in both size and volume, new vascularization must occur in the bone.5 the new vascularization takes place through a process of angiogenesis triggered by several factors, one of which is bfgf.15 bfgf acts as an intermediate between new bone vascularization and chondrocyte and osteoblast differentiations since it emerges in the early phase of proliferation during the bone healing process.6,7 therefore, the healing process of bone damage will be accelerated through the addition of osteoinductive materials, one of which can be derived from marine biota, such as sea cucumbers whose osteoinductive properties can even convert cells in the graft into osteoblast cells composing the bone.16 sea cucumbers (stichopus hermanni) are animals with high economic and nutritional value which also offer bioactive contents, such as glycosaminoglycans (gags), protein and polyunsaturated fatty acid (pufa). the gag content of sea cucumbers has positive effects on the healing of wounds. meanwhile, sulfate chondroitin acts as a bfgf mediator in order to bind the bfgf. thus, the more optimal the sulfate chondroitin, the more it may be assumed to increase the expressions of bfgf involved in the regulation of the angiogenesis process.17 in addition, another content of stichopus hermanni (sh), pufa, consists of arachidonic acid, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) which are useful in the bone healing process.18 the research reported here aims to determine the effects of anadara granosa shells and stichopus hermanni on bfgf expressions and blood vessel counts in the repair of damaged bone. materials and methods this research was fully experimental incorporating a completely randomized design whose samples consisted of twenty-four 5-month old male rats (rattus novergicus) weighing 200–250 grams. the samples were then divided into three groups (n = 8), namely: a negative control group (c) which received no treatment, a treatment group with the administration of ag shells (ag group) and a treatment group administered with ag shells and 10% sh (ag + sh group). preparation of this research was initiated with the manufacture of graft derived from ag shells synthesized into hap powder through calcination, precipitation and sintering processes. sterilization was achieved using an ultraviolet ray unit (a10-uv-30, cleatech, usa). on the other hand, graft material used with another treatment group was made by mixing ag powder and sh powder through a freeze-dried procedure. a milling process incorporating the use of high energy milling elips 3d motion (hem-e3d) by nanotech® indonesia was subsequently carried out in order to obtain micro-sized results.19 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p194–198 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p194-198 196 sari, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 194–198 the wistar rats were acclimatized for seven days, before their femurs were defected/were made defect. they were anesthetized by the intramuscular administering of an 0.11 ml/100 gr bw dose of ketamine and xylazine.20 once the rats had lost consciousness, the fur in the dextral area of the femur to be defaced was shaved off. thereafter, the exposed skin was smeared with antiseptic (10% povidine iodine) for five minutes before a 2 cm-long incision was made with a scalpel in the soft tissues (skin and muscle) and lifted using a periosteal elevator. a defect was made on the dextral and lateral areas of the femoral bone (5 mm from the third trochanter)15 with a straight hand piece, equivalent in depth to half the diameter of a circular bur (mcisinger® germany size 018). after the defect had been created in each rat, the application of the treatment material was conducted according to the division of the group. a pericardium membrane was applied (from the tissue bank at dr. sutomo hospital surabaya). sutures were then used to close the skin and soft tissues by means of cat gut (usp. 3/0) and silk braid (usp. 3/0) both manufactured by dr. sella®. after surgery, the animals received a 0.09 cc/200 gr bw dose of novalgin and a 0.1 cc/100 gr bw dose of interflox in order to control infection and swelling. seven days later, the rats were sacrificed, and their os femur taken to make preparations using separating discs. the discs were then soaked in a 10% formaldehyde buffer solution to prevent the tissues from decomposing, to harden them, to increase the refractive index of various tissue components and to improve the affinity of the tissues against the staining materials used. after completion of tissue fixation, a decalcification process of two months’ duration was performed using ethylenediaminetetraacetic acid (edta). the os femur specimens were subsequently prepared in the form of sagittable piece preparations with he and ihc staining techniques using polyclonal antibodies (anti-bfgf (basic fibroblast growth factor), bs-0217r, by bioss®, massachusetts-usa). the number of blood vessels in the defective areas was then observed by means of a light microscope (olympus® cx21, japan) at 400× magnification. data relating to the osteoblast counts obtained in each group was tabulated. the data was then analyzed statistically by means of a one-way anova parametric test followed by an lsd test. results the observation findings relating to bfgf expressions and the number of blood vessels were evaluated on the seventh day. based on the anova test results, there was a significant difference in the mean of bfgf expressions and blood vessel counts (p < 0.05). the mean of bfgf expressions indicating the positive reaction of fibroblast cells in the negative control group (c) stood at 11.33 ± 2.42, 13 ± 2.37 for the treatment group following the administration of ag, and 32.50 ± 4.14 for the treatment group following that of ag + sh. on the other hand, the mean of blood vessel counts in the negative control group (c) was 6.68 ± 0.82, 10.25 ± 0.38 for the treatment group with the administration ag, and 20.37 ± 0.55 for the treatment group with the administration of ag + sh. blood vessels observed in the course of the research reported here constituted the formation of a lumen surrounded by a layer of endothelial cells seen in the area of the femur os that was defective (figure 1 and 2). figure 2. hpa images of bfgf expressions (red arrows) and blood vessel counts (black arrows) in each group. clinically, the defects in the ag group were slightly closed, and the process of bone formation appeared to have initiated, but not optimally. the lsd test results indicated a significant difference between the ag group and the c group. on the other hand, the defects in the ag+sh group, from a clinical perspective, demonstrated a greater degree of closure, indicating a more optimal healing process. this was supported by the results of the lsd test revealing that there was a significant difference between the ag+sh group as well as both the c group and the ag group (table 1). figure 1. the mean of bfgf expressions and blood vessel counts in each group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p194–198 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p194-198 197197sari, et al./dent. j. (majalah kedokteran gigi) 2017 desember; 50(4): 194–198 discussion angiogenesis is an important stage of the proliferative phase in the bone healing process. during this phase, the endothelial cells migrate to new tissues and undergo proliferation. the new endothelial cells will then attach to each other to form new tubular vessel structures. the interaction between the endothelial cells and the extracellular matrix components plays an important role in regulating the formation of new blood vessels.21 during the normal healing process, angiogenesis initiation usually begins with the local release of both pro and anti-angiogenic growth factors by endothelial cells. this release occurs in response to inflammation caused by injury-inducing inflammation and accumulation of hypoxia-inducible factor-1a (hif-1a) to hypoxia.22 under these conditions of hypoxia, the endothelial cells will trigger bfgf, resulting in micro-vascular growth. bfgf will subsequently begin to produce mature endothelial cells in order to synthesize further new blood vessels. fibroblast growth factor (fgf) and vegf then bind to receptors on the cell surface complemented by tyrosine kinase activity. the activation of the kinase receptor enables the incorporation of signal transduction pathways regulating the proliferation, migration and differentiation of endothelial cells.23 this suggests that during the normal bone healing processes, bfgf expressions remain visible because of the hypoxic conditions of tissue damage (bone). similarly, in the c group, the number of bfgf expressions remained visible without any external treatments (figure 1). moreover, the hap applications studied through this research aimed to serve as a framework or defect filler matrix. hap has insufficient connecting mineral cavities leading to imperfect degradation results. these large, thin cavities then allow bfgf to form new blood vessels (scaffolds). hap and bfgf are influential in the migration process of mesenchyme cell progenitors, including endothelial cell progenitors and also in the stimulation of macrophage cells in the defective areas.24,25 similarly, the expressions of bfgf and the number of blood vessels in the ag group differed significantly from those in the c group. f u r t h e r m o r e , o p t i m a l o s t e o c o n d u c t i o n a n d osteoinduction processes are also essential to the healing of damaged bone. considerable current research on bone substitute materials has, therefore, tried to combine both properties of osteogenesis with a polymer material. 26-29 one of the various polymeric materials used in performing bone grafts is gag. gag consists of sulfate and non-sulfate compounds. sulfate and heparin sulfate are categorized into gag sulfate compounds that can have positive effects on the wound healing process since chondroitin sulfate and heparin sulfate can bind bfgf.17 gag content is found in the flesh of sh.30 on the other hand, hyaluronic acid categorized into non-sulfate compounds of gag plays a complex role in cell adhesion, cell proliferation and cell movement. variations in cell responses induced by hyaluronic acid are proliferation, migration and cytokine synthesis mediated by the determinant molecule-44 cluster (cd44) presented on the cell surface. hyaluronic acid also interacts with a receptor for hyaluronan-mediated motility (rhamm) located on the cell surface to initiate endothelial cell migration.31 the rhamm ligand interaction mainly occurs in endothelial cell motility and interaction with cd44 ligand triggering endothelial cell proliferation. these two receptors then work together to facilitate the formation of new blood vessels.32 the cd44 involvement of depolymerized hyaluronic acid subsequently leads to endogenous bfgf release which, in turn, stimulates the proliferation and growth of new blood vessels.33 in addition to gag, sh also contains high levels of protein and amino acids which play an important role in the inflammatory phase by increasing the immune response and modulating the inflammation to immediately enter the process of bone healing (reparative).34 moreover, sea cucumbers also contain pufa which is useful for mediating control inflammation and regulating cell proliferation, including endothelial cells in the formation of new blood vessels.35 similarly, in the ag+sh group, bfgf expressions and blood vessel counts were significantly different from those in both the c group and the ag group. finally, it can be concluded that the combination of anadara granosa shells and stichopus hermanni used as bone substitute can effectively increase the expressions table 1. results of the post-hoc lsd test for osteoblast dependent variables group (n = 8) ag (group administered anadara granosa paste only) ag+sh (group administered a combination anadara granosa and stichopus hermanni paste) bfgf c (control) 0.411 0.000* ag (anadara granosa paste only) – 0.000* blood vessels c (control) 0.234 0.000* ag (anadara granosa paste only) – 0.001* note: * significant difference if p < 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p194–198 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p194-198 198 sari, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 194–198 of bfgf and the number of blood vessels in the healing of bone damage in wistar rats. references 1. ferdiansyah, rushadi d, rantam fa, aulani’am. regenerasi pada massive bone defect dengan bovine hydroxyapatite sebagai scaffold mesenchymal stem cell. jbp. 2011; 13(3): 179–95. 2. sathyendra v, darowish m. basic science of bone healing. hand clin. 2013: 29: 473–81. 3. la r java h. ora l wound hea ling : cell biology a nd clin ica l management. west sussex: wiley-blackwell; 2012. p. 195–9. 4. lieberman jr, friedlaender ge. bone regeneration and repair: biology and clinical applications. new jersey: humana press; 2005. p. 21–44. 5. zorzi a, de miranda jb. bone grafting. london: intech; 2012. p. 11–38. 6. zhang x, awad ha, o’keefe rj, guldberg re, schwarz em. a perspective: engineering periosteum for structural bone graft healing. clin orthop relat res. 2008; 466(8): 1777–87. 7. athanasopoulos an, schneider d, keiper t, alt v, pendurthi ur, liegibel um, sommer u, nawroth pp, kasperk c, chavakis t. vascular endothelial growth factor (vegf)-induced up-regulation of ccn1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing. j biol chem. 2007; 282(37): 26746–53. 8. mitchell rn, kumar v, abbas ak, fausto n. robbins & cotran buku saku dasar patologis penyakit. 7th ed. jakarta: egc; 2009. p. 62–4. 9. davies j. tissue regeneration from basic biology to clinical application. rijeka: intech; 2012. p. 94–110. 10. keating jf, mcqueen mm. substitutes for autologous bone graft in orthopaedic trauma. j bone joint surg br. 2001; 83-b(1): 3–8. 11. rusnah m, reusmaazran mmy, yusof a. hydroxyapatite from cockle shell as a potential biomaterial for bone graft. regen res. 2014; 3: 52–5. 12. azis y, jamarun n, zultiniar z, arief s, nur h. synthesis of hydroxyapatite by hydrothermal method from cockle shell (anadara granosa). j chem pharm res. 2015; 7: 798–804. 13. tarlton jf, wilkins lj, toscano mj, avery nc, knott l. reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets. bone. 2013; 52: 578–86. 14. mao t, kamakshi v. bone grafts and bone substitutes. int j pharm pharm sci. 2014; 6: 88–91. 15. kopschina mi, marinowic dr, klein cp, araujo ca, freitas ta, hoff g, da silva jb. effect of bone marrow mononuclear cells plus platelet-rich plasma in femoral bone repair model in rats. braz j vet res anim sci. 2012; 49(3): 179–84. 16. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 11th ed. st. louis: elsevier saunders; 2011. p. 824. 17. hendrawan rd, rahayu rp, budhy ti, istiati i. the application of sea cucumber (stichopus hermanni) extract to improve expression fibroblast growth factor-2 (fgf-2), fibroblast cells, and capillary blood vessels over wound healing (cavia cobaya). oral maxillofac pathol j. 2014; 1(1): 7–12. 18. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods--a review. mar drugs. 2011; 9(10): 1761–805. 19. balá p. mechanochemistry in nanoscience and minerals engineering. berlin: springer; 2010. p. 103–32. 20. tanideh n, nazhvani ds, jaberi fm, mehrabani d, rezazadeh s, pakbaz s, tamadon a, nikahval b. the healing effect of bioglue in articular cartilage defect of femoral condyle in experimental rabbit model. iran red crescent med j. 2011; 13(9): 629–36. 21. turley ea, noble pw, bourguignon lyw. signaling properties of hyaluronan receptors. j biol chem. 2002; 277(7): 4589–92. 22. kyzas pa, stefanou d, batistatou a, agnantis nj. hypoxia-induced tumor angiogenic pathway in head and neck cancer: an in vivo study. cancer lett. 2005; 225: 297–304. 23. yin s, ellis de. first-principles investigations of ti-substituted hydroxyapatite electronic structure. phys chem chem phys. 2010; 12: 156–63. 24. bargowo l, ulfah n, putri akn. angiogenesis effect of bone remodeling process due to hydroxyapatite-chitosan natural powder applications in concentration 30:70 and 70:30. periodontic j. 2013; 5(1): 19–25. 25. wu yl. interaction of bone cells with biomimetic hydroxyapatite gelatin nanocomposites towards developing bone tissue engineering. thesis. minnesota: university of minnesota; 2007. p. 1–24. 26. przekora a, palka k, ginalska g. biomedical potential of chitosan/ ha and chitosan/β-1,3-glucan/ha biomaterials as scaffolds for bone regeneration--a comparative study. mater sci eng c. 2016; 58: 891–9. 27. razali kr, nasir nfm, cheng em, mamat n, mazalan m, wahab y, roslan mrm. the effect of gelatin and hydroxyapatite ratios on the scaffolds’ porosity and mechanical properties. in ieee conference of biomedical engineering and sciences (iecbes). kuala lumpur; 2014. p. 256–9. 28 normahira m, raimi rk, fazli mnn, norazian ar, adilah h. biomimetic porosity of gelatin-hydroxyapatite scaffold for bone tissue. adv mater res. 2014; 970: 3–6. 29. mohamed kr, beherei hh, el-rashidy zm. in vitro study of nanohydroxyapatite/chitosan–gelatin composites for bio-applications. j adv res. 2014; 5: 201–8. 30. karnila r. pemanfaatan komponen bioaktif teripang dalam bidang kesehatan. thesis. pekanbaru: university of riau; 2011. p. 100–4. 31. gao f, yang cx, mo w, liu yw, he yq. hyaluronan oligosaccharides are potential stimulators to angiogenesis via rhamm mediated signal pathway in wound healing. clin invest med. 2008; 31(3): e106–16. 32. savani rc, cao g, pooler pm, zaman a, zhou z, delisser hm. differential involvement of the hyaluronan (ha) receptors cd44 and receptor for ha-mediated motility in endothelial cell function and angiogenesis. j biol chem. 2001; 276(39): 36770–8. 33. pardue el, ibrahim s, ramamurthi a. role of hyaluronan in angiogenesis and its utility to angiogenic tissue engineering. organogenesis. 2008; 4(4): 203–14. 34. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219–29. 35. sari rp, wahjuningsih e, soeweondo ik. modulation of fgf2 after topical application of stichopus hermanni gel on traumatic ulcer in wistar rats. dent j (maj ked gigi). 2014; 47(3): 126–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p194–198 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p194-198 126 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 126–129 comparison between probiotic lozenges and drinks towards periodontal status improvement of orthodontic patients natasia melita kohar, victor emmanuel, and luki astuti department of periodontic faculty of dentistry, universitas trisakti jakarta indonesia abstract background: fixed orthodontic appliances may interfere with daily oral hygiene procedure, causing more abundant plaque accumulation, therefore increasing the risk of periodontal disease. probiotic methods represent a breakthrough approach in maintaining oral health and preventing periodontal disease. purpose: the aim of this study was to compare the effect between probiotic lozenges containing lactobacillus reuteri and probiotic drinks containing lactobacillus casei strain shirota towards periodontal status of orthodontic patients. method: fixed orthodontic patients (n=30) from faculty of dentistry, trisakti university dental hospital were included in this clinical trial. periodontal status consisting of plaque index (pli), interdental hygiene index (hyg), and papillary bleeding index (pbi) were then recorded from each patient. all patients received the phase one of periodontal treatment, as well as plaque control instruction. the subjects (n=10/gp) were randomly assigned to one of three groups; control group; probiotic lozenges group (biogaia®); and probiotic drinks group (yakult®). for 14 days, the probiotic groups were instructed to use the probiotic. periodontal index improvement (pli, hyg, and pbi) was found in all groups after 14 days research periode. these indices were then analyzed using kruskal-wallis analysis test. result: it was found that l. reuteri and l. casei strain shirota may improve periodontal status in fixed orthodontic patients. the best results were obtained from probiotic lozenges group. however, the results were not statistically significant (p>0.05). conclusion: it was concluded that probiotics consumption containing l. reuteri and l. casei strain shirota may slightly improve periodontal status in fixed orthodontic patients. keywords: probiotics; lactobacillus reuteri; lactobacillus casei strain shirota; orthodontic correspondence: natasia melita kohar, c/o: departemen periodonsia, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa no. 260, grogol, jakarta 11440, indonesia. e-mail: natasiakohar@gmail.com introduction malocclusion is one of the most common oral health problems. orthodontic treatment may correct malocclusion or prevent them from progressing, but it also holds some potential harm to teeth and periodontal tissues.1 orthodontic brackets and elastics may interfere daily oral hygiene procedure which may lead to plaque accumulation and gingival inflammation.2 plaque retention surrounding orthodontic appliances leads to enamel demineralization caused by organic acids produced by bacteria in the dental plaque.3 this leads to oral cavity changing of orthodontic patients, such as ph reduction, developed larger number of sites available for streptococcus mutans accumulation, and increased accumulation of food particles.4 the changes in the microbial environment after the placement of fixed orthodontic appliances are accompanied by increased gingivitis.5 to prevent harmful risks of orthodontic treatment on periodontal and gingival tissues, better oral hygiene programs during orthodontic treatment were strongly recommended.3 recently, a new method in dentistry is found to preserve oral health, known as probiotics.6 probiotics are defined as living microorganisms which when administered in adequate amounts (in food or as a dietary supplement) confer a health benefit on the host.7 probiotics use in oral health is still a novel method.8 probiotic bacteria maintaining oral health by competing against oral pathogens dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p126-129 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p126-129 127127kohar, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 126–129 for nutrients, growth factors and site of adhesion. once adhered to the oral cavity, probiotic bacteria aggregate and inhibit the adhesion of the harmful microorganisms by producing bacteriocins or other antimicrobial compounds such as acids or peroxides. consequently, probiotics help to prevent the inflammation of oral cavity and the oral tissue destruction by oral pathogens.9 probiotic methods represent a breakthrough approach in maintaining oral health and preventing periodontal disease by using beneficial bacteria, specifically lactic acid bacteria, such as lactobacillus and bifidobacterium.6 probiotics are administered in some products, such as beverages and food, dairy products, or dietary supplements.10 probiotic lozenges containing lactobacillus reuteri (l. reuteri) is one of the edible probiotic products. krasse et al.11 suggested that l. reuteri was effective in reducing both gingivitis and plaque in patients with moderate to severe gingivitis. vivekananda et al.12 reported about plaque inhibition, anti-inflammatory, and antimicrobial effects of probiotic lozenges containing l. reuteri. it can be recommended during non-surgical therapy and maintenance phase of periodontal treatment. the other product that also has the beneficial effect towards oral health is probiotic drink containing lactobacillus casei. a recent study by slawik et al.13 showed that daily consumption of a probiotic milk drink containing l. casei strain shirota (lcs) reduce the effects of plaque-induced gingival inflammation. either probiotic lozenges or probiotic drinks are known for their beneficial effect towards periodontal problems, including inhibition of plaque formation. however, there was no existing research that compares directly the effectiveness between both products. the aim of this study was to compare the effect between probiotic lozenges containing l. reuteri and probiotic drinks containing lcs towards periodontal status of fixed orthodontic patients. materials and methods the subject of the research consists of 30 healthy non-medicating orthodontic patients, with the age range from 18-25 years, who had been undergoing orthodontic treatment used fixed orthodontic appliances for at least 1 year. the subjects volunteered after verbal and written information. all subjects were outpatients at faculty of dentistry trisakti university dental hospital and had signed an informed consent form. habitual consumers of xylitol chewing gums and mouthwash, smokers, pregnant women, and subjects with systemic antibiotic or topical fluoride treatments were excluded from this study. this clinical trial was approved by the ethics committee of trisakti university, jakarta, indonesia. all patients were received the first phase of periodontal treatment, including scaling and root planning, as well as plaque control instruction. the subjects (n=10/gp) were randomly assigned to one of three groups; (i) control group; (ii) probiotic lozenges group (biogaia®); and (iii) probiotic drinks group (yakult®). the periodontal status, such as plaque index (pli), interdental hygiene index (hyg), and papillary bleeding index (pbi) was measured before and after consuming both probiotic products for 14 days. the probiotic lozenges (biogaia prodentis®, sweden) contained l. reuteri prodentis. one lozenge consists of a minimum of 200 million live l. reuteri prodentis. the subjects were instructed to consume 1 lozenge per day at least 1 hour after lunchtime. the other study product was the fermented milk probiotic drink (yakult®, japan), contained lcs. each 65 ml bottle of the fermented milk drink contained a minimally of 6.5 million viable cells of lcs. subjects were advised to consume a bottle per day, slowly taking small sips through a thin straw (3 mm in diameter) at least 1 hour after lunchtime. moreover, the participants were advised to keep their supply refrigerated, in order to preserve the viability of the probiotic bacteria. a leaflet containing information to store the product correctly and how to brush their teeth regularly with bass technique was also provided to the participants. preand post-treatment values are counted by consuming probiotic products within each parameter (pli, hyg, pbi) were compared and analyzed with nonparametric test (kruskal-wallis test). a value of (p<0.05) was considered statistically significant. results all clinical data were collected between octoberdecember 2014 at faculty of dentistry trisakti university dental hospital. primary data collected from examination of the periodontal status of 30 fixed orthodontic patients. as shown on table 1, the mean pli of control group were decreasing from 1.19 to 1.03 while the lozenges group were decreasing further from 1.22 to 0.91. probiotic drinks group were also decreasing from 1.01 to 0.67. the mean hyg of control group were increasing from 0.69 to 0.77, probiotic lozenges group were increasing from 0.64 to 0.79, and probiotic drinks group were increasing from 0.69 to 0.83. the mean pbi of control group were decreasing from 0.82 to 0.58, probiotic lozenges group were decreasing from 1.01 to 0.67, and probiotic drinks group were also decreasing from 0.49 to 0.43. the best results were obtained from probiotic lozenges group. data was analyzed using spss statistics. kruskalwallis test method was applied to the results, statistical analysis showed that there was no significant difference (p>0.05) between the study and control group. discussion treatment with fixed orthodontics appliances usually related to alterations in the oral hygiene and periodontal health. it may increased plaque accumulation and interferes with effective daily oral hygiene procedure. periodontal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p126-129 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p126-129 128 kohar, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 126–129 complications, such as gingivitis and gingival enlargement, are reported to be one of the most collateral damage related to orthodontics.14 plaque considered to be the main aetiological factor in the initial, development, and reoccurrence phase of periodontal disease.15 nowadays, a treatment method towards periodontal health such as probiotic has proposed to modify pathological plaque to biofilm of commensalisms. probiotics defined as a living microorganisms administered in adequate amounts with beneficial health effects on the host.16 probiotics tend to create a biofilm, acting as a defensive lining for oral tissues against oral disease. the mechanisms of probiotic action in the oral cavity are by keeping bacterial pathogens off of oral tissues and compeeting with cariogenic bacteria and periodontal pathogens growth in biofilm.17 fixed orthodontic appliance might render maintaining oral hygiene more difficult. due to these, the patients need to be effectively trained and motivated to achieve an adequate oral hygiene. this is done by placing the patients for regular check ups, improving their oral health during and until orthodontic treatment is finished. preventive procedures such as plaque control, motivation, compliance, and implementation could bring a great impact in maintaining gingival health during orthodontic treatment.18 twetman et al.19 concluded that short-term intake (14 days) of probiotics reduced the pro-inflammatory cytokines in gingival crevicular fluid and due to this reason it could decrease the inflammation in the oral cavity. in this study, both control and study group encounter improving on plaque index score, hygiene index, and bleeding index after 14 days. this may be due to effective plaque control that supports better oral hygiene and periodontal treatment, with or without probiotic consumption. it has been shown that orthodontic patients from the average age of 18-25 years had low risk on periodontal disease if the patients could perform effective plaque control and preserves their oral health optimally. the best improvement of periodontal status was obtained from probiotic lozenges group containing l. reuteri, followed by probiotic drinks group containing lcs and control group. probiotic lozenges showed better improvement among other groups probably because l. reuteri has 2 types of 2 bacteriocyn, such as reuterin and reutericyclin. it contains antibacterial substances that inhibit pathogens growth and anti-inflammatory that inhibit secretion of cytokine pro-inflammatory.20 both probiotic lozenges and probiotic drinks showed better improvement in result than control group. probiotic lozenges are known to reduce production of acid formation, prevent adhesion and invasion of pathogens, and furthermore inhibit the pathogen growth itself.6 probiotic drinks are considered to modulate immune system, lower local ph, inhibit pathogen bacteria, and could adhere on teeth surfaces.21 probiotics inhibit plaque accumulation through producing antioxidants. these antioxidants are able to utilize the free electron needed in mineralization process. this might be one of the probiotic mechanisms to prevent periodontal disease. lactobacillus species may suppress periodontal disease bacteria population growth. the presence of lactobacillus reduces plaque accumulation and gingival inflammation.9 this study requires consuming probiotic lozenges for 14 days based on a previous study by krasse et al.11 that reported 14 days of consuming probiotic chewing gum containing l. reuteri is significantly reduce plaque index and gingivitis severity level. in the present study the result shows reduction in plaque index score, improvement in hyg score, and reduction in pbi score, although not statistically significant. this might be due to the differences use of methods, subject quantity, and population. their study included 59 patients from moderate to severe gingivitis. consumption of probiotic lozenges for 42 days has beneficial effect towards nonsurgical therapy and table 1. mean (± standard deviation) of plaque index, hygiene index, and bleeding index outcome measures at baseline and 14 days on 3 treatment groups (n=30) parameter time point treatment group p-value control biogaia® yakult® srp srp+p1 srp+p2 mean ± sd mean ± sd mean ± sd plaque index baseline 1.19 ± 0.41 1.22 ± 0.46 1.01 ± 0.45 0.173 14 days 1.03 ± 0.29 0.91 ± 0.42 0.76 ± 0.48 hyg baseline 0.69 ± 0.17 0.64 ± 0.22 0.69 ± 0.24 0.196 14 days 0.77 ± 0.14 0.79 ± 0.12 0.83 ± 0.13 pbi baseline 0.82 ± 0.61 1.01 ± 0.62 0.49 ± 0.45 0.053 14 days 0.58 ± 0.36 0.67 ± 0.77 0.43 ± 0.54 explanation: pli= plaque index (silness and löe); hyg= interdental hygiene index; pbi= papillary bleeding index (saxer and mühlemann); srp= scaling and root planning; srp+p1= scaling and root planning + probiotic lozenges; srp+p2= scaling and root planning + probiotic drinks; p-value= (p>0.05) : not significant. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p126-129 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p126-129 129129kohar, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 126–129 maintenance phase in periodontal treatment. probiotic lozenges inhibit the population of periodontopathogen bacteria, such as aggregatibacter actinomycetemcomitans, prevotella intermedia, porphyromonas gingivalis, reduce plaque formation, and have anti inflammation and antimicrobial effect. all subjects received scaling and root planning treatment and probiotic lozenges on 30 chronic periodontitis patients included in the trials. they stated that it gave an effective improvement towards plaque index, gingival index, and papillary bleeding index. the difference in this result might be due to the population and time limit during study. twetman et al.19 mentioned that probiotic chewing gum containing l. reuteri has a positive contribution towards bleeding on probing on gingivitis patients. the study was participated by 42 gingivitis patiens. the subjects were instructed to chew the gum for 10 minutes for 14 days. probiotic drink consumption containing lcs for 14 days can improve plaque index, hygiene index, and bleeding index in all groups but the result was not statistically significant (p>0.05). this study is similar to the previous study by staab et al.22 that indicate the effectiveness of lcs towards periodontal index, such as plaque index and papillary bleeding index in 50 gingivitis patients. lcs may improve towards gingival health by reducing gingivitis severity level though the result was not statistically significant. according to sutula et al.23 on their in vitro studies using probiotic lcs showed an inhibition on the population of periodontopathogens bacteria such as p. gingivalis and fusobacterium nucleatum. it can be concluded that probiotic consumption containing l.reuteri and l.casei strain shirota may slighthy improve periodontal status in fixed orthodontic patients. references 1. lara-carrillo e, montiel-bastida n-m, sańchez-peŕez l, alani śtavira j. effect of orthodontic treatment on saliva, plaque and the levels of streptococcus mutans and lactobacillus. med oral patol oral cir bucal 2010; 15(6): 924-9. 2. alfuriji s, alhazmi n, alhamlan n, al-ehaideb a, alruwaithi m, alkatheeri n, geevarghese a. the effect of orthodontic therapy on periodontal health: a review of the literature. int j dent 2014; 2014: 1-8. 3. türkkahraman h, sayın mö, bozkurt fy, yetkin z, kaya s, önal s. archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. angle orthod 2005; 75(2): 231-6. 4. enita n, dzemidzic v, tiro a, pasic e, hadzic s. antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances. braz j oral sci 2011; 10(2): 79-82. 5. gastel jv, quirynen m, teughels w, carels c. the relationships between malocclusion, fixed orthodontic appliances and periodontal disease. a review of the literature. aust orthod j 2007; 23: 121-9. 6. gupta g. probiotics and periodontal health. j med life 2011; 4(4): 387-94. 7. organization faa, organization wh. probiotics in food health and nutritional properties and guidelines for evaluation: roma, 2006. 8. kamal r, dahiya p, kumar m, tomar v. probiotics in oral health – a new tool in pharmaceutical science. indian j pharm biol res 2013; 1(4): 168-73. 9. jain p, sharma p. probiotics and their efficacy in improving oral health: a review. j appl pharmac sci 2012; 2(11): 151-63. 10. caglar e, b kargu, tanboga i. bacteriotherapy and probiotics’ role on oral health. oral dis 2005; 11: 131-7. 11. krasse p, carlsson b, dahl c, paulsson a, nilsson å, sinkiewicz g. decreased gum bleeding and reduced gingivitis by the probiotic lactobacillus reuteri. swed dent j 2006; 30: 55-60. 12. vivekananda mr, vandana kl, bhat kg. effect of the probiotic lactobacilli reuteri (prodentis) in the management of periodontal disease: a preliminary randomized clinical trial. j oral microbiol 2010; 2: 5344. 13. slawik s, staufenbiel i, schilke r, nicksch s, weinspach k, stiesch m, eberhard j. probiotics affect the clinical inflammatory parameters of experimental gingivitis in humans. eur j clin nutr 2011; 65(7): 857-63. 14. boke f, gazioglu c, akkaya s, akkaya m. relationship between orthodontic treatment and gingival health: a retrospective study. eur j dent 2014; 8(3): 373-80. 15. dannan a. an update on periodontic-orthodontic interrelationships. j indian soc periodontol 2009; 14(1): 66-71. 16. chatterjee a, bhattacharya h, kandwal a. probiotics in periodontal health and disease. j indian soc periodontol 2011; 15(1): 23-8. 17. sheikh s, pallagatti s, kalucha a, kaur h. probiotics. going on the natural way. j clin exp dent 2011; 3(2): 150-4. 18. matic ́ s, ivanovic ́ m, nikolic ́ p. effect of oral hygiene training on the plaque control in patients undergoing treatment with fixed orthodontic appliances. serb dent j 2010; 57: 7-10. 19. twetman s, derawi b, keller m, ekstrand k, yucel-lindberg t, stecksen-blicks c. short-term effect of chewing gums containing probiotic lactobacillus reuteri on the levels of inf lammatory mediators in gingival crevicular fluid. acta odontol scand 2009; 67(1): 19-24. 20. g ä n z le mg, hölt z el a , wa lt e r j, ju ng g, h a m m e s w p. characterization of reutericyclin produced by lactobacillus reuteri lth2584. appl environ microbiol 2000; 66(10): 4325-4333. 21. lima l, motisuki c, spolidorio dmp, santos-pinto l. in vitro evaluation of probiotics microorganisms adhesion to an artificial caries model. eur j clin nutr 2005; 59: 884-886. 22. staab b, eick s, knöfler g, jentsch h. the influence of a probiotic milk drink on the development of gingivitis: a pilot study. j clin periodont 2009; 36(10): 850-856. 23. sutula j, coulthwaite l, thomas l, verran j. the effect of a commercial probiotic drink on oral microbiota in healthy complete denture wearers. microb ecol health dis 2012; 23: 18404. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p126-129 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p126-129 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair.ac.id/ mkg/about/submissions#onlinesubmissions the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) " ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal i want to subscribe the dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) ................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... e-mail: ..................................................................................... date/tanggal: ......................................................................... order description/ keterangan pesanan: ................................ ................................................................................................. ................................................................................................. ................................................................................................. signature/tanda tangan: ....................................................... change of mailling address perubahan alamat surat name/nama: ........................................................................................... address/alamat: ...................................................................................... .................................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... fax: .......................................................................................... e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ country issue* surabaya java island (pulau jawa) outside java island (luar pulau jawa) other countries (negara lain) us $ 30 us $ 60 * quarterly publication (terbit 4 kali setahun) name/nama: .......................................................................... i am paying this journal by: [please tick ( )] institution/institusi: ................................................................... saya membayar jurnal ini dengan: [beri tanda ( )] address/alamat surat: ............................................................ bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... ................................................................................................. ................................................................................................. 2 issue 4 issue idr 200,000 idr 250,000 idr 300,000 idr 400,000 idr 500,000 idr 600,000 account no. : 988.01010.00000.135 bank : bank bni account holder : fkg dental journal nbkbmbi!lfeplufsbo!hjhj faculty of dental medicine, universitas airlangga editorial address c/o: jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone/fax: +6231 5039478 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/mkg/index international subscription not include shipping costs [please tick ( )] for costumers only untuk pelanggan e-mail to: dental_journal@fkg.unair.ac.id dental journal (majalah kedokteran gigi) jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia or fax to: +6231 5039478 vol 52 no 1 jan-mar 2019_new.indd guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . d e ta c h h e re ( p o to n g d i s in i)  subscription order form formulir berlangganan dental journal nbkbmbi!lfeplufsbo!hjhj faculty of dental medicine, universitas airlangga editorial address c/o: jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone/fax: +6231 5039478 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg i want to subscribe the dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) ................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... e-mail: ..................................................................................... date/tanggal: ......................................................................... order description/ keterangan pesanan: ................................ ................................................................................................. ................................................................................................. ................................................................................................. signature/tanda tangan: ....................................................... for costumers only untuk pelanggan e-mail to: dental_journal@fkg.unair.ac.id dental journal (majalah kedokteran gigi) jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia or fax to: +6231 5039478 change of mailling address perubahan alamat surat name/nama: ........................................................................................... address/alamat: ...................................................................................... .................................................................................................................. country/negara: 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this journal by: [please tick ( )] institution/institusi: ................................................................... saya membayar jurnal ini dengan: [beri tanda ( )] address/alamat surat: ............................................................ bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... : 142-00-1613621-9account no. bank : bank mandiri account holder : drg. udijanto tedjosasongko, ph.d., sp.kga ................................................................................................. ................................................................................................. 78 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 78–81 original article effect of formula milk on the roughness and hardness of tooth enamel amaliyah nur irianti, sri kuswandari and al supartinah santoso department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: demineralisation and remineralisation is a natural process in tooth enamel. it is influenced by the content of calcium and phosphorus in saliva, which concentrations are affected by the consumption of food, including formula milk. demineralisation and remineralisation determine the roughness and hardness of the enamel surface. purpose: this study compared the effect of formula milk on the roughness and hardness of tooth enamel. methods: maxillary premolar extracted teeth were demineralised with 37% phosphoric acid for 90 seconds and then divided into four treatment groups. for four days, the teeth were immersed twice a day in cow formula for five and ten minutes (group i and ii) and soy formula for five and ten minutes (group iii and iv). before and after the immersion in milk, the teeth were submerged in artificial saliva. the enamel surface roughness and hardness were measured three times using a surface roughness tester and a vickers microhardness tester, before and after demineralisation and after immersion in milk. data were analysed using kruskal–wallis and post hoc mann–whitney tests. results: there was no significant difference (p=0.88) observed in the roughness reduction among the treatment groups. the highest increase in hardness was noted for the ten-minute cow formula milk group (93.27 ± 16.00). the increase of hardness was higher after immersion for ten minutes. a substantial difference (p=0.03) was seen in the increase of hardness between the treatment groups. conclusion: immersion in cow and soy formula milk for five and ten minutes does not reduce the enamel roughness, but it increases the enamel hardness. keywords: demineralisation; enamel hardness; enamel roughness; milk formula; remineralisation correspondence: sri kuswandari, department of pediatric dentistry, faculty of dentistry, universitas gadjah mada. jl. denta 1, sekip utara, bulaksumur, yogyakarta, 55281 indonesia. email: kuswandarisri@gmail.com introduction enamel is the outermost tooth layer and the hardest tissue in the human body. the components in enamel are inorganic materials (95%), organic materials (1–2%) and water (2–4%). most of the inorganic materials are hydroxyapatite (ca10(po4)6(oh)2) crystals containing mineral ions, especially calcium and phosphate.1 saliva is a source of calcium and phosphate, which maintains the mineral saturation of teeth. its role is to inhibit demineralisation at low ph and to initiate remineralisation when the ph returns to normal.2 demineralisation is the process of removing mineral ions, especially calcium and phosphate, from the hydroxyapatite (ha) crystals of the tooth enamel. remineralisation is the process of restoring the mineral ions in the ha crystals.3 the required calcium and phosphate can be obtained from the consumption of food and drink, including formula milk. formula milk is frequently consumed by children. it contains protein, fat, calcium, phosphorus, zinc and other minerals. the calcium ions that are present in saliva have a buffering effect on dental plaque, preventing demineralisation and promoting remineralisation.4 formula milk is made from animal milk or plants such as soybean. some children are allergic to animal milk lactose, therefore they consume milk derived from plants, such as soy milk.5 soy milk contains more protein, but the amino acids are not as complete as in cow protein. the calcium content is also lower than in cow milk.6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p78–81 mailto:kuswandarisri@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p78-81 79irianti et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 78–81 milk can decrease the ph of plaque ten minutes after being consumed because of the fermentation of carbohydrates. the ph will increase 20 minutes later because hydrolysis of milk protein occurs.7 there are various ways for infants and children to consume formula milk, such as bottle feeding, spoon feeding, drinking using a straw or cup feeding. in this way, a contact is established between milk and teeth. the longer and more frequently milk is consumed, the longer the enamel is exposed to calcium and phosphorus, which results in higher remineralisation.8 surface roughness and hardness indirectly indicate the occurrence of demineralisation and remineralisation of hard tooth tissue.9 demineralisation in enamel starts with the release of minerals, especially calcium and phosphate, which causes the surface to become softer and rougher.10 hardness is defined as the resistance of a solid material to penetration.11 calcium and phosphate ions form ha crystals in enamel and the number of ha crystals formed affects the thickness of the enamel.12 the purpose of this study was to compare the different effects of immersion in cow milk formula and soy formula on the roughness and hardness of the enamel surface. materials and methods this is a quasi-experimental laboratory (in vitro) study using 12 caries-free maxillary premolar extracted teeth. after separating from the root, the crown was cut mesiodistally to obtain the labial and lingual surfaces and then cultivated in resin. in this way, 24 specimens were prepared. the enamel surface was coated with nail polish, except for a circular work area with a diameter of 5 mm.5 the first measurement of roughness and hardness of the enamel surface was done prior to the demineralisation with 37% phosphoric acid (h3po4) for 90 seconds. 13 then, specimens were immersed for three hours in artificial saliva with a ph of 7.0. thereafter, the second measurement was done. the specimens were grouped into four treatment groups. group i was immersed in cow formula milk for five minutes and group ii for ten minutes. group iii was immersed in soy formula milk for five minutes and group iv for ten minutes. for four days, the immersion was done twice a day (07:30 and 16:30) at a temperature of 37 °c.8 the milk was produced by morinaga™ (pt. kalbe morinaga, indonesia). the composition of both milk formulas was similar, namely, calcium, phosphorus, magnesium, iron, zinc and docosahexaenoic acid (dha) in the same proportions, but the soy formula contained also arachidonic acid (aa), phospholipids, nucleotides, and other ingredients. the third measurement was done after the immersion was completed. the surface roughness of the tooth enamel was measured using a surface roughness tester (starrett sr300, taylor hobson, berwyn, pa, usa).13 the value of the roughness was obtained from the motion signal of a diamond-shaped stylus moving along a straight line on the enamel surface. the roughness was expressed as roughness average (ra) and stated in μm. the roughness was measured at three different points on each research object. the magnitude of indentation was determined using a microhardness tester (hmv-2, shimadzu, japan) by looking at the area of the imprint on the indenter. a load of 100 grams was applied for ten seconds onto the enamel surface.8,14 the enamel surface hardness was obtained from the tooth resistance to indentation. the units in the enamel surface hardness test were expressed in vickers hardness (hv). the enamel surface hardness was measured at three different points on each research object and obtained as an average of three measurements. the data were analysed using the kruskal– wallis test (p<0.05) and the post hoc mann –whitney test (p<0.05). results the kruskal–wallis test on mean and standard deviation of surface roughness reduction based on milk type and immersion time (table 1) showed no significant difference (p>0.05) between the treatment groups. the largest average reduction in enamel surface roughness was observed after immersion in soy formula for ten minutes, followed by immersion in cow and soy formula for five minutes. the reduction in surface roughness was lowest after immersion in cow formula for ten minutes, namely 0.21 ± 0.06. the kruskal–wallis test on mean and standard deviation of increased surface hardness based on milk type and immersion time (table 2) exhibited a significant difference (p< 0.05) between the treatment groups. table 1. the averages and standard deviations of the enamel surface roughness based on formula milk and immersion time and the kruskal–wallis test results in μm treatment group n before immersion (x ± sd) after immersion (x ± sd) reduction of enamel surface roughness (x ± sd) kruskal–wallis test x2 df p group i 6 0.98 ± 0.29 0.73 ± 0.22 0.25 ± 0.18 0.66 3 0.82 group ii 6 0.91 ± 0.19 0.70 ± 0.15 0.21 ± 0.06 group iii 6 0.82 ± 0.20 0.57 ± 0.14 0.25 ± 0.13 group iv 6 0.74 ± 0.12 0.48 ± 0.05 0.26 ± 0.12 description: n: number of research objects; df: degrees of freedom; x2: chi square; p: probability dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p78–81 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p78-81 80 irianti et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 78–81 the highest average increase in hardness was observed after immersion in cow formula for ten minutes, namely 93.27 ± 16.00. using the post hoc mann–whitney test, the mean comparison of the increase in enamel surface hardness between the treatment groups (table 3) displayed only significant differences (p<0.05) between the groups of teeth immersed in soy formula for five minutes and those immersed in cow formula for ten minutes and between the groups of teeth immersed in soy formula for five minutes and those immersed in soy milk formula for ten minutes. discussion the study showed that there was no significant difference in enamel surface roughness between the treatment groups using the length of time and milk immersion as variables. however, the enamel surface hardness differed significantly using the same variables. there were considerable differences between the groups of teeth immersed in soy formula for five minutes and those immersed in cow formula for ten minutes and between groups of teeth immersed in soy formula for five minutes and those immersed in soy milk formula for ten minutes. the results agree with those reported by yendriwati et al.,8 who observed that the longer and the more frequently the enamel was exposed to calcium and phosphorus contained in drinks, the more minerals were incorporated into the enamel, increasing its hardness. the increase in enamel surface roughness is the result of increased porosity.15 when remineralisation occurs, calcium and phosphate ions repair the enamel damage caused by demineralisation.16 the formed hydroxyapatite crystals reduce the interprismatic gap and enamel microporosity, which smoothens the surface of the porous part and decreases the surface roughness.17 after five minutes, the average reduction in enamel surface roughness after immersion in cow formula milk was not different compared to that of using soy formula milk. however, after ten minutes, the average reduction in enamel surface roughness after immersion in cow formula milk was lower than that using soy formula milk. in the demineralisation process, hydroxyapatite crystals dissolve and release calcium and phosphate ions. this process results in larger interprismatic gaps and reduces the enamel surface hardness. when remineralisation occurs by incorporating calcium and phosphorus ions, hydroxyapatite crystals are regenerated and fill the voids. according to several studies, the crystals formed are amorphous and differ from previous hydroxyapatite crystals. this may increase the surface hardness of the remineralised enamel, but it changed the hydroxyapatite crystals.18 the study showed that there were significant differences after immersing the teeth in soy formula for five and ten minutes. the longer immersion allows the integration of more calcium and phosphorus ions, which leads to a higher degree of the remineralisation. milk prevents the demineralisation process according to several mechanisms. milk protein can be adsorbed onto the enamel surface and inhibits the enamel demineralisation. in addition, milk fat that is adsorbed onto the enamel surface may act as a protection, and enzymes in the milk can reduce the acidogenic stage of plaque bacteria.12 milk contains proteins that bind calcium and phosphate ions. the remineralisation process starts with the attachment of milk proteins to the enamel surface. the calcium and phosphorus ions in milk can diffuse into the enamel subsurface.19 the ions can occupy the empty ha crystal space because of demineralisation and initiate the remineralisation by forming hydroxyapatite crystals.17,20 the newly formed hydroxyapatite crystals fill the interprismatic gaps of the enamel. this process decreases the roughness and increases the surface hardness of the enamel.14 the following is the general chemical equation for the demineralisation and remineralisation process: 8 h+ + (ca10(po4)6(oh)2) → 6 (hpo4) 2+ 10 ca2+ + 2 h2o (demineralisation); 10 ca2+ + 6 (h2po4) 2+ 14 oh→ ca10(po4)6(oh)2 (remineralisation). table 2. the averages and standard deviations of the enamel surface hardness based on formula milk and immersion time and the kruskal–wallis test results (as hv) treatment group n before immersion (x ± sd) after immersion (x ± sd) increase in enamel surface hardness (x ± sd) kruskal–wallis test x2 df p group i 6 170.43 ± 12.05 248.41 ± 23.31 77.98 ± 13.29 9.34 3 0.025 group ii 6 170.88 ± 24.86 264.15 ± 34.60 93.27 ± 16.00 group iii 6 124.23 ± 23.66 190.45 ± 20.68 66.22 ± 9.32 group iv 6 130.30 ± 37.28 211.53 ± 39.33 81.23 ± 6.31 description: n: number of research objects; df: degrees of freedom; x2: chi square; p: probability table 3. post hoc mann–whitney test comparing the increasing average of the treatment groups comparison between groups post hoc mann–whitney test z p group i group ii -1.92 0.06 group iii -1.60 0.11 group iv -0.48 0.63 group ii group iii -2.40 0.02* group iv -1.12 0.26 group iii group iv -2.25 0.03* description: z: statistical value of z table, p: probability; *: significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p78–81 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p78-81 81irianti et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 78–81 the demineralisation process results in the damage of the ha crystals, which starts with a bond formation between the (po4 3-) ion and the free h+ ion, causing the dissolution of the apatite crystal. the h+ ion binds to an ohion to form h2o and it binds to a po4 3ion to form hpo4 2-. when in contact with the ionic acid, hpo4 2turns into h2po4. 17 the diffusion of calcium and phosphorus ions into the enamel surface can initiate the remineralisation.18 ha crystals can undergo atomic replacement, which leads to a change in the chemical formula ha. it can be concluded that teeth immersed in cow formula and soy formula for five and ten minutes might not decrease the roughness of the enamel surface, but it might increase the hardness of it. the hardness of the enamel surface of teeth immersed in soy formula for ten minutes was higher than that immersed in soy formula for five minutes. references 1. lacruz rs, habelitz s, wright jt, paine ml. dental enamel formation and implications for oral health and disease. physiol rev. 2017; 97(3): 939–93. 2. cummins d. the development and validation of a new technology, based upon 1.5% arginine, an insoluble calcium compound and fluoride, for everyday use in the prevention and treatment of dental caries. j dent. 2013; 41 suppl 2: s1-11. 3. abou neel ea, aljabo a, strange a, ibrahim s, coathup m, young am, bozec l, mudera v. demineralization-remineralization dynamics in teeth and bone. int j nanomedicine. 2016; 11: 4743–63. 4. sharma a, sharma d, singh s, sharma a, sharma m. milk and its products: effect on salivary ph. int healthc res j. 2018; 2(6): 140–5. 5. widanti ha, herda e, damiyanti m. effect of cow and soy milk on enamel hardness of immersed teeth. j phys conf ser. 2017; 884: 012006. 6. maurice-van eijndhoven mht, hiemstra sj, calus mpl. short communication: milk fat composition of 4 cattle breeds in the netherlands. j dairy sci. 2011; 94(2): 1021–5. 7. telgi rl, yadav v, telgi cr, boppana n. in vivo dental plaque ph after consumption of dairy products. gen dent. 2013; 61(3): 56–9. 8. yendriwati, sinaga rm, dennis d. increase of enamel hardness score after cow milk immersion of demineralized tooth: an in vitro study. world j dent. 2018; 9(6): 439–43. 9. r a ha rdjo a , g r a cia e , r isk a g, ad iat m a n m, ma ha r a n i da. potential side effects of whitening toothpaste on enamel roughness and micro hardness. int j clin prev dent. 2015; 11(4): 239–42. 10. carvalho ts, lussi a. combined effect of a fluoride-, stannousand chitosan-containing toothpaste and stannous-containing rinse on the prevention of initial enamel erosion-abrasion. j dent. 2014; 42(4): 450–9. 11. mccabe jf, walls awg. bahan kedokteran gigi. 9th ed. sunarintyas s, editor. jakarta: egc; 2015. p. 19–20. 12. safavi ms, walsh fc, surmeneva ma, surmenev ra, khalilallafi j. electrodeposited hydroxyapatite-based biocoatings: recent progress and future challenges. coatings. 2021; 11(1): 110. 13. makmur sa, utomo rb. pengaruh aplikasi gel theobromine terhadap kekasaran permukaan email gigi desidui pasca demineralisasi. odonto dent j. 2019; 6(2): 95–8. 14. vidyahayati il, utomo rb, soeprihati it. pengaruh konsentrasi gel theobromine terhadap ketahanan kekerasan permukaan email gigi desidui. odonto dent j. 2019; 6(1): 8–13. 15. champigneux p, renault-sentenac c, bourrier d, rossi c, delia m-l, bergel a. effect of surface roughness, porosity and roughened micro-pillar structures on the early formation of microbial anodes. bioelectrochemistry. 2019; 128: 17–29. 16. hamba h, nakamura k, nikaido t, tagami j, muramatsu t. remineralization of enamel subsurface lesions using toothpaste containing tricalcium phosphate and fluoride: an in vitro μct analysis. bmc oral health. 2020; 20(1): 292. 17. mukarromah a, dwiandhono i, imam dna. differences in surface roughness of enamel after whey-extract application and cpp-acp in post extracoronal-tooth bleaching. maj kedokt gigi indones. 2018; 4(1): 15–21. 18. amaechi bt. remineralization therapies for initial caries lesions. curr oral heal reports. 2015; 2(2): 95–101. 19. vandenplas y, castrellon pg, rivas r, gutiérrez cj, garcia ld, jimenez je, anzo a, hegar b, alarcon p. safety of soya-based infant formulas in children. br j nutr. 2014; 111(8): 1340–60. 20. tyagi sp, garg p, singh up, sinha dj. an update on remineralizing agents. j interdiscip dent. 2013; 3(3): 151–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p78–81 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p78-81 �� gingival immunologic defense index: a new indicator for evaluating dental plaque infection risk in allergic children seno pradopo1 and haryono utomo2 1department of pediatric dentistry, faculty of dentistry, airlangga university 2 dental clinic, faculty of dentistry, airlangga university surabaya indonesia abstract there is a possible relationship between dental plaque and children allergic diseases. according to literatures, gingivitis suffered mostly by allergic children than control. case reports also revealed that dental plaque control therapy was able to reduce, even eliminate rhinosinusitis and asthmatic symptoms without additional medications. however, the exact method for confirming the gingivitis-related allergy is still uncertain. allergic diseases have multifactorial etiologies and dental plaque had been proposed as a new trigger of allergic symptoms. nevertheless, since not every child with gingivitis suffered from allergy or vice versa, this uncertain phenomenon may lead to patients or other clinician disbelief. the objective of the present study was to propose a new method, which involving the gingival immunologic defense index (gidi) to evaluate the susceptibility to allergic diseases. gidi is an index that had been developed earlier for evaluating gingival immunologic defense with respect to immunoglobulin a (iga) levels. this index based on the simple count of the inflamed gingival surfaces of a child plus the measurement of salivary iga content. it provides clinicians with important information about the immunologic defense potential of each subject. interestingly, most allergic children also had inherited iga deficiency, thus this concept is likely. based on literatures, gidi could be a potential index for evaluating the risk of allergic diseases through gingival health assessment. however, prior investigation to the value of indonesian gidi index which related to allergy should be conducted. key words: gingival immunity, index, allergy correspondence: seno pradopo, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: pradopo_seno@yahoo.com. telp. (031) 5030255. introduction the connection between periodontal and general health had been studied by abundant literatures. 1,2 nevertheless, the relationship with allergy is still in controversy. evidence-base cases revealed that oral focal infection may cause urticaria,1 rhinitis,3 and sinusitis symptoms;4,5 however, elimination of oral focal infection is not included in allergy management protocol. thus, the involvement of gingival health status that also connected with systemic diseases which may include allergy is often overlooked. unfortunately, it may lead to unnecessarily prolonged medications and treatments, including surgery (i.e. adenoidectomy for chronic sinusitis in children) in allergic children. dental plaque may also act as the source of oral focal infection, even inflammatory reaction in chronic gingivitis is able to elicit systemic pathology.2 nevertheless, until now, the systemic pathology investigated was limited to adult diseases such as cerebrovascular and cardiovascular diseases, diabetes, pregnancy problems etc.1 the systemic effect happened to children was rarely studied. according to several investigators, allergic children, especially rhinitis and asthma had poorer periodontal health. however, these investigations revealed that allergic diseases caused poor oral health (caries and gingivitis) because of dry mouth or mouth breathing and not vice versa.6 several literatures revealed the possible benefit of dental plaque control therapy to reduce allergic symptoms in children.3–5 therefore, comprehensive treatment of allergy should also consider improving children’s oral health, especially reducing dental plaque accumulation which account for gingivitis. through gingivitis, dental plaque may lead to systemic pathology.2 thus, optimal gingival health is considered beneficial to support allergic management, not only medications, immunotherapy or allergen avoidance dependent. regarding to this concept, the importance of gingival immunity which is also related to the occurrence of microorganism invasion, and the role of first line defense in gingival mucosa, the salivary immunoglobulin a (siga),7 should be considered. in 1995, researches from sao paulo university, brasil proposed the gingival immunologic defense index (gidi) for evaluation of gingival immunologic defense with respect to salivary iga levels.8 as the susceptibility of infection also related to allergic patient who also predisposed to lower secretory iga (siga) levels.9 in addition, it is more pronounced in children who their iga level still not full-developed until approximately the age of 10.10 �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 43-46 the objective of this study is to introduce the gidi as a practical indicator of gingival health for evaluating the risk of allergic diseases. hopefully, this concept also encourages medical practitioners to evaluate gingival condition during examination of allergic patients. basic immune system there are two important parts of immune system, the innate immunity and the acquired immunity. the simplest way to avoid infection is to prevent the microorganisms from gaining access to the body. the first line of defense which prevent from infection are skin, sweat, saliva, nasal secretions etc. microbial and other foreign particles trapped within mucus secreted by the lining membranes are removed by mechanical action such as coughing and sneezing.9 for faster recognition and facilitate phagocytosis, antibodies were synthesized in part of the acquired immunity. there are several antibodies in the immune system that termed as immunoglobulins. the immunoglobulin m (igm), igg, iga, igd and ige furthermore, there are two kinds of immunity mechanisms involved, the cell-mediated immunity and humoral immunity. antibodies are mostly produced in humoral immunity which needs the interactions of t-cells and b-cells lymphocytes.10 immune system and allergic disease allergic diseases are related to the subpopulation of t-cells so called the t-helper 2 (th2) cells. these th2 lymphocytes release cytokines upon stimulations that are interleukin 4 (il-4) and il-13 which able to induce antibody production by b-cells. actually, in normal individuals, these cytokines stimulate immunoglobulin m (igm) and igg production which provide defence against bacteremia via opsonization. nevertheless, in allergic individuals, these cytokines convert igm and igg to ige through a mechanism so called “isotype switching”.10 the increase of allergenspecific ige in the circulation which then attached to the fcei receptors of immunocompetent cells such as mast cells and basophils make individuals more sensitive to allergens.1 in addition, il-4 also inactivate macrophages which needed for phagocytosis of bacteria. immunoglobulin a and allergic disease immunoglobulin a (iga) is the predominant immunoglobulin in seromucous secretions such as saliva, colostrums breast milk, tracheobronchial and genitourinary secretions. it is produced by mature b lymphocytes cells and related to the isotype switching mechanism which is regulated by transforming growth factor-b (tgf-b). this cytokine is secreted by t-regulator (treg) lymphocytes. (male) allergy is considered caused by inadequate treg function (romagnani). antigens, that are microorganisms and allergens before attaching to the mucosa are “blanket” by iga, which is the first immunoglobulin involved.11 immunoglobulin a which also a constituent of saliva and termed as salivary iga (siga) is a part of the secretory iga (siga) of the human body.9 failure of iga to protect antigens from adherence to the mucosa leads to further penetration of microorganisms and allergens into the mucosa and may lead to bacteremia.7 in children, iga is the last immunoglobulin that reaches adult level.10 the connection between oral health, systemic diseases and allergy there were abundant literatures related to systemic infection which originated from periodontal or pulpal infections through bacteremia or spread of pro-inflammatory mediators. cerebrovascular, cardiovascular diseases were the most renowned; others are diabetes mellitus and pregnancy problems.1 nevertheless, there were only several studies which related to allergy. most literatures were epidemiological studies and concluded that allergic patients (i.e. rhinitis, asthma) had poorer oral health.6 several case reports revealed that dental plaque control therapy had beneficial effect to children suffered from allergic rhinitis, sinusitis and asthma.3–5 collaborated study which included dental practitioners, pediatrician and children allergic experts revealed that dental plaque therapy without medication lead to disappearing of clinical asthmatic symptoms; even after two months later.11 dental plaque and allergic diseases bacterial dental plaque or dental biofilm consisted of microorganism and organic matrix. it contains grampositive and gram-negative bacteria which may cause bacteremia and trigger immunologic reaction by stimulating immunocompetent cells (i.e. mast cells, macrophages, basophils). bacteria also able to activate the innate immunity, such as the complement system c3 which stimulate mast cell or basophil degranulation via c3a receptor.12 toxins from these bacteria, the peptidoglicans from gram-positive bacteria and lipopolysaccharides from gram-negative bacteria stimulate toll-like receptor-2 (tlr2) and tlr4 respectively. these tlrs are present in immunocompetent cells i.e. mast cells, basophils, macrophages.13 gingival immunological defense index (gidi) the gingival immunologic defense index (gidi), which is based on the simple count of the inflamed gingival surfaces of a child plus the measurement of salivary iga content, provides clinicians with important information about the immunologic defense potential of each subject. the formula of this new index would not be static like the gingival index and that would not take into consideration only the severity of gingival inflammation, but that could be used to evaluate the gingival immunologic defense with respect to salivary content of iga, the major immunoglobulin in the oral cavity, under different gingival conditions.8 to determine the relationship between gingival inflammation and salivary immunoglobulin content we created a new index that we propose to call gidi, an index that seems to be appropriate for the evaluation of the ��pradopo and utomo: gingival immunologic defense index immunologic defense of the oral cavity in the presence of gingival inflammation. this index is the ratio between salivary immunoglobulin level (µg iga/100 ml saliva) and number of inflamed gingival surfaces present in an individual. ultimately, this index reflects the mean level of immunoglobulin secreted per inflamed gingival surface and can be expressed by the following equation: gidi = ig level nigs ig is the immunoglobulin measured (µg) and nigs is the number of inflamed gingival surfaces. the advantage of evaluating gingival immunologic defense by the gidi in relation to simply determining the degree of gingival inflammation by the gi is that the gidi permits the establishment of a prognosis for the evolution of the inflammatory process, leading to a dynamic evaluation of the latter, in contrast to the gi, which is simply a static determination.8 the “neurogenic switching” mechanism this mechanism was introduced by meggs in 1993,14 which hypothesized that local inflammation was able to propagate or to spread to distant location via mast cellnerve fibers interaction. for example, allergic dermatitis may account for rhinitis or vice versa. it was confirmed by researches, i.e. by van der kleij.15 according to lundy and linden,16 this mechanism also occurred in the periodontal tissues, including gingiva. discussion allergic hyperreactivity is defined as an exaggerated immuneresponse typically immunoglobulin e (ige) but also non-ige mediated toward harmless antigenic stimuli.17 a practical method for allergic or hypersensitivity test is the skin prick test (spt).9 nevertheless, the detection of agents or factors which contribute to the severity of allergy is not easily conducted. since allergic reactions mostly manifests as nasal, skin and asthmatic symptoms, the importance of early detection of allergic symptoms via oral cavity is often overlooked. oral mucosa which is a port of entry of antigens inside our body could act as potential indicator or oral tolerance.18 furthermore, poor gingival health in children may account for bacteremia, circulating pro-inflammatory mediators (lie) and the neurogenic switching mechanism may help the assessment of allergic risk in allergic children. allergic children have the propensity to develop lower iga level in several reasons: a) mostly predisposed to iga deficiency; b) have lower or inadequate transforming growth factor-b (tgf-b), cytokines produced by tregulators (tregs) which needed for isotype switching of iga produced by b lymphocytes.19,20 thus it is possible that allergic children are prone to bacterial and other antigens invasion, since they have lower level of salivary iga which actually “blanket” the bacteria and antigens. this condition may lead to gingivitis in children, which in accordance with laurikainen6 study. according to the lower immunity and susceptibility of infection in allergic children, this concept is logical. nevertheless, epidemiological study in adult by friedrich revealed that periodontitis had a protective risk against allergy and asthma, which consistent with the “hygiene hypothesis”.21 it was also in concordance with the oral tolerance concept, that is an inhibition of specific immuneresponsiveness to subsequent parenteral injections of proteins to which an individual or animal has been previously exposed via the oral route. oral tolerance needs tregs system that produces tgf-b and interleukin 10 (il-10) for its development.22 however, a study by mucida et al. showed that tregs were not necessarily needed in oral tolerance.17 it is logical that inflamed tissue, including gingivitis presents more vasodilated and increased permeability blood vessels. other is loosened of epithelial tight junction caused by pro-inflammatory mediators such as tumor necrosis factor a (tnf-a).23 therefore, increased evidence of microorganisms and antigens invasion could be predicted. thus, the importance of first barrier of infection such as the iga is mandatory. the gingival immunologic defense index (gidi) is considered as an “easy to be learned” and “easy to be done” indicator for either dental or medical practitioners, because it is only related to number of inflamed gingival surfaces (nigs), and without specific clinical descriptions. to determine the degree of local immunologic defense conferred on the gingiva by iga, one should not consider the salivary iga content alone as an absolute value, but rather the level of secreted iga per inflamed gingival surface (gidi = iga/nigs).8 the problem is the relative difficulty in salivary iga assessment procedures, which actually needs laboratory test. nevertheless, this stage could be omitted, since iga level in children under 10 is lower than normal. especially allergic children have a predisposition of lower iga level. the most important thing is to conduct a research investigating the average value of gidi in non-allergic and allergic children in indonesia. the value of gidi in allergic screening test could be contradictory with the new concept of oral tolerance which is not tregs dependent.17 however, gingivitis also related to increased amount of bacterial toxins. according to jung et al.,24 in the presence of lps, allergic reaction becomes exaggerated because of its synergistic effect with antigens. the intention of this study is to find a practical indicator for allergic screening test, therefore practical indicator such as the calculation of gidi whish is a simple count of the number of inflamed gingival surfaces of a child, taken �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 43-46 together with the measurement of salivary iga, can provide important information to the clinician in terms of the potential for gingival immunologic defense of the allergic children. early detection of dental plaque infection risk in allergic children could anticipate for unnecessary prolonged allergy medications, treatments, or even surgery. for the concluding remarks in order to increase the collaboration of medical and dental practitioners for preventing the development of children's allergic diseases, it is suggested that collaborated researches and forums should be conducted. in addition, there should be more informations which targetting laymen through print or electronic media which explain the connection between the dental plaque and allergy. references 1. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microbiol rev 2000; 13(4):547–58. 2. scannapieco fa. periodontal inflammation: from gingivitis to systemic disease. compendium 2004; 25(7):s17–s25. 3. utomo h, setijanto d. apakah terapi pengendalian plak gigi dapat menurunkan keparahan rinitis alergika pada anak. majalah kedokteran gigi (dent j) 2005; 38(2):96–102. 4. utomo h. sensitization of sphenopalatine ganglion by periodontal inflammation: a possible etiology for sinusitis and headache in children. majalah kedokteran gigi (dental journal) 2006; 39(2):63–71. 5. utomo h, pradopo s. a practical dental approach in children’s rhinosinusitis management. indonesian dent j 2006; 13(3):133–6. 6. laurikainen k. asthma and oral health: a clinical and epidemiological study. academic dissertation. tampere: tampere university press; 2002. p. 1–182. 7. rabson a, roitt im, delves pj. really essential medical immunology. 2nd ed. carlton: blackwell publishing; p. 8–10. 8. de souza-gugelmin mcm, ito iy, maia campos g. creation of the gingival immunologic defense index (gidi) to evaluate the immunological potential of the gingiva and the possible risk for periodontal diseases. braz dent j 1995; 6(2):95–102. 9. leung dym. in: behrman re, kliegman rm, jenson hb eds. nelson‘s textbook of pediatrics. 17th ed. philadelphia: saunders; 2004. p. 743–77. 10. mcdade tw. life history theory and the immune system: steps toward a human ecological immunology. yearbook of physical immunology 2003; 46:100–25. 11. utomo h. reducing asthmatic symptoms through improving oral health: from imaginary to reality. j indonesian assoc of dentistry 2008; special edition for kongres pdgi xxiii. 12. ali h, panettieri ra. anaphylatoxin c3a receptor in asthma. respir res 2005; 6:19–24. 13. netea mg, van der meer jwm, sutmuller rp, adema gj, kullberg bj. from the th1/th2 aradigm towards a toll-like receptor/t-helper bias. antimicrob ag chemoth 2005; 49(10):3991–6.antimicrob ag chemoth 2005; 49(10):3991–6. 14. meggs wj. neurogenic inflammation and sensitivity to environmental chemicals. environ health perspect 1993; 101:234–8. 15. van der kleij h. mast cell-nerve interaction. academic dissertation. utrecht: universiteit of utrecht; 2002. p. 1–161. 16. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 17. mucida d, kutchukhidze n, erazo a, russo m, lafaille jj, de lafaille mac oral tolerance in the absence of naturally occurring tregs. j clin invest 2005; 115:1923–33. 18. incorvaia c, frati f, sensi l,riario-sforza1 gg, marcucci f. allergic inflammation and the oral mucosa. recent patents on inflammation and allergy drug discovery 2007; 1:35–8. 19. roitt i, brorstoff j, male d. immunology. 6th ed. edinburgh: mosby; 2001. p. 144–5. 20. romagnani s. the increased prevalence of allergy and the hygiene hypothesis: missing immune deviation, reduce immune suppression, or both? j allergy clin immunol 2004; 112:352–63. 21. friedrich n, volzke h, schwahn c, kramer a, junger m, schafer t, et al. inverse association between periodontitis and respiratory allergies. clin exp allergy 2006; 36(4):495–502. 22. dubois b, chapat l, goubier a,, papiernik m, nicolas jf, kaiserlian d. innate cd4+cd25+ regulatory t cells are required for oral tolerance and inhibition of cd8+t cells mediating skin inflammation. blood 2003; 102(9):3295–301. 23. go m, kojima t, takano k, murata m, ichimiya s, tsubota t, et al. expression and function of tight junction in the crypt epithelium of human palatine tonsils. j histochem cytochem 2004; 52(12):1627–38. 24. jung yw, schoeb tr, weaver ct, chaplin dd. antigen and lipopolysaccharide plays synergistic roles in the effector phase of airway inflammation in mice. am j pathol 2006; 168:1425–34. vol 38 no 2-2005 77 respons inflamasi pada pulpa gigi tikus setelah aplikasi ekstrak etanol propolis (eep) (the inflammatory response on rat dental pulp following ethanolic extract of propolis (eep) application) ardo sabir bagian konservasi gigi fakultas kedokteran gigi universitas hasanuddin makassar – indonesia abstract propolis is a resinous hive product collected by bees from tree buds and mixed with secreted bee wax in order to avoid bacterial contamination in the hive, and also to seal it. propolis is employed for the treatment of various infectious diseases because it is wellknown that is has antibacterial and anti-inflammatory properties. the therapeutic use of propolis in dentistry has been done in recent years as in treatment gingivitis and dental caries, in treatment wound healing after surgical procedures, etc. the purpose of this investigation was to study the inflammatory response on rat's dental pulp following application of ethanol extract of propolis (eep). twelve male spraque-dawley rats of 8–16 week old and 200–250 grams in weight were used in this study. the rats were randomly divided into two groups. pulp exposures were performed on the occlusal surface of right maxillary first molars. at the first group, as the control group, zinc oxide-based filler was directly applied on pulp exposure. meanwhile at the second group, as the sample group, pulp exposure was applied with eep. after that, all cavities were filled with glass ionomer cement as permanent filling. animals were sacrificed on the 7th, 14th, and 28th day. the criterion of histological examination was based on the inflammatory responses of the dental pulp, which were the presence of polymorph nuclear leukocytes and macrophages. kruskall-wallis and mann-whitney tests were employed to analyze the data. the results of this research demonstrated that the inflammatory response of eep group was numerically milder compared to the control group, even though statistical analysis showed no significant difference (p > 0.05) between the two groups. key words: ethanol extract of propolis, inflammatory response, dental pulp, rat korespondensi (correspondence): ardo sabir, bagian konservasi gigi, fakultas kedokteran gigi universitas hasanuddin. jln. kandea 5 makassar, indonesia. pendahuluan kata propolis berasal dari bahasa yunani, yaitu pro berarti pertahanan dan polis berarti kota, sehingga propolis bermakna pertahanan kota (atau sarang lebah).1,2 propolis atau lem lebah adalah nama generik yang diberikan untuk bahan resin yang dikumpulkan oleh lebah madu dari berbagai macam jenis tumbuhan, terutama dari bagian kuncup dan daun tumbuhan tersebut.1,3 lebah kemudian mencampur bahan resin ini dengan enzim yang disekresikan dari kelenjar mandibula lebah,4 meskipun demikian komponen yang terdapat di dalam propolis tidak mengalami perubahan.1 lebah menggunakan propolis sebagai: 1) memperkuat sarang lebah;1 2) bahan pelapis untuk melindungi sarangnya dari faktor pengganggu dari luar, misalnya serangga, kumbang, atau tikus;3,5 3) meratakan dinding sarang lebah;4 4) bahan pengisi lubang atau celah dan perekat keretakan yang terdapat pada sarang lebah,5,6 5) melindungi sel sarang tempat ratu lebah menetaskan telurnya sehingga larva lebah terlindungi dari penyakit6 dan 6) antibakteri.7 komposisi propolis sangat bervariasi dan erat hubungannya dengan jenis dan umur tumbuhan di mana propolis tersebut berasal.4,5 umumnya propolis terdiri dari:4 campuran resin dan getah 39–53%, polifenol 1,2–17%, polisakarida 2–3%, lilin (wax) 19–35%, dan bahan lain 8–12%. menurut kaal,6 komposisi propolis meliputi: resin dan balsem ± 50%, lilin ( wax) ± 30%, minyak esensial ± 10%, pollen ± 5%, dan senyawa organik dan mineral ± 5%. penelitian terhadap propolis yang berasal dari 15 daerah yang berbeda di rusia menunjukkan hasil yang hampir sama, yaitu:5 resin 50–55%, lilin (wax) maksimal 30%, minyak esensial ± 8–10%, dan bahan padat ± 5%. jenis senyawa kimia yang terdapat pada propolis sangat kompleks. berdasarkan analisis dengan menggunakan metode gas chromatography-mass spectrometry (gc-ms) yang dilakukan oleh greenaway et al.3 terhadap propolis yang dihimpun oleh lebah yang berasal dari tumbuhan poplar menunjukkan bahwa propolis mengandung berbagai macam senyawa, yaitu: asam amino, asam alifatik dan esternya, asam aromatik dan esternya, alkohol, aldehida, khalkon, dihidrokhalkon, 78 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 77–83 flavanon, flavon, hidrokarbon, keton, dan terpenoid. hasil yang hampir sama juga diperoleh oleh marcucci8 yang menemukan senyawa alkohol, aldehida, asam alifatik dan esternya, asam amino, asam aromatik dan esternya, flavanon, keton, dan glukosa dalam propolis. propolis telah digunakan sejak dahulu kala sebagai obat tradisional, yaitu sebagai bio-kosmetik dan makanan untuk kesehatan. penelitian di bidang kesehatan terhadap propolis telah banyak dilakukan luar negeri, baik secara in vitro maupun in vivo. hasilnya menunjukkan bahwa propolis memiliki beberapa aktivitas biologis dan farmakologis, antara lain: 1) bersifat antibakteri baik terhadap bakteri gram positif 9–11 maupun gram negatif;12 2) bersifat antiinflamasi;9,13,14 3) memiliki aktivitas antijamur, terutama terhadap spesies dermatofita dan kandida;9,10,15 4) propolis meningkatkan regenerasi jaringan tulang dan kartilago,16,17 dan 5) propolis bersifat antioksidan karena mampu menangkap radikal bebas.18 penggunaan propolis di bidang kedokteran gigi baru dilaporkan beberapa tahun terakhir. hasilnya menunjukkan bahwa propolis dapat digunakan sebagai salah satu bahan pengobatan alternatif yakni: 1) dalam perawatan penyakit gingivitis oleh karena mampu mencegah pembentukan plak;19–21 2) digunakan untuk mengobati ulserasi pada rongga mulut;19 3) mencegah terjadinya karies gigi.22,23 hal ini disebabkan karena propolis memiliki aktivitas antibakteri, mengurangi daya adhesi bakteri rongga mulut pada permukaan gigi, menghambat sintesis glukan yang tidak larut dalam air (water-insoluble glucan), dan menghambat enzim glukosiltransferase;20,22–25 4) meningkatkan aktivitas mineralisasi pada permukaan email gigi;26 5) mempercepat perbaikan jaringan dan penyembuhan luka setelah prosedur bedah mulut (pasca ekstraksi maupun terjadinya dry socket),27 dan 6) pada perawatan gangren pulpa28 serta periodontitis,29 namun demikian, pengaruh propolis terhadap pulpa gigi belum diketahui, oleh karena itu timbul suatu permasalahan yakni: bagaimana respons pulpa gigi akibat aplikasi propolis, sehingga tujuan dari penelitian ini adalah untuk mengetahui respons inflamasi yang terjadi pada pulpa gigi tikus setelah aplikasi ekstrak etanol propolis (eep), yang merupakan tahap awal kemungkinan penggunaan eep di bidang endodontik. bahan dan metode jenis penelitian ini adalah eksperimental laboratoris, dan dilakukan di 3 tempat yakni: laboratorium galenika fakultas farmasi, unit pengembangan hewan percobaan fakultas kedokteran hewan, dan laboratorium histologi dan biologi sel fakultas kedokteran universitas gadjah mada, yogyakarta. propolis yang digunakan pada penelitian dikumpulkan dari sarang lebah yang jenis lebahnya adalah trigona sp yang banyak terdapat di kabupaten bulukumba, provinsi sulawesi selatan. adapun teknik pembuatan eep adalah dengan dengan teknik maserasi yakni: propolis dilarutkan dengan larutan etanol 95% dan diaduk sampai rata, kemudian didiamkan selama 5 hari, selanjutnya dilakukan penyaringan untuk memisahkan filtrat dari ampas. filtrat yang diperoleh diuapkan hingga kandungan etanolnya menguap sehingga diperoleh eep dengan konsistensi yang kental. dua belas ekor tikus galur spraque-dawley jantan, umur 8–16 minggu dengan berat badan 200–250 g digunakan dalam penelitian ini. semua tikus diberi pakan standar dan air minum ad libitum. tikus dibagi menjadi 2 kelompok secara random, yaitu: kelompok i terdiri dari 3 ekor tikus sebagai kelompok kontrol dan kelompok ii terdiri dari 9 ekor tikus sebagai kelompok perlakuan (kelompok eep). alat yang akan dipergunakan terlebih dahulu didisinfeksi dengan menggunakan alkohol 95%. semua tikus dianestesi secara intramuskular dengan ketamin (ketalar®, warner lambert, irlandia) (65 mg/kg berat badan) dan xylazine hcl (rompun®, bayer, leverkusen, jerman) (7 mg/kg berat badan) yang dilarutkan dalam phosphat buffered saline (pbs) steril. permukaan oklusal gigi yang akan dibur didisinfeksi dan dibersihkan dengan cotton pellet yang sebelumnya dicelup ke dalam larutan alkohol 70%. suatu kavitas klas i (klasifikasi black) dibuat pada permukaan oklusal gigi molar pertama kanan rahang atas menggunakan handpiece dengan bur intan bundar (diameter 0,84 mm) dengan kecepatan rendah hingga hampir mencapai ruang pulpa. kedalaman preparasi diperkirakan sebesar kepala bur. tindakan perforasi terhadap ruang pulpa dilakukan dengan menggunakan bur intan bundar (diameter bur 0,46 mm). setelah perforasi, kavitas diirigasi dengan larutan salin steril dan dikeringkan dengan cotton pellet. perdarahan yang timbul dihentikan dengan menggunakan ujung paper point steril. pada kelompok i, diaplikasikan zink oxide (dentorit®, dentoria, perancis) sedangkan pada kelompok ii diaplikasikan eep masing-masing ± 0,5 mg. aplikasi bahan pada permukaan pulpa dilakukan dengan menggunakan aplikator (ball aplicator) (diameter ujung aplikator 0,63 mm). semua gigi kemudian ditumpat dengan bahan tumpatan semen ionomer kaca (sik) (fuji ix®, gc tokyo, jepang). tiga ekor tikus kelompok perlakuan dan 1 ekor tikus kelompok kontrol dikorbankan dalam waktu 1, 2, dan 4 minggu setelah perlakuan. setelah tikus didekapitasi, tulang rahang di daerah interdental gigi molar pertama kanan rahang atas diambil. potongan jaringan dimasukkan ke dalam larutan fiksasi (formalin 10%) selama 4 hari pada temperatur kamar, dilanjutkan dengan proses dekalsifikasi dengan menggunakan larutan edta 10% selama ± 30 hari pada temperatur kamar, selanjutnya dilakukan proses dehidrasi terhadap spesimen menggunakan alkohol secara bertingkat. spesimen dimasukkan ke dalam larutan alkohol toluol (1:1), dan dilanjutkan dengan proses penjernihan menggunakan toluol murni, kemudian spesimen dimasukkan ke dalam larutan toluol parafin jenuh. setelah itu, dilakukan proses infiltrasi di dalam oven dengan cara 79sabir: respons inflamasi pada pulpa gigi tikus spesimen dimasukkan ke dalam parafin cair. dilakukan proses embedding terhadap spesimen dan diberi label/ kode. setelah tahap embedding selesai, maka jaringan diiris secara seri dengan menggunakan mikrotom dengan ketebalan ± 6 mm paralel sumbu panjang gigi. untuk melihat ada atau tidaknya sel inflamasi pada pulpa gigi, maka dilakukan pewarnaan hemaktosilin dan eosin (h & e). adapun prosedurnya adalah sebagai berikut: deparafinisasi dengan menggunakan larutan xylol dan alkohol, yang dilanjutkan dengan proses rehidrasi dengan alkohol lalu dicuci dengan air mengalir, dibilas dengan aquades, dan dilap. kemudian, kaca benda dimasukkan ke dalam hematoksilin meyer's dan dicuci dengan air mengalir serta dibilas dengan aquades, selanjutnya proses pewarnaan dilanjutkan dengan memasukkan kaca benda ke dalam eosin dan dibilas dengan aquades, kemudian pewarnaan dinilai di bawah mikroskop cahaya. bila pewarnaan telah dianggap baik, maka langkah selanjutnya ialah proses dehidrasi dengan alkohol secara bertingkat kemudian dilap. setelah itu, dimasukkan ke dalam larutan xylol dan terakhir object glass ditutup dengan deck glass dan dilakukan pengamatan mikroskop cahaya (leitzwetzlar®, jerman). respons inflamasi dievaluasi berdasarkan ada atau tidaknya leukosit polimorfonuklear (polymorphonuclear leukocytes = pmnl), dan sel makrofag. penilaiannya dibagi atas 4 kriteria, yaitu:30 0 = tidak terdapat infiltrasi sel inflamasi; 1 = infiltrasi oleh pmnl dan sel makrofag dalam jumlah sedikit; 2 = infiltrasi oleh pmnl dan sel makrofag dalam jumlah moderat; dan 3 = infiltrasi oleh pmnl dan sel makrofag dalam jumlah banyak. data yang diperoleh merupakan hasil pengamatan secara histologis dari ke-2 kelompok berskala ordinal yang selanjutnya dianalisis dengan menggunakan statistik nonparametrik. untuk mengetahui ada atau tidaknya perbedaan respons inflamasi ke-2 kelompok pada setiap periode waktu, dilakukan analisis dengan uji kruskalwallis, sedangkan untuk mengetahui ada atau tidaknya perbedaan respons inflamasi antara ke-3 periode waktu tiap kelompok dan antara ke-3 periode waktu terhadap kelompok, dilakukan analisis dengan uji mannwhitney.31,32 hasil hasil pengamatan histologis respons inflamasi (jumlah pmnl dan sel makrofag) pada kelompok kontrol dan kelompok eep pada 3 periode waktu dapat dilihat pada tabel 1 dan gambar berikut ini. pada tabel 1 tampak bahwa respons inflamasi (tingkat kepadatan pmnl dan sel makrofag) yang terjadi pada ke-2 kelompok hewan uji cenderung meningkat dengan bertambahnya waktu pengamatan. untuk mengetahui ada atau tidaknya perbedaan respons inflamasi yang timbul antara ke-3 periode waktu pada setiap kelompok, maka dilakukan uji kruskal-wallis dan hasilnya dapat dibaca pada tabel 2 berikut ini. tabel 2. hasil uji kruskal-wallis mengenai perbedaan respons inflamasi antara ke-3 periode waktu pada setiap kelompok mean rank kelompok minggu 1 minggu 2 minggu 4 uji kruskal wallis p kontrol eep 1,00 2,15 2,50 5,17 2,50 7,83 2,00 7,245 0,368 0,027* keterangan: * = signifikan pada p < 0,05 analisis statistik dengan uji kruskal-wallis di atas menunjukkan bahwa pada kelompok kontrol tidak terdapat perbedaan respons inflamasi yang signifikan (p > 0,05) antara ke-3 periode waktu (minggu 1, 2, dan 4), sebaliknya, pada kelompok eep terjadi perbedaan respons inflamasi secara signifikan (p < 0,05) antara ke-3 periode waktu. tabel 1. hasil pengamatan histologis respons inflamasi pada kelompok kontrol dan kelompok eep pada minggu ke-1, ke-2, dan ke-4 respons inflamasi periode waktu (minggu) kelompok jumlah spesimen (n) tidak ada ringan moderat berat 1 kontrol eep 1 3 − 2 1 1 − − − − 2 kontrol eep 1 3 − − − 2 1 1 − − 4 kontrol eep 1 3 − − − 1 1 2 − − 80 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 77–83 analisis lebih lanjut dilakukan dengan menggunakan uji mann-whitney untuk mengetahui respons inflamasi antara ke-3 periode waktu pada kelompok eep (tabel 3). tabel 3. hasil uji mann-whitney mengenai perbedaan respons inflamasi antara ke-3 periode waktu pada kelompok eep pada tabel 3 terlihat bahwa pada kelompok eep terdapat perbedaan respons inflamasi yang signifikan (p < 0,05) antara minggu ke-1 dengan minggu ke-2 dan antara minggu ke-1 dengan minggu ke-4, sebaliknya, tidak terdapat perbedaan respons inflamasi yang signifikan (p > 0,05) antara minggu ke-2 dengan minggu ke-4. untuk mengetahui perbedaan respons inflamasi antara ke-2 kelompok pada setiap periode waktu dapat diketahui dengan melakukan uji mann-whitney dan hasilnya dapat dibaca pada tabel 4. tabel 4. hasil uji mann-whitney mengenai perbedaan respons inflamasi antara ke-2 kelompok pada setiap periode waktu pada tabel 4 tampak bahwa tidak terdapat perbedaan respons inflamasi yang signifikan (p > 0,05) dari kelompok kontrol maupun kelompok eep pada ke-3 periode waktu. gambar 1, 2, dan 3 di bawah ini memperlihatkan respons inflamasi yang terjadi pada pulpa gigi tikus kelompok eep berturut-turut pada minggu ke-1, ke-2, dan minggu ke-4 setelah aplikasi. gambar 1. foto mikroskopik pulpa gigi tikus pada kelompok eep. tidak tampak adanya sel inflamasi 1 minggu setelah aplikasi eep. h & e, 40×. gambar 2. foto mikroskopik respons inflamasi ringan yang terjadi pada pulpa gigi tikus kelompok eep 2 minggu setelah aplikasi. terlihat sel inflamasi (panah) h & e, 40×. gambar 3. foto mikroskopik respons inflamasi moderat yang terjadi pada pulpa gigi tikus kelompok eep 4 minggu setelah aplikasi. terlihat sel inflamasi (panah). h & e, 40×. 81sabir: respons inflamasi pada pulpa gigi tikus pembahasan untuk mengetahui respons inflamasi yang terjadi pada pulpa gigi setelah aplikasi ekstrak etanol propolis (eep), maka pada penelitian ini digunakan tikus sebagai hewan model. hal ini disebabkan karena selain tikus mudah penanganannya dan relatif ekonomis dibandingkan dengan hewan primata, juga yang penting ialah reaksi pulpa gigi tikus terhadap suatu bahan pada prinsipnya mirip dengan reaksi yang terjadi pada pulpa gigi manusia. sementara pemilihan gigi molar pertama pada rahang atas tikus didasarkan atas pertimbangan bahwa struktur dan bentuk anatomi gigi tikus tersebut mirip dengan gigi molar manusia. selain itu, kecepatan atrisi akibat mastikasi pada permukaan oklusal gigi molar tikus lebih lambat dibandingkan dengan permukaan insisal gigi insisivus tikus.30,33 hasil pemeriksaan histologis menunjukkan bahwa 1 minggu setelah aplikasi zink oxide maupun eep respons inflamasi yang terjadi pada pulpa gigi tikus dapat dikatakan hampir sama. pada kelompok kontrol hanya terjadi inflamasi ringan, walaupun hingga saat ini belum diketahui pengaruh zink oxide (dentorit®) terhadap pulpa gigi (tabel 1). pemilihan zink oxide sebagai bahan kontrol pada penelitian ini disebabkan karena zink oxide merupakan bahan yang mempunyai ph yang netral sehingga tidak menimbulkan iritasi terhadap pulpa gigi,34sementara pada kelompok eep, tampak 2 dari 3 spesimen tidak memperlihatkan terjadinya inflamasi, dan hanya 1 spesimen yang mengalami inflamasi ringan (tabel 1). rendahnya respons inflamasi yang terjadi pada minggu ke-1 ini mungkin berhubungan dengan beberapa faktor, antara lain: 1) karakteristik dari bahan tumpatan permanen yang digunakan sik; 2) sifat antibakteri dan antiinflamasi dari eep; dan 3) konsentrasi dari eep. semen ionomer kaca diketahui memiliki sifat antibakteri karena bahan tumpatan ini memiliki ph yang rendah saat mengeras (setting) dan mampu melepaskan ion fluor.35 ion fluor mempengaruhi pertumbuhan bakteri dengan cara menghambat aktivitas enzim glikolitikenolase; penghambatan aktifitas enzim ini dapat dihubungkan dengan penurunan jumlah phosphoenolpyruvate yang dibutuhkan untuk transportasi gula ke dalam sel. sebagai akibatnya, terjadi hambatan pada glikolisis yang akan menghasilkan asam dan sintesis glukan intraselular.36 propolis diketahui memiliki beberapa efek farmakologis yang penting, antara lain sifat antibakteri baik terhadap bakteri gram positif9–11 maupun gram negatif.12 sifat antibakteri dari propolis ini bukan semata-mata disebabkan karena senyawa tunggal, namun karena efek sinergis dari beberapa senyawa yang terdapat pada propolis yang bersifat antibakteri yakni: flavonoid, asam ferulat, ester asam fenol, asam sinamat, dan berbagai ester asam kafeat. 8,37 mekanisme propolis dalam menghambat pertumbuhan bakteri belum sepenuhnya diketahui, namun demikian šimuth et al.38 melaporkan adanya beberapa komponen yang terdapat pada propolis yang mampu mengabsorbsi sinar ultraviolet sehingga menghambat kerja enzim polimerase rna bakteri untuk melekat pada dna sehingga replikasi dna bakteri tidak terjadi. selain itu, komponen tersebut juga menghambat kerja dari enzim endonuklease restriksi sehingga transkripsi tidak terjadi pada rna dan hal ini mengakibatkan pembelahan sel bakteri tidak terjadi karena terganggunya sintesis protein. mekanisme lain dikemukakan oleh takaisi-kikuni dan schilcher39 yang pada penelitiannya mendapatkan bahwa eep bersifat antibakteri terhadap bakteri streptococcus agalactiae melalui beberapa mekanisme, yakni dengan mencegah pembelahan sel bakteri dengan cara menghambat replikasi dna sehingga menyebabkan terbentuknya streptococcus pseudo-multicellular. selain itu eep juga menyebabkan terjadinya disorganisasi dari sitoplasma, membran sitoplasmik, serta dinding sel yang kesemuanya mengakibatkan bakteriolisis parsial dan penghambatan sintesis protein, sehingga dikatakan bahwa mekanisme antibakteri propolis terhadap bakteri sangat kompleks dan tidak dapat dianalogikan dengan cara kerja antibiotika klasik. selain bersifat antibakteri, propolis juga bersifat antiinflamasi.9,13,14 hal ini disebabkan karena adanya kandungan senyawa flavonoid, asam amino, terpen, serta derivate asam sinamat pada propolis.9,14 mekanisme propolis dalam menghambat inflamasi disebabkan karena propolis menghambat sintesis eikosanoid. penghambatan ini akan menyebabkan penurunan kandungan asam arakidonat pada jaringan membran fosfolipid sel40 yang lebih lanjut akan mengakibatkan terhambatnya pelepasan sejumlah mediator inflamasi seperti prostaglandin, leukotrin dan tromboksan.41,42 oleh karena ekstrak etanol propolis yang digunakan pada penelitian ini mempunyai konsistensi yang kental, maka diduga bahwa 1 minggu setelah aplikasi, konsentrasi ekstrak ini relatif masih tinggi. konsentrasi yang masih tinggi ini erat hubungannya dengan kemampuan antibakteri dari ekstrak flavonoid maupun ekstrak non flavonoid. hal ini sesuai dengan pendapat pelzcar dan chan 43 bahwa semakin tinggi konsentrasi bahan antibakteri, maka daya antibakterinyapun semakin besar. hasil pengamatan histologis menunjukkan bahwa pada minggu ke-2 dan ke-4 terjadi peningkatan respons inflamasi yang cukup tinggi pada kelompok eep dibanding minggu ke-1 (tabel 1). hal ini didukung oleh hasil analisis statistik yang menunjukkan perbedaan yang signifikan (p < 0,05) respons inflamasi yang terjadi antara minggu ke-1 dengan minggu ke-2 dan ke-4 pada kelompok eep (tabel 3). terjadinya peningkatan respons inflamasi mungkin disebabkan karena: 1) mulai berkurangnya jumlah atau konsentrasi ion fluor yang dilepaskan oleh bahan tumpatan sik;44–46 2) konsentrasi dari eep yang mulai menurun akibat metabolisme dari eep sehingga sifat antibakteri dan antiinflamasinyapun semakin berkurang;47 dan 3) terjadinya kebocoran mikro (microleakage) antara 82 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 77–83 permukaan email gigi dengan permukaan tumpatan sik walaupun secara visual hal ini tidak terlihat. kebocoran mikro yang terjadi pada pertemuan antara permukaan tumpatan dengan permukaan email gigi dapat meningkatkan kemungkinan terjadinya kontaminasi bakteri pada pulpa gigi, karena bakteri dapat berkembang biak di bawah tumpatan sik. kondisi ini dapat disebabkan karena kekuatan dan resistensi untuk terjadinya fraktur pada bahan tumpatan sik sangat rendah48 akibat sifat sik yang kurang menguntungkan, yaitu rapuh dan daya regang (tensile strength) yang rendah.49 dari penelitian ini dapat disimpulkan bahwa respons inflamasi yang terjadi pada pulpa gigi tikus kelompok eep secara numerik lebih ringan dibanding kelompok kontrol, walaupun secara statistik tidak menunjukkan perbedaan yang signifikan antara ke-2 kelompok (p>0,05). perlu dilakukan penelitian lebih lanjut untuk mengetahui kemungkinan penggunaan eep di bidang endodontik. daftar pustaka 1. ghisalberti el. propolis: a review. bee world 1979; 60: 59–84. 2. dadant cc. the hive and the honey bee. illinois: dadant and sons; 1984. p. 25–35. 3. greenaway w, scaysbrook t, whatley fr. the composition and plant origins of propolis: a report of work at oxford, bee world 1990; 71: 107–18. 4. chen y. apiculture in china. 1st ed. agricultural publishing house; 1993. p. 96–7. 5. hill r. propolis: the natural antibiotic. 6th ed. wellingborough: thorsons publishers ltd; 1981. p. 7–8. 6. kaal j. natural medicine from honey bees (apitherapy). amsterdam: kaal's printing house; 1991. p. 9–12. 7. sammataro d. avitabile a. the beekeeper's handbook. michigan: peach mountain press ltd. 1978; p. 15. 8. marcucci mc. propolis: chemical composition, biological properties and therapeutic activity. apidologie 1995; 26: 83–99. 9. dobrowolski jw, vohora sb, sharma k, shah sa, naqvi sah, dandiya pc. antibacterial, antifungal, antiamoebic, antiinflamatory and antipyretic studies on propolis bee products. j ethnopharmacol 1991; 35: 77–82. 10. kujumgiev a, tsvetkova i, serkedjieva y, bankova v, cristov r, popov s. antibacterial, antifungal and antiviral activity of propolis of different geographic origin. j ethno-pharmacol 1999; 64: 235–40. 11. moreno min, isla mi, cudmani ng, vattuone ma, sampietro ar. screening of antibacterial activity of amaicha del valle (tucumán, argentina) propolis. j ethno-pharmacol 1999; 69: 97–102. 12. grange jm, davey rw. antibacterial properties of propolis (bee glue). j r soc med 1990; 83(3): 159–60. 13. park eh, kim sh, park ss. anti-inflammatory activity of propolis. arch pharm res 1996; 19(3): 337–41. 14. khayyal mt, el ghazaly ma, el khatib as. mechanisms involved in the antiinflammatory effect of propolis extract. drugs exp clin res 1993; 19(5): 197–203. 15. cafarchia c, de laurentis n, milillo ma, losacco v, puccini v. antifungal activity of apulia region propolis. parassitologia 1999; 41: 587–90. 16. scheller s, stojko a, szwarnowiecka i, tustanowski j, obuszko z. biological properties and clinical application of propolis. vi. investigation of the influence of the ethanol extracts of propolis (eep) on cartilaginous tissue regeneration. arzneim-forsch 1977; 27(2): 2138–40. 17. stojko a, scheller s, szwarnowiecka i, tustanowski j, obuszko z. biological properties and clinical application of propolis. viii. experimental observation on the influence of ethanol extract of propolis (eep) on the regeneration of bone tissue. arzneim-forsch 1978; 28(1): 35–7. 18. scheller s, wilczok t, imielski s, krol w, gabrys j, shani j. free radical scavenging by ethanol extract of propolis. int j radiat biol 1990; 57(3): 461–5. 19. martinez-silveira g, gou-godoy a, ona-torriente r, palmer-ortiz mc, falcon-cuellar ma. preliminary study of the effect of propolis in the treatment of chronic gingivitis and oral ulceration. rev cubana estomatol 1988; 25(3): 36–44. 20. murray mc, worthington hv, blinkhorn as. a study to investigate the effect of a propolis-containing mouthrinse on the inhibition of de novo plaque formation. j clin periodontol 1997; 24: 796–8. 21. horax s. efek antimikroba obat kumur propolis terhadap penderita gingivitis. disertasi. ujung pandang: universitas hasanuddin; 2000. h.1. 22. ikeno k, ikeno t, miyazawa c. effects of propolis on dental caries in rats. caries res 1991; 25(5): 347–51. 23. koo h, rosalen pl, cury ja, park yk, ikegaki m, sattler a. effect of apis mellifera propolis from two brazilian regions on caries development in desalivated rats. caries res 1999; 33: 393–400. 24. park yk, koo mh, abreu jas, ikegaki m, cury ja, rosalen pl. antimicrobial activity of propolis on oral microorganisms. current microbiol 1998; 36: 24–8. 25. koo h, gomes bpfa, rosalen pl, ambrosano gmb, park yk, cury ja. in vitro antimicrobial activity of propolis and arnica montana against oral pathogens. arch oral biol 2000; 45: 141–8. 26. giamalia i, steinberg d, grobler s, gedalia i, the effect of propolis exposure on microhardness of human enamel in vitro. j oral rehab 1999; 26: 941–3. 27. magro-filho o, de carvalho ac. topical effect of propolis in the repair of sulcoplasties by the modified kazanjian technique. j nihon univ sch dent 1994; 36(2): 102–11. 28. gafar m, sacalus a, david n, david e. treatment of simple pulp gangrene with the apitherapy product propolis. stomatologie 1986; 33: 115–7. 29. kosenko sv, kosovich ti. the treatment of periodontitis with prolonged-action propolis preparations (clinical x-ray research). stomatologia-mosk 1990; 69(2): 27–9. 30. kirk eej, lim kc, khan mog.a comparison of dentinogenesis on pulp capping with calcium hydroxide in paste and cement form. oral surg oral med oral path 1989; 68(2): 210–9. 31. dayan a. pengantar metode statistik. jilid ii. cetakan ke-10. jakarta: lp3es; 1986. h. 27–35. 32. daniel ww. statistika nonparametrik terapan. jakarta: pt gramedia; 1989. h. 25-31. 33. lim kc, kirk eej. direct pulp capping: a review. endod dent traumatol 1987; 3: 213–9. 34. brosur dentorit®. france: dentoria; 2001. 35. mc comb d, ericson d. antimicrobial action of new proprietary lining cements. j dent res 1987; 66: 1026–8. 36. roth gi, calmes r. oral biology. st louis: the cv mosby co; 1981. p. 434–5. 37. bonvehi js, coll fv, jordà re. the composition, active components and bacteriostatic activity of propolis in dietetics. jaocs 1994; 71(5): 529–32. 38. šimúth j, trnovsk j, jeloková j. inhibition of bacterial dnadependent rna polymerases and restriction endonuclease by uvabsorbing components from propolis. pharmazie 1986; 41(3): 131–2. 39. takaisi-kikuni nb, schilcher h. electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propolis provenance. planta med 1994; 60(3): 222–7. 40. landolfi r, mower rl, steiner m. modification of platelet function and arachidonic acid metabolism by bioflavonoids. biochem pharmacol 1984; 33(9): 1525–30. 83sabir: respons inflamasi pada pulpa gigi tikus 41. yoshimoto t, furukawa m, yamamoto s, horie t, watanabekohno s. flavonoids: potent inhibitors of arachidonate 5lipoxygenase. biochem biophys res communs 1983; 116(2): 612–18. 42. tordera m, ferrandiz ml, alcaraz mj. influence of antiinflamatory flavonoids on granulation and arachidonic acid release in rat neutrophils. z naturforsch 1994; 49: 235–40. 43. pelzcar mj, chan ecs. dasar-dasar mikrobiologi. jakarta: universitas indonesia press; 1977. h. 450–8. 44. hatibovic-kofman s, koch g. fluoride release from glass ionomer cement in vivo and in vitro. swed dent j 1991; 15: 253–8. 45. seppä l, forss h, øgaard b. the effect of fluoride application on fluoride release and the antibacterial action of glass ionomers. j dent res 1993; 72(9): 1310–4. 46. diaz-arnold am, holmes dc, wistrom dw, swift jr ej. shortterm fluoride release/uptake of glass ionomer restoratives. dent mater 1995; 11: 96–101. 47. havsteen bh. the biochemistry and medical significance of flavonoids. pharmacol ther 2002; 96: 67–202. 48. mathis rs, ferracane jl. properties of a glass-ionomer/resincomposite hybrid material. dent mater 1989; 5: 355–8. 49. van de voorde a, gerdts gj, murchison df. clinical uses of glass ionomer cement: a literature review. quintessence 1988; 19(1): 53–61. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 44 no 3 sept 2011.indd 117 vol. 44. no. 3 september 2011 research report the role of probiotic on alveolar bone resorption desi sandra sari1, zahara meilawaty2, and m. nurul amin2 1 department of periodontics 2 department of biomedicine faculty of dentistry, jember university jember indonesia abstract background: probiotics are microbes derived from the group of lactic acid bacteria that work to maintain the health of hosts. probiotics can also be used to improve oral health. periodontal disease is usually marked with gingival inflammation and alveolar bone resorption. gram negative anaerobic bacteria that play important role in human periodontal disease are porphyromonas gingivalis. (p. gingivalis). p. gingivalis is a virulent bacteria in vivo or in vitro, and mostly found in subgingival plaque of periodontitis patients. purpose: this study is aimed to know the role of probiotics to inhibit the resorption of alveolar bone induced with p. gingivalis. methods: this study used male wistar rats divided into 4 groups. group i was control group (without treatment); group ii was induced with p. gingivalis atcc 33277 for 5 days; group iii was induced with p. gingivalis atcc 33277 and also injected with probiotics (lactobacillus casei atcc 4224) for 5 days simultaneously; and group iv was induced with p. gingivalis atcc 33277 for 5 days and also injected by probiotics (lactobacillus casei atcc 4224) in the next 5 days. after that, the samples were decapitated, taken their alveolar bone, and then were examined by immunohistochemistry to observe osteoclast activity in alveolar bone resorption by using tartrate-resistant acid phosphatase (trap) expression. all data were then analyzed statistically. results: it is known that there were significant differences of trap expression among all those treatment groups (p < 0.05). conclusion: it then can be concluded that probiotics can decrease osteoclast activity in periodontal tissue of wistar rats, so it can inhibit alveolar bone resorption. key words: probiotics, porphyromonas gingivalis, lactobacillus casei, tartrate-resistant acid phosphatase, osteoclast abstrak latar belakang: probiotik adalah mikroba dari golongan bakteri asam laktat yang bekerja mempertahankan kesehatan host dan probiotik dapat digunakan untuk meningkatkan kesehatan rongga mulut. penyakit periodontal ditandai dengan adanya keradangan pada gingiva dan resobsi tulang alveolar. bakteri gram negatif anaerob yang sangat berperan dengan penyakit periodontal pada manusia adalah porphyromonas gingivalis (p. gingivalis). p. gingivalis merupakan bakteri yang virulen, baik diuji secara in vivo maupun in vitro, dan banyak ditemukan pada plak subgingiva penderita periodontitis. tujuan: penelitian ini bertujuan untuk mengetahui peran probiotik dalam menghambat resorbsi tulang alveolar yang diinduksi p.gingivalis. metode: penelitian ini memakai tikus jenis wistar jantan sebagai sampel dan dibagi menjadi 4 kelompok: kelompok i yaitu kontrol tanpa perlakuan; kelompok ii di induksi p.gingivalis atcc 33277 selama 5 hari; kelompok iii di induksi p.gingivalis atcc 33277 ditambah suntikan probiotik (lactobacillus casei atcc 4224) selama 5 hari secara bersamaan; dan kelompok iv di induksi p.gingivalis atcc 33277 selama 5 hari ditambah suntikan probiotik (lactobacillus casei atcc 4224) 5 hari selanjutnya. setelah itu sampel didekaputasi, diambil tulang alveolar dilakukan pemeriksaan imunohistokimia untuk melihat aktivitas osteoklas dalam resopsi tulang alveolar dengan mendeteksi tartrate-resistant acid phosphatase (trap). data yang dikumpulkan dianalisis secara statistik. hasil: terdapat perbedaan yang bermakna antar kelompok untuk ekspresi trap (p < 0.05). kesimpulan: dapat disimpulkan bahwa probiotik dapat menurunkan aktivitas osteoklas pada wistar tikus yang mengalami periodontitis sehingga dapat mencegah resorbsi tulang alveol. kata kunci: probiotik, porphyromonas gingivalis, lactobacillus casei, tartrate-resistant acid phosphatase, osteoklas correspondence: desi sandra sari, c/o: bagian periodonsia, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: desisandrasari@yahoo.com, telp/fax : (0331) 333536/(0331) 33199. 118 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 117–121 introduction nowadays, dental treatment of oral periodontal disease is highly needed although the success rate of the treatment is still low. during the active phase of this disease there will be gingival bleeding and exudation which can cause quick damage, such as alveolar bone loss within weeks or months then causing tooth loss and tooth-movement.1 periodontal diseases caused by lypopolysaccharide (lps), can cause the occurrence of alveolar bone destruction. if the periodontium disease occurred during the eruption, it can cause the increasing of osteoclasts in alveolar bones which then causes premature eruption. the loss of bone substance is actually caused by the excessive activity of osteoclasts. organic acids secreted by osteoclasts can dissolve bone mineral density resulting in degradation of collagen. unfortunately, the giving of bone graft is only used for repairing bone damage that already exists, but can not be used for prevention. the procedure of bone graft therapy takes a very long time and complex.2 in recent years, many of probiotic have continually developed to be an actual research topic since the potential of probiotics in the future is enormous. the study of krasse et al.,3 showed the decreasing of gingival bleeding and the reducing level of moderate and severe gingivitis after therapy with probiotics on days 0 and 14 compared to that with placebo. nevertheless, the damage of the alveolar bone is still not proven. it is because probiotic bacteria can stimulate the immune systems, such as improving the function of macrophage phagocytosis, natural killer cells, monocytes and neutrophils, and can also stimulate the secretion of igm and increase the production of iga with the final result that can increase the production of immunoglobulin.4 oral cavity is the first part of gastrointestinal tract so that some actions of probiotics also play a role in the ecosystem of the oral cavity. lactobacillus in the oral cavity derived from healthy and unhealthy periodontal tissues showed antimicrobial activity against bacteria that causes periodontitis such as actinobacillus actinomycetemcomitans, porphyromonas gingivalis (p.gingivalis), and prevotella intermedia. lactobacillus can also produce antimicrobial components, such as organic acids, hydrogen peroxide, carbon peroxide, diacetil, antimicrobial substances which have low molecular weight and bacteriocin, as well as low ph.5 osteoclasts produce tartrate resistant acid phosphatase (trap) enzyme in large numbers which is an enzyme produced in osteoclast precursorsionly, called as a marker of osteoclasts. mature osteoclasts then express receptors for calcitonin and prostaglandins which are inhibitors of hormones or growth factors although not directly.6 therefore, the purpose of this study is to see the expression of trap in experimental animals, induced with p. gingivalis after the administration of probiotics. materials and methods the research was conducted on male wistar rats in the age of three months with 170–200 grams of body weight. those rats were fed with the same food. next, the acclimatization was conducted for a week before they were subjected to adaptation to location and food. the samples were devided into 4 groups, 10 samples each: group i is a control group without any treatment; group ii is a treatment group induced with p. gingivalis; group iii is a treatment group induced with p. gingivalis and at the same time also injected with probiotic bacteria; group iv is a treatment group induced with p. gingivalis for 5 days, and then followed with the injection of probiotic bacteria after 5 days. lactobacillus casei (l. casei) (atcc 4224) was conducted first on media with procedures in accordance with protocols from the factory. then, the infection of periodontal tissues by using p. gingivalis (atcc 33277) injected at the junctional epithelium at the gingival sulcus in the first incisive teeth of the right mandibular labial part, with 2×108 cfu dose, once a day for 5 days. next, the administration of probiotic bacteria was conducted by injecting the same area at the time of p.gingivalis induction. the dose used was 2×108 cfu/ml given once a day for 5 days. the administration of probiotic bacteria was then conducted in 2 ways, group iii given simultaneously and group iv administered after the induction of p.gingivalis for 5 days. the experimental animals either in the control groups or in the treatment groups, would be decapitated for their alveolar bone and gingival region of their first insisive of their right mandibular labial part. decalcified samples was conducted in order to release inorganic material in bone without damaging the protein by giving 5% citric acid for 5 days. fixation (paraffin embedding) was conducted followed with cutting process by using microtome with transverse direction (mesio-distal direction), where the thickness of the cuts was related with the needs. meanwhile, to see resorption caused by osteoclast activity as a result of periodontal infection, trap detection was conducted in order to be used as a cytochemical marker. for analyzing data, kruskal-wallis test was conducted, if there is difference, the test would be followed by mannwhitney test. results it is known that the lowest expression of trap occurred in group 4 induced with p. gingivalis for 5 days, and then induced also with probiotic l. casei (table 1). based on the mean data of trap expression, normality and homogeneity tests are then conducted by using kolmogorov smirnov and levene test. from the test results, it is known that the 119sari: the role of probiotic on alveolar bone table 1. the mean results of trap expression no. groups n mean standard deviation 1 i 10 4.54 1.34 2 ii 10 2.75 1.83 3 iii 10 1.85 1.08 4 iv 10 1.15 0.58 note: group i : control; group ii: induced with p. gingivalis; group iii: induced with p. gingivalis and l. casei at the same time; group iv: induced with p. gingivalis and followed with l. casei. figure 1. the graph of mann-whitney test result of the mean of trap expression. *: significant difference (p < 0.05) group i: control, group ii: induced with probiotic bacteria, group iii: induced with p. gingivalis and l. casei at the same time, group iv: induced with p. gingivalis and followed with l. casei. mean data is not normal. therefore, non-parametric test is then performed. non-parametric test conducted is kruskalwallis test. kruskal-wallis test results shows a significant difference (p < 0.005). it indicates that the administration of probiotics affects the expression of trap. further testing is then conducted, namely man-whitney test. the results of mann-whitney test also shows significant differences in all of those treatment groups (p < 0.005) (figure 1). the results of trap staining expression by using immunohistochemical techniques in the region of the dental alveolar bone tissue of the first insisive of the right mandibular labial part of wistar rats can be seen in figure 2. discussion the results of this study demonstrated the influence of probiotics on the expression of trap-induced p. gingivalis. trap is used as marker of osteoclasts and macrophages. the results showed that the decreasing of osteoclast formation in the surrounding of alveolar bone was characterized by the reducing of trap expression. the results of this study also indicated that the lowest mean of the expression of trap was showed in group iv, the treatment group of male wistar rats induced with lps of p.gingivalis, for five days, and then also injected by probiotic bacteria, lactobacillus casei, for next five days. moreover, lps of p. gingivalis induced in the anterior alveolar bone where there is a seed of rat incisors in order to obtain the condition of periodontitis. p. gingivalis had several virulence factors which are lps, fimbriae, toxic metabolism results, and proteases. these virulence factors could lead to diseases either directly or indirectly by activating host cells to release mediators of lps inflamatory.7 p. gingivalis is actually an important etiological factor in periodontitis that can induce inflammation and cause periodontal tissue damage. lps of p.gingivalis can even stimulate inflammatory cytokine expression in monocytes and gingival fibroblasts, and also induce bone resorption activity.8 periodontal disease caused by lps showed the occurrence of alveolar bone destruction. if periodontal disease occurred during the eruption, it can cause the increasing of osteoclasts in alveolar bone which can cause premature eruption.9 lps is an endotoxin which can bind surface receptors, cluster of differentiation-14 (cd14), on macrophages and monocytes. toll-like receptor-4 (tlr4) of macrophages and monocytes that bind with bacteria by the presence of cd14 then would induce the secretion of cytokines and other i ii iii iv groups t r a p e xp re ss io n 5 4,5 4 ,5 ,5 2 ,5 1 0,5 0 3 * * * * 120 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 117–121 inflammatory mediators, proinflammatory cytokines and prostaglandin e-2 (pge2). those mediators then stimulated osteoclast formation derived from stromal/osteoblast cells through cell-to-cell binding which is receptor activator for nfκ b ligand (rankl) in osteoblasts by receptor activator for nf-κ b (rank) on osteoclast progenitor. lps would stimulate the increasing of osteoblast rankl that functions are for the formation of osteoclasts, so osteoblasts in having differentiation and proliferation for multiplying themselves could not be impaired. mice injected with lps e. coli in their first maxillary molar mucosa region would have the increasing number and size of osteoclasts in each additional doses of lps, and later would cause alveolar bone resorption. bone resorption is actually caused by the degradation of the crystal structure of hydroxyapatite (ha) and the organic structure of collagen due to the low ph, from ±3.0 to 4.5, caused by the activity of osteoclasts.9 in the treatment group given lps of p. gingivalis, the expression of trap in alveolar bone was increased due to the level of lps on gingival crevicular fluid associated with the increasing of gingivitis severity. fine et al. cit. kusumawadini10 showed that lps levels were correlated with the percentage of gram-negative bacteria in healthy periodontal tissue and periodontitis. it also suggested that lps has biological activity contributing to the pathogenesis of periodontal diseases. the production of lactic acid by lactobacillus can make ph low and also inhibit the growth of pathogenic bacteria. lactobacillus is a probiotic bacterium that can prevent the growth of black-pigmented anaerobic bacteria considered to play a role in periodontal diseases in subgingival area.11 one of the factors triggering periodontal diseases is gram-negative bacteria on tooth root surface, a biofilm. lps and other compounds can improve access to the figure 2. immunohistochemistry figure of the alveolar bone of the first incisive of the right mandibular (labial part) of wistar rats. trap expression shows brown spots around the the alveolar bone (shown with arrows). a) group i: control; b) group ii: induced with p. gingivalis; c) group iii: induced with p. gingivalis and l. casei at the same time; d) group iv: induced with p. gingivalis and followed with l. casei. a b c d 121sari: the role of probiotic on alveolar bone gingival tissues, initiate and lead immunoinflammation causing the production of pro-inflammatory cytokines in high levels, which can induce the production of metalloproteinase matrix resulting in the destructions of tissue, periodontal ligament, and alveolar bone resorption.12 l. casei of probiotic bacteria, can regulate the balance of local and systemic immune responses against infection by releasing proinflammatory cytokines and activating natural killer (nk) cells in order to eliminate pathogenic bacteria.13 probiotic bacteria can also form biofilms as oral mucosal defense layer against oral diseases. this biofilm layer can prevent pathogenic bacteria invading the tissue by filling the empty cavities of the tissue that can be entered by pathogenic bacteria. in addition, biofilms may prevent the growth of cariogenic bacteria and other bacteria causing periodontal diseases.14 lactobacilli, produce several antimicrobial compounds including organic acids (lactic acid, acetic acid, succinic acid), hydrogen peroxide, and bacteriosin as well as adhesive inhibitors that may affect oral bacteria. inflammation of the gingiva is one of the effects of food spoilage caused by pathogenic bacteria. probiotics control the growth of pathogenic bacteria in order to prevent gingivitis. probiotics have low ph so that bacteria can not form dental plaque and calculus plaque causing periodontal diseases.15 probiotics also produce antioxidants which can prevent the stain and plaque formations by neutralizing free electrons required for the formation of minerals or calculus. besides that, probiotics can also damage putrescence odors by fixating toxic gas and turning it into gas needed for metabolism.16 l. casei can be used for the treatment of vascular endothelial cells in mice with coronary arteritis.17 l. casei is indicated to be able to stimulate pmn cells on endothelial cells, and also able to increase intercellular adhesion molecule-1 (icam-1). the increasing of these materials can reduce inflammation in coronary arteries of studied animals. probiotics, can also control the growth of pathogenic bacteria in order to prevent gingivitis. probiotics actually have low ph so that bacteria can not form dental plaque and calculus plaque causing periodontal diseases. it can be concluded that probiotics can influence the decreasing of osteoclast activity in periodontal tissue of wistar rats, so it can inhibit alveolar bone resorption. acknowledgement the authours would like to express their special gratitude to ministry of national education of the republic of indonesia, in this case through dipa, research of jember university who has funded this study and research institute of the university of jember who facilitated this study. references 1. novak kf, novak mj, newman mg, takei hh, klokkevold pr. aggressive periodontitis in clinical periodontology. 10th ed. philadelphia: wb saunders; 2006. p. 506–11. 2. lindhe j. clinical periodontology and implant dentistry. 4th ed. blackwell munksgaard. 2003. p. 133–9. 3. krasse p, carisson b, dahl c, paulsson a, nilsson a, sinkiewicz g. decreased gum bleeding ang reduced gingivitis by the probiotic lactobacillus reuteri. swed dent j 2006; 30(2): 55–60. 4. soebijanto. ranuh r. konsep dasar penggunaan prebiotik-probiotik di dalam susu formula bayi dan susu formula khusus alergi. 2006. available at: http://www.pediatrik.com. accessed may 30, 2007. 5. sugano n, matsuoka t, koga y, ito k. effects of probiotics on periodontal disease. dentistry in japan. 2007; 43: 123–6. 6. arnett t. bone structure and bone remodelling. london: university college london; 2003. p. 1-10. 7. sugawara s, nemoto e, tada h, miyake k, imamura t, takada h. proteolysis of human monocyte cd14 by cystein proteinase (gingipains) from porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. j of immunol 2000; 165: 411–8. 8. suryono, kido j, hayashi n, kataoka m, nagata t. calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-α, and interleukin-1β. j periodontol 2005; 76: 437–42. 9. indahyani de, santoso as, utoro t. pengaruh induksi lipopolisakarida (lps) terhadap osteopontin tulang alveolaris tikus pada masa erupsi gigi. ind j dent 2007; 14(1): 2–7. 10. kusumawardani b. pengaruh pajanan lipopolisakarida bakteri gram negatif terhadap viabilitas sel pada kultur fibroblas gingiva. j stomatognatic 2005; 2(3): 14-8. 11. sari ds. pengaruh probiotik terhadap penyakit periodontal. j stomagtonatik 2008; 5(2): 65–140. 12. roeslan bo. aspek imunologik hubungan beberapa penyakit periodntal dan penyakit sistemik. majalah ilmiah kedokteran gigi 2002; edisi khusus foril: 15–21. 13. winkler p, ghadimi d, schrezenmeir j, kraehenbuhl jp. molecular and cellular basis of microflora-host interactions. j nutr 2007; 137: 756s–72s. 14. flichy-fernández aj, alegre-domingo t, peñarrocha-oltra d, peñarrocha-diago m. probiotic treatment in the oral cavity: an update. j oral med pathology 2010; 15(5): 272–5. 15. kusumawati n, jenie bsl, setyahadi s, hariyadi rd. aktivitas antibakteri laktobasili asal makanan fermentasi indonesia terhadap patogen dan pengaruhnya terhadap mikroflora usus tikus. j obat bahan alam 2008; 7(1): 69–75. 16. deepa d, mehta ds. is the role of probiotics friendly in the treatment of periodontal diseases?. j ind soc periodontol 2009; 13(1): 30–1. 17. fuller r. probiotic 2: applications and practical aspects. united kingdom: chapman & hall; 1997. p. 10–39. << 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 154 dental journal (majalah kedokteran gigi) 2023 september; 56(3):154–159 original article surface roughness assessment with fluoride varnish application: an in vitro study anie apriani1, silvia naliani2, rudy djuanda3, shania hysan teanindar4, jessica quiteria florenthe4, ferri baharudin4 1department of pediatric dentistry, faculty of dentistry, maranatha christian university, bandung, indonesia 2department of prosthodontics, faculty of dentistry maranatha christian university, bandung, indonesia 3department of conservation dentistry, faculty of dentistry, maranatha christian university, bandung, indonesia 4students of dentistry, faculty of dentistry, maranatha christian university, bandung, indonesia abstract background: the cause of cavities is initially due to roughness on the tooth surface, requiring fluoride varnish to prevent caries, as the varnish applies a fluoride compound to the tooth surface. fluoride varnish reacts with the tooth enamel surface to form calcium fluoride and fluorapatite, thus making the enamel surface more resistant to demineralization and damage. purpose: this study aims to compare the roughness of tooth enamel surfaces among three fluoride varnishes under acidic conditions. methods: the research method uses three fluoride varnish materials: sodium fluoride 5% + tricalcium phosphate, calcium fluoride, and sodium fluoride 5% + casein phosphopeptide-amorphous calcium phosphate 2%. samples of 81 teeth were divided into three groups (group 1 without fluoride varnish application, group 2 application of fluoride varnish with ph 3, and group 3 application of fluoride varnish with ph 5). the teeth were tested before and after application of the varnishes using the scanning electron microscope and surface roughness tests. results: the results showed a significant difference in the mean surface roughness of the enamel of the anterior deciduous teeth tested with fluoride varnish. the before and after comparisons in the ph 3 and ph 5 groups were very significant (p-value 0.000). the comparison results in each ph group after fluoride varnish administration showed no significant difference (ph 3 p-value 0.074 and ph 5 p-value 0.196). the tooth surfaces appear to be rougher after administration of an all-acid solution. conclusion: there is a difference in surface roughness of primary teeth after being given fluoride varnish in low ph 3 immersion for 24 hours. keywords: anterior deciduous teeth; fluoride varnish; surface roughness article history: received 8 june 2022; revised 21 december 2022; accepted 14 february 2023; published 1 september 2023 correspondence: anie apriani, department of pediatric dentistry, faculty of dentistry, maranatha christian university. jl. surya sumantri no. 65 bandung, 40164, indonesia. email: anie.apriani@dent.maranatha.edu introduction dental caries is the most common health problem affecting about 60%–90% of children and adults in the world’s population.1 in the early stages of the caries process, bacterial fermentation of carbohydrates can lower the local ph level below its standard value (ph >5.5). critical ph (<5.5) results in minerals dissolution on the enamel surface, in a process called demineralization.2 continued demineralization will result in cavities on the enamel surface.3 the success of topical fluoride in reducing the incidence of dental caries has been widely demonstrated. in recent years, studies have supported the idea that fluoride varnish, especially 5% sodium fluoride varnish, not only prevents caries but can also stop early caries lesions.4 five percent fluoride with added tricalcium phosphate (tcp) has a more significant remineralization increase than conventional 5% sodium fluoride.5 sodium fluoride 5% added with tcp is specially designed for pediatric use and has minimal toxicity.6 fluoride varnish material such as calcium fluoride is one of the materials used in dentistry and can be found in fluoride varnish to prevent caries. calcium fluoride is considered the best source of fluoride when compared to sodium fluoride (naf) when the same concentration is used in the enamel with the gel form. when rinsed, calcium fluoride resulted in seven times greater fluoride deposition in healthy enamel when compared to naf.7 in addition, calcium fluoride has the advantage of demineralizing and remineralizing enamel copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 mailto:anie.apriani@dent.maranatha.edu https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 155apriani et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 154–159 and dentin. calcium fluoride nanoparticles are thought to increase tooth strength, although further studies are needed. the release of fluoride ions in calcium fluoride prevents the process of demineralization of enamel and dentin, which in turn prevents secondary caries.8 c a s e i n p h o s p h o p e p t i d e a m o r p h o u s c a l c i u m phosphate (cpp-acp) is an anticariogenic agent that can remineralize lesions on enamel and dentin surfaces. routine administration of cpp-acp can replace calcium lost due to demineralization.9 the insoluble nature of cppacp, its crystal structure at neutral ph, can hold calcium and phosphate that can enter under the surface of tooth enamel. high concentrations of calcium and phosphate ions in plaque can reduce demineralization and promote remineralization.10 surface roughness is a test method used to evaluate changes in the surface of the hard tissues of the teeth. this is important to know because a rough surface is a medium that facilitates the attachment of bacteria and debris. the increase of surface roughness shows demineralization of enamel, and it can allow decay on the tooth.11 research studies have shown that demineralization, the degradation of changes in surface roughness of the enamel surface of primary teeth, can be evaluated through a scanning electron microscope (sem).12 a sem has the advantage of an objective magnification reaching ten nanometers. the value of the surface roughness of the enamel can be measured using the surface roughness tester. the purpose of this study was to determine the effect before and after the application of various fluoride varnishes on the surface roughness of primary tooth enamel under acidic conditions. materials and methods eighty-one maxillary or mandibular anterior primary teeth have been cleaned and cut where the primary teeth have no caries or restorations, and no anatomical deformities. the materials used in the research are fluoride varnish (sodium fluoride 5% + tricalcium phosphate [3m™ clinpro™ white varnish], calcium fluoride [for-lux] and cpp-acp [mi from gc corporation, japan] [sodium fluoride 5% w/w and cpp-acp 2% w/w]). distillation solution, ph 3 and ph 5 were used as a solution for soaking. a solution of ph 3 was obtained by adding 142.5 ml of acetic acid to 107.5 ml of water, while a solution of ph 5 was obtained by adding 15 grams of sodium acetate to 500 ml of water and adding 500 ml of 0.1 m acetic acid. the ph solution was measured using ph meter paper, and 25 ml of each solution was put into a different plastic vial. the tool used to measure roughness was the surface roughness test carried out at the dental material testing & center of research laboratory, faculty of dentistry, trisakti university, jakarta. the sem test was carried out at the faculty of mathematics and natural sciences at institut teknologi bandung. this research methodology is in vitro experimental with preand post-test research design. a total of 81 anterior primary teeth were cleaned and polished to remove debris. each tooth sample was embedded in 2x2x1 cm acrylic resin, with the labial surface facing upward to stabilize the placement of the tooth sample during roughness measurement. the sample was divided into 3 groups, each consisting of 27 samples. the first group was the control group which was not given any treatment; the second group was the group that was immersed in a ph 3 solution; the third group was the group which was immersed in a ph 5 solution. after 24 hours of soaking, the varnish layer was cleaned using a scalpel knife and cotton swab soaked in acetone (table 1). the surface roughness of the anterior deciduous teeth was measured using a surface roughness tester then samples from each group were evaluated for roughness using sem with a scan area of 200 x 200 µm and a magnification of 1100x.13,14 two measurements were made for each sample. the data analysis method used in this study was a paired t-test to find the statistical differences in the increase in roughness of each group (groups 1, 2, and 3). the significance level was set at (p < 0.05). the one-way anova and tukey analysis methods were also used to determine differences in enamel surface roughness between test groups. results comparison of the results of enamel surface roughness measurements between control groups and the fluoride varnish application showed an increase in enamel surface roughness based on applied with different ph solution. the highest mean in the control group that was not given fluoride varnish with ph 3 immersion was 1.415 µm (1b); the same was also found in the control group, which was not given fluoride varnish with ph 5 immersion 1.0815 µm (1c). the treatment group with the best fluoride varnish was found in the calcium fluoride group with the lowest average enamel surface roughness of 0.9815 µm (2b) by table 1. distribution of control group and fluoride varnish group group n control 1a. distillate water 1b. ph 3 1c. ph 5 9 9 9 ph 3 2a. sodium fluoride 5% + tricalcium phosphate 2b. calcium fluoride 2c. naf 5% + cpp acp 9 9 9 ph 5 3a. sodium fluoride 5% + tricalcium phosphate 3b. calcium fluoride 3c. naf 5% + cpp acp 9 9 9 total 81 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 156 apriani et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 154–159 table 2. average surface roughness of primary tooth enamel using fluoride varnish and control at ph 3 and ph 5 group before after mean (µm) sd mean (µm) sd control (group 1) 1a. distillate water 0.3593 0.0846 0.3778 0.1106 1b. ph 3 0.4778 0.1236 1.415 0.487 1c. ph 5 0.4889 0.1213 1.0815 0.2887 ph 3 (group 2) 2a. sodium fluoride 5% + tricalcium phosphate 0.3481 0.1094 1.374 0.1106 2b. calcium fluoride 0.4593 0.1051 0.9815 0.2376 2c. naf 5% + cpp acp 0.4778 0.1067 1.341 0.414 ph 5 (group 3) 3a. sodium fluoride 5% + tricalcium phosphate 0.4481 0.1144 0.9074 0.2350 3b. calcium fluoride 0.4852 0.1334 0.9370 0.2058 3c. naf 5% + cpp acp 0.4481 0.0747 0.8481 0.1804 table 3. comparative test results of surface roughness of primary teeth before and after immersion by group group n gain t-count t-table p-value control (group 1) 1a. distillate water 9 0.019 -0.852 -2.306 0.416 1b. ph 3 9 0.937 -6.014 -2.306 0.000* 1c. ph 5 9 0.589 -6.011 -2.306 0.000* ph 3 (group 2) 2a. sodium fluoride 5% + tricalcium phosphate 9 0.893 -9.041 -2.306 0.000* 2b. calcium fluoride 9 0.522 -9.695 -2.306 0.000* 2c. naf 5% + cpp acp) 9 0.863 -6.388 -2.306 0.000* ph 5 (group 3) 3a. sodium fluoride 5% + tricalcium phosphate 9 0.459 -6.239 -2.306 0.000* 3b. calcium fluoride 9 0.452 -6.180 -2.306 0.000* 3c. naf 5% + cpp acp 9 0.400 -7.083 -2.306 0.000* * p<0.005 table 4. result of comparison of surface roughness of primary teeth before and after immersion between groups group n sd f-value p-value ph 3 (group 2) + control 72 0.277493 25.29 0.000* ph 5 (group 3) + control 72 0.277493 18.87 0.000* *p<0.005 table 5. comparison of surface roughness of primary teeth after application of fluoride varnish immersion ph 3 and ph 5 group n f-value p-value ph 3 (group 2) + control 36 2.54 0.074 ph 5 (group 3) + control 36 1.66 0.196 immersion at ph 3, while the treatment group with the best cpp-acp fluoride varnish with the lowest average enamel surface roughness was 0.8481 µm (3c) at immersion with ph 5 (table 2). the statistical test results with the t-test resulted in a significant difference (p<0.005) in the entire test group except for the distilled water sample (table 3). the test results based on immersion in a ph 3 solution in the study by comparing the fluoride varnish used showed a significant difference in the enamel roughness of primary teeth before and after application (p<0.000). additionally, immersion in a ph 5 solution also demonstrated significant results in this group (table 4). in contrast to the test results above, tests in group 2 and group 3, utilizing tukey’s test showed no significant difference between the three fluoride varnish materials used in the study. this result shows that the three varnish fluoride materials have similarities in the roughness of the primary tooth enamel that has been immersed in ph 3 and ph 5 (table 5). the results of observations using sem in groups 1, 2, and 3 demonstrated increased roughness of tooth enamel after the teeth were immersed in a low-ph solution. however, in contrast, the teeth immersed in distilled water did not show an increase in tooth enamel roughness (figures 1–3). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 157apriani et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 154–159 a b c d e f figure 1. overview of enamel roughness of primary teeth by sem testing at 1100x magnification. control group (group 1) before and after immersion without fluoride varnish application. (a and b distilled water, c and d ph 3, e and f ph 5). a b c d e f figure 2. an overview of the enamel roughness of primary teeth by sem testing at 1100x magnification. group ph 3 (group 2) before and after immersion in fluoride varnish application. (a and b sodium fluoride 5% + tricalcium phosphate (tcp), c and d calcium fluoride, e and f naf 5% + cpp-acp). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 158 apriani et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 154–159 discussion tooth surface roughness is considered one of the determinants in describing the effect of remineralization and demineralization processes on the surface; continuous remineralization can reduce tooth decay.13 several studies have proven the potential of fluoride varnish as an effective anti-caries agent. when used correctly, fluoride varnish can reduce the incidence of caries by 40–56% and reduce enamel surface roughness.14 research that has been done previously found that fluoride varnish was able to reduce fissure caries by 36%, effected a reduction of 66% for noncarious fissure surfaces, as well as demonstrated a 51% reversal of tooth decalcification structure and a 35–21% reduction in enamel demineralization.15 in this study, there were differences in the surface roughness of the primary tooth enamel, which had been soaked in a solution of ph 3 and ph 5 accompanied by the application of fluoride varnish (sodium fluoride 5% + tricalcium phosphate group, calcium fluoride group and naf 5% + casein phosphopeptide-amorphous calcium phosphate group) (table 1). there was an increase in the surface roughness of the primary tooth enamel after immersion in a solution of ph 3 and ph 5 for 24 hours, even though fluoride varnish was applied; this was due to the high value of surface roughness in the ph 3 group due to low ph which could trigger the demineralization process in the teeth. what happens can cause the release of calcium ions in tooth enamel. the release of calcium ions in tooth enamel can cause microporosity resulting in roughness on the tooth surface. the lower the ph level in the oral cavity, the faster tooth decay occurs.16,17 this is because a low ph will cause a continuous increase in hydrogen ions in the tooth enamel; these ions can damage hydroxyapatite in tooth enamel and dissolve enamel crystals, therefore the roughness of the teeth increases.18 in the groups of ph 3 and ph 5, the increase in surface roughness still occurred even though fluoride varnish was applied. according to the results of research conducted by lippert f, it was stated that fluoride varnishes have a susceptibility to loss of fluoride ions at low ph, so the varnish cannot work optimally.19 however, when compared with the results of surface roughness in the control group or the group without fluoride varnish, the increase in roughness was higher, with a value of 0.937 at ph 3 and 0.589 at ph 5, meaning that the administration of fluoride varnish could increase the ability of primary teeth to withstand the demineralization process caused by the ph cycle and reduce the level of roughness on the tooth surface. these results can be found in a study conducted by baothman and assery.4 immersion in ph solution was carried out for 24 hours in the treated group, simulating the consumption of sweet drinks or foods that can cause an acidic atmosphere for 4 minutes every day for 1 year (4 minutes x 30 days x 12 months = 1440 minutes = 24 hours).20 the american dental association council on scientific affairs concluded that fluoride varnish should be applied every six months a b c d e f figure 3. overview of enamel roughness of primary teeth by sem testing at 1100x magnification. ph 5 group (group 3) before and after immersion in fluoride varnish application. (a and b sodium fluoride 5% + tricalcium phosphate (tcp), c and d calcium fluoride, e and f naf 5% + cpp-acp). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 159apriani et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 154–159 because it is effective in reducing the prevalence of caries and also preventing caries in primary and permanent teeth in children and adolescents.21 the application of fluoride varnish in this study was only carried out once in 24 hours, which simulated the application of fluoride varnish, which is carried out once a year. however, the surface roughness of the enamel still experienced an increase in roughness after fluoride varnish was applied to the teeth compared to before immersion. this study showed that immersion of the teeth using ph 3 resulted in more roughness on the enamel surface compared to ph 5 and controls. this is also the same in all treatments using fluoride varnish. surface roughness can be seen in the examination using sem (figures 1–3); from this, it can be seen that the application of fluoride varnish, which is only done once, is not enough and needs to be repeated. fluoride varnish application should be repeated every three or six months per year.22 when the ph in the oral cavity is below a critical ph, demineralization can quickly occur. in this situation, the inorganic elements of the enamel become soluble. fluoride varnish plays an essential role in protecting teeth, especially in preventing demineralization of tooth enamel. fluoride varnish is believed to form an intraoral fluoride reservoir via calcium ions.2 each fluoride varnish used in this study has different advantages and effectiveness. however, based on the results of research carried out and tested with statistical tests, there are significant differences before and after treatment with fluoride varnish application by immersing ph 3 and ph 5 (table 4). when compared between fluoride varnishes, namely between the sodium fluoride 5% + tricalcium phosphate group, the calcium fluoride group, and the naf 5% + casein phosphopeptide-amorphous calcium phosphate (cpp-acp) group, there was no significant difference in surface roughness of the primary tooth enamel (table 5). the result is in line with the study conducted by baothman and assery, which stated that there was no difference in enamel roughness in primary teeth after applying fluoride varnish.4 from the research that has been done, it is evident that there are differences in the surface roughness of the primary teeth before and after fluoride varnish. the increase in surface roughness occurred in all study groups. however, in the ph 3 group where calcium fluoride varnish was applied, the increase in roughness was lower than without calcium fluoride varnish. in contrast, in the ph 5 group, the naf 5% + cpp-acp varnish was better, and the enamel surface roughness was minor compared to the group with another varnish. it proves fluoride varnish can reduce the demineralization process in primary teeth under acidic conditions to prevent early caries. references 1. marinho vc, worthington h v, walsh t, clarkson je. fluoride varnishes for preventing dental caries in children and adolescents. cochrane database syst rev. 2013; (7): cd002279. 2. tuloglu n, bayrak s, tunc e sen, ozer f. effect of fluoride varnish with added casein phosphopeptide-amorphous calcium phosphate on the acid resistance of the primary enamel. bmc oral health. 2016; 16(1): 103. 3. kawashita y, kitamura m, saito t. early childhood caries. int j dent. 2011; 2011: 725320. 4. ba ot h m a n a , a s s e r y m. e f fe ct of mo d i f ie d 5% so d iu m fluoride on the surface roughness and hardness of the enamel of primary incisors: an in vitro study. saudi j oral sci. 2017; 4(1): 28–32. 5. alamoudi sa, pani sc, alomari m. the effect of the addition of tricalcium phosphate to 5% sodium fluoride varnishes on the microhardness of enamel of primary teeth. int j dent. 2013; 2013: 486358. 6. kathariya md, patil sk, fatangare m, jadhav rg, shinde gr, pawar ss. caries preventive effect of sodium fluoride varnish on deciduous dentition: a clinical trial. j contemp dent pract. 2017; 18(12): 1190–3. 7. ghafar h, khan mi, sarwar hs, yaqoob s, hussain sz, tariq i, madni au, shahnaz g, sohail mf. development and characterization of bioadhesive film embedded with lignocaine and calcium fluoride nanoparticles. aaps pharmscitech. 2020; 21(2): 60. 8. nayak ak, mazumder s, ara tj, ansari mt, hasnain ms. calcium fluoride-based dental nanocomposites. in: applications of nanocomposite materials in dentistry. elsevier; 2019. p. 27–45. 9. martins l, pereira k, costa s, traebert e, lunardelli s, lunardelli a, traebert j. impact of dental caries on quality of life of school children. pesqui bras odontopediatria clin integr. 2016; 16(1): 307–12. 10. fajriani f, handini ad. topical applications effect of casein phospho peptide-amorphous calcium phosphate and sodium fluoride on salivary mutans streptococci in children. dent j. 2014; 47(2): 110–4. 11. abreu lg, paiva sm, pretti h, lages emb, júnior jbn, ferreira ran. comparative study of the effect of acid etching on enamel surface roughness between pumiced and non-pumiced teeth. j int oral heal jioh. 2015; 7(9): 1–6. 12. ahmad akhoundi ms, aghajani f, chalipa j, sadrhaghighi ah. the effect of remin pro and mi paste plus on bleached enamel surface roughness. j dent (tehran). 2014; 11(2): 216–24. 13. abdil-nafaa s, qasim a. the effect of silver diamine fluoride and fluoride varnish on roughness of primary teeth enamel (an in vitro study). al-rafidain dent j. 2020; 20(2): 296–307. 14. mullan f, austin rs, parkinson cr, hasan a, bartlett dw. measurement of surface roughness changes of unpolished and polished enamel following erosion. ranjitkar s, editor. plos one. 2017; 12(8): e0182406. 15. vaikuntam j. fluoride varnishes: should we be using them? pediatr dent. 2000; 22(6): 513–6. 16. ham r un n, ka r tika d. tingkat keasaman m inuman r ingan mempengaruhi kelarutan mineral gigi. makassar dent j. 2018; 1(1): 9–15. 17. panigoro s, pangemanan dhc, juliatri. kadar kalsium gigi yang terlarut pada perendaman minuman isotonik. e-gigi. 2015; 3(2): 356–60. 18. widyaningtyas v, rahayu yc, barid i. the analysis of enamel remineralization increase in pure soy milk immersion using scanning electron microscope (sem). j pustaka kesehat. 2014; 2(2): 258–62. 19. lippert f. fluoride release from fluoride varnishes under acidic conditions. j clin pediatr dent. 2014; 39(1): 35–9. 20. sundari i. perbedaan kekasaran permukaan gic tanpa dan dengan penambahan kitosan setelah perendaman minuman isotonik. j mater kedokt gigi. 2016; 1(5): 49–55. 21. virupaxi sg. comparative evaluation of longevity of fluoride release from three different fluoride varnishes – an in vitro study. j clin diagnostic res. 2016; 10(8): zc33-6. 22. soares les, de carvalho filho acb. protective effect of fluoride varnish and fluoride gel on enamel erosion: roughness, sem-eds, and µ-edxrf studies. microsc res tech. 2015; 78(3): 240–8. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p154–159 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p154-159 98 volume 47, number 2, june 2014 koreksi gigitan terbalik posterior dan anterior dengan alat cekat rapid maxillary expansion dan elastik intermaksila (correction of posterior and anterior crossbite using fixed orthodontic appliance with rapid maxillary expansion and intermaxillary elastic) retno dewati, teguh budi wibowo, dan masyithah departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: children with anterior and posterior crossbite usually have a complaint in aesthetic and masticatory function. it could caused by bad habits and hereditary factors which made worse condition. purpose: the purpose of this case report was to report the use of orthodontic appliance rapid maxillary expansion (rpe) and intermaxillary elastic to correct posterior and anterior crossbite in teenage patient. case: a fourteen years-old teenage female patient came to dental hospital dentistry universitas airlangga with case of anterior posterior cross bite and unerupted permanent teeth. case management: the case was treated using orthodontic fixed appliance rapid maxillary expansion (rpe) and followed by intermaxillary elastics. the posterior cross bite treatment took 4 weeks used of orthodontic fixed appliance rpe, while, treatment of anterior cross bite which used intermaxillary elactic was done within three month to achieved normal occlusion. conclusion: this case report showed that the orthodontic appliance rapid maxillary expansion (rpe) and intermaxillary elastic could be used to correct posterior and anterior crossbite. key words: anterior posterior cross bite, rapid maxillary expansion, intermaxillary elastic abstrak latar belakang: anak dengan gigitan terbalik anterior dan posterior pada umumnya mempunyai keluhan dalam hal estetik dan fungsi pengunyahan. kondisi gigitan terbalik biasanya disebabkan oleh adanya kebiasaan buruk dan faktor keturunan yang semakin memperparah keadaan tersebut. tujuan: laporan kasus ini melaporkan pemakaian alat cekat rapid maxillary expansion (rpe) dan elastik intermaksila untuk mengkoreksi gigitan terbalik posterior dan anterior pada anak remaja. kasus: pasien remaja perempuan berusia 14 tahun datang ke rumah sakit gigi dan mulut fakultas kedokteran gigi universitas airlangga surabaya dengan kasus gigitan terbalik anterior posterior dan terdapat gigi permanen yang tidak tumbuh. tatalaksana kasus: perawatan yang dilakukan adalah koreksi gigitan terbalik dengan menggunakan alat ortodonsia cekat rapid maxillary expansion (rpe) dan dilanjutkan dengan pemasangan elastik intermaksila. perawatan koreksi gigitan terbalik posterior memerlukan waktu 4 minggu menggunakan alat ortodonti cekat rpe, sedangkan koreksi gigitan terbalik anterior dilakukan dalam 3 bulan untuk mencapai oklusi normal. simpulan: laporan kasus ini menunjukkan bahwa pemakaian alat cekat rapid maxillary expansion (rpe) dan elastik intermaksila dapat mengkoreksi gigitan terbalik posterior dan anterior. kata kunci: gigitan terbalik anterior posterior, rapid maxillary expansion, elastik intermaksila korespondensi (correspondence): retno dewati, departemen ilmu kedokteran gigi anak, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhew_bale@yahoo.com research report 99dewati, et al.: koreksi gigitan terbalik posterior dan anterior pendahuluan gigitan terbalik posterior adalah maloklusi yang melibatkan gigi kaninus, premolar, dan molar yang ditandai dengan cups bukal gigi posterior rahang atas berada lebih ke lingual daripada cups bukal gigi posterior rahang bawah pada saat beroklusi.1 sebagian besar kasus gigitan terbalik posterior diakibatkan oleh lengkung rahang atas yang sempit diikuti dengan adanya pergeseran mandibula, mengakibatkan deviasi garis median. faktor lain yang termasuk etiologi gigitan terbalik posterior adalah faktor herediter, kebiasaan menghisap jempol, dan bernafas melalui mulut oleh karena pembesaran tonsil dan adenoid.2 untuk menentukan rencana perawatan gigitan terbalik posterior, beberapa hal yang harus ditentukan adalah ada tidaknya pergeseran mandibula saat gerakan menutup mulut; gigitan terbalik bilateral atau unilateral; kelainan dental, skeletal, atau kombinasi keduanya; dan kelainan hanya pada maksila atau pada kedua rahang. pada kasus gigitan posterior unilateral, satu hal yang dapat diperhatikan adalah pada keadaan oklusi sentris terjadi pergeseran garis median gigi rahang bawah terhadap garis median gigi rahang atas dan garis median wajah. pilihan perawatan gigitan terbalik posterior satu sisi tipe dentoalveolar dapat menggunakan piranti lepasan atau cekat untuk menggerakkan gigi yang bermasalah, sedangkan untuk gigitan silang posterior bilateral tipe skelatal salah satu pilihan perawatannya adalah separasi sutura midpalatal.3 gigitan terbalik anterior adalah suatu keadaan di mana terdapat satu atau lebih gigi anterior atas yang pada keadaan oklusi posisinya lebih ke lingual daripada gigi rahang bawah (overjet bernilai negatif). keadaan ini dapat melibatkan satu gigi atau semua gigi anterior. gigitan terbalik anterior yang melibatkan semua gigi anterior berhubungan dengan maloklusi klas iii skeletal.4 gigitan terbalik anterior dapat diakibatkan oleh satu atau kombinasi beberapa faktor etiologi, antara lain trauma pada gigi sulung anterior yang mengakibatkan benih gigi permanen mengalami displacement ke arah lingual; persistensi gigi sulung anterior; gigi tambahan (supernumerary tooth) yang terletak di labial benih gigi permanen anterior; kehilangan prematur gigi sulung yang mengakibatkan sklerosis tulang atau jaringan ikat fibrous; kebiasaan buruk; dan lengkung rahang yang inadekuat oleh karena erupsi gigi permaenen rahang atas ke arah lingual.5 prevalensi gigitan terbalik anterior adalah sebesar 2,2-11,9%, hal ini tergantung pada usia anak yang diperiksa. pilihan perawatan untuk mengkoreksi gigitan terbalik anterior antara lain adalah menggunakan piranti lepasan atau cekat, yang bekerja secara langsung pada gigi malposisi.6 laporan kasus ini melaporkan pemakaian alat orthodonti cekat rapid maxillary expansion (rpe) dan elastik intermaksila untuk mengkoreksi gigitan terbalik posterior dan anterior pada anak remaja. kasus pasien remaja perempuan berusia 14 tahun datang ke rumah sakit gigi dan mulut universitas airlangga surabaya, dengan keluhan utama gigi depan gigitan gambar 1. foto ekstra oral. (a) tampak depan; (b) oklusi sentris; (c) tampak samping kanan. gambar 2. foto intra oral (a) samping kanan; (b) depan; (c) samping kiri. 8 (a) (b) (c) gambar 1. foto ekstra oral. (a) tampak depan; (b) oklusi sentris; (c) tampak samping kanan. (a) (b) (c) gambar 2. foto intra oral (a) samping kanan; (b) depan; (c) samping kiri. gambar 3. gigi 21 dan 22 impaksi. a b c 8 (a) (b) (c) gambar 1. foto ekstra oral. (a) tampak depan; (b) oklusi sentris; (c) tampak samping kanan. (a) (b) (c) gambar 2. foto intra oral (a) samping kanan; (b) depan; (c) samping kiri. gambar 3. gigi 21 dan 22 impaksi. a b c 100 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 98–102 terbalik dan terdapat gigi permanen yang tidak tumbuh. pasien meminta giginya dirapikan. setahun sebelumnya pasien pernah menjalani perawatan pencabutan gigi #61 dan #62 serta pengambilan odontoma di rsgm fakultas kedokteran gigi universitas airlangga. saat itu telah disarankan untuk melanjutkan perawatan ortodonsia dengan pemasangan alat cekat namun orang tua pasien menolak. setelah setahun pasien datang lagi ke rsgm fakultas kedokteran gigi universitas airlangga untuk merapikan gigi dengan alat cekat. pada pemeriksaan ektra oral ditemukan adanya asimetris wajah dan profil pasien cekung (gambar 1a, b, c). pada pemeriksaan intra oral dan hasil analisa model studi ditemukan sebagai berikut: relasi molar pertama permanen klas 1 angle; gigi #21 dan #22 tidak tumbuh; gigitan silang anterior dan posterior kanan; overjet -4 mm, overbite 7 mm; pergeseran garis median rahang bawah ke kanan 2,5 mm; serta rahang atas kekurangan tempat 8 mm dan rahangkekurangan tempat 8 mm dan rahang bawah kekurangan tempat 3 mm (gambar 2a, b, c). hasilgambar 2a, b, c). hasil pemeriksaan radiografi panoramik menunjukkan gigi #21 dan #22 impaksi (gambar 3) dan hasil analisis radiografi sefalometri memberikan kesimpulan adanya maloklusi klas i skeletal (gambar 4). diagnosa hasil pemeriksaan klinis ekstra oral, intra oral, analisis model studi, dan analisis radiografi adalah maloklusi klas i angle disertai gigitan terbalik posterior dan gigitan terbalik anterior. tatalaksana kasus perawatan dimulai dengan penjelasan rencana perawatan kepada orang tua pasien dan persetujuan lembar informed consent. selanjutnya dilakukan pemasangan alat ortodontik cekat rahang bawah dengan molar band pada #36 dan #46 dan bracket mini roth 0,18 pada gigi #35, #34, #33, #32, #31, #21, #22, #23, #24, #25. wire menggunakan niti 0.12 dan power o, selanjutnya dilakukan band back pada ujung wire (gambar 5a). kemudian dilakukan cetak model kerja untuk pembuatan rpe dan pemasangan separator pada mesial distal #14, #16 dan #24, #26. satu minggu kemudian dilakukan pasang coba rpe dilanjutkan penyemenan rpe (gambar 5b) dan aktivasi rpe seperempat putaran. orangtua pasien diinstruksikan untuk melakukan aktivasi di rumah setiap pagi hari dengan memutar skrew sebesar seperempat putaran. empat minggu setelah pemakaian rpe gigitan terbalik posterior kanan gambar 3. gigi 21 dan 22 impaksi. 8 (a) (b) (c) gambar 1. foto ekstra oral. (a) tampak depan; (b) oklusi sentris; (c) tampak samping kanan. (a) (b) (c) gambar 2. foto intra oral (a) samping kanan; (b) depan; (c) samping kiri. gambar 3. gigi 21 dan 22 impaksi. gambar 4. radiografi sefalometri. gambar 5. (a) pemasangan molar band dan bracket pada rahang bawah; (b) insersi rpe pada rahang atas. 9 gambar 4. radiografi sefalometri. (a) (b) gambar 5. (a) pemasangan molar band dan bracket pada rahang bawah; (b) insersi rpe pada rahang atas. (a) (b) gambar 6. (a) relasi gigi anterior setelah perawatan; (b) profil pasien setelah perawatan. sna: 80,5 snb: 89 anb: -8,5 a-m: 12 mm b-m: 0 mm wits: 1 mm go-gn-sn: 33º fma: 23º i-sn: 108º i-mp : 96º i-i : 120º naso labial angle : 90º 9 gambar 4. radiografi sefalometri. (a) (b) gambar 5. (a) pemasangan molar band dan bracket pada rahang bawah; (b) insersi rpe pada rahang atas. (a) (b) gambar 6. (a) relasi gigi anterior setelah perawatan; (b) profil pasien setelah perawatan. sna: 80,5 snb: 89 anb: -8,5 a-m: 12 mm b-m: 0 mm wits: 1 mm go-gn-sn: 33º fma: 23º i-sn: 108º i-mp : 96º i-i : 120º naso labial angle : 90º a b 9 gambar 4. radiografi sefalometri. (a) (b) gambar 5. (a) pemasangan molar band dan bracket pada rahang bawah; (b) insersi rpe pada rahang atas. (a) (b) gambar 6. (a) relasi gigi anterior setelah perawatan; (b) profil pasien setelah perawatan. sna: 80,5 snb: 89 anb: -8,5 a-m: 12 mm b-m: 0 mm wits: 1 mm go-gn-sn: 33º fma: 23º i-sn: 108º i-mp : 96º i-i : 120º naso labial angle : 90º 101dewati, et al.: koreksi gigitan terbalik posterior dan anterior terkoreksi sehingga aktivasi dihentikan. rpe dibiarkan tetap terpasang dalam kondisi tidak aktif selama dua bulan untuk mendapat stabilitas ekspansi. piranti rpe yang digunakan dimodifikasi dengan menambahkan stopper pada sisi yang tidak mengalami kelainan. stopper berupa plat akrilik yang meluas menutupi sisi lingual gigi posterior rahang bawah. dua bulan kemudian, rpe dilepas dan perawatan dilanjutkan dengan pemasangan orto cekat rahang atas dengan molar band pada #26 dan #36, bracket mini roth 0,18 pada gigi #15, #14, #13, #12, #11, #21, #22, #23, #24, #25, wire niti 0,12 dan power o. kemudian diberikan peninggian gigit posterior dengan fuji vii pada oklusal #36 dan #46 untuk menghilangkan blocking gigi anterior pada saat relasi sentris. setelah tahap levelling dan aligning selesai dilakukan penggantian wire menggunakan rectangular 0.16 x 0.22 pada rahang atas dan rahang bawah, ikatan 8 pada #16 #26 dan #36 #46. kemudian dilakukan pemasangan elastik ukuran 3/16 dari gigi #16 ke gigi #43 dan gigi #26 ke gigi #33 (intermaksila) untuk koreksi gigitan terbalik anterior. pasien dianjurkan untuk memakai elastik setiap hari dan diganti satu kali sehari saat malam hari. setelah 3 bulan pemakaian elastik intermaksila, gigitan terbalik anterior terkoreksi. profil penderita tampak lebih cembung dari sebelum perawatan. selanjutnya peninggian gigit posterior dihilangkan (gambar 6a, 6b). pembahasan peranti ekspansi ortodonti cekat yang digunakan pada rahang atas dibedakan menjadi 2 kategori: (1) peranti yang menghasilkan separasi sutura midpalatal; (2) peranti yang tidak menghasilkan separasi pada midpalatal. peranti rpe tipe hass dan hyrax termasuk dalam kategori pertama dan digunakan untuk koreksi gigitan terbalik melalui separasi dari sutura midpalatal. sementara piranti tipe quad helices dan porter atau w-arch menghasilkan ekspansi melalui pergerakan dentoalveolar.3 pada kasus ini digunakan peranti rpe tipe hyrax dimana konstruksinya terdiri dari skrew ekspansi yang dilekatkan dengan empat buah band: 2 pada molar pertama dan 2 pada premolar pertama (gambar 5b). antara skrew ekspansi dan band dihubungkan dengan wire bulat berukuran besar (wire disolder ke permukaan palatal dari band). modifikasi dilakukan pada piranti rpe yang digunakan, yakni dengan menambahkan plat akrilik pada sisi posterior kiri ke arah inferior menutupi permukaan lingual gigi posterior rahang bawah. hal ini bertujuan mencegah ekspansi transversal ke sisi kiri oleh karena pada kasus ini gigitan terbalik hanya terjadi pada sisi kanan (unilateral). piranti rpe diaktifkan dengan cara memasukkan kunci ke dalam lubang pada bagian tengah skrew ekspansi kemudian digerakkan ke depan sampai pada posisi paling anterior sehingga didapatkan pergerakan penuh ke arah transversal. sehari setelah insersi dan aktivasi pertama, skrew ekspansi diaktivasi setiap hari pada pagi hari sebesar seperempat putaran (45º). aktivasi ini akan menghasilkan pergerakan transversal sebesar 0,5 mm per hari.3 aktivasi dilakukan oleh pasien di rumah dan dibantu oleh orang tua. aktivasi dilakukan secara rutin sampai didapatkan gigitan fisura luar rahang atas. setelah 3 bulan perawatan, gigitan terbalik posterior terkoreksi dan didapatkan gigitan fisura luar rahang atas sehingga aktivasi dihentikan. binder3 menyebutkan untuk rpe tipe hass, setelah gigitan terbalik posterior terkoreksi, alat tidak dilepas selama 3–4 bulan sampai didapatkan stabilitas ekspansi. pada kasus ini, setelah gigitan terbalik posterior terkoreksi, alat tidak dilepas selama dua bulan dalam keadaan tidak aktif. perawatan selanjutnya adalah koreksi gigitan terbalik anterior. elastik intermaksila yang dipasang dari gigi molar pertama rahang atas ke kaninus bawah dapat digunakan untuk koreksi gigitan terbalik anterior.7 efek yang diharapkan adalah dapat memberikan labial root torque pada archwire dan terjadi distalisasi gigi anterior rahang bawah dan protraksi gigi anterior rahang atas. pada saat berbicara pasien dapat membuka mulut sebesar 10 mm, keadaan ini akan memberikan gaya yang bervariasi bergantung pada angulasi elastik intermaksila dan efeknya pun akan bervariasi terhadap lengkung rahang atas dan rahang bawah. pada rahang atas, gaya horizontal untuk mendorong gigi anterior lebih besar dibandingkan dengan gaya vertikal yang menyebabkan gigi ekstrusi. akan tetapi pada siang hari, elastik intermaksila menghasilkan gaya gambar 6. (a) relasi gigi anterior setelah perawatan; (b) profil pasien setelah perawatan. 9 gambar 4. radiografi sefalometri. (a) (b) gambar 5. (a) pemasangan molar band dan bracket pada rahang bawah; (b) insersi rpe pada rahang atas. (a) (b) gambar 6. (a) relasi gigi anterior setelah perawatan; (b) profil pasien setelah perawatan. sna: 80,5 snb: 89 anb: -8,5 a-m: 12 mm b-m: 0 mm wits: 1 mm go-gn-sn: 33º fma: 23º i-sn: 108º i-mp : 96º i-i : 120º naso labial angle : 90º a b 102 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 98–102 vertikal yang lebih besar oleh karena pengaruh gerakan fungsional (pengunyahan dan berbicara). pasien dianjurkan memakai elastik setiap hari dan elastik harus diganti setiap hari dan dapat diganti satu sampai tiga kali sehari.8 hasil yang memuaskan dicapai dalam waktu 3 bulan di mana gigitan terbalik anterior telah terkoreksi. perawatan dilanjutkan untuk menyediakan tempat bagi gigi #21 dan #22 impaksi, surgical exposre kemudian dilanjutkan traksi gigi #21 dan #22, finishing, dan retensi. pada kasus ini koreksi gigitan terbalik posterior dilakukan selama 4 minggu sampai diperoleh gigitan fisura luar rahang atas dan gigitan terbalik anterior terkoreksi dalam 3 bulan. laporan kasus ini menunjukkan bahwa maloklusi klas i angle disertai dengan gigitan terbalik posterior kanan dan gigitan terbalik anterior dapat dikoreksi dengan piranti rpe dan penggunaan alat ortodonsia cekat dengan elastik intermaksila. daftar pustaka 1. andrade as, gavião mbd, gameiro gh, rossi md. characteristics of masticatory muscles in children with unilateral posterior crossbite. braz oral res 2010; 24(2): 204-10. 2. pfitzinger w. poterior crossbite in children. st. louis: university school of dentistry; 2008. 3. binder re. correction of posterior crossbites: diagnosis and treatment. pediatr dent 2004; 26(3): 226–72. 4. dowsing p, sandler pj. how to effectively use a 2 x 4 appliance. j orthodontics 2004; 31(3): 248–58. 5. randall s, asher, curtis g, kuster, erickson l. anterior dental crossbite correction using a simple fixed appliance: case report. am academic of pediatr dent 1986; 8(1): 53–5 6. rosa m, lucchi p, mariani l, caprioglio a. spontaneous correction of anterior crossbite by rpe anchored on deciduous teeth in the early mixed dentition. eur j pediatr dentist 2012; 13(3): 176–80. 7. fu ps. unilaterally impacted maxillary central incisor and canine with ipsilateral transposed canine-lateral incisor. angle orthodontist 2013; 83(5): 920-2. 8. bratu cd, fleser c, glavan f. the effect of intermaxillary elastics in orthodontic therapy. tmj 2004; 54(4): 406–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �6 genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast bhk-2� cell cultures ceples dian kartika w.p,1 sri kunarti,2 and ari subiyanto2 1state department of health, regency of banyuwangi, indonesia 2department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: health industry has always used natural products as an alternative. propolis, a natural antibiotic, is a resinous yellow brown or dark brown substance derived from honey bees (apis mellifera). the main chemical compounds contained in propolis are flavonoids, phenolics and other various aromatic compounds. flavonoids are well known plant compounds that have antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory proprieties. propolis is expected to be an alternative used for root canal treatment with lower toxicity compared to calcium hydroxide (ca(oh)2. over the last decade, a new material, mineral trioxide aggregate (mta) was developed, and has been used as the gold standard. all materials used in mouth should be biocompatible. the initial level of material biocompatibility evaluation involves toxicity and genotoxicity tests. purpose: this research is aimed to conduct comparison test of genotoxicity effect of propolis extract, mta and ca(oh)2 on fibroblast bhk-21 cell culture. methods: this research was conducted with single-cell gel electrophoresis method. results: the results indicate that propolis extract cannot cause dna damage, while mta can cause apoptosis and ca(oh)2 can cause neucrosis. conclusion: it can be concluded that propolis extract has genotoxicity effect lower than mta and ca(oh)2, but mta has lower effect on fibroblast bhk-21 cell culture. keywords: propolis extract; mineral trioxide aggregate; ca(oh)2; genotoxicity; single-cell gel electrophoresis correspondence: ceples dian kartika w.p, c/o: dinas kesehatan pemerintah kabupaten banyuwangi. jl. letkol istiqlah no 42 banyuwangi, indonesia. e-mail: ceples_dk@yahoo.co.id research report introduction currently, calcium hydroxide (ca(oh)2) becomes the most common drug used in endodontics since its ability has been proven by many scientific researches. ca(oh)2 is an excellent therapeutic material widely used as a medicine in endodontic therapy. ca(oh)2 was used as a medicine in endodontic therapy for the first time in 1920. the drug is used in dental care, such as pulp capping, pulpotomy, apexification, root perforation, and internal or external resorption. ca(oh)2 does not cause dna damage at a concentration of 20-100 µg/ ml based on the result of genotoxicity test.1,2 on the other hand, the biocompatibility of mineral trioxide aggregate (mta) has been developed. several in vivo and in vitro studies show that mta has sealing ability and excellent biocompatibility. mta is used in dentistry as a root canal filling material. it means that mta can be considered as potential and ideal material for repairing perforation, apical barrier to teeth with an open apex, pulp capping and pulpotomy of young permanent teeth.3 many researches on genotoxicity of mta show that mta does not cause dna damage at a concentration of 1-1000 µg/ ml.1,4,5 in the twelve century, propolis was used by egyptians, greeks, and romans as a medicine to cure skin bruises and wounds, to regenerate tissue, to treat mouth and throath infections, as well as to cure dental caries since it has antiinflammatory, antiseptic, and antimicotic effects.7 there have been many studies on propolis showing that propolis has antioxidant, antibacterial, antifungal, antiviral and antiinflammatory effects. since propolis has anti-inflammatory dental journal (majalah kedokteran gigi) 20�5 march; 48(�): �6–2� �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��kartika, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 16–21 effect, it is known that propolis can inhibit prostaglandin synthesis, can increase both body’s resistance to the presence of phagocytic activity and self-healing, and can also stimulate immune cells. propolis also contains iron and zinc considered as essential elements for the synthesis of collagen. recently, many researches have studied on the use of propolis in dentistry, especially related with antimicrobial and antiinflammatory activities in kariology, oral surgery, pathology, periodontics and endodontics.8 however propolis from piracicaba, brazil can be considered as toxic at a concentration of 2 mg/ ml on fibroblast cells. 9 toxicity test on propolis extract, apis mellifera l, applied on fibroblast bhk-21 cells at a concentration of 1.5 mg/ ml cannot be considered as toxic.10 furthermore, propolis has more advantages than ca(oh)2 used as pulp capping agent in vital pulp therapy. thus, propolis is expected to be an alternative new compound used in root canal treatment since it has lower toxicity than ca(oh)2. 11 actually, all materials used in mouth should be biocompatible. biocompatibility is an ability of a material to show a response to the host in particular. evaluation of the biocompatibility of a material is the initial level of toxicity and genotoxicity tests. toxicity test is conducted by simply placing the material to be tested directly on the membrane tissue or cell culture. genotoxicity test is still required to see whether there is any change in dna of either human or non-mammalian cells caused by certain materials.12 therefore, this research conducted a comparison test of genotoxicity effect on propolis extract, mta and ca(oh)2 using cell culture with single-cell gel electrophoresis method. finally, this research is aimed to evaluate the genotoxicity effects of propolis extract, mta and ch in order to see fragmentation (damage) of dna from fibroblasts, and to determine the genotoxicity ratio of propolis extract, mta, and ca(oh)2. materials and methods fibroblast bhk-21cell culture derived from fibroblasts of hamster’s kidney was used in this research. fibroblast bhk-21cell culture was prepared at the central laboratory of veterinary farma (pusvetma), while dna extraction and electrophoresis were conducted in the laboratory of tissue culture tropical desease centre (tdc). this research can actually be considered as a laboratory experimental observational research with 30 samples for each treatment based on the formula. fibroblast cells were then divided into five groups, namely group 1 as media control consisted of 3 wells; group 2 as cell control consisted of 3 wells; group 3 treated with propolis extract as much as 1.5 mg/ml; group 4 treated with mta with the ratio of powder: liquid about 3: 1; and group 5 treated with 50% ca(oh)2 as control group. g e n o t o x i c i t y t e s t w a s c o n d u c e d w i t h d n a electrophoresis examination. dna was extracted from cells with spin coloumn invitrogen method. the concentration of dna was measured. electrophoresis was conducted to see whether there was dna cut or not. propolis extract was prepared with 11 g/100 ml of phenol as well as organic solvent required to dissolve the phenol. phenol should be gradually diluted with buffer solves solution (bss). the propolis extract was applied into each sample, about 0.5 ml. in other words, the total of the propolis extract used for 30 samples with a concentration of 1.5 mg.was 2.1 ml comparable with 15 ml of propolis extract with a concentration of 1.5 mg. ca(oh)2 powder was prepared with a concentration of 50% (50 g/ 100 ml). it was then applied after it was dissolved in 100 ml of sterile distilled water again with a ratio of 1: 1. fibroblast bhk-21 cel culture was prepared until the samples of bhk-21 in the form of frozen liquid nitrogen (-800 c) were taken and cashed 30 minutes with a stream of water, and then centrifuged at 500 rpm. fibroblast cells were transferred into four small roux bottles each of which was filled with 10 ml eagle medium containing 10% bovine serum, and then covered with aluminum foil. bottle was put into an incubator at a temperature of 370 c for 24 hours. confluence cells were removed and washed with 15 ml of pbs solution for three times. one ml of 0.25% trypsin-versene was given, then shaken and sprayed into the wall of the bottle for 5-10 minutes until the cells were separated from the wall of the bottle. the rest of the cells in the roux was added with 10 ml of medium eagle and 10% bovine serum, and then shaken until all cells were separated from the wall of the bottle and also from the bonds between cells. the cells were moved into four microplates (24 wells), each of which was filled with 1 ml of medium and 0.5 ml of the cell. those microplates were closed and incubated for 24 hours. microplates were observed under a light microscope to see whether fibroblast cells that had been grown in each of the wells were enough or not for the research. propolis extract, mta and calcium hydroxide were prepared to be applied in each well, and then incubated again for 24 hours at a temperature of 370 c. after that, the samples were taken, and the cells were washed with pbs three times. trypsination was conducted by adding trypsineversene and 1% edta, and then waited for a while. another eagle's medium was then given in order to obtain a cell suspension. the cells harvested were then packed into eppendorf tubes that had been sterilized twice and washed with pbs once. each eppendorf tube was put based on treatment group for dna electrophoresis after dna extraction was conducted. dna extraction with spin coloumn invitrogen method was conducted. 200 ml of cell culture samples were put into eppendorf tube, and then added with 20 ml of proteinase k and 20 ml of rnase a. the mixtures were divortexed and incubated for two minutes at room temperature, than were added with 200 ml of pure genomic linktm lysis/ binding buffer, and then vortexed. those were incubated in water bath at a temperature of 55 ° water bath for ten minutes. 200 ml of 96-100% ethanol were added, and vortexed for dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �8 kartika, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 16–21 ± five seconds. those were put into coloumn spin, and then centrifuged at 10,000 g for 1 minute at room temperature. the coloumn spin was added with 500 ml of wash buffer i, and then centrifuged 10,000 g for 1 minute. the coloumn spin was added with 500 ml of wash buffer ii, and then centrifuged at maximum speed for three minutes at room temperature. the coloumn spin was transferred to a new eppendorf, and then added with 25-200 ml of elution buffer. it was incubated for 1 minute, and then centrifuged at maximum speed. visualization of dna was conducted with electrophoresis method by making 12.5% acrylamide gel consisted of bisacrylamid, 10% aps, 0.5 x tbe and temed. main gel was poured until the upper limit of main gel line. the stacking gel was poured on the top of maingel. wells were molded in the stacking gel, and dna samples were prepared to be electrophased. loading dye was added into the dna samples with a ratio of dna samples and dye loading, about 5 : 2. the gel was put into the buffer, and then the electrophoresis tools were set. dna samples were put into those electrophoresis wells. the electrophoresis was conducted at 120 v for 90 minutes. the gel was removed, and then staining was conducted by putting the gel into the drying solution for 5 minutes, and into fixer solution for 15 minutes. agno3 staining was conducted for 1 hour. the gel was washed three times, and then added with develop solution until the band appeared. finally, the results were read with the help of three skilled staff. results this research was conducted to determine genotoxicity effect leading to cell degeneration through dna fragmentation (dna damage) in fibroblast bhk-21 cell culture due to the apllications of propolis extract, mta, and ca(oh)2. based on the reading results of dna electrophoresis visualization in media control and cell control, it is known that there was no result in media control because there was no cell in media control. meanwhile, there were two cells fragmented in cell control, both at 130 bp (figure 1). moreover, based on the visualization of dna electrophoresis, it is known that in the applicaton of mta there was non-random dna fragmentation, namely dna ladder called as apoptosis (figure 2), while in the application of ca(oh)2, there was dna smear/necrosis (figure 3). it is also known that in the application of propolis extract, there was no fragmentation (figure 4). based on the results of kruskal-wallis test, moreover, it is known that there was significant difference in those three samples in each group, namely fragmentation, apoptosis, and necrosis (p <0.05). kruskal-wallis test is a figure 1. dna electrophoresis visualization in media control and cell control. note: m = marker (100-1500bp). figure 2. dna electrophoresis visualization in mta (high apoptosis). note: m = marker (100-1500bp); mta= samples of mineral trioxide aggregat application. 11 figure 1. the example of dna electrophoresis visualization in media control and cell control. note: m = marker (100-1500bp). figure 2. the example of dna electrophoresis visualization in mta (high apoptosis). note: m = marker (100-1500bp); mta= samples of mineral trioxide aggregat application. m cell control media control 100 130 bp 200 300 500 1500 m mta mta 900 bp (high apoptosis) 11 figure 1. the example of dna electrophoresis visualization in media control and cell control. note: m = marker (100-1500bp). figure 2. the example of dna electrophoresis visualization in mta (high apoptosis). note: m = marker (100-1500bp); mta= samples of mineral trioxide aggregat application. m cell control media control 100 130 bp 200 300 500 1500 m mta mta 900 bp (high apoptosis) figure 3. dna electrophoresis visualization in ca(oh)2 (necrosis). note: m = marker (100-1500bp); ca(oh)2 = samples of ca(oh)2 application. figure 4. dna electrophoresis visualization in propolis extract (good dna). note: m = marker (100-1500bp); p= samples of propolis extract application. 12 figure 3. the example of dna electrophoresis visualization in ca(oh)2 (necrosis). note: m = marker (100-1500bp); ca(oh)2 = samples of ca(oh)2 application. figure 4. the example of dna electrophoresis visualization in propolis extract (good dna). note: m = marker (100-1500bp); p= samples of propolis extract application. caoh m caoh dna smear/ necrosis no fragmentation/dna cannot be cut (above 1500 bp) propolis m p 12 figure 3. the example of dna electrophoresis visualization in ca(oh)2 (necrosis). note: m = marker (100-1500bp); ca(oh)2 = samples of ca(oh)2 application. figure 4. the example of dna electrophoresis visualization in propolis extract (good dna). note: m = marker (100-1500bp); p= samples of propolis extract application. caoh m caoh dna smear/ necrosis no fragmentation/dna cannot be cut (above 1500 bp) propolis m p �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��kartika, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 16–21 one-way variance analysis with a non-parametric method used to test whether the samples come from the same distribution. in other words, it is used to compare more than two independent or unrelated samples. when kruskalwallis test leads to significant results, mann-whitney test then must be conducted with three or more groups. mann-whitney will help analyze specific sample pair for a significant difference. the difference between two samples in each group can be considered significant if p<0.05. the result analysis of kruskal-wallis test on the occurrence of fragmentation in cultured bhk-21 cells with the application of propolis extract, mta and ca(oh)2 showed that there were significant difference within them (significant, p = 0.000) (table 1). the result analysis of mann-whitney test showed that there was significant diference between propolis and mta with p = 0.000. there was also significant diference between propolis and ca(oh)2 with p = 0.000 and between mta and ca(oh)2 with p = 0.000 (table 2). second, the result analysis of kruskal-wallis test on the occurrence of necrosis in cultured bhk-21 cells with the application of propolis extract, mta and ca(oh)2 showed that there were significant difference within them (significant, p = 0.000) (table 1). then the result analysis of mann-whitney test showed that there was no significant diference between propolis and mta with p = 0.317. but, there was significant diference between propolis and ca(oh)2 with p = 0.000, and between mta and ca(oh)2 with p = 0.000 ( table 2). finally, the result analysis of kruskal-wallis test on the occurrence of apoptosis in cultured bhk-21 cells with the application of propolis extract, mta and ca(oh)2 showed that there were significant difference within them (significant, p = 0.0005) (table 1). then the result analysis of mann-whitney test showed that there was no significant diference between propolis and mta with p = 0094. there was significant diference between propolis and (caoh)2 with p = 0.000, and between mta and ca(oh)2 with p = 0.000 (table 2). discussion one of the requirements of dental materials that can be applied in oral cavity is that it must be biocompatible and does not contain toxic, irritation, inflammation, allergy, genotoxic, or carcinogenic substances.12,15 based on the iso-1099315, there are three kinds of genotoxicity testing, ie gene mutations, chromosomal aberrations, and dna effects. dna effect test is used to detect the presence of damaged cells. there are actually several methods for detecting dna damage, such as single-cell electrophoresis gel. if these materials are genotoxic, there will be both nonrandom dna fragmentation as dna ladder, commonly called apoptosis appears, and random dna fragmentation that is not clearly visible spread throughout dna smear, commonly called necrosis. in this research, to test the genotoxicity effect of propolis extract, mta and ca(oh)2, fibroblast bhk-21 cells were used. this is because fibroblast cells are important cells in dental pulp, periodontal ligament and gingiva.17 bhk-21 cell culture, derived from fibroblasts of hamsters’ kidney, has been chosen to be used to biocompatibility test because their passage can do more than 50-70 times with high-speed cell growth (2 x 105 cells/cm3 of the surface of the culture), and can also maintain cell integrity, as well as because the cells are able to divide themselves and multiply in suspense, thus increasing the efficiency of cell culture.18 based on the test results on genotoxicity effect, it is known that there was significant difference of the fragmentation of fibroblast bhk-21 cell culture whithin the applications of propolis extract, mta and ca(oh)2. it also known that the application of propolis extract in fibroblast bhk-21 cell culture did not cause dna damage as shown in the visualization of dna electrophoresis, dna found was not cut cut and above 1500 bp. propolis extract used in this research is propolis extract from lawang, east java, where the largest composition of propolis extract in lawang is phenylic acid, which has the basic element of flavonoid. flavonoid is a class of compounds that has antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory properties. propolis, furthermore, is found to be highly effective against gram-possitive bacteria,19 especially against staphylococcus aureus,20 and against gram-negative bacteria, especially against salmonella.21 flavonoids and caffeic acids in propolis are known to play an important role in reducing inflammatory response of lipoxygenase by inhibiting working mechanism of arachidonic acid. table 1. kruskal-wallis test on the fragmentation, necrosis, and apoptosis of propolis extract, mta, and ca(oh)2 applied in fibroblast bhk-21 cell culture kruskal-wallis propolis extract-mta-ca(oh)2 significance of fragmentation p=0.000* significance of necrosis p=0.000* significance of apoptosis p=0.000* note: *p<0.05 = there was significant difference. table 2. mann-whitney test on the fragmentation, necrosis, and apoptosis of propolis extract, mta, and ca(oh)2 applied in fibroblast bhk-21 cell culture propolis ca(oh)2 mta propolis f: p=0.000* a: p=0.000* f: p=0.000* n: p=0.317 a: p=0.094 ca(oh)2 n: p=0.000* f: p=0.000* n: p=0.000* a: p=0.000* note: f = fragmentation; n = necrosis; a = apoptosis; *p<0.05 = there was significant difference. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 20 kartika, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 16–21 flavonoids and caffeic acid also helps immune system to promote phagocytic activity, and stimulate cellular immunity. propolis helps the process of hard tissue formation, the stimulation of various enzyme systems and cell metabolism, as well as the circulation and. formation of collagen that helps in wound healing. this effect has been caused by arginine, vitamin c, provitamin a, b complex and minerals, such as copper, iron, zinc and bioflavonoids contained in propolis. propolis is a good antimicrobial agent because it prevents bacterial cell division as well as breaks down the cell walls of bacteria and sitoplasma.22 moreover, the results of a research on the antibacterial effectiveness of the three intracanal materials commonly used against enterococcus faecalis (e. faecalis) show that in vitro propolis has antibacterial activity against e. faecalis in the root canal, so propolis can be used as an alternative intercanal material.23 another in vivo research on the effectiveness of propolis and ca(oh)2 used as a short term intracanal medication against e. faecalis shows that propolis is more effective as an intracanal medication than calcium hydroxide.24 propolis compared with other experimental materials is the most irritant material as well as one of the valuable alternative endodontic materials.21 based on the results of this research, it is known that there was significant difference of the occurrence of necrosis whithin the application of propolis extract, mta and ca(oh)2. it is known that the application of ca(oh)2 caused necrosis more than propolis and mta. moreover, it is known that the application of propolis extract did not cause any damage to dna because the dna found was not cut and was above 1500 bp. in addition, it is also known that the application of mta did not cause necrosis, but caused apoptosis with a low level of damage. thus, it can be said that the application of mta caused apoptosis more than the application of propolis extract and ca(oh)2, but the occurrence of apoptosis between in the application of propolis and in the application of mta did not differ significantly since some of the applications of propolis extract can cause apoptosis at the level of damage that is not much different from mta. mta is a mixture of smooth portland cement and bismuth oxide. it is also known that mta contains a number of sio2, cao, mgo, k2so4 and na2so4. portland cement mainly contains a mixture of dicalcium silicate, tricalcium silicate, tricalcium aluminate, gypsum, and tetracalcium aluminoferrite. it is known mta has biocompatibility, excellent sealing, as well as antibacterial and low cytotoxic effect. mta does not cause any sitomorphology change in fibroblast cells and osteoblastic cells. it also known that mta has an ability to induce the release of bioactive dentine matrix protein. therefore, the initiation of hard tissue bridge (in coronal or apical) can stimulate cell proliferation and cell migration, then differentiating, as a result stimulating the hard tissue and periodontal tissue regeneration.17,26 it can be concluded that propolis extract has lower genotoxicity effect than mta and ca(oh)2. mta has lower effect than ca(oh)2 on fibroblast bhk-21 cell culture. it is suggested for further research that the genotoxicity effect of propolis extract is needed to be analysed on fibroblast cells in vivo in order to know the appropriate dose and shape of propolis extract used in dental therapies, especially in the field of endodontics. references 1. ribeiro da, marques mea, salvadori dmf. lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay. j endod 2004; 30(8): 593-6. 2. ribeiro da, scolastici c, de lima pla, marques mea, salvadori dmf. genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in chinese hamster ovary (cho) cells. oral surg oral med oral pathol oral radiol endod 2005; 99(5): 637-40. 3. torabinejad m, chivian n: clinical applications of mineral trioxide aggregate. j endod 1999; 25(3): 197-205. 4. braz mg, camargo ea, salvadori dmf, marques mea, ribeiro da. evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and portland cements. j oral rehabil 2006; 33(3): 234-9. 5. ribeiro da, duarte mah, matsumoto ma, marques mea, salvadori dmf. biocompatibility in vitro tests of mineral trioxide aggregate and regular and white portland cements. j endod 2005; 31(8): 605-7. 6. tran xv, gorin c, willig c, baroukh b, pellat b, decup f, opsahl vs, chaussain c, boukpessi t. effect of calcium-silicate-based restorative cement on pulp repair. j dent res 2012; 91(12): 116671. 7. rai r, jain s, sharma v, batra m. propolis in oral health: a natural remedy. world j pharm sci. 2014; 2(1): 90-4. 8. park yk, moraes, cleber s, daugsch, andreas, hongzhen l, rhim js. comparative antiproliferation of human prostate cancer cells by ethanolic extracts of two groups of brazilian propolis. ciênc. tecnol. aliment. campinas apr./june 2010. 9. al-shaher a, wallace j, agarwal s, bretz w, baugh d. effect of propolis on human fibroblas from the pulp and periodontal ligament. j endod 2004; 30(5): 358-61. 10. linda ya. uji toksisitas ekstrak propolis apis mellifera l pada sel fibroblast bhk-21. thesis. surabaya: departement konservasi gigi fakultas kedokteran gigi universitas airlangga; 2013. 11. ahangari z, naseri m, jalili m, mansouri y, mashhadiabbas f, torkaman pharm a. effect of propolis on dentin regeneration and the potential role of dental pulp stem cell in guinea pigs. cell j 2012; 13(4): 223-8. 12. widyastuti. uji genotoksisitas allograf dan senograf pada kultur sel fibroblas. karya tulis akhir program pendidikan dokter gigi spesialis. surabaya: fakultas kedokteran gigi universitas airlangga; 2004. 13. lemeshow s, homer dw, klal j, lwanga sk. adequancy of sample size in health studies. new york: john willey & sona; 1997. p. 40. 14. freshney ri. culture of animals cell: a manual of basic technique. 2nd ed. new york: alan r. liss inc; 2000. p. 7-12, 71, 128, 239. 15. anussavice kj. phillips’ science of dental materials. 11st ed. elsevier science, saunders. 2003. p. 172-94. 16. iso10993.1998. a practical guide iso 10993-3: genotoxicity, medical device and diagnostic industry magazine (mddi) article index. 17. walton, torabinejad. prinsip dan praktik ilmu endodonsi. jakarta: egc; 2006. p. 41-2. 18. dewi ik, mandojo r, ruslan e. pengaruh dosis dan lama pemberian pulperyl terhadap kematian sel fibroblast. jbp 2006; 8(2): 87-92. 19. seidel v, peyfoon e, watson dg, fearnley j. comparative study 2� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 2�kartika, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 16–21 of the antibacterial activity of propolis from different geographical and climatic zones. phytother res 2008; 22(9): 1256-63. 20. velazguez c, navarro m, acosta a, angulo a, dominguez z, robles r, robleszepeda r, lugo e, goycoolea fm, velazquez ef, astiazaran h, hernandez j. antibacterial and free-radical scavenging activities of sonoran propolis. j appl microbiol 2007; 103(5): 1747-56. 21. orsi ro, sforcin jm, rall vlm, funari src, barbosa l, fernandes jr a. susceptibility profile of salmonella agaist the antibacterial activity of propolis produced in two regions of brazil. j venomous anim toxins including trop 2005; dis 11: 109-16. 22. parolia a, manuel s, thomas, kundabala m, mohan m. propolis and its potential uses in oral health. int j medicine and medical sci 2010; 2(7): 210-5. 23. oncag o, cogulu d, uzel a, sorkun k. efficacy of propolis as an intracanal medicament against enterococcus faecalis. general dentistry 2008; 54(5): 319-22. 24. awawdeh l, al-beitawi m, hammad m. effectiveness of propolis and calcium hydroxide as a short-term intracanal medicament against enterococcus faecalis: a laboratory study. aust endod j 2009; 35(2): 52-8. 25. silva fb, almeida jm, sousa sm. natural medicaments in endodontics-a comparative study of the anti-inflammatory action. braz oral res 2004; 18(2): 1749. 26. bakland lk, andreasen jo. will mineral trioxide aggregate replace calcium hydroxide in treating pulpal and periodontal healing complications subsequent to dental trauma? a review. dental traumatology 2011; 10: 1600-9657. � the in vitro assessment of anti proliferation activity of crude diethyl ether extract of dendrophthoe species against to myeloma culture cell mochamad lazuardi veterinary faculty airlangga university surabaya indonesia abstract herb medicine have an active substances that can dissolved on polar, semi polar and non polar liquid extract. the methanol and ethyl acetate as a polar and semi polar extract liquid were used to study of herb medicine such as dendrophthoe species. diethyl ether as a non polar liquid extract was never use to study the dendrophthoe species. the aim of this study was to investigate the anti proliferation activity of dendrophthoe species to myeloma culture cells after extracted by non-polar extract solution (diethyl ether). the post test only control group design was used for this research. a thirty six of microtiterplates wells were used for myeloma culture cells in rpmi medium. the wells were devided placing in two groups: treatment groups and controls groups. each three wells of six treatment subgroups added with 100 µl of 1.1; 5.5; 11; 22; 33 and 44 µg/ml crude diethyl ether extract series. a rpmi solution at similar method and volume were used as control substances. the cells were assessed by inverted microscope in 200x magnified two days after. the cells were quantified analyzed for anti proliferation activity by using 1:1 methylene blue solution. the results showed that started from 11.0 µg/ml of crude diethyl ether extract of dendrophthoe species have been anti proliferation abilities of myeloma culture cells (p<0.05). in conclusion, actives substances of dendrophthoe species where dissolved in non polar liquid as diethyl ether has anti proliferation activities to cancer cell in vitro. key words: cancer, herb medicine, myeloma, dendrophthoe species, anti proliferation correspondence: mochamad lazuardi, c/o: veterinary faculty, airlangga university. jln. mulyorejo (kampus c) universitas airlangga surabaya 60115, indonesia. e-mail:e-mail: ardiunair@yahoo.co.uk introduction the cases of oral squamous cell carcinoma (oscc) were known highly in the worldwide at about 80% to 90% of malignant tumor in oral cavity. the mortality rate remains high about 5% in worldwide and 2.4% to 3.57% in indonesia.1 some of chemotherapeutics for reduced development of proliferates cell as carcinoma like an oscc were seen expensive and not a lot of kinds. a practical solution to this problems as described at above is using traditional plant remedies for reduce highly cost of medication. dendrophthoe species (benalu duku) is a traditional indonesian herbal remedy which grows to a height of 20 cm to 30 cm (figure 1). that trees have a some specific traditional name as follows; pasilan on depok (jakarta) and dendrophthoe on palembang (south of sumatera). in vivo and in vitro experimentally, the hot water extract of leaves of dendrophthoe species were shown have an anti proliferation of myeloma cells.2,3 figure 1. the leaves ofthe leaves of dendrophthoe species (benalu duku) from muara enim (south of sumatera island). � dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 5-9 the ethanol, methanol and chloroform extract of dendrophthoe species were known too, have an anti proliferation ability for myeloma culture cell.4–6 the phenomenon at above showed that leaves of dendrophthoe species have a something substances dissolved in polar solution with anti proliferation ability against to cancer culture cell or analogues as an anti cancer against to carcinoma cells. nuraini et al.,7 reported that ethyl acetate extract of leaves of dendrophthoe species as a semi polar solvent had a anti proliferation activities to development of myeloma culture cells. but we did not known yet, potency of anti proliferation substances of leaves of dendrophthoe species where dissolved in non-polar solvent. diethyl ether, one of non-polar solvent was known commonly use for extract solvent of unknown herbal medicinal.8 by these problems as described at above, we will be exploring potency of diethyl ether extract of leaves of dendrophthoe species as anti proliferation cancer culture cell to obtain concentration data where have a minimum anti proliferation ability against to in vitro cancer culture cell. material and methods research design of this work were using experimentally, by post test only control group design with inhibition ability activities as dependent variable and crude extract of dendrophthoe species and myeloma cells as dependent variable. the air-dried stem barks of dendrophthoe species leaves were collected from muara enim, south of sumatera, indonesia. a voucher specimen of dendrophthoe species has been identified and deposit in peer groups researcher of herb medicines of department medical-pharmacy, medical faculty airlangga university. the leaves of dendrophthoe species were pulverization and weighing carefully at about 850 g for dissolved in 2.5 l of diethyl ether p.a during the days. the mixture were processed by macerate method and filtrating with buchner filter for obtained filtrate of diethyl ether. the residues were processing on similar method at 3 times iterates. 7.5 l filtrates were pooled on one bottle and evaporating by for obtained at about 4–5 g dried filtrate as stock sample. the sample stocks were diluted with rose well park memorial institute (rpmi) medium and performed on series samples as follows; a 100 mg of dried stock samples dissolved up to 100 ml of rpmi medium (100 ppm) and performed to 1.1; 5.5; 11; 22; 33.0, and 44.0 ppm of rpmi. small volume of sample series and medium of rpmi were tested by thioglicolate and incubate during the two days (37° c). the myeloma cell lines (p3ui type) were obtained from the center of veterinary pharmaceutics, directorate of livestock services, agriculture department, surabaya (indonesia). from storage of – 80° c, the cells were thawing and revival to propagates in 100 ml roux’s bottle at least 9 × 107 cells/ml as stock cells. the stock cells were distributing to 2 ml of 36 microtiterplates wells at least 2 × 104 cells/ml each well. a 100 µl crude extract of dendrophthoe species at ranging 1 to 40 µg/ml were inserting onto 18 wells of microtiterplates and it was consists of 2 ml of rpmi medium with 10% of fetal bovine serum (fbs) and myeloma culture cells. a similar procedure of 100 µl rpmi solution as control substance were using for treat of myeloma in micro titer plates at control group wells. all samples were following propagates and inserting into co2 incubator during the 48 hours. two days later, the cells of all samples in microtiterplates were re-suspended. a 5 µl of the samples were mixture with 1:1 of 10% methylene blue solution (w/v) and dropped on the top of haemocytometer thoma. by inverted microscope at 200×× magnified, the viable cell were counting referred to equation 1 for obtained total viable cell data as illustrated of anti proliferate ability of crude diethyl ether extract. total viable cells each well = amount of viable cell in 5 µl × 2095 µl equation i 5 µl the criterion of anti proliferation ability of crude diethyl ether extract of dendrophthoe species were described in table 1.6 the data were comparing each other by minitab 13.3 (treatment group vs., control group) at series concentration of 1.1 ppm to 44.0 ppm. the conclusions of these researches were performed from research analyzed of independent t test at 5% significance of treatment groups vs., control groups. table 1. the anti proliferation ability of crude diethyl ether extract ofcrude diethyl ether extract of dendrophthoe species the 24 wells of microtiterplate on 2 ml rpmi medium amount of viable cell each ml inhibition criteria code > 1.00 × 105 0.95–1.00 × 105 < 0.95 × 105 the activity not appearance the lowest activity the strongest activity – ± + �lazuardi: the in vitro asessment of anti proliferation activity result two days later, the samples and medium were appeared still sterile and suitable for research material. result research showed as follows; the diethyl ether extract of pulverized leaf of benalu duku were produced anti proliferation activity to myeloma culture cell as illustrate at table 2 at below. at 1.1 µg/ml of the crude extract of diethyl ether of dendrophthoe species leaves, was not appeared an anti proliferation activities yet on myeloma figure 2. the myeloma culture cell after treats by crude extract diethyl ether of dendrophthoe species at 11.0 µg/ml (200×). table 2. the analysis of crude extract diethyl ether of dendrophthoe species at ranging 1.1 µg/ml to 44 µg/ml, against myeloma culture cell concentr. diethyl ether rpmi solution available p* 1.1 (µg/ml) 1.79 × 105 1.81 × 105 1.78 × 105 1.80 × 105 1.81 × 105 1.82 × 105 – – – p > 0.05 5.5 (µg/ml) 1.77 × 105 1.77 × 105 1.76 × 105 1.60 × 105 1.78 × 105 1.84 × 105 – – – p > 0.05 11.0 (µg/ml) 5.17 × 104 4.80 × 104 4.78 × 104 1.87 × 105 1.81 × 105 1.80 × 105 + + + p < 0.05 22.0 (µg/ml) 4.76 × 104 3.12 × 104 3.09 × 104 1.81 × 105 1.82 × 105 1.83 × 105 + + + p < 0.05 33.0 (µg/ml) 3.10 × 104 3.07 × 104 3.11 × 104 1.80 × 105 1.79 × 105 1.78 × 105 + + + p < 0.05 44.0 (µg/ml) 3.09 × 104 3.08 × 104 3.07 × 104 1.86 × 105 1.84 × 105 1.81 × 105 + + + p < 0.05 * compared by independent t test of minitab 13.3 figure 3. the myeloma culture cell after treated by rpmi medium as control (200×). culture cell. similar phenomena was seen on concentration of 5.5 µg/ml. but start on 11 µg/ml, the crude extract of diethyl ether of dendrophthoe species leaf were seen have an anti proliferation activities against to myeloma culture cell. at figure 2 appeared clearly that since of 11.0 µg/ml extract dendrophthoe species. leaves have anti proliferation activities to myeloma cells (p < 0.05). figure 3 showed that the rpmi solution was not produced anti proliferation activities on myeloma cells as control groups. � dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 5-9 the crude extract of 22.0; 33.0 and 44.0 µg/ml were showed significance inhibit ability activities as illustrated table 2 at below. in contras, the crude extracts diethyl ether of dendrophthoe species at 1.1 and 5.5 µg/ml were not showed inhibit ability activities yet. discussion the sample beginning at 11.0 µg/ml has a specials unknown substance with inhibition abilities to myeloma culture cell. the unknown substances were dissolved in non-polar solution (diethyl ether). some indonesia researcher predicted that substances with inhibition ability activities of dendrophthoe species were equal to mistellectin and viscotoxine substances. that unknown substances better identified as mistellectin and viscotoxine like effects referred to mentioned by ratna et al., and roostantia et al.11,12 we predicted that substances where dissolved in non polar liquid extraction were appeared a complex molecules at waterproof substances of the leaves. the waterproof substances were obtained on chlorophyll region of the dendrophthoe species at about 10%. on chlorophyll region, there are some other actives substances like a phloem, chloroplast and stoma of the leaf at about 65%. other substances perhaps an un identifying substances with anti proliferation activities as described by hahn.13,14 arifa et al.,15 by fourier transform infra-red spectrophotometer (ft-ir) have been investigated that unknown substances have a specific profile as follows; the wavelength number as stretching vibration of c = alkana was appeared at ranging 2926.28 to 2854.9 cm–1. the wavelength number as stretching vibration of c = aldehide was illustrated at 1736.09 cm–1. the wavelength number of c = alkena and n–h with sharp intensity were identified at ranging 1602.99 to 1658.93 cm–1. the aromatic substances were appeared at 1496.9 cm–1 wavelength number as stretching vibration. the absorbance peak of c–h amine, c–o, acid compounds, carboxylate and esther were founded at 1060.94 to 1359.94 cm–1 wavelength number as stretching vibration.16 ratna et al.,11 predicted that unknown substances have been performing as a new compound with amino acid substances for resulted antiproliferation ability activity. but from the result research predicted that unknown substances is lead substances as futuristic compound for anticancer purpose that where consist of amino acid, organic substances and aromatic substances. the pharmacokinetic and pharmacodynamic of unknown substances that dissolved in diethyl ether of dendrophthoe species on human and animal experiments was not seen before. but it was predicted that duration of action from unknown substances of dendrophthoe species dissolved in hot water on rat with infected of myeloma still available at ranging 8 to 12 hours. the elimination half-life (t1/2β) might be lowest than 1 hours as referrer to nuraini et al.3 in conclusion, that result showed the available of absorption, distribution, metabolism and excretion (adme) of dendrophthoe species as herb medicine on infected subject was shortest than synthetics medicine.17,18 the initial of action of unknown substances of dendrophthoe species after extracted by diethyl ether against to myeloma cell at this test was obtained on a view minutes after dropped. the duration of action at in vitro test of dendrophthoe species was appeared at 24 to 48 hours after dropped. these result showed that the pharmacodynamic action of unknown substances of dendrophthoe species after extracted by diethyl ether on in vitro test was longer than in vivo test as explained by nuraini et al.,3 (the duration of action at in vivo test at ranging 6 to 12 hours after treatment). but unknown substances of dendrophthoe species that dissolved on hot water or other polar solvent at in vitro test as described by roostantia et al.,2 and nuraini19 were showed similar of other unknown substances of herb medicine as an anti cancer as referred to table 3. the phytochemical analysis of crude extract has revealed macromolecules of dendrophthoe species to be among the chemical constituent contained within it. the macromolecules were shown to produced anti cancer activities.2 however, the anti cancer effect of plants containing macromolecules actually depends on type and content of their substances in plants as described by nuraini.19 the active substances of dendrophthoe species from south of sumatera (muara enim) was shown strongest than dendrophthoe species from magelang. the other substances of macromolecules as tannins perhaps have an anti cancer activity. tannins are polyphenolic compounds and already available on herb medicine.23 tannins have an antiproliferation as an interfere substance of biosynthesis of eukaryotic cell by uncoupling oxidative phosphorylation.24,25 table 3. the pharmacodynamic activities of herb medicine against to cancer cell at in vitro test herb medicine cancer cell the duration of action by incubation time dependent references physallis angulata l eurycoma longifolia j cola nitida myeloma cell hela cell, widr, raji breast cancer cell (mcf-7) 48 hours 48 hours 24 to 72 hours maryati, sutrisna20 dinar et al.21 susi et al.22 �lazuardi: the in vitro asessment of anti proliferation activity acknowledgements the author is grateful thank to rector of airlangga university, department of education, republic of indonesia, for giving the sponsorship of this research by loan of institutional income non-tax at programs 2006/2007. i also thank to director of the center of veterinary pharmaceutics, directorate of livestock services, agriculture department, surabaya (indonesia) for enabling this work to be undertaken. references 1. agus p. the pattern of p53 gene mutations on oral squamous cell carcinoma. dent j (majalah kedokteran gigi) 2007; 40(3):119–22. 2. roostantia i, lazuardi m, ratna sm. the comparison of the efficacythe comparison of the efficacy to inhibit myeloma cells between benalu duku and benalu tea versus metotrexate. ebers papyrus 2000; 6(1):13–21. 3. nuraini f, lazuardi m, farida s. the study of anti cancer effect of benalu duku (dendrophthoe spec.,) infusion to the myeloma induced rat. j ked yarsi 2000; 8(1):59–71. 4. lazuardi m. antiproliferation activity of the crude ethanol extracts of dendrophthoe petandra l. miq against myeloma culture cell. the indon. j nat prod. 2007; 6(3):103–6. 5. lazuardi m. antiproliferative activity of methanol extract of benalu duku leaf (dendrophthoe spec.,) against to in vitro myeloma cell. j trad med 2007; 11(39):9–13. 6. widiyatno tv, sianita n, farida n, susilo s, lazuardi m. in vitro antiproliferative effect of benalu duku on myeloma cells. vet med j 2007; 23(2):96–101. 7. nuraini f, ratna sm, roostantia i, lazuardi m. the in vitro study of antiproliferation activities ethyl acetate extract of benalu duku leaves against to myeloma cells. final report. surabaya (tx): airlangga university. office research and social work; 2007. oct. report no. 678/j03.2/pg/2007. p. 1–40. 8. caroline. the antioxidant and free radicals test and determination of ec50 from leaf of green cincao (cyclea barbata miers). j nat med 2005; 4(1):11–18. 9. roostantia i, ratna sm, lazuardi m. the explored in vitro test of tripamidium. medika eksakta 2003; 4(3):170–5. 10. world health organization. health research methodology: a guide for training in research method. basel, switzerland: world health organization; 1999. p. 94–103. 11. ratna sm, roostantia i, wahyudi tw, lazuardi m. the amino acid surveillance of benalu duku leaf extract. j trad med 2007; 11(39):14–8. 12. roostantia i, ratna sm, lazuardi m. the determination of 50% lethal dose infuse of benalu duku in mice (mus musculus albinos/balb-c) by oral administration. j nat med 2007; 6(1): 38–4. 13. hahn petra. apoptoseinduzierende und antimutagene wirkung von viscum album l. auf humane zellkulturen. dissertation. swedish: fachbereich biologie, universität kaiserslauteren; 2000. p. 61–134. 14. alvin n. textbook of modern biology. new york, london, sidney: john willey and sons; 2000. p. 59–95. 15. arifa m, roostantia i, ratna sm. identification function substanceidentification function substance extract of benalu duku by fourier spectrophotometer infra red. final report. surabaya (tx); medical faculty airlangga university. office medical research unit; 2007 march. report no. 10. p. 3–25. 16. lazuardi m. optimizing profile of amino acid analysis of benalu duku leaf (dendrophthoe species) using chromatography. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university; p. 112–9. 17. wahyu d, sumi w, nelly cs. the stimulant influence of ethanol extract of pule cortex (alstonia scholaris (l).br.) on many kind of concentrations on mice. j nat med 2006; 5(1): 44–9. 18. suherman j, susi e, fauziah o, asmah r, wan nor izzah wz. the influence of kola fruit extracted against to enzyme activity of rat liver after induced by liver cancer. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university; p. 140–7. 19. nuraini f. an in vitro analysis of antiproliferation activity of methanol extract of leaves of benalu duku from magelang and from muara enim. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university. p 1–10. 20. maryati, sutrisna em. cytotoxic potention of ceplukan trees (physallis angulata l) to myeloma cell. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university; p. 133–9. 21. dinar scw, subagus w, mae sh. characterization and in vitro cytotoxic assay isolate from akar pasak bumi (euricoma longifolia jack) from west kalimantan jungle. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university; p. 63–70. 22. susi e, himmi m, suherman j, fauziah o, asmah r, wan noor izah wz. the determination of action mechanism of kola fruits extract (cola nitida) on breast cancer culture cell (mcf-7) by flow cytometri. proceeding on national congress of multi discipline research development of herbal in indonesia, march of 6th, 2008. yogyakarta: working groups of allergy and immunology association, traditional medicine research groups of medical faculty gadjah mada university and medical faculty gadjah mada university; p. 147–54. 23. bate smith ec. the phenolic constituents of plants and their taxonomic significance 1, dicotyledons. j linn soc bot 1962; 58:95–73. 24. martin rj. mode of action of anthelmintic drugs. vet j 1997;vet j 1997; 154:11–34. 25. sulaiman mm, mamman m, aliu yo, ajanusi jo. anthelminticanthelmintic activity of the crude methanol extracts of xylopia aethiopica against nippostrongylus brasiliensis in rats. veterinarski arhiv 2005;veterinarski arhiv 2005; 75(6):487–95. vol 38 no 3 2005 108 kekambuhan gingivitis hiperplasi setelah gingivektomi (recurrent of hyperplastic gingivitis after gingivectomy) iwan ruhadi* dan izzatul aini** ** bagian periodonsia ** mahasiswa ppdgs periodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract the inflammatory enlargement is clinically called hyperthropic gingivitis or gingival hyperplasia and generally related to local or systemic factors. they could be edematous or fibrous. the former is treated by scaling, but the latter that could not be treated by scaling only has to be removed by gingivectomy. there are some cases of gingivectomy resulting in recurrences. the writer wanted to find out the cause of the recurrences. the types of research were clinical and laboratories observational studies. the criteria of sample were: male or female patient who came to periodontal clinic of faculty of dentistry airlangga university. they were diagnosed gingivitis hyperplasia; had no systemic diseases; did not wear the orthodontic appliances, prosthesis, and crown and bridge; do not smoke. the indicated teeth to be observed were the labial side of maxillary front teeth. the teeth had score hyperplastic index (hi) = 2 at the 2nd weeks after scaling. there were 7 samples taken selectively. the results of the studies were based on the comparison of 1) hyperplasia index (hi); 2) the number and percentage of monosite and leucocytes from white blood impedance coutl (wic) and white blood optical coutl (woc); 3) plaque index; and 4) gingival index. the result of gingivectomy was reevaluated on the 30th, 45th, 60th, 90th day. the research concluded that the number of monosite was normal, but the dental plaque still accumulated and eventually caused the recurrences of the inflammation. key words: gingivitis hyperplasia, edematous, fibrous, observational, recurrent korespondensi (correspondence): iwan ruhadi, bagian periodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan hiperplasi gingiva merupakan ciri adanya penyakit gingiva, disebut juga dengan inflammatory enlargement terjadi karena adanya plak gigi, faktor yang memudahkan terjadinya akumulasi dan perlekatan plak. di klinik istilah yang digunakan adalah hyperthropic gingivitis atau gingival hiperplasia sebagai keradangan gingiva yang konotasinya mengarah pada patologis.1 pada proses radang kronis monosit melalui sirkulasi darah akan migrasi ke tempat terjadinya keradangan, menjadi makrofag.2,3 aktifasi sistem imun spesifik akibat keradangan akan mengaktifkan makrofag untuk memproduksi sejumlah sitokin dan faktor pertumbuhan yang berperan pada pembentukan fibrosis.4,5 ada dua tipe dasar respons jaringan terhadap pembesaran gingiva yang mengalami keradangan yaitu edematous dengan tanda gingiva halus, mengkilat, lunak dan merah, serta fibrous dengan tanda gingiva lebih kenyal, hilangnya stippling dan buram, biasanya lebih tebal, pinggiran tampak membulat.1 perawatan periodontal diawali dengan fase perawatan tahap awal yang meliputi dental health education (dhe), supra dan subgingival scaling, dan polishing.1,5,6 pada gingivitis hiperplasi dapat dirawat dengan scaling, bila gingiva tampak lunak dan ada perubahan warna, terutama bila terjadi edema dan infiltrasi seluler, dengan syarat ukuran pembesaran tidak mengganggu pengambilan deposits pada permukaan gigi. apabila gingivitis hiperplasi terdiri dari komponen fibrotik yang tidak bisa mengecil setelah dilakukan perawatan scaling atau ukuran pembesaran gingiva menutupi deposits pada permukaan gigi, dan mengganggu akses pengambilan deposits, maka perawatannya adalah pengambilan secara bedah (gingivektomi).1 gingivektomi adalah pemotongan jaringan gingiva dengan membuang dinding lateral poket yang bertujuan untuk menghilangkan poket dan keradangan gingiva sehingga didapat gingiva yang fisiologis, fungsional dan estetik baik.1,7,8 keuntungan teknik gingivektomi adalah teknik sederhana, dapat mengeliminasi poket secara sempurna, lapangan penglihatan baik, morfologi gingiva dapat diramalkan sesuai keinginan.8 setelah 12–24 jam, sel epitel pinggiran luka mulai migrasi ke atas jaringan granulasi. epitelisasi permukaan pada umumnya selesai setelah 5–14 hari. selama 4 minggu pertama setelah gingivektomi keratinisasi akan berkurang,6 keratinisasi permukaan mungkin tidak tampak hingga hari ke 28–42 setelah operasi.9 repair epithel selesai sekitar satu bulan, repair jaringan ikat selesai sekitar 7 minggu setelah gingivektomi. vasodilatasi dan vaskularisasi mulai berkurang setelah hari keempat penyembuhan dan tampak 109ruhadi dan aini: kekambuhan gingivitis hiperplasi hampir normal pada hari keenam belas.6 enam minggu setelah gingivektomi, gingiva tampak sehat, berwarna merah muda dan kenyal.9 kenyataannya secara klinis perawatan gingivitis hiperplasi dengan perawatan gingivektomi sering menimbulkan kekambuhan. berdasarkan pendapat tersebut di atas penulis ingin mengetahui penyebab terjadinya kekambuhan gingivitis hiperplasi setelah dilakukan gingivektomi secara klinis. bahan dan metode jenis penelitian yang digunakan adalah studi observasional secara klinis dan laboratoris. kriteria dan pengambilan sampel: penderita pria atau wanita yang datang ke klinik periodonsia fakultas kedokteran gigi unair dengan diagnosa klinis gingivitis hiperplasi, tidak mempunyai riwayat penyakit sistemik, tidak memakai alat ortodonti, protesa, bridge, dan crown, tidak merokok. sampel yang diindikasikan adalah gigi front rahang atas 11, 12, 13, 21, 22, 23 sebelah labial dengan skor hyperplasia index (hi) tertinggi (hi ≥ 2) pada 2 minggu setelah dilakukan scaling. pengambilan sampel dilakukan secara selektif. berdasarkan kriteria tersebut di atas diharapkan adanya homogenitas sampel, sehingga besar sampel yang digunakan dalam penelitian ini ditentukan sebesar 7 sampel. alat dan bahan: sarung tangan, masker, peralatan gingivektomi, kaca mulut, pinset dan pocket probe; disposible spuit 2 cc, 5 cc, periodontal probe dari who, pehacain ampul, ultrasonic scaler, scaler manual, periodontal pack, tourniquet, alkohol 70%, tabung reaksi, darah vena, kapas, plester, anti koagulan edta, cell–dyn 3700 system, cell–dyn 3700 system yang merupakan alat autoanaliser untuk pemeriksaan hematologi yang menggunakan teknologi multi angles polarization scatter separation (mapss) dan hydrodynamic focusing, dapat digunakan untuk menghitung jumlah monosit dan sel darah yang lain dengan menggunakan dua metode berbeda yang saling mendukung yaitu metode white blood impedance coutl (wic) dan white blood optical coutl (woc). cara kerja untuk melihat penyebab terjadinya kekambuhan gingivitis hiperplasi dilakukan pemeriksaan dengan white blood optical coutl (woc) dan white blood impedance coutl (wic) serta plaque index (pli). sampel yang sudah memenuhi kriteria diambil darahnya dari vena cubiti sebanyak 2–3 ml kemudian dimasukkan ke tabung reaksi yang telah diisi dengan 2 mg edta. setelah itu tabung reaksi ditutup dengan karet penutup. tahap selanjutnya tabung reaksi dikocok dengan membolak-balikkan tabung kira-kira 10 kali secara perlahan sampai campuran merata dan kemudian tabung reaksi dimasukkan ke dalam cell-dyn 3700 untuk menghitung jumlah monosit dengan 2 metode yaitu: 1) white blood optical coutl (woc) dilakukan dengan cara polarisasi, maka sel darah dapat dibedakan melalui ukuran, struktur, dan morfologi permukaan sel sehingga dapat dihitung jumlahnya dan 2) white blood impedance coutl (wic), dapat dihitung jumlah dari masing-masing sel darah tersebut melalui beda potensial. data hasil wic kemudian dibandingkan dengan data hasil woc. hasil akhir terlihat jumlah dan persentase monosit dan sel leukosit lainnya. setelah itu data akumulasi plak gigi didapat dengan menggunakan plaque index (pli) dari silness-loe.10 plaque index (pli) diukur pada 4 permukaan tiap gigi (sisi bukal yang meliputi mesial, mid, distal, dan sisi lingual). semua skor dijumlah dan dibagi dengan jumlah permukaan yang diperiksa. skor sebagai berikut: 0 = tidak ada plak pada gingiva, dites dengan menggeser probe sepanjang permukaan gigi; 1 = tidak ada plak yang bisa diamati dengan mata telanjang, tetapi plak tampak pada ujung probe setelah probe digerakkan sepanjang permukaan gigi; 2 = gingiva ditutupi dengan selapis tipis plak sampai sedang yang tampak dengan mata telanjang; 3 = penumpukan yang banyak dari deposit lunak didalam saku gingiva dan/atau tepi ginggiva dan permukaan gigi yang berbatasan. hyperplasia index (hi) dan gingival index (gi) digunakan untuk melihat kekambuhan gingivitis hiperplasi dipakai parameter. untuk melihat kekambuhan gingivitis hiperplasi melalui pembesaran gingiva menggunakan hyperplasia index (hi) menurut seymour11 dengan skor sebagai berikut: 0 = tidak ada pembesaran interdental papil ke permukaan gigi; 1 = sedikit pembesaran interdental papil, ujung papil tampak membulat; 2 = pembesaran sedang, papil mengembang meliputi bagian lateral melintas permukaan bukal gigi kurang dari ¼ ketebalan gigi; 3 = tanda pembesaran papil, yaitu lebih dari ¼ ketebalan gigi. bentuk normal papil hilang. menilai pembesaran gingiva terhadap permukaan gigi yang berdekatan untuk sebuah unit gingiva (bila ada jarak antara gigi yang bersebelahan, maka diberikan skor hi tertinggi). prevalensi gingival overgrowth yang memerlukan tindakan bedah, ditetapkan pada hi dengan skor klinis ≥ 2.11 gambar 1. skor hiperplasia indeks (hi). gingival index (gi): digunakan untuk melihat keradangan pada gingiva di data dengan menggunakan pengukuran dilakukan pada empat area pada tiap gingival unit (sisi bukal yang meliputi mesial, mid, distal, dan sisi lingual), kemudian skor yang didapat dijumlah dan dibagi 4. untuk pemeriksaan klinis probe masuk kira-kira sedalam 1–2 mm dari margin gingiva dengan tekanan aksial sedang dan dijalankan dari interproksimal ke interproksimal sepanjang aspek bukal dan lingual gigi dengan skor sebagai berikut:1 0 = tidak ada keradangan pada gingiva; 1 = keradangan ringan pada gingiva, sedikit perubahan pada warna dan tekstur, tidak ada perdarahan 110 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 108–111 pada probing; 2 = keradangan sedang pada gingiva, kemerahan, edema dan mengkilat, ada perdarahan pada probing; 3 = keradangan parah pada gingiva, tanda kemerahan, edema dan ulserasi. cenderung terjadi perdarahan spontan. setelah itu semua, baru dilakukan gingivektomi dengan pemasangan periodontal pack. satu minggu setelah gingivektomi surgical pack dibuka, bila epitelisasi permukaan luka belum sempurna, luka ditutup kembali dengan surgical pack selama satu minggu. pada hari ke 30, 45, 60, dan 90 setelah gingivektomi dievaluasi kembali dengan parameter klinis pli, gi, dan hi. hasil skor hi diambil yang tertinggi pada penderita gingivitis hiperplasi, kemudian pada skor hi tertinggi tersebut ditentukan juga jumlah monosit/cmm dalam darah, skor pli, gi. hasil terlihat pada tabel di bawah ini. tabel 1. jumlah monosit pada 7 penderita gingivitis hiperplasi sebelum gingivektomi no urut penderita 1 2 3 4 5 6 7 jumlah monosit/cmm 160 568 195 826 249 690 320 pada tabel 1 menunjukkan seluruh penderita dengan jumlah monosit dalam batas nilai normal (n = 100–900/ cmm). tabel 2. skor plaque index pada 7 penderita gingivitis hiperplasi sebelum gingivektomi 30, 45, 60 dan 90 hari setelah gingivektomi no urut penderita 1 2 3 4 5 6 7 sebelum gingivektomi 1 1 1 1 1 1 1 30 hari setelah gingivektomi 1 1 2 1 1 1 1 45 hari setelah gingivektomi 2 2 2 2 1 1 1 60 hari setelah gingivektomi 2 2 2 2 1 2 1 90 hari setelah gingivektomi 1 1 1 2 1 2 1 pada tabel 2 terdapat 1 penderita mengalami peningkatan skor plak pada 30 hari setelah gingivektomi. tiga penderita mengalami peningkatan skor plak pada 45 hari setelah gingivektomi. satu penderita mengalami peningkatan skor plak pada 60 hari setelah gingivektomi. pada 90 hari setelah gingivektomi 3 penderita mengalami penurunan skor plak. tabel 3. skor gingiva index pada 7 penderita gingivitis hiperplasi sebelum gingivektomi, 30, 45, 60 dan 90 hari setelah gingivektomi no urut penderita 1 2 3 4 5 6 7 sebelum gingivektomi 30 hari setelah gingivektomi 45 hari setelah gingivektomi 60 hari setelah gingivektomi 90 hari setelah gingivektomi 2 0 1 2 0 2 0 1 1 1 2 0 1 2 0 2 0 1 1 1 2 0 0 0 0 2 0 1 1 2 2 0 0 0 0 pada tabel 3 seluruh penderita mengalami penurunan skor gi = 0 pada 30 hari setelah gingivektomi. lima penderita mengalami peningkatan skor gi = 1 pada 45 hari setelah gingivektomi. dua penderita mengalami peningkatan skor gi = 2 pada 60 hari setelah gingivektomi. dua penderita mengalami penurunan skor gi = 0 dan 1 penderita mengalami peningkatan skor gi = 2 pada 90 hari setelah gingivektomi. tabel 4. skor hyperplasia index pada 7 penderita gingivitis hiperplasi sebelum gingivektomi, dan 30, 45, 60 dan 90 hari setelah gingivektomi no urut penderita 1 2 3 4 5 6 7 sebelum gingivektomi 2 2 3 2 3 3 2 30 hari setelah gingivektomi 0 0 0 0 0 0 0 45 hari setelah gingivektomi 0 1 0 1 0 0 0 60 hari setelah gingivektomi 1 1 1 1 0 1 0 90 hari setelah gingivektomi 0 1 0 2 0 2 0 pada tabel 4 seluruh penderita mengalami penurunan skor hi = 0 pada 30 hari setelah gingivektomi. dua penderita mengalami peningkatan skor hi = 1 pada 45 hari setelah gingivektomi. tiga penderita mengalami peningkatan skor hi = 1 pada 60 hari setelah gingivektomi. dua penderita mengalami penurunan skor hi = 0 dan 2 penderita mengalami peningkatan skor hi = 2 pada 90 hari setelah gingivektomi. pembahasan tujuh penderita yang diperiksa adalah penderita lakilaki, usia 22 sampai 35 tahun. dari hasil penelitian pada 30 hari setelah gingivektomi, didapatkan 1 penderita dengan kontrol plak kurang optimal dengan pli = 2 (tabel 2), tetapi tidak tampak adanya tanda keradangan pada gingiva berdasarkan pemeriksaan parameter klinis gi, dan hi. pemeriksaan 45 hari setelah gingivektomi terdapat 4 penderita dengan peningkatan skor pli = 2 111ruhadi dan aini: kekambuhan gingivitis hiperplasi (tabel 2), dan mengalami keradangan ringan pada gingiva. hal ini dapat dibuktikan dengan adanya peningkatan skor gi = 1 (tabel 3). dua penderita mengalami kekambuhan dengan skor hi = 1 (tabel 4), dan kekambuhan tidak terjadi pada 2 penderita lainnya. meskipun pada pemeriksaan laboratoris dari data hasil penelitian yang telah dilakukan terhadap 7 penderita gingivitis hiperplasi jumlah monosit penderita dalam batas nilai normal (n = 100–900/cmm) (tabel 1). hal ini mungkin karena adanya perbedaan respons host terhadap akumulasi bakteri plak, dan gingivitis mencerminkan adanya respons keradangan pada free gingiva terhadap akumulasi plak pada gingiva.4 pemeriksaan 60 hari setelah gingivektomi terdapat 5 penderita mengalami kekambuhan dengan skor hi = 1 (tabel 4), terjadi pada penderita yang mengalami peningkatan skor pli = 2 (tabel 2). tiga penderita mengalami keradangan ringan pada gingiva, yang dibuktikan dengan adanya skor gi = 1 (tabel 3). dua penderita mengalami keradangan sedang pada gingiva, yang dibuktikan dengan adanya peningkatan skor gi = 2 (tabel 3). pemeriksaan 90 hari setelah gingivektomi didapatkan adanya penyembuhan spontan dari kekambuhan yang dialami pada hari ke-60 setelah gingivektomi (tabel 4). proses penyembuhan ini juga dibarengi dengan terjadinya penurunan skor pli = 1 (tabel 2) dan skor gi = 0 (tabel 3), yang menunjukkan tidak terjadinya keradangan pada gingiva. terjadinya penurunan skor pli = 1 (tabel 2) karena adanya kontrol plak yang lebih optimal oleh penderita. pada setiap kunjungan peneliti selalu mengingatkan kembali pada penderita pentingnya kontrol plak yang dilakukan secara optimal untuk keberhasilan perawatan dan menjaga kesehatan jaringan periodontal. hari ke-90 setelah gingivektomi 2 penderita mengalami kekambuhan dengan peningkatan skor hi = 2 (tabel 4), disertai skor pli = 2 (tabel 2). satu penderita mengalami keradangan sedang pada gingiva, yang dibuktikan dengan adanya peningkatan skor gi = 2 (tabel 3). satu penderita lainnya mengalami keradangan ringan pada gingiva, yang dibuktikan dengan skor gi = 1 (tabel 3). satu penderita mengalami kekambuhan dengan skor hi = 1 (tabel 4), dan mengalami penurunan skor pli = 1 (tabel 2), dengan keradangan ringan pada gingiva, dibuktikan dengan skor gi = 1 (tabel 3). penelitian ini menunjukkan tampak jelas adanya faktor lokal sebagai pemicu terjadinya kekambuhan pada proses penyembuhan. kontrol plak yang tidak optimal menyebabkan terjadinya penumpukan bakteri plak supragingiva yang menimbulkan keradangan pada gingiva didekatnya. keradangan yang terjadi menyebabkan terjadinya kekambuhan atau hiperplasi gingiva, oleh karena itu selama masa penyembuhan diperlukan oral hygiene yang baik.12 penyebab utama penyakit keradangan pada jaringan periodontal adalah bakteri plak, tanpa kontrol plak kesehatan periodontal tidak akan pernah tercapai. sebenarnya aspek keberhasilan perawatan dokter gigi tergantung pada kontrol plak.2 tidak optimalnya kontrol plak yang berhubungan dengan penumpukan bakteri plak setelah perawatan gingivektomi telah menimbulkan kekambuhan, meskipun telah dilakukan dhe, scaling dan root planing terhadap setiap sampel penderita pada terapi awal atau 2 minggu sebelum gingivektomi. kontrol plak dikategorikan ke dalam kontrol plak yang dikerjakan oleh dokter gigi dan kontrol plak yang dilakukan oleh penderita. kontrol plak yang dikerjakan oleh dokter gigi memang penting, tapi kontrol plak yang dilakukan oleh penderita sendiri seharihari untuk pemeliharaan merupakan faktor yang lebih penting terhadap keberhasilan perawatan.3 berdasarkan pembahasan awal kekambuhan hiperplastik gingivitis dapat terjadi pada 45 hari setelah gingivektomi dan kemudian meningkat sampai hari ke 90. mengingat bahwa semua sampel pada penelitian ini jumlah monositnya normal, maka dapat disimpulkan pula bahwa kontrol plak memegang peranan penting, sehingga apabila pelaksanaan menjaga kebersihan mulut kurang bagus, maka masih terjadi kekambuhan hiperplastik gingivitis. daftar pustaka 1. newman mg, takei hh, carranza fa. clinical periodontology. 9th ed. philadelphia: wb saunders co; 2002. p. 74–94, 263–9, 432–53, 631–50, 749–61. 2. fedi pf, vermino ar, gray jl. the periodontic syllabus. 4th ed. lippincott williams and wilkins; 2000. p. 41–50. 3. mcmahon rft, sloan p. essentials of pathology for dentistry. harcourt publishers limited; 2000. p. 26–35. 4. axelsson p. diagnosis and risk prediction of periodontal disease. volume 3. quintessence publishing co inc; 2002. p. 317–24. 5. claffey n, loos b, gantes b, martin m, egelberg j. probing depth at re-evaluation following initial periodontal therapy to indicate the initial response to treatment. j clin periodontology 1989; 16:229–33. 6. kantarci a, cebeci i, tuncer o, carin m, firatli e. clinical effects of periodontal therapy on the severity of cyclosporin a-induced gingival hyperplasia. j periodontology 1999; 70:587–93. 7. goldman hm, cohen dw. periodontal therapy. 6th ed. the cv mosby company; 1980. p. 640–90, 773–93. 8. trijani s. evaluasi kesembuhan klinis setelah tindakan gingivektomi dengan atau tanpa peck periodontal pada kasus gingivitis pubertas. timnas 1996; 416–23. 9. lies zbs. gingivektomi sebagai tindakan bedah preprostetik (laporan kasus). jurnal kedokteran gigi universitas indonesia 1997; 4:295–301. 10. ellis js, seymour ra, robertson p, butler tj, thomason jm. photographic scoring of gingival overgrowth. j clin periodontology 2001; 28:81–85. 11. seymour ra, smith dg, turnbull dn. the effects of phenytoin & sodium valproate on the periodontal health of adult epileptic patients. j clin periodontology 1985; 12:413–9. 12. manson jd, eley bm. outline of periodontics. 4th ed. dorset: keytec typesetting ltd; 2000. p. 87–102. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 130 volume 47, number 3, september 2014 perbandingan sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas (citotoxicity comparation of three types of algyrogel on fibroblast cells) stefany elan saktiyawardani,1 hardono jaya lauson,1 anugerah pekerti astamurtiningrum,1 mahadna aulia rahmah,1 pramana pananja putra2 dan juni handajani1 1 bagian biologi mulut, fakultas kedokteran gigi, universitas gadjah mada 2 fakultas kedokteran, universitas gadjah mada yogyakarta indonesia abstract background: the prevalence of osteoporosis and bone defects in the world and indonesia increase each year. algyrogel is a alginate-based hydrogel material recently very popular used as a bone substitute through injectable bone substitute (ibs) method. one of advantages algyrogel is biocompatibility to the body tissues. some natural-based polymer (alginate, chitosan, dan hyaluronat) have different biocompatibility. purpose: the study was aimed to determine the cytotoxicity of three types algyrogel against fibroblast cells. methods: this study used three types algyrogel, there were algyrogel containing alginate (alg), a mixture of alginate with chitosan (alg/ch), and a mixture of alginate with hyaluronic acid (alg/ha). each type algyrogel was exposed in the vero cell line of fibroblast with volume 2 μl; 4 μl; 6 μl; 8 μl; 10 μl; 12 μl; 14 μl; 16 μl; 18 μl; and 20 μl respectively. the cytotoxicity performed by mtt method to determine the percentage of cell death. the optical density was measured by an elisa plate reader then data was analyzed using anova and probit. results: the percentage of cell death in all groups of algyrogel have below 30%. lc50 value at alg was 651.017 mg/ml (21.56%), alg/ch was 280.478 mg/ml (20.91%), and alg/ha was 1054.094 mg/ ml (17.2%). conclusion: alg/ha has the lowest cytotoxicity on fibroblast cells and may have potential as an osteoconductor-synthetic bone product. key words: algyrogel, cytotoxicity, fibroblasts, bone synthesis abstrak latar belakang: prevalensi osteoporosis dan cacat tulang di indonesia maupun dunia semakin meningkat tiap tahunnya. algyrogel sebagai bahan hidrogel yang berbasis alginat akhir-akhir ini sangat popular digunakan sebagai bahan pengganti tulang melalui metode injectable bone substitute. kelebihan algyrogel antara lain biokompatibel terhadap jaringan tubuh. beberapa hidrogel berbasis polimer alami (alginat, chitosan, dan hyaluronat) memiliki perbedaan biokompatibilitas. tujuan: studi ini bertujuan meneliti sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas. metode: penelitian ini menggunakan 3 jenis algyrogel yaitu algyrogel dengan kandungan alginat (alg), campuran alginat dengan chitosan (alg/ch), dan campuran alginat dengan asam hyaluronat (alg/ha). setiap jenis algyrogel dipaparkan pada sel fibroblas jenis vero cell line dengan volume 2 μl; 4 μl; 6 μl; 8 μl; 10 μl; 12 μl; 14 μl; 16 μl; 18 μl; dan 20 μl. pengujian sitotoksisitas dilakukan dengan metode mtt untuk mengetahui persentase kematian sel. penghitungan optical density menggunakan elisa plate reader lalu data diuji anova dan probit. hasil: persentase kematian sel pada semua jenis algyrogel memiliki angka dibawah 30%. nilai lc50 pada alg sebesar 651,017 μg/μl (21,56%), alg/ch sebesar 280,478 μg/μl (20.91%), dan research report 131saktiyawardani, et al.: perbandingan sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas pendahuluan tindakan pencabutan gigi sering dilakukan dalam bidang kedokteran gigi dan dapat menimbulkan perlukaan pada tulang alveolar maupun mukosa rongga mulut. pencabutan gigi dapat menyebabkan kehilangan tulang secara progresif.1 kehilangan tulang juga dapat disebabkan osteoporosis maupun kecacatan tulang. prevalensi kehilangan tulang di indonesia dan dunia menunjukkan peningkatan prevalensi setiap tahunnya. hasil analisis data dan resiko osteoporosis yang dilakukan departemen kesehatan ri bersama dengan pt. fonterea brands indonesia tahun 2006 menunjukkan prevalensi osteoporosis di indonesia telah mencapai 41,75%. hal ini berarti setiap 2 dari 5 penduduk beresiko terkena osteoporosis, lebih tinggi dari prevalensi dunia yang hanya 1 dari 3 berisiko osteoporosis.2 data sistem informasi rumah sakit (sirs) pada tahun 2010 menunjukkan angka insidensi patah tulang paha atas tercatat sekitar 200/100.000 kasus, pada wanita dan pria usia 40 tahun atau lebih yang diakibatkan oleh osteoporosis. world health organization (who) menunjukkan bahwa 50% patah tulang paha atas akan menimbulkan kecacatan seumur hidup dan menyebabkan angka kematian mencapai 30% pada tahun pertama akibat komplikasi imobilisasi. data ini belum termasuk patah tulang belakang dan lengan bawah serta yang tidak memperoleh perawatan medis di rumah sakit. selain itu, hasil review retrospektif yang dilakukan pada 59 pasien fraktur maksilofasial usia 60 tahun atau lebih di sebuah trauma centre antara tahun 1989 dan 2000, diketahui bahwa semakin parah kondisi osteoporosis, semakin besar kemungkinan jumlah fraktur maksilofasial yang dialami. benturan ringan akibat terjatuh pada penderita osteoporosis yang parah dapat menimbulkan fraktur maksilofasial multipel sebagaimana yang terjadi pada kecelakaan kendaraan bermotor.3 bahan pengganti tulang sangat dibutuhkan untuk merekonstruksi morfologi anatomi dan memulihkan stabilitas tulang yang rusak.4 kebutuhan bahan pengganti tulang saat ini dikembangkan dalam bentuk sediaan injeksi sebagai pengisi rongga tulang. kelebihan sediaan bentuk injeksi antara lain dapat dibentuk sesuai dengan bentuk rongga tulang yang akan diisi, steril, dan siap pakai.5 bahan hidrogel saat ini sedang dikembangkan untuk membuat bahan pengganti tulang sintesis dalam sediaan injeksi.6 syarat hidrogel sediaan injeksi antara lain hanya menimbulkan respon inflamasi maupun respon imun ringan, menyerupai sifat mekanis jaringan tubuh, membantu infiltrasi dan interaksi sel yang dibutuhkan, menyediakan sinyal selular melalui molekul bioaktif, dan dapat didegradasikan seiring perbaikan jaringan.7 hidrogel berbasis polimer alami bersifat biokompatibel dan hanya memiliki stimulasi ringan terhadap respon inflamasi maupun respon imun tubuh. polimer alami yang sering digunakan dalam bidang medis antara lain, alginat, chitosan, dan hyaluronat.8,9 ketiga polimer tersebut dibantu dengan bahan crosslinking dapat memenuhi syarat dari hidrogel sediaan injeksi. algyrogel merupakan bahan hidrogel berbasis alginat. komponen yang dapat ditambahkan dalam algyrogel antara lain cerium (ce), chitosan (ch), dan hidroksiapatit (ha). cerium diketahui dapat meningkatkan aktivitas antimikrobial dari hidrogel. ha merupakan komponen utama jaringan tulang dan gigi. sifat utama ha antara lain memiliki biokompatibilitas, dapat berikatan dengan ikatan tulang, dan dapat tumbuh serta berkembang bersama-sama dengan tulang atau regenerasi tulang yang baik.6,10,11 hidrogel berbasis alginat yang dikombinasikan dengan polisakarida alami seringkali dikaji dalam berbagai publikasi. baysal et al.,12 mengkaji pembuatan, karakterisasi, dan aplikasi dari hidrogel crosslinked berbasis kombinasi alginat dan chitosan. morais6 dalam penelitiannya melaporkan perbandingan sitotoksisitas alginate (alg), alginate/ chitosan (alg/ch), dan alginate/ hyaluronat (alg/ha) terhadap sel mg63 sebagai sel osteoblas manusia. pada penelitian tersebut ke dalam sumuran yang berisi sel mg63 diberikan masing-masing 0,6 μl alg, alg/ch, dan alg/ha. dari hasil penelitian ketiga jenis algyrogel terhadap sel mg63 diketahui bahwa alg/ha memiliki sitotoksisitas terendah. beberapa hasil penelitian belum menyebutkan konsentrasi yang aman atau sitotoksisitas yang paling rendah dari ketiga jenis algyrogel terhadap sel fibroblast, yaitu sel yang memproduksi serat kolagen pada lapisan periosteum dan dapat diketahui perubahannya baik yang fisiologis maupun patologis.10 oleh karena itu penelitian ini bertujuan untuk membandingkan sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas. bahan dan metode penelitian ini merupakan penelitian eksperimental labolatoris dengan subjek penelitian berupa kultur sel fibroblas jenis vero cell line yang diperoleh dari laboratorium penelitian dan pengujian terpadu (lppt) alg/ha sebesar 1054,094 μg/μl (17,2%). simpulan: alg/ha memiliki sitotoksisitas paling rendah pada sel fibroblas sehingga memiliki potensi sebagai produk osteokonduktor sintesis tulang. kata kunci: algyrogel, sitotoksisitas, fibroblas, sintesis tulang korespondensi (correspondence): juni handajani, bagian biologi mulut, fakultas kedokteran gigi, universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: junihandajani@yahoo.com 132 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 130–134 unit 2 universitas gadjah mada (ugm). prosedur penelitian telah mendapat persetujuan dari tim etik penelitian fakultas kedokteran gigi universitas gadjah mada yogyakarta (no. 594/kkep/fkg-ugm/ec/2012 tanggal 27 maret 2014). bahan penelitian yang digunakan adalah natrium alginat, natrium hialuronat, chitosan hcl, ha sintetis bovine (laboratorium bioceramics ugm), cerium (iii) nitrat (ce(no3)3), dan cacl2. pembuatan 3 jenis algyrogel sesuai morais6 diawali dengan pembuatan larutan crosslink yaitu larutan penstabil menggunakan cacl2 dan ce(no3)3. alg dibuat dengan cara menambahkan larutan crosslink pada natrium alginat 7% dengan perbandingan 1:4. alg/ha dibuat dengan menambahkan natrium hyaluronat 7% pada larutan crosslink kemudian larutan natrium alginat dengan perbandingan 4:1:4. chitosan hcl 0,5% ditambahkan pada larutan crosslink kemudian larutan natrium alginat dengan perbandingan 4:1:4 untuk menghasilkan alg/ch. pembuatan injectable bone substitute (ibs) dengan cara menambahkan ha pada ketiga jenis algyrogel dengan proporsi 41% algyrogel dan 59% ha. setelah itu masingmasing ibs dengan volume 0,1 μl; 0,2 μl; 0,3 μl; 0,4 μl; 0,5 μl; 0,6 μl; 0,7 μl; 0,8 μl; 0,9 μl; dan 1 μl diencerkan 20 kali dengan menggunakan akuades bersuhu 600 c. pada penelitian ini menggunakan hidroksiapatit sintetis bovine yang berasal dari tulang sapi produksi laboratorium bioceramic fakultas teknik universitas gadjah mada. pengujian mtt assay dimulai dengan memasukkan sel fibroblas ke dalam sumuran plate 96 well sebanyak 2 x 104 sel/ml dalam suspensi sebanyak 100 μl. ketiga algyrogel dimasukkan ke dalam sumuran yang berbeda dengan volume 2 μl; 4 μl; 6 μl; 8 μl; 10 μl; 12 μl; 14 μl; 16 μl; 18 μl; 20 μl dan dilakukan triplet. uji tersebut dilakukan dengan lama perlakuan 24 jam. jika waktu perlakuan telah tercapai, maka dilakukan uji mtt pada sel fibroblas dengan menambahkan reagen mtt, lalu diinkubasi selama 5 jam pada suhu 37o c. medium dibuang perlahan dan ditambahkan 200 μl dimethyl sulfoxide (dmso) ke setiap sumuran lalu dibaca pada λ 570 nm menggunakan elisa reader, sehingga diperoleh optical density. data yang diperoleh dianalisis menggunakan uji-t untuk mengetahui perbandingan sitotoksisitas ketiga gambar 1. rerata dan standar deviasi tiga jenis algyrogel terhadap prosentase sel fibroblas hidup. algyrogel. selanjutnya data dilakukan analisis probit utuk mengetahui nilai lethal concentration 50% (lc50). analisis probit merupakan salah satu tipe dari analisis regresi yang digunakan untuk menentukan toksisitas relatif suatu bahan kimia pada makhluk hidup.14 menurut iso 109993-5,15 suatu bahan dapat dikategorikan tidak toksik apabila jumlah kematian sel kurang dari 30%, sehingga algyrogel aman jika prosentase kehidupan sel lebih dari 70 %. hasil hasil rerata dan standar deviasi prosentase sel fibroblas yang hidup sesuai jenis algyrogel ditampilkan pada gambar 1. hasil penelitian menunjukkan prosentase sel fibroblas yang hidup, terendah terlihat pada jenis alg, sedangkan pada jenis alg/ha tampak prosentase sel fibroblas yang hidup diperoleh lebih tinggi daripada ketiga jenis algyrogel (gambar 1). pada penelitian ini diuji 10 konsentrasi terendah pada masing-masing algyrogel (2 μg/ gambar 2. grafik perbandingan konsentrasi jenis algyrogel terhadap sitotoksisitas sel fibroblas. tampak ketiga jenis algyrogel memiliki pola grafik yang sama antara konsentrasi algyrogel terhadap viabilitas sel fibroblas. 6 yang paling rendah terhadap sel fibroblas. hasil pengujian prosentase sel fibroblas hidup pada masing-masing konsentrasi dan hasil uji-t perbandingan masing-masing jenis algyrogel ditampilkan pada gambar 2 dan tabel 1. gambar 2. grafik perbandingan konsentrasi jenis algyrogel terhadap sitotoksisitas sel fibroblas. tampak ketiga jenis algyrogel memiliki pola grafik yang sama antara konsentrasi algyrogel terhadap viabilitas sel fibroblas. tabel 1. hasil uji-t (p) perbandingan 3 jenis algyrogel terhadap viabilitas sel fibroblas jenis algyrogel alg/ha alg/ch alg alg/ha 0,68 0,03 alg/ch 0,68 0,75 alg 0,03 0,75 perbandingan viabilitas sel fibroblas antara alg/ha dan alg menunjukkan hasil bermakna, maka asam hyaluronat maupun chitosan berpengaruh terhadap tingkat kehidupan sel fibroblas (tabel 1). perbandingan viabilitas sel fibroblas antara alg/ch dengan alg maupun alg/ha menunjukkan hasil tidak bermakna. hasil perhitungan nilai lc50 (lethal concentration 50%) dari setiap jenis hidrogel dihitung dengan analisis probit (tabel 2). grafik perbandingan konsentrasi terhadap sel hidup tiap algyrogel konsentrasi algyrogel (mg/ml) tabel 1. hasil uji-t (p) perbandingan 3 jenis algyrogel terhadap viabilitas sel fibroblas jenis algyrogel alg/ha alg/ch alg alg/ha 0,68 0,03 alg/ch 0,68 0,75 alg 0,03 0,75 tabel 2. nilai lc50 masing-masing jenis algyrogel menggunakan analisis probit. jenis algyrogel nilai lc50 (μg/μl) alg 651,017 alg/ch 280,478 alg/ha 1054,094 133saktiyawardani, et al.: perbandingan sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas μl; 4 μg/μl; 6μg/μl; 8μg/μl; 10μg/μl; 12μg/μl; 14μg/μl; 16μg/μl; dan 20μg/μl) untuk mengetahui dosis yang aman atau toksisitas yang paling rendah terhadap sel fibroblas. hasil pengujian prosentase sel fibroblas hidup pada masing-masing konsentrasi dan hasil uji-t perbandingan masing-masing jenis algyrogel ditampilkan pada gambar 2 dan tabel 1. perbandingan viabilitas sel fibroblas antara alg/ha dan alg menunjukkan hasil bermakna, maka asam hyaluronat maupun chitosan berpengaruh terhadap tingkat kehidupan sel fibroblas (tabel 1). perbandingan viabilitas sel fibroblas antara alg/ch dengan alg maupun alg/ha menunjukkan hasil tidak bermakna. hasil perhitungan nilai lc50 (lethal concentration 50%) dari setiap jenis hidrogel dihitung dengan analisis probit (tabel 2). hasil tabel 2 menunjukkan semua jenis algyrogel memiliki lc50 >60 μg/μl, sehingga dapat dikatakan bahwa ketiga algyrogel memiliki tingkat sitotoksisitas yang rendah terhadap sel fibroblas. hasil gambar 1 dan tabel 2 mengindikasikan semua jenis algyrogel yang digunakan pada penelitian ini dapat dikategorikan aman digunakan untuk sel fibroblas. pembahasan sel fibroblas diketahui sebagai sel yang memproduksi serabut kolagen pada lapisan periosteum, merupakan sel normal yang dapat diketahui perubahannya baik yang fisiologis maupun patologis.10 rerata prosentase sel fibroblas hidup tertinggi diperoleh pada jenis algyrogel alg/ha kemudian diikuti alg/ch sedangkan yang terendah pada jenis algyrogel alg (gambar 1). hasil tersebut mengindikasikan bahwa penambahan material asam hyaluronat maupun chitosan diduga dapat meningkatkan viabilitas kehidupan sel fibroblas. seperti dikemukakan oleh andersen11 bahwa alginat memiliki sifat biokompatibel pada tulang karena dapat didegradasi dengan baik. namun, alginat tidak dapat memicu perlekatan sel, sehingga sel yang membutuhkan substrat untuk perlekatan tidak dapat bertahan dalam gel alginat. penambahan chitosan dan hyaluronat pada algyrogel dapat meningkatkan viabilitas sel karena keunggulan kedua bahan tersebut yang tahan terhadap perubahan kimia dan enzimatik, bertindak sebagai adesif karena muatan positif nya pada ph fisiologis, biodegradable, biokompatibel, dan siap diolah menjadi berbagai bentuk. chitosan memiliki karakteristik non-toksik, non-antigenik, biofungsional dan osteokonduktif. selain itu, chitosan mampu memicu proliferasi dan diferensiasi sel. hyaluronat dapat ditemukan pada jaringan ikat, ditemukan dalam konsentrasi tinggi pada kalus, dan dapat menghambat diferensiasi dari osteoklas. polimer ini merupakan penyusun utama dari matriks ekstraseluler tubuh. penggunaan cacl2 dan ce(no3)3 pada larutan crosslink bertujuan membuat sediaan gel menjadi stabil dengan cacl2, sedangkan ce(no3)3 memiliki fungsi sebagai antimikroba.6,12,13 hasil persentase kematian sel pada semua kelompok algyrogel dan pada semua konsentrasi menunjukkan angka dibawah 30%. iso 109993-5 menyatakan bahwa suatu bahan dapat dikategorikan tidak toksik apabila jumlah kematian sel kurang dari 30%.15 hasil ini menunjukkan bahwa algyrogel dengan menggunakan ha sintetis bovine yang berasal dari tulang sapi produksi laboratorium bioceramic fakultas teknik universitas gadjah mada aman digunakan pada sel fibroblas. lethal concentration 50% (lc50) adalah konsentrasi suatu bahan pada suatu media yang dapat membunuh 50% sel yang diuji.17 nilai lc50 dapat ditentukan dengan menggunakan analisis probit.14 hidrogel termasuk kategori aman jika memiliki sitotoksisitas (lc50) sebesar 60 μg/μl atau lebih.16 hasil penelitian ini menunjukkan semua algyrogel (alg, alg/ch, dan alg/ha) memiliki lc50>60 μg/μl sehingga aman bagi tubuh. berdasarkan hasil yang tersaji pada tabel 2, hidrogel campuran alginat/hyaluronat (alg/ha) memiliki tingkat keamanan yang paling tinggi, ditandai dengan lc50 terbesar. 15,16 mekanisme hidrogel alg/ha memiliki tingkat keamanan yang paling tinggi diduga karena peran biologis dari hyaluronat (ha) sebagai hasil interaksi dengan sejumlah ha-binding protein yang disebut hyaladherins. hyaluronat diketahui dapat berikatan dengan reseptor spesifik cell-surface antara lain cd44, receptor for ha-mediated motility (rhamm) dan intercellular adhesion molecule-1 (icam-1). interaksi ha dengan reseptor tersebut dapat menginduksi transduksi berbagai signal intraseluler untuk pengaturan berbagai proses seluler seperti morfogenesis, penyembuhan luka, dan inflamasi serta keterlibatannya pada saat kondisi patologis. dari hasil penelitian ini diduga hyaluronat memiliki interaksi dengan reseptor spesifik cell-surface pada fibroblas sehingga terjadi ha-binding dengan fibroblas. beberapa reseptor yang dibutuhkan pada saat ha-binding diketahui dapat diekspresikan oleh fibroblas antara lain rhamm dan icam-1.18 penelitian ini menunjukkan bahwa hidrogel alg/ha memiliki sitotoksisitas terhadap sel fibroblas yang paling rendah dibandingkan hidrogel alg/ch maupun hidrogel alg sehingga memiliki potensi menjadi produk osteokonduktor sintesis tulang yang aman dan bermanfaat. ucapan terima kasih terimakasih kepada departemen pendidikan tinggi atas dana program kreativitas mahasiswa bidang penelitian (pkm-p) 2014. terimakasih kepada drg. heni susilowati, m.kes., ph.d, fakultas kedokteran gigi, universitas gadjah mada atas masukan dan bimbingannya. 134 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 130–134 daftar pustaka 1. calixto rfe, teófilo jm, brentegani lg, lamano-carvalho tl. alveolar wound healing after implantation with a pool of commercially available bovine bone morphogenetic proteins (bmps) a histometric study in rats. braz dent j. 2007; 18(1): 2933. 2. sihombing hc. karakteristik kasus menopause osteoporosis di makmal terpadu imunoendrokrinologi fk ui tahun 2006-2008. jakarta: universitas indonesia; 2009. p.25-30. 3. werning jw, downey nm, brinker ra, khuder sa, davis wj, rubin am, elsamaloty hm. the impact of osteoporosis on patients with maxillofacial trauma. arch otolaryngol head neck surg. 2004; 130: 353-6. 4. bintarti tw, izak jr, ady j. sintesis dan karakterisasi bone graft berbasis hidroksiapatit dan alginat, jft. 2013; 1(2): 108-24. 5. warastuti y, abbas b. sintesis dan karakterisasi pasta injectable bone substitute iradiasi berbasis hidroksiapatit, j ilmiah apl isotop radiasi. 2011; 7(2): 74-5. 6. morais ds. rodrigues ma, lopes ma, coelho mj, mauricio ac, gomes r, amorim i, ferraz mp, santos jd, botelho cm. biological evaluation of alginate-based hydrogels, with antimicrobial features by ce(iii) incorporation, as vehicles for a bone substitute. j mater sci mater med. 2013; 24: 2145–55. 7. overstreet dj, duta d, stabenfeldt se, vernon bl. injectable hydrogels. polymer physics. 2012; 50: 881-903. 8. zao w, jin x, chong y, liu y, fu j. degradable natural polymer hydrogels for articular cartilage tissue engineering. j chem technol biotechnol. 2013; 88: 327–39. 9. rahman k. pengaruh lama milling pada sintesis biokeramik hydroxyapatite (ha) dengan metode solid-state reaction terhadap kekristalan, mikrostruktur, dan kuat tekan. available at: http://karyailmiah.um.ac.id/index.php/fisika/article/view/21069. diakses pada 12 oktober 2013. 10. junqueira lc, carneiro j. basic histology text & atlas. edisi 11. usa: mcgrawhill; 2005. p. 95. 11. andersen t, strand f, alsberg c. alginates as biomaterials in tissue ngineering, j carbohydrate chem. 2012; 37: 227-58. 12. baysal k, aroguz a, adiguzel z, baysal b. chitosan/alginate crosslinked hydrogels: preparation, characterization and application for cell growth purposes, int j biol macromol. 2013: 59: 342–48. 13. mu z z a r el l i r a a . c h it o s a n c om p o sit e s w it h i no rga n ics , morphogenetic proteins and stem cells, for bone regeneration, carbohydrate polymers. 2011; 83(4): 1433-45. 14. vincent, k. probit analysis. avaliable at: http://userwww.sfsu. edu/efc/classes/biol710/probit/probitanalysis.pdf. diakses pada 11 oktober 2014. 15. iso 10993-5. biological evaluation of medical devices – part 5: test for in vitro cytotoxicity, international organization for standardization, geneva. 2009. 16. xu x, jha ak, harrington da, carson mcf, jia x. hyaluronic acid-based hydrogels: form a natural polyssacharide to complex networks, soft matter. 2012; 8(12): 3280-94. 17. sharma pd, environmental biology & toxicology. india: rastogi publications: 2005. p.122. 18. turley ea, noble pw, bourguignon lyw. signaling properties of hyaluronan receptors. j biol chem 2002; 277: 4589-92. 89 study of chemical bond strength of methyl methacrylate (mma) based bonding agent on type i dentin collagen at various humidity adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract one of the basic agents used in dentin bonding solution is methyl methacrylate (mma). this bonding agent is widely used in dentistry. it have been proved that the adhesion between dentin bonding agent and collagen fibril is chemically bond; though the chemical bonding contribution is smaller than physical mechanical bond. the purpose of the research was to examined the chemical bonding strength of mma based dentin bonding on type i dentin collagen at various humidity. samples of treatment group were put into desiccator with 60%, 70%, 80%, and 90% humidity, while for control groups at room humidity (65%). chemical bond of pure mma and mma mixed with collagen were measured by ftir. the lower the value of mma carbonyl, the higher absorbance band speak of chemical bond strength between mma and collagen. data was statistically analyzed with one-way anova at 95% confidence level continued with tukey-hsd test. the result showed that the highest chemical bond strength was at 65% humidity (p ≤ 0.05). in conclusion, many esther carbonyl mma molecules reacted with amino collagen at 65% humidity. this can be shown by the lowest peak's value of the mma carbonyl absorbance at ftir. key words: ftir, humidity, mma, type i dentin collagen, carbonyl group correspondence: adioro soetojo, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction science development of operative dentistry made bonding agents have been used excessively to restore dental caries. these agents are ideal to restore class v erosion which involving dentin, especially for the elderly patients. since the bonding agents have hydrophilic characteristic, it can adhere well to dentin.1,2,3 recently there are many bonding agents with hydrophilic and hydrophobic characteristic have been produced; such as bisphenyl dimethacrylate (bpdm), 4-methacryloxyethyl trimellitic anhydride (4-meta) and others. bonding agents with hydrophilic characteristic will adhere to dentin collagen, while the one hydrophobic characteristic adhere on composite resin.3,4 good wetting characteristics of bonding agents due to their low viscosity which increase surface energy. surface energy is the potency of an agent surface to pull another substance surface. the adhesion between two agents occur if there is adhesive force at the interface area. the increase energy of each unit area is related with surface energy and surface tension.3 there are several monomer bonding agents available such as hydroxyethyl methacrylate (hema), bpdm, glutaraldehyde, 4-meta, methyl methacrylate (mma), etc. in order to make clinical application easier, usually the bonding agent is added with photo-initiator (camphoroquinone) which absorb the blue region of visible light spectrum at the wave-length from 400 to 500 nm.3,6 accelerators from amine groups, which well interact with camphoroquinone such as dimethylaminoethyl methacrylate 0.15%, are often added to bonding agents. for long storage, the bonding agent is added with inhibitor agent, butylated hydroxytoluene 0.01%. the adhesion of bonding agent on dentin surface occurs physical-mechanically or chemically. the physicalmechanical bonding is caused by penetration of bonding agent into nano space of collagen fibrils; resin penetration in dentin tubules; microscopically retention on dentin surface (undercut, crack, micro space) and vander-waals force based on dipolar attraction.2,3,4 chemical bonding is caused by interaction of esther carbonyl groups of dentin bonding with amine groups at collagen which produce amide groups [c (o)nh]. this bonding is strong due to of its covalent characteristics called as inter-atomic primer bond.7 the bonding process of restoration materials on dental structure are a complex matter including mma based resin bonding. the resin bonding failure are caused by some factors, such as smear layer on dental surface, topical application of fluoride, unhomogeneous tooth composition (organic and inorganic agents are very different in enamel and dentin), and saliva or blood contamination. dentin is life tissues which containing approximately 60% inorganic components (hydroxyapatite) ca10 (po4) 6 (oh)2; 30% organic components and 10% water. those 90 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 89–93 organic components are 90% collagen, and 10% noncollagenous. most of those collagens are type i and few of them are type v.9 type i collagen which frequently used as research agent are a sequence of amino acid: prolin, prolin, glisin {h2n-(pro-pro-gly)5-cooh}. 10,11 dentin bonding can bond well to collagen fibril if the collagen is in active or permeable condition. the permeability of collagen is highly influenced by humidity of dentin surface. if the humidity is too high or too low, the bonding agent will hardly bonds on collagen fibril. therefore, it requires optimum humidity condition to obtained a maximum bond of resin bonding agent to collagen.5 the humidity used in the research were in the range of 60 to 90%. the other factors that influence the bond of dentin bonding to collagen are viscosity, type and concentration of dentin bonding agents, application of acid agents as conditioner and temperature of surrounding collagen.12–15 this research used resin monomer mma c5h8o2. the mma solution is a clear and transparent liquid at room temperature. the physical characteristics as follows: melting point at –48°c, boilling point at 100,8°c, density = 0,945 g/ml (20°c), heat of polymerization = 12,9 kcal/mol.3 the aim of the research was to examined the chemical bond strength of mma based bonding on type i dentin collagen at various humidity. material and methods material which were used: bovine type i collagen (sigma chemical, st. louis, usa; batch # 031k7054), methyl methacrylate liquid (vertex; dentimex, holland). instruments which were used: desiccators with vacuum faucet (dsc, china), hygrometer (haar. synth-hygro, germany), air suction (schuco, usa), micro injection (hamilton, usa), fourier transformed infra red instrument (ftir, jasco ft/ir 5300, japan). the research was conducted in laboratorium dasar bersama (ldb) airlangga university with room humidity of 65% and room temperature at 25 ± 2°c. the method of this research has been presented in detail in a previous research.17 to arrange 90% of humidity, 150 cc water were put in the bottom part of desiccators, then calibrated hygrometer was set. in this condition, the humidity seen from hygrometer was 94–95%. then from faucet located above desiccators, the air is pumped out with air suction until humidity reached to 90% and the pump directly closed. for 80% humidity, the air in desiccators is pumped out until humidity reached to 80%. in order to fasten the work, we put the silica gel which is activied first in to desiccators. afterwards the air was pumped out until the humidity in desiccators reached to 60%. to prepared kalium bromide (kbr) pellets: 300 mg kbr powder were dropped with 10 ml mma (9.45 mg of weight) using micro-injection. molecular weight of mma was 0.945 g/ml. total weight of kbr powder and mma were 309.45 mg, then all of the materials were crushed with mortar and pestle made from agate stone. after mixed, 50 mg mixed-powder were put in to kbr die and compressed until 10 ton while it’s was vacuumed. the final result of the process was a clear pellet. the pellets were observed into ftir. to prepared collagen-mma mixture, 2 mg collagen were dropped with 10µl mma (9.45 mg of weight) and kbr powder were added with until the weight were 309.45 mg. the mixed powders were compressed, and then 50 mg of the mixtures were put it in to kbr die. next, the samples are compressed until 10 ton while it has been vacuumed. that pellets were finally observed in to ftir. to make samples at various humidity: 2 mg collagen fibers were packed into desiccators with hygrometer that has been calibrated. then the humidity of desiccators were arranged to 60%, 70%, 80%, and 90%. as soon as release from the desiccators, the collagen was dropped with mma and added with kbr powder until reached 309, 45 mg of weight. the mixed powders were compressed, and then 50 mg of the mixtures were put it in to kbr die. next, the samples are compressed until 10 ton while it has been vacuumed. the peak of carbonyl absorbance band (c = 0) of treatment groups and control groups was measured. the method to calculate the peak of carbonyl (p) absorbance band as below.18 a 100 transmittance = t (%) absorbance band c=o (carbonyl wave number) b 0 c figure 1. peak of the mma carbonyl absorbance band. description: p = (bc/ab) × 100; (ab and bc measured in centimeter). the calculation is accurately enough and trustworthy if the intensity of absorbance band at transmittance, t = 30% to 60%. 91soetojo: study of chemical bond strength results figure 2. the ir ray spectrum of mma and collagen (kbr pellets). value of the peak carbonyl absorbance band is 33,3; point 7 (arrow). humidity: 80%. figure 3. the ir ray spectrum of mma + collagen (kbr pellets). value of the peak carbonyl absorbance band is 29,2; point 11 (arrow) humidity: 90%. chemical bond measurement is done by using kbr pellets in which then irradiated by the ir ray. after that the peak value of the mma esther carbonyl absorbance band was recorded. when mma carbonyl groups excessively bond toward collagen amino groups, the peak value of the mma carbonyl absorbance band decreased. the more mma carbonyl groups bond with amino collagen groups the bond between those agents will increase. mean value of mma carbonyl absorbance band and standard deviation were shown at table 1. mean value of the mma carbonyl absorbance was 48.7 ± 3.7 then decreased at 60%, 65%, 70%, 80%, and 90% humidity. to know whether the chemical bond data was normal, one sample of kolmogorov smirnov test was done. the p value at the experimental groups at 60–90% humidity showed the higher number than 0.05 (p > 0.05). it means that the data of chemical bonds experimental groups at 60–90% humidity were normal. to know the homogeneity of experimental groups, homogeneity of variance test was done. the result showed that the value of p was 0.179 (p > 0.05). it means that the experiment of chemical bonds groups at 60–90% humidity was homogen. table 1. mean of the peak of mma carbonyl absorbance and standard deviation at various humidity humidity n x (mean) sd mma 5 48.7200 3.73992 60% 5 27.3600 2.21314 65% (control) 5 19.1800 1.74557 70% 5 20.5000 1.33791 80% 5 30.9400 1.56301 90% 5 34.5000 3.09031 description: n = amount of sample; x = mean of the peak of the mma carbonyl absorbance band; sd = standart deviation. table 2. the tukey-hsd test in carbonyl absorbance band of mma at various humidity dentin bonding agent based on mma significance level mma 60% humidity 65% 70% 80% 90% 21.360* 29.540* 28.220* 17.780* 14.220* 60% humidity mma 65% 70% 80% 90% – 21.360* 8.180* 6.860* – 3.580* – 7.140* 65% humidity mma 60% 70% 80% 90% – 29.540* – 8.180* – 1.320* – 11.760* – 15.320* 70% humidity mma 60% 65% 80% 90% – 28.220* – 6.860* 1.320* – 10.440* – 14.000* 80% humidity mma 60% 65% 70% 90% – 17.780* 3.580* 11.760* 10.440* – 3.560* 90% humidity mma 60% 65% 70% 80% – 14.220* 7.140* 15.320* 14.000* 3.560* note: *) : significant difference at a = 0.05 92 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 89–93 anova test was used to know the influence of humidity towards chemical bonds between mma and dentin collagen. there was a significant difference among the experimental groups (p < 0.05). turkey-hsd test was done to give evidence of distinctions for every experimental group. peak value of the mma carbonyl absorbance band among the experimental groups at 60%, 65%, and 70% humidity showed significant difference (p < 0.05). there was no significant difference (p > 0.05) between 80% and 90% humidity in experimental groups. experimental groups with 70% humidity compared with 60%, 80% and 90% humidity showed significant difference (p < 0.05) and if compared with 65% humidity showed no significant difference (p > 0.05). furthermore, in experimental group with 90% compared with 60%, 65%, and 70% humidity showed significant difference (p < 0.05), but not for 80% humidity (p > 0.05). discussion infra red spectrum also can be used to count the quantitative value of the ofmixing matter components. however the number of each factor definitely must be calculated accurately in order to get accurate and trustworthy datas. if collagen fiber mixes with mma, chemical reaction between amino group of collagen and esther carbonyl group of mma will occur. in fact, not all of the esther carbonyl group of mma fully reacted with amino collagen groups. this matter can be seen at peak of the mixture of mma carbonyl and amino collagen absorbance band which is decrease but not disaper. esther carbonyl wave number (bc=o) absorbance ranging around 1700 to 1750 cm –1. in this research, the peak of esther carbonyl at mma absorbance band is used as the control or comparison with the peak of esther carbonyl absorbance band at experimental groups by various kind of humidity treatments. if many carbonyl groups at mma react with amino collagen groups, the ir ray spectrum will occur the reduction of peak of the mixture of esther carbonyl at mma and collagen absorbance band. so it can be assumed that if there is great reduction of peak of the mma carbonyl absorbance band, it means that will be there are many esther carbonyl groups bond with amino groups. this means that chemical bond between mma and collagen can be stronger than ever. chemical bond between bonding agents mma based and collagen fibril should be considered even though from the previous research chemical bond only 30% compared with physical-mechanical bond strength. the chemical bond strength depends on the condition of surrounding collagen, such as humidity and temperature.5,19 the study used constant temperature (25 ± 2 °c). at an optimum humidity, the condition of collagen fibril is very permeable, not only made a strong bonding between mma carbonyl group and collagen amino group, but also a maximum hydrogen bond inside molecule chain, or among collagen molecules. moreover a good penetration of mma into nano interfibriler space will occur and polymerization of mma will form a mechanical retention. if the humidity of surrounding dentin is too high, mma solution difficult to react with collagen fibril, because many water molecules in surrounding fibril will block the mma penetration. an excessive hydrogen bond between water molecule and dentin collagen made bonding between collagen molecule and mma can not chemically occur. reis et al.20 reported that the use of air stream is not effective to remove water from mixture of bonding agents and water. the location of the highest water concentration is predicted in the deepest collagen fibril (profunda site), so it is difficult to remove water using air stream and the bonding agents is difficult to diffuse. a study of microraman spectroscopy showed that monomer concentration decrease from the upper side to hybrid layer base which formed by adhesive resin. for example, bonding agents concentration will decrease almost 55% at 2 mm under surface region and decrease almost 21% at hybrid layer base. but if the surrounding dentin is very dry (minimum humidity) the collagen will collapsed. it will make the hydrogen chain of inter and intra molecule collagen are broken and collagen amino group are covered with remain of collagen fibril. the condition not only makes amino collagen groups can not react with mma carbonyl groups, but also interaction between functional groups of mma and fungtional group (carboxylate, amino, amide) collagen do not occur and no complex reaction between ca++ dentin and mma. it has been reported that in water free region (dry area), the polymer chain formation is weaker and unstable. the ph of monomer solution plays role in chemical bond strength between bonding agent and collagen.22 the experiment, proved that high bonding strength between hema and collagen occur at ph 2. if ph rise up to 6.6, the bonding strength will decrease. however if ph is rise from ph 9.0 to 12.5, the bonding strength will increase again. bonding strength is studied using method of collagen functional groups dissociation, such as, carboxylate and amino. if carbocylate acid dissociation or amino group is being inhibited. the hydrogen bond between resin and collagen will increase. if ph of collagen suspension is low, carboxylate acid will not be dissociated, so the interaction of resin and collagen will increase. the ph of mma solution in the study was 5.0. nakabayashi and pashly5 reported that the process of collagens collapse known as passive theory. passive theory is demineralized collagen network will float or suspended in water. each fibril will be separated one another by water molecule in the space between fibril. prior to water molecule the space is filled by apatite crystal. at drainage process, the water which supports collagen network will be eliminated, collagen will close and aggregate three dimensionly. this condition is called collapse or shrinkage. the fibril bonding becoming stiff and creating the interfibriler hygrogen bond which interact electrostatically or hydrophobically.5 water additional changed the condition into passive 93soetojo: study of chemical bond strength re-expand. water molecule can periform hydrogen bond with collagen peptide, breaking interfebriler hydrogen bond so the collagen network is re-expand. the re-expand network functions as hydrogel, where water osmotically entering interfibriler space. in this research, pure mma solution is used to examine role of mma on chemical bond to pure collagen. mean of band’s peak of the mma carbonyl absorbance is 48.7 ± 3.7, this value decrease at 60% humidity (27.3 ± 2.2 ); 65% humidity (19.1 ± 1.7); 70% (20.5 ± 1.3); 80% (30.9 ± 1.5) and 90% (34.5 ± 3.1). the lower the band’s peak value of the mma carbonyl absorbance the greater the bonding of mma carbonyl groups to collagen amino groups. it makes the bonding strength between two agents increase. the highest strength of chemical bonds occur at 65% humidity, since the water molecule surrounding around fibril in optimum condition. water molecule makes collagen re-expand, and collagen become more active and permeable to mma. comparing with previous research, the bond strength of bonding agents hema based and mma based are different. the highest bonding strenght of hema based, is at 70% humidity, while bonding agent mma based is at 65% humidity. it may be caused by the difference of agent density. hema’s density is 1.07 g/ml while mma density is 0.945 g/ml. table 1 showed that mean of the band peak’s value of mma carbonyl absorbance from 60% to 90% humidity are significantly increase. it caused chemical bond strength decrease. the increase of humidity makes water molecules surround fibril rise and the penetration of mma solution to collagen fibril is blocked. if we compare the 65% and 60% humidity, chemical bond strength at 65% humidity is higher than 60%, since at 60% humidity, the condition of fibril is drier, so as the results fibril collapsed and low chemical interaction between mma and collagen occur. the research suggest that maximum chemical bond strength between mma carbonyl groups and amino collagen groups occur at optimum humidity of 65%. references 1. accorinte mlr, loquercio ad, reis a, muench a. adverse effect of human pulps after direct pulp-capping with the different components from a total etch, three step adhesive system. dent mat 2005; 21:599–607. 2. summitt jb, robbins jw, hilton tj, schwartz r. fundamentals of operative dentistry. 3rd ed. chicago: quintess publ co, inc; 2006. p. 183–242. 3. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia: wb saunders co; 2003. p. 21–395. 4. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 5. nakabayashi np, pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 6. brackett mg, brackett ww, haish ld. microleakage of class v resin composites placed using self-etching resins. quintess int 2006; 37:109–13. 7. xu j, stangel i, butler is, gilson dfr. an ft raman spectroscopy investigation of dentin and collagen surfaces modified by hema. j dent res 1997; 76:596–601. 8. carrilho mr, tay fr. pashley dh. mechanical stability of resindentin bond components. dent mat 2005; 21:232–41. 9. cohen s, burns rc. pathways of the pulp. 8th ed. st louis, london, philadelphia: mosby inc; 2002. p. 411–54. 10. nishiyama n, asakura t, suzuki k. adhesion mechanism of resin to etched dentin primed with n-mgly studied by 13c nmr. j biomed mater res 1998; 40:458–63. 11. nishiyama n, asakura t, suzuki k, komatsu k. bond strength of resin to acid-etched dentin studied by 13nmr. j dent res 2000; 79:806–11. 12. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32:19–26. 13. breschi l, gobbi p, marzotti g, falconi m. high resolution sem evaluation of dentin etched with maleic and citric acid. dent mat 2002; 18:26–35. 14. finger wj, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4:277–82. 15. zohairy aa, de gee aj, mohsen m. effect of conditioning time of self-etching primers on dentin bond strength of three adhesive resin cements. dent mat 2005; 21: 83–93. 16. frankenberger r, tay fr. self etch vs etch-and-rinse adhesive: effect of thermo-mechanical fatique loading on marginal quality of bonded resin compositerestorations. dent mat. 2005; 21:397–412. 17. soetojo a. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada berbagai kelembaban. disertation. surabaya: airlangga university; 2006. p. 66-9. 18. kemp w. organic spectroscopy. 2nd ed. edinburgh: elbs/macmillan ltd; 1988. p. 12–81. 19. noort rv. introduction to dental materials. 2nd ed. edinburgh, london, new york, oxford: cv mosby co; 2002. p. 11–78. 20. reis a, alessandro d, loguercio, carvalho rm, grande rhm. durability of resin dentin interface: effects of surface moisture and adhesive solvent component. j dent mat 2004; 20:669–76. 21. renzo md, ellis th. chemical reactions between dentin and bonding agents. j adhesion 1994; 47:115–21. 22. nishiyama n, suzuki k, nagatsuka a, nemoto k. dissociation states of collagen functional groups and their effects on priming efficacy of hema bonded to collagen. j dent res 2003; 82:257–61. 143143 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 143–149 original article introduction dental and skeletal malocclusions can occur in a sagittal, vertical or transverse direction. in the sagittal direction they can occur in anterior crossbite, deep bite or open bite. vertical and transverse directions can occur in scissor bite or posterior crossbite.1 occlusal interference due to malocclusion abrupt the effectiveness of mastication on each side of the jaw causing changes in masticatory patterns as individuals employ a more effective and comfortable side.2 angle’s classification divides dental malocclusions into three categories. angle class i malocclusions are defined by the mesiobuccal cusp of the permanent maxillary first molar coming into contact with the mesiobuccal groove of the permanent mandibular first molar. angle class ii malocclusions are specified when the mesiobuccal groove of the permanent mandibular first molar is distal when compared to the mesiobuccal cusp of the permanent maxillary first molar. angle class iii malocclusions are denoted by the mesiobuccal groove of the permanent mandibular first molar being more medially located than the mesiobuccal cusp of the permanent maxillary first molar.3 the presence of one or more posterior group teeth in an irregular bucco-lingual or bucco-palatal relationship with one or more opposing teeth in centric occlusion is defined as posterior crossbite. posterior crossbite, especially unilateral dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 malocclusion with posterior unilateral crossbite affects superficial masseter and anterior temporal muscle activity during mastication yona pricilia anggi siregar, christnawati and darmawan soetantyo department of orthodontics, faculty of dentistry, universitas gadjah mada (ugm), yogyakarta, indonesia abstract background: mastication patterns due to malocclusion with unilateral posterior crossbite may permanently change. purpose: this study aimed to examine the effect of malocclusion with unilateral posterior crossbite of the superficial masseter and anterior temporal muscles on the crossbite and non-crossbite sides during mastication. methods: thirty subjects (8 males and 22 females) between the ages of 17 and 30 years who were students of the 2017–2019 dentistry and dental hygiene study program, faculty of dentistry, ugm and who had at least two posterior teeth with unilateral posterior crossbite were divided into 10 subjects with angle’s class i, 10 subjects with class ii and 10 subjects with class iii malocclusions. the amplitude of the superficial masseter and temporal anterior muscles was performed during mastication using surface electromyography (semg). the mean difference between the groups of malocclusion on the crossbite and non-crossbite sides of the superficial masseter and temporal anterior was analysed by a two-way analysis of variance (anova). results: the results indicated a difference in amplitude mean between the malocclusion types on the crossbite sides and non-crossbite sides of the superficial masseter and temporal anterior muscles (p<0.05). this study confirmed there was a decrease in superficial masseter and anterior temporal muscle activity on the crossbite side rather than in the non-crossbite side in angle’s class i and class ii. however, there was an increase in activity of the superficial masseter and anterior temporal muscles on the crossbite side for class iii. conclusion: malocclusion with unilateral posterior crossbite affects masticatory activity of the superficial masseter and temporal anterior muscles on the crossbite side. keywords: malocclusion; masseter muscle; posterior crossbite unilateral; semg; temporal muscle correspondence: christnawati, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. email: christnawati_fkg@ugm.ac.id mailto:christnawati_fkg@ugm.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 144 siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 posterior crossbite, can cause mandibular shift, postural changes and has a possible link to temporomandibular joint (tmj) disorders.4 the most common type of posterior crossbite is unilateral and bilateral posterior crossbite. several factors causing posterior crossbite were found in this study such as tooth loss, maxillary contraction, persistence of deciduous teeth, occlusal disorders, hereditary factors, delayed eruption and premature loss.5 in brazil, 8–22 % of orthodontic patients and 5–15 % of the general population have posterior crossbite in the primary and early mixed dentition.6 soft tissue is crucial in relation to orthodontic treatment because it has an effect on the dental arch shape that is the etiology of malocclusion, and this can affect the stability of the treatment. facial muscles develop at birth and are the earliest muscles of the body to form to allow airways to be maintained. at the same time, tooth eruption, mastication, facial expressions, swallowing and speech are also developing.7 facial muscles are associated with relapse. in addition, unstable muscles from orthodontic treatment can lead to a relapse.8 electromyography (emg) is used to diagnose facial muscles during orthodontic treatment by using a neuromuscular approach. emg can be recorded in two ways: surface and intramuscular. surface electromyography (semg) is typically used to assess muscle function by recording activity from the skin’s surface over the muscle using a pair of electrodes. surface emg allows for the noninvasive study of the bioelectrical phenomena of muscular contraction.9 kinematic patterns and muscle activity changes in the adjustment of the chewing load capacity can be observed through neuromuscular characteristics using semg.10 mastication is the most essential function of the stomatognathic system. muscles, ligaments, bones and tooth structures are responsible for mastication function and control of the central nervous system. when the muscle is activated, electric signals are generated by ions crossing the muscle cell membrane and this is recorded and presented for emg analysis. the masseter muscle is a thick and intensely strong muscle mass. it is rectangular-shaped originating from the temporal bone and extending to the angle of the mandible.11 the masseter muscle serves as an elevator of the mandible in the protrusive movement and stabilises the condyle of the articular eminence. the temporal muscle is a large, fan-shaped muscle that fills the temporal fossa. the temporal muscle functions as an elevator of the mandible.12 muscle examination and evaluation at the orthodontic clinic, such as accurate muscle activity measurements using semg, has not been widely developed as a diagnostic procedure. the purpose of this research is to examine the effect of malocclusion with unilateral posterior crossbite of the superficial masseter and anterior temporal muscles on the crossbite and non-crossbite sides during mastication. materials and methods this is an analytical cross-sectional research study. the subjects in this study were dentistry and dental hygiene students from the faculty of dentistry at ugm, who met the following inclusion criteria: the correct type of dental malocclusion, including class i, ii and iii and unilateral posterior crossbite involving at least two posterior teeth in centric relation and centric occlusion (maximum intercuspation); had no mandibular shift when opening and closing the mouth; complete number of teeth except for the third molars; no accidental trauma to the face and jaw; no clicking, crepitus or pain in the tmj when opening and closing the mouth; did not use dentures and/or occlusal splints; had never had orthodontic or orthognathic treatment before and who had no history of systemic disease manifestations in the oral cavity. in this study, 30 subjects were selected from the selection consisting of 55 research subjects with unilateral posterior crossbite who did match the inclusion requirements. research was then carried out using a purposive sampling technique, namely the selection inclusion criteria of the 30 suitable subjects were adjusted to comprise of 10 subjects with angle’s class i malocclusion, 10 subjects with angle’s class ii malocclusion and 10 subjects with angle’s class iii malocclusion. the 30 participants were separated into two groups: crossbite with class i, class ii and class iii malocclusions, and non-crossbite with class i, class ii and class iii malocclusions. this research has been approved by the ethical commission of ugm number 00682/kkep/fkg-ugm/ ec/2021. the research subject selection has been qualified by the faculty of dentistry, ugm ethics committee. eligible subjects received an explanation of the research procedure after filling out a written informed consent form. subjects who met the inclusion criteria signed the written informed consent form and after having their history of malocclusion assessed were scheduled to visit the electromedical outpatient unit at dr. sardjito general hospital, yogyakarta. each research subject spent 20 minutes at the electromedical unit. the subject’s details were registered in the emg device system and electrodes were attached. this study employed semg using three electrodes. the electrodes were attached to the masseter and/or temporal muscles, nose (reference) and forehead (ground) using lubricants and adhesive tape above the skin that had been cleaned with a special scrub. emg electrodes have no side effects or risks and since it is not an invasive method it was considered safe to implement it in this study. the initial stage of the study did not record muscle activity. subjects were directed to chew four peanuts on each side of their mouth one by one following a sequence of muscle measurements. measurements were performed respectively through emg recordings on the crossbite side of the superficial masseter (mc), the anterior temporal of the crossbite side (tc), the non-crossbite side dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 145siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 of the superficial masseter (mn) and the non-crossbite side of the anterior temporal (tn) (figure 1). the study course was initiated by delivering instructions to the participants for relaxation of the mind, facial muscles and lips. participants were required to sit upright on a chair. the measurement of muscle activity started with the rightside of the superficial masseter muscle. subjects were required to rest for two minutes before being instructed to chew nuts for 20 seconds during measurements. this treatment was repeated by measuring activity of the left superficial masseter muscle, right side anterior temporal muscle and left side temporal muscle. electrodes were removed after all the measurements had been completed.13 an interpretation of the results was conducted by an electromyographer by synchronising the measurement area based on observation time intervals of 0–5 seconds, 5–10 s, 10–15 s and 15–20 s and discovering the highest amplitude during that time interval. the classification of these time intervals is a result of the motor unit potential (mup), which records muscle action potentials, having only four assessment windows on the device monitor. the observation area is measured not only for the highest amplitude in each interval but also at one screen per interval.14 all statistical analysis data obtained were examined by averages using the statistical package for the social sciences (spss) 24.0 version (ibm corporation, illinois, chicago, us). a two-way analysis of variance (anova) (p<0.05) was carried out to examine the significant difference between the groups. results angle’s class i and ii malocclusions had lower crossbite side superficial masseter muscle activity than the noncrossbite side. in contrast, class iii malocclusion had higher crossbite side superficial masseter muscle activity than the non-crossbite side. in class i and ii malocclusions, anterior temporal muscle activity was lower on the crossbite side than on the non-crossbite side. conversely, anterior temporal muscle activity was higher on the crossbite side than on the non-crossbite side in the class iii malocclusion. the masticatory activity of class i malocclusion in the superficial masseter muscle average activity is displayed in figure 2. the mean anterior temporal muscle activity presented lower amplitude on the crossbite side than the non-crossbite side in class i and class ii malocclusions (figure 3). a. b. figure 1. (a) electrode and subject position during temporal muscle examination; (b) electrode and subject position during masseter muscle examination. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 class i class ii class iii am pl it u d o m ea n e m g (m v) crossbite non-crossbite figure 2. mean and standard deviation of superficial masseter muscle activity in angle’s class i, ii and iii with posterior unilateral crossbite. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 146 siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 the results of the two-way anova test (tables 1 and 2) indicated significant differences in the activity of anterior superficial and temporal masseter in the crossbite groups and between the malocclusions, and in the interaction of the crossbite and malocclusion groups (p<0.05). the post hoc least significant difference (lsd) test results between the groups (tables 3 and 4) revealed significant variations in the activity of the anterior superficial and temporal masseter muscles between class i, ii and iii malocclusions in the crossbite side and the non-crossbite side (p>0.05). in class i malocclusion groups on the crossbite side and class ii on the non-crossbite side, the activation of the superficial masseter muscle was not substantially different across the groups (p>0.05). the anterior temporalis muscle activity was not significantly different between class i malocclusion groups on the crossbite side and class ii groups on the non-crossbite side (p>0.05), nor between the groups in class ii malocclusions on the non-crossbite side and class iii malocclusions on the non-crossbite side (p>0.05). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 class i class ii class iii am pl it u d o m ea n e m g (m v) crossbite non-crossbite figure 3. mean and standard deviation of temporal muscle activity in angle’s class i, ii and iii malocclusions with posterior unilateral crossbite. table 1. two-way anova test of superficial masseter muscle activity in angle’s class i, ii and iii malocclusions with posterior unilateral crossbite variables p-value crossbite side (masseter) 0.003* malocclusion ≤0.001* crossbite side* malocclusion ≤0.001* notes: * significant at p<0.05 table 2. two-way anova test of temporal masseter muscle activity in angle’s class i, ii and iii malocclusions with posterior unilateral crossbite variables p-value crossbite side (masseter) 0.011* malocclusion ≤0.001* crossbite side* malocclusion ≤0.001* notes: * significant at p<0.05 table 3. post hoc lsd test of superficial masseter muscle activity in angle’s class i, ii and iii malocclusions with posterior unilateral crossbite group class1n class2n class3n class1x class2x class3x class1n class2n <0.001* class3n <0.001* <0.001* class1x <0.001* 0.797 <0.001* class2x <0.001* <0.001* <0.001* <0.001* class3x <0.001* <0.001* <0.001* <0.001* <0.001* notes: * significant differences p<0.05; class1x: class i malocclusions crossbite side; class2x: class ii malocclusions crossbite side; class3x: class iii malocclusions crossbite side; class1n: class i malocclusions non-crossbite side; class2n: class ii malocclusions non-crossbite side; class3n: class iii malocclusions non-crossbite side table 4. post hoc lsd test of temporal masseter muscle activity in angle’s class i, ii and iii malocclusions with posterior unilateral crossbite group class1n class2n class3n class1x class2x class3x class1n class2n <0.001* class3n <0.001* 0.406 class1x <0.001* 0.148 0.025* class2x <0.001* <0.001* <0.001* 0.002* class3x 0.018* <0.001* <0.001* <0.001* <0.001* notes: * significant differences p<0.05; class1x: class i malocclusions crossbite side; class2x: class ii malocclusions crossbite side; class3x: class iii malocclusions crossbite side; class1n: class i malocclusions non-crossbite side; class2n: class ii malocclusions non-crossbite side; class3n: class iii malocclusions non-crossbite side dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 147siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 discussion the results reported low activity of the superficial masseter muscle on the crossbite side during mastication in class i malocclusions throughout semg recordings. the masticatory activity of class i malocclusion in the superficial masseter muscle revealed that the mean crossbite side had less activity than the non-crossbite side. the low activity of the superficial masseter muscle on the side of the crossbite during mastication was influenced by the surface chewing area, the duration, and the strength of each individual.13 the emg results of class i malocclusion subjects measured on the crossbite side of the superficial masseter muscle during mastication reported a wide and short amplitude. the recorded amplitude value was lower than the maximum value limit (2mv calibration). the normal amplitude of the manual mup emg is 100 µv–2 mv. at the non-crossbite side, the surface emg results presented narrower peak amplitude. the shape and height of the different amplitudes between the crossbite and noncrossbite sides were influenced by muscle contraction due to masticatory movements.14 increasing amplitude on the non-crossbite side was caused by maximal muscle contractions that simultaneously occurred in the absence of occlusal disturbances. meanwhile, the minimal chewing area of the affected teeth, influenced by the decrease in crossbite muscle activity and the size and thickness of the masticatory muscles, were examined.2 occlusal disturbances decrease masticatory strength on the side of the crossbite and affect the activity of the superficial masseter muscle during a contraction. the presence of occlusal disturbances results in a smaller surface chewing area, thereby reducing the duration and strength of mastication.10 the action potential that occurs during mastication contractions is influenced by the surface chewing area, the duration of mastication and the texture of the food.15 the section of chewing area is represented by molars and premolars that have crossbite, and by the position of molars that do not occlude normally. the electromyogram on the monitor displayed frequencies with high and low amplitudes on the crossbite, while on the non-crossbite side there were several amplitude peaks that increased with an attenuated shape as they approached the calibration limit.14 the class ii malocclusion had a molar disocclusion relationship that slowed down muscle contraction while the acceleration of muscle contraction during mastication of hard food was influenced by linear works of duration and the surface chewing area.15 these results indicated that the non-crossbite side had greater occlusal stability, thereby causing pressure sustained by the muscles during mastication to be greater that the crossbite of the class ii malocclusion. the results of this research are supported by sandhu et al.13 who studied the class ii masseter and anterior temporal muscles of patients in a relaxed state with no movement to support the mandible. this was in contrast to the class iii malocclusion study. patients with class ii malocclusions associated with a high lip line and no stiffness factor in the perioral muscles resulted in decreased masticatory muscle activity.16 the study demonstrated an increasing superficial masseter muscle activity on the crossbite side during mastication in class iii malocclusions through semg recordings. the masticatory activity in class iii malocclusions of the superficial masseter muscle suggested that the average crossbite side was more active than the non-crossbite side. class iii malocclusion with unilateral posterior crossbite had an influence on the activity of the superficial masseter muscle during mastication, including greater activity on the crossbite side compared to the non-crossbite side. class iii malocclusions were determined by mandibular length, mesiocclusion molar relation and muscle hyperactivity to prevent forward mandibular movement.13 the study’s findings indicated a decrease in anterior temporal muscle activity on the crossbite side in class i and class ii malocclusions during mastication compared to the non-crossbite side. in class i and class ii malocclusions, the mean anterior temporal muscle activity had a smaller amplitude on the crossbite side compared to the noncrossbite side. activity in the anterior temporal muscle on the side of the crossbite during mastication differed significantly between class i and class ii malocclusions. this finding revealed that the effect of class i malocclusions with unilateral posterior crossbite on anterior temporal muscle activity during mastication caused decreased activity in the anterior temporal muscle on the crossbite side compared to the non-crossbite side. similarly, activity in the class ii malocclusion anterior temporal muscle on the crossbite side decreased compared to the non-crossbite side.14 there was a decrease in anterior temporal muscle activity caused by occlusal disturbances that occurred due to posterior crossbite with class i and class ii malocclusions. this was due to a decreased acceleration of the anterior temporal muscle contraction during jaw closure that affected the amplitude frequency and was in contrast with the non-crossbite side.10 a study on the type of class iii malocclusions indicated increased muscle activity during mastication in the anterior temporal part of the crossbite compared to the non-crossbite. the mean anterior temporal muscle activity in class iii malocclusions presented a higher amplitude on the crossbite side than the non-crossbite side. there was a significant difference between the activity of the anterior temporal muscle on the side of the crossbite during mastication in class iii. this finding suggested that class iii malocclusions with unilateral posterior crossbite affects the activity of the anterior temporalis muscle during mastication. this includes increased anterior temporal muscle activity during mastication in class iii with unilateral posterior crossbite, which has a higher activity on the crossbite compared to the non-crossbite side. an increase in anterior temporal muscle activity was due to the class iii malocclusions displaying higher activity compared to class i and class ii. in class iii cases, the superficial and anterior temporal masseter dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 148 siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 muscles are hyperactive to resist the forward movement of the mandible.13 mastication of food in the premolar area is less efficient compared to the molar area due to its narrow surface. the efficiency of mastication is not only determined by strength and pattern but also by the occlusal conditions that receive the chewing load. in addition, the size and morphology of teeth in each individual is different which can affect the chewing area during the mastication process.15 the number of unilateral posterior crossbite samples with unequal sex distribution may affect the study result. the imbalance of sex distribution can affect the masticatory strength during mastication on both the crossbite and noncrossbite sides.3 there were significant differences in superficial masseter muscle activity in all types of malocclusions on the crossbite and non-crossbite sides, meanwhile the activity of the superficial masseter muscle was not significantly different between groups in class i malocclusion on the crossbite side and class ii malocclusion on the non-crossbite side. these results indicated that the non-crossbite side class ii malocclusion had the same muscle activity as the crossbite side class i malocclusion but with lower than normal activity on the non-crossbite side of the class i malocclusion. the influencing factor of these results was the distocclusion of the molar relationship and the high lip line, which caused perioral muscle weakness in the class ii malocclusion on the non-crossbite side.16 the activity of the anterior temporal muscles was significantly different in all types of malocclusions on the crossbite and non-crossbite sides. exceptions included the activity of the anterior temporal muscle between the class i malocclusion on the crossbite side and class ii malocclusion on the non-crossbite side, and the class ii malocclusion on the non-crossbite side with the class iii malocclusion on the non-crossbite side. these results confirmed that both the cross-bite side in class i malocclusions and class ii malocclusions on the non-crossbite side had the same influencing factors as the masseter muscle. class ii malocclusions in the non-crossbite side along with class iii malocclusions at the non-crossbite side had the same muscle activity caused by occlusal disturbances that interfered with the outcome of muscle activity.2 in this study, significant differences between crossbite and non-crossbite sides during mastication in class i, ii and iii malocclusion types have highlighted how important utilising an semg is. to evaluate the asymmetry activity of the superficial masseter muscle and anterior temporal muscle during mastication with types of malocclusions among cases of unilateral posterior crossbite during mastication, an semg is necessary to assist the diagnosis process and to promote successful orthodontic treatment. evaluation of muscle activity disharmony since the beginning of orthodontic treatment using semg provides accurate data in the management of orthodontic treatment combined with effective masticatory muscle therapy.17 the limitation of this study used a cross-sectional approach with the aim of obtaining a relationship between exposure (type of malocclusion with unilateral posterior crossbite) and risk factors (activity of the superficial and anterior temporal masseter muscles during mastication). this type of research is analytical; therefore, confounding factors, such as skeletal factors, mastication, muscle shape and anatomy, morphology and teeth size, and bad habits during mastication, are not controlled. class i malocclusions with unilateral posterior crossbite displayed reduced activity on the side of the crossbite on the superficial masseter muscle and anterior temporal muscle during mastication. the superficial masseter muscle and the anterior temporal muscle on the side of the crossbite were less active in class ii malocclusions with unilateral posterior crossbite. class iii malocclusions with unilateral posterior crossbite had a higher activity of superficial masseter muscle and anterior temporal muscle on the crossbite side during mastication. furthermore, there was interaction between class i, ii and iii malocclusions with unilateral posterior crossbite toward the activity of the superficial masseter muscle and the anterior temporal muscle during mastication. further research on malocclusion with unilateral posterior crossbite by controlling masticatory factors, morphology and size of the teeth, area of mastication, sex distribution, bad habits and involvement of skeletal factors on muscle activity both at rest, biting food and during mastication, is needed. references 1. premkumar s. textbook of orthodontics. new delhi: elsevier; 2015. p. 89, 102–9. 2. zamanlu m, khamnei s, salarilak s, oskoee ss, shakouri sk, houshyar y, salekzamani y. chewing side preference in first and all mastication cycles for hard and soft morsels. int j clin exp med. 2012; 5(4): 326–31. 3. nishi se, basri r, alam mk, komatsu s, komori a, sugita y, maeda h. evaluation of masticatory muscles function in different malocclusion cases using surface electromyography. j hard tissue biol. 2017; 26(1): 23–8. 4. iodice g, danzi g, cimino r, paduano s, michelotti a. association between posterior crossbite, skeletal, and muscle asymmetry: a systematic review. eur j orthod. 2016; 38(6): 638–51. 5. sultana n, hassan gs, jha d, nashrin t, nahar l, naim ma. p revalence of cross bite among the or thodontic patients in bangabandhu sheikh mujib medical university. bangladesh j med. 2015; 26(1): 9–12. 6. de sousa rv, ribeiro gla, firmino rt, martins cc, granvillegarcia af, paiva sm. prevalence and associated factors for the development of anterior open bite and posterior crossbite in the primary dentition. braz dent j. 2014; 25(4): 336–42. 7. mitchell l. an introduction to orthodontics. 4th ed. oxford: oxford university press; 2013. p. 11, 34–5, 44. 8. alam mk. a to z orthodontics: retention and relapse. kota bharu: ppsp publication; 2012. p. 14. 9. nishi se, basri r, alam mk. uses of electromyography in dentistry: an overview with meta-analysis. eur j dent. 2016; 10(3): 419–25. 10. woźn i a k k , p iąt kowsk a d, l ip sk i m , me h r k . su r fa c e electromyography in orthodontics a literature review. med sci monit. 2013; 19: 416–23. 11. fox si. human physiology. 14th ed. new york: mcgraw-hill education; 2015. p. 360–76. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 149siregar et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 143–149 12. okeson jp. management of temporomandibular disorders and occlusion. 7th ed. st. louis: elsevier mosby; 2012. p. 234–7. 13. sandhu ss, utreja a, prabhakar s, sandhu n, kashyap r. a study of electromyographic activity of masseter and temporalis muscles and maximum bite force in patients with various malocclusions. singh g, editor. j indian orthod soc. 2013; 47(2): 53–61. 14. merletti r, farina d. surface electromyography: physiology, engineering, and applications. canada: wiley-ieee press; 2016. p. 54–8; 89, 100–23. 15. tomonari h, seong c, kwon s, miyawaki s. electromyographic activity of superficial masseter and anterior temporal muscles during unilateral mastication of artificial test foods with different textures in healthy subjects. clin oral investig. 2019; 23(9): 3445–55. 16. lapatki bg, mager as, schulte-moenting j, jonas ie. the importance of the level of the lip line and resting lip pressure in class ii, division 2 malocclusion. j dent res. 2002; 81(5): 323–8. 17. michelotti a, rongo r, valentino r, d’antò v, bucci r, danzi g, cioffi i. evaluation of masticatory muscle activity in patients with unilateral posterior crossbite before and after rapid maxillary expansion. eur j orthod. 2019; 41(1): 46–53. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p143–149 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p143-149 mkgs vol 44 no 2 april-juni 2011.indd dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 169169 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 169–173 case report management of pericoronitis for partial eruption of second permanent molar in a pediatric patient tengku natasha eleena binti tengku ahmad noor1, james lian yoon chen2, mohd safwani affan alli3 and mohd hosni bin mahmood4 1dental officer of 609 armed forces dental clinic, kuching, sarawak, malaysia 2dental officer of 624 armed forces dental clinic, sibu, sarawak, malaysia 3pediatric dental specialist of duchess of kent hospital, sandakan, sabah, malaysia 4restorative specialist of 609 armed forces dental clinic, kuching, sarawak, malaysia abstract background: this article discussed the management of pericoronitis for partial eruption of molar on pediatric patients. purpose: this case report was to discuss how to manage a pediatric patient with a partially erupted second molar by using an electrosurgery method as the last alternative; also, it assessed whether or not the treatment facilitates spontaneous tooth eruption in respect to incomplete treatments. case: a 9-years-old girl visited the kuching armed forces dental clinic with her parents and complained of recurrent swelling on her lower right jaw in the last six months and noticed a tooth-like white lump under it. after a deliberate examination, the dentists came up with a diagnosis of pericoronitis because of a partially erupted second molar. case management: in managing a pediatric patient, a systematic desensitization was performed whereby the first visit was more of non-invasive treatment such as oral health instructions, a proper tooth brushing technique, and local scaling and debridement. operculectomy using the electrosurgery had been carried out in the fourth visit and reviewed after one-week treatment that shows uneventful healing. conclusion: to prevent and treat oral problems, it is imperative to develop child’s interest and willingness in using dental services. hence, an early diagnosis is critical, especially as parents seek for the best treatment duration and treatment methods with the least number of consequences. the food impaction and the recurrent swelling because of pericoronitis have a major impact on the patient; therefore, treatment is provided regardless of patient’s age. keywords: electrosurgery; operculectomy; pericoronitis; pediatric; second molar correspondence: tengku natasha eleena binti tengku ahmad noor, malaysian armed forces dental officer, 609 armed forces dental clinic, kem semenggo, kuching, sarawak, malaysia. email: tengkunatashaeleena@gmail.com introduction pericoronitis is an inflammatory condition that affects the soft tissues surrounding a newly erupted tooth. the inflammation is quickly progressive and commonly acute in nature in addition to being subacute or chronic if it lasts for a long time or recurs.1 pericoronitis is diagnosed mostly through clinical examination with three distinct diagnostic categories: acute pericoronitis, subacute pericoronitis, and chronic pericoronitis. these classifications depend on the way individual cases fall under the three major clinical groups arbitrarily.2 pericoronitis is frequently associated with tooth impaction, and it is more common in impacted third molars because of tooth tissue discrepancy.3 there have been very few reports on the prevalence of pericoronitis in first and second permanent molars compared to third molars.2 pericoronitis may be linked to an unimpacted third molar. yamalik and bozkaya4 researched and verified non-third molar related pericoronitis among nigerian children, whereby none of the teeth involved in the study was impacted, but partially erupted with softtissue coverage.2,4 the failure of permanent molar eruption is a rather uncommon occurrence. interference with physiological tooth development could be the reason. according to baccetti5, about 0.03% to 0.58% of mandibular molars and 0.04% to 0.08% of maxillary molars have their second permanent molars retained. second permanent molar dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p169–173 mailto:tengkunatashaeleena@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p169-173 170 noor et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 169–173 impaction is a rare case. according to bondemark and tsiopa6 and fu et al.7, it generally occurs in the mandibular arch at 0.06% to 0.3% of occurrence. according to abate et al.8, the failure of eruption is classified into three clinical conditions: impaction, the halting of the eruption process because of a physical obstruction that can be detected radiographically or clinically; primary retention, a disturbance in the eruption process before the tooth has surfaced in the oral cavity; and secondary retention when the eruption process has already begun, and the tooth has already breached the gingiva without any physical obstruction.8,9 the aetiologies of maxillary and mandibular molar eruption problems are considered to be associated with the several conditions such as mechanical obstructions (tumors, hyperdontia, mesial eruption, cysts, and subsequent impaction into the distal part of a neighboring tooth); dentoalveolar disparity; increased space between the first and second molars10 aberrant from the eruption path9; location of third molar germ that limits the second permanent molar eruption pathway11; a viral infection that affects the physiological eruption process by altering local innervation6,7; and genetic predisposition.9,12 the risk of resorption of adjoining teeth, periodontal problems, and caries, malocclusion, follicular cysts, issues addressing deep bite, peri-coronal infections, and pain are all indications for treating impacted and retained second permanent molars.13 according to proffit14, second permanent molars commonly erupt around twelve years of age. hence, the best age to treat disturbances in second permanent molar eruption is between 11 and 14 years of age because the roots are still under development, and the third molars are usually germs.15 an unerupted second permanent molar often needs a multidisciplinary treatment approach. surgical approaches, orthodontic solutions, and surgical and orthodontic treatment altogether are some other choices apart from the multidisciplinary treatments.16 surgical exposure of the second permanent molar and extraction of the third molar are two of the most typical treatment options mentioned.16 taking into consideration, the diagnosis and position of the second permanent molars are some recommendations for the treatment.17 an operculectomy is a minimally invasive surgery that exposes the second permanent molar by removing the operculum or tissue flap over a partially erupted tooth.18 this surgery leaves an easy-to-clean region, which prevents plaque formation and inflammation.18 operculectomy can be performed with a surgical scalpel, electrosurgery, laser, or traditional caustic substances, e.g., trichloroacetic acid.18 an electrosurgical equipment can be used to perform an operculectomy as it can ensure tissue hemostasis and appropriate contouring.19 the use of electrosurgery also allows for a simple tissue incision with a significant hemostatic result.20 it is, however, not recommended for patients who have a cardiac pacemaker.20 any contact with cementum or bone must be avoided at all costs as irreversible damage will follow.20 for enlarged tissue removal, a needle electrode is used, while diamond or ovoid-shaped electrodes are used for shaping and festooning.19,20 during the cutting phase, the electrode is energized in a brief shaving motion, making short contact with the tissues.20 the controlled passage of high-frequency waveforms or currents into the body tissues to generate a controllable surgical impact is known as electrosurgery.20 for children, a dental visit is a unique behavioral and psychological experience, which may scare them and pose a difficulty for the dentist to provide treatment.21 besides, from a professional standpoint, the primary goal of dental therapy is to promote community oral and dental health by encouraging the development of healthy dental attitudes.22 with these goals, dental practitioners must achieve specific standards to deliver successful treatment. therefore, knowledge of childhood’s behavioral, physical, and psychological demands is essential; nevertheless, the existence of contemporary and emerging technology, particularly electrosurgery, cannot be overseen. hence, the purpose of this case study was to show how pericoronitis was managed when it came to a pediatric patient treatment through electrosurgery after successful systemic desensitization. figure 1. orthopantomograph on the first visit showing an vertically angulated lower right second permanent molar covered by thick-soft tissue. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p169–173 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p169-173 171noor et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 169–173 case a 9-years-old female child visited the kuching armed forces dental clinic and complained of recurrent gum swelling on her lower right posterior region for the past week. the patient claimed that she could not eat or chew on the right side. to relieve the pain, she had to take an analgesic. although she had cleaned the side with a toothbrush, her tooth constantly was painful. in this site, she often had food stuck, and the tooth was sometimes bleeding when she accidentally bit the area. after a clinical examination was performed, an edematous operculum was found covering the partially erupted lower right second permanent molar. the operculum was hyperemic, swollen, and bleeding spontaneously upon probing. its antagonist, the upper right second permanent molar was clinically unerupted. however, it was in contact with the right upper first permanent molar and the operculum when the patient occluded. the patient had good oral hygiene, and she was cooperative towards dental treatment. the patient was diagnosed with pericoronitis on the partially erupted lower right second permanent molar exacerbated by secondary trauma during mastication as well as food impaction. orthopantomograph (opg) (figure 1) taken showed the partially erupted vertically angulated lower right second permanent molar covered by thick, soft tissue on the distal part of its crown. case management systemic desensitization has been applied, while oral hygiene instruction and treatment options were given to the patient and parents on the first visit. proper method, time, frequency, and duration of tooth brushing were also explained. brushing the area thoroughly and using chlorhexidine mouthwash as an anti-bacterial agent were recommended for the patient to accomplish. full mouth scaling and polishing were performed to eliminate food impaction in the area. no medication was given as the physician prescribed antibiotics and analgesics before she visited the clinic. a review was done after one-week treatment showed the absence of inflammation and swelling. thereafter, the patient felt better and maintained her oral hygiene. however, a year later, she came back with the same complaint. the same treatment was performed with full mouth scaling and debridement, oral hygiene enforcement, and analgesic. subsequently, the patient was given an appointment for the removal of the operculum during the next visit if signs and symptoms were still present. operculectomy was carried out using the electrosurgery on the fourth visit or two months after the third visit since the initial non-invasive treatments were not successful. clinically, the swelling had subsided, and lesser inflammation was noted (figure 2). the surgical area was cleaned using povidone-iodine at concentration of 10%. figure 2. edematous operculum partially erupted lower right second permanent molar during the second visit. figure 3. haemostasis achieved post operculectomy using electrosurgery. figure 4. operculum of lower right second permanent molar one week after operculectomy. figure 5. second review post-operculectomy of lower right second permanent molar. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p169–173 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p169-173 172 noor et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 169–173 topical anesthesia was applied to the gingiva, followed by infiltration and intraligamentary injection using lidocaine hcl 2% with epinephrine 1:100,000. scaling was done after anesthesia, and excision was performed using a loop electrode. subsequently, with a straight electrode, gingivoplasty was carried out to contour the soft tissue, while a ball electrode was utilized to achieve hemostasis (figure 3). these electrodes were inserted carefully to avoid contact with the bone as this would result in irreversible damage. saline and povidone-iodine at 10% of concentrations were used to irrigate the surgical area. a post-operative instruction was then given to the patient that had to go on a soft diet for three days and vigorous toothbrushing on the surgical site. ibuprofen 250 mg thrice a day was prescribed together with a hyaluronic acid gel that was applied to the surgical site to aid the healing process. after a week of treatment, the review was done again. it turned out that the patient did not feel any pain. as figure 4 shows, the operculum on the lower right second permanent molar was no longer swollen and inflamed. only distal cusps were partially covered. localized scaling was then performed to improve the hygiene of the site. the patient was instructed to chew solid food on the right side to give more pressure which then hastened the eruption of 47. five months later, the second review was completed to monitor the eruption of the lower right second permanent molar. the patient had no complaint of her tooth as the lower right second permanent molar had fully erupted (figure 5). discussion operculectomy performed with an electrosurgery is one of the pericoronitis treatment of partially erupted second permanent molar in a pediatric patient. to respond to such common cases, in 2005 the council on clinical affairs developed some treatment alternatives for pediatric oral surgery and oral pathology.23 supervising children’ hygiene behavior during the surgical and perioperative periods is difficult. to reduce anxiety, methods other than nitrous oxide/oxygen inhalation and local anesthetic benefit many children.24,25 before surgery, the social, emotional, and psychological condition, as well as the cognitive level of the juvenile patient should be carefully assessed.25 in this report, systemic desensitization was used to shape positive attitudes, and the patient were only treated with the electrosurgery on the fourth visit after having some unsuccessful conservative treatments. untreated odontogenic infections may result in pain, difficulty in eating or drinking, cellulitis, airway compromise, abscess, septicemia, and life-threatening infections.26 another review was conducted when the lower right second permanent molar was not well-aligned due to the distal half being tilted buccally. according to proffit14, on average the second permanent molar will begin erupting in 11-13 years of age. this current case is exceptional. although the patient was 9 years old, she had already had her second permanent molar erupting. however, a thick operculum covered the tooth and thus hindered its eruption by mechanical obstruction. according to the american association of paediatric dentistry (aapd), the early orthodontic intervention of the affected teeth should be avoided.27,28 additional considerations could be either maintaining space, repositioning adjacent teeth that have tipped into the sites, preventing overeruption in the opposite arch, or changing lateral tongue push habits.27,28 to provide functional occlusion, multidisciplinary treatment options such as selective extractions followed by implants, single tooth, or segmental osteotomies with rapid traction can be considered once the molar growth is complete.29 pericoronitis treatment mainly focuses on the elimination of the acute phase, followed by the chronic phase.30 full mouth scaling and polishing were done to improve the oral hygiene at the site and act as part of the systemic desensitization. systematic desensitization is a psychological approach that is possibly utilized in the dentistry environment to change the behaviors of nervous patients.31 anxiety over several appointments usually occurs when patients are gradually exposed to aspects of the dentist appointment .32 operculectomy with the electrosurgery was planned on the subsequent visits after the patient was more comfortable. electrosurgery was chosen for several advantages. coagulation and plugging of micro diameter blood vessels are advantages of electrocautery, creating a surgical field that is free of blood and minimal of postoperative oedema.33 the obliteration of the sealing of free nerve endings and dendrites also results in less postoperative discomfort.33 collectively, the electrosurgery can also push rapid and stable hemostasis, self-disinfecting tip, lesser postoperative pain, reduced oedema, and lesser scarring.33 in addition to this finding, a study carried out by bhatsange et al.33 found that electrosurgery has the lowest post-operative pain intensity compared to mucosal incision using a scalpel and laser. such electrosurgery is ideal in pediatric patients as it may be less painful. however, electrocautery has several drawbacks, including heat dissipation that causes lateral thermal tissue injury, delayed healing, an inescapable burning-flesh odor, a lack of tactile sensitivity, and incompatibility for insertion near bones which may lead to bone necrosis.34 hence, laser treatment is recommended to avoid such a side effect, considering its function for better wound healing.33 the patient was advised to practice good oral hygiene, and thus she could receive full restoration of periodontal support. both postoperative complications and periodontal problems were not detected on the treated tooth or the adjacent first permanent molar. at the time of writing, rarely treatment guidelines were found for eruptive disturbances of partially erupted or retained second permanent molar. the only possible treatment is through surgical exposure which is a minimal invasive, reliable procedure for soft tissue impaction dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p169–173 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p169-173 173noor et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 169–173 treatment of permanent molars. electrosurgery is more commonly utilized compared to other surgical techniques since it can achieve good homeostasis and less painful. even though new electrosurgery applications, equipment features, difficulties, and solutions come into practice, the use of electrosurgery in dentistry has relatively remained constant. regular professional oral prophylaxis and patient compliance are required for a good outcomes. to get a predictable result, oral hygiene instructions along with positive motivation should be early treatment in pediatric patients. acknowledgements the authors would like to extend their gratitude to dental services of malaysian armed forces and chief of 3rd brigade of armed forces malaysia, brigadier general dato’ azhar bin hj ahmad. references 1. hupp jr, tucker mr, ellis e. contemporary oral and maxillofacial surgery. 6th ed. st louis missouri: mosby elsevier; 2013. p. 718. 2. folayan mo, ozeigbe eo, onyejaeka n, chukwumah nm, oyedele t. non-third molar related pericoronitis in a sub-urban nigeria population of children. niger j clin pract. 2014; 17(1): 18–22. 3. galvão el, da silveira em, de oliveira es, da cruz tmm, flecha od, falci sgm, gonçalves pf. association between mandibular third molar position and the occurrence of pericoronitis: a systematic review and meta-analysis. arch oral biol. 2019; 107: 104486. 4. yamalik k, bozkaya s. the predictivity of mandibular third molar position as a risk indicator for pericoronitis. clin oral investig. 2008; 12(1): 9–14. 5. baccetti t. tooth anomalies associated with failure of eruption of first and second permanent molars. am j orthod dentofacial orthop. 2000; 118(6): 608–10. 6. bondemark l, tsiopa j. prevalence of ectopic eruption, impaction, retention and agenesis of the permanent second molar. angle orthod. 2007; 77(5): 773–8. 7. fu p-s, wang j-c, wu y-m, huang t-k, chen w-c, tseng y-c, tseng c-h, hung c-c. impacted mandibular second molars. angle orthod. 2012; 82(4): 670–5. 8. becktor kb, bangstrup mi, rølling s, kjaer i. unilateral primary or secondary retention of permanent teeth, and dental malformations. eur j orthod. 2002; 24(2): 205–14. 9. abate a, cavagnetto d, fama a, matarese m, bellincioni f, assandri f. efficacy of operculectomy in the treatment of 145 cases with unerupted second molars: a retrospective case-control study. dent j. 2020; 8(3): 65. 10. sawicka m, racka-pilszak b, rosnowska-mazurk iewicz a. uprighting partially impacted permanent second molars. angle orthod. 2007; 77(1): 148–54. 11. shapira y, finkelstein t, shpack n, lai yh, kuftinec mm, vardimon a. mandibular second molar impaction. part i: genetic traits and characteristics. am j orthod dentofacial orthop. 2011; 140(1): 32–7. 12. maspero c, fama a, cavagnetto d, abate a, farronato m. treatment of dental dilacerations. j biol regul homeost agents. 2019; 33(5): 1623–7. 13. alligri a, putrino a, cassetta m, silvestri a, barbato e, galluccio g. the mandibular permanent second molars and their risk of impaction: a retrospective study. eur j paediatr dent. 2015; 16(3): 246–50. 14. proffit wr, fields hw, larson b, sarver dm. contemporary orthodontics. 6th ed. st. louis: mosby; 2018. p. 744. 15. tamer i̇, öztaş e, marşan g. up-to-date approach in the treatment of impacted mandibular molars: a literature review. turkish j orthod. 2020; 33(3): 183–91. 16. lin s-y, chang c, roberts we. simple mechanics to upright horizontally impacted molars with ramus screws. orthod j nepal. 2015; 5(2): 42–7. 17. lau ck, whang czy, bister d. orthodontic uprighting of severely impacted mandibular second molars. am j orthod dentofacial orthop. 2013; 143(1): 116–24. 18. indrasari sd. management of pericoronitis of newly-erupted permanent tooth using electrosurgery – a case report. cermin dunia kedokt. 2016; 43(8): 597–600. 19. gomathi p, kavitha j, rajasekar s, srinivasan s, lakshmisree s. treatment of chronic inflammatory gingival enlargement using electrosurgery: a case report. iosr j dent med sci. 2020; 19(3): 17–20. 20. ravishankar pl, mannem s. electro surgery: a review on its application and biocompatibility on periodontium. indian joural dent adv. 2011; 3(2): 492–8. 21. ramazani n, ahmadi r, daryaeian m. oral and dental laser treatments for children: applications, advantages and considerations. j lasers med sci. 2012; 3(1): 44–9. 22. dean j, avery d, mcdonald r. mcdonald and avery dentistry for the child and adolescent. 9th ed. st. louis: mosby; 2011. p. 720. 23. a m e r ica n aca d emy of pe d ia t r ic d ent ist r y. m a n agem ent considerations for pediatric oral surgery and oral pathology. ref man pediatr dent chicago, iii am acad pediatr dent. 2020; : 433–42. 24. american academy of pediatric dentistry. informed consent. ref man pediatr dent chicago, iii am acad pediatr dent. 2020; : 470–3. 25. ferneini em, bennett j. oral surgery for the pediatric patient. in: dean j, editor. mcdonald and avery’s dentistry for the child and adolescent. 10th ed. st. louis: mosby; 2015. p. 627–44. 26. baker sr, mat a, robinson pg. what psychosocial factors influence adolescents’ oral health? j dent res. 2010; 89(11): 1230–5. 27. frazier-bowers sa, long s, tucker m. primary failure of eruption and other eruption disorders—considerations for management by the orthodontist and oral surgeon. semin orthod. 2016; 22(1): 34–44. 28. grippaudo c, cafiero c, d’apolito i, ricci b, frazier-bowers sa. primary failure of eruption: clinical and genetic findings in the mixed dentition. angle orthod. 2018; 88(3): 275–82. 29. rhoads sg, hendricks hm, frazier-bowers sa. establishing the diagnostic criteria for eruption disorders based on genetic and clinical data. am j orthod dentofacial orthop. 2013; 144(2): 194–202. 30. melnick pr, takei hh. treatment of periodontal abscess. in: newman mg, takei h, klokkevold pr, carranza fa, editors. carranza’s clinical periodontology. 11th ed. st. louis: elsevier; 2012. p. 443–7. 31. nelson tm, sheller b, friedman cs, bernier r. educational and therapeutic behavioral approaches to providing dental care for patients with autism spectrum disorder. spec care dentist. 2015; 35(3): 105–13. 32. american academy of pediatric dentistry. behavior guidance for the pediatric dental patient. ref man pediatr dent chicago, iii am acad pediatr dent. 2020; : 292–310. 33. bhatsange a, meshram ep, waghamare a, shiggaon l, mehetre v, shende a. a clinical and histological comparison of mucosal incisions produced by scalpel, electrocautery, and diode laser: a pilot study. j dent lasers. 2016; 10(2): 37–42. 34. funde s, baburaj md, pimpale sk. comparison between laser, electrocautery and scalpel in the treatment of drug-induced gingival overgrowth: a case report. ijss case reports rev. 2015; 1(10): 27–30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p169–173 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p169-173 vol 38 no 2-2005 88 hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran kotamadya samarinda provinsi kalimantan timur (the relation of frequency of teeth brush with oral hygiene of state elementary school children in palaran area district of samarinda province of east kalimantan) silvia anitasari* dan nina endang rahayu** * bagian mikrobiologi fakultas kedokteran universitas mulawarman ** dinas kesehatan kota samarinda provinsi kalimantan timur abstract the aim of this study was to get information about the relation of frequency of teeth brush to oral hygiene of elementary school children in palaran samarinda. the samples were 1650 first to sixth grade elementary school children taken from 10 state elementary schools at palaran samarinda. oral hygiene was measured by ohi-s index, and frequency of teeth brush was measured by questionnaire. statistical analysis was performed with chi-square test. the results showed that 6.73% have good oral hygiene, 59.03 % moderate and 34.24% poor. the frequency of teeth brush showed that 18% at 1x, 34.24 % at 2x, 61.88 % at 3x; 1.70% at 4x. with statistical analysis showed that there were relation of frequency of teeth brush to oral hygiene school children (x2 = 98.5, df = 6 and p = 12.59. this study concluded that oral health knowledge and exercise become successful in children from state elementary school at palaran. key words: teeth brush, elementary school, oral hygiene korespondensi (correspondence): silvia anitasari, bagian mikrobiologi, fakultas kedokteran universitas mulawarman. jln. kerayan kampus gn. kelua samarinda kalimantan timur indonesia. pendahuluan kesehatan gigi dan mulut masyarakat indonesia masih merupakan hal yang perlu mendapat perhatian serius dari tenaga kesehatan, baik dokter maupun perawat gigi, hal ini terlihat bahwa penyakit gigi dan mulut masih diderita oleh 90% penduduk indonesia.1 penyakit gigi dan mulut yang banyak diderita masyarakat di indonesia adalah penyakit jaringan penyangga gigi dan karies gigi, sumber dari kedua penyakit tersebut akibat terabaikannya kebersihan gigi dan mulut, sehingga terjadilah akumulasi plak. plak adalah lapisan tipis yang melekat erat di permukaan gigi serta mengandung kumpulan bakteri.1–4 berdasarkan teori blum, status kesehatan gigi dan mulut seseorang atau masyarakat dipengaruhi oleh empat faktor penting yaitu keturunan, lingkungan (fisik maupun sosial budaya), perilaku, dan pelayanan kesehatan. dari keempat faktor tersebut, perilaku memegang peranan yang penting dalam mempengaruhi status kesehatan gigi dan mulut. di samping mempengaruhi status kesehatan gigi dan mulut secara langsung, perilaku dapat juga mempengaruhi faktor lingkungan dan pelayanan kesehatan.2,3,5 sehubungan dengan pendapat di atas, maka frekuensi membersihkan gigi dan mulut sebagai bentuk perilaku akan mempengaruhi baik atau buruknya kebersihan gigi dan mulut, di mana akan mempengaruhi juga angka karies dan penyakit penyangga gigi. namun jarang sekali dilakukan penelitian mengenai hubungan perilaku dengan tingkat kebersihan gigi dan mulut. di samarinda penyakit gigi dan mulut termasuk dalam 10 besar penyakit yang banyak diderita penduduk samarinda dan sampai sekarang belum adanya penelitian yang dilakukan untuk mengetahui faktor yang mempengaruhi terhadap kesehatan gigi dan mulut.6 kecamatan palaran terletak di daerah pinggiran samarinda dengan jarak ± 45 km dari kota samarinda atau 1 jam menyeberangi sungai mahakam. penduduk daerah palaran sebagian besar adalah petani dan buruh pabrik kayu dengan penghasilan cukup untuk memenuhi kebutuhan sehari-hari.6 di kecamatan palaran terdapat 1 puskesmas induk dan 5 puskesmas pembantu. salah satu program puskesmas yaitu penyuluhan dan program sikat gigi masal yang selalu diadakan tiap tahun, tetapi tidak mengetahui sejauh mana keberhasilan dari program yang dilaksanakan tersebut. untuk itu dilakukan suatu penelitian untuk mengetahui ada atau tidaknya hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran kotamadya samarinda kalimantan timur. 89anitasari: hubungan frekuensi menyikat gigi adapun tujuan penelitian ini adalah untuk mengetahui ada tidaknya hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut pada siswa sekolah dasar negeri di kecamatan palaran kotamadya samarinda kalimantan timur.6 diharapkan hasil penelitian ini dapat dijadikan sumber informasi ilmiah bagi dinas kesehatan kota setempat dalam menyusun program kesehatan gigi dan dunia ilmu pengetahuan kedokteran gigi pada umumnya serta menjadi salah satu aspek bagi pengembangan penelitian lebih lanjut. bahan dan metode jenis penelitian ini adalah observasional yang dilakukan di sekolah dasar negeri di kecamatan palaran kotamadya samarinda.7 populasi penelitian adalah siswa kelas 1–6 sdn di kecamatan palaran kotamadya samarinda. sdn yang diambil adalah sdn yang berada di kecamatan palaran dan mewakili tiap gugus yang ada, berdasarkan kriteria tersebut terpilih 10 sdn dan dari tiap sdn tersebut semua populasinya dipilih sebagai sampel penelitian.8 variabel frekuensi menyikat gigi diukur dengan menggunakan kuesioner sedangkan variabel tingkat kebersihan mulut diukur dengan menggunakan indeks oral hygiene index simplified (ohi-s).1 kriteria indeks ohi-s indeks angka kriteria ohi-s 0–1,2 1,3–3 3,1– 6 baik sedang buruk hasil penelitian yang diperoleh dianalisis secara statistik dengan analisis chi-square untuk mengetahui ada atau tidaknya hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran. hipotesa uji yang dipakai adalah: ho = tidak ada hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran, h1 = ada hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sekolah dasar negeri di kecamatan palaran. hasil hasil penilaian tingkat kebersihan mulut dengan menggunakan indeks ohi-s pada 1650 siswa sekolah dasar negeri kelas 1–6 kecamatan palaran samarinda di dapatkan 6,73% siswa keadaan kebersihan gigi dan mulut baik; 59,03% sedang; 34,24% buruk (tabel 1). oral hygiene index simplified (ohi-s) rata-rata adalah 3 termasuk kebersihan gigi dan mulut sedang. tabel 1. tingkat kebersihan gigi dan mulut siswa kelas 1–6 sdn kecamatan palaran samarinda tahun 2004 n persentase tingkat kebersihan gigi dan mulut baik 111 6,73% sedang 974 59,03% buruk 565 34,24% hasil pengukuran frekuensi menyikat gigi siswa sekolah dasar negeri kecamatan palaran, frekuensi menyikat gigi 1 kali sebanyak 2,18%; 2 kali sebanyak 34,24%; 3 kali sebanyak 61,88%; sedangkan 4 kali sebanyak 1,70% (tabel 2). tabel 2. frekuensi menyikat gigi siswa kelas 1–6 sdn kecamatan palaran samarinda tahun 2004 1 kali 2 kali 3 kali 4 kali persentase frekuensi menyikat gigi n = 36 2,18% n = 565 34,24% n = 1021 61,88% n = 28 1,70% bila frekuensi menyikat gigi dihubungan dengan tingkat kebersihan mulut terlihat pada frekuensi menyikat gigi 1 kali, persentase kebersihan gigi dan mulutnya baik 8,33%, sedang 36,11%, buruk 6,37%. pada frekuensi menyikat gigi 2 kali, persentase kebersihan gigi dan mulutnya baik 6,37%, sedang 46,73%, buruk 46,90%. pada frekuensi menyikat gigi 3 kali, persentase kebersihan gigi dan mulut baik 1,57%, sedang 67,38%, buruk 26,24%. pada frekuensi menyikat gigi 4 kali, persentase kebersihan gigi dan mulutnya baik 25%, sedang 32,14%, buruk 42,86% (tabel 3). tabel 3. hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa kelas 1–6 sdn kecamatan palaran samarinda tahun 2004 tingkat kebersihan mulut baik sedang buruk total 1 kali 3 (8,33%) 13 (36,11%) 20 (6,37%) 36 2 kali 36 (6,37%) 264 (46,73%) 265 (46,90%) 565 3 kali 65 (1,57%) 688 (67,38%) 268 (26,24%) 1021 4 kali 7 (25%) 9 (32,14%) 12 (42,86%) 28 frekuensi menyikat gigi total 111 (6,73%) 974 (59,03%) 565 (34,24%) 1650 berdasarkan perhitungan statistik diperoleh x2hitung = 98,42 sedangkan dari tabel chi-square dengan db = (3-1) (4-1) = 6 dan taraf kepercayaan 95%, nilai x2 tabel = 12,59, sehingga x2 hitung = x2 tabel maka ho ditolak, artinya ada 90 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 88–90 hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut. pembahasan usaha pemerintah untuk meningkatkan kesehatan gigi dan mulut masyarakat indonesia sangat membutuhkan peranserta masyarakat sendiri terutama perubahan perilaku, melalui program penyuluhan dan pelatihan. program penyuluhan kesehatan gigi dan mulut dan pelatihan sikat gigi masal merupakan suatu program yang dilakukan oleh pemerintah melalui puskesmas setiap tahun.6 dari hasil penelitian kesehatan gigi dan mulut siswa kelas 1–6 sdn kecamatan palaran yang sudah pernah mendapatkan penyuluhan dan pelatihan cara menyikat gigi yang baik dan benar, didapatkan tingkat kebersihan mulut (ohi-s) mereka rata-rata 3 dengan kriteria sedang (tabel 1), sedangkan dalam hal menyikat gigi sebagian besar siswa ini mengerti bahwa frekuensi menyikat gigi yang baik adalah 2–3 kali sehari (tabel 2). hal ini menunjukkan program penyuluhan kesehatan gigi dan mulut serta sikat gigi masal mengenai target tujuan. jika kita hubungkan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sdn palaran, berdasarkan hasil statistik ada pengaruh frekuensi menyikat gigi dengan tingkat kebersihan mulut siswa tersebut. hal ini terlihat secara jelas pada (tabel 3), di mana siswa yang menyikat gigi dengan frekuensi 4 kali dengan tingkat kebersihan gigi dan mulut baik persentasenya lebih tinggi dibandingkan dengan frekuensi menyikat gigi 1 kali, 2 kali dan 3 kali. berdasarkan penelitian hawkins, 2 pendidikan kesehatan yang diberikan beserta dengan pelatihan akan memberikan hasil yang optimal. hal ini terbukti pada penelitian terhadap siswa sdn di kecamatan palaran, di mana penyuluhan dan sikat gigi masal yang dilaksanakan setiap tahun, mempengaruhi perilaku mereka dalam menyikat gigi (tabel 3). walaupun terdapat hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sdn di kecamatan palaran tetapi tingkat kebersihan gigi dan mulutnya rata-rata sedang bukan baik (tabel 1). dari hasil penelitian ini, dapat disimpulkan terdapat hubungan frekuensi menyikat gigi dengan tingkat kebersihan gigi dan mulut siswa sdn palaran samarinda provinsi kalimantan timur. ucapan terima kasih penelitian ini dapat terlaksana karena dukungan berbagai pihak. untuk itu, pada kesempatan ini peneliti mengucapkan terima kasih kepada dr. h. hatmoko, kepala puskesmas dan drg. liliwati, penanggung jawab poli gigi puskesmas palaran beserta seluruh staf atas kerja sama yang telah diberikan. daftar pustaka 1. departemen kesehatan ri. profil kesehatan gigi dan mulut di indonesia pada pelita v. jakarta: departemen kesehatan ri; 1994. 2. hawkins rj, et al. oral hygiene knowledge of high-risk grade one children: an evaluation of two methods of dental health education. j community dentistry and epidemiology 2000; 28: 336–43. 3. spolsky vw. epidemiology of gingival and periodontal disease in: carranza fa, newman mg, editors. clinical periodontology. 9th ed. philadelphia: wb saunders co; 2000. p. 84. 4. veld hi. ilmu kedokteran gigi pencegahan. sutatmi suryo. yogyakarta: universitas gadjah mada; 1993. h. 59. 5. notoatmodjo s. pendidikan dan perilaku kesehatan. edisi 1. jakarta: rineka cipta; 2003. h. 120–34. 6. dinas kesehatan kota samarinda. profil kesehatan kotamadya samarinda. samarinda: dinas kesehatan kota; 2004. 7. bhisma m. prinsip dan metode riset epidemiologi. edisi 1. yogyakarta: universitas gadjah mada; 1967. 8. lameshow s. adequacy of sample size in health studies. chichester: who-john wiley & sons; 1990. p. 42. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 122 the differences in root canal smear layer removal between 6,25% pineapple (ananas comocus l. merr.) peel extract and 17% ethylene diamine tetra-acetic acid nirawati pribadi, karlina samadi, meliavita n. k. astuti, hendy j. kurniawan, adelina k. tandadjaja and ratna puspita hadi department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: the smear layer is attached to dentine and occludes the orifice and, consequently, must be removed in order to improve the success of inroot canal treatment. the ideal irrigation material removes both the organic and inorganic smear layers. ethylene diamine tetra-acetic acid (edta) is one of the most commonly used root canal irrigation materials, but removes only inorganic smear layer. to overcome this problem, pineapple (ananas comosus l. merr.) peel extract, which contains saponins, bromelain, polyphenol and flavonoid, is used during root canal irrigation. purpose: the study aimed to analyze the difference in smear layer removal between the use of 6.25% pineapple peel extract and 17% edta. methods: 27 samples of mandibular premolar teeth with straight root canals were divided randomly into three groups (n = 9) and subsequently prepared using protaper. irrigation was performed on the control group (aquadest), group i (17% edta) and group ii (6.25% pineapple peel extract). the samples were dried, temporarily compressed and cut horizontally from the apical to the coronal. samples were fixed with holder before the smear layer was observed through a scanning electron microscope (sem). the resulting data was analyzed by means of an anova test. results: the highest score of root canal hygiene was recorded by group ii, followed by group i and, finally, the control group. there were significant differences between the groups (p< 0.000). conclusion: 6.25% pineapple peel extract produces a higher smear layer removal effect than 17% edta on the apical 1/3 of the root canal. keywords: edta; pineapple peel extract; root canal irrigation; smear layer correspondence: nirawati pribadi, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia, e-mail: nirawati-p@fkg.unair.ac.id introduction root canal preparation can be performed using mechanical and irrigation instrumentation. mechanical instrumentation is used to shape and enlarge the root canal in order to facilitate its irrigation and obturation.1 this form of root canal instrumentation creates an organic and inorganic layer refered to as the “smear layer”.2 this is an amorphous and irregular thin layer composed of both organic materials (bacteria and bacterial products) and inorganic materials (calcium hydroxyapatite and tricalium phosphate). it can cover the prepared root canal walls and occludes the orifices of the dentinal tubules.3 this layer will be firmly attached to the dentin and can potentially compromise the success of root canal treatment. the thickness, composition, and morphology of the smear layer depends on the instrumentation process and the location of the dentin from which it was formed.4 the layer can act as a substrate for bacteria, thereby enabling them to survive and proliferate into the dentinal tubules. it may also interfere with the adaptation to and penetration of the filler into the dentinal tubules which will cause microleakage of 1/3 of the apical section of the root canal.1,5 smear layer removal requires what is termed irrigation material. the ideal irrigation material should be able to remove the organic and inorganic smear layers without causing erosive effects on dentine, while also producing an antibacterial effect.1,4 ethylene diamine tetra-acetic acid dental journal (majalah kedokteran gigi) 2019 september; 52(3): 122–125 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p122-125 123pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3):122–125 (edta) is one of the most commonly used root canal irrigation materials. at a concentration of 17%, edta constitutes a chelating agent effective at removing the inorganic smear layer.3,6 this material is non-toxic and, consequently, demonstrates an inability to remove organic smear layer, has a low antibacterial effect, and causes erosion of dentin.1 edta proves effective in removing the smear layer in the coronal area and the 1/2 part of the root canal, but is ineffective in the 1/3 apical part of the root canal. application of edta as the main irrigation material can lead to a significant reduction in dentin microhardness.7 in order to overcome these disadvantages, pineapple (ananas comosus l. merr) peel extract is employed as a root canal irrigation material. pineapple peel extract contains active substances such as saponins, bromelain, polyphenol, and flavonoids. these active substances can lower the surface tension to remove organic and inorganic smear layer resulting in enhanced root canal hygiene. the minimum concentration capable of destroying enterococcus faecalis (e. faecalis), thereby creating sterile conditions in the root canal is one of 6.25%.1 therefore, this study sought to analyze the difference in smear layer removal between the application of 6.25% pineapple peel extract and 17% edta. materials and methods the material used in this research consisted of yellow-green pineapple peel extract from 1 kilogram of honey pineappleskin the 60-day old pineapples whose peel had been dried for 24 hours had been derived from a plantation in blitar, east java. the pineapple peel was immersed in ethanol 96% in the agitator (the original resinator og®, usa), agitated, covered with aluminum foil, allowed to stand for 24 hours and, finally, filtered to produce filtrate 1 and residue. the latter was subsequently added to ethanol solvent 96% and the resulting material was submerged in the agitator for two hours before being filtered to produce filtrate 2. at this point, filtrate 1 and 2 were combined, covered with aluminum foil and allowed to stand for 24 hours before being filtered to produce 1000ml of filtrate. the filtrate was collected a second time and then concentrated through the use of an evaporator (rufouz hitek engineers pvt, india) at a temperature of 50-60oc. employing this process extracted, 500 ml of liquid-free ethanol. a total of 27 permanent mandibular premolars satisfying the criteria of being caries-free and possessing closed apical foramen and straight root canals were immersed in 20 mm of saline solution. the samples were divided into three groups of nine randomly selected samples. sample preparation was initiated by opening access through the use of a high speed drill (dendia dental, germany). the working length of the entire sample was measured using a k-file no.10 (dentsply sirona, usa). root canal preparation used k-files no.s 10 and 15 in sequence with a push-pull motion that inserted them up to 2/3 of their working length before extracting them in a straight motion. root canal preparation was continued by means of a protaper for hand use (dentsply sirona, usa) using a pressureless crowndown technique, up to 2/3 of the working length of an s1 file and and a k-file no.15 for recapitulation and irrigation. if the working length was appropriate, preparation was continued with files ranging from s1 to f3. each tool replacement was irrigated with 3 ml aquadest depending on the treatment group. the control group was irrigated with aquadest, group i was irrigated with 17% edta and group ii with 6.25% pineapple peel extract. irrigation was performed using an anoxygen tube at1 atm pressure (1.033 kg/cm2). the root canal was dried with sterile paper points and cotton swabs and temporarily compressed in order to keep it dry. all the prepared and irrigated samples were contoured on the lingual, buccal, mesial, and distal surfaces using a diamond disc bur (dendia dental, germany). as cutting guidance, the contour was traced as close as possible to the root canal to facilitate the cutting process. samples were subsequently cut 6 mm horizontally from the apical to the coronal with the pieces being split into two using a chisel. the sample was fixed in the sample holder and the smear layer observed using a scanning electron microscope (zeiss, germany) at 1000x magnification. the root canal hygiene rating was determined based on the average hygiene score using the schafer criteria as follows:8 score 1 no smear layer and dentinal tubules are unobstructed and clearly visible; score 2: < 25% of areas have smear layer and dentinal tubules are unobstructed; score 3: more than 50% of the area has an unevenly spread smear layer; score 4: the entire area is covered with a thin and homogenous smear layer; and score 5: the whole area is covered with a thick and non homogeneous smear layer. all data was statistically analyzed using both anova and tukey tests as comparative tests used to investigate the differences between all groups which showed a significant difference (p <0.05). results in this study, the mean score result relating to root canal hygiene was obtained using the schafer criterium in the control group, the group of teeth irrigated with 17% edta (group 1) and the group of teeth irrigated with pineapple peel extract (group ii). the highest root canal hygiene score was obtained from the control group with a mean score of 4.097 followed by group i with a mean score of 2.483, while the lowest score was obtained from group ii with a mean score of 1.484 (figure 1). the result of an anova test showed that the p value = 0.0001 indicating that significant differences existed between the treatment groups (p <0.05). data analysis continued with the administering of a multiple comparison test in the form of a tukey hsd to compare the two groups. the contents of table 1 indicate that there were significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p122-125 124 pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3):122–125 differences between the control groups and groups i and ii, while a significant difference (p <0.05) also existed between the latter two groups. discussion in this study, root canal hygiene in the smear layer was assessed by comparing the smear layer removal effect between 6.25% pineapple peel extract and 17% edta as root canal irrigation material. based on the results obtained, the lowest score was recorded by group i compared to the 17% edta group and the control group in terms of removal the smear layer on the 1/3 apical part of the root canal. this indicated that the irrigated samples group using pineapple peel extract experienced a smear layer removal effect higher than that of the other two groups, the 17% edta group and the control group (aquadest), on the 1/3 apical section of the root canal. the high smear layer removal effect of 6.25% pineapple peel extract is influenced by potentially active substances such as saponins (2.48%), bromelain (1.44%), polyphenol (2.88%), and flavonoids (1.25%).9 saponins are substances with four hydrocarbon rings, divided into triterpenoids and steroid glycosides, that have properties as surfactants. partial molecules of the surfactant constitute the active component of saponin in lowering the surface tension, whereas other surfactant molecules create a micelle ring. the low surface tension allows the irrigation material to reach 1/3 of the apical section of the root canal and increase the contact of the irrigation material with the dentine wall, thus enabling the smear layer to be removed and the dentinal tubule opened. saponins also have a chemical structure consisting of glycosides (polar compounds) possessing hydrophilic properties and pentacyclic triterpenoids (nonpolar compounds) that demonstrate hydrophobic properties. this non-polar saponin compound will dissolve the inorganic components of dentine which are predominantly composed of calcium hydroxyapatite and tricalcium phosphate. these hydrophilic and hydrophobic properties create a ring known as the micelle ring.1 other elements contained within the pineapple peel include; bromelain, polyphenol and flavonoid which synergize with saponin to remove the smear layer by reducing the valence number of calcium ions that it contains. the decreasing valence number will create a metal attachment or an inorganic smear layer material on an unstable root canal which further renders this material loose and soluble. this loose inorganic smear layer will subsequently be surrounded by saponin molecules which create a micelle ring. at that point, the removal process initiated, namely; the absorption of the smear layer into the center of the micelle, thereby transforming it into a water soluble substance. consequently, saponin content supported by the bromelain properties, polyphenols and flavonoids contained in the pineapple peel extract can remove the inorganic smear layer by releasing the bond and wrapping the smear layer. this results in enhanced root canal hygiene.10,11 a study conducted by mancini et al. (2009) argued that, when employed as root canal irrigation material, edta proves ineffective at removing the smear layer on 1/3 of a b c figure 1. (a) group control (aquadest): many smear layers remain on 1/3 apical of the root canal. (b) group i (17% edta): the smear layer appears slightly reduced. (c) group ii (6.25% pineapple peel extract) the smear layer has been removed. table 1. the results of different treatment groups using multiple comparisons tukey hsd group group i (17% edta) group ii (6.25% pineapple peel extract) control group 0.001* 0.001* group i (17%edta) 0.001* * indicated there was a significant difference (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p122-125 125pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3):122–125 the apical section of the root canal.12 17% edta has a high surface tension of 0.0783 n/m. consequently, it is difficult to gain access to the 1/3 apical section of the root canal. in addition, edta can also reduce the mineral components and noncollagenous components (ncps) in dentine with the result that edta not only removes calcium ions, but also prevents calcium-bonding with ncps.13 because of the ncp content reduction in 1/3 of the root, the ability of edta to decalcify that area is compromised. this finding corresponds with the theory proposed by ozdemir et al. (2012) that edta proves ineffective at smear layer removal from the dentin of both young and old root canals.14 therefore, in this study, 17% edta proved less effective as an irrigation material compared to 6.25% pineapple (ananas comosus l. merr) peel extract during smear layer removal from 1/3 apical of the root canal. based on the results of this study, it can be concluded that 6,25% pineapple peel extract proves more effective at higher smear layer removal compared to 17% edta on the 1/3 apical section of the root canal. references 1. dennis ny, prasetia w. the ability of root canal irrigant with ethanol extract of lerak fruit (sapindus rarak dc) in removing root canal smear layer (a sem study). iosr j dent med sci. 2017; 16(1): 24–30. 2. ahmetoglu f, keles a, yalcin m, simsek n. effectiveness of different irrigation systems on smear layer removal: a scanning electron microscopic study. eur j dent. 2014; 8(1): 53–7. 3. kandil he, labib ah, alhadainy ha. effect of different irrigant solutions on microhardness and smear layer removal of root canal dentin. tanta dent j. 2014; 11(1): 1–11. 4. neri jr, passos vf, viana fb, rodrigues lka, de paulo aragão saboia v, santiago sl. efficacy of smear layer removal by cavity cleaning solutions: an atomic force microscopy study. rev odonto cienc. 2011; 26(3): 253–7. 5. dayem r, tameesh m. a new concept in hybridization: bromelain enzyme for deproteinizing dentin before application of adhesive system. contemp clin dent. 2013; 4(4): 421–6. 6. scelza mfz, pierro vs da s, chagas ma, da silva le, scelza p. evaluation of inflammatory response of edta, edta-t, and citric acid in animal model. j endod. 2010; 36(3): 515–9. 7. dua a, dua d, uppin v. evaluation of the effect of duration of application of smear clear in removing intracanal smear layer: sem study. saudi endod j. 2015; 5(1): 26–32. 8. giraki m, harapetian e, ruttermann s, gerhardt-szep s. shaping ability of rotary instrumentation techniques and their limitations in simulated root canals. j dent probl solut. 2019; 6(2): 49–55. 9. varghese v, valarselvan. phytoconstituens of different maturity period fruits of ananascomocus (pineapple). inter j compr res biol sci. 2015; 2(7): 10–5. 10. ha r ipya ree a, guneshwor k, da maya nti m. evaluation of antioxidant properties of phenolics extracted from ananas comosus l. not sci biol. 2010; 2(2): 68–71. 11. ezeabara c. determination of saponin content of various parts of six citrus species. int res j pure appl chem. 2014; 4(1): 137–43. 12. mancini m, armellin e, casaglia a, cerroni l, cianconi l. a comparative study of smear layer removal and erosion in apical intraradicular dentine with three irrigating solutions: a scanning electron microscopy evaluation. j endod. 2009; 35(6): 900–3. 13. srikanth p, krishna ag, srinivas s, reddy es, battu s, aravelli s. minimal apical enlargement for penetration of irrigants to the apical third of root canal system: a scanning electron microscope study. j int oral heal. 2015; 7(6): 92–6. 14. ozdemir ho, buzoglu hd, çalt s, çehreli zc, varol e, temel a. chemical and ultramorphologic effects of ethylenediaminetetraacetic acid and sodium hypochlorite in young and old root canal dentin. j endod. 2012; 38(2): 204–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p122-125 158 volume 47, number 3, september 2014 penatalaksanaan impaksi caninus permanen rahang atas dengan surgical exposure (the management of impacted permanent canine with surgical exposure) syeh brata wijaya dan rinaldi budi utomo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas gadjah mada yogyakarta – indonesia abstract background: impacted tooth is often unidentified because there is no symptom. it is found when patient is examined by dentist. the maxillary canine should be retained for strength masticatory function, esthetics and child development. purpose: the article was aimed to report treatment options of impacted canine in the 13 years old child. case: thirteen years-old girl came to the universitas gadjah mada dental hospital with complaints of the upper right permanent canine had not erupted, with no history of pain. periapical radiograph showed the impacted position of tooth #13 mesioangular. the shift sketch technique radiograph showed the impacted canine located at the palatal site. case management: surgical exposure the upper right maxillary canine was done, followed by orthodontic treatment to direct tooth position into occlusal line. fixed orthodontic appliance used was roth bracket with straight wire technique. after surgery and orthodontic treatment, #13 was in normal occlusion. conclusion: the surgical exposure followed by orthodontic treatment could be done successfully with special consideration to the patient’s age, the dental space, location of dental crowns, dental inclination, the apical root form of impacted tooth and patient cooperation. key words: impacted, surgical exposure, orthodontic fixed appliance abstrak latar belakang: terjadinya gigi impaksi biasanya diketahui setelah melakukan pemeriksaan ke dokter gigi karena jarang menimbulkan keluhan. gigi caninus rahang atas sebaiknya dipertahankan untuk kekuatan fungsi pengunyahan, estetik dan tumbuh kembang anak. tujuan: artikel ini bertujuan untuk melaporkan perawatan impaksi gigi kaninus atas pada anak 13 tahun. kasus: anak perempuan usia 13 tahun datang ke rumah sakit gigi dan mulut fakultas kedokteran gigi universitas gadjah mada dengan keluhan gigi kaninus permanen kanan atas yang belum erupsi, tanpa ada riwayat sakit di area tersebut. hasil radiografi periapikal menunjukkan posisi gigi #13 impaksi mesioangular. hasil radiografi dengan teknik shift sketch menunjukkan gigi kaninus yang impaksi terletak di palatal. tatalaksana kasus: dilakukan perawatan exposure surgical pada gigi #13, dilanjutkan dengan perawatan ortodontik untuk menempatkan posisi gigi ke arah oklusal. alat ortodontik cekat yang digunakan adalah braket roth dengan teknik straight wire. setelah dilakukan tindakan bedah dan penarikan ortodontik, gigi #13 berada pada ruang yang telah disediakan dan sudah masuk pada posisi oklusi. simpulan: surgical exposure yang dilanjutkan perawatan ortodontik dapat dilakukan dengan sukses dengan perhatian khusus pada usia pasien, ruang gigi, letak mahkota gigi, inklinasi gigi dan bentuk apeks akar gigi yang impaksi. kata kunci: impaksi, exposure surgical, alat ortodontik cekat korespondensi (correspondence): syeh brata wijaya, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: wsyekh@yahoo.com case report 159wijaya dan utomo: penatalaksanaan impaksi caninus permanen rahang atas dengan surgical exposure pendahuluan gigi geligi dalam rongga mulut akan mengalami erupsi menurut urutan waktu erupsi masing-masing jenis gigi, mulai dari fase gigi sulung sampai mengalami pergantian menjadi fase gigi permanen. proses erupsi masing-masing gigi baik pada fase gigi sulung maupun permanen akan terjadi secara fisiologis dan jarang sekali mengalami gangguan. gangguan erupsi pada umumnya akibat inflamasi kronis yang meyebabkan fibrosis mukosa di sekitarnya, ruangan yang tidak cukup karena perkembangan rahang yang tidak sempurna atau karena retensi geligi sulung, premature loss gigi sulung, dan nekrosis karena adanya infeksi.1 menurut bishara2 etiologi gigi impaksi dapat disebabkan oleh faktor primer dan faktor sekunder. faktor primer meliputi trauma pada gigi sulung, benih gigi tanggal prematur gigi sulung, dan erupsi gigi kaninus dalam celah pada kasus celah langit-langit. faktor sekunder meliputi kelainan endokrin, defisiensi vitamin d, dan febrile diseases. gigi kaninus merupakan gigi kedua setelah gigi molar ketiga yang berfrekuensi tinggi untuk mengalami impaksi meskipun demikian gigi anterior di rahang atas lainnya seperti gigi insisivus pertama dan kedua rahang atas juga dapat mengalami kesulitan tumbuh akibat terletak salah di dalam rahang. frekuensi terjadinya kaninus impaksi sebesar 0,8–2,8 persen.3 ditinjau dari letaknya, 85 persen posisi gigi kaninus yang impaksi terletak di daerah palatal lengkung gigi, sedangkan 15 persen nya terletak di bagian labial atau bukal.4 ada beberapa bukti yang menyatakan, bahwa penderita dengan maloklusi kelas ii divisi 2 dan gigi aplasia merupakan kelompok yang mempunyai risiko tinggi untuk terjadinya kaninus ektopik.5 untuk mengamati pergerakan gigi kaninus rahang atas dan menghitung jarak gerakan yang terjadi dapat digunakan metode pengamatan secara tiga dimensi dengan menggunakan foto sefalometri, periapikal dengan shift sketch dan foto oklusal.6 untuk mendapatkan hasil yang maksimal pada perawatan ortodonsi dengan kasus sukar diperlukan diagnosis dan rencana perawatan yang tepat. salah satu contohnya adalah perawatan ortodonsi dengan gigi kaninus rahang atas ektopik. khususnya kasus caninus impkasi krn masih tumbuh kembang,foramen apikal belum tertutup sehingga gigi masih bisa bergerak gigi kaninus impaksi dapat terletak ektopik dan sering dijumpai dalam praktek sehari-hari. kejadian impaksi dengan letak ektopik ini belum diketahui penyebabnya yang pasti, dimungkinkan oleh karena sebab yang multifaktorial. salah satu kemungkinan adalah jalan erupsi gigi kaninus yang lebih panjang bila dibandingkan dengan gigi permanen lainnya7. adanya diskrepansi panjang lengkung, gigi berdesakan, diastema antar gigi, dan trauma pada gigi anterior di awal usia pertumbuhan dapat pula merupakan penyebab terjadinya gigi kaninus ektopik8. keterlambatan proses eksfoliasi pada gigi kaninus sulung dapat pula menyebabkan terjadinya pergerakan gigi kaninus permanen ke arah palatal9,10 . ada dugaan bahwa frekuensi terjadinya kaninus ektopik dapat terjadi pada anak yang mengalami gangguan pada proses erupsi.1 laporan kasus ini bertujuan melaporkan perawatan impaksi gigi caninus rahang atas pada anak usia 13 tahun. kasus anak perempuan berusia 13 tahun datang ke rumah sakit gigi mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada, dengan keluhan gigi taring permanen kanan atas yang belum tumbuh dan gigi taring susu belum tanggal sedangkan gigi taring permanen kiri atas sudah tumbuh. keadaan umum pasien baik. pada pemeriksaan ekstra oral (gambar 1) menunjukan profil 3 kaninus rahang atas ektopik. khususnya kasus caninus impkasi krn masih tumbuh kembang,foramen apikal belum tertutup sehingga gigi masih bisa bergerak gigi kaninus impaksi dapat terletak ektopik dan sering dijumpai dalam praktek seharihari. kejadian impaksi dengan letak ektopik ini belum diketahui penyebabnya yang pasti, dimungkinkan oleh karena sebab yang multifaktorial. salah satu kemungkinan adalah jalan erupsi gigi kaninus yang lebih panjang bila dibandingkan dengan gigi permanen lainnya7. adanya diskrepansi panjang lengkung, gigi berdesakan, diastema antar gigi, dan trauma pada gigi anterior di awal usia pertumbuhan dapat pula merupakan penyebab terjadinya gigi kaninus ektopik8. keterlambatan proses eksfoliasi pada gigi kaninus sulung dapat pula menyebabkan terjadinya pergerakan gigi kaninus permanen ke arah palatal9,10 . ada dugaan bahwa frekuensi terjadinya kaninus ektopik dapat terjadi pada anak yang mengalami gangguan pada proses erupsi.1 laporan kasus ini bertujuan melaporkan perawatan impaksi gigi caninus rahang atas pada anak usia 13 tahun. kasus anak perempuan berusia 13 tahun datang ke rumah sakit gigi mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada, dengan keluhan gigi taring permanen kanan atas yang belum tumbuh dan gigi taring susu belum tanggal sedangkan gigi taring permanen kiri atas sudah tumbuh. keadaan umum pasien baik. pada pemeriksaan ekstra oral (gambar 1) menunjukan profil pasien cembung dan pada pemeriksaan intra oral (gambar 2a,b,c) semua gigi permanen sudah tumbuh kecuali kaninus kanan. tulang pada bagian palatal regio gigi #13 menonjol dan teraba keras saat palpasi. relasi molar kelas i angle dengan jarak gigit 3 mm dan tumpang gigit 3 mm. gambar 1. pemeriksaan ekstra oral, profil wajah. gambar 1. pemeriksaan ekstra oral, profil wajah. 4 gambar 2. pemeriksaan intra oral (a) tampak samping kanan; (b) tampak depan; (c) tampak samping kiri gambaran radiologis panoramik menunjukkan bahwa gigi #13 terletak di apikal gigi 51 dengan posisi mendorong apikal gigi 51 ke mesial (gambar 3). gambar 3. gambaran radiografi panoramik. gambar 4. foto periapikal gigi impaksi #13. foramen apikal belum menutup gambar 2. pemeriksaan intra oral (a) tampak samping kanan; (b) tampak depan; (c) tampak samping kiri. a b c 160 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 158–163 pasien cembung dan pada pemeriksaan intra oral (gambar 2a,b,c) semua gigi permanen sudah tumbuh kecuali kaninus kanan. tulang pada bagian palatal regio gigi #13 menonjol dan teraba keras saat palpasi. relasi molar kelas i angle dengan jarak gigit 3 mm dan tumpang gigit 3 mm. gambaran radiologis panoramik menunjukkan bahwa gigi #13 terletak di apikal gigi 51 dengan posisi mendorong apikal gigi 51 ke mesial (gambar 3). diagnosis dari keluhan pasien adalah impaksi gigi #13 klas 1 menurut archer. rencana perawatan meliputi dental health education (dhe), bedah exposure, dan perawatan ortodontik melakukan traksi gigi #13. tatalaksana kasus pada kunjungan pertama di klinik gigi anak, rumah sakit gigi dan mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada telah dilakukan dhe, scaling and root planning, pencetakan gigi rahang atas dan bawah untuk keperluan model studi, pengambilan radiografik panoramik, sefalometri dan periapikal (gambar 4 dan 5). pada kunjungan kedua dilakukan pemasangan alat ortodontik cekat teknik straight wire dengan 2 x 4 mini roth braces diameter slot 0.22 cm, kawat niti 0,12 dan menggunakan open coil pada regio #23 untuk mempertahankan ruang tempat erupsi gigi #23. pada kunjungan ketiga dilakukan bedah exposure gigi #23 oleh tim bedah minor gigi anak di klinik gigi anak, rumah sakit gigi dan mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada. teknik bedah exposure dengan flap tertutup berbentuk trapesium. prosedur bedah exposure gigi #13 dilakukan sebagai berikut, pertama, operator mendudukkan pasien di kursi gigi dan memeriksa tanda vital pasien (tekanan darah, denyut nadi, pernafasan dan suhu tubuh). pemberian antiseptik oral di dalam dan luar rongga mulut dengan povidone iodine (gambar 6a). dilakukan anestesi lokal infiltrasi pada bagian labial dan palatinal regio gigi #13 (gambar 6b), setelah 10 menit dibuat flap pada bagian palatal. insisi flap dibuat sepanjang bone crest melalui periosteum dari servikal palatal gigi #11-#14 (gambar 6c), kemudian dilakukan pemisahan jaringan periosteum dengan insisi vertikal pada palatal (gambar 6d). setelah kaninus terlihat lakukan penghilangan lapisan tipis pada lapisan tulang alveolar. tulang pada bukal ridge sampai cingulum dibuang dengan menggunakan bur tulang low speed. setelah terbebas dan gigi #13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahap-tahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi #14 yang telah diligasi dengan gigi #15 #16 untuk menarik gigi #13 ke bagian insisal (gambar 6f). flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal #11 dan distal #12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. kontrol bedah dilakukan pada hari ketujuh yang menunjukkan penyembuhan luka yang baik dan selanjutnya dilakukan pengambilan jahitan. tiga bulan pasca 4 gambar 2. pemeriksaan intra oral (a) tampak samping kanan; (b) tampak depan; (c) tampak samping kiri gambaran radiologis panoramik menunjukkan bahwa gigi #13 terletak di apikal gigi 51 dengan posisi mendorong apikal gigi 51 ke mesial (gambar 3). gambar 3. gambaran radiografi panoramik. gambar 4. foto periapikal gigi impaksi #13. foramen apikal belum menutup gambar 3. gambaran radiografi panoramik. 5 gambar 5. foto oklusal gigi impaksi #13. diagnosis dari keluhan pasien adalah impaksi gigi #13 klas 1 menurut archer. rencana perawatan meliputi dental health education (dhe), bedah exposure, dan perawatan ortodontik melakukan traksi gigi #13. tatalaksana kasus pada kunjungan pertama di klinik gigi anak, rumah sakit gigi dan mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada telah dilakukan dhe, scaling and root planning, pencetakan gigi rahang atas dan bawah untuk keperluan model studi, pengambilan radiografik panoramik, sefalometri dan periapikal (gambar 4 dan 5). pada kunjungan kedua dilakukan pemasangan alat ortodontik cekat teknik straight wire dengan 2 x 4 mini roth braces diameter slot 0.22 cm, kawat niti 0,12 dan menggunakan open coil pada regio #23 untuk mempertahankan ruang tempat erupsi gigi #23. pada kunjungan ketiga dilakukan bedah exposure gigi #23 oleh tim bedah minor gigi anak di klinik gigi anak, rumah sakit gigi dan mulut prof. soedomo, fakultas kedokteran gigi, universitas gadjah mada. teknik bedah exposure dengan flap tertutup berbentuk trapesium. prosedur bedah exposure gigi #13 (gambar 6a) dilakukan sebagai berikut, pertama, operator mendudukkan pasien di kursi gigi dan memeriksa tanda vital pasien (tekanan darah, denyut nadi, pernafasan dan suhu tubuh). pemberian antiseptik oral di dalam dan luar rongga mulut dengan povidone iodine (gambar 6a). dilakukan anestesi lokal infiltrasi pada bagian labial dan palatinal regio #gigi 13 (gambar 6b), setelah 10 menit dibuat flap pada bagian palatal. insisi flap dibuat sepanjang bone crest melalui periosteum dari servikal palatal gigi 11-14 (gambar 6c), kemudian dilakukan pemisahan jaringan periosteum dengan insisi vertikal pada palatal (gambar 6d). setelah kaninus terlihat lakukan penghilangan lapisan tipis pada lapisan tulang alveolar. tulang pada bukal ridge sampai cingulum dibuang dengan menggunakan bur tulang gambar 4. foto periapikal gigi impaksi #13. terlihat foramen apikal yang belum menutup (tanda panah). gambar 5. foto oklusal gigi impaksi #13. 4 gambar 2. pemeriksaan intra oral (a) tampak samping kanan; (b) tampak depan; (c) tampak samping kiri gambaran radiologis panoramik menunjukkan bahwa gigi #13 terletak di apikal gigi 51 dengan posisi mendorong apikal gigi 51 ke mesial (gambar 3). gambar 3. gambaran radiografi panoramik. gambar 4. foto periapikal gigi impaksi #13. 161wijaya dan utomo: penatalaksanaan impaksi caninus permanen rahang atas dengan surgical exposure pembedahan, gigi kaninus mulai terlihat turun ke bawah. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penggantian button dengan braket 2 x 4 kaninus untuk mengkoreksi posisi gigi kaninus masuk dalam lengkung gigi yang benar. selanjutnya kawat diganti sesuai dengan ketentuan alignment dan leveling sampai gigi kaninus terletak pada tempat yang benar. gambar 8 menunjukkan kondisi saat kontrol bulan ke-3 (a); bulan ke-5 (b); bulan ke-6 (c) dan bulan ke-9 (d). pembahasan gigi kaninus mempunyai peran penting dalam penampilan wajah, estetika gigi, perkembangan lengkung gigi dan fungsional oklusi,10 sehingga harus dipertahankan gambar 6. prosedur bedah exposure gigi #13. (a) pemberian antiseptik pevidone iodine, (b)anestesi lokal pada labial dan palatinal #13, (c) insisi flap sepanjang bone crest, (d) pemisahan jaringan periosteum, (e) pemasangan button pada bagian labial, (f) pemberian power chain 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. gambar 7. flap dikembalikan ke posisi semula dan dijahit dengan metode interrupted gambar 8. kondisi gigi pada saat kontrol: (a) bulan ke-3, (b) bulan ke-5, (c) bulan ke-6, (d) bulan ke-9. 7 kontrol bedah dilakukan pada hari ketujuh yang menunjukkan penyembuhan luka yang baik dan selanjutnya dilakukan pengambilan jahitan. tiga bulan pasca pembedahan, gigi kaninus mulai terlihat turun ke bawah. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penggantian button dengan braket 2 x 4 kaninus untuk mengkoreksi posisi gigi kaninus masuk dalam lengkung gigi yang benar. selanjutnya kawat diganti sesuai dengan ketentuan alignment dan leveling sampai gigi kaninus terletak pada tempat yang benar (gambar 8). gambar 8. kontrol bulan ke-3, bulan ke-5, bulan ke-6, bulan ke-9. pembahasan gigi kaninus mempunyai peran penting dalam penampilan wajah, estetika gigi, perkembangan lengkung gigi dan fungsional oklusi,10 sehingga harus dipertahankan dalam rongga mulut. mekanisme pasti terjadinya impaksi sebenarnya belum dapat dijelaskan secara pasti.11-13 pada erupsi normal, gigi kaninus permanen maksila akan turun di antara distal insisivus lateral permanen dan mesial premolar pertama, mengikuti apeks gigi kaninus desidui. inklinasi kaninus ke midline meningkat lebih ke mesial maksimal sampai usia 9 tahun.14 jika gigi kaninus menunjukkan inklinasi mesial sangat besar atau overlap dengan akar insisivus berdasarkan pemeriksaan klinis dan radiografis, berarti gigi kaninus memiliki kecenderungan untuk erupsi tidak benar. oleh karena itu perlu dilakukan pendekatan preventive untuk mengurangi resiko impaksi gigi kaninus dan resorbsi gigi permanen di sekitarnya.15-17 deteksi awal dan pencegahan impaksi kaninus maksila adalah hal fundamental dan penting untuk mencegah komplikasi, waktu perawatan dan biaya perawatan tambahan untuk mereposisi gigi impaksi. ada beberapa pilihan dalam perawatan gigi impaksi, antara lain: pencabutan atau pengambilan gigi impaksi, reposisi, bedah exposure dan ortodontik, serta replantasi. perawatan konvensional untuk gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga 7 kontrol bedah dilakukan pada hari ketujuh yang menunjukkan penyembuhan luka yang baik dan selanjutnya dilakukan pengambilan jahitan. tiga bulan pasca pembedahan, gigi kaninus mulai terlihat turun ke bawah. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penggantian button dengan braket 2 x 4 kaninus untuk mengkoreksi posisi gigi kaninus masuk dalam lengkung gigi yang benar. selanjutnya kawat diganti sesuai dengan ketentuan alignment dan leveling sampai gigi kaninus terletak pada tempat yang benar (gambar 8). gambar 8. kontrol bulan ke-3, bulan ke-5, bulan ke-6, bulan ke-9. pembahasan gigi kaninus mempunyai peran penting dalam penampilan wajah, estetika gigi, perkembangan lengkung gigi dan fungsional oklusi,10 sehingga harus dipertahankan dalam rongga mulut. mekanisme pasti terjadinya impaksi sebenarnya belum dapat dijelaskan secara pasti.11-13 pada erupsi normal, gigi kaninus permanen maksila akan turun di antara distal insisivus lateral permanen dan mesial premolar pertama, mengikuti apeks gigi kaninus desidui. inklinasi kaninus ke midline meningkat lebih ke mesial maksimal sampai usia 9 tahun.14 jika gigi kaninus menunjukkan inklinasi mesial sangat besar atau overlap dengan akar insisivus berdasarkan pemeriksaan klinis dan radiografis, berarti gigi kaninus memiliki kecenderungan untuk erupsi tidak benar. oleh karena itu perlu dilakukan pendekatan preventive untuk mengurangi resiko impaksi gigi kaninus dan resorbsi gigi permanen di sekitarnya.15-17 deteksi awal dan pencegahan impaksi kaninus maksila adalah hal fundamental dan penting untuk mencegah komplikasi, waktu perawatan dan biaya perawatan tambahan untuk mereposisi gigi impaksi. ada beberapa pilihan dalam perawatan gigi impaksi, antara lain: pencabutan atau pengambilan gigi impaksi, reposisi, bedah exposure dan ortodontik, serta replantasi. perawatan konvensional untuk gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga 7 kontrol bedah dilakukan pada hari ketujuh yang menunjukkan penyembuhan luka yang baik dan selanjutnya dilakukan pengambilan jahitan. tiga bulan pasca pembedahan, gigi kaninus mulai terlihat turun ke bawah. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penggantian button dengan braket 2 x 4 kaninus untuk mengkoreksi posisi gigi kaninus masuk dalam lengkung gigi yang benar. selanjutnya kawat diganti sesuai dengan ketentuan alignment dan leveling sampai gigi kaninus terletak pada tempat yang benar (gambar 8). gambar 8. kontrol bulan ke-3, bulan ke-5, bulan ke-6, bulan ke-9. pembahasan gigi kaninus mempunyai peran penting dalam penampilan wajah, estetika gigi, perkembangan lengkung gigi dan fungsional oklusi,10 sehingga harus dipertahankan dalam rongga mulut. mekanisme pasti terjadinya impaksi sebenarnya belum dapat dijelaskan secara pasti.11-13 pada erupsi normal, gigi kaninus permanen maksila akan turun di antara distal insisivus lateral permanen dan mesial premolar pertama, mengikuti apeks gigi kaninus desidui. inklinasi kaninus ke midline meningkat lebih ke mesial maksimal sampai usia 9 tahun.14 jika gigi kaninus menunjukkan inklinasi mesial sangat besar atau overlap dengan akar insisivus berdasarkan pemeriksaan klinis dan radiografis, berarti gigi kaninus memiliki kecenderungan untuk erupsi tidak benar. oleh karena itu perlu dilakukan pendekatan preventive untuk mengurangi resiko impaksi gigi kaninus dan resorbsi gigi permanen di sekitarnya.15-17 deteksi awal dan pencegahan impaksi kaninus maksila adalah hal fundamental dan penting untuk mencegah komplikasi, waktu perawatan dan biaya perawatan tambahan untuk mereposisi gigi impaksi. ada beberapa pilihan dalam perawatan gigi impaksi, antara lain: pencabutan atau pengambilan gigi impaksi, reposisi, bedah exposure dan ortodontik, serta replantasi. perawatan konvensional untuk gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga 7 kontrol bedah dilakukan pada hari ketujuh yang menunjukkan penyembuhan luka yang baik dan selanjutnya dilakukan pengambilan jahitan. tiga bulan pasca pembedahan, gigi kaninus mulai terlihat turun ke bawah. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penggantian button dengan braket 2 x 4 kaninus untuk mengkoreksi posisi gigi kaninus masuk dalam lengkung gigi yang benar. selanjutnya kawat diganti sesuai dengan ketentuan alignment dan leveling sampai gigi kaninus terletak pada tempat yang benar (gambar 8). gambar 8. kontrol bulan ke-3, bulan ke-5, bulan ke-6, bulan ke-9. pembahasan gigi kaninus mempunyai peran penting dalam penampilan wajah, estetika gigi, perkembangan lengkung gigi dan fungsional oklusi,10 sehingga harus dipertahankan dalam rongga mulut. mekanisme pasti terjadinya impaksi sebenarnya belum dapat dijelaskan secara pasti.11-13 pada erupsi normal, gigi kaninus permanen maksila akan turun di antara distal insisivus lateral permanen dan mesial premolar pertama, mengikuti apeks gigi kaninus desidui. inklinasi kaninus ke midline meningkat lebih ke mesial maksimal sampai usia 9 tahun.14 jika gigi kaninus menunjukkan inklinasi mesial sangat besar atau overlap dengan akar insisivus berdasarkan pemeriksaan klinis dan radiografis, berarti gigi kaninus memiliki kecenderungan untuk erupsi tidak benar. oleh karena itu perlu dilakukan pendekatan preventive untuk mengurangi resiko impaksi gigi kaninus dan resorbsi gigi permanen di sekitarnya.15-17 deteksi awal dan pencegahan impaksi kaninus maksila adalah hal fundamental dan penting untuk mencegah komplikasi, waktu perawatan dan biaya perawatan tambahan untuk mereposisi gigi impaksi. ada beberapa pilihan dalam perawatan gigi impaksi, antara lain: pencabutan atau pengambilan gigi impaksi, reposisi, bedah exposure dan ortodontik, serta replantasi. perawatan konvensional untuk gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga a b c d 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. 6 low speed. setelah terbebas dan gigi 13 terlihat, dilakukan isolasi sekitar gigi kemudian dilakukan pemasangan bagian labial dengan button (gambar 6e). dengan mematuhi tahaptahap dari pemasangan braket. pada button kemudian diberi power chain yang dikaitkan pada gigi 14 yang telah diligasi dengan gigi 15 16 untuk menarik gigi 13 ke bagian insisal (gambar 6f). gambar 6 a-f. prosedur bedah exposure gigi #13. flap dikembalikan pada posisi semula dan dijahit dengan menggunakan metode interrupted pada distal 11 dan distal 12 (gambar 7). luka ditutup dengan tampon dan evaluasi perdarahan sekitar 10 menit. tampon selanjutnya dilepas 30 menit dengan instruksi pada pasien yaitu tidak boleh berkumur terlalu sering, tidak menghisap luka, dan tidak memainkan dengan lidah pada bekas operasi. setelah bedah, pasien diberikan medikasi oral antibiotik, analgesik dan anti-inflamasi. foto saaat operasi, cara inicisi, peletakkan braket. gambar 7. flap dijahit. a b c d e f 162 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 158–163 dalam rongga mulut. mekanisme pasti terjadinya impaksi sebenarnya belum dapat dijelaskan secara pasti.11-13 pada erupsi normal, gigi kaninus permanen maksila akan turun di antara distal insisivus lateral permanen dan mesial premolar pertama, mengikuti apeks gigi kaninus desidui. inklinasi kaninus ke midline meningkat lebih ke mesial maksimal sampai usia 9 tahun.14 jika gigi kaninus menunjukkan inklinasi mesial sangat besar atau overlap dengan akar insisivus berdasarkan pemeriksaan klinis dan radiografis, berarti gigi kaninus memiliki kecenderungan untuk erupsi tidak benar. oleh karena itu perlu dilakukan pendekatan preventive untuk mengurangi resiko impaksi gigi kaninus dan resorbsi gigi permanen di sekitarnya.15-17 deteksi awal dan pencegahan impaksi kaninus maksila adalah hal fundamental dan penting untuk mencegah komplikasi, waktu perawatan dan biaya perawatan tambahan untuk mereposisi gigi impaksi. ada beberapa pilihan dalam perawatan gigi impaksi, antara lain: pencabutan atau pengambilan gigi impaksi, reposisi, bedah exposure dan ortodontik, serta replantasi. perawatan konvensional untuk gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga dapat menempati lengkung gigi yang benar tergantung dari beberapa faktor. faktor tersebut meliputi, usia penderita, adanya diastema atau ruang, adanya gigi yang berdesakan, dimensi vertikal, terbalik atau tidaknya letak mahkota, inklinasi letak gigi terhadap garis media wajah (tidak lebih dari 45 derajat), mengalami ankylosis atau mempunyai akar yang bengkok.18-19 pada kasus ini, prognosis perawatan baik karena usia pasien masih muda, adanya ruang untuk erupsinya gigi impaksi kaninus setelah dilakukan traksi dan juga untuk mengkoreksi gigi-gigi berjejal adalah cukup, inklinasi gigi kaninus tidak lebih dari 45o ataupun mahkotanya tidak terbalik dan akar tidak bengkok. perawatan harus segera dilakukan karena dilihat dari radiografik menunjukkan gigi kaninus yang impaksi telah mendesak akar gigi insisivus lateral ke mesial sehingga beresiko meresorbsi akar gigi permanen insisivus lateral. erupsi spontan gigi impaksi biasanya terjadi setelah membuka ruang untuk erupsi dengan bedah sebelum perawatan ortodontik, menghilangkan faktor etiologi, exposure gigi yang impaksi dan mempertahankan ruang erupsi. jika hal ini tidak dilakukan, gigi impaksi akan membutuhkan waktu yang lama untuk erupsi sekitar 3 tahun atau lebih. oleh karena itu pemasangan alat ortodontik cekat sangat penting untuk merawat gigi yang impaksi sangat perlu.20 teknik bedah exposure untuk erupsinya gigi kaninus pada kasus ini adalah dengan flap tertutup, dengan pertimbangan agar tidak terjadi kerusakan jaringan gingiva yang lebih besar dan estetis yang lebih baik, rasa sakit pasca bedah minimal dan lebih singkat.21-22 penggunaan braket cekat lebih menguntungkan karena kontrol pembukaan akses gigi impaksi lebih mudah dan kekuatan tarik dapat dikontrol, dibandingkan dengan alat ortodontik lepasan. keuntungan lainnya lebih nyaman, pasien tidak harus kooperatif, kontrol pergerakan braket lebih baik, dan memungkinkan pergerakan gigi dalam tiga gerakan secara langsung.23 pada pemakaian pertama braket, kawat yang digunakan adalah jenis nickel titanium karena memiliki elastisitas bagus dan dapat memperbaiki posisi gigi yang tidak beraturan ke dalam lengkung gigi yang benar. setelah beberapa kali kontrol, jenis kawat diganti dengan dengan ukuran yang lebih besar untuk mencegah terjadi perubahan posisi pada gigi yang lain. keberhasilan atau prognosis perawatan ortodonsi dengan disertai tindakan bedah exposure akan tergantung antara lain pada kerja sama pasien dan dokter gigi, karena perawatan akan dalam waktu yang lebih lama, selain itu perlu memperhatikan usia penderita, adanya ruang gigi, adanya gigi yang berjejal, terbalik atau tidaknya letak mahkota, inklinasi letak gigi terhadap garis media wajah, ada tidaknya ankylosis dan ujung akar gigi yang impaksi sudah terbentuk ataupun bengkok. deteksi awal dokter gigi terhadap erupsi gigi kaninus permanen rahang atas dapat mengurangi waktu perawatan, kompleksitas, komplikasi dan biaya. dapat disimppulkan bahwa surgical exposure yang dilanjutkan perawatan ortodontik dapat dilakukan dengan baik dengan pertimbangan khusus pada usia pasien, ruang gigi, letak mahkota, inklinasi, dan bentuk apeks gigi yang impaksi. daftar pustaka 1. shah rm, boyd ma, vakil tf. study of permanent toot anomaly in 7886 cannadians individuals. j canad dent assoc 1978; 44: 262–64. 2. bishara se. management of impacted canines. am j orthod 1976; 69(4): 371–87. 3. kindelan j, cook p. the ectopic maxillary canine: a case report. br j orthod 1998; 25(3): 179–80. 4. ericson s, kurol j. radiographic examination of ectopically erupting maxillary canine. am j orthod dentofacial orthop 1987; 91(6): 483–92. 5. moose pa, campbell hm, luffingham jk. the palatal canine and adjacent lateral incisor: a study of a west of scotland population. br j orthod 1994; 21(2): 268–74. 6. coulter j, richardson a. normal eruption of the maxillary canine quantified in three dimension. eur j orthod 1997; 18: 449–56. 7. jacob h. the etiology of maxillary canine impactions. am j orthod 1983; 84: 125–39. 8. brencheley z,oliver rg. morphology of anterior teeth associated with displaced canines. br j orthod 1997; 24(1): 41–5. 9. mcsherry p, richardson a. ectopic eruption of the maxillary canine quantified in three dimensions on chephalometric radiographs between the ages of 5 and 15 years. eur j orthod 1999; 21(1): 41-8. 10. rachmawati v. kasus kaninus impaksi rahang atas di klinik bedah mulut dan maksilofasial serta klinik ortodontia fakultas kedokteran gigi universitas airlangga periode januari 2008 desember 2011. penelitian deskriptif observasional. p. 1. 11. shapira y, kuftinec mm. early diagnosis and interception of potential maxillary canine impaction. j am dent assoc 1998; 129(10): 1450-4. 12. kurol j. early treatment of tooth-eruption disturbances. am j orthod dentofacial orthop 2002; 121(6): 588-91. 13. warford jh jr, grandhi rk, tira de. prediction of maxillary canine impaction using sectors and angular measurement. am j orthod dentofacial orthop 2003; 124(6): 651-5. 163wijaya dan utomo: penatalaksanaan impaksi caninus permanen rahang atas dengan surgical exposure 14. kurol j, ericson s, andreasen jo. the impacted maxillary canine. in: andreasen jo, petersen jk, laskin dm, editors. textbook and colour atlas of tooth impactions: diagnosis, treatment, prevention. copenhagen, denmark: munskgaard; 1997. p. 124-64. 15. fernández e, bravo la, canteras m. eruption of the permanent upper canine: a radiologic study. am j orthod dentofacial orthop 1998; 113(4): 414-20. 16. tsai hh. eruption process of upper permanent canine j clin pediatr dent 2001; 25(3): 175-9. 17. alessandri bonetti g, zanarini m, danesi m, incerti parenti s, gatto mr. percentiles relative to maxillary permanent canines inclination by age: a radiologic study. am j orthod dentofacial orthop 2009; 36: 486.e1-6. 18. brencheley z, oliver rg. morphology of anterior teeth associated with displaced canines. br j orthod 1997; 24(1):41-5. 19. mcsherry p, richardson a. ectopic eruption of the maxillary canine quantified in three dimensions on chephalometric radiographs between the ages of 5 and 15 years. eur j orthod 1999; 21(1): 41-8. 20. be cker. ea rly t reat ment for i mpact e d ma x i l la r y i ncisor s. international symposium of early orthodontic treatment february 2002; p. 8-10, 21. dowsing p, sandler pj. how to effectively use a 2 x 4 appliance. j orthod 2004; 31(3): 248-58. 22. proffit, wr. contemporary orthodontics. 4th ed. mosby elsevier; 2007. p. 361-5. 23. nirwan ac. frenectomy combined with a laterally displaced pedicle graft. indian j dent sci 2010; 2(3): 1-2. vol 51 no 1 jan-mrt 2018.indd 3333 analysis of anti-streptococcus sanguinis igy ability to inhibit streptococcus sanguinis adherence suryani hutomo,1 heni susilowati,2 dewi agustina,3 and widya asmara4 1 department of microbiology, faculty of medicine, universitas kristen duta wacana 2 department of oral biology, faculty of dentistry, universitas gadjah mada 3 department of oral medicine, faculty of dentistry, universitas gadjah mada 4 department of microbiology, faculty of veterinary medicine, universitas gadjah mada yogyakarta indonesia abstract background: streptococcus sanguinis (s. sanguinis), an oral commensal bacterium, is often implicated in infective endocarditis. its adherence to the tooth surface is the initial step in dental plaque formation. in addition to the important role of s. sanguinis in systemic disease and antimicrobial resistance, it is necessary to develop methods to control dental plaque formation. immunoglobulin y (igy) has been used to prevent bacterial infection. purpose: the purpose of this study is to analyze the ability of anti-s. sanguinis igy antibodies to inhibit s. sanguinis adherence to hydroxyapatite (ha) discs as a model of the tooth surface. methods: antibodies were produced by immunizing hens with s. sanguinis suspension. boosters were given three times following the first injection. an agar gel precipitation test (agpt) was used to detect the presence of anti-s. sanguinis igy. a bacterial adherence assay was performed twice to analyze the ability of igy and the optimal concentration required to inhibit bacterial adherence. results: the formation of a precipitation line using agpt confirmed the presence of the antibody. in addition, it was shown that the anti-s. sanguinis igy antibody could inhibit bacterial adherence to ha. statistical analysis using one-way anova revealed a significant difference in the optical density (od) value between the groups (p<0.05). the results of electron microscopy scanning confirmed the quantitative analysis by means of a bacterial adherence test. conclusion: anti-s. sanguinis igy has the ability to inhibit adherence of s. sanguinis to ha discs at an optimal concentration of 30%. the inhibitive effect was stronger in the presence of saliva. keywords: streptococcus sanguinis; igy; bacterial adherence correspondence: suryani hutomo, department of microbiology, faculty of medicine, universitas kristen duta wacana, jl. dr. wahidin sudirohusodo no. 5-25, kotabaru, gondokusuman, yogyakarta 55224, indonesia. e-mail: suryanihutomo_drg@yahoo.com research report introduction streptococcus sanguinis (s. sanguinis) is known as a primary colonizer of the tooth surface. these bacteria can attach to the tooth surface and the oral epithelium by means of adhesin, a cell wall protein component.1 it attaches to the hydroxyapatite (ha) surface which is the main component of tooth enamel through interaction with salivary glycoprotein in the acquired pellicle.2 maturation of dental plaque occurs in the presence of gram negative bacteria such as veillonella, fusobacterium nucleatum and porphyromonas gingivalis.3 s. sanguinis is often implicated in infective endocarditis and among the viridans group of streptococci it is, in fact, the most commonly involved.4 it becomes attached to the fibrin and platelet vegetation that occurs in the heart valve, forming a biofilm that may cause bacteremia.5 poor quality of dental care is a predisposing factor in infective endocarditis, although the bacteria can also enter the body through the daily diet.6 besides its important role in systemic infection, control of dental plaque is necessary to obtain healthy gingival tissue and, thus, prevent systemic bacterial invasion. due to the possibility of bacteremia as the result of dental care procedure, the prescribing of prophylactic antibiotics is sometimes necessary, especially in high risk patients. however, such prophylactic measures are not always effective. previous studies reported the occurrence of amoxicillin-resistant streptococcus dental journal (majalah kedokteran gigi) 2018 march; 51(1): 33–36 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p33–36 mailto:suryanihutomo_drg@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p33-36 34 hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 33–36 viridans (s. viridans),7 and also fluoroquinolone-resistant s. viridans which are responsible as the cause of bacteremia in neutropenic cancer patients.8 therefore, development of effective prophylaxis measures is necessary. hens produce polyclonal antibodies which play an important medical role. immunoglobulin y (igy) is the predominant immunoglobulin in the serum of hens, amphibians and reptiles that is transferred from serum to egg yolks. its function is to provide passive immunity to the embryo and the neonate. immunoglobulin y is similar in function to immunoglobulin g (igg) in mammals.9 in general, the molecular structure of igy is the same as igg, but the molecular weight of igy is 180 kda, heavier than that of igg at 150 kda.10 in veterinary medicine, igy is also used in therapeutic technology targeting enteric bacterial infection.11 specific igy antibodies are obtained by immunizing the hen with the antigen of interest. in this study, hens were immunized with s. sanguinis to produce the anti-s. sanguinis igy antibody. the aim of this study is to analyze the potential of the anti-s. sanguinis igy antibody to inhibit s. sanguinis adherence to the ha discs as a model of the tooth surface. the final goal of the anti-s. sanguinis igy antibody in this research is the control of dental plaque formation. materials and methods initially, s. sanguinis atcc 10556 was cultured in brain-hearth infusion broth (bhi; oxoid, hampshire, uk). a 100 μl heat-killed suspension of s. sanguinis in freund complete adjuvant (sigma aldrich, saint louis usa) was then subcutaneously injected into hens in order to produce an antibody response. three booster immunizations with heat-killed suspension of s. sanguinis in freund incomplete adjuvant (sigma-aldrich, saint louis, usa) were administered over a time span of two weeks. purification of igy was completed according to the chicken igy purification kit protocol (sigma-aldrich, saint louis, usa). finally, the presence of antibodies was detected by performing an agar gel precipitation test (agpt). human saliva was collected from three healthy volunteers who, before pooling their saliva, rinsed their mouths with water to decrease bacterial contamination. the saliva provided was then centrifuged for 15 minutes at 3000 g and 4° c. the supernatant was stored at -80° c prior to use.12 the ha discs (10mm in diameter, 1.2 mm in thickness) were created by placing 500 mg of hydroxyapatite powder in a mould and pressed at 120 mpa. finally, discs were sintered for two hours at 1300° c. the sterilization of the hydroxyapatite discs was achieved by keeping the discs in the autoclave for 15 minutes at 100° c.13 bacterial adherence assay was performed by modification of the johansen method.14 saliva-coated ha discs were incubated with various concentrations of anti-s. sanguinis igy antibody for 30 minutes at 37° c, then stimulated with 100μl of 1.5 ×108 colony forming unit (cfu) s. sanguinis suspension and incubated at 37° c overnight. after incubation, ha discs were washed with phosphate buffer saline (pbs) solution and fixed with 250μl absolute methanol for 15 minutes. adherent bacteria were stained with 0.1% crystal violet and washed twice with pbs. stained adherent bacteria were extracted from the disks using 96% ethanol and transferred to a fresh 96-well plate. the absorbance of the extract from stained adherent bacteria was measured at 595 nm using a microplate reader (thermo scientific, rockford, illionis, usa).14,15 to further analyze the ability of anti-s. sanguinis igy to inhibit bacterial adherence in the presence and absence of saliva, additional bacterial adherence assays were performed using optimum igy concentration in the four different treatment groups. the ha discs in groups i and ii were coated with saliva, while in groups iii and iv they were left uncoated. in group i, anti-s. sanguinis igy antibodies were incubated with s. sanguinis before ha disks were added, whereas in group ii ha disks were cultured with anti-s. sanguinis igy antibodies before bacterial inoculation. the treatment for group iii was the same as group ii, while in group iv ha disks were cultured with s. sanguinis without antibodies. observation by means of a scanning electron microscope (jeol jed-2300; jeol, tokyo, japan) was performed to provide a general overview of bacterial adherence to the ha disks.16 results anti-s. sanguinis igy antibodies were detected using agpt. the s. sanguinis antigens in the center well and the antibodies in the outer wells each diffused outward and became bound to each other, forming an antigen-antibody complex. this complex precipitated in the gel, forming a white precipitation line, while there was no such formation between the s. sanguinis and igy from non-immunized hens (figure 1). figure 1. the result of agpt. arrows indicate the precipitation lines which formed between the s. sanguinis (center well) and the anti-s. sanguinis antibodies (wells 1, 2, 3, 4) while there was no such formation between s. sanguinis and igy from non-immunized hens (wells 5 and 6). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p33–36 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p33-36 35hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 33–36 the bacterial adherence assay demonstrated that inhibition occurred at concentrations of 20%, 25% and 30%, characterized by a decreased optical density (od) value. the inhibition of bacterial adherence can be observed in the treatment of 40% and 50% antibodies, but not to the same degree as the inhibition effect of 30% antibodies as seen in figure 2a. based on the results of the first bacterial adherence assay, the optimum concentration of antibodies for effective bacterial inhibition was 30%. consequently, this concentration was employed for the second assay. the result of the second bacterial adherence assay demonstrated that the lowest od, and hence the greatest bacterial inhibition, occurred in group ii, followed by groups iii, i and iv respectively (figure 2b). the data was analyzed using one-way anova at a significance level of 0.05 and there was a significant difference between groups (p=0.000). observation through sem confirmed these results. numerous bacteria and matrix plaque were both present in group i, while ha crystals were not visible. in group ii, bacteria and matrix plaque were present and ha crystals were also visible. bacteria and matrix plaque could be observed but less so than in group i or group iii. considerable numbers of bacteria and thick matrix plaque were found in group iv (figure 3). this confirms that anti-s. sanguinis igy could more effectively inhibit s. sanguinis adherence to ha disks in the presence of saliva. discussion the bacterial adherence assay demonstrated that inhibition began at concentrations of 20% and increased at those of 25% and 30%. the inhibition of bacterial adherence still occurred through the administering of 40% and 50% antibody, but not as much as at a concentration of 30%. this phenomenon might occur if antibody concentrations above 40% result in a saturation of antigen-antibody bonds, rendering further adherence inhibition impossible. a similar dose response was observed in a study conducted by fujibayashi et al. using anti-candida spp igy.17 the result of the second bacterial adherence assay demonstrated that the i s. sanguinis group that was exposed to the igy anti-s. sanguinis antibody subsequently bound to each other to form an antigen-antibody complex. proteases produced by the bacteria broke down the antibodies so that they were unable to bind to the bacteria.18 in this group, free bacteria adhered to saliva-coated ha discs initiating the formation of a biofilm. the lowest extent of bacterial adherence to disks occurred in group ii. this was due to a strong affinity between the saliva and ha disks, and also between the saliva and antibodies. the tooth surface is negatively charged which means that positive salivary ions such as ca2+, na+ and k+ are able to form strong bonds.19 saliva-tooth surface binding also occurred due to salivary glycoprotein on the tooth surface which was absorbed and figure 2. a) optical density value of s. sanguinis adherence at various concentrations; b) optical density value of s. sanguinis adherence in four different treatment groups. figure 3. scanning electron microscopy of ha surface colonized by bacteria. group i (a): lots of bacteria were present (1-round shapes) as well as matrix plaque (2dense mass, irregular shape), ha crystals were not visible. group ii (b): some bacteria (1) and matrix plaque (2), ha crystals were visible (3regular solid mass with sharp borders). group iii (c): bacteria and matrix plaque were seen but fewer than in a. group iv (d) lots of bacteria and thick matrix plaque. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p33–36 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p33-36 36 hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 33–36 rearranged by covalent hydrogen and ionic bonds.20 the primary mechanism through which igy limits the pathogen is by inhibition of adhesion such as blocking the surface epitopes required for binding and interfering with binding to mucin.10 antibodies in group iii were able to block surface epitopes on the ha disc, but not as effectively as group ii because, while the igy was able to cover the ha discs, it was unable to bond. bacteria were, therefore, able to penetrate and adhere to the ha discs. in group iv, bacteria could adhere and form a biofilm on the ha discs in the absence of saliva and antibodies. in order to be able to colonize the tooth surface, the bacteria must adhere directly to it or other cells that can bind to the teeth.21 the results of this study demonstrate that anti-s. sanguinis igy can effectively inhibit bacterial adherence to saliva coated ha discs as a model of the tooth surface, thus limiting dental plaque formation. in conclusion, this study confirms that anti-s. sanguinis igy antibody can inhibit s. sanguinis adherence to ha discs at an optimum concentration of 30%. the inhibition effect was stronger in the presence of saliva. this antibody can be recommended as a potential topical application in the oral cavity to control dental plaque formation. acknowledgement the authors would like to thank the faculty of medicine, universitas duta wacana christian, yogyakarta, indonesia for funding of the research reported here. references 1. kreth j, merritt j, qi f. bacterial and host interactions of oral streptococci. dna cell biol. 2009; 28(8): 397–403. 2. okahashi n, nakata m, sakurai a, terao y, hoshino t, yamaguchi m, isoda r, sumitomo t, nakano k, kawabata s, ooshima t. pili of oral streptococcus sanguinis bind to fibronectin and contribute to cell adhesion. biochem biophys res commun. 2010; 391(2): 1192–6. 3. díaz pi, kolenbrander pe. subgingival biofilm communities in health and disease. rev clínica periodoncia, implantol rehabil oral. 2009; 2(3): 187–92. 4. westl ing k. vi r ida ns g roup st reptococci septicaem ia a nd endocarditis. thesis. stockholm: karolinska institutet; 2005. p. 9–31. 5. ekdahl c. infective endocarditis – aspects of pathophysiology, epidemiology, management and prognosis. thesis. linköping: linköping university; 2008. p. 7–36. 6. lockhart pb, brennan mt, sasser hc, fox pc, paster bj, bahranimougeot fk. bacteremia associated with toothbrushing and dental extraction. circulation. 2008; 117(24): 3118–25. 7. masuda k, nemoto h, nakano k, naka s, nomura r, ooshima t. amoxicillin-resistant oral streptococci identified in dental plaque specimens from healthy japanese adults. j cardiol. 2012; 59(3): 285–90. 8. sahasrabhojane p, galloway-peña j, velazquez l, saldaña m, horstmann n, tarrand j, shelburne sa. species-level assessment of the molecular basis of fluoroquinolone resistance among viridans group streptococci causing bacteraemia in cancer patients. int j antimicrob agents. 2014; 43(6): 558–62. 9. wen j, zhao s, he d, yang y, li y, zhu s. preparation and characterization of egg yolk immunoglobulin y specific to influenza b virus. antiviral res. 2012; 93(1): 154–9. 10. xu y, li x, jin l, zhen y, lu y, li s, you j, wang l. application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: a review. biotechnol adv. 2011; 29(6): 860–8. 11. chalghoumi r, beckers y, portetelle d, théwis a. hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infections in chicken: a review. biotechnol agron soc env. 2009; 13(2): 295–308. 12. vanessa b, virginie m, nathalie q, marie-hélène r, christine i. hartmannella vermiformis can promote proliferation of candida spp. in tap-water. water res. 2012; 46(17): 5707–14. 13. siswomihardjo w, sunarintyas s, tontowi ae. the effect of zirconia in hydroxyapatite on staphylococcus epidermidis growth. int j biomater. 2012; 2012: 1–4. 14. johansen t, agdestein a, olsen i, nilsen s, holstad g, djønne b. biofilm formation by mycobacterium avium isolates originating from humans, swine and birds. bmc microbiol. 2009; 9: 159. 15. raja af, ali f, khan ia, shawl as, arora ds. acetyl-11-keto-βboswellic acid (akba); targeting oral cavity pathogens. bmc res notes. 2011; 4: 406. 16. setiawati s, nuryastuti t, ngatidjan n, mustofa m, jumina j, fitriastuti d. in vitro antifungal activity of (1)-n-2-methoxybenzyl-1, 10-phenanthrolinium bromide against candida albicans and its effects on membrane integrity. mycobiology. 2017; 45: 25–30. 17. fujibayashi t, nakamura m, tominaga a, satoh n, kawarai t, narisawa n, shinozuka o, watanabe h, yamazaki t, senpuku h. effects of igy against candida albicans and candida spp. adherence and biofilm formation. jpn j infect dis. 2009; 62(5): 337–42. 18. brezski rj, jordan re. cleavage of iggs by proteases associated with invasive diseases: an evasion tactic against host immunity? mabs. 2010; 2(3): 212–20. 19. amerongen an, verman eci, abyono r. ludah dan kelenjar ludah: arti bagi kesehatan gigi. yogyakarta: gadjah mada university press; 1991. p. 269. 20. huang r, li m, gregory rl. bacterial interactions in dental biofilm. virulence. 2011; 2(5): 435–44. 21. jakubovics ns. intermicrobial interactions as a driver for community composition and stratification of oral biofilms. j mol biol. 2015; 427(23): 3662–75. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p33–36 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p33-36 �� volume 47, number 1, march 2014 research report the expressions of nf-kb and tgfb-� on odontoblast-like cells of human dental pulp injected with propolis extracts ira widjiastuti, ketut suardita and widya saraswati department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: propolis is known to have beneficial effects, namely antibacterial, anti-viral, anti-inflammatory, antioxidant, and immunomodulatory. propolis extracts with anti-inflammatory properties are expected to be useful in treating inflamed pulp tissue with a diagnosis of reversible pulpitis. the inflammation of pulp tissue is caused by bacteria, namely lactobacillus acidophilus. this research used odontoblast like cells derived from pulp tissue of human third molars. odontoblast like cells exposed to lactobacillus achidophilus were used as a model of proinflammatory cytokine signaling. this research examined the effects of propolis extracts on odontoblast like cells exposed to lactobacillus acidophilus. purpose: this research was aimed to determine the effectiveness of propolis extracts on the activities of odontoblast-like cells exposed to lactobacillus acidophillus by measuring the expressions of nfkb and tgfb1. methods: first, pulp odontoblast cultures were derived from human dental pulp tissues of impacted third molars removed by using digestion method. next, odontoblast-like cells exposed to inactive lactobacillus acidophilus bacteria were given propolis extract. finally, the activities of odontoblast-like cells were monitored by measuring the expressions of nf-kb and tgfb-1 with immunocytochemistry technique. results: a decline nf-kb expression and on increase of tgfb-1 expression on odontoblast like cells exposed to inactive lactobacillus acidophilus. conclusion: propolis extracts inhibit the expression of nf-kb, and increase the expression of tgf-b1 in pulp odontoblast-like cells exposed to inactive lactobacillus acidophillus. key words: propolis extracts, odontoblast-like cells, lactobacillus acidophillus, nf-kb, tgfb-1 abstrak latar belakang: propolis dilaporkan mempunyai efek menguntungkan yaitu bersifat anti bakteri, anti virus, anti inflamasi, anti oksidan, dan imunomodulator. ekstrak propolis dengan sifat anti inflamasi diharapkan bermanfaat untuk mengobati jaringan pulpa yang mengalami inflamasi dengan diagnosis pulpitis reversibel. inflamasi jaringan pulpa disebabkan oleh bakteri diantaranya adalah lactobacillus acidophilus. pada penelitian ini digunakan odontoblast like cells yang berasal dari jaringan pulpa dari gigi molar ke tiga manusia. odontoblast like cells dipapar lactobacillus acidophilus digunakan sebagai model signaling sitokin proinflamasi. studi ini, meneliti pengaruh pemberian ekstrak propolis pada odontoblast like cells yang dipajan lactobacillus acidophilus. tujuan: penelitian untuk mengetahui efektifitas ekstrak propolis terhadap aktifitas odontoblast like cells yang dipajan lactobacillus acidophillus dengan mengukur ekspresi nf-kb dan tgf-b1. metode: pembuatan kultur odontoblas pulpa berasal dari jaringan pulpa gigi molar ke tiga impaksi yang dicabut menggunakan metode digesti. odontoblast like cells dipajan bakteri lactobacillus acidophilus inaktif, diberi ekstrak propolis dan aktifitas dari odontoblast like cells diukur melalui ekspresi nf-kb dan tgfb-1 secara imunositokimia. hasil: terjadi penurunan ekspresi nf-kb, dan peningkatan ekspresi tgfb-1 pada kultur odontoblas yang dipapar bakteri lactobacillus �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 13–18 acidophilus inaktif. simpulan: ekstrak propolis menghambat ekspresi nf-kb, dan meningkatkan ekspresi tgf-b1 pada odontoblast like cells pulpa yang dipajan bakteri lactobacillus acidophillus inaktif. kata kunci: propolis extract, odontoblast like cells, lactobacillus acidophillus, nf-kb, tgfb-1 correspondence: ira widjiastuti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: irawidji@yahoo.com introduction in enamel and dentin caries will likely to stimulate, the innate immune response of dental pulp through the diffusion of bacterial products into the dentinal tubules. this excessive pathogen invasion will cause irreversible inflammation, destruct either immune response or pulp tissue necrosis.1 odontoblasts located in peripheral area of the dentine is the first cells received bacterial injury, and also can be considered as the first dental defense process.2 it is because odontoblasts have a role in dental immune response. this is supported by a research explaining that odontoblasts consistently produce both innate immunity components and adaptive immunity components,3,4 and they can also be induced to express cytokines and chemokin.3 in dentin caries, moreover, lactobacillus acidophilus as gram-positive bacteria with a virulence component, namely lipoteichoic acid (lta), are commonly found. as a result, odontoblasts with the presence of gram-positive bacteria will initiate, develop, maintain, and terminate the dental pulp immune response. detector components of gram-positive bacteria, including lipoteichoic acid (lta) and diacylated/triacylated lipopeptides primarily are produced through toll-like receptor 2 (tlr2). the involvement of odontoblast cells in vitro can make lta trigger tlr2 to stimulate the regulation and translocation of nf-kb producing proinflammatory of chemokines and cytokines. thus, this is a potential target to interfere cascade signal ultimately leading to the excessive inflammation of the pulp. actually, the bacteria can stimulate bioactive molecules, namely transformasi growth factor-beta (tgfb1) or bone morphogenic protein (bmp) that will induce dentin formation on the surface of the pulp tissue. however, the formation will be disrupted if there is inflammation of the pulp. several strategies should be conducted to inhibit both inflammation via tlr2 and pro-inflammatory of intracellular signal transduction and chemokine. consequently, a biocompatible material that can prevent or treat inflammation of the pulp tissue is needed. material that has been used is calcium hydroxide, but this material has disadvantages, such as causing both tunnel defects in dentin formation and necrosis of the pulp tissue surface. as an alternative, propolis can be chosen since it has antiinflammatory, anti-bacterial, anti-viral, anti-oxidant, and immunomodulatory properties. propolis is also contain a lot of resin and bioactive ingredients, such as bioflavonoids, artepilin, apigenin, and caffeic acid phenethil esther (cape) participating in body’s immune response to inflammatory, can be used as antioxidants, antibacteria, and antivirus and can be considered as immunomodulator, as well as can stimulate the healing process of tissue.5 thus, propolis extracts are expected to be useful as a pulp capping medicine used as pulp protection. pulp capping is used to protect the pulp by stimulating the formation of reparative dentin, and to maintain the vitality of the pulp tissue. therefore, this study was aimed to determine the effectiveness of propolis extracts on the activities of odontoblast-like cells due to lactobacillus acidophillus by measuring the expressions of nf-kb and tgf-b1. materials and methods all procedures performed in this research have been legalized with ethical clearance issued by ethics committee of the faculty of dentistry, universitas airlangga. the procedures consisted of culturing lactobacillus acidophilus, making pulp cell culture, and conducting immunocytochemistry examination by using monoclonal antibodies to determine the expressions of nf-kb and tgf-b1. pulp cell culture was made from dental pulp tissues of impacted third molars that was taken from patients aged 14-19 years. the teeth were disinfected by using 0.3% chlorhexidine gel put into 30% hydrogen peroxide for 30 to 120 seconds. the pulp was opened by conducting preparation using a sterile fissure bur and pulp cell cultures were made by using digestion method. differentiation of pulp fibroblast was conducted by doing supplementation of 10 nm dexamethasone, 50 mg/ml ascorbic acid and 10 mm-glycerophosphate (100200 ng/ml) on prolifresi medium (dmem + 10% fbs + penicillin/ streptomycin) to create odontoblast-like cells. during differentiation process, odontoblasts secreted specific matrix, ie dentin matrix protein 1 (dmp-1). the identification of dmp1 was conducted by using immunocytochemistry and anti-dmp1 (santacruz) based on immunostaining assay kit (biocare) instructions. then, characterization of odontoblast-like phenotype was conducted. ��widjiastuti, et al.,: the expressions of nf-kb and tgfb-1 on odontoblast-like cells figure 1. odontoblast culture (aec staining with magnification 400x). cells expressing nfkb (arrow) distributed in cell nucleus (red color). a) odontoblasts induced with inactive lactobacillus acidopilus; b) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 1,5µg/ml; c) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 3 µg/ml; d) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 6 µg/ml. tabel 1. the mean and standard deviation of nf-kb expressions in odontoblast culture by using anova test group x (%) sd anova control (-) 4.2a 2.39 f=31.751 control (+) 23.60b 3.84 p=000 1,5µg/ml of propolis 25.20c 4.14 3µg/ml of propolis 19c 3.16 6 µg/ml of propolis 12.40d 3.36 note: the different superscripts indicate that there was significant difference among the groups (p<0.005) table 2. the mean and standard deviation of tgf-b1 expressions in odontoblast culture by using anova test group x (%) sd anova control (-) 21.4a 5.86 f=10.731 control (+) 12.20b 3.27 p=0.001 1,5µg/ml of propolis 10.20b.c 2.38 3µg/ml of propolis 15.40a.b.c 3.64 6 µg/ml of propolis 23.4a 3.50 note: the different superscripts indicate that there was significant difference among the groups (p<0.005) figure 2. odontoblast culture (aec staining with magnification 200x). cells expressing tgf-b1 (arrow) distributed in the cytoplasm of odontoblasts (red color). a) odontoblasts induced with inactive lactobacillus acidopilus; b) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 1,5µg/ml; c) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 3µg/ml; d) odontoblasts induced with inactive lactobacillus acidopilus and propolis extracts 6 µg/ml. before odntoblast-like cells were induced with lactobacillus acidophilus, lactobacillus acidophilus had been inactivated by heat killed method. in this process, lactobacillus acidophilus was heated at 121o c for 5 minutes. the determination of an effective dose of bacterial exposure based a certain ratio of cells and bacteria, namely 1: 25, incubated for 24 hours (an incubator at 37° c).6 finally, after lactobacillus acidophilus was inactvated, propolis extracts derived from raw propolis produced by apis meliferra bees of lawang, east java, indonesia was taken. propolis extracts were made by using maceration method using 70% ethanol. results the expression of nf-kb was determined by using immunocytochemical examination. the result can be seen in table 1. immunocytochemistry examination was conducted to determine the number of nf-kb expressions by immunostaining method using antibody, i.e. anti-nfkb. the results showed that nf-kb expressions (red) were distributed in the cell nucleus as shown in figure 1. it is also known that the distribution of cells expressing nf-kb was decreased. next, tgf-b1 expressions in odontoblast cultured induced with inactive lactobacillus acidophilus were identified by using immunocytochemistry examination as seen in table 2 and figure 2. �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 13–18 discussion in this research, odontoblast like cell culture was induced with lactobacillus acidophilus inactive. the induction of inactive lactobacillus acidophilus into the odontoblast cultures has induced tlr2 receptors as transmembrane receptors, the receptors of gram-positive bacteria binding to lta, then has passed the transduction signal into cells, and later has induced nf-kb to become activated and go into the cell nucleus. as a result, the transcription of the target genes induces into tnf-α and tgf-b1. it means that odontoblasts as the main cells that form the peripheral layer of the pulp tissue in vitro have typical cellular morphology, and can be induced to express cytokines and chemokin.3 an in vitro research on odontoblasts exposed to lta binding tlr2 even shows that the increasing of the odontoblasts can activate the transcription factor of nf-kb, so it will diffuse from the cytoplasm to the nucleus and then secrete proinflamatory cytokines.7 thus, all of these conditions then potentially lead to the inflammation of the pulp. nf-kb is a transcription factor, which will diffuse into the nucleus, and then activate the transcription of various target genes. the activation of nf-kb protein caused by bacterial products can secrete a variety of cytokines, including proinflammatory cytokines that cause cell damage,8,9 leading to the damage of the odontoblast-like cells. next, phosphorylation of the serine residues usually occurs on the responsive signal (srr) of classical ikbs by leading to ikkb of ikb ubiquitination and proteosomal degradation secreted from nf-kb dimers, which then diffuses into the nucleus, induces gene target transcription,10,11 and secretes proinflammatory cytokines, such as tnf-α, il-1, il-6 and il-12.12 similarly, in this research, nf-kb expressions were increased after the odontoblast cultures were exposed with inactive lactobacillus acidophilus. it is because nfkb p65 as a part of nf-kb inhibitor with canonical line secreted proinflammatory cytokines and mediated signal transduction, or due to the induction of tnf-α, il-1, lps or lta and the use of a variety of adapters to signals involved ikk activities. human dentin contains tgf-b1, which has a dual role in the formation and repairing of dentin-pulp complexes. these cytokines also act as a regulator for the activation of immunocompetent cells, such as lymphocytes, macrophages and granulocytes, to control the initiation and resolution of inflammatory response.13 therefore, pulp capping treatment with tgf-b1 can increase and accelerate the synthesis of collagen type 1, as a result, mineralization occurs, then perforation is closed, and reparative dentinogenesis occurs.14 several strategies can be applied to heal pulp inflammation, including blocking or inhibiting the transduction of intracellular signal through tlr2 and pro-inflammatory cytokine/chemokine in odontoblasts. therefore, with a better understanding of molecular mechanisms in odontoblast-like cells exposed to the bacteria, the effective therapeutic components that modulate pulp cell can also be designed to contribute in healing and repairing processes through the formation of reparative dentin.7 the formation of reparative dentin is usually started with the binding of progenitor cells into the pulp cells differentiated into odontoblast-like cells and migrated to the injury area.1 tgf-b1 has a role in regulating cell cycle. tgf-b synthesizes proteins, p15 and p21, that inhibit cyclin-cdk complexes in g1 phase of the cell cycle. tgf-b then regulates the proliferation process, so differentiation process occurs.15 the propolis extracts were then chosen as a treatment for inflamed dental pulp tissue since it has anti-inflammatory properties. propolis extract can also be considered as a complex substance that has an anti-inflammatory factor.16 propolis extracts, furthermore, contain various ingredients depending on the type of bees and plants, but the main component contained in the extracts are bioactive, namely phenolics and flavonoids. the other components contained in propolis extract are anti-inflammatory, such as caffeic acid, quercetin, naringenin, and cape. 17 the main compositions of propolis extracts are phenolic acids and esters, flavonoids (flavones, flavonones, flavonols, dihydroflavonols, chalcones), terpenes, b steroids, aromatic aldehydes, alcohols, sesquiterpenes, naphthalene, stilbene derivatives of benzopyran, benzophenone, caffeic acid, cinnamic acid derivatives and benzoic acid.17 for these reasons, in this research propolis extracts were used together with ethanol solvent containing polyphenols, proleinetin, flavonoids, tertepenoid, galangin, quercetin, minecetin, oligotinperginetin, nikobaleen a and b as well as cape. anti-inflammatory activities of propolis seem to be associated with flavonoid, especially galangin and quercetin. flavonoids inhibit the activities of cyclooxygenase and lipoxygenase, as well as reduce and release of pge2 and cox-2 isoform (cox-2) expressions.18 cape can also be considered as an active component in propolis that inhibit the production of cytokines and chemokines, the proliferation of t cells and the production of lymphokine resulting in the decreasing of inflammatory process. its mechanism is through signaling pathways of nf-kb.19 it means cape is a potent inhibitor of nf-kb.20 ansorge et al.22 studied the effects and functions of some of the components of propolis that can activate the immunity in human blood cells, synthesize dna and cytokine production in vitro by detecting the production of il-1ß and il-12 by macrophages, as well as produce il-2, il-4 , il-10 and tgf-b growth factor. propolis contains flavonoids and caffeic acid that have anti-inflammatory properties by inhibiting lipoksiginase line by arakidonik acid. it is also known that propolis affects immune system by stimulating both the activities of phagocytosis and cellular immunity and the formation of collagen, which will affect dentin bridge formation. propolis also contains compounds arginine, vitamin c, provitamina, b complex, trace minerals and bioflavonoids as well as antibacterial properties, which can accelerate healing process.22,23 ��widjiastuti, et al.,: the expressions of nf-kb and tgfb-1 on odontoblast-like cells propolis extracts can suppress the expressions of proinflammatory cytokines better than quercetin, hesperidin, and cape since the propolis extracts have a synergistic effect in inhibiting proinflammatory cytokines.21 like the previous result, the results of this research also showed that the expressions of nf-kb in the odontoblast cultures induced with inactive lactobacillus acidophilus and exposed to the propolis extracts were decreased, meanwhile the expressions of tgf-b1 were increased. it means that the change of propolis extracts also altered the expressions of nf-kb and tgf-b1. in other words, the induction of the propolis extracts inhibits the activations of nf-kb and tnf-α and also induce the secretion of tgf-b1.6 like previous researches, this research also shows that propolis extracts could inhibit nf-kbp65 expressions on odontoblast culture induced with inactive lactobacillus acidophilus. this is supported by the results of several previous researches that the active ingredient, namely cape, contained in propolis can significantly inhibit the constitutive expressions of cox-2. in other words, cape is a potent and specific inhibitor that inhibits the activation of nf-kb. histopathological examination conducted in this research also showed that cape significantly suppressed inflammation. caffeic acid phenethil esther specifically blocked the activation of nf-kb caused by various inflammatory agents, including tnf-α and h2o2, inflammatory cytokines (il-1, tnf-α), bacterial products, and oxidative stress. caffeic acid phenethil esther, furthermore, does not only inhibit transcription factors, but also reduces the production of il-8 and chemotactic monosit proteins.9 thus, propolis containing cape can inhibit phosphorylation of ikbα and activation of nf-kb, but not through phosphorylation of mitogen-activated protein kinase (mapk) in human monocyte-derived dendritic cells (modcs).24 the results of this research were also supported by the results of a research conducted by aviello et al.25 showing that caffeic acid, quercetin, hesperidin and flavonoids contained in propolis can inhibit dna synthesis and inflammatory cytokine production depended on the concentration of propolis. but, the production of tgf-b, a mediator of immunosuppression, was increased. these findings indicate that certain components contained in the propolis extracts could give direct effects on immune cell function settings, and could also be used as alternative natural ingredients that have anti-inflammatory effects. similarly, a research on the biological activity of propolis shows that cape and artepillin c can be isolated from propolis, which can potentially be used as medicine. in this research, it is known that the higher the dosage of propolis extracts, the more nf-kb expressions will be inhibited and the more tgf-b expression of will be increased. this indicates that the propolis extracts can stimulate odontoblasts in the dental pulp to secrete tgfb1, which can stimulate proliferation and differentiation. propolis has an ability to stimulate tgf-b1 considered as an important factor in the differentiation of odontoblasts in human dental pulp.22 based on the results of the research, it can be concluded that the propolis extracts not only can inhibit nf-kb expressions, but can also increase the expression of tgf-b1 in odontoblast culture induced with inactive lactobacillus acidophillus. references 1. smith aj. vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. j dent educ 2003; 67(6): 678-89. 2. mitsiadis ta, rahiotis c. parallels between tooth development and repair: conserved molecular mechanisms following carious and dental injury. j dent res 2004; 83(12): 896-902. 3. veerayutthwilai o, byers mr, pham tt, darveau rp, dale ba. differential regulation of immune responses by odontoblasts. oral microbiol immunol 2007; 22(1): 5-13. 4. dommisch h, winter j, acil y, dunsche a, tiemann m, jepsen s. human betadefensin (hbd-1, -2) expression in dental pulp. oral microbiol immunol 2005; 20: 163–6. 5. katircioglu h, mercan n. antimicrobial activity and chemical compositions of turkish propolis from different regions. afr j biotec 2006; 5: 1151-3. 6. widjiastuti i. mekanisme molekuler stimulasi ekstrak propolis pada odontoblast like cells yang dipapar lactobacillus acidophilus inaktif dalam menginduksi diferensiasi fibroblas jaringan pulpa. disertasi. surabaya: universitas airlangga; 2012. 7. farges jc. understanding dental pulp innate immunity-a basis for identifying new targets for therapeutic agents that dampen inflammation. j appl oral sci 2009; 17(3): s1678. 8. alliot lb, hurtrel d, gregoire m. characterization of a-smooth muscle actin positive cells in mineralized human dental pulp cultures. arch oral biol 2001; 46(3): 221-8. 9. lee kw, chun ks, lee js, kang ks, surh yj, lee hj. inhibition of cyclooxygenase-2 expression and restoration of gap junction intercellular communication in h-ras-transformed rat liver epithelial cells by caffeic acid phenethyl ester. ann ny acad sci 2004; 1030: 501-7. 10. wong et, tergaonkar v. roles of nf-kb in health and disease: mechanisms and therapeutic potential. clin sci (lond) 2009; 116(6): 451-65. 11. hayden ms, west ap, ghosh s. nf-kb and the immune response. oncogene 2006; 25(51): 6758-80. 12. oeckinghaus a, ghosh s. the nf-kb family of transcription factors and oral bacteria. j dent res 2009; 88: 333-8. 13. li ll, wang zy, bai zc, mao y, gao b, xin ht, zhou b, zhang y, liu b. three dimensional finite element analysis of weakened roots restored with different cements in combination with titanium alloy posts. chin med j (engl) 2006; 119(4): 305-11. 14. kunarti s. stimulasi aktivitas fibroblas pulpa dengan pemberian tgf-b1 sebagai bahan perawatan direct pulp caping. disertasi. surabaya: universitas airlangga; 2005. 15. buchaille r, couble ml, magloire h, bleicher f. a substractive pcrbased cdna library from human odontoblast cells: identification of novel genes expressed in tooth forming cells. matrix biology j int society for matrix biology 2000; 27: 19-23. 16. krol w, scheller s, shani j, pietsz g, czuba z. synergistic effect of ethanolic extract of propolis and antibiotics on the growth of staphylococcus aureus. arzneimittelforschung. 1993; 43(5): 6079. 17. mirzoeva ok, calder pc. the effect of propolis and its components on eicosanoid production during the inf lammatory response. prostaglandins leukot essent fatty acids 1996; 55(6): 441-9. 18. shimoi k, saka n, kaji k, nozawa r. metabolic fate of luteolin and its functional activity on focal site. biofactors 2000; 12(1-4): 181-6. 19. wang l, lin y, liang y, yang y, lee j, yu h, wu w, chiang b. the effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells. bmc immunol 2009; 10: 39. �8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 13–18 20. raso gm, meli r, di carlo g, pacilio m, di carlo r. inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage j774a.1. life sci 2001; 68(8): 921-31. 21. schva rzbeyn j, huleihel m. effect of propolis a nd caffeic acid phenethyl ester (cape) on nfκb activation by htlv-1 tax. antiviral res 2011; 90(3): 108-15. 22. ansorgen ar, einhold d, lendckel u. propolis and some of its constituents and inflammatory cytokine production, but induce tgf-b1 production of human immune cells. z naturforsch c 2003; 58(7-8): 580-9. 23. park yk, alencar sm, aguiar, cl. botanical origin and chemical composition of brazilian propolis. journal of agricultural and food chemistry 2002; 50: 2502-6. 24. castro ml, nascimento am, ikegaki m, costa-neto cm, alencar sm, rosalen pl. identification of a bioactive compound isolated from brazilian propolis type 6. bioorg med chem 2009; 17(14): 5332-5. 25. aveillo g, scalisi c, fillecia r, capusso r, romano g, 1220 aa, borelli f. inhibitory effect of caffeic acid phenethyl ester, a plantderived polyphenolic compound, on rat intestinal contractility. eur j pharmacol 2010, 640 (1-3): 163-7. 8181 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 81–85 research report t h e a c t i v i t y o f p o l y c l o n a l i g y d e r i v e d f r o m a g g r e g a t i b a c t e r actinomycetemcomitans and porphyromonas gingivalis in inhibiting colonization of fusobacterium nucleatum and streptococcus sanguinis oktaviani suci lestari, rini devijanti ridwan, tuti kusumaningsih, and s. sidarningsih department of oral biology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: fusobacterium nucleatum (f. nucleatum) and streptococcus sanguinis (s. sanguinis) play a role in dental plaque formation which leads to periodontitis. immunoglobulin y (igy) is present in both serum and egg yolk and can bind to the surface components of bacteria. f. nucleatum and s. sanguinis feature the same type of iv pili as aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans). saliva binding protein (ssab) in s. sanguinis is a fima homolog. fima constitutes a surface element of porphyromonas gingivalis (p. gingivalis). f. nucleatum and p. gingivalis possess the same outer membrane protein (omp) molecular mass. purpose: the study aimed to determine the activity of a. actinomycetemcomitans and p. gingivalis polyclonal igy present in serum and egg yolk that can inhibit colonization of f. nucleatum and s. sanguinis. methods: igy samples were diluted with phosphate buffer saline (pbs). several holes were made in the nutrient medium with 10 μl antigen (f. nucleatum/s. sanguinis) being inserted into the center hole. 10 μl pbs, 1:1, 1:2, 1:4, 1:8, 1:16 a. actinomycetemcomitans or p. gingivalis polyclonal igy were subsequently introduced into the surrounding holes. the results of incubation at 37°c were observed after 24-48 hours. kruskal wallis and mannwhitney tests were administered to analyse the data. results: a. actinomycetemcomitans and p. gingivalis polyclonal igy groups in serum showed a precipitation line at dilution ratios of 1:1 and 1:2, whereas in egg yolk this occurred only at a 1:1 dilution ratio with f. nucleatum and s. sanguinis bacteria in this study. no significant differences were evident between each dilution (p>0.05) and none existed between serum and egg yolk (p>0.05). conclusion: igy polyclonal of a. actinomycetemcomitans and p. gingivalis in both serum and egg yolk initiate activities that can inhibit colonization of f. nucleatum and s. sanguinis. keywords: aggregatibacter actinomycetemcomitans; fusobacterium nucleatum; igy; porphyromonas gingivalis; streptococcus sanguinis correspondence: rini devijanti ridwan, department of oral biology, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: rini-d-r@fkg.unair.ac.id introduction gingivitis generally occurs between 10 and 21 days after dental plaque formation under poor and untreated oral hygiene conditions and can develop into periodontitis.1 the latter condition results from ecological imbalances between microbial communities in dental biofilms that support the growth of streptococcus pathogenic bacteria.2 streptococcus sanguinis (s. sanguinis) constitutes a pioneer bacterium on the tooth surface which plays an important role in plaque maturation due to its ability to aggregate with other bacteria resulting in periodontal disease.3 the initiation and development of periodontitis is caused by aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans), porphyromonas gingivalis (p. gingivalis), tannerella forsythia (t. forsythia), treponema denticola (t. denticola), prevotella intermedia (p. intermedia) and fusobacterium nucleatum (f. nucleatum) bacteria.4 inhibition of plaque matrix formation and initiation of bacterial aggregation can prevent initial colonization with the result that final dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p81-85 82 lestari, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 81–85 colonization does not occur.5 f. nucleatum and s. sanguinis bacteria promote the formation of dental plaque that can cause periodontitis. over the past two decades, dentists have applied antibiotic therapy to periodontal disease treatment.6 in addition, mouthwash, the main component of which is chlorhexidine, can prevent plaque formation.7 to reduce antibiotic resistance and the side effects of chlorhexidine gluconate mouthwash such as taste disorders, oral irritation and local allergy symptoms, an unprecedented therapeutic approach is required. one potential strategy is to explore passive oral immunotherapy using immunoglobulin y (igy) as a control method in inhibiting dental plaque.7,8 igy is one class of antibody contained in the blood serum and egg yolk of amphibian, reptilian and poultry groups.9 igy constitutes a polyclonal antibody that has been shown to be effective in the prevention and treatment of several diseases.10 moreover, its low cost and simple production process renders this antibody suitable for research and diagnosis.11 igy can inhibit the attachment of bacteria to host cells12 since igy antibacterium binds to certain components on the target bacteria surface such as outer membrane protein (omp), lipopolysaccharide (lps), flagella and fimbriae.13 it has been reported that a. actinomycetemcomitans contains type iv pili.14 type iv pili are also expressed by other gram-positive pathogens such as clostridium perfringens (c. perfringens) and s. sanguinis.15 phenotyping screening also confirms type iv pili to be present on the cell surface of f. nucleatum.16 p. gingivalis pili contains fima.17 s. sanguinis bacteria carry ssab, a fima homolog, on their surface. bacterial adhesion molecules play an important role in the adhesion between bacteria and host cell.18 p. gingivalis and f. nucleatum have an outer membrane protein with a molecular mass of 40-kda.19 against this background, the research aim was to determine whether igy anti a. actinomycetemcomitans and p. gingivalis can be employed to inhibit colonization of f. nucleatum and s. sanguinis bacteria. materials and methods the type of research employed an experimental laboratory methodology. the samples analyzed comprised polyclonal igy anti-a. actinomycetemcomitans and polyclonal igy anti-p. gingivalis in serum and egg yolk. more than three replications were produced, their number being determined by the federer formula. the polyclonal igy contained in the serum and egg yolk produced by hens previously injected four times (booster) with a. actinomycetemcomitans serotype b strain y4 atcc 4371 and p. gingivalis atcc 3327 was subsequently analyzed using elisa.11 the research method employed was granted ethical clearance (certificate number: 294/hrecc.fodm/xi/2018) by the faculty of dental medicine, universitas airlangga. serum was diluted with phosphate buffer saline (pbs) at a ratio of 1:16. each vial/bottle contained 20 μl of assay buffer. the contents of the first vial were mixed with 20 μl serum samples at a dilution level of 1:1. 20 μl of the 1:1 sample dilution were subsequently transferred to a second vial where they were further diluted at a ratio of 1:2. 20 μl of the sample were transferred to a third vial where the dilution rate was one of 1:4. 20 μl of the sample was transferred to a fourth vial at a dilution rate of 1:8. 20 μl of the sample were transferred to the fifth vial and diluted at a rate of 1:16. this procedure was repeated for the igy samples in egg yolk.20 the nutrient medium solution was cooled to 50-60℃, placed in 15 ml petri dishes on a horizontal surface and figure 1. the precipitation line of a. actinomycetemcomitans polyclonal igy groups in serum and egg yolk in f. nucleatum and s. sanguinis. (s/s: s. sanguinis/ igy in serum; s/t: s. sanguinis/igy in egg yolk; f/s: f. nucletum/igy in serum; f/t: f. nucleatum/igy in egg yolk). figure 2. result diagrams of the a. actinomycetemcomitans polyclonal igy group in bacteria: a) f. nucleatum; b) s. sanguinis; a) serum preparation; b) egg yolk preparation. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p81-85 83lestari, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 81–85 table 1. data analysis of a. actinomycetemcomitans polyclonal igy group and f. nucleatum antigen aa1 (1:1) aa2 (1:2) aa3 (1:4) aa4 (1:8) aa5 (1:16) aa6 (pbs) p value f/s + + 0.051 + + f/t + 0.051 + table 2. data analysis of a. actinomycetemcomitans polyclonal igy group and s. sanguinis antigen aa1 (1:1) aa2 (1:2) aa3 (1:4) aa4 (1:8) aa5 (1:16) aa6 (pbs) p value s/s + + 0.051+ + s/t + 0.051 + note: +: there is a precipitation line; -: there is no precipitation line; *: significance (p value <0.05) table 3. comparison of the results of a.actinomycetemcomitans polyclonal igy group in the serum and egg yolk and f.nucleatum and s.sanguinis antigen p value f/s f/t 0.514 s/s s/t 0.514 note: *significance (p value <0.05) table 4. data analysis of p. gingivalis polyclonal igy and f. nucleatum antigen pg1 (1:1) pg2 (1:2) pg3 (1:4) pg4 (1:8) pg5 (1:16) pg6 (pbs) p value f/s + + 0.051 + + f/t + 0.051 + table 5. data analysis of p. gingivalis polyclonal igy and s. sanguinis antigen pg1 (1:1) pg2 (1:2) pg3 (1:4) pg4 (1:8) pg5 (1:16) pg6 (pbs) p value s/s + + 0.051 + + s/t + 0.051 + note: +: there is a precipitation line; -: there is no precipitation line; *: significance (p value <0.05) table 6. comparison of the result of igy p.gingivalis group in the serum and egg yolk and f.nucleatum and s.sanguinis antigen p value f/s f/t 0.514 s/s s/t 0.514 note: *: significance (p value <0.05) left to stand for 30 minutes. the nutrient medium was perforated inappropriate sites in the marked section in order to accommodate this sample. 10 μl of the pbs and serum samples at dilutions of 1:1, 1:2, 1:4, 1:8, 1:16 were deposited in each of the holes on the outer rim of the medium. 10 μl of the antigen (f.nucleatum/s.sanguinis) were inserted into the center hole of each petri dish and incubated at 37℃ for 24 hours. after incubation, the line of precipitation between the antigen hole and the igy serum holes was observed. this procedure was repeated for the igy samples in egg yolk.20,21 results this study confirmed inhibition of the colonization of f. nucleatum and s. sanguinis bacteria in the a. actinomycetemcomitans polyclonal igy group present in serum and egg yolk (figure 1). based on these results, the igy anti-a. actinomycetemcomitan group in the serum showed a precipitation line against f. nucleatum and s. sanguinis at 1:1 and 1:2 dilutions of the treatment groups. in contrast, the igy anti-a. actinomycetemcomitan group in the egg yolk showed the presence of precipitation lines dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p81-85 84 lestari, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 81–85 against f. nucleatum and s. sanguinis during 1:1 dilution treatment. data analysis was performed with a kruskal wallis test, the results of which are contained in tables 1 and 2, and a mann-whitney test whose results are shown in table 3. based on the contents of tables 1 and 2, it is evident that none of the f/s, f/t, s/s and s/t groups showed significant value because the p value equaled 0.051 which was greater than 0.05 (p> 0.05). this figure indicated that the concentration had no effect on the results. the data in table 3 indicates that the value of f/s with f/t was 0.514, while that of s/s with s/t was 0.514. these results, arrived at through statistical calculation, indicated no difference between the polyclonal igy group of a. actinomycetemcomitans in serum and egg yolk and f. nucleatum and s. sanguinis. they were considered to be insignificant with a p value >0.05. this study confirmed colonization inhibition of f. nucleatum and s. sanguinis bacteria of the igy anti-p. gingivalis group in serum and egg yolk which can be characterized by the precipitation line shown in figure 3. these results of the igy anti p. gingivalis group in serum showed a precipitation line for the f. nucleatum and s. sanguinis bacteria occurring at 1:1 and 1:2 dilutions in the treatment groups. by contrast, in the p. gingivalis polyclonal igy group, egg yolk showed the presence of precipitation lines in the f. nucleatum and s. sanguinis bacteria in the 1:1 dilution treatment group. data analysis was performed by means of a kruskal wallis test, the results of which are shown in table 4 and 5, and a mann-whitney test whose results appear in table 6. based on contents of tables 4 and 5, it is known that none of the f/s, f/t, s/s and s/t group showed significant values because the p value equaled 0.051, indicating that it was higher than 0.05 (p>0.05). this indicated that concentration had no effect on the results. the contents of table 6 show that the value of f/s with f/t was 0.514, figure 3. the precipitation line of igy anti p. gingivalis group in the serum and egg yolk in f. nucleatum and s. sanguinis. (s/s: s. sanguinis/igy in serum; s/t: s. sanguinis/igy in egg yolk; f/s: f. nucletum/igy in serum: f. nucleatum/igy in egg yolk). figure 4. result diagrams of igy anti p. gingivalis group in f. nucleatum and s. sanguinis; a) serum preparation; b) egg yolks preparation. while that of s/s with s/t was 0.514. these statistical calculation-based results showed no difference between the polyclonal igy group of p. gingivalis in serum and egg yolk in the f. nucleatum and s.sanguinis bacteria due to insignificant results since the p value >0.05. discussion this study used igy anti-a. actinomycetemcomitans and igy anti-p. gingivalis in serum and egg yolk to be tested against two other bacteria, i.e. f. nucleatum and s. sanguinis, using a double immunodiffusion method. a. actinomycetemcomitans polyclonal igy and p. gingivalis polyclonal igy were obtained from chickens previously immunized with specific antigens i.e. a. actinomycetemcomitans or p. gingivalis prior to serum being taken. testing of the two polyclonal igy was conducted using a double immunodifusion method. the results of the data from the igy antia. actinomycetemcomitans group for serum in the f. nucleatum and s. sanguinis bacteria, found a formation precipitation line at 1:1 and 1:2 dilutions. in contrast, for the igy anti-a. actinomycetemcomitans group in egg yolk formation of a precipitation line occurred at 1:1 dilution in the f. nucleatum and s. sanguinis bacteria. similar results were found in the igy anti p. gingivalis group. the results of the igy anti-p. gingivalis group in serum for f. nucleatum and s. sanguinis bacteria found the precipitation line at 1:1 and 1:2 dilutions. contrastingly, in the igy anti-p. gingivalis group in egg yolk, a precipitation line formed at 1:1 dilution in f. nucleatum and s. sanguinis bacteria. these results show that precipitation lines are formed at low dilutions and that the concentration of antibodies in dilution is higher than that of other dilution groups, meaning that the group with low dilution levels is of an appropriate concentration to be able to interact with dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p81-85 85lestari, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 81–85 the antigen. excessively low antibody concentration will produce negative results22 similar to those of previous research conducted by sharon et al.20 in which the precipitation line occurred at 1:2 dilution. the precipitation lines formed between antigens and antibodies, both in the igy anti-a. actinomycetemcomitans group in serum and egg yolk for f. nucleatum and s. sanguinis bacteria, and igy anti-p. gingivalis group in serum and egg yolk for f. nucleatum and s. sanguinis bacteria, indicated some form of activity between the igy anti-a. actinomycetemcomitans with f. nucleatum and s. sanguinis, as well as between the igy anti-p. gingivalis with f. nucleatum and s. sanguinis. the activity took the form of binding between antibodies and antigens, possibly because igy is a polyclonal antibody that can bind to various epitope antigens. igy anti-a. actinomycetemcomitans can bind to pili from bacteria, such as f. nucleatum and s. sanguinis, because all have type iv pili. polyclonal igy p. gingivalis can also bind to the homologous element found on the surface of s. sanguinis, namely ssab, and can bind to omp f. nucleatum because it possesses the same molecular mass. this binding can inhibit the colonization of f. nucleatum and s. sanguinis bacteria due to the disruption of the function of the surface components of these bacteria which can be useful for their adhesion. previous research conducted by lee et al.23 showed that salmonella specific igy binds to salmonella surface molecules with the result that it can inhibit homologous salmonella growth. igy can affect colonization of salmonella enteritidis and salmonella typhimurium by binding to omp. omp salmonella is useful for adhesion and mucosal invasion. the binding causes disruption of omp biological function, with the result that invasive salmonella is reduced due to the loss of ability to colonize the digestive tract.24 the results of the statistical analysis indicated no difference between the inhibitory colonization in the polyclonal igy a. actinomycetemcomitans dilution group and the polyclonal igy p. gingivalis group. this is consistent with the theory that this test is less sensitive because the formation of precipitation lines depends on the equivalent concentration of antigen antibodies and specific antibodies.25 it can be concluded that the activities of a. actinomycetemcomitans and p. gingivalis polyclonal igy in serum and egg yolk can inhibit colonization of f. nucleatum and s. sanguinis. references 1. ha n k-j, sh i n s c. a cl i n ica l st udy on t he i n f luence of immunoglobulin y-containing chewing gum on the periodontium. int j clin prev dent. 2016; 12(1): 37–44. 2. xu y, selerio-poely t, ye x. clinical and microbiological effects of egg yolk antibody against porphyromonas gingivalis as an adjunct in the treatment of moderate to severe chronic periodontitis: a randomized placebo-controlled clinical trial. j periodontal implant sci. 2018; 48(1): 47–59. 3. safitri ci, soeroso y, sunarto h, pontoh ds, bachtiar b. association of salivary count of streptococcus sanguinis with the periodontal status of coronary heart disease patients: a quantitative study. j int dent med res. 2017; 10(special issue): 782–8. 4. stingu c-s, eschrich k, rodloff ac, schaumann r, jentsch h. periodontitis is associated with a loss of colonization by streptococcus sanguinis. j med microbiol. 2008; 57(4): 495–9. 5. hayati m, herman h, rezano a. the effect of probiotic lactobacillus casei supplementation on the secretory immunoglobulin a level in the saliva of wistar rats. bali med j. 2018; 7(3): 727–31. 6. kapoor a, malhotra r, grover v, grover d. systemic antibiotic therapy in periodontics. dent res j (isfahan). 2012; 9(5): 505–15. 7. kumar sb. chlorhexidine mouthwasha review. j pharm sci res. 2017; 9(9): 1450–2. 8. rahman s, van nguyen s, icatlo jr. fc, umeda k, kodama y. oral passive igy-based immunotherapeutics. hum vaccin immunother. 2013; 9(5): 1039–48. 9. cahyaningsih t. aplikasi igy spesifik staphylococcus aureus sebagai pencegahan staphylococcosis pada kelinci. thesis. bogor: institut pertanian bogor; 2016. p. 19. 10. zeynalian h, kazerouni f, ebrahimi f, rahimipour a, bakhshi m, samadi r. assessment of the safety of chicken egg yolk antibody (igy) consumption by measuring the activity of antioxidant enzymes (superoxide dismutase‚ catalase‚ glutathione peroxidase) and malondialdehyde concentration as a lipid peroxidation marker in mice. j paramed sci. 2017; 8(3): 29–35. 11. amro wa, al-qaisi w, al-razem f. production and purification of igy antibodies from chicken egg yolk. j genet eng biotechnol. 2018; 16(1): 99–103. 12. barati b, ebrahimi f, nazarian s. egg yolk antibodies for disease prevention. j bacteriol mycol. 2016; 3(2): 219–22. 13. chalghoumi r, beckers y, portetelle d, théwis a. hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infections in chicken: a review. biotechnol agron soc env. 2009; 13(2): 295–308. 14. kinane df, mombelli a. periodontal disease. basel: karger; 2012. p. 12. 15. kline ka, fälker s, dahlberg s, normark s, henriques-normark b. bacterial adhesins in host-microbe interactions. cell host microbe. 2009; 5(6): 580–92. 16. zijnge v, ammann t, thurnheer t, gmür r. subgingival biofilm structure. front oral biol. 2011; 15: 1–16. 17. hospenthal mk, costa t, waksman g. pilus biogenesis at the inner and outer membranes of gram-negative bacteria. nature reviews microbiology. 2017. 18. interpro. adhesion lipoprotein (ipr006128). 2018. available from: https://www.ebi.ac.uk/interpro/entry/ipr006128. accessed 2018 sep 25. 19. namikoshi j, maeba s, yamamoto m, hayakawa m, abiko y, otake s. nasal immunization with p. gingivalis surface protein antigen and cholera toxin adjuvant induces t helper 2 responses in both mucosal and systemic compartments. int j oral-medical sci. 2003; 1(2): 90–6. 20. sha r on i , g om ez l a , va n i c , m ich a el a. i solat ion a nd characterization of butanol-tolerant staphylococcus aureus using japanese quail. int j life sci pharma res. 2016; 6(4): 45–52. 21. himedia laboratories. hiper® ouchterlony double diffusion teaching kit (antibody titration). mumbai: himedia; 2012. p. 1–6. 22. thermo scientific. immunodiffusion plates. rockford: thermo fisher scientific inc.; 2011. p. 1–3. 23. lee en, sunwoo hh, menninen k, sim js. in vitro studies of chicken egg yolk antibody (igy) against salmonella enteritidis and salmonella typhimurium. poult sci. 2002; 81(5): 632–41. 24. naidu as. natural food antimicrobial systems. florida: crc press; 2000. p. 235. 25. salazar lf, jayasinghe u. double diffusion test in gels (ouchterlony). in: techniques in plant virology. lima, peru: international potato center; 2018. p. 1–3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p81–85 https://www.ebi.ac.uk/interpro/entry/ipr006128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p81-85 vol 52 no 1 jan-mar 2019_new.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 52, number 1, march 2019 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2019 january 2nd – december 31st, 2019 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (department of dental health techniques, faculty of vocational studies, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); sri oktawati (department of periodontic, faculty of dentistry, universitas hasanuddin, indonesia); irna sufiawati (department of oral medicine, faculty of dentistry, universitas sumatera utara, indonesia); m. rubianto (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontology, faculty of dental medicine, universitas airlangga, indonesia); eha renwi astuti (department of dental radiology, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); gusti aju wahju ardani (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); titien hary agustantina (department of dental material science and technology, faculty of dental medicine, universitas airlangga, indonesia); tania saskianti (department of pediatric dentistry, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 52, number 1, march 2019 p-issn: 1978-3728 e-issn: 2442-9740 1. evaluation of orthodontic tooth movement by 3d micro-computed tomography (μ-ct) following caffeine administration herniyati, happy harmono, leliana sandra devi, and sri hernawati ...................................... 1–7 2. the treatment of gingival recession with coronally advanced flap with platelet-rich fibrin asti rosmala dewi, agus susanto, and yanti rusyanti ............................................................... 8–12 3. effects of hydroxyapatite gypsum puger scaffold applied to rat alveolar bone sockets on osteoclasts, osteoblasts and the trabecular bone area amiyatun naini, i ketut sudiana, moh. rubianto, utari kresnoadi, and faurier dzar eljabbar latief ................................................................................................... 13–17 4. musculoskeletal disorder risk level evaluation of posterior maxillary tooth extraction procedures anggy prayudha, roberto m. simandjuntak, and ni putu mira sumarta ................................ 18–23 5. the effect of udma and bis-gma irradiation period on residual monomers in resin packable composite jayanti rosha, adioro soetojo, putu dewi purnama s.b, and m. mudjiono ............................. 24–26 6. the difference between porcelain and composite resin shear bond strength in the administration of 4% and 19.81% silane ira widjiastuti, dwina rahmawati junaedi, and ruslan effendy .............................................. 27–31 7. the different symptoms determining management of hand foot and mouth disease and primary varicella zoster infection maharani laillyza apriasari .......................................................................................................... 32–35 8. comparative in vitro study of the cytotoxicity of gelatine and alginate to human umbilical cord mesenchymal stem cells nike hendrijantini ........................................................................................................................... 36–40 9. the difference in microleakage levels of nanohybrid composite resin using eighth-generation ethanol and isopropanol solvent bonding materials under moist and dry conditions (in vitro study) irfan dwiandhono, setiadi w. logamarta, and taura dhanurdara .......................................... 41–44 10. the role of kuniran (u. moluccensis) and gurami (o. goramy) fish thorns and scales in increasing salivary leukocyte and monocyte cells viability against streptococcus mutans i dewa ayu ratna dewanti, i dewa ayu susilawati, p. purwanto, pujiana endah lestari, roedy budirahardjo, dyah setyorini, ristya widi endah yani, erawati wulandari, and melok aris wahyukundari ...................................................................................................... 45–50 11. protection against periodontal destruction in diabetic condition with sardinella longiceps fish oil: expression of matrix-metalloproteinase 8 and tissue inhibitor of metalloproteinase 1 dian widya damaiyanti, dian mulawarmanti, and kristanti parisihni .................................... 51–56 152 volume 46, number 3, september 2013 the effect of chitosan gel concentration on neutrophyl and macrophage in gingival ulcer of sprague dawley rat tasya adistya,1 fajar kumalasari,1 anne handrini dewi2 and mayu winnie rachmawati2 1faculty of dentistry, universitas gadjah mada 2departement of biomedical dentistry, faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract bacground: chitosan is polysacharide that extracted from crustaceae, widely used as a wound healing agent. it accelerates the polimorphonuclear cells infitration and increase the macrophage migration. purpose: the aims of this study was to determine the effect of chitosan gel concentration on neutrophyl and macrophage in gingival ulcer healing process of sprague dawley rats. methods: twenty subjects were divided into treatment groups, a, b and c which was given 1%, 2% and 3% chitosan gel respectively and group d as control group. the ulcer was made by applicating the 2 x 2 mm2 whatmann number 1 filter paper which had been soaked into the 98% acetic acid for 5 minutes on the gingival surface below the interdental of the lower incisivus of the rats for 40 seconds. one drop chitosan gel was applicated on the ulcer, twice a day for three days. the subjects were sacrificed and its gingival tissue was taken for histologically processed and stained with hematoxylin eosin. results: the one way anova test showed that significant difference neutrophyl and macrophage among all of group (p<0.05). the pearson correlation showed that there was a strong correlation (0,979) between chitosan concentration and macrophage density. chitosan gel with 1%, 2%, and 3% concentration influenced significantly to neutrophyl and macrophage density. the higher concentration of chitosan gel the power of neutrophyl number and the higher of macrophage number. conclusion: these result indicated that the chitosan gel influence both of neutrofil and macrophage in gingival ulcer healing process and chitosan gel 3% has a better effect than 1% and 2% concentration. key words: chitosan gel, wound healing, gingival ulcer, neutrophyl, macrophage abstrak latar belakang: kitosan, polisakarida hasil ekstraksi dari golongan krustasea, dikenal sebagai agen pemacu penyembuhan luka. kitosan dapat memacu infiltrasi sel-sel polimorfonuklear dan mempercepat migrasi sel makrofag. tujuan: penelitian ini bertujuan untuk meneliti efek gel kitosan dalam meningkatkan jumlah neutrofil dan makrofag selama proses penyembuhan luka buatan pada gingiva mulut tikus sprague dawley. metode: duapuluh subyek dibagi atas 4 grup. grup a dioles dengan 1% gel kitosan, grup b 2% dan grup c 3%, sedangkan grup d sebagai kontrol tidak mendapatkan perlakuan apapun. ulkus dibuat dengan cara mengaplikasikan kertas saring whatmann nomer 1 ukuran 2 x 2 mm2 yang telah dibasahi dengan 98% asam asetat selama 5 menit dan diletakkan pada gingiva di bawah interdental gigi anterior mandibula selama 40 detik. satu tetes gel kitosan diaplikasikan 2 kali sehari selama 3 hari. tikus dikorbankan pada hari ketiga dan jaringan gingivanya diambil untuk dibuat preparat histologi dengan pewarnaan he. jumlah neutrofil dan makrofag dianalisa dengan anova satu jalur. hasil: anova satu jalur menunjukkan adanya perbedaan bermakna pada neutrofil maupun makrofag antar grup (p<0,05). korelasi pearson menunjukkan ada hubungan positif antara konsentrasi gel kitosan dengan jumlah makrofag. gel kitosan dengan konsentrasi 1, 2 dan 3% secara bermakna mampu menurunkan jumlah neutrofil dan meningkatkan jumlah makrofag pada hari ke 3 dibandingkan kelompok kontrol. semakin tinggi konsentrasi gel kitosan maka jumlah neutrofil semakin menurun sedangkan jumlah makrofag semakin meningkat. hal ini membuktikan adanya sifat antimikrobial dari gel research report 153adistya, et al.: the effect of chitosan gel concentration on neutrophyl introduction the wound healing process are divided into three phases: inflammatory, proliferation, and scar maturation, that set in within minutes after skin injury to several months finally enters the maturation phase. the first inflammatory cell are leukocytes, namely neutrophils, monocytes and lymphocytes are recruited at different steps.1 neutrophils are important members of the innate immune and they play a role in the inflammation process. neutrophils contain defisins, proteins that have a broad range of antibiotic activity for bacteria and fungi.2 neutrophils migrate across endothelial from local blood vessels and initiate wound healing by releasing early-response proinflammatory cytokines, such as tnf-α, il-1-α and il-1-β. the other leukocytes cell is monocytes that migrate from blood into tissue and differentiate into macrophages. activating macrophages release many growth factors and cytokines including platelet-derived growth factor (pdgf), transforming growth factor beta 1 (tgf)-β1, as well as tnf-α and il-1, which upregulate the ecm production. neutrophils and macrophages are active in phagocytosis some bacteria, fungi and dispose of dead matter.1-3 chitosan is natural polymers composed by randomly β-(1-4)-linked d-glucosamine (deacetylated unit) and n-aceytl-d-glucosamine (acetylated unit).4 chitosan is soluble only at aqueous acidic solution with ph<6.5. negative characteristic of chitosan is its poor solubility at physiological ph.5 chitin and chitosan have been widely studied in both engineering and medicine due to its low cost, large scale availability, high biocompatibility, biodegradability and wound healing properties.6,7 several studies report the effects of chitin and chitosan on tissue reaction involved in wound healing.3,4,8,9 they accelerate infiltration of inflammatory cells, stimulate angiogenesis, induce the rapid formation of vascular granulating tissue, the disappearance of purulence and they promote skin regeneration with minimal scar formation. they also have candidacidal and bactericidal activity.4 in the oral application, both chitin and chitosan, can be used in tooth paste, mouth washes, chewing gum, freshen the breath and prevent the formation of plaque and caries.10 due to its biocompatibility and biodegradability, chitosan have a great potential to be a drug delivery system (dds).11 among the various approaches to modifying dds, aiming to increase the ability to remain attached to mucous membranes such in oral mucous with complex condition including salivary system and ph changes. this study tried to develop chitosan property as the oral ulcer healing substance. the ideal treatment for oral ulcer should improve stimulating mucosal cell growth and removing bacterial cells that otherwise retard the healing process. the aim of this study was to determine the effect of chitosan gel concentration on the neutrophyl and macrophage in gingival ulcer healing process of sprague dawley rats. materials and methods chitosan powder 95% degree of deasetilation (food grade) and 1% acetic acid solution were prepared as materials for chitosan gel. chitosan powder that has been weighed was put into measuring glass then were added 1% acetic acid solution till 100 ml in volume, stirred well. the comparison between chitosan powder and 1% acetic acid solution within chitosan gel 1%, 2% and 3% concentration was done. this research was done an experimental laboratory and was done in histology laboratorium, faculty of dentistry, universitas gadjah mada. the use of animal protocol was approved by the bio-ethics committee of the faculty of dentistry, universitas gadjah mada. national guidelines for the care and use of laboratory animals were applied during the study for 1 month. the animals were housed in cage located at the integrated research and testing laboratory, universitas gadjah mada. twenty healthy male spraguedawley rats, 2-2,5 month old, weighing 200-250 gr were used for this study. four groups randomly a, b, c which applicated with 1%, 2%, 3% chitosan gel respectively, and group d as negative control have prepared well. surgery was performed under general anesthesia by intramuscular injection of ketamine 50 mg/kg body weight in combination with xylasin 0.5-1.1 mg/kg body weight on left upper leg intramusculary. to reduce the risk of perioperative infection, the rats were treated with antibiotics, interflox-100 (interchemix, holland) at 10 ml/20-40 kg intramusculary during 3 days after injury created. whatmann filter paper no. 1 with size 2 x 2 mm soaked into 98% acetic acid solution for 5 minutes.12 the lower lip was retracted and the whatmann filter paper was applicated to labial gingival below the interdental both of anterior mandibular for 40 seconds with no pressure. kitosan. simpulan: gel kitosan terbukti mampu menurunkan neutrofil dan meningkatkan makrofag pada proses penyembuhan ulkus mulut. konsentrasi gel kitosan 3% mempunyai efek lebih baik dibandingkan dengan konsentrasi 1% dan 2%. kata kunci: gel kitosan, penyembuan luka, ulkus gingiva, neutrofil, makrofag correspondence: anne handrini dewi, bagian biomedika kedokteran gigi, fakultas kedokteran gigi, universitas gadjah mada. jl. sekip utara no 1 bulaksumur, sleman, jogjakarta 55281, indonesia. email: anne_ikgd@ugm.ac.id 154 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 152–157 gingival ulcer was formed two days after the acetic acid application. one drop chitosan gel applicated for the treatment groups twice a day, in the morning (6 a.m) and afternoon (4 p.m) for 3 days. when the gel was applicated, the lower lip was retracted and kept retracted for about 1 minute until the gel penetrated well into gingiva tissue. on the 3rd day all of the rats were sacrificed and its gingival tissue were taken for histologically processed and stained with hematoxillin eosin. the data was collected by counting the amount of neutrophyl and macrophage under the light microscope. the result was ratio data and were statistically analyzed for neutrophyl by one way anova and lsd, meanwhile for the macrophage by one way anova, lsd, and pearson correlations. results microscopic analysis on the rats gingival ulcers slides showed a high infiltration of neutrophyl pmn as the bluish purple cells with 2-5 loby nucleus and red granuled cytoplasm. macrophage was round to oval in shape and has a nonflat border. the excentric nucleus of macrophage was smaller than its on fibroblast and stained darker because of the phagocytosed particles. macrophage’s nucleus is ovoid in shape and has a fold on a side like kidney-shaped.13,14 table 1 for the group c (3% chitosan gel) showed that the lowest of neutrophyl but the highest of macrophage. the chitosan gel with concentration 1%, 2% and 3% for three days aplication influenced the amount of neutrophyl and macrophage on the ulcer healing of rats’ gingiva. the result of the one way anova showed that there was a difference among groups 0.001 (p<0.05) for neutrophyl and macrophage. meanwhile, the pearson corellation test showed that there is positive and strong correlation (0.979) between the concentration of chitosan gel and the amount of macrophage (figure 1). the result of the lsd analysis showed that there was a significant difference (p<0.005) for both of neutrophyl and macrophage among each group (table 2). from the data analysis above, it showed that chitosan gel with 1%, 2% and 3% concentration influence the amount of neutrophyl and macrophage. the higher concentration of chitosan the lower number of neutrophyl and the higher number of macrophage histological examination result was show in figure 2 at days 3 after application. histological examination result was shown in figure 2. discussion chitosan is a linear polysaccaride composed of randomly distributed of glucosamine and n-acetylglucosamine units linked by 1-4 glucosidic bonds. it is obtained by ndeacetylation of chitin, which is the second most naturally occuring biopolymer after cellulose.13 chitosan is distinct from other polysaccarides due to the presence of nitrogen in its molecular structure that makes to be cationic charge and its capacity to form polyelectrolyte complexes. this cationic side allows it become water soluble after the formation with kind of carboxylate salts (formate, acetate, lactate, malate, citrate, glyoxylate, pyruvat, glycolate and ascorbate).14 the wound healing process is complex interaction among cells, extracellular matrix component and signaling pathway between them. to achieve optimal good healing, maintaining a moist wound healing environment, preventing and managing infection are very important. the local delivery of therapeutics to the mouth can be used to treat a number of diseases, such as periodontal disease, stomatitis, fungal or viral infection and oral ulcer like oral mucositis. ulcer is a kind of wound that mostly happened because of many agents, such as physical trauma, chemical substance, allergy, infection, neoplasma, systemic disease and imunity disorder.15 in oral cavity due to many bacteria and masticatoria activity, it will increase a risk of infection. in addition, drug administration through the oral mucose must consider to flushing action of saliva and should be formulated to prolong retention of the drug in the oral cavity. the use of chitosan in this experiment was in gel form. shemer et al.,16 used a ‘gel like’ pharmaceutical to treat aphthous ulcers and it is applied directly on the ulcer. the application of chitosan gel may effectively interact with and protect the wound, ensuring a good, moist healing environment.17 bioadhesive polymers have been utilized in a gel forms to prolong the retention on oral mucose. due to table 1. average and standar deviation of neutrophyl and macrophage group average ± sd neutrophyl macrophage a (1%) 500.361 ± 11.312 176.39 ± 18.8 b (2%) 284.217 ± 3.081 278.25 ± 17.0 c (3%) 139.759 ± 5.418 401.56 ± 19.6 d (control) 656.807 ± 21.529 119.55 ± 5.00 table 2. t h e l s d a n a l y s i s o f t h e n e u t r o p h y l a n d macrophage group significantly a-b 0.001* a-c 0.001* a-d 0.001* b-c 0.001* b-d 0.001* c-d 0.001* 155adistya, et al.: the effect of chitosan gel concentration on neutrophyl muco adhesive properties, chitosan have been recognized as excellent candidates for oral delivery system.14,18 sinha et al.17 reported that the chitosan membrane was found to adhere uniformly over artificial created wound on the dorsum of the mice and also adsorbed the exudates from the wound surface. based on table 1, neutrophyl cells on group c (chitosan 3%) after application twice a day for 3 days, has the lowest compared with group a (chitosan 1%), b (chitosan 2%) and control group. the inflammation process set in within minutes after injury. on the 3rd day, amount of neutrophyl cells will decreased gradually by physiologycal process and the neutrophyl cells on chitosan group were lower than control group. the finding was similar to elassad19 that reported when chitosan was implanted in the rat’s peritoneal cavity, it induced a significant celluler response with recruitment of inflammatory cells as well as the other immune cells to the wound site at 4 hours after implantation. the percentage of pmn was estimated to be 28.4% for chitosan group, greater than 18.5% for control group by gelfoam®. this indicated that chitosan accelerate pmn cell to wound site better than control group. at 3 days after chitosan application, the result showed the decreased of neutrophyl. we suggest that chitosan can accelerate elimination debris substance and any bacteria that is done by neutrophyl. c h i t o s a n a n d c h i t i n h a v e b e e n o b s e r v e d t o accelerate wound healing by stimulate the migration of polymorphonuclear and mononuclear cells. it acts as chemo attraction and activates neutrophyl and macrophages to initiate the healing process.3,4,20 kojima et al.,8 reported that at 2nd day, the inflammatory cells increased within implant of the chitin and chitosan groups but not in the control group. chitosan was also reported has antimicrobial activity that against different groups of microorganism such as bacteria, figure 2. histological examination of group a, b, c and d, showed infiltration of neutrophyl and macrophage on the third day with 400x magnification. figure 1. the average of neutrophyl and macrophage after 3 days aplication of chitosan gel. a b c d 156 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 152–157 yeast, and fungi. there are two main mechanism have been suggested related of antimicrobial of chitosan. the first is interactions between both positive and negative charged molecules leads to increase the permiability of the outer and inner membranes of bacteria and resulting disruption of membrane integrity make releasing of the content of the cells.21 it has been postulated that antimicrobial nature of chitosan is due to surface-surface interaction between the biopolymer chains and microbial cell walls.22 the second mechanism suggest that chitosan may inhibit microbial growth by acting as chelating agent rendering metals, trace elements or essential nutrients that are needed for the organism to grow at the normal rate.21 due to antimicrobial activity, chitosan can enhance elimination activity for some bacterial in the ulcer area. this action can reduce a number of neutrophyl since elimination of injurious agent on the ulcer tissue is done by neutrophyl phagocytosis. increasing the content of the chitosan would enhance the anti microbial capacity.23 chitosan molecules was reported promoted migration of the inflammatory cells which were capable of the production and secretion of large proinflammatory product and growth factor at early phase of healing.3 chitosan stimulates interleukin 8 (il-8) production which chemoattracs and recruits neutrophyls to the wound.19 this result, at 3 days, the macrophages cell showed the highest number by chitosan 3%. macrophage plays an important role in the wound healing process, that is phagocyting the debris4,23 and stimulates the secretion of growth factors, cytokines, and inflammatory mediators that is induce the activation of other cells like endotellium cells, epitelium cells and fibroblasts.3,4 the growth factors produced by macrophage is tgf-α, bfgf, pdgf, hb-egf and vegf that can accelerate the cell proliferation and the synthesis the extracellular matrix. lack of macrophage on the wound tissue will make the cleaning of the wound less optimal, delayed fibroblast proliferation, inadequate angiogenesis and bad fibrosis.1,3 activated macrophages are important in host immune defenses, but their uncontrolled activation can lead to septic shock and death.4 chitosan accelerates the migration of pmn and induces macrophages,4 so the group c (3% chitosan) showed the highest of macrophage than the other groups and pearson correlation analysis showed that there was a positive correlation between the level of chitosan gel concentration and the increase of macrophage. normally, the proliferation phase proceed over the next 5-14 days to initial process for repair both epidermal and dermal layer. macrophages, fibroblast and vascular tissue together enter to the wound and begin to make the formation of granulation tissue that further to re-epithelialization process.3 kojima et al.8 reported that many polykaryocytes from the fusion of macrophages were observed in implant with chitin at 7 days compared to the control group. chitin at higher concentration stimulate platelets and macrophages, resulting in the release of pdgf and tgf-β but the phenomenon suggests influence locally only, not systematically. in addition, based on their study, mori et al.,4 suggest that chitosan was found to induce macrophage apoptosis. it took 6 hr to indice apoptosis in 50% of the macrophages in cell culture. the gradual apotosis needed to control wound healing acceleration and inflamation. chitosan activates macrophages for tumoricidal activity and for the production of interleukin-1. oligochitosan had an in vitro stimulatory effect on the release of tumor necrosis factor-α and interleukin-1β.3 besides, chitosan has also in vivo stimulatory effect on both nitric oxide production, chemiotaxis, and modulates the peroxide production. chitin or chitosan oligomers generated by chemo-enzymatic degradation in the wound environment, exert significant biochemical effects, migration of the mouse peritoneal macrophages was enhanced significantlyby chitin/chitosan oligomers.4 in conclusion, chitosan is a biomaterial already used in various medical devices. as the oral ulcer healing substance, it were prepared using acetic acid to make a gel solution with 1%, 2% and 3% concentration. all concentration group influence to decrease the amount of neutrophyl and increase the amount of macrophage on the rats’ gingival ulcer healing in three days after injury. the lowest of neutrophyl are founded in a highest concentration of chitosan. this is related to antimicrobial properties of chitosan. interaction between anionic groups on the cell surface of microorganism and polycationic charge of chitosan decreased a number of bacteria. meanwhile, this elimination process also are done by antimicrobial activity of chitosan, so that amount of neutrophyl cells on the ulcer area for treatment groups were lower than control group. the result showed that by 3% concentration, whether the lowest of neutrophyl, but the macrophage were the highest. chitosan can stimulate the chemotatic process of the macrophages due to its n-acetyl glucosamin group. at higher concentration it stimulates platelets and macrophages, resulting in the release of pdgf and tgf-β. this study suggested that chitosan gel with 3% concentration has a better effect than 1% and 2% concentration to enhance gingival ulcer healing in the rat. chitosan can accelerate wound healing related to amount neutrophyls cells and macrophages at days 3 after application and have a good adhesive properties on oral mucous of sprague dawley rats. further studies to develop chitosan as drug delivery system are needed for antibiotic or anti inflammation drug in oral application within membran shape to get a long contact with oral mucous. since solubility of chitosan only in acidic condition, it is very important to consider physiologic ph in oral environment. acknowledgement we are grateful to faculty of dentistry, universitas gadjah mada to support this research well. 157adistya, et al.: the effect of chitosan gel concentration on neutrophyl references 1. na ldin i a, ca r ra ro f. role of in f la m mator y mediators in angiogenesis. current drug targets-inflammation & allergy 2005; 4(1): 3-8. 2. telser ag, young jk, baldwin km. elsevier’s integrated histology. 1st ed. mosby elsevier; 2007. p. 219-21. 3. muzzarelli raa. chitin and chitosans for the repair of wounded skin, nerve, cartilage and bone. carbohydrate polymer 2009; 76(2): 167-82. 4. mori t, murakami m, okumura m, kadosawa t, uede t, fujinaga t. mechanism of macrophage activation by chitin derivatives. j vet med sci 2005; 67(1): 51-6. 5. turan so, akbuga j, sezer ad. topical application on antisense oligonucleotide-loade chitosan nanoparticles to rats. oligonucleotides 2010; 20(3): 147-53. 6. kong l, ao q, xi j, zhang l, gong y, zhao n, zhang x. proliferation and differentiation of mc 3t3-e1 cells cultured on nanohydroxiapatite/chitosan composite scaffolds. chin j biotech 2007; 23(2): 262-7. 7. mohamed kr, mostafa aa. preparation and bioactivity evaluation of hydroxyapatite-titania/chitosan-gelatin polymeric biocomposites. mater sci engineer 2008; 28(7): 1087-99. 8. kojima k, okamoto y, kojima k, miyatake k, fujise h, shigemasa y, minami s. effects of chitin and chitosan on collagen synthesis in wound healing. j vet med sci 2004; 66(12): 1595-98. 9. khan ta. a preliminary investigation of chitosan film as dressing for punch biopsy wound in rats. j pharm pharmaceut sci 2003; 6(1): 20-6. 10. dutta pk, dutta j, tripathi vs. chitin and chitosan: chemistry, properties and applications. j sci ind res 2004; 63(1): 20-31. 11. keegan gm, smart jd, ingram mj, barnes lm, burnett gr, rees gd. chitosan microparticles for the controlled delivery of fluoride. j dent 2012; 40(3): 229-40. 12. novianty ra, chrismawaty be, subagyo g. effect of allicin for reepithelialization during healing in oral ulcer model. the indonesian j dent res 2011; 1(2): 89-95. 13. pan zh, cai hp, jiang pp, fan qy. properties of calcium phosphate cement synergistically reinforced by chitosan fiber and gelatin. j polymer research 2006; 13(14): 323-7. 14. bhattarai n, gunn j, zhang m. chitosan-based hydrogels for controlled, localized drug delivery. adv drug deliv rev 2010; 62(1): 83-99. 15. regezi ja, sciubba j. oral pathlogy, clinical pathology correlations. 6th ed. st. louis: elsevier saunders; 2012. p. 22-73. 16. shemer a, amichai b, trau h, nathansohn n, mizrahi b, domb aj. efficacy of a mucoadhesive patch compared with an oral solution for treatment of aphthous stomatitis. drugs r d 2008; 9(1): 29-35. 17. si n ha m, ba n i k r m, ha ld a r c, ma it i p. development of ciprofloxacin hydrochloride loaded poly(ethlene glycol)/chitosan scaffold as wound dressing. j porous mater 2013; 20(4): 799-807. 18. perchyonok vt, zhang s, oberholzer t. chitosan and gelatin base prototype delivery systems for the treatment of oral mucositis: from material to perfomance in vitro. curr drug deliv 2013; 10(1): 14450. 19. elassad ams. mechanism involved in the effect of chitosan in enhancing healing. thesis. michigan state university; 2006: p. 66-75. 20. jayakumar r, prabaharan m, sudheesh kumar pt, nair sv, tamura h. biomaterials based on chitin and chitosan in wound dressing applications. biotechnol adv 2011; 29(3): 322-7. 21. aranaz i, mengibar m, harris r, parlos i, miralles b, acosta n, galed g, heras a. functional characterization of chitin and chitosan. current chemical biology 2006; 3(2): 203-30. 22. raafat d, sahl hg. chitosan and its antimicrobial potential-a critical literature review. microbial biotech 2009; 2(2): 186-201. 23. wang lc, chen xg, zhong dy, xu qc. study on poly (vinyl alcohol)/carboxymethyl-chitosan blend film as local drug delivery system. j mater sci mater med. 2007; 18(6): 1125-33. 6 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 6–9 research report the different effects of preheating and heat treatment on the surface microhardness of nanohybrid resin composite brelian elok septyarini,1 irfan dwiandhono1 and dian n. agus imam2 1department of conservative dentistry, faculty of medicine, universitas jenderal soedirman, 2department of orthodontics, faculty of medicine, universitas jenderal soedirman, purwokerto – indonesia abstract background: a composite resin is used as restorative material in dentistry because it has the same colour as dental enamel, is easy to use in an oral cavity and offers good biocompatibility. based on the size of filler, composite resin is divided into types, one of which is a composite resin nanohybrid. an important mechanical property of restorative material is microhardness. the mechanical properties of restorative material is highly affected by both polymerisation and heating process. there are many methods to improve composite resin’s microhardness, including preheating and heat treatment. purpose: the purpose of this study was to evaluate different effects of preheating and heat treatment on the microhardness of nanohybrid composite resin. methods: this study is an experimental laboratory research with post-test-only group design. samples were divided into six groups: preheating groups at temperatures of 37oc and 60oc, heat treatment groups at temperatures of 120oc and 170oc, a negative control group and a positive control group. afterwards, the resulting data were analysed using one-way anova. results: the result based on the one-way anova test indicated that there was a difference in microhardness in each group with a significance of 0.000 (p<0.005) between preheating and heat treatment. conclusion: the conclusion of this study was the best microhardness of composite resin nanohybrid is the heat treatment group at temperature 170oc. keywords: heat treatment; microhardness; nanohybrid resin composite; preheating correspondence: brelian elok septyarini, dental medicine, faculty of medicine, universitas jenderal soedirman. jl. dr. soeparno karangwangkal, purwokerto, 53123, indonesia. email: brelianeloks@yahoo.com introduction nanohybrid composite resin is a composite resin with nano-sized particles of 0.005-0.020 µm in a resin matrix.1 the advantages of nanohybrid composite resin includes good colour stability, good surface finish and less cure shrinkage.2 nanohybrid composite resins have additional components in the resin matrix, namely nanoparticles and nanocluster.3 this combination can reduce particle interstitial distance, thereby increasing filler resistance, physical properties, mechanical properties and retention.4,5 nanohybrid composite resin also offers mechanical properties such as high compression strength, good surface hardness, resistance to fracture, abrasion resistance and high diametral tensile strength.4,6 the mechanical properties of composite resins are influenced by polymerisation, heat treatment and a mixture of both.7 the polymerisation process determines the number of changes in the double bond of the monomer to a single bond of polymer called the degree of conversion.8 ideally, the dental restorative resin would have all of its monomer converted to polymer, but, in reality, polymerised composite resins only have a 55–75% degree of conversion.9 composites with insufficient hardness will be easily cracked.10 the method used to improve the mechanical properties of composite resins is preheating and heat treatment. preheating is a method of heating composite resin using a composite warmer or an oven at a certain temperature before curing.11 preheating can reduce the viscosity of composite resins, thus reducing microleakage and increasing adaptation to the edge of the lift.12 the method of preheating has an effect on increasing the degree of conversion.13 according to some studies, radical and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p6–9 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p6-9 mailto:brelianeloks@yahoo.com http://e-journal.unair.ac.id/index.php/mkg 7septyarini, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 6–9 monomer mobility will increase after preheating,11 affecting the degree of polymerisation, which can increase polymer crosslink.14 heat treatment is secondary curing in the laboratory, which aims to improve the mechanical and physical properties of a material.15 some tools that can be used for heat treatment include conventional ovens, autoclaves and porcelain furnaces.16 the results of heat treatment can increase the degree of conversion and the mechanical properties of composite resins, as well as minimise polymerisation shrinkage.13 studies comparing preheating and precooling at certain temperatures have indicated the flexural strength and flexural modulus of composite resins. the results of this study demonstrate that preheating at 45oc using a composite warmer for 15 minutes can increase the flexural strength and flexural modulus of nanohybrid composite resins.13 other studies have suggested that preheating at 37oc and 60oc can reduce shrinkage during polymerisation and increase the surface hardness of a nanohybrid composite resin.17 preheating composite resins at 37oc and 54oc using an oven for 30 minutes can increase the resin’s hardness.11 research into nanohybrid composite resins with heat treatment at 170oc has indicated an increase in the surface hardness of composite resins and flexural strength. other studies have shown that a heat treatment temperature of 170oc for 10 minutes for composite resins can reduce water absorption and solubility, enabling the composite resins to become more flexible.15 heat treatment with a temperature of 120oc for 10 minutes on composite resins can increase the flexural strength and hardness of composite resins.19 to date, there is no research comparing treatments between preheating and heat treatment on the microhardness of nanohybrid composite resins. hence, the purpose of this study was to evaluate the different effects of preheating and heat treatment on the microhardness of a nanohybrid composite resin. the temperatures used for preheating in this study were 37oc and 60oc. the temperatures used for heat treatment in this study were 120oc and 170oc. materials and methods this study is an experimental laboratory research with posttest-only group design. according to the american society for testing materials (astm) e384 standard, a cylindrical specimen was used with a diameter of 5 mm and a thickness of 2 mm. the total specimens used in the study were 48, divided into six groups such as 1a nanohybrid composite resin (dentsply, indonesia) preheating 37oc, 1b nanohybrid composite resin preheating 60oc, 2a nanohybrid composite resin heat treatment 120oc, 2b nanohybrid composite resin heat treatment 170oc, 3a negative control with resin nanohybrid composite resin and 3b positive control with a laboratory composite resin as a microhybrid composite resin. preheating groups were treated for 30 minutes in the oven before curing; they were then cured for 20 seconds and put into an incubator for 48 hours at 37oc. the heat treatment group was cured for 20 seconds first; thereafter, the heat treatment group was put into the porcelain furnace for 10 minutes and incubated at 37oc for 48 hours. the negative control group was put into an incubator at 37oc for 48 hours after being lightly cured. the positive control group was incubated at 37oc for 48 hours and then put into a porcelain furnace for 10 minutes at 170oc. the microhardness composite resin test was carried out using a vickers microhardness test with a load of 100gf in 15 seconds. the unit value of the surface hardness of the composite resin was kg/mm2. the research data were tested for normality using the shapiro-wilk test because the number of samples in the study was fewer than 50, and homogeneity tests were carried out using the levene test. the researcher used the one-way anova test to compare each preheating and heat treatment group. furthermore, the data were tested additionally: namely, the post hoc test used the lsd test to determine the value of comparisons between groups. table 1. one-way anova test results: preheating and heat treatment of microhardness of nanohybrid composite resins groups n δrq sig mean sb 1a 8 22.73 3.11 0.000* 1b 8 49.98 4.69 2a 8 28.43 2.66 2b 8 51.75 4.54 3a 8 19.95 2.40 table 2. post hoc preheating and heat treatment test results for microhardness of nanohybrid composite resins using the lsd methods groups 1a 1b 2a 2b 3a 3b 1a 0.000* 0.005* 0.000* 0.183 0.000* 1b 0.000* 0.376 0.000* 0.011* 2a 0.000* 0.000* 0.000* 2b 0.000* 0.001* 3a 0.000* 3b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p6–9 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p6-9 http://e-journal.unair.ac.id/index.php/mkg 8 septyarini, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 6–9 results the results of the nanohybrid composite resin microhardness test via the vickers microhardness tester can be seen in table 1. per the one-way anova test results, there were significant differences in microhardness of the nanohybrid composite resin (p <0.05). next, regarding the post hoc test using the lsd test, the test summary can be seen in table 2. the lsd test results showed significant differences between several groups, such as group 1a with groups 1b, 2a, 2b and 3b (p <0.05). in contrast, group 1b with group 2b and group 1a with 3a showed no significant difference (p> 0.05). discussion heat treatment can reduce the amount of residual monomer in the composite resin which causes an increased degree conversion.18 the heat treatment process will remove some carbon double-bond monomers that cannot react by evaporation because heat and some other carbon double monomers will be covalently bonded to the polymer network.20 the temperature used in the heat treatment affects the increasing degree of conversion and the large amount of residual monomer present in the composite resin.15 heat treatment at a temperature of 170oc is closer to the standard glass transition temperature (tg), so it is very effective in homogenising and modifying the polymer structure, increasing the number of cross bonds and condensing the polymer to become stronger.15 temperatures closer to the glass transition temperature (tg) can reduce stress during polymerisation when free radicals still trapped in the molecule will react again to form more cross bonds. such polymer crosslinking has small groups of atoms on both sides. when the sides of the polymer are close to electrons, they will form covalent bonds that will join. this polymer crosslinking will improve the mechanical properties of the composite resin better.21 irregular filler particles can cause shrinkage during polymerisation, causing a decrease in the mechanical properties of composite resins.22 the heat treatment can homogenise the filler particles, reducing the stress of shrinkage during polymerisation. high temperatures in the heat treatment process can increase radical mobility and the degree of polymerisation, thereby affecting crosslink density and producing dimethacrylate monomers.23 preheating can change high viscosity to low by melting viscosity due to the vitrification of the polymer. polymer vitrification is a polymer melting process. vitrification of the polymer occurs because the time required for the rate of diffusion reaction is reduced by the formation of the polymer. the reduced speed of the polymerisation process aims to determine the final result of the degree of conversion.24 as a result of vitrification, free radicals are trapped in the polymer, but this will not stop the mobility of free radicals because some free radicals are still present in the polymer. due to increased system mobility caused by temperature, free radicals will still react with the remaining double bonds and continue to polymerise. in addition to the free radicals trapped in the polymer due to vitrification, other molecules, such as residual monomers and photoinitiators, are also trapped inside the polymer so that they can influence biological properties such as the surface roughness of composite resins and mechanical properties such as surface hardness, flexural strength and compression strength of composite resins.25 polymer vitrification will stop when the preheating temperature used has reached the standard glass transition temperature of the polymerisation. the reaction rate will decrease significantly after vitrification, and any subsequent reaction will be slower. this process will determine the end result of polymerisation.26 preheating can increase the mobility of polymer chain molecules, delay diffusion reactions and increase the degree of conversion.11 the number of monomers will decrease with the increase in preheating temperature.27 increasing the temperature during preheating can increase the degree of conversion. this is because the polymerisation process of composite resins involves free radicals that convert material from high viscosity to low viscosity. during this process, a change in the c = c double bond becomes a covalent c-c single bond between the methacrylate monomers and changes in the rate of diffusion of free radicals.22 an increase in preheating temperature can increase the amount of monomer dimethacrylate, but only to a certain temperature limit. after the preheating temperature limit is reached, the monomer conversion decreases despite the subsequent temperature rise.28 the effective temperature for changing the viscosity of composite resins is between 54oc and 68oc.29 this is due to reactant evaporation and photoinitiator degradation. in addition, there are other influences, such as the time lag between removing the composite resin from the syringe into the mould and smoothing the composite resin before curing.27 the temperature received by the composite resin was not achieved as expected because the temperature could drop 2–4oc from the temperature it was supposed to be acquired.30 after preheating, the time needed to irradiate the composite resin is five minutes, so that it would increase 52–64% degree of conversion. this research did not observe the time needed to place the composite resin after preheating until printing.31 several factors can cause differences in the surface hardness of composite resins, including the type of composite resin. the positive control group used laboratory composite resins, namely the microhybrid composite resin, and the negative control group used nanohybrid composite resins. differences in the organic matrix composites of resins such as monomers and photoinitiators can affect the microhardness of composite resins.18 another influence is that nanohybrid composite resins have additional components in the form of nanoparticles and nanocluster in dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p6–9 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p6-9 http://e-journal.unair.ac.id/index.php/mkg 9septyarini, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 6–9 the matrix resin. the combination of these two ingredients can reduce the interstitial distance of particles, thus increasing filler loading, offering better physical properties and increasing retention.7 in conclusion, this study has determined that the microhardness of the nanohybrid composite resin heat treatment group is higher than the preheating treatment group. references 1. hatrick cd, eakle ws. dental materials: clinical applications for dental assistants and dental hygienists. 3rd ed. st. louise: elsevier; 2015. p. 65–9. 2. istianah i, aryati ekoningtyas e, benyamin b. perbedaan pengaruh hidrogen peroksida 35% dan karbamid peroksida 35% terhadap microleakage pada resin komposit nanohybrid. odonto dent j. 2015; 2(1): 20–4. 3. ferooz m, basri f, negahdari k, bagheri r. fracture toughness evaluation of hybrid and nano-hybrid resin composites after ageing under acidic environment. j dent biomater. 2015; 2(1): 18–23. 4. jain n, wadkar a. effect of nanofiller technology on surface properties of nanofilled and nanohybrid composites. int j dent oral heal. 2015; 1(1): 1–5. 5. sakaguchi rl, powers jm. craig’s restorative dental materials. 13th ed. st. louis: mosby elsevier; 2012. p. 73, 83, 164, 165, 167. 6. ilie n, hickel r. resin composite restorative materials. aust dent j. 2011; 56(suppl. 1): 59–66. 7. mei ml, so syc, li h, chu ch. effect of heat treatment on the physical properties of provisional crowns during polymerization: an in vitro study. materials (basel). 2015; 8(4): 1766–77. 8. aryanto m, armilia m, aripin d. compressive strength resin komp osit hybr id p ost cu r i ng denga n l ight em it t i ng d io de menggunakan tiga ukuran lightbox yang berbeda. dent j (majalah kedokt gigi). 2013; 46(2): 101–6. 9. mohamad d, young rj, mann ab, watts dc. post-polymerization of dental resin composite evaluated with nanoindentation and microraman spectroscopy. arch orofac sci. 2007; 2: 26–31. 10. allorerung j, anindita ps, gunawan pn. uji kekerasan resin komposit aktivasi sinar dengan berbagai jarak penyinaran. e-gigi. 2015; 3(2): 444–8. 11. kashi tsj, fereidouni f, khoshroo k, heidari s, masaeli r, mohammadian m. effect of preheating on the microhardness of nanohybrid resinbased composites. front biomed technol. 2015; 2(1): 15–22. 12. tauböck tt, tarle z, marovic d, attin t. pre-heating of highviscosity bulk-fill resin composites: effects on shrinkage force and monomer conversion. j dent. 2015; 43(11): 1358–64. 13. sharafeddin f, motamedi m, fattah z. effect of preheating and precooling on the flexural strength and modulus of elasticity of nanohybrid and silorane-based composite. j dent (shiraz, iran). 2015; 16(3 suppl): 224–9. 14. nada k, el-mowafy o. effect of precuring warming on mechanical properties of restorative composites. int j dent. 2011; 2011: 1–5. 15. muniz gr, souza e, raposo c, santana i. influence of heat treatment on the sorption and solubility of direct composite resins. indian j dent res. 2013; 24(6): 708–12. 16. miyazaki cl, santana il, do rosário matos j, filho ler. heat treatment of a direct composite resin: influence on flexural strength. braz oral res. 2009; 23(3): 241–7. 17. didron pp, ellakwa a, swain m v. effect of preheat temperatures on mechanical properties and polymerization contraction stress of dental composites. mater sci appl. 2013; 4(6): 374–85. 18. santana il, mendes jg, corrêa cs, gonçalves lmh, souza em, de sousa rc. effects of heat treatment on the microhardness of direct composites at different depths of restoration. rev odonto cienc. 2012; 27(1): 36–40. 19. junior l de om, mota jml de f, vaz rr, campos wr da c. evaluation of the mechanical properties of light-cure composite resins submitted to post-cure. rfo, passo fundo. 2010; 15(3): 275–80. 20. magne p, malta damp, monteiro-junior s, enciso r. heat treatment influences monomer conversion and bond strength of indirect composite resin restorations. j adhes dent. 2015; 17(6): 559–66. 21. anusavice kj, phillips rw, shen c, rawls hr. phillips’ science of dental materials. 12th ed. philadelphia: saunders; 2012. p. 64–5, 99, 100–4, 278–9, 280–3. 22. hambire uv, tripathi vk. experimental evaluation of different fillers in dental composites in terms of mechanical properties. arpn j eng appl sci. 2012; 7(2): 147–51. 23. dall’oca s, papacchini f, radovic i, polimeni a, ferrari m. repair potential of a laboratory-processed nano-hybrid resin composite. j oral sci. 2008; 50(4): 403–12. 24. soliman em, elgayar il, kamar a aa. effect of preheating on mikroleakage and microhardness of composite resin (an in vitro study). alexandria dent j. 2010; 41: 4–11. 25. leprince jg, palin wm, hadis ma, devaux j, leloup g. progress in dimethacrylate-based dental composite technology and curing efficiency. in: dental materials. 2013. p. 139–56. 26. jin mu, kim sk. effect of pre-heating on some physical properties of composite resin. j korean acad conserv dent. 2009; 34(1): 30–7. 27. daronch m, rueggeberg fa, de goes mf. monomer conversion of pre-heated composite. j dent res. 2005; 84(7): 663–7. 28. choudhary n, kamat s, mangala tm, thomas m. effect of pre-heating composite resin on gap formation at three different temperatures. j conserv dent. 2011; 14(2): 191–5. 29. walter r, swift ej, sheikh h, ferracane jl. effects of temperature on composite resin shrinkage. quintessence int. 2009; 40(10): 843–7. 30. el-deeb ha, abd el-aziz s, mobarak eh. effect of preheating of low shrinking resin composite on intrapulpal temperature and microtensile bond strength to dentin. j adv res. 2015; 6(3): 471–8. 31. fróes-salgado nr, silva lm, kawano y, francci c, reis a, loguercio ad. composite pre-heating: effects on marginal adaptation, degree of conversion and mechanical properties. dent mater. 2010; 26(9): 908–14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p6–9 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p6-9 http://e-journal.unair.ac.id/index.php/mkg 9797 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 97–101 research report the inhibition of streptococcus mutans glucosyltransferase enzyme activity by mangosteen pericarp extract nirawati pribadi, yovita yonas, and widya saraswati department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: streptococcus mutans (s. mutans) is a bacterium that plays an important role in the pathogenesis of dental caries. streptococcus mutans produces the glucosyltransferase enzyme which is capable of catalyzing glucan synthesis in the progression of dental caries. certain treatments involving traditional plant use have been developed to eradicate streptococcus mutans as a means of preventing the formation of dental caries. one of these is mangosteen pericarp extract containing a number of polyphenols that have the capacity to act as antibacterial agents, namely; tannin, mangostin, and flavonoid. purpose: the study aimed to investigate the inhibitory power of mangosteen pericarp extract against streptococcus mutans producing the glucosyltransferase enzyme. methods: the research used mangosteen pericarp extract at concentrations of 0.39% and 0.78% as the treatments, while 0.12% chlorhexidine gluconate was used as a positive control, and distilled water as a negative control. each group consisted of six samples. mangosteen peels extracted with 96% ethanol (maceration method) and mangosteen extract constituted 5% of the total weight of the mangosteen pericarp. supernatant containing gtf enzyme produced from a culture medium and centrifuged at 1500 rpm for 10 minutes at 4o c. glucosyltransferase enzyme activity was measured by analyzing the extensive fructose area by means of high performance liquid chromatography (hplc). the extensive fructose area was determined according to time retention in each group. results: mangosteen peel extract at concentrations of 0.39% and 0.78% demonstrated greater ability than the negative control group (sterile aquades) and similar ability to the positive group (chlorhexidine 0.12%) to inhibit the activity of the gtf enzyme or s. mutans bacteria. conclusion: mangosteen pericarp extract has the ability to inhibit the activity of streptococcus mutans in producing glucosyltransferase enzyme. keywords: glucosyltransferase enzyme; inhibitory power; mangosteen pericarp extract; streptococcus mutans. correspondence: nirawati pribadi, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: nirawatipribadi@gmail.com. introduction according to the basic health research (riskesdas) project of 2013, the dmf-t index of the indonesian population was 4.6, categorized as ‘high’ by the world health organization (who). the high prevalence of dental caries cannot, in fact, be separated from the virulent properties of cariogenic bacteria in the oral cavity.1 cariogenic bacteria, together with fermentable carbohydrates and saliva, contributes to the demineralization cycle and dental remineralization. the main cariogenic bacterium responsible for dental caries is streptococcus mutans (s. mutans), also considered to be the dominant bacterium in the oral cavity.2 s. mutans produces the glucosyltransferase (gtf) enzyme, a virulent factor in dental caries pathogens. the gtf enzyme can catalyze the formation of soluble and insoluble glucan derived from sucrose, while also playing a significant role in the polysaccharide matrix composition of dental plaque. this is because glucan can support both the attachment to and accumulation on tooth surfaces of cariogenic s. mutans bacteria. as a result, the activity of the gtf enzyme generated by s. mutans bacteria should be inhibited in order to prevent dental caries.3 in the field of dentistry, certain antiseptics and other substances, including chlorhexidine, triclosan, and sanguinarine, are often used as dental plaque control dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p97–101 mailto:nirawatipribadi@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p97-101 98 pribadi, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 97–101 agents. one antibacterial material considered to be the gold standard in the field of dentistry is chlorhexidine. nevertheless, it still manifests several deficiencies leading to tooth discoloration and discomfort. consequently, further research should focus on the development of a new, specific agent capable of inhibiting gtf enzyme activity as a means of controlling the formation of dental plaque with minimal side effects on the oral cavity.4 new agents the medical benefits of which have been investigated are naturally occurring materials enjoying wide availability and limited side effects. one frequently used natural material whose benefits are particularly wellknown throughout southeast asia is mangosteen (garcinia mangostana l.). this fruit or, more specifically, its peel provides many benefits contains several compounds producing a range of pharmacological benefits, including; anti-aging, anti-bacterial, anti-viral and anti-hypertension.5 the results of laboratory tests confirm that mangosteen peel extract contains a range of elements, including; xhantone (10.70%), saponin (3.82%), tannins (5.92%), αα-mangostin (2.82%), b mangostin (7.88%), flavonoids (1.88%), and mangostanin (11.88%).6 s. mutans produce three gtf enzymes: gtfb synthesizes the insoluble glucan alpha (1-3) polymer, gtfc synthesizes a mixture of insoluble glucan alpha (1-3) and non-alpha soluble (1-6), whereas gtfd synthesizes soluble glucan alpha (1-6). the content of flavonoids and alpha ααmangostin in mangosteen pericarp extract is effective in inhibiting gtfb and gtfc enzymes restricting their activity by up to 70%. however, the main one is gtfb because it can catalyze the formation of insoluble, alfa-linked glucan.7 inhibition of enzyme activity is also known to be caused by tannin which has an inhibitory power of 31.39%.8 in addition, flavonoids also actively limit the activity of the gtf enzyme as well as promoting antibacterial activity. nevertheless, such compounds have been shown to be effective in inhibiting the activity of gtf enzyme. unfortunately, no research has yet been conducted into the inhibitory power of a natural material containing all three compounds with regard to the activity of gtf enzymes. thus, mangosteen peel, containing flavonoids, α-mangostin, and tannins, is thought to be effective in inhibiting the activity of gtf enzymes. mangosteen pericarp extract is also known to be capable of inhibiting and killing s. mutans bacteria. the minimum inhibitory concentration (mic) of the mangosteen pericarp extract on s. mutans bacteria is 0.39%, while its minimum bactericidal concentration (mbc) is 0.78%.6 further investigations would be most appropriately focused on the mic and mbc of the mangosteen pericarp extract since it is expected to be an alternative ingredient of mouthwash within its mic range. moreover, this research was also expected to analyze the mangosteen pericarp extract within the concentration range of mic and mbc. therefore, the activity of the gtf enzyme can still continue even though s.mutans bacteria have died within the concentration range of mbc.9 materials and methods the research reported here was an experimental laboratory-based study with randomized control group post test-only design. research samples consisted of s. mutans bacteria obtained from the laboratory of microbiology, faculty of dental medicine, universitas airlangga, indonesia. the number of samples totaled six in accordance with federer’s formula (1963). independent variables consisted of the mangosteen pericarp extract at concentrations of 0.39% and 0.78%, while the dependent variable was the activity of gtf enzyme generated by s. mutans bacteria. mangosteen pericarp extract was prepared at materia medika, batu, east java, indonesia. meanwhile, the first preparation of s. mutans bacteria was conducted in the laboratory of microbiology, faculty of dental medicine, universitas airlangga, indonesia. subsequently, the second preparation of s. mutans bacteria was performed at institute of tropical disease, universitas airlangga, indonesia. thereafter, gtf enzyme was extracted from s. mutans bacteria and tested at central testing services, faculty of pharmacy, universitas airlangga, indonesia. mangosteen pericarp extract was prepared by treating dried mangosteen pericarp with 96% ethanol solvent and water using a maceration method. consequently, all less polar, semi polar and polar chemicals could be extracted as far as possible. the ratio of 96% ethanol solvent to mangosteen peel powder used was 1:2. the maceration method is a filtration process of simplicia derived from solvent by agitating the latter several times and then stirring it with a gerhardt thermoshaker, germany (2 x 24 hours) at room temperature. the simplica was then filtered, the resulting clear red filtrates being evaporated with a vacuum evaporator (at 60o c) until the ethanol separated out. brown mangosteen pericarp extract accounting for 5% of the total weight of the mangosteen pericarp was then produced. the s. mutans bacteria used in this research were drawn from s. mutans stock and then injected into bhib (brain heart infusion broth) media before being incubated for 24 hours at 37° c. after cultivation for 24 hours, a sterile tube containing s. mutans bacteria was prepared in 7 ml of bhib, incubated at 37° c for 24 hours and, finally, vibrated at 150 rpm with a gerhardt thermoshaker, germany.10 next, the culture media were centrifuged at 1500 rpm using an ultra sentrifugator hermle z36hk for 10 min at 4° c, generating supernatants containing the gtf enzyme.11 twenty-four test tubes were subsequently utilised during this investigation, six of which were used for each research group, namely the positive control group, the negative control group, the treatment group using the mangosteen pericarp extract at a concentration of 0.39%, and the treatment group using the extract at a concentration of 0.78%. the positive control tubes contained 0.875 ml of 0.25 m sucrose in 0.2 m phosphate buffer with ph of 7, then supplemented with 0.1 ml of gtf enzyme solution and 0.025 ml of 0.12% chlorhexidine. the negative control tubes, on dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p97–101 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p97-101 9999pribadi, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 97–101 the other hand, contained 0.875 ml of 0.25 m sucrose in 0.2 m phosphate buffer with ph of 7, then supplemented with 0.1 ml of gtf enzyme solution and 0.025 ml of sterile aquadest. in addition, tubes in the first treatment group contained 0.875 ml of 0.25 m sucrose in a 0.2 m phosphate buffer with ph 7, then supplemented with 0.1 ml of the gtf enzyme solution and 0.025 ml of the mangosteen peel extract at a concentration of 0.39%. meanwhile, tubes in the second treatment group contained 0.875 ml of 0.25 m sucrose in a 0.2 m phosphate buffer with ph 7, then supplemented with 0.1 ml of the gtf enzyme solution and 0.025 ml of the mangosteen peel extract at a concentration of 0.78%. all such treatment and control materials were then incubated at 370 c for two hours. after incubation and filtering with 0.45 μm, filter papers, the fructose concentration was tested by means of a high performance liquid chromatography (hplc agilent, usa), i.e. by injecting 20 μl of the treatment solution or control solution to reveal their retention time. fructose levels were then calculated by reading the area of fructose in the standard solution as follows:12 concentration (%) = note: ac = sample area as = standard area vic = volume of sample injection vis = volume of standard injection ks = standard concentration fp = dilution factor the results of the fructose standard solution test on hplc in this research showed that the retention time for fructose was approximately 2.8 minutes. the area of fructose generated in each sample can be indicated by the retention time shown on the chromatogram. the normality test is performed to calculate normally distributed data, with levene’s test being subsequently performed to calculate the homogeneity of the data. the next calculation uses the kruskal walles test to calculate the differences between groups. the value of significance between groups is established through application of the mann-witney test. results the results of the reading and measuring of fructose levels with hplc are divided into the control groups and the treatment groups using the mangosteen pericarp extract (figure 1). the results of the normality test revealed the significance value to be greater than 0.05. this suggests that the data for all research groups was normally distributed. meanwhile, the levene’s test results showed a significance value of 0.000 (p<0.05) indicating that the data was not homogeneous. the results of the kruskal-wallis test indicated a significance value of 0.001 (p<0.05), indicating that there were significant differences between the research groups. to reveal the significance value in each group, a mannwhitney test was performed the results of which can be seen in table 1. the negative control group (sterile aquades) table 1. results of the mann-whitney test negative control positive control treatment group 1 treatment group 2 negative control (aquades sterile) – p value= 0,002* p value= 0.002* p value= 0,002* positive control (chlorhexidine 0,12%) – – p value= 0,065 p value= 0,180 treatment group 1 (0,39%) – – – p value= 0,180 treatment group 2 (0,78%) – – – note: (+) = the positive control group using 0.12% chlorhexidine (-) = the negative control group using sterile aquadest i = the treatment group i using mangosteen pericarp extract at the concentrations of 0.39% ii = the treatment group i using mangosteen pericarp extract at the concentrations of 0.78% 120 100 80 60 40 20 0 % control (+) control (–) mean (%) treatment 1 treatment 2 figure 1. the mean and sd level of fructose in the control and treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p97–101 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p97-101 100 pribadi, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 97–101 ability’s to inhibit gtf s. mutans enzyme (p=0.002) was significantly lower than that of the positive control group (chlorhexidine 0.12%), and the 0.78% mangosteen pericarp extract group. the negative control group’s (sterile aquades) ability to inhibit gtf s.mutans enzyme (p=0.002) was significantly lower than that of the treatment group of 0.39% mangosteen pericarp extract. the mangosteen pericarp extract concentration group of 0.39% and 0.78% manifested the same ability as the positive control group (chlorhexidine 0.12%) as a substance which could inhibit the bacterial gtf s. mutans. discussion the research results showed that the concentration of fructose in the negative control group had a higher value (94.5%) than that the other treatment groups. the elevated concentration of fructose indicated the level of activity of s. mutans gtf enzyme in catalyzing the breakdown of sucrose. this was due to the solution in the reaction tube of the negative control containing sucrose, gtf enzyme, and aquadest. since aquadest was neutral, the enzyme broke the sucrose down into fructose and glucose without difficulty. polyphenol compounds in mangosteen pericarp extract are, moreover, reported as inhibiting the activity of the gtf enzyme. polyphenol compounds consist of flavonoids, tannins, and α mangostin and can inhibit the activity of the gtf enzyme by destroying other enzymes and microbial proteins. therefore, this enzyme which is mostly composed of proteins can be denatured by the polyphenol compounds generated by the mangosteen pericarp extract.13 flavonoids playing a role in the inhibition of the gtf enzyme are flavones and flavonols since both compounds have double bonds between c-2 and c-3 atoms in their chemical structure chains. the existence of this double bond provides a space for nucleophilic addition (a tendency to donate electrons or react with fewer electron sites, such as protons). the side chain of the gtf enzyme in the form of aspartic acid (ch2cooh) then possibly acts as a nucleophile and reacts with flavones and flavonols, thereby causing the gtf enzyme to be inhibited.3 another polyphenolic compound is tannin, which features a hydroxyl group in its structure, which potentially leads to a redoxive reaction with the gtf enzyme. this reaction, in turn, triggers the inhibition of gtf enzyme activity. the redoxive reaction is an oxidation-reduction reaction involving the exchange of electrons between two chemical structures. tannins can inhibit gtf enzyme activity by 31.93%.8 meanwhile, the third polyphenol compound in the mangosteen pericarp extract, α mangostin, inhibiting the activity of the gtf enzyme was investigated by means of docking studies. such studies predict the affinity and conformity of a molecule against a target protein. the molecule studied was α mangostin, while the target protein was the gtf enzyme. in the results of that research, amino acids in the chain of the gtf enzyme were found to be strongly and stably bound with α mangostin, indicating the effects of α mangostin on the gtf enzyme.7 based on the results of the analysis of those treatment groups using mangosteen pericarp extract, it could be seen that this extract at a concentration of 0.78% had greater inhibitory power against the activity of the gtf enzyme (with a fructose level of 32.63%) than the mangosteen pericarp extract at a concentration of 0.39% (with a fructose level of 49.77%). nevertheless, the results of the mannwhitney test showed there to be no significant difference between the two treatment groups. as a result, it could be concluded that the mangosteen pericarp extract at both concentrations had almost the same inhibitory power. meanwhile, the positive control group used 0.12% chlorhexidine since this material is the gold standard of material used in mouthwash. chlorhexidine is an antibacterial active ingredient that is relatively effective compared to other antibacterial agents. in addition, chlorhexidine also can inhibit the activity of the gtf enzyme. chlorhexidine at bacteriostatic concentrations can even inhibit membrane enzymes and disrupt the interaction between lipids and proteins in the membrane. consequently, chlorhexidine can inhibit the s. mutans glucosyltransferase enzyme and denature the enzyme protein.14 similarly, in this research, the fructose level was significantly lower than in the negative control group, indicating that 0.12% chlorhexidine was effective in inhibiting the activity of the gtf enzyme. the fructose level in the positive control group was 25.87%, lower than that in the treatment groups using the mangosteen pericarp extract. this suggests that chlorhexidine had an inhibitory effect against gtf enzymes superior to that of the mangosteen pericarp extract. in addition, the results of the mann-whitney test showed that the significance value between the positive control group and treatment group i was 0.065 (p>0.05), while that between the positive control group and treatment group ii was 0.180 (p>0.05). this means that there was no significant difference between the positive control group and those two treatment groups using the mangosteen pericarp extract at respective concentrations of 0.39% of 0.78%. in other words, the ability of mangosteen pericarp extract at these concentrations was equivalent to that of chlorhexidine at the concentration of 0.12% used as the positive control. in conclusion, mangosteen pericarp extract (at concentrations of 0.39% and 0.78%) demonstrated inhibitory power against the activity of s. mutans producing gtf enzyme. however, it proved less effective in this regard than 0.12% chlorhexidine. references 1. hall-stoodley l, stoodley p. evolving concepts in biofilm infections. cell microbiol. 2009; 11(7): 1034–43. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p97–101 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p97-101 101101pribadi, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 97–101 2. featherstone jdb. dental caries: a dynamic disease process. aust dent j. 2008; 53(3): 286–91. 3. koo h, rosalen pl, cury ja, park yk, bowen wh. effects of compounds found in propolis on streptococcus mutans growth and on glucosyltransferase activity. antimicrob agents chemother. 2002; 46(5): 1302–9. 4. torrungruang k, vichienroj p, chutimaworapan s. antibacterial activity of ma ngosteen per ica r p extract aga inst ca r iogenic streptococcus mutans. cu dent j. 2007; 30: 1–10. 5. poeloengan m, praptiwi p. uji aktivitas antibakteri ekstrak kulit buah manggis (garcinia mangostana linn). media penelitian dan pengembangan kesehatan. 2010; 20(2): 65–9. 6. chaovanalikit a, mingmuang a, kitbunluewit t, choldumrongkool n, sondee j, chupratum s. anthocyanin and total phenolics content of mangosteen and effect of processing on the quality of mangosteen products. int food res j. 2012; 19(3): 1047–53. 7. nguyen ptm, falsetta ml, hwang g, gonzalez-begne m, koo h. α-mangostin disrupts the development of streptococcus mutans biofilms and facilitates its mechanical removal. plos one. 2014; 9(10): 1–12. 8. sendamangalam v. antibiofouling effect of polyphenols on streptococcus biofilms. university of toledo; 2010. p. 29-33. 9. bowen wh, koo h. biology of streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. caries res. 2011; 45(1): 69–86. 10. ahmed a, dachang w, lei z, jianjun l, juanjuan q, yi x. effect of lactobacillus species on streptococcus mutans biofilm formation. pak j pharm sci. 2014; 27(5): 1523–8. 11. fujiwara t, hoshino t, ooshima t, sobue s, hamada s. purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from streptococcus oralis. infect immun. 2000; 68(5): 2475–83. 12. isnarianti r, wahyudi ia, puspita rm. muntingia calabura l leaves extract inhibits glucosyltransferase activity of streptococcus mutans. j dent indones. 2013; 20(3): 59–63. 13. petti s, scully c. polyphenols, oral health and disease: a review. j dent. 2009; 37(6): 413–23. 14. fejerskov o, kidd e. dental caries: the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p. 265-76. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p97–101 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p97-101 �� volume 47, number 1, march 2014 research report oral hygiene and number of oral mucosal lesion correlate with oral health-related quality of life in elderly communities dewi agustina department of oral medicine faculty of dentistry, universitas gadjah mada yogyakarta-indonesia abstract background: quality of life assessment mostly is based on general health. deterioration of physiologic condition, polypharmacy and the high occurrence of chronic disease in elderly may manifest in oral cavity that can affect oral function, in turn it will affect quality of life of elderly. purpose: this study was aimed to determine the correlation of oral health status and oral health-related quality of life (ohrqol) in elderly communities of yogyakarta city. method: seventy three elders were subjects of this study. data of ohrqol and oral health status were obtained from modification of questionnaire of dental impact of daily living (didl) index and from intraoral examination, respectively. intraoral examination comprised oral mucosal lesion amount, oral hygiene, dmft index and periodontal tissue status. the data then were analyzed statistically using pearson product moment correlation. result: the results showed that mean of dmft index was 16.9 and 63% of subjects were found with gingivitis, most subject had moderate oral hygiene and each subject at least had two oral mucosal lesions. mean score of quality of life was 27.2 and classified as satisfying. oral hygiene and number of oral mucosal lesion had correlation with ohrqol with r were -0.236 (sig. : 0.045) and -0.288 (sig. : 0.013), respectively. conclusion: the study suggested that oral hygiene and number of oral mucosal lesion correlate with oral health related-quality of life in elderly communities of yogyakarta city. key words: oral health, quality of life, elderly abstrak latar belakang: penilaian kualitas hidup terutama didasarkan pada kesehatan umum. memburuknya kondisi fisiologis, polifarmasi dan tingginya kejadian penyakit kronis pada lansia dapat termanifestasi di dalam rongga mulut sehingga dapat mempengaruhi fungsi mulut yang pada gilirannya akan mempengaruhi kualitas hidup lansia. tujuan: penelitian ini bertujuan untuk meneliti hubungan antara status kesehatan mulut dan kualitas hidup berdasarkan kesehatan mulut pada masyarakat lanjut usia di kota yogyakarta. metode: tujuhpuluh tiga lansia sebagai subjek dalam penelitian ini. data kualitas hidup berdasarkan kesehatan mulut dan status kesehatan mulut diperoleh dari modifikasi kuesioner indeks dampak kesehatan gigi terhadap kehidupan sehari-hari dan dari pemeriksaan intraoral. pemeriksaan intra oral terdiri atas jumlah lesi mukosa rongga mulut, kebersihan mulut, indeks dmft dan status jaringan periodontal. data kemudian dianalisis secara statistik menggunakan pearson product moment correlation. hasil: hasil penelitian menunjukkan bahwa rata-rata indeks dmft adalah 16,9 dan 63% subjek ditemukan dengan gingivitis, subjek rata-rata memiliki kebersihan mulut yang cukup dan setiap subjek rata-rata memiliki dua lesi mukosa mulut. rerata kualitas hidup berdasarkan kesehatan mulut adalah 27,2 dan tergolong memuaskan. kebersihan mulut dan jumlah lesi mukosa mulut memiliki korelasi dengan kualitas hidup berbasis kesehatan rongga mulut dengan masing-masing r adalah -0,236 (sig. : 0,045) dan -0,288 (sig. : 0,013). simpulan: dapat �8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 57–61 disimpulkan bahwa kebersihan mulut dan jumlah lesi mukosa mulut berkorelasi dengan kualitas hidup berbasis kesehatan rongga mulut pada masyarakat lanjut usia di kota yogyakarta. kata kunci: kesehatan mulut, kualitas hidup, lansia correspondence: dewi agustina, c/o: bagian ilmu penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: dewiagustina2004@yahoo.com introduction proportion of elderly (older than ≥ 60 years) worldwide dramatically increased recently. the reasons for this is increasing of life expectancy because of quality of life improvement and development of health aspect.1 problems of elderly health are various either as results of physiologic or pathologic process. to cope with those problems, the best thing to do is improving quality of life of elderly by giving an adequate health management. so far the quality of life assessment is mostly based on general health of people. quality of life itself can not be separated from the whole condition of human being that consists of general and oral health. deterioration of physiologic condition, polypharmacy and the high occurence of chronic disease in elderly may manifest in oral cavity that can affect oral function. health and function of mouth deteriorate as long as getting older.2 several oral findings could be detected in elderly population. all these findings would give badly impact for daily life of elderly. however, maintenance of oral health is still ignored by elderly. the ignorance of oral health results in decreasing of self confidence and social life that eventually affect the quality of life. research correlated between oral health and oral health-based quality of life in elderly is essential to conduct in indonesia since indonesian population can be categorised into old structure because of the percentage of elderly has reached up to 7%. yogyakarta is a province in indonesia with the highest amount of elderly (13.20%).3 yogyakarta is a city with the longest life expectancy in indonesia, so many elderly communities grow rapidly here. the community is a place for elders to share ideas, thoughts and is a pleasure place to intensively communicate each other with a goal to get a better quality of life. to assess quality of life is something so difficult since concept of quality of life is elusive and abstract. quality of life assessment is full of life values.4 there are many parameters to assess the quality of life related to elderly. however, from 20 instruments available, only 7 instruments meet with criteria of measurement, quantification and qualification including didl.5 in fact, parameters used in those instruments are not always suitable if applied in every country. therefore, in this study didl parameters6 has been modified based on the preliminary study. dental impact of daily living was chosen since the alternative statements given in this index is easier to be understood compared with other indexes that usually using likert scale. the aim of this study was to determine the correlation between oral health status and oral health related quality of life in elderly communities of yogyakarta city. the significance of this study were: (a) giving inputs to competent institutions to increase the elderly health management based on the oral disorders found in this study; (b) can be used as a basic reference for the oral health related quality of life studies in elderly. materials and methods seventy three elderly from five elderly communities (gowongan, tegal panggung, danurejan, suryatmajan and universitas negeri yogyakarta) in yogyakarta city were subjects of this study. the subjects received intraoral examination to observe: the number of oral mucosal lesion, periodontal tissue condition using modified periodontal index from russel, general oral hygiene and dmft index (dental caries/decay, missing and restored teeth). all data were recorded in oral health status form. assessment of quality of life based on modified didl was performed and recorded in ohrqol form. principally, didl is an instrument to describe the quality of life based on perception towards oral health. there are 36 alternative statements that can be met with the perception of oral health. someone should judge the statements with "yes" or "no". the score of quality of life is determined by amount of score that should be had by someone with satisfied quality of life. the total score of the statements determines grading of ohrqol with the following classifications: (a) score of 25-36 is satisfied; (b) score 13-24 is relative satisfied; and (c) score 1-12 is unsatisfied. the data were descriptively and statistically analysed using pearson product moment correlation test to determinepearson product moment correlation test to determinedetermine the correlations between independent variables (number of oral mucosal lesion, oral hygiene, periodontal tissue condition, dmft index) with dependent variable (oral health related quality of life). if the correlation existed, so the simple linier regression analysis was conducted with ci = 95% ((α = 0.05) to determine the influence of independentdetermine the influence of independent variables towards dependent variable or to determine the correlation using software of spss of 16 version.software of spss of 16 version. ��agustina: oral hygiene and number of oral mucosal lesion correlate with oral health-related quality of life results this study involved 73 elderly from five elderly communities (gowongan, tegal panggung, danurejan, suryatmajan and universitas negeri yogyakarta) in yogyakarta city. the subjects consisted of 11 males and city. the subjects consisted of 11 males and 62 females aged 61-80 years. fifty three subjects with educational qualification of less than senior high school and twenty subjects with that of higher than senior high school. seventy subjects were married and the other three were unmarried. seventy one subjects were javanese and the other two were other ethnics. sixty seven subjects were independent for activity daily living (adl) and the other six required some help when doing adl. occupational backgrounds were house wife (30 subjects), merchant (4 subjects), employee (4 subjects), retired (25 subjects), entrepreneur (4 subjects) and others (6 subjects). the results of oral health examination can be demonstrated on table 1. according to table 1, it was clear that 82% of elders had oral mucosal lesion. in this study, fissured tongue is the most found lesion, 51% of all elders with this lesion. other oral mucosal lesions found in these subjects were ulcerative lesions (20.5%), keratosis (17,8%), coated tongue (8.2%) and other lesions (2.5%). from linear regression analysis of variables with correlation i.e. oral hygiene and number of oral mucosal lesion, the following equations were obtained: ŷ = 29,451 – 2,337x (ŷ = ohrqol and x = oral hygiene) dan ŷ = 29,418 – 1,500x (ŷ = ohrqol and x = number of oral mucosal lesion). the mean score of dmft index in all subjects was 16.9. these indexes were catagorised as moderate to high, since classification of dmft index i.e. dmft index >13.9 is high and 9.0-13.9 is moderate. 11 in this study, there was no correlation between dmft index with quality of life (table 2). discussion although the status of oral health of elderly generally was not good enough (table 1), however, they had satisfying quality of life. this results probably were affected by aging concept. in this concept, principally elderly will sincerely accept this decreasing condition that is assumed as natural process and it always happens for all elders.7 this concept makes elderly never look for solution and it does not much influence the quality of life perception. one thing needs to be considered is parameters or indicators used to assess the status of oral health. on the other hand, validity for quality of life measurement is difficult to determine. so far, instruments used to measure quality of life is determined by professionals based on their own standard and definition. whereas perception of quality of life is subjective and what someone deems that it is important for his/her life, not always true for someone else.8 the result showed on table 1 was supported by a finding that fissured tongue is the most common tongue condition (5.24%) compared to other tongue abnormalities.9 on the other hand, another study also found that fissured tongue was the most common tongue lesion diagnosed in 11.5% of the subjects comprised of 2,000 dental out patients that have been screened for tongue lesions.10 the older someone the more fissured tongue may be found that might be caused by reduction of elastic fibers in oral tissue. this lesion is benign and does not need special treatment, only required to increase tongue hygiene.10 table 1. summary of intraoral examination from all subjects aspects no. of subjects percentage mean oral mucosal lesion : present absent 60 13 82% 18% 2.02 oral hygiene : good fair poor 14 47 12 19% 64% 16% periodontal tissue : healthy gingivitis with tooth mobility 13 46 14 18% 63% 19% dmft index 16.9 score of ohrqol 27.2 table 2. summary of correlation analyses between oral hygiene, number of oral mucosal lesion, dmft and periodontal status with ohrqol in all subjects oral hygiene σ of oral mucosal lesionoral mucosal lesion mucosal lesion dmft periodontal tissueeriodontal tissue tissue coefficient correlation of pearson (r) pearson (r) -0.236.236236 -0.288.288288 -0.026.026026 -0.066.066066 p-value (sig) 0.045.045045 0.013.013013 0.828.828828 0.580.580580 conclusion weak correlation and negative weak correlation and negative no correlation no correlation �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 57–61 table 2 results was not in line with the previous study that found there was a weak correlation between dmft index with quality of life measured by whoqolold with r = 0.135 and with mean score of dmft was 15.24.12 there was a strong correlation between oral hygiene and ohrqol with r = -0.236 (table 2) and it was highest correlation compared to other correlations, so it indicated that role of oral hygiene as a contributory factor for ohrqol is essential. that is why education and information about oral hygiene for frail elders should be intensified, since many oral diseases are originated from bad oral hygiene. bad oral hygiene in elderly is life-threatening condition since it may indirectly cause malnutrition and dehidration,13 brain abscess,14 arthritis,15 cardiovascular disease16,17 and pneumonia18 those are originated from periodontal disease. this statement was supported by studies at many institutions of elderly in scotland.19 they found that majority of elders had bad oral hygiene especially elders with denture, even the denture wearers sufferred from denture-induced pathology. by regularly increasing of oral hygiene in institutionalised elderly can minimise incidence of pneumonia.20 the two most oral diseases are dental caries and periodontal disease. these two diseases are originated from bad oral hygiene. it is assumed that development of dental plaque is faster in elderly compared to young people.13 one contributory factor for this bad oral hygiene is xerostomia, since the prevalence of xerostomia in elderly is higher than in young people.21 it is caused as well by systemic disease and side effect of medication.22 about 500 medications of 42 categories contribute to xerostomia in elderly.21 in this study not all indicators had correlation with ohrqol. only number of oral mucosal lesions and oral hygiene correlated with ohrqol. it is need to be studied further whether these indicators above are representative enough to asses ohrqol. probably, needs a study to determine standard indicators that are representative to assess ohrqol for each population. according to systematic review carried out towards 1.726 articles about ohrqol, only four studies showed significant correlations between oral health status with ohrqol.23 so, it is clear that currently no adequate instrument available for oral health assessment. the results met with locker’s idea that quality of life was elusive and abstract. assessment of quality of life was full of life values in which vary for each person.4 quality of life is a perception of life related with culture and rules that it is suitable with a place where someone lives and associated with aim, expectation, standard and care of life.11 in this study was observed also that hypertension, diabetes mellitus and gastritis were the three most found systemic diseases/conditions in elders. the occurence of diabetes mellitus itself may be associated with effect of bad oral hygiene that leads to high prevalence of periodontal disease. periodontitis is more common and severe among patients with diabetes mellitus type-2 than healthy patients.24 it was assummed that diabetes mellitus type-2 might initiate or worsen peridontitis and vice versa. a study support that appropriate treatment for periodontitis will improve glycemic control in diabetes mellitus type-2 patients.25,26 oral hygiene itself can be caused by decreasing of oral self cleansing that it can be associated with xerostomia. it is well known that many antihypertension medications have xerogenic effect.27,28 from above discussion can be withdrawn some points as follows. although the ohrqol of both group of elders could be still categorised satisfying, however, based on the oral findings in this study, improvement of elders’ condition was compulsory since oral condition can affect general health. someone with oral problems tend to consume soft and non-fibre foods that make inadequate nutritional fulfillment. finally, it can be correlated with stroke and malignant risks.17, 29 dentist and dental hygienist should be recruited as part of elderly health team. almost majority of oral problems come from bad oral hygiene. besides that, bad oral hygiene makes someone prones to oral infection that can be focal infection for other parts of body. therefore, communication, information and education about oral hygiene is very essential and need to be intensified that it expectedly may increase oral health-based quality of life in elders. there are two main suggestions arised from this study i.e.: (a) it needs to create an instrument for ohrqol measurement that it is suitable for indonesian elders; (b) it needs to create standard parameters to determine oral health status for indonesian elders. the study suggested that oral hygiene and number of oral mucosal lesion correlate with oral health related-quality of life in elderly communities of yogyakarta city. acknowledgement the authors gratefully acknowledge the funding provided by dana masyarakat faculty of dentistry, universitas gadjah mada and sincere gratitude to drs. b. esti chrismawaty and sri budiarti for their involvement in subject’s examination. references 1. yellowitz ja. providing oral cancer examinations for older adults. j calif dent assoc 1999; 27(9): 718-23. 2. peterson pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health programme. community dent oral epidemiol 2005; 33: 81-92. 3. badan pusat statistik. available from: http://www. etd.eprints.ums. ac.id/14760/4/03_bab_i.pdf. accessed november 8, 2011. 4. locker d. concepts of oral health, disease and the quality of life. in: slade gd, editor. measuring oral health and quality of life. usa: dept. of dental ecology, school of dentistry, university of north carolina; 1997. p. 11-24. 5. hebling e, pereira ac. oral health-related quality of life: a critical appraisal of assessment tools used in elderly people. gerodontology 2007; 24(3): 151-61. ��agustina: oral hygiene and number of oral mucosal lesion correlate with oral health-related quality of life 6. leao a, sheiham a. relation between clinical dental status and subjective impacts of daily living. j dent res 1995; 74(7): 140813. 7. iversen tn, larsen l, solem pe. a conceptual analysis of ageism. nordic psychology 2009; 61: 4-22. 8. xavier fmf, ferraz mpt, marc n, escosteguy nu, moriguchi eh. elderly people’s definition of quality of life. rev bras psiquiatr 2003; 25(1): 31-9. 9. gaphor sm, abdullah mj. prevalence, sex distribution of oral lesions in patients attending an oral diagnosis clinic in sulaimani university. j bagh college dentistry 2011; 23(3): 67-73. 10. darwazeh amg, almelaih aa. tongue lesions in a jordanian population: prevalence, symptoms, subject’s knowledge and treatment provided. med oral patol oralo cir bucal 2011; 16(6): e745-9. 11. peterson pe. priorities for research for oral health in the 21st centurythe approach of the who global oral health programme. community dent health 2005; 22: 71-4. 12. wangsarahardja k, dharmawan ov, kasim e. hubungan antara status kesehatan mulut dan kualitas hidup pada lanjut usia. universa medicina 2007; 26(4): 186-94. 13. shay k, ship j. the importance of oral health in the older patient. j am geriatr soc 1995; 43(2): 1414-22. 14. andrews m, farnum s. brain abscess secondary to dental infection. general dentistry 1990; 38: 224-5. 15. bartzokas c, johnson r, jane m, martin m, pearce p, saw y. relation between mouth and haematogenous infection in total joint replacements. br med j 1994; 309(6953): 506-8. 16. fiehn ne, gutschik e, larsen t, bangsborg jm. identity of streptococcal blood isolates and oral isolates from two patients with infective endocarditis. j clin microbiol 1995; 3395): 1399-401. 17. joshipura kj, rimm e, douglass c, trichopoulos d, ascheriio a, willett w. poor oral health and coronary heart disease. j dental res 1996; 75(9): 1631-6. 18. scannapieco f. role of oral bacteria in respiratory infection. j periodontol 1999; 7097): 793-802. 19. ettinger r, manderson r. dental care of the elderly. nursing times 1975; 10: 1003-6. 20. yoneyama t, yoshida m, ohrui t, mukaiyama h, okamoto h, hoshiba k, ihara s, yanagisawa s, ariumi s, morita t, mizuno y, ohsawa t, akagawa y, hashimoto k, sasaki h. oral care reduces pneumonia in older patients in nursing homes. j am geriatr soc 2001; 50(3): 430-3. 21. ship ja, pillemer s, baum b. xerostomia and the geriatric patient. j am geriatr soc 2002; 50(3): 535-43. 22. schwartz m. the oral health of the long-term care patient. ann long-term care 2002; 8: 41-6. 23. naito m, yuasa h, nomura y, nakayama t, hamajima n, hanada n. oral health status and health-related quality of life : a systematic review. j oral sci 2006; 48(1): 1-7. 24. taylor gw, borgnakke ws. periodontitis prevalence and severity in indonesia with type 2 diabetes. j periodontol 2008; 82(4): 550-7. 25. darré l, vergnes jn, gourdy p, sixou m. efficacy of periodontal treatment on glycaemic control in diabetic patients: a meta-analysis of interventional studies. diabetes metab 2008; 3495): 497-506. 26. o'connell pa, taba m, nomizo a, foss freitas mc, suaid fa, uyemura sa, trevisan gl, novaes ab, souza sl, palioto db, grisi mf. effects of periodontal therapy on glycemic control and inflammatory markers. j periodontol 2008; 79(5): 774-83. 27. scully c. drug effects on salivary glands: dry mouth. oral dis 2003; 9(4): 165-76. 28. scully c, bagan jv. adverse drug reactions in the orofacial region. crit rev oral biol med 2004; 15(4): 221-39. 29. terry p, giovannucci e, michels kb, bergkvist l, hansen h, holmberg l, wolk a. fruit, vegetables, dietary fiber, and risk of colorectal cancer. j natl cancer inst 2001; 93(7): 525-33. 28 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 28–31 anti-glucan effects of propolis ethanol extract on lactobacillus acidophillus ira widjiastuti, adioro soetojo, and febriastuti cahyani department of conservative dentistry faculty of dental medicne, universitas airlangga surabaya – indonesia abstract background: in deep dentinal caries cases, bacteria mostly found are lactobacillus acidophilus classified as gram positive bacteria and as facultative aerobes producing glucosyltransferase (gtf) enzyme. gtf enzyme can alter sucrose into glucans. glucan is sticky and insoluble in water. as a result, gtf enzyme can facilitate plaque formation and microorganism colonization on tooth surface. in addition, lactobacillus acidophilus also can form acid leading to demineralization of organic and inorganic materials, resulting in dental caries. multidrug-resistant phenomena, on the other hand, have led to the use of natural resources, one of which is propolis as an antimicrobial material and as a new anti-infective therapeutic strategy. propolis is a resinous substances collected by worker bees (apismellifera) from barks and leaves of plants. propolis has a complex chemical composition and biological properties, such as antibacterial, antiviral, antifungal, anti-inflammatory, and antitumor. purpose: this research aimed to reveal anti-glucan effects of propolis ethanol extract generated from honey bee, apis mellifera spp on lactobacillus acidophilus bacteria. method: before antiglucan test was conducted, glucan-formation test was performed on lactobacillus acidophilus bacteria using sdspage. meanwhile, anti-glucan adhesion test on lactobacillus acidophilus bacteria was carried by culturing the bacteria at 37ºc temperature in a jar with 10% co2. test tubes were placed at an angle of 30º for 18 hours to review the attachment of bacteria at the glass surfaces. after the incubation, the culture of bacteria was vibrated using a mixer vortex for a few minutes, and then cultured in solid mrs a media. bacteria grown were measured by using colony counter. result: the ethanol extract of propolis with a concentration of 1.56% was the lowest concentration inhibiting the attachment of glucan to lactobacillus acidophilus bacteria. conclusion: the ethanol extract of propolis with a concentration of 1.56% can be used as an anti-glucan material for lactobacillus acidophilus bacteria. keywords: insoluble glucan; lactobacillus acidophilus; propolis extract ethanol correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicne, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction dental caries is a multifactorial infectious disease caused by an interaction between teeth, biofilms, carbohydrates, and time, resulting in severe tissue damage. the process of damage to the hard tissue of a tooth triggered by a chemical reaction of bacteria begins with an inorganic breakdown, and then continues in the organic part.1 bacteria, as a result, can be considered to play an important role in the process of dental caries since the absence of bacteria will not trigger dental caries. various species of bacteria colonize in the oral cavity, especially on dental plaque. those bacteria are able to produce acid, causing the process of demineralization of dental hard tissues. caries begins from the enamel surface, and then will progress into deep dentinal caries. microorganisms involved in the caries process are complex. bacterial transition occurs in the development of carious lesions. in early caries lesions, bacteria are in the form of aerobic facultative one, while anaerobic bacteria in deeper caries lesions. in a study of 65 deep dentinal caries samples, the most common bacteria found were lactobacillus acidophilus bacteria.2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p28-31 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p28-31 2929widjiastuti et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 28–31 several researches have shown a positive correlation between the number of lactobacillus acidophilus bacteria in dental plaque and the prevalence of dental caries. this is due to some characteristics of the lactobacillus acidophilus bacteria that are acidophilus, able to synthesize insoluble polysaccharide of the extracellular glucan bond, α (1-3), produce lactic acid through homofermentation process, and form a colony attached tightly to the surface of the tooth. lactobacillus acidophilus bacteria also produce glucosyltransferase (gtf) enzyme that can convert sucrose and produce glucans. glucans are sticky and insoluble in water. g l u c a n s c a n f a c i l i t a t e f o r m a t i o n o f p l a q u e a n d colonization of microorganisms on the tooth surface. lactobacillus acidophilus bacteria can form acids that result in demineralization of organic and inorganic materials in tooth. if caries reaches to dentine and leaves a thin layer of dentine or pulp perforation, it is necessary to take care of the capsule pulp. pulp capping is a treatment using a biocompatible material that serves to protect the pulp from mechanical, chemical, and bacterial irritants, so the inflammatory pulp tissue can be repaired and recovered, as well as healthy.3 nevertheless, fault rate of pulp capping is still high due to opened pulp triggered by caries that is equal to 66.7%, whereas 7.8% is caused by mechanical ones.4-6 medicine commonly used is ca (oh)2, but this material still has deficiency in dentin bridge formation as well as tunnel defect resulting in re-infection of bacteria leading to tooth necrosis. emergence of multidrug-resistant phenomena has led to increased attention to find new antimicrobial agents and new anti-infective therapeutic strategies. in recent years, many direct resources from nature have been explored, one of which is propolis. propolis is a resin substance collected by worker bees (apismellifera) from barks and leaves of trees. propolis has a complex chemical composition as well as biological properties, such as antibacterial, antiviral, antifungal, anti-inflammatory, and antitumor. however, these compositions depend on the surrounding plants. propolis also has strong local antibiotics as well as antifungal properties. in addition, propolis has antibacterial activities against gram-positive and gram-negative bacteria. propolis also has been known to be effective against gram-positive and gram-negative bacteria. a previous research shows that the antibacterial compounds of propolis are also effective against oral bacteria, such as peptostreptococcus anaerobius, lactobacillus acidophilus, actinomyces naeslundii, prevotella oralis, prevotella melaninogenica, porphyromonas gingivalis, and fusobacterium nucleatum.7 another previous research conducted by parolia et al also claims that the antibacterial compounds of propolis also can be effective for veillonella parvula.8 propolis extract derived from different extraction methods can provide minimal inhibitory concentration and minimal bactericidal concentration.9 in the other words, those previous researches have proven that propolis has antibacterial activities against specific caries bacteria. based on the properties of propolis, resin contained in propolis is expected to prevent colonization of lactobacillus acidophilus on the surface of teeth. therefore, this research aimed to reveal anti-glucan effects of propolis ethanol extract generated from honey bee, apis mellifera spp on the attachment of lactobacillus acidophilus bacteria. materials and method this research used propolis generated from apis mellifera spp bees. tools used in research were sterilized in autoclave at a temperature of 121o c for 30 minutes. ethanol extract of propolis was obtained by using maceration method with 70% ethanol solvent. before revealing inhibitory effects of the ethanol extract of propolis on glucan formation in lactobacillus acidophillus, glucan formation test was conducted using sds page. lactobacillus acidophilus was isolated from frozen stock, grown in mrs-b with 5% glucose at 37°c for 24 hours. the sds-page test then was performed in several stages. gel preparation was conducted, gel plates were made by arranging two glass plates with a spacing of 1 mm plate. gel was made with two layers, namely gel as a place of collection of samples (stacking gel) and gel as a medium 9 figure 1 . results of the glucan measurement using sd-spage. table 1. results of the anti-glucan test of the ethanol propolis extract on the number of lactobacillus acidophilus bacteria group mean x¯ (cfu) standard deviation p control 100 1.988 p= 0.000 1.56% eep 5.57 4.082 note: eep: ethanol extract of propolis; p<0.005 is significantly different marker a b c d e f g h i j figure 1. results of the glucan measurement using sdspage. table 1. results of the anti-glucan test of the ethanol propolis extract on the number of lactobacillus acidophilus bacteria group mean x (cfu) standard deviation p control 100 1.988 p= 0.000 1.56% eep 5.57 4.082 note: eep: ethanol extract of propolis; p<0.005 is significantly different dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p28-31 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p28-31 30 widjiastuti et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 28–31 for separation of protein (separating gel). the separating gel mixture was carefully inserted into the plates using the micropipette. those plates were settled for 10-30 minutes until the gel was formed. the stacking gel was poured over the separating gel while fitted with a comb until gel and its well were formed. they were settled for for 30 minutes. after the gel was formed, the comb was removed. the plates were mounted on electrophoresis device. the buffer was poured on the electrophoresis vessel. afterwards, preparation for sample injection was performed by providing 10 µl of protein isolate samples added with 10 µl of tris-cl + 20 µl of reducing sample buffer (rsb), inserted into microtube and then heated in a water bath at 100 °c for 3 minutes. after cooled, the samples were put into the gel wells, about 20 µl for each well. the anode was connected to the lower reservoir, while the cathode was connected to the upper reservoir. the power supply was turned on with an electric current of 30 ma and 130 v. the running process then was stopped after the blue color of the marker reached a height of 0.5 cm from the bottom of the gel plate. staining was conducted by soaking the gel in staining solution for 30-60 minutes. color removal then was performed by soaking the gel in the destaining solution while shaking with an automatic shake until the gel became clear. results of the electrophoresis process was scanned. determination of molecular weight then was carried out by comparing the results of sample electrophoresis with protein marker. minimum inhibitory concentration (mic) of the ethanol extract of propolis was used to determine concentration level of propolis which could be used as an antigen (concentration 1.56%, 1.95% and 2.34%). anti-glucan adhesion test was performed by culturing the bacteria in a jar at 37ºc with 10% co2. reaction tubes containing the bacterial media and propolis then were placed at 30º angle for 18 hours.11 mic could be used to detect bacterial growth. after incubation, the bacterial culture was vibrated using a vortex mixer for several minutes, and cultured on solid mrs a media. the bacteria grown then were measured by using colony counter. the concentration level of propolis that could inhibit bacterial attachment was defined as the lowest concentration where there was no visible attachment to the glass surface.10 results based on results of the preliminary research on the measurement of the resistance zone of lactobacillus acidophilus growth by the ethanol extract of propolis, the ethanol extract of propolis is known to have inhibitory effects on the growth of lactobacillus acidophilus bacteria. in additiion, based on results of sodium dodecyl sulfate poly acrylamide gel electrophoresis (sds-page) test, the total amount of lactobacillus acidophilus protein molecules was 140,864 kda (figure 1). this result is closed to the result of a previous research conducted by mattos-graner et al12, showing that the formation of insoluble glucan has a molecular weight in the range between 150-160 kda. as a result, insoluble glucans formed in this research can be considered as places of bacterial attachment. the number of lactobacillus acidophilus bacteria was calculated based on the anti-glucan effects of the propolis ethanol extract on the number of lactobacillus acidophilus bacteria (table 1). based on the research data, the ethanol extract of propolis with a concentration of 1.56% can inhibit the formation of glucan in lactobacillus acidophilus bacteria. discussion dental biofilms containing 99% of bacteria consist of various types of bacterial cell species attached to the surface, binding to form a series of matrixes. biofilm formation is triggered by population density. various mechanisms can occur through adhesion, generally considered as reversible and irreversible stages. the reversible stage is an early stage that begins with hydrophobic interactions, electrostatic interactions, or van der waals bonds, influenced by hydrodynamic temperature or bonds.13 such activity is sufficient to create bacterial bonds and usually followed by irreversible adherence that begins with a specific host cell or bacterial cell receptor. the attachment of proteins to receptors is generally and clinically associated with biofilmforming bacteria and also associated with early attachment. in addition, this attachment also is considered as bacterial co-aggregation in the same or different sequences. cell surface proteins, such as pili, fimbriae, or flagella, are generally adhesion proteins and may bind to specific receptors or form hydrophobic bonds with surfaces.14 the attachment of bacteria to a surface, then forming biofilms, will be affected by the physical properties of the surface. rough surface causes more bacterial colonization.15 mechanism of lactobacillus acidophilus in attaching to tooth enamel or tooth-plaque surfaces consists of two attcahments, namely sucrose-independent attachment and sucrose-dependent attachment. the sucrose-dependent attachment on the glass surface is mediated by gtfi and gtfsi. lactobacillus acidophilus actually produces three gtf enzymes, namely gtfi encoded as glucosyltransferase b (gtfb), gtfsi encoded as glucosyltransferase c (gtfc), and gtfs encoded as glucosyltransferase d (gtfd). the attcahment of lactobacillus acidophilus onto tooth surfaces with glucan intermediates, in which insoluble glucan production (insoluble in water) serves as an important virulence factor. it indicates that lactobacillus acidophilus has a function in the accumulation and formation of plaque. lactobacillus acidophilus has acidogenic and aciduric properties triggering an ability to synthesize glucan as a major factor in the formation of cariogenic biofilms. glucan is synthesized from sucrose by lactobacillus acidophilus on tooth surfaces. streptococcus mutants and lactobacillus dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p28-31 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p28-31 3131widjiastuti et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 28–31 acidophilus produce three types of gtf, namely gtf-b synthesizing most of insoluble glucans, gtf-c synthesizing soluble and insoluble glucans, and gtf-d synthesizing soluble glucans. the inhibition of glucosyltransferase enzyme activity then causes lactobacillus acidophilus cannot convert glucose, especially sucrose to glucan, so it cannot be attached to tooth surface.11 based on the results of this research, the ethanol extract of propolis with a concentration of 1.56% could inhibit glucan adhesion on glass surface. propolis is a natural ingredient that has antimicrobial properties, namely apigenin and tt-farnesol. apigenin has a role in inhibiting the activities of gtf b and gtf c, but apigenin has no antibacterial properties. by inhibiting the activities of gtf b and gtf c, apigenin can affect the activity of fructosyltransferase. besides, apigenin can effectively inhibit insoluble glucan synthesis. in other words, apigenin is a unique therapeutic substance that affects the activity and expression of the gtf enzyme without showing antibacterial activity. apigenin is a potent non-competitive inhibitor against the activities of gtf b and gtf c.11 on the other hand, tt-farnesol exhibits barriers to bacterial growth and metabolism by destroying bacterial cell membranes, thereby affecting the process of glucan synthesis. if apigenin affects the permeability of the cell membrane, how tt-farnesol in the propolis extract inhibits the synthesis of glucan may be triggered by its effects on the cell membranes more than its effects on enzyme activity since tt-farnesol is a poor gtf inhibitor. nevertheless, the chemical structure and lipophilic properties of tt-farnesol that support membrane localization can trigger changes in the permeability and instability of cell membranes. consequently, cell membrane will damage, not only reducing bacterial metabolism, but also affecting the synthesis of glucan by lactobacillus acidophilus. apigenin and tt-farnesol can be considered as non-toxic materials, both in vitro and in vivo. to suppress the amount of glucan production, some experts then recommend the use of natural ingredients rather than broad-spectrum antimicrobial agents since they will affect the normal flora of the oral cavity. this is also supported by the results of various previous researches determining the effectiveness of anti-plaque or anti-caries.11 it can be concluded that ethanol extract of propolis with a concentration of of 1.56% can be used as an anti-glucan material for lactobacillus acidophilus bacteria. references 1. heymann h, swift e, jr. ritter a. sturdevant’s art and science of operative dentistry. 6th ed. 2012. mosby. p. 41-2 2. martin fe, nadkarni ma, jaques na, hunter n, quantitative microbiological study of human carious dentine by culture and realtime pcr: association of anaerobes with histopathological changes in chronic pulpitis. j clin microbiol 2002; 40: 1698-704. 3. bergenholtz g, bindslev ph, reit c. text books of endodontology. 2nd ed, blackwell publishing ltd. 2010. p. 10-2. 4. barthel cr, rosenkranz b, leuenberg a, roulet jf. pulp capping of carious exposures: treatment outcome after 5 and 10 years: a retrospective study. j endod 2000; 26: 525-8. 5. al-hiyasat as, nusair kmb, al omari ma, the radiographic outcomes of direct pulp-capping procedures performed by dental students. a retrospective study. jada 2006; 137: 1699-705. 6. ritter av, andre v. direct pulp-capping performed by dental student has a succes rate close to 60 % direct capping of carious pulp exposures is significantly less succesful (33%) than direct pulp capping of mechanical pulp exposure (92%). j ev bas dent prac 2007; 7: 165-6. 7. koru of, toksoy ch. in vitro antimicrobial activity of propolis samples from different geographical origins against certain oral pathogens. anaerobe 2007; 13 (3-4): 140–5. 8. parolia a, kundabala m, rao nn, acharya sr, agrawal p, mohan m, thomas m. a comparative histological analysis of human pulp following direct pulp capping with propolis, mineral trioxide aggregate and dycal. australian dental journal 2010; 55: 59–64. 9. soley arslan, s s, duygu p, ayşe n k, özgür er.s, arslan, s. silici, d. perçin, a. n. koç, ö. er.. turk j biol 2012; 65(36): 65-73. 10. duarte s, gregoire s, singh ap, vorsa n, schaich k, bowen wh, koo h. inhibitory effects of cranberry polyphenols on formation and acidogenicity of streptococcus mutans biofilms. fems microbiol lett 2006a; 257: 50–6. 11. koo h, schobel b, scott-anne k, watson g, bowen wh, cury ja, rosalen pl, park yk. apigenin and tt-farnesol with fluoride on s. mutans biofilm and dental caries. j dent res 2005; 84(11): 101620. 12. mattos-graner ro, smith dj, king wf, mayer mp. water-insoluble glucan synthesis by mutans streptococcal strains correlates with caries incidence in 12to 30-month-old children. j dent res 2000; 79: 1371–7. 13. dunne wm jr. bacterial adhesion: seen any good biofilms lately?. j clin microbiol 2002; 15(2): 155-66. 14. o’toole g, kaplan hb, kolter r. biofilm formation as microbial development. annu rev microbiol 2000; 54: 49-79. 15. donlan rm. biofilms: microbial life on surfaces. eid journal 2000; 8(9): 881-90. 16. koo h, hayacibara mf, schobel bd, cury ja, rosalen pl, park yk. vacca-smith am. bowen wh. inhibition of streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol . j antimicrob chemother 2003; 52(5): 782-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p28-31 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p28-31 110 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 110–114 changes in taste sensation of sour, salty, sweet, bitter, umami, and spicy, as well as levels of malondialdehyde serum in radiographers agniz nur aulia, jenny sunariani, and ester arijani r departement of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: radiation used for any purpose certainly contains potential danger to humans. radiographers are given a task, authority, and responsibility by the competent authority to conduct radiography and imaging in health services unit. some researches on the effects of radiation on cancer patients show that radiation can cause an increase in bitterness and metal taste [in cancer patients] leading to discomfort in the oral cavity. in body, free radicals then can cause lipid peroxidation process. lipid peroxidation is an oxidative destruction of polyunsaturated fatty acid producing malondialdehyde (mda). purpose: this study aimed to determine the effects of radiation on changes in the taste sensation of sour, salty, sweet, bitter, umami, and spicy as well as the levels of mda serum in radiographers. method: this study was an observational laboratory research using posttest control design. samples were selected using simple random sampling technique. the samples were seven radiographers who have been working for five years in the laboratory and radiographic units in surabaya. result: based on the results of statistical tests, it showed that there were no differences in the sensitivity of all tastes between the groups tested. moreover, the results also depicted considerable value for the sour taste was 0.550, the saltiness was 0.775, the sweetness was 0.294, the bitter taste was 0.065, the umami taste was 0.705, and the spicy taste was 0.319 (p>0.05). however, the dramatic increase was higlighted in levels of mda serum with a significant value of 0.065 (p>0.005). conclusion. there were no changes in the sensitivity of sour, salty, sweet, bitter, umami, and spicy tastes, but there was a significant increased in level of mda serum in the radiographers compared to the control group. keywords: radiographers; taste buds; mda serum level correspondence: jenny sunariani, departement of oral biology, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: jennyagoes@gmail.com introduction there are some senses of taste in the oral cavity, namely salty, sour, sweet, bitter, and umami tastes. the emergence of a sense of bitter taste is due to the bonding between chemicals as bitter taste stimulants in receptors. this reaction results in g-protein release unit α, which is the sensory receptors of bitter taste referred to as gustducin. gustducin then activates the enzyme, in these circumstances causing closure of channel k+ then stimulating phospholipase c (plc) to activate phosphatidylinositol phosphate (pip) into inositol triphosphate (ip3). inositol triphosphate makes ca2+ released from the endoplasmic reticulum, causing depolarization. consequently, increasing concentrations of ca2+ in the receptor cells of bitter taste causes an increase in the sense of bitter taste then forwarded to the memory inside the brain.1 taste buds on the tongue contain pores, known as taste pores containing microvilli that carry gustatori cells that will be stimulated by various chemical liquids. microvilli are surface receptors for taste. sensory nerve fibers from the taste buds on the anterior part of the tongue then will deliver impulses to the brain stem through chorda tympani (branch of the facial nerve). the posterior part of the tongue, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p110-114 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p110-114 111111aulia, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 110–114 on the other hand, will deliver impulses to the brain stem through glossopharyngeal nerve, while the taste buds in the pharynx and epiglottis innervated by the vagus nerve to interpret the taste.1 utilization of radiology in various fields, especially in the health field can be used for both diagnosis and treatment of patients. radiation used for any purpose, nevertheless, will certainly contain potential danger to humans. radiation safety, as a result, is an effort made to create conditions in which dose of ionizing radiation on humans and environment does not exceed a specified limit. harmful effects of ionizing radiation are known as somatic effects when suffered by people exposed to radiation, and called as genetic effects when experienced by their offsprings.2 the basic unit of biological tissue is cell. if the ionizing radiation penetrates the tissue, it may result in ionization and produce free radicals, such as hydroxyl free radical (oh •), which consists of oxygen atoms and hydrogen atoms. in chemistry, free radicals are highly reactive and can alter important molecules in the cell. radiation can ionize molecules of dna directly causing chemical changes in dna and if the dna interacts with the hydroxyl free radical will cause adverse biological effects, such as cancer or abnormal genetics.3 in the body, moreover, free radicals can trigger lipid peroxidation process. lipid peroxidation exposed to oxygen then is responsible for the destruction of body tissue in vivo, causing various diseases, such as cancer, atherosclerosis, aging, and others. lipid peroxidation is a chain reaction with various damaging effects.4 lipid peroxidation is an oxidative destruction of polyunsaturated fatty acids, which have a long-chain producing mda compound. mda is also a component of cell metabolites produced by free radicals. therefore, a high concentration of mda serum indicates oxidation process in the cell membrane. mda can be used as an index measuring the activity of free radicals in the body. high levels of mda in the body can be caused by increased activity of free radicals.5 for those reasons, this research aimed to analyze how the senses of taste of radiographers almost daily exposed to radiation even in small doses, but continually in the long term was. specifically, this research focused on the effects of radiation on changes in radiographers’ sensation of sour, salty, sweet, bitter, umami, and spicy tastes as well as in their mda serum levels. materials and method this research was an observational laboratory research using post test control study design. this research was conducted by performing a test on taste sensitivity and mda serum levels in samples that had been exposed to radiation. samples in this research were radiographers at the radiology units of universitas airlangga hospital, surabaya surgical hospital, and the clinical laboratory of mitra husada surabaya hospital. criteria for the samples of radiographers classified into the treatment group in this research were as follows: a) working in medical radiography for 5 years or more; b) men aged between 18-44 years (who); c) no history of systemic disease; d) do not smoke; e) no impairment/sores in the oral cavity. meanwhile, criteria for the samples of non-radigraphers classified into the control group were as follows: a) men aged between 18-44 years (who); b) no history of systemic disease; c) do not smoke; d) no impairment/sores in the oral cavity. this research focused on taste sensitivity and mda serum levels. this research was conducted at the laboratory of biochemistry, faculty of medicine, universitas airlangga from august to november 2015. the number of samples then was determined using lemeshow’s formula. the total of samples in this research was seven people. a sensitivity test was performed on each sample of the treatment group and the control group by applying citric acid solutions with different concentration of 0.0044 m, 0.0057 m, 0.007 m, 0.0096 m, 0.0125 m, 0.0162 m, 0.0211 m, and 0.0275 m on the taste buds of sour taste located in the lateral part of their tongue.6 nacl solutions with a concentrations of 0.003 m, 0.01 m, 0.0013 m, 0.0017 m, and 0.022 m also were applied on the taste buds of salty taste located in the lateral part of their tongue.7 sucrose solutions with a concentrations of 0.01 m, 0.013 m, 0.017 m, 0.022 m, 0.029 m, 0.038 m, 0.049 m, and 0.064 m were applied on the taste buds of sweet taste located in the anterior part of their tongue.8 in addition, quinine solutions with a concentrations of 0.031%, 0.063%, 0.125%, 0.25%, 0.5%, 1%, and 2% also were applied on the taste buds of bitter taste located in the posterior part of their tongue.6 l-glutamate solutions with a concentrations of 1%, 2%, 3%, 4%, 5%, and 6% then was applied on the taste buds of umami taste located in the center of their tongue.6 capsaisin solutions with a concentrations of 3.13%, 6.25%, 12.5%, 25%, 50%, and 100% also were applied on the taste buds of spicy taste located in the anterior part of their tongue.6 furthermore, a test were carried out on mda serum levels by taking blood from their samples and then centrifuged them to obtain serum. the test was conducted in the laboratory. mda serum levels were measured with thiobarbituric acid (tba) reagent through nucleophilic addition reactions to form mda-tba compounds. 1,1,3,3-tetraetoksipropana or malondialdehyde tetrebutylammonium salt compound then was used to make a standard curve since 1,1,3,3-tetraetoksipropana can be oxidized in acidic aldehyde compound that can react with tba. although this method is not specific, but this method is accepted as a marker of lipid peroxidation for many researchers.16 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p110-114 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p110-114 112 aulia, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 110–114 results this research was conducted using two research groups, namely control group consisted of non-radiographers and the treatment group consisted of radiographers. the sensitivity test was performed on each group including taste buds for sour, salty, sweet, bitter, umami, and spicy tastes. mda serum levels also were measured by taking blood samples from each group. the total of samples in this research was seven samples as based on a minimum sample of lameshow’s formula. the results of the sensitivity test in each group can be seen in figure 1, while the measurement results of their mda serum levels can be seen in figure 2 as attached in the appendix. the data of obtained from the results of the sensitivity test on each group then were tested using mann whitney test. mann whitney test was performed to determine the significance of difference between the two groups of samples. the results of mann whitney test showed that there was no significant difference between the two groups. the significant value for the bitter taste sensitivity was 0065, while for the umami taste sensitivity was 0.705. the significant value for the sweet taste sensitivity was 0.294, while the salty taste sensitivity was 0.775. and, the significant value for the sour taste sensitivity was 0.550, while for the spicy taste sensitivity was 0.319 (p>0.05). therefore, it means that there was no significant difference in taste sensitivity between the two groups. in the control group consisted of non-radiographers, furthermore, the average levels of mda serum in the control group was 2.619 nmol/ml. on the other hand, in the treatment group consisted of radiographers the average levels of mda serum was 11.525 nmol/ml. the data about the levels of mda serum then was tested using independent t test. independent test t test was conducted to determine the significance of difference between the two groups. before conducting the test, however, there was a requirement to test the data for their normally distribution. thus, kolmogorovsmirnov statistic test was performed. the results of the kolmogorov-smirnov test showed the significance value of those two groups was more than 0.005 (p>0.005). it indicates that the data obtained in those two groups were normally distributed. therfore, the independent t test then was performed. the results of the independent t test showed the significance value of those two groups was 0.065 (p>0.005). it means that there were significant differences in mda serum levels between the two groups. discussion in humans, the senses of taste are very significant since with the senses of taste they can taste scrumptious and delicious food and drink. the sensation of taste arises from the chemicals binding to the receptor senses of taste (taste buds) mostly located on the surface of the tongue and soft palate. however, only the chemical in solution or solids that have been dissolved in saliva can bind to receptor cells.8 after each research group was tested for their sensitivity of flavors, including senses of bitter, umami, sweet, salty, sour, and spicy tastes, it is known that there was no significant difference, either decrease or increase, in the sensitivity of taste between those two groups. this is possible because the radiation exposure received by the radiographers was very small doses, very short period, and good radiation protection, so that the biological effects including changes in taste sensitivity have not happened yet. factors known to influence the onset of biological effects from exposure to the outside are absorbed dose, exposure distribution in the body, distribution of exposure time and age.912 figure 1. graph of taste sensitivity test results on each sample the graph above shows the total score obtained from the amount of concentration that can be perceived by the samples. x-axis is the sense of taste, namely: "1" sense of bitter taste "4" sense of salty taste "2" sense of umami taste "5" sense of sour taste "3" sense of sweet taste "6" sense of spicy taste meanwhile, the y-axis is the total score, the group "a" is the control group control group and "b" is the sample group of radiographers. "1" sense of bitter taste "4" sense of salty taste "2" sense of umami taste "5" sense of sour taste "3" sense of sweet taste "6" sense of spicy taste figure 2. curve of mda serum levels in the control group and in the sample group of radiographers. the curve above shows the results of the absorbance measurements of mda serum level in the research groups. the x-axis is the serum levels of mda (nmol / ml), y-axis is the research groups, namely "a" is the control group and "b" is the sample group of radiographers. 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 1 2 3 4 5 6 7 a b figure 1. graph of taste sensitivity test results on each sample the graph above shows the total score obtained from the amount of concentration that can be perceived by the samples. x-axis is the sense of taste, namely: “1” sense of bitter taste “4” sense of salty taste “2” sense of umami taste “5” sense of sour taste “3” sense of sweet taste “6” sense of spicy taste meanwhile, the y-axis is the total score, the group “a” is the control group control group and “b” is the sample group of radiographers. “1” sense of bitter taste “4” sense of salty taste “2” sense of umami taste “5” sense of sour taste “3” sense of sweet taste “6” sense of spicy taste 12 figure 1. graph of taste sensitivity test results on each sample the graph above shows the total score obtained from the amount of concentration that can be perceived by the samples. x-axis is the sense of taste, namely: "1" sense of bitter taste "4" sense of salty taste "2" sense of umami taste "5" sense of sour taste "3" sense of sweet taste "6" sense of spicy taste meanwhile, the y-axis is the total score, the group "a" is the control group control group and "b" is the sample group of radiographers. "1" sense of bitter taste "4" sense of salty taste "2" sense of umami taste "5" sense of sour taste "3" sense of sweet taste "6" sense of spicy taste figure 2. curve of mda serum levels in the control group and in the sample group of radiographers. the curve above shows the results of the absorbance measurements of mda serum level in the research groups. the x-axis is the serum levels of mda (nmol / ml), y-axis is the research groups, namely "a" is the control group and "b" is the sample group of radiographers. 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 1 2 3 4 5 6 7 a b figure 2. curve of mda serum levels in the control group and in the sample group of radiographers. the curve above shows the results of the absorbance measurements of mda serum level in the research groups. the x-axis is the serum levels of mda (nmol / ml), y-axis is the research groups, namely “a” is the control group and “b” is the sample group of radiographers. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p110-114 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p110-114 113113aulia, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 110–114 in the sense of bitter taste, there was no increase in this sensation. this sense of bitter taste actually can arise because of a bond between the solutions of quinine as stimulants for bitter taste receptors. this reaction makes g-protein in the plasma membrane release α unit, which in sensory receptors of bitter taste is referred to gustducin. gustducin activates phospholipase enzyme, as a result, in these circumstances k+ channels are locked, then stimulating phospolipase c (plc) to activate phospatidylinositol 4,5-biphosphate (pip2) into inositol triphosphate (ip3) and diacylglycerol (dag). next, inositol triphosphate makes ca2+ released from the endoplasmic reticulum causing depolarization and then neurotransmitters, such as serotonin released that generates sensitive post-synaptic axon atp innervating senses of taste and connecting organs and peripheral sensory neurons in the back of the brain triggering a bitter taste that can be tasted.6 in the sense of sour taste, moreover, there was no decrease in this sensation of the radiographers. this is most likely due to the presence of acid (citric acid solution), which makes excitation applied on sour taste receptors in tongue and then arises a bond of h+ ions leading to the closure of k+ channels. the closing of k+ channel stimulates membrane depolarization and generates action potentials. the hydrogen ions then will trigger the sensation of sour taste.10 similarly, in the sense of salty taste, there also was no decrease in this sensation of the radiographers. it is probably because when nacl is applied on the tongue, it can trigger depolarization of the salty taste receptor cells through na+ channels similar to channels of sodium in epithelial cells (ephitelial-type na+ channels [enac]). ephitelial-type na + channels are specific sodium receptors involved in tasting salty taste. because of nacl solution in sufficient concentration, the achievement of threshold can occur so that salty taste can be felt.10 like in salty tatse, in sweet taste there also was no decrease in this sensitivity of taste. it’s likely caused when sucrose solution is applied on the tongue, the substance of sweet taste is then bound to g-protein-coupled receptors (gpcrs) that binds to g-protein gustducin found on the cell surface. gprotein complex is named gustducin because of the similarity of structure and action to transducin. next, this gprotein complex activates adenylyl cyclase as the second messenger to trigger adenosine triphosphate (atp) turning into cyclic adenosine 3’5’-monophosphate (camp) which then activates phosphokinase a until an ion channel phosphorylation occurs. k ion channel then will be closed, and depolarization occurs so that neurotransmitters are released and the stimulation of sensory neurons occurs, so sweet taste can be felt.11 in addition, in the sense of umami taste, there also was no decrease in this sensitivity of taste. this is possible because at the moment there is a solution of l-glutamate on the tongue. this substance is soluble in saliva and then can diffuse into pore taste through fluid layer to create a relationship with both receptor membrane on microvilli and apical membrane. mechanism of the substance reacting with taste villi starts receptor potential by binding chemicals to receptor molecules of umami taste, such as (metabotropic glutamate receptors 4 [mglur4], metabotropic glutamate receptors 1 [mglur1]), and dimer receptor (taste 1 receptor 1 [t1r1] + taste 1 receptor 3 [t1r3]) undergoing intracellular reaction and causing excitation of primary sensory afferents facilitating change of pip2 into ip3 and diacylglycerol. inositol triphosphate increases the activity of intracellular calcium and the release of calcium into cytoplasm. the release of intracellular calcium then stimulates the release of neurotransmitters which in turn causes sensitive post-synaptic axon atp innervates senses of taste and connects organs and peripheral sensory neurons behind the brain.6 simiarly, in the sense of spicy taste there also was no decrease in this sensitivity of taste since capsaisin solution applied on the tongue will be perceived by taste buds contained in papillae of the tongue by vanilloid receptor 1 (vr-1) or taste receptor potential vanilloid 12 (trpv-12), as a component of a molecule mediating the flow of hotactivated in nociceptors c, thus modulating the activity of cacade second meszinker in taste receptor cells (trcs). free nerve ending receptors on the taste buds are served by chorda tympani nerve and send spicy taste to the brain so that the brain will receive and interpret it as pain.6 in the normal mechanisms of stimulation to the senses of taste buds in radiographers, there are actually no distractions, so there are no changes in sensation of sour, salty, sweet, bitter, umami, and spicy. in addition, the taste bud cells also experience a change in growth, death, and regeneration. the taste bud cells are continuously replaced through mitotic division process of cells around, so some of them are young cells and more mature cells located towards the middle of the senses of taste and will soon break down and dissolve. the system is not only influenced by the taste buds cells, but also taste receptor cells and taste nerves.9) 8 the ability to taste in a person is also influenced by several things, including individual factors, threshold value, and concentration.12 it also can affect whether there is any change in the perceived sensitivity of taste on the tongue. based on the research results obtained, there was a significant difference in mda serum levels between the group of radiographers and the control group consisted of non-radiographers. the levels of mda serum in the group of radiographers were higher than in the control group consisted of non-radiographer since the blood sample taken has a high radiosensitivity and are considered as the cells that are sensitive to biological effects derived from radiation exposure.15 in addition, most of the human body is composed of water. radiation interacts with atoms or other molecules in the cells especially water, which then will produce free radicals. in the body, free radicals can cause lipid peroxidation process. in chemistry, free radicals are highly reactive and can alter important molecules in the cells.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p110-114 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p110-114 114 aulia, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 110–114 free radicals can also initiate lipid peroxidation directly against polyunsaturated fatty acid on the cell wall. free radicals will cause lipid peroxidation of cell membranes and produce mda compounds. lipid peroxides will be formed in the chain that is longer and can damage the cell membrane organization.5 since the radiation exposure received by the radiograhers was more often than the control group consisted of nonradiograhers, the mda serum levels in the group of radiograhers were higher. the high mda level may indicate the high level of free radicals in the body. the high level of free radicals in the body is very dangerous since it can cause damage to the cells by direct ionizing of dna molecules, causing chemical changes in the dna. the chemical changes in dna occur indirectly if the dna interacts with the hydroxyl free radicals. consequently, this condition then can cause adverse biological effects, such as damage to the cells of the taste buds and damage to acinar cells in salivary gland triggering discomfort in the oral cavity.13 the damage to cells of the taste buds may result in a decrease in sensitivity to sour, salty, sweet, umami, and spicy tastes. in addition, the radiation also can cause damage to acinar cells in salivary gland. those cells are very sensitive to radiation. the main effect of radiation on the salivary gland is xerostomia characterized by a decrease in the volume of saliva. the decline in the volume of saliva will cause disruption in distributing stimuli / impulses of the senses to the brain.2 the low radiation dose is still risky since changes to the biological system, both molecular and cellular, eventually will develop and lead to a severe effect on health, such as malignancy. thus, even a low dose of radiation exposure on tissue could increase the risk of cancer, which statistically can still be detectable in a population, but not necessarily associated with individual exposures. in this research, though there was no impairment of sensitivity of sensory taste, however, there was a significant increase in mda serum levels. this is because blood cells are more sensitive to radiation than the cells of taste buds. in addition, the biological changes in the body also can be affected by the absorbed dose, exposure distribution in the body, as well as the distribution of exposure time and age.9 the use of precise radiation dose and the right application of radiation protection are good for radiographers and also necessary to avoid long term negative biological effects. it can be concluded that there was no increase or reduction in the sensitivity of bitter, sour, sweet, salty, umami, and spicy tastes, but there was an increase in mda serum levels in the group of radiographers compared to the control group. references 1. nadhia ar, sunariani j, irmawati a. penurunan sensitivitas rasa manis akibat pemakaian pasta gigi yang mengandung sodium lauryl sulphate 5%. jurnal pdgi 2009; 58(2): 10-3. 2. mayerni a, ahmad a, abidin z. dampak radiasi terhadap kesehatan pekerja radiasi di rsud arifin achmad, rs santa maria dan rs awal bros. jurnal ilmu lingkungan. 2013; 7(1): 114-27. 3. henriksen th, maillie d, korchin rs. radiation and health. am assoc phys med 2003; 30(10): 264–87. 4. murray rk. biokimia harper (harper’s biochemistry). andry hartono, editor. edisi ke-25. jakarta: egc; 2003. p. 157-9, 619. 5. winarsi h. antioksidan alami dan radikal bebas; potensi dan aplikasinya dalam kesehatan. yogyakarta: kanisius; 2007. p. 57-5. 6. sunariani j. indera rasa pengecap di dalam rongga mulut. edisi ke-2. surabaya: gajah mada printing; 2014. p. 8-10. 7. mossman kl. taste acuity, plasma zinc, and weight loss during radiotherapy. radiology 1983; 146(3): 856-7. 8. sunariani j, yuliati, bestari a. perbedaan persepsi pengecap rasa asin antara usia subur dan usia lanjut. majalah ilmu faal indonesia 2007; 6(3): 182-91. 9. alatas z. efek radiasi pengion dan non pengion pada manusia. buletin alara 2004; 5(2-3): 99–112. 10. ganong wf. buku ajar fisologi kedokteran. edisi ke-22. djauhari widjajahkusuma, editor. jakarta: penerbit buku kedokteran egc; 2005. p. 182-5. 11. yunus b, dharmautama m. penilaian penempatan implan sebelum dan sesudah pemasangan implan gigi dengan pemeriksaan radiografi periapikal. dentofasial 2009; 8(2): 88-94. 12. puspitawati i, iriani ih, ratna ds. psikologi faal; tinjauan psikologi dan fisiologi dalam memahami perilaku manusia. edisi ke-1. bandung: pt remaja rosdakarya; 2012. p. 174-9. 13. fransiska tricia, pudji rahaju, rus suheryanto. hubungan status nutrisi penderita karsinoma nasofaring stadium lanjut dengan kejadian mukositis sesudah radioterapi. orli 2012; 42 (1): 53-63. 14. hong jh, pinar oo, brian ts. taste and odor abnormalities in cancer patients. j support oncol 2009; (7): 58–65. 15. nan su, victor c, miriam g. taste disorders: a review. j can dent assoc 2013; 79: d86. 16. prayitno g. perbedaan kadar haemoglobin, jumlah leukosit dan jumlah trombosit darah akibat paparan radiasi antara petugas radioterapi dan petugas radiodiagnostik (studi kasus di rsup dr. kariadi semarang). disertasi. semarang: program pasca sarjana universitas diponegoro; 2001. 17. lameshow s, hosmer kw, klar j, lwanga sk. adequacy of sample size in health studies. brisbane: john willey & sons; 1990. p. 42-4, 52-4, 64, 71-83. 18. woroprobosari nr, sunariani j, astuti er. vegf expression and new blood vessel after dental x-ray irradiation on fractured tooth extraction wound. dental journal (majalah kedokteran gigi) 2015; 48(3): 159-64. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p110-114 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p110-114 26 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 26–32 original article tumor necrosis factor-α and osterix expression after the transplantation of a hydroxyapatite scaffold from crab shell (portunus pelagicus) in the post-extraction socket of cavia cobaya irvan salim,1 michael josef kridanto kamadjaja2, agus dahlan2 1prosthodontics resident, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: socket preservation using bone graft is one way to minimise resorption and maximise the bone formation process. tumor necrosis factor-α (tnf-α) is an inflammatory cytokine that affects bone regenerating osteoblast activity, while osterix (osx) is an osteoblast-specific transcription factor that activates gene receptors during pre-osteoblast differentiation. the hydroxyapatite (ha) scaffold from crab shells (portunus pelagicus) has osteoconduction properties. purpose: to analyse the decrease of tnf-α expression and the increase of osx expression and the correlation between these two in the post-extraction socket after the transplantation of a crab shell ha scaffold. methods: the lower left incisors of cavia cobaya (n = 24) were extracted and divided into four groups: the first and second groups were control groups on day 7 and day 14 (k7 and k14), the third and fourth groups were treatment groups (p7 and p14). the statistical analysis used was a multivariate analysis of variance (manova) with a significance level of 0.05. results: a manova test showed that the use of crab shell ha scaffolds led to a significant difference (p < 0.05) in tnf-α expression (p = 0.01) and osx expression (p = 0.01). a pearson correlation test result showed a strong inverse correlation between tnf-α and osx expressions (p = 0.00 and r = -0.78). conclusion: the transplantation of ha scaffolds from crab shells can decrease tnf-α expression but increase osx expression in the post-extraction socket of c. cobayas. furthermore, an inverse correlation was found between tnf-α and osx. keywords: osterix; portunus pelagicus; scaffold; socket preservation; tumor necrosis factor-α correspondence: michael josef kridanto kamadjaja, department of prosthodontics, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47, surabaya, 60132, indonesia. email: michael-j-k-k@fkg.unair.ac.id introduction the procedure of denture fabrication needs a healthy alveolar bone and an ideal shape for the retention of the denture. tooth extraction without follow-up treatment can cause alveolar bone resorption; therefore, the aesthetic aspect and function of retention cannot be achieved. the alveolar bone changes shape not only in a vertical direction, but also in the lingual/palatal direction from the initial position, which causes the alveolar bone to become low, rounded or flat. this phenomenon is called residual ridge resorption.1 this could be avoided by preserving the socket using hydroxyapatite (ha), which can be obtained from crab shells. ha has been proven to have good biocompatibility and osteoconductive properties, meaning that it is well tolerated by the tissues of the human oral cavity and is able to stimulate osteoblast differentiation.2 ha also has the ability to induce mesenchymal cells to differentiate towards osteoblasts, which makes it a scaffold material for bone tissue engineering.3 ha has long-term biodegradable properties that slow down the bone repair process. a scaffold with a polymer matrix of natural materials, namely gelatine, is needed. in this study, gelatine was chosen as a scaffold material because of its excellent biocompatibility, biodegradability and porosity. hagelatine scaffolds are expected to increase the bioactivity, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 mailto:michael-j-k-k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 27salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 biocompatibility and mechanical properties of scaffolds and stimulate the growth of the bone tissue without affecting other mixing substances.4,5 the bone-remodelling process that is regulated by inflammatory mediators, osteoclasts and osteoblasts occurs after the procedure of a tooth extraction. activated macrophage cells (m1) produce tumor necrosis factor-α (tnf-α), which can affect the activity of osteoblasts and osteoclasts through the binding activity of the receptor activator of nf-κb (rank) and receptor activator of nf-κb ligand (rankl).6,7 osteoblast differentiation is a complex process involving the transcription factor osterix (osx) for the processes of osteoblast differentiation and bone formation. osx is an osteoblast-specific transcription factor that activates gene receptors during pre-osteoblast differentiation. it is known that preosteoblasts express transcription osx factors, and their number can affect osteoclasts’ activity in bone resorption. osx is also known as sp7 transcription factor. the absence of osx disrupts the formation of cortical bone and trabecular bone, both in intramembranous and endochondral ossification.8 bone remodelling is a process that occurs over time. in this process, old bone is removed, which is also known as the bone resorption process, and new bone is added, which is also known as the bone formation process. the cycle of normal bone remodelling can only happen if the bone resorption and bone formation processes occur in a coordinated manner, and this depends on the activation of osteoclasts and osteoblasts. the osteogenic response occurs between day 7 and day 25, while bone remodelling and maturation occur between day 14 and day 35. therefore, the examination of tnf-α and osx expressions in this study was carried out on day 7 and day 14.9,10 based on this background, this study’s aim is to analyse the decrease in tnf-α expression and the increase in osx expression and the correlation between the two in the post-extraction sockets of cavia cobaya that had been given ha scaffolds derived from portunus pelagicus. materials and methods this research received an ethical permit from the faculty of dental medicine, universitas airlangga, no. 547/ hrecc.fodm/viii/2019. the first step was to prepare ha powder from crab shells. crab shells were purchased from the pabean market in surabaya. they were then cleaned using distilled water (otsuka®, lawang, indonesia) and submersed in a chlorine solution (5 litres of water: 30 ml chlorine). the submersion was continued with 3% h2o2 (onemed ®, sidoarjo, indonesia) for 24 hours, and then the shells were dried at room temperature. the shell calcination process was carried out with a furnace at 1000°c for approximately 2 hours. the characterisation of ha compounds was done using a scanning electron microscope-energy dispersive x-ray (sem-edx; inspect™ s50 type tp 2017/12, fei company, hillsboro, or, usa) with a size of ± 150 µm.11 after the ha powder was created from the crab shells, the scaffold was prepared. five grams of gelatine (sigma-aldrich®, st. louis, mo, usa) were added to distilled water (otsuka®, lawang, indonesia) at 40°c and stirred for 1 hour. an ha-gelatine scaffold was made by adding 1.5 grams of crab shell ha powder to 0.5 grams of gelatine solution, stirring for 6 hours, and then centrifuging (hettich®, tuttlingen, germany) for 10 minutes to separate the water and the gel. the gel solution was placed on a custom resin acrylic mould (ortho resin®, england) with a diameter of 5 mm, a height of 2 mm and a pore size of 150 µm. it was then put in a freezer with a temperature of -80°c for 24 hours. furthermore, freeze drying (christ® beta 1-8 lscplus, osterode am harz, germany) was carried out for 24 hours.5,12 for the experimental animals, male c. cobaya (n = 24) aged 3–3.5 months that had a body weight of 300–350 grams and were healthy, with no injuries or disabilities, were obtained from the biochemistry laboratory of the faculty of medicine at universitas airlangga. each group of c. cobaya was placed in a cage measuring 50 x 70 x 50 cm and placed in a room with sufficient airflow and light. food was given ad libitum, with an emphasis on foods that contain a great deal of crude fibre, tubers, corn and other greens, every morning and evening. mineral water was provided in 300 ml bottles that were equipped with small pipes. the experimental animals were allowed to adapt for three days to obtain good general health and adaptation to the environment. the cage was placed in a place that was shady but got enough sunlight in the morning. the cage was placed a bit away from noise so that the experimental animals could be calm. the cages were placed in a dry place so as to prevent disease. the cages were free from the direct influence of strong winds, rain and scorching sun. the experimental animals were weighed to ensure they met the sample criteria.13 the left lower incisors of c. cobaya were extracted with a sterile needle holder, and the animals divided into four groups. each group contained six samples. group i was a control group that received no treatment until day 7 (k7). group ii was a control group that received no treatment until day 14 (k14). group iii was a treatment group in which the extraction socket was given a crab shell ha scaffold until day 7 (p7). group iv was a treatment group in which the extraction socket was also given a crab shell ha scaffold until day 14 (p14). the concentrations of the active ingredient processed from crab shells were 1.5 grams of ha and 0.5 grams of gelatine. the combination ha-gelatine scaffold was implanted in the c. cobaya socket, and then the post-extraction wound was sutured in the control groups and the treatment groups with polyamide monofilament suture thread, ds 12 3/8 c, 12 mm, 6/10 meth, 0.7 sterile (braun aesculap®, melsungen, germany).14 for the tissue samplings, groups i and iii were terminated on the 7th day after extraction to observe tnf-α dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 28 salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 and osx expressions. groups ii and iv were terminated on day 14 after extraction to compare the total tnf-α and osx expressions on day 7 and day 14. termination was carried out using ketamine (pfizer®, ny, usa) at a dose of 0.2 ml. the mandible was removed.15 the preparations of c. cobaya mandibular tissue were decalcified with ethylenediaminetetraacetic acid for 60 days. after that, they were processed using a paraffinembedding technique, and the samples were then sliced in a mesial-distal direction with a thickness of 4 µm. evaluation of the calculation of the number of tnf-α and osx expressions was carried out with immunohistochemical methods using tnf-α and osx monoclonal primary antibody and secondary antibody, catalog: anti-osx antibody (f-3): sc-393325, anti-tnf-α antibody (52b83): sc-52746 (santa cruz biotechnology, inc, dallas, tx, usa); each of the antibodies had 1:100 concentration. visualisation of immunohistochemical results was conducted with crftm anti-polyvalent hrp polymer (dab) lab pack, cpp-125 (scytek laboratories, inc, west logan, ut, usa) by counting the amount of tnf-α expressed by the cytoplasm of macrophage cells that had a brownish colour at the base of the extraction socket, and whether the macrophages exhibited an amoeboid, elongated spindle-like, or round shape depending on their lamellipodial extensions.16 also, the amount of osx transcription factor expressed by the cytoplasm of osteoblasts that had a brownish colour at the base of the extraction socket was measured. in terms of morphology, osteoblasts are cuboidal cells that are found at the interface of newly synthesised bone and are strongly basophilic in their cytoplasm.17 observations and counts of tnf-α and osx expressions were carried out using a microscope (nikon® e100) and sony® α 7 as a camera attachment with 1000x magnification and 20 fields of view, and they were done by one person. statistical analysis was conducted using statistical package for social science (spss) software version 21 (spss inc, chicago, il, usa). when examining the research data, the first step was to test for normality using the shapiro-wilk test. after the normality test was carried out, the research data were then processed for homogeneity using levene’s test. a multivariate analysis of variance (manova) test was conducted to analyse tnf-α and osx expressions in the post-extraction sockets of c. cobaya after the transplantation of ha scaffolds from crab shells. analysis was then continued using the tukey honest significant different method to detect the differences in the parameters of each group. a pearson correlation test was performed to determine the correlation between the tnf-α and osx expressions with a significance level of p < 0.05. results the histological appearance of the osteoblasts and osteoclasts after the transplantation of an ha scaffold from crab shells (p. pelagicus) in the post-extraction sockets of c. cobaya can be seen in figures 1 and 2. according to the statistical analysis all of the data were normally distributed and come from a homogenous population (p > 0.05). the transplantation of an ha scaffold from crab shells (p. pelagicus) showed the lowest mean tnf-α expression in the control group on day 14 (k14). in the control and treatment groups, the amount of tnf-α expression continued to decrease from day 7 to day 14. however, when the two treatment groups (p7 and p14) were compared, there was no significant decrease in tnf-α expression from day 7 to day 14. a manova test showed that the transplantation of the ha scaffold from crab shells (p. pelagicus) had the effect of decreasing tnf-α expression, with a significant difference in each group (table 1). the histological appearance of the tnf-α in the osteoblast can be seen in figure 3. figure 1. the histological appearance of the bottom of the extraction socket in the control group under 100x, 400x and 1000x magnification. red arrow = osteoclast, black arrow = osteoblast, and yellow arrow = macrophage. figure 2. the histological appearance of the bottom of the extraction socket in the treatment group under 100x, 400x and 1000x magnification. red arrow = osteoclast, black arrow = osteoblast, and yellow arrow = macrophage. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 29salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 table 1. mean of expression of tnf-α parameter control groups (mean ± sd) treatment groups (mean ± sd) p (manova p < 0.05) k7 k14 p7 p14 tnf-α 15.54 ± 2.460 13.68 ± 2.857 6.86 ± 2.300 6.25 ± 2.092 0.001* * significant at p < 0.05 k7 k14 p7 p14 figure 3. the histological appearance of tnf-α (indicated by red arrows) in the post-extraction socket under 1000x magnification. k7 = control group without treatment until day 7; k14 = control group without treatment until day 14; p7 = treatment group in which the extraction socket was given a crab shell ha scaffold until day 7; p14 = treatment group in which the extraction socket was also given a crab shell ha scaffold until day 14. k7 k14 p7 p14 figure 4. the histological appearance of the osx (indicated by red arrows) in a post-extraction socket under 1000x magnification. k7 = control group without treatment until day 7; k14 = control group without treatment until day 14; p7 = treatment group in which the extraction socket was given a crab shell ha scaffold until day 7; p14 = treatment group in which the extraction socket was also given a crab shell ha scaffold until day 14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 30 salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 the histological appearance of the osx in the osteoblast can be seen in figure 4. the transplantation of an ha scaffold from crab shells (p. pelagicus) showed the highest mean osx expression in the control group on day 7 (k7). in the control and treatment groups, the amount of osx expression continued to increase from day 7 to day 14. a manova test showed that the transplantation of an ha scaffold from crab shells (p. pelagicus) had an effect on the increase in osx expression with significant differences in each group (table 2). the results of the correlation test show that the mean tnf-α expression is inversely proportional to the osx expression (p = 0.00 and r = -0.78), which means there is a strong negative correlation. this shows that an increase in the expression of osx causes a decrease in the expression of tnf-α (table 3). a comparison of the inflammation value and the apposition value shows that the apposition value is more dominant. the expression of osx, which is a marker of apposition, is inversely proportional to the expression of tnf-α as a marker of inflammation. increasing the expression of osx will decrease the expression of tnf-α (figure 5). discussion ha is an inorganic biomaterial that composes about 67% of the mineral content in bone. ha is used because of its excellent biocompatibility to hard tissue in humans because chemically and physically, its mineral content is the same as bones and teeth in humans. the formation of chemical bonds with good tissue provides an advantage in the clinical application of ha as a bone substitute material.18 ha has been known to regenerate bone by conduction or by working as a scaffold to fill bone defects. ha is one of the materials that has osseointegration properties: osteoconduction, osteoinduction and osteogenesis; therefore, it can be used for a bone graft. the definition of osteoconduction is that in the ha function as a scaffold, it is able to induce and stimulate mesenchymal stem cells and osteoblasts to proliferate and differentiate in the formation of new bone or in the process of bone regeneration. meanwhile, gelatine is derived from collagen and has good biological properties. because the main organic portion of hard tissue is made of collagen, it has potential medical applications. among the advantages of gelatine are ductility and high efficiency, which can facilitate manufacturing.18–21 in this study, the variables that are focused on are tnf-α and osx, and the correlation between them. by studying these variables, inflammation and apposition in postextraction sockets can be compared. the research results of the biomarker tnf-α showed a significant difference between the control group (k7 and k14) and the treatment group (p7 and p14) after the transplantation of an ha scaffold from crab shells (p. pelagicus). the correlation test results showed that the transplantation of ha caused an increase in osx expression and a decrease in tnf-α expression. these results are in accordance with the research hypothesis, which stated that the transplantation ha scaffold from crab shells (p. pelagicus) would decrease tnf-α expression in the postextraction sockets. in this research, we used a combination of ha and gelatine. although the gelatine in this study was used only as a binding agent, we can further improve the effectiveness table 3. correlation test results of tnf-α and osx expressions control variables osx tnf-α groups osx correlation 1.000 -.779 significance (2-tailed) . .0000 df 0 25 tnfα correlation -.779 1.000 significance (2-tailed) .000 . df 25 0 figure 5. graph of tnf-α and osx expressions. table 2. mean of expression of osx parameter control groups (mean ± sd) treatment groups(mean ± sd) p (manova p < 0.05) k7 k14 p7 p14 osx 6.29 ± 2.44 6.00 ± 3.54 17.29 ± 4.04 13.07 ± 1.18 0.001* * significant at p < 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 31salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 of this treatment by adding silver. as demonstrated in previous studies, which used a combination of ha and silver to reduce the release of nitric oxide and decrease the secretion of il-1 and tnf-α in cells that were stimulated with lipopolysaccharide (lps), lps stimulates monocytes/ macrophages through tlr4, resulting in the activation of a series of signalling events that potentiate the production of inflammatory mediators.22,23 there is a correlation between the particle size of ha and the secretion of tnf-α. a previous study showed that a smaller ha particle size (especially a diameter of 1-2 µm and a pore size of 10-50å) stimulates a higher macrophage tnf-α secretion. in this study, we used an ha particle size of 150-350 µm; therefore, this material exhibited a lower macrophage tnf-α secretion. the mechanism of this result is not yet understood, but it requires the internalisation of crystal microaggregates into phagocytic vacuoles and the recruitment of protein kinase c to the vacuole membrane.24 at 10 days post-extraction, tnf-α expression was almost entirely absent, which means that inflammation was not prolonged. tnf-α plays an important role in bone resorption. several previous studies have shown that tnf-α increases because the host response is stimulated by plaque, bacterial products and increased osteoclast activity, consequently accelerating bone resorption and periodontal destruction. in other words, a decrease in tnf-α can reduce the host response, reducing the expression of cytokines that stimulate bone resorption, resulting in less bone loss.25 the acute inflammatory response peaks in the first 24 hours, although the proinflammatory molecules later also play an important role in regeneration, while tnf-α concentrations peaked in the first 24 hours and returned to baseline within 3 days post-trauma.26 a previous study investigated the potential impact of tnf-α on the teeth of zucker diabetic fatty rats with diabetes and periodontitis, which was induced by ligature for 7 days. the results of the study indicated that the tnf-α from the test group reached a peak on the third day, and tnf-α expression decreased gradually on the following days. therefore, it is advisable to conduct research with tnf-α biomarkers, and further examinations need to be added on day 1 and day 3.27 in contrast, in the study with the osx biomarker and the data from day 7 and day 14, the highest mean number of osx expression was found in the treatment group on day 7, but this number began to decline on day 14. this is because osx is a transcription factor that is important for osteoblast differentiation in the early stages, but it inhibits osteoblast differentiation at a late stage. however, the function of osx at the late stage of osteoblast differentiation is not fully elucidated; instead, runt-related transcription factor 2 (runx2) plays an important role in inhibiting osteoblast differentiation at a late stage.28,29 however, there was a significant difference between the control groups (k7 and k14) and the treatment groups (p7 and p14). ha is able to stimulate osteoblast differentiation; ha made from crab shells (p. pelagicus) also contains a high level of calcium carbonate (caco3) of around 40–70%. calcium carbonate has properties that are easily absorbed by tissues, easily biodegradable and osteoconductive; as a result, it can support the process of forming new bone.2,30 these results are in accordance with the research hypothesis, which stated that the transplantation of an ha scaffold from crab shells (p. pelagicus) would increase osx expression in the postextraction sockets. in previous studies, the transplantation of an ha scaffold from crab shells (p. pelagicus) showed positive results for reducing the osteoclasts’ number and increasing the osteoblasts’ number in the post-extraction socket when examined on days 14 and 28. it can increase the expression of osteoprotegerin (opg), osteocalcin, collagen type 1 and transforming growth factor-β1 (tgf-β1) and decrease rankl expression.31–36 in conclusion, the transplantation of an ha scaffold from crab shells (p. pelagicus) can reduce tnf-α expression in the post-extraction socket on the day 7. there was an inversely proportional correlation between tnf-α and osx after providing an ha scaffold from crab shells (p. pelagicus). however, for further studies that evaluate tnf-α biomarkers, it is important to add silver and also to include examinations on day 1 and day 3. references 1. silvia p, nasution id. bentuk residual ridge dan hubungannya dengan retensi gigi tiruan penuh. cakradonya dent j. 2016; 8(1): 55–60. 2. adventa y, zubaidah n. the role of hydroxyapatite materials on collagen synthesis in alveolar bone defects healing. conserv dent j. 2021; 11(1): 24–7. 3. wang c, duan y, markovic b, barbara j, rolfe howlett c, zhang x, zreiqat h. proliferation and bone-related gene expression of osteoblasts grown on hydroxyapatite ceramics sintered at different temperature. biomaterials. 2004; 25(15): 2949–56. 4. hoque me, nuge t, yeow tk, nordin n, prasad rgs v. gelatin based scaffolds for tissue engineering – a review. polym res j. 2014; 9(1): 15–32. 5. kim h-w, knowles jc, kim h-e. hydroxyapatite and gelatin composite foams processed via novel freeze-drying and crosslinking for use as temporary hard tissue scaffolds. j biomed mater res a. 2005; 72(2): 136–45. 6. abbas ak, lichtman ah, pillai s. cellular and molecular immunology. 9th ed. philadelphia: eli lilly & company and the national center for advancing translational sciences; 2004-; 2017. p. 608. 7. bradley jr. tnf-mediated inflammatory disease. j pathol. 2008; 214(2): 149–60. 8. nakashima k, zhou x, kunkel g, zhang z, deng jm, behringer rr, de crombrugghe b. the novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. cell. 2002; 108(1): 17–29. 9. kini u, nandeesh b. radionuclide and hybrid bone imaging: physiology of bone formation, remodeling, and metabolism. fogelman i, gnanasegaran g, van der wall h, editors. radionuclide and hybrid bone imaging. berlin: springer-verlag berlin heidelberg; 2012. p. 1046. 10. z hou f h , fost e r bk , sa nde r g, x ia n c j. e xp r ession of proinflammatory cytokines and growth factors at the injured growth plate cartilage in young rats. bone. 2004; 35(6): 1307–15. 11. raya i, mayasari e, yahya a, syahrul m, latunra ai. shynthesis and characterizations of calcium hydroxyapatite derived from crabs shells (portunus pelagicus) and its potency in safeguard against to dental demineralizations. int j biomater. 2015; 2015: 469176. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 32 salim et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 26–32 12. hendrijantini n, kresnoadi u, salim s, agustono b, retnowati e, syahrial i, mulawardhana p, wardhana mp, pramono c, rantam fa. study biocompatibility and osteogenic differentiation potential of human umbilical cord mesenchymal stem cells (hucmscs) with gelatin solvent. j biomed sci eng. 2015; 8(7): 420–8. 13. kusumawati d. bersahabat dengan hewan coba. yogyakarta: gadjah mada university press; 2004. p. 5–22. 14. k resnoadi u. the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast due to the induction of the combination of aloe vera and 2% xenograft concelous bovine. dent j (majalah kedokt gigi). 2012; 45(4): 228–33. 15. waynforth h, flecknell p. experimental and surgical technique in the rat. 2nd ed. academic press; 1992. p. 398. 16. lee h-s, stachelek sj, tomczyk n, finley mj, composto rj, eckmann dm. correlating macrophage morphology and cytokine production resulting from biomaterial contact. j biomed mater res a. 2013; 101(1): 203–12. 17. rutkovskiy a, stensløkken k-o, vaage ij. osteoblast differentiation at a glance. med sci monit basic res. 2016; 22: 95–106. 18. ardhiyanto hb. peran hidroksiapatit sebagai bone graft dalam proses penyembuhan tulang. stomatognatic. 2011; 8(2): 118–21. 19. kusrini e, sontang m. characterization of x-ray diffraction and electron spin resonance: effects of sintering time and temperature on bovine hydroxyapatite. radiat phys chem. 2012; 81(2): 118–25. 20. de santis r, russo a, gloria a, d’amora u, russo t, panseri s, sandri m, tampieri a, marcacci m, dediu va, wilde cj, ambrosio l. towards the design of 3d fiber-deposited poly(ε-caprolactone)/ iron-doped hydroxyapatite nanocomposite magnetic scaffolds for bone regeneration. j biomed nanotechnol. 2015; 11(7): 1236–46. 21. chen l, ma l, zhou m, liu y, zhang y. effects of pressure on gelatinization of collagen and properties of extracted gelatins. food hydrocoll. 2014; 36: 316–22. 22. martínez-sanmiguel jj, g zarate-triviño d, hernandez-delgadillo r, giraldo-betancur al, pineda-aguilar n, galindo-rodríguez sa, franco-molina ma, hernández-martínez sp, rodríguez-padilla c. anti-inf lammatory and antimicrobial activity of bioactive hydroxyapatite/silver nanocomposites. j biomater appl. 2019; 33(10): 1314–26. 23. fang h, pengal ra, cao x, ganesan lp, wewers md, marsh cb, tridandapani s. lipopolysaccharide-induced macrophage inflammatory response is regulated by ship. j immunol. 2004; 173(1): 360–6. 24. nadra i, boccaccini ar, philippidis p, whelan lc, mccarthy gm, ha sk a rd do, l a nd is rc. ef fe ct of pa r t icle si z e on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages. atherosclerosis. 2008; 196(1): 98–105. 25. kim d-j, cha j, yang c, cho a, lee j-s, jung u, kim c, lee s, choi s. changes in periodontium after extraction of a periodontallyinvolved tooth in rats. j periodontal implant sci. 2012; 42(5): 158–65. 26. marsell r, einhorn ta. the biology of fracture healing. injury. 2011; 42(6): 551–5. 27. liu r, bal hs, desta t, krothapalli n, alyassi m, luan q, graves dt. periodontal bone loss through enhanced resorption and. j dent res. 2006; 85(6): 510–4. 28. neve a, corrado a, cantatore fp. osteoblast physiology in normal and pathological conditions. cell tissue res. 2011; 343(2): 289–302. 29. yoshida ca, komori h, maruyama z, miyazaki t, kawasaki k, furuichi t, fukuyama r, mori m, yamana k, nakamura k, liu w, toyosawa s, moriishi t, kawaguchi h, takada k, komori t. sp7 inhibits osteoblast differentiation at a late stage in mice. plos one. 2012; 7(3): e32364. 30. dahlan a, hidayati he, hardianti sp. collagen fiber increase due to hydroxyapatite from crab shells (portunus pelagicus) application in post tooth extraction in wistar rats. eurasian j biosci. 2020; 14(2): 3785–9. 31. gatia nas, laksono h. penurunan jumlah sel osteoklas pada pemberian hidroksiapatit berbasis cangkang kepiting (portunus pelagicus) pasca ekstraksi gigi tikus wistar. thesis. universitas airlangga: surabaya; 2018. p. 1–62. 32. novitananda a, kamadjaja mjk. peningkatan jumlah sel osteoblas pada pemberian hidroksiapatit berbasis cangkang kepiting (portunus pelagicus) pasca ekstraksi gigi tikus wistar. thesis. universitas airlangga: surabaya; 2018. p. 1–68. 33. chumaidi g, kamadjaja mjk. ekspresi osteokalsin pasca pemberian scaffold hidroksiapatit dari cangkang kepiting (portunus pelagicus) pada soket pasca pencabutan gigi marmut (cavia cobaya). thesis. universitas airlangga: surabaya; 2019. p. 1–51. 34. herdianti nc, kamadjaja mjk. ekspresi kolagen tipe-1 paska pemberian hidroksiapatit scaffold dari cangkang kepiting (portunus pelagicus) pada soket pasca pencabutan gigi marmut (cavia cobaya). thesis. universitas airlangga: surabaya; 2019. p. 1–53. 35. maharmardoyo sk, kamadjaja mjk, soekobagiono s. ekspresi transforming growth factor-β1 (tgf-β1) paska pemberian scaffold hidroksiapatit dari cangkang kepiting (portunus pelagicus) pada soket pasca pencabutan gigi marmut (cavia cobaya). thesis. universitas airlangga: surabaya; 2019. p. 1–56. 36. k a madjaja m j k , sa l i m s, subia k to bds. appl icat ion of hydroxyapatite scaffold from portunus pelagicus on opg and rankl expression after tooth extraction of cavia cobaya. res j pharm technol. 2021; 14(9): 4647–51. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p26–32 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p26-32 130 correlation between magnesium and alkaline phosphatase from gingival crevicular fluid on periodontal diseases nila kasuma department of oral biology faculty of dentistry, universitas andalas padang– indonesia abstract background: magnesium is one of the alkaline phosphatase (alp) cofactor. the amount of magnesium contained infoods affect alp activity. increased alp activity will indicate the level of inflammation in periodontal disease. elevated inflammation in periodontal disease will change gingivitis to periodontitis, where there has been damage to the bone ssupporting the teeth, and an increasing number of gingival crevicular fluid (gcf). the content of gcf consists of enzymatic and non-enzymatic. changes in the composition of gcf occurs when the inflammation gets worse. purpose: this study was aimed to prove the correlation between magnesium and alp from gcf on periodontal disease. method: this research involved 60 minangkabaunese people with 20 healthy samples, 20 mild gingivitis samples, and 20 mild periodontitis samples. gcf was collected by absorbing method. then alp level in gcf was measured by using elisa technique. magnesium level in minangkabaunese food was tested by food frequency questionnaire (ffq).univariate analysis was performed to describe each variable. to see a normal distribution, kolmogorov smirnof test was used (p>0.05). unpaired t-test and pearson correlation test was used to see correlation between alp and magnesium level in minangkabaunese food. result: there is a significant correlation between the levels of alp and magnesium level in minangkabaunese food with periodontal disease (p=0.005). alp is higest on mild periodontitis (137.74±23.01 ng/dl). magnesium level normal control group is highest (250.14±32.34 mg) and in mild periodontitis is the lowest (110.83±21.04 mg). corelation between alp and magnesium level indicates strong correlation with negative direction (r=0.907). conclusion: there is correlation between the levels of alkaline phosphatase and magnesium level on periodontal disease. increasing inflamation rate will elevate the alp level. keywords: alkaline phosphatase; magnesium; periodontal disease correspondence: kasuma n, c/o: departemen biologi oral; fakultas kedokteran gigi universitas andalas. jln. perintis kemerdekaan no. 77 padang, indonesia. e-mail: nilakasuma10@gmail.com research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 130–134 introduction alkaline phosphatase (alp) is produced by many cells. alp is the major source of polymorphonuclear (pmn), bacteria in supragingival plaque and subgingival and activity of osteoblasts and fibroblasts, and a small contribution to the serum.1 alp predominantly found in the pocket epithelium. if the main source of alp in periodontal disease is pmn, the alp is a potential marker for inflammation.2 increased accumulation of plaque and inflammation will improve periodontal tissues pathological inflammation. it triggers bacterial endotoxin to activate prostaglandins , which in can activate osteoclasts . in this condition b cells are stimulated to produce il-1b and tnfa to destroy bone, then it causes bone loss. increased activity in periodontal disease is due to increased inflammation and bone turnover rate. periodontal disease progress will be equal with alp activation. due to the increased severity of the inflammation, bone turnover rate elevates.3,4 one of alp cofactor is magnesium. most studies5 have agreed on the correlation of magnesium and alp activity. the results show that assays of alp activity in homogenised tissue samples will give better responses if both mg2+and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p130-134 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p130-134 kasuma/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 130–134 131131 zn2+ ions are included in the reactions. the purpose of this study is to see the relationship between magnesium level and concentration of alp in gingival crevicular fluid on normal, mild gingivitis and periodontitis group. materials and methods this study is a cross sectional comparative study, dependent and independent variable is examined on the same time to see the magnesium level in 3 groups of sample. samples were taken by consecutive sampling technique based on exclusion and inclusion criteria. population is patients who seek for treatment in dental polyclinic of rsud padang–west sumatera–indonesia.the sample consisted of ethnic minangkabaunese who consume minangkabau food daily.ages ranged between 17-30 years. to minimalize the local factor which can influence the activity of alp, at the time of examination , patients are systemically were sistemically healthy and didn’t consume antibiotics during the last 3 month. patients also didnt have any teeth caries. if samples consume antibiotics and antiinflamatory during the last 3 months, have any teeth caries, smokers, pregnant, menstruation, have a systemic disorders such as diabetes melitus,and got a history of periodontal treatment during the last 3 months are excluded from this research. informed consent was obtained from all patients. the first examination of periodontal tissues using the periodontal disease index (pdi) according to ramfjord.6 examination using the instrument a periodontal probe. this tool is used to measure the depth of pockets. normal depth of the pockets around 0-3 mm. scores pdi are: 0=healthy gingiva, absence of signs of inflammation no bleeding and no attachment loss, with coral pink colored gums; 1=mild to moderate inflammatory changes not extending around the tooth; 2=mild to moderately severe gingivitis extending all around the tooth; 3=severe gingivitis characterized by marked redness, swelling, tendency to bleed and ulceration; 4=mild periodontitis, assigned if the loss of attachment is 3 mm or less; 5=is assigned if the loss of attachment is greater than 3 mm but less than 6 mm; 6=if the loss of attachment is 6 mm or more, a score of 6 is given to a particular tooth. six measurement criteria teeth region (16, 21, 24, 36, 41 and 44) on mesial, bucal, distal, and lingual site represent periodontal disease indexes. if a ramfjord tooth was missing, a substitute tooth which was selected is teeth numbers 17, 11, 25, 37, 31, 45. in this study only 3 groups (healthy control, mild gingivitis, and mild periodontitis) were taken. selection of 3 groups of pdi is to examine changes on healthy, mild gingivitis, mild periodontitis group. clinical charactheristic of mild gingivitis are erythema and minimal bleeding on probing, the same as the characteristic of stage ii of gingivitis, the early lession. comparison of alp level on the three groups illustrates the increase of alp level is detectable on mild gingivitis and mild periodontitis state. according to sanikop, alp level increases in mild gingivitis due to tissue alteration as a result of host-parasite reaction or host-bacterial interplay as gingivitis is an inflammatory process. during progression of the disease to mild periodontitis, enzymes are released from dead and dying cells of the periodontium, pmns, inflammatory, epithelial, and connective tissue cells of the affected sites, so the level in mild periodontitis is highest among the three groups.1 increased alp level in early inflammatory can expect the early detection of periodontal disease. each group consisted of 20 persons. gingival crevicular fluid (gcf) was collected using absorbent paper (2.55 mm wide, 0.16 mm thick, and 14.19 mm long; 8.07 mm of the length was identified as a handling area with a blue tape).8 gcf sample was stored at -20c. reagent use is elisa kit for alp,9 homosapiens (human), se91472hu with detection range 3.12-200 ng/ml and sensitivity1.36 ng/ml, uscn product by spectrophotometer variant hemoglobin testing-in2it analyzer – bio rad. the gcf was diluted 20 times, 10µl is added to 190µl rd510 calibrator diluent. gcf then diluted 40 times that 10µl sample was added to 390µl calibrator diluent. diluted gcf is taken 50 ml and then was diluted 7 times with 150 ml of normal saline, to the make 200µl of sample. the diluted gcf was then centrifuged for 3–5 minutes at 3500–4500 rotations per minute (rpm) in a microcentrifuge; 140µl of the clear supernatant was utilized for the analysis of alp level.the enzyme level was recorded using elisa readerat a wavelength of 450 nm. gcf was collected in the morning at 08.00 am according to circardian periodicity. collecting area was minimalized of plaque. to equalize the conditions and minimize the involvement of oral bacteria instructed the patient to rinse using a solution of 2% chlorhexidine. then the lips retracted and isolated using cotton roll. absorbent paper is inserted by using the technique of superficial intracrevicular and left for 30 seconds.10 sample which is contaminated by blood will not be taken. absorbent paper is taken and placed in eppendorf tube that has been filled with 1 ml phosphate buffer solution. specimens are labeled. the samples taken is stored at -20 c and be analyzed using elisa. then participant answered a food frequency questionnaire consist of minangkabau food. before answering questionnaire, participant is shown 220 kinds food dummies to help remembering food they consume daily. portion, kind, and size of food has been determined. in this study, we use foods which contain magnesium. on the ffq interview, sample selected dummy food. this is useful for reducing bias in answering this questionaire. in this study, the relationship between magnesium level in food and alp level is tested at the same time. sample was divided into three groups of healthy , mild gingivitis and mild periodontitis. then gcf is collected to measure alp level. patients were interviewed by a professional nutritionist using ffq method which is equipped with a dummy table of all 220 kinds of food. magnesium level measurement is carried out from ffq in dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p130-134 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p130-134 132 kasuma/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 130–134 220 kinds of minangkabaunese food with way of cook like curry, frying, boiling, grilling, and stir-frying. alp and magnesium level were tabulated and tested statistically using the kolmogorov smirnof test to determine normal distribution of data. pearson correlation test was used to see then correlation of alp and magnesium. results samples were taken from patients who visited the hospital dental clinic rasidin municipal padang for 4 months from june to december 2014. the results of the examination of 200 prospective research subjects than 1200 visitors to the hospital dental clinic rasidin municipal padang, has netted 60 subjects consisted of 20 healthy subjects, 20 subjects with mild gingivitis and 20 subjects with mild periodontitis minangkabaunese foods and met the inclusion criteria through consecutive sampling method. based on table 1 there is significant difference in the levels of alp on the terms of the pdi group, which is highest in the mild periodontitis with mean=137.74± 23.01ng/dl. the table above shows the mild gingivitis patients likely to have elevated levels of alp 3,6 -fold compared to healthy condition, while the condition of mild peridontitis rose 5,3-fold compared to healthy conditions. based on table 2, there is significant difference in the levels of magnesium level on the terms of the pdi group, which is lowest in the mild periodontitis with mean=110.83 ±21.04 mg. the table shows the mild gingivitis patients likely to have elevated levels with mean=164±18.31 mg. in healthy condition with mean=250.14±22.34 mg. statistic analyze by using pearson correlation test shows that coefficient of pearson correlation (r) is -0.907 with significance level (p) 0.000 (p<0.05) between alp enzyme and magnesium. discussion the results of statistical tests in this study found a significant difference between the levels of alp in gcf with periodontal disease (p<0.05). alp in gcf was significantly higher in periodontitis compared to healthy samples and gingivitis. amongst the host enzymes in gcf, alp was one of the first to be identified. alp is a membrane bound glycoprotein produced by many cells within the area of periodontium and gingival crevice. it is released from polymorphonuclear neutrophils during inflamation, osteoblast during bone formation, periodontal ligament fibroblast during periodontal regeneration.7 alp activity is valuable to clinicians because enzymatic modification occure at the gcf level earlier than clinically evident modification. alp is synthesized and secreted by osteoblasts during the period of phenotypic maturation of osteoblasts, three consecutive phases of proliferation, extracellular matrix maturation and mineralization in bone tissue.11 thus it makes sense that osteoblasts synthesize and secrete alkaline phosphates creating a local bone environment of alkalinity by splitting of phosphorus (an acidic mineral) creating an alkaline ph to help bone mineralization. alp is normally presents in high concentration in growing bones and it is also known as zinc dependent enzyme. increased activity in periodontal disease will equal with alp activation. due to the increased severity of the inflammation, bone turnover rate elevates.4,2,12 bone turnover is a process in which the circulation of bone osteoclasts break down bone, and osteoblasts work rebuilding bone. in severe periodontal disease, the increased bone turnover intensifies work of destroying osteoclasts in bone.13-15 alp is an enzyme the which is synthesized by the liver, bone, and less amount by the intestines and kidney. as the name implies, this enzyme works best at an alkaline table 1. alkaline phosphatase levels (ng/dl) in gingival crevicular fluid on healthy, mild gingivitis and mild periodontitis group enzyme pdi f mean sd p alp healthy (score 0) 20 25.78 19.69 0.00 mild to moderate inflammation on gingiva (score 1) 20 92.92 19.23 mild periodontitis (score 4) 20 137.74 23.01 total 60 85.48 61.93 table 2. magnesium levels (mg) from ffq on healthy, mild gingivitis and mild periodontitis group pdi f mean sd p magnesium healthy (score 0) 20 250.14 22.34 0.00 mild to moderate inflammation on gingiva (score 1) 20 164 18.31 mild periodontitis (score 4) 20 110.83 21.04 total 60 174.99 61.69 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p130-134 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p130-134 kasuma/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 130–134 133133 ph (ph≥10), and thus the enzyme itself is inactive in the blood. alp removes phosphate groups (known as dephosphorylation) from many types of molecules, including nucleotides, proteins, and alkaloids.1,2 alp contains two magnesium ion (mg2+) and four zinc ion (zn+). thus, these metal ions are required in defined optimal ratio. magnesium ion is usually employed as the only cofactor of alp. the alp from various mammalian tissues are among the enzymes known to be activated by magnesium in isolated systems.5 in this study, magnesium level is the lowest on mild periodontitis, and highest on healthy control group. meisel et al. investigated the association between magnesium status and periodontal health in a population based analysis. increased serum magnesium was significantly associated with reduced probing depth, attachment loss, and high number of remaining teeth. a decrease of magnesium will be followed by a decrease in alp activity. long-term loss of magnesium from the bone causes disturbances of bone modeling, remodeling, and turnover, with resultant bone abnormalities.nutritional magnesium supplementation may improve periodontal health.16 magnesium is the fourth most abundant mineral and the second most abundant intracellular divalent cation and has been recognized as a cofactor for >300 metabolic reactions in the body. some of the processes in which magnesium is a cofactor include, but are not limited to, protein synthesis, cellular energy production and storage, reproduction, dna and rna synthesis, and stabilizing mitochondrial membranes. magnesium also plays a critical role in nerve transmission, cardiac excitability, neuromuscular conduction, muscular contraction, vasomotor tone, blood pressure, and glucose and insulin metabolism.17 because of magnesium’s many functions within the body, it plays a major role in disease prevention and overall health. low levels of magnesium have been associated with a number of chronic diseases including migraine headaches, alzheimer’s disease, cerebrovascular accident (stroke), hypertension, cardiovascular disease, and type 2 diabetes mellitus. good food sources of magnesium include unrefined (whole) grains, spinach, nuts, legumes, and white potatoes (tubers). this review presents recent research in the areas of magnesium and chronic disease, with the goal of emphasizing magnesium’s role in disease prevention and overall health.18 the body of most animals contains ∼0.4 g magnesium/kg.14 the total magnesium content of the human body is reported to be ∼20 mmol/kg of fat-free tissue. in other words, total magnesium in the average 70 kg adult with 20% (w/w) fat is ∼1000 to 1120 mmol or ∼24 g. about 99% of total body magnesium is located in bone, muscles and non-muscular soft tissue.19 magnesium concentration can affect the stimulation o f p h o s p h a t a s e a c t i v i t y . 1 9 t h i s s u g g e s t s t h a t magnesiummediates stabilization and destabilization of the catalytically active structure of alp concentrations at low and high respectively. magnesium is thought to have a regulatory effect on the expression of catalytic activity and maintenance of the structural integrity of the enzyme.5 magnesium alone does not activate the apoenzyme but it regulates the nature of the zinc dependent restoration of catalytic activity to apophosphatase, increasing the activity of the enzyme containing 2 g atoms of zinc five fold and that of enzyme containing 4 g atoms of zinc 1.4-fold. hence magnesium, the which is specifically bound to the enzyme, both stabilizes the structure and dynamic protein regulates the expression of catalytic activity by zinc in alkaline phosphatase.20 magnesium acts as an activator within optimal concentrations but became inhibitory at higher concentration. one possible explanation for this result is that excess of magnesium ions displaced zinc ion from the catalytic site since both metal ion can bind to the same site. the higher magnesium intake the lower alp activity will be.5 thus periodontal disease progression also decrease. practitioner can also provide appropriate additional therapy by giving magnesium supplement to suppress the periodontal inflammation.20 in conclusion, there is correlation between the levels of alkaline phosphatase and magnesium level on periodontal disease. increasing inflamation rate will elevate the alp level. references 1. sanikop s, patil s, agrawal p. gingival crevicular fluid alkaline phosphatase as a potential diagnostic marker of periodontal disease. j indian society of periodontology 2012; 16(4): 513-8. 2. malhotra r, grover v, kapoor a, kapur r. alkaline phosphatase as a periodontal disease marker. indian j dent res 2010; 21: 531-6. 3. guerrera, mp, stella lv, jun jm. therapeutic uses of magnesium. am fam physician 2009; 80(2): 157-62. 4. al-rawi na, rashad jm. salivary alkaline phosphatase and periodontal disease. j bagh college dentistry 2013; 25(1): 137-9. 5. lorunniji fj, adedoyin i, joseph oa, rotimi oa, sylvia om. cofactor interactions in the activation of tissue non-specific alkaline phosphatase: synergistic effects of zn2+ and mg2+ ions . biokemistri 2007; 19(2): 43-8. 6. ramfjord sp. the periodontal diesase index (pdi). j periodontol 1967; 38: 602-10. 7. carranza fa. the periodontal pocket. in: newman mg, takei hh, klokkevold pr, carranza fa, eds. clinical periodontology, 11th ed. st. louis: saunders-elsevier; 2012. p. 434-51. 8. silva fga, sabrina cg. validation of an alternative absorbent paper for collecting gingival crevicular fluid. periodontia 2009; 19(3): 85-90. 9. kim hj, kwak j. electrochemical determination of total alkaline phosphatase in human blood with a micropatterned ito film. j electroanalytical chemistry 2005; 577(2): 243-8. 10. guentsch a, kramesberger m, sroka a, pfister w, potempa j, eick s. comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis. j periodontol 2011; 82(7): 1051-60. 11. perinetti g, paolantonio m, femminella b, serra e, spoto g. gingival crevicular fluid alkaline phosphatase activity reflects periodontal healing/recurrent inf lammation phases in chronic periodontitis patients. j periodontol 2008; 79: 1200. table 3. correlation analysis of alp and magnesium level in minangkabaunese food level of alp level of magnesium r p -0.907 0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p130-134 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p130-134 134 kasuma/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 130–134 12. üsal b, işıl s, özlem d, bal b, bolu e. the relationship between periodontal status and alkaline phosphatase kadars in gingival crevicular fluid in men with hypergonadotropic hypogonadism. yonsei med j 2008; 49(1): 71–8. 13. neve a, addolorata c, francesco pc.osteoblast physiology in normal and pathological conditions. 2010. cell tissue res. 14. seibel mj. biochemical markers of bone turnover part i: biochemistry and variability. clin biochem rev 2005; 26(4): 97–122. 15. rani k, sanchi d, rachita r. brief review on alkaline phosphatases. int j microbiol and bioinformatics 2012; 2(issue 1): 1-4. 16. meisel f, schwan c, luedeman j, john u, kroener hk, kocher t. magnesium deficiency is associated with periodontal disease. j dnt res 2005; 84(10): 937-41. 17. grober u, joachim s, klaus k. magnesium in prevention and therapy. nutrients 2015; 7(9): 8199-226. 18. volpe sl. magnesium in disease prevention and overall health. adv nutr 2013; 4: 378s-383s. 19. wilhelm jd, markus k. magnesium basics. clin kidney j 2012; 5(suppl 1): i3-i14. 20. farah hs, ali aa, gaber ms. explanation of the decrease in alkaline phosphatase (alp) activity in hemolysed blood samples from the clinical point of view: in vitro study. j of biological sci 2012; 5(2): 125-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p130-134 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p130-134 213 volume 46, number 4, december 2013 research report prevotella intermedia and porphyromonas gingivalis in dental caries with periapical granuloma risya cilmiaty,1 afiono agung prasetyo,2,3,4 khilyat ulin nur zaini,3,4 mandojo rukmo,5 suhartono taat putra6 and widya asmara7 1department of dental and oral disease, faculty of medicine, universitas sebelas maret, surakarta-indonesia 2department of microbiology, faculty of medicine universitas sebelas maret, surakartaindonesia 3a-igic research group, universitas sebelas maret, surakarta-indonesia 4center of biotechnology and biodiversity research and development, universitas sebelas maret, surakarta-indonesia 5department of conservative dentistry, faculty of dentistry universitas airlangga, surabaya-indonesia 6department of pathobiology, faculty of medicine universitas airlangga, surabaya-indonesia 7 department of microbiology, faculty of veterinary medicine universitas gadjah mada, yogyakarta-indonesia abstract background: dental caries with necrotic pulp is a multifactorial disease that attacks enamel involving tooth pulp. the anaerobic bacteria infection in the pulp chamber could induce the formation of periapical granuloma. however, the presence of the most frequently anaerobic bacteria identified in apical periodontitis, porphyromonas gingivalis and prevotella intermedia, in periapical granuloma have not been confirmed. purpose: the aims of study were to determine the presence of porphyromonas gingivalis and prevotella intermedia in dental caries with necrotic pulp and to determine its relation to periapical granuloma. methods: thirty-six patients of dental caries with necrotic pulp in dr. moewardi general hospital in surakarta, indonesia were involved and classified into two groups, the group of patients with periapical granuloma and the group of patients without periapical granuloma. the caries tooth was extracted, and the chronic periapical tissue was swabbed and cultured on blood agar medium in anaerobic condition. the bacterial dna was extracted from the positive cultures and subjected for polymerase chain reaction (pcr). results: periapical granuloma was more likely found in women (or 5.5, 95% ci=1.277-23.693; rr 2.5, 95% ci= 1.025-6.100). black colonies bacteria were associated with periapical granuloma (or 2.2, 95% ci=0.517-9.594; rr 1.5, 95% ci=0.655-3.623). porphyromonas gingivalis and prevotella intermedia were detected in group with or without periapical granuloma, however, only prevotella intermedia was associated with periapical granuloma (or 1.6, 95% ci=0.418-5.903; rr 1.3, 95% ci=0.653-2.393). conclusion: the presence of porphyromonas gingivalis and prevotella intermedia in periapical granuloma were confirmed, however, only prevotella intermedia were associated with periapical granuloma. key words: dental caries, necrotic pulp, periapical granuloma, prevotella intermedia, porphyromonas gingivalis abstrak latar belakang: karies gigi dengan pulpa nekrosis adalah penyakit multifaktorial yang menyerang enamel hingga ruang pulpa gigi. infeksi bakteri anaerob di pulpa nekrosis dapat menginduksi pembentukan granuloma periapikal. namun, keberadaan bakteri anaerob yang paling banyak ditemukan di periodontitis apikal, porphyromonas gingivalis dan prevotella intermedia, di granuloma periapikal masih perlu diteliti . tujuan: penelitian ini bertujuan meneliti keberadaan bakteri porphyromonas gingivalis dan prevotella intermedia, di karies gigi dengan pulpa nekrosis dan menganalisis kaitannya dengan granuloma periapikal. metode: tiga puluh enam pasien karies gigi dengan pulpa nekrosis di rumah sakit umum dr. moewardi di surakarta indonesia dilibatkan dan diklasifikasikan dalam dua kelompok yaitu kelompok pasien dengan granuloma periapikal dan kelompok pasien tanpa granuloma periapikal. gigi karies diekstraksi dan jaringan periapikal kronis diusap dan dikultur di media agar darah dalam kondisi anaerob. dna bakteri diekstrak dari kultur yang positif dan dilakukan pemeriksaan dengan polymerase chain reaction (pcr). hasil: granuloma periapikal lebih banyak ditemukan pada wanita (or 5,5, 95% ci= 1,277-23,693; rr 2,5, 95% ci= 1,025-6,100). koloni bakteri berwarna hitam diasosiasikan dengan granuloma periapikal (or 2,2, 95% ci= 0,517-9,594; rr 1,5, 95% ci= 0,655-3,623). porphyromonas gingivalis dan prevotella 214 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 213–217 figure 1. the radiographic of tooth (a) without periapical granuloma, (b) with periapical granuloma (radiolucent). intermedia terdeteksi di jaringan karies dental dengan atau tanpa granuloma periapikal, namun hanya prevotella intermedia yang diasosiasikan dengan granuloma periapikal (or 1,6, 95% ci= 0,418-5,903; rr 1,3, 95% ci= 0,653-2,393). simpulan: porphyromonas gingivalis dan prevotella intermedia ditemukan di granuloma periapikal, namun hanya prevotella intermedia yang terkait langsung dengan granuloma periapikal. kata kunci: karies gigi, pulpa nekrosis, granuloma periapikal, prevotella intermedia, porphyromonas gingivalis correspondence: afiono agung prasetyo, c/o: laboratorium mikrobiologi fakultas kedokteran universitas sebelas maret. jl. ir. sutami 36 a surakarta 57126, indonesia. email: afie.agp.la@gmail.com atau afieagp@yahoo.com introduction dental caries is characterized by the progressive demineralization of enamel, following acid metabolism by the bacteria.1 the indonesian population in range of age 15 years old or older, 71.2% had caries and 52.3% cases are untreated.2 the untreated dental caries could lead into necrotic pulp, and infection of the root canal system resulting a disruption at the apical.3-5 in some case, the tooth become sensitive that is caused by hyperemia, edema and inflammation of the apical periodontal.6 endodontic and periodontal infections are commonly found preceded by caries process and associated with anaerobic bacteria, including that of the black-pigmented bacteria, porphyromonas spp. and prevotella spp.7-9 the most frequently identified in acute and chronic apical periodontitis are porphyromonas gingivalis and prevotella intermedia.9,10as gram-negative bacteria, both bacteria have lipopolysaccharide (lps) in the cell wall, which can induce macrophage to release pro-inflammatory cytokines such as interleukin-8 (il-8), il-1β, interferon gamma (ifn-γ) and tumor necrosis factor alpha (tnf-α), causing tissue inflammation and bone resorption.7,11 the bacteria also have exopolysaccharides (eps) which can enhance their virulence.12 porphyromonas gingivalis (p. gingivalis) has occurred between family members13 and the bacteria presentation in periodontitis tissue also indicative of alveolar bone loss.14 prevotella intermedia (p. intermedia) has ability to invade and evade the host innate response, so the possibility of infection will be increased.14 however, the role of both bacteria in periapical granuloma have not been elucidated. the presence of periodontal pathogen may affected by ethnic and geography.13 in indonesia, the presence of p. gingivalis and p. intermedia are quite high in oral cavity, but there is lack information concerning the bacteria of chronic periapical tissue due to dental caries. the aims of study were to determine the presence of porphyromonas gingivalis and prevotella intermedia in dental caries with necrotic pulp and to determine its relation to periapical granuloma. materials and methods chronic periapical tissue of permanent teeth from dental caries patients aged 17-57 years old with normal albumin levels and no anemia at dr. moewardi general hospital, in surakarta, indonesia were assayed in the study. all patients had no antibiotic and or immunosuppressant therapy prior the tooth extraction. none patient had systemic disease. the sample size was 9 in every group, calculated as previously published.16 the sample used was doubled for each group. finally, the total sample used was 36, were classified into two groups, with and without periapical granuloma, based on following dental radiographic criteria: well-circumscribed radiolucent periapical lesion attached to the root apex and measured less than 1 cm6,17 (figure 1a and 1b). 9 table 1. age, gender, dental element and radiographic of dental caries patients in general hospital dr. moewardi in surakarta. with periapical granuloma (n= 18) without periapical granuloma (n= 18) n % n % age <21 5 27.8 4 22.2 21-30 1 5.6 2 11.1 31-40 8 44.4 2 11.1 41-50 2 11.1 8 44.4 >50 2 11.1 2 11.1 gender men 4 22.2 11 61.1 women 14 77.8 7 38.9 element quadrant 1 0 1 5.6 quadrant 2 4 22.2 7 38.9 quadrant 3 7 38.9 3 16.7 quadrant 4 7 38.9 7 38.9 figure 1. the radiographic of tooth (a) without periapical granuloma, (b) with periapical granuloma (radiolucent). periapical granuloma a b 215cilmiaty, et al.: prevotella intermedia and porphyromonas gingivalis in dental caries figure 2. a sample of electrophoresis result from pcr assay to detect the presentation of p.. gingivalis’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis, selangor, malaysia). p. gingivalis’s dna was detected in sample no. 19-22 and 24-25. igure 3. a sample of electrophoresis result from pcr assay to detect the presentation of p. intermedia’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis). p. intermedia’s dna was detected in sample no. 19-22 and 24-25. table 1. age, gender, dental element and radiographic of dental caries patients in general hospital dr. moewardi in surakarta. with periapical granuloma (n= 18) without periapical granuloma (n= 18) n % n % age <21 5 27.8 4 22.2 21-30 1 5.6 2 11.1 31-40 8 44.4 2 11.1 41-50 2 11.1 8 44.4 >50 2 11.1 2 11.1 gender men 4 22.2 11 61.1 women 14 77.8 7 38.9 element quadrant 1 0 1 5.6 quadrant 2 4 22.2 7 38.9 quadrant 3 7 38.9 3 16.7 quadrant 4 7 38.9 7 38.9 10 figure 2. a sample of electrophoresis result from pcr assay to detect the presentation of p.. gingivalis’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis, selangor, malaysia). p. gingivalis’s dna was detected in sample no. 19-22 and 24-25. figure 3. a sample of electrophoresis result from pcr assay to detect the presentation of p. intermedia’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis). p. intermedia’s dna was detected in sample no. 19-22 and 2425. 600 bp m 19 20 21 22 23 24 25 m 19 20 21 22 23 24 25 400 bp 10 figure 2. a sample of electrophoresis result from pcr assay to detect the presentation of p.. gingivalis’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis, selangor, malaysia). p. gingivalis’s dna was detected in sample no. 19-22 and 24-25. figure 3. a sample of electrophoresis result from pcr assay to detect the presentation of p. intermedia’s dna. pcr results from sample no. 19-25 were shown. m= vc 100bp plus dna ladder (vivantis). p. intermedia’s dna was detected in sample no. 19-22 and 2425. 600 bp m 19 20 21 22 23 24 25 m 19 20 21 22 23 24 25 400 bp 216 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 213–217 approval was obtained from institutional ethical committee review boards of the faculty of medicine of universitas sebelas maret and dr. moewardi general hospital in surakarta, indonesia. informed consent was obtained from all patients involved in the study. data including age, gender, dental elements and dental radiographic were obtained from all patients. the caries tooth from each patient was extracted and the chronic periapical tissue was swabbed. the swab was then smeared on blood agar medium and incorporated into anaerobic jar with gas generating kit (thermo scientific, hampshire, united kingdom) at 37° c for 7-14 days. the present study was focused on p. gingivalis and p. intermedia, so only the black-pigmented bacteria colonies’ dna were extracted using a high pure pcr template preparation kit (roche molecular diagnostics, mannheim, germany), according to the manufacturer’s instructions. the p. gingivalis’ dna was detected using 5’ agg ctt cag gcc ata ctg cg 3’ and 5’ act gtt agc aac tac cga tgt 3’ set primer. briefly, the pcr reaction was performed by initial denaturation at 94°c for 5 minutes, 40 cycles of 94° c for 45 seconds, 66°c for 1 minute and 72° c for 2 minutes, followed by a final extension at 72° c for 10 minutes. the p. intermedia’s dna was detected using 5’ ttt gtt ggg ggg gag taa agc 3’ and 5’ tca aca tct ctg tat cct gcg t 3’ set primer. the pcr reaction was performed by initial denaturation at 94° c for 5 minutes, 40 cycles of 94° c for 45 seconds, 62°c for 1 minute and 72° c for 2 minutes followed by a final extension at 72° c for 10 minutes. all pcr reaction was performed using ready to go pcr bead (promega, buckinghamshire, united kingdom). the pcr products were then subjected to electrophoresis in 1% agarose gels, stained with ethidium bromide, and visualized under ultraviolet illumination. the data was analyzed by spss version 16 software (spss, chicago, il). a 95% confidence interval (ci) was used for all data analysis. results thirty-six dental caries patients (19 women and 17 men) with necrotic pulp were agreed to participate. the mean age of the patients studied was 35.7 years old (in range of 17 to 57 years old) and the most frequent element was derived from 4th quadrant (38.9%, 14/36). periapical granuloma was found in 77.8% (14/18) women (or 5.5, 95% ci= 1.27723.693; rr 2.5, 95% ci= 1.025-6.100) and 22.2% (4/18) men (or 0.2; 95% ci= 0.042-0.783; rr 0.400; 95% ci= 0.164-0.976) (table 1). twenty-five of the 36 (69.4%) samples had blackpigmented bacteria. these bacteria were identified in 77.8% (14/18) periapical granuloma’s samples (or 2.2, 95% ci= 0.517-9.594; rr 1.3, 95% ci=0.817-1.983) and 61.1% (11/18) of non periapical granuloma’s samples (or 0.4, 95% ci= 0.104-1.934; rr 0.8, 95% ci= 0.504-1.224). p. gingivalis’s dna was detected (figure 2) in 64.3% (9/14) of black colonies from periapical granuloma’s samples and 63.6% (7/11) of black colonies from non periapical granuloma’s samples (p= 1.00) and had no association with periapical granuloma formation (or 1.0, 95% ci= 0.2713.694). p. intermedia’s dna was detected (figure 3) in 57.1% (8/14) of black colonies from periapical granuloma’s samples (or 1.6, 95%ci= 0.418-5.903; rr 1.3, 95% ci=0.653-2.393). co-infection of p. gingivalis and p. intermedia was detected only in 33.3% (6/18) periapical granuloma’s samples (or 1.0, 95% ci= 0.250-3.999; rr 1.0, 95% ci= 0.500-1.999). discussion dental caries with necrotic pulp is a multi-causal disease that attacks the enamel and reach tooth pulp chamber, causing destruction structure of the tooth, and open the door to bacterial infection.1,4 the bacterial infection can activate host immune response to isolate and eradicate microorganisms or chronic irritants, causing periapical granuloma, an inflammatory reaction in the apex of non-vital tooth. the inflammatory tissue of periapical granuloma (containing macrophages, polymorphonuclear leukocytes and lymphocytes) is covered by epithelial cells and ultimately results in destruction of the alveolar bone surrounding the tooth.7,8 the p. gingivalis and p. intermedia already known dominant in primary endodontic infection due to its content, lps.8 lps induces pulp fibroblast and osteoblast to produce il-8. the il-8 then attracts and activates polymorphonuclear leukocytes to surround and kills the bacteria, stimulates osteoclast activity and causes pain symptom in periapical lesion. the lps also activates macrophages through cd14 receptor to produce pro-inflammatory mediators, il-1 and tnf-α. il-1 then induces the production of il-12, tnf-α, ifn-γ and il-1 itself. in human periapical lesion, il-1β, one variant of il-1, is predominant and stimulates t-lymphocytes; enhances bone resorption and inhibits bone formation. the macrophages and t-lymphocytes in the inflammatory tissue also produce tnf-α. the tnf-α activates the macrophages, t-lymphocytes and natural killer cells and also stimulates bone resorption. however, if the bacteria (p. gingivalis and or p. intermedia) are difficult to be eliminated, t-lymphocyte would lead b-lymphocyte activation, to synthesize the antibody to bacterial antigen. the bacterial-antibody formation will attract activatedmacrophages to surround and phagocyte the formation. finally, all reaction will causes periapical granuloma formation in the root of the tooth.7,18-20 p. gingivalis and p. intermedia are frequently detected in intraoral7-9 and the numbers of these bacteria are increase in the disease site compared with healthy site.9 in the present study, p. gingivalis and p. intermedia could be isolated from dental caries tissue, both of with and without periapical granuloma, consistent with previous reports,7,8,21 217cilmiaty, et al.: prevotella intermedia and porphyromonas gingivalis in dental caries however, only p. intermedia was associated with periapical granuloma. in the present study, periapical granuloma was more likely found in women than in men. lps of bacteria induces production of il-1β and tnf-α.11 these proinflammatory cytokines could be influenced by sex hormone. sex hormone, especially estrogen, already known significantly reduces p. gingivalis bacteria compared with p. intermedia.21 testosterone also could reduce the expression of tnf-α and il-1β, so reduce the inflammation process.9,21-23 the study revealed that the presence of p. gingivalis and p. intermedia were confirmed in periapical granuloma and could be isolated in dental caries tissue; however, only p. intermedia had association with periapical granuloma. further studies are needed to confirm and extend our findings. acknowledgement this work was supported partially by grants from the apbn/dipa uns (no. 159a/un27.11/pn/2013) and boptn uns (no. 165/un27.11/pn/2013). references 1. deljo e, cavaljuga s, mescovic b. prevalence of dental caries in the municipality gorazde during the period 2007-2012. mater sociomed 2013; 25(3): 163-6. 2. the indonesian household heath survey. health profile of indonesian 2004. jakarta: ministry of health republic of indonesia; 2006. p. 19. 3. bjørndal l. the caries process and its effect on the pulp: the science is changing and so is our understanding. pediatr dent 2008; 30(3): 192-6. 4. ma r tin f, nadka r ni m, jacques n, hunter n. qua ntitative microbiological study of human carious dentine by culture and realtime pcr: association of anaerobes with histopathological changes in chronic pulpitis. j clin microbiol 2002; 40(5): 1698-704. 5. gomes gb, sarkis-onofre r, bonow ml, etges a, jacinto rc. an investigation of the presence of specific anaerobic species in necrotic primary teeth. braz oral res 2013; 27(2): 149-55. 6. rajendran r. shafer’s textbook of oral pathology 6th ed. india: elsevier; 2009. p. 482. 7. garcia cc, sempere fv, diago mp, bowen em. the post-endodontic periapical lesion: histologic and etiopathogenic aspects. med oral patol oral cir bucal 2007; 12(8): 585-90. 8. narayanan l, vaishnavi c. endodontic microbiology. j conserv dent 2010; 13(4): 233-9. 9. estrela cr, pimenta fc, alencar ah, ruiz lf, estrela c. detection of selected bacterial species in intraoral sites of patients with chronic periodontitis using multiplex polymerase chain reaction. j appl oral sci 2010; 18(4): 426-31. 10. ge x, rodriguez r, trinh m, gunsolley j, xu p. oral microbiome of deep and shallow dental pockets in chronic periodontitis. plos one 2013; 8(6): e65520. 11. corcoran mp, meydeni m, lichtenstein ah, schaefer ej, dillar a, lamon-fava s. sex hormone modulation of proinflammatory cytokine and c-reactive protein expression in macrophages from older men and postmenopausal women. j endocrinol 2010; 206(2): 217-24. 12. yamanaka t, yamane k, furukawa t, matshumoto-mashimo c, sugimori c, nambu t, obata n, walker cb, leung k, fukushima h. comparison of the virulence of exopolysaccharide-producing p revotella inter media to exopolysaccha r ide non-producing periodontopathic organism. bmc infect dis 2011; 11: 228. 13. van winkelhoff aj, rijnsburger mc, abbas f, timmerman mf, van der weijden ga, winkle eg, van der velden u. java project on periodontal disease: a study on transmission of porphyromonas gingivalis in a remote indonesian population. j clin periodontol 2007; 34(6): 480-4. 14. chaves es, jeffcoat mk, ryerson cc, snyder b. persistent bacterial colonization of porphyromonas gingivalis, prevotella intermedia and actinobacillus actinomycetemcomitans in periodontitis and its association with alveolar bone loss after 6 months of therapy. 2000; 27(12): 897-903. 15. kononen e, paju s, pussinen pj, hyvonen m, tella pd, suominentaipe l, knuuttila m. population-based study of salivary carriage of periodontal pathogens in adults. j clin microbiol 2007; 45(8): 2446-51. 16. sastroasmoro s, ismael s. dasar-dasar metodologi penelitian klinis. edisi 2. jakarta: sagung seto; 2002. p. 258-64. 17. langland oe, anglais rp, preece jw. principle of dental imaging. 2nd ed. philadelphia: lippincott wiliams & wilkins; 2002. p. 413. 18. nair pnr. pathogenesis of apical periodontitis and causes of endodontic failures. crit rev oral biol med 2004; 15(6): 348-81. 19. yang lc, huang fm, lin cs, liu cm, lai cc, chang yc. induction of interleukin-8 gene expression by black-pigmented bacteroides in human pulp fibroblast and osteoblast. int endod j 2003; 36(11): 774-9. 20. graunaite i, lodiene g, maciulskiene v. pathogenesis of apical periodontitis: a literature review. j oral maxillofac res 2011; 3(4): e1. 21. tarkkila l, kari k, furuholm j, tiitinen a, meurman j. periodontal disease-associated micro-organisms in peri-menopausal and postmenopausal women using or not using hormone replacement therapy. a two-year follow-up study. bmc oral health 2010; 10: 10. 22. blasco-baque v, serino m, vergnes j, riant e, loubieres p, arnal j, gourdy p, sixou m, burcelin, r, kemoun p. high-fat diet induces periodontitis in mice through lipopolysaccharides (lps) receptor signaling: protective action of estrogens. plos one. 2012; 7(11): e48220. 23. sa it oa a , i n a ga k i s, i sh i h a r a k . d i f fe r e nt ia l a bi l it y of periodontopathic bacteria to modulate invasion. microb pathog 2009; 47(6): 329-33. �� vol. 45. no. 1 march 2012 degrees of chitosan deacetylation from white shrimp shell waste as dental biomaterials sularsih1, anita yuliati2, and coen pramono d3 1 department of dental material, faculty of dentistry, hang tuah university 2 department of dental material, faculty of dentistry, airlangga university 3 department of oral and maxilofacial surgery, faculty of dentistry, airlangga university surabaya indonesia abstract background: chitosan is biomaterial improved for various dentistry applications because it is biocompatible, degradable, nontoxic, and not carcinogenic. the main parameter affecting the characteristics of chitosan is deacetylation degree. purpose: this study is aimed to determine the degree of deacetylated of chitosan derived from white shrimp shell waste used as dental biomaterial. methods: white shrimp shells were crushed into powder. next, deproteination process was conducted with 3.5% naoh solution, demineralized with 1n hcl solution, and then depigmented with 90% acetone solution into chitin powder. deacetylation process was then conducted by soaking the chitin powder in 50% naoh solution for 6 h at 65° c to produce white powder of chitosan. afterwards, deacetylation degree test was conducted by using fourier transform infrared spectrophotometer (ftir) to calculate the ratio of the absorption bands between the absorbance peak of amide group about 1655 cm–1 and the absorbance peak of hydroxyl group about 3450 cm–1. results: the result of the deacetylation degree test on the chitosan powder derived from white shrimp shell waste was high, about 85.165%, and had the eligible form, solubility, and ph. conclusion: it can be concluded that the deacetylation degree of chitosan from white shrimp shells could reach 85.165%. key words: chitosan, shrimp shell waste, deacetylation degree abstrak latar belakang: kitosan merupakan biomaterial yang dikembangkan untuk berbagai aplikasi kedokteran gigi karena biokompatibel, dapat didegradasi, tidak toksik dan tidak karsinogenik. parameter utama yang mempengaruhi karakteristik kitosan adalah derajat deasetilasi. tujuan: tujuan dari penelitian ini adalah mengetahui derajat deasetilasi kitosan dari limbah kulit udang putih sebagai biomaterial kedokteran gigi. metode: kulit udang putih dihaluskan menjadi serbuk. setelah itu dilakukan proses deproteinasi dengan larutan naoh 3,5%, demineralisasi dengan larutan hcl 1n, depigmentasi dengan larutan aseton 90% sehingga menjadi serbuk kitin. proses deasetilasi dilakukan dengan merendam serbuk kitin dalam larutan naoh 50% selama 6 jam pada suhu 65° c sehingga dihasilkan serbuk putih kitosan. uji derajat deasetilisasi menggunakan metode spektrofotometer fourier transform inframerah (ftir) dengan menghitung nilai perbandingan pita serapan antara puncak absorbansi gugus amida sekitar 1655 cm–1, dan puncak absorbansi gugus hidroksil sekitar 3450 cm–1. hasil: hasil uji derajat deasetilasi serbuk kitosan dari limbah kulit udang putih adalah tinggi yaitu sebesar 85.165% dan memiliki bentuk, kelarutan dan ph yang memenuhi syarat. kesimpulan: dapat disimpulkan derajat deasitilasi kitosan dari kulit udang putih adalah 85,165%. kata kunci: kitosan, limbah kulit udang, derajat deasetilasi correspondence: sularsih, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas hang tuah. jl. arif rachman hakim 150 surabaya 60111. e-mail: l4rs_dentist@yahoo.co.id research report �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 17–21 introduction indonesian marine are actually considered as the shell source of marine invertebrate animals (crustaceans) containing a lot of chitin. chitin contained in crustaceans can be in high levels ranging from 20–60% depend on the species. actually, indonesia currently produces chitin derived from approximately 56,200 tons of wastes per year.1 shrimp is one of indonesia’s main commodities for nonoil exports. the average of world consumers for shrimp per year has even been increasing.2 one of nine shrimp species which has high commercial value and is very popular is white shrimp (peneaus merguiensis). the results of shrimp processing are shell and head wastes. however, these wastes have still not been utilized properly and efficiently, and most of them even participate in polluting environment. thus, chitin processing as an alternative effort is needed to utilize the shrimp shell wastes in order to make them have high value.3 chitin is the major polysaccharide found in shrimp and crab shell wastes, but it can actually be derived from fungi and insect exoskeleton. chitosan with the structure of b-(1-4)-2amino-2-deoxy-d-glucose is a natural product derived from chitin polysaccharide. chitosan is produced from deacetylation process of chitin. the deacetylation process of chitin is a process in which most of acetyl groups in chitin is substituted by hydrogen into amide group. the percentage of acetyl groups (coo-) replaced by amide (-nh2) then shows the magnitude of the deacetylation degree of chitosan.3,4 the deacetylation degree of chitosan is a quality parameter of chitosan. the deacetylation degree is related to the ability of chitosan to form isoelectric interaction with other molecules. high degree of chitosan deacetylation will make more amide group formed, so chitosan will become increasingly active.3,5,6 chitosan with the structure of b-(1-4)-2-amino-2deoxy-d-glucose, is one of the abundant natural polymers dispersed in nature.5 chitosan is a cationic polymer with 2000-3000 monomers, which are biocompatible, degradable, and less toxic to the ld 50 = 16 g/kg of body weight. chitosan can interact with charged materials, such as proteins, anionic polysaccharides, fatty acids, bile acids, and phospholipid.6 chitin and chitosan are actually beneficial for health and industry, such as textiles, photography, medicine, fungicides, cosmetics, food processing, and waste handling.4 the use of chitosan as biomaterial for medical applications has rapidly been growing as shown by many studies. chitosan does not only have a role in closing wound, prompting bone regeneration, and accelerating healing process of burns, but is also considered as antibacterial, antitumor, anti-cholesterol, antioxidant, antidiabetic, antihiv, anti-inflammatory, and matrix metalloproteinase (mmp) inhibition.7 the study results even conclude that chitosan is able not only to stimulate macrophage cells, but also to increase transforming growth factor increased bheta 1 (tgf b1), platelets release transforming growth factor (pdgf), fibroblasts growth factor 2 (fgf-2),8 bone morphogenetic protein expression of mrna on the seventh day,9 and deacetylation degrees of chitosan concentration 1% (w/v), over 80%, that can stimulate collagen synthesis in incision wound healing.10 in dentistry, the use of chitosan as biomaterial has also been growing rapidly. the results of antimicrobial chitosan test with high deacetylation degree even show that the growth of streptococcus mutans (s. mutans) and candida albicans (c. albicans) is little.11 chitosan at the concentration of 2% is able to remove plaques that stick to maxillary and mandibulary complete denture, about 9,9169 µg and 9,3021 µg, after immersion for 60 minutes.12 chitosan with molecular weight of 5-6kda and deacetylation degree of 50-50% could inhibit the initial adhesion of s. mutans on tooth surface.13 applications of chitosan in the medical field are determined by the specification of the deacetylation degree. thus, the higher the deacetylation degree of chitosan is, the more active chitosan is. the purpose of this study, therefore was to investigate the characteristics of the deacetylation degree of chitosan derived from white shrimp shell wastes in order to be used as biomaterials in dentistry field. materials and methods to make chitosan, 1 kg of shrimps were washed and boiled in boiling water for 15 minutes. they were dried for 3 hours, and then crushed and mashed. then they were sieved by using sieve with size of 60 mesh, so powder obtained was about 200 mg. after that the extraction of chitin involving deproteination, demineralization, and depigmentation processes was conducted. at the stage of deproteination, white shrimp shell powders were sieved, put into beaker glasses, added with 3.5% naoh solution with a ratio of 1:10 (w/v), and then stirred for 2 hours at 65° c. tthe solution was filtered with filter paper, and then precipitation was obtained in the form of pellets. pellets were washed with distilled water until ph became neutral, and then dried in oven at 65° c for 24 hours. the results obtained were chitin located on shells of the head and chest. at the stage of demineralization, the following steps were conducted: chitin located on shells of the head and chest was put into beaker glasses, added with 1 n hcl solution with a ratio of 1:15 (w/v), and then stirred for 1 hour at room temperature. the results of the solution were filtered with filter paper. the precipitation obtained in the form of pellets was washed with distilled water until ph became neutral, and then dried in oven at 65° c for 24 hours until it was dry and produced chitin powders. at the stage of depigmentation, those chitin powders were soaked in 200 ml of 90% acetone for less 20 hours. the results of the immersion were washed with distilled water until they were clean and then filtered by using filter paper. the precipitation obtained then was 36 grams of white chitin powders. ��sularsih, et al.: the degrees of chitosan deacetylated to extract chitosan, 36 grams of chitin powders were soaked in solution of 50% naoh with a ratio of 1:10 (w/v) for 6 hours at 65° c, and then washed with distilled water and filtered with filter paper. the precipitation obtained then was dried in oven at 80° c. chitosan obtained later was about 23 grams. next, chitosan obtained was stored in a clean and dry room at room temperature until it was ready to be tested by deacetylation degree test. for deacetylation degree test, 10 mg of chitosan powders were grounded with 50 mg of kbr powders until they were smooth, and then pressed with a hydraulic pressure of 1 atm. the mixture of chitosan and kbr powders were analyzed by using a spectrophotometer method, fourier transform infrared (ftir). how to determine the degree of deacetylation was then calculated with the ratio of the absorption band between the absorbance peak of amide group, about 1655cm-1, and the absorbance peak of the hydroxyl group, about 3450cm-1. the comparison of those two groups was determined by making a straight line from 1800 cm-1 to 1600 cm-1 as a baseline for the band of amide group, and another straight line from 4000 cm-1 to 2500 cm-1 as a baseline for the band of hydroxyl group. the degree of deacetylation was then calculated by the following equation.14 the degree of deacetylation (dd) = 100 [(a amide/a hydroxyl) √ 115] a3450 = log (t 0 hydroxyl/t hydroxyl) a1655 = log (t 0 amide/t amide) the next stage, chitosan powders already known for their deacetylation degrees were finally analyzed for their characteristics concerning with their shape, solubility, and ph. results the deacetylation degrees of chitosan derived from white shrimp shells were tested by using ftir spectrophotometry method, and the results were shown in figure 1. chitosan has hydroxyl group and amide group. the degree of deacetylation was then calculated with the ratio of the absorbance band between the peak absorption of amide group and the peak absorbance of hydroxyl group. it is known that the peak of hydroxyl group was at 3432.53 cm-1 (point b). meanwhile, the peak of amide group was at 1652.64 cm-1 (point e). based on the results of quantitative analysis by using ftir spectrophotometry, it is finally known that the deacetylation degree of chitosan derived from white shrimp shells was 85.165%. the results of the characteristics of chitosan with that deacetylation degree, 85.165%, were in the following form, solubility, ph as shown in table 1. table 1. the characteristics of chitosan derived from white shrimp shells with 85.165% of deacetylation degree no characteristics requirements observation results 1 form slightly yellow-white powders, odorless, and tasteless qualified 2 solubility not soluble in water, but soluble in acetic acid qualified 3 ph 7.0 – 9.0 7.4 4 deacetylation degree more than 70 % 85.165 % chitosan.sp 3551 4000 450 53 81 4 %t 3 0 ref 4000 75 2000 80 3925 75 3884 75 3736 74 3432 53 2923 61 2874 61 2139 79 1735 75 1652 64 1599 65 1422 65 1381 63 1322 66 1260 68 1155 57 1076 53 1030 54 896 65 663 65 573 64 figure 1. graph of deacetylation degree test results. �0 dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 17–21 discussion chitin is mostly found in insects, microorganisms, and vertebrates’ heads and shells, such as shrimps, crabs, oysters, and squids. chitosan is made of shrimp shells classified into crustacean group containing a lot of chitin. chitosan is a compound with chemical structure of poly (2-amino-2dioksi-b-d-glucose) that can be derived from hydrolysis process of chitin by using strong alkaline. nowadays, there are more than 200 applications of chitin and chitosan as well as their derivatives in food industry, food processing, biotechnology, agriculture, pharmaceuticals, healthcare, and environment.3,5 during the deproteination process, the making process of chitosan derived from chitin, used a strong alkaline (naoh) to remove protein content. meanwhile, during the demineralization process, a strong acid (hcl), was used to remove mineral contents. calcium compounds then would react with chloride acid soluble in water. protein, fat, phosphorus, magnesium, and iron were actually also wasted in this process. in the depigmentation process, 90% acetone solution was finally used to deacetylate with a strong alkaline, naoh solution, in order to produce chitosan. in the process of deacetylation, furthermore, most of acetyl group (coo-) on chitin was then substituted by hydrogen to amide group (-nh2). the percentage of the acetyl group replaced by the amide one indicates the magnitude of the deacetylation degree (dd) of chitosan since the main parameter affecting the characteristics of chitosan is deacetylation degree.14 therefore, the deacetylation degree of chitosan can be considered as a quality parameter indicating the percentage of acetyl group that can be removed from the yield of chitin and chitosan. in other words, the higher the deacetylation degree of chitosan is, the lower the chitosan acetyl groups is. as a result, the interaction between the ions and their hydrogen bonds will be stronger. it means that the release of the acetyl group of chitosan causes positive chitosan that is able to bind negative compounds, such as proteins, polysaccharide anion, to form neutral ions.4 the degree of deacetylation that is more than 75% is actually considered as the high degree of deacetylation.3,5 and, the characteristics of the deacetylation degree of chitosan obtained in this study were high, about 85.165%. this is because the researchers observed several factors affecting the deacetylation degree of chitosan, such as temperature, the concentration of naoh solution used, and the duration of chitin powder immersion into naoh solution.3 in this study, the deacetylation process of chitin powder used 50% naoh solution with a ratio of 1:10 (w/v) for 6 hours at 65° c to produce chitosan powder with the degree of deacetylation more than 75% because the higher the concentration of naoh is, the more increasing the degree of deacetylation (dd) is. temperature and the duration of naoh immersion, furthermore, can affect the molecular chain of chitin. the use of high temperature, above 150° c, for example, can cause the breaking of polymer bond (depolymerization) of chitosan molecular chains resulting in lowering molecular weight chitosan. the high concentration of naoh solution, more than 40%, can break the bonds of carboxyl group and nitrogen atoms of chitin that have thick and long crystal structure. the high concentration of naoh then can lead to the functional group of amine (nh3 +) substituting the acetyl groups of chitin, so the solution in the system is more active, and the deacetylation is better.3,15,16 therefore, the use of higher concentrations of naoh can make the degree of deacetylation greater, but this does not always make the degree of deacetylation significantly increasing. in the largest concentration of naoh, 60%, the degree of deacetylation even becomes decreasing. this is because in 60% naoh the solution becomes more viscous resulting in imperfect mixing process which means that some parts of chitin can not perfectly react with naoh solution, so the amino group formed is little or has decreased dd value.17 chitosan is actually a polysaccharide that is very hard to be immersed at neutral ph, like in water, since chitosan contains high density of polymer chains that are bonded to each other with very strong hydrogen bonds. chitosan solution is considered as strong alkaline solution, so it will be more soluble in the aqueous solution, acetic acid. acetic acid is actually classified as weak carboxylic acid containing carboxyl group (-cooh). carboxyl group contains carbonyl group and hydroxyl group. it even reaches its boiling point at 118° c and its smell is very sharp. the increasing of the solution is even linear to the increasing of deacetylation degree. this is because the acetyl group in chitin cut by the deacetylation process will leave the amine group. h ions on the amine group of chitosan, as a result, will lead to easily interact with water through hydrogen bonding. in this study 2% acetic acid used to dissolve chitosan powder so that it becomes gel. it is because the higher the degree of deacetylation is, the higher the solubility of chitosan in acetic acid solution is. it means that there is hydrogen interaction between the carboxyl group in acetic acid and the amide group in chitosan.3,15 therefore, the solubility of chitosan is also influenced by the type of crustacean that is used. this type of crustaceans, such as white shrimp, used in this study, as a result, could produce high deacetylation degree of chitosan. another type of crustaceans, such as crab shell, can also make amide group formed little, less reactive, and unpredictable for the duration of deacetylation process.3,15,18 some studies on chitosan derived from shrimp shells have been conducted concerning with the benefit of dentistry. based on the results, it is known that there is no toxicity effect on the cell culture test,19 and for the biocompatibility test, skin patch test is needed to be used. chitosan with concentration of 1–4%, as a result, does not cause allergic reactions in individuals with allergic or not allergic to seafood history. the results of another study even concludes that the number of osteoblast like cell and ��sularsih, et al.: the degrees of chitosan deacetylated collagen synthesis type i in the formation of reparative dentin is increased in direct pulp calping on rattus norvegicus teeth by using chitosan.20 it indicates that 1% chitosan gel with deacetylation degree, about 85.165%, can increase the proliferation of fibroblasts, osteoblasts, and collagen type 1 in healing wounds of tooth extraction of rattus norvegicus at 7 and 14 days of the observation.21 in other words, chitosan with higher deacetylation degree will make more amide group of chitosan formed, so it will become more active, have high chemical reactivity, and be able to interact with proteins and other organic matrices, such as anionic glycosaminoglycans and proteoglycans, and extracellular matrix macromolecule. in addition, the positive chitosan is able to react with the surface of negative anionic polymer, so it can facilitate the migration of inflammatory cells.22,23 based on several studies on chitosan as described above, it can be said that chitosan still needs to be developed since it has some potential functional characters for broad use in dentistry. finally, it can be concluded that the deacetylation degree of chitosan derived from white shrimp shells could reach 85.165%. references 1. departemen kelautan dan perikanan. statistik data perikanan. jakarta: departemen kelautan dan perikanan; 2009. available at: http://www.jaringandata.perikanan-diy.info. accessed january 2, 2010. 2. bahtiar r. pengelolaan budidaya udang indonesia. 2006. available at: http://www.brawijaya.ac.id. accessed april 24, 2006. 3. rochima e, maggy s, dahrul s, sugiyono. viskositas dan berat molekol kitosan hasil reaksi enzimatis kitin deasetilase isolate. prosiding seminar nasional dan kongres perhimpunan ahil tehnologi pangan indonesia (patpi), bandung 17-18 juli 2007; 2007. p. 29–34 4. savitri e, soeseno n, adiarto t. sintesis kitosan, poli(2-amino2-deoksi-d-glukosa), skala pilot project dari limbah kulit udang sebagai bahan baku alternatif pembuatan biopolimer. prosiding seminar nasional teknik kimia “kejuangan” pengembangan teknologi kimia untuk pengolahan sumber daya alam indonesia, yogyakarta 26 januari 2010; 2010. p. 388–97. 5. sugihartini l. pengaruh konsentrasi asam klorida dan waktu demineralisasi kitin terhadap mutu kitosan dari cangkang rajungan. thesis. bogor: program studi teknologi hasil perikanan, fpik, ipb. 2001. 6. wibowo s. produksi kitin kitosan secara komersial. prosiding seminar nasional kitin kitosan, 2006; p. 52–64. 7. suptijah p. deskripsi karakteristik fungsional dan aplikasi kitin kitosan. prosiding seminar nasional kitin kitosan 2006; 2006. p. 14–24. 8. kim s. neuroprotective properties of chitosan and its derivatives. marine drug j 2010; 8(10): 2117–28 9. ueno h, nakamura f, mukarami m, okumura m, kadosawa t, fujinaga t. evaluatione effects of chitosan for the extracellular matrix production by fibroblasts and growth factors production by macrophages. j biomaterials 2001; 22: 2125–30. 10. matsunaga s, yanagiguchi k, yamada s, ohara h. chitosan monomer promotes tissue regeneration on dental pulp wounds. j biomed mater res 2005; 76a: 711–20. 11. astiti t. efek derajat deasetilasi dan konsentrasi kitosan terhadap daya hambat streptococcus mutan dan candida albicans. thesis. surabaya: fakultas kedokteran gigi. universitas airlangga; 2009. 12. ariani md. uji alergi dan aktivitas dari cangkang penaeus monodon dalam melepas plak gigi tiruan lengkap akrilik. thesis. surabaya: fakultas kedokteran gigi universitas airlangga; 2009. 13. sano h, shibasaki k, matsukubo t, takaesu y. effect of rinsing with phosphorilated chitosan on four day plaque regrowth. bull tokyo dent coll 2001; 42(5): 251–6. 14. domszy jg, roberts gaf. evaluation of infrared spectroscopic techniques for analyzing chitosan. makromol chem 1985; 186: 671–7. 15. kojima k. effect of chitin and chitosan on collagen synthesis in wound healing. j vet med sci 2004; 66(12): 1595–98. 16. siregar m. pengaruh berat molekol nanopartikel untuk menurunkan kadar logam besi (fe) dan zat warna pada limbah industry tekstil jeans. thesis. medan: pascasarjana universitas sumatra utara; 2009. 17. patel. s. review of pharmaceutical significance of chitosan. pharmaceutical journal 2006; 4: 6–14. 18. khan ta, peh kk, ching hs. mechanical bioadhesive strength and biological evaluations of chitosanfilm for wound dressing. j pharm pharmaceut sci 2000; 3(3): 303–11. 19. maretaningtias da, anita y, tokok a. toxicity testing of chitosan from tiger prawn shell wate on cell culture. maj kedokteran gigi (dent j ) 2009; 42(1): 15–20. 20. prananingrum w. jumlah osteoblast like cell dan sintesa kolagen tipe i pada pembentukan dentin reparatif dengan perawatan direct pulp calping gigi tikus rattus norvegicus dengan kitosan. thesis. surabaya: fakultas kedokteran gigi. universitas airlangga; 2010. 21. sularsih. penggunaan kitosan dalam proses penyembuhan luka pencabutan gigi rattus norvegicus. thesis. surabaya: fakultas kedokteran gigi. universitas airlangga; 2011. 22. hargono, abdullah, sumantri i. pembuatan kitosan dari limbah cangkang udang serta aplikasinya dalam mereduksi kolesterol lemak kambing. jurnal tehnik kimia undip (reaktor) 2008; 12(1): 53–7. 23. chin l, halim as. in vitro models in biocompability assessment for biomedical-grade chitosan derivatives in wound management. j molecular science 2009; 10(3): 1300–13. vol 50 no 4 desember 2017.indd 171171 effects of herbal medicine components on the physical properties of trial denture adhesives kenichiro nakai,1 takeshi maeda,1 guang hong,2 tadafumi kurogi,3 and joji okazaki1 1department of removable prosthodontics and occlusion, graduate school of dentistry, osaka dental university, hirakata-japan 2liaison center for innovative dentistry, tohoku university graduate school of dentistry, sendai-japan 3department of prosthetic dentistry, graduate school of biomedical sciences, nagasaki university, nagasaki-japan abstract background: denture adhesive is widely used in elderly people who wear complete dentures. chinese herbal medicine has long been used for the treatment of oral disease. the addition of herbal medicine to denture adhesive might be used to develop an adhesive which is effective for xerostomia patients. purpose: the purpose of this study was to evaluate physical properties and cytototoxicity of herbal medicine components in denture adhesive. methods: this study used a combination of 35wt% pvm-ma, 20wt% cmc, 40wt% white petrolatum and 5wt% liquid paraffin as base materials. three kinds of herbal medicine components: maimendong (ophiopogon), fuling (hoelen) and dongkuizi (cluster mallow seed) were added to base materials of 1wt%, 5wt% and 10wt%. the initial viscosity was measured using a controlled-stress rheometer (ar-g2). the adhesive strength was quantified according to iso-10873 recommended procedures. all data was analyzed independently by one-way anova combined with a turkey’s multiple comparison test at a 5% level of significance. results: significant differences were observed between materials in initial viscosity (p < 0.05). specifically, samples containing 5wt% and 10wt% of maimendong (ophiopogon) showed higher values compared with the control samples. the larger the amounts of herbal medicine components, the greater the changes in the adhesive strength of denture adhesives over time. the denture adhesives containing herbal medicine components do not have a cytotoxic effect and are safe for use in actual clinical practice. conclusion: the study showed that the addition of herbal medicine components does not affect physical properties (i.e. initial viscosity and adhesive strength) of denture adhesive and cytotoxicity in fibroblast cells. keywords: ophiogon; hoelen; cluster mallow seed; herbal medicine; denture adhesives; initial viscosity; adhesion strength; cytotoxicity correspondence: guang hong, liaison center for innovative dentistry, graduate school of dentistry, tohoku university, 4–1 seiryo-machi, aoba-ku, sendai, 980-8575, japan. e-mail: hong@m.tohoku.ac.jp dental journal (majalah kedokteran gigi) 2017 december; 50(4): 171–177 research report introduction according to the 2017 annual report on the aging society in japan,1 the over-65s account for 27.3% of the entire country’s population. this constitutes the highest number of elderly in its history. japan has, undisputedly, become regarded as a super-aging society, with the number of japanese above the age of 80 surpassing 10 million. many elderly people wear partial or complete dentures, with the rate of complete denture use known to increase with age.2 denture function begins from the moment dentures are placed in the oral cavity. therefore, having comfortable dentures is crucial to improving the quality of life of the elderly. however, the latter face numerous problems, such as xerostomia, anatomical limitations and psychological anxiety. the number of patients using denture adhesives to stabilize and maintain dentures is increasing worldwide. according to a census of american academic prosthodontists regarding denture adhesives, the general attitude toward their use has changed greatly over the last 10 years.3 despite the range of disagreements regarding the clinical significance of denture adhesives, the fact remains that improvements in denture function, stability and maintenance have been observed following their application.4–7 denture adhesives are broadly divided into two types, liner (home reliners) and glue, based on their mechanism of action.8 liner type denture adhesives fill the gap between the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 mailto:hong@m.tohoku.ac.jp 172 nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 denture and the oral mucosa, thereby improving adhesion. however, the damage (i.e. deviation, bite raising) caused by these materials has long raised concerns regarding their use.9–12 in addition, liner type denture adhesive can change the occlusal vertical dimension and promote ridge resorption, with the result that dental professionals pay little attention to such materials. on the other hand, glue type denture adhesives are mainly composed of water-soluble polymers that absorb the saliva between the denture base and the oral mucosa, thereby improving adhesion. these types of denture adhesives are broadly divided into powder-form, creamform and sheet-form. of these, the cream-form and powderform varieties are advantageous in that they are thin and do not affect the occlusal vertical dimension. cream-form denture adhesives are also advantageous in terms of their durability.13 xerostomia, which is particularly common among complete denture users, is treated with humectants. currently, no other treatments are available and no radical solution has been identified. however, symptoms such as xerostomia have long been treated with chinese herbal medicines. in particular, the herbal medicines maimendong (ophiopogon), fuling (hoelen) and dongkuizi (cluster mallow seed), as written in shennong bencao jing (the classic of herbal medicine) and bencao gangmu (the compendium of materia medica), have long been considered effective in curing illnesses characterized by dry mouth or tongue, conditions wherein the tongue dries and takes on a burnt color, and in treating the dry mouth in patients with head and neck cancers.14 in addition, maimendong (ophiopogon) has been used to improve xerostomia in patients with sjögren’s syndrome.15 many researchers reporting the use of denture adhesive in vitro and in vivo have confirm its effectiveness in improving denture retention and stability, increasing bite force, improving taste discrimination and taste perception among other benefits.16–20 a number investigated the initial viscosity and adhesive strength of denture adhesives and the effect of components on the mechanical properties of denture adhesives in vitro.21,22 however, the effect of herbal medicine on the initial viscosity and adhesive strength remains unclear. a combination of herbal medicine and denture adhesive might be used to develop denture adhesive which proves effective in cases of xerostomia. the purpose of the present study was to examine the effects of herbal medicine components on the initial viscosity and strength of denture adhesive, to examine the cytotoxicity of herbal medicine components on the fibroblast cell and to develop cream-form denture adhesives containing herbal medicines. the null hypothesis adopted was that the addition of herbal medicine components neither affects initial viscosity and adhesive strength nor induces cytotoxicity in fibroblast cells. materials and methods the primary denture adhesive components used in the study consisted of polyvinyl methyl ether-maleic acid (pvm-ma) copolymer alkali salt and sodium carboxymethyl cellulose (cmc). in addition, white petrolatum and liquid paraffin were used as ointment bases. the composition, formulation and code are shown in table 1 and table 2, respectively. after stirring a proportion of 40wt% white petrolatum and 5wt% liquid paraffin, 35 wt% pvm-ma and 20 wt% cmc were added. the ingredients were mixed for two minutes using a vacuum mixer (vm-ii, gc co., tokyo, japan) to produce a control sample for use as a base. a powdered herbal medicine was subsequently added to the prepared bases at different concentrations of 1wt%, 5wt%, and 10wt% (table 2). the vacuum-mixed products were transferred to hermetically sealed containers and placed in a dark room at 23°c for 24 hours to yield table 1. primary denture adhesive components used polymer manufacturer lot. no. methoxy ethylene maleic anhydride copolymer (pvm-ma) isp japan ltd., tokyo, japan cc600150446 sodium carboxymethyl cellulose (cmc) daiichi kogyo seiyaku co., ltd., kyoto, japan 353847 white petrolatum (wpl) nikko pharmaceutical co., ltd., hatori, japan 669319 liquid paraffin (lp) sigma-aldrich japan co. llc., tokyo, japan a7568 table 2. formulations of components code base materials herbal medicine components bmt01 pvm-ma 35wt%; cmc 20wt% wpl 40wt%; lp 5wt% maimendong (ophiopogon) 1wt% bmt05 maimendong (ophiopogon) 5wt% bmt10 maimendong (ophiopogon) 10wt% fkr01 pvm-ma 35wt%; cmc 20wt% wpl 40wt%; lp 5wt% fuling (hoelen) 1wt% fkr05 fuling (hoelen) 5wt% fkr10 fuling (hoelen) 10wt% tks01 pvm-ma 35wt%; cmc 20wt% wpl 40wt%; lp 5wt% dongkuizi (cluster mallow seed) 1wt% tks05 dongkuizi (cluster mallow seed) 5wt% tks10 dongkuizi (cluster mallow seed) 10wt% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 173nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 experimental samples containing each of the three herbal medicines maimendong (ophiopogon), fuling (hoelen), and dongkuizi (cluster mallow seed); (uchida wakanyaku ltd., tokyo, japan) (table 2). in this study, the initial viscosity was measured using a stress-controlled rheometer (ar-g2, ta instruments ltd., tokyo, japan) and a parallel plate measuring 2 cm in diameter witha gap between the plates of 54 μm (figure 1). the instrument was used in a constant strain mode with an angular velocity of 10 rad/s at 37 ± 2°c. five specimens were measured for each material. measurements were obtained using the iso 108738 prescribed sample holder i and a pressure-sensitive axle (figure 2). sample holder i was fabricated from methacrylic resin (acron lot. no. powder 0308011; liquid 0308092, gc co., tokyo, japan). the hole of sample holder i was filled with 500 ± 5 mg of sample which was subsequently immersed in 300 ml of distilled water at 37 ± 2°c for 0, 1, 10, 30, 60, 180 or 360 minutes. a load of 9.8 ± 0.2 n was applied to the sample using a constant load compression testing machine (a-001, japan mecc co. ltd, tokyo, japan) at a pressurizing velocity of 5 mm/min using a pressure sensitive knob for 30 seconds. the sample was then pulled in the reverse direction with tensile velocity using a materials testing machine (model 5565, instron co., canton, ma, usa) at a crosshead speed of 5 mm/min. measurements were taken five times for each combination. a methacrylate board prescribed by iso 108738 and measuring 5.0 x 5.0 cm was fabricated using heatpolymerized denture base resin (acron lot. no. powder 0308011; liquid 0308092, gc co., tokyo, japan). samples were applied evenly on the methacrylate board, immersed in distilled water at 37 ± 2°c for one hour and, finally, scrubbed 20 times with a denture brush under running water. the methacrylate board was subsequently observed visually and the presence or absence of residue clumps assessed five times. the sample was considered to have passed the test if no clumps of residue were observed during at least four of the five assessments. following degradation of 1.00 ± 0.05 gram of sample by the addition of 5.00 ± 0.05 g of propylene glycol, 300 ml of distilled water were stirred in, before the sample was agitated further. then, the electrode of a ph meter (series 2368, beckman coulter inc., tokyo, japan) was inserted and the ph value displayed three minutes later was recorded. the same measurement was recorded five times and the values were averaged. following culture with a human gingival fibroblast cell line (p10-13) in a 24-well plate (1104/well) for 24 hours, the sample was placed in cell culture inserts so that it would account for 10% of the entire culture medium (1500 μl/well). culture was performed again at 37°c under 5% co2 for 24 hours. then, od at 450 nm spectrophotometry (n = 5) was measured using a wst-8 cell counting kit (sigma-aldrich japan, tokyo, japan). for the apoptosis measurement, the cells (1104 cells/well) were exposed to specimens for 24 hours in a 24-well plate. following exposure, the cells were trypsinized, centrifuged and resuspended in 50 μl fbs containing 10 μg/ml hoechst 33342 and 10 μg/ml propidium iodide (sigma, mo, usa). after incubation for 30 minutes in the dark at room temperature, the cells were spread onto microscope slides. a minimum of 300 cells were counted using a fluorescence microscope (bz-9000, keyence, japan) with an excitation filter 340–380 nm, and cells were classified as apoptotic, necrotic or viable. all data was statistically analyzed using one-way analysis of variance (anova) combined with a tukey’s multiple comparison test at a 5% level of significance. all analyses were computed with pasw statistics for windows (pasw statistics 18, spss japan inc., tokyo, japan). results the initial respective viscosity of each sample is shown in figure 3. significant differences (p < 0.05) were observed between materials. more specifically, samples containing 5wt% and 10wt% of maimendong (bkt05 and bkt10) showed higher values compared with the control samples, while samples containing dongkuizi (tks01, tks05 and tks10) showed lower viscosity values compared with the control samples. in general, the addition of herbal medicines tended to increase the initial viscosity. the adhesive strength prior to immersion for all samples is shown in figure 4. all samples indicated values higher than 5kpa, which is the value recommended by the iso figure 1. block diagram of viscosity test jig. figure 2. block diagram of sample holder for adhesive strength test.8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 174 nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 standard for denture adhesives.8 significant differences were observed between materials (p < 0.05). specifically, fkr01 and tks01 showed higher values compared with those of the control samples. the adhesive strength tended to decrease when the proportion of herbal medicine components in the sample exceeded 5wt%. changes in the adhesive strength for each sample are shown in figure 5. compared with the samples containing herbal medicine components, the control samples showed higher values over time. the adhesive strength was more stable in those samples with a lower proportion of herbal medicine components. all samples indicated favorable cleanability. none of the methacrylate boards exhibited clumps of residue in any of the five assessments. all samples showed weakly acidic to neutral ph values (6–7) conforming to iso standards8 (figure 6). no significant differences were observed between materials (p > 0.05). figure 7 shows the cell survival rates relative to those for the control sample which was established as 100%. all samples with herbal medicine components showed high cell figure 4. adhesive strength before immersion. figure 6. ph value of materials. figure 7. results of cytotoxicity test figure 3. mean initial viscosity with standard deviation. survival rates, but there were no differences in cytotoxicity when compared with the control samples. the proportions of necrotic cells are shown in figure 7. the values for all samples were lower than 5%, with no significant differences between the experimental and control samples (p > 0.05). discussion the null hypothesis that the addition of herbal medicine components affects neither initial viscosity nor adhesive strength and lacks cytotoxicity for fibroblast cells was accepted. in the study reported here, the effects of herbal medicine components possessing therapeutic effects against xerostomia on the physical properties of denture adhesive materials were examined. the development of creamform denture adhesives containing herbal medicines that possess therapeutic effects against xerostomia was found to be feasible. denture adhesives, widely used by the elderly for improving stability and retention of complete dentures23, figure 5. variations in adhesive strength. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 175nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 polymers, which may explain why the samples containing herbal medicines demonstrated a high initial viscosity. such initial viscosity is closely related to the application of denture adhesives. highly viscous materials compromise the application because it becomes difficult to squeeze the denture adhesive from the tube and apply it evenly on the mucosal surface of the dentures. thus, adding only a small amount of herbal medicine is desirable. samples containing herbal medicine components not only demonstrated a less pronounced water immersionassociated increase in adhesive strength compared with the control sample, but also manifested a comparatively lower adhesive strength. when a denture adhesive is immersed in water, the water-soluble polymers are degraded, thereby increasing its viscosity and adhesive strength.7,21,22 however, the samples containing herbal medicine components consisted of base material (the control group) with herbal medicine. when the adhesive strength was measured using the same amounts of material, the total amount of water-soluble polymer was, apparently, smaller than that in the control samples, resulting in a small water solubility-associated increase in the viscosity. this may have accounted for the marginal increase in adhesive strength. according to iso 10873,8 only those denture adhesives that exhibit the recommended standard values for adhesive strength, washing performance (cleanability) and ph can be used. all the herbal medicine-containing cream-form denture adhesives assessed in the present study complied fully with all iso standards, indicating that these trial denture adhesives can be used in actual clinical practice. because denture adhesives are applied to the mucosal surface of dentures and are in direct contact with the oral mucosa, they must be biologically safe.8 none of the trial cream-form denture adhesives used in the present study demonstrated cytotoxicity, which confirms that they are safe for use in actual clinical practice. chinese herbs are generally classified as superior, middle and inferior. in shennong bencao jing (the classic of herbal medicine), the oldest chinese text of pharmacology, superior herbs are described as medicines that nourish life, cause no side effects or harm and can be consumed safely, even in large quantities over extended periods. however, middle and inferior herbs entail the risk of side effects and other damage and must be used with caution. the three herbal medicines used in the present study are all classified as superior herbs. this may be a reason why no materials used in this study indicated the presence of cytotoxicity. chinese medicine is not suited to acute illnesses or symptoms identified by modern medicine. however, in recent years, it has been frequently used to support the postoperative recovery of physical strength. in an increasing number of cases, chinese medicine and modern medical science are being used in combination. chinese herbs are used for patients who complain of xerostomia. in fact, in these patients, chinese herbs are used in far greater amounts compared with those used in the present study. are broadly divided into two types: liner and glue.8 when patients use glue type denture adhesives (classified into cream-form, powder-form and sheet-form denture adhesives) under a dentist’s supervision, these prove effective in improving denture stability and retention.4–7 the primary components of cream-form denture adhesives are water-soluble polymers and white petrolatum. in the oral cavity, the water-soluble polymers absorb saliva, thereby increasing viscosity and adhesion strength.22,24 pvm-ma and cmc, the main polymers used in denture adhesives, are the most common water-soluble polymers. made from natural pulp, they are extremely safe and have various applications in situations where environmental and personal safety is of importance.25 however, the watersoluble polymers used in current commercially available cream-form denture adhesives are degraded during use in the oral cavity, resulting in an early decrease in their adhesion strength.25 therefore, the adhesion strength of denture adhesives should be more durable. han et al.21 examined combinations of water-soluble polymers to improve the durability of the adhesion strength and found consistent effects. therefore, for the base material in the present study, we used a combination of 35wt% pvm-ma, 20wt% cmc, 40wt% white petrolatum, and 5wt% liquid paraffin. this formulation was shown to be the most durable combination in our previous study, with strong, stable, adhesive action and the promise of commercial viability as a long-acting cream-type denture adhesive.21 xerostomia greatly affects oral health and causes various oral diseases. in the west, it is particularly pronounced in the elderly, with more than 20% of such individuals being reported as exhibiting subjective symptoms of xerostomia.26,27 in contrast, in japan, nearly 20-40% elderly individuals reportedly exhibit subjective symptoms of this condition.28 currently, in the absence of a radical solution, the main treatment for xerostomia involves the use of humectants. herbal medicines such as maimendong (ophiopogon), fuling (hoelen), and dongkuizi (cluster mallow seed) are considered effective in curing illnesses characterized by dry mouth or tongue. the initial viscosity, adhesion strength, cleanability and ph are the routinely assessed physical properties of denture adhesives. in this study, the experimental samples containing herbal medicines tended to demonstrate significantly higher initial viscosity values compared with those of the control samples. the initial viscosity of a denture adhesive is the viscosity prior to the absorption of water by the material.22,24 it represents properties unique to the composition of the material. here, viscosity increased with a proportional increase in the herbal medicine components. this condition was particularly pronounced in samples containing maimendong (ophiopogon) which were more viscous than samples containing other herbal medicines. maimendong (ophiopogon) is a herbal medicine more viscous than either fuling (hoelen) or dongkuizi (cluster mallow seed). in addition, powdered herbal medicines are more viscous than white petrolatumor water-soluble dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 176 nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 therefore, both physical examination and a prescription from a physician are required which makes it difficult to use chinese herbs on a larger scale. in addition, because denture adhesives can be purchased at pharmacies without a prescription, it is impractical for them to incorporate large amounts of herbal medicines. from these perspectives, it is desirable to include only small amounts of herbal medicines. chinese medicines typically consist of two or more herbal components used in combination. thus, chinese prescriptions include many components. the combination of multiple medicines also enhances potency, while reducing undesirable side-effects. therefore, multiple herbal medicines should be used in combination, rather than a single medicine. in this study, the effects of herbal medicines on the physical properties of trial denture adhesives, each containing only one type of herbal medicine, has been examined. even a 10% concentration of chinese herbal medicines did not result in cell damage. however, since this study only tested the effect of the herbal medicines after a 24-hour period of exposure, testing of longer duration involving the use of animal subjects are needed to ensure their safe use. in addition to explaining the action mechanisms of herbal medicines in animal experiments, future studies should also examine how combinations of multiple herbal medicines affect the physical properties of materials and both the water channels in the luminal membrane of salivary ducts and saliva secretion in test animals. from the standpoint of physical properties, smaller amounts of herbal medicine components are associated with a lower viscosity and, consequently, superior operability of denture adhesives. denture adhesives containing maimendong (ophiopogon), fuling (hoelen), or dongkuizi (cluster mallow seed) are non-cytotoxic and are, therefore, safe for use in real clinical practice. the study showed that the addition of herbal medicine components does not affect initial viscosity or adhesive strength and does not cause cytotoxicity in fibroblast cells. it may be, therefore, be feasible to develop cream-form denture adhesives containing herbal medicines. acknowledgements we would like to extend our deep gratitude to professor hiroshi murata of the department of prosthetic dentistry, graduate school of biomedical sciences, nagasaki university for his guidance on all aspects of the present study. this research was supported by the national natural science foundation of china (project no. 81400560). references 1. cabinet office, government of japan. aged society white paper 2017. tokyo: cabinet office, government of japan; 2017. p. 2–6. 2. ministry of health, labour and welfare of japan. survey on actual conditions of dental diseases 2016. tokyo: ministry of health, labour and welfare of japan; 2016. p. 17. 3. slaughter a, katz r v, grasso je. professional attitudes toward denture adhesives: a delphi technique survey of academic prosthodontists. j prosthet dent. 1999; 82: 80–9. 4. quiney d, nishio ayre w, milward p. the effectiveness of adhesives on the retention of mandibular free end saddle partial dentures: an in vitro study. j dent. 2017; 62: 64–71. 5. abdelnabi mh, swelem aa, al-dharrab aa. influence of denture adhesives on occlusion and disocclusion times. j prosthet dent. 2016; 115(3): 306–12. 6. kore dr, kattadiyil mt, hall db, bahjri k. in vitro comparison of the tensile bond strength of denture adhesives on denture bases. j prosthet dent. 2013; 110(6): 488–93. 7. ozcan m, kulak y, de baat c, arikan a, ucankale m. the effect of a new denture adhesive on bite force until denture dislodgement. j prosthodont. 2005; 14(2): 122–6. 8. iso 10873:2010. dentistry -denture adhesives. 1st ed. geneva: international organization for standardization; 2010. p. 1–5. 9. woelfel jb, kreider ja, berg t. deformed lower ridge caused by the relining of a denture by a patient. j am dent assoc. 1962; 64: 763–9. 10. papadiochou s, emmanouil i, papadiochos i. denture adhesives: a systematic review. j prosthet dent. 2015; 113(5): 391–7.e2. 11. means cr. a report of a user of home reliner materials. j prosthet dent. 1964; 14(5): 935–8. 12 shay k. denture adhesives. choosing the right powders and pastes. j am dent assoc. 1991; 122: 70–6. 13. chew cl. retention of denture adhesives--an in vitro study. j oral rehabil. 1990; 17(5): 425–34. 14. yanying h, juan y, feili l. effect study for xerostomia in patients with nasopharyngeal carcinoma after radiotherapy by gargle of traditional chinese medicine combined with acupoint application. j nurses train. 2016; 31(15): 1348–50. 15. tsuchiya y, jinbu y, kusama m. clinical effect of bakumon-doutou on xerostomia. j japanese soc oral mucous membr. 2006; 12: 1–4. 16. munoz ca, gendreau l, shanga g, magnuszewski t, fernandez p, durocher j. a clinical study to evaluate denture adhesive use in well-fitting dentures. j prosthodont. 2012; 21(2): 123–9. 17. farzin m, golchin a, badie a, ghapanchi j, zamani a, rezazadeh f, kalantarim mh. the effect of two types of denture adhesive on the satisfaction parameters of complete denture wearers. j dent biomater. 2017; 4(3): 425–30. 18. kalra p, nadiger r, shah fk. an investigation into the effect of denture adhesives on incisal bite force of complete denture wearers using pressure transducers a clinical study. j adv prosthodont. 2012; 4(2): 97–102. 19. psillakis jj, wright rf, grbic jt, lamster ib. in-practice evaluation of a denture adhesive using a gnathometer. j prosthodont. 2004; 13(4): 244–50. 20. koronis s, pizatos e, polyzois g, lagouvardos p. clinical evaluation of three denture cushion adhesives by complete denture wearers. gerodontology. 2012; 29(2): e161–9. 21. han jm, hong g, hayashida k, maeda t, murata h, sasaki k. inf luence of composition on the adhesive strength and initial viscosity of denture adhesives. dent mater j. 2014; 33: 98–103. 22. han jm, hong g, dilinuer m, lin h, zheng g, wang x-z, sasaki k. the adhesive strength and initial viscosity of denture adhesives. acta odontol scand. 2014; 72(8): 839–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 177nakai, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 171–177 23. grasso je, rendell j, gay t. effect of denture adhesive on the retention and stability of maxillary dentures. j prosthet dent. 1994; 72(4): 399–405. 24. hong g, tsuka h, dilinuer m, wang w, sasaki k. the initial viscosity and adhesive strength of denture adhesives and oral moisturizers. asian pacific j dent. 2011; 11: 45–50. 25. grasso je. denture adhesives: changing attitudes. j am dent assoc. 1996; 127: 90–6. 26. osterberg t, landahl s, hedegård b. salivary flow, saliva, ph and buffering capacity in 70-year-old men and women. correlation to dental health, dryness in the mouth, disease and drug treatment. j oral rehabil. 1984; 11(2): 157–70. 27. thomson wm, chalmers jm, spencer aj, ketabi m. the occurrence of xerostomia and salivary gland hypofunction in a populationbased sample of older south australians. spec care dent. 1999; 19: 20–3. 28. kakinoki y. disease condition and treatment of dry mouth. ann japan prosthodont soc. 2015; 7(2): 136–41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p171–177 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p171-177 8787 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 87–91 original article pomegranate (punica granatum l.) gel extract as an antioxidant on the shear bond strength of a resin composite post-bleaching application with 40% hydrogen peroxide indes rosmalisa suratno1, irfan dwiandhono1 and ryana budi purnama2 1department of conservative dentistry, 2department of dental materials, faculty of medicine, universitas jenderal soedirman, purwokerto – indonesia abstract background: tooth discoloration can be treated with tooth bleaching. bleaching using 40% hydrogen peroxide can reduce the shear bond strength of resin composite because there are free radicals on the tooth surface, so it can delay the restoration. the application of antioxidants can eliminate free radicals after the bleaching procedure and increase the shear bond strength of the composite resin. the common antioxidants are ascorbic acid and natural ingredients, such as pomegranate (punica granatum l.). purpose: to determine the effect of pomegranate extract gel on the shear bond strength of composite resin after 40% hydrogen peroxide bleaching application. methods: this research used 32 maxillary first premolars that were divided into four groups. the samples were bleached, then the labial was prepared and antioxidant gel was applied: group p1 pomegranate gel extract of 5%, group p2 pomegranate gel extract of 10%, group k1 positive control ascorbic acid gel of 10% and group k2 as the negative control. the samples were restored with a nanohybrid composite resin. the shear bond strength was tested using a universal testing machine. the data were tested using a one-way anova followed by a post-hoc lsd test. results: the pomegranate gel extract increased the shear bond strength of the composite resin after the bleaching procedure of 40% hydrogen peroxide compared with the ascorbic acid gel group and the negative control group. the one-way anova test showed a significant difference (p<0.05). the post-hoc lsd test showed significant differences between the treatment and negative control groups (p<0.05). conclusion: the pomegranate gel extract as an antioxidant increased the shear bond strength of the composite resin restoration after the 40% hydrogen peroxide bleaching application. keywords: antioxidant; bleaching; pomegranate extract; shear bond strength correspondence: indes rosmalisa suratno, department of conservative dentistry, faculty of medicine, universitas jenderal soedirman. jl. dr soeparno, karangwangkal, purwokerto, 53123, indonesia. email: indes.rosmalisa@gmail.com introduction tooth discoloration is a problem that can occur in teeth due to intrinsic or extrinsic factors. intrinsic tooth discoloration is of internal origin, such as tooth necrosis and drug consumption. meanwhile, extrinsic discoloration is a colour change that is influenced by external factors, such as the consumption of tea or coffee, cigarette stains and mouthwash that leaves colouring on the tooth surface.1 the treatment for tooth discoloration is tooth bleaching. bleaching is widely chosen because it is easy, fast and does not cause a reduction of the hard tissue in teeth.2 tooth bleaching is classified into internal and external. external bleaching is divided into in-office and homebleaching. internal bleaching or walking bleach is a procedure performed on non-vital teeth.3 the bleaching materials commonly used in in-office bleaching procedures are 30-50% hydrogen peroxide or 35–37% carbamide peroxide.4 using bleaching agents can have adverse effects, one of which is the reduction of the shear bond strength of the tooth surface with composite resin restorations. the bond strength of composite resin is very important for increasing mechanic and physical quality so that won’t easily released dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p87–91 mailto:indes.rosmalisa@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p87-91 88 suratno et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 87–91 if it given preassure.5 the bond strength of composite resin can be seen from the shear bond strength of the attachment, which is the maximum resistance of a material to withstand loads that cause shear movements before the composite resin material is released.6 several methods have been suggested to increase the bond strength after the bleaching procedure, including delaying the restoration procedure for 24 hours to three weeks.7 the delay of restoration procedures can be accelerated by eliminating free-radical peroxides, which are free radicals formed by residual peroxide from bleaching materials that were left on the tooth surface. a waiting period may not always be possible for many reasons, such as lack of time, personal circumstances or aesthetic reasons. many techniques have therefore been proposed to decrease the bond strength that is caused by the bleaching enamel, such as suggesting the use of adhesives containing organic solvents. the use of an antioxidant has been proven in many studies to be safe and effective to increase the bond strength directly after bleaching, with no significant difference with unbleached teeth.8 natural antioxidants have benefits in the field of dentistry; for example, they can increase the bonding strength of the composite resin fill, which decreases after the bleaching procedure.9 sodium bicarbonate, rosemary extract and aloe vera are already used to remove free radicals of hydrogen peroxide after whitening. some vitamins, like α-tocopherol (an active component of the vitamin e complex) and sodium ascorbate (sa) or ascorbic acid (vitamin c), are known as neutral, biocompatible and potent antioxidants that have the ability to reduce various oxidative compounds.8 the antioxidant material that is often used to eliminate free radicals after the bleaching procedure is 10% of ascorbic acid before the restoration procedure. this compound has high antioxidant potential, is biocompatible and has low toxicity.10 one of the fruits that is used as an antioxidant is pomegranate (punica granatum l.) because it has active alkaloid compounds, flavonoids, saponins, tannins and triterpenoids.11 previous studies by mukka et al.12 and sharafeddin and farshad13 have shown that the shear bond strength of composite resins increased after 5% and 10% of pomegranate peel extract was applied. their research has encouraged prospective researchers to conduct research on the effect of pomegranate extract gel (punica granatum l.) at 5% and 10% as an antioxidant. pomegranate extract gel was chosen because it has active compounds that can be used as antioxidants, such as pomegranate peel extract at 5% and 10%, which have been shown to increase the shear bond strength of composite resins after a bleaching procedure using 40% hydrogen peroxide. materials and methods this research received the approval from the health research ethics commission of faculty of medicine, universitas jenderal soedirman (number 201/kepk/ ix/2020). the type of research was true experimental laboratory using a simple randomised sampling technique using a posttest-only control group design. the materials used were pomegranate, 40% of hydrogen peroxide and a universal testing machine. the samples used were 32 maxillary first premolar teeth. the first premolar teeth were chosen because they represent the anterior teeth. the samples were divided into four groups, namely p1 (treatment group 1, the sample was applied with a concentration of 5% pomegranate extract gel after the application of 40% hydrogen peroxide), p2 (treatment group 2, the sample was a 10% concentration of pomegranate extract gel, which was applied after the application of 40% hydrogen peroxide), k1 (positive control group, the sample was applied 10% ascorbic acid extract gel after 40% hydrogen peroxide application) and k2 (negative control group, the sample was not given any antioxidant after the application of 40% hydrogen peroxide). the ascorbic acid was used because it has been used in previous journals as a positive control.14 the data used in this study are primary data in the form of material or a collection of facts that were collected by the researcher during the research. the methods of data collection in this study can be divided into getting ethical clearance, managing the permits, determining the pomegranate growth, the extracting and gelling of the pomegranate extract, making samples, treating the sample groups and testing the shear bond strength using the pearson panke universal testing machine (utm) (pearson panke, ltd. london, uk). the extract gel was made by macerating 1500 grams of pomegranate, which was mashed and soaked in six litres of a 96% ethanol solvent for two x 24 hours with stirring every 24 hours. stirring aims to accelerate the contact between the sample and the solvent. the solvent will penetrate the cell wall and enter the cell cavity so that the active substance dissolves. the immersion results were evaporated with a rotary evaporator at a temperature of 78 ± 1°c at a speed of 55 rpm. the gel was made by mixing pomegranate extract and natrium carboxymethyl cellulose (na-cmc), which were the ingredients of the gel base. the gel had to be physically evaluated through a homogeneity test, an adhesiveness test and a spreadability test before the application to ensure that the gel preparations had the same physical appearance after the preparation was made and that it still met the positive criteria parameters during storage. the criteria of a good gel are when the homogeneity test results show that the gel is homogeneous because it shows a homogeneous structure; there are no visible or coarse grains. the results of the gel adhesion test in each group showed that all the groups met the adhesion requirements, namely having an adhesion time of more than one second. the longer the stickiness the longer the active substance is attached so that it will have a more optimal effect. the spreadability test was carried out to determine the gel’s ability to spread when applied. the dispersibility dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p87–91 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p87-91 89suratno et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 87–91 requirements for the topical preparations were 5–7 cm and the three groups of preparations met the dispersibility requirements for the topical preparations. the preparations that are difficult to spread will reduce the comfort level of use and the effectiveness of using the preparation, while preparations that are too diluted will cause their adhesion to decrease so that the contact time of the active substance with the target is reduced.15 a sample preparation was done by making a resin block mould measuring 1.5 x 1.5 x 1 cm. the mould was used to fix the crown of the first maxillary premolar, which had been prepared in a labial position facing upwards. the resin block mould is shown in figure 1. the samples were treated by applying an opalescence boost pf 40% hydrogen peroxide gel (ultradent product inc., usa) to the labial surface of the teeth by as much as 1 mg for two x 20 minutes. the next sample was prepared according to iso / ts 11405: 2003 using silicon carbide abrasive paper size p600 under running water on the labial surface of not more than 1mm, while ensuring that all the surfaces were level and flat. after that, each sample group was subjected to treatment, namely groups p1 (sample applied pomegranate extract gel with a concentration of 5% 1 mg for 10 minutes), p2 (sample applied pomegranate extract gel with 10% concentration of 1 mg for 10 minutes), k1 (the sample was applied 1 mg of 10% ascorbic acid extract gel for 10 minutes) and k2 (negative control group, antioxidants were not applied to the sample after the 40% hydrogen peroxide application). all the sample groups were then subjected to restoration procedures using a nanohybrid composite resin to form the block moulds measuring 2 x 2 x 2mm. they were then subjected to light curing for 20 seconds. the entire sample group was immersed in distilled water for 24 hours at room temperature. the sample test was conducted by the shear bond strength test with the utm. a total of 32 tooth samples were subjected to a shear test to determine the value of the composite resin adhesion. the samples that had been implanted in acrylic were mapped on pearson panke equipment with a speed of 2.28 seconds/milli16 and then the shear bond strength was tested with the lowest strength. the resulting (f) in newton (n) units is the shear strength inputted with the shear bonding strength formula t = f/a, where t = shear adhesion strength (mpa), f = shear force (n) and a = cross-sectional area (mm2).17 the data analysis was performed using ibm’s spss statistics version 23 (armonk, new york). the resulting data were stated to be normally distributed (p> 0.05) and homogeneous after the data transformation was conducted. a parametric test using one-way anova with a confidence level of 95% (p = 0.05) was carried out to determine the significant differences in each group. the research data showed that there were significant differences (p <0.5). the data were analysed using the least significant difference (lsd) test to establish any significant differences. results the results of the determination test show that pomegranate comes from punica genus, the punica granatum l. species. the pomegranate extraction resulted in a thick extract of 300 g. the pomegranate extract and the ascorbic acid were then made into a gel based on na-cmc, which were evaluated to determine its physical condition. the evaluation of the gel preparations was carried out before the application to ensure that they had the same physical appearance after the preparation was made and that they still met the good criteria parameters during storage. the evaluation results of f1 (pomegranate extract gel 5%), f2 (pomegranate extract gel 10%) and f3 (ascorbic acid gel 10%) showed homogeneous results. the results of the adhesiveness evaluation of the formulas were 20, 32 and 25 seconds. the results of the dispersibility evaluation of the formulas were in the range of 5–7 cm. the shear bond strength of the composite resin that was tested using the utm had mean values, which can be seen in table 1. the results showed that the mean value in the p2 group (treated with 10% pomegranate extract) had the highest value, namely 42.5 mpa. the result of the oneway anova parametric test demonstrated a significant difference of p 0.000 (p <0.05).figure 1. resin block mould. table 1. one-way anova test result: shear bond strength group mean standard deviation p1 11.56 mpa 3.5197 p2 42.50 mpa 19.7303 k1 15.31 mpa 3.6443 k2 4.50 mpa 2.9881 note: p1: gel treatment pomegranate extract 5%; p2: 10% pomegranate extract gel treatment; k1: positive control 10% ascorbic acid gel; k2: negative control. table 2. post-hoc lsd test result: shear bond strength group p1 p2 k1 k2 p1 0.000 ** 0.154 0.002 ** p2 0.000 ** 0.000 ** k1 0.000 ** k2 ** = there is a significant difference (p <0.01) between the groups. source: primary data, 2020. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p87–91 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p87-91 90 suratno et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 87–91 the data were then tested post hoc using the lsd to compare the differences between the groups. the results of the post-hoc lsd test (table 2) showed that there were significant differences in each treatment group, except for the pomegranate extract gel group at 5% (p1) with the positive control group (k1). the result is said to have no significant difference because it has a value of p> 0.01. discussion the application of the 5% pomegranate extract gel, the 10% pomegranate extract gel and the 10% ascorbic acid gel was proven to increase the shear bond strength of the composite resin restoration. these results indicate that the gel is capable of being an antioxidant agent that can remove free radicals that are left on the tooth surface after the bleaching procedure using 40% of hydrogen peroxide. the 10% pomegranate extract gel treatment group (p2) had higher strength than all the groups, and there was a significant difference, which means that it had better antioxidant effectiveness and ability than other antioxidant ingredients. different test results in the 5% pomegranate extract gel treatment group (p2) with the negative control (k2) had a significant difference, which proves that the 5% pomegranate extract gel could be an antioxidant agent, but the different test results with the 10% ascorbic acid gel (k1) did not have a significant difference; it proved that the 5% pomegranate extract gel with 10% ascorbic acid gel (k1) did not show any difference, which means that the use of the 5% pomegranate extract gel was no better than the 10% ascorbic acid gel. a bleaching procedure can cause the shear bond strength of the composite resin of the enamel to decrease if the restoration procedure is carried out immediately after the bleaching process because there is peroxide residue that interferes with the resin adhesion, which inhibits the resin polymerisation.13 the reduction in bond strength is also caused by the loss of calcium caused by residual free radicals after the residual bleaching application; the peroxide ion is granular and porous with a bubble appearance and were trapped in the enamel.18 antioxidant agents are effective in increasing the shear adhesion strength and surface tension of the composite resin restorations after bleaching because they aim to deactivate free radicals. this accelerates the delay time of the restoration procedure because the antioxidants stabilise the electrons so that the enamel surface is uniform and nonporous. pomegranate peel extract has been shown to be effective removing free radicals and reducing oxidative stress by donating hydrogen atoms to prevent chain reactions of converting superoxide to hydrogen superoxide.19 the high antioxidant activity of pomegranate extract is related to the potency contained in the extracted content. one of the contents in the extract are tannin compounds; for example, punicalagin, which is classified as an ellagitannin, which is a donor of antioxidant activity in pomegranate extract gel compared to other ingredients. the 10% pomegranate extract gel and ascorbic acid gel can be an antioxidant agent that is capable of eliminating residual hydrogen peroxide after the bleaching procedure because the higher the extract concentration, the higher the antioxidant activity. the application of the gel will affect the polymerisation of the adhesive material so that the composite resin can penetrate the dentinal tubules. the enamel gap is also deeper; therefore, the bond between the tooth and the adhesive material is stronger. this is in line with previous research, which stated that the antioxidant agent applied after the bleaching procedure can bind free radicals from the effect of the bleaching procedure to minimise the occurrence of a bubble appearance, which can result in a decrease in the strength of the composite resin on teeth after the bleaching procedure using 40% hydrogen peroxide.14 it can be concluded that the pomegranate gel extract as an antioxidant increased the shear bond strength of the composite resin restoration after the 40% hydrogen peroxide bleaching application. references 1. greenwall l. tooth whitening techniques. 2nd ed. boca raton: crc press; 2017. p. 1–2. 2. tarigan r. perawatan pulpa gigi (endodonti). 3rd ed. jakarta: egc; 2013. p. 208–217. 3. perdigão j. tooth whitening: an evidence-based perspective. minneapolis: springer; 2016. p. 3–21; 101–169. 4. tarigan ra, hudiyati m, negara mc. pengaruh ekstrak bunga rosella (hibiscus sabdariffa l.) terhadap kekuatan rekat geser resin komposit pada gigi pasca bleaching. j mater kedokt gigi. 2014; 3(1): 1–7. 5. susra w, nur dl, puspita s. perbedaan kekuatan geser dan kekuatan tarik pada restorasi resin komposit microhybrid dengan bonding generasi v dan bonding generasi vii. indones dent j. 2013; 2(2): 68–75. 6. lijaya va, santosa p, dayinah hs. perbedaan kekuatan geser perlekatan resin komposit pada dentin menggunakan bonding total etch dan self etch dengan dan tanpa aplikasi klorheksidin diglukonat. j kedokt gigi. 2013; 4(2): 156–62. 7. khoroushi m, aghelinejad s. effect of postbleaching application of an antioxidant on enamel bond strength of three different adhesives. med oral patol oral cir bucal. 2011; 16(7): e990–6. 8. ghaleb m, orsini g, putignano a, dabbagh s, haber g, hardan l. the effect of different bleaching protocols, used with and without sodium ascorbate, on bond strength between composite and enamel. mater (basel, switzerland). 2020; 13(12): 2710. 9. wiwekowati, walianto s. aktivitas antioksidan ekstrak propolis dari yogyakarta dalam kedokteran gigi. semin nas ris inov. 2017; 5: 105–9. 10. danesh-sani sa, esmaili m. effect of 10% sodium ascorbate hydrogel and delayed bonding on shear bond strength of composite resin and resin-modified glass ionomer to bleached enamel. j conserv dent. 2011; 14(3): 241–6. 11. dkhil ma, al-quraishy s, abdel moneim ae. effect of pomegranate (punica granatum l.) juice and methanolic peel extract on testis of male rats. pak j zool. 2013; 45(5): 1343–9. 12. mukka pk, komineni nk, pola s, soujanya e, karne ar, nenavath b, shiva s, vuppunuthula p. an in-vitro comparative study of shear bond strength of composite resin to bleached enamel using three herbal antioxidants. j clin diagn res. 2016; 10(10): zc89–92. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p87–91 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p87-91 91suratno et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 87–91 13. sharafeddin f, farshad f. the effect of aloe vera, pomegranate peel, grape seed extract, green tea, and sodium ascorbate as antioxidants on the shear bond strength of composite resin to home-bleached enamel. j dent (shiraz, iran). 2015; 16(4): 296–301. 14. amiria f, harwoko h, widodo ahb. efek gel ekstrak kulit buah manggis (garcinia mangostana) pada perlekatan komposit pasca in-office bleaching. maj kedokt gigi indones. 2015; 1(1): 32–7. 15. aponno j v, yamlean pvy, supriati hs. uji efektivitas sediaan gel ekstrak etanol daun jambu biji (psidium guajava linn) terhadap penyembuhan luka yang terinfeksi bakteri staphylococcus aureus pada kelinci (orytolagus cuniculus). pharmacon j ilm farm – unsrat. 2014; 3(3): 279–86. 16. sayuti na. formulasi dan uji stabilitas fisik sediaan gel ekstrak daun ketepeng cina (cassia alata l.). j kefarmasian indones. 2015; 5(2): 74–82. 17. puspitasari d, soufyan a, herda e. aplikasi klorheksidin glukonat 2% pada dentin tidak mempengaruhi kuat rekat geser komposit resin yang menggunakan sistem adesif self etch. j dentomaxillofacial sci. 2014; 13(1): 7–12. 18. whang h-j, shin d-h. effects of applying antioxidants on bond strength of bleached bovine dentin. restor dent endod. 2015; 40(1): 37–43. 19. aprila, intan fitri. d& aa. aktivitas superoxyde dismutase (sod) tikus putih (rattus novergicus) wistar diabetes setelah pemberian ekstrak kulit buah delima merah (punica granatum l.). pros semin nas biot. 2018; 5(1): 643–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p87–91 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p87-91 mkgs vol 44 no 1 jan-mar 2011.indd editorial board of dental journal (majalah kedokteran gigi) sk: 2847/h3.1.2/kd/2009 june 1st, 2009 − june 1st, 2011 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. dr. m. rubianto, drg, m.s., sp. perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d.,fds. (conservative dentistry university of guy's dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery university science malaysia, malaysia); prof. widowati witjaksono, dds, ph.d. (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds.,ph.d. (pediatric – university of hiroshima, japan); prof. dr. a.g. m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg, m.s., sp. kga. (pediatric dentistry indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic jember university) managing editors: prof. dr. arifzan razak, drg, msc, sp.pros. (prosthodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp.kg. (conservative dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg., su., sp.bm. (oral and maxillofacial surgery – airlangga university); prof. dr. mieke sylvia m.a.r., drg, m.s., sp.ort. (orthodontic – airlangga university); prof. dr. istiati, drg, m.s. (oral biology – airlangga university); prof. dr. anita yuliati, drg, m.kes. (dental material – airlangga university); prof. seno pradopo, drg, s.u., ph.d. sp.kga. (pediatric dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d., sp.ort. (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes. (dental public health – airlangga university); dr. elly munadziroh, drg., ms. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); udijanto tedjosasongko, drg, ph.d., sp.kga. (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes. (oral medicine – airlangga university); prof. dr. diah savitri ernawati, drg., m.si. (oral medicine – airlangga university); bagus soebadi, drg, mhped. sp.pm (oral medicine – airlangga university); endang pudjirochani, drg, m.s., sp.pros. (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes. (microbiology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); ira widjiastuti, drg, m.kes. sp.kg. (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms. (orthodontic – airlangga university); devi rianti, drg, m.kes. (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio. (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros. (prosthodontic – airlangga university); an'nissa chusida, drg., m.kes. (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg. (conservative dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 44 no. 1 march 2011: prof. dr. regina titi christinawati tandelilin, drg., m.sc (oral biology – gadjah mada university) armasastra, drg., ph.d (dental public health – indonesia university) h.m. bernad ongki iskandar, drg., sp.kg., ficcpe., ficd (conservative dentistry – trisakti university) sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) dr. daniel haryono utomo, drg., sp.ort (dental clinic faculty of dentistry, airlangga university) retno palupi, drg., m.kes. (dental public health – airlangga university) eka fitria, drg., sp.perio (perodontic – airlangga university) hendrik setiabudi, drg., m.kes (oral biology – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 83/dikti/kep/2009 design cover photo by setyabudi, drg., mars., sp.kg issn 1978 3728volume 44 number 1 march 2011 contents page printed by: airlangga university press. (131/09.11/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 44 number 1 march 2011 issn 1978 3728 1. posterior transverse interarch discrepancy on hbe β thalassemia patients yuniar zen and loes d. sjahruddin .............................................................................................. 1–6 2. cytotoxicity difference of 316l stainless steel and titanium reconstruction plate ni putu mira sumarta, coen pramono danudiningrat, ester arijani rachmat, and pratiwi soesilawati ........................................................................................................................... 7–11 3. calcium hydroxide as intracanal dressing for teeth with apical periodontitis sari dewiyani .................................................................................................................................. 12–16 4. titanium ceramic restoration: how to improve the binding between titanium and ceramic harry laksono ................................................................................................................................. 17–24 5. acupuncture analgesia: the complementary pain management in dentistry abdurachman ................................................................................................................................... 25–29 6. management of anterior teeth damage caused by complex caries through aesthetic endorestoration nanik zubaidah ............................................................................................................................... 30–34 7. changes of the sweet taste sensitivity due to aerobic physical exercise ni luh putu ayu wardhani, anis irmawati, and jenny sunariani ............................................ 35–38 8. plaque index between blind and deaf children after dental health education cynthia carissa, jakobus runkat, and yetty herdiyati ............................................................. 39–42 9. cost effectiveness and quality of life assessment on dental filling and tooth extraction in balongsari public health center taufan bramantoro and thinni nurul r ....................................................................................... 43–48 10. tnf-α expression on rats after candida albicans inoculation and neem (azadirachta indica) extract feeding i dewa ayu ratna dewanti ............................................................................................................ 49–53 11. expression of matrix metalloproteinase-8 gene in fixed orthodontic patients susilowati, mansjur nasir, imam mudjari, and thalca hamid .................................................. 54–58 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38 no 2-2005 84 ekspresi produk gen laten virus epstein-barr pada karsinoma sel skuamosa rongga mulut (the expressions of latent gene product of epstein-barr virus in oral squamous cell carcinoma) theresia indah budhy s bagian biologi oral fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract squamous cell carcinoma (scc) is a type of cancer often found in oral cavity and the area of head and neck at about 90%. based on the geographical incidence oral scc (oscc) has many types of different emerging. this case probably has connection with ethnic group, habit and social and economical condition. in east java, the incidence is about 2.64% and it increases every year. the virus is known as one of the main factors that result in this disease. epstein-barr virus (ebv) has potential capability of carcinogenesis. ebv is the family of herpesviridae that can infect cell through the linking of cd 21 receptor of the epithel with glycol protein 350/220 of the virus capsule. after primary infection, the virus will form latent-gene in human cell. periodically, the latent-gene product can disturb proliferation and apoptotic regulator. in indonesia, the expression of ebv latent geneproduct in the oscc has not been reported yet. this study wanted to know the expression of ebv latent gene product found in the oscc. this study found 25 cases of oscc in which 17 were infected by ebv. detection of ebv infection could be done by insitu hybridization to identify rna ebv (eber). to find the expression of ebv latent gene product, immunohistochemical analysis was done. the conclusion was that the emerging of expression of ebv latent gene product in oscc were latent membrane protein-1 (lmp-1), ebv nuclear antigen-1 (ebna-1) and rna ebv (eber). they were 28.28%, 25.26% and 46.47%. it was suggested to do the following research on oscc infected by ebv and the emerging of expression of ebv latent gene product with regulator gene of proliferation and apoptotic in oscc. key words: epstein-barr virus (ebv), oral squamous cell carcinoma, lmp-1, ebna-1, eber korespondensi (correspondence): theresia indah budhy s, bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan epstein-barr virus (ebv) merupakan herpes virus gamma yang termasuk dalam famili herpesviridae dengan besar genom 172 kb. dari seluruh gen yang ada 9 gen di antaranya diekspresikan pada limfosit b yang terinfeksi. virus ini sering dikaitkan dengan karsinoma nasofaring, burkitt's lymphoma. pada karsinoma tersebut akan diekspresikan berbagai gen laten. produk gen laten yang diekspresikan dapat mempengaruhi gen pengatur pada proliferasi dan apoptosis sel.1 di rongga mulut kanker yang sering didapatkan adalah jenis karsinoma. menurut higa et al.2 karsinoma sel skuamosa rongga mulut yang terjadi pada penduduk okinawa jepang terkait dengan infeksi ebv. sejauh ini di indonesia belum ada laporan produk gen laten ebv yang diekspresikan pada karsinoma sel skuamosa rongga mulut (kssrm). mengingat hal tersebut, maka peneliti ingin mengkaji ekspresi gen laten ebv pada kssrm di jawa timur. epstein-barr virus (ebv) yang telah menginfeksi epitel akan menetap secara laten dan secara periodik menjadi aktif. hal ini didukung peneliti terdahulu hu and li3 bahwa pada hairy leukoplakia ditemukan partikel ebv pada hampir 100%. genom ebv yang berada pada sel inang berbentuk latent episome.4 latent episome ini terdiri dari beberapa produk gen yang akan diekspresikan. produk gen tersebut akan direplikasikan selama pembelahan sel inang. mengingat hal tersebut, maka gen ebv akan menyebabkan ebv persisten di dalam sel. hal tersebut mempengaruhi sistem pembelahan sel. bila keadaan ini berlanjut terus tanpa ada mekanisme protektif dan preventif maka menyebabkan progresivitas kanker semakin cepat. pada latent episome ebv terdapat berbagai gen antara lain latent membrane protein (lmp) 1, 2a, 2b, ebv nuclear antigen (ebna) 1, 2, 3, 4, 5, 6, lp dan ebv-rna (eber) 1, 2. pada jenis kanker yang berbeda biasanya akan diekspresikan gen ebv yang berbeda. dilaporkan oleh shimakage et al.5 bahwa eber banyak terkait dengan keganasan karena dapat menghambat apoptosis. sebelumnya dilaporkan oleh nicholson et al,6 bahwa ekspresi lmp-1 pada human cell line menyebabkan transformasi sel, sedangkan middeldorp et al.1 mengatakan bahwa ebna-1 banyak terkait dengan keganasan karena dapat meningkatkan aktivitas faktor transkripsi. berdasar permasalahan tersebut maka didapatkan suatu konsep bahwa di rongga mulut keganasan yang paling 85budhy: ekspresi produk gen laten sering dijumpai adalah karsinoma sel skuamosa. terkait dengan infeksi ebv, yang terdapat pada karsinoma rongga mulut akan diekspresikan berbagai produk gen laten antara lain eber, lmp-1 dan ebna-1. dengan mengkaji keragaman ekspresi gen ebv pada karsinoma sel skuamosa rongga mulut diharapkan dapat digunakan sebagai upaya pencegahan dini. bahan dan metode jenis penelitian yang dilakukan adalah eksploratif dengan menggunakan penderita karsinoma sel skuamosa rongga mulut (kssrm) sebagai obyek penelitian. pada penelitian ini diperiksa sebagai variabel adalah ekspresi produk gen laten virus epstein-barr yaitu: latent membrane protein-1 (lmp-1), ebv nuclear antigen-1 (ebna-1) dan ebv rna (eber). untuk mendapatkan sampel dilakukan secara purposive, didasarkan pada kriteria klinis dan hasil pemeriksaan histopatologis dengan diagnosis karsinoma sel skuamosa rongga mulut. penentuan ekspresi eber dengan menggunakan metode insitu hibridisasi. penentuan ekspresi latent membrane protein-1 (lmp-1) dengan metode imunohistokimia menggunakan antibodi monoklonal lmp-1 produksi novo, ekspresi ebv nuclear antigen-1 (ebna-1) dengan metode imunohistokimia menggunakan antibodi monoklonal ebna-1 produksi novo. penelitian ini dilakukan di rsu. dr. soetomo surabaya jawa timur sebagai sentra rumah sakit di indonesia bagian timur, laboratorium histopatologi di bagian patologi anatomi fakultas kedokteran unair untuk pemeriksaan histopatologi dengan pengecatan hematoksilin eosin guna menentukan diagnosis karsinoma sel skuamosa rongga mulut, gedung bedah pusat terpadu (gbpt) rsu. dr. soetomo untuk melakukan biopsi operasi, laboratorium patobiologi di gramik fakultas kedokteran unair untuk penyimpanan jaringan segar dengan nitrogen cair dan pemeriksaan imunohistokimia. pemeriksaan insitu hibridisasi dilakukan di laboratorium biomolekuler histologi fakultas kedokteran universitas gadjah mada. semua penderita yang memenuhi kriteria sebagai karsinoma sel skuamosa rongga mulut diikutsertakan dalam penelitian. penderita kemudian dilakukan biopsi operasi oleh ahli bedah kanker. semua penderita yang dilakukan biopsi operasi telah menandatangani lembar persetujuan (informedconcent). unit analisis penelitian berupa jaringan hasil biopsi operasi dengan ukuran volume sampel minimal 1 × 1 × 1 mm. kriteria sampel: 1) umur antara 30–70 tahun; 2) bangsa indonesia suku jawa; 3) tidak menderita tumor dan keganasan lain; 4) secara klinis tidak menderita infeksi dan kondisi umum baik; 5) penderita dari instalasi bedah onkologi kepala leher rsu dr. soetomo yang datang untuk pertama kali dengan diagnosa karsinoma sel skuamosa rongga mulut. cara kerja: pengambilan spesimen biopsi kanker rongga mulut dilakukan di gedung bedah pusat terpadu (gbpt) rsu dr. soetomo surabaya, oleh tim dokter bedah kepala dan leher yang diberi wewenang membantu penelitian. hasil biopsi segera dimasukkan ke dalam nitrogen cair dengan suhu -170° c dan menunggu proses pemeriksaan. pewarnaan imunohistokimia: 1) spesimen biopsi mukosa rongga mulut setelah difiksasi dengan buffer formalin selanjutnya dilakukan dehidrasi dan pembuatan blok parafin menggunakan metode baku. selanjutnya disiapkan kaca obyek yang telah dilapisi poli l lisin, pemotongan blok parafin menggunakan mikrotom dengan ketebalan 5 μm, potongan jaringan ditebarkan atau diapungkan pada permukaan bak pemanas berisi air hangat, selanjutnya jaringan yang telah menempel pada slide dilakukan deparafinisasi dan sediaan siap untuk dilakukan pewarnaan; 2) pewarnaan dilakukan dengan menggunakan antibodi monoklonal lmp-1 dan ebna-1 dengan teknik avidin biotin complex (novo castra biotin system), bertujuan supaya hasil pewarnaan menjadi lebih kuat dan jelas karena telah teramplifikasi. teknik insitu hibridisasi sesuai dengan metode yang dipublikasikan oleh dako, untuk menganalisis rna. teknik produk dako yaitu menggunakan probe peptida nucleic acid (pna) ebv-rna (eber) yang telah dicoba dengan jaringan atau sel yang berisi ebv, cmv, hhv, hsv, vzv, hbv namun hanya positif pada jaringan yang terinfeksi ebv. pembuatan slide preparat berasal dari jaringan segar, dicelupkan dalam cryomatix, kemudian dilakukan potong beku setebal 5 μm. teknik ini didasarkan pada pembentukan dupleks antara dua untai asam nukleat yang komplementer, kemudian terjadi pasangan secara tepat antara dua untai dna rna yang komplementer. ada 3 unsur yang penting yaitu: dna pelacak, dna target dan deteksi signal. langkah kerja: 1) denaturasi fragmen dna sehingga menjadi rantai tunggal dengan memberi 150 μl proteinase k terlarut tbs; 2) kemudian dilakukan hibridisasi dengan memberi fluorescent conjugated pna probe maka dna pelacak mencari pasangannya yang komplementer, dengan memasukkan slide dalam inkubator temperatur 55° c selama 1,5 jam; 3) tahap berikutnya adalah deteksi signal, sebelumnya harus dicuci dengan astringent wash solution selama 25 menit pada suhu 55° c, deteksi signal ini dengan memberi enzim biotinfosfatase (anti fitc/ap). ap ini yang akan mengubah substrat menjadi senyawa yang memancarkan sinar yang tidak larut dan tetap ada pada tempat dna hibrid. hasil hibridisasi ini dapat stabil selama 1 tahun pada suhu kamar, selain itu dapat dihitung perubahan warna atau sinar yang terpancar dalam beberapa jam. teknik ini sangat peka oleh karena hanya bereaksi pada dna target saja. penilaian hasil pewarnaan secara mikroskopis dilakukan secara kualitatif dengan melihat intensitas penyerapan warna, serta secara kuantitatif dengan menghitung jumlah sel yang positif menyerap warna. setelah pemeriksaan mikroskopis, selanjutnya dilakukan pengambilan dokumentasi mikrofoto dengan kamera digital pentax optio 230 2.0 mega pixel, film kodak asa 200 dicetak pada kertas dengan pantulan cahaya minimal. 86 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 84–87 etik penelitian dilaksanakan sesuai dengan kaidah yang berlaku. ethical clearance dari komisi etik yang memenuhi declaration of helsinski. hasil mengingat pengambilan sampel secara purposive maka semua kasus yang memenuhi kriteria diikutkan dalam penelitian. didapatkan 25 kasus kssrm yang teridiri 17 kasus terinfeksi ebv, 8 kasus tidak terinfeksi. tabel 1. perbedaan ekspresi lmp-1, eber dan ebna-1 pada karsinoma sel skuamosa rongga mulut yang terinfeksi dan tidak terinfeksi dari hasil penelitian terhadap 17 kasus karsinoma sel skuamosa rongga mulut yang terinfeksi ebv. ditemukan ekspresi gen latent membrane protein-1 (lmp-1) 28,28% (gambar 2), eber (gambar 1) sebesar 46,47% dan ebv nuclear antigen-1 (ebna-1) 25,26% (gambar 3), dapat dilihat pada tabel 1. tabel 2. jumlah anggota sampel kssrm dengan ekspresi gen ebv (lmp-1, eber dan ebna-1). berdasarkan keragaman genetik ebv yang diekspresikan, tampak terdapat perbedaan kuantitatif ekspresi genetik tersebut. dari 17 kasus di atas, sebanyak 5 kasus kssrm diekspresikan 3 ragam ekspresi gen laten ebv baik lmp-1 maupun ebna-1 dan eber, 2 kasus diekspresikan lmp-1 saja, dan 2 kasus hanya dieskpresikan ebna-1, sedangkan yang diekspresikan eber sebanyak 4 kasus, 2 kasus diekspresikan dua gen laten yaitu lmp-1 dan eber. gambar 1. sel kanker dengan insitu hybridization eber, inti berwarna coklat kehitaman dan kecil (pembesaran 400 kali). gambar 2. sel kanker dengan ekspresi lmp-1, menggunakan monoclonal antibody lmp-1, pada membran inti berwarna coklat kehitaman (pembesaran 400 kali). gambar 3. sel kanker dengan ekspresi ebna-1, menggunakan monoclonal antibody ebna-1, pada inti berwarna coklat kehitaman (pembesaran 400 kali). 87budhy: ekspresi produk gen laten pembahasan berdasarkan hasil penelitian yang dilakukan di rumah sakit dr. soetomo surabaya yang merupakan sentra seluruh rumah sakit di indonesia bagian timur, ditemukan 3 ragam produk gen laten ebv yang diekspresikan yaitu lmp-1, eber dan ebna-1 pada kssrm. hal ini sesuai dengan pendapat nicholson et al.6 bahwa lmp-1 dapat menyebabkan transformasi sel. demikian pula laporan gonzales et al.7 bahwa pada kssrm penduduk eropa ditemukan ekspresi lmp-1. walaupun jumlah yang ditemukan di eropa lebih banyak dibandingkan temuan peneliti kemungkinan berbagai faktor lain yang mempengaruhi. ekspresi isolat ebv juga dipengaruhi oleh faktor ras genetik dan geografis.8 ragam genetik ebv lain yang diekspresikan pada kssrm yang ditemukan di rsu. dr. soetomo adalah ebv nuclear antigen-1 (ebna-1). dibandingkan dengan jumlah lmp-1, ternyata ebna-1 lebih kecil diekspresikan pada kssrm. keadaan ini mendukung laporan terdahulu oleh middeldorp et al.,1 bahwa ebna-1 banyak terkait dengan keganasan karena dapat meningkatkan aktivitas faktor transkripsi, namun ekspresi gen ini sangat spesifik hanya terdapat pada jenis kanker tertentu saja. mengingat hal itu, maka diduka kssrm adalah salah satu jenis kanker yang dipergunakan oleh ekspresi ebna-1. dengan diketemukan ekspresi ebna-1 pada kssrm merupakan hal baru. gen ebna-1 ini menyandi faktor transaktivator yang dibutuhkan untuk replikasi dna, selain itu dapat berikatan dengan rna melalui regio pengulangan glyala. keberadaan ebna-1 secara laten akan mempertahankan hidup ebv. ebna-1 juga akan mengaktifkan faktor transkripsi, baik untuk ebna-1 sendiri maupun ebna yang lain, seperti ebna 2a, 2b.3 pada penelitian ini didapatkan ekspresi eber yang paling banyak dibanding gen laten lainnya. hal ini dapat dimengerti bahwa eber pada fase laten akan ditranskripsi dalam jumlah banyak yaitu 106–107 per sel. eber mempunyai kemampuan mengaktifkan transkripsi terhadap genom virus yang lain yaitu, latent membran protein (lmp) dan ebv nuclear antigen (ebna). mengingat hal itu maka keberadaan eber di dalam sel inang akan semakin meningkatkan aktivitas virus. selain itu eber berperan terhadap transkripsi dan menghambat apoptosis. hal ini didukung oleh komano and takada9 bahwa ekspresi eber meningkatkan ekspresi bcl-2. peningkatan ekspresi bcl-2 ini akan menghambat pelepasan cytochrom-c sehingga tidak terjadi cascade caspase. hal ini menyebabkan tidak terjadi apoptosis. pada penelitian ini juga ditemukan kasus kssrm dengan dua ragam genetik ebv yang diekspresikan secara bersama. keadaan ini semakin memperkuat laporan sebelumnya bahwa genom ebv bila berada pada sel inang berbentuk latent episome terdiri dari beberapa ragam genetik yang akan diekspresikan. keberadaan keragaman gen laten ebv tersebut akan menyebabkan proliferasi sel inang berlebihan. hal ini memicu karsinogenesis dan mempercepat progresivitas. karsinogenesis terjadi melalui berbagai tahapan atau multistep dan multi hits.10,11,12 berbagai faktor penyebab yang kompleks juga merupakan hal yang perlu dikaji lebih jauh. selain itu berbagai faktor genom regulator yang mengatur proses proliferasi sel dan apoptosis sangat penting peranan terhadap karsinogenesis. mengingat hal tersebut maka perlu dilakukan penelitian lanjutan untuk mengetahui peran tiap ekspresi gen laten ebv terhadap gen regulator proliferasi dan apoptosis. seperti onkogen, supresor gen, gen repair. hal ini untuk membuktikan pendapat king10 bahwa karsinogenesis terjadi karena ketidak seimbangan antara proliferasi dan apoptosis sel, serta keberadaan mutasi gen. dapat disimpulkan bahwa pada karsinoma sel skuamosa rongga mulut yang ditemukan di rsu. dr. soetomo yang merupakan sentra seluruh rumah sakit di indonesia timur diekspresikan tiga ragam gen ebv yaitu latent membrane protein-1 (lmp-1), ebv rna (eber) dan epstein barr nuclear antigen-1 (ebna-1). disarankan perlu dilakukan penelitian lanjutan untuk mengetahui peran ekspresi genetik ebv terhadap gen regulator proliferasi dan apoptosis sel kanker. daftar pustaka 1. middeldorp jm, antoinette atp. brink, adrian jc van den brule, chris jlm meiger. pathogenic roles for epstein-barr virus (ebv) gene product in ebv– associated proliverative disorder. oncol– hematology 2003; 45: 1–36. 2. higa m, kinjo t, kamiyama k, iwamasa t, hamada t, iyama k. epstein-barr virus subtype in ebv related oral squamous cell carcinoma in okinawa, a sub tropical island in southern japan, compared with kitakyusu and kumamoto in mainland japan. j clin pathol 2002; 55: 414–23. 3. hu, li fu. nasopharyngeal carcinoma and epstein-barr virus. microbiology and tumor biology center, karolinska institute. stokholm 1996; 15–25. 4. aitken c, senggupta sk, aedes c, moss dj, scully tb. heterogeneity within the epstein-barr virus nuclear antigen 2 gene ini different strain of epstein-barr virus. j gen virol 1994; 75: 95–100. 5. shimakage m, horri k, tempaku a, kakudo k, shirosaka t, sasagawa t. association of ebv with oral cancers. hum pathol 2002 jun; 33(6): 608–14. 6. nicholson lj, hopwood p, johannessen i, salisbury jr, codd j, thorley-lawson d, crawford h. epstein-barr virus latent membrane protein does inhibit differentiation and induces tumorigenicity of human epithelial cells. oncogenesis 1997; 15: 275–83. 7. gonzales, m, gutirezz, rodriquez, avila r, archilla r. epsteinbarr virus latent membrane protein-1 (lpm-1) expression in oral squamous cell carcinoma laryngoscope 2002; 112(3): 482–87. 8. degreef h and the famciclovir herpes clinical study group. famciclovir, a new oral antiherpes drugs: result of the first controlled clinical study demonstrating its efficacy and safety in the treatment of uncomplicated herpes zoster in immunocompetent patients. int j antimocrob agent 1994; 4: 241–6. 9. komano jun, kenzo takada. role of bcl-2 in epstein-barr virusinduced malignant conversion of burkitt's lymphoma cell line akata. j of virology 2001 feb; 1561–4. 10. mendelson j, howley pm, israel m, liotta la. tumor suppressor genes. in the molecular basis of cancer. philadelphia: wb saunders co; 1995. p. 86–100. 11. king, roger jb. growth: a balance of proliferation, death and differentiation in cancer biology. 2nd ed. prentice hall; 2000. p. 146–70. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� expression toll-like receptors in the oral mucosa of patients with recurrent aphthous stomatitis diah savitri ernawati department of oral medicine faculty of dentistry airlangga university surabaya indonesia abstract toll-like receptors (tlrs) have recently emerged as key receptors of the innate immune system. they recognize specific pathogenassociated molecular patterns initiating a host defence response. the oral mucosa epithelium encounters potential pathogens like bacteria and viruses in inspired air, and the discovery of tlrs on epithelial cells suggest that the epithelium has a role in the mucosal immune system. the aim of this study was to discover recurrent apthous stomatitis (ras) using etiopathogenetic molecular approach by observing the tlrs. immunohistochemistry using monoclonal antibodies anti-tlr-2, tlr-3, tlr-4, tlr-5, tlr-7 and tlr-9 were used in this study. these antibodies are specific toward tlr on the surface of epithelial cells membrane and macrophages in patients with major and minor ras. tlr was expressed on the surface of epithelial cells membrane of oral mucosa and macrophages in both major and minor ras patients. tlrs was not expressed specifically in non-ras patients. the results above showed indication, that functional tlrs expression by epithelial cells in oral mucosa had remarkable implication on natural immune response and disease pathogenesis. the expression of tlr was found in the oral mucous membrane on epithelial cells surface and macrophages patients with ras. key words: major and minor ras, toll-like receptors correspondence: diah savitri ernawati, c/o: bagian oral medicine, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction the oral mucosal epithelium is an important interface with the environment, and represents a dynamic system for innate host defense. in addition to providing a barrier to the entry of pathogens, epithelial cells lining oral mucosal have been shown to response the presence of microorganisms by producing natural antimicrobial factors and mounting an inflammatory response.1,2 the innate immune system also plays a role in several human diseases. the primary function of tolllike receptors (tlrs) is to recognized pathogens, tlrs can also specifically recognize pathogen-associated molecular patterns (pamps), tranduce signals into cells and initiate complex signal cascade leading to activation of the transcriptional factors such as nuclear factor kappa b (nf-kb) and interferon regulatory factor.3–5 since 1996, 11 tlrs (tlrs1-11) have been found in both mammal and human dendrite, mononuclear and epithelial cells of intestine and bronchus, endothelial cells of blood vessels and epithelial cells as well as cells from various organ system. toll-like receptors are transmembrane proteins, all of which have common extracellular leuinerich domain and conserved cytoplasmic domain. the cytoplasmic domain of tlr is homologous to the il-1 and il-18 receptors and contains the toll/il-1 receptor (tir) homology domain common to these receptors.3–8 recurrent aphthous stomatitis (ras) are the most common oral disease characterized by repeated development of painful ulcers. the aetiology of ras is still unknown. many local and systemic factors such as bacterial or viral infection, genetic factor, hormonal, immune system disorders, hematinic deficiency, and systemic disease could be involved in pathogenesis of ras.9–11 previous studies have also suggested that this inflammatory disease is a result of abnormal immune response directed towards the oral mucosa. activation of the immune system in aphthous lesions and peripheral blood was observed in ras.3,13 tissue-specific autoimmunity is one of the most probable mechanism of ras development, with possible activation of th1 lymphocyte profile of cytokine production.12–14 stimulation of internal and external factors could induce multiplication process in proliferation cell phase. it eventually could be continuously express protein anomaly, because the superficial character is different from normal oral mucosa.15,16 decrease ability of oral tolerance activities on inflammation of disease pathogenesis showed very impressively reduced of t helper, th-2, and th-3. genetic and entvironment may contribute to lower tolerance caused cytotoxic effect by oral epithelial.17 the objective of this study was to disclose or discover ras using etiopathogenetic molecular approach by observing the tlrs. main objectives of this study was to prove �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 47-51 the presence of tlrs on the surface of epithelial cells membrane and macrophages and to identify predominant tlrs types at those sites in patients with major and minor ras. materials and methods patients and samples inform concernt approved by the research ethics committee was obtained from all the participants. the study population consisted of 21 samples major ras (10 women, 11 men) and 18 samples minor from ras patients (7 men, 11 women, mean age 21 years; range 19–55 years) who did not have any systemic or inflammatory disease and 11 healthy individuals. ras patients were defined as those having at least one aphthous lesion per month during the preceding years. tissue samples were obtained from patients in an active stage of ras (1–3 days old ulcers) and from controls were collected by using fine needle aspiration biopsy (fnab) on ulcer marginal edge and scrapped of oral epithelial biopsy. five ml of venous blood were drawn from the anterior arm, anti-coagulated with heparin and diluted 1:1 with hank’s liquid. then the mixture was gently added to a centrifuge tube containing 5 ml of lymphocyte-separating liquid and centrifuged at 700 r/min for 20 minutes. the cells in gray middle layer were collected and washed twice with pbs. the deposit contained about 25% monocytes and 75% lymphocytes.17 immunohistochemistry scrapped specimen of oral epithelial biopsy in oral mucosa fixed on an object glass with 90% alcohol, and incubated in refrigerator or directly blocked with 1% bovine serum albumin (bsa 1%) and incubated in co2 at the temperature 37° c for 45 min. after being washed with pbs, sample is reacted with monoclonal antibody tlrs anti tlr-2, tlr-3, tlr-4, tlr-5, tlr-7 and tlr-9, re-incubated in co2 incubator at 37° c for one hour. after being washed with pbs, the sample was analyzed using immunofluorescence microscope by magnifying with 40×. results a study has been conducted to 21 and 18 patients with major and minor ras, respectively, and to 10 non-ras patients as control in order to identify the presence of protein like receptors (tlrs) in epithelial cells and macrophages of patients with ras. in this study, it was found that tlrs was expressed on the surface of epithelial cells membrane of oral mucosa and macrophages in both major and minor ras patients. tlr was not expressed specifically in non-ras patients. the patients with positive tlrs-2 were 41.02%, revealed among minor ras patients, while in major ras tlrs-2 was 43.58% expressed in (figure 1). the samples which expressed tlrs-3 were mostly major ras patients as much as 43.58% from 38 ras patients, while those with minor ras positive tlrs-3 was 17.94% (figure 2). ras patients with positive tlrs-4 was 48.71%, found in major ras patients, while in minor ras tlrs-4 was 38.46% expressed (figure 3). samples of ras patients expressing tlrs-5 was 7.69% in major ras patients, and 10.25% was expressed mostly in minor ras patients. immunofluorescent analysis showed that tlrs-5 was not well expressed in epithelial cell surface and macrophage the expression of tlrs-7 in ras cases that had been analyzed immunohistochemically showed no specific expression either on the surface of cell membrane or cytoplasm. only 20.51% showed positively against tlrs-7. ras patients with positive tlrs-9 were 33.33%. for major ras cases, there were 17.94% with positive tlrs-9 and, similarly, there were also 17.94% positively minor ras cases (figure 4). figure 1. immunohistochemical detection of tlrs in minor ras patients with scrapped oral epithelial biopsy. tlrs-2 expression on cytoplasmic and surface of epithelial cell membrane. figure 2. tlrs-3 expression on surface of epithelial cells membrane in the oral mucosal major ras patients reacted with tlrs-3 monoclonal antibody. ras major and minor visualized with dab-chromogen ��ernawati: expression toll-like receptors in the oral mucosa figure 3. a) tlrs-4 expression in this minor ras was not well ditributed in all cells, either on the cells membrane surface or in the cytoplasms. b) tlrs-4 expression in major ras was found on epithelial surface and macrophages cells surface. the results above showed indication that tlr expression by epithelial cells in oral mucosa had remarkable implication on innate immune response and disease pathogenesis. discussion tlrs are type i transmembrane protein involved in innate immunity. their structures are characterized by extracellular leucine-rich repeats and an intracellular motif with a high degree of homology to the intra-cellular domain of the il-1 receptor.3,4 tlrs can specifically recognize pathogen-associated molecular pattern (pamps), transducer signals into cells and initiate complex signal cascades leading to activation of the transcriptional factors such as nuclear factor kappa b (nf-kb) and interferon regulatory factor. subsequently, the inflammatory mediators such as il-1a/b, il-6, il-8 and tnf-a are synthesized and released to activate neutrophils and lymphocytes. this result in the initiation of innate and adaptive immune response. deficiency of tlrs may result in corresponding pathogen recognition failure and susceptibility to certain pathological microbes. hyper-expression of tlrs in infected tissues may promote excessive inflammation.18 in the family of 11 tlrs, the function of tlrs-10 remains unknown and the expression of tlrs 11 is limited to urethral epithelial cells. to further investigate the expression of tlrs in oral mucosa epithelium, the expression of tlr1-9 were studied. molecule of pathogen can express some pamps, it is recognized of different tlrs, that heredity redundancy on immune system for blocking of microbial infection. p a m p s c o m e f r o m p o s i t i v e g r a m o r g a n i s m (peptydoglycan and lipoprotein), it is recognized tlrs-2. the other member subfamily of tlrs-2 is tlrs-1 and tlrs-6. tlrs-2 combined with tlrs-1 and tlrs-6, that have a signal response to many kind of microbial pathogen as well as mycobacterium. tlrs-2 signal is needed for fragmentation of m. tuberculosis in macrophage. tlrs-2 in mouse has important role because the individual more sensitive to m tuberculosis infection if they have decrease of tlrs-2. on the otherhand, tlrs-2 also can bind components of herpes virus, which binds in turn to cd14 on the cell surface.19 ras in dental hospital of faculty of dentistry at airlangga university more 43,7% is found in mayor ras. this phenomenon is very important in oral medicine because the signaling through tlrs occurs through a well described pathway in which receptor binding generates a signal through an adaptor molecule, myd88, that leads to intracellular associated with il-1 receptorassociated kinase. tlrs-4 can mediated lps as signaling from bacteria of gram negative that activated macrophage through tlrs-2. base on the type of agent of microbial and their pamps that the tlrs can recognize direct to plasma membrane as well as tlrs-4 or direct in the phagosome like tlrs-2. tlrs response can appear in different cell expression. epithelial gut cells using lps that can associated with tlrs-4 in golgy complex in plasma membrane. this statement like with tlrs which expressed on mouth epithelial cells. however, on the basis of studies with tlrs-4 is similar figure 4. scrapped oral epithelial biopsy in major ras patients after reacted with tlrs-9 monoclonal antibody labelled with fitc. �0 dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 47-51 but not identical to the signaling pathway activated by other tlrs. also the activation of cytokine production by tlrs plays an important role in recruiting other components of innate host defense against molecule pathogen as well.19 it showed here that tlrs-2 and tlrs-4 cells were present in ulcer tissues from both healthy control and subject with major and minor ras. the presentation of tlrs-2 expression in major and minor ras patients could be related with the presence of bacterial infection/lps. immunohistochemically, tlrs-2 is an inflammation signaling in oral mucosa induced by gram-positive bacteria. bacterial molecules that can induce tlrs-2 expression are outer membrane and polysaccharides. these molecules apparently had important role in ras patients visiting dental outpatient clinic. therefore, in oral cavity, the presence of bacterial infection should be noticed although the bacteria are not the predominant cause of ras.2,3,6,8 there are some alternative of tlrs-2 and tlrs-4 that cause inflammatory process in correlation with ras. the first alternative is through hyper secretion of cytokine, also can active an pro inflammation molecule as well as tnf-a, il-1b, il-6 and il-12. the other molecules are activated too as accessories cells are neutrophil, dendritic cells using signaling molecule rantes, mip-1a and mip-1b. the second alternative is inflammatory process began by special pathway, that called myd88 dependent pathway using activated tiram and then induce irak-4 and trap6 and activated mapkkk, these process as became signaling innate immunity through map3 and protein (p38), finally inducing af-1 to make inflammation.8,19,20 especially tlr2 has two possibilities of signaling, through rac1 and myd88. the signaling is through myd88 so it can be activated fadd and then caspase and cause fragmented of dna known as apoptosis. the signaling that through rac1 pathway were activated through p13k and akt to induce signal nf-kb (nuclear factor kb) caused inflammatory. this pathway is predominant to cause ras like tlrs-9 too used always myd88 dependent pathway. ras identification using immunohistochemically examination revealed that tlrs-2 was expressed at the surface of epithelial cells and macrophage, in addition to intra cytoplasm of epithelial cells. this was likely related to nf-kβ activated through ifn-gamma, which finally lead to the occurrence of hypercytochemistry and ended with severe inflammation, resulting in ras.13–15,20 the properties of the tlrs-3 a is different with tlrs-2, tlrs-4 with activity tlrs-9 because tlrs-3 can protect viral infection. the mechanism is began activation of trip and then activated tbk1 to give signal to irf3 which finally secretie ifn-a, b. interaction between lps and receptor tlrs-4 induced complex signal that begin by activating of factors transcription such as nf-kb, and then stimulated and activated inflammation gene as tnf-a, il-6 and il-8. tlr2 and tlr4 are known as mediate inflammatory responses to bacterial components.21 tlrs-2 is responsible for the recognition of lta, whereas tlrs-4 recognizes lps in immune competent cells such as macrophages, the response to lps is mediated by interaction with tlrs-4 in conjunction with tlrs-4 accessory protein md-2 and cd 14, and transducers intracellular signals followed by the activation of tlrs-associated adapter protein, myeloid differentiation factor 88 (myd88), leading to the activation of nf-kb.22 the samples which expressed tlrs-3 were mostly major ras was 43.58% from 38 rau patients, while those with minor ras positive tlrs-3 was 17.94%. immunohistochemically examination using chromogene revealed that the samples showed significant reaction against tlrs-3, as it was clearly expressed at the surface of epithelial cells and macrophage, as it was also expressed within cytoplasm and intra cellular (intra nuclear). this indicated that the predominant inductor was a type of viral infection, with the result that cellular activities at molecular level, such as interleukin/the cytokines increased as well. tlrs-3 uses multiple mechanism to enhance and sustain the antiviral response more strongly than tlrs-4. ras patients with positive tlrs-4 was 48.71%, found in major ras patients, while in minor ras tlrs4 was expressed 38.46%. tlrs-4 could be detected on the surface of epithelial cells and cytoplasm from oral mucosal epithelium in either major or minor ras patients. in major ras patients, the tlrs-4 expression was more predominant in the surface of epithelial cell and macrophage. this seemed since the oral mucosal epithelium of patients with ulceration of oral mucosa can respond the presence of bacterial endotoxin by activating tlrs-2, tlrs-3, and tlrs-4. tnf-alpha is an inflammatory cytokine produced through tlrs activation in its response against bacteria.23 tlrs with transmembrane protein and cell surface receptor and intra cytoplasms signal area may likely play more important role in this intracellular signaling. tlrs-4 blocking by neutralizing antibody anti-tlrs-4 apparently inhibited tnf-alpha after lps administration.23 a previous study using immunohistochemistry also showed that tlrs-2 and tlrs-4 cells numbers increase in the inflamed mucosal ulcers. although level of transcript were not quantitated. expression of toll-like receptors it has been proved immunohistochemically that tlrs can be detected on the surface of oral mucosal epithelium and cytoplasm of ras patients. tlrs localization has been widely related with immune and inflammation cells. epithelial cells in oral mucosa may relate with many potential pathogens, and tlrs expression will be relevant with immunity of oral mucosa. epithelium is the primary target of infectious agents. therefore, these epithelial cells play an important role in inflammation for production of various cytokines and pro inflammatory cytokines. preservation of il-10, while il-1β and tnf-α are down regulated, could play a protective role against inflammatory ��ernawati: expression toll-like receptors in the oral mucosa tissue destruction. on otherhand il-1β, tnf-alfa and il-6 apart from being inflammatory mediators, also can facilitate soft and hard tissue destruction and favor the pathogenesis of ras.14 convergence of intracellular signaling pathways by tlrs and inflammatory cytokines (il-1β and tnf-α) along with reprogramming of signal transduction and gene transcription, may explain to certain extent the cytokine profile during endotoxin tolerance. the regulation of tlrs expression on macrophages in vitro is consistent with the inflammatory cytokine response. we showed that il-1β, tnf-α, il-6, il-8 and il-10 levels increase on initial lps stimulus but were not equally susceptible to a decrease on lps challenge. most notably il-10 and il-8 were refractory to endotoxin tolerance. tlrs mediate tolerance obtunds the inflammatory cytokine response. tlrs activation culminates in transcription of inflammatory cytokine which finally cause ras.5,14,20,25 in addition it was found that expressed tlrs were not only from one type, but also from the others. the predominant tlrs expressed in ras patients were tlr-2, –3 and –4. in connection with the result of previous studies in its clinical implications, the causing agents or inducers were not only one type, but an accumulation of molecules in oral mucosa that played mutual role as predominant inducer. therefore, tlr was expressed not only on the surface of cell or macrophages, but also found in intra cytoplasms, surface membrane cells and even intra nucleus.20,24,25 the conclusions that tlrs expressed on the surface of oral mucosal epithelial cells and macrophages in ras patients, either major or minor. tlrs was not specifically expressed in non-ras. tlrs-2, tlrs-3 and tlrs-4 has been expressed on the surface of cells membrane and macrophages in minor and major ras. tlrs was more predominantly expressed in major ras compared to minor ras. functional tlr expression by oral mucosa epithelial cells had higher implications towards natural immune response and disease pathogenesis. it is suggested to undertake molecular characterization to determine specific tlr against specific disease agents, so that it might be easy to identify the causing agent, with the result that ras management can be established comprehensively. acknowledgement the author would like to thank to badan litbangkes departemen kesehatan ri and lembaga biologi molekuler eijkmann which had given the sponsorship to do this research. we also thanks to prof. dr. yoes prijatna dachlan, dr., m.sc and prof. dr. fedik a rantam, drh. for valuable comments and discussions and mrs. helen for skillful technical help. references 1. diamond gd, legarda a, ryan ik. the innate immune response ofthe innate immune response of the respiratory epithelium. immunol rev 2004; 173:27–38. 2. sugawara s, uehara a, tamai r, takada h. innate immune responses in oral mucosa. j endotoxin res 2002; 8(6):465–8.j endotoxin res 2002; 8(6):465–8. 3. takeda k, akira s. tlr signaling pathway. semin immunol 2004; 16:3–9. 4. akira s, hemmi h. recognation of pathogen-associated molecular pattern by tlr family. immunol lett 2003; 85:85–89. 5. lewkowicz n, lewkowicz p, kumatowska a. innate immune system is implicated in recurrent aphthous ulcer pathogenesis. j.oral pathol med 2003; 32:475–81. 6. delneste y, beauvillain c, jeannin p. innate immunity: structure and function of tlrs. med sci 2007; 23(1):67–73. 7. beutler b, rehli m. evolution of the tir, tolls and tlrs functional inferences from computational biology. curr top microbial immunol 2002; 270:1–21. 8. medzhitov r. toll-like receptor and innate immunity. nat rev immunol 2002; 1:135–45. 9. lorenz e. tlr2 and tlr-4 expression during bacterial infection. curr pharm 2006; 12(32):4185–93. 10. mc. nally. recurrent aphthous stomatitis and precieved stress: preliminary study. available at: http:/www.aphthous.stress study. tripod.com/resed.doc. accessed march 18, 2003. 11. scully c, gorsky m, lozada-nur f. the diagnosis and management of recurrent aphthous stomatitis a consensus approach. j am dent 2003; 134(2):200. 12. porter s, scully c. aphthous ulcers (recurrent). clin evid 2004; (11):1766–73. 13. natah ss, hayrinen-immonen r, hietanen j, malmstromm, konttinen yt. ������������������ ��� t����� ����������� ����� �� ���. �� ���������������������� ��� t����� ����������� ����� �� ���. �� ���� pathol med 2000; 29:19–25. 14. sun a, chang yf, chia js, chiang cp. serum il-8 level is a more sensitive marker than serum il-6 level in monitoring the disease ����v��y ��� ���. �� ���� p��h�� m�d 2004; 33:133–9. 15. hasan a, shinnick t, mizushima y, van der zee r, lehner t. defining t-cell epitope within hsp65 in ras. clin exp immunol 2002; 128:318–25. 16. ernawati ds. immune response 65 kda protein in recurrent aphthous ulceration (rau). the international journal of oral health 2004; 1:53. abstract. 17. ernawati ds. the molecular analysis on the expression of protein anomaly oral mucosa in ras patients. indonesian journal of dentistry 2006; 13(special edition kppikg xiv). 18. borra rc, andrade pm, silva idcg, morgan a, et al. the th1/th2the th1/th2 immune-type response of the recurrent aphthous ulceration analyzed by cdna microarray. j oral pathol med 2004; 33:140–6. 19. tosi mf. innate immune responses to infection. j allergy clin immunol 2005; 116(2):241–7. 20. wu xin-yi, gao jian-lu and ren mei-yu. expression profiles and function of toll-like receptors in human corneal epithelia. chinese medical journal 2007; 120(10): 893–7. 21. anderson kv. toll signaling pathways in the innate immune response. curr opin immunol 2003; 12:13–19. 22. kaisho t, akira s. toll-like receptors as adjuvant receptors. biochem biophys acta 2002; 1589:1–13. 23. xu z, dzarski r, wang q, swartz k, sakamoto km, gupta d. bacterial peptidoglycan-induced tnf-alpha transcription is mediated through the transcription factors egr-1, elk-1 and nf-kappa beta. j immunol 2001; 167:6975–82. 24. stefan b, gunther h. toll-like receptors (tlrs) and innate immunity. handbook of experimental pharmacology 183. springer-verlag berlin heidelberg; 2008. p. 1–14. 25. cario e, gerken g, podolsby dk. toll like receptors-2 controltoll like receptors-2 control mucosal inflammation by regulating epithelial barrier function. gastroenterology 2007; 132(4):1359–74. 94 the effect of voltage and time of exposure on surface hardness of resin modified glass ionomer cement titien hary agustantina department of dental material and technology dentistry faculty of dentistry airlangga university surabaya indonesia abstract light intensity can affect the polymerization of resin-based material therefore it will also affect surface hardness. to compensate reduction of light intensity becaused of the input of low voltage (200 and 210 volt), the exposure time was prolonged to be 40 and 60 seconds. the purpose of this study was to know the effect of voltage and time of exposure of resin modified glass ionomer cement on surface hardness. this study was completed with seventy two samples of resin modified glass ionomer cement 5 mm in diameter and 2 mm in height, exposed by visible light curing for 20, 40, and 60 seconds with voltage input 220, 210 and 200 volt. micro vickers hardness tester was used to test surface hardness on the upper surface of sample. two-way anova test and least significant difference (lsd) were used in data analysis with 5% level of significance. the result showed that lower input (210 and 210 volt) would decrease surface hardness of resin modified glass ionomer cement, longer exposure time (40 and 60 seconds) would increase the surface hardness of resin modified glass ionomer cement. the conclusion is the prolong of exposure time from 20 seconds to 40 and 60 seconds in all voltage input could increase surface hardness of resin modified glass ionomer cement. key words: voltage, exposure time, resin modified glass ionomer cement, surface hardness correspondence: titien hary agustantina, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the tooth restorative material of glass ionomer cement has been widely developed and the development is directed to hybrid form. these materials consist of the component of a glass ionomer cement modified by the inclusion of a small quantity of additional resin which perform an activation to visible light and commonly called resin modified glass ionomer cement. resin modified glass ionomer cement is developed with the purpose to overcome various problems on the character of glass ionomer cement for example: the sensitivity to humidity and dryness, low strength, bad surface hardness.1,2,3 resin modified glass ionomer cement is immediate hardened after being exposed using visible light so water contamination could be eliminated. cement which is hardened more quickly might be resistant to water absorption and solubility in water while the acid base reaction would continue.2 resin modified glass ionomer cement is available in the form of powder and liquid. the composition of powder in glass ionomer cement consist of glass particles of calcium fluoroaluminocilicate which could release ion and initiator to enable the curing process chemically or with visible light.1 the liquid contain of 5–15% resin component in the form of hydroxyethyl methacrylate [hema] together with less than 1% polymerisable group and photo initiator which consist of camphoroquinone.2 the cement also contains of water and polyacrylic acid or polyacrylic acid with carboxylic group modified with methacrylate and hema monomer. the some additional resin and photo initiator in resin modified glass ionomer cement might cause the restorative material harder and quickly hardened (setting).1 resin modified glass ionomer cement could polymerize in two days: dual-cure and tri-cure,1,2,4 and it could harden without radiation of visible light through base-acid reaction with radiation of visible light.5,6 polymerization of restorative material which performs an activation with visible light really depends on light intensity resulted from visible light curing unit. electricity which is the energy source of curing is frequently fluctuated in which the voltage would be up and down. based on the previous study done by using light intensity measurement cure rite visible curing light meter showed input of 200, 220 and 235 volt light intensity: 213.3; 354.6; 388.6; mw/cm2. this condition showed that the change of voltage could alter light intensity resulted from visible light curing unit and possibly would affect the polymerization of resin modified glass ionomer cement. the increase voltage of light exposure until 250 volt will not show significant difference with voltage of light exposure 235 volt or 220 volt toward surface hardness of composite resin.7 in the previous study suggested the data that resin modified glass ionomer cement exposed with visible light for 20 seconds with input, 220, 210, and 200 volt would produce surface hardness value: 36.11, 25.99, and 18.96 95agustantina: the effect of voltage vhn. the result of study demonstrated the decrease of voltage directed to visible light curing unit might significantly lower the surface hardness of ionomer cement with decreased level 47.49%. visible light curing unit in general is completed with regulator to regulate visible light producing constant results, but various light intensity could occur and the length of effective wave is not always constant. to compensate the decrease of light intensity due to the decrease of voltage input, exposure time of composite restorative resin could done until two or three times longer exposure time than which has been recommended. to add the exposure time is expected to overcome the inadequate polymerization process.1,4 the standard of exposure time of polymerized restorative material was 20 seconds. the exposure time which is done longer than 60 seconds tends to be inefficient.5 inadequate visible light curing would produce the polymerization of composite resin uncomplete which could contribute some problems such as decrease of wear resistance: abrasion, atrition, and erosion.4 one of the indicators of wear resistance could be detected by measuring surface hardness.5 based on the above background some ideas arise whether the voltage and time of exposure would affect on the surface hardness of resin modified glass ionomer cement. this study is aimed to know the effect of voltege and time of exposure of resin modified glass ionomer cement on surface hardness. material and method the material of the study is resin modified glass ionomer cement fuji ii a 3,5 (gc japan) and the tools are plastic ring 5 mm in diameter and 2 mm height,9 visible light curing unit (litex), voltage regulator, voltmeter, stopwatch, plastic spatula, glass slab, matrix strip, weighing 0.5 kg, micro vickers hardness tester (matsuzawa mxt70). sample was made as follows: the plastic ring was fixed on glass slab. one spoon powder of resin modified glass ionomer cement was mix with two drops of liquid using plastic spatula on mixing pad. it was done until homogenous for 25 seconds (according to manufacturer recommend). homogenous material was put into the plastic ring started from the edge part and repeatedly knocked on the table for 5 times.10 the upper surface covered with the matrix strip and glass slab was completed with 0.5 kg weighing for 30 seconds.11 the weighing and the glass slab were lifted and the sample surface was exposed with visible light curing unit on matrix strip. in this study there were 9 groups of samples and individual sample groups consisted of 8 samples: group 1: samples with 220 volt input, 20 seconds (control); group 2: samples with 220 volt input, 40 seconds; group 3: samples with 220 volt input, 60 seconds; group 4: samples with 210 volt input, 20 seconds; group 5: samples with 210 volt input, 40 seconds; group 6: samples with 210 volt input, 60 seconds; group 7: samples with 200 volt input, 20 seconds; group 8: samples with 200 volt input, 40 seconds; group 9: samples with 200 volt input, 60 seconds. after sample exposed with visible light, sample was taken out from the mold and the sample result showed cylinder form and should follow the criteria: porous was invisible, the surface was fine, smooth and parallel. surface hardness test was done by measuring the upper surface which was directly exposed by visible light using micro vickers hardness tester completed with 0.5 gr weighing pressure.12 sample was put on the middle part of the table of micro vickers hardness tester and it was activated so that the tip of diamond penetration would leave indentation of sample surface. the result of indentation was observed through the lens of 400 magnifications so that it would show rhomboid form. the diagonal was measured by giving 2 lines to mark both diagonal ends. the read switch was pushed and the data of surface hardness value would appear. measuring the surface hardness was performed in three different places and the mean would be measured. the data was then tabulated and statistical analysis was done using two-way anova and continued by least significant difference (lsd). result the mean and the standard deviation of surface hardness test of resin modified glass ionomer cement which has been cured for 20, 40, and 60 second with voltage input 220, 210, 200 volt could be shown on the table 1. table 1 shows the highest value of surface hardness of resin modified glass ionomer cement is the groups of 60 second exposure with 220 volt input. the lowest value of surface hardness of resin modified glass ionomer cement is the group of 20 second exposure with 200 volt input. it is necessary to perform data normality test using kolmogorov smirnov prior to parametric test. the result of data normality test shows all probability value is higher than 0.05 (p < 0.05) which means all distribution data is normal therefore it is fulfilled the requirement to perform parametric test using two-way anova. t h e r e s u l t o f t w o w a y a n o v a t e s t s h o w s surface hardness was found between time of exposure (p = 0.001) and also the different surface hardness was found in interaction between time of exposure and voltage of light exposure. the result of data analysis can also mean that there is significant different resulted from the change of either time or voltage of light exposure of resin modified glass ionomer cement toward surface hardness. the time of exposure is longer resulting the increase of surface hardness of resin modified glass ionomer cement. the decrease of voltage resulting in the decrease of surface hardness of resin modified glass ionomer cement. lsd test is done in order to know the significance of between individual sample group. 96 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 94–97 the result of lsd test shows: there is no significant difference, between sample group control: group 2 and 3, 3 and 5, 4 and 8, 4 and 9, and 6, 5 and 8, 5 and 9, 6 and 9, 8 and 9. on table 2 shows that all control groups have significant difference toward group 1 (control groups). it is means that duration of lighting of resin modified glass ionomer cement with various electric voltage input will influence the value of surface hardness. discussion table 1 show that the input of voltage of exposure is lower resulting in the decrease of surface hardness of resin modified glass ionomer cement. the result of this is similar to the previous study suggests that the decrease of surface hardness of resin modified glass ionomer cement due to the decrease of voltage in which would lower the output of visible light curing unit consequently the result of light intensity would also decrease.8 the decrease of light intensity would affect photo activation and polymerization and further it would directly affect the number of free radical which initiates the occurrence of polymerization.1,13 excessive low light intensity might contribute the necessary level of energy formation in free radical formation would decrease as a result the polymerization process is not perfectly done. inadequate polymerization would cause a part of monomer could not react to be polymer chain and it would increase residual monomer. the increase number of residual monomer would decrease the degree of polymerization and relative molecule mass consequently the mechanical properties would decrease and in this case the decrease of surface hardness would occur.14 two-way anova test result indicates that significant difference was found between the time of exposure and the surface hardness of resin modified glass ionomer cement. table 1. the mean and the standard deviation of surface hardness test of resin modified glass ionomer cement which has been cured for 20, 40, and 60 second with voltage input 220, 210, 200 volt (vhn) electric voltage (volt) n second 20 40 60 mean sd x sd x sd 220 210 220 8 8 8 36.11 25.99 18.96 0.90 2.27 2.61 38.07 27.35 26.62 0.97 2.29 1.17 38.91 28.74 27.55 1.93 1.83 1.14 table 2. the result of lsd test between sample groups of resin modified glass ionomer cement toward surface hardness group1 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 0.32* 0.001* 0.325 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.135 0.001* 0.001* 0.001* 0.003 1.23 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.484 0.420 0.021 0.001* 0.001* 0.001* 0.001* 0.085 0.87 0.187 0.001* 0.300 note * = there is significant difference group 1 : sample with 220 volt input, 20 seconds (control) group 2 : sample with 220 volt input, 40 seconds group 3 : sample with 220 volt input, 60 seconds group 4 : sample with 210 volt input, 20 seconds group 5 : sample with 210 volt input, 40 seconds group 6 : sample with 210 volt input, 60 seconds group 7 : sample with 200 volt input, 20 seconds group 8 : sample with 200 volt input, 40 seconds group 9 : sample with 200 volt input, 60 seconds 97agustantina: the effect of voltage the prolong exposure time would increase the surface hardness of resin modified glass ionomer cement because the light energy which is needed to activate initiator during polymerization process would increase.15 the decrease of light energy would result the number of photons which initiate the formation of free radical would increase therefore the degree of polymerization and mechanic character i.e surface hardness would increase. table 2 shows that group of voltage of light exposure 220, 210 and 200 volt with exposure time 40 seconds comparing to 60 seconds, the surface hardness value of resin modified glass ionomer cement statistically is not significantly different, in this case it might be due to the addition of resin in resin modified glass ionomer cement is relatively small about hema 5–15%. in relatively small amount of resin, even though the time of exposure is prolonged from 40 seconds to 60 seconds the increase of surface hardness is not significantly different. the result of this study is similar to previous study which stated that low degree of polymerization was possibly caused by the small amount of resin. further it was stated that resin polymerization in resin modified glass ionomer cement might be affected by acid-base reaction.16 during the process of powder and liquid resin modified glass ionomer cement were mixed, the acid-base reaction would immediately occur similarly to conventional glass ionomer cement. a part of cement which reacted acid-base was expected to inhibit polymerization process by visible light, therefore even though the time of exposure was prolonged from 40 to 60 seconds with the same voltage, it will not show significant increase of surface hardness of resin modified glass ionomer cement. light intensity produced by visible light curing could affect the depth and the time which is need for complete polymerization in composite resin.17 the result of this study shows that to prolong the time of exposure until 60 seconds with low voltage (200 and 210 volt) cannot increase the surface hardness of resin modified glass ionomer cement similar to surface hardness of control group. this condition would show that light intensity has more essential role in activating light initiator found in light modified glass ionomer cement. inadequate light intensity due to low voltage (200 and 210 volt) could not activate optimally photo initiator material of resin modified glass ionomer cement therefore the polymerization is not as good as voltage of light exposure 200 volt. control group produced surface hardness of resin modified glass ionomer cement: 36.11 vhn. the result of this study is similar to the previous study which stated that resin modified glass ionomer cement with time of exposure 60 seconds produced surface hardness 36.2 vhn.18 the sample group with input 200 volt and time of exposure 60 seconds produced surface hardness of resin modified glass ionomer cement 27.55 vhn. the result of this study shows that time of exposure was prolonged until 60 seconds with low voltage input could not produce surface hardness of resin modified glass ionomer cement similar to control group. the conclusion is the prolong of exposure time from 20 seconds to 40, 60 seconds in all voltage input can increase surface hardness of resin modified glass ionomer cement. references 1. anusavice. phillips’ science of dental materials. 11th ed. st louis: wb saunders co; 2003. p. 96–8, 399–417, 482–4. 2. mount gj, hume wr. preservation and restoration of tooth structure. london, philadelphia, st louis: mosby co; 1998. p. 55-105, 196–7. 3. abate pf, polack ma, macchi rl. barcoll hardness of resinmodified glass ionomer cements and a compomer. quint int 1997; 28:345-8. 4. craig rg, powers jm. restorative dental materials. 11th ed. st. louis, london, philadelphia: mosby co; 2002. p. 249–51, 212–7, 614–8. 5. van noort r. introduction of dental materials. 2nd ed. edinburgh, london, new york mosby co; 2002. p. 124–46. 6. ellyza h. tinjauan terhadap suatu material restorasi: resin-modified glass ionomer. j ked gigi ui 1997; 4:468–78. 7. budi sl. pengaruh tegangan listrik, intensitas sinar dan waktu penyinaran terhadap kekerasan permukaan resin komposit sinar tampak. tesis. surabaya: pascasarjana universitas airlangga; 1993. p. 42–64. 8. titien ha. pengaruh penurunan tegangan listrik penyinaran pada semen ionomer gelas modifikasi resin terhadap kekerasan permukaan. majalah kedokteran gigi (dent j) 2003; 36:143–6. 9. marais jt, dannheimer mf, germishuys pj, borman jw. depth of cure of light-cured composite resin with light-curing units of different intensity (abstract). j dent assoc s afr 1997; 52:403–7. 10. nirawati p. daya serap air dan kelarutan garam na pada semen perekat ionomer gelas yang mendapat tekanan dan tidak mendapat tekanan. thesis. surabaya: universitas airlangga indonesia. 2000; p. 22–4. 11. ribeiro ap, serra mc, paulillo lams, rodrigues ca. effectiveness of surface protection for resin-modified glass-ionomer materials. quint int 1999; 30:427–31. 12. anita y. pengaruh jarak dan lama penyinaran lampu penerang dental unit terhadap sifat fisik, mekanik dan kimia resin komposit sinar tampak. thesis. surabaya: universitas airlangga indonesia. 1995; p. 41–3. 13. fan pl, wozniak wt, reyes wd, stanford jw. irradiance of visible light-curing unit and voltage variation effect. jada 1987; 115: 442–5. 14. ruslan e. pengaruh suhu dan lama penyimpanan resin komposit terhadap sifat kimia, sifat mekanik dan biokompatibilitas. dissertation. surabaya: universitas airlangga indonesia.1993; p. 140–3. 15. sakaguchi rl, douglas wh, peter mcrb. curing light performance and polymerization of composite restorative materials. j dent 1992; 20:183–8. 16. ikeda k, fujishima a, yamamoto m, inoue m, suzuki m, miyazaki t, sasa r. measurment of degree of cure for resin-modified glass ionomer cements by ir spectroscopic analysis. dent in japan 2002; 38:95–100. 17. poulos jg, styner di. curing lights: changes in intensity output with use over time. gen dent (abstract) 1997; 45:70–3. guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 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country issue* 6 month 1 year surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 22 the effect of silanated and impregnated fiber on the tensile strength of e-glass fiber reinforced composite retainer niswati fathmah rosyida,1 siti sunarintyas,2 and pinandi sri pudyani1 1department of orthodontics 2department of biomaterial faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: fiber reinforced composite (frc) is can be used in dentistry as an orthodontic retainer. frc still has a limitations because of to a weak bonding between fibers and matrix. purpose: this research was aimed to evaluate the effect of silane as coupling agent and fiber impregnation on the tensile strength of e-glass frc. methods: the samples of this research were classified into two groups each of which consisted of three subgroups, namely the impregnated fiber group (original, 1x addition of silane, 2x addition of silane) and the non-impregnated fiber group (original, 1x addition of silane, 2x addition of silane). the tensile strength was measured by a universal testing machine. the averages of the tensile strength in all groups then were compared by using kruskal wallis and mann whitney post hoc tests. results: the averages of the tensile strength (mpa) in the impregnated fiber group can be known as follow; original impregnated fiber (26.60±0.51), 1x addition of silane (43.38±4.42), and 2x addition of silane (36.22±7.23). the averages of tensile strength (mpa) in the non-impregnated fiber group can also be known as follow; original non-impregnated fiber (29.38±1.08), 1x addition of silane (29.38±1.08), 2x addition of silane (12.48±2.37). kruskal wallis test showed that there was a significant difference between the impregnated fiber group and the non-impregnated fiber group (p<0.05). based on the results of post hoc test, it is also known that the addition of silane in the impregnated fiber group had a significant effect on the increasing of the tensile strength of e-glass frc (p<0.05), while the addition of silane in the non-impregnated fiber group had a significant effect on the decreasing of the tensile strength of e-glass frc. conclusion: it can be concluded that the addition of silane in the non-silanated fiber group can increase the tensile strength of e-glass frc, but the addition of silane in the silanated fiber group can decrease the tensile strength of e-glass frc. it is also known that the impregnation of fiber can increase the tensile strength of e-glass frc. keywords: silane; impregnation; tensile strength; fiber reinforced composite. correspondence: niswati fathmah rosyida, departemen ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: niswatifathmah.fkg@ugm.ac.id introduction the need for orthodontic treatment in various countries including in indonesia has continued to increase. many people are interested in orthodontic treatment to improve the condition of teeth.1 after the orthodontic treatment is completed, a retainer is necessary needed to maintain the results of orthodontic treatment. this is because the teeth will have a tendency to return to their original position, and they also require a relatively long time to remain in the same position.2 the main material usually used to make the retainer is metal. however, metal still has limitations, such as high rigidity level hindering stabilization process of teeth during retention period,3 has poor esthetic,4 and allergens.5 therefore, a new material with better properties than metal is necessary to be considered, especially for patients who require high aesthetics and have allergy to metals. fiber reinforced composite (frc) is a composite with fiber reinforcement widely used in dentistry. the use of frc for orthodontic retainer actually has been developed, either using polyethylene fiber6 or using fiber glass.3,7 eglass frc retainer even can be an alternative retainer with research report dental journal (majalah kedokteran gigi) 20�5 march; 48(�): 22–25 2� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 2�rosyida, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 22–25 several advantages, such as having high aesthetics, easy to be used, and easy to be made (only needs one visit).8 nevertheless, e-glass frc retainer can cause fracture due to weak bond between resin matrix composite and fiber glass. it can be seen on how fibers are released from the frc after tested mechanically.9 the addition of silane as coupling agent is expected to bind fiber and matrix chemically to improve the adhesion between the fiber and the matrix resin. furthermore, impregnation process that can unify e-glass fiber bundles in a single frc system will improve adhesion system and increase the strength of the material. tensile strength test can be used to test the bond between two materials including the bond between the fiber and the matrix in the frc. tensile strength is one of important indicators to ensure whether the retainer was able to stabilize teeth in a relative long period.10,11 therefore, this research is aimed to examine the effect of the addition of silane and the impregnation of fiber on the tensile strength of e-glass frc. materials and methods materials used in this research were e-glass fiber, impregnated fiber (everstick®ortho, stick tech ltd, finland), non-impregnated fiber (ahlstrom fiberglass r338-2400/ v/ p®, finland), flowable composite (tetric flow chroma, ivoclar vivadent, liechtenstein), and silane (monobond-s, ivoclar vivadent, liechtenstein). samples in this research were classified into two groups, each of which consisted of three subgroups based on the type of fiber and the frequency of silane provision, namely subgroup i: frc with original impregnated fiber; subgroup ii: frc with impregnated fiber and 1x addition of silane; subgroup iii: frc with impregnated fiber and 2x addition of silane; subgroup iv: frc with original non-impregnated fiber; subgroup v: frc with non-impregnated fiber and 1x addition of silane; and subgroup vi: frc with nonimpregnated fiber and 2x addition of silane. samples were made using acrylic molds (60 x 40 x 6 mm) with a modified cavity in the central (30 x 5 x 2 mm) and a narrowing area (12 x 3 x 2 mm). those acrylic molds were marked on their edges as a marker for setting resin and fiber. composite resin was injected up to 0.5 mm to the bottom of the molds. both impregnated fiber and nonimpregnated fiber were cut to a length of 30 mm. silane was applied using microbrush on impregnated fiber in subgroup ii and iii and on non-impregnated fiber in subgroup v and vi. those fibers were allowed to stand for 60 seconds, and then dried with an air spray for 5 seconds. those fibers were placed on the molds, which have been filled by flowable composite. flowable composite was added again until the molds were fully filled. the surface of frcs was then covered using a celluloid strip followed by irradiation with light curing unit perpendicular, 1 mm distance to the samples. frcs were released from the molds. a tensile strength test was conducted using universal testing machine with a speed of 1 mm/ min. the tensile strength of each group was calculated by dividing the load by the cross sectional area of the material after the fracture. the data were then analyzed statistically with kruskal wallis test and post hoc mean whitney test. results the results showed the comparison of the averages of the tensile strength among the groups. it is known that the tensile strength in the group with 1x addition of silane and the group with 2x addition of silane was decreased about 13% and 50%/. the average of the tensile strength and the standard deviation values for all groups can be seen in figure 1. based on the results of kolmogorov-smirnov test, it was known that the distribution of the data obtained was normal. based on the results of lavene homogeneity test, it is also known that the data obtained were not homogeneous. thus, non-parametric kruskal-wallis test was required to analyze the difference of the average of the tensile strength among all groups. based on the results of kruskal wallis test, it is known that the addition of silane did not affect the tensile strength of frc, while the type of fiber affected the tensile strength of frc. therefore, it can be said that the fiber impregnation significantly affects the increasing of the tensile strength of frc. figure 1. the average and standard deviation of tensile strength (mpa) of e-glass frc. table 1. the results of post hoc test on the tensile strength of e-glass frc 19. group interaction z sig impregnated fiber original silane 1x -2.31 0.02* silane 2x -1.73 0.08 silane 1x silane 2x -1.15 0.24 nonimpregnated fiber original silane 1x -2.31 0.02* silane 2x -2.31 0.02* silane 1x silane 2x -2.31 0.02* note: *= significance (p<0.05) 29.38 24.53 12.48 26.6 43.38 36.22 original silane 1x silane 2x 0 10 20 30 40 50 sd = 7,23 sd = 2,37 sd = 4,42 sd = 0,84sd = 0,51 silanization f r c w i t h n o n i m p r e g n a t e d f i b e r f r c w i t h i m p r e g n a t e d f i b e r te ns ile s tre ng th, m pa sd = 1,08 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 24 rosyida, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 22–25 the results of post hoc mann whitney test (table 1) showed that in the group with impregnated fiber, the addition of silane significantly increased the tensile strength of e-glass frc. meanwhile, in the group with non-impregnated fiber, the addition of silane significantly reduced the tensile strength of e-glass frc. discussion the results showed that the type of fiber significantly has affected the increasing of the tensile strength of frc. it is because impregnated fiber used in the research has already been coated with bis-gma and pmma matrix in its production process.12 as a result, bis-gma and pmma will form the semi structure of interpenetrating polymer network (ipn) bringing fibers together into a single bundle and increasing the bond of the fibers to the other materials.13 it means that the process of coating with polymer matrix by the manufacturer guarantees the perfect impregnation of each fiber to produce fibers that easily bonded to the other materials and to increase the mechanical strength of frc. in the impregnated fiber group, it is known that the 1x addition of silane significantly increased the tensile strength, while the 2x addition of silane slightly lowered the tensile strength of frc. silane is a material that support and enhance the chemical bonding between inorganic materials (fiber) and organic material (composite matrix). thus, alkoxy silane hydrolyzed will react with the surface of the hydroxyl group of the fiber, and then form cross bonding. the functional group of silane will bond with the functional group of bis-gma resin composite, >c=c<. the combination of cross bonding with the functional group of silane will improve the bond and hydrolytic stability of siloxane coating (si-o-si) between the composite resin and fiber glass so that the tensile strength of frc will be increased.14 the bonding that occurs between bis-gma, silane, and fiber are shown in figure 2. addition of silane in fiber during post endodontic treatment can improve the tensile strength of the frc significantly.15 however, the tensile strength can be decreased after the 2x addition of silane on the frc (16% compared to the 1x addition of silane). it means that the excessive use of silane can decrease the strength of frc. this is because the excess silane molecules will form polysilane which is a covalent bond among silane molecules.16 the presence of polysilane will weaken the bond between the fibers and the resin with an indication of decline in the flexural strength of the composite as the increasing number of polysilane.17 it is known that the tensile strength of non-impregnated fiber which had not given with silane was slightly higher than the tensile strength of impregnated fiber which had not given with silane. the non-impregnated fiber used in this research was unidirectional fiber that had given with saline by the manufacturer. the surface of the non-impregnated fiber coated with silane became one of the factors causing the increasing of the tensile strength of the fiber since silane can improve the chemical bonding between the fiber and the matrix.14 in the non-impregnated fiber, the addition of silane caused the decreasing of the strength of the frc. this condition is due to the formation of silsesquioxane weakening the bond between the fibers and the resin.19 the formation of silsesquioxane may occur between the beginning of the provision of saline and the 1x addition of silane since there are solvent and water resulted from the process. the solvent and water can cause the hydrolysis of silane and help to form a condensation polymerization process of silsesquioxane.17 the use of silanated fiber can make unperfect bonding, because fiber has a little functional group, so it is less reactive to silane solution applied. as a result, there will o o h o o h o o h ch 2 c h 3 o o s i g las s f ib er + o o h o o h o o h c h 2 c h 3 o o s i ch 2 c h 3 o o o o h c h 3 ch 3 o o c h 2 c h 3 o o h g las s f ib er ch 2 c h 3 o o o o h c h 3 ch 3 o o c h 2 ch 3 o o h re a k s i la n ju t m e m b e n tu k p o li b is g m a figure 2. the bond between bis-gma, silane, and fiber. 25 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 25rosyida, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 22–25 be no chemical bond between the composite resin and the fiber so that the mechanical strength becomes weak. fibercontaining epoxy silane is also less reactive to silane, so it cannot act as a coupling agent between the fiber and the composite matrix.18 the surface of fiber, however, will be more reactive after micromechanical modification on the surface, such as hydrogen peroxide before silane is given.19 similarly, a research conducted by perdigao et al.,18 showed that the addition of silane would not affect the increasing of the shear and tensile strength between the matrix composites and the fiber which have been given with silane by the manufacturer. it can be concluded that the addition of silane in nonsilanated fiber can increase the tensile strength of e-glass frc, while the addition of silane in silanated fiber can lower the tensile strength of e-glass frc. in other words, it can be said that fiber impregnation can increase the tensile strength of e-glass frc. acknowledgment this research is supported by the beasiswa unggulan of ditjen dikti 2012 for s2 program on the behalf of nfr and dana masyarakat of dentistry faculty, ugm in the fiscal year of 2014 no. 5994/ kg/ pp/ 2014. references 1. thilander b, pena l, infante c, parad ss, mayorga d. prevalence of malocclusion and orthodontic treatment need in children and adolescent in bogota, colombia. an epidemiological study related to different stages of dental development. eur j orthod 2001; 23: 153-67. 2. proffit wr, fields hw, sarver dm. contemporary orthodontics. 4th ed. st. louis: missouri; 2007. p. 617. 3. silvestr ini-biavati a, a ngiero f, gibk h kelli f, signore a, benedicenti s. in vitro determination of the mechanical and chemical properties of a fibre orthodontic retainer. eur j orthod 2012; 34(6): 693-7. 4. milheiro a, kleverlaan c, muris j, feilzer a, pallav p. nickel release from orthodontic retention wires the action of mechanical loading and ph. dent mater 2012; 28(5): 548-53. 5. valiathan a, dhar s. fiber reinforced composite arch wire in orthodontics: function meet esthetics. trends biomater artif organs 2006; 20(1): 16-9. 6. scribante a, sfondrini mf, broggini s, d’allocco m, gandini p. efficacy of esthetic retainers: clinical comparison between multistranded wires and direct-bond glass fiber reinforced composite splints. int j dent 2011; 2011: 1-5. 7. tacken mpe, cosyn j, de wilde w, aerts j, govaerts e, vannet bv. glass fibre reinforced versus multistranded bonded orthodontic retainers: a 2 year prospective multi-centre study. eur j orthod 2010; 32: 117–23. 8. geserick m, ball j, wichelhaus a. bonding fiber -reinforced lingual retainers with color-reactivating flowable composite. j clin orthod 2004; 38(10): 560-2. 9. brauchli l, pintus s, steineck m, lüthy h, wichelhaus a. shear modulus of 5 flowable composites to the everstick ortho fiber -reinforced composite retainer: an in-vitro study. am j orthod dentofacial orthop 2009; 135(1): 54-8. 10. pothan la, george j, thomas s. effect of surface treatments on the matrix interaction in banana reinforced polyester composites. composite interfaces 2002; 9(4): 335–53. 11. powers jm, sakaguchi rl. craig’s restorative dental materials 12th ed. missouri: mosby an imprint of elsevier; 2006. p. 58-59. 12. valittu pk, fiber reinforced composite for dental application. in: richard c, timothy w. dental biomaterials imaging testing and modelling. cambridge: woodheas publ ltd; 2008. p. 241-2. 13. lung cyk, matinlinna jp. aspects of silane coupling agents and surface conditioning in dentistry: an overview. dent mater 2012; 28(5): 467-77. 14. goraccia c, raffaellia o, monticellia f, balleria b, bertellib e, ferrari m. the adhesion between prefabricated frc posts and composite resin cores: microtensile bond strength with and without post-silanization. dent mater 2005; 21(5): 437-44. 15. sideridou id, karabela mm. effect of the amount of 3 methacylo xypropyltrimethoxylane coupling agent on physical properties of dental resin nanocomposie. dent mater 2009; 25(11): 1315-24. 16. antonucci jm, dickens sh, fowler bo, xu hhk, mc donough w. chemistry of silane: interfaces in dental polymers and composites. j res natl inst stand technol 2005; 110: 541-58. 17. perdigao j, gomes g, lee kl. the effect of silane on the bond strengths of fiber. dent mater 2006; 22(8): 752-8. 18. mosharraf r, ranjbarian p. effect of post surface conditioning before silanization on bond strength between fiber post and resin cement. j adv prosthodont 2013; 5(2): 126-32. 126 volume 47, number 3, september 2014 modulation of fgf2 after topical application of stichopus hermanii gel on traumatic ulcer in wistar rats rima parwati sari,1 endahwahjuningsih1dan isidora karsini soeweondo2 1bagian biologi mulut 2bagian ilmu penyakit mulut fakultas kedokteran gigi universitas hang tuah surabaya-indonesia abstract background: stichopus hermanii (golden sea cucumber) is one of the many types of marine organisms containing glycosaminoglycans (gags), a polysaccharide that promote wound healing. the content of this gags, mainly dermatan sulfate, chondroitin sulfate and heparan sulfate has the ability to modulate fgf2. fgf2 many found in the oral mucosa to activate fibroblast proliferation. purpose: the study was aimed to determine the modulation of fgf2after topical application of stichopus hemanii gel on traumatic ulcers in wistar rat. methods: the sample was 36 male wistar rats which were divided into 6 groups. c1 and c2 group was placebo gel, sc1 and sc2 group was stichopus hermanii gel, ha1 and ha2 was hyaluronic acid a gel. the gel was given shortly after the traumatic ulcer (tu) formed and 24 hour later. then all rats were sacrificed lips mucosa were taken and elisa examination was done. results: all data were analyzed by anova test followed by tukey hsd. test results show significant differences between sc1-sc2 with c1c2 group, while the ha1-ha2 with c1-c2 group showed no significant difference. conclusion: the study showed that modulation of fgf2 increased after topical application of stichopus hermanii gel on traumatic ulcers in wistar rats. keywords: fgf2, stichopus hermanii gel, traumatic ulcer abstrak latar belakang: stichopus hermanii (teripang emas) merupakan salah satu jenis biota laut yang banyak mengandung gag, suatu polisakarida yang sangat bermanfaat dalam proses penyembuhan luka. kandungan gag ini, terutama dermatan sulfat, chondroitin sulfat dan heparan sulfat memiliki kemampuan untuk memodulasi fgf2. fgf2 banyak didapatkan pada mukosa rongga mulut untuk mengaktifkan proliferasi fibroblas. tujuan: studi ini bertujuan meneliti modulasi fgf2setelah pemberian aplikasi topikal gel stichopus hermanii pada ulkus traumatikus pada tikus wistar. metode: sampel penelitian ini adalah 36 ekor tikus wistar jantan yang dibagi dalam 6 kelompok. kelompok c1 dan c2 merupakan kelompok kontrol negatif (gel plasebo), kelompok sc1 dan sc2 merupakan kelompok pemberian gel stichopus hermanii (sc), serta kelompok asam hyaluronat ha1 dan ha2 merupakan kelompok pemberian gel asam hialuronat (ah). pemberian gel dilakukan sesaat setelah pembuatan ulkus traumatikus dan 24 jam kemudian. kemudian semua tikus dikorbankan untuk diambil mukosa bibirnya dan dilakukan pemeriksaan elisa. hasil: semua data dilakukan analisa dengan uji anova dan dilanjutkan dengan uji tukey hsd yang hasilnya menunjukkan adanya perbedaan bermakna antara kelompok sc1-sc2 dengan kelompok c1-c2, sedangkan kelompok ha1-ha2 dengan kelompok c1-c2 tidak menunjukkan perbedaan yang bermakna. simpulan: modulasi fgf2 meningkat setelah pemberian aplikasi topikal gel stichopus hermanii pada ulkus traumatikus tikus wistar. kata kunci: fgf2, gel stichopus hermanii, ulkus traumatikus korespondensi (correspondence): rima parwati sari, bagianbiologi oral, fakultaskedokteran gigi universitas hang tuah. jl. arifrahman hakim no. 150 surabaya 60111, indonesia. e-mail: rima.sari@yahoo.com research report 127sari, et al.: modulation of fgf2 after topical application of stichopus hermanii gel introduction aphtous ulcers have been known as oral lesions round or oval shaped, covered by white-yellowish fibrinous exudate with redness border.1 it has been reported that one in five person in the population suffered from aphtous ulcers. these high incedence of cases used to be diagnozed as traumatic ulcer (tu) and recurrent aphtous stomatitis (ras), which ras prevalence is higher 83.6%.2 the difference between tu and ras relied on the history of trauma in tu, while in ras occured without any specific cause but could be triggered from trauma.1 mechanical trauma is the most common cause, could be from denture or accidental trauma bite of the teeth on the mucosa or physiologic trauma on patient who have bite habit on cheeck and lips. banuarea3 stated that ras caused by trauma prevalence were 50,27%, while trauma of bite were the most common cause at 64,17%. the study also stated that the most common location was labial mucosa at 45,25%. trauma of tu also caused by chemical trauma as missused of drug as aspirin attach on the mucosa, teeth and on mouth rinse. 1,4 other cause of tu is thermal trauma occured when having hot meals.5 pain resulted on ulcers is annoying and cause trouble on mastication, speaking and swallowing, even bother patient's emotional stability so that tu and ras affect not only oral function but also systemic in general.6 treatment on tu need to be done by eliminating the cause factors, reducing the pain and accelerate the wound healing process. antibiotic sometimes applied to prevent secondary infection, usually on the case of chronic tu. severe tu could be applied topical therapy, for example corticosteroid.1 therapeutical agents to accelerate wound healing have been developed recently, one of it was hyaluronic acid (ha). hyaluronic acid is one of major glycosaminoglycan (gag) secreted on tissue repair which is the integral part of extracellular matix. ha produced by fibroblast during proliferative phase on wound healing to induce migration an mitosis of fibroblast and epithelial cells.7 it increases the activity of growth factor (gf) to induce migration and fibroblast proliferation and collagen deposition on the ulcer.8 gf is a key component in wound healing which give chemical signalling to regulate biological response and tissue differentiation. one of representative potential growth factor which have the role in repair and tissue regeneration is fibroblast growth factor (fgf). fgf bind and then activate fibroblast growth factor receptor (fgfrs) which dominant in ras/ map (mitogen-activated protein) kinase pathway with high affinity. regarding to its potential function, fgfs have been used as indicator in detecting regenerative process of damaged tissue, including skin, blood vessel, muscle, adipose, tendon/ ligament, cartilage, bone, teeth, nerves. some studies reported that fgf have many potential functions in proliferation, migration, diferentiation and angiogenesis in many cells and tissue. fgf2 is a family of fgf that have been found in all that functions. fgf2 has the target on set up the proliferations of preadipose, endothelial cell, epithelial cells, fibroblast and neural stem cells. in arranging migration cell process, the target of fgf is on myogenic cells, in differentiation process its target is on neuroepithelial cells while in angiogenesis function, its role is on the development of endothelial cells.9 sea cucumber is a valuable natural source that has not been explored, specially in dentistry. among 1.250 types of sea cucumbers, the stichopus hermanii is the species that contains gag components the most.it contains less saponin compare to holothuriae scabra species, and also contain more arachidonic acid (aa), eicosapentaenoic acid (epa) dan docosahexaenoic acid (dha).10 in addition to ha, stichopus hermanii also contain chondroitin sulphate, heparin sulphate and dermatan sulphate which have the ability to activate and bind to gf specially fgf2.11,12 unsaturated fatty acids also found as content in stichopus hermanii are key mediators to control inflammation process in wound healing, arranging fibroblast proliferation and collagen synthesis to produce healthy collagen. another advantage of the presence of this unsaturated fatty acid is to minimalize the scar formation and increase the strength of connective tissue.13,14 the study was aimed to determine the effect of topical application of stichopus hermanii gel in modulating fgf2 on traumatic ulcer of wistar rats. materials and methods the study was true experimental study with post test only control group design. fgf2 were examined in the early phase of traumatic ulcer. samples were healthy male 36 wistar rats, weight 200-300 grams, age 3 months15,16 and were divided randomly into 6 groups each consisted of 6 rats, i.e : negative control group given placebo gel right after tu formed for one day (c1), negative control group given placebo gel 24 hour after tu formed for one day (c2), treatment group given 60%stichopus hermanii extract (sc) gel right after tu formed for one day (sc1), treatment group given 60% stichopus hermanii extract gel 24 hours after tu induction for one day (sc2), treatment group given hyaluronic acid (ha) gel right after tu induction for one day (ha1), treatment group given given hyaluronic acid (ha) gel 24 hours after tu formed or one day(ha2). wistar rats were being adapted for one week, feed and drink daily. research were performed in biochemistry laboratory faculty of dentistry hang tuah university fresh stichopus hermanii were cleaned from dirt, put under running water in a mesh sized 30-40 mesh, squeezed to remove saponin in foam form, washed then put on blender, performed freeze drying resulted in rough extract particle.17 extract were performed by sohlet method then added mucoadhesive agent.18 128 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 126–129 wistar rats were given ketamine-xylazin then traumatic ulcer were induced on labial mucosa. sterile amalgam stopper was flammed in 45 second, touched in labial mucosa for 1 second.16,19,20 after the ulcer formed, gels were applied as its groups of treatment for 24 and 48 hours, then being terminated to obtain the labial mucosa samples on elisa test. samples were rinsed in cold pbs (0,01 mol/l, ph 7,07,2) to remove excessive bleeding and weighed. tissue were chopped in small cut then being homogenized in 5-10 ml pbs above the ice. suspension were sonicated with ultrasonic to disrupt cells or performed freeze-thaw cycle twice to disrupt cell membrane, sentrifuged for 5 minute on 5000×g. supernatan were released then being examined immediately or stored in -20oc. e l i s a t e s t w e r e d o n e u s i n g e n z y m e l i n k e d immunosorbent assay (elisa) kit for untuk fgf2 mouse (uscn life science inc, wuhan, china). reagent and 96 well strip plate were prepared as manufacturer standard and incubated for 25 minutes. absorbance were measured in microplate reader infinite® m200 pro (tecan deutschland gmbh, crailsheim, germany) on wave length 450 nm. all samples were replicated on measure. result topical application of placebo gel, stichopus hermanii extract gel (sc) and hyaluronic acid gel (ha) were evaluated on first and second day after the formation of ulcer (figure 1). anova result showed significant difference on mean of fgf2 protein level (p=0,000).result on sc group was better than placebo group but not significant compare to ha group (p>0.05) (table 1). discussion fgf2 is the key factor on promoting fibroblast proliferation. the fgf main pathway is ras/ map kinase which contain many protein signaling. an important event of fgf signal pathway is tyrosine residue phosphorilation on protein docking. fgf substrat 2α (frs2α) receptor prepare the new binding site to take direct or indirectly protein responsible for signalling.21,22 frs2α rectruit complex of adaptor protein, guanine nucleotide exchange factor 2 (grb2), son of sevenless (sos), the tyrosine phosphatase (shp2) and protein docking grb2-associated binding protein 1 (gab1). signalling complex frs2 resulted in activation of ras/ map kinase and pi3 kinase/ akt pathway.23 many studies stated that ras/ map kinase pathway involved in cell growth and differentiation. one of target molecule for fgfr is phospholipase c gamma (plcγ), which bind to phosphorilated tyr-766 result on its receptor and then become phosphorilated tyrosin plcγ and activate plcγ. activation of plcγ hidrolyze phosphatidylinosito and produce inocyol triphosphate (ip3) dan diacylglycerol (dag).24 ip3 is second massenger which facilitate calcium release from endoplasmic reticulum. increasing level of calcium in cytosol and dag together activate protein kinase c (pkc).physiologic relevance of this pathway has not been described in details on mitogenesis process or cell differentiation. result of fgf2 expression showed the protein level produced on the first day after tu formed generally higher compared to those on second day (figure 1), although lsd result showed no significant difference for fgf2 protein level on each group (k1 and k2; sc1 and sc2; ha1 and ha2) as shown on table 1. level of fgf2 in control group were higher on the first day application right after injury performed and tend to decrease when given after the ulcer formed. this result was related to numata et al.24 stated that one day after injury the level of fgf2 increased and re increased on the third day but the later increasement was less than on the earlier on the beginning of the injury. treatment with sc resulted in increasing of fgf2 compare to other groups and slightly reduced on the second day (p>0.05). sc contain more type of gags compare to ha which have role in increasing the level of fgf2. figure 1. mean of fgf2protein level on control and treatment groups. 5 prepared as manufacturer standard and incubated for 25 minutes. absorbance were measured in microplate reader infinite® m200 pro (tecan deutschland gmbh, crailsheim, germany) on wave length 450 nm. all samples were replicated on measure. result topical application of placebo gel, stichopus hermanii extract gel (sc) and hyaluronic acid gel (ha) were evaluated on first and second day after the formation of ulcer. anova result showed significant difference on mean of fgf2protein level (p=0,000).result on sc group was better than placebo group but not significant compare to ha group (p>0.05) fig 1. mean of fgf2protein level on control and treatment groups table 1. lsd test result dependent variable treatment group k2 sc1 sc2 ha1 ha2 fgf2 k 1 0,931 0,004* 0,024* 0,302 1,000 k 2 0,000* 0,001* 0,016 0,742 sc1 0,999 0,726 0,015* sc2 0,968 0,071 ha1 0,556 note: *significant different discussion fgf2is the key factor on promoting fibroblast proliferation. the fgf main pathway is ras/ map kinase which contain many protein signaling. an important event of fgf c1 sc1 ha1 ha2 sc2 c2 table 1. lsd test result dependent variable treatment group k2 sc1 sc2 ha1 ha2 fgf2 k 1 0,931 0,004* 0,024* 0,302 1,000 k 2 0,000* 0,001* 0,016 0,742 sc1 0,999 0,726 0,015* sc2 0,968 0,071 ha1 0,556 note: *significant different 129sari, et al.: modulation of fgf2 after topical application of stichopus hermanii gel chondroitin sulphate is needed in organizing granulation tissue formation during wound healing. chondroitin sulphate is able to bind to fgf2 and maintaining the level of fgf2 in the tissue and implicated in triggering fibroblast proliferation.11heparan sulphate and dermatan sulphate are great component in wound liquid and become dissolved. the dissolved dermatan sulphate have the ability to activate growth factors like fgf2 and fgf7 which activate map kinase pathway and increase the level of ca+ and then trigger differentiation process and cell mitogenesis.9,12 ha have its role on proliferation, migration and cytokine synthesis mediated by cd44 on the cell surface. along with the increasing of inflammatory cell on ha treatment, the inflammation process accelerated so fgf2 production was not inhibited dan ready to perform the signalling in map kinase pathway. but the treatment given on the second day was resulted on delayed of fgf2 increasement. the fgf2 level on ha2 was lower than in sc group, even not significant different. stichopus hermanii is marine biota that contain gag which have the important role in increasing and binding of fgf2.by that active component, the level of fgf2 after topical appication of sc were increased compared to control group and ha group in first day, and compared to control group in second day. it is concluded that fgf2 modulation was increased after topical application of stichopus extract gel on traumatic ulcers of wistar rats. daftar pustaka 1. regezi ja, sciubba jj,jordan rck. oral pathologic clinical pathologic correlations. 5thed. st. louis: wb sauders; 2008. p. 214. 2. delong l, burkhart n. general and oral pathology for the dental hygienist. philadelphia, us: lippincott williams & wilkins; 2008. p. 295-7. 3. banuarea thp. prevalensi terjadinya stomatitis aftosa rekuren (sar) pada mahasiswa universitas sumatera utara yang berpengalaman sar. perpustakaan universitas sumatera utara. 2009. p. 18-30 4. cunningham sj, quinn fb. ulcerative lesions of the oral cavity. 2002. available from http://www.utmb.edu/otoref/grnds/ulcer-oral021016/ulcer-oral-021016-slidesb.pdf. accessed april 14, 2012. 5. heerden vwfp, boy sc. diagnosis and management of common non-viral oral ulcerations. sa fam pract2007; 49(8): 20-6. 6. scully c, gorsky m, lozada-nur f. the diagnosis and management of reccurent aphtous stomatitis. jamdentassoc2003; 134:200-7. 7. mackay d, miller al. nutritional support for wound healing. alterative medicine review 2003; 8(4):359-77. 8. mariggio ma, cassano a, vinella a, vincenti a, fumarulo r, muzio llo, maiorano e, ribatti d, favia g. enhancement of fibroblast proliferation, collagen biosynthesis and production of growth factors as a result of combining sodium hyaluronate and amino acids. int j immunopathology and pharmacology 2009; 22(2): 485-92. 9. yun yr, won je, jeon e, lee s, kang w, jo h, jang jh, shin us, kim hw. fibroblast growth factors: biology, function, and application for tissue regeneration. j tissue eng 2010; 2010:218142. 10. bordbar s. anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods-a review. marine drugs 2012; 9: 1761-1805. 11. zou xh, foong wc, cao t, bay bh, ouyang hw, yip gw. chondroitin sulfate in palatal wound healing. jdent res 2004; 83(11): 880-5. 12. lee pha, trowbridge jm, taylor kr, morhen vb, gallo rl. dermatansulfate proteoglycan and glycosaminoglycansynthesisis induced in fibroblasts by transfer to a three-dimensionalextracellular environment. jbiological chemistry2004;279(47): 48640–6. 13. collins n, sulewski c. omega-3 fatty acids and wound healing. ostomy wound management august 2011; 57(8): 10-3. 14. mcdaniel jc, belury m, ahijevych k, blakely w. ω-3 fatty acids effect on wound healing. wound repair regen2008; 16(3): 337–45. 15. andreollo na, santos ef, araujo mr, lopes lr. rat’s age versus human’s age: what is the relationship?. arq bras cir dig 2012;25(1):49-51. 16. kusumawati d. biologi hewan coba. bersahabat dengan hewan coba. yogyakarta: gadjah mada university press;2004.p.20-26 17. ridzwan bh, leong tc, idid sz. the antinociceptive effect of water exracts from sea cucumber holothuria leucospileta brandt, bohadscia marmorata vitiensi jaeger and coelomic f luid from stichopus hermanii. pakistan jbiological sci2003; 6(24): 2068-72. 18. nicolazzo ja, finnin bc. in vivo and in vitro models for assessing dr ug absor ption across the buccal mucosa. biotechnology: pharmaceutical aspects2008; 7:89-111. 19. ali zh, dahmoush hm. propolis versus daktarin in mucosal wound healing. life sci j2012; 9(2): 624-36. 20. wong a, lamothe b, lee a, schlessinger j, lax i. frs2α attenuates fgf receptor signaling by grb2-mediatedrecruitment of the ubiquitin ligase cbl. proceedings of thenational academy of sciences of the united states of america, 2002; 99(10):6684–9. 21. lax i, wong a, lamothe b. the docking proteinfrs2α controls a map kinase-mediated negative feedbackmechanism for signaling by fgf receptors. molecular cell 2002; 10(4):709–19. 22. l a mot he b, ya ma d a m, scha ep er u, bi rch meier w, l a x i,schlessinger j. the docking protein gab1 is an essentialcomponent of an indirect mechanism for fibroblast growthfactor stimulation of the phosphatidylinositol 3-kinase/aktantiapoptotic pathway. molecular and cellular biology 2004;24(13): 5657–66. 23. mohammadi m, honegger am, rotin d.a tyrosinephosphorylatedc arboxy-terminal peptide of the fibroblastgrowth factor receptor (flg) is a binding site for the sh2domain of phospholipase c-γ1.molecular and cellularbiology 1991;11(10):5068–78. 24. numata y, terui t, okuyama r, hirasawa n, sugiura y, miyoshi i, watanabe t, kuramasu a, tagami h, ohtsu h. the accelerating effect of histamine on the cutaneous wound-healing process through the action of basic fibroblast growth factor. j investigative dermatology2006; 126: 1403–9. vol 52 no 1 jan-mar 2019_new.indd 24 the effect of udma and bis-gma irradiation period on residual monomers in resin packable composite jayanti rosha, adioro soetojo, putu dewi purnama sari budha, and m. mudjiono department of conservative dentistry faculty of dentistry, universitas airlangga, surabaya, indonesia abstract background: residual monomers are non-polymerized monomers which can cause clinical harm, for example inflammation, to oral cavity tissue while the remaining monomers can potentially be carcinogenic. the more residual monomers that remain due to an imperfect polymerization processes, the lower the compressive strength level and the higher the number of micro slits that can cause secondary caries and tooth sensitivity. urethane dimethacrylate (udma) and bisphenol a glycol dimethacrylate (bis-gma) constitute two of the resins most frequently used in packable composites. during the short irradiaton period forming part of the polymerization process, udma and bis-gma have the potential to produce residual monomers. purpose: this study aimed to compare the number of residual monomers in packable composite resin following irradiation lasting 1x20 seconds and 2x20 seconds. methods: 28 samples of cylindrical packable composite with a thickness of 2 mm and a diameter of 5 mm were divided into four groups. groups 1 and 2 were irradiated for 1x20 seconds, and groups 3 and 4 for 2x20 seconds with the composite subsequently being immersed in ethanol solution for 24 hours. the number of residual monomers using high-performance liquid chromatography (hplc) devices was calculated and the results statistically analyzed using a mann-whitney test. results: repeated irradiation had no effect on the amount of residual monomers in packable composite resins. however, there were differences in the number of residual monomers in the material contained in packable composite resins bis-gma and udma, while the remaining monomers in udma outnumbered those in bis-gma. conclusion: the number of residual monomers in bis-gma is lower than in the remaining udma after 1x20 seconds irradiation, while the number of residual monomers in bis-gma and udma following 2x20 seconds irradiation was no different to that after irradiation of 1x20 seconds duration. keywords: irradiation; packable composite; residual monomer correspondence: adioro soetojo, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo 47, surabaya 60132, indonesia. e-mail: adioros@yahoo.com. dental journal (majalah kedokteran gigi) 2019 march; 52(1): 24–26 research report introduction composite resin constitutes a material indispensable to anterior and posterior tooth restoration.1 certain of the existing literature states that the definition of a composite is a mixture of two or more materials which enhances the properties of the material.2 materials are classified as either packable composite resins or flowable composite resins according to the volume of composite resin filler they contain. due to their utility, packable composites were introduced in the late 1990s. packable composite resin is a di-methacrylate resin which contains several fillers of 66-67% with a particle size of 0.7-2 nm.3 the advantages of this composite include a reduction in shrinkage during polymerization.4 for process efficiency and maximum restoration results, more beam-activated composite resins are employed.5 currently, quartz tungsten-halogens (qth) and lightemitting diodes (led) constitute the light sources most frequently used for hardening composite resin as the photon suppliers which activate photon-initiators. composite polymerization is initiated by exposure to light produced by a light curing unit (lcu).6 the polymerization process consists of four stages: induction, propagation, chain dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p24–26 mailto:adioros@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p24-26 25rosha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 24–26 transfer and termination.7 several factors potentially affecting polymerization include filler type, size, material content, thickness, color of the restoration material, effectiveness of light transmission, exposure time, the distance between rays and restoration materials and light intensity.6 after photoactivation, monomer conversion to a simplified polymer will occur in relation to the degree of conversion (dc). the higher the value of dc monomer crosslinking, the more extensive the polymer network formed.8 the dc constitutes an important parameter in determining the final properties of active composite resin irradiation including: physical, mechanical and biological properties the light wavelength and depth employed, ray tip size, photo activation method, distribution, number of inorganic fillers, composite resin color, and the exposure period which is directly affected by an increase in the irradiation period.5 a low degree of conversion causes decreasing resin properties and the number of non-reactive (trapped or free) residual monomers. one strategy to maximize dc and minimize monomer elution as a means of providing sufficient energy to the material is that of prolonging the hardening time.9 the main problem often encountered in composite resin stacks is that of incomplete polymerization. the more residual monomers remain due to imperfect polymerization, thereby reducing the compressive strength and gap microstructure that can lead to secondary caries and tooth sensitivity. based on previous research, post-curing in a ultraviolet (uv) lightbox proved capable of enhancing the polymerization process due to more evenly distributed light reflection which, in turn, increased the solidity of composite resin. post-curing is an additional polymerization technique implemented after initial irradiation with the aim of increasing the degree of conversion as a means of perfecting the polymerization process.10 the purpose of this study is to explain the effect of the urethane dimethacrylate (udma) and bisphenol a glycol dimethacrylate (bis– gma) irradiation period on residual monomers in packable resin composite. materials and methods the investigative method applied constituted laboratorybased experimental research incorporating a posttest-only control group design. there were four treatment groups. group 1: packable composite with bis-gma base materials 1x irradiation, group 2: packable composite with udma base material with 1x irradiation, group 3: packable composite with bis-gma base materials 2x irradiation, and group 4: packable composite udma base material with 2x irradiation. the total sample size consisted of 28 packable composites. composite resin filtex z350 xt packable (3m espe) samples were produced in 2 mm-thick, plastic, cylindrical composite molds, 5 mm in diameter. packable composite materials were applied using plastic filling instruments. led light curing was subsequently performed (woodpecker dte curing light led) on the members of the four groups at a light intensity of > 500mw/cm2. the samples produced were immersed in ethanol solution for 24 hours in an incubator at 37oc. following immersion, the number of residual monomers was calculated using an hplc (agilent 1100 series, korea).11 the data produced was analyzed using a kolmogorovsmirnov test in order to establish whether data was normally distributed, while a levene`s test was conducted to determine the homogeneity of the samples. both a mannwhitney test and an independent t test were completed on the homogeneous and non-homogeneous groups as a means of identifying any differences between them. all data produced was analized statistically at a degree of confidence of p=0.05. results the results of this research into monomer residues after bis-gma and udma irradiation were calculated with hplc as shown in table 1. the contents of table 2, the results of a statistical calculation conducted, indicated that udma and bis-gma subjected to one-time and two-time irradiation both demonstrated significant differences with respective p-values 0.003 and 0.001. the udma group exposed to one-time irradiation, with a p-value of 0.180, showed no significant differences compared to the udma subjected to illumination on two occasions. there were no significant differences in the bis-gma group with a p-value 0.848 that had been subjected to onetime irradiation, compared to the bis-gma group that had been irradiated twice. table 2 contains the results of the different test treatment groups. table 1. average amount of monomer residue calculated with hplc. monomer residue (%)ngroup 19.99171 58.43072 10.30473 39.15574 table 2. test treatment groups testp-valuetreatment groups one-time udma and onetime bis-gma 0.003* mann-whitney test two-times udma and twotimes bis-gma 0.001* independent t test one-time udma and twotimes udma 0.180 mann-whitney test one-time bis-gma and two-times bis-gma 0.848 mann-whitney test *the differences were significant (p-value< 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p24–26 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p24-26 26 rosha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 24–26 discussions from the various test results, it could be seen that repeated irradiation produced no effect on the number of residual monomers in packable composite resins. this is possibly due to the activator material present in the composite resin in the form of champhoroquinon having already reacted during the first polymerization process. consequently, when a second polymerization occurred, no monomer was polymerized. champhoroquinon absorbs visible light in the region of 467 nm.12 however, there are differences in the number of residual monomers, namely bis-gma and udma, in the material contained in packable composite resins. the residual monomers in udma are more numerous than the remaining monomers in bis-gma due to the difference in molecular weight between udma and bis-gma. the molecular weight of udma is lower than that of bis-gma with the result that udma is easily lifted and its morphology is small, enabling its easy detection on hplc. udma also demonstrates high affinity, namely the ability to react with other chemicals, together with strong solubility properties. in bis-gma, the cross density is high compared to that of udma, and its high molecular weight is more stable and not easily biodegradable.13 previous studies have argued that polymerization is influenced by several factors, including molecular weight and affinity.14 the more numerous the molecules polarized, the fewer the residual monomers which, in turn, reduces the likely occurrence of allergies in oral tissue, toxicity to residual monomers and contamination of pulp space due to residual monomers. it is anticipated that two 20-second exposures can reduce the number of residual monomers that are unpolymerized for several reasons. the molecular shape of the bis-gma, which is large and solid, could render the monomers crowded and less able to absorb irradiation, while it is also influenced by molecules that are slow to form polymer bonds. statistically, the two groups have no significant differences (p<0.05). from this study, it can be concluded that the number of residual monomers on bis-gma is lower than that on the remaining udma at 1x20 seconds irradiation, while there is no difference in the number of residual monomers on bis-gma and udma at 2x20 seconds compared to 1 x 20 seconds irradiation. references 1. putriyanti f, herda e, soufyan a. pengaruh saliva buatan terhadap diametral tensile strength micro fine hybrid resin composite yang direndam dalam minuman isotonic. j pdgi. 2012; 61: 43–7. 2. soetojo a. penggunaan resin komposit dalam bidang konservasi gigi. surabaya: revka petra media; 2013. p. 23–112. 3. sakaguchi rl, powers jm. craig’s restorative dental materials. 13th ed. missouri: elsevier mosby; 2012. p. 161–98. 4. nurhapsari a. perbandingan kebocoran tepi antara restorasi resin komposit tipe bulk-fill dan tipe packable dengan penggunaan sistem adhesif total etch dan self etch. odonto dent j. 2016; 3: 8–13. 5. scotti n, venturello a, borga fac, pasqualini d, paolino ds, geobaldo f, berutti e. post-curing conversion kinetics as functions of the irradiation time and increment thickness. j appl oral sci. 2013; 21(2): 190–5. 6. santini a, gallegos it, felix cm. photoinitiators in dentistry: a review. prim dent j. 2013; 2(4): 30–3. 7. anusavice kj, phillips rw, shen c, rawls hr. phillips’ science of dental materials. 12th ed. philadelphia: saunders; 2012. p. 63–5. 8. karabela mm, sideridou id. synthesis and study of properties of dental resin composites with different nanosilica particles size. dent mater. 2011; 27(8): 825–35. 9. karina e, riolina a, krisnawan n. pengaruh lama penyinaran resin komposit nanofil packable terhadap kekuatan tekan (compressive strength) bahan restorasi. j ilmiah fakultas kedokteran gigi univ muhammadiyah surakarta. 2014; 1: 3–8. 10. aryanto m, armilia m, aripin d. compressive strength resin komp osit hybr id p ost cu r i ng denga n l ig ht em it t i ng d io de menggunakan tiga ukuran lightbox yang berbeda. dent j (maj ked gigi). 2013; 46(2): 101–6. 11. ubaldini alm, baesso ml, sehn e, sato f, benetti ar, pascotto rc. fourier transform infrared photoacoustic spectroscopy study of physicochemical interaction between human dentin and etch-&-rinse adhesives in a simulated moist bond technique. j biomed opt. 2012; 17(6): 1–5. 12. kamoun ea, menzel h. crosslinking behavior of dextran modified with hydroxyethyl methacrylate upon irradiation with visible light effect of concentration, coinitiator type, and solvent. j appl polym sci. 2010; 117(6): 3128–38. 13. papakonstantinou ae, eliades t, cellesi f, watts dc, silikas n. evaluation of udma’s potential as a substitute for bis-gma in orthodontic adhesives. dent mater. 2013; 29(8): 898–905. 14. cornelio rb, wikant a, mjøsund h, kopperud hm, haasum j, gedde uw, örtengren ut. the influence of bis-ema vs bis gma on the degree of conversion and water susceptibility of experimental composite materials. acta odontol scand. 2014; 72(6): 440–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p24–26 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p24-26 �0 coen’s ascending ramus fixator use for repositioning the ascending ramus during mandible reconstruction coen pramono d department of oral and maxillofacial surgery faculty of dentistry, airlangga university/dr. soetomo public hospital surabaya indonesia abstract the aim of mandible reconstruction using reconstruction plate after resection is to restore aesthetic and function for muscles attachment and allow mandible movement during normal function and free from joint problem. temporomandibular joint (tmj) is an area of concern during mandible reconstruction using reconstruction plate as misalignment on placing of the reconstruction plate may cause the joint place in distortion to the glenoid fossa. loss large part of mandible bone structure may lead problems during mandible reconstruction procedure because the surgeon may lose in orientation during forming the reconstruction plate into a horseshoe-shaped form of the mandible as well as during plate placement. the plate can only be well adapted when the position of two distal ends of the resected mandible bone are in a stable position. simple ascending ramus fixator (carf) to fix the ascending ramus in its stable original position to allow easy mandible reconstruction was created. those carf were designed in two types which have one and two fixator stems used to stabilize one or both sites of the ascending rami and showed its effectiveness. key words: mandible reconstruction, ascending ramus, ascending ramus fixator, ascending ramus reposition, mandible joint angle, mandible joint prosthesis correspondence: coen pramono d, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: coen_pram@yahoo.com introduction the mandible bone is movable and has no bony articulation with the skull. it is the heaviest and strongest bone of the head and serves as a framework for the floor of the mouth. the mandible constructed as horizontal portion or descending ramus and two vertical portions or ascending rami which join at an obtuse angle. the articular portion of the mandible is connected with the ascending ramus at the inferior portion of the joint i.e. the neck of condyle and the superior portion or articular portion fitting into the mandibular fossa of the temporal bone articulating surface. mandible has horseshoe-shaped form and serves the airway passage by maintaining the position of the tongue, suprahyoid muscles permitting the mastication, articulation, deglutition, and respiration, supports the teeth at the lower dental arch. it also plays an important role in defining the contour of the lower third of the face. interruption of the mandible continuity therefore produces both functional impairment and cosmetic deformity. the result of functional disturbances after the lost part of the mandible bone due to resection may even be worst when the condyle is involved. loss of mandibular continuity results in deviation of the mandible toward the resection site due to the unopposed pull of the remaining muscles of mastication and soft tissue contracture and scar formation. in mandible resection involving both lateral and central part of mandible (lcl = lateral central lateral), the mandible may lose its function for maintaining the position of the tongue and the suprahyoid muscles. all of the suprahyoid soft tissues will be immediately collapse posteriorly and may cause an airway obstruction after resection in this region is the possible complication should be avoided. therefore mandible reconstruction after resection is necessary to be performed as it is important to restore the mandible contour and necessary for muscles re-attachment. mandible reconstruction using plates can be difficult in cases of: diminished dental occlusion; block resection which left only the ascending ramus as a free bone at the distal end; resection of the mandible involving the lateral and central part of the mandible bone or in fashion of lcl and no occlusion is available; and in edentulous jaws. temporomandibular joint (tmj) is an area of concern in mandible reconstruction using reconstruction plate. a common concern is placement of the plate may torque the position of the ascending rami and the condyle relative to the glenoid fossa. alteration of the condyle position may induce a tmj morphology change and may adversely contribute possible problems. the risk of developing tmj internal derangement or inducing a progression of internal derangement after mandible resection caused by the loss of ascending ramus position should always be taken as one of important considerations. in case of large resection of the mandible part which the surgery will be only left the ascending ramus section, recording of the original position of the ascending rami before resection is presumed to be ��pramono: coen's ascending ramus fixator use for repositioning the ascending ramus very important step. this step can be achieved by placement of fixation device of coen’s ascending ramus fixator (carf). the carf was designed very simple using the principle of intermaxillary splint made by acrylic and a fixator made from stainless steel used for holding the ascending ramus attached on it using screws. the position of the fixator is made according to the position of the rest healthy ascending ramus available after resection. the present paper describes cases of mandible reconstruction after resection. two cases of patients suffered from ameloblastoma in the lower jaw which had grown into large dimension and had been treated with mandible resection and followed with primary reconstruction using coen’s reconstruction plates. the large tumor dimension in these cases presented involved important structures i.e. descending ramus which necessary to maintain the anatomical position of the ascending rami, which were seen collapse immediately after resection of the descending ramus. this situation may lead difficulty for reconstruction procedure using reconstruction plate. procedure of mandible reconstruction would be difficult when the position of the ascending ramus is in unstable position. the free or movable ascending ramus is related directly with the condyle position and this situation may lead five possible perspective condyle positions: anterior, posterior, lateral, medial and caudal, therefore recording the position of the ascending ramus in its original position become necessary to prevent the ascending ramus losing its position. misplacement of the ascending ramus can be related to alteration of tmj morphology and therefore may contribute adversely possible problem in mandible kinetic movement. case case 1: a-34 year old man asked for treatment due to mandible swelling which has been marked since five years ago and reported gradually increased in its diameter and changing his facial appearance. panoramic radiograph shows a multicystic radiolucent lesion in the region of 37 to 47 (figure 1-a). histopathologic finding was reported as an ameloblastoma with plexiform type. the treatment was planned with mandible resection from the region of 38 to 48 followed by immediate reconstruction using plate reconstruction. difficulty during reconstruction procedure was predicted before surgery as the ascending rami will become free movable bones because no dental occlusion is available after mandible resection. mandible reconstruction procedure was planned including the preparation of carf. the carf which has two fixator stems and was designed using a set of gypsum model and mandible simulation model made from transparent plastic sheet taken by tracing from the panoramic radiograph. the mandible model then attached in front of the gypsum model following the mandibular arch and the position of teeth as the guidance (figure 1-c). a multi holes stainless plates were used for fixator stems and fixed into the acrylic splint had been prepared using two stainless screws in each site. the positions of those two stems were adjusted according to the position of predicted available healthy ascending rami and can be found easily when simulation model is made (figure 1-c, d & e). the carf was seen working properly to fix both ascending rami (figure 1-f) facilitated an easy forming and placement of the reconstruction plate (figure 1-e). the reconstruction plate was adapted forming the mandibular arch and fixed with five screws in each site (figure 1-b & 1-g) and followed by releasing the carf. case 2: a-16 year old boy visited our oral and maxillofacial surgery department due to enlarged mandible swelling since past few years. panoramic radiograph shows a large unicystic lesion in the region of 38 to 43 (figure 2-a). histopathology examination confirmed with ameloblastoma with plexyform type. resection of the mandible was planned from the region of 44 to 38. resection a huge part of the mandible bone was done and planned with primary reconstruction using reconstruction plate (figure 2-b). carf with unilateral fixator stem was prepared nearly with same procedure as shown in case 1 and planned to be used only for fixing the left ascending ramus. reposition of the right site of the ascending ramus can be figure 1. panoramic radiograph of: a) a large dimension of ameloblastoma in the mandible in lcl fashion: loss of the ascending rami position due to surgical resection was predicted. b) postoperative panoramic view: plate reconstruction fixed into proper available space between the mandible rami. �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 10-14 achieved to be guided by the right side dental occlusion. placement of crf with unilateral stem type fixed to the left ascending ramus and the intact dental occlusion on the right side gives an adequate and stable space between two ascending rami facilitated an easy reconstruction plate adaptation and placement (figure 2-c). case management two cases of ameloblastoma in the mandible were treated in our oral and maxillofacial surgery department, faculty of dentistry, airlangga university. figure 1. c) mandible simulation model made from transparent plastic sheet taken by tracing from panoramic radiograph used for adjusting the position of the fixator stems. d) the carf with bilateral stems shows from posterior view. figure 1. e) the carf made from acrylic with intermaxillary splint principle: two stems attached using stainless steel screws. f) the carf with bilateral stems fixed the ascending rami properly in lcl case: facilitate an easy placement of reconstruction plate. g) reconstruction plate placed in anatomical space between two mandible rami stabilized using carf with two stems type. figure 2. a) panoramic x-ray: ameloblastoma of the mandible in lcl fashion. the occlusion of the left mandible in the region of molar left intact. b) resection a huge part of mandible bone: immediate reconstruction is obligatory to be proceeded. ��pramono: coen's ascending ramus fixator use for repositioning the ascending ramus mandible resections followed by primary reconstruction using reconstruction plate were done in both cases. the reconstruction procedures were predicted to be difficult in both cases as the tumors had grown into large dimension and the surgical intervention may cause the ascending rami lose its connection with the horizontal portion or the descending ramus. the two vertical portions or ascending rami which actually should be joined at an obtuse angle become free portions and lose its anatomical positions after resection. two type of ramus fixators (carf) were used temporarily for maintaining the ascending rami positions after mandible resection and were applied in both cases respectively to allow an easy procedure during reconstruction plate forming, adaptation and placement. discussion the effect of mandible reconstruction on the temporomandibular joint (tmj) is an area of concern as well as the plate position and its acceptable horseshoe-shaped form. the common concern is that the plate placement may torque the condyle position relative to the glenoid fossa. alteration of tmj morphology may adversely contribute possible problem in mandible kinetic movement. bilateral sagittal split osteotomy (bsso) is the best example of surgical intervention which may contribute an alteration of tmj morphology leading to tmj disorders. the change of the ascending rami position after sagittally split from the descending ramus followed by setback or advancement of the descending ramus and reposition of the ascending rami including the condyle segments may introduce altered condyle segments position. loss of a large segment of the mandible bone in the central segment due to resection may lead the same problem can be shown in bsso procedure while the distal mandible bone segment become left unfixed and therefore it may loss its original position. alder et al.1 analyzed short-term in condylar position changes after bsso on 21 treated patients which had been repositioned and fixated rigidly. pre and postoperative condyles positions were observed using a computed tomography as their tool for investigation. the angular angle of the condyle position after bsso are found changing in all individuals had been investigated. they found an increased and decreased in condylar angle angulation. the majority of cases reported an increased angle, which would lead anterior movement of the medial and posterior movement of the lateral pose. detailed data given that all condyles observed had some displacement and that movement occurred in all directions which presented as average displacements in the positive and negative direction. in the sagittal dimension, the majority of condyles moved posteriorly (67%), superiorly (60%) and demonstrated an inferior or distal distal rotation of the proximal segment (61%). the remaind were displaced anteriorly to the same average of distance, inferiorly to an average of 1.2 mm and rotated superiorly to an average of 3.2 degrees. rotskoff et al.2 compared cases of bsso and fixed with rigid internal fixation (rif) and treated with and without a condyle proximal segment-positioning device (cpd) used at the time of surgery. a total of 20 patients were treated and analyzed with axially corrected sagittal tomography. tracing of the tmjs taken preoperatively and one day postoperatively, were superimposed using the squamotympanic fissure, glenoid fossa, articular eminence, and posterior aspect of the condyle and ramus as constant anatomic landmark. they reported that the average condyles were displaced posteriorly, inferiorly with forward rotation of the condyle. displaced was less pronounced in the group the cpd were used. stoster et al.3 used submentovertex radiograph to assess changes in the transverse dimension. they included fifty three samples who had undergone for bsso and they found condyle rotation, this involved rotation of the medial aspect posteriorlyand the lateral aspect anteriorly due to the segments fixation. schultes et al.4 also analyzed changes in angulation after ramus surgery using the technique of bsso. their study used 3d models of the mandible constructed from 3d ct data. the result taken from sample of 31 patients underwent ramus surgery for mandibular advancement. the result showed that the intercondylar angle was decreased by 2.5 degrees on average, which would require rotation of the medial pole posteriorly and the lateral pole anteriorly. intercondylar distance was increased by about 2 mm. although the carf can be used for maintaining the position of ascending rami in case of loss of large part of the descending ramus, the relative risk of developing tmj internal derangement after mandible reconstruction using plate is important issue in treatment planning and in obtaining informed consent. figure 2. c) the placement of crf with unilateral stem type fixed to the left ascending ramus and the intact dental occlusion on the right side gives an adequate and stable space between two ascending rami. �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 10-14 the philosophy of successful surgical result in mandible surgery involving the tmjs with prediction of altered joints is free from the joints problem postoperatively. those two type of carfs presented in these reported cases were proved to be useful in maintaining an adequate anatomical space or distance between two distal mandible bone ends or between two free ascending rami and can be made in low cost and easy to prepare preoperatively. this procedure can be used as an alternative surgical procedure as it had been proven that the stability of the ascending rami can be achieved properly and also helpful to facilitate an easy procedure during reconstruction plate forming and placement, and should be taken as consideration in a routine surgical protocol for eliminating postoperative tmj problems. references 1. alder me, deahl st, matteson sr, van sickles je, tinner bd, rugh jd. short-term changes of condylar position after sagittal split osteotomy for mandibular advancement. j oral surg oral med oral pathol oral radiol endod 1999; 37:41–45. 2. rotskoff ks, herbosa eg, villla p. maintanance of condyle-proximal segment position in orthogntathic surgery. j oral maxillofac surg 1991; 49:2–7. 3. stoster tg, pangrazio-kulbersh v. assessment of condylar position following bilateral sagittal split ramus osteotomy with wire fixation or rigid fixation. int j adult orthod orthognath surg 1994; 9:55–63. 4. schultes g, gaggl a, karcher h. changes in the dimension of milled mandibular models after sagittal split osteotomies. br j oral and maxillofac surg 1998; 36:196–201. 164 volume 47, number 3, september 2014 daya hambat xylitol dan nistation terhadap pertumbuhan candida albicans (in vitro) (inhibition effect of xylitol and nistatin combination on candida albicans growth (in vitro)) sarah kartimah djajusman, udijanto tedjosasongko, dan irmawati 1departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: the growth of candida albicans can be controlled by using antifungal such as nystatin. these days we found that using antifungal is not enough to control candida albicans, we also have to control the intake of sugar by using xylitol. purpose: purpose of the study was to determine the optimal inhibitory concentration of xylitol-nystatin in the candida albicans growth. methods: this was an in-vitro study using an antimicrobial test of serial dilution with xylitol-nystatin and sucrose–nystatin consentration of 1%, 3%, 5%, 7%, 9%, and 10%.growth inhibition of c. albicans was determined by the inhibition zone of xylitol + nystatin on c. albicans culture media (in vitro) results: the result of study was the inhibitory consentration of xylitol-nystatin to inhibit candida albicans growth was 3%-10%. conclusion: the study showed that combination of xylitol and nystation could inhibit the growth of candida albicans. key words: candida albicans, xylitol, sucrose, nystatin, inhibitory concentration abstrak latar belakang: pertumbuhan candida albicans dapat dikontrol dengan menggunakan antijamur seperti nistatin. penggunakan antijamur saja tidak cukup untuk mengontrol candida albicans, namun perlu pula mengontrol asupan gula dengan menggunakan xylitol. tujuan: tujuan dari penelitian ini adalah untuk menentukan konsentrasi hambat optimal xylitol-nistatin dalam pertumbuhan candida albicans. metode: penelitian ini merupakan penelitian in vitro menggunakan uji antimikroba pengenceran serial dengan xylitol-nistatin dan nystatin-sukrosa konsentrasi 1%, 3 %, 5 %, 7%, 9%, dan 10%. daya hambat pertumbuhan c. albicans diukur dari zona hambat xylitol + nistatin pada media kultur c. albicans (in vitro) hasil: konsentrasi penghambatan xylitol-nistatin untuk menghambat pertumbuhan candida albicans adalah 3-10%. simpulan: hasil penelitian menunjukkan bahwa kombinasi xylitol dan nystation bisa menghambat pertumbuhan candida albicans. kata kunci: candida albicans, xylitol, sukrosa, nistatin, konsentrai daya hambat korespondensi (correspondence): sarah kartimah djajusman, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: udijanto@gmail.com research report 165djajusman, et al.: daya hambat xylitol dan nistation terhadap pertumbuhan candida albicans (in vitro) pendahuluan anak-anak yang menjalani rawat inap di rumah sakit umumnya kurang memperhatikan kondisi rongga mulut sehingga pemberian antibiotik selama perawatan ± 5 hari dapat memicu timbulnya efek samping. efek yang paling sering muncul adalah oral candidiasis karena antibiotika membunuh bakteri yang menguntungkan dan merugikan.1 keseimbangan rongga mulut menjadi terganggu karena tidak ada bakteri menguntungkan yang dapat mengontrol pertumbuhan candida albicans (c. albicans).2 nistatin merupakan antijamur yang dianjurkan untuk terapi oral candidiasis. nistatin efektif untuk jamur dan ragi namun tidak efektif pada bakteri, protozoa dan virus. obat ini aman untuk digunakan oleh anak-anak karena tidak diabsorbsi langsung oleh darah.3 namun dalam nistatin berbentuk obat tetes terkandung sukrosa agar anak-anak tidak menolak obat tersebut. sukrosa diketahui dapat membantu pertumbuhan c. albicans, agar pengobatan lebih optimal hendaknya sukrosa diganti dengan xylitol.4 xylitol merupakan pemanis natural yang dapat ditemukan pada jagung, strawberry, dan plums. xylitol terdiri dari lima rantai karbon yang bersifat antimikrobial mencegah timbulkan bakteri dan jamur termasuk c. albicans, mencegah plak, remineralisasi enamel gigi, aman untuk penderita diabetes dan hipoglikemia.5,6 berdasarkan hal tersebut maka kemungkinan ada pengaruh penambahan xylitol-nistatin dan sukrosa-nistatin terhadap pertumbuhan c. albicans dan berapakah konsentrasi dari xylitol-nistatin yang dapat menghambat pertumbuhan c. albicans. penelitian ini bertujuan meneliti pertumbuhan candida albicans setelah penambahan xylitol dalam nistatin, meneliti dosis optimal xylitol dalam nistatin yang dapat menghambat pertumbuhan c. albicans dan membandingkan pertumbuhan c. albicans setelah penambahan xylitolnistatin dengan penambahan sukrosa-nistatin. diharapkan dari penelitian ini xylitol dapat digunakan sebagai obat pendamping untuk pasien anak-anak yang menderita oral candidiasis dan dosis xylitol yang ditemukan dapat digunakan untuk pencampuran sediaan antifungal bagi anak-anak yang dapat dijual dipasaran bebas. bahan dan metode c. albicans diperoleh melalui swab pasien anak pada poli rawat inap anak rsud nganjuk. swab dilakukan pada pasien yang menerima antibiotik ciprofloxacin dan telah dirawat selama ± 5 hari karena menderita demam thypoid. hasil swab di streak pada saboroud dextrose broth kemudian diinkubasi selama 48 jam pada inkubator di laboratorium rsud nganjuk. candida yang telah tumbuh kemudian dibawa ke laboratorium mikrobiologi fakultas kedokteran gigi universitas airlangga untuk dilakukan tes glukosa untuk mengetahui jenis candida. kemudian hasil identifikasi ditemukan c. albicans dan dibiakkan lebih lanjut. penelitian ini menggunakan rancangan penelitian percobaan the postest only controlled group design, dilakukan di laboratorium mikrobiologi mulut fakultas gigi universitas airlangga sebagai berikut, disiapkan 6 tabung yang berisi xylitol 1%; 3%; 5%; 7%; 9%; dan 10% serta 6 tabung berisi sukrosa 1%; 3%; 5%; 7%; 9%; dan 10% kesemuanya kemudian diencerkan dengan aquades sampai mencapai 10 cc dan ditambahkan nistatin 1 cc. c. albicans yang telah ditipiskan kemudian distreak pada media sdb yang telah disiapkan. xylitol dan sukrosa diteteskan pada paper disc yang terpasang pada sbd yang telah terulas c. albicans. inkubasi pada suhu 37º c selama 48 jam. dilakukan pengamatan dan penghitungan zona hambat c. albicans pada kelompok kontrol dan kelompok perlakuan kemudian dilakukan analisa data. hasil nilai rerata daya hambat xylitol-nistatin, sukrosanistatin terhadap c. albicans tampak pada gambar 1. hasil komlogorov smirnov test menunjukkan bahwa secara keseluruhan nilai daya hambat terhadap candida albicans pada kelompok xylitol dan nistatin dan kelompok glukosa dan nistatin mempunyai distribusi data yang normal. gambar 1. rerata nilai daya hambat xylitol-nistatin, sukrosanistatin, dan nistatin terhadap c. albicans. tabel 1. nilai p hasil independent t-test nilai daya hambat terhadap c. albicans konsentrasi kelompok independent ttest 1% xylitol dan nistatin p = 0,617 sukrosa dan nistatin 3% xylitol dan nistatin p = 0,001 sukrosa dan nistatin 5% xylitol dan nistatin p = 0,001 sukrosa dan nistatin 7% xylitol dan nistatin p = 0,001 sukrosa dan nistatin 9% xylitol dan nistatin p = 0,001 sukrosa dan nistatin 10% xylitol dan nistatin p = 0,001 sukrosa dan nistatin 166 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 164–167 hasil levene test menunjukkan bahwa nilai daya hambat terhadap c. albicans pada kelompok xylitol dan nistatin dan kelompok sukrosa dan nistatin mempunyai varians yang tidak homogen, sehingga dilakukan independent t -test. hasil uji beda nilai daya hambat xylitol-nistatin terhadap c. albicans hampir secara keseluruhan mempunyai nilai p < 0,05 hal ini menunjukkan bahwa ada perbedaan yang bermakna kecuali pada kelompok 1% dengan kontrol dan 3% dengan 5% tidak ada perbedaan yang bermakna. untuk mengetahui perbedaan nilai daya hambat antara xylitol-nistatin dan sukrosa-nistatin terhadap c. albicans pada konsentrasi yang sama dilakukan dengan independent ttest (tabel 1). pada tabel 1 dapat kita ketahui hasil uji beda nilai daya hambat xylitol-nistatin dan sukrosa-nistatin terhadap c. albicans dimana hampir secara keseluruhan mempunyai nilai p < 0,05, kecuali pada kelompok 1%. hal ini menunjukkan bahwa ada perbedaan yang bermakna nilai daya hambat xylitol-nistatin dan sukrosa-nistatin terhadap candida albicans, sedangkan pada kelompok yang 1% mempunyai nilai p > 0,05. hal ini menunjukkan bahwa tidak ada perbedaan yang bermakna nilai daya hambat terhadap candida albicans antara kelompok xylitol dan nistatin dengan kelompok sukrosa dan nistatin. pembahasan pada analisa data terlihat pada xylitol-nistatin seiring dengan kenaikan konsentrasinya terjadi pula kenaikan nilai daya hambat terhadap pertumbuhan c. albicans, sedangkan pada sukrosa-nistatin semakin besar konsentrasinya semakin kecil rerata daya hambat terhadap c. albicans. hasil dari penelitian ini sesuai dengan penelitian yang pernah dilakukan oleh vargas et al.7 yang menemukan bahwa tikus yang mendapat xylitol mengalami penurunan pertumbuhan c. albicans pada gastro-intestinalnya lima kali lebih cepat daripada tikus yang menerima sukrosa. hasil ini juga didukung oleh penelitian santelmann and john8 yang menemukan bahwa pasien polisimptomatik yang mendapatkan nistatin dengan diet gula dan ragi mendapat kesembuhan 35% lebih baik daripada yang tidak melakukan diet. penelitian munita et al.9 juga menemukan hal yang sama yaitu pada pasien recurrent oral candidiasis diinstruksi untuk mengganti sukrosa yang biasa dikonsumsi sehari-hari dengan xylitol dan menghindari segala makanan dan minuman yang mengandung sukrosa dan hasilnya dalam waktu 14 hari ditemukan pengurangan lesi c. albicans sebesar 70%. faktor yang ikut mempengaruhi besar konsentrasi xylitol dalam menghambat pertumbuhan c. albicans diantaranya adalah pengkombinasian xylitol dengan bahan lain yang mempunyai sifat menghambat pertumbuhan candida dalam hal ini berupa nistatin. mekanisme kerja xylitol terhadap candida, prosedur penelitian serta asal isolat dari c. albicans yang didapat melalui swab pada lidah pasien demam thypoid yang menerima perawatan antibiotik ciprofloxacin. pada penelitian ini xylitol dikombinasikan dengan nistatin. nistatin sebagai antijamur yang ditambahkan berfungsi untuk menghalangi jalan candida melekat pada sel inang karena candida dalam mekanisme perlekatannya memerlukan bantuan dari karbohidrat, ß glucan, chitin dan mannoprotein.10 mekanisme nistatin dalam menghalangi perlekatan yaitu dengan mengikat ergosterol sehingga terjadi perubahan pada permeabilitas membran sel akbatnya sel kehilangan berbagai molekul adesin yaitu mannoprotein yang diperlukan untuk membantu perlekatan pada sel inangnya.11 xylitol memerlukan waktu yang cukup lama sampai akhirnya dapat menjadi glukosa yang diperlukan oleh candida dimana xylitol dioksidasi oleh nad (nicotinamideadenine-di-nucleotida) xylitol dehidrogenase menjadi d-xylulosa kemudian fosforilasi oleh d-xylulosa kinase menjadi d-xylulosa-5-fosfat lalu dibantu oleh pentosa fosfat menjadi fruktosa-6-fosfat (2 molekul) dan glyceraldehyde fosfat (1 molekul). fruktosa-6-fosfat dapat dirubah menjadi glukosa dan glycogen dan glyceraldehydes fosfat dapat diubah menjadi glukosa, glycogen dan laktat. dari proses oksidasi ini glukosa yang dihasilkan hanya sekitar 50% dan dalam waktu 165 detik sudah hilang, sehingga candida tidak dapat memanfaatkannya untuk membentuk glycomannoprotein yang dapat merangsang perlekatan candida pada sel hostnya.12,13 pada manusia xylitol memerlukan waktu yang lebih lama untuk melakukan metabolisme dibandingkan pada hewan tikus maupun anjing.12 hal ini juga diperkuat oleh penelitian vargas et al.7 yang menyatakan candida tidak dapat melekat pada sel inang karena metabolisme naddehydrogenase yang lambat sehingga daya serap untuk karbohidrat menjadi lemah sehingga tidak terjadi proses glikolisis yang diperlukan untuk candida agar dapat bertahan hidup karena tidak terjadi pula proses perlekatan pada sel inang sehingga proses kolonisasi akan menurun pula. penelitian yang dilakukan pires et al.14 menemukan hal yang cukup menarik, selain membuktikan bahwa xylitol berpengaruh untuk menghambat pertumbuhan c. albicans, ditemukan pula bahwa c. albicans lebih menderita karena kekurangan glukosa daripada kehadiran xylitol. hal ini juga sesuai dengan hasil penelitian ying et al.13 yang menyatakan bahwa hasil metabolisme xylitol hanya menghasilkan 50% glukosa. dalam penelitian yang dilakukan who12 menyimpulkan untuk mengontrol pertumbuhan candida dapat dilakukan dengan membatasi karbohidrat dan sukrosa dapat meningkatkan pertumbuhan c. albicans. konsentrasi sukrosa yang tinggi dapat meningkatkan pertumbuhan c. albicans dan kenaikan sukrosa juga menyebabkan terjadinya stomatitis pada manusia. menurut weig et al.15 infeksi yang terjadi karena c. albicans pasti dipicu oleh sukrosa. penelitian dilakukan dengan meminta 28 sukarelawan untuk mengkonsumsi makanan tinggi karbohidrat dan gula dalam 11 minggu yang terbagi menjadi 3 periode dan ditemukan 78,6% dari subyek ditemukan 167djajusman, et al.: daya hambat xylitol dan nistation terhadap pertumbuhan candida albicans (in vitro) c. albicans dalam rongga mulutnya dan 71,4% dalam feses14 juga menemukan dalam media kultur yang kaya akan glukosa, sukrosa, maltosa, galaktosa meningkatkan pertumbuhan candida dalam sel epithelial dan dental akrilik. dari hasil analisa data diketahui bahwa dosis minimum xylitol yang berpengaruh dalam membantu nistatin adalah konsentrasi 3%. hasil penelitian ini menunjukkan bahwa dosis xylitol yang dapat membantu nistatin untuk menghambat pertumbuhan c. albicans mulai dari 3-10%. hasil penelitian ini sesuai dengan penelitian yang dilakukan munita et al.9 yang menyatakan bahwa jumlah c. albicans pada penderita aids yang terkena oral candidiasis dengan terapi nistatin 500.000 unit per hari berkurang lebih banyak pada penderita yang diberi xylitol 5% dan 10% dibandingkan yang menerima glukosa 5% dan 10%. ada sedikit perbedaan hasil karena dalam penelitian yang dilakukan oleh penulis konsentrasi pemberian xylitol dimulai dari 1% kemudian meningkat sampai 10% sedangkan dalam penelitian yang dilakukan oleh munita et al.9 hanya memberikan xylitol dalam konsentrasi 5% dan 10% saja. namun perlu diingat pula bahwa dalam hasil penelitian penulis ditemukan tidak ada perbedaan bermakna antara konsentrasi 3% dan 5%. dosis xylitol yang dianjurkan untuk dikonsumsi seharihari oleh sebuah produk permen xylitol nature provision6 yang diterbitkan dalam websitenya yaitu 4-10gr atau dalam bentuk permen karet sekitar 5-12.6 namun perlu diingat pula dosis xylitol yang terlalu banyak dikonsumsi seharihari dapat menimbulkan efek samping berupa diare karena duapertiga dari metabolisme xylitol dalam tubuh diambil oleh bakteri di usus pencernaan guna memecah asam lemak menjadi rantai pendek.16 dari hasil penelitian ini diharapkan untuk selanjutnya dosis xylitol yang sudah ditemukan dapat dikombinasikan dengan nistatin dalam bentuk sediaan obat. karena selama ini nistatin yang beredar dipasaran mengandung pemanis yang dalam penggunaan jangka panjang bisa menyebabkan karies pada anak sehingga penggunaan topical fluor dianjurkan setelah terapi nistatin selesai.17 diharapkan pemanis yang terkandung dalam nistatin dapat diganti dengan xylitol sehingga perawatan dapat efektif dan masa terapi dapat lebih cepat dari 10-14 hari serta efek karies dapat seminimal mungkin. penelitian ini menunjukkan bahwa kombinasi xylitol dan nystation bisa menghambat pertumbuhan c. albicans. daftar pustaka 1. sukanto, pradopo s, yuliati a. daya hambat ekstrak kulit buah delima putih terhadap pertumbuhan candida albicans. majalah kedokteran gigi (dent j) 2002; 35(4): 161-3. 2. vicki k. candida over growth/ yeast hypersensitivity. stonyfield yogurt-moos release. 2007. available from: www. stonyfield.com. accessed march 29, 2007. 3. boroch a. candida-the silent epidemic. 2007. available from: www. annboroch.org. accessed march 29, 2007. 4. levine sa, larry j. candida albicans (yeast infection). springboard. sb3.com. the nutrition notebook. 2004. available from: www. springboardhealth.com. accessed march 29, 2007. 5. sellman s. xylitol: our sweet salvation. the spectrum 2003; 4(8): 23-30. 6. nature provision. xylitol all natural health resources by nature’s provison. 2006. available from: www. xylipro.com. accessed november 16, 2008. 7. vargas sl, christian cp, gregory da, walter th. modulating effect of dietry carbohydrate supplementation on candida albicans colonization in a neutropenic mouse model. infection and immunity 1993; 61(2): 619-26. 8. santelmann h, john mh. yeast metabolic product, yeast antigens and yeast as possible triggers for irritable bowel syndrome. eur j gastroenterol hepatol 2005; 17(1): 21-6. 9. munita ls, julita p, markku v, tammy p, david s. use of polyols in combating yeast infection and polyol preparations for said use. freepatentsonline 2002. 10. cannon rd, chaffin wl. oral colonization by candida albicans. crit rev oral biol and med 1999; 10(3): 359-83. 11. ganiswara sg. farmakologi dan terapi. edisi ke 4. jakarta: gaya baru hal; 2000. h. 567-68. 12. world health organization. xylitol evaluation for accetable daily intake. 1977. available from: www.inchem.org/documents/jecfa. htm. accessed april 11, 2009. 13. ying hc, fulcher c. c. albicans pathway: xylitol degradation. 2007. available from: www. metacyc.com. accessed april 11, 2009. 14. pires, maria de fc, benedito c, walderez g, claudete rp. experimental model of candida albicans (serotypes a and b) adherence in vitro. braz j microbiol 2001; 32(3). 15. weig me, mathias f, heinrich k. limited effect of refined ca rb ohyd r at e d iet a r y supplement at ion on colon i zat ion of gastrointestinal tract of healthy subject by candida albicans. american journal clinical nutrition 1999; 69: 1170-3 16. cronin jr. xylitol a sweet for healthy teeth and more. alternative and complementary therapies 2003; 9(3): 139-41. 17. mcdonald re, david a, jeffrey d. dentistry for the child and adolescent. eight edition. usa: mosby; 2004. p. 423. 167 volume 45 number 3 september 2012 shear bond strength of self-adhering flowable composite on dentin surface as a result of scrubbing pressure and duration ferry jaya1, siti triaminingsih2, andi soufyan s2 and yosi kusuma eriwati2 1 postgraduate student of dental materials science 2 department of dental materials science faculty of dentistry, universitas indonesia jakarta indonesia abstract background: self-adhering flowable composite is a combination of composite resin and adhesive material. its application needs scrubbing process on the dentin surface, but sometimes it is difficult to determine the pressure and duration of scrubbing. purpose: this study was aimed to analyze the effect of scrubbing pressure and duration on shear bond strength of self-adhering flowable composite to dentin surface methods: fifty four mandibulary third molar were cut to get the dentin surface and divided into nine groups (n = 6). dentin surface was scrubbed with 1, 2, and 3 grams of scrubbing pressure, each for 15, 20, and 25 seconds respectively.dentin surface was scrubbed with 1, 2, and 3 grams of scrubbing pressure, each for 15, 20, and 25 seconds respectively. composite resin was applied incrementally and polymerized for 20 seconds. all specimens were immersed in saline solution at 37º c for 24 hours. shear bond strength was tested for all specimens by using universal testing machine (shimadzu ag-5000e, japan) at a crosshead speed of 1 mm/minute and analyzed by anova and post hoc test bonferonni. the interface between self-adhering flowablethe interface between self-adhering flowable composite and dentin was observed with a scanning electron microscope (jeol jsm 6510la). results: the highest shear bond strength was obtained by 3 grams scrubbing pressure for 25 seconds or equal to applying the brush applicator in 0º relative to dentin surface. conclusion: increasing the scrubbing pressure and duration will increase the shear bond strength of self adhering flowable composite resin to dentinal surface. the highest shear bond strength was obtained when the applicator in 0º relative to dentin surface. key words: scrubbing technique, shear bond strength, self-adhering flowable composite abstrak latar belakang: self-adhering flowable composite merupakan gabungan resin komposit dengan material adhesif yang dalam penggunaannya memerlukan teknik scrubbing pada permukaan dentin, namun sulit untuk menentukan besar tekanan yang tepat saat scrubbing. tujuan: tujuan dari penelitian ini adalah untuk menganalisa pengaruh tekanan dan lama scrubbing saat aplikasi selfadhering flowable composite terhadap shear bond strength pada permukaan dentin. metode: 54 gigi molar-3 rahang bawah dipotong sampai permukaan dentin dan dibagi atas 9 kelompok (n = 6). scrubbing pada permukaan dentin dilakukan dengan besar tekanan 1, 2, dan). scrubbing pada permukaan dentin dilakukan dengan besar tekanan 1, 2, dan 3 gram, masing-masing selama 15, 20, dan 25 detik. resin komposit diaplikasikan secara inkremental dan dipolimerisasi dengan sinar selama 20 detik. spesimen direndam dalam larutan salin dan disimpan dalam inkubator pada suhu 37º c selama 24 jam. shear bond strength diuji menggunakan universal testing machine. analisa data melalui uji anova diikuti post hoc test bonferonni. antarmuka self-adhering flowable composite resin dan dentin diamati melalui scanning electron microscope (sem). hasil: shear bond strength tertinggi antara self-adhering flowable composite resin dan dentin diperoleh pada tekanan scrubbing 3 gram selama 25 detik atau kuas membentuk sudut 0º dengan permukaan dentin. kesimpulan: semakin besar tekanan dan lama waktu scrubbing akan meningkatkan shear bond strength material self-adhering flowable composite resin terhadap permukaan dentin. kekuatan paling tinggi diperoleh bila kuas aplikator membentuk sudut 0º dengan permukaan dentin selama proses scrubbing. kata kunci: teknik scrubbing, shear bond strength, self-adhering flowable composite correspondence: siti triaminingsih, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta 10430, indonesia. e-mail: ami_permana@yahoo.com. research report 168 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 167–171 introduction the latest adhesive system on dentin surface is a combination of etchant, primer, and bonding agent in one product or one step application.1 self-adhering flowable composite that combines the resin technology of composite and adhesive has also developed into one product. this self-adhering flowable composite eliminates the need for a separate bonding application step with composite for direct restorative procedures. this material contain of glycerol phosphate dimethacrylate (gpdm) that act as an etchant and dentin bonding agent in the same time. the bonding to dentin is a result of chemical reaction between phosphate group and calcium ion in tooth structure and mechanically as a result of hybrid layer formation. self-adhering flowable composite is indicated for small class i and base or liner for class i and ii restorations. additional indications include pit and fissure sealant, repair of enamel defects, blocking of undercuts, incisal abrasions, and porcelain repairs.the application of this material on the dentin surface needs scrubbing process with moderate pressure using brush applicator.2 the application of self-etch adhesive materials with scrubbing technique on dentin surface will increase the bonding strength to dentin surface by modified the smear layer, and increase chemical interaction between selfadhesive materials anddentin.3–5 the same reason expected in application of self-adhering flowable composite. however practically it is hard to determine the pressure and duration of scrubbing process using brush applicator to reach the optimal bonding strength. the objective of this study were to analyze the effect of scrubbing pressure and duration on shear bond strength of self-adhering flowable composite to dentin surface and that, to develop a guideline of brush applicator usage to obtain the appropriate scrubbing pressure. materials and methods fifty four mandibulary third molars were obtained under a protocol approved by the faculty of dentistry's ethics committee at universitas indonesia. all specimens were sectioned to reach the dentin surface and embedded in resin with the dentin surfaces on the outside. the dentin surface was ground with a mechanical grinder (struer labopol, denmark) and polished with wet silicon carbide paper #600 to obtain a flat surface. all specimens were observed under a light optical stereo microscope to ensure the dentin cleanness with no defect. a plastic sheet with 5 mm in diameter was applied on dentin surface and surrounding dentin surface was varnished with nail polish to border the tested area. thereafter a gelatin ring with same diameter was applied encircle the tested area.6 specimens were randomly divided into nine groups (n = 6). self-adhering flowable composite (table 1) was applied on dentin surface by scrubbing technique for 1, 2, and 3 grams of scrubbing pressure each for 15, 20, and 25 seconds. the scrubbing pressure was controlled by digital balance. according to the previews study, applying the brush applicator with an angle of 0 degree relative to dentin surface was equal to 3 grams, 30 degrees relative to dentin surface equal to 2 grams, and 60 degrees relative to dentin surface equal to 1 gram. polymerization of self-adhering flowable composite was done using led max hilux 450 (benlioglu, turkey) with intensity of 600 mwcm–2 for 20 seconds. composite resin (filtex z350, 3m espe) was applied incrementally(filtex z350, 3m espe) was applied incrementallye) was applied incrementally into the gelatin ring and polymerized for 20 seconds. all specimens were then immersed in saline solution at 37º c for 24 hours. shear bond strength was tested for all specimens with an universal testing machine (shimadzu ag-5000e, japan) at a crosshead speed of 1 mm/minute. the shear bond strength data were statistically analyzed by one-way anova and post hoc test bonferonni. thereafter by two-way anova for analyze the interaction between groups. the surface fracture area of specimens were sectioned and immersed in hcl 37% for 30 seconds to demineralized the dentin, then immersed in naocl 1% for 10 minutes to dissolve the demineralized matrix dentin.7,8 thereafter the interface between self-adhering flowable composite and dentin was observed with a scanning electron microscope (jeol jsm 6510la) at magnification of 1000×. results the mean value of shear bond strength of self-adhering flowable composite (dyad flow, kerr) to dentin surface with scrubbing pressure 1, 2, and 3 grams each for 15, 20, and 25 seconds are shown in table 2. table 1. composition and application procedures of self-adhering flowable composite brand composition ph procedures dyad flow (kerr) gpdm (glycerol phosphate dimethacrylate), barium glass filler, colloidal silica, ytterbium fluoride 1.9 use provide brush to apply dyad flow with moderate pressure for 15–20 seconds to obtain a thin layer (< 0.5 mm). light cure for 20 seconds source: manual product of dyad flow, kerrmanual product of dyad flow, kerr 169jaya, et al.: shear bond strength of self-adhering figure 1. morphology interface between self-adhering flowable composite and dentin surface. a) 1 g scrubbing pressure for 15 sec; b) 1 g scrubbing pressure for 20 sec; c) 1 g scrubbing pressure for 25 sec. figure 2. morphology interface between self-adhering flowable composite and dentin surface. a) 2 g scrubbing pressure for 15 sec; b) 2 g scrubbing pressure for 20 sec; c) 2 g scrubbing pressure for 25 sec. figure 3. morphology interface between self-adhering flowable composite and dentin surface. a) 3 g scrubbing pressure for 15 sec; b) 3 g scrubbing pressure for 20 sec; c) 3 g scrubbing pressure for 25 sec. table 2. mean shear bond strength of self-adhering flowable composite to dentine surface based on scrubbing pressure and duration (mpa) scrubbing pressure scrubbing duration 15 sec 20 sec 25 sec 1 g 3.30 ± 0.92 3.70 ± 1.19 4.33 ± 1.66 2 g 3.77 ± 1.47 4.36 ± 1.44 5.23 ± 1.25 3 g 4.99 ± 0.82 5.25 ± 2.04 7.22 ± 0.68 it was showed that the shear bond strength increased simultaneously with scrubbing pressure and duration. statistically showed the normal data distribution, therefore the analysis continue by two-way anova that showed the differences between scrubbing pressure groups and between scrubbing duration groups. the statistic analysis was supported by the sem images of morphology between dentin surface and self-adhering flowable composite with scrubbing pressure 1, 2, and 3 grams each for 15, 20, and 25 seconds. it was showed that the formation of hybrid layer gradually appears in line with increasing in scrubbing pressure and duration (figure1, 2, 3). 170 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 167–171 discussion the scrubbing pressure guideline to obtain the optimal shear bond strength of self-adhering flowable composite to dentin surface was found in this study. applying the brush applicator with an angle of 0 degree relative to dentin surface was equal to 3 grams scrubbing pressure. the highest shear bond strength of self-adhering flowable composite to dentin was 7.22 mpa. this is value lower than shear bond strength of self-etch adhesive to dentin that studied by al qahtani9 with mean value of 12.61 mpa. in self-etch adhesive system, the etching process and the infiltration of resin occur in the same time. the mechanism of self-adhering flowable composite bonds to dentin through the chemical bond between the phosphate functional group of gpdm monomer and calcium ion of the tooth structure. furthermore, through a micromechanical bond as a result of an interpenetrating network formed between the polymerized monomers and collagen fibers as well as the smear layer of dentin, it was known as hybrid layer. 2,10 it was expected that both self-adhering flowable composite and self-etch adhesive have a similar shear bond strength to dentin, however it was not found in this study. it can be showed that there was the different demineralization potential between self-adhering flowable composite and self-etch adhesive materials. factors that influence the demineralization potential of adhesive materials were ph, duration of application process, wettability, viscosity, and water content in materials.11selfadhering flowable composite has ph of 1.92 or moderate acidity (ph 1.5–2.0),8 which can only remove part of smear layer. the remaining smear layer will prevent the adhesive to infiltrate to the dentinal tubuli and decrease the bond strength. the bond strength to dentin significantly decreased on dentin with thick smear layer.12 on the other hand, the thickness of hybrid layer is not influenced by smear layer thickness.13 the bonding mechanism of adhesive material on dentin surface obtained through the hybrid layer. during demineralization process the hybrid layer formed by infiltration of resin into collagen fibrils. the scrubbing process modified the smear layer into thicker hybrid layer that will increase the bond strength of adhesive.14 in this study application of self-adhering flowable composite was done by scrubbing technique to form the hybris layer. this condition simultaneously increase the shear bond strength of self-adhering flowable composite to dentin surface based on scrubbing pressure and duration. chan et al.,15 stated that bond strength of self-etch adhesive on dentin surface significantly increased after scrubbing process on thick smear layer. the scanning electron microscopy analysis showed that the formation of hybrid layer gradually appears in line with the scrubbing pressure and duration increment. it was expected that increasing in scrubbing pressure and duration would modified the smear layer into more qualified hybrid layer. the bond strength to dentin is not dependent on the thickness of hybrid layer but it depends on the quality of the hybrid layer.16 the important thing in the application of self-adhering flowable composite is the scrubbing process. this process will accelerate the evaporation of solvent contained in adhesive material and increase the diffusion potential of monomer into the dentinal tissue. it will also increase the bond strength of adhesive materials to dentin surface.17,18 this study showed that higher scrubbing pressure will increase the shear bond strength of self-adhering flowable composite to dentin surface. scrubbing pressure will increase the kinetic energy so that increase the monomer diffusion. scrubbing pressure caused the collagen tissues to collapse but when the pressure was released, the collagen will relapse and at that time the monomer will penetrate between the collagen tissues and form the hybrid layer.19 this study also showed that longer scrubbing process increased the shear bond strength of self-adhering flowable composite to dentin surface. longer in scrubbing duration will increase the demineralization potential of phosphate group contained in gpdm and increase the penetration of monomer into the dentinal tissue. zohairy8 explained that longer contact duration of adhesive materials will increase the bond strength to dentin. self-adhering flowable composite has higher viscosity than self-etch adhesive because of filler content. the higher filler content will increase viscosity which can reduce wettability and penetration into dentinal collagen. adhesive materials should have good wettability and low molecular weight to obtain optimal bond strength to dentin surface,20 but self-adhering flowable composite does not have these properties although the scrubbing technique has been used. therefore, self-adhering flowable composite as an adhesive material or a restorative material without separate bonding agent need to be considered. it can be concluded that increasing the scrubbing pressure and duration will increase the shear bond strength of self adhering flowable composite resin to dentin surface. meanwhile the highest shear bond strength was obtained when the applicator in 0º relative to dentin surface during scrubbing process. acknowledgements this study was supported by research grant desentralization dikti dipa ui, 2012. references 1. soderholm kj, guelmann m, bimstein e. shear bond strength of one 4th and two 7th generation bonding agents when used by operations with different bonding experience. j adhes dent 2005; 7(1): 57–64. 171jaya, et al.: shear bond strength of self-adhering 2. kerr corporation (unpublished, 2010) kerr product manual dyad flow self-adhering flowable composite. lit no. 35017. 3. vanajasan pp, dhakshinamoorthy m, rao cs. factors affecting the bond strength of self-etch adhesive: a meta analysis of literature. j conserv dent2011; 14(1): 62–7. 4. pupo ym, michel md, gomes omm, lepienski cm, gomes jc. effect of the regional variability of dentinal substrate and mode application of adhesive systems on the mechanical properties of the adhesive layer. j conserv dent 2012; 15(2): 132–6. 5. miyazaki, platt j, onose h, moore b. influence of dentin primer application methods on dentin bond strength. oper dent 1996; 21(4): 167–72. 6. mark a l, william t n, charles f s. bond strength of composite to dentin and enamel using self-etching adhesive system. general dentistry 2009; 57(3): 257–9 7. nakabayashi n, pashley dh. hybridization of dental hard tissues. tokyo: quintessence; 1998; p. 17–82. 8. zohairy aae, gee ajd, mohsen mm, feilzer aj. effect of conditioning time of self-etching primers on dentin bond strength of three adhesive resin cements. j dent mat 2005; 21: 83–93. 9. vichi a gc, ferrari m. clinical study of the self-adhering flowable composite resin vertise flow in class i restoration; six month followup. international dentistry sa. 2009; 12(1): 14–23. 10. al qahtani mq, al shethri se. shear bond strength of one-step self-etch adhesives with different co-solvent ingredients to dry or moist dentin. the saudi dental j 2010; 22(4): 171–5. 11. vinay s, shivanna v. comparative evaluation of microleakage of fifth, sixth, and seventh generation dentin bonding agent. jcd 2010; 13(3): 136–40. 12. tay f, carvalho r, sano h,. dp. effect of smear layers on the bonding of a self-etching primer to dentin. j adhes dent 2000; 2(2): 99–116. 13. kenshima s, francci c, reis a, loquercio ad, filho ler. conditioning effect on dentin, resin tags and hybrid layer of different acidity self-etch adhesives applied to thick and thin smear layer. j dent 2006 nov; 34(10): 775–83. 14. christian g. self-etching primers are here. j am dent assoc 2001; 132: 1041–3. 15. chan k, tay f, king n, imazato s, pashley d. bonding of mild self-etching primers/adhesives to dentin with thick smear layers. am j sent 2003; 16(5): 340–6. 16. albaladejo a, osorio r, toledano m, ferrari m. hybrid layers of etch-and-rinse versus self-etching adhesive systems. med oral patol oral ci bucal 2010; 15(1): 112–8. 17. bianco k, pellizzaro a, patzlaft r. effects of moisture degree and rubbing action on the immediate resin-dentin bond strength. dental material 2006; 22(12): 1150–6. 18. bansal s, pandit i, srivastava n, gugnani n. technique-sensitivity of dentin-bonding agent application: the effect on shear bond strength using one-step self-etch adhesive in primary molar: an in vitro study. j indian soc pedod prev dent 2010; 28(3): 183–88. 19. reis a, pellizzaro a, dal-bianco k. impact of adhesive application to wet and dry dentin on long-term resin-dentin bond strengths. oper dent 2007; 32(4): 380-7. 20. powers jm, sakaguchi rl. craig's restorative dental materials. 12th ed. london: elsevier; 2006. p. 213–26. vol 38 no 2-2005 91 determination of fluoride in black, green and herbal teas by ionselective electrode using a standard-addition method mochammad yuwono department of pharmaceutical chemistry faculty of pharmacy, airlangga university surabaya indonesia abstract tea leaves are very rich in fluoride, since tea plants take up fluoride from the soil and accumulate in its leaves. some of this fluoride is released into the infusion, which is drunk as tea. fluoride in tea could be beneficial for the prevention of dental caries, but it may result in excessive intake and lead to enamel fluorosis. the purpose of this work was to determine the fluoride levels in 12 different brands and types of tea by means of a computer-controlled ion-selective electrode potentiometry using a standard-addition method. it is a rapid method which showed good accuracy and precision. fluoride contents of tea infusions after 5 min ranged from 0.95 to 4.73 mg/l for black teas; from 0.70 to 1.00 mg/l for green teas, and from 0.26 to 0.27 mg/l for herbal teas. it was concluded that black teas and green teas examined may be important contributors to the total daily fluoride intake. however, the ingestion of some black teas that were found to have high fluoride content by children at the age of risk to dental fluorosis should be avoided. key words: fluoride, tea, ion-selective electrode, potentiometry korespondensi (correspondence): mochammad yuwono, c/o: laboratorium analisis farmasi, fakultas farmasi universitas airlangga. jln. dharmawangsa dalam, surabaya 60286 indonesia. e-mail: mochammadyuwono@hotmail.com introduction fluoride is the anionic form of fluorine. it is an important anion, since a small amount of fluoride has beneficial effects on the teeth by reducing the incidence of caries.1 fluoride interacts with hydroxyapatite by replacing the hydroxyl ions to form a new more crystalline phase. this phase, fluoroapatite, is more resistant to errosion by plaque acid and demonstrates a lower surface energy thus making plaque adhesion more difficult.1,2 fluoride may also increase the rate of enamel remineralization, so that calcium and phosphate ions are protected and not lost during demineralization. moreover, fluoride may reduce oral concentration of cariogenic bacteria or reduce the metabolism of bacteria in plaque.1,3 for this reason, the intake of fluoride is needed to promote good dental health. in some countries fluoridated drinking water seems to be the main dietary source of fluoride, and the optimal amount of fluoride for prevention of dental caries has been suggested as 1.0 ppm. tea is also considered a major source of fluoride, since tea plants camellia sinensis take up fluoride from the soil and accumulate in its leaves. epidemiological surveys have reported that some populations who drink tea on a regular basis have a reduced number of carious teeth.4–6 due to the fluoride content, tea should be an effective vehicle for delivering fluoride to the oral cavity helping to prevent dental decay. during recent years several papers have been published on the fluoride content of tea leaves.7–10 however, the results obtained are often in poor agreement. the accuracy and the precision of the analytical method used may give the contribution on the variation of the results. feldheim and miehe11 reported the total fluoride content in tea leaves to range from 40 to 330 ppm. fung et al.12 reported that the highest fluoride contents are found in fallen leaves. fluoride accumulated in old leaves over 2000 mg/kg and young leaves ranged from 250 to 300 mg/kg. some of this fluoride is released into the infusion, which is drunk as tea.4,5 chan and koh13 reported the fluoride concentration in caffeinated and decaffeinated tea infusion ranged from 0.34 to 3.71 ppm and 1.01 to 5.20 mg/l, respectively. based on the reported fluoride content from black teas, the benefits of fluoride in tea especially for children have been debated. some authors agree that fluoride in tea is beneficial since it could account for a significant portion of total dietary fluoride intake. however, the fluoride in tea may result in excessive intake and lead to enamel fluorosis.1,4 fluoride may cause mottled teeth at around 1 ppm when it is present in water. for that reason, the determination of fluoride content as a trace element in tea infusions is important in order to assess any possible health hazards. determination of fluoride in tea is usually carried out by direct potentiometric methods using an ion-selective electrode. the calculation of the results is obtained using a calibration curve, which shows that the electrode potential is linear to the logarithmic of the ionic acitivity. it is a simple procedure without any sample preparation for measuring aqueous samples. however, any problems appear when the sample matrix is complex, such as tea that contains polysaccharides, volatile oils, vitamins, 92 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 91–95 minerals, purines, alkaloids, and polyphenols. in this case, the electrode potential is often not linear to the logarithmic of the ionic acitivity due to the interfering molecules and the fluoride contents may be found to be somewhat lower than the levels in real samples.14,15 to overcome this problem, a standard-addition method should give good accuracy and precision.16 therefore, the purpose of this work was to develop an accurate method for the determination of fluoride in infusions of black, green and herbal teas by ion-selective electrode using a computercontrolled standard-addition technique. material and methods the analytical method used in the present work incorporates a fluoride ion-selective electrode (metrohm ag, switzerland). the membrane of the electrode is lanthanum fluoride doped with europeum fluoride that interacts specifically with fluoride ion in the sample, allowing the electrical potential. a double junction electrode ag/agcl (metrohm model 90–02) was used as reference electrode. for the measurement of the potentials the ion-meter with millivolt readability to ± 0.1 mv (wsa, würzburg germany) was used combined with an automatic burette (dosimat model e655) with stirrer unit model e649 and titration tube (metrohm ag). the potential values were transmitted every second to a work station.16 the mean value over a 10s period was calculated and displayed in monitor. the difference between the two mean potential values over a period of 10 s was successively monitored. the equilibrium potential was defined as the mean potential value when the potential difference decreased to within 0.10 mv. the standard fluoride solutions were made from naf supra pure reagent. tisab (total ionic strength adjustment buffer) solution contains 58.5 g of sodium chloride, 57.0 ml of glacial acetic acid, 61.5 g of sodium acetate and 5.0 g of cdta (trans-1,2-cyclohexanediamino n,n, n',n'-tetra acetic acid) which were dissolved in milliq water and diluted to 500.0 ml. ph of this solution was adjusted to 5.0–5.5 with 6 m of sodium hydroxide and diluted to 1000.0 ml with milli-q water. the solutions were stored in screw top polyethylene containers. stock solutions were made up as 500 ppm from sodium fluoride. standard solutions of 10 ppm and 100 ppm were prepared by sequential dilution of the stock solution. both standard and stock solutions were placed in polyethylene bottles. twelve different brands and types of tea (see table 2) were purchased from supermarkets in wuerzburg, germany. the samples were obtained in the form of a tea bag and fine powder. a sample bag of tea (1.75–2.25 g) or 1.0 g of fine powder was infused in about 90 ml of boiling milli-q water for 5 minute. the flask was then gently swirled and the solution was filtered using a 0.45 μm millipore filter. the solution was then diluted with milli-q water up to 100.0 ml and bottled in polyethylene containers. the sample solutions were analyzed immediately by ion-selective electrode potentiometry at 25 ° c. all measurements were done in six replicates. a 10.0 ml of sample solution was transferred to the titration tube thermostatted at (25.0 ± 0.1) ° c and mixed with 10.00 ml of tisab solution. the solution was stirred during the analysis. the potential (exo) was recorded after 3 minute. to the sample solution was then added 1/10 volume of about 10 times more concentrated, buffered natrium fluoride standard solution and the new potential (ex1) was recorded. the addition of the standard solution was repeated six times. the fluoride concentration of the sample was then calculated by gran's plot.16–18 by this computer-controlled instrument the standard addition was done in such a way that equidistant intervals of the potential steps to be measured were achieved. the analysis was performed automatically using a computer-controlled potentiometry equipped with wsa software (self developed by department of pharmacy, university of würzburg, germany). results figure 1. a typical gran's plot for the analysis of fluoride in order to investigate the accuracy and precision of the method the samples were spiked with standard solutions and then analyzed by the described method. the accuracy expressed as percent recovery was obtained by comparing the results between the fluoride found and the fluoride added. table 1 presents the results of the accuracy and precision experiment (n = 6). 93yuwono: determination of fluoride in black table 1. the accuracy and precision of the method tea fluoride added (mg/l) added fluoride found (mg/l) recovery (%) precision (rsd) hochland mischung hochland mischung china chun mee kamillentee 0.5 2.0 0.2 0.1 0.4923 1.9820 0.2020 0.1010 98.46 99.10 101.62 101.02 0.52 0.11 3.68 2.41 the effect of brewing time on the fluoride release from three different brands of tea was also studied in this work and the results are shown in figure 2. figure 2. effect of brewing time of the fluoride release from tea (n = 6). the amount of fluoride in 12 brands and type of tea expressed as mg/ml and μg/g sample is summarized in table 2. discussion tea is a popular beverage, which is made from the leaves of the plant species, camellia sinensis. black tea is produced from leaves that are withered, rolled, fermented and dried. when the withered leaves are steamed and rolled before the process of drying and firing to prevent the fermentation of the leaves, the product is called green tea. recently, herbal tea is also available that is simply the combination of boiling water and dried fruit, flower or herb. the fluoride content in three different kinds of tea was presented in the present work. the method used for the determination of fluoride was a potentiometry using an ion-selective electrode. the electrode responds selectively to fluoride ion in the presense of other ions due to the crystal of laf3 doped with euf2 as membrane of the electrode. the analysis was done automatically using a computer-controlled potentiometry for providing the gran's plot and calculating the result, which is based on known additions of standard fluoride ion. the values of unknown fluoride concentration can be obtained by gran's plot, in which the practical values of ion-selective electrode slopes must be known prior or by computer-controlled, especially for the multiple standard-addition. when all points of the gran's plot lie on the straight line, the intercept on the abscissa yields the concentration of the unknown sample16 (see figure 1). as shown in table 1, the accuracy of the method calculated as recovery of the standard fluoride found in three different brands of teas ranged from 98.46 to 101.62%, while the precision of the method expressed by rsd (relative standard deviation) was between 0.11 and 3.68%. the results showed that the described method demonstrated a good accuracy and precision. with increase table 2. fluoride content in tea infusions, expressed in μg/l and μg/g sample fluoride content variety of tea form weight (g) mg/l μg/g black tea darjeeling tea ceylon hochland assam nr. 4 (148) lipton-best english ceylon-indien mischung ceylon-assam mischung darjeeling, messmer green tea japan sencha china chun mee nr. 24 herbal tea pfefferminze hagebuttentee kamillentee open bag open bag bag bag bag open open bag bag bag 1.00 1.75 1.04 1.75 2.25 2.25 1.75 1.50 1.06 1.75 2.85 1.50 0.95 ± 0.07 3.91 ± 0.05 1.09 ± 0.01 4.73 ± 0.15 4.43 ± 0.12 4.10 ± 0.17 1.74 ± 0.07 0.70 ± 0.02 1.00 ± 0.07 0.27 ± 0.00 0.26 ± 0.00 0.27 ± 0.01 93.50 ± 7.17 223.66 ± 2.92 104.45 ± 9.50 270.23 ± 8.45 252.97 ± 6.57 234.37 ± 9.87 99.41 ± 3.86 78.11 ± 0.77 51.40 ± 0.93 15.60 ± 0.15 9.08 ± 0.30 18.16 ± 0.57 94 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 91–95 in length of infusion time there was steady, slight increase in fluoride release from black and green teas. the herbal tea showed a constant fluoride release (see figure 2). the usual practice of tea preparation is to infuse tea for 5 min, which produces the best flavor with a little extraction of tannin. for this reason, the infusion time of the sample for 5 min was used in the present work. the fluoride concentration of tea infusions prepared from black teas ranged from 0.95 to 4.73 mg/l. the fluoride contents of infusion prepared from green tea and herbal tea ranged from 0.70 to 1.00 mg/l and 0.26 to 0.27 mg/l, respectively. the fluoride contents expressed as μg/g of sample were found to be (93.50–270.23) μg/g for black tea, (51.40– 78.11) μg/g for green tea and (9.08–18.16) ìg/g for herbal teas. the values resulted from the present study are comparable to those from previous reports.7,8,13 results obtained in this study showed that depending on the brand, the kind of tea, the fluoride content in tea infusions may vary over severalfold. the variation may be due to the difference of the sample sources such as leaf age, maturity and genetics of the plant, type of soil etc. the low fluoride content of herbal tea is a reflection of its ingredient not from regular tea but from herb, flowers or fruit. it seems that most plants are found to be poor sources of fluoride, even when grown in fluoride-rich soils. tea plants, which grow in relatively acidic soils, are exceptional for taking up fluoride. from the present study it is evident that black and green tea infusions are rich in fluoride. the mean fluoride content from black tea is higher than of green tea. drinking water appears to be the main dietary source of fluoride. the optimal of fluoride for prevention of dental caries in fluoridated drinking water has been suggested as 1.0 ppm. in extremely hot climate, recommended fluoride concentration is as high as 0.8 ppm whilst in cold climate, a concentration of 1.2 ppm is recommended.19,20 on other hand, mottled teeth have occurred where the water supply is above 1.0 ppm21 or above 1.5 ppm.19 this is possibly caused by an inadequate diet or by the inclusion in the diet of some contributor of fluoride, such as fish or tea. a survey performed in india22 reported that mildest grade of mottling in 10% of the population was reported due to the constant use of fluoridated water to the extent of 1 ppm. as the fluoride level increases, the effects get worse. the mildest grade of mottling in 100% of the population was observed when the fluoride concentration reached 6 ppm. the optimum fluoride intake in human is 0.07mg/kg body weight/day.19,23 based on the fluoride content, the amount of fluoride in black tea and green tea may significantly contribute to the daily fluoride intake. if it is considered that a black tea bag containing 1.75–2.25 g of finely powdered black tea is brewed with 250 ml of boiled milli-q water for 5 minutes, the fluoride content varies from 0.4 mg to 1.18 mg. this may be no great harm for human adults when just one or two tea bags are consumed per day. some concern has been raised over excessive intakes of fluoride from black tea infusion that contains high fluoride content such as lipton-best english. the ingestion of some black teas that were found to have high fluoride content by children at the age of risk to dental fluorosis should be avoided. for example, the tolerable upper limits (ul) for children aged one to three years is 1.3 mg fluoride. in this case, two cups of a black tea consumed just one a day may provide over the upper limit of the ranges of estimated believed to be associated with risk of enamel fluorosis. in addition, the level of fluoride may be much higher, when other fluoride-containing food, beverage or toothpastes are consumed. concerning with any possible health hazards especially for children while still growing, the fluoride content in black tea products should be informed by tea manufacturing companies on their labels of the products. it was concluded that fluoride contents of tea infusions after 5 min ranged from 0.95 to 4.73 mg/l for black teas, from 0.70 to 1.00 mg/l for green teas, and from 0.26 to 0.27 mg/l for herbal teas. black teas and green teas examined in this study may be important contributors to the total daily fluoride intake. however, the ingestion of some black teas that were found to have high fluoride content by children at the age of risk to dental fluorosis should be avoided. acknowlegment this work was part of dissertation of m. yuwono, university of würzburg, germany; supervisor: prof. dr. s. ebel. the author would like to thank prof. dr. gunawan indrayanto for his valuable comments of the manuscript. references 1. mutschler e. arzneimittelwirkungen-lehrbuch der pharmakologie und toxikologie. 7 th ed. stuttgart: wissenschaftliche verlagsgesellschaft; 1997. p. 622–32. 2. lehmann km, hellwig e. einführung in die restaurative zahnheilkunde. 8th ed. münchen: urban & schwarzenberg; 1998. p. 133. 3. hellwig e. fluorid: chemie und biochemie. dtsch zahnaerzl z 1996; 51: 649–53. 4. schmidt cw. fluoride-schwarzer tee-gesundheit. zärztl fortbild 1984; 78: 905–7. 5. berlitz hd, grosch w. 1982. lehrbuch der lebensmittelchemie. heidelberg: springer-verlag; p. 214–6. 6. waldbott gl. fluoride in food. am j clin nutrit 1963; 12: 144–6. 7. farsam h, ahmad n. fluorine content of teas consumed in iran. journal of food science 1978; 43: 274–5. 8. harrison mf. fluorine content of tea consumed in new zealand. brit j nutrit 1978; 3: 162–6. 9. pres maf, dantas esk, munita cs. fluoride content of some teas consumed in sao paolo. fluoride 1996; 29: 144–6. 10. strübig w, gülzow. fluoridgehalt verschiedener teesorten. dtsch zahnärztl z 1981; 36: 379–81. 11. feldheim w, miehe so. fluoridgehalt in teeblättern. z lebensm unters forsch 1979; 169: 453–6. 12. fung k, zhang z, wong j, wong m. fluoride contents in tea and soil from tea plantations and the release of fluoride into tea liquor during infusion. environ pollut 1999; 104: 197–205. 95yuwono: determination of fluoride in black 13. chan jt, koh sh. fluoride content in caffeinated, decaffeinated and herbal teas. caries research 1996; 30: 88–92. 14. ebel s, parzefall w. experimentelle einführung in die potentiometrie. weinheim: verlag chemie; 1975. p. 24–52. 15. oehme f. ionselektive elektroden. git fachz lab 1988; 8: 854– 63. 16. ebel s, becht u. rechnergesteuerte multiple standard-zumischmethode beim arbeiten mit ionensensitiven elektroden. fresenius z anal chem 1985; 320: 117–20. 17. phillips ka, rix cj. microprocessor-controlled determination of fluoride in the presence of magnesium and calcium in sea water and blood plasma by a fluoride-selective electrode with standard addition. anal chim acta 1985; 169: 263–71. 18. liberti a, maschini m. anion determination with ion selective electrodes using gran’s plot. anal chem 1969; 41: 677–9. 19. world health organization. water sanitation and health (wsh). available at: http:// www.who.int/water_sanitation_health/ naturalhazards/en/index2.html. accessed april 2005. 20. who. fluorine and fluorides in environmental health criteria. se2005,ries 36, geneva: world health organization; 1984. p. 25–26. 21. katzung bg. basic and clinical pharmacology. 9th ed. usa: prentice hall international; 1998. p. 712. 22. ahuja b. a novel approach for treating fluorosis stains. available at: http://www.healthmantra.com/ypb/apr01/fluorosis.htm. accesed 2005. 23. burt ba. the changing patterns of systemic fluoride intake. j dent res 1992; 71: 1228–37. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2525 biological changes after dental panoramic exposure: conventional versus digital rurie ratna shantiningsih and silviana farrah diba department of dentomaxillofacial radiology faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: dental digital radiography is more practical and requires fewer doses of radiation than conventional radiography. because ionizing radiation has a biological effect on exposed tissue, concerns regarding its stochastic effect merit greater attention. in a previous study, it was found that biological changes and increases in the micronucleus occurred after conventional panoramic exposure to gingival crevicular fluid (gcf). purpose: the purpose of this study is to investigate the difference in biological effects after digital panoramic exposure compared with conventional exposure. methods: twenty subjects were classified into two groups according to the radiographic technique employed. the techniques consisted of ten subjects undergoing digital panoramic radiograph exposure and ten others being subjected to conventional exposure. gcf calculated in mm3 was collected by applying paper strips to the anterior maxillary labial gingival sulcus for one minute prior to and ten minutes after exposure. a micronucleus was obtained from a gingival smear on the same quadrant ten days after panoramic exposure. results: there was a significant difference in the number of micronucleus between conventional and digital panoramic radiographs both before and after exposure (p=0.000). in contrast, increased gcf volume was not statistically significant (p=0.506) before or after digital panoramic exposure, while the significant difference of conventional panoramic exposure was p=0.017. conclusion: digital panoramic radiograph exposure induced a biological change only in terms of an increase in the number of micronucleus but not in the volume of gcf. keywords: digital panoramic; gingival crevicular fluid; micronucleus correspondence: rurie ratna shantiningsih, department of dentomaxillofacial radiology, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, yogyakarta 55281. e-mail: rr_shantin@yahoo.com research report introduction dental digital radiography is gaining rapidly in popularity. the advantages of using dental digital radiography include: optimization of image contrast and sharpness, ease of processing, less prolonged storage and the enhanced practicality of carrying out diagnostic procedures.1 digital radiographs can reduce the effective dose by up to 50% compared with conventional radiography.2 this attempt represents one radiation protection procedure because, although dental radiography administers extremely limited doses, it is not completely without risk.3 theoretically, the biological effects of dental panoramic exposure can damage cells through oxidative reaction, including the formation of micronucleus.4,5 a micronucleus is often employed as a biomarker of chromosomal damage that, in its early stages, contributes to carcinogenesis.6 research has confirmed the increase in micronucleus after conventional panoramic radiograph exposure which has a correlation with 8-oxo-dg expression as a marker of dna adduct.7 in addition to the micronucleus, the volume of gingival crevicular fluid (gcf) also increases after conventional panoramic radiograph exposure. a previous study confirmed a significant increase in the volume of a patient’s gcf due to conventional panoramic radiograph exposure.8 the increase in gcf volume is related to the enhanced permeability of the blood vessels after exposure to radiography. the findings of these studies indicate the incidence of both cytogenetic and genetic damage due to dental journal (majalah kedokteran gigi) 2018 march; 51(1): 25–28 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p25–28 mailto:rr_shantin@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p25-28 26 shantiningsih and diba/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 25–28 panoramic radiograph exposure. the sulcular epithelial of the oral mucosa produces gcf which contains plasma protein and has antimicrobial properties. conditions that increase the production of gcf include: rough food mastication, brushing of the teeth, gingival massage, smoking, periodontal disease and contraceptive use. the amount of gcf also increases in correlation with the severity of inflammation.9 endothelial vascular cells are radiosensitive to x-rays whose effect on vascular permeability constitutes an early symptom of ionizing radiation toxicity.10 vascular permeability increases in normal tissue immediately after radiation before gradually decreasing after two to three days.11 increased gcf volume may occur due to enhanced vascular permeability in the inflammatory state. plasma fluids leaving the blood vessels are released into the gingival crevicular region through the junctional epithelium.12 because of the numerous risks of conventional panoramic radiograph exposure, it is also necessary to conduct research on its effects, especially in the form of an increase in the number of micronucleus in the gingival mucosa and gcf using digital panoramic radiography. the purpose of this study is to investigate the different biological effects after digital panoramic exposure compared with conventional treatment. material and methods sampling was conducted randomly and included an experimental and control group. the research was conducted at the dentomaxillofacial radiology laboratory, faculty of dentistry, universitas gadjah mada after ethical clearance from the faculty of dentistry universitas gadjah mada (no.001099/kkep/fkg-ugm/ec/2017) had been obtained. the subjects were patients who, on the basis of his/her dentist’s recommendation, underwent dental panoramic digital or conventional radiography. informed consent was obtained from willing participants whose suitability for this study was based on the inclusion criteria. preferred subjects were those free from systemic disease, who were able to return for a gingival mucosa smear on day 10, had not been exposed to x-rays during the 14 days prior to the start of the study, were gingivitis-free at the research site, did not smoke and were not alcoholics. two groups participated in this study, one undergoing digital panoramic radiography and the other conventional radiography, each consisting of ten subjects selected by purposive sampling technique (referred to in the previous study).8 the volume of gcf on the labial anterior maxillary around teeth 12 to 22 was measured both prior to and ten minutes after exposure. paper strips were inserted into the labial gingival sulcus for one minute, before a drop of 2% ninhydrin solution was added. after the paper had turned purple, measurements were taken to quantify the volume of gcf in mm3.8 analysis of the increase in the number of micronucleus was conducted by swabbing the anterior gingival mucosa using a cervical brush and subsequently smearing the material obtained onto a microscope slide. the swabbed cells subjected to micronucleus analysis had been obtained from the same region as the gcf whose volume was measured. the swabbed cells were stained with schiff’s reagent for 90 minutes following the feulgen-rossenbeck method and a counter stain was performed with 1% fastgreen for one minute. the micronucleus were examined under a light microscope at 400x magnification. a micronucleus was defined as a cell having an additional nucleus around the main nucleus, while possessing the same colour and a diameter approximately 1/3 that of the main nucleus. based on the results of a saphiro wilk test, the normally distributed data was further analyzed by means of an independent t-test to examine the difference in the number of micronucleus on the exposed gingival mucosa between conventional and digital panoramic techniques. paired t-tests were conducted to analyze the data relating to gcf volume both before and after digital and conventional panoramic exposure. results the results of this study confirmed the mean number of micronucleus before conventional panoramic radiography exposure as 0.40 ±0.15, increasing post-exposure to 12.4±3.08. on the other hand, the mean number of micronucleus pre-digital exposure was 0.60±0.23 and post-exposure 7±1.78 (figure 1). these numbers confirmed the mean of the increase in micronucleus to be higher in conventional panoramic radiography exposure than digital exposure. the micronucleus presented in figure 2 possess a smaller extranucleus around the main nucleus and similar staining to the main nucleus. the appearance of micronucleus as the result of digital and conventional panoramic radiography has a similar character. the volume of gcf was measured preand postexposure by means of both digital panoramic radiography figure 1. the differences mean of micronucleus increasing between digital and conventional panoramic radiography at day 10 after exposure. digital before after conventional dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p25–28 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p25-28 27shantiningsih and diba/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 25–28 exposure and conventional technique. there was a higher increase in the volume of gcf post-digital panoramic exposure with a mean of 0.246±0.050 compared with pre-exposure (0.233±0.052). similar to this result, a conventional panoramic technique showed a higher increase post-exposure (0.321±0.067), compared to pre-exposure (0.235±0.047) (figure 3). a statistical analysis of the number of micronucleus by means of paired t-test preand post-conventional and digital panoramic exposure revealed significant differences (table 1). moreover, an independent t-test of the differences in the micronucleus increasing between digital and conventional panoramic radiography is shown in table 2 indicates a significant difference (p=0.000) between digital and conventional panoramic radiographic exposures. a statistical analysis of gcf volume preand postexposure by using paired t-test was conducted. table 3 shows that there were significant differences in gcf volumes before and after conventional panoramic exposure (p<0.05). in contrast, the gcf volumes before and after digital panoramic exposure were not significantly different (p>0.05). table 4 shows that there was a significant difference in gcf volume (p<0.05) between the number of digital and conventional panoramic exposures. discussion in this study, the pre-exposure number of micronucleus was considerably lower than the post-exposure number in both digital and conventional panoramic radiography. the post-exposure increase in micronucleus was lower in patients undergoing digital panoramic exposure than those of conventional ones (figure 1). the micronucleus detected pre-exposure represented the positive control because they had been swabbed without exposure. the micronucleus figure 2. a representative cell with micronucleus after panoramic radiography exposure (arrow). table 1. paired t-test analysis before and after penoramic exposure in digital panoramic. parameters micronucleus number conventional digital std error mean sig. std error mean sig. before 0.163 0.000* 0.221 0.000 after 1.002 0.526 *significant difference <0.05 figure 3. the means of gcf volume between before and after digital and conventional panoramic radiography exposure. table 2. the result of independent t-test of micronucleus increasing between digital and conventional panoramic radiography. micronucleus number n std.error mean f sig. digital 10 1.0022 0.545 0.000* conventional 10 0.6110 * significant difference <0.05 table 3. paired t-test analysis before and after penoramic exposure in digital panoramic. parameters gcf volume conventional digital std error mean sig. std error mean sig. before 0.1836 0.017* 0.1488 0.506 after 0.1632 0.2114 * significant difference <0.05 table 4. independent t-test analysis between gcf volume of digital and panoramic exposure. csg volume n std.error mean f sig. digital 10 0.5162 1.572 0.012* conventional 10 0.6686 * significant difference <0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p25–28 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p25-28 28 shantiningsih and diba/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 25–28 in the digital and conventional panoramic radiography appeared with smaller extranuclei, approximately 1/3 the size of the main nucleus with which they appeared same colour (figure 2). it is believed that digital radiography can reduce the effective dose by up to 50% compared with conventional radiography. therefore, it is assumed that the effect of digital radiography will decrease.6 this result was in line with the statistical analysis results (table 1 and 2) that showed the significant differences (p<0.05) between both groups and might be related to the theory that digital radiography techniques constitute an effort to reduce dose exposure, compared with conventional techniques.2 since image receptors in digital techniques are more sensitive to x-rays, this allows a reduction in the dose administered to patients.2 the volume of gcf increased when both techniques were used, although more so with conventional radiography exposure than digital exposure (figure 3). a paired t-test only produced a significant difference (p<0,05) between preand post-panoramic radiography exposure with a conventional technique (table 3.). the samples of the pre-exposure group represented the control because they had not yet been exposed to x-ray. the results indicated that the effects of digital panoramic radiography exposure still cause cytotoxic and genotoxic damage, although they do not increase blood vessel permeability. while digital radiography is believed to produce lower doses of radiation protection, there is still a significant increase in micronucleus between the preand post-digital panoramic exposure. in this study, the increasing number of micronucleus along with a greater volume of gcf due to digital panoramic exposure was confirmed. the result of an independent t-test indicated that the gcf volume between conventional and digital panoramic exposure differed significantly (p<0.05) (table 4). digital radiography exposure is estimated to reduce the effective dose by up to 50% compared to conventional radiography, thereby avoiding dilatation of the capillary blood vessels in the gingival mucosa.2 the volume of cgf in cancer patients was found to be statistically higher after they had undergone head and neck radiotherapy.13 similiarly, a recent study conducted by zuelkevin indicated a significant increase in gcf volume after conventional panoramic radiography exposure.8 blood vessels consist of x-ray radiosensitive cells so that panoramic exposure will increase vascular capillary permeability.10 vascular plexus that secretes gcf is very sensitive to various types of stimulants.14 these results confirm that the effects of digital and conventional panoramic radiography exposure are stochastic and there is no dose-limiting value.3 thus, the use of radiography exposure should be noted and guided by the principle that radiological protection should be as low as reasonably achievable (alara). an extremely limited dose does not mean a complete absence of effect in exposed patients.3 dental digital radiography could reduce dose exposure, but still trigger cytotoxicity due to dna damage. this was proved by the formation of markers related to the early mechanism of carcinogenesis in the form of an increase in the micronucleus. based on this study, it was found that the increase in the number of micronucleus and the volume of gcf identified through conventional panoramic radiography was significantly different to that established through digital panoramic radiography. the conclusion of this study is that digital panoramic radiography exposure induced biological change only in terms of an increase in the micronucleus, but not in the volume of gcf. acknowledgement the author would like to thank the “dana masyarakat” of universitas gadjah mada for research funding. references 1. dhir p, david cm, keerthi g, sharma v, girdhar v. digital imaging in dentistry: an overview. ijdms. 2011; 23(6): 62–8. 2. diwakar nr, kamakshi ss. recent advancements in dental digital radiography. j med radiol pathol surg. 2015; 1: 11–6. 3. whaites e, drage n. essentials of dental radiography and radiology. 5th ed. china: churchill livingstone; 2013. p. 68–72. 4. iannucci j, howerton lj. dental radiography : principles and techniques. 4th ed. st. louis: saunders; 2011. p. 35, 152-3, 256-7. 5. ribeiro da. cytogenetic biomonitoring in oral mucosa cells following dental x-ray. dentomaxillofacial radiol. 2012; 41(3): 181–4. 6. cerqueira emm, meireles jrc, lopes ma, junqueira vc, gomesfilho is, trindade s, machado-santelli gm. genotoxic effects of x-rays on keratinized mucosa cells during panoramic dental radiography. dentomaxillofacial radiol. 2008; 37(7): 398–403. 7. shantiningsih rr, suwaldi s, astuti i, mudjosemedi m. korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg akibat paparan radiografi panoramic (the correlation of micronucleus formation and 8-oxo-dg expression due to the panoramic radiography exposure). dent j (maj ked gigi). 2013; 46(3): 119–23. 8. zuelkevin z. efek paparan radiografi panoramik terhadap volume cairan sulkus gingiva (csg). thesis. yogyakarta: universitas gadjah mada; 2015. p. 29–36. 9. talwar gp, hasnaian se, sarin s kumar. textbook of biochemistry, biotechnology, allied and molecular medicine. 4th ed. new delhi: prentice-hall of india; 2016. p. 416. 10. white sc, pharoah mj. oral radiology : principles and interpretation. 7th ed. missouri: mosby; 2013. p. 32–6. 11. park hj, griffin rj, hui s, levitt sh, song cw. radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (sbrt and srs). radiat res. 2012; 177(3): 311–27. 12. barros sp, williams r, offenbacher s, morelli t. gingival crevicular fluid as a source of biomarkers for periodontitis. periodontol 2000. 2016; 70(1): 53–64. 13. mardhiyah i. perubahan volume cairan sulkus gingiva pada penderita kanker kepala dan leher yang menjalani radioterapi. thesis. yogyakarta: universitas gadjah mada; 2010. p. 22–5. 14. berkovitz bkb, moxham bj, linden rwa, sloan aj. master dentistry volume 3 oral biology: oral anatomy, histology, physiology and biochemistry. china: churchill livingstone; 2010. p. 235–50. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p25–28 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p25-28 158 volume 46, number 3, september 2013 kadar leptin saliva dan kejadian karies gigi anak obesitas (salivary leptin levels and caries incidence in obese children) elfrida atzmaryanni dan mochamad fahlevi rizal departemen ilmu kedokteran gigi anak fakultas kedokteran gigi, universitas indonesia jakarta – indonesia abstract background: children with obesity have a lower incidence of caries. salivary leptin levels of obese children is higher than normal children. leptin is protein hormone, contained in saliva. salivary proteins maintain the balance of the ecosystem in the mouth. purpose: the article was aimed to study the correlation of salivary leptin levels with caries incidence in obese children. review: mouth is reflection of the health status and so many changes occur as a weight gain. child with obesity has a low incidence of caries than normal. this condition is associated with changes in oral cavity, especially the increase in salivary leptin. caries is a disease of hard tissues cause by the activty of microorganisms, especially streptococcus mutans. salivary proteins maintain the balance of the ecosystem in the mouth. leptin is a protein saliva, produced predominantly in adipose tissue and conduct active transport to saliva. salivary leptin works in two ways: as an antimicrobial which prevents the attachment of bacteria on tooth surface or by inducing cytokine that affect the immune system in oral cavity. conclusion: salivary leptin is higher in obese children than in normal children. the low incidence of caries on obesity is associated with salivary leptin. alteration in salivary composition and flow rate also decreased caries in obesity. key words: obesity, salivary leptin, dental caries, children abstrak latar belakang: anak yang mengalami obesitas memiliki insiden karies yang rendah. kadar leptin saliva anak obesitas lebih tinggi dari anak normal. leptin merupakan salah satu protein hormon yang terdapat di saliva. protein saliva berfungsi untuk menjaga keseimbangan ekosistem di mulut. tujuan: artikel ini bertujuan mempelajari hubungan antara kadar leptin di dalam saliva dengan kejadian karies anak obesitas. tinjauan pustaka: rongga mulut merupakan cerminan dari status kesehatan dan banyak perubahan yang terjadi seiring peningkatan berat badan seseorang. anak obesitas memiliki insiden karies yang rendah jika dibandingkan anak normal. kondisi ini berhubungan dengan perubahan keadaan rongga mulut terutama peningkatan kadar leptin di saliva. karies adalah penyakit jaringan keras yang disebabkan oleh aktivitas dari mikroorganisme, terutama streptococcus mutans. protein di saliva berfungsi menjaga ekosistem rongga mulut. leptin merupakan protein saliva, leptin terutama di sintesis pada sel adiposa dan melakukan transport aktif sehingga dapat ditemukan di saliva. leptin di saliva bekerja dengan dua cara yaitu sebagai antimikroba yang mencegah perlekatan bakteri di permukaan gigi atau dengan cara menginduksi sitokin yang mempengaruhi sistem imun dalam rongga mulut. simpulan: kadar leptin dalam saliva anak obesitas lebih tinggi dibanding anak normal. rendahnya insiden karies anak obesitas berhubungan dengan kadar leptin di dalam saliva anak obesitas yang lebih tinggi dibandingkan anak normal. perubahan komposisi saliva dan laju alir saliva pada anak obesitas juga menyebabkan rendahnya insiden karies dibandingkan dengan anak normal. kata kunci: obesitas, leptin saliva, karies gigi, anak korespondensi (correspondence): elfrida atzmaryanni, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta pusat, indonesia. e-mail: frida_atz@yahoo.com literature reviews 159atzmaryanni dan rizal: kadar leptin saliva dan kejadian karies gigi anak obesitas pendahuluan leptin (ob), merupakan polipeptida dari sebuah gen obese, dengan berat molekul 16 kda. leptin berasal dari bahasa yunani leptos yang berarti kurus.1,2 leptin terletak pada kromosom no 7 manusia.2 leptin disintesis terutama oleh sel adiposa yang berfungsi sebagai regulasi berat badan, sistem hematopoetik dan remodelling tulang.1-3 reseptor utama leptin berada di hipotalamus, dan berfungsi untuk menghambat asupan makanan seta meningkatkan penggunaan energi.1,3 kadar leptin dipengaruhi oleh jumlah lemak di dalam tubuh manusia, sehingga kadar leptin akan meningkat seiring kenaikan berat badan.4-6 rongga mulut merupakan cerminan dari status kesehatan dan banyak perubahan yang terjadi di rongga mulut seiring peningkatan berat badan seseorang.4,6,7 hal ini dikarenakan rongga mulut memegang peranan utama dalam transmisi mikroorganisme dan reinfeksi penyakit pada tubuh.7,8 leptin merupakan salah satu protein hormon yang terdapat di saliva.6 saliva adalah cairan yang mengelilingi seluruh jaringan lunak dan keras di dalam rongga mulut dan dapat berperan mengurangi jumlah mikroorganisme.9,10 pada umumnya, terjadi perubahan komposisi dan jumlah saliva pada seseorang yang mengalami peningkatan berat badan.11 karies adalah penyakit jaringan keras yang disebabkan oleh aktivitas dari mikroorganisme, terutama streptococcus mutans (s. mutans) dan merupakan salah satu masalah yang sering terjadi di rongga mulut.11,12 aktivitas dari mikroorganisme ini sangat dipengaruhi oleh keadaan rongga mulut seperti diet, keadaan saliva dan giginya.12 anak yang mengalami obesitas, terlihat indeks karies yang rendah dibandingkan dengan anak yang normal.11,13,14 hal ini terjadi seiring dengan peningkatan kadar leptin di saliva.11 leptin sebagai salah satu protein hormon dapat mempengaruhi kolonisasi bakteri rongga mulut yang menyebabkan terjadinya karies.13 leptin di saliva dapat menginduksi sitokin yang dapat mencegah terjadinya proses karies di rongga mulut.14 dalam makalah ini mempelajari hubungan antara kadar leptin saliva dengan kejadian karies pada anak atau anak yang mengalami obesitas. leptin di rongga mulut leptin merupakan nonglycosylated peptide yang dihasilkan oleh gen obese dan bekerja di reseptor neural pada susunan saraf pusat, yaitu di hipotalamus.1,15 leptin memegang peranan penting sebagai energi untuk homoestatis dan disintesis terutama oleh sel adiposa dan dalam kuantitas kecil dapat pula disekresi oleh plasenta, t sel, osteoblas, otot skeletal, otak, kelenjar pituitari dan gastric epithelium yang berfungsi sebagai regulasi berat badan, keseimbangan energi dan osteogenesis.3,15 modulasi fungsi fisiologis lainnya seperti regulasi neuroendokrin, reproduksi untuk mengatur ovarium, termogenesis, metabolisme lipid, sistem hematopoetik.1,3 produksi leptin distimulai oleh glukokortikoid dan regulasinya dipengaruhi oleh hormon insulin.15 kadar leptin pada manusia sangat dipengaruhi oleh berat badan, jumlah lemak dalam tubuh, jenis kelamin, masa pubertal dan testosterone.4 beberapa penelitian sebelumnya menunjukkan bahwa orang yang mempunyai berat badan berlebih biasanya mempunyai kadar leptin yang meningkat.11 hal ini disebabkan oleh resistensi leptin di dalam lemak tubuh.4 produksi leptin pada orang obesitas lebih banyak 2 kali lipat per gram, karena sel lemak pada obesitas membesar 2-4 kali daripada orang normal.5 kadar leptin dapat dipengaruhi oleh kebiasaan atau jadwal makan, di mana kadar leptin akan naik ketika kita makan yang diikuti dengan meningkatnya kadar insulin,4 sedangkan kadar leptin di serum akan menurun ketika menjelang sore hari setelah 6 jam mengkonsumsi makanan dan ketika mengkonsumsi makanan terus menerus maka kadar leptin dapat meningkat sebesar 40%.4,16 leptin selain ditemukan pada serum dan plasma, bisa juga didapatkan di rongga mulut.15 cairan di rongga mulut yang biasa digunakan untuk mengetahui kadar leptin yaitu saliva dan cairan krevikular gingiva atau gingival crevicular fluid (gcf).1,15,17 akhir-akhir ini penggunaan saliva untuk menegakkan diagnosis semakin berkembang, hal ini bisa terjadi karena sampel saliva relatif mudah untuk didapatkan pada manusia di setiap tingkatan usia dan cara penyimpanannya yang mudah.1,18 protein hormon/polipeptida biasanya tidak dapat terdeteksi di saliva kecuali disekresi pada kelenjar saliva.17 leptin ternyata bisa ditemukan dan dipisahkan dari komposisi saliva lainnya, sehingga ditemukan di saliva.1 hal ini disebabkan adanya transpor aktif dan peredaran darah ke kelenjar saliva. tetapi masih diperlukan penelitian lebih lanjut mengenai bagaimana leptin melakukan transpor aktif dari hipotalamus ke kelenjar saliva.17,19 kadar leptin pada saliva normal 6.19±2.10μg/l, sementara pada penelitian sebelumnya terlihat perbedaan kadar leptin pada pria 5.93±1.94μg/l dan pada wanita 6.47±2.29μg/l.18 sama seperti kadar leptin pada tubuh manusia, kadar leptin pada saliva juga dipengaruhi oleh berbagai faktor. faktor yang mempengaruhi bisa dari luar ataupun dari dalam. faktor dari luar seperti metode dan waktu pengambilan saliva.9,17,18 faktor dari dalam terutama karena irama tubuh dari setiap orang berbeda. irama tubuh meliputi kebiasaan makan dan hormon.15 bila saliva terstimulasi dengan asam sitrat akan menurunkan konsentrasi leptin walaupun volume saliva meningkat.2 selain terdapat di saliva, ternyata leptin dapat ditemukan juga di gcf. gingival cervicular fluid merupakan cairan yang keluar diantara permukaan gigi dan jaringan epitel, merupakan cairan kompleks mencakup serum, cairan interstisial, produk bakteri, mediator inflamasi dan enzim.1,2 leptin dapat ditemukan pada gcf walaupun tidak ada sel adiposa di gingiva yang hal ini mungkin disebabkan oleh transpor aktif leptin ke gingiva melalui pembuluh darah.1,10,11,17 obesitas obesitas merupakan suatu penyakit multifaktorial yang diduga disebabkan oleh karena interaksi antara faktor 160 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 158–161 genetik dan faktor lingkungan, antara lain aktifitas, gaya hidup, sosial ekonomi dan nutrisional.20 leptin adalah salah satu indikator biologis yang dapat digunakan untuk mengukur tingkat obesitas. individu dengan jaringan lemak yang berukuran besar akan mengandung lebih banyak leptin dibandingkan dengan jaringan lemak yang lebih kecil.21,22 terjadi perbedaan bermakna pada penelitian mengenai kadar leptin pada anak normal dan obesitas.23,24 prevalensi obesitas semakin meningkat dari tahun ke tahun, baik itu di negara maju maupun negara berkembang.20 perubahan pola makan dan gaya hidup selain berakibat ke obesitas, juga berakibat ke dalam keadaan rongga mulut. hal ini dikarenakan rongga mulut memegang peranan utama dalam transmisi mikroorganisme dan reinfeksi penyakit pada tubuh.7,8 karies karies merupakan kerusakan gigi yang progresif dari email dan dentin yang dimulai saat mikroorganisme pada permukaan gigi.12,24 karies dimulai ketika saliva dan karbohidrat menutupi permukaan gigi dan membentuk asam organik yang mengganggu mineralisasi gigi.25 proses patologis ini berkelanjutan, mulai dari demineralisasi email tingkat mikroskopik sampai kerusakan jaringan luas secara makroskopik membentuk kavitas. karies merupakan masalah yang kompleks dan multifaktorial.12,24,25 saliva berperan dalam menjaga ekosistem di dalam mulut dengan menjaga integritas jaringan lunak dan keras, melindungi jaringan lunak mulut dari infeksi bakteri,jamur dan virus.9 saliva memiliki fungsi, diantaranya untuk membasahi jaringan mukosa, kapasitas buffer dan sebagai ion reservoir yang memfasilitasi remineralisasi pada gigi.9,10 saliva mampu untuk melakukan fungsinya sebagai anti mikroba yang ada di dalam mulut karena protein tertentu. beberapa protein diantaranya mampu memodifikasi metabolisme bakteri dan mempengaruhi kemampuan bakteri menempel pada permukaan gigi.9 bakteri s. mutans merupakan penyebab utama dari karies dengan berkolonisasi pada permukaan gigi.25 bakteri s. mutans dapat menfermentasi sukrosa dan dengan cepat mengkonversi menjadi produk asam (asam laktat), secara signifikan dapat tumbuh dan berkembang pada kondisi asam. sifat asidogenik pada bakteri ini yang dapat mengawali proses terjadinya karies karena larutnya email terjadi pada kondisi ph yang rendah.26,27 bakteri s. mutans mempunyai kemampuan untuk adhesi ke permukaan gigi dan pembentukan biofilm.28 pada biofilm terdapat dua macam polisakarida yaitu polisakarida ekstraseluler yang mendukung terjadinya akumulasi bakteri di dalam gigi serta polisakarida intraseluler yang menjadi sumber karbohidrat endogen.29 peran polisakarida ekstraselular terutama glukan adalah memperkuat ikatan dan akumulasi s. mutans dan streptococcus lainnya pada permukaan gigi, memperkuat stabilitas matriks ekstraseluler yang dapat meningkatkan kepadatan biofilm, melindungi mikroorganisme dari pengaruh mikroba atau lingkungan lain dan sebagai sumber energi cadangan.30 adhesin berfungsi melekatkan s. mutans secara awal pada pelikel di permukaan gigi melalui sel reseptor saliva dan berperan dalam ko-agegrasi dengan bakteri lain.31-33glukan-binding protein merupakan faktor virulensi dari s. mutans yang bertindak sebagai mediasi pengikat glukan yang dihasilkan oleh gtf.31 pembahasan perubahan yang terjadi pada rongga mulut anak obesitas terutama terjadi pada saliva. anak obesitas memiliki total protein saliva yang lebih tinggi dibandingkan dengan anak normal. protein dapat berpengaruh terhadap perlekatan bakteri pada gigi.11,13,14 protein saliva dapat mempengaruhi dan mengontrol kolonisasi s. mutans dengan cara mengurangi adhesi awal bakteri ke permukaan gigi sekaligus menetralisir enzim ekstraseluler.11 leptin sebagai salah satu protein hormon dari saliva, dapat mencegah perlekatan antara s. mutans dengan gigi dengan menghambat polisakarida ekstraselular sehingga s. mutans tidak bisa berkembang dan menempel di permukaan gigi.2,33 aliran saliva sangat berpengaruh terhadap perlekatan bakteri ke permukaan gigi.11 laju alir saliva yang normal memberi efek perlindungan yang kuat dalam melawan karies gigi.29 peningkatan laju alir saliva akan membuat peningkatan ph dengan meningkatnya ion bikarbonat sehingga kapasitas dapar dapat optimal.11,29 protein juga sebagai efek antimikroba berfungsi mencegah kolonisasi bakteri pada gigi dan memodulasi kalsium fosfat (capo4). selain itu, anak obesitas terjadi perubahan komposisi saliva dengan kadar leptin yang meningkat dan laju alir saliva yang lebih tinggi maka dapat mencegah awalnya terjadinya perlekatan bakteri.11 leptin dapat menginduksi produksi sitokin yang mempengaruhi sistem imun dalam rongga mulut.14 leptin bekerja terutama di hipotalamus, namun juga bekerja pada jaringan lain dengan cara berikatan dengan reseptor leptin yang spesifik seperti reseptor sitokin.5 sitokin merupakan protein pembawa pesan kimiawi, atau perantara dalam komunikasi antar sel yang sangat poten.5,26 sitokin berperan sebagai autocrine hormone. protein hormon dapat masuk ke sirkulasi ini, dan dapat berinteraksi dengan sel imun, walaupun berada jauh dari tempat asalnya.26 sitokin berperan dalam aktivasi sel t, sel b, monosit, makrofag, inflamasi dan induksi sitotoksisitas. siga merupakan produk dari mucosa immune system (mis) yang terdiri dari limfosit t dan b. hambatan kolonisasi s. mutans oleh siga secara in vitro, diperkirakan karena siga menghambat kerja gtf sehingga glukan tidak terbentuk, akibatnya tidak terjadi perlekatan kuman pada mekanisme pembentukan plak gigi.20 penelitian terdahulu tentang kadar leptin selalu dikaitkan dengan anak obesitas. anak obesitas dikatakan memiliki kadar leptin yang lebih tinggi dan indeks karies yang rendah. karies disebabkan oleh multifaktoral, oleh 161atzmaryanni dan rizal: kadar leptin saliva dan kejadian karies gigi anak obesitas karena itu perlu dilakukan penelitian pada anak yang memiliki berat badan normal terhadap jumlah karies yang dialami serta kadar saliva. penelitian tersebut diharapkan dapat menjelaskan kaitan antara hubungan kadar leptin saliva dengan kejadian karies. berdasarkan pembahasan di atas dapat disimpulkan bahwa anak obesitas memiliki kadar leptin yang lebih tinggi daripada anak normal. kadar leptin saliva yang tinggi menyebabkan anak obesitas memiliki insiden karies yang rendah. leptin saliva bekerja dengan dua cara, dapat mencegah perlekatan antara s. mutans dengan gigi dengan menghambat polisakarida ekstraselular sehingga s. mutans tidak bisa berkembang dan menempel di permukaan gigi selain itu leptin dapat menginduksi produksi sitokin yang mempengaruhi sistem imun dalam rongga mulut. perubahan komposisi saliva dan peningkatan laju alir saliva pada anak obesitas juga menyebabkan rendahnya insiden karies dibandingkan dengan anak normal. daftar pustaka 1. karthikeyan bv. estimation of leptin levels in gingival crevicular fluid and serum in periodontal health and disease. disertasi. india. 2006. p. 26-30. 2. karthikeyan bv. leptin levels in gingival crevicular f luid in periodontal health and disease. j periodont res 2007; 42(40): 300-4. 3. um s, choi jr, lee jh, zhang q, seo b. effect of leptin on differentiation of human dental stem cells. j oral diseases 2011; 17(7): 662-9. 4. werner f, englaro p, hanitsch s, juul a, herte nt, muller j, niels es, mark lh, martin b, andrea m, wielan k, wolfgang r. plasma leptin levels in healthy children and adolescents: dependence on body mass index, body fat mass, gender, pubertal stage, and testosterone. j clin endocrinology and metabolism 1997; 82: 2904-10. 5. fried sk, matthew r, colleen d, blandine l. regulation of leptin production in humans. j nutrition 2000; 130: 3127-31. 6. kaufman e, lamster ib. the diagnostic application of saliva. int and am assoc for dent res 2002; 13(2): 197-212. 7. palmer ca. dental caries and obesity in children different problems, related causes. quintessence int 2005; 36(6): 457-61. 8. takashi n. microbial ecosystem in the oral cavity: metabolic diversity in an ecological niche and its relationship with oral disease. jurnal of international congress series. 2005: 1284: 103-12. 9. chiappin s, antonelli g, gatti r, de palo ef. saliva speciment: a new laboratory tool for diagnostic and basic investigation. clin chim acta 2007; 383(1-2):30-40. 10. llena-puy c. the role of saliva maintaining oral health and as an aid to diagnosis. med oral patol oral cir bucal 2006; 11(5): e449-55. 11. alsaidi a, djab bs, majid ay. caries experience and salivary constituents among overweight children agef 6-11 years in baghdad, iraq. j bagh college dentistry 2010; 22(2): 75-80. 12. pinkham jr, casamassimo ps, field hw. pediatric dentistry: infancy through adolescence. 4th ed. philadelphia:wb saunders company; 2005; p. 266-7. 13. raymond jt, timothy w, melinda ab. increase serum leptin and decreased dental caries associated with overweight or obesity in children. the federation of american society for experimental biology j 2008; 22: 880. 14. raymond jt, timothy w, melinda ab. obesity-induced leptin inhibits streptococcus mutans but not immunoestimulator y cytokines. dental research in review 2009; 41. 15. randeva hs, karteris e, lewandowski kc, sailesh s, o’hare p, hillhouse ew. circadian rhythmicity of salivary leptin in healthy subjects. mol genet metab 2003; 78(3):229-35. 16. myers mg jr. leptin receptor signaling and the regulation of mammalian physiology. recent prog horm res 2004; 59: 287-304. 17. gröschl m, manfred r, roland w, winfried n, markus m, gultekin t, johannes zennk, werner fb, dotsch j. identification of leptin in human saliva. j endocrinology and metabolism 2001; 86(11): 5234-39. 18. aydin s, halifeoglu i, ozercan ih, erman f, kilic n, aydin s, ilhan n, ilhan n, ozkan y, akpolat n, sert l, caylak e. a comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects. peptides 2005; 26(4): 647-52. 19. mi j, munkonda mn, li m, zhang mx, zhao xy, fouejeu pc, cianflone k. adiponectin and leptin metabolic biomarkers in chinese children and adolescents. j obes 2010; 2010: 892081. 20. pudjiadi s. ilmu gizi klinis pada anak. 4th ed. jakarta: balai penerbit fk ui; 2005. h. 141-9. 21. ahima rs, flier js. adipose tissue as an endocrine organ. trends endocrinol metab 2000; 11(8): 327-32. 22. miner jl. the adipocyte as an endocrine cell. j anim sci 2004; 82(3): 935-41. 23. permatasari r. hubungan kadar leptin saliva dan tingkat tumbuh kembang gigi anak obesitas. thesis. jakarta: fkg ui; 2012. h. 33-35 24. mc donald re, avery dr, dean ja. dentistry for the children and adolscent. 8th ed. missouri: mosby inc; 2011. p. 205-7. 25. nisengard, newman. oral microbiology and immunology. 2nd ed. philadelphia: wb saunder; 1998. p. 36-8. 26. marsh p, martin mv. oral microbilogy. 4th ed. london: wright; 1999. p. 20-2. 27. matsumoto-nakano m, fujita k, ooshima t. comparison of glucanbinding proteins in cariogenicity of streptococcus mutans. oral microbiol immunol 2007; 22(1): 30-5. 28. wang b, kuramitsu hk. a pleiotropic regulator, frp, affects exopolysaccharide synthesis, biofilm formation, and competence development in streptococcus mutans. infect immun 2006; 74(8): 4581-9. 29. zero dt, van houte j, russo j. enamel demineralization by acid produced from endogenous substrate in oral streptococci. arch oral biol 1986; 31(4): 229-34. 30. gopinath vk, arzreanee ar. saliva as a diagnostic tool for assessment of dental caries. archifes of orofacial science 2006;1: 57-59. 31. nakano k, nomura r, nakagawa i, hamada s, ooshima t. role of glucose side chains with serotype-specific polysaccharide in the cariogenicity of streptococcus mutans. caries res 2005; 39(4): 262-8. 32. pecharki d, petersen fc, assev s, scheie aa. involvement of antigen i/ii surface protein in streptococcus mutans and streptococcus intermedius biofilm formation. oral microbiol immunol 2005; 20(6): 366-71. 33. koo h, nino de guzman p, schobel bd, vacca smith av, bowen wh. influence of cranberry juice on glucan-mediated processes involved in streptococcus mutans biofilm development. caries res 2006; 40(1): 20-7. 172 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 172–176 research report the expression of nuclear factor of activated t cell c1 and receptor activator of nuclear factor kappa β induced by enterococcus faecalis in osteoclastogenesis (laboratory experiment on wistar rats) nirawati pribadi, rosita rahmawati, mandojo rukmo, adelina kristanti tandadjaja, hendy jaya kurniawan and ratna puspita hadi department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: enterococcus faecalis (e. faecalis) is the most common bacteria species in persistent endodontic infection of teeth undergoing root canal treatment at a prevalence of 38%. the virulence factor of this bacterium is lipoteichoic acid (lta) which can be recognized by toll-like receptors-4 (tlr-4) that produce a stimulus and provoke an immune response. inflammation results in bone defects that feature multiple cytokines and interactions between different cell types. bone loss within a periapical tooth is characterized by osteoclast formation (osteoclastogenesis) in the bone. purpose: this study aimed to determine the expression of nuclear factor of activated t cell c1 (nfatc1) and receptor activator of nuclear factor kappa β (rank) which played a role in osteoclastogenesis at different time intervals. methods: 36 upper molar teeth of the research subjects were induced with 106 cfu enterococcus faecalis and subsequently observed for 7 and 21 days with the nfatc1 and rank being counted microscopically at 1000x magnification across 20 viewing fields. thereafter, the data was examined and analyzed by means of an independent t test using spss. results: nfatc1 and rank expression were higher in the group including e. faecalis on days 7 and 21 than in the control group. there were significant differences between the treatment group and control group with regard to nfatc1 and rank expression (p<0.05). conclusion: the study showed that the expression of nfatc1 and rank, which plays a role in osteoclastogenesis, was higher in periapical bone defects in wistar rats induced by e. faecalis than those which were not induced. keywords: enterococcus faecalis; endodontic infection; lipotheicholic acid; nfatc1; rank, wistar rats correspondence: nirawati pribadi, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: nirawati-p@fkg.unair.ac.id introduction secondary root canal infections can occur due to inadequate management of primary root canal infection during treatment. the complex anatomy of root canal systems often results in incomplete root canal preparation. cleansing and shaping steps involving the use of disinfectant cannot completely eliminate bacteria, especially in the apical area, rendering the site liable to re-infection.1 gijo et al. (2015)2 proved that in cases of unsuccessful root canal treatment enterococcus faecalis (e. faecalis) was confirmed as the most common bacterium causing persistent endodontic infection in teeth undergoing root canal treatment with a prevalence of 38%. e. faecalis was detected in obturated root canals and was responsible for 77% of periapical deformities.3 e. faecalis represents one bacterium that causes periapical disease due to significant virulence factors, the main one being lipoteichoic acid (lta) which is a major constituent of the outer envelope of gram-positive bacteria.2 lta can be recognized by specific signaling molecules on the surface of host cells called toll-like receptors-4 (tlr-4) that produce a stimulus and provoke an immune response.4 the interface between the pulp and periapical region may cause the remaining bacteria in the root canal to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p1172–176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p172-176 173pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 172–176 inflame periapical tissue which, in turn, can cause bone defects in the periapical region. inflammation resulting in bone defects constitutes a complex regulatory process involving multiple cytokines and interactions between different cell types. the primary cells responsible for bone resorption are osteoclasts.5 bone loss in a periapical tooth is characterized by osteoclast formation (osteoclastogenesis) within the bone. during osteoclastogenesis, osteoclast differentiation factors produce a bonding reaction with the receptor.6 osteoclast differentiation and the subsequent bone resorption are initiated by the nuclear factor of activated t cells c1 (nfatc1). activation of nfatc1 will induce trap + osteoclast formation which produce mature osteoclasts and, furthermore, form an active osteoclast. the higher production of active osteoclast promoted greater bone resorption.7 receptor activator of nuclear factor kappa β (rank) is also referred to as tnfrsf11a/trancer2 which forms part of the tumor necrosis factor (tnf) receptor. rank can be found in osteoclasts and precursors, hematopoietic precursors, dendritic cells and mammary epithelial precursors as type i transmembrane receptors.8 rank can be activated when binding to rankl.6–8 rankl is detected in osteoblasts, t cells, dendritic cells and its precursor as type ii transmembrane proteins. rankl can activate rank, while rank can, in turn, activate rankl. rank and rankl play a critical role in osteoclast formation. rank activity provides signals that will activate nf-κb, nfatc1 and p38. if rankl is blocked, rank cannot be activated alone and osteoclastogenesis will not occur.8 this study aims to determine the level of expression of rank and nfatc1 that can activate osteoclastogenesis after induction of e. faecalis bacteria in the periapical teeth of wistar rats at different time intervals. materials and methods the study reported here was approved and supervised by the universitas airlangga faculty of dental medicine research ethical clearance commission with number 38/kkepk.fkg/iii/2015 and constituted a laboratorybased experiment. the subjects were 8-12 weeks old, adult, male, rattus norvegicus, weighing between 120 and150 grams, in good physical condition with fully developed molar teeth, supplied by the faculty of veterinary medicine, universitas airlangga. each sample consisted of the right upper molars of nine wistar rats, the total sample comprising 36 subjects. each subject was first secured to the jaw retractor board, prior to the pulp chamber roof of the maxillary molar being perforated with a nozzle bur (1/4) (dica®, austria). subjects satisfying the requirements were injected with intra-peritoneal anesthesia consisting of 80 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine. (onemed®, indonesia).9 after being induced with 106 cfu e. faecalis atcc212, the cavity was sealed with gic resin. the research involved the participation of two study groups: treatment group a, as the control group, on whose members cavity preparation was performed until perforation of the pulp chamber roof had been effected, prior to sterile brain heart infusion broth (bhib) being injected. in treatment group b, cavity preparation was undertaken until the pulp chamber roof had been perforated, with 10μl bhib containing 106 cfu bacteria e. faecalis atcc212 subsequently being injected by micropipette. both groups a and b were composed of two sub-groups, namely; a day 7 and a day 21, and b day 7 and b day 21. the subjects were then sacrificed by means of cervical dislocation. the jaw slices were separated and fixed with 10% neutral formaldehyde buffer for 24 hours, and decalcified with 4% edta for 30 days, before being made into paraffin block preparations. at that point, samples were made into hpa preparations, enabling periapical bone defects to be observed. immunohistochemical imaging was performed using anti-nfatc1 antibodies (biolegend # 649601, united states) and rank, in order to facilitate observation from 20 fields of view with a light microscope at 1000x magnification and calculation of the number of osteoclast cells expressing nfatc1 and rank in the periapical. for inferential purposes, a normality test was performed using a kolmogorov-smirnov test. a levene’s test of homogeneity and a one-way t-test to evaluate the effect of e. faecalis induction on the difference between nfatc1 and rank cell numbers in control group (a) and treatment group (b). p value <0.05 indicated a significant difference. results nfatc1 and rank expression data were obtained through observation of osteoclast cell numbers in the periapical bone, given that positive reactions to anti-nfatc1 and antirank monoclonal antibodies with immunohistochemical methods were characterized by brown coloration of the cytoplasm in the control and treatment group (figure 1 and 2). the number of nfatc1 and rank expressions can be seen in table 1. following calculation of the nfatc1 expression number, it was discovered that nfatc1 table 1. the average of nfatc1 and rank expression results in the control and treatment groups group n x nfatc1 sd nfatc1 x rank sd rank k7 control 9 8.56 2.297 4.22 2.386 k21 control 9 12.22 1.922 11.78 2.489 k7 treatment 9 15.56 2.449 12.56 1.236 k21 treatment 9 19.89 1.537 19.67 2.291 note: x: the average of nfatc-1 and rank expression; sd: standard deviation; n: number of samples per group dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p172–176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p172-176 174 pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 172–176 a b c d figure 2. imaging of rank expression (indicated by brown coloration and an arrow) in the immunohistochemical preparation of ra wistar rat molars a) control group on day 7; b) control group on day 21; c) treatment group on day 7; d) treatment group on day 21; (1000x magnification per 20 field of view). the expression of rank became higher on day 21 than on day 7 in the control and treatment groups. table 2. independent t test results for nftac1 and rank expression in each treatment group group p value expression of nftac1 expression of rank control day 7 0.000* 0.000* treatment day 7 control day 21 0.000* 0.000* treatment day 21 *indicated a significant statistical difference (p < 0.05) a b c d figure 1. imaging of nfatc1 expression (indicated by brown coloration and an arrow) in the immunohistochemical preparation of ra wistar rat molars a) control group at day 7; b) control group at day 21; c) treatment group at day 7; d) treatment group in day 21; (1000x magnification per 20 field of view). the expression of nfatc1 becomes higher on day 21 than on day 7 in the control and treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p172–176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p172-176 175pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 172–176 expression in the treatment group was higher than in the control group. in the control and treatment groups, nfatc1 expression increased between day 7 and day 21. based on the calculation of the rank expression number, it was discovered that rank expression in the control group increased between days 7 and 21, while in the treatment group it also increased steadily during the same period. normality test results using a kolmogorovsmirnoff test for nfatc1 and rank expressions showed the research data to be normally distributed (p> 0.05). data analysis was subsequently processed by means of an independent t-test to show whether any difference between the groups existed. the independent t-test results are contained in table 2. the results of an independent t-test on nfatc1 expression showed significant differences between the control group and the treatment group on day 7 and day 21. similar results were also derived for rank expression. the test results using the independent t-test of rank expression also showed significant differences between the control group and the treatment group on days 7 and 21. discussion the remaining bacteria present during root canal treatment represent a major etiology of endodontic treatment failure since they will cause inflammation of the periapical tissue. e. faecalis is a bacterium that often leads to endodontic treatment failure and causes re-infection.3 the connection between the root canal and apical causes inflammation of the periapical and high virulence of e. faecalis, with the presence of lta, will lead to bone defects.4 such bone defects in the periapical are characterized by the formation of osteoclasts (osteoclastogenesis) within the periapical bone. this study used wistar rats as research subjects due to their possessing a genome similar or almost homologous to that of humans, while also representing an experimental model (animal) for periodontal disease. the time periods adopted for this research were day 7 and day 21. their selection was based on the inflammation theory that posits days 0-7 as representing the recognition and activation phase, days 7-14 as constituting the activation and effector phase, while days 14-30 form the homeostasis phase during which restoration occurred.10 osteoprotegerin (opg) is a rank and rankl bonding inhibitor which enables the homeostasis phase to occur. if rankl is larger than opg it will bind to rank, resulting in osteoclastogenesis and bone resorption. in the inflammatory phase, the rankl ratio will increase in the periapical tissue and stimulate osteoclast activity, thereby initiating bone resorption.8 according to the research results, nfatc1 and rank expression in the control group increased between days 7 and 21 due to the activation of body molecules secreted from the damaged tissue and damage-associated molecular pattern (damp) including heat shock protein (hsp70). under normal conditions, hsp70 exists in small concentrations. however, environmental stimuli (ultraviolet radiation, heat, heavy metals and amino acids), pathological stimuli (viruses, bacteria, fever or parasitic infections and inflammation), or physiological stimuli (growth factor, cell differentiation, hormonal stimulation and tissue development) affect the increase in hsp70 synthesis.11,12 in the positive control group, dental perforation and bhib application of 10μl represented the stimuli in the formation of hsp70. there was a significant increase in nfatc1 and rank expression in the treatment group from day 7 to day 21. this indicates that e. faecalis possesses a strong virulence factor that cannot be compensated by a homeostasis mechanism. therefore, the expression of nfatc1 and rank is an important factor in the continuous increase in osteoclastogenesis. statistically, there was a significant difference in the nfatc1 and rank expression results between the control and treatment groups, where the treatment group expression was higher than that of the control group.7,13 e. faecalis, with the presence of lta, will activate monocyte/macrophage which releases pro-inflammatory cytokines such as il-1α, il-1β, tnfα, il-10, and il-6. these cytokines act as pro-osteoclastogenic factors inducing rankl production that will activate nfatc1. the expressed nfatc1 will induce trap+ osteoclast formation resulting in osteoclast maturation which will further form the active osteoclast (ruffled border osteoclast). higher production of active osteoclasts means greater bone resorption.7,13 the study results of nfatc1 and rank expressions between the control and treatment groups confirmed a significant increase. this result is similar to that of the study by park et al. (2015) which states that e. faecalis inhibits osteoblast formation. moreover, it is also supported by the research of tian et al. (2013) which proves that lta from e. faecalis inhibits the proliferation of osteoblasts and induces apoptosis of human-osteoblast-like cells. therefore, osteoblasts become undeveloped, resulting in high levels of osteoclasts and more extensive bone damage.14,15 the results of nfatc1 and rank expressions indicated that e. faecalis bacteria cause damage to periapical bone. this finding is in accordance with that of the study conducted by wang et al. (2015),8 which posited that e. faecalis contributes to bone resorption in apical periodontitis by promoting osteoclastogenesis through an increase in the regulation of osteoclast-specific marker expression, one such marker being nfatc1 and rank. it can be concluded that the number of nfatc1 and rank expression cells in periapical bone defects is higher in wistar rats induced by e. faealis than those not induced. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p172–176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p172-176 176 pribadi, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 172–176 references 1. tabassum s, khan fr. failure of endodontic treatment: the usual suspects. eur j dent. 2016; 10(1): 144–7. 2. gijo j, kumar kp, gopal ss, kumari s, reddy bk. enterococcus faecalis, a nightmare to endodontist: a systematic review. african j microbiol res. 2015; 9(13): 898–908. 3. jhajharia k, mehta l, parolia a, shetty kv. biofilm in endodontics: a review. j int soc prev community dent. 2015; 5: 1–12. 4. gomes bpf de a, her rera dr. etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology. braz oral res. 2018; 32: 82–110. 5. shlezinger m, friedman m, houri-haddad y, hazan r, beyth n. phages in a thermoreversible sustainedrelease formulation targeting e. faecalis in vitro and in vivo. plos one. 2019; 14(7): 1–19. 6. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015; 2015: 1–10. 7. chen x, wang z, duan n, zhu g, schwarz em, xie c. osteoblast– osteoclast interactions. connect tissue res. 2018; 59(2): 99–107. 8. wang s, deng z, seneviratne cj, cheung gs, jin l, zhao b, zhang c. enterococcus faecalis promotes osteoclastogenesis and semaphorin 4d expression. innate immun. 2015; 21(7): 726–35. 9. stashenko p, gonçalves rb, lipkin b, ficarelli a, sasaki h, campos-neto a. th1 immune response promotes severe bone resorption caused by porphyromonas gingivalis. am j pathol. 2007; 170(1): 203–13. 10. abbas ak, lichtman ah, pober js. cellular and molecular immunology. 4th ed. philadelphia: w.b. saunders; 2000. p. 553. 11. jung yk, kang ym, han s. osteoclasts in the inflammatory arthritis: implications for pathologic osteolysis. immune netw. 2019; 19: 1–13. 12. chai rc, kouspou mm, lang bj, nguyen ch, van der kraan agj, vieusseux jl, lim rc, gillespie mt, benjamin ij, quinn jmw, price jt. molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner. j biol chem. 2014; 289(19): 13602–14. 13. xu z, tong z, neelakantan p, cai y, wei x. enterococcus faecalis immunoregulates osteoclastogenesis of macrophages. exp cell res. 2018; 362(1): 152–8. 14. park oj, yang j, kim j, yun ch, han sh. enterococcus faecalis attenuates the differentiation of macrophages into osteoclasts. j endod. 2015; 41(5): 658–62. 15. tian y, zhang x, zhang k, song z, wang r, huang s, lin z. effect of enterococcus faecalis lipoteichoic acid on apoptosis in human osteoblast-like cells. j endod. 2013; 39(5): 632–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p172–176 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p172-176 185 volume 46, number 4, december 2013 minyak ikan lemuru (sardinella longicep) menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (fish oil of lemuru (sardinella longicep) reduced the osteoblast apoptosis in wistar rat alveolar bone) didin erma indahyani bagian biologi oral fakultas kedokteran gigi universitas jember jember indonesia abstract background: periodontal disease is caused by periodontopatogen bacteria resulting the alveolar bone damage. the decrease of osteoblasts and the increased of osteoclasts can cause bone destruction. the decrease of osteoblasts, due to a disturbance of differentiation, proliferation and apoptosis. inflammatory mediators are prostaglandin e2 (pge2), interleukin-1 (il-1), il-6 also tumor necrosis alpha (tnf-α) stimulates osteoblast apoptosis through gene expression, signaling molecules and receptor-forming osteoblasts. fish oil of lemuru, which is widely encountered in indonesian coast, containing n-3 poly unsaturated fatty acids (n-3 pufas) are quite high. consumption of fish oil shown to reduce the expression of pge2, il-1, il-6 and tnf-α. purpose: the purpose of this study was to examine the effect of lemuru (sardinella longicep) fish oil on osteoblast apoptosis of rat alveolar bone induced periodontal infection. methods: thirty wistar rats, male, age 5 days, divided into 3 groups: group i rats induced with normal saline, group ii rats induced by lps, and group iii rats induced with lemuru fish oil and lps. each group was divided into 2 sub-groups that would be sacrified at 13 days and 21 days of age. fish oil was given at a dose 1ml/300-350 grams. lipopolysaccharide (lps) induced with the purpose to cause periodontal infection in the maxillary buccal fold molar region with dose 5μl lps/pbs 0.03 ml. after decapitation and decalcification, the maxilla was cut in 5μm thickness. apoptosis was analyzed on dna and detected by tunel reaction (transferase-mediated digoxigenin-deoxy-utp nick end labeling). results: the results showed that apoptosis of osteoblast cells was significantly smaller in rats induced by lemuru fish oil. conclusion: the study showed that lemuru fish oil reduced the osteoblast apoptosis of rats alveolar bone induced periodontal infection by lps. key words: fish oil, sardinella longicep, osteoblast, apoptosis, n-3 pufa, periodontal disesase, bone resorption abstrak latar belakang: penyakit periodontal akibat bakteri peridontopatogen, menyebabkan terjadinya kerusakan tulang alveolar. penurunan jumlah osteoblas dan peningkatan jumlah osteoklas mengakibatkan kerusakan tulang. penurunan jumlah osteoblas disebabkan terjadinya gangguan diferensiasi maupun proliferasi juga apoptosis. apoptosis osteoblas dimodulasi oleh mediator-mediator inflamatori yaitu prostaglandin e2 (pge2), interleukin-1 (il-1), il-6 juga tumor nekrosis alfa (tnf-α), melalui pengaruhnya pada ekspresi gen, molekul-molekul signaling maupun reseptor pembentukan osteoblas. minyak ikan lemuru yang banyak di pesisir indonesia, banyak mengandung n-3 poly unsaturated fatty acid (n-3 pufa). konsumsi minyak ikan terbukti menurunkan ekspresi pge2, il-1, il-6 maupun tnf alfa. tujuan: tujuan penelitian ini adalah untuk meneliti pengaruh minyak ikan lemuru (sardinella longicep) pada apoptosis osteoblas pada tulang alveolar tikus yang diinduksi infeksi periodontal. metode: tiga puluh ekor tikus wistar, jantan, umur 5 hari, dibagi menjadi 3 kelompok yaitu: kelompok i tikus diinduksi dengan salin normal, kelompok ii tikus diinduksi dengan lipopolisakarida (lps), dan kelompok iii tikus dinduksi dengan minyak ikan lemuru dan lps. masing-masing kelompok dibagi menjadi research report 186 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 185–189 2 sub kelompok yaitu kelompok yang akan didekapitasi pada umur 13 hari dan umur 21 hari. minyak ikan lemuru diberikan dengan dosis 1ml/300-350 gram. lipopolisakarida (lps) diinduksikan dengan tujuan untuk menyebabkan infeksi periodontal pada buccal fold regio molar rahang atas, dengan dosis 5μl lps/0,03pbs (konsentrasi 0,02 mg). setelah didekapitasi dan dekalsifikasi, rahang atas dipotong dengan ketebalan 5μm. apoptosis dianalisis pada dna dan dideteksi dengan tunel reaction (transferase-mediated digoxigenin-deoxy-utp nick end labeling). hasil: hasil penelitian menunjukkan bahwa apoptosis sel osteoblas secara bermakna lebih kecil pada tikus yang diinduksi dengan minyak ikan. simpulan: penelitian ini menunjukkan bahwa minyak ikan lemuru (sardinella longicep) mampu menurunkan apoptosis sel osteoblas pada tikus wistar yang diinduksi infeksi periodontal dengan lps. kata kunci: minyak ikan, sardinella longicep, osteoblas, apoptosis, n-3 pufa, penyakit periodontal, resorbsi tulang korespondensi (correspondence): didin erma indahyani, bagian biologi oral, fakultas kedokteran gigi, universitas jember. jl. kalimantan no. 35 jember 68121, indonesia. e-mail: didinermae@yahoo.com pendahuluan apoptosis osteoblas merupakan komponen penting yang terlibat dalam osteogenesis secara normal dan patologis. di dalam skeletal pada masa post natal dan dewasa, apoptosis merupakan bagian integral terhadap fisiologi turnover tulang, repair dan regenerasi. keseimbangan proliferasi, diferensiasi dan apoptosis osteoblas menentukan ukuran populasi osteoblas pada waktu tertentu.1 tingkat pembentukan tulang ditentukan oleh jumlah osteoblas, replikasi progenitornya dan life-span dari sel yang matur. hal tersebut dapat mencerminkan waktu kematian sel oleh apoptosis. telah dibuktikan bahwa apoptosis menentukan jumlah osteoblas, maka perubahan dalam prevalensi apoptosis pada osteoblas dapat mengubah laju pembentukan tulang.2 periodontopatogen menyebabkan inflamasi dan destruksi tulang alveolaris. mekanisme destruksi tulang merupakan proses kompleks yang melibatkan 2 aksi sel osteoklas dan osteoblas. pada proses inflamasi, sel pembentuk tulang yaitu osteoblas mengalami penurunan jumlah maupun aktivitasnya, sedangkan sel osteoklas akan meningkat. penurunan jumlah sel osteoblas diakibatkan oleh berkurangnya proliferasi atau tingginya apoptosis osteoblas ataupun sel prekursornya, yang keduanya dipengaruhi oleh adanya inflamasi.3 peristiwa tersebut diawali adanya respon sistem imun alami yaitu toll-like receptors (tlrs) pada sel epitel gingiva mendeteksi dan merespon struktur mikroba misalnya lipopolysaccharide (lps), peptidoglycan, dna bakteri, double-stranded rna, dan lipoprotein. struktur mikroba tersebut dikenal dengan pathogen-associated molecular patterns (pamps). tlrs yang terdapat di permukaan sel host, mengenali pamps, dan menyebabkan aktivasi beberapa faktor transkripsi yaitu nuclear factor-𝜅b (nf𝜅b) dan aktivator protein 1 (ap-1) melalui mitogen-activated protein kinase (mak) cascade.4 selain itu respon sistem imun alami, akan mengaktivasi sitokin proinflamatori, chemokin serta eikosanoid yang berperan penting pada apoptosis sel osteoblas. sitokin proinflammatory, misalnya il-1β dan tnf-α, secara langsung menstimulasi apoptosis osteoblas ataupun prekursor osteoblas atau secara tidak langsung mempengaruhi stimulasi ekspresi fas yaitu mediator proapoptosis yang potensial.5 eikosanoid yaitu prostaglandin e-2 (pge-2) berperan penting untuk menurunkan produksi osteoblas. pge-2 menginduksi osteoblas memproduksi receptor activated nuclear kappa-𝛽 ligand (rankl) dan menurunkan produksi osteoprotegerin (opg). selain itu pge2 mempertinggi ikatan antara rankl dan receptor activated nuclear kappa-𝛽 (rank) pada prekursor osteoklas. penurunan opg yang berfungsi mengikat rankl untuk membentuk osteoblas, mengakibatkan rankl berikatan dengan rank yang menyebabkan pembentukan osteoklas. keadaan tersebut menyebabkan jumlah osteoblas menurun.6 minyak ikan lemuru berasal dari ikan lemuru (sardinella longicep) banyak di temukan di pesisir indonesia, terutama pulau jawa. produksinya melimpah, sehingga harganya sangat murah. selama ini ikan lemuru selain di buat minyak ikan juga di buat tepung ikan sebagai makanan ternak. minyak ikan lemuru mengandung n-3 polyunsaturated fatty acid (pufa) yaitu eicosapentaenoic acid 13,70% (epa) dan docohexasonoic acid (dha) 8,91%.7 n-3 pufa adalah prekursor eikosanoid yang terlibat dalam metabolisme tulang, yaitu prostaglandin (pg) dan leukotrienes. diet epa dan dha akan mengganti n-6 pufa dalam membran platelet, eritrosit, monosit dan sel hati. ini berperan pada perubahan rasio n-6/n3 pufa dalam membran yang menyebabkan terjadinya perubahan sifat dan fungsi. perubahan ini berperan penting pada penurunan produksi il-1, il-6 dan tnf-alfa.8 konsumsi n-3 pufa menurunkan tnf-alpha. penurunan sitokin proinflamatori maupun eikosanoid oleh karena n-3 pufa, menyebabkan konsumsi minyak ikan akan menyebabkan terjadinya peningkatkan pembentukan tulang, dan menurunkan destruksi tulang.9 penelitian ini bertujuan untuk meneliti pengaruh minyak ikan lemuru pada apoptosis sel osteoblas pada tulang alveolar tikus wistar yang diinduksi infeksi periodontal. 187indahyani: minyak ikan lemuru (sardinella longicep) bahan dan metode tiga puluh ekor tikus wistar jantan, umur 5 hari, dibagi menjadi 3 kelompok yaitu: kelompok i tikus diinduksi dengan salin normal, kelompok ii tikus diinduksi dengan lps, dan kelompok iii tikus dinduksi dengan minyak ikan lemuru (produksi muncar banyuwangi) dan lps. masingmasing kelompok dibagi menjadi 2 sub kelompok yaitu kelompok yang didekapitasi pada umur 13 hari dan umur 21 hari. waktu dekapitasi 13 hari setelah induksi minyak ikan, saat baru terjadi pergantian n-6 pufa dengan n-3 pufa pada membran sel dan pada 21 hari saat pergantian asam lemak tersebut sudah berlipat.10 minyak ikan lemuru diberikan dengan dosis 1ml/300350 gram berat badan tikus wistar, secara peroral, menggunakan sonde lambung, dan diberikan tiap hari (yang dimulai 3 hari setelah induksi lps) sampai tikus dilakukan dekapitasi. induksi lps selama 3 x 24 jam telah menyebabkan peningkatan jumlah osteoklas pada tulang alveolaris. induksi lps dilakukan di bukal fold regio molar rahang atas, dengan dosis 5µl lps/0,03pbs (konsentrasi lps 0,02 mg), yang dilakukan 24 jam sekali sebanyak 8 kali.11 tikus didekapitasi setelah berumur 13 dan 21 hari. tikus yang telah didekapitasi diambil rahang atas kanannya, kemudian difiksasi dengan bouin’s fixative 4ºc semalam. spesimen didemineralisasi menggunakan asam asetat/formal salin dan ditanam dalam blok parafin kemudian di potong dengan ketebalan 5µm. spesimen dilakukan fragmentasi dna dan dideteksi dengan transferase-mediated digoxigenin-deoxy-utp nick end labeling (tunel reaction), untuk menganalisa apoptosis sel osteoblas, yang secara singkat adalah sebagai berikut. irisan dikonterstain dengan 3% metil green, kemudian diinkubasi selama 1-2 menit dengan 0,15% cuso4 dalam 0,9% nacl. tunel reactions nampak pada nukleus sel dan sel yang nukleusnya nampak coklat gelap yang jelas adalah positif. tunel rections yang positif adalah sel yang mengalami proses apoptosis.12 hasil apoptosis osteoblas secara bermakna (p<0,05) lebih besar pada tikus yang diinduksi lps yang berumur 13 hari maupun 21 hari bila dibandingkan dengan kontrol. tikus yang diinduksi lps kemudian di beri minyak ikan mempunyai tingkat apoptosis yang lebih rendah secara signifikan (p<0,05) pada umur 13 maupun 21 hari bila dibandingkan dengan kontrol (tabel 1). gambaran mikroskopis pada apoptosis osteoklas dan osteoblas dapat dilihat pada gambar 1a, b, c. pembahasan induksi lps mengakibatkan apoptosis sel osteoblas pada tikus umur 13 hari maupun 21 hari. sel osteoblas yang mengalami apoptosis lebih banyak secara signifikan pada tikus yang hanya di induksi lps. lps bersifat endotoksin karena lps mengikat reseptor cluster of differentiation 14 (cd14) di permukaan sel makrofag dan monosit. toll-like receptor-4 (tlr4) makrofag dan monosit yang berikatan dengan bakteri oleh karena adanya cd14 akan menginduksi sekresi sitokin dan mediator lipid inflammation. sitokin dan mediator inflamasi tersebut termasuk il-1, tnf-α juga pge-2. mediator tersebut berperan pada diferensiasi dan aktifitas osteoklas dan menekan jumlah osteoblas. mediator tersebut memacu terbentuknya osteoklas dari sel stromal/ osteoblas melalui ikatan sel ke sel yaitu rankl dalam osteoblas dengan rank pada progenitor osteoklas.13 pemberian minyak ikan lemuru mengakibatkan peningkatan jumlah osteoblas secara signifikan (tabel 1). minyak ikan lemuru mengandung epa dan dha. konsumsi minyak ikan tersebut mengakibatkan terjadi peningkatan komposisi epa dan dha serta rendahnya asam arachidonat (aa) dalam membran sel. asam arachidonat adalah sumber utama pembentukan pge2, leukotrin, lipoksin dan p45 akibat terjadinya oksigenasi gambar 1. gambaran apoptosis osteoblas, dengan tunel (pembesaran 1000x). keterangan: sel yang menunjukan warna hijau merupakan sel yang survive, sedangkan sel yang berwarna coklat merupakan sel yang mengalami apoptosis. a. tikus kontrol (tampak sel osteoblas dalam keadaan survive), b tikus yang diinduksi lps dan di beri minyak ikan (sel osteoblas terlihat lebih banyak yang survive). c, tikus yang diinduksi lps (sel osteoblas banyak mengalami apoptosis). ob (osteoblas), oc (osteoklas). 10    tabel 1. jumlah apoptosis sel osteoblas n apoptosis osteoblas mean std. deviation kontrol 13 hari 5 1.80 .83 kontrol 21 hari 5 1.20 1.30 lps 13 hari 5 5.80 3.96 lps 21 hari 4 10.00 5.22 lps mi 13 hari 5 3.80 2.28 lps mi 21 hari 5 2.60 2.70 total 29 4.00 3.95 keterangan: lps: lipopolysacharide, n: jumlah ulangan gambar 1. gambaran apoptosis osteoblas, dengan tunel (pembesaran 1000x). keterangan: sel yang menunjukan warna hijau merupakan sel yang survive, sedangkan sel yang berwarna coklat merupakan sel yang mengalami apoptosis. a. tikus kontrol (tampak sel osteoblas dalam keadaan survive), b tikus yang diinduksi lps dan di beri minyak ikan (sel osteoblas terlihat lebih banyak yang survive). c, tikus yang diinduksi lps (sel osteoblas banyak mengalami apoptosis). ob (osteoblas), oc (osteoklas).   oc  ob  ob  ob ob  oc  oc  10    tabel 1. jumlah apoptosis sel osteoblas n apoptosis osteoblas mean std. deviation kontrol 13 hari 5 1.80 .83 kontrol 21 hari 5 1.20 1.30 lps 13 hari 5 5.80 3.96 lps 21 hari 4 10.00 5.22 lps mi 13 hari 5 3.80 2.28 lps mi 21 hari 5 2.60 2.70 total 29 4.00 3.95 keterangan: lps: lipopolysacharide, n: jumlah ulangan gambar 1. gambaran apoptosis osteoblas, dengan tunel (pembesaran 1000x). keterangan: sel yang menunjukan warna hijau merupakan sel yang survive, sedangkan sel yang berwarna coklat merupakan sel yang mengalami apoptosis. a. tikus kontrol (tampak sel osteoblas dalam keadaan survive), b tikus yang diinduksi lps dan di beri minyak ikan (sel osteoblas terlihat lebih banyak yang survive). c, tikus yang diinduksi lps (sel osteoblas banyak mengalami apoptosis). ob (osteoblas), oc (osteoklas).   oc  ob  ob  ob ob  oc  oc  10    tabel 1. jumlah apoptosis sel osteoblas n apoptosis osteoblas mean std. deviation kontrol 13 hari 5 1.80 .83 kontrol 21 hari 5 1.20 1.30 lps 13 hari 5 5.80 3.96 lps 21 hari 4 10.00 5.22 lps mi 13 hari 5 3.80 2.28 lps mi 21 hari 5 2.60 2.70 total 29 4.00 3.95 keterangan: lps: lipopolysacharide, n: jumlah ulangan gambar 1. gambaran apoptosis osteoblas, dengan tunel (pembesaran 1000x). keterangan: sel yang menunjukan warna hijau merupakan sel yang survive, sedangkan sel yang berwarna coklat merupakan sel yang mengalami apoptosis. a. tikus kontrol (tampak sel osteoblas dalam keadaan survive), b tikus yang diinduksi lps dan di beri minyak ikan (sel osteoblas terlihat lebih banyak yang survive). c, tikus yang diinduksi lps (sel osteoblas banyak mengalami apoptosis). ob (osteoblas), oc (osteoklas).   oc  ob  ob  ob ob  oc  oc  a b c 188 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 185–189 oleh enzim-enzim lipoksigenase, siklooksigenase dan epoksigenase yang berasal dari n-6 pufa.8 konsumsi n-3 pufa, selama 2 minggu mengakibatkan peningkatan αlinolenic acid (ala) sebanyak 3-4 × lipat, epa 3 × lipat dan dha 1,5 × lipat dalam membran sel. peningkatan membran sel dengan n-3 pufa tersebut menyebabkan penurunan produksi pge2, tetapi akan meningkatkan pge3 yang berfungsi sebagai anti inflamasi.13 rendahnya pge2 mempengaruhi pembentukan osteoblas, karena fungsi pge2 yang menstimulasi ekspresi rankl oleh prekursor osteoblas dan osteoblas matur akan menurun dan meningkatkan produksi opg. opg yang berikatan dengan rankl berperan penting untuk terjadinya pembentukan osteoblas, akan tetapi apabila rankl berikatan dengan rank menyebabkan apoptosis osteoblas.8 n-3 pufa mempengaruhi penurunan sitokin proinflamatori yaitu il-1, il-6 maupun tnf-α. hal ini dihubungkan dengan penurunan aktivitas antigen precenting cells (apc), yaitu dengan berkurangnya ekspresi molekul mayor histocompatibility cell klas ii (mhc klas ii) dan intercellular adhesion molecule (icam). ekspresi mhc klas ii diperlukan untuk berfungsinya apc, sedangkan icam merupakan molekul reseptor yang terdapat pada apc. tanpa adanya mhc klas ii dan icam, apc tidak akan bisa aktif memapar antigen, misalnya lps. apc yang aktif menstimulasi ekspresi sitokin proinflamatori. pge-2, menyebabkan sitokin proinflamatori (il-1 dan tnf-α) juga menurun. penurunan sitokin proinflamatori berkaitan erat dengan penurunan jumlah osteoklas, dan peningkatan jumlah osteoblas. sitokin proinflamatori bisa secara langsung menstimulasi apoptosis osteoblas dan prekursornya, atau secara tidak langsung dengan menstimulasi ekspresi fas. tnf-α menginduksi apoptosis sel ligamen periodontal yang berperan sebagai sumber prekursor osteoblas. apoptosis yang distimulasi oleh sitokin proinflamatori, mempengaruhi faktor transkripsi pro apoptosis yaitu forkhead box-o1 (foxo1).5 foxo1 meregulasi ekspresi gen proapoptosis yaitu fas-associated, via death domain (fadd) dan caspases-3, -8, and -9. peranan map kinase mempengaruhi signal pro inflamatori.14 upstream regulators p38 map kinase, mkk3 and mkk6, diperlukan il-1beta dan tnfα menginduksi ekpsresi rank ligand dalam stromal bone marrow.3 n-3 pufa berperan menurunkan fas melalui penurunan tnf-α, yang mempengaruhi transkripsi gen apoptosis dan menurunkan ekpresi rankl akibat stimulasi dari il-1 dan tnf-α. lipopolisakarida di ketahui berperan penting pada apoptosis osteoblas. lipopolikarida menginduksi osteoblas untuk mengekspresikan nod1 dan nod2, yaitu dua kelompok nucleotide-binding domain dan leucine-rich repeat region yang mengandung kelompok protein reseptor biasa disebut nlrs, yang bertindak sebagai sensor intraselular untuk bakteri peptidoglikan dan menginisiasi produksi mediator proinflamatori. nlr family card domain yang terdiri dari 4 (nlrc4, yang saat ini dikenal sebagai ipaf, card12, atau clan) dan nlr family pyrin domain yang terdiri dari 3 (nlrp3, yaitu dikenal sebagai cias1, cryopyrin, pypaf1, atau nalp3) telah diimplikasikan dalam menginduksi kematian sel dalam merespon bakteri dan komponennya15,16 kedua molekul tersebut dapat berhubungan dengan protein adaptor yaitu apoptosis-associated speck-like protein (asc) untuk menstimulasi aktivasi caspase-1 and caspase-8 yaitu enzim yang menunjukan peningkatan aktivitas osteoblas setelah terinduksi bakteri. aktivasi caspase-1 dan caspase 8 menginduksi apoptosis osteoblas.17,18 apoptosis osteoblas juga diakibatkan meningkatnya produksi nitric oxide (no) akibat induksi lipopolisakarida. lipopolisakarida dan sitokin proinflamatori, terbukti menstimulasi peningkatan inos. kuzushima, dkk.,19 menyatakan bahwa tnf-α, interleukin-1β and interferon-γ menyebabkan kematian sel osteoblas yang di mediai oleh apoptosis bukan nekrosis. sitokin terbukti menghasilkan peningkatan inducible nitric-oxide synthase (inos) mrna dan nitric-oxide (no) dalam sel.19 no menginduksi apoptosis osteoblas melalui synthesis bax protein.20 selain itu no menyebabkan penekanan pada viabilitas sel, potensial membran mitokondria dan sintesis atp, yang mengakibatkan gangguan pada fungsi mitokondria, reaksi spesies oksigen intraseluler dan protein bcl-2 yang berperan penting pada apoptosis osteoblas.21 rendahnya sitokin tersebut berperan untuk menghambat pembentukan no yang tinggi dan juga menghambat stimulasi kelompok protein reseptor yaitu nlrs yang berperan pada apoptosis osteoblas.10 n-3 pufa yang berperan pada penurunan ekspresi sitokin proinflamatori dan eikosanoid, berpengaruh pada penurunan ekspresi inos.22 penelitian ini menunjukkan bahwa minyak ikan lemuru dengan kandungan n-3 pufa dengan kandungan 12,5% n-3 pufa yaitu epa dan dha menurunkan apoptosis sel osteoblas pada tikus wistar yang diinduksi lps. ucapan terima kasih terima kasih kepada pemberi dana untuk pelaksanaan penelitian ini yaitu dp2m dikti pada penelitian fundamental. tabel 1. jumlah apoptosis sel osteoblas n apoptosis osteoblas mean std. deviation kontrol 13 hari 5 1.80 .83 kontrol 21 hari 5 1.20 1.30 lps 13 hari 5 5.80 3.96 lps 21 hari 4 10.00 5.22 lps mi 13 hari 5 3.80 2.28 lps mi 21 hari 5 2.60 2.70 total 29 4.00 3.95 keterangan: lps: lipopolysacharide, n: jumlah ulangan 189indahyani: minyak ikan lemuru (sardinella longicep) daftar pustaka 1. hock jm, krishnan v, onyia je, bidwell jp, milas j, stanislaus d. osteoblast apoptosis and bone turnover. j bone miner res 2001; 16(6): 975-84. 2. robert lj, robert s, weinstein tb, paula ra, michael p, manolagas sc. increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. j clin invest 1999; 104: 439-46. 3. graves dt, li j. cochran dl. inflammation and uncoupling as mechanisms of periodontal bone loss. j dent res 2011; 90(2): 14353. 4. benedetto ad, gigante i, colucci s, maria g. periodontal disease: linking the primary inflammation to bone loss. clin and develop immunol 2013: 7. 5. behl y, siqueira m, ortiz j, li j, desta t, faibish d. activation of the acquired immune response reduces coupled bone formation in response to a periodontal pathogen. j immunol 2008; 181: 871118. 6. kotake s, toru y, manabu k,yuki n. effects of nsaids on differentiation and function of human and murine osteoclasts– crucial ‘human osteoclastology’. pharmaceuticals 2010; 3: 1394410. 7. estiasih t. mikroenkapsulasi konsentrat asam lemak omega 3 dari limbah cair pengalengan ikan lemuru. tesis. yogyakarta: pasca sarjana universitas gadjah mada; 1996. h. 12-6. 8. kajarabille n, díaz-castro j, hijano s, lópez-frías m, lópezaliaga i, ochoa jj. a new insight to bone turnover: role of 𝜔-3 polyunsaturated fatty acids. the sci world j 2013: 16. 9. mahmoud maa, safar mm, agha am, el-shabrawy oam, yassin naz. effect of linseed oil, fish oil and alendronate sodium on ovariectomy-induced osteoporosis in female rats. j of appl sci res 2013; 9(3): 2119-125. 10. montzioris e, cleland lg, gibson ar, neumann ma, demasi m, james mj. biochemical effects of a diet containing foods enriched with n-3 fatty acids. am j clin nutr 2000; 72: 42-8. 11. i n d a h y a n i d e , s a n t o s o a l s , u t o r o t, s o e s a t y o m h . lipopolysa cha r ide ( lps) i nt roduct ion du r i ng g row t h a nd development period of rat’s tooth toward the occurrence of enamel hypoplasia. dent j (maj ked gigi) 2007; 40(2): 85-8. 12. weinstein rs, jilka rl, parfitt m, manolagas sc, weinstein rs, jilka rl, parfitt m, manolagas sc. inhibition of osteoblastogenesis and promotion of apoptosis of osteoblast and osteocytes by glucocorticoids. j clin invest 1998; 102: 274-82. 13. suda t, takahashi n, udagawa n, jimi e, gillespie mt, martin tj. modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. endocr rev 1999; 20(3): 345-57. 14. rogers je, li f, coatney dd, otremba j, kriegl jm, protter ta. a p38 mitogen-activated protein kinase inhibitor arrests active alveolar bone loss in a rat periodontitis model. j periodontol 2007; 78: 1992-98. 15. gumucio dl, diaz a, schaner p, richards n, babcock c, schaller m, cesena t. fire and ice: the role of pyrin domain-containing proteins in inflammation and apoptosis. clin exp rheumatol 2002; (suppl 26): s45–s53. 16. sutterwala fs, ogura y, szczepanik m, lara-tejero m, lichtenberger gs, grant ep, bertin j, coyle aj, galan je, askenase pw, flavell ra. critical role for nalp3/cias1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. immunity 2006; 24: 317–27. 17. ma r iat hasa n s. asc, ipa f a nd cr yopyr in / na lp3: bona f ide intracellular adapters of the caspase-1 inflammasome. microbes infect 2007; 9: 664–71. 18. mccall sh, sahraei m, young ab, worley sc, duncan ja, pan-yun ting j, marriott i. osteoblasts express nlrp3, a nucleotide-binding domain and leucine-rich repeat region containing receptor implicated in bacterially induced cell death. j bone and min res 2008; 23: 1. 19. kuzushima m, mogi m, togari a. cytokine-induced nitric-oxidedependent apoptosis in mouse osteoblastic cells: involvement of p38map kinase. arc of oral biol 2006; 51(11): 1048-53. 20. mungrue ln,bredt ds, stewart dj, husain m. from molecules to mammals: what’s nos got to do with it. acta physiol scand 2003; 179: 123-35. 21. ruei-ming chen, ta-liang chen, wen-ta chiu, chia-chen chang. molecular mechanism of nitric oxide-induced osteoblast apoptosis. j of orth res 2005; 23(2): 462–68. 22. sargi sc, dalalio mmo, moraes ag, visentainer jel, morais dr, visentainer jv. role of omega-3 polyunsaturated fatty acids in the production of prostaglandin e2 and nitric oxide during experimental murine paracoccidioidomycosis. biomed res int 2013: 6. 76 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 76–79 research report effects of strong bite force on the facial vertical dimension of pembarong performers christina, 1 achmad sjafei2, and ida bagus narmada2 1d-art dental clinic 2department of orthodontics, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: a pembarong performer is a reog dancer who bites on a piece of wood inserted into his/her mouth in order to support a 60 kg barongan or dadak merak mask. the teeth supporting this large and heavy mask are directly affected, as the strong bite force exerted during a dance could affect their vertical and sagital facial dimensions. purpose: this study aimed to examine the influence of the bite force of pembarong performers due to their vertical and sagital facial dimensions. methods: the study reported here involved fifteen pembarong performers and thirteen individuals with normal occlusion (with specific criteria). the bite force of these subjects was measured with a dental prescale sensor during its centric occlusion. a cephalometric variation measurement was subsequently performed on all subjects with its effects on their vertical and sagital facial dimensions being measured. results: the bite force value of the pembarong performers was 394.3816 ± 7.68787 newtons, while the normal occlusion was 371.7784 ± 4.77791 newtons. there was no correlation between the bite force and the facial sagital dimension of these subjects. however, a significant correlation did exist between bite force and lower facial height/total facial height (lfh/tfh) ratio (p = 0.013). conversely, no significant correlation between bite force and posterior facial height/total facial height (pfh/tfh) ratio (p = 0.785) was detected. there was an inverse correlation between bite force and lfh/tfh ratio (r = -.464). conclusion: bite force is directly related to the decrease in lfh/tfh ratio. occlusal pressure exerted by the posterior teeth on the alveolar bone may increase bone density at the endosteal surface of cortical bone. keywords: bite force; facial vertical dimension; pembarong correspondence: ida bagus narmada, department of orthodontics, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ida-b-n@fkg.unair.ac.id introduction the pattern of human growth and development differs from one individual to another. facial growth, for instance, can be easily identified if linked with craniofacial shape. vertical facial growth can also be influenced by many factors, including facial and masticating muscle function. these soft tissues play a role in determining the shape and morphology of the face itself.1 bite force can be defined as a force applied by the masticating muscles in dental occlusion resulting from the combined force of the various components within the masticatory system which acts on individual teeth. bite force is also considered to be an indicator of the functional state of the masticatory system due to the activities of the masseter, medial pterygoid and lateral pterigoid muscles, as well as the biomechanical jaw and biomechanical reflex. thus, bite force value varies from one individual to another depending on many factors, leading to changed face height and dental status. an increase or decrease in face height dimension due to bite force may be influenced by several factors, such as age, gender, and periodontal tissue condition.2 bite force may also serve as an indicator of masticatory function and tooth load.3 the presence of bite force and the role of the mastication muscles have the potential to alter craniofacial shape, especially with regard to alveolar bone mass and the thickness of the mandibular.4,5 in addition, bite dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p76-79 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p76-79 7777christina, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 76–79 force can affect facial morphology through the development of a condition in which anterior facial height appears lower than that of its posterior counterpart.6 chewing pencils, pacifiers, and other hard objects may cause dento-facial disorders. the force with which hard objects are bitten is considered to be mechanical in nature and one inducing alveolar bone changes.7 reog ponorogo is a culture originating in east java more precisely the ponorogo district. the pembarong performer who bites the 60 kg mask (figure 1) during a performance may, unfortunately, find his facial height affected by this habit.1,7 in producing position treatment results, an orthodontist should have knowledge of an individual’s bite force which provides information about facial morphology and influences the choice of the most appropriate type of mechanic for the subsequent selected treatment. in addition, a strong bite produces vertical force affecting the maintenance period of a malocclusion, especially with the use of class ii elastics. this negates the orthodontic force but further improves the bite force during mastication.8 the study examined, using cephalometric methods, the strong bite force of pembarong performers that has potentially influenced their facial development. materials and methods twenty-eight patients, consisting of fifteen pembarong performers and thirteen subjects, served as the control group. before the study was conducted, an ethical fit test was carried out on the team in accordance with the research ethical code of the faculty of dental medicine, universitas airlangga, surabaya. this research was then conducted at the clinic of orthodontics, faculty of dental medicine, universitas airlangga. the criteria applied to the pembarong performers consisted of their being male, aged between 25 and 40 years, with at least 3 years’ professional experience of delivering 12-24 reog shows annually. the control group criteria were those of being male, 25-40 years old, demonstrating angle’s class i malocclusion (i.e. light tight, without bilateral or unilateral molar mutilation, no deep or open bite, no root canal region treatment, no bruxism, and no abnormal tmj). a value for the bite force during centric occlusion was obtained using a modified sensor dental prescale (tekscan inc., south boston, massachusetts). bite force values were measured by asking subjects to bite down three times on a rubber-coated sensor at 30-second intervals. the mean value of the bite force was then calculated.3 the cephalometric analysis of vertical dimensions was undertaken by calculating the ratio of the linear distance measurement of the anterior facial height (n-me) to the posterior facial height (s-go). the lower facial height measurement represented the distance from the palatal plane (ans-pns) to the me point (figure 2). all measurements were collected using a ruler (mm) and a ratio of 100%. 8 figure 4. cephalometric landmark points of the subjects (s, n, ans, pns, p, go, gn, me). figure 5. tracing tools. figure 6. pfh of the pembarong performer (left) was shorter than that of the control group (right). figure 2. cephalometric landmark points of the subjects (s, n, ans, pns, p, go, gn, me). 7 figure 1. barongan (a tiger mask) and peacock dadak (peacock feathers). figure 2. cakotan (a wooden board). figure 3. digital scale and calibration tool (resistance meter) as a modified sensor dental prescale. 7 figure 1. barongan (a tiger mask) and peacock dadak (peacock feathers). figure 2. cakotan (a wooden board). figure 3. digital scale and calibration tool (resistance meter) as a modified sensor dental prescale. figure 1. barongan (a) with cakotan, a wooden board (b), the 60kg tiger mask that bite by a pembarong performer a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p76-79 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p76-79 78 christina, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 76–79 the lateral cephalogram measurements for the pembarong performers and the control were processed using statistical product and service solution (spss) sofware version 17 (ibm company, armonk, new york, as). the data was then analysed by means of a kolmogorov-smirnov test to ascertain its distribution. at the next stage, an independent t-test was carried out to identify potential differences between the pembarong performers and the control group. a correlation test was subsequently conducted to determine the effects of bite force on cephalometric variation measurement. bite force value is specific to each individual depending on his or her day-to-day activities. the bite force of pembarong performers (n=15) and control (n=13) were measured, the purpose being to compare the effects of bite force on the cephalometric variation measurement of the pembarong performers with those of the control. the tools used to measure the bite force were calibrated at the department of material and metallurgical engineering, faculty of industrial technology, institute of technology surabaya. the calibration equation obtained was y = 4383.421x with y representing bite force (newton) and x the resistance (ohms). the variables of bite force were sna, snb, anb, y-axis, mp-pp, mpa, wits apraisal, pfh/afh ratio, and lfh/tfh ratio. all were normally distributed, resulting in homogeneous data with a p value >0.05. a difference test was subsequently conducted on each variable. results the average value bite force of the pembarong performers was 394.3816 newtons, while that of the control group stood at 371.7784 newtons. the cephalometric measurement indicated that there was no correlation between bite force and sna, snb, anb, y-axis, mp-pp, mpa, wits appraisal, as well as pfh/tfh ratio (all p values were >0.05). however, correlation did exist between bite force and lfh/tfh ratio (p = 0.13). the correlation coefficient (pearson) obtained was -.464 confirming the existence of an inverse correlation between bite force and lfh/tfh ratio. in other words, if the bite force value is significant, then the lfh/tfh ratio will be small. in addition, the regression model of lfh/tfh was 124.756-0.197 (bite force). this indicates that the lfh/tfh ratio can be identified based on the regression constant of 124.756 0.197 (bite force obtained). consequently, the ratio of lfh/tfh can be measured only by identifying the value of bite force and vice versa. discussion bite force is usually employed as an indicator of masticatory function and tooth load, which is relatively and clinically measurable.3 the average value of bite force of the pembarong performer was 394.38 newtons, while that of the control group was 371.78 newtons. nanda and kapila similarly found there to be a considerable contrast in bite force with the normal occlusion of 423.27 ± 113.92 newtons.9 this discrepancy depends on racial type since the caucasian race, for example, differs from its deutromalay counterpart. the former has different social habits and facial patterns that have endured over a long period. for 8 figure 4. cephalometric landmark points of the subjects (s, n, ans, pns, p, go, gn, me). figure 5. tracing tools. figure 6. pfh of the pembarong performer (left) was shorter than that of the control group (right). figure 3. pfh of the pembarong performer (left) was shorter than that of the control group (right). table 1. mean, standard deviation (sd), and significance values (p) of the bite force of pembarong performers and control based on results of the t-test maximum bite force n mean sd p pembarong 15 394.3816 7.68787 .000 control 13 371.7784 4.77791 table 2. results of the correlation test with pearson values (r) and significance values (p) no. variables correlation test r p 1 sna -.74 .707 2 snb -.126 .523 3 anb .057 .774 4 y-axis .136 .491 5 mp-pp .108 .583 6 mpa .044 .823 7 wits ap .198 .313 8 pfh/afh .054 .785 9 lfh/tfh -.464 .003 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p76-79 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p76-79 7979christina, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 76–79 instance, the cranial-facial morphology of the inuit features hypertrophy of the muscular mass in the mandibular angular region due to their dietary patterns.10 other differences result from the sensitivity and accuracy of the measuring instrument employed.2 the bite force of the pembarong subjects studied was significant due to their habit of biting down on the support incorporated into a 60 kg barongan and dadap peacock mask during dances and exercise. bite force that is exerted comparatively often with a specific frequency in normal daily functions will result in a relatively constant value.8 the complex dentofacial growth system is related to three components, namely; muscle function, skeletal growth, and tooth development.4 biting a mask weighed 60 kg can lead to excessive muscle contractions in the face, neck, and teeth. when a pembarong performer bites the mask, the bones of his head and face areas will be subjected to considerable pressure due to the excessive contraction of the facial muscles (temporal, masseter, bucinator, oral floor muscles). the resulting facial and head muscle function will then interfere with and hinder normal bone development which results from the stimulation of muscle function forwarded to the bone.1 masseter and temporalis muscle contractions occur unilaterally when biting with maximum force.11 there was no significant difference in the ratio of the posterior facial height the anterior facial height between pembarong performers and the control group.1 the low correlation between bite force and the anterior-posterior facial height ratio indicates that bite force does not directly affect the cephalometric variation measurement.8 there was a significant difference in the ratio of lfh/ tfh (figure 3). the posterior facial height of the pembarong performer was shorter than that of the control group. this is possibly due to tooth wear leading to shortening of the posterior facial height. bite force affects the shortening of muscle mass.2 bite force will generate constant occlusal force, resulting in the incisors appearing elongated and the overbite increasing, as a consequence shortening the posterior facial height.8 moreover, individuals with shorter faces tend to exert greater bite force than that of those with longer faces. this difference is statistically significant because of the increase in the tooth occlusal contact area in short-faced people.12 powerful bite force will probably render the lfh/tfh ratio slight because of the compactness of the microscopic alveolar bone structure. therefore, the bite force will induce pressure in the surrounding alveolar bone. under normal circumstances, a physiological regulation occurs within the bone, with pressure side osteoclasts being formed directly on the periodontal ligaments.4 moreover, the power applied when biting something is more than the normal average capacity. this can lead to bone atrophy due to an increase in bone remodeling and inhibition of the formation of osteoblasts, with the result that the trabecular bone may disappear and the thickness of the cortical bone may increase starting with the endosteal surface.10 similarly, reduced alveolar bone trabecular density will occur in persons with a powerful bite force.13,14 a significant correlation also existed between bite force and alveolar bone thickness during mastication (during the alveolar bone remodeling phase). therefore, if the mastication function proves adequate, the development of the mandible, especially the alveolar bone, will be stimulated.4 high pressure during the mastication process will then improve bone remodeling. the pressure along the bone generated by the bite force, decreasing from the cervical to the tooth root, can reduce the thickness of the alveolar bone.4 in conclusion, bite force, while having no effect on the sagittal dimension, does affect the vertical dimension. there is bite force impact on the lfh/tfh ratio, namely a negative correlation. the more powerful the bite force, the smaller the lfh/tfh ratio will be. this means that the posterior facial height will be smaller due to the shortening of alveolar bone mass. references 1. goenharto s, usman p. perbedaan tinggi wajah pembarong dan bukan pembarong pada kesenian reog ponorogo. maj ked gigi (dent j). 2003; 36(2): 42–4. 2. koc d, dogan a, bek b. bite force and influential factors on bite force measurements: a literature review. eur j dent. 2010; 4(2): 223–32. 3. bakke m. bite force and occlusion. semin orthod. 2006; 12(2): 120–6. 4. lemos ad, gambareli fr, serra md, pocztaruk r de l, gavião mbd. chewing performance and bite force in children. brazilian j oral sci. 2006; 5(18): 1101–8. 5. thongudomporn u, chongsuvivatwong v, geater a. the effect of maximum bite force on alveolar bone morphology. orthod craniofac res. 2009; 12(1): 1–8. 6. ash mm, ramfjord s. occlusion. 4th ed. philadelphia: w.b. saunders company; 1995. p. 87-8. 7. salzman ja. orthodontics in daily practice. pennsylvania: lippincott williams and wilkins; 1974. p. 75-6. 8. braun s, bantleon hp, hnat wp, freudenthaler jw, marcotte mr, johnson be. a study of bite force, part 1: relationship to various physical characteristics. angle orthod. 1995; 65(5): 367–72. 9. nanda r, kapila s. current therapy in orthodontics. st louismissouri: mosby elsevier; 2010. p. 227-35. 10. graber tm, vanarsdall rl. orthodontics : current principles and techniques. missouri: mosby inc; 2000. p. 663. 11. bakke m, michler l, han k, möller e. clinical significance of isometric bite force versus electrical activity in temporal and masseter muscles. scand j dent res. 1989; 97(6): 539–51. 12. proffit wr, fields hw, sarver dm. contemporary orthodontics. 4th ed. st louis-missouri: mosby elsevier; 2007. p. 151. 13. braun s, bantleon hp, hnat wp, freudenthaler jw, marcotte mr, johnson be. a study of bite force, part 2: relationship to various cephalometric measurements. angle orthod. 1995; 65(5): 373–7. 14. kamegai t, tatsuki t, nagano h, mitsuhashi h, kumeta j, tatsuki y, kamegai t, inaba d. a determination of bite force in northern japanese children. eur j orthod. 2005; 27(1): 53–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p76-79 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p76-79 137137 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 137–142 original article introduction tooth eruption is a normal physiological process, but it is considered to be abnormal if there is interference or delay. as teeth reach their functional position in the jaw arch, the tooth germs move through three distinct phases, including the pre-eruptive phase, the eruption phase and the posteruptive phase. in the post-eruptive phase, the teeth are in the functional position.1 as in humans, a rat’s teeth develop through interactions between dental epithelium and neural crest cells. a rat’s first molar develops through multiple stages, including dental sheet proliferation, the bud stage, the cap stage, the bell stage and finally, the eruption phase.2 there are various factors that affect tooth eruption, such as root development, alveolar bone remodelling and periodontal ligaments and other predisposing factors, such as endocrine hormones, vascular changes and enzymatic degradation.3 in humans, the onset of diabetes during pregnancy is known as gestational diabetes mellitus (gdm), and it can lead to negative effects on the child’s tooth eruption. a women with gdm has insulin deficiencies that can cause metabolic abnormalities and lead to malnutrition in the fetus.4,5 severe and prolonged malnutrition in the early life of a fetus can later cause delays in the tooth eruption phase.1 diabetes is usually treated by controlling glucose levels in the blood with healthy diet and exercise, but prescription drugs, such as metformin, can also be used to control glucose levels by absorbing glucose through glucose transporters (gluts).6 metformin has gastrointestinal side effects, such as diarrhoea, nausea and vomiting, and these side effects occur in approximately 50% of patients.7 to reduce dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 comparison of rat tooth eruption in rats born from diabetic mothers salsabila qotrunnada, dina z. ummah and mei syafriadi laboratory of oral pathology, department of biomedical sciences, faculty of dentistry, university of jember, jember, indonesia abstract background: tooth eruption begins after crown and root formation and may be delayed by gestational diabetes mellitus. metformin can control blood glucose levels through gluconeogenesis inhibition, and consuming thymoquinone for diabetic treatment will regenerate pancreatic β cells and reduce oxidative stress. purpose: the objective of this study is to compare the tooth eruption in rats that were born with diabetes and are being treated with either metformin or thymoquinone. methods: this study used 48 wistar rats (rattus norvegicus l.), and the rat sample was divided into four groups, including rats who were born from healthy mothers, rats who were born from untreated diabetic mothers, rats who were born from diabetic mothers that were treated with metformin and rats who were born from diabetic mothers that were treated with thymoquinone. diabetes was induced by intraperitoneal injection of a single dose of streptozotocin (40 mg/kg bb). each rat sample was taken with simple random sampling from different mothers, and body weight, blood glucose levels and levels of tooth eruption were recorded. eruptions of the maxillary right first molar were measured from the cusp of the tooth to the alveolar epithelial lining. results: based on the measurements of tooth eruption, it was found that groups a, c and d were closer to mucosa on day 1, 7 and 14 than group b. based on statistical analysis, there were significant differences (p = 0.03) between group b and groups c and d. conclusions: rats born from untreated diabetic mothers have more delays in tooth eruption than those born from diabetic mothers who are treated with metformin and thymoquinone. thymoquinone has the potential to be an alternative to metformin because it has been shown to be similarly effective. keywords: diabetes; eruption; thymoquinone; tooth correspondence: mei syafriadi , department of biomedical sciences, faculty of dentistry, university of jember. jl. kalimantan i no. 37, jember 68121, indonesia. email: didiriadihsb@gmail.com mailto:didiriadihsb@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 138 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 137–142 the side effects of metformin, this researcher tried using thymoquinone (nigella sativa l.; derived from black cumin seeds). it is bioactive, and it can control blood glucose levels and regenerate pancreatic β cells.8 thymoquinone is a terpenoid compound (essential oil) and has volatile properties that are difficult to dissolve in water, therefore, it is recommended that thymoquinone be dissolved in an oil solvent (herbal or olive oil). many functions of thymoquinone offer health benefits,9 but its effectiveness in preventing tooth eruption disruption has not yet been reported. other side effects have also have been reported, such as allergic rash, stomach problems and, with long-term use, hyperlipidaemia,10 but these need further research. the purpose of this study is to examine tooth eruption in rats born from untreated diabetic mothers and in rats born from mothers treated with either metformin or thymoquinone. materials and methods this study was conducted in vivo with a post-test as the only control group., it was approved by the medical research ethics committee at the faculty of dentistry, university of jember, no.586/un25.8/kepk/dl/2019, and it was carried out in a biomedical and animal science laboratory. sixteen pregnant wistar rats were divided into four groups with each group consisting of four samples. group a consisted of normal pregnant rats with blood glucose levels < 126 mg/dl, group b consisted of pregnant diabetic rats who received no treatment, group c consisted of pregnant diabetic rats who received metformin treatment, and group d consisted of pregnant diabetic rats who received thymoquinone treatment. the pregnant wistar rats from groups b, c and d were induced with diabetes using streptozotocin (stz) obtained from bioworld (genelinx international inc., dublin, ohio, usa) on day 10 of gestation by dissolving 40 mg/kg/bw stz powder in 50 mg/ml 0.1 m citric acid buffer solution with a ph of 4.5.11,12 one day after stz injection, blood glucose levels were measured at > 126 mg/dl, and the rats were categorized as diabetic. after inducing diabetes in groups b, c, and d, group c was given metformin obtained from hexapharm jaya laboratories (kalbe company, bekasi, indonesia) at a dose of 100 mg/kg/bw dissolved in 1.5 ml of distilled water and administered intragastrically using an intragastric tube twice a day (every morning and evening). group d was given thymoquinone obtained from merck (sigma aldrich inc., st. louis, missouri, usa) at a dose of 80 mg/kg/bw13 dissolved in 1.5 ml of olive oil and administered intragastrically once a day.14 rats in groups c and d received their respective doses everyday up until 14 days postnatal. after each of the rats gave birth, a research sample was obtained by taking one baby rat from each of the mother rats in all four groups on days 1,7 and 14 postnatal using simple random sampling. this provided 48 baby wistar rats whose teeth were observed (figure 1). rats induced with diabetes by stz on the 10th day of pregnancy figure 1. grouping and sampling diagram. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 139qotrunnada et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 137–142 postnatal rats from each group were euthanized on days 1, 7 and 14 with ketamine obtained from kepro bv (maagdenburgstraat, za, deventer, netherlands) using the overdose method. in each of the rats, the molar region of the right maxillary was removed and placed in a pot with 10% formalin solution for 24 hours and then decalcified using a 10% formic acid solution for 7 days to remove inorganic material from the bones. the tissue was then processed using the paraffin-embedded tissue technique. the paraffin block was cut using microtome with a thickness of 6 μm, and hematoxillin & eosin was used for staining. histopathological appearance was observed under a microscope (olympus cx-21) at 40x magnification that was connected to an optilab advanced v2 camera and computer obtained from miconos (yogyakarta, indonesia) to view histological images. the distance from the outer enamel epithelium of the tooth germ to the surface of the epithelial of the alveolar epithelial lining on the maxillary right first molar was measured using raster image processor software (miconos, yogyakarta, indonesia), and pre-dentin/pre-enamel formations, hertwig’s epithelial root sheath (hers) formations, tooth root formations, and bifurcation formations were also observed.2,15 statistical package for the social sciences (spss) software (version 26, ibm, new york, usa) was used. the normality test of the data used shapiro wilk and the homogeneity test used the levene test (p ≤ 0.05). if the statistical test results were not normally distributed and not homogeneous, then the data was tested using the mann-whitney and kruskal-wallis tests (nonparametric; p ≤ 0.05). results the results showed increases in blood glucose levels in groups b, c, and d after stz injections (blood glucose ≥ 126 mg/dl; table 1). based on the mann-whitney test, there were significant differences (p ≤ 0.05) between all the groups across all observation days (table 2). average bodyweights of the rats 1 day postnatal showed that group b had the lowest average bodyweight, and based on statistical tests between all groups, only group a and b were significant at 1 day postnatal (p = 0.01). in groups c and group d at 14 days postnatal, the average bodyweights differed but not significantly (p = 0.04; table 3). table 1. pregnant diabetic rat average blood glucose levels (mg/dl) group stz post injection 7th day 14th day a 96±5.4 (without stz injection) 66.75±13.5 66.75±13.5 b 411.5±77.9 250.75±233.6 169.75±99.6 c 144.6±31.7 299.00±260.8 149.00±12.2 d 23.3±172.2 141.00±18.3 97.50±1.2 table 2. statistical test results (mann-whitney) for blood glucose levels of pregnant diabetic rats group/day a b c d 1 7 14 1 7 14 1 7 14 1 7 14 a 1 .021 .034 .050 7 .020 .032 .032 14 .042 .020 .060 b 1 .021 .289 .480 7 .020 .372 1.000 14 .042 1.000 .355 c 1 .034 .289 .827 7 .032 .372 .275 14 .020 1.000 .240 d 1 .050 .480 .827 7 .032 1.000 .275 14 .060 .355 .240 a: normal group; b: negative control group; c: metformin group; d: thymoquinone group table 3. the average bodyweight of postnatal rats group postnatal weights (g) 1st day 7th day 14th day a 6.50±0.6 p=0.01 10.25±2.9 18.00±4.3 b 4.75±0.5 8.25±2.1 18.00±5.0 c 6.33±1.2 8.67±1.2 15.00±1.0 p=0.04 d 6.00±1.0 9.33±2.5 18.00±0.0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 140 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 137–142 at 1 day postnatal, the development of the maxillary right first molar of each of the rats was in the bell stage and pre-dentin/pre-enamel was detected, except for one sample in group b that had growth retardation and was still in the cap stage. on day 7 postnatal, observations showed that all samples had entered the stage of apposition and calcification with the formation of hers, except for one sample in group b that was still in the bell stage. the growth and development of teeth on day 14 postnatal in groups a and c showed that all samples had entered the eruption stage, marked by the formation of roots and root bifurcation. in group b, 50% of the samples were in the eruption stage, and in group d, 66% of the samples were in the eruption stage (figure 2). based on measurements of the distance from the cusp of the maxillary right first molar to the alveolar epithelial table 4. measurements of eruption distance of right maxillary first molars (μm) group 1st day 7th day 14th day a 348.00±69.9 322.00±88.5 196.77±69.7 b 339.58±81.9 397.00±95.1 259.23±32.3 c 323.58±45.3 368.16±175.4 153.61±74.3 d 267.98±8.4 344.00±129.3 152.03±47.4 table 5. the statistical test results (mann-whitney) for the measurements from maxillary right first molar cusps to the alveolars epithelial lining group/day a b c d 1 7 14 1 7 14 1 7 14 1 7 14 a 1 .773 .480 .289 7 .248 1.000 .480 14 .248 .480 .480 b 1 .773 1.000 .157 7 .248 .724 .289 14 .248 .034 .034 c 1 .480 1.000 .827 7 1.000 .724 .593 14 .480 .034 .513 d 1 .289 .157 .827 7 .480 .289 .593 14 .480 .034 .513 lining, it was observed that the distances decreased from day 7 to day 14 (table 4). statistical analyses showed that there were significant differences between group b and group c and significant differences between group b and group d (significance of 0.03; p ≤ 0.05; table 5). figure 2. histological features of a right maxillary m1 in a postnatal rat. a: delayed tooth development shows the cap stage (sample from group b; day 1 postnatal); b: a histological picture of the bell stage (groups a, c and d; day 1 postnatal); c: the apposition and calcification stage on day 7 postnatal; d: shows eruption (grey arrows indicate inner enamel epithelium, black arrows indicate outer enamel epithelium, hashtags indicate hers and stars indicate root formation). notes: dp: dental papilla; sr: stellate reticulum; d: dentine; e: enamel; r: reduce enamel epithelium; b: bifurcation. (a-c: he staining 40x magnification; d: he staining 5x magnification) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 141qotrunnada et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 137–142 discussion diabetes induced by stz injection can affect glucose oxidation and decrease biosynthesis and insulin secretion through the glut-2 glucose transporter. this leads to decreased sensitivity of peripheral insulin receptors and can increase insulin resistance and blood glucose levels.10,11 there were decreases in blood glucose levels in the negative control (group b) who had not received treatment. a study showed that this may be due to spontaneous selfrepair mechanisms because, although streptozotocin is an alkylating agent and can damage dna and pancreatic β-cells, it is dose-dependent, and the study showed that after eight hours of exposure to stz, 55% of mitochondrial dna cells repaired themselves and rose to 70% in 24 hours. 12 meanwhile , the increase in blood glucose levels on day 7 in the rats treated with metformin was thought to be due to damage of pancreatic β cells from the stz injection. the effects of metformin were seen after day 14 when there were decreases in blood sugar levels, but normal levels were not reached due to metformin’s ability to reduce glucose absorption through the glut and inhibit gluconeogenesis.13 decreased blood glucose levels were found in group d because thymoquinone can stimulate insulin release by inhibiting oxidative stress, and it stimulates the regeneration of pancreatic cells.14 untreated diabetes in pregnant rats has significant effects on the bodyweight of their offspring. the rats in the negative control (group b) had lower bodyweights on day 1 when compared to the other groups. this was perhaps due to the stress of endoplasmic reticulum placenta (erp) in utero. it is known that the function of erp is related to nutrient transportation, and stress to the erp usually results in phosphorylation from translational initiation factor 2 and eukaryotic initiation factor 2 α (eif2α) that are targets for several serine kinases that phosphorylate serine and lead to the inhibition of protein translation and the signalling of mammalian target of rapamycin (mtor) that functions as a protein kinase serine/threonine that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy and transcription. it also functions as a protein kinase tyrosine that activates insulin receptors and insulin-like growth factor 1 (igf-1) receptors, and this can inhibit transportation of nutrients to the fetus.16,18 disruption in placental function can cause intrauterine growth restriction (igr) that can result in a baby being born with low birthweight.19 from day 7 to day 14, the bodyweight of each rat in the four groups increased in line with their ability to eat. this might be the rat born from diabetic mother rat without treatment also able to achieve the same weight as rat born from a normal rat. the rats in group d appeared healthy, had normal bodyweights and showed normal growth and development when compared to the rats in group a who were born from healthy mothers. it can be concluded that thymoquinone, when given to diabetic mothers during pregnancy, can function as an anti-hyperglycaemic medication.20 this was shown at the end of the study on day 14 when the mothers’ glucose reached normal levels and breastfeeding was sufficient for normal growth and development. nevertheless, on the day 14, it was found that, although the rats in group c gained weight consistently up to day 14, they still gained weight at a slower rate than the rats in other groups. this may be due to the fact that their mothers were consuming metformin. it has been reported that metformin works on the central nervous system and can reduce appetite. 21 loss of appetite in the mother rat can affect nutritional adequacy during breastfeeding and may reduce their offspring’s bodyweight.21 rats born from diabetic mothers may experience growth retardation and delays in the growth and development of their teeth. severe diabetes in pregnant rats can also cause metabolic disorders and protein-energy malnutrition in the fetus due to oxidative stress that is known to disturb cell signalling during growth and development. in the absence of antioxidant activity, oxidative stress continues to increase and can cause extensive cell damage to formed protein, dna, and lipids.9 it was reported that a mother’s nutrition during pregnancy is directly related to the primary teeth eruption of her offspring.22 through thymoquinone therapy, delayed eruption can be prevented because thymoquinone is an antioxidant that can protect pancreatic β cells by decreasing oxidative stress and increasing the production of endogenous antioxidants in the body, such as glutathione peroxidase, superoxide dismutase and catalase, and improve metabolic disorders that cause malnutrition of proteinenergy in the fetus.22,23 in diabetic pregnant rats, metformin therapy can improve insulin sensitivity because it improves blood glucose levels, and developmental tooth disorders can be prevented. the teeth development process continues into the eruption stage. tooth eruption is a process that begins immediately after the crown is formed, followed by the formation of roots that are regulated by hers.1 the process of eruption could be seen in each rat on day 7 through day 14 by observing the shortening of the distance between the tooth cusp (outer enamel epithelium) and the alveolar epithelial lining. in general, a rat tooth will start to erupt when a crown reaches two thirds the length of its root.3 on the day 14 of observation, the negative control group was seen to have delayed eruption which was indicated by the distance of the tooth cusp to the alveolar epithelial lining which was deeper than the other groups. statistical tests showed that there were significant differences between the negative control and group c and significant differences between the negative control and group d (p = 0.03; p ≤ 0.05). the rates of tooth eruption in groups c and d (treated groups) were not significantly different. it was reported that root development takes place once hers has formed. 24 hers is an epithelial bilayer that extends to the apical below the cervical margin. the formed hers proliferates and enters the cervical margin area to form a barrier between the dental papilla and the dental follicle (periodontium).24 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 142 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 137–142 the formation of hers was signalled by the secreted protein sonic hedgehog (ssh/msx2; msh-like gene invertebrates) and igf-1 that have significant roles in tooth growth and development.24 it was also reported that a lack of igf-1 can result in stunted tooth growth and development, 25 and this is directly related to the tooth germs of rats born from diabetic mothers who have delayed eruption phases caused by a reduction of igf-1. from this study, it can be concluded that rats born from untreated diabetic mothers have more delays in tooth eruption when compared to those born from mothers treated with metformin or thymoquinone. thymoquinone has the potential to be an alternative to metformin because is similarly effective. acknowledgments the authors would like to thank all those who have assisted us during this study. we would also like to thank the chancellor of the university of jember and the research and community services institute (lp2m) who funded this study (keris grant no. 2763/un25.3.1/lt/2020; student final project publication grant no. 2618/un25.3.1/ lt/2020). we declare that there were no conflicts of interest before, during or after this study. references 1. choukroune c. tooth eruption disorders associated with systemic and genetic diseases: clinical guide. j dentofac anomalies orthod. 2017; 20(4): 402. 2. al qahtani sj, hector mp, liversidge hm. brief communication: the london atlas of human tooth development and eruption. am j phys anthropol. 2010; 142(3): 481–90. 3. kjær i. mechanism of human tooth eruption: review article including a new theory for future studies on the eruption process. scientifica (cairo). 2014; 2014(3): 189–209. 4. ghapanchi j, kamali f, siavash z, ebrahimi h, pourshahidi s, ranjbar z. the relationship between gestational diabetes, enamel hypoplasia and dmft in children: a clinical study in southern iran. br j med med res. 2015; 10(9): 1–6. 5. dewi n. lebar benih gigi anak tikus yang dilahirkan oleh induk tikus pengidap diabetes mellitus gestasional. dentino j kedokt gigi. 2014; 2(1): 46–50. 6. yang x, xu z, zhang c, cai z, zhang j. metformin, beyond an insulin sensitizer, targeting heart and pancreatic β cells. biochim biophys acta mol basis dis. 2017; 1863(8): 1984–90. 7. sanchez-rangel e, inzucchi se. metformin: clinical use in type 2 diabetes. diabetologia. 2017; 60(9): 1586–93. 8. yenita y. uji efektivitas pemberian minyak jintan hitam (nigella sativa l.) terhadap kadar gula darah mencit diabetes melitus yang diberi aloksan. bul farmatera. 2017; 2(2): 101–15. 9. dolatk hah n, hajifaraji m, shakouri sk. nutrition therapy i n m a n a g i ng p r eg n a nt wom e n w it h ge st a t io n a l d i a b e t e s mellitus: a literature review. j fam reprod heal. 2018; 12(2): 57–72. 10. balbaa m, el-zeftawi m, abdulmalek as, shahin ry. healthpromoting activities of nigella sativa fixed oil. in: ramadan fm editor. black cumin (nigella sativa) seeds: chemistry, technology, functionality and applications. springer. nature switzerland ag. 2021. p. 361–77. 11. firdaus f, rimbawan r, marliyati sa, roosita k. model tikus diabetes yang diinduksi streptozotonic-sukrosa untuk pendekatan penelitian diabetes melitus gestasional. media kesehat masy indones. 2016; 12(1): 29–34. 12. damasceno dc, netto ao, iessi il, gallego fq, corvino sb, dallaqua b, sinzato yk, bueno a, calderon imp, rudge mvc. streptozotocin-induced diabetes models: pathophysiological mechanisms and fetal outcomes. biomed res int. 2014; 2014: 819065. 13. diani a, pulungan ab. tata laksana metformin diabetes mellitus tipe 2 pada anak dibandingkan dengan obat anti diabetes oral yang lain. sari pediatr. 2016; 11(6): 395–400. 14. gray jp, burgos dz, yuan t, seeram n, rebar r, follmer r, heart ea. thymoquinone, a bioactive component of nigella sativa, normalizes insulin secretion from pancreatic β-cells under glucose overload via regulation of malonyl-coa. am j physiol endocrinol metab. 2016; 310(6): e394–404. 15. li j, parada c, chai y. cellular and molecular mechanisms of tooth root development. development. 2017; 144(3): 374–84. 16. tuval-kochen l, paglin s, keshet g, lerenthal y, nakar c, golani t, toren a, yahalom j, pfeffer r, lawrence y. eukaryotic initiation factor 2α--a downstream effector of mammalian target of rapamycin-modulates dna repair and cancer response to treatment. plos one. 2013; 8(10): e77260. 17. yin y, hua h, li m, liu s, kong q, shao t, wang j, luo y, wang q, luo t, jiang y. mtorc2 promotes type i insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mtor. cell res. 2016; 26(1): 46–65. 18. castillo-castrejon m, powell tl. corrigendum: placental nutrient transport in gestational diabetic pregnancies. front endocrinol (lausanne). 2019; 10: 5. 19. nasution yf, lipoeto ni, yulizawati y. hubungan kadar insulin-like growth factor 1 serum maternal dengan berat badan dan panjang badan bayi baru lahir pada ibu hamil kek. maj kedokt andalas. 2019; 42(3s): 19–29. 20. a l s a ’a i d i j a a , k a r e e m h m a , a l -ta m e e m i w t m . antihyperclycemic effects of thymoquinone in diabetic rats. basrah j vet res. 2014; 13(2): 180–92. 21. malin sk, kashyap sr. effects of metformin on weight loss: potential mechanisms. curr opin endocrinol diabetes obes. 2014; 21(5): 323–9. 22. badruddin ia, khansa m, darwita rr, rahardjo a. the relation of mothers’ nutritional status to primary teeth dental caries. int j appl pharm. 2017; 9(special issue 2): 141–3. 23. kusu ma a e , susia nt i s, ok t a r ia d. penga r u h p emb er ia n thymoquinone terhadap gambaran histopatologi jantung tikus putih (rattus norvegicus) galur sprague dawley yang diinduksi asap rokok. major med j lampung univ. 2019; 8(1): 84–9. 24. lungová v, radlanski rj, tucker as, renz h, míšek i, matalová e. tooth-bone morphogenesis during postnatal stages of mouse first molar development. j anat. 2011; 218(6): 699–716. 25. alkaff mn, syafriadi m. effect of diabetes during pregnancy to fetal tooth germ growth and development. j int dent med res. 2019; 12(4): 1328–34. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p137–142 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p137-142 163163 dental journal (majalah kedokteran gigi) 2019 september; 52(3): 163–167 case report reccurent trauma-induced aphthous stomatitis in adjustment disorder patients y. yuliana, saka winias, hening tuti hendarti and bagus soebadi department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: adjustment disorder is a temporary psychological condition related to emotional responses or behaviour in reaction to stress resulting from certain changes in a specific period of an individual’s life yet which does not significantly affect his/her daily life. recurrent aphthous stomatitis (ras) constitutes recurrent inflammation of the oral mucosa, in the form of an ulcer, frequently associated with psychological stress. occasionally, a patient does not realise that she/he is suffering from a psychological disorder until the emergence of clinical symptoms, among them recurrent ulcers the causes of which are unknown. purpose: this article presents a case of adjustment disorder diagnosed from symptoms observable in the oral cavity. case: the case involved a 21-year-old student who presented with the symptoms of large, painful ulcers on her tongue, the inside of her cheek, and the floor of her mouth. these symptoms had been observable for one month but remained untreated. the patient only ate once a day or even once every two days. an introverted personality, she did not associate with other people. case management: the procedure covered anamnesis, clinical examination, blood laboratory tests, total immunoglobulin e (ige), an antinuclear antibody (ana) test, bacterial and fungal culture in ulcus, questionnaire screening on psychological disorders, cortisol level examination and referral to a psychiatrist. symptomatic therapy administered to the patient led to recovery of the ulcer in 29 days. conclusion: ras can be triggered by psychological stress which induces changes in the immune system and oral mucosa tissue. keywords: adjustment disorder; cortisol level; reccurent aphthous stomatitis; ulser correspondence: saka winias, department of oral medicine, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: saka.winias@fkg.unair.ac.id introduction reccurent aphthous stomatitis (ras), repetitive inflammation of the oral mucosa, takes the form of a yellowish white shallow single or multiple ulcer surrounded by an erythematous halo. ras can develop in non-ceratine oral cavities such as the buccal, labial, lateral and ventral tongue mucosa in addition to the mucous membrane in the floor of the mouth, soft palatum and oropharynx. the condition usually presents as a burning sensation lasting for 24 to 48 hours before the development of the ulcers sufficiently painful as to impede activities such as talking and eating.1,2 the prevalence of ras amounts to approximately 20% of the population. ras begins in late childhood, between the ages of 10 and 20 years of age, and continues through to adulthood, the condition having been found to be more common among females than males.1–3 the etiology of ras remains unclear but has been tentatively attributed to environmental and genetic factors, including; stress, chemical or physical trauma, infection, systemic diseases, hormonal disfunction, nutrition deficiency (zing, folat and vitamin b12), food allergies (for example;chocolate, coffee, peanuts, cereals, almonds, and cheese).4–6 a stress-induced decline in the immune system seriously compromises the system itself; subsequently resulting in ras. furthermore, when a patient is under stress, local trauma caused by a bite can occur more frequently. stress constitutes a condition where a person is unable to cope with stressors which then negatively impacts affect his/her dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p163–167 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p163-167 164 yuliana, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 163–167 a b c d figure 1. a-d) painful ulcers, multiple, sizes: 2 x 3.5 mm; 5 x 8.5 mm; 6 x 6.5 mm; 6.5 x 5.5 mm; 6.5 x 8.8 mm; yellowish white centres surrounded by red areas. the surface is slimy with a clear border and a irregular edge. physical or mental condition. in cases of physical changes, stress will induce the body to release chemical substances that trigger the discharge of hormones and neurotransmitters into the bloodstream.6,7 adjustment disorder, a condition frequently related to either acute or chronic stress, is neither a permanent nor a psychotic disorder. rather, this condition results from the failure of various aspects of the patient’s general functioning caused by emotional responses when managing stress or specific changes.8,9 epidemiological studies show that between 2% and 8% of the total number of sufferers from adjustment disorder are female, a percentage twice as high as that of their male conterpartss. interestingly, single women run a high risk. adjustment disorder usually occurs three months after the emergence of stressors which can be recurrent or continuous in character.8,10 at the time of writing, no studies reporting cases of oral cavity problems related to adjustment disorder, such as ras, exist. this paper aims to report the case procedure for trauma-induced ras in patients with adjustment disorder. case the patient, a 21 year-old female student, visited the department of oral medicine of the dental hospital at universitas airlangga on 14 august 2019. the various symptoms she reported included ulcers on the tip and sides of the tongue, the floor of the mouth and the left and right cheeks which had suddenly appeared a month before and had yet to recover due to their not having been treated. the patient had presented the same symptoms six months previously which had necessitated a two-month period of recovery. the patient consumed only vitamin c and a refreshing drink to cure her ulcer. she did not report suffering any allergies, but seldom consumed fruit and vegetables and ate irregularly, i.e. once a day or even once every two days. she complained of the ulcer causing her discomfort when eating or speaking. in terms of her social history, the patient claimed that she was shy, chose to live a quiet life and lacked definite life goals. moreover, she was lacking in self-confidence, found it difficult to socialize with new acquaintances and, on those occasions when willing to associate with others, feared rejection. during the anamnesis period, the patient appeared restless and worried. her hands were cold and the answers she gave to questions were delivered with flat intonation. clinical extraoral examination of the patient’s left and right lymph nodes revealed no abnormality and she reported no pain. furthermore, intraoral examination revealed multiple painful yellowish-white ulcers with a clear border surrounded by red areas. the dimensions of the ulcers were 6.5 mm x 5.5 mm on the right mucosa, 2 mm x 3.5 mm on the left mucosa; 6 mm x 6.5 mm on the lateral of tongue; 5 mm x 8.5 mm on the tip of the tongue; and 6.5 mm x 8.5 mm on the floor of the mouth (figure 1). the provisional diagnosis was one of suspected aphthous stomatitis with a differential diagnosis of muchous membrane pemphigoid (mmp). case management the prescribed treatment consisted of benzydamine hcl 0,15% mouthwash used four times a day and theragran-m taken once a day. the patient was referred to the of pathology clinic laboratory for a comprehensive blood examination, total ig e test, and ana test, in addition to the periodontics clinic for tartar descaling. a depression anxiety stress scale (dass 42) examination indicated that the patient’s level of anxiety was severe (score 17), while that of her depression was moderate (score 14). these findings resulted in the patient being referred for a cortisol level test. the patient was instructed to take medicines in accordance with the prescribed schedule, maintain oral hygiene, consume vegetables and fruit, eat more regularly, and manage her anxiety. on her second visit to the hospital on the fifth day, the patient reported a reduction in pain and the complete healing of the ulcer on her right buccal mucosa, while that on the left had reduced in size. conversely, the ulcer located on the border of the tongue had continued to increase in size and the one on the tip of the tongue was swollen and reddish in colour. the result of a complete blood examination produced normal scores for total ig e and an ana test. with the exception of the eosinophil which was 30/ul (normal 80-360 /ul), the level of cortisol in the afternoon dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p163–167 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p163-167 165yuliana, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 163–167 a b c d figure 2. a-b) size of painless ulcers 6.5 x 1.5 mm; 5.5 x 6.5 mm; c-d) size of erosion ø 0.5 mm; 2 x 3 mm. a b c d figure 3. a-d) healed ulcer of patients (day 29). was 17.39 ug/dl (normal 2.69 – 10.4 ug/dl). the previous symptomatic therapy was discontinued and replaced with a new prescription, namely; a 0.5 mg dexamethasone tablet in powder form mixed with water and gargled three times a day, while the once-daily administration of theragran-m was continued. furthermore, the patient was referred to the microbiology laboratory for bacteria and fungus analysis, as well as to a psychiatrist . on the third visit (day 21), the patient reported that the ulcers were no longer painful and that her general mood had improved following her discussions with the psychiatrist. the microbiology examination results for bacteria culture and fungus were both negative. the intraoral image result indicated lesion erosion on the floor of the mouth, left buccal mucosa and tip of the tongue. the ulcer on the edge of the tongue had already reduced in size (figure 2). based on the minnesota multiphasic personality inventory (mmpi) psychiatric examination result, the patient was diagnosed as suffering from adjustment disorder (f43.20). no pharmacological therapy was undertaken, merely the provision of supportive recommendations that the patient should strive to manage both her anxiety and stress. psycho-education was also provided by suggesting a change of lifestyle, including an attempt to gain weight and undertake regular exercise. the diagnosis in this case was one of aphthous stomatitis with a differential diagnosis of ras. by the fourth visit (day 29), the ulcers had completely recovered (figure 3). the patient reported her anxiety as being reduced and that she felt more confident and comfortable. however, she looked less restless, only gradually opening up when required to converse, but claimed to be eating more frequently (3-4 times a day) and to have gained three kilograms in weight since her first visit to the hospital. one month after her most recent check-up, the patient reported that two small ulcers had developed on the tip of her tongue. she admitted to feeling under pressure due to having to prepare a research proposal. the final diagnosis arrived at was one of trauma-induced ras within a case of adjustment disorder (ad). the patient was instructed to continue the use of mouthwash, maintain oral hygiene, follow a healthy diet and manage her stress levels. discussion a diagnosis of trauma-induced recurrent aphthous stomatitis in this individual who was suffering from adjustment disorder had been arrived at on the basis of anamnesis, clinical description and supporting examination. with regard to anamnesis, the patient acknowledged being depressed and anxious about her brother-in-law, a new member of the family who had married her sister eight months before. she was experiencing difficulty in adapting to this new situation and even experienced feelings of terror when attempting to communicate with him. this situation led her to pay only infrequent visits to her home town in order to avoid meeting this individual. the primary conflict that the patient experienced was between her genuine willingness to socialize and the fear of not being appreciated, and possibly even rejected, by others. the patient preferred to spend her time alone reading books about outer space, dinosaurs and superstitions. her thoughts dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p163–167 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p163-167 166 yuliana, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 163–167 were dominated by the invisible or intangible. she appeared unconcerned about her own well-being, eating only once a day or once every two days due to having forgotten to do so. at times, she felt her life to be empty, aimless, and hypoactive. the results of dass 42 showed her levels of anxiety, depression and stress to be high, moderate and normal respectively. examination of the subject’s cortisol level in the afternoon confirmed it as high. aphthous stomatitis constituted the initial diagnosis because the frequency with which ulcers occurred was twice every 6 months. therefore, the subject’s prevailing condition could not be classified as ras. an ana test produced a negative result, indicating that the prediction of autoimmune disease such as mmp, which constituted the differential diagnosis, was negative. the image of the tip of the tongue appeared to indicate a case of mmp. this led to a preliminary diagnosis of autoimmunity. examination of total ig e, bacteria culture and fungus produced negative results. consequently, the prediction of allergy, infection, bacteria and fungus was disproved. eosinopenia, a condition whereby the amount of eosinophil produced is extremely low, highlighted such hazards as consuming steroid-based medicines, excessive cortisol production, and overdulgence in alcohol.11 one of the quality-tested instruments employed to measure stress levels is dass 42 which consists of 42 questions designed to measure the level of negative emotions such as anxiety, depression, and stress suffered by a patient.9 in this case study, a psychiatric condition was diagnosed since the patient suffered from adjustment disorder as confirmed by an mmpi examination. the patient was recommended to adopt a new lifestyle, gain weight by eating more frequently, and take exercise, for example jogging, on a daily basis. adjustment disorder is maladaptive reaction to an identifiable psychosocial stressor occurring three months after its emergence. the reaction to one or two stressors in a person’s life is more extreme than the normal reactions of others to those same stress factors. these symptoms are supposed to dissipate six months after the stressors appear but may persist for longer if they are eradicated. psychiatry normally helps patients with adjustment disorder directly by providing support during critical periods and seeking to encourage the patient to manage their stressors through coping mechanisms. psychotherapy is the preferred choice of treatment for adjustment disorder.8,10 stressors induce a drastic increase in the level of stress hormones while, under normal conditions, the amount released daily into the body is extremely limited. first, the hypothalamus in the brain releases corticotrophin releasing factor (crf) into the bloodstream, which eventually reachest he pituitary gland located below the hypothalamus. crf stimulates the release of adenocorticotrophin hormone (acth) which, in turn, induces the adrenaline gland to discharge various hormones, including cortisol. cortisol circulates within the body and plays important role in the coping mechanism. the secretion of cortisol can increase 20-fold when an individual under stress, anxious and/or depressed. a high level of cortisol will intensify the regulation activity of the immune system through inflammation by increasing the quantity and quality of leucocytes. when stressed, patients do not consciously move the jaw, tongue, lips or cheeks which can result in the trauma of accidental self-inflicted bites. the tongue tends to be active and, therefore, it is most often affected by trauma or friction from the teeth which necessitates a lengthy recovery period. under stable emotional conditions, effective coping mechanisms send signals to the brain instructing it to stop releasing crf.7 gargling with benzidamine hcl, an anti-inflammation nonsteroid (nsaid) medicine with analgesic effects as a local anaesthetic during the patient’s first hospital visit helped to reduce pain in her ulcerated mouth. benzydamine hcl 0.15% consists of 22.5 mg benzydamine hcl per every 15 ml of solution.12 in addition, the patient was also administered a theragran-m tablet on a daily basis which is a supplement consisting of multivitamins and minerals used to enhance the immune system and accelerate wound recovery. the ulcerated area inside the mouth improved significantly after provision of dexamethasone 0.5 mg contained in mouthwash. dexamethasone is a corticosteroid of the glucocorticoid class which induces an adequate anti-inflammation effect by pressuring inflammation mediators such as tnf-α, il-6 dan il-1.13 curcuma is a food supplement containing 20 mg of curcuma xanthorrhiza extract whose function is to enhance the appetite and promote the metabolism. chlorine dioxide mouthwash is an antibacterial whose function is to strengthen mucosa cell walls in order to accelerate wound recovery.14 the final visit report stated that all ulcers had been eradicated and only reappeared after one month, thereby proving that the ulcers were already in the recurrence stage. this is in line with the theory stating that ras usually starts in sufferers at the age of 10-20 years.1–3 ulceration as a result of the psychological disorders suffered by this patient was supported by the dass result: high levels of cortisol and, conversely, low levels of eosinophil. therefore, the final diagnosis arrived at was one of trauma-induced ras as part of adjustment disorder. it can be concluded that oral ulcers may be the result of psychological disorders which can also induce changes in the immune system and oral mucous tissue. eliminating etiology is used as a therapy to prevent recurrence of ras. dentists play an important role in detecting psychological disorder-related oral abnormalities. references 1. bruch jm, treister ns. clinical oral medicine and pathology. new delhi: humana press; 2010. p. 1–169. 2. gallo c de b, mimura mam, sugaya nn. psychological stress and recurrent aphthous stomatitis. clinics. 2009; 64(7): 645–8. 3. ślebiod a z , szpona r e , kowa lska a. et iopat hogenesis of recurrent aphthous stomatitis and the role of immunologic aspects: dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p163–167 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p163-167 167yuliana, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 163–167 literature review. vol. 62, archivum immunologiae et therapiae experimentalis. birkhauser verlag ag; 2014. p. 205–15. 4. george s, baby joseph b. a study on aphthous ulcer and its association with stress among medical students of an indian medical institution. int j contemp med res. 2016; 3(6): 1692–5. 5. glick m, feagans wm. burket’s oral medicine. 12th ed. shelton: people’s medical publishing house; 2015. p. 733. 6. sari rk, ernawati ds, soebadi b. recurrent aphthous stomatitis related to psychological stress, food allergy and gerd. odonto dent j. 2019; 6: 45–51. 7. lisdiana. regulasi kortisol pada kondisi stres dan addiction. biosantifika. 2012; 4(1): 18–26. 8. carta mg, balestrieri m, murru a, hardoy mc. adjustment disorder: epidemiology, diagnosis and treatment. clin pract epidemiol ment heal. 2009; 5: 1–15. 9. ali i. adjustment disorders (stress related or psychiatric disorder). j psychiatry. 2015; 18(5): 1–2. 10. o’donnell ml, agathos ja, metcalf o, gibson k, lau w. adjustment disorder: current developments and future directions. int j environ res public health. 2019; 16(14): 2537. 11. kementerian kesehatan republik indonesia. pedoman interpretasi data klinis. jakarta: kementerian kesehatan republik indonesia; 2011. p. 1–83. 12. firza ta, umar n, ihsan m. perbandingan obat kumur benzydamine hydrochloride 22,5 mg dan ketamin 40 mg dalam mengurangi nyeri tenggorok dan suara serak akibat intubasi endotrakeal. j anestesi perioper. 2017; 5(1): 57–66. 13. erlangga me, sitanggang rh, bisri t. perbandingan pemberian deksametason 10 mg dengan 15 mg intravena sebagai adjuvan analgetik terhadap skala nyeri pascabedah pada pasien yang dilakukan radikal mastektomi termodifikasi. j anestesi perioper. 2015; 3(3): 146–54. 14. setiadhi r, firman dr. obat di bidang penyakit mulut dan penulisan resepnya. bandung: unpad press; 2017. p. 1–61. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p163–167 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p163-167 vol 49 no 1 jan-mrt 2016.indd 11 case report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 1–4 methisoprinol as an immunomodulator for treating infectious mononucleosis maharani laillyza apriasari departement of oral medicine faculty of dentistry, universitas lambung mangkurat banjarmasin indonesia abstract background: infectious mononucleosis (im) is the self limiting disease that associated with primary epstein barr virus (ebv). it is a gamma herpes virus. ebv infection is follows saliva-transfer by kissing or sexual intercourse. the most clinical manifestation in im consists mainly of the specific sign: pharyngitis, fever, and lymphadenopathy. the main therapy is supportive treatment. actually the antiviral therapy is required for the host with high response immune. purpose: the aimed of this study was to report the therapy of im using methisoprinol. case: the woman patient, 33 years old, came to hospital by suffering pharyngitis and swolen on left neck. it had been since 3 days ago. case management: she had come to puskesmas that were given amoxycillin capsul 500 mg three times a day for three days and paracetamol tablet 500mg three times a day for three days, but she was still ill. then she came to rsgm hasan aman banjarmasin. she was diagnosed as im. the instruction were isolation and bed rest for a week. she had to eat sofly and drink water highly. the therapy were amoxycillin capsul 500 mg three times a day for seven days, methisoprinol caplet 500 mg three times a day for seven days, natrium dikofenak tablet 50 mg three times a day for seven days. she was asked to see the dentist next 7 days. in this case, she were not given acyclovir. conclusion: im is self limiting disease. im is the disease with spesific clinical syndrome that associated with primary ebv infection. depend on the base of clinical experiences, the supportive treatment is adviced for patient of im. methisoprinol has both immuno modulator and antiviral properties. keywords: epstein barr virus; immunomodulator; infectious mononucleosis; methisoprinol correspondence: maharani laillyza apriasari, department of oral medicine, faculty of dentistry, universitas lambung mangkurat, jl. veteran 128 b banjarmasin, south borneo, indonesia. e-mail: maharaniroxy@gmail.com introduction infectious mononucleosis (im) is a disease with clinical syndrom associated with primary epstein–barr virus (ebv) infection. ebv is a gamma herpes virus that has the double stranded dna genome of about 172 kb. ebv infection occurs in humans that make the results in a life long infection. im is one of an acute self limiting disease.1 approximately 90% of ebv infects adults permanently. ebv often be transmitted subclinically between the children through saliva. in adolescents, it is clinical manifestation of im that are pharyngitis, cervical lymphadenopathy, fever, and malaise.2 ebv infection through transfering saliva by kissing or sexual intercourse.2-4 infants and children show an asymptomatic or mild manifestation of ebv infection. it is different from adolescents with the age of 15–25 years that have the highest incidence of im in the united states, japan, united kingdom, and europe.5 each year, approximately 10 to 20% of people become infected. im is happened in 30 to 50% of these patients. there are no obvious annual cycles and no specific season in incidence. there is no affected by the basis of sex.1 the most clinical manifestation in im shows the specific signs: fever, pharyngitis, and lymphadenopathy. the laboratory results of im patients show the lymphocytosis with atypical lymphocytes.6 im by ebv infection show approximately 0.5% of the total lymphocyte population that is infected. fever, cervical lymphadenopathy, sore throat, sometime show the dusky soft exudate around the tonsillar rings, pain, tender left upper quadrant splenomegaly, and atypical lymphocytosis distinguish glandular fever. ebv replicates t and b lymphocyt cells in salivary gland. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p1-4 2 apriasari/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 1–4 during the viremia of explosive primary infection, liver, thyroid, brain, meninges, myocardium, and pericardium may be affected. ebv may be also transferred by blood transfusion.5 primary infections in young children are sometime showed as non specific disease, because the typical signs of im are unclear. im sometime affects people that have primary ebv infection during or after the second decade of life. economic and sanitary conditions have improved over past decades, ebv infection in early childhood has become less common, and more children are risk suspectable as they are adolescence.1 the incubation period between exposure and manifestation of the symptoms can happen in 30 to 60 days, so that make the identification of the initial exposure is difficult.4 this study reports the therapy of im using methisoprinol as different therapy besides acyclovir. methisoprinol has both immuno modulator and antiviral properties. case the woman, 33 years old came by suffering pharyngitis and the swolen under her left ear. she had been febris for three days and suffering pharyngitis. after three days, there was the swolen and painful under her left ear. she was going to puskesmas that given amoxycillin capsul 500 mg three times a day for three days and paracetamol tablet 500 mg three times for three days. case management 1st visit (4 days): the drugs from puskesmas was finished. she was still subfebris. there were the swolen and painful under her left ear bigger than before. she got disphagia, so she did not eat. the extraoral examination was showed the swolen under her left ear, normal color, pain, hard, unmovable and diffuse border on her left neck (figure 1). the intraoral examination showed the swolen, red and painful on her left oropharyng (figure 2). the patient was diagnosed as im. it based on mainly manifestation of the specific signs such as pharyngitis, fever, and lymphadenopathy. the instruction were isolation and bed rest for a week. she had to eat the smooth meals and much water. the therapy were given amoxycillin capsul 500 mg three times a days for seven days, methisoprinol caplet 500 mg three times a day for seven days, natrium diklofenac tablet 50 mg three times a day for seven days. she was asked to see the dentist next 7 days. 2nd visit (11 days): the patient was cured. the drugs was finished. she regularly took the prescribed medicine. the swolen under left ear was gone. the pharyng was not painful, so she could eat again. the extraoral examination of her neck showed the normal condition (figure 3) and so did the intraoral examination (figure 4). she could swollen well without pain, so that could eat again. figure 1. the extraoral examination showed swolen, painful, normal color, unmovable, and diffuse border on her left neck. figure 2. the intraoral examination showed swolen, painful, red, and clear border on her left oropharyng. figure 3. the normal condition without swolen and pain was on her left neck. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p1-4 33apriasari/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 1–4 discussion differential diagnosis of im are pharyngitis by group a streptococcus. it has the same of im symptoms such as sore throat and fever. it can be definited by the bacterial culture examination. diagnostic testing of im are determined by ebv antibody serologies as ig m , ig g, and ebv nuclear antigen antibody (ebna). unfortunately, the ebv antibody peak with in two to six weaks after the onset of the symptoms.4 it is important to treat patient of im based on the specific clinical examination as malaise, fever, sore throath, exudate upon the tonsillar rings, and lymphadenopathy. by anamnesis and clinical manifestation, the final diagnosis can be determined immediately then the patient can be treat. the lymphocytes in im are promoted by the composed of the mixture of cd8+ cytotoxic suppressor t cells, nk cells, and cd4+ helper t-cells. the most population is the cd8+ t cells, which have a role in the suppression of viral replication and have cytotoxic activity against the virus that infect b cells. increased numbers of cd8+ cytotoxic suppressor t cells also have been showed in other virus infections, including hiv, cytomegalovirus, and hepatitis c infections. the virus penetrates through the oropharyngeal epithelium. it replicates in the oropharyngeal epithelium cells especially b-lymphocytes. ebv binds to b-lymphocytes, through cd21 their antigen promotes their transformation and proliferation. in the course of infection, the blood examination shows the large number of atypical lymphocytes resulting from the polyclonal activation of cytotoxic suppressor cd8 cells. they limit the excessive transformation and proliferation of b cells. in the event of inefficacious t cell immune response, it can develop persistent infection and uncontrolled b cell proliferation that is in the basis of the ebv oncogenic potential.7 the unlimit stimulation of ebv to b cells and the general condition under the low immune response make the cells proliferation for being neoplastic b cells. it will stimulate the burkitt’s lymphoma.7,8 base on the clinical examination, supportive care is recommended for patients with im. acetaminophen or non steroidal anti inflammatory drugs are recommended to manage fever, sore throat, and malaise. adequate fluid and nutrition intake should be given. the adequate rest is required. using acyclovir did not significantly reduce the peripheral blood of ebv levels or the duration and severity of clinical symptoms.9 some control trials on treatment with acyclovir in patients with ebv have shown that treatment never decrease the severity of clinical signs nor their duration.10 in cases, the diagnosis of im is unclear. ebv specific serologic testing may be used to definitively diagnose primary ebv infection. the main therapy is supportive treatment. actually the antiviral therapy is required for the host with high response immune. corticosteroid drugs are not indicated.9 the majority of patients with im recover without sequelae and return to normal activities within 2 months after the onset of symptoms. im is the self limiting disease, so that was depend on the host immune response.1,8 acyclovir did not use to treat it, because this drug only prevents virus replication. acyclovir works through three mechanisms.the first is the phosphorylation of the drug within the cell to the phosphate derivative by viral thymidine kinase. since acyclovir is a poor substrate for healthy cell’s thymidine kinases this step happens much more rapidly in infected cells. further metabolism via a cellular enzyme that is present in all cells called guanosine monophosphate kinaseresults in di-and tri-phosphate derivatives. the second mechanism of action for acyclovir is the inhibition of dna polymerase by the active acyclovir. since acyclovir triphosphate is an acyclic nucleoside analog that competes with dgtp it becomes incorporated into the viral dna chain during synthesis in the nucleus. the inhibition of dna polymerase is due to the fact that the drug lacks essential groups that the normal building blocksof the viral dna have. cyclic sugars are missing in acyclovir triphosphate and cause chain elongation termination.11 this case report that the patient was given methisoprinol 500 mg three times a day for seven days. methisoprinol has both immuno modulator and antiviral properties. methisoprinol is a well known immunostimulator that has been used for years. the active agents of isoprinosine are the compound of inosine and 1-(dimethylamino/2-propanol/4acetamidobenzene at a ratio of 1:3). it has a stimulatory effect on cellular and humoral defense mechanisms in both humans and animals. it activates t and b lymphocytes increasing the capacity of their proliferative response to antigens and mitogens, particularly in individuals with lowered resistance. methisoprinol also increases the proliferation of macrophages and their phagocyte activity. additionally, it induces the excretion of interferon and corrects the ability of cells under the influence of immunosuppresants to synthesize it.12 methisoprinol is the value treatment of acute and chronic viral infections, figure 4. the normal condition without swolen and pain was on her left oropharyng. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p1-4 4 apriasari/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 1–4 and also as a prophylactic. it works on the immune system to repair the impaired mediated cell immune response for getting normal cells.13 methisoprinol also has the direct antiviral activity. it will decrease the intensity of symptoms and stop the duration of a viral infection. in addition, the incidence of complications is reduced. it is the frequency and severity of im recurrences. the drug can be prescribed during the disease as a prophylaxis against reactivation of latent viral infections such as herpes simplex or varicella zooster. it is also used for the treatment or management of other secondary viral infections. methisoprinol is the drug with a synthetic purine derivative of immunomodulatory and antiviral properties, which result from an unconnected in vivo increasing of host immune responses.13 the action of methisoprinol can be normalizes the cell mediated immunity by promoting the differentiation of t lymphocytes into t cytotoxic cells and t helper cells, and increasing lymphokine production, production of il-1, il-2 and ifn-gamma, and nk cell functions. it is also enhancing the humoral immune response by promoting the differentiation of b lymphocytes into plasma cells and increasing the antibody production, the number of igg and complement surface markers, neutrophil cells, monocyte cells, macrophage chemotaxis and phagocytosis. it can inhibit the viral growth by suppressing the viral rna synthesis while potentiating depressed lymphocytic working.12,13 in conclusion im is self limiting disease. im is a specific signs that is sometime associated with primary ebv infection. base on the clinical examination, the supportive treatment ahall be given to im patients. the therapy by using acyclovir did not significantly reduce the severity of clinical symptoms. this case used methisoprinol that has both immuno modulator and antiviral properties. references 1. luzuriaga k, sullivan jl. infectious mononucleosis. n engl j med 2010; 362(21): 1993-2000. 2. balfour hh jr, dunmire sk, hogquist ka. infectious mononucleosis. clin transl immunology 2015; 4(2): e33. 3. macsween kf, higgins cd, mcaulay ka, williams h, harrison n, swerdlow aj, crawford dh. infectious mononucleosis in university students in the united kingdom: evaluation of the clinical features and consequences of the disease. clin infect dis 2010; 50(5): 699706. 4. rogers me. acute infectious mononucleosis: a review for urgent care physicians. american journal of clinical medicine 2012; 9(2): 88-91. 5. lerner am, beqaj sh, gill k, edington j, fitzgerald jt, deeter rg. an update on the management of glandular fever (infectious mononucleosis) and its sequelae caused by epstein barr virus (hhv-4): new and emerging treatment strategies. virus adaptation and treatment 2010; 2: 136-45. 6. kunimatsu j, watanabe r, yoshizawa a. eipstein-barr virus infectious mononucleosis in a splenectomized patient. j med cases 2013; 4(6): 353-6. 7. karcheva m, lukanov t, gecheva s, slavcheva v, veleva g, nachev r. infectious mononucleosis – diagnostic potentials. journal of imab annual proceeding 2008; 14(1): 9-13. 8. apriasari ml, baharuddin em. penyakit infeksi rongga mulut. surakarta: yuma pustaka; 2012. p. 15-6. 9. apriasari ml. kumpulan kasus penyakit mulut. jakarta: salemba medika; 2013. p. 19-20. 10. busch d, hilswicht s, schöb ds, trotha kt, junge k, gassler n, truong s, neumann up, binnebösel m. fulminant eipstein-barr virus infection mononucleosis in an adult with liver failure, splenic rupture, and spotaneous esophageal bleeding with ensuing esophageal necrosis: a case report. j med case rep 2014; 8: 35. 11. lemke tl, williams da, roche vf, zito w. foye’s principles of medicinal chemistry. 6th ed. baltimore: wolters kluwer health/ lipincott williams & wilkins; 2008. p. 122-5. 12. kazun b, siwicki ak. impact with bioimmune with methisoprinol on non-specific cellular and humoral defense mechanisms and resistance of african catfish (clarias gariepinus) to experimental infection with iridovirus. arch pol fish 2013; 21: 301-14. 13. sardana v, sharma d, agrawal s. subacute sclerosing panencephalitis revisited. international journal of basic and applied medical sciences 2013; 3(1): 225-41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p1-4 108 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 108–112 review article the role of family history as a risk factor for non-syndromic cleft lip and/or palate with multifactorial inheritance agung sosiawan1, mala kurniati2, coen pramono danudiningrat3, dian agustin wahjuningrum4 and indra mulyawan3 1department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia. 2department of biology, faculty of medicine, universitas malahayati, lampung, indonesia. 3department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya, indonesia. 4department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia. abstract background: cleft lip with or without cleft palate (cl/p) is a facial growth ‘disorder that occurs during gestation and has multifactorial causes owing to both genetic and environmental factors. several factors can increase the likelihood of cl/p, and one of them is family history. differences in results obtained from studies conducted across several countries concerning family history as a risk factor for cl/p suggest there is no consensus on how the condition is inherited. purpose: this study aims to review the literature on the role of family history as a risk factor contributing to the incidence of non-syndromic cl/p (nscl/p). review: this review discusses the etiology of cl/p and the risk factors influencing the incidence of cl/p. the review also examines the criteria for inheriting multifactorial disorders to calculate the risks involved should there be a recurrence of the condition based on family history. conclusion: cl/p is a type of multifactorial disorder with unclear etiology. therefore, it is important to investigate the risk factors stemming from family history (which play an important role) related to the recurrence risk. additionally, there should be focus on increasing genetic education and offering counselling to parents and pregnant women. keywords: cleft lip with or without cleft palate (cl/p); family history; multifactorial; recurrence correspondence: agung sosiawan, department of dental public health, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47, surabaya, 60132 indonesia. email: agung-s@fkg.unair.ac.id introduction cleft lip with or without cleft palate (cl/p) is a facial growth disorder that occurs during gestation and has multifactorial causes owing to both genetic and environmental factors.1–3 cl/p has been the subject of many genetic studies, but there has been no consensus on how the condition is inherited.4 cl/p disorders can interfere with speech, nutrition, hearing, and psychological development.5,6 the prevalence of cl/p cases varies according to ethnicity and region7 and is estimated at 1/600 births worldwide. differences in race, geographical origin, and gender, including the impact of genetic factors, influence the prevalence of cl/p.8,9 the highest prevalence is observed in asian populations (1/500 births), and the lowest prevalence is observed in african populations (1/2,500 births). the prevalence of cl/p in the caucasian race is 1/1,000 births.10,11 family history as a risk factor with the potential to cause cl/p is an interesting area of study. calculating the recurrence risk in cl/p patients having a family history of cl/p concerns multifactorial inheritance, which is different from the mendelian inheritance pattern (single gene), because cl/p is a multifactorial and polygenic disorder. until now, the genes involved in causing cl/p continue to be studied; however, researchers have not found the dominant gene responsible for causing cl/p. therefore, the authors are interested in studying the literature on the role of family history in increasing the risk of cl/p, especially from the perspective of multifactorial inheritance. epidemiology the prevalence of cl/p worldwide is 1/500–1/2,500 births, with the highest incidence in asian races at 1/500 births.12 the ratio of occurrence of cl/p in men and women is 1:2; dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p108–112 mailto:agung-s@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p108-112 109sosiawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 108–112 more women suffer from this disorder than men.3 a study conducted in north america reported a prevalence rate of between 0.6 and 3.92 per 1,000 births. studies conducted in europe reported prevalence rates ranging from 1.02 to 1.94 per 1,000 births. studies carried out in oceania reported prevalence rates in white people ranging from 1.21 to 1.73 per 1,000 births many low-income countries do not have a birth control system yet and a system to register birth defects, especially cl/p. this prevalence is indicated in table 1. international collaborative research on craniofacial malformations in developing countries, under the world health organization (who), is currently being carried out. cases of cl/p are also being recorded in these studies.13 etiology of cl/p the underlying etiology of cl/p is unknown. however, the complex embryogenesis of the lips and palate makes the tissue surrounding these areas susceptible to various disorders that can potentially cause malformations during the developmental stage. as shown in figure 1, the etiology of cl/p is a complex and multifactorial interaction, involving various genetic and environmental factors and gene–environment interactions.4,10,14,15 from a genetic perspective, the etiology of cl/p has been studied for many years. a literature study reveals that the heritability of non-syndromic cleft lip with or without cleft palate (nscl/p) is 70%.16 studies conducted on twins followed by further segregation analysis confirmed the role of genetics in the etiology of cl/p.2,17 the risk of cl/p increases when there is a family history of cl/p. parents with cl/p disorders can have children facing a 3–5% risk of having cl/p.16 the role of environmental factors leading to cl/p is very influential. previous studies have revealed the increasing prevalence of cl/p in patients whose mothers smoked, consumed alcohol, were administered antiepileptic drugs and corticosteroids, had nutritional deficiencies (folic acid), and were afflicted with infectious diseases during pregnancy.all of these factors affected the intrauterine environment.18 these environmental factors were found to increase the risk of nscl/p. recent studies have shown that maternal diseases (e.g. hyperthermia, parental occupation, diabetes mellitus, and obesity) present risk factors for cl/p.19 it is vital to examine the interaction and understand the nature of relationship between genes and the environment because cl/p occurs due to the involvement of many genes and environmental factors. maternal smoking and folic acid deficiency are two factors that can increase the genetic risk of developing cl/p. a study has suggested that there is a gene–environment interaction taking place between mothers who smoke and changes in genetic variants of the growth factor gene, the muscle segment homeobox, and the retinoic acid receptor gene.20,21 table 1. geographical variation in birth prevalence of orofacial clefts as per continent.13 continent (location) numbers of cl/p number of live births birth prevalence (per 1,000 live births) 95% confidence interval asia 15,646 9,965,084 1.57 1.54–1.60 north america 18,276 11,728,914 1.56 1.53–1.59 europe 5,028 3,236,253 1.55 1.52–1.58 oceania 2,822 2,125,912 1.33 1.30–1.36 south america 3,205 3,229,179 0.99 0.96–1.02 africa 216 380,273 0.57 0.54–0.60 total 45,193 30,665,615 1.47 1.44–1.50 genetic factors environmental factors mca, clefts caused by teratogens, some isolated case syndromes, chromosomal aberrations majority of “isolated” clefts, i.e. child has no other anomalies cleft lip, cleft lip and palate, cleft palate only clefts with other anomalies which do not assemble a syndrome, fas, hydantoin embryopathy, rubella embryopathy, … velocardiofacial sy, stickler sy, apert sy, teacher collins sy, down sy, trisomy 18, trisomy 13, … figure 1. etiology of cl/p.15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p108–112 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p108-112 110 sosiawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 108–112 single gene disorders multifactorial/polygenic chromosomal disorders congenital malformation adult-onset conditions criteria for inheritance 1. cleft lip/palate 2. congenital dislocation of the hip 3. congenital heart defect 4. neural tube 5. pyloric stenosis 1. diabetes mellitus 2. hypertension 3. obesity 4. epilepsy 5. ischemic heart disease 6. schizophrenia 1. recurrence risk as a function of relatedness 2. recurrence risk as a function of prior offspring 3. recurrence risk by severity 4. recurrence risk by sex type of genetic diseases risk factors involved in the occurrence of cl/p many factors are associated with the occurrence of cl/p, but the susceptibility of cl/p can increase if triggered at the right time, place, and moment of morphogenesis and facial formation.19 risk factors that can cause cl/p disorders include geographic location, family history, alcohol and tobacco consumption, inadequate nutrition, drug intake during pregnancy, miscellaneous infections, and occupational hazards affecting pregnant women. the first set of factors responsible for the occurrence of cl/p are the geographical location, the climate and the continental differences in the world. asian races are at the greatest risk marked at 14 cases in 10,000 births. this is followed by the caucasian race, who face a risk of 10 cases in 10,000 births. finally, the african americans face a risk of 4 cases in 10,000 births.19 the second risk factor is family history. family members with cl/p are at a greater risk of transmitting the condition to their offspring.22 therefore, clinicians and parents should realise the importance of genetic counselling. the third factor leading to cl/p is consuming alcohol during pregnancy. when alcohol is combined with other factors (e.g. consumption of tobacco and drugs, along with other socio-geographical factors), the risk of developing cl/p is extremely high.23,24 the fourth factor leading to the occurrence of cl/p is nutrition. folic acid, vitamins, zinc, and other micro elements create a great impact on pregnancy. several studies have shown that consuming soda and tea can influence pregnancy.25,26 the fifth factor leading to the occurrence of cl/p is the consumption of certain drugs during pregnancy. drugs, corticosteroids, antibiotics, and local and general agents administered during pregnancy can strongly influence the occurrence of cl/p.27,28 the sixth factor that can lead to the occurrence of cl/p, which is quite important, is the health status and the presence of viral infection in pregnant women. viral infections and diseases associated with an increase in body temperature play a major role in causing hereditary diseases.29,30 the seventh factor leading to the occurrence of cl/p is the nature of work that pregnant women are involved in. factors related to work (e.g. radiation, exposure to high temperatures, chemicals, light and electromagnetic fields, and some other elements can affect a women’s health in the early stages of pregnancy).31–33 discussion the etiology of cl/p is multifactorial, involving many unknown genes that have not yet been thoroughly studied. thus, the possibility of recurrence in a family is based on case experience.14 an interesting risk factor that can be investigated is the relationship between family history and the occurrence of cl/p. family history is one of the genetic factors that can increase the occurrence of cl/p and is polygenic or multifactorial. in certain multifactorial disorders, certain phenotypes are passed from one generation to another, indicating intermittent variation (as shown in figure 2). recurrence of cl/p ranges from 1–5%. this disorder usually has an incidence of about 1 per 1,000 live births and involves a single organ system or an embryologicallyrelated organ system. there are several criteria for inheriting polygenic or multifactorial disorders: recurrence risk as a function of relatedness, recurrence risk as a function of prior offspring, recurrence risk by severity, and recurrence risk by sex.34,35 an individual has two copies of their parent’s genes: one from the mother and the figure 2. criteria for polygenic or multifactorial inheritance. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p108–112 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p108-112 111sosiawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 108–112 second from the father.27 genetic variation occurs due to gene interactions (not spontaneously) and is passed down from generation to generation.36 the incidence of cl/p does not involve only one hereditary factor arising from one or both parents. cl/p is a concurrent condition.28 this explains why the probability of passing down cl/p to subsequent offspring is relatively low. the probability that the next child will also inherit the same combination of several genes and be exposed to certain environmental factors ranges from 3 to 5%.37,38 it can be concluded that if the prevalence of the condition in a population is a, the recurrence risk in offspring and siblings is the root of a (√a).34,35 in polygenic or multifactorial inheritance, the concordance rate of monozygotic twins with cl/p disorders is higher (40–60%) than in dizygotic (dz) twins (3–5%). in dz and non-twin siblings, the recurrence risk can be approximated through the square root of incidence in a population.39 in contrast to mendelian inheritance, the recurrence risk increases empirically after more than one offspring has the disorder. the risk rarely approaches the expected 25% for the recessive trait and the expected 50% for the dominant trait. however, the risk is extremely high (15–20%) after three offspring are affected.34 the study conducted by sivertsen et al.37 found a strong specificity of recurrence risk for two main types of clefts, suggesting that they had different causes. the risk is similar among children of affected fathers, children of affected mothers, and affected siblings. this pattern suggests that autosomal fetal genes play a major role in risk recurrence, with a small additional contribution from the inherited aspects of the maternal phenotype.38,40 the study carried out by martelli et al.38 in brazil aimed to determine the incidence of family nscl/p. the results revealed that there were differences observed between the types of cl/p disorders and family history in 185 patients (p<0.001).38 jamilian et al.28 conducted a study with a sample size of 187 people with the aim of understanding the link between parental risk factors and the incidence of malformations (cl/p). the results revealed that the risk factors involved when considering family history variables were an odds ratio of 7.4 and a 95% confidence interval and an odds ratio of 3.2 and a 95% confidence interval in consanguineous marriages. these factors increased the incidence of cl/p.28 acuña-gonzález et al.41 conducted a study with the aim of understanding the relationship between family history and socio-demographic risk factors in the incidence of nscl/p. the results indicated that the risk factors associated with family history and those associated with the incidence of cl/p were 1) the occurrence of past nscl/p cases in the father’s or mother’s family and 2) having a sibling with cl/p.41 complex inherited diseases can be influenced by interactions between the influence of one or several genes that increase or decrease susceptibility to a disease combined with triggers (e.g. environmental exposure) that can accelerate, exacerbate, or protect an individual against a disease. the new paradigm of genetic engineering today has brought freshness in diagnosing and analysing congenital disorders. previously, the mendel’s law theory was applied to estimate the risk of recurrence in single-gene diseases. however, in multifactorial diseases, chromosomal disorders, and diseases whose etiology is unknown, the empirical method of calculating the recurrence risk is an important tool for evaluating multifactorial disorders. although, in general, the empirical recurrence risk can be inaccurate (either due to differences in gene frequency and environmental factors among populations or due to the heterogeneity of a disease), population studies of family history, computer programmes, genotyping technology, genome-wide association studies, and single nucleotide polymorphisms can be employed to estimate the recurrence risk of multifactorial disorders.42 it is important to emphasise the need to enhance our knowledge about the potential risk factors that can lead to the occurrence of cl/p, especially genetic education and counselling to parents and mothers regarding the right behavioural lifestyle to follow before and during pregnancy. there is a potential to reduce the incidence of cl/p by spreading awareness and educating people. there are several criteria that lead to the high recurrence risk of cl/p, especially in multifactorial inherited diseases (including cl/p): 1) more than one family member is affected with cl/p; (2) the disease expression in the proband is more severe; (3) the proband belongs to the less commonly affected gender; and (4) the recurrence risk usually decreases rapidly in more distant relatives. acknowledgements we would like to thank the directorate of research and community services, the deputy director of research and development reinforcement, and the national research and innovation agency of the republic of indonesia for supporting this research. references 1. dixon mj, marazita ml, beaty th, murray jc. cleft lip and palate: understanding genetic and environmental influences. nat rev genet. 2011; 12(3): 167–78. 2. funato n, nakamura m. identification of shared and unique gene families associated with oral clefts. int j oral sci. 2017; 9(2): 104–9. 3. burg ml, chai y, yao ca, magee w, figueiredo jc. epidemiology, etiology, and treatment of isolated cleft palate. front physiol. 2016; 7(mar): 67. 4. muhamad ah, azzaldeen a, nezar w, firas k. the multifactorial factors influencing cleft lip-literature review. int j clin med res. 2014; 1(3): 90–6. 5. mcgarry a. the influence of genetics on syndromic and nonsyndromic cases of cleft lip and cleft palate. washington: the george washington university; 2017. p. 1–23. 6. nagase y, natsume n, kato t, hayakawa t. epidemiological analysis of cleft lip and/or palate by cleft pattern. j maxillofac oral surg. 2010; 9(4): 389–95. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p108–112 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p108-112 112 sosiawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 108–112 7. muhamad ah, azzaldeen a, watted n. cleft lip and palate; a comprehensive review. int j basic appl med sci. 2014; 4(1): 338–55. 8. martelli drb, machado ra, swerts mso, rodrigues lam, de aquino sn, martelli júnior h. non syndromic cleft lip and palate: relationship between sex and clinical extension. braz j otorhinolaryngol. 2012; 78(5): 116–20. 9. lesmana s, auerkari ei. genes contributing in cleft lip and cleft palate: a literature review. j int dent med res. 2016; 9(special issue: universitas indonesia): 441–8. 10. rafik a, nadifi s. updating genetics polymorphisms of nonsyndromic clefts lip-palates. am j mol biol. 2018; 8: 178–85. 11. mossey p, castilla e. global registry and database on craniofacial anomalies. in: who registry meeting on craniofacial anomalies. geneva: world health organization; 2003. p. 101. 12. chen q, wang h, hetmanski jb, zhang t, ruczinski i, schwender h, liang ky, fallin md, redett rj, raymond g v., wu chou y-h, chen pk-t, yeow v, chong ss, cheah fsh, jabs ew, scott af, beaty th. bmp4 was associated with nscl/p in an asian population. plos one. 2012; 7(4): e35347. 13. panamonta v, pradubwong s, panamonta m, chowchuen b. global birth prevalence of orofacial clefts: a systematic review. j med assoc thai. 2015; 98 suppl 7(7): s11-21. 14. allam e, windsor lj, stone c. cleft lip and palate: etiology, epidemiology, preventive and intervention strategies. anat physiol. 2013; 4(3): 1000150. 15. tolarova mm, al-kharafi l, tolar m, boyd c. pediatric cleft lip and palate. medscape. 2020. available from: https://emedicine. medscape.com/article/995535-overview. accessed 2021 feb 21. 16. mahamad irfanulla khan an, prashanth cs, srinath n. genetic etiology of cleft lip and cleft palate. aims mol sci. 2020; 7(4): 328–48. 17. leslie ej, carlson jc, cooper me, christensen k, weinberg sm, marazita ml. exploring subclinical phenotypic features in twin pairs discordant for cleft lip and palate. cleft palate-craniofacial j. 2017; 54(1): 90–3. 18. beaty th, marazita ml, leslie ej. genetic factors influencing risk to orofacial clefts: today’s challenges and tomorrow’s opportunities. f1000research. 2016; 5(0): 2800. 19. kawalec a, nelke k, pawlas k, gerber h. risk factors involved in orofacial cleft predisposition review. open med (warsaw, poland). 2015; 10(1): 163–75. 20. martelli drb, da cruz kw, de barros lm, silveira mf, swerts mso, martelli júnior h. maternal and paternal age, birth order and interpregnancy interval evaluation for cleft lip-palate. braz j otorhinolaryngol. 2010; 76(1): 107–12. 21. chen q, wang h, schwender h, zhang t, hetmanski jb, chou y-hw, ye x, yeow v, chong ss, zhang b, jabs ew, parker mm, scott af, beaty th. joint testing of genotypic and geneenvironment interaction identified novel association for bmp4 with non-syndromic cl/p in an asian population using data from an international cleft consortium. plos one. 2014; 9(10): e109038. 22. kempa i. identification of candidate genes involved in the etiology of non-syndromic cleft lip with or without cleft palate and isolated cleft palate. riga: rīga stradiņš university; 2013. p. 1–167. 23. deroo la, wilcox aj, lie rt, romitti pa, pedersen da, munger rg, moreno uribe lm, wehby gl. maternal alcohol binge-drinking in the first trimester and the risk of orofacial clefts in offspring: a large population-based pooling study. eur j epidemiol. 2016; 31(10): 1021–34. 24. yin x, li j, li y, zou s. maternal alcohol consumption and oral clefts: a meta-analysis. br j oral maxillofac surg. 2019; 57(9): 839–46. 25. agarwal d, gopalan tr. maternal nutrition and prevention of oral clefts. int j med public heal. 2011; 1(1): 43–5. 26. mckinney cm, chowchuen b, pitiphat w, derouen t, pisek a, godfrey k. micronutrients and oral clefts: a case-control study. j dent res. 2013; 92(12): 1089–94. 27. jackson m, marks l, may ghw, wilson jb. the genetic basis of disease. essays biochem. 2018; 62(5): 643–723. 28. jamilian a, sarkarat f, jafari m, neshandar m, amini e, khosravi s, ghassemi a. family history and risk factors for cleft lip and palate patients and their associated anomalies. stomatologija. 2017; 19(3): 78–83. 29. curcio am, shekhawat p, reynolds as, thakur kt. neurologic infections during pregnancy. steegers ea., cipolla m., miller e., editors. handb clin neurol. 3rd ed. 2020; 172(january): 79–104. 30. angulocastro e, acosta-alfa ro lf, guadron-llanos a m, canizalez-román a, gonzalez-ibarra f, osuna-ramírez i, murillollanes j. maternal risk factors associated with the development of cleft lip and cleft palate in mexico: a case-control study. iran j otorhinolaryngol. 2017; 29(93): 189–95. 31. ács l, bányai d, nemes b, nagy k, ács n, bánhidy f, rózsa n. maternal-related factors in the origin of isolated cleft palate-a population-based case-control study. orthod craniofac res. 2020; 23(2): 174–80. 32. spinder n, bergman jeh, boezen hm, vermeulen rch, kromhout h, de walle hek. maternal occupational exposure and oral clefts in offspring. environ health. 2017; 16(1): 83. 33. suhl j, romitti pa, rocheleau c, cao y, burns tl, conway k, bell em, stewa r t p, langlois p, national bir th defects prevention study. parental occupational pesticide exposure and nonsyndromic orofacial clefts. j occup environ hyg. 2018; 15(9): 641–53. 34. simpson jl. polygenic or multifactorial inheritance. glob libr women’s med. 2012; : 10344. 35. lvovs d, favorova oo, favorov a v. a polygenic approach to the study of polygenic diseases. acta naturae. 2012; 4(3): 59–71. 36. scherer a, christensen gb. concepts and relevance of genome-wide association studies. sci prog. 2016; 99(pt 1): 59–67. 37. sivertsen a, wilcox aj, skjaerven r, vindenes ha, abyholm f, harville e, lie rt. familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. bmj. 2008; 336(7641): 432–4. 38. martelli d-r, bonan p-r-f, soares m-c, paranaíba l-r, martellijúnior h. analysis of familial incidence of non-syndromic cleft lip and palate in a brazilian population. med oral patol oral cir bucal. 2010; 15(6): e898-901. 39. leslie ej, marazita ml. genetics of cleft lip and cleft palate. am j med genet c semin med genet. 2013; 163c(4): 246–58. 40. basha m, demeer b, revencu n, helaers r, theys s, bou saba s, boute o, devauchelle b, francois g, bayet b, vikkula m. whole exome sequencing identifies mutations in 10% of patients with familial non-syndromic cleft lip and/or palate in genes mutated in well-known syndromes. j med genet. 2018; 55(7): 449–58. 41. acuña-gonzález g, medina-solís ce, maupomé g, escoffieramírez m, hernández-romano j, márquez-corona m de l, islas-márquez aj, villalobos-rodelo jj. family history and socioeconomic risk factors for non-syndromic cleft lip and palate: a matched case-control study in a less developed country. biomedica. 2011; 31(3): 381–91. 42. bijanzadeh m. the recurrence risk of genetic complex diseases. j res med sci. 2017; 22(23): 32. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p108–112 https://emedicine https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p108-112 173 volume 46, number 4, december 2013 research report ekspresi cox-2 setelah pemberian ekstrak etanolik kulit manggis (garcinia mangostana linn) pada tikus wistar (cox-2 expression after mangosteen rind (garcinia mangostana linn) etanolic extract administration in wistar rats) rendra chriestedy prasetya,1 tetiana haniastuti,2 dan nunuk purwanti3 1 bagian biomedik, fakultas kedokteran gigi universitas jember-indonesia 2 bagian biologi mulut, fakultas kedokteran gigi universitas gadjah mada, jogjakarta-indonesia 3 bagian biomedik, fakultas kedokteran gigi universitas gadjah mada, jogjakarta-indonesia abstract background: cyclooxygenase is an enzyme for prostaglandins (pgs) synthesis from arachidonic acid. cyclooxygenase have been characterized and named as cox-1 and cox-2. cox-1 is responsible for constitutive pgs production under physiological condition and maintains normal function. on the other hand, while cox-2 expression is inducible by cytokines and endotoxin. periodontitis is a chronic inflammatory disease caused by anaerobic bacteria especially gram negative bacteria. the periodontitis occurrence is followed by increased of cox-2 expression. mangosteen rind (garcinia mangostana linn) contains gamma mangostin which inhibits the synthesis of pge2 through inhibition of cox-2 expression. purpose: this research was aimed to study cox-2 expression in experimental-induced periodontitis in wistar rats after mangosteen rind etanolic extract administration. methods: forty eight male wistar rats were induced periodontitis by putting silk ligature subgingivally around the cervical of the anterior lower teeth for 7 days. after the ligation was taken out, the rats were divided into 4 groups, and treated orally with mangosteen rind extract 60 mg/kg bb, 30 mg/kg bb, ibuprofen and saline respectively. the rats were sacrificed on the 1st, 3rd, 4th, 7th day after the treatment. the rats’ anterior lower jaws were processed for paraffin embedded tissue, cut serially and stained with immunohistochemistry. cox-2 expression were observed and counted under the microscope (400x). the data were analyzed using kruskall wallis test. results: kruskal wallis test showed a significant difference cox-2 expression among group indicating that mangosteen rind etanolic extract affected cox-2 expression. conclusion: mangosteen rind etanolic extract reduced cox-2 expression in periodontitis rats. key words: periodontitis, garcinia mangostana linn, mangosteen rind etanolic extract, cyclooxygenase-2, wistar rats abstrak latar belakang: siklooksigenase adalah enzim yang mensintesis prostaglandin (pg) dari asam arakhidonat. siklooksigenase dibagi menjadi 2 yaitu cox-1 dan cox-2. cox-1 bertanggung jawab pada sintesis pg dalam kondisi fisiologis dan mempertahankan fungsi normal, sedangkan ekspresi cox-2 dapat terinduksi oleh sitokin dan endotoksin. periodontitis adalah penyakit peradangan kronis yang disebabkan oleh bakteri anaerob terutama bakteri gram negatif. terjadinya periodontitis diikuti oleh peningkatan ekspresi cox-2. kulit buah manggis (garcinia mangostana linn) mengandung mangostin gamma yang menghambat sintesis pge2 melalui penghambatan cox-2. tujuan: penelitian ini bertujuan untuk meneliti ekspresi cox-2 pada tikus wistar jantan yang diinduksi periodontitis setelah pemberian ekstrak etanolik kulit manggis. metode: empat puluh delapan ekor tikus wistar jantan diinduksi periodontitis dengan meletakkan ligatur sutra pada subgingiva sevikal gigi anterior rahang bawah selama 7 hari. setelah ligatur dilepas, tikus dibagi dalam 4 kelompok yaitu ekstrak kulit manggis dosis 60 mg/kg bb, 30 mg/kg bb, ibuprofen dan saline dengan pemberian secara peroral. tikus didekapitasi pada hari ke-1,3, 5 dan 7 setelah perlakuan. rahang bawah gigi depan dilakukan pemrosesan menjadi blok paraffin, dipotong serial dan dilakukan pewarnaan imunohistokimia. ekspresi cox-2 diamati di bawah mikroskop dengan perbesaran 400x. 174 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 173–178 pendahuluan siklooksigenase dikenal juga prostaglandin h synthase merupakan enzim kunci dalam sintesa prostaglandin.1 siklooksigenase disintesis dari membran fosfolipid yang didegradasi oleh enzim fosfolipase a2 (pla2). asam arakidonat diubah menjadi prostaglandin h2 (pg-h2) oleh enzim siklooksigenase dan sintesis pge2 oleh enzim prostaglandin sintase (pges).2 siklooksigenase terdiri dari 2 isoform yaitu cox-1 dan cox-2. siklooksigenase-1 merupakan enzim utama yang ditemukan di banyak jaringan dan bertanggung jawab dalam menjaga fungsi normal tubuh termasuk keutuhan mukosa lambung dan pengaturan aliran darah ginjal.3 siklooksigenase 2 merupakan enzim yang diekspresikan sebagai respon terhadap agen proinflamasi seperti sitokin dan endotoksin. enzim ini berperan dalam pembentukan prostaglandin yang diikuti oleh proses patofisiologis seperti edema, hiperalgesia, dan demam.4 periodontitis merupakan inflamasi kronis yang mengenai jaringan periodontal gigi. penyebab utama terjadinya periodontitis adalah invasi bakteri dan produknya terutama bakteri gram negatif anaerob.5 gambaran klinis periodontitis adalah kemerahan, terjadi perubahan kontur tekstur halus mengkilat, kedalaman probing yang dalam diikuti dengan hilangnya attachment dan kerusakan tulang alveolar. apabila kerusakan tulang alveolar semakin parah akan menyebabkan kehilangan gigi (tooth loss).6 salah satu gambaran histologis periodontitis adalah meningkatnya infiltrasi sel inflamasi terutama makrofag dan limfosit. bakteri dan produknya akan menginduksi sel untuk mensintesis interleukin-1 dan tumor necrosis factor-α (tnf-α). interleukin-1 dan tnf-α akan mempengaruhi kepada membran sel untuk mensintesis siklooksigenase-2 melalui metabolisme asam arakhidonat.7 saat ini perawatan periodontitis adalah perawatan mekanis yang ditunjang dengan penggunaan antibiotik dan anti inflamasi. perawatan mekanis meliputi skaling dan rootplaning serta perawatan bedah yang bertujuan untuk mengurangi akumulasi plak dan kalkulus. pemberian antibiotik dan anti inflamasi sebagai terapi tambahan dalam perawatan periodontitis bertujuan untuk menghambat pertumbuhan bakteri, mengurangi bertambah parahnya periodontitis serta menurunkan infiltrasi sel inflamasi.8 manggis merupakan tumbuhan yang berasal dari asia tenggara meliputi indonesia, malaysia, thailand. manggis merupakan buah yang fungsional, buahnya dipakai untuk buah kaleng, sirup atau sari buah. secara tradisional buah manggis digunakan sebagai obat sariawan, wasir dan luka. kulit buah dimanfaatkan sebagai pewarna termasuk untuk tekstil dan air rebusannya dimanfaatkan sebagai obat tradisional. batang pohon dipakai sebagai bahan bangunan, kayu bakar atau kerajinan.9 kulit buah manggis (garcinia mangostana linn) telah dimanfaatkan oleh masyarakat sebagai obat antiinflamasi. khasiat anti inflamasi kulit buah manggis diduga berasal dari senyawa golongan xanton yang termasuk di dalamnya α-mangostin dan γ-mangostin.10 hasil penelitian chen et al11 menunjukkan bahwa alfa mangostin secara signifkan menghambat produksi nitrit oksida (no), prostaglandin e2 (pge2), tumor necrosis factor(tnf)-α dan inducible nos (inos) pada sel raw 264.7 yang diinduksi lipopolisakarida.11 selain itu hasil penelitian nakatani et al12 menunjukkan bahwa gamma mangostin mampu menghambat pelepasan pge2 dengan menghambat ekspresi cox-2 dan mrna pada sel glioma tikus c6 yang diinduksi ca2+ ionophore a23187 (in vitro). penelitian ini bertujuan meneliti pengaruh ekstrak kulit manggis terhadap ekspresi siklooksigenase-2 pada gingiva tikus wistar yang diinduksi periodontitis. bahan dan metode penelitian ini menggunakan 48 ekor tikus wistar jantan usia 2 bulan dengan berat badan 175-200 gram. tikus dianastesi dengan diinjeksi ketamine hcl secara intramuskular pada otot paha belakang dengan dosis 0,2 ml/200 gram berat bb. kulit manggis diidentifikasi di laboratorium biologi farmasi fakultas farmasi universitas gadjah mada. pembuatan ekstrak kulit manggis dilakukan di lppt unit i ugm yogyakarta dengan metode ekstraksi yang digunakan adalah metode perkolasi dengan pelarut etanol. induksi periodontitis dilakukan dengan mengikat benang sutra (silk ligature) ukuran 3,0 pada daerah subgingiva di servikal gigi incisivus rahang bawah. pada hari ke-7 ligasi dilepas kemudian tikus dibagi menjadi 4 kelompok perlakuan yaitu kelompok yang diberi ekstrak kulit manggis dosis 60 mg/kg bb, kelompok yang diberi ekstrak kulit manggis dosis 30 mg/kg bb, kelompok kontrol positif yang diberi ibuprofen 9 mg/kg bb dan kelompok kontrol negatif yang diberi saline 0,5 ml. pemberian data pengamatan dianalisa dengan uji kruskall wallis. hasil: uji kruskall wallis menunjukkan terdapat perbedaan bermakna ekspresi cox-2 diantara kelompok perlakuan yang mengindikasikan bahwa ekstrak kulit manggis mempengaruhi ekspresi cox-2. simpulan: ektrak etanolik kulit manggis menurunkan ekspresi cox-2 pada tikus dengan periodontitis. kata kunci: periodontitis, garcinia mangostana linn, ekstrak etanolik kulit manggis, tikus wistar korespondensi (correspondence): rendra chriestedy prasetya, bagian biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: rendrachriestedy@gmail.com 175prasetya, et al.: ekspresi cox-2 setelah pemberian ekstrak etanolik kulit manggis hari ke-1 hari ke-3 hari ke-5 hari ke-7 ekstrak kulit manggis 60 mg/kg bb ekstrak kulit manggis 30 mg/kg bb ibuprofen saline gambar 1. ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7. sel yang mengekspresikan sel epitel basal ( ), fibroblast ( ) dan makrofag ( ). hari ke-1 hari ke-3 hari ke-5 hari ke-7 ekstrak kulit manggis 60 mg/kg bb ekstrak kulit manggis 30 mg/kg bb ibuprofen saline gambar 1. ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7. sel yang mengekspresikan sel epitel basal ( ), fibroblast ( ) dan makrofag ( ). hari ke-1 hari ke-3 hari ke-5 hari ke-7 ekstrak kulit manggis 60 mg/kg bb ekstrak kulit manggis 30 mg/kg bb ibuprofen saline gambar 1. ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7. sel yang mengekspresikan sel epitel basal ( ), fibroblast ( ) dan makrofag ( ). hari ke-1 hari ke-3 hari ke-5 hari ke-7 ekstrak kulit manggis 60 mg/kg bb ekstrak kulit manggis 30 mg/kg bb ibuprofen saline gambar 1. ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7. sel yang mengekspresikan sel epitel basal ( ), fibroblast ( ) dan makrofag ( ). hari ke-1 hari ke-3 hari ke-5 hari ke-7 ekstrak kulit manggis 60 mg/kg bb ekstrak kulit manggis 30 mg/kg bb ibuprofen saline gambar 1. ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7. sel yang mengekspresikan sel epitel basal ( ), fibroblast ( ) dan makrofag ( ). 176 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 173–178 perlakuan secara per oral sehari 3 kali dengan menggunakan oral gavage pada masing-masing kelompok tikus. pada hari ke-1, 3, 5, dan 7 setelah perlakuan, hewan didekapitasi. rahang bawah pada bagian gigi anterior yang telah diberi perlakuan diambil dan difiksasi dengan buffered formalin 10% selama 24 jam. spesimen kemudian didekalsifikasi menggunakan edta 10% ph 7,4 selama 6 minggu pada suhu 4°c. setelah lunak, spesimen ditanam dalam parafin, dan dipotong serial dengan ketebalan 3 µm untuk dilakukan pengecatan imunihistokimia menggunakan antibodi cox-2 (lab vision rb 9072-po). ekspresi cox-2 diamati dengan menggunakan skor persentase sel positif dan intensitas warna cox-2 yang diamati pada tiga lapangan pandang berbeda pada daerah sulkus gingiva dan jaringan ikat di bawah epitel junctional dan epitel sulcular. pengolahan data statitistik menggunakan kruskall wallis test dilanjutkan dengan uji mann whitney test dengan tingkat signifikansi p<0,05. hasil pada penelitian ini diperoleh data hasil untuk masingmasing kelompok perlakuan, yang terbagi dalam 4 kelompok perlakuan yaitu kelompok ekstrak kulit manggis 60 mg/kg bb, kelompok ekstrak kulit manggis 30 mg/kg bb, kelompok kontrol (+), kelompok kontrol (-). ekspresi cox-2 pada gingiva tikus periodontitis hari ke-1,3,5 dan 7 setelah pemberian ekstrak kulit manggis 60 mg/kg bb, ekstrak kulit manggis 30 mg/kg bb, ibuprofen dan saline. ekspresi cox-2 tampak di daerah sulkus gingiva meluas di jaringan ikat di bawah epitel junctional dan sulkuler. tampak ekspresi cox-2 dengan intensitas kuat pada kelompok ekstrak kulit manggis 60 mg/kg bb pada hari ke-1 dan terus menurun dengan intensitas lemah pada hari ke-7. ekspresi cox-2 dengan intensitas kuat pada kelompok saline hari ke-1 dan ke-7(gambar 1). ekspresi cox-2 pada tikus dengan periodontitis setelah pemberian ekstrak kulit manggis dihitung dengan gambar 2. modus skor persentase area positif cox-2 berdasarkan kelompok perlakuan dan waktu dekapitasi gambar 3. modus skor intensitas warna cox-2 berdasarkan kelompok perlakuan dan waktu dekapitasi tabel 1. hasil uji kruskal-wallis antar kelompok perlakuan terhadap perubahan skor persentase area positif cox2 berdasarkan kelompok perlakuan kelompok mean rank sig. manggis 60mg/kg bb 12,33 0,00* manggis 30 mg/kg bb 24,33 ibuprofen 25,50 saline 35,83 keterangan: * = berbeda bermakna (p<0,05) tabel 2. hasil uji mann-whitney antar kelompok perlakuan terhadap perubahan skor persentase area positif cox2 berdasarkan kelompok perlakuan kelompok mean rank sig. manggis 60 mg/kg bb 7,29 0,00* saline 17,71 manggis 30 mg/kg bb 9,38 0,02* saline 15,62 ibuprofen 9,50 0,02* saline 15,50 manggis 60 mg/kg bb 8,88 0,01* ibuprofen 16,12 manggis 30 mg/kg bb 12,12 0,78 ibuprofen 12,88 manggis 60 mg/kg bb 9,17 0,02* manggis 30 mg/kg bb 15,83 keterangan: * = berbeda bermakna (p<0,05) tabel 3. hasil uji kruskal-wallis antar kelompok perlakuan terhadap perubahan skor intensitas warna cox-2 berdasarkan kelompok perlakuan kelompok mean rank sig. manggis 60 mg/kg bb 22,17 0,04* manggis 30 mg/kg bb 19,25 ibuprofen 23,92 saline 32,67 keterangan: * = berbeda bermakna (p<0,05) 177prasetya, et al.: ekspresi cox-2 setelah pemberian ekstrak etanolik kulit manggis menggunakan modus skor persentase area positif dan intensitas warna cox-2. modus skor persentase area positif cox-2 ditunjukkan dalam grafik pada gambar 2. dari gambar 2 tampak bahwa terjadi penurunan modus skor persentase area positif cox-2 pada kelompok yang diberi ekstrak kulit manggis 60 mg/kg bb, 30 mg/kg bb, ibuprofen serta saline dengan bertambahnya waktu. skor persentase area positif cox-2 terendah tampak pada kelompok yang diberi ekstrak kulit manggis 60 mg/kg bb. data hasil pengamatan dianalisis dengan uji kruskall wallis untuk mengetahui perbedaan skor persentase area positif cox-2 antar kelompok tikus perlakuan (tabel 1). hasil uji kruskall wallis dengan nilai sig. 0,00 menunjukkan bahwa pemberian ekstrak kulit manggis berpengaruh terhadap penurunan skor persentase area cox2 antara kelompok yang diberi ekstrak kulit manggis 60 mg/ kg bb, 30 mg/kg bb, ibuprofen dan saline. selanjutnya dilakukan uji mann-whitney untuk mengetahui perbedaan skor persentase area positif cox-2 antara masing-masing kelompok (tabel 2). hasil uji mann-whitney menunjukkan adanya perbedaan skor persentase area positif cox-2 yang bermakna (p<0,05) antara semua kelompok perlakuan. intensitas warna sel yang mengekspresikan cox-2 diukur dengan kriteria ekspresi dengan intensitas lemah (skor 1), sedang (skor 2) dan kuat (skor 3). gambar 3 menunjukkan modus skor intensitas warna cox-2 tampak mengalami penurunan pada seluruh kelompok perlakuan pada hari ke-5 dan hari ke-7. data hasil pengamatan dianalisis dengan uji kruskall wallis untuk mengetahui perbedaan skor intensitas warna cox-2 antar kelompok tikus perlakuan. hasil uji kruskall wallis menunjukkan adanya perbedaan intensitas warna cox-2 yang bermakna (p<0,05) antar kelompok yang diberi ekstrak kulit manggis 60 mg/kg bb, 30 mg/kg bb, ibuprofen dan saline. hal ini berarti pemberian ekstrak kulit manggis berpengaruh tabel 4. hasil uji mann-whitney antar kelompok perlakuan terhadap perubahan skor intensitas warna cox-2 berdasarkan kelompok perlakuan kelompok mean rank sig. manggis 60 mg/kg bb 13,21 0,048* saline 15,08 manggis 30 mg/kg bb 9,17 0,01* saline 15,83 ibuprofen 10,25 0,09 saline 14,75 manggis 60 mg/kg bb 12,04 0,07 ibuprofen 12,96 manggis 30 mg/kg bb 11,29 0,34 ibuprofen 13,71 manggis 60 mg/kg bb 13,21 0,58 manggis 30 mg/kg bb 11,79 keterangan: * = berbeda bermakna (p<0,05) terhadap penurunan intensitas warna cox-2. selanjutnya dilakukan uji mann-whitney untuk mengetahui perbedaan intensitas warna cox-2 antara masing-masing kelompok (tabel 3). hasil uji mann-whitney menunjukkan adanya perbedaan intensitas warna cox-2 yang bermakna (p<0,05) antara kelompok yang diberi ekstrak kulit manggis 30 mg/kg bb dan manggis 60 mg/kg bb dibandingkan dengan kelompok saline. tidak terdapat perbedaan intensitas warna cox-2 yang bermakna (p>0,05) antara kelompok yang diberi ekstrak kulit manggis 60 mg/kg bb dan 30 mg/kg bb dibandingkan dengan ibuprofen, serta kelompok yang diberi ekstrak kulit manggis 60 mg/kg bb dibandingkan yang diberi ekstrak kulit manggis 30 mg/kg bb (tabel 4). pembahasan pada penelitian ini ekspresi cox-2 diukur melalui skor persentase area positif dan intensitas warna. induksi periodontitis dilakukan dengan cara mengikatkan benang sutra (silk ligature) ukuran 3,0 pada daerah subgingiva di sekeliling gigi incisivus anterior rahang bawah.13 ligasi bertujuan agar terjadi akumulasi dental plak yang akan menginduksi terjadinya periodontitis. setelah ligasi akan terbentuk dental plak yang tersusun antara lain oleh bakteri veilonella parvula, parmivonas micra, streptococcus mitis.14 hasil pewarnaan dengan teknik imunohistokimia menggunakan antibodi anti cox-2 tampak warna coklat pada sitoplasma sel fibroblas, makrofag, neutrofil serta lapisan basal epitel dengan intensitas yang bervariasi mengindikasikan ekspresi positif cox-2. semakin kuat intensitas warna semakin kuat ekspresi cox-2. hal ini sesuai dengan hasil penelitian15 bahwa protein cox-2 terekspresi pada sel inflamasi, sel endotel, fibroblas gingiva dan sel epitel pada gingiva yang terinflamasi. ekspresi cox-2 pada kelompok ekstrak kulit manggis 60 dan 30 mg/kg bb lebih rendah dibandingkan kelompok ibuprofen (kontrol positif) maupun kelompok saline (kontrol negatif). sedangkan ekspresi cox-2 diantara ekstrak kulit manggis 60 dan 30 mg/kg bb menunjukkan penurunan pada hari ke-3, 5 dan 7 dibandingkan hari ke-1 baik pada skor persentase area positif dan intensitas warna. peningkatan intensitas warna hari ke-3 hanya pada kelompok ekstrak kulit manggis 30 mg/kg bb dibandingkan hari ke-1. lipopolisakarida (lps) akan menginduksi terjadinya inflamasi pada jaringan periodontal gigi. lipopolisakarida akan berikatan dengan makrofag melalui toll like receptor 4. ikatan tersebut akan memicu nf-kb di dalam makrofag yang selanjutnya menginduksi sekresi sitokin proinflamasi antara lain il-1 dan tnf-α.8 interleukin-1 dan tnf-α yang dihasilkan oleh makrofag juga akan menyebabkan lepasnya fosfolipid dari membran sel epitel gingiva, fibroblas, sel mast, neutrofil, makrofag, limfosit sehingga terjadi metabolisme asam arakhidonat oleh kerja enzim 178 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 173–178 fosfolipase a2. siklooksigenase (cox) merupakan enzim yang disintesis dari metabolisme asam arakhidonat. siklooksigenase berperan pertama kali mengkatalisis 2 tahap biosintesis prostaglandin dan terdapat dalam 2 bentuk yaitu cox-1 dan cox-2. siklooksigenase 1 berperan dalam proses homeostasis sedangkan cox-2 jumlahnya meningkat saat terjadi inflamasi dan berperan dalam sintesis prostaglandin terutama pge2.16 peningkatan ekspresi cox-2 akan meningkatkan sintesis pge2. peningkatan sintesis pge2 akan menyebabkan peningkatan vasodilatasi dan permeabilitas endotelium yang berakibat meningkatkan infiltrasi sel inflamasi.5 penurunan ekspresi cox-2 pada kelompok yang diberi ekstrak kulit manggis lebih baik dibandingkan kelompok ibuprofen (kontrol positif) maupun kelompok saline (kontrol negatif), hal ini diduga disebabkan oleh kandungan kulit manggis sebagai bahan antiinflamasi. kandungan kulit buah manggis adalah golongan xanton yang termasuk di dalamnya alfa mangostin dan gamma mangostin.10 gamma mangostin dari kulit buah manggis mampu menghambat ekspresi mapk, nf-kb dan ap-1 dalam makrofag.17 hasil penelitian liu et al18 secara in vitro alfa mangostin kulit manggis terbukti mampu menurunkan induksi lipopolisakarida (lps) terhadap sintesi sitokin pro inflamasi tnfα dan il-4 melalui penghambatan ekspresi gen oncostatin m pada jalur mapk pada kultur sel u937. penurunan sekresi il-1 dan tnf-α akan menurunkan ekspresi cox-2 karena hambatan sinyal dari il-1 dan tnf-α untuk lepasnya fosfolipid dari membran sel, sedangkan ekspresi cox-2 pada kelompok kontrol negatif lebih tinggi. jumlah mrna dan protein cox-2 gingiva pada subyek dengan periodontitis kronis lebih tinggi dibandingkan yang sehat.19 hal ini diperkuat hasil penelitian mesa et al20 bahwa ekspresi cox-2 pada pasien gingivitis ataupun periodontitis lebih tinggi dibandingkan dengan gingiva yang sehat. hasil penelitian ini juga menunjukkan ekspresi cox-2 pada kelompok ekstrak kulit manggis 60 mg/kg bb lebih rendah dibandingkan kelompok esktrak kulit manggis 30 mg/kg bb. hal ini kemungkinan disebabkan oleh komponen senyawa yang terkandung dalam kulit manggis terdiri dari gabungan beberapa senyawa kimia yang saling bersinergi.21 komponen yang terkandung dalam kulit manggis antara lain triterpenoid, xanton, vitamin b1, kalsium dan zat besi. senyawa paling utama adalah golongan xanton yaitu alfa mangostin, beta mangostin, gamma mangostin, mangostanol, garcinon dan e gartanin.10 semakin besar dosis yang diberikan semakin banyak komponen-komponen yang bekerja sinergis satu sama lainnya juga semakin kuat bekerja sebagai antiinflamasi sehingga infiltrasi sel inflamasi dan ekspresi cox-2 yang terhambat juga besar.22 penelitian ini menunjukkan bahwa ekstrak kulit manggis mampu menurunkan ekspresi cox-2 pada tikus wistar yang diinduksi periodontitis. daftar pustaka 1. khan a, ladaroda m, yang ht, dionne ra. expression of cox-1 and cox-2 in a clinical model of acute inflammation. j pain 2007; 8(4): 349-54. 2. inada y, ikeda k, tojo k, sakamoto m, takada y, tajima n. possible involvenment of corticotropin release factor receptor of signaling on vascular inflammation. peptides 2006; 5: 142-47. 3. zhang wy, yang x, jin d, zhu x. expression and enzyme activity determination of human cox-1 and 2 in baculovirus-insect cell system. acta pharmacologica 2004; 25(8): 1000-6. 4. fracon nr, teofilo mj, satin br, lamanot. prostaglandin and bone: potential risk and benefit related to the use of nonsteroidal anti-inflamatory drugs in clinical dentistry. j oral sci 2008; 50: 247-52. 5. carranza f, henry h, newman, michael, g clinical periodontology 10th edition. new york: wb saunders; 2006. p. 66-70. 6. reddy ds. the role of neurosteroid in the pathophysiology and treatment of catamenial epilepsy. epilepsy res 2009; 3: 127-29. 7. rose fl, mealey lb, genco jb, rose wd. periodontics medicine and surgery. missouri: mosby; 2004. p. 38-43. 8. kumar v, abbas a, fausto n. pathologic basis of disease. 8th ed. new york: elsevier; 2006. p. 45-9. 9. nugroho ae. manggis (garcinia mangostana l.): dari kulit buah yang terbuang hingga menjadi kandidat suatu obat. majalah obat tradisional 2011; 16(2): 64-9. 10. chin yw, jung, ha, chai h, keller wj, kinghorn ad. xanthones with quinone reductase-inducing activity from the fruits of garcinia mangostana (mangosteen). phytochem 2008; 69: 754–58. 11. chen lg, yang ll, wang cc. anti inf lamator y activity of mangostins from garcinia mangostana. j food chem toxicol 2006; 10: 1016. 12. nakatani k, yamakuni t, kondo n, arakawa t, oosawa k, shimura s, inoue h, ohizumi y. gamma mangostin inhibitor kb kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gen expression in c6 rat glioma cells. j mol pharmacol 2004; 66267. 13. tsagareli w. ultrastructural aspect of gingival soft tissue cells population under experimental gingivitis. georgia: med news 2005; 120: 71-4. 14. duarte mp, tezolim rk, figueiredo cl, feres m, bastus pm. microbial profile of ligature-induced periodontitis in rats. arch oral biol 2010; 55: 1142-47. 15. gitlin mj, loftin dc. cyclooxygenase-2 inhibition increases lipopolysacharide-induced atherosclerosis in mice. cardiovasculer research 2009; 81: 400-7. 16. porth mc, matfin g. pathophysiology concept of altered health science. 8 th ed. new york: mosby; 2009. p. 66-71. 17. bungrumpert a, kalpravidch r, chuang c, overman a, martinez k, kennedy a, mcintosh c. xanthones from mangosteen inhibit inflammation in human macrophages and in human adipocytes exposed to macrophage condition media. j nutr 2010; 16: 342-47. 18. liu sh, lee lt, huu ny, huange kk, shih yc, mukenazu l, li jm, chou ty, wang wh, chen ts. effect of alpha mangostin on the expression of anti inflammatory genes in u937 cells. chinese med 2012; 7-9. 19. zahng f, engebretson sp, morton rs, cavanaugh pf, subbaranaiah k, dannenberg aj. the overexpression of cyclo-oxygenase-2 in chronic periodontitis. jada 2013; 134: 61-7. 20. mesa f, aguilar m, galindo-moreno p, bravo m, valle of. cox-2 expression in gingival biopsies from periodontal patient is correlated with connective tissue loss. j periodontol 2012; 11: 561. 21. pasaribu f, sitorus p, bahri s. the test of ethanol extract of mangosteen (garcinia mangostana l) to decrease blood glucose level. j pharm pharmacol 2012; 1: 1-8. 22. wijaya a, santoningsih d, setyawati s. pengaruh ekstrak kulit buah manggis (garcinia mangostana linn) terhadap penurunan jumlah foam cell pada aorta tikus (rattus novergicus) model aterogenik, tesis. malang: universitas brawijaya 2011. 132132 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 132–138 original article degradation of fusobacterium nucleatum biofilm and quantity of reactive oxygen species due to a combination of photodynamic therapy and 2.5% sodium hypochlorite nanik zubaidah1, sukaton1, sri kunarti1, meidi kurnia ariani1, dawailatur rahman setiady1, dur muhammad lashari2 1department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2department of oral biology, bolan university of medical and health sciences, quetta, pakistan abstract background: the persistence of microorganisms in the root canal system is one of the leading causes of root canal treatment failure. biofilms of putative pathogens hidden inside dentin tubules and other root canal ramifications may limit current disinfection protocols. photodynamic therapy (pdt) with a wavelength of 628 nm can be used as an antimicrobial strategy that uses low-power laser energy to activate a non-toxic photosensitizer to produce singlet oxygen with the ability to kill microorganisms in root canals. fusobacterium nucleatum was used because this bacterium is one of the bacteria involved in root canal infection. purpose: the aim of this study was to compare the bactericidal efficacy of sodium hypochlorite (naocl) 2.5%, pdt, and a combination of pdt and naocl 2.5% against fusobacterium nucleatum. methods: mature biofilm fusobacterium nucleatum was divided into four groups according to the protocol of decontamination: k1 (negative control – biofilm), k2 (naocl 2.5%), k3 (pdt), and k4 (naocl 2.5% + pdt). biofilm degradation was observed using optical density (od) at 570 nm using a microplate reader. a reactive oxygen species quantity check was carried out using a nitroblue tetrazolium test, and od observation was done with a microplate reader at 540 nm. results: group 4 (naocl 2.5% + pdt) showed more biofilm bacteria elimination than the other groups. conclusion: a combination of pdt and naocl 2.5% can be considered an effective protocol for the elimination of fusobacterium nucleatum. there is a potentiation relationship between naocl 2.5% and pdt fotosan. biofilm degradation occurs because of the effect of antibacterial naocl 2.5% and the irradiation effect of the toluidine blue o photosensitizer. keywords: dentistry; fusobacterium nucleatum; medicine; photodynamic therapy; sodium hypochlorite article history: received 13 june 2023; revised 19 september 2022; accepted 17 october 2022 correspondence: nanik zubaidah, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl mayjen prof. dr. moestopo no. 47, surabaya 60132, indonesia. email: nanik-z@fkg.unair.ac.id introduction biofilms are matrices of polysaccharides that cover populations of bacteria that are attached to each other or attached to surfaces or between surfaces. a biofilm is a thin layer in microorganisms that can consist of bacteria, fungi, and protozoa. floating bacteria are also known as planktonic bacteria, which are prerequisites for the formation of biofilms. bacteria in planktonic form are found inside and outside the biofilm. the composition of the biofilms consists of microorganism cells, extracellular products, and polysaccharides as adhesive materials, and water is the main constituent material of biofilms with a content of up to 97%. biofilm matrices are quite complex and can contain a variety of non-biofilm materials such as mineral crystals, blood components, or soil components. the main component of biofilms other than microbial cells is extracellular polysaccharide substances that constitute up to 50–90% of biofilms.1 the microbial cells in the biofilm communicate using a system called quorum sensing. quorum sensing is the ability of microbes to measure cell density (the number of microbes) by measuring the amount of accumulated secretion of molecular signals produced by cells. this quorum sensing ability can provide bioluminescent capabilities, biofilm formation, or exoenzyme production in bacteria.2,3 polysaccharides produced by microbes to form biofilms include extracellular matrix polymers copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 mailto:nanik-z@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 133 zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 (emp), i.e., polysaccharides removed from within cells. emp synthesized by microbial cells differ in composition and chemical and physical properties. the physiology of biofilms is currently characterized using a system that has been simplified to single, dual, and multi-species bacterialcommunity-containing organisms.4 microorganisms are the cause of pulp necrosis in 98.5% of cases while 1.5% are caused by trauma and chemical irritation.5 the treatment indicated for cases of pulp necrosis is endodontic treatment.6 microorganisms in root canals can cause endodontic infections.7 in general, various types of anaerobic bacteria predominate in endodontic infections. lee et al.8 have found that 70.3% of the bacteria in root canals are anaerobic bacteria and 29.7% are aerobic bacteria. in this study, fusobacterium nucleatum was used because this bacterium is one of the bacteria involved in root canal infection. f. nucleatum is an anaerobic bacterium in the form of non-spore, non-motile, and gram-negative bacteria. these bacteria are associated with spontaneous pain, tenderness to percussion, tenderness to palpation, gum swelling, hemorrhagic exudates, tooth mobility, inadequate restorations, and inadequate obturation.9 the irrigation material that is often used in endodontic treatments is sodium hypochlorite (naocl) 2.5%. sodium hypochlorite neutralizes amino acids to form water and salt. hypochlorous acid is a component contained in the solution of sodium hypochlorite. when in contact with organic tissue, hypochlorous acid will act as a solvent and will free chlorine. the liberated chlorine will join the amino protein group and form chloramine. hypochlorous acid (hocl-) and hypochlorite ions (ocl-) induce amino acid degradation and hydrolysis. the chloramination reaction between chlorine and the amino group forms chloramine that disrupts cell metabolism. chlorine is a powerful oxidizing agent that provides antibacterial properties that inhibit bacterial enzymes by forming irreversible oxidation of sulfhydryl groups, essential enzymes of bacteria.10,11 at a ph between 4 and 7, most of the chlorine will take the form of hocl, the active and responsible part in bacterial inactivation, whereas, at a ph above 9, it will be dominated by ocl-, whose nature is less active.12 saponification, neutralization of amino acids, and chloramine reactions that occur in microorganisms and organic tissues will provide antimicrobial effects and tissue dissolution processes.11 in addition, hypochlorite preparations are sporicidal and virucidal in nature, thus will produce a greater dissolving effect on necrotic tissues than in vital tissues. this underlies the use of sodium hypochlorite solution as the irrigation material.12 the photosensitizer is a cation (positively charged) that will bind to the bacterial cell wall that is an anion (negatively charged). from this bond, there will be an electrostatic interaction between the photosensitizer and the bacterial cell wall, namely the release of ca2+ and mg2+ ions from the cell so that the cell wall is weaker and its permeability increases. the increase in the permeability of the bacterial cell wall causes the photosensitizer cation to enter the cytoplasmic membrane of the bacteria so that there is a deeper disorganization of the permeability barrier. this will increase the absorption and binding of photosensitizer cations with bacterial plasma membranes so that photosensitizer bonds occur with bacterial plasma membranes.13,14 the irradiation in the photosensitizer will be absorbed, which produces two types of mechanisms. in mechanism type i, electron transfer occurs between the photosensitizer and the substrate so that it will produce radical ions called reactive oxygen species (ros) that consist of superoxide anions, hydroxyl radicals, and hydrogen peroxide. these ions are oxidative to cells. in mechanism type ii, there is an electron transfer between the photosensitizer and the oxygen receptor that produces a singlet of oxygen, which is a reactive form of oxygen and a powerful oxidative agent.13,14 the results of both mechanisms can cause several effects, including crosslink lengthening of plasma membrane proteins, inactivation of the enzyme nicotinamide adenine dinucleotide + hydrogen succinate, and lactate dehydrogenase, damaging the balance of k+ ions and other ions and damaging the dna of bacterial cells. as a result of some of these effects, the growth of bacteria can be inhibited so that the target bacteria will die.13,15,16 ros is one of the free radicals derived from oxygen.17 ros is a radical form of an unpaired atom. ros is often used in biomedical free radical terms. included in the ros category are not only free radicals carrying oxygen but also molecules that do not have paired electrons such as hydrogen peroxide, hypochlorous acid, and peroxynitrite anion acid (onoo-). such ros, especially superoxide radicals, are constantly produced by the body.18 superoxide radicals are the most widely produced free radicals in the body and are derived from the reduction of one unpaired free electron in the outer shell layer.19 these radicals are produced by phagocytic cells and serve to kill bacteria. in addition to the formation of superoxide radicals in macrophage and neutrophil cells, production of extracellular also occurs in small quantities as intercellular signaling molecules by several other cell types such as endothelial cells, lymphocytes, and fibroblasts.18 photodynamic inactivation is a therapy modality that uses a photosensitizer agent, a light source, and oxygen to produce ros.20 photodynamic therapy (pdt) can be used as an antimicrobial strategy that uses low-power laser energy to activate a non-toxic photosensitizer to produce singlet oxygen with the ability to kill microorganisms in root canals.21 research conducted by neves et al. stated that the combination of pdt with irrigation agents was more effective in killing bacteria than either naocl alone or pdt alone.22 pdt has significant effectiveness in the elimination of bacterial biofilms when combined with a disinfecting agent. pdt can help reach root canal areas of teeth that are not touched by mechanical preparation of endodontic instruments or naocl irrigating copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 134zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 solutions in conventional standard root canal preparation procedures.21–23 absorption by photosensitizer is a photophysical process to produce ros and singlet oxygen. the laser energy absorbed by the photosensitizer molecule will then activate the occurrence of photochemical reactions, resulting in a radical product that damages the bacterial cell. the larger the photon intensity and the longer the exposure, the more photosensitizer will be activated to produce various ros that has an effect on the number of bacterial deaths.24 one of the most common and frequently used root canal irrigation agents to date is 2.5% naocl. bacteria can penetrate the root dentinal tubules to a depth of 1000 µm, while the irrigation disinfection material only reaches a depth of 100 µm. this allows re-infection and causes root canal treatment failure.25,26 therefore, new methods in endodontic treatment are needed to eliminate pathogenic bacteria to achieve successful root canal treatment.27,28 based on the description above, this research was conducted to determine the biofilm degradation and the quantity of ros in the f. nucleatum biofilm due to the combination of pdt and 2.5% naocl irrigation. materials and methods ethical clearance certificate: 027/hrecc.fodm/i/2021. this study was approved by the ethics committee of the faculty of dental medicine, universitas airlangga. the culture of f. nucleatum atcc 25586 was obtained from the f. nucleatum bacterial stock at the faculty of dental medicine research center, airlangga university, surabaya. the bacterial preparations were incubated at 37°c in an anaerobic atmosphere for 24–48 hours. the bacterial culture was diluted into tryptic soy broth (tsb) media and equated with the mcfarland standard of 1.5 x 108 cfu/ml, then 200 µl was placed into a 96-well microtiter plate. the ros was calculated using a nitroblue tetrazolium (nbt) test with a microplate reader.29 group 1: the untreated control group contained only the f. nucleatum biofilm. group 2: 100 µl of 2.5% naocl irrigation solution was dripped into the well containing the f. nucleatum biofilm. 100 µl (1 mg ml-1) of nbt solution was then dripped into the well, and incubation was carried out for 30 minutes at a temperature of 37oc. next, 100 µl of tsb was dripped into the well, followed by 20 µl of hydrochloric acid (hcl) (0.1 m), and finally, 50 µl of dimethyl sulfoxide (dmso) was dripped into the well. the 96-well microtiter plate was inserted into a microplate reader with a wavelength of 570 nm for od observations. group 3: a photosensitizer in the form of 100 µl of toluidine blue o liquid was dripped into the well for 60 seconds and then irradiated with pdt using fotosan for 50 seconds. 100 µl (1 mg ml-1) of nbt solution was then dripped into the well, and incubation was carried out for 30 minutes at a temperature of 37oc. 100 µl of tsb was dripped into the well, followed by 20 µl of hcl (0.1 m), and finally, 50 µl of dmso was dripped into the well. the 96-well microtiter plate was then inserted into a microplate reader with a wavelength of 570 nm for od observations. a total of two 96-well microtiter plates were used to observe the biofilm degradation and quantities of ros. the 96-well microtiter plates were grouped into four groups, with each group containing eight samples. group 1 was the control group and contained only the f. nucleatum biofilm. group 2 contained f. nucleatum biofilm irrigated with 2.5% naocl. group 3 contained f. nucleatum biofilm and was given a toluidine blue o photosensitizer and pdt fotosan irradiation. group 4 contained f. nucleatum biofilm, and 100 µl of 2.5% naocl irrigation solution was dripped into the well. a photosensitizer in the form of 100 µl of toluidine blue o liquid was dripped into the well for 60 seconds and then irradiated with pdt fotosan for 50 seconds. 100 µl (1 mg ml-1) of nbt solution was dripped into the well, and incubation was carried out for 30 minutes at a temperature of 37oc. next, 100 µl of tsb was dripped into the well, followed by 20 µl of hcl (0.1 m), and finally, 50 µl of dmso was dripped into the well. for the results of the study, the means and standard deviations of each group were calculated. the shapiro–wilk test for normality was used to determine the population data distribution of each group. after concluding that the data were normally distributed, levene’s test for homogeneity was carried out to determine the similarity of the variations in the sample groups. to compare the differences across each group, tukey’s hsd test was followed using an independent t-test for the differences in the two group tests. results the data obtained come from the od observations through a microplate reader of each bacterium in each group. the bar chart for the means and standard deviations of biofilm degradation and the quantities of ros in each group can be seen in figure 1. in the degradation biofilm group before data analysis, normality and homogeneity tests were performed. the shapiro–wilk normality test was performed and a p-value of p = 0.674 (p > 0.05) was found for the control treatment group, p = 0.958 for the naocl treatment group, p = 0.821 for the pdt fotosan treatment group, and p = 0.940 for the combination treatment group, meaning that all data were normally distributed. levene’s test was then followed to determine the homogeneity of the data. the results of levene’s test showed p = 0.001 (p < 0.05). this shows that the treatment group had unequal homogeneity of variance (not homogeneous). from the results above, it was found that all treatment groups were normally distributed and had unequal variances (not homogeneous), thus the independent sample statistical test was carried out. as shown in table 1, the biofilm degradation of all treatment groups had a p-value of p < 0.05. this indicates that there was a copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 135 zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 significant difference in the biofilm degradation across all treatment groups. in the ros quantity group, normality and homogeneity tests were carried out. the shapiro–wilk normality test was performed and a p-value of p = 0.767 (p > 0.05) was found for the control treatment group, p = 0.734 for the naocl treatment group, p = 0.317 for the pdt fotosan treatment group, and p = 0.340 for the combination treatment group, meaning that all data were normally distributed. next, levene’s test was followed to determine the homogeneity of the data. the results of levene’s test showed p = 0.999 (p > 0.05). this indicates that the treatment groups had the same homogeneity of variance (homogeneous). to determine the difference in the quantities of ros across treatment groups, the anova statistical test was performed. the results of the anova test obtained a p-value of p = 0.001 (p < 0.05). this indicates that there was a difference in the quantities of ros across the treatment groups. to find out the differences across treatment groups, tukey’s hsd test was carried out statistically. the results of tukey’s hsd statistical test can be seen in table 2. the quantities of ros across all treatment groups had a p-value of p < 0.05. this indicates that there was a significant difference in the quantities of ros across all treatment groups. discussion in this study, fusobacterium nucleatum was used because this bacterium is one of the bacteria involved in root canal infection. fusobacterium nucleatum is an anaerobic bacterium in the form of non-spore, non-motile, and gram-negative bacteria. these bacteria are associated with spontaneous pain, tenderness to percussion, tenderness to palpation, gum swelling, hemorrhagic exudates, tooth mobility, inadequate restorations, and inadequate obturation.9 the results of the statistical analysis show that the average biofilm degradation of the naocl-only group (0.68) was lower than the control group (1.06). this is in accordance with the results of the research conducted by canga and subashi,30 which stated that 2.5% naocl has a better antibacterial effect than 2% chlorhexidine and can denature bacterial toxins and dissolve organic tissue. in addition, sahebi et al.31 also stated that naocl had better bacterial inhibition than aloe vera and normal saline. a study conducted by janani et al.32 showed that 2.5% naocl was more effective in eliminating bacteria from infected root canals than pdt. the results of this study showed a significant decrease in the number of bacteria in the naocl group compared to the control group, and almost no bacteria were detected in the naocl group after using the polymerase chain reaction technique.32 1.06 ±0.0232 0.68 ±0.0069 0.78 ±0.0060 0.46 ±0.0084 0.28 ±0.0085 0.68 ±0.0092 0.79 ±0.0080 0.99 ±0.0096 0 0.2 0.4 0.6 0.8 1 1.2 i (control) ii (naocl) iii (pdt) iv (naocl+pdt) od biofilm od ros figure 1. the mean and standard deviation of biofilm degradation and ros. table 1. the results of the independent sample test for biofilm degradation naocl pdt fotosan combination control p = 0.001 p = 0.001 p = 0.001 naocl p = 0.001 p = 0.001 pdt fotosan p = 0.001 p = 0.001 table 2. the results of tukey’s hsd test for quantities of ros naocl pdt fotosan combination control p = 0.001 p = 0.001 p = 0.001 naocl p = 0.001 p = 0.001 pdt fotosan p = 0.001 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 136zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 the mean quantity of ros in the naocl-only group (0.68) was higher than the control group (0.28). this is in accordance with the statement by zhang et al.,33 who said that exposure to disinfecting agents can induce increased levels of ros, bacterial membrane damage, ros-mediated dna damage, and an increased stress response. in addition, according to harris34 and mohmmed et al.,35 an increase in ros causes oxidative stress in cells that causes lipid peroxides, impaired protein synthesis, and dna damage. naocl is an irrigation agent used in endodontic procedures that has antimicrobial properties and can dissolve organic tissue.36 naocl produces hypochlorous acid, which is an oxidizing agent that acts as a solvent. when naocl comes into contact with tissue it will produce hydroxyl ions and hypochlorous acid.37 in addition, naocl has a high ph, which triggers the release of hydroxyl ions.34–36 the release of hydroxyl ions can cause cell death through two mechanisms, namely by increasing ros directly or by decreasing adenosine triphosphate.38 however, this antimicrobial mechanism of naocl becomes ineffective against pathogenic bacteria in the anatomical area that is difficult to reach by irrigation solutions or mechanical preparation by endodontic instruments at the root canal cleaning stage. in addition, dentinal tubules have a narrow lumen (1–2 um) and are 2–3 mm in length, making them a challenge for disinfection materials. the minimal and maximal bacterial penetration depths into the dentinal tubules were 1 µm and 1480 µm, respectively, with a mean of 167 µm.39 thus, a complementary/supportive technique to increase the effectiveness of root canal treatment is needed.40 the average biofilm degradation in the pdt-only group (0.78) was lower than the control group (1.06). the results of this study showed that pdt only could eliminate fusobacterium nucleatum biofilm. this is in accordance with a study conducted by bibova et al.,41 which stated that fotosan can be considered as an additional procedure to kill bacteria in the root canal system after standard endodontic treatment. in this study, pdt fotosan was used to disinfect root canals. fotosan uses red light with a wavelength of 630 nm. photodynamic therapy might be useful as an alternative approach for antimicrobial treatment. the photosensitizer used in this study was toluidine blue o. the toluidine blue o photosensitizer contains phenothiazine. phenothiazine is a cation that will bind to an anion bacterial cell wall. from this bond, there will be an electrostatic interaction increasing the permeability of bacterial cell walls that causes the toluidine blue o cation to permeate more into the bacterial cytoplasmic membrane to disorganize the barrier of permeability further. toluidine blue o research shows that it also has antibacterial power because it can interact with lipopolysaccharides of bacterial cell membranes even without irradiation. when irradiating with a wavelength of 630 nm, there will be a maximum absorption of photosensitizer fluid so that pdt photo cations occur to kill bacteria better compared to the use of photosensitizer fluid without irradiation. the mean ros quantity in the pdt-only group (0.79) was higher than the control group (0.28). this is in accordance with the statement by abrahamse and hamblin42 that said that pdt uses a non-toxic photoactive dye called a toluidine blue o photosensitizer that is activated with visible light to produce ros. photodynamic mechanisms with toluidine blue o involve the interaction of light with agents that produce oxygen. the irradiation with light at a certain wavelength according to the absorption peak of the photosensitizer will produce energy.43 the effectiveness of quantum yield for producing a particular ros type depends on the photosensitizer, the availability of oxygen, and the reaction environment.44 the energy transferred from the activated photosensitizer will be forwarded to the available oxygen so that it is transformed into singlet oxygen as a very reactive and toxic oxygen species. contact between the singlet oxygen and bacterial cell walls will cause oxidative damage to bacterial cells by inducing ros production. ros is a free radical of oxygen that can damage the microorganism membrane and accelerate the death of microorganisms.45 the concentration of radical ions and many oxygen singlets will cause damage to the lysosomes, mitochondria, and plasma membranes of larger bacterial cells, leading to more dead bacterial cells.42 however, pdt alone was less effective in eliminating bacteria. this is in accordance with a study conducted by damasceno and araújo46; pdt is a supporting technique to improve root canal disinfection after biomechanical preparation of endodontic treatment. in addition, according to souza et al.,47 the main approach for bacterial elimination is conventional chemomechanical preparation with the addition of chemicals such as naocl. thus, further research was conducted on the combination of pdt and naocl. the average ros quantity of the combination group (0.99) was higher than the control group (0.28), the single naocl group (0.68), and the single pdt group (0.79). research conducted by vaziri et al.40 and souza et al.47 said that the combination of pdt and 2.5% naocl was the best choice to maximize disinfection. vaziri et al.40 conducted a study of 60 single-rooted teeth and found that after the combined treatment of pdt and 2.5% naocl, no live bacteria were found. research conducted by ng et al.48 said that the combination of 6% naocl and pdt with toluidine blue o was better than 6% naocl alone. in their study, ng et al.48 used 52 necrotic teeth and radiographically showed apical periodontitis. the results showed that 86.5% of the root canals were free of bacteria after the combination of the chemomechanical method and pdt with toluidine blue o, while in the chemomechanical-only group, only 49% were free of bacteria.48 according to bumb et al.,49 pdt has the ability to penetrate the dentinal tubules in the root canal wall to a depth of 890–900 µm, while naocl was only able to penetrate to a depth of 60–150 µm. research conducted by hopp and biffar50 showed that exposure to red light copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 137 zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 with a wavelength of 628 nm can activate toluidine blue o to produce ros. pdt has significant effectiveness in the elimination of bacterial biofilms when combined with a disinfecting agent. pdt can help reach areas that are not touched by mechanical preparation or naocl irrigating solutions in conventional procedures. in conclusion, there is a potentition relationship between naocl 2.5% and pdt fotosan. biofilm degradation occurs because of the effect of antibacterial naocl 2.5%, and the irradiation effect of the toluidine blue o photosensitizer means that there is a transfer of electrons between the photosensitizer and substrate. ros increases due to the electron configuration of the oxygen molecule being in an excited (unstable) state. excited oxygen tends to strive for a stable electron state; therefore, this oxygen will interact with the surrounding biological system. the interaction that occurs between the excited oxygen and biological systems such as bacterial cells will damage these systems and cell structures. research conducted by hopp and biffar50 showed that exposure to red light with a wavelength of 628 nm can activate toluidine blue o to produce ros. the resulting ros produced is very reactive, such as superoxide oxygen singlets and hydroxyl radicals that destroy bacteria.51 in addition, the resulting ros can target and destroy biomolecules in the bacterial cell wall.52 pdt has significant effectiveness in the elimination of bacterial biofilm when combined with disinfection. pdt can help reach the root canal area that is not touched by mechanical preparation of endodontic instruments or naocl irrigation solutions in the preparation of root canal treatments. acknowledgment the authors gratefully acknowledge laboratory support from the dental research center of faculty of dental medicine, universitas airlangga. references 1. homenta h. infeksi biofilm bakterial. j e-biomedik. 2016; 4(1): 1–11. 2. solano c, echeverz m, lasa i. biofilm dispersion and quorum sensing. curr opin microbiol. 2014; 18: 96–104. 3. wang y, bian z, wang y. biofilm formation and inhibition mediated by bacterial quorum sensing. appl microbiol biotechnol. 2022; 106(19–20): 6365–81. 4. tambayong aj, widjiastuti i, lunardhi cgj. differences in photodynamic therapy exposure time and staphylococcus aureus counts. dent j. 2018; 51(2): 95–8. 5. balakrishna n, moogi p, kumar gv, prashanth b, shetty n, rao k. effect of conventional irrigation and photoactivated disinfection on enterococcus faecalis in root canals: an in vitro study. j conserv dent. 2017; 20(2): 125–8. 6. garg n, garg a. textbook of operative dentistry. 3rd ed. new delhi: jaypee brother medical publishers; 2015. p. 241, 502. 7. martinho fc, leite frm, nóbrega lmm, endo ms, nascimento gg, darveau rp, gomes bpfa. comparison of fusobacterium nucleatum and porphyromonas gingivalis lipopolysaccharides clinically isolated from root canal infection in the induction of pro-inflammatory cytokines secretion. braz dent j. 2016; 27(2): 202–7. 8. lee l-w, lee y-l, hsiao s-h, lin h-p. bacteria in the apical root canals of teeth with apical periodontitis. j formos med assoc. 2017; 116(6): 448–56. 9. gomes bpfa, bronzato jd, almeida-gomes rf, pinheiro et, sousa elr, jacinto rc. identification of fusobacterium nucleatum in primary and secondary endodontic infections and its association with clinical features by using two different methods. clin oral investig. 2021; 25(11): 6249–58. 10. hsieh s-c, teng n-c, chu cc, chu y-t, chen c-h, chang l-y, hsu c-y, huang c-s, hsiao gy-w, yang j-c. the antibacterial efficacy and in vivo toxicity of sodium hypochlorite and electrolyzed oxidizing (eo) water-based endodontic ir rigating solutions. materials (basel). 2020; 13(2): 260. 11. kandaswamy d, venkateshbabu n. root canal irrigants. j conserv dent. 2010; 13(4): 256–64. 12. jaju s, jaju pp. newer root canal irrigants in horizon: a review. int j dent. 2011; 2011: 851359. 13. shrestha a, cordova m, kishen a. photoactivated polycationic bioactive chitosan nanoparticles inactivate bacterial endotoxins. j endod. 2015; 41(5): 686–91. 14. diogo p, fernandes c, caramelo f, mota m, miranda im, faustino maf, neves mgpms, uliana mp, de oliveira kt, santos jm, gonçalves t. a ntim icrobial photodynam ic therapy aga inst endodontic enterococcus faecalis and candida albicans mono and mixed biofilms in the presence of photosensitizers: a comparative study with classical endodontic irrigants. front microbiol. 2017; 8: 498. 15. jiménez a, palmero-cámara c, gonzález-santos mj, gonzálezbernal j, jiménez-eguizábal ja. the impact of educational levels on formal and informal entrepreneurship. brq bus res q. 2015; 18(3): 204–12. 16. bago jurič i, plečko v, anić i, pleško s, jakovljević s, rocca jp, medioni e. antimicrobial efficacy of photodynamic therapy, nd:yag laser and qmix solution against enterococcus faecalis biofilm. photodiagnosis photodyn ther. 2016; 13: 238–43. 17. keshari ak, verma ak, kumar t, srivastava r. oxidative stress: a review. int j sci technoledge. 2015; 3(7): 155–62. 18. correia jh, rodrigues ja, pimenta s, dong t, yang z. photodynamic therapy review: principles, photosensitizers, applications, and future directions. pharmaceutics. 2021; 13(9): 1332. 19. birben e, sahiner um, sackesen c, erzurum s, kalayci o. oxidative stress and antioxidant defense. world allergy organ j. 2012; 5(1): 9–19. 20. astuti sd, mawaddah a, nasution am, mahmud af, fitriyah n, kusumawati i, abdurachman, puspita ps, suhariningsih. effectiveness of photodynamic inactivation with exogenous photosensitizer curcuma longa extract activated by laser diode 403 nm on staphylococcus aureus. j int dent med res. 2020; 13(1): 155–61. 21. paqué f, boessler c, zehnder m. accumulated hard tissue debris levels in mesial roots of mandibular molars after sequential irrigation steps. int endod j. 2011; 44(2): 148–53. 22. neves g de v, dos santos ksa, rocha eal de ss, de moura rq, barros dgm, gominho lf, gomes dq de c. antibacterial effect of photodynamic therapy on root canal disinfection combined with different irrigation protocols. iran endod j. 2020; 15(2): 90–5. 23. plotino g, grande nm, mercade m. photodynamic therapy in endodontics. int endod j. 2019; 52(6): 760–74. 24. astuti sd, hafidiana, rulaningtyas r, abdurachman, putra ap, samian, arifianto d. the efficacy of photodynamic inactivation with laser diode on staphylococcus aureus biofilm with various ages of biofilm. infect dis rep. 2020; 12(11): 8736. 25. tișler c-e, badea m-e, buduru s, kui a, floria m, popescu ștefan, mitaru m, negucioiu m. biofilm inactivation using photodynamic therapy in dentistry: a review of literature. dogaru g, editor. balneo res j. 2020; 11(3): 279–87. 26. lindawati y, primasari a, suryanto d. fusobacterium nucleatum : bakteri anaerob pada lingkungan kaya oksigen (dihubungkan dengan staterin saliva). talent conf ser trop med. 2018; 1(1): 181–8. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 138zubaidah et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 132–138 27. xhevdet a, stubljar d, kriznar i, jukic t, skvarc m, veranic p, ihan a. the disinfecting efficacy of root canals with laser photodynamic therapy. j lasers med sci. 2014; 5(1): 19–26. 28. lins ccsa, melo ars, silva cc, oliveira jb, lima ga, castro cmmb, diniz fa. photodynamic therapy application in endodontic aerobic microorganisms and facultative anaerobic. formatex. 2015; : 559–63. 29. arneiro ras, nakano rd, antunes laa, ferreira gb, fontes kbfc, antunes ls. efficacy of antimicrobial photodynamic therapy for root canals infected with enterococcus faecalis. j oral sci. 2014; 56(4): 277–85. 30. canga m, subashi b. antibacterial effectiveness of naocl and chlorexidine in infected root canal. int j sci res. 2015; 4(3): 678–80. 31. sahebi s, khosravifar n, sedighshamsi m, motamedifar m. comparison of the antibacterial effect of sodium hypochlorite and aloe vera solutions as root canal irrigants in human extracted teeth contaminated with enterococcus faecalis. j dent (shiraz, iran). 2014; 15(1): 39–43. 32. janani m, jafari f, samiei m, lotfipour f, nakhlband a, ghasemi n, salari t. evaluation of antibacterial efficacy of photodynamic therapy vs. 2.5% naocl against e. faecalis-infected root canals using real-time pcr technique. j clin exp dent. 2017; 9(4): e539–44. 33. zhang s, wang y, lu j, yu z, song h, bond pl, guo j. chlorine disinfection facilitates natural transformation through rosmediated oxidative stress. isme j. 2021; 15(10): 2969–85. 34. ha r r is j. compa r ison of st er i plex t m hc a nd sod ium hypochlorite cytotoxicity on primary human gingival fibroblasts. virginia commonwealth university, school of dentistry; 2012. p. 1–25. 35. mohmmed sa, vianna me, penny mr, hilton st, knowles jc. the effect of sodium hypochlorite concentration and irrigation needle extension on biofilm removal from a simulated root canal model. aust endod j. 2017; 43(3): 102–9. 36. uğur aydin z, akpinar ke, hepokur c, erdönmez d. assessment of toxicity and oxidative dna damage of sodium hypochlorite, chitosan and propolis on fibroblast cells. braz oral res. 2018; 32: e119. 37. chandra bs, gopikrishna v. grossman’s endodontic practice. 13th ed. new delhi: wolters kluwer; 2014. p. 55–7. 38. ok e, adanir n, hakki s. comparison of cytotoxicity of various concentrations origanum extract solution with 2% chlorhexidine gluconate and 5.25% sodium hypochlorite. eur j dent. 2015; 9(1): 6–10. 39. rosen e, elbahary s, haj-yahya s, jammal l, shemesh h, tsesis i. the invasion of bacterial biofilms into the dentinal tubules of extracted teeth retrofilled with fluorescently labeled retrograde filling materials. appl sci. 2020; 10(19): 6996. 40. vaziri s, kangarlou a, shahbazi r, nazari nasab a, naseri m. comparison of the bactericidal efficacy of photodynamic therapy, 2.5% sodium hypochlorite, and 2% chlorhexidine against enterococcous faecalis in root canals; an in vitro study. dent res j (isfahan). 2012; 9(5): 613–8. 41. bibova n, simeonova e, dimitrova s, dakov k, denkova z, yanakieva v. photoactivated disinfection of root canal system with fotosan. iosr j dent med sci. 2019; 18(9): 81–4. 42. abrahamse h, hamblin mr. new photosensitizers for photodynamic therapy. biochem j. 2016; 473(4): 347–64. 43. astuti sd, wibowo ra, abdurachman, triyana k. antimicrobial photodynamic effects of polychromatic light activated by magnetic fields to bacterial viability. j int dent med res. 2017; 10(1): 111–7. 44. dai t, fuchs bb, coleman jj, prates ra, astrakas c, st. denis tg, ribeiro ms, mylonakis e, hamblin mr, tegos gp. concepts and principles of photodynamic therapy as an alternative antifungal discovery platform. front microbiol. 2012; 3: 120. 45. pou rhajibag he r m , ba ha dor a. adju nct ive a nt i m ic r obia l photodyna m ic t herapy to convent iona l chemo -mecha n ica l debridement of infected root canal systems: a systematic review and meta-analysis. photodiagnosis photodyn ther. 2019; 26: 19–26. 46. damasceno mcf, araújo mm do c. photodynamic therapy in endodontics: literature review. brazilian j implantol heal sci. 2021; 3(3): 27–40. 47. souza ma, steier l, rossi-fedele g, barth júnior v, de oliveira sd, de figueiredo jap. effectiveness of photodynamic therapy and sodium hypochlorite on root canal system infected with enterococcus faecalis – an in vitro study. rev odonto ciência. 2016; 31(3): 114. 48. ng r, singh f, papamanou da, song x, patel c, holewa c, patel n, klepac-ceraj v, fontana cr, kent r, pagonis tc, stashenko pp, soukos ns. endodontic photodynamic therapy ex vivo. j endod. 2011; 37(2): 217–22. 49. bumb ss, bhaskar dj, agali cr, punia h, gupta v, singh v, kadtane s, chandra s. assessment of photodynamic therapy (pdt) in disinfection of deeper dentinal tubules in a root canal system: an in vitro study. j clin diagnostic res. 2014; 8(11): zc67–71. 50. hopp m, biffar r. photodynamic therapies – blue versus green. laser. 2013; 1: 10–25. 51. olivi g, olivi m. lasers in restorative dentistry. berlin, heidelberg: springer berlin heidelberg; 2015. p. 274. 52. dąbrowski jm. reactive oxygen species in photodynamic therapy: mechanisms of their generation and potentiation. in: van eldik r, hubbard cd, editors. advances in inorganic chemistry: inorganic reaction mechanisms. elsevier academic press; 2017. p. 343–94. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p132–138 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p132-138 7171 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 71–75 research report the potential effect of moringa oleifera leaves extract on vascular endothelial growth factor expression in wistar rat oral cancer cells dwicha rahma nuriska hartono, theresia indah budhy sulisetyawati, and edhi jularso department of oral and maxillofacial pathology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: cancer is a disease characterized by abnormal and uncontrolled growth of tissue cells. in indonesia, cancer ranks as the fifth largest cause of mortality, while it is the second largest worldwide. cancer affected by angiogenesis, the process of forming new blood vessels to provide the nutrient and oxygen necessary for tumor growth. vascular endothelial growth factor (vegf) represents a pro-angiogenic factor. moringa oleifera leaf extract can be used as an anticancer agent by reducing the expression of vegf. purpose: the study aimed to prove that moringa oleifera leaf extract can reduce vegf expression in benzopyrene-induced oral cancer cells of wistar rats. methods: this was an experimental laboratory research with posttest-only control group design. all experimental subjects presented symptoms of cancer following induction with 8 mg/kgbw of benzopyrene. the sample consisted of 28 wistar rats, divided into four groups, namely; a control group (k) whose members were administered with only aquadest and three treatment groups (p) treated with moringa oleifera leaves extract at percentages of 3.125% (p1), 4.6875% (p2) and 6.25% (p3) respectively. observation of vegf expression was undertaken by means of immunohistochemical staining. results: a decrease in vegf expression occurred in all treatment groups when compared with the control group. a significant difference existed between the control group (k) and the treatment group (p1), while there were no significant differences between the treatment groups (p1, p2 and p3). conclusion: moringa oleifera leaf extract with a percentage of 3.125% proved most effective at reducing vegf expression in oral cancer cells in wistar rats. keywords: isothiocyanate; moringa oleifera extract; oral cancer; vegf expression correspondence: theresia indah budhy sulisetyawati, department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo no. 47 surabaya 60312, indonesia. email: terebudhy@gmail.com introduction oral cancer represents one of the six most common malignancies in asia with 274,300 new cases annually.1 cancer ranks as the fifth most common cause of mortality in indonesia and the second largest worldwide with 7.6 million deaths in 2014, a figure projected to increase to 13.1 million in 2030.2,3 most cancer-related deaths occur due to its progressive development and metastasis,4 both of which can be affected by angiogenesis. therefore, analyzing the formation of new blood vessels can play a role in determining the prognosis and physiological and pathological management of cancer patients.5,6 research has indicated that vascular endothelial growth factor (vegf) is the main factor involved in angiogenesis and that its expression in neoplastic epithelium is higher than dysplastic and normal epithelium.7 termination of vegf signaling pathway is now approved and used as an alternative cancer therapy.5 cancer treatment often involves the use of chemotherapy8 which has the disadvantages of being expensive and producing several side effects such as dizziness, nausea, fatigue, mucositis, nerve damage, neutropenia and infection.9 these side effects are caused by chemotherapy drugs which do not selectively destroy cancer cells, but also the surrounding normal cells such as hair follicle cells, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p71–75 mailto:terebudhy@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p71-75 72 hartono, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 71–75 gastrointestinal mucosa cells and bone marrow cells that proliferate rapidly.10 for these reasons, herbal medicines tend to be taken as a form of cancer treatment. not only because of its affordable price, but also its convenience and minimal side effects.3 one herbal treatment product is moringa oleifera, also known as kelor, which has high nutrient content in all its parts, ranging from the leaves, stems, flowers and fruit to the roots. moringa oleifera leaves contain isothiocyanate which the research conducted by gupta et al.3 showed to have anticancer properties. these properties render it effective as a treatment for cancer.11 isothiocyanate, as an anticancer treatment, possesses a mechanism capable of inhibiting the process of angiogenesis.12 during the development of a cancer, angiogenesis can be induced by various factors, one of which is hypoxia due to limited oxygen diffusion.5 a low concentration of oxygen induces the expression of various vasculogenic and angiogenic factors, including vegf.13 the research conducted by boreddy et al.14 stated that isothiocyanate can inhibit hif-1α signal transduction activity and decrease vegf expression, while that carried out by nararya15 also asserted that moringa oleifera leaf extract demonstrates biocompatible properties at concentrations between 0.406% and 3.125%. these concentrations can be interpreted as non-toxic and moringa oleifera leaf extract can, therefore, be used as a herbal medicine.15 based on the data above, the authors intend to undertake further examination of the potential of moringa oleifera leaf extract to treat vegf expression in oral cancer. the study reported here was conducted using a glycoside test to observe the decrease in vegf expression after administration of moringa oleifera leaf extract that contained 6.92% isothiocyanate. materials and methods the research constituted a laboratory-based experimental investigation with posttest-only control group design, approved by the ethical clearance for health research committee, faculty of dental medicine, universitas airlangga (no.121/hrecc.fodm/vii/ 2018). the research sample comprised 2-3 month old, healthy, male, rattus novergicus with a body weight between 130 and 150 grams. the 28 wistar rats in the sample were divided into four groups, namely: the control group (k) which was given only aquadest and three treatment groups (p) treated with moringa oleifera leaves extract at respective percentages of 3.125% (p1), 4.6875% (p2) and 6.25% (p3).15 moringa oleifera leaf extract was produced by the research institute and industrial consultation laboratory in surabaya. moringa oleifera leaves were dried for 24 hours before being mashed in a blender. the resulting powder was then soaked in 96% ethanol in a closed container over a period of two days. the resulting solution was filtered with maceration subsequently carried out until clear results were obtained and evaporated at 40oc in a rotary vacuum evaporator. furthermore, moringa oleifera leaves extract was diluted with aquadest to obtain concentrations of 3.125%, 4.6875% and 6.25%.15 the maceration result subsequently underwent phytochemical screening using a glycoside test to determine the amount of its isothiocyanate phytochemical content. the first step involved producing a trial solution. the filtrate was filtered three times with a 20 ml mixture of chloroform p and isopropanol p at a volume of 3:2. sodium sulfate hydrate was added to the solution, filtered and evaporated at 50oc with the remaining filtrate being diluted with 2 ml of methanol p.15 approximately 0.1 ml of trial solution was evaporated in a water bath and the remaining solution diluted in 5 ml of anhydrous acetic acid p. sulfuric acid p was dripped into the solution ten times. the resulting blue or green color indicated the presence of glycosides (liebermann reaction). another 0.1 ml of trial solution was inserted into a test tube and subsequently evaporated in a water bath. the remaining solution was added to 2 ml of water, two drops of molish lp and 2 ml of sulfuric acid p. a purple ring was formed at the edge of the liquid indicating the presence of sugar bond (molish reaction).15 oral cancer was induced to all the experimental subjects by administering a dose of benzopyrene in powder form amounting to 8 mg/kg of bodyweight which was dissolved in oleum olivarum at a ratio of 2:1.16 the administration of 0.2 ml benzopyrene was performed through extraoral injection into the right cheek of the wistar rat subjects three times a week over a period of four consecutive weeks. after having been induced with cancer, all experimental subjects in the treatment group were administered with moringa oleifera leaf extract in the following approximate amounts: the treatment group 1 (p1) at a concentration of 3.125%; treatment group 2 (p2) at a concentration of 4.6875% and treatment group 3 (p3) at a concentration of 6.25%. based on the research of nararya,15 the 2 ml extract was managed intraorally by means of an insulin sonde, once a day for 20 days. meanwhile, the control group was administered only with aquadest. all experimental subjects were sacrificed through chloroform inhalation. the tissue was excised using surgical scissors and blades, then fixed using 10% neutral buffered formalin (nbf) solution (ph 6.5-7.5). the tissue was processed and 4µm-thick paraffin blocks were produced. vegf expression was examined by immunohistochemical (ihc) staining involving the use of vegf antibodies. the counterstain employed was haematoxylin mayer with the result that the positive cell was indicated by the presence of a brownish color.15 the data obtained was analyzed using one-way anova and post-hoc tukey hsd tests to determine its respective significance in each group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p71-75 73hartono, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 71–75 results observation and calculation of vegf expression using a light microscope at 400x magnification in five fields of view produced the following results. based on the contents of table 1, the respective vegf expressions of p1, p2 and p3 had lower numbers than the control (k). the result of the vegf expression in a field of view using a light microscope is also shown in figure 1. the significance of vegf expression is presented in the p1 group and the control group (k) which have p values lower than 0.05 (table 2). discussion this research aimed to discover the potential of moringa oleifera leaf extract to decrease the vegf expression in the oral cancer cells of wistar rats induced by benzopyrene. benzopyrene is an organic compound with a specific molecular formula, c20h12, which is a member of the extremely toxic class of polycyclic aromatic hydrocarbons (pah) and represents a carcinogen proven to cause tumors in experimental subjects.17 the research conducted by juliyarsi and melia18 indicated that subcutaneous injection of benzopyrene can induce cancer in mice because it constitutes a hydrophobic compound lacking methyl groups or other reactive groups that can be converted into more polar compounds.18 as a result, the body experiences considerable difficulty in excreting these compounds. furthermore, benzopyrene demonstrates structural similarities with nucleobases such as adenosine, thymine, guanine and cytosine. this renders inserting itself into dna strands and potentially disrupting their transcription process relatively straightforward for benzopyrene. failure to repair the resulting damage caused by this process will culminate in the development of cancerous cells.19 for the purposes of this research, ethanol was extracted from moringa oleifera leaves by means of a process involving maceration which employs organic solvents to extract the desired compound from a solution.20 this process is extremely beneficial due to being both economic and comparatively straightforward to complete. it causes table 1. differences in the one-way anova test results of vegf expression group mean ± sd anova (p) k 9.571 ± 5.2782 0.008 p1 3.343 ± 0.8772 p2 6.657 ± 2.2052 p3 5.514 ± 2.0359 table 2. post-hoc tukey hsd test results group k p1 p2 p3 k 0.005* 0.308 0.090 p1 0.208 0.557 p2 0.897 p3 a b c d figure 1. the area designated by an arrow () constitutes the vegf expression in endothelial cancer cells in the control group (a), treatment group 1 (b), treatment group 2 (c), treatment group 3 (d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p71-75 74 hartono, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 71–75 cell walls and membranes to rupture because of differences between the internal and external pressure affecting the cell. consequently, secondary metabolites in the cytoplasm dissolve in organic solvents.21 solvent selection during the maceration process can significantly enhance its effectiveness by prioritizing the solubility of natural material compounds in these solvents.22 since moringa oleifera leaves contain numerous metabolites which are generally polar, during this research polar solvents such as ethanol were employed.23 vongsak et al.24 posited that maceration using ethanol represents the recommended extraction method to promote further treatment product development. moringa oleifera is a plant rich in chemical compounds, one of them being isothiocyanate which is a substance that can act as a chemopreventive agent in cancer cells.11,25 the research of cavell et al.12 has shown isothiocyanate to possess potential chemopreventive and chemotherapeutic properties through a inhibitive mechanism in both in vitro and in vivo angiogenesis processes. angiogenesis constitutes the process of new blood vessel formation within the human body and a prerequisite of tumor development.12 the main mediator of tumor angiogenesis is vegf.26 as tumors grow in size, the distance between cells and their closest blood vessels also increases. this causes tumor cells to experience hypoxia resulting in areas with low oxygen levels. in order to compensate for this, tumor cells will produce endothelial growth factors, including vegf. as a result of this mechanism, vegf expression increases in tumors, specifically in necrotic areas with low oxygen levels. therefore, vegf overexpression can be considered a compensation mechanism that allows tumor tissue to increase oxygen uptake through endothelial proliferation.27 lalla et al.28 state that the increase in vegf expression is proportional to the greater density of micro blood vessels in a variety of tumors. according to smith et al.29 vegf overexpression is the most influential factor in the deficient prognosis of oral squamous cell carcinoma. in addition, the results showed that cancer associated with high vegf levels had a much shorter recurrence period than those with low levels. these results indicate that vegf affects not only the development of cancer and angiogenesis but also their prognosis.27 in general, the research results indicate that the average vegf expression in the group treated with moringa oleifera leaf extract had a lower value than that of the control group. such results are consistent with those of the research conducted by cavell et al.12 which posited that the antiangiogenic activity of isothiocyanate is associated with decreased production of proangiogenic factors, including vegf. isothiocyanate can reduce the amount of vegf by means of inhibitory pathways in the transcription of hypoxia inducible factor (hif).12 the results showed that moringa oleifera leaf extract reduce vegf expression. cancer cells can produce various proangiogenic factors, including vegf, which act to increase the survival and proliferation of endothelial cells, leading to the initiation of angiogenesis. therefore, administration of substances with the potential to act as antiangiogenic agents can suppress the number of proangiogenic factors such as vegf. a balance between pro and antiangiogenic factors will occur which reduces the level of angiogenesis.12 data significance analysis using a post-hoc tukey hsd test indicated the absence of significant differences between groups treated with moringa oleifera leaf extract at concentrations of 3.125%, 4.6875% and 6.25%. this result shows that doses of these three respective concentrations demonstrate almost equal effectiveness. oral provision of certain drugs or other substances will result in their absorption, distribution, metabolism and excretion. drugs reaching target cells will have an effect dependent upon the absorption and distribution processes involved. these processes are influenced by several factors such as the physiological condition of the gastrointestinal tract, absorption mechanisms including active transport, protein carriers (transporters) and individual pathological conditions.30 polyglyco-protein (pgp) is one of the transporters that functions as a repellent to chemical compounds or drugs in the duodenum and ileum. within the body, pgp can be found in the cells of the intestines, liver, kidney tubules, and endothelial capillaries.31 in organ systems, pgp affects the absorption, distribution and elimination of drugs32 and can reduce the amount of chemical compounds or drugs absorbed. pgp can also prevent anticancer compounds entering cancerous tissue, thereby reducing their effectiveness. in cancer therapy, other substances capable of inhibiting pgp are required to enhance the effectiveness of anticancer compounds.33 in this research, moringa oleifera leaf extract was administered as a single substance. the role of pgp can still influence the effect of isothiocyanate on target cells, thus producing insignificant results. therefore, further research is required in this area.34 non-significant results can also be produced due to the individual immune system which affects the acceleration or retardation of the progression of the cancer. weakening of the immune system can cause tumor cells to develop more easily and affect the effectiveness of anticancer drug compounds on target cells.35 the conclusion of this research was that moringa oleifera leaf extract at a concentration of 3.125% containing 6.92% isothiocyanate, as confirmed by phytochemical glycoside screening, was the optimum means of decreasing vegf expression in benzopyreneinduced oral cancer cells of wistar rats. references 1. epstein j, elad s. oral and oropharyngeal cancer. in: glick m, editor. burket’s oral medicine. 12th ed. raleigh: people’s medical publishing house-usa; 2015. p. 173–99. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p71-75 75hartono, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 71–75 2. aulawi t. hubungan konsumsi daging merah dan gaya hidup terhadap risiko kanker kolon. kutubkhanah. 2013; 16: 37–45. 3. gupta p, kim b, kim s-h, srivastava sk. molecular targets of isothiocyanates in cancer: recent advances. mol nutr food res. 2014; 58(8): 1685–707. 4. a b du l-a z i z m a , a m i n a k , e lro uby dh , sh a ke r o g. lymphangiogenesis in oral squamous cell carcinoma: correlation with vegf-c expression and lymph node metastasis. int j dent. 2017; 2017: 1–10. 5. kresno sb. ilmu dasar onkologi. 3rd ed. jakarta: badan penerbit fakultas kedokteran universitas indonesia; 2011. p. 344–67. 6. niu g, chen x. vascular endothelial growth factor as an antiangiogenic target for cancer therapy. curr drug targets. 2010; 11(8): 1000–17. 7. mărgăritescu c, pirici d, stîngă a, simionescu c, raica m, mogoantă l, stepan a, ribatti d. vegf expression and angiogenesis in oral squamous cell carcinoma: an immunohistochemical and morphometric study. clin exp med. 2010; 10(4): 209–14. 8. ahimsa gk. aktivitas antikanker ekstrak kelor (moringa oleifera l.) terhadap cell line kanker serviks hela dengan uji sitotoksisitas, apoptosis, dan jalur induksi apoptosis berdasarkan ekspresi gen p53. thesis. yogyakarta: univeritas gadjah mada; 2014. p. 1–13. 9. loprinzi c, bensinger w, peterson d. understanding and managing chemotherapy side effects. new jersey: cancer care; 2014. p. 4–7. 10. chu e, devita jr vt. physicians’ cancer chemotherapy: drug manual 2015. 15th ed. vol. 111, the british journal of psychiatry. burlington: jones & bartlett learning; 2015. p. 4. 11. anwar f, latif s, ashraf m, gilani ah. moringa oleifera: a food plant with multiple medicinal uses. phyther res. 2007; 21(1): 17–25. 12. cavell be, syed alwi ss, donlevy a, packham g. anti-angiogenic effects of dietary isothiocyanates: mechanisms of action and implications for human health. biochem pharmacol. 2011; 81(3): 327–36. 13. nussenbaum f, herman im. tumor angiogenesis: insights and innovations. j oncol. 2010; 2010: 1–24. 14. boreddy sr, sahu rp, srivastava sk. benzyl isothiocyanate suppresses pancreatic tumor angiogenesis and invasion by inhibiting hif-α/vegf/rho-gtpases: pivotal role of stat-3. plos one. 2011; 6(10): e25799. 15. nararya sa. uji toksisitas daun kelor (moringa oleifera) terhadap sel fibroblas gingiva menggunakan uji mtt assay. thesis. surabaya: universitas airlangga; 2018. p. 1–7. 16. budhy ti, istiati, sumaryono b, arundina i, khrisnanthi rs. hedyotiscorymbosa (l.) lamk the potential inhibitor extract of oral cancer cell progressivity in benzopyrene induced rattus novergicus. j int dent med res. 2018; 11: 312–7. 17. anggraini dr. ekspresi imunohistokimia ki-67 pada tumor payudara tikus wistar yang diinokulasi kanker terinduksi benzo(α) pyrene dengan pemberian ekstrak benalu teh. thesis. medan: universitas sumatera utara; 2013. p. 15. 18. juliyarsi i, melia s. dadih susu sapi mutan lactococcus lactis sebagai food healthy dalam menghambat kanker. padang; 2007. report no.: 001/sp2h/pp/dp2m/iii/2007. 19. elisabeth hjt, donald s. polycyclic aromatic hydrocarbon (pah): kaitannya dengan minyak sawit dan kesehatan. war pus penelit kelapa sawit. 2000; 8(1): 1–9. 20. ibrahim s, marham s. teknik laboratorium kimia organik. yogyakarta: graha ilmu; 2013. p. 16. 21. koirewoa ya, fatimawali f, wiyono w. isolasi dan identifikasi senyawa f lavonoid dalam daun beluntas (pluchea indica l.). pharmacon. 2012; 1(1): 47–52. 22. yulianingtyas a, kusmartono b. optimasi volume pelarut dan waktu maserasi pengambilan flavonoid daun belimbing wuluh (averrhoa bilimbi l.). j tek kim. 2016; 10(2): 58–64. 23. rizkayanti r, diah awm, jura mr. uji aktivitas antioksidan ekstrak air dan ekstrak etanol daun kelor (moringa oleifera lam). j akad kim. 2017; 6(2): 125–31. 24. vongsak b, sithisa r n p, mangmool s, thongpraditchote s, wongkrajang y, gritsanapan w. maximizing total phenolics, total flavonoids contents and antioxidant activity of moringa oleifera leaf extract by the appropriate extraction method. ind crops prod. 2013; 44: 566–71. 25. fahey jw, zalcmann at, talalay p. the chemical diversity and distribution of glucosinolates and isothiocyanates among plants. phytochemistry. 2001; 56(1): 5–51. 26. kerbel rs. tumor angiogenesis. n engl j med. 2008; 358(19): 2039–49. 27. kim s-k, park s-g, kim k-w. expression of vascular endothelial growth factor in oral squamous cell carcinoma. j korean assoc oral maxillofac surg. 2015; 41: 11–8. 28. lalla r v., boisoneau ds, spiro jd, kreutzer dl. expression of vascular endothelial growth factor receptors on tumor cells in head and neck squamous cell carcinoma. arch otolaryngol neck surg. 2003; 129(8): 882–8. 29. smith bd, smith gl, carter d, sasaki ct, haffty bg. prognostic significance of vascular endothelial growth factor protein levels in oral and oropharyngeal squamous cell carcinoma. j clin oncol. 2000; 18(10): 2046–52. 30. hakim l. farmakokinetik klinik. yogyakarta: bursa ilmu; 2012. p. 216–9. 31. deng l, lin-lee y-c, claret f-x, kuo mt. 2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate nf-kappa b pathway. j biol chem. 2001; 276: 413–20. 32. matheny cj, lamb mw, brouwer klr, pollack gm. pharmacokinetic and pharmacodynamic implications of p-glycoprotein modulation. pharmacotherapy. 2001; 21(7): 778–96. 33. marchetti s, mazzanti r, beijnen jh, schellens jhm. concise review: clinical relevance of drug drug and herb drug interactions mediated by the abc transporter abcb1 (mdr1, p-glycoprotein). oncologist. 2007; 12(8): 927–41. 34. nugrahaningsih, sarjadi, dharmana e, subagio hw. ekspresi vegf sel adenokarsinoma mamma pada pemberian oral ekstrak andrographis paniculata. j ilmu kefarmasian indones. 2015; 13: 29–34. 35. kumar c, kohli s, bapsy pp, vaid ak, jain m, attili vss, sharan b. immune modulation by dendritic-cell-based cancer vaccines. j biosci. 2017; 42(1): 161–73. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p71-75 58 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 58–62 original article pain parameters for buffered and non-buffered anesthetic injections in children undergoing dental procedures theodora erlin puspitasari1, iwan ahmad musnamirwan1, kirana lina gunawan2, meirina gartika1 1department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, bandung, indonesia 2department of anesthetics, faculty of dentistry, universitas padjadjaran, bandung, indonesia abstract background: dental procedures, such as injections, usually cause pain and make children uncomfortable and uncooperative. one approach for reducing pain is the use of buffered anesthetics. purpose: the research objective was to assess the pain parameters between buffered and non-buffered anesthetic injections, based on oxygen saturation, pulse rate, and the self-reporting of pain by the children. methods: the research method was quasi-experimental, with purposive sampling of 19 children. pain parameters, based on oxygen saturation and pulse rate, were measured using a pulse oximeter. the self-reporting of pain used the wong–baker faces® pain rating scale. statistical analysis used a t-test and mann–whitney test with p < 0.01 taken as statistically significant. results: the results showed a significant difference in oxygen saturation before and after the injection of buffered and non-buffered anesthetics (p = 0.0002). delivering the buffered anesthetics were reported to be less painful than non-buffered anesthetics. the oxygen saturation and pulse rate were inversely proportional to the self-reporting of pain in children. statistical analysis showed no significant difference between oxygen saturation (p = 0.5) and pulse rate (p = 0.4886) in those receiving buffered and non-buffered anesthetics. however, there was a significant difference in the self-reporting of pain between the two groups (p = 0.00000262). conclusion: pain parameters could be measured physiologically and psychologically. this research concludes that physiologically, there was no difference in pain parameters, based on oxygen saturation and children’s pulse rate. psychologically, there was a difference in the self-reporting of pain; 14 children reported that delivering the buffered anesthetic was painless. keywords: pain parameters; oxygen saturation; pulse rate; self-report; buffered anesthetics; non-buffered anesthetics article history: received 15 may 2022, revised 9 july 2022, accepted 15 july 2022 correspondence: meirina gartika, department of pediatric dentistry, faculty of dentistry universitas padjadjaran. jl. sekeloa selatan no.1 bandung, 40132, indonesia. email: meirina.gartika@fkg.unpad.ac.id introduction children show unique variations in maturity, personality, temperament, and emotions according to their vulnerability and ability to cope with dental treatment situations. therefore, pediatric dental care remains a challenge. the largest percentage of children brought by parents for a first dental visit was between 6–9 years of age.1,2 herawati’s3 study on children aged 7–11 years at cimahi elementary school, west java, showed that the prevalence of premature loss of primary teeth was 36.4%. these children have speech and cognitive abilities, so they can report what they feel, marked by an increase in the acceptance of responsibility for oral hygiene, even though parental involvement is still needed. dental care procedures often make children uncomfortable and uncooperative because tooth preparation, rubber dam placement, pulp treatment, injection, and tooth extraction can cause pain.4 pain is a complex and unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage.5 painful stimulation can cause physiological and psychological reactions to protect the body from tissue damage. patients can generally self-report pain experiences, except for toddlers and children with special needs. behavioral problems and a refusal to attend dental treatment, such as preparation and injection, suggest that children are afraid, especially about feeling discomfort and pain.6 pain control during dental care procedures is essential in pediatric dentistry7,8 and primarily involves the use of local anesthesia.7 the fear copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p58–62 mailto:meirina.gartika@fkg.unpad.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p58-62 59puspitasari et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 58–62 and pain of injecting local anesthetics can impact a child psychologically during the visit and influence attitudes in subsequent sessions.9,10 local anesthetics commonly used in pediatric dentistry are amide-type agents, e.g., 2% lidocaine with 1: 100,000 epinephrine. this agent is used because it rarely causes allergies and has excellent potential at low concentrations. local anesthesia causes pain when pricking the mucosa with a needle when administering local anesthetics and a burning sensation from the acidity of the anesthetics that causes local irritation.11 injection into dense tissue, such as the palate, is considered one of the most painful injections. this injection requires pressures of up to 660 psi. a computercontrolled local anesthetic delivery (cclad) system, the wand (milestone scientific, 1998), was developed to minimize pain sensations. pediatric dentistry literature also reports other efforts to reduce pain using anesthetic solution patches, chemically modifying anesthetic agents, buffering anesthetics, or warming anesthetics.11 research by malamed,11 guo,12 chopra,13 and phero,14 analyzed various attempts to administer local anesthetics more conveniently. buffering the anesthetic agent is recommended by adding a sodium bicarbonate (nahco3) base solution to the local anesthetic so that the ph is nearly neutral.7 malamed10 stated that using lidocaine with epinephrine, buffered to a physiological ph immediately before injection, significantly accelerated the onset of action and increased injection comfort. khatri7 reported that buffered lidocaine could reduce pain during the injection of inferior alveolar nerve blocks in children aged 5–10 years. based on these reports, this study aims to assess pain parameters between buffered and non-buffered anesthetic injections, based on oxygen saturation, pulse rate, and the self-reporting of pain in children. materials and methods ethics clearance was obtained from the research ethics committee of the faculty of medicine, universitas padjadjaran no. 194/un6.kep/ec/2020. this study used a quasi-experimental study design. the study population was children who came to the polyclinic of pediatric dentistry, dental and oral hospital of universitas padjadjaran from january to march 2020. nineteen children were selected by purposive sampling. the inclusion criteria were children aged 6–9 years who had primary molars on both sides of the maxilla (left and right), with mobility grades 0, 1, and 2 for extraction procedures, and whose parents gave informed consent. frank’s behavioral scale requirements were level three (positive) or four (very positive). the exclusion criteria were children allergic to local anesthetic solutions and with a history of special needs and systemic diseases. the study was conducted on three visits, and the interval between visits was one week. the tell–show–do method was used to explain the procedure during each visit before carrying it out. a dental anesthesiologist administered the injection using an anterior, middle superior alveolar technique immediately after mixing the anesthetic solution using a cclad system, i.e., the wand® (milestone scientific, livingston, nj, usa) (figure 1). at the first visit, the right side was injected using a buffered anesthetic, followed by the right primary first molar extraction. a buffered anesthetic solution was prepared by mixing a basic solution of 8.4% nahco3 with 2% lidocaine + 1:100,000 epinephrine using the onpharma® onset mixing pen.15 a week later, a right-sided post-extraction control was performed, and the procedure was continued for the left side using a non-buffered anesthetic followed by extraction of the left primary first molar. the third visit was made one week later for a leftsided post-extraction control. pain at the time of injection (using buffered and nonbuffered anesthetics) was assessed by oxygen saturation and pulse rate using a handheld pulse oximeter (ge healthcare company, usa) and self-report of pain using the wong– baker faces® pain rating scale (wong–baker faces foundation, oklahoma city, ok, usa). differences in pain parameters based on oxygen saturation and pulse rate were analyzed using paired and unpaired t-tests. differences in pain parameters, based on self-reported pain, were analyzed using the mann–whitney test. results table 1 shows the results of the paired t-tests for oxygen saturation. the p-value is 0.0002, indicating a significant difference in oxygen saturation (p < 0.001) before and after figure 1. a. before injection. b. after injection. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p58–62 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p58-62 60 puspitasari et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 58–62 each treatment. however, the oxygen saturation results using the unpaired t-test in table 2 showed a p-value = 0.5, indicating no significant difference in oxygen saturation (p > 0.01) between buffered and non-buffered anesthetic injections. table 3 shows the results of the paired t-tests for pulse rates. the results of the pulse rates using buffered anesthetic have a p-value = 0.00000832, while for the non-buffered anesthetic, the p-value was 0.0000438, indicating a highly significant difference (p < 0.001) in pulse rate before and after buffered and non-buffered anesthetic injections. the results for the pulse rates using the unpaired t-test (table 4) showed a p-value of 0.4886 (p < 0.001), indicating no significant difference in pulse rate between buffered and non-buffered anesthetic injections. table 5 shows the self-report results based on the wong–baker faces® pain rating scale. with buffered anesthetics, 74% had a pain value of 0, whereas 26% had a pain value of 2. for non-buffered anesthetic, 5% had a pain value of 2, whereas 47% had a pain value of 4. the mann–whitney test (table 6) showed a p-value of 0.00000262, indicating a highly significant difference in self-reported pain between buffered and non-buffered anesthetics (p < 0.001). table 1. the difference in oxygen saturation before and after injection of buffered and non-buffered anesthetics using the paired t-test treatment n oxygen saturation + sd t-count p-value before after buffered 19 97.53 + 1.61 97.89 + 1.37 4.44 0.0002 **) non-buffered 19 98.00 + 1.00 98.37 + 0.96 4.44 0.0002 **) note: n = sample; sd = standard deviation; a p-value < 0.001 was considered statistically significant table 2. the difference in oxygen saturation between buffered and non-buffered anesthetics using the unpaired t-test treatment n mean (difference) oxygen saturation + sd t-count p-value buffered 19 0.68 + 0.67 0 0.5 non-buffered 19 0.68 + 0.67 note: n = sample; sd = standard deviation; a p-value < 0.001 was considered statistically significant table 3. the difference in pulse rate before and after injection of buffered and non-buffered anesthetics using the paired t-test treatment n pulse rate + sd t-count p-value before after buffered 19 92.42 + 15.29 100.16 + 12.47 5.81 8.32e-06 **) non-buffered 19 94.26 + 9.73 103.16 + 13.11 5.03 4.38e-05 **) note: n = sample; sd = standard deviation; a p-value < 0.001 was considered statistically significant table 4. the difference in pulse rate between buffered and non-buffered anesthetics using the unpaired t-test variable n mean (difference) pulse rate + sd t-count p-value buffered 19 10.68 + 8.01 0.03 0.4886 non-buffered 19 10.16 + 8.81 note: n = sample; sd = standard deviation; a p-value < 0.001 was considered statistically significant table 5. self-report of pain on the wong–baker faces® pain rating scale for buffered and non-buffered anesthetics pain level buffered non-bufferedn % n % 0 14 74 1 5 2 5 26 9 47 4 0 0 9 47 6 0 0 0 0 8 0 0 0 0 10 0 0 0 0 total 19 100 19 100 note: n = sample table 6. the difference in self-reported pain results between buffered and non-buffered anesthetics using the mann–whitney test variable n mean sum of ranks sd z p-value buffered 19 11.8 224.5 + 32.06 4.55 2.62e-06 **) non-buffered 19 27.2 516.5 note: n = sample; sd = standard deviation; z = corrected for ties; a p-value < 0.001 was considered statistically significant copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p58–62 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p58-62 61puspitasari et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 58–62 discussion table 1 shows no change in oxygen saturation in seven children who received buffered anesthetics and nine who received non-buffered anesthetics. the remaining children had slight changes in oxygen saturation during the administration of local anesthetics, though in the normal range, which according to the world health organization (who), 16 must be 95–100%. rayen et al.17 reported that the oxygen saturation value did not change during dental treatment. the use of local anesthetic agents in dental care can result in changes in physiological responses.18 table 2 shows that oxygen saturation variations were the same with buffered and non-buffered anesthetics. saatchi et al.19,20 reported a similar result, i.e., no significant difference for the inferior alveolar nerve block using buffered and nonbuffered anesthetics. table 3 shows that pain parameters based on pulse rate varied significantly before and after injection using buffered and non-buffered anesthetics. most children showed an increase in pulse rate after the injection. two children experienced a decrease in pulse rate while administering both buffered and non-buffered anesthetic injections. according to the who, the normal pulse rate for children aged 6–9 years is 60–140 beats per minute. yagesh kumar et al.21 reported a smaller increase in pulse rate, pain, and disruptive behavior when using cclad compared to conventional syringes. rayen et al.17 reported a rise in the pulse rate of children in all dental care procedures. table 4 shows no significant difference in pulse rate between buffered and non-buffered anesthetics. buffered and non-buffered anesthetics increased the pulse rate but within the normal range. this study is inconsistent with studies in adults by senthoor et al.,22 which showed a significant decrease in pulse rate using buffered anesthetic injection after previously having been given an injection using non-buffered anesthetics. in this study, buffered anesthetics did not cause significant physiological changes. malek et al.23 stated that monitoring pain by observing changes in vital signs was not recommended. monitoring oxygen saturation and pulse rate in pediatric dental care is recommended to prevent cardiovascular accidents. cowen et al.24 states that an objective method of pain assessment is to observe changes in the autonomic nervous system, such as pulse rate, blood pressure, sweating, and pupillary responses. a pulse oximeter monitors pulse rate and oxygen saturation while administering local anesthetics. table 5 shows that most children reported pain values of 0, i.e., no pain during the injection of buffered anesthetics. the self-reported results are consistent with those of malamed,11 guoet al.,12 chopraet al.,13 and pheroet al.,14 who reported that buffered anesthetics could reduce pain during anesthetic administration. table 6 shows a difference in the value of pain parameters, based on selfreports using the wong–baker faces® scale. fourteen children reported buffered anesthetics as less painful than non-buffered anesthetics. one child said there was no pain with both buffered and non-buffered anesthetics. the wong–baker faces® facial pain rating scale used in this study complies with the american academy of pediatric dentistry guidelines on pain assessment. ethnic, cultural, and language factors can influence the expression of pain and its assessment.5 the experience of pain can shape children’s perception of pain in the future. selfreported pain assessment is the gold standard for pain assessment.25 this study’s results agree with those found by afsal et al.7 who studied healthy children aged 5–10 years using the wong–baker pain rating scale. it showed a significant difference in the injection of 2% buffered lidocaine compared to 2% non-buffered lidocaine and 4% articaine. gupta et al.,26 and kashyap et al.27 also found that buffering anesthetics could reduce pain during injection. malamed10 noted that a lower anesthetic ph tended to produce a burning sensation at the injection site and a slightly slower onset of action. lidocaine cartridges have an acidic ph due to the addition of hydrochloric acid (hcl) to extend their shelf life.28,29 adding a buffer solution can increase the ph of the anesthetic solution from about 3.5–5.5 to 6.5–7.3.30 when added to local anesthetics, there is a reaction between nahco3 and hcl, which produces water and carbon dioxide (co2). 8 davoudi et al.8 reported that co2 provided an independent anesthetic effect that increased the anesthetic action sevenfold. buffered lidocaine uses 0.5 mmol/ ml nahco3 in a ratio of 9:1, which is the most common method for reducing pain when administering local anesthetics.30 goodchild and donaslon,31 in compendium continuing education in dentistry (2019), examined three buffered anesthetic ratios of 9:1, 19:1, and 18:1. an anesthetic buffer ratio of 9:1 produces an average ph of 6.97 ± 0.06, while anesthetic buffer ratios of 19:1 and 18:1 produce a slightly lower ph of 6.77 ± 0.12 and 6.82 ± 0,05, respectively. this study used a 19:1 buffered anesthetic ratio according to the instructions for the onpharma® onset15 mixing pen. the chemistry laboratory of universitas padjadjaran measured the anesthetic ph in this study. the ph of the non-buffered and buffered anesthetics were 4.16 and 6.82, respectively. although adding nahco3 shortens the shelf life of the anesthetic, the solution can still be used at room temperature without reducing the effects of lidocaine.30 the researchers ensured uniformity in administering injections via the quasi-experimental, non-randomized sampling method. however, the samples did not all have the same probability, which is a limitation of this study. researchers also did not provide a specific time to calculate the onset and duration of the action of anesthesia. certain patients in the study sample required additional intraligament injections.32 this study concluded that there was no difference in pain parameters, based on oxygen saturation and pulse rate, between injections using buffered and noncopyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p58–62 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p58-62 62 puspitasari et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 58–62 buffered anesthetics. however, there were differences in pain parameters according to self-reported feedback. pain parameters can be measured physiologically and psychologically. physiologically, there was no difference in the oxygen saturation and pulse rate between buffered and non-buffered anesthetics. psychologically, based on self-reported feedback, 14 children in this study reported that a buffered anesthetic was painless. references 1. sanguida a, vinothini v, prathima gs, santhadevy a, premlal k, kavitha m. age and reasons for first dental visit and knowledge and attitude of parents toward dental procedures for puducherry children aged 0-9 years. j pharm bioallied sci. 2019; 11(suppl 2): s413–9. 2. murshid ez. children’s ages and reasons for receiving their first dental visit in a saudi community. saudi dent j. 2016; 28(3): 142–7. 3. herawati h, sukma n, utami rd. relationship between deciduous teeth premature loss and malocclusion incidence in elementary school in cimahi. j med heal. 2015; 1(2): 156–69. 4. romito lm, mcdonald jl. nutritional considerations for the pediatric dental patient. in: dean ja, editor. mcdonald and avery’s dentistry for the child and adolescent. 10th ed. elsevier; 2016. p. 138–54. 5. the reference manual of pediatric dentistry. pain management in infants, children, adolescents and individuals with special health care needs. pediatr dent. 2018; 40(6): 321–9. 6. son tm, nhu ngoc vt, tran pt, nguyen np, luong hm, nguyen h-t, sharma k, van tu p, ha ls, ha vn, van huy p, thimiri govinda raj db, chu d-t. prevalence of dental fear and its relationship with primary dental caries in 7-year-old-children. pediatr dent j. 2019; 29(2): 84–9. 7. afsal mm, khatri a, kalra n, tyagi r, khandelwal d. pain perception and efficacy of local analgesia using 2% lignocaine, buffered lignocaine, and 4% articaine in pediatric dental procedures. j dent anesth pain med. 2019; 19(2): 101–9. 8. davoudi a, rismanchian m, akhavan a, nosouhian s, bajoghli f, haghighat a, arbabzadeh f, samimi p, fiez a, shadmehr e, tabari k, jahadi s. a brief review on the efficacy of different possible and nonpharmacological techniques in eliminating discomfort of local anesthesia injection during dental procedures. anesth essays res. 2016; 10(1): 13. 9. davidian d. using buffered anesthesia and injection techniques to reduce pain and improve effectiveness. in: cdeworld ce ebook continuing dental education. dental learning systems, llc; 2017. p. 1–9. 10. malamed sf. handbook of local anasthesia. 7th ed. mosby elsevier; 2019. p. 464. 11. malamed sf, tavana s, falkel m. faster onset and more comfortable injection with alkalinized 2% lidocaine with epinephrine 1:100,000. compend contin educ dent. 2013; 34(spec no 1): 10–20. 12. guo j, yin k, roges r, enciso r. efficacy of sodium bicarbonate buffered versus non-buffered lidocaine with epinephrine in inferior alveolar nerve block: a meta-analysis. j dent anesth pain med. 2018; 18(3): 129–42. 13. chopra r, jindal g, sachdev v, sandhu m. double-blind crossover study to compare pain experience during inferior alveolar nerve block administration using buffered two percent lidocaine in children. pediatr dent. 2016; 38(1): 25–9. 14. phero ja, nelson b, davis b, dunlop n, phillips c, reside g, tikunov ap, white rp. buffered versus non-buffered lidocaine with epinephrine for mandibular nerve block: clinical outcomes. j oral maxillofac surg. 2017; 75(4): 688–93. 15. onpharma company. onset® by onpharmatm. 2017. available from: https://onpharma.com/. accessed 16. world health organization. pulse oximetry training manual. who library cataloguing-in-publication data. geneva: who press; 2011. p. 1–23. 17. rayen r, muthu ms, chandrasek har rao r, sivakumar n. evaluation of physiological and behavioral measures in relation to dental anxiety during sequential dental visits in children. indian j dent res. 2006; 17(1): 27–34. 18. patil sb, bondarbe pa, patil pm, mujawar sm. incident and extent of pulse rate and oxygen saturation alteration during local anesthesia in children. austin pediatr. 2016; 3(2): 1034. 19. saatchi m, khademi a, baghaei b, noormohammadi h. effect of sodium bicarbonate–buffered lidocaine on the success of inferior alveolar nerve block for teeth with symptomatic irreversible pulpitis: a prospective, randomized double-blind study. j endod. 2015; 41(1): 33–5. 20. saatchi m, farhad ar, shenasa n, haghighi sk. effect of sodium bicarbonate buccal infiltration on the success of inferior alveolar nerve block in mandibular first molars with symptomatic irreversible pulpitis: a prospective, randomized double-blind study. j endod. 2016; 42(10): 1458–61. 21. yogesh kumar td, baby john j, asokan s, geetha priya pr, punithavathy r, praburajan v. behavioral response and pain perception to computer controlled local anesthetic delivery system and cartridge syringe. j indian soc pedod prev dent. 2015; 33(3): 223–8. 22. senthoor p, janani k, ravindran c. a prospective, randomized double-blinded study to evaluate the efficacy of buffered local anesthetics in infected and inflamed pulp and periapical tissues. j maxillofac oral surg. 2020; 19(2): 246–50. 23. málek j, ševčík p, bejšovec d, gabrhelík t, hnilicová m, křikava i, kubricht v, lejčko j, mach d, mixa v. postoperative pain management. praha: mlada fronta; 2017. 24. cowen r, stasiowska mk, laycock h, bantel c. assessing pain objectively: the use of physiological markers. anaesthesia. 2015; 70(7): 828–47. 25. shindova m, belcheva a. pain assessment methods among pediatric patients in medical and dental research. sci technol med biol stud clin stud soc med heal care. 2016; vi(1): 16–23. 26. gupta s, mandlik g, padhye mn, kini yk, kakkar s, hire av ija. combating inadequate anesthesia in periapical infections, with sodium bicarbonate: a clinical double blind study. oral maxillofac surg. 2014; 18(3): 325–9. 27. kashyap vm, desai r, reddy pb, menon s. effect of alkalinisation of lignocaine for intraoral nerve block on pain during injection, and speed of onset of anaesthesia. br j oral maxillofac surg. 2011; 49(8): e72–5. 28. malamed sf, falkel m. advances in local anesthetics: ph buffering and dissolved co2. dent today. 2012; 31(5): 88–93; quiz 94–5. 29. brandis k. alkalinisation of local anaesthetic solutions. aust prescr. 2011; 34(6): 173–5. 30. finsen v. reduced pain when injecting lidocaine. tidsskr nor laegeforen. 2017; 137(9): 629–30. 31. goodchild jh, donaldson m. novel direct injection chairside buffering technique for local anesthetic use in dentistry. compend contin educ dent. 2019; 40(7): e1–10. 32. peedikayil fc, vijayan a. an update on local anesthesia for pediatric dental patients. anesth essays res. 2013; 7(1): 4–9. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p58–62 https://onpharma.com/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p58-62 �� volume 47, number 1, march 2014 research report spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket rostiny, mefina kuntjoro, ratri maya sitalaksmi and sherman salim department of prosthodontics faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: prominent residual ridge is necessary to gain retention and stabilility for succesful prosthodontic treatment such as removable, fixed or implant. spirulina is a natural substance that can help tissue healing and chitosan also a natural substance that reported to have the ability to help bone remodelling. the combination gel of spirulina and chitosan could be considered as an alternative material to maintain residual ridge height after tooth extraction. purpose: the aim of study was to examine the effect of combination gel of spirulina and chitosan on healing process of cavia cobaya post tooth extraction socket by counting the amount of osteoclast, osteoblast and colagen as an indicator. methods: twenty eight cavia cobaya were divided into 4 groups. insisive mandible extraction was done and the sockets were filled with 3% cmcna for control groups, 3% spirulina chitosan 200 mg for group 1, 6% spirulina chitosan 200 mg for group 2, 12% spirulina chitosan 200 mg for group 3. after 30 days, histopathology examination was done by using microscope to count the amount of osteoclast, osteoblast and collagen. results: data was analyzed by using anova and tukey hsd. for osteoclast, there was no significant different between every groups, while for osteoblast and collagen there was significant different between groups. the results showed that induction of combination gel spirulina chitosan was able to accumulate collagen fiber and resulting faster wound healing. conclusion: combination 12% gel spirulina chitosan 200 mg could be used as an alternative material for better bone remodeling after tooth extraction. key words: spirulina, chitosan, bone remodeling, tissue healing, cavia cobaya abstrak latar belakang: residual ridge yang prominen sangat dibutuhkan untuk mendapatkan retensi dan stabilitas untuk menunjang keberhasilan perawatan di bidang prostodonsia seperti pada kasus removable, fixed atau implant. tindakan pencabutan gigi dapat merusak jaringan periodontal, sementum dan tulang alveolar yang mengakibatkan resorbsi ridge yang besar. spirulina telah terbukti mempunyai kemampuan untuk membantu penyembuhan tulang sedangkan kitosan mempunyai kemampuan untuk membantu proses pembentukan tulang. kombinasi kedua bahan ini diharapkan dapat menjadi bahan alternatif untuk mempercepat proses penyembuhan luka dan pembentukan tulang. tujuan: penelitian ini bertujuan meneliti efek induksi kombinasi gel dari spirulina dan kitosan terhadap proses penyembuhan soket pasca ekstraksi gigi cavia cobaya dengan indikator jumlah osteoklas, osteoblas dan kolagen. metode: penelitian ini menggunakan 28 marmot yang dibagi menjadi 4 kelompok penelitian. pencabutan dilakukan pada incisive rahang bawah kemudian soket pencabutan diisi dengan cmcna 3% pada kelompok control; spirulina 3% chitosan 200 mg pada kelompok perlakuan 1; spirulina 6 % chitosan 200 mg pada kelompok perlakuan 2, dan spirulina 12% citosan 200 mg pada kelompok perlakuan 3. pada hari ke 30 dilakukan pemeriksaan histopatologi menggunakan mikroskop untuk menghitung jumlah osteoblas, osteoklas dan kolagen. hasil: data dianalisis dengan anova dan tukey hsd. jumlah osteoklas tidak berbeda secara signifikan antara setiap kelompok, sedangkan jumlah osteoblas dan kolagen terdapat perbedaan yang signifikan antara kelompok. hasil penelitian menunjukkan bahwa �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 19–24 induksi kombinasi gel spirulina chitosan mampu mengakumulasi serat kolagen dan menghasilkan penyembuhan luka lebih cepat. simpulan: kombinasi gel spirulina 12% chitosan 200 mg dapat digunakan sebagai bahan alternatif untuk remodeling tulang yang lebih baik setelah pencabutan gigi. kata kunci: spirulina, kitosan, penyembuhan luka, pembentukan tulang, cavia cobaya correspondence: rostiny, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tikaratri@yahoo.com introduction tooth extraction is a procedure that cause damage to the part of the tooth socket which includes periodontal tissues such as gingival, cementum, periodontal ligament and alveolar bone. inflammatory process, which is the main result of tooth extraction, always followed by bone remodeling and tissue repair. healing process need sterile state and materials containing anti-inflammatory, antibacterial, anti-mycotic, insecticidal, antiseptic and antiparasitic to accelerate this process.1 in general, healing process consists of three phases, inflammatory phase, proliferative phase and remodeling phase. inflammatory phase begins immediately after tooth extraction until 3 to 5 days. cardinal signs such as rubor, tumor, calor, dolor and fungsiolaesa always happen in this phase. production of histamine, kinin and prostaglandin by leukocytes increase.2 proliferative phase lasts for 3 days to 3 weeks. in this phase the tooth sockets filled with inflammatory cells, fibroblasts, collagen matrix, and hyaluronic acid which serve for the formation of granulation tissue.3 remodeling phase is also called maturation phase. in this phase of osteoblast cells will aggregate the intercellular substance of bone that contains collagen to form new collagen fibers. activity of osteoblasts and osteoclasts will change immature bone (woven bone) become mature bone (lamellar bone). the states of the bones become stronger so that osteoclasts can penetrate tissue and debris in the injured area followed by osteoblasts that will fill the gap between the new bones. this happened a few months or even years until alveolar bone become its original form.4 currently pharmaceutical technology development has focused worldwide attention on the ingredients derived from nature because relatively safe compared to chemical drugs. spirulina has many nutritional benefits to the human body, such as c-phycocyanin, b-carotenoids, vitamin e, zinc and many trace elements and other natural phytochemicals . one of the ingredients derived from nature that has been researched and proven as an antiinflammatory and antioxidant in wound healing process is c-phycocyanin or blue substance.5 gel concentration of 12% spirulina most effectively to increase the number of fibroblast cells after tooth extraction guinea pig (cavia cobaya).6 chitosan, product of chitin derivatives with the formula n-acetyl-d glucosamine, is a cationic polymer that has number of monomers around 2000-3000 monomeric, non-toxic and molecular weight about 800 kd. chitosan is produced from chitin deacetylation under alkaline conditions. chitin can be obtained from the shells of crustaceans, insects and other sources. this biopolymer has good character, biodegradable, biocompatible, antibacterial properties and safe for humans.7,8 chitosan has been used as a drug delivery system, orthopedic implants and periodontal wound healing management, and scaffolds for tissue regeneration. in the field of wound healing, chitosan proved to activate immune cells, inflammatory cells such as pmn, macrophages, fibroblasts and cells angioendotelial. natural healing of chitosan derived from its ability to stimulate the production of fibroblasts by affecting fibroblast growth factor.9 ariani et al.,10 in his research 2013 using chitosan 200 mg said that chitosan has a porosity structure and good retention to support proliferation osteoblast cell. the aim of study was to examine the effect of combination gel of spirolina and chitosan on healiny process of cavia cobaya post tooth extraction secret by counting the amount of osteodast, osteoblast and colagen as an indicator. materials and methods this research was an experimental laboratory by using the draft post-test only control group design. experimental animals used in this study were cavia cobaya, 2-3 months old, male, average body weight of 300 grams. total of 28 cavia cobaya divided into 4 treatment groups, each group has consist of 7 animals. experimental animals maintained for 3 days to adapt in the cages measuring 60 cm x 65 cm x 80 cm (7 animals per cage) and placed in the room light enough to avoid moisture, away from the noise and not exposed to direct sunlight. the food provided is corn and fresh carrots. spirulina was in powder form produced by wellness usa. chitosan was in powder derived from the shells of shrimp produced by soetomo hospital tissue bank. combination gel was the result of mixing spirulina powder and chitosan powder with base gel cmc na 3% to produce stable gel consistency. base gel 3% na cmc does not affect the gel function, viscosity so it’s as treatment to control group. chitosan that used for each treatment group is 200 grams. the combination of gel for this study were as follows, treatment group 1, 3% of spirulina from 300 mg spirulina, 9.5 g of cmc na 3% and 200 mg chitosan, ��rostiny, et al.,: spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket tabel 1. multiple comparison test tukey lsd osteoblast, osteoclast and collagen control group 1 group 2 group 3 osteoclast control group 1 * * group 2 * group 3 * osteoblast control * group 1 * * * group 2 * group 3 * collagen control * group 1 group 2 group 3 * treatment group 2, 6% of spirulina from 600 mg spirulina, 9.2 g of cmc na 3% and 200 mg chitosan, treatment group 3, 12% of spirulina from 1200 mg spirulina, 8.6 g of cmc na 3% and 200 mg chitosan. treatment was done by extracted left mandibular incisors cavia cobaya using modification of the needle holder under anesthesia 10% inhalation. after extraction, socket was filled with combination gel using 0.1 cc syringe then closed by former revocation stitched using silk threads 3/0. animal was feeding as usual until day 30 then cavia cobaya was executed with 10% ether anesthesia to remove the mandible then performed decalcification with 2.5% nitric acid for 2 days. after mandibular bone tissue becomes soft, cutting incisor socket area was done in rectangular shaped cuts in the sagittal direction. results of the pieces was immersed in 10% buffered formalin for 24 hours. further processed for making preparat histopathological anatomy (hpa) with haematocylin eosin staining (he). observations were made on preparat hpa and divided into three random visual fields by counting technique using a beta counter. region that will be calculated was the socket that filled with combination gel. counting the number of osteoclasts, osteoblasts and collagen was conducted with binocular microscope lens with magnification 1000x connected directly to the computer. collagen (figure 1a) the most fiber in the human body, was observed shaped thick, sinuous, consisting of inelastic collagen protein (white fibers), pink color was obtained as the staining was using he. osteoblast cells (figure 1b) was observed cuboidal or cylindrical-shaped short, have a cell nucleus, cytoplasm red and blue in microscopically. osteoclasts (figure 1c) was observed multinucleus form giant cells, round or oval, blue and red cytoplasmic surface located on the side of the resorbed bone slight rough. results the results of counting the number of osteoclasts, osteoblasts and collagen can be seen in the figure 2. the results of osteoclasts numbers was increased in treatment group 1 compared to the control group, but in treatment groups 2 and 3 osteoclasts decreased compared with the control group. the results of osteoblasts numbers appears that the highest number of osteoblasts were in treatment group 1, while the lowest number of osteoblasts were found in treatment group 3. the results of counting the numbers of collagen appears that cells in the treatment group 1, 2 and 3 increased compared to the control group. a b c figure 1. histological examination with he staining (a) collagen, (b) osteoblast, and (c) osteoclast. figure 2. mean and standard deviation of osteoblast, osteoclast and collagen of cavia cobaya socket after treatment with spirulina chitosan gel. control group 1 group 2 group 3 osteoclast osteoblast colagen �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 19–24 the results of the kolmogorov and smirnov test statistic lavene showed that all data were normally distributed and homogeneous then proceed with the anova test. to determine differences in the number of osteoclasts, osteoblasts and collagen in each treatment group and control group least squares different test was used (table 1). discussion cavia cobaya was chosen as experimental animals as its metabolic systems anatomically and physiologically similar to humans. lower incisor was chosen because the tooth sockets was deeper and larger than the other teeth, always calcified and continuously erupting so that the tooth crown can be as high as elongated molars. lower incisor shaped like a segment of scissors or a cutting tool that resembles a pair of scissors. it is easier for researchers to incorporate material combination of spirulina and chitosan gel in a tooth socket after a tooth extraction so that the socket wound healing process can be observed.11 this research used male cavia cobaya because its hormonal system more stable compare to female. hormonal systems in female cavia cobaya will affect growth hormones production such as epidermal growth factor (egf), which is a hormone compound derived from blood platelets in addition to pdgf and tgf that have important role in wound healing. counting the number of osteoblast, osteoclast and collagen was done on third cervical tooth socket as it was the healing center. based on table 1, treatment group 1 (combination of spirulina 3% and 200 mg of chitosan) numbers of osteoclasts was increased compared to the control group, where as in treatment group 2 (combination spirulina 6% and chitosan 200 mg) and treatment group 3 (combination spirulina 12% and 200 mg of chitosan) was decreased. considering only with these results, there is unmatch between existing theories and this research which actually increased the number of osteoclasts. in the process of bone remodeling there is a close relationship between osteoclasts and osteoblasts because these two cells cooperate together in bone remodeling process. therefore, its important to consider the number of osteoblasts in the number of osteoclasts. it turns out that the number of osteoblasts directly proportional to the number of osteoclasts in this study. it was proven that bone remodeling happened because of the balance amount of osteoblasts and osteoclasts. the results of this research showed that osteoblast cells increased by induction of a combination spirulina and chitosan gel. osteoblast in treatment group 1 increased compared with the control group, but in treatment group 2 osteoblast significantly decreased compared with the treatment group 1. in treatment group 3 osteoblast also decreased although not significantly different from treatment group 2. this happened because observation was done in day 30th and the combination gel can regenerate bone remodeling. therefore osteoblast numbers become less because its change into bone matrix and this was proven by observation in the microscope that in treatment group 2 was seen less granulation tissue compared with treatment group 1. observations in the treatment groups 3 showed osteoblasts was getting more difficult to find as well as the formation of islands of bone growth or spicules are fused and form a branching to make nets bone hence granulation tissue at this stage has not looked. this can be seen from histological examination in figure 3a and 3b. the result was clearly visible on the socket preparations that were observed under a microscope. in the control group visible scars of tooth extraction socket area was still a lot of granulation tissue and little bone was formed. in the treatment group 1 visible granulation tissue began to decrease replaced by bone matrix so that the number of osteoblasts and osteoclasts increased. this indicate that bone remodeling process was going on. in the treatment group 2 appeared to have less granulation tissue and bone figure 3. histological section of healing socket 30 days after ekstraction with he staining. a) minimal granulation tissue, newly formation bone can be seen, b) granulation tissue with minimal bone formation. a b ��rostiny, et al.,: spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket matrix which was quite a lot, in the 3 treatment groups were seen socket begins to fill with the bone matrix of the surrounding granulation tissue (figure 2). this indicates that the treatment group 2 and 3 bone remodeling occurs faster than the control group and the treatment group 1. the number of osteoclasts and osteoblasts were decreased in treatment groups 2 and 3 due to the phase formation of osteoclasts and osteoblasts peak had passed and many formed bone in the socket. according miloro,12 osteoclasts begin to resorb alveolar crest in the first week and the second week will be even greater resorption. while the number of osteoblasts peaked at 6-8th week.13 amler et al., cit. mezzomo et al.14 stated that human alveolar bone can cured histologically without any drug, and after 4 weeks of tooth extraction will occur naturally in the process of osteoblastic bone tissue formation. in this study it appears that the islands have been formed bone growth (spicules) are fused and form a branching to create webs of the former bone in tooth extraction sockets within 4 weeks. this suggests that administration of a combination of spirulina and chitosan gel capable to speeding up the process of bone remodeling. the results of the study showed that the induction of combination gel spirulina 12% chitosan 200 mg showed significant results compared with the control group. this suggests that this combination gel able to accumulate collagen fiber and resulting faster wound healing. in this study, collagen density was used as an indicator in wound healing because collagen plays an active role in the proliferation stage which starts up with the maturation phase. collagen was first detected on 3rd day after injury and increased until 3rd week. collagen fibers will continue to accumulate until 3 months. specific function of collagen is to make the new tissue (connective tissue matrix) and the release of substrates by fibroblast cells will give a mark on macrophage cells and new blood vessels and fibroblasts as well as one unit in order to enter the area of the wound so that the process of granulation is formed.15 spirulina has a high alkaline properties, about ph 9 to 11.16 chitosan has a ph of 6.2 to 7.17 mixing the two materials make the ph becomes slightly more alkaline. slightly more alkaline atmosphere necessary for alkaline phosphatase activity that contributes to mineralization.4 in the bone remodeling process, osteoblast has an important role. osteoblasts are a major component in this process to synthesize new bone tissue resulting in the formation of the alveolar bone.18 tooth extraction can lead to complications such as inflammation in the tooth socket. an inflammation can also occur in the healing phase socket, this happened because the first defense cells were activated such as macrophage. macrophages are phagocytic cells that are produced in the spinal cord that plays an important role in inflammation, such as bacteria digest and remove the unwanted cell lysis or that have been damaged. these macrophages would trigger the secretion of proinflammatory cytokines such as tumor necrosis factor (tnf), interleukin 1 (il-1) and interleukin 6 (il-6) as a mediator of inflammation to amplify the immune response and increase in metabolic processes. along with this, macrophages activate nuclear factor-kappab (nfkb). nfkb transcription factor is a protein in macrophages which are activated as a result of a bacterial toxin. this process will lead to an increase in proinflammatory mediators such as tnf, il-1,and il-6. if nfkb increases, three proinflammatory cytokine genes will also increase, because tnf, il-1 and il-6 are interconnected to stimulate inflammation.19 according aranaz et al.,9 dai et al.,20 and pinto,21 chitosan is able to improve the function of inflammatory cells such as polymorphonuclear leukocytes (pmn), macrophages, fibroblasts and osteoblasts to help bone formation. spirulina contains phycocyanin as an anti-inflammatory which will suppress excessive inflammatory reaction after tooth extraction. phycocyanin and carotenoids work on macrophages via toll like receptor (tlr) by suppressing the activity and inhibit the translocation of nfkb, which will reduce the excessive expression of proinflammatory cytokines such as tnf σ, il-1 (interleukin-1) and il-6. osteoblasts express osteoprotegerin (opg), which serves as the receptor binding of rankl (receptor activator of nf-kappab ligand) that blocks rankl binds to rank. opg binds to rankl, thus preventing the activation of osteoclasts. decreasing the amount of production of proinflammatory cytokines (tnf-α, il-1 and il 6) led to decreased rankl is also expressed. increasing opg and rankl will lead to a decreased in active osteoclasts. decreased osteoclast resorption will reduce during bone remodeling.22-24 the study suggested that combination gel 12% spirulina chitosan 200 mg could be used as an alternative material for better bone remodeling after tooth extraction. references 1. topazian rg, goldberg mh, hupp jr. oral and maxillofacial infections. 4th ed. usa: elsevier saunders; 2002. p. 2-157. 2. hupp jr. wound repair. in: hupp jr, ellis e, tucker mr, eds. contemporary oral and maxillofacial surgery. 5th ed. st. louis: mosby yearbook inc; 2008. p. 47-54. 3. peterson a, ellis e, hupp jr, tucker t. contemporary oral and maxillofacial surgery 3th ed. philadelphia: mosby inc; 1998. p. 772. 4. lieberman jr, friedlaender ge. bone regeneration and repair. totowa, new jersey: humana press; 2005. p. 6-9, 21-4. 5. romay ch, gonzález r, ledón n, remirez d, rimbau v. cphycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. curr protein pept sci 2003; 4(3): 207-16. 6. rahmitasari f. the effect of spirulina gel on fibroblast cell number after wound healing process. dent j (maj ked gigi) 2011; 44(4): 192-5. 7. honakar h, barikani m. application of biopolymers 1: chitosan. monatsh chem. 2009; 140: 1403-20. 8. tangsadthakun c, kanokpanont s, sanchavanakit n, pichyangkura r, banaprasert t, tabata y, damrongsakkul s. the influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds. j biomater sci polym ed 2007; 18(2): 147-63. �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 19–24 9. aranaz i, mengibar m, harris r, panos i, miralles b, acosta n, galed g, heras a: functional characterization of chitin and chitosan. current chemical biology 2009; 3(2): 203-30. 10. ariani md, matsuura a, hirata i, kubo t, kato k, akagawa y. new development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. dent mater j 2013; 32(2): 317-25. 11. hadinata f. kitosan sebagai stimulator makrofag pada proses penyembuhan luka pada pencabutan gigi cavia cobaya. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2001. 12. miloro m. peterson’s principles of oral and maxillo surgery. 2nd ed. london: bc decker inc; 2004. p. 3-8. 13. trombelli l, farina r, marzola a, bozzi l, liljenberg b, lindhe j. modeling and remodeling of human extaction sockets. j clin periodontol 2008; 35(7): 630-9. 14. mezzomo la, shinkai rs, mardas n, donos n. alveolar ridge preservation after dental extraction and before implant placement: a literature review. rev odonto cienc 2011; 26(1): 77-83. 15. kalangi sjr. peran kolagen pada penyembuhan luka. dexa media 2004; 4: 168-74. 16. ogbonda kh, aminigo re, abu go. optimization studies of biomass production and protein biosynthesis in a spirulina sp. bioresour technol 2007; 98(11): 2207-11. 17. de alvarenga es. characterization and properties of chitosan. biotechnology of biopolymers. 2011. p. 91-108. 18. clarke b. normal bone anatomy and physiology. clin j am soc nephrol 2008; 3 suppl 3:s131-9. 19. bronner f, carson mcf, rubin j. bone resorption. usa: spinger; 2005. 2; 17 20. dai t, tanaka m, huang y, hamblin mr. chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. expert review of anti infective therapy 2011; 9(7): 857-79. 21. pinto ar, reis rl, neves nm. scaffold based bone tissue engineering: the role of chitosan. tissue eng part b rev 2011; 17(5): 331-47. 22. cherng sc, cheng sn, tarn a, chou tc. anti-inflamatory activity of c-phycocyanin in lipopolysaccaride-stimulated raw 264.7 macrophages. life sci 2007; 81(19-20):1431-5. 23. ku cs, phamt, park y, kim b, shin ms, kang j, lee j. edible bluegreen algae reduce the production of pro-inf lammatory cytokines by inhibiting nfkb pathway in macrophages and splenocytes. biochim biophys acta 2013; 1830(4): 2981-8. 24. soontormchaiboon w, joo ss, kim sm. anti-inflammatory effects of violaxanthin isolated from microalgae in raw 264.7 macrophages biol pharm bull 2012; 35(7): 1137-44. vol 38 no 3 2005 142 perawatan gigi impaksi anterior rahang atas pada remaja (the treatment of maxillary anterior impacted teeth in adolescent) herdi eko pranjoto* dan jusuf sjamsudin** ** bagian ilmu bedah mulut ** bagian ortodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract the incidence of impacted tooth in adolescent is usually found by oral surgeon or orthodontist during their sequence of treatments. the maxillary anterior teeth, especially canine and third lower molar are the most common impacted teeth found as the result of their eruption disturbances. the surgical technique principle is to facilitate the impacted tooth, so that it can be erupted by creating a window and take the bone obstructed surrounding the tooth crown surgically and afterward it is orthodontically tracted. in a case of third molar germ which is predicted to be impacted in mesio version position, germinectomy is preferred to prevent anterior mechanical drive during its development which may cause mal-alignment of the mandibular teeth. germinectomy which is usually easier than odontectomy, the germ position, and less complication after the surgical intervention are three factors that should be considered in making decision. key words: impacted, germinectomy, third molar, canine korespondensi (correspondence): herdi eko pranjoto, bagian ilmu bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan gigi geligi dalam rongga mulut akan mengalami erupsi menurut urutan waktu erupsi masing-masing jenis gigi, mulai dari fase gigi sulung sampai mengalami pergantian menjadi fase gigi permanen. proses erupsi masing-masing gigi baik pada fase gigi sulung maupun permanen akan terjadi secara fisiologis dan jarang sekali mengalami gangguan. gangguan erupsi pada umumnya terjadi pada fase pergantian dari gigi sulung menuju fase gigi permanen, sehingga gigi permanen tertentu tidak dapat mengalami erupsi. gigi kaninus merupakan gigi kedua setelah gigi molar ketiga yang berfrekuensi tinggi untuk mengalami impaksi,1 meskipun demikian gigi anterior di rahang atas lainnya seperti gigi insisivus pertama dan kedua rahang atas juga dapat mengalami kesulitan tumbuh akibat terletak salah di dalam rahang. frekuensi terjadinya kaninus impaksi sebesar 0,8–2,8 persen.2 ditinjau dari letaknya, 85 persen posisi gigi kaninus yang impaksi terletak di daerah palatal lengkung gigi, sedangkan 15 persen nya terletak di bagian labial atau bukal.3 ada beberapa bukti yang menyatakan, bahwa penderita dengan maloklusi kelas ii divisi 2 dan gigi aplasia merupakan kelompok yang mempunyai risiko tinggi untuk terjadinya kaninus ektopik.4 untuk mengamati pergerakan gigi kaninus rahang atas dan menghitung jarak gerakan yang terjadi dapat digunakan metode pengamatan secara tiga dimensi dengan menggunakan foto lateral, sefalometri dan foto antero posterior. foto diambil secara berkala pada usia penderita 5 tahun sampai 15 tahun. pada perhitungan ini dapat ditunjukan bahwa gigi kaninus akan bergerak sebesar 22 milimeter. sedangkan pada proyeksi foto lateral dapat dilihat bahwa pada usia 10–12 tahun pergerakan gigi menuju ke arah bukal. sebelum menginjak usia 10–12 tahun pergerakan gigi menuju ke arah palatal.5 untuk mendapatkan hasil yang maksimal pada perawatan ortodonsi dengan kasus sukar diperlukan diagnosis dan rencana perawatan yang tepat. salah satu contoh adalah perawatan ortodonsi dengan gigi kaninus rahang atas ektopik. pertumbuhan gigi molar ketiga permanen rahang bawah juga memerlukan perhatian khusus pada penderita anak sampai remaja. gigi molar ketiga rahang bawah yang belum erupsi akan dapat mempunyai posisi yang sedemikian sehingga pada proses pertumbuhannya dapat diperkirakan akan dapat menimbulkan gangguan pada alignment gigi di rahang bawah oleh karena daya dorong erupsi gigi tersebut ke arah anterior. pada posisi benih gigi molar ketiga rahang bawah yang diperhitungkan terletak miring, terutama dalam posisi mesio versi, tindakan germinectomy pada benih gigi molar ketiga tersebut perlu dipertimbangkan agar pada proses pertumbuhan selanjutnya tidak menimbulkan kelainan terhadap posisi gigi di sebelah anteriornya. menurut bishara 5 etiologi gigi impaksi dapat disebabkan oleh faktor primer dan faktor sekunder. faktor primer meliputi trauma pada gigi sulung, benih gigi rotasi, tanggal prematur gigi sulung, dan erupsi gigi kaninus dalam celah pada kasus celah langit-langit. faktor sekunder 143prajanto dan sjamsudin: perawatan gigi impaksi anterior rahang atas meliputi kelainan endokrin, defisiensi vitamin d, dan febrile diseases. gigi kaninus impaksi dapat terletak ektopik dan sering dijumpai dalam praktek sehari-hari. kejadian impaksi dengan letak ektopik ini belum diketahui penyebabnya yang pasti, dimungkinkan oleh karena sebab yang multifaktorial. salah satu kemungkinan adalah jalan erupsi gigi kaninus yang lebih panjang bila dibandingkan dengan gigi permanen lainnya.6 adanya diskrepansi panjang lengkung, gigi berdesakan, diastema antar gigi, dan trauma pada gigi anterior di awal usia pertumbuhan dapat pula merupakan penyebab terjadinya gigi kaninus ektopik.7 keterlambatan proses eksfoliasi pada gigi kaninus sulung dapat pula menyebabkan terjadinya pergerakan gigi kaninus permanen ke arah palatal.8,9 ada dugaan bahwa frekuensi terjadinya kaninus ektopik dapat terjadi pada anak yang mengalami gangguan pada proses erupsi.1 kasus 1 penderita wanita usia 16 tahun datang ke tempat praktek ortodontis atas rujukan dari dokter gigi umum dengan dugaan kedua gigi kaninus rahang atas ektopik. pemeriksaan ekstra oral menunjukan profil penderita agak cembung dan pada pemeriksaan intra oral semua gigi permanen sudah tumbuh kecuali kaninus kiri dan kanan. tulang pada bagian bukal gigi posterior kiri dan kanan kelihatan menonjol. relasi molar kelas i angle dengan jarak gigit 5 mm dan tumpang gigit 4 mm. gambaran radiologis menunjukkan bahwa gigi 13 dan 23 terletak di atas apikal gigi 14 dan 24 dengan posisi hampir horizontal (gambar 1). gambar 1. gambaran radiologi kasus 1. gambar 2. kaninus kanan rahang atas gambar 3. kaninus kiri rahang atas tatalaksana kasus 1 perawatan yang dilakukan pertamakali adalah pencabutan gigi 14 dan 24 untuk tempat gigi 13 dan 23 serta perbaikan gigi anterior yang agak protrusif. setelah itu dilakukan pemasangan peranti cekat ortodontik dengan menggunakan breket edgewise standar. untuk proses leveling dan unrevelling pertama kali digunakan busur multistrand dengan diameter 0.155, satu bulan kemudian dilakukan penggantian busur labial dengan niti 0.15. setelah semua gigi dalam keadaan satu level maka busur labial diganti dengan niti 0.16 x 0.22 dan di bagian belakang tube molar pertama rahang atas kiri dan kanan dibengkokan. selanjutnya dilakukan tindakan bedah untuk melakukan surgical exposure disertai pemasangan button pada bagian labial gigi kaninus kiri dan kanan. agar gigi kaninus yang ektopik dapat turun ke bawah, pada button 144 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 142–145 diikatkan ligature wire yang dikaitkan pada busur labial tanpa disertai tarikan. sehari pasca pembedahan penderita didatangkan untuk dilakukan pemeriksaan bekas pembedahan dan dilakukan penarikan. dua bulan pasca pembedahan gigi kaninus mulai terlihat turun ke bawah, akan tetapi masih terlihat dalam posisi agak horizontal dan terlihat tidak dapat bergerak turun. untuk mengarahkan ke tempat seperti yang diinginkan sulit oleh karena terhalang oleh tulang bagian bukal yang tebal. pada kasus ini tindakan pembedahan untuk menuntun letak gigi kaninus agar dapat terletak pada posisinya diperlukan sampai tiga kali. setelah separuh bagian labial mahkota gigi kaninus keluar, dilakukan penarikan dengan menggunakan elastic threat sampai tonjol gigi kaninus menyentuh busur. selanjutnya busur labial diganti lagi dengan niti 0.16 untuk leveling sampai gigi kaninus terletak pada tempat yang benar (gambar 2 dan 3). kasus 2 penderita laki-laki usia 15 tahun datang ke klinik bedah mulut fakultas kedokteran gigi universitas airlangga atas rujukan dari bagian ortodonsia dengan impaksi total gigi 11 untuk dilakukan surgical exposure. pemeriksaan ekstra oral pada kasus ini tidak terdapat kelainan, sedangkan pada pemeriksaan intra oral regio bukal gigi 11 pada palpasi teraba tonjolan insisal dengan arah horisontal. pada rontgenogram tampak gigi 11 impaksi total dengan posisi horisontal, terletak di atas apikal 21 dengan ujung akar bengkok (gambar 4, 5). gambar 4. foto panoramik kasus 2. tata laksana kasus rencana terapi dikonsultasikan untuk dilakukan odontektomi dengan pertimbangan apabila dilakukan terapi ortodontik akan mengalami kesulitan karena lokasi gigi yang dalam serta bentuk akar yang abnormal. dengan latar belakang pemeriksaan klinis dan rontgenologis maka diputuskan untuk dilakukan tindakan odontektomi pada gigi 11 tersebut (gambar 6), sekaligus disertai pengambilan odontoma pada regio tersebut yang kebetulan ditemukan pada saat dilakukan foto panoramik. gambar 5. foto lokal gigi 11 impaksi total (tampak gambaran ujung akar yang bengkok). gambar 6. gigi setelah dikeluarkan (hasil odontektomi). pembahasan terapi konvensional suatu gigi anterior impaksi adalah surgical exposure dan traksi secara ortodontik. penanganan gigi kaninus dengan letak yang ektopik sering lebih sukar dibandingkan gigi anterior di rahang atas lainnya. hal ini disebabkan letaknya yang sedemikian sehingga dalam proses penarikannya gigi kaninus impaksi tersebut sering terbentur dengan jaringan tulang yang keras. keadaan ini akan dapat menghentikan laju erupsi gigi, sehingga tindakan bedah untuk pengambil bagian tulang yang menghambat tersebut perlu dilakukan. prognosis untuk keberhasilan penempatan gigi kaninus ektopik sehingga dapat menempati lengkung gigi yang benar tergantung dari beberapa faktor. faktor tersebut meliputi, usia penderita, adanya diastema, adanya gigi yang berdesakan serta dimensi vertikal, dan anteroposterior, terbalik atau tidaknya letak mahkota. jika 145prajanto dan sjamsudin: perawatan gigi impaksi anterior rahang atas inklinasi letak gigi terhadap garis media wajah lebih dari 45 derajat, maka akan mempunyai prognosis yang jelek untuk dapat erupsi.8 semakin dekat letak gigi terhadap garis median, maka semakin jelek prognosisnya. demikian pula gigi kaninus ektopik yang telah mengalami ankylosis atau mempunyai akar yang bengkok.8,9 gigi molar ketiga rahang bawah pada anak di usia antara 9 sampai 12 tahun mulai dapat terlihat, bahwa gigi tersebut tidak akan dapat mengalami erupsi sehingga akan mengalami impaksi. keadaan ini pada proses pertumbuhannya akan dapat menyebabkan dorongan ke arah anterior sehingga akan menyebabkan gigi di depannya mengalami perubahan letak. pada kasus 1 telah dilakukan perawatan secara bedah pada dua gigi kaninus rahang atas ektopik dan bekerja sama dengan ortodontis sehingga memberikan hasil yang baik. dikarenakan tingginya letak gigi kaninus ektopik dan tebalnya tulang yang menutup gigi tersebut maka tindakan surgical exposure dilakukan secara bertahap untuk membimbing keluarnya kedua gigi kaninus rahang atas tersebut agar dapat erupsi dan terletak pada tempat yang diharapkan. gigi molar ketiga di rahang bawah yang diperhitungkan akan impaksi sebaiknya tindakan bedah untuk mengeluarkan gigi tersebut dilakukan lebih awal sehingga diharapkan dapat mencegah terjadinya dorongan terhadap gigi di bagian anterior gigi molar ketiga dan tindakan pembedahannya dapat dilakukan dengan lebih mudah dan jarang terjadi komplikasi pasca bedah. tindakan germinectomy pada gigi impaksi lebih menguntungkan dibandingkan dengan odontektomi dengan alasan komplikasi lebih rendah sebab trauma yang terjadi lebih kecil oleh karena proses pengambilannya lebih mudah serta proses kesembuhannya lebih cepat. tindakan tersebut lebih baik dilakukan pada saat usia anak-anak atau remaja dikarenakan suplai darah pada anak-anak atau remaja lebih baik dibandingkan pada usia dewasa. hal ini dilakukan untuk mencegah terjadinya malposisi pada gigi sebelahnya serta mencegah kemungkinan terjadinya kelainan yang lain, misalnya kista dentigerous atau rusaknya gigi sebelahnya. berdasarkan pembahasan tersebut di atas, dapat disimpulkan bahwa keberhasilan perawatan ortodonsi dengan disertai tindakan surgical exposure akan bergantung pula dari pengalaman serta kerja sama penderita dan ortodontis, karena perawatan akan memakan waktu yang lebih lama. hal lain yang mendukung keberhasilan adalah perawatan gigi ektopik dilakukan pada usia remaja. tindakan odontektomi ataupun germinectomy dilakukan pada gigi anterior impaksi yang disertai dengan anomali bentuk akar serta lokasi yang tidak memungkinkan. tindakan surgical exposure pada gigi yang terletak di palatal gigi yang lain tidak mungkin dilakukan karena menyulitkan dalam perawatan ortodontiknya. daftar pustaka 1. shah rm, boyd ma, vakil tf. study of permanent toot anomaly in 7886 cannadians individuals. j canad dent assoc 1978; 44:262–64. 2. kindelan j, cook p. the ectopic maxillary canine: a case report. br j orthod 1998; 25:179–80. 3. ericson s, kurol j. radiographic examination of ectopically erupting maxillary canine. am j orthod 1987; 91:483–92. 4. moose pa, campbell hm, luffingham jk. the palatal canine and adjacent lateral incisor: a study of a west of scotland population. br j orthod 1994; 21:268–74. 5. bishara se. management of impacted canines. am j orthod 1976; 69:371–87. 6. coulter j, richardson a. normal eruption of the maxillary canine quantified in three dimension. eur j orthod 1997; 18:449–56. 7. jacob h. the etiology of maxillary canine impactions. am j orthod 1983; 83:125–32. 8. brencheley z,oliver rg. morphology of anterior teeth associated with displaced canines. br j orthod 1997; 24:41–5. 9. mc. sherry pf. ectopic eruption of the maxillary canine quantified in three dimensions on chephalometric radiographs between the ages of 5 and 15 years. eur j orthod 1996; 20:501–8. << 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 155155 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 155–159 case report the treatment of covid tongue in an isolation unit dwi setianingtyas1,2, nafiah1,2, cane lukisari1,2, paulus budi teguh2,3, felicia eda haryanto4 and erni marlina5 1department of oral medicine, faculty of dentistry, hang tuah university, surabaya, indonesia 2dental department of dr. ramelan naval hospital, surabaya, indonesia 3department of prosthodontics, faculty of dentistry, hang tuah university, surabaya, indonesia 4general practitioner, dental clinic, samarinda, indonesia 5department of oral medicine, faculty of dentistry, hasanudin university, makassar, indonesia abstract background: in 2019, a viral disease spread from wuhan, hubei province in china. the disease was caused by severe acute respiratory syndrome related to coronavirus 2 (sars-cov-2 virus), which was named by the coronavirus study group of the international committee on taxonomy of viruses. purpose: this article reports the multi-discipline treatment of covid tongue and exfoliative cheilitis with a main diagnosis of bilateral pneumonia caused by sars-cov-2 infection. case: a female patient, 70 years old was referred with a diagnosis of bilateral pneumonia by a pulmonologist. she complained of painful wounds all over her mouth. an intraoral clinical examination revealed white-thick lesion and multiple ulcerations, whilst an extraoral exam for exfoliative dermatitis has not been described. the working diagnosis was covid tongue mixed with exfoliative cheilitis. case management: the procedure was performed by teledentistry and direct visits to implement oral health care by asepsis, debridement and the application of oxygene gel. conclusion: the lesion was treated successfully due to the multidisciplinary approaches by an internist and pulmonologist by implementing integrated knowledge and was supported hugely by patient cooperation. keywords: covid-19; oral health care; covid tongue correspondence: dwi setianingtyas, department of oral medicine, faculty of dentistry, hang tuah university. jl. arif rahman hakim no. 150 surabaya, 60111 indonesia. email: dwi.setianingtyas.anik@gmail.com introduction it is more than a year since the coronavirus disease 2019 (covid-19) spread from wuhan, hubei province in china. the disease is caused by severe acute respiratory syndrome related to coronavirus 2 (sars-cov-2 virus), which was named by the coronavirus study group of the international committee on taxonomy of viruses.1 coronaviruses were responsible of the two previous respiratory diseases: the severe acute respiratory syndrome (sars) in 2002 and 2003, and the middle east respiratory syndrome (mers) in 2012.2 although the respiratory organ is considered as the main target for sars-cov-2 infection, co-infection of another organ such as the oral cavity has been reported elsewhere.3 dysgeusia, xerostomia, geographic tongue and covid tongue are some of the reported oral symptoms for this infection.4 one reason for oral manifestation is the distributed angiotensin-converting enzyme 2 (ace2) receptors on the oral epithelial cells from glandular epithelial to some lining area in the mouth.3 unfortunately, during the first covid-19 pandemic, most dentists were unable to manage oral diseases, especially those related to covid-19 infection. these led to oral-related covid-19 lesions being overlooked and poorly understood. the management poses a big challenging to clinicians; therefore, it sometimes needs an inter-disciplinary approach.5 this article reports the multidiscipline management of covid tongue with exfoliative cheilitis in an isolated patient with lung pneumonic bilateral caused by sars-cov-2 infection at the high care unit (hcu) of dr. ramelan naval hospital, surabaya. case on 25th january, a 70-year-old woman was referred to the oral medicine outpatient clinic of the department of oral dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p155–159 mailto:dwi.setianingtyas.anik@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p155-159 156 setianingtyas et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 155–159 medicine at dr. ramelan naval hospital in surabaya, indonesia. the patient was referred by a pulmonologist from an isolation hcu dedicated to covid-19 patients after being hospitalized for 12 days. her chief complaint was unbearable pain all over the mouth with a sharp pain mainly on the tongue. the patient also complained of dry lips and seromucous fluid from her mouth, which was sometimes mixed with blood. further investigation of medical history revealed chronic diabetes mellitus, along with a treatment of antrain injection and ranitidine. as a first step for management, more information was acquired from the information system of hospital management (ishm). it was confirmed that the patient had been hospitalised almost 24 days previously, initially at siti khotijah hospital, where she had stayed for nine days. due to unresolved fever, the patient was subsequently instructed for polymerase chain reaction (pcr) on 5th january. the result was positive for covid19 with a cycle threshold (ct) of 29.75. the patient was hospitalised and released after nine days. however, she then developed breathing difficulties and was brought to the dr. ramelan naval hospital on 14th january. the pulmonologist subsequently referred her for a routine blood examination and thorax radiography. based on the examination results, it was concluded as bilateral pneumonia. as a first measurement, we asked a responsible nurse to help us take an intraoral photograph (figure 1a). she reported that the patient was attached to oxygen and cardiogram devices. all daily activities were performed on the bed. extra oral images by photograph revealed two crusts on the midline superior labial, yellowish black in colour, with an irregular margin of 0.4 and 0.5 mm with a marked border. the lesion was still bleeding and painful and the lips look dry and cracked. intraoral examination demonstrated pseudomembranous plaque lesion with a range between 0.3 mm and 3 cm in width. additionally, there were multiple exophytic ulcerations. based on her medical history and the clinical examination, we then concluded covid tongue with exfoliative cheilitis as our working diagnosis. on the ishm, we prescribed chlorhexidine gluconate 0.2% to help reduce the inflammation of the tongue. case management the procedure was performed by teledentistry and direct visits to implement oral health care by asepsis, debridement and the application of oxygene gel. on the first day, which was a televisit on 25th january, on the ishm, we prescribed chlorhexidine gluconate 0.2% to help reduce inflammation of the tongue. on the second day, by direct visit, the patient was given a comprehensive assessment of other oral areas such as gingiva, tongue, palate and all oral soft tissue. a dental assistant prepared a cotton bud, sodium chloride, chlorhexidine gluconate 0.2% and oxygene gel, which contained oxygene, zinc, folic acid, xylitol, aloe vera extract and chamomile. after washing the patient’s mouth with water, lesion debridement and sanitation in the oral environment was carried out using a pz solutionimmersed cotton swab. this step was then followed up by the application of chlorhexidine gluconate 0.2% liquid to the lips and tongue as a double protective sanitation, and wrapped up with oxygene dental gel. to reduce dryness on the lips, borax glycerin liquid was then applied. for the subsequent oral health care, the patient’s granddaughter, who was a nurse at the isolation hcu, then instructed her to improve her oral health by performing oral hygiene a d b f c e figure 1. oral involvement with covid-19 infection. a) day 1. the conditions of the patient’s oral cavity from hcu isolation nurse’s photo, with pseudomembranous plaque; b) day 4. thin pseudomembranous with ulcer lesions and surrounded by an erythematous on the dorsal of the tongue and upper lips, with crusts that did not bleed easily; c) day 7. dorsal tongue: the size of pseudomembranous had reduced and they were surrounded by an erythematous area; d) day 10. no more lesions on the upper or lower lips and the patient did not wear the oxygen canule; e) day 11. all previous complaints were eliminated; f) day 12. oral health care was done by oral medicine personnel. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p155–159 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p155-159 157setianingtyas et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 155–159 four times a day. we also instructed a nutritionist to prepare a soft diet with high calories and high protein and with small but regular portions. on the third day, a virtual visit was performed. anamnesis revealed that pain intensity was reduced. the lesions on the tongue looked the same, but some ulcerations had diminished. because the patient felt fresh and comfortable after every oral health care was performed, she was asked to do her oral hygiene by herself. we then decided that aloe vera extract gel would be used to speed up the wound healing process on the tongue area. during a direct visit on the fourth day, the patient felt uncomfortable and reported a pain scale of around seven. an intraoral examination demonstrated that the white pseudomembranous plaque lesion had become thinner, with some ulcers surrounded by an erythematous area (figure 1b). chlorhexidine gluconate 0.2% liquid was applied to the lips and tongue as a double protective sanitation, and wrapped up with aloe vera extract gel. painless crust was still found on the labial superior. on the virtual visit on the fifth day, the patient felt healthier in general. however, the pulmonologist and the internist did not allow her out of hospital because of a high glucose level of 300 ml/dl and oxygen saturation of 95%. she could eat comfortably and agreed to do her oral hygiene diligently. mouthwash was changed to povidone iodine 0.1% with nystatin oral suspension as per package instructions. on the virtual visit on the sixth day, the patient’s general condition was clearly better with no oral complaint. it was planned to refer her for tongue swab microbiology to validate suspected oral candidiasis microorganism. on the tenth visit, oral health care was still being undertaken and the patient was instructed to maintain her achieved level of oral health. the patient could finish her food, which indicated that her metabolism, appetite and condition were better. the patient could spontaneously breathe and intermittently loosen her oxygen equipment. all previous prescriptions were continued. the progress of the oral lesion was normal. when the patient’s pcr examination was negative with 98% saturation, she was allowed to leave the hospital. when she was released from the hospital, we prescribed her with povidone iodine gargle and borax glycerin liquid to maintain her oral health. figure 1 shows the intraoral series management photograph of the patient. discussion the covid-19 pandemic was declared by the world health organization (who) in february 2020, and the first infection case in indonesia was in march 2020. from that date, the number of covid-19 infections in indonesia continuously escalated with a fast dissemination throughout the country. having been labelled as a life-threatening disease mainly from infection affecting the lungs, this disease also demonstrated a range of non-specific symptoms including the one found manifested in the oral cavity. xerostomia, dysgeusia and anosmia were commonly reported signs. recently, there was a ‘new clinical entity’ called covid tongue.4 the main characteristics of the new entity were glossitis with lateral clefts, anterior ‘temporary’ lingual papillitis due to swelling of the tongue and friction with the teeth, and glossitis with patchy depapillation. others reported geographic tongue coined as the covid tongue.5 the latter was identified through the covid-19 symptoms study, where participants submit symptom reports on a daily basis. either way, the definition of covid tongue is still far from clear.4 it is still debatable whether the oral manifestations of covid-19 are due to the primary infection of this virus, or whether they are merely direct systemic effects or secondary to the infection, such as drug-associated effects. one thing for certain is that the sars cov-2, as the etiologic microorganism of covid-19 infection, has the capability to attach to and and infect epithelial cells layering the salivary gland, which can potentially modify the quality and quantity of the secreted saliva.6 changes in saliva quality and quantity will lead to dysbiosis in the oral environment with the end outcome triggering other opportunistic infections such as candidiasis. in the oral cavity, fungal infections have long been known as immunosuppressed indicators and have been reported in both symptomatic and asymptomatic covid-19 patients.7 hence, the role of dentists, especially oral medicine specialists, has become significant not only to confirm the oral candidiasis as a covid-19 manifestation but also to be involved in managing the disease since oral candidiasis may transform into a life-threatening condition for covid19 patients. therefore, the oral health care management should address oral opportunistic infection to prevent it from growing into a more serious complication. covid-19 is very infectious; the virus was initially thought to be spread by droplets, but recently, it was discovered that it can be spread by airborne and aerosol transmission, so it is easily spread.8 if covid-19 infects someone, it can be fatal, and quite a few health workers have become victims of covid-19.9 to avoid this, the indonesian ministry of health (ministry of health of the republic of indonesia) issued se menkes hk.02.01/menkes/303/2020 regarding the use of information technology in the context of preventing covid-19 by providing services to patients in the form of telemedicine, which has a legal basis. after telemedicine, several other terms such as teledentistry, televisit and teleconsultation were introduced.9,10 televisit services and the availability of the hospital management information system called sistem informasi manajemen rumah sakit (simrs) at dr. ramelan naval hospital, surabaya, make integrated multidisciplinary care easier. this service makes it possible to find out patients’ conditions and to give them drugs without direct contact, so the transmission of the virus through direct contact can be avoided. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p155–159 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p155-159 158 setianingtyas et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 155–159 during the first televisit of the case in this study, chlorhexidine gluconate mouthwash 0.2% was given, which is a topical drug used to prevent secondary infection in immunocompromised patients.7 however, when looking at the results of the photos, it is clear that this therapy was not enough, and that however, secondary infection was prevented by sanitising the lesion, carrying out debridement and administering the drug directly. during the second visit, oral health care was immediately carried out by giving pz solution and chlorhexidine gluconate solution, so that the lesions in the oral cavity were free from impurities including all microorganisms. finally, oxygene dental gel was applied. this drug functions as a wound healer and contains zinc, folic acid and herbal aloe vera.11 at that time, the lesion was not swabbed because the patient experienced severe pain with bleeding. the hcu isolation nurse was asked to carry out this procedure four times a day. on the third televisit, maintenance therapy was replaced by providing aloe vera extract mouthwash for the oral cavity and aloe vera extract gel for the lips. the use of mouthwash was intended so that enough lesions on the tongue could be rinsed with one rinse, but could not cover the lip area. the aloe vera extract contains extract aloe vera polyvinylpyrrolidone (pvp), sodium hyaluronate and dipotassium glycyrrhizate. the drug works by forming a protective film that covers the innervation of a lesion in order to avoid irritation and to reduce pain. the ultimate goal was to be able to accelerate healing and overcome pain.11 on the fifth televisits, the drug was replaced with povidone iodine mouthwash and nystatin oral suspension. at the beginning, the patient received a nystatin oral suspension therapy but it did not produce a positive response. this suggests that the use of nystatin oral suspension as a single application did not give a positive response. however, when it was used together with povidone iodine mouthwash, it improved the lesion.12 in the management of the covid tongue case, an oral medicine specialist played a role in overcoming pain and discomfort that could affect the patient’s quality of life. providing only topical drugs to patient was one of the methods used, as the use of topical drugs had several advantages over systemic drugs. among these, topical drugs can come into direct contact with oral lesions, allowing them to increase their therapeutic effect and have fewer side effects.9 generally, patients with systemic illnesses (geriatric condition and diabetes mellites) generate more risk of infection due to the immune system dysregulation.13 in addition, the patient had received various systemic drugs to treat her main disease, including immunosuppressant and corticosteroid, which contributed to an increased risk of infection in the oral cavity.13 oral health care management is very important to keep sars cov-2 from replicating in the oral cavity and to prevent secondary infection.13 with this prolonged therapy, the patient felt less pain and could get their nutritional intake by eating. getting enough nutrition strengthened the immune system.14 the patient was very cooperative in carrying out instructions from the operator. the patient also had a cheerful personality and did not give up easily, demonstrating a strong desire for recovery. she also received support from her family, as her grandchildren took good care of her while she was sick. another important factor was that the management at dr. ramelan naval hospital, surabaya, fully supported the drugs that were not covered by the national health insurance from health social security agency (bpjs kesehatan indonesia) used by the patient, including aloe vera extract mouthwash and aloe vera extract gel. this current report described a hospitalised covid-19 patient with covid tongue and exfoliative cheilitis who was treated in dr. ramelan naval hospital. whether the oral manifestation was caused by direct infection of the sars-cov-2 virus, a drug associated with the disease, or was secondary to the severity of the general infection, the oral health care improvement was the first step to reducing the patient’s complaint. moreover, patient education is also important to speed up the reduction of an oral complaint.15 in conclusion, the lesions were treated successfully and with optimal results due to the multidisciplinary approach by an internist and pulmonologist, by implementing integrated knowledge and supported hugely by patient cooperation. references 1. bergmann cc, silverman rh. covid-19: coronavirus replication, pathogenesis, and therapeutic strategies. cleve clin j med. 2020; 87(6): 321–7. 2. aiello f, gallo aff litto g, mancino r, li j-po, cesareo m, giannini c, nucci c. coronavirus disease 2019 (sars-cov-2) and colonization of ocular tissues and secretions: a systematic review. eye. 2020; 34(7): 1206–11. 3. xu h, zhong l, deng j, peng j, dan h, zeng x, li t, chen q. high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa. int j oral sci. 2020; 12: 8. 4. pérez-sayáns m, ortega kl, braz-silva ph, martín carreras-presas c, blanco carrión a. can “covid-19 tongue” be considered a pathognomonic finding in sars-cov-2 infection? oral dis. 2021; 00: 1–2. 5. dos santos ja, normando agc, da silva rlc, de paula rm, cembranel ac, santos-silva ar, guerra ens. oral mucosal lesions in a covid-19 patient: new signs or secondary manifestations? int j infect dis. 2020; 97: 326–8. 6. galván casas c, català a, carretero hernández g, rodríguezjiménez p, fernández-nieto d, rodríguez-villa lario a, navarro fernández i, ruiz-villaverde r, falkenhain-lópez d, llamas velasco m, garcía-gavín j, baniandrés o, gonzález-cruz c, morillas-lahuerta v, cubiró x, figueras nart i, selda-enriquez g, romaní j, fustà-novell x, melian-olivera a, roncero riesco m, burgos-blasco p, sola ortigosa j, feito rodriguez m, garcía-doval i. classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases. br j dermatol. 2020; 183(1): 71–7. 7. ruslijanto h, amtha r, meiyanti, marwati e, febrina s. obat topikal untuk lesi mulut: pemilihan dan cara aplikasi. jakarta: egc; 2019. p. 31–44. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p155–159 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p155-159 159setianingtyas et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 155–159 8. dal’belo se, gaspar lr, maia campos pmbg. moisturizing effect of cosmetic formulations containing aloe vera extract in different concentrations assessed by skin bioengineering techniques. skin res technol. 2006; 12(4): 241–6. 9. setiadhi r, firman dr. obat di bidang penyakit mulut dan penulisan resepnya. bandung: unpad press; 2017. p. 1–61. 10. jawad h, hodson na, nixon pj. a review of dental treatment of head and neck cancer patients, before, during and after radiotherapy: part 1. br dent j. 2015; 218(2): 65–8. 11. argoff ce, kaur m, donnelly k. topical analgesics. in: deer tr, leong ms, gordin v, editors. treatment of chronic pain by medical approaches. new york: springer; 2015. p. 77–87. 12. al-hashimi i, schifter m, lockhart pb, wray d, brennan m, migliorati ca, axéll t, bruce aj, carpenter w, eisenberg e, epstein jb, holmstrup p, jontell m, lozada-nur f, nair r, silverman b, thongprasom k, thornhill m, warnakulasuriya s, va n der wa a l i. o ra l l ichen pla nus a nd ora l l ichenoid lesions: diagnostic and therapeutic considerations. oral surg oral med oral pathol oral radiol endod. 2007; 103 suppl: s25.e1-12. 13. imai k, tanaka h. sars-cov-2 infection and significance of oral health management in the era of “the new normal with covid-19”. int j mol sci. 2021; 22(12): 6527. 14. coke cj, davison b, fields n, fletcher j, rollings j, roberson l, challagundla kb, sampath c, cade j, farmer-dixon c, gangula pr. sars-cov-2 infection and oral health: therapeutic opportunities and challenges. j clin med. 2021; 10(1): 156. 15. la rosa grm, libra m, de pasquale r, ferlito s, pedullà e. association of viral infections with oral cavity lesions: role of sars-cov-2 infection. front med. 2021; 7: 571214. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p155–159 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p155-159 mkgs vol 44 no 1 jan-mar 2011.indd dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 98 pin-retain for restoration of widely tooth damaged ira widjiastuti department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abtract widely damaged tooth involves enamel as well as dentin could be due to caries or other causes, resulted the preparation of retention could not be fulfilled so that additional pin is used as retention. pin is made of titanium and used as retention for restoration with the aim to unite restorative material in tooth cavity by filling the hole that is prepared in the dentin. the number of pins that are needed is according to the width of the lost tooth tissue. restoration with pin functions as retention has conservative advantage to the tooth structure, increases retention, resistance, period of treatment is short, and the cost is cheaper. key words: retention, pin, restoration correspondence: ira widjiastuti, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132 introduction restoration failure can be due to various factors in which one of them is releasing restoration because of lack of retention. wide loss of tooth structure involves enamel and dentin causing insufficient resistance and retention consequently releasing restoration or fracture of tooth structure might occur.1 to reach good restoration, a dentist should be able to determine the tooth structure that should be preserved or evacuated so that the tooth could be well restored, the restoration is not easily released and the residual of tooth tissue would be resistant to mastication forces. box, undercut, or grove retention could not be made when tooth damage is widely. in this case, pin is needed as retention to avoid releasing restoration and residual of tooth tissue is strong to resist mastication forces. pin placement is intended to attach restorative material on tooth cavity that has been prepared and the number of pins that are needed should be according to the width of the lost tooth tissue. pin placement is performed as alternative consideration to conserve tooth vitality and to prevent extended disease. restoration with pin as retention can be done in one visit. furthermore, the cost is more economical compared to extra or intra indirect restoration coronal with alloy material.2 the use of pin the use of pin on restoration is to unite restorative material with the prepared tooth and to increase retention. the use of pin in excessive number will weaken the strength of restoration toward mastication, therefore to obtain restoration with pin retention requiring the number of pin in accordance with the width of tooth damage.2 the use of excessive number of pins can cause the distance of pin smaller therefore the compression of restorative material into the cavity will be difficult, dentin will be easily fracture and broken so that the restoration is not strong enough to bear mastication forces. some factors that influence pin retention are type, surface form, parallel or bending, and the number of pin. pin gives retention and relatives retention value. the number of inserted pin should be in accordance with the necessity. one pin is needed for every missing cusp and for every missing proximal tooth structure.1 types of pins and techniques for pin insertion in tooth with large damage, the pin that will be used is inserted into dentin hole which has been prepared. pin is made of stainless steel that polished with gold and gradually pin is made of titanium.2 in general, there are two types of pin: non-parallel and parallel pin.3 the pin that is frequently used and easily inserted is non parallel pin (figure 1). there are three types of non parallel pins: cemented pin, friction-locked pin (unitek) and self-threading pin (tms, whaledent ). this type of pin is frequently used.1,2,3 figure 1. various non-parallel pin. (a) cemented pin, (b) friction locked pin, (c) selfthreading pin. cemented pin is a pin that inserted by cementation. the pin surface is serated and inserted into pinhole with diameter 0,001–0,002 inch (0,025–0.05 mm) larger than 99widjiastuti: pin-retain for restoration the diameter of the pin. friction-locked pin (unitek) is a pin that implanted by taped to place. pin is inserted into pinhole with diameter 0.001 inch or 0.025 mm smaller than the diameter of the pin. retention of this pin depends on the resiliency of dentin. self threading pin (tms, whaledent) is a type of pin which inserted by screwing. pin is inserted into the pinhole with diameter 0.038–0.1 mm smaller than pin diameter, retention is made by gripping and depends on resiliency of dentin. stabilok pin is self-threading pin that combined in the form of screw bur design, in which pin is united with latch type mandrel (mandrel pin) which can be implanted on contra angel hand piece. the diameter of small pin is 0.021 mm (yellow), while medium pin is 0.027 mm (orange) that available in a kit completed with twist drill. tooth with wide damage and residual tissue is not strong enough such as: lost one or two of cusps, in order to achieve optimal retention the tooth should prepared in the form of cavity of pulp wall and gingival wall forming perpendicular angle, making axial split, while gingival wall is not enlarged in sub gingival.2 the requirement to obtain restorative result with pin as additional retention is thick dentin should be available (dentin thickness is 1.5 mm between pin and enamel surface and root) perpendicular and parallel with tooth axial. the part of pin on tooth surface is 2 mm and the thickness of restorative material is 2 mm between the tip of pin with occlusal surface of restorative material (figure 2-a). a b figure 2. location of pin.2 (a) pin position in dentin, (b) pin position for cavity. before inserting the pin, pinhole should be made in dentin by using depth limiting twist drill and could be inserted according to the type of the pin. to decide the pinhole, some factors should be considered such as pulp anatomy, the contour of tooth surface (convex or concave), dentin thickness, periodontal pocket, and the age of patient. pinhole should be placed on the flat surface and half of the distance between outer part of tooth and pulp. pin insertion should be required 0.5 mm from axial wall and 0.5 mm from dentino-enamel junction or the root surface, far from tooth bifurcation, parallel with tooth outer surface in order to prevent from effecting periodontal tissue, located in restorative material mass, and closed to proximal line (one pin per one missing cusp) (figure 2-b). performing pin insertion in tooth, tooth anatomy should be paid closed attention. prominensia concavity is found on the middle part of upper first molar, pulp horn in mesio buccal side is found on the upper and lower first molar, on the lower first and second molar found convexity on mesial and buccal side while molar teeth are found on buccal, mesial and distal side. this condition should be seriously considered otherwise restoration failure might happen such as: the occurrence of perforation either on the pulp or periodontal tissue.2,4 after pin insertion has been completed, it is continued by manipulation using restorative material while amalgam, resin composite4 can be used as restorative material. if proximal contact is opened, matrix band and wedge are obligatorily by used and if multiple cusp has lost, greenstick might be applied to achieve matrix stabilization.4 by the development of science, pin implantation is combined by giving adhesive material to increase restoration strength. treatment and prognosis to forming restoration on vital tooth with pin retention, tooth condition should be paid closed attention. if caries in the dentin is found and remains in thin layer, pulp capping is the first thing to be done. further treatment is done to release symptomatic preoperative complaint to avoid the increase of sensitivity and prolonged complaint.2,3,5 on posterior tooth with crown damage affected more than one cusp which is classified as total damage required complex restoration treatment, and the use of pin as retention should be considered in order to keep pulp vitality which means the treatment is more conservative and the prognosis is good (figure 3).6,7 figure 3. pin retaine for complex restoration.7 (a) before treatment, (b) tooth preparation, and pin inccertion, (c) after restoration, (d) after used for 5 years. 100 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 98–100 discussion the effort of performing tooth restoration is one of the efforts to conserve the tooth in oral cavity, to maintain the function for mastication, phonetic, and aesthetic according to stomatognatic function. it’s difficult to achieve good retention for vital tooth with widely lost hard structure tissue affecting enamel and dentin due to caries or other causes, so additional retention is required by using pin which is inserted in dentin and compressed restorative material in adjacent area.1 pin is the alternative to increase retention of restored tooth with the loss of the one cusp even with totally damaged crown and to preserve the pulp vitality so the restoration of tooth with the loss of more than one cusp can be done only in one visit. meanwhile in tooth with totally damaged crown, x-ray radiograph is needed to know the thickness of dentin and if the thickness is adequate (1.5 mm) pin can be used as additional retention so pulp vitality can be preserved and the tooth is restored by using composite resin or amalgam as the core, finally followed by crown restoration.2,5 pin hole preparation is conservative treatment compared with slot and lock as well as tooth preparation for indirect extra or intra coronal restoration with alloy due to more dentin tissue evacuation. pin insertion into cavity increase retention and resistance, further more the treatment can be done one visit, relatively inexpensive, and suitable for elderly patient or patient with mental trouble.2 to increase retention of restorative material in tooth cavity, before pin insertion, cavity wall should be polished by adhesive material. uyera et al.6 and davis & overton8 suggested that the combination between dentin adhesive material and pin insertion can give better strength compared with giving pin only or dentin adhesive material only. rosen9 suggested that tooth with amalgam complex restoration will be stronger if prior to filling should be polished with bonding agent and pin is used as additional retention. bonding material will either chemically or physically attach the dentin finally will mechanically attach restorative amalgam so it will increase attachment strength and will reduce the possibility of leakage in the edge part of amalgam restoration.10 pin as an additional retention is rarely used for composite resin restoration in anterior tooth due to etch technique and bonding, however in the case of tooth with widely damaged crown in class vi cavity, it is necessary to perform additional retention to stabilize restoration besides the treatment can be done only in one visit.2 pin which is used as additional retention can keep the structure of tooth tissue without excessive evacuation of dentin tissue, therefore restoration can be done in widely damaged crown, pulp vitality can be restored, retention factor and tooth resistance will increase, period of treatment will be short. the cost of treatment will be unexpensive compared to indirect extra or intra coronal restoration using either alloy or porcelain. references 1. baum l, phillips rw, lund mr. buku ajar ilmu konservasi gigi. edisi ke-2. jakarta: egc; 1995. p. 456–86. 2. sturdevant cm, roberson tm, heyman ho, sturdevant jr. the art science of operative dentistry 3rd ed. st louis, toronto, priceton: the cv mosby company; 1995. p. 504–12. 3. howard ww. atlas of operative dentistry. 2nd ed. st. louis: the cv mosby company; 1973. p. 141–6. 4. wassel rw, smart er, george st. crowns and other extra coronal restoration: cores for teeth with vital pulps. br dent j 2002; 192:499–509. 5. soetanto s. perbedaan kekuatan tarik pin ulir pada dentin dengan cara pemasangan menggunakan hand piece putaran rendah dan uliran tangan. maj. ked. gigi (dent j) 1995; 29:135–8. 6. uyehara my, davis rd, overton jd. cuspal reinforcement in endodontically. j op dent 1999; 24:364–70. 7. summit jb, burgeess jb, berry tg, robbins jw. the performance of bonded vs. pin retained complex amalgam restorations. a five year clinical evaluation. j am dent assoc 2001; 132:923–31. 8. davis rd, overton jd. efficacy of bonded and non bonded amalgam in the treatment of teeth with incomplete fractures. j am dent assoc 2000; 131:469–78. 9. rosen at. resistance of bonded complex amalgam restoration with and without pins. j dent res 1998; 77:153. 10. setcos jc, staninec m, wilson nhf. the development of resin bonding for amalgam restorations. br dent j 1999; 186:328–32. 153 the management of oral erythema multiforme in juvenile patient diah savitri ernawati department of oral medicine faculty of dentistry, airlangga university surabaya indonesia abstract erythema multiforme is an acute inflammatory disease of the skin and mucous membranes that causes a variety of the skin lesionhence the name ‘multiforme’.the oral mucosa looks severely inflamed, but the feature are non specific and usually a biopsy is required in order to confirm the diagnosis. cracked, bleeding, crusted, swollen and ulcers of the lips is very characteristic of erythema multiforme, and lip involvement may cause significant morbidity. em is assumed as an immune complex disorder which rises as a result of an immune response to an external agent such as herpes simplex virus or various drugs. we reported: 14-year girl, complained she suffered from painful oral ulceration for one week. one weeks advance the patient received a treatment of paracetamol and paramex for febris, headache and cough. clinical examination of the skin showed no signs of cutaneous involvement. other site such as the conjunctival, and genital were also free of lesions. the patients had several red-based superficial erosions on the upper and lower lips accompanied by crusting and bleeding. intra oral findings showed multiple irregular erosions, ulcers and intense erythematous areas, mainly on the labial mucosa. the clinical diagnosis of em was concluded by anamnesis and clinical appearance, with differential diagnosis of secondary herpes infection (herpes labialis) and pemphigus vulgaris. systemic and topical corticosteroid therapy is frequently used to treat em although it may partially suppress the disease. objective: this report explains and describes the management of patients with em which may help dentists to determine an accurate diagnosis to avoid further complication and to give medical intervention to the disease. conclusion: early recognition of this disease may prevent delayed diagnosis and incorrect treatment. key words: oral erythema multiforme, children, oral management correspondence: diah savitri ernawati, c/o: bagian oral medicine, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: savitri_glx@yahoo.com introduction erythema multiforme (em) is an acute inflammatory disease of the skin and mucous membranes. the main feature of an attack is the sudden development of widespread erosions of the oral mucosa which characteristically involving the lips. the erosions are produced by the disintegration of sub epidermal bullae, lesions that only rarely last long enough to become a diagnostic feature.1 erythema multifome is an immune-mediated disease that may be initiated either by deposition of immune complexes in the superficial micro vessels of skin and mucosa, or cell-mediated immunity. the most common triggers for episodes of em are herpes simplex virus and drug eruption. the drugs that most frequently associated with em reactions are oxycam non steroidal anti inflamatory drugs (nsaids), sulfonamides; anticonvulsants such as carbamazepine; phenobarbital; and phenytoin; trimethoprim-sulfonmide combinations, allopurinol and penicillin.1,2 this report explains and describe a case of oral erythema multiforme in juvenile patients and its management. erythema multiforme is most frequently detected in children and young adults and is rarely found after the age of 50 years. it has an acute or even an explosive onset and generalized symptoms such as fever and malaise, which appear in severe cases. a patient may also be asymptomatic and in less than 24 hours have extensive lesions of the skin and mucosa. erythema multiforme is a self-limiting form of the disease and is characterized by macules and papules 0.5–2 cm in diameter, appearing in a symmetric distribution. the most common skin areas involved are the hands, feet and extensor surfaces of the elbows and knees. the face and the neck are commonly involved.1,2,3 typical skin lesions of em may be non specific macules, papules and vesicles. more typical skin lesions contain petechiae in the center of the lesions. the pathognomonic lesion is the target or “iris” lesions, which consists of central bulla or pale clearing area surrounded by edema and erythematous bands. em is classified as stevens-johnson’s syndrome when the generalized vesicles and bullae involve the skin, mouth, eyes and genitals.1,2,3 the diagnosis is made on the basis of the total clinical picture, including the rapid onset of lesion. the oral lesion starts as bullae on an erythematous base, but intact bullae are rarely seen by the clinician because they break rapidly into irregular ulcers. em lesion is larger and deeper than common ulcers and often bleeds spontaneously or at the slightest touch. lesions may occur any where on the oral mucosa, but involvement of the lips is especially prominent, and gingival involvement is rare. this is an 154 dent. j. (maj. ked. gigi), vol. 40. no. 4 october–december 2007: 153–156 important criterion for distinguishing em from primary herpes simplex infection. in full blown clinical cases, the lips are extensively eroded, and large portions of the oral mucosa are denuded of epithelium. the patient can hardly eat or even swallow and tend to drool blood-tinged saliva. within 2 or 3 days, the labial lesions begin to crust. healing occurs within two weeks in majority of cases, but in some severe cases extensive disease may continue for several weeks.2,3 histopathological examination of ulcer reveals intense chronic inflammatory infiltrate. degenerative changes in the epithelium are associated with infiltration by inflammatory cells which also involve the corium and may have a perivascular distribution. leakage of immunoglobulins from blood vessels has been reported, but vasculitis is not seen histologically.1 treatment of the case is restricted to the mouth area depending on the use of local or systemic steroids, to which there is usually a rapid response. a steroid mouthwash is likely to give symptomatic relief and effectively reverse the process in a few days. when the skin or oral lesions are severely damaged or when the eye or other mucous membranes are affected, a short course of systemic steroids may be necessary to shorten the attack. an initial dose of 30 mg/day to 50 mg/day of prednisone or methyl prednisolone for several days, which is then tapered, is helpful to quicken the healing time of em, particularly when therapy started early in the course of the disease.2,3 cases case 1: a 14-year girl was referred to the oral medicine clinic, school of dentistry of airlangga university, with a one week history of painful oral ulceration. a week in advance the patient received a treatment of paracetamol and paramex for febris and headache and cough. the oral lesions appeared a few days later, causing considerable discomfort and affected her normal oral function. personal and family histories and laboratory tests were uneventful. clinical examination of the skin showed no signs of cutaneous involvement. other site such as the conjunctival, and genital were also free of lesions. the patients had several red-based superficial erosions on the upper and lower lips accompanied by crusting and bleeding. intra oral findings showed multiple irregular erosions, ulcers and intense erythematous areas, mainly on the labial mucosa (figure 1). the clinical diagnosis of em was concluded by anamnesis and clinical appearance, with differential diagnosis of secondary herpes infection (herpes labialis) and pemphigus vulgaris. the patient was treated with local and systemic prednisone (30 mg/day). the application of topical steroids was necessary due to the severity of the lesions in the upper and lower lip. the disease showed a marked improvement in two weeks. case 2: a 7-year old boy was referred to the oral medicine clinic, faculty of dentistry airlangga university, after two week history of painful oral ulceration. one week in advance, the patient received a treatment of 250 mg of amoxicillin and 250 mg of paracetamol for laryngitis. the oral lesions appeared a few days later, causing considerable discomfort and affected his normal oral function. he also reported weight loss during this time since the ulcers caused eating difficulty. clinical examination, intra oral findings showed multiple irregular erosions, ulcers and intense erythematous areas, mainly on the labial mucosa. the skin showed no signs of cutaneous involvement. others site such as the conjunctival, and genital were also free of lesions. the patients had several red-based superficial erosions on the upper and lower lips accompanied by crusting and bleeding (figure 2). the patient was commenced on systemic corticosteroids (prednisolone) at an initial dose 30 mg/day. the application of topical steroids was necessary due to the severity of the lesions in the upper and lower lip. the disease showed a marked improvement in two weeks. figure 1. (a) erythema multiforme. on the first visit: ulceration of the vermillion border of the lip with bleeding, swelling and crusting is characteristic. (b) twelve days after the fisrt visit, patients came with much better condition. 155ernawati: the management of oral erythema multiforme in juvenile patient 0.25 gram of lanoline, 0.1 gram of kemicitine, and 10 gram of vaseline, which should be applied on the oral lesions. other medication are systemic intermediateacting corticosteroid 30 mg per day for 5 days and multivitamin.2,4 six days later the patient showed up with less complaint. the prescribed medicine was consumed as instructed. pain on the lips and mouth lessened. clinical examination revealed improved condition. crust on upper lips have mostly eroded, the swelling lessened, and ulcer in the labial mucosa has significantly healed. the systemic prednisone dose was then tapered while the topical corticosteroid was maintained. six days after the 1st control, or twelve days after the 1st visit, patient came with much better condition. crusts on the upper lip had completely eroded, leaving the lips in slight erythematous condition, sensitive but not painful. the lower lip appeared normal. on the 3rd visit, a week later, the lesions had completely healed and the lips appear normal (figure 3). discussion erythema multiforme is an acute, self-limiting, mucocutaneous disorder with symmetrically distributed, erythematous skin lesions, some with concentric colour changes (target lesions), which resolve within 1 to 6 weeks and show compatible histology. there exists a subgroup of patients with recurrent em in whom frequent episodes of the disease over several years cause significant morbidity. the experience 2 or more attacks per year. prodromal symptoms include malaise, fever, headache, sore throat, rhinorrhoea and cough which may occur approximately 1 week before the onset of em. the typical primary lesions of em is a round, erythematous macule that rapidly becomes popular or urticarial. individual oedematous papules may enlarge to small plaques and may also develop concentric alteration in morphology and colour. the concentric changes produce characteristic lesions with either a central blister or a central area of necrosis resulting in target lesions. as the skin lesions resolve, they may develop some scaling but typically heal without theraphy.3,5 the definite etiology remains unclear. no convincing mechanism has been proposed, nevertheless the disease may be a reaction to various causes. infection, particularly herpetic, may serve as triggering factors. drugs, particularly sulphonamides and barbiturate, have also been reported as a trigger. a positive drug history is also rare. even when drugs have been taken, coincidence cannot always be excluded and in most patients no precipitating cause can be found.3,6 the main feature of an attack is the sudden development of widespread erosions of the oral mucosa, characteristically involving the lips. the erosions are produced by the disintegration of sub epidermal bullae, lesions that only figure 2. erythema multiforme. ulceration of the vermillion border of the lip with bleeding, swelling and crusting is characteristic. figure 3. on the 3rd visit, a week later, the lesions had completely healed and the lips appear normal. case management on the first visit, the lips were sanitized with sterile gauze and antiseptic solution. patient was prescribed a topical cream consisting 0.1 gram of hidrocortisone, 156 dent. j. (maj. ked. gigi), vol. 40. no. 4 october–december 2007: 153–156 rarely last long enough to become a diagnostic feature. the erosions on the lips (especially the lower lip) are accompanied by crusting and bleeding and are, if not absolutely diagnostic, strong pointers to the nature of the condition. there is often a cervical lymphadenitis with pyrexia and the patient feels unwell. the lesions are typically symmetrical and occur commonly on the dorsal surfaces of the hands and extensor aspects of the extremities. mucosal involvement occurs in 25% to 60% of cases either simultaneously or preceding it by several days. the durations from onset to healing is less than 4 weeks (–2 weeks).3,6,7 the initial diagnosis is entirely clinical, the important differential diagnosis being from a primary herpetic stomatitis may be confidently excluded since this is an isolated event in immuno-competent individuals. the involvement of the lips is a strong indication of the diagnosis of em and the presence of “target” lesions of the skin can be taken as almost conclusive evidence for the diagnosis. the histological appearances are variable. widespread necrosis of keratinocytes with eosinophilic colloid change in the superficial epithelium may can be conspicuous. this may progress to intraepithelial vesicle or bulla formation. degenerative changes in the epithelium are associated with infiltration by inflammatory cells which also involve the corium and may have a perivascular distribution. leakage of immunoglobulin from blood vessels has been reported, but vasculitis not seen histologically.1 patients should be warned of the possibility of recurrences but the disease usually runs a limited course. as with adult, em in the juvenile patient is managed with systemic corticosteroids.4 treatment of the case is restricted to the mouth, depending on the use of local or systemic steroids. the use of systemic steroids for em remains controversial due to the immune-suppressing side effect, it may give symptomatic relief. an initial dose of 30 to 50 mg per day of prednisone or methyl-prednisolone for several days, which is then tapered, is helpful in shortening the healing time of em, particularly when therapy is started early in the course of the disease.2,3,4,6 antibiotics are often also prescribed in severe cases with the idea of preventing secondary infection. there are two cases of em described among children with varying forms of treatment and evolution. the diagnosis can be made based on characteristic clinical appearance accompanied by general complaints and medical history of the patient. corticosteroids, topical and systemic, in spite of its immune-suppressing side effect, remains the drug of choice against this disease. references 1. cawson ra, odell ew. essentials of oral pathology and oral medicine. 7th ed. toronto: churchill livingstone; 2005. p. 206–7. 2. wray d, lowe gordon do, dagg jh, felix dh, scully c. textbook of general and oral medicine. edinburgh, london, newyork, philadelphia st louis, sydney, toronto: churchill livingstone; 2001. p. 238–40. 3. sen p, chua sh. a case of recurrent erythema multiforme and its therapeutic complication: case report. j ann acad med singapore 2004; 33:793–6. 4. pereira cm, gaspaetto pf, aires mp. pempigus vulgaris in a juvenile patient: case report. j oral med oral diagnosis 2006 july-agustus; 262–3. 5. farthing pm, maragaou p, coates m, et al. characteristics of the oral lesions in patients with cutaneous recurrent erythema multiforme. j oral pathol med 1995; 24:9–13. 6. greenberg m. burket’s oral medicine diagnosis and treatment. 10th ed. philadelphia: lippincott-reven; 2003. p. 57–59. 7. gandolfo s, scully c, carrozo m. oral medicine. edinburgh london, newyork, philadelphia, st louis, sydney, toronto: churchill livingstone; 2006. p. 72, 154. 218 volume 46, number 4, december 2013 antifungal effect of sticophus hermanii and holothuria atra extract and its cytotoxicity on gingiva-derived mesenchymal stem cell kristanti parisihni and syamsulina revianti department of oral biology faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: sea cucumber had been acknowledged to have some medical properties sticophus hermanii and holothuria atra are species of sea cucumber which has been known to have antifungal properties thus potentially explored as therapeutic agent in oral candidiasis. purpose: the aim of this study was to examine the antifungal property sticophus hermanii and holothuria atra extract against candida albicans and its cytotoxicity to human gingiva-derived mesenchymal stem cell. methods: the study was an experimental laboratories research with post test only control group design. methanolic extract of sticophus hermanii and holothuria atra in concentrations of 1%, 0.5%; 0.25%; 0.13%, 0.07%; 0.03%, 0.02% and 0.01%; were tested its cytotoxicity on gingiva-derived mesenchymal stem cell. cell viability were measured by mtt assay. the antifungal property against candida albicans was tested by disk diffusion method. data were analyzed by anova followed by lsd. results: extract of sticophus hermanii showed no cytotoxicity in all concentrations (p>0.05), while holothuria atra showed toxicity in the concentration of 1% and not cytotoxic in the concentrations below (p<0.05). both sea cucumber extract could inhibit the growth candida albicans, in vitro, proved by the clear zone around the disc in all concentrations (p<0.05). conclusion: stichopus hermanii and holothuria atra extract had the antifungal effect against candida albicans. sea cucumber extract were not cytotoxic togingiva-derived mesenchymal stem cell in the concentration of sticophus hermanii ≤ 1% and holothuria atra ≤ 0.5%. key words: sticophus hermanii, holothuria atra, cytotoxicity, gingival, mesenchymal stem cell abstrak latar belakang: teripang telah diketahui mempunyai berbagai khasiat medis. sticophus hermanii dan holothuria atra adalah spesies teripang yang telah diketahui mempunyai sifat anti jamur sehingga santat potensial untuk diekplorasi sebagai agen terapeutik pada infeksi di rongga mulut. tujuan: tujuan dari penelitian ini adalah untuk meneliti sifat anti jamur ekstrak sticophus hermanii and holothuria atra terhadap candida albicans dan sitotoksisitasnya terhadap stem sel mesenkimal yang berasal dari gingiva manusia. metode: penelitian ini merupakan penelitian eksperimental laboratoris dengan rancangan post test only control group design. ekstrak metanol sticophus hermanii dan holothuria atra pada konsentrasi 1%, 0,5%; 0,25%; 0,13%, 0,07%; 0,03%, 0,02% and 0,01% diuji sitoksisitasnya terhadap stem sel mesenkimal yang berasal dari gingiva. viabilitas sel diukur dengan menggunakan metode mtt. sifat anti jamur terhadap candida albicans diuji dengan metode difusi. data dianalisis dengan anova dan lsd. hasil: ekstrak sticophus hermanii tidak menunjukkan adanya toksisitas pada seluruh konsentrasi (p>0,05), sedangkan holothuria atra menunjukkan adanya toksisitas pada konsentrasi 1% dan tidak toksik pada konsentrasi di bawahnya (p<0,05). kedua ekstrak mampu menghambat research report 219parisihni dan revianti: antifungal effect of sticophus hermanii and holothuria atra extract pertumbuhan candida albicans in vitro yang ditunjukkan dengan adanya zona jernih disekitar disk pada semua konsentrasi (p<0,05). simpulan: ekstrak stichopus hermanii dan holothuria atra mempunyai daya anti jamur terhadap candida albicans. ekstrak teripang bersifat tidak toksik terhadap stem sel mesenkimal yang berasal dari gingiva masing-masing pada konsentrasi sticophus hermanii ≤ 1%, dan holothuria atra ≤ 0,5%. kata kunci: sticophus hermanii, holothuria atra, sitotoksisitas, gingiva, stem sel mesenkimal correspondence: kristanti parisihni, c/o: bagian biologi oral, fakultaskedokteran gigi universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: tanti_kris@yahoo.co.id introduction sea cucumbers belong to the phylum echinodermata, meaning that, they are spiny-skinned, under the class holothuridea. sea cucumbers are important components of the marine ecosystem. sea cucumbers, informally named as bêche-de-mer, or gamat, have long been used for food and folk medicine in the communities of asia and middle east.1-6 marine biota is the source of structurally unique natural products that are mainly accumulated in living organisms, not just as food consumption and industrial need but later has been known to have biomedical properties. therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array of bioactives especially triterpene glycosides (saponins), chondroitin sulfates, glycosaminoglycan (gags), sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, cerberosides, lectins, peptides, glycoprotein, glycosphingolipids and essential fatty acids. nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as vitamin a, vitamin b1 (thiamine), vitamin b2 (riboflavin), vitamin b3 (niacin), and minerals, especially calcium,magnesium, iron and zinc.4,7 generally, most species of sea cucumber share the same bioactive compound mentioned above but in different level contain.4,7 regarding to its contents, the aqueous and organic extracts from some sea cucumber species has been proved to have antioxidant and antiproliferative activities,3,4,7 immunomodulator1,4 while the other has been known to have antimicrobial properties on gram negative, gram positive bacteria2,4,8 and antifungal action.4,9,10,11 candidiasis is the most common fungal infection in oral cavity caused by candida albicans, which its prevalence raised specially along with the raise prevalence of hivaids.12,13 a natural source of antifungal agent could become the novel alternative solution in therapy of oral candidiasis. considering to the bioactive compound, the extract of sticophus hermanii and holothuria atra are potentially explored its antifungal property to candida albicans and as the potential candidatetherapeutic agent in oral candidiasis,8,14 its cytotoxicity should be well identified to assure the biocompatibility to oral cells.15 during the last years, the interest of in vitro systems as an alternative to animal experiments in toxicological research has been steadily increasing.16–18 stem cells and their derivatives represent a promising opportunity for developing in vitro, human cell assays that would ultimately replace, enhance, or surpass the current models that are used for predictive toxicology.19–23 in this paper, two sea cucumber extract sticophus hermanii and holothuria atra were studied its antifungal property to candida albicans and its cytotoxicity to gingiva-derived mesenchymal stem cell. these two species of sea cucumber are found plenty in karimun jawa coastal and so far had been explored mostly for food consumption.12 considering to the bioactive compound, there are some opportunities to explore sea cucumber for medical properties and yield more great value on it specially in oral disease. the aim of this study was to examine the antifungal property and cytotoxicity of various concentration of sticophus hermanii and holothuria atra extract onhumangingiva-derived mesenchymal stem cell. the result of this study could be served as preliminary data to be continued in preclinical and clinical research with marine natural products which will probably result in novel therapeutic agents for the treatment of fungal infection in oral disease. materials and methods two sea cucumber species: sticophus hermanii and holothuria atra were collected from karimun jawa coastal region. adult sea cucumber were selected to get the best extract result considering to its maximum secondary metabolit contents. the collected samples were cleaned from dirt, immersed in water for one night to get rid of salt and parasite then dried in dryer machine. sea cucumber then splitted, the inner abdomen were removed then cleaned and washed, so only the flesh of the body proceed to next process. each samples were cut in small piece of 3-10 cm, the wet weight then measured then dried up in solar dryer for 3-4 days to reduce the water content. the dried sea cucumber then cut into smaller pieces of 1 cm, mashed by blender the the weight were measured and ready for the maceration process. two hundred and fifty (250) gram 220 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 218–223 mashed dry sea cucumber sample immersed until soaked in 500 ml methanol solvent for 24 hours at room temperature, then filtered with filter paper to separate filtrate and residue. residue then reimmersed in 500 ml methanol solvent for 24 hours, again filtered with filter paper to separate filtrate and residue, resulted in maceration filtrate with the ratio of 250 gram sample / 1000 ml solvent (1:4 w/v). methanol (polar) filtrate got homogenized with 1000 ml hexane solvent (non polar) then performed partition with separatory funnel the each of the filtrate layer of methanol and hexane solvent were separated. methanol (polar) filtrate then got re-homogenized with 1000 ml chloroform solvent (semi polar), performed partition with separatory funnel the each of the filtrate layer of methanol and chloroform solvent were separated. each filtrate were separated by its solvent with rotary evaporator until extract produced. the evaporated extract then placed in the vial and stored in -30°c until the next analysis. candida albicans were cultured in sabouraud dextrose agar, suspensión were prepared by inoculating one single loop of fungal colony to sabouraud broth medium, incubated in 37°c for 24 hours and adjusted its turbidity to standard mcfarland 0,5. the samples were divided into 5 groups each consisted of 6 samples i.e: positive control was given nystatin oral solution 100.000 iu, negative control was given dmso 1%, treatment group were given sticophus hermanii and holothuria atra extract separately, each diluted by dmso 1% with concentration of 20%, 40% and 80%. antifungal activity test was performed by disk diffusion method on mueller hinton agar. fungal supension of candida albican sequal to 0,5mc farland was swabbed on to muller hinton agar plate. sterile paper disks were immersed for 15 second into each concentration of extracts for treatment groups, for control negative groups in dmso 1%, each, and for the positive control group in nystatin oral solution, then put on to muller hinton agar, gently pressed for a while and leave, incubated in 37°c for 48 hours. the clear zone around the disk showed inhibition effect to the growth of candida albicans. diameter of inhibition zone was measured with digital caliper. t h e m t t 3 ( 4 , 5 d i m e t h y l t h i a l 2 y l ) 2 , 5-diphenyltetrazalium bromide) cytotoxicity test is tests for in vitro cytotoxicity, was performed to evaluate the viability of gingiva-derived mesenchymal stem cell after treated with sticophus hermanii and holothuria atra extracts which diluted with dmso 1% into 9 groups concentration of 1%, 0,5%; 0,25%; 0,125%, 0,006%; 0,003%, 0,0015%, 0,0007%; 0,0003%. the gingiva-derived mesenchymal stem cellwere obtained from itd, surabaya. after the thawing process, the cell were resuspended with culture medium of α alpha minimum essential medium (mem) (gibco, invitrogen co, new york, usa), sentrifuged for 5 minute in 1500 rpm, repeated for 3 times then get cultured, passaged every 4 days. in the second passage monolayer was formed and ready to be performed the cytotoxicity test. the cells were seeded into 96-well microplates (iwaki, asahi glass co, tokyo, japan) each containing 200 µlwit the density of 5 × 104 in α mem medium incubated at 37° c for 24 hours. medium then replaced then extract were added as amount of 200 µl and incubated 37° c for 20 hours. control positive cells were also prepared containing cells in culture medium, assumed to be viable 100%. microplates then taken out from the incubator, added the solution of mtt 5 mg/ml in pbs 25 µl for each well, incubated for 4 hour. the medium then discarded, replaced with 200 µl dmso in each well. cell viability was assessed by the mitochondrial dependent reduction of yellow mtt (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) to purple formazan. after the exposure, the formazan formations were determined for each treatment concentration by elisa reader at a wavelength of 595 nm. the relative viability of the treated cells as compared to the control cells were expressed as the % cytoviability, using the following formula: % cell viability = [asample/acontrol] x 100% note: a sample is mean value of the measured optical density of the treated cells;a control is mean value of them easured optical density of the control cells.16-18 the data were presented as means + standard deviation (sd). statistical analysis was performed using analysis of variance (anova) to determine the effect of sea cucumber extract concentration on the gingiva-derived mesechymal stem cells cytoviability. results antifungal activity showed by the inhibition zones around the disk were observed in all treatment groups and positive control but not the negative control. all treatment groups in all concentrations showed inhibition zones but the diameter were less than nystatin as positive control. both sticophus hermanii and holothuria atra extracts showed that the increasing concentration tested on candida albicans resulted in the increasing diameter of inhibition zone as shown in figure 1. further statistical analysis by two way anova test and lsd multiple comparison test at 5% significance level presented in table 1 showed the significant difference on all concentration of sticophus hermanii and holothuria atra extract compared to negative and positive control group (p<0.05). result on cytotoxicity test showed that all concentrations of sticophus hermanii extract were not cytotoxic to gingivaderived mesenchymal stem, showed by the average of cell viability above 50%, while holothuria atra extract were 221parisihni dan revianti: antifungal effect of sticophus hermanii and holothuria atra extract figure 2. cytoviability percentage of gingiva-derived mesenchymal stem cell treated by various concentration of sticophus hermanii and holothuria atra extract. figure 1. the inhibition zone of sticophus hermanii and holothuria atra extracts on serial concentration to candida albicans compared to control groups. table 2. anova summary of cytotoxicity of sticophus hermanii and holothuria atra extracts concentrations on gingiva-derived mesenchymal stem cell group f sig sticophus hermanii extract between groups 2.056 0.086 within groups total holothuriaatra extract between groups 20.326 0.000 within groups total cytotoxic on the concentration of 1% and not cytotoxic on the concentration below. the result of relative viability of the treated cells as compared to the control cells was showed in figure 2. the increased concentration of the sticophus hermanii and holothuria atra extract exposure on the cells resulted in the decreasing of cytoviability percentage of gingivatable 1. anova and lsd summary of inhibition zone of sticophus hermanii and holothuria atra extracts on serial concentration extracts to candida albicans compared to control groups group aquaeous nystatin holothuria atra 20% holothuria atra 40% holothuria atra 80% stichopus hermanii 20% stichopus hermanii 40% stichopus hermanii 80% aquaeous 0.000* 0.032* 0.001* 0.002* 0.117 0.006* 0.000* nystatin 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* holothuria atra 20% 0.238 0.307 0.547 0.53 0.023* holothuria atra 40% 0.872 0.077 0.578 0.263 holothuria atra 80% 0.107 0.692 0.201 stichopus hermanii 20% 0.221 0.005* stichopus hermanii 40% 0.096 derived mesenchymal stem cell. the less concentrations of the two sea cucumber extract applied to gingiva-derived mesenchymal stem cell, the more cells viability increased. further statistical analysis by anova as shown in table 2 described that there was not any significant influence of the treated extracts concentration on the cytoviability of gingiva-derived mesenchymal stem cell (p>0.05). sticophus hermanii extracts showed no cytotoxic effect where the cells were viable more than 50% after the treatment in all extract concentrations (p>0.05). holothuria atra showed cytotoxic effect to the gingiva-derived mesenchymal stem cellin concentration of 1% where the the viable cells appeared less than 50% after the treatment, but it showed no cytotoxic effect on the treatment of the extract in lower concentration of 0.5-0.0001% (p<0.05). discussion result of antifungal sensitivity test showed inhibition zone around the disk in all treatment group and in control positive group, means that both extract of sticophus in hi bi ti on z on e c yt ov ia bi li ty 222 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 218–223 hermanii and holothuria atra has antifungal effect to c. albicans in vitro.the largest diameter of inhibition zone of treatment group was in the concentration of 80 mg/ml but still less than in the control group of nystatin (p<0.05). nystatin is a polyene antifungal drug to which many molds and yeasts are sensitive, including candida spp, used as the positive control for its the common topical antifungal agent therapy on oral candidiasis. nystatin exerts its antifungal activity by binding to ergosterol found in fungal cell membranes. binding to ergosterol causes the formation of pores in the membrane. potassium and other cellular constituents leak from the pores causing cell death.12,13 sea cucumber extract have been known to have the antifungal property, assumed to be related to its content of alcaloid, saponin and triterpen glycoside.1,4,9,11result showed both sticophus hermanii and holothuria atra has antifungal activity against c. albicans according to the study stated that generally, most species of sea cucumber share the same bioactive compound mentioned above but in different level contain.4,7 saponin were identified in the content of sea cucumber extract.1,4,9 it is secondary metabolites of glycosidic nature widely distributed in higher plants and marine invertebrates resulted as the defend mechanism also has the biological properties i.e. the ability to lyse erythrocytes or to foam. it form complexes with cell membrane cholesterol leading in consequence to pore formation and cell permeabilization, alterations in the negatively charged carbohydrate portions on the cell surface.24,25 saponin performed its antifungal activity by the interaction with sterol membrane of c. albicans and disrupting the cell wall‘s integrity caused the cell death, similar with the mechanism action of nystatin. sticophus hermanii and holothuria atra have been extracted by methanolic extract.14 the antibacterial compound of sea cucumber assumed to be polar for it is dissolved in methanol solvent and have been proven to have the antibacterial, antifungal and cytotoxic agent on some studies.2,3,4,9 regarding to antifungal property of both sea cucumber extract to c. albicans, cytotoxicity test must be performed to examine its biocompatibility prior to explore its potency as treatment in oral candidiasis. cell culture can be used to screen for toxicity both by estimation of the basal functions of the cell or by tests on specialized cell functions.19,20 recently, toxicity test have been developed and stem cells were explored regarding to some basic consideration in some advantage in the technique and result. human stem cells are potentially attractive reagents for predictive toxicology, particularly if they can be shown to be a reliable, large-scale source of differentiated human cells. the use of human cells could increase the correlation between safety studies and clinical trials, an important benefit since conventional animal models of toxicity are not always predictive of human responses. stem cells that are generated from adult tissues (ips cells) could allow models to be created from individuals with a diverse range of drug susceptibilities, resistances or disease, which could reduce the rate of adverse effects within patient subpopulations.21 in this research, cytotoxicity test of sticophus hermanii and holothuria atra extracts were performed on gingiva-derived mesenchymal stem cell. gingiva-derived mesenchymal stem cells (gmscs) were employed on this study considering to its accessibility and potency for further application in oral theraphy. gmsc are stem cells from human gingiva, a tissue source easily accessible from the oral cavity, which exhibited clonogenicity, selfrenewal, and multipotent differentiation capacities. most importantly, gmscs were capable of immunomodulatory functions and able to induce osteogenic, chondrogenic, and adipogenic differentiation.22,25 the cytotoxicity test of sea cucumber extract on gmsc will be a proper predictor regarding to the potency of the cell. once the extract is not cytotoxic to gmsc the greater possibility of oral application without degrading the potency of stem cell attained. cytotoxicity testing includes numerous methods, both qualitative and quantitative. in this study we used indirect test, in which the rate of cell growth (cell number) and the metabolic activity (mtt) have indicated the degree of cytotoxicity of sea cucumber extract. result showed cytotoxic activity on gingiva-derived mesenchymal stem cell after treated with holothuria atra extract in concentration of 1%, shown by the cell viability less than 50% (p<0.05). the cytotoxic effect to gingiva-derived mesenchymal stem cellassumed related to the content of saponin in sea cucumber extract. it has been stated that the bioactive compound of holothuria atra are mostly triterpene glycoside (saponin). saponins are secondary metabolites of glycosidic nature widely distributed in higher plants but also found in some animal sources, like e.g. marine invertebrates. saponins have large structural diversity, but these compounds share some unique biological properties like the ability to lyse erythrocytes or to foam. the latter contributed to naming this group saponins, which is derived from latin sapo meaning soap. haemolysis of red blood cells seems to result from saponin ability to form complexes with cell membrane cholesterol leading in consequence to pore formation and cell permeabilization, and also to cause alterations in the negatively charged carbohydrate portions on the cell surface. surface activity responsible for foaming properties, as well as some other biological functions including haemolytic activity, are attributed to characteristic structural features of saponins and their amphiphilic nature which results from the presence of a hydrophilic sugar moiety and a hydrophobic genin (called sapogenin). it seems that for all saponins both aglycone and sugar part play an important role for cytotoxic activity. with respect to cytotoxic mechanisms of bothtriterpene and steroid saponins a wide variety of these was reported. cytotoxic effect of most of the reviewed saponins was due to their ability to stimulate apoptotic process in tumor cells, usually through its intrinsic pathway. moreover, non apoptotic processes were also involved in saponin cytotoxic 223parisihni dan revianti: antifungal effect of sticophus hermanii and holothuria atra extract activity, such as cell cycle arrestment, autophagic cell death stimulation, inhibiting of metastasis and cytoskeleton disintegration.23,24 result showed no cytotoxic activity on gingiva-derived mesenchymal stem cell after treated with all concentration of sticophus hermanii extract (p>0.05). regarding to its bioactive component, the saponin content probably have less role than in holothuria atra extract, but prominently it has been known that sticophus hermanii mostly contained polyunsaturated fatty acids (pufa): arachidonic acid (aa c20:4 n-6), eicosapentaenoic acid (epa c20:5 n-3), docosa hexaenoic acid (dha c22:6 n-3)4. it has been stated that fatty acids including arachidonic acid (aa c20:4), eicosa pentaenoic acid (epa c20:5), and docosa hexaenoic acid (dha c22:6) can play a potential role in tissue repair and wound healing. an appreciable amount of epa in sea cucumbers might be linked well with the ability of these echinoderms to initiate tissue repair.4 it related to the traditional medicine which believed that direct use of sea cucumber can reduce wound recovery time and help new tissue formation and regeneration in human just as the sea cucumber’s abilityto quickly regenerate its own body tissue when damaged. the conclusion of this research is that stichopus hermanii and holothuria atra extract had the antifungal effect against candida albicans on the concentration of 80 mg/ml. sea cucumber extract were not cytotoxic to gingiva-derived mesenchymal stem cell in the concentration of sticophus hermanii ≤1% and holothuria atra ≤0.5%. acknowledgement this research was supported by a grant from fundamental research program, funded by ministry of education and culture indonesia. references 1. mayer ams, rodrigues ad, berlinck rgs, hamann mt. marine pharmacology in 2005-6: marine compounds with anthelminthic, antibacter ial, anticoagulant, antifungal, anti-inf lam mator y, antimalarial, antiprotozoal, antituberculosis, antiviral activities; affecting the cardiovascular immune and nervous system and other miscelanous mechanism of action. biochim biophys acta 2009;1790(5) : 283-308. 2. abraham tj, nagarajan j, shanmugam sa. antimicrobial substances of potential biomedical importance from holothurian species. indian j marine sci 2002; 31(2):161-4. 3. althunibat oy,hashim r, taher m, daud jm, ikeda ma, zali bi. in vitro antioxidant and antiproliferative activities of three malaysian sea cucumber species. eur j sci res 2009; 37(3): 376-87. 4. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods-a review mar. drugs 2011; 9(10): 1761-805. 5. dang nh, thanh nv, kiem pv, huong le m, minh cv, kim yh. two new triterpen glycosides from the vietnamese sea cucumber holothuriascabra. arch pharm res 2007; 30(11): 1387-91. 6. farouk ae, ghouse fah, ridzwan bh. new bacterial species isolated from malaysian sea cucumbers with optimized secreted antibacterial activity. am j biochemistry and biotechnology 2007; 3(2): 60. 7. sendih sg. keajaiban teripang penyembuh mujarab dari laut. jakarta: agromedia pustaka; 2006. p.13-45. 8. pringgenies d, ali ridlo, kemal taj. the potency antibacterial of bioactive compound of holothuria atra extract from territorial water of bandengan. world ocean conference. manado 2009. 9. pranoto en, maruf ws, pringgenies d. kajian aktivitas bioaktif ekstrak teripang pasir (holothuriascabra) terhadap jamur candida albicans. jurnal pengolahan dan bioteknologi hasil perikanan 2012; 1(1): 1-8. 10. a m i n i n dl , p i neg i n bv, p ichug i na lv, z ap oroz het s ts, agafonova ig, boguslavski vm, silchenko as, avilov sa. stonik va. immuno modulatory properties of cumaside. international immunopharmacology 2006; 6: 1070-82. 11. han h, yi yh, li l, liu bs, la mp, zhang hw. antifungal active triterpene glycosides from sea cucumber holothuriascabra. yao xuexuebao 2009;44(6):620-4. 12. williams d, lewis m. pathogenesis and treatment of oral candidosis. j oral microbiol 2011; 3. 13. shalini k, kumar n, drabu s, sharma pk. advances in synthetic approach toand antifungal activity of triazoles. beilstein j org chem 2011; 7 : 668–677 14. pringgenies d, ocky kr, sabdono a, hartati r, widianingsih. penerapan teknologi budidaya teripang dalam meningkatkan produksinya dan bioprospek teripang sebagai sumber senyawa antimikroba untuk kesehatan. laporan penelitian. hibah kemitraan hi-link; 2008. p. 65. 15. gallagher me. toxicity testing requirements, methods and proposed alternatives. evirons 2003; 26(2): 253-73. 16. sunarintyas s, siswomihardjo w, tontowi ae. cytotoxicity of criculatriphenestrata cocoon extract on human fibroblasts. int j biomater 2012; 2012: 493075. 17. dufrane d, cornu o, verraes t, schecroun n, banse x, schneider yj, delloye c. in vitro evaluation of acute cytotoxicity of human chemically treated allografts. eur cell mater 2001; 1: 52-8. 18. cao t, saw ty, heng bc, liu h, yap au, ng ml. comparison of different test models for the assessment of cytotoxicity of composite resins. j appl toxicol 2005; 25(2): 101-8. 19. lausriat d, gide j, peschanski m. human pluripotent stem cells in drug discovery and predictive toxicology. biochem soc trans 2010; 38(4): 1051-7. 20. mitrano ti, grob ms, carrión f, nova-lamperti e, luz pa, fierro fs,quintero a, chaparro a, sanz a. culture and characterization of mesenchymal stem cells from human gingival tissue. j periodontol 2010; 81(6): 917-25. 21. jeon km. international review of cell and molecular biology. 1st ed. san diego: elsevier academic press; 2009. p. 161-202. 22. junqueira c, carneiro j. basic histology: text and atlas. 11th ed. philadelphia: mcgraw-hill; 2005. p. 106-7. 23. z ha ng q, sh i s, liu y, uya n ne j, sh i y, sh i s, l e a d. mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. j immunol 2009; 183(12): 7787–98. 24. podolak i, galanty a, sobolewska d. saponins as cytotoxic agents: a review. phytochem rev 2010; 9(3): 425-74. 25. wojtkielewicz a, długosz m, maj j, morzycki jw, nowakowski m, renkiewicz j, strnad m, swaczynová j, wilczewska az, wójcik j. newanalogues of the potentcy to toxic saponin osw-1. j med chem 2007; 50(15): 3667-73. �� vol. 45. no. 1 march 2012 antimicrobial effect of chlorine dioxide on actinobacillus actinomycetemcomitans in diabetes mellitus rats treated with insulin tantin ermawati1 and kwartarini murdiastuti2 1 department of biomedicine, faculty of dentistry, jember university, jember indonesia 2 department of periodontics, faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: periodontitis is a chronic inflammatory disease of periodontal tissues. etiology of periodontal disease includes actinobacillus actinomycetemcomitans (a. actinomycetemcomitans) which is the most predominant disease-causing bacteria found in the gingival sulcus. periodontitis can be exacerbated by the systemic disease, such as diabetes mellitus considered as a metabolic disease characterized by hyperglycemia due to insulin deficiency. treatment of periodontitis is then required in patients with type i diabetes to avoid radical reaction that can not only cause bleeding, but can also prevent infection, as a result, topical antimicrobial therapy and blood glucose control are required. topical antimicrobial chlorine dioxide is a disinfectant that is effective in killing a. actinomycetemcomitans. purpose: this study is aimed to determine the effects of topical antimicrobial chlorine dioxide gel or rinse on the number of a. actinomycetemcomitans in dm rats treated with insulin. methods: 20 three month old male wistar rats with weight of 170–200 grams were divided into four groups. first, periodontitis and dm were manipulated into all groups through aloksan injection with dose of 170 mg/kg. those rats in group i were treated with insulin and chlorine dioxide gel, those in group ii were treated with insulin and chlorine dioxide rinse, those in group iii were treated with insulin only, and those in group iv were without treatment. in the third and seventh weeks, the number of a. actinomycetemcomitans was measured. the data was tested by using one-way anova test followed by lsd test. results: the study showed that chlorine dioxide gel has a greater ability in reducing the number of a. actinomycetemcomitans than chlorine dioxide rinse although both are antimicrobials. conclusion: it can be concluded that the use of chlorine dioxide gel can more effective to decrease the number of a. actinomycetemcomitans than chlorine dioxide rinse in dm rats treated with insulin therapy. key words: periodontitis, actinobacillus actinomycetemcomitans, diabetes mellitus, insulin, chlorine dioxide abstrak latar belakang: periodontitis adalah suatu penyakit inflamasi kronis jaringan periodontal. etiologi penyakit periodontal diantaranya adalah a. actinomycetemcomitans yang merupakan bakteri paling dominan penyebab penyakit yang terdapat padaa. actinomycetemcomitans yang merupakan bakteri paling dominan penyebab penyakit yang terdapat pada sulkus gingiva. periodontitis yang terjadi diperparah adanya penyakit sistemik yaitu diabetes mellitus (dm) yang merupakan penyakit metabolik yang ditandai dengan hiperglikemi akibat defisiensi insulin. perawatan periodontitis pada penderita dm tipe i adalah untuk perawatan periodontitis pada penderita dm tipe i adalah untuk menghindari tindakan radikal yang dapat menyebabkan perdarahan dan mencegah terjadinya infeksi, sehingga digunakan terapi antimikroba topikal serta kontrol glukosa darah. antimikroba topikal chlorine dioxide merupakan desinfektan yang efektif dalam membunuh a. actinomycetemcomitans. tujuan: penelitian ini bertujuan untuk mengetahui efek antimikroba topikal chlorine dioxide gel atau rinse terhadap jumlah a. actinomycetemcomitans pada tikus dm dengan insulin. metode: dua puluh ekor tikus wistar, jantan, usia 3 bulan, berat 170–200 gram, dibagi menjadi empat kelompok. semua kelompok sebelumnya dimanipulasi periodontitis serta dm dengan injeksi aloksan dosis 170 mg/kgbb. kelompok i adalah tikus yang diterapi insulin dan chlorine dioxide gel, kelompok ii diterapi insulin dan chlorine dioxide rinse, kelompok iii diterapi insulin, serta kelompok iv adalah tikus tanpa dilakukan terapi apapun. pada minggu ke-3 dan ke-7 dilakukan penghitungan jumlah a. actinomycetemcomitans. hasil pengukuran di analisis menggunakan uji anava 1 jalur dilanjutkan dengan uji lsd. hasil: penelitian menunjukkan bahwa chlorine dioxide gel mempunyai kemampuan lebih besar dalam menurunkan jumlah a. actinomycetemcomitans dibandingkan chlorine dioxide rinse, walaupun keduanya bersifat antimikroba. research report ��ermawati and murdiastuti: antimicrobial effect of chlorine dioxide on actinobacillus actinomycetemcomitans kesimpulan: dapat disimpulkan bahwa pemberian chlorine dioxide gel lebih efektif menurunkan jumlah a. actinomycetemcomitans dibandingkan kelompok yang diberi chlorine dioxide rinse pada tikus dm dengan terapi insulin. kata kunci: periodontitis, actinobacillus actinomycetemcomitans, diabetes mellitus, insulin, chlorine dioxide correspondence: tantin ermawati, c/o: departemen biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: tantin.ermawati@gmail.com. telp. (0331) 333536. introduction periodontitis is common disease in oral cavity and often associated with various diseases is periodontitis. periodontitis can be defined as an infectious disease leading to local inflammation of tissues supporting teeth that later can cause the destruction of periodontal ligament and alveolar bone.1 bacteria that have a major role on such periodontal tissue destruction are actinobacillus actinomycetemcomitans (a. actinomycetemcomitans), porphyromonas gingivalis (p. gingivalis), prevotella intermedia, and fusobacterium nucleatum.2 early symptoms of periodontitis is caused by the colonization of pathogenic species in periodontal tissue. the fact that bacteria or their products move into the tissue then can cause destruction due to their direct interaction with hospes cells.3 periodontal disease can be exacerbated by systemic diseases, such as diabetes mellitus (dm). the severity of periodontitis in patients with dm is greater than those wihout dm, especially with poor glycemic control, indicated by the increasing of plaque index, gingival index, probing depth, attachment damage, and dental loss. these conditions of patients with dm then lead to the decreasing function of polymorphonuclear (pmn) that can increase the severity of periodontal tissue destruction. under these conditions, there will be impaired fatty acid metabolism later that causes the damage of the cell functions and homeostasis.4,5 as a result, the content of glucose in gingival crevicular fluid of dm patients is higher than in that of non-dm ones. this increasing then may not only alter the microflora environment of oral cavity, and but also be considered as a good growing medium for bacteria, such as a. actinomycetemcomitans. bacteria are actually also found in non-dm patients, but their number is lower than in dm ones.6 the level of virulence of a. actinomycetemcomitans, furthermore, is characterized by the presence of inflammation modulation, tissue damage induction, and tissue repair inhibition. a. actinomycetemcomitans actually produces a virulence factor by producing leucotoxin in the form of lipoproteins that are secreted, especially for killing pmn and macrophages by producing citolethal distending toxin (cdt).7,8 the purpose of periodontitis treatment in type i dm patients is not only to avoid radical reaction that can cause bleeding, but also to prevent infection either by eliminating plaque and calculus, or by using antibacterial drugs and blood glucose control.9 thus, systemic therapy for type i dm patients is insulin therapy that can make blood glucose level normal. dm rats treated with insulin can decrease their blood glucose levels close to normal and also the number of bacterial colonies of a. actinomycetemcomitans in the gingival sulcus fluid. it means that there is an improvement for periodontal condition, but the healing process of the tissue takes longer time. thus, antimicrobial therapy is required to accelerate the improvement of the periodontal tissue conditions.10,11 topical antimicrobial drug used is chlorine dioxide. this antimicrobial material is effective to kill bacteria in oral cavity, especially gram-negative bacteria causing periodontitis. chlorine dioxide used for periodontal therapy is offered in the forms of gel, rinse, solution, or paste. however, antimicrobial therapy used is often in the form of gel or rinse.12 therefore, this study is aimed to determine the effects of topical antimicrobial agents, chlorine dioxide gel or rinse, on the number of a. actinomycetemcomitans in diabetes mellitus rats treated with insulin therapy. materials and methods this study is a experimental study using 20 three-month old wistar rats with weighing between 170–200 grams. to create periodontitis condition, all of those rats were then bond around the cervical of their lower incisors for 7 days. to prevent the loss of the binding, the thread was sewed on the gingiva. after 7 days, the thread was removed and evaluated in order to know whether there had been any signs of periodontitis involving the changes of tooth servical color into darker red (compared with the normal one, pink), the enlargement or swelling of the gingiva, and the increasing of the depth of the gingival sulcus. the identification of bacteria was then conducted by using sample cultures of pure a. actinomycetemcomitans (lab. veterianary faculty, gadjah mada university). the medium used was a modified agar-tood hewit broth + bacitracin 5 units/ml media, in which biochemical test was then conducted for identifying a. actinomycetemcomitans. diabetic rats were then obtained by injecting aloxan with dose of 170 mg/kg dissolved in 0.9% nacl (physiological saline), and then given subcutaneously. next, those rats were fasted for 12 hours before and after the injection of aloksan.13 �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 22–27 indicated suffered from dm. those rats in group i were then injected with insulin (5 iu dose of suspension-insulin isophan) subcutaneously every day for 28 days, and then treated with chlorine dioxide gel (oxygene dental gel) with dose of 0.27 ml in the gingival sulcus. meanwhile, those rats in group ii were injected with insulin, and then treated with chlorine dioxide rinse (oxygene mouthrinse) with dose of 0.27 ml. those rats in group iii, moreover, were injected with insulin, while those in group iv, as control group, were not treated with any therapies. the number of bacteria was measured twice, in the third week when those rats were indicated to suffer from dm rats (before being treated with any therapies), and in the seventh week after the rats were treated. initially, those rats were anesthetized by using ketamine, and then a sterile paperpoint was inserted into the gingival sulcus of the incisors for 10 seconds. samples were suspended into vortex for 10 seconds. each 0.1 ml of the solution was diluted into 10-2 ml. 1 ml of the dilution result was then planted in agar tood-hewit broth + bacitracin 5 units/ml (modification) media. next it is known that small round-shaped, convex, and translucent white color a. actinomycetemcomitans attached to agar tood-hewit broth + bacitracin 5 units/ml (modification) media. then, table 1. the mean and standard deviation of the number of a. actinomycetemcomitans (cfu/ml) based on the treatment groups and the observation week groups the mean and standard deviation of the number of a. actinomycetemcomitans decreasing week 3 week 7 i 65.000 ± 28.133 1.000 ± 1.414 64.000 ± 28.204 ii 58.200 ± 30.449 28.000 ± 5.147 30.200 ± 25.606 iii 70.000 ± 16.324 51.000 ± 12.708 19.000 ± 13.583 iv 109.400 ± 18.270 198.800 ± 8.467 -89.400 ± 20.354 note: group i : dm rats treated with insulin + chlorine dioxide gel group ii : dm rats treated with insulin + chlorine dioxide rinse group iii : dm rats treated only with insulin group iv : dm rats treated without any therapy those rats were then divided into four groups, each of which consisted of 5 rats (group i, ii, iii, iv) randomly. after 2 weeks, blood glucose levels were measured. when blood glucose levels had reached ≥ 250 mg/dl, then the rats figure 1. the colonies of a. actinomycetemcomitans after 28 days of the treatment. note: a) the colonies of a. actinomycetemcomitans in dm rats treated with insulin and chlorine dioxide gel; b) the colonies of a. actinomycetemcomitans in dm rats treated with insulin and chlorine dioxide rinse; c) the colonies of a. actinomycetemcomitans in dm rats treated without any therapy. b c a ��ermawati and murdiastuti: antimicrobial effect of chlorine dioxide on actinobacillus actinomycetemcomitans to simplify the calculation process of the number of a. actinomycetemcomitans, each petridish was divided into four parts. the number of bacteria was then calculated in each petridish. data obtained from the observations were finally analyzed by using one-way anova test and then with least significant different (lsd) test. results calculating the number of a. actinomycetemcomitans in dm rats was conducted twice, in the third week (before the therapy) and in the seventh week (after the therapy). the purpose of this calculating of the number of the bacteria is to determine the effects of insulin therapy using chlorine dioxide gel or rinse on the number of a. actinomycetemcomitans. the morphology of a. actinomycetemcomitans is round, slightly convex, and transparent white color as seen in each treatment group in figure 1. the mean of the calculating results of the number of a. actinomycetemcomitans can be seen in table 1. the results show that there were the differences of the number of a. actinomycetemcomitans in the group treated with insulin and chlorine dioxide gel, that treated with insulin and chlorine dioxide rinse, and that treated with insulin therapy alone. the mean values of the reduction were 64.000 cfu/ml; 30.200 cfu/ml; and 19.000 cfu/ ml. unlike those groups, group iv had a mean value of the increasing number of the bacteria about 89.400 cfu/ml indicating that those dm rats with treatments in group i, ii, and iii had lower number of the bacteria than those without any treatment. to determine the effects of the therapy given to the number of a. actinomycetemcomitans in the gingival sulcus, one-way anova test was conducted as shown in table 2. the statistical test results then showed that there was the highly significant difference of the decreasing number of a. actinomycetemcomitans in the gingival sulcus (p <0.05) among those different treatment groups. next, lsd test was conducted to determine different effects occurred in each treatment group (table 3). based on the obtained results of lsd test, it is known that there was significant difference of the mean of the decreasing number of a. actinomycetemcomitans among the groups in the third week and in the seventh week (p<0.05), except in the group (ii-iii), since the mean obtained was not significantly different. discussion based on the data obtained in this study, it is known that the severity of periodontal disease that occurred in those rats was caused by systemic diseases, namely dm. although it is known that the main factor of periodontal disease was bacterial plaque, the presence of systemic disease could also decrease the immunity of oral cavity, so it could affect the progression of periodontal disease.3 those dm rats were then treated with systemic therapies, which were insulin therapy and topical antimicrobial therapy by using chlorine dioxide gel or chlorine dioxide rinse. the working mechanism of insulin was that after being synthesized, insulin was secreted into the blood circulation in the free forms, and then moved to the target cells. insulin actually would work after binding to specific receptors. these insulin receptors have two main functions: to distinguish other ingredients from insulin, and then to tie them fast and reversibly, to form complex insulin-receptor formation which could stimulate a series of intracellular events leading to the cellular effects of characteristic insulin.14 insulin therapy given regularly to those dm rats could make their metabolism and glucose transport smooth, so their blood glucose levels could be normal and be within safe limits. those dm rats which were not treated with insulin therapy would get hyperglycemia condition resulting in the formation of advanced glycation end-product (ages) leading to endothelial oxidative stress, so the disruption of blood vessels in periodontal tissues could occur. without insulin therapy, people with diabetes will easily get infection because there have been damages to the function of neutrophils and monocytes, hypofunction of immune cells, and neuropathy (micro and macro circulation disorders) exacerbating periodontal disease. therefore, blood glucose control with insulin therapy can prevent infection and complication.15,16 insulin therapy actually also plays a role in lowering the number of a. actinomycetemcomitans since with a controlled blood glucose levels the function of leukocytes (pmn) and macrophages as body defense could become normal, so the metabolism of the body could become normal again. this table 2. the summary of test results of one-way anova concerning with the decreasing of the number of a. actinomycetemcomitans source of variance jk dk ms f p inter groups 66098.950 3 22032.983 42.991 .001* within groups 8200.000 16 512.500 total 74298.950 19 note: jk = sum of square, dk = degree of freedom, ms = variance, p = level of significance; * = significant table 3. the summary of lsd test results concerning with the decreasing of the number of a. actinomycetemcomitans groups i ii iii iv i – 33.800* 45.000* 153.400* ii – 11.200 119.600* iii – 108.400* iv – note: * = significant �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 22–27 condition then causes a. actinomycetemcomitans become blocked and lack of supports from environment, so its number in the gingival sulcus become lowered. thus, it indicates that the systemic insulin therapy conducted could also improve the health condition and periodontal tissues of those dm rats, but it would require very long time. besides that, periodontal treatment given to people with diabetes could also avoid radical acts lead to infection. therefore, local therapy was conducted by using topical antimicrobial agents, chlorine dioxide gel and chlorine dioxide rinse. these materials are actually the most widely used disinfectants for periodontitis therapy.11,17 chlorine dioxide gel and rinse actually contain the same basic material, chlorine dioxide. chlorine dioxide is antimicrobial, the base material of gel and rinse with high redox capacity.17 it is known that there was the difference of the reduction of the number of a. actinomycetemcomitans between in chlorine dioxide gel and in chlorine dioxide rinse (table 3). this difference may be caused by the fact that chlorine dioxide therapy is actually more effective in the form of gel applied into the periodontal pocket. another reason may also be caused by the fact that chlorine dioxide gel containing chlorine dioxide (stabilized chlorine dioxide) can kill pathogenic bacteria, especially a. actinomycetemcomitans. the working mechanism of chlorine dioxide, furthermore, causes reaction with natural organic substances contained in the cell walls of bacteria leading to the impaired cellular mechanisms of those bacteria. it is because chlorine dioxide reacts directly with amino acids and rna causing the inhibition of protein production in bacterial cells. chlorine dioxide then affects the bacterial cell membrane by altering protein and fat membranes as well as by inhibiting the process of bacterial respiration.18 this fact is also supported by a research conducted by bayaty et al.,19 showing that topical antimicrobial agent, chlorine dioxide gel, is very effective for killing aerobic and anaerobic bacteria in either supra or subgingival plaque. there are two reasons explaining how chlorine dioxide gel can kill bacteria or viruses. first, chlorine dioxide gel can react with specific biomolecules. second, the effects of chlorine dioxide gel on bacteria are through physiological function since chlorine dioxide gel is a topical antimicrobial agent that is very powerful in killing plaque bacteria. therefore, the topical antimicrobial agent, chlorine dioxide gel, is better in reducing the number of a. actinomycetemcomitans that chlorine dioxide rinse. based on its application mechanism, this gel material gives greater advantages in its use that is more resistant in pockets because its consistency is more concentrated towards the flow of gingival and saliva creviculer fluids due to oral activities, such as mastication, speaking, and so forth. the ability of periodotophatic bacteria, especially a. actinomycetemcomitans, in conducting tissue attachment and penetration is actually very high, and is even able to penetrate into the gingival connective tissue. it then leads to more resistant periodontal disease. therefore, chlorine dioxide therapy in the form of gel is considered as disinfectant that either can be resorbed or can kill bacteria inside and outside the tissues, so the reaction of the material with bacteria is more increasing, and then can damage bacterial cell membranes.12,20 chlorine dioxide rinse, on the other hand, can neutralize vscs in oral cavity. amino acids, cysteine and methionin (precursors of vscs), can even be removed by oxidation reactions. chemically, chlorine dioxide produces oxygen that can reduce both vscs production and bad breath by breaking the bonds of sulfide hydrogen and mercaptan methyl.21 chlorine dioxide rinse is applied into the gingival sulcus by spraying, so the material will be more soluble due to the flow of gingival and saliva creviculer fluids which generally have a short duration. this then causes chlorine dioxide rinse has limited ability to kill bacteria. according to greenstein et al.,22 the use of antimicrobials in the form of mouth rinse is less effective not only because it is difficult for the antimicrobial agent to achieve the gingival sulcus or periodontal pocket, but also because the agent will be quickly cleared by gingival or saliva creviculer fluids from the mucosal surface. this then causes chlorine dioxide in the form of rinse is less effective in killing a. actinomycetemcomitans than that in the form of gel. it can be concluded that the induction of topical antimicrobial chlorine dioxide gel can reduce the number of a. actinomycetemcomitans more than the induction of chlorine dioxide rinse in dm rats treated with insulin therapy. references 1. draidi yma. differences in amount & architecture of alveolar bone loss in chronic & aggressive periodontitis assessed throught panoramic radiographs. pakistan oral and dental journal 2009; 29(1): 59–62. 2. amel y. microbiological study of periodontitis in the west of algeria. world journal of medical science 2010; 5(1): 7–12. 3. newman mg, ta kei hh, ca r ranza fa. ca r ranza’s clinical periodontology. 9th ed. philadelphia: wb saunders co; 2002. p. 67–8. 4. oedijani. mekanisme biokimia dan biomolekular komplikasi diabetes mellitus dan periodontitis. jurnal kedokteran gigi ui 2003; 10(edisi khusus): 578–85. 5. btaiche, alaniz. hyperglycemia and infection in critically iii patients: pathogenesis of hyperglycemia-associated infection. pharmacotherapy 2005; 25(7): 963–97. 6. herlina, hernawan i. hubungan antara kadar glukosa darah dan kadar glukosa saliva pada penderita diabetes mellitus. maj ked gigi (dent j) 2003; 36(2): 64–7i. 7. henderson b, wilson m, sharp l, ward jm. actinobacillus actinomycetemcomitans. j med microbiol 2002; 51(12): 1013-20. 8. kaplan jb, schreiner hc, furgang d, fine dh. population a structure and genetic diversity of actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients. j of clin microbiol 2002; 40(4): 1181–7. 9. southerland jh, taylor gw, offenbacher s. diabetes and periodontal infection: making the connection. clinical diabetes 2005; 23: 171–8. 10. suwandi t. efek klinis aplikasi subgingival racikan gel metronidazole 25% dan larutan povidone-iodine 10% sebagai terapi penunjang skaling-penghalusan a ka r pada per iodontitis k ronis. jur nal kedokteran gigi ui 2003; 10(edisi khusus): 669–74. ��ermawati and murdiastuti: antimicrobial effect of chlorine dioxide on actinobacillus actinomycetemcomitans 11. nur a. pengaruh terapi insulin pada tikus dengan diabetes melitus terhadap jumlah koloni actinobacillus actinomycetemcomitans dalam sulkus gingiva. thesis. yogyakarta: program pascasarjana ugm; 2003. 12. krismariono a. perawatan infeksi periodontal dengan pemberian antibiotik secara local. jurnal pdgi 2005; 55(edisi khusus): 37–42. 13. jelodar ga, maleki m, motadayen mh, sirus s. effect of fenugreek, onion and garlic on blood glucose and histopathology of pancreas of alloxan-induced diabetic rats. j indian med sci 2005; 59(2): 64–9. 14. bowen r. physiologic effects of insulin. 2009. available at: http:// www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/ insulin_phys.html accessed march 4, 2012. 15. rodrigues dc, taba m, novaes ab, souza sls, grisi mfm. effect of non-surgical periodontal therapy on glycemic control in patiens with type 2 diabetes mellitus. j periodontol 2003; 74(9): 1361–79. 16. mirza baq, izhar asf, khan aa. bidirectional relationship between diabetes and periodontal disease: review of evidence. j pak med assoc 2010; 60(9): 766–8. 17. stier re. oral care compositions comprising stabilized chlorine dioxide. available at: http://www.docstoc.com/docs/53120996/oralcare-compositions-comprising-stabilized-chlorine-dioxide--patent-6582682. 2002. accessed march 5, 2012. 18. russell ad. similarities and differences in the responses of microorganism to biocide. journal of antimicrobial chemotherapy 2003; 52: 750–63. 19. bayaty fa, ali tt, abdulla ma, hashim f. antibacterial effect of chlorine dioxide and hyaluronate on dental biofilm. african journal of microbiology research 2010; 4 (14): 1525–31. 20. winata is, rahmat m, dwiraharjo b. pengaruh pemberian chlorine oxygene dental gel pada soket gigi pasca operasi molar ketiga bawah terhadap pencegahan terjadinya alveolar osteitis. jurnal kedokteran gigi ui 2010; 1(3): 104–9. 21. perruzzo dc, jandiroba pfcb, filho grn. use of 0.1% chlorine dioxide to inhibit the formation of morning volatile sulphur compounds (vsc). braz oral res 2007; 21(1): 70–4. 22. greenstein g. the role of supra and subgingival irrigation in the treatment of periodontal diseases. j periodontol 2005; 76: 2015–27. vol 51 no 1 jan-mrt 2018.indd 14 the rankl expression and osteoclast in alveolar bone of rat diabetic model at different mechanical force application nuzulul hikmah, amandia dewi permana shita, and hafiedz maulana department of biomedics faculty of dentistry, universitas jember jember indonesia abstract background: diabetes is a serious and important public health problem, especially in relation to dental treatment. because of its complications in periodontal tissue, diabetes can be contraindicated in patients undergoing orthodontic treatment. the receptor activator of nuclear factor-κb ligand (rankl) is an essential cytokine inducing osteoclastogenesis. osteoblasts produce this cytokine which has been suggested to play an integral role in osteoclast activation during bone remodeling of orthodontic tooth movement. purpose: the aim of this study was to determine the correlation between rankl expression of osteoblast and the number of osteoclasts in the alveolar bone of diabetic rat models at different mechanical force application. methods: this study used animal subjects, white rats (rattus norvegicus) of the wistar strain (n=24) divided into six groups. the mechanical force to which they were subjected ranged between 10, 20, and 30 gramforce (grf). the animal models with diabetes were injected with a stratified dose of streptozotocin. an orthodontic appliance was inserted in both the maxillary incisors for seven days. the tissue was subjected to histological analysis of osteoclasts and immunohistochemistry analysis of rankl expression on the pressure and tension side of the alveolar bone. results: the results of this study showed that the increase in mechanical force produced a rise in rankl expression and osteoclast number on the pressure and tension side of the alveolar bone of diabetic rat models. conclusion: there was a correlation between the rankl of osteoblast and osteoclast numbers in the alveolar bone of diabetic models with different mechanical force application. keywords: rankl expression; osteoclast; rat diabetic model; mechanical force correspondence: nuzulul hikmah, department of biomedics, faculty of dentistry, universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: nuzulul.drg@gmail.com research report introduction diabetes is a chronic disease resulting from the inadequate production of insulin, a pancreatic hormone regulating blood glucose levels, or ineffective insulin use.1 diabetes is a major global medical condition representing a complicating factor in dental treatment, especially the branch of orthodontics. this complicating factor results from elevated blood glucose levels that can induce vital organ failure and tissue damage in the heart, blood vessels, eyes, kidneys, nerves, and periodontal tissues.2,3 for this reason, orthodontics represent a contradictive treatment for diabetics. during orthodontic treatment, the mechanical force applied to the teeth affects periodontal tissue, included alveolar bone remodeling. there are two bone cell activities in alveolar bone remodeling, osteoclast activation leading to bone resorption and osteoblast activation resulting in bone formation.4 both bone cell activities involve certain cytokines. the receptor activator of nuclear factor-κβ ligand (rankl) is an essential cytokine produced by osteoblast and constitutes a tumor necrosis factor (tnf)related ligand which plays a role in the osteoclastogenesis process, including osteoclast formation and activation during orthodontic tooth movement.5 diabetes can influence orthodontic treatment. certain studies have argued that diabetes affects the expression of cytokines which play a role in the osteoclastogenesis process. previous studies using mice models indicated that the mechanical force of a 35-gram orthodontic appliance dental journal (majalah kedokteran gigi) 2018 march; 51(1): 14–19 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 mailto:nuzulul.drg@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 15hikmah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 14–19 increased the expressions of receptor activator of nuclear factor-κb (rank), rankl, matrix metalloproteinase-13 (mmp-13) and colony stimulating factor-1 (csf-1) in the periodontal tissues of diabetic models.6 furthermore, previous studies described how uncontrolled type 2 diabetes patients with chronic periodontitis expressed higher rankl and osteoprotegerin (opg) than both control group patients and healthy individuals.7,8 the aim of this study was to determine the correlation between rankl expression of osteoblast and the number of osteoclast in alveolar bone of diabetic models under different mechanical force applications. from the application of varying mechanical forces, the optimal force that can be applied during the orthodontic treatment of diabetics could be estimated. materials and methods this study was approved by the ethics committee of the medical faculty, universitas brawijaya, malang, east java, indonesia. this study used animal subjects, namely; white wistar strain rats (rattus norvegicus). the criteria for the animal subjects comprised the following: healthy, 4-month old males, weighing 250-300 grams. the animal subjects (n=24) were divided into six groups (table 1). the orthodontic appliance, which was inserted into both maxillary incisor teeth of the subjects and used to apply mechanical force was fabricated from stainless steel 0.012u wire (classone orthodontics, usa), configured into a special shape. the configuration was a coil, 2 mm in diameter, with two 10 mm-long arms connected by a round matrix band 2 mm in diameter (meba, germany) at the end of wire arms (figure 1). the mechanical force applied to the animal models amounted to 10, 20, and 30 grf. the forces were measured using structural modeling design tools (the ansys software ver.14) and set by the richmond orthodontics stress and tension gauge (ormco, usa). the animal models were injected intraperitoneally with streptozotocin (nacalaitesque inc., kyoto japan, code 32238-91). the dose of streptozotocin administered was initially 40 mg/kgbw on the first day, with respective doses of 35, 30, 25 and 20 mg/kgbw being subsequently given up to the fifth day. after receiving an injection of streptozotocin, the diabetic animal subjects were incubated for 14 days during which period the blood glucose levels of samples taken from their tail vein were analyzed. the presence of diabetes was confirmed when the blood glucose levels of the subjects were above 300 mg/dl. before insertion of the orthodontic appliance, the animal subjects were anesthetized by intraperitoneal administration of 10 mg/kgbw ketamine hcl (anasject®, danpac pharma, indonesia). the orthodontic appliance was inserted in both the maxillary incisor teeth, perpendicular to the teeth axis a c b a b figure 1. the orthodontic appliance configured in the special shape (a) and inserted into both maxillary incisor teeth (red arrow) of animal models (b). a) arms; b) matrix bands; c) coil. table 1. types and periods of the treatment applied to the groups timetreatmentsgroups 14 days-control groups i (cg i)control groups 14 daysdiabetescontrol groups ii (cg ii) 7 daysappliance 30 grfcontrol groups iii (cg iii) 21 daysdiabetes + appliance 10 grfexperimental groups i (eg i)experimental groups 21 daysdiabetes + appliance 20 grfexperimental groups ii (eg ii) 21 daysdiabetes + appliance 30 grfexperimental groups iii (eg iii) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 16 hikmah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 14–19 for seven days. the stabilization of orthodontic appliance was achieved by means of type ix glass ionomer cement (fuji ix, gc, japan). the animal subjects were sacrificed by the administering of an overdose of an anesthetic agent. thereafter, their incisor teeth in the maxillae were removed and fixed in 10% formalin for 24 hours to serve as tissue samples. after the fixation time had elapsed, the tissue samples were decalcified in 14% ethylenediaminetetraacetic acid (edta) for 30 days at room temperature. the tissue samples were subsequently embedded in paraffin. for the osteoclast analysis using a histological method, the tissue sample that had been embedded in paraffin was cut into slices of 5 mm thickness longitudinally and placed on object glass. thereafter, the sliced samples on object glass were deparaffinized, rehydrated, and stained with hematoxylin-eosin for five minutes at room temperature. the samples were then dehydrated and mounted on a cover glass. the number of osteoclasts in the pressure and tension side of the alveolar bone area was counted using a light microscope at 400x magnification. the counting was completed on 4 slides from five selected areas. for rankl expression using immunohistochemistry analysis, the tissue samples embedded in paraffin were sliced to 3 mm of thickness longitudinally and placed on poly l-lysine slides (microscope, usa). after that, the slides were deparaffinized and incubated in 3% h2o2 dissolved in methanol for 15 minutes at room temperature. these procedures were undertaken to inhibit endogen peroxide activity. the slides were then washed with phosphate-buffered saline (pbs) and incubated with background sniper (starr trek universal hrp detection system, biocare medical, usa) for 30 minutes, before being stained with rankl antibody (n-19) (sc-7628, santa cruz biotechnology, usa; working dilution, 1:100) for 60 minutes at room temperature. after washing in pbs, the sections were incubated with secondary antibody, mouse antigoat igg-b (sc-53799, santa cruz biotechnology, usa; working dilution, 1:100) for 60 minutes at room temperature. rankl was stained using an immunohistochemistry staining kit (starr trek universal hrp detection system, biocare medical, usa) following the manufacturer’s instructions. the sections were rinsed with pbs and the final color reactions performed using a 3.3’diaminobenzidine (dab) chromogen and buffer (working dilution, 1:200) for 15 minutes at room temperature. the sections were then counterstained with mayer hematoxylin (working dilution 1:10) for 5 minutes. the rankl expression was counted using the light microscope at 400x magnification in the pressure and tension side of the alveolar bone. the counting was completed on 4 slides from five selected areas. all of the data were statistically analyzed using the spss 20.0 software program (ibm-spss inc., chicago, usa). the statistical analyses was the pearson correlation (p<0.05) and regression analysis. results immunohistochemistry images of rankl expression and histological images of osteoclast in the pressure and tension sides of diabetic rat subjects were shown in figure 2. the average of osteoclast number and rankl expression was shown in table 2. from table 2, it can be seen that the averages demonstrated a similar pattern, tending to be higher in the diabetes group with a higher mechanical force. according to correlation analysis, there was an association between rankl expression of osteoblast and the osteoclast number in the pressure and tension sides of alveolar bone (p<0.05) (table 3). moreover, diabetic models subjected to for free of various mechanical forces table 2. the number of osteoclasts and rankl expressions in the pressure and tension sides of subjects’ alveolar bone the study group the number of osteoclasts (mean + standard deviation) rankl expressions (mean + standard deviation) tension sidepressure sidetension sidepressure side gg i 0.8 + 0.4 0.6 ± 0.01.0 ± 0.10.7 + 0.4 cg ii 1.3 + 0.4 4.6 ± 0.54.9 ± 0.61.2 + 0.4 cg iii 1.7 + 0.3 2.3 ± 0.31.9 ± 0.31.2 + 0.3 eg i 4.5 + 0.8 7.5 ± 0.95.6 ± 1.33.9 + 0.6 eg ii 5.2 + 0.5 9.7 ± 1.07.3 ± 0.94.1 + 0.4 eg iii 6.8 + 1.3 12.9 ± 1.512.6 ± 1.45.5 + 0.9 table 3. the correlation and regression test between the rankl expression of osteoblast with the number of osteoclast on the pressure and tension sides of the alveolar bone of a diabetic rat model the study group pressure side tension side r r2 rp value r 2 p value 0.00*experimental grups 0.835 0.698 0.00* 0.896 0.802 * = the correlation was significant with p value<0.05; r = the pearson’s correlation; r2 = the r square dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 17hikmah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 14–19 demonstrated a similar blood glucose level of more than 300 mg/dl for the duration of the experiment. discussion the correlation between rankl expression of osteoblast and the osteoclast number in the alveolar bone area of diabetic subjects under different mechanical force application can serve illustrate alterations to alveolar bone remodeling during the orthodontic treatment of diabetics. osteoblast, osteoclast and rankl are the major components playing a pivotal role in the periodontal tissue remodeling process, while osteoblast plays a role in the bone formation and osteoclast involved in bone resorption. the rankl-rank system regulates alveolar bone and periodontal tissue remodeling during orthodontic treatment.5 when mechanical force was applied to both incisors, there were pressure and tension sides in the periodontal tissue which involved the periodontal ligament and alveolar bone. the pressure side is an area of tooth movement direction located on the distal side of the incisors after mechanical force application. while the tension side is the opposite area to the pressure side located at the mesial side of the incisors after mechanical force application. this study showed that the increase in mechanical force influenced the enhancement of rankl expression and osteoclast number in the pressure and tension side of the alveolar bone area of the diabetic subject. this study corresponded to a previous one in which diabetes altered alveolar bone turnover through an imbalance in osteoblast/ osteoclast activity and enhancement of pro-inflammatory mediator levels which induce increased bone resorption and tooth movement.6 in diabetes, osteoblast experiences apoptosis easily due to the activity of advanced glycation end products (ages) via the mitogen-activated protein (map) kinase and cytosolic apoptotic pathways.9 according to statistical analysis, there was a significant correlation between rankl expression of osteoblast and osteoclast numbers on the pressure and tension side of the alveolar bone area in diabetic subjects. this meant that rankl expressions of osteoblast and osteoclast were the major factors in increased tooth movement in diabetic subjects undergoing orthodontic treatments. osteoclast activation and differentiation occurred when rankl binds to rank, a key preliminary step in downstream signaling is binding of tumor necrosis factor receptor-associated factors (trafs) to specific siter within the cytoplasmic domain of rank, which is a transmembrane protein. however, not only did trafs play a role in the activation and differentiation of osteoclast. at least seven signaling c d ab pdl t t pdl ab ab ab t t pdl pdl a b figure 2. light microscopy images showing rankl expression of osteoblast (black arrow) in the pressure (a) and tension (b) side and osteoclast (red arrow) in the pressure (c) and tension (d) side of the diabetic model with mechanical force application. ab, alveolar bone; pdl, periodontal ligament; t, teeth. a c b d dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 18 hikmah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 14–19 pathways are activated by rank-mediated protein kinase signaling, four of them directly mediate osteoclastogenesis (inhibitor of nuclear factor κb (nf-κb) kinase/nf-κb, c-jun amino-terminal kinase/activator protein-1, c-myc, and calcineurin/nuclear factor of activated t cells [nfat] c1), while three mediate osteoclast activation (src and mkk6/p38/mitf) and survival (src and extracellular signal-regulated kinase).10 mechanical force also caused periodontal ligament cells to produce prostaglandin e2 (pge2) and stimulate osteoclastogenesis via rankl in osteoblast.11 diabetes induces up-regulation of osteoclastogenic factor which stimulates osteoclast activation and differentiation. inflammatory mediators such as tnfα and il-1β incline in diabetes which can exert an influence on osteoblast to express rankl protein, stimulate osteoclast differentiation and, finally, cause alveolar bone resorption in diabetes.12,13 both diabetes and mechanical force can induce inflammation which activates t cells to produce inflammatory cytokine (tnfα, il-1, il-6) leading to enhancement of rankl expression on osteoblast and bone marrow. t cells caused bone resorption directly via rankl expression and indirectly via pro-inflammatory cytokine that mediated rankl expression on non-t cells.14–16 recent studies have also shown that blood glucose levels in the diabetes group and diabetes group with different orthodontic force were more than 300 mg/dl, causing hyperglycemia, during the experimental period. hyperglycemia in diabetes drives the irreversible formation of advanced glycation end products (ages) that can have direct pro-inflammatory and pro-oxidant effects on cells. when ages bind their signaling receptor, advanced glycation end product (rage), cellular phenotype and function are critically impacted and enhance inflammation, oxidative stress, and tissue repair impairment.12,17 ages can lead to cellular stress by exerting pro-inflammatory/ oxidant effects directly, or through interaction with cellsurface receptors.12 hyperglycemia also contributes to enhancement of reactive oxygen species (ros) levels and a state of oxidative stress, both directly and indirectly through the age/rage axis, promoting quantitative and qualitative shifts in cytokine profiles.12 ros stimulates pro-inflammatory cytokine production through activation of intracellular signaling pathways such as map kinase, nf-κb and the nalp3 inflammasome.18,19 ros also have more wide-ranging effects, including those on bone formation and recently revealed pathways involving the interaction of ros, wnt signaling and activation of foxo transcription factors in the regulation of osteoblast activity suggest another novel pathway which may link periodontitis and diabetes.20,21 previous studies of the relationship between diabetes and periodontal diseases suggested that there were many factors related to the osteoclastogenesis process and reported that there was enhancement of rankl level in diabetes. the age-rage axis might contribute to the osteoclastogenesis process. rage is involved in osteoclast and in reorganization, adhesion and activation, thereby contributing to reduced bone mass in diabetes. ages increased mrna levels of rage and rankl in osteoblasts, thus further suggesting the active participation of rage in osteoclastogenesis.22,23 rankl/opg ratio also is modulated by hyperglycemia in diabetes directly and indirectly, resulting in the stimulation of inflammation and tissue destruction.12 from this study, it can be concluded that mechanical force application in a diabetic rat model affects rankl expression and osteoclast number in the pressure and tension side of alveolar bone area. the increased rankl expression in osteoblast was caused by the application of increased mechanical force, the effect on the increase in the number of osteoclast in the pressure and tension side of the alveolar bone of diabetic rat models. based on these results, the application of low mechanical force in orthodontic treatment under diabetic conditions is recommended. references 1. world health organization. global report on diabetes. geneva: who press; 2016. p. 11-9. 2. leite rs, marlow nm, fernandes jk. oral health and type 2 diabetes. am j med sci. 2013; 345(4): 271–3. 3. fowler mj. microvascular and macrovascular complications of diabetes. clin diabetes. 2008; 26(2): 77–82. 4. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015; 2015: 1–10. 5. yamaguchi m. rank/rankl/opg during orthodontic tooth movement. orthod craniofac res. 2009; 12(2): 113–9. 6. braga smg, taddei sr, andrade i, queiroz-junior cm, garlet gp, repeke ce, teixeira mm, da silva ta. effect of diabetes on orthodontic tooth movement in a mouse model. eur j oral sci. 2011; 119: 7–14. 7. santos vr, lima ja, gonçalves ted, bastos mf, figueiredo lc, shibli ja, duarte pm. receptor activator of nuclear factor-kappa b ligand/osteoprotegerin ratio in sites of chronic periodontitis of subjects with poorly and well-controlled type 2 diabetes. j periodontol. 2010; 81(10): 1455–65. 8. ribeiro fv, de mendonça ac, santos vr, bastos mf, figueiredo lc, duarte pm. cytokines and bone-related factors in systemically healthy patients with chronic periodontitis and patients with type 2 diabetes and chronic periodontitis. j periodontol. 2011; 82(8): 1187–96. 9. alikhani m, alikhani z, boyd c, maclellan cm, raptis m, liu r, pischon n, trackman pc, gerstenfeld l, graves dt. advanced glycation end products stimulate osteoblast apoptosis via the map kinase and cytosolic apoptotic pathways. bone. 2007; 40(2): 345–53. 10. boyce bf, xing l. biology of rank, rankl, and osteoprotegerin. arthritis res ther. 2007; 9(suppl 1): s1. 11. mayahara k, yamaguchi a, takenouchi h, kariya t, taguchi h, shimizu n. osteoblasts stimulate osteoclastogenesis via rankl expression more strongly than periodontal ligament cells do in response to pge2. arch oral biol. 2012; 57(10): 1377–84. 12. taylor jj, preshaw pm, lalla e. a review of the evidence for pathogenic mechanisms that may link periodontitis and diabetes. j clin periodontol. 2013; 40(suppl 14): s113–34. 13. cochran dl. inflammation and bone loss in periodontal disease. j periodontol. 2008; 79(8 suppl): 1569–76. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 19hikmah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 14–19 14. wu c-c, sytwu h-k, lu k-c, lin y-f. role of t cells in type 2 diabetic nephropathy. exp diabetes res. 2011; 2011: 1–9. 15. yan y, liu f, kou x, liu d, yang r, wang x, song y, he d, gan y, zhou y. t cells are required for orthodontic tooth movement. j dent res. 2015; 94(10): 1463–70. 16. kohli s, kohli v. role of r a nk l-r a nk /osteoproteger in molecular complex in bone remodeling and its immunopathologic implications. indian j endocrinol metab. 2011; 15(3): 175–81. 17. giacco f, brownlee m. oxidative stress and diabetic complications. circ res. 2010; 107(9): 1058–70. 18. graves dt, kayal ra. diabetic complications and dysregulated innate immunity. front biosci. 2008; 13: 1227–39. 19. martinon f. signaling by ros drives inflammasome activation. eur j immunol. 2010; 40(3): 616–9. 20. almeida m, han l, ambrogini e, weinstein rs, manolagas sc. glucocorticoids and tumor necrosis factor α increase oxidative stress and suppress wnt protein signaling in osteoblasts. j biol chem. 2011; 286(52): 44326–35. 21. galli c, passeri g, macaluso gm. foxos, wnts and oxidative stress-induced bone loss: new players in the periodontitis arena? j periodontal res. 2011; 46(4): 397–406. 22. yamagishi s. role of advanced glycation end products (ages) in osteoporosis in diabetes. curr drug targets. 2011; 12(14): 2096–102. 23. yoshida t, flegler a, kozlov a, stern ph. direct inhibitory and indirect stimulatory effects of rage ligand s100 on sranklinduced osteoclastogenesis. j cell biochem. 2009; 107(5): 917–25. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p14–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p14-19 96 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 96–101 original article facial height proportion based on angle’s malocclusion in deutero-malayids aulia rohadatul aisy, avi laviana and gita gayatri department of orthodontics, faculty of dentistry, universitas padjadjaran, bandung – indonesia abstract background: facial aesthetics are closely related to the harmonious proportions of the facial components. one of the components is facial height. the reference of facial height proportion of certain racial groups needs to be known by orthodontists and surgeons to create treatment outcomes that can be specifically designed for these particular demographics. one of the factors that can affect facial height proportion is malocclusion. purpose: this study aimed to determine facial height proportion based on angle’s classification of malocclusion in deutero-malayids. methods: this study used a descriptive cross-sectional method, which was conducted on 116 deutero-malayid subjects. the subjects’ malocclusion was first examined using angle’s classification of malocclusion. upper and lower facial height were then measured to determine the proportion of these dimensions. the results were then grouped based on each malocclusion class. results: it was found that the upper and lower facial height proportions in the class i malocclusion group were 46.74% and 53.26% in males and 47.52% and 52.48% in females, respectively. the upper and lower facial height proportions in the class ii malocclusion group were 48.46% and 51.54% in females. upper and lower facial height proportions in the class iii malocclusion group were 45.31% and 54.69% in males and 46.29% and 53.71% in females, respectively. conclusion: the largest proportion of upper facial height in deutero-malayids was seen in the class ii malocclusion group, followed by class i and class iii. the largest proportion of lower facial height in deutero-malayids was seen in the class iii malocclusion group, followed by class i and class ii. keywords: angle’s classification of malocclusion; anthropometry; deutero-malayid; facial height proportion correspondence: aulia rohadatul aisy, department of orthodontics, faculty of dentistry, universitas padjadjaran. jl. sekeloa selatan no. 1 bandung, 40132 indonesia. email: aulia16013@mail.unpad.ac.id introduction malocclusion is defined as the malrelation of molars or teeth between the dental arches beyond the range of what is accepted as normal.1 according to the world health organization, the incidence of malocclusion is third largest after caries and periodontal disease, and has rather high prevalence, ranging from 20% to 100% in different populations in the world.2–4 although malocclusion is not life-threatening, it has a negative impact on quality of life,3,5–7 and for those who experience the greatest negative impact, it can result in physical pain and psychological discomfort.7 psychological discomfort is often linked with lowered self-esteem and the dissatisfaction of psychosocial well-being.8,9 physical appearance plays a key role in self-esteem, and according to a previous study, malocclusion can affect that.8 facial aesthetics are closely related to the harmonious proportioning of the facial components.10,11 orthodontists and surgeons have emphasised the importance of seeing the face in proportion, and this includes facial height.10,12 the assessment of facial height is a part of clinical evaluation, and it is important in diagnosis and treatment planning in orthodontics.5,13,14 the quantitative evaluation of soft tissue by using the anthropometric method is essential and has come to prominence currently because the evaluation of hard tissue alone often does not bring satisfactory results, and as seen clinically, soft tissue determines facial appearance.11,12,15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 mailto:aulia16013@mail.unpad.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 97aisy et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 96–101 according to facial anthropometry, there are two types of facial height. the first one, physiognomic facial height, divides the face into the upper face (trichionglabella), middle face (glabella-subnasale), and lower face (subnasale-gnathion). the second type, morphological facial height, divides the face into the upper face (nasionsubnasale) and lower face (subnasale-gnathion).12,16 facial height proportion has been studied and used as a guideline for treatment planning in many fields.12 it can also be used to distinguish a wide variety of races or ethnic groups among populations since these groups will have different norms and facial features.14,17,18 malocclusion can alter facial height; hence, it can affect facial proportion. for instance, a deep bite resulting from malocclusion class ii division 2 will create the appearance of a short face.10 in this study, the researchers seek to investigate facial height proportion in every type of malocclusion. angle’s classification of malocclusion is used since this is still widely used to determine malocclusion because of its simplicity and practicality.19,20 angle divided malocclusion into three classes based on the relationship between the maxillary and mandibular first molar. class i describes a normal relationship between the molars, class ii describes a lower molar that is distally positioned relative to the upper molar, and class iii describes a lower molar that is mesially positioned relative to the upper molar.5 ethnicity is an interesting concept in studying human variations, as different race and ethnic groups will have different gene pools that exhibit different characteristics. according to the two layers theory, there were two racial migrations to indonesia through asia, namely mongoloid and austromelanesoid. the fusion between mongoloid and australomelanesoid produced proto-malayid and the fusion between proto-malayid and mongoloid produced deutero-malayid, which now inhabits most of indonesia.21–23 this includes ethnic groups such as the aceh, minangkabau, bugis, makassar, sasak, bali, malay, jawa, betawi, sunda, madura, and manado communities.24,25 it is important to know the reference of facial proportion of a certain ethnic group as a guideline for treatment planning.12 orthodontists and surgeons need to adjust the treatment planning based on the facial pattern that is endemic to where the individual lives to help create a natural and aesthetically acceptable facial appearance relative to the their demographic’s standard.14 unfortunately, to date, there have been no available data of facial height proportion for the deutero-malayids. therefore, this study was carried out to determine facial height proportion based on angle’s classification of malocclusion in deutero-malayids. it is hoped that the findings in this study can give an overview of facial height proportion based on angle’s classification of malocclusion and provide a reference for treatment planning in achieving the ideal facial height proportion for deuteromalayids. materials and methods this study was descriptive research with a cross-sectional design. samples were sourced using a total sampling technique that included all the undergraduate students of the faculty of dentistry, universitas padjadjaran, specifically those who were of deutero-malayid descent in the 2016–2019 cohort. the number in the sample that met the criteria of this study was 116 subjects. ethical exemption was obtained from the research ethics committee of the faculty of medicine, universitas padjadjaran. the reference number is 1466/un6.kep/ec/2019. the study procedure began with the collection of data by questionnaire from all dental students in order to select subjects according to the inclusion criteria, which were as follows: eligible subjects (1) were of deutero-malayid descendent within the past two generations, (2) had angle’s malocclusion class i, ii, or iii, (3) had complete permanent teeth except for their third molars, (4) were 18 years or older, and (5) were willing to participate in this study. exclusion criteria were (1) having abnormal tooth shape or size; (2) having a previous history of facial trauma and/ or fracture; (3) currently undergoing (or with a history of) previous orthodontic treatment, orthognathic surgery, or dentocraniofacial surgery; (4) having a previous history of syndromic disorders’ and (5) having a previous history of germinectomy. intra-observer and inter-observer measurements were performed on 23 students prior to measurements on all subjects to ensure the reliability of the measurement. intraobserver measurements were carried out by an observer on 23 subjects once each day for a total of two days. inter-observer measurements were carried out by three observers on 23 subjects once on the same day. all eligible subjects were given oral and written information regarding this research and then asked to sign an informed consent prior to the procedure, thereby confirming their agreement. the intraclass correlation coefficient (icc) of all data obtained was subsequently tested using ibm spss statistics. the results of intra-observer measurement for the icc with regard to the upper face and lower face measurements were 0.917 and 0.896, respectively. the results of inter-observer measurement for the icc regarding upper face and lower face measurements were 0.982 and 0.936, respectively. both of the tests showed excellent reliability. angle’s classification of malocclusion was used to determine the type of the subject’s malocclusion in this study. the landmarks for this study were defined as follows: (1) nasion, the sagittal midline point of the nasal root at the nasofrontal suture; (2) subnasale, the midpoint of the columella base at the apex of the angle where the lower border of the nasal septum and the upper lip meet; and (3) gnathion, the lowest median landmark on the lower border of the mandible, which is identified by palpation and is identical to the bony gnathion.11 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 98 aisy et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 96–101 the subject was instructed to sit in an upright relaxed position with their teeth making contact in centric occlusion. malocclusion of the subject was determined by assessing the relationship between the maxillary and mandibular first molar. the subject was asked to keep their head straight and parallel to the ground so that the researcher could palpate and identify facial landmarks. prior to taking the standard measurements, surface landmarks were marked on the face with a non-toxic marker. upper and lower face height were then measured using a digital vernier caliper while the subject was in centric occlusion. upper face height (ufh) was determined by measuring the distance between the nasion to the subnasale (figure 1), and lower face height (lfh) was determined by measuring the distance between the subnasale to the gnathion (figure 2). the sum of upper facial height and lower facial height is the total facial height. the data were subsequently computed to determine the proportion of upper facial height and lower facial height using following formula.26 𝑈𝑝𝑝𝑒𝑟 𝑓𝑎𝑐𝑒 𝑝𝑟𝑜𝑝𝑜𝑟𝑡𝑖𝑜𝑛 = 𝑈𝑝𝑝𝑒𝑟 𝑓𝑎𝑐𝑖𝑎𝑙 ℎ𝑒𝑖𝑔ℎ𝑡 (𝑈𝐹𝐻) 𝑥 100% 𝑇𝑜𝑡𝑎𝑙 𝑓𝑎𝑐𝑖𝑎𝑙 ℎ𝑒𝑖𝑔ℎ𝑡 (𝑇𝐹𝐻) 𝐿𝑜𝑤𝑒𝑟 𝑓𝑎𝑐𝑒 𝑝𝑟𝑜𝑝𝑜𝑟𝑡𝑖𝑜𝑛 = 𝐿𝑜𝑤𝑒𝑟 𝑓𝑎𝑐𝑖𝑎𝑙 ℎ𝑒𝑖𝑔ℎ𝑡 (𝐿𝐹𝐻) 𝑥 100% 𝑇𝑜𝑡𝑎𝑙 𝑓𝑎𝑐𝑖𝑎𝑙 ℎ𝑒𝑖𝑔ℎ𝑡 (𝑇𝐹𝐻) both facial height and facial height proportion were then grouped into class i, ii, and iii of angle’s classification of malocclusion and differentiated based on sex. data is expressed as mean ± sd. the result provided an overview of facial height proportion. results of the 626 undergraduate students in the faculty of dentistry, universitas padjadjaran, 116 met our inclusion criteria: 17 males (14.66%) and 99 females (85.34%) aged 18–23 years with an average age of 20. table 1 shows the distribution of malocclusion in both sexes, and it was found class i malocclusion was the most common among all subjects, followed by class iii, and class ii malocclusion respectively. table 2 provides the average of facial height based on angle’s classification of malocclusion among males figure 2. measurement of lower facial height. figure 1. measurement of upper facial height. table 1. distribution of malocclusion in both sexes (n=116) molar relation male female totaln % n % class i 13 11.21 92 79.31 90.52% class ii 0 0 3 2.59 2.59% class iii 4 3.45 4 3.45 6.9% total 17 14.66 99 85.34 100% table 2. distribution of facial height based on angle’s classification of malocclusion in both sexes (n=116) angle’s classification of malocclusion upper facial height lower facial height male female male female n mm n mm n mm n mm class i 13 57.23 ± 4.05 92 53.47 ± 3.86 13 65.22 ± 4.08 92 59.08 ± 4.23 class ii 0 3 54.52 ± 4.94 0 3 57.97 ± 4.78 class iii 4 54.92 ± 1 4 51.48 ± 2.15 4 66.38 ± 3.71 4 59.80 ± 3.94 table 3. distribution of upper and lower facial height proportion based on angle’s classification of malocclusion in both sexes (n=116) angle’s classification of malocclusion upper facial height proportion lower facial height proportion male female male female n % n % n % n % class i 13 46.74 ± 2.20 92 47.52 ± 2.20 13 53.26 ± 2.20 92 52.48 ± 2.20 class ii 0 3 48.46 ± 3.99 0 3 51.54 ± 3.99 class iii 4 45.31 ± 1.56 4 46.29 ± 1.42 4 54.69 ± 1.56 4 53.71 ± 1.42 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 99aisy et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 96–101 and females who were deutero-malayids. it was found that upper and lower facial height in all malocclusion groups were greater in deutero-malayid males compared to female deutero-malayids. upper facial height in deutero-malayid males with class i malocclusion was greater than those with class iii malocclusion. on the other hand, lower facial height in deutero-malayid males with class iii malocclusion was greater than those with class i malocclusion. deutero-malayid females with class ii malocclusion had the greatest upper facial height, followed by class i and class iii malocclusion groups. meanwhile, deutero-malayid females with class iii malocclusion had the greatest lower facial height, followed by class i and class ii malocclusion. the linear measurements from table 2 were then used to calculate upper and lower facial height proportion and the result is shown in table 3. discussion observing that the human face is in proportion has been emphasised as it creates a harmonious appearance.10,12 anthropometric measurement of facial height itself is an essential part of clinical evaluation and it is important in diagnosis as well as treatment planning in orthodontics.5,13,14 the facial height proportion for deutero-malayids needs to be known as a reference. this study aimed to determine the facial height proportion in deutero-malayids based on angle’s classification of malocclusion and the study was done by assessing malocclusion as well as measuring the upper facial height and lower facial height of the subjects. this study found that the lower facial height proportion in deutero-malayids was greater than the upper facial height proportion in all types of malocclusion class in both sexes. given that class i malocclusion is the most common and the molar relation is considered as normal, it was found that in deutero-malayids, the ideal upper facial height proportion was 47.13% (46.74% in males and 47.52% in females), and the ideal lower facial height proportion was 52.87% (53.26% in males and 52.48% in females), which is slightly different to what we used to believe was the ideal proportion (45% for ufh and 55% for lfh).12,27 the ideal proportion of facial height in class ii and class iii malocclusion has not been discussed in the previous studies. however, in this study, it was found that upper and lower facial height proportion were respectively 48.46% and 51.54% in class ii malocclusion and 45.8% and 54.2% in class iii malocclusion. previous studies have also conducted similar research on different populations. in the study that was conducted by farkas et al.18 on various ethnic groups and races, it was found that facial height proportion in hungarian males (46.50% for ufh and 53.50% for lfh) almost resembled that of the deutero-malayids. however, this was very different from the african american male population, where the proportion of lower facial height (62.66%) far exceeded the proportion of upper facial height (37.33%). the proportion of facial height in deutero-malayid females showed similarities to the research conducted by farkas et al.18 on portuguese females (the proportion of ufh was 46.86% and the proportion of lfh was 53.13%) and russian females (the proportion of ufh was 46.23% and the proportion of lfh was 53.76%), whereas this was very different from african american females, where the proportion of lower facial height (61.37%) far exceeded the proportion of upper facial height (38.62%). another study of facial height proportion was also done by sheikh et al.12 in bangladesh and it was found that the facial proportion of bangladeshi males (45.32% for ufh and 54.68% for lfh) almost resembled deutero-malayid males. however, the facial proportion of bangladeshi females (44.27% for ufh and 55.73% for lfh) was slightly different from deutero-malayid females because bangladeshi females had an upper facial height proportion that was less than 45% and a lower facial height proportion that was more than 55%.12 this result also corroborates findings by baral et al.26 regarding the rai population in nepal where the upper facial height proportion was smaller (43.2% in males and 43.1% in females) than deutero-malayids, and the lower facial height proportion (56.8% in males and 56.9% in females) was greater than deutero-malayids.26 research conducted by farkas et al.,18 baral et al.,26 and sheikh et al.12 had similarities with the present study in terms of landmark points and the inclusion criteria that were used. the difference, however, was in the number of samples. farkas et al.18 had 60 subjects consisting of 30 males and 30 females, baral et al.26 had 208 subjects, and sheikh et al.12 had 500 subjects. another difference is that these studies did not specify as a requirement the subjects’ type of malocclusion.12,18,26 the similarities and differences in facial height proportion between deuteromalayids and other populations may also be influenced by the diversification of ethnicity and genetics.10,12 the calculation of facial height proportion based on the angle’s classification of malocclusion in table 3 shows that the proportion of upper facial height in the class ii malocclusion group (48.46%) was greater than that of the class i malocclusion group (47.52%). it was also found that the proportion of upper facial height in the class iii malocclusion group (45.31% in males and 46.29% in females) was smaller than the class i malocclusion group (46.74% in males and 47.52% in females). in contrast, the proportion of lower facial height in the class ii malocclusion group (51.54%) was smaller than the class i malocclusion group (52.48%) and the proportion of lower facial height in the class iii malocclusion group (54.69% in males and 53.71% in females) was greater than the class i malocclusion group (53.26% in males and 52.48% in females). in the present study, the result of facial height proportion and its correlation with malocclusion are in line with the study that was conducted by ifwandi et al. with the aceh ethnic group. his study showed that the lower facial height proportion in class i malocclusion (52.97%) was greater dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 100 aisy et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 96–101 than the lower facial height proportion in class ii, division 2 malocclusion (51.84%). the difference from this present study was that ifwandi did not have deutero-malayid descent and a class iii malocclusion group as inclusion criteria. the difference of facial height proportion in different malocclusion groups can be a result of jaw rotation growth. individuals with a short face who are characterised by a significantly smaller lower facial height experienced an increased internal rotation during growth, resulting in forward rotation in which the posterior growth is greater than anterior growth. this type of rotation is usually accompanied by a deep bite malocclusion, which can be seen in class ii division 2 malocclusion. individuals with a long face who are characterised by greater lower facial height have a palatal plane that rotates posteriorly downward, creating a negative inclination. this results in a backward rotation in which the anterior growth will be greater than the posterior growth. this type of rotation is usually associated with an anterior open bite malocclusion.5 the association between malocclusion and facial morphology was also examined in the study conducted by siriwat and jarabak.28 these authors conducted a cephalometric analysis on 500 cephalographs and suggested that class ii malocclusion is the dominant malocclusion group in the hypodivergent growth pattern where the face tends to grow horizontally resulting in smaller lower facial height, while class iii malocclusion is the dominant malocclusion in the hyperdivergent growth pattern where the face experiences downward rotation resulting in an increased lower facial height. this study was conducted to determine the proportion of facial height based on angle’s malocclusion classification. the limitations of this study lie in the application of angle’s classification of malocclusion as a classification system. even though it is one of the classifications that is frequently used, it still has some drawbacks. angle only considered malocclusion in the anteroposterior plane, and there is no differentiation between dental and skeletal malocclusions. the proportion of facial height involves the dimension of the face in a vertical plane; therefore, it is necessary to examine and consider the subject’s skeletal relationship. another limitation in this study is the absence of a male sample in class ii malocclusion, which meant that we were unable to identify and compare facial height proportion from that specific class. it can be concluded that the largest proportion of upper facial height in deutero-malayids was seen in the class ii, class i, and class iii malocclusion groups, respectively. the largest proportion of lower facial height in deutero-malayids was seen in the class iii, class i and class ii malocclusion groups, respectively. references 1. bhullar mk, nirola a. malocclusion pattern in orthodontic patients. indian j dent sci. 2012; 4(4): 20–3. 2. haralur sb, addas mk, othman hi, shah fk, el-malki ai, al-qahtani ma. prevalence of malocclusion, its association with occlusal interferences and temporomandibular disorders among the saudi sub-population. oral health dent manag. 2014; 13(2): 164–9. 3. tak m, nagarajappa r, sharda aj, asawa k, tak a, jalihal s, kakatkar g. prevalence of malocclusion and orthodontic treatment needs among 12-15 years old school children of udaipur, india. eur j dent. 2013; 7(suppl 1): s045–53. 4. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10(1): 7. 5. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st. louis: mosby elsevier; 2013. p. 3–114. 6. scapini a, feldens ca, ardenghi tm, kramer pf. malocclusion impacts adolescents’ oral health-related quality of life. angle orthod. 2013; 83(3): 512–8. 7. neto afc, pinho rcm, de sousa rv, vajgel b de cf, cimões r. the impact of malocclusion on quality of life and life satisfaction. int med soc. 2017; 10(222): 1–10. 8. taibah sm, al-hummayani fm. effect of malocclusion on the selfesteem of adolescents. j orthod sci. 2017; 6(4): 123–8. 9. perillo l, esposito m, caprioglio a, attanasio s, santini ac, carotenuto m. orthodontic treatment need for adolescents in the campania region: the malocclusion impact on self-concept. patient prefer adherence. 2014; 8: 353–9. 10. ifwandi, rahmayani l, maylanda a. proporsi tinggi wajah pada relasi molar klas i dan klas ii divisi 2 angle mahasiswa fakultas kedokteran gigi universitas syiah kuala. j syiah kuala dent soc. 2016; 1(2): 153–60. 11. jagadish chandra h, ravi ms, sharma sm, rajendra prasad b. standards of facial esthetics: an anthropometric study. j maxillofac oral surg. 2012; 11(4): 384–9. 12. sheikh ma, chowdhury gm, jolly f, zaman m. an anthropometric evaluation of morphological facial height in bangladeshi young adult. j armed forces med coll bangladesh. 2014; 10(2): 33–8. 13. al-jassim nh, fathallah zf, abdullah nm. anthropometric measurements of human face in basrah. basrah j surg. 2014; 20(2): 29–40. 14. zacharopoulos g v, manios a, kau ch, velagrakis g, tzanakakis gn, de bree e. anthropometric analysis of the face. j craniofac surg. 2016; 27(1): e71-5. 15. costa mcc, e barbosa m de c, bittencourt mav. evaluation of facial proportions in the vertical plane to investigate the relationship between skeletal and soft tissue dimensions. dental press j orthod. 2011; 16(1): 99–106. 16. hall jg, allenson je, gripp kw, slavotinek am. handbook of physical measurements. 2nd ed. new york: oxford university press; 2006. p. 100–5. 17. ajami s, zarif najafi h, mahdavi s. angular photogrammetric analysis of the soft tissue facial profile of iranian young adults. iran j orthod. 2015; 10(2): 1–8. 18. farkas lg, katic mj, forrest cr, alt kw, bagic i, baltadjiev g, cunha e, cvicelová m, davies s, erasmus i, gillett-netting r, hajnis k, kemkes-grottenthaler a, khomyakova i, kumi a, kgamphe js, kayo-daigo n, le t, malinowski a, negasheva m, manolis s, ogetürk m, parvizrad r, rösing f, sahu p, sforza c, sivkov s, sultanova n, tomazo-ravnik t, tóth g, uzun a, yahia e. international anthropometric study of facial morphology in various ethnic groups/races. j craniofac surg. 2005; 16(4): 615–46. 19. villada-castro m, rueda zv, botero-mariaca pm. level of knowledge on classification systems of malocclusions among dentists and orthodontists. j educ ethics dent. 2017; 7(2): 37–43. 20. bugaighis i, karanth d. the prevalence of malocclusion in urban libyan schoolchildren. j orthod sci. 2013; 2(1): 1–6. 21. primasari a, angelia v, agara d. differences in head size and shape during the growth of deutero malay children. padjadjaran j dent. 2019; 31(1): 53–9. 22. irsa r, syaifullah td. variasi kefalometri pada beberapa suku di sumatera barat. j biol univ andalas. 2013; 2(2): 130–7. 23. rieuwpassa ie, hamrun n, riksavianti f. ukuran mesiodistal dan servikoinsisal gigi insisivus sentralis suku bugis, makassar, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 101aisy et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 96–101 dan toraja tidak menunjukkan perbedaan yang bermakna. j dentomaxillofacial sci. 2013; 12(1): 1–4. 24. chairani cn, rahmi e. korelasi antara dimensi vertikal oklusi dengan panjang jari kelingking pada sub-ras deutro melayu. maj kedokt gigi indones. 2016; 2(3): 155–63. 25. harahap mn. hubungan sudut interinsisal dengan profil jaringan lunak wajah menurut analisis holdaway pada mahasiswa fkg usu ras campuran proto dan deutro melayu. dentika dent j. 2013; 17(4): 314–8. 26. baral p, lobo sw, menezes rg, kanchan t, krishan k, bhattacharya s, hiremath ss. an anthropometric study of facial height among four endogamous communities in the sunsari district of nepal. singapore med j. 2010; 51(3): 212–5. 27. rakosi t. an atlas and manual of cephalometric radiography. great britain: wolfe medical publications; 1982. p. 71. 28. siriwat pp, jarabak jr. malocclusion and facial morphology is there a relationship? an epidemiologic study. angle orthod. 1985; 55(2): 127–38. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p96–101 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p96-101 mkgs vol 45 no 2 april-juni 2012.indd dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 135 volume 47, number 3, september 2014 pengaruh lama pemberian aspirin pada ekspresi protein ki-67 dan ketebalan epitel mukosa rongga mulut tikus wistar jantan (the effect of aspirin administration period on ki-67 expression protein and oral epithelial mucosal thickness in male wistar mice) dian yosi arinawati,1 heni susilowati1 dan supriatno2 1 departemen biologi mulut, 2 departemen ilmu penyakit mulut, fakultas kedokteran gigi, universitas gadjah mada yogyakarta indonesia abstract background: aspirin has been widely used as an anti-inflammatory, antipyretic, and analgesics drugs. it has seriously side effects like gastrointestinal ulceration, delayed healing ulcer, and oral mucosal ulceration when aspirin is administered for long time. purpose: the aim of study was to examine the effect of aspirin administration period on kiehl-67 (ki-67) protein expressions and oral mucosal epithelial thickness in male wistar mice. methods: experimental laboratory study with post-test only control group design was performed and 40 male wistar mice were used in this experiment. the samples were divided into 2 groups. group i was treated with aspirin, whereas group ii was receive aquadest. each group was divided into 5 subgroups for assessment of the length administration effect. all of mice were sacrificed on day 1, 3, 5, 7 and 10 after treatment. aspirin was orally administrated with doses of 9 mg/kg body weight. the buccal right of mice oral mucosal tissue was sliced and delivered for immunohistochemistry staining using anti-ki-67. hematoxylin-eosin (he) staining was performed to measure the oral epithelial thickness. examination of ki-67 expressions and oral epithelial thickness were performed by using imagej software. two-way anova and kruskall-wallis test were carried-out for data analysis with significant level of 95%. results: the results revealed that the administration of aspirin in mice on day 1, 3, 5, 7, and 10 was markedly decreased in the ki-67 protein expressions and oral epithelial thickness compared with that of control (p<0.05), otherwise the duration of aspirin administration did not affect mucosal epithelial thickness. conclusion: aspirin administration period has the potential to suppress the ki-67 protein expression within 10 days; the effect in line with the length of duration. the epithelial thickness was not influenced by the length of aspirin administration. key words: aspirin, ki-67 expression, oral mucosal epithelial thickness, wistar mice abstrak latar belakang: aspirin digunakan sebagai anti inflamasi, anti demam, dan anti nyeri. aspirin merupakan obat yang aman, namun dilaporkan menimbulkan efek samping berupa kerusakan gastrointestinal dan kerusakan mukosa rongga mulut apabila dikonsumsi dalam jangka panjang. tujuan: penelitian ini bertujuan untuk mengetahui pengaruh lama pemberian aspirin terhadap ekspresi ki-67 dan ketebalan epitel rongga mulut tikus galur wistar. metode: jenis penelitian eksperimental laboratories dan menggunakan 40 tikus jantan galur wistar. hewan coba dibagi menjadi 2 kelompok, yaitu kelompok perlakuan aspirin dan kontrol akuades. masing-masing kelompok dibagi menjadi 5 subkelompok berdasarkan lama pemberian aspirin, yaitu 1, 3, 5, 7 dan 10 hari. dosis yang diberikan 9 mg/kg berat badan sekali per hari. mukosa bukal tikus kemudian dipotong untuk pengecatan ki-67 dan hematoxylin-eosin (he). ekspresi ki-67 dan ketebalan epitel diukur menggunakan software imagej. data dianalisis menggunakan two-way anova and kruskallwallis dengan tingkat kepercayaan 95%. hasil: lama pemberian aspirin hari ke 1, 3, 5, 7 dan 10 dapat menurunkan ekspresi ki-67 research report 136 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 135–140 (p<0.05) dan ketebalan epitel dibandingkan kontrol, namun lama pemberian tidak berpengaruh terhadap mukosa epitel. aspirin dapat menurunkan ketebalan epitel rongga mulut dibandingkan kontrol (p<0.05). simpulan: pemberian aspirin dapat menurunkan ekspresi ki-67 pada sel epitel mukosa rongga mulut tikus galur wistar; efek tersebut berbanding lurus dengan durasi pemberian sampai hari ke-10. lama pemberian aspirin tidak berpengaruh terhadap ketebalan mukosa rongga mulut tikus galur wistar. kata kunci: aspirin, ekspresi ki-67, ketebalan mukosa rongga mulut, tikus wistar korespondensi (correspondence): dian yosi arinawati, departemen biologi mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara, yogyakarta 55281. email: dianyosi@gmail.com. pendahuluan jaringan lunak rongga mulut dilindungi oleh mukosa yang merupakan lapisan terluar rongga mulut. mukosa melindungi jaringan dibawahnya dari kerusakan dan masuknya mikroorganisme serta agen berbahaya. lapisan terluar mukosa dilindungi oleh epitel skuamosa berlapis yang mempunyai mekanisme adaptasi pertahanan yang berbeda-beda tergantung letaknya.1 jaringan epitel rongga mulut mempunyai struktur tidak stabil yang secara teratur selalu beregenerasi melalui aktivitas pembelahan sel. pembelahan sel jaringan epitel berlapis terjadi pada lapisan germinal, yaitu sel-sel yang paling dekat dengan lamina basalis, selanjutnya sel akan meninggalkan lapisan basalis dan masuk ke tahap diferensiasi.2 aktivitas pembelahan sel dipengaruhi oleh beberapa faktor diantaranya infiltrasi ringan sel inflamasi subepitel yang akan menstimulasi pembelahan sel, sedangkan inflamasi berat menyebabkan penurunan aktifitas proliferasi. proliferasi sel epitel distimulasi oleh peptide growth factor yang disebut sitokin, yaitu epidermal growth factor (egf), transforming growth factor-α (tgf-α), platelet derived growth factor (pdgf), dan interleukin 1 (il-1). obat-obatan dan radiasi juga dapat membatasi aktivitas proliferasi epitel sehingga menjadi lebih tipis dan memudahkan terbentuknya ulkus.1,2 kerusakan mukosa rongga mulut yang terjadi akibat penggunaan obat topikal maupun obat per oral salah satunya aspirin yang digunakan untuk mengatasi nyeri gigi telah banyak dilaporkan. gejala yang timbul antara lain rasa terbakar, atau nekrosis koagulasi yang ditandai dengan terbentuknya mukosa berwarna putih yang berangsur-angsur mengelupas membentuk lesi ulseratif berwarna merah. aspirin merupakan golongan obat nsaid yang sering digunakan untuk pereda atau penghilang nyeri. efek samping penggunaan obat aspirin banyak dilaporkan pada kasus saluran gastrointestinal. penggunaan dosis aspirin 500 mg/kg pada tikus satu kali sehari secara per oral dapat menimbulkan ulkus di lambung dengan ulcer index sebesar 3,2.3 mekanisme kerja aspirin, yaitu dengan menghambat jalur cyclooxigenase (cox) dan sistesis prostaglandin. penghambatan cox dapat menurunkan sekresi cairan mukus dan sekresi bikarbonat, menyebabkan kerusakan vaskular, pembentukan akumulasi leukosit, dan menghambat diferensiasi sel.4 prostaglandin (pge2) saliva berkurang selama tahap ulseratif dari stomatitis.5 peranan pge2 pada epitel mukosa lambung dan epitel mukosa rongga mulut diduga karena adanya persamaan struktur. indikasi lama pemberian aspirin disarankan tidak lebih dari 10 hari untuk mengatasi nyeri.6 aspirin dapat menyebabkan penghambatan regenerasi mukosa.7 dalam keadaan normal, sel basalis dapat berproliferasi secara berkelanjutan, kemudian sel tersebut menggantikan sel di lapisan permukaan yang hilang, sehingga integritas mukosa tetap terjaga. penghambatan aktifitas proliferasi sel menyebabkan epitel menjadi tipis dan terbentuk ulkus.1,2 proliferasi sel pada lapisan suprabasalis dapat diamati menggunakan marker ki-67.8 protein ki-67 terdeteksi di semua siklus sel kecuali fase g0 dan mencapai puncak tertinggi saat terjadi pembelahan sel.9 tujuan penelitian ini untuk menguji pengaruh lama pemberian aspirin terhadap ekspresi ki-67 dan ketebalan epitel mukosa rongga mulut tikus galur wistar. bahan dan metode penelitian ini telah mendapat persetujuan dari komisi etik fakultas kedokteran gigi, universitas gadjah mada no. 561/kkep/fkg-ugm/ec/2014. jenis penelitian yang digunakan adalah penelitian eksperimental laboratorik dengan rancangan penelitian post-test only control group design. penelitian ini menggunakan 40 ekor tikus putih jantan galur wistar, umur 3 bulan, berat badan 200-300 gram yang diperoleh dari laboratorium farmakologi, fakultas kedokteran, universitas gadjah mada, yogyakarta. tikus dibagi menjadi 2 kelompok secara acak. kelompok perlakuan, terdiri dari 20 ekor tikus wistar yang diberi aspirin per oral dalam dosis 9 mg/kg bb satu kali per hari selama 10 hari. kelompok kontrol negatif, terdiri dari 20 ekor tikus wistar yang diberi akuades per oral. masing-masing 4 ekor tikus dari kelompok perlakukan dan kontrol dikorbankan pada hari ke-1, 3, 5, 7 dan 10 setelah perlakuan. tahap selanjutnya adalah pengorbanan tikus dari kelompok kontrol maupun perlakuan pada hari ke-1, hari ke-3, hari ke-5, hari ke-7, dan hari ke-10 setelah dilakukan perlakuan. tahap selanjutnya diambil sampel jaringan pada mukosa bukal sepanjang gigi molar pertama sampai terakhir dilanjutkan difiksasi menggunakan larutan pbs formalin 10% dan dilakukan pembuatan sediaan 137arinawati, et al.: pengaruh lama pemberian aspirin pada ekspresi protein ki-67 histologis. pewarnaan dengan metode imunohistokimia dilakukan untuk mengamati ekspresi ki-67, sementara itu hematoxylin eosin untuk pengamatan ketebalan epitel. pengamatan ekspresi ki-67 dilakukan di bawah mikroskop cahaya dengan perbesaran 400x pada daerah basalis dan suprabasalis dengan 3 lapang pandang yang berbeda di sepanjang area preparat. sel yang positif dihitung pada masing-masing lapang pandang kemudian dijumlahkan dan dibagi 3. rerata sel yang positif mengekspresikan ki-67 kemudian disajikan dalam bentuk persentase (%). pengukuran epitel dilakukan pada ketebalan maksimal epitel, yaitu jarak paling panjang yang diukur dari batas terbawah lapisan sel basal sampai dengan lapisan terluar sel superfisial dan ketebalan minimal epitel. hasil pengukuran ketebalan lapisan epitel didapat dari penjumlahan ketebalan maksimal dan minimal kemudian dibagi dua. data yang diperoleh dari hasil pengamatan ekspresi ki-67 dan ketebalan epitel berskala ratio. hasil pengamatan diuji secara statistik dengan menggunakan analisis parametrik two way anova dilanjutkan uji lsd dan analisis non parametrik kruskall-wallis. analisis data dilakukan dengan level signifikansi 95%. hasil pengukuran ekspresi ki-67 dilakukan setelah prosedur histologis dan teknik imunohistokimia menggunakan a n t i b o d y k i 6 7 . r e p r e s e n t a t i f j u m l a h s e l y a n g mengekspresikan ki-67 pada sel epitel mukosa yang diberi akuades lebih banyak dibandingkan dengan kelompok aspirin (gambar 1 a, b). rerata ekspresi ki-67 pada kelompok perlakuan aspirin yang semakin menurun dari pemberian hari ke-1 sampai hari ke-10, sedangkan perlakuan menggunakan akuades menunjukkan kondisi yang relatif konstan (gambar 2). hasil uji normalitas dan homogenitas menunjukkan bahwa data yang diuji mempunyai nilai signifikansi lebih besar dari 0,05 (p>0,05) berarti data terdistribusi normal dan homogen sehingga memenuhi syarat untuk dilakukan uji parametrik dengan menggunakan uji two way anova. hasil uji two way anova menunjukkan bahwa terdapat perbedaan bermakna antara ekspresi ki-67 pada masingmasing hari dan kelompok perlakuan (p<0,05). hasil ini berarti bahwa hari pengamatan, kelompok perlakuan dan interaksi antara hari pengamatan dan kelompok perlakuan berbeda bermakna terhadap ekspresi ki-67. selanjutnya untuk mengetahui perbandingan ekspresi ki-67 antara masing-masing subkelompok hari perlakuan, dilakukan uji least significant different (lsd). hasil analisis statistik menggunakan uji lsd menunjukkan bahwa sebagian besar subkelompok mempunyai nilai probabilitas kurang dari 0,05 (p<0,05), hal tersebut mengindikasikan sebagian besar kelompok mempunyai perbedaan yang bermakna antar hari perlakuan. pengukuran ketebalan epitel dilakukan menggunakan software imagej setelah pengecatan preparat menggunakan hematoxylin-eosin dan pengambilan gambar mikroskopis menggunakan kamera optilab. ketebalan epitel maksimal dan minimal pada mukosa yang diberi aspirin dapat dilihat pada gambar 3a-b, sedang ketebalan epitel maksimal gambar 1. a) ekspresi ki-67 pada lapisan basalis dan suprabasalis setelah pemberian aquades setelah hari ke-1; b) ekspresi ki-67 pada lapisan basalis dan suprabasalis setelah pemberian aspirin setelah hari ke-1. a b gambar 2. rerata ekspresi ki-67 pada kelompok perlakuan dan hari perlakuan. 138 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 135–140 dan minimal pada mukosa yang diberi akudes dapat dilihat pada gambar 4a-b. perbandingan rerata ketebalan epitel kelompok perlakuan dan kontrol dapat dilihat pada gambar 5. hasil uji normalitas menunjukkan bahwa data yang diuji mempunyai nilai signifikansi lebih dari 0,05 (p>0,05) berarti data terdistribusi normal. hasil uji homogenitas menunjukkan bahwa data yang diuji mempunyai signifikansi kurang dari 0,05 (p<0,05) mengindikasikan data tidak homogen sehingga tidak memenuhi syarat untuk dilakukan uji parametrik dengan menggunakan two way anova. analisis dilanjutkan menggunakan uji non parametrik menggunakan kruskal-wallis. hasil uji kruskal-wallis menunjukkan bahwa terdapat perbedaan bermakna antara kelompok perlakuan aspirin dibandingkan akuades (p<0,05). hasil ini menunjukkan bahwa semua subkelompok pada perlakuan aspirin dibandingkan akuades mempunyai perbedaan bermakna terhadap ketebalan epitel. kelompok perlakuan akuades mempunyai epitel yang lebih tebal dibandingkan kelompok perlakuan aspirin, sedangkan nilai ketebalan epitel antar subkelompok berdasarkan lama perlakuan menunjukkan perbedaan yang tidak bermakna (p>0,05). hasil ini mengindikasikan bahwa hari pengamatan tidak berpengaruh secara signifikan terhadap ketebalan epitel. pembahasan aspirin merupakan golongan asam asetil salisilat yang banyak digunakan sebagai pereda nyeri, peradangan atau anti trombosis. hasil penelitian menunjukkan bahwa pemberian aspirin dosis 9 mg/kg bb peroral pada tikus galur wistar dapat menurunkan jumlah ekspresi ki-67 gambar 3. a) ketebalan epitel maksimal dan minimal setelah pemberian aspirin hari ke-1; b) ketebalan epitel maksimal dan minimal setelah pemberian aspirin hari ke-10. gambar 4. a) ketebalan epitel maksimal dan minimal setelah pemberian akuades setelah hari ke-1; b) ketebalan epitel maksimal dan minimal setelah pemberian akuades setelah hari ke-10. gambar 5. rerata ketebalan epitel mukosa bukal rongga mulut tikus galur wistar pada kelompok perlakuan dan kontrol. a b a b 139arinawati, et al.: pengaruh lama pemberian aspirin pada ekspresi protein ki-67 dibandingkan dengan kontrol (akuades). hasil penelitian menunjukkan bahwa terdapat penurunan indeks proliferasi dari pemberian hari pertama sampai hari kesepuluh. aspirin dilaporkan mempunyai efek anti proliferasi disebabkan oleh menurunnya faktor-faktor pertumbuhan (growth factors) dalam sel. penghambatan proliferasi sel disebabkan mekanismenya dalam menghambat platelet-derived growth factor (pdgf). dilaporkan pula bahwa ekspresi transforming growth factor (tgf-β) menurun sebanding dengan penghambatan beberapa growth factors lainnya.10 tgf-β merupakan molekul yang dibutuhkan oleh sel dalam melakukan proliferasi sel di lambung, usus, kulit, rongga mulut, dan mekanisme penyembuhan luka. aspirin dilaporkan dapat menyebabkan perdarahan lambung dan penurunan integritas mukosa.6 kegagalan pemeliharaan integritas mukosa terjadi akibat penurunan ekspresi prostaglandin. penurunan prostaglandin diakibatkan oleh penghambatan enzim cyclooxigenase. cyclooxigenase (cox) merupakan enzim yang berperan penting sebagai katalisator konversi asam arakhidonat menjadi cyclicprostaglandin endoperoxides. prostaglandin (pge2) adalah suatu agen penting dalam saliva yang mempunyai peran melindungi mukosa mulut. dilaporkan bahwa penderita stomatitis mempunyai kadar pge2 yang menurun. 5 prostaglandin dalam saliva mempunyai peran sebagai mekanisme pertahanan mukosa mulut seperti peran prostaglandin dalam pertahanan mukosa di lambung. penghambatan cox-1 pada mukosa lambung memicu terjadinya penurunan mukus dan bikarbonat pada lambung, mengurangi aliran darah mukosa, kerusakan vaskular, akumulasi leukosit, penurunan kemampuan turnover, dan semua faktor yang berkontribusi pada perbaikan mukosa.4 hasil penelitian ini menunjukkan bahwa terjadi penurunan ekspresi ki-67 dari hari pertama pemberian aspirin sampai hari kesepuluh. hal ini kemungkinan disebabkan karena protein ki-67 responsif terhadap aspirin. protein ki-67 terekspresi pada semua sel selama berlangsungnya siklus sel. ekspresi tersebut terdeteksi pada semua fase siklus sel pada g1, s, g2, dan m (mitosis), kecuali fase g0.11 pengaruh aspirin terhadap ekspresi ki67 terdeteksi paling tinggi pada fase g1. aspirin menahan fase g1 sehingga proses pembelahan sel terhambat.12 hasil penelitian ini juga menunjukkan bahwa hari pengamatan tidak berpengaruh secara signifikan terhadap ketebalan epitel. pemberian aspirin hari pertama sampai hari kesepuluh tidak menyebabkan perubahan ketebalan epitel yang bermakna. data membuktikan bahwa aspirin dapat menurunkan ketebalan epitel mukosa bukal tikus galur wistar, tetapi lama pemberiannya tidak berpengaruh secara signifikan. data tersebut sesuai dengan penelitian lacy dkk. bahwa pemberian salin pada mukosa lambung tikus tidak mengakibatkan perubahan morfologi, fisiologi, maupun ketebalan epitel.13 sebaliknya mukosa lambung yang mendapat iritan dalam jangka waktu 2 minggu menyebabkan penurunan ketebalan epitel sebesar 50%. penurunan ketebalan terjadi akibat iritasi kronis pada permukaan epitel mukosa lambung.13 aspirin mempunyai efek lokal yang lebih tinggi pada mukosa lambung dibandingkan mukosa lain,14 sehingga meskipun dilaporkan terjadi penurunan ketebalan epitel mukosa lambung, ketebalan epitel mukosa bukal rongga mulut tikus galur wistar tidak terpengaruh. penggunaan aspirin per oral dapat diabsorbsi sebanyak 70% di lambung dan sisanya diabsorbsi di usus halus bagian atas. aspirin akan menyebar ke seluruh tubuh dan cairan transeluler setelah diabsorbsi, seperti cairan sinovial, cairan spinal, saliva dan air susu.14 respon mukosa rongga mulut tidak sama dengan mukosa lambung karena efek lokal di lambung tidak sama seperti efek secara sistemik. rongga mulut subjek pada penelitian ini adalah normal. rongga mulut dan lambung mempunyai persamaan struktur penyusunnya, yaitu sama-sama dilapisi oleh membran mukosa pada lapisan terluarnya, lamina propia pada lapisan dibawahnya serta jaringan yang dapat memproduksi kelenjar. perbedaan membran mukosa keduanya terletak pada lapisan penyusun epitel. lapisan mukosa lambung tersusun atas epitel selapis kolumner (simple columnar epithelium), sedangkan rongga mulut tersusun atas epitel berlapis gepeng (stratified squamous epithelium). secara fisiologi, keduanya mempunyai persamaan dalam fungsi pencernaan dan motorik.1 aspirin dapat menurunkan ekspresi ki-67 pada penelitian ini kemungkinan karena persamaan struktur dan fisiologi jaringan lambung dan rongga mulut, sedang lama pemberian aspirin tidak menurunkan ketebalan epitel kemungkinan karena perbedaan lapisan epitel yang menyusun kedua jaringan tersebut. penelitian ini menunjukkan bahwa aspirin dalam waktu pemberian 10 hari mempengaruhi ekspresi ki-67, namun tidak berpengaruh pada ketebalan epitel mukosa bukal. daftar pustaka 1. squire ca, mary jk. biology of oral mucosal and esophagus. j of nat can ins mon 2001; 29: 7-15. 2. junqueira lc, carneiro j, kelley ro. jaringan epitel dalam histologi dasar. sugiarto k, santoso a, editors. jakarta: egc; 1995. h. 6290. 3. shah js, patel jr. short communication: anti-ulcer activity of lucer against experimentally induced gastric ulcers in rats. ayu j2012; 33(2): 314-6. 4. halter f, tarnawski as, schmassmann a, peskar bm. cyclooxygenase 2implications on maintenance of gastric mucosall integrity and ulcer healing: controversial issues and perspectives. gut 2001; 49(3): 443-53. 5. wang cyw, patel m, feng j, milles m, wang sl. decreased levels of salivary prostaglandin e2 and epidermal growth factor in recurrent aphtous stomatitis. archs oral biol 1995; 40 (2): 1093-8. 6. yagiela ja, dowd fj, neidle ea. pharmacology and therapeutics for dentistry. 5th ed. missousi: mosby; 2004. p. 337-52. 7. seleem hs, ghobashy ha, zolfakar as. effect of aspirin versus aspirin and vitamin c on gastric mucosal (fundus) of adult male albino rats. histological and morphometric study egypt j histol 2010; 33(2): 313-26. 140 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 135–140 8. gonzales-moles, bravo, ruiz-avila, acebal f, gil-monyoya ja, brener s, esteban f. ki-67 expression in non-tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumours. j oral dis 2009; 16(1): 68-75. 9. jonat w, arnold n. is the ki-67 labelling index ready for clinical use?. ann oncol 2011; 22(3): 500-2. 10. hoefer ie, grundman s, schirmer s, royen vn, meder b, bode c, piek jj, buschmann ir. aspirin, but not clopidogrel, reduces collateral conductance in a rabbit model of femoral artery occlusion. j am coll cardiol 2005; 46(6): 994-1001. 11. scholzen t, gerdes j. the ki-67 protein: from the known and the unknown. j cell physiol 2000; 182(3): 311-22. 12. redondo s, santos-gallego cg, ganado p, garcía m, rico l, del rio m, tejerina t. acetylsalicylic acid inhibits cell proliferation by involving transforming growth factor-β. circulation 2003; 107(4): 626-9. 13. lacy er, cowart k s, king js, delvalle, j, smolka aj. epithelial response of the rat gastric mucosal to chronic superficial injury. yale j biol med 1996; 69(2): 105-8. 14. wimana ff. analgesik-antipiretik analgesik anti-inflamasi steroid dan obat pirai. jakarta: fakultas kedokteran universitas indonesia; 1995. h. 207-22. 172 volume 45 number 3 september 2012 facial reconstruction using polypropylene mesh after resection of maxillary ossifying fibroma r. soesanto department of oral and maxillofacial surgery faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: ossifying fibroma is a variant of fibrous dysplasia and catagorized as osteofibrosis lesion. it commonly affects long bones but occasionally involves jaws. mandible affected more common than maxilla. the treatment of ossifying fibroma include excision and resection. excision of lesion and resection of maxilla could cause facial defect which cannot be reconstructed with bone graft. purpose: the aim of this case report is to report the potential use of polypropylene mesh in facial reconstruction after hemimaxillectomy in patients diagnosed with ossifying fibroma of the maxilla. case: a 17-years-old female patient came to oral and maxillofacial surgery clinic, dental hospital, faculty of dentistry, airlangga university, with chief complaint of swelling in the upper left cheek of 2 years duration which was not related to any history of toothache. patient also complained of the itchiness and pain on the swelling area occasionally as well as salty discharge from the mass. the fna was done and the citology result indicated a benign mesenchimal tumor. incisonal biopsy was subsequently performed and the histopathology report confirmed the diagnosis of cementifying fibroma. case management: left hemimaxillectomy was done, and post surgical defect in the facial and buccal aspect was immediately reconstructed using three layers of polypropylene mesh. on follow-up, eleven months post-operatively, the patient was well and there was no facial deformity or asymmetry. conclusion: polypropylene mesh is a potential material for facial reconstruction as it can reduce the risk of facial deformity after hemimaxillectomy of patients with tumor of the maxilla. key words: polypropylene mesh, maxilla reconstruction, hemimaxillectomy, ossifying fibroma abstrak latar belakang: ossifying fibroma merupakan varian dari fibrous dysplasia dan termasuk di dalam kategori lesi osteofibrosis. umumnya mengenai tulang panjang tetapi dapat juga mengenai rahang dan lebih banyak menyerang tulang mandibula dibanding maksila. perawatan ossifying fibroma dilakukan dengan eksisi atau reseksi. perawatan eksisi atau reseksi tulang maksila dapat mengakibatkan deformitas wajah yang tidak dapat direkontruksi dengan bone graft. tujuan: laporan kasus ini bertujuan untuk melaporkan penggunaan polipropylene mesh pada rekonstruksi wajah setelah hemimaxillectomy pada pasien dengan diagnose ossifying fibroma pada maksila. kasus: penderita wanita berusia 17 tahun datang ke klinik bedah mulut dan maksilofasial, rumah sakit gigi dan mulut fakultas kedokteran gigi universitas airlangga, dengan keluhan utama pembengkakan pada pipi kiri atas yang muncul sejak 2 tahun yang lalu tanpa ada riwayat keluhan sakit gigi. penderita mengeluh gatal, kadang sakit dan keluarnya cairan asin pada daerah benjolan. hasil fna dan test sitologi menunjukkan gambaran tumor jinak mesensimal. pemeriksaan dengan biopsi dan histopatologi mengkonfirmasi diagnosa cementifying fibroma. tatalaksana kasus: dilakukan hemimaksilektomy pada sebelah kiri, dan defek pasca pembedahan pada sisi fasial dan bukal segera direkontruksi dengan pemasangan polipropylene mesh 3 lapis. sebelas bulan setelah operasi kondisi pasien baik dan tidak ada deformitas atau asimetri wajah. kesimpulan: polypropylene mesh merupakan bahan yang potensial untuk rekonstruksi wajah karena dapat mengurangi terjadinya deformitas wajah setelah hemimaxillectomy pada pasien dengan tumor maksila. kata kunci: polypropylene mesh, rekostruksi maksila, maksilektomi, ossifying fibroma correspondence: r. soesanto, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: soesantoraden@yahoo.com case report 173soesanto: facial reconstruction using polypropylene introduction ossifying fibroma is a neoplasm consisting of fibrous tissue that contains a mixture of trabecular bone, cementum as spherules or both.1 ossifying fibroma or cementoossifying fibroma or cementifying fibroma, are also called osteogenic neoplasms. it may be a variant of fibrous dysplasia and included in the category of osteofibrosis lesion. the jaw lesions are wellcircumscribed and slowly growing. because of the slow growth, the cortical plates of the bone and the overlying mucosa or skin are invariably intact. they are generally asymptomatic until the growth produces a noticeable swelling and mild deformity; displacement of teeth may be an early clinical sign. pain and parathesia are rarely associated with an ossifying fibroma.1-4 most often found in the third and fourth decades, frequently occur in female radiographically, at an early stage shows well demarcated radiolucent area appearance, which turn rapidly into calcified lesions, and the lesion edges become less clear. overall, radiographic view describes radiolucent area containing various level of radiopacity along with resorption and diverging of tooth apical. rare case shows fully radiopaque area with radiolucent edge. ossifying fibroma was treated with wide excision or enucleation. treatment includes surgical removal of the lesion including the periosteum which reduces the high recurrence rate.5 resection of the maxilla, however, would usually result in noticeable facial defect. the defects are usually reconstructed directly with surgical obturators. they are usually constructed from base plate made of acrylic splint using the remaining contralateral teeth as retainers. they serve to support the defect obturator usually made of silicone impression material. however, in cases where all the maxillary teeth are retained after tumor resection, it is not possible to use such methods. therefore, certain materials are required to support the facial defect which do not use palatal splint. polypropylene mesh is prosthetic, non absorbable, standard flat mesh made from polypropylene which has a tensile strength that is physiologically required.6 polypropylene material was commonly used as prostesis for hernia repair to close the abdominal wall defect. polypropylene mesh was also used on reconstruction of auricular defect as alloplast.7–12 the following case is ossifying fibroma which has extended to buccal part of maxilla and a half part of zygoma, so that the tumor resection had resulted in a facial deformity. to compensate for the deformity, the authors performed a reconstruction where the inside of the maxillary buccal wall part was supported with 3 layers of foldable polypropylene mesh material. in the author's opinion this is a novel technique in that this material has never been used for facial reconstruction. the purpose of this case report is to present the potential use of polypropylene mesh as one of reconstruction materials in oral and maxillofacial tumor surgeries. case figure 1. preoperative extraoral view. on frontal view (left) a ill-defined border, smooth surfaced mass was seen on the left cheek and maxilla, which has normal color. on left view (right), a mass was noted over left zygomatic bone. a 17-years-old female patient came to oral and maxillofacial surgery clinic, dental hospital, faculty of dentistry, airlangga university, with chief complaint of swelling in upper left cheek which has been growing for the past 2 years, and not related to any history of toothache. patient also complained of itchiness and pain on the swelling area occasionally, and there was salty discharge from the mass. on the previous treatment by hospital in tuban, analgesic and antibiotics were prescribed. extra oral examination showed facial asymmetry, a mass was noted over the left cheek with unclear border, showing smooth surface and normal color (figure 1). intra oral examination showed elevated nasolabial sulcus, a mass was seen over both the palatal and buccal aspect from canine to upper first molar, 4 × 6 cm in size, solid hard and tender on palpation. there was a pus and salty discharge from the mass via drainage in the buccal mass. there were no loose teeth and no dental caries observed (figure 2). figure 2. preoperative intra oral view. noted over left cheek, a mass with unclear border and elevated of nasolabial sulcus. 174 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 172–176 ct scan examination showed a solid mass in the left sinus maxilary, 6.35 × 5.27 × 6.08 cm in size, with calcified components inside causing erosion of the left maxillary sinus bone walls to the base of the left orbital cavity and extends to the right causing nasal septal deviation to the right. water's radiographic showed radiopaque area in left maxilla (figure 3). open biopsy was performed preoperatively and the histopathology report showed cementifying fibroma with no signs of malignancy. case management since the tumor has spread to the left eye, joint operation with opthalmologist was performed to ensure tumor free margin in the inferior border of orbita. hemimaxillectomy was performed with ferguson weber design incision to obtain a wide view of zygoma area. the tumor, on the buccal aspect, was found to have extended to the zygomatic area. the tumor and the surrounding maxillary bone was then resected. after tumor resection a large facial and buccal defect was found causing noticeable assymetry. to overcome the defect, three layers of foldable polypropylene mesh were placed inside the wall of maxillarybuccal area, and sutured into surrounding tissue. since these material are relatively thick and rigid. they were able to support the bulk of facial and buccal soft tissue thus resulted in facial convexity and symmetry (figure 4). histopathology examination of the lesion showed a benign neoplastic tissue which was composed of proliferating fibroblast, with spindle-shaped, uniform nuclei showing fine chromatin. calcifiying bone trabeculae were seen. there was no sign of malignancy. a b figure 3. preoperative ct scan and water's radiographic view. ct scan showed a solid mass in the left sinus maxilary with calcified components inside and nasal septal deviation to the right (a). water's radiographic showed radiopaque area in left maxilla (b). figure 4. application of propylene mesh in defect area. the mass was sutured into near tissue (left). suturing postoperative (right). 175soesanto: facial reconstruction using polypropylene at follow-up 11 months postoperatively, the patient was well and there was minimum facial asymmetry. intra orally, there was no signs of material rejection and wound seemed to have healed properly (figure 7). discussion from clinical and radiographic examination of the case above, the tumor may be diagnosed as ossifying fibroma or fibrous dysplasia due to the same view. however the treatment of both cases are quite difference of which ossifing fibroma is usually treated radically with wide excision or enucleation because of the recurrency rate, whereas fibrous dysplasia was treated with a surgical contouring of the mass to restore to normal. bone retrieval is easy to do because the bone is generally soft and bone retrieval was performed to obtain better healing. therefore, histopathology diagnosis is required with a careful examination because will determine the treatment plan of the case. maxillary tumor surgery was performed with two techniques; according to weber-incision pattern-longmire verguson, and patterns according midfacial degloving incision.5 in this case, the hemimaxillectomy was performed using incision pattern of verguson weber, therefore a wide view of zygoma area could be obtained. on the walls of the maxillary buccal area, there is an extension of the tumor in the part of zygoma, so that the ressection would be resulting a facial deformity. to reduce the deformity, the authors performed the reconstruction of the maxillary buccal wall part using 3 layers of foldable polypropylene mesh material. the material was thick and stiff,6,13–15 therefore it could support the bulk of soft tissue of the face and cheek, reducing the facial deformity. polypropylene mesh material has never been used for post maxillectomy reconstruction. polypropylene mesh was chosen because of its strong ideal material protesis, flexible, non-allergenic, inert, non-biodegradable, noncarcinogenic and should stimulate fibroblastic activity for optimal tissue healing, connecting to normal tissue. polypropylene material was commonly used as protesis for hernia repair to close the abdominal wall defect. polypropylene mesh was also used on reconstruction of auricular defect as alloplast.7–12 this is in accordance with the result of this case report. at follow up review 11 months postoperatively, polypropylene mesh material that was used in this case did not cause any complaints from the patient nor sign of material rejection. it is, therefore, considered biocompatible for use as permanent biomaterial in oral and maxillofacial region. it can be concluded that polypropylene mesh can be used as reconstruction material after hemimaxillectomy on patient with ossifying fibroma. the material can support the facial and buccal soft tissue to reduce the resulting facial deformity. besides, polypropylene mesh is clinically safe and biocompatible to be used as a permanent alloplastic material in oral and maxillofacial tumor surgery. figure 5. day-9 postoperative extra oral view. from frontal and lower view showed no difference between left and right facial and zygomatic area. figure 6. day 9 postoperative intra oral view. the mucosa had normal color and well sutured. figure 7. eleventh month postoperative. extraoral view showed minimum asymetry (left). intraoral view showed normal mucosa color (right). post surgery evaluation showed a satisfactory result. day-9 post surgery showed that there was no facial assymetry (figure 5). intra orally, there was no signs of soft tissue inflammation which would indicate that there was no rejection against polypropylene mesh material (figure 6). 176 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 172–176 references 1. knutsen bm, larheim t a, johannessen s, hillestad j, solheim t, stromme koppang h. case report recurent conventional cementoossifying fibroma of the mandible. dentomaxilofacial radiology 2002; 31: 65–8. 2. canger em. familial ossifying fibromas: report of two cases. journal of oral science 2004; 46(1): 61–4. 3. tamiolakis de, tomaidis va, tsamis io. cemento-ossifying fibroma of the maxilla: a case report. acta stomatology croat 2005; 39: 319–21. 4. neville bw, damm dd, allen c, bouquot j. oral and maxillofacial pathology. 2nd ed. philadelphia: wb saunders; 2002. p. 563–4. 5. converse jm. rekonstructive plastic surgery. 2nd ed. philadelphia: wb saunders co; 1977. p. 2578–86. 6. vrijland ww. mesh repair of hernias of the abdominal wall. rotterdam: educost publishers;. 2003. p. 43–50. 7. soames jv, joutham jc. oral pathology. 3rd ed. oxford: oxford university press; 1999. p. 292–5. 8. parra ja, revuelta s, gallego t. pictorial review: prosthetic mesh used for inguinal and ventral hernia repair: normal appearance and complications in ultrasound and ct. brit j rad. 2004; 77: 261–5. 9. lin g, lawson w. complications using grafts and implants in rhinoplasty. op tech otolaryng. 2007; 18: 315–23. 10. doctor hg. symposium: evaluation of various prosthetic materials and newer meshes for hernia repairs. j min acc surg 2006; 2(3): 110–6. 11. klosterhalfen b, junge k, klinge u. review: the lightweight and large porous mesh concept for hernia repair. expert rev med devicess 2005; 2(1): 1–15. 12. paula e, silva e, toledo oa. tissue repair after orbital reconstruction using polypropylene mesh implants: a histological study in dogs. rev odonto scienc 2009; 24(4): 396–400. 13. paula e, silva e, rosa els, barbosa sv. tissue reactions to polypropylene mesh used in maxillofacial trauma. braz dent j 2001; 12(2): 121–5. 14. schmidbauer s, ladurner r, hallfeldt kk, mussack t. heavy-weight versus low-weight polypropylene meshes for open sublay mesh repair of incisional hernia. eur j med res 2005; 10: 247–53. 15. conze j, kingsnorth an, flament jb. randomized clinical trial: randomized clinical trial comparing lightweight composite mesh with polyester or polypropylene mesh for incisional hernia repair. brit j surg 2005; 92: 1488–93. 154 dental journal (majalah kedokteran gigi) 2017 september; 50(3): 154–159 case report gingival enlargement as oral manifestation in acute myeloid leukemia patient sandra olivia kuswandani, yuniarti soeroso, and sri lelyati c. masulili department of periodontics faculty of dentistry, universitas indonesia jakarta indonesia abstract background: acute myeloid leukemia (aml) is a bone marrow cancer, a malignant disease that triggering the cells develops into different types of blood cells. it is widely recognized that the main manifestation of aml could be gingival hyperplasia and bleeding. occasionally, an initial diagnosis of leukemia is made after a dental examination. in relation to systemic diseases, gingival enlargement could constitute the intensification of an existing inflammation initiated by dental plaque, or a manifestation of the systemic disease independent of the inflammatory condition of the gingiva. gingival enlargement negatively affects the quality of life, especially nutritional intake. purpose: this study aimed to report on gingival enlargement in aml patients, dental management of this condition and considerations when treating patients. case: a 46 year-old woman diagnosed with aml who chiefly complained of gingival enlargement in all parts of the mouth which restricted her nutritional intake. case management: the subject attended the clinic twice where nonsurgical treatment for the gingival enlargement, supragingival scaling and dental health education to maintain her oral hygiene was carried out. unfortunately, she did not return for follow-up appointments due to having already passed away. information about aml and its relation to gingival enlargement contained in the literature is also reviewed. conclusion: in conclusion, gingival enlargement represents one oral manifestation of aml. this condition is related to and affects the nutritional intake of the patient. keywords: acute myeloid leukemia; gingival enlargement; blast cells; periodontal systemic disease related correspondence: sandra olivia kuswandani, department of periodontics, faculty of dentistry, universitas indonesia. jl. salemba raya jakarta 10430, indonesia. e-mail: drg.sandra@gmail.com introduction acute myeloid leukemia (aml) is a bone marrow cancer, a malignant disease that triggering the cells develops into different types of blood cells. generally, aml disease afflicts older person and is remarkably in individuals below the age of 45. the average age of a patient with aml is approximately 67 years. aml is commonest in men than women, but the average lifetime risk for both sexes is less than half of 1%. the site of aml disease is bone marrow where new blood cells are produced and it usually circulates quickly into the blood. consequently, it can sometimes spread to other parts of the body, including the central nervous system (brain and spinal cord), liver, lymph nodes, spleen, and testicles. in term of the acute leukemia, the immature blood cells of cancerous cells are called blasts. these normal blast cells divide quickly which cause the leukemias fast-growing. leukemia cells do not stop dividing when normal blast cells would.1,2 it presents with marrow failure and cytopenia. symptoms include fever, fatigue, pallor, mucosal bleeding, petechiae, and local infections.3 aml presents oral manifestations which represent a number of the diagnostic indicators of the disease. the oral symptoms of acute leukemia include the swollen gingiva, petechiae, mucosal anemic, oral ulceration, spontaneous gingival bleeding candidacies and herpetic infections.4 the first diagnosis of leukemia to be made after a dental examination might be because of the main oral appearance of aml are the bleeding and hyperplasia of gingiva.5–7 from the surveillance, epidemiology, and end results (seer) data in year 2011 for aml during were estimated to be 17.5 per 100,000 (n=7,245) among the ≥65-year-old population and 1.8 per 100,000 (n=4,864) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 mailto:drg.sandra@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 155155kuswandani, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 154–159 for those <65 years old.5 the term of gingival hyperplasia is commonest in acute, rather than chronic, leukemia. whilst, it is unpredictable in any individual patient for progress of gingival infiltration.8 the enlargement of the gingiva could constitute the compounding of an existing inflammation initiated by dental plaque or a manifestation of a systemic disease which is independent of the inflammatory status of the gingiva.9 oral manifestations of leukemia may include bleeding, oral ulcerations, leukemic infiltration until periodontal infections. the expression of these symptoms is less in chronic than acute and subacute forms of leukemia.9 a dentist’s role in identifying oral manifestation of aml is central. firstly, a diagnosis of leukemia is occasionally established after a dental examination. the dentist’s role is to identify leukemic disease by means of oral examination, and referral to an internist or oncology department. second, a dentist plays an important role in patient education about the importance of maintaining oral health and advising about the oral manifestation that may affect the quality of life.10 the case of a female patient diagnosed with aml and complaining chiefly of generalized gingival enlargement which affected her nutritional intake was reported. the internist referred the patient for a dental consultation to locate the focus of infection and gum infiltration. the purpose of this report was to explain the cases of gingival enlargement in aml patients, dental management of this condition and considerations connected with treating the patient. case a 46 year-old-female attended the periodontics specialist clinic, faculty of dentistry, universitas indonesia, following referral by an internist at cipto mangunkusumo hospital in order to locate the focus of infection and gum infiltration. the individual concerned was diagnosed with aml, her chief complaint being bleeding gums during the previous month. she presented symptoms including: an enlarged gum, difficulty in swallowing and constant pain. according to her medical history, prior to the previous three months, when she had suffered from fever, mild weight loss and loss of appetite the patient had appeared normal. in the objective examination, the patient was in a good general condition, good communication, and had signs of anemia with the skin look pale. on extraoral examination, there was no abnormality. on intraoral examination, generalized gingival enlargement was noticed in upper and lower arches, buccally and lingually or palatally, and reached half of teeth surface. the color was pink, with plaque and supra and subgingival calculus (figure 1). case management on the patient’s first consultant of october 2nd 2015, an initial therapy for periodontal treatment including dental health education, supra-gingival scaling, and antimicrobial therapy using chlorhexidine 0.12% to improve oral hygiene was conducted. the patient was referred to an oral medicine specialist for an oral lesion examination and sent back to the internist to potentially undergo a gingivectomy. based on the findings of the systemic and intraoral examinations, the patient was advised of the desirability of a routine blood test. on october 22nd, 2015, the results of a blood check and flow cytometry were reported by the department of pathology clinic (table 1). the contents of the table show that the highest percentage of leucocyte types was that of blast cells (73%). the result of the flow cytometry confirmed the presence of a positive marker in cd 33, cd 34, cd 117, hla-dr, cd 13 (figure 2). the interpretation from the department of pathology was myeloid lineage. figure 1. clinical view of gingival enlargement in aml patient. (a) right side; (b) left side; (c) frontal view; (d) maxilla occlusal; (e) mandible occlusal. from a clinical viewpoint, the condition of almost all the tooth surface in premolar and molar region were covered by enlargement of the gingiva. the color was bluish red in the gingival margin. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 156 kuswandani, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 154–159 the patient was subsequently scheduled for chemotherapy treatment. on december 9th, 2015, the patient presented the symptom of almost all premolar and molar region tooth surfaces being covered by an enlargement of the gingiva. the color was bluish red in the gingival margin. the patient was recommended to maintain oral hygiene and a consultation letter was sent to her internist doctor enquiring about the possibility of conducting a gingivectomy if prechemotherapy hematologic values reached the minimum required levels. however, unfortunately, the patient had already passed away. discussion aml in other term used as acute myelogenous leukemia, acute myeloblastic leukemia, acute granulocytic leukemia or acute nonlymphocytic leukemia, is a bone marrow fast-growing cancer. the fast growing means aml occurs when bone marrow produces blasts, cells that have not yet wholly matured, which normally develop into white blood cells. unfortunately, these cells do not grow and are unable to ward off infection. chemotherapy, radiation therapy, stem cell transplant and immunotherapy are treatment choices of aml.1–3 in 1997, based on who classification, aml was sub-divided into four groups which are aml with recurrent cytogenetic translocations, aml with myelodysplasia-related features, therapy-related aml and myelodysplastic syndromes and non-specific aml.12 several forms of treatment may be applied for people suffering from the condition. chemotherapy is the primary treatment for aml, occasionally in tandem with a targeted therapy drug. other drugs may also be used to treat people with acute promyelocytic leukemia (apl). in extraordinary condition, surgery and radiation therapy may be resorted.1 gingival enlargement in aml patients is one case that a dentist should understand and treat with appropriate remedial measures. swelling of the gingiva is a standard clinical feature of the condition. the term “gingival enlargement” merely describes the clinical finding, rather than also seeking to offer an explanation of its fundamental nature.3 the clinical findings made from periodontal examination in patient majority lead to diagnosis of aml as oral manifestations of the disease.12 in some instances, aml could first be identified by a dentist or periodontist, following a chief complaint of gingival enlargement.13 without the sharing of knowledge of the patient’s condition, the dentist will apply treatment potentially aggravating it, thereby possibly exacerbating acute symptoms. in the most extensive review of the topic, observation of gingival enlargement in aml patient range in the frequency of 3% to 5% among 1,076 patients receiving anti-leukemia chemotherapy at a referral center.8 oral manifestations of leukemia may include, bleeding, oral ulcerations, leukemic infiltration until periodontal infections. infiltration of leukemic cells into the gingiva and, but less frequently to the alveolar bone. the table 1. the result of hematologic findings in molecular diagnostic of leucocytes type of leucocytes result normal value unit basophil 0.0 0 – 1 % eosinophil 0.0 % neutrophil rod 0.0 2 – 6 % neutrophil segment 1.5 50 – 70 % limphocyte 7.0 20 – 40 % monocyte 2.5 2 – 8 % blast 73.0 % pro mielocyte 0.0 % pro limphocyte 0.0 % pro monocyte 0.0 % mielocyte 0.0 % metamielocyte 0.0 % rubriblast 0.0 % prorubricyte 1.0 % rubricyte 4.0 % metarubricyte 3.0 % plasmocyte 7.5 % histiocyte 0.5 % dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 157157kuswandani, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 154–159 accumulation of immature blast cells in the gingiva is related to tooth surface with bacterial plaque. leukemic gingival enlargement consists of a primary infiltration of the gingival corium by leukemic cells, this increases the gingival thickness resulting gingival pockets in which bacterial plaque accumulates. this condition initiating a secondary inflammatory lesion that role to the enlargement of the gingiva.9 the observation result of aml-m4 and m5 subtypes patients that are the leukemic cells are monocytic. monocytes has strong chemo attractant ability to infiltrate tissues.6 in this study, the case was mild periodontitis, but low-level antigens derived from periodontal bacteria acted as chemo attractants for myelomonocytic leukemic cells. in fact, the observation of notable mucosal enlargement only appears in the gingiva. the gingival enlargement manifested from leukemia may involve the accumulation of blast cells in the gingiva by chemo attractants derived from periodontal pathogens. gingival enlargement as a rule be more severe in patients with severe periodontitis, which is often observed in aml-m4 and m5.6 under microscopic examination, the gingiva was observed to be predominantly infiltrated by immature leucocytes in the marginal gingiva, and also attached gingiva. indication of ectopic hematopoiesis could be seen as mitotic figures. the leukemic cells supplant normal connective tissue components of the gingiva. the feature of the cells depends on the type of leukemia. the cellular accumulation is denser in all layers of reticular connective tissue. the epithelium presents various changes and may be thinned or hyperplastic.9 the planning of periodontal treatment in aml patients should consider the hematologic values of the patient. the role of the dentist should be conducted at three different stages: pre-antineoplastic treatment, during antineoplastic treatment, and post-antineoplastic treatment.14 in this case, the patient was undergoing pre-antineoplastic treatment that necessitates proper nutritional intake to promote an excellent health condition. in the pre-antineoplastic treatment of aml, dental figure 2. the results of flow cytometry show the positivity of cd 33, cd 34, cd 117, hla dr and cd 13 which represent the myeloid lineage cell. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 158 kuswandani, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 154–159 treatment is based on priorities and should be directed towards meeting acute needs. the identifiable aims of dental treatment include: preventing potential infection from an oral condition, education for maintaining oral health and raising awareness about the possibility of specific issues relating to oral tissue such as gingival enlargement.10 shankarapillai et al. reported their research into the periodontal health parameters in young adults with aml in kerala, south india. the research confirmed around three-quarters of the patients to be suffering from either reasonable or poor oral hygiene. the association between dental plaque levels and both gingival overgrowth and periodontal index was observed resulted statistically significant (p<0.001). the resulting conclusion was that poor oral hygiene is a risk factor for leukemic gingival overgrowth and defect in periodontal disease.15 in the case of the patient reported here, the improvement of oral hygiene proved critical to periodontal healing. a decision to undertake invasive procedure gingivectomy will be considered if the minimum hematologic values have reached the required level. with regard to the risk of bleeding and severe infection associated with invasive procedures in the oral cavity, there are certain protocols that emphasize the importance of evaluating individual hematological indices, mainly: neutrophils and platelets. the us national cancer institute argues that interventions at this stage should be directed at the treatment of lesions in the oral mucosa, the lesions of carious and endodontic, periodontal disease, unfitting dentures, orthodontic appliances, temporomandibular joint disorder and dysfunction of the saliva.16 the minimum hematologic values in pre-chemotherapy treatment patients of the us national cancer institute featured in table 2, show minimum platelet counts >30,000 cells/mm3 and minimum neutrophil counts <1,000 cells/mm3. according to haytac et al. a neutrophil count of 1,500 cells/mm3 and platelets of 40,000 cells/mm3 are required in order for periodontal probing or extractions to be performed. the procedures must be carried out under antibiotic cover and at least three days before the start of chemotherapy (approximately ten days before the granulocyte count falls below 500 cells/ mm3). in cases when this is not possible, dental treatment should be postponed until the hematological indices increase.17 a dentist or periodontist could play the most significant role in the systemic condition of the patient as, potentially, the first medical staff who diagnoses leukemia. as soon as the potential conditions are detected, the patients should be referred to a hematologist-oncologist for early treatment to cure the leukemia. a dentist should be involved at three different stages of the dental management of the patient which are pre-antineoplastic treatment, ongoing antineoplastic treatment and post-treatment care. the primary goal of dental treatment of an aml patient is oral hygiene maintenance. invasive procedures, including periodontal probing, should be completed if minimum hematologic values are required. in conclusion, gingival enlargement represents one oral manifestation of aml. this condition is related to and affects the nutritional intake of the patient. references 1. american cancer society. cancer facts & figures 2016. atlanta: american cancer society inc; 2016. p. 13–14. 2. niederhuber j, armitage j, doroshow j, kastan m, tepper j. abeloff’s clinical oncology. 5th ed. philadelphia: elsevier; 2014. p. 1890–906. 3. chavan m, subramaniam a, jhaveri h, khedkar s, durkar sd, agrwal a. acute myeloid leukemia: a case report with palatal and lingual gingival alterations. braz j oral sci. 2010; 9(1): 67–9. 4. gowda tm, thomas r, shanmukhappa sm, agarwal g, mehta d. gingival en largement as an early diagnostic indicator in therapyrelated acute myeloid leukemia: a rare case report and review of literature. j indian soc periodontol. 2013; 17(2): 248–52. 5. howlader n, noone am, krapcho m, garshell j, miller d, altekruse sf, kosary cl, yu m, ruhl j, tatalovich z, mariotto a, lewis dr, chen hs, feuer ej, cronin ka. seer cancer statistics review, 1975-2011. bethesda (md): national cancer institute (us); 2014. p. 1–6. 6. sonoi n, soga y, maeda h, ichimura k, yoshino t, aoyama k, fujii n, maeda y, tanimoto m, logan r, raber-durlacher j, takashiba s. histological and immunohistochemical features of gingival enlargement in a patient with aml. odontology. 2012; 100(2): 254–7. 7. matsushita k, abe t, takeda y, takashima h, takada a, ogawa y, sato h, mukai m, fujiwara t. granulocytic sarcoma of the gingiva: two case reports. quintessence int. 2007; 38(10): 817–20. 8. deliverska eg, krasteva a. oral signs of leukimia and dental management – literature data and case report. j of imab. 2013; 19(4): 388–91. 9. newman mg, takei hh, klokkevold pr, carranza fas. carranza’s clinical periodontology. 11th ed. st. louis: saunders elsevier; 2012. p. 84–95, 310–2. table 2. minimum haematological values for performance of invasive procedures in pre-chemotherapy treatment patients according to the us national cancer institute, 20115 platelet counts neutrophil counts >60,000 cell/mm3: without additional support. >2,000 cell/mm3: without the need for antibiotic prophylaxis. 30,000 to 60,000 cell/mm3 : optional transfusion for noninvasive procedure. 1,000 to 2,000 cell/mm3: antibiotic prophylaxis (low risk). <30,000 cell/mm3: platelets should be transfused 1 h before the procedure. obtain immediate post-infusion platelet count; transfuse regularly to maintain counts >30,000–40,000 cell/mm3 until healing begins. <1,000 cell/mm3: antibiotic prophylaxis with amikacin 150 mg/ m2 1 h before surgery and ticarcillin 75 mg/kg iv 1 h before surgery. repeat both 6 h post-operative. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 159159kuswandani, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 154–159 10. zimmermann c, meurer mi, grando lj, moral jagd, da silva rath ib, tavares ss. dental treatment in patients with leukemia. j oncol. 2015; 2015: 1–14. 11. mani a, lee da. leukemic gingival infiltration. n engl j med. 2008; 358(3): 274. 12. soheylifar s, vahedi m, kadkhodazadeh m, bidgoli mj. a case of gingival enlargement in acute myeloid leukemia. j periodontol implant dent. 2009; 1(1): 48–50. 13. babu spkk, kashyap v, sivaranjani p, agila s. an undiagnosed case of acute myeloid leukemia. j indian soc periodontol. 2014; 18(1): 95–7. 14. elad s, raber-durlacher je, brennan mt, saunders dp, mank ap, zadik y, quinn b, epstein jb, blijlevens nma, waltimo t, passweg jr, correa mep, dahllöf g, garming-legert kue, logan rm, potting cmj, shapira my, soga y, stringer j, stokman ma, vokurka s, wallhult e, yarom n, jensen sb. basic oral care for hematology–oncology patients and hematopoietic stem cell transplantation recipients: a position paper from the joint task force of the multinational association of supportive care in cancer/ international society of oral oncology (mascc/isoo) and the european society for blood and marrow transplantation (ebmt). support care cancer. 2015; 23(1): 223–36. 15. shankarapillai r, nair ma, george r, walsh lj. periodontal and gingival parameters in young adults with acute myeloid leukaemia in kerala, south india. oral health prev dent. 2010; 8(4): 395–400. 16. pdq suppor tive and palliative ca re editor ial boa rd. oral complications of chemotherapy and head/neck radiation (pdq®): health professional version. pdq cancer information summaries. bethesda (md): national cancer institute (us); 2002. p.12–16. 17. haytac mc, dogan mc, antmen b. the results of a preventive dental program for pediatric patients with hematologic malignancies. oral health prev dent. 2004; 2(1): 59–65. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p154-159 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p154-159 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 26 the effect of nanochitosan hydrogel membrane on absorbtion of nickel, inhibition of streptococcus mutans and candida albicans andi triawan,1 pinandi sri pudyani,2 soesatyo marsetyawan hne,3 and sismindari4 1universitas gadjah mada hospital 2department of orthodontics, faculty of dentistry, universitas gadjah mada 3faculty of medicine, universitas gadjah mada 4faculty of pharmacy, universitas gadjah mada yogyakarta indonesia abstract background: the use of fixed orthodontic appliance for a long time can potentially cause nickel ion release, increase in the growth of streptococcus mutans (s. mutans) and candida albicans (c. albicans). chitosan has the ability to bind metal, antibacterial and antifungal. physical modification of chitosan into the nanoparticles size will expand the surface of the chitosan so that the absorption of nickel ions and the inhibition of growth bacteria and fungi can be increased. purpose: the purpose of the study was to determine the effect of nanochitosan hydrogel membrane to the absorption of nickel ions, and the inhibition of s. mutans and c. albicans growth. methods: nanochitosan hydrogel membrane with chitosan weight variation of 0.6; 0.8 and 1 g immersed in artificial saliva containing nickel 0.075 mg / l for 15, 30 and 45 minutes. the nanochitosan hydrogel membrane was tested for nickel ion absorption by atomic absorption spectrophotometry, whereas the antibacterial and antifungal tests were done by exposing the nanochitosan hydrogel membrane nickel on s. mutans and c. albicans in the wells of plate. results: demonstrated that absorption of nickel ions was related with the increase in weight of chitosan and soaking time. inhibition of growth of s. mutans and c. albicans showed a positive correlation with the increase in weight of chitosan. conclusion: variation on chitosan weight on hydrogel membrane and variation on immersion time have effect on nickel ion absorption, inhibition of s. mutans and c. albicans growth. keywords: nanochitosan; nickel ion; streptococcus mutans; candida albicans correspondence: andi triawan, c/o: rumah sakit universitas gadjah mada, jl. kabupaten (ring road), kronggahan, trihanggo, gamping, sleman, yogyakarta 55291 indonesia telp. (0274) 4530404, fax. (0274). e-mail: andi.triawan@ugm.ac.id introduction orthodontic appliance has three basic components i.e braces, archwires, buccal tubes, molar bands and accessories, which are made of stainless steel alloys. to get the best result of orthodontic treatment usually takes a long time, ranging from 1 to 3 years. during the period of orthodontic treatment, side effects can occur from the use of fixed orthodontic appliances.1 some of the reported side effects include corrosion of metal alloy,2 an increase in the accumulation of dental plaque,3 an increase in the growth of some bacteria4 and candida that causes disease dental hard tissues and soft tissues of the oral cavity.5 the main results of corrosion on metal alloy orthodontic appliance is iron (fe), chromium (cr), and nickel (ni).6 although all three components of the potentially harmful effects, ni has been reported to cause allergic, toxic and carcinogenic reactions.7 several studies have reported that exposure to nickel ions result of 2.521 ± 1.764 mg/l can cause dna damage and cell death by apoptosis, as shown by the growing number of comet cells and apoptotic cells.2 increasing accumulation of plaque in orthodontic treatment will be a problem for the health of the teeth and mouth.8 colonies of bacteria were initially formed gram positive colonies, namely streptococcus, neisseria and research report dental journal (majalah kedokteran gigi) 20�5 march; 48(�): 26–�0 2� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 2�triawan, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 26–30 actinomices. gram positive predominant bacteria that causes dental caries is streptococcus mutans (s. mutans).9 beside the increased accumulation of s. mutans, candida is also encountered during orthodontic treatment. research on the effects of orthodontic treatment on the increased growth of candida albicans (c. albicans) stated that c. albicans are found mainly on the surface of the orthodontic braces.5 according to the study on 60 orthodontic patients, 15 orthodontic patients (25%) were found with increase c. albicans colonies in the oral mucosal cells. increased growth of c. albicans in orthodontic treatment can trigger candidiasis and angular cheilitis.10 one of the natural ingredients that have the ability to absorb metal,11 antibacterial and antifungal is chitosan.12 the basic principle in the binding mechanism between chitosan and metal is the principle of ion exchange. especially nitrogen in the amine group of chitosan will react and bind metals from solution.11 chitosan is able to inhibit the growth of various types of bacteria and fungi as chitosan has a polycationic natural shape, so chitosan can act as an antibacterial agent against bacteria and fungi through ionic interactions on the cell wall of bacteria, which can even damage the cell wall.12 chitosan is expected to be the active ingredients that can reduce the negative effects of the use of orthodontic appliances. chitosan was modified into the nanoparticle size, in order to increase the surface area so that the amine group increases and form a nanochitosan preparations to be a nanochitosan hydrogel membrane with the aim to improve the absorption of nickel ions, capable of inhibiting the growth of s. mutans and c. albicans. the purpose of this study was to determine the effect of nanochitosan hydrogel membrane to the absorption of nickel ions, and the inhibition of growth of s. mutans and c. albicans. in previous studies it is known that chitosan 0.8 g can absorb zn ions well. 13 in this experiment, chitosan weight ratio of 0.6 ; 0.8 and 1 g were used to determine the pattern of optimal absorption of nickel ions. the results of this study are expected to be useful nanokitosan hydrogel membrane to reduce the risk of release of metal ions and inhibit the growth of bacteria s. mutans and c. albicans during orthodontic treatment. materials and methods this research use chitosan from crab shells (sigmaaldrich, st. louis, mo, usa), sodium acetate 1% 5 mm (brand), sodium phosphate (fluka) 50 mm, 1 m naoh solution, concentrated nh3 pa (merck), gelatin powder sigma from bovine skin type b (nitta), preparations of c. albicans 108 cfu/ ml, mueller hilton agar (mha), brain heart infusion (bhi), preparation of s. mutans, brown agar, nickel solution (laboratorium penelitian dan pengujian terpadu ugm), artificial saliva. a total of 0.6; 0.8 and 1 g of chitosan, dissolved in 500 ml (0.75 ml of acetic acid and distilled water add. 500 ml) was stirred until evenly dispersed chitosan. each solution of chitosan 0.6; 0.8 and 1 g was added 0.8 ml of nh3 (21%) dropped slowly over the stirrer while checked with a ph meter, until the ph reached 6.2 to 6.3. the solution became cloudy whitish color, then put in ultrasonic bacth for ± 1 hour, then allowed to stand for 30 minutes at room temperature. gelatin 1.8 g and 30 ml nanochitosan (each of the weight chitosan of 0.6; 0.8 and 1 g) was dissolved into 60 ml of distilled water and then subsequently homogenized using a magnetic stirrer for 3 hours, then allowed to stand for 30 minutes. after swelling it was diluted with water bath 370 c. nanochitosan gelatin liquid was inserted into the mold (petri dish with a diameter of 6 cm). nanochitosan hidrogel was then put in the refrigerator at a temperature of 40 c for 7 days to dry. to obtain crosslinked gelatin hydrogel dihydrothermal (dht) was done with a vacuum oven at integrated laboratory in faculty of dentistry universitas gadjah mada at a temperature of 1400 c to 1600 c for 48 hours. after the hydrogel membranes dry out and form a thin sheet, the hydrogel membrane was cut to the size of 20 x 20 mm. nanochitosan hydrogel membrane (chitosan weight of 0.6 g) was soaked into 50 ml artificial saliva containing nickel 0.075 mg/l for 15 minutes.13 then nanochitosan hydrogel membranes were analyzed using absorption atomic spectrometry (aas) to measure the concentration of nickel metal are absorbed in the membrane. nanochitosan hydrogel membranes that contained nickel was diluted with distilled water. nanochitosan hydrogel membrane solution was diluted 1000 ppm to 100 ppm, then diluted further to a concentration of 3-12 ppm. after all the solution is ready, the computer is turned on by using aas analysis program, namely gbc version 1.33. conduct program settings was done by adjusting the nickel element to be analyzed. then the tool was turned on, the program will ask for blank and all standard solutions sequentially. capillary tube was inserted into the solution, the program will read the content of nickel ions contained in the solution. after that will come a calibration graph of the results of analysis of nickel element contained within the solution. the same thing is done in the nanochitosan hydrogel membrane that containing chitosan weight 0.8 and 1 g. nanochitosan hydrogel membrane is soaked into 50 ml of artificial saliva containing nickel 0.075 mg/l for 15 minutes.14 then nanochitosan hydrogel membranes were analyzed using aas to measure the concentration of nickel metal are absorbed in the membrane. the same thing was done with a variation of contacts 30 and 45 minutes. s. mutans was obtained from cultures that had been available in the laboratory of microbiology, faculty of veterinary medicine universitas gadjah mada. this test used a medium brown agar. pure cultures of s. mutans were inoculated into medium brown agar using a sterile cotton swab, allowed to stand for 10 minutes. blank disc was inserted into the nanochitosan hydrogel membrane solution-nickel and allowed to stand for 1 hour. the discs were then placed on the surface of the brown agar using dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 28 triawan, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 26–30 sterile tweezers. the media put in the incubator for 24 hours at 370 c. after incubation for 24 hours would appear the presence of bacterial inhibition zone. measurements were performed by measuring the inhibition zone using a digital caliper accuracy of 0.01 mm. suspension of c. albicans was obtained from cultures that had been available in the laboratory of microbiology, faculty of veterinary medicine ugm. this test uses mueller hinton agar (mha) medium. pure cultures of c. albicans were inoculated into medium mha agar using a sterile cotton swab, allowed to stand for 10 minutes. blank disc was inserted into the nanochitosan hydrogel membrane solution nickel and allowed to stand for 1 hour. the discs were then placed on the surface of the brown agar using sterile tweezers. the media put in the incubator for 24 hours at 370 c. after incubation for 24 hours would appear the presence of fungal inhibition zone. measurements were performed by measuring the inhibition zone using a digital caliper accuracy of 0.01 mm. results results of the nickel ions absorption study showed that nanochitosan hydrogel membrane containing 1 g of chitosan with a 45 minute soak time is able to absorb the highest nickel ions are 0,0674 ± 0,002 ppm (table 1). nickel ions absorption pattern shows the pattern increases with increasing chitosan content in the nanochitosan hydrogel membrane (figure 1). variations on immersion time of 15, 30 and 45 minutes in all variations of weight chitosan showed an increasing pattern. fifteen minutes soaking time the nanochitosan hydrogel membranes 0.6 shows the average nickel ion absorption by 0.0280 ± 0.003 ppm, while the highest absorption of nickel ions on a 45-minute immersion in the nanochitosan hydrogel membrane 1 g of 0.0674 ± 0.002 ppm. all treatment groups of nanochitosan hydrogel membrane, the highest nickel ion absorption is still below the absorption of nickel ions in the control group of nanochitosan membrane. based on a two-ways anova test showed that the weight variation of chitosan on nanochitosan hydrogel membranes and variation of soaking time significantly affect the absorption of nickel ions. the results of the s. mutans growth inhibition studies showed that the higher more weight of chitosan on nanochitosan hydrogel membranes, the greater inhibition zone s. mutans. nanochitosan hydrogel membrane 1 g has the highest inhibition zone diameter of 1.018 ± 0.034 mm (table 2). based on the variation of soaking time, the highest inhibition zone diameter at 15 minutes soaking time of 1.018 ± 0.034 mm (figure 2). based on a two-ways anova test, significance value of p<0.05 in all groups so that it can be concluded that the variation of the weight of chitosan on nanochitosan hydrogel membranes and variation of soaking time significantly effect on s. mutans inhibition zone. the results of inhibition of c. albicans growth studies showed heavier chitosan on nanochitosan hydrogel table 1. the mean nickel ion absorption by the nanochitosan hydrogel membrane no materials sample nickel ion absorption (ppm) 15 minutes 30 minutes 45 minutes 1 nch membrane 9 0.048±0.003 0.061±0.002 0.068±0.002 2 nch hgel membrane 0.6 9 0.027±0.002 0.035±0.002 0.040±0.037 3 nch hgel membrane 0.8 9 0.039±0.002 0.051±0.002 0.061±0.002 4 nch hgel membrane 1.0 9 0.048±0.002 0.060±0.002 0.067±0.002 table 2. average inhibition zone growth of streptococcus mutans no materials sample inhibition zone of streptococcus mutans (mm) 15 minutes 30 minutes 45 minutes 1 nch membrane 9 1.056± 0.017 0.992± 0.011 0.930± 0.023 2 nch hgel membrane 0.6 9 0.873 ± 0.042 0.838 ± 0.031 0.817 ± 0.042 3 nch hgel membrane 0.8 9 0.922 ± 0.020 0.899 ± 0.018 0.855 ± 0.025 4 nch hgel membrane 1.0 9 1.018 ± 0.034 0.960 ± 0.012 0.904 ± 0.028 nickel ions absorption by nanochitosan hydrogel membranes 0 .0 5 0 .0 4 0 .0 5 0 .0 6 0 .0 5 0 .0 6 0 .0 7 0 .0 5 0 .0 6 0 .0 7 0 .0 3 0 .0 4 n a n o c h ito s a n (n c ) n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 nanochitosan hydrogel membranes n ic k e l a b s o rp ti o n 1 5 m in u t e 3 0 m in u t e 4 5 m in u t eminutes minutes minutes nickel ions absorption by nanochitosan hydrogel membranes 0 .0 5 0 .0 4 0 .0 5 0 .0 6 0 .0 5 0 .0 6 0 .0 7 0 .0 5 0 .0 6 0 .0 7 0 .0 3 0 .0 4 n a n o c h ito s a n (n c ) n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 nanochitosan hydrogel membranes n ic k e l a b s o rp ti o n 1 5 m in u t e 3 0 m in u t e 4 5 m in u t efigure 1. the average of nickel ions absorption by nanochitosan hydrogel membrane. 2� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 2�triawan, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 26–30 membranes make up the greater inhibition zone c. albicans. nanochitosan hydrogel membrane 1 g has the highest inhibition zone diameter 1.541 ± 0.028 mm (table 3). for a variation of soaking time 15, 30 and 45 minutes in all variations of chitosan weight showed a declining pattern. the highest inhibition zone diameter at 15 minutes soaking time 1.541 ± 0.028 mm (figure 3). based on a twoways anova test obtained significance value of p<0.05 so that it can be concluded that the variation of the weight of chitosan on nanochitosan hyrogel membranes and variation of soaking time significantly have effect on c. albicans inhibition zone. discussion the basic principle in the binding mechanism between chitosan and metal contained in the solution is the principle of ion exchange.11 especially nitrogen in the amine group of chitosan will react and bind metals from solution. increased absorption of nickel ions occurs because of the greater weight of chitosan that make nanochitosan, nanochitosan surface area is increased and the amine group formed the more so the higher the ability to absorb metals.14 chitosan has a very good the properties of metal ion absorption due to a) the nature of the hydrophilic chitosan; b) primary amine group with high activity; c) chitosan polymer chain structure which can form a flexible configuration of chitosan to bond with metal ions.16 in a variation of chitosan weight of 0.6; 0.8 and 1 g contained in nanochitosan hydrogel membrane known that nickel absorption increases with the increase chitosan weight that contained in nanochitosan hydrogel membrane. chitosan adsorption ability can be improved by modifying both physically and chemically. modification of physics related to the crystallinity structure that reduces the size of chitosan to be nanoparticles. nanoparticles are particulate material with at least one dimension smaller than 1000 nanometers. one nanometer is 10-9 m, so that the nanoparticles have a greater surface area to volume ratio. the more surface area of chitosan by changing its size to nanoscale, then getting bigger too absorptive capacity.17 absorption of nickel by nanochitosan hydrogel membrane as the highest in the weight of 1 g chitosan. increased absorption of nickel ions occurs due to the greater weight of chitosan that compile nanochitosan the increasingly widespread nanochitosan surface amine groups formed led to more and more, so the ability to absorb the metal increase.16,17 based on the variation of immersion time 15, 30 and 45 minutes, data show the nanochitosan hydrogel membrane 1 g with contact time 45 had the greatest absorption. at 15 minutes soaking time is not optimal absorption, because the process of adsorption and the formation of bridges between the particles is not perfect. whereas at 30 and 45 minutes absorption of nickel ions is increased, due to the amine group on nanochitosan hydrogel membrane still able to bind nickel ions. in the 45minute contact time highest absorption occurs in the weight variation nanochitosan 1 g. compared with the control group nanochitosan membrane then the value of nickel ion absorption by the nanochitosan hydrogel membrane 1 g is almost the same. this is probably due to the composition of gelatin contained in nanochitosan hydrogel membrane, which can also bind to gelatin amine and hydroxyl groups of chitosan. nanochitosan hydrogel membrane can bind nickel ions via the amine group (nh3), this amine group is also involved in the antimicrobial mechanism of chitosan against bacteria and fungi.18 the results showed nanochitosan hydrogel table 3. average inhibition zone growth of candida albicans no materials sample inhibition zone of candida albicans (mm) 15 minutes 30 minutes 45 minutes 1 nch membrane 9 1.594± 0.014 1.554± 0.013 1.511± 0.013 2 nch hgel membrane 0.6 9 1.456± 0.014 1.423± 0.013 1.378± 0.013 3 nch hgel membrane 0.8 9 1.504± 0.016 1.474 ± 0.016 1.448 ± 0.019 4 nch hgel membrane 1.0 9 1.541± 0.028 1.504± 0.020 1.489± 0.018 inhibition zone of streptococcus mutans 0 .8 7 0 .9 2 1 .0 2 1 .0 6 0 .8 4 0 .9 0 0 .9 6 0 .9 9 0 .8 2 0 .8 6 0 .9 0 0 .9 3 n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 n a n o c h ito s a n (n c ) nanochitosan membrane in h ib it io n z o n e 1 5 min u te 3 0 min u te 4 5 min u te nickel ions absorption by nanochitosan hydrogel membranes 0 .0 5 0 .0 4 0 .0 5 0 .0 6 0 .0 5 0 .0 6 0 .0 7 0 .0 5 0 .0 6 0 .0 7 0 .0 3 0 .0 4 n a n o c h ito s a n (n c ) n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 nanochitosan hydrogel membranes n ic k e l a b s o rp ti o n 1 5 m in u t e 3 0 m in u t e 4 5 m in u t eminutes minutes minutes figure 2. the average of streptococcus mutans inhibition zone by nanochitosan hydrogel membrane.inhibition zone of candida albican 1 .4 6 1 .5 0 1 .5 4 1 .5 9 1 .4 2 1 .4 7 1 .5 0 1 .5 5 1 .3 8 1 .4 5 1 .4 9 1 .5 1 n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 n a n o c h ito s a n (n c ) nanochitosan membrane in h ib it io n z o n e 1 5 min u te 3 0 min u te 4 5 min u te nickel ions absorption by nanochitosan hydrogel membranes 0 .0 5 0 .0 4 0 .0 5 0 .0 6 0 .0 5 0 .0 6 0 .0 7 0 .0 5 0 .0 6 0 .0 7 0 .0 3 0 .0 4 n a n o c h ito s a n (n c ) n c -h g me m b ra n e 0 ,6 n c -h g me m b ra n e 0 ,8 n c -h g me m b ra n e 1 nanochitosan hydrogel membranes n ic k e l a b s o rp ti o n 1 5 m in u t e 3 0 m in u t e 4 5 m in u t eminutes minutes minutes figure 3. the average of candida albicans inhibition zone by nanochitosan hydrogel membrane. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �0 triawan, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 26–30 membrane with variation of chitosan weight and variation of immersion time can inhibit the growth of s. mutans and c. albicans. the higher weight of chitosan that contained in nanochitosan hydrogel membrane, the greater inhibition zone of s. mutans and c. albicans, but by the variation of immersion time, the longer soaking time, inhibition zone of s. mutans and c. albicans decreases. s. mutans and c. albicans inhibition zone is still high due to the nickel ion is still not completely absorbed by nanochitosan hydrogel membrane, so the ability of the amine group to destroy the bacterial membrane can be optimized. amine functional group (-nh2) on chitosan, which is a very strong positive charge, may be able to bind to the bacterial cell wall which negatively charged. this bond may occur in electronegative sites on the surface of the bacterial cell wall. in addition, because -nh2 also has a lone pair, then amina group can bind ca2+ minerals contained in the cell wall of bacteria to form a coordinate covalent bond.18 at the variation of immersion time, the longer soaking of the amine group on the nanochitosan hydrogel membrane will increasingly bind to nickel ions, so the ability to damage the cell walls of bacteria decreased. mechanism between antimicroba and antifungal is same, that through the interaction between the charge positive group nh3 + on the unit glucosamine chitosan and the negative charge on the cell membrane of microbes resulting in electrostatic interactions that cause changes in the permeability of the membrane walls of microbes that alter the osmotic balance internally that can inhibit microbial growth, and hydrolysis of peptidoglycan in the wall of microbes resulting in loss of intracellular electrolytes, proteins, nucleic acids and glucose in microbes.18,19 nh3 group plays an important role in the process of absorption of nickel ions and the antibacterial/ antifungal. if the absorption of nickel ions is not optimal, there is still an active nh3 group so that the process antibacterial and antifungal mechanism can be improved.15 if the nh3 group has bonded optimum with nickel ions, nanochitosan hydrogel membrans still able to be an antibacterial and antifungal because of the presence of acetic acid content in the solution during the process of making nanochitosan membrane. acetic acid has the ability to inhibit bacterial or fungal.17 in conclusion, variation of chitosan weight in the nanochitosan hydrogel membrane and variations of soaking time in artificial saliva have effect on the absorption of nickel ions, the inhibition of growth of s. mutans and c. albicans. the nanochitosan hydrogel membrane is able to absorb the nickel ions in artificial saliva, with the highest absorption of nickel ions on the nanochitosan hydrogel membrane containing 1 g chitosan. the ability of nanochitosan hydrogel membrane-nickel in inhibiting the growth of c. albicans and s. mutants is the highest on nanochitosan hydrogel membranes 1g-nickel but the highest effect in the first 15 minutes of soaking time. references 1. jonsson a, malmgen o, levander e. long-term follow-up of tooth mobility in maxillary incisors with orthodontically induced apical root resorption. eur j orthod 2007; 29: 482–7. 2. hafez hs, selim em, kamel fh, tawfik wa, al-ashkar ea, mostafa ya. cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study. am j orthod dentofac orthop 2011; 140(3): 298-308. 3. julien kc, buschang ph, campbell pm. prevalence of white spot lesion formation during orthodontic treatment. angle orthod 2013; 83(4): 641-7. 4. joon ahna s, soon limb b, cheol yang h, il chang i. quantitative analysis of the adhesion of cariogenic streptococci to orthodontic metal brackets. angle orthod 2005; 75(4): 666-71. 5. rammohan sn, juvvadi sr, gandikota cs, challa p, manne r, mathur a. adherence of streptococcus mutans and candida albicans to different bracket materials. j pharm bio sci 2012; 4(6): 216-7 6. hafez hs, nassef em, kamel eid fh, tawfik wa, al-ashkar ea, mostafa ya. cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study. am j orthod dentofac orthop 2011; 40(3): 298–308. 7. ortiz aj, fernández e, vicente a, calvo jl, ortiz c. metallic ions released from stainless steel, nickel-free, and titanium orthodontic alloys: toxicity and dna damage. am j orthod dentofac orthop 2011; 140(3): 115-22. 8. al-anezi sa, harradine nw. quantifying plaque during orthodontic treatment. angle orthod 2012; 82(4): 748-53. 9. davies tm, shaw wc, worthington hv, addy m, dummer p, kingdon a. the effect of orthodontic treatment on plaque and gingivitis. am j orthod dentofac orthop 1991; 99(2): 155-61. 10. carrillo el, montiel nm, pérez ls, tavira ja. effect of orthodontic treatment on saliva, plaque and the levels of streptococcus mutans and lactobacillus. med oral patol oral cir bucal 2010; 15(6): 924-9. 11. yassaei s, kafaie p. four cases of angular cheilitis in orthodontic patients. j shahid sadaughi univ 2006; 14(2): 77-81. 12. divakaran r, paul aj, anoop kk, kuriakose vj, rajesh r. adsorption of nickel (ii) and chromium (vi) ions by chitin and chitosan from aqueous solutions containing both ions. int j sci tech res 2012; 1(1): 43-50. 13. kamelia s. pengaruh derajat deasetilasi nanokitosan untuk menyerap zn 2+ dari limbah cair industri karet. tesis. medan: universitas sumatera utara; 2009. 14. eldin ms, soliman e, hashem a, tamer tm. antibacterial activity of chitosan chemically modified with new technique. trends biomat artif organs 2008; 22(3): 121-33. 15. gürsoy s, güngör a, şeşen ç. comparison of metal release from new and recycled bracket-archwire combinations. angle orthod 2005; 75(1): 92-4. 16. guibal e, touraud e, roussy j. chitosan interactions with metal ions and dyes: dissolved-state vs. solid-state application. world j microbiol biotechnol 2005; 21(6-7): 913-20. 17. ramisz ab, pajak aw, pilarczyk b, ramisz a, laurans l. antibacterial and antifungal activity of chitosan. j pharm sci 2005; 2(1) : 406-8. 18. oliveira ap, ribeiro mp, oliveira pr, gaspar c, oliveira ps. anticandida activity of a chitosan hydrogel: mechanism of action and cytotoxicity profile. obstet invest 2010; 70(4): 322-7. 19. adewuyi s, kareem kt, atayese ao, amolegbe sa, akinremi ca. chitosan-cobalt (ii) and nickel (ii) chelates as antibacterial agents. int j biol macromol 2011; 48(2): 301-3. 159159 contrasting efficacy of cocoa pod husk extract and 8% propolis extract in maintaining of root canal wall cleanliness tamara yuanita, uli sasi andari, mandojo rukmo, s. sukaton and deavita dinari department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: the existence of the smear layer, which can be produced during root canal instrumentation, may compromise the bond between filling material and the root canal walls. therefore, the use of an effective root canal irrigation solution, a commonly employed form of which is sodium hypochloride (naocl), is important. sodium hypochloride has several positive properties including effectiveness as a disinfectant agent and its ability to promote tissue-dissolution, although it is ineffective at cleaning the smear layer. there have been numerous recent studies of the application of phytomedicines in endodontics due to their advantages such as minimum toxicity and cost effectiveness. the saponin contained in both the propolis and cocoa pod husk acts as a surfactant that may lower surface tension and dissolve debris containing organic and anorganic materials. purpose: the study aimed to provide evidence of the differences between root canal wall cleanliness when treated with 8% propolis extract and different concentrations of cocoa pod husk extract. methods: 25 extracted teeth with single straight root canals were randomly divided into five categories (n=5). sample preparation was performed using a rotary file and irrigated with different solutions. the first group was administered 2.5% naocl, the second group 8% propolis, the third group 3.12% cocoa pod husk extract, the fourth group 6.25% cocoa pod husk extract, and the fifth group 12.50% cocoa pod husk extract. the samples were then dissected into two sections at the apical third and their cleanliness scores subjected to a mann-whitney test with a significance level of p=0.05. results: a significant difference was identified between all groups (p<0.05) and on the median control test, the highest value of 1.6 was recorded by the 6.25% cocoa pod husk extract, compared to the other four groups conclusion: cocoa pod husk extract demonstrates greater efficacy at cleaning root canal walls compared to 8% propolis extract. keywords: cocoa pod husk extract; propolis extract; root canal irrigation correspondence: tamara yuanita, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132. indonesia. e-mail: tamara-y@fkg.unair.ac.id introduction the main purpose of root canal treatment is to disinfect the entire canal by eliminating microorganisms and other microbe components in order to prevent reinfection both during and after the treatment. chemo-mechanical debridement can achieve this objective during root canal treatment.1 the debridement process includes cleaning and shaping of the canal to remove necrotic tissue residue, bacteria and the smear layer in order to facilitate sterilization and obturation of the canal.2 chemical debridement is important for teeth with a challenging anatomy, such as fins or other irregularities, that might be undetected by instrumentation.3 the ideal properties of irrigation solution comprise: a broad antimicrobial spectrum, low toxicity, the ability to solve the problems of necrotic tissue and debris, and low surface tension, while also being capable of dissolving the smear layer.4 sodium hypochlorite (naocl) is a commonly used irrigation solution, mainly because of the fact that it constitutes a cheap antiseptic lubricant possessing the ability to dissolve necrotic materials. the major disadvantages of naocl are its cytotoxicity on dental journal (majalah kedokteran gigi) 2019 september; 52(3): 159–162 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p159–162 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p159-162 160 yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 159–162 entering the periradicular tissue and its inability to dissolve the smear layer. moreover, both its odor and the taste are unpleasant.5 a considerable amount of research into natural ingredients as an alternative to conventional dentistry materials has recently been undertaken because of their advantages compared to commonly used chemical materials, including: widespread availability, cost effectiveness, low toxicity, and also their lower susceptibility to microbial resistance.6 one natural ingredient commonly used for research purposes is propolis, a bee-produced material containing a mixed complex consisting of wax, a small amount of sugar, and tree sap collected by honey bees (apis mellifera).7 at a concentration of 8%, propolis is more effective at cleaning the root canal walls of smear layer compared to 2.5% naocl.8 in addition to propolis, another natural ingredient whose use is rapidly gaining in popularity is cocoa pod husk extract (theobroma cacao) which contains in excess of 500 different chemicals and has traditionally been used as an antioxidant, anticarcinogenic, immunomodulator, vasodilator, antimicrobial, and analgesic.9 cocoa pod husk extract can impede the formation of enterococcus faecalis (e. faecalis) bacterial biofilm at a minimum biofilm inhibitory concentration (mbic) of 3.12% and possesses significant potential as an alternative root canal irrigation agent. the other concentrations of 6.25% and 12.50% are selected because the higher the concentration, the lower the optical density value of biofilm.10 this research was undertaken in order to obtain knowledge about the relative cleaning effectiveness of cocoa pod husk extract and extract of 8% propolis in relation to the root canal walls. materials and methods the experiment employed 25 human mandible premolar samples, previously extracted for orthodontic reasons, which satisfied the following criteria: single canal teeth with an average length of 20 ± 2 mm; a post-access gauging process using a niti file #8, #10, or maximum #20; a good fit at the apex, and defect-free closure of the apical foramen. the mandible premolar teeth meeting the criteria were soaked in saline and divided into five groups, each containing five teeth. access opening was completed by means of a high-speed endo access bur. files no. 8 to 10 were used to determine the working length of each sample, supported by a gauging process. preparation of the root canal involved the use of rotary files (protapper next, dentsply sirona, tulsa) combined with an endomotor (x-smart plus, destsply sirona, tulsa). each file preparation took approximately ten seconds. during file exchange, the irrigation solution was divided as follows: the first group was supplied with 2.5% naocl, the second group 8% propolis extract, the third group 3.12% cocoa pod husk extract, the fourth group 6.25% cocoa pod husk extract, and the fifth group 12.50% cocoa pod husk extract. the irrigation process was performed with an instrument set up in such a manner that the air pressure was at 1 atm (1033kg/cm2). the 3ml of irrigation solution were applied for ten seconds on each occasion. therefore, the total amount of irrigation solution for each sample was 12 ml. during the final irrigation process, the canal was activated by means of eddy (vdw, germany) before, finally, being dried with sterile paper points and closed with temporary restoration. all samples from each group were marked on the lingual and buccal side using a high-speed diamond disc as a cutting guide, prior to being cut horizontally through the apical third of the tooth (4mm from the apex) by means of a disc bur. the samples were then bisected with a chisel and affixed to a sample holder (stub) with the surface of the root canal upward facing using a specific glue (araldite®, switzerland) that had been mixed with aluminium powder. having been allowed to air dry for a day, the surfaces of the samples were coated for approximately one hour with pure gold or carbon for later observation with a vacuum evaporator. at that point, the samples were ready for observation by means of a scanning electron microscope (sem). the samples were individually inserted into the sem for observation of their middle sections at a magnification of 150x. this section was subsequently magnified again at 1000x, having been set up for the particular contrast and lighting. evaluation of the photomicrograph was completed by two observers. the field of view was divided into nine boxes identical in size (three cubes).11 evaluation of the sem image was conducted with the following scoring system for each box:6 score 1 indicated the absence of a smear layer and that dentinal tubule surfaces were clean, score 2 signified that 25% of the root canal wall surface was covered by a smear layer, score 3 denoted that 25% to 50% of the root canal wall surface was covered by a smear layer, score 4 showed that 50% to 75% of the root canal wall surface was covered by a smear layer, score 5 indicated that more than 75% of the root canal wall surface was covered by a smear layer. a nonparametric kruskal-wallsis test was performed to establish the difference for all groups followed by a mann-whitney test to identify the difference between each group with a p-value lower than 0.05. in such cases, the difference was considered to be significant. results the cleanliness scores recorded 25 samples of teeth divided into five groups are shown in table 1. from the statistical analysis, the average cleanliness score of the first group was the lowest, indicating that it contained a smear layer covering between 50% and 75% of the root canal walls. in contrast, the highest cleanliness score was recorded by the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p159–162 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p159-162 161yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 159–162 fourth group signifying the absence of a smear layer and clean dentinal tubules. figure 1 contains the sem image of each group which shows the cleanliness of root canal walls free of smear layer. the result of a kruskal-wallis test indicated that the significance level was 0.000 (p-value<0.005) meaning that a significant difference existed between all groups. the mann-whitney test identified the differences between each group and the results contained in table 2 show how the groups compare to each other. all of the numbers represent a value lower than 0.05 which indicates that significant data discrepancies existed between each group. table 1. cleanliness score for each group group cleanliness score 2.5% naocl 4.7 8% propolis 3.4 3.12% cocoa 5 6.25% cocoa 1.6 12.50% cocoa 2.5 table 2. results of differences between group group 2.5% naocl 8% propolis 3.12% cocoa 6.25% cocoa 12.50% cocoa 2.5% naocl 8% propolis 0.009* 3.12 % cocoa 0.044* 0.005* 6.25 % cocoa 0.009* 0.009* 0.005* 12.50 % cocoa 0.009* 0.036* 0.005* 0.009* * p-value < 0.05 means the the data has a significant data discrepancy. b c a d e figure 1. (a) sem image for control group 2.5% naocl; (b) sem image for 8% propolis; (c) sem image for 3.12% cocoa; (d) sem image for 6.25% cocoa; (e) sem image for 12.50% cocoa. discussion root canal cleanliness constitutes one of the parameters for effective root canal treatment since research has shown that the smear layer on the root canal walls covers the dentinal tubules. a smear layer constitutes a mixture of organic and inorganic particles that can accumulate during the preparation process relating to the root canal walls8 and whose cleanliness is observable under sem, thereby enabling calculation of the number of clean and unclogged dentinal tubules.11,12 a significant body of recent research has been undertaken into the natural ingredient phytomedicine because of its advantages when compared to the chemical agents currently widely employed within the field of dentistry.6 from statistical analysis data, the average cleanliness score of the control group which uses naocl as irrigating solution was considered to be high. this was due to the inability of naocl to dissolve the inorganic substance and its high surface tension which render it more difficult to clean and disinfect the entire root canal system with the result that the smear layer covers the root canal walls.13 the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p159–162 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p159-162 162 yuanita, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 159–162 8% propolis extract produced an average cleanliness result and in comparison to 3.12% cocoa pod husk extract was more effective at cleaning the root canal walls. however, the result was different when compared to 6.25% and 12.50% cocoa pod husk extract because both concentrations are more effective at cleaning the root canal walls. both extract of propolis and cocoa pod husk contain saponin, although at differing concentrations. according to the examination results produced by the surabaya research center and industry consultant laboratory the concentration of saponin in the propolis extract is lower (0.88%) than that of cocoa pod husk extract (2.18%). consequently, the efficacy of saponin in the propolis is reduced. the extract of 3.12% cocoa pod husk recorded the highest root canal wall cleanliness score indicating that it had not yet reached the critical micelle concentration (cmc) at which point a surfactant initiates the formation of micell capable of dissolving fat and oil. this means that at a concentration of 3.12%, the amount of saponin in cocoa pod husk extract was insufficiently effective to clean the smear layer from root canal walls. 6.25% cocoa pod husk extract recorded the lowest cleanliness score among all the other treated groups. this signified that a concentration of 6.25% was the most effective at cleaning the smear layer from root canal walls compared to other treatment groups. based on these results, once that concentration of cmc had been reached the micelle started to form and was able to clean inorganic material and smear layer from the the root canal walls. according to the statistical analysis results, a significant difference existed between the fourth and fifth groups, where the average score of the fourth group was lower, which means that at a concentration of 6.25% the cocoa pod husk extract was more effective than at one of 12.50%. this possibly occurred because of the theobromin content demonstrating an ability to increase enamel hardness by substituting for the hydroxyapatite crystal lost through the demineralisation process. for example, since theobromin crystal size is smaller than that of hydroxyapatite, this facilitates its penetrating the microtunnel of enamel or dentin and exchanging the apatite ions. this explains why cocoa pod husk extract (12.5%) had a lower cleanliness score at higher concentrations than 6.25% cococa pod husk extract.14,15 therefore, the higher concentration of cocoa pod husk did not invariably induce efficacy of cleanliness in root canal walls due to its theobromin content. in conclusion, the efficacy of root canal wall cleanliness differed between 8% propolis extract and cocoa pod husk extract. cocoa pod husk at 6.25% demonstrated the greatest efficacy in promoting root canal wall cleanliness compared to 8% propolis extract. references 1. plotino g, cortese t, grande nm, leonardi dp, di giorgio g, testarelli l, gambarini g. new technologies to improve root canal disinfection. braz dent j. 2016; 27(1): 3–8. 2. torabinejad m, walton re. endodontics: principles and practice. 5th ed. st louis missouri: saunders/elsevier; 2014. p. 29–30. 3. ruqshan anjum mg, sujatha i, sharath chandra sm. antimicrobial efficacy of various irrigating solutions on e . faecalis in root canals: an in-vitro study. int j appl dent sci. 2015; 1(4): 94–7. 4. garg n, garg a. textbook of endodontics. 3rd ed. new delhi: jaypee brothers medical publishers; 2014. p. 150–3. 5. torabinejad m. root ca na l i r r iga nts a nd disinfecta nts. i n: endodontics: colleagues for excellence. chicago: american association of endodontics; 2011. p. 2–8. 6. jena a, sahoo sk, govind s. root canal irrigants: a review of their interactions, benefits, and limitations. compend contin educ dent. 2015; 36(4): 256–61. 7. silva-carvalho r, baltazar f, almeida-aguiar c. propolis: a complex natural product with a plethora of biological activities that can be explored for drug development. evidence-based complement altern med. 2015; 2015: 1–29. 8. yuanita t. the cleanliness differences of root canal walls after irrigated with east java propolis extract and sodium hypoclorite solutions. dent j (majalah kedokt gigi). 2017; 50(1): 6–9. 9. santos rx, oliveira da, sodré ga, gosmann g, brendel m, pungartnik c. antimicrobial activity of fermented theobroma cacao pod husk extract. genet mol res. 2014; 13(3): 7725–35. 10. yuanita t, vergeina d, rukmo m, zubaidah n, wahjuningrum da, kunarti s. antibiofilm power of cocoa bean pod husk extract (theobroma cacao) against entercoccus faecalis bacteria (in vitro). in: international medical device and technology conference. johor bahru: universiti teknologi malaysia; 2017. p. 129–31. 11. rao, rao rn, kumar vs, babannavar r, muniyappa m, naman s. root canal cleanliness after preparation with ultrasonic handpiece and hand instruments: an in vitro comparative scanning electron microscope study. endodontology. 2018; 30(1): 32–7. 12. dohaithem a, tovar n, coelho p, alnazhan s, almansouri s, bafail a. a scanning electron microscopy evaluation of the cleanliness of un-instrumented areas of canal walls after root canal preparation. saudi endod j. 2015; 5(2): 114–9. 13. gusiyska a, gyulbenkiyan e, vassileva r, dyulgerova e, mironova j. effective root canal irrigantion a key factor of endodontic treatment review of the literature. int j rescent sci res. 2016; 7(4): 9962–70. 14. jena a, govind s, sahoo sk. gift of nature to endodontics as root canal irrigant: a review. world j pharm res. 2015; 4(9): 471–81. 15. sailaja d, srilakshmi p, puneeth k, krishna cr, srilakshmi p. estimation of protein content and phytochemicals studies in cocoa fruit outer covering. int j plant, anim environ sci. 2015; 1(2): 111–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p159–162 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p159-162 91 dental journal (majalah kedokteran gigi) 2018 june; 51(2): 91–94 research report acid fast bacilli detected in the oral swab sample of a pulmonary tuberculosis patient reiska kumala bakti,1 ni made mertaniasih,2 diah savitri ernawati,1 bagus soebadi,1 and priyo hadi1 1department of oral medicine, faculty of dental medicine, universitas airlangga 2department of clinical microbiology, faculty of medicine, universitas airlangga surabaya indonesia abstract background: tuberculosis (tb) is an infectious disease that persists as a health problem worldwide. mycobacterium tuberculosis, as an etiological agent, is transmitted from infected to uninfected individuals via airborne droplet nuclei. oral health care workers or dental practitioners may be at high risk of tb infection because of their close proximity to infected individuals during treatment procedures. simple and rapid screening of mycobacterium tuberculosis in the oral cavity is necessary in order to prevent transmission of infection. purpose: to investigate the presence of acid-fast bacilli in the buccal mucosa of pulmonary tb patients. methods: nineteen pulmonary tb patients of both sexes, ranging in age from 19 to 74 years old participated in this study. the diagnosis of tuberculosis was performed by clinical symptom assessment and supporting examination, including acid-fast bacilli on sputum examination. two buccal mucosa swabs taken from pulmonary tb patients were collected for acid fast bacilli direct smear by ziehl neelsen staining. results: with regard to mycobacterium tuberculosis, acid-fast bacilli presented in 10.5% of the oral buccal mucosa swabs of subjects, whereas in the sputum specimens, bacilli were found in 52.6% of subjects. conclusion: acid-fast bacilli can be found in the buccal epithelial mucosa of pulmonary tuberculosis patients, although its presence was very limited. keywords: tuberculosis (tb); oral; buccal mucosa; acid fast bacilli; mycobacterium tuberculosis correspondence: reiska kumala bakti, department of oral medicine, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: reiska91@gmail.com introduction tuberculosis (tb) is a disease caused by mycobacterium tuberculosis (m. tuberculosis) that remains a global health problem. who estimates m. tubercolusis in 2015, there were 10.4 million new cases of tb worldwide, resulting in the deaths of 1.8 million people. indonesia, together with india and china, is one of three countries with the highest incidence rate of tb since 2014 and one of six countries accounting for 60% of new tb cases. of these, china, india and indonesia together accounted for 45% of global cases in 2015.1,2 transmission of tuberculosis occurs from an infectious patient to other individuals via droplet nuclei that contain mycobacterium tuberculosis.3 droplet nuclei are small particles, approximately 1–5 micrometers in diameter that can remain airborne for minutes to hours after expectoration through coughing, sneezing or talking by carriers of pulmonary tb.4,5 in dental clinics, oral health care workers and dental practitioners may be at high risk of mycobacterium tuberculosis infection because of their close proximity to patients producing infectious secretions and exposure to the aerosol used during the dental treatment process. the potential for transmission of mycobacterium tuberculosis cannot be discounted since patients and oral health care workers share common air space. the probable transmission of mycobacterium tuberculosis from infected patients to two oral health care workers has been documented, while evidence exists of tb transmission from an oral surgeon with active bilateral pulmonary tuberculosis to 15 patients post-extraction.6 patients suffering from tb may visit dental clinics because of oral problems necessitating prevention of the transmission of infection. simple and rapid screening for mycobacterium tuberculosis in oral samples is required, given that this microorganism can be transmitted to others, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p91–94 mailto:reiska91@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p91-94 92bakti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 91–94 both oral health care workers and fellow dental patients. various oral samples could be used, such as mixed saliva, dental plaque, caries lesions, denture plaque, oral wash and buccal swabs. mycobacterium tuberculosis might be found in the oral cavity since the bacilli is passed from the lungs and airways to the oral cavity of pulmonary tb patients and then accumulated on the buccal mucosa.7–9 this study was aimed to investigate acid-fast bacilli detection on the buccal mucosa epithelium of pulmonary tb patients. an oral mucosa swab was selected because, naturally, mycobacterium species are more commonly associated with surfaces than with fluid matrices.8 materials and methods this study was conducted at universitas airlangga hospital from september to october 2016 and at dr. soetomo general hospital between july and october 2017. a total of 19 subjects participated, ranging in age between 19 and 74 years old. the participants consisted of ten males and nine females. tuberculosis in the patients were diagnosed by pulmonologists at the pulmonary outpatient unit of universitas airlangga hospital and the tuberculosis outpatient unit of dr. soetomo general hospital. a diagnosis of tuberculosis was established by clinical symptom assessment and supporting examination, both bacteriological and radiographic, while confirmation of diagnosis was provided by an outpatient unit pulmonologist. bacteriological examination was performed three times on three sputum specimens: ‘spot’, ‘morning’, and ‘spot’. one of the tb patients was hiv-positive, but tuberculosis constituted the only other systemic disease then affecting the other subjects. a swab specimen was collected from the buccal mucosa of subjects immediately after a positive diagnosis. this involved the use of a cotton swab stick under standardized conditions at least one hour after eating and the completion of oral hygiene procedures. the swabs were brushed along the inside of each subject’s cheek 7–8 times for about 10 seconds on each occasion in order to collect specimens uniform in both volume and composition. the swabs were then applied to glass slides for direct smear. the slides were processed and stained with ziehl neelsen acid-fast stain and examined under a microscope at 1000 to establish the presence, or otherwise, of acid-fast bacilli. the acid-fast bacilli on the slides were counted and subsequently evaluated using the international union against tuberculosis and lung diseases (iuatld) scale (table 1). the amount of acid-fast bacilli present on the buccal mucosa of tb patients was compared with the previous acid-fast bacilli contained in the sputum. results according to the iuatld scale, there were five levels of bacilli number (table 1). based on their medical records, nine of the 19 subjects (47.4%) presented negative results in the acid-fast bacilli of sputum specimens. one patient presented large numbers of acid-fast bacilli (3+ on all three sputum specimens), while others presented varied levels of bacilli number. on the other hand, most subjects (89.5%) presented negative acid-fast bacilli of buccal mucosa swab specimens, with the exception of two subjects with weak results. the presence of 2–9 bacilli in 100 fields was observed in both the sputum and buccal mucosa swab specimens and considered slightly positive. the systematic result could be seen in table 2. compared to the sputum specimens, the identification of acid fast bacilli in oral swab specimens occurred in those table 1. international union against tuberculosis and lung disease scale10 microscope examination smear result smear interpretation infectiousness of patient no acid fast bacilli was found in 100 fields negative negative probably not infectious 1–9 acid fast bacilli in 100 fields number of acid fast bacilli moderately positive probably infectious 10–99 acid fast bacilli in 100 fields 1+ moderately positive probably infectious 1–10 acid fast bacilli 1 fields 2+ strongly positive probably very infectious >10 acid fast bacilli 1 fields 3+ strongly positive probably very infectious table 2. acid fast bacilli presence on sputum and oral swabs no. of subjects (sex) acid fast bacilli sputum oral swabs 1 (m) (–) (–) 2 (f) (–) (–) 3 (f) (–) (–) 4 (m) (–) (–) 5 (m) (2+) (3) 6 (f) (–) (–) 7 (f) (3+) (4) 8 (m) (1+) (–) 9 (m) (5) (–) 10 (m) (1+) (–) 11 (f) (–) (–) 12 (f) (–) (–) 13(f) (1+) (–) 14 (f) (1+) (–) 15 (m) (2+) (–) 16 (m) (–) (–) 17 (m) (1+) (–) 18 (m) (–) (–) 19 (f) (7) (–) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p91–94 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p91-94 93 bakti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 91–94 patients with a significant number of acid-fast bacilli in their sputum. as described above,10 the interpretation of the results of these patients with regard to their sputum could be classified as strongly positive, while the patients themselves were probably highly infectious. seven patients with moderately positive results in their sputum examination presented negative results in a buccal swab examination, whereas subjects with negative sputum examination results presented the same results in buccal swab specimens. discussion tuberculosis is an infectious disease caused by a pathogenic agent, mycobacterium tuberculosis complex. this complex includes m. tuberculosis, m. bovis, m. caprae, m. africanum, m. microti, m. pinnipedii, m. mungi, m. orygis and m. canetti, with the species most commonly affecting humans being m. tuberculosis.3 this bacteria is bacillus shaped, approximately 0.4–0.5  3 µm in size and aerobic. mycobacterium tuberculosis could not be classified as a positive or negative gram bacteria, due to the difficulty of identifying it in gram staining.3,11,12 not all individuals with tb present symptoms, and as a result, diagnosis of the disease can be delayed with the result that the patient may remain ill and possibly infectious to others for a prolonged period.10 a diagnosis of tuberculosis can be confirmed by means of microscopic examination with ziehl neelsen staining, culture, radiographic examination and a tuberculin test.13 bacteriological examination which identifies the presence of mycobacterium tuberculosis is crucial to diagnosis. specimens used for such examinations include: sputum, pleural fluid, cerebro-spinal fluid, joint fluid, gastric washings, blood and other tissues.11 a variety of clinical specimens other than sputum may be submitted for examination when extrapulmonary tb disease is suspected.10 based on its natural history, tuberculosis is transmitted by air-borne droplet nuclei containing bacteria from an infected person while coughing, sneezing, speaking and singing.6 the tb bacilli in sputum from the lungs may be deposited passively in the oral cavity during expectoration. consequently, self-inoculation of the oral mucosa may occur and oral lesions as a manifestation of tuberculosis would be present.14,15 the diagnostic method used for the last 15 years as the implementation and expansion of a dots strategy program is the examination of direct smear of acid fast bacilli on sputum.13,16 at least three consecutive sputum specimens are required, each collected at 8 to 24-hour intervals, with at least one being an early morning specimen.10 the specimens are then referred to as ‘spot’, ‘morning’, and ‘spot’ specimens.17 the identification of acid-fast bacilli in sputum underpins a presumptive diagnosis of tuberculosis and indicates that the patient is capable of transmitting the disease. conversely, the absence of acid-fast bacilli in sputum smears has been taken as an indication that such patients are relatively less infectious, although they should not be regarded as immune to tb.18 smear results show a number of acid-fast bacilli observed at 1000× magnification that are categorized into five levels as described in the iuatld scale (table 1). in short, the greater the number, the more infectious the patient.10 in this study, the number of bacteria in sputum and buccal swabs were compared. it was observed that acid-fast bacilli were present on the buccal swab of subjects with 2+ and 3+ direct smear test results of sputum. the acid-fast bacillus smear status of the source case provides a strong indication of which patients are the most contagious. as an airborne transmitted disease, it has been estimated that 10 secondary infections arise annually from untreated smear-positive cases of tuberculosis.19 this study was undertaken to detect the presence of acid-fast bacilli in the oral cavity, and buccal swabs were used as an oral sample, since they were considered as smaller samples, more uniform in volume and composition, and less viscous and heterogenous.8 acid-fast bacilli were found in two oral buccal swab samples. this study supports a previous one conducted by yassen et al.,7 into tuberculosis bacilli detection in the oral cavity which suggested that approximately 60% of patients presented a positive result in acid-fast identification of tuberculosis patient saliva. the study also suggested that there was no acidfast bacilli present in the parotid saliva of 25 pulmonary tuberculosis patients who had recorded a positive acid-fast bacilli result in their sputum. it was, therefore, suspected there had been contact between infectious sputum and oral epithelial tissue.7 shenai suggested that out of 26 cases of culture–positive pulmonary tuberculosis, only 10 subjects provided mtb positive saliva samples when tested with the xpert assay.20 both samples, saliva and buccal swab, are easily and simply collected with minimal discomfort.8 another study suggested that in countries such as india, where tuberculosis, especially active pulmonary tuberculosis is endemic, tuberculosis bacilli deposition in the oral cavity might subsequently contaminate the lips, tongue, gingiva, oral mucosa and saliva during expectoration.21 wood also proposed that there might be adherence tuberculosis bacilli on buccal epithelial mucosa, because mycobacterium tuberculosis dna can be detected in buccal mucosa samples collected from 90% of active pulmonary tuberculosis patient.8 oral cavity sample-based research has also been conducted on dental plaque and caries lesions. eguchi table 3. comparation of acid fast bacilli on sputum and buccal swab specimens sputum specimens oral buccal swab specimens strongly positive moderately positive negative strongly positive 0 2 1 moderately positive 0 0 7 negative 0 0 9 total 0 (0%) 2 (10.5%) 17 (89.5%) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p91–94 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p91-94 94bakti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 91–94 compared the culture examination technique, as the gold standard method, and polymerase chain reaction (pcr) technique on several oral cavity samples. saliva-based culture examination confirmed a sensitivity of about 17.3%, a figure relatively low when compared to pcr, which reached 98%.9 another study comparing salivary and dental plaque samples by the pcr method showed that the sensitivity and specificity of this method in detecting salivary m. tuberculosis were 92% and 88% respectively, while the pcr results on m. tuberculosis plaques showed lower sensitivity but higher specificity.21 eguchi shows that the results of pcr dental plaque are the same at about 92%, but in contrast with the results of a culture (2%). the examination plaque attached to denture showed the results of a sensitivity of 0% on culture and 100% using pcr.9 this study showed that, in comparison to the pcr method, the sensitivity of direct smear of buccal mucosa swab test for acid-fast bacilli against sputum was 20%, although the sensitivity in pulmonary tuberculosis patients was only 10.5%. therefore, molecular-based diagnostic tests were more effective at measuring sensitivity and specificity when detecting mycobacterium tuberculosis in the oral samples. one point to remember here is that the major disadvantage of pcr methods is that they cannot differentiate live from dead cells. pcr technique is based on the bacterium genome, while the staining method is based on bacterial morphology.22 another study suggests that mycobacterium tuberculosis survive less successfully in the oral cavity because of certain inhibitory factors there which have bactericidal features inimical to m. tuberculosis growth. this theory is supported by an in vitro study, where mycobacterium tuberculosis isolate was inhibited by a. naeslundii, p. gingivalis, and f. nucleatum, the bacteria commonly found in the oral cavity.9 other oral defense systems include: saliva, intact mucosa, enzymes and tissue antibodies that also play a role in resisting the development of tuberculosis. because of these factors, the incidence rate of oral tuberculosis is extremely rare, approximately 0.05–5% worldwide.23,24 in conclusion, the results of this study show that acid-fast bacilli can be detected in a buccal epithelial mucosa sample, although their number was extremely low. the acid-fast bacilli bacterium detected in the oral sample during this study indicates, firstly, that the oral cavity might be a source of tuberculosis and, secondly, it is possible for the bacteria to be transmitted to other individuals. however, further studies in this area are necessary. references 1. floyd k. global tuberculosis report 2015. who press. france; 2015. p. 1–3. 2. who. global tuberculosis report 2016. who press. geneva; 2016. p. 1–3. 3. kasper dl, fauci as, hauser sl, longo dl, jameson jl, loscalzo j. harrison’s principles of internal medicine. 19th ed. new york: mcgraw hill education medical; 2015. p. 1102–22. 4. cadmus si, okoje vn, taiwo bo, van soolingen d. exposure of dentists to mycobacterium tuberculosis, ibadan, nigeria. emerg infect dis. 2010; 16(9): 1479–81. 5. cleveland jl, robison va, panlilio al. tuberculosis epidemiology, diagnosis and infection control recommendations for dental settings: an update on the centers for disease control and prevention guidelines. j am dent assoc. 2009; 140(9): 1092–9. 6. porteous nb, terézhalmy gt. exposure control issues for oral healthcare workers. dental continuing education course. new york: american dental association; 2013. p. 1–24. 7. yassen g, noori j, yas ns. detection of acid fast bacilli in the saliva of patients having pulmonary tuberculosis. j bagh coll dent. 2012; 24(3): 59–62. 8. wood rc, luabeya ak, weigel km, wilbur ak, jones-engel l, hatherill m, cangelosi ga. detection of mycobacterium tuberculosis dna on the oral mucosa of tuberculosis patients. sci rep. 2015; 5: 1–5. 9. eguchi j, ishihara k, watanabe a, fukumoto y, okuda k. pcr method is essential for detecting mycobacterium tuberculosis in oral cavity samples. oral microbiol immunol. 2003; 18(3): 156–9. 10. cdc. chapter 4: diagnosis of tuberculosis disease. in: core curriculum on tuberculosis: what the clinician should know. 6th ed. united states of america: cdc; 2012. p. 75–108. 11. brooks gf, carroll kc, butel js, morse sa, mietzner ta. jawetz, melnick & adelberg’s medical microbiology. 26th ed. new york: mcgraw-hill medical; 2013. p. 313–21. 12. parija sc. textbook of microbiology & immunology. 2nd ed. india: elsevier; 2012. p. 345-57. 13. chang k, lu w, wang j, zhang k, jia s, li f, deng s, chen m. rapid and effective diagnosis of tuberculosis and rifampicin resistance with xpert mtb/rif assay: a meta-analysis. j infect. 2012; 64(6): 580–8. 14. jain p, jain i. oral manifestations of tuberculosis: step towards early diagnosis. j clin diagn res. 2014; 8(12): ze18-21. 15. gill js, sandhu s, gill s. primary tuberculosis masquerading as gingival enlargement. br dent j. 2010; 208(8): 343–5. 16. leung e, minion j, benedetti a, pai m, menzies d. microcolony culture techniques for tuberculosis diagnosis: a systematic review. int j tuberc lung dis. 2012; 16: 16–23. 17. stinson kw, eisenach k, kayes s, matsumoto m, siddiqi s, nakashima s, hashizume h, timm j, morrissey a, mendoza m, mathai p. mycobacteriology laboratory manual. japan: global laboratory initiative; 2014. p. 1–154. 18. behr ma, warren sa, salamon h, hopewell pc, ponce de leon a, daley cl, small pm. transmission of mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. lancet. 1999; 353(9151): 444–9. 19. craft dw, jones mc, blanchet cn, hopfer rl. value of examining three acid-fast bacillus sputum smears for removal of patients suspected of having tuberculosis from the “airborne precautions” category. j clin microbiol. 2000; 38(11): 4285–7. 20. shenai s, amisano d, ronacher k, kriel m, banada pp, song t, lee m, joh js, winter j, thayer r, via le, kim s, barry ce, walzl g, alland d. exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in hiv-negative patients by use of the genexpert mtb/rif assay. j clin microbiol. 2013; 51(12): 4161–6. 21. palakuru sk, lakshman vk, bhat kg. microbiological analysis of oral samples for detection of mycobacterium tuberculosis by nested polymerase chain reaction in tuberculosis patients with periodontitis. dent res j. 2012; 9(6): 688–93. 22. caulfield aj, wengenack nl. diagnosis of active tuberculosis disease: from microscopy to molecular techniques. j clin tuberc other mycobact dis. 2016; 4: 33–43. 23. sansare k, gupta a, khanna v, karjodkar f. oral tuberculosis: unusual radiographic findings. dentomaxillofacial radiol. 2011; 40(4): 251–6. 24. nanda kds, mehta a, marwaha m, kalra m, nanda j. a disguised tuberculosis in oral buccal mucosa. dent res j. 2011; 8(3): 154–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p91–94 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p91-94 104 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 104–109 antibacterial effect of 70% ethanol and water extract of cacao beans (theobroma cacao l.) on aggregatibacter actinomycetemcomitans ayu rafania atikah, hendrik setia budi, and tuti kusumaningsih departement of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans) is a gram negative bacteria that form a subgingival plaque causing periodontitis. nowadays, many natural resources can be used as a basic ingredient of drugs. one of the resources used as an antibacterial material is cacao bean. it contains of polyphenol flavonoids, such as catechin, epicatechin, anthocyanin, and proanthocyanidin. chemical compounds contained in ethanol extract and water extract are different in quantity from those in cocoa beans. purpose: this research aimed to find out difference in antibacterial activity between the 70% ethanol and water extract of cacao beans (theobroma cacao l.) on a. actinomycetemcomitans. method: this research was an in vitro laboratory experiment. the serial dilutions was performed on the 70% ethanol and water extract of cacao beans a concentration of 100% to 3.125%. at each concentration, the 70% ethanol and water extract of cacao beans were added with grown bacterial suspension of a. actinomycetemcomitans. after they were incubated for 24 hours, the bacteria grown on luria berthani media were observed. bacteria colonies then were measured in cfu/ml. result: there were significant differences in bacterial colonies grown at the concentrations of 6.25% and 3.125% between the 70% ethanol extract of cacao beans and the water extract of cacao beans as p-value = 0.000 (p<0.05). conclusion: 70% ethanol beans and water extract of cacao beans have antibacterial activity against a. actinomycetemcomitans. the concentrations of mic and mbc extracts were 6.25% and 12.5% respectively. keywords: aggregatibacter actinomycetemcomitans; cacao beans; antibacterial effect correspondence: hendrik setia budi, department of oral biology, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: hendriksetiabudi@gmail.com introduction periodontal disease is an infectious disease of the oral cavity that can be suffered by teens and adults. based on health research (riskesdas) in 2013, the prevalence of oral and dental problems, including periodontal disease reached 25.9%. the most common periodontal tissue diseases are gingivitis and periodontitis.1-3 periodontitis is an infectious disease resulting in inflammation of the tissues supporting teeth, gingival attachment loss progressively, and bone loss. the disease is caused by the induction of facultative anaerobic bacteria and gram-negative bacteria. one of the gram-negative anaerobic bacteria that plays a role in the formation of subgingival plaque causing periodontitis is aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans).1,4 a. actinomycetemcomitans are gram-negative bacteria that have a small, non-motile, capnophilic, fermentative coccobacillus form. a. actinomycetemcomitans can be found in dental plaque, periodontal pockets, and buccal mucosa in 36% of patients in the normal population.1 a.actinomycetemcomitans release virulence factors, deliver adhesion on oral surfaces, inactivate the host immune response, as well as induce inflammation and tissue dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 105105atikah, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 104–109 damage. a. actinomycetemcomitans are also frequently detected in periodontal tissues of healthy people and then can be grouped into a part of the normal flora in oral cavity.5 currently, some herbal ingredients have been developed as an alternative to medicine ingredients, one of which is cocoa beans. indonesia ranks on the third position in the world cocoa producers. cocoa production in indonesia has the potential to be improved, but it depends on political and local economic factors.6 the size of cocoa growing areas in indonesia in 2002 has reached 776, 900 hectares scattered in all provinces, except in the capital city of jakarta (dki).7 cocoa is composed of stems, fruits and seeds, flowers, roots, bark seeds, and leaves. cocoa (theobroma cacao l.) has polyphenolic compounds, such as catechin, epicatechin, anthocyanin, proanthocyanidin, phenolic acid, condensed tannins, flavonoids, and other small compounds.8 cocoa polyphenols are useful as an antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial.9 generally, the majority of people consume cocoa only on the seeds alone. several researches also have shown that cocoa bean skin and cocoa beans can be used as antibacterial ingredients.7 cocoa beans contain polyphenolic flavonoid of flavonol class, namely monomeric catechin and epicatechin flavonoids as well as proanthocyanidin polymerized flavonoids.10 flavonols are classified into a flavonoid group composed of several molecules of phenols (polyphenols).11 flavonols contained in cocoa stimulate peripheral blood mononuclear cells to secrete interleukin-5 (il-5 ) and stimulate immunoglobulin a (iga) production that protects the oral cavity from streptococcus mutans.8 cocoa beans can also inhibit the growth of streptococcus mutans at an effective concentration of 12.5%.12 the extract water of cocoa beans and the 70% ethanol extract of cocoa beans even have antibacterial activity against escherichia coli and bacillus subtilis. a research on cocoa beans extracted with the ethanol 70% shows a higher antimicrobial activity against streptococcus mutans and c. albicans bacteria than with water.13 cocoa, furthermore, will generate a different antibacterial activity when extracted with different polarities of its solvents.14 extraction method varies depending on the samples to be tested. several different solvents used include water, 70% ethanol, 70% methanol, acetonitrile, diethyl ether, and acetone. 70% ethanol (ethyl alcohol) is a solvent which has a low boiling point and is widely used by industries. 70% ethanol has a boiling point of 70° c so that extraction temperature is used to attract all the components in the basic materials.15 70% ethanol is widely used as a solvent of various chemical or natural materials devoted to drugs. in in vitro research, 70% ethanol can disrupt transport ions, namely na +, k +, and atp. the use of 70% ethanol in antibacterial material has a risk to human health, especially in liver because of the residual concentration and ethanol exposure.16 polyphenol level, furthermore, will be higher when extracted with a lower solvent polarity. solvent water will tend to dissolve inorganic compounds and salts of acids or bases.14 the use of water compared to the 70% ethanol as cosolvent can generate higher residual polyphenols, so the solvent water is considered to be better because it can maintain a high quantity of beneficial compounds.17 this study aimed to determine whether there were differences in antibacterial activity between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against a. actinomycetemcomitan bacteria. materials and method this study was an in vitro laboratory experimental research using randomized post test only control group design. this study was conducted at the laboratory of microbiology, faculty of dental medicine, universitas airlangga from june to october 2015. samples were a. actinomycetemcomitans bacteria taken from the microbiology laboratory, faculty of dental medicine, universitas airlangga, and then cultured in brain heart infusion broth media (bhib). minimal sample size eligible to be analyzed is determined by lemeshow formula, as many as five. manufacture of 70% ethanol extract of cocoa beans and water extract of cocoa beans was conducted. the 70% ethanol extract of cocoa beans and the water extract of cocoa beans were obtained from integrated service unit (upt) materia medika batu, east java. cocoa beans used were non-fermented cocoa beans and macerated using 70% ethanol and water solvent. having obtained the 70% ethanol extract of cocoa beans and the water extract of cocoa beans, the preparation of a. actinomycetemcomitans bacteria then was performed by suspending the bhib media until turbidity standard was equivalent to 0.5 mc farland (1.5 × 108 cfu/ ml). manufacture of the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at various concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% was performed using serial dilution method. the determination of minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against a. actinomycetemcomitan bacteria was started from the preparation of the test tube as much as 9 tube. six test tubes were filled with bacterial suspensions of a. actinomycetemcomitans as much as 0.05 ml that had been standardized to 0.5 mc farland and mixed with bhib media as well as the extracts at the various concentrations (100%, 50%, 25%, 12.5%, 6.25 %, and 3.125). the test tube k + (as positive control) containing 0.05 ml bacterial suspension of a. actinomycetemcomitans were planted in the bhib media, while the test tube k(as negative control) contained only media bhib and control extracts. control is useful to make sure that no bacterial contamination in the media. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 106 atikah, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 104–109 the reading of the results of antibacterial activity in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against the growth of a. actinomycetemcomitans bacteria was conducted by measuring the number of colonies grown on bacteria subculture as much as 0.1 ml of each tube as well as a positive control and a negative control on the luria berthani media. planting on the luria berthani media was conducted using spreader, and incubated at 37 ° c for 24 hours. the results showed 90% of a. actinomycetemcomitans bacterial growth was inhibited compared with the positive control used as mic. the results also indicated 99.9% of a. actinomycetemcomitans bacterial death compared with the positive control was used as mbc. mic and mbc were determined by measuring the number of colonies grown on the luria berthani media manually and expressed as cfu/ ml. the calculation was repeated five times by three different observers, and then the mean values were measured. processing data was conducted using a statistical analysis test for normality with kolmogorov-smirnov test to see whether the data obtained were normally distributed or not. next, homogeneity test using levene test was performed. a statistical test for antibacterial power difference in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration then was carried out using independent t-test. results this research used a. actinomycetemcomitan bacteria incubated for 1 x 24 hours at 37° c in the bhib media, synchronized with the 0.5 mcfarland standard. materials used were non-fermented cocoa beans from kota batu, malang. next, cocoa extracts were made in upt materia medika batu in east java by maceration method using 70% ethanol and water. based on the analysis results of chemical compounds contained in the 70% ethanol extract of cocoa beans as well as in the water extract of cocoa beans, there were different dosages of flavonoids, catechins, epicatechin, anthocyanin, and proanthocyanidin contained (table 1). catechin, epicatechin, and anthocyanin contained in the 70% ethanol extract of cocoa beans were higher than in the water extract of cocoa beans. meanwhile, flavonoids and proanthocyanidin contained in the water extract of cocoa beans were higher than in the 70% ethanol extract of cocoa beans. serial dilution method is a method usually used in a comparative research of mic and mbc in both the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against a.actinomycetemcomitan bacteria (figure 1). during the observation of mic and mbc in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration, the results of serial table 1. the analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans no. chemical compounds 70% ethanol extract of cocoa beans water extract of cocoa beans 1. flavonoids 1.32% 2.18% 2. catechins 2.01% 1.22% 3. epicatechin 1.36% 1.08% 4. anthocyanins 1.74% 1.67% 5. proanthocyanidin 1.93% 2.51% 11 table 1. the analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans figure 1. serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans against a. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. figure 2. a) streak results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; b) streak results of the water extract of cocoa beans against a.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; d) replanting results of the water extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media. no. chemical compounds 70% ethanol extract of cocoa beans water extract of cocoa beans 1. flavonoids 1.32% 2.18% 2. catechins 2.01% 1.22% 3. epicatechin 1.36% 1.08% 4. anthocyanins 1.74% 1.67% 5. proanthocyanidin 1.93% 2.51% 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% 11 table 1. the analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans figure 1. serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans against a. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. figure 2. a) streak results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; b) streak results of the water extract of cocoa beans against a.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; d) replanting results of the water extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media. no. chemical compounds 70% ethanol extract of cocoa beans water extract of cocoa beans 1. flavonoids 1.32% 2.18% 2. catechins 2.01% 1.22% 3. epicatechin 1.36% 1.08% 4. anthocyanins 1.74% 1.67% 5. proanthocyanidin 1.93% 2.51% 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% a b figure 1. serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans against a. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. dilution were planted in luria berthani agar media using streak technique as a cross-check to see the growth of bacterial colonies. it was necessary to do because of dark-colored extract material and turbidity occured at any concentration. furthermore, based on the observation results on the number of bacterial colonies grown in the luria berthani media expressed in colony forming units (cfu), 70% ethanol extract of cocoa beans and water extract of cocoa beans could be able to inhibit and kill a. actinomycetemcomitan bacteria (table 2). the comparative test on the mic and mbc in the 70% ethanol extract cocoa beans and the water extract of cocoa beans was conducted using serial dilution method. the results showed that the extracts at the concentrations of 100%, 50%, 25%, and 12.5% contained no bacterial growth. meanwhile, the mean bacterial growth in the 70% ethanol extract cocoa beans at the concentration of 6.25% was 11.4. on the other hand, the mean bacterial growth in the water extract of cocoa beans at the same concentration was 7.8 (table 2). before the antibacterial power difference analysis test on the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against a. actinomycetemcomitan bacteria was performed, the normality test using kolmogorovsmirnov test as well as the homogeneity test using levene test were conducted in each group. the results of the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 107107atikah, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 104–109 normality and homogeneity tests showed that data obtained in the water extracts of cocoa beans at the concentrations of 6.25% and 3.125% were normally distributed and homogeneous (p>0.05). whereas, data obtained in the 70% ethanol extract of cocoa beans at the concentration of 6.25% were not normally distributed (p <0.05), and at the concentration of 3.125% was not homogeneous (p<0.05). 11 table 1. the analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans figure 1. serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans against a. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. figure 2. a) streak results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; b) streak results of the water extract of cocoa beans against a.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; d) replanting results of the water extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media. no. chemical compounds 70% ethanol extract of cocoa beans water extract of cocoa beans 1. flavonoids 1.32% 2.18% 2. catechins 2.01% 1.22% 3. epicatechin 1.36% 1.08% 4. anthocyanins 1.74% 1.67% 5. proanthocyanidin 1.93% 2.51% 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% a 11 table 1. the analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans figure 1. serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans against a. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. figure 2. a) streak results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; b) streak results of the water extract of cocoa beans against a.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; d) replanting results of the water extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media. no. chemical compounds 70% ethanol extract of cocoa beans water extract of cocoa beans 1. flavonoids 1.32% 2.18% 2. catechins 2.01% 1.22% 3. epicatechin 1.36% 1.08% 4. anthocyanins 1.74% 1.67% 5. proanthocyanidin 1.93% 2.51% 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% b c d figure 2. a) streak results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; b) streak results of the water extract of cocoa beans against a.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media; d) replanting results of the water extract of cocoa beans against a. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the luria berthani media. table 2. the number of a. actinomycetemcomitan bacterial colonies grown in the luria berthani media at various concentrations of 70% ethanol extract of cocoa beans and water extract of cocoa beans treatment group n the means number of the bacterial colonies (x108 cfu/ml) 70% ethanol extract water extract positive control negative control at the concentration of 100% 5 0 0 109 0 at the concentration of 50% 5 0 0 at the concentration of 25% 5 0 0 at the concentration of 12.5% 5 0 0 at the concentration of 6.25% 5 11.4 7.8 at the concentration of 3.125% 5 27.2 18 note: n: number of replication; 0: no growth on the negative control; 109: the number of bacterial colonies grown as the positive control table 3. the mean and standard deviation of the number of bacterial colonies of a. actinomycetemcomitans treatment control mean ± sd significance 70% ethanol extract water extract at the concentration of 6.25% 11.4 ± 0.54772 a 7.8 ± 0.83666 b p = 0.000 at the concentration of 3.125% 27.2 ± 1.64317 a 18.0 ± 1.22474 b p = 0.000 note: a b: significant difference next, kruskal-wallis test was carried out to test significant differences in antibacterial activities between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans since there were groups which were not normally distributed and not homogeneous. the results showed value of p was less than 0.05 (p = 0.000), indicating that there was a difference in antibacterial activity between the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 108 atikah, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 104–109 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentrations of 6.25% and 3.125% (table 3). discussion this research was an in vitro research aimed to see the difference in antibacterial power beween the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against a. actinomycetemcomitan bacteria. antibacterial agents used in this research were non-fermented cocoa beans since non-fermented cocoa beans actually have higher antibacterial effects than fermented ones. fermentation and drying processes, however, can reduce the active components of natural materials, such as polyphenol.18 this research also used a serial dilution method with the concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. a. actinomycetemcomitan bacteria grown on the bhib media were considered as the positive control (+), whlie the bacteria grown on the bhib media and the control extracts were considered as as the negative control (-). serial dilution method was used because it can give the significant calculation results of microorganism colonies at small concentrations. thus, if the serial dilution method is not conducted, there will be a number of colonies stacked so that the calculation of the colonies will be not accurate.19 serial dilution method has widely been used for many years.20 in the antibacterial activity test, properties owned by the cell wall of bacteria may affect the ability of cocoa bean extracts to inhibit tested bacterial growth.21 a.actinomycetemcomitan bacteria are gram-negative bacteria that have a cell wall in the form of peptidoglycan surrounded by a membrane structure, called as outer membrane. outer membrane of gram-negative bacteria has lipopolysaccharide or endotoxin components that can prevent an antibacterial agent to penetrate into the cells.22,23 in the layers of lipopolysaccharide, gram-negative bacteria have a system of selection against strange substances so that gram negative bacteria are more resistant to strange substances than gram positive bacteria.21 the presence of polyphenols, such as flavonoids in cocoa beans can penetrate the membrane of gram-negative bacteria to mediate the cell response.24 flavonoids have antibacterial activity through the barrier function of bacterial dna gyrase so that replication capabilities and bacterial translation can be inhibited.25 flavonoids may cause damage to the permeability of bacterial cell wall, microsomes, and lysosomes as a result of the interaction of flavonoids with bacterial dna.26 catechins, moreover, are natural polyphenolic compounds and secondary metabolites included in the constituent group of tannins.21 catechins may damage cytoplasmic membrane causing the release of important metabolites that inactivate bacterial enzyme system. proanthocyanidin, furthermore, is condensed tannins that can inhibit the formation of cell walls and lead to termination of the crosslinking peptide that combines the glycan chains of peptidoglycan in another chain, causing cell membrane damage.28 how to cultivate antibacterial materials, thus, is a thing that needs attention. antibacterial material can be extracted by using various methods, one of which is by solvent extraction (maceration). solvents widely used for polyphenol extraction method are water and 70% ethanol. cocoa, on the other hand, will yield a different antibacterial activity when extracted with a different solvent polarity.14 ethanol is a polar solvent that dissolves polar compounds, while water is an inorganic solvent that dissolves inorganic materials. in addition, cacao bean is an inorganic material because it is derived from plants. to dissolve the cocoa beans using ethanol, therefore, will take a long process because of evaporation process. the process of evaporation can affect the polyphenol content in the extract. water, on the other hand, is a solvent that can maintain a high quantity of essential compounds contained in natural materials.17 water solvent will dissolve inorganic compounds and salts of acids and bases.14 cocoa beans diluted with water is more soluble and requires a shorter time than cocoa diluted with ethanol. the analysis results of chemical compounds in the 70% ethanol extract of cocoa beans contain flavonoids (1.32%), catechin (2.01%), epicatechin (1.36%), anthocyanin (1.74%), and proanthocyanidin (1.93%). the water extract of cocoa beans contain flavonoids (2.18%), catechin (1.22%), epicatechin (1.08%), anthocyanin (1.67%), and proanthocyanidin (2.51%). proanthocyanidin contained in cocoa beans was 58%, catechin amounted to 37%, and anthocyanin amounted to 4%, thus indicating that the water extract of cocoa beans contain more useful chemical compounds than the ethanol extract of cocoa beans.10 in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 6.25%, moreover, there was still bacterial colony growth on the luria berthani media after incubated for 1x24 hours. it means that not all bacteria die, or there are barriers to the growth of colonies by 90%.28 nevertheless, in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 12.5%, there was no colony growth. it indicates that the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at this concentration could kill bacteria by 99.9% of the total average bacteria that managed to grow on the positive control and were considered as bactericide.29,30 it can be concluded that water and 70% ethanol extract of cocoa beans have antibacterial activities against a. actinomycetemcomitans. both the extracts also have the same mic and mbc at concentrations of 6.25 and 12.5%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 109109atikah, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 104–109 references 1. carranza p, klokkevold f, newman m, takei h. carranza’s clinical periodontology. st. louis, mo: saunders elsevier; 2009. p. 187192 2. riset kesehatan dasar (riskesdas) nasional. jakarta: badan penelitan dan pengembangan kesehatan, departemen kesehatan republik indonesia; 2013. p. 113. 3. chauhan vs, chauhan rs, devkar n, vibhute a, more s. gingival and periodontitis diseases in children and adolescents. j dent & allie sci 2012 ; 1(1): 26. 4. newman m, takei h, klokkevold p, carranza f. carranza’s clinical periodontology. st. louis, mo: saunders elsevier; 2012. p. 18593. 5. umeda ej, priscila ll, maria rl, marcia pam. differential t r a n s c r i p t i o n o f v i r u l e n c e g e n e s i n a g g r e g a t i b a c t e r actinomycetemcom ita ns serotypes. brazil: depa r tement of microbiology, institute of biomedical sciences, university of sao paulo; 2013. p. 4-6. 6. gu f, tan l , wu h, fang y , xu f , chu z, wang q. comparison of cocoa beans from china, indonesia and papua new guinea. foods 2013; 2(2): 183-97. 7. misnawi. pemanfaatan biji kakao sebagai sumber antioksidan alami. jember: pusat penelitian kopi dan kakao indonesia; 2005. p. 6. 8. arlorio m, coisson jd, travaglia f, varsaldi f, miglio g, lombardi g, martelli a. antioxidant and biological activity of phenolic pigments from theobroma cacao hulls extracted with supercritical co2. food research international 2005; 38(8-9): 1009-14. 9. hii cl, law cl, suzannah s, misnawi, cloke m. polyphenols in cocoa (theobroma cacao l.). as j food ag-ind 2009; 2(04): 70222. 10. sulistyowati, misnawi. effects of alkali concentration and conching temperature on antioxidant activity and physical properties of chocolate. international food research journal 2008; 15(3): 297304. 11. lee sy, yoo ss, lee mj, kwon ib, dan pyun yr. optimization of nib roasting in cocoa bean processing with lotte-better taste and color process. food sci biotechnol 2001; 10: 286-93. 12. purnamasari da, munadziroh e, yogiartono rm. konsentrasi ekstrak biji kakao sebagai material alam dalam menghambat pertumbuhan streptococcus mutans. jurnal pdgi 2010; 59(1): 14-8. 13. gianmaria f. ferrazzano, ivana amato, aniello ingenito, antonino de natale, antonino pollio. anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). fitoterapia 2009; 80(5): 255–262 14. john na, fang z, kebitsamang jm, mohamed lb, camel l. quantification of total polyphenolic content and antimicrobial activity of cocoa (theobroma cacao l.) bean shells. pakistan journal of nutrition 2012; 11(7): 574-579 15. manuchair ebadi. pharmacodynamic basic of herbal medicine. 2nd ed. new york: taylor & francis; 2007.p. 647. 16. darmono. farmasi forensik dan toksikologi. jakarta: ui press; 2009. p. 23. 17. boakye s. levels of selected pesticide residues in cocoa beans from ashanti and brong ahafo regions of ghana. dissertation. ghana: faculty of physicial science college of science; 2012. 18. smullen j, koutsou ga, foster ha, zumbe a,storey dm. the antibacterial activity of plant extract containing polyphenols against streptococcus mutans. caries res 2007; 41: 342-9. 19. mejos jay al, caraignj.w, de pano j, labador av, macapagal em. bacterial colony isolation using serial dilution techniques. quezon city: institute of biology, college of science, university of the phillipines; 2010. p. 6. 20. seeley hw, vandemark pj. selected exercises from microbes in action a laboratory manual of microbiology. 3 rd ed. sanfrancisco: freeman and company; 1981. p. 37-41. 21. v.s.t. saito, t.f. dos santos, c.g. vinderola, c. romano, j.r. nicoli, l.s. araújo, m.m. costa, j.l. andrioli, a.p.t. uetanabaro. viability and resistance of lactobacilli isolated from cocoa fermentation to simulated gastrointestinal digestive steps in soy yogurt. journal of food and science 2014; 79(2): doi. 10.1111/1750-3841.12326. 22. kenneth t. structure and function of bacterial cells. online textbook of bacteriology2009; 5. 23. zainal h, made a, kasno, anggraini ad. uji aktivitas antibakteri propolis lebah madu trigona spp. prosiding seminar nasional indonesia, 2006; p. 204-14. 24. ariza bts, mufida d, fatima n. in vitro antibacterial activity of cocoa ethanolic extract against escherichia coli. international food research journal 2014; 21(3): 935-40. 25. misnawi, wulandari p, suswati e, rianul a. antibacterial effect of ethanol extract cocoa beans (theobroma cacao) on growth in vitro by shigella dysentriae. jurnal medika planta 2012; 1(5): 73. 26. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). majalah kedokteran gigi (dental journal) 2005; 38(2): 135-41. 27. chinami hirao, eisaku nishimura, masanori kamei, tomoko ohshima, nobuko maeda. antibacterial effects of cocoa on periodontal pathogenic bacteria. journal of oral biosciences 2010; 52(3): 283-291. 28. duskova m, karpiskova r. antimicrobial resistance of lactobacilli isolated from food. czech j food sci 2013; 31(1): 27-32. 29. surbhi leekha, christine l. terrell, and randall s. edson. general principles of antimicrobial therapy. mayo clin proc 2011;86(2): 156–167. 30. akinyemi ko, oluwa ok, omomigbehin eo. antimicrobial activity of cr ude extracts of three medicinal plants used in south-west nigerian folk medicine on some food borne bacterial pathogens. african journal of traditional, complementary and alternative medicines 2006; 3(4): 13-22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p104-109 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p104-109 contents page printed by: airlangga university press. (070/06.11/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 43 number 4 december 2010 issn 1978 3728 dental journal majalah kedokteran gigi 1. clinical consideration of thrombocytopenia in children s. ratna laksmiastuti ..................................................................................................................... 163–167 2. the increasing of odontoblast-like cell number on direct pulp capping of rattus norvegicus using chitosan widyasri prananingrum ................................................................................................................. 168–171 3. minor modification of millard's surgical technique for correction of complete unilateral cleft lip coen pramono d .............................................................................................................................. 172–175 4. biocompatibility and osteoconductivity of injectable bone xenograft, hydroxyapatite and hydroxyapatite-chitosan on osteoblast culture bachtiar ew, bachtiar bm, abas b, harsas na, sadaqah nf, and aprilia r ......................... 176–180 5. alveolar ridge rehabilitation to increase full denture retention and stability mefina kuntjoro, rostiny, and wahjuni widajati ....................................................................... 181–185 6. pulp nerve fibers distribution of human carious teeth: an immunohistochemical study tetiana haniastuti ........................................................................................................................... 186–189 7. the management of dental fracture on tooth 61 in a child with attention deficit hyperactivity disorders veranica and mochamad fahlevi rizal ......................................................................................... 190–194 8. multidisciplinary management of a mandibular buccal plate perforation yuli nugraeni and chiquita prahasanti ......................................................................................... 195–200 9 candida albicans adherence on acrylic resin plates immersed in black tea steeping soebagio ........................................................................................................................................... 201–204 10 a combination of endodontic therapy and root resection in furcation involvement case ernie maduratna setiawati ............................................................................................................. 205–209 11 interleukin-1b expression on periodontitis patients in surabaya chiquita prahasanti ......................................................................................................................... 210–214 128 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 128–131 original article the effect of x-ray irradiation to the formation of polychromatic e r y t h r o c y t e c e l l m i c r o n u c l e u s i n w i s t a r r a t s ( r a t t u s norvegicus) eha renwi astuti1, hutojo djajakusuma1, indeswati diyatri2 and nastiti faradilla ramadhani1,3 1department of dentomaxillofacial radiology, 2department of oral biology, 3graduate student of dental health science faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: panoramic and cephalometric radiography is very important for diagnosis, treatment plan, and evaluation of orthodontic treatment results. panoramic and cephalometric radiography are frequently performed at the same time, causing dna damage and chromosome aberration. purpose: this study aims to analyse the effect of x-ray exposure in panoramic and cephalometric radiography on micronuclei cell numbers. methods: laboratory-based analytical study with 60 healthy-male wistar rats weighing 200–300 grams divided into 6 treatment groups (n=10). the control group: without radiographic exposure, the treatment group 2: using panoramic radiographic exposure followed by cephalometric, and the treatment group 3: using panoramic radiographic exposure and 24 hours later performed cephalometric radiographic. the unit of analysis was the polychromatic erythrocytes of mice cell, were examined 24 hours and 48 hours after irradiation had been finished. the polychromatic erythrocytes were examined using may-gruenwald-giemsa staining and 100x magnification under a microscope with 2000 cells per view. data obtained were analysed using the statistical package for social science (spss) 20 version software. the mean and standard deviations were calculated for each clinical parameter, and a one-way anova statistical test of significance was used. statistical significance was set at p<0.05. results: the analysis showed a significant increase (p<0.05) in the number of micronucleus in groups that used panoramic radiographic exposure followed by cephalometric. conclusion: x-ray radiation can increase the number of micronucleus in polychromatic erythrocyte cells in rats. keywords: cephalometric x-ray; micronucleus; panoramic x-ray; polychromatic erythrocyte cell correspondence: eha renwi astuti, department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47, surabaya 60132 indonesia. email: eha-r-a@fkg.unair.ac.id introduction radiographic examination denotes essential steps in dentistry, especially in clinical orthodontics.1 panoramic imaging and cephalometric analysis are required before treatment to help make an established diagnosis and consider several treatment options.2 the processes of panoramic and cephalometric radiography are mostly done in succession to save the patients time and energy.3 besides, taking panoramic and cephalometric imaging at the same time also hasten diagnosis establishment and treatment planning process. however, this decision gives rise to several undesirable effects toward cells in the body such as the dna single-strand break, dna double-strand break, dna cross-link, and chromosome aberrations.4 chromosome aberration is an immediate effect of radiation exposure. therefore, radiation protection is needed for patients and operators.5 chromosome aberrations can affect one or more genes in a complex manner by changing the regulation of gene expression, disrupting exons, and creating fusion genes.5 the examination of chromosome aberration can be done using a micronuclei test. in the telophase stage, the chromosome fragment and chromatin inside the cell will fall behind in the cytoplasm forming a nucleus-like structure, with sizes ranging from 1/20 to 1/5 of the nuclei diameter, called dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p128–131 mailto:eha-r-a@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p128-131 129astuti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 128–131 micronucleus.6 the micronucleus formation denotes an indicator of mutagenic activity that may damage the chromosome, leading to cancer.7 a simple method to detect micronuclei can be done by examining polychromatic erythrocyte cells from the mice bone marrow swab.8 this study aims to analyse the effect of x-ray exposure in panoramic and cephalometric radiography on micronuclei cell numbers. materials and methods all the procedures in this study have been reviewed and approved by the faculty of dental medicine, universitas airlangga ethical committee, with ethical certificate number 395/hrecc.fodm/vi/2019. this was a laboratory-based analytical experiment involving 60 male wistar rats, three to four months old, weighing 200–300 g. the sampling technique from the rat’s right femur was determined by lemeshow’s method. the rats were acclimatized for a week and randomly divided into six groups, each containing ten samples, namely a1, b1, a2, b2, a3, and b3. a1 and b1 act as the control groups receiving no radiation; while a2 and b2 groups were exposed to x-ray radiation from panoramic and cephalometric radiographs consecutively. the a3 and b3 groups were exposed to x-ray radiation from a panoramic radiograph and were re-exposed to x-ray radiation from a cephalometric radiograph after 24 hours. the observation of micronuclei for groups a1, a2, and a3 was conducted 24 hours after radiation exposure while the observation for groups b1, b2, and b3 was done 48 hours after radiation exposure. the rats were kept in a plastic cage at room temperature with a 12-hour light-dark cycle at a constant temperature of 23°c and fed a standard pellet diet (expanded pellets, stepfield, witham, essex, uk) with tap water ad libitum. the x-ray exposure was done according to the group allocation with doses of 0.3 msv for panoramic radiograph and 0.03 msv for cephalometric radiograph using the x-ray machine orthopantomograph® op100 (instrumentarium corporation, u.s) with a capacity of 77 kvp 12 ma. after being exposed to the x-ray radiation, the rats were put back in the cage. twenty-four hours after the x-ray exposure, rats in groups a1, a2, and a3 were euthanised by means of cervical dislocation while the rats in groups b1, b2, and b3 were euthanised 48 hours after, in the same manner. bone marrow was aspirated with a 5-ml syringe slowly from the femur, moved to a micro-tube, and centrifuged consecutively. after a suspension was formed, a drop of the suspension was put on an object-glass, dried, and stained using may-gruenwald-giemsa. the bone marrow slides were observed after oil immersion under a light microscope (bx 53 upright microscope, olympus corporation, tokyo, japan) at 100x magnification. the micronuclei in polychromatic erythrocyte counting were conducted using the score blind method by means of a cell counter. in this study, micronuclei from 2000 polychromatic erythrocytes were counted by three researchers after calibration between the researchers. the data obtained were analysed using spss version 20 for windows (ibm corp, chicago, usa). the assumption of the normality data was assessed by the shapiro-wilk test, the test for the homogeneity of variances using levene statistics, and the one-way anova test at 0.05 significance to find any difference between the groups. results the result of this study, observing the effect of x-ray radiation towards micronuclei in polychromatic erythrocyte cells of male wistar rats bone marrow, revealed that the control groups (a1 and b1) had the least number of micronuclei compared to the other groups while the most micronuclei recorded was from a2, followed by b2, a3, and b3 groups (figure 1). a1 a3 a2 b1 b3 b2 figure 1. micronuclei (red arrows) from polychromatic erythrocyte cells from each experimental group with 100x magnification. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p128–131 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p128-131 130 astuti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 128–131 generally, micronuclei count was found less in the groups exposed to both radiation after a 24-hour interlude compared to the groups exposed to both radiations on the same day. the data obtained are presented in mean and standard deviation (table 1). based on those observational results, it can be seen that a time interval between two x-ray exposure results in fewer micronuclei. it is supported by statistical analysis that shows a significant difference between groups (p<0.05). discussion ionizing radiation is known to have an undesirable side effect that results in various changes and damages in the cell.1 the cell damage, as a consequence of radiation, may be both reversible and irreversible.9 the damaged cells due to x-ray radiation may recover through a cell repair process, yet it depends on the cell type and radiation dose received.10 cells are constantly exposed to internal and external harmful agents such as viruses and chemicals which can lead to changes in cells function and structure. these factors can cause cell necrosis or changes in nucleus genetic information.11 x-ray is one of the electromagnetic forms that can cause changes in organisms, and it is widely used for diagnosis and treatment in medicine and dentistry.12 results of this study show a significant difference in micronuclei cell numbers before and after x-ray exposure. a previous study by preethi et al.13 used the cytology method to evaluate the x-ray effects on cells after panoramic and lateral cephalometric radiography. preethi et al.13 investigated the genotoxicity effects of panoramic radiography in gingival epithelial cells and showed that x-ray increased genotoxicity in these cells that caused chromosomal damages. micronucleus index reflects genomic instability, and an increase in the number of micronuclei shows an increased risk for cancer.7 the damage because of micronuclei formation happens in epithelial basal cells where mitosis happened. epithelial cell turnover brings them to the surface thus most rates of micronuclei formation happens in mucosal surface one to three weeks after genotoxic factors exposure.13 the radiation used in this study was acquired from a single dose panoramic and cephalometric radiograph. a significant increase of micronuclei on polychromatic erythrocyte cells in rats was recorded on groups exposed to both radiations on the same day, observed 24 (a2) and 48 hours (b2) after exposure. the number of micronuclei from both groups was significantly higher compared to both control groups (a1 and b1), which did not receive any radiation exposure. the micronucleus found in both control groups a1 and b1 denotes micronuclei that are naturally formed without any influence from mutagenic substances exposure.14 meanwhile, micronucleus found in treatment groups a2 and b2 are formed due to x-ray exposure from panoramic and cephalometric radiographs. based on the microscopic examination, it can be seen that in the control group only one to three micronuclei were found in each sample. the result is in accordance with the previous research conducted by reisz et al.10 which observed 2000 polychromatic erythrocytes and found about one to two micronuclei in the control group. the formation of natural micronuclei may happen due to several factors such as stress level, which in this study is the experimental animal stress level. stress may lead to a hypoxic condition that may further affect the production of red blood cells. the red blood cells formation may be impaired or stopped due to cell cycle disruption. interference in the interphase stage may lead to a decrease in red blood cell production in accordance with the blood cell progenitor sensitivity. meanwhile, disrupted anaphase or telophase stage may lead to the formation of micronucleus. micronuclei formation due to x-ray exposure in panoramic and cephalometric radiography is caused by the ionization process from x-ray which leads to dna damage and chromosome aberration. the interaction between ionizing radiation and biological substance may give rise to biological side effects immediately after exposure. an x-ray exposure may disrupt the mitotic process by inhibiting the formation of spindle fibres and further causing incomplete chromosome segregation.7 the micronucleus form in the metaphase-anaphase transition period due to either acentric chromosome fragments or whole chromosome fragments loss during cell division. x-ray exposure may induce the formation of acentric chromosome and chromosome misaggregation. acentric chromosome fragment and malsegregated whole chromosome fail to interact with spindle fibre, thus, resulting in chromosome instability in the daughter cell. the remaining chromosome fragments will form a micronucleus separated from the daughter cell.14 the micronucleus observation in the groups with a 24-hour interlude between panoramic and cephalometric radiograph, both in 24-hours (a3) and 48-hours (b3) observation, are showing significantly fewer micronucleus compared to the groups exposed to both radiations on the same day (a2 and b2). the fewer micronucleus is due to the ionizing radiation from x-ray which may cause a homeostasis process in the cell cycle. the cell cycle may stop, which inhibits cells from entering the g1, s, and g2 table 1. mean and standard deviation of micronuclei count on experimental groups 24 hours (a) and 48 hours (b) after radiation exposure groups micronuclei number p-value mean ± sd a1 3.10 ± 8.75 0.000* a2 20.30 ± 1.56 a3 11.30 ± 1.33 b1 3.40 ± 96 b2 15.90 ± 87 b3 6.40 ± 51 *significant (<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p128–131 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p128-131 131astuti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 128–131 phases, to allow the cell to repair and regenerate, preventing a cell mutation.15,16 a study by kalsbeek et al.17 proved that a delayed cell cycle may result in fewer micronucleus formations. based on the result of this study, there is a significant difference in the number of micronucleus in erythrocytes. this result showed that the mature erythrocytes are morphologically more resistant to radiation. the result is supported by previous research, which stated that microscopic damage of erythrocytes will be visible after radiation exposure, yet the mature erythrocytes are more resistant to the exposure.17 another aspect observed in this study is the time interval between the next radiation exposure. a 24-hour interlude can result in fewer micronucleus formations. the micronuclei formed in b3 were significantly lower than a3 groups. this can be caused by the regeneration process of the erythrocyte; an interlude may give chance to the erythrocyte to do a cell repair. this result is in accordance with the previous research which stated that cell damage due to radiation exposure may be reversible through a cell repair process depending on the cell type and the dose of radiation.10 from the results obtained in this study, it can be concluded that x-ray radiation can affect the formation of micronucleus in polychromatic erythrocyte cells in rats or increase the number of micronucleus in the polychromatic erythrocyte. references 1. sha h n, bansal n, logani a. recent advances in imaging technologies in dentistry. world j radiol. 2014; 6(10): 794–807. 2. heil a, lazo gonzalez e, hilgenfeld t, kickingereder p, bendszus m, heiland s, ozga a-k, sommer a, lux cj, zingler s. lateral cephalometric analysis for treatment planning in orthodontics based on mri compared with radiographs: a feasibility study in children and adolescents. plos one. 2017; 12(3): e0174524. 3. wrzesień m, olszewski j. absorbed doses for patients undergoing panoramic radiography, cephalometric radiography and cbct. int j occup med environ health. 2017; 30(5): 705–13. 4. bogdanova n v, jguburia n, ramachandran d, nischik n, stemwedel k, stamm g, werncke t, wacker f, dörk t, christiansen h. persistent dna double-strand breaks after repeated diagnostic ct scans in breast epithelial cells and lymphocytes. front oncol. 2021; 11: 634389. 5. kloosterman wp, hochstenbach r. deciphering the pathogenic consequences of chromosomal aberrations in human genetic disease. mol cytogenet. 2014; 7(1): 100. 6. pla madea la c, wojci k a, crea nga d. m icronuclei versus chromosomal aberrations induced by x-ray in radiosensitive m a m m a l i a n c e l l s . i r a n j p u b l i c h e a l t h . 2 015; 4 4 (3): 325–31. 7. luzhna l, kathiria p, kovalchuk o. micronuclei in genotoxicity assessment: from genetics to epigenetics and beyond. front genet. 2013; 4: 131. 8. jain ak, pandey ak. in vivo micronucleus assay in mouse bone marrow. methods mol biol. 2019; 2031: 135–46. 9. roos wp, thomas ad, kaina b. dna damage and the balance between survival and death in cancer biology. nat rev cancer. 2016; 16(1): 20–33. 10. reisz ja, bansal n, qian j, zhao w, furdui cm. effects of ionizing radiation on biological molecules--mechanisms of damage and emerging methods of detection. antioxid redox signal. 2014; 21(2): 260–92. 11. miller ma, zachary jf. mechanisms and morphology of cellular injury, adaptation, and death. in: pathologic basis of veterinary disease. elsevier; 2017. p. 2-43.e19. 12. white sc, pharoah mj. oral radiology: principles and interpretation. 7th ed. st. louis: mosby; 2013. p. 154. 13. preethi n, chikkanarasaiah n, bethur ss. genotoxic effects of x-rays in buccal mucosal cells in children subjected to dental radiographs. bdj open. 2016; 2: 16001. 14. kirkland d, kasper p, martus h-j, müller l, van benthem j, madia f, corvi r. updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. mutat res genet toxicol environ mutagen. 2016; 795: 7–30. 15. broustas cg, xu y, ha rken a d, ga r ty g, amundson sa. compa r ison of gene exp r e ssion r e sp on se to neut r on a nd x-ray irradiation using mouse blood. bmc genomics. 2017; 18(1): 2. 16. yamamori t, sasagawa t, ichii o, hiyoshi m, bo t, yasui h, kon y, inanami o. analysis of the mechanism of radiation-induced upregulation of mitochondrial abundance in mouse fibroblasts. j radiat res. 2017; 58(3): 292–301. 17. kalsbeek d, golsteyn rm. g2/m-phase checkpoint adaptation a nd m icronuclei for mat ion as mecha n isms t hat cont r ibute to genomic instability in human cells. int j mol sci. 2017; 18(11): 2344. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p128–131 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p128-131 92 volume 47, number 2, june 2014 koreksi dimensi vertikal oklusal dengan modifikasi restorasi mahkota logam pada kasus severe early childhood caries (correcting occlusal vertical dimension using modified stainless steel crown restoration in severe early childhood caries case) amrita widyagarini dan sarworini b budiardjo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas indonesia jakarta indonesia abstract background: severe early childhood caries (secc) describes progressively dental caries in primary dentition among children aged less than 3 to 5-year-old. loss of occlusal vertical dimension (ovd) with deep bite in clinical feature found 2.5 times more often in s-ecc children than others. it leads discrepancies of vertical development of permanent dentition, hence creates malocclusion. purpose: the aim of this paper was to report consideration and correction of ovd in secc child. case: a 5-year-old girl was accompanied by her mother came to pediatric dental clinic faculty of dentistry universitas indonesia. patient’s chief complaint was decay on all her teeth. clinical examination revealed caries on all primary dentition, tooth #16, #46 have not yet been occluded, 26 erupted partially and it occluded with 36, anterior deep bite. diagnose was anterior deep bite caused by secc. case management: operative-rehabilitative treatment restores ovd to prevent malocclusion by modifying height of ssc in primary molars. correcting deep bite using stainless steel crown (ssc) modified in posterior was done. first, restoration with glass-ionomer cement for raising the bite followed by a week evaluation to observe masticatory function and functional analysis of temporomandibular joint. second, ssc were placed in primary molars. conclusion: modifying height of ssc in primary molars could corrected ovd in secc child. permanent first molars eruption could be guided to completely occlusion and prevent early malocclusion. key words: occlusal vertical dimension, severe early childhood caries, early malocclusion, stainless steel crown, operative-rehabilitative treatment abstrak latar belakang: severe early childhood caries (secc) menunjukan pola karies gigi sulung yang progresif dan menyeluruh pada anak usia di bawah 3 hingga 5 tahun. kehilangan dimensi vertikal oklusal (dvo), dengan gambaran gigitan dalam pada periode gigi sulung dilaporkan terjadi 2,5 kali lebih banyak pada anak secc. hal ini dapat menyebabkan gangguan perkembangan vertikal gigi permanen yang nantinya menyebabkan maloklusi. tujuan: laporan kasus ini melaporkan mengenai pertimbangan dan koreksi dimensi vertikal pada anak secc. kasus: anak perempuan, 5 tahun, diantar ibunya ke klinik gigi anak fakultas kedokteran gigi universitas indonesia dengan keluhan semua giginya karies. klinis, seluruh gigi sulung karies, #16, #46 belum mencapai oklusi, #26 erupsi sebagian dan beroklusi dengan #36, gigitan dalam regio anterior. diagnosis adalah gigitan dalam regio anterior karena secc. tatalaksana kasus: perawatan operatif-rehabilitatif bertujuan mengembalikan dvo guna mencegah maloklusi, antara lain dengan memodifikasi restorasi mahkota logam gigi posterior. dilakukan koreksi gigitan dalam regio anterior dengan modifikasi restorasi mahkota logam gigi posterior. tahap pertama, restorasi glass-ionomer cement dengan meninggikan gigitan gigi posterior. adaptasi gigitan selama 1 minggu dan pengamatan fungsi pengunyahan serta analisa fungsional temporomandibular joint. kedua, restorasi mahkota logam gigi molar, dengan mempertahankan tinggi gigit tahap pertama, dilanjutkan restorasi mahkota untuk gigi anterior. case report 93widyagarini dan budiardjo: koreksi dimensi vertikal oklusal dengan modifikasi restorasi mahkota logam simpulan: modifikasi tinggi restorasi mahkota logam seluruh gigi molar sulung kasus secc, dapat mengembalikan dvo, sehingga erupsi empat gigi molar satu permanen dapat mencapai oklusi sempurna, dan mencegah maloklusi dini. kata kunci: dimensi vertikal oklusal, severe early childhood caries maloklusi dini, restorasi mahkota logam, perawatan operatifrehabilitatif korespondensi (correspondence): amrita widyagarini, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta pusat 10430, indonesia. e-mail: amrita.w@gmail.com pendahuluan severe early childhood caries (secc) memiliki gambaran klinis berupa karies meluas di gigi sulung dengan perkembangan lesi cepat yang terjadi pada anak usia di bawah 3 hingga 5 tahun.1 karies yang meluas pada sebagian besar jumlah gigi dapat mengakibatkan penurunan tinggi mahkota klinis, menyebabkan gambaran klinis deepbite. kedua hal ini merupakan faktor yang mengindikasikan hilangnya dimensi vertikal oklusal (dvo). kehilangan dvo pada periode gigi sulung dilaporkan terjadi 2,5 kali lebih banyak pada anak dengan secc.1,2 pengamatan perubahan oklusi dan dimensi vertikal secara klinis perlu diamati untuk mencegah gangguan perkembangan vertikal gigi permanen yang nantinya menyebabkan maloklusi.2,3 penilaian dan koreksi dimensi vertikal pada perawatan secc perlu dilakukan sebagai bagian dari perawatan operatif-rehabilitatif. pengembalian dimensi vertikal oklusal dalam perawatan rehabilitasi anak dengan secc dapat memberikan pola dan panduan erupsi gigi permanen sekaligus memberikan ruang yang cukup untuk penempatan bahan restorasi.4-6 koreksi dvo dapat dilakukan dengan meninggikan gigit yang menggunakan restorasi cekat atau lepasan.6,7 salah satu restorasi cekat pada anak untuk meninggikan gigit adalah dengan modifikasi restorasi metal crown dengan memodifikasi tinggi mahkota. laporan kasus ini melaporkan mengenai pertimbangan dan koreksi dimensi vertikal pada anak secc. kasus seorang anak perempuan usia 5 tahun 5 bulan (bb 29 kg; tb 112,5 cm) datang ke klinik ilmu kedokteran gigi anak fakultas kedokteran gigi universitas indonesia diantar oleh ibunya untuk memeriksakan gigi karena gigi banyak yang berlubang. ibu mengeluhkan semua gigi anaknya karies. anak mulai dibiasakan menyikat gigi sejak usia 1 tahun. kebiasaan anak sikat gigi kadang-kadang, waktu pagi sebelum sarapan dan mandi sore. saat ini anak menyikat gigi sendiri. anak memiliki kebiasaan minum susu botol saat tidur malam hari dan siang hari. pemeriksaan ekstra oral menunjukan wajah simetris, kelenjar getah bening submandibula kanan dan kiri teraba, keras, dan sakit. pemeriksaan tmj didapat data tidak ada bunyi sendi, palpasi sendi tidak sakit, deviasi ke kanan saat buka mulut. pemeriksaan intraoral menunjukan kebersihan mulut buruk, kalkulus pada gigi #64, #65, #26, #75 dan #36, gingivitis pada regio gigi #55, #54, #65, #26, #36, #75, #74, #84, #85, #46. hubungan vertikal molar satu permanen (m1) kanan suspek kelas i dengan kondisi klinis #16 dan #46 belum mencapai oklusi. gigi #26 erupsi sebagian dan beroklusi dengan #36 sehingga hubungan vertikal molar kiri ditentukan oleh hubungan vertikal molar dua sulung (dm2) kiri, yaitu mesial step. gigi #16, #46 belum mencapai oklusi, #26 erupsi sebagian dan beroklusi dengan #36. gigitan dalam regio anterior, susunan gigi tidak berjejal, gigi jarang di regio anterior atas dan bawah. pemeriksaan gambar 1. foto intra oral pada kunjungan pertama (setelah dilakukan aplikasi disclosing solution). a) tampak depan; b) tampak kanan; c) tampak kiri; d) rahang bawah; e) rahang atas. 9 gambar 1. foto intra oral pada kunjungan pertama, 14 juni 2012 (setelah dilakukan aplikasi disclosing solution). gambar 2. foto panoramik sebelum perawatan. gambar 3. pengukuran proporsional keseimbangan wajah. a b c d e 94 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 92–97 status geligi terdapat karies mencapai pulpa (kmp) gigi #75, #61 dan #62; karies dentin (kd) #55, #54, #53, #52, #63, #64, #65, #74, #73, #72, #71, #81, #82, #83, #84, #85; #51 dan #52 pasca psa; #16, #36, #46 pit fisur dalam (gambar 1) . interpretasi radiografik gigi #75 menunjukan gambaran radiopak mencapai kamar pulpa, resorpsi akar gigi sulung mencapai 1/3 apikal, radiolusensi di interadikular, pembentukan benih gigi tetap mencapai 1/3 servikal, benih gigi tetap belum menembus tulang. interpretasi radiografik foto panoramik menunjukan #51, #52 terdapat gambaran radiopak sepanjang saluran akar, resorpsi akar gigi sulung 1/3 apikal, pembentukan benih gigi tetap mencapai 1/3 servikal, benih gigi tetap belum menembus tulang; #61, #62 radiolusensi di mahkota mencapai kamar pulpa, resorpsi akar gigi sulung 1/3 apikal, pembentukan benih gigi tetap mencapai 1/3 servikal, benih gigi tetap belum menembus tulang; mandibula kanan dan kiri simetris, kondil kanan dan kiri simetris, kondil kanan dan kiri terletak di dalam fossa glenoid (gambar 2). tatalaksana kasus tujuan utama rencana perawatan adalah meninggikan dimensi vertikal dengan memodifikasi restorasi mahkota logam melalui peningkatan tinggi mahkota klinis molar. rencana perawatan menyeluruh pada pasien ini terbagi dua bagian, yaitu tahap awal dan lanjut. tahap awal adalah tindakan promotif dan preventif, sedangkan tahap lanjut meliputi tindakan operatif-rehabilltatif. gigi yang mengalami kmp direncanakan perawatan endodontik sedangkan gigi dengan kd direncanakan restorasi sesuai indikasi. koreksi dimensi vertikal didahului dengan menentukan pengukuran dvo yang akan dikoreksi. pengukuran dilakukan dengan menggunakan proporsi keseimbangan 1/3 wajah dan metode pengucapan huruf ‘m’. penghitungan keseimbangan wajah 1/3 wajah atas (trichion ke glabella) = 53 mm, 1/3 wajah tengah (glabella ke subnasal) = 52 mm, 1/3 wajah bawah (subnasal ke menton) = 50 mm. pengukuran dimensi vertikal istirahat dengan metode pengucapan huruf ‘m’ dari titik di bawah hidung ke dagu = 54 mm. tinggi dimensi vertikal saat gigi beroklusi = 50 mm. ditetapkan peninggian gigit sebanyak 2 mm. dilakukan pengecekan dimensi vertikal oklusal baru melalui ukuran jarak glabella ke subnasion berukuran sama dengan jarak subnasion ke dagu (gambar 3). gambar 2. foto panoramik sebelum perawatan. 9 gambar 1. foto intra oral pada kunjungan pertama, 14 juni 2012 (setelah dilakukan aplikasi disclosing solution). gambar 2. foto panoramik sebelum perawatan. gambar 3. pengukuran proporsional keseimbangan wajah. gambar 3. pengukuran proporsional keseimbangan wajah. a) profile tampak depan; b) pengukuran 1/3 wajah atas (trichion ke glabella); c) pengukuran 1/3 wajah tengah (glabella ke sub nasal); d) pengukuran 1/3 wajah bawah (sub nasal ke meuton). gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. a) tampak depan; b) tampak kanan; c) tampak kiri. 9 gambar 1. foto intra oral pada kunjungan pertama, 14 juni 2012 (setelah dilakukan aplikasi disclosing solution). gambar 2. foto panoramik sebelum perawatan. gambar 3. pengukuran proporsional keseimbangan wajah. a b c d 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c b ca 95widyagarini dan budiardjo: koreksi dimensi vertikal oklusal dengan modifikasi restorasi mahkota logam gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. a) sisi kanan; b) sisi kiri. tahapan pertama adalah dilakukan peninggian gigit posterior dengan restorasi glass ionomer cement (gic) pada permukaan oklusal gigi posterior. kontrol 1 minggu setelahnya menunjukan tidak ada keluhan dan pasien dapat menggunakan kedua rahang untuk mengunyah. tahap kedua adalah dilakukan restorasi mahkota logam pada seluruh gigi molar sesuai dengan tinggi mahkota setelah peninggian gigit (gambar 4 dan 5). kontrol 2 bulan setelah koreksi dvo menunjukan tidak ada keluhan subjektif, pasien dapat mengunyah dengan kedua sisi, pemeriksaan tmj tidak ada kelainan, buka mulut normal, erupsi 26 dan 36 yang tadinya mengalami hambatan sudah terkoreksi (gambar 6). kontrol 9 bulan setelah koreksi menunjukan hubungan m1 kanan dan kiri kelas i angle (gambar 7). 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j a b 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c a b c 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c 10 gambar 4. keadaan intra oral sebelum dilakukan restorasi sementara gic untuk peninggian dvo. gambar 5. restorasi transisi dengan gic untuk peniggian gigit (a-e). a b d e c d e 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j i j 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j f 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j g h 96 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 92–97 gambar 7. oklusi pada saat kontrol 9 bulan setelah perawatan. 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j pembahasan pemeriksaan klinis pasien anak usia 5 tahun 5 bulan menunjukan kebersihan mulut buruk akibat kebiasaan sikat gigi yang belum teratur. seluruh gigi sulung mengalami karies dengan gambaran karies di daerah servikal gigi pada gigi anterior serta di daerah oklusal dan proksimal pada gigi molar. kedalaman karies sebagian besar adalah karies dentin. pasien disimpulkan mengalami secc dengan tingkat keparahan sedang. sesuai dengan literatur bahwa secc dijumpai pada anak usia di bawah 3 hingga 5 tahun dengan gambaran klinis karies meluas di seluruh gigi sulung hingga meliputi gigi anterior bawah apabila sudah mencapai tahap lanjut.1 perawatan secc merupakan perawatan menyeluruh yang terbagi dalam perawatan non operatif dan perawatan operatif-rehabilitatif. perawatan non operatif yang dapat dilakukan adalah tindakan promotif dan preventif berupa edukasi dan motivasi orangtua, pengasuh, dan pasien untuk membersihkan gigi dan mulut, kontrol diet, pemeriksaan faktor risiko karies, dan aplikasi fluoride. perawatan operatif-rehabiliatif dilakukan untuk merestorasi gigi yang mengalami kerusakan akibat karies dengan tujuan mengembalikan fungsi gigi, estetik, sekaligus mengkoreksi penurunan dimensi vertikal yang terjadi.4,5 pasien pada awal kunjungan secara klinis terlihat mengalami penurunan dimensi vertikal dengan gambaran klinis deepbite di regio anterior yang diakibatkan oleh kehilangan mahkota gigi insisif rahang atas dan kehilangan tonjol gigi molar sulung bawah. sesuai dengan literatur bahwa sebagian besar anak dengan secc menunjukan penurunan dimensi vertikal.2 adanya penurunan dimensi vertikal perlu diperhatikan dalam merencanakan tahap restorasi. jika ditinjau dari segi restorasi, peningkatan dimensi vertikal perlu dilakukan sebelum restorasi definitif agar tersedia ruang bagi penempatan bahan restorasi.6 jika ditinjau dari segi tumbuh kembang, perawatan rehabilitatif dengan memperbaiki dimensi vertikal pada anak-anak dapat membentuk dan memandu oklusi gigi permanen.4,5 dimensi vertikal secara umum dapat didefinisikan sebagai sepertiga panjang wajah bagian bawah.8 dimensi vertikal oklusal pada pasien ini ditentukan melalui pengukuran keseimbangan 1/3 wajah. pengecekan dimensi vertikal oklusal dilakukan dengan mengukur jarak dari glabela ke subnasion berukuran sama dengan subnasion ke dagu. pengukuran wajah merupakan metode sederhana yang tidak memerlukan alat khusus.9 hal ini dapat diaplikasikan pada anak usia 5 tahun. metode pengucapan huruf ‘m’ untuk mengukur dimensi vertikal istirahat juga dilakukan untuk membantu penentuan ukuran peninggian gigit. terdapat berbagai pilihan teknik untuk menentukan kehilangan dimensi vertikal oklusal. semua teknik dapat digunakan akan tetapi tidak ada teknik yang terbukti secara ilimiah lebih akurat dibandingkan teknik lainnya. hal ini menyebabkan teknik yang digunakan sebaiknya lebih dari satu untuk meningkatkan keakuratan prosedur pencatatan.6 koreksi peninggian dimensi vertikal ditetapkan 2 mm. koreksi ini sesuai dengan ukuran freeway space. menurut literatur, peninggian dimensi vertikal oklusal dapat dilakukan dalam ukuran freeway space sehingga dimensi vertikal oklusal baru tidak melebihi ukuran dimensi vertikal istirahat dan dimensi vertikal baru dapat terbentuk tanpa gejala.10 terdapat berbagai pertimbangan sebelum melakukan koreksi dimensi vertikal. evaluasi adanya gejala dan tanda tmd dilakukan sebelum perawatan.6 pasien ini tidak menunjukan adanya keluhan nyeri sendi atau otot mastikasi walaupun dia memiliki kebiasaan mengunyah satu sisi, terdapat deepbite, dan deviasi saat buka mulut. pemeriksaan foto panoramik menunjukan kesimetrisan mandibula kanan dan kiri dan kondil kanan dan kiri. koreksi dimensi vertikal dengan meninggikan dimensi vertikal oklusal yang dilakukan pada anak yang sehat tidak akan meningkatkan risiko gangguan tmj. 11 keadaan intraoral klinis juga menjadi faktor pertimbangan. pasien ini menunjukan gambaran klinis periode gigi bercampur tahap awal, keempat molar pertama tetap sudah erupsi walaupun belum mencapai oklusi maksimal. gigi #26 erupsi parsial dan berkontak dengan oklusal lawan saat pasien oklusi. penurunan dimensi vertikal oklusal yang ada membuat gigi #26 dikhawatirkan tidak dapat erupsi maksimal mencapai oklusi yang tepat. sesuai literatur, penyebab adanya dental deepbite adalah infraoklusi gigi molar sehingga kondisi ini harus diperbaiki agar tidak terjadi deepbite pada oklusi gigi permanen.12 perbaikan kehilangan dimensi vertikal yang dilakukan pada anak dapat menjadi pola atau panduan untuk gigi permanen erupsi. 5 11 gambar 5. pasca koreksi dimensi vertikal dengan modifikasi tinggi restorasi mc pada gigi molar sulung serta restorasi mc facing compomer pada gigi 53 dan 63 (f–j). gambar 6. kondisi rongga mulut setelah 2 bulan koreksi dimensi vertikal oklusal. gambar 7. oklusi pada saat kontrol tanggal 11 april 2013. f g h i j 97widyagarini dan budiardjo: koreksi dimensi vertikal oklusal dengan modifikasi restorasi mahkota logam tujuan utama rencana perawatan dalam rehabilitasi total adalah meninggikan dimensi vertikal melalui peningkatan tinggi molar.5 perawatan ini dapat dilakukan melalui modifikasi restorasi metal crown pada gigi molar sulung. peningkatan tinggi molar dengan restorasi memiliki efek seperti peninggian gigit pada alat ortodontik lepasan. alat peninggi gigit mengubah secara sementara perkembangan dentoalveolar dengan didukung oleh peningkatan sementara di wajah bagian bawah dan diikuti oleh erupsi cepat gigi lain untuk membangun kontak oklusal.11 tahapan pertama melakukan koreksi dimensi vertikal dengan restorasi modifikasi metal crown pada pasien ini adalah dengan membuat peninggian gigit dari gic sebagai restorasi transisi pada permukaan oklusal gigi posterior sulung. pemilihan gic sebagai restorasi sementara adalah bahan ini mudah diaplikasikan, memiliki seal yang baik sehingga dapat melapisi basis pasca perawatan endodontik, berikatan dengan email dan dentin, dan mengeluarkan fluoride.4 tujuan dari restorasi sementara ini adalah agar anak dapat beradaptasi terhadap koreksi dimensi vertikal. sesuai dengan laporan terdahulu bahwa peninggian dvo dilakukan bertahap agar otot-otot mastikasi dapat beradaptasi terhadap dvo baru.13 satu minggu kemudian saat pasien datang kontrol, pasien tidak ada keluhan terhadap peninggian gigit, kedua rahang dapat dipakai mengunyah. keluhan yang biasa terjadi adalah adanya nyeri pada tmj dan rasa pegal pada otot mastikasi.7 tahapan kedua koreksi dimensi vertikal adalah dengan restorasi definitif menggunakan mahkota logam pada gigi molar sulung yang telah dilakukan peninggian gigit dengan gic. mahkota logam dapat mengembalikan bentuk anatomis gigi posterior sehingga mengembalikan bentuk tonjol gigi molar, mampu memperbaiki oklusi, dan membantu fungsi pengunyahan, memiliki retensi yang baik, tahan lama, dan dapat melindungi gigi sulung. restorasi ini juga memiliki permukaan yang halus sehingga dapat berkontribusi mengurangi akumulasi biofilm bakteri sehingga meningkatkan kebersihan mulut pada pasien dengan risiko karies tinggi. 14 gigi posterior dilakukan restorasi lebih dulu daripada gigi anterior karena kunci oklusi berada pada gigi posterior. kestabilan oklusi dari gigi posterior harus didapatkan sebelum merestorasi gigi anterior.5 gigi molar permanen tidak dilakukan restorasi koreksi dimensi vertikal karena gigi molar permanen diharapkan akan erupsi mencapai oklusi sesuai ruang yang telah dibentuk dari peninggian dimensi vertikal oklusal gigi sulung. setelah koreksi dvo, pasien tidak mengalami kesulitan mengunyah dengan kedua sisi, deviasi buka mulut hilang, tidak ada keluhan subjektif tmj. kontrol pasca peninggian dimensi vertikal dengan modifikasi restorasi metal crown menunjukan bahwa gigi molar permanen dapat melanjutkan erupsi untuk mencapai oklusi. laporan kasus ini menunjukkan bahwa modifikasi tinggi restorasi mahkota logam seluruh gigi molar sulung kasus secc, dapat mengembalikan dvo, sehingga erupsi empat gigi molar satu permanen dapat mencapai oklusi sempurna, dan mencegah maloklusi dini. daftar pustaka 1. losso em, tavares mc, da silva jy uca. severe early childhood caries: an integral approach. j pediatr (rio j) 2009; 85(4): 295300. 2. robke fj. effects of nursing bottle misuse on oral health. prevalence of caries, tooth malalignments and malocclusions in north-german preschool children. j orofac orthop 2008; 69(1): 5-19. 3. kim js, akimoto s, shinji s, sato. importance of vertical dimension and cant of occlusal plane in craniofacial development. j stomat occ med 2009; 2: 114-21. 4. koch g, poulsen s. pediatric dentistry a clinical approach. 2nd ed. west sussex: blackwell publishing ltd; 2009. p. 116-26. 5. gopal y, mallabadi r. full mouth rehabilitation. j clin and diagn res 2007; 1: 143-6. 6. abduo j, lyons k. clinical considerations for increasing occlusal vertical dimension: a review. aust den j 2012; 57(1): 2-10. 7. abduo j. safety of increasing vertical dimension of occlusion: a systematic review. quintessence int 2012; 43(5): 369-80. 8. nelson sj, ash jr mm. wheeler’s dental anatomy, physiology, and occlusion. 9th ed. st louis: saunders elsevier; 2010. p. 302-3. 9. misch ce. clinical indications for altering vertical dimension of occlusion. objective vs subjective methods for determining vertical dimension of occlusion. quinterssence int 2000; 31(4): 280-2. 10. chander ng, venkat r. an appraisal on increasing the occlusal vertical dimension in full occlusal rehabilitation and its outcome. j indian prosthodont soc 2011; 11(2): 77–81. 11. innes np. the hall technique; a randomized controlled clincal trial of a novel method of managing carious primary molars in general dental practice acceptability of the technique and outcomes at 23 months. bmc oral health 2007; 7-18. 12. sreedhar c, baratam s. deep overbite a review. anual essen dent 2009; 1(1): 8-25. 13. djulaeha e, sukaedi. the management of over closured anterior teeth due to attrition. dent j (maj. ked. gigi) 2009; 42(4): 194-8. 14. parisotto tm, souza-e-silva cm, steiner-oliveira c, nobre-dossantos m, gaviao mbd. prosthetic rehabilitation in a four-year-old child with severe early childhood caries: a case report. j contemp dent pract 2009; 10(2): 90-7. �� the role of transforming growth factor beta in tertiary dentinogenesis tetiana haniastuti1, phides nunez2, and ariadna a. djais3 1department of oral biology, gadjah mada university, indonesia 2division of oral ecology in health and infection, niigata university, japan 3department of oral biology, university of indonesia, indonesia abstract the most visible repair response to pulp injury is the deposition of a tertiary dentin matrix over the dentinal tubules of the primary or secondary dentin. tertiary dentin is distinguished as reactionary and reparative dentin, depending on the severity of the initiating response and the conditions under which the newly deposited dentin matrix was elaborated. transforming growth factor beta (tgf-b) superfamily is a large group of growth factors that serve important roles in regulating cell growth, differentiation, and function. members of this superfamily have been implicated in the repair process of the dental tissue after injury. although numerous studies have proved that those bioactive molecules carry out an important role in the formation of tertiary dentin, comprehensive report regarding that phenomenon is not yet available. this review article aimed to summarize the role of tgf-b on tertiary dentinogenesis during the progression of a carious lesion. key words: transforming growth factor beta, tertiary dentinogenesis, reactionary dentin, reparative dentin correspondence: tetiana haniastuti, c/o: bagian biologi oral, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. 1, sekip utara yogyakarta 55281, indonesia. e-mail: haniastuti@yahoo.com introduction the complex structural composition of teeth provides hardness and durability as a barrier against bacterial infection. when a carious lesion breaks down this barrier, repair takes place to prevent further caries progression; hence, preventing invasion of the pulp chamber by the bacteria. the capacity of pulp cells to resist and repair injuries is fundamental to maintain the integrity and homeostasis of the dental organ. in an adult pulp, cell division and the secretory activity of odontoblasts are limited, but these processes may be re-activated after injury. the most common and well-known feature of pulp repair is the formation of tertiary dentin,1 which can be classified as being reactionary or reparative in origin, depending on the severity of the initiating response and the conditions under which the newly deposited dentin matrix was formed.2 a number of studies have reported the presence of tertiary dentin in 63.6% of teeth with carious lesions and found that it often occurs in combination with dentinal sclerosis.3 the formation of the tertiary dentin on the pulpal aspect of stimulated dentinal tubules occurs in human deciduous teeth4 as well as in permanent teeth.5 it starts during the active stage of the carious process and continues after lesion arrest.6 the rate of carious attack seems to be an influencing factor since more dentin is formed in response to a slowly progressing chronic caries than to a rapidly advancing acute caries.3 transforming growth factor beta (tgf-β) is a multifunctional regulator of a variety of cellular functions, such as cell proliferation, differentiation, and matrix synthesis. recent studies showed that this substance has a significant role in the immune response7 and tissue repair8 of the dental pulp. tgf-β has also been implicated in induction of odontoblast-like cell differentiation and in signaling primary odontoblasts during dentin repair. several in vivo studies have demonstrated the effects of dentin matrix preparation containing this bioactive molecule on both reactionary and reparative dentinogenesis.9–11 recently, numerous studies regarding tertiary dentinogenesis and various aspects related to it have already been reported. however, there is not any comprehensive report discussing this phenomenon. this review article will briefly summarize the role of tgf-β on tertiary dentinogenesis during the progression of a carious lesion. tgf-β the tgf-β super family comprises over 40 different proteins, most of which can be classified into three broad groupings of tgf-βs (isoforms tgf-β1, tgf-β2, tgf-β3, tgf-β5), bmp-bone morphogenetic proteins (bmp-2, bmp-3, bmp-4, bmp-5, bmp-6, bmp7 and bmp-8), and the activins/inhibins. each of them is capable of regulating a fascinating array of cellular processes including cell proliferation, lineage determination, differentiation, motility, adhesion, and death. expressed in complex temporal and tissue-specific patterns, tgf-β and related factors play prominent roles in the development, homeostasis, and repair of virtually all tissues in organisms.12 in addition, these molecules also have mitogenic effects and �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 15-20 a regulatory role in matrix biosynthesis. collectively, these factors account for a substantial portion of the intercellular signals governing cell fate.13 tgf-β has been implicated as a key mediator in odontoblast differentiation and dentin mineralization. members of this super family have been implicated both in aspects of tooth development and repair of the dental tissue after injury.9 a previous study by hu et al.14 showed that tgf-β as a pulp-capping medicament enhances reparative dentin formation in rat molars. the presence of tgf-β in the dentin matrix has been reported15 and measured by elisa.16 tgf-β also has been immunolocalized in the dentin matrix at the ultra structural level.17 in humans, odontoblasts and other cells of the pulp show the presence of both tgf-β receptors i and ii with odontoblasts showing the strongest expression.18 mclachlan et al.19 found that genes of the tgf-β family members and their receptors are predominantly expressed by odontoblasts in healthy teeth and generally show higher levels of expression in odontoblasts and pulpal tissue from carious teeth. sloan et al.20 reported that tgf-β is normally expressed in both healthy and carious molar teeth in humans. they have identified all three tgf-β isoforms in mature human odontoblasts. in their study, odontoblasts cells, pulpal fibroblasts, and endothelial cells were stained to varying degrees for tgf-β1, 2 and 3, with tgf-β3 showing the greatest intensity and tgf-β1 the weakest intensity; however, tgf-β1 showed significantly increased staining intensity within the odontoblast and pulpal cells of the carious teeth. in addition, a study by piatelli et al.21 showed that in the odontoblastic and subodontoblastic layer of specimens with irreversible pulpitis, there was a higher expression of tgf-β1 than in normal healthy specimens. tertiary dentinogenesis the response of tertiary dentinogenesis is one of repair or wound-healing as observed after injury to many other tissues of the body. when a carious lesion has invaded dentin, the pulp usually responds by depositing a layer of tertiary dentin over the dentinal tubules of the primary or secondary dentin that communicate with the carious lesion. secretion of tertiary dentin matrix is restricted to those odontoblasts directly affected by the injury process.19 tertiary dentin differs morphologically from primary dentin reflecting their variations in molecular mechanism of formation.22 according to olgart and bergenholtz,23 compared with primary dentin, tertiary dentin is less sensitive to thermal, osmotic, and evaporative stimuli. the tubules of tertiary dentin tend to be more irregular with larger lumina. in some cases no tubules are formed. the degree of irregularity of tertiary dentin is determined by factors such as the amount of inflammation present, the extent of cellular injury, and the state of differentiation of the replacement odontoblasts. in addition, tertiary dentin is less permeable to externally derived matter than primary dentin. along the border zone between primary and tertiary dentin, the walls of dentinal tubules are thickened and the tubules are frequently occluded with material resembling peritubular dentin. the border zone thus appears to be considerably less permeable than ordinary dentin and may serve as a barrier to the ingress of bacteria and their products.23 a study by kim et al.3 revealed that the accumulation of pulpal dendritic cells is reduced after tertiary dentin formation, which may indicate the reduction of incoming bacterial antigens. tertiary dentin has been defined as the dentin deposited on the pulpal aspects of primary or secondary dentin at sites corresponding to areas of external irritations.4 a number of terms have been used to describe this dentin such as irregular secondary dentin, reactionary dentin, reparative dentin, and irritation dentin, all reflecting its varied etiology. smith et al.2 has re-defined it in relation to the nature of the injury to attempt a better discrimination of the responses occurring in the dentin-pulp complex. this has led to adoption of the terms reactionary and reparative dentin to subdivide tertiary dentinogenesis into the responses seen after either survival or death of the primary odontoblast population, respectively. reactionary dentinogenesis the term reactionary dentinogenesis has been adopted to describe the secretion of a tertiary dentin matrix by primary odontoblasts which have survived injury to the tooth. typically, this type of dentin will be seen with injury of mild intensity, such as in precavitational stages of active enamel caries and in slowly progressing dentinal lesions.2 slowly progressing carious lesions are characterized by an early increased mineralization of the affected dentin. this hyper mineralization occurs when the caries process is located in the enamel, before it reaches the dentin. by the time the carious lesion reaches the dentin, some of the dissolved mineral salts will reprecipitate within the tubules and form a hyper mineralized transparent zone in the dentin subjacent to the demineralized carious dentin.24 relatively small changes are observed histological in the odontoblast-predentin region associated with slowly progressing caries, but an increased formation of reactionary dentin is apparent. most of the original odontoblasts survive although they may be somewhat shortened. the reduction of the height of the odontoblasts that form reactionary dentin is not compatible with the increased matrix production. increased matrix production should result in an increase of intracellular organelles and therefore larger formative cells. it is likely that the subodontoblastic cells and a new generation of odontoblastlike cells contribute to the matrix production under this condition. if the original odontoblast continues to form dentin, the tubules extend from the primary dentin into secondary and tertiary dentin; thus, these pathways to the pulp are kept open. the subodontoblastic region remains virtually unaltered morphologically, but the cell-free zone is often absent, possibly due to the altered physiology of the area. other components normally present are found, ��haniastuti, et al.: the role of transforming growth factor beta in tertiary dentinogenesis including fibroblasts, undifferentiated cells, and dendritic cells.24 tertiary dentin formed in the case of a superficial carious lesion may resemble primary dentin in terms of tubularity and degree of mineralization. in general, the tubules of reactionary dentin continue with those of secondary dentin, while the thickness of the newly formed layer is related to the intensity and duration of the stimulus. the reactionary dentin possesses an organic matrix as well as a mineral content similar to those found in primary and secondary dentin.25 reparative dentinogenesis reparative dentinogenesis is a term used to describe the secretion of tertiary dentin after the death of the primary odontoblasts underlying the injury. reparative dentin would arise after an injury of greater intensity and represents a much more complex sequence of biological events, involving progenitor cell recruitment and differentiation as well as an up-regulation of cell secretion. in relation to caries, the formation of reparative dentin can be observed in deep dentinal lesions where clinically, a change in lesion activity has occurred.26 in rapid progression of carious lesions, the structure of the tertiary dentin that normally forms in the pulp-predentin region varies, depending on the severity of the attack. sometimes the reparative tissue has cellular inclusions or atubular like fibro dentin. this is the dentin that is referred to as interface dentin. provided that this reaction prevents the ingress of excessive reactive agents from the carious lesion, the interface dentin has a barrier effect that may be an essential part of the defense mechanisms in the tooth. interface dentin with irregular, often atubular dentin forms a barrier between the physiologic secondary dentin and the tertiary dentin. this barrier, which corresponds to the hyaline zone of the dead tract, reduces the permeability of the affected dentin and may make it impermeable because the tubules from primary dentin do not cross the interface dentin. the reparative dentin usually continues to form as tubular dentin after the interface dentin has formed although its structure is more irregular, less mineralized, softer, and contains more organic material than primary dentin.24 reparative dentin is in the majority of cases quite different morphologically from reactionary dentin. it may contain cellular inclusions, which resemble the osteocytes from bone. in addition, its extra cellular matrix contains some noncollagenous proteins that are more typical for bone than for dentin. for these reasons, sometimes the reparative dentin shows an osteodentin appearance.25 in rapidly progressing caries, the odontoblasts are often destroyed. odontoblasts are post mitotic terminally differentiated cells that cannot proliferate to replace subjacent irreversibly injured odontoblasts. consequently, these primary cells must be replaced by a new generation of odontoblast-like cells.27 therefore, the formation of reparative dentin is a complex sequence of biological process, which is dependent on multiple factors, including the presence of responsive progenitor cells as well as the appropriate inductive molecular signals for induction of proliferation, migration, and differentiation of the new generation of odontoblast-like cells. the derivation of the progenitor cells for these odontoblast-like cells remains unclear although they may be variable. the undifferentiated mesenchymal cells in the cell rich zone adjacent to the odontoblast layer are attractive candidates since they will have experienced a developmental history similar to the primary odontoblasts. other pulpal cells, including perivascular cells, undifferentiated mesenchymal cells, fibroblasts etc., have also been implicated as progenitors for the odontoblast-like cell.28 discussion odontoblasts are the cells responsible for the formation of dentin as well as provide an innate immune barrier for the tooth.29 the main task of odontoblasts is to synthesize and secrete collagens and several non-collagenous proteins from which the dentin organic matrix is formed. odontoblasts control dentin matrix mineralization by determining the nature of the extra cellular matrix and by controlling the influx of mineral ions.25 odontoblasts secrete dentin matrix components which contain a reservoir of biologically active molecules, such as platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, metalloproteinase and also tgf-β super family members. during dentinogenesis, the extra cellular matrix of the dentin is mineralized by deposition of hydroxyapatite onto the fibrous matrix. after mineralization the dentin molecules remain trapped in the mineralized phase bound to matrix components or to hydroxyapatite crystals.30 diffusion of acids from bacterial metabolism during caries could solubilize those bioactive molecules from the soluble tissue compartment of dentin matrix and unmask those bound in the insoluble tissue compartment. subsequent bacterial proteolytic action during more advanced stages of caries might also contribute to, and possibly modulate, such process. further, those molecules initiate the healing process, providing chemotactic cues to recruit inflammatory cells and undifferentiated pulpal cells to the injury site, stimulating the angiogenic response, and initiating the subsequent tissue movement for repair.31,32 release of tgf-β from the dentin matrix requires its diffusion, presumably mainly through the dentinal tubules, to the pulpal cells for signaling events to occur. several factors may influence the ability of this agent to participate in such events, including their release in a biologically active form, their interaction with dentin matrix components, either in solubilized form or by immobilization on the insoluble matrix, and the diffusion distance along the dentinal tubules. much of the tgf-β in dentin matrix �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 15-20 appears to be in an active form and to be associated with dentin matrix components, which may regulate its availability and biological activity.33 a study by lucchini et al.34 showed that differentiated odontoblasts secrete and deposit tgf-β into the dentin matrix and can respond to it, thus enabling possible autocrine modes of action. the detection of tgf-β receptors i and ii on odontoblasts and other pulpal cells demonstrates that an active signaling complex for signal transduction exists on these cells. the target cells for signaling molecules released during carious demineralization will vary, but in terms of repair, the odontoblast will be of prime importance.18 tgf-β initiates its actions trough smad signaling pathway by binding to two different classes of serine/ threonine kinase receptors, termed type i and type ii, which have been demonstrated in odontoblasts and pulp cells.35 ligand-induced phosphorylation of the type i receptor by the type ii receptor results in activation of the type i receptor kinases, which in turn phosphorylate a receptor-regulated smads 2/3. the phosphorylated smads 2/3 then associate with smad4, a common-mediator smad. these heteromeric complexes are translocated to the nucleus, where they regulate gene transcription by either association with dnabinding protein or direct binding to promoter sequences of target genes.36 smad6 and smad7 belong to the inhibitory smads and act as inhibitors of the tgf-β family signaling pathway, probably by competitive interaction with the type i receptor or with smad4.37 during reactionary dentinogenesis (figure 1), solubilized tgf-βs may be able to diffuse to the odontoblasts and pulpal cells localized beneath the damaged region and signal the up-regulation of these cells’ secretory activity.27 this upregulation of surviving odontoblasts will lead to focal secretion of new matrices at the pulp-dentin interface.2 quite a few studies have demonstrated that tgf-β stimulates the synthesis of extracellular matrices and also initiates odontoblast cytodifferentiation in vitro38 and in vivo.39 an in vivo study by denbesten et al.40 using transgenic mice overexpressing tgf-β2 showed an increase in their dentin mineral apposition relative to their wild-type littermates. tgf-β2 has been shown to be present in mature dentin; therefore, it seems that tgf-β2 also stimulates odontoblast differentiation in maturing dentin to increase the dentin mineral apposition rate.15 an in vitro study by sloan and smith41 revealed that tgf-β1 and tgf-β3 increase the predentin thickness of the cultured tooth in the region immediately adjacent to agarose beads containing those growth factors. the localized increase in the predentin thickness reflects reactionary dentinogenic effect. tgf-β also carries out a significant role in modulating the soft and hard tissue repair following a carious lesion, enhancing reparative dentin formation. the formation of reparative dentin (figure 2) results from the recruitment and proliferation of the pulp cells which have stem cell properties. a previous study by pasavant et al.42 revealed that tgf-β induces the synthesis of secreted protein acidic, rich in protein, which in turn induces dental pulp cells migration toward the odontoblastic layer.43 the dental pulp stem cells are attracted to the injury site and differentiate into a second generation of odontoblasts or odontoblast-like cells to replace irreversibly injured odontoblasts.44,45 tgfβ1 may play a role in the differentiation of the pulp cells into preodontoblasts and odontoblasts and in the reparative dentinogenesis process after tissue injury.46 a previous in vivo study showed that tgf-β induces the differentiation of pre-odontoblasts and formation of functional odontoblastlike cells.47 several studies reported that tgf-β1 and tgfβ3 stimulate proliferation of the cells of the subodontoblast figure 1. schematic representation of reactionary dentin formation induced by tgf-β. (a) dentin injured by a small carious lesion. (b) release of tgf-β from dentin matrix. (c) deposition of reactionary dentin by primary odontoblasts. figure 2. schematic representation of reparative dentin formation induced by tgf-β. (a) pulp exposed by carious lesion. (b) release of tgf-β from dentin matrix . (c) proliferation, migration and differentiation of odontoblast-like precursor cells into odontoblastlike cells. (d) deposition of reparative dentin by newly differentiated odontoblast-like cells. ��haniastuti, et al.: the role of transforming growth factor beta in tertiary dentinogenesis layer and formation of odontoblast-like cells.13,41,48 in addition, tgf-β1 induces type i collagen production by the odontoblastic/subodontoblastic pulp cells.49 huojia et al.50 reported that tgf-β3 induces ectopic mineralization through upregulation of osteocalcin and type i collagen expression in the dental pulp cells and may regulate the differentiation of the dental pulp stem cells to odontoblasts. such activities might be important during reparative processes in the dentin-pulp complex after tissue injury. in conclusion, the dentin-pulp complex has ability to respond to a carious lesion by localized deposition of a tertiary dentin matrix which can be classified as being reactionary or reparative in origin, depending on the severity of the initiating response and the conditions under which the newly deposited dentin matrix was generated. tgf-β super family which is captured in the dentin matrix, may be solubilized or exposed during carious demineralization and provides the molecular signaling to initiate tertiary dentinogenesis. in reactionary dentinogenesis, tgf-βs stimulate the upregulation of odontoblast cells’ synthetic and secretory activities to secrete a reactionary dentin matrix. tgf-βs also have the ability to induce the proliferation, migration, and differentiation of odontoblastlike cells from the pulp cells leading to the secretion of reparative dentin. references 1. mitsiadis ta, rahiotis c. parallels between tooth development and repair: conserved molecular mechanisms following carious and dental injury. j dent res 2004; 83:896–902. 2. smith aj, cassidy n, perry h, begue-kirn c, ruch j-v, lesot h. reactionary dentinogenesis. int j dev biol 1995; 39:273–80. 3. kim s, trowbridge h, suda h. pulpal reaction to caries. in: cohen s, burns rc, editors. pathway of the pulp. 8th ed. st louis: mosby, 2002. p. 574. 4. klinge rf. a microradiographic and electron microscopic study of tertiary dentin in human deciduous teeth. acta odontol scand 1999; 57:87–92. 5. stanley hr, pereira jc, spiegel e, broom c, schultz m. the detection and prevalence of reactive and physiologic sclerotic dentin, reparative dentin and dead tracts beneath various type of dental lesions according to tooth surface and age. j pathol 1983; 12:257–89. 6. massler m. pulpal reactions to dental caries. int dent j 1967; 17:441–60. 7. yongchaitrakul t, pasavant p. transforming growth factor-β1 upregulates the expression of nerve growth factor through mitogenactivated protein kinase signaling pathways in dental pulp cells. eur j sci 2007; 115:57–63. 8. farges jc, romeas a, melin m, pin jj, lebecque s, lucchini m, bleicher f, magloire h. tgf-β1 induces accumulation of dendritic cells in the odontoblast layer. j dent res 2003; 82:652–6. 9. massague j. tgf-β signal transduction. annu rev biochem 1998; 67:753–91. 10. duque c, hebling j, smith aj, giro ema, oliviera mf, costa cads. reactionary dentinogenesis after applying restorative materials and bioactive dentin matrix molecules as liners in deep cavities prepared in nonhuman primate teeth. j oral rehabil 2006; 33:452–61. 11. smith aj, tobias rs, murray pe. transdentinal stimulation of reactionary dentinogenesis in ferrets by dentin matrix components. j dent 2001; 29:341–6. 12. nakashima m, toyono t, murakami t, akamine a. transforming growth factor-beta superfamily members expressed in rat incisor pulp. arch oral biol 1998; 43:745–75. 13. tziafas d, papadimitrious s. role of exogenous tgf-β in induction of reparative dentinogenesis in vivo. eur j oral sci 1998; 106(suppl.1):192–6. 14. hu cc, zhang c, qian q, tatum nb. reparative dentin formation in rat molars after direct pulp capping with growth factors. j endod 1998; 24:744–51. 15. finkelman rd, mohan s, jennings jc, taylor ak, jepsen s, baylink d. quantitation of growth factors igf-1, sgf/igf-11 and tgf-β in human dentin. j bone miner res 1990; 5:717–23. 16. cassidy n, fahey m, prime ss, smith aj. comparative analysis of transforming growth factor-beta (tgf-β) isoforms 1-3 in human and rabbit dentine matrices. arch oral biol 1997; 42:219–23. 17. zhao s, sloan aj, murray pe, lumley pj, smith aj. ultrastructural localization of tgf-β exposure in dentine by chemical treatment. histochem j 2000; 32:489–94. 18. sloan aj, matthews jb, smith aj. tgf-beta receptor expression in human odontoblasts and pulpal cells. histochem j 1999; 31:565–9. 19. mclachlan jl, smith aj, sloan aj, cooper pr. gene expression analysis in cells of the dentine-pulp complex in healthy and carious teeth. arch oral biol 2003; 48:273–83. 20. sloan aj, perry jb, matthews jb, smith aj. transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth. histochem j 2000; 32:247–52. 21. piatelli a, rubini c, fioroni m, tripodi d, strocchi r. transforming growth factor-beta 1 (tgf-beta 1) expression in normal healthy pulps and in those with irreversible pulpitis. int endod j 2004; 37:114–9. 22. moses kd, butler wt, qin c. immunohistochemical study of small integrin-binding ligand, n-linked glycoproteins in reactionary dentin of rat molars at different ages. eur j oral sci 2006; 114:216–22. 23. olgart l, bergenholtz g. the dentin-pulp complex: responses to adverse influences. in: bergenholtz g, horsted-bindslev p, reit c, editors. textbook of endodontology. oxford: blackwell publishing company; 2003. p. 23. 24. mjor ia. pulp-dentin biology in restorative denstistry. chicago: quintessence publishing company; 2002. p. 98–9. 25. arana-chavez ve. massa lf. odontoblasts: the cells forming and maintaining dentine. int j biochem cell bio 2004; 36:1367–73. 26. smith aj. pulpal responses to caries and dental repair. caries res 2002; 36:223–32. 27. murray pe, windsor lj, smyth tw, hafez aa, cox cf. analysis of pulpal reactions to restorative procedures, materials, pulp capping, and future therapies. crit rev oral biol med 2002; 13:509–20. 28. fitzgerald m, chiego jr dj, heys dr. autoradiographic analysis of odontoblast replacement following pulp exposure in primate teeth. arch oral biol 1990; 35:707–15. 29. veerayutthwilai o, byers mr, pham ttt, darveau rp, dale ba. differential regulation of immune responses by odontoblasts. oral microbiol immunol 2007; 22:5–13. 30. silva ta, rosa al, lara vs. dentin matrix proteins and soluble factors: intrinsic regulatory signals for healing and resorption of dental and periodontal tissues? oral diseases 2004; 10:63–74. 31. smith aj, murray pe, sloan aj, matthews jb, zhao, s. transdentinal stimulation of tertiary dentinogenesis. adv dent res 2001; 15:51–4. 32. lara vs, figueiredo f, silva ta, cunha fq. dentin induced in vivodentin induced in vivo inflammatory response and in vitro activation of murine macrophages. j dent res 2003; 82:460–5. 33. smith aj, matthews jb, hall rc. transforming growth factor β1 (tgf β1) in dentine matrix: ligand activation and receptor expression. eur j oral sci 1998; 106:179–84. 34. lucchini m, romeas a, couble ml, bleicher f, magloire h, farges jc. tgf-β1 signaling and stimulation of osteoadherin in human odontoblasts in vitro. connect tissue res 2002; 43:345–53. 35. he w, niu z, zhao s, jin w, gao j, smith aj. tgf-β activated smad signaling leads to a smad3-mediated down-regulation of dspp in an odontoblast cell line. arch oral biol 2004; 49:911–8. �0 dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 15-20 36. wrana jl, attisano l, wieser r, ventura f, massague j. mechanism of activation of the tgf beta receptor. nature 1994; 370:341–7. 37. robert ab, russo a, felici a, flanders kc. smad3: a key player in pathogenetic mechanisms dependent on tgf-β. ann n y acad sci 2003; 995:1–10. 38. begue-kirn c, smith aj, ruch jv, wozney jm, purchio a, hartman d, et al. effects of dentin proteins, transforming growth factor beta 1 (tgf beta 1) and bone morphogenetic protein 2 (bmp2) on the differentiation of odontoblast in vitro. int j dev biol 1992; 36:491– 503. 39. leonard cm, fuld hm, frenz da, downie sa, massague j, newman sa. role of transforming growth factor beta in chondrogenic pattern formation in the embryogenic limb: stimulation of mesenchymal condensation and fibronection gene expression by exogeneous tgfbeta and evidence for endogenous tgf-beta-like activity. dev biol 2000; 145:99–109. 40. denbesten pk, machule d, gallagher r, marshall jr gw, mathews c, filvaroff e. the effect of tgf-β2 on dentin apposition and hardness in transgenic mice. adv dent res 2001; 15:39–41. 41. sloan aj. smith aj. stimulation of the dentine-pulp complex of rat incisor teeth by transforming growth factor-β isoforms 1-3 in vitro. arch oral biol 1999; 44:149–56. 42. pasavant p, yongchaitrakul t, pattamapun k, arksornnukit m. the synergistic effect of tgf-beta and 1,25-dihydroxyvitamin d3 on sparc synthesis and alkaline phosphatase activity in human pulp fibroblasts. arch oral biol 2003; 48:717–22. 43. pasavant p, yongchaitrakul t. secreted protein acidic, rich in cysteine induces pulp cells migration via αvβ3 integrin and extracellular signal-regulated kinase. oral diseases [published article online] 2007 jul 27. available from: url:http://www.blackwellmunksgaard. com. accessed december 26, 2007. 44. téclès o, laurent p, zygouritsas s, burger as, camps j, dejou j, about i. activtion of human dental pulp progenitor/stem cells in response to odontoblast injury. arch oral biol 2005; 50:103–8. 45. liu j, jin t, ritchie hh, smith aj, clarkson bh. in vitro differentiation and mineralization of human dental pulp cells induced by dentin extract. in vitro cellular dev biol 2005; 41:232–8. 46. tziafas d. the future role of a molecular approach to pulp-dentinal regeneration. caries res 2004;38:314–20. 47. tziafas d. induction of reparative dentinogenesis in vivo: a synthesis of experimental observation. connect tissue res 1995; 32:297– 301. 48. deng m, shi j, smith aj, jin y. effects of transforming growth factor β1 (tgfβ-1) and dentin non-collagenous proteins (dncp) on human embryonic ectomesenchymal cells in a three-dimensional culture system. arch oral biol 2005; 50:937–45. 49. melin m, joffre-romeas a, farges jc, couble ml, magloire h, bleicher f. effects of tgf-beta 1 on dental pulp cells in cultured human tooth slices. j dent res 2000; 79:1689–96. 50. huojia m, muraoka n, yoshizaki k, fukumoto s, nakashima m, akamine a, et al. tnf-b induces ectopic mineralization in fetal mouse dental pulp during tooth germ development. develop growth differ 2005; 47:141–52. vol 51 no 3 jul sep 2018_pus.indd 108108 research report dental journal (majalah kedokteran gigi) 2018 september; 51(3): 108–113 cytotoxicity test of binjai leaf (mangifera caesia) ethanol extract in relation to vero cells fifi dwidhanti,1 irham taufiqurrahman,1 and bayu indra sukmana2 1department of oral and maxillofacial surgery 2department of radiology faculty of dentistry, universitas lambung mangkurat banjarmasin indonesia abstract background: binjai leaves (mangifera caesia) constitute one part of a medicinal plant from south borneo that contains potential anticancer and antioxidant flavonoids. before using medicinal plants as adjuvant therapy material, a cytotoxicity test of a material extract needs to be conducted in order to establish the safety of natural ingredients that will be used in the production of medicinal products. purpose: this research aimed to determine whether the ethanol extract of binjai leaves proved cytotoxic to vero cells and determine the value of ic50 after the administering of ethanol extract of binjai leaves by means of an mtt assay method. methods: this research incorporated a true experimental method with posttest-only control design that consisted of ten groups. the binjai leaf ethanol extract of varying concentrations was administered to eight groups, namely;1.25μg/ml, 62.5μg/ml, 125μg/ml, 250μg/ml, 500μg/ml, 1000μg/ml, 2000μg/ml and 4000μg/ml. the control groups consisted of two groups, one cell control group and one media control group. the cell viability percentage was calculated by an absorbent of elisa reader. results: the probit analysis result had an ic50 value of 2498.48μg/ml (ic50 >1000μg/ml constituted a non-toxic category). conclusion: ethanol extract of binjai leaves is not cytotoxic to vero cells as shown by an assay mtt method which produced an ic50 value of 2498.48μg/ml. keywords: cytotoxicity; ethanol extract of binjai (mangifera caesia) leaves; flavonoid; mtt assay; vero cell correspondence: fifi dwidhanti, departement of oral maxillofacial surgery, faculty of dentistry, universitas lambung mangkurat, jl. veteran no. 128b, banjarmasin 70232, indonesia. e-mail: fifidwidhanti02@gmail.com introduction cancer is a disease resulting from damage to and abnormal development of body tissue cells inducing genetic mutations with the potential to produce cancer cells.1,2 in 2013, according to riset kesehatan dasar (riskesdas), the prevalence of cancer in indonesia stood at 1.4 per 1000 people or approximately 347,000 individuals. globally, indonesia ranked in fifth position, while south borneo was the region that placed twelfth within the country in terms of cancer prevalence at 1.6%.3 cancer of the oral cavity and throat placed sixth with regard to all forms of the disease around the world. in indonesia, oral cancer represents about 3-4% of all cases of the disease, producing mortality rate of 2-3%.4 cancer can be managed effectively with conventional treatments such as surgery, chemotherapy and radiotherapy.5 surgery is considered ineffective, particularly in those cases where the cancer has metastasized. chemotherapy and radiotherapy treatments are also less selective in their effect, one side-effect of prolonged use of the former being its toxicity to healthy tissue.6 due to the ineffectiveness and limited selectivity of conventional treatments, medicinal plants are currently being used as adjuvant therapy or secondary treatment for cancer. medicinal plants are currently in demand within society as an alternative adjuvant therapy to traditional treatment because it is relatively safe and ubiquitous.5 one of the medicinal plants utilized by the people of south borneo is the binjai plant (mangifera caesia). binjai dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 mailto:fifidwidhanti02@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 109 dwidhanti, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 108–113 is commonly consumed in south borneo for mixed sauce, a melange of pickles which is consumed with freshwater fish.7 binjai, including mangifera, has secondary metabolites such us tannins, alkaloidss, triterpenoid and flavonoids. binjai leaves contain flavonoid which has potential as an anticancer agent8 since has been proven to inhibit the proliferation of certain cancer cells, while demonstrating low cytotoxicity or even non-toxicity in relation to normal cells.6 previous research has proved that ethanol extract of binjai leaves is effective in protecting against the mortality of artemia salina leach larvae within a brine shrimp lethal test (bslt) method because its value of lc50 <1000 mg/l was 489.059 mg/l.8 a number of natural ingredients can be utilized within adjuvan therapy and, therefore, need to be subjected to a test of material extract cytotoxicity to establish the safety of those that will become medicinal products. the method employed is that of methylthiazol-2-yl-2,5-diphenyl tetrazolium (mtt) assay because it is relatively rapid, accurate and sensitive.9 mtt assay method constitutes an in vitro cytotoxicity test using a cell culture. the cell culture identical to normal human cells is reported to consist of vero cells which are normal cells isolated from the kidneys of african green apes (cercopithecus aethiops) which were first extracted by t. yasamura and t. kwalata at the university of chiba, japan in 1967.10–12 against this background, a cytotoxicity test of binjai leaves ethanol extract in relation to vero cells by mtt assay method had not yet been undertaken. the cytotoxicity of binjai leaves ethanol extract against vero cells was, therefore, established to determine the safe concentration of an adjuvan therapy material made from natural ingredients. materials and methods the research process began with a request for the necessary research permit and ethical clearance to be issued the ethics committee of the faculty of dentistry, university of lambung mangkurat no. 034/kepkg-fkgulm/ec/ ix/2017. the research methodology was true experimental with posttest-only in nature with a design control. the research population was divided into ten groups; eight groups administered with binjai leaves ethanol extract at varying concentrations (31.25μg/ml, 62.5μg/ml, 125μg/ ml, 250μg/ml, 500μg/ml, 1000μg/ml, 2000μg/ml and 4000μg/ml), one cell control group and one media control group. a minimum number of three repetitions in each group was obtained through application of the federer formula.13 the instruments used for making binjai leaves ethanol extract comprised a maceration vessel, plastic container, strainer, micro-pipette, volume pipette, glass beaker, test tube, shelf test tube, volumetric flask, stirring bar, blender, digital balance, water bath and vacuum rotary evaporator. the cytotoxicity test of vero cells in vitro used a 96-well microplate, co2 incubator, laminary air flow cabinet, pipette, flask, centrifugal, inverted microscope, 15ml conical tube, yellow tip, elisa plate reader, electric pipette and a hemasitometer and counter. the production materials for binjai leaf ethanol extract consisted of binjai leaves (obtained from sungai lulut village, east banjarmasin sub-district, banjarmasin district, south borneo), 70% ethanol solvent and filter paper. the vero cells used in the cytotoxicity test, namely: m199 media, foetal bovine serum (fbs) 10% tripsin-edta, dimethyl sulfoxide (dmso) and reagent methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide were grown at the dengue laboratory of airlangga university. preparation and production of extract using a maceration method.8 the binjai leaves (figure 1) used consisted of mature samples (the fourth leaf from the apex to the fifth leaf from the basal)14 selected by the biology laboratory, faculty of mathematics and natural sciences, universitas lambung mangkurat (125/lb.labdasar/ix/2017). leaves were separated from their branches, the dirty specimens being subsequently washed in water. at the extraction stage, 1 kg of wet binjai leaves were cut into small pieces, dried in the open air and pulverized in a blender to form dried simplicia, 430g of which was quantified by means of an analytical balance. the dried simplicia was inserted into a maceration vessel and soaked in 2.5 l of ethanol solvent 70%. maceration was conducted over three days, with the liquid protected from sunlight, while being agitated occasionally with a stirring bar to ensure that all the simplicia powder would dissolve in the solvent to produce the required solution concentration. after maceration, the resulting liquid was passed through whatman number 1 filter paper to remove the filtrate which was subsequently concentrated using a rotary evaporator at 50oc. the filtrate was poured into a vaporizer cup, before being evaporated in a waterbath. the final result of the thick ethanol extract of binjai leaves (figure 2) was calculated for yield using the formula below:15 figure 1. binjai leaves. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 110dwidhanti, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 108–113 cytotoxicity test against vero cell vero cells from the dengue laboratory of universitas airlangga, were grown in an m199 medium and incubated in a co2 5% incubator at a temperature of 37°c. the vero cell condition while in the co2 incubator was closely observed. if the cell is 80% confluent, it is washed twice with phosphate bufer saline (pbs) and added to tripsinedta 0.2% in order to release cells from the flask. cell density was calculated using a hemasitometer.16 vero cells were transferred to a 96-well microplate at a density of 2x104 cell in 100μl. binjai leaf ethanol extract at varying concentrations: 31.25μg/ml, 62.5μg/ml, 125μg/ml, 250μg/ml, 500μg/ml, 1000μg/ml, 2000μg/ ml and 4000μg/ml was then added – a process repeated for three times. 25μl reagent mtt was inserted in each microplate, including the control medium and incubated for four hours. 240μl stopper solution dmso 0.01% was inserted in each microplate after four hours of incubation. the 96-well microplate was absorbed by the elisa reader at a wave length 595nm with the resulting data being used to calculate the viability percentage of vero cells with the formulation below:17 % viability = note: abs = absorbent the percentage value of vero cell viability is analysed and an inhibitory concentration 50 (ic50) value obtained. the ic50 value was arrive at by means of probit analysis using spss 23.0 for windows. results the extraction of simplicia from binjai leaves used a maceration method with ethanol 70% as solvent with 430g pf simplicia being obtained from 73.43g of leaves. the resulting thick extract was dark brown in color. using the formula above, the calculation of the yield produced a figure of 17.076%. a cytotoxicity test of binjai leaf ethanol extract in relation to vero cells produced a viability percentage as shown in figure 3. in figure 3 shows that the life percentage of vero cell a concentration of 31.25μg/ml was 98%; at 62.5μg/ml 85%; 125μg/ml 83%; 250μg/ml 77%; 500μg/ml 66%; 1000μg/ml 61%; 2000μg/ml 56% and 4000μg/ ml 45.7%. the vero cell was viewed through an inverted microscope at 100x magnification in order to observe the morphological changes in cell death caused by the ethanol extract of binjai leaves.17 normal vero cell observed in figure 4 and 5. the morphological changes in cell death can see figure 6a and figure 7. the result data of percentage cell viability was analysed and the inhibitory concentration 50 (ic50) value calculated on the basis of the data obtained from three repetitions. the ic50 value, based on probit analysis using spss 23.0 for windows, was 2498.48 μg/ml with a minimum level of 1715.843μg/ml and an upper level of 4131.846μg/ml. discussion this research aimed to establish whether binjai leaf ethanol extract proved cytotoxic against vero cells and to determine the presence of ic50 after its administering by means of mtt assay method. according to the cytotoxicity figure 2 ethanol extract of binjai leaves. figure 4 morphological observation of normal vero cells. before being induced with extract and being administered with mtt (indicated by the arrow). figure 3 binjai leaves ethanol extract and the average. percentage of vero cell viability. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 111 dwidhanti, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 108–113 test result based on this method, the ethanol extract of the binjai leaves had an ic50 value of 2.498.48μg/ml. ic50 can show the cytotoxic potential of the compound. with regard to the cytotoxicity of natural ingredients, in cases where ic50 > 1000μg/ml, they are classified as non-toxic. 18 the final cytotoxicity test result provided information about the percentage of cells which had been able to survive.15 this data proved that ethanol extract of binjai leaves administered by mtt assay method was not cytotoxic to vero cells. in figure 6b, vero cellhad been able to survive by ability >50%. the screening results of the phytochemicals contained in ethanol extract of mangifera foetida confirmed the presence of secondary metabolites, namely: phenols, tannins, alkaloids, saponins, triterpenoids and flavonoids.19 mangifera caesia which included genus mangifera was considered to have the same compound content as mangifera foetida. phenol promoted antioxidant activity and played the role of maintaining free radical attack potentially harmful to dna.20 tannin is a phenol compound promoting antioxidant activity that is able to protect itself against oxidative damage.21 at high concentrations it can prove toxic to cells22 by damaging the cell membrane by shrinking the cell wall, thereby reducing its permeability. consequently, the continued viability of the cell is so severely compromised that it dies.23 in figure 3, 4000μg/ml constituted the highest concentration of ethanol extract of figure 5 (a) vero cell after being inducted with concentration extract of 4000. μg/ml (indicated by arrow). (b) vero cell after being inducted with concentrated extract of 2000 μg/ml and before the administering of mtt (indicated by the arrows). a b figure 6 morphological change of a vero cell after being induced with binjai leaves ethanol extract and mtt resulting in a formazan. crystal (indicated by arrows). (a) inhibition vero cell <50% at a concentration of 4000 μg/ml and (b) inhibition of a vero cell >50% at a concentration of 2000 μg/ml. a b figure 7 death of vero cell at a concentration of 4000. μg/ml. dead vero cells resulted in a loss of citoplasma fluid because of cell membrane damage which produces black and dark colors (indicated by the arrow).10 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 112dwidhanti, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 108–113 binjai leaves. therefore, it was estimated that tannin played a role in the concentration and promoted a percentage cell viability <50%. alkaloids, saponins and triterpenoids all have chemoprotective potential and in non-enzymatic conditions can inhibit lipid peroxide.24 the previous result confirmed that binjai leaves contain flavonoid8 which is reported to promote antioxidant activity capable of preventing injury because of the activity of free radical scavengers.25 these work by donating hydrogen ions to hydroxyl radicals and peroxyls in the b ring hydroxyl flavonoid group. the flavonoid hydroxyl group renders free radicals inactive with the result that they become more stable and acts as antioxidant radicals.25,26 antioxidant radicals are formed through a resonance process within the structure of an aromatic ring hindering their involvement in a reaction with other radicals with the result that they detoxify free radicals, prevent cell damage and promote cell viability.25 flavonoid could have an effect through interaction with metabolite enzyme phase i (cytochrome p450) within the cell. phase i metabolite enzymes activate numbers of procarsinogens to reactivate intermediates and substances which interacte with cellular nucleophiles thereby nullifying their capacity to initiate carcinogenesis. other mechanisms include flavonoid stimulated phase ii metabolism enzymes such as glutathione-s-transferase, quinone reductase, and udp-glucuronyl transferase in which carcinogens was detoxified.27 phase ii detoxification substances can catalyze the reaction which increases excretion of toxic compounds or carcinogenic chemical material in the body.28 therefore, the flavonoid in ethanol extract of binjai leaves is able to contribute to the concentration of the extract militating against the viability of vero cells. in this research, the method of binjai leaf extraction employed was that of meseration using ethanol solvent 70%. ethanol 70% was selected as the solvent in extraction because it pulled the compound in simplicia of binjai leaves. the compound capable of being pulled by ethanol is flavanoid which has antioxidant, chemoprotective and cytotoxicity potential through a mechanism of cycle arrest or apoptosis.29 extraction with ethanol 70% involved cytotoxic activity caused by compound varieties such as polar, semi polar or non-polar results in toxins affecting each other.30 non-polar compounds impede the pulling process of flavonoids because the defatting process did not occur and they caused a decrease in flavonoid activity. based on the research findings, it can be concluded that ethanol extract of binjai leaves is not cytotoxic in relation to vero cells as proved by an mtt assay method with an ic50 value of 2498.48 μg/ml. acknowledgements the authors acknowledge the support of the faculty of dentistry, university of lambung mangkurat for this research. they also express gratitude to the pharmacy and biology laboratory, faculty of mathematics and natural sciences, university of lambung mangkurat; dengue laboratory, university of airlangga; and laboratory of stem cell research and development center, university of airlangga for permitting use of its laboratory facilities and providing support during this experimental research. references pamungkas dw. uji toksisitas ekstrak buah debregeasia longifolia1. (burm.f.) wedd. terhadap larva artemia salina leach. dengan metode brine shrimp lethality test (bslt). universitas sebelas maret. 2016; : 1–11. witantri rg, ruspendi eca, saputro ds. keanekaragaman pohon2. berpotensi obat antikanker di kawasan kampus kentingan universitas sebelas maret, surakarta, jawa tengah. pros sem nas masy biodiv indon. 2015; 1(3): 477–83. badan penelitian dan pengembangan kesehatan. riset kesehatan3. dasar. jakarta: kementerian kesehatan republik indonesia; 2013. p. 85-7. sirait am. faktor risiko tumor/kanker rongga mulut dan tenggorokan4. di indonesia (analisis riskesdas 2007). media litbangkes. 2013; 23(3): 122–9. oratmangun sa, fatimawali f, bodhi w. uji toksisitas ekstrak5. tanaman patah tulang (euphorbia tirucalli l.) terhadap artemia salina l. dengan metode brine shirmp lethality test (bslt) sebagai studi pendahuluan potensi anti kanker. pharmacon j ilmu farmasi unsrat. 2014; 3(3): 316–24. mardiyaningsih a, ismiyati n. cytotoxic activity of ethanolic extraxt6. persea americana mill. leaves on hela cervical cancer cell. tradit med. 2014; 19: 24–8. rosyidah k, siska s, astuti md. isolasi senyawa antioksidan dari7. kulit batang tumbuhan binjai (mangifera caesia). j ilmu berkala sains dan terapan kimia. 2011; 5: 8–14. syahdana nl, taufiqurrahman i, wydiamala e. uji efektivitas8. ekstrak etanol daun binjai (mangifera caesia) terhadap mortalitas larva artemia salina leach. dentino j kedokteran gigi. 2017; 1: 39–44. listyowati y, nurkhasanah n. efek sitotoksik dan pemacuan9. apoptosis fraksi petroleum eter ekstrak etanol daun tapak liman (elephantopus scaber linn) terhadap sel hela. pharmaciana. 2013; 3(2): 1–7. 10. aulia an, salamah e, purwaningsih s. pengujian toksisitas ekstrak keong matah merah (cerithidea obtusa) terhadap artemia salina dan sel vero. bogor agricultural university (ipb); 2016. p. 1-56. 11. triatmoko b, hertiani t, yuswanto a. sitotoksisitas minyak mesoyi (cryptocarya massoy) terhadap sel vero. e-jurnal pustaka kesehatan. 2016; 4(2): 263–6. 12. nurani lh. uji sitotoksitas dan antiproliferatif sel kanker payudara t47d dan sel vero biji nigella sativa l. j ilmu kefarmasian. 2012; 2: 17–29. 13. wijaya j, salenussa j, marantika j. potensi ekstrak heksan daun kapur (harmsiopanax aculeatus, harms) sebagai obat antimalaria. in: program kreativitas mahasiswa-penelitian (pkm-p). 2014. p. 1–9. 14. anwar k, rahmanto b, triyasmono l, rizki mi, halwany w, lestari f. the influence of leaf age on total phenolic, flavonoids, and free radical scavenging capacity of aquilaria beccariana. res j pharm biol chem sci. 2017; 8: 129–33. 15. turalely r, hadanu r, mahulete f. uji aktivitas sitotoksik dan analisis fitokimia ekstrak daun kapur (harmisiopanax aculetus harms.). in: prosiding insinas. 2012. p. 98–103. 16. freshney ri. culture of animal cells: a manual of basic technique and specialized applications. 6th ed. new jersey: john wiley & sons; 2011. p. 187-94. 17. samarghandian s, boskabady mh, davoodi s. use of in vitro assays to assess the potential antiproliferative and cytotoxic effects of saffron (crocus sativus l.) in human lung cancer cell line. pharmacogn mag. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 113 dwidhanti, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 108–113 2010; 6(24): 309–14. 18. mardja te, rahmi f, rusmawati e, adriany r, setijanti hb, usia t. riset sitotoksik campuran ekstrak daun sirsak (annona muricata l.) dan kulit buah manggis (garcinia mangostana l.) pada sel vero dan aml 12. j trop pharm chem. 2016; 3(4): 284–90. 19. nuryanto a, luliana s, armyanti i. uji aktivitas antibakteri ekstrak etanol daun mangga bacang (mangifera foetida l.) terhadap escherichia coli secara in vitro. j mahasiswa pspd fk universitas tanjungpura. 2014; 1: 1–15. 20. falah s, haryadi d, kurniatin pa, syaefudin s. komponen fitokimia ekstrak daun suren (toona sinensis) serta uji sitotoksisitasnya terhadap sel vero dan mcf-7. j ilmu kefarmasian indonesia. 2015; 13(2): 174–80. 21. kusumawardhani ad, kalsum u, rini is. pengaruh sediaan salep ekstrak daun sirih (piper betle linn.) terhadap jumlah fibroblas luka bakar derajat iia pada tikus putih (rattus norvegicus) galur wistar. majalah kesehatan fkub. 2015; 2: 16–28. 22. carabelly an, putra st, suardita k. the toxicity of methanol extract of mauli banana stem (musa acuminate) against bone marrow mesenchymal stem cell in vitro. dentino j kedokteran gigi. 2017; 2: 24–8. 23. harsini h, hertama afn. pengaruh variansi konsentrasi ekstrak kulit batang jambu mete terhadap sitotoksikitas sel fibroblas. maj ked gi ind. 2017; 2: 6–12. 24. sudirman s. aktivitas antioksidan dan komponen bioaktif kangkung air (ipomoea aquatica forsk.). thesis. bogor: institut pertanian bogor; 2011. p. 68-76. 25. astuti s. isoflavon kedelai dan potensinya sebagai penangkap radikal bebas. j teknologi industri dan hasil pertanian. 2008; 13(2): 126–36. 26. tang lic, ling apk, koh ry, chye sm, voon kgl. screening of anti-dengue activity in methanolic extracts of medicinal plants. bmc complement altern med. 2012; 12(3): 1–10. 27. katyal p, bhardwaj n, khajuria r. flavonoids and their therapeutic potential as anti cancer agents: biosynthesis, metabolism and regulation. world j pharm pharm sci. 2014; 3(6): 2188–216. 28. sayuti k, yenrina r. antioksidan alami dan sintetik. padang: andalas university press; 2015. p. 70-1. 29. puspitasari e, umayah ulfa e. uji sitotoksisitas ekstrak metanol buah buni (antidesma bunius (l) spreng) terhadap sel hela. j ilmu dasar. 2009; 10(2): 181–5. 30. djajanegara i, wahyudi p. pemakaian sel hela dalam uji sitotoksisitas fraksi kloroform dan etanol ekstrak daun annona squamosa. j ilmu kefarmasian indonesia. 2009; 7: 7–11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p108–113 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p108-113 192 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 192–196 research report microleakage difference between total-etch and self-etch bonding in bulk fill packable composite restoration after carbonic acid immersion widya saraswati, dian pramita ayu kumalasari and adioro soetojo department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: bulk fill packable composite that can be applied to a depth of 4mm in cavities is widely used in posterior teeth restoration. unfortunately, this composite is subject to potential microleakage which occurs due to erosion resulting from the consumption of carbonated drinks containing carbonic acid. nevertheless, microleakage can be reduced by bonding applications the etch technique of which is divided into two forms; self-etch bonding and total-etch bonding. purpose: this study aims to determine the difference in microleakage between total-etch and self-etch bonding in bulk fill packable composite following carbonic acid immersion. methods: this study constitutes experimental laboratory research utilizing 28 incisors bovine teeth which were cleaned, immersed in 0.01% nacl, and randomly divided into four groups. the cervical area of the teeth of all groups were prepared through the creation of cylindrical shapes 2mm in diameter and 3 mm deep. groups i and iii used total-etch bonding, while groups ii and iv used self-etch bonding. groups iii and iv were control groups, whereas groups i and ii were treatment groups (immersed in carbonic acid) for 24 hours. thermocycling was carried out in all groups which were subsequently immersed in 1% methylene blue for 24 hours after which the teeth were cut in a buccolingual direction using a diamond disc wheel. microleakage was subsequently evaluated by calculating the amount of methylene blue passing between the restoration wall and cavity using a stereomicroscope and scanning electron microscope (sem). the data was then analyzed using kruskal wallis and mann whitney u tests. results: there was a significant difference between the control groups and treatment groups (p<0.05). the microleakage in group 2 was higher than that of other groups. conclusion: the microleakage of total-etch bonding was lower than self-etch bonding in bulk fill packable composite after carbonic acid immersion. keywords: bulk fill packable composite; carbonic acid; microleakage; self-etch bonding; total-etch bonding correspondence: widya saraswati, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: widya-s@fkg.unair.ac.id introduction composite resin is considered a popular restorative material since it not only has high aesthetic value and strong physical mechanical properties, but also demonstrates long-term stability and can be employed in the treatment of almost all classifications of gv black carious lesions.1,2 a number of innovations in the development of composite materials have been introduced, one of which is to intended to simplify its restoration technique while maintaining its physical properties. bulkfill composites are ones that can be applied up to a depth of 4mm and are consequently capable of reducing processing time. another characteristic of bulk fill composites is that they are not only effective against polymerization shrinkage, durable, and not easily fractured, but also possess sound dimensional stability.3 bulk fill packable composite resins not only have high viscosity which enables them to adapt quickly to dentin, but also considerable mechanical strength rendering them suitable for use in posterior teeth.4,5 nevertheless, it is known that microleakage in bulk fill packable composites is greater than in flowable composites which can be prevented through the administration of dentin bonding.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p192–196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p192-196 193saraswati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 192–196 microleakage is caused by dimensional changes and unfavorable adaptation to the cavity wall.6 a bonding material is one that increases the bond strength between the composite resin and tooth structure, enhances retention of restorations, and reduces microleakage along the surface between dentin and composite resin.7 bonding applications can be classified into one of two etching techniques, namely; total-etch and self-etch.8 one required characteristic of a restoration is the ability to resist degradation which in the oral cavity is a complex phenomenon related to the disintegration and dissolution of the restorative material present there.9 degradation in composite resins can also take the form of material loss due to erosion.10,11 significantly, acidic drinks with a ph below the critical level for the demineralization of enamel (4.55.5) have the potential to cause erosion.11 carbonated drinks are one of the non-alcoholic varieties with a high acidity level. a major ingredient of carbonated drinks is carbonic acid which has a ph of 2.37 and a concentration of 56.693 mg/ml.12 composite resins are said to be degraded due to acid exposure in artificial saliva with a low ph.12 unfortunately, it is not yet certain which bonding technique demonstrates the optimum attachment to dentine. a previous study posited that total-etch bonding has more minimal microleakage than the self-etch variety.13 in contrast, another prior investigation stated that the selfetch bonding demonstrates less microleakage than the total-etch variety.14 meanwhile, another study revealed that no significant difference exists in microleakage resulting from the total-etch dentin bonding or its self-etch counterpart.15 hence, this study aims to determine the difference in microleakage between total-etch and self-etch bonding in bulk fill packable composite after carbonic acid immersion. materials and methods this study constitutes an in vitro laboratory experimental research with post-only control group design which was granted ethical eligibility by the ethics commission of the faculty of dental medicine, universitas airlangga (no. 368/hrecc.fodm/vi/2019). certain stages were conducted in the course of this study. first, 28 samples of cariesand fracture-free bovine teeth (mandibular incisors) were removed intact from the jaw.16 they were subsequently cleaned and soaked in solution before being randomly divided into four groups, each of which contained seven samples. all the samples were then prepared using low speed round and cylindrical diamond burs (nsk, usa) cylindrical in shape, 3 mm in diameter and 2 mm deep which were applied to the cervical area of the teeth. cavity depth was examined by means of a straight probe.17 in the fifth step, the members of group i and group iii were treated with total-etch bonding technique (ivoclar vivadent, n-etch® schaan, liechtensein, germany) and separate bonding. meanwhile, group ii and group iv were treated with a self-etch bonding technique using universal bonding materials (ivoclar vivadent, tetric® n-bond universal, schaan, liechtensein, germany). all groups were restored using bulk fill packable composite (ivoclar vivadent, tetric® n-ceram bulk fill, schaan, liechtensein, germany) and irradiated for 20 seconds by means of a curing unit (woodpecker® light curing led.c wireless, usa). the samples were then prepared for thermocycling and carbonic acid immersion. thermocycling was carried out in all sample groups for 120 cycles at 5o and 55o c. the samples were covered apically with dental wax to prevent further penetration. the coronal part was then coated twice with nail polish in an area up to 1 mm around the restoration. group i and group ii were subsequently immersed in carbonic acid with a ph of 2.33 or a concentration of 56.693 mg/ml for 24 hours while the control groups, group iii and group iv were immersed in distilled water for 24 hours. all sample groups were dried with tissue paper before immersion in 1% methylene blue solution for 24 hours. following immersion, a buccolingual direction was cut in the middle of the restoration using a diamond disc wheel. (intensive® diamond dental disc wheels, switzerland). microleakage in restoration was evaluated by examining the penetration of 1% methylene blue solution at the margins of the overlap in the occlusal and gingival walls. each sample was then assessed by three observers by means of a stereomicroscope (zeiss® stremi dv4, germany) at 15x magnification. the microleakage was then evaluated with a scoring method based on that proposed by didron et al. (2013),18 namely; 0 = no colour penetration; 1 = colour penetration up to half of the cavity wall; 2 = total colour penetration of the cavity wall; 3 = colour penetration up to half of the axial wall; and 4 = colour penetration more than half of the axial wall (figure 1). more detailed observations of microleakage were conducted with a scanning electron microscope (sem) (zeiss® evo ma 10, germany) before the data was analyzed using a kruskal wallis test and a mann whitney u test with spss software (ibm® spss statistics 20, ibm, usa) with a p value of <0.05. figure 1. microleakage scoring criteria. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p192–196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p192-196 194 saraswati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 192–196 table 1. results of the difference test on the microleakage of total-etch bonding and self-etch bonding on bulk fill packable composite restoration after carbonic acid immersion for 24 hours groups n* mean standard deviation p i 7 1.7143 ± 0.48795 0.000 ii 7 3.8571 ± 0.37796 iii 7 1.0000 ± 0.81650 iv 7 1.8571 ± 0.69007 note: *number of samples table 2. result of mann-whitney test groups i ii iii iv i 0.001* 0.080 0.705 ii 0.001* 0.001* iii 0.065 iv note: *significant difference i ii iii iv bonding dentin dentin bonding mikroleakage bonding dentin dentin mikroleakage bonding figure 2. sem results at 1000x magnification. (i) the total-etch bonding group with carbonic acid immersion. (ii) the self-etch bonding group with carbonic acid immersion. (iii) the total-etch bonding control group. (iv) the self-etch bonding control group. arrows indicate microleakage on a border between bonding and dentin. results the contents of table 1 shows that the highest mean score of microleakage was recorded by group i with self-etch bonding technique after carbonic acid immersion. according to the kruskall wallis test results, a significant difference existed in all groups with a p value of 0.000 (p<0.005). however, the results of the mann whitney test (table 2) indicated a significant difference between group i with the total-etch bonding technique and group ii with the selfetch bonding technique after carbonic acid immersion. no significant differences existed between group i employing the total-etch bonding technique; group iii employing the total-etch bonding technique, but without carbonic acid immersion; and group iv employing the self-etch bonding technique, but without soaking in carbonic acid. moreover, group ii employing the self-etch bonding technique demonstrated a significant difference from group iv with the self-etch bonding technique, but without carbonic acid immersion. meanwhile, no significant difference existed between control group iii and control group iv. the microleakage in samples observed using a buccolingual 15x magnification stereomicroscopes that based on metylene blue as color penetration, the lowest microleakage score with a score of 1 occurred in group iii. meanwhile, group ii recorded the highest microleakage score with a score of 4. group i and group iv produced almost identical microleakage scores with a score of 2. furthermore, sem observation results indicated that microleakage as a gap or cavity with an irregular shape and darker color than the surrounding tissue structure, (yellow arrows) was predominantly identified in group ii and group iv. contrastingly, no microleakage occurred in group i and group iii (figure 2). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p192–196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p192-196 195saraswati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 192–196 discussion the results of this study indicated a significant difference in microleakage between total-etch bonding and self-etch bonding after carbonic acid immersion. the bonding material employed was of a universal adhesive type which contains 10-30% more water than a conventional bonding variety. this caused greater ionization of acid monomers resulting in an accompanying increase in the depth of the demineralized dentin. therefore, deeper resin penetration was possible in producing adequate adhesion.19 the results of this study also revealed that group i had fewer microleakage values than the other groups due to separate etching and rinsing application processes within the total-etch bonding. therefore, the smear layer produced in the etching process would be eradicated during rinsing. the clean surface of the smear layer substrate is an indication of the exposure of the hydroxyl enamel group to hydroxyapatite crystals which renders the substrate hydrophilic with the result that wettability increases and results in deeper and stronger penetration of the bonding material monomer.20 this process will then increase marginal adaptation culminating in the edge of the leakage that occurs in the total-etch bonding technique being smaller than that in its self-etch counterpart.21 a previous study conducted by tsujimoto et al. (2016),22 similarly argued that the base surface of the enamel covered by the smear layer is hydrophobic resulting in a reduction in wettability. nevertheless, the acid etching application procedure can culminate in increased wettability in the enamel causing the formation of a small phi angle. as a result, bonding and composite resin material easily penetrates the entire cavity and good marginal adaptation is formed.21 the increased wettability of the enamel will also alter the hydrophobic characteristics to hydrophilic ones through exposure to groups of cavities, thereby increasing chemical bonds on the substrate involved in the adhesive process.23 ultimately, the surface area and surface energy available for binding which are derived from the interaction between the composite resin and collagen dentin (hybrid layer) are greater. therefore, the restoration is more resistant to thermocycling and erosion due to carbonic acid immersion. as with the results of this research, those of a previous study conducted by el sayed et al. (2014)7 argued that total-etch bonding experiences less extensive microleakage than self-etch bonding. furthermore, the results of this study found that group ii (using the self-etch bonding technique with carbonic acid immersion) was the group with the highest leakage. the self-etch bonding technique group did not go through the process of etching and rinsing and, consequently, the hydroxyapatite decalcification results (smear layer) were still present on the dentin surface. the smear layer chemically bonded with the functional monomer of acrylic phosphonic acid to form a smear plug (0.5 μm-5 μm) of total-etch bonding. these chemical bonds are stable but weak, leading to the formation of lower marginal adaptations.24 37%, phosphoric acid, is the etching material most frequently used to produce consistent microporosity. the etching process with weak acid, in this case present in self-etch bonding material, will cause irregularly formed microporosity of insufficient depth, thereby causing inadequate micromechanical retention and susceptible edge leakage.23 the influence of thermocycling and erosion caused by carbonic acid immersion causes not only self-etch bonding to demonstrate higher demineralization, but also induces the appearance of a gap allowing the low ph of the carbonic acid in carbonated drinks to soften ca10 (po4) 6 (oh) 2 hydroxyapatite crystals. of the subsequently decomposing complexes formed, one is ca+2 which will then be bound by carbonate ions to form caco3. 25 demineralization occurs continuously during consumption of carbonated drinks which have a low ph of 2.37. this condition, in turn, provides an opportunity for carbonic acid to penetrate and react to form a gap between the cavity and tooth restoration, resulting in the development of enamel porosity at the cast edge.22,24 finally, the results of this study also confirmed no statistically significant difference in microleakage between group iii and group iv (p>0.005). this is because both total-etch and self-etch techniques make equally effective marginal adaptations to the tooth structure surface. similarly, a previous study conducted by perdigão et al. (2003)15 stated that both of these adhesive techniques produce microleakage that is not significantly different. moreover, the application of universal bonding material in the two sample groups provided optimum adhesive strength to minimize microleakage (p<0.05). in conclusion, the level of microleakage in bulk fill packable composite restorations in total-etch bonding after carbonic acid immersion was lower than that in self-etch bonding. references 1. cramer nb, stansbury jw, bowman cn. recent advances and developments in composite dental restorative materials. j dent reseacrh. 2011; 90(4): 402–16. 2. anusavice kj, phillips rw, shen c, rawls hr. phillips’ science of dental materials. 12th ed. budiman ja, purwoko s, editors. jakarta: egc; 2013. p. 275–306. 3. van ende a, de munck j, lise dp, van meerbeek b. bulk-fill composites: a review of the current literature. j adhes dent. 2017; 19(2): 95–109. 4. jackson rd. posterior composites and the new bulk-fill materials. insid dent. 2014; 10(8): 68–75. 5. orłowski m, tarczydło b, chałas r. evaluation of marginal integrity of four bulk-fill dental composite materials: in vitro study. sci world j. 2015; 2015: 1–8. 6. lokhande na, padmai as, rathore vps, shingane s, jayashankar dn, sharma u. effectiveness of flowable resin composite in reducing microleakage an in vitro study. j int oral heal jioh. 2014; 6(3): 111–4. 7. el sayed hy, abdalla ai, shalby me. marginal microleakage of composite resin restorations bonded by desensitizing one step self etch adhesive. tanta dent j. 2014; 11(3): 180–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p192–196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p192-196 196 saraswati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 192–196 8. muñoz ma, luque i, hass v, reis a, loguercio ad, bombarda nhc. immediate bonding properties of universal adhesives to dentine. j dent. 2013; 41(5): 404–11. 9. khan aa, siddiqui az, al-kheraif aa, zahid a, divakar dd. effect of different ph solvents on micro-hardness and surface topography of dental nano-composite: an in vitro analysis. pakistan j med sci. 2015; 31(4): 854–9. 10. sabdi s, zaripah w, bakar w, husein a. assessment of microleakage of few restorative materials after erosion by acidic solution. arch orofac sci sci. 2011; 6(2): 66–72. 11. amaechi bt. dental erosion and its clinical management. london: springer international publishing; 2015. p. 70, 100–10. 12. felix kr, bressan mc, kanis la, de oliveira mt. assessment of acid neutralizing capacity in cola-based drinks and energy beverages by artificial saliva. j contemp dent pract. 2013; 14(4): 578–81. 13. hegde m, hegde pn, ravi chandra c. morphological evaluation of new total etching and self etching adhesive system interfaces with dentin. j conserv dent. 2012; 15(2): 151–5. 14. gupta a, tavane p, gupta pk, tejolatha b, lakhani aa, tiwari r, kashyap s, garg g. evaluation of microleakage with total etch, self etch and universal adhesive systems in class v restorations: an in vitro study. j clin diagnostic res. 2017; 11(4): zc53–6. 15. perdigão j, geraldeli s, hodges js. total-etch versus self-etch adhesive: effect on postoperative sensitivity. j am dent assoc. 2003; 134(12): 1621–9. 16. nurhapsari a. perbandingan kebocoran tepi antara restorasi resin komposit tipe bulk-fill dan tipe packable dengan penggunaan sistem adhesif total etch dan self etch. odonto dent j. 2016; 3: 8–13. 17. yuan h, li m, gao y, liu hl, li j, guo b. evaluation of microtensile bond strength and microleakage of a self-adhering f lowable composite. j adhes dent. 2015; 17(6): 535–43. 18. didron pp, chrzanowski w, ellakwa a. effect of temperatures on polymerization stress and microleakage of class v composite restorations. open j compos mater. 2013; 03(04): 107–12. 19. ritter a v., boushell lw, walter r, sturdevant cm. sturdevant’s art and science of operative dentistry. 7th ed. st. louise: elsevier mosby; 2018. p. 417–18. 20. soares cj, faria-e-silva al, rodrigues m de p, fernandes vilela ab, pfeifer cs, tantbirojn d, versluis a. polymerization shrinkage stress of composite resins and resin cements what do we need to know? vol. 31, brazilian oral research. sociedade brasileira de hematologia e hemoterapia; 2017. p. 49–63. 21. soetojo a. penggunaan resin komposit dalam bidang konservasi gigi. surabaya: pt. revka petra media; 2013. p. 45–7. 22. tsujimoto a, barkmeier w, takamizawa t, latta m, miyazaki m. the effect of phosphoric acid pre-etching times on bonding performance and surface free energy with single-step self-etch adhesives. oper dent. 2016; 41(4): 441–9. 23. d’alpino php, da rocha svizero n, carrilho m. self-adhering composites. in: dental composite materials for direct restorations. springer international publishing; 2018. p. 129–51. 24. laurensia ek, untara te, daradjati s. perbedaan kebocoran mikro tumpatan resin komposit flowable bulkfill yang menggunakan bahan bonding total etch dan self etch. j kedokt gigi. 2014; 5(2): 56–62. 25. prasetyo ea. keasaman minuman ringan menurunkan kekerasan permukaan gigi (acidity of soft drink decrease the surface hardness of tooth). dent j (majalah kedokt gigi). 2005; 38(2): 60–3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p192–196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p192-196 179 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 179–185 case report management of a complete denture in the flat mandibular ridge using a semi-adjustable articulator along with an effective suction method muhammad dimas aditya ari1, harry laksono1, valerian laksono2, real akbar aucky sanjaya3, tasya regita pramesti3, ratri maya sitalaksmi1 1department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2resident of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: complete dentures can improve the quality of life of edentulous patients. the selection of a suitable articulator and an impression technique is important to construct a stable and retentive complete denture with good occlusion. the use of a semiadjustable articulator ensures that the dentures can be constructed such that their movement closely resembles the patient’s physiological movements. the effective suction method can ensure the development of the border seal according to the patient’s anatomical condition. the use of a semi-adjustable articulator along with an effective suction method is expected to provide good results in the construction of a complete denture. purpose: this report aimed to describe the management of individual complete dentures in the flat mandibular ridge using a semi-adjustable articulator along with an effective suction method. case: a 69-year-old female patient came with a chief complaint of her old dentures being unusable and wanted new dentures made. the patient’s general condition was good, and the last extraction was done three months prior to the patient’s arrival. the mandibular posterior alveolar ridge showed dextral tapering and sinistral flatness. case management: a complete denture with an acrylic base was fabricated using a semi-adjustable articulator along with an effective suction method. conclusion: flat ridge case management using a semi-adjustable articulator with an effective suction method can improve complete denture retention and stability. keywords: complete denture; effective suction method; semi-adjustable articulator correspondence: muhammad dimas aditya ari, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. e-mail: dimasadityadrg@fkg.unair.ac.id introduction the prevalence of tooth loss has increased in the past few decades, especially in elderly patients. the loss of teeth causes a decrease in the masticatory, phonetic and aesthetic functions of the patient, thus resulting in a reduction of the patient’s quality of life.1 a replacement of the missing teeth with complete dentures is needed to restore the patient’s masticatory, phonetic and aesthetic functions, which can improve the patient’s quality of life.2 retention and stability are important factors in the successful fabrication of complete dentures. retention and stability of mandibular dentures is relatively difficult to achieve due to the surface area of the mandible being much smaller than that of the maxilla and the active movement of muscles resulting from the presence of the tongue and floor of the mouth.3 this makes obtaining a border seal more difficult in the lower jaw compared to that in the upper jaw. an effective suction method can be used to generate an adequate border seal for the dentures to be more retentive and stable, which is not easily achievable by conventional methods.4 in the case of flat ridges, maximum retention and stability must be achieved in order to increase the comfort and function of the complete denture. this case report aims to describe the management of individual complete dentures in the flat mandibular ridge using a semi-adjustable articulator along with effective suction methods. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 mailto:dimasadityadrg@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 180ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 case a 69-year-old female patient arrived at the prosthodontic specialist clinic at the universitas airlangga dental hospital to get a new complete denture made. the patient was seeking an improvement in the dental appearance and masticatory functions because her old dentures were no longer usable due to the extraction of the remaining teeth. the general condition of the patient was good, there was no history of systemic disease, and the last extraction was performed approximately three months prior to the patients arrival. no abnormalities were found on extraoral clinical examination. the patient profile is shown in figure 1. intraoral clinical examination (figure 2) revealed the missing maxillary and mandibular teeth to be accompanied by remnants of the root of the 12th tooth. the ridge could be seen to be ovoid in the maxilla and anterior mandible, a b figure 1. patient profile: (a) front view, (b) side view. a b c d e figure 2. clinical intraoral examination of the patient: (a) frontal, (b) right side, (c) left side, (d) maxillary occlusal and (e) mandibular occlusal. figure 3. panoramic radiograph. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 181 ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 ba figure 4. the first facebow transfer with the universal transferbow system (uts) stratos300® and centric tray: (a) frontal view and (b) lateral view. tapering on the right posterior of the mandible (cawood and howell classification of edentulous jaws class iv) and flat on the left posterior of the mandible (cawood and howell classification of edentulous jaws class v). a normal ridge relation was seen, and no exostosis was found. the retro-mylohyoid area was deep. radiographic examination (figure 3) showed a radiopaque appearance in the region of the tooth, the impression of the remaining roots and the maxillary and mandibular edentulous ridges. the diagnosis obtained from all examinations performed was chronic apical periodontitis e.c gangrene radix of tooth 12 and edentulous ridge of the maxilla and mandible. case management treatment of this case was carried out by making complete dentures with an acrylic base using an effective suction method. the treatment began with the informed consent of the patient and continued with the preliminary impression of the upper and lower jaws using the accu-tray (accudent®) stock tray with an irreversible hydrocolloid (alginate) impression material and was followed by the pouring of gypsum type iii (dental stone) to obtain study models. subsequently, the patient was referred for a radiograph. from the results of all examinations, the operator established a diagnosis, made a treatment plan, and developed a denture design. the patient was referred for the extraction of the remaining root of tooth 12. prosthodontic treatment was initiated by measuring the patient’s preliminary vertical dimension occlusion (vdo) using the niswonger method. subsequently, a preliminary bite registration was carried out using an irreversible hydrocolloid material that was applied to the centric tray. the centric tray was inserted into the patient’s mouth and the patient was instructed to close the mouth slowly until the predetermined vdo was reached. next, the facebow transfer was attached to the patient using the universal transferbow system (uts). the study model was mounted on a semi-adjustable articulator (stratos 300®) with the aid of the centric tray and was further developed with the manufacture of maxillary and mandibular individual trays with bite rim mounts (figure 4). figure 5. results of intraoral gothic arch tracing on the maxillary registration plate. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 182ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 a b figure 6. mounting the working cast on the semi-adjustable articulator from frontal (a) and lateral (b) sides. figure 7. polished acrylic complete denture ready for patient insertion. a b c d fe figure 8. acrylic complete denture used in the patient looks clinically extraoral from the frontal (a) and lateral (c) sides when the patient smiles (b) and looks clinically intraoral from the right lateral side (d), frontal (e) and left lateral side (f). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 183 ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 the stability, flange, median lines and alignment on individual trays were checked. border molding was then performed on the maxilla, starting with applying adhesive material to all edges of the maxillary individual tray, which was followed by injection of polyvinyl siloxane medium body material on all its edges. the maxillary individual tray was intraorally inserted, and the patient was instructed to say ‘ah’ firmly in an open mouth position. mandibular individual trays were inserted into the patient’s mouth and then the patient was instructed to close her mandibula. the patient was instructed to pronounce the words ‘wee’ and ‘woo’ while maintaining the last position. the patient was then instructed to perform a thumb sucking motion and wait for the material to set. subsequently, the individual tray was removed, and all excess border molding material was cut and eliminated. the procedure was continued by performing border molding on the mandibular ridge. the same procedure was repeated for the maxilla except that the patient was instructed to stick the tongue forward and right and perform a motion of swallowing saliva. the next step was to acquire the final impression of the maxillary and mandibular ridge to obtain a working cast. adhesive material was applied to the anatomical surface of the individual tray followed by an injection of polyvinyl siloxane light body material. the individual tray was inserted intraorally, and the patient was instructed to perform the same movements as done for border molding. the impression was initiated with the maxilla and the excess material was cut after the setting of the material. the impression then continued with the same procedure for the mandible. the vdo was corrected by replacing the bite rims mounts on the maxillary individual tray with a maxillary registration plate and the bite rims mounts on the mandibular individual tray with a mandibular registration plate having a gothic arch registration stylus with pliers. the patient’s vdo was measured using the niswonger method and the individual trays were then placed on the maxillary and mandibular ridge. the vdo was adjusted by rotating the stylus on the individual tray. after getting the right vdo, the stylus was locked by dripping liquid wax. the horizontal relation was determined using a gothic arch traced with an m-gnathometer. the maxillary registration plate was given a marker and the patient was instructed to practice opening and closing the mouth in a consistent position that had been given a marker as the starting point. the patient was instructed to move the mandible anteriorly (protrusive movement) beginning from the starting point and returning to it several times. similar movements were also performed for the right (right lateral movement) and the left side (left lateral movement). gothic arch tracing was confirmed by examining the results of the mandibular movements recorded on the marker on the maxillary registration plate as shown in figure 5. a transparent plastic fixation was installed with one of its holes located at the starting point so that when the patient closed her mouth, the stylus was locked in the correct starting point position. once locked, o-bite® polyvinyl siloxane medium body bite registration material was injected in the gap formed between the maxillary and mandibular registration plates. the patient’s median, canine and smile lines were assigned on individual trays with the marker. subsequently, a facebow transfer was performed on the patient using the uts and was followed by the selection of the type and colour of the elements of the teeth. the final impression was boxed and poured with gypsum type iii (dental stone) to get a working cast. the maxillary working cast was mounted on a semi-adjustable stratos 300® articulator using a facebow transfer guide. the working cast of the mandibular was mounted using a bite registration guide (figure 6). the denture elements were arranged with the setting of a two-dimensional template based on the centric occlusion, protrusive movement and lateral movement. complete denture wax was used for a trial denture for the patient. the patient’s profile, retention, stability and occlusion were checked. the complete denture wax was subjected to contouring, flasking, packing, and initial polishing (figure 7). the denture was tried in for the patient and the patient’s profile, retention, stability and occlusion were checked. subsequently, an insertion of the denture was performed (figure 8). this was followed by a session of information and advice for the patient. discussion at the patient’s initial visit, a preliminary impression was performed to produce a study cast that was used to assist clinicians in conducting examinations, establishing a diagnosis and determining a treatment plan, and for being the basis for making custom impression trays.5 the preliminary impression was made using the accu-tray (accu-dent®) stock tray with alginate material. the accutray has advantages over conventional stock trays owing to the extra flange that duplicates the depth of the vestibule and the extended distal part that duplicates the retromolar pad area more efficiently.6 irreversible hydrocolloid material was chosen because it can produce a good impression with detailed anatomical landmarks and has a lower cost.5,7 however, this material often exerts excessive pressure on the patient’s vestibule, resulting in an inaccurate model. the pressure on the vestibule usually results in an overextended study model.8 the impressions made with irreversible material are susceptible to damage from water content in the surrounding environment. if the impression is placed in a wet environment, the mold absorbs the water, which results in the occurrence of syneresis, whereas if the impression is placed in a dry environment, it releases its water content, resulting in the occurrence of an imbibition process. therefore, the preliminary impression must be filled with gypsum immediately to avoid any changes in the dimensions.9 according to the ansi/ada specification dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 184ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 no. 25, gypsum type i and ii are capable of reproducing grooves with a width of 75mm, while gypsum type iii, iv and v are capable of reproducing grooves with a width of 50mm. therefore, gypsum type iii, iv and v are capable of producing more accurate duplication as compared to that produced by type i and ii. gypsum type iii has a compressive strength lesser than gypsum type iv and v.10 material needed to produce a study model has to be accurate, however, it does not require very high strength. therefore, gypsum type iii (dental stone) was chosen as the material of choice for filling the preliminary mold. the use of a centric tray also facilitates the process of making complete dentures. the centric tray can be used to obtain an initial vdo on the patient. this provides the operator with a reference to the patient’s vdo, which results in minimal adjustment being required at the stage of determining the occlusion. in addition, the use of a centric tray can assist the operator in mounting the study cast in the articulator in a centric relation, which can enable an analysis related to the patient’s occlusion relationship to be carried out.4 the cast was mounted on the articulator with the aim to simulate the relationship and the movement of the patient’s jaw extraorally. articulators are classified into three types, namely non-adjustable, semi-adjustable and fully-adjustable articulators.11 in this case, a semiadjustable articulator (stratos 300®) was used because some plane orientations and angles on the semi-adjustable articulator could be adjusted according to the anatomical conditions and physiology of the patient. this facilitated the production of dentures that had occlusion and articulation (individualized prosthesis) similar to that of the patient, which resulted in an increase in the patient comfort.4,5 a facebow transfer was used for this case in addition to the semi-adjustable articulator. according to the glossary of prosthodontics terms (2017), facebow transfer is a process of transferring the spatial relationship of the maxillary arch to anatomical points on an articulator using certain instruments.12 the facebow transfer on the patient was expected to result in the orientation of the maxillary study model to the axis of rotation of the articulator being close to that between the patient’s maxilla and the patient’s transverse horizontal axis, namely the tmj. this allows the turning radius of the articulator to be more similar to the patient’s arc closure, thereby minimizing errors.13 several factors influence the success of denture prosthesis, such as the retention and stability of the denture, which can both be achieved properly if the denture has an adequate border seal.14 according to the glossary of prosthodontic terms, 9th edition, a border seal is the contact of the denture edge against the underlying tissue to prevent air or any other materials from entering the denture.12 to obtain an adequate border seal, the final impression must be ensured to be accurate at the boundaries of the movable and immovable mucosa. if the edge does not reach the movable and immovable mucosa and underextends the mucosal margin, the denture bearing area will be reduced and a border seal will not form. if the edge exceeds the border of the moving and immovable mucosa (overextension), the muscle movement will result in the denture breaking the border seal.15 individual trays and border molding are required to obtain a precise denture border area at the boundaries of the movable and immovable mucosa.16,17 peri-compound wax is a material that is often used for this purpose. however, this material needs to be used in sectional parts and is, therefore, considered time consuming and uncomfortable for the patient.18 an alternative method to obtain an adequate border seal is to perform a functional impression using polyvinyl siloxane material along with an effective suction method. the impression is initiated with the maxillary arch because the surface area of the maxillary denture base is wider than that of the mandible, which makes it easier for the impression material to displace. if the displacement occurs in the individual trays, the predetermined occlusal position cannot be reproduced accurately.4 the impression obtained using the effective suction method is almost the same as that obtained by the close mouth method. this method mainly involves the patient performing a suction movement with their mouth during the impression. this allows for an adequate denture border seal to be obtained due to the active movement of muscle trimming performed by the patient. this impression method is a patient-oriented method for obtaining an adequate denture border seal and a more stable and retentive complete denture.4,19 the individual tray used for this case was also equipped with an m-gnathometer. the m-gnathometer is an instrument used to assist in tracing the intraoral gothic arch. gothic arch tracing is a scalable method in which the entire range of mandibular movement is recorded on a registration plate to determine the horizontal relation of the jaws. gothic arch tracing helps the operator to obtain a stable tapping point that is at the right point of occlusion and to determine an accurate horizontal relationship. the use of gothic arch tracing can minimize the presence of occlusal discrepancies that subsequently need to be corrected after the acrylic dentures are inserted into the patient’s mouth. this gothic arch tracing procedure facilitated the fabrication of the complete denture with optimal aesthetics, function, and phonetics.20–23 from this case report, it can be concluded that flat ridge case management using a semi-adjustable articulator along with an effective suction method can be used to manufacture individual complete dentures. references 1. emami e, de souza rf, kabawat m, feine js. the impact of edentulism on oral and general health. int j dent. 2013; 2013: 498305. 2. kaushik k, dhawan p, tandan p, jain m. oral health-related quality of life among patients after complete denture rehabilitation: a 12-month follow-up study. int j appl basic med res. 2018; 8(3): 169–73. 3. jain p, rathee m. stability in mandibular denture. statpearls. treasure island (fl): statpearls publishing; 2022. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 185 ari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 179–185 4. abe j, kokubo k, sato k. mandibular suction-effective denture and bps: a complete guide. quintessence pub; 2012. p. 291. 5. rahn ao, ivanhoe jr, plummer kd. textbook of complete dentures. 6th ed. usa: pmph-usa; 2009. p. 446. 6. saini v, singla r. biofunctional prosthetic system: a new era complete denture. j pharm bioallied sci. 2011; 3(1): 170–2. 7. rao s, chowdhary r, mahoorkar s. a systematic review of impression technique for conventional complete denture. j indian prosthodont soc. 2010; 10(2): 105–11. 8. johnson t, wood dj. techniques in complete denture technology. uk: wiley-blackwell; 2012. p. 112. 9. mccabe jf, walls a. applied dental materials. 9th ed. uk: wileyblackwell; 2008. p. 312. 10. sakaguchi rl, powers jm. craig’s restorative dental materials. thirteenth. sakaguchi rl, powers jm, editors. saint louis: mosby; 2012. p. 150–2. 11. gross m. the science and art of occlusion and oral rehabilitation. uk: quintessence publishing; 2015. p. 544. 12. driscoll cf, freilich ma, guckes ad, knoernschild kl, mcgarry tj, goldstein g, goodacre c, guckes a, mors, rosenstiel s, vanblarcom c. the glossary of prosthodontic terms: ninth edition. j prosthet dent. 2017; 117(5s): e1–105. 13. sh i l l i ngbu rg jr. h t, sat her da, wi lson jr. e l , ca i n j r, mitchell dl, blanco lj, kessler jc. fundamentals of fixed prosthodontics. 4th ed. usa: quintessence publishing; 2012. p. 584. 14. patel j, jablonski ry, morrow la. complete dentures: an update on clinical assessment and management: part 1. br dent j. 2018; 225(8): 707–14. 15. pridana s, danial nasution i, nasution i, welda utami ritonga p. effect of border molding materials and techniques on peripheral tissue morphology and retention of denture bases in edentulous patients at rsgm usu. int j oral heal dent. 2019; 5(1): 14–9. 16. kaur s, datta k, gupta sk, suman n. comparative analysis of the retention of maxillary denture base with and without border molding using zinc oxide eugenol impression paste. indian j dent. 2016; 7(1): 1–5. 17. özkan yk. complete denture prosthodontics. cham: springer international publishing; 2018. p. 290. 18. qureshi i, rashid s, qureshi s, rehman au. critical evaluation of material and procedures used for the functional peripheral moulding. j pakistan dent assoc. 2010; 19(2): 129–32. 19. gosavi s, nalawade k, gosavi s. use of innovative suction device to improve the retention in denture wearer patients a pilot study. int j appl dent sci. 2016; 2(2): 24–7. 20. abe j, iwaki k, sudo t, kokubo k. mandibular suction-effective denture “the professional”: clinical and laboratory technique for class i/ii/iii with aesthetics. tokyo: quintessence publishing; 2019. p. 188. 21. rubel b, hill ee. intraoral gothic arch tracing. n y state dent j. 2011; 77(5): 40–3. 22. hayakawa i. principles and practices of complete dentures: creating the mental image of a denture. tokyo: quintessence publishing; 1999. p. 255. 23. zarb ga, hobkirk j, eckert s, jacob r. prosthodontic treatment for edentulous patients: complete dentures and implant-supported prostheses. 13th ed. st. louis: elsevier health sciences; 2012. p. 466. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p179–185 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p179-185 6161 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 61–65 research report topical application of snail mucin gel enhances the number of osteoblasts in periodontitis rat model h. hendrawati, hanindya noor agustha, and rezmelia sari department of periodontics faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: repair of bone damage represents a fundamental issue in the treatment of periodontitis. the important indicator employed to monitor the bone damage repair process is the number of osteoblast cells. achatina fulica snail mucin (sm) contains glycosaminoglycans which have the potential to increase their number. however, the use of sm in dentistry remains limited. purpose: to determine and prove the effect of sm gel in increasing the number of osteoblasts in rat models suffering from periodontitis. methods: this study used 36 rat models divided into three groups, namely; a treatment group (t: 20% snail mucin gel, n = 12), a positive-control group (p: hyaluronic acid gel, n = 12) and a negative-control group (n: cmc-na gel, n = 12). 0.2 ml of all material was applied to a pocket by means of a tuberculin syringe once a day for 14 days. histologic observations using haematoxylin-eosin staining were carried out on days 3, 5, 7 and 14. data was analyzed by two-way anova followed by a post-hoc lsd. results: a significant difference existed between the number of osteoblasts in the test groups. the highest number of osteoblasts observed was consistently that in the treatment group. conclusion: the application of 20% snail mucin gel was effective in enhancing the number of osteoblasts in rats suffering from periodontitis. keywords: bone repair; osteoblast; periodontitis; snail mucin (achatina fulica) correspondence: rezmelia sari, department of periodontics, faculty of dentistry, universitas gadjah mada, jl. denta no. 1, sekip utara, yogyakarta 55281, indonesia. email: rezmelia_sari@mail.ugm.ac.id introduction periodontal disease constitutes an inflammation affecting the tissues surrounding the teeth, i.e. the gingiva, periodontal ligament, cementum and alveolar bone.1 research conducted in 497 districts/cities across indonesia has indicated that the prevalence of periodontal disease is one of 95.21%.2 this situation pertains as the result of persistent infection and inflammatory responses to periodontal pathogens which subsequently cause progressive changes in and damage to the gingiva, periodontal ligaments and alveolar bone around the teethresulting in tooth mobility.1,3 bone healing represents an important objective of the treatment of periodontal disease. the healing process in alveolar bone consists of three phases, namely: inflammation, proliferation and bone remodeling. osteoblasts, as one indicator of bone healing, play a role in the remodeling phase by secreting osteoid (bone matrix). at the outset of bone formation, osteoblasts synthesize basic substances and collagen which undergo a polymerization process resulting in the formation of collagen and tissue fibers and, subsequently, osteoid which mineralizes to become bone.4 one common therapy in the treatment of periodontitis is the topical application of antimicrobial therapy and hyaluronic acid.5,6 hyaluronic acid (ha) can weaken the bonds of chronic inflammatory tissue cells with the result that they are readily released and replaced through the regeneration of healthy cells. it also produces antimicrobial effects on aggregatibacter actinomycetemcomitans, provotela oris, porphyromonas gingivalis and staphylococcus aureus.7 the availability of snails in indonesia has long promoted the use of snail mucin as a traditional medicine by groups of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p61–65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p61-65 62 hendrawati, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 61–65 herbal sellers from solo to manage tooth-related infections by dripping it onto perforated teeth. snail mucin contains glycosaminoglycans consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid that play many important roles within biological systems.8 during bone regeneration, ha induces the stages of osteogenesis as a continuous extracellular matrix component.9 the interaction between heparane sulfate and bone morphogenetic protein (bmp) antagonists will inhibit the activity of inhibitors and potentiate bmp activity during bone healing.10 heparin functions in an almost identical manner to heparane sulfate in that it affects the activity of bmp.11 interestingly, snail mucin also contains antibacterial achasin. the study described below was undertaken to determine and prove the effect of snail mucin gel in increasing the number of osteoblasts in rat models suffering from periodontitis. materials and methods characteristic of test groups: the experimental protocol of this study was approved by the research ethics committee, faculty of dentistry, universitas gadjah mada (ugm) no. 001279/kkep/fkg-ugm/ec/2018. the research was conducted over two months between january and march 2018 and involved 36 periodontitis rat models aged 2.5-3 months which were fed pellets and mineral water on a regular basis. induction of periodontitis was effected by injecting 0.2 ml of aggregatibacter actinomycetemcomitans bacteria intragingivally. bacteria were injected into the labial mandibular incisors for seven consecutive days. the clinical signs of periodontitis in rats were observed as redness and enlargement of the gingiva and apically positioning of the gingival margin (gingival recession). the test population was divided into three groups: a treatment group (t: 20% snail mucin gel, n = 12), a positive-control group (p: hyaluronic acid gel/gengigel®, n = 12) and a negative-control group (n: cmc-na gel, n = 12). snail mucin processing: the snails (achatina fulica) identified at the animal systematics laboratory, faculty of biology, ugm were 5-10 cms in length and weighed 33 grams. mucin gel was obtained from 20 subjects by means of a looped ligature being inserted in and pulled through their bodies. the gel obtained in this manner was collected in a glass beaker, its volume being measured before processing. snail mucin preparation: snail mucin gel was made with 2% cmc-na (2 grams of cmc-na dissolved in 100ml of distilled water). a concentration of 20% was obtained by mixing 20ml of snail mucin with up to 100 ml of 2% cmcna and stirred for ten minutes before being transferred to a container and cooled to produce a gel subsequently sterilized with uv light. the application of gel: 0.2 ml of the materials (20% snail mucin gel, hyaluronic acid/gengigel®, cmc-na gel) was topically applied to inflamed gingiva using a tuberculin syringe. the frequency of application was once daily for 14 days. observation and data analysis: the sample was stained with haematoxylin-eosin. the number of osteoblasts was observed on days 3, 5, 7 and 14 in five visual fields by two observers using a calibrated light microscope at 400x magnification. the normality of data was established by a shapiro-wilk test, while normality was evaluated by means of a levene’s test. all data was normally distributed and homogenous. a two-way anova test was subsequently conducted to determine whether the test material, observation time or interaction between test material and observation time had any effect on the number of alveolar bone osteoblasts. the difference in the number of osteoblasts on days 3, 5, 7 and 14 was analyzed with a post hoc lsd test. results the results of histological observation in the treatment (t), positive control (p) and negative control (n) groups on days 3 (a), 5 (b), 7 (c) and 14 (d) can be observed in figure 1. the number of osteoblasts in the study groups shown in table 1 indicate that it increased between days 3 and 14. at all observation points, the highest number of osteoblasts was found in the treatment group. the lowest average number of osteoblasts was on day 3, while the highest was on day 14. the results of an anova test indicated statistically significant differences between tested groups (p=0.000). the post hoc test confirmed that the highest number of osteoblasts was found in the treatment group followed by the positive control group. discussion osteoblast as a crucial indicator of bone healing is expressed during the embryogenesis, remodelling and the bone healing process.4 morphologically, osteoblasts possess special characteristics in that they are cuboidal in shape and located at the interface of newly synthesized bone and strongly basophilic.12 on the initiation of bone formation, osteoblasts synthesize basic substances and collagen which undergo polymerization which forms collagen fibers and tissue and, subsequently, osteoid. osteoid constitutes a non-calcified matrix which does not yet contain minerals but which, shortly after deposition, is mineralized to become bone.4 therefore, the higher the number of osteoblasts present, the more rapid the bone healing process will be. in this study, the results indicate that the number of osteoblasts increased during the observation period. this may, potentially, be due to the proliferation phase having started on day 3 when osteoblasts became active in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p61–65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p61-65 63hendrawati, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 61–65 t.c 7 p.c 7 n.c t.d p.d n.d 4 t.a n.a p.a n.b p.b t.b figure 1. osteoblasts of treatment group (t), positive control group (p), and negative control group (n) on day 3 (a), day 5 (b), day 7 (c), and day 14 (d). osteoblasts are marked by white arrows. picture taken at 400x magnification. table 1. the number of osteoblast (mean and standard deviation) in the treatment (t), positive control (p), and negative control (n) groups time (day) the number of osteoblasts (x ± sd) p (intergroup) t p n p (intragroup) t-p t-n p-n 3 8.60±0.40 6.80±0.53 4.20±0.20 0.000* 0.000* 0.000* 0.000* 5 10.33±0.42 8.73±0.46 5.00±0.35 0.000* 0.000* 0.000* 0.000* 7 11.20±0.40 9.67±0.31 7.13± 0.58 0.000* 0.000* 0.000* 0.000* 14 12.13±0.31 11.20± 0.20 9.13±0.64 0.000* 0.000* 0.000* 0.000* *statistically significant (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p61–65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p61-65 64 hendrawati, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 61–65 bone formation process,13 reaching its peak on day 14 as indicated by an increase in collagen and extracellular matrix deposition.4 the proliferative or granulation phase does not occur at a distinct time but, rather, continuously in the background. from day 5 to day 7, the fibroblasts started to lay down new collagen and glycosaminoglycans. these proteoglycans form the core of the wound helping to stabilize it. this phase lasts until the third week.14 pdgf and tgf-β will help accelerate bone tissue formation by stimulating mitogenic activity and through the differentiation of osteoblasts and periodontal ligament fibroblasts.15 polypeptides of the tgf-β family are crucial to controlling cell activity and metabolism during ontogenic development in humans. these tgf-β family attributes are established during the bone healing process and considered to recapitulate embryonic intra-cartilaginous ossification.16 in addition, pdgf and tgf-β will stimulate bone matrix deposition by osteoblasts. when detecting wounds, the body will automatically release bone morphogenetic proteins (bmps). disruption of this signaling will affect various skeletal and extra-skeletal anomalies.15 the highest number of osteoblasts was found both in the treatment and positive control groups. this contrast may have been due to the hyaluronic acid-containing gel contained in both these two groups. ha plays an important role in various biological cycles, including wound healing, chondrogenesis, immune response, cell migration17,18 and osteogenesis.9 its oeteoinductive activity promotes growth factors such as bone morphogenetic proteins.19 interestingly, the number of osteoblasts in the treatment group was higher than positive-contol group. this might be due to other active substances contained in snail mucin gel such as glycosaminoglycans and glycoprotein. glycosaminoglycans contain hyaluronic acid, heparan sulfate, heparin, chondroitin sulfate and hyaluronan sulfate, while glycoproteins contain achasin.8,20 heparan sulfate (hs) is a membrane-bound proteoglycan featuring two main structures, namely; core protein and highly sulfated glycosaminoglycan side chains of d-glucuronic acid-n-acetyl-d-glucosamine repeats.10,21 heparin sulfate is a receptor for many substances such as coagulation enzymes, molecular adhesions, cytokines and proteases. this causes the heparane sulfate to execute a wide range of functions from supporting simple mechanical activities to supporting more complex processes such as cell proliferation and differentiation.22 in the process of bone formation, heparane sulfate works by binding to bone morphogenetic protein (bmp). bmp ligands (e.g. bmp2, bmp4, bmp7) and their antagonist (noggin) can bind to heparane sulfate because of their negatively charged side chains structure. these ligands and antagonists possess anti-and pro-osteogenic properties in bone. the process of binding hs with bmp and its antagonists can occur in one of two ways. the first is through restricted diffusion, during which bmp is transported from cell to cell by heparin sulfate. the second is heparin binding to the bmp antagonist to cause the inverse function of bmp. increased bmp activity during bone healing will occur because these interactions can block the activity of inhibitors.10 another snail mucin gel with the same structure and function as heparane sulfate is heparin. heparin represents a glycosaminoglycan with a high sulfate content and extremely negatively charged molecules whose function is to influence bmp activity thereby affecting the bone formation process. heparin can be found on the cell surface and in the extracellular matrix (ecm).11 chondroitin sulfate is a component of sulfated glycosaminoglycans with two molecular structures, namely; n-acetylgalactosamine and glucuronic acid. with this molecular structure chondroitin sulfate can bind with various molecules such as growth factors, cytokines, chemokines, adhesion molecules and lipoproteins. the existence of these bonds supports the important role of chondroitin sulfate in cell growth, nerve development and tissue integration which supports anti-inflammatory activity and promotes the absorption of nutrients to cells.23,24 previous research has shown that chondroitin sulfate and sulfated hyaluronan can inhibit sclerostin and support bone regeneration in diabetic rats.25 based on the results of a paper disc diffusion test, achasin could be seen to act as an antimicrobial against aggregatibacter actinomycetemcomitans and streptococcus mutans bacteria.26 the benefits of the antimicrobial properties of achasin were observable on day 5 when pus was detected in the negative control group, but not in either the treatment or positive control group. cmc-na present in the negative control group produced no antibacterial effect.27 previous research has also demonstrated that snail mucus promotes an increase in the density of collagen fibers during the gingival wound healing process due to the presence of glycosaminoglycans and glycoproteins.28 this study also confirmed that the topical application of 20% snail mucin gel induced osteoblast proliferation more rapidly than in other groups. the number of osteoblasts in the positive-control group on days 5, 7 and 14 was similar to that in the snail mucin gel on days 3, 5 and 7. furthermore, the number of osteoblasts in the negative-control groups on day 14 was similar to that in snail mucus gel on day 3. within the limitations of this study, it is suggested that the topical application of 20% snail mucin (achatina fulica) gel can increase the number of osteoblasts in a rat models with periodontitis. however, additional histological and clinical research is required to establish the role of this gel in bone regeneration in order that this gel can be used as an adjunctive therapy for periodontitis. references 1. lamster ib, pagan m. periodontal disease and the metabolic syndrome. int dent j. 2017; 67(2): 67–77. 2. notohartojo it, sihombing m. faktor risiko pada penyakit jaringan periodontal gigi di indonesia (riskesdas 2013). bul penelit sist kesehat. 2015; 18(1): 87–94. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p61–65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p61-65 65hendrawati, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 61–65 3. kayal ra. the role of osteoimmunology in periodontal disease. biomed res int. 2013; 2013: 1–12. 4. mescher al. junqueira’s basic histology: text and atlas. 14th ed. usa: mcgraw-hill; 2015. p. 560. 5. hatipoglu m, aytekin z, daltaban o, felek r, firat mz, ustun k. the effect of diode laser as an adjunct to periodontal treatment on clinical periodontal parameters and halitosis: a randomized controlled clinical trial. cumhur dent j. 2017; 20(3): 152–60. 6. yellanki sk, singh j, manvi f v. formulation, characterization and evaluation of metronidazole gel for local treatment of periodontitis. int j pharma bio sci. 2010; 1(2): 1–9. 7. gupta r, pandit n, makkar a. role of professionally applied 0.8% hyaluronic acid gel in managing inf lammation in periodontal disease. baba farid univ dent j. 2011; 2(2): 117–20. 8. park y, zhang z, laremore tn, li b, sim j-s, im a-r, ahn my, kim ys, linhardt rj. variation of acharan sulfate and monosaccharide composition and analysis of neutral n-glycans in african giant snail (achatina fulica). glycoconj j. 2008; 25(9): 863–77. 9. patterson j, siew r, herring sw, lin asp, guldberg r, stayton ps. hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration. biomaterials. 2010; 31(26): 6772–81. 10. gdalevitch m, kasaai b, alam n, dohin b, lauzier d, hamdy rc. the effect of heparan sulfate application on bone formation during distraction osteogenesis. plos one. 2013; 8(2): 1–9. 11. brkljacic j, pauk m, erjavec i, cipcic a, grgurevic l, zadro r, inman gj, vukicevic s. exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity. int orthop. 2013; 37(3): 529–41. 12. rutkovskiy a, stensløkken k-o, vaage ij. osteoblast differentiation at a glance. med sci monit basic res. 2016; 22: 95–106. 13. sintessa s, soemarko hm, suprapti l, hernawan i. hambatan prostaglandin pada pemberian oains dan non-oains pasca pemakaian alat ortodontik. j exp life sci. 2013; 3(2): 65–75. 14. ninan n, thomas s, grohens y. wound healing in urology. adv drug deliv rev. 2015; 82–83: 93–105. 15. beederman m, lamplot jd, nan g, wang j, liu x, yin l, li r, shui w, zhang h, kim sh, zhang w, zhang j, kong y, denduluri s, rogers mr, pratt a, haydon rc, luu hh, angeles j, shi ll, he t-c. bmp signaling in mesenchymal stem cell differentiation and bone formation. j biomed sci eng. 2013; 06(08): 32–52. 16. pon iatowsk i ła, wojd a siewicz p, ga si k r , sz u k iewicz d. transforming growth factor beta family: insight into the role of growth factors in regulation of fracture healing biology and potential clinical applications. mediators inflamm. 2015; 2015: 1–17. 17. bertl k, bruckmann c, isberg p-e, klinge b, gotfredsen k, stavropoulos a. hyaluronan in non-surgical and surgical periodontal therapy: a systematic review. j clin periodontol. 2015; 42(3): 236–46. 18. de brito bezerra b, mendes brazão ma, de campos mlg, casati mz, sallum ea, sallum aw. association of hyaluronic acid with a collagen scaffold may improve bone healing in critical-size bone defects. clin oral implants res. 2012; 23(8): 938–42. 19. arpağ of, damlar i, altan a, tatli u, günay a. to what extent does hyaluronic acid affect healing of xenografts? a histomorphometric study in a rabbit model. j appl oral sci. 2018; 26: 1–8. 20. hardhani pr, lastianny sp, herawati d. pengaruh penambahan platelet-rich plasma pada cangkok tulang terhadap kadar osteocalcin cairan sulkus gingiva pada terapi poket infraboni. j pdgi. 2013; 62(3): 75–82. 21. chiodelli p, bugatti a, urbinati c, rusnati m. heparin/heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use. molecules. 2015; 20(4): 6342–88. 22. mansouri r, jouan y, hay e, blin-wakkach c, frain m, ostertag a, le henaff c, marty c, geoffroy v, marie pj, cohen-solal m, modrowski d. osteoblastic heparan sulfate glycosaminoglycans control bone remodeling by regulating wnt signaling and the crosstalk between bone surface and marrow cells. cell death dis. 2017; 8(6): 1–10. 23. haylock-jacobs s, keough mb, lau l, yong vw. chondroitin sulphate proteoglycans: extracellular matrix proteins that regulate immunity of the central nervous system. autoimmun rev. 2011; 10(12): 766–72. 24. salbach j, rachner td, rauner m, hempel u, anderegg u, franz s, simon j-c, hof bauer lc. regenerative potential of glycosaminoglycans for skin and bone. j mol med. 2012; 90(6): 625–35. 25. picke a-k, salbach-hirsch j, hintze v, rother s, rauner m, kascholke c, möller s, bernhardt r, rammelt s, pisabarro mt, ruiz-gómez g, schnabelrauch m, schulz-siegmund m, hacker mc, scharnweber d, hofbauer c, hofbauer lc. sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function. biomaterials. 2016; 96: 11–23. 26. mafranenda hd, kriswandini il, arijani re. antimicrobial proteins of snail mucus (achatina fulica) against streptococcus mutans and aggregatibacter actinomycetemcomitans. dent j (majalah kedokt gigi). 2014; 47(1): 31–6. 27. syafril dsn, astuti iy, suparman s. uji sifat fisis gel antiacne ekstrak daun gambir (uncaria gambir roxb) dalam basis na cmc dan uji aktivitas antibakteri terhadap staphylococcus aureus. pharm j farm indones (pharmaceutical j indones. 2012; 9(2): 118–27. 28. rahmatillah gata. pengaruh aplikasi gel lendir bekicot (achatina fulica) 20% terhadap kepadatan serabut kolagen pada proses penyembuhan luka gingiva (kajian pada rattus norvegicus). thesis. yogyakarta: universitas gadjah mada; 2016. p. 1–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p61–65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p61-65 30 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 30–35 research report correlation of the vertical dimension of occlusion with five distances between facial landmarks among those of batak toba ethnicity rehulina ginting and debora lovelisa hinson simbolon departement of oral biology, faculty of dentistry, universitas sumatera utara, medan – indonesia abstract background: the normal vertical dimension of occlusion (vdo) results in orofacial and temporomandibular joint (tmj) biomechanical balance. if the vdo changes due to attrition, full edentulism, accidents involving the lower third of the face and even improper denture manufacturing will result in the disruption of mastication, speech and aesthetic functions. therefore, the right technique is needed to predict the correct vdo. purpose: to identify the correlation values and regression equation of the vdo for five distances between facial landmarks among people of batak toba ethnicity. methods: this research is an analytical study with a cross-sectional design. a purposive-sampling technique obtained 30 batak toba subjects, consisting of 15 males and 15 females aged 19–24 years. the data were analysed by an independent t-test, one-way anova, the pearson correlation, and linear regression (p<0.05). results: a significant difference distance in the vdo (p=0.0001, p<0.05) was observed between male subjects (72.96±3.75mm) and female subjects (65.24±5.12mm). a positive and significant correlation was observed between the vdo distance and the facial landmark distances, where the criteria for significant correlation were the ro–pu distance being {r male=0.723(p=0.02) and female=0.650(p=0.09)} and the oc– ro distance being {r male=0.689(p=0.004) and female=0.615(p=0.015)}; the moderate correlation criteria were the oc–ic distance being {r male=0.476(p=0.045) and female=0.428(p=0.043)}, the e–e being {r male=0.435(p=0.043) and female=0.458(p=0.047)}, and the eh being {r male=0.398(p=0.051) and female=0.414(p=0.051)}. the regression equation for the vdo distance in males is {[22.694 + 0.673 (ro–pu)], [24.371 + 0.642 (oc–ro} and in females is {[23.017 + 0.616 (ro–pu)], [21.795 + 0.632 (oc–ro)]}. conclusion: the distances of ro–pu and oc–ro have the strongest correlation with the vdo in people of batak toba ethnicity. keywords: correlation value; facial landmark distance; vertical dimension of occlusion correspondence: debora lovelisa hinson simbolon, department of oral biology, universitas sumatera utara, jl. alumni no. 2 medan 20155, indonesia. e-mail: debora.lovelisa@gmail.com introduction the 2017 glossary of prosthodontic terms defines the vertical dimension of occlusion (vdo) as the vertical distance measured between two anatomical points, one on the maxilla and the other on the mandible, when the mouth is closed and the teeth are in maximum intercuspal occlusion.1 theoretically, measurement of the vdo distance can be performed in two ways: measuring the distance between the most prominent point on the nose (the pronasale) to the most prominent point on the chin (the gnathion) and measuring the distance between the base of the nose (the subnasale) to the base of the mandible (the menton). in this study, the vdo measurement method is the distance between the pronasale and the gnathion. the vdo distance is affected by ethnicity due to the genetics involved in jaw and bone growth, eruption and occlusion of the teeth and the position of the temporomandibular joint (tmj), which provides a good balance between orofacial biomechanics and the tmj for mastication, phonetic and aesthetic functions.2–4 in cases of severe dental attrition, full edentulism and accidents involving the lower third of the face, there can be a loss of the normal vdo distance, meaning that it is necessary dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 mailto:debora.lovelisa@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 31ginting, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 30–35 to determine the correct vdo to restore the mastication, phonetic and aesthetic functions.2–4 determination of the vdo can be done during pre-extraction or post-extraction. the pre–extraction method can be performed by facial photographs, facial silhouettes, cephalometric radiographs, articulation models or the swenson method, while the postextraction method can be performed by the niswonger method, electromyography, biometrics, the willis method, using the closest speaking space, ingestion, or using finger length or the distance between facial landmarks. in this study, the chosen method for determining the vdo was the one associated with the five distances between facial landmarks.2–7 this method was chosen based on the theory that faces remain relatively unchanged throughout the course of a life, as well as the fact that it is non-invasive, simple, low-risk and inexpensive; it also does not require special tools and does not involve radiation exposure, so it can be recommended for daily practice as a guide for reconstructing the lower third of the face.2, 4 there are 14 known distances between facial landmarks that can be correlated to the vdo distance. the study of majeed et al.3 in saudi arabia showed that only seven landmarks have a correlation to the vdo in both males and females, but in the study of basnet et al.2 it is recommended that only five facial landmarks have a strong correlation to the vdo distance, which are: the distance of the rima oris to the pupil (ro–pu), the outer canthus of the right eye to the inner canthus of left eye (oc–ic), the outer canthus of eye to the rima oris (oc–ro), the outer canthus eye to the external auditory meatus ear (e–e), and the ear length (eh). a regression equation can then be obtained to predict the vdo distance. the study of majeed et al.3 on the population of saudi arabia obtained the following regression equation in men: vdo = [27.07 + 0.655 (ro–pu)], vdo = [20,323 + 0.675 (oc–ro)], vdo = [42.12 + 0.402 (oc–ic)], vdo = [45.31 + 0.354 (e–e)], and the following in women: vdo = [34.91 + 0.347 (ro–pu], vdo = [24.22 + 0.471 (oc–ro)], vdo = [45.63 + 0.207 (oc–ic)], vdo = [39.54 + 0.263 (e–e)], vdo = [42.72 + 0.238 (eh)]. based on the previous description that ethnicity is one of the factors affecting the vdo and the fact that this research has never been done on batak toba people, researchers are interested in conducting studies into the correlation value and conversion of the vdo of five distances between facial landmarks of batak toba people aged 19–24 years. the purpose of this study is to identify the correlation values and regression equation of the vdo to five facial landmarks among those of batak toba ethnicity. materials and methods this is an analytical study with a cross-sectional design. the research was conducted in the laboratory of the department of oral biology, faculty of dentistry, universitas sumatera utara (usu). the hypothesis test formula obtained 30 samples, divided into 15 male and 15 female and consisting of students at the faculty of dentistry, universitas sumatera utara. the sample was then selected using a purposivesampling technique that matches the inclusion criteria. the inclusion criteria were: being aged 19–24 years; being of batak toba ethnicity for two generations; possessing a complete set of 28 teeth, including molar 2–molar 2 in the maxilla and mandible; having a straight face-profile; having an angle’s class i occlusion; and having had no restoration on the incisal and occlusal surfaces. the exclusion criteria were: having attrition of more than ⅓ of the incisal or occlusal surfaces; having a bruxism habit; having large, carious lesions on the occlusal surface; having malocclusion; having tmj abnormalities; having intraoral or extraoral abnormalities; a history of severe trauma, surgery, or facial abnormalities in the area of the eyes, nose, lips or ears; and having used or currently be using orthodontic and prosthodontic treatments. ethical clearance was obtained from the usu medical faculty health research ethics commission (no. 849/tgl/kepk fk usu-rsup ham/2019). clinical examination was performed by looking at the condition of the teeth and the relationship of the molar occlusion. then, in subjects who fit the criteria, measurements were taken with digital callipers of the vdo distance (figure 1) and the five distances between facial landmarks (figure 2). the data were analysed using spss version 20.0 software (student edition, ibm america) and were tested by an independent t-test, one-way anova, the pearson correlation, and linear regression (p<0.05). results the purpose of this study on 30 people of batak toba ethnicity with angle’s class i occlusion between 19–24 years of age was to obtain: the mean value of the vdo and the five distances between facial landmarks, the conversion value, the correlation value and the regression equation of the vdo distance with the five distances between facial landmarks. there was no significant difference in the mean vdo distance and the five distances between facial landmarks between each age group (p>0.05) (table 1), but b a figure 1. the vdo measurement: a. pronasale–gnathion and b. subnasale–menton. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 32 ginting, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 30–35 a b e c d figure 2. the facial landmark measurements: a. ro–pu, b. oc–ic, c. oc–ro, d. e–e, e. eh. table 1. the mean vdo distance and distances between five facial landmarks in those of batak toba ethnicity aged 19–24 years with angle’s class i occlusion character age (years) x ±sd (mm) p-value vdo 19 70.84±8.31 0.451 20 68.61±4.73 21 64.83±6.69 22 69.87±5.67 23 73.52±4.98 24 70.43±7.14 ro–pu 19 71.91±6.71 0.218 20 71.91±4.79 21 68.27±5.55 22 72.15±5.63 23 77.97±2.51 24 67.01±8.39 oc–ic 19 71.67±3.79 0.130 20 70.88±4.72 21 67.25±3.90 22 72.39±4.50 23 75.07±0.34 24 65.77±7.38 oc–ro 19 73.03±7.36 0.352 20 72.45±4.89 21 69.45±5.47 22 72.83±5.39 23 77.45±4.25 24 66.89±8.56 e–e 19 80.17±3.39 0.622 20 76.52±4.79 21 77.34±2.59 22 76.70±7.52 23 79.52±2.01 24 81.09±1.39 eh 19 80.17±3.39 0.622 20 76.52±4.79 21 77.34±2.59 22 76.70±7.52 23 79.52±2.01 24 81.09±1.39 table 2. the mean vdo distance and five distances between facial landmarks in those of batak toba ethnicity aged 19–24 years with angle’s class i occlusion, separated by gender character male x ±sd (mm) female x ±sd (mm) p-value vdo 72.96±3.75 65.24±5.12 0.0001* ro–pu 74.69±4.03 68.54±5.40 0.001* oc–ic 73.39±3.28 68.03±4.48 0.001* oc–ro 75.66±4.02 68.78±4.98 0.0001* e–e 80.13±2.96 75.43±4.76 0.003* eh 59.07±3.24 58.12±5.04 0.545 table 3. pearson’s correlation coefficient between the vdo distance and the distances between five facial landmarks of those of batak toba ethnicity with angle’s class i occlusion, separated by gender character male female pearson’s® p-value pearson’s® p-value ro–pu 0.723 0.002* 0.650 0.009* oc–ic 0.476 0.045* 0.428 0.043* oc–ro 0.689 0.004* 0.615 0.015* e–e 0.435 0.043* 0.458 0.047* eh 0.398 0.051* 0.414 0.050* table 4. conversion values the vdo distance to the five facial landmark distances in the batak toba ethic aged 19-24 years with angle’s class i occlusion age conversion value ro–pu oc–ic oc–ro e–e eh 19-24 69.10±4.93 69.09±4.60 69.10±4.82 69.10±4.58 69.12±4.54 19 69.35±6.33 69.71±4.79 69.63±6.48 70.57±4.66 70.86±4.12 20 69.10±4.47 69.00±4.66 69.09±4.39 68.13±4.87 68.57±4.79 21 66.61±5.28 66.51±4.64 67.01±5.13 68.31±3.89 67.27±4.44 22 69.71±3.95 70.56±4.32 69.80±3.69 69.02±6.57 69.74±4.86 23 73.85±3.97 72.53±2.46 72.99±4.64 70.85±2.48 71.12±3.04 24 66.21±4.93 66.05±7.53 65.73±7.74 69.10±4.58 68.68±8.77 p-value 0.462 0.479 0.579 0.890 0.837 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 33ginting, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 30–35 there were significant differences between the results for males and females (p<0.05), except for the eh distances, which showed no differences based on gender (p>0.05) (table 2). there was significant positive correlation between the vdo distance and the facial landmark distances; the strong correlation criteria (r=0.50–0.75) were the distance of the ro–pu and the oc–ro, while the moderate correlation criteria (r=0.26–0.50) were the distances of the oc–ic, the e–e and the eh (table 3). in this study, there was no significant difference in the conversion value of the vdo distance with the five distances between facial landmarks in each age group 19–24 years (p> 0.05) (table 4), while based on gender there was a significant difference, as in men it was significantly longer than in women (p< 0.05) (table 5). there was a difference in the regression equation of the vdo distance with the five distances between facial landmarks between male and female (table 6). discussion the results of this study, based on 15 men and 15 women of batak toba ethnicity aged 19–24 years with a class i occlusion, show no significant difference in the mean vdo distance and the five distances between facial landmarks between each age group. this might have happened due to the theory of laksmapappa that states that the maximum bone-growth rate will stop at the age of 18 years.8 after reaching the age at which bone growth is complete, the size of the bones, including those in the face, will not change and will remain stable until age 24 (as an estimate) is reached. in addition, the bones in the head and face area, except for the mandible, are connected with connective tissue fibrosis (synarthrosis), meaning that the skull bones do not allow movement and that dead joints can stimulate osteoblasts. this means that when the growth of the head and face bones is complete, there will be no change, or only a minimum amount of shrinkage throughout life.9, 10 farkas also states that the growth of the ear’s length reaches a peak and stops at the age of 15, meaning that between 19 and 24 years of age, the size of the ear does not increase.11 according to khanehzad et al., at the age of 25 and over, the vdo distance has begun to decrease due to attrition of use of the incisal and occlusal surfaces of the teeth, resulting in the shortening of the incisal and occlusal surfaces of the teeth, followed by reduction in the intercuspal distance, which in turn makes the vdo distance shorter.12 in this study, the subjects have angle’s class i occlusion, which is a normal occlusion with maximum intercuspation, meaning that the mandibular condyle is in the centre of the mandibular fossa, where all muscle and innervation functions are expected to work properly; the normal pattern of mandibular bone growth results in a normal lower third of the face.13 this is why there is no difference in the vdo distance and the five distances between facial landmarks between age groups. there were differences in the mean values of the vdo, ro–pu, oc–ic, oc–ro and e–e distances between gender, as for men they are significantly greater than for women. the results of this study are consistent with previous studies that mention that the vdo distance in men is significantly greater than in women.2–4, 16 sex is one of the most important factors in influencing the process of growth and development, especially in the bones, due to the role of sexual hormones.14 according to hauspie, generally, bones in men are bigger than in women. genetically, men are dominated by testosterone, which increases the speed of protein synthesis in the body for the formation of bones’ organic matrix secreted by osteoblasts during the process of bone mineralisation as part of bone growth and bone mass formation more than women, meaning that the bones in men become larger.14,15 on the other hand, women are table 5. conversion values the vdo distance to the five facial landmark distances in the batak toba ethic aged 19-24 years with angle’s class i occlusion based on gender character conversion value p-value male x ±sd (mm) female x ±sd (mm) ro–pu 72.97±2.71 65.24±3.33 0.0001* oc–ic 72.96±2.16 65.23±2.70 0.0001* oc–ro 72.94±2.58 65.26±3.15 0.0001* e–e 72.98±1.98 65.23±2.69 0.0001* eh 72.98±1.90 65.25±2.63 0.0001* table 6. regression equation the vdo distance with the five facial landmark distances in the batak toba ethnic with angle’s class i occlusion based on gender gender v. dependent v. independent regression equation male vdo ro–pu y=22.694+0.673 (ro–pu) oc–ic y=24.593+0.659 (oc–ic) oc–ro y=24.371+0.642 (oc–ro) e–e y=19.373+0.669 (e–e) eh y=38.254+0.588 (eh) female vdo ro–pu y=23.017+0.616 (ro–pu) oc–ic y=24.210+0.603 (oc–ic) oc–ro y=21.795+0.632 (oc–ro) e–e y=22.534+0.566 (e–e) eh y=34.910+0.522 (eh) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 34 ginting, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 30–35 dominated by oestrogen, which is important for menstrual regulation and the reproductive cycle and stimulates the proliferation of breast-glandular epithelial cells; its role in the process of bone growth is that it is responsible for epiphyseal closure, so that bone growth stops faster in women and cause bones in women to be smaller than in men because there is no increase in size due to the closure of the epiphyses. thus, all bones, including facial bones, are smaller in women than in men.13, 16 this causes a significant difference between the distances of the vdo, ro–pu, oc–ic, oc– ro and e–e in men and women. in this study, there was no significant difference in the distance of eh between men and women. the results of this study are consistent with the statement of alexander et al., that the length of the ear is not affected by gender, but is influenced by genetic factors inherited from both parents and ethnicity.11 therefore, the eh distance in this study is no different between male and female because the subjects of this study are mongoloid. there was no significant difference in the conversion value between the vdo distances of the five distances between facial landmarks for each age group, while based on gender there was a significant difference: the distances in men are significantly longer than in women. this result is in accordance with the data above showing that the vdo distance was not significantly different between each age group, but there was a significant difference between men and women, which is known to be related to hormonal factors.16 in this study, the ro–pu and oc–ro distances have a strong correlation with the vdo distance, while the oc–ic, e–e and eh distances have a moderate correlation based on gender. this is supported by leonardo da vinci's theory which states that face height vertically consists of the upper, middle, and lower third faces in the same ratio. the author assumes that the distance of ro-pu and oc-ro is in the middle third of the face and both have the same height in other words that the distance of ro-pu and oc-ro is considered almost close to the same result, then the vdo distance is the bottom third of the face . this statement is appropriate because the proportion of the middle third of the face (ro-pu and oc-ro distance) with the lower third of the face (vdo distance) is the same, therefore the correlation becomes stronger.3, 17-19 the distances of oc–ic, e–e and eh have a moderate correlation with the vdo distance. there is no existing study that explains the reasons why the oc–ic, e–e and eh distances do not have too strong a correlation to the vdo distance. therefore, further research is needed regarding the correlation between the distances of oc–ic, e–e and eh to the vdo distances. however, researchers tend to assume that this is because the oc–ic distance is affected by the varying conditions of people’s eyelid shapes, the e–e distance is affected by facial convexity and the thickness of the soft tissue that protects the zygomatic bones or cheeks, and the eh distance is affected by the varying anatomic shape and position of the earlobe in each individual, so that the correlation with face height is moderate.10 the regression equation is obtained to determine the distance of the vdo using five distances between facial landmarks, but it cannot be used as a standard in determining the vdo distance in the batak toba ethnicity. this is because the number of samples in this study, 15 men and 15 women, is still too small. in addition, this study is limited to those who have two-generation batak toba ethnicity, whereas most researchers set limitations of using research subjects with three or more generations of batak toba ethnicity to obtain a purer ethnicity. this has happened because this research was conducted in a city where many intermarriages have taken place, making it difficult to obtain subjects with pure batak toba ethnicity of three generations or more. therefore, further research needs to be done on the conversion of the vdo distances with five distances between facial landmarks in those of batak toba ethnicity with more samples, based on age, gender, diet and having three or more generations of batak toba ethnicity. in conclusion, the five facial landmark distances have a correlation to the vdo in those with batak toba ethnicity: the ro–pu and oc–ro distances have a strong correlation, while the oc–ic, e–e and eh distances have a moderate correlation. references 1. the glossary of prosthodontic terms. 9th ed. j prosthet dent. 2017; 117(55): e63, e77, e86, e90. 2. basnet bb, singh rk, parajuli pk, shrestha p. cor relation between facial measurements and occlusal vertical dimension: an anthropometric study in two ethnic groups of nepal. int j dent scie res 2014; 2(6): 172-4. 3. majeed mi, haralur sb, khan mf, al-ahmari ma, al-shahrani nf, shaik s. an anthropometric study of cranio-facial measurements and their correlation with vertical dimension of occlusion among saudi arabian subpopulations. open access maced j med sci 2018; 6(4): 680-6. 4. alhajj mn, khalifa n, amran a. eye-rima oris distance and its relation to the vertical dimension of occlusion measured by two methods: anthropometric study in a sample of yememi dental students. european j dent 2016; 10(1): 29-33. 5. prakash v, gupta r. concise prosthodontics. 2nd ed. new delhi: elsevier; 2017;308-27. 6. sarandha dl, hussain z, uthkarsh. textbook of complete denture prosthodontics. new delhi: jaypee brothers; 2007;80-5. 7. veeraiyan dn, ramalingam k, bhat v. textbook of prosthodontics. new delhi: jaypee brother 2003; 19-20, 129-39. 8. marshall sd, caspersen m, hardinger r r, franciscus rg, aquilino sa, southard te. development of the curve of spee. american journal of orthodontics and dentofacial orthopedics 2008;134(3):344-52. 9. moore lf, dalley af. anatomi berorientasi klinis: kepala, leher, saraf-saraf kranial. 5th ed. hartanto h, translator. jakarta: erlangga 2013;2-7,49. 10. snell rs. anatomi klinis berdasarkan sistem. alih bahasa. sugiharto l. jakarta: egc; 2015;286-95. 11. alexander ks, stott dj, sivakumar b, kang n. a morphometric study of the human ear. j plastic recons aest surg 2011; 64: 41-7. 12. khanehzad m, madadi s, tahmasebi f, kazemzadeh s, hassanzadeh g. the correlation between occlusal vertical dimension, length of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 35ginting, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 30–35 the thumb and facial landmarks measurements: an anthropometric student of iranian university students. global j human anatomy physiology res 2018; 4: 1-6. 13. ifwandi, rahmayani l, maylanda a. proporsi tinggi wajah pada relasi molar klas i dan klas ii divisi 2 angle mahasiswa fakultas kedokteran gigi universitas syiah kuala. j syiah kuala dent soc 2016; 1(2): 153-60. 14. enikawati m, soenawan h, suharsini m. panjang maksila dan mandibula pada anak usia 10-16 tahun. j fkg ui 2013: 1-15. 15. entie rs, hastuti tp, triredjeki h. hubungan status gizi dengan perkembangan anak usia 1 sampai 5 tahun di kelurahan tidar utara kota magelang. j kep soedirman 2017; 12(1): 27-37. 16. ladda r, bhandari aj, kasat vo, angadi gs. a new technique to determine vertical dimension of occlusion from anthropometric measurements of fingers. indian j dent res 2013; 24(3): 316-20. 17. bhalajhi si. orthodontics the art and sciences. 7th ed. new delhi: arya; 2004;168. 18. bajunaid so, baras b, alhathlol n, ghamdi aa. evaluating the reliability of facial and hand measurements in determining the vertical dimension of occlusion. int j med pharm 2017; 5(1): 1-11. 19. nagpal a, parkash h, bhargava a, chittaranjan b. reliability of different facial measurements for determination of vertical dimension of occlusion in edentulous using accepted facial dimensions recorded from dentulous subjects. j indian pros soc 2014; 14(3): 233-42. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p30–35 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p30-35 vol 49 no 1 jan-mrt 2016.indd 4343 research report effect of mangosteen peel extract combined with demineralized freezed-dried bovine bone xenograft on osteoblast and osteoclast formation in post tooth extraction socket utari kresnoadi, yurike hadisoesanto, and harly prabowo department of prosthodontics faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: tooth extraction, a common procedure in dentistry, can cause bone resorption during socket healing. therefore, it is important to perform socket preservation procedure to maintain alveolar bone. providing a combination of mangosteen peel extract with demineralized freezed-dried bovine bone xenograft (dfdbbx) in tooth extraction socket was expected to accelerate alveol bone formation. purpose: this study aims to determine the effect of mangosteen peel extract combined with dfdbbx introduced into the socket of post tooth extraction on the formation of osteoblasts and osteoclasts. method: twenty-eight (28) cavia cobayas were divided into four groups. extraction to the lower left incisor of cavia cobaya was performed. the extraction socket was filled with 25 gram of peg (group i) as a control, active materials consisted of mangosteen peel extract and dfdbbx 0.5% (group ii), active materials consisted of mangosteen peel extract and dfdbbx 1% (group iii), and active materials consisted of mangosteen peel extract and dfdbbx 2% (group iv). after thirty days, those cavia cobayas were sacrificed. by using he on histopatological examination, the number of osteoblasts and osteoclasts were measured by light microscope with 400 times of magnification. the statistical analysis was then performed using oneway anova & tukeyhsd test. result: the component active materials consisted of mangosteen peel extract and dfdbbx 2% had the most significant results related to the formation of osteoblasts and osteoclasts. conclusion: mangosteen peel extract combined with dfdbbx can increase osteoblasts and decrease osteoclasts in the socket of tooth extraction in cavia cobaya. the combination of mangosteen peel extract and dfdbbx 2% is the most effective material in increasing osteoblast and decreasing osteoclast. keywords: dfdbbx; mangosteen peel extract; osteoblast; osteoclast correspondence: utari kresnoadi, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ut.kres@yahoo.com introduction tooth extraction is a common procedure performed in the field of dentistry. tooth extraction followed socket healing process usually leads to alveolar bone deformities, including the reduction of residual ridge height and width. poor bone healing pattern will then cause problems for dentists, especially related to aesthetic problems in the manufacture of dental implants or conventional prostheses as well as in the placement of dental implants.1 therefore, it is important to perform socket preservation procedures to maintain alveolar bone. one of socket preservation procedures is to put a bone graft in the socket immediately after tooth extraction.2 one of bone graft materials used is demineralized freezeddried bovine bone xenograft (dfdbbx). dfdbbx are often used due to their osteoconductive inorganic matrix components, which serve to provide scaffold for bone regeneration without getting involved in bone formation dental journal (majalah kedokteran gigi) 2016 march; 49(1): 44–49 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48 44 kresnoadi, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 43–48 itself. however, the bone graft healing process is a complex process involving many factors, such as bone metabolism, hormonal balance changes, and external factors for a long time.3 thus, an innovative material is needed to induce osteogenesis activity in order to accelerate bone formation. in the current technological development, a lot of various natural materials has been used to assist or accelerate the wound healing process. one of them is mangosteen (garcinia mangostana l.). the results of previous researches showed that mangosteen is rich of nutrients, namely xanton that are abundant in the peel. the functions of xanton in human body are as anti-oxidant, antiproliferation/anti-cancer, anti-inflammatory, anti-microbe, anti-histamine, anti-fungus, heart disease treatment, hiv treatment, and sugar blood-lowering.4 in the previous research on toxicity test of mangosteen peel extract, mangosteen extract at certain concentration, from 200 μg/ ml to 800 μg/ml, is not toxic to gingival cell cultures of human fibroblasts.5 during tooth extraction, trauma may occur which will lead to inflammation. inflammation will cause the infiltration of inflammatory cells, including macrophages, to the traumatized area. as a result, it will activate proinflammatory cytokines, such as il-1, tnf-α, and pge. il-1 and tnf-α in this case can trigger production of rankl and binding of rankl in rank receptor on the surface of the pre-osteoblasts that can lead to activation of nf-kb triggering the formation of osteoclasts, while pge plays a role in vascular changes, and when injected into bone surfaces, it can cause bone resorption.6,7 therefore, this research aims to determine the effect of the combination of mangosteen peel extract and dfdbbx on the number of osteoblasts and osteoclasts in tooth extraction sockets. materials and methods this research is an experimental research with randomized factorial design (true eksperimental design). this research also has already passed eligible ethics from the ethics committee of faculty of dental medicine, universitas airlangga with number 44/kkepk.fkg/iv/ 2015. subjects in this research were male cavia cobayas weighed 300-350 g and aged 3-3.5 months. those animals were healthy and active, had normal appetite, body temperature and five senses, and suffered no injuries in the limbs and skin as well as no deformed limbs or limp, as well as normal body temperature. those animals were obtained from the test animal unit of biochemistry laboratory, faculty of medicine, universitas airlangga. materials used were mangosteen peel extract, distilled sterile, dfdbbx produced by batan with a size of 10 mesh/2000 microns, polyethilen glycol suspension (peg) 400 and 4000, 100 mg of ketamine, blue nylon 5-0 non cutting sewing thread, sterile cotton, alcohol absolute 99%, 95%, 90%, 80%, and 70%, reagent for haemotoxilin eosin (he) staining, formalin buffer 10%, paraffin solution, ethanol 96%, xylol, ethilen diamine tetra acetate (edta), and he dye. the research was conducted in several places, namely biology laboratory in faculty of science and technology for identifying mangosteen peel; laboratory of research and industry consultation in research & industry consultation unit, surabaya for manufacturing mangosteen peel extract; chemical analysis laboratory of pharmacy faculty, universitas airlangga for mixing mangosteen peel extract, dfdbbx, and peg; biochemistry laboratory of faculty of medicine, universitas airlangga for preparing and treating cavia cobaya; anatomical pathology laboratory of hospital dr. soetomo for preparing preparations; and biochemistry and biomolecular engineering laboratory of faculty of medicine, universitas brawijaya for observing and measuring the number of osteoblasts and osteoclasts. twenty-eight (28) cavia cobaya were divided into four groups, each of which consisted of seven animals. in group i, the lower left incisors of those cavia cobaya were extracted and then given with 25 grams of peg into the socket (control). in group ii, the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx + 99 grams of peg (at the active substance concentration of 0.5%). in group iii, the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx + 49 grams of peg (at the active substance concentration of 1%). in group iv, the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx + 24 grams of peg (at the active substance concentration of 2%).8 after 30 days of treatment, those cavia cobayas were sacrificed, and then their mandible was cut to be decalcified with edta for 30 days. manufacture of paraffin blocks was performed and cut with a rotary microtome with a thickness of 4 microns, and then deparaffinized by dissolving into xylol for 2 x 3 minutes. residual xylol was washed with absolute alcohol, 99%, 95%, 90%, 80%, and 70% respectively for 2 x 1 minutes. residual alcohol was washed with running water. afterward, he staining was performed for 30 seconds, and then rinsed with water. staining with eosin was then conducted for 1-2 minutes, and washed with alcohol 70%, 80%, 90%, 95%, 99%, and absolute for 2 x 1 minutes. meanwhile, xylol was washed for 2 x 2 minutes, and then glass cover previously dripped with canada balsam covered them. osteoblast cells, round cells located on the edge of trabecular bone, and osteoclasts, large cells with multiple nuclei, were observed. the number of osteoblasts and osteoclasts was measured using a light microscope with 400 times of magnification.9 statistical test was performed on the data obtained using kolmogorov-smirnov test. to know the difference between the groups, oneway anova test was conducted, followed with multifactorial comparison test using tukey hsd test after levene homogeneity statistic test was previously performed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48doi: 10.20473/j.djmkg.v48.i4.p177-182 4545kresnoadi, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 43–48 results the calculation results of averages and standard deviations of the number of osteoblasts and osteoclasts in each treatment group and the control group can be seen in figure 1. the figure shows that there were differences in the averages of the number of osteoblasts in the extraction sockets after the treatments with the extract of mangosteen peel and dfdbbx with various concentrations of active ingredient (0.5%/1%/2%). the highest average of the number of osteoblasts was found in the fourth group at the concentration of 2%, while the lowest average of the number of osteoblasts was found in the control group. on the other hand, there were differences in the averages of the number of osteoclasts in the extraction sockets after the treatments with the extract of mangosteen peel and dfdbbx with various concentrations of active ingredient (0.5%/1%/2%). the highest average of the number of osteoclasts was found in the control group, while the lowest average of the number of osteoclasts was found in the fourth group at the concentration of 2%. before the analysis of the test results conducted on the groups, normality test was conducted on each group using kolmogorov-smirnov test. in this research, all the research groups had p value greater than 0.05, which means that data from all the research groups had normal distribution. based on the results of homogeneity test, all the research groups had significance value greater than 0.05. it indicates that all the groups had the same variance (homogeneous). with the prerequisite of normal and homogeneous distribution, oneway anova test was then conducted to see the significance among the groups. based on the results of the one way table 2. the results of tukey hsd test on the number of osteoblasts and osteoclasts in each treatment group osteoblasts group 2 group 3 group 4 group1 0.022* 0.000* 0.000* group 2 0.078 0.000* group 3 0.000* osteoclasts group 1 0.000* 0.000* 0.000* group 2 0.002* 0.000* group 3 0.000* * = there is a significant difference (p<0.05) note: group i: the lower left incisors of those cavia cobaya were extracted and then given with 25 grams of peg into the socket; group ii: the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx +99 g peg (at the active substance concentration of 0.5%); group iii: the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx +49g peg (at the active substance concentration of 1%); group iv:the lower left incisors of those cavia cobaya were extracted and then given with mangosteen peel extract and dfdbbx + 24 gpeg (at the active substance concentration of 2%). control 0.5% dose 1% dose 2% dose osteoblast osteocla figure 1. the graphs display the averages and standard deviations of the number of osteoblasts and osteoclasts after 30 days of the treatment. y-axis shows the averages of the number of osteoblasts and osteoclasts. x-axis shows the sockets filled with peg (the control group), the sockets filled with a dose of 0.5%, the sockets filled with a dose of 1% dose, and the sockets filled with a dose of 2%. osteoclast a ve ra ge dose dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48 46 kresnoadi, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 43–48 anova test, the significance value obtained was 0.000, smaller than α = 0.05. it means that there was a significant difference in the number of osteoblasts and osteoclasts between the control group and the treatment groups. the results of tuckey hsd test showed that there was a significant difference in the number of osteoblasts and osteoclasts between the control group and the treatment groups i, ii, iii and iv with significance p = <0.05. but, there was no significant difference between group ii and group iii with p = 0.78. the microscopic description of osteoblast cells can be seen in figure 2. the microscopic description of osteoclast cells can be seen in figure 3. a c b d figure 2. the histological examination in identifying osteoblast cells (he staining using light microscope with a magnification of 400x). control and treatment groups. (a) control group; (b) active substance group with a concentration of 0.5%; (c) active substance group with a concentration of 1%; (d) active substance group with a concentration of active substance group with a concentration of 2%. a (a) c b (b) d figure 3. the histological examination in identifying osteoclast cells (he staining using light microscope with a magnifi cation of 400x). control and treatment groups. (a) control group; (b) active substance group with a concentration of 0.5%; (c) active substance group with a concentration of 1%; (d) active substance group with a concentration of active substance group with a concentration of 2%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48 4747kresnoadi, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 43–48 discussion based on the results of the data analysis, it is known that the number of osteoblasts increased, while the number of osteoblasts declined significantly between the control group and the treatment groups at the concentrations of 0.5%, 1%, and 2%. it indicates that the combination of mangosteen peel extract, dfdbbx, and peg has significant effect on the formation of osteoblasts and osteoclasts in the extraction socket since the content of mangosteen peel extract plays a role as graft material. mangosteen peel contains at least about 40 kinds of xanton, such as mangostin, mangostenol, mangostinon a, mangostenon b, trapezifolixanthone, tovophyllin b, α-mangostin, β-mangostin, garcinon b, mangostanol, flavonoid epicatechin, epicatechin, garciniafuran, mangoxanthone, and gartanin. among those kinds of xanton, α-mangostin and γ-mangostin are the most useful substance.10 xanton in the human body plays a role as anti-oxidant, anti-proliferation, anti-inflammatory, and anti-microbe. xanton is a powerful antioxidant, which is necessary for balancing the pro-oxidants in the body and the environment, known as free radical.4 γ-mangostin is able to stop inflammation by inhibiting production of cyclooxygenase-2 enzyme (cox-2) causing inflammation, inhibiting activities of ikappa b kinase enzyme directly, preventing the process of gene transcription of cox-2 (the target gene of nf kappab), and then lowering the production of pge-2 during inflammation process.11,12 the administration of mangosteen peel ethanolic extract can reduce infiltration of inflammatory cells and expression of cox-2 in mice induced with periodontitis.13 α and γ-mangostin are bioactive substances that have antiinflammatory effects by inhibiting the production of no and pge-2.14 α and γ-mangostin have anti-inflammatory properties based on the previous research on mice. α and γ-mangostin are also able to decrease lps-induced inflammatory gene, including tnf-α, il-1, il-6, il-8, monocyte chemoattractant protein-1, and toll-like receptor2.15 the use of garcinia mangostana as a medicine for inflammatory diseases is associated with its ability to inhibit the release of no and pge-2, even control tnf-α and il-4 moderately. 15 in this research, mangosteen peel extract was used to lower inflammation caused by tooth extraction trauma by lowering the growth of osteoclasts and increasing the growth of osteoblasts. this is in line with a research conducted by port and martin in 2009 explaining that inflammation will trigger macrophages to induce the synthesis of pro-inflammatory cytokines, il-1 and tnf-α leading to the release of phospholipids of fibroblast cell membranes, mast cells, neutrophils, macrophages, and lymphocytes; as a result, the metabolism of arachidonic acid will be triggered by phospholipase a2 enzyme, and cyclooxygenase (cox-2) enzyme will be produced.6 the increasing of cox-2 can stimulate the synthesis of prostaglandins, especially pge-2, which can lead to the increasing of vasodilation and endothelial permeability, thereby, increasing the infiltration of inflammatory cells.16 in addition, proinflammatory cytokines (il-1, tnf-α) and pge-2 (prostaglandin e2) also stimulates osteoclast formation, either directly or via the rankl (receptor activator of nuclear factor kβ ligand), resulting in differentiation and fusion of osteoclast precursors into osteoclasts. pge-2 induces the occurrence of bone resorption intensively.7 a research conducted by hakozaki et al.18 shows that rankl is a key mediator in the formation of osteoclasts. protein bound on this membrane is part of tumor necrosis factor, expressed in a variety of cell types including osteoblasts, fibroblasts, and t cells during normal bone metabolism, rankl are expressed by osteoblasts. however, the inflammatory part of rankl is also expressed by immune cells, such as t lymphocytes. expression of rankl is also controlled by other modulators in the bone metabolism, such as parathyroid hormone, vitamin d3, and interleukin-11. binding of rankl on rank receptor on the surface of pre-osteoblast can cause activation of jun terminal kinase and nuclear factor-kappab, which leads to the formation osteoclasts.18 similarly, a research conducted by chang et al.19 also explains that the activation of nf-kb can increase osteoclast activity and bone resorption, and will simultaneously inhibit the function of osteoblasts.18 rankl also plays an important role in osteoimmunology. rankl production is set in conjunction with the presence of inflammatory cytokines, such as tnf-α and il-119. consequently, since mangosteen peel extract inhibited cox-2 and decreased proinflammatory cytokines (il-1, tnf-α) and pge-2, the formation of osteoclasts, either directly or indirectly by rankl (receptor activator of nuclear factor kβ ligand) was inhibited. as a result, the differentiation and fusion of osteoclast precursors into osteoclasts did not occur. in addition, the binding of the rankl on rank receptor on the surface of preosteoblasts was also inhibited. thus, the activation of nuclear factor-kappab (nf-kb) was inhibited. therefore, the number of osteoclasts decreased. for those reasons, it can be said that there is an inverse relation between osteoblasts and osteoclasts. the decreasing of osteoclast cells will occurs when the growth of osteoblast cells increases according to homeostasis in bone cells. some researchers even state that osteoclasts are responsible for bone resorption, while osteoblasts are responsible for new bone formation.20 similarly, the results of this research also shows that the administration of the combination of mangosteen peel extract and dfdbbx increased the number of osteoblasts, but decreased the number of osteoclasts. another cause that also affected the increasing of osteoblast cells in this research was the participation of a graft material combined with mangosteen peel extract. graft material used in this study was dfdbbx, a type of xenograft production of batan. xenografts are useful for stimulating proliferation of osteoblasts, fibroblasts and endothelial cells.21 dfdbbx dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48 48 kresnoadi, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 43–48 contain osteoconductive inorganic matrix component, serving to provide scaffold for bone regeneration without getting involved in bone formation itself.22 the combination of mangosteen peel extract and dfdbbx, consequently, could reduce inflammation, decrease osteoclasts resulting in decreasing bone resorption, as well as stimulate and induce osteoblasts needed in alveolar bone formation. finally, it can be concluded that the combination of mangosteen peel extract and dfdbbx can reduce osteoclasts and increase osteoblasts. the most effective dose of the active substance was 2%. references 1. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc 2007; 72(10): 917-22. 2. kotsakis g, markou n, chrepa v, krompa v, kotsakis a. alveolar ridge preservation utilizing the ‘socket plug’ technique. int.j oral implantol clin res 2012; 3(1): 24-30. 3. habal mb, reddi ah. an update on bone grafting and bone substituting. adv ast reconstr surg 1987; 3: 147-210. 4. yatman e. kulit buah manggis mangandung xanton yang berkhasiat tinggi. wawasan 2012; 29(324). 5. hayyu ns. sitotoksisitas ektrak kulit garcinia mangostana linn terhadap sel fibroblas gingiva manusia. skripsi. fakultas kedokteran gigi, universitas airlangga 2013:24. 6. porth mc, matfin g. pathophysiology concepts of alterred health science. 8th ed. new york: mosby; 2009. p. 320. 7. wei s, kitaura h, zhou p, ross fp, teitelbaum sl. il1 mediates tnf induced osteoclastogenesis. j clin invest 2005; 115(2): 28290. 8. kresnoadi u. toll-like receptor 2 sebagai signaling pathway osteogenesis tulang alveol yang diinduksi kombinasi aloe vera dan graft. disertasi. surabaya: fakultas kedokteran, universitas airlangga; 2012. p. 42-4. 9. vindani d. efektivitas kombinasi ekstrak jinten hitam (nigella sativa) dan graft terhadap peningkatan osteoblas tulang alveol pada cavia cobaya. tesis. surabaya: ppdgs fkg unair; 2013. p. 24-35. 10. mardiana l. ramuan dan khasiat kulit manggis. cetakan v. jakarta: penebar swadaya; 2013. p. 38. 11. nakatani k, nakahata n, arawaka t, yasuda h, ohizumi y. inhibition of and prostaglandin e2 syinthesis by gamma-mangostin, a xanthone derivative cyclooxygenesa in mangosteen, in c6 rat glioma cell. biochem pharmacol 2002; 63(1):73-9. 12. nakatani k, yamakuni t, kondo n, arakawa t, oosawa k, shimura s, inoue h, ohizumi y. gammamangostin inhibits inhibitorkappab kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in c6 rat glioma cells. mol pharmacol 2004; 66(3): 667–74. 13. prasetya rc. ekspresi siklooksigenase-2 dan inflitrasi sel inflamasi gingiva pada tikus yang diinduksi periodontitis setelah pemberian ekstrak etanolik kulit manggis. tesis. yogyakarta: universitas gajah mada; 201. p. 34. 14. chen lg, yang ll, wang cc. anti-inflammatory activity of mangostins from garcinia mangostana. food chem toxicol 2008; 46(2): 688-93. 15. bumrungpert a, kalpravidh rw, chitchumroonchokchai c, chuang cc, west t, kennedy a, mcintosh m, xanthones from mangosteen prevent lipopolysaccharide-mediated inflammation and insulin resistance in primary cultures of human adipocytes. j nutr 2009; 139(6): 1185–91. 16. tewtrakul s, wattanapiromsakul c, mahabusarakam w. effects of compounds from garcinia mangostana on infl ammatory mediators in raw264.7 macrophage cells. j ethnopharmacol 2009; 121(3): 379–82. 17. carranza f, henry h, newman, michael g. clinical periodontology. 10th ed. new york: wb saunders; 2006. p. 5-7. 18. hakozaki a, yoda m, tohmonda t, furukawa m, hikata t, uchikawa s, takaishi h, matsumoto m, chiba k, horiuchi k, toyama y. receptor activator of nf-kb (rank) ligand induces ectodomain shedding of rank in murine raw264.7 macrophages. j immunol 2010; 184(5): 2442-8. 19. chang j, wang z, tang e, fan z, mccauley l, franceschi r, guan k, krebsbach ph, wang cy. inhibition of osteoblast function by ikk/nf-kb in osteoporosis. nat med 2009; 15(6): 682-9. 20. lorenzo j, horowitz m, choi y. osteoimmunology: interaction of the bone and immune system. endocr rev 2008; 29(4): 403-40. 21. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc 2007; 73(6): 513. 22. ghamdi sh, mokeem as, anil s. current concepts in alveolar bone augmentation: a critical appraisal. the saudi dentistry journal 2007; 19(2): 74-85. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p43-48 vol 38 no 2-2005 96 apakah terapi pengendalian plak dapat menurunkan keparahan rinitis alergika pada anak? (does oral plaque control therapy reduce severity of allergic rhinitis in children?) haryono utomo* dan darmawan setijanto** *klinik vip rsgm **bagian ilmu kesehatan gigi masyarakat fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract allergic rhinitis is one of the most common ailments in children. in clinical practice approximately 50% of patients with symptoms of rhinitis are diagnosed with non-allergic rhinitis. positive skin prick test or specific ige in vitro tests that are relevant to aeroallergens are conclusive diagnostic of allergic rhinitis. however, simple diagnostic method such as "sneezing sign" has already proved to be reliable. hypersensitive children have humoral immune system (th2) which release inflammatory factors in the presence of allergen or infection that contribute to allergic response. immunological reactions occurred and antibodies concentration arise, especially specific ige instead of igg because of the isotype switching. a lot of procedures such as allergen avoidance, medication and immunotherapy were done in allergic rhinitis management. however, oral plaque controls were not the point of interest in this case. the aim of this study is to find out the effectiveness of oral plaque control in the reduction of severity of allergic rhinitis symptoms using symptom scores. fifty children, male and female aged 8–14 years, subjective diagnosed as allergic rhinitis using "sneezing sign" were included in this study. oral plaque control procedures were done by polishing and flossing followed by 4 days of gargling with 1% povidone iodine. clinical result showed that after 3 days, oral plaque control 2.925 times more effective than control group. the conclusion was oral plaque control is effective reducing the severity of allergic rhinitis symptoms. key words: rhinitis, children, oral plaque control korespondensi (correspondence): haryono utomo, klinik vip spesialis terpadu, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo 47 surabaya 60132, indonesia. telp. (031) 5951935. pendahuluan penelitian di amerika serikat menunjukkan bahwa rinitis alergika merupakan penyakit yang sangat sering ditemui dan kurang lebih 2,5% kunjungan ke dokter merupakan penderita rinitis alergika. dalam setahun terjadi kehilangan dua juta hari sekolah dan enam juta hari kerja sedangkan biaya yang dikeluarkan untuk obat anti alergi kurang lebih us$ 2,4 milyar dan us$ 1,1 milyar untuk biaya periksa dokter. kurang lebih 10–20% penduduk amerika serikat menderita rinitis alergika dan prevalensi pada daerah perkotaan makin meningkat. prevalensi terendah terdapat pada anak usia kurang dari 5 tahun dan meningkat pada usia remaja hingga prevalensinya mencapai 24%, pada usia dewasa prevalensi menurun lagi.1 pada literatur yang berhubungan dengan rinitis alergika, penatalaksanaan terutama berupa penghindaran terhadap alergen atau iritan yang dicurigai sebagai penyebab gejala rinitis alergika. selain itu juga pengobatan simptomatis dengan memberikan antara lain antihistamin, dekongestan, kortikosteroid lokal, dan munoterapi.1 sampai saat ini belum ada informasi mengenai tindakan penatalaksaanaan rinitis alergika dengan prosedur perawatan kesehatan gigi dan mulut. rinitis adalah suatu keradangan dari lapisan mukosa hidung yang disebabkan mekanisme alergi atau non alergi. pada seorang yang mempunyai predisposisi genetik, paparan pada bahan tertentu dapat menimbulkan terbentuknya antibodi imunoglobulin e (ige) terhadap alergen spesifik, kemudian terjadi reaksi imunologi yang merupakan tahap awal rinitis alergika (gambar 1).1–3 gambar 1. patofisiologi rinitis alergika (paparan pertama pada alergen).3 gambar 1 menunjukkan bila penderita rinitis alergi terpapar dengan alergen misalnya serbuk bunga, antigen dari serbuk bunga yang masuk ke mukosa hidung akan diproses oleh antigen presenting cell (apc) dan dipresentasikan pada sel th (pada penderita alergi adalah sel t helper 2). sel th2 yang teraktivasi akan melepaskan sitokin il-4 dan il-13 (homolog il-4) yang menimbulkan reaksi isotype switching igg menjadi ige pada sel b.1 97utomo: apakah terapi pengendalian plak pada paparan ulang, alergen akan berikatan dengan ige spesifik pada membran mastosit dan menimbulkan degranulasi mastosit yang akan melepaskan beberapa mediator, histamin, prostaglandin dan leukotrien yang menyebabkan timbulnya reaksi alergi pada hidung, kulit dan paru (gambar 2 dan 3).1–3 gambar 2. patofisiologi alergi (rinitis, eczema, asma) paparan alergen pertama dan selanjutnya.3 gambar 2 menunjukkan pada paparan alergen yang pertama, ige spesifik akan berikatan dengan reseptor pada membran mastosit sehingga pada paparan berikutnya alergen tersebut akan dengan cepat berikatan dengan mastosit sehingga menimbulkan degranulasi.1–3 patofisiologi rinitis alergika dimulai dengan earlyphase nasal reaction yaitu reaksi mediator kimiawi (histamin, pgd2, dll) yang dilepaskan mastosit dengan mukosa hidung dengan gejala gatal, bersin, rhinorrhea dan hidung buntu. setelah 2 sampai 6 jam mastosit terjadi lagi pelepasan mediator kimiawi (leukotrien b4) dan sitokin yang memicu keradangan dan disebut sebagai late phase reaction dengan gejala hidung buntu, hiperreaktivitas dan anosmia (gambar 3).1,2 gambar 3 menunjukkan alergen diproses oleh apc dan dipresentasikan pada limfosit th2. pada penderita atopi proses ini memicu produksi sitokin il4, il-5, il-6, il-9, il-10 dan il-13 oleh th2. interleukin-4 dan il-13 selanjutnya akan menstimulasi terjadinya isotype switching igg menjadi ige pada limfosit b. imunoglobulin e yang dilepaskan oleh sel plasma akan berikatan dengan mastosit dan menyebabkan degranulasi. pelepasan mediator oleh mastosit akan memicu early-phase nasal reaction. pelepasan mediator berikutnya terjadi 2–6 jam kemudian sehingga terjadi kekambuhan. late-phase nasal reaction merupakan keradangan yang terjadi karena pelepasan faktor kemotaksis oleh mastosit dan sitokin baik oleh sel th2 juga mastosit.1 diagnosa rinitis alergika ditetapkan berdasarkan hasil positif uji skin prick atau ige spesifik. dalam praktek sehari-hari 50% penderita dengan gejala rinitis tergolong non alergi dengan gejala hidung buntu yang tidak jelas penyebabnya.2 kesulitan mendiagnosa secara tepat antara rinitis alergika dan non alergika juga menimbulkan masalah karena perlu pemeriksaan laboratoris yang lebih akurat. dapat pula dilakukan pemeriksaan sederhana dengan menghitung jumlah eosinofil pada sekret hidung, anamnesa tanda bersin (sneezing sign) di samping ada riwayat keluarga.4,6 pada negara yang sudah maju, pemeriksaan perawatan kesehatan gigi dan mulut sudah mendapat prioritas utama sehingga faktor fokal infeksi rongga mulut sebagai sumber penyakit sistemik dapat dieliminasi. di indonesia, orang tua kurang memperhatikan kesehatan gigi sulung penderita anak, karena banyak yang berpendapat bahwa gigi sulung akan digantikan oleh gigi permanen sehingga perawatannya tidak terlalu penting. pada anak semua imunoglobulin belum mengalami maturasi optimal sampai usia 4 tahun termasuk sekretori immunoglobulin a. sekretori iga pada mukosa rongga mulut berfungsi sebagai penghambat perlekatan bakteri atau virus pada permukaan epitel dan juga mengaglutinasi antigen, sehingga pada anak sistem pertahanan mukosa terhadap alergen termasuk bakteri dan endotoksin belum maksimal.7 kegagalan siga sebagai pertahanan pertama menyebabkan infectious agent menembus membran mukosa.8 infeksi fokal rongga mulut dapat berasal dari infeksi pulpa gigi atau jaringan periodontal. infeksi ini berhubungan dengan mikroflora kompleks yang terdiri dari kurang lebih 200 spesies pada periodontitis apikalis dan lebih dari 500 spesies pada periodontitis marginalis. faktor penyebab terbanyak adalah bakteri anaerobik bentuk gambar 3. patofisiologi rinitis alergika (early and late phase reaction).1 98 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 96–102 batang (rod) gram negatif. mikroflora tersebut akan masuk ke dalam dengan aliran darah dan mempermudah terjadinya bakteremia serta penyebaran sistemik produk bakteri, komponen bakteri, dan kompleks imun.9–11 masuknya mikroorganisme rongga mulut ke dalam pembuluh darah terjadi lebih kurang dari satu menit setelah prosedur perawatan gigi (perawatan saraf gigi, pembersihan karang gigi) bahkan menyikat gigi dapat meningkatkan bakteremia (17 sampai 40%) dan dapat menuju ke jantung, paru serta sistem pembuluh darah kapiler perifer.6,9–11 plak gigi yang merupakan tempat akumulasi bakteri komensal ataupun patogen dapat merupakan faktor penyebab iritasi kronis pada gingiva yang menyebabkan penyakit periodontal. pada keradangan gingiva terjadi perubahan permeabilitas kapiler sehingga mempermudah bakteri dan endotoksin masuk ke pembuluh darah sehingga akan menimbulkan reaksi imunologis dan produksi imunoglobulin. pada sistem imun humoral, imunoglobulin yang berperan adalah ige yang bila terakumulasi berlebihan akan memperparah reaksi alergi penderita rinitis alergika.5 jaringan periodontal disebut sebagai reservoir sitokin (cytokine reservoir) karena sulkus gingiva merupakan tempat penampungan produk imunologi apabila terjadi keradangan periodontal termasuk il-1, tnfα, ifnγ, dan prostaglandin e2. 11–14 prostaglandin e2 merupakan hasil metabolisme asam arakidonat dengan perantaraan enzim cyclooxygenase (cox 1 dan cox 2) (gambar 4).12,15,16 gambar 4. mekanisme produksi pge2 dari fosfolipid membran sel.12,15 pada gambar 4 menunjukkan bahwa fosfolipid di membran plasma dengan perantaraan enzim fosfolipase a2 akan diubah menjadi asam arakidonat. asam arakidonat dengan perantaraan enzim cyclooxygenase 1 dan 2 (cox 1 dan cox 2) akan diubah menjadi prostaglandin e2 (pge2). peningkatan konsentrasi pge2 antara lain karena rangsangan dari endotoksin bakteri gram negatif (lps) pada makrofag yang kemudian akan melepaskan sitokin proinflammatory yaitu il-1 dan tnf-α yang akan meningkatkan produksi cox 2, yang reaksinya dapat dilihat pada gambar 5.10,11 pada gambar 5 menunjukkan lps mempengaruhi secara langung produksi cox 2 atau secara tidak langsung melalui aktivasi sel makrofag yang akan meningkatkan produksi pge2 seperti pada penjelasan gambar 4. prostaglandin e2 berfungsi sebagai bahan imunosupresif umum (general immunosuppresant) yang dapat menurunkan daya tahan tubuh.14 akumulasi pge2 secara sistemik akan mengakibatkan penghambatan pembentukan sitokin sistem imun jalur th1 (th 1 pathway) yaitu il-2 dan ifnγ sehingga sistem imun lebih kearah jalur th2 (th2 pathway), pge2 dalam hal ini secara tidak langsung merangsang produksi sitokin il-4, il-5 dan il-10 oleh sel th2.14,15-17 gambar 5. pengaruh endotoksin (lps) terhadap produksi pge2.12, pada sistem imun terdapat sekelompok sel heterogen yang disebut antigen presenting cells (apc), yaitu: dendritic cell, makrofag, dan sel b. dendritic cell akan membawa antigen dari jaringan perifer ke limfonoduli sehingga akan berinteraksi dengan reseptor pada sel t helper (cd4+).18 apabila terjadi invasi bakteri, protozoa intraseluler atau virus maka dendritic cell meningkatkan produksi il-12. produksi il-12 oleh dendritic cell ini membantu sel th0 (naïve th cell) berkembang menjadi sel subset th1, sebaliknya kurangnya rangsangan dari il-12 atau adanya prostaglandin e2 (pge2) akan mendorong perkembangan ke arah sel subset th2 (gambar 6).8,18 gambar 6. reaksi sistem imun seluler (th1) dan humoral (th2) pada paparan antigen.18 99utomo: apakah terapi pengendalian plak hygiene hypothesis pertama kali dikemukakan oleh strachan cit. romagnani 20 dan didukung oleh romagnani20 menyatakan bahwa anak yang pada usia dini saat perkembangan sistem imunnya belum matur, kurang mendapat paparan infeksi akan lebih mudah menderita alergi. ini disebabkan karena sistem imun tubuhnya lebih ke arah tipe humoral (jalur th 2), sehingga respons pertahanan tubuh terhadap alergen memproduksi bahan kimiawi (histamin) dan sitokin (il-4 dan il-5) yang dapat menimbulkan reaksi alergi.2,5,15,19,20 indonesia merupakan negara yang penduduknya banyak tinggal di lingkungan yang mudah terpapar infeksi dini, seperti pasar dengan fasilitas mandi umum, air sarana kebutuhan sehari-hari kurang memenuhi syarat kesehatan dan lain sebagainya sehingga membuat seseorang lebih kebal terhadap imunogen. walaupun demikian, dengan kemajuan sosioekonomi terutama di kota besar anak usia dini jarang terpapar dengan infeksi dari luar karena sudah tersedia sarana televisi, videogame dan hiburan lain yang membuat anak betah tinggal di rumah. perubahan pola kehidupan tersebut tidak dapat dihindari, maka untuk tidak lebih memperparah bakat alergi yang sudah dimiliki, sebaiknya memperhatikan kesehatan tubuh secara keseluruhan, antara lain dengan mengurangi kemungkinan terjadinya penyakit infeksi, terutama yang bersumber dari bagian tubuh sendiri (infeksi fokal) antara lain dengan perbaikan kesehatan rongga mulut.4,9–10 tujuan penelitian pendahuluan ini adalah untuk mengetahui hubungan kesehatan rongga mulut dengan tingkat keparahan rinitis alergika dan efektifitas penatalaksanaan rhinitis alergika dengan profilaksis pengendalian plak. dengan adanya perbaikan kesehatan rongga mulut diharapkan faktor infeksi fokal sebagai salah satu pencetus infeksi sistemik dapat dikurangi. manfaatnya bagi masyarakat adalah untuk dapat meningkatkan kesadaran akan pentingnya kesehatan rongga mulut dalam mendukung kesehatan tubuh secara keseluruhan. bagi dunia kedokteran adalah untuk lebih mempererat hubungan antara ilmu kedokteran umum dan ilmu kedokteran gigi dalam upaya menyembuhkan penyakit penderita, khususnya dalam penatalaksanaan rinitis alergika. bahan dan metode jenis penelitian ini adalah epidemiologi klinis untuk menilai probabilitas keberhasilan terapi dengan rancangan prospektif kohort. keseluruhan sampel adalah penderita maloklusi yang sedang dalam perawatan ortodonsi peranti lepasan dan cekat. kriteria pemilihan sampel adalah penderita yang dengan anamnesa tanda bersin positif terhadap debu dan bersin tiap pagi sampai siang hari. penderita bebas karies atau bila terdapat karies dilakukan penumpatan terlebih dahulu. sampel terdiri dari 50 penderita, 19 pria dan 31 wanita. subyek adalah penderita anak yang sedang dalam perawatan ortodonti, berusia 8 sampai 14 tahun. sampel dibagi menjadi 2 kelompok. kelompok pertama adalah kelompok yang mendapat perlakuan pengendalian plak rongga mulut, kelompok ini disebut kelompok perlakuan, sedangkan kelompok kedua adalah kelompok yang tidak dikenai perlakuan sebagai kelompok kontrol. kedua kelompok telah disetarakan (matching) kondisi keadaan umum subyek. pengambilan sampel dan pengelompokan dilakukan secara random. penelitian dilakukan sejak oktober 2004 sampai februari 2005 di klinik praktek swasta perorangan jalan dharmahusada indah utara i no 31 surabaya. kedua kelompok sampel telah menandatangani informed consent dan persetujuan tindakan medik. penilaian tingkat keparahan gejala rinitis yaitu bersin, rhinnorhea, hidung buntu, hidung dan mata gatal (5 gejala), dilakukan secara skoring.2,21 kriteria skoring sebagai berikut: 0 = tidak ada gejala; 1 = ada sedikit gejala, yaitu hanya bersin saja di pagi hari; 2 = gejala sedang, mengalami sedikitnya dua gejala dan terjadi paruh hari saja serta tidak mengganggu kehidupan sehari-hari (gangguan tidur, kegiatan sehari-hari, waktu santai, olahraga, sekolah atau kerja); dan 3 = gejala parah, lebih dari dua gejala rinitis alergika (gejala timbul hampir sepanjang hari dan secara bermakna mengganggu kehidupan sehari-hari).2 pada kelompok perlakuan dilakukan pemulasan keseluruhan permukaan gigi rahang atas dan bawah dengan menggunakan sikat pada contra-angle handpiece putaran rendah dan pumis. kemudian dilakukan pembersihan interdental keseluruhan gigi dengan benang gigi dan dilanjutkan dengan irigasi dengan povidon iodin 1%. untuk perawatan di rumah dianjurkan untuk berkumur dengan obat kumur povidon iodine 1% selama 4 hari. kelompok kontrol tidak dilakukan pengendalian plak rongga mulut. evaluasi hasil perawatan dilakukan pada hari ke 3 dengan menanyakan pada orang tua penderita mengenai tingkat keparahan bersin penderita melalui telepon. kepada orang tua telah diberi instruksi untuk memantau tingkat keparahan rinitis dengan tanda bersin setiap hari mulai dari pagi hari. efektifitas pengendalian plak rongga mulut dianalisis dengan menggunakan fourfold table, uji chi-square, fischer's exact test dan perhitungan relative risk.22 hasil pada penelitian ini oral hygiene index (ohi) tidak diperiksa karena semua sampel dianggap sama derajat kebersihan mulutnya pada waktu penelitian, yaitu dengan dilakukan pengendalian plak rongga mulut (pemulasan gigi dengan sikat dan pumis). untuk mengetahui hasil penatalaksanaan rinitis alergika dengan pengendalian plak rongga mulut dibuat perbandingan skoring sebelum dan sesudah 3 hari. hasil penatalaksanaan rinitis berdasarkan anamnesa subyektif tanda bersin penderita dengan rinitis alergika dapat dilihat pada tabel 1. 100 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 96–102 tabel 1. perbandingan kesembuhan antara kelompok perlakuan dengan kelompok kontrol sembuh dalam 3 hari kelompok sembuh tidak sembuh total perlakuan 26 (81,3%) 6 (18,8%) 32 (100%) kontrol 5 (27,8%) 13 (72,2%) 18 (100%) 31 (62,2%) 19 (38,0%) 50 (100%) tabel 1 menunjukkan bahwa dengan uji statistik chisquare didapatkan p = 0,001 dan relative risk = 2,925 (1,363-6,725 ci=95%). hal ini menunjukkan bahwa terdapat perbedaan kesembuhan yang bermakna antara kelompok perlakuan dengan kelompok kontrol. sedangkan kemungkinan sembuh kelompok perlakuan 2,925 kali lipat dibandingkan kelompok kontrol. tabel 2. perbandingan kesembuhan antara kelompok perlakuan dengan kelompok kontrol pasien pria sembuh dalam 3 hari kelompok sembuh tidak sembuh total perlakuan 9 (69,2%) 4 (30,8%) 13 (100%) kontrol 2 (33,3%) 4 (66,7%) 6 (100%) 11 (57,9%) 8 (42,1%) 19 (100%) tabel 2 menunjukkan bahwa dengan uji statistik fischer exact didapatkan p = 0,319 dan relative risk = 2,077 (0,633-6,815 ci = 95%). hal ini menunjukkan bahwa terdapat perbedaan kesembuhan yang tidak bermakna antara kelompok perlakuan dengan kelompok kontrol. sedangkan kemungkinan sembuh kelompok perlakuan 2,077 kali lipat dibandingkan kelompok kontrol. oleh karena terdapat perbedaan kesembuhan yang tidak bermakna, maka efektifitas perlakuan adalah terjadi karena kebetulan saja (by chance). tabel 3. perbandingan kesembuhan antara kelompok perlakuan dengan kelompok kontrol pasien wanita sembuh dalam 3 hari kelompok sembuh tidak sembuh total perlakuan 17 (89,5%) 2 (10,5%) 19 (100%) kontrol 3 (25,0%) 9 (75,0%) 12 (100%) 20 (64,5%) 11 (35,5%) 31 (100%) tabel 3 menunjukkan bahwa dengan uji statistik fischer's exact didapatkan p = 0,001 dan relative risk = 3,579 (1,327-9,651 ci=95%). hal ini menunjukkan bahwa terdapat perbedaan kesembuhan yang bermakna antara kelompok perlakuan dengan kelompok kontrol. sedangkan kemungkinan sembuh kelompok perlakuan 3,579 kali lipat dibandingkan kelompok kontrol. pembahasan banyak penderita alergi ternyata dipengaruhi faktor keturunan dan paparan infeksi pada waktu bayi atau anak usia dini. paparan infeksi waktu bayi dan usia dini akan merangsang aktivasi respons imun seluler (th1) karena terjadi stimulasi produksi sitokin il-12 oleh dendiritic cell sehingga limfosit th1 sebagai pengatur pertahanan terhadap infeksi akan lebih berperan (gambar 6).8,19,20 lebih berperannya th1 pada imunitas tubuh akan mengurangi peranan th2 yang banyak mensintesa sitokin (interleukin 4, 5, 13) yang merangsang limfosit b untuk memproduksi ig e yang menyebabkan proses terjadinya alergi. reaksi keseimbangan sistim imun ini banyak dibahas dalam hygiene hypothesis yang dikemukakan pertama kali oleh david.2,19,20 perkembangan antibodi pada anak belum matur dan jumlahnya tidak seperti orang dewasa, terutama iga sebagai pertahanan pertama mukosa terhadap invasi bakteri, virus dan alergen. pada anak iga belum mengalami maturasi sebelum umur kurang lebih 4 tahun7 sehingga perkembangan pertahanan mukosa belum sempurna. hal ini memudahkan terjadinya penetrasi bakteri dan endotoksin yang akan menimbulkan terjadinya reaksi imunologi di dalam gingiva dan pada penderita alergi akan banyak terjadi akumulasi ige, sitokin proinflammatory dan mediator kimiawi lainnya. imunoglobulin e, sitokin proinflammatory, kompleks imun, bakteri beserta produknya dan mediator kimiawi lain yang berhubungan dengan reaksi alergi dapat masuk ke aliran darah sehingga kemungkinan dapat menimbulkan reaksi alergi di organ lain yang banyak mengandung mastosit, antara lain hidung, paru atau kulit. sampel penelitian berusia 8–14 tahun, kelompok usia tersebut mungkin juga dipengaruhi oleh faktor hormonal (puberty gingivitis) juga kesembuhan dipengaruhi oleh faktor internal (daya tahan tubuh) dan eksternal (gizi dan lingkungan). hasil penelitian menunjukkan bahwa dari 50 subyek anak yang didiagnosa sebagai penderita rinitis alergika dengan anamnese tanda bersin, 32 orang dilakukan tindakan pengendalian plak rongga mulut. setelah dilakukan tindakan pengendalian plak rongga mulut ternyata 26 subyek (81,3%) hilang gejala bersin sebelum atau paling lambat pada hari ketiga. enam penderita (18,8%) hanya mengalami pengurangan gejala atau sama sekali tidak berkurang. sedangkan hasil uji chisquare menghasilkan p = 0,01 (<0,05), hal ini menunjukkan efektifitas pengendalian plak rongga mulut yang bermakna. persentase kesembuhan pada subyek wanita (19 subyek) adalah 89,5%, dengan hasil perhitungan statistik fischer exact didapatkan p = 0,001 (< 0,05) sehingga hasil penatalaksanaan rinitis alergika berhasil secara signifikan. pada penderita pria (13 subyek) didapatkan tingkat kesembuhan 69,2%, dengan hasil 101utomo: apakah terapi pengendalian plak p = 0,319 (> 0,05) sehingga hasil tindakan profilaksis tidak signifikan. keberhasilan penatalaksanaan rinitis sesuai dengan yang perkirakan karena plak sebagai penyebab utama sudah sebagian besar dihilangkan dengan pemulasan gigi, sehingga bakteri dan endotoksin sebagai penyebab keradangan dan bakteremia sudah berkurang. kurang berhasilnya penatalaksanaan rinitis dengan tindakan profilaksis pada gigi dapat disebabkan antara lain: a) tidak berhasilnya pengurangan keradangan pada gingiva terutama daerah interdental yang sulit dibersihkan; b) subyek tidak mengikuti instruksi perawatan kesehatan rongga mulut di rumah; c) gejala rinitis alergika terkontaminasi dengan infeksi (upper respiratory infection/ispa); d) keadaan umum subyek pada waktu pengamatan dilakukan (sedang menderita penyakit yang menurunkan daya tahan tubuh, aktivitas fisik yang berlebihan dan sebagainya); e) terdapat gigi yang sudah mengalami kelainan periapikal walau sudah dilakukan penumpatan tetap merupakan sumber infeksi lokal. pada penderita alergi, mekanisme pertahanan tubuh terhadap bakteri dan endotoksin dapat meningkatkan konsentrasi ige karena adanya isotype switching igg menjadi ige.1,8 endotoksin (lps) juga dapat mengaktivasi makrofag atau monosit untuk meningkatkan sintesa pge2 dari asam arakidonat,9,12,13,15,16 akibatnya konsentrasi ige dan pge2 serum akan meningkat. dengan berkurangnya gingivitis maka konsentrasi sistemik pge2 yang merupakan imunosupresan berkurang sehingga keseimbangan respons imun akan lebih mengarah ke respons imun seluler (th1), akibatnya kepekaan penderita rinitis alergika terhadap rangsangan alergen juga berkurang. pada penderita rinitis alergika terjadi vasodilatasi dan perubahan permeabilitas pembuluh darah kapiler mukosa hidung. seharusnya setelah alergen hilang maka gejala rinitis alergika akan berkurang dan menghilang, akan tetapi dengan adanya perubahan permeabilitas pembuluh darah maka endotoksin dalam darah atau kompleks imun yang melibatkan ige dapat berikatan dengan mastosit yang banyak terdapat pada mukosa hidung. reaksi inilah yang mungkin akan menyebabkan terjadinya gejala rinitis alergika yang berkesinambungan, penyebabnya bukan alergen dari luar tubuh tetapi dari dalam pembuluh darah sendiri, antara lain dari endotoksin (lipopolisakarida) yang berasal dari infeksi fokal rongga mulut antara lain gingivitis. dari bahasan di atas dapat disimpulkan bahwa pengendalian plak rongga mulut pada penderita dengan cara pemulasan gigi, menggunakan benang gigi dan berkumur dengan povidon iodine 1% dapat mengurangi faktor infeksi fokal yang menyebabkan rinitis alergika sering kambuh. namun, faktor infeksi fokal lain yang berhubungan dengan gigi dan mulut seperti karies yang tidak terawat harus dihilangkan terlebih dahulu. di samping itu, kesehatan tubuh secara umum dan faktor kebersihan lingkungan juga perlu diperhatikan dalam penatalaksanaan rinitis alergika. perlu dilakukan penelitian lebih lanjut apakah sebenarnya penyebab utama berkurangnya keparahan rinitis alergika setelah dilakukan perbaikan kesehatan mulut dan kemungkinan adanya pengaruh kadar iga serum umumnya dan kadar sekretori iga rongga mulut khususnya. hal lain yang perlu diperhatikan adalah melakukan uji imunologis serum ige spesifik, dan kerok (swab) mukosa hidung untuk mengetahui jumlah eosinofil sebelum dan sesudah penelitian. pemeriksaan ini perlu dilakukan untuk mendapatkan hasil penelitian yang lebih akurat. daftar pustaka 1. goldman l, auisello d. cecil: textbook of medicine. 22nd ed. philadelphia: saunders; 2004. p. 1590–608. 2. hsu py, yang yh, lin ys, chiang bl. serum cationic protein level and disease activity in childhood rhinitis. as pac j allergy immunol 2004; 22:19–24. 3. benjamini e, coico r, sunshine g. immunology: a short course. 4th ed. john wiley & sons. 2000. available at: url http:// www.wiley.com. accessed march 10, 2005. 4. behrman r. nelson: textbook of paediatrics. 16th ed. philadelphia: saunders; 2000. p. 650–3. 5. budiman s. tanda bersin sebagai alat diagnostik alergi debu rumah pada anak. karya tulis akhir ppds ilmu kesehatan anak. surabaya: fakultas kedokteran unair; 2003. h. 4–41. 6. wang dy, yeoh kh. the significance and technical aspects of quantitative measurements of inflammatory mediators in allergic rhinitis. as pac j allergy immunol 1999; 17: 219–28. 7. istiati s. imunitas mukosa rongga mulut pada anak. majalah kedokteran gigi (dental journal) 2005; edisi khusus pertemuan ilmiah nasional ilmu kedokteran gigi anak: h. 67–9. 8. roiti im, delves pj. roitt's essential immunology. 10th ed. london: blackwell; 2001. p. 184, 260. 9. slots j, taubman ma. contemporary oral biology and immunology. 1st ed. st louis: mosby-year book; 1992. p. 500–4. 10. israelson. gum disease: what you need to know. available at: url. http:/www.periodallas.com. accessed november 12, 2004. 11. american medical network. oral bacteria from gum disease can cause ailments elsewhere in the body periodontal disease. available at: url http://www.dental.am. accessed november 10, 2004. 12. lie xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection clinical microbiology. reviews october 2000; 13(4): 547–58. 13. newman mg. carranza's clinical periodontology. 10th ed. philadelphia: saunders; 2002. p. 113–47. 14. miyazaki k. periodontal immunology. available at: url:http// www.dent.ucla.edu/. accessed november 10, 2004. 15. betz m, fox bs. prostaglandin e2 inhibits production of production of th1 lymphokines but not of th2 lymphokines. j immunology 1991;146(1):108–13. 16. trebble tm. prostaglandin e2 production and t-cell function after fish-oil supplementation: response to antioxidant cosupplementation. am j clin nutr 2003; 78(3): 376-382. available from: url http://www.intl.ajcn.org. accessed december 19, 2004. 17. kuroda e, sugiura t, okada k, zeki k, yamashita u. prostaglandin e2 up-regulates macrophage-derived chemokine production but suppresses ifn-inducible protein-10 production by apc. j immunology 2001; 166: 1650–8. available at: url http:// www.jimmunol.com. accessed december 24, 2004. 18. roitt i, brostoff j, male d. immunology. 6th ed. chicago: mosby; 2001. p. 105–7. 102 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 96–102 19. kay ab,rosen fs. allergy and allergic diseases. n eng j med, 2001; (1): 30–6. 20. romagnani s. the increase prevalence of allergy and the hygiene hypothesis: missing immune deviation, reduced immune suppression, or both?. j allergy clin immunol 2004; 113: 395– 400. 21. pawankar r. allergic rhinitis and its impact on asthma: an evidence-based treatment strategy for allergic rhinitis. as pac j allerg immunol 2002; 20: 43–52. 22. rebecca g. knapp, m. clinton miller iii. clinical epidemiology and biostatistic. maryland: baltimore williams & wilkins; 1992. p. 109. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 9292 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 92–97 original article inhibitory test of andaliman (zanthoxylum achantopodium dc) extract mouthwash against dental plaque bacteria martina amalia1, priscillia sekar yosuana2, iqlima salsabila binti mohammad2, filya suri risky nababan3, zulkarnain1, pitu wulandari1, aini hariyani nasution1, armia syahputra1 1department of periodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia 2undergraduate student, faculty of dentistry, universitas sumatera utara, medan, indonesia 3periodontics residency program, department of periodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: andaliman (zanthoxylum achantopodium dc) is an endemic plant that is found in the province of sumatera utara, indonesia. it contains secondary metabolites, such as alkaloids, flavonoids, glycosides, saponins, tannins, and triterpenoids/steroids, which can potentially be used as a mouthwash. streptococcus sanguinis and staphylococcus aureus are the primary colonizing bacteria in plaque formation. bacterial plaque is known to be the main cause of periodontal disease but can be controlled mechanically and chemically using mouthwash. purpose: this study aimed to determine the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of andaliman extract mouthwash (2%, 4%, 8%) against streptococcus sanguinis atcc®10556™ and staphylococcus aureus atcc® 25923™. methods: this is a laboratory study with a post-test control-only design. the sample consists of andaliman extract mouthwash (2%, 4%, 8%), a positive control (chlorhexidine gluconate 0.2%), and a negative control (mouthwash formulation without andaliman extract) with three repetitions for each group. data were analyzed with the one-way anova test and post hoc lsd test. results: the andaliman extract mouthwash with concentrations of 2%, 4%, and 8% significantly reduced the number of streptococcus sanguinis and staphylococcus aureus colonies (p<0.05), and there was a significant difference in the andaliman extract mouthwash with concentrations of 2%, 4%, and 8% compared to the negative control. conclusion: andaliman extract mouthwash with a concentration of 8% was more effective in inhibiting streptococcus sanguinis growth than staphylococcus aureus. the mic values for both bacteria were 2%, but the study could not determine the mbc value. keywords: andaliman; plaque; streptococcus sanguinis; staphylococcus aureus article history: received 13 june 2022; revised 2 august 2022; accepted 23 august 2022 correspondence: martina amalia, department of periodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no.2 medan 20155, indonesia. e-mail: martina.amalia@usu.ac.id. introduction periodontal disease is a chronic inflammatory process accompanied by the destruction of the surrounding connective tissue and alveolar bone as well as tooth loss in some cases.1 in general, periodontal disease is caused by bacterial plaque on a tooth’s surface, where plaque is a thin layer of biofilm containing a collection of pathogenic microorganisms.2 these pathogenic microorganisms can cause direct damage to periodontal tissue by activating the immune–inflammatory response but could also be beneficial bacteria in the periodontal pocket by providing sources of nutrition.3 supragingival plaque plays an important role in the growth, accumulation, and pathogenesis of subgingival plaque, especially in the early stages of the periodontal diseases gingivitis and periodontitis.4 supragingival plaque formation is mediated by bacteria that can form extracellular polysaccharides that allow these bacteria to attach to teeth and interact with other bacteria,5 i.e., streptococcus sanguinis and staphylococcus aureus. streptococcus sanguinis is facultative anaerobic grampositive bacteria and normal flora found in the human oral cavity, and it plays a role in the initial colonization of plaque formation. streptococcus sanguinis is known as a pioneer in forming dental plaque. the attachment of copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 mailto:martina.amalia@usu.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 93 amalia et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 92–97 streptococcus sanguinis is mediated by fimbriae, pilus proteins, lipoproteins, and the enzyme glucosyltransferase, which gives it a greater adhesion ability to participate in the process of plaque maturation that contributes to the development of periodontal disease.6 staphylococcus aureus is a normal bacterial flora, but in certain circumstances, it can turn into a diseasecausing pathogen due to predisposing factors such as poor oral hygiene. staphylococcus aureus can exacerbate periodontitis by getting into the periodontal pocket formed due to abnormal gingival sulcus depth.7 staphylococcus aureus plays an important role in causing periodontal diseases by forming a biofilm on dental plaque and exacerbating periodontal diseases by secreting various pathogenic factors.8 plaque control is vital to prevent plaque formation and reduce the progression of periodontal disease caused by bacteria. it can be performed mechanically with a toothbrush, dental floss, or interdental brush, and chemically by using mouthwash.9 currently, the active ingredient in mouthwash that is widely used in indonesia is chlorhexidine, and 0.2% chlorhexidine gluconate is an antimicrobial and gold standard in reducing plaque formation. the use of chlorhexidine mouthwash has various reversible side effects such as discoloration of teeth and restorations, changes in taste sensation, and it can even trigger desquamative lesions.10 herbal ingredients are currently used as alternative materials that have the antibacterial ability to prevent the formation of dental plaque. one of the herbal plants with antibacterial activity and the potential to be used as a mouthwash is andaliman (zanthoxylum achantopodium dc), which originated in north sumatera, indonesia. it is widely found in dairi, north tapanuli, tobasa, humbang, silindung, and toba holbung. antibacterial compounds identified from andaliman are alkaloids, terpenoids, flavonoids, saponins, and glycosides. andaliman has been reported for its strong antimicrobial activity and acts as a natural preservative to prevent the growth of pathogen bacteria.11 shasti’s research found that testing andaliman extract against staphylococcus aureus showed the largest inhibition zone, 18.98 mm, at 8%.12 the 25 mg/ml and 12.5 mg/ml of andaliman extract also had an inflammatory effect that resulted in a reduction in the tnf-α and il-6 levels of fibroblast infected by streptococcus sanguinis.13 the novelty of this study is to examine andaliman extract formulated into mouthwash, thereby inhibiting streptococcus sanguinis and staphylococcus aureus. this study aims to observe the andaliman extract mouthwash as an antibacterial against streptococcus sanguinis and staphylococcus aureus in terms of its mic and mbc values. materials and methods this is experimental laboratory research (true experimental design) with a post-test-only control group design. this research has received approval from the ethics committee of the university sumatera utara hospital (no. 309/kepk/ usu/2022). andaliman was confirmed by a herbarium medanese (meda) laboratory, medan, indonesia. the sample used in this study was taken from one of the andaliman fruit producers in sipira village, onan punggu district, toba samosir regency. extracts were made using the maceration method: 2.5 kg of andaliman was dried and refined to produce simplicia powder, which was immersed in 96% ethanol solvent (1:10) and left for 18 hours. it was then filtered to obtain a macerate, and the maceration process was repeated twice. the maceration results were combined and concentrated in a rotary vacuum evaporator to obtain a thick extract. phytochemical tests were carried out to identify alkaloids, flavonoids, glycosides, saponins, tannins, steroids, and triterpenoids.14 next, the formulation of andaliman extract mouthwash was performed according to table 1. cultures of streptococcus sanguinis atcc®10556™ and staphylococcus aureus atcc® 25923™ were taken from the microbiology laboratory in the faculty of pharmacy at the university sumatera utara. the bacteria were rejuvenated before conducting the antibacterial test. the bacterial suspension was made with a concentration of 10-3 mcfarland. the antibacterial effectiveness of the andaliman extract mouthwash was carried out using the dilution method to obtain the mic and mbc values. eight ml of nutrient broth media was put into a test tube, then 1 ml of andaliman extract mouthwash with three concentrations (2%, 4%, and 8%) was added along with a positive control (chlorhexidine gluconate 0.2%) and a negative control (mouthwash formulation without andaliman extract). table 1. andaliman extract mouthwash formulation materials negative control concentration (w/w) positive control 2% 4% 8% andaliman extract 2 gr 4 gr 8 gr 0.2% chlorhexidine gluconate glycerin 4 gr 4 gr 4 gr 4 gr sorbitol 9 gr 9 gr 9 gr 9 gr cmc-na 0.3 gr 0.3 gr 0.3 gr 0.3 gr aquadest 86.7 gr 84.7 gr 82.7 gr 78.7 gr total 100 gr 100 gr 100 gr 100 gr copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 94amalia et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 92–97 after that, 1 ml of bacterial suspension was added to each tube and vortexed. all tubes were incubated at 37 °c for 24 hours.15 observations were made on each tube by looking at turbidity levels. the mic value was the lowest concentration that showed a clear area in the tube.16 all test tubes were transferred to solid media. streptococcus sanguinis atcc®10556™ was streaked on the blood agar medium using the streak plate method. at the same time, the tube containing the staphylococcus aureus atcc® 25923™ bacterial suspension was subcultured on the plate count agar (pca) medium using the pour method. after incubation at 37 °c for 24 hours, the number of bacterial colonies was calculated using a colony counter.15 the lowest concentration that did not indicate the presence of bacterial colonies was defined as the mbc value.16 the data were analyzed using the shapiro–wilk test because the results were homogenous and normal, then continued with one-way anova and post hoc least significant difference (lsd) tests to compare the differences between all groups. results the phytochemical tests showed that the metabolite compounds found in andaliman extract are alkaloid, flavonoid, glycoside, saponin, tannin, and triterpenoid/ steroid (table 2). the antibacterial activity of the andaliman extract mouthwash against streptococcus sanguinis and staphylococcus aureus with concentrations of 2%, 4%, and 8%, a positive control, and a negative control was observed. it could be seen that all of the tubes were turbid due to the color of the concentrated andaliman extract mouthwash (figure 1). furthermore, to discover if turbidity was caused by bacterial growth or not and to determine the values of mic and mbc, all the diluted tubes were transferred to solid media and incubated for 24 hours. then, a calculation was carried out on a petri dish with the number of bacterial colonies observed using a colony counter (figures 2 and 3). as the concentration increased, the results showed a decrease in the number of colonies for streptococcus sanguinis and staphylococcus aureus. the highest average number of streptococcus sanguinis and staphylococcus aureus colonies were in the negative control group (mouthwash without andaliman extract) at 1,896.33 cfu/ ml and 4,849 cfu/ml, respectively. the concentration of 2% was the lowest concentration that began to inhibit the growth of streptococcus sanguinis and staphylococcus aureus, with the average number of bacterial colonies being 703.33 cfu/ml and 3,878 cfu/ml, respectively. the comparison of antibacterial activity of andaliman extract mouthwash for each concentration showed more effectiveness in inhibiting the growth of streptococcus sanguinis with a fewer average number of bacterial colonies than staphylococcus aureus with a more significant average number of bacterial colonies (table 3). post hoc lsd tests on antibacterial activity against streptococcus sanguinis revealed that the concentrations of 2%, 4%, 8%, and a positive control had no significant differences (p>0.05) in each concentration, while the negative control group showed a significant difference (p<0.05). meanwhile, lsd tests on antibacterial activity against staphylococcus aureus showed that the concentrations of 2% and 4% and 4% and 8% had no significant difference (p>0.05). at the same time, in the other group, there were table 2. phytochemical screening test results for andaliman extract secondary metabolites result alkaloid + flavonoid + glycoside + saponin + tannin + triterpenoid / steroid + a b figure 1. observation results of the andaliman extract mouthwash dilution test with three repetitions: a. streptococcus sanguinis; b. staphylococcus aureus. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 95 amalia et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 92–97 e c a d b figure 3. results of andaliman extract mouthwash and staphylococcus aureus cultured on pca media: a. 2% concentration; b. 4% concentration; c. 8% concentration; d. positive control e. negative control. a c b d e figure 2. results of andaliman extract mouthwash and streptococcus sanguinis cultured on blood agar media: a. 2% concentration; b. 4% concentration; c. 8% concentration; d. positive control e. negative control. table 3. the average number of staphylococcus aureus and streptococcus sanguinis bacterial colonies concentrations the average number of bacteria (cfu/ml) streptococcus sanguinis staphylococcus aureus mean ± sd anova mean ± sd anova 2% 703.33±105.19 0.001* 3,878 ± 634.863 0.000* 4% 524.67±163.37 3,638.66± 332.726 8% 204.33±108.45 2,982 ± 259.963 pc ( + ) 460.67±137.59 811.33 ± 440.312 nc ( ) 1,896.33±744.23 4,849 ± 203.315 notes: * a significant difference (p<0.05) table 4. the difference in the average number of bacterial colonies in the andaliman extract mouthwash against the growth of streptococcus sanguinis and staphylococcus aureus for each concentration concentrations p-value2% 4% 8% pc (+) nc (-) streptococcus sanguinis 2% 0.549 0.114 0.419 0.002* 4% 0.292 0.829 0.001* 8% 0.394 0.000* pc (+) 0.001* staphylococcus aureus 2% 0.485 0.022* 0.000* 0.015* 4% 0.075 0.000* 0.004* 8% 0.000* 0.000* pc (+) 0.000* notes: post hoc lsd *a significant difference (p<0.05) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 96amalia et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 92–97 significant differences in each concentration (p<0.05). the antibacterial tests for streptococcus sanguinis and staphylococcus aureus showed significant differences in each concentration against the negative control group. the andaliman extract mouthwash effectively inhibited the growth of both streptococcus sanguinis and staphylococcus aureus (table 4). discussion in this study, andaliman extract (zanthoxylum acanthopodium dc) was dissolved and macerated in 96% ethanol solvent. sepriani et al.17 used 96% ethanol solvent because it is a universal solvent that can be polar or non-polar so that the metabolite compounds found in the andaliman plant can be extracted. the formulation of andaliman extract mouthwash consists of four main ingredients: glycerin, cmc-na, sorbitol, and aqua dest. glycerin is a humectant component to prevent the active substances in the andaliman extract mouthwash from evaporating into the air. the addition of cmc-na binds the mouthwash ingredients so that all components will be homogeneous. sorbitol is used to provide a sweet taste to compensate for the bitterness of andaliman. aqua dest is used as a solvent.18 the results of the phytochemical tests on andaliman extract (zanthoxylum acanthopodium dc) in this study showed the presence of alkaloid compounds, flavonoids, glycosides, saponins, tannins, and triterpenoids/steroids. this agrees with muzafri’s research, which states that the results of phytochemical screening of andaliman extract show that it contains alkaloid compounds, flavonoids, glycosides, saponins, tannins, triterpenoids/steroids, and anthraquinone glycosides.19 alkaloids as antibacterial compounds can inhibit bacterial nucleic acid and protein synthesis, metabolism, and efflux pumps.20 the mechanism of flavonoids in inhibiting bacteria is almost similar to glycosides, which can form complex compounds with extracellular proteins, so streptococcus sanguinis and staphylococcus aureus cannot maintain the shape of their cell membranes and pathogenicity, which are essential for bacterial growth.21 saponin compounds can increase membrane permeability, which can inactivate bacterial enzymes in the metabolic process and cause the death of streptococcus sanguinis and staphylococcus aureus. tannins are phenolic compounds that can damage the function of bacterial genetic material and inhibit enzymes so that bacteria cells are not formed.22 terpenoids as antibacterial compounds can form bonds with porins. damaged porins inhibit the nutrient transport process so that bacteria will lack nutrients, which results in bacterial inhibition or death in streptococcus sanguinis and staphylococcus aureus.23 the antibacterial effectiveness test used in this study was the dilution method, which was carried out by calculating the values of mic and mbc. each test tube that had andaliman extract mouthwash with concentrations of 2%, 4%, 8%, a positive control, and a negative control was given a bacteria suspension of streptococcus sanguinis and staphylococcus aureus. the observations showed that each test tube had the same level of turbidity that was difficult to determine via the dilution method (figure 2). this was influenced by the color of the andaliman extract mouthwash, which was concentrated. therefore, this study had to calculate the number of bacterial colonies by continuing the culture of diluted results in solid media, namely, blood agar and pca. the mic value of the andaliman extract mouthwash was determined by a petri dish with a smaller number of bacterial colonies than the petri dish of the negative control group. the mbc value was determined by a petri dish with no bacterial growth. the results showed a decrease in bacterial colonies as the concentration increased. khalishah’s research states that the higher the concentration of antibacterial substances, the higher the ability to inhibit the growth of bacteria.24 the results of this study showed that the mic value was at the concentration of 2% because it was the lowest concentration that began to inhibit the growth of streptococcus sanguinis and staphylococcus aureus compared to the negative control. meanwhile, this study’s mbc value could not be determined because bacterial colonies were still found at the concentration of 8%. the andaliman extract mouthwash inhibited the growth of streptococcus sanguinis and staphylococcus aureus in this study. lubis found that 4% andaliman extract mouthwash was effective because it reduced the number of streptococcus mutants.13 furthermore, research by shasti et al.12 proved the ability of 8% andaliman extract to inhibit the growth of staphylococcus aureus. sitanggang et al.23 researched the inhibitory activity of andaliman extract on the growth of escherichia coli. based on the analysis of inhibitor diameter, the mic value was found at a concentration of 60%. this proves that andaliman extract mouthwash (zanthoxylum acanthopodium dc) has a bacteriostatic effect on gram-positive anaerobic bacteria that cause periodontal disease. the concentration of antibacterial compounds affects the ability to inhibit bacterial growth. darajat stated that the higher the concentration of antibacterial substances, the more antibacterial compounds contained, which means bacteria will be killed quickly at higher concentrations.25 in this study, bacterial growth was found at each concentration of 2%, 4%, and 8%, and several technical and biological factors could cause this in the control group. the technical factors can mainly be controlled by researchers; these include the large inoculum, ph, incubation length, temperature, and medium. biological factors consist of bacterial cell wall structure and resistance.26 grampositive bacteria, such as streptococcus sanguinis and staphylococcus aureus, have thicker peptidoglycan layers on the cell wall than gram-negative bacteria, forming a rigid structure. the presence of wider peptidoglycan structures in gram-positive bacteria makes the antimicrobial copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 97 amalia et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 92–97 compounds more challenging to penetrate gram-positive cell walls than gram-negative cell walls.27 another biological factor is resistance. bacteria are likely to become resistant during antibacterial tests because resistance is an adaptation bacteria naturally make to survive. resistance is a fundamental factor that cannot be controlled.26 in conclusion, there was a decrease in the number of bacterial colonies for both streptococcus sanguinis and staphylococcus aureus from the highest to the lowest concentration. the concentration of 8% was shown to be more effective in inhibiting streptococcus sanguinis growth than staphylococcus aureus. the mic value that began to inhibit bacterial growth for both bacteria was 2%, while the mbc could not be determined in this study. references 1. nugraha ap, sibero mt, nugraha ap, puspitaningrum ms, rizqianti y, rahmadhani d, kharisma vd, ramadhani nf, ridwan rd, noor tne binti ta. anti-periodontopathogenic ability of mangrove leaves (aegiceras corniculatum) ethanol extract: in silico and in vitro study. eur j dent. 2022; . 2. andriani i, medawati a, humanindito mi, nurhasanah m. the effect of antimicrobial peptide gel rise-ap12 on decreasing neutrophil and enhancing macrophage in nicotine-periodontitis wistar rat model. dent j. 2022; 55(2): 93–8. 3. wulandari p, widkaja d, nasution ah, syahputra a, gabrina g. association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients. dent j. 2022; 55(1): 16–20. 4. nugraha ap, ardani igaw, sitalaksmi rm, ramadhani nf, rach maya nti d, kuma la d, k ha r isma v d, ra h mada n i d, puspitaningrum ms, rizqianti y, ari mda, nugraha ap, noor tne binti ta, luthfi m. anti–peri-implantitis bacteria’s ability of robusta green coffee bean (coffea canephora) ethanol extract: an in silico and in vitro study. eur j dent. 2022; . 5. saputra g, nugraha ap, budhy ti, rosari fs, lestari nai, sari aa, gheasani a, valensia t, ramadhani nf, noor tnebta, nugraha ap, nugraha ap, sosiawan a. nanohydroxyapatite-chitosan hydrogel scaffold with platelet rich fibrin and buccal fat pad derived stem cell for aggressive periodontitis treatment: a narrative review. res j pharm technol. 2022; 15(12): 5903–8. 6. ber niyanti t, mahmiyah e. microbiological studies on the production of antimicrobial agent by saponin aloe vera linn against streptococcus sanguinis. res j microbiol. 2015; 10(10): 486–93. 7. busman, edrizal, wirahmi sd. daya hambat ekstrak rimpang temu putih (curcuma zedoaria) terhaadap streptococcus mutans dan staphylococcus aureus. menara ilmu. 2019; 13(6): 19–28. 8. kim gy, lee ch. antimicrobial susceptibility and pathogenic genes of staphylococcus aureus isolated from the oral cavity of patients with periodontitis. j periodontal implant sci. 2015; 45(6): 223–8. 9. yousefimanesh h, amin m, robati m, goodarzi h, otoufi m. comparison of the antibacterial properties of three mouthwashes containing chlorhexidine against oral microbial plaques: an in vitro study. jundishapur j microbiol. 2015; 8(2): e17341. 10. dany ss, mohanty p, tangade p, rajput p, batra m. efficacy of 0.25% lemongrass oil mouthwash: a three arm prospective parallel clinical study. j clin diagnostic res. 2015; 9(10): zc13–7. 11. wijaya ch, napitupulu fi, karnady v, indariani s. a review of the bioactivity and flavor properties of the exotic spice “andaliman” (zanthoxylum acanthopodium dc.). food rev int. 2019; 35(1): 1–19. 12. shasti h, putra siregar ta. uji aktivitas antibiotik ekstrak buah andaliman (zanthoxylum acanthopodiumdc) terhadap pertumbuhan bakteri staphylococcus aureus secara in vitro. j ibnu sina biomedika. 2017; 1(1): 49–56. 13. lelyana s, widyarman as, amtha r. indonesian andaliman fruit (zanthoxylum acanthopodium dc.) extract suppresses the expression of inflammatory mediators on fibroblasts cells in vitro. pharm sci asia. 2021; 48(5): 491–7. 14. direktorat jenderal kefarmasian dan alat kesehatan. farmakope herbal indonesia. 2nd ed. jakarta: kementerian kesehatan republik indonesia; 2017. p. 531–2. 15. blaize jf, suter e, corbo cp. serial dilutions and plating: microbial enumeration. j vis exp sci. 2019; : 1–4. 16. deepa v, gayathri g v, vinayaka am, mehta ds. evaluation & comparison of antibacterial activity of ethanolic extract of aegle marmelos (bilwa) leaf and fruit with 0.2% chlorhexidine on periodontopathic bacteria-an in-vitro study. world j pharm pharm sci. 2020; 9(10): 2182–97. 17. sepriani o, nirhamidah n, handayani d. potensi ekstrak tumbuhan andaliman (zanthoxylum acanthopodium dc) sebagai antibakteri staphylococcus aureus. alotrop. 2019; 4(2): 133–9. 18. mangulkar s. review on dental care preparation. world j pharm res. 2020; 9(7): 236–61. 19. a l muza f r i. uji a k t ivit as a nt i m i k roba ek st ra k a nd a l i ma n (zanthoxylum acanthopodium dc.) pada staphylococus aureus. j sungkai. 2019; 7(1): 122–6. 20. yan y, li x, zhang c, lv l, gao b, li m. research progress on antibacterial activities and mechanisms of natural alkaloids: a review. antibiotics. 2021; 10(3): 318. 21. shamsudin nf, ahmed qu, mahmood s, ali shah sa, khatib a, mukhtar s, alsharif ma, parveen h, zakaria za. antibacterial effects of f lavonoids and their structure-activity relationship study: a comparative interpretation. molecules. 2022; 27(4): 1149. 22. syaputri i, girsang e, chiuman l. test of antioxidant and antibacter ial activity of ethanol extract of andaliman fr uit (zanthoxylum acanthopodium dc.) with dpph (1.1-diphenyl2-picrylhydrazil) trapping method and minimum inhibitory concentration. int j heal pharm. 2022; 2(2): 215–24. 23. sitanggang fmc, duniaji as, pratiwi idpk. daya hambat ekstrak buah andaliman (zanthoxylum achantopodium dc) dalam etil asetat terhadap pertumbuhan escheria coli. j ilmu dan teknol pangan. 2019; 8(3): 257. 24. khalishah n, oktiani bw, adhani r. antibacterial effectiveness test of ramania leaves (bouea macrophylla griffith) flavonoids extract on aggregatibacter actinomycetemcomitans bacteria causing aggressive periodontitis. dentino j kedokt gigi. 2021; 6(1): 25–30. 25. darajat sm, ismail a, kodir a, sitii y. efektivitas ekstrak daun jamblang (syzygium cumini l) terhadap pertumbuhan bakteri streptococcus sanguinis. in: prosiding konstelasi ilmiah mahasiswa unissula (kimu) 7. semarang: universitas islam sultan agung; 2022. p. 155–63. 26. novitasari iw. uji aktivitas antibakteri infusa daun mangga bacang (mangifera foetida l) terhadap pertumbuhan salmonella typhi. j mhs pspd fk univ tanjungpura. 2015; 3(1): 2–19. 27. hidayat s. uji aktivitas antibakteri infusa daun mangga bacang (mangifera foetida l.) terhadap staphylococcus aureus secara in vitro. j mhs pspd fk univ tanjungpura. 2015; 3(1): 2–17. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p92–97 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p92-97 � electro-gene therapy in a human oral tongue cancer cell by intratumoral injection of pcdna3.�-p27kip� wt supriatno department of oral medicine faculty of dentistry, gadjah mada university yogyakarta indonesia abstract oral tongue cancers are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes. also, treatment options for this cancer are limited. however, a new strategy for refractory cancer, gene therapy is watched with keen interest. recently, a novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electro-gene therapy and intratumoral plasmid dna injection. in the present study, a nonviral gene transfer system, in vivo electrogene therapy in human oral tongue cancer cell, sp-c1 xenograft was examined. the aim of the study is to examine the efficiency of transfection of exogenous p27kip1 gene by electroporation and the antitumor activity of p27kip1 gene therapy in human oral tongue cancer xenografts using pcdna3.1-p27kip1 wild type (wt) and pcdna3.1 empty vector with the local application of electric pulses. to evaluate this in vivo gene transfer method, the enhanced green fluorescence protein (egfp) gene was transfected into xenografts by electroporation. the efficiency of transfection of exogenous p27kip1 gene by electroporation was confirmed by western blotting analysis. to estimate the reduction of oral tongue cancer xenografts by this method, the size of sp-c1 xenografts in nude mice after electroporation with wild type p27kip1 gene was measured. the growth of tumors was markedly suppressed by wild type p27kip1 gene transfection by electroporation compared with transfection of empty vector only. moreover, histological specimens revealed apoptotic cell death was increased in wild type p27kip1-transfected tumors than empty vector. these results suggest that it is possible to transfer wild type p27kip1 into human oral tongue cancer xenografts using electroporation. wild type p27kip1 has a high-potencially to suppress the growth of tumors. finally, combination system of pcdna3.1-p27kip1 wt-injected tumor and electroporation might be used for human oral cancer. key words: intratumoral, wild type p27kip1, human oral tongue cancer, electroporation correspondence: supriatno, c/o: bagian penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta 1, sekip utara, yogyakarta 55281, indonesia. e-mail: pridentagama@lycos.com, phone/fax: 0274-515307. introduction oral tongue cancers are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes. moreover, oral tongue cancer frequently shows local recurrence after initial treatment, probably due to microinvasion and/or micrometastasis of tumor cells at the primary site.1 despite advanced in surgery, radiotherapy and chemotherapy, the survival of patients with oral tongue cancer has not significantly improved over the past several decades. also, treatment options for recurrent or refractory oral cancers are limited.2 furthermore, the ratio of mortality or incidence in 1980 and 1990 was 0.48 and 0.47, respectively,3 and the prognosis has not changed during the past 10 years. however, as a new strategy for refractory cancer, gene therapy is watched with keen interest. electro-gene therapy or electroporation has been developed for the purpose of achieving highly efficient in vitro gene or drug transfer.4,5 this system provides markedly higher efficiency transfer compared with other nonviral transfer system, including cationic liposomes.6 electroporation has been applied to in vivo drug transfer for cancer treatment and clinical trial has been started.7 electroporation has become more and more popular as an effective technique for introduction of foreign dna into cells of various kinds of mammalian cells,8,9 for investigation of gene regulation,10 and has been demonstrated to be highly useful in transfecting human hematopoetic stem cells for gene therapy.11 however, the transfection efficiency in mammalian cells using in vivo electroporation has received little attention12 and usually is still low, typically about 0.01–1%.13 because electroporation is a physical method, it has a little biological side effect and is free of chemical toxicity.10 many types of methods and techniques for in vivo gene transfer have been developed, and some of them have already been applied in clinical trials.4 nonviral gene transfer, “naked” plasmid dna is an ideal system for gene transfer. a plasmid mediated method would be economical and easy because use of this system obviates the necessity to construct viral vectors, establish clones of producer cells, assess viral titers and presence of replication-competent helper virus, which has been known to activate passive oncogenes. the transfer procedure could be easily repeated because “naked” plasmid dna has little antigenicity to the host body.14 2 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 1–5 p27kip1 is an universal cyclin-dependent kinase inhibitor that directly inhibits the enzymatic activity of cyclin-cdk complexes, resulting in cell cycle arrest at g1. 15 p27kip1 has an important prognostic factor in various malignancies. recently, decreased expression of p27kip1 has been frequently detected in human cancer.16–18 in addition, loss of p27kip1 has been associated with disease progression and an unfavorable outcome in several malignancies.19 furthermore, mice lacking the p27kip1 gene show an increase in body weight, thymic hypertrophy and hyperplasia of pituitary intermediate lobe adrenocorticotropic hormon cells, adrenal glands and gonadal organ.20) also, malignant human oral cancer cells transfection with p27kip1 gene leads to inhibition of proliferation, invasion and metastasis.21,22 in the present study, the efficiency of transfection of exogenous p27kip1 gene by electroporation and the antitumor activity of p27kip1 gene therapy in human oral tongue cancer xenografts using pcdna3.1-p27kip1 wild type (wt) and pcdna3.1 empty vector with the local application of electric pulses was evaluated. materials and methods cell and cell culture sp-c1 cells were isolated from a cervical lymph-node metastasis of an oral squamous cell carcinoma patient in our laboratory.22 the original tumor of sp-c1 cells was moderately differentiated squamous cell carcinoma of tongue, and was not invasive into muscle layer. cells were maintained in dulbecco’s modified eagle medium (dmem, sigma, st louis, mo, usa) supplemented with 10% fetal calf serum (fcs, moregate biotech, bulimba, australia), 100 g/ml streptomycin, and 100 units/ml penicillin (invitrogen corp., carlsbad, ca, usa). construction of a mammalian expression vector the mammalian expression vectors pcdna3.1p27kip1wt (invitrogen) containing sense oriented human wild type p27kip1 cdna was constructed. briefly, pcdna3.1 (+) was digested with kpn1 (takara biomedicals, kusatsu, japan) and bamh1 (takara), and dephosphorylated by calf intestinal alkaline phosphate (roche diagnostics, mannheim, germany). the human wild type p27kip1 cdna fragment (0.69 kb kpn1 and bamh1 fragment) was obtained as a generous gift from dr. j massague (howard hughes medical institute, memorial sloankettering cancer center, ny). this fragment containing the human wild type p27kip1 open reading frame was ligated to the prepared cloning site of pcdna3.1 (+) by t4 dna ligase (takara). the direction of the ligated fragmen was confirmed by sequencing analysis with a spesific primer (p27kip1-sqp: 5’-atgtcaaacgtggcgagtgtc3’) for human p27kip1 cdna the dna sequence was determined by the dideoxy chain termination method, using fluorescene-labeled primers and a thermo sequenase cycle sequencing kit (amersham pharmacia biotech, sweden). electrophoresis and scanning were performed with a shimadzu dsq-500 dna sequencer (shimadzu, kyoto, japan). tumorigenesis in nude mice and electrotransfection the oral tongue cancer cell line, sp-c1 cells were trypsinized, washed with pbs, and suspended in saline solution at 1 × 106 cells in 0.1 ml. cell suspension (0.1 ml) was injected into each male nude mouse with balb/ca jcl-nu genetic background (clea japan, inc. tokyo, japan) subcutan in the back area. a pair of 1 cm diameter of disc-shaped electrodes (pinsettes-type electrode 449-10 prg, meiwa shoji, tokyo, japan) was used to nip the tumor nodule through the skin. a series of eight electrical pulses with pulse length of 1 msec was delivered with a standard square wave electroporator btx t820 (btx, inc, san diego, ca). the voltage of 100 v/1.0 cm diameters of xenografts was used. then, it delivered an appropriate pulse length and frequency of pulses according to previous report.8,9 immediately after electrical pulsing, 20 g of plasmid cdna or pcdna3.1-p27kip1 wt dissolved in 50 l of tris edta buffer was directly injected into the tumor nodule. this electroporation and injection were performed a total of three times at 3-day intervals. tumor volume and body weight were measured every 3 days from the time electroporation started until the mice were sacrificed. the tumor volume was determined by measuring length (l) and width (w) diameters of the tumor and calculated as v = 0.4 × l × w2.21 detection of repoter gene expression in vivo for fluorescence microscopy, pegfp-c3 vector (bd bioscience clontech)-injected tumors for 48 h were sectioned and mounted in pbs for immediate microscopy. to visualize egfp, a xenon arch lamp and a fitc filter were used on a zeiss axioskop. images were acquired with a color ccd camera and frame-grabbing equipment at identical magnification, light intensity and amplification for each sample pair of tumors from electroporated or nonelectroporated animals, respectively. western blotting analysis cell lysates were prepared from the xenograft tumor tissue. briefly, samples containing equal amounts of protein (50 g) were electrophoresed on a sds-polyacrylamide gel and transferred to a nitrocellulose filter (pvdf membrane: biorad, hercules, ca, usa). the filters were blocked in tbs containing 5% nonfat milk powder at 37º c for 1 hour and then incubated with a 1: 500 dilution of the monoclonal antibody against p27 protein (clone 1b4, monoclonal antibody, novocastra laboratories, new castle, uk) as the primary antibody and an amersham ecl kit (amersham pharmacia biotech). anti- tubulin monoclonal antibody (zymed laboratories, san fransisco, ca, usa) was used for normalization of western blot analysis. tunel method apoptosis was analyzed in situ by the tunel technique using apoptag (oncor, inc., gaithersburg, md, usa), labeling 3’-oh dna ends generated by dna 3supriatno: electro-gene therapy in human oral tongue fragmentation. mice tumors were fixed in 4% formaldehyde in pbs (–). dewaxed paraffin sections were treated with 20 g/ml proteinase k in pbs (–) for 10 minutes to digest protein, then treated with 3% hydrogen peroxide in pbs for 5 minutes to quench endogenous peroxidase activity and equilibrated. tdt enzyme was applied to the cells or sections were incubated at 37ºc for 1 h. after each step, the sections were rinsed with pbs. statistical analysis statistical analysis was performed with a stat work program for macintosh computers (cricket software, philadelphia, pa, usa). data were analyzed for statistical significance of 95% with two-way anova and student’s t-test. results detection of transgene expression in xenograft expression of reporter gene (egfp) after plasmid injection and electroporation in tumor tissue was assessed in fresh tissue sections by light microscopy fluorescence imaging. very few cells were positive when only naked dna without consecutive electroporation was injected. the combination with electroporation resulted in consistenly efficient transduction of a higher number of cells with egfp reporter gene (figure 1). in vivo effect of p27kip1 wt transfection by electroporation on tumor growth the mean relative volume for sp-c1 xenografts treated with an injection of pcdna3.1-p27kip1 wt or pcdna3.1 empty vector was shown in figure 2a. p27kip1-up-regulated tumors (pcdna3.1-p27kip1 wt-injected) became much smaller than pcdna3.1 empty vector-injected tumors (p < 0.01). interestingly, during the experimental period, no loss of body weight was observed in each treatment group, and that no skin region including a burn also was observed (figure 2b). expression of p27kip1 protein in xenografts to evaluate the efficiency of transfection of p27kip1 gene, the expression of p27kip1 protein by westen bloting egfp + + electroporation – + 2.1 ± 0.3 37.5 ± 3.5 figure 1. detection of reporter gene expression. figure 2. (a) growth of tumors formed by transfectants, (b) change of body weight in mice. 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 0 3 6 9 1 2 1 5 e m p t y v e c t o r p 2 7 w t day (mm3) r el at iv e of tu m or v ol um e * * (a) (b) electroporation day (gram) 0 1 0 2 0 3 0 4 0 0 3 6 9 1 2 1 5 e m p t y v e c t o r p 2 7 w t � dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 1–5 was evaluated. as shown in figure 3, up-regulated of p27kip1 protein in pcdna3.1-p27kip1 wt-injected tumors was detected when compared with that in pcdna3.1 empty vector-injected tumors. the expression of -tubulin as an internal control was approximately the same in all of the tumors. detection of apoptosis induces by pcdna3.1-p27kip1 wt to assess the incidence of apoptotic cell death, the internucleosomal dna fragmentation using the tunel method was investigated. the tunel-positive cells were significantly increased in tumors electroporated with pcdna3.1-p27kip1 wt when compared with that in samples from xenografts electroporated with pcdna3.1 empty vector (figure 4). discussion cell membranes electro-gene therapy (electroporation) has been developed for the purpose of achieving highly efficient in vitro gene and/or drug transfer.5 interestingly, the application of electroporation to cultured cells has been well established, but the use of in vivo electroporation has received little attention.12 in vivo electro-gene therapy has just recently been proposed for transdermal drug delivery23 and for electrochemotherapy with bleomycin of superficial tumors.24 recently, successful in vivo transfer of il genes into muscle, and transfer of marker and therapeutic suicide genes into normal tissues and tumors has been reported.25 in the present study, electro-gene therapy with naked plasmid dna was evaluated. the wild type p27kip1 gene figure 3. expression of p27kip1 and  tubulin protein in the pcdna3.1 empty vector and pcdna3.1-p27kip1 wt transfectant tissues. p27kip1 -tubulin pcdna3.1 em pty vector pcdna3.1 -p27 kip1 wt figure 4. tunel, p27 kip1 staining of sp-c1 xenotransplants. pcdna3.1 empty vector pcdna3.1-p27 kip1 wt �supriatno: electro-gene therapy in human oral tongue was used as a transfection gene and was evaluated its antitumor activity in human oral tongue cancer (sp-c1 cell) xenograft. the results of study demonstrated the efficiency of electro-gene therapy was thought to be about 40–65% of cells, determined by egfp expression as shown in figure 1. also, it was demonstrated that the transfection of wild type p27kip1 gene by electro-gene therapy could induce apoptotic cell death (figure 4), and inhibit the growth of oral cancer xenografts (figure 2). also, wild type p27kip1 gene by electro-gene therapy could induce the expression of p27kip1 protein (figure 3), which has the negative regulator function in the cell cycle. therefore, electro-transfer of plasmid dna p27kip1 wt into sp-c1 xenograft can be successfully achieved using disk-shaped electrodes. suggesting that clinical application using this electroporation system for oral cancer may be possible in the future. on the other hands, some disadvantages of this method should be considered. although transfection by electrogene therapy inhibited the growth of sp-c1 xenografts, the target area was limited to local tumors and the growth of multiple metastatic lesions cannot be target for efficient suppression. for that reason, with a view to obtaining more effective gene therapy using electroporation for oral cancer, i plan to attempt gene transfer with several other genes and to use various anticancer agents in combination with gene transfection by this electro-gene therapy system. in conclusion, intratumoral injection of pcdna3.1p27kip1 wt gene following in vivo electro-gene therapy has a highly antitumor activity in oral tongue cancer xenografts. it might be possible to transfer pcdna3.1-p27kip1 wt gene into oral cancer xenograft. in vivo gene transfer method is a simple procedure and can solve some of the critical drawbacks of the present gene transfer techniques, thus providing a new strategy for gene therapy. acknowledgments i t h a n k d r . k o j i h a r a d a , d d s . , p h . d a n d dr. takashi bando, dds., ph.d, second department of oral maxillofacial surgery and oncology, school of dentistry, tokushima university, japan, for their valuable advices and providing cancer cell lines. references 1. ammar a, uchida d, begum nm, tomizuka y, iga h, yoshida h, et al. the clinicopathological significance of the expression of cxcr4 protein in oral squamous cell carcinoma. int j oncol 2004; 25:65–71. 2. inagi k, takahashi h, okamoto m, nakayama m, makoshi t, nagai h. treatment effects in patients with squamous cell carcinoma of the oral cavity. acta otolaryngol 2002; suppl 547:25–29. 3. parkin dm, pisani p, ferlay j. estimates of the worldwide incidence of 25 major cancers in 1990. int j cancer 1999; 80:827–41. 4. nishi t, yoshizato k, yamashiro s, takeshima h, sato k, hamada k, kitamura i. high efficiency in vivo gene transfer using intra arterial plasmid da injection following in vivo electroporation. cancer res 1996; 56:1050–5. 5. matthew ke, dev sb, toneguzzo f, keating a. electroporation for gene therapy. in: nickoloff ja, editor. methods in molecular biology. volume 48. animal cell electroporation protocols, totowa, nj: humana press, inc; 1995. p. 273–80. 6. sukharev si, klenchin va, serov sm, chernomordik lv, chizmadzhev, yu a. electroporation and electroporetic dna transfer into cells: the effect of dna interaction with electropores. biophys j 1992; 63:1320–7. 7. belehradek m, domenge c, luboinski b, orlowski s, belehradek j jr, mir lm. electrochemotherapy, a new antitumor treatment. first clinical phase i-ii trial. cancer 1993; 72:3694–700. 8. chu g, hayakawa h, berg p. electroporation for the efficient expression of mammalian cells with dna. nucleic acids res 1987; 15:1311–26. 9. suzuki t, shin bc, fujikura k, matsuzaki t, takata k. direct gene transfer into rat liver cells by in vivo electroporation. febs lett 1998; 425:436–40. 10. anderson kp, lingrel jb. glucocorticoid and estrogen regulation of a rat t-kininogen gene. nucleic acids res 1989; 17:2835–48. 11. keating a, toneguzzo f. gene transfer by electroporation: a model for gene therapy. prog clin biol res 1990; 333:491–8. 12. titomirov av, sukharev s, kristanova e. in vivo electroporation and stable transformation of skin cells of new born mice by plasmid dna. biochem biophys acta 1991; 1088:131–4. 13. andreason gl, evans ga. optimization of electroporation for transfection of mammalian cells. anal biochem 1989; 180:269–75. 14. jiao s, williams p, berg rk, hodgeman ba, liu l, repetto g. direct gene transfer into non human primate myofibers in vivo. hum gene ther 1992; 3:21–33. 15. sherr c, roberts jm. inhibitors of mammalian g1 cyclin-dependent kinases. genes dev 1995; 9:1149–63. 16. catzavelos c, bhattacharya n, ung yc, wilson ja, roncari l, sandhu c. decreased levels of the cell-cycle inhibitor p27kip1 protein: prognostic implications in primary breast cancer. nat med 1997; 3:227–30. 17. porter pl, malone ke, heagerty pj, alexander gm, gatti la, firpo ej, daling jr, robert jm. expression of cell-cycle regulators p27kip1 and cyclin e, alone and in combination, correlate with survival in young breast cancer patients. nat med 1997; 3:222–5. 18. harada k, supriatno, yoshida h, sato m. low p27kip1 expression is associated with poor prognosis in oral squamous cell carcinoma. anticancer res 2002; 22:2985–9. 19. lloyd rv, erickson la, jin l, kulig e, qian x, cheville jc. p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancer. m j pathol 1999; 154:313–23. 20. nakayama k, ishida n, shirane m, inomata a, inoue t, shishido n. mice lacking p27kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia and pituitary tumors. cell 1996; 85:707–20. 21. supriatno, harada k, kawaguchi s, yoshida h, sato m. effect of p27kip1 on the ability of invasion and metastasis of an oral cancer cell line. oncol rep 2003; 10:527–32. 22. supriatno, harada k, hoque mo, bando t, yoshida h, sato m. overexpression of p27kip1 induces growth arrest and apoptosis in an oral cancer cell line. oral oncol 2002; 38:730–6. 23. powel kt, morgenthaler aw, weaver jc. tissue electroporation. observation of reversible electrical breakdown in viable frog skin. biophys j 1989; 56:1163–71. 24. mir lm, banoun h, paoletti c. introduction of definite amounts of non permanent molecules into living cells after electropermeabilization: direct access to the cytosol. exp cell res 1988; 175:15–25. 25. goto t, nishi t, tamura t, dev sb, takeshima h, kochi m. highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. proc natl acad sci usa 2000; 97:354-9. 165165 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 165–168 review article enamel remineralisation-inducing materials for caries prevention sri kunarti1, widya saraswati1, dur muhammad lashari2, nadhifa salma1 and tasya nafatila1 1department of conservative dentistry, faculty of dental medicine, airlangga university, surabaya, indonesia 2department of oral biology, bolan university of medical and health sciences, quetta, pakistan abstract background: dental caries is a multifactorial disease indicated by the progressive demineralisation process of dental tissue. it is caused by an imbalance between the remineralisation and demineralisation processes. the focus of caries management is on prevention. providing materials that can induce remineralisation is one management of caries prevention. various materials have been or are being researched, such as casein phosphopeptide amorphous calcium phosphate (cpp–acp), tricalcium phosphate (ftcp), bioactive glass (bag), and nanotechnologies such as nano-hydroxyapatite (n-hap) and silver nano fluorine (nsf). purpose: this study aims to review the development of enamel remineralisation inducing materials as a newer approach in caries prevention. review: various ingredients have been shown to increase enamel remineralisation through different mechanisms in preventing the development of carious lesions. conclusion: cpp–acp, ftcp, bag, n-hap, and nsf can induce enamel remineralisation as caries prevention agents. n-hap and nsf are the most effective agents to enhance enamel remineralisation to prevent caries. keywords: caries; enamel; fluoride; remineralisation; bag; cpp–acp; n-hap; nsf; tcp correspondence: sri kunarti, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: sri-k@fkg.unair.ac.id introduction caries is considered one of the most common dental and oral health problems globally, including in indonesia. based on national basic health research (2018), the prevalence of dental and oral health problems in indonesia is 57.6%, while the prevalence of dental caries reaches 88.8%.1 dental caries is a disease with a multifactorial aetiology. caries is caused by the imbalance of the remineralisation and demineralisation processes. it is influenced by pathological factors such as microbes, a carbohydrate diet, poor oral hygiene and time, as well as protective factors such as antimicrobial agents, hosts, saliva and minerals in particular fluor, calcium and phosphate.2 based on the international caries classification and management system (iccms), caries management is based on the level of progress. preventive measures are the most important ways to maintain tooth structure, while restoration and recovery from damage are secondary treatments in dental practice.3 preventive dental treatment will inhibit tooth decay and maintain tooth vitality, one of which is through the principle of remineralisation. the most common therapy for dental remineralisation is fluorine therapy, which is the gold standard in increasing remineralisation.4 the remineralisation ability of fluor relies on mineral ions such as phosphate and calcium found in the oral cavity. physiologically, these ions can be found in saliva, but the amount of phosphate and calcium in the saliva is limited; thus, an extrinsic source is needed. although various studies on alternative materials to improve enamel remineralisation have been carried out, more need to be done to optimise treatment. materials that can be used include casein phosphopeptide amorphous calcium phosphate (cpp–acp), tricalcium phosphate (ftcp), bioactive glass (bag), and most recently, nanoparticles. nano-hydroxyapatite (n-hap) is a nanoparticle ranging in size from 50–1000 nano that is biocompatible with good affinity to the enamel surface.5 differing from conventional restorative approaches, consensus currently states that caries must be detected and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p165–168 mailto:sri-k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p165-168 166 kunarti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 165–168 monitored in the earliest stage when non-invasive methods can still be used, and the development of caries can be prevented. the purpose of this paper is to review enamel remineralisation inducing materials as a newer approach in caries prevention. review caries is the destruction of the hard dental tissue due to the fermentation of food carbohydrates, especially sucrose, by acidic bacteria. caries is a multifactorial disease with three main factors: microorganisms, diet and host, with time as an additional factor.2 caries occurs when there is an imbalance in the remineralisation and demineralisation processes in the oral cavity. remineralisation happens when calcium and phosphate ions in the oral cavity are deposited into enamel crystals. the fluor ion binds with calcium and phosphate ions, forming a fluorapatite unit [ca10(po4)6f2]. fluorapatite then will replace the dissolved enamel hydroxyapatite structure. fluorapatite is more acid resistant, insoluble and stronger than hydroxyapatite.6 fluor antimicrobial properties can inhibit plaque formation on teeth.7 various calcium-phosphate-based remineralisation technologies have been used and are continually being developed. this technology can improve remineralisation, especially when used in conjunction with fluorine and include cpp–acp, ftcp, bag, and n-hap. these materials provide exogenous minerals such as calcium and phosphate ions for the oral cavity. both cpp–acp and ftcp contain calcium and phosphate ions that are needed in the formation of fluoroapatite.8 bag releases calcium, phosphate, sodium and silica ions, which helps enamel remineralisation.9 the material n-hap utilises nanoparticles with morphology and properties that resemble original enamel crystals and can improve enamel integrity.5 another technology developed in the remineralisation approach is biomimetic material. it is developed by adapting the original structure of a tissue, which in this case is enamel. the biomimetic material used to increase remineralisation is amelogenin. amelogenin is one of the enamel matrix proteins that controls the growth and formation of enamel crystals and helps mineral deposition in them.10 discussion fluor affects the chemical and physical properties of apatite minerals by increasing the hardness and stability of enamel and maintaining the apatite structure. fluor can be added through various sources such as toothpaste, varnish, mouthwash, water fluoridation or supplements.11 fluor compounds commonly used in dentistry include sodium fluoride (naf), stannous fluoride and silver diamine fluoride.7 the use of fluor in various dosages and concentrations has been shown to increase enamel remineralisation. however, the anti-caries properties of fluor depend on the availability of phosphate and calcium ions in the oral cavity. the lack of availability of these ions can be a barrier to remineralisation after fluor application. a strategy to improve the anti-caries properties of fluor is therefore needed.4,11 cpp–acp is a material that interferes with the progression of carious lesions by increasing enamel remineralisation. cpp stabilises acp by binding calcium and phosphate ions to form nanoclusters with acp. it is used to maintain a supersaturation state of calcium and phosphate around the enamel to prevent demineralisation and increase remineralisation.12 in vitro studies have shown that topical application of cpp–acp can improve enamel microhardness and reduce lesion depth better than fluor.13,14 however, cpp–acp has the disadvantage of having low solubility in acidic conditions, and this causes a decrease in its ability to retain calcium and phosphate ions.15 combining fluor with cpp–acp to form cpp–acpf can increase its ability to enhance remineralisation. during in vitro studies, cpp–acpf increased enamel microhardness and reduced the depth and area of the lesion.4,16 this increase in remineralisation can be caused by the presence of calcium, phosphate and fluoride ions that will easily form fluorapatite on the enamel surface layer.17 attiguppe et al.12 reported that cpp–acpf varnish (mi varnish) showed superior effects compared to conventional fluor varnish. clinical studies have shown that patients who get fluor varnish, cpp–acp and cpp–acpf can inhibit the development of white spot lesions (wsl), which are the initial lesions of caries. both cpp–acp and cpp–acpf can regress wsl, with the cpp–acpf group showing the greatest percentage of regression.18 tricalcium phosphate in beta form that is then modified to functionalised tcp (ftcp), is another material that provides calcium-phosphate ions, which can then be administered with fluor.19 in vitro studies measuring the depth of a lesion using a polarised light microscope show that dentifrice containing fluorine ftcp can improve enamel remineralisation better than dentifrice containing fluor alone.20,21 dentifrice containing a combination of naf and ftcp showed better enamel surface microhardness than dentifrice with twice the fluor content.21 combining ftcp with fluor reduces the required fluor concentration. incorporating ftcp in fluor varnishes increases enamel microhardness and reduces the incidence of lesions better than fluor varnish alone in both in vitro and clinical studies.21 although naf varnish is considered sufficient to protect tooth enamel from caries, the addition of ftcp in the varnish should be considered. bag is a synthetic material containing ions needed for the remineralisation of enamels such as calcium, phosphate, sodium and silica. these ions will be released by bag when it comes into contact with saliva, and they will directly form a calcium-phosphate layer such as hydroxyapatite on the surface of the enamel and will continuously release mineral dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p165–168 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p165-168 167kunarti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 165–168 ions.22 based on the results of an in vitro study carried out by looking at the surface microhardness of enamel, bag has a better ability to increase enamel resistance to caries development compared to fluor.23 compared with cpp–acp, bag showed a better effect on increasing remineralisation. the two ingredients have a similar effect, and they can both increase remineralisation and prevent enamel demineralisation in the initial carious lesion.24,25 however, regarding the enamel microhardness, compared to tcp, bag has a better remineralisation effect than tcp.26 the most recent remineralisation treatment is nanotechnology that utilises materials with sizes between 1–100 nm. nano-hydroxyapatite (n-hap) has a similar structure and morphology to dental apatite crystals, so it has a good affinity with enamel by forming ionic interactions with it. n-hap acts as a provider of calcium and phosphate ions and maintains the supersaturation of these ions in the oral cavity. the nanoscopic particle size of n-hap allows it to fill in the tiny holes that form when a tooth is demineralised. n-hap will continue to bind calcium and phosphate ions, thereby increasing crystal integrity and growth.27,28 in vitro studies have shown that n-hap has a better remineralisation enhancement effect when compared to fluor. it showed good mineral content as well as increased enamel surface microhardness, and it forms a mineral-rich layer on the enamel surface so that the lost enamel structure can be restored.29,30 the use of n-hap in combination with fluor exhibits a superior effect.31 the material n-hap also showed better remineralisation effects compared to other materials such as cpp–acp, ftcp, and bag.32,33 a clinical trial proved that n-hap, ftcp and fluor varnish exhibited significant remineralising effects on early caries with n-hap use having the best effect.34 another nanotechnology is nanosilver fluorine (nsf), which contains silver nanoparticles, chitosan and fluor. dentifrice containing nsf has a bacteriostatic effect and exhibits a remineralising effect like naf. the use of nsf for enamel remineralisation can improve the performance of fluorides by the antimicrobial action of silver nanoparticles added to this compound.35,36 biomimetic material is a new development in remineralisation therapy. amelogenin is the predominant protein in forming enamel, and it plays a role in modulating and controlling the growth of calcium hydroxide crystals in enamel.37,38 amelogenin is a biomimetic material that is used to induce remineralisation. it can bind calcium and phosphate ions, facilitate ion transport into the enamel, and form a mineral layer on the surface of the enamel to increase enamel remineralisation.39 the use of amelogenin and fluor showed less mineral loss and lesion depth than fluor alone because amelogenin helps bind fluor, calcium and phosphate.40 studies on the comparison of the effectiveness of amelogenin biomimetic material with other remineralisation materials is still not widely done, so it cannot be concluded that amelogenin biomimetic material is the most superior technology. cpp–acp, ftcp, bag, n-hap and nsf are materials that can induce enamel remineralisation as caries prevention. nanotechnologies such as n-hap and nsf are the most effective ingredients to improve enamel remineralisation to prevent caries. their nano-size enhances their ability to remineralise enamel because they can penetrate deeper and form a tighter bond. it is recommended to conduct further studies regarding the development of materials that can induce enamel remineralisation. references 1. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 197, 207. 2. ritter a v. sturdevant’s art and science of operative dentistry. 7th ed. missouri: mosby elsevier; 2019. p. 544. 3. ismail ai, pitts nb, tellez m, authors of international caries classification and management system (iccms), banerjee a, deery c, douglas g, eggertsson h, ekstrand k, ellwood r, gomez j, jablonski-momeni a, kolker j, longbottom c, manton d, martignon s, mcgrady m, rechmann p, ricketts d, sohn w, thompson v, twetman s, weyant r, wolff m, zandona a. the international caries classification and management system (iccmstm) an example of a caries management pathway. bmc oral health. 2015; 15 suppl 1(1): s9. 4. fonta na m. en ha ncing f luor ide: clinical huma n studies of alternatives or boosters for caries management. caries res. 2016; 50(suppl. 1): 22–37. 5. pepla e, besharat lk, palaia g, tenore g, migliau g. nanohydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature. ann stomatol (roma). 2014; 5(3): 108–14. 6. byeon sm, lee mh, bae ts. the effect of different fluoride application methods on the remineralization of initial carious lesions. restor dent endod. 2016; 41(2): 121–9. 7. chu ch, mei ml, lo ecm. use of fluorides in dental caries management. gen dent. 2010; 58(1): 37–43; quiz 44–5, 79–80. 8. ekambaram m, mohd said snb, yiu cky. a review of enamel remineralisation potential of calciumand phosphate-based remineralisation systems. oral heal prev dent. 2017; 15(5): 415–20. 9. kathleen hj, lunardhi cgj, subiyanto a. kemampuan bioaktif glass (novamin) dan casein peptide amorphous calcium phosphate (cpp-acp) terhadap demineralisasi enamel. conserv dent j. 2019; 7(2): 111–9. 10. bronckers aljj. ion transport by ameloblasts during amelogenesis. j dent res. 2017; 96(3): 243–53. 11. eden e. evidence-based caries prevention. switzerland: springer; 2016. p. 189. 12. attiguppe p, malik n, ballal s, naik s v. cpp-acp and fluoride: a synergism to combat caries. int j clin pediatr dent. 2019; 12(2): 120–5. 13. shetty s, hegde m, bopanna t. enamel remineralization assessment after treatment with three different remineralizing agents using surface microhardness: an in vitro study. j conserv dent. 2014; 17(1): 49–52. 14. morales-vadillo r, guevara-canales j-o, garcía-rivera h-p, bazánasencios r-h, robello-malatto j-m, cava-vergiú c-e. in vitro comparison of the remineralizing effect of casein phosphopeptideamorphous calcium phosphate and fluoride varnish on early carious lesions. j int oral heal. 2019; 11(1): 45–9. 15. abidin t, nainggolan m, susi s. the effect of bilimbi (belimbing wuluh) extract (averrhoa bilimbi l) against dental remineralization dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p165–168 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p165-168 168 kunarti et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 165–168 and enamel microstructure (in vitro research). dentika dent j. 2017; 20(1): 20–6. 16. vo truong nhu n, pham thi hong t, le aq, minh st, nguyen thi thu p. the effect of casein phosphopeptide-amorphous calcium fluoride phosphate on the remineralization of artificial caries lesions: an in vitro study. j dent indones. 2017; 24(2): 45–9. 17. llena c, leyda am, forner l. cpp-acp and cpp-acfp versus f luoride varnish in remineralisation of early caries lesions. a prospective study. eur j paediatr dent. 2015; 16(3): 181–6. 18. güçlü za, alaçam a, coleman nj. a 12-week assessment of the treatment of white spot lesions with cpp-acp paste and/or fluoride varnish. biomed res int. 2016; 2016: 8357621. 19. meyer f, amaechi bt, fabritius h-o, enax j. overview of calcium phosphates used in biomimetic oral care. open dent j. 2018; 12(1): 406–23. 20. rirattanapong p, vongsavan k, saengsirinavin c, waidee s. enhancing remineralization of primary enamel lesions with fluoride dentifrice containing tricalcium phosphate. southeast asian j trop med public health. 2017; 48(2): 494–500. 21. rirattanapong p, vongsavan k, saengsirinavin c, pornmahala t. effect of fluoride varnishes containing different calcium phosphate sources on mineralization of initial primary enamel lesions. southeast asian j trop med public health. 2014; 45(6): 1503–10. 22. abbassy m, bakry a, alshehri n, alghamdi t, rafiq s, aljeddawi d, nujaim d, hassan a. 45s5 bioglass paste is capable of protecting the enamel surrounding orthodontic brackets against erosive challenge. j orthod sci. 2019; 8(1): 5. 23. vahid golpayegani m, sohrabi a, biria m, ansari g. remineralization effect of topical novamin versus sodium fluoride (1.1%) on caries-like lesions in permanent teeth. j dent (tehran). 2012; 9(1): 68–75. 24. ku m a r k , sr e e d h a r a n s. c ompa r a t ive eva lu a t ion of t h e rem ineralization potential of monof luorophosphate, casein phosphopeptide-amorphous calcium phosphate and calcium sodium phosphosilicate on demineralized enamel lesions: an in vitro study. cureus. 2018; 10(7): e3059. 25. rajendran r, kunjusankaran rn, sandhya r, anilkumar a, santhosh r, patil sr. comparative evaluation of remineralizing potential of a paste containing bioactive glass and a topical cream containing casein phosphopeptide-amorphous calcium phosphate: an in vitro study. pesqui bras odontopediatria clin integr. 2019; 19(1): 1–10. 26. jagga u, paul u, padmanabhan v, kashyap a, guram g, keswani k. comparative evaluation of remineralizing effect of novamin and tricalcium phosphate on artificial caries:an in vitro study. j contemp dent pract. 2018; 19(1): 109–12. 27. enax j, epple m. synthetic hydroxyapatite as a biomimetic oral care agent. oral health prev dent. 2018; 16(1): 7–19. 28. carrouel f, viennot s, ottolenghi l, gaillard c, bourgeois d. nanopa r ticles as anti-microbial, anti-inf lammator y, and remineralizing agents in oral care cosmetics: a review of the current situation. nanomaterials. 2020; 10(1): 140. 29. grewal n, sharma n, kaur n. surface remineralization potential of nano-hydroxyapatite, sodium monofluorophosphate, and amine fluoride containing dentifrices on primary and permanent enamel surfaces: an in vitro study. j indian soc pedod prev dent. 2018; 36(2): 158–66. 30. amaechi bt, abdulazees pa, alshareif do, shehata ma, lima pp de cs, abdollahi a, kalkhorani ps, evans v. comparative efficacy of a hydroxyapatite and a fluoride toothpaste for prevention and remineralization of dental caries in children. bdj open. 2019; 5(1): 18. 31. souza bm, comar lp, vertuan m, fernandes neto c, buzalaf mar, magalhães ac. effect of an experimental paste with hydroxyapatite nanoparticles and fluoride on dental demineralisation and remineralisation in situ. caries res. 2015; 49(5): 499–507. 32. bajaj m, poornima p, praveen s, nagaveni nb, roopa kb, neena ie, bharath kp. comparison of cpp-acp, tri-calcium phosphate and hydroxyapatite on remineralization of artificial caries like lesions on primary enamel -an in vitro study. j clin pediatr dent. 2016; 40(5): 404–9. 33. mancher y n, john j, nagappan n, subbia h gk, p rem nath p. r e m i n e r a l i z a t i o n p o t e n t i a l o f d e n t i f r i c e c o n t a i n i n g nanohydroxyapatite on artificial carious lesions of enamel: a comparative in vitro study. dent res j (isfahan). 2019; 16(5): 310–7. 34. alhamed m, almalki f, alselami a, alotaibi t, elkwatehy w. effect of different remineralizing agents on the initial carious lesions – a comparative study. saudi dent j. 2020; 32(8): 390–5. 35. teixeira ja, costa e silva av, dos santos ve, de melo pc, arnaud m, lima mg, flores map, stamford tcm, pereira jrd, targino agr, galembeck a, rosenblatt a. effects of a new nano-silver fluoride-containing dentifrice on demineralization of enamel and streptococcus mutans adhesion and acidogenicity. int j dent. 2018; 2018. 36. e. silva avc, teixeira j de a, de melo júnior pc, lima mg de s, de oliveira mota ccb, lins eccc, pereira jrd, gomes asl, targino agr, rosenblatt a. remineralizing potential of nano-silver-fluoride for tooth enamel: an optical coherence tomography analysis. pesqui bras odontopediatria clin integr. 2019; 19(1): 1–13. 37. tanimoto k, le t, zhu l, chen j, featherstone jdb, li w, denbesten p. effects of f luoride on the interactions between amelogenin and apatite crystals. j dent res. 2008; 87(1): 39–44. 38. dennis d, abidin t. role of amelogenin as predominant organic matrix in enamel biomineralization: structural and functional aspects. int j clin dent. 2018; 11(3): 173–8. 39. chu j, feng x, guo h, zhang t, zhao h, zhang q. remineralization efficacy of an amelogenin-based synthetic peptide on carious lesions. front physiol. 2018; 9(jul): 1–11. 40. ding l, han s, wang k, zheng s, zheng w, peng x, niu y, li w, zhang l. remineralization of enamel caries by an amelogeninderived peptide and fluoride in vitro. regen biomater. 2020; 7(3): 283–92. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p165–168 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p165-168 179 volume 46, number 4, december 2013 profil jaringan lunak wajah kasus borderline maloklusi klas i pada perawatan ortodonti dengan dan tanpa pencabutan gigi (facial soft tissue profile on borderline class i malocclusion in orthodontic treatment with or without teeth extraction) pinandi sri pudyani dan yenni hanimastuti bagian ortodonsia fakultas kedokteran gigi universitas gadjah mada yogyakarta indonesia abstract background: determination of orthodontic treatment plan with or without teeth extraction remains controversial, especially in borderline cases, so it requires more data and information to establish appropriate treatment plans in order to obtain optimal treatment results. purpose: the study was aimed to determine the facial soft tissue changes in the borderline class i cases treated with and without tooth extraction on post-orthodontic treatment. methods: the study was conducted on 28 lateral cephalograms, divided into two groups; 13 cases with tooth extraction, and 15 cases without tooth extraction. the subject criterias were as follows; class i malocclusion treated with straightwire technique, skeletal class i, in range of age between 18 to 30 years old, normal overjet 2-4 mm, arch length discrepancy between 2.5 to 5 mm, index of fossa canine (ifc) between 37% to 44%, did not using extraoral devices, and treated with teeth extraction of 4 second premolars or without tooth extraction. the measurement of nasolabial angle, labiomental angle, and linear position of the upper and lower lip to e-ricketts line were done on each cephalogram before and after orthodontic treatment. results: in teeth extraction cases, there was a change on upper and lower lips positions (p<0.05), but there were no changes on nasolabial angle and labiomental angle (p>0.05). in non teeth extraction cases, there were no changes in nasolabial angle, labiomental angle, and lips positions (p>0.05). both of groups also have indicated that there were no changes on linear position of the upper and lower lip (p>0.05). post-orthodontic treatment indicated a significant differences between extraction and nonextraction cases on nasolabial and labiomental angle, and lips position (p<0.05). conclusion: the facial soft tissue profile changes on teeth extraction case was more retruded than nonteeth extraction case. key words: class i malocclusion, borderline cases, facial soft tissue profile, teeth extraction, non teeth extraction abstrak latar belakang: penentuan rencana perawatan ortodonti dengan pencabutan atau tanpa pencabutan masih menjadi kontroversi, terutama pada kasus borderline, sehingga diperlukan lebih banyak data dan informasi untuk menetapkan rencana perawatan yang tepat agar didapatkan hasil perawatan optimal. tujuan: studi ini bertujuan meneliti perubahan profil jaringan lunak wajah sesudah perawatan ortodonti dengan pencabutan dan tanpa pencabutan. metode: pengukuran dilakukan pada 28 sefalogram lateral yang terdiri dari 2 kelompok, yaitu 13 sefalogram lateral untuk kasus dengan pencabutan gigi dan 15 sefalogram lateral untuk kasus tanpa pencabutan gigi. kriteria subjek penelitian adalah maloklusi klas i yang dirawat dengan teknik straightwire, hubungan skeletal klas i, berusia 18–30 tahun, overjet normal antara 2–4 mm, diskrepansi panjang lengkung antara 2,5–5 mm, indeks fossa canina (ifc) antara 37%-44%, tidak menggunakan alat ekstraoral, dan perawatan dengan pencabutan 4 premolar kedua atau tanpa pencabutan. pada tiap sefalogram dilakukan pengukuran sudut nasolabial, sudut labiomental, dan pengukuran linier posisi bibir atas dan bawah terhadap garis e ricketts sebelum dan sesudah perawatan ortodonti. hasil: pada kelompok pencabutan terdapat perubahan posisi research report 180 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 179–184 bibir atas dan bawah terhadap garis e ricketts (p<0,05), namun tidak terdapat perubahan sudut nasolabial dan sudut labiomental (p>0,05). pada kelompok tanpa pencabutan tidak terdapat perubahan pada sudut nasolabial, sudut labiomental, dan posisi bibir (p>0,05). terdapat perbedaan sudut nasolabial, sudut labiomental, dan posisi bibir antara kelompok dengan pencabutan dan tanpa pencabutan sesudah perawatan ortodonti (p<0,05). simpulan: profil jaringan lunak wajah kelompok yang dirawat dengan pencabutan gigi menjadi lebih retrusi daripada profil jaringan lunak wajah kelompok yang dirawat tanpa pencabutan. kata kunci: maloklusi klas i, kasus borderline, profil jaringan lunak wajah, pencabutan gigi, tanpa pencabutan gigi korespondensi (correspondence): pinandi sri pudyani, bagian ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: pinandi@yahoo.com pendahuluan salah satu alasan utama pasien membutuhkan perawatan ortodonti adalah untuk meningkatkan penampilan fasialnya.1 profil fasial yang seimbang dan harmonis menjadi tujuan utama perawatan ortodonti masa kini. penelitian mengenai interaksi antara perawatan ortodonti dan estetika profil fasial telah dilakukan sejak beberapa tahun yang lalu. hasil dari penelitian tersebut menunjukkan bahwa beberapa prosedur ortodonti, termasuk pencabutan gigi, dapat mempengaruhi profil jaringan lunak, namun konsekuensi dari pencabutan masih menjadi kontroversi.2 faktor utama yang menentukan kebutuhan untuk pencabutan premolar dalam perawatan ortodonti diantaranya adalah diskrepansi panjang lengkung, protrusi incisivus mandibula, curve of spee, dan protrusi bibir.3 penentuan rencana perawatan ortodonti dengan pencabutan atau tanpa pencabutan hingga saat ini masih menjadi kontroversi, terutama pada kasus-kasus borderline, sehingga diperlukan lebih banyak data dan informasi untuk menetapkan rencana perawatan yang tepat sehingga didapatkan hasil perawatan yang optimal. penelitian mengenai pengaruh perawatan ortodonti pada kasus borderline maloklusi klas i masih sangat sedikit dilakukan.1 malrelasi transversal ataupun vertikal pada rahang, seperti gigi yang berjejal dan iregularitas merupakan penyebab umum dari maloklusi klas i dan umumnya perawatan dapat dilakukan dengan pencabutan atau tanpa pencabutan gigi-gigi permanen.4 konsep pasien dalam kelompok borderline telah didiskusikan secara luas dalam ilmu ortodonti, namun hanya sedikit penelitian yang dilakukan untuk mendefinisikan istilah tersebut secara tepat.5 carey6 merupakan orang pertama yang menggunakan istilah borderline dalam literatur dan menyarankan pasien dengan diskrepansi panjang lengkung kurang dari 2,5 mm harus dirawat dengan tanpa pencabutan, sementara pasien dengan diskrepansi panjang lengkung lebih dari 5 mm harus dirawat dengan pencabutan empat premolar pertama. pasien intermediet, atau borderline, dengan diskrepansi panjang lengkung 2,5–5 mm dirawat dengan pencabutan empat premolar kedua. gigi berjejal lebih sering terjadi karena defisiensi lebar lengkung daripada defisiensi panjang lengkung. howes menemukan hubungan antara total lebar 12 gigi-gigi dari anterior hingga molar kedua dan lebar lengkung gigi pada regio premolar pertama, sehingga didapatkan rumusan, yaitu indeks fossa canina kurang dari 37% merupakan indikasi perawatan dengan pencabutan, indeks fossa canina lebih dari 44% perawatan kemungkinan dapat dilakukan tanpa pencabutan gigi-gigi, dan indeks fossa canina 37%–44% merupakan kasus borderline.7 kebanyakan penelitian yang dilakukan menilai perubahan profil fasial pada perawatan ortodonti dengan pencabutan dan tanpa pencabutan pada kasus-kasus klas ii, meskipun maloklusi klas i dengan gigi berjejal sering terjadi namun hanya sedikit penelitian yang membandingkan pengaruh perawatan dengan pencabutan dan tanpa pencabutan pada kasus-kasus klas i. masalah yang timbul dalam ortodonti adalah bagaimana merawat pasien-pasien borderline dengan gigi berjejal sedang dan keseimbangan fasial yang baik tanpa menyebabkan pengaruh negatif pada wajah.1,2 beberapa peneliti8,9 melaporkan bahwa pencabutan gigi premolar tidak menyebabkan perubahan atau peningkatan profil fasial, namun beberapa peneliti lainnya menyatakan bahwa pencabutan premolar dapat menyebabkan penampilan fasial yang rata yang tidak diinginkan.2 perbandingan perubahan sefalometri yang terjadi pada kelompok kasus borderline maloklusi klas ii yang dirawat dengan dan tanpa pencabutan premolar menunjukkan bahwa kelompok yang dirawat dengan pencabutan menghasilkan profil 2 mm lebih rata daripada kelompok yang dirawat tanpa pencabutan.10 perawatan kasus borderline maloklusi klas i yang dirawat dengan pencabutan menunjukkan perubahan jaringan lunak yang signifikan pada posisi bibir atas dan bawah, serta sudut nasolabial, sementara pada kelompok tanpa pencabutan menghasilkan retraksi bibir atas dan protraksi bibir bawah yang signifikan.1 penelitian pada subjek klas i dan klas ii menyimpulkan bahwa tidak terdapat perbedaan pada profil dan posisi bibir pada perawatan ortodonti dengan pencabutan ataupun tanpa pencabutan, sehingga pernyataan bahwa pencabutan dapat menyebabkan profil dished-in atau profil yang lebih retrusif tidak dapat diterima.11 analisis sefalometri merupakan salah satu perangkat penunjang untuk menegakkan diagnosis dan rencana perawatan. untuk mengevaluasi pertumbuhan kraniofasial, 181pudyani dan hanimastuti: profil jaringan lunak wajah kasus borderline maloklusi klas i sering dipakai garis-garis rujukan dengan metode yang telah diperkenalkan oleh beberapa ahli, diantaranya adalah ricketts. ricketts telah menganalisis kedudukan bibir terhadap garis estetik. analisis jaringan lunak ini dimaksudkan untuk mengevaluasi keseimbangan jaringan lunak fasial.12 saelens dan de smit13 membandingkan perubahan estetika fasial antara perawatan dengan pencabutan dan tanpa pencabutan. pengukuran sefalometri jaringan lunak diantaranya meliputi pengukuran sudut nasolabial, sudut labiomental, pengukuran jaringan lunak wajah menggunakan garis estetik (e). garis e digunakan sebagai referensi untuk protrusi bibir sesuai ketentuan ricketts, yaitu garis yang menghubungkan jaringan lunak ujung hidung (titik p) ke jaringan lunak pogonion (pog’). nilai normalnya adalah bibir atas 4 mm di belakang garis e dan bibir bawah 2 mm di belakang garis e. sudut nasolabial diukur dari columella (c)-subnasale (sn)-labrale superiorous (ls), sedangkan sudut labiomental diukur dari labrale inferior (li)-titik b jaringan lunak (b’)-pogonion jaringan lunak (pog’). penelitian ini bertujuan untuk mengetahui perubahan profil jaringan lunak pada kasus borderline klas i sesudah perawatan ortodontik dengan pencabutan dan tanpa pencabutan gigi. bahan dan metode sampel penelitian adalah data laporan pasien karyasiswa program pendidikan dokter gigi spesialis program studi ortodonsia fakultas kedokteran gigi universitas gadjah mada yogyakarta tahun 2000–2012 yang telah selesai perawatan ortodonti dengan kriteria sebagai berikut: 1) kasus borderline maloklusi klas i yang dirawat dengan teknik straight wire; 2) usia antara 18–35 tahun; 3) overjet 2–4 mm; 4) dilakukan pencabutan 4 premolar kedua atau perawatan tanpa pencabutan. kasus borderline klas i adalah yang memiliki diskrepansi panjang lengkung 2,5–5 mm dan indeks fossa canina antara 37– 44%, diukur dari studi model. objek penelitian adalah sefalogram lateral sebelum dan sesudah perawatan ortodonti dari pasien yang telah memenuhi kriteria pemilihan subjek penelitian. sefalogram lateral dari masing-masing subjek penelitian diseleksi kelengkapan sebelum dan setelah perawatan ortodonti dan kejelasan bidang dan titik-titik referensi. didapatkan 28 data laporan pasien yang memenuhi kriteria, terdiri dari 15 kasus borderline klas i yang dirawat tanpa pencabutan dan 13 kasus borderline klas i angle yang dirawat dengan pencabutan premolar kedua. dilakukan panapakan pada sefalogram sebelum dan setelah perawatan ortodonti dengan menggunakan kertas asetat dengan pensil 4h di atas illuminator. bidang dan titik-titik yang digunakan dapat dilihat pada tabel 1 dan gambar 1. tabel 1. sefalometri landmarks jaringan keras dan jaringan lunak sefalometri landmarks definisi pronasale (p) titik paling prominen atau paling anterior dari hidung (ujung hidung) pogonion jaringan lunak (pog’) titik terdepan dari jaringan lunak dagu dalam bidang midsagital labrale superior (ls) titik yang mengindikasikan batas mucocutaneus bibir atas, titik terdepan pada kurva bibir atas labrale inferior (li) titik median pada margin bawah dari membran bibir bawah. columella (c) septum nasal, batas bawah hidung subnasale (sn) titik di mana columella (septum nasal) menyatu dengan bibir atas dalam bidang sagital, titik paling atas paling posterior pada cekungan nasolabial titik b jaringan lunak (b’) titik tercekung pada pertengahan antara labrale inferius dan jaringan lunak pogonion. disebut juga labiomental sulcus garis estetik (e) garis yang menghubungkan jaringan lunak ujung hidung (titik p) ke jaringan lunak pogonion (pog’). sudut nasolabial sudut yang diukur dari columella (c)subnasale (sn)-labrale superiorous (ls). sudut labiomental sudut yang diukur dari labrale inferior (li)-titik b jaringan lunak (b’)pogonion jaringan lunak (pog’). gambar 1. pengukuran permukaan jaringan lunak wajah, 1. sudut nasolabial: c-sn-ls, 2. sudut labiomental: li-b’-pog’, 3. posisi bibir atas terhadap garis e, 4. posisi bibir bawah terhadap garis e.13 182 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 179–184 subjek penelitian dibagi dalam dua kelompok, yaitu: 1) kelompok pencabutan dan 2) kelompok tanpa pencabutan. jarak bibir atas dan bibir bawah terhadap garis e, sudut nasolabial, dan sudut labiomental sebelum dan sesudah perawatan diukur pada kedua kelompok menggunakan jangka sorong dan protractor sefalometri pada masingmasing sefalogram (gambar 1). pengukuran dilakukan dua kali oleh peneliti dengan selang waktu satu minggu. hasil dari kedua pengukuran dibandingkan, jika nilainya sama atau kurang dari 0,5 mm (≤ 0,5 mm) untuk pengukuran linier dan sama atau kurang dari 1° (≤ 1°) untuk pengukuran angular, maka dihitung reratanya. penapakan dan pengukuran ketiga dilakukan bila perbedaan hasil kedua pengukuran linier lebih besar dari 0,5 mm (> 0,5 mm) dan pengukuran anguler lebih dari 1° (>1°). hasilnya dibandingkan dengan kedua nilai sebelumnya, kemudian nilai rerata ditentukan dari dua nilai yang terdekat.6 data hasil pengukuran yang telah dikelompokkan, dianalisis dengan menggunakan paired t-test untuk mengetahui perubahan posisi bibir, sudut nasolabial, dan sudut labiomental sebelum dan setelah perawatan pada masing-masing kelompok, serta menggunakan independent t-test untuk membandingkan perubahan posisi bibir, sudut nasolabial, dan sudut labiomental setelah perawatan di antara kedua kelompok. hasil data hasil pengukuran pada kelompok dengan pencabutan dapat dilihat pada tabel 2. data hasil pengukuran pada kelompok tanpa pencabutan dapat dilihat pada tabel 3. uji independent t test perbandingan perubahan posisi bibir atas dan bibir bawah terhadap garis e ricketts, sudut nasolabial, serta sudut labiomental antara perawatan dengan pencabutan dan tanpa pencabutan dapat dilihat pada tabel 4. tabel 4 memperlihatkan perubahan yang bermakna posisi bibir atas, bibir bawah, sudut nasolabial, dan sudut labiomental antara perawatan ortodonti dengan pencabutan dan tanpa pencabutan. rerata perubahan posisi bibir atas terhadap garis e ricketts pada kelompok dengan pencabutan adalah -0,7938 dan pada kelompok tanpa pencabutan adalah 0,2760. tanda positif menunjukkan perubahan posisi bibir berada di depan garis e ricketts, sedangkan tanda negatif menunjukkan perubahan posisi bibir berada di belakang garis e ricketts. rerata perubahan sudut nasolabial pada kelompok dengan pencabutan adalah 3,0769 dan pada kelompok tanpa pencabutan adalah -3,9667. tanda negatif menunjukkan terjadi pengurangan besar sudut nasolabial sesudah perawatan ortodonti aktif pada kelompok tanpa pencabutan, sedangkan tanda positif menunjukkan terjadi peningkatan besar sudut nasolabial sesudah perawatan tabel 2. rerata, simpangan baku dan uji paired t-test sebelum dan sesudah perawatan ortodonti pada kelompok perawatan dengan pencabutan variabel n rerata simpangan baku t sig (p) posisi bibir atas terhadap garis e ricketts sebelum perawatan 13 0,6092 2,80062 3,380 0,005* sesudah perawatan -0,1846 2,79639 posisi bibir bawah terhadap garis e ricketts sebelum perawatan 13 3,4169 3,17221 6,475 0,000* sesudah perawatan 1,2108 2,65649 sudut nasolabial sebelum perawatan 13 90,077 14,1684 -1,623 0,131 sesudah perawatan 93,154 10,5739 sudut labiomental sebelum perawatan 13 133,308 10,6253 -1,893 0,083 sesudah perawatan 136,308 7,8992 keterangan: * : bermakna (p < 0,05) n : jumlah sampel tabel 3. rerata, simpangan baku dan uji paired t-test sebelum dan sesudah perawatan ortodonti pada kelompok perawatan tanpa pencabutan variabel n rerata simpangan baku t sig (p) posisi bibir atas terhadap garis e ricketts sebelum perawatan 15 -0,1793 2,06611 -0,720 0,483 sesudah perawatan 0,0953 2,0047 posisi bibir bawah terhadap garis e ricketts sebelum perawatan 15 2,1460 2,27210 0,260 0,798 sesudah perawatan 2,0727 2,06159 sudut nasolabial sebelum perawatan 15 90,167 12,1091 1,495 0,157 sesudah perawatan 86,200 10,1731 sudut labiomental sebelum perawatan 15 134,633 10,3880 1,341 0,201 sesudah perawatan 132,467 11,9201 keterangan: * : bermakna (p < 0,05) n : jumlah sampel 183pudyani dan hanimastuti: profil jaringan lunak wajah kasus borderline maloklusi klas i ortodonti aktif pada kelompok dengan pencabutan. hasil penelitian ini menunjukkan terjadi perubahan posisi bibir atas, bibir bawah, sudut nasolabial, dan labiomental antara perawatan ortodonti dengan pencabutan dan tanpa pencabutan pada kasus borderline klas i sesudah perawatan ortodonti aktif. pembahasan malrelasi transversal ataupun vertikal pada rahang, seperti gigi yang berjejal dan iregularitas merupakan penyebab umum maloklusi klas i, umumnya perawatan dapat dilakukan dengan pencabutan atau tanpa pencabutan gigi-gigi permanen. pada kasus-kasus borderline penentuan rencana perawatan dengan pencabutan ataupun tanpa pencabutan masih menjadi perdebatan dan kontroversi, kemungkinan profil wajah juga ikut menentukan keputusan untuk dilakukan pencabutan atau tanpa pencabutan. hasil penelitian menunjukkan pada kelompok kasus borderline klas i yang dirawat dengan pencabutan terjadi retraksi posisi bibir atas dan bibir bawah terhadap garis e ricketts sesudah perawatan ortodonti aktif. berdasarkan garis e ricketts, pada kelompok dengan pencabutan sebelum perawatan ortodonti, posisi bibir atas 0,6092 mm di depan garis e ricketts menjadi 0,1846 mm dibelakang garis e ricketts, sementara posisi bibir bawah sebelum perawatan 3,4169 mm di depan garis e ricketts menjadi 1,2108 mm di depan garis e ricketts. terjadi retraksi bibir atas sebesar -0,7938 mm dan retraksi bibir bawah sebesar -2,2061 mm (tabel 2). tanda negatif menunjukkan adanya pengurangan jarak posisi bibir terhadap garis e ricketts sesudah perawatan. hasil penelitian sesuai dengan penelitian sebelumnya yang dilakukan oleh drobocky dan smith,14 kocadereli,15 dan konstantonis.1 terjadinya retraksi bibir atas dan bibir bawah kemungkinan terjadi karena adanya retraksi gigi-gigi anterior untuk menutup ruang sisa bekas pencabutan premolar kedua. posisi bibir atas dan bibir bawah sebelum dan sesudah perawatan ortodonti pada kelompok dengan pencabutan ini masih lebih kecil bila dibandingkan dengan nilai normal dari posisi bibir atas dan bibir bawah terhadap garis e ricketts, yaitu bibir atas 4 mm di belakang garis e ricketts dan bibir bawah 2 mm di belakang garis e ricketts. keadaan tersebut kemungkinan terjadi disebabkan subjek penelitian yang digunakan adalah populasi orang indonesia, dan kebanyakan populasi orang indonesia memiliki tipe wajah yang hampir sama, yaitu protrusif bimaksila.17 terdapat variasi yang luas di antara kelompok rasial, namun kebanyakan penelitian berdasarkan profil wajah jaringan lunak dengan standar subjek ras kulit putih yang memiliki profil wajah yang lebih rata bila dibandingkan dengan profil populasi ras yang ada di asia. retraksi pada bibir atas lebih kecil daripada retraksi bibir bawah, hal ini kemungkinan disebabkan pergerakan pada bibir atas lebih sulit diprediksi karena bibir atas dipengaruhi oleh posisi bibir bawah dan berhubungan dengan adanya perlekatan bibir atas terhadap hidung.16 pada tabel 2 memperlihatkan tidak terjadi perubahan besarnya sudut nasolabial dan sudut labiomental antara sebelum dan sesudah perawatan pada kelompok yang dirawat dengan pencabutan. namun dari tabel 2 terlihat adanya peningkatan rerata sudut nasolabial pada kelompok dengan pencabutan, sebelum perawatan ortodonti sebesar 90,077º dan setelah perawatan ortodonti menjadi 93,154º, meskipun dalam penelitian perubahan sudut nasolabial sebelum dan sesudah perawatan tidak bermakna. perubahan linier pada posisi bibir atas dan bawah tidak diikuti oleh perubahan anguler sudut nasolabial dan labiomanetal. hal ini kemungkinan terjadi karena adanya perubahan pada kedalaman sulkus labial, sesuai dengan hasil penelitian garmec dan tanner2 yang menyatakan terjadi peningkatan kedalaman sulkus labial pada perawatan dengan pencabutan. meningkatnya kedalaman sulcus labial dapat terjadi karena adanya faktor pertumbuhan jaringan lunak pogonion dan resesi relatif dari titik sulkus labial inferior.2 nilai besarnya sudut nasolabial sebelum dan sesudah perawatan masih termasuk dalam rentang ideal sudut nasolabial yaitu antara 90–110º. pada kelompok tanpa pencabutan, tidak terdapat perbedaan posisi bibir atas, bibir bawah, sudut nasolabial, dan sudut labiomental antara sebelum dan sesudah perawatan ortodonti aktif. keadaan tersebut menunjukkan bahwa tidak terjadi perubahan profil wajah jaringan lunak antara sebelum dan sesudah perawatan pada kelompok yang dirawat tanpa pencabutan. berdasarkan tabel 3, tabel 4. uji independent t test antara perawatan dengan pencabutan dan tanpa pencabutan variabel n rerata simpangan baku t sig (p) posisi bibir atas terhadap garis e ricketts dengan pencabutan 13 -0,7938 0,84678 2,299 0,030* tanpa pencabutan 15 0,2760 1,47857 posisi bibir bawah terhadap garis e ricketts dengan pencabutan 13 -2,2062 1,22852 4,686 0,000* tanpa pencabutan 15 -0,0733 1,09063 sudut nasolabial dengan pencabutan 13 3,0769 6,83693 -2,098 0,041* tanpa pencabutan 15 -3,9667 10,27908 sudut labiomental dengan pencabutan 13 3,0000 5,71548 -2,282 0,031* tanpa pencabutan 15 -2,1667 6,25833 ket: * : bermakna (p < 0,05); n : jumlah sampel 184 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 179–184 terjadi sedikit perubahan pada posisi bibir terhadap garis e ricketts, sudut nasolabial, dan sudut labiomental sesudah perawatan pada kelompok tanpa pencabutan yaitu posisi bibir menjadi sedikit lebih maju serta sudut nasolabial dan sudut labiomental menjadi sedikit lebih kecil sehingga tidak mempengaruhi profil wajah secara keseluruhan. hasil penelitian tersebut sesuai dengan penelitian yang dilakukan oleh germec dan taner.2 perubahan hasil perawatan yang sangat kecil pada kelompok tanpa pencabutan tersebut kemungkinan karena hanya terjadi sedikit pergerakan ke labial gigi-gigi anterior sehingga tidak menyebabkan profil yang protrusif di akhir perawatan pada kelompok tanpa pencabutan. hal ini kemungkinan disebabkan adanya efek kompensasi jaringan lunak, keadaan ini juga ditemukan pada kasus dengan pencabutan premolar pertama dan kedua yaitu penutupan ruang bekas pencabutan dapat dilakukan tanpa mempengaruhi profil fasial.17 tabel 4 menunjukkan hasil perbandingan perubahan profil jaringan lunak wajah antara kelompok kasus borderline klas i yang dirawat dengan pencabutan dan tanpa pencabutan terdapat perbedaan dari posisi bibir atas, bibir bawah, sudut nasolabial, dan sudut labiomental. hasil penelitian ini sesuai dengan penelitian paquette dkk.,10 germec dan taner,2 serta konstantonis1 yang menyatakan bahwa pada kasus borderline perawatan dengan pencabutan premolar dapat menyebabkan profil yang retrusi daripada perawatan tanpa pencabutan premolar. secara keseluruhan dari penelitian didapatkan bahwa pada kasus borderline klas i perawatan ortodonti dengan pencabutan menyebabkan profil wajah jaringan lunak menjadi lebih retrusif daripada perawatan tanpa pencabutan. keadaan tersebut kemungkinan terjadi karena adanya gaya resiprokal dari gigi-gigi anterior dan gigi-gigi posterior pada saat penutupan ruang bekas pencabutan, sehingga terjadi retraksi pada gigigigi anterior yang mempengaruhi profil jaringan lunak wajah. pada populasi orang indonesia dengan mayoritas tipe wajah adalah protrusi bimaksila, perawatan ortodonti pada kasus borderline klas i dengan pencabutan 4 premolar kedua yang menghasilkan profil wajah yang lebih retrusif daripada perawatan tanpa pencabutan, kemungkinan akan lebih disukai. studi ini menunjukkan bahwa profil wajah jaringan lunak kasus borderline maloklusi klas i yang dirawat dengan pencabutan profil wajah menjadi lebih retrusi daripada kasus borderline maloklusi klas i yang dirawat dengan tanpa pencabutan. ucapan terima kasih terima kasih kepada fakultas kedokteran gigi universitas gadjah mada atas dana penelitian dana masyarakat 2013, dan program pendidikan dokter gigi spesialis ortodonsia universitas gadjah mada yogyakarta atas ijin penelitian yang diberikan. daftar pustaka 1. konstantonis d. the impact of extraction vs nonextraction treatment on soft tissue changes in class i borderline malocclusions. angle orthod 2011; 82: 209-17. 2. germec d and taner u. effects of extraction and nonextraction therapy with air-rotor stripping on facial esthetic in postadolescent borderline patients. am j orthod dentofac orthop 2008; 133: 539-49. 3. lim hj, ko kt, and hwang hs. esthetic impact of premolar extraction and nonextraction treatments on korean borderline patients. am j orthod dentofac orthop 2008; 133: 524-31. 4. aksu m and kocadereli i. arch width changes in extraction and nonextraction treatment in class i patients. angle orthod 2005; 75: 948-52. 5. xu t, liu y, yang m, and huang w. comparison of extraction versus nonextraction orthodontic treatment outcomes for borderline chinese patients. am j orthod dentofac orthop 2006; 129: 672-7. 6. hanimastuti h, pudyani ps, sutantyo d, profil bibir dan posisi insisivus perawatan kasus borderline klas i dengan pencabutan dan tanpa pencabutan. majalah kedokteran gigi 2013; 20(2): 132-9. 7. bhalajhi si, 2004, orthodontics the art of science, arya, kuwait. p. 69-70, 176-7, 259-61. 8. james rd. a comparative study of facial profiles in extraction and nonextraction treatment. am j orthod dentofac orthop 1998; 114: 265-76. 9. bowman sj, johnston jr le. the esthetic impact of extraction and nonextraction treatments on caucasian patients. angle orthod 2000; 70: 3-10. 10. paquette de, beattie jr, and johnston le jr. a long-term comparison of nonextraction and premolar extraction edgewise therapy in “borderline” class ii patients. am j orthod dentofac orthop 1992; 102: 1-14. 11. bascifti fa, usumez s. effects of extraction and nonextraction treatment on class i and class ii subject. angle orthod 2003; 73: 36-42. 12. kusnoto h. penggunaan cepalometri radiografi dalam bidang ortodonti, bagian ortodonti fkg universitas trisakti, jakarta, 1977; 3-15. 13. saelens na, de smit aa. therapeutic changes in extraction versus non-extraction orthodontic treatment. am j orthod dentofac orthop 1998; 20: 225-36. 14. l ew k. p rof ile cha nge following or thodontic t reatment of bimaxillary protrusion in adult with the begg appliance. eur j orthod 1989; 11: 375-81. 15. drobocky ob, smith rj. changes in facial profile during orthodontic treatment with extraction of four first premolars. am j orthod dentofac orthop 1989; 95: 220-30. 16. kocadereli i. changes in soft tissue profile after orthodontic treatment with and without extractions. am j orthod dentofac orthop 2002; 122: 67-72. 17. kusnoto j, kusnoto h. the effect of anterior tooth retraction on lip position of orthodontically treated adult indonesians, am j orthod dentofac orthop 2001; 120: 304-7. 18. wholley cj, woods mg. the effects of commonly prescribed premolar extraction sequences on the curvature of the upper and lower lips. angle orthod 2003; 73: 386-95. �� volume 47, number 1, march 2014 alkaline phosphatase expression during relapse after orthodontic tooth movement pinandi sri pudyani,1 widya asmara,2 ika dewi ana3 and tita ratya utari4 1 department of orthodontics, faculty of dentistry, universitas gadjah mada 2 department of microbiology, faculty of veterinary medicine, universitas gadjah mada 3 department of dental biomedical sciences, faculty of dentistry, universitas gadjah mada 4 department dental science, faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: the increasing of osteoblast activities during bone formation will be accompanied with the increasing expression of alkaline phosphatase enzyme (alp). alp can be obtained from clear fluid excreted by gingival crevicular fluid (gcf). bone turnover, especially bone formation process, can be monitored through the expression of alp secreted by gcf during orthodontic treatment. thus, retention period is an important period that can be monitored through the level of bone metabolism around teeth. purpose: this research were aimed to determine the relation of distance change caused by tooth relapse and alp activities in gingival crevicular fluid after orthodontic; and to determine alp as a potential biomarker of bone formation during retention period. methods: lower incisors of 25 guinea pigs were moved 3 mm to the distally by using open coil spring. those relapse distance were measured and the gingival crevicular fluid was taken by using paper points to evaluate alp levels on days 0, 3, 7, 14 and 21 respectivelly by using a spectrophotometer (405 nm). t-test and anova test were conducted to determine the difference of alp activities among the time intervals. the correlation regression analysis was conducted to determine the relation of distance change caused by the relapse tooth movement and alp activities. results: the greatest relapse movement was occurred on day 3 after open coil spring was removed. there was significant difference of the average of distance decrease among groups a1-a5 (p<0.05). it was also known that alp level was increased on day 3, but there was no significant difference of the average level of alp among groups a1-a5 (p>0.05). finally, based on the results of correlation analysis between the alp level decreasing and the relapse distance on both right and left of mesial and distal sides, it is known that there was no relation between those two variables (p>0.05). conclusion: it can be concluded that relapse after orthodontic tooth movement occurs rapidly as the teeth are free from orthodontic force. alp level can be detected through gingival crevicular fluid during relapse by using a spectrophotometer. key words: relapse distance, alkaline phosphatase, gingival crevicular fluid, guinea pigs abstrak latar belakang: peningkatan aktivitas osteoblas selama pembentukan tulang akan disertai peningkatan ekspresi enzim alkalin fosfatase (alkaline phosphatase/alp). sumber alp dapat diperoleh dari cairan bening yang diekskresi celah gingiva gigi yang dikenal sebagai cairan krevikuler gingiva (gingival crevicular fluid/gcf). bone turnover terutama proses pembentukan tulang dapat dimonitor melalui ekspresi alp cairan krevikuler gingiva selama perawatan ortodonti. periode retensi merupakan periode yang penting. kesulitan memecahkan masalah retensi akan dapat ditangani dengan memonitor tingkat metabolisme tulang disekitar gigi. tujuan: penelitian ini bertujuan untuk meneliti perubahan jarak relapse gigi dan aktivitas alp pada cairan krevikuler gingiva setelah digerakkan secara ortodonti dan potensi alp sebagai biomarker pembentukan tulang pada periode retensi. metode: gigi insisivus bawah 25 ekor marmot digerakkan ke distal menggunakan opencoil spring sampai mencapai jarak ± 3 mm, diukur gerakan relapse gigi dan pengambilan cairan krevikuler gingiva menggunakan paper point untuk dievaluasi kadar alp pada hari ke 0, 3, 7, 14 dan 21 menggunakan spektrofotometer (405 nm). analisis t-test dan anova untuk mengetahui perbedaan aktivitas alp antar interval waktu dan analisis regresi korelasi untuk mengetahui hubungan besarnya jarak relapse dengan aktivitas alp. hasil: pergerakan relapse yang research report �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 25–30 paling besar terjadi pada hari ke 3 setelah opencoil spring dilepas. terdapat perbedaan rata-rata penurunan jarak antar kelompok a1-a5 yang signifikan (p<0,05). kadar alp mengalami peningkatan pada hari ke 3, namun tidak terdapat perbedaan rata-rata yang signifikan kadar alp antar kelompok a1-a5 (p>0,05). hasil uji korelasi antara penurunan jarak dengan kadar alp pada mesial distal gigi baik kanan maupun kiri tidak menunjukkan adanya hubungan kedua variabel (p>0,05). simpulan: relapse pada perawatan ortodonti terjadi secara cepat ketika gigi terbebas dari gaya ortodonti. kadar alp dapat terdeteksi dari cairan krevikuler gingiva pada pergerakan relapse gigi menggunakan spektrofotometer. kata kunci: jarak relapse, alkaline fosfatase, cairan krevikuler gingiva, guinea pigs correspondence: tita ratya utari, c/o: ilmu kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: tita_utari@yahoo.com introduction tooth movement actually occurs in the same direction as bone remodeling response occurs, such as bone resorption which is in the area of pressure and bone apposition on the area of tension in the periodontal ligament.1 orthodontic tooth movement is based on biological principle, in which the provision of sustained pressure on teeth will cause alveolar bone remodeling, triggering balance between bone formation occurred at the tension area and bone resorption in the pressure area.2 the results of orthodontic treatment will become so unstable that the use of orthodontic retainer is necessary. these devices are aimed to maintain teeth in a new position after the completion of active treatment and orthodontic appliances has been removed.3 therefore, treatment during retention period is as important as active period since there is a tendency to return to the previous positions prior to the treatment or relapse.4 relapse in orthodontic treatment can occur quickly, so it is important to provide immediate retention appliance.5 bone remodeling that occurs during orthodontic tooth movement is a biological process that involves an acute inflammatory response in periodontal tissue.6 in the initial phase of orthodontic tooth movement, an acute inflammatory response is involved together with periodontal vasodilatation and leukocyte migration out of the blood vessels of periodontal ligament.7 thus, bone remodeling can be considered as complex process, including bone resorption and bone formation requiring the coordination of osteoclasts, osteocytes and osteoblasts.8 it means that the provision of orthodontic pressure with continuous force can cause bone resorption and bone formation at the same time since in the pressure area the activities of tartrateresistant acid phosphatase (trap) with positive osteoclasts and osteoblasts will be increasing.9 the increasing of osteoblast activities during bone formation then will be accompanied by the increasing of alkaline phosphatase enzyme expressions (alkaline phosphatase/alp).10 alkaline phosphatase is a hydrolase enzyme that works as a phosphatase removing oxygen groups and phosphate groups from many types of other molecules, including nucleotide, proteins and alkaloids. in addition, this enzyme is called as alkaline phosphatase because it works in alkaline conditions (non-acidic) at ph 10, consequently, sometimes it is referred to basic phosphatase breaking phosphate (mineral acidic) and then creating alkaline ph 11. besides that alkaline phosphatase is synthesized and secreted by osteoblasts during bone formation, it also catalyzes the hydrolysis of ester phosphatase, which is a potent inhibitor of mineralization process in the alkaline ph associated with the formation of tissue calcification.12 alkaline phosphatase can be obtained from clear fluid excreted from gingival crevicular fluid (gcf). this gingival crevicular fluid can describe body’s biological response to periodontal healing process in patients with chronic periodontitis13 or can stimulate orthodontic mechanism6,13,14 and can be obtained in various ways, such as micro pipetted or paper strips.15 gingival crevicular fluid, moreover, is exudate liquid composed of substances derived from various sources, including microbial dental plaque, inflammatory host cells, and host tissue and serum. the last few years, cgf has been used as a diagnostic marker of active tissue destruction in periodontal disease. however, there are only a few researches focusing on the content of gcf involved in bone remodeling during orthodontic tooth movement. thus, it can be said that alp expression can describe biochemical changes occurred in supporting tissues after the provision of orthodontic pressure.16 in some researches, the increasing of alp levels can be detected during orthodontic tooth movement in week 1 to 3.13 alkaline phosphatase activities in gcf during orthodontic treatment can be associated with treatment time and pressure given on periodontal tissue.17 relapse after orthodontic treatment has the same process as orthodontic tooth movement. the positive change of the number of trap cells and their distribution along the alveolar bone and molars moved as well as the tooth next to them will cause bone resorption in relapse direction. simultaneously, new bone formation then will occur in the area opposite to positive trap cell activities. it can be said that relapse is like orthodontic tooth movement since in both processes osteoclast differentiation in pressure area is increasing, while that in tension area is decreasing. this indicates that alveolar bone is an important element ��pudyani, et al.: alkaline phosphatase expression during relapse in relapse process.5 in other words, relapse is affected by occlusal instability, increased mechanical tension influenced by transepted fibers and alveolar bone resorption caused by osteoclasts. thus, relapse will occur if there is bone resorption caused by osteoclasts.18 for those reasons, examining gingival crevicular fluid can be considered as a good way to analyze biochemical process continuously occurred in relation to bone metabolism (bone turnover) during orthodontic tooth movement.19 examining gingival crevicular fluid can also be considered as a biological evaluation of the results of orthodontic force, so management of tools can be based on tissue response individually, and effectiveness of dental treatment can be improved. besides that, difficulty to solve retention problem can be addressed by monitoring the rate of bone metabolism (bone turnover) around the teeth. thus, bone alkaline phosphatase and osteocalcin can be considered as the best marker for bone formation in serum, while deoksipiridinolin and n-telopeptida crosslinks as degradation product of collagen secreted through urine can be considered as the most specific marker for systemic osteoclast activity as a bone marker in gingival crevicular fluid.20 in other words, by selecting the appropriate biomarkers of bone turnover, the progress of each individual treatment can be monitored, and the amount of force applied can be modified to prevent iatrogenic effect.21 thus, bone turnover as primarily a process of bone formation must be monitored through the expression of alp in gingival crevicular fluid during orthodontic treatment.22 the aim of this study were to determine the changes of relapse length and alp activity on the gingival crevicular fluid after orthodontic tooth movement and to assess whether alp can be used as a biomarker of bone remodeling on the retention period. materials and methods this research was an experimental laboratory research using 25 local guinea pigs since they can be taken care easily and can survive with a long time treatment. those animals then became the samples of the research based on certain criteria: weight, male, 6-8 weeks old, 0.5-0.6 kg. then the application of ethical approval was submitted to the research ethics committee of dentistry faculty, universitas gadjah mada. afterwards, acclimatization of those guinea pigs had were conducted for one week before they got treatment for adaptation with the place and food in the laboratory next, all treatments in this research were conducted in in lppt unit iv at universitas gadjah mada. those samples selected were anaesthetized with ketamine (0.1 mg) and xylasin (0.1 mg) by intramuscular injection in thigh of guinea pigs. then, bonding cleat was inserted on the lower incisors of each sample. round steel wire with a diameter of 0.016 and an open coil spring with a length of 1.5 times the inter cleat distance were set between the cleats on each samples (figure 1). open coil spring is commonly used in fixed orthodontic treatment for creating space. niti wire with the smallest size of 0.010 “x 0.045” and a length of 1.5 times the length between the cleats of the lower incisor was also used. with the compression of the open coil about 25%, force resulted will be 0.25 n-1.3 n, while with the compression of the open coil about 50%, force resulted will be about 0.64n-2.9n.23 after the tooth moved and the open coil spring was not in passive condition, it was replaced with the new one with the new inter-incisor distance until the distance was about ± 3 mm (usually takes about 14 days). the distance of ± 3 mm was maintained for one week as a period of stabilization. after one week stabilization, the open coil and the wire were removed. those twenty-five samples were divided into five groups, each of which consisted of five. the relapse distance of each sample in each group was measured (figure 2a). gingival crevicular fluid of each sample in each group was taken on days 0, 3, 7, 14 and 21 respectivelly. gingival crevicular fluid was taken by cleaning the lower incisors with cotton to remove supra-gingival plaque, and then those teeth were isolated with cotton rolls and dried. next, paper points were inserted approximately 1 mm into the gingival sulcus (figure 2b) of each sample for 30 seconds with an interval of 90 seconds to increase the volume of gcf fluid taken each side, and then immediately inserted eppendorf tubes with the size of 1.5 ml containing 350 ml of physiological saline solution. afterwards, those eppendorf tubes were centrifuged for five minutes at 2000 g to elute gcf components completely. alkaline phosphatase levels figure 1. process of tooth movement: a) lower incisors; b) separator setting, c) etchant application; d) bonding cleat setting; e) open coil spring setting; f) tooth movement. �8 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 25–30 contained in the gingival crevicular fluid were measured by using paper points on day 0 (a1), day 3 (a2), day 7 (a3), day 14 (a4) and day 21 (a5). next, paper points were taken, and then the supernatant solution was stored at -80° c for one week.22 examination of alp activities was conducted at laboratory of molecular biology, faculty of medicine, universitas gadjah mada. alkaline phosphatase activities were determined using a spectrophotometer (jenway 6330, uk) at a wavelength of 405 nm. approximately 50 ml of 40 mm carbonate buffer at ph 9.8 mixed with 3 mm mgcl2 was put into eppendorf tubes by using pipettes. fifty ml of gcf samples and 50 ml of 3mm p-nitrophenylphosphate were added to the same tubes. the tubes were then incubated for 30 min at 37° c. the enzymatic reaction was stopped by adding 50 ml of 0.6 m sodium hydroxide, and then the absorbance was measured immediately at a wavelength of 405 nm. the amount of p-nitrophenol formed was measured by using a standard curve prepared from phosphatase subtrate (sigma 104, sigma-aldrich, st. louis, usa). alkaline phosphatase activities were presented in the form of enzyme unit (u). u is defined as the amount of p-nitrophenol (mol) released per minute at 37° c.22 the data were analyzed by using spss, and then the difference of variables between sub-groups was measured with the t-test and anova test. finally, the relation of the relapse distance and alp levels was analyzed by using correlation and regression analyses. results the average of the decreasing of the relapse distance after open coil was removed on day 3 was about 0.8 mm (29.2% from the early distance), while on day 7 was 1 mm (37% from the early distance). moreover, the average f the decreasing of the relapse distance on day 14 was 1.7 mm (51.5% from the early distance), while on day 21 was 1.83 mm (90.1% from the early distance). in other words, it is known that the relapse distance from day 0 to day 3 was 0.8 mm, while from day 3 to day 7 was about 0.2. furthermore, the relapse distance from day 7 to day 14 was 0.7, while from day 14 to day 21 was 0.13 (table 1). therefore, the greatest relapse distance occurred on day 3 after open coil spring was removed (figure 3). to determine alp levels, gingival crevicular fluid was taken from both right and left of mesial and distal sides of those two incisor teeth (table 2). based on the data, figure 2. a) measuring the relapse distance using sliding caliper; b) taking gingival crevicular fluid using paper point. table 1. the average of the decreasing of the relapse distance the average of the decreasing of the relapse distance (mm) from day 0 to day 3 0.8 ± 0.19039 from day 0 to day 7 1 ± 0.15411 from day 0 to day 14 1.7 ± 0.17678 from day 0 to day 21 1.83 ± 0.30332 from day 3 to day 7 0.2 ± 0.24238 from day 7 to day 14 0.7 ± 0.20917 from day 14 to day 21 0.13 ± 0.32519 figure 3. a) tooth movement was finished; b) bracket was removed; c) relapse on day 3. table 2. the average of alp levels alp levels (alkaline phosphatase) (iu/i) ± sd right distal right mesial left distal left mesial day 0 (a1) 0.0944 ± 0.11106 0.111 ± 0.15544 0.0296 ± 0.02074 0.267 ± 0.44658 day 3 (a2) 0.1511 ± 0.24803 0.4568 ± 0.51127 0.071 ± 0.13888 0.2417 ± 0.241 day 7 (a3) 0.0258 ± 0.00645 0.1232 ± 0.21321 0.0808 ± 0.1089 0.0486 ± 0.05167 day 14 (a4) 0.07236 ± 0.06431 0.1722 ± 0.23505 0.1438 ± 0.19187 0.0894 ± 0.08476 day 21 (a5) 0.148 ± 0.13218 0.107 ± 0.15902 0.0622 ± 0.07409 0.1182 ± 0.13054 ��pudyani, et al.: alkaline phosphatase expression during relapse it is known that significance values obtained were 0.726; 0.432; 0.388; dan 0.724 (>0.05). it means that there was no significant difference of alp levels among groups a1-a5 in both right and left of mesial and distal sides. based on the results of the correlation test, it is known that there was no relation between the decreasing of the relapse distance and alp levels in both right and left of mesial and distal sides since the significance value was >0.05. discussion based on three-day observation of relapse, it was known that the quickest and greatest movement was on day 3 after orthodontic appliances removed. however, the relapse rapidly occurred only at the beginning of orthodontic appliances removal, since after the next 3 days both relapse distance and its percentage began to decline gradually. thus, it can be indicated that the same processes occurred at orthodontic relapse movement and orthodontic tooth movement, by increasing of osteoclast differentiation in the pressure area and decreasing of pressure in the tension area. thus, it can be said that alveolar bone can be considered as an important element in the process of relapse.5 it is also known that alkaline phosphatase activities are usually higher in the periodontal ligament than those in the other connective tissue.14 thus, to examine the activities, gingival crevicular fluid was taken to determine the level of alp on day 0 (at the time of cleats and open coil spring removed). the changes occurred directly on the periodontium and the rapid movement in the area of the periodontal ligament were observed to determine the enzyme activities on day 3 after orthodontic appliances removed. it is because on days 7 the enzyme activities are expected to show exactly at the final phase of tooth movement when hyalinisation occurs, and on day 14 and day 21, the enzyme activities are expected to go to the continuation phase or to the final phase.14 in this research, alp levels in gingival crevicular fluid was detected by using a spectrophotometer with pnitrophenol as a standard solution. in general, the enzyme activities generated in this research seemed much less than previous studies likely due to the lack of pressure exerted during the retention period. in this research, acid and alkaline phosphatases were released due to the pressure, injury and death of extracellular tissue fluid. these enzymes produced by the periodontium then diffused in gcf as the result of the application of orthodontic force. thus, it needs to monitor phosphatase activities in gcf causing tissue changes during orthodontic tooth movement. similarly, the results of a research on mice shows that phosphatase activities may reflect bone turnover in orthodontic tooth movement.24 the results of the previous research14 also show that there were significant changes (p<0.05) of alkaline phosphatase activities on days 7, 14 and 21 on both right and left of mesial and distal sides between in the treatment side and in the control side. it is also known that the highest enzyme activities occurred on day 14 in the initial phase, but they were significantly decreased, especially on the mesial side. similarly, the results of a research conducted by yokoya et al.25 showed that osteoclasts on the pressure area are increased on day 7, but then they are decreased rapidly on day 14 when the highest enzyme activities occur. this indicates that alp activities are followed with tooth movement during initial phase. in contrast, in this research there was no significant difference in alp levels between groups a1-a5 on both the right and left of the mesial and distal sides (p>0.05). in this research, it is known that the greatest movement occurred on day 0 to day 3, and alp levels were increased on day 3. however, the decreasing of this relapse distance in this research was not so large since there was no significant difference among groups. in addition, many clinical researches also showed that there was a correlation between alveolar bone remodeling with changes in phosphatase activities contained in gcf.17 in this research, there was no correlation between the decreasing of alp levels on both right and left of mesial and distal sides with the relapse distance (p>0.05). it is also known that the process of bone remodeling with initial resorption activity occurred on days 3-5 days, and repeatedly on days 5-7. the final bone deposition occurred in both the pressure and tension areas on the walls of the alveolar bone on days 7-14. in the initial phase, bone resorption occurs more greatly than bone deposition, but in the next phase, both bone resorption and deposition occurs synchronically.26 alp levels in this research then were measured when the stabilization of the relapse movement had been done for 1 week so that both bone resorption and deposition were probably already equal. thus, there was no significant difference among groups or between the mesial and distal sides. the study showed that orthodontic relapse occurs rapidly when teeth free of orthodontic force. alp levels can be detected from gingival crevicular fluid during relapse tooth movement using spectrophotometer with a wavelength of 405 so that we can used as a potential biomarker of bone formation in the retention periode. there was no correlation between the alp levels with the relapse distance. acknowledgement the author wish to thanks the faculty of dentistry universitas gadjah mada for grand research dana masyarakat 2013. references 1. krishnan v, davidovitch z. on a path to unfolding the biological mechanisms of orthodontic tooth movement. j dent res 2009; 88(7): 597-608. �0 dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 25–30 2. king gj, keeling sd. orthodontic bone remodeling relation to appliance decay. angle orthod 1995; 65(2): 129-40. 3. proffit wr, fields hw jr. contemporary orthodontics. 2nd ed. st. louis: cv mosby co; 2000. p. 455. 4. dyken ra, sadowsky pl, hurst d. orthodontic outcomes assesment using the peer assesment rating index. angle orthod 2001; 71(3): 164-9. 5. franzen tj, brudvik p, vandevska-radunovic v. periodontal tissue reaction during orthodontic relapse in rat molars. eur j orthod 2013; 35(2): 152-9. 6. perinetti g, paolantinio m, d’attilio m, d’archivio d, tripodi d, femminella b, festa f, spoto g. alkaline phosphatase activity in gingival crevicular fluid during human orthodontic tooth movement. am j orthod dentofacial orthop 2002; (5): 548-56. 7. davidovitch z, nicolay of, ngan pw, shanfeld jl. neurotransmitters, cytok ines, a nd the cont rol of a lveola r bone remodeling in orthodontics. dent clin north am 1988; 32(3): 411-35. 8. su m, borke jl, donahue hj, li z, warhawsky nm, russel cm, lewis je. expression of connexin 43 in rat mandibular bone and periodontal ligament (pdl) cells during experimental tooth movement. j dent res 1997; 76(7): 1357-66. 9. bonafe-oliveira l, faltin rm, arana-chavez ve. ultrastructural and histochemical examination of alveolar bone at the pressure areas of rat molars submitted to continuous orthodontic force. eur j oral sci 2003; (5): 410-6. 10. i nt a n z z a, sha h r ul h, rohaya m aw, sa h ida n s, za ida h za. osteoclast and osteoblast development of mus musculus haemopoietic mononucleated cells. j biological sci 2008; 8(3): 506-16. 11. sara f, saygili f. causes of high bone alkaline phosphate. j mol biol 2001; 310(4): 149-54. 12. stucki u, schmid j, hämmerle cf, lang np. temporal and local appearance of alkaline phosphatase activity in early stage of guided bone regeneration. clin oral implants res. 2001; 12(2): 121-7. 13. perinetti g, paolantonio m, serra e, d’archivio d, d’ercole s, festa f, spoto g. longitudinal monitoring of subgingival colonization by actinobacillus actinomycetemcomitans, and crevicular alkaline phosphatase and aspartate aminotransferase activities around orthodontically treated teeth. j clin periodontol 2004; (1): 60-7. 14. batra p, kharbanda o, duggal r, singh n, parkash h. alkaline phosphatase activity in gingival crevicular fluid during canine retraction. orthod craniofac res 2006; 9(1): 44-51. 15. ozmeric n. advance in periodontal disease markers. clin chim acta 2004; 343(1-2): 1-16. 16. dhopatkar aa, sloan aj, rock wp, cooper pr, smith aj. a novel in vitro culture model to investigate the reaction of the dentine-pulp complex to orthodontic force. j orthod 2005; 32(2): 122-32. 17. insoft m, king gj, keeling sd. the measurement of acid and alkalin phosphatase in gingival crevidular fluids during orthodontic tooth movement. am j orthod dentofacial orthop 1996; 109(3): 28796. 18. sadowsky c, schneider bj, begole ea, tahir e. long-term stability after orthodontic treatment: nonextraction with prolonged retention. am j orthod dentofacial orthop. 1994; 106(3): 243-9. 19. isik f, sayinsu k, arun t, ünlüçerçi y. bone marker levels in gingival crevicular fluid during orthodontic intrusive tooth movement: a preliminary study. j contemp dent pract 2005; 6(2): 27-35. 20. eyre dr. bone biomarkers as tools in osteoporosis management. spine (phila pa 1976). 1997; 22(24 suppl): 17s-24s. 21. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofacial orthop 2006; 129(4): 469. 22. asma aaa, megat awr, zainal ash. crevicular alkaline phosphatase activity during orthodontic tooth movement: canine retraction stage. j med sci 2008; 8(3): 228–33. 23. brauchli lm, senn c, ball j, wichelhaus a. force levels of 23 nickel-titanium open-coil springs in compression testing. am j orthod dentofacial orthop 2011; 139(5): 601-5. 24. keeling sd, king gj, mccoy ea, valdez m. serum and alveolar bone phosphatase changes reflect bone turnover during orthodontic tooth movement. am j orthod dentofacial orthop 1993; 103(4): 320-6. 25. yokoya k, sasaki t, shibasaki y. distributional changes of osteoclasts and pre-osteoclastic cells in periodontal tissues during experimental tooth movement as revealed by quantitative immunohistochemistry of h(+)-atpase. j dent res 1997; (1): 580–7. 26. king gj, latta l, rutenberg j, ossi a, keeling sd. alveolar bone turnover and tooth movement in male rats after removal of orthodontic appliances. am j orthod dentofacial orthop 1997; 111(3): 266–75. 80 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 80–85 research report the influence of artificial salivary ph on nickel ion release and the surface morphology of stainless steel bracket-nickel-titanium archwire combinations ida bagus narmada,1 natalya tanri sudarno,1 achmad sjafei,1 and yuli setiyorini2 1department of ortodontics, faculty of dental medicine, universitas airlangga 2department of materials and metalurgical engineering, faculty of technology industry, sepuluh nopember institute of technology surabaya – indonesia abstract background: in the oral cavity, orthodontic appliances come into contact with saliva which may cause corrosion capable of changing their surface morphology due to the release of metal ions. surface roughness can influence the effectiveness of tooth movement. one of the ions possibly released when body fluid comes into contact with brackets and archwire is nickel ion (ni). ni, one of the most popular components of orthodontic appliances, is, however, a toxic element that could potentially increase the likelihood of health problems such as allergic responses during treatment. purpose: the purpose of this study was to investigate the effect of different artificial salivary ph on the ions released and the surface morphology of stainless steel (ss) brackets-nickel-titanium (niti) and archwire combinations. methods: brackets and archwires were analyzed by an energy dispersive x-ray detector system (edx) to determine their composition, while niti archwire compound was examined by means of x-ray diffraction (xrd). the immersion test was performed at artificial salivary ph levels of 4.2; 6.5; and 7.6 at 37°c for 28 days. ni ion release measurement was performed using an atomic absorption spectroscopy (aas). surface morphology was analyzed by means of a scanning electron microscopy (sem). results: the chemical composition of all orthodontic appliances contained ni element. in addition, xrd was depicted phases not only niti but also ni, titanium, silicon and zinc oleate. the immersion test showed that the highest release of ni ions occured at a ph of 4.2, with no significant difference at various levels of ph (p=.092). there were surface morphology changes in the orthodontic appliances. it was revealed that at a ph of 4.2, the surfaces of orthodontic appliances become unhomogenous and rough compared to those at other ph concentrations. conclusion: the reduction of ph in the artificial saliva increases the amount of released ni ions, as well as causing changes to the surface morphology of brackets and archwires. keywords: salivary ph; ni ions release; niti archwires; ss brackets correspondence: ida bagus narmada, department of orthodontics, faculty of dental medicne, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ida-b-n@fkg.unair.ac.id introduction in the present day, the demand for orthodontic treatment is increasing.1 two main components are frequently employed in this form of dental treatment: brackets and archwire. the most common bracket material is stainless steel (ss) because of its affordability and appropriate physical properties. the generally-used archwire material is nickel titanium (niti) due to its favorable results with regard to tooth movement.2 in the oral cavity, orthodontic appliances invariably come into contact with saliva which produces a electrochemical reaction commonly referred to as corrosion.3 this process has the potential to change the chemical structure of orthodontic appliances and can only be measured by analyzing the ions released.4 one of the ions which can potentially be released and often causes allergic reactions is nickel (ni).5,6 the potential negative health effects resulting from exposure to ni and its compounds have been investigated dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 8181narmada, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 80–85 for over a century, with it being established that ni can induce hypersensitivity, dermatitis, and asthma.7 in addition, ni can cause dna damage to oral mucosa, suppression of chemotaxis of leukocytes, and changes in enzyme activity.8,9 ni is known to be an indispensable element for both human beings and animals. in general, the most significant exposure for humans occurs through diet, air, drinking water, and jewellery with the average dietary intake of ni estimated to be 200-300 μg/day.10 there are several factors potentially affecting the release of metal ions from orthodontic appliances, including; alloy composition, salivary ph, and duration of use.9 in this study, an evaluation of the alloy composition was conducted by means of an energy dispersive x-ray detector system (edx) and x-ray diffraction (xrd) in order to identify their compounds. the effect of salivary ph on the ni ions released was analyzed using an atomic absorption spectroscopy (aas). the release of ions can also be observed by examining the surface morphology of orthodontic appliances.11 that of a stainless steel bracketsniti archwire combination was analyzed by means of a scanning electron microscopy (sem). this in vitro study was performed through a classic procedure involving the immersion of the sample in artificial saliva at various ph values over an extended time period. the purpose of this investigation was to examine the influence of salivary ph on the release of ni ions and surface morphology changes in ss brackets-niti archwire combinations. materials and methods the artificial saliva employed consisted of fusuyama meyer artificial saliva (morvabon, tehran, iran) with the following chemical composition: 0.4 gr nacl; 0.4 gr kcl; 0.795 gr cacl2.h2o; 0.78 gr nah2po4.h2o; 0.005 gr na2s.9h2o; 1 gr urea and 1000 ml distilled water at 37 ± 1°c. the artificial saliva solution was adjusted with a small amount of hcl and measured potentiometrically using a ph meter (ebro® electronic pth 810, germany) to provide a certain level of ph. the levels of salivary ph at 4.2, 6.5, and 7.6 simulated conditions within the oral cavity and were monitored at days 14 and 28, thereby simulating the time interval of the appliances fitted in the oral cavity. the ph levels of 6.5 and 7.6 were within the range of naturally-occurring human salivary ph, whereas a ph of 4.2 was intended to simulate acidic conditions that occasionally occurs when one eats or drinks acidic foods or beverages (e.g. lemon, fruit juice, coca-cola).7 brackets (protect, china) and archwires (protect, china) were analyzed using sem (esem quanta 400 feg, fei, japan) equipped with edx (edax teamtm, ametek, japan) and xrd (shimadzu xrd-6000 diffractometer, columbia, usa). an edx uses the x-ray spectrum emitted by an ss bracket or niti archwire to subsequently identify the specific energy of characteristic x-ray peaks of each element. xrd was used to confirm characterization of the material content by analyzing the crystal structure and to compare the results with those contained in a component structures database as a means of determining the compound of the niti archwire.11 this study simulated fixed orthodontics treatment of half of the maxillary and mandibulary arches (from central incisor to first molar). all orthodontic materials used were produced by zhejiang protect medical equipment (china). the bracket meshes were coated with adhesive resin (xeno ortho paste, japan) to protect them from corrosion. three groups represented the focus of this study and were divided into two control groups (bracket and archwire only) and one treatment group (bracket-wire combination). the bracket group consisted of 5 brackets and 1 buccal tube per jaw immersed in artificial saliva at a ph level of 6. the archwires group was composed of 6 cm of 0.021 x 0.025inch wire for the upper and lower jaws each of which was immersed in artificial saliva at respective ph levels of 4.2 and 6.5, whereas the treatment group consisted of a half set of a bracket-archwire combination for both the upper and lower jaws. each 6 cm length of 0.021 x 0.025-inch wire was ligated by means of elastic in the bracket slot and immersed in the artificial saliva at ph levels of 4.2, 6.5, and 7.6. each group had 3 samples for its repetition, which were 3 samples for bracket groups, 6 samples for archwire groups (3 samples respectively for ph 4.2 and 6.5), and 9 samples for treatment groups (3 samples respectively for ph 4.2, 6.5, and 7.6). each sample group was immersed in 180 ml artificial saliva. 10 ml sample of artificial saliva was collected at days 14 and 28, with the release of ni ions in this sample subsequently being analyzed by aas. on day 28, the brackets and wires were removed from the artificial saliva and their respective surface morphologies were subjected to sem analysis. the statistics used for data analysis on a sample resulted from a paired t-test to compare the release of nickel ions during immersion on days 14 and 28. one-way analysis of variance (anova) was conducted to compare the amount of nickel ions released, while a kruskal wallis test was conducted for morphologic surface comparison in the tested ph/groups. results edx analysis showed that the elemental compositions of the ss brackets were c, o, f, al, si, cr, fe, and ni (figure 1), whereas those of niti wires were c, o, zn, al, si, s, cl, ti and ni (figure 2). the xrd analysis showed that niti archwire contains other compounds besides niti such as nickel titanium silicon and zinc oleate (figure 3). in addition, the aas analysis confirmed that the highest release of ni ions occurred at a ph of 4.2 in the artificial saliva immersion, while the lowest was at a ph of 7.6 (table 1). one-way anova statistical analysis confirmed there to be no significant difference in the number of ni ions released at various ph levels (p=.092). when evaluating dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 82 narmada, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 80–85 9 figure 1. edx analysis for ss bracket. figure 2. edx analysis for niti archwire. figure 3. xrd analysis for niti archwire. figure 1. edx analysis for ss bracket. 9 figure 1. edx analysis for ss bracket. figure 2. edx analysis for niti archwire. figure 3. xrd analysis for niti archwire. figure 2. edx analysis for niti archwire. 9 figure 1. edx analysis for ss bracket. figure 2. edx analysis for niti archwire. figure 3. xrd analysis for niti archwire. figure 3. xrd analysis for niti archwire. table 1. the amount of nickel ions released ph ni ions release (ppm) ss bracket attached niti archwire control sample group archwire group bracket (10 brackets+2 buccal tubes) 14 days 28 days 14 days 28 days 14 days 28 days 4.2 0.7995 0.9118 0.2596 0.2669 6.5 0.5580 0.6848 0.0000 0.0000 0.5157 0.8309 7.6 0.1937 0.3974 table 2. the comparison between present study and the other studies references quantity part of whole appliance materials ni ion release time (days)bracket band/ tube wire bracket wire acid ph normal ph present study 10 (protect) 2 (protect) ½ ua, ½ la (protect) ½ ss niti 0.9118 ppm (ph=4.2) 0.6848 ppm (ph=6.5) 28 kuhta et al (2009) 5 (dentaurum) 1 (dentaurum) ½ ua (dentaurum) ¼ ss niti 5430.76 ppb (ph=3.5) 166.88 ppb (ph=6.75) 28 mikulewicz et al (2012) 20 (3m) 4 (3m) 2 (ao) 1 ss ss 573 μg/l (ph=7) 30 * ua=upper arch; la=lower arch; ph=potential of hydrogen; ppb=part per billion; ppm= part per million; and ao, american orthodontics. the effect of time on ni ion release, each treatment group was revealed to increase the volume of nickel ions released across the 14-day and 28-day time intervals, but no statistically significant difference existed (p=.055) (table 1). the sem images showed the surface morphology of brackets and archwires were unhomogeneous and rough due to contact with artificial saliva. the image at a ph of 4.2 revealed rougher scratches than the others (figures 4, 5, 6, 7). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 8383narmada, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 80–85 10 table 1. the amount of nickel ions released ph ni ions release (ppm) ss bracket attached niti archwire control sample group archwire group bracket (10 brackets+2 buccal tubes) 14 days 28 days 14 days 28 days 14 days 28 days 4.2 0.7995 0.9118 0.2596 0.2669 6.5 0.5580 0.6848 0.0000 0.0000 0.5157 0.8309 7.6 0.1937 0.3974 figure 4. the surface morphology of niti archwires in the treatment group after 28 days’ artificial saliva immersion at ph of: a) 4.2; b) 6.5; and c) 7.6. (5000x magnification). figure 5. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 4.2 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. figure 6. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 6.5 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. a b c a b c d a b c d figure 4. the surface morphology of niti archwires in the treatment group after 28 days’ artificial saliva immersion at ph of: a) 4.2; b) 6.5; and c) 7.6. (5000x magnification). 10 table 1. the amount of nickel ions released ph ni ions release (ppm) ss bracket attached niti archwire control sample group archwire group bracket (10 brackets+2 buccal tubes) 14 days 28 days 14 days 28 days 14 days 28 days 4.2 0.7995 0.9118 0.2596 0.2669 6.5 0.5580 0.6848 0.0000 0.0000 0.5157 0.8309 7.6 0.1937 0.3974 figure 4. the surface morphology of niti archwires in the treatment group after 28 days’ artificial saliva immersion at ph of: a) 4.2; b) 6.5; and c) 7.6. (5000x magnification). figure 5. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 4.2 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. figure 6. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 6.5 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. a b c a b c d a b c d figure 5. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 4.2 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. 10 table 1. the amount of nickel ions released ph ni ions release (ppm) ss bracket attached niti archwire control sample group archwire group bracket (10 brackets+2 buccal tubes) 14 days 28 days 14 days 28 days 14 days 28 days 4.2 0.7995 0.9118 0.2596 0.2669 6.5 0.5580 0.6848 0.0000 0.0000 0.5157 0.8309 7.6 0.1937 0.3974 figure 4. the surface morphology of niti archwires in the treatment group after 28 days’ artificial saliva immersion at ph of: a) 4.2; b) 6.5; and c) 7.6. (5000x magnification). figure 5. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 4.2 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. figure 6. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 6.5 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. a b c a b c d a b c d figure 6. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 6.5 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. 11 figure 7. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 7.6 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. ( : saliva deposit) table 2. the comparison between present study and the other studies references quantity part of whole appliance materials ni ion release time (days) bracket band/ tube wire bracket wire acid ph normal ph present study 10 (protect) 2 (protect) ½ ua, ½ la (protect) ½ ss niti 0.9118 ppm (ph=4.2) 0.6848 ppm (ph=6.5) 28 kuhta et al (2009) 5 (dentaurum) 1 (dentaurum) ½ ua (dentaurum) ¼ ss niti 5430.76 ppb (ph=3.5) 166.88 ppb (ph=6.75) 28 mikulewicz et al (2012) 20 (3m) 4 (3m) 2 (ao) 1 ss ss 573 µg/l (ph=7) 30 *ua=upper arch; la=lower arch; ph=potential of hydrogen; ppb=part per billion; ppm= part per million; and ao, american orthodontics. a b c d figure 7. the micromorphologic characteristic of ss brackets slot surfaces in treatment group after 28 days artificial saliva immersion at ph of 7.6 with various magnification. a. 75x, b. 5000x, c. 5000x, d. 5000x. (: saliva deposit) discussion there are many aspects of long-term orthodontic treatment to be considered, including the materials used. metal alloys are the most common whose microstructure, due to the manufacturing process, influences their mechanical properties and corrosion behaviour. this can be a process involving casting, machine-based milling, or metallurgical powder.4 the ph level of the solution can accelerate the corrosion process, as confirmed by the release of metal ions which may, in turn, cause changes in the metal’s surface. the aforementioned corrosion process will release metal ions directly into the solution or damage the protective layer on the metal surface resulting in the release of ions. this process is continuous and cumulative, invisible to the human eye and only measurable by the ions released.4 in this study, the ni ions were released directly from the niti archwire because it contained no tio2 substances acting as a protective layer such as are generally provided in niti alloys as reported in the xrd results (figure 3).12 several previous studies have shown that metal ions can be released by most orthodontic appliances, potentially leading to an inflammatory reaction, irritation or dermatitis.4,13,14 small concentrations of metal ions could affect the mucous membrane cells. it has also been reported that the nickel ions released can accumulate in the cells over time and may endanger them by, for example, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 84 narmada, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 80–85 altering the chemotaxis of leukocytes and the synthesis of dna and enzyme activities.8,9 ni solution (0.05mol/l) may impede polymorphonuclear leukocyte phagocytosis, impair leukocyte chemotaxis, and stimulate neutrophils to become aspherical and move more slowly. according to edx analysis, both ss brackets and niti archwires contain the ni element. the chemical bonding of nickel atoms to inter-metallic compounds is weak, causing an increase in the release of ni ions from alloy which may subsequently compromise the biocompatibility of appliances.16 in addition, during the present study, the release of ni ions was also shown to be influenced by environmental factors, such as salivary ph (replicating oral conditions) and the duration of immersion (simulating the time interval of the appliance in the mouth).9 the measurement of ni ion release in this group revealed that the highest concentration occured in the artificial saliva immersion at a ph of 4.2 (0.9118 ppm/28 days), with the lowest at a ph of 7.6 (0.3974 ppm/28 days). meanwhile, the ni ion release at a ph of 6.5 was 0.6848 ppm. these values are comparable to those obtained by kuhta et al. and mikulewicz et al.9,17 kuhta et al. simulated the orthodontic appliances which consist of incisor to premolar maxillary brackets (dentaurum, germany), molar bands and ligature wire (dentaurum, germany), niti archwire 0.016 x 0.022 over 28 days of observation.9 the research showed that nickel ion release at ph = 6.75 was 166.88 ppb (0.1669 ppm), whereas at ph = 3.5 it was 5430.76 ppb (5.4308 ppm). a similar study was undertaken by mikulewicz et al. using neutral ph=7 and simulated orthodontics appliances with 20 ss brackets (3m unitek, usa), 4 bands (3m unitek, usa), two 0.017” x 0.025” ss archwires (american orthodontics, usa) and 20 metal ligatures (american orthodontics, usa). the study showed that ni ion release was 573 μg/l (0.573 ppm) over 30 days.17 it can be concluded from the present study that the orthodontic appliances incorporating ss bracketsniti archwire combinations were acceptable because the concentration of nickel ions released in acidic condition was not as high as in those referred to above (table 2). the effect of salivary ph on the release of ni ions can be observed in the surface morphological of ss bracketsniti archwire combinations after immersion in the artificial saliva for 28 days. the surface morphology of niti archwire at a ph of 4.2 appeared to be rougher than that of one at 7.6. this finding is in line with the previous study which concluded that the higher the acidity, the rougher the surface of the niti archwire. this condition relates to the volume of ni ions released. under acidic conditions, this increases with the morphology of the surface, consequently, appearing to be rougher.18 to confirm the occurrence of ni ion release during this research, two control groups (bracket group and archwire group) with the artificial saliva immmersion at ph of 6.5 (table 2) were analysed. additional investigation of the niti archwire group was conducted at a ph of 4.2 because no ni ion release was detectable at one of 6.5, whereas at a ph of 4.2 the figure was 0.2669 ppm at 28 days. these results indicate that niti archwire (protect, china) was acceptable at a ph of 6.5 even though the release of ni ions can still occur on the wire by changing the salivary ph to be more acidic. the level of nickel ion release of the ss bracket-niti archwire combination (the upper and lower jaws) at various ph levels over 28 days remained below the toxic level of leukocyte chemotaxis (2,5 ppm).19 this value is still tolerable for human beings because it is below the toxicity level of nickel (30 ppm).13 as a consequence, this orthodontic appliance (protec, china) is safe for use over a period of 28 days. however, further research of greater duration is needed in order to determine the exact period during which the orthodontic appliance can be safely used. in conclusion, decrease in salivary ph increases the corrosion process, thereby releasing ni ions from the metal alloys contained in appliances and affecting the homogeneity of the surface morphology of the ss bracketniti archwire combination. references 1. bilgic f, gelgor ie, celebi aa. malocclusion prevalence and orthodontic treatment need in central anatolian adolescents compared to european and other nations’ adolescents. dental press j orthod. 2015; 20(6): 75–81. 2. quintão cca, portella hiv. orthodontic wires : knowledge ensures clinical optimization. dental press j orthod. 2009; 14(6): 144–57. 3. heravi f, mokhber n, shayan e. galvanic corrosion among different combination of orthodontic archwires and stainless steel brackets. j dent mater tech. 2014; 3(3): 118–22. 4. de menezes lm, quintão cca. the release of ions from metallic orthodontic appliances. semin orthod. 2010; 16(4): 282–92. 5. noble j, ahing si, karaiskos ne, wiltshire wa. nickel allergy and orthodontics, a review and report of two cases. br dent j. 2008; 204(6): 297–300. 6. rahilly g, price n. current products and practice: nickel allergy and orthodontics. j orthod. 2003; 30(2): 171–4. 7. sfondrini mf, cacciafesta v, maffia e, massironi s, scribante a, alberti g, biesuz r, klersy c. chromium release from new stainless steel, recycled and nickel-free orthodontic brackets. angle orthod. 2009; 79(2): 361–7. 8. faccioni f, franceschetti p, cerpelloni m, fracasso me. in vivo study on metal release from fixed orthodontic appliances and dna damage in oral mucosa cells. am j orthod dentofac orthop. 2003; 124(6): 687–94. 9. kuhta m, pavlin d, slaj m, varga s, lapter-varga m, slaj m. type of archwire and level of acidity: effects on the release of metal ions from orthodontic appliances. angle orthod. 2009; 79(1): 102–10. 10. ağaoğlu g, arun t, izgi b, yarat a, izgü b. nickel and chromium levels in the saliva and serum of patients with fixed orthodontic appliances. angle orthod. 2001; 71(5): 375–9. 11. sheikh t, ghorbani m, tahmasbi s, yaghoubnejad y. galvanic corrosion of orthodontic brackets and wires in acidic artificial saliva: part ii. j dent sch. 2015; 33(1): 88–97. 12. huang h-h, wang c-c, chiu s-m, wang j-f, liaw y-c, lee t-h, chen f-l. corrosion behavior of titanium-containing orthodontic archwires in artificial saliva : effects of fluoride ions and plasma immersion ion implantation treatment. chinese dent j. 2005; 24(3): 134–40. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 8585narmada, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 80–85 13. agarwal p, upadhyay u, tandon r, kumar s. nickel allergy and orthodontics. asian j oral heal allied sci. 2011; 1(1): 61–3. 14. gopinath a, admala nr, srinivas d. comparison of metal ion release from different bracket archwire combination. j res adv dent. 2013; 2(1): 58–64. 15. swathi r. effects of nickel leaching from orthodontic appliances in the oral cavity : a review. iosr j dent med sci. 2014; 13(7): 28–9. 16. house k, sernetz f, dymock d, sandy jr, ireland aj. corrosion of orthodontic appliances—should we care? am j orthod dentofac orthop. 2008; 133(4): 584–92. 17. mikulewicz m, chojnacka k, woźniak b, downarowicz p. release of metal ions from orthodontic appliances: an in vitro study. biol trace elem res. 2012; 146(2): 272–80. 18. perinetti g, contardo l, ceschi m, antoniolli f, franchi l, baccetti t, di lenarda r. surface corrosion and fracture resistance of two nickel-titanium-based archwires induced by f luoride, ph, and thermocycling. an in vitro comparative study. eur j orthod. 2012; 34(1): 1–9. 19. nayak rs, khanna b, pasha a, vinay k, narayan a, chaitra k. evaluation of nickel and chromium ion release during fixed orthodontic treatment using inductively coupled plasma-mass spectrometer: an in vivo study. j int oral heal. 2015; 7(8): 14–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p80–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p80-85 vol 38 no 3 2005 146 kekerasan permukaan semen ionomer kaca konvensional tipe ii akibat lama penyimpanan (the surface hardness of type ii conventional glass ionomer cement conventional because of the length of storage) asti meizarini* dan irmawati** ** bagian ilmu material dan teknologi kedokteran gigi ** bagian ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract type ii conventional glass-ionomer cements (gic) are restorative materials consisting of powder and liquid mixed to produce a plastic mass that subsequently sets to a rigid solid. the early gic was marketed without the expired date, although this material has been currently marketed with the expired date, how stable the materials are not yet clear. the purpose of this study was to evaluate the powder and the liquid gic storage on the surface hardness of the samples. six disc samples (5 mm in diameter and 3 mm thick) were made for each of four groups consisting of powder: aged liquid ages, aged powder: new liquid new, new powder new: new liquid new and aged powder: aged liquid ages respectively. all samples prepared followed the manufacturers’ recommendations and were allowed to set for 15 minutes then they were kept for 24 hours at room temperature before testing. vickers microhardness was assessed for measuring the surface hardness at the top of the sample surface. the data were statistically analyzed using an anova and lsd (p < 0.05). the results of this study indicated that the highest surface hardness was on the group of new powder: new liquid new = 52.040 vhn. the other groups were nearly similar, for new powder: aged liquid, aged powder: new liquid, aged powder: aged liquid they were 49.558 vhn; 49.123 vhn; 48.938 vhn respectively. conclusion, powder and liquid of the type ii conventional glass ionomer that had been stored for a long time descreased the surface hardness of the glass ionomer cements. key words: glass ionomer cement, surface hardness, storage korespondensi (correspondence): asti meizarini, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan semen ionomer kaca pertama diperkenalkan oleh wilson dan kent pada tahun 1971, yang merupakan gabungan dari semen silikat dan semen polikarboksilat dengan tujuan untuk mendapatkan sifat translusen, pelepasan fluor dari semen silikat dan kemampuan melekat secara kimia pada struktur gigi dari semen polikarboksilat.1,2 sifat utama semen ionomer kaca adalah kemampuannya untuk melekat pada enamel dan dentin tanpa ada penyusutan atau panas yang bermakna, mempunyai sifat biokompatibilitas dengan jaringan periodontal dan pulpa, ada pelepasan fluor yang beraksi sebagai anti mikroba dan kariostatik, kontraksi volume pada pengerasan sedikit, koefisien ekspansi termal sama dengan struktur gigi.3,4 ada beberapa jenis semen ionomer kaca berdasarkan penggunaannya, tipe i untuk material perekat, tipe ii untuk material restorasi dan tipe iii untuk basis atau pelapis.1,5 semen ionomer kaca tipe ii secara umum mempunyai sifat lebih keras dan kuat dibandingkan tipe i, karena mempunyai rasio powder terhadap liquid lebih tinggi.6 material ini amat berguna dalam merawat pasien gigi anak yang mempunyai risiko karies tinggi karena melepas fluor dan estetik dapat diterima,7 juga untuk restorasi kelas iii dan v pada dewasa.1 sifat semen ionomer kaca adalah cukup keras, tetapi rapuh, kekuatan tekan relatif tinggi, tetapi daya tahan terhadap fraktur dan keausan rendah, sehingga tidak digunakan untuk merestorasi gigi dengan beban besar.6 daya tahan yang rendah terhadap keausan, dipengaruhi oleh sifat kekerasan permukaan, oleh karena itu dalam penelitian ini pengujian dilakukan terhadap kekerasan permukaan semen ionomer kaca. semen ionomer kaca berkembang sejalan dengan waktu, dengan penambahan partikel logam pada powder untuk memperbaiki sifat mekanik, penggantian sebagian komponen agar dapat dikeraskan dengan penyinaran dan banyak lagi modifikasi yang dipakai untuk memperbaiki sifat ionomer kaca. semen ionomer kaca yang tidak dimodifikasi disebut semen ionomer kaca konvensional.8 kemasan semen ionomer kaca konvensional terdiri dari powder dan liquid. powder ionomer kaca adalah kaca kalsium fluoroaluminosilikat yang larut dalam liquid asam. 147meizarini dan irmawati: kekerasan permukaan semen ionomer kaca konvensional tipe ii kandungan powder ionomer kaca komersial adalah silika (sio2), alumina (al2o3), aluminium fluorida (alf3), kalsium fluorida (caf 2), natrium fluorida (naf), aluminium fosfat (alpo4). material dasar ini digabung sehingga membentuk kaca yang seragam dengan cara memanaskan sampai suhu 1100–1500° c. lantanum, sronsium, barium, atau oksida seng ditambahkan untuk mendapatkan sifat radiopak. kemudian kaca digerus menjadi powder dengan ukuran partikel berkisar antara 15–50 μm. perbedaan kegunaan material semen ionomer kaca, terletak pada ukuran partikelnya. material untuk restorasi mempunyai ukuran partikel maksimum 50 μm, sedang ukuran partikel untuk material perekat atau pelapis di bawah 20 μm.2,8 data hasil pengujian powder semen ionomer kaca tipe ii menyatakan bahwa powder bersifat stabil dan tidak akan terjadi polimerisasi.9 liquid untuk semen ionomer kaca adalah larutan dari asam poliakrilat dengan konsentrasi 40–50%. liquid ini agak kental dan cenderung menjadi gel dengan berjalannya waktu. pada semen ionomer kaca yang beredar saat ini, liquid asamnya berada dalam bentuk kopolimer dengan asam itakonik, maleik, atau trikarbosilik. asam ini cenderung meningkatkan reaktivitas dari liquid, mengurangi kekentalan, dan mengurangi kecenderungan menjadi gel. liquid juga mengandung asam tartarik. asam ini memperbaiki karakteristik manipulasi dan meningkatkan waktu kerja, tetapi memperpendek waktu pengerasan. kekentalan dari semen yang mengandung asam tartarik tidak mengalami perubahan dengan berjalannya waktu, tetapi perubahan kekentalan dapat terjadi bila kadaluwarsa.8 komponen semen ionomer kaca di atas mengindikasikan powder dan liquid dapat bertahan cukup lama, mungkin oleh karena itu pada kemasan semen ionomer kaca konvensional tipe ii lama, tidak dicantumkan waktu kadaluwarsa. dalam pemakaian, seringkali powder masih banyak, tetapi liquid sudah habis atau sudah terlalu kental karena botol tidak ditutup rapat, sehingga tidak dapat dipakai lagi dan membutuhkan liquid baru. kemasan semen ionomer kaca yang ada di pasaran sekarang sudah dicantumkan tanggal kadaluwarsa, meskipun demikian peneliti ingin mengetahui keadaan powder dan liquid dengan cara menguji kekerasan permukaan hasil pencampuran antara semen ionomer kaca baru dan semen ionomer kaca yang telah 10 tahun disimpan. manfaat penelitian ini diharapkan dapat memberikan tambahan informasi mengenai sifat kekerasan permukaan pada semen ionomer kaca tipe ii yang telah lama disimpan, sehingga bila memerlukan powder atau liquid baru, dapat dijadikan sebagai bahan pertimbangan apakah masih memenuhi syarat untuk digunakan sebagai material restorasi. bahan dan metode jenis penelitian eksperimental laboratoris, rancangan penelitian post test only control group. penelitian dilakukan di laboratorium fisika, pusat antar universitas, universitas gajah mada yogyakarta pada bulan oktober 2002. material yang digunakan pada penelitian ini adalah 2 paket semen ionomer kaca fuji ii (gc, japan). paket pertama adalah paket lama yang telah disimpan selama 10 tahun dalam suhu kamar, powder batch no. 920221a, liquid batch no. 911212a dan tidak ada expire date. paket kedua merupakan paket baru, powder lot 0012221 exp 2003-12, liquid lot 0003081 exp 2003-03. kedua paket dalam keadaan tertutup rapat dan belum pernah dibuka. alat yang digunakan adalah cetakan sampel dari teflon dengan ukuran diameter dalam 5 mm dan tebal 3 mm,10glass slab, stop watch, spatula plastik, mixing pad, celluloid strip, anak timbangan 1 kg, digital micro hardnesstester-vickers (matsuzawa mxt 70, japan). penelitian ini terdiri dari 4 kelompok yang mempunyai besaran sampel 6 buah: kelompok i: menggunakan powder baru dengan liquid lama; kelompok ii: menggunakan powder lama dengan liquid baru; kelompok iii: menggunakan powder baru dengan liquid baru; kelompok iv: menggunakan powder lama dengan liquid lama. cara pembuatan sampel sebagai berikut: cetakan sampel terbuat dari teflon diberi alas celluloid strip diletakkan di atas glass slab. powder dan liquid sesuai kelompok, diaduk di atas mixing pad dengan perbandingan 1 : 1 (sesuai aturan pabrik = 2,7 : 1,0 g), kemudian dimasukkan ke dalam mold cetakan sampel. bagian atas mold diberi celluloid strip, ditekan dengan beban 1 kg sampai mengeras kurang lebih 15 menit. setelah itu sampel dilepaskan dari cetakan, kelebihan sampel dipotong menggunakan scalpel. sampel disimpan dalam plastik berperekat secara terpisah berdasarkan kelompoknya selama 24 jam.11 uji kekerasan permukaan masing-masing sampel, dilakukan di permukaan atas sampel menggunakan alat micro hardness tester dengan beban 50 gram. sampel diletakkan di bagian tengah dari landasan uji micro hardness tester, diperiksa dengan mikroskop, setelah terlihat ganti lensa mikroskop dengan diamond penetrator. alat diaktifkan, sehingga ujung diamond penetrator turun menekan sampel dan naik kembali, meninggalkan indentasi pada permukaan sampel. bentukan indentasi yang dihasilkan diamati melalui lensa mikroskop dengan pembesaran 400 kali, sehingga akan tampak bentukan belah ketupat. panjang diagonal diukur dengan menempatkan 2 tanda garis yang ada pada alat micro hardness tester pada kedua ujung bentukan diagonal tersebut. selanjutnya tombol baca ditekan, akan muncul data nilai kekerasan permukaan dalam satuan vickers hardness number (vhn). setiap sampel dilakukan pengukuran kekerasan permukaan pada 3 tempat yang berbeda dan hasilnya dirata-rata. data yang diperoleh ditabulasi, kemudian dilakukan analisis statistik menggunakan one-way anova dan dilanjutkan dengan least significant difference (lsd) dengan p > 0,05. 148 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 146–150 hasil nilai rerata kekerasan permukaan semen ionomer kaca konvensional tipe ii, standar deviasi, probabilitas normalitas dapat dilihat pada tabel 1. tabel 1. rerata dan standar deviasi kekerasan permukaan semen ionomer kaca tipe ii (vhn) n x sd p kelompok i powder baru – liquid lama 6 49,56 1,933 0,509 kelompok ii powder lama – liquid baru 6 49,12 2,017 0,674 kelompok iii powder baru – liquid baru 6 52,04 1,984 0,648 kelompok iv powder lama – liquid lama 6 48,94 1,832 0,466 keterangan: n = jumlah sample, x = rerata kekerasan permukaan, sd = standar deviasi, p = probabilitas normalitas kelompok yang menggunakan powder baru dan liquid baru menunjukkan nilai rerata kekerasan permukaan paling tinggi, yaitu 52,04 vhn. kelompok yang menggunakan powder lama dan liquid lama menunjukkan rerata kekerasan permukaan paling rendah, yaitu 48,94 vhn. hasil kekerasan permukaan pada kelompok yang menggunakan powder baru dan liquid lama 49,56 vhn dan kelompok powder lama dan liquid baru 49,12 vhn. probabilitas normalitas pada kolmogorov smirnov test menunjukkan semua kelompok mempunyai distribusi normal, karena didapatkan probabilitas normalitas lebih besar dari 0,05 (p > 0,05). setelah diketahui semua kelompok mempunyai distribusi normal, maka untuk mengetahui adanya perbedaan kekerasan permukaan semen ionomer kaca konvensional tipe ii antar kelompok secara statistik, dilakukan uji parametrik one-way anova dengan taraf kemaknaan 5%. hasil uji anova, didapatkan probabilitas 0,042 (p < 0,05), maka berarti ada perbedaan yang bermakna antar kelompok yang diuji. penentuan perbedaan kemaknaan antar kelompok, dilakukan dengan uji lsd pada α = 0,05 yang dapat dilihat pada tabel 2. kelompok perlakuan yang bermakna adalah yang mempunyai signifikansi kurang dari 0,05 (p < 0,05). hasilnya kelompok yang menggunakan powder baru dan liquid baru berbeda bermakna bila dibandingkan dengan kelompok yang menggunakan powder baru dan liquid lama, powder lama dan liquid baru, powder lama dan liquid lama. tidak ada perbedaan bermakna di antara kelompok semen ionomer kaca konvensional tipe ii yang menggunakan powder baru dan liquid lama, powder lama dan liquid baru, powder lama dan liquid lama. pembahasan material semen ionomer kaca yang dipakai untuk penelitian ini adalah paket powder dan liquid semen ionomer kaca konvensional tipe ii lama yang telah disimpan selama 10 tahun dan paket powder dan liquid semen ionomer kaca tipe ii baru. paket lama dijual tanpa tanggal kadaluwarsa. paket ini masih dalam kemasan yang baik, tertutup rapat dan belum pernah dibuka. pada saat akan dilakukan pembuatan sampel, powder dibuka tampak berwarna putih, secara kasat mata sama dengan powder yang baru dibeli, tetapi liquid telah mengalami perubahan warna, lebih kekuningan dan lebih kental. paket semen ionomer kaca konvensional tipe ii yang baru, tercantum kadaluwarsa powder desember 2003 dan kadaluwarsa liquid maret 2003. pengujian dilakukan untuk mengetahui pengaruh penyimpanan terhadap kekerasan permukaan hasil pencampuran powder dan liquid yang telah disimpan 10 tahun, dibandingkan dengan powder dan liquid semen ionomer kaca belum kadaluwarsa. hasilnya didapatkan kekerasan permukaan pada kelompok yang menggunakan powder baru dan liquid baru paling tinggi (tabel 1), dan berbeda bermakna dibandingkan dengan kelompok lainnya (tabel 2). hasil pengujian kelompok yang menggunakan powder baru dan liquid lama, powder lama dan liquid baru, maupun powder lama dan liquid lama, tidak menunjukkan tabel 2. uji lsd kekerasan permukaan semen ionomer kaca tipe ii kelompok kelompok i powder baru – liquid lama kelompok ii powder lama – liquid baru kelompok iii powder baru – liquid baru kelompok iv powder lama – liquid lama kelompok i powder baru – liquid lama − kelompok ii powder lama – liquid baru tb (0,702) − kelompok iii powder baru – liquid baru b (0,039) β (0,017) − kelompok iv powder lama – liquid lama τβ (0,587) τβ (0,871) b (0,012) − keterangan: b = bermakna, tb = tidak bermakna 149meizarini dan irmawati: kekerasan permukaan semen ionomer kaca konvensional tipe ii perbedaan yang bermakna (tabel 2). hasil ini menunjukkan adanya kemungkinan bahwa penyimpanan powder dan liquid berpengaruh terhadap kekerasan permukaan sampel. kemungkinan disebabkan liquid yang lama disimpan mulai mengental dan berubah warna sedikit kekuningan, meskipun powder yang lama disimpan tidak ada perubahan warna maupun bentuk fisik. penelitian sebelumnya mengenai kekuatan tekan pada material yang sama, didapatkan hasil kekuatan tekan pada kelompok yang menggunakan powder baru dengan liquid baru juga lebih tinggi, meskipun tidak bermakna terhadap kelompok lain.11 hasil dari kedua penelitian di atas sesuai dengan pendapat yang menyebutkan semen ionomer kaca lebih tahan terhadap daya tekan, tetapi rentan terhadap keausan.2 daya kunyah berhubungan dengan kekuatan tekan, sedang kekerasan permukaan berhubungan dengan keausan material, oleh karena itu bila ada penurunan kwalitas dari material, pengaruh pada keausan permukaan semen ionomer kaca akan lebih besar bila dibandingkan pengaruh pada kekuatan tekan. hasil kekerasan permukaan semen ionomer kaca konvensional tipe ii yang menggunakan powder lama dan liquid lama dalam penelitian ini paling rendah = 48,938 vhn, meskipun demikian kekerasan permukaan dalam penelitian ini masih lebih tinggi daripada kekerasan permukaan semen ionomer kaca modifikasi tipe ii dengan aktivasi sinar (merek fuji ii lc) = 36,2 vhn,12 tetapi lebih rendah dari kekerasan permukaan semen ionomer kaca tipe ii dalam kapsul (merek fujicap ii) = 74 vhn.12 komposisi semen ionomer kaca modifikasi tipe ii dengan aktivasi sinar, berbeda dengan semen ionomer kaca konvensional tipe ii, karena ada tambahan resin hidroksietil metakrilat (hema) pada liquidnya. hema bersifat sangat hidrofilik, menyerap air lebih banyak, menyebabkan semen ionomer kaca ekspansi dan menurunkan daya tahan terhadap keausan.13 pada penelitian kekerasan permukaan lain menggunakan semen ionomer kaca modifikasi tipe ii dengan aktivasi sinar warna a3, didapatkan hasil 50,37 vhn.4 adanya perbedaan kekerasan permukaan pada semen ionomer kaca modifikasi tipe ii dengan aktivasi sinar tersebut, dapat disebabkan karena ada perbedaan warna powder semen ionomer kaca, perbedaan alat dan metode penelitian yang dipakai. komposisi semen ionomer kaca tipe ii dalam kapsul sama dengan semen ionomer kaca konvensional tipe ii. kekerasan permukaan semen ionomer kaca dalam kapsul lebih tinggi karena perbandingan powder dan liquid akurat, pencampuran tepat, cepat dan tidak ada masalah kelembaban udara (tertutup rapat dalam kapsul, pencampuran secara mekanik selama 10 detik 4000 rpm), dan dimasukkan ke kavitas langsung dengan menyuntikkan material dari dalam kapsul. hasil pencampurannya tepat dan seragam, dengan rasio powder dan liquid yang tinggi memberikan kekuatan mekanik optimal.1,14 ada tiga bentuk komposisi semen ionomer kaca, yaitu pencampuran powder dan liquid menggunakan air, pencampuran tidak dengan air atau kombinasi keduanya. bentuk pertama, pengerasan semen ionomer kaca dengan pencampuran air. dapat terjadi bila poliasam (terutama poliakrilik dan polimaleik) dikeringkan melalui pembekuan atau hampa udara, dicampur dalam powder. komponen liquidnya adalah air suling atau cairan asam tartarik, sehingga batas kadaluwarsa maksimal, tidak terjadi gelasi atau mengental dan pengerjaan semen mudah. bentuk kedua, pengerasan semen ionomer kaca tidak dengan air. liquidnya mengandung poliasam (umumnya poliakrilik, polimaleik, itakonik dan tartarik). liquid ini sedikit kental dan dapat terjadi gelasi karena perlekatan hidrogen diantara rantai asam poliakrilik, yang dapat tampak dalam waktu 6 minggu atau lebih. salah satu contoh adalah semen ionomer kaca tipe i versi lama (merek fuji). bentuk ketiga, pengerasan semen ionomer kaca kombinasi. terdiri dari powder mengandung asam poliakrilik yang dikeringkan dan liquid yang mengandung asam poliakrilik dan tartarik. bentuk semen ionomer kaca ini mempunyai sifat fisik kekentalan dan waktu kadaluwarsa diantara bentuk pertama dan kedua, contohnya semen ionomer kaca tipe i versi baru (merek fuji). produk dari fuji mengandung sekitar 5% asam poliakrilik yang dikeringkan, ditambahkan pada powder dan asam poliakrilik selebihnya ada pada liquid.6 penjelasan mengenai pencampuran kombinasi atau pengerasan semen ionomer kaca kombinasi, sesuai dengan data powder fuji ii tanggal 3 september 2003 yang menyebutkan komponen powder terdiri dari 3–5% asam poliakrilik dan 90–95% kaca aluminosilikat. powder fuji ii disebutkan juga bersifat stabil, tidak dapat terjadi polimerisasi bila disimpan dalam kelembaban dan temperatur ruang yang normal, selain itu hindarkan panas yang tinggi.9 dari data tersebut dapat diketahui bahwa powder glass ionomer dapat bertahan lama bila disimpan dalam temperatur ruang dan tertutup rapat. perbedaan antara tipe i dan tipe ii terletak dari besarnya partikel,2 sehingga dapat dikatakan bahwa paket fuji ii baru adalah komposisi bentuk ketiga. sedang paket fuji ii lama, yang telah disimpan selama 10 tahun adalah komposisi bentuk kedua, hal ini sesuai juga dengan kondisi liquid lama yang mengental meskipun masih bisa mengalir. perbedaan bentuk komposisi antara paket lama dan baru juga ditunjang dengan adanya perbedaan pada gambar logo kedua kemasan, meskipun kedua paket berasal dari pabrik yang sama. tingginya nilai kekerasan permukaan powder baru dengan liquid baru, kemungkinan disebabkan karena dalam kandungan powder ada 3–5% asam poliakrilik. pada waktu pencampuran powder dan liquid, asam poliakrilik pada powder membantu mempercepat pelarutan, pembentukan gelasi dan pengerasan dengan liquid asam poliakrilik-tartarik pada reaksi dasar asam. pencampuran powder baru dengan liquid lama yang mengandung 150 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 146–150 poliasam berbeda dengan poliasam liquid baru, menyebabkan reaksi yang berbeda, karena kandungan hasil pencampuran tidak sama. selain lama penyimpanan material, adanya perbedaan komposisi powder dan liquid juga mempengaruhi hasil kekerasan permukaan. pada penelitian ini tidak dilakukan analisa kandungan powder dan liquid semen ionomer kaca konvensional tipe ii yang telah lama di simpan, sehingga penyebab penurunan kekerasan perlu penelitian lebih lanjut. kesimpulan penelitian ini adalah pemakaian powder dan liquid semen ionomer kaca konvensional tipe ii yang telah lama disimpan menurunkan kekerasan permukaan. daftar pustaka 1. wilson ad, mclean jw. glass-ionomer cement. chicago: quintessence publ. co; 1988. p. 13, 22, 33–40, 131–6. 2. van noort r. introduction to dental material. 2nd ed. london: cv mosby company; 2003. p. 124–35. 3. hse kmy, leung sk, wei shy. resin-ionomer restorative materials for children. aust dent j 1999; 44:(1):1–11. 4. palma-dibb rg, palma ae, matson e, chinelatti ma, ramos rp. microhardness of esthetic restorative materials at different depths. materials research 2002; 6:(1):85–90. 5. tyas mj, burrow mf. adhesive restorative materials: a review. aust dent j 2004; 49:(3):112–21. 6. webteam. glass-ionomer cements. available at: http:// www.brooks.af.mil/dis/dmnotes/gic.pdf. accesed 8 may, 2005. 7. cameron ac, wilmer rp. handbook of pediatric dentistry. 2nd ed. edinburg: mosby; 2003. p. 51–5. 8. anusavice kj. phillips’ science of dental materials. 11st ed. saunders; 2003. p. 471–7. 9. material safety data sheet (msds)-435021fuji ii powder. available at: http://www.gcamerica.com/msds/435021.htm. accesed may 8, 2005. 10. watts dc, amer om, combe ec. surface hardness development in light cured composites. j dent mater 1987; 3:265–9. 11. wibowo m. pengaruh lama penyimpanan semen ionomer kaca tipe ii terhadap kekuatan tekan diametral. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2002. h. 13, 18–23. 12. o’brien wj. dental materials and their selection. 3rd ed. chicago: quintessence publ co; 2002. p. 379–80. 13. mount gj, patel c, makinson of. resin modified glass-ionomers: strength, cure depth and translucency. aust dent j 2002; 47:(4):339–43. 14. a practical approach to selection is to first decide which restorative, or combination, is best for the indication. available at: http:// www.gcamerica.com/images/pdfs/wchrestorative.pdf. accessed may 8, 2005. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� vol. 45. no. 1 march 2012 analysis of importance level and quality achievement aspect in dental health service (a case study on waru sidoarjo community dental health service) taufan bramantoro and retno palupi department of dental public health faculty of dentistry, airlangga university surabaya indonesia abstract background: patients as customers of health services actually have expectation and assessment of health services perceived. during the initial interview conducted at waru sidoarjo community dental health service (waru sidoarjo cdhs), it is known that one hundred percent of initial respondents were not satisfied with dental care service provided. all of those respondents assessed that waru sidoarjo cdhs still has not met their expectations of service quality factors considered to be important for them. it is even known that there is usually a gap between the expectations of quality dental care service and the assessment of services perceived. as a result, further researches are needed to be conducted regarding the level of importance and achievement-related with factors that affect the quality of health services. purpose: the purpose of this study was to determine the level of importance and achievement of the quality aspects of the health service provided by waru sidoarjo cdhs. methods: this study can be considered as a descriptive observational study. the instrument used in this study was measurement instruments of service quality. respondents in the study were 200 patients who visited to dental care services in waru sidoarjo cdhs in july 2011. results: all of the attributes had a mean value of dominant importance and assessments at four. the attributes of the appearance feasibility of medical staffs had the highest interest, about 4.780. meanwhile, the mean value of the lowest importance was on the attributes of the service suitability, about 4.595. during the observation of the service value, it is also known that the highest mean value was on the non-discriminative services, about 4.600. conclusion: it can be concluded that there were attributes considered to be important for patients, but still not being fully met by health services provided by the service provider or community dental health care. those attributes involving waiting room comfort, service readiness and service preparation attributes which not only had high importance value, but also had a large percentage of respondents who were not satisfied. key words: the expectations of patients, the assessments of patients, the satisfactory of patients, dental care service abstrak latar belakang: pasien sebagai pengguna jasa pelayanan kesehatan, memiliki harapan dan penilaian terhadap pelayanan kesehatan yang diterima. pada wawancara awal yang dilakukan pada pasien balai pengobatan gigi puskesmas waru sidoarjo, didapatkan bahwa seratus persen responden awal merasa tidak puas dengan pelayanan kesehatan gigi. seluruh responden awal menilai pihak bpg puskesmas belum memenuhi harapan mereka terhadap faktor kualitas pelayanan yang mereka nilai penting. terdapat kesenjangan antara harapan mereka terkait kualitas pelayanan kesehatan gigi yang mereka nilai penting dengan penilaian mereka terhadap pelayanan yang diterima, sehingga diperlukan penelitian lebih lanjut mengenai tingkat kepentingan dan pencapaian terkait faktor yang berpengaruh terhadap kualitas pelayanan kesehatan. tujuan: tujuan penelitian ini adalah untuk mengetahui tingkat kepentingan dan pencapaian aspek kualitas pelayanan yang diberikan balai pengobatan gigi puskesmas waru sidoarjo. metode: penelitian ini merupakan penelitian deskriptif observasional. instrumen yang digunakan pada penelitian ini adalah instrumen pengukuran kualitas pelayanan jasa. responden pada penelitian adalah 200 pasien yang berkunjung dan mendapatkan pelayanan kesehatan gigi di balai pengobatan gigi puskesmas waru sidoarjo pada bulan juli 2011. hasil: seluruh atribut memiliki rerata nilai kepentingan dan penilaian yang dominan pada nilai 4. atribut kelayakan penampilan staf medis memiliki nilai kepentingan tertinggi sebesar 4,780. rerata nilai kepentingan terendah pada atribut kesesuaian layanan sebesar 4,595. pada pengamatan nilai penerimaan layanan, rerata nilai tertinggi pada atribut layanan yang tidak diskriminatif sebesar 4,600. rerata nilai terendah didapatkan pada atribut kesiapan layanan sebesar 4,200. kesimpulan: disimpulkan bahwa terdapat atribut yang dinilai penting tetapi belum sepenuhnya terpenuhi oleh research report ��bramantoro and palupi: analysis of importance level and quality achievement introduction waru sidoarjo community dental health service (waru sidoarjo cdhs) is a governmental organization that works to provide community health services. thus, it is always required to constantly improve the quality of its health care services. one of the services organized by the waru sidoarjo cdhs is dental and oral health care. as a service provider organization, it has to pay attention to interaction between structure, process, and outcome; since these three components will affect the health service quality assessment.1 the structure of the public health center also involves physical facilities, supplies and equipment, organization and management, finance, human resources, and resource sharing. the existence of the structure is actually supported by a process, namely as a professional activity carried out by medical and nonmedical resources including the assessment of patients, diagnose, treatment plans, treatment indications, and so on. the final results of the patients’ activity in terms of changes in health status and satisfactory actually involve both positive and negative outcomes, which could impact the short and long term development of health care service.1,2 patient as an individual has different individual expectation and assessment of the health services perceived. the patient will be satisfied when his expectation of the health services is provided. it means that the satisfactory of the patients at waru sidoarjo cdhs can have an implication towards the improvement of dental health care services.3,4 during the initial interview conducted at waru sidoarjo cdhs, it is known that one hundred percent of the initial respondents were not satisfied with dental care services perceived. all of the respondents even stated that the bpg still had not provided their expectations of service quality factors. this condition was actually related to the gap between their expectations of dental care service quality considered to be important for them and their assessment of the dental care services perceived. therefore, further researches are needed regarding the level of the importance and achievement of related factors that affect the quality of health services. the purpose of this study, thus, was to determine the level of the importance and achievement of the aspects of service quality provided by waru sidoarjo cdhs. materials and methods this study is considered as a descriptive observational study. the instrument used in this study was an instrument of service quality measurement, questionnaire, consisting of five aspects of the observations, namely: a) reliability, the ability to deliver promised services immediately, accurately, and satisfying, b) assurance or insurance includes knowledge, compensation, decency and trustworthiness, free from danger, and risk or doubt, c) tangibles or physical evidence includes physical facilities, equipment, as well as personnel and means of communication, d) empathy includes relationships, good communication, as well as attention and understanding of customer needs, e) responsibility or responsiveness, the desire of staffs to assist customers and provide a responsive service. the respondents in the study were 200 patients who visited waru sidoarjo cdhs in july 2011. the respondents then answered 22 questions by circling items based on likert scale score of 1 to 5 concerned with the service quality problems that have been adapted to the condition of the dental health services. the questionnaire consisted of column values of expectations and judgments. the data of the respondents’ response were then recapitulated. afterwards, the comparison of the expectation of the respondents and the assessment of the respondents was conducted. the result was then classified into three groups: the value of the assessment of the respondents was smaller than the value of the expectation of the respondents; the value of the assessment of the respondents was the same as the value of the expectation of the respondents; and the value of the assessment of the respondents was higher than the value of the expectation of the respondents. the mean value of the expectations and assessments of the respondents was then calculated for each topic of questions in order to know the importance of the service quality provided by waru sidoarjo cdhs. pelayanan kesehatan yang diberikan oleh penyedia layanan atau puskesmas. atribut kenyamanan ruang tunggu, kesigapan layanan, dan kesiapan layanan, memiliki nilai kepentingan yang tinggi, tetapi memiliki nilai persentase yang besar pada jumlah responden yang merasa tidak puas. kata kunci: harapan pasien, penilaian pasien, kepuasan pasien, pelayanan kesehatan gigi correspondence: taufan bramantoro, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tbramantoro@yahoo.com �0 dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 48–51 results based on the recapitulation result of the expectation and assessment of the patients in waru sidoarjo cdhs, it is known that more than 50% of respondents in each attribute of health care service quality aspects had the same expectation value as the assessment one concerned with dental care services received. the highest mean value was obtained from the attribute of the appearance feasibility of the medical staffs, about 4.780. meanwhile, the lowest mean value was obtained from the attribute of the dental care service suitability, about 4.595. based on the observation results on the value of the dental care services, the highest mean value was obtained from the attribute of the non-discriminatory services, about 4.600. meanwhile, the lowest mean value was obtained from the attribute of the service readiness, about 4.200. the attribute of the service readiness on responsiveness aspects had the highest mean value of the expectation, about 4.775. it means that the aspect of the service quality had high level of importance. it was contradicted with the percentage of the dissatisfaction value. compared to the other aspects of the service quality, the aspect of the service readiness had the largest percentage of the dissatisfaction value, about 39.5%. the largest percentage value on the service satisfactory was obtained from the attribute of the non-discriminatory service, about 78.5%. this was consistent with the highest mean value of the service reception obtained from the attribute of the non-discriminatory service. it was also followed by other five attributes on the aspect of assurance, which were the attribute of the hospitality services by medical staffs, the attribute of the medical staffs’ skills, the attribute of the reasonable and affordable rates, the attribute of the medical staffs’ thoroughness, and the attribute of the medical staffs’ knowledge, so the aspect of assurance had the highest percentage of the satisfactory of the patients, above 70%, than the aspect of dental service quality. based on the results, it can be indicated that there were some attributes that were considered to be important for the patients or had high expectation value, but still not being fully met by the dental care services provided by the service providers or the public health center. the attributes of waiting room comfort, service readiness, and service preparation actually obtained high interest value from the respondents, but there still was a large percentage of the number of the respondents who were not satisfied. discussion the characteristics of services involved can be related with an activity with intangible elements. in the service process, there are aspects of interaction between service users and service providers although sometimes parties involved are not always aware of the interaction, and do not result in the transfer of owner.1,4,5 quality improvement efforts that have impacts on improving the utilization of services require both of the focus on how to provide needs, demands, and expectations of service users, as well as the integration of all activities of the organization in order to meet the needs, demands, and expectations in providing services. both of them can actually be related with the orientation of the long-term achievement of organizational goals. the process of fulfilling the needs of service users is actually more complicated than the process of purchasing goods. the first stage in the process of fulfilling the needs of service users is concerned with how the customers identify the various alternatives, consider the risks and the advantages, and then make a purchase decision. the next stage is the interaction between service users and service providers. an the last stage is about the assessment of the service users on the services perceived.5,6 based on the observation results of the value of importance and achievement of the service quality of waru sidoarjo cdhs, it is known that there were some attributes including the comfort of waiting room, the readiness of service, and the preparation of service obtaining high importance value from the respondents, but they still obtained a large percentage of the number of respondents who felt unsatisfied. those patients of cdhs felt that the dental care service perceived still had not optimally met their expectation considered to be important for them. the service quality perceived by service users (perceived service quality) is actually the difference of the expectations or wishes of the service users (expected service) and the reality of the service they received (perceived service). based on this difference between the expectations and the reality, the satisfactory of the service users then can be obtained. this condition will finally trigger the loyalty of the service users to the service providers. the level of the ability of the service providers in fulfilling the expectations and needs of the service users is considered as the basis of the service quality.7,8 waru sidoarjo cdhs actually requires efforts to increase the satisfactory of patients with quality of dental care provided. these efforts, therefore, require a fundamental understanding of the expectations of patients. the expectations of patients are greatly influenced by experiences and taste. the expectations of service users are believed to have a major role in determining the quality of service and customer satisfaction. basically there is a close relationship between the determination of quality and customer satisfaction. in evaluating process, it is known that customers will usually use their expectations as a standard or reference. the components of the expectations of patients can become the basic ingredient in the process of mutual interaction between the needs and the providing of the needs of patients. knowing the needs of service users, therefore, can help service providers understand how and why customers react to product delivery. the expectations of patients, however, will continue to evolve to keep pace of time progress. the development of the expectations of patients improves as much information perceived by ��bramantoro and palupi: analysis of importance level and quality achievement patients, and patients become more experienced, and will affect the level of the perceived satisfactory of the patients.9,10 based on the results of the study, it can be concluded that there are attributes that are considered important or have a high expectation value by the respondents, but they are not fully provided by health services conducted by service provider or health center. those high interest value attributes include waiting room comfort, service readiness, and service preparation. however, in this study it is known that there are still a large percentage of respondents who were not satisfied with the services. references 1. supriyanto s, ernawaty. pemasaran jasa industri kesehatan. edisi 1. surabaya: fkm universitas airlangga; 2006. p. 195–216. 2. sabarguna b. pemasaran rumah sakit. konsorsium rsi jateng-diy, 2004. p. 88–93. 3. ka r yd is a, kodova zen it i mk , hat zigeorgiou d, pa n is v. expectations and perceptions of greek patients regarding the quality of dental health care. int j for quality in health care 2001; 13(5): 409–16. 4. muninjaya aa. manajemen kesehatan. jakarta: egc. 2004. p. 235–39. 5. chahal h, sharma rd, gupta m. patient satisfaction in public outpatient health care services. j health management 2004; 6(1): 23–45. available at: http://jhm.sagepub.com. accessed december 11, 2010. 6. cooper br, monson al. patient satisfaction in a restorative functions dental hygiene clinic. j dent educ 2008; 72(12): 1510–5. 7. sur h, hayranl o, yildirim c, mumcu g. patient satisfaction in dental outpatient clinics in turkey. croat med j 2004; 45(5): 651–54. 8. john j, yatim fm, mani sa. measuring service quality of public dental health care facilities in kelantan malasyia. asia pac j public health 2010; 20(10): 1–12. available at: http://aph.sagepub.com. accessed june 12, 2011. 9. patrick jm, alan be. marketing the dental practice: eight steps toward success. j am dent assoc 2006; 137: 1426-33. available at: http://jada.ada.org/cgi/content/full/137/10/1426. accessed november 10, 2010. 10. westaway ms, rheeder p, van zyl dg, seager jr. interpersonal and organizational dimentions of patient satisfaction: the moderating effects of health status. int j for quality in health care 2003; 15(4): 337–44. 157 the citotoxicity of calcium hydroxide intracanal dressing by mtt assay nanik zubaidah department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract calcium hydroxide had been used as the intracanal dressing in endodontic treatment due to its high alkaline and high antimicrobial capacity. it also be able to dissolve the necrotic tissue, prevent the root resorbtion and regenerate a new hard tissue. the aim of this study is to identify the concentration of calcium hydroxide that has the lowest citotoxicity. there are 5 groups, each group had 8 samples with different concentration of calcium hydroxide. group i: 50%, group ii: 55%, group iii: 60%, group iv: 65% and group v: 70%. the citotoxicity test by using enzymatic assay of mtt [3-(4.5-dimethylthiazol-2yl) ]-2.5 diphenyl tetrazolium bromide, against fibroblast cell (bhk-21). the result of susceptibility test was showed by the citotoxicity detection of the survive cell of fibroblast that was measured spectrophotometrically using 595 nm beam. the data was analyzed using one-way anova test with significant difference a = 0.05 and subsequently lsd test. the result showed that in concentration 50%, 55%, 60%, 65%, and 70% calcium hydroxide had low toxicity, but calcium hydroxide 60%, had the lowest toxicity. key words: calcium hydroxide, citotoxicity, mtt assay correspondence: nanik zubaidah, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction to day calcium hydroxide [ca(oh)2] is prefere drug used as intracanal dressing in dentistry especially in endodontic treatment. in 1920 the first time herman used calcium hydroxide to nonvital dental treatment. in modern endodontic treatment, calcium hydroxide is introduced as intracanal dressing which is used in post intracanal preparation to obtain sterile intracanal. the principe of intracanal treatment including preparation, sterilization and filling intracanal in which all of them are interrelated.1,2 the form of calcium hydroxide would be a powder or paste. calcium hydroxide paste is a mixture of calcium hydroxide powder and steril distilled water or combination with other solvent such as: steril physiological saline solution, local anesthetic solution (citanest-octapressin 3%), camphorated monochlorophenol (chkm) etc. calcium hydroxide 50% (50 gram calcium hydroxide powder/ 100 ml sterile distilled water can be clinically used in routine practices (according to the direction of manufactor).3,4 now day endodontic material with calcium hydroxide as raw material is available in the market and it is used for intracanal dressing paste in various trade mark and common by expensive will beneficially in routine application and cheap. pure calcium hydroxide in endodontic treatment is expected to be clinically applied as intracanal dressing.5 cohen and burn6 reported that calcium hydroxide was chosen as intracanal dressing due to the properties such as: antimicrobial capacity and capable to regenerate a new hard tissue, to dissolve pulp necrotic, to penetrate accessories root canal and dentinal tubuli so that it could decrease permeability of dentinal surface, to dry excessive excudate because of the presence of calcium ion so that it could decrease permeability of capillary vessel and also irritation and inflammation on the tooth apical will not occur during excessive filling of calcium hydroxide pasta.7 the above opinion is in contradiction with vajrabhaya’s8 opinion stated that the use of calcium hydroxide pasta in filling intracanal dressing, if it flows out of intracanal would cause tissue irritation, while, stock and nehamer9 suggested the use of excessive calcium hydroxide intracanal dressing resulting irritation on periapical tissue as well as on healthy, survive tissue and could slow down the healing process. calcium hydroxide is dangerous if it flows out of periapical and enters alveolaris inferior canal resulting neurotoxic.10 the prolong use of calcium hydroxide intracanal dressing in root canal, therefore, biocompatibility factor is absolute requirement in addition non toxic, either locally or systemically will not irritate biological environment.11,12 citotoxicity test on a tissue is one of the tests to determine toxic effect of a material or medicine. citotoxicity test is one of methods evaluate to dentistry material for its direct toxicity to cell. enzymatic assay is applied to measure citotoxicity of a material using mtt 3-[4.5-dimethylthiazol2-yl]2.5 diphenyl tetrazolium bromide. mtt enzymatic test is to measure the capability of survive cell based on 158 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 157-160 mitochondria activity of cell culture. the test is mostly used to measure quantitatively cellular proliferation or to measure the number of survive cell.11,13 based on the above description, the problems appear: how is the citotoxicity of calcium hydroxide as intracanal dressing using mtt method? the purpose of the present study is to determine the concentration of calcium hydroxide with low citotoxicity and the advantage is to provide for scientific information dentist and people that proper concentration of calcium hydroxide could be used as intracanal dressing resulting good endodontic treatment. material and method the study is experimental laboratory and the study design is the post test only controlled group design, done in the laboratory of tropical disease center (tdc), airlangga university. performing citotoxicity test calcium hydroxide is in pusat veterina viafarma (pusverma) for processing cell culture in which the cell was taken from baby hamsterkidney-21(bhk-21) specifically from kidney fibroblast of baby hamster and l-929 cell was taken from lung fibroblast of a mouse.14 device and material were :pure calcium hydroxide powder (no. m2047, merck, darmstadt,germany), sterile aquabidest (kimia farma ), fibroblast cell culture (bhk21), stock pusvetma passage 52, trypsin versene solution, media rose well park memorial institute(rpmi)-1640 containing hepes 6gr/lt, pen strep 100ml, bovine serum 10%, phosphatase buffer saline (pbs) solution containing na cl 8 gr, kcl 0.2 gr, na2hpo4 1.15 gr kh2po4 in 0.2 gr steril distilled water 1 lt, mtt(sigma cat. no. m-5655) dimethyl sulfoxide (dmso) (mp biomedical, llc, catalog 190186 lot .r.19953), 96 microplates, roux bottle spectrophotometer 595 nm, micro pipette, eppendorf, cement spatel. sample groups of calcium hydroxide paste were classified into 5 groups for citotoxicity test consisting of group i (calcium hydroxide 50%), group ii (calcium hydroxide 55%), group iii (calcium hydroxide 60%), group iv (calcium hydroxide 65%), and group v(calcium hydroxide 70%). the process of making calcium hydroxide in concentration 50%, 55%, 60%, 65%, 70% was obtained by mixing powder of calcium hydroxide which had been measured according to the desired weight with sterile aquabidest until it become paste form the mixture was done in eppendorf tube and stirred using cement pastel for 1 minute, then, homogenization was done with vortex for 30 seconds.15 calcium hydroxide solution was made ie: various concentration 50%, 55%, 60%, 65% and 70% by having calcium hydroxide and was sterilized using ultra violet for 30 minutes. calcium hydroxide was mixed with sterile distilled water until homogeneous for 1 minute. next, the mixture was put into plate 320 µl and followed by incubating for 1 hour at 37° c. the next step, the mixture was put into 640µl media rpmi-1640 and incubated at 37° c for 24 hours. the solution was filtered with 0.2 µm (ministart). citotoxicity test of calcium hydroxide was done with cell culture (bhk-21) in cell line planted in roux bottle. after confluent, culture was harvested using trypsine versen solution. the harvest was taken little by little replanted in rose well park memorial institute (rpmi-1640) media. which contained 10% albumine serum of vital bovine incubated for 24 hours at 37° c.then cell with moved into roux bottle and was made with 2 × 105 cell/ml density. the cell was ready for sample testing. citotoxicity test using at the bottom of wells of 96 well cell culture plate. citotoxicity test was done according to standard procedure for mtt assay.16 every well contained cell + rpmi 100 ml with 2 × 105 cell/ml density in the amount of 50 ml. prior to the test, calcium hydroxide solution sample was sterilized using ultra violet for 15 minutes, next, 50 ml sample was put into well cell culture plate. on this study, the test was done twice (duplo). then, well cell culture plate was incubated for 20 hours at 37° c. followed by every well was filled 25 ml of 5 mg/ml mtt which had been solved in pbs, incubated for 24 hours at 37° c. then every well was added by 50 ml dmso. finally, well cell culture plate was read on spectrophotometer with 595 nm wave length. the result was stated in optical density (absorbent). the amount of absorption in every well showed the number of survive cell in media culture.17 on this study, the data of survive cell measurement of citotoxicity test. using enzymatic test with method of mtt (3-(4.5-diethyl thiazol-2yl), 2.5 diphenyl tetrazolium bromide. the study sample was divided into 5 groups of calcium hydroxide in different concentration i.e. group i (50%), ii (55%), iii (60%), iv (65%) and v (70%) in which every group consisted of 8 samples. the data was taken from statistical one-way anova test with significance limit α=5%, then followed by lsd. result the result of the study on citotoxicity of calcium hydroxide done on fibroblast cell (bhk-21) using 40 samples classified into 5 groups. each group consisted of 8 sample with different concentration of calcium hydroxide group i: 50%, group ii: 55%, group iii: 60%, group iv: 65% and group v: 70%. the mean and deviation standard of various concentration calcium hydroxide on fibroblast cell bhk-21 could be seen on table1. citotoxicity test shows the calcium hydroxide in various concentration (50%, 55%, 60%, 65% and 70%) on fibroblast cell bhk-21. the result shows that calcium hydroxide in concentration 60% (group iii) has reached the highest number of survive cells comparing with other concentration. it is showed on figure 1. 159zubaidah: the citotoxicity of calcium hydroxide intracanal dressing by mtt assay table 1. the mean deviation standard of citotoxicity test in various concentration calcium hydroxide on fibroblast cell bhk-21 in optical density (od) concentration n x sd group i group ii group iii group iv group v 8 8 8 8 8 0,70500 0,75638 0,77350 0,76400 0,74075 0,075848 0,092657 0,091147 0,070751 0,076282 note : n = the number of samples x = mean of the number survive cell sd = standard deviation figure 1. graphic of mean of survive cell citotoxicity test calcium hydroxide various concentration toward fibroblast. table1 shows that citotoxicity test of calcium hydroxide done on fibroblast cell (bhk-21) in 5 groups, each group consists of 8 samples. group iii (60%) shows the mean of the highest number survive cells: 0.77350, while group i (50%) shows the mean of the lowest number survive cells: 0.70500. the data on table 1 was achieved from homogenecity test with lavene statistic, p = 0.557 (p > 0.05), meaning the data is homogeneous and normal distribution using one sample kolmogorov smirnov test. the citotoxicity effect was analyzed by one-way anova statistically a = 5% (table 2). table 2. the result of one-way anova direction citotoxicity test in various concentration calcium hydroxide toward fibroblast cell bhk-21 ts fl ms c sig inter group on group 0,023 0,204 4 35 0,005756 0,005826 0,988 0,427 total 0,227 39 note: ts : total square; fl : free level; ms : mean square; c : calculation; sig : significant the result of one-way anova test showed no significant difference among control groups p > 0.05. discussion clinical used of pure calcium hydroxide as intracanal dressing on endodontic treatment is generally in the form of paste. mixing calcium hydroxide powder and sterile distilled water as vehicle could make calcium hydroxide paste. calcium hydroxide 50% is generally used at the clinic. besides sterile distilled water as vehicle, calcium hydroxide could be combined with other solution such as: glicerin, methyl celllose, buffer saline, local anesthetic solution etc.3,4 a material that is used as intracanal dressing in endodontic treatment should be antitoxic. chang chao18 supports this idea suggested that biological and toxicological aspects of a material clinically used are very important. to use calcium hydroxide as intracanal dressing in endodontic treatment is always expected to have low toxicity. pissiotis and spangberg7 stated that excessive calcium hydroxide pasta in root canal filling would not cause irritation and no symptom of inflammation was found in apical region. the above idea was against the opinion of vajrabhaya et al.8 stated either intracanal dressing or sealer which flowed out of root canal would not induce periapical tissue irritation, while according to schawrtz10 stated it is dangerous, if calcium hydroxide flows out of alveolaris inferior canal it would cause neurotoxic. the opinion is also supported by wayman et al.19 that 17 of 58 cases found to require periapical surgical treatment due to endodontic failure caused by sealer flowing to periapical. the use of calcium hydroxide as intracanal dressing in various concentrations, up to now the safety factor toward periapical and adjecent tissue in oral cavity is still unknown. one of the requirements for dentistry material which is applied in oral cavity should be biocompatible i.e. no substance with toxic content. to prove that the material is safe and nontoxic, invitro cytotoxicy test should be done on fibroblast cell culture taken from baby hamster kidney (bhk-21) using enzymatic test i.e. mtt method in which fibroblast cell is dominant cell in pulp component, periodontal ligament and gingival. this cell line has been widely used to perform toxicity test on material and medicine in dentistry field. the advantages of using cell line culture are passage 50–70 times could be done, having high cell growth, cell integrity is well preserved and cell is capable to multiply in suspension. enzymatic test with mtt method [3-(4.5 dimethyl thiazol-2yl)-2.5 diphenyl tetrazolium bromide] is a method that is widely used to test citotoxicity of material or medicine in dentistry field and to measure quantitatively cellular proliferation or living cell. the use of this method is based on various reasons such as: accurate and sensitive result could be achieved by using spectrophotometer which could clearly detect cell metabolism change, could be easily manipulated, the 160 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 157-160 device could be easily acquired and available at laboratory, efficient and without isotop radioactive.11,17 mtt method determines the capability of living cell to reduce mtt. the mechanism is tetrazolium (yellow color) would be reduced in cell, which has metabolic activity. mitochondria of living cell would express dehydrogenase enzyme, which has an important role in this process. if mitichondria were not active due to toxicity of a substance, cell metabolism would be disturbed. so dehydrogenase enzyme could not be excreted consequently formazan, which has been formed, would be equal to the enzymatic activity of living cell.11,17,20 table 1 shows: cytotoxic strength of calcium hydroxide as intracanal dressing in fibroblast cell (bhk-21), there is no significant difference in concentration 50%, 55%, 60%, 65%, and 70% calcium hydroxide paste. it means that there is no difference in toxicity in all groups of calcium hydroxide test in various concentrations. calcium hydroxide paste 60% shows optical density of detected survive cell is 0.77350 higher comparing with calcium hydroxide in other concentration. in this concentration the number of survive cells is the highest since calcium hydroxide 60% is still capable to stimulate the cells so calcium hydroxide would release high calcium ion resulting the increase of alkaline phosphatase (alpase) enzyme activity consequently proliferation of fibroblast cell (bhk-21) would increase. the result of study also shows that in calcium hydroxide 65% and 70% the numbers of survive cells decrease. because calcium hydroxide has already reached saturated point toward viscosity so it is difficult to release calcium ion consequently it would decrease the activity of alkaline phosphatase (alpase). some in vitro studies on calcium hydroxide, one of studies done by rashid et al.21 has proved that by adding cacl2 4 mm in human pulp cell which has been measured, resulting the increase of alkaline phosphatase (alpase) activity so pulp cell proliferation would occur, however by adding 0.7 mm decreasing alkaline phosphatase (alpase) activity in pulp cell would cause decreasing the number of pulp cell. collagen synthetisis and alkaline phosphatase would increase simultaneously with the increase of ph medium until 8. calcium hydroxide in water would decompose into calcium ion and hydrixil ion. calcium ion, which is the content of calcium hydroxide, has an important role in cell calcification. chang chao18 suggested calcium hydroxide could produce calcified barrier at the end of apical root done in vivo study, while hasoya22 stated that calcium ion could improve circulation in capillary vessel, which could resist the effect. it is concluded that in concentration 60% calcium hydroxide shows the lowest toxicity comparing with calcium in concentration 50%, 55%, 65%, and 70%. while among the control groups there is no significant difference. sugesstion: it is necessary to perform further study on animal experiment to know the strength of citotoxicity of calcium hydroxide pasta in concentration 60%. references 1. grossman li, oliet s, del rio ce. ilmu endodontik dalam praktek. ebyono r, editor. cetakan i. jakarta: penerbit buku kedokteran egc; 1995. p. 248–50, 256. 2. widodo t. paradigma baru pada perawatan endodontik. kumpulan2. widodo t. paradigma baru pada perawatan endodontik. kumpulan naskah seminar sehari tema ramah tamah perlakuan pada jaringan gigi. ikorgi; 2000; p. 2, 6–7.ikorgi; 2000; p. 2, 6–7. 3. siqueira jf, uzeda m. influence of different vehicle on the antibacterial effect of calcium hydroxide. j endod 1998; 24(10):663. 4. solak h, oztan md. the ph change of four different calciumsolak h, oztan md. the ph change of four different calciumthe ph change of four different calcium hydroxide mixture used for intracanal medication. j oral rehab 2003;2003; 30:436–9. 5. estrela c. calcium hydroxide: study based on scientific evidence.estrela c. calcium hydroxide: study based on scientific evidence. j appt oral sci 2003; 11(4):269–82. 6. cohen s, burn rc. pathways of the pulp. 66. cohen s, burn rc. pathways of the pulp. 6th ed. st louis: mosby; 1994. p. 211–12, 486–507. 7. pissiotis, spangberg isw. biological evaluation of collagen gels containing calcium hydroxide and hydroxiapatite. j endod 1990; 16: 369. 8. vajrabhaya l, sithisam p, wilairat p, leelaphiwat s. comparison8. vajrabhaya l, sithisam p, wilairat p, leelaphiwat s. comparison between sulphorhodamine-b dye staining and 51 release method in cytotoxicity assay of endodontic sealers. j endod 1997; 23(6):355–57. 9. stock cjr, nehammer cf. endodontic in practice: the modern concept of endodontic. 2nd ed. london: british dental association; 1990. p. 1–10, 23–27. 10. schawrtz rg, dreyer m, aydin m. the opinion within this web peg are not our. authors have been credited for the individual post where they are. available at: www.rxroot.com-x-rays. courtesty. dental india home page. accessed may 23, 2005. 11. meizarini a. sitotoksisitas bahan restorasi cyanoacrylate pada variasi perbandingan powder dan liquid menggunakan mtt assay. majalah kedokteran gigi (dental journal) 2005; 38(1):20–24. 12. van noort r. introduction to dental material. 2nd ed. london: cv mosby company; 2003. p. 3–5. 13. fazwishui s, hadijono bs. uji sitotoksisitas dengan esei mtt.uji sitotoksisitas dengan esei mtt. jkgui 2000; 7:28–32. 14. freshney ri, culture of animal cells. a manual of basic technique. 2nd ed. new york: alan r liss inc; 1987. p. 9, 71, 128, 239. 15. estrela c, pimenta fc, ito yoko i, bammann l. in vitro determination of direct antimicrobial effect of calcium hydroxide. j endod 1998; 24(1):15–17 16. dash p. standard protocols mtt assay (kutipan dari yuliati a.dash p. standard protocols mtt assay (kutipan dari yuliati a. majalah kedokteran gigi (dental journal) 2004). available from: http://web.bham.ac.uk/can4psd4/brum/mtt.html. accessed january 8, 2002. 17. yuliati a. viabilitas sel fibroblast bhk-21 pada permukaan resin akrilik rapid heat cured. majalah kedokteran gigi (dental journal) 2005; 38(2):68–71. 18. chang chao y, huang fue m, cheng hm. in vitro evaluation of the cytotoxicity and genotoxicity of root canal medicine on human pulp fibroblast. j endod 1998; 24(9):604–06. 19. wayman be, murata sm, almeida rj, fowler cb. a bacteriological and histological evaluation of 50 periapical lesion. j endod 1992; 18:152–5. 20. telli c, serper a, dogan l, gug d. evaluation of the cytotoxicity of calcium phosphate root canal sealer by mtt assay. j endod 25 1999; 25(12):811–13. 21. rashid f, shiba h, mizuno n. the effect of extracellular calcium ion on gene expression of bone related protein in human pulp cells. j endod 2003; 29(2):104–5. 22. hosoya n, takashi g, arai t, nakamura j. calcium concentration22. hosoya n, takashi g, arai t, nakamura j. calcium concentration and ph of periapical environment after applying calcium hydroxide into root canal in vitro. j endod 2001; 27(5):343–46. 177 volume 45 number 3 september 2012 research report physical characteristic of brown algae (phaeophyta) from madura strait as irreversible hydrocolloid impression material prihartini widiyanti1,2 and siswanto3 1biomedical engineering study program, department of physics, faculty of science and technology, universitas airlangga 2institute of tropical disease, universitas airlangga 3physics study program, department of physics, faculty of science and technology, universitas airlangga surabaya-indonesia abstract background: brown algae is a raw material for producing natrium alginates. one type of brown algae is sargassum sp, a member of phaeophyta division. sargassum sp could be found in madura strait indonesia. natrium alginate can be extracted from sargassum sp. the demand of alginate in indonesia is mainly fulfilled from abroad, meanwhile sargassum sp is abundantly available. purpose: the purpose of study were to explore the potency of brown alga sargassum sp from madura strait as hydrocolloid impression material and to examine its physical characteristic. methods: the methods of research including extraction natrium alginate from sargassum sp, synthesis of dental impression material and the test of porosity, density, viscosity, and water content of impression material which fulfilled the standard of material used in clinical application in dentistry. results: extraction result of sargassum sp was natrium alginate powder with cream colour, odorless, and water soluble. the water content of natrium alginate was 21.64% and the viscosity was 0.7 cps. the best porosity result in the sample with the addition of trinatrium phosphate 4% was 3.61%. density value of impression material was 3 gr/cm3. conclusion: the research suggested that brown algae sargassum sp from madura strait is potential as hydrocolloid impression material, due to its physical properties which close to dental impression material, but still need further research to optimize the physical characteristic. key words: brown algae, madura strait, irreversible hydrocolloid, physical characteristic abstrak latar belakang: alga coklat adalah sumber bahan baku material natrium alginat. salah satu jenis alga coklat adalah sargassum sp yang merupakan anggota divisi phaeophyta. sargassum sp dapat ditemukan di selat madura indonesia. natrium alginat dapat diekstraksi dari sargassum sp. kebutuhan akan bahan ini di indonesia sebagian besar dipenuhi dari impor, padahal ketersediaan sargassum sp di indonesia sangat melimpah. tujuan: penelitian ini bertujuan untuk mengeksplorasi potensi alga coklat sargassum sp dari selat madura sebagai bahan cetak hidrokoloid dan meneliti karakteristik fisiknya. metode: tahap pertama adalah ekstraksi natrium alginat dari sargassum sp, tahap kedua yaitu sintesis bahan cetak gigi dan menguji karakteristik bahan seperti porositas, densitas, viskositas, kadar air, bahan cetak yang memenuhi standar bahan yang digunakan dalam aplikasi klinis bidang kedokteran gigi. hasil: hasil ekstraksi berupa natrium alginat bubuk dengan warna krem, tidak berbau, dan dapat larut dalam air. kadar air natrium alginat sebesar 21,64% dengan viskositas 0,7 cps. porositas terbaik dalam sampel dengan penambahan trinatrium fosfat 4% yaitu 3,61%. nilai densitas bahan cetak 3 gr/cm3. kesimpulan: penelitian ini menunjukkan bahwa alga coklat sargassum sp dari selat madura memiliki potensi sebagai bahan cetak hydrocolloid kedokteran gigi karena memiliki karakter fisik yang mirip dengan bahan cetak kedokteran gigi, namun masih memerlukan penelitian lebih lanjut untuk mengoptimalkan karakter fisiknya. kata kunci: brown algae, selat madura, hidrokoloid ireversibel, karakteristik fisik correspondence: prihartini widiyanti, c/o: departemen fisika, fakultas sains dan teknologi universitas airlangga, jl. mulyorejo surabaya, indonesia. e-mail: drwidiyanti@yahoo.com 178 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 177–180 introduction sargassum sp., the main substance of natrium alginate as the material of irreversible hydrocolloid impression material in dentistry, could grow in the calm or wavy and craggy seas. sargassum sp has cylindrical thallus shape or flattened, with lots of branching resembles land trees. the main stems are rounded, harsh, with discoid shape. the leaves are wide, tapering like a sword, and has solitaire air bubble. the edge of leaves is a bit serrated and wavy with curved or tapered tip. its color is brown, relatively largesized, grow and flourish on strong base substrate. the upper part resembles bilateral symmetrical shaped shrub or radial and equipped with growing part.1 brown algae, sargassum sp grow in the shallow sea territorial. it can be found in many places around madura strait indonesia and can be easily taken from the sea but the availability in the beach is not so abundant. sargassum sp has not been cultivated yet because the people living around the beach still did not a ware the benefit of these algae. one of dental impression materials is alginate. alginate has become the material of choice due to the accuracy of line and shape reproduction, comfortable for patient, and its easy mixing and modification.2 alginate impression material is the irreversible hydrocolloid material. so if alginate is mixed with other substance and the chemistry reaction occurred, the alginate cannot turn back to its original form. the main component of hydrocolloid impression material is natrium alginate. if natrium alginate is mixed with water, it would become solution and as booster could be added calsium sulphate. diatom earth and silica gel was added as filler to increase the strength, hardness, to influence setting time and physical properties of alginate gel. accelerator and retarder material was needed to arrange the setting time. kalium sulphate is act as accelerator. natrium or trinatrium phosphate is act as retarder.3 polyethylene glicol is added to coat impression material powder so that the powder cannot easily steam like dust. research about the influenced of retarder which were trinatrium phosphate and kalium oxalat to the alginate impression material was performed in 2008.2 impression material with addition of trinatrium phosphate 0.3 gram yielded flatter surface, homogen, and the highest decomposition temperature which was 55° c. up to now the availability of alginate is supported by importing the material from abroad. since the brown algae are contain alginate and it can be found easily and abundantly in indonesian seas, sargassum sp has an economical value. sargassum sp has potency to produce natrium alginate which has been known as raw material for alginate dental impression. some research has been done but the result has not been used and produced directly as dental impression material. this research aims were to explore the potency of brown algae sargassum sp from madura strait as hydrocolloid impression material and to examine its physical characteristic. materials and methods material of this research were brown algae sargassum sp. from madura strait, aquades, water, 5% hcl, 4% na2co3, 12% naocl, 10% naoh, isopropanol, calsium sulphate, silica gel, calium sulphat, peg, diatom earth, and trinatrium phosphate. the research process was divided into 2 parts. the first part was the extraction of natrium alginate and second part was the synthesis of irreversible hydrocolloid.4,5 dry brown algae sargassum sp. was immersed in 1% hcl for 1 hour. after immersion in acid solution brown algae was washed and 4% na2co3 was added. the mixture was heated in the temperature of 60° c for 2 hours. brown algae then was diluted with aquades, left for approximately 30 minutes, and filtered. the result then was bleached and stirred with 12% naocl solution. five percent of hcl then was added until ph value reached 2-3 (acid). after foam wadding of alginate acid was gained by filtering the mixture, the foam wadding was washed with water to eliminate dangerous acid sludge and the 10% naoh was added until ph 9 reached. alginate acid converted to natrium alginate then added isopropanol (99%) with the ratio 1: 2 (ipa: acid alginate). separated natrium alginate then was filtered and dried. the extraction result was natrium alginate powder which ready to be composed as impression material. the irreversible hydrocolloid impression material making was done by mixing all the composition material using mortar and pestle. the composition material consist of 19% natrium alginate, 40% calsium sulphate, 15% calium sulphate, 4% diatom earth, 15% silica gel, and 7% peg. there were 5 variations of trinatrium phosphate percentage of impression material sample which were 0% (sample a), 1% (sample b), 2% (sample c), 3% (sample d), and 4% (sample e). physical characteristics was determined by examined the water content, viscosity, porosity and density of materials.4,5 water content examination was done by measuring the initial weight, drying material in oven (thermogravimetri) in high temperature (100–300° c) for 3 minutes to 3 hours or until it reaches constant weight, and the final weight. the difference between the weight before and after the drying process was the amount of water vapor. viscosity measurement was done using kinematic viscometer bath by entering fluid sample, installing the rotor, and then switching on the machine. the viscosity of substance could be read in the scale. the porosity test examined dry weight. the material inserted into water and examined the mass. the porosity value could be known by comparing the dry weight material and the wet material mass. the procedure was done by mixing 0.3 gram of material with 150 µl water, after setting, measured the dry weight sample. the material was dipped into 6 ml water, and measured the wet sample weight. the dry and wet weight difference was calculated. density test was calculated using archimedes method. the first step was sample weight measurement using 179widiyanti: physical characteristic of brown algae analytical scales with accuracy 0.001 gram to determine sample mass w, then measured the weight sample in water media wa (whole sample was drowned). density test was done to characterize sample and gain the density of sample. it is defined as total mass in one unit volume. the denomination was in unit gram per centimeter cubic (g/cm3). the mass in gram was the mass in 1 cm3 water in specific temperature. results extraction result was natrium alginate powder with cream colour, odorless, dissolved in water (figure 1). was the porosity of commercial dental material impression is 1.82%. the porosity of dental impression with addition of 0%, 1%, 2%, 3%, 4% trinatrium fosfat were 6.48%; 6.42%, 6.37%, 6.62% and 3.61% respectively. density value of impression material were with addition of 0%, 1%, 2%, 3%, 4% trinatrium fosfat were subsequently 3.27, 3.35, 3.33, 3.26, 3.39 gr/cm3. the data of porosity and density is shown in figure 2. the water content of natrium alginate was 21.64% and the viscosity was 0.7 cps. discussion extraction of natrium alginate from sargassum sp refers to modified extraction method.4,5 freeze drying method was used for drying process. natrium alginate powder was cream in color, odorless and dissolved in water. this result fulfilled the farmakope requirement 1974.6 maximum water content of natrium alginate which had required by food chemical codex7 is 15%. the content of natrium alginate in food is at least 13%8. according to the research,9 water content in natrium alginate was in the range between 5% to 20%. water content of natrium alginate of sargassum sp was 21.64%. it closed to the range of allowed water content of natrium alginate. there was no significant difference in this data (1.64%). this is because sargassum sp has hygroscopic properties which need long time in drying process. drying process which was used in this research was thermogravimetri method (high heating). this method has some weakness. the first was water the other substance and could evaporated and gone with water vapor. beside that, the reaction could be happened during the heating. material contain substance which capable to bind water strongly will difficult to release water even it had been heated. material which has been dried could be more hygroscopic than the origin material. that is why during cooling process before weighing, material should be placed in closed container like exycator or decycator with water absorbant.10 viscosity value is a measurement of the resistance of a fluid which is being deformed by either shear stress or tensile stress. the lower viscosity, the higher elasticity and the lower permanent deformity of impression material.11 viscosity value of natrium alginate of sargassum sp and control was 7000 cps and 5000 cps respectively. viscosity of natrium alginate from sargassum sp higher than the control group, it showed that the sample's elasticity was lower than control. viscocity of natrium alginat standard value are varied in the range of 10 to 5.000 cps (solution porosity density 1,82 6,75 6,48 3,27 6,42 3,35 6,37 6,62 3,33 3,26 3,61 3,39 0 control a (0% trisodium phosphat) b (1% trisodium phosphat) c (2% trisodium phosphat) d (3% trisodium phosphat) e (4% trisodium phosphat) gr /c m 3 0 1 2 3 4 5 6 7 figure 2. the porosity and density of impression material with natrium alginate from sargassum sp. figure 1. natrium alginate powder of sargassum sp. 180 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 177–180 concentration 1%).9 beside that, there are 3 type of viscocity standard commercial (sigma 2008) which are 14.000 cps (high viscosity), 3.500 cps (medium viscosity) and 250 cps (low viscosity). it has been suggested that the viscosity property of all alginates is affected by method of extraction.12 prolong extraction period with higher na2 c03 concentration also causes degradation of alginates. sargassum habitat is rocky areas and is affected by direct waves, so the concentration of polygalacturonate is higher than alginofit which live in different habitat and has weak holdfast. the viscosity was also determined by the presence of cations such as ca2+ and the residue of galacturonate.13 the difference of viscosity value may cause of the different of sample viability and quality. compare with the commercial viscosity standard of sigma, the viscosity result in this research was in the range of "medium viscosity" and "high viscosity". it showed that sargassum sp has high potential as raw material of natrium alginate processing in indonesia. the density value of sample was much lower than the density value of control which was 6.75 gr/cm3. it might be caused by the procedures of milling and the quality of brown algae. the quality of brown algae is depend on the salinity of sea water, humidity etc. it revealed that we still need more advanced research of natrium alginate from sargassum sp density was supporting the porosity value. the high density, the minimum amount of extractable fraction, and the good mechanical properties of cross-linked alginate were found to be responsible for the elasticity of water-swollen hydro gel hybrid.14 the density standard of natrium alginate were 2.54 g/cm3 (anhydrous), 2.25 g/cm3 (monohydrate), 1.51 g/cm3 (heptahydrate) and 1.46 g/cm3 (decahydrate).15 the porosity value of natrium alginate commercial product was 1.82%. the porosity value of sample without addition of trinatrium phosphate was 6,48%, and with the addition of trinatrium phosphate were 1%, 2%, 3%, 4% were 6.42%, 6.37%, 6.62%, 3.61% respectively. compared with commercial product, impression material with natrium alginate from sargassum sp had higher porosity value, which was not caused by the retarder (trinatrium phosphate). addition of 4% trinatrum phosphate in impression material will decrease the porosity value. in porosity test, the weight of impression material and the volume of water were constant so the porosity value was not influenced by both factors. porosity value is drived by the size of particle. the mixing process of impression material which used mortar and pestle (manual) could be considered as one of the reasons. this manual method provided different pressure to the particle of sample.16 the research suggested that brown algae sargassum sp from madura strait is potential as hydrocolloid impression material, due to its physical properties which close to dental impression material, but still need further research to optimize the physical characteristic. references 1. anggadiredja jt, zatnika a, purwoto h, istini s. rumput laut. jakarta: penebar swadaya; 2010. p. 5–22, 69–83. 2. anusavice jk. philiphs: buku ajar ilmu bahan kedokteran gigi. budiman ja, purwoko s, eds. jakarta: penerbit buku kedokteran (egc); 2003. p. 239–44. 3. situngkir j. pembuatan dan karakterisasi fisikokimia bahan cetak gigi palsu kalsium alginate. thesis. medan: universitas sumatera utara; 2008. 4. juniarto. rendemen dan kualitas alginat hasil ekstraksi alga (sargassum sp.) dari pantai selatan daerah cidaun barat. jurnal bionatura 2006; 8(2): 152–60. 5. rasyid a. ekstraksi natrium alginate dari alga coklat sargassum echinocarphum. jakarta: pusat penelitian oseanografi–lipi; 2010. p. 393–400. 6. tomitro fx, dina ka, rike r. pemanfaatan daun cyclea barbata sebagai alternatif substansi dasar bahan cetak di bidang kedokteran gigi. buletin penalaran mahasiswa ugm 1997; 3(1): 19–22. 7. food chemical codex. food chemical codex. 3rd ed. washington dc: national academic of science; 1981. p. 135–95. 8. cottrell iw, kovacs p. algin. in: graham hr, editor. food colloids. new york. avi publ co connect; 1977. p. 438–63. 9. winarno fg. teknologi pengolahan rumput laut. jakarta: pustaka sinar harapan; 1990. p. 112. 10. winarno fg. kimia pangan dan gizi. jakarta: pt. gramedia pustaka utama; 1997. p. 20–5. 11. powers jm, sakaguchi rl. craig's restorative dental material. 12th ed. missouri: elsevier; 2006. p. 514–6, 524–7. 12. american dental association specification no. 18 for alginate impression material, may 1992. 13. ertesvag h, vall s, skjak-braek g. enzymatic alginate modification. in: rehm bha, editor. alginates: biology and applications. berlin: springer-verlag; 2009. p. 102–22. 14. omidian h, rocca jg, park k. elastic, superporous hydrogel hybrids of polyacrylade and natrium alginate macromol. biosci 2006; 6: 703–10. 15. united nationts environment programme. sodium carbonate. cas no 437-19-8. sids initial assessment report for siam 15. 2003, p. 5 16. mour m, das d, winkler t, hoenig e, mielke g, morlock mm, schilling af. advances in porous biomaterials for dental and orthopaedic applications. materials 2010; 3: 2947–74. 224 volume 46, number 4, december 2013 korelasi indeks morfologi wajah dengan sudut interinsisal dan tinggi wajah secara sefalometri (cephalometric correlation of facial morphology index with interincisal angle and facial height) pricillia priska sianita k dan verenna departemen ortodonsia fakultas kedokteran gigi universitas prof. dr. moestopo (b) jakarta indonesia abstract background: in a disaster or criminal case, comprehensive information is needed for identification process of each victim. especially for some cases that only leave skull without any information that could help the identification process, including face reconstruction that will be needed. one way of identifications is specific face characteristic, race, some head-neck measurements, such as facial morphology index, interincisal angle and facial height. purpose: the aim of study was to determine the correlation of facial morphology index with interincisal angle and facial height through cephalometric measurement. methods: the samples were cephalogram of 31 subjects (deutro-malayid race) who met the inclusive criteria. cephalometric analysis were done to all samples and followed by pearson correlation statistical test. results: the correlation was found between facial morphology index and facial height, but no correlation between facial morphology index and interincisal angle. conclusion: the study showed that the cephalometric measurement of facial morphology index and facial height could be used as the additional information for identification process. key words: facial morphology index, interincisal angle, facial height, identification, facial type abstrak latar belakang: dalam bencana alam atau kasus kriminal informasi yang komprehensif diperlukan untuk proses identifikasi masing korban. khususnya pada beberapa kasus yang hanya meninggalkan tengkorak tanpa informasi yang dapat membantu proses identifikasi, termasuk rekonstruksi wajah yang akan dibutuhkan. salah satu cara identifikasi karakteristik wajah tertentu, ras, beberapa pengukuran kepala leher, seperti indeks morfologi wajah, sudut interincisal dan tinggi wajah. tujuan: penelitian ini bertujuan meneliti korelasi indeks morfologi wajah dengan sudut interincisal dan tinggi wajah melalui pengukuran sefalometrik. metode: sampel penelitian adalah cephalogram dari 31 subyek ras deutro malayid ras yang memenuhi kriteria inklusif. analisis cephalometri dilakukan pada semua sampel dan dilanjutkan dengan uji statistik korelasi pearson. hasil: korelasi ditemukan antara indeks morfologi wajah dan tinggi wajah, tapi tidak ada korelasi antara indeks morfologi wajah dan sudut interincisal. simpulan: hasil penelitian menunjukkan bahwa pengukuran sefalometrik indeks morfologi wajah dan tinggi wajah, dapat digunakan sebagai informasi tambahan untuk proses identifikasi. kata kunci: indeks morfologi wajah, sudut interinsisal, tinggi wajah, identifikasi, tipe wajah korespondensi (correspondence): pricillia priska sianita k, departemen ortodonsia, fakultas kedokteran gigi universitas prof. dr. moestopo (b). jl. bintaro raya 3 jakarta selatan 12330, indonesia. e-mail: ppriska@cbn.net.id research report 225sianita k dan verenna: korelasi indeks morfologi wajah dengan sudut interinsisal dan tinggi wajah pendahuluan dalam proses identifikasi seseorang, diperlukan informasi secara umum, termasuk karakteristik wajah seseorang yang kemudian dapat menunjukkan ciri khas ras seseorang yang diidentifikasi.1 dalam proses menentukan karakteristik wajah seseorang, diperlukan informasi terkait struktur wajah dan susunan gigi geligi. informasi struktur wajah ini meliputi dimensi vertikal wajah yang juga akan dipengaruhi oleh relasi oklusi gigi geligi.2-,4 pembahasan hubungan oklusal ini melibatkan sudut yang juga terlibat dalam pengukuran dimensi vertikal, yaitu sudut interinsisal.5 keanekaragaman pengukuran vertikal wajah akan menghasilkan keanekaragaman tipe vertikal wajah. satu metode pengukuran vertikal wajah adalah indeks morfologi wajah. indeks ini dihasilkan melalui pengukuran jarak dari nasion (n) sampai gnathion (gn) dikalikan 100 dan dibagi dengan jarak dari titik zygion (zy) kiri sampai kanan. hasil pengukuran ini akan menempatkan subyek ke dalam klasifikasi tipe wajah sangat lebar, lebar, sedang, sempit dan sangat sempit (tabel 1).6 sudut interinsisal adalah sudut yang dibentuk oleh perpotongan garis yang melalui sumbu gigi insisif rahang atas dan gigi insisif rahang bawah.5 tinggi vertikal wajah merupakan jarak yang diukur dari nasion (n) sampai gnathion (gn).4,5 tipe-tipe wajah yang dihasilkan dari pengukuran ini sangat khas dan menjadi ciri bagi individu yang bersangkutan, yang tentunya sangat penting dalam kaitan dengan proses identifikasi di bidang forensik. penelitian ini merupakan kajian pada sub ras deutromalayid, salah satu populasi yang banyak tersebar di wilayah indonesia bagian barat.6,7 dengan demikian, diharapkan dapat membantu mempermudah pengambilan sampel subyek penelitian. tujuan penelitian adalah untuk meneliti korelasi antara indeks morfologi wajah dengan sudut interinsisal dan tinggi wajah dalam analisis sefalometri. diharapkan, hasil penelitian ini dapat menjadi masukan dalam pertimbangan pemanfaatannya pada proses identifikasi kerangka tengkorak individu yang membutuhkan rekonstruksi ataupun pembuatan sketsa wajah dengan mengacu pada tipe wajah aslinya berdasarkan hasil pengukuran yang bersifat ilmiah. bahan dan metode sampel penelitian terdiri dari 31 subyek sub ras deutromalayid yang memenuhi kriteria inklusi sebagai berikut, maloklusi klas i angle, diskrepansi panjang lengkung gigi ≤4 mm, usia 20–30 tahun dan belum pernah menjalani perawatan ortodonsi serta bersedia berpartisipasi dengan menandatangani surat persetujuan setelah penjelasan (psp) atau informed consent. adapun kelompok subyek sub ras deutro-malayid ini dipilih melalui wawancara dan pengisian kuesioner tentang identitas diri sampai ke tingkatan dua generasi di atasnya yaitu ibu dan nenek yang termasuk dalam kelompok sub ras deutro-malayid. selanjutnya, dilakukan pembuatan sefalogram lateral di rumah sakit gigi dan mulut fakultas kedokteran gigi universitas prof. dr. moestopo (beragama). analisis sefalometri dan somatometri dilakukan pada sampel penelitian yang diambil dengan cara purposive sampling berdasarkan kriteria inklusi yang telah ditetapkan, antara lain: subyek dengan maloklusi klas i angle, diskrepansi panjang lengkung gigi 4 mm, berusia 20–30 tahun dan belum pernah menjalani perawatan ortodonsi. pengukuran somatometri dilakukan untuk tabel 1. klasifikasi indeks morfologi wajah genap menurut martin6 tipe wajah laki-laki perempuan sangat lebar (hypereuryprosopic) x – 78,9 x – 76,8 lebar (euryprosopic) 79,0 – 83,9 77,0 – 80,9 sedang (mesoprosopic) 84,0 – 87,9 81,0 – 84,9 sempit (leptoprosopic) 88,0 – 92,9 85,0 – 89,9 sangat sempit (hyperlepsoprosopic) 93,0 – x 90,0 – x tabel 2. distribusi sudut interinsisal dan tinggi wajah subyek penelitian descriptive statistic n mean std. deviation min max indeks morfologi wajah i 31 84.6648 6.00852 75.85 98.28 indeks morfologi wajah ii 31 84.9642 6.06908 75.40 98.51 indeks morfologi wajah (rerata) 31 84.8145 5.97828 75.78 98.40 sudut interinsisal (sefalogram lateral) 31 123.3226 10.34211 105.00 147.00 tinggi wajah (sefalogram lateral) 31 126.2258 10.02567 108.00 156.00 226 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 224–228 indeks morfologi wajah: mengukur jarak titik nasion (n) ke titik gnation (gn), dilanjutkan pengukuran jarak bizygion. pengukuran dilakukan dengan kaliper lengkung (gambar 1). pengukuran sudut interinsisal dan tinggi wajah dilakukan pada sefalogram lateral dari 31 subyek penelitian. sudut interinsisal adalah sudut dalam yang dibentuk oleh perpotongan garis sumbu gigi insisif atas dan gigi insisif bawah (gambar 2). tinggi wajah pada sefalogram merupakan jarak dari titik nasion ke gnation. hasil pengukuran dalam penelitian ini berupa skala rasio dan menjalani uji statistik pearson-correlation sebagaimana terlihat dalam tabel 3 dan tabel 4. uji statistik dengan menggunakan pearson-correlation dilakukan pada data hasil pengukuran untuk menilai tabel 3. hasil pengukuran indeks morfologi wajah dengan tinggi wajah indeks morfologi wajah tinggi wajah (sefalogram lateral) indeks morfologi wajah pearson correlation 1 .383* sig. (2-tailed) .033 n 31 31 tinggi morfologi wajah (sefalogram lateral) pearson correlation .383* 1 sig. (2-tailed) .033 n 31 31 keterangan: p<0,05= korelasi bermakna; p>0,05 = korelasi tidak bermakna tabel 4. hasil pengukuran indeks morfologi wajah dengan sudut interinsisal indeks morfologi wajah sudut interinsisal indeks morfologi wajah pearson correlation 1 .218 sig. (2-tailed) .238 n 31 31 sudut interinsisal pearson correlation .218 1 sig. (2-tailed) .238 n 31 31 keterangan: p<0,05 = korelasi bermakna; p>0,05 = korelasi tidak bermakna gambar 1. pengukuran indeks morfologi wajah. a) pengukuran jarak dari titik nasion (n) ke titik gnation (gn); b) pengukuran jarak bi-zygion. korelasi antara hasil pengukuran indeks morfologi wajah dengan sudut interinsisal dan dengan tinggi wajah. hasil ukuran tinggi wajah dan sudut interinsisal dapat dilihat pada tabel 2 sedangkan hasil uji statistik pearsoncorrelation ditunjukkan pada tabel 3 dan 4. hasil penelitian ini membuktikan bahwa tidak terdapat perbedaan bermakna antara sudut interinsisal dan indeks morfologi wajah genap (p>0,05), sementara dengan tinggi wajah yang diukur secara sefalometri dan indeks morfologi wajah genap memperlihatkan korelasi bermakna (p<0,05) (tabel 3). hasil analisis statistik sebagaimana terlihat dalam tabel 3, memperlihatkan adanya perbedaan korelasi hasil pengukuran indeks morfologi wajah dengan sudut interinsisal dan dengan tinggi wajah secara sefalometri pembahasan identifikasi karakteristik seseorang dapat membutuhkan informasi tentang banyak hal, antara lain, tinggi wajah (dimensi vertical wajah), yang berhubungan dengan tipe wajah, mulai dari leptoprosopic sampai euryprosopic, lebar tengkorak dan susunan gigi geligi.1,8 hal ini terutama bila berhadapan dengan sisa jasad yang hanya berwujud tengkorak. sementara korban 227sianita k dan verenna: korelasi indeks morfologi wajah dengan sudut interinsisal dan tinggi wajah putus atau hilang kontak dengan keluarganya, dan data awal yang berfungsi sebagai pembanding dalam proses identifikasi menjadi sulit atau hampir mustahil didapatkan. dengan demikian, pada proses rekonstruksi wajah untuk mengetahui prakiraan sub ras melalui tipe fasial, pengukuran tinggi wajah pada sefalometri lateral dapat menjadi salah satu sumber informasi data ante mortem. di samping itu, data yang diperoleh ini juga bisa menjadi dasar untuk membangun prakiraan sketsa wajah untuk membantu korban menemukan kembali keluarganya atau sebaliknya, pihak keluarga mengenali sketsa korban yang dibuat berdasarkan data perhitungan tersebut9,10. dalam hal ini, tinggi wajah dapat membantu pihak pembuat sketsa untuk menggambarkan korban mendekati rupa aslinya, sementara besar sudut insisal dapat membantu menggambarkan pola konveksitas daerah sepertiga bawah dari tinggi wajah korban.11-13 temuan dalam penelitian ini, sebagaimana tertuang dalam tabel 3, dengan nilai p<0,05 membuktikan bahwa terdapat hubungan bermakna antara indeks morfologi wajah dengan tinggi wajah pada analisis sefalometri lateral. adanya korelasi bermakna ini diharapkan memberikan manfaat aplikatif dalam proses identifikasi individu yang hanya menyisakan tengkorak kepala. sketsa individu dalam ukuran tinggi wajah juga berhubungan dengan klasifikasi tipe fasial, mulai dari tipe leptoprosopic sampai euryprosopic.14 jadi pada korban dalam wujud tengkorak yang tidak memiliki identitas, dimana tim identifikasi tidak memiliki gambaran apapun tentang rupa korban semasa hidup, maka korelasi bermakna antara indeks morfologi wajah dan tinggi wajah secara sefalometri lateral dapat dimanfaatkan untuk menjadi dasar pertimbangan rekonstruksi sketsa wajah korban dalam upaya mengembalikan korban kepada keluarganya. foto sefalogram lateral dapat dibuat pada tengkorak kepala korban untuk mendapatkan tinggi wajahnya dan pengukuran indeks morfologi wajah dapat diukur langsung untuk mendapatkan gambaran tipe fasial untuk kemudian dihubungkan dengan karakteristik tinggi wajah korban. dalam penelitian ini, juga diperoleh korelasi tidak bermakna pada perhitungan statistik antara indeks morfologi wajah dan sudut interinsisal. hal ini diduga disebabkan oleh adanya variasi besar dan relasi sudut interinsisal pada klasifikasi maloklusi yang sama sekalipun (gambar 2). variasi besar dan relasi sudut interinsisal ini bilamana dikaitkan dengan variasi soft tissue thickness yang membungkus kondisi dentoalveolar individu akan semakin mempertegas tingkat kesulitan variabel tersebut sebagai faktor yang dapat membantu proses rekonstruksi prediktif yang bersangkutan. dalam hal ini karakteristik tipe fasial yang diperoleh melalui pengukuran indeks morfologi wajah dapat membantu menggambarkan pola jaringan lunak yang umumnya dimiliki oleh individu tertentu, mulai dari tipe leptoprosopic sampai euryprosopic. kondisi yang lebih menguntungkan mungkin bisa diperoleh bilamana tengkorak yang tersisa masih dilengkapi dengan gigi geligi dalam jumlah memadai, terutama gigi geligi anterior di rahang atas maupun rahang bawah. dengan adanya gigi geligi anterior atas maupun bawah, maka relasi dan gigitan dapat diperoleh untuk kemudian dibuat roentgenogramnya sehingga dapat diukur pola konveksitas ataupun protrusiveness gigi geligi anterior korban.11,14 hasil pengukuran ini dapat gilirannya diharapkan dapat menyumbangkan informasi yang berharga pada tim pembuat sketsa rekonstruktif wajah korban untuk tujuan identifikasi maupun pencarian keluarga korban. pada akhirnya, penelitian ini merupakan langkah awal kolaborasi antara bidang keilmuan ortodonsia, yang memiliki kaitan erat dengan berbagai pengukuran di area kepala dengan menggunakan titik-titik baku anatomis pada jaringan keras di tengkorak, dengan bidang keilmuan forensik odontologi, yang tidak jarang berhadapan dengan sisa jasad korban dalam wujud tengkorak yang bilamana dicermati memiliki banyak sekali titik baku anatomis yang diharapkan dapat menyumbangkan informasi berharga dalam proses menguak identitas individu termasuk korban.15,16 diharapkan ke depannya, akan semakin banyak lagi penelitian-penelitian dalam bentuk kolaborasi bidang keilmuan yang dapat memberikan kontribusi dalam pengembangan ilmu forensik odontologi. melalui penelitian ini, dapat disimpulkan bahwa terdapat korelasi bermakna (p<0,05) antara indeks morfologi wajah dengan tinggi wajah hasil pengukuran pada sefalogram lateral, sedangkan untuk analisis indeks morfologi wajah dengan sudut interinsisal, diperoleh korelasi tidak bermakna, sehingga korelasi bermakna antara indeks morfologi wajah dengan tinggi wajah secara sefalometri diharapkan dapat membantu proses rekonstruksi wajah dalam kaitannya dengan digital imaging tiga dimensi yang biasanya dapat membantu menggambarkan tipe fasial melalui perkiraan beberapa titik anatomis. namun, hasil dari penelitian ini masih memerlukan penelitian lebih lanjut terhadap sub ras lainnya. 1470 1320 1050 gambar 2. kedudukan gigi insisif dan besar sudut interinsisal yang dihasilkan. 228 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 224–228 daftar pustaka 1. hinchliffe j. forensic odontology. part1 dental identification. br dent j 2011; 210(5): 219-24. 2. debi g. asessment of facial type: average, short, or long. cdha j 2007; 23(2): 23-5. 3. foster td. a textbook of orthodontics. 2nd ed. blackwell scientific publications; 1984. p. 85-101. 4. enlow, donald h, mark g hans. essentials of facial growth. philadelphia: wb saunders company; 1997. p. 122-9. 5. jacobson, a, jacobson, rl. radiographic cephalometry. 2nd ed. canada: quintessence publishing; 2006. p. 63-183. 6. glinka j. antropometri & antroposkopi. surabaya: fisip universitas airlangga; 1990. h. 57-68. 7. djoeana kh. antropologi untuk mahasiswa kedokteran gigi. jakarta: universitas trisakti, 2005. h. 6-7, 40-90. 8. korkmaz s, fulya i, göksu t, tülin a. an evaluation of the errors in cephalometric measurements on scanned cephalometric images and conventional tracings. european j orthodontics 2007; 29(1): 105–8. 9. brons r. facial harmoni standarts for orthognathic surgery and orthodontics. canada: quintessence publishing; 1998. p. 69-77. 10. enlow dd. handbook of facial growth. 2nd ed. wb. philadelphia: saunders company; 1982. p. 228-311. 11. sianita k, priska p. buku ajar sefalometri laboratorium orthodonti fa k ultas kedok tera n gigi un iversitas p rof. dr. moestopo (beragama). jakarta: fkg updm (b); 2011. h. 47-60. 12. olmez h. measurement accuracy of a computer-assisted threedimentional analysis and a conventional two-dimensional method. angle orthodontist 2011; 81(3): 375-82. 13. jacobson a. radiographic cephalometry. canada: quintessence publishing; 1995. p. 236-54. 14. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee; 2007. p. 216-23. 15. farkas lg. anthropometry of the head and face in medicine. 2nd ed. new york: raven press; 1994. p. 241-336. 16. herschaft ee. manual of forensic odontology. 4th ed. usa: crc press; 2007. p. 12. mkgs vol 45 no 2 april-juni 2012.indd 59 volume 45 number 2 june 2012 case report the importance of masticatory functional analysis in the diagnostic finding and treatment planning for prosthodontic rehabilitation harry laksono, agus dahlan, and sonya harwasih department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: the masticatory system as a biologic system is subjected to harmful influences of varying severity. almost half of routine patients requesting prosthodontic treatment indicated at least one sign or symptom of temporomandibular disorders. analysis of the masticatory system often neglected by dentist. untreated temporomandibular disorders may significantly implicated in the perpetuation of the disorder and may interfere with routine prosthodontic clinical procedures. it would be resulted unsuccessful long term goal of prosthodontic rehabilitation because of the uncompleted diagnoses and treatment plan. purpose: the purpose of this case report was to give the information of the importance of masticatory functional analysis in the diagnostic finding for treatment planning in the prosthodontic rehabilitation. case: a 45 year old male patient, partial dentate with reduced chewing efficiency, mild pain in right preauricular region in function, left click in opening mouth, severe attrition on all anterior lower teeth with vertical dimension of occlusion decreased due to loss of posterior support. he wanted to make a new denture. case management: record and analyze of active and passive mandibular movement, opening pathway, muscle and temporomandibular joints palpation, load testing, and vertical dimension of occlusion with manual functional analysis (mfa), occlusal condition and radiographic examination. treatment plan was formulated into 3 phases: stabilization of the masticatory system, definitive treatment and periodical control. the result of this treatment excellent for 1 year evaluation after permanent cementation. conclusion: masticatory functional analysis is very important and must be done in the diagnosis finding for treatment planning in every case of prosthodontic rehabilitation. key words: severe attrition, masticatory functional analysis, prosthodontic rehabilitation abstrak latar belakang: sistem pengunyahan sebagai sistem biologis sewaktu-waktu dapat terjadi gangguan dengan berbagai derajat keparahan. hampir setengah dari jumlah pasien yang memerlukan perawatan prostodontik minimal menunjukkan satu tanda atau keluhan dari gangguan temporomandibular. analisis fungsional sistem pengunyahan masih sering dilupakan oleh dokter gigi. gangguan temporomandibular yang tidak dirawat akan terus ada dan mungkin dapat mengganggu prosedur klinis perawatan prostodonsia. hal tersebut akan menyebabkan keberhasilan klinis jangka panjang perawatan prostodonsia tidak dapat tercapai karena diagnosis dan rencana perawatan yang kurang lengkap. tujuan: tujuan dari laporan kasus ini adalah untuk memberikan informasi tentang pentingnya analisis fungsional sistem pengunyahan untuk menegakkan diagnosis dan rencana perawatan pada perawatan prostodonsia. kasus: pasien laki-laki usia 45 tahun, bergigi sebagian merasa sulit untuk mengunyah makanan, nyeri ringan di daerah depan telinga kanan saat fungsi, keletuk sendi kiri saat membuka mulut, atrisi pada seluruh gigi depan rahang bawah disertai penurunan dimensi vertikal oklusi akibat kehilangan dukungan gigi belakang. dia ingin membuat gigi tiruan yang baru. tatalaksana kasus: mencatat dan menganalisis pergerakan aktif dan pasif rahang bawah, arah pergerakan rahang bawah saat membuka mulut, palpasi otot-otot pengunyahan dan sendi temporomandibula, uji beban, dimensi vertikal oklusi dengan metode analisis fungsional secara manual, keadaan oklusal dan radiologis. rencana perawatan dibagi menjadi 3 tahap berupa stabilisasi sistem pengunyahan, perawatan tetap dan kontrol secara periodik. hasil perawatan menunjukkan keberhasilan klinis yang baik setelah dilakukan evaluasi selama 1 tahun 60 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 59–67 introduction masticatory system is the organs and structures primarily functioning in mastication.1 it is an extremely complex and interrelated system of muscles, bones, ligaments, teeth and nerves2 and a dynamic orthopedic system.3 the masticatory system as a biological system is subjected to harmful influences of varying severity.4 the temporomandibular joints (tmjs) is irrevocably connected to the rest of the masticatory system and the teeth. all forces that are applied to the masticatory system are also applied to the tmjs as well.3 some studies and references showed that lost of posterior teeth, inadequate molar support and subtle occlusal interferences are likely to be the response of the neuromuscular system to hold the mandible in a comfortable position and impairment of masticatory function.5-9 this condition known as orthopedic instability and can cause to develop temporomandibular disorders (tmds)2,10 and interfere with routine prosthodontic clinical procedures.3 therefore appropriate methods for analyzing masticatory function are needed. tmds can be subdivided into muscular and articular categories.2,10 the strong relationship between muscular and articular disorders makes accurate diagnosis difficult. it must be determine whether muscle incoordination activated the disc derangement or vice versa, because their treatment is completely different.2 the dental treatment’s long term goal in prosthodontic rehabilitation is to preserve the health of the patient’s masticatory system.10 therefore, programmed diagnosis and treatment is the key.8 in diagnostic findings, screening for a history and systematic analysis of the masticatory system is a wise precaution and can determine whether a more comprehensive evaluation is necessary.11 it is important for the dentist to know what a stable, maintainably healthy masticatory system. in a complete analysis, each part of the system should be analyzed and determining the pathological condition is the main purpose of clinical diagnostic and treatment planning.8,10,12 for adequate diagnosis and treatment plan, a paradigm shift is necessary to change from a mechanical era, where treatment is designed to fix problem caused by neglect, to a biologically based discipline, where the dentist assess an individual patient’s susceptibility or risk to disease progression. the principle is a diagnostically driven, systematic analysis of the individual patient’s periodontal, biomechanical, functional and dentofacial risk level should be the driving force behind treatment.8,13,14 setelah penyemenan tetap. kesimpulan: analisis fungsional sistem pengunyahan sangat penting dan harus dilakukan untuk menegakkan diagnosis dan rencana perawatan pada setiap perawatan prostodonsia. kata kunci: atrisi berat, analisis fungsi pengunyahan, perawatan prostodonsia correspondence: harry laksono, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: harrylaksonol@yahoo.com to develop the functional stability necessary for success of treatment, the dentist must analyze the masticatory function. it is important to observation masticatory function or functional analysis may disclose uncoordinated movements that may indicate biomechanical problems4,11 and will determine individual patient’s treatment plan.8 for this reason, a valid and reliable examination method is needed. one of the methods is manual functional analysis (mfa).2,4,13 functional analysis is a method for evaluating symptoms in the tmjs and muscles jaw movement. it serves further to help recognize the necessity for additional therapeutic measures and should protect both the patient and the dentist from failure.4 recording the functional analysis enables the dentists suggestive of certain disorders or improvement.10 mfa is a functional analysis using manual palpation, a millimeter ruler and a stethoscope execute by a trained examiner.4,13 the mfa was based active mouth opening or range of motion (rom) consist of maximum mouth opening, protrusive, lateral movement and overlap of the central incisors, opening pathway, and some cases these are complemented by passive mouth opening (end feel), then muscles and tmjs palpation, tmjs auscultation, load testing and occlusal evaluation. a decrease rom, deflection or deviation in opening pathway, pain in load testing and palpation, and tmjs noise suggestive of certain disorders.2,4-5,10,15-17 for medical and legal reasons, a masticatory functional analysis should be a routine that dentist perform for all new patients before any definitive prosthodontic treatment should be deferred until the tmds has been properly managed.4,17 for this reason, masticatory functional analysis must be reliable and valid to provide the right diagnosis and treatment plan.13 dentist should realize that almost half of routine patients requesting prosthodontic treatment may clinically indicate at least one sign or symptom of tmds.13 unfortunately, masticatory functional analysis is often neglected by dentist. in fact, a precise and satisfactory prosthetic rehabilitation can only be made for a patient who has a stable masticatory system. the presence of tmds and if not treated properly, could be implicated in the perpetuation of the disorder and may interfere with routine prosthodontic clinical procedures. this conditions would increase the awareness and indicate modifications of diagnosis and treatment plan or at least precautionary measures during the course of the prosthodontic therapy.6,18 61laksono, et al.: the importance of masticatory functional analysis the purpose of this case report is to give the information of the importance of masticatory functional analysis in the diagnostic finding and treatment planning in prosthodontic treatment. case a patient 45 years old man presented to the prosthodontic specialist clinic at airlangga university dental hospital with main complaint about reduced chewing efficiency and had a dull pain in right preauricular region. he wanted to make a new denture. his previous denture was a removable posterior mandibular bare root overdenture and since three month ago he had never used the denture because of the discomfort. the pain began after trying to bite on posterior teeth since a week ago. this pain was aggravated by trying to bite on his right posterior teeth. he had a left click without pain every time he opened the mouth. there was no systemic diseases and patients was in good health. mfa revealed a limitation of maximum mouth opening (32 mm) (normal value 40-50 mm)12 accompanied by a mandibular deflection to the left (3 mm), assisted mouth opening or passive movement 1 mm longer than maximum mouth opening (end feel soft), right lateral movement 6 mm (normal value 7-15 mm),12 left lateral movement 7 mm (normal value 7-15 mm),12 protrusive movement 7 mm (normal value 7-15 mm).12 palpation of his masticatory muscles identified that pain was limited to right medial pterygoid and no pain in rest position. palpation of both lateral tmjs areas were not tender and confirmed a click during mandibular opening in the left side. load testing was no pain. vertical dimension of occlusion (vdo) was assessed by phonetic evaluation (closest speaking distance) 4 mm (normal value 1-2 mm),12,19 interocclusal rest space 6 mm (normal value 2-4 mm),19 and inter cementoenamel junction 14 mm (normal value 18-20 mm).8,20,21 intra oral examination confirmed overbite was 6 mm (normal value 3-5 mm),2 overjet was 5 mm (normal value 3-5 mm),2 wear facets on all anterior lower teeth, and loss of posterior support because of decaputated teeth with glass ionomer cement filling as abutment teeth for overdenture in teeth 35, 37, 38, 44, 45, 46, and 47 (figure 1). metal ceramic fixed partial dentures in all maxillary teeth. none of the teeth were mobile, and there was no significant gingival recession and/or furcation involvement. panoramic radiograph evaluation confirmed missing teeth in 18, 28, 36, and 48; proper root canal treatment in 12, 17, 22, 23, 24, 25, 26, 27, 35, 37, 38, 44, 45, 46, 47, and improper root canal treatment in 46; supernumerary teeth in left mandibular in 35 and 36 region with horizontal and vertical position (figure 2). on the basis of history, clinical and radiographic examination, the patient was diagnosed with local muscle soreness, anterior disk displacement with reduction, deep bite with severe attrition and loss of vdo, edentulous ridge on 18, 28, 36 and 48, post root canal treatment in 12, 17, 22, 23, 24, 25, 26, 27, 35, 37, 38, 44, 45, 46, and 47, and supernumerary teeth. on the basis of diagnosis, the treatment plan formulated and should be broken down into 3 phases. phase 1 treatment should be for the removal of disease, the stabilization of the joint, corresponding musculature and establishing the correct vdo; phase 2 definitive treatment; and phase 3 were maintenance of prosthesis and follow-up. case management this case management begans with analyzing the masticatory function with mfa and then classified the disorders using the classification from american academy figure 2. panoramic radiograph showed decaputated lower posterior teeth with good endodontic treatment axcept tooth no. 46. figure 1. (a) maximum intercuspation showed deep overbite, (b, c) with loss of posterior support. a b c 62 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 59–67 of orofacial pain (aaop). the examination started with measuring the active and passive mandibular movement, opening pathway, muscle and tmjs palpation, measure the vdo, load testing, and occlusal evaluation. the patient was asked to lie back in the dental chair, relax his mandible with it slightly open, and close his teeth together slowly. measuring the active mandibular movement by asking the patient to open as wide as he can then measuring maximum mouth opening from incisal edge to incisal edge. measuring the overlap of the central incisors by asking the patient to close into maximum intercuspation (mi), marked on the facial surface of the mandibular incisor against the maxillary central incisor’s incisal edge, then asking the patient to open and observes the distance from the mandibular central incisor’s incisal edge to the marked on the ruler. measuring the lateral movement by asking the patient to close into mi and asked to move his mandible as far as possible to the right and left side then observes the distance the embrasure on the ruler.10 measuring the passive mandibular movement (end feel) by asking the patient open the mouth as far as possible and at the end of the active movement then assist further opening and measuring from incisal edge to incisal edge. to determine the pathway of the mandible by asking the patient open the mouth as far as possible the observed the position of the midline at the end of maximum opening.10 muscle palpation by using bilaterally palpating for extra-oral, unilaterally for intra-oral, and face the patient during palpation. start by palpating the anterior, middle and posterior region of temporalis muscles, tmjs, masseter muscles and intraorally palpation for lateral pterygoid in the fifth digit along the lateral side of the maxillary alveolar ridge to the most position region of the vestibule, and then medial pterygoid at the insertion site for an inferior alveolar injection and press laterally. the palpation begin with light pressure and slowly increased the force until the patient’s eye or facial expression convey that the patient was experiencing discomfort (figure 3). palpate the tmjs noise by placing the finger’s palmar surface over the tmjs as the patient opens, closes, and moves through lateral and protrusive excursions.10 measuring the vdo using phonetic evaluation by spoken “s” sound and measuring the distance maximal closure line to the “s” sound line (figure 4);12,19 then by using interocclusal rest space measured between nose tip and chin tip;19 and inter cementoenamel junction measured between cementoenamel junction anterior mandibular tooth with upper tooth.8,20,21 verifying healthy condyles using load testing by bilateral manipulation. start with reclined the patient all the way back, stabilized the head, lifted the patient’s chin again to slightly stretch the neck, position the four fingers on the lower border of the mandible and the thumbs in the notch above the symphysis, ask the patient to hinge open and close.12 in phase 1 treatment, the first step was to treat the tmds symptom, loss of vdo, and stabilized the tmjs using hard stabilization splint with smooth flat plane. this splint was made by verified centric relation (cr) bite record with bilateral manipulation then mounting the cast in a semiadjustable articulator with a face bow transfer. the pain was opened 2 mm to sparate all posterior teeth and the coverage area was then waxed up. acrylic processing and insert the splint to the upper arch then observe the occlusal contact (figure 5). the protrusive and lateral movement figure 3. muscle palpation pain in right medial pterygoid with patient’s eye express discomfort. figure 4. measuring the vdo using phonetic evaluation by spoken “s” sound. figure 5. maxillary acrylic splint to an entire occlusal surface: articulating paper marks of centric contacts on an adjusted splint. 63laksono, et al.: the importance of masticatory functional analysis provided 0.5 mm posterior disclusion (figure 6). this splint wears 24 hours a day except to eat for 1 month and observe the result (table 1). the second step was to make a transitional denture with the reorganized approach and designed using mutually protected occlusion (mpo). this denture verified by cr bite record with anterior index (figure 7), then mounting the cast in a semiadjustable articulator. the incisal guidance pin was opened 3 mm for the new vdo. the perfect anterior guidance was formed from the diagnostic wax-up made in articulated model. the process of customizing this anterior guidance is designed the shape and locate the correct incisal edge position by diagnostic wax-up (figure 8). the leading edge of each lower anterior tooth should be formed by a definite labio-incisal line angle contour. then analyzed in cr and move the articulator slightly forward and lateral to achieve a solid stop and posterior disclusion about 0.5 mm. after finishing the diagnostic wax-up, a putty matrix formed from the diagnostic wax-up. this matrix was use for construction the transitional denture in autopolymerizing resin after the teeth are prepared, as a guide to prepare of the mandibular anterior teeth (figure 9), and to communicate table 1. the results of mfa after insertion stabilization splint mfa before treatment (mm) 1 day (mm) 1 week (mm) 2 week (mm) 1 month (mm) maximum mouth opening 32 33 35 37 37 right lateral 6 7 7 8 8 left lateral 7 7 8 8 8 protrusive 7 7 7 7 7 deflection 3 2 0 0 0 muscle palpation 3 2 0 0 0 tmj palpation 0 0 0 0 0 tmj noise + ± ± ± ± load testing 0 0 0 0 0 figure 6. patient occluding on acrylic splint in the neutral position. figure 7. cr bite record used silicone material and anterior index at the selected vdo. figure 8. correct shape and incisal edge position diagnostic wax-up. figure 9. lower anterior teeth are prepared for transitional denture with putty matrix as a guide. 64 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 59–67 with the dental technician about the precise incisal edge position and contours of line angle. this transitional denture was used as a guide for the definitive treatment (figure 10). the transitional denture was used for 1 month to adapt the patient to the new vdo and observe the masticatory functional (table 2). phase 2 treatment was not started until the transitional denture was approved. the first step of this treatment should be made the anterior restoration with perfect anterior guidance formed from the diagnostic wax-up and try-in for 1 week. the next step was to make the posterior restoration and the anterior restoration should not be cemented until all functional excursions have been verified against the upper teeth, try-in for 1 week and observed the masticatory function (table 3), then cemented the definitive restoration (figure 11). in phase 3 treatment, maintenance of prosthesis and follow-up with masticatory functional analysis ware done (table 4). discussion dentist should monitor not only immediate oral problems such as caries or prosthodontic needs, but also disorders affecting the head, neck muscles and tmjs. so, teeth, muscles and tmjs make up an inseparable unit even throughout pathology, therapy and final outcome.22 the primary purpose of the clinical examination is to gather additional information to help confirm or rule out structures involved in a patient’s complaints and other suspected disorders that may contribute to these complaints.10 this case report used mfa for functional analysis because it is easy to learn, cost effective, reliability, figure 10. lower transitional denture are placed with 17 mm inter cementoenamel junction. figure 11. definitive denture copied transitional denture with 17 mm inter cementoenamel junction. table 2. the result of mfa after insertion transitional denture mfa before transitional denture 1 week 1 month maximum mouth opening 37 37 37 right lateral 8 8 8 left lateral 8 8 8 protrusive 7 7 7 deflection 0 0 0 muscle palpation 0 0 0 tmj palpation 0 0 0 tmj noise ± ± 0 load testing 0 0 0 over bite 6 5 5 over jet 5 3 3 table 3. the result of mfa in temporary cementation of definitive denture mfa after transitional denture 1 week maximum mouth opening 37 37 right lateral 8 8 left lateral 8 8 protrusive 7 7 deflection 0 0 muscle palpation 0 0 tmj palpation 0 0 tmj noise 0 0 load testing 0 0 over bite 5 5 over jet 3 3 table 4. the result of mfa after permanent cementation of definitive denture mfa 1 week 1 year maximum mouth opening 37 37 right lateral 8 8 left lateral 8 8 protrusive 7 7 deflection 0 0 muscle palpation 0 0 tmj palpation 0 0 tmj noise 0 0 load testing 0 0 over bite 5 5 over jet 3 3 65laksono, et al.: the importance of masticatory functional analysis internationally accepted,5,13,23,24 valid and sensitivity.5,13,25 for classifying the disorders, this case report used the classification from aaop because this classification is a clinically oriented taxonomic proposal that contains some referrals to the plausible pathogenesis of the different disorders.23 the mfa procedures started with measure active and passive movement. this movement must be measured prior to palpating, because palpation can aggravate the masticatory muscles and/or tmjs, which may cause a decrease in patient’s active movements. for recording maximum mouth opening, millimeter ruler was used instead of three fingers between upper and lower incisors, because the used of three fingers can be considered insufficiently accurate.26 recording the mfa enables the dentist suggestive of certain disorders or improvement.10 in this case report, mfa confirmed that there were restricted mouth opening (32 mm), painless accompanied with deflection to the left (contralateral direction from the involved muscle), end feel soft, lateral movement no deflection in and no restriction in lateral movement (right and left lateral movement in normal value), protrusive movement in normal value, no pain at rest, and muscle pain on palpation in right medial pterygoid. this condition confirmed restriction and interference of mouth opening produced by shortening of the right medial pterygoid muscle.2,4,5 medial pterygoid muscles are one of the masticatory muscles that responsible for mandibular closure and bite force. this shortening of right medial pterygoid muscle may be due to the prolonged chewing movement producing muscle fatique, and the intercuspation of the teeth is not in harmony with the joint-ligament-muscle balance, a stressful and tiresome protective role is forced onto the muscles during mastication.12,13 muscle pain always involves the relationship between the tmjs and occlusal contact. so, the important point is to determine if the tmjs are healthy and complete seating into cr. in this case, with the use of bilateral manipulation technique confirmed painless. this is indicates that the tmjs are healthy and capable complete seating into cr.12 determination the vdo is a critical procedure for a totally or partially edentulous patient,19 and should not be confined to a single technique or consideration.27 in this case report, the vdo assessed with phonetic evaluation (4 mm), interocclusal rest space (6 mm) and inter cementoenamel junction (14 mm) confirmed that there was a decrease vdo about 3 mm. in this case, increasing the vdo will not cause any joint changes because the patient has healthy tmjs.28 in occlusal evaluation, there were severe labio-incisal wear in all anterior mandibular teeth. the possible cause of this condition may be due to the results of interference to the envelope of function, and there were no holding posterior contacts because of loss posterior teeth. the loss of posterior contacts may result in an increase anterior load which, in turn, increase the number and intensity of anterior incisal contact.12 the rehabilitation of partially edentulous patients with severe tooth wear is a complex task, and more information regarding treatment protocols, prosthetic indications and treatment outcome is needed.12,29 for reinstating the teeth back into functional harmony with the masticatory system as a whole, the treatment plan should be broken down into 3 phases with the reorganized approach to occlusal reconstruction in definitive treatment.30 this treatment would be carried out to the reorganized approach because all of the occlusal contacts are going to be changed and reconstructed. the objective of this approach is to provide an occlusion that is more ideal for the teeth, periodontal tissues and tissues of the articulatory system (tmjs and mandibular muscles).6,31 in occlusal reorganization, it is important therefore to know before start treatment whether a patient has an underlying tmds, great care must be taken and try to stabilize the masticatory system. a reorganized occlusion is an occlusion in which the pattern of occlusal contact is deliberately changed or reconstructed.6 in the first step in phase 1 treatment, the stabilization splint with smooth flat surface was designed to offer contacts of all teeth and coordinate elevator muscles contraction. this condition always attempts to seat the condyles up into cr.12 this stabilization splint performs a dual diagnostic and therapeutic role. as a diagnostic aid, it prevent occlusal interference, thereby relaxing the muscle and reducing tension and pain; as a therapeutic aid, it contributes to the general rearrangement of joint structures by acting on the transversal and anteroposterior planes in addition to specific vertical action from the posterior wedge.22,32 cr is the only condylar position that permits an interference-free occlusion and the most musculoskeletally stable position for the mandible.10,12 recording of an accurate cr is critical for the most trouble-free restorative or prosthetic dentistry.12 after insertion the splint, record and observe the masticatory function for 1 day, 1 week, 2 week and 1 month (table 1). in the first row, the mfa is shown for before treatment, while the others for after the use of stabilization splint. it can be seen that the value of maximal mouth opening was increased (37 mm), right lateral movement was increased to normal value (from 6 to 8), left lateral movement was increased to normal value (from 7 to 8), elimination of deflection (from 3 to 0) and elimination of pain during palpation (from 3 to 0), tmj click was decreased (from + to ±). it is suggest that occlusal therapy of tmds with stabilization splint was success and ready for the next step. the success of stabilization splint treatment might be the smooth flat plane contribute to occlusal stability (simultaneous contacts occur upon mandibular closure). freedom in centric increases the occlusal comfort by allowing slight protrusive and laterotrusive tooth-guided mandibular movement along the surface of the splint. it is induced reorganization of intramuscular recruitment patterns unloads those muscle region that are 66 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 59–67 characterized by high stress concentration and overload during mastication.13 these effects may lead to a reduction of muscle pain, tmj click, elimination of deflection, and increase maximal mouth opening. in this case, 37 mm of maximum mouth opening was suggest a normal value of maximum mouth opening of this patient, because this value varies greatly and the result of measure the maximum mouth opening after 2 week using stabilization splint (37 mm) is constant until 1 year post permanent cementation. although some references stated that minimal value of the maximum mouth opening is 40 mm, however one reference stated that some patient can function readily at 35 mm.15 the next step was to make a transitional denture. in this case, the transitional and definitive denture would be changed the vdo and determined by the distance of inter cementoenamel junction as a guide. it is the philosophy for condylar position by bioesthetics, works via a fixed numerical value based on incisal relationship that the distance of inter cementoenamel junction was 18-20 mm.8,20,21 this restoration made with the reorganized approach and designed using mpo with cr bite record. this reorganized approach will be provide posterior stability and establish anterior guidance on transitional denture, the copy the anterior guidance into definitive denture. designed using mpo, because guidance from the anterior teeth will be to protect the posterior teeth from excursive force, wear and posterior teeth supported the bite force12 and creating a synergistic stability.27 the ultimate goal of this procedures was to provide a stable occlusion to maintain the vdo during mi and to create appropriate harmonious gliding tooth contacts or anterior guidance during eccentric movements that do not cause deflection of the mandible or excessive stress to the dentition, muscles, or joints.33,34 in this case, cr bite record was used as the basis for reconstructing a new icp because the patient in situations in which the existing icp is unsatisfactory (loss of posterior support). the anterior index was used for stabilize the jaw in cr because cr registration used a fluid material (silicone). in tooth preparation, the putty matrix was used as a guide to get a precise preparation for transitional and definitive treatment, and to communicate precise incisal edge position and contour (anterior guidance) that have been made from the diagnostic wax-up to the technician.12 it is then copied into the definitive restoration eliminating all guesswork. this guesswork could be destroyed the anterior guidance that have been formed in the articulated model and failure of occlusal treatment causing damage of the masticatory system. after insertion of transitional denture, record and observe the masticatory function for 1 week and 1 month (table 2). it can be seen that rom was stable, no deflection, no pain in muscle and tmj palpation, those was no pain in load testing, elimination of tmj click, patient can tolerate for the new overbite (from 6 to 5 mm), overjet (from 5 to 3 mm) and vdo (from 14 to 17 mm inter cementoenamel junction). it is suggested that the anterior guidance formed from the diagnostic wax-up is correct and occlusal reorganization can provide a stable occlusion to maintain the vdo during mi and do not cause deflection of the mandible. the success of increasing the vdo make be the patient has healthy tmjs.12 for the phase 2 treatment, the anterior restoration should be done before the posterior because anterior guidance is the most important determination that must be made when one is restoring an occlusion. this anterior guidance of definitive denture copied from the transitional denture and then provides definitive posterior restoration. this will create stability in icp and avoid damage in excursions.12 for the type of restoration, we used fixed partial denture because there is a correlation between masticatory efficiency and maximum molar bite force of the masticatory system. it is the greatest bite force and masticatory efficiency values compared to the removable partial dentures,35 and more predictable and results in a higher adaptation level and the negative signs and symptoms were self-limiting.36 for the material, metal-ceramic restoration was used because the longitudinal study of 515 metal-ceramic fixed prostheses by walton37 showed that an overall success rate of 96% at 5 years, 87% at 10 years and 85% at 15 years. before cemented the definitive restoration, should be monitored and complete the mfa to verify that treatment was effective. this monitor is important in this case because the study by abe et al.,38 showed that the position of the main occluding areas (functional cusp) in the fpds tended to be less stable; and some experimental evidence that small acute changes might provoke transient symptoms in subjects with a history of past tmds.39 in temporary cementation of definitive denture, record and observe the masticatory function in 1 week (table 3). it can be seen that rom, overbite, overjet and vdo are stable, no deflection, no pain in muscles and tmjs palpation, no pai in load testing. it is suggest that occlusal reorganization of the definitive denture copied from the transitional denture can provide a stable occlusion and the functional cusps do not cause deflection of the mandible. in periodical control phase, observe with mfa (table 4) showed normal value of rom, no deflection, no pain in muscles and tmjs palpation, no pain in load testing, patient can tolerate for the new denture (feel comfort, chewing better than his old denture, and no speech concern) after 1 year post cemented evaluation. it is suggest that the diagnostic, treatment planning and prosthodontic treatment of this case is correct. from this case, it can be concluded that the application of mfa is very important and must be done in the diagnostic finding for treatment planning in every case of prosthodontic rehabilitation. 67laksono, et al.: the importance of masticatory functional analysis references 1. the academy of prosthodontics. the glossary of prosthodontic term. j prosthet dent 2005; 94(1): 51. 2. okeson jp. management of temporomandibular disorders and occlusion 6th ed. st louis, missouri: mosby elsevier; 2008. p. 25, 72–3, 130–47, 227–41. 3. becker im. comprehensive occlusal concepts in clinical practice. oxford u.k: willey-blackwell publ, ltd; 2011. p. 6 –8, 34–8, 77–121. 4. bumann a, lotzmann u. tmj disorders and orofacial pain. the role of dentistry in a multidisciplinary diagnostic approach. color atlas of dental medicine. stuttgart, new york: thieme; 2002. p. 4, 5, 58–140. 5. dos santos j jr. occlusion. principles and treatment. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque, and warsaw: quintessence publ co, inc; 2007. p. 100–14. 6. wasell r, naru a, steele j, nohl f. applied occlusion. london, berlin, chicago, paris, milan, barcelona, istanbul, sao paulo, tokyo, new delhi, moscow, praque, warsaw: quintessence publ co., ltd; 2008. p. 19, 26, 31–48, 73–85, 97–110. 7. walmsley d, walsh tf, lumley pj, burke fjt, shortall acc, hayes-hall r, pretty ia. restorative dentistry. 2nd ed. edinburgh, london, new york, oxford, philadelphia, st louis, sydney, toronto: churchill livingstone, elsevier; 2007. p. 47–55. 8. cohen m. interdisciplinary treatment planning. principles, design, implementation. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque, and warsaw: quintessence publ co, inc; 2008. p. 190–2, 253–63, 285–6, 314–22. 9. wang mq, xue f, he jj, chen jh, chen cs, raustia a. missing posterior teeth and risk of temporomandibular disorders. j dent res 2009; 88(10): 942–45. 10. wright ef. manual of temporomandibular disorders. 2nd ed. iowa usa: blackwell munksgaard; 2010. p. 33–55, 100 –3, 138– 41, 272–3. 11. de leeuw r. orofacial pain, guidelines for assessment, diagnosis and management. 4th ed. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque and warsaw: quintessence publ co, inc; 2008. p. 25–6, 31–4, 129–38, 172. 12. dawson pe. functional occlusion. from tmj to smile design. st louis, missouri: mosby elsevier; 2007. p. 4–6, 68, 76–80, 271–3, 346–8, 388–91, 430–52. 13. manfredini d. current concepts on temporomandibular disorders. london, berlin, chicago, tokyo, barcelona, istanbul, milan, moscow, new delhi, paris, beijing, praque, sao paulo, seoul and warsaw: quintessence publ; 2010. p. 417–28, 153–8, 179–85. 14. klineberg i, stohler cs. interface 4: occlusion. int j prosthodont 2003; 16(supplement): 71–90. 15. murphy e. managing orofacial pain in practice. london, berlin, paris, milan, barcelona, istanbul, sao paolo, tokyo, new delhi, moscow, praque, warsaw: quintessence publ co ltd; 2008. p. 12–28. 16. gray r, al-ani z. risk management in clinical practice. part 8. temporomandibular disorders. br dent j 2010; 209(9): 433–49. 17. gray r, al-ani z. temporomandibular disorders. a problem-based approach. oxford uk: wiley-blackwell; 2011. p. 29–46. 18. preti g. prosthetic rehabilitation. london, berlin, chicago, tokyo, barcelona, istanbul, milan, moscow, new delhi, paris, beijing, praque, sao paulo, seoul and warsaw: quintessence publ ltd; 2008. p. 43–57. 19. toolson lb, smith de. clinical measurement and evaluation of vertical dimension. j prosthet dent 2006; 95: 33–9. 20. paesani da. bruxism. theory and practice. london, berlin, chicago, tokyo, barcelona, beijing, istanbul, milan, moscow, new delhi, paris, praque, sao paulo, seoul and warsaw: quintessence publ; 2010. p. 415–37. 21. bloom dr, padayachy jn. increasing occlusal vertical dimension – why, when and how. bdj 2006; 200: 251–6. 22. cozzani g. treating the triad. teeth, muscles, tmjs. milan, berlin, chicago, tokyo, barcelona, istanbul, london, moscow, new delhi, paris, peking, praque, sao paulo, seoul, warsaw: quintessenza edizione; 2011. p. 46, 49, 75, 92–3, 126–31. 23. manfredini d, bucci mb, nardini lg. the diagnostic process for temporomandibular disorders. stomatol, baltic dent and maxillofacial j 2007; 9: 35–9. 24. celic r, jerolimov v, zlataric k. relationship of slightly limited mandibular movements to temporomandibular disorders. braz dent j 2004; 15(2): 151–4. 25. slater jjrh, lobbezoo f, chen jj, naeije m. a comparative study between clinical and instrumental methods for the recognition of internal derangements with a clicking sound on condylar movement. j orofac pain 2004; 18(2): 138–47. 26. abou-atme ys, chedid n, melis m, zawawi k h. clinical measurement of normal maximum mouth opening in children. j craniomandib pract 2008; 26(3): 1–6. 27. laney wr, salinas tj, carr ab, koka s, eckert se. diagnosis and treatment in prosthodontic. 2nd ed. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, new delhi, moscow, praque, and warsaw: quintessence publ co, inc; 2011. p. 270–92. 28. rebibo m, darmouni l, jouvin j, orthlieb jd. vertical dimension of occlusion: the keys to decision. j stomat occ med 2009; 2: 147–59. 29. katsoulis j, nikitovic sg, spreng s, neuhaus k, mericske-stern r. prosthetic rehabilitation and treatment outcome of partially edentulous patients with severe tooth wear: 3-years results. j dent 2011; 39(10): 662–71. 30. khan f, young wg. tooth wear. the abc of the worn dentition. the atrium, southern gate, chichester, west sussex. uk: wileyblackwell ltd. 2011; p. 183, 211–9. 31. davies s. conformative, re-organized or unorganized. dental update 2004; 31: 334–45. 32. slavicek g. eight questions on intra-oral splint concept. j stomat occ med 2009; 2: 55–6. 33. lerner j. a systematic approach to full-mouth reconstruction of the severely worn dentition. pract proceed aesthet dent 2008; 20(2): 81–7. 34. song my, park jm, park ej. full mouth rehabilitation of the patient with severely worn dentition: a case report. jadv prosthodont 2010; 2: 106–10. 35. rosa lb, bataglion c, siessere s, palinkas m, mestriner junior w, de freitas o, de rossi m, de oliviera lf, regalo sch. bite force and masticatory efficiency in individuals with different oral rehabilitation. open j stomatol 2012; 2: 21–6. 36. abduo j. safety of increasing vertical dimension of occlusion: a systematic review. quintessence int 2012; 43(5): 369–80. 37. walton tr. an up to 15-year longitudinal study of 515 metal ceramic fpds: part 1.outcome. int j prothodont 2002; 15: 439–45. 38. abe h, miura h, okada d, kato h, shin c. the main occluding area in patients with fixed partial dentures. j med dent sci 2011; 58: 41–8. 39. le bell y, jamsa t, korri s, niemi pm, alanen p. effect of artificial occlusal interferences depends on previous experience of temporomandibular disorders. acta odontol scand 2002; 60: 219–22. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �� the increasing of beta-defensin-2 level in saliva after probiotic lactobacillus reuteri administration tuti kusumaningsih department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: commesal bacteria is an excellent inducer for beta defensin-2 (bd-2). probiotics bacteria lactobacillus reuteri (l. reuteri) as commensal bacteria may play the same role as an excellent inducer for bd-2. beta defensin is natural antimicrobial peptides widely expressed in oral cavity, including in epithelium salivary gland. streptococcus mutans (s. mutans) as the main of bacteria causing caries are sensitive to bd-2. purpose: this research was aimed to determine whether administration of probiotic l. reuteri can increase salivary bd-2 level in wistar rats. methods: this research can be considered as a laboratory experimental research with a randomized control group post test only design. twenty-four male rattus norvegicus wistar strain rats aged 3 months were used. they were randomly divided into four groups, namely two control groups (negative control group that was not induced and positive control group induced with s. mutans), and two treatment groups (k1: induced with l. reuteri for 14 days and s. mutans for 7 days, and k2: induced with l. reuteri and s. mutans simultaneously for 14 days). l. reuteri culture at a concentration of 108 cfu/ml and s. mutans culture at a concentration of 1010cfu/ml were induced into the oral cavity of wistar rats. an examination of bd-2 level was then conducted by using elisa techniques. results: there was significant difference of salivary bd-2 level among those treatment groups (p=0.001). bd-2 level in saliva was increased after the administration of l. reuteri. conclusion: l. reuteri probiotic can increase salivary bd-2 level in wistar rats. keywords: probiotic; l. reuteri; level of bd-2; s. mutans; caries correspondence: tuti kusumaningsih, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction dental caries is a disease mostly found in oral cavity, especially in children. similarly, riset kesehatan dasar conducted by the indonesian ministry of health in 2007 showed that 76% of child population in east java suffered with dental caries, and according to data from dinas kesehatan kota surabaya, it is also known that among 61,214 students, 4,359 students had dental caries.1,2 etiology of dental caries are multifactorial. there are three main factors causing dental caries: carbohydrate diet factors, especially sucrose, bacterial factors, especially streptococcus mutans (s. mutans), and response factors of the host, especially innate immunity.3 in the oral cavity, innate immunity is a part of immune system participating in defense process against pathogen. one of innate immunity, which has an important contribution in maintaining a balance between healthy and sick tissues, is antimicrobial peptides (amp). antimicrobial peptides first identified in the oral cavity are beta defensins (bds), which have antimicrobial activities against gram positive and gram negative bacteria as well as against fungi and viruses.4 bds are antimicrobial peptides, which are small cationic peptides widely expressed in the oral cavity, including gingival epithelium, buccal mucosa, salivary glands, salivary duct and saliva.5 the family of beta defensins consists of four peptides, namely bd-1, bd-2, bd-3, and bd-4, but only bd-1, bd-2, and bd-3 are found in oral cavity. bd-1 is expressed constitutively, while bd-2 and bd-3 are induced by bacteria.5,6 on the research report dental journal (majalah kedokteran gigi) 20�5 march; 48(�): ��–�4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �2 kusumaningsih/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 31–34 other hand, microorganisms playing an important role in the etiology of dental caries are oral streptococci, especially s. mutans and s. sobrinus.7 it is known that those two bacterial species are sensitive to bd-2.6 probiotic administration as a preventive treatment against dental caries has currently been evaluated. according to the who, probiotics can be defined as live microorganisms which when consumed in sufficient quantities, they would be beneficial to health.6 commensal bacteria is excellent inducer for bd-2 in epithelial cells of the oral cavity. it means that probiotic lactobacillus reuteri (l. reuteri) as commensal bacteria can possibly act as an inducer for secreting bd-2 possibly detected in saliva samples.7 thus, this research was aimed to know whether the administration of probiotic l. reuteri into the oral cavity of wistar rats can increase bd-2 level in the saliva inoculated with s. mutans. materials and methods this research can be considered as a true experimental laboratory research with randomized control group post test only design. this research was approved by the commission on health research ethics airworthiness of faculty of dental medicine, universitas airlangga. experimental units in this research were twenty-four white rat rattus norvegicus wistar strain provided by integrated research and development laboratory (lppt) yogyakarta. those wistars were used as animal models for dental caries.9 those rats were divided into four groups: negative control group not induced either by s. mutans or l. reuteri, positive control group induced by s. mutans, treatment group 1 induced by l. reuteri from day 1 to day 14 (for 14 days) and by s. mutans from day 8 to day 14 (for 7 days), treatment group 2 induced by l. reuteri and s. mutans from day 1 to day 14 (simultaneously for 14 days). the sample of this research, furthermore, was saliva. the concentration of l. reuteri used as inducer was 4 x 108 cfu/ ml.10 there are several steps to make l. reuteri culture. tablet “x” which contains probiotic l. reuteri prodentis (dsm 17 938 + atccpta 5289) was put in liquid bhi, and then incubated for 1 x 24 hours in anaerobic gas generating kit (oxoid). after removed from the incubator, it would show the presence of turbidity and sediment showing l. reuteri growth. then it was cultured to mrs (de man, rogosa and sharpe) agar plates (oxoid) by scratching, and put it into the incubator again for 2 x 24 hours. some colonies were cultured in liquid bhi media, and then incubated again for 1 x 24 hours. to know whether the bacterial density had reached 4 x108 cfu/ ml, it needs to be tested by using a spectrophotometer with a wavelength λ= 625, identical to the mc farland 0.5.11 the concentration of s. mutans inoculated in this research was 1010 cfu/ ml.12 s. mutans used was s. mutans serotype c in the form of freeze dry taken about one oasis and then put into liquid bhi medium incubated for 1 x 24 hours. further culturing was conducted in the same way with the making of l. reuteri culture. on day 15, saliva samples (whole saliva) were taken after the stimulation of salivary secretion by inducing 1cc of ketamine hcl 100 mg/ cc and 1cc of diazepam (stezolid) 5 mg/ml into thigh area.4 after 2-3 minutes, the saliva was taken about 50 ml by using a micropipette, and then inserted into 1.5 ml eppendorf tubes (figure 1). after centrifuged at 6000 rpm for 10 min at 40 c, the supernatant was taken with a micropipette and then stored at -800 c. elisa procedures were then conducted based on manual kit rnd system. then 20 µl of saliva samples was added with 80 µl of blocking buffer (0.20% triton x-100 and 5% bsa), put into microplate polycarbonate previously been coated with ab capturing of anti bd-2 (rat monoclonal antibody) (santa-cruize), and then incubated at 40 c for 24 hours. after then it was washed 3 times with wash solution (0.15 m nacl + 0.05% triton x-100 + 0.02 g nan3 in 1 liter of distilled water). next, it was added with ab detection (secondary ab) labeled with biotin, and incubated at room temperature for 2 hours. it was then washed 3 times with wash solution (0.15 m nacl + 0.05% triton x-100 +0.02 g nan3 in 1 liter of distilled water). afterwards, each well was added with 100 µl of ab detection (anti bd-2) labeled with hrp, and incubated at room temperature for 1 hour. it was then washed 3 times with wash solution (0.15 m nacl + 0.05% triton x-100 + 0.02 g nan3 in 1 liter of distilled water). next, it was added with 50 µl of tmb substrate (tetra methyl bensidine), then incubated at room temperature for 40 minutes, and stooped by adding 1 n h2so4. the results of optical density was read by using microplate reader (bio-rad model 680) at 450 nm wavelength. anova/ figure 1. collecting saliva of wistar rat with a micropipette. �� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg ��kusumaningsih/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 31–34 spss test was conducted to find the difference of salivary bd-2 level among groups. results the examination conducted by elisa test was aimed to determine bd-2 level in saliva after induced with probiotics l. reuteri in each group as shown in table 1. the results of anova test showed that there was significant difference in the level of salivary bd-2 in wistars among treatment groups (p=0.001). it can also be seen that the mean level of salivary bd-2 in the positive control group (induced with s. mutans) was declined compared to that in the negative control group, from 11.14 to 9.50. on the other hand, the mean level of salivary bd-2 in group 2 (induced with probiotics l. reuteri for 14 days and also induced with s. mutans for 14 days) was increased compared to group 1 (induced with probiotics l. reuteri for 14 days and induced with s. mutans for 7 days), from 12.53 to 14.96. based on all of the results, it can be said that the difference of bd-2 levels was significant. bd-2 level in the negative control group was significantly different from that in the treatment group 2, whereas bd-2 level in the positive control group was significantly different from that in the treatment group 2. however, the difference of bd-2 levels among the other groups was not significant. discussion bds will universally be expressed in all epithelial cells. the epithelial cells can be found in the gingival salivary gland in the oral mucosa of the oral cavity. it is also known that beta defensin has effective and broad antimicrobial activities against gram positive and gram negative bacteria, such as s. mutans, porphyromonas gingivalis and actinobacillus actinomycetemcomitans.5 bds is secreted in biological fluids, including urine, bronchial fluid, saliva and gingival crevicular fluid (gcf). this peptide shows specific expression pattern. bd-1 is expressed constitutively, while bd-2 and bd3 expressions, which have bacterial components and inflammatory mediators, such as interleukin-1β (il-1β), tumor necrosis factor α (tnf-α) and interleukin-17 (il-17) can be induced by bacteria.13 although bd-2 is induced and expressed only as long as there is inflammation in the epithelial tissue, bd-2 can actually be expressed in healthy epithelial tissues in the oral cavity (gingival tissue is not clinically inflamed).14 based on table 1 and figure 2, it is known that the induction of probiotic l. reuteri bacteria into the oral cavity of wistar rats can increase bd-2 level since bd2 is potentially against s. mutans. it is expected that the number of s. mutans bacteria can be reduced, then resulting the reducing of the occurrence of dental caries in rats. it is because l. reuteri are probiotic bacteria, which active biological molecules are on the surface of their cell wall, called microorganism-associated molecular patterns (mamps) such as peptydoglican (pg) and lipoteichoic acid (lta) potentially activating surface cell receptors, namely pattern recognition receptors (prrs) of the host.15 pattern recognition receptors (prrs), such as toll-like receptor-2 (tlr-2) and nucleotide-binding oligomerizationdomain protein-2 (nod-2) in cytoplasm, furthermore, can recognize a variety of microbial components. peptydoglican (pg) and lipoteichoic acid (lta) derived from the cell wall of l. reuteri function as ligands of tlr-2.16 interaction between peptydoglican (pg) and lipoteichoic acid (lta) with tlr-2 and nod-2 induces a signaling cascade involving nuclear factor-kb (nf-kb) and inhibitor of nf-kb kinase (ikbk). with pg and lta of l. reuteri, the phosphorylation, ubiquitination, and degradation of ikb proteins occur, which cause nf-kb translocation into the nucleus, then resulting the activation of nf-kb and activating the promoter of bd-2. the levels of bd-1 and bd-2 in human saliva actually varies from undetectable to 39 ng/ ml for bd-1 and 33 ng/ ml for bd-2.13 it is known that the average level of bd-2 in the saliva samples from healthy people (n = 60) is 9.5 (1.2 to 21) mg/ l.17 most of protein material in the saliva is produced by acini cells in the salivary gland, so beta defensin does not represent a major component of saliva. beta defensin secreted by the salivary ducts may act locally to attack bacteria, viruses and fungi using salivary gland ducts as their invasion route.18 table 1. mean values and standard deviations bd-2 levels in the saliva of wistar rats (ng/ ml) at each treatment group group mean standard deviation significant*) negative control 11.14 0.32 positive control 9.50 1.10 group 1 12.53 1.37 group 2 14.96 4.13 p = 0.001 9 figure 1. collecting saliva of wistar rat with a micropipette. table 1. mean values and standard deviations bd-2 levels in the saliva of wistar rats (ng/ ml) at each treatment group group mean standard deviation significant*) negatif control 11.14 0.32 positif control 9.50 1.10 group 1 12.53 1.37 group 2 14.96 4.13 p = 0.001 figure 2. mean bd-2 levels in the saliva of wistar rats after diindukssi with probiotics l. reuteri in each treatment group. treatment group negative control positive control group 1 group 2 b d -2 levels in the saliva of w istar rats (ng/ m l ) figure 2. mean bd-2 levels in the saliva of wistar rats after diindukssi with probiotics l. reuteri in each treatment group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg �4 kusumaningsih/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 31–34 the inoculation of s. mutans in the oral cavity of those rats was aimed to make the oral cavity of those rats similar to the human oral cavity since s. mutans was not found in the normal oral cavity of wistar rats. in addition, this research was also aimed to know whether s. mutans can induce bd-2 in saliva. based on table 1 and figure 2, it is known that bd-2 level in the positive control group only induced by s. mutans was the lowest one. it may indicate that s. mutans is not strong to induce bd-2. it may also indicate that s. mutans in the oral cavity of wistar rats can be considered as pathogenic bacteria so that the host will use bd-2 to fight the infection. therefore, in the salivary examination of bd-2 level by using elisa method, it is known that the mean concentration of bd-2 in the positive control group was the lowest one, about 9.50 ng/ ml, but this concentration can already be active because defensins can be active at the concentration of 1 to 10 ng/ ml. 19 based on table 2, the average level of bd-2 in the negative control group was different from that in the treatment group 2 (p = 0.024). in other words, it is known that the average level of bd-2 in the negative control group was significantly increased compared to that in the treatment groups 2, from 11:14 ng/ ml to 14.96 ng/ ml. the increasing of bd-2 level can be considered due to the strong induction of probiotic l. reuteri for 14 days. meanwhile, the level of bd-2 in the control group positive induced with s. mutans for 14 days was only 9.5 ng/ ml. based on the results of this study, it can be concluded that the administration of probiotic l. reuteri can increase bd-2 level in the saliva of wistar rats inoculated with s. mutans. further research is needed to know the effect of the administration of probiotic l. reuteri on bd-2 and bd-3 levels in preschool students to get dental caries prevention. references 1. laporan nasional. riset kesehatan dasar (riskesdas). jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia; 2007. p. 140. 2. laporan riset kesehatan dasar provinsi jawa timur. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia; 2007. p. 143. 3. tao r, jurevic rj, coulton kk, tsutsui mt, roberts mc, kimball jr, wells n, berndt j, dale ba. salivary antimicrobial peptide expression and dental caries experience in children. antimicrob agents chemother 2005; 49(9): 3883-8. 4. dale ba, fredericks lp. antimicrobial peptides in the oral environment: expression and function in health and disease. curr issues mol bio 2005; 7(2): 119-33. 5. dale ba, tao r, kimball jr, jurevic rj. oral antimicobial peptides and control of caries. bmc oral health 2006; 6 suppl 1: s13. 6. pepperney a, michael l, chikindas. antibacterial peptides: opportunities for the prevention and treatment of dental caries. probiotics & antimicro prot 2011; 3: 68-96. 7. forssten sd, björklund m, ouwehand ac. streptococcus mutans, caries and stimulation models. nutrients 2010; 2(3): 290-8. 8. wallace tc, guarner f, madsen k, cabana md, gibson g, hentges e, sander me. human gut microbita and its relationship to health and disease. nutr rev 2011; 69(7): 392-403. 9. lamont rj, burne r a, lantz m s, leblanc d j. the oral environment in oral microbiology and the immune response. american society for microbiology press; 2006. p. 201-29. 10. valeur n, engel p, carbajal n, connolly e, ladefoged k. colonization and immunomodulation by lactobacillus reuteri atcc 55730 in the human gastrointestinal tract. appl environ microbiol 2004; 70(2): 1176-81. 11. sutton s. measurement of microbial cells by optical density. j validation technology 2011; 17(1): 46–9. 12. hasan s, singh k, danisuddin m, verma pk, khan au. inhibition of major virulence pathways of streptococcus mutans by quercitrin and deoxynojirimycin: a synergistic approach of infection control. plos one 2014; 9(3): e91736. 13. hans m, hans vm. epithelial antimicrobial peptides: guardian of the oral cavity. international journal of peptides 2014; (article id 370297): 1-3. 14. eberhard j, pietschmann r, falk w, jepsen s, dommisch h. the immune response of oral epithelial cells induced by single-species and complex naturally formed biofilms. oral microbiol immunol 2009; 24(4): 325-30. 15. lebeer s, vanderleyden j, de keersmaecker sc. host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. nat rev microbiol 2010; 8(3): 171-84. 16. takeda k, akira s. toll-like receptors in innate immunity. int immunol 2005; 17(1): 1-14. 17. ghosh sk, gerken ta, schneider km, feng z, mccormick ts, weinberg a. quantification of human beta defensin-2 and -3 in body fluids: application for studies of innate immunity. clin chem 2007; 53(4): 757-65. 18. farnaud sj, kosti o, getting sj, renshaw d. saliva: physiology and diagnostic potensial in health and disease. scientific world journal 2010; 10: 434-56. 19. yin c, dang hn, gazor f, huang j-gt. mouse salivary glands and human β-defensin-2 as a study model for antimicrobial gene therapy: technical considerations. int j antimicrob agents 2006; 28(4): 352–60. table 2. significance difference test between the study groups using hsd group negative control positive control group 1 group 2 negative control _ 0,765 0,825 0,024* positive control _ 0,113 0,001* group 1 _ 0,214 group 2 _ 111111 research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 111–115 correlation between reactive oxygen species and oral conditions in elderly individuals with hypertension: a preliminary study nanan nur’aeny, wahyu hidayat, and indah suasani wahyuni department of oral medicine faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: the increased number of elderly people in indonesia can be a positive phenomenon if their health is well-preserved. the elderly are influenced by physiological changes, environmental factors, and personal habits making them susceptible to chronic diseases. the oral cavity is also subject to change, one of the causes being reduced salivary flow that is manifested in dental caries and other oral health concerns. this disease is stimulated by oxidative stress in the body due to an imbalance between reactive oxygen species (ros) and the antioxidant defense system. purpose: this preliminary study aimed to investigate ros-related hypertension and the state of oral health of elderly patients. methods: the study constitutes a combination of cross-sectional observation and consecutive sampling. twelve subjects, selected on the basis of inclusive and exclusive criteria, consisted of two males and ten females. physical examinations and blood sampling were performed on all subjects. r version 3.4.1 for windows operating system was used to perform statistical tests. results: the 12 patients shared a history of hypertension, the lowest ros level was 0.87 iu/ml, while the highest was one of 7.20 iu/ml. the correlation between ros and oral conditions showed only ros and tooth loss as having a significant positive correlation. an independent t test showed there to be a mean difference between ros with miyazaki index 1 and 2 but it was statistically insignificant. conclusion: there was no correlation between the ros level in the bloodstream and general oral health, except in the relationship between the ros level in the bloodstream and tooth loss which had a positive correlation. keywords: reactive oxygen species; hypertension; oral condition; elderly correspondence: nanan nur’aeny, department of oral medicine, faculty of dentistry, universitas padjadjaran. jl. sekeloa selatan no. 1 bandung 40132, indonesia. e-mail: nanan.nuraeny@fkg.unpad.ac.id. introduction the increased number of elderly people in indonesia could be a positive phenomenon if their health can be wellpreserved. the elderly are influenced by physiological changes, environmental factors, and personal habits making them susceptible to chronic diseases, such as hypertension.1 the oral cavity might also experience such conditions due to reduced salivary flow that is manifested in dental caries, dry mouth, and numerous other symptoms.2 the human body undergoes a metabolic process involving internal and external stimuli. the external stimuli include lifestyle and personal habits. data from riset kesehatan dasar (riskesdas) in 2013 showed the frequency of brushing their teeth among a group of elderly people to be low. such neglect might result in poor oral hygiene and, ultimately, as is the case with the elderly who experience a high incidence of the condition, tooth loss.3 a coated tongue is also often found in the elderly due to decreased efficiency of the salivary glands resulting from degenerative conditions and others factors including: systemic condition, drugs, or dietary habits.4 all processes in the elderly could generate free radicals in the cell as a consequence of enzymatic and nonenzymatic reactions. free radicals represent any molecular species capable of independent existence that contains unpaired electrons in an atomic orbital.5 a mitochondria cell is an internal source of free radicals, released as a dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p111-115 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p111-115 mailto:nanan.nuraeny@fkg.unpad.ac.id 112 nur’aeny, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 111–115 by-product of energy metabolism when cells produce adenosine tri phosphate (atp). this by-product is generally a reactive oxygen species (ros) which plays a dual role as both a toxic and beneficial compound and is crucial to human physiological and pathophysiological processes.6,7 at low or moderate levels, ros exerts a beneficial effect on cellular responses and immune functions. with regard to host defence, ros contributes directly and indirectly to the killing of microorganisms.8 at high concentrations, they generate oxidative stress. this constitutes a deleterious process potentially prejudicial to the entire cell structure, including: lipids, membranes, protein and dna.9 progressive aging is associated with higher levels of oxidative biomolecules reacting with free radicals.10 the progress of a pathologic condition is stimulated by oxidative stress in the body due to an imbalance between ros and an antioxidant defense system.11 research into ros in elderly patients in indonesia, particularly regarding its relation to changes within the oral cavity, has not been widely conducted. thus, the purpose of this preliminary study is to investigate ros-related hypertension and oral cavity conditions in elderly patients. materials and methods this study constitutes a cross-sectional observation and consecutive sampling conducted at the community health center, ujung berung, bandung. the observers were members of the research team (n=3). the inclusion criteria applied to all patients consisted of the following: male or female, aged 60 years or above, registered as a patient at the ujung berung community health center, a history of hypertension, and a willingness to provide signed confirmation of informed consent. the exclusion criteria comprised: oral candidiasis lesions, oral leukoplakia, multiple ulceration of the tongue, and pre-cancerous ulcer lesions. twelve subjects fitting the inclusion and exclusion criteria included two males and ten females. oral examination and blood sampling were performed on the subjects, from whom 5 ml of blood was taken for ros examination at the clinical pathology laboratorium, hasan sadikin hospital. r version 3.4.1 for windows operating system was used to conduct statistical tests, all of which were performed to a significance level of 0.05. the research reported here received ethical approval from the health research ethics committee, universitas padjadjaran, number 754/un6.c.10/pn/2017, and registration number: 0817050696. results this preliminary study involved the participation of 12 elderly patients with a history of hypertension (table 1 and table 2). a spearman correlation was calculated to measure the correlation between systolic and diastolic table 1. characteristics of patients (n=12) no. characteristics n 1. age range 60–69 7 70–79 5 2. gender female 10 male 2 table 2. blood pressure category no. category blood pressure (mmhg) n 1. high normal 130–139 and/or 85–89 2 2. mild 140–159 and/or 90–99 5 3. moderate 160–179 and/or 100–109 3 4. severe ≥180 and/or ≥110 2 ros (figure 1a and figure 1b). oral cavity anomalies were found to include dental caries, tooth necrosis, plaque and calculus, dental filling, tooth root, tooth loss, and coated tongue (table 3). the lowest ros level was 0.87 iu/ml, while the highest stood at 7.20 iu/ml (table 4). there was no correlation between the ros level in the bloodstream and general oral health, except in the relationship of the ros level in the bloodstream and tooth loss which demonstrated a significant correlation (p= 0.028) (figure 1c-1f). an independent t test showed that there was a mean difference between ros and the miyazaki index 1 and 2, but this was not considered to be statistically significant (figure 1g). clinically, a coated tongue was defined as one with a white-yellowish layer equals in size to oneto two-thirds the area of the posterior dorsum of the tongue (figure 2a and 2b). discussion this research was a preliminary study conducted on a small group of elderly people aged between 60 and 79 years, with the majority falling within the 60-69 years age range. according to the badan pusat statistik (bps), the life expectancy of members of west java’s population continues to increase and in the period 2015-2020 is anticipated to reach 72.8 years.12 the definition of who constitutes an elderly or geriatric person is based on broad and varied age ranges. nevertheless, a common classification system would include: a lower range (65–74 years), a middle range (75–85 years), and an upper range (>85 years).2,13 a limited amount of data in this study (table 1) showed scant difference between the lower and middle range groups, with the number of females higher than that of males. according to the bps, females enjoy a higher life expectancy than males.12 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p111-115 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p111-115 113113nur’aeny, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 111–115 table 3. oral conditions of patients no. dental caries* tooth necrosis* tooth loss* filling plaque/calculus** tooth root* index coated tongue*** 1 2 1 2 none exist 2 2 2 none none 4 none exist 3 2 3 none none 7 none exist 2 2 4 3 none 10 none exist 3 1 5 none 3 10 none exist 3 2 6 6 none 4 none exist 2 2 7 4 none 6 none exist 3 2 8 none none 6 none exist 4 1 9 1 none 5 none exist 2 1 10 none none 9 none exist 7 1 11 none 2 3 none exist 1 2 12 1 none 2 none exist 1 1 note: * number of tooth ** exist = by visual, at least plaque or calculus found in one site of tooth *** index miyazaki; score 0 = not visible, score 1 = <1/3 surface of the tongue covered, score 2 = <2/3 surface of the tongue covered, and score 3 => 2/3 the surface of the tongue is covered. a d c b f e a d c b f e a d c b g f e a d c b g f e a d c b g f e a d c b g f e a d c b g f e figure 1. a. ros level and systolic blood pressure. b. ros level and diastolic blood pressure. c. ros level and dental caries. d. ros level and missing teeth. e. ros level and tooth root. f. ros level and tooth necrosis. g. ros level and miyazaki index. (iu/ml) (iu/ml) (iu/ml) (iu/ml) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p111-115 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p111-115 114 nur’aeny, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 111–115 aging is simply the result of accumulative deteriorative processes such as oxidation.14 biological functions gradually decline with age and may be due to one or more of the following factors: lifestyle, behaviour, diet, and environment.14 oxidants or free radicals are generated internally as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from a variety of cytosolic enzyme systems whereas, externally, ros production is triggered by a certain agent within an indvidual’s lifestyle and/or environment.15 aging is a natural and progressive process producing functional changes in the body due to aforementioned internal and external factors that might manifest themselves as a chronic condition such as hypertension. in this study, patients’ blood pressure largely fell within the mild category (systolic =140-159 and/or diastolic= 90-99 mmhg). a test of the correlation between ros and systolic blood pressure levels confirmed it to be negative (figure 1) but there was a positive diastolic and ros correlation (figure 2). according to the study described here, ros levels were within the lower range, this result being in line with the studies indicating that, in a mild hypertension condition, lipid peroxidation and oxidative stress levels are not increased.16,17 oxidative stress promotes smooth vascular muscle cell proliferation, together with hypertrophy and collagen deposition leading to thickening of the vascular media and narrowing of the vascular lumen. in addition, increased oxidative stress may damage the endothelium and impair endothelium-dependent vascular relaxation and increase vascular contractile activity. all these effects on the vasculature may explain how increased oxidative stress can cause hypertension.18 this result confirmed all patients to have been well cared for as they had received routine monthly treatment from a health programme (chronic disease management program) at the community health center,ujung berung, bandung. such treatment usually included the implementation of a dietary regime. the dietary approaches to ttop hypertension (dash) study confirmed that the dash diet, which was rich in vegetables, fruits and low-fat dairy products, low in fat, and including whole grains, poultry, fish and nuts, and lowered blood pressure.19 other results confirmed the correlation between ros and diastolic levels in this study as positive. although this study did not feature a complete data history of hypertension, the theory mentions the potential occurrence of this condition because ros potentially results in hypertension by inducing vascular contraction (figure 2).20 it is known that superoxide (ros) rapidly deactivates endotheliumderived nitric oxide (no), the most important endogenous vasodilator, thereby promoting vasoconstriction.21 intra-oral findings showed most cases to be dental caries, tooth loss, plaque/calculus, and tooth root. the correlation only produced positive and significant results for ros and tooth loss, at p=0.028 (figure 4). this possibly happened due to the presence of remnants of ros related to previous infections or conditions triggering tooth loss, supported by the presence of plaque and calculus in all patients. this, in turn, meant that there were some forms of gingival inflammation (gingivitis or periodontitis) which induced increased ros in tooth loss.22 other conditions produced a negative correlation between ros for dental caries and tooth necrosis, meaning that when dental caries and tooth necrosis decrease, the level of ros increases. however, this condition requires further investigation involving a greater number of research subjects. oral mucosa conditions also produced a coated tongue, a clinical condition in which a white yellowish layer forms on the dorsum of the tongue caused by the accumulation of food debris, microorganisms, desquamated epithelial cel/keratin, pigment, and mucus. several factors are suspected of being associated with a coated tongue, including: chemotherapy, allergies, vitamin deficiency, radiation therapy, poor oral hygiene, salivary ph change, decreased salivary production, systemic disease, and drug consumption.14 clinically, according to the miyazaki index, the coated tongue of the majority of patients (7) registered a score of 2, signifying that the layer covered an area two-thirds the size of the posterior of the tongue. the t test result showed that the mean difference between ros and the miyazaki index was higher in a group with score 1 than score 2. the data table 4. characteristics of patients and ros level no. age (years) gender ros level (iu/ml) 1. 64 male 0.95 2. 60 female 1.37 3. 69 male 4.71 4. 73 female 4,2 5. 65 female 1,94 6. 73 female 0.87 7. 77 male 2.94 8. 73 female 7.2 9. 73 female 1.14 10. 62 female 4.24 11. 64 female 2.07 12. 64 female 1.28 figure 2. a. 1/3 coated section of the tongue; b. 2/3 coated section of the tongue 11 figure 1a.ros level and systolic blood pressure. 1b. ros level and diastolic blood pressure. 1c. ros level and dental caries. 1d. ros level and missing teeth.1e. ros level and tooth root.1f. ros level and tooth necrosis.1g. ros level and miyazaki index. figure 2a. 1/3 coated section of the tongue. g f e a b 11 figure 1a.ros level and systolic blood pressure. 1b. ros level and diastolic blood pressure. 1c. ros level and dental caries. 1d. ros level and missing teeth.1e. ros level and tooth root.1f. ros level and tooth necrosis.1g. ros level and miyazaki index. figure 2a. 1/3 coated section of the tongue. g f e a b a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p111-115 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p111-115 115115nur’aeny, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 111–115 showed that all patients who recorded a score of 2 also presented considerable tooth loss, plaque, and calculus, thereby indicating poor oral hygiene and, probably, a soft diet resulting in the accumulation of debris on the tongue. decreased salivary flow as physiologic changes occur in elderly might also influence the occurrence of a coated tongue.4 in this study, there was no oral manifestation related with antihypertension drugs, such as xerostomia, lichenoid reactions, burning mouth syndrome, or stomatitis.15 in conclusion, there was no correlation between the ros level in the bloodstream and general oral conditions, except in the relationship between the ros level in the bloodstream and tooth loss which had a positive correlation. acknowledgement this study was supported by internal grant funding from universitas padjadjaran and thanks to the dermatomusculoskeletal (dms) study center of faculty of medicine universitas padjadjaran which has provided an opportunity for the implementation of this research. references 1. veiga n, domingues a, douglas f, rios s, vaz a, coelho c, bexiga f, coelho i. the influence of chronic diseases in the oral health of the elderly. j dent oral health. 2016; 2: 1–6. 2. razak pa, richard kmj, thankachan rp, hafiz kaa, kumar kn, sameer km. geriatric oral health: a review article. j int oral health. 2014; 6(6): 110–6. 3. kementerian kesehatan republik indonesia. riset kesehatan dasar (riskesdas 2013). jakarta: badan penelitian dan pengembangan kesehatan kementerian kesehatan ri; 2013. p. 1–384. 4. nur’aeny n, sari ki. profil lesi mulut pada kelompok lanjut usia di panti sosial tresna wreda senjarawi bandung. maj ked gi ind. 2016; 2(2): 74–9. 5. lobo v, patil a, phatak a, chandra n. free radicals, antioxidants and functional foods: impact on human health. pharmacogn rev. 2010; 4(8): 118–26. 6. pham-huy la, he h, pham-huy c. free radicals, antioxidants in disease and health. int j biomed sci. 2008; 4(2): 89–96. 7. d’autréaux b, toledano mb. ros as signalling molecules: mechanisms that generate specificity in ros homeostasis. nat rev mol cell biol. 2007; 8: 813–24. 8. brieger k, schiavone s, miller jr fj, krause k. reactive oxygen species: from health to disease. swiss med wkly. 2012; 142: 1–14. 9. valko m, leibfritz d, moncol j, cronin mtd, mazur m, telser j. free radicals and antioxidants in normal physiological functions and human disease. int j biochem cell biol. 2007; 39: 44–84. 10. oliveira bf, nogueira-machado ja, chaves mm. the role of oxidative stress in the aging process. sci world j. 2010; 10: 1121–8. 11. moham med mt, kadhim sm, jassimand a m n, abbas si. free radicals and human health. int j innov sci res. 2015; 4(6): 218–23. 12. badan perencanaan pembangunan nasional, badan pusat statistik, united nations population fund. proyeksi penduduk indonesia: indonesia population projection 2010-2035. jakarta: badan pusat statistik; 2013. p. 32–4. 13. gates bj, walker km. physiological changes in older adults and their effect on diabetes treatment. diabetes spectr. 2014; 27(1): 20–9. 14. goldsmith tc. an introduction to biological aging theory. 2nd ed. crownsville: azinet press; 2014. p. 45. 15. rahman t, hosen i, islam mmt, shekhar hu. oxidative stress and human health. adv biosci biotechnol. 2012; 3: 997–1019. 16. national heart foundation of australia. guideline for the diagnosis and management of hypertension in adults 2016. melbourne: national heart foundation of australia; 2016. p. 74. 17. touyz rm, briones am. reactive oxygen species and vascular biology: implications in human hypertension. hypertens res. 2011; 34: 5–14. 18. logan ac, wong c. chronic fatigue syndrome: oxidative stress and dietary modifications. altern med rev. 2001; 6(5): 450–9. 19. vasdev s, singal p, gill v. the antihypertensive effect of cysteine. int j angiol. 2009; 18(1): 7–21. 20. lassègue b, griendling kk. reactive oxygen species in hypertension: an update. am j hypertens. 2004; 17: 852–60. 21. rodrigo r, prat h, passalcqua w, araya j, guichard c, bächler jp. relationship between oxidative stress and essential hypertension. hypertens res. 2007; 30: 1159–67. 22. leong x, ng c, badiah b, das s. association between hypertension and periodontitis: possible mechanisms. sci world j. 2014; 2014: 1–11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p111-115 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p111-115 �� vol. 45. no. 1 march 2012 research report genetic variability of candida albicans in hiv/aids patient with and without arv therapy and non hiv/aids retno puji rahayu1, widiyanti p2, and arfijanto m3 1,2,3 institute of tropical disease, airlangga university 1 faculty of dentistry, airlangga university 2 faculty of science and technology, airlangga university 3 faculty of medicine, airlangga university surabaya indonesia abstract background: oral candidiasis is the mostly found oral manifestation in hiv/aids infected patient caused by immunocompromised especially immunodeficiency. clinical symptoms is severe pain in oral cavity and dry mouth because of xerostomia which cause the loss of appetite. candida albicans (c. albicans) is normal flora in oral cavity which plays as opportunistic pathogen and also the cause of oral candidiasis. almost 90% of hiv–infected patient have oral candidiasis. this condition is clinical problem which has not been well-managed yet. c. albicans colonized oral mucous cavity has different genetic variability for each strain. phenotype of c. albicans has been determined by genetic factor and environtment. this condition stimulate differences of genotype among various strain of c. albicans in the world. purpose: the purpose of this research is to analyze the genetic variability of c.albicans which colonized in the mucous oral cavity of hiv/aids patient in surabaya in the treatment with and without arv therapy and non hiv/aids. methods: this research has been identify and characterize the prevalent strain of c. albicans isolat in surabaya (east java) in hiv/aids infected patient with oral candidiasis by method of iatron candidal check. the highlight of this research including cytology examination by papanicoloau staining, c. albicans culture, spheroplast making, dna isolation and genetic variability checking by randomly amplyfied polymorphism dna (rapd). results: c. albicans colonizing oral mucosa of non-hiv patients had a predisposition of farther genetic relationship (genetic distance of 0.452) with c. albicans colonizing oral mucosa of hiv arv and hiv non-arv patients. the genetic distance was ranging between 0 and 1, where 9 was long genetic distance and 1 was short genetic distance. in contrast, c. albicans colonizing oral mucosa of hiv arv have predisposition of closer genetic relationship (genetic distance of 0.762) with c. albicans colonizing oral mucosa of hiv non-arv patients. conclusion: the conclusion of this research were c.albicans colonizing hiv/aids patiens with and without arv showed no high genetic variability between c.albicans isolate in hiv patients. there fore, the character of c.albicans colonizing hiv arv and hiv non-arv patients had similar genotype predisposition of closer relationship value with c.albicans colonizing oral mucosa non hiv patients. key words: candida albicans, hiv/aids, oral candidosis, rapd abstrak latar belakang: oral candidiasis merupakan manifestasi kelainan rongga mulut yang paling sering timbul pada penderita hiv/aids karena kondisi immunocompromised terutama defisiensi imun. gejala klinisnya berupa nyeri hebat di rongga mulut dan mulut kering karena xerostomia yang menyebabkan hilangnya nafsu makan. candida albicans (c. albicans) berperan sebagai patogen oprtunistik dan merupakan penyebab kandidiasis rongga mulut. hampir 90% penderita terinfeksi hiv mengalami kandidiasis rongga mulut. kondisi ini merupakan masalah klinis yang belum teratasi dengan baik. kolonisasi c. albicans di mukosa rongga mulut mempunyai variabilitas genetic yang berbeda untuk tiap strainnya. fenotip c. albicans ditentukan oleh faktor genetic dan lingkungan. kondisi ini menstimulir perbedaan genotip di antara berbagai strain c. albicans di dunia. tujuan: tujuan penelitian ini adalah meneliti korelasi antara hubungan genetik yang menunjukkan variasi genetik kolonisasi c. albicans pada rongga mulut dan insidens kandidiasis rongga mulut pada penderita hiv/aids dan non-hiv/aids. metode: penelitian ini mengidentifikasi dan mengkarakterisasi strain candida albicans isolat surabaya (jawa timur) pada penderita hiv/aids dengan kandidiasis rongga mulut dengan metode iatron candidal ��rahayu, et al.: genetic variability of candida albicans in hiv/aids check. penekanan dalam penelitian ini termasuk pada pemeriksaan sitologi dengan pengecatan papanicoloau, kultur c. albicans, pembuatan spheroplast, isolasi dna dan pemeriksaan variabilitas genetik dengan randomly amplified polymorphism dna (rapd). hasil: c. albicans yang berkolonisasi di rongga mulut pada penderita non-hiv mempunyai predisposisi hubungan genetik (jarak genetik 0.452) dengan c. albicans yang berkolonisasi di rongga mulut pada penderita hiv yang mendapatkan terapi arv dan non arv. jarak genetic bervariasi antara 0 dan 1, dimana 9 dalah jarak genetik terpanjang and 1 adalah jarak genetik terpendek. sebaliknya, c. albicans yang berkolonisasi di rongga mulut pada penderita hiv yang menerima terapi arv memiliki predisposisi hubungan genetic yang lebih dekat (jarak genetic 0.762) dibandingkan c. albicans yang berkolonisasi di rongga mulut pada penderita hiv non-arv. kesimpulan: sebagai kesimpulan, penelitian ini menunjukkan bahwa c. albicans isolate surabaya yang mengkolonisasi penderita hiv/aids dengan arv dan non arv memiliki hubungan kekerabatan genetik yang sama dibanding dengan pasien non hiv/aids. kata kunci: candida albicans, hiv/aids, kandidiasis rongga mulut, rapd correspondence: retno puji rahayu, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: retnorahayu@yahoo.com; drwidiyanti@yahoo.com introduction hiv/aids cases is increasing and concern about the problem is growing. in indonesia, the total number of hiv/aids cases up to the end of june 2006 was 10.859 cases (4.527 hiv cases and 6.332 aids cases), and the largest proportion, as much as 53%, belonged to age group of 20–29 years.1 candida albicans (c. albicans) causing oral candidiasis are frequently found along the course of hiv infection, resulting from immunocompromised condition that commonly accompany hiv/aids patients, and one of the complications with the highest incidence rate in oral cavity, reaching 90%.2 oral candidiasis occurs due to immunocompromised condition that commonly accompanying hiv/aids patients and may disturb host’s immune response, one of which is the disturbed production of cytokines, such as il-1a and tnf-a. in such condition, abnormalities in polymorphonuclear (pmn) and macrophage phagocytosis function are also found.3,4 reduction of immunity system in the body of hiv/aids patients result in the absence of the host’s defense against c. albicans attachment and colonization to oral mucosal epithelium. in hiv/aids patients, c. albicans infection in oral surface epithelium is recurrent and persistent. such condition remains a clinical problem that cannot be solved satisfactorily up to the moment.5 the increase of fungal infection is reported to raise morbidity and mortality rate of immunocompromised (hiv) patients.1 c. albicans is a commensal organism in oral cavity. within the oral cavity, there are various c. albicans strains with certain phenotype characteristics, determining them as commensal or pathogenic,6 and it was suspected that c. albicans virulence in various strains is affected by genetic variability. the success of c. albicans attachment to oral mucosa epithelial surface is the beginning of c. albicans and oral candidiasis is one of the complications of hiv/aids with the highest incidence rate in oral cavity.7,8 c. albicans infection on epithelial surface of oral mucosa is recurrent and persistent in dm patients. about 90% of hiv/aids population showed the presence of oral candidiasis. such condition is a clinical problem that cannot be satisfactorily overcome,6 since it is still endemic and the therapy has never been effective. c. albicans colonizing oral mucosa have various genetic variations in each strain. profiles of a c. albicans character (profile) are determined by genetic factors and environmental factors.9 such condition results in different genotype among various c. albicans strains. information on genetic correlation among individuals within and between species has several important benefits for improvement of an organism. in studying the outcome of genetic variations, assumption on the presence of genetic correlation will be useful for genotype identification. knowledge on genetic data, such as the presence of genetic variance in c. albicans colonizing oral mucosa of hiv/aids patients based on the level of severity is necessary to improve therapy management of virulent c. albicans infection. so far, correlation between genetic relationship of c. albicans colonizing oral mucosa of hiv/aids patients and the severity of the disease remains unclear. literature studies revealed that c. albicans virulence in various strains are supposedly affected by genetic variability. the objective of this study was analyze the genetic variability of c. albicans in hiv/aids patients in surabaya with and without arv therapy, and patients with no hiv/aids. materials and methods specimen for cytological examination was taken from the scrubbing of the tongue and buccal mucosa, and the result of scrubbing that had been swabbed onto object glass was fixed and stained with papanicoloau procedure and mounted with dpx. surabaya isolate of c. albicans were cultured for 2 √ 24 hours in 37° c in sabouraud dextrose agar (difco). then, gram staining and sugar fermentation test were performed. sugar fermentation test with glucose, maltose, sucrose, and lactose were incubated in incubator 37° c for 3 √ 24 hours. the existence of c. albicans was marked by color change. the obtained c. albicans were grown within sabouraud dextrose broth (difco) media, incubated for 18–20 hours in 37º c in orbital shaking, �0 dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 28–34 centrifuged in 700 g for 5 minutes at 24° c, and washed with pbs 3 times. then the spheroplast production was done by inoculating c. albicans in dextrose-containing yeast pepton dextrose broth (ypd broth, difco laboratories). after aerobic incubation in 37º c for 18 hours in rotary incubator with 100 rev/min. candida cells were harvested and washed 2√ with pbs. then, candida cells were counted using hemocytometer and suspended to become 3.5 √ 107 cell/ml in pbs. pellet was resuspended with 600 ul sorbitol buffer containing 200 u litycase. incubation was performed in 30° c for 1 hour and the produced spheroplasts were harvested with centrifugation in 3000 rpm for 5 minutes. pellet was suspended in 180 ul atl (qiagen) buffer, and added with 20 ul proteinase k into the supernatant and incubated in 55° c overnight. after being incubated for 55° c overnight dna isolation was done. supernatant and 4 ul rnase were added and treated with 200 ul buffer al and incubated in 70° c for 10 minutes. subsequently, 200 ul ethanol 96% was added and vortexed directly to become homogeneous. all supernatants were removed to dneasy spin column 2 ml collection tube and added with 500 ul buffer aw1. the tube was centrifuged in 800 rpm in 4° c for one minute, and dneasy spin column in a new 2 ml collection tube and 500 ul buffer aw2 was added and centrifuged for 3 minutes in high speed. the dneasy spin column was removed to 1.5 ml sterile microcentrifugation tube, and 200 ul ae buffer was pipetted directly to dneasy membrane. it was incubated at room temperature for 1 minute and centrifugated in a speed of 8000 rpm. the ae buffer administration procedure was repeated one time. dna purity and concentration were determined using uv/vis, jasco v-530. p c r w a s d o n e u s i n g e f b 1 g e n e p r i m e r a s internal control for c. albicans using efb1 primer: 5 ’ a t t g a a c g a a t t c t t g g c t g a c 3 ’ 5 ’ catcttcttcaacagcagcttg-3’. the final volume of pcr reaction mixture was 25 ul, comprising 10 x buffer mg free, 25 mm mgcl2, 2.5 mm dntp mix, 25 ng dna, 20 um primer and 100 u taq polymerase (promega), and then mixed and eppendorf pcr was put into master cycler machine (gene amp pcr system 2499, perkin elmer). the pcr condition was as follows: denaturation at 94° c for 1 minute, annealing at 55° c for 1 minute and extension at 72° c for 1 minute, and the final step was extra extension at 72° c for 10 minutes. the number of pcr cycles was 45. random amplified polymorphism dna (rapd) method was done using nt and at primers.11 nt primer: 5’ cccgtcagca 3’ and at primer : 5’ gcgcacgg 3’. to perform amplification in rapd-pcr method, the following materials are needed: sterile distilled water (ddh2o), 10√ buffer (qiagen), dntp (qiagen), q-solution (qiagen), te ph 8, taq polymerase, and dna sample with pcr. pcr-raps conditions were as follows: hot start 94° c for 5 minutes, denaturation in 94° c for 1 minute, annealing in 35° c for 1 minute and extension in 72° c for 2 minutes. extra extension in 72° c for 5 minutes and the number of cycles were 45 cycles. to identify the success of pcr amplification, electrophoresis 2% was performed, marker (dna 174-hae iii digest and 1 kb dna ladder), and transluminator-uv polaroid gel camera. results efb1 gene examination as internal control using primers 5’-attgaacgaattcttggctgac-3’ and 5’-catcttcttcaacagcagcttg-3’ with following pcr conditions: denaturation in 94° c for 1 minute, annealing in 55° c for 1 minute and extension in 72° c for 1 minute and the final step was extra extension in 72° c for 10 minutes. the number of pcr cycles was 45. efb1 gene is a housekeeping gene that acts as an internal control in c. albicans. the result of pcr of efb1 gene in control group, hiv/aids patients with and without arv were apparent in all samples (figure 1). this indicates that candidap sp. samples was c. albicans. the examination of c. albicans genotype infecting oral mucosa of hiv/aids patients with or withour arv and control group was performed using random amplified polymorphic dna (rapd) method. in this method nt primer 5’ cccgtcagca 3’ and at primer 5’ gcgcacgg 3’ with pcr-rapd condition was used as follows: hot start in 94° c for 5 minutes, denaturation 94° c for 1 minute, annealing in 35° c for 2 minutes and extension in 72° c for 2 minutes, extra extension in 72° c for 5 minutes and the number of cycles was 45. the result of rapd was electrophoresized in 2% agarose gel separating c. albicans dna fragment in a range of 250–3000 bp (figure 2). based on unweight pair group method with arithmatic averages (upgma) clustering analysis using mvsp ver 3.1 (kovach computing service) program, by determining similarity value through simple matching coefficient method, indicated that rapd result in genotype examination of c. albicans infecting oral mucosa of hiv/aids patients with and without arv and control group (non-hiv/aids) using nt primer revealed two groups, that the similarity value of group i and ii was 0.705 (70.5%). figure 1. pcr result of efb1 gene. note: lane 3: 1 kb dna marker (ladder intron) lane 24: 1 kb dna marker (ladder intron) ��rahayu, et al.: genetic variability of candida albicans in hiv/aids figure 2. result of rapd with c. albicans nt primer in hiv/aids patients. note: lane 1:1 kb dna marker (ladder intron) lane 12: dna marker 174-hae iii digest figure 3. result of rapd with c. albicans at primer in hiv/aids patients. note: lane 1:1 kb dna marker (ladder intron) lane 16: dna marker 174-hae iii diges figure 4. dendogram from rapd with c. albicans nt primer in hiv/aids patients. notes: a = c. albican in hiv/aids patients with arv, b = c. albican in hiv/aids patients without arv, c = control (c. albicans in non-hiv/aids) �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 28–34 based on upgma clustering analysis using mvsp ver 3.1 (kovach computing service) program by determining similarity value through simple matching coefficient method indicated that rapd result in genotype examination of c. albicans infecting oral mucosa of hiv/aids patients with and without arv using at primer showed that there were three groups. above data indicated that similarity value in group ii and iii was 0.686 (68.6%), while the similarity value in group ii and iii to group i was 0.681 (68.1%). table 1. similarity matrix between sample groups sample groups hiv arv hiv non arv non hiv b c a b 0.762 0.476 c 0.428 a genetic relationship and variability of c. albicans colonizing hiv arv, hiv non-arv and non-hiv, based on the genotype was measured through similarity matrix of dna fragments based on genetic distance. between hiv arv and hiv non-arv the genetic distance was 0.762. between hiv arv and non-hiv the genetic distance was 0.476, and between non-hiv and hiv non-arv the genetic distance was 0.428. phenogram shows that c. albicans colonizing oral mucosa of non-hiv patients had a predisposition of farther genetic relationship (genetic distance of 0.452) with c. albicans colonizing oral mucosa of hiv arv and hiv non-arv patients. the genetic distance was ranging between 0 and 1, where 9 was long genetic distance and 1 was short genetic distance. in contrast, c. albicans colonizing oral mucosa of hiv arv have predisposition of closer genetic relationship (genetic distance of 0.762) with c. albicans colonizing oral mucosa of hiv non-arv patients. figure 5. dendogram from rapd with c. albicans at primer in hiv/aids patients. notes: a = c. albicans in hiv/aids patients with arv, b = c. albicans in hiv/aids patients without arv figure 6. phenogram based on similarity matrix of rapdresulted c. albicans dna fragment. figure 7. distribution of the number of bands resulting from c. albicans rapd. result of statistical analysis revealed that the mean of polymorphism (number of bands) in non-hiv (4.91 ± 2.02) was higher than the mean polymorphism in hiv-arv (3.55 ± 0.82) and hiv non-arv (4.27 ± 0.65). ��rahayu, et al.: genetic variability of candida albicans in hiv/aids discussion oral candidiasis is commonly found anytime during of hiv infection. this is due to immunocompromized condition generally accompanying hiv/aids patients and one of complications with highest rate of incidence in oral cavity. various literatures reported that factors affecting the incidence of c. albicans in oral cavity of hiv/aids patients are the immunodeficiency resulting from immunocompromized condition, so the patients are susceptible to oral infection. in hiv/aids patients, the factor is a predisposition of superficial and systemic infection, including c. albicans infection. this is because immunocompromized condition in host cells results in immunodeficiency and disordered cytokines production, leading to disturbed phagocytic function of pmn and macrophage. in oral cavity live various strains of c. albicans with certain phenotype characteristics that determine its nature as commensal or pathogenic. up to the moment, it remains unclear what cell types and receptor as the primary target of hiv virus in oral mucosa. however, it is reported that the hiv-resulted change of oral epithelial cells causes changes in cd4 t cells in the mucosa and the reduction of th1 cytokine within the saliva of chronic hiv patients. this triggers the occurrence of opportunistic infection. knowledge on genetic data, such as the presence of genetic variations in c. albicans colonizing oral mucosa of hiv/aids patients, should be improved to determine the virulence, both qualitatively and quantitatively. knowledge on genetic variation can be used as a basis for improving the management for solving various infection cases resulting from c. albicans in hiv/aids patients. genotype examination of surabaya isolate c. albicans colonizing oral mucosa in hiv/aids and non-hiv/aids patients was aimed to detect polymorphism at dna level. polymorphism is a different shape from the basic structure to find the variability of c. albicans using molecular epidemiology. dna markers are widely used in studying genetic variations in c. albicans is rapd.9,11,12 rapd technique analysis have several advantages, such as shorter processing time, requiring less dna samples (0.5–50 nm), and need no radioisotope. rapd also needs no early dna sequence information, it has more simple procedure and larger number of samples which can be processed rapidly. profile of c. albicans (phenotype) character depends on genetic factors and environmental factor. such condition result in different genotype in various c. albicans strains worldwide. genotype-based genetic relationship and variability of c. albicans colonizing patients with hiv/aids with arn, non-arv and non-hiv/aids was measured using similarity matrix of dna fragments based on genetic distance. the result of dendogram (figure 4) through upgma clustering analysis using mvsp ver 3.1 program describes that c. albicans colonizing oral mucosa of hiv/aids patients with arv, non-arv with nt primer had predisposition of closer relationship value with genetic distance of 0.705 (70.5%). however, the result of dendogram analysis with at primer (figure 5) indicated that c. albicans colonizing oral mucosa of hiv/aids patients with arv and non-arv had predisposition of relatively closer relationship value as well (genetic distance 0.681). from both results it was apparent that c. albicans isolated from patients with hiv/aids with arv and nonarv, either using at or nt primer, had predisposition of homogeneous genetic distance, ranging between 0.681 and 0.705. this means that c. albicans colonizing hiv/aids patients with and without arv, both using nt and at primers, showed no high genetic variability between c. albicans isolate in hiv patients. therefore, the character of c. albicans colonizing hiv arv and hiv non-arv patients had similar genotype predisposition. the result of phenogram analysis shows that c. albicans colonizing oral mucosa of non-hiv had a predisposition of farther relationship value (genetic distance 0.452) with c. albicans colonizing oral mucosa of hiv arv and hiv non-arv patients. in contrast, c. albicans colonizing oral mucosa of hiv arv patients have a predisposition of closer relationship value (genetic distance 0.762) with c. albicans colonizing oral mucosa of hiv non arv patients (figure 6). the result of statistical analysis shows that the mean polymorphism in non-hiv (4.91 ± 2.02) was higher than the mean of polymorphism in hiv arv (3.55 ± 0.82) and hiv non arv (4.27 ± 0.65) (figure 7). this difference is interesting since arv therapy in hiv patients may possibly influence c. albicans genotype. this was also supported by the effect of oral environmental conditions, such as saliva quality and quantity, diet pattern, nutritional status, and host immune response. nevertheless, it requires further analysis to find the target gene in c. albicans that are subjected to mutation due to the use of arv for hiv/aids patients. several references reported that oral environment condition also facilitates certain strains of c. albicans to colonize oral mucosa. from several genotypes that present as the existence of c. albicans genetic variance, serotype a is reported to have a predisposition of polymorphism higher than that of polymorphism in serotype b. this is possibly because serotype a has higher virulence and adherence capability compared to serotype b through glycomannoprotein receptor that present on c. albicans cell wall. however, patients with immunodeficiency due to immunocompromized condition are highly susceptible to infection, particularly by c. albicans, so that c. albicans may become virulent and have higher adherence capability, facilitating the occurrence of infection in the oral mucosa of hiv/aids patients. the conclusion of this research were c. albicans colonizing hiv/aids patients with and without arv showed no high genetic variability between c. albicans isolate in hiv patients. therefore, the character of c. albicans colonizing hiv arv and hiv non-arv patients had similar genotype predisposition of closer relationship value (genetic distance 0.762) with c. albicans colonizing oral mucosa non hiv patients. �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 28–34 references 1. nasronudin. hiv dan aids. pendekatan biologi molekuler, klinis dan sosial. cetakan pertama. surabaya: airlangga university press; 2007. p. 203–13. 2. willis am, coulter wa, fulton cr, hayes jr, bell pm. the influence of antifungal drugs on virulence properties of candida albicans in patients with diabetes mellitus. oral surg oral med oral pathol oral radiol endod 2001; 91: 317–21. 3. lamey pj. oral candidiasis, in opening comments orofacial disease in the uk the british society for oral medicine web service, 2001; p. 1–8. 4. herring ac, huffnagle gb. innate immunity and fungal infections. in: immunology of infectious diseases. washington: asm press american society for microbiology; 2002. p. 127–34 5. elahi s, clancy r, pang g. a therapeutic vaccine for mucosal candidiasis. vaccine 2001; 19(17–19): 2516–21. 6. felk a, kretschmar m, albrecht a, schaller m, beinhauer s, nichterlein t. candida albicans hyphal formation and the expression of the efg1-regulated proteinases sap4 to sap6 are required for the invasion of parenchymal organs. infection and immunity 2002; 70(7): 3689–700. 7. costa ic, loyola w, gaziri lcj, custodio la, felipe i. low dose of concanavalin-a enhances innate immune response and prevents liver injury in mice infected with candida albicans. fems immunol med microbiol 2007; 49: 330–6. 8. reznik da. perspective oral manifestation of hiv disease. topic in hiv medicine 2005; 13(5): 143–8. 9. munoz c, xavier m, tanaca lv, rodrigues ch. identification of candida spp by randomly amplified polymorphic dna and differentiation between c. albicans and c. dubliniensis by direct pcr methods. j clin microbiology 2003; 41(1): 414–20. 10. regina tl, asmara w. molecular typing of candida albicans isolated from oral cavity of cancer patients using randomly amplified polymorphic dna. indonesian journal of biotechnology 2001; 6: 470–9. 11. widodo i. penggunaan marka molekuler pada seleksi tanaman. disertation. bogor: program pascasarjana institut pertanian bogor; 2003. 12. faqih ar. optimasi analisis hasil keragaman genetik udang metapenaeus monoceros berdasarkan rapd dan rflp. thesis. malang: pascasarjana universitas brawijaya malang; 2004. 168 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 168–171 research report a gender-based comparison of intermolar width conducted at padjajaran university dental hospital, bandung, indonesia adriana azlan, endah mardiati and ida ayu evangelina department of orthodontics, faculty of dentistry, universitas padjadjaran, bandung – indonesia abstract background: evaluation of dental arches is important for both diagnosis and treatment in the fields of orthodontics, prosthodontics, and forensics. the perimeter or circumference affects the gender-specific dimensions of the dental arch. purpose: to identify the inter-gender difference between maxillary and mandibular intermolar width of the first molars in indonesia. methods: this retrospective and comparative analytical study involved a gender-based comparison of maxillary and mandibular intermolar width in the first molars. a purposive sampling technique was employed for data selection. ninety dental cast models were selected according to the inclusion criteria of non-growing patients and perfect dental conditions, with any damaged dental models being rejected. after selection, the dental cast was marked at the maxillary and mandibular first molar central fossae before being measured three times with a digital vernier caliper. the data obtained was subsequently analyzed by means of a kolmogorov-smirnov test, an f-test-snedecor (with p>0.05) and independent sample t-test (with p<0.05). results: the average maxilla intermolar widths for males and females were 49.36mm and 46.75mm respectively, while the average mandibular intermolar widths for males and females were 43.17mm and 40.5mm. an independent sample t-test showed that the maxilla and mandibular intermolar widths were significantly different for males and females (p=0.000, p<0.05), with male subjects possessing a higher value than female subjects. conclusion: a significant gender-based difference existed between the maxillary and mandibular intermolar width of patients attending padjadjaran university dental hospital, bandung, indonesia. keywords: malocclusion; orthodontic; sex correspondence: ida ayu evangelina, department of orthodontics, faculty of dentistry, universitas padjadjaran, jl. sekeloa selatan no.1, bandung 40132, indonesia. e-mail: ida.evangelina@fkg.unpad.ac.id introduction information regarding the arch dimension in human populations can be used for a variety of purposes. the width, length, and depth of dental arches have significant implications for orthodontic diagnosis and treatment planning which affect the available space, dental aesthetics, and dentition stability.1 the arch dimension may be also useful in the field of forensic dentistry since gender determination of skeletal remains constitutes a key element within forensic science.2,3 in prosthodontics, a significant aspect of denture manufacture is closely related to differences in the dimensions of dental arches.4 various landmarks employed in measuring the dental arch have previously been described and discussed by several researchers. for example, the various dental arch widths of contralateral teeth have been measured in numerous ways, such as the arch across the permanent canines, premolars and first molars, or at the cusp tips, central fossae, contact points and the largest distance between buccal surfaces.5 certain research has posited that the arch dimension is affected by gender, while other studies have identified no significant gender-based differences in arch dimensions.6 the contrasting results of these studies could be due to different landmarks, sample sizes, age groups, subject ethnicity, or investigative procedures. moreover, the fact that members of the various ethnic groups present different morphological conditions should induce clinicians to anticipate contrasts in size and form, rather than treating all cases in an identical manner.2,7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p168–171 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p168-171 169azlan, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 168–171 figure 1. a) upper intermolar width measured from the central fossa left m1 to the right m1; b) lower intermolar width measured from the central fossa left m1 to the right m1. there are numerous studies comparing intermolar width in the maxillary and mandibular teeth. however, to date, no study has been carried out to compare the difference in intermolar width of the maxillary and mandibular in indonesians, specifically among non-growing young adults. this knowledge can be used to develop a more comprehensive understanding of this period and enhanced diagnosis and treatment planning in the future. a greater comprehension of these aspects could also influence patient expectations regarding the formulation of treatment and subsequent retention plans by orthodontists. therefore, a comparison of intermolar width in the maxillary and mandibular of male and female students at padjadjaran university dental hospital, bandung, indonesia has been the focus of interest in further research. the objective of the present study is to investigate the difference between maxillary and mandibular intermolar width in the first molars of male and female students at padjadjaran university dental hospital, bandung, indonesia. materials and methods this study constitutes retrospective comparative analytical research where two distinct samples, in this case male and female students at padjadjaran university dental hospital, constituted the subjects of the investigation. the research samples were supplied by the orthodontic laboratory of padjadjaran university between january and march 2017. sample selection employed a purposive sampling technique.8 according to the central limit theorem (clt), a minimum of 30 samples is required in comparison studies. between 2013 and 2015, three batches of dental models were made available by the orthodontic laboratory of padjadjaran university. thus, a total of 90 samples was obtained in the three batches each containing 30 samples equally divided between 15 male and 15 female. in the preparation phase, an ethical letter (registration number 0217030350) was issued by hasan sadikin hospital, bandung and submitted together with a permission letter to the dental hospital administration department and the orthodontics department laboratory of padjadjaran university since this research utilized secondary data in collections. 94 dental models were obtained from the orthodontics department laboratory padjadjaran university and divided into male and female groups equal in size. all dental models selected were required to satisfy the inclusion criteria of dental models of non-growing patients aged between 18 and 24 years old. dental models should also be free of molar rotation, mesial dentition drifting, crowding at the molar region, edentulous regions, or sagittal and transversal discrepancies of molar tooth position. dental models were required to be symmetrical and possess a complete set of teeth. certain exclusion criteria were applied to dental models including; attrited, broken, or extracted teeth; agenesis; malocclusion; a history of orthodontic treatment; and adolescent individuals who were not universitas padjadjaran students. according to the previous study, several materials including pencils, erasers and digital vernier calipers were employed. the dental models were marked with a pencil at the maxillary and mandibular central fossae which acted as an intermolar width distance indicator.7,9,10 a digital vernier caliper (mitutoyo, japan) was used to measure the intermolar width distance according to the stated indicator (figure 1). an internal calibration technique was used during intermolar width measurement, whereby three measurements were taken on different days, to increase accuracy. the experiment was conducted three times on different days by undergraduate students (aa) during internal calibration and supervised by postgraduate teachers (em and ia). after the data had been collected and recorded, its analysis was initiated. the data obtained was analyzed using statistical package for social science (spss) version 20.0 software (student edition, ibm america), while the kolmogorov-smirnov test (p>0.05) was employed as a normality test, a f test–snedecor test (p>0.05) was used to assess homogeneity and an independent sample t-test (p<0.05) was used to quantify the differences in intermolar arch width between the genders. results the research sample was divided into three age groups. group one: patients aged 18-20 years old; group two: dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p168–171 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p168-171 170 azlan, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 168–171 49.36 ± 2.75 43.17 ± 2.92 46.74 ±2.62 40.5 ± 2.58 0 10 20 30 40 50 60 maxilla mandible male female figure 2. gender-based comparison of intermolar width in maxilla and mandible. table 1. result of normality test for maxillary and mandibular intermolar width in first molars among males and females gender result of normality test p-value distribution maxilla female 1,000 normal male 0,817 normal mandible female 0.826 normal male 0.763 normal table 2. result of independent sample t-test for maxillary and mandibular first intermolar width among male and female students at padjadjaran university dental hospital, bandung gender maxillary intermolar width mandibular intermolar width p-value mean sd mean sd female 46.74 2.62 40.50 2.58 0.000* male 49.36 2.75 43.17 2.92 0.000* note: *significance patients aged 20-22 years old; and group three: patients aged 22-24 years old. each group consisted of 30 patients; 15 males and 15 females. table 1 contains the results of a normality test for maxillary and mandibular intermolar width of the first molars of males and females. the results of a kolmogorovsmirnov test (p>0.05) indicated normal distribution across all groups. meanwhile, a homogeneity test using f test– snedecor (p>0.05) revealed that the homogeneity value of the maxillary intermolar width in males and females was 1.1, while the homogeneity value of mandibular intermolar width in both genders was 1.28. these results indicated that there was no significant difference between the samples. in other words, homogeneity prevailed. according to figure 2, the average maxilla intermolar widths for male and female were 49.36 ± 2.75 mm and 46.75 ± 2.62 mm respectively. the average of mandibular intermolar widths of males and females were 43.17 ± 2.92 mm and 40.5 ± 2.58 mm. an independent sample t-test showed that maxilla and mandibular intermolar width between male and female was significantly different (p = 0.000, p<0.05), with male subjects possessing larger values than their female counterparts. according to the contents of table 2, the average maxilla intermolar widths of males and females were 49.36 ± 2.75 mm and 46.75 ± 2.62 mm respectively. the average mandibular intermolar widths of males and females were 43.17 ± 2.92 mm and 40.5 ± 2.58 mm. independent sample t-test showed that the maxilla and mandibular intermolar widths of males and females were significantly different (p=0.000, p<0.05), while male subjects possessed a larger value than female subjects. discussion the study reported here found significant inter-gender differences in maxillary and mandibular intermolar width in indonesia, whereby that of males was greater than that of females. in this discussion, intermolar width in indonesian adults with normal occlusion had been compared with values from previous studies featuring various populations of both males and females from the same group. previous studies have demonstrated the intermolar width of taiwanese people to be wider than that of southern chinese people of both genders by approximately 1.3 mm. although both populations are of the same racial group, different locations and lifestyles produce contrasting results.5 ethnic diversity creates varied results of intermolar width values. environmental factors are equally important, whereas culture and human behavior enhance dental arch dimensions. for example, middle eastern, caucasian and asian people have a different range of intermolar width values. the maxilla intermolar width of iraqi males and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p168–171 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p168-171 171azlan, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 168–171 females are 45.14 mm and 43.46 mm respectively, while the average mandibular intermolar width is 40.76 mm for males and 39.55 mm for females. 1 the respective maxilla intermolar width of spanish males and females are 56.99 mm and 55.19 mm, while the mandibular intermolar width of males and females are 53.74 mm and 52.41 mm. 11 a previous study conducted by pondicherry (2014) cited the respective maxilla intermolar width of males and females to be 48.74 mm and 45.44 mm. in contrast, the mandible intermolar width of males and females were 42.45 mm and 39.53 mm respectively.3 the study from peshawar measured the maxilla intermolar width at the midpoint of the cervical region of the first molar and reported that the maxilla and mandible intermolar widths were 34.67 mm and 32.82 mm.12 another indian researcher reported the maxilla intermolar width of males and females being 53.36 mm and 49.5 mm, whereas the mandible intermolar width for males and females were 46.66 mm and 42.88 mm.13 similarly, another study reported that the maxilla intermolar width of males and females were 49.24 mm and 46.31 mm respectively.9 a study from another state of india, peshawar stated the maxilla intermolar width of males and females to be 47.37 mm and 44.29 mm, whereas the mandible intermolar width for males and females were 41.67mm and 38.06 mm respectively.10 the maxilla intermolar width of saudi arabian males and females were 45.38 mm and 43.42 mm respectively.14 the value of intermolar width in several earlier studies could not be compared with the present research because of the differences in sample selection, difference reference points, and measuring techniques. the results the authors of this article are similar to those of other previous studies which have also observed males to have wider intermolars than females. these findings might be because dental arch width lies on the basal bone which, in general, is larger in males than females, the same might apply to the dental arches. the above differences in results may be explained by several factors such as type of measurement and sample size used. given the variation of results across studies, the environmental and genetic factors may also appear to play an essential role in determining intermolar width, variety of sample size, mastication function and environmental factors such as nutrition.1,2 this study yielded a database about arch dimensions, especially intermolar width, in indonesians. gender considerations relating to space management of intermolar arch width can be considered as underpinning aesthetic considerations and stability in orthodontic treatment. another implication of gender-based intermolar differences can be applied to the manufacture of dentures. moreover, in the forensic field, intermolar arch width may be useful in determining gender from dental remains. the limitations of this study lie in the fact that the ethnicity of the patients included in the sample was restricted to javanese and sundanese and, as such, was not representative of the entire population of indonesia. a comprehensive study of indonesians from all regions across the country is necessary to yield more complete data. nevertheless, in conclusion, it is evident that a significant gender-based difference existed between maxillary and mandibular intermolar width among patients at padjadjaran university dental hospital, bandung, indonesia. references 1. al-taee zh. a comparison of arch width in adults with normal class i occlusion and adults with class ii division1 malocclusion in ramadi city. anb med j. 2012; 10(1): 75–80. 2. mohammad ha, hassan mia, hussain sf. dental arch dimension of malay ethnic group. am j appl sci. 2011; 8(11): 1061–6. 3. daniel mj, khatri m, srinivasan s v., jimsha vk, marak f. comparison of inter-canine and inter-molar width as an aid in gender determination: a preliminary study. j indian acad forensic med. 2014; 36(2): 168–72. 4. louly f, nouer pra, janson g, pinzan a. dental arch dimensions in the mixed dentition: a study of brazilian children from 9 to 12 years of age. j appl oral sci. 2011; 19(2): 169–74. 5. ling jyk, wong rwk. dental arch widths of southern chinese. angle orthod. 2009; 79(1): 54–63. 6. owais ai, abu alhaija es, oweis rr, al-khateeb sn. maxillary and mandibular arch forms in the primary dentition stage. oral health dent manag. 2014; 13(2): 330–5. 7. sitanggang m, boel t. mandibular morphology of the mongoloid race in medan according to age groups. dent j (maj ked gigi). 2018; 51(2): 81–5. 8. sudjana s. metoda statistika. 6th ed. bandung: tarsito; 2002. p. 182–3. 9. gupta j, daniel mj. crown size and arch width dimension as an indicator in gender determination for a puducherry population. j forensic dent sci. 2016; 8(3): 120–5. 10. rao gv, kiran g. sex determination by means of inter-canine and inter-molar widtha study in telangana population. asian pacific j heal sci. 2016; 3(4): 171–5. 11. paulino v, paredes v, cibrian r, gandia jl. dental arch changes from adolescence to adulthood in a spanish population: a crosssectional study. med oral patol oral cir bucal. 2011; 16(4): 607–13. 12. mushtaq n, tajik i, baseer s, shakeel s. intercanine and intermolar widths in angle class i , ii and iii malocclusions. pakistan oral dent j. 2014; 34(1): 83–6. 13. syed m, selarka b, tarsariya v. sexual dimorphism in permanent maxillary and mandibular canines and intermolar arch width: endemic study. j indian acad oral med radiol. 2015; 27(3): 405–11. 14. alkadhi oh, almahfouz sf, tokhtah ha, binhuwaishel la. dental arch dimensions in saudi adults. int j dent. 2018; 2018: 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p168–171 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p168-171 67 mauli banana stem extract application increased expression of nf-kb in traumatic ulcer healing maharani laillyza apriasari,1 retno pudji rahayu,2 and diah savitri ernawati3 1department of oral medicine, faculty of dentistry, universitas lambung mangkurat, banjarmasin indonesia 2department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, surabaya indonesia 3department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: a traumatic ulcer represents one of the most prevalent disorders affecting the oral cavity. ulceration of the oral cavity potentially results in secondary infection requiring topical medication which involves the use of antiseptics to accelerate wound healing. previous research has shown that mauli banana (musa acuminata) stem extract (mbse) contains bioactive material from terpenoid saponin present in ambon bananas. the terpenoid saponin in ambon banana stems will be captured by a g protein receptor in the macrophages, subsequently producing a protein kinase c that activates nuclear factor kappa beta (nf-kb). this increases both the activity and number of macrophages. purpose: to analyze the expression of nf-kb (p50) in traumatic ulcers as an effect of mbse. methods: a true experimental design with a post-test only control group. it involved 40 male rattus norvegicus strain rats as traumatic ulcer models divided into four groups: the negative control group administered gel, and the other treatment groups administered 25%, 37.5% and 50% ethanol extracts of mbse gel respectively. a biopsy was performed on days 3 and 5. the preparation was produced to analyze the expression of nf-kb (p50) by means of immunohistochemistry examination. results: there was a significant difference (p<0.05) in nf-kb (p50) expression (p=0.005) following mbse gel administration of 37.5% concentration on day 3 compared to day 5. conclusion: it can be concluded that mbse gel topical application can increase expression of nf-kb (p50) in traumatic ulcer healing. keywords: expression; mauli banana stem extract; nf-kb; traumatic ulcer; wound healing correspondence: maharani laillyza apriasari, department of oral medicine, faculty of dentistry, universitas lambung mangkurat, jl. veteran 128b, banjarmasin, indonesia. e-mail: maharaniroxy@gmail.com dental journal (majalah kedokteran gigi) 2018 june; 51(2): 67–70 research report introduction traumatic ulcers constitute one of the most prevalent disorders affecting the oral cavity resulting from trauma either physical (mechanical, thermal, electrical) or chemical (acid or base substances, spicy foods) in nature. their prevalence is relatively high as evidenced by several studies showing variations in incidence rates of between 3% and 24% within specific populations and locations.1,2 ulceration of the oral cavity can be potentially subject to secondary infection because of the numerous commensal microorganisms found in the oral cavity. hence, the need for application of antiseptic topical medication capable of accelerating wound healing to the oral cavity. at the time of writing, individuals suffering from this condition often use patent medicine containing aloe vera leaf extract. one of the antiseptic topical drugs used in the field of dentistry to treat oral ulceration, it is difficult to acquire outside java.3–5 previous research has shown that mauli banana stem extract (mbse) gel at a concentration of 25% produces no significantly different effect compared to other patent medicines containing aloe vera extract in terms of accelerating wound healing. this can be seen from the increased number of macrophage cells on day 3.5 the bioactive contents of mauli banana stem consist of 67.59% tannins, 14.49% saponins, 0.34% alkaloids, 0.44% ascorbic acid, 0.25% flavonoids and 0.006% lycopene. mbse is an antioxidant containing hydrogen peroxide and hydroxyl which stimulates heavy metal binding activity, in addition dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p67–70 mailto:maharaniroxy@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p67-70 68apriasari, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 67–70 to lowering malondildehyde (mda) and promoting super oxide dismutase (sod) activities and catalase in the healing process of rat oral mucosa.6,7 previous research has shown that banana stems contain a condensed tannin bioactive material and terpenoid saponins also found in ambon banana.8,9 in addition, mbse contains condensed tannins and terpenoid saponins common to other banana stems. terpenoid saponin in ambon banana stems constitutes an immunomodulator that can increase both the number and activity of macrophages. captured by the g protein receptor in macrophages, it activates nuclear factor kappa beta/nf-kb (p50) through a protein kinase c-yielding process, thereby increasing the number and activity of macrophages.10,11 based on the above statement, the mauli banana extract has the potential to accelerate wound healing as an immunomodulator through an increase in the number of macrophage cells by means of enhancing expression of nf-kb (p50). the purpose of this study is to analyze the expression of nf-kb (p50) in traumatic ulcers as an effect of mbse. materials and methods the materials used in this experiment were 100 gms of mauli banana stems, six liters of 70% ethanol, carbopol, hydroxypropyl cellulose medium (hpmc), propylenglycol, aquadest, aluminum foil, hydroxypropyl methylcellulose, banana stem, candy oil (cv. cahaya kimia), propylene glycol (brataco) and tween 80 (brataco). each gel composition of mauli banana ethanol extract at concentrations of 25%, 37.5%, and 50% respectively was added to 15% hpmc, 1% tween 80, 8% propylenglikol, five drops of candy oil and aquades making up the total weight. the other materials included chemical substances required for immunohistochemistry (xylol, ethanol, pbs, trypsin, alcohol, aquadesilata, streptavidin biotin, 0.5% h2o2, substrate and phosphotase buffer), monocloal anti-mouse antibody to nf-kb (p50) (e-10): sc-8414 (santa cruz biotechnology, inc.) and staining material of haematoxylin eosin (he). samples of the banana stems to be extracted were washed with running water and cut into small pieces, then dried in the oven at a temperature of 40–60 degrees for three days. when dehydrated, the pieces were smoothed in a blender and weighed again before extraction was carried out using maceration method. this involved soaking the extract in 750 ml of 70% ethanol for a period of 72 hours while occasionally agitating it. the resulting liquid was evaporated with a vacuum rotary evaporator at a temperature of 40–50°c until a thick extract was obtained which was subjected to ethanol-free examination. 25%, 37.5% and 50% concentrations of the extracts were subsequently made by means of hydroxypropyl methylcellulose (hpmc). this study received ethical clearance for experimentation on animal subjects (no. 56/kkepk.fkg/vi/2015) from the ethics research committee, faculty of dental medicine, universitas airlangga, surabaya, east java, indonesia. the research type constituted a true experimental study incorporating post-test only control group design using male, 300 gm, wistar strain rattus norvegicus as the models afflicted with a traumatic ulcer. the treatment was initiated with the inhalation of 0.75 ml of diethyl ether for 5–10 minutes to sedate each subject. the right buccal mucosa was then penetrated with a 6 mm diameter biopsy punch to a depth of 1 mm. from a clinical perspective, the traumatic ulcers induced in the subjects’ right buccal mucosa could be seen to have extended into the epithelial tissue, but not into the muscles. this study involved 40, male, wistar strain rattus norvegicus samples divided into a negative control group (k) given gel without mbse every 6–8 hours; a treatment group 1 (p1) given mbse gel of 25% concentration three times a day every 6–8 hours; treatment group 2 (p2) given mbse gel of 37.5% concentration three times a day every 6-8 hours and treatment group 3 (p3) given mbse gel of 50% concentration three times a day every 6–8 hours. the traumatic ulcer tissues of the left buccal mucosa of the subjects were removed for preparation, followed by imunohistochemical staining, in order to analyze nf-κb (p50) expression. results statistical analysis began with a saphiro wilk normality test and continued with a homogeneity test, the results of which both proved normal (p>0.05) and homogeneous (p>0.05). the analytical process continued with anova and post hoc lsd tests whose results are presented in table 1. there was a significant difference (p<0.05) in nf-kb expression (p=0.005) in the mbse gel at 37.5% concentration on day 3 compared to day 5. on day 3, there was a significant difference in nf-kb (p50) expression between the negative control and all treatments (mbse concentrations of 25%, 37.5% and 50%). a significant difference also existed in nf-kb (p50) expression between mbse gel of 25% concentration and all other groups, except with mbse gel of 37.5% concentration. on the same day, there was a significant difference in nf-kb expression between mbse gel of 50% and all other groups (see figure 1). following the application of mbse gel on day 5, there was a significant difference in nf-kb (p50) expression between the negative control group and all treatment groups (mbse concentrations of 25%, 37.5%, and 50%). on day 5, there was both a significant difference in nf-kb (p50) expression between mbse gel of 25% concentration and that of all other groups and between nf-kb (p50) expression and mbse gel concentration of 37.5% in all groups. on the same day there was a significant difference dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p67-70 69 apriasari, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 67–70 administration at all concentrations (25%, 37.5% and 50%) compared to the negative control group on day 3 and day 5. the mbse gel containing terpenoid saponin is absorbed by g protein receptors which then penetrate the cell membranes. the concentration level will depend on how many receptors bind to saponins. the number of receptors, the type of receptor bonds with the ligand and the receptor binding strength of the ligand also determine the concentration of drug required and the subsequent effect produced. the application of low concentrations of drugs will have a limited pharmacological effect. an increase in their concentration will strengthen the pharmacological effects until a maximum level is reached beyond which no further enhancement is possible.9,10 the increase in nf-kb expression will augment cells’ ability to avoid extermination through activation of c-jun n-terminal kinase (jnk). normally, signals from the jnk and erk map kinase path will initiate cell growth. jnk activity plays an important role in the migration of fibroblast cells within the wound healing process.11,12 nf-kb constitutes a family of transcription factors including regulators in the proinflammatory process and transcription of the antiapoptotic gene. this plays a role in the homoeostasis governing the host immune response. nf-kb is mediated by a transcription as the end of a series of reaction complexes initiated by several stimuli from cellular stresses in receptor involvement which mediate 8 anicals for wound healing activity. int res j pharm. 2012; 3(7): 1–7. figure 1. the immunohistochemistry examination results showed nfb (p50) expression in oral buccal mucosal tissue (brownish in the nucleus) in each group on day 3. notes: a. nfb expression in macrophage cells on administration of negative control b. nfb expression in macrophage cells on administration of mbse gel concentration of 25% c. nfb expression in macrophage cells on administration of mbse gel concentration of 37.5% d. nfb expression in macrophage cells on administration of mbse gel concentration of 50% a b c d a b figure 1. the immunohistochemistry examination results showed nf-kb (p50) expression in oral buccal mucosal tissue (brownish in the nucleus) in each group on day 3. notes: a. nf-kb expression in macrophage cells on administration of negative control b. nf-kb expression in macrophage cells on administration of mbse gel concentration of 25% c. nf-kb expression in macrophage cells on administration of mbse gel concentration of 37.5% d. nf-kb expression in macrophage cells on administration of mbse gel concentration of 50% table 1. means of nf-kb (p50) expression in macrophage cells of traumatic ulcer healing group day 3 day 5 p negative control 4.40 ± 1.34a 4.60 ± 1.52a 0.831 mbse 25% 7.20 ± 1.48b 8.00 ± 0.82b 0.369 mbse 37.5% 8.60 ± 1.14b 12.00 ± 1.58c 0.005* mbse 50% 12.60 ± 1.34c 14.80 ± 1.92d 0.069 p 0.000* 0.000* 0.000* notes: mbse = mauli banana stem extract * significant in α = 0,05. abcd the same superscripts showed no difference among groups. between mbse gel concentration of 50% and that of all groups. this can be seen in figure 2. this study showed that mbse gel at 50% concentration can increase the highest nf-kb (p50) expression in traumatic ulcers on days 3 and 5. however, mbse gel at 37.5% concentration provoked the highest nf-kb (p50) expression in traumatic ulcers from day 3 compared to day 5, as can be seen in table 1. discussion the results of this study confirmed an increase in the expression of nf-kb (p50) as the effect of mbse gel 8 anicals for wound healing activity. int res j pharm. 2012; 3(7): 1–7. figure 1. the immunohistochemistry examination results showed nfb (p50) expression in oral buccal mucosal tissue (brownish in the nucleus) in each group on day 3. notes: a. nfb expression in macrophage cells on administration of negative control b. nfb expression in macrophage cells on administration of mbse gel concentration of 25% c. nfb expression in macrophage cells on administration of mbse gel concentration of 37.5% d. nfb expression in macrophage cells on administration of mbse gel concentration of 50% a b c d a b 9 figure 2. the immunohistochemistry examination results showed nfb (p50) expression in oral buccal mucosal tissue (brownish in the nucleus) in each group on day 5. notes: a. nfb expression in macrophage cells on administration of negative control b. nfb expression in macrophage cells on administration of mbse gel concentration of 25% c. nfb expression in macrophage cells on administration of mbse gel concentration of 37.5% d. nfb expression in macrophage cells on administration of mbse gel concentration of 50% table 1. means of nfb (p50) expression in macrophage cells of traumatic ulcer healing. group day 3 day 5 p negative control a 4.60 ± 1.52a 0.831 mbse 25% 7.20 ± 1.48b 8.00 ± 0.82b 0.369 mbse 37.5% 8.60 ± 1.14b 12.00 ± 1.58c 0.005* mbse 50% 12.60 ± 1.34c 14.80 ± 1.92d 0.069 p 0.000* 0.000* 0.000* notes: mbse = mauli banana stem extract * significant in = 0,05. abcd the same superscripts showed no difference among groups. c d figure 2. the immunohistochemistry examination results showed nf-kb (p50) expression in oral buccal mucosal tissue (brownish in the nucleus) in each group on day 5. notes: a. nf-kb expression in macrophage cells on administration of negative control b. nf-kb expression in macrophage cells on administration of mbse gel concentration of 25% c. nf-kb expression in macrophage cells on administration of mbse gel concentration of 37.5% d. nf-kb expression in macrophage cells on administration of mbse gel concentration of 50% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p67-70 70apriasari, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 67–70 innate and adaptive immunity.13 nf-kb in eukaryotic microorganisms represents a family of transcription factors governing the expression of a large variety of genes involved in several processes such as inflammatory, immune, growth and cellular responses. nf-kb transcription factor is activated in response to various signals, including: cytokines, wounds and other stressful conditions. in a stimulated cell, nf-kb is bound to the ikb inhibition protein. the nf-kb and ikb compounds in the cytoplasm will prevent nf-kb from binding to dna. the activation of signaling on nf-kb is initiated by extracellular stimuli.14,15 in this research, mbse gel application was shown to influence the increase of signaling in nf-kb. in previous studies, astragalus plants containing terpenoid saponin were shown to increase nf-kb expression along with that of mrna from il-1k and tnf-k cytokines that produce effects as immunostimulators.16 furthermore, this will increase the number and activity of macrophages as in mbse gel application, a fact showing that mbse gel is also an immunostimulator. macrophages play an important role in wound healing because they produce growth factors and initiate angiogenesis and fibrogenesis. the macrophages released will exterminate bacteria prior to cleaning the tissue debris. in the transition from an inflammatory process to one of wound healing, macrophages stimulate cell migration, proliferation and tissue matrix formation. the growth factors promoting angiogenesis comprise tgfk, vegf and fgf-2.17,18 in a prevailing condition of macrophage deficiency, inhibition of wound healing then ensues. a balance between the number of neutrophils and macrophages in the wound healing process is required. in contrast to a chronic inflammatory state that inhibits wound healing, an excessive increase in neutrophil leads to a surplus of macrophages. this, in turn, causes severe tissue damage and long-term hypoxic conditions.19 previous research has shown that plants containing a n t i o x i d a n t s p o s s e s s t h e p o t e n t i a l t o b e c o m e immunomodulators which have immunostimulator and immunosuppressant effects on conditions influenced by the amount of extract concentration.16 mbse gel has antioxidant and immunostimulator properties at concentrations of 25%, 37.5% and 50%. it can be concluded, that mbse gel topical application can increase the expression of nf-kb (p50) in traumatic ulcer healing. acknowledgements the authors gratefully acknowledge the support provided to this research by the indonesian ministry of research, technology and higher education. references 1. cavalcante gm, de paula rjs, de souza lp, sousa fb, mota mrl, alves apnn. experimental model of traumatic ulcer in the cheek mucosa of rats. acta cirúrgica bras. 2011; 26(3): 227–34. 2. regezi ja, sciubba jj, jordan rck. oral pathology: clinical pathologic correlations. 7th ed. st. louis: saunders; 2016. p. 23–6. 3. majewska i, gendaszewska-darmach e. proangiogenic activity of plant extracts in accelerating wound healing a new face of old phytomedicines. acta biochim pol. 2011; 58(4): 449–60. 4. yadav kch, kumar jr, basha si, deshmukh gr, gujjala r, santhamma b. wound healing activity of topical application of aloe vera gel in experimental animal models. int j pharma bio sci. 2012; 3(2): 63–72. 5. puspitasari d, apriasari ml. analysis of traumatic ulcer healing time under the treatment of the mauli banana (musa acuminata) 25% stem extract gel. padjadjaran j dent. 2017; 29: 21–5. 6. apriasari ml, dachlan yp, ernawati ds. effect of musa acuminata stem by immunohistochemistry test in ulcer. asian j biochem. 2016; 11(3): 135–41. 7. apriasari ml, iskandar i, suhartono e. bioactive compound and antioxidant activity of methanol extract mauli bananas (musa sp) stem. int j biosci biochem bioinforma. 2014; 4(2): 110–5. 8. noora wf, apriantia n, saputra sr, afifah b, apriasari ml, suhartono e. oxidative stress on buccal mucosa wound in rats and rule of topical application of ethanolic extracts of mauli banana (musa acuminata) stem. j trop life sci. 2015; 5(2): 84–7. 9. prasetyo bf, wientarsih i, priosoeryanto bp. aktivitas sediaan salep ekstrak batang pohon pisang ambon (musa paradisiaca var sapientum) dalam proses persembuhan luka pada mencit (mus musculus albinus). majalah obat tradisional. 2010; 15(3): 121–37. 10. ghag sb, shekhawat uks, ganapathi tr. silencing of musaanr1 gene reduces proanthocyanidin content in transgenic banana plants. plant cell tissue organ cult. 2015; 121(3): 693–702. 11. besung i nk. pegagan (centella asiatica) sebagai alter natif pencegahan penyakit infeksi pada ternak. buletin veteriner udayana. 2009; 1(2): 61–7. 12. basori a, zakaria s, purwaningsih s, rochmanti m, setiawati y, indiastuti dn, qotib f, d’arqom a, purba akr. interaksi antar obat. surabaya: department of pharmacology and therapy, faculty of medicine, universitas airlangga; 2014. p. 46–9. 13. morgan mj, liu z. crosstalk of reactive oxygen species and nf-kb signaling. cell res. 2011; 21(1): 103–15. 14. wertz ie, o’rourke km, zhou h, eby m, aravind l, seshagiri s, wu p, wiesmann c, baker r, boone dl, ma a, koonin e v, dixit vm. de-ubiquitination and ubiquitin ligase domains of a20 downregulate nf-kb signalling. nature. 2004; 430: 694–9. 15. courtois g, gilmore td. mutations in the nf-kb signaling pathway: implications for human disease. oncogene. 2006; 25(51): 6831–43. 16. napetschnig j, wu h. molecular basis of nf-kb signaling. annu rev biophys. 2013; 42: 443–68. 17. mukherjee pk, nema nk, bhadra s, mukherjee d, braga fc, matsabisa mg. immunomodulatory leads from medicinal plants. indian j tradit knowl. 2014; 13(2): 235–56. 18. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219–29. 19. soni h, singhai ak. a recent update of botanicals for wound healing activity. int res j pharm. 2012; 3(7): 1–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p67–70 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p67-70 113113 dental journal (majalah kedokteran gigi) 2021 september; 54(3): 113–118 original article formula milk increases lactoferrin levels in 7–9 years old children luthfiani1, dwi suryanto2 and suzanna sungkar3 1postgraduate student, faculty of dentistry, universitas sumatera utara, medan – indonesia 2department of biology, faculty of mathematics and natural sciences, universitas sumatera utara, medan – indonesia 3department of paediatric dentistry, faculty of dentistry, universitas syiah kuala, banda aceh – indonesia abstract background: lactoferrin is known to have a bacteriostatic or bactericidal effect by binding ions in saliva to interfere with the survival of bacteria that need such ions, such as streptococcus mutants. lactoferrin is a whey protein and can be found in formula milk. purpose: this study aimed to analyse lactoferrin levels before and after consuming formula milk and sterilized milk. methods: this study was conducted on 22 students aged 7–9 years at public elementary school (sd negeri) 060817 using purposive sampling, with 11 students consuming formula milk and 11 students consuming sterilized milk. saliva was collected by the spitting method before and after consumption on the first and seventh days. examination of lactoferrin levels was done using the enzyme-linked immunosorbent assays (elisa). differences in lactoferrin levels in each group before treatment on day one and day seven were analysed using the friedman and analysis of variance (anova) tests. differences in lactoferrin levels between the formula milk and sterilized milk groups were analysed using the independent sample t-test and the mann-whitney test (p<0.05). results: the average levels of lactoferrin before consuming formula milk was 0.212 ± 0.034 mg/100ml and increased to 0.222 ± 0.036 mg/100ml and 0.315 ± 0.026 mg/100ml. in the sterilized milk group, lactoferrin levels increased from 0.216 ± 0.033 mg/100ml to 0.225 ± 0.032 mg/100ml and 0.235 ± 0.027 mg/100ml. the increase in lactoferrin levels was more significant in the formula milk group on the seventh day (p=0.001, p<0.05). conclusion: formula milk, which contains whey protein, has a high potential in increasing lactoferrin levels. keywords: dental caries prevention; formula milk; lactoferrin; sterilized milk; whey protein correspondence: luthfiani, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: luthfianismd.dentist@gmail.com introduction the growth and development of a child are influenced by several factors, one of which is the health of the oral cavity. dental caries is a common disease of the oral cavity in childhood.1 research in shimoga city-karnataka, india, showed the prevalence of caries in children aged 5–6 years, 9–10 years, and 14–15 years was 68.8%, 77.2%, and 48.9% respectively, and the overall prevalence of dental caries was 65.3%.2 meanwhile, the national dental health survey conducted by the national institute of health research and development through basic health research or riset kesehatan dasar (riskesdas) 2018 showed that the prevalence of caries in children aged 5–9 years in indonesia was 92.6%. the 5–9 years group showed the highest prevalence of caries among other age groups of children.3 an early clinical sign of caries is the presence of white spot lesions, which is enamel and dentin areas that are demineralized due to biofilms. several factors that interact causing the occurrence of caries are a vulnerable host (teeth and saliva), carbohydrate-rich diets that can ferment, the presence of dental plaque, as well as high amounts of cariogenic microorganisms such as streptococcus mutans and lactobacillus.4,5 saliva has many antibodies as a defence mechanism against infection, including antimicrobial proteins.6–8 lactoferrin is a non-enzymatic antibacterial protein formerly known as lactotransferrin, which is a transferrin group glycoprotein.6 it can sequester the iron content of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 mailto:luthfianismd.dentist@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 114 luthfiani et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 113–118 pathogenic bacteria, inhibiting its growth.8 some research has shown that lactoferrin is also capable of eliminating s. mutants through an independent mechanism of iron.8 iron free lactoferrin, called apolactoferrin, also shows antimicrobial properties through the direct binding of lactoferrin to bacteria and agglutination of s. mutans, thereby facilitating the removal of agglutinated bacteria through the mechanical action of salivary ingestion.9 previous studies also showed the antimicrobial capacity of iron-free lactoferrin (both in human and bovine lactoferrin) and bovine lactoferricin against the growth of some oral streptococcus. both human and bovine lactoferricin had a bacteriostatic effect that corresponds to the concentration of lactoferrin in the streptoccous species tested in the study.10 lactoferrin is also known to be present in whey, which can be found in milk.11,12 however, whey protein content in cow’s milk and formula milk differs significantly. cow’s milk contains 3.5% proteins, which is 80% casein and 20% whey.11,13 this milk can be further processed into different types, one of which is sterilized milk that has undergone a heating process of more than 100°c.14 this heating process can disrupt the stability of protein contents in the milk, including the whey protein, which will be denaturized at 70°c, affecting its solubility and functions.12,14,15 meanwhile, formula milk manufactured by the industry usually contains more whey protein to mimic human’s milk composition, with 40% casein and 60% whey protein.16 these differences in composition lead to an assumption that formula milk, which contains more whey protein than sterilized milk, will be more effective in increasing the lactoferrin levels in saliva. this study aims to analyse the effectiveness of formula milk consumption in increasing lactoferrin levels in saliva in children aged 7–9 years when compared to sterilized milk consumption. materials and methods the type of research used in this study is quasi-experimental with preand post-test control group design. this study was carried out in public elementary school 060817 jl. sakti lubis workshop no.19, medan and the integrated laboratory of the faculty of medicine, universitas sumatera utara, from december 2019 to january 2020. this study was approved by the research ethics commission, universitas sumatera utara (no. 950/tgl/kepk fk usu-rsup ham/2019). the inclusion criteria of this study were children aged 7–9 years old that have 4–9 carious teeth who were allowed by parents to join the research with written informed consent and are generally healthy both physically and mentally. we only included 7–9 years of age children to reduce the variability of dentition status among those included in the study. the exclusion criteria of this study are children who are currently or have been taking antibiotic drugs in the last month, have a systemic disease, use mouthwash or are allergic to milk. the sample size in this study was calculated using the sample size formula for hypothesis testing to detect the mean difference between two paired groups, referred to in a previous study.17 subjects in this study consisted of 22 children, which were later divided into two groups: 11 samples consuming formula milk as a test group and 11 samples consuming sterilized milk as a control group. the study subjects in each group were instructed to consume as much as one glass per day (±200 ml) of formula milk (blenuten®, ordesa, indonesia) and as much as one can per day (±189 ml) of sterilized milk (bearbrand®, nestle, indonesia) at 08.00 am (western indonesia time) for seven days under the supervision of the researchers. the procedures were carried out at this time to make sure that all the children had not had any snacks or beverages beforehand. subjects were asked not to eat or drink for an hour before consuming the formula or sterilized milk. the subjects were also instructed to brush their teeth at home twice a day, in the morning after breakfast and at night before bed. saliva collection was done one hour after consuming formula milk and sterilized milk on the first and seventh day. saliva collection was done by the spitting method.18 before saliva collection began, the subject was instructed to sit in a comfortable position and asked to swallow the saliva present in the oral cavity. then, the subjects were asked to collect their saliva with their lips closed for one minute and then spit it into the provided container. all containers were labelled and stored in an icebox with an ice pack. saliva samples were examined using human lactoferrin elisa kit (fine test, catalogue no. eh0396, wuhan fine biological technology co., ltd., wuhan, china) with the sandwich method and then read by the elisa reader (thermo scientific multiskan go, 100-240v, catalogue no. 51119200 and 51119250, thermo fischer scientific corporation, japan). the procedures were as follows: the plate was washed two times before adding the standard saliva, the sample and the control into the well. a total of 100μl saliva samples were added to each well and incubated for 90 minutes at a temperature of 37°c. then, an additional 100μl biotin-detection antibody solution was added, and the wells were incubated for 60 minutes at a temperature of 37°c. aspiration and rinsing were performed three times. a total of 100μl hrp-streptavidin conjugate (sabc) working solution was then added to each well and incubated for 30 minutes at a temperature of 37oc. aspiration and flushing were carried out five times. then, 90μl tmb substrate was added, and this was incubated for 15–30 minutes at a temperature of 37oc. finally, 50μl stop solution was added and a reading at 450nm wavelength was taken as soon as possible. the calculation of lactoferrin concentration in the sample was done by comparing the value with the standard curve. data analysis was performed using ibm spss statistics for windows, version 25 (new york, usa). the shapirodental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 115luthfiani et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 113–118 wilk test was used to determine the normality of the data. baseline data differences between the formula and sterilized milk groups were analysed with the independent sample t-test. differences in lactoferrin levels in the formula milk group before treatment on days one and seven were analysed first using friedman’s test, then with the wilcoxon test. differences in lactoferrin levels in the sterilized milk group before treatment on days one and seven were analysed using the anova test and then the bonferroni test. differences in lactoferrin levels in both groups were analysed using independent sample t-tests and mann-whitney tests. a p-value <0.05 was considered statistically significant. results most subjects in both groups were eight years old (68.2%). the least age characteristic in both groups was nine years old (4.5%). the overall characteristics of the study subjects by gender were 12 boys (54.5%) and 10 girls (45.5%). most subjects had nine carious teeth, which comprised six subjects (27.3%). in the formula milk group, two subjects (9.1%) had four carious teeth, one subject (4.5%) has five carious teeth, one subject (4.5%) had six carious teeth, two subjects (9.1%) had seven carious teeth, one subject (4.5%) had eight carious teeth and the remaining four subjects (18.2%) had nine carious teeth. the subjects in the sterilized milk group were two children with four carious teeth (9.1%), one child with five carious teeth (4.5%), one child with six carious teeth (4.5%), one child with seven carious teeth (4.5%), four children with eight carious teeth (18.2%) and two children with nine carious teeth (9.1%) (table 1). the average level of lactoferrin before consuming milk in the formula group was 0.212 ± 0.034 mg/100ml while in the sterilized milk group it was 0.216 ± 0.033 mg/100ml. normality test results showed p>0.05, thus the test was continued using an independent sample t-test. results showed that there was no significant difference in the lactoferrin levels between either group at baseline with (p = 0.782; p<0.05) (figure 1). there was an increase in the mean of lactoferrin levels in the formula milk group from 0.212 ± 0.034 mg/100ml before consumption, to 0.222 ± 0.036 mg/100ml on the first day and 0.315 ± 0.026 mg/100ml on the seventh day after consumption. data on lactoferrin levels in the formula milk group were not normally distributed and were analysed using friedman’s test and the wilcoxon test. the wilcoxon test results showed that the consumption of formula milk was effective in increasing lactoferrin levels on the first and seventh days. consumption of formula milk increased the lactoferrin levels significantly on the first (p=0.003; p<0.05) and seventh day (p=0.003; p<0.05) compared to the baseline (figure 1). examination of lactoferrin levels in sterilized milk groups showed an increase from 0.216 ± 0.033 mg/100ml before consumption to 0.225 ± 0.032 mg/100ml on the first day and 0.235 ± 0.027 mg/100ml on the seventh day after consumption. the data were normally distributed table 1. characteristics of study subjects characteristic formula milk sterilized milk total n % n % n % age 7 2 9.1 4 18.2 6 27.3 8 8 36.4 7 31.8 15 68.2 9 1 4.5 0 0 1 4.5 gender women 7 31.8 3 13.6 10 45.4 men 4 18.2 8 36.4 12 54.6 no. of caries 4 2 9.1 2 9.1 4 18.2 5 1 4.5 1 4.5 2 9.1 6 1 4.5 1 4.5 2 9.1 7 2 9.1 1 4.5 3 13.6 8 1 4.5 4 18.2 5 22.7 9 4 18.2 2 9.1 6 27.3 0.212 0.222 0.315 0.216 0.225 0.235 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 baseline h1 h7 la ct of er rin le ve ls (m g/ 10 0m l) formula milk sterile milk consumption time (days) figure 1. mean lactoferrin levels in formula milk and sterilized milk based on consumption time. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 116 luthfiani et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 113–118 and was analysed using the anova test followed by the bonferroni test. the results showed a significant increase in lactoferrin levels in the sterilized milk group after the first day of consumption (p=0.001; p<0.05) and at the seventh day (p=0.001; p<0.05) after consumption (figure 1). an increase in lactoferrin levels from the baseline to the first day was seen, both in formula milk and sterilized milk. however, figure 2 shows that the mean increase was higher in the formula milk group (0.0100 ± 0.0048 mg/100ml) compared to the sterilized milk group (0.0085 ± 0.0054 mg/100ml). the increase in lactoferrin levels from baseline to the seventh day was greater in the formula milk group (0.1027 ± 0.0215 mg/100ml) compared to the sterilized milk group (0.0189 ± 0.0122 mg/100ml). the increase in lactoferrin levels from the first day to the seventh day was also higher in the formula milk group (0.0926 ± 0.0205 mg/100ml) compared to the sterilized milk group (0.0104 ± 0.0111 mg/100ml). a normality test showed that data on the increase in lactoferrin levels from baseline to the first day and the baseline to the seventh day in the formula milk group were normally distributed p-value=0.847 and p=0.997; p>0.05 respectively, so the data was analysed using an independent samples t-test. normality test results on the first to seventh day showed that the data were not normally distributed, so the analysis was done using the mannwhitney test. the results showed that there was a significant difference in the increase of lactoferrin levels from baseline to the first day between the formula and sterilized milk group (p=0.496; p<0.05). there were also significant differences in the increase in lactoferrin levels from the baseline period to the seventh day (p=0.001; p<0.05) and the first day to the seventh day (p=0.001; p<0.05) in the two groups (figure 2). discussion lactoferrin levels before treatment in the formula milk group and the sterilized milk group show no significant difference. this agrees with the research conducted by moslemi et al.,6 which showed no significant differences in lactoferrin levels before treatment in overall samples. lactoferrin level increases with age, so its concentration is expected to be lower in children than adults.19 similar to the research conducted by moslemi et al.,6 this study also included samples with a narrow age range, so it can be assumed that lactoferrin levels between the two sample groups before treatment did not have significant differences. there was a significant increase in the mean lactoferrin levels from the baseline to the first and seventh days in the group consuming formula milk. lactoferrin levels were also seen to increase significantly in the group consuming sterilized milk. the results of this study showed the effect of milk consumption on increased levels of lactoferrin may occur due to the protein content in the milk used in this study. protein content in milk consists of various types, one of which is lactoferrin. the increase in lactoferrin levels in this study might be explained by the study conducted by ye et al.,20 which stated that after centrifugation in the sediment mixture of lactoferrin and heated saliva, there was an increase in lactoferrin content especially at ph 6.8 and ph 3.6. whereas in our study, we did not do salivary ph examinations, so the increase in lactoferrin levels in the saliva is assumed to be due to milk consumption. in this study, both formula and sterilized milk consumed by the children contained whey protein. whey protein in milk includes major and minor proteins, one of which is lactoferrin.21 thus, milk consumption is expected to increase lactoferrin levels in saliva. the immune system is divided into two categories, the innate immune system (natural or non-specific) and the adaptive immune system (specific). the innate immune system is a direct defence against infection when the host is attacked by pathogens (viruses, bacteria, fungi, or parasites). the innate immune system responds before the adaptive one. this type of immunity provides benefits for the body. when pathogens enter the body, the innate immune system controls the development of the incoming pathogens. the antibacterial activity of lactoferrin can be increased by natural factors of immunity.22 the increase in lactoferrin levels in this study is expected because milk consumption can increase immunity for the subject causing the lactoferrin levels to also increase. several studies were conducted to examine lactoferrin levels in saliva. some studies analysed lactoferrin levels with the decay, missing, and filled teeth index. lactoferrin levels in caries and caries-free children before and after treatment of their carious teeth showed there was no significant effect of the treatment performed on changes in lactoferrin levels. in contrast, the study conducted by felizardo et al. showed 0.01 0.1027 0.0926 0.0085 0.0189 0.0104 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 baseline – 1st day difference baseline – 7th day difference 1st day – 7th day difference formula milk sterile milk figure 2. comparison of increased lactoferrin levels between formula milk and sterilized milk. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 117luthfiani et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 113–118 significant changes in lactoferrin levels after treatment in the children with caries group.6,23 the results showed an increase in lactoferrin levels in both the formula and sterilized milk group, but from the mean value, we can conclude that a greater increase occurred in the formula milk group. the increase in lactoferrin levels in this group was likely due to the higher protein content in the formula milk than sterilized milk. the formula milk used in this study contained 100% whey protein.21 most studies, including our study, only investigate one protein at a time. the antimicrobial proteins in saliva are numerous and interact with each other in various ways. the interaction of these antimicrobial proteins can produce additive, synergistic or inhibitory effects. low concentration in one of these proteins can be compensated by other proteins with similar functions.6 study results on the sterilized milk group also showed an increased level of lactoferrin, but it was smaller than the formula milk group. the smaller increase of lactoferrin in the sterilized milk group was assumed to be due to the processing of the milk at 115-120○c for 20-30 minutes. processing milk at high temperatures and long duration can cause denaturation of lactoferrin and reduce its biological activities.22 this was in line with the previous study done by lin et al., which showed that whey protein content in milk processed at high temperatures was significantly reduced by more than 85% when compared to raw milk.24 the formula milk used in this study contained 100% whey protein, so it is reasonable to assume that the higher increases of lactoferrin level in the formula milk group were due to its higher whey protein content. dietary patterns and brushing habits in children were uncontrolled variables in this study. the increase in lactoferrin levels was assumed to be not only due to milk consumption but also influenced by other uncontrolled factors. appropriately consuming formula milk is one alternative to prevent dental caries. another limitation of this study is that we did not measure salivary ph, which can affect the lactoferrin level in saliva.20 based on the results of this study, it can be concluded that formula milk consumption is effective in increasing lactoferrin levels in saliva when compared to sterilized milk. formula milk consumption showed higher increases in lactoferrin levels at baseline to the first day and baseline to the seventh day when compared to the control group. it can be suggested to parents and the community to provide additional foods, such as formula milk containing whey protein, to children as an alternative option to prevent dental caries. it should be noted that the milk should be consumed in the proper way and timing, according to recommendations from doctors and dentists. schools are also expected to educate on the use of formula milk, especially those containing whey protein, as an alternative way to prevent dental caries. references 1. mcdonald re, avery dr, dean ja. dentistry for the child and adolescent. 8th ed. mosby, inc; 2004. p. 1–777. 2. soumya sg, shashibhushan kk, pradeep mc, babaji p, reddy vr. evaluation of oral health status among 5-15-year-old school children in shimoga city, karnataka, india: a cross-sectional study. j clin diagn res. 2017; 11(7): zc42–7. 3. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 1–384. 4. pitts nb, zero dt, marsh pd, ekstrand k, weintraub ja, ramosgomez f, tagami j, twetman s, tsakos g, ismail a. dental caries. nat rev dis prim. 2017; 3: 17030. 5. ayoub hm, gregory rl, tang q, lippert f. influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization. j appl oral sci. 2020; 28(317): 1–8. 6. moslemi m, sattari m, kooshki f, fotuhi f, modarresi n, khalili sadrabad z, shadkar ms. relationship of salivary lactoferrin and lysozyme concentrations with early childhood caries. j dent res dent clin dent prospects. 2015; 9(2): 109–14. 7. de andrade fb, de oliveira jc, yoshie mt, guimarães bm, gonçalves rb, schwarcz wd. antimicrobial activity and synergism of lactoferrin and lysozyme against cariogenic microorganisms. braz dent j. 2014; 25(2): 165–9. 8. lynge pedersen am, belstrøm d. the role of natural salivary defences in maintaining a healthy oral microbiota. j dent. 2019; 80 suppl 1: s3–12. 9. astuti esy, sukrama idm, mahendra an. innate immunity signatures of early childhood caries (ecc) and severe early childhood caries (s-ecc). biomed pharmacol j. 2019; 12(3): 1129–34. 10. roseanu a, florian p, condei m, cristea d, damian m. antibacterial activity of lactoferrin and lactoferricin against oral streptococci. rom biotechnol lett. 2010; 15(6): 5788–92. 11. gupta c, prakash d. therapeutic potential of milk whey. beverages. 2017; 3(4): 31. 12. de wit jn. lecturer’s handbook on whey and whey products. brussels: european whey products association; 2001. p. 1–91. 13. yalçin as. emerging therapeutic potential of whey proteins and peptides. curr pharm des. 2006; 12(13): 1637–43. 14. barraquio vl. which milk is fresh? int j dairy sci process. 2014; 1(201): 109–14. 15. deeth h, lewis m. protein stability in sterilised milk and milk products. in: mcsweeney plh, o’mahony ja, editors. advanced dairy chemistry. 4th ed. new york, ny: springer; 2016. p. 247–86. 16. prosser cg. compositional and functional characteristics of goat milk and relevance as a base for infant formula. j food sci. 2021; 86(2): 257–65. 17. siddiqui m, singh c, masih u, chaudhry k, deepa hegde y, gojanur s. evaluation of streptococcus mutans levels in saliva before and after consumption of probiotic milk: a clinical study. j int oral heal. 2016; 8(2): 195–8. 18. bellagambi fg, lomonaco t, salvo p, vivaldi f, hangouët m, ghimenti s, biagini d, di francesco f, fuoco r, errachid a. saliva sampling: methods and devices. an overview. trac trends anal chem. 2020; 124: 115781. 19. ide m, saruta j, to m, yamamoto y, sugimoto m, fuchida s, yokoyama m, kimoto s, tsukinoki k. relationship between salivary immunoglobulin a, lactoferrin and lysozyme flow rates and lifestyle factors in japanese children: a cross-sectional study. acta odontol scand. 2016; 74(7): 576–83. 20. ye a, streicher c, singh h. interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low ph. j dairy sci. 2011; 94(12): 5842–50. 21. niaz b, saeed f, ahmed a, imran m, maan aa, khan mki, tufail t, anjum fm, hussain s, suleria har. lactoferrin (lf): a natural antimicrobial protein. int j food prop. 2019; 22(1): 1626–41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 118 luthfiani et al./dent. j. (majalah kedokteran gigi) 2021 september; 54(3): 113–118 22. janeway ca, travers p, walport m, shlomchik m. immuno biology, the immune system in health and disease. 5th ed. new york: garland publishing; 2001. p. 1–884. 23. felizardo kr, gonçalves rb, schwarcz wd, poli-frederico rc, maciel sm, de andrade fb. an evaluation of the expression profiles of salivary proteins lactoferrin and lysozyme and their association with caries experience and activity. rev odonto ciência. 2010; 25(4): 344–9. 24. lin s, sun j, cao d, cao j, jiang w. distinction of different heattreated bovine milks by native-page fingerprinting of their whey proteins. food chem. 2010; 121(3): 803–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i3.p113–118 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i3.p113-118 141 volume 47, number 3, september 2014 characterization of lactoferrin in gingival crevicular fluid of chronic periodontitis patient sisca meida wati,1 istiati1 and pratiwi soesilawati2 1department of oral pathology and maxillofacial 2department of oral biology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: human periodontal diseases are inflammatory disorders as the result of complex interactions between periodontopathogens and the host’s immune response. periodontitis results in tooth loss and can even lead to systemic diseases if not treated. gingival crevicular fluid (gcf) reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamated sites. polymorphonuclear leukocyte (pmns) infiltration can be seen in each stage of periodontitis. lactoferrin is one of the pmn specific granules and could be a useful marker of pmn activity. purpose: the aim of this study was to determine the band intensity of lactoferrin used as periodontitis biomarker. methods: gingival crevicular fluid (gcf) samples were collected using paper point no.30 from 40 subjects, 30 periodontitis patients that devide according to the severity (10 mild periodontitis, 10 moderate periodontitis, and 10 severe periodontitis) and 10 healthy controls, ranging in ages from 20 to 35 years. gcf lactoferrin was analyzed by western blot and measured the band intensity by quantity one software (bio-rad). results: the periodontitis sites exhibited significantly greater band intensity of lactoferrin than healthy sites. the band intensity of lactoferrin was positively correlated with the severity of periodontitis (α = 0.05). conclusion: the study showed that the intensity of the lactoferrin protein bands could be used as biomarkers of periodontitis. key words: gingival crevicular fluid, lactoferrin, periodontitis, band intensity abstrak latar belakang: penyakit periodontal adalah gangguan inflamasi yang merupakan hasil dari interaksi yang kompleks antara periodontopathogens dan respon imun host. periodontitis mengakibatkan hilangnya gigi dan bahkan dapat menyebabkan penyakit sistemik jika tidak diobati. cairan sulkus gingiva (gcf) mencerminkan kondisi gingiva dan mengandung protein yang tertransudasi dari serum atau sel pada lokasi radang. infiltrasi polymorphonuclear leukosit (pmn) dapat dilihat pada setiap tahap periodontitis. laktoferin adalah salah satu granula spesifik pmn dan bisa menjadi indicator aktivitas pmn. tujuan: tujuan penelitian ini adalah untuk meneliti intensitas band laktoferin dapat sebagai biomarker periodontitis. metode: cairan sulkus gingiva (gcf) dari tiap sampel dikumpulkan menggunakan paper pint no. 30 dari 40 subjek, 30 pasien dengan periodontitis yang dibagi sesuai dengan tingkat keparahan (10 periodontitis ringan, 10 periodontitis moderat, dan 10 periodontitis parah) dan 10 kontrol, mulai usia 20-35 tahun. gcf lactoferrin dianalisis dengan western blot dan diukur intensitas bandnya dengan quantity one software (bio-rad). hasil: pada jaringan yang mengalami periodontitis menunjukkan intensitas band yang secara signifikan lebih besar dari laktoferin daripada periodontal yang sehat. intensitas band laktoferin berkorelasi positif dengan tingkat keparahan periodontitis (α = 0,05) simpulan: hasil penelitian ini menyimpulkan bahwa intensitas band protein laktoferin dapat digunakan sebagai biomarker periodontitis. kata kunci: cairan sulkus gingiva, laktoferin, periodontitis, intensitas band correspondence: sisca meida wati, c/o: departemen patologi mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: kotakpos.sisca@gmail.com research report 142 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 141–145 introduction periodontitis is a periodontal disease due to the development of the inflammatory process in the gingival tissue that continues to the other support tissues.1 bacterial biofilms are regarded to be the primary aetiological factor in the initiation of gingival inflammation and subsequent destruction of periodontal tissues and three major specific pathogens have been repeatedly identified as etiologic agents, namely aggregatibacter (actinobacillus) actinomycetemcomitans (aa), porphyromonas gingivalis (pg) and tannerella forsythia (tf). although chronic exposure to bacteria and their products is a prerequisite for gingival inflammation and periodontal tissue destruction to occur, the major causative factor of softand hardtissue breakdown associated with periodontitis is currently attributed to the host’s immune-inflammatory response to bacterial challenge. furthermore, the nature of the inflammatory response might determine the destructive character of the disease.2 porphyromonas gingivalis is one of the major pathogens in chronic periodontitis. p. gingivalis has a number of virulence factors such as capsule, fimbriae, lipopolysaccharide (lps) and potent proteolytic enzymes such as gingipains. these factors can induce an inflammatory cascade involving pro-inflammatory cytokines, reactive oxygen species and matrix metalloproteinases (mmps), thus leading to the destruction of supportive soft and hard tissues around the teeth.3 the movement of leukocytes from the blood to the local tissue is the key in process of inflammation. transendothelial migration is a selective interaction between leukocytes and the endothelium resulting in leukocytes out of the bloodstream into the tissues. local damage trigger a variety of inflammatory signals (il-1 β, tnf-α), mainly derived from resident leukocytes such as mast cells. mast cells are an important part which triggered the mobilization of pmn against bacteria and response to anaphylatoxins such as c3a and c5a.1 pmn acts as a barrier between the mass of plaque and crevicular epithelium.4 a diagnosis of periodontitis is established by traditionally used indices, e.g., bleeding on probing and probingdepth, which indicate the loss of periodontal tissue attachment to the teeth. additionally, radiographyvisualizes the loss of periodontal tissue, which supports the diagnosis by determining the amount of bone lossaround the teeth. however, these methods are only useful when attachment loss has occurred to somedegree. for chair-side and single visits, more reliable biomarkers for periodontitis are needed to provide constant classification and customized treatment and for monitoring periodontal diseases.5 gingival crevicular fluid (gcf) is bodily fluid that reflects the condition of the periodontium, contains various components that originate by transudation of the serum or inflammatory factors and are derivedfrom the interaction between the bacterial biofilm and the cells of periodontal tissues. additionally,despite a large variation, the amount of gcf tends to increase with the severity of gingival inflammation. in addition to these characterizations, the simple and noninvasive collection of gcf is required for the discovery of periodontitis biomarkers.5,6 lactoferrin is a glycoprotein with a molecular weight of about 80 kda, which shows high affinity for iron, and classified as a member of the transferrin family, due to its 60% sequence identity with serum transferrin.the ability to keep iron bound even at low ph is important, especially at sites of infection and inflammation where, due to the metabolic activity of bacteria, the ph may fall under 4.5. in such a situation lactoferrin also binds iron released from transferrin, which prevents its further usage for bacterial proliferation.7 lactoferrin can be found in saliva and crevicular fluid and acts bacteriostatic and bactericid. in the crevicular fluid significant higher levels of lactoferrin have been found at periodontitis sites in comparison to healthy sites.6 lactoferrin is one of the pmn specific granules and could be a useful marker of pmn activity.increased lactoferrin levels have been reported in severe infections, for example, meningitis, in burns patients, rheumatoid arthritis, and chronic salivary gland diseases. there are few reports about the quantification of lactoferrin in gcf. lactoferrin of gingival crevicular fluid levels in gingivitis, periodontitis, and localized aggressive periodontitis patients were two-fold higher than in periodontally healthy individuals. gingival crevicular fluid production is a result of the increase in permeability of microvasculatures of the periodontal tissue due to inflammatory reactions. lactoferrin released from pmns into the gcf is a good indicator of periodontal inflammation.8 the objective of this study was to determine the band intensity of lactoferrin used as periodontitis biomarker. materials and methods gcf samples were collected from periodontitis patients and healthy individuals for the identification of periodontitis biomarkers at the department of periodontology, faculty of dental medicine universitas airlangga. the standard protocol was approved by the kkepk (komisi kelaikan etik penelitian kesehatan) of faculty of dental medicine universitas airlangga (kkepk no. 151/kkepk.fkg/ x/2013). informed consent was obtained from all donors. forty male patients in range of age 20 to 45 years old participated in the study. all participants were free of systemic disease and had not taken medication (such as anti-inflammatory agents, antibiotics, immunosuppressants, or contraceptives) that could affect their periodontal status for at least six months prior to the study. radiographic examination and clinical periodontal assessment were performed. to stratify chronic periodontitis patients, the clinical periodontal parameters from average 6 sites per individual were assessed at the initial examination for mean probing depth (pd), clinical attachment loss 143wati, at al.: characterization of lactoferrin in gingival crevicular fluid (cal), and bleeding on probing (bop). according tothe criteria, we divided the samples into 4 groups, healthy, mild periodontitis, moderate periodontitis, and severe periodontitis based on clinical attachment loss (cal), healthy subjects with no cal, mild periodontitis with 1-2 mm of cal, moderate periodontitis with 3-4 mm of cal, and severe periodontitis with more than 5 mm of cal. gcf was taken from the tooth at posterior region of maxilla with the deepest pocket. each tooth was issolated with a cotton roll and the supragingival plaque was carefully removed without touching the marginal gingiva. the gingiva was then gently dried with an air syringe. gingival crevicular fluid was collected using paper point no. 30 at the orifice of the sulcus 1-2 mm subgingivally for 30 seconds. the same procedure was repeated after 1 minute. the paper points werethen stored in 100 μl pbs (ph 7.4) and then stored at -40° c until further analysis the entire sample were calibrated for their total protein concentration using a nano drop before sds-page analysis. sds-page analysis performed to separate proteins based on molecular weight. after sds-page analysis, proteins in the gel were then transferred onto nitrocellulose (nc) membranes. nc membranes were incubated in primary antibody (anti-lactoferrin antibody) at a ratio of 1: 1000 in 1% skim milk solution overnight, then washed with pbst. after the membranes were incubated in secondary antibody labeled with alkaline phosphatase which has a ratio of 1: 2500 for 1 hour, then washed with pbst. the membranes were incubated with western blue substrate solution in the dark until visible color bands or overnight, then washed with distilled water. then the membranes were dried at room temperature. the membranes were documented, furthermore, each image is analyzed to calculate the intensity of bands with quantity one software. data analysis was performed using one-way anova. the correlation among the intensity of lactoferrin band and the severity of chronic periodontitis were assessed using tukey hsd test. results figure 1 showed that protein bands were thicker with a higher color intensity than others. the increase of lactoferrin intensity was shown in figure 2. the mean of the control group was higher than the group with mild periodontitis, moderate and severe, so it can be seen that there is an intensity increase at each level of periodontitis severity (table 1). figure 1. westernblot result of lactoferrin; m = marker; co = kontrol; mi = mild; mo = moderate; se = severe. figure 2. the increase of lactoferrin intensity. table 1. lactoferrin bands intensity of gingival crevicular fluid from each severity of chronic periodontitis no control (int/mm2) mild periodontitis (int/mm2) moderate periodontitis (int/mm2) severe periodontitis (int/mm2) x 12,22* 12,78 13,29 13,87 sd 0,25 0,17 0,2 0,18 *data presented as mean ± sd 144 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 141–145 discussion the role of bacteria in periodontal disease progression has been widely recognized, but in reality the innate immune systems were more responsible for damage to bones, and ligaments that support the teeth and gingiva. initiation of bacteria causing pmn migrate to the site of infection. polymorphonuclear leukocytes (pmn) penetrate the connective tissue into the gingival sulcus through intercellular junctional epithelium,9 and are responsible for detecting and eliminating bacteria. polymorphonuclear leukocytes loaded with enzymes that have a high toxicity called protease. this enzyme is also responsible for the damage caused during a prolonged inflammatory phase that occurs when pmn become hyperactive or chronically activated by bacterial stimulus.10excessive host response may lead to tissue damage that occurs depends on the length of pmn in the tissue. the existence of pmn in the tissue for a long time can lead to tissue damage.9 during phase active of periodontal disease, cell death occurs and intracellular content are released. cell death through apoptosis is essential for maintaining the function and integrity of the tissue. apoptotic cells can help resolve inflammation by sending a signal to the macrophages to engulf pmn and secrete anti-inflammatory cytokines, such as tgf-β. if the pmn did not undergo apoptosis, the number and activity of macrophage will also decrease, so that pmn will continue to release the destructive contents. thus, in addition to killing bacteria, pmn can continue to release products that can cause direct damage to the extracellular matrix and other host cells.9 cytokines such as tnf-α, monocytecolony granulocyte stimulating factor (gm-csf) may cause a delay of pmn apoptosis by increasing the stability of its mitochondria, decrease the activity of caspase 3 and downregulated gene expression of bax which is a pro-apoptotic member of the bcl-2. recent studies also indicate that the products of bacteria isolated from strains of porphyromonas gingivalis can also delay pmn apoptosis.11 pmn is the largest source of lactoferrin in adults. lactoferrin is mostly stored in the specific granules (secondary granules), but can also be found in tertiary granules although in lower concentrations.7,8 pmn go to the site of infection in response to chemotactic molecules that are activated by bacteria, such as complement proteins, interleukin-1, and interleukin-8. bacteria are recognized and engulfed by pmn into the phagosome. phagosome membranes fused with granular cytoplasmic membrane, resulting in release of granule contents (degranulation) into the phagosome and the extracellular environment to kill bacteria.9 lactoferrin is found in the saliva and crevicular fluid, and acts bacteriostatic and bakteriosid.6 in this study, the presence of lactoferrin derived from the saliva have been eliminated at the time of sampling, so that lactoferrin were identified only from the gcf. iron levels in gcf increased in a periodontal disease, iron can increase oxidative stress by catalyzing the formation of ros through the fenton reaction and increase the growth of certain periodontopatogen, so that increased iron in gcf can be harmful to periodontal tissues. iron is important for the survival of the bacteria. lactoferrin binds to iron to prevent the progression of fenton reaction, so the increased levels of gcf lactoferrin in periodontitis may act as an antimicrobial agent and antioxidant preventive.12 gingival crevicular fluid was taken from male patients with chronic periodontitis, do not have a systemic disorder and nonsmokers. the determination of these criteria due to hormonal influences. sex differences have been reported to affect the body’s response to bacteria.13smoking is also associated with the etiology and pathogenesis of periodontal disease, as well as to the results of periodontal treatment.14 smoking also has a detrimental effect on the survival and function of pmn in gcf in populations with healthy periodontal.15 in this research, lactoferrin in gingival crevicular fluid was characterize using western blot method. previous research has shown a positive relationship between the titers of lactoferrin periodontitis in gcf. according to kivadasannavar et al.,16 a statistically significant difference exists between the lf levels before and after calling and root planning and periodontal flap surgery. a correlation has been found between the lactoferrin levels in gcf and the treatment modalities carried out. that result is in accordance with the investigation done by jentsch et al.,6 who conclude that there is a decrease in lactoferrin found in gcf of periodontitis patients during periodontal therapy. another study conducted by glimvall et al.,7 concluded that higher levels of lactoferrin were detected in subjects with chronic periodontitis and correlated with probing pocket depth ≥ 6 mm. although some data have been raised regarding the titers of lactoferrin in gcf of periodontitis patients, but this study is the first to reveal the relationship between the intensity of the band lactoferrin in the gcf of patients with periodontitis severity. the band is formed because there is a specific reaction between antigen with antibody. primary antibodies used in this study is a monoclonal antibody lactoferrin, so that antigen-specific binding protein lactoferrin. the specific proteins can be used for serological diagnosis by conducting further tests to obtain high specificity and sensitivity.18 the calculations using quantity one show that there is an intensity increase of lactoferrin bands in accordance with the increase in periodontitis severity. in the control group appeared lactoferrin band with low intensity. this indicates that the gingival crevicular fluid of healthy periodontal tissues also contained lactoferrin in low concentrations, as in the human oral cavity also contained a constant amount of bacteria in a controlled. continuous influx of pmn into the gingival pockets surrounding periodontal tissues play a major role in the control,10 whereas in the group with periodontitis, lactoferrin band intensity thicken with 145wati, at al.: characterization of lactoferrin in gingival crevicular fluid increasing severity of periodontitis. this is in accordance with the opinion of moosani which states that the pmn was found in the pocket of clinically healthy gingival tissue, and showed an increase in the number of chronic inflammation.9 anova showed a significant difference in lactoferrin band intensity. according kusnoto, pure isolates and a good levels of protein homogenates will produce a good and clear protein bands. there is a dominant protein bands with bold colors and there is expressed by the form of a thin bands. this is due to the good level of purity and protein concentration were sufficient. this means that there are differences in the concentration of lactoferrin in any degree of periodontitis severity.20 tukey test results indicate that there are significant differences between each group of samples. severity increase of chronic periodontitis followed by an increase in the intensity of lactoferrin bands. the increased of band intensity indicates an increase in the concentration of lactoferrin that contained in gingival crevicular fluid of each sample. this illustrates that the increase in the chronic periodontitis severity affects the concentration of lactoferrin in gingival crevicular fluid. these results are in accordance with the opinion of adonogianaki et al.14 and ozdemir et al.19 that there are different levels of lactoferrin in gcf significantly between patients with gingivitis and periodontitis compared with healthy subjects. tsai et al.8 also revealed that there is a strong relationship between periodontal clinical parameters with levels of lactoferrin. increased severity of periodontal inflammation followed by an increase in gcf lactoferrin. similar results have also been reported that myeloperoxidase derived from pmn associated with gcf volume, in addition to the β-glucorodinase also has a relationship with periodontal pocket depth. it may reflect a decrease in neutrophil chemotactic factor in the pocket due to the elimination of plaque and periodontal inflammation.14 gcf production is the result of an increase in microvascular permeability due to periodontal tissue inflammatory reaction. lactoferrin that is released from pmn into the gcf could be a usefulmarker of periodontal inflammation. therefore lactoferrin levels in gcf is a sensitive and objective method for detecting the severity of periodontitis.the results of this study showed that the intensity of the lactoferrin bands could be used as a biomarker of periodontitis. further research needs to be directed towards the developing a chairside diagnostic kit to collect and evaluate the concentration of lactoferrin in the clinic. references 1. newman mg, takei hh, carranza fa, klokkevold pr. clinical periodontology. 10th ed. philadelphia: elsevier saunders; 2006. p. 494. 2. gemmell e, yamazaki k, seymour gj. destructive periodontitis lesions are determined by the nature of the lymphocytic response. crit rev oral biol med 2002; 13(1): 17-34. 3. kuula h, salo t, pirila e, tuomainen am, jauhiainen m, uitto vj, tjaderhane l, pussinen pj, sorsa t. local and systemic responses in matrix metalloproteinase 8-deficient mice during porphyromonas gingivalis-induced periodontitis. infect immun 2009; 77(2): 8509. 4. wilson tg, kornman ks. fundamentals of periodontics. slovakia: quintessence publishing co. inc; 2003. p. 160-6. 5. choi yj, heo sh, lee jm, cho jy. identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid. proteome science 2011; 9: 42. 6. jentsch h, sievert y, go¨cke r. lactoferrin and other markers from gingival crevicular fluid and saliva before and after periodontal treatment. j clin periodontol 2004; (31): 511–4. 7. adlerova l, bartoskova a, faldyna m. lactoferrin: a review. veterinarni medicina 2008; 53(9): 57–468. 8. tsai cc, kao cc, chen cc. gingival crevicular fluid lactoferrin levels in adult periodontitis patients. aust dent j 1998; 43(1): 40-4. 9. moosani a. evaluation of oral neutrophil levels as a quantitative measure of periodontal inflammatory load in patients with special needs. thesis. toronto: department of paediatric dentistry faculty of dentistry university of toronto; 2012. 10. glogauer m. neutrophil research: the future of periodontal diagnosis and treatment. peak of royal college of dental surgeon ontario; 2007. p. 1-8. 11. gamonal j, sanz m, o’connor a, acevedo a, suarez i, sanz a, martı´nez b, silva a. delayed neutrophil apoptosis in chronic periodontitis patients. j clin periodontol 2003; 30(7): 616–23. 12. wei pf, ho ky, ho yp, wu ym, yang yh, tsai cc. the investigation of glutathione peroxidase, lactoferrin, myeloperoxidase and interleukin-1b in gingival crevicular fluid: implications for oxidative stress in human periodontal diseases. j periodont res 2004; 39: 287–93. 13. kowolik mj, dowsett sa, rodriguez j, de la rosa rm, eckert gj. systemic neutrophil response resulting from dental plaque accumulation. j periodontol 2001; 72(2): 146-51. 14. ozdemir b, ozcan g, karaduman b, teoman ai, ayhan e, ozer n, us d. lactoferrin in gingival crevicular fluid and peripheral blood during experimental gingivitis. eur j dent 2009; 3(1): 16-23. 15. guntsch a, erler m, preshaw pm, sigusch bw, klinger g, glockmann e. effect of smoking on crevicular polymorphonuclear neutrophil function in periodontally healthy subjects. j periodontal res 2006; 41(3): 184-8. 16. kivadasannavar av, pushpa sp, sanjeevini ah, soumya sk. estimation of lactoferrin levels in gingival crevicular fluid before and after periodontal therapy in patient with chronic periodontitis. contemp clin dent 2014; 5(1): 25-30. 17. glimvall p, wickstrom c, jansson h. elevated levels of salivary lactoferrin, a marker for chronic periodontitis?. j periodont res 2012; 47(5): 655-60. 18. kresno sb. imunologi: diagnosis dan prosedur laboratorium. edisi kelima. jakarta: fakultas kedokteran universitas indonesia; 2010. p. 508. 19. adonogianaki e, moughal na, kinane df. lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases. j clin periodontol 1993; 20(1): 26-31. 20. kusnoto. karakterisasi molekular protein toxocara cati dan toxocara canis untuk pengembangan diagnostik toxocariasis. disertasi. surabaya: program pascasarjana universitas airlangga; 2008. 168 volume 47, number 3, september 2014 pelepasan ion nikel dan kromium kawat australia dan stainless steel dalam saliva buatan (the release of nickel and chromium ions from australian wire and stainless steel in artificial saliva) nolista indah rasyid, pinandi sri pudyani dan jcp heryumani departemen ortodonsia fakultas kedokteran gigi universitas gajah mada yogyakarta-indonesia abstract background: fixed orthodontic treatment needs several types of wire to produce biomechanical force to move teeth. the use orthodontic wire within the mouth interacts with saliva, causing the release of nickel and chromium ions. purpose: the study was aimed to examine the effect of immersion time in artificial saliva between special type of australian wire and stainless steel on the release of nickel and chromium ions. methods: thirty special type australian wires and 30 stainless steel wires were used in this study, each of which weighed 0.12 grams. the wires were immersed for 1, 7, 28, 35, 42, and 49 days in artificial saliva with a normal ph. the release of ions in saliva was examined using atomic absorption spectrophotometry. results: the result indicated that the release of nickel ions on special type of australian wire was larger than that on stainless steel wire (p<0.005), there were differences in the release of the amount of nickel ions on special type of australia in different immersion time, and there was a correlation between the types of wire and immersion time. nickel ions released from the special type of australian wire detected on the 7th day of immersion and reached its peak on the 35th day, while from stainless steel wire were detected on the 49th day of immersion. the released of chromium ions from the special type of australian wire and stainless steel wire were not detected until the 49th day of immersion. conclusion: the release of nickel ions were highest on the 35th day of immersion in special type of australian wire and they were detected on the 49th day in stainless steel wire. the release of chromium ions were not detected until 49th day of immersion in special type of australian and stainless steel wire. key words: australian wire, stainless steel, immersion time, nickel ion, chromium ion abstrak latar belakang: perawatan ortodonti cekat memerlukan beberapa macam kawat untuk menghasilkan kekuatan biomekanika yang sesuai dalam menggerakkan gigi. pemakaian kawat ortodonti di dalam mulut dapat bereaksi dengan saliva sehingga menyebabkan terjadinya pelepasan ion nikel dan kromium. tujuan: penelitian ini bertujuan untuk meneliti pengaruh waktu perendaman dalam saliva buatan terhadap pelepasan ion nikel dan kromium antara kawat australia tipe spesial dengan kawat stainless steel. metode: penelitian ini menggunakan 30 buah kawat australia tipe spesial dan 30 buah kawat stainless steel dengan berat tiap kawat 0,12 gram. kawat direndam selama 1, 7, 28, 35, 42 dan 49 hari dalam saliva buatan ph normal. pemeriksaan pelepasan ion pada saliva menggunakan spektrofotometri serapan atom. hasil: pelepasan ion nikel pada kawat australia tipe spesial lebih besar dibandingkan dengan kawat stainless steel, terdapat perbedaan pelepasan jumlah ion nikel pada kawat australia tipe spesial antara waktu perendaman dan terdapat interaksi antara jenis kawat dengan waktu perendaman. ion nikel yang terlepas pada kawat australia tipe spesial mulai terdeteksi pada hari ke-7 perendaman dan mencapai jumlah tertinggi pada hari ke-35 sedangkan pada kawat stainless steel mulai terdeteksi pada perendaman hari ke-49. pelepasan ion kromium pada kawat australia tipe spesial dan kawat stainless steel tidak research report 169rasyid, et al.: pelepasan ion nikel dan kromium kawat australia dan stainless steel terdeteksi sampai perendaman hari ke-49. simpulan: pelepasan ion nikel pada kawat australia tipe special terdeteksi paling tinggi pada lama perendaman hari ke tiga puluh lima, kawat stainless steel terdeteksi pada lama perendaman hari ke empat puluh sembilan. pelepasan ion kromium pada kawat australia tipe special dan stainless steel tidak terdeteksi sampai lama perendaman hari ke empat puluh sembilan. kata kunci: kawat australia tipe spesial, stainless steel, lama perendaman, ion nickel, ion chromium korespondensi (correspondence): nolista indah rasyid, program studi spesialis ortodonsia, fakultas kedokteran gigi universtas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: nolista@yahoo.co.id pendahuluan kawat ortodonti merupakan salah satu komponen penting pada perawatan ortodonti. kawat yang sering digunakan adalah kawat austenitic stainless steel dan australia karena sifat fisik dan mekaniknya dibutuhkan dalam perawatan ortodonti yaitu memiliki sifat nontoksik, tahan terhadap korosi, lentur, kekuatan besar, kekerasan dan resilience tinggi.1 kedua kawat tersebut memiliki kandungan kromium dan nikel, kromium merupakan elemen yang memberi sifat tahan karat dan nikel meningkatkan kekuatan baja tahan karat.2 perbedaan kawat stainless steel dengan kawat australia terdapat pada kandungan karbon. kawat australia memiliki kandungan karbon yang lebih tinggi sehingga meningkatkan sifat kekerasan dan menyebabkan kawat lebih rapuh sehingga kemampuan dalam ketahanan terhadap bending berkurang.3 kawat stainless steel lebih banyak digunakan pada mekanisme sliding karena memiliki friksi yang rendah.4 kawat ortodonti perlu memenuhi kriteria berikut, kekuatan tinggi, kekakuan rendah, range tinggi, formability tinggi, serta dapat dilakukan welding dan solder.5 kawat ortodonti pada alat cekat akan selalu berkontak dengan saliva dan jaringan rongga mulut. pada lingkungan rongga mulut, kawat yang digunakan dalam perawatan ortodonti berpotensi mengalami korosi atau pelepasan elemen logam penyusun alloy. hal ini dapat terjadi karena pengaruh perubahan suhu, mikroflora, enzim rongga mulut dan perubahan keasaman (ph) saliva.6,7 elemen logam yang terlepas dari alloy akan bereaksi secara kimia dengan elemen non logam membentuk suatu ikatan logam yang dapat merusak struktur logam itu sendiri sehingga berpengaruh terhadap kualitas, estetika, bentuk fisik dan memperlemah kekuatan alloy logam.8 ion yang terlepas dapat masuk ke dalam tubuh, dan yang memiliki pengaruh paling merugikan bagi tubuh adalah ion nikel dan kromium.9 ion yang terlepas dapat memberikan efek biologi seperti toksisitas, alergi dan mutagenicity. ion nikel paling sering menyebabkan alergi kontak.10 dalam rongga mulut, korosi terjadi dengan lepasnya ion logam positif dari alloy ortodonti ke bentuk senyawa yang lebih stabil seperti klorida, sulfida dan oksida.11 korosi yang terjadi dapat diperiksa dengan beberapa cara yaitu melihat perubahan warna pada permukaan alloy, tes elektrokimia untuk melihat perubahan muatan alloy dan identifikasi elemen yang terlepas dengan menggunakan spektrofotometri emisi atom atau spektrofotometri serapan atom. pemeriksaan korosi pada kawat ortodonti dapat dilakukan dengan identifikasi jumlah elemen yang terlepas dalam cairan saliva dengan alat spektrofotometri serapan atom.10 saliva mengandung komponen organik dan anorganik seperti ion natrium, kalium, klorida, bikarbonat, kalsium, magnesium, hidrogen fosfat, tiosianat dan fluor. ion klorida mempunyai mekanisme perusakan logam melalui lapisan kromium oksida yang dipergunakan sebagai pelindung terhadap korosi. pemakaian kawat ortodonti dalam rongga mulut sangat berpotensi mengalami korosi. lama kawat ortodonti berkontak dengan saliva mempengaruhi pelepasan ion logam. pengaruh lama perendaman terhadap pelepasan ion logam telah dilakukan banyak penelitian. pelepasan ion logam dari alat cekat yaitu pelepasan ion nikel, kromium, kadmium, tembaga, besi, mangan, molibdenum, silikon. konsentrasi ion tertinggi yang terlepas terlihat pada hari pertama.12 pada penelitian lain dengan melakukan perendaman kawat stainless steel dalam saliva buatan menunjukkan hasil konsentrasi ion nikel yang terlepas tertinggi pada minggu ke dua belas.13 kawat australia yang direndam dalam saliva buatan selama tiga puluh lima hari menunjukkan pelepasan ion nikel paling banyak sedangkan ion kromium pada hari ke empat puluh dua.14 pada penelitian ini penambahan lama perendaman dilakukan sampai empat puluh sembilan hari untuk melihat pengaruh lama perendaman terhadap pelepasan ion nikel dan kromium dan untuk melihat pola pelepasan ion nikel dan kromium selanjutnya dari penelitian sebelumnya dengan lama perendaman hanya sampai empat puluh dua hari. pelepasan ion kromium dapat menurunkan ketahanan terhadap korosi karena kromium memberikan sifat tahan karat dan pelepasan ion nikel dapat menurunkan kekuatan baja tahan karat karena nikel berperan dalam meningkatkan kekuatan baja tahan karat.2 penelitian ini bertujuan untuk mengetahui pengaruh lama perendaman terhadap jumlah pelepasan ion nikel dan kromium dari kawat australia tipe spesial dengan kawat stainless steel dalam saliva buatan ph normal selama 1,7, 28, 35, 42 dan 49 hari. hasil penelitian dapat memberikan sumbangan pengetahuan tentang pengaruh lama perendaman terhadap pelepasan ion nikel dan kromium kawat australia tipe spesial dan stainless steel, memberikan pertimbangan waktu bagi ortodontis dalam melakukan pergantian kawat 170 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 168–172 serta pemilihan jenis kawat yang akan digunakan dalam melakukan perawatan ortodonti dengan alat cekat sesuai tujuan perawatan. bahan dan metode bahan yang digunakan adalah kawat australia tipe spesial (a.j. wilcock, g&h® wire company) dan kawat stainless steel (ortho organizers, ortho organizers, inc) diameter 0,016 inci, panjang kawat sebesar 12 cm dengan berat 0,12 gram. potongan kawat australia tipe spesial berbentuk kumparan dimasukkan ke dalam cawan petri dan stainless steel berbentuk kawat lurus dimasukkan ke dalam tabung kaca yang sudah bersih dan kering yang telah diisi saliva buatan sebanyak 5 ml dengan ph 6,75 ± 0,15 agar semua sampel terendam. komposisi saliva buatan terdiri dari 0,4 g nacl; 0,4 kcl; 0,78 g nah2po4. 2h2o; 0,005g na2s. 9h2o; 1g co(nh2)2; 0,81 g potasium klorida, seluruh bahan dicampur dengan aqua destilata sampai 1000 ml kemudian dititrasi dengan hcl dan naoh sehingga diperoleh ph yang diinginkan. semua sampel yang telah direndam dalam saliva buatan disimpan dalam inkubator dengan suhu 37o c selama 1, 7, 28, 35, 42 dan 49 hari. jumlah ion nikel dan ion kromium yang larut dalam saliva buatan hasil perendaman diperiksa dengan alat spektrofotometer metode spektrofotometri serapan atom (perkin elmer model 3100). cara kerja pemeriksaan ion nikel dan kromium dimulai dengan menyiapkan larutan sampel dan standar, larutan standar adalah larutan yang dibuat mengandung unsur nikel dan kromium, kemudian menyiapkan hollow cathoda lamp (lampu katoda berrongga) untuk nikel dan kromium dan alat spektrofotometer dinyalakan. burner dinyalakan untuk mengubah larutan sampel dan larutan standar menjadi uap atom, tombol cont dinyalakan untuk menampilkan serapan yang diukur dan dipilih serapan yang paling stabil, kemudian menyalakan tombol data untuk melakukan pengukuran, selanjutnya masukkan selang injeksi ke dalam wadah pencucian kemudian tekan tombol a/z. pengukuran larutan standar dan larutan sampel dilakukan dengan menyalakan tombol read out, setiap ganti larutan standar dan larutan sampel, selang injeksi harus dimasukkan ke dalam wadah pencucian. data penyerapan radiasi dari larutan standar dan sampel dimasukkan maka akan diketahui jumlah ion nikel dan kromium yang terkandung dalam larutan. hasil pemeriksaan berupa angka dengan satuan ppm. hasil pelepasan ion nikel pada kawat australia tipe spesial mulai terdeteksi pada hari ke tujuh sedangkan pada kawat stainless steel mulai terdeteksi pada hari ke empat puluh sembilan. pelepasan ion kromium tidak terdeteksi sejak hari pertama perendaman sampai hari ke empat puluh sembilan pada kawat australia tipe spesial maupun kawat stainless steel. jumlah pelepasan ion nikel pada kawat australia tipe spesial dan stainless steel dapat dilihat pada tabel 1. pelepasan ion nikel pada kawat australia tipe spesial lebih besar dibandingkan pelepasan ion nikel pada kawat stainless steel. uji kruskal-wallis untuk melihat perbandingan jumlah pelepasan ion nikel antara kawat australia tipe spesial dengan kawat stainless steel menunjukkan terdapat perbedaan yang bermakna dengan p<0,05 (p=0,043). tabel 1. rerata dan simpangan baku pelepasan ion nikel dan kromium dalam saliva buatan (part per million/ppm) waktu perendaman jenis ion rerata + simpangan baku kawat australia kawat stainless steel 1 hari cr tidak terdeteksi tidak terdeteksi 7 hari cr tidak terdeteksi tidak terdeteksi 28 hari cr tidak terdeteksi tidak terdeteksi 35 hari cr tidak terdeteksi tidak terdeteksi 42 hari cr tidak terdeteksi tidak terdeteksi 49 hari cr tidak terdeteksi tidak terdeteksi 1 hari ni tidak terdeteksi tidak terdeteksi 7 hari ni 0,062 + 0,036 tidak terdeteksi 28 hari ni 0,369 + 0,224 tidak terdeteksi 35 hari ni 0,401 + 0,133 tidak terdeteksi 42 hari ni 0,272 + 0,120 tidak terdeteksi 49 hari ni 0,337 + 0,062 0,099 + 0,056 171rasyid, et al.: pelepasan ion nikel dan kromium kawat australia dan stainless steel hasil uji mann-whithney pada tabel 2 menunjukkan terdapat perbedaan pelepasan ion nikel yang signifikan (p<0,05) antara lama perendaman tujuh hari dibanding dua puluh delapan, tiga puluh lima, empat puluh dua, empat puluh sembilan hari dan tiga puluh lima hari dibanding empat puluh dua hari. hasil uji kruskall-wallis menunjukkan terdapat interaksi ion nikel antara jenis kawat australia tipe spesial dengan stainless steel pada lama perendaman empat puluh sembilan hari dengan nilai signifikansi p<0,05 (p=0,004). pembahasan pelepasan ion nikel pada kawat australia tipe spesial mulai terdeteksi pada perendaman 7 hari sedangkan pada kawat stainless steel mulai terdeteksi pada 49 hari. pelepasan ion nikel yang lebih besar pada kawat australia tipe spesial kemungkinan disebabkan karena adanya kandungan karbon yang lebih banyak dibandingkan dengan kawat stainless steel. kawat australia tipe spesial memiliki kandungan karbon 0,04% sedangkan kawat stainless steel memiliki kandungan karbon 0,02%.15 karbon dapat berikatan dengan kromium membentuk senyawa kromium karbida sehingga lapisan kromium oksida pada permukaan logam berkurang, karena kandungan karbon yang tinggi dapat memicu terjadinya korosi.3 kandungan karbon yang lebih tinggi mengakibatkan kawat australia tipe spesial lebih mudah mengalami korosi sehingga pelepasan ion nikel lebih banyak dibandingkan dengan kawat stainless steel. pelepasan ion kromium pada penelitian ini tidak terdeteksi, kemungkinan karena jumlah ion yang terlepas jumlahnya di bawah 0,015 ppm yang merupakan batas deteksi dari alat spektrofotometri serapan atom yang digunakan pada penelitian ini. hasil pada tabel 2 menunjukkan bahwa waktu perendaman mempengaruhi pelepasan jumlah ion nikel kawat australia tipe spesial. ion yang terlepas disebabkan karena adanya kontak antara kawat dengan cairan saliva buatan. cairan saliva buatan merupakan cairan elektrolit yang dapat mengakibatkan terjadinya proses korosi, ion klorida pada saliva dapat merusak lapisan oksida pada permukaan kawat sehingga mengakibatkan terjadinya pelepasan ion logam seperti besi, nikel, kromium, molibdenum dan titanium yang merupakan elemen penting kawat.8,11 ion nikel memiliki tabel 2. hasil uji mann-whitney pelepasan ion nikel pada kawat australia tipe spesial antara lama perendaman 7, 28, 35, 42 dan 49 hari waktu perendaman 7 hari 28 hari 35 hari 42 hari 49 hari 7 hari 0,000* 0,000* 0,000* 0,000* 28 hari 0,250 0.345 0,461 35 hari 0,015* 0,217 42 hari 0,089 49 hari keterangan: *= p<0,05 sifat larut dalam cairan saliva, sehingga lama kawat berkontak dengan cairan mempengaruhi pelepasan ion logam. ion nikel memiliki kecenderungan yang tinggi untuk terlepas, berkaitan dengan struktur elemen pada tingkat atom. atom nikel tidak terikat dengan kuat pada senyawa intermetalik.10 pelepasan ion nikel 7 sampai 35 hari semakin meningkat kemudian mengalami penurunan pada 42 hari kemungkinan terjadi pasivasi yang menghambat pelepasan ion yang intensif setelahnya.12 empat puluh sembilan hari perendaman terjadi peningkatan pelepasan ion nikel akan tetapi dengan jumlah yang lebih rendah dari pelepasan tiga puluh lima hari. jenis kawat berkaitan dengan komposisi dari setiap kawat. komposisi kawat merupakan faktor yang mempengaruhi pelepasan ion logam dengan ion utama yang terlepas adalah besi, kromium dan nikel.10 penelitian menyatakan komposisi ion kromium dan nikel pada kawat australia tipe spesial sebesar 17,2% dan 12,0% sedangkan komposisi ion kromium dan nikel pada kawat stainless steel sebesar 18,0 % dan 9,0%.15 pelepasan ion logam dipengaruhi oleh komposisi kawat, derajat keasaman saliva dan lama perendaman. komposisi kromium pada kawat stainless steel lebih besar dibandingkan kawat australia tipe spesial,16 kromium berfungsi sebagai pelindung terhadap korosi dan nikel berfungsi untuk menambah kekuatan, kelenturan dan memperbaiki daya tahan terhadap korosi.14 pelepasan ion nikel lebih banyak terjadi pada ph asam dibandingkan ph normal.17 pada kondisi asam, jumlah ion h+ akan semakin besar sehingga bersifat korosif dan dapat mengoksidasi logam.18 pelepasan ion nikel yang terjadi pada kawat ortodonti merupakan keadaan yang tidak dapat dicegah karena sangat sulit untuk menemukan material yang sangat stabil. hal yang perlu diperhatikan adalah jumlah ion yang terlepas berkaitan dengan jumlah ion yang aman untuk diterima oleh tubuh.16 organisasi kesehatan dunia (who) merekomendasikan dosis harian ion nikel sebesar 25-35 μg. pada penelitian ini rerata ion nikel terbesar adalah 4,812 μg, nilai ini diperoleh dengan menghitung jumlah ppm dikalikan dengan berat kawat.19 nilai ini masih jauh lebih rendah dari batas yang direkomendasikan oleh who, pada beberapa penelitian menyatakan bahwa sejumlah kecil ion nikel yang terlepas dapat menyebabkan reaksi alergi berupa stomatitis kontak.13 penelitian lain menyatakan bahwa pelepasan ion nikel dari alat ortodonti cekat dapat 172 dent. j. (maj. ked. gigi), volume 47, number 3, september 2014: 168–172 mengakibatkan kerusakan dna pada sel mukosa mulut, dan dapat terjadi reaksi hipersensitivitas pada pasien dengan perawatan ortodonti cekat.20 ion logam yang terlepas pada proses korosi merupakan reaksi kimia alloy yang dapat mempengaruhi kualitas, estetika, bentuk fisik dan memperlemah kekuaan logam.8 berdasarkan pembahasan di atas dapat disimpulkan bahwa pelepasan ion nikel pada kawat australia tipe spesial terdeteksi paling tinggi pada lama perendaman hari ke 35, kawat stainless steel terdeteksi pada lama perendaman hari ke-49. pelepasan ion kromium pada kawat australia dan stainless steel tidak terdeteksi sampai lama perendaman hari ke empat puluh sembilan. daftar pustaka 1. kusy rp. orthodontic biomaterial: from the past to the present. angle orthod 2002; 72 (6): 501-12. 2. martinez cc. degradation of lingual orthodontics archwires. barcelona: department of odontostomatology, faculty of dentistry, university of barcelona; 2007, p. 32-3. 3. pelsue bm, zinelis s, bradley gt. structure, compotition, and mechanical properties of australian orthodontic wire. angle orthod 2009; 79(1): 97-101. 4. marques isv, araujo am, gurgel ja, normando d. debris, roughness and friction of stainless steel archwires following clinical use. angle orthod 2010; 80(3): 521-7. 5. profitt wr. contemporary orthodontics. 4th ed. philadelphia: mosby elsevier; 2007. p. 369, 407-11, 572-3, 592-600, 604-8. 6. brantley wa, eliades t. orthodontic material: scientific and clinical aspects. germany: stuttgart; 2001. p. 77-105, 288. 7. hsiung huang h, hui chiu y, hsin lee t, ching wu s, wen yang h, hsiung su k, chih hsu c. ion release from niti orthodontic wires in artificial saliva with various acidities. biomaterial 2003; 24(20): 3585-92. 8. phillips rw. skinner’s science of dental materials. 11th ed. philadelphia, london, toronto: wb saunders company; 2003. p. 56-9. 9. e l ia de s t, at ha na siou a e . i n v ivo ag i ng of or t ho dont ic alloys: implication for corrosion potential, nickel release and biocompatibility. angle orthod 2002; 72(3): 222-37. 10. schmaltz g, arenholt-bindslev d. biocompatibility of dental materials. berlin: springer-verlag; 2009. p. 224-5. 11. brantley wa, eliades t. orthodontic material: scientific and clinical aspects. germany: stuttgart; 2001. p. 77-105, 288. 12. mikulewicz m, chojnacka k, wolowiec p. release of metal ions from fixed orthodontic appliance an in vitro study in continuous flow system. angle orthod 2013; 84(1): 140-8. 13. oh kt, kim kn. ion release and cytotoxicity of stainless steel wires. eur j orthod 2005; 27(6): 533-40. 14. lenti-canina. pengaruh waktu perendaman dalam saliva buatan terhadap pelepasan ion kawat australia. karya tulis ilmiah. yogyakarta: ppdgs fakultas kedokteran gigi universitas gadjah mada; 2005: 33-45 15. shankar sg, shetty s, karrant hs. a comparative study of physical and mechanical properties of different grades australian steel wires. trends biomater artif organs 2011; 25(2): 67-74. 16. kuhta m, pavlin d, slaj m, varga ml. type of archwire and level of acidity: effect on the release of metal ions from orthodontic appliances. angle orthod 2009; 79(1): 102-10. 17. siti-fatimah. perbandingan pelepasan ion nikel antara empat merek braket stainless steel baru dan daur ulang dalam saliva buatan dengan ph 5, 6 dan 7. karya tulis ilmiah. yogyakarta: ppdgs fakultas kedokteran gigi universitas gadjah mada; 2013: 36-55 18. callister tp. fundamental of materials science and engineering: an integrated approach. 5th ed. new york: john wiley & son, inc; 2012. p. 205-6. 19. mikulewicz m, chojnacka k, wozniak b, downarowicz p. release of metal ions from orthodontic appliances : an in vitro study. biol trace elem res 2012; 146: 272-280. 20. faccioni f, franceschetti p, cerpelloni m, fracasso me. in vivo study on metal release from fixed orthodontic appliances and dna damage in oral mucosa cells. am j orthod dentofac orthop 2003; 124(6): 687-94. 122 vol. 43. no. 3 september 2010 the effectiveness of sharp end and rounded end bristle toothbrush paulus januar�, anastasia susetyo�, and ratih widyastuti� 1 department of dental public health 2 department of periodontology faculty of dentistry, prof. dr. moestopo university jakarta indonesia abstract background: numerous designs of manual toothbrush are available in the market with the claims of superiority in plaque removal. it often makes the public confuse which is the best design. the sharp end bristle toothbrush is a modification that commercially available in the market. purpose: the objective of the study was to compare the effectiveness in plaque removal of the sharp end bristle toothbrush and the rounded end bristle toothbrush. methods: this clinical trial was a double blind crossover design. the subjects were 65 dental students, divided into two groups for comparing the 2 types of toothbrush. on the 1st day, the allocated toothbrushes were distributed to each group according to their designation, and the subjects were instructed to use the toothbrushes according their normal daily practices. on the 1st, 7th, and 14th day, the subjects were scored using the patient hygiene performance index (php index) and the gingival index. based on cross over design, the same procedure was repeated during the 2 week second test periods using different type of toothbrush respectively. results: the mean scores of the 2 groups showed no significant difference on the beginning the study. though minor differences were observed in the effectiveness of toothbrush, but the comparison of the two types of toothbrush showed no statistically significant differences on 7th and 14th day. conclusion: there were no significant differences between sharp end and rounded end bristle toot brusher. there is no manual toothbrush superiorly designed than the others single superior design of manual toothbrush. key words: toothbrush, effectiveness, rounded end bristle, sharp end bristle abstrak latar belakang: berbagai jenis desain sikat gigi saat ini terdapat di pasaran, dengan masing-masing menyatakan keunggulannya dalam membersihkan plak. penelitian ini dilakukan terhadap dua jenis sikat gigi manual yaitu sikat gigi dengan ujung bulu sikat runcing dan ujung bulus sikat bulat. tujuan: penelitian ini dilakukan untuk membandingkan efektivitas membersihkan plak antara 2 jenis sikat gigi. metode: penelitian ini merupakan percobaan klinis dengan desain penyilangan (crossover) secara tertutup ganda (double blind). subyek penelitian 65 mahasiswa dibagi 2 kelompok. pada hari pertama, sikat gigi dibagikan pada masing-masing kelompok yang telah ditentukan jenis sikat giginya, dan diinstruksikan untuk menggunakannya sesuai kebiasaan mereka. pada hari ke 1, 7, dan 14 dilakukan pengukuran indeks php dan indeks gingiva. berdasarkan desain penyilangan, proses yang sama diulangi pada masing-masing kelompok dengan menggunakan jenis sikat gigi yang berbeda. hasil: pada awal penelitian tidak terdapat perbedaan skor pada ke 2 kelompok. meski terdapat sedikit perbedaan, namun tidak terdapat perbedaan yang bermakna dalam efektivitas kedua jenis sikat gigi setelah penggunaan selama 7 dan 14 hari. kesimpulan: tidak ada perbedaan yang bermakna antara sikat gigi jenis ujung bulu sikat bulat dan ujung bulu sikat runcing. tidak terdapat satupun jenis sikat gigi yang paling baik. kata kunci: sikat gigi, efektivitas, ujung bulu sikat bulat, ujung bulu sikat runcing correspondence: paulus januar, c/o: bagian kesehatan gigi masyarakat, fakultas kedokteran gigi universitas prof. dr. moestopo (beragama). jl. bintaro permai no. 3, jakarta selatan 12330, indonesia. e-mail: paulusjanuar@yahoo.com research report 123januar et al.: the effectiveness of sharp end and rounded bristle toothbrush introduction dental caries and periodontal diseases are major problems in dental health. dental plaque is the risk factor of dental caries and periodontal diseases.1 tooth brushing is the regular removal of dental plaque on the teeth and adjacent gingival surface. tooth brushing is the most effective method to maintain healthy conditions for teeth and gingival.2 several types of toothbrush are available in the market with the claims of superiority for a particular design. generally, toothbrushes vary in size and design, diameter, length, hardness, arrangement and material of the bristles as well. the claims of superiority are primary based on plaque removing efficacy and ease of use. however, there is still a lack of data on their efficacy in plaque removal and gingivitis reduction.1,3,4 the various designs of toothbrushes available in the market often confusing the public which is the best design and they often seek professional advice on this matter. the type of the bristles is important consideration in selecting a good toothbrush. because of the varieties of brushes currently available, the dental professional must maintain a high level of knowledge of these products. ideally, clinical study should be carried out to compare the existing models and new designs as they appear on the market. the design of manual toothbrushes has been modified and refined in pursuit of more effective plaque removal and improved oral health.3,5,6 in recommending a particular toothbrush, the effectiveness in plaque removal and the safety from injury, as well as the perception that the brush works well are important considerations.1 the american dental association (ada) recommended that the toothbrush should be able to clean teeth effectively and thoroughly, the bristles are free of sharp or jagged edges and endpoints; the bristles would not fall out with normal use, the toothbrush can be used to provide a significant decrease in mild periodontal disease and plaque, also the handle material has the durability under normal use.7,8 choosing the right bristles is very important in choosing the toothbrush. bristles are important because they directly contact the teeth and gum tissue. the effectiveness of toothbrush bristle is based on shape, type, and arrangement. hard, medium, and soft-bristled toothbrushes all remove plaque; however, hard bristles may cause irreversible damage to the gum, tongue, and cheeks, also can lead to periodontal disease and receding gum lines. studies showed that soft-bristled toothbrushes remove plaque as effectively as medium or hard bristles.3,8 the sharp end bristle toothbrush is a modification that commercially available in the market. the modification is on the bristle of the toothbrush that had a smaller diameter at the top of the bristle (figure 1). even though the bristle has a sharpen shape, but the tip of the bristle is trimmed perfectly round to prevent gum injury. it is assumed that the sharp end bristle has a better cleaning efficacy, because the brushing action can be made more effective. on the marketing process, the manufacturer claimed the effectiveness of the sharp end bristle toothbrush in plaque removal, although there was a lack of its evidence basis. figure �. sharp end bristle toothbrush and rounded end toothbrush. the aim of the study was to compare the sharp end bristle toothbrush with the conventional round end bristle toothbrush on the effectiveness of plaque removal and maintaining gingival health. the identification of the effectiveness of the toothbrushes would guide the dental professionals in recommending the toothbrush of choice for the public. materials and methods the study was a double blind clinical trial, and the cross over design9 was conducted to compare the 2 types of toothbrush commercially available in the market comprise of sharp end bristle toothbrush and rounded end bristle toothbrush. the study was carried out at the dental hospital of prof dr moestopo university, jakarta. the subjects of the study were dental students that at random divided into 2 groups. the criteria for the subjects were good general health, at least 28 functionally good teeth (excluding third molar), good soft oral tissue, no calculus or periodontal diseases, good occlusion, no smoking, no extensive restorations, not using orthodontic appliances, and using no other oral hygiene procedures (mouthwash, tooth whitening, etc). the study used 2 types of toothbrush comprise of sharp end bristle toothbrush and rounded end bristle toothbrush. the two types of toothbrush were similar in all specification, and the difference was only in the bristles (figure 1). the study was conducted over 4 weeks and involved six visits. at the initial day the toothbrushes were distributed to each group according to the designation of the study. the subjects were instructed to use the toothbrush for 2 weeks, according to their daily tooth brushing method, with the same tooth paste. cross overly the same procedure was repeated during the second week second test periods using different type of toothbrush respectively. the plaque reduction were evaluated using patient hygiene performance index (php index) and the gingival 124 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 122–125 condition were evaluated using gingival index.1 the assessment were conducted at the 1st day (initial day), and after the 7th and 14th day using each type of toothbrush respectively. a set of questionnaires were used to access the perception of the subject on the use of the toothbrushes. statistical analysis of the data was carried out using t test, and chi square test. the computer analysis of the data used the statistical package for social sciences (spss) software version 17. the values of p < 0.05 were considered statistically significant. results a total of 65 dental students consist of 54 females and 11 males participated. the ages the subjects mostly were 18 years old, the range was 17 until 19 years old, and the mean age was 17.77 years old. the assessment using the php index and gingival index were carried out at the 1st, 7th and 14th day after using each type of toothbrush. no changes in hard or soft oral tissues were reported post-brushing and no adverse events occurred. the scores of php index and gingival index were displayed on table 1 and 2. at the initial day before using sharp end bristle toothbrushes, the mean scores of the php index and the gingival index of the subjects were 2.005 and 0.140. after using the sharp end bristle toothbrushes at the 7th and the 14th day, the php index mean scores decreased into 1.818 and 1.690, and the gingival index mean scores decreased into 0.094 and 0.070 respectively. at the initial day before using rounded end bristle toothbrushes, the mean scores of php index and gingival index of the subjects were 2.136 and 0.165. after using the rounded end bristled toothbrushes at the 7th and the14th day, the php index mean scores decreased into 1.463 and 1.724, and the gingival index mean scores decreased into 0.092 and 0.061 respectively. the comparisons between 2 types of toothbrush were statistically analyzed using paired t test. at the beginning of the study, between the 2 groups showed no significant difference of the mean scores of the php index (p=0.720) and also the gingival index (p=0.863). at the 7th day there was significant difference on the mean scores of php index between the 2 types of toothbrush (p=0.009), but at the 14th day showed no significant difference. the mean scores of gingival index showed no significant difference between the 2 type of toothbrush at the 7th day (p=0.839), and at the 14th day (p=0.401). the questionnaire was on the perceptions of the subjects on using the toothbrush that comprising of the perception on cleaning efficacy, the comfort in using the toothbrush, also on the hardness and the sharpness of the bristles. the data of the perceptions on using the 2 types of toothbrush were on table 3 and 4. based on the perceptions of the subjects, most of them stated that both toothbrushes were good in cleaning efficacy to their teeth, and statistical analysis using paired t test showed no significant difference between the 2 types of toothbrush (p=0.27). in comparing the 2 types of toothbrush, 25 subjects (38.5%) perceived the sharp end bristle toothbrush was better than the round end bristle toothbrush, but 21 subjects (32.3%) perceived the round end bristle toothbrush was better than the sharp end bristle toothbrush, while 12 subjects (18.5%) perceived there was table �. the php index at the 1st, 7th and 14th day day php index sharp end bristle rounded end bristle p score mean standard deviation mean standard deviation 1 2.005 1.032 2.136 0.937 0.720 7 1.818 0.818 1.463 0.741 0.009 14 1.690 0.772 1.724 0.690 0.987 table �. the gingival index at the 1st, 7th and 14th day day gingival index sharp end bristle rounded end bristle p score mean standard deviation mean standard deviation 1 0.140 0.138 0.165 0.165 0.863 7 0.094 0.092 0.092 0.074 0.839 14 0.070 0.084 0.061 0.070 0.401 table ��. the perceptions on the cleaning efficacy of the toothbrushes cleaning efficacy the bristles of the toothbrush sharp rounded very good 8 (12.3%) 6 (9.4%) good 50 (76.9%) 56 (87.5%) neutral 3 (4.6%) 0 (0%) bad 4 (6.2%) 2 (3.1%) very bad 0 (0%) 0 (0%) σ 65 (100%) 64 (100%) table �. the perception on the comparison of using the toothbrushes perception subject (n) % the sharp end was much better 3 4.6 the sharp end was better 25 38.5 no difference 12 18.5 the round end was better 21 32.3 the round end was much better 4 6.2 σ 65 100 125januar et al.: the effectiveness of sharp end and rounded bristle toothbrush no difference between the 2 types of toothbrush. chi-square test showed no significant difference on the comparison of both types of toothbrush (p=0.101). discussion many researches have been conducted evaluating the effectiveness of many different types of toothbrush. tooth brushing is one of the most studied topic areas in the field of dentistry, with many publications on efficacy, methods of brushing, and types of brushes, documenting the health benefits of mechanical removal.6 this study compared the effectiveness of the sharp end bristle toothbrush with the rounded end toothbrush. the dental students as the subjects of the study were relatively homogenous in the sense of their age and oral hygiene awareness. as dental students, they had good oral hygiene shown in their scores of php index and gingival index. according to wolf et al.9 the cleaning afficacy of different toothbrushes was frequently tasted in a croos over design on the same subjects to control the testing effects. that the subjects keep up in creased oral health awarness during the course of the study founded on the improved oral hygiene. the effectiveness of tooth brushing was showed in the difference of the scores of php index and gingival index between baselines and after using the toothbrushes at the 7th and 14th day. the comparison of the effectiveness of the 2 types of toothbrush showed no significant difference. moreover the subjects perceived that there was no significant difference between the 2 types of toothbrush in cleaning efficacy. opinion regarding the benefit of the toothbrushes on this study was comparable in the same subjects, and they perceived a benefit from both types of toothbrush. based on the results of this study, the 2 types of toothbrush were similar in their effectiveness. significant effectiveness in plaque removal could be achieved regardless of the toothbrush bristles used. the present clinical study was undertaken to find out the claim of the effectiveness of sharp end bristle toothbrush. the results of the study indicated that the sharp end bristle toothbrush significantly reduced the plaque, but yet no significant differences were observed when compared with the conventional rounded end bristle toothbrush. the results were in line with the study of sripriya and ali5 that showed no significant differences in plaque removal between four different bristle designs of toothbrushes. comparative clinical studies are crucial for assessing the relative effectiveness of different toothbrushes. there were few published studies that have investigated the plaque removal effectiveness of various types of toothbrushes, and some of the results had not demonstrated definitively that any one design was absolutely better.11,12 the results are conflicting, with some studies reporting some designs of toothbrushes to be superior, but the general consensus in most of the studies has been that there is no one superior design of toothbrush for plaque removal.4,5,8,10,13 no significant difference between the 2 types of toothbrush bristles in this study was largely due to the complexity of tooth brushing. thus making a specific toothbrush recommendation on the basis of sound science is not easy. toothbrush bristles was only one of several factors that contribute to the effectiveness of tooth brushing. the effectiveness of tooth brushing depended not only on the design and the bristles of the toothbrush, but also depended on various other variables such as knowledge, skill, manual ability, the amount of forces used to brush, attitude, time devoted for tooth brushing, dental arch anatomy, and tooth brushing method.1,6,8 as the conclusions, the sharp end bristle toothbrush and the round end toothbrush, both were significantly effective to reduce plaque and maintain gingival health, but there were no significant differences between those two types of toothbrush. further study on different types of toothbrush is needed in order to invent new innovations for a better toothbrush design. references 1. newman, g, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. tenth edition. st. louis: saunder, elsevier; 2006. p. 115–16, 728–32. 2. chandra s, chandra s. textbook of community dentistry. new delhi: jaypee brothers medical publishers ltd; 2004. p. 123–8. 3. sasan d, thomas b, bhat mk, aithal ks, ramesh pr. toothbrush selection: a dilemma? indian j dent res 2006; 17: 167–70. 4. biesbrock ar, bartizek rd, walters pa. improved plaque removal efficacy with a new manual toothbrush. j contemp dent pract 2008; 9(4): 1–8. 5. sripriya n, ali sh. a comparative study of the efficacy of four different bristle designs of toothbrushes in plaque removal. j indian soc pedod prev dent 2007 june; 76–81. 6. cappelli dp, mobley cc. prevention in clinical oral health care. st. louis, missouri: mosby elsevier; 2008. p. 214–7. 7. american dental association (ada). a look at toothbrushes. j am dent assoc 2007 september; 138: 1288. 8. perry da, beemsterboer pl. periodontology for the dental hygiegienist. st. louis, missouri: mosby elsevier; 2007. p. 239–43. 9. wolff d, pioch t, dorfer ce. effect of a crossover design on the 24hour plaque regrowth. int poster j dent oral med 2005; 7(1): 279. 10. dörfer c, kugel b, von bethlenfalvy e, pioch t. effect of an experimental manual toothbrush on plaque and gingivits reduction. int poster j dent oral med 2004; 6(2): 229. 11. goyal cr, sharma nc, qaqish jg, cusini ma, thompson mc, warren pr, efficacy of a novel brush head in the comparison of two power toothbrushes on removal of plaque and naturally occurring extrinsic stain. journal of dentistry 2005; 33(suppl. 1): 37–43. 12. scheidegger n, lussi a. tooth cleaning with different children’s toothbrushes. a clinical study. schweiz monatsschr zahnmed 2005; 115(2): 100–6. 13. terézhalmy gt, bsoul sa, bartizek rd, biesbrock ar. plaque removal efficacy of a prototype manual toothbrush versus an ada reference manual toothbrush with and without dental floss. j. contemp dent pract 2005; 6(3): 1–13. 1313 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 13–15 original article a comparison of the accuracy of the cervical vertebrae maturation stage method and demirjian’s method on mandibular length growth alfira putriana dewi, seno pradopo, sindy cornelia nelwan department of pediatric dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: malocclusion is one of the most familiar dental problems, with a high prevalence among the population. understanding the patient’s craniofacial growth and development is crucial in diagnosis, as are the planning and subsequent success of the treatment. malocclusion needs to be treated early to optimise the outcome achieved by the treatment. one of the most common types of malocclusions observed in clinics is crowding. the craniofacial bone relevant to the treatment of crowding is the mandible, defined as the mandibular length from the condylion to gnathion areas. when planning treatment, clinicians may experience difficulties in determining the biological age of patient, particularly when supporting diagnostic tools are not available. the indicators of biological age can be observed by the assessment of bone maturation using the cervical vertebrae maturation (cvm) method and by the analysis of tooth maturation using demirjian’s method. however, limited studies are available regarding the accuracy of these methods as diagnostic tools. purpose: this study aims to analyse the accuracy of the cvm method compared with demirjian’s method concerning mandibular length growth. methods: an analytic research method and a cross-sectional design are employed. the research sample comprised 50 lateral cephalometric and panoramic photos of children aged 8 -16 years. data were collected by analysing the maturity level of the cervical vertebrae and the teeth, and measuring the mandible length of the children in the photos. the statistical test used was the wilcoxon test. results: the results of the wilcoxon test for the asymptotic sign had a p-value of 0.116 > 0.05, indicating no significant difference between the cvm and demirjian methods. conclusion: both of the methods noted above yielded equally accurate results for determining mandibular length growth. keywords: biological age; cvm; demirjian’s method; malocclusion; mandibular length growth correspondence: seno pradopo, department of pediatric dentistry, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: seno-p@fkg.unair.ac.id introduction malocclusion is the most common dental problem in paediatric dentistry, with a prevalence of 20%–100%. the most common malocclusions observed in paediatric dentistry clinics include a deep overbite, an excessive overjet, midline deviation, an anterior crossbite, crowding, and an open bite.1 when planning treatment, it is necessary to know the patient’s age, which can be determined using chronological age and biological age. the chronological age only provides general information about the patient’s development, whereas the biological age has more significance for the planning of treatment. to evaluate biological age, the developmental level of the teeth and bones must be assessed.2 several biological factors such as an increase in height, weight gain, cervical vertebrae maturation (cvm), and tooth development must be taken into account.3 during growth and development, the bones, such as cervical vertebrae, undergo changes that can be detected radiographically as wrist radiographs, which can be used to assess bone maturation. however, this technique requires additional x-ray exposure in orthodontic patients already undergoing lateral and panoramic cephalograms. considering these shortcomings, the assessment of cvm and changes in the shape and size of cervical vertebrae dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p13–15 mailto:seno-p@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p13-15 14 are suggested as alternatives to the assessment of skeletal age.3 the stages of cvm are determined based on the morphology of cervical vertebrae bodies (c), namely c2, c3, and c4, which are visualised in a two-dimensional lateral cephalogram, i.e. an analysis of the development of the depression on the inferior edges of c2, c3, and c4 is conducted, and changes in the body shape and size of c3 and c4 are recorded. the bodies of these vertebrae change shape in a characteristic order, progressing from trapezoid to rectangular-horizontal, then to square, and then to rectangular-vertical.2,4 indicators of biological age can also be found in the dental maturity of growing children. demirjian’s method is the most accepted approach for estimating tooth age. the assessment of tooth maturation is based on the classification stages of each tooth. this assessment is divided into eight stages, taking into account the development of the permanent incisors, permanent canines, permanent first and second premolars, and the mandibular first and second permanent molars on the left.5,6 as previously stated, malocclusion is one of the most familiar dental problems, with a high prevalence among the population. an understanding of the patient’s craniofacial growth and development is very important when making a diagnosis and in the planning success of the treatment. it is necessary to treat malocclusion early to obtain optimal results from the treatment. one of the most common types of malocclusions observed in clinics is crowding. the craniofacial bone relevant to the treatment of crowding is the mandible, as measured by the mandibular length.7–10 recent studies showed that cervical vertebral growth is closely related to mandibular maturation and growth. in particular, mandibular growth peaks have been reported to occur concurrently in cvm stages 3 and 4. regarding tooth maturation, demirjian’s method was presented as an alternative to bone maturation assessment. although several studies have reported that tooth maturation can be used as an indicator of development, it has a relative relationship with the occurrence of peak mandibular growth.11,12 this study aims to analyse the accuracy of the cvm method and demirjian’s method when applied to mandibular length growth. materials and methods this research combined analytic observation with a crosssectional design. the population group observed in the study comprised paediatric patients aged from 8–16, who were treated at the airlangga dental and oral hospital, which is part of the airlangga university faculty of dental medicine. the sample inclusion criteria were the availability of clear and sharp lateral cephalometric and panoramic photos, as well as the patient having all their limbs without ever having experienced a fracture. the sample in this study required 50 samples using the total sampling technique. the ethical clearance certificate number is 436/hrecc.fodm/vii/2021, assigned by the ethical clearance commission of the faculty of dental medicine health research, airlangga university. in this study, the independent variable was the age of the child, while the dependent variables were the stage of cvm for c2, c3, and c4, as well as the stage of estimating the age using demirjian’s method, and the mandibular length growth. data were collected by selecting lateral cephalometric and panoramic photos that were clearly visible, and analysing each photo for cervical vertebra bone maturation through cvm, for tooth maturation using demirjian’s method, and by measuring the mandibular length from the condylion point to the gnathion point. each group was determined by the average value of cervical vertebral bone maturation, tooth maturation, and mandibular length. the wilcoxon difference test was conducted to determine which method is more accurate between the cvm method and demirjian’s method for measuring the mandibular length. the statistical analysis test used in this study was wilcoxon’s non parametric difference test. results table 1 shows that the average cvm method is 11.43 and demirjian’s method is 11.73; this meant that the standard deviation of the cvm method was 2.47 and that of the demirjian’s method was 2.02. the average between cvm and demirjian’s method showed a difference of 0.3 and the standard deviation had a difference of 0.45. from the statement, this data demonstrates that there is no significant difference, and that it is necessary to retest the difference. the original data were initially tested for normality. table 2 shows that the cvm method obtained a significance value of 0.000 because a significance value lower than from 0.05 inferred that the data were not normally distributed. table 1. the average method of cervical vertebrae maturation and demirjian’s method. cvm demirjian mean 11.43 11.73 standard deviation 2.47 2.02 table 2. the results of the kolmogorov–smirnov normality test. group significance description cvm 0.000 not normal demirjian 0.200 normal table 3. the results of the wilcoxon difference test. mean rank demirjian 24.97 cvm 25.82 asymptotic significance (demirjian – cvm) 0.116 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p13–15 dewi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 13–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p13-15 15 regarding demirjian’s method, a significance value of 0.200 was obtained because a significance value higher then from 0.05 inferred that the data were normally distributed. table 3 shows that demirjian’s method had a mean rank value of 24.97 and the cvm method had a mean rank value of 25.82. the mean rank value differed by 0.85, which can be observed in the asymptotic significance (demirjian and cvm). asymptotic significance was the probability value (p-value) of the wilcoxon test, and was compared with the alpha value, i.e. 0.05. the results would then show that there was either a significant or no significant difference between the data of the two data methods being compared. a p-value of > 0.05 would refer no significant difference, while a p-value of < 0.05 would indicate a significant difference. in this research, a p-value of 0.116 was found for a result higher than 0.05; this indicated that there was no significant difference between the results obtained using the cvm method and using demirjian’s method. discussion malocclusion is the most common dental problem in paediatric dentistry, with a prevalence of 20%–100%. when planning treatment, it is necessary to know the patient’s age. biological age is more significant than chronological age in the planning of treatment. several biological factors can be used to determine developmental age, e.g. cvm and tooth maturation.1,3 during growth and development, the bones undergo changes that can be detected radiographically in the form of lateral cephalometric radiographs and by observing the maturation of the cervical vertebrae using the cvm method. the stages of cvm were determined based on the body morphology of c2, c3, and c4, i.e. the analysis of the development of the inferior edge basins of c2, c3, and c4, as well as changes in body shape and the sizes of c3 and c4. indicators of biological age can also be observed in the maturity of the teeth using demirjian’s method. the assessment of tooth maturation is based on the classification stages of each tooth. this assessment is divided into eight stages, which are observed using panoramic radiographs.2,3,5 in previous studies, cvm was evaluated using the cvm index, which is applied to observe the concavity of the inferior edge and the shape of the cervical vertebrae, which are subsequently grouped into six stages. these changes are related to the morphological modification of the vertebral shape, as well as the estimated time interval from the peak mandibular growth. in particular, peak mandibular growth has been reported to occur concurrently with cvm stages 3 and 4. the assessment of tooth maturation using demirjian’s method was presented as an alternative to bone maturation assessment. although several studies have reported that tooth maturation can be used as an indicator of development, tooth maturation has a relative relationship to the occurrence of peak mandibular growth.11,12 this study also demonstrated that there is a difference of only 0.3 in the average of the cvm method and that of demirjian’s method, meaning that the standard deviation of the cvm method and demirjian’s methods also had a difference of only 0.45. considering these results, it can be stated that no significant difference was found, and that it is necessary to retest this method. the nonparametric wilcoxon test was applied as a statistical test in the current study because the cvm method included data that was not normally distributed. the results of the wilcoxon difference test indicated that there was a difference in the mean rank value of only 0.85 between demirjian’s method and the cvm method; accordingly, the asymptotic significance (demirjian and cvm) had a p-value of 0.116 for result higher than 0.05, demonstrating that there was no significant difference between the results obtained from the cvm method and demirjian’s method. in conclusion, the two methods discussed herein yielded equally accurate results for determining mandibular length growth. additional research is necessary to determine which of the two methods is the most accurate. references 1. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10(1): 7. 2. szemraj a, wojtaszek-słomińska a, racka-pilszak b. is the cervical vertebral maturation (cvm) method effective enough to replace the hand-wrist maturation (hwm) method in determining skeletal maturation? a systematic review. eur j radiol. 2018; 102: 125–8. 3. mollabashi v, yousefi f, gharebabaei l, amini p. the relation between dental age and cervical vertebral maturation in orthodontic patients aged 8 to 16 years: a cross-sectional study. int orthod. 2019; 17(4): 710–8. 4. mcnamara ja, franchi l. the cervical vertebral maturation method: a user’s guide. angle orthod. 2018; 88(2): 133–43. 5. mini m, thomas v, bose t. correlation between dental naturity by demirjian method and skeletal maturity by cervical vertebral mat u r it y met ho d usi ng pa nora m ic rad iog raph a nd lat era l cephalogram. j indian acad oral med radiol. 2017; 29(4): 362. 6. chinna r, chinna s. dental age estimation by using demirjian method in adultsa review. world j pharm pharm sci. 2019; 8(4): 458–65. 7. verma s, tikku t, khanna r, maurya r, srivastava k, singh v. predictive accuracy of estimating mandibular growth potential by regression equation using cervical vertebral bone age. natl j maxillofac surg. 2021; 12(1): 25–35. 8. cangialosi tj, vives vj. another look at skeletal maturation using hand wrist and cervical vertebrae evaluation. open j orthop. 2018; 8(1): 1–10. 9. al-mohaidaly ms. correlation between cervical vertebral maturation and chronological age in a group of saudi arabian females. ec dent sci. 2016; 3(5): 608–14. 10. enikawati m, soenawan h, suharsini m. panjang maksila dan mandibula pada anak usia 10-16 tahun: kajian sefalometri lateral. jakarta: fakultas kedokteran gigi universitas indonesia; 2013. p. 1–15. 11. perinetti g, braga c, contardo l, primozic j. cervical vertebral maturation: are postpubertal stages attained in all subjects? am j orthod dentofac orthop. 2020; 157(3): 305–12. 12. oyonarte r, sánchez-ugarte f, montt j, cisternas a, morales-huber r, ramirez-lobos v, janson g. diagnostic assessment of tooth maturation of the mandibular second molars as a skeletal maturation indicator: a retrospective longitudinal study. am j orthod dentofac orthop. 2020; 158(3): 383–90. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p13–15 dewi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 13–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p13-15 7171 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 71–75 synergistic effect of the combination of cinnamomum burmanii, vigna unguiculata, and papain exracts derived from carica papaya latex against c. albicans biofilms degradation muhammad luthfi, indah listiana kriswandini, and fitriah hasan zaba departement of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: candidiasis is an opportunistic infection commonly occurs on host with immunodeficiency, organ transplantation, leukopenia, or radiation therapy. biofilms are structures that protect c. albicans from antifungals treatments. c. albicans biofilms display multidrug resistance to antifungal agents. purpose: this study aimed to know whether the combination of cinnamomum burmannii, vigna unguiculata, and papain extracts derived from carica papaya latex has inadequate inhibitory effects against c.albicans biofilms compared to the combination of cinnamomum burmannii and vigna unguiculata extracts. method: c. albicans growing on sda were dissolved in 1 mcfarland of sterile aquadest. micro-plate was filled with 180 µl of sdb, glucose 8%, and 20 µl of c. albicans. suspension was incubated at 37oc overnight. extracts were added and incubated for 24 hours. then, each well was washed with distilled water, and stained with crystal violet 0.1% for 15 minutes. afterward, each well was washed with distilled water and immediately stained with acetic acid. after 15 minutes of staining, the suspension was transferred to a new well, then measured with micro-plate reader at 595 nm. results: the combination of cinnamomum burmanii and vigna unguiculata extracts had adequate inhibitory effects which is equal to 60.75%. inhibition increased to 72.09%, 79.06%, and 79.50% after papain derived from carica papaya latex was added on concentrations of 138 mg/ml, 276 mg/ml, and 552 mg/ml. conclusion: the combination of cinnamomum burmanii (0.25µg/ml), vigna unguiculata (200 µg/ml), and papain (276 µg/ml) extracts showed an optimum synergic inhibition for c. albicans biofilms. keywords: c. albicans biofilm; cinnamomum burmannii; vigna unguiculata; papain correspondence: muhammad luthfi, department of oral biology, faculty of dental medicine universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: m.luthfi7@yahoo.com introduction opportunistic fungal infection has been discussed in this decade. the ability of fungi to be able to infect the host actually depends on the immune response of the host and the presence of xenobiotics. opportunistic fungal infections are mostly caused by candida species infections.1 candida albicans (c. albicans) grows excessively in patients with low immune circumstances, such as human immunodeficiency virus (hiv) infection, organ transplantation, leukopenia, post-surgery, or radiation therapy. thrush or oropharyngeal candidiasis is a fungal infection found on the surface of the oral mucosa, therefore, the ability of c. albicans to form biofilms has a huge impact on its ability to cause disease.2 results of a research on patients with hiv / aids showed that the prevalence of oral candidiasis from 2008 to 2009 in cipto mangunkusumo hospital was approximately 80.8%, while in dr. hasan sadikin hospital about 27% and in h. adam malik hospital about 28.7%. similarly, mccullough said that 70-80% of oral candidiasis is caused by c. albicans.3 laboratory diagnosis and treatment of diseases caused by candida species, especially c. albicans, furthermore, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p71-75 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p71-75 72 luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 71–75 has not given satisfactory results because of the resistance to common antifungal. c. albicans biofilms have more multidrug resistance to fluconazole, amphotericin b, flucytosine, itraconazole, and ketoconazole than c. albicans in free-cells.1 biofilms, moreover, are structured microbial communities, which are bound to the surface and become embedded in an extracellular matrix polymer produced.4 extracellular matrix provides a significant contribution to drug resistance in c. albicans biofilms. extracellular matrix is composed of materials considered as causative factors of resistance. biofilms cannot be penetrated by antifungal for c. albicans cells coated by β-glucans, chitin, and glycoproteins. the realization of this biofilm is a selfdefense form of c. albicans against radical agents.4 in addition, cinnamon (cinnamomum burmannii) is known to have antimicrobial properties. cinnamomum burmannii contains with sinamaldehid, eugenol, cinnamic acid, sesquiterpene, and proanthocyanidin, which have antimicrobial power. extract of cinnamomum burmannii has a minimum inhibitory concentration (mic) of 0.33 mg/ ml against c. albicans.5 papain derived from carica papaya latex and fluconazole, furthermore, has synergistic action in inhibiting the growth of c. albicans. the result is a synergistic effect to degrade the cell wall of c. albicans. papain is responsible as antifungal at a concentration of 138 μg/ml. papain containing a specific content in the form of nasetil-β-dglukosaminidase and α-d-mannosidase is known to have a role in the degradation of c. albicans biofilm matrix .6 tolo bean extract (vigna unguiculata), moreover, is known to contain the β-1,3-glucanase enzyme. vigna unguiculata at a concentration of 200 μg/ml has higher activity of β-1,3-glucanase enzyme that at a concentration of 3.152 u/mg.8 the combination of β-1,3-glucanase enzymes derived from snails and cinnamomum burmannii extract is able to lyse c. albicans biofilm, and the result is the death of c. albicans biofilm cells, about 75%.5 snails have β-1,3-glucanase enzymes, but the enzymes are unstable. it is reported that β-1,3-glucanase enzymes derived from snails and stored at room temperature has glucanase activity decreasing dramatically. similarly, in the storage temperature of 24oc, the activity of β-1,3-glucanase also decreases.6 enzymes from vigna unguiculata have good stability and high glucanase activity.7 in short, cinnamomum burmannii extract will degrade free albicans cells. 8 extracellular matrix of c. albicans biofilms is composed of β-glucan (50-60%), mannoprotein (30-40%), and chitin (0.6 to 9%).4 β 1,3-glucanase enzyme derived from vigna unguiculata extract is able to hydrolyze the components of the extracellular matrix in the glucanase form.7 α-β-d-mannosidase derived from papain is able to hydrolyze the components of the biofilm matrix in the form of mannoprotein, while n-acetyl-β dglukosaminidase of papain is able to hydrolyze components of the biofilm matrix in the form of chitin.6 in other words, the use of cinnamomum burmannii extracts has antimicrobial activity, while the combination of vigna unguiculata and papain extracts derived from carica papaya latex is synergistic in the extracellular matrix of the biofilms hydrolyzing c. albicans. this aim of this study was to know whether the combination of cinnamomum burmannii, vigna unguiculata, and papain extracts derived from carica papaya latex has inadequate inhibitory effects against c. albicans biofilms compared to the combination of cinnamomum burmannii and vigna unguiculata extracts. materials and methods this research is an experimental laboratory research with post-test-only control group design. the research was conducted at rumah sakit khusus infeksi (hospital for infection) universitas airlangga in october-november 2015. samples used in this research were albicans. the number of samples used were determined by using lemeshow formula, about seven samples. the samples were divided into five groups. the control group consisted of c. albicans planktonics and c. albicans biofilms without being treated. group i consisted of c. albicans planktonics and c. albicans biofilms treated with 0.25 mg/ml of cinnamomum burmannii extract and 200 μg/ml of vigna unguiculata extract. group ii consisted of c. albicans planktonics and c. albicans biofilms treated with 0.25 mg/ml cinnamomum burmannii extract, 200 μg/ ml of vigna unguiculata extract, and 138 μg/ml of papain extract. group iii consisted of c. albicans planktonics and c. albicans biofilms treated with 0.25 mg/ml cinnamomum burmannii extract, 200 μg/ml of vigna unguiculata extract, and 276 μg/ml of papain extract. group iv consisted of c. albicans planktonics and c. albicans biofilms treated with 0.25 mg/ml cinnamomum burmannii extract, 200 μg/ml of vigna unguiculata extract, and 552 μg/ml of papain extract. this research was started with the preparation of cinnamomum burmannii, vigna unguiculata, and papain extracts. the extracts of cinnamomum burmannii and vigna unguiculata were obtained from balai penelitian dan konsultasi industri (research and industry consulting center) in surabaya together with aquadest as solvent. meanwhile, papain extract was derived from carica papaya latex obtained from sigma (p3x15-250). next, 25 mg of cinnamomum burmannii extract was dissolved in 100 ml of distilled water. vigna unguiculata extract then was added in a concentration of 200 mg/ml, whereas papain extract was added in concentrations of 138 mg/ml, 276 mg/ml, and 552 mg/ml. c. albicans planktonics and biofilms were prepared by using microtiterdish assay method. c. albicans taken from sda stock was used to make suspension in 1 mcfarland of sterile distilled water (3 x 107 cfu/ml). microplates then were filled with 180 ml of sdb together with 8% glucose and 20 ml of c. albicans to grow c. albicans biofilm. meanwhile, to grow c. albicans planktonics, micro-plates dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p71-75 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p71-75 7373luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 71–75 then were filled with 180 ml of sdb together and 20 ml of c. albicans without 8% glucose. afterwards, those micro-plates were incubated for 24 hours at 37°c. they then were washed with sterile aquadest before the extracts were added into them and incubated for 24 hours. after that, the micro-plates were washed again, and then stained with 0.1% crystal violet for 15 minutes. those micro-plates were washed with sterile distilled water. they then were given with acetic acid for 15 minutes and transferred to a new ones. od values were read using micro plate reader at a wavelength of 595 nm.10,11 inhibition effect then was determined based on the values of od. if od values obtained were getting smaller, inhibition effect would be indicated to be high. the formula used to calculate the inhibitory activity was as follows:11 (mean of od595 control – mean of od595 concentration) × 100% mean of od595 control the normality of data were tested using kolmogorov smirnov test, while the homogeneity of data were tested using levenne’s test. finally, to identify the significance of the difference among the treatment groups, post hoctukey hsd test was conducted.12 results absorbance levels of planktonic and biofilm cells were observed in the value of optical density (od) using a microplate reader with a wavelength of 595 nm. od value obtained then was comparable with c. albicans biofilm formation. to determine the growth of c. albicans biofilms, a preliminary experiment on the relation of c. albicans biofilm growth and incubation time was conducted, and the results showed that the optimal biofilm growth was at 24 hours. after knowing the optimal incubation time for c. albicans biofilm to grow well, a combination of the extracts was used to inhibit the growth of c. albicans biofilms. this aimed to compare the effectiveness of the combination of cinnamomum burmannii, vigna unguiculata, and papain extracts in inhibiting the growth of c. albicans biofilms. in the control group, c. albicans planktonic cells was untreated with c. albicans biofilm. in this research, the concentration of cinnamomum burmannii extract used was 0.25 mg/ml, the concentration of vigna unguiculata extract was also the same or equal to 200 pg/ml, while the concentrations of papain extract were 138 μg/ml, 276 pg/ml, and 552 μg/ml. the results of od values obtained were as follows (table 1). based on table 1 above, it can be seen that the means of od values of c. albicans planktonics was 0.367, while the means of od values of c. albicans biofilm was 0.688. the od value of c. albicans biofilms was higher than the od values of c. albicans planktonics. the od value of c. albicans biofilms dropped to 0.27 after treated with the combination of cinnamomum burmannii and vigna unguiculata extracts. the od value of c. albicans biofilms decreased into 0.192 after treated with the combination of the cinnamomum burmannii, vigna unguiculata, 138 μg/ml of papain extracts. the od value of c. albicans biofilms remained down to 0.144 after papain concentration was increased to 276 mg/ml, and the od value declined into 0.141 after the addition of papain concentration to 552 mg/ml. the comparison of the od values of c. albicans planktonics and c. albicans biofilms in the control group and the od values of c. albicans biofilms treated can be seen in the graph below (figure 1). table 1. the od values of c. albicans biofilms after the treatment treatment groups n means of sd od values planktonic cells 7 0.367 0.530 biofilms 7 0.688 0.174 cb +vu extracts 7 0.270 0.187 cb +vu + p138 extracts 7 0.192 0.304 cb +vu + p276 extracts 7 0.144 0.207 cb +vu + p552 extracts 7 0.141 0.149 note: cb: cinnamomum burmanii extract; vu: vigna unguiculata extract; p138: 138 μg/ml of papain; p276: 276 μg/ ml of papain; p552: 552 μg/ml of papain. 11 table 1. the od values of candida albicans biofilms after the treatment treatment groups n means of sd od values planktonic cells 7 0.367 0.530 biofilms 7 0.688 0.174 cb +vu extracts 7 0.270 0.187 cb +vu + p138 extracts 7 0.192 0.304 cb +vu + p276 extracts 7 0.144 0.207 cb +vu + p552 extracts 7 0.141 0.149 note: cb: cinnamomum burmanii extract; vu: vigna unguiculata extract; p138: 138 μg/ml of papain; p276: 276 μg/ml of papain; p552: 552 μg/ml of papain. figure 1. the graph of od values of candida albicans biofilms with various treatment. figure 1. the graph of od values of c. albicans biofilms with various treatment. table 2. the inhibition percentage of c. albicans biofilms treatment groups n the inhibition percentage cb +vu extracts 7 60.75 % cb +vu +p138 extracts 7 72.09 % cb +vu +p276 extracts 7 79.06 % cb +vu +p552 extracts 7 79.50 % dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p71-75 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p71-75 74 luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 71–75 the inhibition percentage of c. albicans biofilms obtained can be seen in table 2. the inhibition percentage of the combination of cinnamomum burmannii and vigna unguiculata extracts against c. albicans biofilms in the control group was 60.75%. the inhibition percentage of the combination of cinnamomum burmannii, vigna unguiculata, and 138 ug/ml of papain extracts was 72.09%. the inhibition percentage of the combination of cinnamomum burmannii, vigna unguiculata, and 276 mg/ml of papain extracts was 79.06%. meanwhile, the inhibition percentage of the combination of cinnamomum burmannii, vigna unguiculata, and 552 μg/ml of papain extracts was 79.50%. a statistical test was conducted on distribution of data in each group using the kolmogorov-smirnov test. the results of kolmogorov-smirnov test showed that the distribution of data in those treatment groups was normal because p-value in the treatment groups was greater than 0.05. after that, levenne test was conducted to know the homogeneity of data. the results of levenne test showed that p-value obtained was <0.05. it means that the variation of the data was not homogeneous. as a result, kruskal wallis test was performed. the results of kruskal wallis test showed that value obtained was > 0.05. it indicates that there were significant differences between each treatment group. to know the differences of each treatment group, post hoc-tukey hsd test then was carried out. the results of post hoc-tukey hsd test showed that there was a significant difference between the group treated with biofilms and the treatment groups treated with cb + vu extracts, cb + vu + p138 extracts, cb + vu + p276, and cb + vu + p552 extracts. similarly, there was also a significant difference between the group treated with cb + vu extracts and the groups treated with cb + vu + p276 and cb + vu + p552 extracts. discussion in the process of biofilm inhibition, there are some stages of the degradation of the elements of c. albicans biofilm biomass. extracellular matrix is one of the elements composing the biomass of biofilms. one of the elements composing the extracellular matrix of the biofilms is glucan (50.60%). the hydrolysis mechanism of glucanase contained in vigna unguiculata against c. albicans biofilm is related to glucan in the cell walls of fungi that can be utilized by glucanase enzyme as a substrate by cutting the glucose residues of non-reducing end of polymers or oligomers, resulting in forming a glucose monomer.13 cinnamomum burmannii has several compounds that play a role in degradation of c. albicans cells, such as sinamaldehide and eugenol. the ability of sinamaldehide in inhibiting the growth of c. albicans due to the free 3-phenyl group that can bind to aspartic proteases in the wall of c. albicans cells and also bind to oxygen required for the metabolism of c. albicans. these bounds can cause sinamaldehide inhibits the synthesis of enzymes on the wall of c. albicans cells and the metabolism of c. albicans cells, resulting in the death of c. albicans cells.8 eugenol, is known to be lipophylic, which can penetrate between fatty acid chains and layers of bilayer membrane by altering the permeability of cell membranes. if the phenol compound interacts with the cell wall of c. albicans, there will be denaturation of proteins in the cells of c. albicans. the interaction causes a change in the balance of protein molecules, resulting in a change in the structure of the protein and triggering coagulation. protein experiencing coagulation will lose its physiological activities that cannot function properly. changes in the structure of proteins in c. albicans will cause increased permeability of the cells, so the cell growth is inhibited and then the cells will die, thus eugenol has an ability to reduce adherent and to inhibit metabolism of c. albicans biofilms. 14 therefore, in the treatment group, the inhibition was adequate when c. albicans biofilms were treated with combination of cinnamomum burmannii and vigna unguiculata compared to the control group. it means that the combination of cinnamomum burmannii and vigna unguiculata extracts is able to inhibit c. albicans biofilms with the inhibition of 60.75%. in addition, papain contains specific enzymes, namely αd-mannosidase and n-acetyl-β-dglucosaminidase hydrolyzing the extracellular matrix of the biofilms, such as mannoprotein and chitin. glycosidase process of both the enzymes of papain can occur by cutting the polysaccharide chain residues in the extracellular matrix of biofilms.6 in the other treatment group, moreover, the inhibition was adequate when 276 mg/ml of papain was added to the combination of cinnamomum burmannii and vigna unguiculata used to treat c. albicans biofilms. papain extract at that concentration could inhibit c. albicans biofilms with good inhibitory, increasing from 60.75% to 79.06%. it means that there was an increase in the inhibition of 18.31%. the addition of papain extract at the concentrations ranging from 138 mg/ml to 276 pg/ml and 552 mg/ml did not show adequate inhibition. papain can be active when given activator since the enzyme contained in papain can be activated or inhibited. compounds classified as activators of papain are cysteine, sufida and sulfite, as well as a chelator of heavy metals, such as edta and n-bromosuksinimida; whereas compounds classified as inhibitors of papain are pmsf, tlck & tpck, e-64, heavy metals, cystatin, and leupeptin.15 however, papain used in this research was not classified as an activator. thus, od values of the inhibition of c. albicans biofilms obtained were inadequate though the concentration of papain increased. another possibility is c. albicans can develop some mechanisms to overcome the existing antimicrobial agents by producing genetic mutation enzyme and transmission for new generation.16 in the process of extract administration, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p71-75 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p71-75 7575luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 71–75 the incubator temperature was 37°c. this possibility also becomes a factor causing the working of papain on c. albicans biofilms not optimal. concentration level (ph), furthermore, is also considered as a factor that can influence the effectiveness of the activity of the enzyme. the effectiveness of the enzyme showed a gradual increase with increasing ph from ph 3.5 to ph 7.5, whereas at ph 9 resulting in a decrease in the activity of papain.17 meanwhile, in the treatment groups, pbs ph used as a solvent was 7. it can be concluded that the combination of 0.25 mg/ml of cinnamomum burmannii, 200 mg/ml of vigna unguiculata, and 276 mg/ml of papain extracts had an optimal and synergistic effect on the inhibition of c. albicans biofilms. references 1. darouiche ro, mansouri md, kojic em. antifungal activity of antimicrobial-impregnated devices. clin microbiol infect 2006; 12(4): 397-9. 2. richard ml, nobile cj, bruno vm, mitchell ap. c. albicans biofilm defective mutants. eukaryot cell 2005; 4(8): 1493–502. 3. mccullough mj, savage nw. oral candidosis and the therapeutic use of antifungal agents in dentistry. aust dent j 2005; 50(4 suppl 2): s36-9. 4. nett j, lincoln l, marchillo k, massey r, holoyda k, hoff b, vanhandel m, andes d. putative role of beta-1,3 glucans in c. albicans biofilm resistance. antimicrob agents chemother 2007; 51(2): 510-20. 5. krishna kl, paridhavi m, patel ja. review on nutritional, medicinal and pharmacologinal properties of papaya (carica papaya linn.). natural product radiance 2008; 7(4): 364-73. 6. giordani r, siepaio m, moulin-traffort j, régli p. antifungal action of carica papaya latex: isolation of fungal cell wall hydrolysing enzymes. mycoses 1991; 34(11-12): 469-77. 7. oliveira jta, barreto alh, vasconcelos im, eloy yrg, gondim dm f, fer na ndes cf, frei re-fil ho f r. role of a ntioxida nt enzymes, hydrogen peroxide and pr-proteins in the compatible and incompatible interactions of cowpea (vigna unguiculata) genotypes with the fungus colletotrichum gloeosporioides. j plant physiol pathol 2014; 2(3):2-8. 8. erna f, rostiny, sherman s. efektivitas minyak kayu manis dalam menghambat pertumbuhan koloni c. albicans pada resin akrilik. journal of prosthodontic 2010; 11(2): 19-23. 9. o’toole, ga. microtiter dish biofilm formation assay. j vis exp 2011; (47) pii: 2437. 10. mahmoudabadi az, zarrin m, kiasat n. biofilm formation and susceptibility to amphotericin b and fluconazole in c. albicans. j microbiol 2014; 7(7): e17105. 11. bakkiyaraj d, nandhini jr, malathy b, pandian sk. the antibiofilm potential of pomegranate (punica granatum l.) extract against human bacterial and fungal pathogens. biofouling 2013; 29(8): 929-37. 12. kao ls, green c. analysis of variance: is there a difference in means and what does it mean?. j surg res 2008; 144(1): 158-70. 13. el-katatny mh, somitsch w, robra kh, ei-katatny ms, gübitz gm. production of chitinase and β-1,3-glucanase by trichoderma harzianum for control of the phytopathogenic fungus sclerotium rolfsii. j food technol biotechnol 2000; 38(3): 170-80. 14. raja mrc, srinivasan v, selvaraj s, mahapatra sk. versatile and synergistic potential of eugenol: a review. pharm anal acta 2015; 6(5): 367. 15. dongoran ds. pengaruh aktivator sistein dan natrium klorida terhadap aktivitas papain. jurnal sains kimia 2004; 8(1): 30-5. 16. nikaido h. multidrug resistance in bacteria. annu rev biochem 2009; 78: 119-46. 17. omeje ko, eze so, ozougwu ve, ubani cs, osayi e, onyeke cc, chilaka fc. application of papain from paw paw (carica papaya) latex in the hydrolysis of tiger nut (c.esculentus) proteins. mitteilungen klosterneuburg 2014; 64: 1-17. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p71-75 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p71-75 200 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 200–204 original article comparison of maxillary sinus on radiograph among males and females rona aulianisa1, rini widyaningrum2, isti rahayu suryani2, rurie ratna shantiningsih2 and munakhir mudjosemedi2 1faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of dentomaxillofacial radiology, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: an obstacle in forensic odontology is an incomplete body caused by post-mortem damage. the problem can be solved by using lateral cephalometric radiographs for victim identification. sex determination can be performed on the maxillary sinus, which is the largest among the paranasal sinuses. purpose: this study aims to analyse the maxillary sinuses’ width and height on lateral cephalometric radiographs among male and female subjects. methods: the study samples were 60 lateral cephalometric radiographs (30 males and 30 females) between the ages of 20 and 40, with complete permanent dentition (or third molar absence). the height and the width of maxillary sinus measurement were performed using measurement tools of ezdent-i vatech software. results: the average width of the maxillary sinus on males was 40.60 ± 1.56 mm, and the height was 35.02 ± 2.09 mm, while the width and the height on females were 36.93 ± 1.30 mm and 29.72 ± 1.76 mm, respectively. the independent t-test reveals a significant difference (p<0.05) between males and females, both in the maxillary sinus’s width and height on the lateral cephalometric radiograph. conclusion: the maxillary sinus in males is larger than in females, it opening up possibilities for disaster victim identification. keywords: cephalometric; maxillary sinus; sex estimation; width; height correspondence: rona aulianisa, faculty of dentistry, universitas gadjah mada. jl. denta 1, sekip utara, yogyakarta 55281, indonesia. email: rona.a@mail.ugm.ac.id introduction indonesia is a country most vulnerable to natural disasters and is at risk from numerous mortality-related hazards.1 disaster victim identification in human-made and natural disasters requires ante-mortem and post-mortem medical record comparisons. dental records play an essential part in the identification of bodies.2,3 one of the pressing issues in body identification is sex estimation, which can be achieved using either morphological or metric methodologies of the maxillary sinus. the bones for sex estimation, especially the pelvis and cranium, are particularly valuable when the body has been incinerated or has decayed. research and reports show the maxillary sinus remains unaffected even after burns and significant injuries.4 the maxillary sinus is also the largest sinus of the paranasal sinus and, therefore, easy to analyse.4,5 the maxillary sinus can be analysed using 2d and 3d radiographs.6 lateral cephalometric radiograph is one of the 2d radiographs showing the maxillofacial complex such as teeth, soft tissue, the relation of maxilla and mandible, and another part of the cranium comprising the maxillary sinus.7,8 this radiograph is commonly used in assessing, planning, and evaluating orthodontic treatments. it is also used in growth analysis, morphological analysis, and treatment analysis of orthognathic surgeries.8 for sex determination, the maxillary sinus’s combined width and height can be used when the entire skeleton is unavailable. sex determination are generally based on findings of dimorphism between males and females in most human bones. previous studies reveal the maxillary sinus is larger in males than in females in the current human population.2,4,9 the purpose of this study was to analyse the difference in the maxillary sinus’ width and height between male and female subjects using a lateral cephalometric radiograph in the prof. soedomo dental hospital of universitas gadjah mada, yogyakarta, indonesia. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p200–204 mailto:rona.a@mail.ugm.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p200-204 201aulianisa et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 200–204 materials and methods the ethics committee approved the protocol for this study [faculty of dentistry universitas gadjah mada (ref. 00332/kkep/fkg-ugm/ec/2020)]. the study sample comprised 60 lateral cephalometric radiographs collected from 30 males and 30 females 20–40 years old. only lateral cephalometric radiographs from patients with complete permanent dentition were included in this study. however, the samples from patients with missing the third molar were also included. all samples were diagnostically acceptable and presented images of the completely formed maxillary sinus in adequate contrast and density, especially on the facial surface of the maxilla, infraorbital bone, and alveolar process of the maxilla. the quality assessment was conducted by a trained observer and calibrations were made under a radiologist. the radiographic image’s features of the maxillary sinus bearing pathologies or abnormalities were excluded. samples were obtained from the department of dentomaxillofacial radiology in prof. soedomo dental hospital, universitas gadjah mada, yogyakarta, indonesia. all were generated using vatech pax-i pch-2500 (korea), 90 kv, 10 ma. the measurements of the maxillary sinus were performed on these radiographs using ezdent-i vatech software (figure 1) by utilising the grid feature available on the software. first, the width of the maxillary sinus was measured using measurement tools by drawing a horizontal line from the anterior wall of the maxillary sinus (most anterior point of the facial surface of the maxillary bone, point a in figure 1) parallel with the horizontal plane to the posterior wall of the maxillary sinus (infratemporal surface of the maxillary bone, point b in figure 1). subsequently, the height of the maxillary sinus was drawn as a vertical line perpendicular to the centre of the horizontal line. it was drawn from the cranial wall of the maxillary sinus (infraorbital bone, point c in figure 1) to the caudal wall of the maxillary sinus (alveolar and palatine processes of the maxillary bone, point d in figure 1). the reliability of the measurement using lateral cephalometric radiograph depends on the position of the head; hence the radiograph with the standardised head position was taken to minimise the measurement error in this study. twelve radiographs, consisting of six male subjects and six female subjects (20% of the sample), were selected at random and remeasured to determine intraand interobserver reliability. one observer served as the primary, and the reliability of the intra-observer was calculated between measures conducted one week apart. the reference observer with the most experience then served as another observer, and inter-observer reliability was assessed. intra and inter-observer reliability were recorded and calculated by using cronbach’s alpha correlation. the independent sample t-test was used to compare the maxillary sinus measurements between the two groups. results in the male and female groups, the mean value for the maxillary sinus width was 40.60 ± 1.56 mm and 36.93 ± 1.30 mm, respectively. the male group’s mean value for maxillary sinus height was 35.02 ± 2.09 mm, whereas the mean height in the female group was 29.72 ± 1.76 mm. the figure 1. measurement of the width and the height of the maxillary sinus on the lateral cephalometric radiograph. the width of the maxillary sinus was measured on the horizontal line from the anterior wall (a) to the posterior wall of the maxillary sinus (b). the height of the maxillary sinus was drawn as a vertical line from the cranial wall (c) to the caudal wall of the maxillary sinus (d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p200–204 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p200-204 202 aulianisa et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 200–204 normality test shows the significant value of the maxillary sinus width and height, both male and female, as 0.200 (>0.05) and the data were normally distributed. compared with females, the independent sample t-test showed a statistically significant (p<0.05) larger dimension in males (figure 2). based on the cronbach’s alpha correlation, the inter-and intra-observer reliability in this study showed an extremely high correlation (0.998 for maxillary sinus width and 0.995 for maxillary sinus height). discussion sex identification from the remains of human skeletons is one of the most important and difficult forensic procedures. sex determination accuracy has been reported to be 80–90% from the long bones only, 90–95% from both the skull and the long bones, 95% from the pelvis only or the pelvis and the long bones, 98% from both the pelvis and the skull, and 100% from a skeleton. various methods have been demonstrated for sex identification, such as dna analysis, fingerprints, lip grooves, palatal rugae, and morphometric analysis of maxillary sinus.2,4,10 the largest paranasal sinus is the maxillary sinus, which can be observed in various shapes and sizes. the maxillary sinus also makes a major contribution to the formation of facial contours.11 variations are affected by age and sex. result of the study reveals the width and height of the maxillary sinus in males are significantly different from those in females. most studies demonstrate that maxillary sinuses in males are larger than those in women. these have been found in the indian and brazilian populations.2,12,13 the result of this study was in accordance with previous studies, despite being carried out in a distinct population. the maxillary sinus is formed by the lateral wall of the nasal cavity, the infratemporal surface of the maxilla, the facial surface of the maxilla, orbit floor, the palatine process, and the alveolar of the maxilla.11 the maxillary sinus begins to develop and continue with its enlargement after the eruption of deciduous teeth at ten weeks in utero. the maxillary sinus grows most rapidly between one to eight years old. sinus pneumatisation ceases with the completion of permanent teeth eruption by the age of 20 years. the analysis of maxillary sinus is not at all reliable when performed in prepubertal populations, and subjects included in the present study were therefore over the age of 20.12 the maxillary sinus can be assessed using both 2d and 3d radiographs. lateral cephalometric radiograph shows a lateral view of the cranium, which provides diagnostic information for the skeletal, dental and soft tissues anatomic landmarks.4 recent literature mentions that 3d radiography, such as computed tomography and cone-beam computed tomography, provides excellent images; thus, they are more suitable as the gold standard in evaluating paranasal sinuses and craniofacial bones. these techniques are highly effective and able to provide high-definition images as well as three-dimensional information. however, they need a high radiation dosage resulting in a high cost, thus limiting accessibility and its application in the field of forensic medicine and forensic dentistry.13,14 a lateral cephalometric radiograph can be used for morphometric analysis of the maxillary sinus. it can be done using a manual or digital tracing method. the manual tracing or conventional tracing method uses tools such as sliding callipers or graded rulers. however, along with the rapid development of computer radiography, the manual tracing method has been replaced by digital tracing methods. in this study, digital tracing was used because it provides many advantages such as being more effective, efficient, easier to use and reducing personal errors in forensic analysis.15 results reveal that the maxillary sinus in males (n=30) were larger than in females (n=30) on lateral cephalometric radiograph using ezdent-i vatech software. the software was used as a digital tracing facility connected and integrated with the x-ray machine used in the research. the width and height of the maxillary sinus in males showed 40.6 ± 1.56 35.02 ± 2.09 36.93 ± 1.30 29.72 ± 1.76 0 5 10 15 20 25 30 35 40 45 width height t he m ea n va lu es o f m ax ill ar y si nu s male female * * figure 2. the comparison of maxillary sinus measurement between male and female groups. the data represent the mean ± sd values of the width and the height of the maxillary sinus. *p<0.05, based on an independent sample t-test. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p200–204 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p200-204 203aulianisa et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 200–204 an average size of 40.60 ± 1.56 mm and 35.02 ± 2.09 mm, respectively, while the measurements in females were 36.93 ± 1.30 mm and 29.72 ± 1.76 mm, respectively (figure 2). a previous study2 conducted on 50 subjects (25 males and 25 females) in the indian population described the mean width of male and female maxillary sinuses as 38 mm and 37.3 mm, respectively. in addition, the mean height of male and female maxillary sinuses was 30.4 mm and 28.5 mm, respectively. the maxillary sinus in males has been reported to be larger than in females. unlike our findings, however, the difference between the two sexes was not significant.2 another previous study9 comprising 80 iranian subjects, consist of 40 males and 40 females, describe the mean width of male and female maxillary sinuses as 40.31 mm and 37.31 mm, respectively. meanwhile, the mean height of male and female maxillary sinuses among iranian subjects were 40.48 mm and 38.7 mm, respectively. according to the previous study, it is statistically significant, the maxillary sinus in males has been larger than in females, defining the difference between the two groups.9 the previous study16 conducted on ct scan radiographs show the mean width (anteroposterior) of male and female maxillary sinuses was 42.60 ± 3.79 mm and 36.00 ± 4.09 mm, respectively. the mean height (superoinferior) of male and female maxillary sinuses was 38.21 ± 5.77 mm and 33.34 ± 6.57 mm, respectively.16 it can be concluded that in males, the maxillary sinuses are larger than females, and the difference was statistically significant, as assessed in both 2d9 and 3d radiographs.16 the differences between the values obtained in the several studies can be attributed to inclusion criteria, sample size, applied measurement methods, reference points, and differences in the types of radiographs assessed.9,12,16 they can also be explained by the different developmental patterns of the neurocranium and viscerocranium, which are closely linked to functional tissue in a human and are affected by numerous internal and external factors.17 the internal factors include genetics, the pneumatisation process of sinuses and the size of the skeleton may possibly influence the maxillary sinus. a previous study18 found the skeleton’s external facial measurement (palatal length, bimaxillary width, and facial length) was positively and significantly correlated with the maxillary sinus volume.18 it is known males and females have significant differences in growth patterns during a pubertal growth spurt.17 the duration of the pubertal spurt was longer, and the growth velocity was greater for males than for females.19 in our study, the internal factor was uncontrolled because the samples were obtained from the database in our dental hospital. the external factors during adulthood may influence the changes in maxillary sinus size, morphologic pattern, and anatomy, mainly due to the loss of teeth. a fully edentulous maxilla shows an increase in maxillary sinus volume in relation to decreased function and less bone stress that subsequently induce atrophy and degrade the surface of the maxillary sinus.20 it also increased osteoclastic activity, simultaneously with maxillary resorption, hence resulting in maxillary sinus expansion.21 teeth loss also decreases the volume and the surface of the maxillary sinus. this can be caused by the loss of minerals in the bone matrix in all directions of the maxillary sinus wall, which contracts the maxillary sinus and subsequently decreases the maxillary sinus volume.20,21 the difference in the means of maxillary sinus between males and females in our study can be used further to compare the ante-mortem and post-mortem data in forensic cases. when other methods are inconclusive, maxillary sinus in lateral cephalometric radiograph can be used for sex determination. the difference between the left or right maxillary sinuses of the same person were statistically not significant. it has been stated that while the cranium and other bones may be severely disfigured in victims who are incinerated, the maxillary sinus remains intact.9 the lateral cephalometric radiograph is one of the simplest radiographs generally used for assessment and evaluation in orthodontic treatments. nevertheless, it has some application limitations in the forensic area because of the difficulty of placing the remains of bodies in a static position due to rigour mortis and lack of equipment in forensic laboratories.4,9 the superimposition of the right and left maxillary sinus on lateral cephalometric radiograph may also affect the accuracy of the measurements.9 however, the sample in this study was obtained only from patients in prof. soedomo dental hospital. hence, they cannot be considered a genuinely representative sample of the population to generalise the result of this study to javanese and indonesian peoples. besides the fact that indonesia is in a natural disaster-prone area and one of the most ethnically diverse societies, further studies in larger or specific ethnic populations are needed to collect data for sex estimation. in conclusion, the maxillary sinus’s width and height in males are larger than in females. the present study results suggest that maxillary sinus assessment on lateral cephalometric radiograph can be used for sex determination. further studies can be developed with a larger number of samples to determine a discriminant function analysis to facilitate forensic analysis. such studies are crucial to support disaster victim identification. acknowledgement this study is part of an undergraduate thesis conducted by the first author. the authors would like to thank rellyca sola gracea for her assistance during the study. references 1. yofrido fm, harjana lt. social-fairness perception in natural disaster, learn from lombok: a phenomenological report. indones j anesthesiol reanim. 2019; 1(1): 1–7. 2. k haitan t, kabiraj a, ginjupally u, jain r. cephalometric analysis for gender determination using maxillary sinus index: a dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p200–204 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p200-204 204 aulianisa et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 200–204 novel dimension in personal identification. int j dent. 2017; 2017: 1–4. 3. interpol. disaster victim identification. interpol national central bureau. 2011. p. 1–31. 4. sidhu r, chandra s, devi p, taneja n, sah k, kaur n. forensic impor tance of maxilla r y sinus in gender deter m ination: a morphometric analysis from western uttar pradesh, india. eur j gen dent. 2014; 3(1): 53. 5. spradley mk, jantz rl. sex estimation in forensic anthropology: skull versus postcranial elements. j forensic sci. 2011; 56(2): 289–96. 6. gómez o, ibáñez o, valsecchi a, cordón o, kahana t. 3d-2d silhouette-based image registration for comparative radiographybased forensic identification. pattern recognit. 2018; 83: 469–80. 7. badam rk, manjunath m, rani m. determination of sex by discriminant function analysis of lateral radiographic cephalometry. kailasam s, editor. j indian acad oral med radiol. 2011; 23(3): 179–83. 8. white sc, pharoah mj. oral radiology: principles and interpretation. 7th ed. st. louis: mosby; 2013. p. 9, 16,17, 18, 41, 43. 9. abasi p, ghodousi a, ghafari r, abbasi s. comparison of accuracy of the maxillary sinus area and dimensions for sex estimation lateral cephalograms of iranian samples. j forensic radiol imaging. 2019; 17(june): 18–22. 10. nagare sp, chaudhari rs, birangane rs, parkarwar pc. sex determination in forensic identification, a review. j forensic dent sci. 2019; 10(2): 61–6. 11. iwanaga j, wilson c, lachkar s, tomaszewski ka, walocha ja, tubbs rs. clinical anatomy of the maxillary sinus: application to sinus floor augmentation. anat cell biol. 2019; 52(1): 17–24. 12. leao de queiroz c, terada assd, dezem tu, gomes de araújo l, galo r, oliveira-santos c, alves da silva rh. sex determination of adult human maxillary sinuses on panoramic radiographs. acta stomatol croat. 2016; 50(3): 215–21. 13. bangi bb, ginjupally u, nadendla lk, vadla b. 3d evaluation of maxillary sinus using computed tomography: a sexual dimorphic study. int j dent. 2017; 2017: 9017078. 14. putri dr, imanto m, irianto mg. identifikasi jenis kelamin menggunakan sinus maksilaris berdasarkan cone beam computed tomography (cbct). majority. 2018; 7(2): 232–7. 15. darkwah wk, kadri a, adormaa bb, aidoo g. cephalometric study of the relationship between facial morphology and ethnicity: review article. transl res anat. 2018; 12: 20–4. 16. prabhat m, rai s, kaur m, prabhat k, bhatnagar p, panjwani s. computed tomography based forensic gender determination by measuring the size and volume of the maxillary sinuses. j forensic dent sci. 2016; 8(1): 40–6. 17. przystańska a, kulczyk t, rewekant a, sroka a, jończyk-potoczna k, gawriołek k, czajka-jakubowska a. the association between maxillary sinus dimensions and midface parameters during human postnatal growth. biomed res int. 2018; 2018: 1–10. 18. koppe t, weigel c, bärenklau m, kaduk w, bayerlein t, gedrange t. maxillary sinus pneumatization of an adult skull with an untreated bilateral cleft palate. j craniomaxillofac surg. 2006; 34(suppl 2): 91–5. 19. zheng w, suzuki k, yokomichi h, sato m, yamagata z. multilevel longitudinal analysis of sex differences in height gain and growth rate changes in japanese school-aged children. j epidemiol. 2013; 23(4): 275–9. 20. möhlhenrich sc, heussen n, peters f, steiner t, hölzle f, modabber a. is the maxillary sinus really suitable in sex determination? a three-dimensional analysis of maxillary sinus volume and surface depending on sex and dentition. j craniofac surg. 2015; 26(8): e723-6. 21. jasim hh, al-taei ja. computed tomographic measurement of maxillary sinus volume and dimension in correlation to the age and gender (comparative study among individuals with dentate and edentulous maxilla). j baghdad coll dent. 2013; 25(1):87–93. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p200–204 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p200-204 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. all manuscripts submitted to the journal must be written in english. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak1, matsuo hamada2, ninuk hartati3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur, malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima, japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne, australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in original articles should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in review articles should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (original articles, case reports, or review articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission  the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair. ac.id/mkg/about/submissions#onlinesubmissions  the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id �� morphological changes of alveolar bone due to orthodontic movement of maxillary and mandibulary incisors pinandi sri pudyani, darmawan sutantyo, and sri suparwitri department of orthodontic faculty of dentistry gadjah mada university yogyakarta indonesia abstract ideally in orthodontic tooth movement, alveolar bone will follow its movement, therefore, the ratio between bone remodeling and tooth movement is 1:1. the problem whether the ratio is valid for all kinds of tooth movement such as: tipping, torquing, or bodily, or it could be applied in tooth movement of all directions such as facially, lingually or sagitally. various studies also showed many different ideas. some studies state that root dehiscence and fenestration are frequently found in final orthodontic treatment and some other state that bone remodeling can compensate tooth movement. the purpose of this study was to know the changes of alveolar bone morphology caused by anterior tooth movement. the conclusion is remodeling compensation is not matched with the extension of tooth movement, thus there are many cases of root dehiscence and fenestration after orthodontic treatment. key words: alveolar bone, tooth remodeling, orthodontic tooth movement correspondence: pinandi sri pudyani, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jln. denta, sekip utara yogyakarta 55281, indonesia. e-mail: orto_fkgugm@yahoo.com introduction during orthodontic treatment, mechanical force applied in tooth will cause alveolar bone reaction.1 the mechanic force which applied will move the tooth orthodontically and being continued to the entire tissue resulting the occurrence of remodeling process.2 orthodontic force will result in the alteration of regulating alveolar bone function as well as its cell.3 the alteration is including bone formation on tension side and bone resorption on pressure side thus the tooth will move to the new position. the process of bone formation involving osteoclast.4–9 mechanism correlates with cell activation due to mechanical force is still unknown up to now, however, the evidence has shown that electric current appears on compressed tissue.10,11 excessive force will cause the damage of periodontal tissue on pressure region, the adjacent bone will be necrotic followed by undermining resorption.12 similarly, excessive force will cause injury by principle fibers rupture in periodontal ligament, and a part of alveolar bone will be necrotic13–15 due to vessel injury. the pressure which is exceeded than the blood pressure will make capillary blood vessel in periodontal ligament collapse, which can inhibit the blood supply. on the contrary, if maximal pressure applied is lower than the blood pressure, the capillary blood vessel will not collapse, therefore, optimal force to move the tooth should not be higher than capillary blood vessel.16 direct alteration occurs in reconstructive step of alveolaris processus, but, if the tooth is continuously moved tipping toward the palate, alteration will be in cortical palate of alveolar processus,1 therefore, the current study has discussed on the limitation of tooth movement which could be improved by alveolar bone remodeling.13–15 excessive orthodontic force which is frequently applied in orthodontic practice will not move the tooth any furher, but it will cause excessive load on periodontal tissue and as a result it will inhibit tooth movement.7–8 some studies still showed various ideas on the changes of alveolar bone due to anterior tooth movement. it is caused by alveolar tooth remodeling as a response to various tooth movement in every person. if tooth movement area is limited, excessive orthodontic force will cause cortical bone resorption and root exposure will also occur because the tooth loses the supporting tissue i.e. alveolar bone. various opinions appear such as whether the capacity of bone alveolar remodeling can compensate the loss bone.15 some researchers suggested that root dehiscence will occur if the tooth is moved facially.17 others said that incisors retraction and root torque will not change anterior palate width on the root margin. alveolar bone remodeling can occur on half of the root and alveolar margin, if the tooth gets closed to or surpasses lingual cortex. the purpose of the study is to know various morphological changes of alveolar bone due to orthodontic movement of maxillary and mandibular incisors. anatomical form of alveolar bone in malocclusion handelman18 observed the comparison of partial alveolar bone on various cases of malocclusion. the width of alveolar bone of linguo posterior part toward upper incisor apex (up), labioanterior of alveolar bone to upper incisor apex (ua), linguo posterior of alveolar bone (lp), �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 21-24 labioanterior to lower incisor apex (la), superior part to upper incisor apex (uh), and inferior part to lower incisor apex (lh) were observed. the result showed uh and lh in angle class iii malocclusion was bigger than class i. lp in class iii was narrower than class i and ii in group of divergent mandible, up was bigger in low sn (sellanation)-mp (mandibular plane) group while in sn-mp average or high groups, found wider lp. edwards1 did not find any difference in alveolar bone width in divergent mandible case. the effect of incisor facially tipping movement on alveolar bone morphology the changes of alveolar bone and cementum on incisor facially tipping movement have been observed. in this study, extrusion has also occurred. extensive apposition found in mesial, distal and lingual region on extrusive tooth. on lingual and interproximal surface, the distance between cementum enamel junction and alveolar crest is 1 mm longer than controlled group. alveolar dehiscence is found on the facial surface of tooth which is facially moved and extruded.19 in the study done on the monkey, 1.7 mm bone loss was found in the tooth which was moved facially, in controlled group 1.3 mm bone loss was found, but statistically it is not significant.17 the effect of lingual tooth movement on alveolar bone morphology a study has been done on the changes of alveolar bone thickness in retraction of anterior tooth in bimaxillary protrusion with 4 premolar teeth extracted. ct scan and cephalogram examinations were done before treatment and 3 months after incisive tooth retraction. the first step, lingual tipping movement was done in maxillary and mandibulary incisors. the first slice on ct scan was done on labial and lingual region of alveolar bone. the result was: more alveolar bone loss occurred in the middle tooth margin than in apical region. the bone thickness on lingual part of maxillary lateral incisors decreases more than lingual part of maxillary central incisors. this can be due to the force given to the forth incisive is similarly spreading among the teeth. in maxilla, periodontal ligament area of central incisors is bigger than lateral incisors, therefore, the pressure was concentrated on cortical alveolar plate in lateral incisor region so it caused more thickness decrease in lingual cortical plate. in anterior mandible region, each of incisor has the same number of periodontal ligament and would get the same pressure. this case would cause different change of bone thickness in maxilla. in mandible, the quantity of alveolar bone loss is the same with the forth incisors.15 the change of buccal alveolar bone morphology in mandibulary incisors lingual retraction in protrusion case has been observed. the result of the study suggests the increase of buccal alveolar bone height in 58.8% cases, while in the rest, 41.2% cases was decreasing.20 the effect of extrusive and intrusive tooth movement in alveolar bone extrusive tooth movement will cause stretching of supracrestal and principle fibers with bone formation in apical alveolar crest of extrusive tooth. principle fibers will re arrange the position and will return faster to normal condition during retention period, but supracrestal fibers will be constantly stretched in longer time.19 intrusive tooth is still a controversial problem in some literatures, because the occurrence of iatrogenic damage in tooth supporting tissue was reported.21 tooth intrusion causes various changes of alveolar tissue. cementum resorption in tooth apex and reorientation of periodontal fibers direction based on intrusive tooth movement are found.22 intrusive tooth contributes pressure on supra alveolar fibers and the pressure will result in alveolar crest remodeling.23 other study reported the occurrence of resorption in alveolar laminal bone and cementum on experimented animal (monkey) and there was compression in apical periodontal ligament if mandibulary incisor was intruded.24 the stable periodontal tissue was found in intrusive tooth done in extrusive tooth with infra bony disorder.25 the ratio of cortical bone remodeling and tooth movement during maxillary incisors retraction the well known axiom in moving the tooth is that the bone will follow the trace of the moving tooth. if tooth movement occur due to orthodontic force, the bone around the tooth socket will remodel in the same width with tooth movement, so, the ratio between remodeling bone and tooth movement is 1:1. vardimon14 compared the ratio of maxillary incisors retraction with tipping and torque movement. it was found that either tipping or torque movement would not produce ratio 1:1. in tipping movement the ratio was 1:2, meant that if the apex of central incisors moves 3 mm posteriorly, so a point would be retracted 1.5 mm. the ratio for torque movement was figure 1. up, ua, lp, la, uh, lh. ��pudyani, et al.: morphological changes of alveolar bone 1:2.35; meaning if the apex of central incisors moved 5 mm posteriorly, a point would be retracted closely 2 mm. it is said that mechanical component in retraction and tipping movement would decrease the risk of central incisors apex moved closely to labial cortical plate. it is recommended to use the ratio 1:2 to decide the prognosis of point a-p movement or p-a in maxillary central incisors movement. discussion the result of recent study has shown that limitation of tooth movement could be compensated perfectly by remodeling process.13–15 if extensive palatal tooth movement was done, the tooth root would be contacted with palatal cortex of alveolar bone.1 cortex would bend and limited movement would occur. if contact occurred, further movement would cause perforate of cortical plate followed by bone loss, root resorption and relapse. adaptation phenomenon of bone form on pressure showed that bone at any time can change it self by pressure, increasing or decreasing the mass to compensate the force.26 in the condition that cortical plate has been penetrated by root, the surface of buccal root will not be covered by the bone. although osteogenesis process may occur for four months in retention period, it is not sufficient to cover the whole root surface. it is said that repair of root penetration region will only occur if the tooth comes back to it is original position (relapse).27 in the area of limited movement, excessive force will cause the tooth touching the cortical plate of alveolar bone, so, cortical bone resorption and root penetration will appear. tooth movement in limited area can contribute alveolar bone loss and it is still debatable whether the capacity of alveolar bone remodeling can compensate in every case of alveolar bone loss. many experts have an opinion that extensive incisors movement should be avoided to prevent cortex distraction of lingual alveolar bone resulting in tooth supporting tissue loss.15 it has been observed the alteration of alveolar bone resulting from incisors retraction in class ii malocclusion with bimaxillary protrusion. the result of the study stated that even though retraction and torque of tooth root have been done for long, the width of palatal alveolar bone around the tooth will not change. alveolar bone remodeling can occur on half of the root and alveolar margin, apical area is the border of orthodontic tooth movement.1 the opposite study stated that decrease of alveolar bone thickness more frequently occurs in coronal tooth region and the middle of tooth root than in apical.15 some experts found morphological changes of alveolar bone in incisors movement. in narrow simphisis mandible case, sagittal tooth movement and tooth derotation is critical condition and it can cause labial and lingual cortical plate loss and tooth decrease alveolar bone height.28 after oval root tooth derotation, the width of sagittal bone is adequate to support lingual and labial cortical plate. during derotation of root with oval form, two sides affected by pressure and tension. in distally rotation case, both sides are mesiolingual and distolabial root side, so, more root reception is found on that side. to move incisors, optimal stability will be reached if the tooth located in medula region of alveolar bone and in good balance with labial and lingual muscles. positioning incisors perpendicular towards basal bone will increase the support around the root of incisors and will produce good periodontal tissue condition.15 basic axioma states that bone will follow the trace of tooth movement, therefore it is assumed that the ratio between bone remodeling and tooth movement is 1:1. the problem is whether the ratio is the same for anteroposterior, vertical and transversal. on vertical dimension, dehiscence and fenestration of buccal cortical plate in rapid maxillary expansion has been reported. it is assumed that root movement in buccal dental segment will inhibit lateral remodeling bone. in this case, the question is whether the ratio 1:1 can be applied in slow expansion treatment.14 in sagittal dimension, different reaction is found in anterior and posterior segment. in posterior segment, ratio 1:1 still can be applied as long as tooth movement is limited between two cortical plates because it will intermitently influence the cancelous bone.14 in anterior segment, palatal or labial cortical plate is involving in the whole anteroposterior movement either in maxillary or mandibulary anterior tooth. ratio 1:1 will not occur in anterior segment. protraction of maxillary incisors can contribute dehiscence of labial cortical plate29 which is reversible if the tooth returns to its initial position (relapse).14 ratio 1:1 between the number of remodeling bone and tooth movement was not found after 3 months retention period after incisors retraction. at the end of treatment, dehiscence and fenestration were found in coronary region and middle of tooth root. the width of alveolar bone in apical region decreases but the level of alteration was small, therefore bone dehiscence did not occur. based on the result, it is considered that the width of marginal alveolar bone and middle tooth root is similarly important with apical alveolar bone. it is concluded that remodeling bone is not always in the same quantity with the number of tooth movement.15 increasing the height of buccal alveolar bone is reported after retraction of mandibulary central incisors. of the 17 cases, 58.8% showed increasing the bone height in buccal region, while the rest would get decreasing height. increasing bone height in that region is thought not only due to angulations changes between mandibular plane and mandibulary central incisor axis but due to tooth intrution.20 alveolar bone resorption might be caused by too far tooth movement, narrow alveolar bone and symphisis and oval form tooth root. based on the studies done by vardimon,14 sarikaya15 and wehrbein28 it can be conclude �� dent. j. (maj. ked. gigi), vol. 41. no. 1 january-march 2008: 21-24 that compensation of remodeling bone is not matched with the number of tooth movement so there are many dehiscence and fenestration found at the end of orthodontic treatment. to overcome this problem, therefore, prior to orthodontic treatment, evaluation of the bone structure and anterior tooth is necessarily done, thus stable position can be reached after treatment and adverse effect on tooth supporting tissue will not occur. in incisors movement, optimal stability will be reached if the tooth is in medulary region of alveolar bone and in good balance with labial and lingual muscles. perpendicularly positioning incisive towards basal bone will increase the support around the root of incisors and will produce good periodontal tissue condition. references 1. edwards jg. a study of anterior portion of the palate as it relates to orthodontic therapy. am j orthod 1976; 69(3):249–73. 2. yun cho. a histologic study of the alveolar bone remodeling on the periosteal side incident to experimental tooth movement. dent in japan; 33:79–82 3. sandy j, farndale rw, meikle mc. recent advances in understanding mechanically induced bone remodeling and their relevance to orthodontic theory and practice. am j orthod dentofac orthop 1993; 103(3):212–21. 4. noxon js, king gj, gu g, meikle mc. osteoclast clearance fromosteoclast clearance from periodontal tissues during orthodontic tooth movement. am j ortod dentofac orthop 2001; 120(5):466–76. 5. rody w, king gj, gu g, huang g. osteoclast recruitment to sites of compression in orthodontic tooth movement. am j orthop dentofac orthop 2001; 120(5):477–89. 6. kohno s, kaku m, tsutsui k, motokawa m, ohtani j, tenjo k, et al. expression of vascular endothelial growth factor and effectsexpression of vascular endothelial growth factor and effects on bone remodeling during experimental tooth movement. j dent res 2003; 177:177–82. 7. ren y, martha j, hof v, kuijpers jagtman am. optimum force magnitude for orthodontic tooth movement: a mathematic model. am j orthod dentofac orthop 2004; 125(1):71–7. 8. ren y, maltha jc, kuijpers jagtman am. optimum force magnitude for orthodontic movement: a systematic literature review. angle orthod 2003; 73:86–92. 9. cronau m, ihlow d, meesenburg k, fanghanel j, dathe h, nageri h. biomechanical features of the periodontium: an an experimental pilot study in vivo. am j orthod dentofac orthop 2006; 129:599. 10. davidovitch z. bone metabolism associated with tooth eruption and orthodontic tooth movement. j periodontol 1975; 48:22–9. 11. davidovitch z, vinkelson md, stegman s, sfanfeld jl, montgomeri pc, korostoff. elective currents, bone remodeling and orthodonticelective currents, bone remodeling and orthodontic tooth movement part i: the effect of electric currents on periodontal cyclic nucleotides. am j orthod 1980; 77:14–32. 12. melsen b. biological reaction of alveolar bone to orthodontic tooth movement. angle orthod 1999; 69(2):151–58. 13. meikle mc. the dentomaxillary complex and overjet correction in class ii, division 1 malocclusion: objectives of skeletal and alveolar remodeling. am j orthod 1980; 71(2):185–97. 14. vardimon ad, oren e, bassat, yb. cortical bone remodeling/cortical bone remodeling/ tooth movement ratio during maxillary incisor retraction with tip versus torque movements. am j ortod dentofac orthop 1998; 114(5):520–9. 15. sarikaya s, haydar b, ciger s, ariyurek ma. changes in alveolar bone thickness due to retraction of anterior teeth. am j orthod dentofac orthop 2002; 122(1):15–26. 16. choy k, pae ek, park yc, kim kh, burstone j. effect of root and bone morphology on the stress distribution in dental periodontal ligament. am j orthod dentofac orthop 2000; 117(1):98–105. 17. wingard ce, bowers gm. the effect on facial bone from facial tipping of incisors on monkeys. j periodontol 1976; 47(8):480–544. 18. handelman cs. the anterior alveolus: its importance in limiting orthodontic treatment and its influence on the occurrence of iatrogenic squeal. angle orthod 1996; 66(2):95–110. 19. batenhorst kf, bowers gm, williams je. tissue changes resulting from facial tipping and extrusion of incisors in monkeys. j periodontol 1974; 45(9):660–68. 20. bimstein e, crevoisier ra, king dl. changes in the morphology ofchanges in the morphology of the buccal alveolar bone of protruded mandible permanent incisors secondary to orthodontic alignment. am j orthod dentofac orthop 1990; 97(5):427–31. 21. melsen b. tissue reaction following application of extrusive and intrusive forces to teeth in adult monkeys. am j orthod dentofac orthop 1986; 89(6):469–75. 22. melsen b, agerbaek n, markenstam g. intrusion of incisors in adult patients with marginal bone loss. am j orthod dentofac orthop 1989; 96(3):232–41. 23. kanzeki r, daimaruya t, takahashi i, mitani h, sugawara j. remodeling of alveolar bone crest after molar intrusion with skeletal anchorage system in dogs. am j orthod dentofac orthop 2007; 131(3):343–51. 24. murakami t, yokota s, takahama y. periodontal changes after experimentally induced intrusion of the upper incisors in macaca fustata monkeys. am j orthod dentofac orthop 1989; 95(2):115–26. 25. cardaropoli d, re s, corrente g. intrusion of migrated incisors with infrabony defects in adult periodontal patients. am j orthod dentofac orthop 2001; 120(6):671–5. 26. kukihara s, enlow dh. a histochemicaland electron microscopic study of an adhesive type of collagen attachment on resorptive surface of alveolar bone. am j orthod 1980; 77:532, 546. 27. wainwright wm. faciolingual tooth movement: its influence on the root and cortical plate. am j orthod 1973; 64:278–302. 28. wehrbein h, privatdozent, bauer w, diedrich p. mandiblemandible incisors, alveolar bone, and symphysis after orthodontic treatment. a retrospective study. am j orthod dentofac orthop 1996; 110(3):239–46. 29. engelking g, zachrisson bu. effect of repositioning on the monkey periodontium after expansion through the cortical plate. am j orthod 1982; 82:23–32. vol 51 no 4 okt-des 2018.indd 164164 antioxidant activity test of ethyl acetate fraction of binjai (mangifera caesia) leaf ethanol extract k. khairiah,1 irham taufiqurrahman,1 and deby kania tri putri2 1 department of oral and maxillofacial surgery 2 department of oral biology faculty of dentistry, universitas lambung mangkurat banjarmasin – indonesia abstract background: binjai (mangifera caesia) is a herb derived from south kalimantan possessing antioxidant properties which promote wound healing inhibiting oxidation radicals. the natural antioxidants present in binjai leaves can be extracted by fractionation. purpose: this study aimed to analyze the antioxidant activity of ethyl acetate fraction in 96% ethanol extract of binjai leaf. methods: the study constituted a pure experimental study incorporating a post-test design with only random sampling technique consisting of two groups, namely; an ethyl acetate fraction as the treatment group and ascorbic acid as the positive control group. the leaves were treated in accordance with the soxhlet method and subsequently fractionated to extract ethyl acetate fraction. this was used to measure antioxidant activity with dpph radical damping method using a uv-vis spectrophotometer. a linear regression calculation was performed with a standard curve to quantify the ic50 value, before the ethyl acetate fraction underwent a qualitative test of secondary metabolite. results: an independent t-test indicated significant differences between groups, an average value of ic50 in ascorbic acid of 13.812 ppm with 0.996 linearity and a fraction of ethyl acetate 38.526 ppm with a linearity of 0.999. in contrast, at this linearity value ascorbic acid and ethyl fraction acetate demonstrate a very high linear connection between concentration and inhibition. a secondary metabolite test conducted on the ethyl acetate fraction produced positive results for flavonoid, tannins, and phenol. conclusion: based on the ic50 parameters, the fraction of ethyl acetate in 96% ethanol extract of binjai leaf produces very strong antioxidant activity in the content of the compounds in the fraction, namely: flavonoid, tannins and phenol. keywords: antioxidant; binjai leaf; ethyl acetate fraction; ic50 correspondence: khairiah, department of oral and maxillofacial surgery, faculty of dentistry, universitas lambung mangkurat, banjarmasin. jalan kuin selatan no. 25, banjarmasin 70128, indonesia. e-mail: rya.mayang@ymail.com introduction interrupted wound healing after tooth extraction frequently occurs even in cases of healthy patients due to several factors which inhibit the process. there are approximately 11 million patients who experience postoperative conditions such as pain, swelling and bruising on a daily basis. it has been reported that in 1.0-11.5% of cases patients experience inconsistent wound healing.1 a wound is classified as physical damage or anatomical injury caused by microbes, a chemical reaction, temperature, mechanical trauma or surgery resulting in continuous tissue breakdown.2 the healing process itself occurs in three phases, namely: an inflammatory phase, a proliferation or epithelization phase and a remodelling or maturation phase. a fibroblast is a cell that plays a critical role in wound healing. fibroblasts will proliferate and produce collagen to repair damaged tissues in the inflamed wound tissue site.3 wound healing requires antioxidants, substances which protect cells, protein, tissue and body organs from free radicals in order to accelerate the process. the excessive presence of reactive oxygen during the wound healing process can induce a reduction in the collagen production matrix produced by fibroblast proliferation. therefore, antioxidants are required to inhibit the oxidation dental journal (majalah kedokteran gigi) 2018 december; 51(4): 164–168 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p164–168 mailto:rya.mayang@ymail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p164-168 165 khairiah, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 164–168 process. antioxidants work by donating a hydrogen atom to radical substances with the result that they become more stable.4,5 antioxidants present in natural materials can be obtained through extraction. in this study, the researcher chose to adopt a socletation method using ethanol solution 96% because denis, (2017) claimed that it promotes greater antioxidant activity than maceration. after binjai (mangifera caesia) leaf ethanol extract has been obtained, the fractionation process is completed.6 fractionation involves separation of an antioxidant compound based on its degree of solvent polarity in order to produce a fraction with similar secondary metabolite properties.7 according to research conducted by rohman et al, (2010), ethyl acetate fraction produces stronger antioxidant activity compared to methanol and chloroform fraction.8 phang et al, (2011), also compared methanol, n-hexane and aquadest to ethyl acetate, concluding that ethyl acetate fraction promotes stronger antioxidant activity.9 binjai part of the mangifera genus and ancardiaceae family, represents one of the herbs indigenous to south kalimantan which act as antioxidants. the resistance of binjai to pests and disease is evident from the widespread natural presence of its wild variety throughout sumatra and the malayan peninsula. a cultivated form was subsequently produced in locations such as bali, philippines, thailand and certain areas on java where the local populations value binjai as part of the daily diet and an important element of the treatment for diabetes.10,11 antioxidant activity analysis of binjai leaf ethanol extract fraction can be performed using a 1.1diphenyl2-picryhidrazyl (dpph) method. the score of antioxidant activity lower than ic50 recorded by a specific compound leads it to be considered highly active.12 the advantages of the dpph method can be more easily realized because the radical compounds employed are more stable than those of other methods. dpph is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, with the result that the molecules do not dimerise like most other free radicals.13 the activity of bioactive substances contained in the ethyl acetate fraction of binjai leaf extract has been tested by means of secondary metabolite qualitative test using colour performance with tube method.14 the objective of this study was to analyze the antioxidant capacity of ethyl acetate fraction in binjai leaf ethanol extract. materials and methods the research reported here was granted ethical clearance by document no. 031/kepkg-fkgulm/ec/ix/2017 issued by the faculty of dentistry, universitas lambung mangkurat. this research represented a true experiment incorporating a post-test only and control group design. the research sample consisted of binjai leaves selected through simple random sampling using two groups of unpaired comparative numerical formula. the three samples used ascorbic acid as ethyl acetate fraction within a positive control and three samples of binjai leaf ethanol extract as a treatment group. the separation process of ethanolic extract from binjai leaf used soxhlet extraction. the binjai leaf samples were washed and dried in a room free from direct sunlight for four days. after the leaves had been dried, they were chopped and blended to the consistency of a powder which was subsequently sieved through a size 40 mesh until homogeneous and weighed to obtain 91.61 grams of simplicia powder. binjai simplicia was extracted by socletation method using 96% ethanol solution for five hours. the liquid extract was then concentrated and evaporated using a 40°c rotary evaporator and water bath for seven hours in order to produce 11.92 grams of thick extract.6 fractionation was conducted using a separating funnel by suspending the thick binjai leaf extract in aquadest at a ratio of 1:2. first, fractionation was conducted by adding 100ml of n-hexane solution prior to agitation for one minute and settling until it separated into two layers. the bottom layer constituted n-hexane fraction, while the top layer consisted of aquades fraction. ethyl acetate fraction was obtained by adding 100ml ethyl acetate to the aquadest fraction, shaking it for one minute and then allowing it to stand until the ethyl acetate fraction separated from the aquadest fraction. the ethyl acetate fraction was then concentrated, evaporated and weighed using a rotary evaporator and water bath. the ethyl acetate fraction obtained amounted to 0.62 grams.5 in order to analyze quantitative antioxidant activity with dpph assay on ethyl acetate fraction of binjai leaf ethanol extract, the solution sample was made by carefully measuring a 10 mg sample dissolved into 96% p.a. ethanol until its volume reached 10 ml. the researcher extracted 200, 300, 400 and 500 μl samples from the solution each of which was then inserted into a 10 ml volumetric flask. 96% p.a. ethanol was added until the boundary mark was reached in order to obtain concentrations of 20, 30, 40 and 50 ppm. one ml of dpph 0.4 mm solution was added to each 4 ml sample of solution concentrate which were deposited in a dark room for 20 minutes. the solution was found to have absorbed a maximum wavelength of 516 nm.6 in order to measure quantitative antioxidant activity with dpph assay on ascorbic acid, the comparative solution was made by carefully weighing 10 mg of ascorbic acid which was then dissolved into 96% p.a ethanol until it reached a volume of 10 ml. from the solution, the researcher took 40, 80, 120 and 160 ul samples each of which was then placed in a 10 ml volume flask and each 96% p.a. ethanol was added as far as the boundary mark in order to obtain concentrations of 4, 8, 12 and 16 ppm. one millimeter of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p164–168 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p164-168 166khairiah, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 164–168 dpph 0.4 mm solution was added to each 4 ml of standard ascorbic acid solution, before being left in a dark room for 20 minutes. the solution was read its absorbance on maximum wavelength 516 nm.12 secondary metabolite qualitative testing on ethyl acetate fraction of binjai leaf extract involved several tests. in the alkaline reagent test 0.5 mg of flavonoids were dissolved in 100 ml of solvent.1 ml of the sample was then added to some drops of naoh solution. if a yellow color appears and subsequently fades when added to a dilute acid mixture, this represents a positive test of the presence of flavonoids. 0.5 mg of lead acetate test was first dissolved in 100 ml of its solvent before 1 ml was taken as a sample. 1 ml 10% pb acetate was subsequently added to the sample and agitated. if the solution colour changes to yellowish brown, this signifies that it contains flavonoids.15 in a terpenoids test using a libermann burchard test, a 0.5 mg sample was dissolved in chloroform and then strained. the filtrate obtained was added to several drops of concentrated sulfuric acid before being shaken. if a brown ring formed, terpenoid was present. 15 in a tannin test using a gelatin test, a 0.5 mg sample was dissolved in 100 ml of its solvent, 2 ml samples were added to gelatin solution 1% containing nacl. if white sediment formed, this confirmed the presence of tannin.15 in a saponin test using a froth method, a 0.5 mg sample was dissolved in 100 ml of its solvent, with 2 ml samples being taken and agitated in 2 ml water. if the foams are stable, it shows positive result for saponin.15 in an alkaloids test using a dragendroff test a 0.5 mg sample was dissolved in 100 ml of its solvent. a 1 ml sample was added to 1 ml dragendroff reagent (bismuth potassium iodide). if a red sediment was formed, this signified a positive result for alkaloid. a mayer test used a 0.5 mg sample dissolved into 100 ml of its solvent. a 1 ml sample was added to 1 ml of mayer reagent (mercuric potassium iodide). if yellow sediment was formed, this indicated the presence of alkaloids.15 in a steroid test using a libermann burchard test a 0.5mg sample was dissolved in chloroform, before being strained. the resulting filtrate was added to anhydride acetic acid, heated and then allowed to cool. concentrated sulfuric acid was carefully added to the tube wall. if a brown ring formed, this confirmed the presence of steroids.15 in a phenol test using an iron (iii) chloride test a 0.5 mg sample was dissolved in 100 ml of its solvent. a 1 ml sample was added to 1 ml of fecl3 3%. a blue black sediment indicated the presence of phenol.16 following the tally result for antioxidant activity of ethyl acetate fraction on binjai leaf, the data obtained was subjected to a normality test in the form of a shapiro wilk test. the data was then evaluated by means of an independent t-test which confirmed it to be normally distributed (p>0.05). if the data obtained was not normally distributed (p<0.05), a nonparametric test, namely a wilcoxon test, was conducted instead. results based on the research results, the average calculation of the ethyl acetate fraction of binjai leaf ethanol extract and ascorbic acid and the score of comparison on antioxidant activity based on the ic50 parameter were obtained, as shown in figure 1. it can be concluded that the average ic50 in ascorbic acid was 13.812. this score was obtained from the calculation of ascorbic acid solution absorbance made by using the raw relationship curve between the ascorbic acid concentrate and percentage inhibition. the linear regression equation of y= 3.997x – 5.141 was obtained with a correlation coefficient of r= 0.996, in which x is the antioxidant score and y represents the absorbance score. meanwhile, the antioxidant activity of ethyl acetate fraction of binjai leaf ethanol extract produces an ic50 average score of 38.526 ppm obtained from a linear regression calculation of y= 1.395x – 3.720 with a correlation coefficient r= 0.999. figure 1. the comparative scores of antioxidant activity on ascorbic acid and ethyl acetate fraction of injai leaf ethanol extract based on ic50. table 1. the result of secondary metabolites qualitative testing on ethyl acetate fraction of binjai leaf ethanol extract. compounds and reagents classes explanationresult flavonoid: alkaline + hcl pb acetate + fading yellow and brownish yellow saponin: foams no foams– alkaloid: mayer dragendroff no sediment– tannin: gelatin white sediment+ steroid: libermann burchard’s – no brown ring was formed terpenoid: libermann burchard’s – no brown ring was formed phenol: iron (iii) chloride black blue sediment+ dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p164–168 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p164-168 167 khairiah, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 164–168 the antioxidant activity scores of the ethyl acetate fraction of binjai leaf ethanol extract and ascorbic acid can be seen in table 1. this data shows that there were secondary metabolite substances such as flavonoid, tannin, and phenol contained in the ethyl acetate fraction of binjai leaf ethanol extract. a statistical analysis was conducted with normality testing of both groups using a shapiro-wilk. all values showed normal distribution since the score obtained was p>0.005. statistical analysis of the data obtained was followed by independent t-test. the data analysis results had a significance score of p=0.00 (p<0.005). it can be concluded that there was a significant difference between the treatment group of ethyl acetate fraction and the control group of ascorbic acid. eggplant ethanol extract because the antioxidant activity is determined by the compounds extracted which depend on the solution used, namely ethanol. this causes the extracted compound to form part of the polyphenol class because the solvent is polar. meanwhile, other antioxidant compounds such as beta carotene and vitamin c are not being extracted because vitamin c does not dissolve in ethanol.20 ascorbic acid, as a comparative solvent, is a nonenzymatic antioxidant which functions by catching free radical compounds to avoid chained oxidation reaction so that they will not react with other components.12,21 the stronger antioxidant activity in ascorbic acid compared to the ethyl acetate fraction in binjai leaf ethanol extract is also caused by the ascorbic acid itself. ascorbic acid is a pure antioxidant compound which means it only demonstrates antioxidant activity while present in natural ingredient extracts. these are complex compounds which have many activities such as antioxidant anti-inflammatory and anticancer among others.22 moreover, munte et al (2015) stated that ascorbic acid demonstrates highly active antioxidant activity because during its chemical reaction, ascorbic acid holds two hydroxyl atom groups making it easier to donate hydrogen in order to suppress free radicals.23 meanwhile, antioxidant working mechanism on ethyl acetate fraction to supress dpph radicals is caused by the existance of certain compound which able to give hydrogen radicals to unpaired dpph radicals. this results on reaction to compounds which able to supress free radicals, causing the one-electron binding to the electron which donates it and forms diphenylpicrylhidrazin. the formation of reduced dpph into dpph-h radical results on colour decay from purple to yellow.24 secondary metabolite qualitative testing in this research was aimed at determining the secondary metabolite compounds which were found in the ethyl acetate fraction of binjai leaf ethanol extraction. this qualitative testing involved flavonoids testing, terpenoid testing, tannin testing, saponin testing, alkaloids testing, steroid testing and phenol testing. after these tests had been conducted, positive test results were obtained for flavonoids, tannin and phenol, confirming that such compounds are semi-polar. these test results were also in accordance with those of tanaya et al, (2015) which stated that flavonoid and tannin exist in ethyl acetate fraction because those compounds are semi-polar.25 tursiman et al, (2012) mentioned the semi-polar compounds which will be extracted from ethyl acetate solvent based on qualitative testing conducted on ethyl acetate fraction of kandis, contains phenol compounds which acts as antioxidant.26 it can be concluded that even though there is a difference between the ethyl acetate fraction and ascorbic acid, based on ic50 parameter the score of ethyl acetate fraction of binjai leaf ethanol extract and ascorbic acid both have highly active antioxidant activity in preventing free radical since their score were lower than 50 ppm. discussion based on the research conducted, it is argued that antioxidant activity in the ascorbic acid and the ethyl acetate fraction of binjai leaf ethanol extract demonstrate a significant difference. the difference obtained was caused by the antioxidant activity score in ascorbic acid being more active compared to that of ethyl acetate fraction which was 13.812 ppm. meanwhile, the antioxidant activity score in the ethyl acetate fraction of binjai leaf ethanol extract was 38.526 ppm. the parameters used to establish the antioxidant activity in the samples determined the score of (inhibitory concentration) ic50. ic50 is a concentration which causes the loss of 50% of dpph activity. the score of ic50 is considered a valid measurement of the efficiency of the antioxidants of both pure and extract compounds.17 the smaller the absorbance score, the bigger the inhibition percentage score. therefore, lower ic50 scores show that the antioxidant activity compound is more active or stronger.18 anggresani et al., (2017) discussed the level of antioxidant strength based on ic50 parameters: less than 50 ppm is said to be highly active, 50-100 ppm is said to be active, 101-250 ppm is said to be average and 250-500 ppm is said to be weak. based on the research conducted, it is argued that antioxidant activity in the ascorbic acid and ethyl acetate fraction of binjai leaf ethanol extract has significant difference.19 the difference obtained is caused by the antioxidant activity of ascorbic acid being more active compared to that of the ethyl acetate fraction which was 13.812 ppm. meanwhile, the antioxidant activity score on ethyl acetate fraction of binjai leaf ethanol extract was 38.526 ppm. the difference between the antioxidant score activity in ascorbic acid, which was more active compared to ethyl acetate fraction, is in accordance with the findings of martiningsih et al., (2016) who conducted antioxidant activity testing on eggplant ethanol extract which was comparable to ascorbic acid. the research concluded that ascorbic acid activity was very strong compared to that of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p164–168 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p164-168 168khairiah, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 164–168 acknowledgements the authors acknowledge the support of the faculty of dentistry, universitas lambung mangkurat and the phytochemical laboratory, faculty of mathematics and natural sciences, universitas lambung mangkurat for permitting access to laboratory facilities and providing support during the experimental research. references 1. susilawati, khafid m, tiarisna hn, narendra kw, chotimah c. potensi kulit dan biji kelengkeng (euphoria longan) sebagai gel topikal untuk mempercepat penyembuhan luka pasca ekstraksi gigi. berkala ilmiah mahasiswa kedokteran gigi indonesia. 2013; 1(2): 1–3. 2. rupina w, trianto hf, fitrianingrum i. efek salep ekstrak etanol 70% daun karamunting terhadap re-epitelisasi luka insisi kulit tikus wistar. ejournal kedokteran indonesia. 2016; 4: 26–30. 3. kurnia pa, ardhiyanto hb, suhartini. potensi ekstrak teh hijau (camellia sinensis) terhadap peningkatan jumlah sel fibroblas soket pasca pencabutan gigi pada tikus wistar. e-jurnal pustaka kesehatan. 2015; 3: 122–7. 4. nugroho ya, kusnadi j. aplikasi kulit manggis (garicinia mangostana l.) sebagai sumber antioksidan pada es krim. j pangan dan agroindustri. 2015; 3(4): 1263–71. 5. fitriana wd, fatmawati s, ersam t. uji aktivitas antioksidan terhadap dpph dan abts dari fraksi-fraksi daun kelor (moringa oleifera). in: simposium nasional inovasi dan pembelajaran sains. bandung; 2015. p. 657–60. 6. denis dc. perbandingan aktivitas antioksidan antara metode ekstraksi maserasi dengan sokletasi pada ekstrak daun ramania. thesis. banjarmasin: universitas lambung mangkurat; 2017. p. 40-1. 7. chasani m, fitriaji rb, purwati. fraksinasi ekstrak metanol kulit batang ketapang (terminalia catappa linn.) dan uji toksisitasnya dengan metode bslt (brine shirmp lethality test). molekul. 2013; 8: 89–100. 8. rohman a, riyanto s, yuniarti n, saputra wr, utami r, mulatsih w. antioxidant activity, total phenolic, and total flavaonoid of extracts and fractions of red fruit (pandanus conoideus lam). int food res j. 2010; 17: 97–106. 9. phang cw, malek sna, ibrahim h, wahab na. antioxidant properties of crude and fractionated extracts of alpinia mutica rhizomes and their total phenolic content. african j pharm pharmacol. 2011; 5(7): 842–52. 10. mustikasari k, ariyani d. studi potensi binjai (mangifera caesia) dan kasturi (mangifera casturi) sebagai antidiabetes melalui skrining fitokimia pada akar dan batang. j ilmiah berkala sains dan terapan kimia. 2008; 2(2): 64–73. 11. rosyidah k, siska s, astuti md. isolasi senyawa antioksidan dari kulit batang tumbuhan binjai (mangifera caesia). j ilmiah berkala sains dan terapan kimia. 2011; 5: 8–14. 12. pangaribuan fxr, sitorus s, saleh c. uji fitokimia dan aktivitas antioksidan ekstrak daun rambutan (nephelium lappaceum) dengan metode dpph (1,1-diphenyl-2-picryhidrazyl). j atomik. 2016; 1(2): 81–5. 13. kedare sb, singh rp. genesis and development of dpph method of antioxidant assay. j food sci technol. 2011; 48(4): 412–22. 14. setyowati wae, ariani srd, ashadi, mulyani b, rahmawati cp. skrining fitokimia dan identifikasi komponen utama ekstrak metanol kulit durian (durio zibethinus murr.) varietas pektruk. in: seminar nasional kimia dan pendidikan kimia vi. surakarta; 2014. p. 271–80. 15. sutomo, arnida, rizki mi, triyasmono l, nugroho a, mintowati e, salamiah. skrining fitokimia dan uji kualitatif aktivitas antioksidan tumbuhan asal daerah rantau kabupaten tapin kalimantan selatan. j pharmascience. 2016; 3: 66–74. 16. wijaya dp, paendong je, abidjulu j. skrining fitokimia dan uji aktivitas antioksidan dari daun nasi (phrynium capitatum ) dengan metode dpph (1,1-difenil-2-pikrilhidrazil). j mipa unsrat online. 2014; 3: 11–5. 17. pratama m, baits m, yaqin rn. uji aktivitas antioksidan ekstrak etanol daun tomat buah (lycopersicon esculentum mill, var. pyriforme alef) dan daun tomat sayur (lycopersicon esculentum mill, var. commune bailey) dengan metode dpph (1,1-diphenyl2-picryl hydrazil). j fitofarmaka indonesia. 2015; 2: 76–82. 18. rahmi h. review: aktivitas antioksidan dari berbagai sumber buahbuahan di indonesia. j agrotek indonesia. 2017; 2: 34–8. 19. anggresani l, yuliawati, desriyanti e. uji total kandungan flavonoid dan aktivitas antioksidan ekstrak daun kembang bulan (thitonia diversifolia (hemsley) a. gray). riset informasi kesehatan. 2017; 6: 18–22. 20. martiningsih nw, widana gab, kristiyanti plp. skrining fitokimia dan uji aktivitas antioksidan ekstrak etanol daun matoa (pometia pinnata) dengan metode dpph. in: prosiding seminar nasional mipa. singaraja; 2016. p. 332–8. 21. winarsi h. antioksidan alami & radikal bebas. yogyakarta: kanisius; 2007. p. 20-4. 22. matheos h, runtuwene mrj, sudewi s. aktivitas antioksidan dari ekstrak daun kayu bulan (pisonia alba). pharmacon j ilmiah farmasi unsrat. 2014; 3(3): 235–46. 23. munte l, runtuwene mr, citraningtyas g. aktivitas antioksidan da r i ekstra k daun p rasman (eupator ium tr ipliner ve va hl.). pharmacon j ilmiah farmasi unsrat. 2015; 4(3): 41–50. 24. setyaningsih d, pandji c, perwatasari dd. kajian aktivitas antioksidan dan antimikroba fraksi dan ekstrak dari daun dan ranting jarak pagar (jatropha curcas l.) serta pemanfaatannya pada produk personal hygiene. agritech. 2014; 34(2): 126–37. 25. tanaya v, retnowati r, suratmo. fraksi semi polar dari daun mangga kasturi (mangifera casturi kosterm). kimia student j. 2015; 1(1): 778–84. 26. tursiman, ardiningsih p, nofiani r. total fenol fraksi etil asetat dari buah asam kandis (garcinia dioica blume). j kimia khatulistiwa. 2012; 1(1): 45–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p164–168 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p164-168 103 volume 47, number 2, june 2014 p a p a r a n z a t b e s i p a d a e k s p r e s i p r o t e i n s p e s i f i k extracellular polymeric substance biofilm aggregatibacter actinomycetemcomitans (iron exposure to specific protein expression of extracellular polymeric substance of aggregatibacter actinomycetemcomitans biofilm) marchella hendrayanti w dan indah listiana k departemen biologi oral fakultas kedokteran gigi universitas airlangga surabaya-indonesia abstract background: the study of biofilms bacteria could be an alternative of preventive treatment in reducing prevalence of aggressive periodontitis in the community, because biofilm protects the bacteria from environmental conditions, including the attack of immune system and antimicrobial. aggregatibacter actinomycetemcomitans is a major cause of bacterial aggressive periodontitis. purpose: this study aims to examine the iron exposure to specific protein expression of extracellular polymeric substance (eps) of aggregatibacter actinomycetemcomitans biofilm. methods: protein containing eps biofilm was isolated from cultures of a.actinomycetemcomitans. the protein was processed through several procedures: electrophoresis , electroelution , immunization of rabbits , serum isolation , and purification of antibodies. after the western blotting procedure the antibody was used. protein containing eps biofilms exposed to iron, then once again isolated from cultures of a. actinomycetemcomitans. the electrophoresis and western blotting were done on the isolated protein. results: the result showed that the the expression of specific proteins in eps biofilm decreased in response to iron exposure. conclusions: iron exposure could influenced the specific protein expression in eps biofilm of aggregatibacter actinomycetemcomitans. key words:: iron, specific protein, extracellular polymeric substance, biofilm, aggregatibacter actinomycetemcomitans, specific protein, extracellular polymeric substance, biofilm, aggregatibacter actinomycetemcomitans abstrak latar belakang: penelitian terhadap bakteri biofilm dapat menjadi alternatif perawatan preventif dalam menurunkan prevalensi periodontitis agresif di masyarakat, karena biofilm melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba. aggregatibacter actinomycetemcomitans merupakan bakteri penyebab utama periodontitis agresif. tujuan: studi ini bertujuan meneliti paparan zat besi terhadap ekspresi protein spesifik extracellular polymeric substance (eps) aggregatibacter actinomycetemcomitans. metode: protein yang mengandung eps biofilm diisolasi dari kultur a. actinomycetemcomitans. protein yang diisolasi ini kemudian melalui beberapa prosedur: elektroforesis, elektroelusi, imunisasi pada kelinci, isolasi serum, dan purifikasi antibodi. pada prosedur western blotting di sesi penelitian berikutnya antibodi ini digunakan. protein yang mengandung eps biofilm dipapar dengan zat besi, kemudian diisolasi sekali lagi dari kultur a. actinomycetemcomitans. protein yang diisolasi dilakukandilakukan elektroforesis dan western blotting. western blotting. hasil: penelitian ini menunjukkan hasil berupa penurunan ekspresi protein spesifik biofilm eps research report 104 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 103–109 sebagai respon terhadap paparan zat besi. simpulan:: paparan zat besi memberi pengaruh ekspresi protein spesifik biofilm eps aggregatibacter actinomycetemcomitans. kata kunci: zat besi, protein spesifik, extracellular polymeric substance, biofilm, aggregatibacter actinomycetemcomitans, protein spesifik, extracellular polymeric substance, biofilm, aggregatibacter actinomycetemcomitans korespondensi (correspondence): marchella hendrayanti w, departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan periodontitis agresif adalah penyakit periodontal dengan ciri khas early onset, yaitu secara umum menyerang individu berusia di bawah 30 tahun, meskipun terkadang juga menyerang individu berusia lebih dari 30 tahun.1 periodontitis agresif lebih destruktif pada perlekatan periodontal dan tulang alveolar, biasanya muncul pada periode waktu yang relatif singkat dengan minimal akumulasi dari faktor lokal.2 prevalensi periodontitis agresif di indonesia cukup tinggi. menurut cho et al. 3 prevalensi rendah didapatkan di eropa yaitu antara 0,1% dan 0,2%; sedangkan prevalensi tinggi yaitu antara 3% sampai dengan 10% didapatkan di brazil, iraq, indonesia, dan amerika serikat. beberapa mikroorganisme spesifik-aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans), capnocytophaga spp., eikenella corrodens, prevotella intermedia, dan campylobacter rectus sering ditemukan pada pasien periodontitis agresif localized, yang merupakan patogen primer yang berkaitan dengan periodontitis agresif localized adalah a. actinomycetemcomitans.1 a. actinomycetemcomitans bersifat patogen oportunistik dan merupakan bagian flora normal yang berkolonisasi di mukosa rongga mulut, gigi dan orofaring.2 a. actinomycetemcomitans ditemukan dengan frekuensi yang tinggi (sekitar 90%) pada lesi periodontitis agresif localized.1 penelitian yang telah dilakukan lebih difokuskan pada pengamatan efektivitas berbagai bahan alternatif pengganti antibiotika, yaitu menggunakan bahan herbal dan hewani, dalam menghambat atau bahkan membunuh bakteri penyebab permasalahan gigi dan mulut, namun implementasinya dalam praktik kedokteran gigi masih jarang didapatkan. antibiotika masih menjadi pilihan utama dokter gigi dalam mengatasi infeksi bakteri di rongga mulut, termasuk untuk mengatasi a. actinomycetemcomitans. beberapa studi menunjukkan penggunaan tetrasiklin pasca debridement mekanis untuk perawatan pasien periodontitis akibat a. actinomycetemcomitans.1 penelitian terhadap biofilm bakteri dapat menjadi alternatif tindakan preventif dalam rangka menurunkan kejadian periodontitis agresif di masyarakat. perkembangan biofilm merupakan proses kompleks yang mengawali adhesi sel bakteri pada suatu permukaan, yang dilanjutkan dengan pembelahan sel yang subsekuen dan pertumbuhan mikrokoloni permukaan.4 secara alami, pembentukan biofilm dipengaruhi oleh sinyal lingkungan yang beragam, beberapa sudah diidentifikasi, tetapi masih banyak faktor yang yang belum dilakukan penelitian. faktor yang mempengaruhi pembentukan biofilm maupun menghilangkan biofilm yaitu sinyal mekanis, nutrisi, molekul inorganik, osmolaritas, host-derived signals, antimikroba dan quorum signals.5 biofilm merupakan suatu kondisi fisiologis yang melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba.4 pada penelitian ini studi biofilm difokuskan pada komponen utama, yaitu eksopolisakarida, yang disebut juga extracellular polymeric substance (eps). pada a. actinomycetemcomitans, eps mengandung polimer dari β-1,6-n-asetil-d-glukosamin, sering disebut pnag atau pga. pnag merupakan salah satu faktor virulensi a. actinomycetemcomitans, selain juga leukotoksin.6 dengan memfokuskan pada eps biofilm bakteri, penelitian ini diharapkan dapat membantu mencari alternatif untuk menurunkan virulensi bakteri, sehingga kejadian penyakit rongga mulut, khususnya periodontitis agresif, dapat ditekan tanpa penggunaan antibiotik. zat besi merupakan elemen inorganik esensial bagi sebagian besar sistem biologis. zat besi berfungsi sebagai kofaktor sejumlah enzim dan protein redoks yang berperan vital pada proses penting seperti energetika membran dan biosintesis deoxyribonucleic acid (dna) dan ribonucleic acid (rna). ketersediaan zat besi menjadi sinyal yang penting yang meregulasi ekspresi dari banyak faktor virulensi pada bakteri patogen.7 zat besi juga merupakan aktivator pembentukan biofilm, dan pada beberapa kasus, zat besi menghambat pembentukan biofilm.5 rhodes et al.8 menemukan bahwa beberapa senyawa zat besi berpengaruh terhadap pembentukan biofilm a. actinomycetemcomitans. a. actinomycetemcomitans mendapatkan besi melalui interaksi langsung dengan senyawa seperti haemin, haemoglobin, lactoferrin, dan transferrin meskipun tidak memproduksi siderofor.8 pada vertebrata, iron-binding protein seperti transferrin, lactoferrin, hemoglobin, dan ferritin, menjaga free iron pada konsentrasi rendah, sehingga menghambat pertumbuhan bakteri.7 berdasarkan hal ini, penulis meneliti pengaruh paparan zat besi terhadap ekspresi protein spesifik eps biofilm a. actinomycetemcomitans dengan menggunakan zat besi sebagai model untuk iron-supplemented conditions. tujuan penelitian ini adalah mengetahui pengaruh paparan zat besi pada ekspresi protein spesifik eps biofilm a. actinomycetemcomitans. 105hendrayanti, et al.: paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance bahan dan metode pada penelitian ini ada pemberian perlakuan, tetapi belum dilakukan replikasi. penelitian ini dilakukan di laboratorium mikrobiologi fakultas kedokteran dan laboratorium biokimia fakultas matematika dan ilmu pengetahuan alam universitas brawijaya, malang. prosedur penelitian ini dibagi menjadi dua kelompok besar, yaitu prosedur untuk pembuatan antibodi primer terhadap protein 100 kda, yang diidentifikasi sebagai pnag, dan prosedur untuk melihat pengaruh zat besi terhadap ekspresi pnag pada eps biofilm a. actinomycetemcomitans. prosedur untuk pembuatan antibodi primer terhadap pnag meliputi: kultur bakteri, isolasi protein bakteri, elektroforesis, elektroelusi, imunisasi pada hewan coba dan isolasi serum, serta purifikasi antibodi. kultur bakteri dilakukan dengan mengisolasi terpisah untuk mendapatkan koloni murni a. actinomycetemcomitans pada medium triptone soya agar (tsa+). koloni murni a. actinomycetemcomitans pada tsa+ dimasukkan ke dalam anaerobic jar, kemudian dimasukkan ke dalam inkubator dengan suhu 37° c selama 2 x 24 jam. pertumbuhan koloni a. actinomycetemcomitans diamati. diambil 1 koloni murni dengan ose sengkelit secara aseptis, lalu dimasukkan kedalam medium cair triptone soya broth (tsb+), diinkubasikan kembali secara anaerob. pertumbuhan bakteri diamati terhadap standar mcfarland 1. kultur bakteri dicek dengan pewarnaan gram untuk memastikan tidak adanya kontaminan, lalu diamati di bawah mikroskop dengan perbesaran 1000 kali. suspensi bakteri a. actinomycetemcomitans siap digunakan untuk prosedur isolasi protein bakteri. isolasi protein dilakukan dengan pemisahan suspensi bakteri a. actinomycetemcomitans dengan sentrifugasi selama 15 menit dengan kecepatan 3.000 rpm pada suhu 4° c. dari proses sentrifugasi, didapatkan pemisahan antara supernatan dan pellet. supernatan dibuang. pellet dihomogenkan dengan vortex selama 10 menit, lalu ditambahkan phosphate buffered saline tween–phenyl methyl sulfonyl fluoride (pbst-pmsf) sebanyak 5x volume. pellet kemudian dimasukkan ke dalam sonicator selama 10 menit, lalu dipisahkan dengan sentrifugasi selama 15 menit dengan kecepatan 6.000 rpm. endapan yang terbentuk dari proses sentrifugasi ditambahkan dengan etanol absolut dingin dengan perbandingan 1 : 1, lalu dibiarkan selama 12 jam hingga terbentuk endapan, yang kemudian dipisahkan dengan sentrifugasi selama 15 menit dengan kecepatan 10.000 rpm. endapan yang terbentuk dikeringkan di udara bebas hingga bau etanol hilang, lalu ditambahkan buffer tris-hcl dingin 20 mm dengan perbandingan volume 1 : 1. endapan ini merupakan isolat protein yang mengandung eps. prosedur elektroforesis yang dilakukan terhadap isolat protein adalah elektroforesis dengan separating gel 12% dan stacking gel 3%. isolat protein yang mengandung eps ditambahkan dengan 10 µl reducing sample buffer (rsb), lalu dididihkan pada suhu 100° c selama 5 menit. dilakukan running elektroforesis 130 v, 30 ma (2 plates) hingga terbentuk tracking dye 0,5 cm di atas dasar gel. gel hasil running direndam dalam larutan staining, lalu di-shaker selama 30 menit. gel direndam dalam larutan destaining untuk menghilangkan pewarna, ditambahkan kertas saring, lalu di-shaker hingga gel menjadi bening. gel lalu di-scan untuk melihat fraksi-fraksi protein.11 elektroelusi dilakukan dengan memotong gel hasil running sesuai dengan berat molekulnya. gel dengan pnag (protein 100 kda) lalu dimasukkan dalam kantong selofan, kemudian ditambahkan buffer fosfat 0,2 m. gel dalam kantong selofan yang mengandung protein 100 kda dimasukkan ke dalam chamber elektroelusi yang berisi buffer fosfat 0,1 m, lalu dielektroelusi pada 250 v, 20 ma selama 12 jam, kemudian diangkat dari chamber elektroelusi. buffer fosfat dalam kantong selofan ditambahkan dengan etanol absolut dingin dengan perbandingan 1 : 1, dan diinversi. diinkubasikan dalam refrigerator selama 1 jam, lalu disentrifugasi dingin dengan kecepatan 10.000 rpm selama 15 menit. dari proses sentrifugasi, didapatkan pemisahan antara supernatan dan pellet. supernatan dibuang. pellet dikeringkan dengan diangin-anginkan, lalu ditambahkan dengan buffer tris-cl dengan perbandingan 1 : 1, lalu disimpan pada suhu -20º c. pellet siap digunakan untuk prosedur imunisasi pada hewan coba. imunisasi pada hewan coba, yaitu kelinci betina (oryctolagus cunicullus), dilakukan secara subkutan dengan pnag yang telah ditambahkan dengan complete freund ajuvant (cfa) dengan perbandingan 1:1 (lalu divortex hingga terbentuk emulsi). dua minggu kemudian di-booster menggunakan pnag yang telah ditambahkan dengan incomplete freund ajuvant (ifa) dengan perbandingan 1 : 1, dan diimunisasikan secara subkutan. isolasi serum dilakukan sebanyak 3 kali. isolasi serum pertama dilakukan 1 minggu setelah booster dengan ifa, isolasi serum kedua dilakukan 1 minggu setelah isolasi serum pertama, dan isolasi serum ketiga dilakukan 1 minggu setelah isolasi serum kedua. serum yang digunakan untuk penelitian adalah hasil isolasi serum ketiga. prosedur isolasi serum adalah dengan mengambil darah kelinci betina yang sudah diimunisasi dengan pnag sebanyak 2 cc, lalu dibiarkan selama 1-1,5 jam atau hingga darah dan serum terpisah, kemudian disentrifugasi dengan kecepatan 3.000 rpm selama 15 menit pada suhu ruang. dari proses sentrifugasi, didapatkan pemisahan antara presipitat dan supernatan. presipitat (yang berisi sel darah) dibuang, sedangkan supernatan (yang berupa serum) siap digunakan untuk prosedur selanjutnya.10 purifikasi serum dilakukan dengan menambahkan serum dengan saturated ammonium sulfate (sas) 50% dengan perbandingan volume 1 : 1, lalu dibiarkan ± 30 menit, kemudian disentrifugasi dengan kecepatan 10.000 rpm pada suhu 4º c selama 10 menit. dari proses sentrifugasi, didapatkan pemisahan antara supernatan dan presipitat. supernatan dibuang. presipitat dicuci dengan sas 50% 10x volume, lalu di-vortex, kemudian disentrifugasi 106 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 103–109 10.000 rpm pada suhu 4º c selama 10 menit. dari proses sentrifugasi, didapatkan pemisahan antara supernatan dan presipitat. supernatan dibuang. presipitat dilarutkan dalam buffer fosfat 0,2 m, ph 7 dengan perbandingan 1 : 1, lalu dimasukkan dalam kantong selofan, kemudian didialisis menggunakan buffer fosfat 0,1, ph 7 selama 12 jam pada kondisi dingin. buffer fosfat ditambahkan dengan etanol absolut dingin, lalu diinkubasi dalam refrigerator selama 1 jam atau sampai terlihat endapan, kemudian disentrifugasi dingin dengan kecepatan 6.000 rpm selama 15 menit, lalu didiamkan dalam freezer selama ± 15 menit. dari proses sentrifugasi, didapatkan pemisahan antara supernatan dan pellet. supernatan dibuang. pellet dikeringkan dengan diangin-anginkan, lalu ditambahkan dengan buffer tris-cl dengan perbandingan 1 : 1, lalu disimpan pada suhu -20º c. pellet ini merupakan antibodi terhadap pnag, dan siap digunakan untuk prosedur western blotting.10 prosedur untuk melihat pengaruh zat besi terhadap ekspresi pnag pada eps biofilm a. actinomycetemcomitans meliputi: kultur bakteri, isolasi protein bakteri, elektroforesis. kultur bakteri pada prosedur ini dilakukan seperti kultur bakteri pada prosedur sebelumnya, tetapi dengan modifikasi. di tahap ini dibagi menjadi 2 kelompok besar, yaitu: kelompok kontrol (tanpa perlakuan) dan kelompok perlakuan (pada tsb+ ditambahkan zat besi). kelompok perlakuan dibagi menjadi 3, yaitu: kelompok perlakuan i (pada tsb+ ditambahkan 250 µm zat besi), kelompok perlakuan ii (pada tsb+ ditambahkan 300 µm zat besi), dan kelompok perlakuan iii (pada tsb+ ditambahkan 350 µm zat besi). isolasi protein bakteri dan elektroforesis pada prosedur ini dilakukan seperti isolasi protein bakteri dan elektroforesis pada prosedur sebelumnya.7 gel dari elektroforesis dilakukan running dengan sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds page). protein dari gel sds page ditransfer ke membran nitrocellulose dengan alat semi-dry (kertas saring, membran nitrocellulose, dan gel hasil running disusun seperti sandwich dengan komposisi: 9 lembar kertas saring bagian bawah, membran nitrocellulose, gel hasil running, dan 6 kertas saring). dilakukan running selama 2 jam, lalu dimatikan, dan membran nitrocellulose diambil. membran nitrocellulose diblok dalam blocking buffer (pbst skim milk 5%) selama 1 jam, digoyang, lalu dicuci 3 x 5 menit dalam pbst. diinkubasikan dengan antibodi primer pnag dengan perbandingan 1:200 dalam pbst skim selama 12 jam pada temperatur 4° c, lalu dicuci 3 x 5 menit dalam tris buffered saline (tbs). diinkubasikan dengan antibodi sekunder alkali phosphatase conjugated dengan perbandingan 1:2.500 dalam tbs selama 1 jam pada suhu ruang, lalu dicuci 4 x 5 menit dalam phosphate buffered saline tween (pbst). diinkubasi dengan western blue substrate solution dalam ruang gelap selama 12 jam atau sampai terlihat warna band, lalu dicuci dengan aquadest untuk stop reaksi. ekspresi protein pada membran (terlihat dalam bentuk protein band) siap untuk diamati. membran prosedur western blotting yang telah dilakukan lalu difoto dengan instrumen gel documentation dan dianalisis dengan quantitione software (raw data dapat dibaca pada lampiran). hasil dari prosedur elektroforesis didapatkan bahwa eps biofilm a. actinomycetemcomitans memiliki beberapa fraksi protein, selain protein spesifik 100 kda. fraksifraksi protein bisa dilihat melalui prosedur elektroforesis (gambar 1). dari prosedur elektroforesis, ditemukan 5 fraksi protein yang terekspresi dari eps biofilm a. actinomycetemcomitans, yaitu protein dengan berat molekul 100 kda, 61 kda, 55 kda, 47 kda, dan 44 kda. d a r i p r o s e d u r w e s t e r n b l o t t i n g , d i p e r o l e h gambaran ekspresi protein spesifik eps biofilm a. actinomycetemcomitans dengan berat molekul 100 kda (gambar 2). dari hasil penelitian, didapatkan bahwa paparan zat besi berpengaruh pada ekspresi protein spesifik eps biofilm a. actinomycetemcomitans. pada penelitian ini terjadi penurunan ekspresi protein spesifik eps biofilm a. actinomycetemcomitans dengan berat molekul 100 kda, yang diidentifikasi sebagai polimer dari β-1,6-n-asetild-glukosamin (pnag), pasca paparan zat besi. ekspresi protein pada kelompok kontrol (tanpa perlakuan) lebih tinggi daripada kelompok perlakuan dengan paparan zat besi. dari hasil penelitian juga didapatkan bahwa kadar zat besi yang diberikan berpengaruh pada ekspresi pnag. paparan zat besi menyebabkan penurunan ekspresi pnag yang gradual, dengan kelompok perlakuan i (dengan 250 gambar 1. hasil elektroforesis. bm (kda) = berat molekul dalam satuan kilodalton, m = marker protein, 1 = reducing sample buffer (rsb), 2 = sampel protein bakteri a. actinomycetemcomitans. 107hendrayanti, et al.: paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance µm zat besi) memiliki ekspresi tertinggi dan kelompok perlakuan iii (dengan 350 µm zat besi) memiliki ekspresi terendah. dari hasil penelitian, didapatkan bahwa paparan zat besi berpengaruh pada densitas pita protein spesifik eps biofilm a. actinomycetemcomitans (pnag). pada penelitian ini terjadi penurunan densitas pita protein spesifik eps biofilm a. actinomycetemcomitans dengan berat molekul 100 kda, yang diidentifikasi sebagai polimer dari β-1,6n-asetil-d-glukosamin (pnag), pasca paparan zat besi. densitas pnag pada kelompok kontrol (tanpa perlakuan) lebih tinggi daripada kelompok perlakuan dengan paparan zat besi. dari hasil penelitian juga didapatkan bahwa kadar zat besi yang diberikan berpengaruh pada densitas pita pnag. paparan zat besi menyebabkan penurunan densitas pita pnag; dengan kelompok perlakuan i (dengan 250 µm zat besi) memiliki densitas pita protein tertinggi (279,8478274667 int/mm2), kelompok perlakuan ii (dengan 300 µm zat besi) memiliki densitas pita protein lebih rendah dari densitas pita protein kelompok perlakuan i dan lebih gambar 2. hasil western blotting. bm (kda) = berat molekul dalam satuan kilodalton, k = ekspresi protein spesifik 100 kda pada kelompok kontrol, 1 = ekspresi protein spesifik 100 kda pada kelompok perlakuan i, 2 = ekspresi protein spesifik 100 kda pada kelompok perlakuan ii, 3 = ekspresi protein spesifik 100 kda pada kelompok perlakuan iii. tinggi daripada kelompok perlakuan iii (207,1479999351 int/mm2), dan kelompok perlakuan iii (dengan 350 µm zat besi) memiliki densitas pita protein terendah (142,2883007712 int/mm2) (gambar 3 dan tabel 1). pembahasan zat besi memiliki peran yang penting untuk pertumbuhan bakteri, baik sebagai nutrisi maupun katalis pembentukan radikal hidroksil.9 ketersediaan zat besi menjadi sinyal yang penting yang meregulasi ekspresi dari banyak faktor virulensi pada bakteri patogen.7 zat besi juga merupakan aktivator pembentukan biofilm, dan pada beberapa kasus, zat besi menghambat pembentukan biofilm.5 paparan zat besi pada a. actinomycetemcomitans menyebabkan protein fur membentuk kompleks dengan fe2+ yang berikatan pada sekuen kosensus yang spesifik (yang disebut “fur box”) pada srna. ikatan ini menyebabkan mutasi pada srna, yang menyebabkan penurunan transkripsi gen determinan biofilm. penurunan transkripsi gen determinan biofilm menyebabkan penurunan ekspresi pnag eps biofilm a. actinomycetemcomitans. hasil penelitian ini menunjukkan bahwa densitas pita protein pada kelompok kontrol (480,8601678454 int/mm2) lebih tinggi daripada kelompok perlakuan dengan paparan zat besi; dan paparan zat besi menyebabkan penurunan densitas pita pnag dengan kelompok perlakuan i (dengan 250 µm zat besi) memiliki densitas pita protein tertinggi (279,8478274667 int/mm2), kelompok perlakuan ii (dengan 300 µm zat besi) memiliki densitas pita protein lebih rendah dari densitas pita protein kelompok perlakuan i dan lebih gambar 3. diagram data densitas kelompok kontrol, perlakuan i, ii, dan iii. tabel 1. data densitas kelompok kontrol, perlakuan i, ii dan iii name identity density (int/mm2) u1 kelompok kontrol 480,8601678454 u2 kelompok perlakuan i 279,8478274667 u3 kelompok perlakuan ii 207,1479999351 u4 kelompok perlakuan iii 142,2883007712 d en si ty 108 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 103–109 tinggi daripada kelompok perlakuan iii (207,1479999351 int/mm2), dan kelompok perlakuan iii (dengan 350 µm zat besi) memiliki densitas pita protein terendah (142,2883007712 int/mm2). menurut amarasinghe et al.,4 densitas pita protein ini mewakili ekspresi pnag. densitas pita protein kelompok kontrol yang lebih tinggi daripada kelompok perlakuan dengan paparan zat besi menunjukkan bahwa ekspresi pnag menurun akibat paparan zat besi. densitas pita protein yang makin menurun pasca peningkatan kadar zat besi yang diberikan menunjukkan bahwa semakin tinggi kadar zat besi yang dipaparkan menyebabkan semakin rendah ekspresi pnag. paparan zat besi secara signifikan mempengaruhi kuantitas biofilm yang terbentuk. semakin tinggi ekspresi pnag, semakin besar resistensi bakteri terhadap antibiotik dan aktivitas makrofag.4 berdasarkan konteks teori, didapatkan dalam penelitian ini bahwa (a) paparan zat besi menyebabkan penurunan sintesis biofilm dan penurunan resistensi bakteri terhadap antibiotik dan aktivitas makrofag; (b) semakin tinggi kadar zat besi yang diberikan menyebabkan kuantitas biofilm yang terbentuk makin kecil dan resistensi bakteri terhadap antibiotik dan aktivitas makrofag makin rendah. penelitian ini menggunakan western blotting sebagai metode utama karena memiliki spesifisitas dan sensitivitas yang baik. western blotting merupakan metode yang menggunakan spesifisitas dari interaksi antigen-antibodi untuk mendeteksi protein tertentu yang spesifik dari suatu sampel protein. metode lain yang dapat digunakan untuk penelitian protein antara lain imunopresipitasi (ip), imunohistokimia/sitokimia, dan enzyme-linked immunosorbent assay (elisa). metode-metode ini dapat menimbulkan misinterpretasi jika terjadi reaktivitas silang antara antibodi dan protein selain protein yang akan diteliti. prosedur western blotting memiliki keuntungan dengan adanya resolve protein berdasarkan berat molekul (bm), dan protein yang akan diteliti biasanya terpisah dari proteinprotein yang bereaksi silang.10 untuk pembuatan antibodi primer, dilakukan imunisasi pnag dan adjuvant terhadap kelinci untuk merangsang terbentuknya antibodi terhadap pnag, yang kemudian digunakan untuk reaksi antigen-antibodi dalam prosedur western blotting. antibodi terhadap pnag ini perlu diteliti lebih lanjut sebagai informasi untuk pengembangan imunisasi pasif terhadap sejumlah bakteri yang memproduksi protein ini. penelitian terhadap sifat-sifat imunokimia dari pnag juga perlu dilakukan untuk menambah informasi dalam pengembangan vaksin yang efektif terhadap bakteri-bakteri yang memproduksi pnag. bakteri yang memproduksi pnag antara lain staphylococcus epidermidis, staphylococcus aureus, escherichia coli, yersinia spp. (termasuk juga y. pestis), bordetella spp., dan actinobacillus spp. (termasuk juga a. actinomycetemcomitans). dengan mengetahui pengaruh paparan zat besi pada ekspresi pnag eps biofilm a. actinomycetemcomitans diharapkan dapat membantu perkembangan perawatan terhadap periodontitis agresif. dengan meneliti kondisikondisi yang dapat berpengaruh terhadap pembentukan biofilm, diharapkan dapat mengarahkan strategi terapi yang mampu menurunkan pertahanan diri bakteri ini. zat besi merupakan salah satu faktor yang berpengaruh pada pembentukan biofilm a. actinomycetemcomitans. meneliti zat besi saja tidak cukup untuk menyimpulkan pengobatan yang sesuai, karena masih banyak kondisi-kondisi lain yang dapat mempengaruhi pembentukan biofilm a. actinomycetemcomitans. namun, penelitian terhadap fraksi-fraksi protein, selain juga pnag, yang terkekspresi pada eps biofilm a. actinomycetemcomitans juga perlu dikembangkan lebih lanjut. dari prosedur elektroforesis, ditemukan 5 fraksi protein yang terekspresi dari eps biofilm a. actinomycetemcomitans, yaitu protein dengan berat molekul 100 kda, 61 kda, 55 kda, 47 kda, dan 44 kda. identifikasi terhadap masing-masing fraksi protein ini dan fungsinya dapat membantu mengarahkan perkembangan terapi yang lebih signifikan terhadap penyakit periodontitis agresif dan penyakit-penyakit lain yang disebabkan oleh infeksi a. actinomycetemcomitans. penelitian ini menunjukkan bahwa paparan zat besi berpengaruh pada ekspresi spesifik biofilm eps a. actinomycetemcomitans. semakin tinggi kadar zat besi yang dipaparkan menyebabkan densitas biofilm yang terbentuk menurun dan resistensi a. actinomycetemcomitans terhadap antibiotik dan aktivitas makrofag menurun. penelitian lebih lanjut perlu dilakukan terhadap faktor yang berpengaruh terhadap pembentukan biofilm a. actinomycetemcomitans. identifikasi terhadap masing-masing fraksi protein eps biofilm a. actinomycetemcomitans dan fungsinya juga perlu dilakukan untuk membantu mengarahkan perkembangan terapi yang lebih signifikan terhadap infeksi a. actinomycetemcomitans. penelitian lebih lanjut perlu dilakukan terhadap efek zat besi terhadap flora normal dan bakteri oportunistik patogen lain di rongga mulut karena sifat zat besi yang bisa menghambat pembentukan biofilm, tapi pada beberapa kasus, dapat menjadi aktivator pembentukan biofilm. daftar pustaka 1. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 11th ed. st. louis: elsevier saunders; 2012. p. 674, 685, 1929. 2. amalina r. perbedaan jumlah actinobacillus actinomycetemcomitans pada periodontitis agresif berdasarkan jenis kelamin. jurnal majalah ilmiah sultan agung. 2011. 3. cho cm, you hk, jeong sn. the clinical assessment of aggressive periodontitis patients. j periodontal implant sci 2011; 41(3): 1438. 4. amarasinghe jj, scannapieco fa, haase em. transcriptional and translational analysis of biofilm determinants of aggregatibacter actinomycetemcomitans in response to environmental perturbation. infection and immunity 2009; 77(7): 2896–907. 109hendrayanti, et al.: paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance 5. karatan e, watnick p. signals, regulatory networks, and materials that build and break bacterial biofilms. microbiology and molecular biology reviews 2009; 73(2): 310-47. 6. venketaraman v, lin ak, le a, kachlany sc, connell nd, kaplan jb. both leukotoxin and poly-n-acetylglucosamine surface polysaccharide protect aggregatibacter actinomycetemcomitans cells from macrophage killing. microbial pathogenesis 2008; 45 (3): 173–80. 7. haraszthy vi, lally et, haraszthy gg, zambon jj. molecular cloning of the fur gene from actinobacillus actinomycetemcomitans. infection and immunity 2002; 70(6): 3170–9. 8. rhodes er, shoemaker cj, menke sm, edelmann re, actis la. evaluation of different iron sources and their inf luence in biofilm formation by the dental pathogen actinobacillus actinomycetemcomitans. journal of medical microbiology 2007; 56: 119–28. 9. haraszthy vi, jordan sf, zambon jj. identification of fur-regulated genes in actinobacillus actinomycetemcomitans. microbiology 2006; 152(pt 3): 787-96. 10. howard gc, bethell dr. basic methods in antibody production and characterization. boca raton: crc press lcc; 2001. p. 218. 11. mahmood t, young p. western blot: technique. theory and trouble shooting. n am j med sci 2012; 4(1): 134-429. 66 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 66–70 research report assessment of behavioral factors associated with dental caries in pre-school children of high socioeconomic status families bushra rashid noaman department of pedodontics faculty of dentistry, tishk international university erbil, kurdistan region – iraq abstract background: many iraqi children of high socioeconomic status (ses) families attend dental clinics presenting predominantly cavitated and painful multiple carious lesions. the factors responsible for dental caries within this sector of society need to be identified. purpose: the aim of this study is to assess the dental care behavior of mothers and its relationship with the prevalence of dental caries in pre-school age children drawn from high ses families in northern iraq. methods: a study was conducted to assess the prevalence of dental caries and its relationship to oral hygiene habits in 440 pre-schoolers living in erbil, northern iraq. an oral hygiene questionnaire was distributed among the families. dental examination of the children was performed to calculate the dependent factor of decayed, missing and filled teeth due to caries (dmf). all data was analyzed by means of the spss microsoft statistical system using descriptive tables to identify the relationship between the dependent and independent variable dmf indexes. in order to find the significances, a chi-square test, a fisher’s exact test and a likelihood ratio test were used at level of p<0.05. results: 67% of the children in the sample had dental caries (mean dmf=3.25±3.77) with 5-year-olds being more affected by dental caries (74%) than 4-year-olds (60%). a strong correlation was found in this study between the dependent factor, dmf, and the following independent factors: frequency of snack consumption, the need to assist the child during brushing, maternal caries and the mother’s education. conclusion: despite being members of high ses families, the children examined were significantly subject to dental caries, a fact directly correlated with inappropriate behavior on the part of their caregivers in relation to the essential aspects of oral health care. keywords: dental caries; dmf index; oral health; preschoolers; socioeconomic status correspondence: bushra rashid noaman, department of pedodontics, faculty of dentistry, tishk international university, erbil, kurdistan region, iraq. email: bushra.rashid@ishik.edu.iq introduction early childhood is defined as the period between birth and the 71st month during which dental caries affect children.1 an important element within primary care is early examination of the child. dental caries constitute a disease caused by bacteria and, consequently, is transmissible from mother to child thereby negatively affecting the dentition status of the latter.1 dental caries caused by aciduric and acidogenic bacteria transform sugar into lactic acid subsequently resulting in the dissolution of the tooth enamel.2 although high ses exerts a noticeable positive influence on the general and oral health of individuals,3 numerous other factors may cause dental caries, such as poor oral hygiene, the absence of assistance to children during the brushing of their teeth and a high daily consumption of snacks. the incidence of dental caries in young children may be higher due to the frequent intake of snacks, especially sweets. therefore, restricting the frequency of children’s consumption of snacks allied with consistently regular meals is recommended.4 dental caries in early childhood may be the result of bottle feeding or breastfeeding while the infant in question is asleep.2,5 regular visits to the dentist and the professional application of fluoride are essential factors in the prevention of dental caries6 which negatively impact on the psychology of children due to aesthetic problems which can negatively dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p66–70 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p66-70 67bushra rashid noaman/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 66–70 affect their self-esteem and ability to eat when the lesions are cavitated.7 in the usa, it has been proved that caregivers presenting tooth loss due to caries negatively influenced the dental health of their young children who also suffered from the same condition.8 the level of education enjoyed by the mother is an important influencing factor in the incidence of dental caries in a child, together with such variables as income and the frequency of their visits to the dentist.9 the aim of this study was to assess the influence of maternal behavior on the prevalence of dental caries in pre-schoolers drawn from high ses families in northern iraq. materials and methods this study was undertaken in four selected private kindergartens located in the north, south, west and east of erbil, northern iraq. 440 children aged 4-5 years old were examined for dental caries, while information concerning their oral hygiene habits was also collected from their families by means of a questionnaire. all the children were drawn from families in which the parents occupied high income jobs. the number of subjects drawn from the kindergartens totaled 486. after the application of specific inclusion and exclusion criteria, 46 children were rejected, of which 21 failed to submit their questionnaires, six did not attend on the day of the examination, 18 did not return their oral habit questionnaires and one was excluded due to his being asthmatic. the research population of the study, therefore, comprised 440 individuals. all subjects satisfying the following criteria were included in the study: aged 4-5 years, free of systemic disease, parental agreement provided, comprehensive oral habits data and complete dental records which could be reviewed by the observers. failure to meet these criteria was considered justifiable grounds for exclusion. the questionnaire included items concerning the tooth-brushing habits of each subject, the timing and duration of their feeding during infancy, their eating routine, untreated caries and use of fluoride, together with the level of education of his/her mother. an oral hygiene habits questionnaire in kurdish, the regional language, was prepared by the author which included the factors potentially causing dental caries9 (table 1) and distributed among the families who completed the questionnaire one week before the dental examinations were conducted. dental examinations involving the use of a plan mouth mirror and cpi probe were conducted under natural light in the well-lit hall of the kindergartens with the subject seated on an ordinary chair. the dentition status for children, annex 2, who (2013)10 was used to register the examination results. dental caries were recorded when a lesion in a pit or fissure, or on a smooth tooth surface, constituted an observable cavity, damaged enamel due to caries, or a visibly softened floor or wall. temporarily restored teeth, or ones previously restored but also carious, were classified as decayed. a tooth was considered to be present even if only parts of it remained in the mouth. in cases of uncertain tooth presence, caries were not recorded as existing. in order to enhance the reliability of clinical judgments, four dentists were trained to collect the dental examination data. the dmf index was used to measure the prevalence of dental caries. in order to quantify intraobserver validity, a pediatric dentist repeated ten of the observations previously performed by each dentist, thereby acting as a calibrator. a level of intra-observer validity between the pedodontics specialist and each dentist was achieved. the research was initiated after approval by the scientific committee of tishk international university (document no. iu.fa.fr. 137e. decree no. 4, 2018), and that of the families of the subjects had been secured. ibm spss system 22 was used to analyze the data collected. descriptive tables were used to indicate the table 1. oral habits questionnaire no. questions answers q1 does the child suffer from any systemic disease? yes no q2 does the mother have caries? yes no q3 the feeding time of the child during infancy daytime hours only both daytime and nighttime hours q4 does the child demonstrate poor eating habits, such as consuming cakes, sweets, chocolate, soda drinks and potato chips? yes 1-2 times a day >3 times a day no q5 does the child brush his/her own teeth? yes no q6 do you assist your child during tooth-brushing? yes no q7 has the child ever undergone professional fluoride application? yes no q8 education level of the mother or caregiver primary school secondary school undergraduate degree postgraduate degree dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p66–70 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p66-70 68 bushra rashid noaman/dent. j. (majalah kedokteran gigi) 2019 june; 52(2):66–70 total research population together with the gender of each and his/her relationship to the dependent variable dmf index. a chi-squared test, a fisher’s exact test and a likelihood ratio test were used to compare the relationship between the dependent variable (dmf) and the independent variables; tooth-brushing habits, the frequency of feeding during infancy, eating habits, untreated maternal caries, professional fluoride application and the level of education of the mother. the p-value was measured at a level of p<0.05. results 440 pre-school children were examined during this study, 67% of whom had dental caries (mean dmf=3.25±3.77). the 5-year-olds were more affected by dental caries (74%) than their 4-year-old counterparts (60%) with respective dmf indexes of 3.8±4.33 and 2.9±3.4 which represented a statistically significant difference. the contents of table 2 confirm that males were more affected by dental caries (70%), than females (52%) and had a higher dmf index (3.6±3.76). table 2. distribution of age and gender and dmf index in the sample variables factors n caries (%) mean dmf±sd p-value age 4 years 160 60.00 3.8±4.33 0.021* 5 years 280 74.00 2.9±3.40 total 440 67.00 3.25±3.77 gender boys 240 70.00 3.6±3.76 0.25 girls 200 52.00 2.8±3.78 *significant difference at the level p<0.05 table 3. the relationship between mean dmf and tooth brushing habits in the sample variable type n caries (%) mean dmf±sd p-value tooth brushing 1/day 304 63 3.5 ± 3.87 0.2442-3/day 96 40 1.9 ± 2.7 none 40 80 4.6 ± 4.59 table 4. the relationship between mean dmf and brushing assistance habit variable type n caries (%) mean dmf±sd p-value brushing assistance no 240 67 3.93 ± 4.06 0.05* yes 200 50 2.37 ± 3.29 *p-value at the limit of significance table 5. the relationship between the mean dmf and snack consumption variable type n caries (%) mean dmf±sd p value snacking once and twice/ day 232 58.60 2.75 ± 3.02 0.001* >3/day 104 84.00 5.46 ± 4.85 *significant difference at level p<0.05 table 6. the relationship between mean dmf and nursing time during infancy variable type n caries (%) mean dmf±sd p value nursing time day and night 56 85.70 5.42 ± 3.58 0.001* day 384 56.25 2.93 ± 3.71 *significant difference at the level p<0.0 table 7. the relationship between mean dmf and topical fluoride application variable type n caries (%) mean dmf±sd p-value topical fluoride yes 64 62.50 3.75 ±4.18 0.214 no 376 59.50 3.17± 3.71 table 8. the relationship between mean dmf and dental caries among the children’ mothers variable type n caries (%) of children mean dmf±sd p-value mother caries yes 184 60.00 3.4 ± 3.72 0.025* no 256 59.40 3.12 ± 3.83 *significant difference at the level p<0.05 table 9. the relationship between the mothers’ education and dmf in the sample variable type n caries (%) mean dmf±sd p value mother’s education post graduate 48 16.6 0.16±0.38 0.001*bachelor 200 56 2.26±2.6 secondary 120 73.3 5.9±4.7 primary 72 77.7 3.55±3.29 *significant difference at the level p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p66–70 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p66-70 69bushra rashid noaman/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 66–70 the dmf index was higher in the research subjects who did not brush their teeth (4.6±4.59) and who were most susceptible to caries (80%) than those who brushed once or twice a day, but it showed no statistically significant difference (table 3). responses to the question about brushing assistance showed that 240 mothers did not provide this to their children. of the subjects who were not assisted with tooth brushing, 67% had caries with a higher dmf index (3.93 ± 4.06) than those who were assisted (2.37 ± 3.29). however, the difference was not statistically significant (table 4). of 440 children, 232 consumed snacks once or twice a day and 104 more than three times daily. the highest dmf index in subjects was that relating to more than three times (5.45±4.85); 84% were affected by dental caries and the difference was statistically significant (table 5). responses relating to the intensity of breastfeeding showed that 56 mothers breast fed their infants both during the day and at night and 85.7% of their children had dental caries. the other 384 children were breast fed only during the day and 56.25% were affected by dental caries, indicating a respective dmf index of 5.42 ± 3.58 and 2.93 ± 3.71 which constituted a statistically significant difference (table 6). topical fluoride was applied to only 64 subjects in the sample who still recorded a high caries index (dmf = 3.37±4.18). 62.5% were affected by dental caries, almost the same percentage as those who did not received fluoride (59.5%) with no statistically significant difference (table 7). the question concerning whether the mothers were affected by dental caries, showed that 184 had dental caries, while 60% of their children were affected by dental caries with a dmf of 3.4 ±3.72. these was a statistically significant difference in comparison with the caries index of children whose mothers were free of dental caries (mean dmf = 3.12 ± 3.83) (table 8). with regard to the education level of mothers and its relationship to the incidence of dental caries, the results of this study showed that the highest percentage of dental caries was found in those children whose mothers had only gained a primary school education (77.7%, dmf = 3.55±3.29). in contrast, only 16.6% of the children whose mothers had undergone higher education were affected (dmf = 0.16±0.38). there was a highly significant statistical difference between the level of the education of the mother in terms of the prevalence of caries in their children, namely; p<0.05 (table 9). discussion as dental caries develops over time, it can be argued that those detected in the subjects of the current study largely began in infancy due to their inappropriate dietary habits becoming increasingly prominent from the point of weaning until the time of the examination (4-5 years). this viewpoint agrees with that contained in the study conducted by moynihan and petersen.11 caregivers lacked knowledge about the negative impact of frequent snacks. moreover, high ses families in iraq purchase sweets for their children to make them feel content. in addition, there was considerable inconsistency regarding the appropriate point in time to wean infants which rendered dental treatment for the entire family essential. there is a need to introduce this practice in iraq. there are numerous potential causative factors of dental caries which comprise: the host, the mediator and the environment. the primary one is mutans streptococcus (ms) bacteria. the adherence of ms to the tooth surface will result in the formation of plaque. ms ferments sugar and converts it into lactic acid which leads to demineralization of the enamel.12 families need to be aware of the effect of these bacteria in forming dental caries and how to prevent this by tooth brushing, assisting the child during brushing, diet counseling and regular visits to the dentist. pediatric dentists, in particular, have a major role to play in educating mothers to manage the oral health of their children. all dentists who treat adults with caries should ask the patient about the oral health of their children.13 one of the measures to prevent dental caries in infants and toddlers is that of treating the dental caries of their caregivers since these may be induced in the child.14 of the mothers featured in the current study, 46% suffered from dental caries. however, this figure may represent a case of under-reporting which can be considered a limitation of this study. preventive methods should be established such as oral hygiene improvement, fluoride application, the use of pit and fissure sealants, decreased frequency of snacks during the day and educating caregivers.15 in this study, the mothers with caries themselves were found to have children suffering from high levels of this condition. the components of the ses are the level of parental education, house ownership, family income and type of parental occupation.16 in the current study, the families of the research subjects were all owner-occupiers of their homes and earn high incomes, but their children were found to be strongly affected by dental caries. in the current study, one of the factors that directly influenced the dental health of those individuals was the level of their mothers’ education. the highest dmf score was found in children whose mothers had only attended primary school, while the lowest was in those whose mothers had progressed to higher education. several studies2,5,7,8 concur that the education level of a mother influences the advice she gives her child with regard to diet and duration of brushing, as well as the regularity of both her own visits and those of her child to the dentist in order to monitor their respective oral health. educational programs can be introduced into schools and may prove beneficial in increasing the health awareness of school children in iraq17 which will, in turn, have a positive impact on subsequent generations. this study is subject to certain limitations in that it relates only to iraq. despite being drawn from high ses families, the subjects of this study demonstrated a significant incidence of dental caries. the causative factors dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p66–70 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p66-70 70 bushra rashid noaman/dent. j. (majalah kedokteran gigi) 2019 june; 52(2):66–70 underpinning their high rates in these children were related to inappropriate behavior on the part of the caregivers with regard to the essential aspects of oral health care. on this point, the author recommends the introduction of educational programs for all categories of iraqi society using a variety of media and methods and activating community dentistry to increase the awareness of families regarding oral health care. acknowledgements the author wishes to extend her appreciation to the presidency and deanery of dentistry faculty of tishk international university for their financial support of the research reported here, to research assistants, dr. saya hadi rauf, dr. lava sabir and dr. zahraa emad, for their invaluable assistance in examining children during the study and to dr. zhakaw amang for her support in formatting the tables contained in the article. references 1. chandna p, adlakha vk. oral health in children — guidelines for pediatricians. indian pediatr. 2010; 47(4): 323–7. 2. mwakayoka h, masalu jr, kikwilu en. dental caries and associated factors in children aged 2-4 years old in mbeya city, tanzania. j dent (shiraz, iran). 2017; 18(2): 104–11. 3. nicolau b, marcenes w, bartley m, sheiham a. a life course approach to assessing causes of dental caries experience: the relationship between biological, behavioural, socio-economic and psychological conditions and caries in adolescents. caries res. 2003; 37(5): 319–26. 4. jain m, namdev r, bodh m, dutta s, singhal p, kumar a. social and behavioral determinants for early childhood caries among preschool children in india. j dent res dent clin dent prospects. 2015; 9(2): 115–20. 5. meyer f, enax j. early childhood caries: epidemiology, aetiology, and prevention. int j dent. 2018; 2018: 1–7. 6. gomes m, pinto-sarmento t, costa e, martins c, granville-garcia a, paiva s. impact of oral health conditions on the quality of life of preschool children and their families: a cross-sectional study. health qual life outcomes. 2014; 12: 1–12. 7. roberts cr, warren jj, weber-gasparoni k. relationships between caregivers’ responses to oral health screening questions and early childhood caries. j public health dent. 2009; 69(4): 290–3. 8. moimaz sas, fadel cb, lolli lf, garbin cas, garbin ají, saliba na. social aspects of dental caries in the context of mother-child pairs. j appl oral sci. 2014; 22: 73–8. 9. litt md, reisine s, tinanoff n. multidimensional causal model of dental caries development in low-income preschool children. public health rep. 1995; 110(5): 607–17. 10. world health organization. oral health surveys: basic methods. 5th ed. world health organization; 2013. p. 85. 11. moynihan p, petersen pe. diet, nutrition and the prevention of dental diseases. public health nutr. 2004; 7(1a): 201–26. 12. çolak h, dülgergil çt, dalli m, hamidi mm. early childhood caries update: a review of causes, diagnoses, and treatments. j nat sci biol med. 2013; 4: 29–38. 13. weintraub ja, prakash p, shain sg, laccabue m, gansky sa. mothers’ caries increases odds of children’s caries. j dent res. 2010; 89(9): 954–8. 14. lee y. diagnosis and prevention strategies for dental caries. j lifestyle med. 2013; 3(2): 107–9. 15. douglass jm, li y, tinanoff n. association of mutans streptococci between caregivers and their children. pediatr dent. 2008; 30(5): 375–87. 16. adler ne, newman k. socioeconomic disparities in health: pathways and policies. health aff. 2002; 21(2): 60–76. 17. noaman br, rauf sh. initial impact of an educational program on the oral health awareness of iraqi primary school students aged 12 years. j dent heal oral disord ther. 2017; 8(6): 1–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p66–70 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p66-70 vol 49 no 3 juli-sept 2016.indd 153153 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 153–157 compressive strength and porosity tests on bovine hydroxyapatitegelatin-chitosan scaffolds nadia kartikasari,1 anita yuliati,1 and indah listiana2 1department of dental materials 2department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: degenerative diseases, aggressive periodontitis, trauma, jaw resection, and congenital abnormalities can cause defects in jaw bone. the surgical procedure for bone reconstruction currently performed is bone regeneration graft (brg). unfortunately, this procedure still has many disadvantages. thus, tissue engineering approach is necessary to be conducted. the main component used in this tissue engineering is scaffolds. scaffolds used in bone regeneration is expected to have appropriate characteristics with bone, such as high porosity and swelling ratio, low degradation rates, and good mechanical properties. for those reasons, this research used scaffolds made from bovine hydroxyapatite (bha), gelatin (gel), and chitosan (k)/bha-gel-k as one of biomaterial candidates for bone regeneration. purpose: this study aimed to determine compressive strength value and porosity size of bha-gel-k scaffolds. method: compressive strength of bha-gel-k scaffolds was tested using autograph with speed 10 mm/ min with a load cell compress machine of 100 kn. compressive strength was calculated by force divided to surface area. porosity test was measured using sem. scaffold were coated with pb and au, then the porosity size is calculated with sem at 100x magnification. result: bha-gel-k scaffolds had a mean compressive strength value of 174.29 kpa and a porosity size of 31.62 + 147.06 lm. conclusion: it can be concluded that bha-gel-k scaffolds has a good compressive strength, but not yet resemble real bone mass, while porosity of bha-gel-k scaffold is appropriate for bone tissue regeneration application. keywords: scaffolds; bovine hydroxyapatite; gelatin; chitosan; compressive strength; porosity correspondence: anita yuliati, department of dental material, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: nitaruslan@hotmail.com introduction degenerative diseases, aggressive periodontitis, trauma, jaw resection, and congenital abnormalities can cause defects in jaw bone.1,2 in a massive bone defect, gap can emerge. massive bone defect is still a major challenge in the field of dentistry since bone healing process often cannot successfully restore the shape and size of the jaw bone as the same as the previous ones.3 a surgical procedure for bone reconstruction currently performed is bone regeneration graft (brg). unfortunately, this procedure still has many disadvantages. tissue engineering approach is necessary to be conducted. tissue engineering aims to regenerate damaged structures and tissues. the concept of tissue engineering is combining three basic components of a cell, scaffolds, and signal regulator, called as triad tissue engineering.1 scaffolds are three-dimensional structure used as temporary replacement for damaged natural extra cellular matrixes (ecm). consequently, the attachment, anchoring, proliferation, migration, and differentiation of cells as well as regeneration of tissue can occur. scaffolds for bone regeneration have some criteria, such as good mechanical properties, high porosity and swelling ratio, and low degradation.4,5 the mechanical properties of scaffolds are expected equal with mechanical properties in normal bone. the mechanical properties can be measured by several parameters. the most widely used parameter is compressive strength. compressive strength is an ability of a material to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p153-157 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p153-157 154 kartikasari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 153–157 withstand the burden of pressure. meanwhile, porosity of scaffolds is required for cell attachment. porosity can also determine the mechanical properties of scaffolds. the size of the scaffolds porosity can be observed using a scanning electron microscope (sem).6 in the last few years, biomimetic scaffolds made of hydroxyapatite, gelatin, and chitosan scaffolds began to be developed. scaffolds with the composition of hydroxyapatite, gelatin, and chitosan is expected to resemble ecm bone, which consists of inorganic components (70%) and organic components (30%).7 hydroxyapatite is the biggest inorganic component. hydroxyapatite is osteoconductive and biocompatible, that is able to integrate well and strongly in the host bone.8 hydroxyapatite can also be obtained from synthesis and natural materials, such as bovine hydroxyapatite. bovine hydroxyapatite has similarities with hydroxyapatite in humans, and is considered as non-toxic material.3 gelatin is a type-i collagen considered as the major organic component in bone (90%). the main advantage of the gelatin is increasing attachment and growth of cells since gelatin has many arginine-glycine-aspartic acid (rgd) protein chains and sequences. gelatin is also biodegradable and biocompatible, but has low biomechanical properties.9 in addition, chitosan has the same structure as glycosaminoglycans (gag), a noncollagen organic c o m p o n e n t o f b o n e . c h i t o s a n i s b i o c o m p a t i b l e , biodegradable, bioactivity, and osteoconductive. chitosan also has anti-microbial activity. chitosan can help cell attachment, differentiation, and migration.6 integration of hydroxyapatite (ha), gelatin (gel), and chitosan (k) in scaffolds is expected to improve the mechanical and biological properties so that it can be considered as ideal scaffolds used for bone regeneration. gelatin hydroxyapatite, and chitosan scaffolds with a ratio of 70:15:15 (w/ w/ w) is a good biomaterial candidate for tissue engineering in bone.10 unfortunately, there are still no recent researches on gelatin hydroxyapatite, and chitosan scaffolds explaining the origin of the hydroxyapatite used. therefore, this research focused on the use of hydroxyapatite derived from bovine bones developed by bank of tissue in dr. soetomo hospital, surabaya. this study aimed to determine the value of compressive strength and the size of porosity derived from bha-gel-k scaffolds with ratio 70:15:15 (w/ w/ w). bha-gel-k scaffolds are expected to be biomaterials for regenerative therapy development in bone defects in the field of dentistry. materials and methods materials used were bha (a particle size of <150 μm made from bovine bones produced by bank of tissue in dr. soetomo hospital, surabaya), gelatin (rousselot 150 lb 8, ghuangdong, china), chitosan (sigma 448877, st. louis, usa), a deacetylation degree of >81%), 10% naoh (brataco chemica pt., surabaya, east java, indonesia), 2% acetic acid, and distilled aqua (pt. duta farma). bha-gel-k scaffolds conducted in this research were based on a modification of procedures for producing scaffolds from previous research. 9 ml of 2% acetic acid (brataco chemica pt., surabaya, east java, indonesia) was mixed with 0.375 grams of gelatin using magnetic stirrer (dragonlab, ms-pro-h280) (dragonlab, ms-pro-h280, beijing, china). 1.75 grams of bha was mixed with 5 ml of distilled aqua, and allowed to settle. the sedimented bha that had already been wet was soaked into the mixture, and then added 0.375 gram of chitosan and 2 ml of 10% naoh. the mixture was put into scaffolds molds. scaffolds were made with two different sizes diameter of 8 mm and a height of 10 cm, and diameter of 5 mm and a height of 5 mm. scaffolds in molds then was frozen -80° c for 24 hours and then dried using freeze dryer (virtis bech top “k” series, sp scintific pennyslvania, usa) for 2 x 24 hours.10,11 compressive strength test was performed on scaffolds with a diameter of 8 mm and a height of 10 cm. the sizes of scaffolds used were adjusted with the specification of the tools used. the sizes of the surface area of bha-gel-k scaffolds were measured. autograph table (shimadzu ag10 te) was covered with paper. scaffolds were placed in the middle of the table with the vertical axis position, perpendicular to the plane of the samples. autograft tool was switched on, and the samples were pressed with a speed of 10 mm/ min with a load cell compress machine of 100 kn until scaffolds were distorted. the tool then automatically stopped, and the number figured out was noted.12 compressive strength value then was calculated using the following formula:13 compressive strength (n/mm2) = force (newton) surface area (mm2) in this research, the number generated by the autograft tool is in a unit of kgf (kilogram force). the compressive strength value is in a unit of n/ mm2, so this value has to be converted to newton first before compressive strength value is calculated. compressive strength value commonly used is in a standard unit of pascal (pa), so the final compressive strength value then has to be converted into kpa.12,13 scaffolds used for porosity test were scaffolds with a diameter of 5 mm and a height of 5 mm. porosity size measurement procedure was performed by using sem (fei, inspect-s50, hillsboro, oregon, usa). in the initial preparation, sem holders were taken and coated with carbon tape. carbon tape used was double-sided carbon tape. one side was attached to the sem holders, and the other side was attached to the scaffolds. samples in the form of scaffolds are non-conductor materials that has to be coated before using them. coating was conducted using a sputter coater (sc7620, qourum technologies ltd., east sussex, england) by inserting bha-gel-k scaffolds and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p153-157 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p153-157 155155kartikasari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 153–157 their holders in the sputter coater, and vacuum then was performed for 30 minutes. after the vacuum was done, plasma coating was conducted for 3 seconds using au and pb. the samples having coating were put into the sem holders, and vacuum was carried out for 5 minutes. after that, the program for the sem was started, and then sem images appeared on the monitor. pictures were taken at magnifications of 75x, 100x, and 250x. scaffolds porosity measurement was performed on pictures with a magnification of 100x. in one field of view, the largest porosity size was selected corresponded to the number of samples required. porosity size was measured by drawing a line on the selected porosity. results scaffolds were made by mixing powdered bha, gel, and k as seen in figure 1. the results of compressive strength and porosity tests on bha-gel-k scaffolds can be seen in table 1. the mean value of compressive strength was 174.29 + 31.62 kpa, while the mean porosity size was 147.06 μm + 27 02 (n = 7). porosity size was measured based on the pore diameter of the sem image. eleven porosity sizes were measured and used as samples. sem images with a magnification of 75x resulted can be seen in figure 2. with the magnification of 75x, almost the entire porosity surface of bha-gel-k scaffolds could be seen. with a magnification of 100x and 250x, porosity sizes seemed much larger. the blue arrow indicates porosity on the surface of the scaffolds. figure 3 shows sem images at 100x magnification used in this research. in the picture, how porosity measurements conducted can also be seen. porosity measurements were performed by selecting the largest porosity to represent the scaffolds porosity. green lines were drawn from one point to another. the length of the green lines was the size of pores in the scaffolds. the length of the green line then was calculated using a unit of μm. the total number of the measured porosity size was 11. discussion tissue engineering is one of therapeutic methods that have been widely used and developed recently. tissue engineering is regarded as a promising procedure for a biological component of a bone substitute that can be used in all kinds of bone damage.6 scaffold is also considered as an important component in tissue engineering. scaffolds act as substitute for ecm so that bone regeneration can occur. every tissue actually needs scaffolds that has different biomechanical and biological properties from others.5 b figure 1. bha-gel-k scaffolds. table 1. results of compressive strength and porosity tests on bha-gel-k scaffolds maximal valueminimal valuecharacteristics mean + sd* 200.00120.00compressive strength values (kpa) 174.29 + 31.62 porosity size (μ 208.60107.20m) 147.06 + 27.02 * sd: standard deviation a c cb figure 2. (a) results of sem test and porosity measurement test at the magnification of 75x; (b)100x; and (c) 250x. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p153-157 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p153-157 156 kartikasari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 153–157 selection of materials used for composing scaffolds is an important factor in the success of using scaffolds since the materials selected will affect the final and functional characteristics of the tissue formed.14 in this research, scaffold was made of mixed materials derived from bha, gel, and k. hydroxyapatite is an osteoconductive material with the main composition of calcium (ca) and phosphate (p). calcium and phosphate in hydroxyapatite can act as regulators of signal in bone tissue engineering, as well as basic ingredients in formation of new bone tissue.15 in this research, a natural hydroxyapatite used was derived from bovines. bha used was manufactured by bank of tissue in dr. soetomo hospital, surabaya with product validation in accordance with the standards set by the ministry of health and apastb (asian pacific association of surgical tissue bank).16 bha has no toxic properties, but similar to human ha, it has a porosity size of 150-360 μm and high osteoconductivity, as well as can easily integrate with the surrounding bones.17 bha, consequently, has been widely studied and applied for bone tissue regeneration. a previous research even indicates that the use of bha scaffolds planted with msc on rabbit defected bone can regenerate the bone well. it is characterized by an increase in type i collagen and osteocalcin.2 other main components in this research were gelatin and chitosan. the objectives of adding gelatin and chitosan as adhesive materials for binding bha is to improve the effectiveness of bha in binding the active ingredients and to reduce the fragility of bha.12 gelatin is a denatured collagen product that plays an important role in cell adhesion and proliferation. application of gelatin for bone regeneration is generally combined with other materials since gelatin has a low mechanical property.9 scaffolds made of ha-gel is considered as a suitable environment for the growth of periodontal ligament fibroblasts, human mesenchymal stem cells (hmsc), and primary cells from human pelvic bone.18 chitosan, on the other hand, is a deacetylation product of chitin which has similar structure to gag, a non-organic component in bone collagen. gag modulates bone precursor cells to the defect area, and helps cell differentiation to regulate protein that is essential for the bone regeneration.19 consequently, the combination of bha, gel, and chitosan is expected to form scaffolds similar to ecm in the bones. those components of scaffolds interact with each other. organic signals of chitosan and gelatin then will cover hydroxyapatite particles. this is consistent with interactions that occur in normal bone components, in which organic components will cover inorganic components.11 mechanical properties of scaffolds are factors that also must be taken into account in the process of making scaffolds. scaffolds must be strong enough to withstand mechanical stresses derived from the surrounding tissue. therefore, the low mechanical properties on scaffolds can cause dimensional changes in scaffolds.12 the value of compressive strength in this research was lower than the value of compressive strength in canselous bone, 2-12 mpa.20 this can happen because in this research there was no crosslink agent added. the addition of crosslink agent will trigger crosslinking between molecules of the materials forming scaffolds.12 in addition, the materials can also make crosslink bonds between the molecules stronger and prevent molecular bonds between the molecules shifting. the stronger bonds between the molecules then will improve the mechanical and biological properties of scaffolds.21 another important property that should be owned by scaffolds is an appropriate size of porosity. porosity size is related to cell adhesion and migration as well as diffusion of nutrients and removal of metabolic waste.4 in this research, all samples had a pore size of more than 100 μm, and the mean porosity size of bha-gel-k scaffolds was 147.06 μm. this is consistent with a previous research stating that the minimal pore size of scaffolds is 100-150 μm.6 the appropriate porosity size is used for the attachment of msc-sized 17.9-30.4 μm.22 the porosity size that is too small will lead to limited cell migration as well as will disrupt nutrient and metabolic waste diffusion. when this occurs, it will cause necrosis of scaffolds. meanwhile, the porosity size of scaffolds that is too big will cause the cells to be easily separated from the scaffolds.4,6 it can be concluded that the bha-gel-k scaffolds has a good value of compressive strength, but not yet resemble bone mass. therefore, further researches use bha-gel-k scaffolds with addition of cross linking agent should be conducted to increase the value of compressive strength. however, the porosity of bha-gel-k scaffolds is suitable for bone tissue regeneration application. references 1. lanza r, langer r, vacanti j. principles of tissue engineering. 3rd ed. uk: elsevier academic press; 2007. p. 845-56, 861-3, 1095-103. 2. wattanutchariya w, changkowchai w. characterization of porous scaffolds from chitosan-gelatin/hydroxyapatite for bone grafting. figure 3. porosity measurement process of bha-gel-k scaffolds. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p153-157 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p153-157 157157kartikasari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 153–157 international multi-conference of engineers and computer scientists volume ii. march 12–14 2014, hong kong; p. 1-5. 3. ferdiansyah, rushadi d, rantam fa, aulani’am. regenerasi pada massive bone defect dengan bovine hydroxyapatite sebagai scaffolds mesenchymal stem cell. jbp 2011; 13: 3-15. 4. peter m, binulai ns, nair sv, selvamurugan n, tamura h, jayakumar r. novel biodegradable chitosan-gelatin/nano-bioactive glass ceram is composite scaffolds for alveola r bone tissue engineering. chemical engineering j 2010; 353-61. 5. sadeghi d, nazarian h, nazanin m, aghalu f, nojehdehyan h, dastjerdi ev. alkaline phosphatase activity of osteoblast cells on three-dimensional chitosan-gelatin/hydroxyapatite composite scaffolds. journal dental school 2013; 30: 203-9. 6. costa-pinto ar, reis rl, neves nm. scaffolds based bone tissue engineering: the role of chitosan. tissue engineering j 17 (part b): 5-11. 7. sobczak a, kowalski z, wzorek z. preparation of hydroxyapatite from animal bones. acta of bioengineering and biomechanics j 2009; 11: 45-51. 8. li j, dou y, yang j, yin y, zhang h, yao f, wang h, yao k. surface characterization and biocompatibility of microand nanohydroxyapatite/chitosan-gelatin network films. materials science and engineering c j 2009; 29: 1207–15. 9. rodriguez i. tissue engineering composite biomimetic gelatin sponges for bone regeneration. thesis. virginia commonwealth university; 2013. 10. mohamed kr, beherei hh, el-rashidy zm. in vitro study of nanohydroxyapatite/ chitosan–gelatin composites for bio-applications. journal of adv res 2014; 5: 201–8. 11. zhao f, grayson wl, ma t, bunnell b, lu ww. effects of hydroxyapatite in 3-d chitosan–gelatin polymer network on human mesenchymal stem cell construct development. biomaterials j 2006; 1859–67. 12. budiatin as. 2014. pengaruh glutaraldehid sebagai crosslink agent gentasimin dengan gelatin terhadap efektifitas bovine hydroxypatite-gelatin sebagai sistem pengantaran obat dan pengisi tulang. disertasi surabaya: pascasarjana universitas airlangga. 2014. pp 13-65. 13. anusavice kj, shen, rawls. phillips: science of dental materials. 12th ed. china: elsevier; 2013. p. 50-3. 14. qin l, genat hk, griffith jf, leung ks. advanced bioimaging technologies in assessment of the quality of bone and scaffold materials: techniques and applications. germany: springer; 2007. p. 259-68. 15. murphy cm, o’brien fj, little dg, schindeler a. cell-scaffolds interactions in the bone tissue engineering triad. european cell and material j 2013; 26: 120-32. 16. ferdiansyah. ilmu kedokteran regeneratif (regeneratif medicine): inovasi terapi masa depan. sidang universitas airlangga 10 november 2010: dies natalis ke 56. surabaya: airlangga university press; 2010. p. 3-18. 17. ferdiansyah. regenerasi pada massive bone defect dengan bovine hydroxyapatite sebagai scaffolds stem sel mesenkimal. disertasi. surabaya: pascasarjana universitas airlangga; 2010. p. 35-57. 18 rungsiyanont s, dhanesuan n, swasdison s, kasugai s. evaluation of biomimetic scaffolds of gelatin-hydroxyapatite crosslink as a novel scaffolds for tissue engineering: biocompatibility evaluation with human pdl fibroblasts, human mesenchymal stromal cells, and primary bone cells. j biomater appl 2012; 27: 47-54. 19. sa lbach j, r ach ner t d, r au ner m, hemp el u, a nderegg u, franz s, simon jc, hofbauer lc. regenerative potential of glycosaminoglycans for skin and bone. j mol med 2012; 90: 62535. 20. ficai a, andronescu e, voicu g, ficai d. advances in collagen/ hydroxyapatite composite materials: advances in composite materials for medicine and nanotechnology. rijeka: in tech d.o.o; 2011. p. 1-32. 21. haugh mg, murphy cm, mckieman rc, altenbuchner c, o’brien fj. crosslinking and mechanical properties significantly influence cell attachment, proliferation, and migration within collagen glycosaminoglycan scaffolds. tissue engineering: part a 2011; 17: 1201-8. 22. ge j, guo l, wang s, zhang y, cai t, zhao rc, wu y. the size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. stem cell rev j 2014; 10: 295-303. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p153-157 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p153-157 vol 50 no 4 desember 2017.indd 199199 research report dental journal (majalah kedokteran gigi) 2017 december; 50(4): 199–204 antioxidant potency of mangosteen peel extract topical application in reversing reduced orthodontic brackets tensile strength after bleaching ananto ali alhasyimi department of orthodontics faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: nowadays, cosmetic dentistry has become an ever-increasing requirement with interest in it growing over time. bleaching is one of the popular cosmetic treatments that has been proven to diminish the tensile bond strength (tbs) of orthodontic brackets attached to bleached teeth. mangosteen peel (mp) extract contains antioxidants that may potentially reverse the reduction in tbs. purpose: the purpose of this study was to evaluate the effect of mp extract on the postbleaching tbs of brackets. methods: the reported research constitutes an experimental in vitro study conducted on a total of 120 maxillary first premolar teeth randomly divided into six groups (n = 20) as follows: negative-control (nc: no bleaching), positive-control (pc: bleaching + no treatment), and the treatment groups (bleaching + 10% sodium ascorbate (sa), 10% (mp10), 20% (mp20) and 40% (mp40) mp extract gel). post-treatment, the brackets were bonded using transbond xt and tbs testing was performed using a universal testing machine. the ari was examined by means of a stereoscopic microscope, while enamel morphological changes were observed through a scanning electron microscope. the tbs-generated data was analyzed by means of anova and tukey tests. for the adhesive remnant index, a kruskal-wallis analysis test was performed. results: there was a significant tbs difference (p = 0.001) between the various groups. the pc group showed the significantly highest tbs compared to the others (8.33 ± 3.92 mpa), whereas nc demonstrated the lowest (4.15 ± 2.27 mpa). the tbs value of the mp40 group was considerably higher than other groups treated with antioxidants (7.87 ± 3.26 mpa). the failure of orthodontic brackets using mp extract mostly occurred at the adhesive-bracket interfaces. conclusion: topical application of 40% mangosteen peel (mp) extract as an antioxidant after bleaching was effective in reversing the reduced post-bleaching tensile bond strength (tbs) of orthodontic brackets. keywords: brackets; bonding; bleaching; mangosteen peel extract; tensile bond strength correspondence: ananto ali alhasyimi, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: anantoali@ugm.ac.id introduction in contemporary society, popular interest in cosmetic dentistry has increased to the extent that it has become regarded as a necessity.1 tooth whitening or bleaching has become one of the most common procedures in this branch of dentistry, adopted by numerous dentists and their patients as the method of choice to improve tooth appearance due to its being minimally-invasive, easy, efficient and effective.2,3 the number of patients seeking orthodontic treatment who might have a history of tooth bleaching is increasing because, following previous procedures, they usually tend to be conscious of orthodontic problems and desire treatment.4 unfortunately, bleaching may lead to a reduction in orthodontic bracket bond strength.5–7 in-office bleaching can produce a direct result including residual peroxide on the tooth surface which might inhibit the polymerization of the adhesive.8 on the other hand, treating bleached teeth with antioxidants helps in the elimination of free oxygen radicals from the tooth surface dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p199–204 mailto:anantoali@ugm.ac.id http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 200 alhasyimi/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 199–204 before bonding and, as a consequence, will reverse reduced bond strength.9 in vitro study has demonstrated the application of synthetic antioxidants such as sodium ascorbate to be effective in reversing the reduced bond strength of brackets after bleaching.10 the limited routine use of synthetic antioxidants has increased because the efficiency of synthetic antioxidants is lower than that of natural antioxidants.11 moreover, the side-effects of synthetic antioxidants include the presence of residual electrons that are difficult for human tissues to recycle.12 the interest in natural antioxidants of plant origin has developed considerably in recent years. mangosteen is a tropical fruit whose peel is a source of powerful, natural antioxidants. previous in vitro studies demonstrated that mangosteen peel (mp) extract is more efficient at controlling the oxidative reaction of free radical molecules compared to certain commercial antioxidants.13 therefore, the present study was carried out to evaluate the effect of mp extract topical application on the tensile bond strength of orthodontic brackets bonded with resin materials to bleached teeth. materials and methods the research reported here represents an experimental laboratory study. ethical approval was granted in 2016by the research ethics committee, faculty of dentistry, universitas gadjah mada. a total of 120 human maxillary first premolars extracted for orthodontic purposes were collected. the inclusion criteria of the teeth comprised the following: healthy, intact enamel surface, caries-free, devoid of defects, cracks, or restorations and the absence of chemical-agent pre-treatment. the teeth were exposed to scaling using an ultrasonic scaler (woodpecker uds-a w led, china) to remove organic debris, before being cleaned and decontaminated through a week-long immersion in 0.5% chlorine at 25oc, renewed once every two days to limit bacterial contamination. the teeth were mounted in acrylic resin (figure 1a). samples were then randomly distributed between six groups (n = 20) as follows: group 1 consisted of unbleached teeth (negative-control = nc), group 2 consisted of bleached teeth untreated before bracket bonding (positive-control = pc), group 3 were bleached before being treated with 10% sodium ascorbate and undergoing bracket bonding (sa), and groups 4, 5 and 6 were bleached and then topically treated with 10%, 20% and 40% mp (mp-10, mp-20, mp-40) extract gel before bracket bonding. all of the samples were subjected to 40% hydrogen peroxide (opalescence® boost, ultradent, usa) on the enamel surfaces as a bleaching agent in accordance with the manufacturer’s protocol. a uniform thickness of bleaching agent (0.5–1 mm) was applied to the enamel surface of each sample. after 20 minutes, the surfaces were washed with distilled water and gently dried with an air jet for 30 seconds. the bleaching procedure was re-applied twice as per the manufacturer’s instruction to obtain optimal results. groups 3, 4, 5, and 6 were then treated with an antioxidant agent in the following manner: 10% sodium ascorbate gel (manufactured by lppt, indonesia) and 10%, 20%, and 40% mp gel (manufactured from a sample containing mp extract, cmc-na 2%, glycerin, propylene glycol, propylparaben and methylparaben by lppt, indonesia). 0.5–1 mm of antioxidant was attached to the enamel surfaces of the teeth with a sterile brush on conclusion of the bleaching process. after ten minutes, they were washed in distilled water and gently dried with an air syringe. following completion of this exercise, the samples were submerged in artificial saliva solution for 24 hours prior to the initiation of the bonding procedure. 120 stainless steel edgewise premolar brackets (american orthodontics, usa) with a 0.022-inch slot and a bracket base surface area of 10.64 mm2 were used in the study. the brackets were then bonded with transbond xt (3m unitek, usa) before being placed on the mid-buccal surfaces of the teeth using a bracket positioning gauge. furthermore, each bracket was light-cured for 40 seconds in accordance with the manufacturer’s recommendation (10 seconds per side: occlusal, cervical, mesial, and distal) using a light-curing unit (litex 680a, dentamerica, usa). a tensile bond strength (tbs) test was performed using a universal testing machine (pearson panke equipment, london). a mounting jig aligned the bracket base parallel to the bottom of the mould and perpendicular to the force during the tensile strength test. tbs was measured at a crosshead speed of 0.5 mm/min (figure 1b). the results obtained were converted to mega-pascal (mpa) by dividing the debonding force (n) by the bracket base surface area (10.64 mm2). immediately after bracket debonding, the enamel surface of each specimen was examined at 10× magnification with a stereoscopic microscope (smz-2t, nikon, japan) to determine the amount of residual adhesive. adhesive remnant index (ari) scores at the failure sites were noted according to the classification applied by artun and bergland as follows: score 0 no adhesive remained figure 1. (a) sample mounted in self-curing acrylic resin, (b) illustrations of the test construction for determining tensile bond strength. a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p200–204 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 201alhasyimi/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 199–204 on the tooth, 1 less than half of the enamel bonding area was covered with adhesive, 2 more than half of the enamel bonding area was covered with adhesive, and 3 the enamel bonding area was fully covered with adhesive.14 measurement was conducted by three qualified examiners who were unsighted as to the composition of the samples. the examiners’ analysis showed a high level of intraexaminer and inter-examiner agreement and reliability kappa index value (0.81). the structure of enamel was then observed using a scanning electron microscope (jeol, jsm-6510 series, japan) under 5000× magnification. statistical analysis was performed with statistical package for the social sciences (spss) software (version 22.0, chicago, ill). all data is presented as mean ± standard deviation. the data of the groups was subjected to a normality and homogeneity test. with respect to this, one-way anova was used to determine the significance between the groups, while a tukey honestly significant difference post-hoc test was applied to evaluate individual differences (table 1). the ari scores were examined by means of a kruskal–wallis analysis. to determine the differences between the groups, a mann–whitney u test was also performed. the significance for all statistical tests was set at a p-value less than 0.05. results the tensile bond strength of samples in each of the six study groups are shown in table 1 in mpa (mean ± standard deviation). these descriptive statistics clearly indicate the variation in tbs between the six groups with the maximum tbs value being found in the nc group (8.33 ± 3.92 mpa) and the minimum in the pc group (4.15 ± 2.27 mpa). groups sa, mp10, mp20, and mp40, all of which were subjected to post-bleaching antioxidant treatment, show an improvement in tbs compared with the pc group, while the group treated with mp40 shows the highest tbs compared to other groups treated with antioxidants (7.87 ± 3.26 mpa). the results of the anova indicated statistically significant differences between the tested groups (p < 0.05). the tukey test showed that the tbs of the nc group was by far the highest compared to that of other groups. furthermore, no statistical significant difference in tbs value existed between group nc and groups treated with sa and 40% mp extract (p > 0.05). the ari scores for all the tested groups are listed in table 2. the results of the kruskal-wallis test showed significant differences between the groups (p < 0.05). ari scores of 0 and 1 occurred with high frequency, while ari table 2. distribution of the ari scores of 6 groups tested and results of the kruskal-wallis test group ari scores kruskal walis 0 1 2 3 n % n % n % n % chi-square p n 3 (15) 4 (20) 5 (25) 8 (40) 8.162 0,031* p 7 (35) 13 (65) 0 (0) 0 (0) sa 5 (25) 4 (20) 7 (35) 4 (20) mp10 7 (35) 10 (50) 2 (10) 1 (5) mp20 5 (25) 8 (40) 4 (20) 3 (15) mp40 3 (15) 4 (20) 6 (30) 7 (35) values are presented as number (%). *significant differences between groups (p < 0.05). ari, adhesive remnant index. *ari scores: 0, no adhesive left on the tooth; 1 = less than half of the adhesive left on the tooth surface; 2 = half of the adhesive or more left on tooth surface; 3 = all adhesive left on tooth surface table 1. results of the anova and tukey tests comparing the tbs in the 6 groups tested group n tbs (mpa) min. max. sig* p-value pc sa mp-10 mp-20 mp-40 nc 20 8.33±3.92 6.48 12.97 p = 0.001* 0.000* 0.163 0.013* 0.048* 0.798 pc 20 4.15±2.27 2.11 6.02 0.048* 0.012* 0.000* 0.000* sa 20 7.12±3.04 5.83 10.19 0.027* 0.037* 0.048* mp-10 20 6.39±2.86 4.94 7.71 0.082 0.021* mp-20 20 6.41±2.94 5.19 10.03 0.014* mp-40 20 7.87±3.26 5.88 11.38 values are presented as mean ± standard deviation. *significant differences between groups (p < 0.05). anova, analysis of variance; tbs, tensile bond strength. nc: negative-control, pc: positive-control. sa: sodium ascorbate, mp-10: 10% mangosteen peel extract, mp-20: 20% mangosteen peel extract, mp-40: 40% mangosteen peel extract. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p199–204 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 202 alhasyimi/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 199–204 scores of 2 and 3 were seen with less frequency in groups pc, mp10, and mp20. in contrast, the highest frequency of ari scores of 3 were recorded by groups nc and mp40. discussion the results confirmed that there were increases in tbs in groups receiving antioxidant treatment. the nc group showed the highest values of tbs, while the pc group showed the lowest. the reduced tbs in the pc group compared to that of other groups may have been due to the remaining oxygen layer left by the bleaching process. this could have interfered the resin polymerization by free-radical mechanism.15 marković et al.,16 recommended that clinically acceptable mean bond strength values range between 7.10 and17.84 mpa with no enamel fractures. however, al shamsi et al.,17 state that an increased number of enamel fractures occurred when the bond strength passed 13.5 mpa. these findings correspond favourably to the results here that the group treated with mp-40 showed average values within marković’s adequate range (7.87 ± 3.26 mpa). this confirmed that samples subjected to peroxide agents could still resist the stresses generated by orthodontic forces without suffering enamel fractures. groups receiving antioxidant treatments showed significantly higher bond strength than that of the pc group. these findings were in accordance with the fact that the post-bleaching procedure use of antioxidants was effective in reversing compromised bond strength on completion of the elimination of residual oxygen.18 it has been reported that sodium ascorbate (sa) is a potent antioxidant with the potential to relieve the reactive free radicals and neutralize their negative effect. sa is a derivative of ascorbic acid with a neutral ph. it neutralizes the effect of the residual oxygen layer, while enabling free radical polymerization of resinbased materials to proceed without premature termination by restoring the modified redox potential of the oxidized bonding substrate, thus reversing the bond strength.19 in the process following the application of sa, tbs values reached a level almost similar to that of the mp40 groups. however, mp40 groups showed significantly higher bond strength value than that of a group receiving sa treatments which could be associated with the fact that antioxidant present in mp extract is more potent than in the sa. the nc group showed significantly higher bond strength than the other groups. this correlates with the fact that there are no changes in the unbleached teeth surface. previous studies have shown that there is a change in the structure of enamel and the bond strength when the teeth are exposed to bleaching agents.2,3 the reduction in bond strength has been related to morphological changes in mineralized tissues. bleaching agents also affect the collagen network of dentin, resulting in denaturing and relative instability of the dentin organic matrix, thereby decreasing the bond strength.4 in addition, tbs values of a group undergoing 40% mp extract treatment reached a level almost equal to those of the nc group. it is suggested that treatment with mp extract could approximate the bond strength values of teeth which had not experienced the bleaching procedure. the results indicated that application of mp extract on bleached enamel surfaces could neutralize and overcome the negative effect of residual oxygen molecules and significantly increase the tbs of orthodontic brackets. mp also demonstrated potential antioxidant properties stronger than those of its pericarp and leaves.20 antioxidant properties of mp extract can inhibit or delay oxidation by scavenging free radicals (i.e. reactive oxygen species such figure 2. photomicrographs of enamel surface in six groups tested using sem at a magnification of 5.000x; a) nc group: negativecontrol, b) pc group: positive-control, c) sa group: sodium ascorbate, d) mp-10 group: 10% mangosteen peel extract, e) mp-20 group: 20% mangosteen peel extract, and f) mp-40 group: 40% mangosteen peel extract. thick arrows indicate bubble like structures. a e f b c d dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p200–204 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 203alhasyimi/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 199–204 as hydroxyl, superoxide, nitric oxide, thiyl and peroxyl) by donating a hydrogen atom or electron which convert their radicals to more stable products.11 mp extract contains alpha-mangostin has been reported as antioxidant by disintegrating oxygen molecule.21,22 furthermore, mp extract also contains a potent free radical scavenger, in the form of a flavonoid, called epicatechin.23 flavonoids can perform scavenging action on free radicals such as superoxide, hydroxyl, and 1,1-dipheny l-2-picrylhydrazyl (dpph) and have metal chelating properties. the presence of the functional group oh in the structure and its position on the ring of the flavonoid molecule determines its antioxidant capacity.24 the morphology of the surface enamel in six groups was observed using an sem as shown in figure 2. figure 2-a illustrates the lack of change in the morphology of the enamel in group nc with the highest tbs value. conversely, the nc group had the lowest tbs value, showing changes to enamel surfaces, including: depression, cracks, porosity, and erosion (figure 2-b). meanwhile, figure 2-f, demonstrates the morphology of the enamels in the mp40 group with the highest tbs value compared to that of other groups treated with antioxidants, presenting a minimal bubble-like structure formed in the enamel surfaces compared with sa, mp10, and mp20 groups. this finding is supported by the theory stating that the enamel surface of bleached teeth exhibited more extensive nano-leakage in the form of additional bubble-like structures observed there. this, in turn, indicates that the oxygen released by the hydrogen peroxide could be trapped within the adhesive during light-activation, inhibiting its polymerization, consequently decreasing bond strength.25 the ari scores indicated significant differences between the various groups, although ari scores of 0 and 1 were seen with high frequency. in groups pc, mp10, and mp20, there was a higher frequency of ari scores of 1. this means that failures occurred at the enamel-adhesive interfaces. this could be clinically advantageous since, when brackets there fail, the less residual adhesive remains and tooth clean-up is likely to be easier and faster.26,27 in this study, the nc, sa and mp40 groups demonstrated a higher frequency of ari scores of 3 (all the adhesive remained on the enamel surface), implying a relatively stronger bond between the adhesive and the enamel surface. it can be concluded that the application of 40% mangosteen peel extract as an antioxidant was capable of reversing the reduced tensile bond strength of orthodontic brackets in bleached teeth. references 1. avriliyanti f, suparwitri s, alhasyimi aa. rinsing effect of 60% bay leaf (syzygium polyanthum wight) aqueous decoction in inhibiting the accumulation of dental plaque during fixed orthodontic treatment. dent j (maj ked gigi). 2017; 50(1): 1–9. 2. ferreira nds, da rosa pcf, ferreira rdij, valera mc. evaluation of shear bond strength of orthodontic brackets bonded on the tooth surface after internal bleaching. rev odontol da unesp. 2014; 43(3): 209–13. 3. henn-donassollo s, fabris c, gagiolla m, kerber í, caetano v, carboni v, salas mms, donassollo ta, demarco ff. in situ and in vitro effects of two bleaching treatments on human enamel hardness. braz dent j. 2016; 27(1): 56–9. 4. britto far, lucato as, valdrighi hc, vedovello sas. influence of bleaching and desensitizing gel on bond strength of orthodontic brackets. dental press j orthod. 2015; 20(2): 49–54. 5. mullins jm, kao ec, martin ca, gunel e, ngan p. tooth whitening effects on bracket bond strength in vivo. angle orthod. 2009; 79(4): 777–83. 6. machado smm, nascimento dbp, silva rc, loretto sc, normando d. evaluation of metallic brackets adhesion after the use of bleaching gels with and without amorphous calcium phosphate (acp): in vitro study. dental press j orthod. 2013; 18(3): 101–6. 7. do rego mvnn, dos santos r ml, leal lmp, braga cgs. evaluation of the influence of dental bleaching with 35% hydrogen peroxide in orthodontic bracket shear bond strength. dental press j orthod. 2013; 18(2): 95–100. 8. patusco vc, montenegro g, lenza ma, de carvalho aa. bond strength of metallic brackets after dental bleaching. angle orthod. 2009; 79(1): 122–6. 9. muraguchi k, shigenobu s, suzuki s, tanaka t. improvement of bonding to bleached bovine tooth surfaces by ascorbic acid treatment. dent mater j. 2007; 26(6): 875–81. 10. nayak usk, katyal a. shear bond strength of orthodontic brackets after antioxidant treatment on previously bleached teeth: an in vitro study. j orthod res. 2015; 3(2): 96–100. 11. suttirak w, manurakchinakorn s. in vitro antioxidant properties of mangosteen peel extract. j food sci technol. 2014; 51(12): 3546–58. 12. bjelakovic g, nikolova d, gluud ll, simonetti rg, gluud c. mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and metaanalysis. jama. 2007; 297(8): 842–57. 13. weecharangsan w, opanasopit p, sukma m, ngawhirunpat t, sotanaphun u, siripong p. antioxidative and neuroprotective activities of extracts from the fruit hull of mangosteen (garcinia mangostana linn.). med princ pract. 2006; 15(4): 281–7. 14. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984; 85(4): 333–40. 15. kimyai s, valizadeh h. the effect of hydrogel and solution of sodium ascorbate on bond strength in bleached enamel. oper dent. 2006; 31(4): 496–9. 16. markovic e, glišic b, scepan i, markovic d, jokanovic v. bond strength of orthodontic adhesives. metal j metall. 2013; 14(2): 79–88. 17. al shamsi a, cunningham jl, lamey pj, lynch e. shear bond strength and residual adhesive after orthodontic bracket debonding. angle orthod. 2006; 76(4): 694–9. 18. subramonian r, mathai v, christaine angelo jbm, ravi j. effect of three different antioxidants on the shear bond strength of composite resin to bleached enamel: an in vitro study. j conserv dent. 2015; 18(2): 144–8. 19. anil m, ponnappa kc, nitin m, ramesh s, sharanappa k, nishant a. effect of 10% sodium ascorbate on shear bond strength of bleached teeth an in-vitro study. j clin diagn res. 2015; 9(7): zc31-zc33. 20. palakawong c, sophanodora p, pisuchpen s, phongpaichit s. antioxidant and antimicrobial activities of crude extracts from mangosteen (garcinia mangostana l.) parts and some essential oils. int food res j. 2010; 17(3): 583–9. 21. guzmán-beltrán s, orozco-ibarra m, gonzález-cuahutencos o, victoria-mares s, merchand-reyes g, medina-campos on, pedraza-chaverri j. neuroprotective effect and reactive oxygen species scavenging capacity of mangosteen pericarp extract in cultured neurons. curr top nutraceutical res. 2008; 6(3): 149–58. 22. pedraza-chaverri j, cárdenas-rodríguez n, orozco-ibarra m, pérez-rojas jm. medicinal properties of mangosteen (garcinia mangostana). food chem toxicol. 2008; 46(10): 3227–39. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p199–204 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 204 alhasyimi/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 199–204 23. shibata m-a, matoba y, tosa h, iinuma m. effects of mangosteen pericarp extracts against mammary cancer. altern integr med. 2013; 2(8): 1–6. 24. perumalla avs, hettiarachchy ns. green tea and grape seed extracts — potential applications in food safety and quality. food res int. 2011; 44(4): 827–39. 25. kunjappan s, kumaar v, prithiviraj, vasanthan, khalid sa, paul j. the effect of bleaching of teeth on the bond strength of brackets: an in vitro study. j pharm bioallied sci. 2013; 5(suppl 1): s17–20. 26. nascimento gcr, de miranda ca, machado smm, brandão gam, de almeida ha, silva cm. does the time interval after bleaching influence the adhesion of orthodontic brackets? korean j orthod. 2013; 43(5): 242–7. 27. sharma s, tandon p, nagar a, singh gp, singh a, chugh vk. a comparison of shear bond strength of orthodontic brackets bonded with four different orthodontic adhesives. j orthod sci. 2014; 3(2): 29–33. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p199–204 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p199-204 vol 51 no 3 jul sep 2018_pus.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 51, number 3, september 2018 editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 editorial boards of dental journal (majalah kedokteran gigi) sk: 02/un3.1.2/2018 january 2nd – december 31st, 2018 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium);cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states);guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan);kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan);miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany);sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand);samir nammour (department of dental science, faculty of medicine, university of liege, belgium);reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran);hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore);widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia);hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states);harry huiz peeters (laser research center, bandung, indonesia);rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia);elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); retno palupi (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty of vocational studies, universitas airlangga, surabaya); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); febriastuti cahyani (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); siti sunarintyas (department of dental biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); retno widayati (department of orthodontics, faculty of dentistry, universitas indonesia, indonesia); i. b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); mei syafriadi (department of oral and maxillofacial pathology, faculty of dentistry, universitas jember, indonesia); trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); david kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); diah savitri ernawati (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); titien hary agustantina (department of dental material science and technology, faculty of dental medicine, universitas airlangga, indonesia); nurina febriyanti ayuningtyas (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) abdullah mas’udy (faculty of dental medicine, universitas airlangga) contents page printed by: airlangga university press. (rk. 276/07.18/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 51, number 3, september 2018 p-issn: 1978-3728 e-issn: 2442-9740 1. antibacterial potential of ocimum sanctum oils in relation to enterococcus faecalis atcc 29212 diani prisinda, ame suciati setiawan, and fajar fatriadi ........................................................... 104–107 2. cytotoxicity test of binjai leaf (mangifera caesia) ethanol extract in relation to vero cells fifi dwidhanti, irham taufiqurrahman, and bayu indra sukmana .......................................... 108–113 3. the effect of various concentrations of ha-tcp derived from cockle shell synthesis on scaffold porosity reyhan alvaryan ferdynanto, priska evita setia dharmayanti, putu tahlia krisna dewi, and widyasri prananingrum ........................................................................................................... 114–118 4. socioeconomic status and orthodontic treatment need based on the dental health component hilda fitria lubis and hilda paula laturiuw ............................................................................... 119–123 5. potential immunomodulatory activity of phyllanthus niruri aqueous extract on macrophage infected with streptococcus sanguinis suryani hutomo, denise utami putri, yanti ivana suryanto, and heni susilowati ................. 124–128 6. the effect of avocado leaf extract (persea americana mill.) on the fibroblast cells of postextraction dental sockets in wistar rats christian khoswanto, wisnu setyari juliastuti, and karina awanis adla ................................ 129–132 7. cytoxicity test of naocl and mangosteen (garcinia mangostin l.) peel extract used as an irrigation solution in human periodontal ligament fibroblast cells (hpdlfc) tamara yuanita, dina rystiawati and karlina samadi .............................................................. 133–137 8. effects of liquid ionic silver concentration on caspase-3 and p53mt expressions in the oral mucosal epithelium of wistar rats r. aries muharram, i. istiati and pratiwi soesilawati ................................................................. 138–142 9. the effects of breadfruit leaf (artocarpus altilis) extract on fibroblast proliferation in the tooth extraction sockets of wistar rat darin hulwani rinaldi, david b. kamadjaja and ni putu mira sumarta ................................ 143–146 10. oral lesions as a clinical sign of systemic lupus erythematosus eliza kristina m. munthe and irna sufiawati ............................................................................... 147–152 11. herbal-induced stevens-johnson syndrome with oral involvement and management in an hiv patient s. suniti and irna sufiawati ............................................................................................................. 153–157 �0 volume 46 number 1 march 2013 unidentified angular recurrent ulceration responsive to antiviral therapy rahmi amtha1 and siti aliyah pradono2 1department of oral medicine, faculty of dentistry, universitas trisakti 2department of oral medicine, faculty of dentistry, universitas indonesia jakarta indonesia abstract background: recurrent ulcer on angular area is usually called stomatitis angularis. it is caused by many factors such as vertical dimension reduce, vitamin b12, and immune system deficiency, c. albicans and staphylococcus involvement. clinically is characterized by painful fissure with erythematous base without fever. purpose: to describe an unidentified angular ulcer proceeded by recurrent ulcers with no response of topical therapy. case: an 18-years old male came to oral medicine clinic in rscm who complained of angular recurrent ulcers since 3 years ago which developed on skin and bleed easily on mouth opening. patient had fever before the onset of ulcers. large, painful, irregular ulcers covered by red crustae on angular area bilaterally. patient has been treated with various drugs without improvement and lead to mouth opening limitation. intra oral shows herpetiformtype of ulcer and swollen of gingival. case management: provisional diagnosis was established as viral infection thus acyclovir 200 mg five times daily for two weeks and topical anti inflammation gel were administered. blood test for igg/igm of hsv1 and hsv2 were non reactive, however ulceration showed a remarkable improvement. the ulcers healed completely after next 2 weeks with acyclovir. conclusion: the angular ulceration on above patient failed to fulfill the criteria of stomatitis angularis or herpes labialis lesion. however it showed a good response to antiviral. therefore, unidentified angular ulceration was appointed, as the lesion might be triggered by other type of human herpes virus or types of virus that response to acyclovir. key words: unidentified angular ulceration, anti inflammatory gel, acyclovir abstrak latar belakang: ulser rekuren pada sudut mulut biasanya disebut stomatitis angularis. kelainan ini disebabkan oleh banyak faktor seperti berkurangnya dimensi vertikal, defisiensi vitamin b12 dan sistem kekebalan tubuh, infeksi c. albicans serta staphylococcus. secara klinis kelainan ini ditandai dengan fisur sakit pada sudut mulut dengan dasar eritematus tanpa disertai demam. tujuan: melaporkan kasus ulser sudut mulut rekuren yang tidak biasa, tanpa respon terhadap terapi topikal yang biasa diberikan. kasus: seorang lakilaki berusia 18 tahun datang ke klinik penyakit mulut rscm dengan keluhan ulser rekuren pada sudut mulut yang meluas ke kulit sekitarnya sejak 3 tahun yang lalu. ulser mudah berdarah saat pasien membuka mulut dan demam sebelum lesi timbul. ulser membesar, bilateral, sakit, tepi tidak teratur, ditutupi oleh krusta merah. pasien telah diobati dengan berbagai obat, namun tidak menunjukkan perbaikan. intra oral tampak ulser jenis herpetiformis pada gingiva disertai dengan pembengkakan. tatalaksana kasus: diagnosis awal ditegakkan sebagai infeksi virus, oleh karena itu pasien diberikan acyclovir 200 mg lima kali sehari selama dua minggu dan gel anti radang topikal. walaupun hasil darah igg/igm hsv1 dan hsv2 non reaktif, namun ulserasi menunjukkan penyembuhan yang luar biasa dengan anti virus yang diberikan. ulser sembuh sempurna setelah pemakaian acyclovir 2 minggu berikutnya. kesimpulan: ulserasi sudut mulut pada pasien di atas gagal memenuhi kriteria stomatitis angularis atau herpes labialis. namun ulser menunjukkan respon yang amat baik terhadap antivirus. diagnosis ditetapkan sebagai ulserasi sudut mulut yang tidak teridentifikasi. kemungkinan lesi dipicu oleh virus herpes manusia jenis lain atau jenis virus yang memberikan respon terhadap asiklovir. kata kunci: ulser sudut mulut tidak teridentifikasi, gel anti radang, asiklovir correspondence: rahmi amtha, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa grogol, jakarta 11440, indonesia. e-mail: rahmi.amtha@gmail.com case report ��amtha and pradono: unidentified angular recurrent ulceration responsive to antiviral therapy introduction in daily practice, it is common to find an angular ulceration which can be triggered by many factors. cheilitis is a broad term description of inflammation of the lip surface characterized by dry scaling and fissuring. there are some types of cheilitis such as atopic, angular, granulomatous, and actinic. angular cheilitis is commonly seen and it specifically refers to cheilitis that radiates from the commissures or corners of the mouth. other terms synonymous with angular cheilitis are perlèche, commissural cheilitis or angular stomatitis.1 clinically angular cheilitis appears as redness, ulceration and fissuring either unilateral or bilaterally at the corners of the mouth. it can appear alone or in conjuction with another form of candidiasis. cheilitis angularis is a syndrome that involves several factors. the factors may play role individually or interchangeably. candida albicans, staphilococus aureus, vertical dimension decreasing, vit b12 deficiency until immune deficiency (such as in hiv patients) are the established predisposing factors of angular cheilitis.2,3 however, occasionally the clinical manifestation shows unpathognomonic or unspecific clinical appearance with recurrent episodes and involves general conditions. this condition may make the provisional diagnosis sometimes difficult to be established and need further analysis to be able to treat the ulcers. ulceration at the corner of the mouth can be also as the manifestation of secondary herpes simplex infection. typically, lesions are located on the vermillion border of the lips (herpes labialis, “cold sores or “fever blister), but may develop elsewhere in the mouth, on the face or inside the nose. the initial primary episode of herpes labialis occurs 1 to 26 days after inoculation and can appear as multiple blisters, 1–2 mm in size, associated with severe discomfort that lasts for 10 to 14 days.4 recurrent herpes labialis may affect about one-third of the population in the world, with episodes usually occurring from one to six times per year.4 orolabial recurrent herpesvirus infections can be triggered by stimuli such as fever, stress, cold, menstruation and ultraviolet radiation. prodormal symptoms, including paraesthesia, tenderness, pain, burning sensation, tingling or itching sensation at the site of viral re-activation, arise in 46–60% of patients, and last for about 6 hours.5 some autoimmune diseases such as erythema multiforme eruption is known to have an association with those who have history of recurrent herpes simplex infection. erythema multiforme ranges from mild, severe to potentially life-threatening, and can involve acutely painful oral and labial ulcers.6 herpes simplex virus or other viral infections may precipitate erythema multiforme in the oral cavity.6-8 besides herpes simplex virus, there are some other human herpes viruses that may induce the oral ulcerations. eight human herpesvirus species with distinct biological and clinical characteristics have been described: herpes simplex virus-1, herpes simplex virus-2, varicella– zoster virus, epstein–barr virus, human cytomegalovirus, human herpesvirus-6, human herpesvirus-7 and human herpesvirus-8. each herpesvirus subfamily maintains latent infection in specific cell population(s). alpha herpesviruses exhibit a relatively short reproductive cycle, rapid lyses of infected cells and latency in sensory ganglia.9 herpes simplex virus and cytomegalovirus are also reported to be potential pathogens of behcet’s syndrome ulcerations and pemphigus vulgaris (an intraepidermal bullous disease which frequently involves large recalcitrant oral ulcers that precede the onset of skin lesions). further research is needed to determine the extent to which viruses are involved in the oral ulcerogenesis of these and other systemic diseases, including crohn’s disease, ulcerative colitis and neutropenia.9 epstein–barr virus is involved in a great variety of cancers. the virus possesses factors capable of immortalizing b lymphocytes and epithelial cells, contains several potentially oncogenic antigens epstein–barr virus is associated with numerous lymphoid proliferations, including african burkitt's lymphoma, classical hodgkin’s disease and recurrent periodontal disease.10 the epstein–barr virus is present in two-thirds of aids-related lymphomas.11 in the oral cavity, epstein–barr virus has been identified in hodgkin’s lymphoma, burkitt’s lymphoma, cyclosporinerelated post-transplant lymphoproliferative disorder, posttransplant diffuse b-cell lymphoma, follicular lymphoid hyperplasia and plasmablastic lymphoma. demographic, geographic and environmental factors may be important, as most studies showing a herpes viral association with oral tumors originate from asian countries. epstein–barr virus-related nasopharyngeal carcinoma is known to occur with a high relative prevalence in natives of southern china and southeast asia,12 which may be a result of ethnically determined host–virus interactions or distinct epstein–barr genotypes predominating in some asian populations. human cytomegalovirus genome and antigens have been identified in malignant tumors, including colon cancer, malignant glioblastoma. cytomegalovirus is a member of the herpes family of dna viruses. herpes viruses are capable of latency after infection with an acute disease followed by an asymptomatic, quiescent state. fifty to ninety five percent of adults have antibodies against cmv.13 infection with cmv in most immunocompetent hosts is asymptomatic but can present as a mononucleosis-like syndrome.14,15 cytomegalovirus is the virus most frequently isolated from people with aids. ninety percent of patients with aids are infected with cmv, and disseminated cmv is found during autopsy in 93% of patients with aids. there are infrequent reports in the literature of cutaneous cmv infections. this may be because cutaneous cmv infections are uncommon or because making a diagnosis of cmv is difficult as a result of its multiple clinical presentations and subtle histopathological findings. below is the case report of patient who have an recurrent angular ulceration which show a good improvement with antiviral agent. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 30–34 case an eighteen years old man came to oral medicine clinic in cipto mangunkusumo hospital to seek for treatment of recurrent angular ulcer since four days ago that developed to skin and bleed easily on mouth opening. patient had slight fever before the onset of ulcers. large, painful, irregular ulcer covered by red crustae found on bilateral angular area (figure 1). ulcers appear at least twice a year for last three years and always preceded by slight fever and herpetiformis type of ulcer on the commissure, upper and lower lip bilaterally, floor of the mouth and absence of skin ulcers. upper and lower gingival showed pseudomembranous and slightly inflamed on interdental papilla (figure 2). ulcers healed ranging in 2-3 weeks. patient has seen medical doctors and been given antibiotic, analgesic, some topical agent (triamcinolone acetonide ointment, albotyl®, chinese green traditional powder) and variety of mouthwashes. however, the ulcer showed no improvement and lead to a deeper and wider ulcer develop to surrounding skin, easy to bleed and cause limitation of mouth opening. patient stays in dormitory with sufficient facilities and admits no psychological stress. patient’s diet pattern shows normal and likes to eat vegetables and drink a lot of water. patient is non smoking and non alcohol drinking and use sodium lauryl sulfate dental paste. on the first day of visit, patient looked depressed due to unhealed ulcers and showed reluctant in replying question during anamnesis because of painful and bleeding ulcers. hematological results (ordered by previous doctors) showed an increase in erythrocyte sedimentation rate (esr), erythrocyte and hemoglobin and negative of widal test. based on the history of slight fever and the herpetiform type of ulcer preceded the bilateral angular ulcer, two provisional diagnosis were established as herpes labialis or ertythema multiforme triggered by herpes simplex infection (haem). thus, acyclovirs 200 mg five times daily, hyaluronic acid gel and multivitamin once a day for two weeks were administered on the first visit. blood test for igg/igm of hsv1 and hsv2 was ordered. second visit (10 days later): after ten days patient came again for first control. the angular ulcers as well as the surrounding skin showed remarkable improvement (figure 3). there was no red crustae seen over the lesion. thin fissure with yellowish base and desquamation still obvious on the angular area without bleeding tendency every time patient opened his mouth. no other ulcers found on the upper, lower lips and floor of the mouth. the laboratory result showed igg and igm for hsv 1 and hsv 2 were no reactive/normal. acyclovir 200 mg 5 times a day for 2 weeks and multivitamin once daily were continued. fucidin ointment was administered three times a day to help recovery of the skin and prevent further involvement of bacteria (staphylococcus). third visit (15 days later): on the second control, patient looked satisfy that the angular ulcer has completely healed without skin desquamation, scar and soreness. the recovery tissue still looked pale compared to surrounding tissue; however follicle and sebaceous gland near commissural area showed well emerge (figure 4). therefore, patients was discharged with instruction to maintain the oral hygiene and have enough rest. the update interview is done by phone one month before this case is reported, showed that patient has no longer oral ulcers including at the angular area as before. discussion by looking to the clinical manifestation, the angular ulcers in this case cannot be called as a classic cheilitis angularis as the manifestation expressed differently from figure 1. bilateral chronic multiple angular ulcer covered with red crustae which easy to bleed (1st visit). figure 2. slight edematous on upper and lower gingival, coexist with multiple minor ulcer on lower lip (1st visit). figure 3. angular ulcers showed remarkable healing with still desquamation (2nd visit) the 10th days. figure 4. complete healing of the angular ulcers bilaterally (3rd visit) the 15th days. ��amtha and pradono: unidentified angular recurrent ulceration responsive to antiviral therapy the common cheilitis angularis which caused by nutritional deficiencies, reduction of vertical dimension, microorganism involvement or allergy. moreover, the background of recurrent onset with slight fever and do not response to any topical agent (anti ulcer) lead to possibility of recurrent ulcers due infection of human herpes virus, usually herpes simplex (hsv). the severe clinical appearance on the first visit, which showed angular ulcers with tendency of bleed easily coincide with multiple herpetiform ulcers in the mouth is also leading to further autoimmune disease which associated with herpes virus infection. hsv infection is a predominant preceding event in individuals that experience recurrent episodes of erythema multifome (em), and such individuals are labeled as having herpes-associated erythema multifome (haem). however, usually the em skin lesions characteristically occur 1 to 10 days after an episode of herpes labialis or genitalis, which did not appear in this case. it was strengthened by the laboratory results of hsv1 and hsv2 igg/igm that showed normal (non reactive). weston16 has reported that seven of 34 patients with hsv had detectable hsv dna in peripheral blood mononuclear cells (pbmc) isolated, these subjects, however, did not develop em. this led some researchers to believe that hsv is transported to skin lesions via the blood, but further noted that hsv-specific antibody responses and lymphocyte transformation responses to hsv antigens were similar in-group with or without hsv infection. therefore, they found that haem occurred in spite of high immunity. aurelian in year 2005 also reported that subjects with haem have detectable herpes simplex viral particles in their circulating peripheral blood cd34 cells and it presumably destined to be precursors of epidermal langerhans cells.17 the findings found that patients with recurrent herpes labialis or genitals but without episodes of em did not have detectable virus in this type of cells.16 the administration of anti viral for the second visit was still prescribed eventhough the laboratory results of igg/igm of hsv showed negative. the reason was because the ulcers showed a significant improvement with the antiviral given. this is supported the findings by weston16 and aurelian17 regarding the non-specific antigen of hsv found in case of erythema multiforme. therefore the positive response of the ulcers with anti viral agent is in consequence of possibility of triggered by one or more type of human herpes virus (hhv). as we know that hhv has 8 types and their oral manifestation has not been completely elaborated and studied. so that when igm/igg of hsv were tested showed negative. it is assumed that this type of virus did not induce the ulcers and it needs further study. furthermore, it is probable that several of the risk factors for oral ulceration cause lesion outbreak by activating a latent viral infection that is not herpes simplex type of virus. also, some viruses may induce oral ulceration when co-infecting with other viruses. in this case, stress, nutritional deficiency and lack of rest can be the risk factors, even though there was not a specific test done to describe that condition and patient did not admit that he was under pressure in his dorm life of style. widespread and multiple oral ulcers should raise the suspicion of skin disease or vasculitis, particularly if associated with mucocutaneous lesions (e.g. blistering, hyperkeratosis or scarring); and ulcers limited to the commissures (angular cheilitis) have typically a microbial basis (often a candida or staphylococcal infection). history of recurrent angular ulcers and herpetiform ulcerations with fever is the basic of decision of prescribing the anti viral in this patient, besides increasing immune system by instruction of enough rest and multivitamin. the response of using corticosteroid (triamcinolone ointment) showed no improvement of the lesions and actually a contraindication for viral infection except for bell’s palsy type of case with background of hsv reactivation. there is no harmful side effect of giving patient of standard dosage of anti viral as the empirical treatment. as mentioned in previous research that one of eight people showed detectable particle of human herpes virus in their peripheral blood cells but not as the complete genome of the viral, so that the igg/igm found no reactive.16,17 moreover, all the human herpes virus has showed positive response to anti viral acyclovir. the fucidin ointment is an optional treatment to eliminate the involvement of staphylococcus infection on the skin surrounding ulcers. some clinical trial also showed that it helps to regenerate the skin texture damage. in this case, recurrent angular ulcers showed severe ulcers until involve deeper surrounding dermis. finally, as clinicians sometime it is difficult to determine one fixed diagnosis as the clinical appearance of oral ulcers is often not pathognomonic, and several different ulcerogenic conditions of the mouth may currently be lumped together. the best approach can be done is to review the basic pathogenesis of the disease to establish the best treatment. angular cheilitis terminology has a broad meaning and approach of treatment. the variety of clinical appearance and history of onset may lead to some diagnosis which is caused by viral infection. the angular ulceration on above patient failed to fulfill the criteria of stomatitis angularis or herpes labialis lesion. however it showed a good response to antiviral. therefore, unidentified angular ulceration was appointed; as the lesion might be triggered by other type of human herpes virus or types of virus that response to acyclovir. references 1. skinner n, jessie a. junker, flake d. what is angular cheilitis. j fam pract 2005; 54(5): 470–1. 2. scully c. oral and maxillofacial medicine. 2nd ed. edinburgh: churchill livingstone; 2008; 147–9. 3. dangi ys, soni ml, namdeo kp. oral candidiasis: a review. int j pharm pharm sci 2010; 2(4): 36–1. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 30–34 4. cernik c, gallina k, brodell rt. the treatment of herpes simplex infections: an evidence-based review. arch intern med 2008; 168(11): 1137–44. 5. arduino pg, porter sr. herpes simplex virus type 1 infection: overview on relevant clinico-pathological features. j oral pathol med 2008; 37(2): 107–21. 6. al-johani ka, fedele s, porter sr. erythema multiforme and related disorders. oral surg oral med oral patholoral radiol endod 2007; 103(50: 642–54. 7. sinha a, chander j, natarajan s. erythema multiforme presenting as chronic oral ulceration due to unrecognised herpes simplex virus infection. clin exp dermatol 2006; 31(5): 737–8. 8. wanner m, pol-rodriguez m, hinds g, hutt c, husain s, grossman me. persistent erythema multiforme and cmv infection. j drugs dermatol 2007; 6(3): 333–6. 9. slots j. oral viral infections of adults. periodontol 2000, 2009; 49: 60–86. 10. lin y l , li m. huma n cytomega lovi r us a nd epstein-ba r r virus inhibit oral bacteria-induced macrophage activation and phagocytosis. oral microbiol immunol 2009; 24(3): 243-8. 11. saccoccio fm, sauer al, cui x, armstrong ae, habib ese, johnson dc, ryckman bj, klingelhutz aj, adler sp, mcvoy ma. peptides from cytomegalovirus ul130 and ul131 proteins induce high titer antibodies that block viral entry into mucosal epithelial cells. vaccine 2011; 29(15): 2705–11. 12. chang et, adami ho. the enigmatic epidemiology of nasopharyngeal carcinoma. cancer epidemiol biomarkers prev 2006; 15(10): 1765–77. 13. tessme ms, reilly ac, brossay l. salivary gland nk cells are phenotypically and functionally unique. plos pathog 2011; 7(1): e1001254. 14. lambert em, strasswimmer j, lazova r, antaya rj. cytomegalovirus ulcer. arch dermatol 2004; 140(10): 1199–201. 15. juckem lk, boehme kw, feire al, compton t. dofferential i n it iat ion of i n nate i m mu ne responses i nduced by hu ma n cytomegalovirus entry into fibroblast cells. j immunol 2008; 180(7): 4965–77. 16. weston wl. herpes-associated erythema multiforme. j invest derm 2005; 124(6): 15–6. 17. aurelian l, ono f, sharma bk, smith cc, burnett jw. cd34 cells in the peripheral blood transport herpes simplex virus (hsv) dna fragments to the skin of patients with erythema multiforme (haem). j invest dermatol 2005; 124(6): 1215–24. 18. chen j, abatangelo g. functions of hyaluronan in wound repair. wound repair regen 1999; 7(2): 79–89. 92 volume 46 number 2 june 2013 garis estetik menurut ricketts pada mahasiswa fakultas kedokteran gigi universitas airlangga (ricketts esthetic line of dental student of universitas airlangga) nadiya fitriyani,1 i.g.a. wahju ardani2 dan elly rusdiana2 departemen ortodonsia fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: ricketts esthetic line is a line drawn from pronasale (prn) to soft tissue pogonion (pog) and lip prominence with reference to this line is assessed. esthetic line norms, may be spesific to an ethnic group and cannot always be applied to other ethnics. purpose: this study was aimed to determine the standard of esthetic line of javanese population student of faculty of dentistry, universitas airlangga. methods: radiographic sefalometric was taken from twenthy three dental students of 18–25 years old of universitas airlangga who fulfilled criteria sample and selected by three ortodontist and 3 lay person. two references line identified, traced and measured according to ricketts esthetic line. results: the mean of the esthetic line on upper lip was -1.4 mm and on the lower lip was 0.4 mm in males, on upper lip was -1.7 mm and on lower lip was -0.1 mm in females. this study showed there was no significant difference of the esthetic line between males and females. conclusion: the harmonious profile of the student in faculty of dentistry, universitas airlangga when labrale superior and labrale inferior are right or slightly behind the esthetic line. key words: esthetic line, facial profile, javanesse population abstrak latar belakang: garis estetik ricketts adalah garis yang ditarik dari pronasale (prn) ke jaringan lunak pogonion (pog) dan jarak bibir dihitung terhadap garis ini. standar sefalometri lateral spesifik untuk kelompok etnik tertentu dan tidak bisa digunakan pada kelompok etnik lain. tujuan: penelitian ini ditujukan untuk mengetahui nilai baku garis estetik pada populasi jawa mahasiswa fakultas kedokteran gigi universitas airlangga. metode: pengambilan foto sefalometri pada 23 mahasiswa fakultas kedokteran gigi universitas airlangga umur 18-25 tahun yang dipilih berdasarkan penyeleksian sampel, yaitu kuisioner, kriteria sampel dan pemilihan profil wajah sesuai oleh tiga dokter gigi spesialis ortodonti dan 3 orang awam dari foto siluet profil wajah. pengukuran dua garis, penampakan dan pengukuran mengacu pada garis estetik menurut ricketts. hasil: rata-rata garis estetik bibir atas -1.4 mm dan bibir bawah 0.4 mm pada laki-laki, dan pada perempuan bibir atas -1.7 mm dan bibir bawah 0.1 mm. penelitian ini menunjukkan tidak ada perbedaan yang signifikan antara garis estetik laki-laki dan perempuan. simpulan: profil wajah yang harmonis pada mahasiswa fakultas kedokteran gigi universitas airlangga adalah bila bibir atas dan bibir bawah tepat berada atau sedikit di belakang garis estetika. kata kunci: garis estetik, profil wajah, populasi jawa korespondensi (correspondence): nadiya fitriyani, departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: nadiyafitriyani_020911004@yahoo.com research report 93fitriyani, et al.,: garis estetik menurut ricketts pada mahasiswa pendahuluan p e r u b a h a n p a d a t u j u a n p e r a w a t a n o r t o d o n t i menunjukkan perubahan paradigma yang sebelumnya mengutamakan pada hubungan gigi geligi dan skeletal mulai menuju pada pertimbangan jaringan lunak wajah.1 prosedur perencanaan perawatan yang hanya berdasarkan pengukuran jaringan keras dapat menghasilkan perubahan jaringan lunak yang tidak diinginkan dan menyebabkan kekecewaan pasien.2,3 menurut arnett4 dengan munculnya sefalometric headfilm, berbagai analisa dikembangkan dalam upaya untuk meningkatkan kualitas dan kuantitas profil estetika wajah. terdapat perbedaan standar sefalometri antara satu populasi dengan populasi yang lain. dalam menentukan keserasian dan keseimbangan wajah pada perawatan ortodonti, umumnya digunakan standar ras kaukasoid.5,6 hal tersebut kurang tepat jika diterapkan pada ras lain. kelompok etnik yang berbeda cenderung memiliki pola karaktristik wajah berbeda. menurut heryumanni7 orang jawa memiliki ciri tertentu, sehingga perlu dievaluasi profil jaringan lunak pada suku jawa untuk bahan pertimbangan dalam keberhasilan perawatan ortodonti, agar memperoleh profil jaringan lunak yang baik. analisis bibir menurut rickets terdiri atas e-line yang digambarkan dengan garis yang ditarik dari ujung hidung ke jaringan lunak progonion.2 ricketts mengevaluasi posisi anteroposterior bibir, menggunakan garis estetika atau eline, sehingga memperoleh penilaian posisi bibir terhadap garis estetika.8 bibir atas harus terletak 4 mm di belakang garis estetik, dan bibir bawah berada 2 mm di belakang garis estetik.9 pemeriksaan perubahan profil wajah pada populasi dibenarkan untuk mengevaluasi standar individu pada etnik dan ras asli setempat. standar sefalometri untuk kelompok etnik berbeda sehingga dilakukan pengembangan.5, 10,11 penelitian ini bertujuan mengukur nilai rata-rata garis estetika populasi jawa dengan subyek penelitian mahasiswa fakultas kedokteran gigi universitas airlangga. bahan dan metode seleksi subyek dalam penelitian dilakukan dalam tiga tahap. tahap pertama pembagian kuesioner untuk memilih subyek populasi jawa. tahap kedua seleksi subyek melalui kriteria sampel yaitu: overbite normal dan overjet normal; tidak berdesakan atau spacing; tidak pernah perawatan ortodonti dan orthognatic surgery; penutupan bibir kompeten; sedikit atau tidak pernah perawatan restoratif. subyek yang memenuhi kriteria sampel kemudian difoto profil wajah, hasil foto dibuat siluet hitam-putih untuk kemudian dipilih oleh 3 dokter gigi spesialis ortodonti dan 3 orang awam berdasarkan wajah yang sesuai, sampel diterima apabila dipilih paling sedikit oleh 2 dokter gigi spesialis ortodonti dan 2 orang awam. sampel yang telah terpilih dari tiga proses seleksi sampel kemudian di foto sefalometri. foto sefalometri dilakukan di laboratorium pramita surabaya pada 23 mahasiswa fakultas kedokteran gigi universitas airlangga (11 laki-laki dan 12 perempuan) umur 18–25 tahun. penelitian ini telah lulus uji laik etik dari fakultas kedokteran gigi universitas airlangga. foto sefalometri dilakukan dengan posisi bibir menutup dan gigi keadaan oklusi. posisi frankfort horizontal plane, telinga ditahan ear rod. hasil foto sefalometri ditrasing dua kali dengan rentang waktu dua minggu untuk melihat bias pengukuran. titik-titik acuan dalam esthetic line adalah prn (titik pronasale): titik pronasale yang diambil dari ujung tertinggi hidung; pg (titik progenion): titik terendah pada dagu; ls-e: jarak antara bibir atas terhadap garis e-line; li-e: jarak antar bibir bawah terhadap garis e-line. hasil yang di peroleh diolah secara statistik dengan program spss 15.0 for windows untuk mengukur statistik (mean dan standar deviasi). analisa t-test dilakukan terhadap jarak li-e dan ls-e pada seflogram untuk setiap jenis kelamin. untuk uji analisa kesalahan pengukuran dilakukan pengukuran sefalogram dua kali setelah dua minggu dan diuji dengan paired t test. hasil berdasarkan pengukuran yang dilakukan didapatkan hasil pengukuran garis estetik menurut ricketts pada lakilaki dan perempuan mahasiswa fakultas kedokteran gigi universitas airlangga tahun 2009-2011 (tabel 1). rerata garis estetik menurut ricketts laki-laki untuk jarak bibir atas terhadap garis estetik (ls-e) adalah -1.4 mm dan jarak bibir bawah terhadap garis estetik (li-e) adalah 0.4 mm. rerata garis estetik menurut ricketts perempuan untuk jarak bibir atas terhadap garis estetik (ls-e) adalah -1.7 mm dan jarak bibir bawah terhadap garis estetik (li-e) adalah -0.1 mm. hal ini menunjukkan garis estetik menurut ricketts pada laki-laki lebih besar dari perempuan. hasil uji normalitas garis estetik menurut ricketts pada laki-laki bibir atas terhadap garis estetik (ls-e) p=0.629 (>0.05) dan bibir bawah terhadap garis estetik (li-e) (p=0.531>0.05) sehingga data berdistribusi normal. pada uji normalitas garis estetik pada perempuan bibir atas terhadap garis estetik (ls-e) p=0.514 (>0.05) dan bibir bawah terhadap garis estetik (li-e) p=0.502 (>0.05) sehingga data tersebut juga berdistribusi normal. hasil analisis independent t-test dengan derajat kemaknaan 95% pada bibir atas dan bibir bawah memiliki nilai p=0.780 (>0.05) dan p=0.586 (>0.05). hal ini menunjukkan bahwa tidak terdapat perbedaan yang signifikan antara garis estetik menurut ricketts antara laki-laki dan perempuan baik pada bibir atas terhadap garis estetik (ls-e) maupun bibir atas terhadap garis estetik (li-e). dari hasil analisa paired t-test tidak didapatkan perbedaan pengamatan garis estetika baik pada laki-laki dan perempuan setelah 2 minggu. 94 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 92–96 pembahasan profil jaringan lunak memiliki peran yang signifikan dalam diagnosis dan perawatan ortodonti. pertimbangan profil wajah penting karena daya tarik wajah memiliki efek psikososial yang dirasakan oleh pasien.2,3 perawatan ortodonti yang hanya mempertimbangkan hubungan rahang saja belum tentu memberikan profil wajah yang harmonis.2 untuk itu profil wajah menjadi salah satu pertimbangan dalam keberhasilan perawatan ortodonti. keharmonisan profil jaringan lunak merupakan salah satu hasil perawatan yang paling dirasakan pada pasien, wajah yang harmonis akan memberikan kepuasan pada hasil perawatan ortodonti. meskipun seseorang mampu mengenali wajah yang cantik, tetapi menjelaskan konsep cantik menjadi tujuan perawatan merupakan hal yang sulit.4 konsep kecantikan wajah dibatasi oleh berbagai aspek penilaian subjektif, serta dipengaruhi juga oleh berbagai faktor seperti kelompok etnik, umur, jenis kelamin, wilayah dan latar belakang pekerjaan.12 praktisi kesehatan sudah berusaha untuk mendefinisikan konsep cantik yang ideal melalui standarstandar yang bersifat objektif.4 terdapat berbagai metode untuk menganalisa profil jaringan lunak wajah, metode tersebut dikembangkan untuk mengupayakan peningkatan keharmonisan profil wajah setelah perawatan ortodonti. analisa profil jaringan lunak digunakan untuk mendapatkan perubahan wajah yang baik. dalam perawatan ortodonti profil wajah yang paling diperhatikan adalah sepertiga wajah bawah, terutama posisi anteroposterior bibir.8 ricketts memperkenalkan metode untuk mengevaluasi posisi anteroposterior bibir, menggunakan garis estetik atau e-line, garis yang ditarik dari ujung jaringan lunak hidung (titik pronasale) menuju jaringan lunak pogonion.8 posisi estetik didapat dari pengukuran jarak bibir atas dan bawah terhadap garis tersebut.13 bibir atas harus terletak 4 mm di belakang garis estetik, dan bibir bawah berada 2 mm di belakang garis estetik.9 posisi bibir berada di posterior terhadap bidang estetika jarak tersebut dianggap negatif tabel 1. hasil pengukuran garis estetik pada laki-laki dan perempuan pengukuran laki-laki (n = 11) perempuan (n = 12) rerata (mm) simpang baku rerata (mm) simpang baku ls-e -1.4 2.59 -1.7 2.09 li-e 0.4 2.29 -0.1 2.68 tabel 2. hasil paired t-test dua pengamatan pengukuran rerata paired t-test pengamat 1 pengamat 2 pengamatan 1-2 laki-laki -0.52 -0.48 0.723 perempuan -0.94 -0.92 0.553 dan positif bila berada diposisi anterior.13 dengan melihat posisi bibir pada bidang ini akan mendapatkan kesan dari posisi gigi, dan keadaan retrusi atau protrusi bibir. penentuan protrusi dan retrusi bibir membantu klinisi untuk menentukan perlunya perawatan ekstraksi.14 sebagai standar, sefalometri lateral digunakan untuk mendiagnosis, membuat rencana perawatan, serta memprediksi respons jaringan keras dan jaringan lunak pada perawatan ortodonti.3 dengan adanya sefalogram standar dapat dibandingkan keadaan seseorang pada waktu berlainan atau keadaan seseorang dengan populasinya.16 subyek menggunakan populasi jawa yang berada di surabaya karena merupakan salah satu etnis terbesar di indonesia yang menempati urutan etnis terbesar dari seluruh total populasi indonesia (41,7%). kelompok ini paling banyak menempati wilayah provinsi jawa tengah, jawa barat, dan jawa timur (termasuk surabaya).17 selain itu menurut heryumanni7 orang jawa memiliki ciri tertentu, sehingga perlu dievaluasi profil jaringan lunaknya untuk sebagai bahan pertimbangan keberhasilan dalam menentukan perawatan untuk memperoleh profil jaringan lunak yang baik. subyek yang dipilih adalah mahasiswa yang berusia 18–25 tahun karena profil jaringan lunak dipengaruhi oleh umur. pada umur 7 tahun ukuran median hidung laki-laki dan perempuan menunjukkan pertumbuhan yang cepat, kemudian umur 8 dan 11 tahun pertumbuhan menurun, dan mengalami percepatan pada umur 14–17 tahun karena ada prepubertal and pubertal acceleration. proporsi pertumbuhan pada bidang sagital dan vertikal menunjukkan proporsi akhir sampai 100% saat umur 18 tahun.13 pemilihan sampel melalui kriteria untuk menentukan nilai baku garis estetik mahasiswa fakultas kedokteran gigi universitas airlangga yang memiliki wajah menyenangkan dan diterima oleh populasi jawa. penyeleksian oleh lay person dari populasi jawa untuk mendapatkan sampel yang dapat mewakili kriteria wajah yang disukai dan diterima menurut populasi jawa. penilaian estetika bersifat self preference. pemahaman estetika setiap orang dipengaruhi pengalaman personal dan lingkungan sosial. pendapat ortodontis belum tentu diterima oleh persepsi orang awam.18 pengukuran garis estetik pada laki-laki dan perempuan mahasiswa fakultas kedokteran gigi universitas airlangga tahun 2009–2011, garis estetik laki-laki untuk bibir atas berada 1.4 mm di belakang garis estetik dan bibir bawah 0.4 mm di depan garis estetik. garis estetik perempuan, bibir atas berada 1.7 mm di belakang garis estetik dan bibir 95fitriyani, et al.,: garis estetik menurut ricketts pada mahasiswa bawah 0.1 mm di belakang garis estetik (tabel 1). garis estetik laki-laki lebih mendekati garis estetik dibanding perempuan. pada uji statistika dengan independent t-test (tabel 2) menunjukkan bahwa tidak terdapat perbedaan yang signifikan antara garis estetik laki-laki dengan garis estetik pada perempuan. analisa ricketts memberikan perbedaan 3 tipe wajah, yaitu cekung, lurus, dan cembung. cekung apabila posisi bibir berada di belakang garis estetik, lurus bila berada pada standar garis rata-rata estetika yaitu bibir atas berada 4 mm di belakang garis estetika dan bibir bawah berada 2 mm di belakang garis estetika, dan cembung apabila berada di depan atau sedikit menyentuh garis estetika.19 dari hasil penelitian ini rata-rata garis estetika pada lakilaki dan perempuan mahasiswa fakultas kedokteran gigi universitas airlangga tahun 2009–2011 populasi jawa menunjukkan bibir atas dan bibir bawah sedikit menyentuh dan di depan garis estetika maka dapat dikategorikan sebagai cembung. hal ini juga sesuai menurut heryumanni7 bahwa populasi jawa memiliki ciri bibir tebal, hidung yang tidak terlalu mancung, dan dagu tidak terlalu menonjol, sehingga profil wajah cenderung cembung. terdapat perbedaan ukuran protrusi bibir pada ras yang berbeda, sehingga perlu pertimbangan pada perawatan. pasien harus dicocokkan pada profil wajah kelompok suku dan ras yang paling mendekati, bukan pada standar nilai rata-rata.15 melalui pengukuran garis estetik maka akan memberikan kemudahan kepada ortodontis untuk mendapatkan hasil perawatan yang baik melalui pertimbangan dalam keharmonisan jaringan lunak wajah. sudah banyak penelitian yang dilakukan untuk mengukur nilai standar esthetic line (e-line) pada berbagai populasi ras, seperti pada populasi korea, jordan dan pada ras mongolian. pemeriksaan profil wajah pada populasi dibenarkan untuk mengevaluasi standar individu pada etnik dan ras asli setempat. hal ini dilakukan untuk mengembangkan standar seflometri untuk etnik berbeda.5,14 setiap kelompok ras memiliki ciri profil wajah yang berbeda sehingga kurang tepat apabila dalam suatu prosedur diagnosa dan perencanaan perawatan ortodonti dengan standar dari ras lain. analisa profil jaringan lunak digunakan untuk mendapatkan perubahan wajah yang baik. penelitian kim et al.,6 menyatakan bahwa tidak terdapat perbedaan yang terlalu signifikan antara nilai esthetic line korea dan mongolia. nilai estetik wanita korea ls-e -1.66 mm dan li-e -0.04 mm, untuk pria ls-e -1.66 mm dan li-e 0.09 mm. terdapat beberapa perbedaan pada pengukuran jaringan lunak. mongolia memiliki tinggi wajah anterior yang pendek dan prominen. nilai esthetic untuk wanita mongolia ls-e -0.8 mm dan li-e -0.11 mm, untuk pria ls-e -0.83 mm dan li-e -0.21 mm. banyak peneliti juga mengutamakan evaluasi posisi horizontal berkenaan dengan hidung dan dagu pada ras dan etnik berbeda. sebagai contoh, pada orang cina bibir atas dan bibir bawah lebih protrusi daripada ras kaukasoid atau orang hitam, karena fakta dari posisi dagu orang cina lebih rendah dan lebih posterior.15 dari hasil penelitian dapat dilihat bahwa garis estetik pada populasi jawa memiliki nilai yang mendekati nilai garis estetika pada korea dan mongolia. selain dipengaruhi oleh umur dan jenis kelamin, profil jaringan lunak juga dipengaruhi oleh etnik dan ras. populasi jawa termasuk ras deutro malayid. orang jawa merupakan sub-ras sekunder mongoloid yaitu berasal dari subras deutro melayu.20 hamdan et al.,21 meneliti esthetic line pada populasi jordan, didapatkan nilai ls-e pada wanita -6 mm dan pada pria -3.2 mm, sedangkan li-e pada wanita -0.9 mm dan pada pria -3.7 mm. pria jordan lebih protrusif bila dilihat dari nilai garis estetik, labrale inferior dan superior pada perempuan jordan lebih retrusif. prominen di hidung tidak berbeda antara pria dan wanita. tetapi pria memiliki dagu yang lebih prominen, bahkan pria populasi jordan memilki wajah yang lebih cembung dari populasi amerika. garis estetik pada populasi jawa dan populasi jordan berbeda, hal ini dikarenakan ciri profil wajah populasi jawa lebih cembung. garis estetika dipengaruhi oleh tinggi hidung dan dagu, seperti yang disebutkan sebelumnya populasi jawa memiliki hidung yang tidak terlalu mancung dan bibir yang tebal, sedangkan populasi jordan memiliki hidung yang tinggi dan dagu yang prominen. penelitian ini mempunyai keterbatasan yang mungkin dapat mempengaruhi validitas hasil penelitian, diantaranya adalah jumlah sampel yang digunakan terbatas. keterbatasan ini diakibatkan karena kesulitan mencari sampel yang memenuhi kriteria, dan dari 37 sampel yang memenuhi kriteria, kemudian diseleksi kembali oleh dokter gigi spesialis ortodonti dan oleh lay person populasi jawa. hasil penelitian ini menunjukkan bahwa profil wajah yang harmonis pada mahasiswa fakultas kedokteran gigi universitas airlangga adalah bila bibir atas dan bibir bawah tepat berada atau sedikit di belakang garis estetika. hal ini berbeda dengan beberapa penelitian yang dilakukan pada populasi yang berbeda, yang mungkin disebabkan oleh perbedaan jumlah sample. daftar pustaka 1. proffit wr, henry wf, david ms. contemporary ortohodontic. 4th ed. missouri: mosby elsivier; 2007. p. 5. 2. hasan sr, ulfat br. correlation among different profile planes used to evaluate lower lip position. pakistan oral and dental journal 2011; 31(2): 332-5. 3. gianelly aa, henry mg. biologic basis of orthodontic. philadelpia: lea & ferrigier; 1971. p. 3381. 4. arnett w, robert t, bergman. facial keys to orthodontic diagnosis and treatment planning. am j orthod 1993; 103: 299-312. 5. hwang hs, wang sk, james am. ethnic differences in the soft tissue profile of korean and european-american adults with normal occlusion and well-balance faces. angle orthod 2002; 72(1): 7280. 6. kim jh, odontuya g, bazar a, shin jl, tae wk. comparison of cephalometric norms between mongolian and korean adults with normal ocllusion and well balances profile. korean j orthod 2011; 41(1): 42-50. 96 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 92–96 7. heryumani. proporsi sagital wajah laki-laki dan perempuan dewasa etnik jawa. mi kedokteran gigi 2007; 22(1): 22-7. 8. milosevic sa, marina lv, mladen s. possibilities of sofft tissue analysis in orthodontic. acta stomatol croat 2007; 41(3): 251-9. 9. reyneke jp. essentials of or thognathic surger y. k imberly: quistessence publishing; 2003. p. 40-1. 10. hazar s, sercan a, hayal b. soft tissue profile changes in anatolian turkish girls and boys following orthodontic treatment with and without extraction. turk j med sci 2004; 34: 171-8. 11. altemus la. cephalofacial relationship. departemen orthodontist harvard university. 1968. 38(3): 175-85. 12. singh jr. preference of lip profile in varying mandibular sagittal position. j int oral healt 2011; 3(5): 47-58. 13. nanda rs, hanspeter m, sunilk. growth change in the soft tissue facial profile. the angle orthodontist 1989; 60(3): 177-90. 14. english jd. mosby’s orthodontic review. missouri: mosby elsivier; 2009. p. 55, 58, 63, 114. 15. burstone cj, michael rm. problem solving in orthodontic. goal oriented treatment strategies. kimberly: quintesence publishing; 2000. 16. rahardjo p. ortodonti dasar. surabaya: airlangga university press; 2009. h. 164-5. 17. aris a, evi na, bahtiar. ethnicity and ageing in indonesia 20002050. journal of the asian population 2005; 1: 228-43. 18. flores-mir c, silva e, barriga mi, lagravere mo, major pw. lay person’s perception of smile aesthetics in dental and facial views. j orthod 2004; 31(3): 204-9. 19. miksic m, mladen s, senka m. bioprogressive therapy and diagnostic. acta stomatol croat 2003; 37: 461-4. 20. mahyastuti rd, christnawati. perbandingan posisi bibir dan dagu antara laki-laki dan perempuan jawa berdasarkan analisa estetik profil muka menurut bass. m.i kedokteran gigi 2008; 23(1): 1-7. 21. hamdan am. soft tissue morphology of jordanian adolescent. angle orthod 2011; 80(1): 80-4. 209 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 209–214 original article il-17 plasma levels and erythrocyte sedimentation rate on oral candidiasis animal model erna sulistyani1, iin eliana triwahyuni1, happy harmono2, lisa miftakhul janna3, saikha adila azzah3, muchamad ziyad afif3, ainunnusak ayuningtyas3 1department of oral medicine, faculty of dentistry, university of jember, jember, indonesia 2department of biomedical, faculty of dentistry, university of jember, jember, indonesia 3student of faculty of dentistry, university of jember, jember, indonesia abstract background: a study with female animal models is important because the system immune of females is remarkably different from the male because of interaction between sex hormone and immune system. interleukin-17 (il-17) plays an important role in immune response toward candida albicans (c. albicans) infection and erythrocyte sedimentation rate (ers) is an easy and sensitive test to assess the inflammation. purpose: this study aimed to evaluate c. albicans infection, analyse the il-17 levels and esr in a female animal model of oral candidiasis. methods: female wistar rats were used as oral candidiasis animal model. the rats divided into three groups (pre-treatment group (p0), 5th-day post-treatment group (p1) and 8th-day posttreatment group (p1). each group consists of six rats. after the adaptation period, the p0 was sacrificed. the drinking water of the p1 and p2 was added tetracycline hcl 500mg /1l. on the day before and after inoculation c. albicans, methylprednisolone was injected. ten minutes before inoculation with 0.3 ml c. albicans 9.4 x 107/ml, rats were sedated by cpz 0.7 mg im. the rats in p1 group were sacrificed after five days and in p2 were sacrificed eight days after inoculation. the il-17 plasma levels measured by enzyme-linked immunosorbent assay (elisa), decreased on the 5th day but not on the 8th-day post-treatment. the obtained data were analyzed by parametric and non-parametric tests according to normality and homogeneity of the data with p<0.05. results: the colony forming unit (cfu) of c. albicans collected over the mouth on increased almost 8-fold and on 8th-day post-treatment almost 3-fold compared with pre-treatment. the esr increased on the 8th day but not on the 5th-day post-treatment. conclusion: the il-17 level was decreased on the 3rd day, esr increased on eight days after inoculation of c. albicans in female rats’ model of oral candidiasis. keywords: animal model; c. albicans; il-17; infectious disease; medicine correspondence: erna sulistyani, department of oral medicine, faculty of dentistry, university of jember. jl. kalimantan no. 37 jember, 68121 indonesia. email: erna.fkg@unej.ac.id introduction candida albicans (c. albicans) is almost always found as a commensal microorganism in the healthy human oral cavity. the commensal properties of c. albicans are maintained by both innate and adaptive immune systems and normal flora bacteria c. albicans can grow commensals because c. albicans have the capability to avoid the immune system, called immune evasion. c. albicans can escape from the human immune system in various ways, including by regulating the complement cascade either by secreting enzymes secreting aspartyl proteases or by binding to the surface of complement regulators. during infection, complement facilitates the process of phagocytosis by opsonization and then initiates an inflammatory response by modifying the behavior of b cells and t cells.1 c. albicans can also survive if phagocytized by leukocytes and can spread to other parts of the body (vomocytosis) and can cause leukocyte death.2 if there is a change in the defense immune system of the oral cavity, it can trigger the shift of c. albicans from commensal into a pathogen. the prevalence of oral candidiasis in infants is estimated at 5-7%, in acquired immunodeficiency syndrome (aids) patients 9-31%, and up to 20% in cancer patients. c. albicans is the cause of 75% candidiasis overall.3 the incidence of human immunodeficiency virus (hiv) infection which dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 mailto:erna.fkg@unej.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 210sulistyani et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 209–214 caused aids, has increased very rapidly in this decade. the main complication of hiv infection is opportunistic infections including oral candidiasis.4 according to study in surabaya general hospital, 2018, oral candidiasis was found in 57.14% of hiv patients.5 the incidence of oral candidiasis was significantly correlated with the decrease of cd4 count in hiv patient.6 the prevalence of auto inflammatory/autoimmune diseases due to therapy with immunosuppressant and cancer chemotherapy and radiotherapy also contribute for the enhancement of c. albicans opportunistic infections. c. albicans was isolated from 73.3% of patients who received chemotherapy in general hospital hasan sadikin bandung.7 in the past two decades, it has been observed an abnormal overgrowth in the gastrointestinal, urinary and respiratory tracts, not only in immunocompromised patients, but also related to nosocomial infections and even in healthy individuals.8 in addition to the oral cavity, c. albicans infections often occur in the vagina, gastrointestinal tract, skin and systemic infections called invasive candidiasis can also occur in the heart, eyes, intra-abdominal, joints, bones, and brain membranes.9 the immunocompromised patient needs long term antifungal drug as prophylaxis and therapy and this may lead to serious side effects and drug resistance. the prevalence of c. albicans’s antifungal resistance is approximately 56.7% in patients with hiv infection.10 this fact has resulted in the need for research to find alternative drugs that are safer for long-term use and this research requires appropriate animal models previous study believed the estrus cycle in females leads to significant variation response and need to increased sample sizes if using this gender in the study even though this is not proven. female subjects are underrepresented in animal research across disciplines. the biological response, especially the immune response of women is different from that of men because of genes and, hormones can interfere with the immune response.11 the unavailability of research using experimental animals causes a shortage of materials to be tested on women and prevents women from receiving treatment as well as men. on the other hand, various diseases caused by immune response deviations are more common in women.12 in animal model, murine oral candidiasis, deviation of immune response was believed to have an important role in the pathogenesis of oral candidiasis.13 in a previous study using the female murine model that mimics the natural infection in humans, the inoculation of c. albicans 2.5 x107 cells/ml (=106 cells/mouse) in prednisolone, tetracycline, and chlorpromazine treated caused high and stable colonization until seven days.14 in a study conducted by sulistyani (2019), the injection of c. albicans cell wall intraperitoneally in healthy rat highly correlated with increase of il-17 plasma levels.15 il-17 is the most potent inflammatory cytokine and most important cytokine toward c. albicans infection can eliminate c. albicans exposure rapidly.16,17 these results also explain why, in healthy individuals, c. albicans cannot cause infection. the il-17 induces adequate immune response to eliminate the c. albicans. the evaluation of systemic inflammation in animal model of oral candidiasis has never been revealed. the hypothesis of this study was there were a reduction of il-17 plasma level and an elevation esr in the rat model of oral candidiasis. thus, the purposes of the study are to analyze the plasma level of il-17 and the erythrocyte sedimentation rate (esr) in female wistar rat (rattus novergicus) as animal model of oral candidiasis. the esr was chosen because it is an easy and sensitive marker of inflammation. materials and methods the c. albicans atcc 10231 was purchased from biology oral laboratory, faculty of dentistry, universitas hang tuah, surabaya. identification test was performed by culturing in the chromagar and the color of c. albicans colony was a green colony. chromagar technology is a color-based differentiation method. it is based on soluble colorless molecules (called chromogens), composed of a substrate (targeting a specific enzymatic activity) and a chromophore. when the target organism’s enzyme cleaves the colorless chromogenic conjugate, the chromophore is released.18 this strain was cultured in sabouraud 4% dextrose agar (sda) (merck cat. no 1.05438.0500) and then diluted in sabouraud dextrose broth (merck. cat no 1.08339.0500) in concentration 9.4 x 107 cells/ml and stored at −4°c until the experiment was performed. six-week-old female wistar rats (iwan farm, pakis aji, malang) were used for all animal experiments. the rats were kept in cages with sufficient light from the sun, and the environmental temperature was constantly maintained at 26-29°c. one cage was used to keep three rats. rats were given access to food and water ad libitum. before the study began, it was confirmed that the female rats were not pregnant because they had been separated from the male rats since they were 15 day-olds. the two rats, which were randomly selected, were confirmed to be both in a healthy condition and not pregnant by a veterinarian at veterinary clinic, livestock office, jember. all research procedures were approved by the health research ethics committee, faculty of dentistry, university of jember with approval letter number 11280/un25.7/kepk/dl/2021. the research design used was a pre-post separated group design. the experimental animals were divided into three groups, the pre-treatment group (p0), the 5th day posttreatment group (p1), and the 8th day post-treatment group (p2). after adaptation for 10 days, rats in the p0 group were sacrificed and intracardiac blood was taken for the measurement of the variables. the drinking water of the rats in the p1 and p2 groups were given tetracycline hcl (sanbe pharmacy, indonesia) in a concentration of 500 mg/l to reduce the number of oral floras, which have capability to inhibit c. albicans growth. tetracyclines are dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 211 sulistyani et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 209–214 broad-spectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical organisms. on the day before treatment, the rats in the treatment (p1 and p2) groups were injected subcutaneous with methylprednisolone sodium succinate (mp) (phapros ltd, indonesia) in dose 40 mg/kg.bw. on the treatment day, the rats were sedated with chlorpromazine (phapros ltd, indonesia) injection 5 mg/kg.bw im. chlorpromazine is a psychotropic agent indicated for the treatment of schizophrenia. it also exerts sedative and antiemetic activity. after rats were sedated, 0.3 ml suspension of c. albicans atcc 10231 in concentration 9.4 x 107 cells/ ml (3.1 x 106 cell/rat) was inoculated in the oral cavity of the rats with a small plastic brush stick. on a day after inoculation, rats were injected with mp again in the same dose as before treatment. on the 5th day of treatment, the rats in the p1 group were sacrificed and on the 8th day the rats in the p2 group were sacrificed. the sacrifice procedure of rats followed the rules of animal euthanasia policy of university in st louis usa. the rats were anesthetized with ketamine-xylazine cocktail (0.1 ml ketamine 1000mg/10ml+ 0.05ml xylazine 20mg/50ml) and followed by exsanguination.19,20 all procedures are shown in table 1. the infection of c. albicans was determined by microbial evaluation: the whole oral cavity, including the buccal mucosa, tongue, soft palate, and other oral mucosal surfaces, was swabbed using a sterile cotton swab then, the end of the cotton swab was cut off and placed in a tube containing 5 ml sterile saline. to dissolve c. albicans from the swab into the saline, we used a vortex. then, after serial 100-fold dilutions, the cell suspension was incubated on sabouraud dextrose agar + chloramphenicol (condalab cat. no 1134, madrid spain) at 37°c for 20 hr. the colony forming units (cfus) of candida colonies were counted. the number of cfus was used as the marker of c. albicans infection. the esr was measured using an esr fast detector, and 1.25 ml esr vacuum tubes containing sodium citrate (monotes™, zhejiang, china). esr was performed immediately after blood had been drawn to avoid changes in the blood composition due to storage. the blood was put in an esr tube, until the line mark and shaken 90 degrees six times so that the blood was well-mixed. after that, the tube was placed in an esr fast-rack esr rack. the esr rack was confirmed flat by adjusting the mark in the rack. the timer was turned on when the tube had been inserted on the rack. the esr was observed after 30 minutes. the level of il-17 was measured using il-17 rat elisa kit (mybiosource catalog no. mbs164772 r, sandiego, california usa) the blood was inserted into a 3 ml vacutainer containing ethelene diamine tetra-acetic acid (edta) (vaculab™, with lilac color cap dubai united arab emirates) then the plasma was separated using a mini centrifuge. the plasma separation was performed immediately after collection to avoid blood lysis. the obtained plasma was inserted into the mini tubes and stored in a styrofoam box containing ice gel, and the box was kept in a freezer at -4°c until brought to surabaya for analysis. elisa il-17 analysis was carried out at the laboratory of specialised hospital for infection, universitas airlangga. the data were analyzed using statistical package for social science (spss) version 22 (ibm®, spss®, illinois, chicago, us). to analyze the distribution and homogeneity of data, we use shapiro-wilk and levene tests. if the data were normally distributed and homogeneous, the differences between groups were analyzed using a one-way analysis of variance (anova) test followed by a post-hoc least significant difference (lsd) test to compare differences between all groups. if the data were not normally distributed neither homogeneous, the differences between groups were analyzed using the kruskal-wallis test followed by the mann-whitney test when the data were not normally distributed or/and non-homogeneously distributed, the p value was set at <0.05. table 1. the treatment of animal model of oral candidiasis day -2 -1 0 1 2 3 4 5 6 7 8 po sacrificed p1 given drinking water + tetracycline injected with mp injected with cpz + inoculation c. albicans injected with mp sacrificed p2 sacrificed table 2. the mean and standard deviation of cfu number, il-17 level, and esr in the pre-treatment, 5th day post-treatment and 8th day post-treatment groups variable mean ±sd pre-treatment 5th day post-treatment 8th day posttreatment cfu (x 105) 0.125 ± 0.049 9.778 ± 4.259 3.057 ± 0.963 il-17 level (pg./l) 79.98 ± 4.96 55. 62 ± 6.03 52.56 ± 9.97 esr/hour 4.5 ± 0.55 4.83 ± 0.75 5.83 ± 1.17 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 212sulistyani et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 209–214 results the means and standard deviations of cfu in p0, p1, and p2 are shown in table 2. the cfu on the 5th-day posttreatment group increased almost 8-fold compared with the pre-treatment group. on the 8th-day after treatment, the cfu tended to decrease but was still nearly 3fold compared with the pre-treatment group. the distribution and homogeneity test data using shapiro-wilk and levene test showed that the data were in normal distribution and homogenous. the mean differences between groups were analyzed by a one-way anova test, followed by lsd. the result showed that the mean of cfu c. albicans increased in the p1 and declined in the p2 but still more than the p0 group. the test of normality and homogeneity data results showed that the il-17 level data were neither distributed normally nor homogeny. the mean differences of il-17 plasma level analysis between groups were used with a nonparametric test, namely the kruskal-wallis test followed by the post hoc mann-whitney test. the result showed that the levels of il-17 between the pre-treatment and 5th-day post-treatment groups were significantly different. the il-17 levels between the p1 and the p2 were not different significantly (p<0.05). the il-17 levels between the p0 and the p2 group were not different either. from these facts, it can be concluded that the levels of il-17 in female animal wistar rat models of oral candidiasis decreased starting on the 5th day post treatment (p1) and persisted until 8th day post treatment. this fact indicates that the decrease in il-17 levels occurs in animal models of oral candidiasis. the normality and homogeneity esr data test were also neither normally distributed nor showed homogeny. the analysis of the difference test was using the same test as il-17. the results showed a difference between groups p0 and p2 (p<0.05), but not between p0 and p1. this result indicates an elevation of esr in the experimental animal model of oral candidiasis of female wistar rats on the 8th post-treatment group but not on the 5th day post-treatment group. discussion the number of c. albicans colonies from the entire oral cavity on 5th day after inoculation of c. albicans increased almost 8-fold. on 8th day after exposure, the number decreased but was still three times higher than the pretreatment group. this fact indicates that a very significant infection occurred. in five days, 3.1 x 106 cell (spores) of c. albicans had become 9.7 x 105 cfu. in studies using healthy mice exposed to the highly infectious strain of c. albicans sc5314 or its derivatives, the immune response was showed very rapidly and colonization of c. albicans did not occur. effective activation of the immune response toward c. albicans leads to the rapid elimination of c. albicans only in a few days.21 therefore, in producing a model of c. albicans infection, it is necessary to reduce the immune system, and reduce local oral defense factors, which is in this study were done by injection of mp and giving a broad-spectrum antibiotic tetracycline hcl, in drinking water. the result showed that the level of il-17 reduced on 5th day post treatment. this decrease was probably due to the injection of methylprednisolone sodium succinate. methylprednisolone sodium succinate is the sodium succinate salt of a synthetic glucocorticoid receptor agonist with immunosuppressive and anti-inflammatory effects. methylprednisolone sodium succinate is converted into active prednisolone in the body, that diffuses passively across cell membranes and binds to intracellular glucocorticoid receptors. this complex translocates into the nucleus, where it interacts with specific dna sequences, resulting in increased or suppressed transcription of certain genes. the methylprednisolone-glucocorticoid receptor complex binds to and blocks the promoter sites of proinflammatory genes, promotes the expression of anti-inflammatory gene products, and inhibits the synthesis of inflammatory cytokines, primarily by blocking the function of transcription factors, such as nuclear factor-kappa-b (nf-kb).22 considerable evidence in both humans and mice reveals a clear and specific role for il-17 in protection against the c. albicans. 21,23 the natural resistance of mice to c. albicans is highly dependent on the functional il-17 pathway.21 the t-helper (th)-17 response during disseminated fungal infection can be both protective and detrimental. splenocytes isolated from hil-37tg mice with a higher fungal burden produced significantly more il-17 in response to c. albicans pseudohyphae.24 the immune response to c. albicans is initiated by the binds of its pathogen associated molecular pattern (pamps) with the pattern recognition receptors (prr) of immune cells. this binding induces intracellular signaling cascades, then triggers the secretion of various inflammatory cytokines. some interleukins induce differentiation of naive th0 to th17 through the enhancements of signal transducer and activator transcription 3 (stat3) and retinoid-related orphan receptor γt (rorγt). th17 will produce the il-17 that trigger epithelial and mesenchymal cells to express chemokines for recruitment of neutrophils (interleukin-8, c-x-c motif ligand-1 (cxcl1), cxcl5), granulocyte-colony stimulating factor, and antimicrobial peptides (amp) such as defensins and protein s100.25 those chemokine trigger immune cells, particularly neutrophils, move to the site of infection and induce secretion various other inflammation mediators.26,27 excessive il-17 production can trigger various autoimmune and autoinflammatory diseases.15 on the other hand, deficiency in the il-17 pathway can lead to bacterial and fungal infections and promote tumor growth. il-17 deficiency states are associated with susceptibility to infections from c. albicans, staphylococcus aureus, and mycobacterium tuberculosis. 28 examination of the esr is an easy and sensitive examination of changes in the body, particularly dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 213 sulistyani et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 209–214 inflammation condition. in this study, the esr increased on 8th day post-treatment and not yet on the 5thday post-treatment. the increase in esr was mainly due to an increasing level of inflammation-sensitive plasma proteins (isps), a plasma protein that eliminates infection. the plasma proteins usually have a positive electrical static charge. red blood cells in healthy conditions have negatively charged, so they repel each other. the positive electrical static charge of plasma proteins can neutralize the negative charge of red blood cells so that red blood cells easily attach each other and form rouleaux. in the rouleaux form, the erythrocytes will settle faster. than single red blood cells.29 in 8th day post-treatment, the esr increased indicated that inflammation-sensitive plasma proteins (isps) were elevated in blood. isps can reduce the infection. this result was in accordance with the decreased the number of cfu of c. albicans. on the 8th day posttreatment group, the inflammation elevated and caused a decrease in the number of c. albicans cfu. the inflammatory response toward various conditions including fungal infection leads to a significant alteration in the plasma level of several proteins. the measurement of the plasma protein level is applied to determine the normal/abnormal response of the host to tissue injury and some extent, indicate the amount of tissue involved. the inflammatory response leads to a decrease of albumin and transferrin levels, and enhancement of haptoglobin level as a response to blood loss. the level of coagulation factor for example fibrinogen, globulins, and protease inhibitors, observed elevation from 10% until 5-fold the baseline level by the cause of injury and at the stage of the process when the sample was collected.11 several plasma proteins that increase during inflammation are called inflammationsensitive plasma proteins (isps), including fibrinogen, haptoglobin, 1-antitrypsin, serum amyloid a, c-reactive protein, and orosomucoid. 12,13 this study has several limitations such as the separated pre-post design could not accurately describe the pre-post condition, but the study design was chosen because 5 ml of blood to measure all dependent variables is needed. it should be understood that this study was only basic research that provides a new perspective that female laboratory animals can be used as an experimental model. a very severe infection may develop in female laboratory animals receiving the treatment we gave. in conclusion, there was significant infection even up to the 8th day after exposure; the il-17 reduced on both the 5th and the 8th day after treatment. the esr increased on the 8th day after treatment in our oral candidiasis model. further study could use the oral candidiasis animal model for the treatment exploration of oral candidiasis with various methods. references 1. singh dk, tóth r, gácser a. mechanisms of pathogenic candida species to evade the host complement attack. front cell infect microbiol. 2020; 10: 94. 2. seoane pi, may rc. vomocytosis: what we know so far. cell microbiol. 2020; 22(2): 1–6. 3. patil s, rao rs, majumdar b, anil s. clinical appearance of oral candida infection and therapeutic strategies. front microbiol. 2015; 6: 1391. 4. nugraha ap, ernawati ds, parmadiati ae, soebadi b, prasetyo ra, triyono ea, sosiawan a. study of drug utilization within an antifungal therapy for hiv/aids patients presenting oral candidiasis at upipi rsud, dr. soetomo hospital, surabaya. j int dent med res. 2018; 11(1): 131–4. 5. mensana mp, ernawati ds, nugraha ap, soebadi b, triyono ea, husada d, prasetyo ra, utami sb, sufiawati i. oral candidiasis profile of the indonesian hiv-infected pediatric patients at upipi dr. soetomo general hospital, surabaya, indonesia. hiv aids rev. 2018; 17(4): 272–7. 6. nugraha ap, ernawati ds, parmadiati ae, soebadi b, triyono ea, prasetyo ra, utami sb, sosiawan a. prevalence of candida species in oral candidiasis and correlation with cd4+ count in hiv/aids patients at surabaya, indonesia. j int dent med res. 2018; 11(1): 81–5. 7. sufiawati i, pratiwi u, wijaya i, rusdiana t, subarnas a. the relationship between candida albicans colonization and oral hygiene in cancer patients undergoing chemotherapy. mater today proc. 2019; 16: 2122–7. 8. martins n, ferreira icfr, barros l, silva s, henriques m. candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. mycopathologia. 2014; 177(5–6): 223–40. 9. seladi-schulman j, sethi s. about candida albicans: natural yeast and problematic infections. medical news today. 2018. available f rom: ht t ps://www.med ica l newstoday.com /a r t icles/322722. accessed 2021 sep 23. 10. wicaksono s, rezkita f, n. wijaya f, nugraha ap, winias s. ellagic acid: an alternative for antifungal drugs resistance in hiv/aids patients with oropharyngeal candidiasis. hiv aids rev. 2020; 19(3): 153–6. 11. klein sl, flanagan kl. sex differences in immune responses. nat rev immunol. 2016; 16(10): 626–38. 12. beery ak. inclusion of females does not increase variability in rodent research studies. curr opin behav sci. 2018; 23: 143–9. 13. ninomiya k, hayama k, ishijima sa, maruyama n, irie h, kurihara j, abe s. suppression of inflammatory reactions by terpinen-4-ol, a main constituent of tea tree oil, in a murine model of oral candidiasis and its suppressive activity to cytokine production of macrophages in vitro. biol pharm bull. 2013; 36(5): 838–44. 14. takakura n, sato y, ishibashi h, oshima h, uchida k, yamaguchi h, abe s. a novel murine model of oral candidiasis with local symptoms characteristic of oral thrush. microbiol immunol. 2003; 47(5): 321–6. 15. sulistyani e, dachlan yp, putra st. enhancement of il23independendent il17 level on intraperitoneal injection of candida albicans cell wall in wistar male rat. j int dent med res. 2019; 12(4): 1368–71. 16. chaplin dd. overview of the immune response. j allergy clin immunol. 2010; 125(2 suppl 2): s3-23. 17. qin y, zhang l, xu z, zhang j, jiang y-y, cao y, yan t. innate immune cell response upon candida albicans infection. virulence. 2016; 7(5): 512–26. 18. chromagar. chromogenic technology. 2022. available from: ht t ps://www.ch romaga r.com /en /ou rcompa ny/ch romogen ictechnology/. accessed 2022 may 18. 19. university of iowa. anesthesia (guideline). vertebrate animal research website. 2020. p. 1. 20. washington university in st. louis. animal euthanasia policy. usa; 2015. p. 1–4. 21. sparber f, leibundgut-landmann s. interleukin-17 in antifungal immunity. pathog (basel, switzerland). 2019; 8(2): 54. 22. ocejo a, correa r. methylprednisolone. treasure island (fl): statpearls publishing; 2021. 23. conti hr, gaffen sl. il-17-mediated immunity to the opportunistic fungal pathogen candida albicans. j immunol. 2015; 195(3): 780–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 https://www.medicalnewstoday.com/articles/322722 https://www.chromagar.com/en/our-company/chromogenic-technology/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 214sulistyani et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 209–214 24. van de veerdonk fl, gresnigt ms, oosting m, van der meer jwm, joosten lab, netea mg, dinarello ca. protective host defense against disseminated candidiasis is impaired in mice expressing human interleukin-37. front microbiol. 2014; 5: 762. 25. hernández-santos n, gaffen sl. th17 cells in immunity to candida albicans. cell host microbe. 2012; 11(5): 425–35. 26. sokol cl, luster ad. the chemokine system in innate immunity. cold spring harb perspect biol. 2015; 7(5): a016303. 27. zenobia c, hajishengallis g. basic biology and role of interleukin17 in immunity and inflammation. periodontol 2000. 2015; 69(1): 142–59. 28. welch ez, anderson kl, feldman sr. interleukin 17 deficiency and implications in cutaneous and systemic diseases. j dermatology dermatologic surg. 2015; 19(2): 73–9. 29. tishkowski k, gupta v. erythrocyte sedimentation rate. treasure island (fl): statpearls publishing; 2021. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p209–214 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p209-214 65 volume 46 number 2 june 2013 a study of extraction and characterization of alginates obtained from brown macroalgae sargassum duplicatum and sargassum crassifolium from indonesia decky j. indrani1 and emil budianto2 1department of dental materials science, faculty of dentistry, universitas indonesia 2department of chemistry, faculty of mathematics and natural sciences, universitas indonesia jakarta – indonesia abstract background: worldwide commercially available alginate have been used for tissue engineering purposes. the macroalgae sargassum obtained from indonesia have been used for various purposes, however, they have not been applied for tissue engineering scaffolds. purpose: this study was aimed to extract alginate from the macroalgae sargassum from indonesia sea and to characterize in morphology, chemical element and functional groups. methods: macroalgae sargassum duplicatum (s. duplicatum) and sargassum crassifolium (s. crassifolium) were collected from banten, indonesia. extraction of alginates were carried out using the alkaline extraction procedure. scanning electron microscopy as well as x-ray fluorescence and fouirer transform infra-red spectroscopy were used to characterize the extracted powders. obtained data from the extracted powders were compared to those of the commercially available alginate. results: extraction using the alkaline method has resulted in s.duplicatum and s.crassifolium alginate powders. alginate particles were suggested as irregular shapes with various dimension. element components were mainly na and ca, whereas, minor elements were considered as negative impurities. cooand c-o-c groups were evident in the finger print regio. the characteristics of alginates extracted from the macroalgae s.duplicatum and s.crassifolium found similar to those of the commercially available alginate. conclusion: extraction obtained from the macroalgae s.duplicatum and s.crassifolium showed the typical alginate and the morphology, chemical element and functional groups were in agreement with those of the commercially available alginate. key words: alginate extraction, morphology, chemical element, functional groups abstrak latar belakang: alginat dari berbagai penjuru dunia telah digunakan untuk kegunaan rekayasa jaringan. alginat dari alga makro sargassum yang diperoleh dari indonesia telah digunakan untuk berbagai kegunaan, namun ini belum diterapkan untuk scaffold jaringan. tujuan: untuk mengekstrak alginat dari alga makro sargassum perairan indonesia dan untuk memperoleh karakteristik alginat dalam morfologi, unsur kimia dan gugus fungsi. metode: alga makro sargassum dari spesies sargassum duplicatum (s. duplicatum) dan sargassum crassifolium (s. crassifolium) diperoleh dari banten, indonesia. ekstraksi alginat dilakukan dengan menggunakan prosedur ekstraksi alkali. scanning electrone microscope, x-ray fluorescence dan fouirer transform infra-red spektroscope digunakan untuk mengarakterisasi bubuk alginat hasil ekstraksi. data yang diperoleh dari serbuk laginat dibandingkan dengan yang tersedia secara komersial. hasil: ekstraksi menggunakan metode alkali telah menghasilkan serbuk alginat dari s.duplicatum dan s. crassifolium. morfologi partikel alginat terlihat tidak teratur dengan berbagai dimensi. elemen na dan ca muncul sebagai komponen utama, sedangkan, elemen minor dianggap sebagai pengotor. gugus fungsi coo-dan coc terdeteksi di regio sidik jari. karakteristik alginat s.duplicatum dan s.crassifolium ditemukan sesuai dengan karakteristik alginat yang tersedia secara komersial. simpulan: research report 66 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 65–70 serbuk yang diperoleh dari alga makroi s. duplicatum dan s.crassifolium menunjukkan kekhasan alginat dan morfologi, unsur kimia dan kelompok fungsional alginat sesuai dengan yang tersedia secara komersial. kata kunci: ekstraksi alginat, morfologi, elemen kimiawi, gugus fungsi correspondence: decky j. indrani, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 6 jakarta 10430, indonesia. e-mail: decky@ui.ac.id introduction the failure or loss of an organ or tissue is one of the most numerous and costly problems in human health care. tissue engineering, that integrates a variety of science and engineering disciplines to create functional organ or tissues for transplantation, evolved as one of the most promising therapies in regenerative medicine.1 scaffoldguided tissue engineering for cells growth matrix made of biopolymer were appealing due to their structural similarities to the macromolecular-based human tissues.2 among biopolymers, alginate enabled more efficient penetration of cells into scaffold matrices.3,4 to enhance its biological performance, alginate has been combined with other biomaterials. blends of alginate and chitosan have been used to regenerate cell in soft tissues.5,6 composites of alginate and hydroxyapatite seeded with osteoblast have also been observed for bone tissue engineering.7,8 bone tissue engineering may be an advantage, for instance, to increase ridge width due to resorption from tooth loss or for the treatment of edentulous patients if they lack the appropriate bone volume. worldwide alginates are collected from macroalgae of sub-tropical seas. for example, algae species of laminaria from norwegia, france, and japan, as well as, species of macrocystis from north america and australia have produced alginates and made them important industries for food and nutrients,9 and other industries, such as, cosmetics, textile protection, fertilizers, pharmaceuticals, and biotechnology.10 in dentistry, alginates have been applied for impression materials.11 in tropical seas of malaysia, phillipine and indonesia, more than 400 species of macroalgae are widely spreaded and mainly consist of sargassum and turbinaria. indonesia has a lot of islands with broad coral rock, allowing huge supply of macroalgae as raw material for alginate and is a potential renewable marine resource in great abundance.12,13 of the locals observation around pamengpeuk shore, banten, indonesia, the macroalgae sargassum are abundant and floated to the beach, at high tide. the locals had not have much information about the role of alginate in the industry, accept its downstream products, such as for food, syrup and gelling. small industries of alginates have produced cosmetics, textile protection, fertilizers, and pharmaceuticals, limited for thickening, stabilizing and emulsifying agent.13,14 sargassum, as one of the many species of macroalgae from indonesia, are currently being researched. characterization of alginates have been based on requirements for quality standards, i.e. water and ash content, ashing temperature, and biochemistry analysis, such as thickening and stabilizing properties, gel-forming, estimation of content, etc.9,14,15 although pharmaceutical uses from sargassum alginates has not been evaluated fully, they have started an early stage of research as scaffold materiasl for tissue engineering scaffolds. however, neither of their characterizations were available. for tissue engineering, alginates scaffolds should be biocompatible to facilitate the process of cell regeneration.16,17 biocompability of alginate scaffold including the biodegradability and pore size for growth of cells was necessary.18 biodegradability is an essential factor as scaffolds were absorbed by the surrounding tissues. alginate is generally accumulated by minerals from the sea.10 besides, there were also fukosantin pigment and polyphenol bound in the plant that make them become dark brownish colour.9 for this, pure alginate is required. next, a high porosity and adequate pore size which can seed and grow cells are necessary; to secure mechanical properties of the scaffold appropriate for the cells.8,18 for this, alginate should be able to gelled and cross-linked through ionic bonding with divalent cations that act as bridges.19,20 essential factor to cross-linked alginate was the presence of carboxyl and carbonyl functional groups;17 besides, the existance of those functional groups are admired as they mimic the part of the protein group in human.2 as an addition, algiante powder should show morphology typical alginate. characterizations of alginate scaffolds for tissue engineering have not been well addressed. to achieve the goal, therefore, characterization of sargassum powder, i.e. morphology of alginate, chemical element and functional groups, should be carried out procede the characterization of alginate scaffold. furthermore, extraction of alginate from the the macroalgae sargassum was needed. extracted and named by a scottish scientist in 1881, alginic acid was realized as a kind of polysaccharide consisting of copolymers d-mannuronic and l-guluronic acids. they were found in cell walls of macroalgae and composed of 3 kinds of polymers: i.e. alginates, cellulose, and complex heteroglycans. alginates were presented mainly as the ca salt of alginic acid, although mg, k and na salts may also be present; they do not dissolve in water.21 this structural integrity of alginate can be broken down with the use of extraction, to allow the direct transformation of the mixed salts (na+, mg+2+, and ca2+) of alginate into 67indrani: a study of extraction and characterization of alginates obtained sodium alginate, in order to obtain alginate that dissolves in water.15,22 the purpose of the present study, therefore, was to extract alginate obtained from the macroalgae sargassum from indonesia and to characterize the morphology, chemical element and functional groups. the information obtainded from the present study can be used partly to consider the use of the alginate extracted from the macroalgae in preparing scaffold of hydroxyapatite and alginate composite for scaffoled material for tissue engineering. materials and methods all chemicals used for alginate extraction process were analyst grade, obtained from merck (usa). two species of the macroalgae, i.e. sargassum duplicatum jg agardh (s. duplicatum) and sargassum crassifolium jg agardh (s. crassifolium), collected in november 2009 from pamengpeuk shore of banten, indonesia, were obtained from the biotechnology laboratory, ministry of marine affairs and fisheries–ri. a commercially available alginate powder of alginic acid sodium salt from brown algae, purchased from sigma (germany), was used routinely for a comparison. macroalgae were collected by cutting the thallus about 40 cm from the top of the plant and were washed with water to remove impurities, such as sand, etc, and were sun-dried for at least three days. all alginate extraction experiment were conducted on fronds of s.duplicatum and s. crassifolium macroalgae. leaves of the plant were cut into pieces, stored in aerated bags and kept in shaded and ventilated site, until they were taken for extraction. alginate extraction using alkaline protocol used in the present study was a laboratory adaptation of the industrial process and was conducted with a serial step, as described earlier.15–23 first of all, pieces of algae leaves were rinsed and immersed in water until they were expanded and then immersed in a 0.3% hcl solution for one hour. for each extraction experiment, 50 g of algae pieces were rinsed with distilled water and soaked in 1 l of a 4% (w/w) na2co3 solution under continuous stirring a least two hours. at the end of the extraction, supernatant were separated from the solution by means of a vibrating screen and then acidified using hcl 10%. the resulting alginic acid, in the form of fiber foam, were rinsed with water on a filter screen. solution of 10% naoh was added to produce sodium alginate (na-alginate). no decoloration procedure was included to the extraction process. extracted na-alginate fibers were recovered from solution by oven drying. finally, dried na-alginate fibers were milled to obtain smooth powders. samples were powders of the na-alginate, extracted from the macroalgae s. duplicatum (s. duplicatum alginate) and from the macroalgae s. crassifolium (s. crassifolium alginate), and, the commercially available alginate. all samples were then characterized. scanning electron microscopy (sem) was conducted to study the morphology of the alginate samples. alginate powder samples were mounted on a sample holder. the convert into electrically conductive samples, the samples were coated with an ultrathin gold coating deposited on the surface of the samples by low-vacuum ion sputter apparatus. coated samples were moved to scanning electron microscope (zeus, germany) and were then scanned and convert to an image through a monitor to visualize particle shapes. x-ray fluoroscence (xrf) spectroscopy was used to quantify chemical elements of the alginate samples. each sample was prepared in a plastic ring mold (2.5 cm in diameter and 0.5 mm in thickness) and was compressed under a hydraulic press. the na-alginate powder together with the mold was then loaded in xrf spectrometer (jeol jsx-3211, japan). the samples were scanned by xrd spectrometer and x-ray beam would interact with the samples. data collected from the measurements were recorded using a software programme in the xrf spectrometer. measurements were conducted three times. fouirer transform infra-red (ftir) spectroscopy was utilized to confirm the existance of functional groups in the alginate samples. a total of 5% (w/w) sample was mixed with dry potassium bromide (kbr), with respect to the pellet technique. the mixture was ground into a fine powder using an agate mortar and was compressed into a disc under a hydraulic press. each sample was then mounted in and scanned by ftir spectroscopy (spectrumone, perkin elmer, japan) at 4 mm/s over a wave number region of 4000-450cm-1. ftir transmission spectra obtained from the measurement were recorded using a software programme for ftir. measurements were conducted three times in the alginate samples. results alkali extraction used in the extraction of the macroalgae s. duplicatum and s. crassifolium has resulted solid powder of alginates (figure 1). s. duplicatum and s. crassifolium alginates were pale yellow and dark brown, respectively. in contrast, and the commercially available alginate were in white. results from the characterizations showed comparison of s. duplicatum, s. crassifolium and the commercially available alginates in the morphology, chemical elemental and functional groups. morphology of s. duplicatum, s. crassifolium and the commercially available alginates revealed from sem were presented in figure 2. s. duplicatum and s. crassifolium alginates displayed shapes of irregular particles in varied size. some were long, whereas, others were round. some of which form the helical structure extending up to 200 μm and 20 μm in diameter. the variation in morphology and dimension coincided with those seen in the commercially available alginate. 68 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 65–70 quantitative chemical elements detected in s. duplicatum, s. crassifolium and the commercially available alginates yielded by xrf spectroscopy were listed in table 1. it can be seen from table 1 that s and fe elements were seen in low amount in all alginate powders. surprisingly, the quantitative of si (16%) in s. duplicatum alginate and cl (30%) in s. crassifolium alginate were considerable. all chemical elemental were not detected in the commercially available alginate. as expected, the major chemical element components were na and then ca. ftir spectra of the s. duplicatum, s.c rassifolium and the commercially available alginates in the band range of 4000–450 cm-1 were illustrated in figure 3. a broad envelop occured between the band of 3500-3000 cm-1 was assigned to stretching vibration of oh (hydroxyl) group. it is obvious that the presence of ohions around 3000 cm-1 correspond to adsorbed h2o, derived from moist of the samples. in the fingerprint region, the band around 1626-1623 cm-1 were correspond to asymmetrical and symmetrical stretching modes of coo (carboxyl) group. another coo with similar modes also appeared at the band at 1421 cm-1. other charateristic peaks around the band at 1027 cm-1 derived from c-o-c (ether) group, as aliphatic alcohol with c-o (carbonyl) substituent. the functional groups obtained from both extracted alginate were in agreement with those appeared in the commercially available alginate. as an addition, the extraction has resulted na-alginate as ini figure 4. a possible mechanism of the addition of naoh into alginic acid producing na-alginate was because of the weak bond between h+ and coowas broken causing na+ ions, possibly, forming coo-na. (a) (b) (c) figure 1. alginate powders from the macroalgae a) s. duplicatum, b) s. crassifolium and the c) commercially available alginate. (a) (b) (c) figure 2. sem micrographs of the na-alginates obtained from the macroalgae a) s. duplicatum, b) s. crassifolium and c) commercially available. figure 3. ftir spectra of na-alginates obtained from the macroalgae a) s. duplicatum, b) s.crassifolium and c) available commercially. figure 4. schematic diagram of the incorporation of naoh into (a) alginic acid, consisting the m and g groups resulting the (b) na-alginate.9 table 1. chemical elements contained in the macroalgae a) s. duplicatum, b) s. crassifolium and c) available commercially source of alginate chemical elemental wt (%) s.duplicatum na ca s fe si 50,5 12,1 3,5 6,9 16,4 s.crassifolium na ca s fe cl 49,3 10,2 2,6 3,2 30,8 commercially available na ca s fe 73,2 6,7 8,2 5,0 69indrani: a study of extraction and characterization of alginates obtained discussion macroalgae sargassum were used in the present study because they grew abundant in areas near the shore; the collection was far easier compared with other macroalgae, and was routinely accomplished by the ministry of marine affairs and fisheries–ri. the sargassum were identified as s.duplicatum and s. crassifolium by the research centre of oceanography-indonesian institute of sciences. the alkaline extraction procedure used in the present study was possible to to produce s.duplicatum and s. crassifolium alginates. as mentioned previously, that alginic acid in macroalgae were presented mainly as the ca-alginat, a salt of alginic acid which were insoluble in water. the extraction process has converted the insoluble ca salts into s.duplicatum and s. crassifolium alginates which were soluble in water. alkali treatment was necessary for an ion exchange process. the process was carried out by 2 steps; the first was a more efficient extraction by treating the seaweed with dilute mineral acid, as in reaction (1): pre-extraction: ca(alg)2 + 2h+  2halg + ca2+................................ (1) according to several authors,15, 24, 25 when ca-alginate was converted to alginic acid, it was more readily to be extracted with alkali than the original calcium alginate. reaction (1) was continued by extraction using alkali as shown in reaction (2). extraction: 2halg + na+  naalg + ca2+...................................... (2) no coloring agent was given in the extraction process. both s. duplicatum and s. crassifolium alginates showed a distinctive brownish (figure 1). the extremely dark brown colour demonstrated in s. crassifolium alginate powder was probably a reflection of fucosantin pigment, that often contained in certain algae, and probably covered other pigments. white color reflected from the commercially available alginate was ascertained as a result of color treatment at the time of the extraction process; this was always did as an effort to make alginate interesting. during alginate extraction, following the acid pre-treatment, a brown discoloration develops and this carries through the rest of the process resulting in a dark sodium alginate powder.10 previously, it was demonstrated that phenolic compounds are responsible for the discoloration. x-ray fluoroscence results showed chemical elemental contained in s. duplicatum and s. crassifolium alginates (table 1). observing reaction (1), the incorporation of na+ ions from naoh into ca2+ ions from ca-alginate had made them rich with both na+ and ca2+ ions. therefore, the quantitative chemical elemental of ca and na yielded by xrf was high. as na and ca elements were also in a considerable amount found in the commercially available alginate, it implied that the material experienced similar method of alginate extraction and naoh was used in the extraction process. chemical elemental occcured in the sample alginates were impurities. the minor amount of fe and s elements in s. duplicatum and s. crassifolium alginates, as well as, in the commercially available alginate (table 1) were impurities which likely to occure from the xrf instrument at the time of measurement. this possibilitiy was supported by the occurance fe and s elements in the commercialy available alginates, as well. whereas, the existance of the considerable amount of si and cl in s. duplicatum and s. crassifolium alginates, respectively, may due to mineral contaminations that originated from sea environment. it was presumed that they accumulated inside the macroalgae when producing m and g acids. this idea were supported by the absent of either si or cl in the commercialy available alginates. a purification process, actually, may omit impurities in s. duplicatum and s. crassifolium alginates. nevertheless, impurities in alginate were presumed not to cause negative reaction during the overall biocompatibility of the material to human. in fact, bone mineral was known to compose of hydroxyapatite with substitutes, such as ca na, k, cl, fe, f, etc, originated from the welknown biomineralization in human.24 ftir spectra showed the occurance of carboxyl and carbonyl groups in s.duplicatum and s.crassifolium alginates implied the typical alginate (figure 3). these functional groups showed the similarity to human protein, as in most organisms, polysaccharides and amino acids contained carboxylic acid (-cooh). the presence of carboxyl and carbonyl groups in s.duplicatum and s. crassifolium alginates coincided with those revield in the commercial available alginate, as well as, in a commercial available alginate analyzed studied.17 the present study was an initial report describing both s. duplictum and s. crassifolium alginates powders, obtained from macroalgae of banten, indonesia. the use of sem, xrf and ftir have helped in identifying and verifying the material to describe the characteristic of alginate powder. the morphology of alginate, the chemical elemental, as well as the functional groups contained in the material were found similar with those of the commercially available alginate. the commercially available alginate use in the present study has been used as scaffold material for research. it was a great expectation of both s. duplictum and s. crassifolium alginates to be used as alginate for scaffold material. further studies, therefore, need to characterized both s. duplictum and s. crassifolium alginates to explore the posibility of the material to be used as material for scaffolds, procede to further research in application of alginate scaffolds for tissue engineering applications. it is concluded that extraction obtained from the macroalgae s. duplicatum and s. crassifolium showed the typical alginate; the morphology, chemical element and functional groups were in agreement with those of the commercially available alginate. 70 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 65–70 acknowledgement the financial support rendered by the ministry of education through the universitas indonesia is greatly acknowledged. references 1. vacanti ca, bonnassar lj, vacanti jp. structural tissue engineering. in: lanza rp, langer r, vacanti jp, editors. principles of tissue engineering. san diego: academic press; 2000. p. 671–82. 2. james bt, david j. timson§, richard j. reece, hazel m. holden. molecular structure of human galactokinase. the journal of biological chemistry 2005; 280(10): 9662–70. 3. nicola h. an alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture to expedite wound healing. thesis. uk: bioengineering, school of chemical engineering, university of birmingham; 2009. p. 101–4. 4. garagorri n, fermaniana s, thibault r, ambrose wm, scheinb od, chakravarti s, elisseeffa. keratocyte behavior in three-dimensional photopolymerizable poly (ethylene glycol) hydrogels. acta biomater 2008; 4(5): 1139–47. 5. majima t, funakosi t, iwasaki n, yamane s, harada k. alginate and chitosan polyion complex hybrid fibers for scaffolds in ligament and tendon tissue engineering. j orthop sci 2005; 10(3): 302–7. 6. li z, zhang m. chitosan-alginate as scaffolding material for cartilage tissue engineering. j biomed mater res a 2005; 75(2): 485–93. 7. turco g, marsich e, bellomo f, semeraro s, donati i, brun f, grandolfo m, accardo a, paoletti s. alginate/hydroxyapatite biocomposite for bone ingrowth: a trabecular structure with high and isotropic connectivity. biomacromolecules 2009; 10(6): 1575–83. 8. bernhardt a, despang, lode a, demmler a, hanke t, gelinsky m. proliferation and osteogenic differentiation of human bone marrow stromal cells on alginat–gelatine– hydroxyapatit scaffolds with anisotropic pore struture. j tissue engineering regen med 2009; 3(1): 54–62. 9. draget kl, smidsroed o, skjak-braek g. alginates from algae. in: steinbuchel a, rhoe sk. polysaccharides, polyamides, in the food industry, properties, production and patents. kgoa weinbeim: wiley-vch verslag gmbh & co; 2006. p. 30. 10. mchugh dj. a guide to the seaweed industry. fao fisheries technical paper. 2003; 441: 105. 11. ashley m, mccullagh a, sweet c. making a good impression: a ‘how to’ paper on dental alginate. dent update 2005; 32(3): 169–70. 12. yulianto k, setiapermana f, pettipeilohy, mansur a. pengembangan pengolahan rumput laut. laporan triwulan ii. pusat penelitian oseanografi – lipi; 2002. p. 9–12. 13. sulistijo. penelitian rumput laut (algae makro/seaweed) di indonesia. pidato pengukuhan ahli peneliti utama bidang marikultur. jakarta: pusat penelitian oseanografi – lipi; 2006. p. 18–21. 14. zatnika a. proses ekstraksi dan manfaat alginat di bidang farmasi. jurnal sains dan teknologi indonesia 2003; 5: 143–50. 15. pa r t h iba n c , pa r a m e swa r i k , sa r a nya c , hem a la t h a a , anantharaman p. production of sodium alginate from selected seaweeds and their physiochemical and biochemical properties. asian pacific j tropical biomedicine 2012; 1(4): 1–4. 16. andersen t, strand bl, formo k, alsberg e, christensen be. alginates as biomaterials in tissue engineering. carbohydr chem 2012; 37: 227–58. 17. mohan n, nair pd. novel porous, polysaccharide scaffolds for tissue engineering applications. trends biomater artif organs 2005; 18: 219–24. 18. khalil s, sun w. bioprinting endothelial cells with alginate for 3d tissue constructs. j biomech eng 2009; 131(11): 111002. 19. kakita h, kamishima h. some properties of alginate gels derived from algal sodium alginates. j applied phycology 2008; 20(5): 93–9. 20. kuo c, ma px. ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. structure, gelation rate and mechanical properties. biomaterials 2001; 22(6): 511–21. 21. stanford ecc. new substance obtained from some of the comonomer species of marine algae. algin chem news 1881; 47: 254–7. 22. yulianto k. pengaruh konsentrasi naoh terhadap viskositas natrium alginat yang diekstraksi dari sargassum duplicatum jg agardh (phaeophyta). oseanologi dan limnologi di indonesia 2007; 33: 295–306. 23. hernandez cg, mchugh dj, arvizu-higuera dl, rodriguezmontesinos ye. pilot plant scale extraction of alginate from macrocystis pyrifera. conversion of alginic acid to sodium alginate. drying and milling. j appl phycol 2002; 14: 445–51. 24. weiner s, wolfie t, wagner hd. lamellar bone: structure-function relations. j structural biology 1999; 126: 241-55. 192 vol. 44. no. 4 december 2011 the effect of spirulina gel on fibroblast cell number after wound healing fitria rahmitasari1, wisnu setyari j2, and ester arijani rachmat2 1 dental student 2 department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: wound healing treatment after tooth extraction should be an important consideration due to mouth discomfort and pain. spirulina (blue green algae) consists of c-phycocyanin, b–carotenoids, vitamin e, zinc, some other trace elements and natural phytochemical which are believed to act as antioxidant and takes part in wound healing process. purpose: the purpose of this study was to examine the effect of spirulina gel on fibroblast cell number after wound healing process. methods: twenty eight males guinea pig are devided into four group, 7 guinea pig each. they are control group and treatment group which is given 0%, 3%, 6%, and 12% spirulina gel. after tooth extraction, histopathological evaluation was done to count fibroblast cell. the data was analyzed by one-way anova and tukey hs�. results: the research has proven the relation between the increased growth of fibroblast cell and spirulina gel application. the higher the doses, the more cell growth. hence, there has been significant different (p < 0.05) among groups. conclusion: spirulina gel increases the number of fibroblast in wound after tooth extraction and 12% spirulina gel has the most potential ability. key words: spirulina gel, fibroblast cell number, wound healing abstrak latar belakang: proses penyembuhan luka pasca pencabutan gigi merupakan salah satu hal yang penting karena akan menimbulkan rasa nyeri dan tidak nyaman dalam rongga mulut. spirulina (blue green algae) mengandung c-phycocyanin, b-carotenoids, vitamin e, seng, beberapa trace elemen lainnya, dan phytochemical alami yang terbukti dapat berperan sebagai antioksidan dalam proses penyembuhan luka. tujuan: tujuan dari penelitian ini adalah untuk mengetahui efek pemberian gel spirulina terhadap jumlah sel fibroblas pada proses penyembuhan luka pasca pencabutan gigi. metode: �ua puluh delapan ekor guinea pig jantan dibagi dalam 7 kelompok, masing-masing terdiri dari 4 ekor. kelompok tersebut adalah kelompok kontrol dan kelompok perlakuan yang diberikan gel spirulina dengan konsentrasi 0%, 3%, 6%, dan 12%. setelah pencabutan gigi, dilakukan penghitungan sel fibroblas dengan metode histopatologi. hasil: penelitian ini menunjukkan adanya hubungan antara pemberian gel spirulina terhadap peningkatan jumlah sel fibroblas. semakin tinggi dosis gel spirulina akan semakin meningkatkan jumlah sel fibroblas pula. �idapatkan perbedaan yang signifikan antar kelompok (p < 0,05). kesimpulan: gel spirulina meningkatkan jumlah sel fibroblas pada luka bekas pencabutan dan gel spirulina dengan konsentrasi 12% mempunyai kemampuan yang paling potensial. kata kunci: gel spirulina, jumlah sel fibroblas, penyembuhan luka correspondence: wisnu setyari j, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjen prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: wizn_zetya@yahoo.com research report 193rahmitasari, et al.,: the effect of spirulina gel on fibroblast cell number introduction tooth extraction is often done in dentistry. the common reason for tooth extraction is caries teeth with no viable option for treatment, since the decayed teeth can become the source of infection. the other reason is tooth impaction which often cause oral disturbance. the wound healing process post tooth extraction is one of the problems that clinicians need to concern because the impact is pain and inconvenience inside the mouth cavity. it can cause the disturbance in speech and chewing function, until the healing is completed. there are 11 million people per day suffering pain, inflammation and bruise after having a third molar tooth surgery.1 the wound healing process starts a moment after the lesion happens. this process is a complex and systematic series which involves blood cell, tissue, cytokines and growth factor activities.2 if there is any disturbance in one of these phase, the wound healing process cannot complete optimally or potentially creates new problems such as, bleeding, inflammation, dry socket or infection caused by the microorganism inside the mouth cavity.1 recently, the progress of pharmacy-health technology in the world is focused on the natural ingredient because it is safer to use compares to the drug which contains chemical ingredient. spirulina contains a lot of nutrition which are useful for the body, such as c-phycocyanin, b-carotenoids, vitamin e, zinc, traced elements and other natural phytochemical. one of the nature ingredients that has been researched and proven to have the capability as an anti-inflammation and anti-oxidant during the wound healing process is c-phycocyanin or blue cell substance.3 spirulina is included in blue green algae group because it contains 14–20% c-phycocyanin. spirulina is a cyanobacteria class which has the highest blue pigment (c-phycocyanin) compares to other microalgae.4 spirulina is rich in mineral and other important substance which are needed by the body, in addition, the usage of spirulina supplement has spread in the society and has many trademark product, such as powder, tablet and capsule.5 laboratory research has been done previously by using the extract concentration spirulina of 3%, 7% and 10% towards the speed of wound healing of rat skin.6 those three concentrations show the real result in accelerating the wound healing compares to the untreated sample. the purpose of this study was to examine the effect of spirulina gel on fibroblast cell number after wound healing process. the amount of fibroblast cell which is viewed histologically becomes the researched variable because the cell has a role for producing collagen fibers needed for accelerating the wound regeneration. materials and methods the type of researches done is laboratory experimental. the sample used 28 male guinea pigs (cavia cabaya) with weight of 200–300 gram. those samples were divided into four groups which had 7 guinea pigs each. they were the controlled group and treatment group which was given 0%. 3%, 6% and 12% spirulina gel. the tooth extraction was done on the mandibular left incisor using the modification of needle holder under 10% ether anesthesia inhaled. spirulina gel was then applied on the extracted socket of each treatment group. after day five, the mandibular extraction socket was done to the controlled group and treatment group under anesthesia. post extraction was taken for histological examination by using haematoxylin eosin (he) painting. the calculation of fibroblast cell was done by taking the photograph from microscope view after 450 times enlargement.7 the photo was then given square lines which by then the fibroblast cell around the outer box were counted (figure 1). this research data was analysed by one-way anova statistical test and continued with tukey hsd.8,9 figure 1. the calculation of fibroblast cell method (arrow sign) on the surrounding of the outer box. results the amount of the highest fibroblast cell is in the sample that is given 12% spirulina gel, meanwhile, the lowest fibroblast cell is in the controlled group. table 1. mean and standard deviation of fibroblast cell amount on controlled and treatment group group n mean standard deviaton controlled 7 14.00* 1.633 3% concentration 7 18.57* 2.440 6% concentration 7 35.57* 2.149 12% concentration 7 39.14* 1.676 note: *: significant difference before one-way anova test is done, kolmogorovsmirnov statistical test was done and shown that the data is distributed normally and levene test shows that the data 194 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 192–195 was homogeneous. based one-way anova test, there was significant value of 0.001 (p < 0.05). therefore the average of fibroblast cell on each group was significantly increased. table post hoc test from tukey hsd method showed that there were significant differences on each group comparation. discussion guinea pig is chosen as the test animal because it is easy to handle and the socket from the post-extraction has enough width for spirulina gel application. the choice to extract mandibular left incisor is based on the structure and tooth anatomy pattern of guinea pig. male guinea pig is chosen because it is not affected by hormonal system, therefore, its body is more stable compares to the female guinea pig. gel is a semisolid system which contains of suspension made from small inorganic particle or big organic molecule and penetrated by a liquid. the gel is chosen because it is semi solid, soft and elastic so it is easier to apply in the post-extraction socket and can sustain longer in the socket, therefore, it helps the wound healing process. spirulina gel in this research was made by using cmc na 3% material as the thickening and stabilizing material. cmc na is easier to use and it is not influence the function of the condensed substance so it is not affect the result of the research.10 the post extraction socket on the controlled group was also applied with gel with 0% concentration to standardize the physical condition of the animal between the controlled and treatment group. 3%, 6% and 12% spirulina were applied on the wound of post tooth extraction on the guinea pig. this study was done similarly with the previous laboratory research which used 3%, 7%, and 37% concentration spirulina extract on the wound healing of rat skin in which those three concentrations showed the acceleration the of wound healing rather than the controlled goup. this test animal is killed on the fifth day because the fibroblast cell is filling the wound area in day 5 to 7.11 generally, the result of the research showed that the average amount of fibroblast cell on the treatment group was increased along with increasing level of concentration of spirulina gel (table 1). the lowest average amount of fibroblast cell was in the controlled group, while the highest amount was in the test animal which is given 12% spirulina gel concentration (figure 2). there were significant differences (p < 0.05) on the treatment groups which were given three different concentrations (3%, 6% and 12%) if compared to the controlled group. this condition happened because spirulina gel had a few useful contents such as: c-phycocyanin, b-carotenoids, vitamin e (tocopherols) and zinc to accelerate the wound healing.11 c-phycocyanin in the spirulina gel is a binding protein in the form of blue pigment which can be used to accelerate wound healing process and anti-oxindant.3 the previous research showed that the application of c-phycocyanin topically can accelerate the wound healing on the skin.12 c-phycocyanin, b-carotenoids, vitamin e (tocopherols) and zinc have the role as anti-oxidant. the inflammation process is marked by implication of multiple inflammation cell such as eusinophil, neutrophil and macrophag which able to produce reactive oxygen species (ros) which can figure 2. the features of fibroblast cell on each group. a) controlled group, b) 3% concentration group, c) 6% concentration group, d) 12% concentration group. a b c d 195rahmitasari, et al.,: the effect of spirulina gel on fibroblast cell number delay the wound healing process.13 the ros then will be neutralized by the nutrient content, so it has the role to support tissue regeneration such as granulation tissue formation which contains of chronic inflammation cell such as macrophag, limphosit and plasma cell; proliferation of capillary blood vessels and fibroblast and the formation of connective tissue, basalic membrane and matrix between cells and also accelerating wound healing. c-phycocyanin can reduce the functional metabolism activity of neutrophil which cause the slow movement activity. c-phycocyanin can inactivate ros produced from neutrophil as the mediator of inflammation process.14 healing cascades start when wound happens. the whole process needs an interaction from various cell, including fibroblast. proliferation and fibroblast migration is important during the healing process.15 the application of spirulina gel on this research is capable to increase the proliferation and fibroblast cell migration. proliferation and fibroblast migration is driven significantly by cpycocyaninof a protein algae. fibroblas proliferation happens through cyclin-dependent kinase pathway (cdk1 and cdk2), meanwhile the fibroblast migration happens through upa pathway (urokinase-type plasminogen activator) and then upa drives fibroblast migration through kemokin pathway (mdc, rantes, eotaxins, ena-78) and rho-gtpase protein (cdc 42 and rac 1).12,16 there were significant differences on the treatment group which was given 3% spirulina gel concentration and 6% and 12% concentration. this happened because c-phycocyanin concentration in each preparation was the same with spirulina concentration, therefore, the higher spirulina gel concentration, the increasing amount of fibroblast cell will be higher during the wound healing process. there were also significant differences between the group which was given 6% spirulina gel concentration and 12% concentration. it showed that 12% concentration was the most effective concentration to increase the amount of fibroblast cell on the post extraction wound. spirulina which contains c-phycocyanin has low toxicity. on the previous research, the highest concentration of c-phycocyanin (3 grams/kg per oral) was applied on the test animal and it was monitored for 14 days. the result of the research showed that there was no behavioural changes and body weight differences between the treated and the non-treated test animal. the histopathology check is not shown any organ or tissue damages.3 so, the topical application of spirulina gel on the post tooth extraction wound is safe without creating side effect. in conclusion, spirulina gel increase the number of fibroblast and the application of 12% spirulina gel has the potential capability to increase the amount of fibroblast cell on the post extraction wound. references 1. friedman jw. the prophylactic extraction of third molars: a public health hazard. am j public health 2007; 97: 1554–9. available at: http://www.ajph.org/cgi/content /full/97/9/1554. accessed december 30, 2011. 2. mackay d, miller al. nutritional support for wound healing. 2003. available at: www.highwire.standford.edu. accessed may 2, 2011. 3. romay ch, gonzalez r, ledon n, remirez d, rimbau v. cphycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. current protein and peptide science 2003; 4: 207–16. 4. ismet. mikroalga. 2009. available at: http://ismail-jeunib.blogspot. com/2009/11/ mikroalga.html. accessed february 27, 2011. 5. suhaya d. spirulina, “superfood” berprotein tinggi. 2008. available at: http://dedesuhaya.blogspot.com/2008/07/spirulina-superfoodberprotein-tinggi.html. accessed february 27, 2011. 6. panigrahi bb, panda pk, patro vj. wound healing activity of spirulina extracts. int j of pharmaceutical sciences review and research 2011; 6(2): 132–5. 7. kunarti s. stimulasi aktivitas fibroblas pulpa dengan pemberian tgf-ß1 sebagai bahan perawatan direct pulp capping. disertation. surabaya: program pascasarjana universitas airlangga; 2005. 8. lemeshow s, hosmer dw, klar j. adequancy of sample size in health. couries international ltd. 1990. p. 9–11. 9. ghozali i. aplikasi analisis multivariate dengan program spss. semarang: badan penebit universitas diponegoro; 2009. p. 59–70. 10. khoswanto c. the effect of mengkudu gel (orinda citrifolia linn.) in accelerating the ecalation of fibroblast post extraction. maj ked gigi (dent j) 2010; 43(1): 31–4. 11. madhyastha h, nakajima r, omura y, maruyama m. regulation of growth factors associated cell migration by c-phycocyanin scaffold in dermal wound healing. clin exp pharmacol physiol 2011; 39(1): 13–9. 12. maruyama m, madhyastha hk, radha ks, nakajima y, omura s. upa dependent and independent mechanisms of wound healing by c-phycocyanin. j cell mol med 2008; 12(6b): 2691–703. 13. nagata m. inflammatory cells and oxygen radicals. curr drug targets inflamm allergy. 2005; 4(4): 503–4. 14. dartsch pc. antioxidant potential of selected spirulina platensis preparations. germany: wiley inter science; 2008. p. 627–33. 15. tomasek jj, gabbiani g, hinz b, chaponnier c, brown ra. myofibroblasts and mechano-regulation of connective tissue remodelling. nature reviews. molecular cell biology 2002; 3: 349–63. 16. malumbres m, barbacid m. to cycle or not to cycle: a critical decision in cancer. nat rev cancer 2001; 1: 222–31. 14 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 14–18 differences in tensile adhesion strength between hema and nonhema-based dentin bonding applied on superficial and deep dentin surfaces eresha melati kusuma wurdani, adioro soetojo, and devi eka juniarti department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: improvement in dentistry shows some progresses, due to patients awareness on the importance of dental care. cervical lesion is the most common phenomenon which oftenly found 46.36% in man and 38.13% in woman. cervical lesions need composite restoration for treatment to stop the process of tissue damage. the process of adhesion of composite restoration material to the structure of the tooth is not easily separated and it needs optimal function in the oral cavity. application of dentin bonding agents to attach the composite is needed. selection of hema-based bonding material and hema free-based bonding material which have a different solvent in their composition, as applied to the dentin superficial and deep dentin, affect the results of debonding test. debonding test is done to measure the adhesion strength of a bonding material. purpose: the purpose of this study was to analyze differences in tensile bond strength of dentine bonding hema-based and hema-free based after application in superficial and deep dentine surfaces. method: the tooth of the bovine was as samples. a superficial dentine sample was taken from 0.5-1 mm of dentino enamel junction and a deep dentine sample was taken from 0.5 mm culmination of pulp horn. dentine surface area was equal to p x r2 = (3.14 x 22) = 12.56 mm2. six samples of hema-based bonding was applied to the dentine superficial. six samples of hemafree based bonding was applied to the superficial dentine. six samples of hema-based bonding was applied to the deep dentine. six samples of hema-free based bonding was applied to the deep dentine. tensile strength was measured using an autograph ag-10te. result: there were differences tensile bond strength of dentine bonding hema-based and hema-free based after the application on superficial (p=0.000) and deep dentine surfaces (p=0.000). conclusion: there were differences tensile bond strength of dentine bonding hema-based and hema-free based after the application on superficial and deep dentine surfaces. the use of dentine bonding materials hema-free based were better than hema-based after application on different dentine depths. keywords: hema-based; hema-free based; superficial dentine; deep dentin correspondence: eresha melati kusuma wurdani, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: eresha.melati@yahoo.com introduction development in the field of dentistry is growing in line with real demand of patients for dental care. recently, cervical lesions with open cementum and dentin areas that can make teeth experience a sensitive to changes in temperature have commonly been found. the cervical lesions can be caused by toothbrush abrasion. today, the number of erosion on dentin area increases. a previous research showed that the prevalence of cervical lesions in males was 46.36%, while in females it was 38.13%.1 dental fillings applied on the dentin area have been known to have an ability to stop the process of tissue damage, but the attachment process of filling materials to the tooth structure is complex, especially the attachment to the dentine.2 this condition is caused by differences in the density of dentinal tubules and the level of water content in each dentin depth.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p14-18 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p14-18 1515wurdani, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 14–18 the lowest level of water is found in supeficial dentin, while the highest level is found in deep dentin. in the superficial dentin, there are fewer tubules. as a result, when dentin bonding penetrates into intertubular dentin, responsible for providing adhesion strength between dentin and dental restorative materials, the adhesion strength will become lower. in the deep dentin, there are numerous dentin tubules. therefore, the permeability of intratubular resin will have higher adhesion strength.4 dentin is a tissue that is always wet because of the presence of fluid in the dentin tubules. thus, composite resins with hydrophobic properties cannot be attached to the dentin easily. consequently, a bonding material for dentin and composites is required.2 the application of adhesive resin material aims to form the bond between resin composites and tooth structure, derived from bonding system using adhesion technique.5,6 dentin bonding is composed of 2-hydroxyethyl methacrylate (hema)-based material and non-hemabased material. however, the hema-based dentin bonding material is mostly often used because it has several advantages, namely relatively easy to manufacture, surviving long enough because of the addition of a preservative, and serving as hydrophobic and hydrophilic groups.5,7 on the other hand, the non-hema-based dentin bonding generally has monomer, called as urethane dimethacrylate (udma). udma can form a congested cross-link polymer, resulting in increased mechanical strength. each dentin bonding material also has different solvent composition. the composition greatly affects the adhesion strength of dentin bonding to the collagen fibrils.8 therefore, this research aimed to analyze whether there are differences in tensile adhesion strength between hema-based dentin bonding and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin. material and method this research was a laboratory experimental research with a one-way anova design. samples used in this research were teeth (bovine) obtained from a slaughterhouse in pegirian, surabaya. those bovines selected were healthy and plump. after slaughtered, their good incisors were removed and cleaned with a brush and a sharp scalpel under running water. soft tissue that still attached was disposed carefully. those prepared teeth then were soaked in saline and stored in a refrigerator at a temperature 4o c.5 afterwards, superficial dentin located 0.5-1 mm from dentino enamel junction was taken (figure 1). meanwhile, deep dentine located 0.5 mm from the top of pulp horn was taken. dentin surface area used in this research was equal to p x r2 = (3.14 x 22) = 12.56 mm2 (figure 2). samples were divided into four groups. group i was consisted of six samples using hema-based (ivoclar vivadent, inc., united states and canada) dentin bonding applied on the surface of the superficial dentin. group ii was consisted of six samples using non-hema-based (g-ænial bond tm inc., gc europe) dentin bonding applied on the surface of the superficial dentin. group iii was consisted of six samples using hema-based dentin bonding applied on the surface of the deep dentin. group iv was consisted of six samples using non-hema-based dentin bonding applied on the surface of the deep dentin. all of the superficial and deep dentin taken was smeared with 37% phosphoric acid gel (ivoclar vivadent, inc., united states and canada) for 10 seconds. the dentin was washed with water for ± 10 seconds, and then dried by cotton to remove excessive water. two drops (0.02 grams) of the hema-based dentin bonding material were dropped on a disposable brush (according to the manufacturer’s instructions), then smeared on the dentin (twice), and settled for 20 seconds. the dentin then was sprayed with an air spray of chip blower for 5 seconds to remove excessive solvent (solvent contained in the primer solution, namely water), and then curing was carried out for 10 seconds (according to the manufacturer’s instructions). cylinder cast was inserted into lower plunger and then fixed by installing a lock. cylinder cast on the upper plunger then 3 material and method this research was a laboratory experimental research with a one-way anova design. samples used in this research were teeth (bovine) obtained from a slaughterhouse in pegirian, surabaya. those bovines selected were healthy and plump. after slaughtered, their good incisors were removed and cleaned with a brush and a sharp scalpel under running water. next, soft tissue still attached was disposed carefully. those prepared teeth then were soaked in saline and stored in a refrigerator at a temperature 4o c.5 afterwards, superficial dentin located 0.5-1 mm from dentino enamel junction was taken. meanwhile, deep dentine located 0.5 mm from the top of pulp horn was taken. dentin surface area used in this research was equal to p x r2 = (3.14 x 22) = 12.56 mm2. figure 1. illustration of the superficial dentin located 0.5-1 mm from dentino enamel junction (a sign black in the image refers to the superficial dentin). next, samples were divided into four groups. group i was consisted of six samples using hema-based (ivoclar vivadent, inc., united states and canada) dentin bonding applied on the surface of the superficial dentin. group ii was consisted of six samples using non-hema-based (gænial bond tm inc., gc europe) dentin bonding applied on the surface of the superficial dentin. group iii was consisted of six samples using hema-based dentin bonding applied on the surface of the deep dentin. and, group iv was consisted of six samples using non-hema-based dentin bonding applied on the surface of the deep dentin. figure 2. illustration of the deep dentin located 0.5 mm from the top of pulp horn (a sign black in the image refers to the deep dentin). figure 1. illustration of the superficial dentin located 0.5-1 mm from dentino enamel junction (a sign black in the image refers to the superficial dentin). 3 material and method this research was a laboratory experimental research with a one-way anova design. samples used in this research were teeth (bovine) obtained from a slaughterhouse in pegirian, surabaya. those bovines selected were healthy and plump. after slaughtered, their good incisors were removed and cleaned with a brush and a sharp scalpel under running water. next, soft tissue still attached was disposed carefully. those prepared teeth then were soaked in saline and stored in a refrigerator at a temperature 4o c.5 afterwards, superficial dentin located 0.5-1 mm from dentino enamel junction was taken. meanwhile, deep dentine located 0.5 mm from the top of pulp horn was taken. dentin surface area used in this research was equal to p x r2 = (3.14 x 22) = 12.56 mm2. figure 1. illustration of the superficial dentin located 0.5-1 mm from dentino enamel junction (a sign black in the image refers to the superficial dentin). next, samples were divided into four groups. group i was consisted of six samples using hema-based (ivoclar vivadent, inc., united states and canada) dentin bonding applied on the surface of the superficial dentin. group ii was consisted of six samples using non-hema-based (gænial bond tm inc., gc europe) dentin bonding applied on the surface of the superficial dentin. group iii was consisted of six samples using hema-based dentin bonding applied on the surface of the deep dentin. and, group iv was consisted of six samples using non-hema-based dentin bonding applied on the surface of the deep dentin. figure 2. illustration of the deep dentin located 0.5 mm from the top of pulp horn (a sign black in the image refers to the deep dentin). figure 2. illustration of the deep dentin located 0.5 mm from the top of pulp horn (a sign black in the image refers to the deep dentin). superficial dentin deep dentin top of pulp horn dentino enamel junction dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p14-18 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p14-18 16 wurdani, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 14–18 was filled with composite, assembled with the lower plunger, and fixed by installing a lock. irradiation was performed using a light cure for 20 seconds on both sides of the plunger, 10 seconds for each. it would harden within 2 minutes. tensile strength test then was performed using a te autograph (ag-10 shimadzu, japan). in all cases, the results were considered statistically significant with a p value <0.05 results visual illustration of the mean tensile adhesion strength of hema and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin can be seen in figure 3: results of the normality test using the kolmogorovsmirnov test showed a significance value (p) of >0.05. the data obtained were normally distributed. moreover, results of the homogeneity test using the levene test showed a significance value (p) of 0.343 (significance (p) value is (>0.05). therefore, it was indicated that the data obtained were homogeneous. furthermore, results of the difference test using the one-way anova test showed a significance value (p) of 0.000, it means that there were significant differences in tensile adhesion strength between hema-based dentin bonding and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin. consequently, the use of non-hema-based dentin bonding was better than the use of hema-based dentin bonding applied on different dentin depths (table 1). discussion the tensile strength obtained in this research was derived from the adhesion strength between hema based bonding resins and non-hema based bonding resins applied on the surfaces of the superficial and deep dentin. based on figure 1, it can be seen that the first tensile adhesion strength was found in non-hema-based dentin bonding applied on the surface of the deep dentin. the second adhesion strength was found in hema-based dentin bonding applied on the surface of the deep dentin. the third tensile adhesion strength was found in non-hema-based dentin bonding applied on the surface of the superficial dentin. and, the fourth tensile adhesion strength was found in hema-based dentin bonding applied on the surface of the superficial dentin. it can be said that the use of non-hema-based dentin bonding on different dentin depths was better than the use of hema-based dentin bonding. non-hema based bonding generally has monomers, called as urethane dimethacrylate (udma). udma can form a congested cross-link polymer, resulting in increased mechanical strength. when the polymer is stretched or tensed due to polymerization contraction, it can prevent individual chains form sliding over each other. when the polymerization is disappeared during stress condition, the cross-link polymer chains can move back to the starting position, so the object will be back to the previous form. udma also has a higher molecular weight, so udma can increase the degree of polymerization.9,10 in addition, each of dentin bonding materials has a different solvent composition. the composition greatly affects the tensile adhesion strength of dentin bonding to the collagen fibrils. solvents commonly used are acetone and alcohol (ethanol).9 solvents are required to remove water from the surface of dentin to prepare collagen tissue for adhesive resin infiltration.11 priming materials can increase the diffusion of resin into the demineralized and humid dentin, leading to optimal micromechanical bond.12 therefore, in this research, hema-based bonding resin used contains alcohol solvent, while non-hema based bonding resin contains acetone. the application of non-hema based bonding resin contains acetone on the superficial and deep dentin in this research could trigger higher tensile adhesion strength than 4 all of the superficial and deep dentin taken was smeared with 37% phosphoric acid gel (ivoclar vivadent, inc., united states and canada) for 10 seconds. the dentin was washed with water for ± 10 seconds, and then dried by cotton to remove excessive water. afterwards, two drops (0.02 grams) of the hema-based dentin bonding material were dropped on a disposable brush (according to the manufacturer's instructions), then smeared on the dentin (twice), and settled for 20 seconds. next, the dentin then was sprayed with an air spray of chip blower for 5 seconds to remove excessive solvent (solvent contained in the primer solution, namely water), and then curing was carried out for 10 seconds (according to the manufacturer's instructions). afterwards, cylinder cast was inserted into lower plunger and then fixed by installing a lock. cylinder cast on the upper plunger then was filled with composite, assembled with the lower plunger, and fixed by installing a lock. after that, irradiation was performed using a light cure for 20 seconds on both sides of the plunger, 10 seconds for each. next, it would harden within 2 minutes. tensile strength test then was performed using a te autograph (ag-10 shimadzu, japan). in all cases, the results were considered statistically significant with a p value <0.05 results visual illustration of the mean tensile adhesion strength of hema and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin can be seen in the following graph: figure 1. the mean tensile adhesion strength of hema and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin (mpa). results of the normality test using the kolmogorov-smirnov test showed a significance value (p) of >0.05. the data obtained were normally distributed. moreover, results of the homogeneity test using the levene test showed a significance value (p) of 0.343 (significance (p) value is (p) of >0.05). therefore, it was indicated that the data obtained were homogeneous. superficial dentin with hema bonding application superficial dentin with non-hema bonding application deep dentin with hema bonding application deep dentin with non-hema bonding application : superficial dentin with hema bonding application; : superficial dentin with non-hema bonding application; : deep dentin with hema bonding application; : deep dentin with non-hema bonding application figure 3. the mean tensile adhesion strength of hema and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin (mpa). table 1. results of the difference test on the tensile adhesion strength of hema and non-hema-based dentin bonding applied on the surfaces of the superficial and deep dentin (mpa) group n mean sd p i 6 2.2733 0.08506 0.000ii 6 3.0888 0.06769 iii 6 3.9110 0.08340 iv 6 4.5788 0.09750 t en si le a dh es im s tr en gt h dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p14-18 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p14-18 1717wurdani, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 14–18 the application of hema-based bonding resin containing alcohol solvent. that is because acetone contained in the non-hema based bonding resin can rapidly evaporate, causing optimal removal of water from the surface of the dentin, greater sensitivity to dentin humidity, and better dentin wetting than the alcohol solvent. thus, infiltration of resin with collagen fibrils was getting higher. the attachment of non-hema based bonding resin to collagen fibrils then can form more hybrid layers, leading to stronger adhesion properties.6,11 on the other hand, the results of this research also showed that the use of hema-based bonding resin containing alcohol solvent could generate lower tensile strength of the attachment than the use of non-hema based bonding resin. that is because alcohol contained in the hema based bonding resin possess longer evaporate, causing the non-optimal removal of water from the surface of the dentin, lower sensitivity to dentin humidity, and worse dentin wetting than acetone contained in the nonhema based bonding resin. consequently, infiltration of resin with collagen fibrils was getting lower. the attachment of hema based bonding resin to collagen fibrils then can form fewer hybrid layers, leading to weaker adhesion properties.6,11 the adhesion tensile strength of the non-hema-based dentin bonding on different dentin depths was better than the adhesion tensile strength of the hema-based dentin bonding. hema-based bonding, according to some previous researches, clinically has some weaknesses. one of them is that the material can be considered as the most common sensitizer to induce hypersensitivity in the teeth.9 several previous researches even have shown significant cytotoxic effects associated with methacrylate monomers contained in hema-based dentin bonding. therefore, there is a dentin bonding material alternative in the form of non-hema-based dentin bonding that can be considered as a good choice.3 in this research, the adhesion strength value of hemabased bonding in the superficial dentin was 2.2733 mpa. meanwhile, the adhesion strength value of non-hemabased bonding in the superficial dentin was 3.0888 mpa. the adhesion strength value of hema-based bonding in the deep dentin was 3.9110 mpa. meanwhile, the adhesion strength value of non-hema-based bonding in the deep dentin was 4.5788 mpa. those values were quite small when compared with the adhesion strength values of the dentin bonding resin materials in other several previous researches.5 in a previous research, the high tensile adhesion strength values were ranging from 10.02250 mpa to 16.7375 mpa.5 clinically, a good tensile adhesion strength value is about 5 mpa. these low tensile adhesion strength values actually could be caused due to some difficulties found in this research. one of them is because of not considering humidity dentin in detail. to generate the optimal humidity, drying should be performed using dry-bonding technique to prevent over-wet phenomenon. unfortunately, this research did not measure humidity on the dentin scientifically. as a result, there were two possibilities occurred. the dentin was too dry as a result of the drying process. thus, the collagen fibrils were collapse, and the water of the solvent was not enough to make the collagen do re-expansion. the dentin just did not completely dry in the drying process, and then added with water from the solvent. therefore, the phenomenon of over-wet still occurred. both possibilities then could lower the interaction between the bonding resin and the dentin collagen fibrils, resulting in lower adhesion strength as described previously. another difficulty in this research was a limitation on the tool (plunger set) used. this tool was not able to create the same filling procedure with clinical circumstances. in the clinical circumstances, composites were applied layer by layer, and then irradiation was carried out on the entire surface of the composites. meanwhile, in this research, composites were applied only on the upper plunger set, providing a composite cast, and then assembled with the lower plunger set, containing specimens of teeth applied with dentin bonding before. after that, radiation was performed. radiation, unfortunately, could not be performed in all parts of the surface of the composites. radiation was only conducted on the exterior surfaces of the two sides that were visible through the gap of the plunger sets. the results of tensile strength test showed that there were 70% of the dentin bonding resin materials released from the dentin in wet conditions, especially on the area of dentin hybrid layer. this is because the structure of collagen fibrils are very soft and easily cut when compared with the structures of dentin-bonding resin polymerization and composite-bonding resin polymerization. when observed with a microscope, it is known that dentin tubules are not aligned. consequently, the anchoring of the collagen fibrils releasing the bonding resin is located in composite resin area, bonding resin layer, and demineralized dentin (30%).12 the release of the dentin bonding resin materials was expected to occur in the interface area of both the materials. after applying the bonding resin solution, the composite filling material then was applied on the top of it. during its polymerization, the bonding resin then would be covalently attached to the composite, considered as a primary chemical bond, strong enough for both resins as derivatives of methacrylate groups. consequently, the risk of the release of the bonding resin materials in composite resin area, bonding resin layer, and demineralized dentin, not at the interface area of the dentin bonding resins, can be ignored. the use of hema based bonding resin containing alcohol solvent can generate lower tensile adhesion strength than the the use of non-hema based bonding resin containing acetone. the density of dentin tubules and the level of water contained at each dentin depth is also known to have a great effect on the use of both bonding resin on the surfaces of the supeficial and deep dentin. the superficial dentin has lower water level and fewer tubules. the dentin dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p14-18 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p14-18 18 wurdani, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 14–18 bonding penetrating into the intertubular dentin responsible for providing adhesion strength between dentin and dental restorative materials will have lower adhesion strength. on the other hand, the deep dentin has higher water level and more numerous tubules. therefore, the permeability of the intratubular resin has higher adhesion strength.3 finally, it can be concluded that there were differences in the tensile adhesion strength of hema and non-hemabased dentin bonding materials applied on the surfaces of the superficial and deep dentin. the use of non-hemabased dentin bonding material on different dentin depths was also known to be better than the use of non-hemabased dentin bonding material. references 1. al zahawi ar, mahmood ma, talabani rm, mansoor ra. the prevelence and causes of dental non carious cervical lesion in the sulaimani population (cross-sectional study). iosr journal of dental and medical sciences 2015; 14(8): 94-5. 2. anusavice kj. philip’s science of dental material. 11th ed. usa: wb elsevier; 2003. p. 21, 24, 79, 251-9, 227-32. 3. kumari rv, siddaraju k, nagaraj h, poluri rk. evaluation of shear bond strength of newer bonding systems on super cial and deep dentin. j int oral health 2015; 7(9): 31–5. 4. adioro s. tensile bond strength of hydroxyethyl methacrylate (hema) bonding agent to bovine dentine surface at various humadity. dental journal (majalah kedokteran gigi) 2006; 39(2): 59-62. 5. von fraunhofer ja. adhesion and cohesion. international journal of dentistry 2012; 2012: 1-9. 6. craig rg. restorative dental materials. 11th ed. london: mosby; 2002. p. 57, 69-70, 232-40, 261, 269-70. 7. bourbia m. biodegradation of dental resin composite and adhesive by streptococcus mutans: an in vitro study. toronto: proquest; 2013. p. 6-13. 8. papakonstantinou ae, eliades t, cellesi f, watts dc, silikas n. evaluatin of udma’s potential as a substitute for bis-gma in orthodontic adhesives. dent mater 2013; dent mater 2013; 29(8): 898-905. 9. van dijken jwv. a randomized controlled 5-year prospective study of two hema-free adhesives, a 1-step self etching and a 3-step etchand-rinse, in non-carious cervical lesions. sweden: dental materials; 2013. p. 271-80. 10. felizardo kr, lemos lvfm, de carvalho rv, junior ag, lopes mb, moura sk. bond strength of hema-containing versus hemafree self-etch adhesive systems to dentin. braz dent 2011; 22(6): 468-72. 11. cardoso mv, de almeida neves a, mine a, coutinho e, van landuyt k, de munck j, van meerbeek b. current aspects on bonding effectiveness and stability in adhesive dentistry. aust dent j. 2011; 56 (suppl 1): 31-44. 12. hashimoto m, ohno h, kaga m, sano h, endo k, oguchi h. fractured surface characterization: wet versus dry bonding. dent mater 2002; 18(2): 95-102. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p14-18 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p14-18 vol 44 no 3 sept 2011.indd 137 vol. 44. no. 3 september 2011 the effectiveness of nigella sativa seed extract in inhibiting candida albicans on heat cured acrylic resin hanoem eh,1 imam b,1 and kartika purnama pranoto2 1department of prosthodontics 2student faculty of dentistry, airlangga university surabaya indonesia abstract background: indonesia has a variety of plants that can be used for medicines. one of the medicinal plants is nigella sativa. nigella sativa has been used for medicinal purposes, both as medicinal herb and as medicinal oil. it contains saponin and atsiri oils that have antifungal, antimicrobial and antibacterial effects. nigella sativa has been suggested as denture cleansers since it can inhibit the growth of candida albicans (c. albicans) on heat cured acrylic resin. purpose: the aim of this research is to know the effectiveness of nigella sativa seed extract in inhibiting the growth of c. albicans on heat cured acrylic resin. methods: eighteen acrylic samples were divided into three groups. group i was control group, only contaminated with c. albicans without immersing in any solution. group ii was acrylic sample immersed in sterile aquades for one hour. group iii was acrylic sample immersed in nigella sativa seed extract for one hour. results: there were significant differences of c. albicans (p < 0.05) among the three groups. the number of candida albicans was significantly higher in group i, while that in group ii was lower than that in group i, and that in group iii was the lowest. conclusion: nigella sativa seed extract was effective in inhibiting the growth of c. albicans on heat cured acrylic resin. key words: nigella sativa, candida albicans, heat cured acrylic resin abstrak latar belakang: indonesia memiliki berbagai tanaman yang dapat dipakai sebagai obat, salah satu tanaman tersebut adalah jinten hitam (nigella sativa). pada beberapa negara jinten hitam telah digunakan untuk berbagai tujuan, baik sebagai obat herbal maupun sebagai minyak. kandungan jinten hitam adalah saponin dan minyak atsiri yang mempunyai efek anti jamur dan anti mikroba. jinten hitam disarankan sebagai pilihan pembersih gigi tiruan yang dapat menghambat pertumbuhan candida albicans (c. albicans) pada resin akrilik heat cured. tujuan: tujuan penelitian ini adalah mengetahui efektivitas dari ekstrak biji jinten hitam dalam menghambat pertumbuhan c. albicans pada resin akrilik heat cured. metode: delapan belas sampel akrilik heat cured dibagi dalam tiga kelompok, kelompok i sebagai kelompok kontrol, lempeng akrilik tanpa direndam dalam bahan apapun selama 1 jam. kelompok perlakuan ii, lempeng akrilik direndam dalam aquades steril selama 1 jam. kelompok perlakuan iii, lempeng akrilik direndam dalam ekstrak biji jinten hitam selama 1 jam. hasil: ada perbedaan yang bermakna dari c. albicans (p < 0,05) pada ketiga kelompok tersebut. jumlah c. albicans lebih banyak secara bermakna pada kelompok i, pada kelompok ii lebih sedikit daripada kelompok i dan kelompok iii paling sedikit. kesimpulan: ekstrak biji jinten efektif dalam menghambat pertumbuhan c. albicans pada resin akrilik heat cured. kata kunci: jinten hitam, candida albicans, resin akrilik heat cured correspondence: hanoem eh, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. research report 138 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 137–140 introduction acrylic resin is an option for making removable denture base because it is relatively cheap, easy to repair, easy to make denture by using simple equipments, color stable, and easy to be polished.1 acrylic resin material has been known since 1937, and used by dentists since 1946 until now since the materials are well received in the dentistry.2 materials often used as removable denture base is polymethyl metacrylate (pmma) acrylic resin, heat cured type which need heat during polymerization.3 one of acrylic resin properties is absorbing the water when contacting with saliva it will absorb saliva. when inserted, it will be coated with protein-rich saliva so that pellicle is formed. pellicle is able to attach with microorganisms, such as candida albicans (c. albicans).4 after two hours, pellicle will formed plaque, ie a collection of microorganisms, glycoproteins matrix and polysaccharides which attached to the tooth surface. the process of plaque formation is the same while happened on the denture surface.5 plaque and food accumulation then will cause the frequency and density of c. albicans increased. c. albicans can contribute to the occurrence of denture stomatitis. denture stomatitis is an inflammation that occurs on the oral mucosa resulted from using denture.6 removable denture cleansing can be done in two ways: mechanical and chemical ways. one effective way to clean acrylic resin denture is by immersing removable denture in antiseptic liquid/solution or denture cleanser.7 denture cleanser should not cause abrasion or denture dimension changing, therefore, denture cleanser must not contain alcohol.5 denture cleanser with buffer containing alcohol actually can make the surface of the acrylic resin acrylic microcrazed. crazing is the separation of polymer molecular chain caused by mechanical pressure or solvents. crazing consists of small cracks that make acrylic become brittle, known as an early symptom of acrylic fracture. denture cleanser materials on market today are quite expensive for the user, therefore, there is a need to find for an alternative material. indonesia has a rich natural biodiversity. these diverse plants have already been used by indonesian people for a long time, either as food or medicines.9 since ancient times, indonesian ancestors have come to know and used medicinal plants to maintain their health and to treat their diseases. the general term of medicinal herbs is traditional herbal medicine. this is suitable with the recommendation of world health organization (who) that in order to improve and distribute equitable health services for all communities, thus, these effort must be nurtured, and developed in order to be more efficient and effective.8 in indonesia many traditional plants are available to be used as antiseptic materials, one of which is nigella sativa that has been used since thousands of years ago and has been examined by experts as a very useful herbs for health.9 nigella sativa also has antibacterial and antifungal effects of aqueous, methanol and chloroform extracts which is able to fight c. albicans, compared with standard medicines, such as clotrimazole, cloxacillin and gentamicin. it contains saponin that has antimicrobial effect and atsiri oil used as antiseptic, antioxidant, also have activities against several gram-positive, gram-negative bacteria, and anti-fungal properties. scientists in europe have recently stated that habbatus sauda (the black seed) works as an anti-bacterial and anti fungi.10 this research is aimed to examine the effectiveness of nigella sativa in inhibiting the growth of c. albicans on heat cured acrylic resin. therefore, this research is expected to provide information to health professionals and public about the benefits of black nigella sativa especially for health, as an alternative denture cleanser. materials and methods the samples in this research is 18 heat cured acrylic resin plate. the dimension of the samples is 10 mm x 10 mm x 1 mm. the samples were devided into three groups, six samples each. group i was the control group in which the acrylic resin plate was not immersed in any material. group ii was the treatment group in which the acrylic resin plate was immersed in sterile aquades for 1 hour. group iii was the treatment group in which acrylic resin plate was immersed in 20 ml nigella sativa seed extract for 1 hour. the criteria of the sample size: not porous, not polished, not changing in shape, and flat for the surface of the sample. c. albicans was incubated for 2 × 24 hours at temperature of 37° c. a colony was taken and grown on blood serum for 2 hours at 37° c. then, c. albicans as much as a loop was put into sabouraud's broth 5 ml, and incubated for 48 hours at 37° c. after incubated, it was adjusted to the standard of mc.farland 3 which is identical with 900 × 106 c. albicans. this suspension later was used to immerse the acrylic resin. saliva taken from one person as much as 50 ml was put in test tube with certain criteria. saliva was taken from 22 year old health male students who were not smoking, not taking anti-fungal medications, and not taking antibiotics. the saliva was centrifuged for 15–20 minutes at 1000 rpm at 4° c to obtain supernatant which was then filtered and put in sterile test tube for the preparation of pellicle formation on heat-cured acrylic resin base. sterilization of heat-cured acrylic resin base was conducted by using an autoclave at 121° c for 18 minutes. the heat-cured acrylic resin base was put into sterile saliva for 1 hour at room temperature to form pelikel. the heat cured acrylic resin was removed and rinsed with pbs solution 2 times, was put into sabouraud's broth media containing a suspension of c. albicans. afterwards, it was incubated 37° c for 24 hours. each sample was put in a test tube, immersed in 20 cc sterile aquades and 20 cc nigella sativa seed extract until all parts of acrylic plate was immersed for 1 hour. for control group sample was not immersed in any materials. all of the samples were put in sabouraod's broth 10 ml, and vibrated with a vortex for 30 seconds to release 139hanoem: the effectiveness of nigella sativa c. albicans attaching to the samples. 0.1 ml of c. albicans suspension was taken using 1 ml of tuberculin syringe, dripped on sabouraud's dextrose agar, made spreading, and then incubated for 48 hours at temperature of 37° c. the measurement of c. albicans colonies was conducted by using colony counter with unit of cfu/ml. the data was analyzed using anova test. results it was found that the number of c. albicans colonies on heat cured acrylic resin after immersing in 20 cc nigella sativa was low compare after immersing in sterile aquadest or without immersion. table 1. the mean and standard deviation of the number of c. albicans colonies on the heat cured acrylic resins group number mean standard deviation i 6 164.5000 46.16384 ii 6 62.6667 41.12258 iii 6 25.0000 16.26038 prior to statistical calculations, normality test (kolmogorov-smirnov) was conducted. furthermore, to determine whether there are differences among the three groups, anova test was then conducted with significance p = 0.05. to know the difference among each treatment group, least significant difference (lsd) test was conducted. there was significant differences between the number of c. albicans colonies on acrylic resins immersed in nigella sativa seed extract and without immersed (p < 0.05). there was no significant difference of the number of c. albicans colonies on between acrylic resin immersed in nigella sativa seed extract and that immersed in sterile aquades (p > 0.05) (table 2). discussion the colony numbers of c. albicans on heat cured acrylic resin immersed in nigella sativa seed extract, sterile aquades, and without immersed can be detected. the number of c. albicans on heat cured acrylic resin immersed in nigella sativa seed extract, sterile aquades, and without immersed was different among them. this difference was caused by the differences of those materials used to immerse the heat cured acrylic resin. c. albicans can actually be classified into candida species, the most common and most widely found fungus in oral cavity. c. albicans can be found in the entire of oral mucosal surface, especially palatal mucosa and tongue. c. albicans is a pathogenic, opportunistic, dimorphic fungus normally found in oral cavity. in oral cavity, there are actually many different strains of c. albicans with particular phenotype characteristics that determine its character as commensal or pathogenic.6 c. albicans is dangerous, however, if the body's defense is weak, or especially got decreased immune system, then the commensal character of c. albicans can be turned into pathogenic one that can cause infections.10 c. albicans can be found in 66% of denture users who have healthy oral cavity, but the prevalence of c. albicans can be increased on denture users.11 meanwhile, c. albicans in oral cavity of nondenture users will only be considered as normal flora with the prevalence of 45%, whereas that in denture users will be increased to 47.5–55.6%.6 candida is often found on the surface of the maxillary denture because of continuous pressure under the maxillary denture so that saliva antibodies of the area are reduced and the fungus will be able to breed well in between the denture and mucosa.6 as noted in the introductory part, it is already known that one of the characters of acrylic resin is absorbing water when contacting with saliva, and then the acrylic resin will absorb saliva.5 on the other hand, denture is a good place for gathering the remnants of food. thus, people who have poor hygiene and long wearing dentures will possibly a b c figure 1. the number of c. albicans colonies on control group (a), group i (b), and group ii (c). table 2. the results of lsd test on the number of c. albicans colonies on heat cured acrylic resins group i ii iii i p = 0.001 * p = 0.001 * ii p = 0.001 * p = 0.097 iii p = 0.001 * p = 0.097 *: significant differences 140 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 137–140 get plaques on their denture. the buildup of plaque and food scraps then will cause the frequency and density of c. albicans increased. c. albicans will contribute greatly to the occurrence of denture stomatitis. denture stomatitis is an inflammation that occurs in oral mucosa resulted from using denture.6 to decrease the number of c. albicans and prevent denture stomatitis, removable denture cleaning must be conducted. one effective way to clean acrylic resins is by immersing the removable denture in antiseptic liquid/ solution or denture cleanser. research conducted by the faisol in 1993 tries to analyze the use of cleaning material soap to maintain the cleanliness of removable denture in which the denture must be brushed with soap and then immersed in antiseptic solution. faisol then conducted a research on the effect of cleaning material soaps on removable denture base. the result of the research shows that the effective prevention of denture stomatitis can be conducted by using chemical materials, ie immersing the denture in antiseptic solution for 15 minutes, 30 minutes, 1 hour, or all night depended on chemical cleaning materials.7 similarly, zarb et al.,12 also argued that dentures should be cleaned by immersing it in denture cleansers at least 15 minutes at night since 15 minutes is sufficient to kill microorganisms. based on the above literatures, this research then determined 1 hour immersing time for examining the effectiveness of the materials used as denture cleansers in inhibiting the growth of c. albicans. in this research, moreover, nigella sativa seed extract was used as antiseptic solution or denture cleanser. from the data obtained, it is known that the average of c. albicans in group iii (with the immersion in nigella sativa seed extract) was lower than the average of c. albicans in group i and group ii (by immersion in sterile aquades). based on the data and data analysis, it then can also be known that use of nigella sativa seed extract had a very significant effect on the inhibition of growth of c. albicans on heat cured acrylic resin since there was an active compound containing antimicrobial power in nigella sativa seed. thus, the ability of nigella sativa seed in inhibiting the growth of c. albicans was caused by the active compound contained in nigella sativa seed extract. nigella sativa seed actually contains atsiri oils, nigelion and arganin crystals, fatty acid, carotene, and 15 kinds of amino acids, proteins, and carbohydrates. furthermore, it also contains various minerals, like calcium, sodium, potassium, magnesium, selenium, iron, vitamins a, b1, b2, b6, c, e, and niacin. besides that, it contains chemical forms, such as fats and vegetable oils (35%), carbohydrates (32%), protein (21%), water (5%), saponins, nigellin.11 among the contents of nigella sativa seed, the ability of nigella sativa seed as an antimicrobial is caused by saponins and atsiri oils. saponin is a glycoside forming base in water that exist on many kinds of plants. therefore, saponin is considered as a surface active chemical compound that can be detected based on their ability to form base. saponin can be used as an antimicrobial. destruction of microbes by antimicrobials, moreover, is considered as bacteriostatic since it still depends on the immune reaction ability of hosts. the action mechanism of antimicrobial can actually be classified into four main methods, namely the inhibition of cell wall synthesis, the inhibition of cell membrane function, the inhibition of protein synthesis, and the inhibition of the nucleat acid synthesis. saponin then will broke cytoplasmic membrane and kill cells. therefore, saponin can be used as antimicrobial. the mechanism of saponin, furthermore, is by denaturing bacteria and breaking cell membranes that can not be repaired again. since saponin is considered as antimicrobial, saponin then can inhibit the growth of fungi, such as c. albicans which atsiri oils can serve as antiseptics, antioxidants, anti fungi and can be able to attack several gram-positive and gram-negative bacteria. on the immersion in sterile aquades, it is even known that the number of c. albicans was decreased because of sterile aquades, but the decreasing was still less than on the immersion in nigella sativa seed extract. this is probably because nigella sativa seed extract contains active compounds that have antimicrobial power, ie saponins and atsiri oil, while sterile aquades only contains sterile solution. it can be concluded that nigella sativa seed extract effective in inhibiting the growth of c. albicans on heat cured acrylic resin. references 1. annusavice kj. phillips science of dental materials. 11th ed. philadelpia: wb saunders co; 2003. 177, 192, 200–18. 2. craig rg, powers. restorative dental material. 11th ed. st. louis: the mosby co; 2002. p. 636–82. 3. combe ec. notes on dental materials. 6th ed. edinburg: churcil livingstone; 1992. p. 157–63. 4. edgerton m, levine mj. characterization of acquired denture pellicle from healthy stomatitis patient. j prosthet dent 1993; 68: 683–91. 5. abelson dc. denture plaque and denture cleanser. prosphet dent 1981; 45: 376–9. 6. soenartyo h. denture stomatitis: penyebab dan pengelolaannya. maj ked gigi (dent j) 2000; 33(4): 148–51. 7. faisol. pengaruh bahan pembersih sabun terhadap basis gigi tiruan lepasan. jurnal kedokteran gigi pdgi 1993; (3): 72–4. 8. prameswari, zuraida. efek laksatif infusa daun paria (momordica charantia l.) pada mencit. majalah ilmiah kedokteran medika kartika 2005; 3(1): 36–44. 9. yulianti s, junaedi e. sembuhkan penyakit dengan habbatus sauda (jinten hitam). jakarta: agromedia pustaka; 2006. p. 9–34. 10. norman pw. essential dental microbiology. usa: appleton & lange; 1991. p. 240–4. 11. marcos-arias c, vicente jl, sahard ih, eguia a, de-juan a, madariaga l, aguirre jm, eraso e, quindos g. isolation of candida dubliniensis in denture stomatitis. archives of oral biology 2009; 54(2): 127–31. 12. zarb, grasser gn, zander ha. the efficacy of denture-cleansing agents. j of prosthet dentist 1990; 48(5): 515–20. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 229 volume 46, number 4, december 2013 case report orthodontic-surgical treatment of a severe class iii malocclusion pakpahan evie lamtiur department of orthodontic faculty of dentistry, universitas prof dr. moestopo (b) jakarta indonesia abstract background: adult patient with dentofacial deformities usually need surgical orthodontic treatment. although case of class ii dentofacial deformities are more common, the need for treatment and improvement in term of facial profile is generally greater in class iii patients. when a skeletal class iii malocclusion is diagnosed, orthognathic surgery is always considered if the orthodontist and patient desire complete correction of the skeletal discrepancy. purpose: the purpose of this article were to reported a case of severe class iii malocclusion and to showed the positive effect of orthognatic surgical treatment on the patient’s profile. case: this case report describes the surgical-orthodontic treatment of a 20 year old male patient with class iii dentofacial deformity. case managements: to allow adequate surgical movement, both maxillary first premolars were extracted, and the maxillary incisors were retracted. no extractions were performed in the mandibular arch. surgery included a le fort i osteotomy with 8 mm advancement, a bilateral sagittal split osteotomy with the mandibula was set back 13 mm at right side and 11 mm at left side for the correction of dental midline and chin deviation. the genioplasty treatment also was done. conclusion: surgical-orthodontic treatment could be chosen as a treatment option for achieving an acceptable occlusion and a good esthetic result in a patient with a class iii dentofacial deformity. nevertheless, it should be performed by a multidisciplinary team to ensure a satisfactory outcome key words: class iii dentofacial deformity, orthognatic surgery, orthodontic treatment abstrak latar belakang: pasien dewasa dengan deformitas dentofacial biasanya ditangani dengan perawatan bedah orthodonti. walaupun kasus deformitas dentofacial klas ii lebih sering dijumpai, namun kebutuhan perawatan dan keinginan untuk memperbaiki profil muka lebih tinggi pada pasien dengan kasus klas iii. untuk koreksi kelainan skeletal secara menyeluruh pada maloklusi skeletal klas iii maka dibutuhkan perawatan bedah ortodonti. tujuan: laporan kasus ini bertujuan melaporkan penanganan kasus dengan kelainan klas iii maloklusion skeletal serta memperlihatkan hasil perawatan bedah ortognati yang memperbaiki profil pasien. kasus: perawatan bedah ortodonti dilakukan pada pasien laki-laki (20 tahun) dengan kelainan deformitas dentofacial klas iii. tatalaksana kasus: dilakukan pencabutan premolar pertama rahang atas kanan dan kiri, kemudian dilakukan retraksi gigi insisif rahang atas, agar didapatkan pergerakan yang adekuat. tidak dilakukan pencabutan gigi pada rahang bawah. perawatan bedah yang dilakukan adalah le fort i osteotomy dengan memajukan rahang atas sebanyak 8mm, mandibula dimundurkan dengan tehnik bilateral sagittal split ostetomy sebanyak 13 mm disebelah kanan, dan 11 mm disebelah kiri untuk koreksi dental midline dan deviasi dagu. pasien juga mendapatkan perawatan genioplasty. simpulan: bedah ortognatik dapat dipilih sebagai perawatan untuk mendapatkan oklusi dan hasil estetik yang baik pada pasien dengan deformitas dentofasial kelas iii. namun demikian, perawatan perlu dilakukan oleh tim dari multidisiplin untuk mendapatkan hasil yang memuaskan. kata kunci: deformitas dentofasial klas iii, bedah orthognati, perawatan ortodonti correspondence: pakpahan evie lamtiur, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas prof dr. moestopo (b). jl. bintaro permai raya no. 3 jakarta selatan 12330, indonesia. e-mail: evie_lamtiur@yahoo.com 230 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 229–234 introduction many approaches have been performed successfully by orthodontist for the treatment of malocclusion and class iii malocclusion is considered as one of the most complex and difficult orthodontic problem to be diagnosed and treated.1 in reality dental camouflage only leads to an effective improvement of aesthetics in a few situations. more frequently it has no significant influence on facial aesthetics, as in purely orthodontic correction of class iii cases. in an adult with dentoskeletal discrepancy, surgery is the only sure treatment option if the dental defect cannot be corrected by orthodontics alone or if dental camouflage would involve technical or periodontal contraindications, or would not produce a marked aesthetic improvement. when a skeletal class iii malocclusion is diagnosed, orthognatic surgery is almost always a consideration if the orthodontist and the patient desire complete correction of the skeletal discrepancy. individuals with class iii malocclusion frequently show combinations of skeletal and dentoalveolar components. several distinct cephalometric features have been reported in class iii patients, such as a short anterior cranial base length, acute cranial base angle, a short and retrusive maxilla, proclined maxillary incisors, retroclined mandibular incisors, an excessive lower anterior face height and obtuse gonial angle.2 therefore class iii patients are large proportion of those seeking surgical orthodontic treatment. however, the etiology all class iii malocclusion might not be the same. the etiology of class iii malocclusions may involve hereditary factors, environmental influences and even pathology.3 additionally, class iii patients typically have longer and unpredictable facial growth.2 class iii patients can have varying degrees of dental and skeletal abnormalities. an accurate diagnosis is important to treat the malocclusion in order to insure that the treatment plan is directed at correcting the various abnormalities.3 the purpose of this article were to reported a case of severe class iii maloclusion and to showed the positive effect of orthognatic surgical treatment on the patients profile. case a male patient, 20 years old, initially came for orthodontic treatment with the complaint of long face, crossbite and inability to incise. he has family history of class iii malocclusion. the patient has a nonrelevant medical history. after thorough clinical examination and cephalometric analysis, surgical-orthodontic treatment was recommended. extraoral examination showed a concave profile with extremely long lower face height, incompetent lip and a flattened lower lip without labiomental sulcus. frontal view of the face showed chin deviation to the left and long middle third of the face. the lower lip was stretched to compensate for the vertical discrepancy (figure 1). table 1. cephalometric summary measurement initial presurgical postsurgical na 85° 84° 92° snb 96° 96° 90° anb -11° -12° +2° wits -20mm -29mm -10mm napg -20° -22° -2° fma 40° 40° 35° n perp a -3° -5° +4° n perp b +12° +13° +6° n-ans 60 mm 59mm 65mm ans-me 87mm 86mm 75mm faxial axis 66° 65° 62° sn go gn 39° 38° 35° i sn 114° 107° 110° ipp 125° 120° 109° mx i to na 10mm 12mm 8mm 25° 24° 35° mn i to nb 6mm 10mm 7mm 14° 32° 15° i mpa 65° 77° 75° e line bibir atas -5mm -6mm -3mm e line bibir bawah +5mm +5mm +3mm 231lamtiur : orthodontic-surgical treatment of a severe class iii malocclusion the analysis of intraoral confirmed an angle class iii malocclusion with antero posterior discrepancy in the molar relationship was more than 10 cm, and a -13 cm incisor overjet, absence of overbite were measured. the mandibular dental midline were deviated 6mm to the left. there was posterior crossbite on the left side, mild crowding in the lower as well and compensation of incisor inclination in both arches (figure 2). study model analysis showed an angle class iii malocclusion more than 10 mm anteroposterior discrepancy in the molar relationship. the archform were not well coordinated due to severe compensations that led to large anterior and buccal overjet. the lateral cephalometric radiograph revealed class iii skeletal malocclusion (anb = -11°), maxillary a bit protusion (sna = 85°), and mandible protusion (snb = 96°) in relation to the anterior skull base. concave bone profile (napg = -20°), dolichofacial morphological type (facial axis = 66°) and predominance of vertical growth of the face (sngogn = 39° and fma = 40°). the maxillary incisors presented increased to their alveolar (table 1 and figure 3) . 11 figure 1. pretreatment facial photographs. 11 figure 1. pretreatment facial photographs. figure 1. pretreatment facial profiles: a, b. front; c. right side. figure 2. pretreatment intraoral photographs. a) right side; b) left side; c) front; d) maxilla; e) mandibula. figure 3. presurgical cephalometric radiograph. 12 figure 2. pretreatment intraoral photographs. 12 figure 2. pretreatment intraoral photographs. 12 figure 2. pretreatment intraoral photographs. 13 figure 3. presurgical cephalometric radiograph. b c e a b c 12 figure 2. pretreatment intraoral photographs. a 12 figure 2. pretreatment intraoral photographs. d 232 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 229–234 figure 4. a, c, e) presurgical ekstraoral photographs. b, d, f) post surgical ekstraoral photographs-1. figure 5. a, b, c, d, e) presurgical intraoral photographs. f, g, h, i, j) post surgical intraoral photographs copy. case managements preoperative orthodontic preparation was performed with 0.022 appliance. leveling and alignment began with 0.016-in nickel-titanium archwires, followed by 0.018, 0.016 x 0.022-inch nickel-titanium archwires up to 0.19 x 0.025-inch stainless steel rectangular archwires. in the leveling and alignment stage, the archwires were coordinated. to allow adequate surgical movement, the maxillary first premolars on both side was extracted to correct the position of maxillary incisor therefore the maxillary incisors were retracted. no extractions were performed in the mandibular arch because there was minimal crowding. the mandibular incisors were aligned and the arch and the archform were coordinated. after presurgical orthodontic treatment achieved, the orthognatic surgery were performed. surgery included a le fort i osteotomy with 8mm of advancement and it was rotated anterior down 3 mm and posterior up 1 mm at posterior nasal spine (clockwise rotation). the pivoting point is around the first molar. a bilateral sagittal split osteotomy with 13 mm of setback at right and 11 mm at left for the correction of dental midline and chin deviation. the chin therefore moves 11 mm backward. rigid internal fixation with screws and plates was used to stabilize the osteotomy site. the patient was followed up closely after the procedures. postoperatively, to achieve the proper occlusal contacts, vertical intermaxillary and class iii elastic were used. after surgery, it was observed functional occlusion, normal overjet and overbite, and adequate intercuspation, with class ii angle molar relationship and class i canine relationship, coincident midlines. mandibular prognatism and asymmetry were eliminated and facial esthetic was considerably improved. figures 4 and 5 show the results obtained with the orthognatic surgery and orthodontic finishing stage, and postsurgical cephalometric radiograph is shown in figure 6. discussion there are three main treatment options for skeletal class iii malocclusion, which is growth modification, orthodontic therapy, and orthognatic surgery combined with orthodontic treatment. maxillofacial growth modification with dentofacial orthopedic appliance is an effective method for resolving skeletal class iii jaw discrepancies in children. correcting this problem in adults requires orthognathic surgery in conjuction with orthodontic treatment.4 some studies reported the factors that the choice between conventional orthodontic treatment and surgical orthodontic 14 figure 4. left side presurgical ekstraoral photographs. right side post surgical ekstraoral photographs-1. 14 figure 4. left side presurgical ekstraoral photographs. right side post surgical ekstraoral photographs-1. 14 figure 4. left side presurgical ekstraoral photographs. right side post surgical ekstraoral photographs-1. 14 figure 4. left side presurgical ekstraoral photographs. right side post surgical ekstraoral photographs-1. a b c d e f 15 figure 5. left side presurgical intraoral photographs. right side post surgical intraoral photographs copy. f b g c h d i 15 figure 5. left side presurgical intraoral photographs. right side post surgical intraoral photographs copy. a 15 figure 5. left side presurgical intraoral photographs. right side post surgical intraoral photographs copy. e 15 figure 5. left side presurgical intraoral photographs. right side post surgical intraoral photographs copy. j 233lamtiur : orthodontic-surgical treatment of a severe class iii malocclusion treatment. kerr5 et all reported that patients with anb angles of less than -4° and mandibular incisor inclination of less than 83° were more likely to have surgical-orthodontic treatment than conventional orthodontic treatment. a recent study concluded that surgical patients could be distinguished from nonsurgical ones, on the basis of wits measurement, maxillary/mandibular length ratio, gonial angle, and sella-nasion distance.6 miller5 found there were four areas of significantly difference between the surgery and non surgery case, which are: anb angle was significantly more negative in the surgical group.5 surgery was often considerers necessary when anb angle was less than -4°. thus the ratio of maxillary to mandibular lengths was significantly smaller for the surgical patients. also, the lower incisor were more retroclined in the surgical group. the division between the two group was about 83°. the last role is the holdaway angle was larger in the orthodontic group. surgery was almost always carried out when the holdaway angle was less than 3.5°.5 before treatment this patient had anb = -11°, and mandibular incisor inclination of less than 83° which is 65° (table 1 and figure 3) . that measurement indicate for this patient to have orthognatic surgery treatment. it is very important to understand the components of facial asymmetry in order to outline an accurate and effective treatment plan.7-10 the analysis of posteroanterior cephalometric radiographs determines if the asymmetri is related to the maxilla, mandible or both, in the sagittal or transverse direction, and is the anomaly is also associated with dental compensations. most studies have demonstrated that transverse dental compensation figure 6. post surgical cephalometric radiograph copy. 16 figure 6. postsugical cephalometric radiograph copy. is correlated with skeletal asymmetry.11-13 inclination of the occlusal plane greater than 4 and mentum deviations observed in the posteroanterior cephalorgram are important characteristic to determine the presence and extension of facial asymmetry.14-15 the patient of this case presented significant facial asymmetry with mandibular asymmetry and mentum deviation to the left. severt and proffit also haraguchi et al.15 have reported that in patients with dentofacial deformities with mandibular deviation, lateral excursion to the left was present in over 85% of the studied population. he also added that the mandible is more asymmetrical than the maxilla because of its greater to potential. while the mandible is a movable bone, the maxilla is rigidly connected to the adjacent skeletal structures by means of sutures and synchondroses. the patient presented unilateral posterior crossbite on the left side and midline sifting to the left, it results from mandibular deviation. the mandibular deviation and midline shifting was corrected by surgically. the same treatment was taken with similar cases in some case report.17,18 orthodontic treatment to obtain the preoperative alignment of the dental arches was also an important part as the best result with minimum relapse could not be expected without a stable occlusion.19-20 the maxillary premolars on both side were extracted to correct maxillary incisors inclination. the main aim of the presurgical orthodontic phase is to correct the incisor inclinations to normal to allow maximum surgical correction and the less-than-ideal outcomes for the skeletal relationships might have been due to inadequate presurgical orthodontic decompesation of the incisor inclination.20 after surgery the position of maxilla in this patient was +5mm anterior to the nasion frankfort line and the maxillary incisor was +8mm anterior to the nasion frankfort. position maxillary incisor was 109°. when only orthodontic treatment considered, the orthodontist formulates a treatment plan based on the desired final position of the mandibulat incisor. for surgical treatment planning the scenario is reversed; the maxillary incisor final position is used to determine the placement of the facial bones. mcnamara’s nasion frankfort perpendicular is an anteroposterior guideline that can be used to help determine the best placement of the maxillary incisor.21 point a should be positioned as close as possible to the nasion frankfort line, but more importantly, the maxillary incisor should be positioned 5 mm ±2 mm anterior to the nasion frankfort line and at 110° to the palatal plane.22-23 the overall result of the treatmetnt was good (figure 4, 5 and 6). the lower face height (ans-me) was improved cephalometrically from 87 mm to 75 mm. the anb was improved from -11° to +2°. visually, pleasing changes in the frontal and profile views of the face are evident (figure 4). the pretreatment lip incompetency was totally eliminated and the labiomental sulcus was normalized. dental changes resulted in a class ii molar occlusion with a class i canine relationship. the pretreatment maxillary midline deviation to the left was completely 234 dent. j. (maj. ked. gigi), volume 46, number 4, december 2013: 229–234 corrected. all the functional movement of the mandible are without limination and without symptoms. the treatment protocol produced a satisfactory occlusal and esthetic result for the patient and his parents as demonstrated by their attitude. this case report describes the treatment of an adult patient with severe class iii dentofacial deformity. orthognatic surgery treatment was the best option for achieving an acceptable occlusion and a good esthetic result in this patient. an experienced multidisciplinary team approach ensures a satisfactory outcome. presurgical orthodontics removes all the dental compensations and suggests the location and extent of the skeletal discrepancy. normal skeletal base relationship was achieved by osteotomy and setback of the prognathic mandible, postsurgical orthodontics guides the normal occlusal rehabilitation by correcting any emerging dental discrepancies. acknowledgements sincere gratitute to dr. enrina diah, sp.bp for performing the orthognatic surgery in cipto mangunkusomo hospital, jakarta. references 1. mucedero m, coviello a, baccetti t, franchi l,cozza p. stability factors after double-jaw surgery in class iii malocclusion. angle orthodontist 2008; 78(6): 1141-52. 2. katiyar r, singh gk, mehrotra d, singh a. surgical–orthodontic treatment of a skeletal class iii malocclusion. natl j maxillofac surg 2010; 1(2): 143–9. 3. lowenhaupt eb. compromised nonsurgical treatment of a patient with a severe class iii malocclusion. international dentistry 2009; 11: 52-61. 4. seng yc, pan cy, chou st, liao cy, lai st, chen cm, chang hp, yang yh. treatment of adult class iii malocllusion with orthodontic therapy or orthognatic surgery: receiver operating characteristic analysis. am j orthod dentofacial orthop 2011; 139(5): e485-e493. 5. kerr wj, miller s. dawber je. class ii malocclusion: surgery or orthodontics?. br j orthod 1992; 19: 21-4. 6. stellzig-eisenhauer a, lux cj, schuster g. treatment decision in adult patients with class iii malocclusion: orthodontic therapy or orthognatic surgery?. am j orthod dentofacial orthop 2002; 122: 27-37. 7. decker, jd. asymmetric mandibular prognatism: a 30-year retrospective case report. am j orthod dentofacial orthop 2006; 129(3): 436-43. 8. hayashi k, mugutuma t. morphologic characteristic of the dentition and palate in cases of skeletal asymmetry. angle orthod 2004; 74(1): 26-30. 9. ko ew, huang cs, chen yr. characteristic and corrective outcome of face asymmetry by orthognatic surgery. j oral maxillofac surg 2009; 67(10): 2201-9. 10. sekiya t, nakamura y, oikawa t, ishii h, hirashita a, seto k. elimination of transverse dental compensation is critical for treatment of patients with severe facial asymmetry. am j orthod dentofacial orthop 2010; 137(4): 552-62. 11. hayashi k, muguruma t. morphologic characteristics of the dentition and palate in cases of skeletal asymmetry. angle orthod 2004; 74(1): 26-30. 12. kusayama m, motohashi n, kuroda t. relationship between transverse dental anomalies and skeletal asymmetry. am j ortho am j orthod dentofacial orthop 2003; 123(3): 329-37. 13. van elslande dc, russett sj, major pw, flores-mird c. mandibular asymmetry diagnosis with panoramic imaging. am j orthod dentofacial orthop 2008; 134(2): 183-92. 14. padwa bl, kaiser mo, kaban lb. occlusal cant in the frontal plane as a reflection of facial asymmetry. j oral maxillofac surg 1997; 55(8): 811-6. 15. haraguchi s, takada k, yasuda y. facial asymmetry in subjects with skeletal class iii deformity. angle orthod 2002; 7291): 28-35. 16. severt tr, proffit wr. the prevalence of facial asymmetry in the dentofacial deformities population at the university of north carolina. int j adult orthodon orthognath surg 1997; 12(3): 171-6. 17. janson m, janson g, sant ana e. tibolao, martins dr. orthognatic treatment for a patient with class iii malocclusion and surgically restricted mandible. am j orthod j dentofacial 2009; 136(2): 290-8. 18. cho hj. patient with severe skeletal class ii malocclusion and severe open bite treated by orthodontic and orthognathic surgery-a case report. am j orthod dentofac orthop 1996; 110(2): 155-62. 19. nakajima t, kajikawa y, hirose t, tokiwa n, hanada k, fukuhara t. surgical-orthodontic approach to skeletal class iii malocclusion. int j oral surg 1978; 7(4): 274-80. 20. johnston c, burden d, kennedy d. class iii surgical-orthodontic treatment: a cephalometric study. am j orthod dentofacial orthop 2006; 130: 300-9. 21. mc namara ja. a method of cephalometric evaluation. am j orthod 1984; 86(6): 449-69. 22. bilodeau je. vertical consideration in diagnosis amd treatment, a surgical orthodontic case report. am j orthod dentofac orthop 1995; 107(1): 91-100. 23. bilodeau je. correction off a severe class ii malocclusion that required two stage orthognatic procedure: a case report. am j orthod dentofac orthop 1995; 108(4): 421-7. 175 vol. 42. no. 4 october–december 2009 correction parameters in conventional dental radiography for dental implant barunawaty yunus department of radiology faculty of dentistry, hasanuddin university makassar indonesia abstract background: radiographic imaging as a supportive diagnostic tool is the essential component in treatment planning for dental implant. it help dentist to access target area of implant due to recommendation of many inventions in making radiographic imaging previously. along with the progress of science and technology, the increasing demand of easier and simpler treatment method, a modern radiographic diagnostic for dental implant is needed. in fact, makassar, especially in faculty of dentistry hasanuddin university, has only a conventional dental radiography. researcher wants to optimize the equipment that is used to obtain parameters of the jaw that has been corrected to get accurate dental implant. purpose: this study aimed to see the difference of radiographic imaging of dental implant size which is going to be placed in patient before and after correction. method: the type of research is analytical observational with cross sectional design. sampling method is non random sampling. the amount of samples is 30 people, male and female, aged 20–50 years old. the correction value is evaluated from the parameter result of width, height, and thick of the jaw that were corrected with a metal ball by using conventional dental radiography to see the accuracy. data is analyzed using spss 14 for windows program with t-test analysis. result: the result that is obtained by t-test analysis results with significant value which p<0.05 in the width and height of panoramic radiography technique, the width and height of periapical radiography technique, and the thick of occlusal radiography technique before and after correction. conclusion: it can be concluded that there is a significant difference before and after the results of panoramic, periapical, and occlusal radiography is corrected. key words: panoramic radiography, pericapical radiography, occlusal radiography, dental implant, dental radiography correspondence: barunawaty yunus, c/o: bagian radiologi, fakultas kedokteran gigi universitas hasanuddin. jl. perintis kemerdekaanjl. perintis kemerdekaan km.10, talamanrea/jl. kandea no. 5, makassar, indonesia. e-mail: barunawaty@yahoo.combarunawaty@yahoo.com introduction radiographic imaging as a supportive diagnostic tool is the essential component in planning treatment using implant, and has benefit in helping dentist to access target area of implant due to recommendation of many inventions in making radiographic imaging previously. although invention of technology has produced new innovation for dental implant, a conventional dental radiography tools is still the most commonly used to measure the quality and quantity of jawbone.1 periapical and oclusal radiography is a radiography method that produces resolution imaging with a smoother and sharper result. maxillary and mandible periapical radiography are generally used to evaluate the relationship status of teeth and alveolar bone that are still in the mesiodistal direction. this radiography type can also be used to determine the vertical level, form and quality of bone such as bone density, area around cortical bone, and trabecular bone, so that it can be used for the dental implant treatment.2 a radiography that can show mouth tissues wider than periapical film is occlusal radiography. this radiography is able to give information on cross-sectional way, and also used to see the condition of alveolar ridge in mandible with buccolingual and faciolingual direction which is very useful in dental implant treatment.2 panoramic radiographic provides very useful information about the status of teeth in general and the relationship between the alveolar bone, basal bone, and anatomical structure that is not possible to conduct any dental implant. although the research report 176 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 175-178 imaging of panoramic experiences enlargement, but the length and number of dental implant that will be placed on the edentulous area to support the implant still can be estimated. intraoral periapical radiography can help and very important in estimating the mesiodistal dimension that is potential for implant placement and getting the initial estimation of vertical dimension. a combination between intraoral and panoramic imaging is often recommended for initial evaluation of implant target area.1 tooth lost care can be done in various ways, in line with the development of technology in dentistry. dental implant care is progressively popular at this time. dental implant is an alternative treatment that can overcome many limitations of conventional artificial tooth.3–5 dental implant is an artificial tooth that replaces the root and used in prosthodontic to support the restoration of artificial tooth.6 dental implant is an ideal tooth replacement at this time, because its feature and shape can resemble the original tooth. dental implant is made from titanium metal that is biocompatible.7 various modern radiographic imaging diagnostic devices are used for dental implant care, but in makassar, the availability of the equipment is still very limited. in the faculty of dentistry hasanuddin university makassar a modern three-dimensional radiographic imaging is not available. viewing the facts mentioned above, effort is required to optimize and improve the quality of conventional dental radiographic diagnostic information. although it is very simple, either for the quality or quantity of the jawbone for dental implant, parameter still can be obtained accurately by using many conventional dental radiographic diagnostics which are corrected such as panoramic radiography, occlusal radiography, and periapical radiography closely in the implementation of the technique. this effort can lead to more accurate parameters of width, height and the thickness of jaw, so that it may help in determining parameter of dental implant that will be placed in the patients. material and method the design of research is analytical observational with pre-post test approach. the subjects are 30 humans with criteria as follows loss of 1–2 teeth, aged 20–50 years old, and have good bone density. tools used are extraoral radiography set and intraoral radiography by using the panoramic radiography, periapical radiography, and occlusal radiography techniques. film used is pericapical intraoral film with 3 × 4 cm in size, occlusal film with 5.7 × 7.5 cm in size, and panoramic film with 15 × 30 cm in size. a metal ball with 6 mm in size is used for correcting the radiographic results. research is conducted at the mouth and dental education hospital, dentistry faculty of hasanuddin university on february to march 2009. research procedure that is conducted starts with patient that comes to hospital and wants to be treated with dental implant who meets the criteria of research is informed to be subject based on his/her consent. the clinical examination is then conducted on the teeth that will be placed with implant. radiographic diagnostic examination is conducted with the three radiographic techniques namely panoramic radiography, periapical radiography, and occlusal radiography. before the image of target area of implant is taken, the area is given a metal ball with 6 mm in size as a material to make a correction. after the results of three radiographic techniques is obtained, the next step is analyzing the measurement of the width, height, and thick jawbone before and after correction, as an effort to get the accurate size of the dental implant that will be placed in patient. data is analyzed by using spss 14 for windows program. then it is tested using the t-test analysis to get the right parameters from the width, height, and thick of jawbone before and after correction by using conventional dental radiography techniques which are panoramic, periapical, and occlusal radiography. result the research result can be seen in table 2, 3, and 4. the result that is obtained by t-test analysis results with significant value which p < 0.05 in the width and height of panoramic radiography technique (figure 3), the width and height of periapical radiography technique (figure 1), and the thick of occlusal radiography technique (figure 2) before and after correction. figure 1. periapical radiography with a metal ball. figure 2. occlusal radiography with a metal ball. 177yunus: correlation parameters in conventional dental figure 3. panoramic radiography with a metal ball. table 1. correction parameters in conventional radiography no radiography mean (mm) differencebefore correction after correction 1 occlusal 7.46 7.32 0.16 2 periapical (height) 11.30 10.62 0.71 3 periapical (width) 10.56 9.87 0.72 4 panoramic (width) 11.58 10.29 1.32 5 panoramic (height) 13.58 11.78 1.81 table 2. results of panoramic radiography on 30 subjects before and after correction measurement mean (mm) significancebefore correction after correction width 11.58 10.29 0.000*** (p < 0.05) height 13.58 11.78 0.000*** (p < 0.05) table 3. results of periapical radiography on 30 subjects before and after correction measurement mean (mm) significancebefore correction after correction width 10.56 9.87 0.000*** (p < 0.05) height 11.30 10.61 0.000*** (p < 0.05) table 4. results of occlusal radiography on 30 subjects before and after correction measurement mean (mm) significancebefore correction after correction thick 7.46 7.32 0.001*** (p < 0.05) discussion the three radiographic techniques above found radiographic techniques arranged by the accuracy: 1) occlusal radiography technique in thick/buccolingual measurement which is the most precise technique that almost approach the accurate value of the actual jawbone, 2) periapical radiography techniques in height/vertical measurement, 3) in width/mesiodistal measurement, 4) panoramic radiography techniques in width/mesiodistal measurement and 5) in height/vertical measurement. panoramic radiography technique gives a whole maxillomandibular image in one film, can reduce the time consuming, requires little oromaxillofasial radiographic expertise, and it does not give any uncomfortable effect for the patient. but in addition to positive things, one thing that has to be remembered is the magnification. because of magnification, lack of definition and overlapping structure, the diagnosis of panoramic radiography possibly less accurate compare with intraoral radiography. the difference is appeared as a correction parameter value and it is used as a subtracted value (table 1). the size of dental losing area which is measured in certain radiography method is subtracted with the difference value or correction parameter value to get the real size of alveolar bone space. there are a difference between before and after correction on panoramic radiography technique (table 2). both in width/mesiodistal and high/vertical measurement has significant result (p < 0.05). this is because the panoramic radiography technique experiences an enlargement image from the original size. distortion on panoramic radiography technique cannot be avoided because of the illumination towards film, and structure projections which varies in some individuals and between individuals themselves. differences in shape and size of jawbone and teeth, variations in the order of teeth on jaw and asymmetry between the right and left, all of them cause a difference in distortion degree.8 panoramic radiography is considered only as a complement of examination, not as a substitute for pericapical radiography. panoramic radiography should be used in the examination of wide jawbone area, for example the edentulous patient, the patient who does not need intraoral radiography well, or patient with wide pathologic symptom.9 panoramic radiographic provides very useful information about the status of teeth in general and the relationship between the alveolar bone, basal bone, and anatomical structure that is not possible to conduct any dental implant. although the imaging of panoramic experiences enlargement, but the length and number of dental implant that will be placed on the edentulous area to support the implant still can be estimated.1 there are difference of parameter values before and after correction on periapical radiographic technique (table 3). both in width/ mesiodistal and high/vertical measurement of jawbone, has significant result (p < 0.05). this is because the periapical radiography has certain distance between film and x-ray which is 16 inches, and film position and ray source is set in such a way to make it upright. although periapical radiography with a parallel technique has relatively high sharpness and accuracy but image magnification is still 178 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 175-178 inevitable. this is because variation in morphology of residual alveolar ridge.2 periapical radiography is helpful and very important in estimating the width/mesiodistal dimension that is potential for implant placed and getting the initial estimation of height/vertical dimension. a combination between panoramic and periapical radiography is often recommended for initial evaluation of dental implant target area.1 the difference of parameter values before and after correction on occlusal radiography technique was seen on table 4. the thick/buccolingual measurement of the jawbone has significant value result which is in p < 0.05. although this is the most significant and accurate techniques to a correction value but still it has enlargement. this is because only a part of the widest jawbone that can be measured, that is the bottom edge of mandible. in addition, this technique cannot describe the maxillary well because of anatomical limitation.10 in previous research it obtained a significant result before and after radiographic examination of panoramic radiographic, occlusal radiographic, and periapical radiographic with p < 0.05. another thing was also shown by reddy and wang,11 that implant position in jaw alveolus is needed to know by random block design to ensure that the same implant variety doesn’t always get expected position in jaw alveolus. from the results of this research, it can be concluded that there is a difference before and after the results of panoramic, periapical, and occlusal radiography is corrected in a measurement of jaw bone area that will be installed with dental implant. references 1. anil s. a method of gauging dental radiographs during treatment planning for dental impalnts. the journal of contemporary dental practice 2007; 8(6): 1–3. 2. shetty v, benson. bw. orofacial implants. in: white sc, pharoah mj, editors. oral radiology principles and interpretation: 5th ed. st. louis: mosby,; 2004. p. 677–91. 3. block m, kent j, guerra l. implants in dentistry: essential of endosseous implant for maxillofacial reconstruction. philadelphia: wb saunders company; 1997. p. 74–147. 4. misch ce. contemporary impant dentisrty. 2nd ed. st. louis: mosby; 1999. p. 73–118. 5. elsubehi es, attard n, zarb ga. implant prosthodontics for edentulous patients: currents and future directions. in: zarb ga, bolender cl, editors. prosthodontic treatment for edentulous patiens complete dentures and implant-supported protheses. 12th ed. st louis: mosby; 2004. p. 528–38. 6. adipatria a, mastuti i, sejati ir. kegagalan perawatan implan. 2008. available from http://images.bahasajiwa.multiplycom/attachment/0/ seijfgokcoeaaeutsec1/kegagalan%20perawatan%implan. ppt?nmid=98824365. accesed october 10, 2008. 7. weiss mc, weiss a. implant dentistry nomenclature, classification, and examples. principles and practice of implant dentistry. 2001. st louis: mosby; p. 7. 8. mason r. a guide to dental radiography. britain: bristol john wright and sons limited; 1997. p. 20–35. 9. gibilisco ja, turlington eg, del van grevenhof. radiography techniques. stafne’s oral radiographic diagnosis. 5th ed. philadelphia: wb saunders co; 1985. p. 410–43. 10. de lyre wr, johnson on. essential of dental radiography for dental assistants and hygienist. 4th ed. connecticut: appleton & lange; 1990. p. 319–28. 11. reddy ms, wang ic. radiographic determinants of implant performance. adv dent res 1999; 13: 145. vol 49 no 1 jan-mrt 2016.indd 10 potency of stichopus hermanii extract as oral candidiasis treatment on epithelial rat tongue syamsulina revianti and kristanti parisihni department of oral biology faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: oral candidiasis is the most prominent oral fungal infection with candida albicans (c. albicans) as 75% of ethiologic factor. golden sea cucumbers (stichopus hermanii) have been consumed by asian community as folk medicine. it has been known to have antifungal and immunomodulator agent thus potential to be explored as treatment in oral candidiasis. purpose: the aim of this study was to examine the potency of stichopus hermanii extract as oral candidiasis treatment. method: the study was an experimental laboratories research with post test only control group design. thirty male wistar rats were divided into 5 groups i.e negative control, positive control and 3 treatment groups. oral candidiasis condition were induced by spraying c. albicans suspension on dorsal tongue of wistar rats, once in 2 days for 14 days. the treatment groups were given stichopus hermanii extract on the dose of 4.25 ml/ kgbw, 8.5 ml/kgbw, 17 ml/kgbw once daily for 14 days. the expression of anti c. albicans antibody and tnf-α were examined by immunohistochemistry on ephitelial tongue. data was analyzed by manova and lsd test. result: anti c. albicans antibody expression were higher in positive control group than in negative control group while tnf-α expression were lower in positive control group than in negative control group (p<0.05). treatment with stichopus hermanii extract on all doses decreased the expression of anti c. albicans antibody and increased the expression of tnf-α (p<0.05). conclusion: stichopus hermanii extract decreased the expression of anti c. albicans antibody and increased the expression of tnf-α in epithelial rat tongue. keywords: stichopus hermanii extract; oral candidiasis; rat; epithelial tongue correspondence: syamsulina revianti, department of oral biology, faculty of dentistry universitas hang tuah. jl. arif rahman hakim 150 surabaya 60111, indonesia. e-mail: syamsulinarevianti16@gmail.com. research report introduction oral candidiasis is the most prominent oral fungal infection caused by candida albicans (c. albicans), the commensal microflora in human skin, vagina, and intestine. c. albicans also cause infections in condition with underlying diseases such as diabetes, prolonged broad spectrum antibiotic administration, steroidal chemotherapy and aids.1 recently, the incidence of any of candidosis type have been increased, it raised about 50% of oral candidosis cases.1,2 the impairment of the immune system expression is strongly correlated with the virulence of c. albicans in the oral cavity.3 the adherence property,colonization, enzyme production and interactions with host defences plays their role of the pathogenicity.3 the opportunistic fungus c. albicans is a major cause of oral and esophageal infections in immunocompromised patients such as human immunodeficiency virus (hiv)infected individuals and the elderly. other predisposing condition as hyposalivation, diabetes mellitus, prolonged use of antibiotics or immunosuppressive drugs, use of dentures, and poor oral hygiene played the role in oral c. albicans infection. it seems that oral candidiasis is not life threatening but still it caused significant morbidity and were increasing by the time. some drugs such as azole antifungal agents has been used to treat this fungal infection. hiv-positive patients received highly active antiretroviral treatment showed significantly fewer episodes of oral candidiasis than those without highly active antiretroviral therapy. however, drug-resistance and side effects are dental journal (majalah kedokteran gigi) 2016 march; 49(1): 10–16 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 1111revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 something to be consider regarding to long-term treatment with antifungal drugs.2,4 nystatin, ravuconazole, clotrimazole, fluconazole and ketoconazole are antifungal drugs choices commonly used in candidiasis treatment. in recent years, polyenes andazole agents have been used for treating infections caused by c. albicans.4 empirically, some natural plant products have also been used, but the recent recurrent infections have revived interest in the products. it has been known that some plant essential oils are having some health benefits as antifungal, antibacterial, anti-inflammatory and antioxidative properties. anyway, the scientific validation of their use as preventive and therapeutic products still need to be considered before the application in human health. in oral candidiasis, herbal formulations and phytotherapies play the major role.5 in the era of globalization and trading, it has been noticed that sea cucumbers such as stichopus hermanii have increasing high commercial value. sea cucumber have some medicinal benefits regarding to its bioactive compound as triterpene glycosides (saponins), chondroitin sulfates, glycosaminoglycan (gags), sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, cerberosides, lectins, peptides, glycoprotein, glycosphingolipids and essential fatty acids. sea cucumber components and bioactives posses the multiple biological and therapeutic properties of their potential uses for beneficical functional foods and nutraceuticals. recent research stated that sea cucumber extract have its biomedical properties.6 it has been proved that the aqueous and organic extracts from some sea cucumber species have antioxidant activities,4,5,8 immunomodulator6-8 while it also been known to have antimicrobial properties on gram negative, gram positive bacteria6,9,10 and antifungal action.11-13 the biocompatibility to oral cells have to be assured by identified the cytotoxicity. during the last years, the interest of in vitro systems as an alternative to animal experiments in toxicological research has been increased. the current models of predictive toxicology. have been increased by the using of stem cells and their derivatives as the developing in vitro, human cell assays.14,15 considering to the bioactive compound, stichopus hermanii extract is potentially explored its immunomodulator and antifungal property as the potential candidate therapeutic agent in oral candidiasis. sea cucumber extract has been consumed as tonic and traditional medicine, some has been produced as small industry herbal medicine products but the doses of treatment has not been established rather than just once to twice a day consumption. prior to in vivo study to examine the proper doses of treatment in oral candidiasis., we have been studied the antifungal potency of stichopus hermanii extract to c. albicans in vitro and its cytotoxicity to gingival derivedmesenchymal stem cell.16 based on empirical regular consumption of sea cucumber extract and the conversion to animal model, three doses of stichopus hermanii extract as therapeutic agent of oral candidiasis were explored in this research. the aim of this study was to examine the potency of stichopus hermanii extract as oral candidiasis treatment using immunohistochemical technique. the result of this study could be served as preliminary data to be continued in preclinical and clinical research with marine natural products which will probably result in novel therapeutic agents for the treatment of oral disease. materials and methods the design of this research was post-test only control group design. the materials investigated in this study is the golden sea cucumber (sticopus hermanii) ethanol extract which tested for its anti-inflammatory potency in vivo in wistar rats induced by c. albicans for oral candidiasis condition. therapeutic efficacy studies were performed against c. albicans. the culture was stored at -20º c in sabouraud dextrose broth containing 15% glycerol until use. for inoculation, c. albicans was grown on sabouraud dextrose agar plates at 30ºc for 24 h. fungal colonies were then scraped off the agar, washed three times in phosphate buffered saline (pbs) ph 7 and solution was adjusted to appropriate concentration using heamocytometer.4 stichopus hermanii weight of 100-250 grams were taken ± from karimun jawa coastal. sea cucumbers were cleaned, cut into pieces with a size of 3-10 cm, weighed wet weight after it dried in the solar dryer rack for sample until it looks dry (3-4 days) to reduce the water content. samples were dried sea cucumber, cut into pieces ± 1 cm, pulverized in a blender.the extraction process was done by the maceration process, by soaking 250 grams of dried sample in 500 ml of solvent refined methanol until all samples submerged and allowed to stand at room temperature for 24 hours. after filtered with a filter paper to separate the filtrate and residue, it was then soaked again with 500 ml of methanol solvent for 24 hours. after filtered with a filter paper to separate the filtrate and residue. filtrate thus obtained results maceration with 250 gram sample comparison/1000 ml of solvent (1: 4 w/v). filtrate methanol (polar) conducted homogenization with hexane solvent (non-polar) and 1,000 ml done with separatory funel partition, then each layer of the filtrate solvent methanol and hexane solvent separated. methanol filtrate done homogenization back with chloroform solvent (semi-polar) and 1,000 ml done with separatory funel partition, then each layer of the filtrate solvent methanol and chloroform solvent separated. each filtrate was then separated from the solvent using a rotary evaporator to obtain the extract.11,17 the whole experimental plan was approved by faculty of dentistry, universitas hang tuah ethical committee. three months-old, male wistar strain rats weighing approximately 175 g each were used. the rats were housed in 480 x 270 x 200 mm cages. photoperiods were adjusted to 12/12 h light and darkness cycle daily, and the environmental temperature was constantly maintained dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 12 revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 at 21 ± 1° c. access to food and water was allowed ad libitum to rats. sample were 30 male wistar rats, divided into 5 groups i.e negative control, positive control and 3 treatment groups. oral candidiasis condition were induced in all groups except negative control group, performed by spraying c. albicans. suspension to dorsal tongue of wistar rats, once in 2 days for 14 days. the treatment groups were treated by stichopus hermanii extract using feeding tube, consecutively on the dose of 4.25 ml/kgbw, 8.5 ml/kgbw, 17 ml/kgbw. on the 14th day rats were terminated and the tongue were biopzied and fixed in 10% neutral-buffered formalin and embedded in paraffin. tissue was sliced (6 to 10 mm) at the cryostat temperature of around –18 to –20º c. sections were air dried at room temperature for at least 30 minutes, and then immersion-fixed in acetone for 1 to 2 minutes at room temperature. subsequently, endogenous peroxidase activity was blocked for 10 minutes with 1% h2o2 diluted in pbs. slides were then washed with pbs containing 1% bovine serum albumin (bsa) and background staining was blocked with powdered skim milk (3% in phosphate buffered saline). sections were incubated overnight at 4º c with anti c. albicans antibody and anti-tnf-α (santa cruz biotechnology,usa) and then were washed three times for 5 minutes each in pbs plus 1% bsa and incubated at room temperature for 45 minutes with biotinylated antibody. after being washed three times, in pbs plus 1% bsa, for 5 minutes each, sections were incubated with ab complex (vector laboratories,usa) for 45 minutes. sections were washed again and the reaction was revealed by dab (sigma-aldrich,usa) and finally counterstained with mayer’s hematoxylin. the control slide was not incubated with the primary antibody. tissue sections from at least one mouse of each group were processed at the same time. photomicrographs were taken with light microscope (olympus america inc.,usa) equipped with an automatic camera system with magnification of 400x by two histopathologists, blinded to sample type.18,19 data was analyzed with oneway anova and lsd test. data was analyzed by anova and lsd test at 95% significance level. results the data on figure 1 were presented as means + standard deviation. statistical analysis was performed using anova and lsd test to determine the effect of stichopus hermanii extract as oral candidiasis treatment using immunohistochemical technique. results of anova and lsd test showed a significant difference between the control and treatment groups (p<0.05). the result on figure 2 showed the highest anti c. albicans antibody expression in epithelial tongue of positive control group that inoculated with c. albicans compared to negative control group and treatment groups. the expression of anti c. albicans antibody were not found in negative control group which were not induced by c. albicans. the higher dose of stichopus hermanii extract given, the lower anti c. albicans antibody expression resulted. thus indicates that stichopus hermanii extract could decrease anti c. albicans antibody expression with dose of 4.25 ml/kgbw, 8.5 ml/ kgbw, 17 ml/kgbw (p<0.05). the result in figure 3 showed the lowest tnf-α expression in epithelial tongue of positive control group that inoculated with c. albicans compared to negative control group and treatment groups. in treatment groups, the higher dose of stichopus hermanii extract given, the lower tnf-α expression resulted. thus indicates that stichopus hermanii extract could decrease tnf-α expression with dose of 4.25 ml/kgbw, 8.5 ml/kgbw, 17 ml/kgbw (p<0.05). discussion in vivo studies used wistar rats that had been commonly used in oral candidiasis experimental because it has several advantages that are relatively easy to manage and have adequate oral cavity size for inoculation and sampling because of tongue is the most area predilection for candida infection.20 negative control group is a group of normal mice were not inoculated with c. albicans. the result showed that the expression of anti c. albicans antibody were not found in the negative control group, which is in negative control positive control stichopus hermanii extract dose 4.25 ml/kgbw stichopus hermanii extract dose 8.5 ml/kgbw stichopus hermanii extract dose 17 ml/kgbw anti c.albicans antibody expression tnfα after figure 1. the expression of anti c. albicans antibody and tnf-α after addition of stichopus hermanii extract. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 1313revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 line with the assumption that in the normal animals there is no process of candidiasis. c. albicans is not the normal flora of the oral cavity of mice.21 the positive control group and the treated group inoculated with c. albicans for 14 days. stichopus hermanii extract were given orally with three concentrations, compared with positive and negative control group were given only 1% cmc solution. the procedure adopted in the present study exhibited a progressive increase in the number of colonies after inoculation, which demonstrated that infection was successful on the dorsal side of the tongue. after inoculation of c. albicans does not appear to be any significant differences in the clinical manifestations between the normal group and infected group. inoculated with c. albicans for 14 days is not expected to provide significant clinical manifestations appear but the immunohistochemical examination is expected to give an idea about the condition of oral candidiasis by examining the expression of anti c. albicans antibody. based on the presence of the expression of anti c. albicans antibody is assumed that there is the presence of c. albicans in a certain amount of tongue that indicates a condition of oral candidiasis. oropharyngeal candidiasis is the most common infection associated with oral injuries.22 c. albicans is part of the normal microbial flora of mucous surfaces, can be present as acquired defects of cell-mediated immunity. the adherence capacity, colonization, enzyme production and interactions with host defences determine its the pathogenicity.20 the impairment of the immune system is highly related with the expression of c. albicans virulence in the oral cavity. the defense immune mechanisms to fungal infections are various, started from protective mechanisms that were in innate immunity to adaptive immunity mechanisms that are specifically induced during the infection. skin and mucous membranes are the first-line innate defense as physical barriers, which is complemented by cell membranes, some cellular receptors and humoral factors.23 oral epithelial cells has its role on the process of inflammation as protective effort or destructive result, via amplifying signals in infection responses. it considered to play the important role in host defense, including antigen presentation. as response to infection, cytokines/ chemokines were produced, one of those are tnf-α which capable to induce expression of other mediators such as prostaglandins. it could amplify the inflammatory response therefore leads lytic enzymes production and stimulates the production of chemokines. it has been studied that cytokines, such as tnf-α played important roles in the control of oral mucosal infection and can be lead to tissue destruction. based on these findings, the enhancement of tnf-α expression by c. albicans infection appears to be involved in the initiation and/or amplification of inflammatory responses to oral candidiasis.23,24 the antifungal drugs of choice to treat oral candidiasis commonly are nystain, ravuconazole, clotrimazole, fluconazole and ketoconazole. in recent years, polyenes and azole agents have been usedfor treating infections caused by c. albicans.25,26 the existing conventional drugs, however pose several undesirable side effects. a continuous need for new drugs, especially the biocompatible and bio–based drugs is expected to overcome the side effct and resistantance. natural plant products have also been used figure 2. the expression of anti c. albicans antibody intongue ephitelial. expression of anti-c. albicans antibody in rat tongue epithelial with immunohistochemistry technique (400 magnification). brown color shows presence of anti c. albicans antibody expression (→), a) expression of anti c. albicans antibody of positive control group; b) expression of anti c. albicans antibody of treatment group 1 (stichopus hermanii 4.25 ml/kgbw); c) expression of anti c. albicans antibody of treatment group 2 (stichopus hermanii 8.5 ml/kgbw); d) expression of anti c. albicans antibody of treatment group 3 (stichopus hermanii 17 ml/kgbw). figure 3. the expression of tnf-α intongue ephitelial. expression of tnf-α in rat tongue epithelial with immunohistochemistry technique (400 magnification). brown color shows presence of tnf-α expression (→), a) expression of tnf-α of positive control group; b) expression of tnf-α of treatment group 1 (stichopus hermanii 4.25 ml/kgbw); c) expression of tnf-α of treatment group 2 (stichopus hermanii 8.5 ml/kgbw); d) expression of tnf-α of treatment group 3 (stichopus hermanii 17 ml/kgbw). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 14 revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 as folk medicine, but the recent recurrent infections have revived interest in the products. in oral candidiasis, herbal formulations and phytotherapies play a major role.27 sea cucumbers, especially stichopus hermanii have been known to have various health benefits, such as antifungal, antibacterial, anti-inflammatory and antioxidative properties. the lack of scientific validation of their use as preventive and therapeutic products restricts their application in human health. stichopus hermanii extract is known to have anti-fungal properties which contained saponins, alkaloids, and triterpenes that to act as antifungal agent. in earlier laboratory studies using etahnol extract of stichopus hermanii, it was found that the active fraction of the ethanolic extracts were able to control the biofilm formation of candida spp. and more over the extracts were able to kill c. albicans. biofilm formation is one of the pivotal factors in establishing the infections in the host.7,11,17 this study is a continuation of previous studies which have known of the power of its anti fungal agent against c. albicans in vitro.28 in the present study we carried out animal experiments to assess the effect of stichopus hermanii extract as a marine medicinal to treat oral candidiasis. in this study stihopus hermanii extract contains triterpene and saponins as an active anti-fungal agent with ethanol extraction method according to research pranoto et al.11 this antifungal compounds soluble in ethanol as a polar solvent that has antifungal and antibacterial activity.8 triterpenes, which comprise a broad chemical group of active principles, are implicated in the mechanisms of action and pharmacological effects of many medicinal plants used in folk medicine against diseases in which the immune system is implicated.29 the results showed that administration of stichopus hermanii extract in all treatment groups decrease anti c. albicans antibody expression and increase tnf-α expression. it is estimated that there are other possible mechanisms of saponins in the potential anti-fungal and immunomodulatory agent. stichopus hermanii extract are one of the potential marine animals with multiple biological activities and medicinal value. therefore, marine echinoderms can be explored as a sustainable natural source for the discovery of novel antifungal agent. bioactive substances in sea cucumbers, such as triterpene glycosides, enzymes, amyloses, fatty acids, cytotoxins, etc. with potential capabilities to antifungal activities and increase immunity as well as contribute to immunopotentiation. in this study, ethanol extract of stichopus hermanii showed promising antifungal and immunostimulantory activity in vivo. it was shown that stichopus hermanii extracts were effective against c. albicans at a concentration of 4,25 mg/kg bw until 17 mg/kg bw. the inhibition of fungal growth started from the lowest to the highest tested extract concentrations showed that the amount of extract present related with its antifungal activity. saponins produced as a form of chemical defense mechanism for sea cucumbers in nature. in addition to alleged as a defense from predators, also believed to have biological effects, including anti fungal and immune activity.6 saponin is a class of compounds that inhibit or kill microbes by interacting with membrane sterols, contribute as antifungal with membrane sterols that can decrease surface tension of the cell wall of c. albicans, so the permeability was increased,29 similar to the mechanism nystatin action. saponins may also result in apoptosis because it can damage the mitochondrial membrane cell, lowering the transmembrane potential, increase cytosolic calcium and activates apoptosis pathways through calcium.30,31 its capabilities that triggers macrophages in response to an infection in which this mechanism as antifungal. prior research showed that sea cucumber protein contained rich of glycine and arginine, especially produced from its body wall. glycine contributes to enhancing phagocytosis by stimulating production and release of il-2 and b cell antibody, while arginine can enhance cell immunity by promoting activation and proliferation of t-cell. sea cucumbers have remarkable function in immune regulation due to these amino acid components.6 this research showed that the expression of tnf-α were low in the control positive group, suggesting the reduction of immune activity happened in infection of c. albicans. in immuneresponse to c. albicans, tnf-α played the role as primary immunity in immune system regulation. specifically to macrophage, this cytokine increased the activity in killing pathogens, in which this action became an important mediator in inflammation.32 treatment with stichopus hermanii extract could increase tnf-α activity. its related to macrophage phagocytosis activity to c. albicans. function of phagocytosis and tnf-α is playing important role at macrophage level which also influence the degradation of adaptive immune response on c. albicans on the other side with existence of ethanol extract from stichopus hermanii condition of imunosuppresion resulted from c. albicans will be improve and repaired. macrophage as professional phagocyte function to break immunogen as antigen presenting cells (apc) which recognizes microbe through some receptors its to stimulate migration of cell to the place of infection and stimulates the production of substance. improvement mechanism of tnf-α by stichopus hermanii to c. albicans started by the existence toll-like receptor (tlr)-2 and tlr-4. it has been highlighted that tlr-2 and tlr-4 are involved in recognition of candida and aspergillus. fungal wall components zymosan, phospholipomannan and glucuronoxylomannan have been identified as ligands or pathogen associated molecular patterns (pamps) for tlr-2, while glucoronoxylomannan and o-linked mannan are ligands for tlr-4.32 recognition of microorganism by tlr-2 and tlr-4 then activated nuclear factor kappa b (nf-κb) and activator protein-1 (ap-1) through jun kinase via the mitogen-activated protein kinase (mapk) pathway. after the release of i-κb, an increase would occur inthe activity of transcription factor nf-κb which stimulatedgene expression that affected the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 1515revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 production of tnf-α and phagocytic activity. stimulation of gene expression among others affected the production of tnf-α.32,33 it also been stated that c. albicans can evade the host defense through tlr2derived signals. the tlr2-deleted macrophages have been found to have enhanced anticandidal capabilities, while in-vivo study stated that tlr-2 knock out mice are relatively more resistant to disseminated c. albicans infection. hence the tissue-invasive hyphal forms of c. albicans, by means of evading tlr4 in favor of a predominant tlr-2 activation, are able to tilt the balance towards a th2 response33 resulted in changes in inflammatory response, assumed to be related with tnf-α production and activation. the capability of stichopus hermanii extract in increasing the expression of tnf-α facilitate the proper immune response in combating c. albicans infection. the decreasing of antic. albicans antibody expression showed the antifungal property of sticophus hermanii extract and with addition of its property in enhancing the immune response by increasing tnf-α expression will be the advantage of the potential agent for oral candidiasis treatment. it is concluded that ethanol extract from stichopus hermanii has the potency as oral candidiasis treatment by decreasing the expression of anti c. albicans antibody and increasing the expression of tnf-α significantly in epithelial tongue of rats inoculated with c. albicans. acknowledgement this research was supported by a grant from hibah bersaing research program, funded by ministry of research, technology and higher education and culture indonesia 2015-2016. references 1. greenberg m, glick m. burket’s oral medicine: diagnosis and treatment. 10th ed. new york: bc decker inc.; 2003. p. 564-8, 570-2. 2. william d, lewis m. pathogenesis and treatment of oral candidosis. j oral microbiol 2011; 3: 5771. 3. netea mg, brown gd, kullberg j, gow na. an integrated model of the recognition of candida albicans by the innate immune system. nat rev microbiol 2008; 6(1): 67-78. 4. niimi m, firth na, cannon rd. antifungal drug resistance of oral fungi. odontology 2010; 98(1): 15-25. 5. chami n, chami f, bennis s, trouillas j, remmal a. antifungal treatment with ca r vacol and eugenol of oral candidiasis in immunosuppressed rats. braz j infect dis 2004; 8(3): 217-26. 6. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods-a review. mar drugs 2011; 9(10): 1761-805. 7. mayer ams, rodriguez ad, berlinck rgs, hamann mt. marine pharmacology in 2005–6: marine compounds with anthelmintic, antibacter ial, anticoagulant, antifungal, anti-inf lammator y, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. bhiochim biophys acta 2009; 1790(5): 283-308. 8. avilov sa, kalinin vi, silchenko as, aminin dl, agafonova ig, stonik va, collin pd, woodward c, inventor; process for isolating sea cucumber saponin frondoside a, and immunomodulatory methods of use. united states patent us 7,163,702 b1. 2007. 9. abraham tj, nagarajan j, shanmugam sa. antimicrobial substances of potential biomedical importance from holothurian species. indian journal of marine sciences 2002; 31(2): 161-4. 10. pringgenies d, ridlo a, kemal taj. the potency antibacterial of bioactive compound of holothuria atra extract from territorial water of bandengan. manado: world ocean conference; 2009. 11. pranoto en, ma’ruf wf, pringgenies d. kajian aktivitas bioaktif ekstrak teripang pasir (holothuria scabra) terhadap jamur candida albicans. jurnal pengolahan dan bioteknologi hasil perikanan 2012; 1(1): 1-8. 12. hua h, yi yh, li l, liu bs, la mp, zhang hw. antifungal active triterpene glycosides from sea cucumber holothuria scabra. acta pharmaceutica sinica 2009; 44(6): 620-4. 13. pringgenies d, ocky kr, sabdono a, hartati r, widianingsih. penerapan teknologi budidaya teripang dalam meningkatkan produksinya dan bioprospek teripang sebagai sumber senyawa antimikroba untuk kesehatan. laporan penelitian. hibah kemitraan hi-link; 2008. p. 65. 14. jeon km. international review of cell and molecular biology. 1st ed. san diego: elsevier academic press; 2009. p. 161-202. 15. ekwall b, silano v, stammati p, zucco f. toxicity tests with mammalian cell cultures. in: bourdeau p, editor. short-term toxicity tests for non-genotoxic effects. scope: john wiley & sons ltd.; 1990. p. 75-82. 16. parisihni k, revianti s. antifungal effect of sticophus hermanii and holothuria atra extract and its cytotoxicity on gingiva-derived mesenchymal stem cell. dent j (majalah kededokteran gigi) 2013; 46(4): 218-23. 17. rasyid a. identifikasi senyawa metabolit sekunder serta uji aktivitas antibakteri dan antioksidan ekstrak metanol teripang stichopus hermanii. juenal ilmu dan teknologi kelautan tropis 2012; 4(2): 360-8. 18. bouquot je, speight pm, farthing pm. epithelial dysplasia of the oral mucosa-diagnostic problems and prognostic fatures. current diagnostic pathology 2006; 12(1): 11–21. 19. marinho m, monteiro crm, peiro jr, machado gf, oliveira-junior is. tnf-α and il-6 immunohistochemistry in rat renal tissue experimentaly infected with leptospira interrogans serovar canicola. j venom anim toxins incl trop dis 2008; 14(3). 20. samaranayake yh, samaranayake lp. experimental oral candidiasis in animal models. clin microbiol rev 2001; 14(2): 398-429. 21. naglik jr, fidel pl jr, odds fc. animal models of mucosal candida infection. fems microbiol lett 2008; 283(2): 129-39. 22. kamai y, kubota m, kamai y, hosokawa t, fukuoka t, filler sg. new model of oropharyngeal candidiasis in mice. antimicrob agents chemother 2001; 45(11): 3195-7. 23. blanco jl, garcia me. immune response to fungal infections. vet immunol immunopathol 2008; 125(1-2): 47–70. 24. villar cc, kashleva h, mitchell ap, dongari-bagtzoglou a. invasive phenotype of candida albicans affects the host proinflammatory response to infection. infect immun 2005; 73(8): 4588-95. 25. clemons kv, stevens da. treatment of orogastrointestinal candidosis in scid mice with fluconazole alone or incombination with recombinant granulocyte colony-stimulating factor or interferon-gamma. med mycol 2000; 38(3): 213-9. 26. clemons kv, stevens da. efficacy of ravuconazole in treatment of mucosal candidosis in scid mice. antimicrob agents chemother 2001; 45(12): 3433-6. 27. kalemba d, kunicka a. antibacterial and antifungal properties of essential oils. curr med chem 2003; 10(10): 813-29. 28. parisihni k, revianti s, pringgenies d. the antifungal effect of stichopus hermanii extract to candida albicans in vitro. proceeding of 5th hiroshima conference on education and science in dentistry 2013; p. 115. 29. r ío s j l . e f fe c t s of t r it e r p e n e s o n t h e i m m u n e s ys t e m . j ethnopharmacol 2010; 128(1): 1-14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 16 revianti and parisihni/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 10–16 30. podolak i, galanty a, sobolewska d. saponin as cytotoxic agent: a review. phytochem rev 2010; 9(3): 425-74. 31. wojtkielewicz a, długosz m, maj j, morzycki jw, nowakowski m, renkiewicz j, strnad m, swaczynová j, wilczewska az, wójcik j. new analogues of the potent cytotoxic saponin osw-1. j med chem 2007; 50(15): 3667-73. 32. gauglitz gg, callenberg h, weindl g, korting hc. host defence against candida albicans and the role of pattern-recognition receptors. acta derm venereol 2012; 92(3): 291-8. 33. chai ly, netea mg, vonk ag, kullberg bj. fungal strategies for overcoming host innate immune response. med mycol 2009; 47(3): 227-36. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p10-16 mkgs vol 45 no 2 april-juni 2012.indd 68 volume 45 number 2 june 2012 case report the management of chronic traumatic ulcer in oral cavity maharani laillyza apriasari department of oral medicine study program of dentistry, faculty of medicine, lambung mangkurat university kalimantan selatan indonesia abstract background: the traumatic ulcer is one of the most common oral mucosal lesions. the etiology of traumatic ulcer may result from mechanical trauma, as well as chemical, electrical, or thermal stimulus, may also be involved in addition, fractured, malposed, or malformed teeth. the clinical manifestation of traumatic ulcer are ulcer, have a yellowish floor, fibrinous center, red and inflammatory margin without induration. purpose: the purpose of this case report is to present how to manage the patient with the chronic traumatic ulcer in oral cavity. case: this case report is about the patient with chronic ulcer in oral cavity. intra oral examination showed on the right tongue margin appeared the major ulcer, single, diameter 1,5 cm, pain, white color, induration and irreguler margin around the ulcer. the patient had been suffering it for 5 months. she had come to a lot of dentist and the oral maxillofacial surgery, but they could not heal the ulcer. the dental occlusion of the patient, especially 17 and 47 then 15 and 45 teeth was looked bitten the right tongue. it underlied to get the clinical diagnosis as the chronic traumatic ulcer. case management: the main therapy of traumatic ulcer is eliminiting the etiology factor, so that decided to do teeth extraction 45 and 47 that was looked linguversion position on 45 degrees. before doing the teeth extraction, the patient was referred to take complete blood count (cbc), blood glucose examination and biopsy. the monitoring of the ulcer must be done until 2 weeks after the teeth extraction. if the lesion was persistent, it is suspected as malignancy. conclusion: it can be concluded that the main management of chronic traumatic ulcer in oral cavity is removing the etiology factors. if the ulcer is still persistent after 2 weeks from the etiology factor had been removing, it is suspected as the malignancy that is needed biopsy examination to get the final diagnosis. key words: biopsy, chronic, management, traumatic ulcer abstrak latar belakang: ulkus traumatikus adalah salah satu lesi pada mukosa mulut yang sering terjadi. penyebab ulkus traumatikus adalah adanya trauma mekanik, seperti kimia, elektrik atau suhu, selain itu dapat pula terjadi karena fraktur, malposisi atau malformasi gigi. manifestasi klinis dari ukus traumatikus adalah ulser, dasar berwarna kuning, pada bagian tengah tampak fibrin, pinggiran berwarna merah dan mengalami keradangan tanpa adanya indurasi. tujuan: tujuan dari laporan kasus ini adalah untuk melaporkan bagaimana penatalaksanaan pasien dengan ulkus traumatikus kronis pada rongga mulut. kasus: kasus ini melaporkan tentang ulser kronis yang terjadi pada rongga mulut. pemeriksaan pada rongga mulut menunjukkan pada pinggir lidah kanan tampak ulser mayor, tunggal, diameter 1,5 cm, sakit, berwarna putih, pinggiran sekitarnya tampak indurasi dan tidak teratur. ulser terjadi selama 5 bulan. pasien mengunjungi banyak dokter gigi dan spesialis bedah mulut, tetapi ulser tidak dapat disembuhkan. pada saat pasien oklusi, pada gigi, 17 dengan 47 serta gigi 15 dengan 45 tampak lidah sebelah kanan tergigit. hal ini yang mendasari diagnosis sementaranya adalah ulkus traumatikus kronis. tatalaksana kasus: penanganan utama dari ulkus traumatikus adalah menghilangkan faktor penyebab, oleh sebab itu dilakukan ekstraksi pada gigi 45 dan 47 yang terlihat posisi linguoversi 45 derajat. sebelum gigi-gigi tersebut diekstraksi, pasien dirujuk untuk melakukan pemeriksaan darah lengkap, gula darah dan biopsi. ulser harus tetap dimonitor sampai 2 minggu pasca ekstraksi. jika lesi menetap, maka ini diduga squamous cell crsinoma. kesimpulan: dapat disimpulkan bahwa penatalaksanaan utama dari ulkus traumatikus kronis pada rongga mulut adalah dengan menghilangkan faktor penyebab. ulser yang persisten setelah 2 minggu setelah faktor penyebab dihilangkan, maka diduga suatu keganasan yang perlu pemeriksaan biopsi untuk menegakkan diagnosis akhir. kata kunci: biopsi, kronis, penatalaksanaan, ulkus traumatikus 69apriasari: the management of chronic traumatic ulcer correspondence: maharani laillyza apriasari, program studi kedokteran gigi, fakultas kedokteran gigi universitas lambung mangkurat. jl. veteran 128 b banjarmasin, kalimantan selatan, indonesia. e-mail: rany.rakey@gmail.com introduction traumatic ulcer is one of the most common mucosal lesions in oral medicine. the lesion injuries involving the oral cavity may typically lead to the formation of surface ulcerations. the injuries may result from events such as accidentally biting oneself while talking, sleeping, or secondary to mastication. other forms of mechanical trauma, as well as chemical, electrical, or thermal stimulus, may also be involved in addition, fractured, caries, malposed, or malformed teeth. poorly maintained and ill-fitting dental prosthetic appliances may also cause trauma.1 traumatic ulcers are usually caused by a denture and often seen in the buccal or lingual sulcus. the etiology of traumatic ulcers is the accidental injury. the clinical manifestation of uler traumatic are tender to painful, have a yellowish floor, fibrinous center, red and inflammatory margin, and no induration. if caused by the sharp edge of a broken-down tooth, they are usually on the tongue or buccal mucosa. occasionally, a large ulcer is caused by biting the cheek after a dental local anaesthetic. during the healing phase they frequently develop a ‘keratotic halo’.2-4 the differential diagnosis of the traumatic ulcer are recurrent aphthous stomatitis, squamous cell carcinoma, and tuberculosis ulcer.1,5 the diagnosis of traumatic ulcer is usually based on the history and the clinical examination. if the ulcer is still persistent after 2 weeks or the ulcer clinical manifestation suspects the malignancy so that must be done a biopsy that is necessary to rule out malignancy.1,2,5 this case report show the importance of biopsy to help the final diagnosis of chronic ulcer. she had been suffering it for 5 months and had been visiting a lot of doctors as dentist, the oral and maxillofacial surgeon, the neurologist, and the oto laryngologist. actually none of them asked her to do biopsy, although she had been suffering the chronic ulcer more than 1 month. all of them could not heal the chronic ulcer. case the patient, woman, 33 years, had been suffering stomatitis on right of tongue margin since 5 months ago. she was difficult for talking and eating which made the loss body weight until 10 kg. the ulcer had never been cured, pain, numb sensation of the tongue and difficult speaking. the patient had been given 36% policresulen and 0.1% triamcinolone acetonide, but it was not healed. she decided to see the spesialist of oral maxillofacial surgery whom grinding the tooth 47. the tooth was on linguoversion position until 45 degrees that suspected as etiology of the traumatic ulcer. she was given the oral drugs by the dentist as amoxycillin 500 mg three times a day, mefenamic acid 500 mg three times a day, dexamethason 0.5 mg three times a day and the topical drug as 0.1% triamcinolone acetonide, oral base three times a day. actually she was still not cured, so that she decided to visit the oral and maxillofacial surgeon, the oto laryngologist and the neuorologist, but they referred to the dentist. finally all of them could not heal the ulcer. case management on the first visit, the extra oral of clinical examination showed the right submandibula lymphenode was palpable, chewy, swelling and not pain. the intra oral examination showed on the right tongue margin appeared the major ulcer, single, diameter 1.5 cm, pain, white color, induration and irreguler margin around the ulcer (figure 1). the ulcer was bitten by between teeth 16 and 17 with 47 on occlusion position (figure 2). based on this condition, this case was diagnosed as chronic ulcer traumatic. the differential diagnosis was squamous cell carcinoma, it was caused the induration and irreguler margin around the ulcer. for getting the final diagnosis, the patient was reffered to undertake biopsy, complete blood count (cbc) and blood glucose examination. the therapy had been given to the patient was the topical drugs as alloevera gargle. the patient was asked to control after she got the result of biopsy, blood glucose examination and cbc. on visit 2 (1 day after visit 1), the result of cbc showed patient was not severe anemia and normal of blood glucose. the biopsy result from ulcer on the right tongue margin showed distribution of epithel squamous cell with round nucleus, spread cytoplasma, not rough chromatin and reguler nucleus membran. it was meant there was not the malignancy cell. based on the biopsy result, the final diagnosis was the chronic ulcer traumatic. the patient was figure 1. chronic major ulcer, single, diameter 1.5 cm, tender to painful, white, eleveted periphery. 70 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 68–72 asked to do teeth 45 and 47 extration. the therapy was oral drugs as cefadroxil 500 mg 2 x 1, calium diclofenac 50 mg 3 x 1 and multivitamin 1 x 1 for 5 days. on visit 3 (3 days after visit 1), the patient complained that her lips and tongue were pain and chapped after drinking the drugs. the intra oral examination showed lips and tongue were erosion, erytheme, little bleeding, and pain. the clinical diagnosis was the allergic stomatitis because of consume cefadroxil 500 mg. the patient asked to change cefadroxil 500 mg with amoxycillin 500 mg, then continuing to consume calium diclofenac 50 mg 3 x 1 and multivitamin for 5 days. for curing the allergy, the patient was given cetirizine hcl 10 mg 1 x 1 for 5 days. on visit 4 (6 days after visit 1), the result of anamnesis showed the lips and tongue had been cured, the ulcer on right tongue margin was more better, diameter 0.5 mm, erytheme and the tongue might be moved more better (figure 3). the wound post extraction was still pain. the patient instruction stopped to consume cetirizin, but continue to consume amoxicillin 500 mg 3 x 1, calium diclofenac 50 mg 3 x 1 and multivitamin contains zinc for five days. figure 2. the ulcer was lied on the oclusion position between teeth 16 and 17 with 47. figure 3. the lips and tongue had been cured, the ulcer on right tongue margin was more better, diameter 0.5 mm, erytheme and the tongue might be driven more. figure 4. the ulcer on right tongue margin had cured, but there was a new ulcer near the tooth 45, diameter 0.5 mm, pain, white color and surrounded erytheme. figure 5. the ulcer was suspected as traumatic ulcer because it was bitten by the teeth 15 and 45 on occlusion position. figure 6. the wound post extraction of tooth 45 was still pain and not recovered yet, but the ulcer was healed. finally the patient could be cured. 71apriasari: the management of chronic traumatic ulcer on visit 5 (14 days after visit 1), the result of clinical examination showed the ulcer on right tongue margin had cured, but there was a new ulcer near the tooth 45, diameter 0,5 mm, pain, white color and surrounded erytheme (figure 4). the ulcer was suspected as traumatic ulcer because it was bitten by the oclusion of the teeth 15 and 45. the teeth 45 was linguoversion (figure 5). the patient was asked to do teeth 45 extraction and continue to consume amoxycillin 500 mg 3 x 1, calium diclofenac 50 mg 3 x 1 and multivitamin contains zinc 1 x 1. on visit 6 (19 days after visit 1), the result of anamnesis showed ulcer had cured. the wound post extraction of tooth 45 was still pain and not healed yet. the patient was asked for continuing to consume amoxycillin 500 mg 3 x 1, calium diclofenac 50 mg 3 x 1 and multivitamin for 5 days. finally the patient could be cured (figure 6). discussion this case presents about the woman, 33 years old that was suffering the chronic ulcer on right tongue margin. she had been suffering the ulcer for 5 months and never recovered. the patient had come to a lot of dentists, the oral and maxillofacial surgeon, the neurologist, and the oto laryngologist, but the ulcer is still persistent. none of them asked her to do biopsy, although she had been suffering the chronic ulcer more than 2 weeks. the intra oral examination showed in oclusion position, the teeth 17 and 47 then the teeth 15 and 45 was bitting the right tongue. the oral and maxillofacial surgeon had ever been grinding the teeth 47, but the tongue was still bitten. the teeth 45 and 47 were linguoversion on 45 degrees. based on the clinical examination, the diagnosis was chronic traumatic ulcer. the main therapy of traumatic ulcer is removing the etiology factors, so that the patient was referred to do the extraction of teeth 47 and 45. before doing extraction of teeth 45 and 47, the patient was referred to take cbc, blood glucose examination and biopsy. cbc examination will show the anemia condition, because the patient looked skiny and pale. it might because of her difficult condition for eating caused the loss body weight until 10 kg for 5 months. actually the anemia condition caused the oxigen transport and nutrition was disturbed. it made the enzyms activity on mitochondria of red blood cells was not in a good process, so that obstructed the differentiated of epithel cells growth. the terminal differentiated of ephitel cells to stratum corneum was disturbed, then the oral mucous would be thinner. there was not the normal keratinezed, atrophy, and disruption of the healing process.3 the malnutrition condition made the ulcer was difficult to get the healing process, so in this case it aggravated the ulcer for recovering. the result of cbc was showed not severe anemia, so the anemia was not the blood glucose examination of the patient was normal, so it was not the etiology of the chronic ulcer. this examination was done because sometimes the patient with undiagnosed or inadequately treated diabetes suffers xerostomia and candidiasis that cause the stomatitis, sore tongue, and non specific glossitis.4 the clinical manifestation of ulcer on the right tongue margin were major ulcer, diameter 1.5 cm, pain, white color, surrounding induration margin and irreguler. it had been suffering since 5 months ago and never been healed. based on the clinical manifestation the differential diagnosis was squamous cell carsinoma. it was caused some reasons there was the induration margin arround the chronic ulcer and the ulcer had been more than 2 weeks without healing procces. the biopsy must be done to get the final diagnosis as squamous cell carsinoma.1,2,5 the malignant lesion was not determined just by seeing the clinical examination, it needs the others examination to determine it as early detection by 1% toluidine blue aplication, brush biopsy and scalpel biopsy.6 the dentists must know the possibility of lesion changes to be carsinoma. the lesion as chronic ulcer, white or red color, and swelling on mucous membran must be confirmed by biopsy.2 early diagnosis helps the patient with malignancy to get recovery quickly. the small malignancy lesion can spread as extensive lesion rapidly. most carsinoma is found in late condition which is in difficult phase to be cured.6,7,9 the patient was done the brush biopsy by specialist of anatomic pathology. the biopsy result from ulcer scrapping did not show the malignancy cell, so it was diagnosed as the chronic traumatic ulcer. the main therapy was removing etiology factors, so the teeth 47 and 45 had to be done the extraction. the patient was given the oral drugs as amoxycillin 500 mg three times a day and calium diclofenak 50 mg three times a day for 5 days. post extraction had been done by the dentist, the ulcer was cured. after removing the etiology factor of traumatic ulcer, the monitoring of the lesion must be done. if the traumatic ulcer is persistent, although the etiology factors had been removed after 2 weeks, accordingly the patient must be done the biopsy. it may be a carsinoma.5,10 after the lesion treatment had done, the dentists must avoid the trauma because of the denture or the sharp carries teeth, monitor the malignancy lesion appeared, and give the good mental support to the patients.1,2,8 it can be concluded that the main management of chronic traumatic ulcer in oral cavity is removing the etiology factors. if the ulcer is still persistent after 2 weeks from the etiology factor had been removing, it is suspected as the malignancy that is needed biopsy examination to get the final diagnosis. references 1. laskaris g. treatment of oral disease: a concise textbook. new york: thieme, stuttgart; 2005. p. 169. 2. cawson ra, odell ew, porter s. cawson’s essensials of oral pathology and oral medicine. 7th ed. uk: churchill livingstone, elsevier; 2002. p. 251–3. 3. apriasari ml, hendarti ht. stomatitis aftosa rekuren oleh karena anemia. jurnal dentofasial 2010; 9(1): 45. 72 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 68–72 4. field a, longman l. tyldesley’s oral medicine. 5th ed. new york, usa: oxford university press; 2004. p. 51–2. 5. sciubba jj, regezi ja, rogers rs. pdq oral disease: diagnosis and treatment. hamilton, london: bc decker inc; 2002. p. 90–1. 6. guneri p, epstein jb, kaya a, veral a, kazandi a, boyacioglu h. the utility of toluidine blue staining and brush cytology as adjuncts in clinical examination of suspicious oral mucusal lesions. int j oral maxillofac surg 2010; 40(2): 155–61. 7. eipstein jb, guneri p. the adjunctive role of toluidine blue in detection of oral premalignant and malignant lesions. current opinion in otolaryngology and head & neck surgery 2009 april; 17(2): 79–87. 8. scu l ly c. o r a l a nd ma x i l lofa cia l me d ici ne: t he ba sis of diagnosis and treatment. london, british: wright elsevier; 2004. p. 287–310. 9. agus p. prognostic value of molecular markers of oral pre-malignant and malignant lesions. dent j (maj ked gigi) 2009; 42(2): 104. 10. leosari y, hadiati s, agustina d. screening of premalignant lesions in smokers using toluidine blue. dent j (maj ked gigi) 2009; 42(2): 90. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 36 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 36–42 the potentiation of mangifera casturi bark extract on interleukin1β and bone morphogenic protein-2 expressions during bone remodeling after tooth extraction bayu indra sukmana,1 theresia indah budhy,2 and iga wahju ardani3 1department of dental radiology, faculty of dentistry, universitas lambung mangkurat, banjarmasin indonesia 2department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, surabaya-indonesia 3department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: the main oral health problem in indonesia is the high number of tooth decay. tooth extraction is the treatment often received by patients who experience tooth decay and the wound caused by alveolar bone resorption. bark of mangifera casturi has been studied and proven to contain secondary metabolite which has the ability to increase osteoblast’s activity and suppress osteoclast’s activity. purpose: the purpose of this study was to analyze interleukin-1 beta (il-1β) and bone morphogenic protein-2 (bmp-2) activities during bone remodeling after mangifera casturi’s bark extract treatment. method: this study was laboratory experimental research with randomized post-test only control group design. the mangifera casturi bark was extracted using 96% ethanol maceration and n-hexane fractionation. this study used 40 male wistar rats which are divided into 4 groups and the tooth extraction was performed on the rats’ right mandible incisive tooth. the four groups consisted of 6.35%, 12.7%, 25.4% extract treatment group, and a control group. wistar’s mandibles were decapitated on the 7th and 14th day after extraction. antibody staining on preparations for the examination of il-1β and bmp-2 expressions was done using immunohistochemistry. result: there was a significant difference of il-1β and bmp-2 expressions in 6,35%, 12,7%, and 25,4% treatment groups compared to control group with p<0.05. conclusion: mangifera casturi’s bark extract was able to suppress the il-1β expression and increase the bmp-2 expression during bone remodeling after tooth extraction. keywords: bone morphogenic protein-2; bone remodeling; interleukin-1β; mangifera casturi (kosterm.); tooth extraction correspondence: theresia indah budhy, department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: terebudhy@gmail.com introduction bones are the structures built for supporting the body which characteristics are rigid, solid, and have the ability to regenerate and repair themselves.1 injury on bones might be caused by both pathological and physiological conditions.1,2 injury on jaws, especially mandible, is one of the most common injuries of maxillofacial region, its prevalence ranging from 36% to 59%.3 injury on jaw bones can also happen after tooth extraction.1 regeneration and repair of bone tissues are important processes to heal the wounded tissues. bone tissues repair can be classified into four stages: hematoma formation, soft callus formation, hard callus formation, and bone remodeling.1,2 in hematoma formation stage, fibroblast and osteoblast migrate to the fracture site and start reconstructing bones. the amount of bone remodeling regulators in the body will affect osteogenic process post tooth extraction.1,4 these regulators include osteoblasts, osteoclasts, systemic factors such as hormones and vitamins and local factors such as interleukin and bone morphogenic protein (bmp). interleukin-1 beta (il-1β) has a role as inflammatory cytokines that regulate immune system. these cytokines are produced by macrophage and other inflammatory cells. il-1β also gives a chemotactic effect on other inflammatory cells, stimulates extracellular matrix synthesis, angiogenesis, recruits endogen fibrogen cells to the wound site, and in bone resorption stage. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 3737sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 bmp-2 stimulates osteoconductive activities to regulate differentiation and proliferation of mesenchymal cells into osteoblasts.5 bones are the body’s dynamic hard tissues, always undergoing repair when needed. bone tissues repair is mainly mediated by osteoblasts, the bone-forming cells and osteoclasts, which resorb bones. alveolar bones repair after tooth extraction molecularly involves receptor activator of nf-kb ligand (rankl) which stimulates the osteoclast formation, and osteoprotegerin (opg) which would bind rankl, thus decreasing osteoclasts and bone resorption.6 bone tissues repair can be accelerated using either natural or synthetic materials. some of these materials have disadvantaging side effects such as rejection from the recipient’s body, faster bone degradation, osteolysis, and ineffective cost.7 the limitations of these synthetic materials lead many researchers to seek out natural materials which can be easily found around and have fewer side effects. mangifera casturi is the native plant of kalimantan. some of this plant’s parts have been known to have active substances and can be potentially used with medical purposes. the plant was reported to have alkaloids, flavonoids, saponins, steroids, triterpenoids, and tannins.8,9 casturi’s bark contains saponins, steroids, and tannins, which have antibacterial properties against s. mutans,10 s. aureus, and e.coli.11 the tannins contained in 127.42 grams of mangifera casturi bark extract is 8.5%.12 tannins are polyphenols, belonging to the fenolic acid class. tannins were reported to have effect in increasing the osteoblast’s activities and suppressing the osteoclast’s activities.9 the increase of osteoblast’s activities by alkaloids and polyphenols such as tannins is because the induction of il-1β and bmp-2 expressions on wound site.13 secondary substances such as polyphenols in foods and plants, one of them being mangifera casturi, can help bone tissues repair by increasing the osteoblast’s activities and suppressing the osteoclast’s activities.9 terpenoids in casturi bark extract was assumed to play a role in bone regeneration process. triterpenoids have many prominent physiological activities, thus they are used medically in daily life to help cure diabetes, menstruation problems, venomous snake’s bite, skin diseases, liver diseases, and malaria. in plants which contain triterpenoids, there’s an ecological value to it because these substances work as antifungal, insecticide, anti-predator, antibacterial, and antiviral substances.14 the purpose of this study was to examine about the effect of mangifera casturi bark extract on il-1β and bmp-2 expressions during bone remodeling post tooth extraction. materials and methods this study was true experimental laboratory research, using randomized post test only control group design. samples used were counted using lemmeshow equation and each group consisted of 5 samples. before the research was carried out on experimental animals, the design has been approved by ethics committee of the faculty of dental medicine, universitas airlangga. right mandible incisors from the male wistar rats were extracted using forceps. outer surface of the forceps were sharpened to improve the internal pressures so they could clasp firmer around the tooth during extraction. before extraction, rats were sedated intraperitoneally using sodium pentobarbital (50 mg/ kg bb); then they were anesthetized with infiltration technique on lingual fold using 2% lidocaine (1:100.000), to produce the effect of local anesthesia and homeostasis. after extraction, sockets were filled by 6.35%, 12.7% and 25.4% mangifera casturi bark extract. the control group was treated using gel without extract. post-extraction sites were then sutured using 5.0 monofilament sutures. after this treatment, each animal was administered trimethoprim-sulfa 30 mg/ kg subcutaneously, which worked as antibiotics, per 12 hours for 3 days. two days post treatment, animals were given soft food to minimize further trauma and prevent delayed healing. after this period, standard foods were given. foods were given orally using normal saline 5 ml/ kg/ day. after that, rats were sacrificed on day 7 and day 14 through injection of sodium pentobarbital (50 mg/ kg bb). their mandible parts were decalcified using 10% edta. the expressions of il-1β and bmp-2 were observe on day 7 and 14 using immunohistochemistry methods. results examinations were done using 400x zoom/ 15625µ2 in ten fields of view, and the results were calculated to find out the means of each animal. the means were then summed to find out the final means of treatment groups. statistical tests were carried out to assess the significance of each treatment group. the observations of il-1β expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 1 and 2). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mannwhitney test to assess the significance of the differences. the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 20.55 ± 1.761, 20.80 ± 3.222, 14.55 ± 2.911, 13.00 ± 1.806 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. duncan test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group and 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups; meanwhile the il-1β expression in control group dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 38 sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 compared to 6.35% extract treatment group and also in 12.7% extract treatment group compared to 25.4% extract treatment group had no significant differences (table 1). the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 19.55 ± 1.605, 22.90 ± 3.007, 14.90 ± 3.401, 14.85 ± 3.731 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12,7% and 25.4% extract treatment groups; the il-1β expression in 6.35% extract treatment group compared to 12.7%, and 25.4% extract treatment groups had significant difference, but in 12.7% extract treatment group compared to 25.4% extract treatment group, there was no significant difference (table 1). the observations of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 3 and 4). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mann-whitney test to assess the significance of the differences. the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 7.7 ± 2.179, 11.35 ± 2.519, 15.30 ± 2.386, 15.75 ± 2.807 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12.7% and 25.4% extract treatment groups; the bmp-2 expression in 6.35% extract treatment table 1. means and standards deviation of il-1β and bmp-2 expressions group il-1β bmp-2 day 7 day 14 day 7 day 14 control 20.55 ± 1.761a 19.55 ± 1.605b 7.7 ± 2.179a 8,9 ± 1,804a 6.35% extract 20.80 ± 3.222a 22.90 ± 3.007a 11.35 ± 2.519b 10,15 ± 1,387b 12.7% extract 14.55 ± 2.911b 14.90 ± 3.401c 15.30 ± 2.386c 17.40 ± 1.759d 25.4% extract 13.00 ± 1.806b 14.85 ± 3.731c 15.75 ± 2.807c 15.90 ± 3.110c 5 the observations of il-1β expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 2 and 3). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mannwhitney test to assess the significance of the differences. the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 20.55 ± 1.761, 20.80 ± 3.222, 14.55 ± 2.911, 13.00 ± 1.806 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. duncan test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group and 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups; meanwhile the il-1β expression in control group compared to 6.35% extract treatment group and also in 12.7% extract treatment group compared to 25.4% extract treatment group had no significant differences (table 1 and figure 4). figure 2. il-1β expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. 5 the observations of il-1β expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 2 and 3). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mannwhitney test to assess the significance of the differences. the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 20.55 ± 1.761, 20.80 ± 3.222, 14.55 ± 2.911, 13.00 ± 1.806 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. duncan test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group and 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups; meanwhile the il-1β expression in control group compared to 6.35% extract treatment group and also in 12.7% extract treatment group compared to 25.4% extract treatment group had no significant differences (table 1 and figure 4). figure 2. il-1β expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. a b c d figure 1. il-1β expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 3939sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 6 the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 19.55 ± 1.605, 22.90 ± 3.007, 14.90 ± 3.401, 14.85 ± 3.731 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12,7% and 25.4% extract treatment groups; the il-1β expression in 6.35% extract treatment group compared to 12.7%, and 25.4% extract treatment groups had significant difference, but in 12.7% extract treatment group compared to 25.4% extract treatment group, there was no significant difference (table 1 and figure 4). figure 3. il-1β expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. 6 the means of il-1β expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 19.55 ± 1.605, 22.90 ± 3.007, 14.90 ± 3.401, 14.85 ± 3.731 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12,7% and 25.4% extract treatment groups; the il-1β expression in 6.35% extract treatment group compared to 12.7%, and 25.4% extract treatment groups had significant difference, but in 12.7% extract treatment group compared to 25.4% extract treatment group, there was no significant difference (table 1 and figure 4). figure 3. il-1β expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. a b c d figure 2. il-1β expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show il-1β expression. 7 figure 4. means and standards deviation of il-1β expression. the observations of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 5 and 6). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mannwhitney test to assess the significance of the differences. figure 5. bmp-2 expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression. 7 figure 4. means and standards deviation of il-1β expression. the observations of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats were done using immunohistochemistry methods (figure 5 and 6). cells were counted using kruskal-wallis test to assess the differences between groups, and continued by using mannwhitney test to assess the significance of the differences. figure 5. bmp-2 expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression. a b c d figure 3. bmp-2 expression in the post tooth extraction socket on day 7 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 40 sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 group compared to 12.7% and 25.4% extract treatment groups had significant difference, meanwhile in 12.7% extract treatment group compared to 25.4% had no significant difference (table 1). the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 8.9 ± 1.804, 10.15 ± 1.387, 17.40 ± 1.759, and 15.90 ± 3.110 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p < 0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group and 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups; the bmp-2 expression in control group compared to 6.35% extract treatment group had significant difference, and also in 12.7% extract treatment group compared to 25.4% there was a significant difference (table 1). discussion il-1β expression in alveolar sockets on day 7 and 14 after casturi bark extract treatment showed a decrease compared to control group, except for 6.35% extract treatment group which showed an increase. il-1β is involved in osteoclast differentiation. il-1β synergize with rankl to induce osteoclast induction and bone resorption and indirectly boost osteoclastogenesis through the excretion of pge2 and rankl by osteoblasts. il-1β cytokines have important roles on bone destruction by forming osteoclasts and increasing osteoclast’s activities. the main role of il-1β in bone metabolism is to stimulate bone resorption and delay the bone formation. these cytokines work by stimulating t and b lymphocytes to increase inflammatory responses, which is through stimulation of prostaglandin and degradative enzymes, such as colagenase. the bigger the decrease of il1β in remodeling process is, the more effective mangifera casturi extracts help improve post extraction wound site. tooth extraction is a treatment to take out tooth from maxilla and mandible because of dental-related disease, such as decay, periodontal diseases, and trauma.15 this treatment causes wound on alveolar sockets, and this wound will go through healing process, such as hemostasis, inflammation, proliferation, and remodeling. inflammation is the body’s response to clean out wound sites from foreign objects, bacteria, and dead cells thus the healing process can begin.16 the inflammatory process started from neutrophils, which is leukocytes, increasing around wound site. neutrophils work to clear foreign objects and bacteria, and then they would be replaced by macrophages. macrophages also work to synthesize collagen, form granulation tissues with fibroblasts, produce vascular endothelial growth factor (vegf)-a which would form new capillary vessels. il-1β play a role as stimulator of bone resorption before proliferation phase. tgf-β stimulates the production of vegf, which play a part in angiogenesis, a new blood vessels formation process. tgf-β also has an important role in immunoregulation through neurophilin-1 (nrp1, a protein which bind to both active or latent tgf-β). 8 the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 7.7 ± 2.179, 11.35 ± 2.519, 15.30 ± 2.386, 15.75 ± 2.807 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12.7% and 25.4% extract treatment groups; the bmp-2 expression in 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups had significant difference, meanwhile in 12.7% extract treatment group compared to 25.4% had no significant difference (table 1 and figure 7). figure 6. bmp-2 expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 8.9 ± 1.804, 10.15 ± 1.387, 17.40 ± 1.759, and 15.90 ± 3.110 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p < 0.05, proving that there was a significant difference 8 the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 7 were as following: 7.7 ± 2.179, 11.35 ± 2.519, 15.30 ± 2.386, 15.75 ± 2.807 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p<0.05, proving that there was a significant difference between groups. mann-whitney test was carried out next to find out the significance of differences between each treatment group. there was a significant difference of control group compared to 6.35%, 12.7% and 25.4% extract treatment groups; the bmp-2 expression in 6.35% extract treatment group compared to 12.7% and 25.4% extract treatment groups had significant difference, meanwhile in 12.7% extract treatment group compared to 25.4% had no significant difference (table 1 and figure 7). figure 6. bmp-2 expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression the means of bmp-2 expression in post mandibular incisor extraction sockets of wistar rats on day 14 were as following: 8.9 ± 1.804, 10.15 ± 1.387, 17.40 ± 1.759, and 15.90 ± 3.110 in control group, 6.35%, 12.7% and 25.4% mangifera casturi bark extract treatment groups respectively. kruskal-wallis test showed p < 0.05, proving that there was a significant difference a b c d figure 4. bmp-2 expression in the post tooth extraction socket on day 14 using microscope, 400x zoom. (a) control; (b) 6.35% mangifera casturi bark extract; (c) 12.7% mangifera casturi bark extract; (d) 25.4% mangifera casturi bark extract; yellow arrows show bmp-2 expression dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 4141sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 il-1β is the cytokine which play a part in inflammatory process, proliferation, tissue remodeling and maturation. tgf-β collaborate with growth factors, such as connective tissue growth factor (ctgf) to stimulate wound healing by forming fibroblast.17 the higher the expression of il-1β is, the faster inflammatory process resolves. the decrease of il-1β shows the end of inflammatory phase and the start of proliferation. healing on post extraction wound needs repair on both soft and hard tissues, such as alveolar bones. bones in mandible and maxilla are tissues with complex mineralization and always undergo remodeling through bone formation and resorption. repair on alveolar bones after extraction affects growth factors release and one of them plays a part in bone repair, which is bmp-2. proliferation phase is marked by granulation tissues formation on wound sites. new capillary vessels formation or angiogenesis is stimulated by vegf and will also synthesize the endothelial cells formation. new bone tissues formation is heavily influenced by bmp-2. bone formation in post extraction wound is membranous osteogenesis, started by collage secretions and then mineralization.16 bmp-2 is growth factors which is important in new bone formation and can be examined in signaling molecules accelerating bone formation. bmp-2 has in vivo osteoconductive activities compared to other bmp families.18 bmp-2 expression in alveolar socket day on 7 after 6.35%, 12.7%, and 25.4% extract treatments showed the increase of bmp-2 compared to control group. this increase showed a faster bone formation through cellular mechanism. bmp-2 expression on day 7 was dominated by osteoblasts. osteoblasts differentiation through bmp ligands bind to receptors, a pair of bmpr-i and bmprii form heterotetrameric-activated receptor complex protein smad, which is a substrate of bmpr-i, and has a function to relay signal from receptors to target gens in nucleus. dimeric ligand bond to heterometric bmp receptor activates intrinsic serine/threonine kinase and phosphorized r-smad activities. bmp-2 can phosphorize intracellular transducers, smad 1 and 5, which will begin the differentiation of osteoblasts.19 bmp-2 expression in alveolar socket day on 14 after 6.35%, 12.7%, and 25.4% extract treatments still showed the increase of bmp-2 compared to control group. according to references, bmp-2 on day 14 should decreased which shows the healing responses. this result showed that casturi bark extract hasn’t yet optimally help wound healing on day 14. bmp-2 activates tgf-β and boosts fibroblast formation. fibroblasts are cellular elements commonly found in gingival connective tissues which proliferate and actively synthesize matrix in wound healing and repair. fibroblasts are basic substances of scarring and collagens which give the tensile strength in soft tissue wound healing. during inflammation, fibroblasts will migrate to wound site, proliferate and produce collagen matrix to repair tissues.17 the higher the bmp-2 expression is, the faster proliferation can begin, the decrease of bmp-2 showed the end of proliferation and the start of remodeling. remodeling is the last phase in wound healing, in this phase, granulation tissues become mature, which is marked by mechanical strength on formed tissues, the decrease of capillary vessels in wound, the decrease of fibroblasts, and the increase of collagen fibers.15 in conclusion, mangifera casturi’s bark extract was able to suppress the il-1β expression and increase the bmp-2 expression during bone remodeling after tooth extraction references 1. fogelman i, gnanasegaran g, van der wall h. radionuclide and hybrid bone imaging. berlin, germany: springer; 2012. p.29-30 2. balaji sm. textbook of oral and maxillofacial surgery. philadelphia, usa: elsevier; 2007. p.985. 3. ghodke mh, bhoyar sc, shah sv. prevalence of mandibular fractures reported at csmss dental collegem aurangabad from february 2008 to september 2009. j int soc prevent communit dent 2013; 3(2): 51-8. 4. rusnawati m, carnozzi m, moroni e, boltazzi b, peri g, indraccolo s, anadoni a, mantovani a, presta m. selective recognition of fibroblast, growth factor 2 by the long pentraxin ptx-3 inhibits angiogenesis. american society of haematology 2004; 104(1): 92 101. 5. dimitriou r, tsiridis e, giannoudis pv. current concepts of molecular aspects of bone healing. int j care injured 2005; 36(12): 1392–404. 6. belibasakis gn, bostanci n. the rankl-opg system in clinical periodontology. j clin periodontol 2012; 39(3): 239-48. 7. oryan a, alidadi s, moshiri a, maffulli n. bone regenerative medicine: classic options, novel strategies, and future directions. journal of orthopedic surgery and research 2014; 9: 18. 8. suhartonoa e, viani e, rahmadhan ma, gultom is, rakhman mf, indrawardhana d. total flavonoid and antioxidant activity of some selected plants in south kalimantan of indonesia. asiapacific chemical, biological & environmental engineering society procedia 2012; 4: 235-9. 9. hapidin h, abdullah h, soelaiman in. the potential role of quercus infectoria gall extract on osteoblast function and bone metabolism. open journal of endocrine and metabolic diseases, 2012; 2: 82-8. 10. syarifuddin ni. perbandingan daya hambat anitbakteri ekstrak etanol kulit batang kasturi (mangifera casturi kosterm) dan ekstrak daun sirih terhadap pertumbuhan streptococcus mutans strain 2302unr secara in vitro. jurnal pharmascience 2014; 1 (2) : 46-53 11. rosyidah k, nurmuhaimina sa, komari n, astuti md. aktivitas antibakteri fraksi saponin dari kulit batang tumbuhan kasturi. alchemy 2010; 1(2): 53-103. 12. sutomo, agustina n, arnida, fadilaturrahmah. studi farmakognostik dan uji parameter nonspesifik ekstrak metanol kulit batang kasturi (mangifera casturi kosterm.). jurnal pharmascience 2017; 04 (01): 94-101. 13. khan i, kumar n, pant i, naria s, kordaiah p. activation of tgfbeta pathway by areca nut constituents: a possible cause of oral submucous fibrosis. plos one 2012; 7(12): e51806. 14. widiyati e. penentuan adanya senyawa triterpenoid dan uji aktivitas biologis pada beberapa spesies tanaman obat tradisional masyarakat pedesaan bengkulu. jurnal gradien 2006; 2(1): 116-22. 15. olczyk pl, mencner l, vassev kk. the role of the extracellular matrix component in cutaneous wound healing. biomed research international 2014; 1: 1-8. 16. tortora gj. principles of anatomy and physiology. 14th ed. new jersey, usa : wiley; 2013. p. 25-86. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 42 sukmana, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 36–42 17. prud’homme, gj. pathobiology of transforming growth factor β in cancer, fibrosis and immunologic disease, and therapeutic considerations. lab invest 2007; 87(11): 1077-91. 18. zou d, zhang z, ye d, tang a, deng l, han w, zhao j, wang s, zhang w, zhu c, zhou j, he j, wang y, xu f, huang y, jiang x. repair of critical-sized rat calvarial defect using genetically engi neered bone ma r rowder ived mesenchyma l stem cel ls overexpressing hypoxia-inducible factor-1α. stem cells 2011; 29(9): 1380-90. 19. chen, g., c. deng, & y. li. tgf-β and bmp signaling in osteoblast differentiation and bone formation. international journal of biological sciences. 2012. 8(2): 272 88. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p36-42 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p36-42 170 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 170–172 purple sweet potato (ipomea batatas p.) as dentin hypersensitivity desensitization gel chariza hanum mayvita iskandar, hardita bicevani mulya, windy pretyani kusumawati, and andina rizkia putri kusuma department of conservative dentistry faculty of dentistry, universitas islam sultan agung semarang indonesia abstract background: dentin hypersensitivity is a short sharp sense of pain in the teeth when exposed to excitatory stimulus. a total of 74% of world population experiencing dentin hypersensitivity. home treatment topical desensitization is rarely found in indonesia. the use of dentrifice is less practical because it must be done with regular brushing. indonesia has abundant natural resources, one of which is purple sweet potato. purple sweet potato (ipomea batatas p.) has highest potasium ions compared to other foodstuffs. potassium ions can be a solution of dentin hypersensitivity by temporary blocking the suffix pulp nerve impulses. purpose: the research objective was to determine the effectiveness of the 10% purple sweet potato extract gel of the dental pain threshold score. method: an experimental study carried out by dental pain threshold score measurements using vitality tester into the teeth with gum recession. samples included 32 respondents with a single blind and pre-post test control group design. they were divided into treatment group and negative control group. paired t-test and wilcoxon were used as data analysis. result: the results showed dental pain threshold score increasing either in treatment group and negative control, although not as significant as in the treatment group. conclusion: 10% purple sweet potato extract gel containing potassium ions is able to reduce the pain of dentin hypersensitivity. keywords: dentin hypersensitivity; potassium ion; purple sweet potato; desensitization correspondence: chariza hanum mayvita iskandar, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas islam sultan agung. jl. raya kaligawe km. 4, semarang 50112, indonesia. e-mail: chariza.i@std.unissula.ac.id introduction sensitive teeth or dentin hypersensitivity has become one of the problems experienced by many people. the sensation of short and sharp pain in patients with dentine hypersensitivity can reduce their activity comfort that may impact on the productivity and welfare of the individuals.1 dentin hypersensitivity is a hidden complaint to the sufferer, but it should not be ignored because it doesn’t fit the definition of healthy according to world health organization (who). healthy according to who is not only the absence of disease in terms of physical, but also mental, and social well being of individuals.2 people with dentin hypersensitivity in the world reached 74%, while in indonesia reached 45%.3 people with dentin hypersensitivity 67% of whom are women where at the age of 20-40 years are at greater risk of dentin hypersensitivity.4 pain due to dentin hypersensitivity caused by the movement of fluids within the dentinal tubules. triggers of dentinal fluid movement is the presence of external stimuli on the exposed dentin resulting pressure changes in the dentin and activates nerve fibers a delta in the pulp, causing sharp pain.5 desensitization toothpaste is one of the ways that can be used by dentin hypersensitivity patient.6 the solutions already exist today through the invasive and non-invasive treatments. gingivectomy is an invasive surgical treatment. in non-invasive treatment principle, the opened tubules is closed to block the hydrodynamic mechanism. desensitization toothpaste containing potassium ion basic material which capable to block the stimulus (heat, cold, touch, and chemical) to the type a delta nerve to pain respone.7 however, desensitization toothpaste is from synthetic material and less practical because it must be used regularly. indonesia have a lot of natural ingredients dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p170-172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p170-172 171171iskandar, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 170–172 that have a high potassium content and also affordable at a time likes purple sweet potato. when utilized optimally, it can be a solution of dentine hypersensitivity and increase the farmers and national’s income. the aim of this study was to find out the effectivity 10% potassium ions of purple sweet potato extract gel. materials and method this research is an experimental with single blind study design and pretest posttest control group design. the research took place at the laboratory of chemistry and physiology semarang state university and in the faculty of dentistry, universitas islam sultan agung. 10% purple sweet potato extract gel was made by mixing the extract of purple sweet potato with cmc-na, nipagin, and sterile distilled water. purple sweet potato was washed and then squeezed with a pres that the sap flowing in the shelter. sap were then collected in the glass using a funnel. sap was heated in the oven at 50º celcius to remove water. toxicity and inflammation test was carried out to 7 male wistar rats, as much as ±0,3 gram of 10% purple sweet potato extract gel was applied topically on the tooth surface and gingival tissue, it was left for 7 days. in the seventh day no inflammation sign was found on the gingival tissue and all of the male wistar rats was still alive. the sample used was 32 female respondents 20-40 years old with gingival recession. the samples were divided into two groups, the group treated with 10% purple sweet potato extract gel and a negative control group treated with petroleum jelly. inform concent was done before the study started. the measurement of the threshold value of dental pain stimuli was done on a labial surface of normal teeth and sensitive teeth by using a vitality tester with moderate rate. the tip of vitality tester which had been smeared by toothpaste placed on a dried labial surface of cementoenamel junction, hold the button to turned on the stimuli and released when the respondent felt short and sharp pain sensation. the result data obtained in this study were statistically analyzed by paired t-test. results according to the research on 32 samples with application of 10% purple sweet potato extract gel as a treatment (table 1). it shows in the post treatment category of treatment group is higher than the post treatment category in the control group. shapiro-wilk test showed that pre treatment category in all groups (treatment and control) are normally distributed, and also in post treatment category of a control group (p>0.05). then, the pre and post treatment category of treatment group was analyzed by wilcoxon nonparametric test, while the control group category before and after treatment were comparative tested by t-test. there is a significant difference between the pre-test and post-test in the treatment group (p=0.01). according to paired t-test p = 0.01, because p<0.05 it can be concluded there is a significant difference between the pre and post treatment in treatment group that showed significance value which is 0.05 (error tolerance limit), so that there is a difference between the pre and post treatment in control group. discussion dentin hypersensitivity is a common painful sensation, which is rather difficult to treat in spite of the availability of various treatment options. the main principles of dentin hypersensitive treatment are dentinal tubule occlusion and nerve desensitization.7 according to hydrodynamic theory, dentine hypersensitivity occurs due to the excitatory stimulus resulting in fluid rapid movement in the dentinal tubules. the pressure changes result in sensitized of pulp nerve endings in dentin causing brief pain.8 dentinal tubule occlusion was done to inhibit the fluid movement in the dentinal tubule.8 calcium phosphate, strontium chloride, and calcium sodium phosphosilicate are some materials that are usually used as a tubule occlusion.9 potassium ions is one of the material that could block the nerve impuls. in some research that was conducted to evaluate the efficacy of potassium, showed that 10% of potassium ions covered dentinal tubules by crystal formation.10 potassium does not cause tooth color changes, does not irritate the gums, and does not damage the dentin, so it is a proper material to used as a desensitization.11 purple sweet potato that has been extracted and made into a gel formulation contain potassium ion that is the most commonly used active ingredients in desensitizing dentifrices.10 it had an effect to blocked myelinated a-fibers in odontoblast processus of dentinal tubules. increased in extracellular potassium allows for the large concentration to depolarize the nerve fibers. as a result, neural transmission will not occur following exposure to the stimulus and the patient will have no sensation of sensitivity or pain.9 result of the research showed a significant value of dental threshold pain stimuli in the treatment group compared with control group. respondents who have been treated by the application of 10% purple sweet potato table 1. the average of pain threshold value in treatment and control group pain threshold value in control group pain threshold value in treatment group pre treatment post treatment pre treatment post treatment 29.63 µa 29.16 µa 25.94 µa 51.44 µa dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p170-172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p170-172 172 iskandar, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 170–172 extract gel showed a greater effect on the value increased of dental threshold pain stimuli. it means that purple sweet potato extract gel could reduce the pain of dentin hypersensitivity. research that conducted by orchardson et al.10 which evaluate the efficacy of potassium ion that contained in mouthwash and toothpaste showed that potassium produced a significant reduction in sensitivity to tactile and air stimuli. potassium ion could help to reduce the duration and intensity of tooth sensitivity caused by dental bleaching according to some review of several research.11 in conclusion, the present study was conducted to find out the effectivity 10% potassium ions of purple sweet potatos extract gel. it was found that 10% purple sweet potatos extract gel could reduce the pain threshold value of 32 respondent with dentin hypersensitivity. references 1. sischo l, broder hl. oral health-related quality of life what, why, how, and future implications. j dent res 2011; 90(11): 1264–70. 2. al shamrany m. oral health-related quality of life: a broader perspective. east mediterr health j 2006; 12(6): 894-901. 3. dhillon p, govila v, verma s. evaluation of various desensitizing agents in reducing dentin hypersensitivity using scanning electron microscope: a comparative in vitro study. indian dent j science 2014; 6(5): 031-5. 4. torabinejad m, richard ew. endodontics: principles and practice. 4th ed. india: elsevier inc; 2009. p. 259. 5. limeback h. comprehensive preventive dentistry. oxford: willeyblackwell; 2012. p. 218-32. 6. bartold pm. dentinal hypersensitivity: a review. aust dent j 2012; 51(3): 212-8. 7. walters pa. dentinal hypersensitivity: a review. j contemp dent pract 2005; 6(2): 107-17. 8. bedi g. clinical and scanning electron microscopic evaluation of various concentration of potassium nitrat as a desensitization agent. smile dent j 2011; 6(1): 38-45. 9. tjahajawati s, maskoen am, adhita, hd. pengaruh iontoforesis naf 2% dan kcl terhadap kadar mmp-8 pada gingival crevicular fluid (gcf) dentin hipersensitif kelompok usia dewasa. bionatura-jurnal ilmu-ilmu hayati dan fisik 2012; 14(1): 38-44 10. orchardson r, gillam dg. the efficacy of potassium salt as agent for treating dentin hypersensitivity. j orofac pain 2000; 14(1): 9-19. 11. po lh, wilson nw. effect of different desensitizing agents on bleaching treatment. eur j gen dent 2014; 3(2): 93-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p170-172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p170-172 �� volume 47, number 1, march 2014 research report antimicrobial proteins of snail mucus (achatina fulica) a g a i n s t s t r e p t o c o c c u s m u t a n s a n d a g g r e g a t i b a c t e r actinomycetemcomitans herluinus mafranenda dn, indah listiana kriswandini and ester arijani r department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: achasin and mytimacin-af are proteins of snail mucus (achatina fulica) which have antimicrobial activity. snail mucus is suspected to have other proteins which have antimicrobial activity against streptococcus mutans and aggregatibacter actinomycetemcomitans the oral pathologic bacteria. purpose: the study were aimed to characterize the proteins of snail mucus (achatina fulica) that have antimicrobial activities to streptococcus mutans and actinobacillus actinomycetemcomitans, and to compared the antimicrobial effect of achasin and mytimacin-af. methods: the sample of study was the mucus of snails which were taken from yogyakarta province. the isolation and characterization of protein were conducted by using sds-page method, electroelution, and dialysis. nano drop test was conducted to determine protein concentration. the sensitivity test was conducted by using dilution test, and followed by spectrophotometry and paper disc diffusion tests. results: the study showed that proteins successfully characterized from snail mucus (achatina fulica) were proteins with molecular weights of 83.67 kda (achasin), 50.81 kda, 15 kda, 11.45 kda (full amino acid sequence of mytimacin-af) and 9.7 kda (mytimacin-af). based on the dilution test, achasin had better antimicrobial activities against streptococcus mutans, while mytimacin-af had better antimicrobial activities against aggregatibacter actinomycetemcomitans. but the paper disc diffusion test result showed that achasin had antimicrobial activities against streptococcus mutans and aggregatibacter actinomycetemcomitans, while mytimacin-af had no antimicrobial activities. conclusion: the proteins with molecular weights of 50.81 kda, 15 kda, 11.45 kda were considered as new antimicrobial proteins isolated from snail mucus. achasin, had better antimicrobial activities against streptococcus mutans, while mytimacin-af had better antimicrobial activities against aggregatibacter actinomycetemcomitans. key words: achatina fulica, achasin, mytimacin-af, antimicrobial abstrak latar belakang: achasin dan mytimacin-af adalah protein lendir bekicot (achatina fulica) yang memiliki aktivitas antimikroba. lendir bekicot diduga memiliki protein lain yang memiliki aktivitas antimikroba terhadap streptococcus mutans dan actinobacillus actinomycetemcomitans bakteri patologis oral. tujuan: penelitian ini bertujuan untuk mengkarakterisasi protein lendir bekicot (achatina fulica) yang memiliki aktivitas antimikroba terhadap streptococcus mutans dan aggretibacter actinomycetemcomitans, dan membandingkan efek antimikroba protein achasin dan mytimacin-af. metode: sampel penelitian adalah lendir bekicot yang diambil dari provinsi yogyakarta. isolasi dan karakterisasi protein dilakukan dengan metode sds-page, elektro-elusi, dan dialisis. nano drop test dilakukan untuk menentukan konsentrasi protein. uji sensitivitas dilakukan dengan menggunakan uji dilusi, dan diikuti oleh spektrofotometri dan tes difusi kertas cakram. hasil: protein dari lendir bekicot (achatina fulica) yang ditemukan adalah protein dengan berat molekul 83,67 kda (achasin), 50,81 kda, 15 kda, 11,45 kda (urutan asam amino penuh mytimacin-af) dan 9,7 kda (mytimacinaf). berdasarkan uji dilusi, achasin memiliki aktivitas antimikroba yang lebih baik terhadap streptococcus mutans, sedangkan mytimacin-af memiliki aktivitas antimikroba yang lebih baik terhadap aggregatibacter actinomycetemcomitans. namun hasil uji difusi cakram kertas menunjukkan bahwa achasin memiliki aktivitas antimikroba terhadap streptococcus mutans dan �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 31–36 aggegatibacter actinomycetemcomitans, sementara mytimacin-af tidak memiliki kegiatan antimikroba. simpulan: protein dengan berat molekul 50,81 kda, 15 kda, 11,45 kda merupakan protein antimikroba baru diisolasi dari lendir bekicot. achasin, memiliki aktivitas antimikroba yang lebih baik terhadap streptococcus mutans, sedangkan mytimacin-af memiliki aktivitas antimikroba yang lebih baik terhadap actinobacillus actinomycetemcomitans. kata kunci: achatina fulica, achasin, mytimacin-af, antimikroba correspondence: herluinus mafranenda dwi n, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: herluinusmafranenda@yahoo.com introduction antimicrobial materials derived from nature either from animals and plants have commonly been used since they are easily obtained and have low side effects.1 antimicrobial materials derived from phenolic group actually have been considered as a solution. however, some researchers suggest that phenol still has some disadvantages. traditionally, snail mucus has been used by javanese to heal wound.2 achasin and mytimacin af have been extracted from snail mucus and it is reported to have anti microbial effect. achasin contained in snail mucus is known as an effective antimicrobial protein to gram-positive andnegative bacteria that have been successfully extracted since 1982 by iguchi et al.3 besides that, mytimacin-af is also known as a broad-spectrum antimicrobial protein.4 achasin has been identified as a protein with molecular weight of 59.086-150 kda.5 meanwhile, mytimacin-af protein has been identified as a protein with molecular weight of 9.7 kda.4 streptococcus mutans is a gram-positive bacteria in cluster known more as opportunistic pathogenic bacteria in oral cavity, that has an ability to form plaque and cause primary caries in oral cavity.6 moreover, streptococcus mutans are in symbiosis with other pathogenic bacteria causing caries in oral cavity.7 on the other hand, aggregatibacter actinomycetemcomitans is a gram-negative bacterium that also has the properties of opportunistic pathogens in oral cavity. it has virulence factors that can cut igg, immunosuppressive factors, and strong leukotoxin, as well as produce collagenase enzyme, which is harmful to the health of periodontal tissues. thus, aggregatibacter actinomycetemcomitans are usually associated with aggressive periodontitis diseases and other periodontal diseases. aggressive periodontitis disease is caused by the lack of the body’s defense system, and often occurs during pubertal period.8 the study were aimed to characterize the proteins of snail mucus (achatina fulica) that have antimicrobial activities to steptococcus mutans and aggregatibacter actinonycetemcomitans, and to compared its effectiveness with adhesin and mytimacin af. materials and methods this experiment can be considered as a laboratory experiment with post test-only control group design. the samples of this research were obtained by using simple random sampling technique. the samples were snail mucus (achatina fulica) obtained from 36 snails taken from cangkringan village, sleman. however, the snails used as the samples of this experiment had to meet certain requirement, had 4.5 to 10 cm shell length and they could move actively. they had to be quarantined for two days in a plastic box. and, they had to have fasting for 12 hours before their mucus was taken into coconut wood box. snail mucus was taken by using a method with electrical shock from 4.5 to 12 volts at 1.5 amperes for 60 seconds. in the protein isolation phase the mucus obtained was mixed with twice the early volume of water, and then stirred in the stirrer overnight. it was centrifuged at 11,000 g by using beckman model j-21 for 30 minutes, and then supernatant called water soluble fraction (wsf) was produced. the wsf was added with three times the early volume of the wsf, and then centrifuged at 2900 g with yuan kr 422 for 30 minutes. as a result, precipitation known as ethanol precipitated fraction (etp) was obtained. then etp was diluted with 50 mm tris-hcl (ph 8.0) before electrophoresis was performed.9 in the electrophoresis phase, the proteins derived from the snail mucus was characterized by using sds-page technique with both thermo scientific spectra multicolor low range protein ladder, about 4.6-40 kda and thermo scientific spectra multicolor broad range protein ladder, about 10-260 kda. in other words, sds-page technique used in this phase was divided into two sections. the first running by using the low range marker and the second running by using the broad range marker. material used for the low range marker was 16% separating gel, consisting of 5.3 ml of bis-acrylamide, 2.1 ml of ddi h2o, 2.5 ml of buffer gel, and 0.1 ml of 10% sds. material used as the broad range marker was 10% separating gel, consisting of 3.3 ml of bis-acrylamide, 4.1 ml of ddi h2o, 2.5 ml of buffer gel, and 0.1 ml of 10% sds. besides that, 5% stacking gel, consisting of 1.7 ml of bis-acrylamide, 5.7 ��mafranenda, et al.: antimicrobial proteins from snail mucus table 1. the order of the eppendorf tube in dilution test tube number weight of protein molecule the number of proteins in the tube (ml)/ protein number 1 2 3 4 5 6 7 8 9 83.67 0,5 0,25 0,125 0,0625 0,0312 0,0156 0,0078 kp kn 50.81 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 kp kn 15 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 kp kn 11.45 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 kp kn 9.7 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 kp kn tabel 2. the concentration of protein in nanodrop test the number of protein (ml) no. protein absorbance values streptococcus mutans actinobacillus actinomycetemcomitans 1 2 3 4 5 6 7 1 2 3 4 5 6 7 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 0.5 0.25 0.125 0.0625 0.0312 0.0156 0.0078 1 (83.67 kda) 1,504 1.243 1.119 1.292 1.095 0.728 0.832 0.525 0.104 0.597 0.250 0.803 0.332 2 (50.81 kda) 0.934 0.586 1.056 0.815 1.043 1.466 0.636 0.543 0140 0.311 0.713 0.277 3 (15 kda) 0.563 1.063 0.412 -0.355 0.495 0.762 0.454 0.482 0.479 -0.322 0.467 0.156 4 (11.45 kda) 1.459 0.997 1.203 1.504 1.006 0.941 0.726 1.007 -0.284 -0.062 0.384 0.797 0.759 5 (9.7 kda) 0.819 1.106 0.521 1.060 1.040 1.134 0.711 0.410 0.771 0.600 0.770 0.232 -0.045 ml of ddi h2o, 2.5 ml of buffer gel, and 0.1 ml of 10% sds was also used in this phase.10 the separating gels mixed with 0.05 ml of 10% aps and 0.01 ml of temed were used as catalyst, and then put into plates in vertical position reaching the bottom of the comb. after polymerization, the stacking gel was fully inserted and the comb was installed. the plates were set into electrophoresis chamber and poured with electrophoresis buffer (3.03 g tris base, 14.4 g glycine, and 1 g sds). then 50 ml of etp diluted was mixed with the buffer sample (3.55 ml of deionized water, 1.25 ml of 0.5 m tris-hcl at ph 6.8, 2.5 ml glycerol, 2 ml of 10% sds, and 0.2 ml of 0.5% bromophenol blue). meanwhile, 950 ml of the other buffer sample was mixed with 50 ml of 2-merchaptoethanol, and boiled for 4 minutes at 950 c. after the samples were in cold condition, they were loaded into wells using micropipette.11 electrophoresis was then conducted at 200 volts in 39 minutes for the low range marker and 30 minutes for the broad range marker. once the process was completed, the plates were opened, and the gels were stained with comassie brilliant blue staining, and then destained. after the bands were clearly visible, the molecular weight of proteins were calculated by comparing both markers and then determined by using the formula retardation factor (rf) with logarithm derived from the molecular weight of protein markers known. then the value of rf obtained was put into a linear regression equation with the formula: y = ax + b, with y = logarithm of molecular weight and x = rf values. to simplify the calculation, the calculation was conducted by using the microsoft-excel 2007 program. protein production was analogous to proteins that have been identified through previous researches, such as proteins with a molecular weight of 9.7 kda (mytimacinaf) and 11.45 kda which were analogous to amino acid sequence of the full mytimacin-af since they have closest molecular weight to mytimacin-af with a molecular weight of 12 kda. protein with a molecular weight of 15 kda was analogous to protein contained in ictalarus punctatus mucus since they had same antimicrobial activities and same molecular weight, about 15 kda. protein with a molecular weight of 50.81 kda was analogous to protein contained in tinca tinca mucus with a molecular weight of 49 kda since they have almost the same molecular weight, and also analogous to achasin with a molecular weight of 59.086 kda and 83.67 kda as found by achasin ebran et al.4,5 those proteins were characterized by using sdspage technique followed by electro-elution and dialysis techniques. the sds-page technique actually consisted of the same steps as the profiling process, requiring two different wells and four gels for each electrophoresis. those four first gels were used for low range protein marker, and the other four gels were used for broad range protein markers. the first well was used for both low range and broad range markers, while the second well was filled with isolated protein. after the electrophoresis process was completed, staining was conducted until the protein bands could be seen quite clearly. destaining was not necessary until the gel was completely clear. next, the process of protein production was followed by electro-elution process. after the protein bands with certain molecular weight were cut, destaining �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 31–36 table 3. the results of dilution test on spectrophotometric absorbance values protein labels inhibitory factor against s. mutans (cm) inhibitory factor against a. actinomycetemcomitans (cm) 1 2 3 4 mean 1 2 3 4 mean 1 (83.67 kda) 1.640 1.860 1.855 1.950 1.826 1.200 0.990 1.115 1.200 1.126 2 (50.81 kda) 1.840 1.980 1.780 1.905 1.876 1.410 1.295 1.255 1.515 1.369 3 (15 kda) 1.525 1.390 1.665 1.625 1.551 1.365 1.225 1.205 1.140 1.234 4 (11.45 kda) 0 0 0 0 0 1.180 1.065 1.125 1.180 1.138 5 (9.7 kda) 0 0 0 0 0 0 0 0 0 0 table 4. the results of paper disc diffusion test on inhibitory factor protein labels inhibitory factor against s. mutans (cm) inhibitory factor against a. actinomycetemcomitans (cm) 1 2 3 4 mean 1 2 3 4 mean 1 (83. kda) 1.640 1.860 1.855 1.950 1.826 1.200 0.990 1.115 1.200 1.126 2 (50.81 kda) 1.840 1.980 1.780 1.905 1.876 1.410 1.295 1.255 1.515 1.369 3 (15 kda) 1.525 1.390 1.665 1.625 1.551 1.365 1.225 1.205 1.140 1.234 4 (11.45 kda) 0 0 0 0 0 1.180 1.065 1.125 1.180 1.138 5 (9.7 kda) 0 0 0 0 0 0 0 0 0 0 was re-done in a certain time until the gel appeared slightly clear.12 then dialysis process was conducted to purify the protein content. the pellets formed were measured with ohauss scales and dissolved in a solution of 0.5 m tris-cl at ph 7.3. hereafter, the concentration of each protein was measured by using nanodrop test. dilution test was conducted by using brain heart infusion medium (bhi) in eppendorf tubes. the first tube was filled with protein 0.5 ml and bhi 0.5 ml. the number of protein continued was then reduced 50% by dilution from tube 2 to tube 6 as seen in table 1. the preparation then was conducted twice, and made into two groups. the first group was inoculated with streptococcus mutans, and the second group was inoculated with actinobacillus actinomycetemcomitans. the results of the preparations then were incubated under anaerobic conditions at a temperature of 370 c for 24 hours. after 24 hours, the incubation result of streptococcus mutans then was grown in tyc medium, while that of actinobacillus actinomycetemcomitans was grown in luria berthani medium in order to determine the maximum inhibitory factor of proteins against both bacteria. incubation result grown in bhi medium was analyzed for its absorbance values by using spectrophotometric at a wavelength (λ) of 600 nm to determine the level of turbidity of bhi media. thus, the more turbid bhi medium is, the higher the growth of bacteria. absorbance values that appear in the tool indicate the reduction result of the absorbance values of treatment and the absorbance values of negative control. the negative result of the reading can indicate that the turbidity value in the negative control is higher than that in the treatment. it means that there is no growth of bacteria. meanwhile, the positive result of the reading can indicate the presence of bacterial growth. finally, the media in petri dish was then divided into five parts, on each of which bacterial swab was conducted evenly. paper disc that has given protein was placed on each medium. these steps were repeatedly conducted four times. petri dishes were incubated in anaerobic condition at a temperature of 370 c for 24 hours, and then measured for their inhibitory diameter by using calipers. results the samples of this experiments were thirty-six healthy and active snails. the total of snail mucus collected was 150 ml with the average of snail mucus taken from each sample about 3-5 ml. the molecular weight of proteins contained in the snail mucus was measured by using the formula rf. based on the results shown in the low range marker, it is known that there were proteins contained in snail mucus with a molecular weight of 28.59 kda, 15 kda, and 13.3 kda. meanwhile, based on the results shown in the broad range marker, it is known that there were proteins contained in snail mucus with a molecular weight of 83.67 kda, 50.81 kda, 48.1 kda, 35 kda, 28.88 kda, 11.45 kda, and 9.7 kda. the result of reading the value concentration of the isolated crude protein was 10.152 µg/ml, while the results of reading the value concentration of those proteins contained in snail mucus by using nano drop test were different among each other as seen in table 2. it is also known that there was bacterial growth as shown in the result of inoculated subculture on tyc medium. the presence of bacterial growth was expected because of contamination occurred in one treatment to another one due to poor sterilization of spreader. ��mafranenda, et al.: antimicrobial proteins from snail mucus based on the reading results of spectrophotometer, it was known that a protein with a molecular weight of 15 kda has antimicrobial activities against streptococcus mutans with absorbance value about -0.355 at the number of protein about 0.0312. the results also show that a protein with a molecular weight of 9.7 kda (mytimacinaf) had antimicrobial activities against actinobacillus actinomycetemcomitans with absorbance value about 0.045 at the number of protein about 0.0078 ml. it was also known that a protein with a molecular weight of 11.45 kda hadantimicrobial activities against streptococcus mutans with absorbance value about -0.284 at the number of protein about 0.0625 ml and against actinobacillus actinomycetemcomitans with absorbance values about 0.062 at the number of protein about 0.125 ml. meanwhile, a protein with a molecular weight of 83.67 kda (achasin) had antimicrobial activities against streptococcus mutans with the lowest absorbance value about 0.0156 at the number of protein about 0.728 ml and against actinobacillus actinomycetemcomitans with absorbance value about 0.104 at the number of protein about 0.25 ml. this suggests that achasin found in this research did not have a maximum inhibitory factor against streptococcus mutans and actinobacillus actinomycetemcomitans as seen on the results of the dilution test in table 3. based on the results of paper disc diffusion test, it was known that a protein with a molecular weight of 50.81 kda had the greatest inhibitory factor against streptococcus mutans with a mean inhibition of 1.876 cm and actinobacillus actinomycetemcomitans with a mean inhibition of 1.369 cm. it was also known that a protein with a molecular weight of 83.67 kda (achasin) had inhibitory factor against streptococcus mutans and actinobacillus actinomycetemcomitans. proteins with a molecular weight of 11.45 kda and 9.7 kda (mytimacinaf) had no antimicrobial activities against streptococcus mutans. however, although a protein with a molecular weight of 11.45 kda had no antimicrobial activities against streptococcus mutans, but it still had inhibitory factor on the growth of actinobacillus actinomycetemcomitans. in contrary, a protein with a molecular weight of 9.7 kda (mytimacin-af) had no inhibitory factor and antimicrobial activities against streptococcus mutans and actinobacillus actinomycetemcomitans. finally, it was known that a protein with a molecular weight of 15 kda had inhibitory factor about 1.551 cm against streptococcus mutans, and about 1.234 cm against actinobacillus actinomycetemcomitans (table 4). discussion achasin found in this research had a molecular weight of 83.67 kda. this corresponds to a molecular weight range of achasin protein, from 59.086 to 150 kda, ever found in previous researches conducted by venugopal r9 and jyh-yih.13 these various molecular weights can be caused by the differences of snail subspecies and geographical location. based on the results of the protein characterization phase, it was known that achasin had many differences of its band thickness. these differences of its band thickness can affect on its concentration value. thus, the thicker the band of a protein is, the greater the concentration value of the protein is.14 it is because band thickness indicates that there are many levels of protein in the sample. crude protein, for example, as the sample of protein isolated has a concentration of 10.152 µg/ml indicating that there is a great potential source of proteins contained in snail mucus.2,13 furthermore, based on the results of subculture conducted after the inoculation of bacteria in those eppendorfes, it was known that the number of bacteria was increased. it may be caused by bacterial contamination during the inoculation process of bacteria into tyc medium since inoculum contaminated from one treatment to other treatments had caused the increasing of bacterial growth. better reading was found from the reading of the absorbance value. when bacteria lives and breeds, it will produce matrix from planting medium. as a result, the matrix made the planting medium become turbid. thus, it can be said that if the culture medium becomes turbid, there must be bacteria grown. nonetheless, there were still some samples that cannot be read (table 3) because the number of samples did not meet the minimum standard volume (5 ml) specified by spectrophotometer so that the tool could not read their absorbance values . based on the results of dilution test, it was known that only protein with a molecular weight of 15 kda has antibacterial activities against streptococcus mutans and actinobacillus actinomycetemcomitans. similarly, in previous researches, it is known that a protein with a molecular weight of 15 kda, namely hlp-1, taken from catfish mucus (ictalarus punctatus) has been identified as an effective antimicrobial to gram-positive and gramnegative bacteria. this is because the protein works with two mechanisms: forming classic transmembrane channels to become the targeted cell membrane and making the cell membrane become more soluble with carpet like mechanism. these mechanisms have been considered to be effective for both gram-positive and gram-negative bacteria. b e s i d e s t h a t , i t i s a l s o k n o w n t h a t p r o t e i n s with a molecular weight of 11,45 kda and 9,7 kda (mytimacin-af) are only effective against actinobacillus actinomycetemcomitans. unlike the results of previous researches show that mytimacin-af as a broadspectrum antimicrobial protein is more effective on gram-positive bacteria, the result of this research showed that mytimacinaf also had antimicrobial activity against gram-negative bacteria. this is because the mechanism of mytimacin-af as an antimicrobial protein basically consists of damaging cell membrane, interfering cell metabolism, and destroying �� dent. j. (maj. ked. gigi), volume 47, number 1, march 2014: 31–36 cytoplasmic cell components.4 therefore, this mechanism is considered as an effective mechanism for gram-positive and negative bacteria. actually, the main reason of this lack of antimicrobial activity in these proteins with a molecular weight of 11.45 kda and 9.7 kda can be caused by the use of sds-page method during protein production for several reasons. the use of denaturing and dissociating agents in sdspage method can impair protein chain, then causing the loss of some or all its caracterization.14 besides that residual staining materials of comassie brilliant blue can interfere the nature of proteins. nevertheless, the use of sds-page method for protein production is not entirely disadvantageous since the properties owned by the protein did not disappear entirely, but its sensitivity will be not as good when using ion chromatography method.12,15 thus, ion chromatography method is necessary to be conducted in researches related to protein characterization and production. based on the results of the paper disc diffusion test, it was known that a protein with a molecular weight of 15 kda was effective for streptococcus mutans and actinobacillus actinomycetemcomitans, while a protein with a molecular weight of 9.7 kda (mytimacin-af) hadno antibacterial activities. it is also known that a protein with a molecular weight of 11.45 kda is only effective for actinobacillus actinomycetemcomitans. it means that this protein with a molecular weight of 11.45 kda is analogous to the amino acid sequence of the full mytimacin-af, which is effective on gram-negative bacteria as the same as a protein with a molecular weight of 9.7 kda. a protein with a molecular weight of 83.67 kda (achasin) has a better inhibitory factor against streptococcus mutans. nevertheless, based on the results of this experiment, it is known that achasin also has inhibitory factor against actinobacillus actinomycetemcomitans. similarly, previous researches also show that achasin has antimicrobial activities that can inhibit the growth of gram-positive and negative bacteria, but it gives a better inhibition on gram-positive one.3 this is because the mechanism of achasin is aimed to inhibit the formation of peptidoglycan and cytoplasmic membrane. like achasin, a protein with a molecular weight of 50.81 kda also has a greater inhibitory factor against streptococcus mutans than against actinobacillus actinomycetemcomitans. a protein with a molecular weight of 50.81 kda is the closest analogue of the antimicrobial proteins derived from tinca tinca mucus, which has a molecular weight of 49 kda and also has the best inhibitory factor against gram-positive bacteria.5 finally, the study showed that proteins successfully characterized from snail mucus (achatina fulica) were proteins with molecular weights of 83.67 kda (achasin), 50.81 kda, 15 kda, 11.45 kda (full amino acid sequence of mytimacin-af) and 9.7 kda (mytimacin-af). achasin, based on the results of the dilution test, has better antimicrobial activities against streptococcus mutans, while mytimacin-af has better antimicrobial activities against actinobacillus actinomycetemcomitans. achasin, based on the results of the paper disc diffusion test, actually has antimicrobial activities against streptococcus mutans and actinobacillus actinomycetemcomitans, while mytimacinaf has no antimicrobial activities. those proteins with molecular weights of 50.81 kda, 15 kda, 11.45 kda are considered as new antimicrobial proteins isolated from snail mucus. however, their effectiveness need to be studied further. acknowledgement sincere gratitude to the biomedical laboratory of faculty of medicine universitas brawijaya, laboratory of satrep and proteomic institute of tropical disease universitas airlangga, and laboratory of microbiology faculty of dental medicine universitas airlangga surabaya. references 1. wayan s, made m, putu ahw. uji daya hambat ekstrak daun kedondong (lannea grandis engl) terhadap pertumbuhan bakteri erwinia carotovora. buletin veteriner universitas udayana denpasar 2011; 3: 45-50. 2. nastiti r. sukses budidaya bekicot. yogyakarta: penerbit pustaka baru press; 2013. h. 1-5, 12-6 3. titiek b, edy b, suwarno w. biochemical characterization of an antibacterial glycoprotein from achatina fulica ferussac snail mucus local isolate and their implication on bacterial dental infection. indonesian j biotechnology 2007; 12: 943-51. 4. jian z, wenhong w, xiaomei y, xiuwen y, rui l. a novel cysteinerich antimicrobial peptide from the mucus of the snail of achatina fulica. peptides 2013; 39: 1-4. 5. venugopal r, jyh-yih c. aplications of antimicrobial peptides from fish and perspectives for the future. peptides 2011; 32(2): 415-20. 6. philip dm, michael aol, david w. oral microbiology. 5th ed. china: churchill livingstone elsevier; 2009. p. 14-5, 30-2, 36-9, 142-3. 7. arora, hina a. textbook of microbiology for dental students. singapore: alkem company; 2009; p. 364-5. 8. michael gn, henry ht, perry rk. carranza’s clinical periodontology. 11th ed. missouri: elsevier saunders; 2012. p. 43, 69, 107-9. 9. titiek bs. karakterisasi protein lendir bekicot (achasin) isolat lokal sebagai faktor antibakteri. media kedokteran hewan 2007; 23: 139-40, 142-3. 10. rantam fa. metode imunologi. surabaya: airlangga university press; 2003. 11. mary ep, loren w. sds page gel electrophoresis. available from: url: http://ww2.chemistry.gatech.edu/~lw26/bcourse_ information/4581/techniques/gel_elect/page_protein.html accessed desember 14, 2013 . 12. mohammadian t, doosti m, paknejad m, siavoshi f, massarrat s. preparative sds-page electroelution for rapid purification of alkyl hydroperoxide reductase from helicobacter pylori. iranian j publ health 2010; 39: 85-91. 13. raya nd i as. meraup unt ung besa r da r i beter na k bek icot. yogyakarta: enjoy publishing; 2012. p. 1-4, 6-9. 14. weaver rf. molecular biology. 3th ed. new york: mcgraw hill; 2005. p. 91-4. 15. robert cv, margaret l. mini risk assessment giant african snail, achatina fulica bowdich. minnesota: department of entomology university of minnesota; 2004. p. 1. 208 volume 45 number 4 december 2012 research report inhibition of dental plaque formation by toothpaste containing propolis nurin aisyiyah listyasari and oedijani-santoso department of oral and dental health faculty of medicine, universitas diponegoro semarang – indonesia abstract background: plaque is the main cause of caries and periodontal disease. caries and periodontal disease can be prevented by inhibiting dental plaque formation. to inhibit the formation of plaque, teeth must be brushed with toothpaste. according to previous studies, propolis contains apigenin and tt-farnesol classified as flavonoid that can inhibit the formation of dental plaque by inhibiting glucosyltransferase enzym and membrane integrity of streptococcus mutans. purpose: the aim of this study was to determine the effect of toothpaste containing propolis on the formation of dental plaque. methods: post test with only control group design was used. the subjects of this study were 30 boarding school students of hidayatullah, yayasan al-burhan, gedawang, semarang, divided into two groups, randomized control group and treatment group. control group was not treated with toothpaste contanining propolis. meanwhile, treatment group was treated with toothpaste containing propolis. plaque then was measured by using plaque index of sillness and loe method after using toothpaste containing propolis for four hours. afterwards, the data was analyzed by a computer program, mann-whitney test, with its significance p < 0.05. results: the result of mann-whitney test showed a significant difference, 0.002 (p < 0.05), between the control group and the treatment group. the median of the control group was about 3.41, while that of the treatment group was about 0.58. conclusion: the use of toothpaste contaning propolis can prevent dental plaque formation. key words: propolis, tooth paste, dental plaque abstrak latar belakang: plak merupakan penyebab utama terjadinya karies dan penyakit periodontal. karies dan penyakit periodontal dapat dicegah dengan menghambat pembentukan plak gigi. untuk mencegah terbentuknya plak, gigi harus digosok menggunakan pasta gigi. penelitian terdahulu menyebutkan bahwa propolis mengandung flavonoid apigenin dan tt-farnesol yang mampu menghambat aktivitas enzim glukosiltransferase dan menghambat pembentukan membran bakteri streptococcus mutans yang berperan pada pembentukan plak gigi. tujuan: tujuan dari penelitian ini adalah untuk mengetahui pengaruh pasta gigi dengan kandungan propolis terhadap pembentukan plak gigi. metode: menggunakan rancangan post test only control group design. sampel penelitian ini adalah santri pondok pesantren hidayatullah yayasan al-burhan, gedawang, semarang, sebanyak 30 santri dibagi dalam dua kelompok secara acak yaitu kelompok kontrol diberikan pasta gigi tanpa kandungan propolis dan kelompok perlakuan diberikan pasta gigi dengan kandungan propolis. plak diukur dengan menggunakan indeks plak menurut sillness and loe sesudah menggunakan pasta gigi dalam jangka waktu kurang lebih empat jam. data diolah menggunakan program komputer dengan analisis statistik non parametrik mann-whitney dan taraf signifikansi diterima bila p < 0,05. hasil: analisis statistik non parametrik mann-whitney menghasilkan perbedaan rerata bermakna (p < 0,05) antara kelompok kontrol dan perlakuan sebesar 0,002. nilai tengah skor plak pada kelompok kontrol sebesar 3,41 dan pada kelompok perlakuan sebesar 0,58. kesimpulan: penggunaan pasta gigi dengan kandungan propolis dapat menghambat pembentukan plak gigi. kata kunci: propolis, pasta gigi, plak gigi correspondence: oedijani-santoso, c/o: bagian kesehatan gigi dan mulut, fakultas kedokteran universitas diponegoro. jl. dr. sutomo no. 18, semarang 50321, indonesia. e-mail: oediyanisantoso@yahoo.com 209listyasari and santoso: inhibition of dental plaque formation by toothpaste containing propolis introduction dental plaque plays an important role in causing oral health problems. dental plaque is a soft layer that consists of a collection of microorganisms breeding in a matrix. dental plaque is firmly attached to the tooth surface that is not brushed.1 some studies showed that at the beginning of the formation of dental plaque, gram-positive cocci, such as streptococcus mutans (s. mutans), streptococcus sanguis, streptococcus mitis, and streptococcus salivarius, is mostly found.1-3 to prevent dental and oral health problems, brushing the teeth with toothpaste is needed since it can not only help to prevent oral disease, but also to make our permanent teeth strong.4, 5 fluoride contained in toothpaste is a chemical substance that can prevent our teeth from dental cavities, however, the use of fluoride in large amounts over certain period of time can cause enamel fluorosis, tooth enamel with speckled spots caused by fragile tooth enamel with an irreversible blackish brown color.6 the use of natural ingredients actually can reduce the side effects of chemicals on the body, so the addition of natural ingredients in toothpaste can support dental and oral health care program.7 propolis is a natural substance collected by honey bees from various types of plants, especially from buds and leaves. the benefit of propolis in oral health is as an anti-bacterial because of the flavonoid contained in it. apigenin and tt-farnesol are classified as important flavonoids because they can prevent glucosyltransferase enzyme activity and subsequently inhibit the growth of s. mutans causing dental plaque formation.8, 9 the study was aimed to determine the effect of toothpaste containing propolis on the formation of dental plaque, assessed by plaque index measured with sillness and loe method. finally, this research was expected to provide benefits and evidence that toothpaste containing propolis may reduce dental plaque scores, so it can provide an alternative option of effective toothpaste for society and medical personnel to prevent dental plaque formation. materials and methods this research was an experimental research with post test only control group design.10 the population in this research were students of pondok pesantren hidayatullah of �ayasan al-burhan in gedawang, semarang. the samples were obtained by consecutive sampling. based on the calculation, the samples obtained were about 15 respondents in control group and treatment group.11, 12 allocation random was then conducted by using a two-sided currency coin in order to divide the samples into two groups, the control group and the treatment group. inclusion criteria for this research were that the age of the patients must be from 12 years old to 18 years old, with a complete permanent dentition, well aligned or has slightly crowded, as well as that there must be no dental caries and no fixed orthodontic appliances. meanwhile, the exclusion criteria were uncooperate patients, or that they consumed foods other than food provided by the researchers during the treatment. the independent variable, moreover, was giving toothpaste containing with propolis, while dependent variable was dental plaque score. possible confounding variable was the method of brushing teeth. thus, to control this confounding variable, the information about the correct way of brushing teeth was given. the data collected, was primary data derived from plaque score measured in the control group and in the treatment group after the treatment. the research then was started after obtaining ethical clearance from kepk in faculty of medicine, diponegoro university/kariadi hospital, semarang. patients in the control group were asked to brush their teeth without toothpaste containing with propolis, while the patients in the treatment group were asked to brush their teeth by using toothpaste containing with propolis. then, they were also asked to eat food that had been provided by the researchers and to have activities as usual. the measurement of dental plaque scores in the two groups was conducted by using sillness and loe method for next 4 hours.4 the normality of the data obtained from the treatment group was analyzed with shapiro wilk test. based on the results of the normality analysis, it was known that the distribution of the data was not normal, so the transformation of the data was needed. however, since the transformation of the data distribution was not normal, an alternative test, mann-whitney test, was conducted as a non-parametric statistical test with significant value of p < 0.05 (95% confidence level).10 results the age and gender distribution of subject were shown in table 1 and 2. the assessment of dental plaque score in the control group and in the treatment group (by using sillness & loe method) can be seen in figure 1. the results of the statistical analysis of the dental plaque score using a non-parametric test, mann-whitney, show p values about 0.002 with significant value p <0.05. thus, it can be indicated that there were significant differences between the plaque scores in the control group and those in the treatment group. it shows that the plaque scores in the treatment group were lower than those in the control group. furthermore, the results of the statistical analysis of the ages of the patients using a non-parametric test, mann-whitney, show p value about 0.593 with significant value p<0.05. therefore, it can be indicated that there was no significant difference between the ages of the subject with dental plaque in the control group and those in the treatment group. 210 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 208–211 the results of the statistical analysis of gender of the patients using chi-square test show p value about 1 with significant value p<0.05. it can be indicated that there was no relationship between gender of the subject with dental plaque in the control group and those in the treatment group. discussion dental plaque formation is started with the formation of acquired pellicle, a thin layer, formed by deposition of salivary glycoprotein on dental enamel.2,4 microorganisms forming extracellular polysaccharide, s. mutans, adhere to the surface of acquired pellicle.4,13 glucosyltransferase enzyme contained in s. mutans then will change sucrose into extracellular polysaccharide. the polysaccharide will strengthen the adherence of bacteria’s surface on dental pellicle components.2,4,14 the results of this study indicate that toothpaste containing propolis may reduce dental plaque scores, indicated by the results of non-parametric statistical analysis with mann-whitney, p value of 0.002 (p<0.05). it can be concluded that there was the significant difference of the dental plaque score in the control group and that in the treatment group. the dental plaque scores in the treatment group can indicate that patients who brushed their teeth by using toothpaste containing with propolis had lower dental plaque scores than those in the control group. this declining of dental plaque scores in the treatment group can indicate the inhibition of dental plaque formation. propolis is a resin compound produced by bees from various plants. most of the biological activity of propolis comes from flavonoids contained in it.15 the high number of flavonoids in propolis have a role as antibacterial, especially in inhibiting bacterial growth in oral cavity. flavonoids contained in propolis of bee honey can inhibit the growth of s. mutans in vitro.7 propolis derived from honeybee have good potential as antibacterial.16 the content of antibacterial propolis is beneficial for reducing oral bacteria in vitro and in vivo. in other worlds, propolis has antibacterial ability in vitro against oral streptococcus and bacteria in saliva for clinical study.17 mechanism of antibacterial activity of propolis is actually related with flavonoid contained in it. flavonoids contained in propolis contain apigenin and tt-farnesol which can inhibit plaque formation process. mechanism of apigenin activity in preventing plaque formation is through the inhibition of glucosyltransferase enzyme activity in s. mutans that later can inhibit the formation of extracellular polysaccharides caused by bacteria. meanwhile, tt-farnesol has high antibacterial capability to inhibit the growth and metabolism of s. mutans in order to disrupt the formation of bacterial membrane.15,18 both components can inhibit the accumulation and composition of polysaccharide, the biofilm layer of s. mutans, without disrupting the bacterial survival. apigenin and tt-farnesol have bacteriostatic capability so that they can overcome infection without killing normal oral microorganisms and can also not cause bacterial resistance.15 several studies have shown that flavonoids can inhibit the formation of dental plaque. catechin flavonoids contained in apples can inhibit the formation of dental plaque.12 a similar study, strawberries contain catechin flavonoids can inhibit the formation of dental plaque.19 catechin flavonoids contained in grapes can inhibit the formation of dental plaque. the mechanism of catechins in inhibiting the formation of dental plaque is actually by inhibiting the activity of glucosyltransferase enzyme (gtfs) and by killing the growth of bacteria causing dental plaque, such as s.mutans.12,19,20 finally, it can be concluded that the use of toothpaste containing with propolis can inhibit dental plaque formation. table 1. the distribution of the subjects of this research based on the age of the patients in the control group and in the treatment group age (year) control group treatment group number % number % 12 0 0 1 6.25 13 5 35.71 3 18.75 14 2 14.28 7 43.75 15 2 14.28 2 12.5 16 3 21.42 2 12.5 17 2 14.28 1 6.25 s 14 100 16 100 table 2. the distribution of the samples based on gender sex control treatment number % number % male 7 50 8 50 female 7 50 8 50 number 14 100 16 100 figure 1. the assesment results of dental plaque score in the control group and in the treatment group. control treatment tooth paste d en ta l p la gu e s co re 211listyasari and santoso: inhibition of dental plaque formation by toothpaste containing propolis references 1. ariningrum. beberapa cara menjaga kebersihan gigi dan mulut. jakarta: cermin dunia kedokteran; 2008; (45–51). 2. marsh pd, devine da. how is the development of dental biofilms influenced by the host. j clin periodontol 2011; 38 (s11): 28–35. 3. kolenbrander pe, palmer jr., rj, rickard ah, jakubovics ns, chalmers ni, diaz pi. bacterial interactions and successions during plaque development. periodontol 2000, 2006; 42(1): 47–79. 4. putri mh, herijulianti e, nurjannah n. ilmu pencegahan penyakit jaringan keras dan jaringan pendukung gigi. jakarta: egc 2010. p. 54–64, 93-5, 111–2. 5. attin t, hornecker e. tooth brushing and oral health: how frequently and when should tooth brushing be performed. oral health prev dent j. 2005; 3 (135–40) 6. paine ml, slots jorgen, rich sk. fluoride use in periodontal therapy: a review of the literature. jada 2001; 129(2): 69–7. 7. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). majalah kedokteran gigi (dent j) 2005; 38: 135–41. 8. ahuja v, ahuja a. apitherapy: a sweet approach to dental diseases part ii: propolis. j academy adv dent res 2011; 2(2): 1–8. 9. mahmoud l. biological activity of bee propolis in health and disease. asian pacific j cancer prev 2006; 7: 22–31. 10. dahlan ms. statistik untuk kedokteran dan kesehatan. edisi 4. jakarta: salemba medika; 2009. p. 4–20. 11. dahlan ms. besar sampel dalam penelitian kedokteran dan kesehatan. jakarta: pt. arkans; 2006. p. 14-15, 59–63. 12. ayu r. pengaruh pasta gigi dengan kandungan buah apel (pyrus malus) terhadap pembentukan plak gigi. diponegoro university instutional repository2012; 37137(1): 1–15. 13. zijnge v, van leeuwen mbm, degener je, abbas f, thurnheer t, gmur r. oral biofilm architecture on natural teeth. plos one 2010; 5(2): 1–9. 14. marsh pd. dental plaque as a biofilm and a microbial communityimplications for health and disease. bmc oral health 2006; 6(1): 1–7. 15. liberio sa, pereira la, araujo mj, dutra rp, nascimento frf, neto vm. the potential use of propolis as a cariostatic agent and its actions on mutans group streptococci. j ethnopharmacol 2009; 125(1): 1–9. 16. parolia a, thomas ms, kundabala m, mohan m. propolis and its potential uses in oral health. int j medicine and medical sci 2010; 2(7): 210–215. 17. khalid a, afaf d, ameira m. propolis as a natural remedy: an update. saudi dent j 2001; 13: 45–49. 18. koo h, hayacibara mf, schobel bd, cury ja, rosalen pl, park �k. inhibition of streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol. j antimicrobial chemotherapy 2001; 52(5): 782–89. 19. kusumaningsih. pengaruh pasta gigi dengan kandungan buah stroberi (fragaria chiloensis l.) terhadap pembentukan plak gigi. diponegoro university instutional repository 2012; 188(1): 1–18. 20. amiati rd. pengaruh pasta gigi dengan kandungan buah anggur (vitis vinifera) terhadap pembentukan plak gigi. diponegoro university instutional repository 2012; 129 (1): 1–17. �� vol. 45. no. 1 march 2012 effect of robusta coffee beans ointment on full thickness wound healing yorinta putri kenisa1, istiati2, and wisnu setyari j2 1dental student 2department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: traumatic lesions, whether chemical, physical, or thermal in nature, are among the most common lesion in the mouth. wound healing is essential for the maintenance of normal structure, function, and survival of organisms. experiments of robusta coffee powder on rat-induced alloxan incision wound, clinically demonstrated similar healing rate with the povidone iodine 10%. no studies that look directly the effect of coffee extract in ointment form when viewed in terms of histopathology. robusta coffee bean (coffea canephora) consists of chlorogenic acid (cga) and caffeic acid which are belived to act as antioxidant and take part in wound healing process. purpose: the aim of this study was to identify the enhancement of healing process of full-thickness skin wound after robusta coffee beans extract ointment application. methods: sample consisted of 20 cavia cabaya treated with full-thickness with wounds and was given robusta coffee beans extract ointment concentration range of 22.5%, 45%, and 90% except the control group which was given ointment base material. animals were then harvested on the fourth day and made for histopathological preparations. data were calculated and compared by one-way anova test and lsd test. results: the study showed that robusta coffee bean extract ointment can increase the number of lymphocytes, plasma cells, macrophages, fibroblasts, and blood vessels by the presence of chlorogenic acid (cga) and caffeic acid. conclusion: in conclusion robusta coffee bean extract ointment enhance the healing process of fullthickness skin wound of cavia cabaya. key words: robusta coffee bean extract, the healing process, chlorogenic acid, caffeic acid abstrak latar belakang: lesi traumatik, baik akibat rangsang kimia, fisik, atau termal, merupakan lesi yang paling umum terjadi di dalam rongga mulut. penyembuhan luka yang terjadi ini penting untuk pemeliharaan struktur normal, fungsi, dan kelangsungan hidup organisme. percobaan pemberian bubuk kopi robusta terhadap luka sayatan pada tikus yang diinduksi aloksan, secara klinis menunjukkan tingkat penyembuhan yang sama dengan povidone iodine 10%. namun belum ada penelitian yang melihat secara langsung pengaruh ekstrak kopi dalam bentuk salep bila dilihat dari segi histopatologi. biji kopi robusta (coffea canephora) terdiri dari chlorogenic acid (cga) dan caffeic acid yang dipercaya berperan sebagai antioksidan dan mengambil bagian dalam proses penyembuhan luka. tujuan: tujuan penelitian ini adalah untuk mengidentifikasi peningkatan proses penyembuhan luka full-thickness pada kulit setelah pengaplikasian salep ekstrak biji kopi robusta. metode: sampel terdiri dari 20 cavia cabaya yang diberi perlakuan berupa luka sayat full-thickness pada kulit punggung dan diberi salep ekstrak biji kopi robusta dengan beberapa konsentrasi, yaitu 22,5%, 45%, dan 90%, sedangkan kelompok kontrol hanya diberi bahan dasar salep. binatang coba kemudian dieksekusi pada hari keempat dan dibuat sediaan histopatologinya. data dihitung dan dibandingkan dengan uji one-way anova dan uji lsd. hasil: hasil penelitian menunjukkan bahwa salep ekstrak biji kopi robusta dapat meningkatkan jumlah limfosit, sel plasma, makrofag, fibroblas, dan pembuluh darah yang dipengaruhi oleh chlorogenic acid (cga) dan caffeic acid. kesimpulan: disimpulkan bahwa salep ekstrak biji kopi robusta memiliki efek dapat meningkatkan proses penyembuhan luka full-thickness pada kulit cavia cabaya. kata kunci: salep ekstrak biji kopi robusta, proses penyembuhan, chlorogenic acid, caffeic acid research report ��kenisa, et al.: effect of robusta coffee beans ointment introduction wound can be defined as a disability or injury of living tissue caused by physical or thermal disturbance arising both pathologically and physiologically.1 traumatic lesions, whether chemical, physical, or thermal in nature, are among the most common in the mouth. this lession to oral-sot tissue can occur due to accidental, iatrogenic, and factitious traumas. they may present as burns, ulcerations, and gingival recession.2 bastone et al.,3 also described the aetiology of dental trauma from national and international studies as well as the different classifications currently used to report dental injuries. an english study determined the incidence of trauma to permanent incisors and related soft tissues as four cases/100 children/15 months, which was almost twice the incidence of australian study. based on those literatures, wound healing is essential for the maintenance of normal structure, function, and survival of organisms.4 wound healing is a complicated pathophysiological process. although mucosal wounds demonstrate accelerated healing compared to cutaneous wounds, both cutaneous and mucosal wound healing proceed through the same stages.5 wound healing consists of several stages, namely stage of acute inflammation, cell proliferation, and maturation. at the stage of proliferation, cell proliferative activity of fibroblasts in the lesion has a central role to begin the wound healing process. increasing number of fibroblasts in the dermal showed the healing ability.6 wound healing process may be hampered by the presence of reactive oxygen stress (ros) produced by microbes or neutrophils in the wound area, through mechanisms that lead to dna damage. this fact strengthens the opinion that the existence of local antioxidants in wound area became crucial factors that have promoted the acceleration of the healing process.7-9 several studies conducting the process of wound healing using natural materials have been widely applied. the use of natural materials done because it is easy to use, inexpensive, and has an adequate bactericidal or bacteriostatic effect.10 in addition, natural materials rarely cause adverse side effects compared with synthetic materials.11 one of these natural materials is robusta coffee beans. robusta coffee is widely spread on the island of java, sumatra, and sulawesi. price of this coffee is cheaper than other types of coffee and more resistant to diseases that attack the coffee plants. robusta coffee contains various compounds including 42.3% sugars (polysaccharides), 7.5% protein, 11% lipid, 2.4% caffeine, and 6.4% acids.12 also reported that the application powder of raw robusta coffee on rat-induced alloxan incision wound, showed clinical cure rate similar to the application of povidone iodine 10%.13 the polyphenols of coffee, caffeic acid and chlorogenic acid (cga), is believed to promote wound healing. robusta coffee beans have higher number of these polyphenols than arabica coffee beans.14 chlorogenic acid and caffeic acid have antioxidant properties that are significantly more potent than vitamin c and e.15 in addition to having antioxidant potential, robusta coffee has also been investigated to have antibacterial ability against methicillinresistent staphylococcus aureus that can cause opportunistic infections on the injured area. phenolic compounds in coffee also have been studied to reduce the effects of histamine, bradykinin, and leukotrienes as well as to reduce the activity of the complement system.16 research on the potential ointment of robusta coffee bean extract in dosage form in wound healing has not been reported. the extraction is done so that the active substances are needed can be taken optimally. the purpose of this study was to determine the potential of robusta coffee bean extract ointment on the healing process of full-thickness wounds on the skin of male guinea pigs (cavia cabaya) which was evaluated histopathologically. materials and methods this research is an experimental research laboratory. the material used is the ointment of robusta coffee bean extract with a range of concentration of 22.5%, 45%, and 90%. robusta coffee beans that have been roasted and used as a powder, then extracted using ethanol solvent. the extract was mixed with an ointment base material (peg 400 and peg 4000) and is based on the required concentration. this research used 20 male guinea pigs (cavia cabaya), aged 2–3 months, and weighing 200–300 mg. research subjects were divided into 4 groups each consisted of 5 guinea pigs. incision wound of 2.5 cm long with a depth of 2 mm was created on the back skin of each guinea pigs using number 11 scalpel under the effect of 10% ether anesthesia by inhalation. each treatment group was given ointment of robusta coffee bean extract and a control group given only simple ointment base using a syringe at a quantity of 2 cc. then treated in a closed wound with sterile gauze and plaster bandages. all guinea pigs in each group were harvested on the fourth day using 10% ether as sedation. back skin biopsies and subsequent histopathological preparations was done using haematoxylin eosin (he) staining. then calculation of chronic inflammatory cells (macrophages, lymphocytes, plasma cells), capillary blood vessels, and fibroblasts were done. the data obtained from histopathological examination is quantitative data obtained by calculating the number of cells and capillary blood vessels under light microscopy correspondence: wisnu setyari j, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjen prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: wizn_zetya@yahoo.com �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 52–57 performed on five different fields of view with 1000√ magnification. these research data were analyzed with statistical tests of one-way anova and lsd.17 results the largest number of lymphocytes present in the sample group which were given ointrment of 90% robusta coffee beans extract, while the smalles number found in the control group. the largest amount of plasma cells present in the sample group which were given ointment of robusta coffee beans extract concentration of 45%, while the smallest number found in the control group (table 1). the largest number of macrophages present in the control group, while the smallest number of groups present in concentrations of 22.5%. the largest number of fibroblasts present in the sample group which were given ointment of robusta coffee bean extract concentration of 45%, while the smallest number found in the control group. the largest number of capillaries present in the sample group which were given oinment of robusta coffee bean extract concentration of 90%, while the smallest number found in the control group (table 1). obtaining data on the number of cells and capillary blood vessels in each group performed one-way anova test. before the one-way anova test, this study shows that the data are normally distributed after the kolmogorovtable 1. distribution of mean and standard deviation of lymphocytes and plasma cells, macrophages, fibroblasts, and capillary blood vessels on the fourth day after treatment groups x ± sd lymphocytes plasma cells macrophages fibroblasts capillary blood vessels control 13.60 ± 8.735* 2.60 ± 1.817 39.80 ± 21.394 148.20 ± 22.928* 206.00 ± 83.896 g1 (22.5%) 14.40 ± 3.435* 6.00 ± 4.899 19.20 ± 11,189 217.60 ± 57.051* 213.20 ± 68.766 g2 (45%) 25.60 ± 8.649* 8.80 ± 5.020 27.20 ± 8.927 271.00 ± 94.557* 219.80 ± 134.908 g3 (90%) 34.20 ± 19.136* 7.60 ± 2.702 27.80 ± 6.099 173.00 ± 22.226* 238.00 ± 63.075 note: *: significant difference between groups a b c d figure 1. histopathological image on the group: a) control group, b) group 1 (22.5%), c) group 2 (45%), group 3 (90%). note: 1) lymphocytes, 2) capillary blood vessels, 3) fibroblast, 4) macrophages, 5) plasma cells. (he staining; magnification 1000√; olympus bx-50 microscope. pentax optio 230; digital camera 2.0 megapixels). ��kenisa, et al.: effect of robusta coffee beans ointment smirnov test statistic and homogeneous after levene test. one-way anova test showed significant values (p<0.05) in lymphocytes and fibroblasts. the average value of lymphocytes and fibroblasts in the treatment group differed significantly, whereas the other dependent variables such as plasma cells, macrophages, and capillaries found no significant difference. table 2. lsd statistical test of significance figures on the number of lymphocytes and fibroblasts between groups concentrations of ointments compared concentrations sig. (lymphocytes) sig. (fibroblasts) control 22,5% 0.697 0.074 45% 0.133 0.004* 90% 0.012* 0.505 22,5% control 0.697 0.074 45% 0.065 0.161 90% 0.015* 0.238 45% control 0.133 0.004* 22,5% 0.065 0.161 90% 0.273 0.016* 90% control 0.012* 0.505 22,5% 0.015* 0.238 45% 0.273 0.016* * the mean difference or significance value smaller than 0.05 (p <0.05) to determine the effect of differences in test conducted further post hoc test lsd. significant differences in this table are expressed with an asterisk ‘*’ on the mean difference or significance value smaller than 0.05 (p<0.05). in lymphocytes, the data showed significant mean differences in comparisons between the control group with group 3 and group 1 with group 3. whereas in fibroblasts, a significant mean differences found in comparisons between the control group with group 2 and between group 2 with group 3. discussion regeneration process can be seen from the cells that play a role during the wound healing process such as poli morpho nuclear (pmn) cells, lymphocytes, macrophages, plasma cells, fibroblasts, and capillary blood vessels. observation of the results of this study was done the fourth day after treatment for acute inflammatory cells such as pmn, especially neutrophils which will soon be replaced by macrophages on the third day and granulation tissue which enter the slit incision. gap is filled with granulation tissue and maximum vascularization on the fifth day. the observation of the results on this study was conducted on the fourth day so that all cells needed can be seen.4 this study used a range of 22.5%, 45%, and 90% concentration and is a preliminary study using ointment of robusta coffee bean extract. extraction of coffee was done so the active substances can be taken optimally. ethanol was used as extraction solvent because it can withdraw the amount of phenolic acids higher than methanol and pure water.18 the ointment used are made of poly ethylen glycol (peg) because peg is chemically stable. both peg 400 and peg 4000 used in this study are soluble in ethanol. peg does not irritate skin and easy to clean by washing.19 wound healing involves several mechanisms, such as inflammatory phase, proliferation, and maturation. in the inflammatory phase, the objectives are to stop the bleeding and clean the wound area of foreign bodies, dead cells, and bacteria to prepare for the start of healing process. pmn cells migrate into the interstitial area to perform phagocytosis of foreign bodies and bacteria. however, wound healing is enhanced by the presence of stress ros produced by pmn or microbial infection. if ros are produced too much, it can cause cellular and dna damage. cga and caffeic acid are contained in robusta coffee beans extract act as antioxidants to neutralize ros which is the free radicals produced in the process of wound healing. ros can increase lipid peroxidation which is a major cause of damage to the cell membrane so that it can damage the cell structure and function.20 antioxidants have been reported to have a significant role in the process of wound healing and protect tissues from oxidative damage.21 antioxidant mechanism is expected to protect cells that play a role in the process of wound healing. cga and caffeic acid as antioxidants convert free radicals into stable products. the neutralized free radicals can not react on polyunsatured fatty acids (pufas) which generate alcoxyl and peroxyl radicals that responsible for the basic process of membrane cell lipid peroxidation.22 in the initial adhesion process, pmn adhere to the endothelium through the interaction of specific molecules such as selectin and glycosylated protein so that pmn ables to exit the endothelial transmigration as it is called an acute inflammatory process.23 this phase continues as chronic inflammatory cells into the injured area. table 1 showed that the mean number of lymphocytes in group 3 is higher than the control group, group 1, and group 2. according to hung et al.,24 cga was shown to increase lymphocytes proliferation.24 it can be seen from the mean number of increased lymphocytes until the highest (90%). lsd test on lymphocytes showed that there were significant mean differences in comparisons between the control group with group 3 and group 1 with group 3 (table 2). while results for the plasma cells in table 1 showed mean number of the highest plasma cells found in group 2 which did not differ significantly group 3. active substances contained in coffee, namely cga, has been mentioned to have a role in increasing proliferation of lymphocytes. this is probably an indirect effect of plasma cells as these cells is the end product of activation of b lymphocytes activation that have differentiated, then plasma cell produce direct antibody against antigens in inflammation. results of the next calculation is the amount of macrophage cells in control group which showed a higher �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 52–57 mean than group 1, group 2, and group 3. this is presumably due to the treatment group, the phase of chronic inflammation will soon ends characterized by the declining number of macrophages and the beginning phase of proliferation. increasing the mean number of macrophages seen in the treated group. group 3 has the highest mean followed by group 2 and group 1. this is because cga stimulates the mobilization of macrophages, may indirectly increase the ability of macrophage phagocytosis because it affects the secretion of ifn g that act as macrophage activators.22,24 t lymphocytes which are activated by interaction with macrophages that present antigen fragments on the surface of cells can produce ifn g. these cytokines may activate macrophages so that macrophages release other cytokines to activate lymphocytes and causes inflammation where there is a focus of both these cells stimulate each other to destroy the antigen. the next phase is the proliferative phase which involves the proliferation of fibroblasts, collagen synthesis, angiogenesis, granulation tissue formation, and epithelisation.25 an important first step in this phase is the improvement of microcirculation to supply oxygen and nutrients needed to fill the metabolic needs of tissue repair. regeneration of new blood vessels (angiogenesis) is stimulated by hypoxic injury condition as well as several growth factors, particularly vegf-a, fgf-2, tnf-b. at the same time, fibroblasts migrate into the wound in response to cytokines and growth factors produced by inflammatory cells, among which are macrophage.26 that activated macrophages can stimulate growth factors and cytokines (tgf-a, tgf-b, pdgf, vegf -a, and il-1) on the injured area. tgf-b plays a role in angiogenesis, reepithelisation, and connective tissue regeneration. tgf-b which are dominant in cutaneous wound healing is tgf-b1. tgf-b works by activating its receptor on the cell surface and transducing signal on target genes. binding of a tgf-b to its type ii receptor in concert with a type i receptor leads to formation of a receptor complex and phosphorilation of type i receptor. thus activated, the type i receptor subsequently phosphorylates a receptor-regulated smad (r-smad), allowing this protein to associate with smad4 (co-smad) and move into nucleus. in the nucleus, the smad complex associate with a dna-binding partner (fast-1) and this complex binds to a specific enhancers in target genes so that it can activate the gene transcription.27,28 in the injured tissues, extracellular matrix molecules (ecm), namely tenascin-c, expressed during the process of tissue repair. tenascin-c plays a role in proliferation and migration of fibroblasts. this molecule can induce phosphorylation of epidermal growth factor receptor (egfr) and stimulates activation of mitogenic activated protein (map) kinase and mitogenesis of fibroblasts. in addition, tenascin-c can induce migration of fibroblasts through the activation of plcg and m-calpain.29 those growth factors and molecules play a role in cell proliferation and migration of fibroblasts so that the process of wound healing can be achieved. the counting results in graph 2 showed the mean number of fibroblasts in group 2 which is higher than the control group, group 1, and group 3. lsd test on fibroblast cells showed that there were significant mean differences in comparisons between the control group with group 2 and group 2 with group 3 (table 2). the number of decreased fibroblasts in group 3 caused by the proliferation of fibroblasts cells which have reached the optimum effect at a concentration of 45%. the mean results of capillary blood vessels showed that the number of capillaries in group 3 is higher than the control group, group 1 and group 2. an increasing number of these occurred with increasing concentrations of the ointment. this occurs indirectly as the influence of several growth factors such as vegf-a, fgf-2, tnf-b that are produced both by macrophages and fibroblasts. tgf-b1 produced by macrophages can also induce up-regulation of growth factor for angiogenesis such as vegf.27 fibroblasts are actively moving from the network around the wound into the wound area, proliferate and issue some substances (collagen, elastin, hyaluronic acid, fibronectin, and proteoglycans) that play a role in forming new tissue. collagen is a protein substance that increase the surface tension of the wound.25other phenolic compounds in coffee, namely caffeic acid, has also been studied to play a role in the healing process by stimulating the synthesis of collage-like polymer by fibroblasts.6 increased amount of collagen that add strength to the wound surface can avoid the possibility of opened wound.26 in the results of data analysis, the highest levels of robusta coffee bean extract ointment (90%) showed the highest value on the mean number of lymphocytes, macrophages, and capillary blood vessels, but not in plasma cells and fibroblasts, although it is higher than the concentration of 22.5%. while coffee bean extract 45% concentration ointment showed the highest value on the average number of plasma cells and fibroblasts compared to ointment of coffee bean extract 90%. overall, robusta coffee bean extract 45% ointment can give a good effect on wound healing process because at this concentration the number of fibroblasts increased significantly compared with the control group. it can be concluded that ointment of robusta coffee been extract could enhance skin wound healing process of cavia cabaya. references 1. boateng sj, matthews kh, steven hne, eccieston gm. wound healing dressing and drug delivey system: a review. j pharmaceu sci 2007; 97: 2892–923. 2. dilsiz a. self-inflicted oral soft-tissue burn due to local behavior and treatment. clin exp dent 2010; 2(1): 51–2. 3. bastone eb, freer tj, mcnamara jr. epidemiology of dental trauma: a review of the literature. aust dent j 2000; 45(1): 2–5. 4. kumar v, cotran r, robbins sl. buku ajar patologi. 7th ed. philadelphia: wb saunders co; 2007. p. 41–3, 55–60. 5. chen l, arbieva zh, guo s, marucha pt, mustoe ta, dipietro la. potisional differences in the wound transcriptome of skin and oral mucosa. bmc genomic 2010; 11: 471. 6. song hs, park tw, sohn ud, shin yk, choi bc, kim cj, sim ss. the effect of caffeic acid on wound healing in skin-incised mice. koeran j physiol pharmacol 2008; 12: 343. ��kenisa, et al.: effect of robusta coffee beans ointment 7. james tj, hughes ma, hofman d, cher ry gw, taylor rp. antioxidant characteristic chronic wound fluid. br j dermatol 2001; 145: 185–6. 8. gupta a, singh rl, raghubir r. antioxidant status during woung healing in immunocompromised rats. mol cell biochem 2002; 241: 1–7. 9. russo a, longo r, vanella a. antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin. fitoterapia 2002; 73: 21–9. 10. ramos m. propolis: a review of its anti-inflamatory and healing actions. j venom anim toxins incl trop dis 2007; 13: 697–710. 11. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). maj ked gigi (dent j) 2005; 38(3): 135–41. 12. panggabean e. buku pintar kopi. jakarta: agromedia pustaka; 2011. p. 5, 124. 13. susanto y, puradisastra s, ivone j. efek serbuk biji kopi robusta (coffea robusta lindl. ex de willd) terhadap waktu penutupan luka pada mencit jantan galur balb/c yang diinduksi aloksan. jurnal kedokteran maranatha 2009; 8(2): 121. 14. lelyana r. pengaruh kopi terhadap asam urat. thesis. semarang: p rogra m pascasa r ja na magister i lmu biologi k un iversitas diponegoro. 2008. 15. kweon mh, hwang hj, sung hc. identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo (phyllostachys edulis). j agric food chem 2001; 49: 4646. 16. yuwono hs. ilmu bedah vaskular: sains dan pengalaman praktis. bandung: refika aditama; 2010. p. 315–22. 17. ghozali i. aplikasi analisis multivariate dengan program spss. cet a k a n ke empat. sema ra ng: bad a n penerbit un iver sit a s diponegoro; 2009. p. 8–14 18. pinelo m, tress ag, pedersen m, arnous a, meyer as. effect of cellulaces, solvent type, and particle size distribution on the extraction of chlorogenic acid and other phenols from spent coffe grounds. am j food technology 2007; 2(7): 641–51. 19. rowe rc, sheskey pj, quinn me. handbook of pharmaceutical excipients. washington dc: pharmaceutical press and american pharmacist association; 2009. p. 121. 20. kont a s-a sk a r t, a lt ug m e , k a r ap eh l iva n m, at a k isi e , hismiogullari aa. is cape a therapeutic agent for wound healing?. j animal and veterinary advances 2009; 8 (1): 129–33. 21. al-henhena aa, mahmood a, al-magrami ab, nor syuhada aa, zahra md, summaya, ms, suzi, salmah i. histological study of wound healing potential by ethanol leaf extract of strobilanthes crispus in rats. j med plants research 2011; 5(16): 3660–6. 22. morishita h, ohnishi m. absorption, metabolism, and biological activities of chlorogenic acids and related compounds. studies in natural products chemistry 2001; 25: 932. 23. hebeda cb, bolonheis sm, nakasato a, belinati k, souza pd, gouvea dr, lopes np, farsky sh. effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus. j ethnopharmacol 2011. 135(2): 261–9. 24. hung cm, yeh cc, chong ky, chen hl, chen yj, kao st, yen cc, yeh mh, lin ms, chen cm. gingyo-san enhances immunity and potentiates infectious bursal disease vaccination. evidence-based complementary and alternative medicine 2008; 8. 25. rubin r, strayer d. rubin’s pathology: clinicopathology foundations of medicine. 5th ed. philadelphia: lippincott williams and wilkins; 2008. p. 38–70. 26. ismail s. luka dan perawatannya. 2002. available at: images. mailmkes.multiply.multiplycontent.com. accessed december 24, 2010. 27. barrientos s, olivera s, golinko ms, brem h, tomic-canic m. growth factors and cytokines in wound healing. j wound repair and regeneration 2008; 16: 585–601. 28. massague j. tgf-b signal transduction. j ann rev biochem 1998; 67: 753–91. 29. midwood ks, orend g. the role of tenascin-c in tissue injury and tumorigenesis. j cell commun signal 2009; 3(3-4): 287–310. 109 vol. 42. no. 3 july–september 2009 case report introduction of all the non-traumatic ulcerations that affect oral mucosa, recurrent aphthous stomatitis (ras) are the most common lesions found and observed by physicians and dentists.1,2 the term "recurrent aphthous stomatitis" should be reserved for recurrent ulcers confined to the mouth and seen in the absence of systemic disease.3 the disease is characterized by recurring painful ulcers of the mouth that are round or ovoid and have inflammatory halos.3 the condition ranges in severity from ras minor, characterized by occasional and self-limited ulcerations, to a very debilitating form, called ras major. ras minor heal in about one week without scarring. ras major can last from weeks to months and, rarely, extend out to years.4 ras are typically classified as minor, major, and herpetiform types. ras major (also referred as periadenitis mucosa necrotica recurrens or sutton's disease) is the most severe expression of aphthous stomatitis.1 ras major develop deep lesions that are larger than 1 cm in diameter and may reach 5 cm.5 in this type, 1–10 major aphthae may be present simultaneously. ulcers are large and deep, they may coalesce, and they often have a raised and irregular border. on healing, which may take as long as 6 weeks, the ulcers leave extensive scarring.2 occasionally, patients may experience an unremitting course with significant morbidity; systemic health may be compromised secondary to difficulty in eating and psychological stress.1 cross-sectional studies suggest that recurrent aphthous stomatitis is more common in women, in people under the age of 40 years, whites, nonsmokers, and in people of high socioeconomic status.3 ras is the most common oral mucosal disease in north america. ras affect 2–66% of the international population.2 although the lesions may appear at any age, they usually present during the second treatment of recurrent aphthous stomatitis major with metronidazole and ciprofloxacin m. jusri1 and nurdiana2 1 department of oral medicine 2 resident of oral medicine faculty of dentistry, airlangga university surabaya indonesia abstract background: recurrent aphthous stomatitis (ras) are painful oral ulcerations that recur from days to months or even years. it represents the most common lesion of the oral mucosa with prevalence ranging from 15% to 30%. although the clinical characteristics of ras are well defined, the precise etiopathogenesis of ras remains unclear. since the etiology of ras remains unknown, there is no definitive treatment. ras responds quite well to the use of topical or systemic antiinflammatory drugs, particularly corticosteroids. purpose: the objective of this paper is to discuss the treatment of ras with secondary infection. case: this paper reported a case of 22-year-old man with multiple oral ulcers that did not heal for 7 months. case management: these ulcers were diagnosed as ras major with secondary infection that caused by normal oral flora (aerobic and anaerobic bacteria) and treated with metronidazole (topical and oral) and ciprofloxacin (oral). these lesions healed in 3 weeks with scars. conclusion: large ulcer without signs of malignancy that contaminated with normal oral flora will delayed in healing, but with rational treatment ras mayor with secondary infection has good prognosis. key words: recurrent aphthous stomatitis, major ulcer, metronidazole, ciprofloxacin correspondence: m. jusri, c/o: departemen oral medicine, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: m_jusri@yahoo.com 110 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 109-113 and third decades of life.6 development of ras minor usually begins in childhood or adolescence. the onset of ras major is after puberty, and recurrent episodes may continue to develop for up to 20 years or more.7 ras herpetiform first occurs in the second decade of life, 67–85% of persons have onset under 30 years.2 more than 42 percent of patients with ras have first-degree relatives with ras. the likelihood of ras is 90 percent when both parents are affected, but only 20 percent when neither parent has ras.8 treatment of ras should relate to the severity of the disease. pain relief of minor ulcers can be obtained with use of a topical anesthetic agent or topical nsaid. in more severe cases, the use of high-potency topical steroid preparation, such as fluocinonide, betamethasone or clobetasol, placed directly to the lesion after meals and at bedtime. this agent shortens healing time and reduces the size of the ulcers. other topical preparation include amlexanox paste and topical tetracycline, which can be used either as a mouth rinse or applied on gauze sponges.5 systemic corticosteroids, such as prednisone (20–30 mg/ day) and betamethasone (2–3 mg/day) for 4-8 days are very helpful for major or herpetiform ulcers.6 this case reports a patient suffering ras major that did not heal for approximately 7 months. clinically, the lesion showed multiple ulcerations which consist of ras major and ras minor. even though patient has seen several dentists, and use different kind of drugs, such as mefenamic acid, clindamycin, chlorhexidine mouthwash, another mouthwash, and vitamin, but the lesion did not heal. the authors assumed that there was a secondary infection from normal oral flora that caused ras difficult to heal. this case report discusses management of ras major with secondary infection that is treated with antibiotic. case a 22-year-old man was referred to oral medicine department faculty of dentistry airlangga university surabaya by a dentist from madiun with ulcers in right buccal mucosa that did not heal for a long time. from anamnesis known that about 7 months ago the patient had an ulcer in his right backside buccal mucosa. after consuming 'adem sari' the ulcer healed. one week later, an ulcer appeared in the same area but at different place. the patient then took 'adem sari' again but the ulcer did not heal. in a few days several ulcers appeared in this areas and very painful. the lesion was treated by several dentists and given several drugs. afterward, the lesion became smaller and the pain decreased but then the lesion enlarged again and painful. the patient then visited a dentist in haji hospital and treated with mefenamic acid, clindamycin, and chlorhexidine mouthwash, afterward the pain decreased but the lesion remains the same. before this ulcers appeared the patient often experienced an ulcer approximately once in a month, but the patient did not remember when it was. clinical examination revealed that general condition of the patient was good. the patient had smoking habit since approximately 5 years ago. there's no known disorder in patient's medical history and family history. extra oral examination showed mild swelling on right cheek, fissure and desquamation at corner of the lips, and chronic lymphadenitis on the right submandibular gland. intra oral examination showed two ulcers on right lower labial mucosa, irregular shaped ulcer with diameter approximately 8 mm, raised border, surrounded by erythematous base, covered by white pseudomembrane, and painful (figure 1-a) and round shaped ulcer with diameter approximately 6 mm, raised border, surrounded by erythematous halo, covered by white pseudomembrane, and painful (figure 1b). irregular shaped ulcer, was found on right buccal mucosa size approximately 4 × 10 mm, raised center, surrounded by erythematous base, covered by white pseudomembrane, and painful. erosions, fissures and scars (figure 1-c) were also found in this area. on lower buccal fold mucosa appeared ulcer with round irregular shape with diameter approximately 10 mm, clear and raised border, surrounded by erythematous halo, covered by white pseudomembrane, and painful (not seen at figure 1). beside these ulcers, intra oral examination also revealed radix of lower right first molar, dental plaque and calculus on upper and lower right teeth. figure 1. visit 1: multiple ulcers and scars on lower labial mucosa and right buccal mucosa. case management on first visit, according to anamnesis and clinical examination, the clinical diagnosis of this case was ras major with squamous cells carcinoma and noma as the differential diagnosis. scrapping for cytology examination and swab for bacteriology examination were done. the patient was instructed to take examination of complete blood, sgot, sgpt, and blood glucose. then the patient was given 5% extract sanguine + 0.1% polidocanol gel applied to the lesion 3 times daily, chlorhexidine mouthwash 3 times daily, and h2o2 solution as mouthwash twice daily. 111jusri and nurdiana: treatment of recurrent aphthous stomatitis major on second visit (two days later), the patient came for the first control. according to anamnesis it was known the pain has decreased. the result from cytology examination showed eosinofilic amorphous material, squamous cells spread, and cells with round nucleus degeneration. bacteriological examination result found gram (+) cocci, gram (–) rods, and candida. the result of complete blood examination showed an erythrocyte sedimentation rate (esr) value elevation is (33). intra oral examination revealed smaller ulcers on lower labial mucosa with diameter approximately 6 mm (figure 2-a) and 5 mm (figure 2-b). ulcer on buccal mucosa had become smaller, approximately 4 × 8 mm, while erosions, fissures, and scars (figure 2-c), and ulcer on lower labial fold mucosa did not change. on third visit, according to anamnesis there was no pain. intra oral examination revealed ulcers on lower labial mucosa with diameter approximately 5 mm (figure 4-a) and 4 mm (figure 4-b) with erythematous halos. ulcer on buccal mucosa had become smaller, 2 × 6 mm, while erosions, fissures, and scars were still remained but the erythema had decreased (figure 4-c). ulcer on lower labial fold mucosa became smaller with diameter approximately 8 mm. patient was instructed to continue the therapy. figure 4. third visit showed: (4th day) (a) ulcer became smaller (5 mm), (b) ulcer became smaller with erythematous halos (4 mm), (c) erosions, fissures, scars, and decreased erythema. on fourth visit (the third control), eight days later, intra oral examination revealed that all ulcers, erosion and fissure were healed, except irregular shaped ulcer on buccal mucosa, diameter approximately 2 mm, flat border, covered by white pseudomembrane (figure 5). the patient instructed to continue the therapy, added with multivitamin once daily and h2o2 solution as mouthwash twice daily. figure 5. fourth visit showed: (12th day) all ulcers had healed, except ulcer on corner of buccal mucosa (2 mm), flat border, covered by white pseudomembrane, and without erythematous halo (arrow). the patient treated with 500 mg metronidazole 3 times daily, and 500 mg ciprofloxacin twice daily. two tablets of 500 mg metronidazole made into powder and divided into 20 doses, then the powder placed on the ulcers and covered with 5% extract sanguine + 0.1% polidocanol gel applied 3 times daily (figure 3). chlorhexidine mouthwash was continued. figure 2. second visit showed, smaller ulcers but erosions, fissures, and scars did not change. figure 3. second visit (3rd day) showed ulcers after being given metronidazole powder and covered with 5% extract sanguine + 0.1% polidocanol gel. 112 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 109-113 on fifth visit (the fourth control): intra oral examination revealed all ulcers, erosion, and fissure had healed with scars. the patient instructed to continue taking multivitamin once daily. figure 6. comparison of the lesion om the first visit and the fifth (18th day). all lesions had healed with scars. discussion intra oral examination of this patient revealed several ulcers with variation of clinical features. some ulcers had ras characteristic. ras major usually appeared after puberty, they are round or ovoid with clearly defined margins, and the ulcers usually are deeper and larger and last significantly longer than ras minor. these ulcers have a raised irregular border and frequently exceed 1 centimeter in diameter and painful that last for weeks or months and often leave a scar after healing.8 ras usually confined to movable nonkeratinized or poorly keratinized oral mucosa, e.g., buccal mucosa, labial mucosa, tongue, floor of the mouth, soft palate, and uvula.6 another lesion found in intra oral examination was ulcerations without erythematous halo, which is an indication of chronic lesion. extra oral examination found no other lesion. on examination, it was known that ulcer had happened previously, about once a month. according to the clinical features, multiple ulcers with ras characteristic and ulcerations with sign of chronic infection, as well as history of recurrency, the clinical diagnosis of the disease was ras major with squamous cell carcinoma and noma as the differential diagnosis. the cytological examination of this case showed no malignant cells, so the possibility of squamous cells carcinoma can be ruled out. noma also considered as differential diagnosis in this case. noma is rapidly progressive, opportunistic infection caused by components of the normal oral flora that become pathogenic during periods of compromised immune status. noma often begins on the gingiva as necrotizing ulcerative gingivitis, which may extend to involve the adjacent soft tissue and form areas called necrotizing ulcerative mucositis. the necrosis can extend into deeper tissues, over the next few days zones of bluish-black discoloration of the overlying skin surface may develop. these discolored zones break down into areas of yellowish necrosis that also frequently spreads into adjacent bones, with large area of osteomyelitis possible.7 diagnosis of noma was inappropriate because clinically there's neither necrotic area nor bones destruction. histological findings, showed nonspecific ulcers with chronic mixed inflammatory cells. the pseudomembrane covering of ulcer was a combination of oral bacteria and fungi, as well as necrotic keratinocytes and sloughed oral mucosa.2 the result from cytology examination showed eosinophilic amorphous material, squamous cells spread, and cells with round nucleus degeneration, which lead to assumption of chronic infection. the result of complete blood examination showed an erythrocyte sedimentation rate (esr) value elevation, with value of 33 from the normal of less than 15. this result supports the assumption of chronic infection. bacteriology examination result found gram (+) cocci, gram (–) rods, and candida. candida found on this examination did not show candida involvement but more there was lesion contamination by candida which also available in normal oral flora. according to clinical features, history, no involvement of other disease shown by normal complete blood examination, and chronic infection signs shown by cytology examination and elevation of esr value, the final diagnosis for this case was ras major with secondary infection from normal oral flora. we assumed these lesions did not heal for 7 months because there's contamination of the lesion by normal oral flora. this theory supported by the result of bacteriology examination. the bacteria found in bacteriology examination were normal oral flora. normal oral flora consists of aerobic and anaerobic bacteria. the aerobic bacteria found in oral cavity were primarily aerobic gram (+) cocci, and aerobic gram (–) rods. two major groups of anaerobic bacteria in oral cavity are anaerobic gram (+) cocci and anaerobic gram (–) rods.9 typical oral infection is caused by mixture of aerobic and anaerobic bacteria; approximately 70% of these infections are caused by this mixed flora. this fact has major clinical implications. the useful antibiotic for oral 113jusri and nurdiana: treatment of recurrent aphthous stomatitis major infection must be effective against aerobes and anaerobes bacteria.9 ciprofloxacin has 6-fluoro substituent which greatly improve antibacterial potency against gram (+) and gram (–) aerobes organisms.10 these agents interfere with bacterial enzyme critical for dna transcription.8 side effects of ciprofloxacin such as nausea, vomiting, rash, dizziness, and headache are rare.10 the usual adult dose for ciprofloxacin is 500 to 750 mg orally every 12 hours. metronidazole is effective only against anaerobic bacteria including those in the oral cavity.8 these agents diffused into the bacteria where nitro component is reduced. during this reduction process, chemically reactive intermediate component is formed, which inhibit dna synthesis and/or destroying dna resulting in disruption of dna function.10 metronidazole is available for oral, intravenous, intravaginal, and topical use.5 metronidazole is administered orally with usual dosage of 500 mg every 8 hours.8 in this case oral and topical metronidazole were used. because there is no topical metronidazole available we ground powdering metronidazole tablet. this powder then placed on the ulcers and covered with 5% extract sanguine + 0.1% polidocanol gel to get continuous direct contact. treatment of this case was done according to the treatment of ras and secondary infection control. the patient was treated with systemic metronidazole and ciprofloxacin to treat the infection. topical treatment that we used was metronidazole that made into powder and applied to the lesions to get the bactericidal effect through direct contact with the lesions to help improving infection healing. five percent of extract sanguine + 0.1% polidocanol gel were applied as covering agent to reduced pain and improving epithelialization. the author has already used this method to treat several similar cases. treatment with metronidazole powder that applied to the lesions also had been used in the wound treatment of a patient in ear nose throat department rsupn-cm.11 chlorhexidine mouthwash was used to help eliminating and healing the infection. it was concluded that ras major in this case was contaminated by normal oral flora that causing secondary infection of the lesions. so, the treatment of this case was directed to treat the secondary infection. after proper therapy, the lesions healed in 3 weeks. to avoid recurrent of the disease, the patient was suggested to improve the oral condition. the patient was instructed to extract lower right first molar, scaling to remove plaque and calculus, and maintenance his oral hygiene. references 1. sciubba jj. oral mucosal disease in the office setting–part i: aphthous stomatitis and herpes simplex infections. available from: http://www. agd.org/publications/articles/?artid=1858. accessed january 4, 2008. 2. casiglia jm, mirowski gw. aphthous stomatitis. available from: http://www.emedicine.com/derm/topic486. accessed january 8, 2008. 3. scully c. aphthous ulceration available from: http://members.tripod. com.enotes.aphthous_ulcer. accesed august 8, 2007. 4. melamed f. aphthous stomatitis. available from: http://www.med. ucla.edu. accessed january 4, 2008. 5. laskaris g. treatment of oral disease. a concise textbook. 1st ed. stuttgart: thieme; 2005. p. 15–7. 6. scully c, gorsky m, lozada-nur f. the diagnosis and management of recurrent aphthous stomatitis. available from: http://jada.ada. org/cgi/content/full/134/2/200. accessed january 4, 2008. 7. neville bw, damm dd, allen cm, bouquot je. oral and maxillofacial pathology. 2nd ed. philadelphia: saunders co; 2002. p. 178–9. 8. peterson lj. principles of surgical and antimicrobial infection management. in: topazian rg, goldberg mh, hupp jr. oral and maxillofacial infections. 4th ed. philadelphia: wb saunders company; 2002. p. 101–3, 121. 9. greenberg ms. ulcerative, vesicular, and bullous lesions of the oral mucosa. in: greenberg ms, glick m, editors. burket's oral medicine diagnosis & treatment. 10th ed. hamilton: bc decker; 2003. p. 63–5. 10. neal mj. 2005. at a glance pharmacology medis. surapsari j, safitri a, editors. jakarta: penerbit erlangga; 2006, p. 80–1. 11. irna s. identifikasi dan pengendalian faktor resiko mukositis oral selama radioterapi kanker nasofaring. available from: http://resources. unpad.ac.id/unpad-content/uploads/publikasi_dosen/identifikasi dan pengendalian faktor resiko mukositis oral.pdf. accessed june 12, 2009. 81 mandibular morphology of the mongoloid race in medan according to age groups maria sitanggang and trelia boel department of oral and maxillofacial radiology, faculty of dentistry, universitas sumatera utara medan indonesia abstract background: the mandible constitutes part of the craniofacial bone that plays an important role in determining an individual’s facial profile. the mandible grows and develops throughout life from the prenatal phase up to old age when it becomes and edentulous. changes in the mandible can be measured using radiographs. these establish several parameters of mandibular morphology, including: ramus height, condylion height, body length, condylion angle, symphysis height, symphysis width and symphysis angle. purpose: this study aimed to determined differences in the mandibular morphology of members of the mongoloid racial group in medan according to age as measured by cephalometric radiography. methods: this investigation constituted analytical research using cross-sectional study with a total sample of 150 individuals divided according to age: group 1 (aged 4-12 years), group 2 (aged 13-24 years, group 3 (aged 25-34 years), group 4 (aged 35-60 years) and group of 5 (aged > 60 years). the parameters were computerized by means of a digital cephalometric radiograph, the resulting data being analized with one-way anova and lsd. results: the mean value of the highest to the lowest ramus height, and symphysis height from the five age groups, sequentially, were in group 3, group 4, group 5, group 2, and group 1. the mean value from the highest to the lowest of body length, condylion height, condylion angle, and symphysis width, sequentially, were in group 3, group 4, group 2, group 5, and group 1. the mean value from the highest to the lowest of symphysis angle, sequentially, were in group 1, group 3, group 4, group 2, and group 5. conclusion: the mandibular morphology of each age group differs in mongoloid races in medan based on lateral cephalometric radiography in which changes are may be affected by the state of teeth and age. keywords: mandibular morphology; cephalometric radiograph; age group correspondence: maria sitanggang, department of oral and maxillofacial radiology, faculty of dentistry, universitas sumatera utara, jl. alumni no. 2, kampus usu, medan 40132, indonesia. e-mail: manaomi79@gmail.com dental journal (majalah kedokteran gigi) 2018 june; 51(2): 81–85 research report introduction the mandible is a part of the body that can be used in determining race, sex and age of an individual.1,2 skeletal anomalies that often occur are not only caused by the condition of the teeth, but also because of mandibular anomalies.3 in dentistry, especially orthodontics, the mandible is used to determine a person's facial profile. in prosthodontics, particularly in edentulous patients, the determination of vertical dimension is influenced by the maxillary, mandibular, temporomandibular joints and masticatory muscles.4 growth and development that begins at childbirth and continues into old age will produce changes in the mandible. research on growth and development has been widely conducted, indicating that the most rapid and intense changes occurs during infancy and childhood. mandibular vertical growth occurs through bone remodelling along with alveolar process development and dental eruption. posterior borders of the condyle and ramus are very active in mandible growth with new bone deposition and remodelling, while the anterior surface undergoes bone resorption.5 the ramus height, condyle height, condyle angle, mandible length (overall length) and condylion angle are all parameters that undergo changes in the shape and the size of the growing and developing mandible.6–9 after the first year of life, mandibular growth is more selective.10 according to doi: 10.20473/j.djmkg.v51.i2.p81–85 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg mailto:manaomi79@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p81-85 82sitanggang and boel/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 81–85 enlow and harris who conducted a study of 25 mandibles in children aged 4-12 years, the lingual and buccal cortex of the condyle neck is formed by the endosteal bone. the size of the gonial angle is reduced (140°) to provide additional space for permanent teeth formation. the coronoid process will experience movement through the periosteal bone incision that occurs continuously. at the age of 5-6 years, growth of the ramus and mandibular bodies is believed to occur mainly in parallel with changes in the midface. the growth of the ramus increases anteroposteriorally, in that vertical growth in the ramus is associated with maxillary growth and maxillary teeth eruption. liu et al.6 conducted a study of the size of the mandible from the first 6 months until the fifth year of a person’s life. the condyle, posterior ramus, inferior ramus, gonion, posterior body and point "b" will change more in the superior and posterior.6 in general, edentulism affect both general and oral health which, in turn, will influence quality of life. several studies conducted on the mandible suggest that there are morphological and anatomical changes between dentate and edentulous individuals.11,12 changes in the structure and function of the masticatory muscles in edentulous individuals due to old age, indicate a decrease in activity and muscle density rather than individuals who have lost all their teeth.9 this study was conducted to determine differences in the mandibular morphology of mongoloid individuals in medan according to age as identified by means of cephalometric radiography. several parameters of morphology mandibular are ramus height, condylion height, body length, condylion angle, symphysis height, symphysis width and symphysis angle. materials and methods this analytical research was conducted using crosssectional design and carried out at the unit of oralmaxillofacial radiology, faculty of dentistry, universitas sumatera utara. the research population consisted of members of three successive generations of the mongoloid race in medan, divided into groups of 30 individuals. a sample selection method was applied by purposive sampling method adhering to fulfilled inclusion and exclusion criteria. inclusion criteria: group 1 (age 4-12 years): in primary dentition phase: complete primary dentition and never having undergone orthodontic treatment during the mixed dentition period: there is no premature loss of deciduous teeth, no permanent teeth are missing, dentures are not employed and orthodontic treatment has never been applied. group 2 (age 13-24 years): dentures are not employed, and orthodontic treatment has never been applied. during the mixed dentition period, there is no premature loss of deciduous teeth, and no permanent teeth are missing. during the permanent dentition period, the teeth are complete (28 teeth with the exception of the third molars). group 3 (age 25-34 years): the teeth are complete (28 teeth with the exception of the third molars) and orthodontic treatment has never been applied. group 4 (35-60 years): the teeth are complete (28 teeth with the exception of the third molars) and orthodontic treatment has never been applied. group 5 (aged >60 years): fully edentulous and orthodontic treatment has never been applied. exclusion criteria: has suffered from and/or received treatment for systemic diseases, has undergone jaw surgery, odontectomy, has suffered from micrognathia, pathological conditions in the mandible, still possesses deciduous teeth, has experienced deep caries with exposure of the pulp and crown loss, has received treatment of the full crown or space maintainer/space regainer. after completing the informed consent form, a lateral digital cephalometric radiograph (2d) was taken for each sample using an instrumentarium orthopantomograph model qc 200 d 1-4-1, 2012. the parameters were computerized by a digital cephalometric radiograph (figure. 1). a one-way anova test was performed to analyze the calculated data with p<0.05. ethical clearance was obtained from the research ethics committees of the faculty of medicine, universitas sumatera utara no. 345/ tgl/kepk fk usu-rsup ham/2017. results the results of the normality test using a one-sample kolmogorov-smirnov test confirmed that the variables are normally distributed with p>0.05. table 1 shows the mean values and significant differences (multiple comparison lsd test result) of all variables (ramus height, symphysis height, body length, condylion height, condylion angle, symphysis width and symphysis angle) in five age groups (p value <0.05). the mean value of ramus height and symphysis height sequentially from the highest to the lowest were in group 3, group 4, group 5, group 2 and group 1. the mean value of body length, condylion height, condylion angle and symphysis width sequentially from the highest to the lowest were in group 3, group 4, group 2, group 5 and group 1. the mean value of the symphysis angle sequentially from the highest to the lowest were in group 1, group 3, group 4, group 2, and group 5. discussion in this study, the average values of the ramus and symphisis height of the five age groups showing the mean value sequentially from the highest to the lowest as follows: group 3, group 4, group 5, group 2 and group 1. there was a significant difference in ramus and symphisis height between group 1 and the other groups. after birth, the mandibular body gradually extends towards the horizontal especially behind the foramen mentale to produce a site for three permanent teeth. meanwhile, the high vertical dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p81-85 83 sitanggang and boel/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 81–85 table 1. the mean value of the result of the measurement of mandibular morphological parameters mandibular morphological parameters groups mean ± sd post test lsd group 1 group 2 group 3 group 4 group 5 ramus height (ar-go) 1 40.973± 3.599 .000* .000* .000* .000* 2 48.803±4.020 .000* .094 .614 .903 3 51.010±6.828 .000* .094 .241 .121 4 49.467±5.728 .000* .614 .241 .702 5 48.963±4.512 .000* .903 .121 .702 condylion height (co-ar) 1 8.163±1.854 .005* .000* .000* .315 2 9.420±1.516 .005* .004* .260 .065 3 11.143±2.484 .000* .004* .072 .000* 4 10.103±2.260 .000* .260 .072 .003* 5 8.547±1.500 .315 .065 .000* .003* body length (go-gn) 1 67.373±4.078 .000* .000* .000* .000* 2 75.437±4.467 .000* .000* .193 .705 3 80.037±4.162 .000* .000* .008* .000* 4 76.940±4.852 .000* .193 .008* .094 5 75.000±4.674 .000* .705 .000* .094 condylion angle (go-co-me) 1 33.917±3.006 .000* .000* .000* .050* 2 37.150±3.116 .000* .234 .784 .061 3 38.150±2.949 .000* .234 .359 .002 4 37.380±3.698 .000* .784 .359 .032* 5 35.570±3.366 .050* .061 .002 .032* symphysis height 1 12.893±1.638 .000* .000* .000* .000* 2 17.343±1.899 .000* .049* .329 .826 3 18.337±2.274 .000* .049* .316 .079 4 17.833±2.090 .000* .329 .316 .449 5 17.453±1.711 .000* .826 .079 .449 symphysis width 1 12.813±1.513 .011* .000* .001* .153 2 13.747±1.590 .011* .001* .503 .256 3 15.033±1.393 .000* .001* .005* .000* 4 13.990±1.266 .001* .503 .005* .072 5 13.333±1.217 .153 .256 .000* .072 symphysis angle 1 86.453±5.052 .192 .944 .261 .000* 2 84.530±5.960 .192 .217 .856 .000* 3 86.350±5.558 .944 .217 .291 .000* 4 84.797±6.291 .261 .856 .291 .000* 5 75.557±5.468 .000* .000* .000* .000* *multiple comparison lsd test, the mean diff. (mean differences) is significant at the level of 0.05. direction of the ramus increases in accordance with the growth of the alveolar bone that will be the site for the dental root and the development of the permanent tooth. high differences in ramus also occur in patients aged between 11 and 69 years. the highest average score was identified in the 20-29 year age group.13 the mean value of body length, condylion height, condylion angle and symphysis width from the highest to the lowest is, sequentially, in group 3, group 4, group 2, group 5 and group 1. there was a significant difference in body length, condylion height and condylion angle between group 1 and the other groups, and a significant difference in condylion height and symphysis width between group 1 and all other groups, except group 5. other studies on the height of the condylion in non edentulous and edentulous groups using panoramic radiography showed that the mean high value of condyles in the non-edentulous group was greater than in the edentulous group.7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p81-85 84sitanggang and boel/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 81–85 the mean value of symphysis angle from the highest to the lowest was sequentially in group 1, group 3, group 4, group 2,and group 5. the mean value of the symphysis angle showed a significant difference between group 5 and the other groups. as teeth in the mandible erupt, they will move upwards and slightly forward. the normal rotation of the mandible causes that section on the anterior portion to grow upward. this rotation changes the pathway of the eruption of the incisor tending to lead it more posteriorly and the molars to further migrate mesially during growth.14 in this study, not all parameters showed significant differences in each age group which may be due to the age range used being very wide. along with tooth eruption in the mandible, the process of growth and development of the mandible also involves remodeling, anterior and inferior displacement (aposition). the endocondrium of the condyle hardens, thereby affecting the growth of the posterior mandible. apposition and remodeling will cause the mandible to grow larger.8,15 condyle experiences considerable activity as the mandible moves and grows downward and forward. apposition occurs at the posterior boundary of the ramus and the alveolar process. resorption occurs along the anterior border of the ramus extending the alveolar border and maintaining the antero-posterior dimension of the ramus. the cephalometric study shows that the body of the mandible maintains a relatively constant angular relationship with the ramus. a slight change in the gonial angle after muscle activity works properly. the mandible's alveolar process grows upward and outward in a widening arc. this allows the dental arch to accommodate a larger permanent tooth.10 the length of the mandible increase due to the posterior apposition of the ramus and resorption of the anterior ramus. the increase in mandibular height is largely due to growth of the alveolar bone. the chin becomes more prominent as the mandible is elongated and there is only a slight increase of bone in the chin, however, it does not occur again after adolescence.8,15 a considerable body of research has been conducted to evaluate mandibular morphology, such as the measurement of ramus and condyle. post-natal changes in mandibular morphology are thought to be affected by mastication and the age of the patient. age, systemic factors and tooth loss may alter mandibular morphology.6,7,11,16,17 age and changes in the function and structure of the mastication muscles in edentulous patients appear to 8 figure 1. measurements on lateral cephalometric radiography. ramus height: the distance measured from one point ar to go.1 condylion height: the distance measured from the height of the ramus to the most superior point of the condyle.7 body length: the distance measured from go to gn.15 condylion angle: the angle formed by go-co-me.6 symphysis height: the distance from point b to the most inferior point of simphisis1 symphysis width: the distance from the most anterior border to the posterior simphisis.1 symphysis angle: the posterosuperior angle formed by the line through the point me and point b in the mandible plane.1 figure 1. measurements on lateral cephalometric radiography. ramus height: the distance measured from one point ar to go.1 condylion height: the distance measured from the height of the ramus to the most superior point of the condyle.7 body length: the distance measured from go to gn.15 condylion angle: the angle formed by go-co-me.6 symphysis height: the distance from point b to the most inferior point of simphisis1 symphysis width: the distance from the most anterior border to the posterior simphisis.1 symphysis angle: the posterosuperior angle formed by the line through the point me and point b in the mandible plane.1 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p81-85 85 sitanggang and boel/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 81–85 decrease contraction and lower muscle density than in their counterparts who still possess their teeth. because the masseter and medial pterygoideus muscles exert force in the gonial corner area, the contraction strength of these muscles also affects the mandible's basic shape.2,9 the morphology of superficial masseter muscles in the gonial region is wider in dentate subjects than in their edentulous counterparts. muscle mastication changes in function and structure according to the age of the edentulous subject. in computed tomographic scans, the activity of electromyography and lower muscle density is greater in edentulous subjects than dentulous subjects. the occurrence of masticatory muscle atrophy may be one of the factors that cause changes in the mandible.8,11,15–17 another factor likely to induce such changes is the loss of intermaxillary support due to missing teeth, thereby permitting the masseter and medial pterygoid muscles to exert unrestricted tension on the mandible.15 the conclusion of this study is that group 3 has the highest mean value of mandibular morphology. there is a difference in the mandibular morphology of members of the mongoloid race resident in medan according to age identified by cephalometric radiography, in which the morphological differences in the mandible may be affected by the age and condition of the teeth. acknowledgement this research was conducted with the financial support of the talenta grant fund of the universitas sumatera utara no: 325/un5.2.3.1/ppm/kp-talenta usu/2017. references 1. mangla r, singh n, dua v, padmanabhan p, khanna m. evaluation of mandibular morphology in different facial types. contemp clin dent. 2011; 2(3): 200–6. 2. thakur kc, choudhary ak, jain sk, kumar l. racial architecture of human mandible an anthropological study. j evol med dent sci. 2013; 2(23): 4177–88. 3. sharma p, arora a, valiathan a. age changes of jaws and soft tissue profile. sci world j. 2014; 2014: 1–7. 4. basnet bb, parajuli pk, singh rk, suwal p, shrestha p, baral d. an anthropometric study to evaluate the correlation between the occlusal vertical dimension and length of the thumb. clin cosmet investig dent. 2015; 7: 33–9. 5. wolfswinkel em, weathers wm, wirthlin jo, monson la, hollier lh, khechoyan dy. management of pediatric mandible fractures. otolaryngol clin north am. 2013; 46(5): 791–806. 6. liu y, behrents rg, buschang ph. mandibular growth, remodeling, and maturation during infancy and early childhood. angle orthod. 2010; 80: 97–105. 7. huumonen s, sipilä k, haikola b, tapio m, söderholm al, remeslyly t, oikarinen k, raustia am. influence of edentulousness on gonial angle, ramus and condylar height. j oral rehabil. 2010; 37: 34–8. 8. reynolds m, reynolds m, adeeb s, el-bialy t. 3-d volumetric evaluation of human mandibular growth. open biomed eng j. 2011; 5: 83–9. 9. okşayan r, asarkaya b, palta n, simsek i, sökücü o, isman e. effects of edentulism on mandibular morphology: evaluation of panoramic radiographs. sci world j. 2014; 2014: 1–5. 10. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publisher; 2007. p. 7-21. 11. shaw jr rb, katzel eb, koltz pf, kahn dm, girotto ja, langstein hn. aging of the mandible and its aesthetic implications. plast reconstr surg. 2010; 125(1): 332–42. 12. ghaffari r, hosseinzade a, zarabi h, kazemi m. mandibular dimensional changes with aging in three dimensional computed tomographic study in 21 to 50 year old men and women. j dentomaxillofacial radiol pathol surg. 2013; 2: 7–12. 13. al-shamout r, ammoush m, alrbata r, al-habahbah a. age and gender differences in gonial angle, ramus height and bigonial width in dentate subjects. pakistan oral dent j. 2012; 32: 81–7. 14. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st louis-missouri: mosby elsevier; 2012. p. 1-60. 15. ghosh s, vengal m, pai km. remodeling of the human mandible in the gonial angle region: a panoramic, radiographic, cross-sectional study. oral radiol. 2009; 25: 2–5. 16. chole rh, patil rn, balsaraf chole s, gondivkar s, gadbail ar, yuwanati mb. association of mandible anatomy with age, gender, and dental status: a radiographic study. isrn radiol. 2013; 2013: 1–4. 17. shilpa b, srivastava s, sharma rk, sudha c. combined effect of age and sex on the gonial angle of mandible in north-indian population. j surg acad. 2014; 4(2): 14–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p81–85 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p81-85 161 the effect of mastication muscular tone on facial size in patients with down syndrome margaretha suharsini*, josef glinka svd**, and soekotjo djokosalamoen*** *** lecture staff of department of pediatric dentistry, faculty of dentistry, university of indonesia *** professor department of anthropology, faculty of social politics, airlangga university *** professor department of orthodontics, faculty of dentistry, airlangga university abstract muscular hypotonia is one of the clinical signs in patients with down syndrome. as a characteristic of patients with down syndrome, hypotonia is clearly evident in face expression and oral dysfunction. dentocraniofacial growth abnormalities in patients with down syndrome may be influenced by genetic and environmental factors. stomatognathic system musculature as an environmental factor (factor outside the bone) can affect dentocraniofacial growth by orofacial muscles activities when chewing, swallowing, breathing, and speaking. oral dysfunctions commonly seen in patients with down syndrome are open mouth, protruding tongue posture, difficulties when chewing, swallowing, and speaking, drooling, and mouth breathing. the purpose of this study was to observe how the mastication muscular tone affecting the facial size of down syndrome patient. twenty five of 14–18 years old children with down syndrome were diagnosed by clinical characteristic and cytogenetic examination. mastication muscular tone was described by masseter and temporalis muscle synergy and oral function, whereas the facial size consisted of facial size of lateral, anteroposterior and vertical growth. the result of regression test revealed that the degree of mastication muscular tone has a significant effect on facial size of the anteroposterior growth and facial size of vertical growth, but did not significantly influence the facial size of lateral growth. key words: mastication muscular tone, facial size, down syndrome correspondence: margaretha suharsini, c/o: bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jln. salemba raya no. 4 jakarta pusat, indonesia. introduction the down syndrome is one of the most common genetic disorders, which clinical neurologic disorders such as muscular hypotonia and mental retardation occur. muscular hypotonia in children with down syndrome may affect growth, such as motoric growth and speech impediment. hypotonia in children with down syndrome will raise difficulties to move against gravitation, thus resulted in inefficient motoric growth. the manifestations are delay in child’s ability to seat, stand, and walk. children with down syndrome are able to walk more or less in 2 years of age.1 in addition, hypotonia in children with down syndrome can also manifest in weakness of orofacial musculature, i.e lips, tongue, and cheek. it produces an indistinguishable pronunciation, speech impediment, or stutters, because they cannot hold breathe long enough when saying a whole sentence. in this situation, oral muscular therapy as part of speech therapy program is recommended to increase the muscular tone.2 in normal condition, stomatognathic system musculature as an environmental factor (factor outside the bone) may influence dentocraniofacial growth. stomatognathic system musculature that gives effect to dentocraniofacial is orofacial muscles’ activities in chewing, swallowing, breathing, and speaking. these orofacial muscles are referred to mastication, lips and surroundings, tongue, and facial muscles.3,4 hypotonia as a characteristic of patients with down syndrome is clearly evident in face expressions and oral dysfunctions. oral dysfunctions commonly seen in these patients are open mouth, protruding tongue, difficulties in chewing, swallowing, and speaking. in addition, drooling and mouth breathing are frequently observed in these patients.5,6,7 macroglossia is a frequent finding in down syndrome, but it is not clear whether the apparent large tongue in these patients is anatomically enlarge or functionally enlarged as a result of an abnormal forward posture. the narrow maxilla results in lack of space for the tongue may lead the patient to protrude it. this can also gives impression of macroglossia.8 protruded tongue in down syndrome patients is predicted to cause a dull gonion angle in the mandible, as well as anterior openbite. however, fischer-brandies9 in his study of children with down syndrome 0–14 years of age found that gonion angle when they were baby was slightly smaller than normal babies (without down syndrome). moreover, he found no difference in gonion angle during growth to 14 years of age, so he concluded that protruded tongue in children with down syndrome seems to have no effect to gonion angle. this needs a further study, because the likeness of gonion angle between down syndrome 162 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 161–164 patients and normal children can not be assessed only by protruded tongue, yet the forces of mastication muscles need to be considered. it is likely that oral muscular exercises can increase the force of mastication muscles; therefore the craniofacial bone abnormality can be minimized. craniofacial can be divided into two sections: cranial section and facial section. growth of facial section has a closer association with somatic growth and it occurs gradually. begin with lateral growth, continued with anteroposterior growth, and ultimately vertical growth.10,11 explanation above indicates that muscular hypotonia in patients with down syndrome can produce disturbance in oral function, and affecting the bone growth in the region of mouth. therefore, the purpose of this study was to observe the effect of mastication muscular tone on facial size in down syndrome patients. materials and methods in this study, male and female between 14–18 years of age with down syndrome from sekolah luar biasa (special schools) in jakarta was selected. the diagnosis were established clinically by observing the clinical characteristic in children with down syndrome, and by citogenetic examination, that was, a child with trisomy 21 aberration. twenty-five children with down syndrome were chosen, after several approaches and examinations were carried out. mastication muscular tone acted as independent variable, described with:12 1) electromiogram of masseter and temporalis muscle, afterward these muscles were joined as a single variable, that was, masseter-temporalis muscle synergy. 2) oral function, that was total score of these examinations: a) swallowing; b) chewing, the amount of chew before swallowing ± 2 cm2 crackers; c) resting position between upper and lower lip; d) breath habit; e) drooling, involuntary saliva excretion; f) tongue in resting position; g) free way space measurement. both variables of mastication muscular tone were analyzed, to obtain factor variable of muscular tone factor. facial size of lateral, anteroposterior, and vertical growth served as dependent variable:12 1) variables attributed to facial size of lateral growth were: goniongonion (go-go), nose width, maxillary width, mandibular width, face width, inter orbits, zygion-zygion (zy-zy), alae nasi-alae nasi (al-al), endocantion-endocantion (enen); 2) variables attributed to facial size of anteroposterior growth were: maxillary length, mandibular length, sellanasion-point a angle (s-n-a), s-n-b angle, articularegonion-menton angle (ar-go-me); 3) variables attributed to facial size of vertical growth were: ans-menton (ans-me), nasion-ans (n-ans), nasion-gnathion (n-gn), nasion-subnasale (n-sn), ramus mandibulae, sella-gonion (s-go). after factor analysis of all three variable groups was carried out, each factor variable of facial size of lateral growth, facial size of anteroposterior growth, and facial size of vertical growth was obtained. regression test was carried out to observe the effect of degree of mastication muscular tone on each facial size in down syndrome patients. results the outcome of examinations carried out in 25 male and female with down syndrome were mean and standard deviation ( ± sd) of electromyogram of massetertemporalis muscle synergy i.e. 596.28 ± 120.34 mm2/sec, and mean ± sd of oral functions total score, i.e. 11.36 ± 3.25. these muscular tone variables were subjected to factor analysis, to attain factor variable of muscular tone. table 1. results of regression test regarding degree of mastication muscular tone effect on facial size in down syndrome patients results of regression test regarding degree of mastication muscular tone effect on facial size in down syndrome patients were listed in table 1. the test showed that the degree of mastication muscular tone has a significant effect on facial size of anteroposterior growth (p = 0.012) and facial size of vertical growth (p = 0.020), but not significant on facial size of lateral growth (p = 0.527). discussion bone is flexible and may react to functional tension and pressure, thus it can be suggested that form and function are intimately related. functions that performed by muscles include mastication, deglutition, speech, face expressions, and respiration. physiologically, tension and pressure can produce alteration in external form and internal structure of the bone. the amount of alteration within bone depends on the tension and pressure encountered. however, pressure from the muscle is within the limits of its morphogenetic 163suharsini: the effect of mastication muscular tone pattern. the stimulation from muscle to a certain limit (optimal limit) will induce cell growth to become remodeling that may influence bone shape and size.13 the mastication process always relates to activities of mastication muscles. the impact of functional stimulation to craniofacial is mainly derived from mastication muscles activity. this stimulation will produce biomechanical forces and piezo effect to the bone. both effects will produce the distortion of collagen tissue, and will create both direct and indirect electric potential that induce osteoblastic and osteoclastic process.14 kiliaridis15 stated that eating soft diet regularly will reduce the function of mastication, thus fibers of masticatory muscles will be smaller. consequently, mastication or contraction strength will be reduced and produce changes in bone remodeling. this may influence the craniofacial bone growth. moreover, engstrom et al.16 demonstrated that rats fed a soft diet will have diminution in degree of mastication function, which will change bone apposition pattern in cortical bone and upper facial sutures. kiliaridis et al.17 proposed that weakened of mastication muscles may influence the size of gonion angle. in this study, regression test was carried out to observe the effect of degree of mastication muscular tone on facial size in patients with down syndrome. the result of regression test indicates that the degree of mastication muscular tone has a significant effect on facial size of anteroposterior growth (p = 0,012) and vertical growth (p = 0.20), but not significant on facial size of lateral growth (p = 0,527). factors of facial size of anteroposterior growth contain the length of maxilla, length of mandibula, and ar-go-me angle (gonion angle). those parts have a direct correlation to masseter muscle activities when function, considering masseter muscle have insertion on angulus and ramus mandibulae, and its origin is on arcus zygomaticus.18 kiliaridis et al.17 also stated that weakened of mastication muscles will reduce mechanical strength, that compress the mandible and resulting changes to gonion angle. the research performed in patients with myotonic dystrophy revealed that weakened of mastication muscle will reduce mechanical compression to the mandible, so the mandible will be pulled downward easily. the downward movement of mandible may be resulted from gravitation or probably by force of suprahyoid muscle downward. the mouth will likely to open, meanwhile posterior teeth will continue to erupt. thus, anterior open bite will occur. as a result, gonion angle will be larger than normal. a strong mastication muscle will amplify the rotation of mandible to anterior, thus gonion angle will be narrowed. diet alteration will cause changes in dimension of maxilla and mandible. soft diet can diminish the size of mastication muscles. therefore, functional stimulation to bone will decrease, that may cause rotation of mandible backward, resulting the gonionangle larger than normal. the changes of dimension will directly affect both maxilla and mandible, because origin and insertion of mastication muscles are attached to them.19,20 dubrul21 suggested that the site of growth of maxilla complex occurs prominently in several sutures in this region. the suture is running frontward-upward to backward-downward, therefore the maxilla complex grow in up-front direction. this will cause bone apposition in the entire posterior surface of maxilla, thus enlarging maxilla anteroposteriorly. in his study of rats fed a soft diet, engstrom et al.16 demonstrated that soft diet will diminish the degree of mastication function, subsequently bone apposition in upper facial suture will undergo an alteration that affecting the suture closing process. consequently, facial growth in anteroposterior direction is insufficient. based on explanation above, it can be assumed that muscular hypotonia in patients with down syndrome will decrease the function of mastication, resulted in facial growth insufficiency in anteroposterior direction. likewise soft diet will decrease muscle strength, muscular hypotonia in patients with down syndrome most likely to give direct impact to the dimension of maxilla and mandible. in addition, muscular hypotonia will reduce mechanical strength that compress the mandible, thus gonion angle in patients with down syndrome have a tendency to be larger than normal. for that reason, it can be concluded that the degree of mastication muscular tone in down syndrome patients has an impact on the maxillary length, mandibular length, and gonion angle, in which all three of them are facial size of anteroposterior growth. the degree of mastication muscular tone also has a significant impact on facial size of vertical growth (p = 0.020). to describe this, masseter and temporalis muscle activities are open and close the mouth when eating, and speaking as well.18 muscular hypotonia, or weakened of masticatory muscles, in down syndrome patients reduces muscle stimulation to the bone, resulted in insufficiency of facial growth in vertical direction. dubrul21 proposed that vertical facial growth in subnasal region is augmenting, as a result of resorption in nasal floor and aposition in surface of palatum and alveolar bone, concurrently with teeth eruption. muscular hypotonia in down syndrome patients reduces mastication force, so it will also reduce the force from mandibular stroke to alveolar bone in upper jaw and afterward transmitted to palatum. consequently, remodeling process in subnasal region will not be optimal, that will cause a lack of vertical facial size. the result revealed that the degree of mastication muscular tone in down syndrome patients has no influence on facial size of lateral growth (p = 0.527). considering that fibers of masseter muscle run obliquely and temporalis muscle fibers runs vertically and obliquely, and their main functions are to elevate and retract the mandible,18,21 it is most likely that facial size of lateral growth is not influenced by masticatory muscles that largely move vertically in accordance with their fibers direction. according to dubrul,21 there is only small amount of lateral facial growth in the posterior region because it is limited by os sphenoidale. or else, it is likely that lateral 164 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 161–164 facial growth have already completed in 12 years of age. therefore, the muscle activity around facial region is not strong enough to exert influence upon lateral facial growth, what is more in down syndrome patients who have masticatory muscular hypotonia. the result and discussion above revealed that the degree of mastication muscular tone in down syndrome patients may influence the facial size of anteroposterior and vertical growth, but not the facial size of lateral growth. references 1. sidiarto ld. aspek neurologik sindroma down dan penanganannya. jakarta: simposium perkembangan genetika kedokteran fkui; 1991. 2. kumin l. communication skills in children with down syndrome. a guide for parents. rockville: woodbine house inc; 1994. p. 1–33, 129–41, 193–97. 3. nanda ks. the developmental basis of occlusion and malocclusion. chicago: quintessence publish co. ltd; 1983. p. 29–42. 4. y’edynak gj, iscan my. craniofacial evolution and growth. in: iscan my, editor. forensic analysis of the skull. new york: wiley-liss, inc; 1993. p. 11–27. 5. hoyer h, limbrock gj. orofacial regulation therapy in children with down syndrome using the methods and appliances of castillomoralles. j dent child 1990; 442–44. 6. limbrock gj, hoyer h, scheying h. regulation therapy by castillomoralles in children with down syndrome: primary and secondary orofacial pathology. j dent child 1990; 437–41. 7. glatz-noll e, berg r. oral dysfunction in children with down’s syndrome: an evaluation of treatment effects by means of videoregistration. eur j orthod 1991; 13:446–51. 8. kaban lb. pediatric oral and maxillofacial surgery. philadelphia: wb saunders co; 1990. 9. brandies fh. cephalometric comparison between children with and without down’s syndrome. eur j orthod 1988; 10:255–63. 10. proffit wr, fields hw, ackerman jl, thomas pm, tulloch jfc. contemporary orthodontics. st louis: the cv mosby co; 1986. p. 16–94. 11. sperber gh, tobias pv. craniofacial embryology. 2nd ed. bristol: john wright & son ltd; 1976. p 3–5, 57–65, 68–97, 110–120. 12. suharsini m. pengaruh hipotonia otot pengunyah terhadap ukuran dan bentuk kraniofasial penderita sindroma down. disertation. surabaya: universitas airlangga; 1999. p. 63–65. 13. mokhtar m. dasar-dasar ortodonti perkembangan dan pertumbuhan kraniodentofasial. jakarta: yayasan penerbitan ikatan dokter indonesia; 1998. p. 3.1–3.26. 14. enlow dh, hans mg. essentials of facial growth. philadelphia: wb saunders co; 1996. p. 18–38, 57–110, 200–11, 265–80. 15. kiliaridis s. masticatory muscle function and craniofacial morphology. an experimental study in the growing rat fed a soft diet. am j orthod dentofac orthop1986; 92:355–56. 16. engstrom c, kiliaridis s, thilander b. the relationship between masticatory function and craniofasial morphology. ii a histological study in the growing rat fed a soft diet. eur j orthod 1986; 8:271–79. 17. kiliaridis s, mejersjo c, thilander b. muscle function and craniofacial morphology: a clinical study in patients with myotonic dystrophy. eur j orthod 1989; 11:131–38. 18. heartwell cm, rahn ao. syllabus of complete dentures. 4th ed. philadelphia: lea & febiger; 1986. p. 1–13, 30–40. 19. varrela j. effects of attritive diet on cranio facial morphology; a cephalo-metric analysis of a finnish skull sample. eur j orthod 1990; 12:219–23. 20. mieke s. pengaruh pola makan pada morfologi rahang, gigi, dan wajah serta akibatnya pada kejadian maloklusi. disertation. surabaya: universitas airlangga; 1993. p. 98–100. 21. dubrul el. oral anatomy. 7th ed. st louis: cv mosby co; 1980. p. 93–132, 142–73, 200–09. mkg vol 42 no 2 april 2009.indd 99 vol. 42. no. 2 april–june 2009 case report the aesthetic treatment for anterior teeth with lost crown by endorestoration nanik zubaidah department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: the aesthetic has an important role in social life, especially the anterior teeth. the aesthetic abnormality of anterior teeth i.e. discoloration, malpotition or the anterior teeth with crown damage for more than one third or all part of crown is lost due to caries or other causes, will influence its appearance especially during smile. purpose: the aim of this case report, therefore, is to show how teeth with clinical crown lost or only the root left still can be treated by endorestoration treatment in order to reconstruct the shape and function of the teeth similar to the original ones. case: female 52 years old with the lost crown of anterior teeth. the patient did not want her teeth to be extracted. case management: the abnormality of these teeth are still able to be reconstructed by endorestoration i.e. endodontic treatment with post and core insertion in the root canal will increase its retention and recovery by the porcelain crown fused to metal to recover the original formation and aesthetic and thus has the normal refunction. the treatment, it improve the confidence of the patient, and also can function normally. the patient did not feel pain. ronsenography showed the periapical lesion diminished, the neighbor gingival was going better in both function and color. conclusion: endorestoration treatment on the anterior teeth with lost crown could recover the normal function, dental aesthetic and self confidence. key words: endorestoration, aesthetic, anterior teeth correspondence: nanik zubaidah, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: nanikzubaidah@yahoo.com introduction aesthetics and beauty can not be separated from social life. everyone even can enjoy the aesthetics anytime since the aesthetics is part of human life. many patients, thus, are really concerned with their anterior teeth because of aesthetic reason. unfortunately, some of them ignore the chewing function, and more concern with the appearance.1,2 the natural and harmonious appearance, furthermore, is one of aesthetic elements that all patients wish. the basic principles of the treatment in dentistry are actually including power, good function, and satisfying aesthetics. however, the good aesthetics without being supported by the good function and optimal power still can make the patients feel difficult in doing their daily chewing activity. on the other hand, though without being supported by good aesthetics, good function and optimal power can make the patients comfortable in doing their activities, they will still be lack of confidence.3,4 being able to smile with full of confidence, moreover, is a strong indicator that indicates the person is satisfied with himself. this condition then can improve his social relation and support him to reach any success in business or professional life.3 specifically, the anterior teeth are important element during laughing. thus, all abnormalities in the terms of discoloration, malposition, or the anterior teeth with crown damage for more than one third or all part of crown lost due to caries or other causes are the most significant element disturbing the appearance. however, the abnormal teeth still can be treated by endorestoration treatment.1,5 endorestoration treatment actually consists of endodontic treatment and restoration. the determining process of the restoration after endodontic treatment requires some factors to be considered i.e: the degree of tooth damage; the quality 100 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 99−103 of dental supporting tissue; the present of antagonist tooth or not; and the capacity of chewing power.5,6 the success of a restoration, moreover, was determined by retention, stability, and aesthetics of the teeth. thus, the improving process of the appearance, especially in the term of aesthetics, is getting more important in dentistry in this modern era.7 the aim of this case report, therefore, is to show how teeth with clinical crown lost or only the root left still can be treated by endorestoration treatment in order to reconstruct the shape and function of the teeth similar to the original ones. the following report is about a case of anterior teeth with crown damage for more than one third or all part of crown lost due to caries after being treated with aesthetic treatment by endorestoration. case 1 the patient is a fifty two year old woman. the clinical crown of her anterior teeth was lost, or only the root of the teeth was left due to carries. nevertheless, the patient did not want to have an extraction for her teeth. the patient wanted a treatment for her anterior teeth so that she could get the normal shape and the dental aesthetics similar to her original ones. during the intra oral examination on the teeth number 12, 11, 21, 22 and 23 (figure 1), moreover, it was found that the clinical crown was lost or the root left was strongly inside socket and alveolar bone. thus, for conducting diagnosis and treatment plan, panoramic and local radiographic photos must be taken first. the photo result showed radiolucency around periapical area on teeth number 12, 11, 21, 22 and 23. the condition of her teeth was also non vital with a clinical diagnosis of necrotic pulp followed by periapical lesion. figure 1. the condition of the teeth number 12, 11, 21, 22 and 23 before treatment. this endorestoration treatment plan, moreover, consisted of intracanal endodontic treatment with restoration like porcelain jacket crown fused to metal. this treatment also needed retention like pin dowel and core. case management intracanal endodontic treatment was carried out on the teeth number 12, 11, 21, 22, and 23 with some visitation treatment. after that, the root canal of the teeth treated by endodontic treatment was duplicated with elastomers in order to make retention like pin dowel and core as well as to make restoration like a temporary jacket crown appropriate with the normal position in good dental curvature. the aim of the making of the temporary jacket crown was to protect posts and core inserted during the treatment and to describe the normal position of the anterior teeth appropriate with the good dental curvature with normal overbite and overjet. the insertion of pin dowel and core was carried out one by one into the root canal of the teeth number 12, 11, 21, 22, and 23 (figure 2). afterwards, with phosphate zinc cements the preparation of pin dowel and core inserted was carried out to obtain appropriate position and parallel construction so that the setting of the porcelain jacket crown fused to metal could be done easily and created good dental curvature. figure 2. insertion of posts and core into the teeth number 12, 11, 21, 22, and 23. the next stage, the duplicating of the teeth number 12, 11, 21, 22, and 23 was carried out with double impression materials. the biting mold was also made in order to make the position of the teeth similar to the original position. the teeth were then set with the temporary jacket crown which was not only appropriate with the shape but also harmonious with the aesthetics. afterwards, the duplicating result of upper and lower jaw models was sent to dental laboratories in order to make the porcelain jacket crown fused to metal. the final stage of the treatment, the porcelain jacket crown fused to metal was set on the teeth number 12, 11, 21, 22 and 23. since the condition of the initial fit was good, the shape and the color were normal, and there was no premature contact, the permanent insertion was carried out with glass ionomer cement, luting type (figure 3). the patient was then asked to do 1 week, 3 months, 7 months, and 1 year visit treatment. 101zubaidah: the aesthetic treatment for anterior teeth figure 3. the condition of the teeth number 12, 11, 21, 22 and 23 after treatment. case 2 the patient is thirty two year old woman. her number 33 tooth crown was lost due to carries (figure 1). thus, the patient used to feel ashamed whenever she smiled. however, the patient did not want to have an extraction for her tooth. she wanted her tooth to be treated and kept. she wished that her tooth could have had normal function again similar to her original ones. during the intra oral examination, it was found that the condition of the root left on 33 tooth was still strong inside socket and alveolar bone. the result of the roentgen photo showed that there was periapical lesion. the condition of the teeth was non vital with a clinical diagnosis of total necrotic pulp. figure 1. the condition of the 33rd tooth before treatment. case management the tooth number 33 could be treated by intracanal endodontic treatment only with one visit treatment. then, retention like pin dowel and core could be set. a temporary crown which must be fit with the normal position in the good dental curvature could also be made. this aim of these procedures was to protect the core post inserted during the treatment and to describe the position of the normal anterior teeth appropriate with the good dental curvature with normal overbite and overjet. the making of pin dowel and core, thus, must be taken place in dental laboratory. the next stage was insertion of pin dowel and core made of phosphate zinc cement (figure 2). first, the preparation of post was carried out in order to obtain not only the appropriate position and parallel construction, but also the good and harmonious dental curvature, which then was duplicated with double impression materials. afterwards, the insertion of temporary crown appropriate with the harmonious and aesthetic dental curvature was carried out. the duplicating result then was sent to dental laboratories in order to make the porcelain jacket crown fused to metal. figure 2. insertion of post and core on the 33 tooth. the final stage of the treatment was setting porcelain jacket crown fused to metal on the 33 tooth. since the initial fit was good, the shape and the color were appropriate, and there was no premature contact, permanent insertion then was carried out with glass ionomer cements with luting type, insertion of porcelain jacket crown fused to metal (figure 3). the patient then only needed some visits, 1 week, 3 months, 7 months, and 1 year visit treatment. figure 3. the condition of the 33 tooth after treatment. discussion in these cases, the patients needed an aesthetic treatment of anterior teeth since their anterior teeth have got crown 102 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 99−103 damage for more than one third or all part of the crown due to caries, which then have made the patient feel ashamed and less confident during their smiling. on teeth number 12, 11, 21, 22, and 23 (case 1) and number 33 (case 2), moreover, the tooth vitalities were non vital with a clinic diagnosis, necrotic pulp. those teeth, however, still could be treated concerning with the condition of root left which was still appropriate in the terms of length, thickness, and depth of their rooting in the socket and alveolar bone. thus, this condition could probably be treated by endorestoration treatment, which was by intracanal endodontic treatment with supporting pin dowel and core (case 1) and (case 2) in order to improve the tooth retention as well as to make restoration of porcelain jacket crown fused to metal. in other words, this condition is the same as shillingburg’s opinion6 which said that the condition of teeth with the crown damage or the heavy oclusal pressure has an indicator that the crown is with pin dowel. similarly, tohiroh8 and kamizar,9 also said that teeth with short clinic crown condition or without any clinic crown, but still having appropriate roots in the terms of length, thickness, and depth of their rooting in alveolar bone, can be treated well with posts. this treatment, however, is also depend on the condition of the teeth whether the comparison between root and crown follows the requirement, in which the length of posts inserted into root canal at least is the same as the length of the crown. therefore, the selection of post and core design as supporting in root canal must be appropriate with the size of the crown left concerning with the height of oclusal pressure (chewing power), diameter of root canal and tooth location as well as the health of periodontal tissue as supporting to post crown.10 it means that the consideration in selecting kind of post crown can not be separated with how the posts will be designed. the procedures of design selection and root canal preparation, furthermore, must carefully be carried out in order not to cause the weakening of the tooth tissue left and the removing of posts.11 in case 1 (teeth number 12, 11, 21, 22, and 23) and case 2 (tooth number 33), for example, those teeth used pin dowel since the posts have some advantages, like that the posts and the core can not only become unity but can also follow the root canal preparation, so they can be retentive and stable and do not need additional retention like pin.8 then, the setting of pin dowel and core on teeth 12, 11, 21, 22, and 23 (case 1) was done in the same time but one by one. the aim of this procedure is to get an appropriate construction and size based on both the normal size of each and the curvature of the anterior teeth with normal overbite and overjet so that the good and harmonious aesthetics can be created. the principle of the tooth treatment after the endodontic treatment, moreover, is to carry out the restoration of root and crown with post crown and core which is retentive and stable so that it can not only easily remove but can also be used for long in the mouth cavity like the original teeth. however, it must be noticed that teeth which have been treated by the endodontic treatment are relatively more fragile and can easily fracture compared to the healthy teeth since there is an organic and biological changes because of the death of pulp, the reduction of dental internal tissue, and the weakening linkage between enamel and dentin due to the scraping of dentin tissue during the root canal preparation causing the change of the tooth color. for those reasons, comprehensive protection is needed by using supporting pins and core as well as by making restoration of porcelain jacket crown fused to metal in order to prevent the teeth from fracture.8,10 the making of porcelain jacket crown fused to metal, thus, is a good treatment for reconstructing the esthetics, especially the anatomy construction and the color of the teeth which is appropriate with their original color and can function naturally. similarly, hume12 also states that porcelain jacket crown is the best solution to carry out the restoration of the first sensitive tooth with optimal aesthetics. according to qualthrough and burke,13 moreover, from 956 patients there are only 63% who feel satisfied with their outlooks when one of their anterior teeth using dressing crown, and there are 79% who feel satisfied with their outlooks when four or more of their anterior teeth using porcelain jacket crown. the final treatment result of case 1 and 2, however, could be obtained with one week, three month, seven month, and one year visit treatment after the insertion of the porcelain jacket crown fused to metal. visit i (1 week after the treatment): the patients did not complain again about feeling painful (either shooting or sharp painful) on their treated teeth; they also got percussion and unpainful pressure; their gingiva color was still normal; they did not get any inflammation around the tissue; their radiographic photo result was good since their abnormal radiolucent image of periapical on the teeth number 12, 11, 21, 22 and 23 (case 1) and number 33 (case 2) was rather decreasing; and their recovery process was also good. visit ii (3 months after insertion and treatment): the condition of porcelain crown jacket was good enough; there was no pain around the tissue; the color of the gingiva around was normal; and based on the roentgen photo the radiolucent image around periapical area was getting smaller either in case 1 or case 2. visit iii (7 months after insertion): the patients did not feel painful, either hooting or sharp painful, or discomfortable around the teeth number 12, 11, 21, 22, and 23 (case 1) and number 33 (case 2) which had been set with the porcelain jacket crown fused to metal; the abnormality of periapical lesion on their teeth was getting smaller; the color of the gingival around was normal; and the patients were satisfied with their daily outlook as well as they could laugh without feeling lack of confidence anymore. visit iv (one year after treatment): the patients did not feel painful, either hooting or sharp painful, or discomfortable, so they could do their activities again with 103zubaidah: the aesthetic treatment for anterior teeth full of confidence. the roentgen result even showed that the condition around the teeth was good and the color of them was also normal (either in case 1 or case 2). therefore, criteria used in determining the success of the endodontic treatment are not only based on radiograph of periapex tissue but based on the clinic condition of the teeth as well. in other words, a treatment can be determined whether it is successful or not based on the conditions that are no complaint from the patient about the treated teeth and the successful effort in treating the abnormality in the periapex tissue after one year treatment.14 in addition, ingle15 reports that the success of the treatment in elderly group is better than that of the treatment in other age groups. the reason may be because one third of the apex area of other age groups has been completely closed since there is secondary cement or ramification of root canal which has been declined. based on those explanations above, it can be concluded that the anterior teeth either in upper jaw or lower jaw which hadlost their clinical crown due to caries or fracture does not need to have extraction since they still can probably be treated with endorestoration, intracanal endodontic treatment, by inserting posts inside root canal (intrapulp) and core, in order to make them retentive and stable as well as to make porcelain jacket crown fused to metal. thus, they finally can reconstruct the shape and function of the teeth as well as the dental aesthetics, so the construction of the teeth can become normal again similar to the original construction of the teeth. references 1. baum l, phillip rw, lund mr. textbook of operative dentistry. 3rd ed. philadelphia: wb saunders company; 1995. p. 307-9, 677–89. 2. melanie tw. analisis diakronis kedokteran gigi estetik bidang prostodonsia. pidato pengukuhan jabatan guru besar fakultas kedokteran gigi unair. 2002. p. 2. 3. soemantri ess. pengertian dan konsep ilmu kedokteran gigi estetik. journal kedokteran gigi 1996; 8:1. 4. antune rpa, magalhaes f, matsumoto w, orsi ia. anterior estetik rehabilitation of all ceramic crown. quint int 1998; 29:38–40. 5. ismiatin k. restorasi kerusakan mahkota klinis gigi yang luas dengan penguat pasak jadi. majalah kedokteran gigi (dental journal) 2001; 34(4):767–9. 6. shillingburg ht, kessler. restoration of endodontically treated tooth. chicago: quintessence publishing co. inc; 1982. p. 17–8. 7. nanik z. perbaikan estetik pada gigi anterior dengan letak berdesakan disertai karies kompleks. majalah kedokteran gigi (dental journal) 2001; 34(3):116–8. 8. tohiroh dj, rahardo tbw. retensi mahkota pasak berdasarkan desain pasak. kumpulan naskah temu ilmiah nasional i (timnas i) 1998; 145–6. 9. kamizar. etiologi dan pencegahan kasus-kasus iatrogenic dalam restorasi pasca endodontic. jkgui 2000; (edisi khusus):470-4. 10. chan dcn, myers ml. chipped, fractured, or endodontically treated teeth. in: goldstein re, editor. esthetics in dentistry. 2nd ed. hamilton, london: bc decker inc; 2002. p. 537–9. 11. ziebert gj. restoration of endodonticcally treated teeth. in: malone wfp, koth dl, kaiser da, morgano sm, editors. tylman’s theory and practice of fixed prosthodontic. 8th ed. st louis, tokyo: ishiyaku euro america inc; p. 407–17. 12. hume wr. preservationand restoration of tooth structure. london: the cv mosby co; 1998. p. 185–90. 13. qualthrough aje, burke fjt. a lookat dental esthetics. j quintessence international 1994; 25(1):7–9. 14. margono da. etiologi dan pencegahan kasus-kasus iatrogenic dalam perawatan endodontic. jkgui 2000 7; (edisi khusus):464-9. 15. ingle jl. endodontics, modern endodontic therapy. 2nd ed. philadelphia: lea and febriger; 1976. p. 1–56. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� vol. 42. no. 1 january–march 2009 treatment of temporomandibular disorder using occlusal splint agus dahlan department of prosthodontic faculty of dentistry airlangga university surabaya indonesia abstract background: patient suffering from occlusal abnormality is usually detected months or even years when the acute patient visits a dentist, and generally the patient does not receive direct treatment upon his complaints since minimum information is available on this type of treatment. in general, the dentist provides medication only or conducts incorrect selective grinding where in fact, the patient does not feel better from the previous conditions. purpose: the objective of this study is to discuss the treatment on the dysfunctional temporomandibular joint followed by orofacial pain caused by occlusal disorder using occlusal splint. case: in this case, a forty three years old male having trouble with the joint on the left jaw followed by orofacial pain caused by occlusal disorder. case management: initial treatment with occlusal splint makes the patient comfortable and recovers from his complaints since the patient could restructure the chewing muscles. this treatment will be more successful if the dentist has the knowledge to use and choose occlusal splint method properly. occlusal splint could be used as a supporting therapy and consideration as one of the therapies to avoid the unwanted side effects. the use of occlusal splint is meant as an alternative of the main therapy in overcoming the problem of occlusal splint. conclusion: finally, therapy with occlusal splint is very effective as an alternative treatment to handle the dysfunction of temporomandibular joint caused by occlusion. key words: occlusal disorder, temporomandibular joint, occlusal splint correspondence: agus dahlan, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction disorder on temporo mandibular joint often creates symptoms which has become the main complaint for a patient to visit a dentist. symptoms suffered by a patient is usually as follows: stiff neck, headache, facial pain, earache, clicking when a patient opens and close his or her mouth, or even when the symptom has been suffered for too long, it can cause arthritis on the joints. one of the factors that cause this disorder is occlusal splint.1,2 the treatment of occlusal splint which will be conducted on a patient who still has a complete set of teeth or even a patient who has lost some, may be sufficient by adjusting occlusion from the tooth which has been the cause of the occlusion so that the occlusion could be back in line with the chewing system of the patient. if the disorder has been suffered for too long, however, that kind of treatment would not be sufficient since the acute occlusal disorder will cause a joint temporomandibular disorder followed by orofacial pain.2,3 oftenly, a dentist does not take into consideration on the disorder of the jaw joint in doing his treatment since there are limited information on how to deal with the temporomandibular joint disorder. the disorder could be triggered by related multi factors which have reciprocal influence one to another. there are three other supporting factors on the disorder of temporomandibular joint, such as: neuromuscular, skeletal and dental, as well as the existence of stress which is enough to create muscle strain.1,4 the disorder on temporomandibular followed by orofacial pain due to the occlusal disorder has been the disorder often found in the clinic. the occlusal disorder itself could caused by several factors, for example, the acute abrasive teeth caused by bruxism,4,5 the chewing case report �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 31-36 habit with only one side which is often called unilateral chewing, lost of teeth, tooth caries, the imperfect shape and position of teeth, or even the incorrect adjustment of teeth, the occlusal disorder, however, could provide various adaptation to each patient.5 the treatment of the joint temporomandibular disorder caused by occlusal disorder, is less effective if the patient is only given medicine to cure the infection or pain killer without handling the main problem, which is the occlusal disorder itself.6 as an alternative to this treatment, it is needed to conduct preliminary treatment with a temporary tool on occlusal and tooth incision which is called occlusal splint.3,7 occlusal splint is a tool made of acrylic and installed on the top or bottom jaw, the position has to be in contact with the whole teeth surface and fixed on its place since the acrylic is placed deep inside to the undercuts within the teeth proximate. the main objective of the occlusal splint is to eliminate the occlusal disorder by changing the connection between the top and the bottom jaw determined by the intercuspation (figure 1).7 the other use of occlusal splint is to control the pain of the dysfunction temporomandibular joint muscle which is related to the teeth touching disorder.8 occlusion has been of of the most important factors in the dentistry since the success or failure of a dentist depends on the ability to treat physiological occlusion on a patient even if it is only a simple tooth patching which will change the way of a patient to bite which can be uncomfortable to the patient.9 the occlusal disorder can be defined as the deviation from the normal occlusion (both from the shape and function) or an unstable condition which creates pressure at the time of chewing and bruxism pattern as well as pressure on the tongue and lips. the occlusal disorder has also been the deviation from normal occlusion and hypermobility and the result of the occlusal trauma.10,11 the occlusal disorder can cause temporomandibular joint dysfunction. case a male patient, 43 years of age, an entrepreneur, shows up with the pain on the left jaw joint. it has been checked by a dentist and an adjustment has been conducted on the left bottom left region jaw, the jaw radiation and the pain killer has also been given to the patient. the pain, however, remains. the patient has been using portable false teeth for 13 years with the broken false teeth wire. the last extraction was done 5 years ago on the bottom right region. the patient is asking for the jaw joint treatment for the better. on the extra oral examination, on the left temporomandibular, it is found that the clicking occurs when the mouth is opened, the firm palpation and the intermittent pain. the intraoral examination suggests the following lost teeth: 17, 18, 23, 24, 25, 27, 28, 36, 37, 38, 46, 48 teeth with patch up: 14, 16, 26, 35, 44, 45, 47, teeth with attrition: 11, 12, 13, 21, 22, 31, 32, 41, 42, and tooth with rotation: 15 and the panoramic as well (figure 2). figure 2. panoramic rontgenographic of the patient. figure 1. a) occlusal splint in use (intra oral). b) the occlusal splint. a b ��dahlan: treatment of temporomandibular a b figure 3. jig in use (intra oral). a) in centric occlusion; b) in open mouth condition. figure 4. patient position in centric relation. figure 5. wax impression. case management to handle the emergency or the acute pain, the installation of the occlusal pemogram (jig) with cold curing acrylic was conducted on 11, 21. jig was made by the following steps: brushing part of the teeth 11, 21 with sterile vaseline separator. next, dough was made using cold curing acrylic which was then applied directly to the teeth surface in accordance to the jig design. the patient wass then instructed in the protrusive movement to achieve the jig thickness to 2 mm. after the material set, the retruded cusp position was checked in the palatal part, which was adjusted to the movement limit of mandibula and the result was a recorded spot on the functional movement. jig was ready to use, figure 3. then, the wax molding was implemented for the centrically connection and the occlusal molding position the result of the wax molding was actually the biting records along the protrusive and lateral movement needed to be installed on the semi adjustable articulator (dentatus). method of molding: the patient was sitting straight up with the chin pointing up and the jaw in centric relation (figure 4). two plates of red wax is softened and put on the occlusal top jaw (on the working model) which fits with the shape of the palatum of the patient. finally, the wax plate was inserted into the patient’s mouth. the patient was guided to close the jaw so that the top and bottom occlusal teeth could be recorded. the molding of the occlusal wax was then taken out from the mouth. the excessive wax was the cut off with scissors as in figure 5. protrusive molding: the bottom jaw was upright 5 mm, this condition will make angle with approximately 2° of accuracy. lateral molding: the bottom jaw was shifted to the lateral for 5 mm. the non working side also makes angle with approximately 2° of accuracy. to check the relation of the working occlusal model in the articulator starting from the cusp initial occlusion to the protrusive and lateral for 5 mm. putting up the 5 mm red wax in the molding part pacing up to the bottom jaw to mould the protrusive and lateral position on both sides of each part of the non working part. repeating the softened wax molding and inserted to the teeth in the patient’s mouth, to guide the bottom jaw to move protrusive and laterally for each molding process so that condillus shifting exists for 5 mm. the molding is taken out from the mouth and put in the working model of the bottom jaw, and its steadiness was examined. the making of the molding pattern on functional occlusion is based on the occlusion pattern on the patient by marking it with color. the occlusal pattern covers the contact of the teeth on retruded contact position = rcp, on intercuspal contact position = icp, on working side contact position = wscp, on non working side contact position = nwsp and on protrusive contact position = a-pcp or p-pcp (figure 6). this is to differentiate the position of the functional occlusion pattern and can be used �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 31-36 as a guide in the arrangement on occlusion pattern in the articulator, as in figure 7. to conduct facebow transfer, it was needed to make an imaginary line from other canthus ketragus and to determine condilli point which is about 12 mm from the tragus. continue with the installation of bite fork on the top jaw with the help of the red wax and putting the orbital pin pointer at the base of the orbital bone. after the facebow molding was gained, the immediately the facebow transfer is released from the patient. the making of permissive and directive occlusal splint was started with the making of the occlusal splint with the waxing process using the blue wax on the working model in the articulator which was in accordance with the occlusal splint design. then, the planting was conducted in a cuvet, after that the wax was dismissed and the acrylic filling using the transparent heat curing acrylic before the brushing. in inserting occlusal splint, the occlusal has to be stable within the teeth and the patient should not feel any pain since the teeth are not pressed by the occlusion. control i was conducted one day after the insertion with the permissive occlusion. the patient does not longer feel any pain on the joint area, move the jaw for approximately two finger wide, temporomandibular joint shall slide smoothly. entering control ii (one week after the use of the permissive occlusion, the patient should feel calm, there are no worries present, the examination of intra oral and the reading of the panoramic photo on the left joint. no disorder present, and then the replacement of the permissive occlusion with the directive occlusion (figure 8). control iii (one day after the use of the directive occlusion). no complaint and no disorder. the patient should feel that the position of the occlusion was more stable using directive occlusion, movement of the mouth was about three fingers wide. control iv (two weeks after the use of the directive occlusion). no complaint and no disorder. the occlusion position was more stable with the directive occlusal splint and the next step was to provide the definitive proteases in accordance with the guide to directive occlusion (figure 9). discussion based on the complaints, history and treatment that has been conducted to the patient who has been complaining about headache and the jaw joint and at the same time the pain from the teeth which has been adjusted, the patient also complains about the sound which initiated from the joint when the lower jaw is moved, the pain that often exists, sometimes it diminishes, the most common symptoms which make patient seeks for treatment. clinically, in this figure 7. m o d e l i n s e m i a d j u s t a b l em o d e l i n s e m i a d j u s t a b l e articulator. figure 6. result of the pattern of functional occlusion. occlusion.occlusion. figure 8. permissive occlusal splint.permissive occlusal splint.. figure 9. directive occlusal splint.directive occlusal splint.. ��dahlan: treatment of temporomandibular case, the patient has been suffering from temporomandibular joint disorder, the patient sometimes has difficult times in differentiating the source of pain from the adjusted teeth or from other source. each and every person has different pain limit and different pain acceptance, and this condition is caused by psychological factors.11 the edge of the sensitive tooth could initiate a headache and jaw joint, which also might be followed by clicking to the patient. this has to do with the disharmonized occlusion which results in disorder on temporomandibular joint. the complain of the pain is the most often complaint. this condition could occur in the morning, in the middle of the day or during the night when the mouth is opened. the clicking sound also occurs when the mouth is closed and this condition is called reciprocal clicking.5,12 it is found that during the clinical and radiological examination, the patch up is in poor condition on teeth 35, 44, 45. especially, on teeth 44 and 45 the overhanging patch up is found, whereas on tooth 35, the adjustment of the occlusion is detected. this condition does not show the occlusal harmonization. the lost of posterior 17, 18, 24, 25, 27, 28, 36, 37, 38, 46, 48 have caused the excessive closing of the upper jaw. in turn, condilli is pressed to the posterior part. as a result, pain occurs around the joint, and sometimes followed by headache. the excessive muscle contraction could be initiated by the lost of bilateral posterior teeth. spasm could cause pain and limited movement, other than that, the jaw position shall shift so that the teeth do not experience the right occlusion. if this condition keeps occurring, the teeth shall adjust themselves and occupy the new position so that the condyle shall not be in centric relation.6,13 the occlusal disorder could initiate mastikasi muscles along with their nervous system which will result in stomagtognatic dysfunctional system. the chewing system dysfunction could stimulate neuralgia trigeminal.11 predisposition factor is the excessive factor from the chewing muscle which is connected to bruxism for the whole night and followed by jaw stiffness when the patient wakes up and trismus. low intensity pain is suffered during the day due to the activities which sometimes need more attention. muscle contraction has been the manifestation of spasm in the long run. on the above case, other than the mastikasi muscles as the source of pain, other factors such as emotional stress could also be considered as the etiology factor, that is problems on the patient working condition. so, there are several factors related to the joint disorder which are related to each other. based on the anamnesis, clinical examination and radiology, it has been found that the diagnose on the patient with “temporomandibular joint disorder” is caused by occlusion disorder. the initial treatment on this case is focused to overcome the existing pain, which is conducting a biting test using cotton roll for approximately five minutes, until the patient should feel much better from the initial condition. afterwards, ‘jig” is installed on the top anterior jaw using cold cure acrylic. after the pain fade away, immediately replaced by occlusal splint. the tool chosen for the first time is permissive occlusion in which the occlusal surface and incision are slippery so that the friction is smooth, sliding without any obstacle. this tool is to free the intercuspation from the touching teeth, which will make neuromuscular reflex disappear, and muscle shall function according to the regular interaction which could make the cause and the effects of the functional irregularity.7,14 the use of the occlusal splint shall add the vertical dimension to the patient. this shall place condili to the stable support to the fossa glenoid (centrically related), so in turn, it will decrease the pressure on the joint structure and the possibility of decreasing the muscle activities due to the muscle relaxation.1, 17 the position of occlusion is changed, so the accuracy and the certainty of the proper position of condili joint shall be achieved. the occlusal splint changes the position of the lower jaw as opposed to the upper jaw which experiences the intercuspation, that is, rearranging the relation within the teeth by erasing the command to the muscle (muscle de-programmer) which causes the inaccuracy of the relation among the teeth.7,16 the occlusion is used in a relatively short period of time, that is between 5–7 days or should not be more than 6-8 weeks. this is due to the effective time limit of neuromuscular in adapting in the relaxation period.3,15 moreover, the permissive occlusal splint was replaced by directive occlusal splint since the above mentioned occlusal disorder has already been solved and the correct position of the occlusion has also been achieved, and the use of directive occlusion is still needed. the occlusion surface and the incision of this last tool are not slippery. it is, however, in a form of occlusal molding and the opposite tooth incision was given the occlusion so that the occlusal pattern becomes stable and is in line with the chewing system of the patient who is free of occlusal disorder. this directive occlusion is used in a relatively longer period of time than the permissive occlusion to give the chance to the occlusal pattern to adapt to the new occlusal position.3 the patient’s evaluation is conducted regularly. control is carried on until the patient does not have any complaints and disorder around the joint area. when the jaw is opened and close, the temporomandibular is sliding smoothly. this reflects the condyle position is adapting to the new position on fossa glenoid. the professional and consistent treatments are truly the key to success in managing the patient in curing the temporomandibular joint disorder. with the series of comprehensive treatment enable doctors to give opportunity in each and every step of comprehensive treatment to evaluate the next status for every chewing system and to provide time and accurate intervention when needed. based on the above explanation, it can be summarized that the use of occlusal splint could overcome the temporomandibular joint disorder followed by orofacial �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 31-36 pain since the use of occlusal splint would add vertical dimension to the patient. this shall place condyle to the stable position to fossa glenoid (centric relation). this shall also decrease the pressure on the joint structure and opens up the possibility to decrease the muscle activities due to muscle relaxation. the position of occlusion is indirectly changed, so that the accuracy and the certainly of the true condyle joint position are achieved. the objective of the initial treatment with permissive occlusion, followed by occlusal treatment and occlusal adjustment with directive occlusion is to stabilize the occlusion position and to continue diminishing the uncomfortability from the previous treatment, so that the treatment progress is achieved. in addition, definitive prosthetic could be provided by using directive occlusal splint guide if the occlusal disorder has been overcome. references 1. okeson jp. management of temporomandibular disorders and occlusion. st. louis: mosby year book; 1996. p. 190–200. 2. dawson pe. functional occlusion: from tmj to smile design. leawood, kansas: mosby elsevier; 2007. p. 57–61. 3. krisnowati d. penggunaan dan pemilihan belat gesel. maj. ked. gigi 1996; 29(2):41–5. 4. ramfjord sp. bruxism: a clinical and electromyographic study. j am dent assoc 1971; 62(3):21–4. 5. mcdevitt. clinical periodontology: matiscatory system disorder. 9th ed. carranzas: wb saunders co; 2002. p. 384–93. 6. ogus hd, toller pa. gangguan sendi temporomandibula. cetakan i. lilian y, editor. jakarta: hipokrates; 1990. p. 20–42. 7. dawson pe. evaluation, diagnosis, and treatment of occlusal problems. leawood, kansas: mosby co; 1990. p. 183–205. 8. thuan dao. musculoskeletal disorders and the occlusal interface. 2005. 18(4):29–33. 9. ramfjord sp. occlusion. 3rd ed. philadelphia: wb saunders company; 1983. p. 193–9. 10. gross md, mathews jd. oklusi dalam kedokteran gigi restoratif teknik dan teori. krisnowati h, editor. surabaya: airlangga university press; 1991. p. 2–5, 187. 11. satyanegara md. teori dan terapi nyeri kepala bagian bedah saraf rumah sakit pusat pertamina. 1978. p. 9–31. 12. joseph k, leader ms. the influence of mandibular movement on joints sounds in patients with temporomandibular disorders. the journal of prosthetics dentistry 1999; 82(2):186–95. 13. wright ef, schiffman el. treatment alternatives for patient with masticatory myofacial pain. jada 1955; 126(4):28–34. 14. kato t, thie nmr, montplaisir jy, lavigne gj. bruxism and orofacial movement during sleep. dent clin n am 2001; 45(2): 157–84. 15. celia m, rizzati-barbosa. eagle’s syndrome associated with tmj disorders: a clinical report. the journal of prosthetics 1999; 81(4):4951. 16. julian k. prevalence of dental occlusal variables and intra articular temporomandibululat disorders : molar relationship, lateral guidance, and non working side contact. the journal of prosthetic dentistry; 1995. 83(4):53–9. 17. krisnowati d. perubahan morfologi oklusal dan insisal gigi permanen sebagai gejala diagnosis, oklusi fungsional. cetakan i. surabaya: airlangga university press; 1995. p. 55–8. 18. shillingburg ht. fundamentals on fixed prosthodontics. 3rd ed. oklalona city: quintessence publishing co inc; 1997. p. 86–90. 67 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 67–70 original article effects of tooth preparation on the microleakage of fissure sealant gesti kartiko sari1, sri kuswandari2, putri kusuma wardani mahendra2 1paediatric dentistry specialty program, department of paediatric dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of paediatric dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: fissure sealing can be achieved by preparing and sealing the deep pits and fissures in the teeth with a sealant to prevent caries. fissure sealing is performed using resin modified glass ionomer cement (rmgic) and failure is most often due to weak adhesion between the material and the tooth, resulting in microleakage. purpose: the study aimed to determine the effect of a preparation technique with bur and acid application on potential rmgic fissure sealant microleakage. methods: twenty-four extracted maxillary premolars were divided into four treatment groups. group 1 underwent enameloplasty with a round bur and application of 37% phosphoric acid; group 2 with a tapered bur and 37% phosphoric acid; group 3 with a round bur and 10% polyacrylic acid; and group 4 with a tapered bur and 10% polyacrylic acid. the application of 37% phosphoric acid was carried out for 15 seconds, while 10% polyacrylic acid was applied for 20 seconds, before rmgic filling. the teeth were stored in artificial saliva at 37°c for 24 hours, then thermocycled 100 times at 5°c and 55°c for 20 seconds each. the teeth were immersed in a 1% methylene blue solution for 24 hours at 37°c, then cut crosswise. the length of the microleakage was observed with a stereo microscope at 8 times magnification and measured using raster image application. data was analysed with one-way anova. results: significant differences were found between treatment groups (f=562.14; p<0.05). the deepest mean microleakage was in the round bur and 10% polyacrylic acid group (1657.87 ± 78.08) and the shallowest was in the round bur and 37% phosphoric acid group (500.70 ± 38.55). conclusion: the preparation method, type of bur and acid solution have an effect on microleakage potential of rmgic fissure sealing. round bur preparation and 37% phosphoric acid resulted in shallow microleakage. keywords: preparation technique; fissure sealant; microleakage correspondence: sri kuswandari, department of pediatric dentistry, faculty of dentistry, universitas gadjah mada. jl. denta 1, sekip utara, yogyakarta, 55281, indonesia. email: ndaribacrun@ugm.ac.id; kuswandarisri@gmail.com introduction pit and fissure sealant was introduced by buonocore in 1971. he started using an acid etch before sealing fissures for preventing caries. various fissure sealing preparation methods were observed by agarwal, such as air abrasion, increased etching time, air polishing, application of pumice slurry, brushing, and mechanical bur preparation.1 fissure sealing aims to seal the deep fissures of the teeth as a physical barrier to prevents the entry of food debris into those fissures.2 fissure sealing is performed using a low viscosity dental material, namely resin modified glass ionomer cement (rmgic).3 rmgic combines the advantages of glass ionomer materials and composite resins. rmgic releases fluoride which can prevent and control caries. the material hardens upon light curing.4 adhesion of the material will occur after the light curing process, but the literature reveal that the sealant will lose about 5-10% efficacy per year.5 failure of the fissure sealant in the form of microleakage causes bacteria to easily enter through the gap and continue to develop into dental caries. as microleakage occurs due to poor adhesion of the material to the tooth, the preparation method before fissure sealing treatment needs to be considered.6 the adhesion and bond strength between the sealant and the tooth is influenced by the preparation of the tooth before the application of the sealant.7 fissure sealing dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p67–70 mailto:ndaribacrun@ugm.ac.id mailto:kuswandarisri@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p67-70 68sari et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 67–70 preparation, among others, takes the form of prophylaxis with pumice, application of acid to the enamel, air abrasion, or enameloplasty. the preparation is carried out on the enamel surface prior to sealant application. preparation can be divided into two types, namely mechanical and chemical.5 chemical preparation is performed through acid application, which serves for the adhesion of fissure sealants.8 according to the manufacturer’s recommendations, fissure sealing with rmgic is preceded by the application of 10% polyacrylic acid to remove the smear layer.9 chemical preparation can also be carried out with the application of 37% phosphoric acid to increase the mechanical strength of the sealant.5 acid application on the tooth surface can form enamel micropores within which the material will form resin tags. mechanical preparation can create access to deep fissure areas, remove debris, enable deeper sealant penetration, and increase retention.5 one of the commonly used mechanical preparations techniques is enameloplasty. it is a prophylactic procedure that removes enamel in the pits and fissures of the tooth to produce a smooth and sloping surface.10 enameloplasty is performed with a cutting instrument in the form of a bur, which can cut the enamel effectively.11 most dentists use round burs to perform enameloplasty.5 it can also be performed with a tapered bur, often called a fissurotomy bur. enameloplasty can determine the penetration depth of the sealant.5 a combination of mechanical and chemical preparation techniques plays an important role in increasing the bond strength between the tooth and the material, which in turn can affect the success of fissure sealing.7 one indicator of efficacy is the microleakage test. this study aims to determine the effect of the preparation method with various bur and acid applications on the microleakage potential of rmgic fissure sealant. materials and methods the research protocol was reviewed by the ethical committee of faculty of dentistry and was granted ethical clearance with certificate number of 00476/kkep/fkgugm/ec/2020. this experimental laboratory research included 24 extracted human maxillary premolars, free of caries. human premolars extracted for orthodontic reasons were included in this study. the teeth were stored in a saline solution for 6 months extraction and divided into four groups (n=6 each). teeth were randomly selected and distributed into the four experimental groups (coded 1, 2, 3, and 4). groups 1 and 3 underwent enameloplasty with a 1/4 round bur (ss white, lakewood, new jersey), 2 and 4 with a tapered bur (ss white, lakewood, new jersey). the depth of the enameloplasty was adjusted to the diameter and height of the bur head. afterwards, 37% phosphoric acid (3m, saint paul, minnesota) was applied for 15 seconds in groups 1 and 2, while groups 3 and 4 used 10% polyacrylic acid (gc, tokyo, japan) for 20 seconds. fissure sealing was performed with rmgic (gc, tokyo, japan) in all groups after acid application. the teeth were immersed in artificial saliva (mipa ugm laboratory, sleman, indonesia) for 24 hours in an incubator at 37oc, then thermocycled for 100 cycles at 5oc and 55oc. the apical part of the tooth was coated with nail polish and sticky wax before immersing in a 1% methylene blue solution (pudak scientific, bandung, indonesia) for 24 hours at 37oc. the soaked teeth were cleaned from nail polish and wax, then rinsed in water. the teeth were dried and cut crosswise in the mesial-distal centre using a benchtop micro milling machine (proxxon, hickory, north carolina). microleakage was observed with a binocular microscope (olympus, tokyo, japan) at 8 times magnification and measured in micrometres (µm) using image raster application version 3 (miconos, sleman, indonesia). the data was then statistically tested with one-way anova, spss version 22 (ibm, new york, usa). results the mean results obtained in each group are presented in table 1. the deepest microleakage (mean value) was in the round bur enameloplasty and 10% polyacrylic acid application group. the shallowest microleakage (mean value) was in the group with round bur enameloplasty and 37% phosphoric acid application group. the one-way anova test was used to determine the effect of the type of bur and acid in the preparation on rmgic fissure sealant microleakage; results can be seen in table 2. results show an f value of 562.14 (p<0.05), table 1. mean and standard deviation of rmgic fissure sealant microleakage based on preparation method (µm) treatment group n mean + sd (µm) round bur and 37% phosphoric acid 6 500.70 ± 38.55 tapered bur and 37% phosphoric acid 6 900.55 ± 10.53 round bur and 10% polyacrylic acid 6 1,657.87 ± 78.08 tapered bur and 10% polyacrylic acid 6 998.39 ± 46.29 table 2. one-way anova test between treatment groups sum of square degree of freedom mean square f p between groups 4,146,928.49 3 1,382,309.49 562.14 <0.001 within groups 49,180.03 20 2,459.00 total 4,196,108.52 23 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p67–70 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p67-70 69 sari et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 67–70 so it can be assumed that the preparation method has an influence on the microleakage potential of the fissure sealant. comparative analysis of the study groups by a post hoc lsd test (table 3) showed significant differences (p<0.05) between treatment groups. discussion deep pits and fissures are a reason why teeth are prone to caries, so prevention is necessary. fissure sealing functions as a physical barrier to the entry of food debris and to plaque retention into the dental fissures.2 the material used for fissure sealing in this study is rmgic. excess rmgic can bind to the hard tooth tissue directly. mechanical (enameloplasty) and chemical (acid application) preparations in the fissure sealing procedure are carried out to reduce the possibility of microleakage.12 the results showed that the type of enameloplasty bur and acid application had a significant effect on the microleakage of the rmgic fissure sealing. the shallowest leakage was observed after a preparation method using a round bur and 37% phosphoric acid. results in this group were better than with a tapered bur and 37% phosphoric acid group due to the difference in the preparation surface area, which is wider with a round bur than with a tapered bur. it can be determined by the following calculation: surface area of round bur = (ω x 4 x π x r2) + (π x d x t) = (2 x 3.14 x 0.352) + (3.14 x 0.7 x 0.77) x mm2 = 2.46 mm2 surface area of tapered bur = π x r x s = 3.14 x 0.39 mm x 1.86 mm = 2.27 mm2 the fissure surface of the teeth prepared with a round bur (2.46 mm2) is wider than the surface of the fissure prepared with a tapered bur (2.27 mm2), so more resin tags will form in the sealant applied to the fissure after a round bur preparation. this is in accordance with parihar and pilania, stating that the number of resin tags can affect the retention of the bond between the sealant and the tooth; the higher the tagging, the stronger the bond will be. 13 the “u” shaped fissure resulting from the round bur preparation also facilitates material filling due to the blunt fissure base. the round bur creates a fissure shape with the same diameter from top to bottom, in contrast to the shape of the fissure after a tapered bur preparation, which gets narrower towards the bottom. this is in accordance with findings from tzifa et al. that increasing the width of the fissure makes the penetration of the material more optimal.5 results from this study showed that the shallowest microleakage occurred in teeth with the application of 37% phosphoric acid after preparation with a round bur, while the deepest followed the application of 10% polyacrylic acid and the use of a round bur. this is because 37% phosphoric acid can dissolve the prism core and form a type 1 enamel etching pattern, while polyacrylic acid does not change the configuration of tooth enamel.14 the enamel etching pattern produced by 37% phosphoric acid causes the filling material to enter the enamel prism, and increases the bond strength between sealant and enamel. as such, it can reduce the risk of microleakage.15 application of 37% phosphoric acid can also produce a coarse and porous layer as deep as 5-50 µm.16 chemical preparation with 10% polyacrylic acid only produces an average micro-tag depth of about 8.73 µm.17 the use of 37% phosphoric acid produces deeper micro-tags, so the retention and bond strength between the material and the tooth will be better than with 10% polyacrylic acid.16 ion exchange between glass ionomer and tooth coating without a smear layer will result in a stronger bond, whereas bonding to a smear layer would mean weaker tooth structure.8 hydroxyapatite in the tooth will interact with the material. demineralisation of the dentin and the submicron interdiffusion layer creates micromechanical retention for the cement against the tooth.18 another factor that can influence microleakage is the storage period of the tooth from extraction to study, which is different for each tooth. all research objects were obtained from the extraction of premolars within a period of 6 months with the same storage media (saline).19 according to research conducted by secilmis et al.,20 dental minerals can dissolve quickly in saline. the calcium mineral of the teeth will decrease with increasing storage time and cause decreasing tooth hardness, which will affect the occurrence of microleakage. based on this research, if using the manufacturer’s recommendation of rmgic with 10% polyacrylic acid application, mechanical preparation with a tapered bur (fissurotomy) is advised; however, for the best results, it is recommended to use rmgic after tooth preparation with a combination of round bur and 37% phosphoric acid before sealant application. references 1. agrawal a, shigli a. comparison of six different methods of cleaning and preparing occlusal fissure surface before placement of pit and fissure sealant: an in vitro study. j indian soc pedod prev dent. 2012; 30(1): 51–5. 2. wright jt, tampi mp, graham l, estrich c, crall jj, fontana m, gillette ej, nový bb, dhar v, donly k, hewlett er, quinonez rb, chaffin j, crespin m, iafolla t, siegal md, carrasco-labra a. sealants for preventing and arresting pit-and-fissure occlusal table 3. lsd test between treatment groups between groups mean difference p 1 and 2 -399.85 0.000 1 and 3 -1,157.17 0.000 1 and 4 -497.69 0.000 2 and 3 -757.32 0.000 2 and 4 -97.84 0.033 3 and 4 659.47 0.000 group information: 1= round bur and 37% phosphoric acid 2= tapered bur and 37% phosphoric acid 3= round bur and 10% polyacrylic acid 4= tapered bur and 10% polyacrylic acid dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p67–70 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p67-70 70sari et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 67–70 caries in primary and permanent molars. pediatr dent. 2016; 38(4): 282–308. 3. asefi s, eskandarion s, hamidiaval s. fissure sealant materials: wear resistance of flowable composite resins. j dent res dent clin dent prospects. 2016; 10(3): 194–9. 4. sakaguchi rl, powers jm. craig’s restorative dental materials. thirteenth. sakaguchi rl, powers jm, editors. saint louis: mosby; 2012. p. 150–2. 5. tzifa v, arhakis a. sealant retention in pits and fissures: preparation and application techniques. a literature review. balk j stomatol. 2013; 17: 9–17. 6. rahimian-imam s, ramazani n, fayazi mr. marginal microleakage of conventional fissure sealants and self-adhering flowable composite as fissure sealant in permanent teeth. j dent (tehran). 2015; 12(6): 430–5. 7. hatirli h, yasa b, yasa e. microleakage and penetration depth of different fissure sealant materials after cyclic thermo-mechanic and brushing simulation. dent mater j. 2018; 37(1): 15–23. 8. zhang l, tang t, zhang z, liang b, wang x, fu b. improvement of enamel bond strengths for conventional and resin-modified glass ionomers: acid-etching vs. conditioning. j zhejiang univ sci b. 2013; 14(11): 1013–24. 9. burrer p, dang h, par m, attin t, tauböck tt. effect of over-etching and prolonged application time of a universal adhesive on dentin bond strength. polymers (basel). 2020; 12(12): 2902. 10. gopikrishna v. preclinical manual of conservative dentistry and endodontics. 2nd ed. chennai: elsevier; 2015. p. 206. 11. dixit k, dixit kk, pandey r. minimal intervention tooth prepration: a new era of dentistry. j dent sci oral rehabil. 2012; 3(4): 4–7. 12. ugurlu m. bonding of a resin-modified glass ionomer cement to dentin using universal adhesives. restor dent endod. 2020; 45(3): e36. 13. parihar n, pilania m. sem evaluation of effect of 37% phosphoric acid gel, 24% edta gel and 10% maleic acid gel on the enamel and dentin for 15 and 60 seconds: an in-vitro study. int dent j students res. 2012; 1(2): 29–41. 14. justus r. iatrogenic effects of orthodontic treatment: decisionmaking in prevention, diagnosis, and treatment. cham: springer international publishing; 2015. p. 130. 15. k hurshid z, najeeb s, zafar ms, sefat f. advanced dental biomaterials. duxford: woodhead publishing; 2019. p. 232. 16. nanjannawar lg, nanjannawar gs. effects of a self-etching primer and 37% phosphoric acid etching on enamel: a scanning electron microscopic study. j contemp dent pract. 2012; 13(3): 280–4. 17. bhandari dpk, anbuselvan gj, karthi m. evaluation of resin penetration depth in enamel surface for orthodontic bonding exposed to five types of enamel conditioning methods: a scanning electron microscopic study. j pharm bioallied sci. 2019; 11(suppl 2): s221–7. 18. sauro s, faus-matoses v, makeeva i, nuñez martí jm, gonzalez martínez r, garcía bautista ja, faus-llácer v. effects of polyacrylic acid pre-treatment on bonded-dentine interfaces created with a modern bioactive resin-modified glass ionomer cement and subjected to cycling mechanical stress. mater (basel, switzerland). 2018; 11(10). 19. reena rk, gill s, miglani a. storage media: a neglected variable for in vitro studies. j indian orthod soc. 2011; 45(1): 5–8. 20. secilmis a, dilber e, gokmen f, ozturk n, telatar t. effects of storage solutions on mineral contents of dentin. j dent sci. 2011; 6(4): 189–94. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p67–70 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p67-70 p-issn: 1978-3728 e-issn: 2442-9740 volume 48, number 3, september 2015 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2015 january 2nd – december 31st, 2015 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga) editorial boards: prof. dr. pinandi sri pudyani, drg., su., sp.ort(k) (department of orthodontics faculty of dentistry, universitas gadjah mada); prof. dr. anita yuliati, drg., m.kes (department of dental material – faculty of dental medicine, universitas airlangga); prof. dr. jenny sunariani, drg., ms (department of oral biologyfaculty of dental medicine, universitas gadjah mada); prof. dr. adioro soetojo, drg., ms., sp.kg(k) (department of conservative dentistryfaculty of dental medicine, universitas gadjah mada); prof. dr. sri kunarti, drg., ms., sp.kg(k) (department of conservative dentistryfaculty of dental medicine, universitas gadjah mada); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (department of oral medicine faculty of dental medicine, universitas airlangga); kus harijanti, drg., ms., sp.pm(k) (department of oral medicine faculty of dental medicine, universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (department of periodontics faculty of dental medicine, universitas airlangga); dr. retno indrawati, drg., m.si (department of oral biologyfaculty of dental medicine, universitas gadjah mada); dr. retno pudji rahayu, drg., m.kes (department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga); dr. theresia indah budhy, drg., m.kes (department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga); dr. indah listiana kriswandini, drg., m.kes (department of oral biologyfaculty of dental medicine, universitas gadjah mada); dr. ernie maduratna setiawati, drg., m.kes., sp.perio(k) (department of periodontics faculty of dental medicine, universitas airlangga); dr. agung krismariono, drg., m.kes., sp.perio (department of periodontics faculty of dental medicine, universitas airlangga); dr. pratiwi soesilowati, drg., m.kes (department of oral biologyfaculty of dental medicine, universitas gadjah mada); wisnu setyari, drg., m.kes (department of oral biologyfaculty of dental medicine, universitas gadjah mada) managing editors: dr. susy kristiani, drg., m.kes (department of odontology forensic – faculty of dental medicine, universitas airlangga); priyawan rachmadi, drg., ph.d (department of dental material – faculty of dental medicine, universitas airlangga); udijanto tedjosasongko, drg., ph.d., sp.kga (department of pediatric dentistry – faculty of dental medicine, universitas airlangga); dr. hendrik setia budi, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); sianiwati goenharto, drg., ms (department of orthodontics – faculty of dental medicine, universitas airlangga); dr. anis irmawati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); yuliati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); eric priyo prasetyo, drg., m.kes., sp.kg (department of conservative dentistry – faculty of dental medicine, universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 contents page printed by: airlangga university press. (rk 063/02.16/aup-b3e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 48, number 3, september 2015 p-issn: 1978-3728 e-issn: 2442-9740 1. xerostomia severity difference between elderly using alcohol and non alcohol-containing mouthwash hendri susanto ................................................................................................................................ 109–112 2. serum c-reactive protein and c-reactive gene (-717c>t) polymorphism are not associated with periodontitis in indonesian male patients antonius winoto suhartono, benso sulijaya, niniarty zeiroeddin djamal, sri lelyati chaidar masulili, christopher talbot, and elza ibrahim auerkari ....................... 113–118 3. potential of jatropha multifida sap against traumatic ulcer basri a. gani, abdillah imron nasution, nazaruddin, lidya sartika, and rahmat kurniawan alam ....................................................................................................... 119–125 4. comparison between probiotic lozenges and drinks towards periodontal status improvement of orthodontic patients natasia melita kohar, victor emmanuel, and luki astuti ......................................................... 126–129 5. correlation between magnesium and alkaline phosphatase from gingival crevicular fluid on periodontal diseases nila kasuma ..................................................................................................................................... 130–134 6. apoptosis of rattus novergicus gingival fibroblasts caused by silver nano-particles gel exposure kharinna widowati, titiek berniyanti, and retno pudji rahayu .............................................. 135–138 7. autogenous tooth transplantation: an alternative to replace extracted tooth david b. kamadjaja ........................................................................................................................ 139–143 8. the effect of ethyl acetate fraction of citrus limon peel on mesenchymal cell proliferation and polybacterial growth astrid marinna, priyo hadi, and desiana radithia ...................................................................... 144–149 9. the effect of 25% mauli banana stem extract gel to increase the epithel thickness of wound healing process in oral mucosa maharani laillyza apriasari, ariska endariantari, and ika kustiyah oktaviyanti ................. 150–153 10. experimental comparative study and fracture resistance simulation with irrigation solution of 0.2% chitosan, 2.5% naocl and 17% edta ernani, trimurni abidin, and indra ............................................................................................. 154–158 11. vegf expression and new blood vessel after dental x-ray irradiation on fractured tooth extraction wound niluh ringga woroprobosari, jenny sunariani, and eha renwi astuti .................................... 159–164 141 vol. 42. no. 3 july–september 2009 review article the role of msx1 and pax9 in pathogenetic mechanisms of tooth agenesis yani corvianindya rahayu1 and dyah setyorini2 1 departement of oral biology 2 departement of pediatric dentistry faculty of dentistry university of jember jember indonesia abstract background: tooth agenesis is one of the most common developmental anomalies in human, which one or a few teeth are absent because they have never formed, may cause cosmetic or occlusal harm, while severe agenesis which are relatively rare require clinical, may cause cosmetic or occlusal harm, while severe agenesis which are relatively rare require clinical attention to support and maintain the dental function. molecular studies have demonstrated that tooth development is under strict genetic control. purpose: this article want to review the genetic regulating that are responsible for tooth agenesis especially the role of msx1 and pax9 in pathogenetic mechanisms of tooth agenesis. review: tooth agenesis is a consequence of a qualitatively or quantitativelyooth agenesis is a consequence of a qualitatively or quantitatively impaired function of genetic networks, which regulate tooth development. mutations in msx1 and pax9 genes are dominant for tooth agenesis in humans. the pax9 gene, which codes for a paired domain-containing transcription factor that plays an essential role in the development of mammal dentition, has been associated with selective tooth agenesis in humans and mice. conclusion: reduced amount of functional msx1 or pax9 protein in the tooth forming cells is able to cause severe and selective tooth agenesis. there are differences in the frequency of agenesis of specific teeth associated with the defects in msx1 and defects in pax9. key words: tooth agenesis, genetic regulation, pathogenetic mechanisms, msx1, pax9 correspondence: yani corvianindya rahayu, c/o: bagian biologi oral, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. e-mail: ryanicorvianindya@yahoo.com introduction during the last decades after the advent of molecular biology and genetics, the new technologies have been extensively used to elucidate developmental mechanisms and the genetic regulation of tooth development. the positional cloning of several genes that cause different developmental dental anomalies, have contributed to understanding of the genetic regulation of developmental and patterning of the human dentition.1 agenesis of one or more teeth is one of the most common of human developmental anomalies. the term oligodontia refers to congenital absence of many but not all teeth whereas the term hypodontia implies the absence of only a few teeth. in the permanent dentition, hypodontia has a prevalence of 1.6% to 9.6%, excluding agenesis of the third molars. oligodontia has a population prevalence of 0.3% in the permanent dentition. it occurs more frequently in girls at a ratio of 3:2. agenesis of only the third molars has prevalence between 9% and 37%. in the deciduous dentition, hypodontia occurs less often (0.1%-0.9%) and has no significant sex distribution.2 both environmental and genetic factors can cause failure of tooth development. numerous different genes have been implicated in tooth development by genes expression and experimental studies in the mouse, and any of these genes may cause tooth agenesis. variability in expression includes the number and region of missing teeth, and various other dental features associated with the trait.3,4 the present study confirmed the results of garn and lewis5 showing other physical dental traits associated with the occurrence of tooth agenesis. it contributes to mount the evidence that agenesis and its associated abnormalities are under genetic control. the possible explanation is that a single genetic defect may give rise to different anomalies, so that two or more dental anomalies in the same patient may present a common genetic origin. studies of families, as well as investigations of the association of agenesis and 142 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 141-146 other types of dental anomalies, previously highlighted the role played by genetic mechanisms in the etiology of various dental anomalies.5,6 dental anomalies are ideal conditions for the geneticist to study the hereditary factors involved in their pathogenesis. this article reviewed the genetic regulating that are responsible for tooth agenesis especially the role of msx1 and pax9 in pathogenetic mechanisms of tooth agenesis. tooth morphogenesis in the late bud stage, a group of cells at the tip of the epithelial bud, the primary enamel knot stop to proliferate and then removed by apoptosis. the enamel knot deviates significantly from the surrounding epithelium because of its gene expression. it expresses several transcription factors and numerous signalling molecules as well as signalling inhibitors with a specific schedule of appearance, thus having potential to act as a signalling centre that orchestrates the development of the surrounding tissues. the primary enamel knot is apparently induced and maintained by signals emanating from the underlying mesenchyme. on the other hand, formation of the primary knot seems to be a prerequisite for the advancing of the tooth development to the cap stage.7 the dental lamina in 6th week, and later the enamel organs, represent the epithelial portion of the oral cavity with potential capacity to generate the ectodermal components of the teeth. in subsequent development, the adjacent mesenchymal tissue will proliferates and condenses to form other components and portions of the future teeth. the permanent-teeth germs are developed later. they originate from the accessory dental lamina, in the case of molars, or from growth of the free edge of the dental lamina on its lingual side for the remaining permanent teeth. the emergence or eruption of permanent dentition takes place over an extended period ranging from 7 to 12 years, apart from the third molars, which erupt between 13 and 25 years, although sometimes they fail to appear at all. most dental anomalies are more frequent in permanent than in deciduous dentition. with regard to permanent dentition, the lack of one or more teeth is evident in about 1–2 % of the population.8 molecular regulation of tooth development the technologies of molecular biology and genetics have been extensively used to elucidate developmental mechanisms and the genetic regulation of tooth development. the most usual model has been the mandibular molar teeth of the mouse, the most practical laboratory animal that develops teeth. immunohistology and in situ hybridization have been used to study gene expression during mouse tooth development and differentiation. natural and transgenic mutant mice have been utilized to reveal gene function. tissue culture of whole tooth or jaw explants as well as culture of recombined tissues has been used to study effects of proteins and mutations. this knowledge is applicable to humans and other mammals because of the conservation of the basic genetic and developmental mechanisms.1,9 molecular studies have revealed that the instructive and permissive tissue interactions during mouse tooth development described above are mainly mediated by growth factor signalling. development from initiation to eruption is governed by a sequential and reciprocal signalling process rather than simple one-way messages. the signalling involves all major signalling pathways, including transforming growth factor b (tgfb), fibroblast growth factor (fgf), sonic heghehog (shh), anhidrotic ectodermal dysplasia (eda), and epidermal growth factor (egf) signalling, and studies with mouse mutants have shown that they are needed simultaneously during critical stages of development.9 msx1 and pax9 are transcription factors intimately involved in the genetic networks regulating tooth development. msx1 contains a homeobox which binds to specific target sequences in the dna but is also capable to proteins interaction. msx1 has often been considered rather as a repressor than activator of gene expression. pax9 belongs to the paired-box containing transcription factor family, and is one of the earliest mesenchymal markers of the future tooth forming positions in mouse. pax9 is regulated by epithelial signals, especially fgf8, and it apparently regulates reciprocal signalling from the mesenchyme. in mice with hypomorphic pax9 mutations, a partial failure of tooth development was observed, affecting in a dose-dependent manner the third molars and incisors and to a smaller extent the other molars. the ameloblast differentiation and dentinogenesis were also affected.10 it has been suggested that the key role of msx1 and pax9 is to facilitate the bud to cap stage transition. there is signals emanating from the epithelium and mesenchymal during tooth development and molecular regulation (figure 1). mesenchymal msx1 expression is initially activated by the epithelial bone figure 1. tooth development and molecular regulation. signals emanating from the epithelium are shown above and signals from the mesenchyme below the scheme.1 143rahayu: the role msx1 and pax9 in pathogenetic morphogenetic protein 4 (bmp4) signal, and needed for a reciprocal bmp4 signal from the mesenchyme. bmp4 and msx1 thus form an autoregulatory loop. bmp4 signal to the epithelium is crucial for the formation of the epithelial signalling centre, the enamel knot, and the arrest of the development in msx1 null mutant teeth can be rescued by external bmp4 or transgenically activated bmp4 expression. the expression of pax9 is apparently needed to maintain and, by the synergism with msx1, to enhance this loop and also needed later in tooth development.11 incidence of tooth agenesis the partial absence of dental germs is a congenital defect of hereditary or acquired origins. dental agenesis candental agenesis can be defined as any situation in which one or more teeth are missing because they have never formed. this can also be called oligodontia, dental aplasia, and congenital absence of teeth or hypodontia. the term “oligodontia” is usually limited to those cases in which three or more teeth are missing; anodontia is the type of agenesis in which all the teeth are missing. when agenesis is of one or a few teeth, it tends to be present more distally.9,12 congenital agenesis of one or more permanent teeth, also known as hypodontia, is among the most wellrecognized morphologic anomalies in humans, and yet the etiology is largely unknown (figure 2). oligodontia has been defined as agenesis of more than 6 permanent teeth. in caucasians, tooth agenesis most commonly involves third molars, with from 10 to 25% of the population affected. reports on the overall incidence of missing permanent teeth, excluding third molars, vary substantially, from 2% to 10%. in caucasians, approximately 80% of tooth agenesis cases involve only one or two teeth.9 etiology and pathogenesis of tooth agenesiss evidence supporting a genetic etiology for tooth agenesis is well established reviewed. tooth agenesis usually presents as an isolated anomaly. however, it is known to occur in association with syndromes or inherited disorders, many of which have known genetic defects.13 tooth agenesis is one of the most commonooth agenesis is one of the most common developmental problems in children. the congenital absence of teeth results from disturbances during the initial stages of tooth formation: initiation and proliferation. missing teeth may occur in isolation, or as part of a syndrome. isolated cases of missing teeth can be familiar or sporadic in nature. familiar tooth agenesis is transmitted as an autosomal dominant, autosomal recessive, or x-linked genetic condition.9 while several potential and verified environmental (post genetic) etiological factors at tooth agenesis have been presented, there is definitive proof that genetic factors play a major role in the etiology. the role of the genetic factors was suggested by observed familial occurrence, prevalence differences between populations, and association with heritable syndromes as well as by twin and family studies, but definitive evidence has been acquired during the molecular genetic era: defects in several genes have been shown to cause agenesis and anomalies in size and morphology. tooth agenesis and tooth size reductions have been related to trauma, maternal systemic disease and various external factors. among the maternal systemic disease, diabetes and different infections have been suggested. for example, developmental dental anomalies and tooth size reduction have been described in association of maternal rubella infection during pregnancy.14 the pathogenesis of human tooth agenesis is perhaps best understood in anhidrotic ectodermal dysplasias. in this case the molecular pathogenesis and the phenotypes in human patients and mouse mutants are directly comparable. the gene defects in anhidrotic ectodermal dysplasia (eda), eda-receptor (edar), immunoglobulin k-gamma (ikky) and their mouse homologs, i.e. the signalling ligand, its receptor and the intracellular mediators of the signalling, cause complete inactivation of this signalling pathway. in the mutant mice, incisors and third molars commonly fail to develop and first molars are hypoplastic, while in the patients with anhidrotic ectodermal dysplasia, both dentitions are severely affected and tooth morphology is simplified. the phenotypes of the mice with impairment or over expression of eda signalling suggest that early defects of ectodermal placodes and, in teeth, the enamel knots would explain the ectodermal defects in human patients. thus, failure of signalling at an early stage leads to anomalies that are present also in the deciduous dentition. on the other hand, as the mutant and disease phenotypes are caused by complete inactivation of the figure 2. the case of permanent teeth agenesis at 17 th year old woman. 144 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 141-146 signalling pathway, the partial albeit severe tooth agenesis phenotypes suggest redundancy in the function of the signalling pathways, i.e. that different signalling pathways have overlapping functions. this redundancy adds a further element explaining how different gene defects may cause partial agenesis.15 the congenital absence of teeth is one of the commonest developmental abnormalities seen in human populations. familial hypodontia or oligodontia represents an absence of varying numbers of primary and/or secondary teeth as an isolated trait. while much progress has been made in understanding the developmental basis of tooth formation, knowledge of the aetiological basis of inherited tooth loss remains poor. the study of mouse genetics has uncovered a large number of candidate genes for this condition, but mutations in only three have been identified in human pedigrees with familial hypodontia or oligodontia: msx1, pax9 and axin2. this suggests that these conditions may represent a more complex multifactorial trait, influenced by a combination of gene function, environmental interaction and developmental timing.16 the most compelling evidence for the genetic etiology of tooth agenesis has been provided by the identification of gene defects associated with different types of tooth agenesis. dominant defects in msx1, pax9 and axin2 have been described in families with isolated severe tooth agenesis. however, in association with defects in msx1, nail dysplasia and some patients with oral clefts have each been described in single families. in addition to causing severe tooth agenesis phenotype, a defect in axin2 also predisposed to colorectal cancer. recently, two defects that affected only dentition were also described in eda. all these gene defects cause severe types of agenesis. however, evidence for association of specific intragenic polymorphisms to tooth agenesis, apparently consisting mostly of common types of tooth agenesis, has been presented for msx1, pax9, axin2, tgfa, irf6 and fgfr1.17 the role of genes in tooth agenesis identifying a hereditary dental pathology and defining its unique characteristics are the first steps toward the dissection of its genetic basis. a thorough interview of the patient and his or her relatives is the next step to defining the trait as familial; if it proves to be so, it is imperative to define the pattern of inheritance of the anomaly.18 in this respect, several genes that are pivotal inin this respect, several genes that are pivotal in initiating the development of teeth have been subjected to intense study in the past decade. mutations in a number of genes were found to interrupt tooth development in mice. however, to date there are only three genes associated with the nonsyndromic form of human tooth agenesis: axin2, msx1, and pax9. among them, msx1 and pax9 was more intensively studied. recently, the general structure of the pax paired domain was described and the phylogenetics and relation between the several members of the pax family were established. in addition, both gene expression and molecular pathogenesis of msx1 and pax9 have been relatively well characterized, making it a special candidate to explain at least part of primate tooth variation (figure 3).19 figure 3. the mutation of two genes tooth development (msx1 and pax9) which can lead to tooth agenesis. darkness of the colour expresses the frequency of agenesis.19 discussion there is considerable evidence suggesting that genes play a fundamental role in the etiology of tooth agenesis. moreover, there seems to be a genetic relationship in the determination of different dental anomalies, considering the high frequency of patterns of association. a single genetic defect may result in different phenotypic expressions, including such various traits as tooth agenesis, microdontia, ectopic tooth position, and delayed development of different teeth. as with many other organs, tooth development involves sequential and reciprocal signalling processes between epithelial and mesenchymal cell layers that are orchestrated by a hierarchy of genes encoding secreted growth factors, extra cellular matrix components, and transcriptional regulators. because the regulatory genes required for tooth formation are common components of signalling cascades involved in development of other embryonic structures. among the transcriptional regulatory genes required for tooth formation, the msx1 homeobox gene is highly expressed in the dental mesenchyme and is essential for tooth development, since targeted gene disruption results in arrested tooth formation at an early stage in msx1. in addition to its expression in the tooth primordia, msx1 expression is prominent in regions of epithelialmesenchymal interactions in several other embryonic structures, including other craniofacial structures and the limb. these findings have led to the hypothesis that msx1 is an important component in the signalling events that occur between epithelial and mesenchymal tissues.19 145rahayu: the role msx1 and pax9 in pathogenetic both msx1 and pax9 are also needed for the mesenchymal cell condensation around the growing epithelial bud. the reduced condensation which is also seen in the pax9 hypomorphic mutans, perhaps indicating a decreased amount of committed dental mesenchymal cells, may be related to tooth agenesis. as msx1 is known to be important for the commitment of neural crest, an early defect in the migration of neural crest cells could also be responsible for the tooth agenesis, if it caused a reduction in the amount of competent ectomesenchymal cells.20 one of the earliest placodal markers, edar, is originally expressed throughout the oral epithelium and epidermis, but becomes limited to the placodes at an early stage. when eda was over expressed in the epithelium, the hair and tooth placodes become larger, probably due to an increased amount of the cells destined to become placode cells. thus eda signalling probably acts rather as a modulator of ectodermal placodeformation than as an initiator. eda signalling may be important as a mediator of effects of shh and bmps. mutation in the eda and edar genes in human cause x-linked and autosomal anhidrotic ectodermal dysplasia characterized by failure of sweat development, tooth agenesis and size reduction of teeth.15 tooth agenesis is a consequence of a qualitatively and quantitatively impaired function of genetic networks, which regulate tooth development. impaired function of genetic networks are reflected as reduced signalling or impaired signal regulation, cell proliferation, migration and differentiation. the most critical are the stages of formation of signalling centres that have an organizing role for the future development. the reduction of the “tooth forming potential” may follow from a reduced functional activity of a single gene as in the case of defects in msx1 and pax9.21 the number and type of teeth are strictly controlled during odontogenesis. msx1 and pax9 form a signalling cascade during tooth development. mutations in msx1 and pax9 genes are dominant for tooth agenesis in humans. the gene pax9 was found to be localized in chromosome 14 (14q12-q13). the disruption of dna-binding ability of pax9 that causes hypodontia. nonsense mutation in exon 1 of msx1 in chromosome 4 was found to be heterozygous in all affected family members. nieminen have identified there was gene deletions in msx1 and pax9, missense mutation r196p of msx1 and missense l21p of pax9.21,22 the key role of msx1 and pax9 is to facilitate the bud to cap stage transition. mesenchymal msx1 expression is initially activated by the epithelial bmp4 signal. loss of function defects in msx1 and pax9 in humans cause partial failure of tooth development, tooth agenesis. defects in msx1 associate especially with agenesis of second premolars and third molars, whereas the defects in pax9 affect particularly the permanent molars. the size of the permanent teeth may also be reduced. in one of the families with a defect in msx1, some patients also presented with nail dysplasia and in another family with oral clefts. several other sequence changes in msx1 have also been described in connection with oral clefting. in addition, a micro satellite allele in the intron of msx1 has been associated with both tooth agenesis and oral clefting, and two promoter region snp alleles of pax9 with tooth agenesis.17,23 in the case of msx1 and pax9, tooth agenesis has been related to critical function of the mouse homologues of these genes in the formation of the enamel knot and the subsequent transition from bud to cap stages. the msx1 haploinsufficiency, however, appear to affect only secondary teeth and permanent molars, and it is not obvious how a weakened enamel knot function, which presumably follows from a reduced amount of functional msx1 protein, is linked to impaired secondary tooth development. it is possible that the late developing teeth are more sensitive to impaired enamel knot function. the development of these teeth normally is a long lasting process and happens surrounded by the alveolar bone. it can also be speculated that enamel knots may regulate the program leading to the secondary tooth formation.23 it is concluded that based on the molecular andbased on the molecular and genetic studies of tooth development, tooth agenesis is a consequence of a qualitatively or quantitatively impaired function of genetic networks, which regulate tooth development. reduced amount of functional msx1 or pax9 protein in the tooth forming cells is able to cause severe and selective tooth agenesis. another conclusion, based on the analysis of the phenotypes associated with the known defects in these genes, is that the phenotypes associated with the defects in msx1 and those associated with the defects in pax9 are different. despite the similarities, there are clearcut differences in the frequency of agenesis of specific teeth. references 1. nieminen p. molecular genetics of tooth agenesis. dissertation. finlandia: department of orthodontics institute of dentistry and institute of biotechnology and department of biological and environmental sciences faculty of biosciences university of helsinki; 2007. p. 48–60. 2. londhe sm, viswambaran m, kumar p. multidisciplinary management of oligodontia. mjafi 2008; 64: 67–69. 3. arte s. phenotypic and genotypic features of familial hypodontia. thesis. helsinki: department of oral and maxillofacial disease university of helsinki; 2001. p. 203–87. 4. näsman m, forsberg cm, dahllöf g. long-term dental development in children after treatment for malignant disease. eur j orthod 1997; 19: 151–9. 5. vastardis h. the genetics of human tooth agenesis: new discoveriesastardis h. the genetics of human tooth agenesis: new discoveries for understanding dental anomalies. am j orthod dentofac orthop 2000; 117: 650–6. 6. peck s, peck l, kataja m. mandibular lateral incisor-canine transposition, concomitant dental anomalies, and genetic control. am j orthod dentofac orthop 2000; 117: 650–6. 7. thesleff i, keränen s, jernvall j. enamel knots as signalling centres thesleff i, keränen s, jernvall j. enamel knots as signalling centres linking tooth morphogenesis and odontoblast differentiation. adv dent res 2001; 15: 14–8. 8. moore kl, persaud tvn. embriología clínica. 7th ed. madrid: elsevier;madrid: elsevier; 2004. p. 30–7. 9. thesleff i. genetic basis of tooth development and dental defects.thesleff i. genetic basis of tooth development and dental defects. acta odontol scand 2000; 58: 191–4. 146 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 141-146 10. yüksel s, uçem t. the effect of tooth agenesis on dentofacial structures. eur j orthod 1997; 19: 71–8. 11. peters h, balling r. teeth-where and how to make them. trends genet 1999; 15: 59–65. 12. jiménez e, castellanos a, carmona1 cj, herrera c. variations in the number of human permanent teeth: hypodontia. eur j anat 2005; 9(1): 23–7. 13. lidral ac, reising bc. the role of msx1 in human tooth agenesis..the role of msx1 in human tooth agenesis.. j dent res 2002; 81(4): 274–8. 14. fekonja a. hypodontia in orthodontically treated children. eur j of orthod 2005; 27: 457–60. 15. mikkola ml, thesleff i. ectodysplasin signalling in development. cytokine growth factor rev 2003; 14: 211–24. 16. cobourne mt, hardcastle z, sharpe pt. sonic hedgehog regulates epithelial proliferation and cell survival in the developing tooth germ. j dent res 2001; 80: 1974–9. 17. vieira ar, meira r, modesto a, murray jc. msx1, pax9, andmsx1, pax9, and tgfa contribute to tooth agenesis in humans. j dent res 2004; 83: 723–7. 18. trevor p, pemberton j, gee j, pragna i. gene discovery for dental anomalies: a primer for the dental professional. j am dent assoc 2006; 137: 743–52. 19. pereira tv, salzano fm, mostowska a. natural selection andpereira tv, salzano fm, mostowska a. natural selection and molecular evolution in primate pax9 gene, a major determinant of tooth development. proc natl acad sci usa 2006; 103(15): 5676–81. 20. kist r, watson m, wang x, cairns p, miles c, reid dj, peters h. reduction of pax9 gene dosage in an alleli series of mouse mutant causes hypodontia and oligodontia. hum mol genet 2005; 11: 181–90. 21. lammi, halonenk, pirinen s, thesleff i, arte s, nieminen p. a missense mutation in pax9 in a family with distinct phenotype of oligodontia. eur j hum genet 2003; 11: 866–71. 22. nieminen p, arte s, tanner d, paulin l, alaluusas, thesleff i, pirinen s. identification of a nonsense mutation in the msx1 and pax9 gene in molar oligodontia. eur j hum genet. 2001; 9: 743–6. 23. thesleff i. the genetic basis of tooth development and dental defects. am j med genet 2006; 140: 2530–5. 126 vol. 43. no. 3 september 2010 contact hypersensitivity after tongue piercing ananta herachakri p�, afrini puspita�, feby aryani�, and hendri susanto� 1 dental practitioner 2 department of oral medicine faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: recently tongue piercing has become increasingly popular in the society. several case reports have presented various complications of tongue piercing. however, there is no scientific evidence about contact hypersensitivity to tongue piercing. purpose: the aim of this study was to investigate the contact hypersensitivity after using tongue piercing. methods: nineteen male rattus norvegicus were divided into three groups: group a treated with vaseline on the back and dorsum tongue (control group), group b (i) treated with hgcl2 10% cream on the tongue dorsum, group b (ii) treated with tongue piercing for 10 days and group c with hgcl 2 10% cream on the back, ear lobe, and tongue, then re-exposure with same materials on ear, back and tongue for 24 and 48 hours. before and after 24 and 48 hours applications, ear width was measured with sliding caliper. at the end of treatments, the rats were sacrificed. all tissue specimens were made for hematoylin eosine (h&e) staining examination. the number of mononuclear cells was counted under light microscope data was analyzed with one-way anova followed by lsd (p<0.05). results: the result of this study showed that there were a significant difference of the thickness of ear lobe and the number of mononuclear cells (lymphocyte and monocyte) among all groups. conclusion: it is concluded that tongue piercing induce contact hypersensitivity. key words: contact hypersensitivity, tongue piercing, mononuclear cell abstrak latar belakang: saat ini pemakaian tongue piercing sangat popular di masyarakat. beberapa laporan kasus menunjukkan bahwabeberapa laporan kasus menunjukkan bahwa tongue piercing menimbulkan beberapa komplikasi. namun, belum ada bukti ilmiah mengenai reaksi hipesensitivitas tongue piercing. tujuan: untuk mengetahui reaksi hipersensitivitas setelah menggunakan tongue piercing. metode: sembilan belas tikus jantan rattus novergicus yang dibagi dalam tiga kemlompok yaitu: grup a diberi perlakuan dengan vaselin pada punggung dan dorsum lidah, grup b (i) diberi perlakuan dengan krim hgcl2 10% pada dorsum lidah dan b (ii) perlakuan tongue piercing selama 10 hari. grup c diberi perlakuan dengan hgcl2 pada punggung, daun telinga, dan dorsum lidah, kemudian diberi perlakuan ulang dengan bahan dan tempat yang sama selama 24 dan 48 jam. sebelum dan setelah perlakuan selama 24 dan 48 jam ketebalan telinga diukur dengan sliding caliper. setalah perlakuan tikus didekapitasi kemudian dibuat preparat jaringan untuk pemriksaan hematoxilin & eosin (h & e). perhitungan jumlah sel mononuclear dilakukan menggunakan mikroskop cahaya. hasil: penelitian ini menunjukkan bahwa terdapat perbedaan ketebalan telinga dan jumlah sel mononuklear yang bermakna setelah perlakuan antar kelompok pada hasil analisa dengan menggunakan anova dan lsd (p<0.05). kesimpulan: tongue piercing dapat menginduksi reaksi hipersensitivitas kontak. kata kunci: hypersensitivitas kontak, tongue piercing, mononuclear cell correspondence: hendri susanto, c/o: bagian ilmu penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta identa i sekip utara yogyakarta 55281, indonesia. e-mail: drghendri@ugm.ac.id research report 127herachakri et al.: contact hypersensitivity after tongue piercing introduction body piercing is an art of the human body that has existed since many centuries ago and became a symbol of pride of tribes.1 in recent years, body piercing tends to increase in most communities.2 tongue piercing is one of the oral piercing which is the most increasingly used by teenagers to express his or her identity.3-5 oral piercing is the insertion of metal which has a barbell with varying in size 12-18 mm intraoral and perioral.6 in general, the metal in tongue piercing is made from stainless steel7 and can be also derived from surgical stainless steel,6 silver, gold-plated surgical stainlees steel, and plastic.8 various case reports related to the use of tongue piercing has shown the existence of various complications. allergic reactions have been reported in some cases of oral piercing, especially in nickel-contained metal.9 silver-contained metal can release abrasive material that may cause infection and allergic reactions,8 the most complication of tongue piercing was contact dermatitis.4,10,11 however there have never been studies that prove the contact hypersensitivity to the metal of tongue piercing. hypersensitivity reaction to the tongue piercing may associate with the metallic contained of material in a tongue piercing. metal contained-tongue piercing that may induce allergy is nickel, or alloy containing nickel and cobalt. chromates have also been reported as a metal that causes allergies.7 contact hypersensitivity has been also known as type of slow hypersensitivity, cell mediated immunity (cmi), delayed type hypersensitivity (dth), cell mediated immunity (cmi), delayed type hypersensitivity (dth) or a reaction to the tuberculin which is established more than 24 hours after the body exposed to allergens. contact hypersensitivity is a response of t cells that have been desensitized to the particular antigen. this resulted in sensitized t cells that will release lymphokine which acts as a mediator of delayed type hypersensitivity. the manifestations of this reaction are infiltration of monocytes and lymphocytes, or macrophages and cause tissue swelling at the site of antigen.12 in animal models, manifestation of hypersensitivity can be seen from the swelling in the ear lobe.13 wistar rat (rattus norvegicus) is one of animal model which widely used for research in dentistry and medical science. many studies have used wistar rats (rattus norvegicus) as animal model such as hypersensitivity reactions to mercury (hg),13 the influence of cold cured acrylic resin monomer14 and the contact hypersensitivity of aloe vera’s gel.15 wistar rats have biological system which relatively similar with human body. another consideration was the wistar rat is bigger than mice and more easily to handle.13,15,16 previous study of tongue piercing had been done in sprague dawley,16 and other studies used beagles dog,17 but the study of contact hypersensitivity of tongue piercing has never been reported. this aim of this study is to investigate contact hypersensitivity after the use of tongue piercing. this research may benefit for development of science, particularly in oral medicine, information for clinician, tongue piercing users and the community about the potential harmful effect of the use of tongue piercing on immune system of body. materials and methods the method of this study was similar which is described in previous study with some modifications.13-16 nineteen male rats wistar (rattus norvegicus) were divided into three groups: negative control (a), treatment (tongue piercing) (b), and positive control (hgcl2) (c). there are three phases of the treatment of this study, namely phase of sensitization to tongue piercing and hgcl2, phase of re-exposure on the back and right ear, and determination phase hypersensitivity reaction after 24 hours (group b1 & c1) and 48 hours (group b2 & c2) of re-exposure. group a was treated with vaseline (negative control group). group b was the treatment group (tongue piercing). group c was a positive control group with treated with hgcl2. all treatments carried out in wistar rats for 10 days for sensitization then performed the same treatment on the back and ear lobe after 24 hours (day 11) and 48 hours (day 12). the thickness of the ear was measured before and after re-exposure 24 and 48 hours. six hours later, all rats were decapitated. tissue specimen from ears, back and tongue’s wistar in each group were taken and made a tissue slide for he staining examination to count mononuclear cells (lymphocytes and monocytes). mononuclear cells mean were obtained from seven different views using a light microscope with a magnification of 100x. all procedure of this study has been approved by the committee of ethics of medical research and health faculty of medicine gadjah mada university yogyakarta. the mean difference of ear lobe thickness and the number of cells mononuclear (lymphocytes and monocytes) in all groups were analyzed with one-way anova and least significant difference (lsd). results clinically, contact hypersensitivity can be shown in the difference of the ear lobe thickness after treatment (tongue piercing and hgcl2). the ear lobe thickness increased after tongue piercing for 24 hours (figure 1a) and for 48 hours (figure 1b). the ear lobe increased after hgcl2 treatment for 24 hours (figure 1b). the thickness of ear lobe after treatment showed a significant higher than before treatment (table 1 & 2). the significant difference of the thickness of ear lobe was shown between tongue piercing treatment for 24 hours (group b1), tongue piercing treatment for 48 hours (group b2), and hgcl2 treatment for 24 hours (group c1). after comparing all groups, the result of lsd analysis (table 128 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 126–130 tongue piercing treatment for 24 hours (group b1) and hgcl2 treatment for 48 hours (group c2), between tongue piercing treatment for 48 hours (group b2) and hgcl2 treatment for 24hours (group c1), between tongue piercing treatment for 48 hours and hgcl2 treatment for 48 hours (group c2). contact hypersensitivity also can be shown by the infiltration of mononuclear cells (arrow) in treatment group (b1 & b2) and positive control group (c1 & c2) (figure 2). the infiltration of mononuclear cells (monocytes) was shown in the site of tongue piercing. the picture was an example that the increased number of monocytes (arrow) was shown in the connective tissue as response to the tongue piercing treatment. according to one-way anova, there were a significant difference in the number of mononuclear cells mean between all treatment groups (b), negative control group (a) and positive control group (c) (p<0.05). table 3 showed that the means of monocytes between tongue piercing treatment groups (group b) were higher than hgcl2 treatment groups (group c). the result of anova showed that there was a significant difference (p<0.05) of the number of monocyte between all treatment groups in every part of treatment site. table 4 showed that the means of lymphocytes between tongue piercing treatment groups (group b) were higher than hgcl2 treatment groups (group c). the result of anova showed that there was a significant difference a b c figure �. infiltration of mononuclear cells (black arrows). a) back, b) ear, c) tongue after tongue piercing treatment 24 hours (microscope, magnification 100×, he). table �. comparison of ear lobe thickness before ad after treatment in all groups (mm) group before after mean difference + sdmean + sd mean + sd group a 0.63 + 0.05 0.62 + 0.02 0.01 + 0.02 group b1 0.61 + 0.02 0.75 + 0.05 0.14 + 0.14* group b2 0.61 + 0.02 0.90 + 0.14 0.29 + 0.29* group c1 0.61 + 0.07 0.70 + 0.07 0.09 + 0.10* group c2 0.62 + 0.02 0.61 + 0.02 0.01 + 0.02 * significantly difference (p<0.05) table �. lsd test result between all groups group p a and b2 *0.01 b1 and b2 *0.02 b1 and c2 *0.05 b2 and c1 *0.01 b2 and c2 *0.01 * significantly difference (p<0.05) 2) showed there were significant difference of ear lobe thickness between control group (group a) and tongue piercing treatment for 48 hours (group b2), between tongue piercing treatment for 24 hours (group b1) and tongue piercing treatment for 48 hours (group b2), between figure �. changes in the thickness of the ear before and after treatment. a) 24 hours in group b and c, b) 48 hours in group b and c. e ar lo be th ic kn es s (m m ) time : 24 hours group b group c a e ar lo be th ic kn es s (m m ) time : 48 hours group b group c b 129herachakri et al.: contact hypersensitivity after tongue piercing (p<0.05) of the number of lymphocytes between all treatment groups in every part of treatment site. the results of least significant difference (lsd) showed there were significant differences between both control groups and treatment group (table 3, 4 & 5). table ��. the mean of monocyte in all groups treatment back ear tongue mean + sd mean + sd mean + sd group a 0.01 + 0.010 0.01 + 0.010 0.01 + 0.010 group b1 2.39 + 1.07 2.21 + 0.99 2.39 + 1.07 group b2 2.89 + 0.69 1.36 + 1.28 2.21 + 1.17 group c1 0.96 + 0.79 1.07 + 0.77 1.14 + 0.80 group c2 2.00 + 1.52 1.36 + 1.10 1.36 + 1.06 p 0.01 0.01 0.01 table �. the mean of lymphocyte in all groups treatment back ear tongue mean + sd mean + sd mean + sd group a 0.01 + 0.010 0.01 + 0.010 0.01 + 0.010 group b1 2.43 + 1.10 2.79 + 1.20 2.68 + 1.12 group b2 3.07 + 0.86 1.68 + 1.16 3.29 + 1.41 group c1 1.15 + 2.29 2.68 + 1.09 3.21 + 0.88 group c2 1.25 + 1.40 1.11 + 1.20 1.39 + 1.13 p 0.01 0.01 0.01 table 5. lsd test result between groups p a and b2 *0.01 b1 and b2 *0.02 b1 and c2 *0.05 b2 and c1 *0.01 b2 and c2 *0.01 * p = significantly difference (p<0.05) after comparing all groups, the result of lsd analysis (table 5) showed there were significant difference of between control group (group a) and tongue piercing treatment for 24 hours (group b1), between control group (group a) and tongue piercing treatment for 48 hours (group b2), between control group (group a) and hgcl2 treatment for 24 hours (group c1), between control group (group a) and hgcl2 treatment for 48 hours (group c2), between tongue piercing treatment for 24 hours (group b1) and hgcl2 treatment for 48 hours (group c2), between tongue piercing treatment fro 48 hours (group b2) and hgcl2 treatment for 48 hours (group c2). discussion the results of this study showed changes of thickness of the ear lobe before and after treatment of tongue piercing (group b) and hgcl2 10% (group c) (table 1). this finding supported previous study that the manifestation of delayed type hypersensitivity reaction in experimental animals can be seen through swelling in the ear lobe.13 the swelling of ear lobe is the clinical sign of tissue inflammation in hypersensitivity reaction.18 re-exposure in the same area will result in vasodilatation of blood vessels locally then will cause the excessive blood flow.19 vasodilatation causes coagulation activation, result in the formation of fibrin then accumulate in the site of inflammation. deposit fibrin cause induration or swelling,18 and the swelling causes increased thickness of the ear. sensitization phase can occur 7–10 days after the first contact with allergens. slow reactions usually appear 24–72 hours after re-exposure to allergen.15 in this study, increase thickness of the ear lobe was seen in group b and c (figure 1). sensitization phase begins with the exposure hapten on the body, then bind with protein structural to form a hapten-carrier complex. hapten-carrier complex is then recognized by the langerhans cells which function as antigen presenting cells (apc). these langerhans cells and their maturation then migrate to the lymph nodes, particularly to the lymph node, and presenting antigens to lymphocytes t.18 normally, sensitization phase occur in 10–14 days.19,20 after antigen presented to the cells of cd4+ t helper (th) proliferation followed by clonal expansion to be antigen specific (memory) t cells.21,23 tongue piercing may result in a trauma on the tongue,21 which triggering the inflammatory response as an attempt of the body to maintain homeostasis under the influence of adverse environmental effect.19 there was infiltration of mononuclear cells, monocytes and lymphocytes in the histological he tissue slides of group b and group c. mononuclear cell infiltration shown in group b were treated by the insertion of tongue piercing and the group c was treated with exposure to hgcl2 (figure 1, 3 and 4). the group treated by tongue piercing showed a reaction which is same with the group treated with hgcl2. the results are consistent with previous research which showed infiltration of mononuclear cells (lymphocytes and macrophages) in the tongue piercing in rats, sprague dawley up to 12 weeks,14 and similar with the result of study in beagles dogs.17 in addition, similar results are also shown in this study in tongue piercing for 2 weeks. lymphocytes and granulation tissue predominantly found in the area around the tongue piercing.14,21 exposure to hgcl2 can induce the contacts hypersensitivity that also showed infiltration of mononuclear cells.13 mercury (hg) is an alloy which is one component of amalgam in dentistry and hg in the hgcl2 is reported to be toxic.22 mercury is also a strong allergen and can induce polymorphonuclear cells and macrophages infiltration in rats 13. it was related to the results of previous study which showed infiltration of mononuclear cells in rats tongue after 6 hours of re-exposure with tongue piercing. repeated exposure of allergen directly recognized by t cell effectors and the cells release lymphokines as a signal for mononuclear cells in order to attract the cells to the exposed area to phagocyte the allergens. the initial symptoms of contact hypersensitivity can be seen 4-6 hours 130 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 126–130 after re-exposure, which showed mononuclear cells such as lymphocytes and monocytes out of the blood vessels and move between endothelial cells to the injury site. mononuclear cells would be dominant in the area of injury that seen in the histological tissue slide.18,19 type iv hypersensitivity is a slow reaction, which taking place in 24-48 hours. t lymphocytes provide receptors on the macrophages to bind the antigen. this resulted in sensitized t cells that will release lymphokine which acts as a mediator of delayed type hypersensitivity. manifestations of this reaction are infiltration of monocytes and lymphocytes, or macrophages and cause tissue swelling at the site of antigen. the release of lymphokines by sensitized of t cells will cause the accumulation of large numbers of macrophages and cells epitheloid who will develop giant cells. tissue damage further due to the cytotoxicity of macrophages and perhaps natural killer cells is activated by lymphokines or limphotoxin.12,19 reaction similar suggested caused swelling of the ear lobe and the infiltration of mononuclear cells in the tongue piercing, so the possible mechanism also occurs in hypersensitivity contacts of tongue piercing. the result of anova and lsd indicate that there were significant difference of the number of mononuclear cells in each treatment between groups (p<0.05). this is probably due the increase of duration the contact hypersensitivity reaches the maximum intensity. the manifestation of contact hypersensitivity can be seen microscopically through increased infiltration of mononuclear cells although sometimes not directly proportional to the clinical manifestation macroscopically15,16 and this is accordance with the theory that the exposure of foreign materials in a long time can cause the cellular reaction of body which is dominated by mononuclear cells in the area of the injury.14,19,23 it was concluded that tongue piercing induce contact hypersensitivity in male wistar rats (rattus norvegicus). it is characterized clinically by increasing the ear lobe thickness, histologically by the infiltration of monocyte and lymphocyte. further research needed to determine cytokines in specific immune reaction that indicate the body’s reaction to the tongue piercing. references 1. botchway c, kuc i. tongue piercing and associated tooth fracture. j can dent assoc 1998; 64(11): 803–5. 2. meltzer di. complications of body piercing. am fam physician 2005; 72: 2029–36. 3. scully c. oral piercing in adolescents. cpd dentistry 2001; 2(3): 79–81. 4. abramovits w, stevenson lc. hand eczema in a 22-year old woman with piercings. bumc proceedings 2004; 17(2): 211–3. 5. rosivack rg, kao jy. prolonged bleeding following tongue piercing: a case report and review of complications. pediatr dent 2003; 25: 154–6. 6. farah cs, harmon dm. tongue piercing: case report and review of literature. aus dent j 1998; 43(6): 387–9. 7. gawkrodger dj. metal sensitivities dan orthopaedic implants revisited: the potential for metal allergy with the new metal to metal joint prostheses. bdj 2003; 148: 1-5. 8. peticolas t, tillis tsi, cross-poline gn. oral and perioral piercing: an unique form of self-expression. j contemp dent pract 2000; 1(3): 1–10. 9. chambrone l, chambrone la. gingival recessions caused by lip piercing: a case report. j can dent assoc 2003; 69(8): 505–8. 10. ross sc. complications of body piercing. proceeding of ucla healthcare 2000; 4(4): 21–4. 11. plemons jm. from piercing to periodontics: oral medicine in the new millenium [serial online] 2004. available at: http:// www.db.uth.tmc. edu/cont-ed/. accessed november 5, 2004. 12. roeslan bo. imunologi oral: kelainan di dalam rongga mulut. jakarta:jakarta: balai penerbit fk ui; 2002. p. 87–96. 13. sumiwi yaa, sosroseno w, soesatyo mhne. uji hipersensitivitas kontak dan spesifisitas terhadap merkuri (hg) pada tikus wistar. berkala ilmu kedokteran 1998; 30(1): 1–5. 14. kusumadewi u, tandelilin rtc. pengaruh induksi monomer resin akrilik cold cured terhadap hipersensitivitas kontak pada mukosa bukal tikus wistar. dent j 2003; (edisi khusus): 88–92 15. marsudi ka, tandelilin rtc, haniastuti t. hipersensitivitas kontak pada mukosa bukal tikus wistar setelah induksi gel aloe vera. dent j 2005; edisi khusus: 203–7. 16. aryani f, puspita a, susanto h. reaksi radang pada lidah dengan tongue piercing (kajian in vivo pada lidah tikus jantan sprague dawley). indonesian journal of dentistry 2007; 14(3): 223–9. 17. jornet pl, ortega vv, gascon cy, hidalgo ac, lajarin lp, ballesta cg, banos ma. clinicopathological characteristic of tongue piercing:clinicopathological characteristic of tongue piercing: an experimental study. j oral pathol med 2004; 33: 340–5. 18. abbas ak, lichtman ah. cellular and mollecular immunology. 5th ed. philadelphia: wb saunders company; 2003. p. 201–16. 19. cotran rs, kumar v, robbins sl. basics pathology. 8basics pathology. 8th ed. philadelphia: wb saunders co; 1999. p. 1–50 20. kresno sb. imunologi: diagnosis dan prosedur laboratorium. edisi ke-4. jakarta: balai penerbit fk ui; 2001. p. 136–60. 21. theodossy ta. complication of tongue piercing: a case report and review of literature. bdj 2003; 194(10): 551–2. 22. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby; 2002. p. 110. 23. watanabe h, unger m, tuvel b, wang b, sauder d. contact hypersensitivity: the mechanisms of immune responses and t cell balance. j interf cytok res 2002; 22: 407–12. 97 volume 46 number 2 june 2013 dental student’s perception to aesthetic component of iotn and demand for orthodontic treatment wees kaolinni, thalca hamid and ervina r. winoto department of orthodontic faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: the aims of orthodontic treatment are to achieve the best interdigitation in centric occlusion in relation with good facial aesthetic and to get a pleasant dento-facial looked with good function as well. nowadays, people who are seeking orthodontic treatment to fulfill those need come to orthodontist, mostly for aesthetic reason, for good teeth arrangement that will be more aesthetically favorable. however, not all people with malocclusion seek the orthodontic treatment due to the fact, they do not realize that they have orthodontic problem. on the other hand, some other feel they need orthodontic treatment but they can not afford it. purpose: to determine the knowledge of malocclusion and demand for orthodontic treatment in related to aesthetic component (ac) iotn. methods: the samples were 107 college students of faculty of dentistry airlangga university divided into two as 1st and 7th semester group. both of the groups were given questionnaire about their knowledge of malocclusion and demand of orthodontic treatment. then, they were asked to examine themselves using ac iotn, then researcher and orthodontist examine them. results: there was a significant difference on knowledge of malocclusion between both groups, no significant difference on demand of orthodontic treatment between both groups, and no correlation between demand and ac iotn in both groups. there was a significant difference in ac iotn score examined by respondent, researcher and orthodontist. conclusion: knowledge of malocclusion was not the only factor that determines demand of orthodontic treatment. key words: malocclusion, demand, orthodontic treatment, aesthetic component iotn abstrak latar belakang: tujuan perawatan ortodonti adalah untuk mendapatkan hubungan interdigitasi yang baik berhubungan dengan penampilan estetik wajah untuk mendapatkan fungsi dan penampilan dento-fasial yang baik. saat ini, banyak orang mencari perawatan ortodonti untuk memenuhi kebutuhan fungsi dan penampilan. tujuannya adalah susunan gigi yang rata yang lebih dapat diterima secara estetik. namun, tidak semua orang dengan maloklusi mencari perawatan ortodonti, karena sebagian dari mereka tidak menyadari bahwa mereka memiliki masalah ortodonti, sedangkan sebagian lainnya merasa mereka membutuhkan perawatan ortodonti tetapi tidak bisa membiayainya. tujuan: mengetahui hubungan pengetahuan maloklusi dan demand perawatan ortodonti dengan aesthetic component (ac) iotn. metode: sampel adalah 107 mahasiswa fakultas kedokteran gigi universitas airlangga yang dibagi ke dalam 2 kelompok, semester 1 dan semester 7. kedua kelompok kemudian diberi kuesioner tentang pengetahuan maloklusi dan demand perawatan ortodonti. kemudian responden diminta untuk menilai diri mereka sendiri menggunakan ac iotn, setelah itu responden dinilai peneliti dan ortodontis. hasil: terdapat perbedaan pengetahuan maloklusi yang signifikan antara kedua kelompok, tidak ada perbedaan demand perawatan ortodonti yang signifikan pada kedua kelompok, tidak ada hubungan antara demand dan ac iotn pada kedua kelompok. ada perbedaan yang signifikan pada skor ac iotn yang diperiksa oleh responden, peneliti dan ortodontis. simpulan: pengetahuan maloklusi bukan satu-satunya faktor yang mempengaruhi demand perawatan ortodonti. kata kunci: maloklusi, demand, perawatan ortodonti, aesthetic component iotn correspondence: wees kaolinni, c/o: departemen ortodonsia, fakultas kedokteran gigi unversitas airlangga. jl. mayjen. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: thalcaia@gmail.com research report 98 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 97–100 introduction protrusion and crowded teeth have been a problem for some individuals since long, the efforts to correct the condition has been found since 1000 years bc. in the early of the 21st century, the objective of orthodontic treatment had changed. nowadays, the need to have the good appearance of dental and facial performance has increased rapidly. this trend shown the awareness of the patient and the parents towards the orthodontic treatment since the dentofacial appearance and the psychosocial problem related to the one’s appearance have great effect to a person’s quality of life. not all patients with malocclusion are seeking help for orthodontic treatment. some patients do not even realized that they have problem and some other patients feel that they need treatment but not be able to afford it. the need and demand for orthodontic treatment varies due to the social condition and culture. in recent days there is a great number of people who need to improve appearance as well as the dental esthetic, especially in young adult group. they are seeking treatment to repair their dental appearance which is considered imperfect. they feel the change of appearance could assist them to get a better self-esteem.1 as a consequence, the trend which developed nowadays in the society is to have fixed orthodontic treatment.2 one of malocclusion index could be used to measure the need of orthodontic treatment is the index of orthodontic treatment need (iotn). iotn is malocclusion index designed based on malocclusion effect that disturb the facial esthetic and dental health. iotn has two components, the aesthetic component and dental health component. aesthetic component iotn consists of scale from 10 pictures of colorful anterior dental looked with different level of severities.3 iotn has been widely used recently since its validity, reliabilities and its user friendliness. aesthetic component iotn can be used to measure ones perception towards dental appearance from aesthetical point of view.4 patient’s opinion on their dental appearance (especially from aesthetical point of view) is very important to consider before deciding the orthodontic treatment for the patient.5 the most factors that influences the patient to seek orthodontic treatment is the knowledge and education. the aim of study was to determine the demand of orthodontic treatment and the aesthetic component of the iotn is corelated significantly. materials and methods this study was an analytic observational with the sample of 107 students from the faculty of dentistry universitas airlangga. the criteria of the subject were firstly and most important, they never had the orthodontic treatment. respondents consisted of two groups, the first group was students from the first semester (56 students) and the second group was students from the seventh semester (51 students). the sample were then asked to fill out the questioners to find out about their knowledge on malocclusion and the demand for orthodontic treatment. then they are being asked as well to judge their own dental appearance based on aesthetic component of iotn using a mirror. for direct assessment aesthetic component of iotn consisted of scale from 10 pictures of colorful anterior dental appearance with different level of severities3 (figure 1) were used, then alternatively taking turns, the researchers and orthodontist also judged them regarding their dental appearance using aesthetic component of iotn as a guide. mann-whitney analysis was carried on, to test the difference between the knowledge of malocclusion and the demand for orthodontic treatment on each population. spearman test was conducted to see the correlation between the demand for orthodontic treatment and the score of the aesthetic component of iotn on each population. the friedman test was also used to see the difference between the score of aesthetic component iotn based on judgment from respondents, researchers and orthodontist. results the knowledge score of the students from the first semester were varied ranging from the highest to the lowest. students on the seventh semester, on the other hand, showed high knowledge score (figure 2). using mann-whitney test, it found that there were a significant difference between the knowledge of students from the first and the seventh semester (p=0.001). most of students from the first semester had a moderate demand for orthodontic treatment. the students from the seventh semester had high demand for orthodontic treatment (figure 3). figure 1. aesthetic component iotn.6 99kaolinni, et al.,: dental student’s perception to aesthetic component of iotn there was no significant difference between the demand between students from semester 1 and students from semester 7, based on mann-whitney test to see the difference in demand on both population (p=0.506). spearman test was used to know the difference between correlation demand on orthodontic treatment and aesthetic component iotn. the results shown that there was no significant correlation between demand on orthodontic treatment and aesthetic component iotn on both population. p value acquired on students from semester 1 and 7 are respectively 0.243 and 0.916. this showed that there was no significant difference between demand on orthodontic treatment and aesthetic component iotn. however, a significant score difference on aesthetic component iotn among researchers and orthodontist (p=0.01) due to the fact that there was individual subjectivity in judging their own aesthetical dental aesthetic. discussion many factors influenced the demand on orthodontic treatment instead of knowledge on malocclusion. demand or the willingness to seek orthodontic treatment is one example of health behaviors, which most likely based on knowledge. no difference found between demand in orthodontic treatment between students from semester 1 and 7 could be due to the fact that, nowadays, orthodontic treatment has been widely popular. during the last few years, the demand on orthodontic treatment has increased significantly in accordance with the increase of standard of living in indonesia. the perception of demand on orthodontic treatment is influenced by many factors.6 one of it, that widespread of information about orthodontic treatment through media that can be assessed anytime, so the students either from semester 1 and 7 could retrieve information eventhough they have not yet taken any classes on orthodontics. perception in the society could also become a factor that could influence the demand of treatment. these days orthodontic treatment has been the favorite treatment in the society. this could also influence an individual perception towards the orthodontic treatment. people with malocclusion, who seek orthodontic treatment was not depends on the patient’s characteristic, parents, dentist, orthodontist and the health care system. study conducted previously on different population in many countries showed that the dentofacial appearance was the strongest motivator to seek orthodontic treatment.7 the variation on orthodontic treatment on the sample in this study could be due by many factors that influence attitude and influence the demand on orthodontic treatment as well. this is due to the fact that those individuals were not perfect in judging their own dental aesthetic before their visit to orthodontist. some people with moderate or severe malocclusion feel satisfied with their appearance. on the other hand, others with minor discrepancy or even without discrepancy fell unsatisfied with their dental formation. perception and confidence towards accepted dental appearance also influenced the demand request of orthodontic treatment. other than that, there also many factors influenced the demand of orthodontic treatment, that could be explored. there were many patients who do not really understand on the knowledge, they understand about the information about dental health but maybe would have different perception (in this case, the score of aesthetic component of iotn), which leads to different attitude and perception.8 this attitude difference leads to the difference in behavior to what they knows and how they perceived of aesthetic in dental health. the scoring of aesthetic component of iotn on each individual to themselves could be different and very subjective, and it does not always influence on the demand of orthodontic treatment. on the low score of aesthetic component of iotn, individuals in age group 17-24 years old (young adult) tends to seek orthodontic treatment. this showed the high demand on orthodontic treatment does not depend on the severity of the malocclusion viewed from the aesthetical point of view of the person.9 there was no guarantee for individual with a good knowledge of dental health would have good attitude and figure 2. malocclusion knowledge on students from semester 1 and semester 7. figure 3. students demand or orthodontic treatment in semester 1 and semester 7. h ig h m o d e ra te lo w h ig h m o d e ra te lo w h ig h m o d e ra te lo w h ig h m o d e ra te lo w 100 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 97–100 perception or behavior in dental treatment. knowledge could influence attitude. however, this could not predict the individual’s behavior with high accuracy to asses themselves.10 good attitude towards dental health was the tendency to take action. an individual with a positive attitude, however, in this matter was not always be guaranteed to have a positive behavior. therefore, knowledge on malocclusion was not the only factor that could influence the demand of orthodontic treatment. demand (high or low) on orthodontic treatment also affected by an individual’s environment and the level of education. the perception on aesthetic component of iotn by a respondent is highly affected by each individual’s subjectivity. therefore, the aesthetic component score judging by the student respondent could easily be affected by their own perception. the use of aesthetic component to judge the aesthetic from an individual dental aesthetic performance could lead to subjectivity so that the perception of aesthetic component by respondent, examiner and orthodontist could also be different. the results from an orthodontist is more objective compared to that of examiner and respondent towards themselves. the judgment on dental performance is subjective and related to the demographic background of the scorer. the judgment of aesthetical dental formation were very complex and varied on each individual. the dental aesthetic performance that could be affected by an individual may not be accepted by others.5 in addition, an orthodontist’s perception in aesthetic component was different from examiner and respondents with less experience. this could also lead to discrepancy in results. therefore, the study suggested that the knowledge on malocclusion is not the only factor that influences the demand for orthodontic treatment. many factors affected the demand for orthodontic treatment, such as, perception, confidence, willingness to seek the treatment, motivation, and attitude. references 1. proffit wr. contemporary orthodontics. 4th ed. missouri: mosby elsevier; 2007; p. 3-6, 16-22, 218-25. 2. kruger e, tennant m. accessing government subsidized specialist orthodontic services in western australia. aust dent j 2006; 51(1): 29-32. 3. hagg u, mcgrath c, zhang m. quality of life and orthodontic treatment need related to occlusal indices. the hong kong med diary 2007; 12(10): 8-12. 4. hosseinzadeh nt, nourozi s, kharazi fard mj, noorozi h. the relationship between patient, parent and orthodontic treatment need and demand in 17-year-old students residing in abade/iran. j dent tehran university of medical sciences 2007; 4(3): 107-14. 5. johansson ma, follin me. evaluation of the aesthetic component of the index of orthodontic treatment need by swedish orthodontist. eur j orthod 2005; 27: 160-6. 6. agusni, t. the need and demand for orthodontic treatment in urban and rural schoolchildren in surabaya, east java – indonesia. thesis. sydney: university of sydney; 1998. 7. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publishers (p) ltd; 2007. p. 204-5 8. jin j, sklar ge, oh vms, li sc. factors affecting therapeutic compliance: a review from the patient’s perspective. therapeutic and clinical risk management 2008; 4(1): 269-86. 9. hunt o, hepper p, johnston c, stevenson m, burden d. the aesthetic component of the index of orthodontic treatment need validated against lay opinion. eur j orthod 2002; 24: 53-9. 10. yonan h, widyanti n, priyono b. hubungan antara pengetahuan, persepsi, dan sikap terhadap kesehatan gigi dengan status kesehatan gigi pada siswa tuna netra di panti sosial bina netra (psbn) wyata guna. bandung: sains kesehatan; 2005; 18(2). vol 51 no 3 jul sep 2018_pus.indd guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english. • abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english. • correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research 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bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita �� volume 46 number 1 march 2013 the inhibition of malignant epithelial cells in mucosal injury in the oral cavity of strains by pomegranate fruit extract (punica granatum linn) through bcl-� expression sri hernawati department of oral medicine faculty of dentistry, universitas jember jemberindonesia abstract background: squamous cell carcinoma of oral cavity is a malignant neoplasms derived from epithelia. the malignant neoplasms are cells that have changed their structure and function, and their number becomes increasing abnormally, invasive, and metastatic. carcinoma can be caused by the resistance of malignant cell apoptosis. bcl-2 is a proto-oncogene of bcl family that inhibits the process of cell apoptosis and suppresses bax protein (pro-apoptotic). the management efforts of cancer diseases, however, still have many obstacles. thus, the researcher was triggered to explore more herbal plants, namely pomegranate. pomegranate as a medicinal plant is accessible and cheap. ellagic acid (ea) is a single active compound derived from whole pomegranate fruit extract (pgl), which has anti-cancer activity as in vitro, but ea is low concentration in plasma, low water solubility, and insoluble in intestinal. these facts prompted the researcher to compare between pomegranate extract, which consists of several active compounds, and that, which only consists of ellagic acid. thus, this research is expected to know how some active compounds can work synergistically in the pgl, so the effect can be more potent. purpose: the purpose of this research, therefore, was to compare ea with pgl in reducing the expression of bcl-2. methods: this laboratory experimental research was used 32 male mice (balb/c) in the age of 5 months. they were randomly divided into 4 groups: 2 control groups (k0: which was not exposed with benzopirene and also untreated and k1: which was exposed with benzopirene and also untreated), 2 treatment groups (p1: which was exposed with benzopirene and also treated with ea and p2: which was exposed with benzopirene and also treated with the pgl). next, an examination was conducted by using immunohistochemical techniques. results: the results then showed that the provision of the pgl could decrease the expression of bcl-2 significantly higher than that of ea in the malignant epithelial cells of the oral mucosa of those mice. conclusion: it may be concluded that the provision of the pgl can kill malignant cells in the oral cavity of mice by increasing apoptosis through decreasing bcl-2 expression that was higher than the provision of ea. key words: pomegranate fruit extract, malignant cells, bcl-2, ellagic acid abstrak latar belakang: karsinoma sel skuamosa rongga mulut adalah istilah yang digunakan menyebut neoplasma ganas berasal dari epitel. neoplasma ganas adalah sel yang telah berubah struktur dan fungsi, sehingga mengalami peningkatan jumlah secara abnormal,invasif dan metastasis. terjadinya karsinoma salah satu disebabkan oleh karena hambatan apoptosis terhadap sel ganas. bcl-2 adalah protoonkogen keluarga bcl yang berperan menghambat proses apoptosis sel dan bekerja menekan protein bax (pro apoptosis). berbagai upaya penatalaksanaan penyakit kanker masih banyak menemui kendala,sehingga peneliti menggali tanaman obat yaitu buah delima. buah delima sebagai tanaman obat, mudah didapat dan harganya murah. ellagic acid (ea) senyawa tunggal bahan aktif dari ekstrak buah delima yang memiliki aktivitas sebagai anti kanker secara in-vitro tetapi ea aktivitas dan konsentrasinya dalam plasma rendah, kelarutan dalam air rendah, metabolisme (ea) tidak larut dalam intestinal. fakta ini mendorong peneliti untuk membandingkan dengan whole ekstrak delima (pgl) yang terdiri dari beberapa senyawa bahan aktif, tidak hanya ellagic acid, memungkinkan beberapa senyawa bahan aktif pada pgl bisa bekerja sinergis, sehingga efeknya lebih poten. tujuan: tujuan penelitian ini membandingkan antara ea, dengan whole ekstrak buah delima (pgl) dalam menurunkan ekspresi bcl-2. metode: metode penelitian yang digunakan research report �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 35–38 adalah eksperimental laboratories, 32 ekor mencit (balb/c), jantan, umur 5 bulan dibagi secara random menjadi 4 kelompok, 2 kelompok kontrol (k0: tidak dipapar benzopirene dan tidak diberi perlakuan, k1: dipapar benzopirene dan tidak diberi perlakuan), 2 kelompok perlakuan (p1: dipapar benzopirene dan diberi ea, p2: dipapar benzopirene dan diberi pgl). pemeriksaan dengan menggunakan teknik imunohistokimia. hasil: hasil penelitian menunjukkan bahwa pemberian pgl dapat menurunkan ekspresi bcl-2 lebih tinggi dibandingkan ea pada sel epitel ganas mukosa rongga mulut mencit. kesimpulan: kesimpulan penelitian ini adalah pemberian pgl dapat membunuh sel ganas pada rongga mulut mencit dengan jalan meningkatkan apoptosis melalui penurunan ekspresi bcl-2 lebih tinggi dibandingkan pemberian ea. kata kunci: ekstrak buah delima, sel ganas, bcl-2, ellagic acid correspondence: sri hernawati, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: srihernawati.drg5@yahoo.com introduction cancer or malignant neoplasms is still a major health problem in industrialized countries and also in developing countries. in 2005 in the united states there were approximately 1.372 million people diagnosed with cancer, and 570,280 people died of cancer.1 oral squamous cell carcinoma is cancer ranked sixth in the world. in india there are 75,000–80,000 new cases reported every year. in singapore and other asian countries the number of the same cases is also high.2 several studies in southeast asia, moreover, also show that an area of buccal mucosa is the most common area of squamous cell carcinoma, that is equal to 50–72%. squamous cell carcinoma of the oral cavity usually causes no complaints at an early stage. thus, among 68% patients with squamous cell carcinoma, 48% of them spread to lymph node.3 it is also known that patient survival index continues to decline over the improvement in the diagnosis and treatment of cancer.4 microscopic picture of squamous cell carcinoma showed that the proliferation of cells squamous epithelial cells, got atipia then followed with the changes of rete peg processus, the formation of abnormal keratin, the increasing of basaloid cells, the irregular structure of the cells, and the formation of tumor nest infiltrating into the surrounding tissue.5 the growth of squamous cell carcinoma in the oral cavity was actually influenced by exogenous and endogenous factors, which can make protein function abnormal due to gene mutation. the failure of apoptosis also contributes significantly to the growth and development of squamous cell carcinoma.6 apoptosis can occur physiologically and pathologically. physiological apoptosis is a cell death process in order to maintain the overall integrity of the body and also to maintain homeostasis. meanwhile, pathological apoptosis is to limit cell proliferation required, including malignant cells. pathological apoptosis is an efficient mechanism to eliminate cells that are unnecessary and harmful. on malignant cells, the apoptosis will usually get interference or obstacles.7 one of proteins that plays an important role in the malignant process is bcl-2 (protooncogene). bcl-2 will act as an anti-apoptosis, so the increasing of bcl-2 protein will inhibit bax (pro-apoptotic).8 bcl-2 is a member of the bcl family that has a function as an apoptosis inhibitor. thus, if the number of bcl-2 protein is decreased, then the pro-apoptotic protein (bax) will increase and induce apoptosis.6 however, various management efforts of cancer diseases have still got many obstacles, which cause the lack of success in preventing and treating malignancies. one treatment that has been initiated is the use of phytopharmaca, in which the contents of chemical elements in plants that can potentially be used as drugs are explored. one of the medicinal plants that has been used is pomegranate punica granatum linn. (pgl). the main phytochemical group contained in pgl is polyphenol, which consists of flavonoids (flavonols, flavonols and anthocyanins), hydrolyzable tannins (ellagitannins and gallotannins), and condensed tannins (proanthocyanidins). based on the previous researches, it is also known that pomegranates have therapeutic efficacy, such as anti-bacterial, anti-viral, anti-cancer, and anti-inflammatory.9 similarly, based on other researches, the pgl with standardized ingredients involving 40% ellagic acid (ea) can both inhibit the growth of cancer cells, anti-proliferation, and induce apoptosis and anti-oxidants in vitro.10 therefore, it is believed that the pomegranate extract can increase apoptosis in vitro in cultured human tongue squamous cell carcinoma with a dose of 250 ug/ml.11 thus, the standardized ingredient with 40% ea can indicate that 40% could describe the strength of the pomegranate extract, which is responsible for the pharmacological activity.12 the pgl used in this research contained the active ingredient ea. ellagic acid in the whole pomegranate fruit extract is in the free form as ellagic acid-glycosides or bounded in the form of ellagitannins.13 ellagic acid in vitro has a function as an anti-cancer, but still rarely studied in vivo. actually, the low activity and concentration of ea in plasma is caused by a low solubility in water, in addition, ea can easily transformate and degrade before being absorbed.13 thus, the advantage of the pgl is that it has several active ingredients that are likely to work synergistically, such as polyphenol that can improve the solubility and absorption of ellagic acid, as a result, the pgl has potential anti-cancer effects.13 for the reasons, if the effects of the pgl on strain swiss webster (balb/c) ��hernawati: the inhibition of malignant epithelial cells in mucosal injury can be revealed, then the pgl can be used as an alternative treatment for squamous cell carcinoma of the oral cavity. so, the aim of the study was to compare ea with pgl in reducing the expression of bcl-2. materials and methods this research was considered as an experimental laboratory research. animals used in this research were five month male strains swiss webster (balb/c) with the weight of 30–50 gram. those animals were obtained from the unit of animal testing, universitas gajah mada, yogyakarta. moreover, those animals were divided into 4 groups, which were 2 control groups, k0 (not exposed with benzopirene and also not treated with ea and pgl) and k1 (exposed with benzopirene and also not treated with ea and pgl), and 2 treatment groups, p1 (exposed with benzopirene and also treated with ea) and p2 (exposed with benzopirene and also treated with pgl). for each group, there were eight strains. pgl used in this research was obtained by extracting all parts of pomegranate fruit into powder and then standardized its ingredient by using 40% of ea, produced by late biof xi biotechnology co. ltd. (room 1–1111, high-tech venture park, n0. 69 jinye distric of rood gaoxin xi’an, people republic of china). ea is a white crystalline used as one component of the active ingredient of pgl derived from the same company with pgl. next, those strains were exposed with benzopiren (0.04 mg)/olium olivarum (0.04 ml) orally 3 times a week for 4 weeks on the right buccal mucosa in oral cavity of those strains. at the end of the 9 weeks, the oral mucosal tissues of those strains were biopsied, and then sacrificed. those which were considered to have squamous cell carcinoma were those (balb/c) which had suffered from malignancy in their epithelia due to benzopirene exposure with the microscopic picture showing cell proliferation-atypical squamous epithelium followed with the changes of rete peg processus shape, the formation of abnormal keratin, and the irregular structure of the cells. afterwards, the pgl, ea was given orally every day for 4 weeks. the dose of the pgl, ea used was 75 mg/kg/bw/day dissolved in 0.3% cmc-na. then, the examination for the expression of bcl-2 was conducted by using immunohistochemical. the procedure of immunohistochemical conducted on the expression of bcl-2 involved (1) reagent preparation: the fixation stage of the working solution, dab, (2) staining, (3) washing, (4) labeling, and (5) reading. materials used for immunohistochemical examination in this research were 3% h202, 0.025% trypsin, pbs, aquadestilata, substrate buffer, xylol, absolute ethanol, methanol, water, anti-bcl-2 (mouse antirat) antibodies, enzymes, glass poly l-lysine object, formalin buffer, labeled antiglobulin, secondary antibodies, and streptavidin. the procedures of immunohistochemical examination involved preparation of reagents, staining, washing, labeling, and reading. the results of immunohistochemical examination showed that cells that did not express bcl-2 protein did not have brown color (transparent). afterwards, the calculating of the cells was conducted on 10 fields of view with a microscope using 400x magnification, presented their own means. finally, analysis of the research data was tested by using normality test, homogeneity test, anova test, and lsd test. analysis result between treatment groups was conducted by using lsd test. results the results of preparation examination by using immunohistochemical techniques on the expression of bcl-2 can be seen table 1. the results of preparation examination with immunohistochemical techniques showed that the administration of standardized pgl (p2/benzopirene + pgl) could decrease the expression of bcl-2 in malignant squamous epithelial cells of those strains. the decreasing of the expression of bcl-2 in group p2 (benzopirene + pgl) was about 0.016 ± 0.040 higher than that in group p1 (benzopirene + ea), which was about 0.083 ± 0.075). discussion the results of this research indicate that the standardized pgl could decrease the expression of bcl–2, which was higher than ea. bcl-2 gene, an antiapoptotic, that encodes protein is considered as protooncogene group. it is because bcl-2 can suppress the function of bax or proapoptotic, and can also inhibit c–myc, which function is to induce apoptosis. thus, the increasing of the expression of bcl2 has a very important role in resisting the apoptosis of malignant cells.14 the results showed that the decreasing of the expression of bcl-2 by pgl was stronger than that by the standardized ea. it indicates that there were additional or synergistic effects of the other active ingredients in the pomegranate extract.15 one of the possible mechanisms of malignant cells to increase the expression of bcl-2 is by a process in which bcl-2 forms a pore in the membrane where it steps on, table 1. the mean and standard deviation of cells expressing bcl-2 on those strains group (n = 6) the number of cells expressing bcl-2 ( ± sd) control (–)/k0 0.001 ± 0.001a control (+)/k1 (benzopirene + cmc) 0.367 ± 0.103c p1/(benzopirene + ea) 0.083 ± 0.075b p2/(benzopirene + pgl) 0.016 ± 0.040ab *) different superscripts in the same column were significantly different (p< 0.05) �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 35–38 and then interacts with various types of other intracellular proteins that are directly or indirectly involved in apoptotic process. this interaction shows one of the roles of bcl-2 protein in providing a place for others, so the cellular activity of those proteins stop (eg. bax, its activities will be stopped). bcl-2, furthermore, also control checkpoint of caspase activation pathway, so the possibility of bcl2 to control apoptosis pathway is either dependent or independent of caspase (intrinsic pathway and extrinsic pathway).6 bh3 protein is a protein that has a function to receive stimulus from outside the cell like a drug. the stimulus then makes bh3 proteins active and work directly to free bond between bax and bcl-2 proteins, and also decreases the expression of bcl-2.17 if the expression of bcl-2 decreases, bax protein then has a greater chance of binding to bh3, which is the initiation of apoptosis. the decreasing of the expression of bcl-2 then can cause the increasing of bax activity (proapoptotic of malignant epithelial cells). next, bax has a role in opening pt-pore so that cytochrome-c can be out of mitochondria. afterwards, cytochrome-c activates apaf 1, and then apaf-1 activates caspase cascade, so it causes cell death (apoptosis).6 biovaibilitas of the whole pomegranate fruit extract was better than that of the single compound one. this illustrates the multifactorial and synergistic effects of various compounds in the pgl.16 the presence of polyphenols in the pgl can actually improve the solubility and absorption of ea in the digestive tract. besides, polyphenols contained in the whole pomegranate fruit extract also has an ability to inhibit the metabolism of ea by intestinal microflora through antibacterial activity possessed by the pgl so that the pgl has the ability to decrease the expression of bcl -2 stronger than ea.15 polyphenols found in the pgl has a function as an antibacterial so that ea in the intestinal tract is not metabolized by intestinal microflora. ellagic acid, as a result, is not subject to degradation and transformation before being absorbed. thus, this condition would provide a stronger effect in reducing the expression of bcl-2. low activity and concentration of ea in the plasma was due to its low solubility in water. besides that, it is allegedly caused by the fact that ea was susceptible to transformation and degradation before absorbed.13 finally, the results in group k0 (not exposed with benzopirene and also not treated with ea and pgl) showed that the expression of bcl-2 (0.001) was not expressed. this was due to a physiological system in an individual cell growth that is regulated by a balance system between apoptosis and proliferation. the balance between bcl-2 and bax was occured.8 based on the results, it may be concluded that the whole pomegranate fruit extract can increase apoptosis of malignant epithelial cells through a decreased expression of bcl-2 and it was higher than the provision of ea. references 1. widyasari a, retnoningsih d, lassie n. ekstrak etanol biji mahkota dewa (phaleria macrocarpa (scheff) boerl) meningkatkan ekspresi caspase-3 aktif pada cell line ca colon widr. jurnal farmakologi 6(2). 2. epstein w. oral cancer. texbook of oral medicine. burket eleventh edition. bc decker inc hamilton press; 2008. p. 153–67. 3. wahyuni f. karsinoma sel skuamosa yang didahului inflamasi kronis non-spesifik. medan: departemen penyakit mulut, universitas sumatera; 2010. p. 3–9. 4. mehrotra r, yadav s. oral squamous cell carcinoma, etiology phatogenesis and prognostic of genomic alteration. indian j cancer. 2006; 43(2): 60–6. 5. safriadi m. patologi mulut tumor neoplastik dan non neoplastik rongga mulut. jogyakarta: cv andi; 2008. p. 73–83. 6. kresno sb. texbook ilmu dasar onkologi. edisi kedua. jakarta: badan penerbit fakultas kedokteran universitas indonesi; 2011. p. 66–129. 7. salido, rosado ja. apoptosis; involvement of oxidative stress and intracellular ca 2+ homeostasis. spain: dept of physiology. universty of extremadure; 2009. p. 229–35. 8. sudiana ik. patobiologi molekuler kanker. jakarta: penerbit salemba medika; 2008. p. 27–90. 9. lansky ep, newman ra. punica granatum (pomegranate) and its potential for preventif and treatment of inflammation and cancer. j ethnopharmacol 2007: 109(2): 177–206. 10. jurenka j. therapeutic applicationof pomegranate (punica granatum l). a review altern med rev 2008; 13(2): 128–44. 11. kholifah m. pengaruh konsentrasi ekstrak etanol buah delima (punica granatum linn) terhadap peningkatan apoptosis sel kanker lidah manusia sp-c1 invitro. biomedika 2011; 2(2): 72–80. 12. saifudin a, rahayu v, teruna hy. texbook standardisasi bahan obat alam. edisi pertama. graha ilmu. 2011. p. 1–26. 13. lumangga f. apoptosis. dissertation. medan: pascasarjana universitas sumatera utara; 2008. p. 1–7. 14. seeram np, schulman rn, heber d. pomegranate ancient roots to modern medicine. 1st ed. new york: taylor and francis group; 2006. p. 2–99. 15. seeram np, adam ls, henning sm, niu y, zhang y, nair mg, heber d. invitro antiproliferative, apoptosis and antioxidant activitics of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenol as found in pomegranate juice. j nutr biochem 2005; 16(6): 360–7. 16. marzo l, naval j. bcl-2 family members as molecular targets in cancer therapy. biochem pharmacol 2008; 76(8): 939–46. 7676 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 76–80 original article introduction cleft lip and palate is one of the most common congenital anomalies in newborn babies, with a worldwide prevalence range from 3.4 to 22.9 per 10,000 births.1 according to the indonesia basic health research report (riskesdas), there was a nationally increasing incidence from 2013 to 2018 (0.08 to 0.12%).2,3 cleft occurs as a result of prominent tissues not merging correctly during embryonic lip-palate formation. most people with clefts will often have speech difficulties and hyper-nasal speech, malocclusion, mouth breathing, abnormal tongue and lip postures, and ear problems like otitis media. this condition, especially cleft palate, affects a baby’s nutritional intake and can consequently cause malnutrition.4 cleft can be syndromic or non-syndromic. cleft is caused by multiple factors, genetic and environmental. according to the global burden of disease, the worldwide estimated prevalence of non-chromosomal orofacial cleft is 1.25 per 1000 births and 1.36 per 1000 births in southeast asia.1 environmental factors include maternal nutritional deficiencies, alcoholic beverages, cigarette smoke inhalation, medicine like antiepileptic and antiretroviral, radiation, and toxic chemicals such as pesticides.5,6 nutritional deficiency such as micronutrient deficiency, especially folate/folic acid, has been linked to a higher likelihood of developing cleft in some studies. folate or folic acid is required for dna synthesis during embryonic development. folate or folic acid act as a one-carbon donor in the form of tetrahydrofolate during deoxythymidine monophosphate (dtmp) nucleotide, which is required for dna synthesis. the increased cell proliferation during pregnancy requires more dna, and the need for folate also increases.7,8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p76–80 association of maternal folic acid supplementation and incidence of non-syndromic cleft lip and palate hendry rusdy1, isnandar1, indra basar siregar1, rizkiani cahya putri sinaga2 1department of oral and maxillofacial surgery, faculty of dentistry, universitas sumatera utara, medan, indonesia 2undergraduate student, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: cleft lip and palate as a frequent congenital defect is caused by genetic and environmental factors. micronutrient folic acid as an environmental factor has shown a role as a cleft lip and palate protective factor in several previous studies. purpose: the purpose of this study was to determine the distribution of adequacy of folic acid supplementation and the association between folic acid supplementation during the first trimester of pregnancy with the incidence of cleft lip and palate at mitra sejati hospital, medan, north sumatra, indonesia. methods: this study used analytical research with a case-control design and questionnaire. a chi-square test was used to observe the association between the folic acid supplement intake during the first trimester with the incidence of cleft. the p-value ≤ 0.05 was deemed to be significant. results: the study found that 47 mothers (51.09%) had insufficient, while 45 mothers (48.91%) had adequate folic acid supplementation. this study did not show a significant association between supplement use and all cleft incidence (p>0.05), but a significant result was found between inadequate supplementation (<400µ/day) and cleft lip with or without cleft palate occurrence (p=0.043; or 2.4[1.022-5.625]). conclusion: the present study showed that most pregnant women did not have sufficient folic acid supplement in the first trimester. furthermore, inadequate maternal folic acid supplementation (<400µ/day) during the first trimester of pregnancy increased the tendency for cleft lip and cleft palate (with or without cleft lip) to occur significantly. keywords: cleft lip and palate; folic acid; 400mcg; first trimester; pregnancy correspondence: hendry rusdy, department of oral and maxillofacial surgery, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: hendry_rusdy@yahoo.co.id mailto:hendry_rusdy@yahoo.co.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p76-80 77 rusdy et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 76–80 several studies have shown an association between folic acid and the occurrence of cleft lip and palate, but other studies have shown no significant association in certain types of cleft.1 a study conducted by kelly et al.9 and kunjana10 showed a significant association betweefolic acid consumption during the first trimester and cleft, while song li et al.11 showed that taking folic acid was consistently associated with a lower risk of giving birth to babies with cleft lip and palate, whereas neogi et al.12 showed no significant results. subsequently, most of the previous studies that showed significant results still used mothers of patients with a family history of clefts, which is a factor that is difficult to prevent.9–13 the results of this study motivated the researchers to determine the association between the two events (variables), especially among patients with no family history of cleft lip and palate. folate in food varies widely and tends to be unstable and easily damaged by heat during the cooking process.14 folic acid supplements, which have a more stable form than folate, have been recommended for pregnant women to get enough folate during foetal development. since the facial development process starts from the fourth week, the role of folate in normal lip and palate formation is most important in the first trimester of pregnancy.7,15,16 the who recommends that all pregnant women take a folic acid supplement of 400µg daily.17 in indonesia, the government has determined that every pregnant woman is to be given a minimum of 90 tablets of 400µg folic acid supplements.18 according to the indonesian basic health research report,3 around 87.6% of pregnant women have received folic acid supplements in indonesia, and only 70.9% in north sumatra. it is necessary to evaluate folic acid supplementation in this area, especially in hospitals that handle cleft lip and palate cases, such as the mitra sejati hospital in medan, north sumatra. mitra sejati hospital sees many patients with cleft lip and palate, handling approximately 144 cases per year. we wanted to see a comparison of the sufficient folic acid supplementation in biological mothers of children with cleft lip and palate with mothers of children without the condition, to see if the two groups differed significantly. data on the distribution of adequacy of maternal folic acid supplementation was then needed to assess the association between the two variables. therefore, this case-control analysis is needed to assess the association of sufficient folic acid with the incidence of cleft lip and palate in north sumatra, especially in mitra sejati hospital, medan. materials and methods this was a retrospective case-control study conducted in a hospital. this study took place between july and august 2021 at mitra sejati hospital, medan, north sumatra, indonesia, as one of the referral hospitals for cleft lip and palate care and was part of smile train, a nongovernmental organization that provides free treatment for orofacial clefts. this study was approved by mitra sejati hospital (no. 993/un5.2.1.6/ssa/2021) and the research ethics commission of universitas sumatera utara (no. 685/kep/ usu/2021). according to the standard of selection (purposive sampling), 92 patients (46 cases) were enrolled in the study. cases were paediatric patients presenting cleft lip only (cl), cleft lip and palate (clp), and cleft palate only (cpo), not associated with any other birth defects or syndromes (non-syndromic cleft lip/palate patients). the exclusion criteria included children whose biological mother was dead, who had been adopted, who had a family history of cleft, and children who were not the first child born with a cleft. controls were hospitalised paediatric patients without a congenital disorder or systemic disease. all patients were paediatric patients aged 18 years and under (according to the definition of a child). mothers of paediatric patients were debriefed by the same researcher. the questionnaire was used to investigate information on demographic characteristics, such as the first-trimester maternal antenatal visits, the first three months and preconception period (at least one month before pregnancy) maternal folic acid supplement use, folic acid supplement dosage daily consumption, and cleft incidence among first-and second-degree family relatives. they were asked to give more details about the folic acid supplement brand, dosage, duration and when they used the supplement. adequacy of folic acid supplementation was divided into sufficient and insufficient. women who consumed at least 400µ/day during the first trimester were added to the sufficient supplementation group, and those who had never and those who were not consistent in taking supplements were placed in the insufficient group. if the mother during the first trimester consumed at least 400µ of folic acid most days day but ever missed it, then the mother was still categorised as a subject with insufficient supplementation. all mothers of the patients in the study agreed to participate. only the data from one pregnancy was provided by each woman for this study. this study was conducted using secondary data in the form of medical record status at mitra sejati hospital between the years 2017 to 2021. the maternal ages were divided into five group respectively: <19; 20–24; 25–29; 30–34; and ≥35 years of age. folic acid supplementation was divided into none/ any use during the first trimester, adequate folic acid consumption (≥400µg/day) during the first trimester, none/ any pre-pregnancy use (preconception). an ibm spss statistics for windows version, 20.0 (armonk, new york, usa) software application was used to analyse the data, and the descriptive statistics of epidemiology were presented by frequency and percentage in the table. the association of two variables was analysed using the chi-square test. the analyses were done as odds ratio (ors) with 95% confidence intervals (ci), and a p value of <0.05 was deemed significant. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p76–80 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p76-80 78rusdy et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 76–80 results after collecting >150 medical records as the population, several of these were excluded due to exclusion criteria and maternal unavailability as study subjects. then, 92 subjects were obtained that matched the inclusion criteria. the study subjects, as the parents of cases and control patients, gave informed consent; hence, they filled out the questionnaire honestly. the study subjects filled out the questionnaire, assisted by the researcher’s explanation beforehand. in conformity with the requirements of selection, 92 patients (46 cases and 46 controls) opted to participate in this study. table 1 represents the frequency of cleft-control status based on gender. cleft lip with cleft palate showed the highest number of cases (73.91%), followed by cleft lip only (19.57%), and cleft palate only (6.52%) (table 1). male patients were the more dominant part of sample (55.43%), compared to girls (44.57%). there were 24 boys and 19 girls in cleft and palate cases. there is no significant difference in the incidence of all cleft between boys and girls, and in this study the number of cases in boys was only slightly higher than in girls (table 2 and figure 1). however, cleft palate only was not found in boys in this study (figure 1). cleft lip only (cl) were figure 1. number of cleft cases by gender. table 1. distribution of case-controls status by gender male n (%) female n (%) total (%) cases 24 (52.17) 22 (47.83) 46 cl 5 (10.87) 4 (8.7) 9 (19.57) cl+p 19 (41.3) 15 (32.61) 34 (73.91) cpo 3(6.52) 3 (6.52) controls 27 (58.7) 19 (41.3) 46 total 51 (55.43) 41 (44.57) 92 notes: cl/p: cleft lip with or without cleft palate; cpo: cleft palate only table 2. chi-square test results of gender and occurrence of cleft gender cl/p all cleft controlsn or 95% ci p n or 95% ci p n (%) male 24 0.889 0.383-2.060 0.784a 24 0.768 0.337-1.750 0.529a 27 (58.7)female 19 22 19 (41.3) notes: a statistical evaluation by the pearson’s chi-square; p-value <0.05 statistically significant table 3. maternal characteristics with non-syndromic cleft lip/palate maternal characteristic cl/p cpo all cleft n (%) control n (%) total n (%) age at birth ≤ 19 3 0 3 (6.52) 2 (4.35) 5 (5.43) 20-24 12 1 13 (28.26) 14 (30.43) 27 (29.35) 25-29 9 1 10 (21.74) 18 (39.13) 28 (30.43) 30-34 10 0 10 (21.74) 8 (17.4) 18 (19.57) ≥35 9 1 10 (21.74) 4 (8.7) 14 (15.22) first trimester antenatal care none 7 1 8 (17.4) 7 (15.22) 15 (16.3) any visit 36 2 38 (82.6) 39 (84.78) 77 (83.7) first trimester folic acid use none 13 1 14 (30.43) 7 (15.22) 21 (22.83) any use 30 2 32 (69.57) 39 (84.78) 71 (77.17) periconception daily use <400µg/day 27 1 28 (60.87) 19 (41.3) 47 (51.09) ≥400µg/day 16 2 18 (39.13) 27 (58.7) 45 (48.91) preconception (before pregnant) <400µg/day 41 3 44 (95.65) 45 (97.83) 89 (96.74) ≥400µg/day 2 0 2 (4.35) 1 (2.17) 3 (3.26) recorded for 20.93% of cl/p (cleft lip with or without cleft palate) cases. the association of gender with the occurrence of cleft is depicted in table 2. the distribution of cases and control status by maternal characteristics can be seen in table 3. the average maternal age in the case group was 28.46, and 26.78 years in the control group. a maternal age between <19 years and ≥35 years at birth was more common in the case group (28.26%) than in the control group (13.04%). there was dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p76–80 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p76-80 79 rusdy et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 76–80 no significant difference in the number of mothers who had visited antenatal care in the case group (82.6%) and the control group (84.78%). it was found that 77.17% of subjects had obtained folic acid supplements during the first trimester, while another 22.83% of subjects had never done so. subjects who had consumed folic acid supplements during this period were more common in the control group (84.78%) than in the cases group (69.57%). only 48.91% of subjects had sufficient daily doses of folic acid. subjects who had ≥400µg/day were more common in the control group (58.7%) than in the cl/p group (37.21%) and the all cleft group (39.13%). only 3.26% of subjects who took adequate folic acid supplements before pregnancy, and none of the subjects from the cleft palate only group had taken folic acid supplements before pregnancy. table 4 shows that the association between the history of taking folic acid supplements during the first trimester and the incidence of all cleft and cl/p was not significant in this study. the association of the history of maternal folic acid use during the first trimester with the incidence of all cleft was assessed, but no statistically significant results were found. however, a significant association was found (p=0.043) between the adequate intake of folic acid supplements (≤400µg/day) in the first three months and the incidence of cl/p with x2 = 4.108 which showed a significant association (>3.841) and contingency coefficient = 0.21. discussion this study used secondary data from mitra sejati hospital medical records to reduce face-to-face interactions during the covid-19 pandemic and to facilitate the identification of samples that could meet the required criteria. subjects and patients in this study came from medan and surrounding areas in the north sumatra region. no study has been conducted to examine the relationship between adequate folic acid intake during the first trimester and cleft without family history in north sumatra, indonesia. the indonesian government has required folic acid supplements for pregnant women to be at least 90 tablets.18 the who recommendations state that both pregnant women and women who are planning a pregnancy should use a folic acid supplement of 400µg/day from at least approximately four weeks before conception until 12 weeks after conception.17 folic acid supplements in the form of blood-boosting tablets (tablet tambah darah) can be obtained freely at the nearest community health hub (puskesmas) and (posyandu). usually, folic acid supplements are given by health workers during antenatal care visits (anc).18,19 this study showed that 16.3% of the subjects never visited antenatal care during the first trimester of their pregnancy. a further 22.83% of subjects never took supplements during the first trimester. kelly et al.9 have shown that mothers with a low income; mothers in lower occupational class; multigravida mothers aged less than 18 years, and mothers with very low education were less likely to take supplements. there was a difference in the number of subjects who had ever taken supplements (77.17%) with subjects who consumed at least 400µg/day (48.91%). this shows that 36.62% of the subjects who had taken supplements during the first trimester were not disciplined enough to take them every day. these mothers’ indiscipline was influenced by a low motivation to take supplements for a long time and the presence of nausea/vomiting, which is common in early pregnancy and makes it difficult for mothers to take supplements. the human chorionic gonadotropin (hcg) hormone produced by the placenta stimulates the production of progesterone and oestrogen, which triggers the production of unused hydrochloric acid and a slowdown in gastrointestinal emptying.20 the results show that the highest incidence of cleft is cleft lip with cleft palate (73.91%), followed by cleft lip only (19.57%) and cleft palate only (6.52%). these results are supported by other studies which still show that cl/p are the most common, whereas cleft lip only and cleft palate only differ from place to place.1,4,11,13 table 4 shows the association of adequate folic acid supplement intake with the occurrence of non-syndromic cleft lip/palate (p=0.043) and an odd ratio (or= 2.4 [95%ci 1.022-5.625]) which means that mothers who do not take or consume folic acid supplements less than 400µg/day have 2.4 times greater tendency to have a child with cleft lip with or without cleft palate than those with adequate supplementation during the first trimester. then it became less significant when related to all incidences of clefts in which cleft palate only cases were included (p=0.061). this is probably because the small number of cpo cases does not represent the cpo group and is difficult to test statistically separately. it may also be influenced by the adequacy of other vitamins not measured in this study, such as b6 and table 4. chi-square test result of folic acid supplement consumption and occurrence of cleft cl/p all cleft controls n or 95% ci p n or 95% ci p n (%) folic acid use none 13 2.414 0.858-6.795 0.149b 14 2.438 0.878-6.764 0.082a 7 (15.22)any use 30 32 39 (84.78) ≥400µg/day no 27 2.398 1.022-5.625 0.043a* 28 2.211 0.960-5.088 0.061a 19 (51.09)yes 16 18 27 (48.91) notes: a statistical evaluation by the pearson’s chi-square; b statistical evaluation by; * p-value ≤ 0.05 statistically significant are displayed in bold dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p76–80 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p76-80 80rusdy et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 76–80 b12, which are required during folic acid metabolism and any folate antagonist drugs such as phenytoin.7,15,21 there was no association between gender and the occurrence of non-syndromic cleft lip and/or cleft palate in this study, although other studies have suggested that boys tend to have cleft lip and cleft palate (with or without cleft lip) .1,4,6,11,13 however, the results in this study are supported by eshete et al.,22 golalipur et al.,23 and taghavi et al.24 in this study, the association of folic acid supplement intake with cleft palate only could not be assessed because the number of cases was small, making it difficult to test statistically. the association of supplement intake before pregnancy could not be tested because the number of mothers who had taken folic acid supplements before pregnancy was very small. this study also shows that mothers who gave birth at an older age showed an increased risk of giving birth to children with a cleft lip and palate. the study’s limitations were that it did not look at cases of stillborn babies; and we did not gather details on maternal smoking history, other maternal vitamin supplement consumption, drug use, alcohol consumption, consumption of foods high in folate or maternal malabsorption disease. moreover, smoking and drinking habits during pregnancy were uncommon among indonesian women. in conclusion, this study showed a significant association between the consumption of folic acid supplements of at least 400µg/day during the first trimester with the incidence of non-syndromic cleft lip with or without cleft palate in cases with no family history. the odd ratio was 2.4, which means that mothers who did not consume supplements with folic acid of at least 400µg/day during the first trimester had a 2.4 tendency times to have children with cleft lip only, or cleft lip and cleft palate. and in this study, gender did not show an association with the incidence of non-syndromic cleft lip and palate. acknowledgements the authors wanted to express profound thanks to director mitra sejati hospital from medan, who granted the permission to conduct this research; all the staff members of the paediatric patient ward; and anita, one of the smile train social workers in mitra sejati hospital. references 1. mossey pa, modell b. epidemiology of oral clefts 2012: an international perspective. in: cobourne mt, editor. cleft lip and palate: epidemiology, aetiology and treatment. front oral biol. basel: karger; 2012. p. 1–18. 2. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2013. jakarta: kementerian kesehatan republik indonesia; 2013. p. 230. 3. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 197, 207. 4. hupp j, tucker m, ellis e. contemporary oral and maxillofacial surgery. 7th ed. philadelphia: elsevier; 2018. p. 608–616. 5. martinelli m, palmieri a, carinci f, scapoli l. non-syndromic cleft palate: an overview on human genetic and environmental risk factors. front cell dev biol. 2020; 8: 592271. 6. spinder n, bergman jeh, boezen hm, vermeulen rch, kromhout h, de walle hek. maternal occupational exposure and oral clefts in offspring. environ health. 2017; 16(1): 83. 7. lammi-keefe cj, couch sc, kirwan jp. handbook of nutrition and pregnancy. 2nd ed. medicine & science in sports & exercise. switzerland: springer nature, hummana press; 2018. p. 139. 8. timotius kh, kurniadi i, rahayu i. metabolisme purin & pirimidin: gangguna & dampaknya bagi kesehatan. risanto e, editor. yogyakarta: penerbit andi; 2019. p. 59,68. 9. kelly d, o’dowd t, reulbach u. use of folic acid supplements and risk of cleft lip and palate in infants: a population-based cohort study. br j gen pract. 2012; 62(600): 466–72. 10. kunjana t, zuliyanto a. studi komparatif kejadian celah orofasial menurut tingkat konsumsi suplemen asam folat. sainteks. 2017; 14(2): 159–68. 11. li s, chao a, li z, moore ca, liu y, zhu j, erickson jd, hao l, berry rj. folic acid use and nonsyndromic orofacial clefts in china: a prospective cohort study. epidemiology. 2012; 23(3): 423–32. 12. neogi sb, singh s, pallepogula dr, pant h, kolli sr, bharti p, datta v, gosla sr, bonanthaya k, ness a, kinra s, doyle p, gudlavalleti vsm. risk factors for orofacial clefts in india: a case–control study. birth defects res. 2017; 109(16): 1284–91. 13. lin y, shu s, tang s. a case-control study of environmental exposures for nonsyndromic cleft of the lip and/or palate in eastern guangdong, china. int j pediatr otorhinolaryngol. 2014; 78(3): 545–51. 14. nix s. williams’ basic nutrition and diet therapy. 15th ed. st. louis, missouri: elsevier mosby; 2017. p. 99–101, 150–1. 15. s c a g l i o n e f, p a n z a vo l t a g . f o l a t e , f o l i c a c i d a n d 5 methyltetrahydrofolate are not the same thing. xenobiotica. 2014; 44(5): 480–8. 16. berkowitz s. cleft lip and palate: diagnosis and management. 3rd ed. berlin heidelberg: springer-verlag; 2013. p. 17, 299, 788, 803. 17. world health organization. guideline: daily iron and folic acid supplementation in pregnant women. switzerland: world health organization; 2012. p. 32. 18. kementerian kesehatan republik indonesia. peraturan menteri kesehatan republik indonesia nomor 97 tahun 2014 tentang pelayanan kesehatan masa sebelum hamil, masa hamil, persalinan, dan masa sesuda h mela hirkan, penyelengga raan pelayanan kontrasepsi, serta pelayanan kesehatan seksual. jakarta; 2014 p. 33, 37, 40, 43. 19. achadi e, latief d, briawan d, dillon dhs, muslimatun s, marudut, probhoyekti d, santika o, suroto, usman y. pedoman penatalaksanaan pemberian tablet tambah darah. adil m, marlina l, rossa d, samkani h, kusumaningtias i, editors. jakar ta: kementerian kesehatan republik indonesia; 2021. p. 13,15. 20. bustos m, venkataramanan r, caritis s. nausea and vomiting of pregnancy what’s new? auton neurosci. 2017; 202: 62–72. 21. hernández-díaz s, smith cr, shen a, mittendorf r, hauser wa, yerby m, holmes lb, north american aed pregnancy registry, north american aed pregnancy registry. comparative safety of antiepileptic drugs during pregnancy. neurology. 2012; 78(21): 1692–9. 22. eshete m, butali a, abate f, hailu t, hailu a, degu s, demissie y, gravem pe, derbew m, mossey p, bush t, deressa w. the role of environmental factors in the etiology of nonsyndromic orofacial clefts. j craniofac surg. 2020; 31(1): 113–6. 23. golalipour mj, kaviany n, qorbani m, mobasheri e. maternal risk factors for oral clefts: a case-control study. iran j otorhinolaryngol. 2012; 24(69): 187–92. 24. taghavi n, mollaian m, alizadeh p, moshref m, modabernia s, akbarzadeh ar. orofacial clefts and risk factors in tehran, iran: a case control study. iran red crescent med j. 2012; 14(1): 25–30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p76–80 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p76-80 196 vol. 44. no. 4 december 2011 research report the difference of acrylic resin residual monomer levels with various polymerization method sherman salim department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: after polymerization process, heat cured acrylic resin denture base actually still contains residual monomers that can become potential irritants later in oral cavity. polymerization process is essential to obtain acrylic resin which can meet the requirements of the biocompatible and good physical properties. to meet the requirements, there are several methods of polymerization process used. purpose: the purpose of this study was to determine the differences of the residual monomer levels of acrylic resin processed by various polymerization methods. methods: acrylic resin powder and liquid were mixed based on the rules of factory, and sample was made with size of 30 mm × 50 mm × 3 mm and then polymerized by using microwave at 70° c for 24 hours based on the methods of japan industrial standard (jis). each group of samples was cut with weight of ± 0.2 g, dissolved in 5 ml of methyl ethyl ketone in test tubes, and then stored at ± 5° c for four days. residual monomer level was conducted by using gas chromatograph mass spectrometer. �ata obtained were then analyzed by using one-way anova test with p < 0.05. results: after the level of polymerizing residual monomer with jis method was compared to that at 70° c for 24 hours using microwave, it is known that there were significant differences (p < 0.05). conclusion: the highest level of residual monomer of acrylic resin was that polymerized at 70° c for 24 hours. key words: acrylic resin, residual monomer, gas chromatograph abstract latar belakang: basis gigi tiruan yang berbahan dasar resin akrilik jenis heat cured setelah proses polimerisasi selesai masih mengandung monomer sisa yang berpotensi sebagai bahan iritan dalam rongga mulut. proses polimerisasi sangat penting untuk mendapatkan resin akrilik yang memenuhi persyaratan biokompatibilitas dan fisik yang baik. untuk persyaratan tersebut digunakan berbagai macam proses polimerisasi. tujuan: penelitian ini bertujuan untuk menentukan kadar monomer sisa resin akrilik yang diproses dengan metode polimerisasi berbeda menggunakan gas chromatograph mass spectrometer. metode: bubuk dan cairan resin akrilik dicampur sesuai aturan pabrik dan sampel dibuat berukuran 30 mm × 50 mm × 3 mm dipolimerisasi berdasarkan metode japan industrial standard (jis), suhu 70° c selama 24 jam, dan microwave. setiap kelompok sampel dipotong seberat ± 0,2 gram dilarutkan 5 ml dalam metil etil keton pada tabung uji dan disimpan suhu ± 5° c selama empat hari, dilakukan analisis kadar monomer sisa dengan gas chromatograph mass spectrometer. �ata yang diperoleh dianalis anova satu arah dengan nilai p < 0,05. hasil: kadar monomer sisa resin akrilik yang berpolimerisasi metode jis dibandingkan polimerisasi suhu 70° c selama 24 jam dan microwave terdapat perbedaan bermakna (p < 0,05). kesimpulan: kadar monomer sisa paling tinggi pada resin akrilik yang dipolimerisasi suhu 70° c selama 24 jam. kata kunci: resin akrilik, monomer sisa, gas chromatograph correspondence: sherman salim, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: sherman.salim@yahoo.com 197salim: the difference of acrylic resin residual monomer levels introduction polymethyl methacrylate is the basic material of heat cured acrylic resin a major choice in the prosthodontics for making the denture base. it is still the main choice of dentists although metallic materials or polyamide-mixed materials have widely been used as removable denture base. acrylic resin is easily manipulated and repaired if fracture occurs, it can meet aesthetic needs because its translucent and color stability are good enough, it is not toxic and not soluble in oral fluids, and its absorption is relatively low and relatively cheap price. in general, acrylic resin actually has low strength, either impact power or resistance strength to fracture, but it is still quite flexible. another character is that acrylic resin can cause dimensional changes occured during polymerization process and crack that might be found on the surface of acrylic resin.1,2 acrylic resin material used in dentistry is generally available in powder and liquid. some polymerization methods and curing processes, such as chemical activation, visible light, water heating, microwave energy, are then needed to manipulate the material into a solid material. this type of activation is used to initiate the formation of free radicals as the initial polymerization process of acrylic resin.3 the polymerization process is important to obtain acrylic resin which can meet the requirement of the physical character which is biocompatible to the tissues of oral cavity. thus, to meet the requirement, there are several methods of polymerization process that can be used. thermal polymerization method or conventional one is most often used in addition to using microwave. polymerization method using microwave has the advantages of saving time, clean, being not porous, with physical mechanical properties and dimensional changes similar to that processed conventionally.4 japan industrial standard (jis) is also introduced to the polymerization of acrylic resin, which is heated in water for 90 min at 70° c and was continued at 100° c for 30 minutes.5 the polymerization reaction of acrylic resin actually tends not to produce perfect unresidual monomer, but it still leaves residual monomer that has still not reacted yet, called the residual monomer of methyl methacrylate.3 the selection of the proper method of polymerization, as a result, is very important because it deals with the levels of residual monomer which will affect the quality of acrylic resin denture base. the residual monomer of acrylic resin may also be released into the water or saliva potentially causing irritation, inflammation, hypersensitivity, and allergic responses in mucosal tissue.1,6 therefore, it is necessary to know the residual monomer levels of acrylic resin which is processed by several polymerization techniques to avoid unacceptable impacts. the residual monomer levels of acrylic resin can actually be determined by several kinds of methods, such as by using infrared spectroscopy, gas chromatography, high performance liquid chromatography (hplc) and fluorescent flow injection.3 gas chromatography is a very precise and quick method to separate very complex mixtures. the duration needed varies from a few seconds for a simple mix up to hours for a complex mixture so that the method is widely used by researchers to analyze the residual monomer levels. therefore, the purpose of this study was to determine the differences of the residual monomer levels of acrylic resin processed by various polymerization methods. materials and methods gypsum made of water and gypsum with ratio of 24ml: 10grams was stirred on vibrator, and then poured into the cuvette. master model measuring 30 mm × 50 mm × 3 mm was placed in the middle of cuvette already containing the hard gypsum (new plastone gc, japan), and then was abandoned until the gypsum got harder. after the gypsum got harder, could mould seal was applied on the surface, and the top of cuvette was filled with the dough on vibrator. the master model that had been planted in the cuvette was abandoned for 24 hours. then, the master model in the cuvette was taken, so the mould was obtained. the cuvette was then filled with acrylic resin (bioresin, shofu japan; and acron mc, gc dental industrial japan), with a ratio of 10 grams of powder: 4.5 ml of fluid (based on the manufacturer’s instructions). after 20 minutes, the dough reached the dough stage. the mould on which separator had already been applied was filled with acrylic resin dough. before the cuvette was closed, acrylic was coated with celluloid plastics and pressed gently with hydraulic bench press (yoshida, japan). next, the cuvette was reopened, and the excess of acrylic resin was cut and then closed again. afterwards, pressing was conducted with pressure of 2200 psi or 50 kg/cm2. this procedure was repeated three times, and then transferred to the clamp abandoned for 24 hours. next, curing process was then conducted by heating water at 70° c for 90 min based on jis, and then followed at 100° c for 30 minutes. this procedure was repeated for the other treatment groups, but the polymerization was conducted by heating water at 70° c for 24 hours. making samples curing by microwave used a ratio of 10 grams of powder: 4.3 ml of fluid (based on the factory’s instructions). polymerization process was then conducted with microwave oven (500 watts) for 3 minutes. group i was acrylic resin polymerizing by using jis method. group ii was acrylic resin polymerizing at 70° c for 24 hours. group iii was acrylic resin polymerizing with microwave. after all of the polymerization processes were completed, the samples were abandoned until got cool. the samples in each treatment group consisted of six pieces, so the total samples were eighteen pieces. prior to being tested, the samples were stored in distilled water 37° c ± 1° c for 48 hours.5 the determination of the residual monomer levels of acrylic resin was then conducted by using gas chromatograph 198 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 196–199 mass spectrometer (jeol jms dx 303) and mass analysis/ data processing system (jeol jma da 5000) (jeol ltd., japan). the samples that had already polymerized were cut into small pieces weighing ± 0.2 grams. each treatment group sample was then dissolved in 5 ml of methyl ethyl ketone (merck) in a test tube, and kept at ± 5o c for four days (96 hours). the determination of the residual monomer was conducted based on the operating conditions of gas chromatograph mass spectrometer instrument as follows: helium carrier gas (p = 0.5 kg/cm2, flow ± 1 ml/min) using supelcowax column 10, fused silica capilary column (= carbowax 20 m = sp 1000), film thickness injector: spitless (60 seconds), ionization voltage 70 ev; current 100 a, acceleration 3 kv, chamber temperature 150o c, injector temperature 190o c, and program t1 (initial) 40° c and t2 70° c with the increasing of 4o c/minute. data obtained were then analyzed by using one-way anova test with p < 0.05. results it is known that the polymerization of acrylic resin at 70o c for 24 hours has the highest residual monomer level compared to the other polymerization methods (table 1). table 1. the mean and standard deviation of the residual monomer levels of acrylic resin with different polymerization methods (mg/l) treatment groups mean ± sd polymerization with jis method 0.3700 ± 0.1128 polymerization at 70° c for 24 hours 2.0717 ± 0.3100 polymerization with microwave 0.6917 ± 0.0572 after being tested with one sample kolmogorovsmirnov test (p > 0.05), it is known that the data obtained were homogeneous and normally distributed. to find out the differences of the residual monomer levels of acrylic resin processed by different polymerization methods, statistical calculations was conducted by using one-way anova. it is then known that there were significant differences with p value < 0.05. furthermore, to determine the differences of the polymerization methods towards the residual monomer levels of acrylic resin, lsd test was conducted (table 2). there were significant differences on the residual monomer level of acrylic resin polymerized with jis method compared to that polymerized at 70o c for 24 hours and that polymerized with microwave. discussion acrylic resin is a polymer that is popularly used in dentistry. it is known that ninety-five percents of patients use heat cured acrylic resin denture.7 heat cured acrylic resin heat is a mixture of methyl methacrylate monomer and poly methyl methacrylate polymer through polymerization process conducted after heating. in this case, acrylic resin polymerizes additionally. if at the time of the polymerization, temperature increases to 60° c, then bensoil peroxide which acts as initiator will decompose into free radicals. free radicals will then further react with monomer to form new free radicals resulting in from the occurence of propagation reaction to the occurence of termination. polymerization process at too low temperature or with too short duration even will cause weak acrylic resin due to short chain polymer.1 method of acrylic resin polymerization used in this study showed the significant differences of the residual monomer levels. these results are also supported by the results of the previous researches.8 some researchers also reported that the composition and the process of polymerization of denture base made of acrylic resin could affect on the release of residual monomer.9 the residual monomer levels of acrylic resin polymerize at 70° c for 24 hours was higher than that with jis method and microwave. this is due to temperature used below the temperature of tg (glass transition temperature), as a result, the polymerization of methyl methacrylate monomer will only slightly occur since methyl methacrylate during the polymerization is not mobilized, therefore, there are many residual monomer.10 another opinion states that the residual monomer level can be reduced if the duration of the polymerization process is extended and the temperature is enhanced.11 as a results, the polymerization process of acrylic resin using too low temperature or too short duration of curing process will generate residual monomer and high porosity that can reduce the strength of denture base made of acrylic resin which later can facilitate the occurrence of fractures. the table 2. the results of lsd test on the residual monomer levels of acrylic resin processed by different polymerization methods (mg/l) polymerization methods polymerization with jis method polymerization at 70° c for 24 hours polymerization with microwave polymerization with jis method * * polymerization at 70o c for 24 hours * polymerization with microwave note: *: significant 199salim: the difference of acrylic resin residual monomer levels residual monomer that remains in the acrylic resin denture base will adversely affect the mechanical properties since the residual monomer acts as plasticizer that makes acrylic resin denture base soft and flexible.12 moreover, the residual methyl methacrylate monomer can also come out into the surrounding tissues13 which later may cause side effects, such as the hypersensitivity of oral tissue and the changing of color stability.14 clinical signs and symptoms are often reported including erythema, erosion, oral mucosa, and burning sensation in mucosa and tongue.6 according to iso 1567, the maximum residual monomer of denture base material shall not be more than 2.2% of weight.14 the residual monomer levels of acrylic resin used as a denture base, therefore, showed be reduced after the polymerization process. some researchers even recommend to soak it in water for at least 24 hours before inserting it into patients in order to make the residual monomer become not potentially toxic. other researchers, moreover, said that after polymerization, acrylic resin can be soaked in water at 50o c for one day to reduce the release of the residual monomer because the water heated can cause the monomer molecules more rapidly spread, so it can enhance the reaction of polymerization. 6 polymerization of acrylic resin with jis method can produce the lower residual monomer than that with other polymerization methods. acrylic resin polymerizing can usually reach the boiling point of monomer, and can also produce the lower residual monomer than that polymerizing without reaching the boiling point.15 it means that the use of high heat will cause the mobility of the molecule chain that facilitate the conversion of monomers into polymers.6 thus, it is known that there was significant difference between the residual monomer levels of acrylic resin polymerizing with microwave and that polymerizing at 70° c for 24 hours. it is because the polymerization process with microwave can produce internal heat due to the high frequency of electromagnetic waves causing the faster monomer molecule movement and the more complete polymerization reaction, consequently, the residual monomer was reduced. previous researchers even concluded that the polymerization of acrylic resins with microwave compared to that with the pressure and injection molding methods can cause the significant reduction of the residual methylmethacrylate monomer.3 finally, it can be concluded that the highest residual monomer level of acrylic resin is that polymerized at 70° c for 24 hours. references 1. craig rg, powers jm. restorative dental materials. 11ed ed. st louis: mosby; 2002. p. 636–89. 2. ayad nm, badawi mf, fatah aa. effect of reinforcement of highimpact acrylic resin with zirconia on some physical and mechanical properties. rev clín pesq odontol 2008; 4(3): 145–51. 3. celebi n, yuzugullu b, canay s, yucei u. effect of polymerization methods on the residual monomer level of acrylic resin denture base polymers. polym adv tehnol 2008; 19: 201–6. 4. del bel cury aa, rached rn, ganzarolli sm. microwave cured acrylic resins and silicone-gypsum moulding technique. j oral rehab 2001; 28: 433-8. 5. salim s. various curing methods on transverse strength of acrylic resin. dent j (maj ked gigi) 2010; 43(1): 40–3. 6. jorge jh, giampaolo et, vergani ce, machado al, pavarina ac, carlos iz. cytotoxicity of denture base resins: effect of water bath and microwave post plimerization heat treatment. int j prosthodont 2004;17(3): 340–4. 7. combe ec. notes on dental materials. 6th ed. edinburgh: churchil livingstone; 1992. p. 157–63. 8. bayraktar g, guvener b, bural c, uresin y. influence of polymerization method, curing process, and length of time of storage in water on the residual methyl methacrylate content in dental acrylic resins. j biomed mater res b appl biomater 2006; 76(2): 340–5. 9. lasilla lvj, vallittu pk. denture base polymer aldent sinomer: mechanical properties, water sorption and release of residual compounds. j oral rehabil 2001; 28(7): 607–13. 10. sadoon mm, mohammed nz, al–omary ao. residual monomer and transverse strength evaluation of auto polymerized acrylic resin with different polymerization treatment. al–rafidain dent j 2007; 7: 30–4. 11. mohamed sh, al-jadi am, ajaal t. using of hplc analysis for evaluation of residual monomer content in denture base material and their effect on mechanical properties. j physical science 2008; 19(2): 127–35. 12. barbosa db, souza rf, pero ac, marra j, compagnoni ma. flextural strength of acrylic resins polymerized by different cycles. j appl oral sci 2007; 15(5): 428–8. 13. abdi k, mandegary a, amini m, bagheri m, gerami-panah f. determination of residual methylmethacrylate monomer in denture base resins by gas chromatography. iranian journal of pharmaccutical research (ijpr) 2005; 5: 227–32. 14. golbidi f, asghari g. the level of residual monomer in acrylic denture base materials. j biol sci 2009; 2(issue 2): 244–9. 15. anussavice kj. phillip’s science of dental materials. 11th ed. missouri: elsevier science; 2003. p. 163–70. 71 volume 46 number 2 june 2013 research report effectiveness of various sterilization methods of contaminated post-fitted molar band anggia tridianti, krisnawati and nia ayu ismaniati department of orthodontics faculty of dentistry, universitas indonesia jakarta-indonesia abstract background: molar band as anchoring device may be contaminated during the fitting process. thus, decontamination process is essential to prevent cross-infection between patients. purpose: the objective of this research was to determine the amount of bacteria in molar band post-fitted on the patient teeth, after previously undergone pre-sterilization using alcohol and ultrasonic cleaning bath followed by sterilization using dry heat oven and steam autoclave, in order to find the best method in decontamination of post-fitted molar band. methods: four molar bands which already fitted on one patient then divided evenly into two groups. the first group was pre-sterilized using alcohol. one of the bands then sterilized using dry heat oven, while the other one was sterilized using steam autoclave. the second group was pre-sterilized using ultrasonic cleaning bath. one band from this group then sterilized using dry heat oven and the other was sterilized using steam autoclave. the next step was to immerse all the bands in a phosphate-buffered saline solution. using micropipette, the solution was retrieved and dropped upon a petri dish containing brain heart infusion broth. the dish was then stored in an incubator for 24 hours, prior to counting the number of bacteria existed. the same methods were used to the rest of the patients, with total 128 molar bands from 32 patients. results: there was a profound difference in numbers of bacteria found between those methods of sterilization. however, there was a non significant difference between the two groups which were at the alcohol-steam autoclave group and at the ultrasonic cleaning bath-steam autoclave group. conclusion: this study showed that steam autoclave is better than for sterilizing molar band, as it left the minimal amount of bacteria in post-fitted molar band. key words: molar band, dry heat oven, steam autoclave, sterilization abstrak latar belakang: molar band merupakan suatu alat penjangkaran yang dapat mengalami kontaminasi selama proses fitting band, sehingga perlu dilakukan suatu proses dekontaminasi untuk menghindari terjadinya cross-infection pada pasien. tujuan: penelitian ini bertujuan untuk mengetahui perbedaan jumlah bakteri pada molar band pasca fitting band setelah sterilisasi dry heat oven dan steam autoclave yang sebelumnya telah dilakukan pre-sterilisasi alcohol dan ultrasonic cleaning bath, sehingga dapat ditentukan metode sterilisasi yang terbaik dalam dekontaminasi molar band. metode: empat molar band yang telah melalui proses fitting band pada seorang pasien dibagi dalam dua kelompok. pada kelompok pertama, dua molar band dilakukan pre-sterilisasi dengan alkohol, kemudian satu band dilakukan sterilisasi dengan dry heat oven dan satu band lainnya dengan steam autoclave. kelompok kedua, dua molar band dilakukan pre-sterilisasi dengan ultrasonic cleaning bath, kemudian satu band dilakukan sterilisasi dengan dry heat oven dan satu band lainnya dengan steam autoclave. molar band tersebut masing-masing kemudian dimasukkan ke dalam cairan phosphate-buffered saline, dengan micropipette cairan diambil dan dituangkan ke cawan petri yang berisi brain heart infusion. kemudian dimasukkan ke dalam inkubator selama 24 jam dan dihitung jumlah bakterinya. metode yang sama dilakukan terhadap molar band lainnya, dengan total 128 molar band dari 32 pasien. hasil: terdapat perbedaan jumlah bakteri yang bermakna antara beberapa kelompok metode sterilisasi dan terdapat satu kelompok dengan perbedaan tidak bermakna, yaitu kelompok alcohol-steam autoclave dengan ultrasonic 72 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 71–74 cleaning bath-steam autoclave. simpulan: hasil penelitian menunjukkan bahwa steam autoclave merupakan metode sterilisasi yang terbaik pada molar band yang telah melalui proses fitting band karena menunjukkan jumlah bakteri yang paling minimal. kata kunci: molar band, dry heat oven, steam autoclave, sterilisasi correspondence: anggia tridianti, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta 10430, indonesia. e-mail: myaidanna@yahoo.com introduction stainless steel molar bands are often selected as an component anchorage in orthodontic treatment, particularly if difficulties encountered when using bucal tubes which have to be bonded on the surface of the molar teeth. thus, it may be detached accidentally from its place due to pressure of chewing.1 in determining the suitable molar band, the process is often had to be carried out several times to find an appropriate size.1,2 molar bands are quite expensive, thus orthodontists choose not to throw it out if the size does not fit to a patient, as it may be suitable for others. in the process of fitting, the molar bands usually come in contact with contaminated saliva or blood, that caused by the injury to the gums on the subgingival area. these areas contain the anaerobic gram-negative bacteria, such as porphyromonas gingivalis and actinobacillus actinomicetemcomitans.2,3 while supragingival plaque composed of gram-positive bacteria, such as streptococcus sanguis, streptococcus mutans, streptococcus mitis, streptococcus salivarius and lactobacillus. these bacteria play an important role in dentistry, as they are sources of mouth diseases, such as caries and periodontitis.4 inadequate cleaning process and sterilization may result in remaining of potentially harmful blood-borne agents or contaminants on the orthodontic band surface. the contaminated molar bands can lead to a spreading of diseases or causing cross-infection to other patients, such as subacute bacterial endocarditis, herpes, hepatitis b, hepatitis c and hiv. such diseases have a high mortality rate, thus it is necessary to do preventive action to avoid transmission of pathogenic microorganisms from infected individuals.5 sterilization is a process of destroying all forms of life, including spores. on the other hand, disinfection is a process to destruct most of microorganisms but it does not include spores. usually, this process needs a solution like phenol, alcohol, chlorine or iodine that is applied to the instrument.6,7 health workers should always use personal protective equipment (ppe) such as disposable gloves, masks and goggles. it has to be done as a form of infection control so there would be no cross-infection to other patients.6,8 there are three stages in the decontamination process, which are pre-sterilization stage, sterilization stage and storage stage.9,10 recommendation for the initial cleaning process include removal the contaminant by hand, the use of disinfectant enzyme-based cleaning solution or alcohol, the use of instrument washer such as ultrasonic cleaning bath, and then followed by drying it using hot air or a sponge, this step is important to avoid any damages on the instruments during the sterilization process. methods of sterilization can be done using steam autoclave, chemical, dry heat ovens, boiling water, salt or glass bead sterilizer, and hyperbaric gas (ethylene oxide) sterilization.6,10,11 according to surveys, most of orthodontists in the united kingdom always clean and disinfect the band prior to reuse on other patients. due to the availability of various types of sterilization method, until today there is no reliable method to deliver the best results in decontamination.9 the purpose of this research is to find the best method in decontamination of post-fitted molar band. materials and methods on this research, there were 128 molar bands that came from 32 patients where each of them used 4 bands. the four molar bands that came from the first patient divided evenly into two groups. the first two groups were pre-sterilized using alcohol, one molar band then sterilized using dry heat oven at 150⁰ c for 20 minutes (group a), while the second one was using steam autoclave with a temperature at 130⁰ c for 1 hour (group b). the last two groups were pre-sterilized using ultrasonic cleaning bath, one molar band then sterilized using dry heat oven at 150⁰ c for 20 minutes (group c), and the other one was applying steam autoclave at 130⁰ c for 1 hour (group d). the molar bands in all groups were soaked in a medicine bottle containing 5 ml phosphate-buffered saline (pbs), then they were inserted into a shaker for 30 minutes, and left at room temperature for 15 minutes. ten μl of pbs was taken with a micropipette and poured onto a petri dish, contained with brain heart infusion agar. the pbs solution was then swiped to spread it out evenly. the dish was then placed into an incubator for 24 hours with co2 in it. the same methods were also used to the other 31 patients. the number of bacteria that appeared on the media was then calculated. the researcher also calculated the bacteria from 5 new molar bands that were taken straight out of the box and 5 molar bands that have gone through random fitting process, without applying any decontamination process. these procedure were conducted to examine the amount of bacteria from new molar band before fitting and the amount of bacteria prior to decontamination. 73tridianti, et al.,: effectiveness of various sterilization methods in addition of this research, another examination has been applied on randomly taken molar bands, to determine availability of gram positive or negative bacteria from the molar bands. this examination was performed by staining the bacteria using crystal violet 10% liquid and self-ranin liquid. the bacteria were then viewed under microscope. data that have been obtained were processed to see its normality. by using the shapiro-wilk test, abnormal distribution can be observed. the kruskal-wallis test was also performed to see if all values have significant difference between each other. to examine comparative difference between two groups, the mann-whitney tests was done. results from 5 new molar bands that were taken directly from the box, the number of bacteria that was found was 0 cfu/ ml at the minimum, while at the maximum was 2 cfu/ ml. the number of bacteria on molar bands, which were taken directly from the patients without conducting any decontamination process, at the minimum was 32 cfu/ ml, and at the maximum was 49 cfu/ ml (table 1). the results showed significant differences between the groups and there was one group that has a non-significant difference, it was the group which applying method of alcohol-steam autoclave with ultrasonic cleaning bathsteam autoclave (group b-d). which means, the two groups of sterilization method provides equally good results in table 1. the number of bacteria from molar band before contaminated and prior to decontamination no 5 new molar band taken out of the box (cfu/ml) 5 molar bands taken from 5 randomized patients who have not been through decontamination (cfu/ml) 1 1 45 2 1 32 3 0 49 4 2 41 5 1 38 table 2. the number of bacteria at minimum, maximum from each method of sterilization methods of sterilization number of samples minimum number of bacteria (cfu/ml) maximum number of bacteria (cfu/ml) alcohol+dry heat oven (a) 32 10 31 alcohol+steam autoclave(b) 32 0 7 ultrasoniccleaning bath+dry heat oven (c) 32 6 18 ultrasoniccleaning bath+steam autoclave (d) 32 0 6 table 3. post-hoc analysis of the 2 sterilization methods groups were compared comparison of two groups of methods of sterilization p alcohol-dry heat oven compared to alcohol-steam autoclave (group a-b) alcohol-dry heat oven compared to ultrasonic cleaning bath-dry heat oven (group a-c) alcoholdry heat oven compared to ultrasonic cleaning bath-steam autoclave (group a-d) alcohol-steam autoclave compared to ultrasonic cleaning bath-dry heat oven (group b-c) alcohol-steam autoclave compared to ultrasonic cleaning bath-steam autoclave (group b-d) ultrasonic cleaning bath-dry heat oven compared to ultrasonic cleaning bath-steam autoclave (group c-d) .000* .000* .000* .000* .182 .000* * p <0.05 means that there is a difference table 4. staining the bacteria to determine the gram positive or negative bacteria randomly taken sample staining results 1 blue 2 red 3 red 4 red 5 red note: red: gram negative; blue: gram positive the decontamination of bacteria on the molar band (table 2 and 3). the bacterial determination on result showed that 4 out of 5 sample were found as gram negative bacterial (table 4). 74 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 71–74 discussion among 4 groups that have undergone process of decontamination, the minimal amount of bacteria of 0 cfu/ml, was found in a steam autoclave alcohol group (group b) and the ultrasonic cleaning bath with a steam autoclave group (group d), which showed similar results with the brand new molar bands that were taken out straight from the box. while the maximum number of bacteria, found in the group of alcohol-dry heat oven (group a), that was 31 cfu/ml (table 2), not much in difference when compared to the minimal number of bacteria on the molar bands which has not been through decontamination process of 32 cfu/ml (table 1). there are significant differences between the 2 groups of sterilization method, between group a and b, between group a and c, between group a and d, between group b with c, and between group c with d. while there were no significant differences found between group of alcoholsteam autoclave (group b) with the ultrasonic cleaning bath-steam autoclave (group d). this is due to the two groups using a steam autoclave sterilization method that have minimum number of bacteria 0 cfu/ ml. some studies showed that steam autoclave sterilization is the method of choice by health workers because it provides the best results in eliminating all forms of microorganisms.9-11 according to dowsing and benson,9 most orthodontist in the united kingdom were using a conventional steam autoclave sterilization as a method for the prevention of cross-infection on the orthodontic instruments. as for the sterilization of fitted-in molar bands, they used different types of steam autoclave. in addition to conventional steam autoclave they also used vacuumphase autoclave. there are many different opinions about the best way of sterilizing orthodontic instruments. some experts argue that dry heat oven is better than steam autoclave, which can cause corrosion on the instrument, thus reducing its effectiveness in cutting the wire, and also cause corrosion on the joint. vendrell rj et al.,12 conducted a study to compare the effects of steam autoclave with dry heat oven at ligature cutting pliers, the results of this study showed that both methods of sterilization are equally effective and does not cause rust and corrosion, as long as the orthodontic instruments are made ​​of stainless steel. in this research, the other examination had been done to determine what kind of bacteria on the molar band if the bacteria are gram positive or negative. under the microscope, the results obtained from 5 samples there were 4 red colors and 1 blue color. the red color were formed because the bacteria bind to self-ranin liquid which indicates bacteria are gram-negative. while the blue color was formed because the bacteria bind to the crystal violet 10% liquid which indicates the bacteria are gram-positive. based on these samples the majority of bacteria that have been founded are gram-negative. the gram-negative bacteria are anaerobic bacteria, commonly found in subgingival plaque, which can lead to periodontitis.3,4 this finding fits with a research conducted by huser et al,13 which stated that the usage of molar bands can increase the number of fusobacterium, spirocheta and spirilla, that are usually found on periodontitis. the steam autoclave sterilization provides the best result in decontamination of post fitted molar band, assuming gramnegative bacteria also. the decontamination prevents crossinfections between patients as in dowsing and benson’s research.9 furthermore, according to bda advisory service10and mccarthy et al.,11 the steam autoclave sterilization is the method of choice for orthodontic instrument decontamination. however, this method is not recommended for ligature cutting plier or joint pliers that is not made of stainless steel. based on this research, any method of sterilization combined with steam autoclave gave the best result in reducing bacteria, so it can be concluded that effective method in decontamination of post fitted molar band is the steam autoclave. references 1. benson pe, douglas cwi. decontamination of orthodontic bands following size determination and cleaning. j orthod 2007; 34(1): 18-24. 2. fulford mr, ireland aj, main bg. decontamination of tried-in orthodontic molar bands. eur j orthod 2003; 25(6): 621-2. 3. casaccia gr, gomes jc, alviano ds, ruellas ac, anna ef. microbiological evaluation of elastomeric chains. angle orthod 2007; 77(5): 890-3. 4. jawetz e, melnick jl, adelberg ea. 1954. mikrobiologi kedokteran. nugroho e, maulany rf, editors. medical microbiology. jakarta: penerbit buku kedokteran egc; 1995. p. 38-47. 5. lucas vs, omar j, vieira a, roberts gj. the relationship between odontogenic bacteraemia and orthodontic treatment procedures. eur j orthod 2002; 24(3): 293-301. 6. kuramitsu hk, he x, lux r, anderson mh, shi w. interspecies interactions within oral microbial communities. am soc microbiol 2007; 71(4): 653-70. 7. whitworth cl, martin mv, gallagher m, worthington hv. a comparison of decontamination methods used for dental burs. br dent j 2004; 197(10): 635-40. 8. hoesin s, herda e, damiyanti m, odang r, susanti l, yuniastuti m. pedoman pendidikan dokter gigi fkg ui. jakarta: upkg fkg-ui; 2007 p. 79-87. 9. dowsing p, benson pe. molar band re-use and decontamination: a survey of specialists. j orthod 2006; 33(1): 30-7. 10. bda advisory service. infection control in dentistry, advice sheet a12. london: british dental association; 2003. p.7-9. 11. mccarthy gm, mamandras ah, macdonald jk. infection control in the orthodontic office in canada. am j orthod dentofac orthop 1997; 112(3): 275-81. 12. vendrell rj, hayden cl, taloumis lj. effect of steam versus dry heat sterilization on the wear of orthodontic ligature-cutting pliers. am j orthod dentofac orthop 2002; 121(5): 467-71. 13. huser mc, baehni pc, lang r. effects of orthodontic bands on microbiologic and clinical parameters. am j orthod dentofac orthop 1990; 97(3): 213-08. 36 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 36–40 original article knowledge of orofacial pain in students of the dental professional program faculty of dental medicine, universitas airlangga desvia nuzela qurzani hariyadi1, ari hapsari tri wardani2, saka winias3, fatma yasmin mahdani3, adiastuti endah parmadiati3, nurina febriyanti ayuningtyas3, meircurius dwi condro surboyo3 1undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2oral medicine specialist degree, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3oral medicine department, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: orofacial pain is associated with the hard and soft tissues of the head, face, and neck. knowledge of orofacial pain helps in getting information on clinical symptoms, trigger factors, and risks of orofacial pain, as well as clinical and supportive examinations for identifying the pain. the dental professional students’ knowledge influences the ability to diagnose and decide on an appropriate treatment plan. this knowledge is seen from the intelligence level in obtaining information about orofacial pain. purpose: to describe the level of knowledge of orofacial pain in the students of the dental professional program, faculty of dental medicine, universitas airlangga, based on intelligence level. methods: this research was a descriptive study. data was taken using a questionnaire on google form with a simple random sampling data technique and was analyzed using spss version 25. results: the majority understand the general description of postherpetic neuralgia (phn), as well as the risk factors and triggers (93.4% and 87.8%, respectively) associated with it. further, 90.6% understand burning mouth syndrome (bms) and the clinical symptoms of phn. bms based on clinical examination and support is understood by 96.1%, and 82.9% know how to manage trigeminal neuralgia (tn). intelligence level is divided into three categories, namely source of knowledge (55.8% are from lectures, journals, and textbooks), material repetition (77.3% never repeated), and retention of material (65.2% no retention). conclusion: students’ knowledge of orofacial pain is good, but the relationship between the level of intelligence and knowledge is not yet known. keyword: intelligence level; knowledge; orofacial pain article history: received 28 january 2022, revised 19 july 2022, accepted 29 august 2022 correspondence: saka winias, oral medicine department, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: saka.winias@fkg.unair.ac.id introduction orofacial pain is the pain associated with the hard and soft tissues of the head, face, and neck. pain stimulation through tissues such as skin, blood vessels, teeth, glands, or muscles sends impulses through the trigeminal nerve, which is transmitted through a special nerve network to the central nervous system, interpreted as pain or discomfort.1 in the united states, the prevalence of orofacial pain, according to a study by lipton et al.,2 is as follows: the most common orofacial pain originating from toothache was reported by 12.2% of the population, 5.3% complained about temporomandibular joint (tmj) pain, and 1.4% complained about pain related to face or cheeks. more than 81% of patients suffer from orofacial pain originating from trigeminal system.1 orofacial pain is usually characterized or described as burning, sharp, episodic, or continuous pain, which can also be accompanied by a headache. pain associated with headaches is felt in the teeth, but the origin of the pain is hard to identify. there are degrees of orofacial pain ranging from mild to moderate to severe. some patients also complain of tinnitus, vertigo, paresthesias, hyperalgesia, and allodynia.3 the causes of orofacial pain can be local, namely from teeth, starting from caries, periodontal disease, musculoskeletal disease, and neuropathology. these are some of the most common causes of orofacial pain.4 other causes of orofacial pain include vascular disorders, post-traumatic pain, and lesions on the ears, nose, and oral cavity.5 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p36–40 mailto:saka.winias@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p36-40 37hariyadi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 36–40 trigger and risk factors of orofacial pain are anxiety disorders, systemic disorders, trauma, and behavioral disorder.6 clinical and supporting examinations were performed to help determine the diagnosis. clinical and supporting examinations for orofacial pain include palpation of joints and muscles, dental examination, and panoramic examination or cone beam computed tomography (cbct) examination.1 according to a study conducted by ziegeler et al.,7 it is said that in practice, it is very difficult to diagnose orofacial pain because the clinical symptoms of each disease related to orofacial pain are the same. this makes it difficult for dental students and professional dentists to determine the correct diagnosis and treatment plan. therefore, if dental students lack the theoretical knowledge of orofacial pain, it may later get difficult for them to treat orofacial pain patients.7 according to research by borromeo and trinca,8 in dental education, the topic of orofacial pain, according to the international association for the study of pain (iasp), includes the definition, mechanism, and assessment of pain. this topic is a basis for a dentist’s knowledge about orofacial pain. along with clinical and supporting examinations, knowledge of orofacial pain is used to make an accurate diagnosis, which is obtained by examining clinical symptoms, triggers, and risk factors. based on research conducted by ziegeler et al.7 in 2018 on 533 general and specialist dentists and 130 dental students, it was found that 92% of dental students stated that they feel either “not at all” (56%) or only “somewhat” (36%) prepared for the diagnosis or treatment of non-dental orofacial pain. only 23% of the dentists reported “good” or “very good” confidence in diagnosing non-dental orofacial pain. the knowledge attained by dental students during their undergraduate dental education influences their capability of diagnosing and carrying out appropriate treatment plans. intelligence is the ability to understand and solve problems with the obtained knowledge.9 one type of intelligence is crystalized intelligence that improves with the increase in knowledge, experience, and skills possessed by an individual.10 based on this theory, knowledge can be determined by the level of intelligence. so here, the author wanted to describe the level of knowledge of orofacial pain in the students of the dental professional program, faculty of dental medicine, universitas airlangga. materials and methods in this observational descriptive study, dental professional students’ of batch 2018 and 2019 of the dental medicine faculty, universitas airlangga, participated voluntarily. by applying the slovin formula (e=0.05), the minimum number of samples needed was 175, with the proportion of 90 dental professional students from batch 2018 and 91 dental professional students from batch 2019, due to a slight difference in the number of dental professional students from each batch. there are two inclusion criteria: first, a student of the faculty of dental professional program, faculty of dental medicine, universitas airlangga. second, willingness to fill out informed consent and a questionnaire. meanwhile, the exclusion criteria are students of the faculty of dental professional program, faculty of dental medicine, universitas airlangga, who have completed all the stations. the research was conducted online from the end of january 2021 until early february 2021. health research ethical clearance commission approved it in the faculty of dental medicine, universitas airlangga, with registration number 458/hrecc.fodm/x/2020 (approval date: october 09, 2020). the research was done using a 20-item questionnaire, which tested the validity and reliability of the questionnaire with statistical package for the social sciences (spss) version 25 for windows (ibm, new york, usa). the questionnaire was divided into two groups: respondents’ sociodemographic data and the items to measure respondents’ knowledge level. the questionnaire had five domains, and each domain consisted of four questions representing four diseases related to orofacial pain and three regarding intelligence levels. the data were collected through a google form questionnaire. some comprehensive and adequate explanations about the research aim were also given. the link was shared via social media. respondents willing to participate had to give consent by ticking the provided check box before entering the questionnaire section. respondent’s answers were measured using the guttman scale, in which respondents have to provide answers in the form of “true” or “false.” the answers to the questionnaire were assessed on a 0–1 scale, with each correct answer getting a value of 1 and the wrong or no answer 0. results out of 310 dental professional students in the faculty of dental medicine, universitas airlangga, who were given the questionnaire’s link, 181 decided to participate. the result not only fulfilled the minimum number of respondents needed but also got a 58% of response rate. the sociodemographic characteristic of the respondents is described in table 1. the majority of respondents were 23 years old, with as many as 79 respondents (43.6%). most table 1. frequency distribution of respondents based on sociodemographic characteristics categories n % age 20 1 0.6 21 7 3.9 22 64 35.4 23 79 43.6 24 25 13.8 gender male 30 16.6female 151 83.4 batch 2018 90 49.72019 91 50.3 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p36–40 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p36-40 38 hariyadi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 36–40 table 2. distribution of knowledge based on an overview disease question answer total true false n (%) n (%) n (%) trigeminal neuralgia trigeminal neuralgia often occurs at the age of 40–70 years 162 89.5 19 10.5 181 100 burning mouth syndrome burning mouth syndrome is rarely located on the tongue 160 88.4 21 11.6 181 100 postherpetic neuralgia postherpetic neuralgia due to reactivation of the latent virus 169 93.4 12 6.6 181 100 atypical facial pain atypical facial pain is idiopathic pain 163 90.1 18 9.9 181 100 table 3. distribution of knowledge based on clinical symptoms disease question answer totaltrue false n (%) n (%) n (%) trigeminal neuralgia trigeminal neuralgia is often unilateral 146 80.7 35 19.3 181 100 burning mouth syndrome pain intensity of burning mouth syndrome can be severe 164 90.6 17 9.4 181 100 postherpetic neuralgia postherpetic neuralgia is characterized by hyperalgesia 164 90.6 17 9.4 181 100 atypical facial pain atypical facial pain is usually characterized by long-lasting pain 138 76.2 43 23.8 181 100 table 4. distribution of knowledge based on risk factors and triggers disease question answer totaltrue false n (%) n (%) n (%) trigeminal neuralgia chewing movements, talking, and smiling can trigger attacks of pain in trigeminal neuralgia 145 80.1 36 19.9 181 100 burning mouth syndrome burning mouth syndrome cannot be triggered by gastrointestinal disorders 157 86.7 24 13.3 181 100 postherpetic neuralgia immunosuppressive conditions can trigger postherpetic neuralgia 159 87.8 22 12.2 181 100 atypical facial pain atypical facial pain is not triggered by a depressive disorder 143 79.0 38 21.0 181 100 table 5. distribution of knowledge based on clinical and supporting examination disease question answer totaltrue false n (%) n (%) n (%) trigeminal neuralgia in trigeminal neuralgia, ct scan or mri cannot be performed 130 71.8 51 28.2 181 100 burning mouth syndrome examination of burning mouth syndrome is also examined by the condition of the mucosa 174 96.1 7 3.9 181 100 postherpetic neuralgia intensity and quality tests were carried out with a pain scale for postherpetic neuralgia examination 173 95.6 8 4.4 181 100 atypical facial pain examination of atypical facial pain does not require examination of the head and neck 170 93.9 11 6.1 181 100 table 6. distribution of knowledge based on management disease question answer totaltrue false n (%) n (%) n (%) trigeminal neuralgia trigeminal neuralgia can only be treated with lamotrigine 150 82.9 31 17.1 181 100 burning mouth syndrome the drug, clonazepam, can be given for burning mouth syndrome 145 80.1 36 19.9 181 100 postherpetic neuralgia postherpetic neuralgia contraindications to treatment with topical lidocaine 92 50.8 89 49.2 181 100 atypical facial pain atypical facial pain contraindications to treatment with tricyclic depressants 100 55.2 81 44.8 181 100 of the respondents were female, and about 50.3% were from batch 2019. table 2 shows the knowledge of orofacial pain based on the general description of orofacial pain. respondents’ answers exceeded 80%; the highest percentage of correct answers was found in the general description of postherpetic neuralgia, which was 93.4%. while table 3. shows that dental professional students can recognize clinical symptoms of orofacial pain well, especially in burning mouth syndrome and postherpetic neuralgia, which have the highest percentage in this question, which is 90.6%. in the item asking about the risk factors and triggers of orofacial pain, 159 respondents (87.8%) correctly answered the risk factors and triggers copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p36–40 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p36-40 39hariyadi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 36–40 of postherpetic neuralgia, which can be seen in table 4. burning mouth syndrome still occupies the highest correct answers on the item about clinical and supporting examinations. as many as 174 respondents (96.1%) answered correctly (table 5). however, the management of postherpetic neuralgia turned out to have the lowest percentage in table 6, where only 92 respondents (50.8%) answered correctly. table 7 describes knowledge from the respondents based on intelligence level, divided into three categories: source of knowledge, material repetition, and retention of material. the majority of respondents’ knowledge about orofacial pain was obtained from lectures, journals, and textbooks (55.8%). regarding the repetition of orofacial pain material, most respondents, namely 140 people (77.3%), stated that they had never repeated the material. the answer “not easy to understand” regarding orofacial pain was also given by 118 respondents (65.2%). discussion knowledge of orofacial pain was assessed under five domains: general description, clinical symptoms, risk factors and triggers, clinical and supporting examinations, and management. each domain of knowledge included four orofacial pain diseases: trigeminal neuralgia, burning mouth syndrome, postherpetic neuralgia, and atypical facial pain. in the first domain, knowledge based on the general description found that postherpetic neuralgia got the highest percentage of correct answers, namely 93.4%, where respondents agreed that the incidence of postherpetic neuralgia was due to reactivation of a latent virus. this is because postherpetic neuralgia is pain that occurs due to the reactivation of the varicella-zoster virus (vzv). the virus begins to settle in the body after a primary varicella infection (chickenpox) that may have occurred decades ago, ultimately causing postherpetic neuralgia.11 dental professional students’ knowledge of orofacial pain is not limited to the general description. they can also give correct answers with a percentage as high as 90.6% on clinical symptoms, especially postherpetic neuralgia and burning mouth syndrome. one of the clinical signs of postherpetic neuralgia is hyperalgesia. meanwhile, the pain intensity can become more severe in burning mouth syndrome. pain in the burning mouth syndrome occurs in the morning and continues to aggravate to the maximum intensity by night.3 in the third domain, namely, knowledge based on risk factors and triggers of four orofacial pain diseases, postherpetic neuralgia still ranks first in the percentage of correct answers (87.8%) given by dental professional students with burning mouth syndrome ranking second (86.7%). dental professional students know that an immunocompromised host condition is one of the triggering factors for postherpetic neuralgia. this is in accordance with a study conducted by muñoz-quiles12 that patients with immunocompromised conditions have a higher risk of developing herpes zoster, recurrence of herpes zoster, and also complications of postherpetic neuralgia. in the fourth domain, namely, knowledge based on clinical and supporting examinations of four orofacial pain diseases, the majority of respondents answered correctly, especially on burning mouth syndrome (96.1%) and postherpetic neuralgia (95.6%). one of the clinical examinations for burning mouth syndrome is to examine the condition of the mucosa because certain patients describe burning mouth syndrome as a burning sensation in the oral mucosa without any obvious mucosal changes.13, therefore, mucosa should be examined to identify or rule out factors that may be contributing to the disease.14 whereas in postherpetic neuralgia, it is necessary to test the intensity and quality with a pain scale where patients with postherpetic neuralgia can experience three main types of pain: 1) constant pain without a stimulus, which is often described as burning, aching, or throbbing; 2) intermittent pain without a stimulus, which feels like getting stabbed or an electric shock; 3) pain caused by the stimulus but not proportional to the stimulus received (hyperalgesia).10 in the fifth domain, namely, knowledge based on management, it was found that there were two orofacial pain diseases where the respondents gave almost similar numbers of correct and incorrect answers. among them is postherpetic neuralgia, with 45 correct answers—50.8% correct answers. dental professional students answered questions about postherpetic neuralgia contraindications to treatment using topical lidocaine with the correct answer: disagree. there was almost a balance between right and wrong answers to questions regarding postherpetic neuralgia. there is a balance between correct and incorrect answers because, in the drugs for first-line postherpetic neuralgia based on the recommendations of the american academy of neurology (2004), the international association for the study of pain (2007), and the european federation of neurological societies (2010) are tricyclic antidepressants that are gabapentin and pregabalin, and topical lidocaine 5%. however, the uk and canadian guidelines place topical lidocaine as a second-line drug.15 since topical lidocaine is also an anesthetic, many dental professional students’ answers agreed with the question that topical lidocaine is contraindicated with treatment contraindications. table 7. distribution of knowledge based on intelligence level categories n % source of knowledge course material 80 44.2 lecture materials, journals, and textbooks 101 55.8 material repetition yes 41 22.7 no 140 77.3 retention of material yes 63 34.8 no 118 65.2 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p36–40 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p36-40 40 hariyadi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 36–40 multiple teaching strategies, such as theoretical, preclinical, and clinical education, are used by dental schools. assessment of these strategies has a crucial role in evaluating the efficiency of the provided education methods and the achievement of the intended learning outcomes of future graduate dentists.16,17 to obtain knowledge about orofacial pain, dental professional students obtain and collect information. in this study, the researcher asked the students about the source from which they received information on orofacial pain. around 44.2% of students stated that they got information from the lecture material. furthermore, 55.8% stated that they got information from lecture materials, journals, and textbooks. in obtaining information, the more sources obtained, the better the knowledge obtained. in addition to the source of knowledge, the distribution of the material in the study found that around 22.7% of students often revised study material on orofacial pain, and 77.3% said that they did not often revise. revising study material enables a person’s ability to recall experiences or information stored in short-term memory. there are many ways to revise existing information, including revising the material with a list of presentations sequenced correctly according to the serial recall category, revising the material that is freely unstructured (free recall), and revising the material with instructions such as given essay questions (cued recall).18 regarding understanding the material on orofacial pain, in a study, around 34.8% of dental professional students said it was easy to understand, and 77.2% said it was not easy to understand. understanding is a level of knowledge defined as a person’s ability to interpret material correctly. a person with a good understanding can explain, conclude, and give examples of the material being studied. the study results showed that many students found orofacial pain material hard to understand because they could not recall the previously learned study material.19 the research results showed that many students found orofacial pain material hard to understand because to determine or conclude the etiology and management of orofacial pain, one must understand and know clinical symptoms such as pain onset, duration, and location, and not just a clinical picture. there is a need to understand and identify the risk factors associated with pain or the origin and triggers of pain.20 understanding chronic pain etiology and treatment has been challenging for all health fields. therefore, raising the standards of required competencies in dental training is essential to reduce the population burden of chronic pain and improve the clinical care of dental patients.20 the importance of understanding orofacial pain for students of the dental profession is to be able to determine the diagnosis and management of orofacial pain management. so they must understand the terminology and concepts of orofacial pain.8 the limitation of this research is that there is no relationship between intelligence and knowledge. in conclusion, students’ knowledge of orofacial pain is good, but the relationship between the level of intelligence and knowledge is not yet known. references 1. sessle bj, lavigne gj, lund jp, dubner r. orofacial pain: from basic science to clinical management. 2nd ed. quintessence publishing; 2009. p. 264. 2. lipton j, ship j, larach-robinson d. estimated prevalence and distribution of reported orofacial pain in the united states. j am dent assoc. 1993; 124(10): 115–21. 3. gilkey sj, plaza-villegas f. evaluation and management of orofacial pain. j am acad physician assist. 2017; 30(5): 16–22. 4. badel t. undergraduate students’ knowledge on temporomandibular disorders in croatia. acta clin croat. 2017; 56(3): 460–8. 5. srinivasan a, de cruz p. review article: a practical approach to the clinical management of nsaid enteropathy. scand j gastroenterol. 2017; 5(9): 1–7. 6. crandall ja. an introduction to orofacial pain. dent clin north am. 2018; 62(4): 511–23. 7. ziegeler c, wasiljeff k, may a. nondental orofacial pain in dental practices – diagnosis, therapy and self-assessment of german dentists and dental students. eur j pain. 2019; 23(1): 66–71. 8. borromeo gl, trinca j. understanding of basic concepts of orofacial pain among dental students and a cohort of general dentists. pain med. 2012; 13(5): 631–9. 9. iman s. hubungan antara adversiti dan intelegensi dengan kreativitas. j psikol. 2011; 9(1): 1–8. 10. cattell rb. intelligence: its structure, growth and action. elsevier; 1986. p. 694. (advances in psychology; vol. 35). 11. mallick-searle t, snodgrass b, brant j. postherpetic neuralgia: epidemiology, pathophysiology, and pain management pharmacology. j multidiscip healthc. 2016; 9: 447–54. 12. muñoz-quiles c, lópez-lacort m, díez-domingo j, orrico-sánchez a. herpes zoster risk and burden of disease in immunocompromised populations: a population-based study using health system integrated databases, 2009-2014. bmc infect dis. 2020; 20(1): 905. 13. ghom ag, ghom sa. textbook of oral medicine. 3rd ed. new delhi: jaypee brothers medical publishers; 2014. p. 1119. 14. bender sd. burning mouth syndrome. dent clin north am. 2018; 62(4): 585–96. 15. nalamachu s, morley-forster p. diagnosing and managing postherpetic neuralgia. drugs aging. 2012; 29(11): 863–9. 16. koole s, van den brulle s, christiaens v, jacquet w, cosyn j, de bruyn h. competence profiles in undergraduate dental education: a comparison between theory and reality. bmc oral health. 2017; 17(1): 109. 17. chouchene f, taktak n, masmoudi f, baaziz a, maatouk f, ghedira h. competency assessment of final-year dental students in tunisia. educ res int. 2020; 2020: 8862487. 18. dewi nnai, omegantini ms, dian npj. efektivitas media gambar terhadap recall memory pada mata pelajaran ips bagi siswa kelas iii sd. j psikol mandala. 2017; 1(1): 53–61. 19. retnaningsih r. hubungan pengetahuan dan sikap tentang alat pelindung telinga dengan penggunaannya pada pekerja di pt. x. j ind hyg occup heal. 2016; 1(1): 67. 20. costa ym, de koninck bp, elsaraj sm, exposto fg, herrero babiloni a, kapos fp, sharma s, shimada a. orofacial pain education in dentistry: a path to improving patient care and reducing the population burden of chronic pain. j dent educ. 2021; 85(3): 349–58. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p36–40 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p36-40 isi vol 39 no 3 juli-september 2006.pmd 126 anticarcinogenesis effect of gynura procumbens (lour) merr on tongue carcinogenesis in 4nqo-induced rat d. agustina*, wasito**, s.m. haryana***, and a. supartinah **** * oral medicine department, faculty of dentistry, gadjah mada university ** pathology department, faculty of veterinary medicine, gadjah mada university *** histology and molecular biology department, faculty of medicine, gadjah mada university **** pedodontics department, faculty of dentistry, gadjah mada university jogjakarta indonesia abstract in indonesia gynura procumbens (lour) merr leaves have been long used as various cancers medication. many in vitro and in vivo studies have demonstrated anticarcinogenesis of ethanol extract of gynura procumbens leaves. the aim of this study was to investigate the anticarcinogenesis of the ethanol extract of gynura procumbens leaves on 4 nitroquinoline 1-oxide (4nqo)-induced rat tongue carcinogenesis. fifty six 4 week old male sprague dawley rats were used in this study and divided into 7 groups. group 1, 2 and 3 were lingually induced by 4nqo for 8 weeks. in groups 2 and 3 the extract was given simultaneously with or after 4nqo induction finished, each for 10 weeks and 26 weeks, respectively. groups 4, 5 and 6 were induced by 4nqo for 16 weeks. however, in groups 5 and 6 the extract was given as well simultaneously with or after the 4nqo induction, each for 18 weeks, respectively. group 7 served as the as untreated control group. the results from microscopical assessment showed that tongue squamous cell carcinomas (scc) developed in 100% (3/3) of group 1. however, only 33.3% (2/6) and 25% (2/8) of rats in groups 2 and 3, respectively demonstrated tongue scc. among groups 4, 5 and 6, no significant difference of tongue scc incidence was observed. from these results it is apparent that the ethanol extract of gynura procumbens leaves could inhibit the progression of 4nqoinduced rat tongue carcinogenesis in the initiation phase. key words: gynura procumbens, 4nqo, rat, tongue carcinogenesis correspondence: d. agustina, c/o: bagian penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jln. denta sekip utara jogjakarta 55281, indonesia. according to interview with local residents in magelang, jawa tengah, gynura procumbens (lour) merr has been long used to treat several cancers such as leukaemia, uteric and breast cancers.3 this plant is easily found in indonesia and in various parts of south east asia. three fresh leaves are consumed daily for certain period of time, depending on the disease severity. previous study reported that consumption of the ethanol extract of gynura procumbens leaves might inhibit the occurrence of lung cancer up to 23% in newborn mice after being induced by benzopyrene on the first, eighth and fifteenth days. the extract dosage was equivalent to 100 mg simplisia of gynura procumbens leaves given twice weekly for 8 weeks.4 in vitro study also showed that the ethanol extract of gynura procumbens leaves had a cytotoxic effect on myeloma cell culture with lc50 of 72 μg/ml.5 recent data demonstrated that the extract was also effective in inhibiting the pre-initiation and initiation phases of stomach carcinogenesis in swiss mice after induction of benzopyrene, which is showed by, the percentage of animals bearing tumour and the number of tumour nodules.6 a similar result also occurred which is in mammary carcinoma of sprague dawley rat induced by dmba for introduction oral cancer is a serious public health problem although the incidence of this disease is much lower in comparison with, other malignancies such as prostate, colon, breast and uteric cancers. overall mortality rate of oral cancer remains high, at approximately 50%, despite of modern medical services, which is probably due to the advanced stage of the disease at presentation.1 around 86.8% of patients seeking medication usually have developed advanced condition or even metastatic.2 those patients tend to have poor prognosis and low survival rate, resulting a short life expectancy. conventional cancer treatment such as radiotherapy and chemotherapy often generate unfavorable effects to the patients, such as lowering the patient‘s immune system due to the destruction of normal cells. the extent of involved areas causing surgical treatment to create disfigurement and dysfunction of oral and facial tissues, which further leads to quality of life decrease in oral cancer patient. based on these facts, currently more indonesians return to the use of traditional herbal remedies as an empirical alternative therapy which is potential to cure cancers by loweing the side effects with a lower cost. 127agustina et al.: anticarcinogenesis effect of gynura procumbens (lour) merr 10 times with a dosage of 20 mg/kg body weight during the initiation phase. the incidence of mammary carcinoma was reduced up to 40% until the end of the experiment.7 the most recent study showed an anti angiogenic effect of the ethanol extract of gynura procumbens leaves on the chorioallantois membrane of chicken after bfgf induction.8 those findings promote the anti carcinogenic potency of gynura procumbens leaves for cancer therapy which should be elucidated in more detail. current literatures, report no previous study or research on anti carcinogenic effect of the ethanol extract of gynura procumbens leaves in oral carcinogenesis. in the light of different etiology that leads to different pathways in oral carcinogenesis, it is interesting to investigate the role of the ethanol extract of gynura procumbens in inhibiting the oral carcinogenic process. the goal of the present study is to elucidate the inhibitory effect of the ethanol extract of gynura procumbens leaves in oral carcinogenesis by comparing clinical and histopathological appearances in rat tongues between rats given 4 nitroquinoline 1-oxide (4nqo) and the extract with those given by 4nqo alone. 4 nitroquinoline 1-oxide (4nqo) is a full carcinogenic agent. it can function either as an initiator or a promoter of cancer process. it can also be classified as an indirect carcinogen since it must be metabolically activated in vitro to produce “ultimate carcinogens”, which are involved in the induction of tumours.9 previous in vitro studies have been used 4nqo to induce malignancy in oral cavity, either by water drinking or direct application on oral mucosal tissue for a certain a period of time.10-14 squamous cell carcinoma (scc) produced by 4nqo behaved in a similar way to those of humans.11 according to nauta et al.15 creating an oral carcinogenic model in rat by inducing 4nqo is appropriate to mimic a human oral carcinogenesis since they have similarities either in histopathologic or immunologic characteristics. the significance of this study may serve as scientific evidence for the industrial development of gynura procumbens leaves as anti oral carcinogenic agent. materials and methods animals used in this study were 56 male sprague dawley rats (f54), aged 4 weeks with average body weight of 63 g (ppom, food and drug research center, jakarta, indonesia). carcinogen agent used was 4-nitroquinoline 1-oxide (4nqo) (sigma chemical company, australia), dissolved in propane-1,2-diol (pd) (sigma aldrich chemic gmbh, germany) to a final concentration of 0.5% (m/v). ethanol extract of gynura procumbens (lour) merr leaves was obtained by soxhletation method, with 80% ethanol and suspended in 0.5 ml aquadest. 4 nitroquinoline 1-oxide (4nqo) solution was applied using a no. 2 artists’ brush which delivered a relatively constant volume (0.15 mg). a single brush stroke was applied from posterior to anterior of the dorsal surface of rat tongue three times weekly. the extract was given through oral intubation twice weekly with a dosage per administration equivalent to 3.5 g dry leaves (simplisia)/kg body weight. figure 1. design of the study. ................................................................................................................................................................ ................................................................................................................................................................ ................................................................................................................................................................ ................................................................................................................................................................ ................................................................................................................................................................ 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........................................................... ........................................................... ........................................................... .......................................................... .......................................................... .......................................................... 0 1 9 10 17 18 36 wks g-1 g-2 g-3 g-4 g-5 g-6 g-7 : 4nqo application administration : diet and tap water : ethanol extract of ad libitum gynura procumbens leaves .............................. 128 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:126–132 design of the study (figure 1): group 1 (n = 6) consisted of rats given 4nqo alone for 8 weeks (4nqo-8 wk); group 2 (n = 9) consisted of rats given 4nqo for 8 weeks and the extract simultaneously. the ethanol extract of gynura procumbens was administered for 10 weeks commencing one week before the 4nqo exposure and terminated one week after 4nqo induction finished (4nqo-8 wk + extract); group 3 (n = 11) consisted of rats given 4nqo for 8 weeks and the extract afterwards. the extract was administered for 26 weeks, starting 1 week after the cessation of 4nqo treatment until the end of the experiment (4nqo-8 wk → extract); group 4 (n = 9) consisted of rats given 4nqo alone for 16 weeks (4nqo-16 wk); group 5 (n = 9) consisted of rats given 4nqo for 16 weeks and the extract simultaneously. the ethanol extract of gynura procumbens was administered for 18 weeks commencing one week before the 4nqo exposure and terminated one week after 4nqo induction finished (4nqo-16 wk + extract); group 6 (n = 6): rats were given 4 nqo for 16 weeks and the extract afterwards. the extract was administered for 18 weeks, starting 1 week after the cessation of 4nqo treatment until the end of the experiment (4nqo-16 wk → extract); group 7 (n = 6) served as an untreated control group. animal experimentation: every 2-4 rats were housed in stainless steel and plastic cages at constant humidity (50–55%) and temperature (± 25 º c) with 12 hours light in the animal house of pharmacology and toxicology department, faculty of pharmacy, gadjah mada university, jogjakarta, indonesia. they were fed ad-ii pellets (pt japfa, sidoarjo, indonesia) and had tap water available ad libitum. during the course of study, the animals were weighed biweekly and general inspection of the general health was carried out daily to record any macroscopic changes. should the general condition deteriorate significantly, the rat was terminated by overdosed ether. to observe the progression of tongue carcinogenesis, three rats from groups 1, 2, 3, 4, 5, and 7 were terminated in the middle of the experiment period (the 19th week) to allow histological examination. at the end of the 36th week, all animals were terminated and all tongues and other organs of rats detected with pathological lesions were removed. for histological examination, tissues and gross lesions were fixed in 10% buffered formalin, embedded in paraffin blocks and stained with hematoxylin-eosin (h-e). tongue lesions were diagnosed according to the criteria of who: “histological typing of cancer and precancer of the oral mucosa”.16 results at the beginning of the experiment, both the experimental and control groups appeared to be in good health assessed by subjective assessment of their physical mobility and fur luster. there was no significant difference of the body weight among the groups of the experiment at the end of the study. in general, the health of the experimental rats throughout the experimental period appeared poorer than that of the untreated rats. three rats from groups 1, 2, 3, 4, 5, and 7 (total: 18 rats) were terminated in the middle of the experiment for observation of carcinogenesis progression. histological assessment to those tongues demonstrated no dysplastic changes occurred in groups 1 (4nqo-8 wk), 2 (4nqo-8 wk + extract) and 3 (4nqo-8 wk → extract). however, each one of three rats from groups 4 (4nqo-16 wk) and 5 (4nqo-16 wk + extract) had tongue scc. the tongue of three rats from group 7 (untreated control) remained normal. nineteen (50%) rats survived until the end of the experiment (the 36th week), however the other nineteen (50%) had to be necropsied before the end of the experiment. three of group 2, five of group 3, two of group 4, five of group 5 and four of group 6 failed to survive until the 36th week. possible causes of deaths were the presence of tongue scc (10 of 19 rats, 53%) and systemic problems (9 of 19 rats, 47%) such as chronic respiratory disease (crd), splenomegaly and liver infection of taenia crassicollis. those systemic problems were also noted in the survivors (17 of 19 rats, 89%). determination of systemic problems presence was based on histopathological table 1. histopathological examination of 38 rats’s tongues from rats necropsied either before or at the end of the experiment histopathological examination group no. of rats n h mid mod sed cis miescc wellscc scc incidence (%) 1 2 3 4 5 6 7 3 6 8 6 6 6 3 0 0 0 0 0 0 3 0 3 3 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 0 2 2 2 5 5 5 0 100 33.3 25 100 100 83.3 0 group 1: 4nqo-8 wk; group 2: 4nqo-8 wk + extract; group 3: 4nqo-8 wk → extract; group 4: 4nqo-16 wk; group 5: 4nqo-16 wk + extract; group 6: 4nqo-16 wk → extract; group 7: untreated control; n: normal; h: hyperplasia; mid: mild dysplasia; mod: moderate dysplasia; sed: severe dysplasia; cis: carcinoma in situ; micscc: micro-invasive squamous cell carcinoma; wellscc: well differentiated squamous cell carcinoma; for scc incidence, cis, micscc and wellscc were categorized as scc. 129agustina et al.: anticarcinogenesis effect of gynura procumbens (lour) merr examination with he staining. macroscopical or clinical appearances of 38 rat tongues showed hyperkeratosis, white, granular/nodular, thickening (figure 3), ulcerative (figure 4) lesions or the presence of tumor mass (figure 5) on the posterior dorsal surface of the tongue, which could be clearly differentiated from the normal condition (figure 2). lesion diagnosed as well differentiated scc might have macroscopical appearance as hyperkeratosis, granular/nodular, thickening, ulcerative lesions or tumor mass. results histopathological examination of he sections on 38 rats' tongues from rats necropsied either before or after the experiment are shown in table 1 (note: 18 rats necropsied in the middle of the experiment were not included). percentage according to histopathological diagnosis in groups of study is demonstrated in table 2 and on figure 6 in which mild, moderate and severe dysplasia were categorized as dysplasia and carcinoma in situ (cis), micro-invasive scc and well differentiated scc were grouped together in scc. normal histological appearance of the dorsal surface of the rat tongue is characterized by regular epithelial stratification. there is no epithelial down growth and polarity of the basal cells is significantly present (figure 7). moderate hyperplasia was especially demonstrated by epithelial down growth and basal and spinous cells proliferation. on the other hand, bulbous rete ridges was relatively prominent as well (figure 8). severe dysplasia showed that more severe atypic cells involving the whole thickness of epithelial layer, irregular epithelial stratification and loss of intercellular adherence (figure 9). well differentiated scc was characterized by the prominent appearance of keratinization on tumor cells more than 75% and many tumor nests infiltrated into lamina propria even deeper to musculatory area (figure 10). scc: squamous cell carcinoma table 2. percentage according to histopathological diagnosis in groups of study group of study diagnosis 1 2 3 4 5 6 7 normal hyperplasia dysplasia scc 0 0 0 100 0 50 16.7 33.3 0 37.5 37.5 25 0 0 0 100 0 0 0 100 0 0 16.7 83.3 100 0 0 0 figure 2. macroscopical appearances of dorsal surface of rat tongue indicated as normal appearance from untreated control group. figure 3. a white thickening lesion (an arrow) taken from a rat from group 2 (4nqo-8 wk + extract) that has been histologically diagnosed as hyperplasia. figure 4. an ulcerative lesion (an arrow) taken from a rat from group 3 (4nqo-8 wk → extract) that has been histologically diagnosed as well differentiated scc. figure 5. a tumor mass (an arrow), a pedunculated lesion taken from a rat from group 4 (4nqo-16 wk) that has been histologically diagnosed as well differentiated scc. 130 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:126–132 figure 7. microscopical appearances of dorsal surface of rat tongue indicated as normal appearance from untreated control group, h&e, 100x. figure 8. moderate hyperplasia, h&e, 100x taken from a rat from group 3 (4nqo-8 wk → extract). figure 9. severe dysplasia, h&e, 200x taken from a rat from group 3 (4nqo-8 wk → extract). figure 10. well differentiated scc, h&e, 100x taken from a rat from group 1 (4nqo-8 wk). percentage according to histopathological diagnosis in groups of study 100 80 60 normal hyperplasia40 dysplasia 20 scc groups of study figure 6. percentage based on the histopathological assessment of lingual mucosa of 7 groups of rats used in this study. 131agustina et al.: anticarcinogenesis effect of gynura procumbens (lour) merr discussion decreased general health in treated rats was especially the result of the 4nqo treatment. 4nqo treatment led to the presence of a big tumor mass on the dorsal surface of the tongue caused rats to have difficulties in eating, causing limitation of food consumption. on the other hand, the long duration of the experiment caused rats more susceptible to many infection from their environment. this assumption was based on the finding that almost 50% of unsurviving rats and 89% of surviving rats had crd, splenomegaly or liver infection of worm cyst such as taenia crassicollis. this incidence suggests that the longer the experimental duration the more susceptible the animals are to infectious agents. the severity of infection was indicated by the number of animals suffering splenomegalies. other factors that contribute to crd and liver infection by taenia crassicollis were sanitation, maintenance and humidity of the bedding. in this study, replacement of the cage, drinking bottle and bedding were carried out once a week. infection might be prevented by more frequent sanitation maintenance.17 histopathological examination on rat tongue in the middle of the experiment (the 19th week) to observe the progression of tongue carcinogenesis indicated that the longer the 4nqo induction the earlier tongue scc will develop. this statement is supported by the finding that no scc developed until the 19th week in rats given 4nqo for 8 weeks. however, two out of 6 rats developed tongue scc by the 19th week in groups induced by 4nqo for 16 weeks. this means that longer 4nqo induction might cause more genetical changes, leading to a faster progression of the carcinogenic process. according to tanaka et al.,18 the initiation phase in 4nqo-induced tongue carcinogenesis in rat was achieved by 4nqo induction for ≤ 10 weeks. the data on the incidence of tongue neoplasms among the groups 1, 2 and 3 (table 1) indicated that administration of ethanol extract of gynura procumbens leaves on the initiation phase (8 wk 4nqo induction) could suppress the progression of tongue carcinogenesis in the models used. however, the inhibition was more effective if the extract was given in a longer period than 4nqo induction after the animals have been initiated as conducted in group 3 in which the reduction of tongue scc incidence was up to 75%. if the administration of the extract was calculated, rats in group 2 were only given the extract for 10 weeks, but rats in group 3 were given the extract for 26 weeks. it suggests that the longer the administration of the extract against the induction of carcinogen, the more the suppression on oral carcinogenesis leading to a lower incidence of tongue scc. the above assumption was proven by the results of groups 4, 5 and 6 were compared. it was clear that in the groups 5 and 6, the duration of 4nqo induction compared to that of the extract administration was not so different (the 4nqo: 16 wk, the extract: 18 wk), so the inhibition of tongue carcinogenesis could not be achieved. apparently, more frequency of extract administration was needed to be able to inhibit the progression of tongue carcinogenesis. our study suggests that gynura procumbens leaves are possible new dietary preventive agents against tongue carcinogenesis. anticarcinogenic component contained in the leaves is especially flavonoids. flavonoids are found include in polyphenolic compounds that might be able to dysregulate cancer development. these include antioxidant activities,19 the scavenging effect on activated carcinogens and mutagens,20,21 the action on proteins that control cell cycle progression,22 and altered gene expression.23 the core structure of the flavonoids, 2-phenyl-4h-1-benzopyran-4one (flavone) affects proliferation, differentiation, and apoptosis in human colon carcinoma cell line. even its apoptotic inducer is stronger than clinically established anti tumour agent, camptothecin.24 the results of the study corroborated the results of the previous investigations that administration of ethanol extract of gynura procumbens leaves could block the pre-initiation and initiation phases of stomach carcinogenesis in swiss mice induced with benzopyrene 6 and that of mammary carcinoma in sprague dawley rat induced by dmba7 and the extract could reduce the occurrence of lung tumor up to 23% in new born mice after being of after induction with benzopyrene.4 in conclusion, the results of our study demonstrate the inhibitory effect of ethanol extract of gynura procumbens (lour) merr leaves in the initiation phase of 4nqo-induced tongue carcinogenesis in rats especially viewed from clinical and histopathological aspects. further experiments to elucidate the inhibitory mechanism of gynura procumbens leaves in molecular level is strongly recommended for the development of chemopreventive agent of gynura procumbens in oral carcinogenesis. acknowledgements this study was a part of ad’s ph.d. research partially supported by bpps funding of the indonesian government, hibah bersaing xii project of the indonesian government (2004), human resources development of gadjah mada university and by research institution of gadjah mada university (dipa). references 1. walker dm, boey g, mcdonald la. the pathology of oral cancer. pathology 2003; 35(5): 376–83. 2. sajid m. frequency of the oral cancer at the dental clinic of the dr. sardjito hospital during 3 years. scientific abstracts-free paper session on first international congress on oral cancer and jaw tumours, singapore, 1987. 3. sudarto b. daya antibakteri minyak atsiri daun dewa (gynura procumbens (lour) merr. laporan penelitian. 1,3. jogjakarta: fakultas farmasi ugm; 1991. h. 3. 4. sugiyanto, sudarto b, meiyanto e. efek penghambatan karsinogenisitas benzo(a)piren oleh preparat tradisional tanaman gynura sp. dan identifikasi awal senyawa yang berkhasiat. laporan penelitian p4m ditjen dikti. jogjakarta: fakultas farmasi ugm; 1993. h. 29–51. 132 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:126–132 5. arianti s. aktivitas biologis ekstrak etanol daun gynura procumbens (lour) merr terhadap sel vero dan sel mieloma. skripsi. jogjakarta: fakultas farmasi ugm; 1998. h. 26–46. 6. ulfa em. pengaruh pemberian ekstrak etanol daun gynura procumbens (lour) merr pada pre-inisiasi sampai fase inisiasi pertumbuhan tumor lambung mencit karena benzo(a)piren. skripsi. jogjakarta: fakultas farmasi ugm; 2000. h. 39–57. 7. susilowati s. efek kemopreventif ekstrak etanolik daun gynura procumbens (lour) merr terhadap kanker payudara tikus yang diinduksi 7,12-dimetil benz(a) antrasen (dmba). tesis. jogjakarta: pascasarjana ugm; 2004. h. 44–70. 8. jenie ri, meiyanto e, murwanti r. efek antiangiogenik ekstrak etanolik daun sambung nyawa (gynura procumbens (lour) merr) pada membran korio alantois (cam) embrio ayam. majalah farmasi indonesia 2006; 17(1):50–5. 9. miller ec. some current perspectives on chemical carcinogenesis in humans and experimental animals: presidential address. cancer res 1978; 38:1479–96. 10. wallenius k, lekholm u. oral cancer in rat induced by the watersoluble carcinogen 4-nitroquinoline n-oxide. odont revy 1973; 24:39–48. 11. steidler ne, reade pc. experimental induction of oral squamous cell carcinomas in mice with 4-nitroquinoline 1-oxide. j oral surg 1984; 57:524–31. 12. steidler ne, rich am, reade pc. experimental induction of preneoplastic and neoplastic changes in the lingual mucosa of rats. j biol buccale 1985; 13:339–46. 13. ohne m, satoh t, yamada s, takai h. experimental tongue carcinoma of rats induced by oral administration of 4-nitroquinoline 1-oxide (4nqo) in drinking water. oral surg oral med oral pathol 1985; 59:600–7. 14. prime ss, malamos d, rosser t, scully c. oral epithelial atypia and acantholytic dyskeratosis in rats painted with 4-nitroquinoline n-oxide. j oral pathol 1986; 15:280–83. 15. nauta jm, roodenburg jln, nikkels pgj, witjes mjh, vermey a. comparison of epithelial dysplasia: the 4nqo rat palate model and human oral mucosa. int j oral maxillofac surg 1995; 24:53–58. 16. pindborg jj, reichart pa, cj smith cj, van der waal i. world health organization international histological classification of tumours: histological typing of cancer and precancer of the oral mucosa. 2nd ed. berlin: springer; 1997. p. 11–13, 24–26, 28, 44–46, 68–69, 81. 17. smith jb, mangkoewidjoyo. pemeliharaan, pembiakan dan penggunaan hewan percobaan di daerah tropis. 1st ed. jakarta: ui press; 1988. p. 49–54. 18. tanaka t, kohno h, sakata k, yamada y, hirose y, sugie s, mori h. modifying effects of dietary capsaicin and rotenone on 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. carcinogenesis 2002; 23(8):1361–67. 19. duthie sj, dobson vl. dietary flavonoids protect human colonocyte dna from oxidative attack in vitro. eur j nutr 1999; 38:28–34. 20. williamson g, faulkner k, plumb gw. glucosinolates and phenolics as antioxidants from plant foods. eur j cancer prev 1998; 7:17–21. 21. calomme m, pieters l, vlietinck a, vanden berghe d. inhibition of bacterial mutagenesis by citrus flavonoids. planta med 1996; 62:222–6. 22. plaumann b, fritsche m, rimpler h, brandner g, hess rd. flavonoids activate wild-type p53. oncogene 1996; 13:1605–14. 23. gerritsen me. flavonoids: inhibitors of cytokine induced gene expression. adv exp med biol 1998; 439:183–90. 24. wenzel u, kuntz s, brendel md, daniel h. dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. cancer res 2000; 60:3823–31. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 49 no 3 juli-sept 2016.indd 158 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 158–162 inhibitory effects of siwak (salvadora persica. l) extract on the growth of enterococcus faecalis planktonics and biofilms in vitro ika rhisty cendana sari,1 rini devijanti ridwan,2 and diah savitri ernawati3 1badan penyelenggara jaminan sosial (bpjs) kesehatan, malang-indonesia 2department of biology oral, faculty of dental medicine, universitas airlangga, surabaya indonesia 3department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: enterococcus faecalis (e. faecalis) is one of the most persistent gram positive bacteria in root canal, resulting in secondary infection after endodontic treatment. e. faecalis pathogenicity is caused by overgrowth of e. faecalis planktonics and biofilms. e. faecalis planktonics produce lipoteichoid acid (lta) as a virulence factor that can defend their permeability cell. on the other hand, e. faecalis biofilms produce protease, such as esp (enterococcal surface protein), gele (gelatinase), and spre (serin protease), that have quorum-sensing mechanism as an adhesion factor to form extracellular polysaccharide substance (eps) and increase the growth of the biofilms themselves. siwak (salvadora persica l.) has active components, namely benzylisothio-cyanate, trimethylamine, and salvadorine that can inhibit the growth of e. faecalis planktonics and biofilms. purpose: this study aimed to measure inhibitory effects of siwak extract on the growth of e. faecalis planktonics and biofilms. method: this research was an antimicrobial research on the culture of e.faecalis incubated in a tsb medium. siwak extract was diluted into different concentrations, namely 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 100%. the extract then was placed into the e. faecalis’s colony and planted into trypticase soy agar medium. after incubated for 24 hours at 37°c, the colony would be measured and compared with the control (+) and control (-). as an antibiofilm research, this research used biofilm microtitter assay method to form e. faecalis biofilms incubated in a well-plate medium containing tsb and 0.1 % glucose. siwak extract then was diluted into the same range concentration as in first method, and placed into the colony of e. faecalis to form biofilms. the biofilms were measured and compared to the control (+) given siwak extract and the control (-) given 0.1% chlorhexidine. after the incubation, they were washed three times, and staining process was conducted using chrystal violet. the optical density then was measured by elisa reader 595 nm. result: siwak extract could inhibit the growth of e. faecalis planktonics at the concentration of 35% as a minimum inhibitory concentration as well as the growth of e. faecalis biofilms at the concentration of 45% as a minimum biofilm inhibitory concentration. conclusion: siwak extract has an inhibitory effect, particularly at a concentration of 35% on the growth of e. faecalis planktonics and at the concentration of 45% on the growth of e. faecalis biofilms. keywords: enterococcus faecalis; siwak; antibacterial; antibiofilm; benzylisothio-cyanate; trimethylamine; salvadorine correspondence: rini devijanti ridwan, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: devi.rini@yahoo.co.id dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p158-162 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p158-162 159159sari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 158–162 introduction enterococcus faecalis (e. faecalis) is one of the most resistant gram-positive bacteria in root canal. e. faecalis can cause a secondary infection of the root canal after endodontic treatment. e. faecalis bacterial pathogenicity can be triggered by the growth of e. faecalis planktonics and biofilms.1 one of the herbs currently widely used by society in maintaining their oral health is siwak rod (salvadora persica.l). siwak is known to have a wide variety of useful components. trimethylamine and benzyl-isothiocyanate in siwak serve to reduce the occurrence of adhesion to the tooth surface and inhibit the accumulation of plaque. antimicrobial and cleaning effects of siwak have been indicated by chemical content variations, such as high benzyl-isothiocyanate, specific alkaloid salvadorine, trimethylamine, saponins, flavonoids, tannins, vitamin c, plant sterols, silica, sodium chloride, resins, and potassium chloride (salvadourea).2 moreover, siwak extract with distilled solvent, based on a previous research conducted by abdelrahman, can inhibit the growth of streptococcus mutans and streptococcus faecalis at a concentration of 50%. another previous research conducted by almas also shows that siwak extract with ethanol at a concentration of 25% can lead to the absence of the bacterial growth of s. mutans and streptococcus sanguinis.3,4 those bacteria are gram-positive bacteria commonly found in endhodontic infections.5 those results of the previous researches underlie the objective of this research to measure the effectiveness of siwak extract in inhibiting the growth of e. faecalis biofilms since siwak extract contains potential substances that can be used as an irrigation and antiseptic material. in other words, this research aimed to reveal whether siwak extract (salvadora persica.l) could inhibit the growth of e. faecalis and its biofilms by in vitro. materials and method this research was an experimental study with true experiment-post test only control group design. this research was conducted at the laboratory of rspti (hospital of tropical and infectious diseases) in surabaya form june 2014 to october 2015. the research samples used for the inhibition test of the growth of bacteria were e. faecalis bacterial cultures obtained from the laboratory of hospital of tropical and infectious diseases in surabaya. inoculum of each bacterium was taken from the cultures, incubated on trypticase soy broth at 37° c temperature for 18 hours, and then diluted with 0.85% sterile nacl solution (sigma-aldrich inc., st. louis, mo, usa) to achieve suspension turbidity equivalent to 0.5 mc.farland standard.1,5 in addition, the research samples used for the inhibition test of the growth of bacteria were e. faecalis bacterial cultures that could form biofilms indicated through 100 μl of cultured bacteria suspension in polyprophylene tube containing 2 ml of trypticase soy broth (tsb) (merck milipore inc., darmstadt, germany) with the addition of 1% glucose for 48 hours at a temperature of 37° c. after 48 hours of the incubation, biofilm cells were harvested by removing the culture medium, and then the tube was rinsed three times with 200 μl of phosphate buffer saline (ph 7.2) (sigma-aldrich inc., st. louis, mo, usa) to remove non-adhesive part of the bacteria. meanwhile, the adhesive part of the bacteria was harvested through vortex and centrifugation processes. the pellets then were suspended in pbs to be conditioned in 0.5 mcfarland turbidity standard (cloudiness).1 the number of samples in each type of treatment, moreover, was four (4) samples. those samples in each type of treatment were treated using the siwak extract through sokhletation process at the concentrations of 100% (1 g/ ml), 75% (0.75 g/ ml), 70% (0.7 g/ ml), 65% (0.65 g/ ml), 60% (0.6 g/ ml), 55% (0.55 g/ ml), 50% (0.5 g/ ml), 45% (0.45 g/ ml ), 40% (0.4 g/ ml), 35% (0.35 g/ ml), 30% (0.3 g/ ml), and 25% (0.25 g/ ml) obtained through dilution method. 10 ml of the siwak extract was planted on each culture in a petri dish using a micropipette, and then incubated on solid mueller hinton media (biomerieux industry, inc., philadelphia, pa, usa) for 24 h at 37° c. the number of colonies then was counted using a colony counter. to measure inhibitory effects on the bacterial biofilm growth, each well containing 100 μl of tsb + 1% glucose was added with 100 μl of e. faecalis bacterial cultures proven to produce biofilms. 100 μl of the siwak extract then was added into the wells of each treatment, and incubated for 48 hours at 37° c. after that the well-plates were placed in an anaerobic jar for 24 hours. the wells then were washed 3x with 0.2 ml of pbs, and then staining process was conducted using 0.2 ml of crystal violet. they were rinsed again with distilled water, and then added with 0.2 ml of sterile acidified isopropanolol (honeywell international inc., morristown, nj, usa).1 optical density (od) values in the wells of each treatment were read using elisa reader with a wavelength of 595 nm (bio-rad laboratories, inc., berkeley, ca, usa).1 results the results showed that the growth of the bacteria declined as the increasing of the concentration of the siwak extract used. the growth of e. faecalis bacteria was detected after the provision of the siwak extract at the concentrations of 25% to 35%. meanwhile, there was no bacterial colony grown after the provision of the siwak extract at the concentration of 40%. furthermore, there was also no bacterial colony grown after the provision of the siwak extract at the concentrations of 45% to 100%. the results of the anova test, furthermore, indicated that there were significant differences between the research dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p158-162 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p158-162 160 sari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 158–162 table 1. results of lsd test on bacterial inhibitory effects concentration (%) number of samples sd 25 4 102.956 30 4 0.68981 35 4 0.76158 40 4 .00000c 45 4 .00000c 50 4 .00000c 55 4 .00000c 60 4 .00000c 65 4 .00000c 70 4 .00000c 75 4 .00000c 100 4 .00000c control (+) 4 373.363 control (-) 4 .00000c table 2. results of lsd test on bacterial biofilm inhibitory effects concentration (%) number of samples sd 25 4 0.0133 30 4 0.01794 35 4 0.03244 40 4 0.0466 45 4 0.01117 50 4 0.02193 55 4 0.01439 60 4 0.03063 65 4 0.01192 70 4 0.022411 75 4 0.0229 100 4 0.00569 control (-) 4 0.02723 (chlorhexidine) control (+) medium + bacteria) 4 0.11877 extract control 4 0.0637 (only extract) staining control 4 0.00356 (only medium) groups (p value <0.05). thus, least square differences (lsd) test was performed. the results of the lsd test demonstrated that there was no significant difference between the provision of the extract at the concentration of 35% and that at the higher concentration. therefore, the siwak extract at the concentration of 35% was considered as the minimum inhibitory concentration against the growth of e. faecalis bacteria. in addition, the results of the optical density readings indicated that the growth of bacterial biofilms tended to decrease, in line with the increasing of the concentration of the siwak extract used, characterized by the decreased values of optical density and the adhesive cell attachment at the less visible staining. the results of the lsd test, moreover, showed that there was no significant difference between the provision of the siwak extract at the concentration of 25% and at the concentration of 40%. there was a significant difference between the provision of the siwak extract at the concentration of 45% and at the lower concentration. the concentration of 45%, as a result, can be considered as the minimum inhibitory concentration for biofilms. further analysis found that there was no significant difference between the concentration of 45% and the higher ones, namely 50%, 55%, 60%, 65%, 70%, and 75%. in other words, the lowest concentration of the siwak extract to inhibit the growth of biofims was 45%. however, siwak extract at the concentration of 100% was not significantly different from the extract at the concentrations of 50% to 75%. discussion the growth of e. faecalis bacteria can continually occurs by maintaining the resistance of the bacterial cell wall and cell membrane in the presence of lipoteichoid acid (lta), capable of performing defense of external bacterial membrane permeability and peptidoglycan playing a role in maintaining the cell shape.6 a previous research even shows that siwak extract with distilled solvent can inhibit the growth of s. mutans and s. faecalis bacteria from a low concentration to a concentration of 50%.3,4 another in vitro research shows that the use of ethanol in siwak extract at a concentration of 25% triggers no growth of s. mutans and s. sanguinis bacteria classified as cocci-shaped gram-positive bacteria, similar to e. faecalis, involved in endodontic infections. consequently, this research used the same material extract.3,4 in this research, ethanol at a concentration of 35% used in the siwak extract significantly inhibited the growth of e. faecalis bacteria. the concentration of 35% could be considered as the minimum inhibitory concentration. it means that siwak extract had an anti-bacterial ability, almost similar to the result of the previous research, but it requires a slightly higher concentration to inhibit e. faecalis bacteria than to inhibit other gram-positive bacteria. in other words, siwak, extracted using alcohol solvent, is an effective antibacterial material to inhibit e. faecalis bacteria.4,7 this inhibitory effects on the growth of e. faecalis bacteria indicate that there are disturbances in mechanisms of peptidoglycan and lipoteichoic acid (lta) as well as metabolism and virulence of e. faecalis. siwak extract is considered as an effective anti-bacterial material due to benzylisothio-cyanate, salvadorine, and trimethylamine dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p158-162 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p158-162 161161sari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 158–162 contained.8 a previous research using mass spectroscopy shows that benzylisothio-cyanate is the largest component contained in the siwak extract. benzylisothio-cyanate can trigger oxidase process, thus inhibiting the mechanism of bacterial metabolism. meanwhile, salvadorine as one of specific alkaloids in siwak can inhibit both activity of enzymes produced by bacteria in synthesizing protein and synthesis of cell walls, interfere metabolic processes of bacteria, as well as produce organic anionic group resulting in unstable bacterial cell membrane leading to lysis and cell death.8 the mechanism of siwak extract in inhibiting the growth of e. faecalis bacteria correlates with a previous research showing that 17% edta, caoh2, and 0.2% chlorhexidine also have an anti-bacterial ability against e. faecalis. 17% edta alter cell membrane permeability, thus destroying the bacterial cell wall. chlorhexidine works by damaging the cytoplasmic membrane of bacterial cells so the intracellular matrix secreted from the cells, leading to lysis.9 caoh2 can also be considered as an antibacterial material due to its ability to damage lta, known as a major virulence factor in gram-positive bacteria. in other words, the mechanism of siwak extract is similar to the endodontic treatment material that inhibits the growth of e. faecalis bacteria.8 the growth of e. faecalis is known to generate biofilms. biofilms can form and grow due to adhesion factors and aggregation substance, such as exopolysaccharide, as well as a variety of biofilm associated protein. one of proteins associated to the biofilm formation is esp which acts as a specific protein in inducing bacterial colonization and bacterial colony attachment on the surface of the substrate. esp together with other specific proteins, such as serine protease (spre) can initiate e. faecalis biofilm formation, both in vitro and in vivo. the process of e. faecalis biofilm formation is also triggered by both ace (collagen binding antigen) that has strong adhesion to dentin, as well as gele that plays a role in the process of bacterial quorum-sensing and induces extracellular matrix as a response to biofilm formation.10 the whole of virulence factors that are able to form extracellular polymeric substances (eps) are attached to the bacteria themselves and correlated to the formation of bacterial biofilms. adhesions occur in stages. the first stage occurs in a reversible way, then becoming into permanent (irreversible) and undergoing maturation and aggregation to form biofilms. biofilm formation itself is not essential to trigger pathogen infection, but increased bacterial resistance in bacteria when forming biofilms can trigger virulence factor in bacteria to work maximally, leading to increased pathogenicity.6 this eps formation process is influenced by signal transduction mechanisms, called as quorum-sensing. bacterial adhesions occur in the surface of the matrix, in which bacteria form stable microcolonies and emit chemical signals as communication between cells. when communication between these cells reach threshold intensity level, eps production will be activated so that the attachment of microcolonies multiplies and thickens resulting in the attachment of macrocolonies to the extracellular matrix. in other words, the thicker the layer forming biofilms is, the lower the nutrient and ph conditions in the surface of the matrix is, encouraging the release of a colony form the outermost layer of the matrix to alter again into bacterial planktonics.11,12 with the increasing of planktonic cells due to the release of the colony, the virulence and pathogenicity of e. faecalis bacteria will increase.13,14 in the second previous research, siwak extract is tested on e. faecalis biofilms. the main elements contain in siwak extract, such as benzylisothio-cyanate, trimethylamine, and salvadorine, also have effectiveness against bacterial biofilms. trimethylamine in siwak can inhibit the adhesion factor on the surface of the substrate thereby inhibiting the formation of bacterial biofilms. benzylisothio-cyanate, on the other hand, can react to sulfhydryl groups contained in the protein enzyme associated to biofilm formation, leading to cell death and biofilm-forming inhibition. benzylisothiocyanate in siwak has led to anti-quorum sensing capability of the enzyme protein produced by e. faecalis. 6,15 in another previous research on e.faecalis biofilms, microtiter biofilm assay method is used to see the effects of fsr gene on the activation of gelatinase-serine protease (gele-spre) operon expressions. the mechanism of gele-spre operon also becomes quorum-sensing in the formation of e. faecalis biofilms. thus, it can be concluded that the result of this previous research indicates that this method are relevant to the activities of specific genes in the formation and growth of e. faecalis biofilms resulting in tissue destruction causing endodontic infection.13,14 by using microtiter assay method, the optical density (od) values of the bacterial biofilms obtained was read by elisa reader with a wavelength of 595 nm. od values measured based on turbidity colors absorbed by the biofilms have several complicating factors, such as turbidity of extracts used that can affect the thickness of the biofilms contained.6 consequently, in this research staining and turbidity were controlled, so the staining and turbidity of siwak extract did not significantly affect. thus, in this research the smaller the concentration of siwak extract was used, the higher the od values were. in other words, the larger the od values were obtained, the thicker the biofilms were generated. the thicker biofilms indicates the greater resistance to anti-bacterial material. it means that anti-bacterial material will be more difficult to penetrate into the biofilms. as a result, the growth and formation of the biofilms will be more difficult to inhibit. bacteria that can form biofilms have a slowdown in their metabolic rate and bacterial planktonic colony growth.6 this slow metabolic rate contributes to bacterial resistance to antibiotics, one of which is ß-lactam. antibiotics work by targeting growth factors working during the high bacterial growth, so they become not optimum and resistant to antibiotics.6 similarly, the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p158-162 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p158-162 162 sari, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 158–162 results of this research showed that siwak extract as an alternative for anti-biofilm material that could disturb the mechanism of bacterial resistance and inhibit the growth of the bacteria. therefore, it can be said that siwak extract has effectiveness as anti-biofilm agent against e. faecalis bacteria at a minimum concentration of 45%. nevertheless, it took a greater concentration to inhibit the growth of e. faecalis biofilms than to inhibit the growth of e. faecalis planctonics in this research. similarly, previous researches and literatures also show that e. faecalis biofilms are more resistant to antibacterial than e. faecalis planctonics. bacterial biofilms reported to have a resistance of 10 to 1000-fold to antibiotics in the process of phagocytosis compared to bacterial planktonics.16 to compare to another antimicrobial agent frequently used as a root canal irrigation to reduce the growth of bacterial biofilms, chlorhexidine at a concentration of 0.1% was used. according to a previous research, chlorhexidine at the concentration of 0.1% is effective to suppress the number of aerobic and anaerobic bacteria in oral cavity up to 97%. however, chlorhexidine at the concentration of 0.1% is more effective against gram-positive bacteria than against gram-negative bacteria. chlorhexidine at the concentration of 0.1 to 2%, moreover, is also considered as anti-bacteriostastic material, but its activities depend on a condition of ph. its activities also trigger a damage to cells forming periodontal ligament tissue, oral mucosal irritation, burning sensation, and changes in taste perception.17 on the other side, a previous research conducted by sukkarwalla et al.4 reports that siwak does not destruct periodontal tissues, and has a better tolerance level with both changes in the perception of taste and burning sensation. for those reasons, chlorhexidine at the lowest concentration was used in this research since it was expected to be effective as an antimicrobial in this antibiofilm test, ie 0.1%. based on the analysis of data, the use of siwak extract at the concentrations of 70%, 75%, and 100% did not have significant differences from the use of chlorhexidine at the concentration of 0.1% (table 2). as a result, it can be said that siwak extract has an ability to inhibit the growth of the biofilm with the level of effectiveness almost equal to chlorhexidine.17 besides compared to the root canal irrigation material, siwak was also compared to another root canal treatment material since it has antibacterial and antibiofilm activities. in a previous research conducted by zhang,9 cetrimide at a concentration of 0.2% reduce adhesion factor that can inhibit the formation and growth of bacterial biofilms. cetrimide at the concentration of 0.2% with the ability to reduce the adhesion factor also can allow for the attachment of bacterial colonies apart so that the number of bacterial planktonics increase. similarly, trimethylamine contained in siwak extract can inhibit bacterial adhesion factors. other elements contained in siwak extract, such as benzylisothio jointcyanate and salvadorine, are also capable of inhibiting the formation of acids and enzymes as products of bacterial metabolism, initiating the formation and defense of bacteria cell wall. with those capabilities, siwak extract can reduce the growth of bacterial planktonics and biofilms. therefore, siwak extract can be used as an alternative to root canal treatment since it is not only safe for periodontal tissue, either as a root canal irrigation material or as a root canal sterilization material, but also effective to inhibit the growth of e. faecalis bacteria often found in cases of endhodontics.9 finally, it can be concluded that the active components of siwak can inhibit the growth of e. faecalis bacteria, both e. faecalis planktonics and biofilms. siwak can also be considered as an antibiofilm agent derived from natural materials. references 1. oli ak, raju s, rajeshwari, nagaveni s, kelmani c. biofilm formation by multidrug resistant enterococcus faecalis (mdef) originated from clinical samples. journal microbiol biotech res 2012; 2(2): 284-8. 2. halawany hs. a review on miswak (salvadora persica) and it’s effect on various aspects of oral health. the saudi dental journal 2012; 24: 63-9. 3. haque m. a review of the therapeutic effects of using miswak (salvador persica) on oral health. saudi medical journal 2015; 36(5): 530-43. 4. sukkarwalla a, salima m, pranee l, farzeen t. efficacy of miswak on oral pathogens. dent res j (isfahan) 2013; 10(3): 314-20. 5. masadeh mm, shadi g, karem a. antimicrobial activity of common mouthwash solutions on multidrug-resistance bacterial biofilms. j clin med res 2013; 5(5): 389-94. 6. skogman, m. a platform for anti-biofilm assays. proceedings pharmaceutical sciences department of biosciences abo akademi university finland, 2012; p. 14 – 63. 7. naseem s, khurseed h, fatimah f, shaheen s. in vitro evaluation of antimicrobial effect of miswak against common oral pathogens. pak j med sci 2014; 30(2): 398-403. 8. o’toole ga. microtiter dish biofilm formation assay. j vis exp 2011; (47): 2437. 9. zhang, r, min c, yang l, xiangjun g, ligeng w. antibacterial a nd residual a ntim icrobial activities aga inst enterococcus faecalis biofilm: a comparison between edta, chlorhexidine, cetrimide, mtad and qmix. journal scientific reports 2015; 5(12944): 1–5. 10. li yh, tian x. quorum sensing and bacterial interactions in biofilms. sensors (basel); 12(3): 2519–2538. 11. archer nk, mazaitis mj, john wc. staphylococcus aureus biofilms properties, regulation and role in human diseases. journal landes bioscience virulence 2011; 2(5): 445-59. 12. gupta a. biofilm quantification and comparative analysis of mic and mbic value for different antibiotic against e.coli. int journal curr microbiol app sci india 2015; 4(2): 198-224. 13. duggan jm, sedgley cm. biofilm formation of oral and endodontic enterococcus faecalis. j endod 2007; 33(7): 815-8. 14. mohamed ja, david h. biofilm formation by enterococci. journal of medical microbiology 2007; 56: 1581-8. 15. rezaei a, glenn o, virgilio b. molecular screening of anti-quorum sensing capability of salvadora persica on enterococcus faecalis. journal of hard tissue biology 2011; 20(20): 115-24 . 16. czaczyk k, myszka k. biosynthesis of extracellular polymeric substances (eps) and it’s role in microbial biofilm formation. polish journal of environment studies university of poznan poland 2007; 16(6): 799-806. 17. al-azzawi aj. the antibacterial effect of herbal alternative, green tea and salvadora persica (siwak) extracts on enterococcus faecalis. j bagh college dentistry 2015; 27(2): 1-4. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p158-162 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p158-162 195195 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 195–200 aggregatibacter actinomycetemcomitans sensitivity towards chlorophyll of moringa leaf after activated by diode laser i gde bagus yatna wibawa,1 suryani dyah astuti,2 and ernie maduratna setiawati3 1biomedical engineering magister program, post graduate school, universitas airlangga 2department of physics, faculty of science and technology, universitas airlangga 3department of periodontics, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: regular brushing teeth with scaling and root planning (srp) cannot effectively decrease the periopatogen bacterial colonies. even with the addition of antibiotics to support srp, such as tetracycline given with low doses and for a long time may cause bacteria to become resistant and the effectiveness to eliminate colonies of bacteria being reduced. photodynamic is a treatment modality that does not cause resistance and potentially to eliminate the growth of bacterial colonies. moringa oleifera is a plant that can be easily found in indonesia, by extracting chlorophyll of moringa oleifera leaves, it can be used as a photosensitizer agent to increase the absorption of light on photodynamic method. purpose: this study aimed to determine the potential photodynamic inactivation therapy to inactivate (eliminate) periopatogen bacterium. method: this study used aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans). laser diode 660nm as a light source with 8mm optical fiber to guide the beam, also used 20% extract chlorophyll of moringa oleifera leaf as photosensitizer. four diode lasers energy density exposures (2,5j.cm-2, 5j.cm-2, 7,5j.cm-2, and 10j.cm-2) are used from both at the in vitro photodynamic inactivation test. result: the highest percentage of deaths occurred in the group treated with addition of photosensitizer and exposed by 660 nm diode laser with 10j.cm-2 energy density, which is 83.01%, compared to the results obtained in the group without addition of the photosensitizer. conclusion: chlorophyll of moringa oleifera leaf after activated by diode laser effectively eliminates a. actinomycetemcomitans. keywords: photodynamic inactivation; laser diode; aggregatibacter actinomycetemcomitans; chlorophyll; moringa oleifera correspondence: i gde bagus yatna wibawa, biomedical engineering magister program, post graduate school, universitas airlangga. jl. airlangga no. 4-6, gubeng, surabaya 60115, indonesia. e-mail: gde.yatna@gmail.com introduction the antimicrobial effects of photodynamic method first described by oscar raab in 1900 when observing the lethal effect of acridine red combine with light on infusoria (malariacausing protozoa).1 photodynamic therapy which used to reduce the growth of bacteria, known as photoinactivation or photodynamic antimicrobial chemotherapy (pact), and nowadays known as antimicrobial photodynamic therapy (apdt).2,3 photodynamic therapy requires three main elements, including light-sensitive substance (photosensitizer), a harmless light source, and oxygen availability.3 bacteria had their light-sensitive substance that called endogenous porphyrin, studies have demonstrated that bacteria containing porphyrins are sensitive to visible light, in the blue as well as the red spectral region. study shows aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans) have protoporphyrin ix with 405nm soret band spectrum and peak q band at 510 nm, 545 nm, 580 nm, 630 nm, 670 nm, and 700 nm.4,5 antimicrobial effect is a phototoxic response due to singlet oxygen which caused oxidative damage to cell dna and changes in molecular mass of some cell membrane proteins and plasma membrane.2,7,6 oxidative damage in bacterial cells generally occurs in the dna cells and other cell organelles, but damage to the cell organelles can differ, depended on types of bacteria and photosensitizer used.8 the addition of exogenous photosensitizer has their role dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 196 wibawa, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 195–200 in improving the absorption of a light photon, where the photosensitizer is localized to the plasma membrane till enter the cell organelles such as mitochondria, lysosomes and endoplasmic reticulum causes photosensitization occurred in the area photosensitizer absorbed.9,10 there are numerous studies on the antibacterial effects of photodynamic method; for instance, researches on several periopathogenes, such as aggregatibacter actinomycetemcomitans (a.a), fusobacterium nucleatum (f.n), porphyromonas gingivalis (p.g), prevotella intermedia (p.i), and streptococcus sanguis (s.s).11 based on those previous researches, the highest bacterial (p.i, p.g, s.s, a.a and f.n) death are triggered by an exposure of a diode laser (665nm 100mw diode laser for 60 seconds at 21.2j.cm-2 energy density) by administration of methylene blue (mb) as a photosensitizer.11 another photodynamic research using diode laser 660 nm 30 mw combined with administration of malachite green (mg) as a photosensitizer can produce a bactericidal effect on a.a, which bacterial deaths up to 97.2% by three minute exposure time (5.4j. cm-2 energy density) and up to 99.9% by five minute exposure time (9j.cm-2 energy density).12 another in vitro photodynamic research using 405 nm laser diode by 75 second exposure time (25j.cm-2 energy density) combined with administration of chlorophyll as a photosensitizer with 1,5 cm distance can cause the s. mutant bacteria death, up to 74%.13 this study was perform to obtain an appropriate energy density of 660nm diode laser in photodynamic inactivation in vitro test with exposure time less than 1 minute using a. actinomycetemcomitans bacteria. this research also aimed to analyze the optimization of bactericide by adding chlorophyll extract derived from moringa oleifera leaves as an exogenous photosensitizer to increase the ability to absorb light. a. actinomycetemcomitans bacteria were selected as the test targets since they are flora in the oral cavity that potentially induce periodontal disease, especially localized aggressive periodontitis.14 a. actinomycetemcomitans bacteria classified intohaemophilus spp, a. actinomycetemcomitans, cardiobacterium hominis, eikenellacorrodens, and kingella kingae (hacek) group of pathogens which potentially triggering endocarditis infections.15,16 materials dan method a strain of bacteria used as samples in this research was a. actinomycetemcomitans (43718 atcc, usa). materials and laboratory equipment were used including chlorophyll extract derived from moringa oleifera leaves, tryptic soy agar (tsa) oxoid cm0131 (thermo fisher scientific inc. uk) and tryptic soy broth (tsb) caso broth (merck millipore, germany) as a medium to growing bacteria, 660 nm diode laser instruments, digital thermometer (th3 sanwa electric instrument co. japan), millimeter paper block, monochromator (ct-10 jasco inc. usa ) and power meter (pm100d thorlabs inc. usa). this study examined the effects of photosensitizer and diode laser spectrum with four (4) different energy densities. there were a group with administration of photosensitizer, and another group without administration of photosensitizer for further comparison. the bacterial strain, were grown in sterile tsb media then incubated in anaerobic jars containing candles, sealed for 24 hours at a temperature of 37o c. as explained above, there were two groups, namely a treatment group without the administration of photosensitizer and a treatment group with the administration of photosensitizer. the sample group with the photosensitizer was a bacterial culture treated with the administration of chlorophyll extract (moringa oleifera) with a concentration of 20%. a.actinomycetemcomitans bacteria were cultured in tsb medium then incubated in anaerobic jars (used candles to burn down oxygen) sealed at a temperature of 370 c for 24 hours. after the bacterial colonies grew, dilution was performed. 0.1 ml of each dilution tube were taken then put on tsa media to be incubated in anaerobic jars at 370 c for 24 hours. observation was carried out using total plate counting (tpc) method to obtain eligible samples (most close to 300 colonies).17 laser irradiation was performed at 1 cm distance from the surface of the sample in a pcr tube. 660 nm diode laser can produce 15.3 mw, 25.7 mw, 35 mw, and 45.5 mw power outputs (measured after guided by 8mm multimode optical fiber from 1cm distance). four energy densities (2.5j. cm-2, 5j. cm-2, 7.5j. cm-2, and 10j. cm-2) were used in the both treatment groups as shown in figure 1. this research used quadruplication technique for each treatment. calculation was performed using tpc method. to obtain percentage of viability of bacterial colonies, the following formula was used.13 ∑ colonies of treatment – colonies of control x 100% colonies of control the exposure area characterization was conducted to ensure the exposure area of the diode laser which guided by 8mm multimode optical fiber. the results showed that the exposure area of the 660 nm diode laser was 0.162 ± 0.01 cm2. the temperature characterization of the diode laser exposure beam guided by 8mm multimode optical fiber with 1cm distance using digital thermometer for one minute show the maximum temperature reached by each use of power output as shown in table 1. photosensitizer used in this research was chlorophyll derived from moringa oleifera leaf extract with a concentration of 20%. to ensure the ability of chlorophyll as a photosensitizer, light absorbance characterization was carried out using a uv-vis spectrophotometer (genesys 10s thermo fisher scientific inc. usa) light spectrum dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 197197wibawa, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 195–200 absorbance characterization result of chlorophyll derived from moringa oleifera leaf extract at the concentration of 20% are shown in figure 2. by using light spectrum absorbance characterization data and notice the diode laser wavelength, the percentage of energy absorbance (quantum yield) can be calculated using the following formula.13 % absorbance = (110-abs) x 100% the 660 nm diode laser beam had 81,84% energy absorbance. exposure time can be calculated to obtain the energy density which can be absorbed by using power output data of the diode laser and the absorbance percentage of the photosensitizer as shown in table 2. one-way anova statistical test was conducted to analyze the effects of the photosensitizer on the two treatment groups to ensure that the photosensitizer given was not toxic when not exposed with the laser diodes. 4 figure 1. the treatment group a. without the addition of photosensitizer, b. with addition of photosensitizer this research used quadruplication technique for each treatment. calculation was performed using tpc method. to obtain percentage of viability of bacterial colonies, the following formula was used.13 ∑ colonies of treatment – colonies of control x 100% colonies of control the exposure area characterization was conducted to ensure the exposure area of the diode laser which guided by 8mm multimode optical fiber. the results showed that the exposure area of the 660 nm diode laser was 0.162 ± 0.01 cm2. the temperature characterization of the diode laser exposure beam guided by 8mm multimode optical fiber with 1cm distance using digital thermometer for one minute show the maximum temperature reached by each use of power output as shown in table 1. table 1. temperature characterization of 660nm diode laser output power (mw) max. temperature (oc) 15.3 30 25.7 32 35 34 45.5 35 a b figure 1. the treatment group a) without the addition of photosensitizer; b) with addition of photosensitizer. table 1. temperature characterization of 660 nm diode laser output power (mw) max. temperature (oc) 15.3 30 25.7 32 35 34 45.5 35 table 2. calculation of energy density wavelength (nm) output power (mw) exposure area (cm2) exposure time (s) quantum yield (%) absorbed energy density (j.cm-2) exposure energy density (j.cm-2) 660 15.3 0.162 32.3 81.84 2.5 3.06 25.7 38.5 5 6.11 35 42.5 7.5 9.16 45.5 43.5 10 12.22 5 photosensitizer used in this research was chlorophyll derived from moringa oleifera leaf extract with a concentration of 20%. to ensure the ability of chlorophyll as a photosensitizer, light absorbance characterization was carried out using a uv-vis spectrophotometer (genesys 10s thermo fisher scientific inc. usa)light spectrum absorbance characterization result of chlorophyll derived from moringa oleifera leaf extract at the concentration of 20% are shown in figure 2. figure 2. light spectrum absorbance of chlorophyll derived from moringa oleifera leaf extract with the concentration of 20%. by using light spectrum absorbance characterization data and notice the diode laser wavelength, the percentage of energy absorbance (quantum yield) can be calculated using the following formula.13 % absorbance = (110-abs) x 100% the 660nm diode laser beam had 81,84% energy absorbance. exposure time can be calculated to obtain the energy density which can be absorbed by using power output data of the diode laser and the absorbance percentage of the photosensitizer as shown in table 2. table 1. calculation of energy density one-way anova statistical test was conducted to analyze the effects of the photosensitizer on the two treatment groups to ensure that the photosensitizer given was not toxic when not exposed with the laser diodes. wavelength (nm) output power (mw) exposure area (cm2) exposure time (s) quantum yield (%) absorbed energy density (j.cm-2) exposure energy density (j.cm-2) 660 15.3 0.162 32.3 81.84 2.5 3.06 25.7 38.5 5 6.11 35 42.5 7.5 9.16 45.5 43.5 10 12.22 figure 2. light spectrum absorbance of chlorophyll derived from moringa oleifera leaf extract with the concentration of 20%. results certain of energy density were used in the treatment group with the photosensitizer and the treatment group without the photosensitizer. results of tpc are shown in table 3, while the percentages of the bacteria viability are shown in table 4. a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 198 wibawa, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 195–200 table 3 shows the number of a.actinomycetemcomitans colonies in the treatment group with the photosensitizer are fewer than in the treatment group without the photosensitizer. the results of the one-way anova statistical test conducted to analyze the effects of the photosensitizer on the number of a.actinomycetemcomitans bacterial colonies in the two treatment groups, that showed there was no significant difference between the treatment group with administration of photosensitizer and the treatment group without administration of photosensitizer with a 0.069 (p>0.05) significance value. it can be assumed that chlorophyll photosensitizer derived from moringa oleifera leaf extract with the concentration of 20% was not toxic to a. actinomycetemcomitans bacteria. the chlorophyll photosensitizer potentially increase the energy density absorbed to improve the process of photodynamic inactivation. t a b l e 4 s h o w s t h e p e r c e n t a g e s o f t h e a . actinomycetemcomitans bacterial viability. negative signs indicate reduction in the growth of a. actinomycetemcomitans or bacterial death due to treatment or photodynamic inactivation. the highest bacterial death occurred in the treatment group with administration of photosensitizer and 660nm diode laser exposure at 10 j.cm-2 energy density, reaching 83.01%. discussion the death of the a. actinomycetemcomitans bacteria in this research was influenced by the photophysics, photochemical, and photobiology processes due to the diode laser exposures, optimized with chlorophyll as a photosensitizer derived from moringa oleifera leaf extract to increase the number of photons absorbed. the photophysics process due to exposure of the 660nm diode lasers can affect endogenous porphyrin (in the treatment group without the administration of photosensitizer) and chlorophyll moringa (in the treatment group with the administration of photosensitizer).18 the higher photon energy absorption occurred in the group treated with the exogenous photosensitizer than in the treatment group without the exogenous photosensitizer (only bacterial endogenous porphyrin). chlorophyll moringa which localized to the cell membrane or in the presence of bacteria endogenous porphyrin will be active when irradiated with diode laser then triggering the photochemical process which do the energy transfer to another molecules especially local oxygen that will generate oxidative agents, including radical hydroxyl, excited singlet oxygen, hydrogen peroxide, and superoxide anions, triggering photobiology process that aggressively oxidize cell membranes and organelles resulting in a damage to lipid membranes, proteins, and bacterial dna cell leading to bacterial death.19-22 a.actinomycetemcomitans grows well at 20-42º c.23 the exposure of 660 nm diode laser at 2,5j.cm-2, 5j.cm-2, 7,5j.cm-2 and 10j.cm-2 energy density produce the optimum temperature for a.actinomycetemcomitans growth shown at table 1, thus concluded that bacterial killed should not occurs due to photothermal effect. this study using <100 w.cm-2 laser diode power density and <103 s exposure time, so the light interaction that occurs is the photochemical reaction.19 table 3. bacterial colonies count at both treatment groups energy density (j.cm-2) colonies count in the treatment group without administration of photosensitizer (cfu.ml-1) colonies count in the treatment group with administration of photosensitizer (cfu.ml-1) 0 (control) 5.23.1010 ± 1.1.109 4.76.1010 ± 4.1.109 2.5 3.79.1010 ± 2.6.109 1.19.1010 ± 3.2.109 5 3.69.1010 ± 2.5.109 1.14.1010 ± 1.7.109 7.5 2.69.1010 ± 3.6.109 1.12.1010 ± 2.1.109 10 2.44.1010 ± 3.6.109 8.05.109 ± 9.7.108 table 4. viability of the bacteria at both treatment groups energy density (j.cm-2) viability percentages of the treatment group without administration of photosensitizer (%) viability percentages of the treatment group with administration of photosensitizer (%) 2.5 -27.47 ± 6 -75.22 ± 5.6 5 -29.49 ± 5.3 -75.72 ± 5.4 7.5 -48.5± 7.6 -76.48 ± 3.9 10 -53.42 ± 7 -83.01 ± 2.4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 199199wibawa, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 195–200 in this research, the highest percentage of killed bacteria was obtained in the group treated with the administration of photosensitizer by 10j.cm-2 energy density, which is -83.01 ± 2.4 %. the bacteria death at group treated without the administration of photosensitizer by 10j.cm-2 energy density only reaching -53.42 ± 7%. the viability of bacteria by 2,5j.cm-2, 5 j.cm-2, 7,5 j.cm-2 energy density also showed more bacteria death in the treatment group with the addition of photosensitizer compared to the group without photosensitizer. tukey hsd comparison test between energy density and the a.actinomycetemcomitans bacterial viability showed there was no significant difference between 2,5j.cm-2 and 5 j.cm-2 energy density (sig.0,745), which is by using the lower energy density (2,5j.cm-2) it can obtain bacterial death reaching 75% at treatment group with administration of photosensitizer. energy density, exposure time and wavelenght of light source with the administration of photosensitizer have a very important role in inactivation of bacteria, the result at this research showed exposure energy density of diode laser and administration of photosensitizer plays important role in inactivation that occurs at a.actinomycetemcomitan death.3,10-13,20,22 photodynamic therapy effectively killing pathogens, including strains that have resistance to antibiotics therefore the photodynamic method is more effective than giving antibiotics.24 ecologically, commensal microorganisms and pathogenic bacteria can be found in mouth.25-27 commensal microorganisms are bacteria found on the surface of epithelial cells at human body, including oral epithelium, which in normal condition is favorable be the barrier and contributes to homeostasis as well as the host’s body defense.26,27 pathogenic bacteria tend to give inflammatory responses through virulence factors expressed.25 two bacteria with two different behaviors in the mouth ecosystem which mutually pressing (antagonistic) resulting no excessive growth of pathogenic bacteria in periodontal tissues. conversely, excessive growth of pathogenic bacteria can cause damage to teeth supporting collagen.28 it can be concluded that chlorophyll extract derived from moringa oleifera leaves showed no direct toxic effect for a. actinomycetemcomitans. chlorophyll of moringa oleifera leaf after activated by diode laser effectively eliminates a. actinomycetemcomitans. acknowledgement we would like to express our gratitude to indra brahma prasaja for his assistance during this research. references 1. ackroyd r, kelty c, brown n, reed m. the history of photodetection and photodynamic therapy. photochem photobiol 2001; 74(5): 656669. 2. wainwright m. photodynamic antimicrobial chemotherapy (pact). j antimicrob chemother 1998; 42(1): 13-28. 3. or uba z , łabuz p, macyk w, gajewska mc. a ntim icrobia l photodynamic therapy-a discovery originating from the preantibiotic era in a novel periodontal therapy. photodiagnosis photodyn ther 2015; 12(4): 612-618. 4. fyrestam, jonas, nadja bjurshammar, elin paulsson, and annsofi johannsen. 2015. “determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.” analytical and bioanalytical chemistry 407(23): 7013–7023. 5. torezan, luís, ane beatriz mautari niwa, and cyro festa neto. 20 09. “terapia fotodinâmica em der matologia: p r incípios básicos e aplicações.” anais brasileiros de dermatologia 84(5): 445–459 6. bhatti m, macrobert a, meghji s, henderson b, wilson m. a study of the uptake of toluidine blue o by porphyromonas gingivalis and the mechanism of lethal photosensitization. photochem photobiol 1998; 68(3): 370-376. 7. bhatti m, nair sp, macrobert aj, henderson b, shepherd p, cridland j, & wilson m. identification of photolabile outer membrane proteins of porphyromonas gingivalis. curr microbiol 2001; 43(2): 96-99. 8. harris f, chatfield lk, phoenix da. phenothiazinium based photosensitisers-photodynamic agents with a multiplicity of cellular targets and clinical applications. curr drug targets 2005; 6(5): 615627. 9. castano, ana p., tatiana n. demidova, and michael r. hamblin. 20 04. “mechanisms in photodynamic therapy: par t one — photosensitizers, photochemistry and cellular localization.” photodiagnosis and photodynamic therapy 1(4)(december): 279–293. 10. de paula eduardo, c., de freitas, p. m., esteves-oliveira, m., aranha, a. c. c., ramalho, k. m., simões, a., bello-silva, m.s., tunér, j. (2010). laser phototherapy in the treatment of periodontal disease. a review. lasers in medical science, 25(6), 781–792. http:// doi.org/10.1007/s10103-010-0812-y 11. chan y, lai ch. bactericidal effects of different laser wavelengths on periodontopathic germs in photodynamic therapy. lasers med sci 2003; 18(1): 51-55. 12. prates ra, yamada am jr, suzuki lc, eiko hashimoto mc, cai s, gouw-soares s, gomes l, ribeiro ms. bactericidal effect of ma lach ite g reen a nd red laser on acti nobacil lus actinomycetemcomitans. j photochem photobiol b biol 2007; 86(1): 70-76. 13. astuti sd, zaidan a, setiawati em, suhariningsih. chlorophyll mediated photodynamic inactivation of blue laser on streptococcus mutans. 5th international conference and workshop on basic and applied sciences (icowobas 2015) 2016; 120001:1-8. 14. fine dh, markowitz k, furgang d, fairlie k, ferrandiz j, nasri c, mckiernan m, gunsolley j. aggregatibacter actinomycetemcomitans and its relationship to initiation of localized aggressive periodontitis: longitudinal cohort study of initially healthy adolescents. j clin microbiol 2007; 45(12): 3859-3869. 15. fine dh, kaplan jb, kachlany sc, schreiner hc. how we got attached to actinobacillus actinomycetemcomitans: a model for infectious diseases. periodontol 2000. 2006; 42(1): 114-157. 16. das md, badley ad, cockerill fr, steckelberg jm, wilson wr. infective endocarditis caused by hacek microorganisms. annu rev med 1997; 48(1): 25-33. 17. basalamah, raina. potensi pemaparan light emitting diode (led) inframerah untuk fotoinaktivasi bakteri bacillus subtilis. diss. universitas airlangga, 2015. 18. gábor f, szocs k, maillard p, csík g. photobiological activity of exogenous and endogenous porphyrin derivatives in escherichia coli and enterococcus hirae cells. radiat environ biophys 2001; 40(2): 145-151. 19. niemz mh. laser-tissue interactions, fundamentals and applications. 3rd ed. heidelberg: springer-verlag; 2007. p. 316. 20. plaetzer k, krammer b, berlanda j, berr f, kiesslich t. photophysics and photochemistry of photodynamic therapy: fundamental aspects. lasers med sci 2009; 24(2): 259-268. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 200 wibawa, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 195–200 21. cox b. introduction to laser-tissue interactions. phas 4886. 2007; (march): 1-61. 22. grossweiner li, grossweiner jb, rogers bhg. the science of phototherapy: an introduction. jones lr, editor. dordrecht, the netherlands: springer; 2005. p. 299-327. 23. kesić, ljiljana, milica petrović, radmila obradović, and ana pejčić. 2009. “the importance of aggregatibacter actinomycetemcomitans in etiology of periodontal disease mini review.” acta medica medianae 48 no.3: 35–37. 24. pedigo la, gibbs aj, scott rj, street cn. absence of bacterial resistance following repeat exposure to photodynamic therapy. 12th world congr int photodyn assoc photodyn ther back to futur 2009; 7380: 73803h-73803h-7. 25. gupta g. probiotics and periodontal health. j med life 2011; 4(4): 387-394. 26. krisanaprakornkit s, kimball jr, weinberg a, darveau rp, bainbridge bw, dale ba. inducible expression of human β-defensin 2 by fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier. infect immun 2000; 68(5): 2907-2915. 27. brestoff jr, artis d. commensal bacteria at the interface of host metabolism and the immune system. nat immunol 2013; 14(7): 676-684. 28. sari dn, nawawi s, alif r. perbedaan pengaruh antara probiotik a, b, da n c t erhad ap d aya ha mbat p er t u mbu ha n ba k t er i aggregatibacter actinomycetemcomitans (kajian in vitro). diss. universitas muhammadiyah surakarta, 2014. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p195-200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p195-200 165 prevalence of oral habits in homeless children under care of yayasan bahtera bandung gildasya*, eriska riyanti** and syarief hidayat** ** student ** department of pediatric dentistry faculty of dentistry padjadjaran university bandung indonesia abstract oral habits, comprising of thumb and lip sucking, lip and nail biting, tongue thrusting and mouth breathing are commonly caused by disturbance in mental development. these persistent habits may lead to disturbance in physical growth, causing disorders such as malocclusion. homeless children are prone to this condition. the purpose of this descriptive research was to present the prevalence of oral habits in homeless children under care of yayasan bahtera bandung, by survey technique. the sample consisted of 92 children collected by purposive sampling. this research was conducted through interviews and clinical examnations of the oral cavity to elucidate signs of oral habits. the result showed 50% of homeless children performed oral habits, consisting of 26 boys (55,32%) and 20 girls (44.44%). the prevalence of thumb sucking habit was 21.74%, lip sucking or biting was 17.37%, tongue thrusting was 4,35%, nail biting and mouth breathing tied at 3,26%. the research concluded that a part of homeless children had oral habits, with boys as slight majority, and thumb sucking was the most performed. key words: oral habits, homeless children correspondence: gildasya, c/o: mahasiswa sarjana kedokteran gigi fakultas kedokteran gigi universitas padjadjaran. jln. sekeloa selatan no.1 bandung 40133, indonesia. introduction children’s physical growth and mental development is influenced by surrounding social environment. family holds the biggest role in shaping children’s personality. correct education applied by parents helps children facing their future environmental challenges. parents’ mistakes, especially the mother’s, during educating and caring for their children will later cause asocial, physically and mentally pathologic children.1 intestinal colic, facial twitching and persistent habits such as bed wetting, thumb sucking and eating disorders are often the cause of parents’ -especially mothers’incompetence in caring for their children, such as parent-child relationship disharmony, insufficient pleasure during oral phase, early breast-feeding cessation, emotional disturbances, anatomical malformation and diseases.1,2,3 a habit is defined as automatic repetitive action as a result of complex natural process involving muscle contraction.2 normal habits may serve as constructive dentofacial function and hold an important role in normal facial growth and occlusal physiology. in contrary, abnormal habits may promote disturbances in dentofacial growth pattern. on the more specific definition, every habit that cause abnormal stress on dentofacial structure which allow a malformation of a structure and inter-structural connection, is defined as a bad habit, and also referred to oral habit.5 oral habit is a repetitive action which provokes disorders in teeth and the surrounding supportive tissues, such as thumb and lip sucking, lip and nail biting, tongue thrusting and mouth breathing.2,6 these habits are normally temporary, but may also be persistent as the children grow older, causing oral structure growth disturbance which may lead to local disorders such as malocclusion.7 oralhabit-induced malocclusion is dependent on the frequency, intensity and duration of habit action. homeless children are a pathetic image of nation’s generation. children are normally related to laughter, games, carefree-ness and problem-less. reality proves that beautiful childhood does not apply to every individual. some children, like the homeless ones, are already forced to work for living at relatively infant age. these children are the product of economic issues, broken homes, parental divorce and parent-child relationship disharmony. environmental harassment with lack of family protection is their expected and ordinary threats. many studies report that lack of protection against life’s cruelness may cause severe damage in children’s personality development.8,9 according to the references on homeless children, these individuals are victims of family harassment who are forced to leave their families. although the cruel life on the street appears to give more promises than the cruel life in their family, homeless children still long for parents’ love and care, especially from a mother. instead, they must face the cruel street life which obliges them to earn living by 166 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 165–167 begging, singing, selling small stuffs et cetera, to provide their basic needs. they often call a day with empty stomach, causing insufficient nutritional intake.9 during the pilot survey, the researcher revealed oral habit phenomenon among the population of homeless children. this manifestation is probably caused by bad treatment from their original family, lack of parents’ love, emotional disturbance and famine. there has not been any study on the oral habits prevalence in homeless children, so the researcher wanted to elucidate the prevalence of the manifestation among the homeless children under care of yayasan bahtera bandung, age ranging from 6 to 12 years old. this research may serve as a raw data for further planning on oral health development, especially in preventing oral habits in homeless children, which may later increase the national level of oral health. materials and methods the study was assessed by descriptive method with survey technique. research population consisted of homeless children under care of yayasan bahtera bandung. ninety-two samples matching the criteria of homeless children under care of yayasan bahtera bandung, between 6–12 years old, agreed to participate in the study (informed consent) and cooperative, was taken by purposive sampling technique, consisting of 47 boys and 45 girls. instruments and materials used in this study were oral mirrors, semilunar explorers, pincers, alcohol, cotton rolls, masks, gloves, flashlights, informed consent forms, questionnaire sheets and writing instruments. research was assessed questionnaire-guided interview, which was followed by clinical examination to observe the lesions on the oral soft and hard tissue which were probably cause by oral habits. the collected data was later summarized in tables and diagrams of percentage. results the research conducted on 92 homeless children under care of yayasan bahtera bandung, age ranging from 6–12 years old resulted in: the result showed that 50% of the homeless children population had oral habits, while the other 50% did not (table 1). table 1. prevalence of oral habits in homeless children both sex types have equal risks in practicing oral habits. the study showed that the prevalence is higher in boys (55.32%) than in girls (44.44%) (table 2). table 2. prevalence of oral habits in homeless boys and girls actions defined as oral habits in this study are finger sucking, lip sucking or biting, tongue thrusting, nail biting and mouth breathing. the result showed finger sucking in the highest position (43.48%) while mouth breathing and nail biting tied in the lowest position (6.52%). discussion kharbada10 reported that 25.5% of school children in delhi practicing oral habits. children in this range of age are on the phase of learning to control their emotion. emotional disturbance such as lack of care and love with too much fear and anxiety is a factor to oral habits.7,11 research showed the prevalence of this action in homeless children under care of yayasan bahtera bandung is 50% (table 1), showing that a part of homeless children adopt thus habit. in homeless children, oral habits occur as a manifestation of several factors. the most striking factor is the insufficiency of basic needs, e.g. food, clothing and home, provided by their parents. questionnaire showed that 76.09% of population only had one meal per day, therefore oral habits such as sucking was adopted. according to sigmund freud, oral activity can give sufficient pleasure to distract children from hunger.1,3 according to gunarsa,3 disharmony between children and parents is also a factor to the adoption of oral habits. this study confirmed the theory by the interview results 69.57% of homeless children admitted of being afraid to their parents, because they are often angry when their children failed to earn sufficient money. these children often serve as victims of their parents’ anger and frustration towards life burden. this causes a disharmonic parent-child relationship. oral habits are often performed after children being punished or grounded. table 2 showed that homeless boys had bigger prevalence of oral habits than girls (55.32% to 44.44%). according to massler, oral habits in boys are more persistent in longer period than girls because boys tend to openly fight against family’s or surrounding society’s rules than girls, including when they are told to stop practicing oral habits.9,12,13 this research defined oral habits as finger sucking, lip sucking or biting, tongue thrusting, mouth breathing and 167gildasya: prevalence of oral habits in homeless children nail biting. finger sucking is a very common oral habit in children, and as shown in table 3 and figure 1, finger sucking held the topmost position (43,48%). cumley stated that persistent finger sucking habit until over 4 years old suggests the presence of emotional disturbance, leading children to suck their fingers to obtain pleasure.14 finger sucking is also performed when feeling insecure to attract attention after being punished or yelled at.5,15 most homeless children performed this action as a manifestation of insecure feeling and fear against anger and punishment. tabel 3. types of oral habits performed in homeless children oral habits may cause disorders on teeth and supportive tissues, depending on intensity or how often the action is performed, frequency or how often the action is repeated per day, and duration or how long the action has been performed.7 malocclusions present as anterior open-bite, anterior protrusion, anterior-posterior cross-bite, hightapered palate and crowded dentition may be caused by finger sucking, tongue thrusting and mouth breathing. clinical examination of the homeless children showed that those habits had caused disorders in dentofacial structures as mentioned and also oral soft tissue disorders such as abnormal dentition marking on the labial and buccal mucosa. according to mcdonald and avery,14 there is a relevancy between class ii malocclusion and finger sucking habit in various ages. malocclusions increase from 21.5% at the age of 3–4 years old to 41.9% at the age of 12 years old. malocclusions increase when habit duration increases. studies reported that effect on the occlusion is temporary when habits are controlled and stopped before 6 years of age. interview result showed that homeless children of 6 to 12 years of age performed oral habits for over than 1 year period of time with more than once a day frequency as it helps to provide pleasure and convenience. acknowledgment the researcher would like to thank the principal of yayasan bina sejahtera indonesia (bahtera), field coordinator, and all the children participating in the research. references 1. kartono k. psikologi anak (psikologi perkembangan). bandung: mandar maju; 1995. p. 44–7. 2. finn sb. clinical pedodontics. 4th ed. philadelphia, london and tokyo: wb saunders company; 2003. p. 310–9. 3. gunarsa sd. perkembangan psikologi anak dan remaja. jakarta: bpk gunung mulia; 1983. p. 9–10, 43–4, 50–1. 4. dorland wa, newman. kamus kedokteran dorland. edisi ke-29. huriawati hartanto, dkk. jakarta: penerbit buku kedokteran egc; 2002. 5. moyers re. handbook of orthodontics. 4th ed. chicago, london, boca raton: year book medical publ; 1988. p. 152–6. 6. boucher co. current clinical dental terminology. 2nd ed. st louis: the cv mosby co; 1974. p. 182. 7. christensen j, henry w fields, adair sm. oral habits. in: pinkham jr, editor. pediatric dentistry infancy through adolescence. 4th ed. philadelphia: elsevier saunders; 2005. p. 431–7, 470. 8. terloit aj. konsep diri anak jalanan usia remaja yang mengalami abuse dan tidak mengalami abuse. skripsi. depok: fakultas psikologi universitas indonesia; 2001. available from: url:http//www. kompas.com/compas-cetak/0507/23/swara/1916829.htm. accessed july 23, 2005. 9. irwanto dkk. anak yang membutuhkan perlindungan khusus di indonesia: analisis situasi. jakarta: pkpm unika atma jaya jakarta, departemen sosial, unicef; 1999. h. 99–113. 10. kharbanda op, sidhu ss, et al. oral habits in school going children of delhi: a prevalence study. j indian soc. pedodontics prevention dentristry 2003; 21(3):120–4. 11. graber tm. orthodontics principles and practice. 2nd ed. philadelphia, london and tokyo: wb saunders company; 1970. p. 296–313, 628–91. 12. massler m. oral habits : development and management. the journal of pedodontics 1983; 7(2):109–19. 13. polyakov e. digit sucking before the age 4.5: interpretation and some management considerations. international pediatrics 2002; 17(4): 203–8. 14. mcdonald re, dr avery. dentistry for the child and adolescent. 6th ed. st louis: the cv mosby co; 1994. p. 776–84. 15. foster td. buku ajar ortodonsi. edisi ketiga. lilian yuwono. jakarta: penerbit buku kedokteran egc; 1997. p. 110–3. tongue thrusting, 8.70% nail biting, 6.52% m outh breathing, 6.52% lip sucking or biting 34.78% finger sucking, 43.48% figure 1. prevalence of various oral habits in street children. no oral habits, 50% finger sucking, 21.74% lip sucking or biting, 17.39% tongue thrusting, 4.35%nail biting, 3.26% mouth breathing, 3.26% figure 2. distribution of various oral habits in street children. vol 50 no 4 desember 2017.indd 188 research report dental journal (majalah kedokteran gigi) 2017 december; 50(4): 188–193 the role of active ingredients nanopowder stichopus hermanii gel to bone resorption in tension area of orthodontic tooth movement noengki prameswari and arya brahmanta departemen of orthodontics faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: orthodontic tooth movement is a continual and balanced process between bone deposition and bone resorption in pressure and tension sites. stichopus hermanii is one of the best fishery commodities in indonesia. it is natural and contains various active ingredients such as hyaluronic acid, chondroitin sulphate, cell growth factor, eicosa pentaenoic acid (epa) docosa hexaenoic acid (dha) and flavonoid that potentially play a role in orthodontic tooth movement. purpose: the aim of this study was to investigate the active ingredients of nanopowder stichopus hermanii promoting bone resorption in tension area orthodontic tooth movement. methods: a quantitative test for active ingredients of stichopus hermanii was conducted. thirty two male cavia cobaya were divisibled became four groups. k (–) groups as a negative control group (without treatment), k (+) groups as a positive control group which were provided with a separator rubber for orthodontic tooth movement, and p1, p2 groups, which were treated with 3% and 3.5% stichopus hermanii for orthodontic tooth movement. after treatment the cavia cobaya were sacrificed. trap-6 expression as a osteoclast marker was examined by means of an immunohistochemistry method. results: a one-way anova test confirmed that trap-6 expression was significantly increased with p = 0.00 (p≤0,05) in p2 compared to k (+). p2 to k (–), p2 to p1 and p1 to k (+) had no significant differences conclusion: nanopowder stichopus hermanii 3.5% has an active ingredient that could increase osteoclast activity to resorb periodontal ligament and alveolar bone in tension areas of orthodontic tooth movement. keywords: nanopowder; stichopus hermanii; resorption; trap-6; orthodontic tooth movement correspondence: noengki prameswari, department of orthodontic, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim 150 surabaya, indonesia. e-mail: noengki.prameswari@hangtuah.ac.id introduction according to riskesdas data of 2013, malocclusion is the third most common oral disease.1 orthodontic treatment is generally associated with malocclusion that causes esthetic problems.2 orthodontic tooth movement involving the use of orthodontic appliances is characterized by remodeling changes of the alveolar bone and a reaction of the periodontal ligament (pdl) to mechanical stimuli. tooth movement occurs in the direction of force when there is a multifaceted bone remodelling response, with bone resorption on the compression side and bone apposition on the tension side of the periodontal ligament.3 orthodontic treatment for malocclusion correction requires a period of 1–2 years. mechanically induced periodontal ligament and bone remodelling is still not fully understood. the role of the periodontal ligament has been questioned since tooth movement occurs. as long as an orthodontic appliance is applied, orthodontic movement occurs which can effect sequential reactions as respons of periodontal tissue and alveolar bone in remodeling and releasing of numerous mediators and substances from the periodontal and alveolar tissues and surrounding structures.4,5 after orthodontic mechanical pressure with both physical and biological characteristics, orthodontic tooth movement can occur either slowly or rapidly depending on its biological response.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 mailto:noengki.prameswari@hangtuah.ac.id http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 189189prameswari and brahmanta/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 188–193 an orthodontic appliance can be defined as a mechanical stimulus resulting in biological celular response. under orthodontic mechanical force, periodontal ligament responds with subsequent bone resorption in the area of pressure application and, conversely, under tension force will result in bone formation. an early response in the pressure area is one of periodontal ligament inflammation. when inflammation occurs, many cells are produced, including: cytokine, t-cell, b-cell, and matrix metalloproteinases (mmps).6 tissue reactions is also initiated immediately after force application in pressure and tension periodontal ligament. the extravasation and chemoattraction of numerous inflammatory cells begins, and followed by complex process of recruitment of osteoclast and osteoblast progenitors.3 orthodontic appliances with varying degrees of frequency, magnitude, and duration of mechanical loading, result in extensive macroscopic and microscopic changes to the bone adjacent to periodontal tissues. mechanical loading also alters periodontal tissue vascularity, metabolic process and blood flow resulting in the local synthesis and release of various molecules such as arachidonic acid, cytokines, growth factor, and colony-stimulating factors until tooth movement occurs.7 the released molecules as cellular responses in the various cell types in and around teeth, such as fibroblasts, osteoblasts, cementoblasts, and vascular cells as a stressor response of mechanical forces, provide a favourable microenvironment for tissue apposition or resorption in orthodontic tooth movement.3 when orthodontic forces are applied to the tooth, the resulting pressure will induce fibroblast cells, osteoclasts and osteoblas in periodontal ligament as a response to mechanical pressure. orthodontic tooth movement is mediated by coupling mechanism between resorption and apposition process in the pressure and tension area of periodontal ligament and alveolar bone.3 periodontal ligaments increase widening and induce bone remodeling so that orthodontic tooth movement occurs.8 extracellular changes and crevicular gingival fluid as a biomarker orthodontic response occured.7 collagen became the most important tissue in periodontal ligament remodeling. the ligament itself undergoes remodeling and the role of mmps with their natural inhibitors, tissue inhibitors of metalloproteinases (timps), are clearly of importance.6 during orthodontic tooth movement, periodontal ligament remodeling followed with bone remodeling. several enzymes such as alkaline phosphatase to induce osteoblast cell, and growth factors such as fibroblast growth factor-2 (fgf-2) to increase fibroblast proliferation, and bone morphogenetic protein-2 (bmp-2) as a mature osteoblast marker were present. osteoblast plays a direct role in bone formation, especially in bone matrix formation, involving non-collagenous protein and growth factors.9 acceleration in bone remodeling will increase orthodontic tooth movement.10 bone resorption is a crucial process during which orthodontic tooth movement by resorbing alveolar bone occurs as orthodontic forces respond. in this process, osteoclast is the most important cell involved in cell mediation. osteoclasts are found in physiologic periodontal ligaments as mature condition cells. osteoclasts seems appear within days when orthodontic mechanical force is applied to produce tooth movement.3 osteoclasts differentiate from stem cells pathways in haemopoietic lineage and the early precursors of osteoclasts are granulocyte-macrophage colony-forming units. resorption process cascade involves several steps directed toward removing organic and anorganic structures of alveolar bone matrix by osteoclast so that orthodontic tooth movement occurs. after that, the unmineralized bone surface is replaced by apposition process with lining osteoblasts. several enzymes such as mmps, collagenases and gelatinases is produced by osteoblasts which have role in accessing mineralized bone. local and systemic factors are important for osteoclast activation and induce the production of hydrogen ions, proteolytic enzymes in vacuole under the ruffled border.3 tartrate-resistant acid phosphatase (trap-6) positive multinuclear cells are an enzyme which is localized within the ruffled border area as a osteoclast mature biomarker.11 orthodontic tooth movement produces both a pressure area and a tension area. in the pressure area, periodontal ligament shows disorganization and diminution of ligament fiber production and vascular contriction. in the tension area, stimulation produced by the stretching of periodontal ligament fiber bundles results in an increase in fiber production.3,8 osteoclasts appear in the pressure area and osteoblasts in the tension area. osteoclasts in the tension area remain poorly understood.11 the utilization of marine biota for dental treatment has been developed. stichopus hermanii is well recognized as a human food source. stichopus hermanii contain active ingredients such as proteins 86% (80% collagens), glucosaminoglycans including hyaluronic acid, chondroitin sulphate, cell growth factor, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) that are important for tissue regeneration.12,13 previous research showed that 3% stichopus hermanii can decrease relapse biometric and increase fgf-2 and collagen type 1 expression in relapse orthodontic compared with 2.5%. stichopus hermanii can act as an anticandidal.10,14 another research showed that stichopus hermanii modulates the inflammatory responses, stimulates the activation and proliferation of fibroblasts and enhances the rapid production of collagen fiber networks with shorter healing times. the level of proinflammatory cytokines; il-1α, il-1β, and il-6, are significantly reduced in stichopus hermanii-treated wounds and stimulation tissue regeneration.15 the role of stichopus hermanii in bone resorption in tension area orthodontic tooth movement has yet not been fully investigated. the aim of this study was to investigate the active ingredients of nanopowder stichopus hermanii in bone resorption in tension area orthodontic tooth movement of cavia cobaya. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 190 prameswari and brahmanta/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 188–193 materials and methods the study was conducted at experimental laboratories with completely randomized control group post-test only design. ethical permission was obtained from the ethics and scientific research committee of experimental animal use at the faculty of dentistry, universitas hang tuah no 125/kepk/i/2016. thirty-two male guinea pigs (cavia cobaya) aged 2.5 months and weighing 200–300 grams, fed a standard pellet diet and tap water ad libitum, were randomly divided into four groups each consisting of eight guinea pigs. based on previous research, the optimum stichopus hermanii concentration used were 3%, while attempts to add concentrations of 3.5% stichopus hermanii were made.10 a 10% ketamine injection was administered as an anesthetic, with 0.1–0.2 ml/kg for acepromazine 0.5 ml, 10% buffered formalin and cotton.8 in this research, stichopus hermanii were taken from raas island sumenep, east java indonesia and was cleaned by longitudinal incision using a scalpel without damaging the internal organs. the stichopus hermanii were dried in ovens (u type, memmert, wisconsin, usa) at 28 degrees centigrade, blended (model hgbtwt, waring commercial, usa) and reduced to nanopowder (20 nm) by means of a high energy milling (puspitek, tangerang, indonesia) method. quantitative analysis of stichopus hermanii active ingredients involved uv vis transmittance (t872) spectrophotometry (intertek ptl, pittsfield, usa) to examine flavonoid, gas chromatography (agillent gc-ms 5975c, palo alto, ca, usa) to examine epa and dha and a reversed phase high performance liquid chromatography (hplc j.t baker, united states) with uv detection to examine chondroitin sulphate.10 to prepare nanopowder, 3% stichopus hermanii gel was made from 0.3 gr stichopus hermanii powder diluted with natrium carboxy methylcellulose (nacmc) 2% in 10 ml of dimethyl sulfoxide (dmso) 5%. nanopowder 3.5% stichopus hermanii gel was made from 0.35 g stichopus hermanii powder diluted with nacmc 2% in 10 ml of dmso 5%.10 the procedure within this study began with the acclimatization of 32 guinea pigs for 48 hours. the guinea pigs were divisibled became four groups of eight subjects: k(–) group as a negative control group (without treatment), k(+) group as a positive control group whose orthodontic tooth movement was triggered by means of an elastic separator at a force of 0.0474 kn, measured with a gauge name autograph (ags-x series, shimadzu, kyoto, japan) during experiment and p1, p2 groups which were given with both orthodontic pressure and stichopus hermanii 3% and 3.5% over 14 days. 0.025 ml of stichopus hermanii gel was applied to the gingival sulcus tension area once per day with an insulin syringe.10 the research was conducted at the biochemistry laboratory medical faculty of universitas airlangga. the guinea pigs were monitored during the experiment all of, with all of the groups being sacrificed on the fourteenth day of the experiment. the maxillary incisive teeth were dissected and placed in 10% buffered formaline.10 histological sections were subsequently prepared with trap-6 immunohistochemistry as an osteoclast marker and then observed by using a light microscope (nikon optiphot 2, japan). the expression of trap-6 as osteoclast marker in the periodontal ligament on 1/3 apical in the tension area was observed. photographs using an optilab advance (miconos, yogyakarta, indonesia) were taken to measure the osteoclasts (trap-6) expression seen through a microscope at 400x enlargement. each histological section was observed and calculated.10 finally, the data was statistically measured using a statistical package for the social science (spss) version 20. the resulting research data was tabulated, the statistical hypothesis being conducted with a standard analytic significance of 95% (p = 0.05) by one-way anova test (analysis of variants) to analyze the difference of each variable compared with the control. the data was tested with lsd test (p < 0.05). results the data obtained from the quantitative analysis of stichopus hermanii showed that the percentage of flavonoid was higher than the other active ingredients examined (table 1). the data resulting from orthodontic tooth movement measurement showed that there were differences in orthodontic tooth movement width within each group. in the k(+) group, the mean was 0.45 mm, while the mean of the p1 group was 0.496 mm, and the mean of p2 group was 0.498 mm (table 2 and figure 1). moreover, the data also showed that the trap-6 expression as a osteoclast marker increased in p1 and p2 groups. the highest number found in the p2 group treated table 1. active ingredients of nanopowder stichopus hermanii no. active ingredients result method 1 flavonoid (%) 5.3218 (%) spectrophotometry (saura-calixto,1998; larrauri et al, 1997) 2 decosahexaenoic acid (dha) (mg/100g) no detection (< 1.20) gas chromatography 3 eicosapentaenoic (epa) (mg/100g) 17.10 mg/100 g gas chromatography 4 condroitin sulphate (mg/100g) 706.15 mg/100 g 18-5-56/mu/smm-sig, hplc dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 191191prameswari and brahmanta/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 188–193 with nanopowder stichopus hermanii 3.5% was 13.17 cell/ field of view. meanwhile, in the negative control group, the mean was 9.67, and in k(+) group the mean was 8.5 (table 3 and figure 3). trap-6 expression is showed in figure 2. the statistical results with one-way anova test confirmed that there were significant differences between all groups (p≤0.05). the statistical results of one-way anova and lsd showed that there was a significant difference of trap-6 expression as a osteoclast marker in the tension area between k(-), k(+) groups and the p1 and p2 groups. trap-6 expression was significantly increased in p2 compare to k(+), p2 to k(-), p2 to p1, but p1 to k(+) had no significantly differences as seen in table 4. discussion the results showed that the p2 group that was administered the orthodontic force separator rubber and stichopus hermanii 3.5% had the widest orthodontic tooth movement (otm) among the groups. group p2 also had the highest trap-6 expression in the tension site compared to the k(–), k(+) groups. orthodontic tooth movement is a useful model for understanding the mechanism of bone remodeling induced by mechanical loading. osteoclasts play an important role in otm where osteoclast have a bone resorption function.11 osteoclastogenesis mechanisms at a physiologic level have table 2. descriptive mean and standard deviation of orthodontic tooth movement maxillary left central incisive (mm) group mean standard deviation k(-) 0 0 k(+) 0.45 0.022 p1 0.496 0.008 p2 0.498 0.013 table 3. descriptive mean and standard deviation of trap-6 expression in tension area (cell/field of view) group trap-6 expression mean standard deviation k(-) 9.67 2.73 k(+) 8.5 1.22 p1 11.5 1.87 p2 13.17 2.13 table 4. one-way anova test of trap-6 expression in tension area variable one-way anova test f sig trap-6 expression between group 5.933 0.005 within group total table 5. lsd test of trap-6 expression in tension area variable lsd test sig trap-6 expression k(-) k(+) 0.763 p1 0.434 p2 0.038 k(+) p1 0.087 p2 0.004 p1 p2 0.514 figure 2. imunohistological section of trap-6 expression in the control group (a), in the orthodontic group (b), in the orthodontic + stichopus 3% group (c),), in the orthodontic + stichopus 3.5% group (d) under a light microscope at 400× magnification. figure 1. line chart mean of orthodontic tooth movement. figure 3. mean and sd of trap-6 in tension area orthodontic tooth movement. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 192 prameswari and brahmanta/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 188–193 a function through the macrophage-colony stimulating factor (m-csf) that induces osteoclast when there are no growth factor involved.3 in the tension area, trap-6 is found as positive multinuclear cells are localized within the ruffled border area as a osteoclast mature biomarker which has a relation with matrix remodeling.16 osteoclasts which formed by chondrocytes or osteoblasts resorb the bone mineralized matrix. osteoclasts are multinucleated giant cells dervied from hematopoietic cells. osteoclast differentiation is dependent on two cytokines, a tumor necrosis factor (tnf) family cytokine, receptor activator of nuclear factorκb (nf-κb) ligand (rankl) and m-csf. m-csf can stimulate monocyte proliferation. the cytokine receptor activator of nuclear factor-κb ligand (rankl), which is secreted by mesenchymal stem cell and osteoblasts, stimulates monocyte differentiation into osteoclasts.16,17 rankl produced by osteoblasts or their precursors plays a role in osteoclast formation, thereby relation between bone formation to resorption.17 the interaction of rankl with its receptor rank results in a cascade of intracellular events including: nf-κb, mitogen-activated protein kinases (mapks), ionic calcium and calcium/ calmodulin-dependent kinase by recruiting the adaptor signal protein tnf receptor associated factor (traf6). as a result, a number of osteoclast-related marker genes, including: trap-6, calcitonin receptor (ctr), cathepsin k (ctsk) and nuclear factor of activated t cells (nfatc1) are upregulated.16 research with a mouse strain without rankl, which can be conditionally deleted and made a series of cre-deleter strains to showed that rankl that controls mineralized bone resorption and bone remodeling produced by hypertrophic chondrocytes and osteocytes, where both embedded in bone matrix. besides osteoblast, osteocyte rankl have role for the bone loss associated with unloading.17 in the research reported here, the highest expression of trap-6 occurred in the p2 group that experienced the highest osteoclast activity resulting in matrix remodeling in the group given nanopowder stichopus hermanii 3.5%. the p1 group administered with stichopus hermanii 3% demonstrated no significant relationship with k(-) meaning that osteoclast for matrix remodeling in p1 group approached to normal physiologic condition. osteoclastogenesis has a crucial role in bone homeostasis. bone is preserved by active remodeling through the equilibrium between bone resorption by osteoclasts and bone apposition by osteoblasts.18 previous research by blummer into trap deficient mice confirmed that the formation of distinct bone relevant proteins and type i collagen were initiated at an earlier point in time. osteopontin, another bone specific marker in trap deficient mice, directly modulates bone formation in a response to mechanical stress which is independent of its effect on osteoclasts. runt related transcription factor 2 (runx2) expression occurred at the same point in trap deficient mice and proved crucial for osteoblast differentiation.19,20 this indicated that trap-6 has a role in bone and matrix formation. bone apposition can occur after trap-6 function in bone matrix remodeling.4 nanopowder stichopus hermanii contains various active ingredients such as flavonoid, epa, dha, triterpene, and glycosaminoglycans.13 epa as a component of: nanopowder stichopus hermanii is known to have a function in osteoclast differentiation. osteoclast differentiation takes places through several steps, including: progenitor growth, differentiation to mononuclear pre-osteoclasts, cell fusion to multinuclear osteoclasts and the activation of osteoclasts to unique ability to resorb bone and epa accelerated osteoclast fusion. in other ways, dha can prevent osteoclastogenesis is also related to cell-cell fusion, as shown by mononuclear trap-positive osteoclasts.18,21 osteoclastogenesis is also regulated by ap-1 as a transcription factor. ap-1 is a cell biosensor that can change extracellular signaling for cell function. when there is no ap-1 expressed in osteoblast, this can induce osteoclastogenesis through trap-6.22 flavonoid is one active ingredient of stichopus hermanii that can inhibit ap-1 and induce osteoclastogenesis.23–25 the application of nanopowder stichopus hermanii in the tension area could induce m-csf due to the effect of triterpene. triterpene can induce caspase-3 and caspase-9.26 activated caspases play an important role in the degradation of specific nuclear proteins and induce osteoblastic differentiation.27 accelerated bone formation in the tension area will induce bone resorption indirectly into the pressure area, thereby increasing biometric orthodontic tooth movement.28 chondroitin sulphate is one glycosaminoglycan which have effect on another functionally with cytokines, kemokins, and growth factors in the alveolar bone element and structures where osteoblasts and osteoclasts cooperate to coordinate the process of bone remodeling. glycosaminoglycan may help control osteoclastogenesis in microenvironments where osteoblasts/osteoclasts inherent. glycosaminoglicans-bound rankl block the interaction between rankl and rank.29 glycosaminoglycan has affinity for rankl and significantly prevents ranklinduced osteoclastogenesis by activating erk pathway. local interaction between bone cells is crucial factor for control bone remodeling and formation. the overall effect of glycosaminoglycan on osteoblasts is stimulatory, together with the ability of this glycosaminoglycan to prevent osteoclastogenesis. glycosaminoglycan shifts the homeostasis of bone remodeling tends towards bone formation by preferencing osteoblastogenesis while antagonizing osteoclastogenesis.30 the stimulatory effect of bone formation in the tension area can induce bone resorption in the pressure area as a response to physiological mechanical stress.11 in conclusion, active ingredients of nanopowder stichopus hermanii gel play a role in bone resorption in tension areas of orthodontic tooth movement. nanopowder stichopus hermanii 3.5% represented the optimum dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 193193prameswari and brahmanta/dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 188–193 concentration containing the active ingredient flavonoid, epa that can increase trap-6 expression as osteoclast activity to resorb periodontal ligament and alveolar bone in tension area orthodontic tooth movement. references 1. kementerian kesehatan republik indonesia. riset kesehatan dasar (riskesdas) 2013. jakarta: badan penelitian dan pengembangan kesehatan kementerian kesehatan ri; 2013. p. 143–5. 2. de almeida ab, leite icg, melgaço ca, marques ls. dissatisfaction with dentofacial appearance and the normative need for orthodontic treatment: determinant factors. dental press j orthod. 2014; 19(3): 120–6. 3. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofac orthop. 2006; 129(4): 469.e1–32. 4. ariffin shz, yamamoto z, abidin izz, wahab rma, ariffin zz. cellular and molecular changes in orthodontic tooth movement. sci world j. 2011; 11: 1788–803. 5. proffit wr, fields hw, sarver dm. contemporary orthodontics. 4th ed. st louis-missouri: mosby elsevier; 2007. p. 75–84. 6. susilowati s. peran matriks metaloproteinase-8 pada cairan krevikuler gingiva selama pergerakan gigi ortodontik. dentofasial. 2010; 9(1): 47–54. 7. alhadlaq am. biomarkers of orthodontic tooth movement in gingival crevicular fluid: a systematic review. j contemp dent pract. 2015; 16(7): 578–87. 8. prameswari n. the response of periodontal ligament collagen fibres and the thickness of inserting periodontal ligament fibre bundles at cementum pressure sites of fixed orthodontic appliances. dent j (maj ked gigi). 2007; 40(2): 70–5. 9. mohamed am. an overview of bone cells and their regulating factors of differentiation. malays j med sci. 2008; 15(1): 4–12. 10. prameswari n, soetjipto s, rahayu rp. osteogenesis at tension site by stichopus hermanii application as relapse orthodontic prevention. int j chemtech res. 2016; 9(6): 686–93. 11. kitaura h, kimura k, ishida m, sugisawa h, kohara h, yoshimatsu m, takano-yamamoto t. effect of cytokines on osteoclast formation and bone resorption during mechanical force loading of the periodontal membrane. sci world j. 2014; 2014: 1–7. 12. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods--a review. mar drugs. 2011; 9: 1761–805. 13. sendih s, gunawan. keajaiban teripang: penyembuh mujarab dari laut. jakarta: agro media pustaka; 2006. p. 13–49. 14. revianti s, soetjipto s, rahayu rp, parisihni k. protective role of sticophus hermanii ethanol extract supplementation to oxidative stress and oral hyperkeratosis in smoking exposed rats. int j chemtech res. 2016; 9(5): 408–17. 15. zohdi rm, zakaria zab, yusof n, mustapha nm, abdullah mnh. sea cucumber (stichopus hermanii) based hydrogel to treat burn wounds in rats. j biomed mater res part b appl biomater. 2011; 98(1): 30–7. 16. li j, zeng l, xie j, yue z, deng h, ma x, zheng c, wu x, luo j, liu m. inhibition of osteoclastogenesis and bone resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops. sci rep. 2015; 5: 1-14. 17. xiong j, onal m, jilka rl, weinstein rs, manolagas sc, o’brien ca. matrix-embedded cells control osteoclast formation. nat med. 2012; 17(10): 1235–41. 18. akiyama m, nakahama k, morita i. impact of docosahexaenoic acid on gene expression during osteoclastogenesis in vitro--a comprehensive analysis. nutrients. 2013; 5: 3151–62. 19. blumer mjf, hausott b, schwarzer c, hayman ar, stempel j, fritsch h. role of tartrate-resistant acid phosphatase (trap) in long bone development. mech dev. 2012; 129: 162–76. 20. rodriguez-carballo e, gámez b, ventura f. p38 mapk signaling in osteoblast differentiation. front cell dev biol. 2016; 4: 1–20. 21. rahman mm, bhattacharya a, fernandes g. docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in raw 264.7 cells than eicosapentaenoic acid. j cell physiol. 2008; 214: 201–9. 22. braun t, zwerina j. positive regulators of osteoclastogenesis and bone resorption in rheumatoid arthritis. arthritis res ther. 2011; 13: 1–11. 23. shu s. immunological effects of complementary and alternative medicine in allergy and astma. disertation. california: university of california; 2008. p. 1–20. 24. chi l, gao w, shu x, lu x. a natural flavonoid glucoside, icariin, regulates th17 and alleviates rheumatoid arthritis in a murine model. mediators inflamm. 2014; 2014: 1-10. 25. osta b, lavocat f, eljaafari a, miossec p. effects of interleukin-17a on osteogenic differentiation of isolated human mesenchymal stem cells. front immunol. 2014; 5: 1–8. 26. reyes-zurita fj, pachón-peña g, lizárraga d, rufino-palomares ee, cascante m, lupiáñez ja. the natural triterpene maslinic acid induces apoptosis in ht29 colon cancer cells by a jnk-p53dependent mechanism. bmc cancer. 2011; 11: 1–13. 27. bell rav, megeney la. evolution of caspase-mediated cell death and differentiation: twins separated at birth. cell death differ. 2017; 24: 1359–68. 28. nimeri g, kau ch, kheir nsa, corona r. acceleration of tooth movement during orthodontic treatment a frontier in orthodontics. progress in orthodontics. 2013; 14: 42. 29. savage jr, pulsipher a, rao nv., kennedy tp, prestwich gd, ryan me, lee wy. a modified glycosaminoglycan, gm-0111, inhibits molecular signaling involved in periodontitis. plos one. 2016; 11(6): 1–20. 30. l i ng l , mu r a l i s, st e i n g s, va n wijn e n a j, c o ol sm . glycosaminoglycans modulate rankl-induced osteoclastogenesis. j cell biochem. 2010; 109(6): 1222–31. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p188–193 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p188-193 81 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 81–87 original article introduction periodontal dressings are commonly applied to open wounds after periodontal treatment.1 the periodontal dressings used in dentistry today do not contain compounds that can accelerate wound healing, only protecting wound tissue rather than providing healing factors.2 gum damage can occur due to periodontal disease, trauma, tooth extraction or oral surgery.3 in surgical procedures such as gingivectomy and depigmentation, an incision in the gum tissue is made to provide access and field of view and repair morphological and anatomical damage.4 restoring the integrity of damaged tissue and maintaining homeostasis is an important procedure in healing gum wounds.3 a periodontal dressing is a physical barrier that serves to protect patients from pain due to contact of the wound with food or with the tongue during chewing, provides comfort to the patient, allows the tissues to adapt to the process of wound closure, and minimises postoperative bleeding and the possibility of infection.4 the healing process takes place in several phases, including the proliferative phase. an indicator of wound healing in the proliferative phase is characterised by an increase in the number of fibroblasts during this phase. fibroblasts are cellular components commonly found in connective tissue. fibroblasts are responsible for the formation of collagen, the main constituent of the extracellular matrix, which is useful for strengthening scar tissue, cell contraction, influencing the re-epithelialisation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 the effect of lime (citrus aurantifola swingle) peel extract in periodontal dressings on the number of fibroblasts in the gingival wound healing process malianawati fauzia, audia putri dewanti department of periodontics, faculty of dentistry, universitas brawijaya, malang, indonesia abstract background: periodontal dressing commonly used in dentistry today does not contain compounds that can accelerate wound healing. lime (citrus aurantifolia swingle) peel contains flavonoids that play a role in increasing fibroblast cells so that they can accelerate the healing process. periodontal dressings supplemented with lime (citrus aurantifolia swingle) peel extract are expected to provide an alternative material that can accelerate wound healing in addition to closure. purpose: the study aims to determine the effect of adding lime (citrus aurantifolia swingle) peel extract to periodontal dressings on the increase in the number of fibroblasts in the gingival healing process. methods: the study was conducted in an experimental laboratory in vivo. the study used a post-randomised control group of 32 rabbits with lesions of the mandibular gingiva using a 2 mm diameter punch biopsy. the experimental animals were divided into 8 groups, namely the control group, which was treated with periodontal dressings without the addition of lime (citrus aurantifolia swingle) peel extract, and the treatment group, which was treated with periodontal dressings with the addition of the extract. histological observations of the tissues were performed with he staining to count the number of fibroblasts. results: statistical test results showed that there was a significant difference in the number of fibroblasts between the control group and the treatment group on day 3 and day 5 (anova, p <0.05). conclusion: adding extra lime (citrus aurantifolia swingle) peel to the periodontal dressing increases the number of fibroblast cells after gum injury. keywords: fibroblasts; lime (citrus aurantifolia swingle) peel extract; periodontal dressing; wound healing correspondence: malianawati fauzia, department of periodontics, faculty of dentistry, universitas brawijaya. jl. veteran, malang, 65145, indonesia. email: meli_fkg@ub.ac.id mailto:meli_fkg@ub.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 82 process, and forming granulation tissue in the process of angiogenesis. thus, fibroblast cells play an important role in the wound-healing process.5 the use of natural ingredients is increasing, both in medicine and for other purposes. natural ingredients have the following advantages: easy to obtain; inexpensive; minimal side effects; and, generally, a plant can have more than one pharmacological effect. lime (citrus aurantifolia swingle) is one of the herbal remedies that can be used as an additional ingredient in periodontal dressings.6 linden (citrus aurantifolia swingle) is a type of herbaceous plant widely cultivated in indonesia, both in gardens and plantations. compared to other types of citrus, citrus aurantifolia has more variations and applications, so it is often referred to as a versatile fruit.7 lime (citrus aurantifolia swingle) peel contains beneficial compounds such as flavonoids, alkaloids, tannins and saponins, which can be used for wound healing.8 the peel has a higher concentration of flavonoids compared to other parts, such as seeds, fruits, and lime juice.9 the flavonoids are believed to have anti-inflammatory properties that inhibit prostaglandins, which are inflammatory mediators, thus reducing the number of inflammatory cells that migrate to the area.10 in addition, the flavonoid compounds have antibacterial and antioxidant properties, so they are effective in accelerating the wound healing process.11 a previous study was conducted on the effectiveness of lime (citrus aurantifolia swingle) peel extract in accelerating the healing process of post-extraction alveolar wounds on rats’ teeth. the results indicated a significant increase in the number of fibroblast cells on the third and fifth days after topical application of peel extract gel in the post-extraction socket.12 these findings motivated the authors to investigate the effects of adding lime (citrus aurantifolia swingle) peel extract to the periodontal dressing on increasing the count of fibroblasts in the healing process of gum wounds. materials and methods this research is an in vivo laboratory experiment using the post test-only control group research method. the study was approved by the animal care and use committee, brawijaya university, malang, with the ethics committee number 051-kep-ub-2021. the sample used in this study was the new zealand white rabbit. the sample crown was selected using the simple random sampling technique. the samples were of 32 individuals and were divided into 8 groups. the control groups (k1, k2) had no peel extract added to the periodontal dressing. the treatment groups (p1, p4) had 5% peel extract added to the periodontal dressing; the treatment groups (p2, p5) had 10% peel extract added to the periodontal dressing, and the treatment groups (p3, p6) had 15% peel extract added to the periodontal dressing. inclusion criteria for the sample were: healthy, male new zealand white rabbits, 4-5 months old, with body weights of 3–4 kg, without previous treatment or exposure, and not disabled. the exclusion criteria were rabbits that died during the study. this research was conducted for about four months in the stem cell laboratory of the university of airlangga. the experimental animals were selected according to the criteria of the sample and then adapted for 7 days. the production of lime (citrus aurantifolia swingle) peel extract employed the maceration method using 70% ethanol solvent and a thick extract preparation was obtained. a periodontal dressing according to baer’s formulation consists of a powder and a paste. on a paper stirrer using a wooden spatula, make a powder dressing by mixing 28.5 g of rosin and 21.5 g of zinc oxide until homogeneous, additionally, make a paste dressing by mixing 47.5g of hydrogenated fat and 2.5g of zinc oxide until blended on a paper stirrer using a wooden spatula. then gradually mix 50 mg of powder and 50 mg of paste until homogeneous and 100 mg of each group is obtained (figure 1a).11 then baer’s formulation of periodontal dressing (mg) was mixed with lime (citrus aurantifolia swingle)peel extract (mg) into groups as shown in table 1. then the operator performed surgery on the test animals, first disinfecting the operating area with 70% alcohol. they were then anaesthetised by injecting a combination of ketamine and xylazine intramuscularly. the dose of ketamine used was 25 mg/kg body weight, followed by an injection of xylazine at a dose of 3 mg/kg body weight. each rabbit underwent surgery on the lower incisor region of the attached labial gingival mucosa with fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 figure 1. the manipulation of the periodontal pack with lime peel extract (a) and the performed punch biopsy on the rabbit gingiva (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 83 fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 table 1. baer’s formulation periodontal dressing with and without added lime peel extract group baer’s formulation of periodontal dressing (mg) lime peel extract (mg) day-3 k1 0% 100 0 p1 5% 95 5 p2 10% 90 10 p3 15% 85 15 day-5 k2 0% 100 0 p4 5% 95 5 p5 10% 90 10 p6 15% 85 15 the same control treatment, namely: using a punch biopsy tool with a diameter of 2 mm and pressing the depth of the wound to reach the alveolar bone but not to damage it, so as not to cause bone injury (figure 1b). then, the wound was cleaned with a solution of 0.9 ml nacl and 3% h2o2.13 this was followed by the application of a periodontal dressing to the wound area (figure 2a). the periodontal dressing had lime (citrus aurantifolia swingle) peel extract added for the treatment group and was without added peel extract for the control group. the periodontal dressing is shaped according to the shape of the wound. after application, the periodontal dressing was gently squeezed to cover the gum wound area using an excavator. to increase the retention of the periodontal dressing so that it would not come off, sutures were made between the lips and the gingival lining of the lower jaw using a 5.0 floss thread (figure 2b). sampling was done on the third and fifth days to evaluate the number of fibroblast cells in the gingival granulation tissue of each experimental group. experimental animals were euthanised under anaesthesia using a lethal dose of ketamine, i.e. 200 mg/kg body weight, by intramuscular injection. then, the gingival granulation tissue was placed in a container with a 10% formalin solution.14 tissue preparations were made with eosin haematoxylin staining. observation of fibroblast cells was performed with an olympus digital optical microscope at 400x magnification from 5 different fields of view.15 shapiro wilk’s statistical test and levene’s test were performed because the data results were homogeneous and normal and then continued with one-way anova, post-hoc tukey, and independent t tests. results this study showed that the number of fibroblast cells on day 3 (figure 3) and day 5 (figure 4) in the control group was lower than in the treatment group. this is because the treatment group received lime (citrus aurantifolia swingle) peel extract to speed up wound healing. in figure 5, it can be seen that in a peel extract of 5% and 10%, the average number of fibroblasts in the gum healing process on day 3 showed an increase, but it showed a decrease in a concentration of 15%. results on day 5 were the same. overall, the administration of the 10% peel extract showed the highest average number of fibroblasts on day 3 and day 5 compared to other groups. thirty-two rabbits, divided into 8 groups, were observed by counting the number of fibroblasts on histological preparations at a 400x magnification. the data obtained from the calculation of the number of fibroblast cells was then processed using the one-way anova test (table 2). before performing the one-way anova test, a normality test was calculated by performing the shapiro-wilk test, which indicated a significance (p > 0.05). this means that research data is distributed normally. figure 2. application of periodontal pack on the gingival wounds (a) and wound closure with sutures (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 84 a b c d figure 3. day 3 fibroblast cells with he staining, magnification 400x. a) (k1) periodontal dressing without lime peel extract, b) (p1) periodontal dressing with 5% lime peel extract, c) (p2) periodontal dressing with 10% lime peel extract, d) (p3) periodontal dressing with 15% lime peel extract. a b c d figure 4. day 5 fibroblast cells with he staining, magnification 400x. a) (k2) periodontal dressing without lime peel extract, b) (p4) periodontal dressing with 5% lime peel extract, c) (p5) periodontal dressing with 10% lime peel extract, d) (p6) periodontal dressing with 15% lime peel extract. 0 20 40 60 80 100 120 140 160 180 10% lime peel extract m ea n fi br ob la st day 3 day 5 control 79.85 67.5 119.55 164.25 138.65 111.95 59.75 41.453 15% lime peel extract 5% lime peel extract fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 figure 5. the average of fibroblast cells by preparation. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 85 fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 the results of the one-way anova test (table 2) on days 3 and 5 obtained sig 0.000 (sig < 0.05), which indicates the effect of adding lime (citrus aurantifolia swingle) peel extract to periodontal dressings by the increase in the number of fibroblasts in the gingival healing process on day 3 and day 5. the post-hoc tukey test was performed separately between time points due to the sheer effect of each day of treatment. the results of the post-hoc tukey test –the difference in the number of fibroblasts in the gingival healing process on the 3rd and 5th days – indicated that each group showed a significant difference (sig < 0.05), which can be seen in table 3. this proves that adding lime (citrus aurantifolia swingle) peel extract to the periodontal dressing significantly increased the number of fibroblasts in the gingival healing process on day 5, with a concentration of 10% resulting in the greatest increase in the number of fibroblasts, followed by a concentration of 5%, and then by a concentration of 15%. to find out if there was difference in the number of fibroblasts in the gingival healing process between observations, an independent t test was performed. the results of the independent t test at concentrations of 5%, 10% and 15% of lime (citrus aurantifolia swingle) peel extract showed a significant increase in the number of fibroblasts (sig < 0.05) from day 3 to day 5 (table 4). discussion this study indicates that compared to the control group, the treatment group that received a periodontal dressing with the addition of lime (citrus aurantifolia swingle) peel extract in concentrations of 5%, 10% and 15% was able to accelerate the healing response in rabbits previously treated with the labial gingiva of the lower jaw. the increased acceleration of the wound healing response was indicated by the increased number of fibroblast cells in the wound area in the treatment group. in observation of the histological preparations of the post-injured rabbit gingival granulation tissue, fibroblast cells were observed on the 3rd and 5th days. this is consistent with chasya et al.’s16 statement that active fibroblast cells proliferate or experience a significant increase from day 3 to day 7 after injury. in a study conducted by nguyen et al.,17 it was found that fibroblast cell proliferation occurred for 7 days and reached its peak on day 5. after which, the more the number of days, the more fibroblast proliferation decreases, indicating that there has been progress in the healing process.16 in this study, baer’s periodontal dressing formula was applied because it is commonly used for research purposes and is a pure composition of periodontal dressing without the addition of other ingredients. the advantage of this periodontal dressing is that it is safe to use during the wound healing process as it does not contain eugenol that can cause soft tissue irritation or necrosis, so it does not interfere with fibroblast formation.18 in the control group, the number of fibroblasts was the least because the control group had not received any stimulus that could increase the activation of macrophages to stimulate growth factor components, so the group selfhealed, unlike the treatment group, which showed an increase in the number of fibroblast cells caused by the presence of active substances contained in the lime (citrus aurantifolia swingle) peel extract to speed up the healing process of gum wounds by, for example, the intermediary role of macrophages that stimulate growth factors.12 based on the study’s phytochemical tests, krismaya et al.12 showed that the highest saponin content in lime (citrus aurantifolia swingle) peel was 3.05, which had antiseptic, antioxidant and antibacterial properties. next is the flavonoid content of 2.78% with its anti-inflammatory, antioxidant and antibacterial properties. in addition, lime (citrus aurantifolia swingle) peel also contains 2.14% of tannic compounds with antioxidant and antibacterial properties, and 1.86% of alkaloids with antibacterial properties.12 so that the most dominant chemical activity is saponins and flavonoids. the ability of the active substances in lime (citrus aurantifolia swingle) peel extract to increase the average number of fibroblast cells in the treatment group is due to the anti-inflammatory effect of the flavonoids that work by directly inhibiting the activity of cox enzymes and lipoxygenase, which cause release inhibition of a table 2. data of one-way anova fibroblast cells number of fibroblast cells sig. day 3 between groups .000 within groups total day 5 between groups .000 within groups total table 3. post-hoc tukey (hsd) test day 3 and 5 day test group treatment sig day 3 k1 – p1 0.000 k1 – p2 0.000 k1 – p3 0.000 p1 – p2 0.016 p1 – p3 0.000 p2 – p3 0.000 day 5 k2 – p4 0.000 k2 – p5 0.000 k2 – p6 0.000 p4 – p5 0.000 p4 – p6 0.000 table 4. independent t test group observation time mean sig 5% lime peel extract day 3 111.95 0.000 day 5 138.65 10% lime peel extract day 3 119.55 0.000 day 5 164.25 15% lime peel extract day 3 67.50 0.000 day 5 79.85 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 86 number of inflammatory mediators such as prostaglandins, thromboxane and leukotrienes, leading to a decrease in the inflammatory response in the wound area. flavonoids have the ability, as immunomodulators, to activate macrophages to perform phagocytosis, tissue repair, and work to activate t cells to differentiate and proliferate into th1, th2, and th3. th3 cells produce growth hormones (growth factors) such as the epidermal growth factor (egf), transforming growth factor-β (tgf-β) and fibroblast growth factor (fgf), which play a role in stimulating fibroblast proliferation and increasing the migration of fibroblasts, smooth muscle cells, and endothelial cells in the wound area.18 the increase in the average number of fibroblast cells in the treatment group can also be caused by antibacterial properties. alkaloids play an important role because they have antibacterial properties that work by inhibiting the formation of protein synthesis so that they can interfere with bacterial metabolism, while flavonoids act as antibacterial with bacteriostatic properties. flavonoid compounds can form complex compounds with proteins through hydrogen bonds, so that the tertiary structure of the protein is disrupted and the protein can no longer function in accelerating wound healing.17 tannins also have antibacterial properties that work by binding to any of the membrane proteins possessed by bacteria and can then damage the availability of receptors on the surface of bacterial cells or interfere with cell permeability so that it interferes with cellular metabolic processes and cells can suffer death.18 in addition, saponins also have antibacterial properties by binding to sterols (bacterial proteins) on the surface of the bacterial cell membrane, which can increase the permeability of the bacterial cell membrane so that it can change the structure and function of the membrane causing protein denaturation, so that the cell membrane will be damaged and lysis.18 bacterial death results in a reduction in bacterial phagocytosis by pmn leukocytes. this results in a brief inflammatory phase so that the proliferative phase takes place earlier.19 the increase in the average number of fibroblast cells in the treatment group can also be caused by the antioxidant capacity of tannins. tannins have an antioxidant feature that acts as an anti-inflammatory by inhibiting the production of oxidants (o2) by neutrophils, monocytes and macrophages. this high antioxidant activity can accelerate wound healing as it can stimulate the production of endogenous antioxidants at the wound site and provide a conducive environment for wound healing.19 compared to previous studies, testing the periodontal dressing with the addition of cinnamon extract that showed that the average number of fibroblasts on days 3 and 5 respectively in the control group was 21 and 16, whereas in this study the average number of fibroblasts in the control group on days 3 and 5 respectively was 41 and 59. in the treatment group, the cinnamon extract added at 5% showed average results of 22 and 20 while this study obtained averages of 111 and 138. in the treatment group, 10% of added cinnamon extract showed average scores of 26 and 23, while in this study averages of 119 and 164 were obtained. in the treatment group, when 15% of cinnamon extract was added, it showed average scores of 32 and 28, while in this study averages of 67 and 79 were obtained.19 this shows that adding lime (citrus aurantifolia swingle) peel extract to periodontal dressings is more effective in accelerating wound healing compared to adding cinnamon extract to periodontal dressings, because the active content of 1 lime (citrus aurantifolia swingle) peel extract can inhibit and significantly decrease the inflammatory response. this is in line with the statement by sloane et al.21 on the efficacy test of lime (citrus aurantifolia swingle) peel extract cream at a concentration of 10% that was found to be 96% effective in healing burns in the skin of white mice. in this study, the results showed that the average increase in the number of optimal fibroblast cells was obtained in the treatment group that received the periodontal dressing with baer’s formula with the addition of lime (citrus aurantifolia swingle) peel extract to a concentration of 10%, while at a concentration of 15%, the average number of fibroblasts was the lowest. indeed, the mixture of materials is not as homogeneous as in the mixture of a periodontal dressing with the addition of extract concentrations of 5% and 10%. with the addition of a concentration of 15%, the preparation of the periodontal dressing becomes more humid and difficult to position, so that the substances contained in the peel do not work optimally at a concentration of 15%. meanwhile, adding an extract with a 5% concentration increased the number of fibroblast cells, which was less than optimal compared to adding a 10% concentration. this is probably caused by the lack of active lime (citrus aurantifolia swingle) peel content added to the periodontal dressing, causing less than optimal results.13 we can conclude that adding lime peel extract (citrus aurantifolia swingle) to the periodontal dressing has the effect of increasing the number of fibroblasts in the gum healing process. the number of fibroblast cells in the periodontal dressing treatment group with the addition of lime (citrus aurantifolia swingle) peel extract was higher than that in the group without the addition of the extract, and there is a relationship between the number of fibroblasts and the lime (citrus aurantifolia swingle) peel extract in the healing process of gum wounds. references 1. baghani z, kadkhodazadeh m. periodontal dressing: a review article. j dent res dent clin dent prospects. 2013; 7(4): 183–91. 2. newman m, takei h, klokkevold p, carranza f. newman and carranza’s clinical periodontology. 13th ed. st louis: saunders elsevier; 2018. p. 944. 3. novitasari aim, indraswary r, pratiwi r. pengaruh aplikasi gel ekstrak membran kulit telur bebek 10% terhadap kepadatan serabut kolagen pada proses penyembuhan luka gingiva. odonto dent j. 2017; 4(1): 13–20. 4. budisidharta y, syaify a, lastianny sp. the effects of zinc oxide non-eugenol and cellulose as periodontal dressings on open wounds fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 87 fauzia and dewanti/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 81–87 after periodontal surgery. dent j (majalah kedokt gigi). 2020; 53(1): 45–9. 5. sumbaya k em. fibroblas: st r uktur da n pera na n nya da la m penyembuhan luka. j kedokt meditek. 2015; 21(6): 1–6. 6. willianti e, theodora, parmasari wd. analisa aktivitas antibakteri rebusan daun sir ih dengan rebusan daun kemangi terhadap pertumbuhan bakteri streptococcus mutans. hang tuah med j. 2020; 18(1): 36–46. 7. gaol lal, meiriani, purba e. respons pertumbuhan setek jeruk nipis (citrus aurantifolia swingle) pada berbagai bahan tanam dan konsentrasi iba (indole butyric acid). j agroekoteknologi. 2015; 4(1): 1815–21. 8. pratiwi d, suswati i, abdullah m. efek anti bakteri ekstrak kulit jeruk nipis (citrus aurantifolia) terhadap salmonella typhi secara in vitro. saintika med. 2013; 9(2): 110–4. 9. wardani r, jekti dsd, sedijani p. uji aktivitas antibakteri ekstrak kulit buah jeruk nipis (citrus aurantifolia swingle) terhadap pertumbuhan bakteri isolat klinis. j penelit pendidik ipa. 2018; 5(1): 10–7. 10. rahmadhani n, yudaniayanti is, saputro al, triakoso n, wibawati pa, yudhana a. efektivitas k rim ekstrak buah naga merah (hylocereus polyrhizus) dalam meningkatkan jumlah sel fibroblas luka bakar derajat ii pada tikus putih (rattus norvegicus). j med vet. 2020; 3(1): 65–75. 11. pradita au, dhartono ap, ramadhany ca, taqwim a. periodontal dressing-containing green tea epigallocathechin gallate increases fibroblasts number in gingival artifical wound model. j dent indones. 2013; 20(3): 68–72. 12. krismaya idgan, pramudya r, sati pyi, kamadjaja db. effects of lime (citrus aurantifolia christm. swingle) peel extract on fibroblast proliferation and angiogenesis in rat’s tooth extraction sockets. biochem cell arch. 2019; 19(suppl. 2): 4917–9. 13. naba’atin i, wahyukundari ma, harmono h. penambahan ekstrak kulit buah kakao (theobroma cacao l.) pada periodontal dressing terhadap kepadatan kolagen luka gingiva kelinci. indones dent student j. 2015; 3(2): 28–38. 14. widyastomo, wulan ka, sari ip. pengaruh jus buah belimbing manis (averrhoa carambola linn.) terhadap peningkatan jumlah fibroblas pada soket tikus wistar pasca ekstraksi gigi. prodenta j dent. 2013; 1(2): 62–70. 15. sorongan rs, siagian, v k. efektivitas perasan daun pepaya terhadap aktivitas fibroblas pasca pencabutan gigi pada tikus wistar jantan. pharmacon. 2015; 4(4): 52–7. 16. chasya sa, munawir a, sulistyaningsih e. pengaruh pemberian gel doksisiklin terhadap jumlah fibroblas pada proses penyembuhan dermatitis paederus akibat racun kumbang tomcat (paederus sp.) pada mencit. pustaka kesehat. 2016; 4(2): 200–4. 17. nguyen pa, pham tav. effects of platelet-rich plasma on human gingival fibroblast proliferation and migration in vitro. j appl oral sci. 2018; 26: e20180077. 18. prastiwi ss, ferdiansyah f. kandungan dan aktivitas farmakologi jeruk nipis (citrus aurantifolia swing.). farmaka. 2013; 15(2): 1–8. 19. liliawanti, siswanto fm. krim ekstrak daun binahong (anredera condifolia (ten) steenis) mempercepat penyembuhan luka insisi tikus wistar jantan. j media sains. 2019; 3(2): 63–70. 20. vavata ml, lisda v.e nlpb, ramadhana s, ari susanti dn. penga r uh cinnamaldehyde da r i kayu manis (cinnamomum burmanii) pada periodontal dressing terhadap sel fibroblas pada luka gingiva kelinci. interdental j kedokt gigi. 2019; 15(2): 45–9. 21. ulfa am, marcellia s, rositasari e. efektivitas formulasi krim ekstrak kulit jer uk nipis (citr us aurantifoliaper icappium) sebagai pengobatan luka sayat stadium ii pada tikus putih (rattus novergicus). j farm malahayati. 2020; 3(1): 42–52. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p81–87 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p81-87 1919 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 19–22 the correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in surabaya puspa dila rohmaniar,1 titiek berniyanti,2 and retno pudji rahayu3 1department of dental public health, faculty of dentistry, insitut ilmu kesehatan bhakti wiyata, kediri indonesia 2department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 3department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: exposure of metals among dental technicians that come from the working environment can lead to the formation reactive oxygen species (ros). ros can cause mutations in the p53 gene (p53). the mutation is transversion mutation guaninethymine. p53 mutations can lead to low expression of the wild-type p53 protein (p53). wild-type p53 involved in many biological processes such as regulation of genes involved in cell cycle, cell growth after dna damage, and apoptosis. however, exposure to metals among dental technicians can be prevented through the use of personal protective equipment (ppe) during work. purpose: the purpose of this study was to analyze the correlation between the use of personal protective equipment to wild-type p53 protein levels among dental technicians in surabaya. method: this study was observational analytic with cross sectional approach. 40 samples were taken by random sampling. data were retrieved through interviews and observations. wild-type p53 was analyzed from saliva with indirect elisa method. analysis of data used kolmogorov smirnov normality test and a pearson correlation test. value significance was p<0.05 (95% confidence level). result: there was a significant association between the use of personal protective equipment with wild-type p53 levels with p=0.002 conclusion: the use ppe properly is positively correlated with the wild-type p53 protein levels of dental technicians in surabaya. keywords: personal protective equipment; dental technician; p53 wild correspondence: titiek berniyanti, department of dental public health, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: berniyanti@gmail.com introduction dental technician should be exposed to various physical agents, chemical, and biological derived from the work environment. that exposure by inhalation, ingestion or skin contact.1 exposure of dust or smoke to the dental technician are coming from grinding of dental restoration materials during processing.2 there is a study that reported high concentrations of cobalt metal, nickel, chromium in blood of dental technicians in surabaya, such as: levels of cobalt: 27 g/ l, nickel 37 g/ l, and chromium 117 mcg/ l.3 other research in northern jordan also reported high levels of cobalt and chromium in blood of dental technicians. 2 exposure may result in potential lung diseases such as bronchial asthma, cancer, mesothelioma and pneumoconiosis depends on the duration of exposure.4 the prevalences of contact dermatitis among dental technicians are 22% in australia and 43% in denmark.5 the prevalence of pnumoconis among dental technicians in ankara is 10.1% and the prevalence of dermatitis kontakta of the dental technicians in germany is 16%.4 it is important for dental technicians to obey the standards and safety procedures. dental technicians must wear personal protective equipment (ppe) including work wear, protective mask, protective gloves and goggles, and ventilate the workplace. if ventilation, exhauster, adequate and adequately filter will reduce the level of chromium, cobalt, and nickel in the air.1 genotoxic metal exposure may increase the number of reactive oxygen species (ros). the metal ions of chromium, cobalt, and nickel can produce hydroxyl radicals (oh) through the fenton and haber-weis reaction. the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p19-22 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p19-22 20 rohmaniar, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 19–22 hydroxyl radicals can cause dna damage.6 typical damage caused by dna oxidation is a transversion mutations in guanine.7 p53 protein is involved in various biological processes such as regulation of genes involved in cell cycle, cell growth after dna damage, and apoptosis.8 p53 is considered as the gene most commonly mutated in human malignant tumors. p53 tumor suppressor gene is mutated in 50% of human tumors in various organs. with the development of molecular biology techniques can explain that one of the causes of the malignant process is a failure or inactivation of tumor suppressor genes p53.7,9 cells that have mutations or loss of wild-type p53 gene, the p53 protein expression wild does not occur or occurs wild-type p53 protein expression but can not function as activating gene transcription in some target.10 p53 protein expressed by tumor suppressor p53 gene can be detected through saliva. saliva is a diagnostic medium for the detection of various molecules contained in the blood; saliva can provide similar information about the human status as obtained from a blood test without invasive procedures.11 the purpose of this study was to analyze the correlation between the use of personal protective equipment and wild-type p53 protein levels among the dental technicians in surabaya. material and methods the ethics committee of the faculty of dental medicine, universitas airlangga has approved the implementation of this study, and all respondents in this study had signed a written consent. this study was conducted among 40 dental technicians in surabaya. sampling was done by random sampling with sample criteria namely; working on dentures with metal-containing material mixed ni, co and, cr for more than 3 years.3 all the participants were accepted all procedures such as saliva examination, answering questions and using ppe through a questionnaire. the criteria gender are not differentiated because gender differences have no significant effects on the levels of p53.12,13 the use of ppe during working hours by participants is via observations and questions using questionnaires and then do the scoring. examinations of ppe include the frequency and manner of use of masks, gloves, goggles, laboratories work clothes, and shoes by a dental technician. before taking saliva samples, subjects were instructed to not eat, smoke, antiseptic gargle one hour earlier.14 each saliva sample was taken between at 10.00 a.m -13.00 p.m.12 the participants were asked to first collect their saliva and accumulated it in their mouth then instructed to spit 3cc into a tube. each sample of saliva collected was centrifuged 2000 rpm for 10 minutes in order to obtain supernatan.12 the level of p53 wild was analysed at the institute of tropical disease, universitas airlangga. p53 saliva levels were checked by indirect elisa method using human tp53 (tumor protein p53) elisa kit (elabscience biotechnology co., wuhan, hubei, china ). rating score of ppe based on exposure categories main lines of metal that enters the body. masks and gloves have the highest percentage due to exposure to the metal enters the body through three main channels; respiratory, oral, and skin.15 how to score ppe is based on research conducted by risdayanti.16 categories score of ppe usage frequenty score questionnaires are divided into: always, rarely, and never with respective scores of 100, 50, and 0. categories score of ppe usage procedures are divided into: true, is not always true, and not true with respective scores of 100, 50, and 0. summing scores ppe usage frequency and usage procedures ppe score. the score was multiplied by the percentage weighting of ppe. the percentages of weighting were 30% for mask , 25% gloves, 20% for goggles, 15% for laboratories work clothes and 10% for shoes. results based on observations and questionnaires the use of ppe and p53 levels among dental technician in surabaya. in table 1 shows the average score ppe dental technicians in surabaya is 86.19 ± 27.41. the average p53 wild score 10 table 1. mean and standard deviation score the use of ppe and levels of p53 wild score ppe p53 n 40 40 mean ± sd 86.19 ± 27.41 0.27 ± 0.28 figure 1. relationship p53wild levels with a score of use ppe. table 2. relationship scores the use of ppe and levels of wild p53 protein among dental technicians in surabaya independent variable dependent variable p r score ppe level p53 0,002 0,466 * p<0.05 = significant correlation figure 1. relationship p53wild levels with a score of use ppe. to ta l s co re a pd dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p19-22 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p19-22 2121rohmaniar, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 19–22 dental technicians is 0.27 ± 0.28. figure 1 shows that levels p53 increase as increasing score of ppe. table 2 shows the significance correlation test pearson between score of ppe and level wild-type p53 saliva among dental technicians is p= 0,002 with significance value of p<0.05. the correlation pearson value is r = 0.446 discussion based on the results of this study showed there were positive correlation between the levels of wild-type p53 with a score of use of personal protective equipment at the dental technicians in surabaya. if dental technician used ppe appropriately during work, so the levels of wild-type p53 was higher too. because protective clothing, protective mask, protective gloves and goggles. can prevent metal exposure during work.1 this metal exposure can increase the number ros endogenous.17 increasing ros can oxidize dna and produce 8-hidroksi-deoksiguanosin (8-oh-dg). this products induce transversions mutation guanine into thymin,18 the same typical mutations occurs in p53 gene. 7 cells that have mutations or loss of the p53 gene, the expression of p53 (p53 protein) might or might not occur p53 protein expression but if occurred it could not be functioning as an activating gene transcription in some target.10 the dental technicians in surabaya, on average, did not use ppe properly during the work, so the wild-type p53 levels in saliva became lower than normal.12,19 this occurs because of exposure to the metal derived from a dental technician working environment can not be prevented through the use of personal protective equipments which include the use of masks, gloves, goggles, clothing and lab work, special shoes. when the use of personal protective equipment is adequate then it will reduce the level of chromium, cobalt, and nickel in blood.1 masks can minimize the chemical exposure entering the respiratory tract. the results showed that the efficiency of face masks appropriate to reduce exposure to chemicals are inhaled to reach 70% -95%.20 disposible latex gloves can inhibit the penetration of chemicals through the skin.21 .goggles, protective clothing and shoes can reduce chemical exposure. 22,23,24 nickel, chromium, and cobalt can enter to the body through of inhalation, oral and skin contact. inhalation exposure is exposure to the metal through the respiratory tract. inhalation exposure become the main route of exposure entry of metal into the human body.15,25 digestion and inhalation exposure can accumulate in the oral cavity. the trachea and bronchi are covered by the ciliated epithelium and coated by a thin layer of mucus secreted from goblet cells. cilia and mucus in the bronchi and trachea epithelial layer can be pushing up the particles that accumulated toward to the mouth surface. particles that containing mucus then discharged from the respiratory tract with spit or swallow. some particles fagosited by macrophages, but some are absorbed through the epithelial tissue and then diffuses and circulating into the blood vessel.26,27 molecular components in the blood vessels can entry into saliva through transcellular pathway (passive diffusion and active transport) and paracellular pathways (ultrafiltration). the composition of saliva and then is secreted into transport molecules from blood to saliva.11 the other hand, nickel may activate hypoxia-signaling pathways by mediating transcription factor hypoxia induced factor-1 (hif-1). nickel plays a role in inactivating the enzyme prolyl hydroxylase, resulting in hipoksia.28 hif-1 is downregulated by tumor suppressor protein p53 homeodomain-interacting protein kinase-2 (hipk2).29 genotoxic metal exposure can increase the number ros endogenous. the metal ions of chromium, cobalt, and nickel can produce hydroxyl radicals (oh) through the fenton and haberweis reaction. fenton reaction is the reaction of the transition metal ion with h2o2 to generate oh radicals and metal ions are oxidized. chromium, cobalt and nickel are type reagent fenton. haberweiss reaction is a reaction consisting of oxidation of the metal ions are reduced by o2 and then react with h2o2 to produce ohradicals. 17 the hydroxyl radical fenton reaction and results from haber-weiss can cause dna damage.6 typical damage caused by oxidation of dna are guanine transversion into thymine in p53 (p53 gene).7 cells that have mutations or loss of the p53 gene, the expression of p53 (p53 protein) does not occur or occurs p53 protein expression but can not function as activating gene transcription in some target.10 it can be concluded that the use ppe properly was positively correlated with the wild-type p53 protein levels in dental technicians, the use of ppe appropriately during work, prevented metal exposure, and decreased levels of p53. the contribution of ppe on the level of p53 wild on dental technicians was really significant. table 1. mean and standard deviation score the use of ppe and levels of wild-type p53 score ppe p53 n 40 40 mean ± sd 86.19 ± 27.41 0.27 ± 0.28 table 2. relationship scores the use of ppe and levels of wild-type p53 protein among dental technicians in surabaya independent variable dependent variable p r score ppe level p53 0,002 0,466 * p<0.05 = significant correlation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p19-22 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p19-22 22 rohmaniar, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 19–22 acknowledgement gratitude to higher education which has provided assistance through the supervisor to facilitate this research and to all dental technicians in surabaya to participate. references 1. anusavice kj, shen c, rawls hr. phillips’ science of dental materials. 12th ed. st. louis: saunders; 2013. p. 8. 2. al-hourani z. chromium and cobalt levels among dental technicians in the northern jordan. european scientific journal 2013; 9(21): 130. 3. hariyani n, berniyanti t, setyowati d. effects of occupational environmental controls on the level of co, ni and cr among dental technicians. international journal of environmental science and development 2015; 6(9): 1-4. 4. ergün d, ergün r, ozdemir c, oziş tn, yilmaz h, akkurt i. pneumoconiosis and respiratory problems in dental laboratory technicians: analysis of 893 dental technicians. int j occup med environ health 2014; 27(5): 785-96. 5. petroviü d, kruniü n, kostiü m. risk factors and preventive measures for occupational diseases in dental technicians .vojnosanit pregl 2013; 70(10): 959–63. 6. valko m, morris h, cronin mt. metals, toxicity and oxidative stress. curr med chem 2005; 12(10): p. 1161-208. 7. lu h, shi x, costa m, huang c. carcinogenic effect of nickel compounds. mol cell biochem 2005; 279(1-2): 45-67 8. hassan nm, tada m, hamada j. presence of dominant negative mutation of tp53 is a risk of early recurrence of oral cancer. cancer lett 2008; 270(1): 108-19. 9. munir d, lutan r, hasibuan m, henny f. ekspresi protein p53 mutan pada karsinoma nasofaring. majalah kedokteran nusantara 2007; 40(3): 168. 10. kumar v, robbins, neoplasia ls. in: robbins & cotran pathologic basis of disease. 8th ed. philadelphia: saunders elsevier; 2010. p. 269-342. 11. istindiah hn, auerkari ei. penggunaan saliva untuk mendeteksi kanker. jurnal kedokteran gigi universitas indonesia 2003; vol 10( edisi khusus): 279 -81. 12. hozzeini fa, dizgah im, zarandi ns. unstimulated salivary p53 in patients with oral lichen planus and squamous cell carcinoma. acta medica iranica 2015; 53(7): 440-43. 13. lee jj, kuo my, cheng sj, chiang cp, jeng jh, chang hh, kuo ys, lan wh, kok sh. higher expressions of p53 and proliferating cell nuclear antigen (pcna) in atrophic oral lichen planus and patients with areca quid chewing. oral surg oral med oral pathol oral radiol endod. 2005;99(4): p. 471-8. engelen l, de wijk ra, prinz jf, van der bilt a, bosman f. the relation between saliva flow after different stimulations and the perception of flavor and texture attributes in custard desserts. physiol behav 2003; 78(1): 165-9. 14. costa m. molecular mechanism of nickel carcinogenesis. biol chem 2002; 383(6): p 69-75 15. risdayanti a. perilaku pemakaian alat pelindung diri pada teknisi gigi terhadap paparan bahan kimia di laboratorium gigi surabaya. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2010. 16. stephen l, harris g, xianlin. metalinduced oxidative stress and signal transduction. free radical biology and medicine 2004; 37(12): 1921-42. 17. sudjarwo .8-hidroksi-deoksiguanosin sebagai salah satu indikator infertilitas pria. berk. penel. hayati; 2004: vol 10. p. 43–47 18. streckfus c, bigler l, tucci, thigpen jt. a preliminary study of cal5-3, c-erbb-2, epidermal growth factor receptor, cathepsin-d, and p53 in saliva among women with breast carcinoma. cancer investigation 2000; 18(2): 101-9. 19. kundie, fam. mohamed, sh. issaid, ma. omran, a. evaluation of dental technicians awareness of health and safety rule in dental laboratories at some cities in libya. international journal of engineering; 2010: 7(2). p. 126. 20. phalen, rn. le, t. wong, wk. changes in chemical permeation of disposable latex, nitrile and vinyl gloves exposed to simulated movement. j occup environ hyg 2014; 11(11): p. 716–721 21. sunarto. keselamatan dan kesehatan kerja laboratorium kimia. yogyakarta: fmipa uny; 2008. p. 3. 22. tran, tad. arnold, m. schacher, l. adolphe, dc. reys, g. development of personal protection equipment for medical staff: case of dental surgeon. autex research journal 2015; 15(4): pp. 280-287. 23. yurdasal, b. bozkurt, n. bozkurt, ai. yilmaz, o. the evaluation of the dust-related occupational respiratory disorders of dental laboratory technicians working in denizli province. annals of thoracic medicine; 2015: 10(4). pp. 249-255. 24. susanto da. pnumokoniosis. j. indon med assoc. 2011; 61(12): 503-50. 25. wirasuta img, niruri r. buku ajar toksikologi umum. denpasar: farmakologi universitas udayana; 2010. p. 12. 26. djojodibroto d. respirologi. jakarta: penerbit buku kedokteran egc; 2007. p. 11-12. 27. todd davidson, qunwei zhang, lung chi chen, weichen su, and max costa. the involvement of hypoxia-inducible transcription factor-1-dependent in nickel carcinogenesis. cancer research 2003; vol 63: p 3524–3530. 28. obacz j, pastorekova s, vojtesek, and hrstka r. cross-talk between hif and p53 as mediators of molecular responses to physiological and genotoxic stresses. mol cancer 2013; 12(93): p 2-7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p19-22 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p19-22 vol 51 no 4 okt-des 2018.indd 169 dental journal (majalah kedokteran gigi) 2018 december; 51(4): 169–172 the difference between residual monomer dentin bonding hema and udma with acetone and ethanol solvent after binding to type i collagen n. normayanti, adioro soetojo, and nirawati pribadi department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: in caries and non-caries lesions involving dentine, it is necessary to provide dentine-bonding material to help improve retention between the composite resin and the tooth surface. composite resin attachment to dentine is influenced by bonding polymerization reactions. in several studies, researchers found that polymerized monomers will experience volume shrinkage because not all will fully polymerize but, rather, become residual monomers that can cause post-operative pain. purpose: this study aimed to identify the difference in the amount of residual monomers between hemaand udma-based dentin bonding materials with acetone and ethanol solvents after binding to type i collagen. methods: four groups featured in this study: hema with acetone solvent and type i collagen , hema with ethanol solvent and type i collagen , udma with acetone solvent and type i collagen and udma with ethanol solvent and type i collagen . all groups were checked by high performance liquid chromatography (hplc) to quantify the remaining amount of monomers. results: the percentage of residual monomers of dentine bonding hema with acetone solvent and type i collagen was 10.69%, hema with ethanol solvent and type i collagen was 13.93%, udma with acetone solvent and type i collagen was 2.89% and udma with ethanol solvent and type i collagen was 7.48%. conclusion: hema with ethanol solvent has the highest number of residual monomers, while udma with acetone solvent has the lowest. keywords: acetone; ethanol; hema; residual monomer; udma correspondence: adioro soetojo, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132 indonesia. e-mail: adioro-s@fkg.unair.ac.id introduction dentin is a perpetually wet hard tissue because it contains dentinal tubular fluid that renders composite resin with hydrophobic properties incapable of attaching to dentine. therefore, a bonding material is required to glue dentin to composite resin. in widespread cervical lesions extending as far as the dentine and/or near the cementum dentin bonding is necessary to help increase retention between the composite resin and the tooth surface.1 the adhesiveness of dentin bonding to dentine collagen fibrils also constitutes an important interaction. dentin bonding can penetrate the nano interfibrillar cavities before polymerizing to mechanically form anchorage. polymerized monomers will experience volume reduction because not all monomers undergo complete polymerization becoming residual monomers, namely ones which do not react after polymerization is in process.2 common bonding is generally based on 2-hydroxyethylmethacrylate (hema), but recently many non-hema based bonding materials have been developed. generally, hema substitutes present in non-hema based bonding materials are monomer dimethacrylates such as urethane dimethacrylate (udma).3 in order to promote deeper monomers penetration of the dentine, the bonding material contains solvents which play a role in transporting the monomers to the tooth, promoting dissolution of the thick monomers and facilitating penetration of the demineralized dentin. acetone is known to have a very high vapor pressure, while that of ethanol is lower.4 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p169–172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p169-172 170normayanti, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4):169–172 determination of the number of residual monomers eluted from the dental material is usually carried out with high-performance liquid chromatography (hplc) which constitutes an extremely strong separation method.5 in several studies, hplc has been used to measure residual monomers in resin materials because they are capable of detecting soluble and non-volatile reactive compounds such as bisphenol-a glycidyl methacrylate (bis-gma), urethane dimethacrylate (udma) and triethylene glycol dimethacrylate (tegdma).6 the purpose of this study was to prove the existence of monomer differences in residual hema and udma dentin bonding with acetone and ethanol solvent after binding to type i collagen. materials and methods the research sample consisted of acetone-coated hema dentin bonding material (solobond m, voco, germany), ethanol-based hema (tetric n-bond, ivoclar vivadent, liechtenstein), udma acetone (spectrum bond, dentsply, germany), ethanol-coated udma (tetric n-bond, ivoclar vivadent, liechtenstein) and collagen type i (sigma chemical, st. louis, usa). for the purposes of this study, the subjects were divided into four groups, group 1: hema dentin bonding with acetone + type i collagen, group 2: hema dentin bonding with ethanol + type i collagen, group 3: udma dentin bonding with acetone + type i collagen and group 4: udma dentin bonding with ethanol + type i collagen . in order to conduct the research, 100 μl standard solution was produced from pure hema (sigma chemical, st. louis, usa) and udma (sigma chemical, st. louis, usa) and added to 900 μl methanol (methyl alcohol, mallinckrodt chemical, usa) because pure hema and udma can only dissolve in methanol. this standard solution is only a standard reference for monomers that will be detected on inspection by high performance liquid chromatography (hplc) devices. 1 ml of dentin bonding material was added to 100 mg of type i collagen before each sample was irradiated for 20 seconds by means of a light curing unit (dba, guilin woodpecker, china) at a wavelength of 550nm and then immersed in 10 ml ethanol. standard solutions and samples were taken using a 1 ml syringe and then injected into a filter holder previously filled with nylon membrane and accommodated in a closed vial. in the final step, they were analyzed using hplc (agilent 1100 series, agilent, germany). the time at which a specific sample is eluted is referred to as the retention time. the samples measured by hplc for the analysis of residual monomers require a standard reference from the monomers to be detected and compared with existing standards to determine the monomers to be measured. the hplc results in the form of a chromatogram provide information about the retention time and sample area (figure 1). reading the chromatogram involves looking at the treatment area results. the results of the sample area are compared to those of the standard area before being calculated using a formula in order that quantitative results are obtained. c b d ab = area height cd = width of area figure 1. illustration of hplc calculation result the results of the remaining monomers were calculated by percentage. to determine the percentage of residual monomers from the research sample after treatment the following formula can be used:7 results based on the results of the study, it can be seen that the udma with acetone solvent + type i collagen group produces the lowest number of residual monomers, while the hema with ethanol solvent + type i collagen group produces the highest (table 1). to identify the distribution of data in the study groups, a kolmogrov-smirnov test was conducted. of the four groups tested statistically, all showed a value of p> 0.05 signifying normal data distribution. furthermore, in the four study groups, homogeneity tests conducted by means of a levene test produced a result of p = 0.003 (p> 0.05) indicating that the samples were not homogeneous. these were then subjected to a kruskal-wallis test as a means of identifying differences between groups, the p = 0.000 (p <0.05) result obtained confirmed differences between the four study groups. in order to establish the differences in each experimental group a tukey hsd analysis test was conducted. the test results had a value of p <0.05 across all experimental groups confirming significant differences between them. this showed that the amount of residual dentin bonding monomers of udma acetone + type i collagen, udma ethanol + type i collagen, hema acetone + type i collagen, and hema ethanol + type i collagen had significant differences (table 2). % residual monomer = sample area standard area (ab x cd) x 100% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p169–172 a http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p169-172 171 normayanti, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4):169–172 discussion dentin is a hard tissue containing approximately 60% inorganic ingredients, 30% organic matter and 10% water. organic matter consists of 90% collagen, while the remaining 10% is non-collagen material.8 dentin bonding is an agent used as a material to combine composite restorations with dental tissues. it is usually employed in combination with composite resins to reduce the occurrence of microleakage between the material lodged and the tooth surface, while also increasing retention of the filling material.2 several studies of dentin bonding state that specific functional monomers can interact chemically with dental tissues.9 the polymerization of resin-dentin bonding is obtained using visible light from a light curing unit. this resin can polymerize with visible light because it contains a photo-initiator, camphorquinone.2 various dentin bonding, for example hema and udma10, containing a range of basic materials has been available on the market. solvents are often added to bonding adhesive materials, serving to dilute thick monomers and help monomers penetrate demineralized dentine. the most commonly used solvents are acetone and ethanol because they have the optimum physical and chemical properties compared to other solvents.4 this research is based on the existence of a number of clinical phenomena potentially causing postoperative pain after filling with composite resin. several previous studies have posited that the free monomers in dentin bonding material are believed to be one source of pain. it is important to realize that complete polymerization rarely occurs and that residual monomers emerge, consequently causing postoperative pain.11 the results showed that ethanol-coated hema dentin bonding material contained the largest amount of residual monomer, while acetone-coated udma dentin bonding material contained the smallest compared to acetone-coated udma and ethanol-coated hema dentin bonding material in this study group. an important factor influencing the release of residual monomers is the size of monomers in resin materials since more smaller molecules than larger ones are released.5 hema possesses small, heavy molecules12, although udma has a greater molecular weight.13 solvents play an important role in the process of penetrating dentin bonding to collagen.4 monomers that use solvents have a higher shear strength than those that do not. this is due to the acetone and ethanol solvents having strong evaporation power which helps to both evaporate the moisture content of the dentine surface and penetrate the monomers into collagen fibrils.14 acetone, also known as a water-chaser2, is an effective solvent which helps remove water from dentine. because the evaporation pressure of acetone is high the remaining water on the dentine surface is reduced, causing the monomer material to be easier to penetrate into collagen. the more monomers that bind to collagen, the stronger the resulting chemical bonds so that the adhesive strength is also greater.15 unlike acetone, the water-chasing ability of ethanol is weak.2 because ethanol evaporation is not as great as that of acetone, considerable amounts of water remains on the surface of the dentine and it becomes difficult for the monomer material to penetrate the collagen fibrils. the fewer the monomers that bind to collagen, the weaker the chemical bonds that occur with the result that the adhesive strength is also lower.15 it can be concluded that ethanolbased hema dentin bonding produces the largest number of residual monomers, while acetone-coated udma produces the smallest. table 1. mean and standard deviation of monomers of residual hema and udma dentin bonding with acetone and ethanol solvent (%). sdmean (%)nsampel 0.2026010.69147hema acetone + type i collagen 0.2449813.93147hema ethanol + type i collagen 0.826252.88717udma acetone + type i collagen 0.171747.48437udma ethanol + type i collagen table 2. multiple comparisons of tukey hsd data on hema and udma dentin bonding with acetone and ethanol solvent. group udma acetone + type i collagen udma ethanol + type i collagen hema acetone + type i collagen hema ethanol + type i collagen 0.0000.000*_udma acetone + type i collagen * 0.000* 0.000_udma ethanol + type i collagen * 0.000* 0.000_hema acetone + type i collagen * hema ethanol + type i collagen _ * p ≤ 0.05 = there are significant differences dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p169–172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p169-172 172normayanti, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4):169–172 references 1. anusavice kj, phillips rw, shen c, rawls hr. phillips’ science of dental materials. 12th ed. st louis missouri: saunders; 2012. p. 176-258. 2. soetojo a. penggunaan resin komposit dalam bidang konservasi gigi. surabaya: revka petra media; 2013. p. 23-112. 3. sogata op, lunardhi cgj, subiwahjudi a. perbedaan kebocoran mikro pada restorasi tumpatan dengan bonding berbasis hema dan non-hema. conserv dent j. 2016; 6: 45–50. 4. ekambaram m, yiu cky, matinlinna jp. an overview of solvents in resin-dentin bonding. int j adhes adhes. 2015; 57: 22–33. 5. botsali ms, kuşgöz a, altintaş sh, ülker he, tanriver m, kiliç s, başak f, ülker m. residual hema and tegdma release and cytotoxicity evaluation of resin-modified glass ionomer cement and compomers cured with different light sources. sci world j. 2014; 2014: 1–7. 6. altunsoy m, botsali ms, tosun g, yasar a. effect of increased exposure times on amount of residual monomer released from singlestep self-etch adhesives. j appl biomater funct mater. 2015; 13(3): e287–92. 7. charasseangpaisarn t, wiwatwarrapan c, leklerssiriwong n. ultrasonic cleaning reduces the residual monomer in acrylic resins. j dent sci. 2016; 11(4): 443–8. 8. hargreaves km, berman lh, rotstein i. cohen’s pathways of the pulp. 11th ed. st. louis: mosby elsevier; 2015. p. 411-54. 9. ubaldini alm, baesso ml, sehn e, sato f, benetti ar, pascotto rc. fourier transform infrared photoacoustic spectroscopy study of physicochemical interaction between human dentin and etch-&-rinse adhesives in a simulated moist bond technique. j biomed opt. 2012; 17(6): 1–5. 10. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a, coutinho e, suzuki k, lambrechts p, van meerbeek b. systematic review of the chemical composition of contemporary dental adhesives. biomaterials. 2007; 28(26): 3757–85. 11. bakir ş, bakir ep, yildirim zs. biocompatibility of dental adhesives. adv dent oral heal. 2017; 4(4): 1–6. 12. moreira f do cl, antoniosi filho nr, de souza jb, lopes lg. sorption, solubility and residual monomers of a dental adhesive cured by different light-curing units. braz dent j. 2010; 21(5): 432–8. 13. papakonstantinou ae, eliades t, cellesi f, watts dc, silikas n. evaluation of udma’s potential as a substitute for bis-gma in orthodontic adhesives. dent mater. 2013; 29(8): 898–905. 14. koliniotou-koumpia e, kouros p, koumpia e, helvatzoglouantoniades m. shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems. braz j oral sci. 2014; 13: 64–9. 15. silva e souza mh, carneiro kgk, lobato mf, silva e souza pdar, de góes mf. adhesive systems: important aspects related to their composition and clinical use. j appl oral sci. 2010; 18(3): 207–14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p169–172 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p169-172 169 vol. 44. no. 4 december 2011 research report betel leaf toothpastes inhibit dental plaque formation on fixed orthodontic patients rizka amelia mayasari1, sianiwati goenharto2, and ahmad sjafei2 1 dental student 2 department of orthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: brackets, archwires, ligatures, and other fixed orthodontic appliance components complicate the use of conventional oral-hygiene measures. this often results in significant plaque accumulation around the bracket bases. the addition of betel leaf extract in toothpaste is expected to inhibit the growth of dental plaque. purpose: the purpose of this study was to evaluate the effect of betel leaf toothpaste in inhibiting plaque formation on the fixed orthodontic patients. methods: this study was done on dental student of airlangga university aged 18–24 years, have been wearing fixed orthodontic appliances for 1–2 years, have no systemic diseases. the samples were divided into two groups, consisting of 20 samples. first group of samples brushed their teeth with betel group of samples brushed their teeth with betel brushed their teeth with betel leaf toothpaste and the second using placebo. the subjects were instructed to brush their teeth using scrub method until reaching zero (0) scor of orthodontic plaque index (opi). plaque scores were taken again 4 hours after brushing. the statistical analysis was done by using paired t test. results: the average of accumulated plaque on group that use betel leaf toothpaste was 25.54 and placebo was 41.09. the result showed that there was significant difference in plaque accumulation between the group with betel leaf toothpaste and placebo 4 hours after brushing (p = 0.001). conclusion: in conclusion, betel leaf toothpaste is effective in inhibiting the dental plaque formation on the fixed orthodontic patients. key words: betel leaf toothpaste, dental plaque, fixed orthodontic appliancesappliances abstrak latar belakang: bracket, kawat busur, kawat ligatur dan komponen peranti ortodonti cekat yang lain mempersulit pembersihan gigi secara konvensional. hal ini sering menyebabkan terjadinya akumulasi plak di sekitar dasar braket. penambahan ekstrak daun sirih yang mempunyai efek bakterisid pada pasta gigi diharapkan dapat menghambat pertumbuhan plak. tujuan: tujuan penelitian ini adalah untuk mengevaluasi efek pasta gigi mengandung ekstrak daun sirih dalam menghambat pembentukan plak pada pemakai peranti ortodonti cekat. metode: penelitian dilakukan pada mahasiswa fakultas kedokteran gigi universitas airlangga berusia 18–24 tahun, sudah memakai peranti cekat ortodonti selama 1–2 tahun, tidak mempunyai penyakit sistemik. sampel dibagi dalamsampel dibagi dalam dua kelompok, masing-masing 20 sampel. sampel kelompok pertama menyikat gigi dengan pasta gigi yang mengandung daun sirih dan kelompok kedua dengan pasta gigi placebo memakai metode scrub. pembersihan gigi dilakukan sampai didapatkan skor nol (0) pembersihan gigi dilakukan sampai didapatkan skor nol (0)sampai didapatkan skor nol (0) orthodontic plaque index. empat jam setelah itu skor plak diukur kembali. �ata dianalisis dengan uji t berpasangan.. �ata dianalisis dengan uji t berpasangan. hasil: rerata pengumpulan plak pada pemakai pasta gigi mengandung daun sirih adalah 25,54 dan placebo adalah 41,09. analisis data menunjukkan adanya perbedaan bermakna antara kedua kelompok (p = 0,001). kesimpulan: �apat disimpulkan bahwa pasta gigi mengandung daun sirih efektif dalam menghambat pertumbuhan plak pada pemakai peranti ortodonti cekat. kata kunci: pasta gigi daun sirih, plak gigi, peranti ortodonti cekat correspondence: sianiwati goenharto, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: sianiwati.goenharto@yahoo.co.id 170 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 169–172 introduction orthodontic fixed appliances is appliances that are attached to the patient’s teeth so it can not be removed by the patient. orthodontic treatment with fixed appliances alters the oral environment, increases plaque amount, changes the composition of the flora, and complicates the cleaning for the patients.1 fixed orthodontic patients usually have difficulty in cleaning their teeth because there are certain parts of teeth that are difficult to clean. whereas in patients using fixed orthodontic appliances, it is very important to maintain and improve oral hygiene, considering the device is attached in such a way that will facilitate the formation of bacterial accumulation in the area.2 brackets, archwires, ligatures, and other orthodontic appliances complicate the use of conventional oralhygiene measures. this often results in significant plaque accumulation arround the bracket bases.3 the presence of a fixed orthodontic appliance greatly inhibits oral hygiene and creates new retentive area for plaque and debris, which in turn predisposes to increased carriage of microbes and subsequent infection.4 brushing teeth helps controling plaque and is the first step to control caries and periodontal disease. currently plaque control is equipped with additional types of active component that contain the basic ingredients of natural or synthetic material as antibacterial. antibacterial agents are available in the form of mouth rinses and toothpaste.5 toothpaste contains antimicrobial agents such as triclosan and chlorhexidine as an active component that may provide direct inhibitory effect of plaque formation. due to the progress of science and technology, many innovations to add other substances that are beneficial to dental health. one of the substances added to the toothpaste is betel leaf extract. betel leaf has been known by the people of indonesia for a long time, as an ingredient that will strengthen teeth, stop gingival bleeding, and as a mouthwash remedy. betel leaf could stop the bleeding (styptic), skinwound healing (vulnerary), gastrointestinal drugs (stomachic), and clear the throat. betel leaf also has antiseptic, antioxidant, and antifungal effect. essential oil and its extract are able to fight some gram-positive and negative bacteria.6 betel leaf toothpaste contains essential oils with the active substances are phenol and cavicol. toothpaste containing essential oil of betel leaf is considered relatively new. but actually the efficacy of betel leaf as antibacterial agent has long been known and proven. the addition of betel leaf extract in toothpaste is expected to inhibit the growth of dental plaque. this is related to the ability of betel leaf as an antibacterial agent.7 this study was undertaken to evaluate the effectivity of betel leaf toothpaste in inhibiting plaque formation on the fixed orthodontic patients. the benefits of this study to offer an alternative method for fixed orthodontic patients in maintaining their oral hygiene. materials and methods this study was done on dental student of airlangga university aged 18-24 years, wearing upper and lower fixed applliances, used elastomeric modules, without caries, doesd elastomeric modules, without caries, does not have systemic diseases, acute or chronic diseases in the oral cavity. the sample was determined through purposive random sampling of 20 students. on the first visit, subjects were asked to brush their teeth using betel leaf toothpaste with scrub technique to obtain score zero (0). score was measured after applying disclosing solution with microbrush on labial or buccal surface of the teeth. for four hours after brushing, the subject was not allowed to eat anything and gargling. after four hours of fasting, plaque score was measured again. figure 1. tooth surface was divided into three areas. i: cervical, ii: central, iii: occlusal measurement of plaque used the orthodontic plaque index (opi).8 tooth surface that being examined is all cervical ∑ … 2× central ∑ … 3× occlusal ∑ … 1× 7 6 5 4 3 2 1 1 2 3 4 5 6 7 teeth occlusal ∑ … 1× central ∑ … 3× cervical ∑ … 2× figure 2. plaque record. 171mayasari, et al.,: betel leaf toothpastes inhibit dental plaque formation part of the labial and buccal teeth with brackets exceptwith brackets except for posterior teeth which had bands. tooth surface being examined was divided into three areas by dividing the tooth surface horizontally (figure 1). the plaque of every area was recorded (figure 2). area iii (occlusal) was multiplied with 1, area i (cervical) was multiplied with 2 and area ii (central) was multiplied with 3, and then the total score were found. the opi were determined as follows: total score = opi total tooth being examined × 6 plaque score was opi multiplied 100. the criteria were: good: 0-25, average: 26-50, poor: >50 on the second visit, three days after the first visit, subjects were asked to brush their teeth with placebo tooth paste until got the score zero (0). like the first visit, after four hours of fasting, plaque score was measured again. data was collected and statistical analysis was done bystatistical analysis was done by using paired t test. resultss the result of this study could indicate that most of samples were in average criterion (60% of samples brushed with betel leaf toothpastes and 85% of samples brushed with placebo). good criteria were found on 40% samples brushed with betel leaf toothpastes and only 5% samples brushed with placebo (table 1). table 1. criteria of plaque with orthodontic plaque index (opi) criteriariteria betel leaf toothpaste placebo good 8 (40%) 1 (5%) average 12 (60%) 17 (85%) poor 2 (10%) total 20 (100%) 20 (100%) it was shown that the mean of plaque scores in group brushed with placebo was higher (41.09) than the group brushed with betel leaf toothpastes (25.54) (table 2). before the paired t test was done to determine the significance of difference, this data needed to conduct normality test using kolmogorov-smirnov test. the result for group brushed with placebo was 0.978 (p > 0.05) and the group brushed with betel leaf toothpastes was p = 0.980 (p > 0.05). it means that both of the groups had normal distribution. table 2. average plaque score in each group group total mean betel leaf toothpaste 20 25.54 placebo 20 41.09 from the result of paired t test, it is known that the p value is 0.001 < 0.05 so the data was significant differrent. based on this analysis it can be seen that there was significant different between the plaque score of subject who brush their teeth using betel leaf toothpaste and placebo. the plaque score in subject using placebo was higher than subject who brushed with betel leaf toothpaste. discussion this study was done on 20 subjects with fixed orthodontic appliances who were asked to brush their teeth until got the score zero (0) to make homogenized samples before treatment. dental plaque can be formed within 4 hour after tooth cleaning,9 so in this study, samples was asked to wait for 4 hours without eating, rinsing or drinking before the second measurement of plaque scores were done. it was shown that the mean of plaque scores in group brushed with placebo was higher (41.09) than the group brushed with betel leaf toothpastes (25.54). from the paired t test analysis, it is known that the p value = 0.001 (p < 0.05), so the results showed that the plaque formation on fixed orthodontic patients brushed with placebo was significantly higher than the subject brushed with betell leaf toothpaste. the result are consistent with previous study that says that the betel leaf extract may inhibit the formation of colonies of streptococcus mutans in plaque.10 betel leaf extract also reduced the adherence of bacteria to the biofilm contained a layer on the surface of the tooth. this reduction is possible due to alteration of protein on the cell surface of bacteria by extract of betel leaf. based on laboratory test results, it was found that betel leaf extract in the toothpaste had chemical components as much as 31.80% flavonoids, alkaloids as much as 17.05%, polyphenate as much as 21.41%, anthocyanin as much as 8.62%, and oil essential as much as 2.86%. one of the largest classes of naturally-occuring polyphenolic compounds are flavonoids. a number of flavonoid, shows antibacterial and antiviral activity.11 they are known to be synthesized bythey are known to be synthesized by plants in response to microbial infection and found in vitro to be effective antimicrobial substances against a wide array of microorganisms. their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. more lipophilic flavonoids may also disrupt microbial membranes.12,132,13 alkaloids are heterocyclic nitrogen compounds.133 purified alkaloids as well as their synthetic derivatives are used as medicinal agents for their various biological 172 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 169–172 effects such as analgesic, antispasmodic and bactericidal.12 anthocyanins and polyphenate is one kind of phenol which is both antibacterial and antioxidant.13 several mechanisms of action in the growth inhibition of bacteria are involved, such as destabilization of cytoplasmic membrane, permeabilization of plasma membrane, inhibition of extracellular microbial enzymes, direct actions on microbial metabolism and deprivation of the substrates required for microbial growth.15 essential oil of betel leaf is an effective antibacterial agent.16 essential oil of betel leaf with phenol components include: carvacol, cineol, cariofilen, eugenol, and chavicol. components of these phenols have a very strong antiseptic power. bacterisid properties of carvacol efficacy has five times more powerful than other phenol component. carvacol and cineol has the same efficacy as eugenol, which is antiseptic and topical analgesic. cariofilen is antiseptic and local anesthetic.17 chavicol is a major phenolic compound present in the aqueous extract of the piper betle leaf. the compound is better known for its antioxidant and anticancer properties.18 chavicol can behave as a desinfectant and antifungal. this is aninfluential component in inhbiting the growth of dental plaque.19 from the statement above, betel leaf has many ingredients with antibacterial, antiseptic, disinfectant, and anti fungal effect, which makes the plaque formation on fixed orthodontic patients brushed with betel leaf toothpaste was more difficult than with placebo. placebo also can reduce plaque accumulation in orthodontic fixed patients. this can be caused by mechanical process that occurs, ie brushing using a toothbrush. another possibillity would be caused by detergent (sodium lauryl sulfate) and abrasive (calcium carbonate) contained in placebo. detergent makes foam and also has antibacterial effect. however, the use of placebo in reducing plaque on fixed orthodontic patients are not as effective as the betel leaf toothpaste. in conclusion, betel leaf toothpastes can inhibit dental plaque formation on fixed orthodontic patients. mecanical prevention of dental plaque formation on fixed orthodontic patients by tooth brushing betel leaf toothpaste is effective. references 1. ay zy, sayin mo, ozat y, goster t, atilla ao, bozkurt fy. appropiate oral hygiene motivation method for patient with fixed appliances. angle orthod. 2007; 77(6): 1085–9. 2. yohana w. pentingnya kesehatan mulut pada pemakai alat ortodonti cekat. available at: http://pustaka.unpad.ac.id. accessed on march 22, 2010. 3. derks a, kuijpers-jagtman am, frencken je, hof mav, katsaros c. caries preventive measures used in orthodontic practices: an evidence-base decision?. am j orthod dentofac orthop 2007; 132(2): 165–70. 4. hagg u, kaveewatcharanont p, samaranayake yh, samaranayake lp. the effect of fixed orthodontic appliances on the oral carriage of candida species and enterobactericeae. eur j orthod 2004; 26: 623–9. 5. pratiwi r. perbedaan daya hambat terhadapdaya hambat terhadapaya hambat terhadaphambat terhadapambat terhadap streptococcus mutans dari beberapa pasta gigi yang mengandung herbal. maj ked gigibeberapa pasta gigi yang mengandung herbal. maj ked gigieberapa pasta gigi yang mengandung herbal. maj ked gigipasta gigi yang mengandung herbal. maj ked gigiasta gigi yang mengandung herbal. maj ked gigigigi yang mengandung herbal. maj ked gigiigi yang mengandung herbal. maj ked gigimengandung herbal. maj ked gigiengandung herbal. maj ked gigiherbal. maj ked gigierbal. maj ked gigi. maj ked gigi (dent j) 2005; 38(2): 64–7. 6. moeljanto, rini d. khasiat dan manfaat daun sirih: obat mujarab dari masa ke masa. jakarta: pt argomedia pustaka; 2003. p. 7–12. 7. sasmita is, pertiwi asp, hali m. gambaran efek pasta gigi yang mengandung herbal terhadap penurunan indeks plak. available at: http://pustaka.unpad.ac.id. accessed on march 22, 2010. 8. heintze sd. oral health for orthodontic patient. london: quintessence pub; 1999. p. 65–87. 9. ruhadi i. efektivitas pasta gigi yang mengandung bahan bubuk kayu siwak dalam menghambat pembentukan plak gigi. maj ked gigikan plak gigi. maj ked gigian plak gigi. maj ked gigi (dent j) 2004; 37(1): 24–7. 10. nalina t, rahim zah. effect of piper betle l. leaf extract on the virulence activity of streptococcus mutans-an in vitro study. pak j biol sci 2006; 9(8): 1473. 11. bylka w, matlawska i, pilewski na. natural flavonoids as antimicrobial agents. jana 2004; 72(2): 24–31. 12. khullar n. antimicrobials from plants and their use in therapeutics and drug discovery. iioab 2010; 1(3): 31–7. 13. kaur gj, daljit sa. antibacterial and phytochemical screening of gj, daljit sa. antibacterial and phytochemical screening of anethum graveolens, foeniculum vulgare and trachyspermum ammi. bmc complementary and alternative medicine 2009; 9: 30. 14. xia eq, deng gf, guo yj, li hb. biological activities of polyphenols from grapes. int j mol sci 2010; 11: 622–46. 15. burdulis d, sarkinas a, jasutiene i, stackeviciene e, nikolajevas l, janulis v. comparative study of anthocyanin composition, antimicrobial and antioxidant activity in bilberry (vaccinium corymbosum l.) fruit. acta poloniac pharmaceutica–drug study. 2009; 66(4): 400. 16. caburian ab, osi mo. characterization and evaluation of antimicrobial activity of the essential oil from the leaves of piper betle l. international scientific study journal 2010; 2(1): 2–13. 17. agustin d. perbedaan khasiat antibakteri bahan irigasi antara hidrogenkhasiat antibakteri bahan irigasi antara hidrogenhasiat antibakteri bahan irigasi antara hidrogenantibakteri bahan irigasi antara hidrogenntibakteri bahan irigasi antara hidrogenbahan irigasi antara hidrogenahan irigasi antara hidrogenirigasi antara hidrogenrigasi antara hidrogenhidrogenidrogen peroksida 3% dan infusum daun sirih 20% terhadap bakteri mix. majeroksida 3% dan infusum daun sirih 20% terhadap bakteri mix. majinfusum daun sirih 20% terhadap bakteri mix. majnfusum daun sirih 20% terhadap bakteri mix. majdaun sirih 20% terhadap bakteri mix. majaun sirih 20% terhadap bakteri mix. majsirih 20% terhadap bakteri mix. majirih 20% terhadap bakteri mix. majbakteri mix. majakteri mix. majmix. majix. maj. majmaj ked gigi (dent j) 2005; 38(1): 45–7. 18. sharma s, khan ia, ali f, kumar m, kumar a, johri rk, abdullah st, bani s, pandey a, suri ka, gupta bd, satti nk, dutt p, qazi gn. evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent. antimicrobial agents and chemotherapy 2009; 53(1): 216–22. 19. prahasanti c. pengaruh pasta gigi yang mengandung ekstrak daun sirih terhadap pertumbuhan plak gigi. maj ked gigi (dent j) 2000; 33(4): 127–8. dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 114 vol. 42. no. 3 july–september 2009 mechanical properties of carving wax with various ca-bentolite filter composition widjijono, purwanto agustiono, and dyah irnawati department of dental biomaterials faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: the carving wax is used as a medium in dental anatomy study. this wax composes of many waxes and sometimes a filler is added. carving wax is not sold in indonesian market. whereas the gradients of carving wax such as beeswax, paraffin and bentonite are abundant in indonesia. based on that fact, to make high quality and standard,the exact composition if this carving wax should be known. purpose: the aim of this study was to investigate the effect of carving wax composition with ca-bentonite filler on the melting point, hardness, and thermal expansion. methods: five carving wax compositions were made with paraffin, ca-bentonite, carnauba wax, and beeswax in ratio (% weight): 50:20:25:5 (ki), 55:15:25:5 (kii), 60:10:25:5 (kiii), 65:5:25:5 (kiv), 70:0:25:5(kv). all components were melted, then poured into the melting point, hardness, and thermal expansion moulds (n = 5). three carving wax properties were tested: melting point by melting point apparatus; hardness by penetrometer; thermal expansion by digital sliding caliper. the data were analyzed statistically using one-way anova and lsd0.05. result: the ca-bentonite addition influenced the melting point and thermal expansion of carving wax with significant differences between ki and other groups (p < 0.05). ca-bentonite addition influenced the carving wax hardness and the mean differences among the groups were significant (p < 0.05). conclusion: ca-bentonite filler addition on the composition of carving wax influenced the physical and mechanical properties. the carving wax with high ca-bentonite concentration had high melting point and hardness, but low thermal expansion. key words: carving wax, ca-bentonite, melting point, hardness, thermal expansion correspondence: widjijono, c/o: bagian biomaterial kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara yogyakarta 55281, indonesia. e-mail: widji_biomat@yahoo.com research report introduction a thorough knowledge of the surface anatomy of each tooth is essential in restorative dentistry. three approaches to study tooth form are tooth drawing, wax block carving and tooth wax-up. carving wax is used by dental students for dental anatomy study. carving wax is not sold in indonesian market. whereas the gradients of carving wax such as beeswax, paraffin and bentonite are abundant in indonesia. the production of carving wax does not need complicated method and equipments. based on that fact, to produce high quality and standard, the exact composition of this carving wax should be known. wax block carving is to produce a wax tooth by carving it from a rectangular piece of wax.1 carving wax consists of high quality waxes similar to inlay wax.2 generally, the main ingredient of inlay waxes is paraffin.3 inlay wax may contain of 60% paraffin, 25% carnauba, 10% ceresin, and 5% beeswax.4,5 this mixture is carried out to produce a material with the required properties for a specific application.6 paraffin is a mixture of solid hydrocarbon, obtained from petroleum.7 paraffin consists of straight-chained hydrocarbon with 26–30 carbon atoms and has melting range between 40 and 71° c and thermal expansion coefficient of 307 × 10–6/° c between temperature of 20 and 27.8 degree celcius.5 the hardness of paraffin is about 9–16 mm of penetration depth measured by astm d-1321.8 in indonesia, paraffin is produced by pertamina in cepu, cilacap, and balikpapan unit productions. 115widjijono, et al.: melting point, hardness, and thermal expansion carnauba wax is an exudates from the pores of the leaves of brazilian wax palm tree (copernicia prunifera).7 carnauba wax is composed of straight-chain esters, alcohols, acids, and hydrocarbons. this wax has melting range between 84 and 91° c and thermal expansion coefficient of 156 × 10–6/° c between temperatures of 22 and 52 degree celcius.5 the hardness of carnauba wax is relatively high, 2–8 mm penetration depth.8 carnauba wax is used for increasing melting point and hardness of paraffin.5 although brazilian wax palm tree is not grown in indonesia, carnauba wax product is imported by chemical distributors. beeswax is a substance obtained from bee honeycombs and consists of ester complex mixture, saturated and unsaturated hydrocarbons, and organic acid with high molecules weight.7 two kind of beeswaxes are used in dentistry, yellow and bleached beeswax.10 beeswax has melting range between 63 and 70° c and thermal expansion coefficient 344 × 10–6/° c (between temperatures of 22–41.2° c) for yellow beeswax and 271 × 10–6/° c (temperature 22–38.6° c) for bleached beeswax.5 the hardness of beeswax is between 8–23 mm penetration depth.8 beeswax is produced from many honey bee centres in some region of indonesia.11 the accuracy and ultimate utility of the casting resultant depend largely on the accuracy and fine detail of the wax pattern. inlay wax must possess certain, very important, physical properties. the desirable properties of inlay wax are; a) when softened, the wax should be uniform; b) the color should be contrast compared with the die material or prepared tooth; c) the wax should be no flakiness or similar surface roughening when the wax is bent and molded after softening; d) the inlay wax should leave no solid residue in excess of 0.10% of the original weight when vaporized at 500° c; e) the wax should be carved to a very thin layer; and f) the wax should be completely rigid and dimensionally stable at all time until it is eliminated.3 inlay wax should have a low thermal contraction, correct flow properties, and easy to carve without chipping or flaking.12 inlay wax may be softened over a flame or water at 54–60° c.3 manufacturer could control the melting point and softening temperature of dental waxes by blending many wax components from mineral, animals, and plants.6 natural resins may be added to paraffin waxes to improve their tough, film forming characteristics, and melting ranges.5 some inlay wax formulation contain a compatible filler to control expansion and shrinkage of the wax products.3 inorganic filler can act as an effective hardener for natural wax blended for dental applications. the addition of in organic silica filler up to 10% in paraffin and beeswax blend could increase hardness and decrease melting point.13 filler addition in resins and waxes will reduce plasticity of matrix, increase hardness, and reduce thermal expansion.14 bentonite is one of inorganic filler. bentonite usually formed from weathering of volcanic ash, most often in the presence of water.15 bentonite is used as a bleaching material, additive material, filler, and drilling mud.16 bentonite can be used in cement, adhesives, ceramic bodies, and cat litter. bentonite is also used as a binding agent in steel making industry and a therapeutic face pack for the treatment of acne/oily skin.15 for industrial purposes, two main classes of bentonite exist: wyoming type bentonite (na-bentonite) and meta bentonite (sub bentonite, cabentonite). natural ca-bentonite expand less but after activated by acid has good absorbent properties, dispersed in water, and ion-exchange properties (especially by calcium and magnesium ions).16 in general, filler materials are added to certain composition in purpose to increase the hardness of mixture, increase toughness quality, avoid bubbles, avoid flaking, smoother carving, improve accuracy, and free of tackiness to models and instruments.17 the application of ca-bentonite as a filler in carving wax would give chemical reaction between calcium and magnesium ions with hydrogen atoms from hydrocarbon chain of paraffin to form calcium salt bond and consequently increase physical and mechanical properties of carving wax. the aim of this study was to investigate the effect of carving wax composition with ca-bentonite filler toward the melting point, hardness, and linear thermal expansion properties. materials and methods the materials used in this research were paraffin (pertamina, indonesia), yellow beeswax (sea, indonesia), carnauba wax (bratachem, indonesia), inlay wax (gc, japan), ca-bentonite (bratachem, indonesia). five groups of carving wax with ca-bentonite filler were composed (table 1). two thousands grams of each composition of carving wax was prepared. the carving waxes were prepared by melting the ingredients together under continuous stirring table 1. carving wax compositions composition ingredients concentration (% weight) paraffin carnauba ca-bentonitee beeswax k i k ii k iii k iv k v 50 55 60 65 70 25 25 25 25 25 20 15 10 5 0 5 5 5 5 5 116 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 114-117 at the temperature below 100° c on the hot plate. then, the mixtures were poured into the moulds and stored at room temperature for 24 hours prior to testing. the mixtures were poured into cylindrical metal mould for hardness specimens (35 mm height and 55 mm diameter), bar metal mould (22.2 × 305 × 14.3 mm), and plate polycarbonate mould for melting point (50 × 10 × 2 mm). five specimens were made from each composition for each tests. commercial inlay wax product was melted and 5 specimens were prepared for linear thermal expansion testing. the melting point was measured by melting point apparatus (fischer john, uk). the carving wax plate was cut into 5 × 5 × 2 mm, then put on the specimen plate and the temperature when the wax was melted is observed in 0.1 degrees celcius accuracy. hardness was measured based on astm d 1321 standards using penetrometer (setamatic, uk).9 specimens were immersed in waterbath at 25° c for 1.5 hours, then, moved into the waterbath under the penetrator needles. the penetration were measured by moving down the penetrator needle to the wax surface until the needle cannot penetrate the wax anymore. the penetration depth was measured in 0.1 mm accuracy. linear thermal expansion was measured based on ansi/ada specification number 24.18 after the specimens is prepared, it was stored at 37° c for 24 hours before testing. the specimens were immersed in bath water at 25° c for 20 minutes, then the specimens length were measured by digital calipers (0.01 mm accuracy). after that, the specimens were immersed again in bath water at 40° c for 20 minutes and the specimens length were measured again. the percentage of linear thermal expansion were calculated from the subtraction of two length measurements divided by the initial length of specimens at 25° c.19 the data were analyzed statistically by anova and lsd0.05. result five composition of carving waxes with different concentration of ca-bentonite filler were tested for melting point, hardness (penetration depth), and linear thermal expansion. the means and average of melting point, hardness, and linear thermal expansion showed in table 2. the linear thermal expansion of commercial inlay wax products was 0.57 ± 0.06%. the composition of carving wax influence the melting point, hardness, and linear thermal expansion significantly (table 3). the lsd0.05 results of carving wax melting point showed significant differences between ki group and kii, kiii, kiv, kv groups. the lsd0.05 results of carving wax hardness (penetration depth) showed significant differences among all groups. the lsd0.05 results of carving wax linear thermal expansion showed significant differences between ki group and other groups. discussion the results showed the variation in melting point average of carving waxes. the group without filler had the lowest melting point and the group with 20% filler had the highest melting point. those value were lower than the commercial inlay wax product (gc, japan) from the previous study (59.20° c).20 but, the melting point of carving waxes were still in the range of desirable softening points (54–60° c).3 the result of this research was in accordance with kotsiomiti & mccabe13 results that the filler addition up to 10% prohibit the melting properties of wax mixtures. based on that research, the higher temperature was needed in the melting of wax mixtures with filler. the inorganic filler particles in wax mixtures had function as seeds to form gel structure. the energy that was accepted by the table 2. mean and average of melting point, penetration depth and linear thermal expansion of carving wax carving wax compositions melting point (° c) penetration depth (mm) linear thermal expansion (%) ki (50:20:25:5)50:20:25:5) kii (55:15:25:5)55:15:25:5) kiii (60:10:25:5)60:10:25:5) kiv (65:5:25:5)65:5:25:5) kv (70:0:25:5)70:0:25:5) 55.00 ± 1.36° c 52.40 ± 1.16° c 53.06 ± 0.59° c 53.13 ± 1.12° c 52.93 ± 0.75° c 912 ± 0.39± 0.39 10.60 ± 0.98± 0.98 12.43 ± 0.95± 0.95 14.06 ± 0.38± 0.38 20.13 ± 1.56± 1.56 0.17 ± 0.01% 0.37 ± 0.04% 0.38 ± 0.03% 0.41 ± 0.06% 0.44 ± 0.04% table 3. anova results f significancy melting point penetration depth linear thermal expansion 4.554 98.246 31.408 0.009 0.000 0.000 117widjijono, et al.: melting point, hardness, and thermal expansion wax was absorbed by gel structure of filler particles, so the amount of heat absorbed by paraffin wax was decreased. this caused the increasing of wax melting point. the average penetration depth of carving wax were in the range of 20.13 to 9.12 mm. the higher filler concentration showed the increasing of carving wax hardness, that expressed by lower penetration depth. the hardness value of the group without filler was lower than the commercial inlay wax (gc, japan) from irnawati's study (14.12 mm).20 the results was similar with previous study on series of filler content of composite restorative materials that showed the filler influence with strong positive correlation on the elastic properties.21 the carving wax with 5% filler showed higher hardness than the previous study. some formulation of inlay wax contain a compatible filler to control expansion and shrinkage of the wax product.3 silica as in organic filler effectively played important role in the increasing of wax mixture hardness.13 the linear thermal expansion of carving waxes were in variation but lower than the inlay wax product (gc, japan). those value were also lower than the typical inlay wax (0.45%).3 the linear thermal expansion of carving waxes fulfilled the ansi/ada specification. no. 4 standard (0.6%) and also the iso specification (0.8%) from temperature 25 to 40° c.18,22 paraffin wax structure consists of covalent bonds with the non polar coordination. the non polar bond with other molecules had weakness properties, making the other molecules moved easily. the smaller the amount of non polar bond in the compound caused the smaller the expansion when the material was heated.22 in general, the filler was mixed in physicochemically with wax while decreasing the paraffin volume. this phenomenon caused the decreasing of expansion in heating and contraction in cooling of carving wax. it was comcluded that serums was with high ca-bentolite filter composition had high melting point and hardness, but low linear thermal expansion. references 1. benson hj, kipp re. dental science laboratory guide. 4th ed. iowa: wmc brow company publisher; 1973. p. 74–79. 2. anonymous. metrodent carving wax. available from: http://www. metrodent. com/pdfs /modelling. pdf. accessed january 15, 2005. 3. anusavice kj. phillips' science of dental materials. 11st ed. st. louis: elsevier science; 2003. p. 283–93. 4. american dental association (ada). dentist' desk reference; materials, instruments and equipment. 2nd ed. chicago: ada; 1983. p. 241–9. 5. craig rg, powers jm. restorative dental materials. 11th ed. st louis: mosby co; 2002. p. 423–48. 6. mccabe jf, walls awg. applied dental materials. 8th ed. cambridge: blackwell science ltd; 1998. p. 36–40. 7. budavari s. the merck index. 12th ed. new jersey: merck research laboratories division of merck & co., inc; 1996. p. 170–1, 302, 1206. 8. strahl, pitsch. beeswax. available from: http://www.spwax.com. accessed october 28, 2004. 9. american society for testing materials (astm). annual book of astm standards 2001. section 5 petroleum products, lubricants, and fossil fuels.vol. 05.01 petroleum products and lubricants(1) d56-d25962001. baltimore: astm; 2001. p. 504–6. 10. craig rg, eick jj, peyton fa. properties of natural waxes used in dentistry. j dent res 1965; 44(6): 1308–16.j dent res 1965; 44(6): 1308–16. 11. departemen kehutanan. perlebahan di indonesia. 2005. available at: http://www/ dephut.go.id /informasi/humas/lebah.htm. accessedaccessed april 29, 2009. 12. combe ec. notes on dental materials. 6th ed. edinburgh: churchil livingstone; 1992. p. 194–7. 13. kotsiomiti e, mccabe jf. experimental wax mixtures for dental use. j oral rehabilitation 1997; 24: 517–21. 14. manappallil jj. basic dental materials. 2nd ed. new delhi: jaypee brothers medical pub; 2003. p. 149–50, 276. 15. wikipedia. bentonite. available from: http: //en.wikipedia.org/wiki/ bentonite. accessed january 23, 2009. 16. arifin m, sudrajat a. bentonit. in: suhala s, arifin m, editors. bahan galian industri. bandung: pusat penelitian & pengembangan teknologi mineral; 1997. p. 124–6. 17. anonymous. mdm corporation expanding dealership, network,mdm corporation expanding dealership, network, inquiries, solicited, available from: http:/indiamart.com. accessed february 28, 2007. 18. american national standard/merican dental association. dental base plate wax. specification no. 24–1991. 1991. p. 1–9. 19. sears fw. mekanika panas dan bunyi. edisi iv. jakarta: bina cipta; 1980. p. 355. 20. irnawati d. pengaruh rasio malam parafin dengan malam carnaubaarafin dengan malam carnauba terhadap titik leleh dan kekerasan malam ukir. laporan penelitian.laporan penelitian. fkg–ugm; 2007. 21. masouras k, sikkas n, watts d. corelation of filler content and elastic properties of resin-composites. dental materials 2008; 24(7):dental materials 2008; 24(7): 932–39. 22. noort rv. introduction to dental materials. 3rd ed. eidenburg: mosbyeidenburg: mosby elsevier; 2008. p. 54–56. vol 44 no 3 sept 2011.indd 141 vol. 44. no. 3 september 2011 research report efficacy of various topical agents to prevent enamel demineralization priska lestari hendrawan1, erwin siregar2, and krisnawati2 1orthodontic resident 2 department of orthodontics faculty of dentistry, university of indonesia jakarta indonesia abstract background: enamel demineralization is a common and undesirable side effect of fixed appliance orthodontic treatment. many sudies showed that the prevalence varied between 2–96%. there are many ways to prevent demineralization and increased remineralization such as oral hygiene instruction and by topical application such as acidulated phosphate fluor (apf) casein phospo peptide-amorphous calcium phosphate (cpp-acp), casein phospo peptide-amorphous calcium phosphate plus (cpp-acpf). purpose: the purpose of this in-vitro study was to evaluate the efficacy of various topical agents to prevent enamel demineralization. methods: fourty extracted human premolars were allocated to 1 of 4 groups: 1.23% apf gel; 10% cpp-acp paste; 10% cpp-acpf paste; and untreated control. all samples were subjected to ph cycling treatment for 12 days through a daily procedure of demineralization solution with ph 4 for 6 hours and remineralization solution with ph 7 for 18 hours. microhardness testing were done before and after ph cycling and the delta hardness values were determined. results: apf, cpp-acp and cpp-acpf application significantly prevent lowering of enamel microhardness value compared with untreated control group. kruskal-wallis, anova, mann-whitney u, tukey and bonferroni post-hoc multiple comparison test showed significant difference between mean delta microhardness value of cpp-acpf and cpp-acp group with apf group, but there is no significant difference between mean delta microhardness value of cpp-acpf and cpp-acp group. conclusion: apf, cpp-acp and cpp-acpf prevent enamel demineralization. cpp-acp and cpp-acpf prevent demineralization more than apf. key words: enamel demineralzation, topical agents, enamel microhardness testing abstrak latar belakang: demineralisasi email merupakan efek samping negatif yang sering dijumpai pada perawatan ortodontik cekat. beberapa penelitian menyatakan bahwa prevalensinya bervariasi 2–96 persen. ada beberapa cara untuk mencegah demineralisasi dan meningkatkan remineralisasi, misalnya dengan instruksi kebersihan mulut dan menggunakan bahan topical aplikasi seperti acidulated phosphate fluor (apf) casein phospo peptide-amorphous calcium phosphate (cpp-acp), casein phospo peptide-amorphous calcium phosphate plus (cpp-acpf). tujuan: tujuan penelitian in vitro ini adalah untuk mengevaluasi efektivitas berbagai agen topikal untuk mencegah demineralisi email yang dilihat dengan uji kekerasan mikro permukaan email. metode: emapat puluh gigi premolar yang sudah di ekstraksi dibagi dalam 4 kelompok: aplikasi gel 1,23% apf; aplikasi psta 10% cpp-acp; aplikasi pasta 10% cpp-acpf dan kelompok kontrol. semua sampel diberikan perlakuan siklus ph selama 12 hari yang terdiri dari perendaman dalam larutan demineralisasi dengan ph 4 selama 6 jan dilanjutkan dengan perendaman dalam larutan remineralisasi dengan ph 7 selama 18 jam. uji kekerasan dilaku kan sebelum dan sesudah perlakuan serta diperoleh juga data delta kekerasan. hasil: semua aplikasi agen topikal tersebut dapat mencegah demineralisasi email secara signifikan dibandingkan kelompok kontrol. kesimpulan: apf, cpp, acp, dan cpd-acpf mencegah demineralisasi enamel. cpp-acp dan cpd-acpf mencegah demineralisasi lebih baik dibanding apf. kata kunci: demineralisasi email, agen topikal, uji kekerasan permukaan email correspondence: krisnawati, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas indonesia. jl. salemba raya. no. 4. jakarta 10430, indonesia. e-mail: krisnawati.61@ui.ac.id 142 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 141–144 introduction enamel is the hardest tissue of the human body consist of 96% hydroxyapatite, 4% organic content and water. enamel hardness is influenced by the total mineral content and structural characteristics of the prisms.1 microhardness value for sound enamel varied between 292–390 knoop hardness number (khn).2,3 enamel demineralization around orthodontic brackets is a common and undesirable side effect of fixed appliance orthodontic treatment, with incidence and prevalence varied between 2–96%.4,5 clinically, early enamel demineralization lesion appear as white spot, which is a white opaque area and is softer than the surrounding enamel. carbohydrate consumption will cause the saliva and plaque fluid ph to fall to critical ph (between 4.5–5.5) as acids are produced from fermentation of the carbohydrate by bacteries such as streptococcus mutans (s. mutans) and lactobacillus which then will cause hydroxyapatit to dissolve. s. mutans and lactobacillus levels were found to be significantly elevated during active orthodontic treatment. saliva is the most important defense mechanism against formation of demineralization lesion. saliva parameters such as flow rate, ph, and buffer capacity influence the dynamics between demineralization and remineralization.6 bracket placement increases plaque retention sites and limiting salivary flow access, especially on enamel area between brackets and gingival margins and below orthodontic wire. bracket placement also complicates tooth cleansing by the patient.5 preventive measures that can be done to reduce the risk of demineralization during orthodontic treatment are patient education and oral hygiene instruction, routine professional cleaning, and giving additional topical agents that contain fluoride and casein phospho peptide–amorphous calcium phosphate (cpp-acp).7,8 the efficacy of fluoride topical agents to prevent enamel demineralization during orthodontic treatment has been proven as a fact. there are several methods to deliver fluoride to teeth during orthodontic treatment which include toothpaste with a higher fluoride content, fluoride rinse, gel/foam and varnish application.8 previous researches have shown that cpp-acp also can reduce demineralization risk because of its ability to stabilize free calcium and phosphate ions in plaque surrounding the enamel thereby helping to maintain a state of supersaturation with respect to enamel and its ability to buffer plaque ph, thus promoting remineralization and depressing demineralization. cpp-acp at 0.5–1.0% w/v produced a reduction in caries activity similar to that of the 500 ppm fluoride containing solution.9–11 products that contain cpp-acp that are available in the indonesian market is gc tooth moussetm (gc corporation). cpp-acp and fluoride have been shown to have a synergistic effect enhancing the potential to depress demineralization and promote remineralization.9,12,13 recently, a new variant of the gc tooth mousse which is the gc tooth mousse plustm is introduced to the indonesian market, it contained additional 0.2% naf (900 ppm). this product is expected to have added benefit of the synergistic effect between cpp-acp and fluoride. until this research is done, the writer has not encountered any research that tested the efficacy of the casein phospho peptide–amorphous calcium phosphate paste (cpp-acpf) in reducing demineralization risk on enamel surrounding orthodontic brackets. microhardness testing is a quick, simple, and nondestructive test. knoop microhardness test is the most common test to determine the microhardness value of surface enamel, with 1–1000 g indenter load. there is a linear relationship between the square root of khn and the mineral content of dental tissues; therefore the demineralization process of enamel could be detected by the reduction of enamel microhardness. loss of enamel mineral will increase its porosity thereby reducing its ability to resist the load of the indenter causing deeper and wider indentation mark.2,14 the purpose of this research is to analyze the efficacy of topical fluor application such as apf, cpp-acp and cpp-acpf to prevent enamel demineralization using microhardness testing and to know which topical agent is the most effective. materials and methods forty extracted human premolars that never been bonded with orthodontic bracket, free of filling, stain, white spot and carious lesion, and without structural enamel defects are cleaned and stored in saline solution until it is time to be used in the research. roots were sectioned at the cement-enamel junction using low speed hand piece and diamond disc. the crowns were then fixated on a decorative self-curing resin using 20 mm in diameter and 10 mm thick pipe as mold with the buccal/lingual side facing upward. each specimen is polished with silicone carbide paper no. 1500 and 2000 continued with 1μm alumina polishing solution and polishing cloth on top of a polishing machine. each specimen is polished until maximum of 200 μm of enamel thickness is taken. after polishing, microhardness testing is done with knoop indentation to determine inital surface microhardness value of each specimen. forty specimens were allocated to 1 of 4 groups: 1.23% apf gel; 10% cpp-acpf (gc tooth mouse); 10% cpp-acp (gc tooth mouse plus); and untreated control. specimens from application group were applied with 0.5 ml of corresponding topical agents for 5 minutes. specimens from untreated group receive no topical agent application. all specimens were then placed in 20 ml 37° c, ph 4 demineralizing solution (containing 2.2 mm/l cacl2, 2.2 mm/l kh2po4 dan 50 mm/l acetic acid) 16 for 6 hours. after that the specimens were removed and placed in 20ml 37° c, ph 7 remineralizing solution (containing 1.5 mm/l cacl2, 0.9 mm/l kh2po4, dan 130 mm/l kcl) 16 for 18 hours. after that, all specimens were removed and rinsed with aquadest and dried using paper towel. this cycle was 143hendrawan et al.: efficacy of different topical agents repeated 12 times. specimens from cpp-acp and cppacpf group were reapplied with 0.5 ml of corresponding topical agents for 5 minutes after each cycle, whereas specimens from apf group were not. after 12 cycles, microhardness testing was done with knoop indentation to determine final surface microhardness value of each specimen. initial and final microhardness value of each specimen is a mean value from 3 indentations. delta microhardness value is the difference between initial and final microhardness value. the data of these result was analyzed using anova and tukey test. results table 1. enamel microhardness of control and treated groups group n x initial hardness x final hardness x δ (delta) control 10 359.9 ± 9.63 73.3 ± 18.07 286.6 ± 20.75 apf 10 359.9 ± 15.1 241.4 ± 25.27 118.5 ± 23.78 cpp-acpf 10 359.9 ± 11.32 332 ± 15.08 27.9 ± 12.77 cpp-acp 10 359.9 ± 13.51 329 ± 11.78 30.9 ± 12.77 * showed significant differences with control group (p < 0.05). apf, cpp-acp and cpp-acpf application significantly prevent lowering of enamel microhardness value compared with untreated control group (table 1). this means that all topical application agents is effective in preventing enamel demineralization compared to untreated control group. anova and tukey test showed significant difference between mean delta microhardness value of cpp-acpf and cpp-acp group with apf group, but there is no significant difference between mean delta microhardness value of cppacpf and cpp-acp group (table 2 and 3). table 2. anova test of delta microhardness value group n x ∆ (khn) anova f p control 10 286.6 ± 20.75 444.973 0.001 apf 10 118.5 ± 23.78 cpp-acpf 10 27.9 ± 12.77 cpp-acp* 10 30.9 ± 12.77* * showed no statistical difference, significant at p < 0.05 table 3. tukey test of delta microhardness value group mean ∆ (khn) group mean ∆ (khn) tukey (p) control 286.6 apf 118.5 p < 0.05 cpp-acpf 27.9 p < 0.05 apf 118.5 control 286.6 p < 0.05 cpp-acpf 27.9 p < 0.05 cpp-acpf 27.9 control 286.6 p < 0.05 apf 118.5 p < 0.05 discussion there are a linear relationship between the square root of khn and the mineral content of enamel and dentin.14 fujimaru et al.2 and white et al.,17 compared contact microradiography (cmr), which is the gold standard to measure mineral content of dental tissues, with microhardness testing and found the result from both methods showed insignifi cant differences and strong correlation (p < 0.01, r2 = 0.94) thus enamel microhardness testing can be used as a simpler alternative to evaluate enamel remineralization and demineralization process.16 it is more valuable to a clinician to know enamel surface microhardness than to know its mineral content. enamel is not a homogen material, it consists of inorganic and organic materials, therefore the mean of 3 indentations was calculated for each specimen to increase the accuracy of the hardness value. the best way to simulate the process of demineralization and remineralization in vivo, in which demineralization occurs in an neutral ph.18–20 the in vitro ph cycling model can be varied in the time intervals of the demineralization and remineralization phase and the number of cycles.16–18 in this research, 12 cycles were done with each cycle consisting of 6 hours demieralization followed by 18 hours remeralization. this model was showned as the best model to simulate the mineral loss that occurs in vivo around orthodontic brackets.19 the 5 minutes topical agent application is in accordance with recommendation from american dental association (ada).21 apf were applied only once before the 12 cycles to simulate professional application which was done every 3 to 6 months, whereas cpp-acp and cpp-acpf pastes were re-applied at the beginning of each cycle to simulate daily use by the patient. therefore, the in vitro method used in this research may represent the in vivo conditions of the demineralization challenge experienced by a patient during fi xed orthodontic treatment. 144 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 141–144 delta microhardness value of cpp-acp group is not distributed normally caused by one speciment with a khn way above mean. the probable explanation for this is a possible nonuniform time of topical agent application or length of demineralization/remineralization cycle. eventhough the time had been strictly scheduled and monitored, this can still happen considering the number of specimens. the efficacy of topical fluoride to prevent enamel demineralization.17 the effi cacy of cpp-acp paste to prevent enamel demineralization is in accordance with studies done by oshiro et al.,10 hodnett and sato et al.,22 reynolds et al.,13 and kumar et al.,9 found a synergistic effect between cpp-acp and topical fl uoride agent which decreases enamel demineralization and enhances enamel repair with remineralization process and furthermore the combination effect of the two is better than just using either one. recently, a new variant of gc tooth moussetm (contatining 10% w/v cpp-acp), the gc tooth mousse plustm (containing additional 0.2% 900 ppm naf), is introduced to the indonesian market. this product is expected to have the combined effect of topical fl uoride and cpp-acp within one product. until this research is done, the writer has not encountered any research that tested the effi cacy of the gc tooth mousse plustm (cpp-acpf) in reducing demineralization risk on enamel surrounding orthodontic brackets. the result of this research showed cpp-acpf and cpp-acp application prevent demineralization more signifi cantly than 1.23% apf, which is in accordance with the result of a study done by sato et al.,22 statistical analysis showed no signifi cant difference between cpp-acpf and cpp-acp even though delta microhardness value of cpp-acp group is less than cppacpf group (table 1). further in vitro and in vivo research is needed to compare the efficacy of cpp-acpf and cppacp paste in preventing enamel demineralization especially in patients undergoing fixed orthodontic treatment. in conclusion topical agents such as apf gel, cpp-acp paste and cpp-acpf could prevent demineralization. cpp-acp paste and cpp-acpf application to prevent demineralization were more effective then apf. references 1. ten cate ar. oral histology. 7th ed. st. louise: the mosby co; 2008. p. 232–8. 2. fujimaru t, ishiyaki re, hayman, nemoto k. microhardness testing to evaluate remineralization of tooth enamel. http://iadr.confex. com/iadr/2003goteborg/techprogram/abstract_35853.htm. accessed january 14th, 2009. 3. gutierrez-salazar mp, reyes-gasga j. enamel hardness and caries susceptibility in human teeth. http://www.scielo.org.ve/scielo. php?script=sci_arttext&pid=s0255-69522001000200007&lng=pt &nrm=iso. accessed january 29th, 2009. 4. gorelick l, geiger am, gwinett at. incidence of white spot formation after bonding and banding. am j orthod 2004 march; 81: 93–8. 5. chang hs, walsh lj, freer tj. enamel demineralization during orthodontic treatment. aetiology and prevention. aust dent j 1997; 42(5): 322–7. 6. rosenbloom rg, norman. salivary streptococcus mutans levels in patients before, during, and after orthodontic treatment. orthod dentofac orthop 2008; 100: 35–7. 7. sudjalim tr, woods mg, manton dj prevention of white spot lesions in orthodontic practice: a contemporary review. aust dent j 2006; 51: 284–9. 8. benson pe, shah aa, millett dt, dyer f, parkin n, vine rs. fluorides, orthodontics and demineralization: a systematic review. j orthod 2005; 32(2): 102–14. 9. kumar vln, ittagarun a, king nm. the effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: an in vitro study. aust dent j 2008; 53: 34–40. 10. oshiro m, yamaguchi k, takamisawa t, inage h, watanabe t, irokawa a, miyazaki m. effect of cpp-acp paste on tooth mineralization: a fe-sem study. j of oral science 2007; 49(2): 115–20. 11. yamanaka k, yoshii e. caries prevention potential of tooth-coating material containing cpp-acp. iadr, general session, goteborg. http://iadr.confex.com/iadr/2003goteborg/techprogram/abstract_ 33123.htm. accessed january 25th, 2009. 12. sudjalim tr, woods mg, manton dj. prevention of demineralization around orthodontic brackets in vitro. am j orthod dentofac orthop 2007; 131: 705.e1–e9. 13. reynolds ec, shen p. fluoride and cpp-acp. j dent res 2008; 87(4): 344–8. 14. kodaka t, debari k, yamada m, kuroiwa m. correlation between microhardness and mineral content in sound human enamel. caries res 2009 november; 26: 139–41. 15. hodnett s. the protective potential of paste containing cpp-acp as measured by confocal microscopy: an in vitro study. thesis. west virgina university. 2007. 16. argenta e, mark bockeoue. a modified ph-cycling model to evaluate fluoride effect on enamel demineralization. prequi odontol bras 2003; 17(3): 241–6. 17. delbem acb, brigenti fl,vira ae de m, cury ja. in vitro comparison of the cariostatic effect between topical application of fluoride gels and fluoride toothpaste. j appl oral sci 2004; 12(2): 121–6. 18. dogan f, civelek arsu, oktay. effect of different fluoride concentrations on remineralization of demineralized enamel: an in vitro ph-cycling study. ohdmbsc 2004; 3(1). 19. o ’ r e i l l y m m , f e a t h e r s t o n e j d b . d e m i n e r a l i z a t i o n a n d remineralization around orthodontic appliances: an in vivo study. am j orthod dentofac orthop 2004; 92: 33–40. 20. wei h, featherstone jdb. prevention of enamel demineralization: an in-vitro study using light-cured filled sealant. am j orthod dentofac orthop 2005; 128: 592–600. 21. the ada council on scientific affairs. professionally applied topical fluoride-excecutive summary of evidence-based clinical recommendations. 2006. available at: http://www.ada.org/prof/ resources/pubs/jada/reports/report_fluoride_exec.pdf accessed online on september 7th 2008. 22. sato t, yamanaka k, yoshii e. caries prevention potential of a tooth coating material contaning casein phosphopeptide-amorphous calcium phosphate. iadr, general session, goteborg. available at: http://iadr. confex.com/iadr/2003goteborg/techprogram/abstract_33123.htm. accessed online on january 25th 2009. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 212 volume 45 number 4 december 2012 inhibition effect of cashew stem bark extract (anacardium occidentale l.) on biofilm formation of streptococcus sanguinis rizni amaliah, sri larnani and ivan arie wahyudi department of dental biomedical sciences faculty of dentistry, universitas gadjah mada �ogyakarta – indonesia abstract background: biofilm is communities of microorganisms attached to solid surface and enclosed in extracellular matrix that protected microorganisms from antibacterial agents and host defense. one of bacteria might have a role in initial colonization of biofilm formation is streptococcus sanguinis (s. sanguinis). previous studies showed that cashew stem bark extract (anacardium occidentale l.) can inhibit the growth of streptococcus strains. purpose: the purpose of this study was to determine the inhibition effect of cashew (anacardium occidentale l.) stem bark ethanol extract on biofilm formation of s. sanguinis. methods: streptococcus sanguinis grown in brain heart infusion (bhi) + 2% sucrose medium by using microplate polystyrene 96 wells. the samples were divided into 3 groups, 5% polyethyleneglycol (peg) as negative control, cashew stem bark extract (concentration 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml), and 0.12% chlorhexidine (as positive control). biofilm was stained by 1% crystal violet. afterwards, optical density (od) of samples were measured by microplate reader λ 595 nm. the data of biofilm formation inhibition percentage were analyzed by one way anova and then continued by least significant difference (lsd) test. results: the result of one way anova showed that there were significant differences in inhibition of s. sanguinis biofilm formation (p<0.05). lsd test showed that concentration extract 3.125 mg/ml had significant difference with concentration 9.375 mg/ml and 12.5 mg/ml. reciprocally, concentration 6.25 mg/ml had significant difference with concentration 9.375 mg/ml and 12.5 mg/ml. conclusion: cashew stem bark extract was able to inhibit biofilm formation of s. sanguinis. key words: inhibition of biofilm formation, streptococcus sanguinis, cashew stem bark abstrak latar belakang: biofilm merupakan sekumpulan mikroorganisme yang melekat pada permukaan solid dan diselubungi oleh matriks ekstraseluler yang melindungi mikroorganisme dari bahan-bahan antibakteri dan sel-sel pertahanan tubuh. salah satu bakteri yang berperan pada awal pembentukan biofilm adalah streptococcus sanguinis (s. sanguinis). beberapa penelitian menunjukkan bahwa ekstrak kulit batang jambu mete (anacardium occidentale l.) dapat menghambat pertumbuhan bakteri strain streptococcus. tujuan: penelitian ini bertujuan untuk mengetahui pengaruh ekstrak etanol kulit batang jambu mete (anacardium occidentale l.) terhadap pembentukan biofilm s. sanguinis. metode: media pertumbuhan s. sanguinis menggunakan brain heart infusion (bhi) + 2% sukrosa yang ditumbuhkan pada microplate polystyrene 96 wells. kelompok perlakuan dibagi menjadi tiga kelompok yaitu peg 5% (kontrol negatif), ekstrak kulit batang jambu mete (konsentrasi 3,125 mg/ml, 6,25 mg/ml, 9,375 mg/ml, dan 12,5 mg/ml), dan klorheksidin 0,12% (kontrol positif). biofilm yang terbentuk diwarnai dengan crystal violet 1%. kemudian optical density (od) sampel diukur menggunakan microplate reader λ 595 nm. data berupa persentase penghambatan pembentukan biofilm dianalisis menggunakan uji one way anova dan dilanjutkan dengan uji least significant difference (lsd). hasil: uji one way anova menunjukkan terdapat perbedaan daya hambat pembentukan biofilm s. sanguinis yang signifikan (p<0,05). hasil uji lsd menunjukkan konsentrasi 3,125 mg/ml memiliki perbedaan yang signifikan dengan konsentrasi 9,375 mg/ml dan konsentrasi 12,5 mg/ml. begitu juga dengan konsentrasi 6,25 mg/ml research report 213amaliah, et al.: inhibition effect of cashew stem bark extract memiliki perbedaan yang signifikan dengan konsentrasi 9,375 mg/ml dan konsentrasi 12,5 mg/ml. kesimpulan: ekstrak kulit batang jambu mete dapat menghambat pembentukan biofilm s. sanguinis. kata kunci: daya hambat pembentukan biofilm, streptococcus sanguinis, kulit batang jambu mete correspondence: rizni amaliah, c/o: bagian biomedik, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara, �ogyakarta 55281, indonesia. email address: rizni.amaliah@gmail.com introduction the bacteria can form dental plaque and cause periodontal disease.1-2 since 1996, dental plaque is not only recognized as the etiologic factor of periodontal disease, but also considered as a biofilm.3 biofilms are a community of bacteria that have extracellular matrix, circulation, and communication system. biofilms grow in moist area and attach to solid surface such as tooth, dental restoration, prosthesis, and dental implant.2-3 the formation of biofilm begins with the attachment of bacteria such as neisseria and streptococcus, at over tooth surface which dominated by mitis groups such as s. sanguinis.4 the bacteria in biofilm will grow and become maturate, then it form microcolonies.2,5 an extracellular polymeric matrix is a thick layer surrounded the cells that form biofilm.4 this layer is a biofilm barrier against antibiotics, antimicrobials, and immunity cells. the main component is exopolysaccharides (eps).6 exopolysaccharides is mostly formed by bacteria that produced glucosyltransferase (gtf), such as s. sanguinis.6-7 this bacteria plays an important role in initial colonization of biofilm formation.6 s. sanguinis have some adhesin that bind to tooth surface that was layered by saliva. beside that, iga1 protease that is produced by colony of s. sanguinis, enable this bacteria grow and proliferate over tooth surface.4 the cashew excessively have been widely used such as from its wood, bark, leaf, fruit, and seed.8 cashew was reported might have antidiabetic, antibacterial, and antiinflammation.9 cashew’s leaf could have inhibited bacterial growth such as klebsiella pneumoniae, staphylococcus aureus, bacillus subtilis, salmonella typhy, escherichia coli, and candida albicans.10 the stem bark was also used for medication. from previously report, the stem bark could inhibit staphylococcus aureus growth.11 the aim of the research was to determine the effect of cashew stem bark ethanol extract (anacardium occidentale l.) on inhibition biofilm formation of s. sanguinis. materials and methods cashew stem bark (anacardium occidentale l.) was taken from cashew trees at kasiutri plantation, imogiri. plant identification and extraction processed at unit ii laboratory of pharmaceutical biology, faculty of pharmacy, universitas gadjah mada. one kilogram of cashew stem bark was dried using a drying cabinet at 40500c for 48 hours. after drying, the bark was made into powder using machine pollinators. the 300g of powdered cashew stem bark was then extracted using 3l of 70% ethanol by maceration method then stirred for 30 minutes and allowed to stand for 24 hours. powdered bark that was extracted then filtered using a buchner funnel. ethanol in filtrate was evaporated using stove for 3-4 hours until the stiff extract obtained. the materials used for the inhibition of biofilm formation test were 2.5% polyethileneglycol (peg), 0.12% chlorhexidine (as positive control), bhi media containing 2% sucrose, s. sanguinis mcfarland standard v (15 x 108), 1% crystal violet and 96% ethanol. culture of s. sanguinis was from balai laboratorium kesehatan (blk), �ogyakarta. bacteria were prepared in mcfarland v suspension (15 x 108cfu/ml) using densicheck. plate was divided into two, one test plate and one blank plate. the test plate was containing extract solution/peg and bhi + 2% sucrose with the addition of 10% v/v suspension of bacteria, while the blank plate containing extract solution/peg and bhi + 2% sucrose with the addition of 10% v/v saline. according to pereira et al.,14 cashew stem bark extract was starting inhibit s. sanguinis growth in concentration 3.12% (mg/ml), the antimicrobial activity was carried out on solid media plates by a diffusion method for the screening and determination of minimum inhibitory concentration (mic) of the extract on bacterial. whereas in this study, biofilm inhibition assay did with microdilution method. extract concentrations was 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml and bhi + 2% sucrose added to the polystyrene u bottom microplate with a total volume of each well 90 μl. chlorhexidine is the most effective antibacterial agent for oral use.4 as a positive control using 0.12% chlorhexidine (v/v) each well while the negative control using 5% peg. afterward, the microplate was incubated for 24 hours at 370 c. after the incubation, the microplate was washed with water three times. then a solution of 1% crystal violet in 125 μl each well was added, and incubated at room temperature for 15 minutes, and then microplate was washed with water three times. two hundred μl 96% ethanol added to each well using a 50-200 μl micropippet and incubated at room temperature for 15 minutes. furthermore, as each 150 μl solution was transferred into a microplate flat bottom polystyrene 96 wells. od readings 214 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 212–216 table 1. mean and standard deviation of the inhibition of biofilm formation in 5% peg, cashew stem bark extract (3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml and 12.5 mg/ml), and 0,12% chlorhexidine concentration mean (%) ± sd 5% peg cashew stem bark extract 3.125 mg/ml cashew stem bark extract 6.25 mg/ml cashew stem bark extract 9.375 mg/ml cashew stem bark extract 12.5 mg/ml 0,12% chlorhexidine -27.35 ± 1.11 67.22 ± 4.80 73.22 ± 1.45 87.24 ± 9.51 94.20 ± 5.71 89.55 ± 4.85 sd: standard deviation table 2. lsd test results 5% peg, cashew stem bark extract, and 0.12% chlorhexidine groups control (–) 3.125 mg/ml 6.125 mg/ml 9.375 mg/ml 12.5 mg/ml 0.12% chlorhexidine control (–) – 0.001* 0.001* 0.001* 0.001* 0.001* 3.125 mg/ml – – 0.131 0.001* 0.001* 0.001* 6.125 mg/ml – – – 0.002* 0.001* 0.001* 9.375 mg/ml – – – – 0.084 0.552 12.5 mg/ml – – – – – 0.236 0.12% chlorhexidine – – – – – – control –: 5% peg * there were significant differences figure 1. mean of the inhibition of biofilm formation peg 5%, cashew stem bark extract (3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml), and chlorhexidine 0.12%. used biorad benchmark® microplate reader with 595 nm wavelength at the laboratory of parasitology faculty of medicine, universitas gadjah mada. based on quave et al.12 research modified by ardani et al.13 the percentage inhibition of biofilm formation was calculated using the formula: % inhibition = od sample: optical density extract and bacterial suspension; od blank sample: optical density extract and saline; od vehicle: optical density peg and bacterial suspension; od blank vehicle: optical density peg and saline. the data of biofilm inhibition percentage assay were tested for normality and homogenity. to determine the mean difference between groups statistically analyzed using one way anova test and then proceed using lsd test. results the inhibition of biofilm formation was done by calculating the percentage of inhibition of s. sanguinis biofilm formation after adding cashew stem bark extract. biofilms will absorb color crystal violet. the percentage of inhibition of biofilm formation values obtained with the four values substituted into the equation od percentage inhibition. the results of the calculation of the percentage of inhibition of biofilm formation can be seen in table 1. table 1 showed that 5% peg did not inhibit biofilm formation because it had a negative mean. as the increasing concentrations of cashew stem bark extract, the inhibition of biofilm formation also increased. cashew stem bark extract concentration 12.5 mg/ml was the highest average inhibition biofilm formation if compared with concentration 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml and 0.12% chlorhexidine. to make clearing up the results in table 1, the mean of each group would presented in figure 1. odsample odblank sample odvehicleodblank vehicle 0.12% chlorhexidine 215amaliah, et al.: inhibition effect of cashew stem bark extract based on one way anova result, the treatment between groups had significant difference (p<0.05). furthermore, to find a group that has a significant difference, the lsd test was using with a level of 95%. least significant difference (lsd) analysis results can be seen in table 2. based on lsd test, the inhibition of formation 5% peg groups had a significant differences with the cashew stem bark extract concentration of 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml. this was due to cashew stem bark extract had substances that act as an antibacterial, while peg did not have antibacterial substances. the results were consistent with studied of pereira et al.,14 who showed that cashew stem bark extract had antibacterial power against streptococcus mutans, s. sanguinis, and streptococcus mitis. discussion biofilm is a bacterial community that attach to surface that covered by extracellular matrix. this matrix has a main component, that is exopolysaccharides (eps). exopolysaccharides is formed by bacterial that produce gtf such as s. sanguinis.6,15 this study used bhi with 2% sucrose as growth medium. addition 2% sucrose in bhi because of sucrose is substrate that will breakdown by s. sanguinis to form eps.6 in this study, biofilm of s. sanguinis attached to wall of microplate polystyrene wells. according to ge et al.,16 this surface is one of abiotic surface that biofilm can attach. biofilm that have attached, was stained with crystal violet. assessment of crystal violet-biofilm bonding quantitatively was using an elisa reader (microplate reader).17 the result showed that cashew (anacardium occidentale l.) stem bark extract capable of inhibiting biofilm formation of s. sanguinis. anacardium occidentale l. stem bark extract had antibacterial activity against s. sanguinis.14 anacardium occidentale l. had some antibacterial agent such as tannin and flavonoid that can disturb metabolism of s. sanguinis. beside that, these bacterial agent can also to deactivated enzyme.18,19 the high concentration of anacardium occidentale l. extract more inhibit biofilm formation than others. this is due to the high concentration has more antibacterial agent, so that make an effect to number of percentage of inhibiting biofilm formation. increase in the concentration of cashew stem bark extract will increase the antibacterial of s. sanguinis.14 in this study, chlorhexidine had antibacterial power greater than the concentration of 3.125 mg/ml, 6.25 mg/ml, and 9.375 mg/ml and lower than the cashew stem bark extract concentration 12.5mg/ml. chlorhexidine against some bacterials and fungi because of this agent increased cell membrane permeability then followed by cytoplasm macromolecule coagulating. chlorhexidine against some bacterials and fungi because of this agent increased cell membrane permeability then followed by cytoplasm macromolecule coagulating.20 however, the results of our study showed that chlorhexidine 0.12% inhibition of biofilm formation was lower than the cashew stem bark extract concentration of 12.5 mg/ml. this might be due to the concentration of 12.5 mg/ml was too thick so it settles on the surface of the base well and stained with crystal violet. this caused the difference between the od test well (12.5 mg/ml + bhi + sucrose 2% + s. sanguinis) to the od blank sample well (extract 12.5 mg/ml + bhi + sucrose 2% + saline) very small compared with the difference in od control vehicle (2.5% peg). therefore, the inhibition of biofilm formation was intense. in addition, the antibacterial microdilution test required agent in small volume.21 cashew stem bark extract concentration 12.5 mg/ml with a volume 2.5 ml per well seem too many that are less appropriate to be used in the microdilution test. broadly, cashew bark extract can inhibit the formation of biofilms that was formed by s. sanguinis. this might be caused by the phenolic compounds such as tannins and flavonoids contained in cashew stem bark extract. phenolic compounds can provide an antibacterial effect by disrupting the cell wall and membrane, precipitate proteins, and deactivate the enzyme.22 one of enzyme activity might be inhibited by phenolic compounds was gtf enzymes. some phenolic compounds could inhibit enzyme activity gtf.23 if the gtf enzyme activity was interrupted, as a matrix of eps biofilm formation also declined. thus, the formation of biofilm s. sanguinis can be inhibited. in conclusion, cashew stem bark extract (anacardium occidentale l.) can inhibit streptococcus sanguinis biofilm formation. references 1. socransky ss, haffajee hd. evidence of bacterial etiology: a historical perspective. periodontology 2000, 1994; 5: 7–25. 2. nield-gehrig js, willmann de. foundation of periodontics for dental hygienist. 2nd ed. new �ork: lippincote williams and wilkins; 2008. p. 71–5. 3. slavk i n hc. biof i l m , m ic robia l e colog y a nd a nton i va n leeuwenhoek. j am dent assoc 1997; 128(4): 492–5. 4. marsh p, martin mv. oral microbiology. 4th ed. oxford: wright publ; 2002. p. 51, 58, 61, 98. 5. costerton jw, stewart ps, greenberg ep. bacterial biofilms: a common cause of persisten infection. science 1999; 248(5418): 1318–22. 6. venkataramaiah pd, biradar b. plaque biofilm. in: panagakos fs, davies rm, eds. gingival disease-their aetiology, prevention, and treatment. rijeka: intech; 2011. p. 23, 24, 26, 27. 7. russell, roy rb. bacterial polysaccharides in dental plaque. in: ullrich m, editor. bacterial polisaccharides. norfolk: caister academic press; 2009. p. 150. 8. dalimartha s. atlas tumbuhan obat indonesia. jilid 2. jakarta: trubus agriwidya; 2008. p. 79–80. 9. age d a h ce , bawo, dds, nya na nyo bl . ident i f icat ion of antimicrobial properties of cashew, anacardium occidentale l. (family anacardiaceae). j appl sci environ manag 2010; 14(3): 25–7. 10. ayepola oo, ishola ro. evaluation of antimicrobial activity of anacardium occidentale (linn.). amds 2009; 3(1): 1–3. 216 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 212–216 11. harsini. pengaruh ekstrak etanolik kulit batang jambu mete (anacardium occidentale) dalam obat kumur terhadap pertumbuhan staphylococcus aureus (in vitro). majalah kedoktera gigi 2009; 16(1): 13–8. 12. quave cl, plano lr, pantuso t, bennett bc. effects of extracts from italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant staphylococcus aureus. j ethnopharmacol 2008; 118(3): 418–28. 13. ardani m, pratiwi sut, hertiani t. efek campuran minyak atsiri daun cengkeh dan kulit batang kayu manis sebagai antiplak gigi. majalah farmasi indonesia 2010; 21(3): 191–201. 14. pereira jv, sampaio fc, pereira msv, melo afm, higino js, carvalho aat. in vitro antimicrobial activity of an extract from anacardium occidentale linn. on streptococcus mitis, streptococcus mutans, and streptococcus sanguis. odontolgia 2006; 5(2): 137–42. 15. russell, roy rb. bacterial polysaccharides in dental plaque. in: ullrich m, editor. bacterial polisaccharides. norfolk: caister academic press; 2009. p. 150. 16. ge x, kitten t, chen z, lee sp, munro cl, xu p. identification of streptococcus sanguinis genes required for biofilm formation and examination of their role in endocarditis virulence. infect and immun 2008; 76(6): 2551–9. 17. al-ouqaili mt, al-quhli sq, al-izzy m�. the role of milleri streptococci in the formation of cariogenic biofilm: bacteriological aspects. jjbs 2011; 4(3): 165–72. 18. cowan mm. plant products as antimicrobial agents. clin microbiol rev 1999;12(4): 564–82. 19. chambers hf. berbagai macam antimikroba, desinfektan dan sterilan. in: katzung bg, editor. farmakologi dasar dan klinik. jakarta: salemba medika; 2004. p. 166-8, 170. 20. eley bm, manson jd. periodontics. 5th ed. london: wright; 2004. p. 134, 138, 139, 210. 21. winn wc, allen sd, janda wm, koneman ew, procop gw, schreckenberger pc, woods gl. koneman’s color atlas and textbook of diagnostic microbiology. philadelphia: lippincott williams and wilkins; 2006. p. 989. 22. chambers hf. berbagai macam antimikroba, desinfektan dan sterilan. in: katzung bg, editor. farmakologi dasar dan klinik. jakarta: salemba medika; 2004. p. 166–8, 170. 23. tomczyk m, pleszczynska m, wiater a. va r iation in total polyphenolics contents of aerial parts of potentilla species and their anticariogenic activity. molecules 2010; 15: 4639–51. 179 vol. 42. no. 4 october–december 2009 evaluation of seat and non-seat post preparation design using conventional and computational methods g. subrata1, z. hasratiningsih2, e. kurnikasari1, and t. dirgantara3 1 prosthodontic department, dental faculty, university of padjadjaran 2 dental material department, dental faculty, university of padjadjaran 3 lightweight structures research groups, faculty of mechanical and aerospace engineering, institut teknologi bandung indonesia abstract background: design of root canal preparation especially in cervical-third area of the root, is one of many factors involved in the success of post-core restoration. seat design that is used in prosthodontics installation, faculty of dentistry, university of padjadjaran, is in the contrary to minimal preparation design. the root fracture resistance of this design has not been proven yet. purpose: the aim of this study was to evaluate the root fracture resistance of seat compare to non-seat design, with two different research methods: experimental laboratory and computer simulation with finite element method (fem). method: the experimental laboratory investigation used 20 upper central incisors: 10 used seat design and 10 non-seats, with the cast posts cemented in the preparation. the specimens were tested by using universal testing machine with compressive force until the root fracture. the fem used 2d digital models: seat and non-seat design of maxillary central incisors using a finite element software. the distribution of internal stress caused by static loading 110n at 135° angle with longitudinal axis of the tooth was evaluated. result: the results of the fracture strength test showed a significant difference (p = 0.05) between the non-seat group (852.27n ± 112.6n) and the seat group (495.78n ± 82.90n). the fem showed a lower stress concentration in non-seat compare to seat group. this study proved that non-seat distributes stress better than seat design. conclusion: it can be concluded that the fem confirmed the result of the laboratory method. stress concentration will cause fracture, therefore root fracture resistance in the non-seat design was higher than the seat design. key words: post preparation design, stress distribution, finite element method (fem), root fracture resistance correspondence: gantini subrata, c/o: jln. setiabudhi 438 bandung 40143. e-mail: gantinisubrata@yahoo.com research report introduction in vitro studies in dentistry which deal with designs and structures of prostheses or appliances used in oral environment are complicated procedures. many obstacles are found e.g. identical samples collection, procedures of making the research specimen, making jigs for mechanical properties test, giving the identical treatments for every specimen, and evaluating the test results and especially if the intention is to study the mechanism of internal stress distribution in the hard tissue in oral cavity (i.e.: enamel, dentine, bone, and restorative materials) while in physiological function of mastication system.1 all of these make the studies become more complicated, expensive, and time consuming. therefore nowadays a research method that are quick, accurate, and inexpensive is developed in form of computer simulation technique of a design or structure under various treatments, using finite element method (fem) or also called finite element analysis (fea). the method is very helpful in overcoming the difficulties caused by the conventional technique, at the same time lowering the cost of the study and still produce very accurate results.2–4 in prosthodontics department at faculty of dentistry, university of padjadjaran, undergraduate students are required to make a post-core crown restoration with cast post. the post alloys that usually used are cuzn alloy. this alloy is beneficial for both patient and operator because it is inexpensive and easy to manipulate, but it is also considered as a weak alloy compared to gold alloys type iii/iv according to adas for post metal.5 180 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 179-184 martanto6 offered a technique to solve a weak alloy problem in the clinic. in his experience he found that if a post is bent or fractured, the bending location is always at the cervical area of the tooth. according to him even though the post is weak, it can be prevented from bending by making a suitable preparation design at the cervical area which is called seat design. this design can enhance the fracture resistance of the post material. in seat design more tooth structure is prepared from the root, so this design in is the contrary with other authors who recommend to preserve as much intact dentine structure as possible to prevent the root of the tooth from fracture.7–11 in fact the advantage that has been described about seat design has not been proven scientifically. whereas in evidence based medicine era, any guidelines which are suggested to be used, have to be supported with research findings, literature reviews, and retrospective clinical studies, before all those things can be used in the clinics. the success of the post-core restoration is when the tooth structure is preserved and not only the post. it is totally no point in preventing the post from fracturing and bending, while the root itself is vulnerable to fracture. when making a treatment plan for post-core crown, the construction and relation between post-core and dentine root structure have to be planned so that the stress can be distributed evenly in the post-core material and dentine, which can prevent root fracture when receiving normal chewing function. one of the factors influenced in stress distribution of post-core material and root is the root canal preparation design at cervical area of the root.11,12 in vitro study to examine whether there is a difference of root fracture resistance between seat and non-seat preparation design, in combination with the usage of a weaker alloy of cuzn has been performed recently.13 computer simulation using finite element method to investigate whether there is a difference in stress distribution (which at the end will influence root fracture resistance) between seat and design in combination with the usage of cuzn alloy has also been done.14 this study will evaluate the effect of post preparation designs: seat and non-seat on the root fracture resistance of central maxillary incisors with cuzn post-core placement under simulated mastication force, using two different research methods: conventional fracture strength test and numeric method using 2d (2 dimensional) fem. evaluating the seat and non-seat design is crucial because the seat design is still used until now in our installation without knowing the disadvantages. comparing these two techniques is also important, to introduce and to develop fem for dental researches especially in faculty of dentistry, university of padjadjaran. it will make the researches of material structures and designs can be done continuously, accurately, quickly, and inexpensively. material and method conventional laboratory fracture strength test13 and numeric technique using finite element method were done in thus study.14 twenty extracted, intact single-rooted maxillary central incisors were selected for investigation and preserved in physiologic saline solution. teeth were selected for similarity in size, shape, and root anatomy. the teeth were visually inspected to ensure the absence of caries, surface cracks, and fractures. the teeth were randomly divided into two groups of 10, and were decoronated until 2 mm above the cemento enamel junction, perpendicular to their long axis using a water spray-cooled diamond bur at high speed. the first group was given non-seat design (group 1) whereas the other was given seat design (group 2). for non-seat design (group 1), the root canal of each tooth was prepared with a peeso reamer (maillefer ballaigues, switzerland) followed by tapered fissure diamond 1.6 mm. post space diameter was made round; the depth was 10 mm similar with the length of the tapered fissure diamond 1.6 mm (figure 1).11 the accuracy of post space preparation was examined with compound impression. figure 1. non-seat preparation design. (a) root canal preparation: incisal view, (b) root canal preparation: labial view. a b for seat design (group 2) initially the root canal of each tooth was prepared similar with the group 1. then the preparation was continued by making a seat that was prepared at the cervical part of the root, with the depth of 1 mm and the width of 0.7 mm, similar with the width of fissure cylinder diamond bur diameter 0.7 mm encircling the tooth. (figure 2).6 the accuracy of the preparation was then examined with compound impression. figure 2. seat preparation design. (a) root canal preparation: incisal view, (b) root canal preparation: labial view. a 10 b 10 181subrata, et al.: evaluation of seat and non-seat post preparation after that, the post-core wax pattern was made and casted. after trying in the cast post-core into the root canal, all dowels were luted with zinc phosphate cement (mixed according to the manuals) and the cement was also put in the channels then pressed with the thumb for one minute until the cement set. the teeth were stored in normal saline at 37° c before embedding it in a resin blocks jigs for testing procedures. each tooth was embedded in a self curing acrylic resin in a cylinder mould, so that the long axis of the tooth was parallel to the cylinder walls and the acrylic resin covered the root, leaving 2 mm above the remaining dentin. a b figure 3. making the jigs. (a) specimens, (b) specimens in the test position. the specimens were then mounted in universal testing machine (shimadzu japan). a continuous increasing compressive force was applied at a point in the middle of the lingual part and 2mm below the incisal edge of the core. the load was applied at an angle of 135° to the long axis of the tooth at a crosshead speed of 0.5mm/min until failure occurred (figure 3).7,9,15 failure loads (measured in newton) from each tooth preparation designs were recorded and statistically analyzed for significant correlation between designs and failure loads using independent t test. this study is a numerical technique using finite element method (fem) to analyze stress distribution of cu-zn cast post metal toward tooth structure, under mastication force simulation, on non-seat and seat root canal preparation design in cervical area using two dimensional (2-d) models. a personal computers and commercial finite element software were used.4,16,17 picture of 2-d sagital sectioned model of maxillary central incisor that were prepared with non-seat and seat designs were restored with cuzn cast post metal, respectively. the research procedures consisted of several phases: pre-processing, solution/solving, and post-processing (post-solution), convergence test, and data analyzes. in pre-processing phase, a structure geometry of tooth model (creating geometry) was constructed.3,16,17,18 the normal geometry data’s of an intact maxillary central incisor were quoted from the reference.19 the type of element used in this study was triangular. the model was then divided into small symmetrical elements (meshing) and the material was created.17 the model consisted of 3 types of materials: dentin, guttapercha, and alloys cuzn. the modulus of elasticity (e) and poisson ratio (m) were quoted from literatures20–22 except for alloys cuzn the elastic modulus data of alloy cuzn were found by bending test, done in institute technology bandung.5 the tooth was restrainted by all of the nodes on the outer surface of the root toward translation, compression, and rotation force to all direction.17 the static load of 110 n, which resembled biting force on incisor region, was applied. the direction of force to the axial long axis was 135° on the palatal surface of the core, to simulate the normal chewing condition.7,9,15 the solution in structural problem was to figure out the displacement of nodes and stress value on each element which had been loaded. at post-processing phase, the location of the deformation and the value of maximal stress on the structure were interpreted and analyzed, so the result could be used in analyzing the stress distribution and to choose the proper preparation design. on 2-d model, the structure was assumed as a surface or a plane structure, so that the stress in thickness direction was not taken into account. the data was then analyzed by qualitative analysis to determine the stress distribution through the color change on 2-d digital model of maxillary central incisor after treatment and quantitative analysis to identify the comparison of the maximal stress value in the critical area between non-seat and seat design. results from the conventional fracture strength test it was found that the mean compressive load for non-seat and seat designs was as follow: table 1. t-test data for maxillary central incisors with non-seat and seat design preparation designs n x p nonseat 10 852.27 8.062 seat 10 495.78 the statistical analysis showed a significant result (p < 0.05); the mean fracture load in non-seat design was significantly higher than seat design. in group with seat design, root fractured happened in all teeth and none of the post was bent. whereas in non-seat design, all teeth underwent the root fracture and 8 posts were bent and came off from the root canal, at the mean fracture load of 852.27n. the bending location of the post was at the 1/3 apical of the post and not on the border between post and core in cervical area. it was clear that the root fracture locations were always located at the cervical area of the root in both designs. from qualitative analysis result of the finite element method it was found that the color change pattern which related to the stress distribution pattern scattered through out the 2-d model of maxillary central incisor in both nonseat and seat design. the higher stress could be detected 182 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 179-184 from the range color of blue until red, when other location which shows white color has the lowest stress. compare to the laboratory test, on the finite element method of 2d maxillary central incisor simulated the seat and non-seat design using the normal biting force 110 n showed the change in color code pattern that support the laboratory test work. the change in color was initiated on the location of loading and spread to the cervical area (figure 4). both design can be calculate as a ratio of maximum stress value between seat and non-seat design. maximum stress ratio = s seat design = 1520 = 3,69 s non seat design 412 from this comparison of the maximum stress value between seat and non-seat design, can be seen that the maximum stress value of seat design was 3.69 higher than non-seat design. the higher the stress concentration in certain location, the higher the possibility of tooth fracture. discussion the laboratory test results showed that the average compressive forces which can be resisted by the seat design of maxillary central incisor root canal preparation was 495.78n, while for non-seat design was 852.27n (p = 0.05). the statistic calculation of the data showed a significant difference. this result indicated that the thickness of remaining dentin root structure influence the root fracture resistance. the thicker the remaining dentin root structure, the higher the root fracture resistance. those findings were in accordance to the studies which have been done by other investigators. they proposed that a large root structure lost can weaken the strength of the tooth itself and increase the facture risk.7,9,23 within the seat design group, which was tested using universal testing machine at laboratory, the whole specimens, in other words 10 teeth were fractured on the cervical area of the root but no posts were bent. the location of fractures were found around the cervical area, mostly on the labial site (6 teeth), while the rest of the specimens fractured on the linguo-cervical and proximo-cervical. on the other hand, at the non-seat design which can resist higher force, showed not only they fracture around the cervical area but also most of the posts were bent on one third of the apical area as well. the bending was not on post-core border around the cervical area, this condition maybe caused by some reasons, possibly because the compressive strength of the cast post alloys were higher than 495.78 n and lower than 852.27 n. all the specimens were imbedded in self curing acrylic and did simulate the supporting tissue of natural tooth, so that the roots were defended from fracture. on the other hand, the supporting tissue could act as a shock absorber, which could reduce the actual stress applied on the surrounding root. actually the normal biting force is not as big as the force found in this study and it does not mean that for the tooth to fracture needs those big forces above. the dentin preparation work for creating the post space and cervical design, especially in this non-vital root structure may produce small crack around the preparation and can propagate further through the root structure. the stress distribution on the cervical area which has been identified as critical area showed that at the seat area figure 4. stress distribution pattern on non-seat and seat design. figure 5. comparison of stress distribution on the root. (a1) non-seat design; (a2) seat design. comparison ofcomparison of stress distribution at the cervical region (b1) non-seat design; (b2) seat design. this color spectrum on both the loading area and cervical area showed that the stress were concentrated on that location. around the cervical area the concentrated stress was mostly located on the labial part. the color spectrum on seat design (red) was higher than non-seat area (blue). focusing the stress distribution on the cervical or critical area, the color spectrum on seat design (red) was higher compare to non-seat design (blue) (figure 5). as from the fem quantitative analysis: the internal stress value can easily be seen through finite element method, where the loading on the model will produce stress detected by clinical or laboratory test. from this result, a comparison between the maximal stress value in 183subrata, et al.: evaluation of seat and non-seat post preparation the maximum stress value was 3.69 times higher than nonseat area (see maximum stress ratio above). this was due to that in seat design there were more tooth structure removed, so it had the tendency to transfer stress differently. the stress distribution process to the dentine is more complex when the dentine structure is thin.24,25 the higher stress on seat design means that the stress was not distributed evenly, therefore a high stress is concentrated on one location. in contrary, at non seat design, the external forces were distributed through the large remaining root volume on cervical area, which was wider and thicker, so that the localized stress becomes smaller. besides, it is also proven that too much preparation of tooth structure will have influence to the increase of stress concentration in root and root fracture can easily happens in tooth with minimal coronal structure.25 fracture is due to the inability of the material to resist the concentration of stress located in certain location. therefore an evenly stress distribution is expected and has to be arranged in planning a treatment to prevent the tooth from fracture. all the findings from the conventional fracture strength test and finite element method confirm the recommendations that preserving more dentine around the post will give strength and resistance to tooth fracture.13,14 these findings confirmed other author findings which recommended that the operator have to preserve as much tooth structure as possible to increase the fracture resistance of the tooth.9,15,23,24,26,27 nevertheless, as in all other in vitro studies, the result of this study can not be directly applied in the clinic, because many of clinical parameters are not simulated here i.e. periodontal ligament, supporting bone, the condition of tooth structure, differences in mastication system etc. long term clinical study has to be done to evaluate the influence of the post preparation design at the cervical area to the fracture resistance of the remaining tooth. oral rehabilitation is a difficult procedure since the functional forces in oral cavity will results in complicated response in oral tissue.24,26 a difference in preparation design will result in different stress distribution patterns. the stress distribution is related to the restorative materials, in this case cu-zn alloy and tooth structure. in biomechanical function, the important thing is to detect stress that will cause tooth fracture. the results of numeric simulation using computational method14 also confirm the results of conventional laboratory test that has been done in advance13 and both concluded that preserving more dentine structure will minimalize the risk of tooth fracture. considering all the findings it is recommended to use a non-seat than a seat design. about the method comparison between conventional and computational method, the internal stress value can easily be observed through finite element method where the given loading will result in calculation of stress value which distributed in every nodes and difficult to be detected by conventional or clinical studies.28 fem is a good method to test and predict the mechnical properties of a prosthesis or devices. fem develops to overcome the laboratory and clinical research about material structures and designs which are relatively high in cost, difficult in procedures, and many other technical obstacles. this research used 2d model of tooth structure and static load. forces in oral cavity is very complex which constantly change in direction, quantity, and location, therefore this study used only one clinical parameter which was static load with constant direction, which have been used in several investigation.7,9,11,23,28,29 three dimensional 3d model is more valid, but it needs more time and cost compared to 2d model.30 although the usage of 2d model is not fully representing the real condition, but the result was representatif enough for certain clinical conditions.31 therefore, further investigation about the usage of this method, in more complex situation imitating the condition in the oral cavity, has to be done. other clinical parameters like periodontal tissue, bone support, and dynamic loading forces have to be involved. within the limitation of these studies, it can be concluded that non-seat design group showed a higher fracture resistance compare to the seat group. no post are bent in seat design, did not prove that the design was good. possibly it was due to the fracture of the root in advance, even before the post bent and came out. non-seat design can distribute the stress more evenly than seat design. the more tooth structure remains in the cervical area, the better the fracture resistance of the tooth restored with cast post. this finite element method study confirm the result of the conventional study, therefore it can be used as an alternative method in studying the structures and designs of material used in dentistry. acknowledgement we wish to express our sincerest thanks to the chiefs of laboratories metallurgy pt pindad and laboratorium struktur ringan institut teknologi bandung who are permitting us to use their research facilities. our thanks are also due to rikfi kania and aldilla miranda, our intelligent students, for their technical assistance in preparing the work. special thanks are extended to program hibah kompetisi a2 fkg unpad for funding part of this work. references 1. subrata g. penggunaan finite element analysis dalam penelitian di bidang kedokteran gigi. kumpulan makalah pertemuan ilmiah ilmu kedokteran gigi iprosi i, bandung; 2007. p. 192–200. 2. dejak b. mlotkowski a, romanowicz m. finite element analysis of stress in molars during clenching and mastication. j prosthet dentj prosthet dent 2003; 90(6): 591–7.. 3. widas p. introduction to finite element analysis. available at: http:// digilib.itb.ac.id/gdl.php?mod=browse&node=2604. accessed april 10, 2007. 184 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 179-184 4. roensch sj. finite element method: a four-article series. 2006: available at http://www.wikipedia.com. accessed january 20, 2008. 5. siregar na. uji komposisi, kekuatan tarik dan modulus elastisitas logam paduan orden sebagai bahan pasak cor. skripsi. bandung: fkg unpad; 2008. p. 45. 6. martanto p. teori dan praktek ilmu mahkota dan jembatan jilid ii. 1st ed. bandung: penerbit alumni; 1982. p. 40–58. 7. pereira jr, de omelas f, conti pcr, do valle al. effect of a crown ferrule on the fracture resistance of endodontically treated teeth restored with prefabricated posts. j prosthet dent 1995; 95:50-4. 8. shillingburg h.t, hobo s, whitsett ld, jacobi r, brackett se. fundamental of fixed prosthodontics. 33th ed. chicago: quintessence publishing co, inc; 1997. p. 335–418. 9. zhi-yue l, yu xing z. effects of post core design and ferrule on fracture resistance of endodontically treated mallary central incisors. j prosthet dent 2003; 89: 368–373. 10. cheung w. a review of the management of endodontically treated teeth. j am dent 2005; 136: 611-8. 11. tan plb, aquilino sa, gratton dg, syanford cm, tan sc, johnson wt, dawson d. in vitro fracture resistance of endodontically treated central incisors with varying ferrule heights and configurations. j prosthet dent 2005; 93: 331–6. 12. weine fs. endodontic therapy. 6th ed. st. louis: mosby co; 2004. p. 546–62. 13. pramanik rk. uji ketahanan fraktur akar dan ketahanan pasak cor terhadap bengkok dan lepas pada preparasi saluran akar menggunakan seat dan tanpa seat. skripsi. bandung: fkg unpad; 2007. p. 11,skripsi. bandung: fkg unpad; 2007. p. 11, 35–41. 14. miranda a. analisis distribusi tegangan desain preparasi saluran pasak cor seat dan non-seat pada gigi insisivus pertama atas menggunakan metode elemen hingga. skripsi. bandung: fkg unpad; 2008. p. 39–45. 15. kutesa-mutebi a, osman y. effect of the ferrule on fracture resistance of teeth restored with prefabricated posts and composite cores. african health sci 2004; 4(2): 131–5. 16. logan dl. a first course in the finite element method. 3rd ed. usa: wadsworth group; 2002. p. 1–21. 17. febrinaldy f. finite elements model and verifications using msc patran/nastran. bandung: creative create corp; 2007. p. 3–37. 18. roensch sj. finite element analysis. available at: www.finiteelement. com/fea.html. accessed april 10, 2007. 19. ash mm. wheeler’s dental anatomy, physiology & occlusion. philadelpia: wb saunders co; 1993. p. 130. 20. sertgoz a. finite element analysis study of the effect of superstructure material on stress distribution in an implant-supported fixed prosthesis. int j prosthodont 1997; 10(1): 19–27. 21. seymour kg, cerukara gp, samarawickrama dyd. stress within porcelain veneers and the composite lute using different preparation designs. j prosthodont 2001; 10(1): 16–21. 22. o’brien wj. dental materials and their selection. 3rd ed. chicago: quintessence publishing co inc; 2002. p. 326–72. 23. dikbas i, tanalp j, ozel e, koksal t, ersoy m. evaluation of the effect of different ferrule designs on the fracture resistance of endodontically treated maxillary central incisors in incorporating fiber posts, composite cores and crown restorations. j contemp dent pract 2007; 8(7): 1–10. 24. roberson tm, heyman ho, swift ej. indirect restorative dental materials. in: sturdevant’s art & science of operative dentistry. 4th ed. st. louis: cv mosby; 2002. p. 214–6. 25. gutmann jl, lovdahl pe. problem solving in endodontic prevention, identification and management. missouri: mosby co; 1997. p. 333. 26. ferrari m, scott, r. fiber posts. milano: masson; 2002. p. 7–13, 39–49. 27. o’sullivan m. fixed prosthodontics in dental practice. london: quintessence publishing co, inc; 2005. p. 139–52. 28. eskitaciolu g, belli s, kalkan m. evaluation of two post-coreevaluation of two post-core system using two different methods (fracture strength test and a finite elemental stress analysis). j endod 2002; 28(9): 629–33. 29. martínez-insua a, da silva l, rilo b, santana u. comparison of the fracture resistance of pulpless teeth restored with a cast post and core or carbon-fiber post with a composite core. j prosthet dent 1998; 80(5): 527–32. 30. asmussen e, peutzfeldt a, sahafi a. finite element analysis of stresses in endodontically treated, dowel-restored teeth. j prosthet dent 2005; 94(4): 321–9. 31. yang hs, lang la, felton da. finite element stress analysis on the effect of splinting in fixed partial dentures. j prosthet dent 1999; 81(6): 721–8. 133133 dental journal (majalah kedokteran gigi) 2019 september; 52(3): 133–137 research report hyperplasia of wistar rat tongue mucosa due to exposure to cigarette side-stream smoke nurina febriyanti ayuningtyas,1 grahania octaviono mahardika,1 bagus soebadi,1 adiastuti endah parmadiati,1 saka winias,1 hening tuti hendarti1 and rosnah binti zain2 1 department of oral medicine, faculty of dental medicine airlangga university, surabaya – indonesia 2 department of oral pathology an oral medicine, faculty of dentistry, mahsa university, bandar saujana putra – malaysia abstract background: hyperplasia, a condition whereby an increasing number of cells are produced due to their uncontrolled division, represents a common symptom of carcinogenesis. cancer is a physical manifestation of cell malignancy resulting from abnormal proliferation. globally, oral cancer currently constitutes the sixth largest lethal form of the condition. the most common etiology of oral cancer is tobacco of which cigarettes are the most popular related product. the health risks associated with cigarette smoke not only affect active smokers but also individuals who ingest it passively. sidestream smoke comes from the lighted end of a burning tobacco product such as a cigarette, pipe or cigar and contains nicotine and many harmful cancer-causing chemicals. inhaling sidestream smoke increases the risk of lung and other types of cancer. purpose: the purpose of this study was to understand how sidestream cigarette smoke initiates precancerous changes, in this case hyperplasia, in the oral mucosa epithelium of wistar rats. methods: the subjects were divided into three groups, a 4-week treatment group (p1), an 8-week treatment group (p2), and a control group (k), each consisting of ten subjects. the subjects were exposed to a daily two-cigarette dose of smoke. the experiment used a post-test only control group design. all samples were sacrificed during the fourth and eighth weeks. haematoxylin-eosin staining was performed on the tongues of the wistar rats to establish the presence of hyperplasia. data was analyzed using a one-way anova test. results: after the wistar rats had been exposed to cigarette smoke, an increased degree of epithelial cell proliferation (hyperplasia) showed a significant difference with a p-value <0.05 during the eighth week. conclusion: exposure to cigarette sidestream smoke induces increased epithelial cell proliferation (hyperplasia) in wistar rats. keywords: cigarette smoke; hyperplasia; oral cancer correspondence: nurina febriyanti ayuningtyas, department of oral medicine, faculty of dental medicine, airlangga university, jalan mayjen. prof. dr. moestopo 47, surabaya 60132. e-mail: nurina.ayoe@gmail.com introduction body cells undergo a process of proliferation and growth constituting a well-regulated gene regulation phenomenon. when damage occurs, cell growth increases as part of the body’s defense mechanism. the cells surrounding those damaged accelerate their growth to restore normal tissue structure and function. after this has occurred, the tissue cells will die through a process known as apoptosis. the body will subsequently produce replacement cells for those which have died.1 the process of cell progression and apoptosis does not invariably progress in a uniform manner due to certain risk factors such as stress, chemicals, toxins, bacteria, viruses, parasites, fungi, and genetics. however, cells can survive through a mechanism of adaptation which may occur due to atrophy, hypertrophy, hyperplasia, dysplasia, and metaplasia.1–5 hyperplasia, also defined as the abnormal proliferation of cells resulting from continuous multiplication1, can be regarded as a symptom of the onset of carcinogenesis. cancer represents a form of malignancy involving abnormal cell proliferation with head and neck cancer constituting the sixth most common form in humans. 48% of cases are located in the oral cavity, with 90% of these being oral squamous cell carcinomas (osccs). the common etiology of oscc is tobacco use in its various forms (22%).6–9 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p133–137 mailto:nurina.ayoe@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p133-137 134 ayuningtyas, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 133–137 figure 1. the process of handling experiment subjects exposed to cigarette smoke by means of a full-body exposure device. a length=64.384µm length=178.042µm b length=68.970 µm length=208.938µm length=77.971µm length=233.308µm c figure 2. histopathological features of rat tongue mucosa (one field of view). (a) the thickness of the epithelium and stratum corneum in the k group; (b) epithelial and stratum corneum thickening in the p1 group; (c) thickening of the epithelium and stratum corneum in the p2 group (at 1000x magnification). *the yellow line indicates hyperkeratosis, while the red line denotes hyperplasia. table 1. average and standard deviations of increased thickness of the epithelium and stratum corneum layer k (μm) p1 (μm) p2 (μm) epithelium 179.9±23.15 206.02±28.55 231.46±22.79 stratum corneum 64.38±11.33 68.97±8.94 77.97±9.48 the inhaling of second-hand cigarette smoke by nonsmokers is termed passive smoking. for passive smokers, the risk of oral cancer increases by 87% compared to that for non-smokers who are not exposed to cigarette smoke.8–11 if the source of smoke inhaled is the cigarette itself then the smoke is called side stream smoke, while the smoke exhaled by the smoker is termed exhaled mainstream smoke.6,7,12,13 cigarette smoke contains approximately 60 carcinogenic substances. the processes induced by cigarette smoke which have been identified as causing cancer are those of free radical metabolism and dna damage within the human body which induces gene mutation. in the oscc, a premalignant lesion develops in the upper aerodigestive tract (uadt). such lesions experience an increase in epithelial proliferation that can be interpreted as a symptom of the early onset of oscc.5 the purpose of this study was to comprehend the manner in which side-stream cigarette smoke triggers precancerous changes, in this case hyperplasia, in the oral mucosa epithelium of wistar rats. materials and methods this research was received ethical approval from the ethics committee of the faculty of dental medicine, universitas airlangga, with number 175/hrecc.fodm/ix/2017. this research represented a laboratory experimental research with post-test only control group design and used a sample of 30 male wistar rats (rattus novergicus), aged three months, with a body weight of 170 grams (± 10%). the animals were randomly divided into three groups, consisted of two treatment groups (p1 group treated for four weeks and p2 group treated for eight weeks), and one control group (k group), each group consisting of ten rats. the research material consisted of clove cigarettes. exposure to their smoke was effected using a device referred to as a smoking pump. each subject underwent a single daily exposure to the smoke derived from a maximum of two cigarettes.14 this level of exposure was maintained up to the time at which the subject was sacrificed. the control group subjects were placed in separate tubes to ensure their simultaneous exposure to the air. in order to ensure that the difference in smoke exposure between the tubes in the smoking pump was consistent, the placement of subjects in each tube was rotated (figure 1). at the end of the fourth and eighth weeks, each subject was sacrificed, its tongue being removed and subjected to histopathological examination by means of hematoxylineosin (he) staining. microscopic assessment comparing the k, p1, and p2 groups was conducted with a light microscope at 1000x magnification across five random visual fields. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p133-137 135ayuningtyas, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 133–137 a kolmogorov-smirnov test was performed to discover whether the data obtained was normally distributed (value p>0.05). the data of the research treatment groups was then subjected to a levene’s homogeneity test followed by a one-way anova test. normally distributed data subsequently underwent a tukey hsd to determine the differences between groups (value p<0.05). results the contents of table 1 show that an increase in the thickness of the epithelium and stratum corneum of the subject’s tongue occurred. increases in the epithelial and stratum corneum thickness were observed in the k group compared to the p1 and the p2 groups. thickening of the epithelium indicated hyperplasia, while stratum corneum thickness constituted a symptom of hyperkeratosis. the tukey hsd test result indicated a significant increase in terms of hyperplasia in the k group compared to the p2 group with a p-value of 0.000 (p<0.05). meanwhile, the increases in hyperplasia in the k group compared to the p1 group and in the p1 group compared to the p2 group as indicated by p-values were 0.067 and 0.076 respectively. in terms of hyperkeratosis, there was significant difference in the k group compared to the p2 group with a p value of p=0.014 (p<0.05). with regard to hyperplasia, a significant increase occurred in the k group in comparison to the p1 and in the p1 group compared to the p2 group as indicated by their respective p-values of p=0.566 and p=0.127. figure 2 shows the histopathological features of the tongue mucosa of the subjects. from the one representative field of view, it can be observed that there is increased hyperplasia and hyperkeratosis in the k group compared to the p groups. discussion cancer constitutes a form of cell malignancy in which proliferation occurring through the process of carcinogenesis is uncontrolled. the early stage of the condition is characterized by hyperplasia which is often accompanied by hyperkeratosis. oral cancer is the sixth most common fatal form of the condition in the world. its most common etiology consists of tobacco and cigarettes, products most commonly found in the community environment.1,3–5 the study conducted was based on a simple hypothesis about the potential for hyperplasia to occur in wistar rats exposed to cigarette smoke over a certain period of time. in this study, male wistar rats (rattus norvegicus) were used due to their being unaffected by hormonal conditions. their body weight was maintained at an ideal level of 170 grams (± 10%) during a week-long process of adaptation. wistar rats were selected because of the similarity of their immune system, oral mucosa, and lymphatic drainage system with that of humans. their response to tumor antigens with t-lymphocytes is also comparable. the oral epithelium on the inferior tongue surface of rats is much thinner (8-12 layers) than that of humans (20-30 layers) with the same degree of rete ridge.3,15 previous research into cigarette smoke involving the use of wistar rats has been carried out using three treatment methods, namely; inhalation exposure via the respiratory tract, whole body exposure, and nicotine injection. nasal inhalation methods require oxygen masks, but these may be easily damaged by rats which belong to the rodent species. moreover, the hyperplasia histology involving nasal inhalation method results are not significant. based on these considerations, most researchers opt for the wholebody exposure method which provides sufficient space for the subject during treatment. however, the exposure dose of cigarette smoke is not administered via the oral cavity but, rather, throughout the entire body. nevertheless, the histology results produced by the later method remain significant. accordingly, this study adopted the whole-body exposure method with certain modification.16 the research material selected for this study consisted of clove cigarettes on the basis of their nicotine level being twice as high as the standard cigarettes commonly consumed by the public. the nicotine content of the latter is 1.1 mg, while that of clove cigarettes, which contain 60% tobacco and 40% cloves, amounts to 2 mg. there are several different ingredients of such cigarettes compared to white varieties. clove cigarettes contain five additional ingredients, namely; eugenol, acetyl eugenol, β-caryophyllene, α-humulene, and caryophyllene epoxide.6,16,17 in a previous study, the oral cavity of in vivo male wistar rats exposed to cigarette smoke for 60 days presented premalignant lesions. due to the absence of p53 protein expression, theis time period proved of insufficient duration to cause cancer. however, a ki-67 analysis showed an increase in epithelial proliferation resulting from the damage response.16 therefore, the researchers decided to adopt point of exposures of 4 and 8 weeks duration. the tongues of wistar rats were selected because previous studies had confirmed these areas to have frequently presented significant symptoms of hyperplasia and hyperkeratosis. moreover, 40% of oscc cases affect the tongue. the three groups presented differences in the effect of cigarette smoke on the thickness of the epithelium and stratum corneum.3,16,18 based on the results of histopathological examination of the k, p1, and p2 groups, a significant difference was identified in the epithelial thickness of the epithelium and stratum corneum. as indicated by the contents of table 1, the k group which was not exposed to cigarette smoke presented an average hyperplasia value of 179.89μm and an average hyperkeratosis value of 64.38μm. when compared with the p1 and p2 groups, the k group was the thinnest, while the p2 group was the thickest. the p1 group recorded a hyperplasia value as high as 206.01μm and a hyperkeratosis value of 68.97μm, while the p2 group registered hyperplasia dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p133-137 136 ayuningtyas, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 133–137 and hyperkeratosis values of 231.46μm and 77.97μm, respectively. based on these results, it can be argued that clove cigarette smoke affects the proliferation occurring in the epithelium and stratum corneum. when the oral mucosal epithelium is exposed continuously to clove cigarette smoke its structure and function may be negatively impacted. the nicotine in cigarette smoke contains tobacco-specific n-nitrosamines (tsna) derivatives which, in turn, contain carcinogen substances in the form of 4-(methylnitrosamino )-1-( 3-pyridyl )-1-butanone (nnk) and n-nitrosonornicotine (nnn). cigarette smoke molecules can induce epidermal growth factor receptor (egfr) phosphorylation which mediates cytoplasmic tail (ct) mucin-1 (muc1) phosphorylation. this, in turn, induces the cleavage of β-catenin from e-cadherin to form the β-catenin/muc1-ct complex of the wnt/β-catenin canonical pathway. the resulting complex translocates from the cytosol to the nucleus and joins the family of t-cell factor/lymphoid enhancer-binding factor (tcf/lef) and transcription factors leading to the transactivation of genes that drive cell cycles (e,g, c-myc, cyclin-d).19–21 egfr, which continuously binds to the molecular components of cigarette smoke, causes an increase in epithelial cell proliferation and decreased attachment between cells which will subsequently experience abnormal migration. this occurs because the activation of egfr by cigarette smoke causes the loss of the e-cadherin/βcatenin complex which mediates adherence between cells. decreased levels of e-cadherin cause metastatic cancer. thus, cigarette smoke can increase epithelial proliferation and migration through the activation of egfr and reduction of e-cadherin leading to the development of cancer.19,21 cigarette smoke also contains free radicals such as reactive oxygen species (ros) and reactive nitrogen species (rns) which increase oxidative stress in the body. excessive oxidative cells can trigger epithelial cell activity through various signaling pathways, among others erk1/2, p38, jnk, and nf-κβ. the pathway will lead to the secretion of proinflammatory cytokines. exposure to cigarette smoke will also stimulate the expression of ki67 in the mucosal tissues of the oral cavity. ki67 is a protein crucial to cell cycle progression an increase of which leads to higher cell proliferation. judging from the level of cell proliferation following exposure to cigarette smoke, a mechanism of resistance to apoptosis operates. oral epithelial cells repeatedly exposed to cigarette smoke show a decrease in bax expression (proapoptotic protein) and an increase in bcl-2 (antiapoptotic protein).22–26 exposure to cigarette smoke that modulates epithelial cell proliferation may have an effect on keratin protein expression. cigarette smoke reduces the expression of keratin1, keratin5, keratin10, keratin16 proteins and stimulates that of their keratin6 and keratin14 counterparts. cigarette smoke that modulates gene expression related to keratin is often associated with protein production. increasing keratin14 protein, indicates an increase in stratum basal cell proliferation. conversely, a decrease in keratin10 expression due to cigarette smoke which can increase cell proliferation demonstrates the keratin10 function as a negative modulator in the cell progression cycle.18,25 the disrupted cell cycle and cytoskeleton protein result in the abnormal orientation of basal and suprabasal epithelial cells which experience increased proliferation and migration. continuously activated egfr will result in greater uncontrolled proliferation and migration of cells without being offset by apoptosis. consequently, cells will metastasize and become malignant. the process of proliferation enhancement that occurs is one of pathological hyperplasia and the subsequent involvement of keratin cytoskeleton protein facilitates hyperkeratosis.25,26 the research results reported here were in line with those of previous studies. exposing mice to cigarette smoke for one month, a period considered to constitute longlasting, chronic exposure, may induce a thickening of the airway epithelium as the body’s defenses against cigarette smoke compounds intensify.21 against this background, the ratio of k to p1 and of p1 to p2 become insignificant in the course of 28 days because of the ability of the subjects to tolerate cigarette smoke compounds. however, a significant comparison between the k and p2 groups occurred because the p2 group demonstrated mucosal defense against cigarette smoke and simultaneously adapted to exposure to it over a period of eight weeks. this was due to proliferation enhancement resulting from a combination of pathological hyperplasia and hyperkeratosis. thus, the statistical results confirmed that the k group showed significant differences to the p2 group, while the k group showed no significant difference to the p1 group and the p1 group showed no significant difference to the p2 group. since hyperplasia is a reversible condition, further experiments are required to observe any histopathological changes due to extended exposure to sidestream cigarette smoke which support a hypothesis of hyperplasia, characterized by increased proliferation of cells resulting from chronic irritation as a symptom of the onset of carcinogenesis.1,3–5,26 taken together, it can be concluded that sidestream cigarette smoke exposure induces chronic irritation which may lead to prolonged inflammation and precancerous changes in the oral mucosa of the tongue of a wistar rat, thereby inducing a response of pathological hyperplasia together with hyperkeratosis. references 1. bisen ps, khan z, bundela s. biology of oral cancer: key apoptosis regulators. boca raton: crc press; 2014. p. 1–6, 21–22, 37–46, 49–195. 2. sembulingam k, sembulingam p. essential of medical physiology. 6th ed. new delhi: jaypee brothers medical publishers; 2012. p. 20, 351–6. 3. tanaka t, ishigamori r. understanding carcinogenesis for fighting oral cancer. j oncol. 2011; 2011: 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p133-137 137ayuningtyas, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 133–137 4. watanabe n, ohkubo t, shimizu m, tanaka t. preneoplasia and carcinogenesis of the oral cavity. oncol discov. 2015; 3(1): 1–12. 5. kuriakose ma. contemporary oral oncology. new york: springer; 2016. p. 31–6. 6. behera sn, xian h, balasubramanian r. human health risk associated with exposure to toxic elements in mainstream and sidestream cigarette smoke. sci total environ. 2014; 472: 947–56. 7. fujimoto h, tsuji h, okubo c, fukuda i, nishino t, lee km, renne r, yoshimura h. biological responses in rats exposed to mainstream smoke from a heated cigarette compared to a conventional reference cigarette. inhal toxicol. 2015; 27(4): 224–36. 8. kusuma arp. pengaruh merokok terhadap kesehatan gigi dan rongga mulut. maj ilm sultan agung. 2011; 49(124): 1–8. 9. oral health foundation. mouth cancer risk factors. available from: https://www.dentalhealth.org/mouth-cancer-risk-factors. accessed 2018 aug 25. 10. glick m. burket’s oral medicine. sheffield: people’s medical publishing house; 2015. p. 173–201. 11. arifa beegom a. passive smoking and oral cancer risk: a case report. kerala med j. 2014; 7(3): 74–8. 12. ibuki y, toyooka t, zhao x, yoshida i. cigarette sidestream smoke induces histone h3 phosphorylation via jnk and pi3k/akt pathways, leading to the expression of proto-oncogenes. carcinogenesis. 2014; 35(6): 1228–37. 13. travers m, nayak n, annigeri v, billava n. indoor air quality due to secondhand smoke: signals from selected hospitality locations in rural and urban areas of bangalore and dharwad districts in karnataka, india. indian j cancer. 2015; 52(4): 708–13. 14. teague s v., pinkerton ke, goldsmith m, gebremichael a, chang s, jenkins ra, moneyhun jh. sidestream cigarette smoke generation and exposure system for environmental tobacco smoke studies. inhal toxicol. 1994; 6(1): 79–93. 15. thirion-delalande c, gervais f. comparative analysis of the oral mucosae from rodents and non-rodents: application to the nonclinical evaluation of sublingual immunotherapy products. plos one. 2017; 12(9): 1–18. 16. de oliveira semenzati g, de souza salgado b, rocha ns, michelin matheus sm, de carvalho lr, garcia martins rh. histological and immunohistochemical study of the expression of p53 and ki-67 proteins in the mucosa of the tongue, pharynx and larynx of rats exposed to cigarette smoke. inhal toxicol. 2012; 24(11): 723–31. 17. husein a. penga r uh rokok terhadap peningkatan frekuensi pembentukan mikronukleus pada mukosa mulut. thesis. universitas diponegoro: semarang; 2013. p. 8–15. 18. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. université laval: québec; 2015. p. 22–24, 42. 19. wee p, wang z. epidermal growth factor receptor cell proliferation signaling pathways. cancers (basel). 2017; 9(5): 1–45. 20. ackers i, malgor r. interrelationship of canonical and non-canonical wnt signalling pathways in chronic metabolic diseases. diabetes vasc dis res. 2018; 15(1): 3–13. 21. chen yt, gallup m, nikulina k, lazarev s, zlock l, finkbeiner w, mcnamara n. cigarette smoke induces epidermal growth factor receptor-dependent redistribution of apical muc1 and junctional β-catenin in polarized human airway epithelial cells. am j pathol. 2010; 177(3): 1255–64. 22. dwivedi n, chandra s, kashyap b, raj v, agarwal a. suprabasal expression of ki-67 as a marker for the severity of oral epithelial dysplasia and oral squamous cell carcinoma. contemp clin dent. 2013; 4(1): 7–12. 23. lee j, taneja v, vassallo r. cigarette smoking and inflammation: cellular and molecular mechanisms. j dent res. 2012; 91(2): 142–9. 24. fitria f, triandini r, mangimbulude jc, karwur ff. merokok dan oksidasi dna. sains med. 2014; 5(2): 121–7. 25. alharbi ia, rouabhia m. repeated exposure to whole cigarette smoke promotes primary human gingival epithelial cell growth and modulates keratin expression. j periodontal res. 2016; 51(5): 630–8. 26. geng h, zhao l, liang z, zhang z, xie d, bi l, wang y, zhang t, cheng l, yu d, zhong c. cigarette smoke extract-induced proliferation of normal human urothelial cells via the mapk/ap-1 pathway. oncol lett. 2017; 13(1): 469–75. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p133–137 https://www.dentalhealth.org/mouth-cancer-risk-factors http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p133-137 vol 51 no 4 okt-des 2018.indd 173 a comparison between orthodontic model analysis using conventional methods and imodelanalysis vita previa indirayana, gita gayatri, and n. r. yuliawati zenab departement of orthodontics faculty of dentistry, universitas padjadjaran bandung – indonesia abstract background: model analysis constitutes an essential aspect of orthodontic diagnostic practice. pavan has developed an application to simplify the mathematical calculations employed in orthodontic model analysis. purpose: this study was conducted to obtain the differences in results and time periods of model analysis using conventional means and imodelanalysis. methods: the research represented a comparative analytic study. the populations comprised dental casts dating from 2014 in the orthodontics laboratory of padjadjaran university. the samples comprised 31 dental casts which were subjected to a total sampling method consisting of two treatments; a conventional method calculation and one using imodelanalysis. a normality test was conducted and processed using a paired t-test with α=0.05. results: the means of arch length discrepancies were 1.64±2.63 mm and 1.37±3.07 mm for the conventional methods and 1.65±2.43mm and 1.42±3.04mm for imodelanalysis. the results of a bolton analysis for conventional methods were 78.05±2.69% and 91.93±1.29%, while those for imodelanalysis were 77.91±2.70% and 91.96±2.13%. a howes analysis of conventional methods produced a result of 45.56±2.83%, while for an imodelanalysis one of 45.56±2.85%. pont analysis for conventional methods was 39.35±0.04 mm and 49.17±2.55 mm, while for imodelanalysis it was 39.35±0.07 mm and 49.19±2.57mm. the mean of the duration of analysis using conventional methods was 1703.81±56.46 seconds, while for imodelanalysis it was 990.06±34.87 seconds. a normality test confirmed that the data was normally distributed (p>0.05). the results of a paired sample t-test with p>0.05 showed that there was no significant difference between the results of each analysis, while there was significant difference in the time period of analysis. conclusion: there was no difference in the analysis results. however, there was difference in the time period of analysis between conventional methods and that of imodelanalysis. keywords: conventional; imodelanalysis; result of analysis; time period of analysis correspondence: vita previa indirayana, department of orthodontics, faculty of dentistry, universitas padjadjaran, no.1 jl. sekeloa selatan. bandung 40132, indonesia. e-mail: vita14003@mail.unpad.ac.id introduction study model analysis has been the gold standard for diagnostic procedures and dental treatment for many years. various methods have been used for measuring and analyzing plaster models as study models, including calipers, rulers and other measuring tools. the data from the measurement was subsequently calculated to relevant formulas to produce the results of the analysis.1 in the modern era, the use of electronic devices such as smartphones and tablets, often referred to as gadgets, is increasing because they are extremely portable. this consistent development has also been observed by healthcare practitioners active in the orthodontic field. many applications for tooth ratio calculations within model analysis are available on google play store for android and apple’s app store for ios to facilitate treatment for both dentists and patients.2,3 over the last ten years, dental technology has developed considerably in the area of model analysis, for example in diagnosis using a digital model. experts are developing computerbased analyzes that can simplify the work of dentists. computer-based analyzes were employed to scan the model for analysis, but not to measure it. although they may facilitate the practice of dentists, their use is rare because dental journal (majalah kedokteran gigi) 2018 december; 51(4): 173–178 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 174indirayana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 173–178 the devices are limited from the perspective of obtaining accurate results. such devices for digital modelling are still being developed in order to obtain more accurate results and are only produced in limited numbers because of the difficulty and cost of doing so.4 analysis of the study model was not only performed using conventional methods or computers utilising expensive digital models, but also by means of the applications available to dentists on smartphones or other portable electronic devices. one such application for performing model analysis available through google play store and apple’s app store is imodelanalysis. this downloadable application is available free of charge and facilitates mathematical calculations of model study analysis.5 according to mamillapalli et al. (2012) who were its creators, imodelanalysis performs mathematical calculations easily and accurately as part of model study analysis. model analysis employing the conventional method is a relatively time-consuming process so the imodelanalysis application is expected to be more efficient than conventional methods.5 the purpose of this study was to obtain the differences in results and time periods of models analysis using conventional and imodelanalysis. materials and methods before conducting the research, the authors applied for a permit to conduct research at the orthodontics laboratory of padjadjaran university and a letter of approval from the ethics committee. these documents were required since the research employed dental casts constituting the personal data of patients. the letter of ethical exemption no:1248/un6.c.10/pn/2017 contained the registration number 0217121360. this research was comparative analytical in nature and conducted to identify any differences in terms of duration and results between model analysis adhering to conventional methods and imodelanalysis application on smartphones. this research was conducted using a conventional study model whose inclusion and exclusion criteria were adopted when samples were collected (figure 1a). the collected study model samples were measured using both conventional and imodelanalysis methods. the inclusion criteria included: the gips model having fully erupted teeth from the first left molar to the first right molar (12 teeth on each jaw), none having been extracted from the 12 teeth in the study model, the study model being in a good condition without defects and the impression of the teeth anatomy being well-defined. the exclusion criteria comprised the study model having caries so severe that the crown structure was missing from the 12 teeth, anomalies in the teeth, and the gips model being fractured, broken or eroded. during the measuring process, random sampling was performed in an effort to reduce the error rate of measurement (error method). based on the results of this study conducted in the laboratory of padjadjaran university on 31 pairs of padjadjaran university dentistry students of the study model class of 2014. measurements were taken once for each analysis by one participant using a conventional method and imodelanalysis, while the other assisted in the preparation of the study. the tools and materials required for this research included study models, pencils or marker pens, calipers, rulers and paper on which to write the measurements taken, android or apple-based smartphones with an imodelanalysis application and a stopwatch. the application named imodelanalysis can be run on androidbased smartphones and ios iphones. the measurements of the model analysis to be performed included an arch length discrepancy (ald) analysis, bolton analysis, howes analysis and a pont analysis. the analysis is frequently conducted in daily dental practice and forms part of dental college syllabi in indonesia. the duration of the count model analysis using conventional methods was recorded with a stopwatch. ald analysis involves measuring the mesiodistal of each tooth with calipers starting from regio 1 on the study model provided and recording the measurements on paper. the teeth measured included 12 maxilla (16 -26) and 12 mandibles (36-46) (figure 1b). the length of the jaw arch was subsequently measured by dividing the jaw into six segments each consisting of two teeth from the first right molar to the left first molar. the length of each segment was measured with the calipers and added together (figure 1c). the results were then calculated by looking at the difference between the number of mesiodistal 12 teeth and the length of the jaw arch.6 bolton analysis was conducted using calipers to measure the mesiodistal of the teeth in the same manner as that used to take ald measurements starting from region 1 in the study model provided. the teeth measured were 12 maxilla (16-26) and 12 mandible (36-46). the measurement data was entered in the formula, the result calculated and then recorded.6 howes analysis measured the mesiodistal of teeth 16-26 in the study model provided. the width of the jaw arch, the apical base diameter and the distance between the deepest point of the right and left right fossa (apex tip of the tooth 14-24) were measured from the forward direction of the tooth model using a caliper (figure 1d), before the length was quantified. the measurement result was entered into the formula available and the result calculated. a record was kept of the analysis results obtained through the application of a howes formula.6 pont analysis measures the mesiodistal of four maxillary anterior teeth in the study model. the premolar region, the distance from the distal pit of upper right and left first premolar on the occlusal surface (figure 1e) and the molar region and the distance from the mesial pit of upper right and left first molar on the occlusal surface (figure 1f) were then measured with calipers. the width of the dental arch in the ideal premolar and molar region was calculated by using the pont formula. the stopwatch was stopped and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 175 indirayana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 173–178 the time required to get the results of the analysis using conventional methods recorded.6 a model analysis using an imodelanalysis commenced with the recording of the time using a stopwatch. this model analysis does not need to include measurement results in the analytical formula as it features an automated system for calculating the analysis. firstly, the mesiodistal of each tooth starting from 12 teeth of the maxilla (1626) and 12 teeth of the mandible (36-46) was measured using calipers. the measurements were inputted in the imodelanalysis application to process the results of the analysis (figure 2).5 the ald calculation results were obtained after measuring the amount of available space in the jaw or the length of the jaw arch by means of an imodelanalysis. bolton’s analysis of the results will be obtained after the data relating to the 12 teeth of the maxilla and the 12 teeth of the mandible are inputed into the imodelanalysis. a howes analysis measured the distance between the deepest point of the right and left right fossa (apex tip of teeth 14-24) and the distance between the buccal tops of teeth 14-24 measured from the occlusal direction. the pont analysis calculation was performed by including the width of the first upper premolar (14 to 24) in the distal pit and the width between the maxillary first molars (16 to 26) in the mesial pit region. the stopwatch was stopped and the time required to obtain the results of the analysis using imodelanalysis recorded. all data obtained was subsequently subjected to normality and paired t-test tests using “statistical package for the social sciences of international business machines” or a ibm spss statistics version 20 program developed at the international business machine corperation (ibm corperation) office new york, usa in 2016. a kolmogorov smirnov normality test with a significance level equal to 0.05 with p>0.05. the data was normally distributed and homogen with p>0.05 leading to the conducting of a parameter test, specifically a paired t-test, intended to determine whether the mean value of the data was statistically different. figure 1. a) one of the study models for this research, b) the conventional method of measuring tooth dimensions c) measuring a segment of the arch during ald analysis, d) measuring the apical base for howes analysis, e) measuring the premolar regio during pont analysis and f) measuring the molar regio during pont analysis figure 2. the measurement of the model study inputed in imodelanalysis application and the orthodontic model analysis in imodelanalysis. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 176indirayana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 173–178 table 1. the mean of all analysis and time period between conventional method and imodelanalysis on the smartphone. results mean ± sd imodelanalysisconventional ald analysis in maxilla 1.652 ± 2.432 mm1.635 ± 2.636 mm ald analysis in mandible 1.416 ± 3.046 mm1.374 ± 3.073 mm bolton analysis in anterior ratio 77.910 ± 2.706%78.048 ± 2.698 % bolton analysis in total ratio 91.961 ± 2.135%91.929 ± 1.297 % 45.561 ± 2.853%45.558 ± 2.839 %howes analysis pont analysis in premolar 39.348 ± 0.071mm39.345 ± 0.045 mm pont analysis in molar 49.190± 2.572 mm49.174 ± 2.557 mm time period of the analysis 990.06 ± 34.870 s1703.81 ± 56.464 s table 2. normality test results imodelanalysisconventionalkolmogorov smirnov z sig. of ald analysis in maxilla 0.2350.331 sig. of ald analysis in madible 0.4380.388 sig. of bolton analysis in anterior ratio 0.2240.492 sig. of bolton analysis in anterior ratio 0.9020.478 0.9990.986sig. of howes analysis sig. of pont analysis in premolar regio 0.6800.629 sig. of pont analysis in molar regio 0.7770.567 sig. of time period of the analysis 0.8980.951 sig: probability sig p > 0.05 table 3. the results of paired data t-test. df sig. 2 tailed t tab.t hit. 2.045-0.5250.60429ald analysis in maxilla ald analysis in mandible 2.045-0.7870.43429 bolton analysis in anterior ratio 2.0451.1250.26929 bolton analysis in total ratio 2.045-0.1990.84329 2.0451.9620.05929howes analysis pont analysis in premolar regio 2.045-0.1120.91129 pont analysis in molar regio 2.0450.0620.95029 2.04566.6390.000*29time period of analysis *significant if p < 0.05 df = degree of freedom sig. 2 tailed = the probability of paired data t-test t hit = t count t tab. = t table sig p > 0.05 results all data obtained from the research confirmed its normal distribution with p>0.05 indicating that it was spread evenly when the amount of data above and below the mean or average was equal. once the average difference of two groups within the same sample had been established, a t-test of paired data with a significance level of 0.05 with p>0.05 was conducted. objectively, measuring the results of analysis did not show any significant differences, while for the period of analysis there was a significant difference between conventional methods and imodelanalysis (table 1). data probability values were, above all, more than 0.05 (p>0.05), signifying that the data was evenly spread and that the existing sample can represent the actual population (table 2). after confirming that the data was normally distributed (p>0.05), a parameter test was performed using a paired sample t-test. the results of a paired sample t-test with p>0.05 showed that all analyses indicated no significant difference of results between conventional methods and an imodelanalysis. in contrast, there was a significant period of analysis between conventional methods and imodelanalysis where the working time of the latter was shorter (table 3). discussion model analysis is an important step because it is one source of information in conducting an orthodontic diagnosis. a complete, clear and accurate diagnosis will determine the comprehensiveness of the treatment plan capable of maximizing the success of the orthodontic treatment undertaken. in addition to the study model, the analysis also utilizes other tools, such as measuring tools, radiographic features and approximate tables. analysis can be performed either manually or using a digital system each of which has both advantages and disadvantages. analyses of the study model varied, but one was selected according to its applicability to the experiences of the patient.6 the results of the analysis model showed no significant difference between the conventional and imodelanalysis methods. the average results of the bolton analysis conducted for this study showed an ideal overbite and overjet relationship which optimised the anterior ratio and total ratio. this result was related to the statement from premkumar that the ideal values are 77.2% ± 1.65 in anterior ratios and 91.3% ± 1.91 in the total ratio.7 other devices dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 177 indirayana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 173–178 that can be used to accurately conduct a pont analysis in addition to an imodelanalysis are the laser assembly scanners at the department of orthodontics of the faculty of dentistry at the university of indonesia and at the school of electrical engineering and informatics at the bandung institute of technology used for the measurement and analysis of the upper transverse dental arch using a pont analysis 3-dimensional digital model study case of medium to heavy dental teeth.8 the results of this study matched the opinion of singh (2013) who was seeking a wide range of applications available to practitioners and orthodontic patients on four types of smartphones. the search results confirmed the existence of 32 orthodontics-related applications which can be downloaded from android and 57 applications that can be downloaded from apple. a number of these applications provide invalid and unsupported information, while only some have been recognized such as bolton calc, carriere ortho 3d, faq fix, imodelanalysis which align on time. imodelanalysis was rated at 4.5 out of 5 by users because it provides an easy-to-execute model analysis.3 overall, digital models have been widely used for diagnostic purposes either by using a plaster model or directly involving the patient in question. the respective accuracy of measurements taken using digital and plaster models remains a frequently-researched issue.4 other research provides a systematic review of comparisons between digital model measurements and those taken by measuring instruments on plaster models. seven digital model systems are used in fleming research, namely: orthocad, emodel, c3d-builder, conoprobe, easy3d scan, digimodels and cecile. the results of this study state that “digital models offer the same level of validity as compared with direct measurements on the plaster model, but the quality of the difference in outcomes is clinically acceptable, due to inadequate samples, and standard errors due to different techniques.”9 (2011:14) the results of a hypothesis test comparing all the calculations of a study model analysis of conventional methods with an imodelanalysis on smartphones using paired data t-test showed no statistically significant difference. the main factor in performing model analysis is the different form of measurement. measurement by conventional methods involves the use of calipers and committing the results to paper before the overall amount is quantified. in contrast, imodelanalysis measures all data inputted, produces the measurements directly and then calculates the results. the differences in model analysis using conventional methods should be calculated using the existing formula for each analysis, whereas imodelanalysis-generated calculation results will be produced automatically when the data inputted.4 factors such as the ability and experience of the researcher in performing measurements contribute to the emergence of differences when comparing the two methods of measurement model analysis.10 competent researchers will provide more accurate results compared to their counterparts lacking experience. another factor evident in performing model analysis consists of the tools and methods employed. contemporary highly developed computer-based applications or systems and tools utilize digital tools.10 the digital analysis conducted by wan hassan et al. found no statistically significant differences between methods and operators. bland-altman plots showed that the mean biases were close to zero, while 95% of the limits of agreement were within 0.50 mm.11 leifert also conducted a study using orthocad which compared space analysis results by means of a digital model with conventional gypsum models. this research yielded a slightly significant difference (0.4 mm) in spatial analysis for the maxillary model and no significant difference in the mandibular model between the digital and gypsum models. the accuracy of the digital model produced by orthocad software is clinically acceptable for the evaluation of space analysis.12 one study argued that the digital measurements obtained from study models produced by orthoproof® (cbct-imaging) systems and digimodel software are as accurate as those obtained manually through traditional study models.1 although computerized model analyzes have evolved up to the present, conventional model analysis is still commonly conducted by orthodontic practitioners because it involves the use of simple, accessible and affordable tools such as symmetographs, manual calipers with sharp edges, rulers, digital calipers and a sliding range. data storage systems are generally still performed manually, while the study model is stored in a tailor-made storage facility.13,14 the ever-increasing number of tools and devices devised and developed are expected to be readily applicable to model analysis, providing accurate results. nevertheless, given the proliferation of tools and devices created for model analysis, examination of their accuracy is ongoing.14,15 the times taken to conduct an analysis using each method show that the average period of analysis using the conventional method is 1703.81 seconds or about 28 minutes 24 seconds and the average working time using imodelanalysis is 990.06 seconds or about 16 minutes 30 seconds. the result of a hypothesis test shows that there is a significant difference in the time period of analysis between a conventional method and imodelanalysis whose working time is shorter. the results of this research show no difference in the model analysis calculation result between conventional methods and an imodelanalysis using a smartphone. however, there was a significant difference in the time period of analysis between the conventional method and the smartphone-based imodelanalysis. the data confirmed that this imodelanalysis application provides accurate results more quickly and efficiently. the significant time difference between conventional methods and imodelanalysis can occur because in model analysis, two tasks must be undertaken, namely; measurement and calculation. the conventional method of model analysis dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 178indirayana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 173–178 involves initial measurement followed by calculation of the result through application of the formula for each analysis. since imodelanalysis requires only the measurement results which exit automatically from its system, this results in more rapid processing.4,14 this conclusion matches that of gupta and vaid (2017) which states that of the various existing smartphone applications available to orthodontic practitioners the most appropriate is imodelanalysis because it facilitates the mathematical calculation of results from model analysis and renders research more efficient.2 it concluded that no difference exists between the analysis results of conventional methods and imodelanalysis so that the application of the latter can be used in calculating model analysis and producing a result equal to that of conventional methods. however, the respective duration of analysis in conventional methods and imodelanalysis provides a difference, the time required in for imodelanalysis is much shorter than analysis by conventional methods, rendering it more efficient. recommendations for further research include calculations being produced more than once. undertaking calculations twice or three times in one model analysis should ensure that the results produced are unbiased and more accurate. acknowledgements the authors would like to express gratitude to doctors nina djustiana, azhari and annisa for critism and suggestions of the research also providing guidance on the article, as well as laboratorium preclinic orthodontic dentistry of padjadjaran university for enabling use of the dental casts and facilitating laboratory research. the authors have read and approved the manuscript, take full responsibility for its content and declare no conflict of interest in regard to their research or its funding. references 1. lippold c, kirschneck c, schreiber k, abukiress s, tahvildari a, moiseenko t, danesh g. methodological accuracy of digital and manual model analysis in orthodontics a retrospective clinical study. comput biol med. 2015; 62: 103–9. 2. gupta g, vaid nr. the world of orthodontic apps. apos trends orthod. 2017; 7(2): 73–9. 3. singh p. orthodontic apps for smartphones. j orthod. 2013; 40(3): 249–55. 4. rossini g, parrini s, castroflorio t, deregibus a, debernardi cl. diagnostic accuracy and measurement sensitivity of digital models for orthodontic purposes: a systematic review. am j orthod dentofac orthop. 2016; 149(2): 161–70. 5. mamillapalli pk, neela pk, sesham vm. model analysis on a smartphone. j clin orthodontics. 2012; 46(6): 356–8. 6. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st louis-missouri: mosby elsevier; 2012. p. 247-78. 7. premkumar s. textbook of orthodontics. new delhi: elsevier; 2015. p. 227. 8. laksmihadiati td, ismaniati na, krisnawati. akurasi pengukuran lengkung gigi rahang atas arah transversal hasil pemindaian laser model studi digital 3 dimensi. j pdgi. 2015; 64(2): 116–28. 9. fleming ps, marinho v, johal a. orthodontic measurements on digital study models compared with plaster models: a systematic review. orthod craniofacial res. 2011; 14: 1–16. 10. sousa mvs, vasconcelos ec, janson g, garib d, pinzan a. accuracy and reproducibility of 3-dimensional digital model measurements. am j orthod dentofac orthop. 2012; 142(2): 269–73. 11. wan hassan wn, othman sa, chan cs, ahmad r, ali sna, abd rohim a. assessing agreement in measurements of orthodontic study models: digital caliper on plaster models vs 3-dimensional software on models scanned by structured-light scanner. am j orthod dentofac orthop. 2016; 150(5): 886–95. 12. leifert mf, leifert mm, efstratiadis ss, cangialosi tj. comparison of space analysis evaluations with digital models and plaster dental casts. am j orthod dentofac orthop. 2009; 136: 16.e1-16.e4. 13. phulari bs. orthodontics : principles and practice. new delhi: jaypee brother medical publishers; 2011. p. 172-80. 14. laviana a. analisis model studi, sumber informasi penting bagi diagnosis ortodonti. thesis. bandung: universitas padjadjaran; 2008. p. 1-18. 15. thilander b, bjerklin k, bondemark l. essential orthodontics. hoboken, nj: wiley-blackwell; 2017. p. 89. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p173–178 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p173-178 147 vol. 42. no. 3 july–september 2009 research report the use of holmium-yttrium aluminum garnet laser as pit and fissure cleaner armasastra bahar faculty of dentistry, universitas indonesia jakarta indonesia abstract background: the prevention and management of pit and fissure caries has become relatively more important in recent times. there is a need for an effective preventive measure against pit and fissure caries. purpose: the purpose of this study was to investigate the effect of laser beam as a cleaning method of pits and fissures. methods: ho-yag laser which has a wavelength of 2.1 µm was used in this experiment. the specimens were extracted human teeth. the effect of three cleaning methods was examined comparatively by scoring the cleaned area of fissure, namely laser irradiation with ho-yag laser, chemico-mechanical with combination of 10% naocl and ultrasonic scaler and mechanical with ultrasonic scaler. vertico-bucco-lingual serial ground sections of each tooth were observed under light microscopy. scoring the depth of cleaned area was performed by comparing the depth of fissure. result: progressive result was obtained on the cleaning effect of three methods laser irradiation methods which was the most effective compared to other methods but statistically was not significant. cleaned area of laser irradiation method was 48.91%, chemico-mechanical method was 41.77% and mechanical method was 36.78%. conclusion: holmium -yttrium aluminum garner laser is a relatively new method for pit and fissure cleaning even though the effectivity is not yet maximal. more research is needed to maximize the use of this laser. key words: laser, cleaning methods, pit and fissure correspondence: armasastra bahar, c/o: fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta, indonesia. e-mail: armsbah@hotmail.com introduction the factor concerned in the initial destruction of tooth is a biomechanical process resulting from the activity of microbiological agents existing in the environment concerned. pit and fissure caries is therefore primarily of environmental origin, although its initial progression may be governed by the enamel structure. relative caries incidence is low on smooth, self cleaning surfaces but increase significantly on interproximal and occlusal surface. several investigations have shown that the oral surfaces are especially susceptible to dental decay accounting for nearly 50% of all dental caries. the prevention and management of pit and fissure caries has become relatively more important in recent times. there is a need for an effective preventive measure against pit and fissure caries, which now account for more than 90% of the total caries experience in some child populations.1 the immature permanent teeth, especially the first molar shows a high caries rate prior to the start of their functioning as teeth. many previous studies have reported in prevention of pit and fissure caries such as drilled pit and fissure to have a better shape of pit and fissure. however, this method damaged to the tooth structures. some investigators used fluoride application, but penetration was difficult to the area of pit and fissure. more recently various adhesive materials that act as agents to seal the fissures from oral environment have been investigated. pit and fissure sealant is known to prevent occlusal surface caries.2 while a sealant is generally applied as part of the system for the prevention of caries, the cleaning of tooth surface, pit and fissure is very important. for obtaining the better retention of sealant, necessity of cleaning of pit and fissure is widely recognized. aoki3 have used the solution of 1n hcl, 50% h3po4, 1n naoh and 148 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 147-150 10% naocl, ultrasonic and combination of ultrasonic and naocl to clean pit and fissure. the use of co2 laser in dental caries prevention and the effects of co2 laser in combination with fluoride have been investigated.4 co2 laser irradiation can decrease enamel demineralization, it has still not been clarified which laser wavelength and which irradiation conditions represent the optimum parameters for application as preventive treatment. scanning electron microscopy examination did not reveal any obvious damage caused by the laser irradiation.5 prior to the sealant therapy, it is also possible to clean up a pit and fissure with the yttrium aluminum garnet (yag) laser to remove organic and inorganic debris. myers & myers used a pulsed yag laser beam on the technique effectively vaporized the organic and debris.6 in reaching the depth of pit and fissure using laser irradiation is needed in order to remove fissure of the contents and to facilitate direct penetration without any alterations of size and shape of the fissure. there is no detailed study has been reported yet concerned about the effectiveness of laser irradiation through pits and fissures. holmium-yttrium aluminum garnet (ho-yag) laser was saved used in the human teeth. laboratory findings by roy7 suggested that when used with collateral water spray, the ho-yag laser can ablate human dentine in a controlled manner without adverse thermal effects. ho-yag laser at a wavelength of 2.12 microns can be safely and effectively used for photoconditioning of the dental surfaces of teeth in clinical conditions.8 in this study, the effect of ho-yag laser on cleaning of pit and fissure was compared to other methods. materials and methods fifty extracted human teeth, particularly molars and premolars were used in this study. samples without any lesions in the area of pit and fissure were carefully selected. since we measure the percentage of cleaned area of fissure content, the depth and shape of fissures were exclusive criteria. the tooth specimens were fixed with formalin and caco3, washed in distilled water for 15 minutes, cleaned with brush cone and placed in ultrasonic bath for 15 minutes. samples were divided into 4 groups. each 10 teeth treated with laser irradiation, chemico-mechanical with 10% naocl and ultrasonic scaler for 2 minutes, mechanical with ultrasonic scaler for 2 minutes and no treatment after cleaned with brush cone for control group. 200 µm diameter of tip of scaler was used for chemico-mechanical and mechanical methods. the longitudinal ground sections were prepared buccolingual from the fissure area of each group. the specimens were fixed into slide glass and processed for examinations under light microscope. scoring the depth of cleaned area was performed by comparing to the depth of fissure. ho-yag laser with a wavelength of 2.1 mm was used in this study. cleaning of pit and fissure with ho-yag laser irradiation which has a repetition rates of 10 pps with an energy of 40 joule/cm2. the 200 mm diameter of fiber was used. the direction of laser beam for irradiation was vertical to the occlusal surface, pit and fissure of enamel. black ink was applied into the fissure before laser irradiation to have better absorption of laser beam. the cleaning effect of pit and fissure was evaluated by determining the distance between the entrance of the pits and fissures and the top of residues. percentage of cleaned area was calculated by comparison between length of cleaned area and depth of fissure × 100%. anova test was used to reveal any significant differences in the cleaning effect of the pits and fissures among the four groups. result cleaning effects of pit and fissure with laser irradiation, chemico-mechanical and mechanical are presented in table 1 shows a cleaned area of control group. the comparison of cleaning effect of laser irradiation, chemicomechanical method, mechanical method and cleaning with brush cone only as a control is shown in table 2. percentage of cleaned area of fissure after ho-yag laser irradiation (mean = 48.91%), cleaning with 10% naocl and ultrasonic scaler (mean = 41.77%), cleaning with ultrasonic scaler (36.78%) and cleaning with brush cone only as control (17.11%) (tabel 1). there are no significant different of cleaning effect of pit and fissure content between laser application and chemico-mechanical method (p = 1.00), laser application and mechanical method (p = 0.85), but significant different of cleaning effect of pit and fissure content between laser application and cleaning with brush cone only as control group (p = 0.002). there are no significant different of cleaning effect of pit and fissure content between chemicomechanical method and mechanical method (p = 1.00), but significant different of cleaning effect of pit and fissure table 1. cleaning effect of pit and fissure with laser irradiation, chemico-mechanical method, mechanical method compared to control no. cleaning method n mean of cleaned area standar deviation 1. ho-yag laser 10 48.91 22.60 2. chemico-mechanical method (10% naocl + ultrasonic scaler) 10 41.77 16.96 3. mechanical method (ultrasonic scaler) 10 36.78 19.25 4. control (brush cone) 10 17.11 21.03 149bahar: the effect of holmium-yttrium aluminum garnet laser table 2. the comparison of cleaning effect of laser irradiation, chemico-mechanical method, mechanical method and control group (cleaning with brush cone only) p value laser chemico-mechanical mechanical control laser 1.00 0.85 0.002* chemico-mechanical 1.00 0.025* mechanical 0.12 control a = 0.05 figure 2. c l e a n i n g o f fissure content. figure 1. a s c h e m a t i c o f comparison between cleaned area and depth of fissure. ef = entrance of fissure; ca = cleaned area; df = depth of fissure. figure 3. cleaning of fissure c o n t e n t w i t h 1 0 % with laser irradiation. naocl + ultrasonic scaler. figure 4. cleaning of fissure content. figure 5. cleaning with brush cone (control), with ultrasonic scaler. content between chemico-mechanical method and cleaning with brush cone only as control group (p = 0.025). there is no significant different of cleaning effect of pit and fissure content between mechanical and cleaning with brush cone only as control group (p = 0.12). the findings showed that the cleaning effect of laser irradiation resulted was seemingly most effective than other methods. the findings showed that the cleaning effect of laser irradiation resulted 48.91% of cleaned area. chemicomechanical method was 41.77% and mechanical method was 36.78% compared to control was 17.11%. condition of fissure’s content after exposure to laser irradiation, cleaning with chemico-mechanical, mechanical and control were demonstrated in figure 2, 3, 4, and 5 cleaning effect of laser irradiation was seemingly more effective than other methods, however, laser irradiation and other methods was not statistically different. discussion since stern and sognnaes9 showed in their pioneering work that laser irradiation of enamel with a ruby laser increased its acid resistance, many attempts have been made to apply lasers in preventive dentistry. stern and sognnaes10 also demonstrated in vivo that enamel subjected to 10 to 15 j/cm2 showed a greater resistant to dental caries than the controls. 150 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 147-150 pit and fissure sealant is known to prevent occlusal surface caries. while a sealant is generally applied as part of the system for the prevention of caries, the cleaning of tooth surface, pit and fissure is very important. for obtaining the better retention of sealant, necessity of cleaning of pit and fissure is widely recognized. prior to the sealant therapy, it is also possible to clean up a pit and fissure with the yag laser to remove organic and inorganic debris.6 most of researcher investigated the effect of laser irradiation in the smooth surface of enamel. the prevention of pits and fissures caries has become relatively important to be investigated. lenz et al.11 has been suggested the co2 laser irradiation for sealing of enamel defects. walsh and perkins12 demonstrated the technique for enamel fusion using co2 laser has potential application for sealing pits and fissures and producing physico chemical alterations in enamel which may have preventive benefits. preliminary findings by myers6 showed that the yag laser has the potential to remove organic and inorganic debris from pits and fissures without causing pulpal or enamel injury due to minimal laser energy. ho-yag laser were used in this study for cleaning of pits and fissures and tried to compare with chemicomechanical and mechanical methods. the progressive results were obtained that the cleaning effects of three approaches, laser irradiation was seemingly most effective compared to other methods and control (table 2). scoring the depth of cleaned area was performed by comparing to the depth of fissure. the differences of cleaned area of chemico-mechanical and mechanical method was not significant compared to laser irradiation method. however, cleaning method with chemico-mechanical and mechanical had consequence in the alteration of the shape of fissure and damage the enamel surfaces of fissures. the cleaned area supposed to be higher in percentage if the black ink as a removable absorptive coating which was used in this study could penetrates deeply into the fissure to have better absorption of laser beam. the study by tagomori and morioka13 has shows that the use of black ink on enamel greatly enhances nd-yag laser absorption in it. because of the shape of fissures, application of black paint into the enamel surface of fissure in this study may be was not penetrate deeply into the fissures. on the other hand, effect of laser irradiation also has the potential to increase acid resistance of the enamel. many previous studies have reported that the lased enamel surface was more resistant to demineralization of the enamel.9,10,14,15 consequently besides effect of cleansing, laser irradiation has also increasing acid resistance on the area of pit and fissure. two hundred µm diameter of fiber of laser beam was used in this study, whereas generally width of fissure’s entrance is less than 200 µm. the result should be better if its possible to use fiber with diameter smaller than 200 µm, because there will be better adaptation to the entrance of fissure. cleaning effects of laser irradiation was seemingly more effective compared to chemico-mechanical method. cleaned area of chemico-mechanical and mechanical method was not statistically significant different compared to laser irradiation method. however, cleaning method with chemico-mechanical and mechanical had consequence in the alteration of the shape of fissure and damage the enamel surfaces of fissures. laser irradiation has also increasing acid resistance on the area of pit and fissure. the cleaned area supposed to be higher in percentage of the black ink as removable absorptive coating could penetrates deeply into the fissure to have better absorption of laser beam. for better adaptation to the entrance of fissure, using fiber with a diameter smaller than 200 mm is recommended. in conclusion, holmium -yttrium aluminum garner laser is a relatively new method for pit and fissure cleaning even though the effectivity is not yet maximal. more research is needed to maximize the use of this laser. acknowledgement this work was partially supported by faculty of dentistry, niigata university, japan. references 1. brunelle ja, carlos jp. changes in the prevalence of dental caries inchanges in the prevalence of dental caries in us schoolchildren. j dent res 1982; 61(sp.iss):1346–51. 2. weintraub ja. the effectiveness of pit and fissure sealant. j. public health dent 1989; 49:317–30 3. aoki k. a study on cleansing of fissure. j oral pathology (in japanese) 1974; 41(3):225–32. 4. rodriguesa lka, santosb mn, pereira d, assafb av, pardib v. carbon dioxide laser in dental caries prevention. journal of dentistry 2004; 32(7):531–40. 5. oliveira me, zezell dm, meister j, franzen r, stanzel s, lampert f, et al. co2 laser (10.6 µm) parameters for caries prevention in dental enamel. caries res 2009, 43:261–8. 6. myers td, myers wd. the use of laser for debridement of incipient caries. j of prosthet dent 1985; 53(6):776–9. 7. roy g, walsh, laurence j. coaxial water mist spray alters the ablation properties of human radicular dentin for the holmium: yag laser. journal of oral laser application 2007; 7(4):225–31. 8. raleigh ah, robert ne. holmium: yag laser in dentistry: photoconditioning of dental surfaces. proc spie 2009; 2128: 308–18. 9. stern rh, soggnaes rf. laser beam effect on dental hard tissue. a preliminary report. js calcif. dent assoc 1965; 33:17–9. 10. stern rh, soggnaes rf. laser irradiation of dental caries suggested by first test in vivo. j am dent assoc 1972; 85:1087. 11. lenz p, gilde h, walz r. untersuchungen zur schmelzversiegelung mit dem co2 laser. dtsh zahnarztl z 1982; 37:469–78. 12. walsh lj, perham sj. enamel fusion using a carbon dioxide laser. a technique for sealing pits and fissures. clinical prevent dent 1991; 13(3):16–20. 13. tagomori s, morioka t. combined effect of laser and fluoride on acid resistance of human dental enamel. caries res 1989; 23:225–31. 14. oho t, morioka t. a possible mechanism of acquired acid resistance of human dental enamel by laser irradiation. caries res 1990; 24:86–92. 15. yamamoto h, ooya k. potential of yttrium aluminum garnet laser in caries prevention. j oral pathol 1974; 3:715. mkgs vol 45 no 2 april-juni 2012.indd 73 volume 45 number 2 june 2012 literature reviews simplified digital infra red photography: an alternative tool in bite mark forensic investigation haryono utomo and mieke sylvia department of forensic odontology faculty of dentistry, airlangga university surabaya indonesia abstract background: decades ago, documentation of forensics evidences such as bitemarks, bloodstains and others which required sophisticated photographic techniques and equipments such as infrared (ir) and ultraviolet (uv) photography, became a problem since they only use film that must be developed. therefore, direct evaluation of the photographic result could not be directly visualized. the equipments prices were relatively high. moreover, most of the equipments were still not available and relatively expensive; and converted ir digital camera could not use for regular photography. recently, digital camera made image documentation and editing easier. purpose: this review was aimed to explore the different characteristics and benefits of regular digital camera in ir forensic photography as well as to simplify the equipments needed. reviews: ir photography becomes easier since certain digital cameras could capture the ir image by using ir filters or to be switched to ir camera. the regular non-slr digital camera had certain advantages compared to slrs, such in focusing. however, since not every digital camera has the ability to capture ir light, laser pointer or tv remote could be used as a tester. conclusion: knowledge about ir bite mark photography, characteristics of regular digital camera and its accesories could reduce the budget for an ideal standard forensic photographic equipments by modifications. key words: digital photography, infrared, bite mark, forensic abstrak latar belakang: puluhan tahun silam, dokumentasi bukti forensik seperti teraan gigit, bercak darah dan sebagainya yang memerlukan teknik dan peralatan yang canggih seperti fotografi infra merah (ir) dan ultraviolet (uv) merupakan masalah karena memakai film yang harus diproses terlebih dahulu untuk mengetahui hasilnya. akibatnya, hasil pemotretan tidak bisa langsung dievaluasi, selain itu harganya relatif mahal, kamera digital yang diubah menjadi kamera ir tidak bisa untuk pemotretan biasa. saat ini kamera digital dan program komputer mempermudah dokumentasi dan penyuntingan. tujuan: tujuan dari tulisan ini adalah untuk menjabarkan sifat dan keuntungan kamera digital dalam fotografi ir forensik dan menyederhanakan peralatan yang dibutuhkan. tinjauan pustaka: fotografi ir dipermudah karena kamera digital tertentu dapat menangkap gambar ir dengan menggunakan filter ir atau diubah menjadi kamera digital ir. kamera biasa non-slr mempunyai beberapa keuntungan dibandingkan slr antara lain dalam memfokuskan obyek. walaupun demikian, karena tidak semua kamera digital biasa dapat menangkap sinar ir, dapat dilakukan pengujian dengan penunjuk laser atau pengatur jarak jauh televisi. kesimpulan: pengetahuan tentang fotografi ir, kamera digital biasa dan peralatan tambahannya dapat mengurangi biaya peralatan fotografi ir standar forensik dengan cara modifikasi. kata kunci: fotografi dijital, infra merah, teraan gigit, forensik correspondence: haryono utomo, c/o: departemen forensik, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132. indonesia. e-mail: dhoetomo@indo.net.id 74 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 73–78 introduction photography often represents the best method to collect and preserve evidence in forensic cases. this is especially true in forensic odontology with cases involving dental identification, human abuse and, perhaps most significantly, bitemark cases. basic visible light photography is adequate in most dental identification cases; however, full spectrum digital photography is best utilized to collect all available evidence in cases of human abuse and bite marks. it captures the forensic injuries using special techniques recording the injuries in each of the four resultant events that occur when light strikes skin.1 however, decades ago when developed acetate film were used, direct visualization of the image was impossible, thus “trial and error” photography such as bracketing exposures to get the best image. therefore, a lot of films were needed as well as difficulties in film developing.1,2 in most forensic odontology cases, evidence collection and preservation using photography is a crucial aspect in the management of cases as they move forward to become part of a future legal proceeding.3-5 full spectrum (infra red, visible light and ultra violet) forensic photography is very important in cases involving dental identification, human abuse and bite marks. by understanding the individual techniques associated with full spectrum digital photography, complete evidence collection becomes routine when a forensic dentist is faced with the collection and preservation of the evidence.3,5-8 in dental identification, it may be necessary to take ultraviolet (uv) photographs of loose teeth found 5 indicating a non-natural avulsion. similarly, in human abuse or bitemark cases it may be advantageous to use alternate light imaging (ali) photography to document injuries to the skin that are not visible to the naked eye. infrared (ir) photographs can be useful to locate and document bleeding below the surface of the skin or to enhance detail of tattoos in decomposing or mummified skin.3,7 in all cases, the investigator should take typical visible light photographs, as well as employ special non-visible spectrum photographic techniques, so that the images are captured using the full spectrum of light.1,9 nevertheless, these techniques needed relatively expensive equipments such as special camera, filters as well as special light source. this review was aimed to explore the different characteristics and benefits of regular digital camera in infra red forensic photography as well as to simplify the equipments needed. standard photographic technique ensuring accuracy during the process of photographing evidence such as bite mark injuries requires a thorough understanding of the basic principles of image capture, including a familiarization with the camera’s features, limitations, and other equipment necessary for the task. attempting to achieve success without first comprehending the fundamentals of photography is just like playing golf in the dark.1 familiarization with the essentials should occur long before the photographer ever finds him/herself employed in a real-time situation so that he/she knows exactly what camera settings, filters, and light sources are appropriate for each different protocol. a standard technique for crime scene photo-documentation includes proper orientation shots, close-up (macro) photography, correct angulation of the lens of the camera to the plane of injury, and inclusion of a scale with identifiers for each case.5 orientation photos orientation shots are for the purpose of showing the location of the bite mark. these are usually captured from three to five feet from the subject and include enough information in the frame to see exactly where on the body the bite occurred (figure 1).1,8 inclusion of a scale is not mandatory; however, it is a good idea to acquire a few images with a scale in place from this distance for data reference which can be included on a label attached to the scale. one recommended scale that is readily accepted by the forensic scientific community is the american board of forensic odontologist (abfo) #2 scale available from lightning powder corporation.1 abfo #2 scale is an lshaped scale with two arms perpendicular to each other. it includes millimeter indices, neutral grey color blocks, and perfect circles placed at the ends and intersection of each arm. the inclusion of the scale allows the user to determine photographic distortion if any, the ability to correct it later with imaging software such as adobe photoshop, and facilitates enlargement of the injury to life-sized proportion. the protocol for image handling could be read in digital analysis of bite mark evidence published in 2002.9 electromagnetic radiation and skin the full spectrum of electromagnetic radiation ranges from extremely short wave lengths (200–375 nm), which is ultraviolet light, through the visible spectrum (400–700 nm) to the longer infrared wavelengths of 700–900 nm (figure 2). regular camera is incapable of seeing outside figure 1. bite marks on victim body with abfo #2 scale.1 75utomo and sylvia: simplified digital infra red photography the visible light spectrum; therefore, special photographic techniques are utilized to create images in the non-visible zones of electromagnetic radiation such that they can be seen with the human eye.5,10 when light strikes skin, there are four simultaneous events that occur: reflection; absorption, fluorescence, and scattering of the light within the skin (also known as diffusion). reflection occurs as the shorter wavelengths of light strike the surface of the skin. depending on the racial characteristics of an individual, the incident angle, and concentration of radiation, up to 50% of shortwave lengths do not penetrate the surface of the skin and are reflected back. conversely, the longer wavelengths of light (700–900 nm) can penetrate the skin up to 3mm. other wavelengths of light strike the skin and diffuse throughout the layers of the skin such that they dissipate without being absorbed or reflected.1,5,10 one final event that occurs when light strikes skin is a molecular-level excitation within the skin that increases the resting state energy of the molecules within the skin, which is known as biofluoresence. almost anything can be made to fluorescence.7 however, the laws of physics require objects to maintain a resting state (neutral) energy level or risk being destroyed. therefore, when light energy is applied to skin, the molecules must get rid of the extra energy. the skin removes the energy of the molecular excitation by re-emitting the energy at a lower wave fluorescent level glow that lasts only 10-9 s. skin reaches peak fluorescence at 450 nm incident light, but it is such a lower energy event that lasts such a short time, it cannot be seen without employing special photographic techniques such as ali illumination.2,11,12 the role of dentists in forensic denstry forensic odontology involves the management, examination, evaluation and presentation of dental evidence in criminal or civil proceedings, all in the interest of justice. the forensic odontologist assists legal authorities by examining dental evidence in different situations. the subject can be divided roughly into 3 major fields of activity: civil or noncriminal, criminal and research.4,13-15 eventhough general dentists do not involved directly to forensic dentistry, each practitioner has a responsibility to understand the forensic implications associated with the practice of his or her profession. appreciation of the forensic field should give the dental clinician another reason to maintain legible and legally acceptable records, and assist legal authorities in the identification of victims and suspects.16,17 the dental record is a legal document owned by the dentist, and contains subjective and objective information about the patient. results of the physical examination of the dentition and supporting oral and surrounding structures must be recorded. in addition, the results of clinical laboratory tests, study casts, photographs and radiographs become components of the record, and should be kept for 7 to 10 years.18,19 all entries should be signed or initialled by recording personnel. changes in the record should not be erased, but corrected with a single line drawn through the incorrect material. this method permits the original entry to remain readable and removes any questions about fraudulent intent to alter recorded information. recently, computergenerated dental records are becoming more common for dental records. the obvious advantage of the electronic record is that it can be easily networked and transferred for routine professional consultation or forensic cases requiring dental records for identification.4,14,16 bite mark a bite mark can be generally defined as a pattern made by teeth in a substrate. since the teeth can be of human or animal origin and the substrate can be skin, food, or a firm but compressible substance, more specific definitions are needed. most bite marks of forensic interest involve the contact between human teeth and skin.20-22 the american board of forensic odontology defines the human cutaneous bitemark as follows: “an injury in skin caused by contacting teeth (with or without the lips or tongue) which shows the representational pattern of the oral structures”. the definition excludes other nonpatterned injuries made by teeth contacting skin such as might be encountered by a fist to the mouth. it also excludes the closing action of jaws during intended biting if a recognizable pattern is not produced.20,23-26 these other tooth-to-skin interactions are still important even if not distinguished by the term “bite mark” because they can be responsible for infection, tissue destruction, or transmissible diseases, and they can transfer dna in saliva.11 however, by convention, the term “bite mark” signifies to the forensic odontologist an injury that, by its pattern, helps establish its origin from teeth.11, 26-27 therefore, not all of these marks should be called bite marks. those marks that occur as a result of objects or surfaces striking the teeth are more accurately called teeth marks. bitemarks are created by the dynamic actions of the mouth and jaw complex of a person or animal. in human interactions, biting is known to occur in situations ranging from play to lovemaking and, more malevolently, in violent interchanges, such as fights and frays, and criminal activities from assaults to homicides.20,24,25 in a pathologist perspective, a patterned injury such as a bite mark is made visible not by a transfer of material and figure 2. penetration of different wavelengths of electromagnetic radiation to the skin.5 76 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 73–78 not often by indentations, but rather by a vital response of the bitten tissue. it may include the superficial scraping of epithelium by the contacting teeth (abrasion), the bleeding within skin by the pressure of the teeth (contusion), or the tearing of skin by teeth (laceration) that renders the bite mark visible.20 bleeding or scraping of skin under assault is not obliged to conform precisely to the anatomy of the object that produced it. the bleeding can extend beyond the tooth marks. conversely, bitten skin may not have been sufficiently damaged to react at all.20,25 the bite mark is not an imprint or impression, but rather a reactive response generated by injured skin that is invariably less precise than a direct recording.20,26,27 since bite mark will fade or changes its color with time, standardized forensic photography should be done as soon as possible. the use of uv and/or ir photographic equipments are able to enhance the captured image of “old” bite mark. decades ago, conventional camera which used films, special filters and lighting were the main tools.1,27 conventional vs. digital camera for uv and ir photography prior to the introduction of uvir digital cameras, all images were film based. special ir film is still available for purchase, however there are specific handling, developing, and focusing requirements that must be employed for successful image capture. unfortunately most digital cameras are designed for capturing images using visible light only.1 their manufacturing process includes software designed for only the visible part of the light spectrum and a special filter in front of the electronic sensor that blocks the uv and ir ends of the spectrum.1,27, 28 therefore, in order to acquire ir images with a digital camera, one must either have a camera that has been produced specifically for uv and ir capture, or modified to accomplish the task. fujifilm of north america was the first company to produce a digital camera with these capabilities. nevertheless, these cameras are no longer being produced. many a fine art photographer has modified an older, retired digital camera and resurrected it for use specifically for ir imaging.29 the same modification allows the forensic photographer to shoot images in ir and uv.1,5 since uv photography needs more sophisticated equipments, this article limits only on infrared photography. infrared (ir) photography digital infrared photography can be tricky when it comes to record bite mark in that one must adjust for a focal shift due to the longer wavelength of light reflecting back to the sensor. focal shift changes can be eliminated with a quartz lens, a subject that will be discussed in the uv section of this paper. an ir filter must also be placed over the lens so that only the ir part of the spectrum is transmitted through the lens to the sensor. there are several types of ir filters to choose from, however a #87glass or gelatin filter will suffice for this application.1 lighting for ir is generally not an issue, as most ambient or room light will be adequate for exposures. infrared photography also requires the use of a full spectrum or modified ir digital camera and lens. additional armamentarium necessary includes an ir band pass filter.1,5,27 in bite mark photography, the ir range of the spectrum shows the viewer the deepest part of the bruise pattern, well into the dermis and underlying vascular tissue. results are often mixed, with ir photos showing less detail than ali and visible techniques.28-30 however, one very useful area where ir application outperforms visible light techniques is in tattoo documentation when the original tattoo is either occluded or has faded considerably. ir also has the ability to see through blood.10,27,28 with practice, the forensic photographer should be able to repeatedly get good results using the ir technique. finally, there must be an ir light source illuminating the patterned injury. there are several types of ir light sources on the market, ranging from typical flash units modified to emit ir light to specialized ir led light sources. one of the simplest way in lighting setup for ir photography is using overhead fluorescent and tungsten room lights which create adequate illumination under normal room lighting (figure 3).6 single lens reflex (slr) vs non-slr in ir digital photography for newcomers in digital ir photography it should be a prime question why an expert said that the digital slr camera was not the best choice for ir, that is for two main reasons. first, the light metering in digital slrs is not done by the image sensor itself (like in non-slr models), but by a separate set of sensors, which may have a different response to ir. one cannot rely on camera’s autoexposure, although it may be able to correct a given camera/filter combination. second, many cameras in lower type of slr camera do not offer real-time electronic preview, because the light from the lens reaches the sensor only during the actual exposure.12,29 this means that you have to put the camera on a tripod, compose the picture without the ir filter, then put it on and shoot blind; most camera makers offer now models with the live view. this is why it may be easier to do ir photography using an advanced non-slr model, or an electronic-finder camera.1,7,12 figure 3. infrared reflected photography setup.6 77utomo and sylvia: simplified digital infra red photography simplified digital infrared (ir) photography eventhough the best digital ir photography is a”true” ir camera followed by ir conversion camera, there are several ways to make regular digital camera to produce ir imaging, such as using ir filter, creates simple ir light source from ir or regular flashlight covered with thin plastics; and ir computer software such adobe photoshop and special ir conversion softwares. these simple steps could be done to regular digital camera to capture ir images; a better way than digital ir conversion cameras which cannot make regular pictures.1, 5,12 infrared photography with regular digital camera regular digital camera is able to catch infrared spectrum by attaching infrared filter and replacing the ir blocking filter with a visible light blocking filter. however, this kind of camera needs long exposure if using infrared filter. therefore, the second choice is more practical, because unconverted cameras need long exposures, thus tripod use was mandatory; or strong infrared light source that sometimes not easily found. replacing ir blocking filter can be done by experts in this techniques. simple test to reveal that a digital camera is sensitive to infrared spectrum is just by pointing tv remote control towards camera lens and see in the lcd panel.1,5 simple infrared light source the “true” ir light source in figure 4 is relatively expensive. other ir light source is surefire™ illuminator that is aroung usd 160.30 a brilliant idea by creating simple ir source using regular led flashlight or ir leds is cheaper. the more or the bigger led’s will give more illumination; however, the simple ir light source bt using black plastic disk from 3.5” computer disk on the front of led flashlight was considered adequate to illuminate limited area of skin (figure 4). nevertheless, the darker the environment light may help enhancing the illumination. other light source such as halogen bulb or light white bulb could be used, but these thin disk may not withstand the heat produced by these light sources. therefore, making led light source from special led’s which emit infrared light is a better idea.31 to the human eye, the 950 nm ir leds looked completely black, even when held up to a bright light source, but when viewed by a video camera, appear perfectly clear, even when not powered. the reason the camera can see right through the leds is because the plastic is made up of materials that only pass infrared light, creating a band pass filter that blocks most of the light that is not within the specified wavelength. so, if this led emits light between 800 and 1000 nms, the band pass filter may help cut all unwanted light except for the output close to 950 nms as specified in the datasheet. other leds are perfectly clear, or have slightly blue tinted plastic bodies.31 other difficulty in ir photography with non-converted ir camera, that is by attaching ir filter is in focusing. firstly because autofocus will not work well in attached ir filter, because of ir filter is very dark. secondly, if the photographic object is not well illuminated, some digital camera that doesn’t have preflash illuminator and through the lens (ttl) flash metering may not have a problem in autofocusing. nevertheless, it could be solved by fixing the camera on the tripod use manual focus and attach the filter after sharp focus is accomplished. if you have a ring flash, just attaches it on the flash unit screw in and bring it tightly closed to the lens after focusing, but it must be sure that no visible light enters.6,32 discussion difficulties in infrared film photography, according to schneider,12 who quote chuck mckern, an experienced ir expert, “it’s difficult getting the film, difficult shooting with it, and difficult processing it.” infrared film must be handled and processed in total darkness, is susceptible to static markings in low humidity, and requires a series of tests using heavy filtration such as a no. 87, 87c, or 89b filter that blocks all uv radiation and visible light to determine the best exposure. in forensic photography, photographing evidence using full spectrum digital camera was still complicated, mostly because the uv/ir camera and special lens such as nikon uv 105 mm were discontinued. additionally, it was quite expensive for newcomers, approximately usd 2000-3000 used.1 therefore, some modifications or “simple inventions” should be found to fulfill the requirements. in this article, the discussion is limited to simplified ir bitemark photography using mostly available digital camera and simple ir light source. it is interesting that on the contrary with uv light source, which could be found as fake money detector, finding simple “true” infrared light source is more challenging. “true” infra red light source such as produced for the military, surefire™ is the best choice but it is also expensive.30 capturing bite marks and teeth marks in considered “new” or “old” ones was different. a new bite mark, which happened within hours is best captured with uv because it produced sharper images; figure 4. ir illumination with led flashlight covered with floppy disk. 78 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 73–78 neverttheless, bite mark up to two weeks is best captured with ir since it could reach deeper tissue 6,9 concerning the difference of slr and non-slr regular digital cameras for ir photography, several characteristics, eventhough the non-slr cameras seems smaller than slr, are superior than slrs such as in easier autofocusing for ir photography. autofocus (af) is performed in the image sensor plane, by contrast detection. this means the circuitry will properly detect when the image is in focus, regardless of the light type. there may be a problem with the amount of light available for the job, especially if ir filter is used, but not with its kind; the af action may be slower and less reliable, but there will be no systematic shift. if your camera is capable of autofocusing in low light down to exposure compensation 0 (ev0) or not much above, you’ll be just fine.12,32 other inferiority of digital slrs in ir photography in autofocusing is because the af is done by dedicated sensors behind a system of mirrors. these sensors are at the same effective distance from the lens as the imager. this should, in principle, functional, as both the af sensors and the imager are getting ir light only. there may be, however, some inaccuracy caused by the fact that both sensors are receiving somewhat different kind of ir, so both focus planes will be shifted with respect to each other: what the af sensor will see as in focus, the imager may see somewhat out-of-focus. nevertheless, it does not mean that non-slr digital camera always more superior, because the lens quality as well as the camera type, which should not a “point and shoot”, or the very basic non-slr digital camera that has poor image quality.12,30-3 for the concluding remarks, knowledge about ir bite mark photography, characteristics of regular digital camera and its accesories could reduce the budget for an ideal standard forensic photographic equipments by modifications. references 1. wright fd, golden gs. the use of full spectrum digital photography for evidence collection and preservation in cases involving forensic odontology. forens sci int 2010; 201: 59–67. 2. sanders ma. history of forensic imaging. in: robinson em, editor. crime scene photography. 2nd ed. burlington, ma, usa: elsevier; 2010. p. 1–16. 3. robinson em. legal issues related to photograph and digital images. in: robinson em, editor. crime scene photography. 2nd ed. burlington, ma, usa: elsevier; 2010. p. 584. 4. avon sl. forensic odontology: the roles and responsibilities of the dentist. j can dent assoc 2004; 70(7): 453–8. 5. wright fd, golden gs. forensic dental photography. in: senn d, stimson pg, editors. forensic dentistry. 2nd ed. boca raton fl: crc press taylor & francis group; 2010. p. 206, 212. 6. davidhazy a. overview of infrared and ultraviolet photography theory, techniques and practice. new york: crc press taylor and franas; 2005. p. 199–211 7. robinson em. ultra violet, infrared and fluorescence. in: robinson em, editor. crime scene photography. 2nd ed. 2010. burlington, ma, usa: elsevier; p. 368–87. 8. metcalf rd. yet another method for marking incisal edges of teeth for bitemark analysis. j forens sci 2008; 53(2): 426–9. 9. bowers cm, johansen jd, johansen rj. photographic evidence protocol: the use of digital imaging methods to rectify angular distortion and create life size reproductions of bite mark evidence. j forens sci 2002; 47(1): 178–85. 10. farrar a, porter g, renshaw a. detection of latent bloodstains beneath painted surfaces using reflected infrared photography. j forensic sci 2012 sep; 57(5): 1150–7 11. stavrianos c, vasiliadis l, papadopoulos c, pantazis a, petalotis. a case report of facial bitemarks: reference of methods of analysis. res j med sci 2011; 5(3): 126–32. 12. baker bw, reinholz ad, espinoza eo. digital near-infrared photography as a tool in forensic snake skin identification. herpetol j 2012; 22(2): 79–82 13. shamim t, varughese v, shameena pm, sudha p. forensicodontology – a new perspective. medico-legal update 2006; 6(1): 8–12. 14. stavrianos c, kokkas a, eliades a, andreopoulos e. applications of forensic dentistry: part i. res j med sci 2010; 4(3): 179–86. 15. stavrianos c, kokkas a, eliades a, andreopoulos e. applications of forensic dentistry: part ii. res j med sci 2010; 4(3): 187–94. 16. american society of forensic odontology. introduction to forensic odontology. in: herschaft, alder, ord, rawson & smith, editors. manual of forensic odontology. 4th ed. new york: impress printing & graphics; 2007. p. 1–6. 17. al amad sh. forensic odontology. smile dent j 2009; 4(1): 2–5. 18. adams bj. establishing personal identification based on specific patterns of missing, filled, and unrestored teeth. j forens sci 2003; 48(3): 487–96. 19. kavitha b, einstein a, sivapathasundharam b, saraswathi tr. limitations in forensic odontology. j forens dent sci 2009; 1(1): 8–11. 20. bernstein ml. the nature of bitemarks. in: dorion bj, editor. bitemark evidence a color atlas and text. 1st ed. new york: crc press taylor and francis; 2011. p. 53–5. 21. sorin h, cristian cg, dan d, mugurel r. bitemark analysis in legal medicine-literature review. rom j leg med 2008; 16(4): 289–98. 22. aggarwal a. bite marks as evidence in crime investigation. j indopacific acad forens odontol 2011; 2(1): 27–30. 23. freeman aj, senn dr, arendt dm. seven hundred seventy eight bite mark: analysis by anatomic location, victim, and biter demographics, type of crime, and legal disposition. j forens sci 2005; 50: 1436–43. 24. miller rg, bush pj, dorion rbj, bush ma. uniqueness of the dentition as impressed in human skin: a cadaver model. j forens sci 2009; 54: 909–14. 25. bush ma, miller rg, bush pj, dorion rbj. biomechanical factors in human dermal bite marks in a cadaver model. j forens sci 2009; 54: 167–76. 26. shamim t, varghese vi, shameena pm, sudha s. human bite marks: the tool marks of the oral cavity. jiafm 2006; 28(2): 0971–3. 27. wright fd, golden gs. photography. in: dorion bj, editor. bitemark evidence a color atlas and text. 1st ed. new york: crc press taylor and francis; 2011. p. 74–98. 28. golden gs. standards and practices for bite mark photography. j forens odonto-stomatol 2011; 29(2): 13–20. 29. mesloh c, henych m, wolf r., gallatin k. infrared beacon evaluation: applications for law enforcement. 1st ed. florida: national institute of justice; 2008. p. 20. 30. graham b, mcgowan k. how to make infrared light source. available online at url http://www.lucidscience.com/pro-simple %20infrared%20illuminator-2.aspx. accessed june 20, 2012. 31. rowan p, hill m, gresham ga, goodall e, moore t. the use of infrared aided photography in identification of sites of bruises after evidence of the bruise is absent to the naked eye. j forensic leg med 2010; 17(6): 293–7. 32. andrew farrar a. detection of latent bloodstains beneath painted surfaces using reflected infrared 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 217217 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 217–222 the effect of peer support education on dental caries prevention behavior in school age children at age 10-11 years old debby syahru romadlon,1 taufan bramantoro,2 and muhammad luthfi3 1politeknik kesehatan kemenkes malang, malang-indonesia 2department of dental public health, faculty of dental medicine, universitas airlangg, surabaya indonesia 3department of oral biology, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: dental caries is an infectious disease and it is preceded by a progressive demineralization in hard tissue surface of the crown. dental caries is one of the most common diseases that experienced by school-age children. health education on the prevention of dental caries and peer support education method can improve the behavior of caries prevention. purpose: this study aimed to determine the effect of peer support education on dental caries prevention behavior in children of school age. method: this study is a quasi-experimental design with pretest and posttest control group design. the sampling technique is simple random sampling. the study was conducted in sdn bandulan 3 sukun malang with 35 respondents (experimental group) and in sdn bandulan 2 sukun malang with 35 respondents (control group). the experimental group was treated using peer support education while the control group was given health education with the lecture method. the data of student’s dental caries prevention behavior (knowledge and attitudes) is obtained through a questionnaire. data were analyzed using paired t-test and independent t-tests with significant p value <0.05. result: the results showed the average score of knowledge in the experimental group at 20.48 and knowledge among respondents in the control group amounted to 18.02, and the average total score of the attitude of the respondents in the experimental group at 20.08 and in control group of 17.77. results of independent t test analysis test that there is a significant difference in the average scores of knowledge and attitudes of respondents in the experimental group and the control group (p value 0.000, <0.05). conclusion: health education with peer support education method is effective in improving dental caries prevention behavior in children at school age. keywords: peer support education; school age children; dental caries prevention behavior correspondence: taufan bramantoro, department of dental public health, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 60132, indonesia. e-mail: tbramantoro@gmail.com. introduction dental caries are existed around the world without looking at age, race or status. research in european countries, us and asia, including indonesia, showed that 80-95% of children under 18 years old are suffered of dental caries. world-widely, 90% of school age children ever suffered of dental caries.1 data from the ministry of health in 2010 showed that the prevalence of caries in indonesia reaches 60-80% of the population, and at the 6th ranks as the most disease that suffer.1 in indonesia there is an increase in the prevalence of dental caries in 2007, from 43.4% to 53.2% in 2013. that means in indonesia there are approximately 93,998,727 people who suffer dental caries.1 one way to prevent caries through health education with the current methods is peer support education. the method of prevention includes primary prevention, secondary, and tertiary. one of the strategies is community empowerment. kreisberg describe that empowerment is a process of formation of knowledge and skills that can enhance one’s mastery over the decisions that affect their lives. community empowerment at schools, in an effort to control caries disease can be carried out by empowering school students in the form of empowering peer educators approach peer support education. school-age children dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 218 romadlon, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 217–222 usually start a lot of activity outside the home, at school and play with peers outside the home. this condition leads to school-age children begin to have a close emotional relationship with a peer group than family.2 peer support education can be identified as sharing experiences and learn from a group of people who have similarities such as age, gender, culture or a place to stay that is effective in improving knowledge, attitudes and positive behavior.3 peer support education conducted in school-age children more effectively done in school compared to other institutions, because the school is well organized, easy to reach the target group because legally the child must attend school and do an evaluation. the results of a review of research by mellanby et al showed that health education in school age children through peer support education is more effective than the education that directed to adult.4 the same fact was also shown from the results of research on the influence of biological peer support education on the behavior of children of school age in determining a healthy snack in lhokseumawe in 2009. the results showed that an increase in behaviors (knowledge, attitudes and skills) are significant in children of school age in determining a healthy snack in the experimental group after a given peer support education, and there are differences in the behavior of children of school age in determining a more healthy snacks both in the experimental group compared with the control group after given by peer support education.5 health education to school-age children typically use the lecture method with presenters who are experts in their field that tends to make passive students, just listening to what the presenters and lack of feedback between the speakers and the students were given materials, so in this study used a control group health education lecture method. based on the phenomenon, which is supported by the results of research on peer support education, it is necessary to study how the effect of peer support education on dental caries prevention behavior in school age children. materials and methods this study is a quasi-experimental design with pretest and posttest control group design. the sampling technique is simple random sampling. the study used two groups: the experimental group received peer support education treatment and control groups were treated with a health education lecture method. the location of this study in sdn bandulan 3 sukun malang for the experimental group and sdn bandulan 2 sukun malang for the control group. respondents in this study is 70 students (35 respondents for the experimental group and 35 respondents to the control group) with the inclusion criteria: 1) registered officially in the schools studied; 2) school-age children aged 10-11 years; 3) following the research activities of the initial stage to the final stage; 4) obtain consent from the parents to get involved as the respondents of this study. research on the experimental group begins with the selection of 8 students as peer educator candidates that chosen directly by the teacher. then they got training to increase the prevention of dental caries for 4 days, so that they can forward the information on peer support education to participants in a small-group (peer group) members who have been determined at random by teacher and researchers, each group contains 4-5 children. peer support education process hold a total of 10 sessions of meetings for approximately 4 weeks with several method such discussions, demonstrations, singing and games, whereas in the control group received health counseling methods lectures in twice meeting by a dentist. data were collected using a questionnaire of dental caries prevention behavior that previously consulted by a dentist, lecturer of psychology, and lecturer of elementary school education. according to the experts the questionnaire was appropriate for school age children. questionnaire in this study consists of two aspects, namely the knowledge and attitude of prevention of dental caries. measurements of behavior at pretest (before treatment) and one week after the treatment are completed (post test). data analysis using spss program with paired t test and independent t test with significance level p<0.05. results table 1 shows that the knowledge of the respondent before the treatment between the experimental and the control group do not have any differences with p=0.626 (p>0.05). similarly table 2 shows the attitude of the respondent before the treatment between the experimental and the control group is not different p=0.918 (p>0.05). the results in table 3 shown that average score of knowledge of respondents on experimental group before peer support education is 16.11, and score knowledge after peer support education was conducted 20.48. there is a difference in the average score it is 4.37. the average score of attitude of respondents on experimental group before peer support education is 15.97, and score attitude after peer support education was conducted 20.08. there is a difference in the average score table 1. results of test behavior analysis on knowledge of the experimental and control group before the treatment (pre test) with p = 0,626 (p>0.05) such discussions, demonstrations, singing and games, whereas in the control group received health counseling methods lectures in twice meeting by a dentist. data were collected using a questionnaire of dental caries prevention behavior that previously consulted by a dentist, lecturer of psychology, and lecturer of elementary school education. according to the experts the questionnaire was appropriate for school age children. questionnaire in this study consists of two aspects, namely the knowledge and attitude of prevention of dental caries. measurements of behavior at pretest (before treatment) and one week after the treatment is completed (post test). data analysis using spss program with paired t test and independent t test with significance level p<0.05. results table 1 shows that the knowledge of the respondent before the treatment between the experimental and the control group do not have any differences with p=0.626 (p>0.05). similarly table 2 shows the attitude of the respondent before the treatment between the experimental and the control group is not different p=0.918 (p>0.05). table 1. results of test behavior analysis on knowledge of the experimental and control group before the treatment (pre test) with p = 0,626 (p>0.05) table 2. results of test behavior analysis on attitude of the experimental and control group before the treatment (pre test) with p=0.918 (p>0.05) 0 5 10 15 20 25 30 35 40 experiment control n mean sd dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 219219romadlon, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 217–222 table 2. results of test behavior analysis on attitude of the experimental and control group before the treatment (pre test) with p=0.918 (p>0.05) the results in table 3 shown that average score of knowledge of respondents on experimental group before peer support education is 16.11, and score knowledge after peer support education was conducted 20.48. there is a difference in the average score it is 4.37. the average score of attitude of respondents on experimental group before peer support education is 15.97, and score attitude after peer support education was conducted 20.08. there is a difference in the average score of 4.11 attitude. based on the results of further analysis it can be concluded that there is a meaningful difference in the knowledge and attitude of the respondent after the given peer support education better than before given peer support education on experimental group p = 0.000 (p<0.05). table 3. results of analysis of the difference in behavior (knowledge and attitudes) before and after the treatment of the experimental and control group group variable n mean sd p experiment knowledge : before 35 16.11 2.78 0.000 after 35 20.48 2.00 difference 4.37 0.78 attitude : before 35 15.97 3.60 0.000 after 35 20.08 2.14 difference 4.11 1.46 control knowledge : before 35 15.77 3.00 0.000 after 35 18.02 2.12 difference 2.25 0.88 attitude : before 35 15.88 3.30 0.000 after 35 17.77 2.63 0 5 10 15 20 25 30 35 40 experiment control n mean sd table 4. results of test behavior analysis of knowledge between the experimental and the control group after the treatment (post test) with p=0,000 (p>0.05) difference 1.89 0.67 note: *significant on p<0.05 the mean score in the knowledge of control group before treatment is 15.77 and after treatment is 18.02. there is a difference in the mean score for knowledge amounted to 2.25. based on the results of further analysis it can be concluded that there is a difference in the knowledge of the respondents after treatment is better than before p=0.000 (p<0.05). the same conditions from the results of the analysis of the mean score of attitudes in the control group before treatment is 15.88 and score attitude after treatment is 17.77. there is a difference in the mean score it is 1.89. table 4. results of test behavior analysis of knowledge between the experimental and the control group after the treatment (post test) with p=0,000 (p>0.05) table 5. results of test behavior analysis of attitude between the experimental and the control group after the treatment (post test) with p=0.000 (p>0.05) 0 5 10 15 20 25 30 35 40 experiment control n mean sd 0 5 10 15 20 25 30 35 40 experiment control n mean sd table 3. results of analysis of the difference in behavior (knowledge and attitudes) before and after the treatment of the experimental and control group group variable n mean sd p experiment knowledge : before 35 16.11 2.78 0.000 after 35 20.48 2.00 difference 4.37 0.78 attitude : before 35 15.97 3.60 0.000 after 35 20.08 2.14 difference 4.11 1.46 control knowledge : before 35 15.77 3.00 0.000 after 35 18.02 2.12 difference 2.25 0.88 attitude : before 35 15.88 3.30 0.000 after 35 17.77 2.63 difference 1.89 0.67 note: *significant on p<0.05 of 4.11 attitude. based on the results of further analysis it can be concluded that there is a meaningful difference in the knowledge and attitude of the respondent after the given peer support education better than before given peer support education on experimental group p = 0.000 (p<0.05). the mean score in the knowledge of control group before treatment is 15.77 and after treatment is 18.02. there is a difference in the mean score for knowledge amounted to 2.25. based on the results of further analysis it can be concluded that there is a difference in the knowledge of the respondents after treatment is better than before p=0.000 (p<0.05). the same conditions from the results of the analysis of the mean score of attitudes in the control group before treatment is 15.88 and score attitude after treatment is 17.77. there is a difference in the mean score it is 1.89. based on the results of further analysis in table 4 and 5 it can be inferred that there is a meaningful difference to the mean score of the knowledge and attitude of respondents on experimental group and control group after treatment p=0.000 (p<0.05). after further analyzed it can be inferred that there is a meaningful difference to the average score of respondents knowledge and attitude before and after the given peer support education between experimental and control group p=0.000 (p<0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 220 romadlon, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 217–222 the research results obtained in accordance with research done earlier by hayati that indicates an increase in the average score of knowledge about healthy snacks that are meaningful in the group that given peer support education p=0.000 (p<0.05).5 bleeker also describe that the results of the meta-analysis of the abuse of drugs, show improvement in aspects meaningful knowledge at 143 people adolescents who participate in peer support education.7 peer support education is one approach that is often used to change knowledge on the level of group.8 changes on knowledge that occur are influenced by the communicator and the content of the message. a communicator of messages that originate from the group itself has a strong influence in attracting the attention of a group of.3 if the communicator is an adult will be able to give a gap in the use of language, terms, and the speech could hinder the understanding of the meaning of the message and the cause of the condition is less interactive learning. this opinion also supported the zioleny et al. 9 stated that the utilization of media such as videos and popular culture’s approach through the game and sing in the implementation of the peer support education sessions can increase interest and better absorption material in children. increased knowledge in the control group is certainly influenced by health education given by dentists in the control group twice. the granting of information on prevention of dental caries by the method of lecturing in the control group will enhance the knowledge in the control group on the prevention of dental caries. the results of this research has a similarity with another assumption as expressed in the who in notoatmodjo that one of the strategies for behavior change is the giving of information to enhance the knowledge that can rise the awareness so that in the end people will behave in accordance with his knowledge. one of the method is by giving information through outreach. the increase in the average score a meaningful stance in experimental group was influenced by the presence of educator in the form of sharing, play with the media of video and game material. other similar research conducted by ergene et al.10 that aims to see the change of knowledge and attitude of students in turkey. the study also showed the same results, in the form of a meaningful increase in positive attitudes in the prevention of hiv/aids on peer support education. behavioral changing that occurred in this study, obtained as a result of change of knowledge school age children from peer support education process. peer support education process conducted during the 4-week increase contacts or interactions that continuously between one and the other in peer group members particularly in the school environment, which ultimately affects the score of schoolaged children. score changing that is affected by the grant of a peer support education provides learning experiences among one another on a group of school age children. attitudes are shaped by a specific value can be learned gradually, perceived as a way and the response shown to family, peers, and social influence.11 table 5. results of test behavior analysis of attitude between the experimental and the control group after the treatment (post test) with p=0.000 (p>0.05) difference 1.89 0.67 note: *significant on p<0.05 the mean score in the knowledge of control group before treatment is 15.77 and after treatment is 18.02. there is a difference in the mean score for knowledge amounted to 2.25. based on the results of further analysis it can be concluded that there is a difference in the knowledge of the respondents after treatment is better than before p=0.000 (p<0.05). the same conditions from the results of the analysis of the mean score of attitudes in the control group before treatment is 15.88 and score attitude after treatment is 17.77. there is a difference in the mean score it is 1.89. table 4. results of test behavior analysis of knowledge between the experimental and the control group after the treatment (post test) with p=0,000 (p>0.05) table 5. results of test behavior analysis of attitude between the experimental and the control group after the treatment (post test) with p=0.000 (p>0.05) 0 5 10 15 20 25 30 35 40 experiment control n mean sd 0 5 10 15 20 25 30 35 40 experiment control n mean sd table 6. results of the analysis of the difference in behavior of knowledge between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) based on the results of further analysis in table 4 and 5 it can be inferred that there is a meaningful difference to the mean score of the knowledge and attitude of respondents on experimental group and control group after treatment p=0.000 (p<0.05). table 6. results of the analysis of the difference in behavior of knowledge between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) table 7. results of the analysis of the difference in behavior of attitudes between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) after further analyzed it can be inferred that there is a meaningful difference to the average score of respondents knowledge and attitude before and after the given peer support education between experimental and control group p=0.000 (p<0.05). discussion the results of the analysis show that there is an increase in the average score a meaningful knowledge between before and after peer support education in experimental group 0 5 10 15 20 25 30 35 40 experiment control n mean sd 0 5 10 15 20 25 30 35 40 experiment control n mean sd table 7. results of the analysis of the difference in behavior of attitudes between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) based on the results of further analysis in table 4 and 5 it can be inferred that there is a meaningful difference to the mean score of the knowledge and attitude of respondents on experimental group and control group after treatment p=0.000 (p<0.05). table 6. results of the analysis of the difference in behavior of knowledge between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) table 7. results of the analysis of the difference in behavior of attitudes between the experimental and the control group after the treatment (post test) with p=0.000 (p<0.05) after further analyzed it can be inferred that there is a meaningful difference to the average score of respondents knowledge and attitude before and after the given peer support education between experimental and control group p=0.000 (p<0.05). discussion the results of the analysis show that there is an increase in the average score a meaningful knowledge between before and after peer support education in experimental group 0 5 10 15 20 25 30 35 40 experiment control n mean sd 0 5 10 15 20 25 30 35 40 experiment control n mean sd discussion the results of the analysis show that there is an increase in the average score a meaningful knowledge between before and after peer support education in experimental group with average 16.11 (before treatment) become 20.48 (after treatment) and p=0.000 (p<0.05). in the analysis of control group show that there is an increase in the average score of knowledge before and after the research with an average 15.77 (before treatment) become 18.02 (after treatment) and p=0.000 (p<0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 221221romadlon, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 217–222 improvement of the attitude in the control group o influenced by outreach health care that provided by the dentist in the control group twice. the information on prevention of dental caries by the method of lecturing in the control group will improve the attitude in the control group on the prevention of dental caries. notoatmodjo also stated that a change in attitude is basically influenced by the factors of the knowledge and belief/trust that is derived from the results of sensing, one of which is obtained through the education or learning process.6 the difference in score on knowledge after the peer support education between experiment and control groups influenced the existence of peer support education. mcdonald et al stated that based on a review of research has shown that peer support education is effective in improving knowledge.3 knowledge is a process of thinking, remembering and recognizing knowledge that can enhance the capabilities and skills of a person.11 peer support education is effective in increasing knowledge. peer support education is a method of empowerment group, so in school-aged children who have the new system in the form of peers also have the same interest with the teenagers, if the information provided is derived from their peer group.3 it is also supported by the opinion of wawan and dewi that increased a person’s knowledge can be influenced by the characteristics of the physical and non-physical environment such as family, peers, interacting with the child spontaneously and continuously. characteristics of non physical form of peer support education treatment gets experiments such interact and affect each other between the members of the group, resulting in the change of knowledge.12 on the peer support education process in this study went success, peers educator and children who were given education in a healthy state and calm in the peer support education process. peers educator very motivated in providing material to participants of dental caries prevention, children who were given education give their attention to peers educator in providing the material to the prevention of dental caries. in the control group given health counseling of specialists, namely dentists tend to make students look passive such only listening to what was said by the presenters and the lack of feedback between presenters and students. based on the results of the research which has been described and some of the concepts that support, stating there is a meaningful difference in knowledge of school age children having given peer support education between the experimental and the control group. significant differences in average score between the control group and the experimental group after treatment influenced by the process of improving knowledge and value through peer support education on experiments group. this is in line with the opinion of usoro that stating that a change in attitude can be obtained from the learning process continuously and can vary.13 peer support education is an approach to give health information through peer groups, that sometimes can be more successful than education through professional, due to members in a group of peers influence each other and interacting with a powerful.7 peer support education in this research can take place interactively so that it can increase the interest of the group in following the education process. this is demonstrated by the increase of the peer group members outside the group who researched after a peer support education sessions running. the increased interest and consistency of school age children in the following peer support education on this research, pointed out that way and the message appealing to school-aged children. this condition also occurs on the research that shows zok garcia and an increase in positive attitudes towards the importance of making healthy food choices in children in the community. this research also proves the existence of an increased participation of the members of the group from 35% to 50%, the increase in children that become educator was from 2 to 35 people. this condition caused by the presence of interaction and the influence of peer group members who previously have been following education to other peers.14 peer support education about prevention of dental caries by peers educator can take place in accordance with the ordinance, the culture and habits of school-aged children, making it easier in acceptance and understanding of dental caries prevention information provided. dental caries prevention information that given by peers educator, considered it a meaningful and important to be accepted and understood by members of the group, thereby increasing the attachment between the members of the group responsible for the increases in knowledge and understanding of the peer group. the results of the research and concepts that have been described there is a meaningful difference expressed knowledge of school-aged children between the experimental and control group before and after the given peer support education. the results of the analysis in this study also shows that there is a difference between the average total score of attitudes before and after peer support education between the experimental and the control group with p=0.000 (p<0.05). the results of this research are aligned with the results of research conducted by dianita15 that there is an increase in clean and healthy living behaviors in schoolaged children after peer support education done. behavioral changing on this research, it is a success that gained from the implementation of the peer support education is done with various methods and media by using media such as videos, games, pictures and guess which are considered relevant to the development of school-aged children. in addition the implementation of peer support education in the form of structured encounter sessions in schools makes it easy to support changes in the skills of school age children due to remind each other, teach and support each other between group members. this suggests that peer support education session conducted at the school is the right choice, because in addition to be able to reach a large number of peer groups target school-aged children, the relationship of the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 222 romadlon, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 217–222 child became very closely with the school environment especially peers.7 the same opinion also showed by stanhope and lancaster that school age children in daily is a lot to learn and play with peers at school and going on a few things meaningful social interaction processes of school age children with peers. first children’s ability to provide increasing appreciation of different views from my peers. the views of the different school age children about the prevention of dental caries can be facilitated and influenced by peers educator as a new information, in which group members can receive it without forced. second, the increasing sensitivity of school-aged children against the rules and the pressure from the peer group. peer support education can increase the motivation of children to apply the same motivation as peer educator.16 the results of this study showed that there is a significant difference in behavior (knowledge and attitude) of school age children before and after peer support education between the experimental and the control group. it concluded that health education with peer support education method is more effective in improving dental caries prevention behavior in school age children rather than health counseling with lectures method. references 1. balitbang kemenkes ri. riset kesehatan dasar; riskesdas. jakarta: balitbang kemenkes ri; 2013. p. 48-67. 2. edelman m. health promotion; throught the lifespan. 6th ed. mosby: st louis; 2006. p. 89-109. 3. mcdonald j, roche am, durbridge m. peer support education from evidenced to practice: an alcohol & other drugs primer. 2015. p. 75-85. 4. mellanby ar, newcomb rg, rees j, tripp jh. a comparative study of peer-led and adult-led school sex study education. health educ res 2001; 16(4): 481-92. 5. hayati m. pengaruh peer edukasi tentang jajanan sehat terhadap perilaku anak usia sekolah di kota lhokseumawe provinsi naggroe aceh darussalam. tesis. depok: pascasarjana universitas indonesia; 2009. 6. notoatmodjo s. ilmu perilaku kesehatan. jakarta: rineka cipta; 2010. p. 23-30. 7. bleeker a. presentation for the second international drugs and young people conference. 2011. p. 102-15. 8. population council. peer education and hiv/aids: past experience, future direction. popcouncil. 2012. p. 18-23. 9. zioleny r, kimzeke g, stakic s, bruyn md. peer support education training of trainers manual: youth peer support education elektronik resources.). aidsmark 2011. p. 54 65 10. ergene t, cok f, tuner a, unal s. a controlled-study of preventive effects of peer support education and single session lectures on hiv/ aids knowledge and attitudes among university students in turkey. 2010. p. 45-50. 11. allender ja, spradley bw. communnity health nursing: promoting and protecting the public’s health. 6th ed. philadelphia: lippincott williams & wilkins; 2005. p. 301-12. 12. wawan a, dewi m. teori dan pengukuran pengetahuan, sikap, dan perilaku. yogyakarta: nuha medika; 2010. p. 90-109. 13. usoro a. attitudes as a factor for the use of information and communication technology for global planning. computing and information system. 2000. p. 154-60. 14. garcia ac, zock a. peer support education in nutrition for childrents: canadian journal of dietetic practice and research 2015; 34-43. 15. fitriani d. pengaruh edukasi sebaya terhadap perilaku hidup bersih dan sehat (phbs) pada agregat anak usia sekolah yang beresiko kecacingan di desa baru kecamatan manggar belitung timur. tesis. depok: keperawatan universitas indonesia; 2011. 16. stanhope m, lancaster j. community and public health nursing. 6th ed. mosby: st louis; 2014. p. 446-56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p217-222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p217-222 16 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 16–20 original article association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients pitu wulandari1, dody widkaja2, aini hariyani nasution1, armia syahputra1, gebby gabrina3 1department of periodontology, faculty of dentistry, universitas sumatera utara, medan, indonesia 2undergraduate student, faculty of dentistry, universitas sumatera utara, medan, indonesia 3universitas sumatera utara dental hospital, medan, indonesia abstract background: as individuals grow older, they may be more susceptible to chronic diseases, which can affect their overall health. periodontitis, for instance, is one of the most common oral diseases that result from prolonged exposure to pathogens. the main etiology of periodontitis is dental biofilms, but several risk factors may also affect the progression of the disease. purpose: the study aimed to determine whether there was an association between age, gender and education level and the severity of periodontitis using the 2017 american academy of periodontology (aap) classification. methods: a retrospective analytical study was used to determine the stage and grade of periodontitis in relation to age, gender and education level. secondary data in the form of medical record status was analysed using software applications. results: the distribution of periodontitis was 66.7% in pre-elderly patients (45–59 years), 61.5% in male subjects and 39.7% in individuals with middle education levels. using chi-square analysis, the severity of periodontitis showed a statistically significant correlation with age (p=0.01) and gender (p=0.003). in contrast, the level of education was not statistically correlated to the severity of periodontitis (p=0.887). the percentage of stage iv grade b was highest in elderly patients (65.38%), while stage iv grade c was the highest in male subjects (41.67%). conclusion: the study showed that age and gender significantly influenced the occurrence and severity of periodontitis, while education level showed the opposite. keywords: ageing population; periodontitis; severity correspondence: pitu wulandari, department of periodontology, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: pitu.wulandari@usu.ac.id; puput_seven@yahoo.co.id introduction according to the central bureau of statistics (bps), in 2020, the population of indonesia began to age, with the percentage of elderly people recorded at 9.92% (approximately 26.82 million people) and projected to reach almost one-fifth of the total population by 2045. this is occurring as a result of the improvements in health facilities, nutrition and socioeconomic conditions.1 nonetheless, compared with other age groups, elderly people are more vulnerable to chronic inflammation, including periodontitis. the increased susceptibility to periodontitis among the elderly may be due to prolonged exposure to periodontal pathogens as well as changes in the immuno-inflammatory status of the periodontal tissue.2 globally, periodontitis in severe form affects more than 10% of the population. in 2010, it was ranked the sixth most prevalent condition, with approximately 743 million people affected.3 as one of the major causes of tooth loss in the adult population, periodontitis may lead to multiple edentulous and masticatory dysfunctions, which affect nutrition, quality of life and self-esteem while also imposing high socioeconomic and healthcare costs.4 according to one finding in 2010, direct and indirect costs due to dental diseases amounted to usd442 billion worldwide, with 83% of the direct treatment costs being attributable to highincome countries (e.g. north america, western europe and high-income asia-pacific).5 in line with that, between 1990 and 2010, the global burden of periodontitis increased by 57.3%. these numbers are likely to rise due to increased dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p16–20 mailto:pitu.wulandari@usu.ac.id mailto:puput_seven@yahoo.co.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p16-20 17 tooth retention globally and as many populations continue to grow and age.4 meanwhile, in indonesia, the 2018 basic health research (riskesdas) report showed a high prevalence of periodontitis (74.1%), with the highest prevalence in preelderly patients (77.8%) and decreasing gradually in elderly patients (66.0%).6 this may be due to an unawareness of periodontal diseases symptoms, and many individuals tend to seek dental care only when they can no longer bear the pain. similarly, tooth loss is regarded as an unavoidable outcome of the ageing process in many cultures. these cultural beliefs can have a negative impact on oral and periodontal health.7 the accumulation of dental biofilm as a result of poor oral hygiene habits initiates the colonisation of microorganisms in periodontal tissue. these biofilms can cause direct damage to periodontal tissue by impairing polymorphonuclear leukocyte (pmn) functions, such as chemotaxis, phagocytosis and intracellular killing.8 however, their primary role in periodontal pathogenesis is to activate immune-inflammatory responses, which result in tissue damage and may be beneficial to the bacteria in the periodontal pockets by providing nutrient sources.9 a possible mechanism is the release of pro-inflammatory cytokines, such as interleukin-1 (il-1) and tumor-necrotising factor (tnf-α), due to the immune-inflammatory response to bacterial invasion, resulting in the progressive destruction of periodontal tissue characterised by clinical attachment loss, followed by an increase in pocket depth and changes in the density and height of the alveolar bone. although periodontal diseases are initiated by dental biofilms, several risk factors include socio-demographic characteristics, such as age, gender, educational level and income. risk behaviours like smoking are also important factors that influence the progression and severity of the disease.9,10 in response to new scientific evidence, the classification of periodontitis has been repeatedly modified in the last 30 years. in 2017, the american academy of periodontology (aap) published an updated classification of periodontal and peri-implant diseases and conditions.11 however, the latest classification of periodontitis is still not commonly used in indonesia, especially in relation to the medical records of universitas sumatera utara dental hospital. therefore, this study aims to provide data on the distribution of periodontitis in universitas sumatera utara dental hospital and identify the association between several risk factors, such as age, gender and education level, that affect the severity of periodontitis by using the new approach for classification. materials and methods this retrospective analytical study was carried out between may and july 2021 at the periodontics clinic of universitas sumatera utara dental hospital, one of the referral places for treating periodontitis patients in medan. the research ethics commission of universitas sumatera utara approved this study (no.392/kep/usu/2021) in accordance with the medical and ethics protocols of the declaration of helsinki. sample selection was made using the purposive sampling technique corresponding to inclusion and exclusion criteria. the inclusion criteria were medical records with the visit years 2015–2019, which included information on age, gender, education level, clinical attachment loss (cal), pocket depth (pd), number of missing teeth and the diagnosis of periodontitis. patients under 45 years old, patients with systemic conditions and incomplete or unreadable medical record data were excluded. during the recording of medical records in 2015–2018, the diagnosis of periodontitis was still based on the 1999 classification of periodontal diseases and conditions; hence, the conversion to the 2017 classification of periodontal and peri-implant diseases and conditions was performed for this study. periodontitis was classified as stage i to stage iv and determined by cal, pd, percentage of bone loss and the number of missing teeth stated in the medical record data. cal 1–2 mm was defined as stage i, 3–4 mm as stage ii and ≥5 mm as stage iii to stage iv. an evaluation of the complexity of management was also performed, wherein stage ii was reclassified to stage iii if the maximum pd was ≥6 mm and stage iii was reclassified to stage iv if there were less than 20 remaining teeth or 10 opposing pairs. in addition to the severity and extent of the disease, grading systems were used to determine the likelihood that periodontitis would progress at a faster rate than the majority of the population or respond less predictably to standard therapy. it was classified into three groups: grade a (slow rate of progression), grade b (moderate rate of progression) and grade c (rapid rate of progression). age was classified into two groups: pre-elderly (45–59 years) and elderly (≥60 years), while the education level was categorised into low (elementary to junior high school), middle (secondary high school) and high (diploma) levels. the association between age, gender, educational level and the severity of periodontitis was determined by using chi-square analysis. data analysis was performed using software applications and presented in the table, where the significance level was established at p-value <0.05. results the present study was conducted using secondary data in the form of medical records with the visit years 2015–2019 from the periodontics clinic of universitas sumatera utara dental hospital. after 297 medical records were collected, several of them were excluded due to incomplete or unreadable sections; hence, 78 subjects were obtained that matched the inclusion criteria. the characteristics of the study subjects are shown in table 1. it was found that the percentage of periodontitis in the pre-elderly age group was higher (66.7%) than in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p16–20 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p16-20 18 elderly age group (33.3%). males were the dominant part of the sample (61.5%), compared with females (38.5%). the frequencies of the study subjects were 34.6%, 39.7% and 25.6% among the three education level groups, respectively. the highest percentage of periodontitis corresponding to age group, based on the 2017 aap classification, was stage iv grade b (where the cal was 5 mm or greater with a moderate rate of the disease’s progression) in the elderly age group (65.38%). when categorised by gender, the highest percentage of periodontitis was stage iv grade c (where the cal was 5 mm or greater with a rapid rate of the disease’s progression) in male subjects (41.67%). based on the education level, stage iv grade b was found to have the highest percentage in low-education level subjects (37.04%) (table 2). table 3 shows the association between the variables and the severity of periodontitis. when categorised by age, stage iv was more prevalent than stage iii in the pre-elderly age group (52.9% and 47.1%, respectively), while the elderly age group suffered from stage iv the most (85.2%), and it was statistically significant (p=0.01). using chi-square analysis, males were found to be more likely to have stage iv periodontitis (79.2%), while stage iii periodontitis was found to be more prevalent in female subjects (56.7%) (p=0.003). in contrast, there was no significant difference (p>0.05) observed in the education level. discussion this study was conducted using secondary data in the form of medical record status because the relationship among variables within a population that has not been previously analysed can be examined, potentially yielding important new findings to advance science, especially in the periodontology field. in addition, using and interpreting data that are already available may be helpful to uncover new information that can be used to improve education, health services and health policy.12 table 1. characteristics of subjects by socio-demographic variables (n=78) variables subject (n=78) percentage (%) age pre-elderly (45–59 years) 52 66.7 elderly (≥60 years) 26 33.3 gender male 48 61.5 female 30 38.5 education level low 27 34.6 middle 31 39.7 high 20 25.6 table 2. staging and grading based on age, gender and education level at the periodontics clinic of universitas sumatera utara dental and oral hospital in medan variables (n=78) periodontitis totalstage iii stage iv grade a grade b grade c grade a grade b grade c n % n % n % n % n % n % n % age pre-elderly 1 1.92 14 26.92 10 19.23 0 0 9 17.31 18 34.62 52 100 elderly 0 0 1 3.85 2 7.69 0 0 17 65.38 6 23.08 26 100 gender male 1 2.08 3 6.25 6 12.50 0 0 18 37.50 20 41.67 48 100 female 0 0 12 40.0 6 20.0 0 0 8 26.67 4 13.33 30 100 education level low 0 0 5 18.52 5 18.52 0 0 10 37.04 7 25.93 27 100 middle 0 0 7 22.58 4 12.90 0 0 9 29.03 11 35.48 31 100 high 1 5.0 3 15.0 3 15.0 0 0 7 35.0 6 30.0 20 100 table 3. distribution of the severity of periodontitis based on age, gender and education level variables (n=78) severity of periodontitis total p-valuestage iii stage iv age pre-elderly 24 (47.1%) 27 (52.9%) 51 (100%) 0.01 a * elderly 4 (14.8%) 23 (85.2%) 27 (100%) gender male 10 (20.8%) 38 (79.2%) 48 (100%) 0.003 a * female 17 (56.7%) 13 (43.3%) 30 (100%) education level low 11 (37.9%) 18 (62.1%) 29 (100%) 0.887 bmiddle 10 (33.3%) 20 (66.7%) 30 (100%) high 6 (31.6%) 13 (68.4%) 19 (100%) astatistical evaluation by the continuity correction test; bstatistical evalution by the pearson’s chi-square test; *p-value <0.05 statistically significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p16–20 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p16-20 19 there are currently no studies that show the association of the severity of periodontitis with age, gender and education level using the 2017 aap classification in indonesia. this classification describes periodontitis more specifically because it considers the severity of periodontitis progression, history, the possible risk of periodontitis progression in the future and the assessment of the risk of diseases that affect the general condition of the body.11 it is well documented that chronic inflammation (such as periodontitis) may play a role in other disease conditions in which inflammation is a major component. once periodontal pathogens and their metabolic enzymes have access to blood circulation, they produce inflammatory mediators, which are responsible for systemic inflammation.13 in this study, periodontitis was more prevalent in preelderly patients, males and subjects with middle education levels (table 1). the result was similar to a study conducted by machado et al.,14 wherein chronic periodontitis mostly occurred in the 45–65 years age group and individuals with elementary to middle education levels. subsequently, males were more likely to experience greater cal and deeper pd than females (p<0.05). lower percentages of elderly patients may occur due to their high adaptability; these individuals tend not to seek treatment simply because they do not experience pain or other noticeable symptoms. in line with that, spinler et al.15 concluded that the frequency of dental visits declines with age in older adults. greater cal and deeper pd in males than females may be due to the role of sex chromosomes in mediating the differences in the immune response. monocyte production of il-12 and regulatory t cells, which have anti-inflammatory properties, have been found at increased levels in men.16 when it comes to gender, females show more positive attitudes about dental visits, higher oral health literacy and better oral health behaviours than males. in addition, hormonal mediators (i.e. estrogen, progesterone and testosterone) have been shown to have an effect on innate and adaptive immunity. a sustained level of estrogen has been demonstrated to reduce pro-inflammatory cytokine production, while testosterone suppresses the immune response.17,16 table 1 also shows that the percentages of periodontitis are higher in subjects with low to middle education levels. similarly, a cohort study from sweden showed that elderly subjects with less than high school education are twice as likely to have periodontitis compared with those with more than high school education.18 stage iv periodontitis was found in higher percentages among all variables (table 2), showing that patients who came to the periodontics clinic at universitas sumatera utara dental hospital encountered cal≥5 mm. based on age, the highest occurrence of periodontitis was stage iv grade b in elderly patients, which indicated a moderate rate of the disease’s progression, where the destruction of periodontal tissue corresponded to the deposits of biofilms with age.19 in line with that, a study by eke et al.20 showed that more than 50% of individuals had cal≥5 mm and 20% of cal ≥7 mm were found in the elderly. when stratified by gender, stage iv grade c was found with the highest percentages in male subjects. the rapid progression of the disease was consistent with the effect of smoking on periodontal tissue. meanwhile, the highest occurrence of periodontitis according to the education level was stage iv grade b in low-education level subjects. this may be due to poor oral hygiene habits as a result of lower levels of dental service use, higher body mass index and irregular oral self-care practices directly or indirectly caused by a lower education level.21 a statistically significant correlation between age and the severity of periodontitis in the present study (table 3) was similarly shown in several studies.22,23,24 as a result of the host’s innate and adaptive immune-response systems perceiving microbial transitions in biofilms, the prevalence and severity of periodontitis in the ageing population become increasingly high. in addition to the immune system, genetic and epigenetic factors play a role in periodontal changes in the elderly, although the mechanism is still not clearly understood.25 the present study shows that gender and the severity of periodontitis are significantly correlated, but not the education level (table 3). similarly, a study by paksoy et al.26 showed that gender is significantly associated with the severity of periodontitis, with males more likely to suffer from severe periodontitis. smoking is one possible reason for the increased prevalence and severity of periodontitis, since smoking is vastly more prevalent in males than females in indonesia.6 however, the number of smokers is not shown, which makes it one of the limitations in the present study. different plausible mechanisms on the effect of smoking towards periodontal tissue include pathogenic subgingival flora, diminished microcirculation, neutrophil dysfunction, pro-inflammatory cytokine production and increased levels of pathogenic t-cells, although there is no clear evidence that points to one particular mechanism as being of greater importance.27 in conclusion, the present study shows that the severity of periodontitis in pre-elderly and elderly patients is significantly related to age and gender but not with the level of education. the majority of patients who visited the periodontics clinic at universitas sumatera utara dental hospital had stage iv periodontitis, which may further lead to edentulous if not treated appropriately. the treatment of periodontitis includes mechanical therapy like scaling and root planing. however, in deep and periodontal pockets, such treatments are difficult. in response, systemic or local drugs need to be administered because periodontitis is mainly caused by bacterial deposits.28 this study, however, had several limitations when it came to determining the stage and grade of periodontitis. the data regarding the cause of tooth loss were not recorded; hence, the prevalence of stage iv periodontitis was possibly overrated due to other reasons for missing teeth. in addition, medical records data did not show the radiographic bone loss over a five-year period, which could have assisted in determining the grade of periodontitis. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p16–20 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p16-20 20 nonetheless, the percentages of bone loss per age were examined carefully. acknowledgements the authors would like to express their sincere gratitude to the director of universitas sumatera utara dental hospital, who granted permission for this research. we also thank all the staff members at the department of periodontology, faculty of dentistry, universitas sumatera utara for their support in writing this article. references 1. badan pusat statistik. statistik penduduk lanjut usia. jakarta: badan pusat statistik; 2020. p. 13–24. 2. hajishengallis g. aging and its impact on innate immunity and inflammation: implications for periodontitis. j oral biosci. 2014; 56(1): 30–7. 3. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cjl, marcenes w. global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. j dent res. 2014; 93(11): 1045–53. 4. tonetti ms, jepsen s, jin l, otomo-corgel j. impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: a call for global action. j clin periodontol. 2017; 44(5): 456–62. 5. listl s, galloway j, mossey pa, marcenes w. global economic impact of dental diseases. j dent res. 2015; 94(10): 1355–61. 6. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 201–204. 7. jin lj, armitage gc, klinge b, lang np, tonetti m, williams rc. global oral health inequalities: : task group—periodontal disease. adv dent res. 2011; 23(2): 221–6. 8. hasan a, palmer rm. a clinical guide to periodontology: pathology of periodontal disease. br dent j. 2014; 216(8): 457–61. 9. preshaw pm. periodontal disease pathogenesis. in: newman mg, takei h, klokkevold pr, carranza fa, editors. newman and carranza’s clinical periodontology. 13th ed. philadelphia: elsevier inc; 2014. p. 434–63. 10. aljehani ya. risk factors of periodontal disease: review of the literature. int j dent. 2014; 2014: 1–9. 11. caton jg, armitage g, berglundh t, chapple ilc, jepsen s, kornman ks, mealey bl, papapanou pn, sanz m, tonetti ms. a new classification scheme for periodontal and peri-implant diseases and conditions introduction and key changes from the 1999 classification. j periodontol. 2018; 89(suppl 1): s1–8. 12. o’connor s. secondary data analysis in nursing research: a contemporary discussion. clin nurs res. 2020; 29(5): 279–84. 13. jain p, hassan n, khatoon k, mirza ma, naseef pp, kuruniyan ms, iqbal z. periodontitis and systemic disorder—an overview of relation and novel treatment modalities. pharmaceutics. 2021; 13(8): 1175. 14. machado v, botelho j, amaral a, proença l, alves r, rua j, cavacas ma, delgado as, mendes jj. prevalence and extent of chronic periodontitis and its risk factors in a portuguese subpopulation: a retrospective cross-sectional study and analysis of clinical attachment loss. peerj. 2018; 6: e5258. 15. spinler k, aarabi g, valdez r, kofahl c, heydecke g, könig h-h, hajek a. prevalence and determinants of dental visits among older adults: findings of a nationally representative longitudinal study. bmc health serv res. 2019; 19(1): 590. 16. ioannidou e. the sex and gender intersection in chronic periodontitis. front public heal. 2017; 5: 1–8. 17. lipsky ms, su s, crespo cj, hung m. men and oral health: a review of sex and gender differences. am j mens health. 2021; 15(3): 15579883211016360. 18. borrell ln, crawford nd. socioeconomic position indicators and periodontitis: examining the evidence. periodontol 2000. 2012; 58(1): 69–83. 19. to n e t t i m s , g r e e nwe l l h , k o r n m a n k s . s t a g i n g a n d grading of periodontitis: framework and proposal of a new classification and case definition. j periodontol. 2018; 89(suppl 1): s159–72. 20. eke pi, thornton-evans go, wei l, borgnakke ws, dye ba, genco rj. periodontitis in us adults: national health and nutrition examination survey 2009-2014. j am dent assoc. 2018; 149(7): 576-588.e6. 21. boillot a, el halabi b, batty gd, rangé h, czernichow s, bouchard p. education as a predictor of chronic periodontitis: a systematic review with meta-analysis population-based studies. plos one. 2011; 6(7): e21508. 22. tadjoedin fm, fitri ah, kuswandani so, sulijaya b, soeroso y. the correlation between age and periodontal diseases. j int dent med res. 2017; 10(2): 327–32. 23. abbass mms, rady d, radwan ia, el moshy s, abubakr n, ramadan m, yussif n, al jawaldeh a. the occurrence of periodontal diseases and its correlation with different risk factors among a convenient sample of adult egyptian population: a cross-sectional study. f1000research. 2019; 8: 1740. 24. jiao j, jing w, si y, feng x, tai b, hu d, lin h, wang b, wang c, zheng s, liu x, rong w, wang w, li w, meng h, wang x. the prevalence and severity of periodontal disease in mainland china: data from the fourth national oral health survey (2015-2016). j clin periodontol. 2021; 48(2): 168–79. 25. ebersole jl, graves cl, gonzalez oa, dawson d, morford la, huja pe, hartsfield jk, huja ss, pandruvada s, wallet sm. aging, inflammation, immunity and periodontal disease. periodontol 2000. 2016; 72(1): 54–75. 26. paksoy t, ustaoğlu g, peker k. association of socio-demographic, behavioral, and comorbidity-related factors with severity of periodontitis in turkish patients. aging male. 2020; 23(3): 232– 41. 27. genco rj, borgnakke ws. risk factors for periodontal disease. periodontol 2000. 2013; 62(1): 59–94. 28. lubis p, nasution r, syafruddin i, ervina i, ameta p. effectiveness of 1% curcumin gel subgingival application on interleukin-6 levels in chronic periodontitis patients. int j appl dent sci. 2020; 6(3): 513–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p16–20 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p16-20 9898 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 98–103 original article antibiofilm activity of neem leaf (azadirachta indica a. juss) ethanolic extracts against enterococcus faecalis in vitro suhartono suhartono,1 cut soraya,2 putri shabira1 1department of biology, faculty of mathematics and natural sciences, universitas syiah kuala, banda aceh, indonesia 2department of conservative dentistry and endodontics, faculty of dentistry, universitas syiah kuala, banda aceh, indonesia abstract background: enterococcus faecalis commonly infects root canals by forming a biofilm. extracts from neem leaves (azadirachta indica a. juss) have been shown to have antibacterial properties, indicating their potential in preventing or treating biofilm formation caused by bacteria. purpose: this study aims to investigate the phytochemical compounds present in neem leaves (azadirachta indica a. juss) and establish the concentration of ethanol-based neem leaf extract that can effectively inhibit the in vitro growth of enterococcus faecalis biofilm. methods: this study employed the maceration technique for extraction, gas chromatography mass spectroscopy for the analysis of plant chemicals, and a microtiter plate assay for measuring biofilm formation with treatment concentrations of 6.25%, 12.5%, 25%, 50%, and 75%, with a positive control of 0.2% chlorhexidine. results: a phytochemical analysis revealed that the ethanol extract of neem leaves contained 22 different metabolites, mainly terpenoids and fatty acids. the extract demonstrated antibiofilm activity only at a concentration of 12.5% with an average biofilm inhibition of 36.85%. however, lower concentrations of 6.25%, 25%, 50%, and 75% had the opposite effect, promoting biofilm formation in enterococcus faecalis. conclusion: phytochemical metabolite contained in the ethanolic extracts of neem leaves might contribute a promising agent in treating a biofilm-mediated root canal infection of enterococcus faecalis. keywords: antibiofilm; azadirachta indica a. juss; enterococcus faecalis; ethanol extract article history: received 9 july 2022; revised 12 august 2022; accepted 20 october 2022 correspondence: suhartono suhartono, department of biology, faculty of mathematics and natural sciences, universitas syiah kuala. jl. tgk. syech abdurrauf no. 3 banda aceh, indonesia. email: suhartono@unsyiah.ac.id introduction root canal infections are generally caused by microorganisms. the presence of microorganisms penetrating dental pulps followed by the colonization of the root canal system is a major cause of periapical pathology that leads to the failure of root canal treatment.1 one of the bacterium often isolated in root canal treatment is enterococcus faecalis (e. faecalis). it is a group of gram-positive enterococci that have a cocci shape and are classified as lactic acid bacteria that can be found in the root canals of teeth along with other pathogens, including streptococcus, actinomyces, staphylococcus, and lactobacillus.2 root canal treatment aims to repair the infected tooth by restoring the infected tissue, eliminating the pathogenic bacteria, and preventing recontamination after root canal treatment.3 the pathogenic bacteria causing root canal infections are developing resistance, which poses major challenges in treatment. e. faecalis can survive in a variety of environmental conditions, including environments with extreme alkaline ph;4 they remain in root canals after disinfection using sodium hypochlorite and chlorhexidine (chx).5 furthermore, root canal infections may be difficult to treat due to bacterial infiltration into the dentin, accompanied by virulence factors and biofilm production. virulence factors with the ability to form biofilms, such as gelatinase production, limit the penetration of antibiotics to achieve effective concentrations at the target site, which hinders the healing process.6 a biofilm is a cooperative association of microorganisms that adhere to surfaces, biotic and abiotic, using extracellular polymeric substances (eps) and glycocalyx, and they communicate through a quorum sensing (qs) system.7 biofilms forming in the intracanal and periapical dentin copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 mailto:suhartono@unsyiah.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 99 suhartono et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 98–103 allow e. faecalis to be more resistant to phagocytes, antibodies, and antibiotics compared with bacteria that do not form biofilms.8 in vitro studies have shown different stages of e. faecalis biofilm development on root canal dentin.9 increasing bacterial resistance to drugs requires efforts to find antimicrobial agents that are effective against pathogenic bacteria. alternative treatments to overcome bacterial resistance due to the formation of biofilms include searching for natural compounds derived from plants that are able to inhibit the formation of biofilms, like neem leaves (azadirachta indica a. juss). the neem plant has long been used to maintain oral hygiene and prevent cavities as well as prevent gingival disease and periodontitis. neem leaves have several active compounds in the form of alkaloids, tannins, essential oils, and flavonoids that have potential as antimicrobials.10 previous research reported that neem leaves have antibiofilm properties, which strengthen the possibility of using neem extract in eradicating biofilm-mediated infections.11 certain concentrations of neem extract show a significant reduction in the number of exopolysaccharides, changing the biofilm structure, which could facilitate the penetration of antibiotics into the bacterial community.12 therefore, this study aims to identify the phytochemical compounds present in neem leaves (azadirachta indica a. juss) and determine the concentration of the ethanol extract of neem leaves that can effectively prevent the in vitro formation of biofilm by e. faecalis. materials and methods the extraction was conducted following the method previously described.13 neem leaves (azadirachta indica a. juss) were collected from limpok, darussalam, aceh besar, indonesia. a total of 2 kg of fresh neem leaves were washed thoroughly and dried at room temperature. dried neem leaves were mashed using a blender, filtered to get a finer powder, and then weighed. the neem leaves were then extracted using the maceration method using 96% ethanol as a solvent, and the ratio between ethanol and the simplicia solvent was 1:10. the initial stage of maceration was carried out by soaking simplicia in 7 liters (l) of 96% ethanol solvent (7/10 of 10 l) for five days. the container used for maceration was covered with aluminum foil and stored in a location shielded from the sun. after that, filter paper was utilized to separate the filtrate and dregs. the dregs were again immersed in 3 l of 96% ethanol (3/10 of 10 l) for five days. the mixed extract and solvent were stirred occasionally every day. extract yield was calculated using the following formula: yield (%) = extract weight dried simplicia weight x 100% the analysis of the phytochemical compounds in the ethanol extract of neem leaves was performed using gas chromatography mass spectroscopy (gc/ms) equipment (shimadzu gcms-qp 2010 ultra) with reference to the method developed by a previous study.14 sample preparation was carried out by diluting 1 g of the sample in ethanol with a ratio of 1:2. moreover, 5 µl of the sample was injected into the gc/ms system in splitless mode. the stationary phase used in this study was rxi-1ms (100% dimethyl polysiloxane), which had a column length of 30 mm and a diameter of 0.25 mm. the carrier gas used was helium conditioned at a pressure of 37.1 kpa and a flow rate of 0.72 ml/min. the injector temperature, the ion source temperature, and the surface temperature were set at 250°c, 230°c, and 230°c, respectively, and the split mode was 10. the column temperature used was 70°c–270°c, with an increase program as follows: the temperature was increased from 70°c to 230°c, with an increase rate of 10°c /minutes, and then held for three minutes before finally being raised again until it reached a final temperature of 270°c. bacterial cultures of e. faecalis were grown on agar plates and broth media. the bacterial culture was inoculated on a tryptone soya agar (tsa) medium, which was then incubated at 37°c for 24 hours. furthermore, isolates were inoculated from the tsa medium into a tryptone soya broth (tsb) media containing 2% sucrose in a volume of 50 ml, followed by a 24-hour incubation at room temperature on an orbital shaker.15 antibiofilm assay for e. faecalis was carried out using the microtiter plate biofilm essay method.16 a suspension of e. faecalis in a 2% tsb sucrose medium was prepared. a 25 µl bacterial suspension was then inoculated into each well on a round bottom microplate. the ethanolic extract of neem leaves was diluted in 2% tsb sucrose media to obtain concentrations of 6.25%, 12.5%, 25%, 50%, and 75% (w/v). a 0.2% chx was used as a positive control. a 100 µl of each series concentration of neem leaf ethanol extract was added to the wells containing the bacterial suspension. a 100 µl 0.2% chx was also added as a positive control well, whereas, in the negative control, no extracts nor chx were added. the microtiter plate was then incubated for 72 hours at 37°c. the microplate was then washed three times using a sterile 200 µl of phosphate buffered saline. the washed microplate was then added with 200 µl of 96% ethanol for 15 minutes before it was drained and dried. a 200 µl of 0.1% crystal violet was then added to the dry microplate for 15 minutes before it was washed using sterile distilled water three times and dried for several minutes. a 125 µl of 30% glacial acetic acid was added and allowed to stand for 15 minutes. a total of 125 µl of 30% glacial acetic acid was then transferred to a new flat bottom microplate to determine its optical density (od) using a microtiter plate reader at a wavelength of 570 nm, and the biofilm inhibition was then calculated using the following formula: % inhibition = od negative control − od experiment od negative control x 100% quantitative data was collected for the percentage value of the area of the phytochemical component with gc/ms and the od value of the percentage of antibiofilm.16 od values were analyzed using one-way analysis of variance copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 100suhartono et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 98–103 (anova) (p ≤ 0.05 on two-tailed) and further analyzed using the turkey test to determine the differences in each ethanolic extract concentration of neem leaves. microsoft excel and spss 18 were used to tabulate and analyze the data. results the dried neem leaves were extracted using the maceration method with 96% ethanol as a solvent. the ethanol extract of neem leaves produced from 1 kg of simplicia was 114.63 g with a total extract yield of 11.46%. the resulting extracts were dark green with a thick consistency. the gc/ms analysis showed that there were 22 phytochemical compounds detected from the ethanol extract of neem leaves in this study. the detected phytochemical compounds were characterized by the presence of 22 eluted peaks starting from the fourth minute to the 29th minute (figure 1). the detected compounds generally belong to the group of alkaloids, acetals, terpenoids, and fatty acids that have an area between 0.41% and 45.33% (table 1). phytol; (e)-9-octadecenoic acid ethyl ester; and hexadecanoic acid, ethyl ester (cas) ethyl palmitate were the three most phytochemical compounds in the ethanolic extracts of the neem leaves in this study. they accounted for 45.33%, 8.35%, and 7.97%, respectively. phytol compounds had the highest peak, with an area of 45.33% and retention of 20.973 minutes, whereas (e)-9-octadecenoic acid ethyl ester had an area of 8.35% and a retention time of 21.362 minutes. the ethyl ester (cas) ethyl palmitate detected had an area of 7.97% and a retention time of 19.746 minutes. the results of the antibiofilm test of neem leaf ethanol extract against e. faecalis bacteria using the microtiter plate biofilm assay method showed the potential to inhibit the formation of biofilms. the od value of the percentage of antibiofilm obtained was analyzed using the one-way anova test (p < 0.05), which showed a significant difference in the od value of each treatment with the concentration of neem leaf ethanol extracts. further tests using the turkey analytical test were carried out to determine the concentration of neem leaf ethanol extract that has potential as an antibiofilm compared with the positive control (0.2% chx). it is evident that only the ethanolic extract of neem leaves at a concentration of 12.5% showed significant antibiofilm activity of 36.85% (table 2). table 2 also shows that the concentration of 12.5% has a relatively high average percentage of antibiofilm compared with concentrations of 6.25%, 25%, 50%, and 75%. the positive control (0.2% chx) showed a higher percentage of antibiofilm than the concentration of 12.5%, which was 68.28%. there was no significant difference between the ethanolic extracts of neem leaves at a 12.5% concentration and the positive control. figure 1. chromatogram of gc/ms analysis of neem leaf (azadirachta indica a. juss) ethanol extracts. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 101 suhartono et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 98–103 table 1. the gc/ms analysis of phytochemical compounds in the ethanolic extracts of neem leaves (azadirachta indica a. juss) peak# retention time (min) area (%) compound name chemical formula molecular weight (g/mol) 1 4.28 2.18 pyrrolidine-2,2,5,5-d4 c4h5d4n 71 2 6.494 1.25 1,1-diethoxypentane c9h20o2 160 3 12.188 1.18 1-(1-hydroxy-1-methyl-ethyl)-cyclobutanecarboxylic acid c8h14o3 158 4 15.405 3.64 (-)-caryophyllene oxide c15h24o 220 5 16.242 1.78 epiglobulol c15h26o 222 6 17.358 0.86 tetradecanoic acid (cas) myristic acid c14h28o2 228 7 17.684 1.6 tetradecanoic acid, ethyl ester (cas) ethyl myristate c16h32o2 256 8 18.317 3.72 9-hexadecenoic acid, phenylmethyl ester, (z)c23h36o2 344 9 19.053 4.25 hexadecanoic acid, methyl ester c17h34o2 270 10 19.5 5.03 n-hexadecanoic acid c16h32o2 256 11 19.746 7.97 hexadecanoic acid, ethyl ester (cas) ethyl palmitate c18h36o2 284 12 20.681 2.73 9,12,15-octadecatrienoic acid, methyl ester, (z,z,z)c19h32o2 292 13 20.72 0.95 9-octadecenoic acid, methyl ester, (e)c19h36o2 296 14 20.973 45.33 phytol c20h40o 296 15 21.31 0.92 9,12,15-octadecatrienoic acid, methyl ester, (z,z,z)c19h32o2 292 16 21.362 8.35 (e)-9-octadecenoic acid ethyl ester c20h38o2 310 17 21.603 1.87 octadecanoic acid, ethyl ester c20h40o2 312 18 24.073 0.41 hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester c19h38o4 330 19 24.373 2.25 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester (cas) bis(2-ethylhexyl) phthalate c24h38o4 390 20 25.452 0.5 9-octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester c21h40o4 356 21 28.177 1.55 oxalic acid, hexadecyl 1-menthyl ester c28h52o4 452 22 29.258 1.68 lanosterol c30h50o 426 table 2. average antibiofilm percentage of neem leaf (azadirachta indica a. juss) ethanol extract against enterococcus faecalis treatments antibiofilm activity (%) ± sd 6.25% -29.06c ± 27.66 12.5% 36.85d ± 5.53 25% -106.29b ± 24.53 50% -110.19b ± 8.43 75% -287.28a ± 29.197 positive control (0.2% chx) 68.28d ± 2.702 notes: a, b, c, and d’s different notations show a significant difference at the 95% confidence interval. discussion the search for natural-based compounds that can be applied as an antibiofilm to overcome biofilm-related infections like root canal diseases is increasing. this study elucidates the phytochemical compounds of ethanolic extracts of neem leaves (azadirachta indica a. juss) and shows their potential as an antibiofilm against e. faecalis. the extraction efficiency using 96% ethanol as a solvent in this study was quite high (i.e., 11.46%). this result is higher than other studies demonstrating a total extract yield of 9.75%17 or 3.5%.18 the different percentage values of the total extract yield indicate how large the amount of phytochemical content is in simplicia. the outcome of the chemical extraction process is influenced by the choice of solvent, duration of the extraction, temperature, ratio of the sample to solvent, and the chemical and physical properties of the sample.19 the variety in extraction efficiency is also attributed to differences in the polarity of compounds present in plants. the yield of extracts with high phytochemical compounds can be produced with the right maceration time. phytochemical compounds do not completely dissolve into the solvent if the maceration time is too short and will be degraded if the maceration time is too long.20 the high extract yield value was also influenced by the size of the dried simplicia leaves. simplicia with a small size can increase the surface area so that more contact with the solvent occurs during the soaking process.21 based on the gc/ms analysis, the phytochemical components contained in the ethanolic extract of neem leaves in this study were primarily terpenoid and fatty acid groups. phytol, (e)-9-octadecenoic acid ethyl ester, and hexadecanoic acid, ethyl ester (cas) ethyl palmitate were detected as the three most phytochemical compounds in the ethanolic extracts of the neem leaves in this study. phytol is a compound of the diterpenoid group, whereas copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 102suhartono et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 98–103 (e)-9-octadecenoic acid ethyl ester and hexadecanoic acid, ethyl ester (cas) ethyl palmitate are compounds of the fatty acid group. previous investigations found the presence of alkaloids, terpenoids, flavonoids, tannins, saponins, glycosides, and phenolics in the phytochemical screening results of neem leaves.22 internal and environmental factors can influence the diverse secondary metabolite composition of a plant. internal parameters are determined by genetic material, while external factors include nutrient content, altitude, temperature, light intensity, humidity, and ph. the observed differences in the presence of phytochemicals can also be attributed to differences in solvent polarity.17 phytol (3,7,11,15-tetramethyl-2-hexadecen-l-ol) is a type of diterpenoid that acts as a plant metabolite and can be found in vitamins k and e, as well as other forms of tocopherols. previous studies have demonstrated that phytol possesses antibacterial properties against clostridium sporogenes, e. faecalis, and sarcina lutea.23 this organic compound showed a greater ability to inhibit the growth of e. faecalis (with a minimum inhibitory concentration (mic) of less than 2 g/ml) compared with gentamicin and ampicillin (mic of 5 and 16 g/ml respectively). phytol also has the ability to reduce biofilm thickness, alter the shape of the biofilm, and significantly inhibit the production of eps in serratia marcescens.24 fatty acids are reported to have antibacterial and antibiofilm effects. palmitic acid and stearic acid have antibacterial activity of vancomycin-resistant e. faecalis with mics of 2 g/ml and 0.5 g/ml, respectively.25 there are two molecular mechanisms that can explain the antimicrobial activity of fatty acids, namely changes in biochemical functions and the loss of viability through specific interactions with sites within microorganisms or disruptions in the structure of microorganisms through nonspecific interactions, inhibiting the physiological functions of microorganisms.26 the ethanolic extract of neem leaves at a concentration of 12.5% showed the presence of antibiofilm activity (table 2). previously, studies reported the effectiveness of neem leaf extract against streptococcus sanguis biofilm in the oral cavity by reducing the plaque index and bacterial count significantly compared with the control group.27 moreover, another study using neem leaf extracts showed biofilm inhibition, a decrease in the percentage of hydrophobicity, and a decrease in the adhesion ability of pseudomonas aeruginosa using neem leaf extract.28 antibiofilm activity against e. faecalis bacteria could be caused by the phytochemical content contained in the ethanolic extract of neem leaves, such as terpenoids, fatty acids, and alkaloids. terpenoids are able to damage planktonic cells in biofilms and disrupt the integrity of bacterial cell membranes.29 medium-chain fatty acids resemble qs molecules, and they have antibiofilm activity by inhibiting qs signaling.30 alkaloids with a small composition in the ethanolic extract of neem leaves are also thought to have an antibiofilm role. alkaloids can effectively eliminate biofilms and inhibit their formation (antibiofilm activity) on both gram-positive and gramnegative bacteria.31 the concentration of neem leaf extract that was lower than 12.5% ( i.e., 6.25%) had an average antibiofilm percentage of -29.06%. this shows that low concentrations cannot inhibit the formation of biofilms. the lack of antibiofilm properties of neem leaf extracts in the test could be caused by the low concentration of bioactive compounds at low concentrations. similar findings found in other investigations signify increased biofilm development in pseudomonas aeruginosa 27853 with the addition of neem extracts.32 furthermore, other findings showed that the clinical isolate of pseudomonas aeruginosa in sub-mic of biocidal agents, such as chx, savlon, and deconex, showed biofilm induction.33 moreover, antibiotics might induce biofilm formation, particularly at low concentrations, as the drugs trigger a bacterial cell response to environmental stress that has a role in bacterial protection.34 the ethanol extract of neem leaves at higher concentrations of 25%, 50%, and 75% did not show any antibiofilm activity but were thought to induce the formation of e. faecalis biofilms, with an average antibiofilm percentage of -106.29%, 110.19%, and -287.28%, respectively. there was no significant difference between ethanolic extracts of neem leaves and the positive control. a 0.2% chx was used as a positive control because it is the gold standard of root canal irrigation solutions with the ability to degrade bacterial biofilm.35 both extract concentrations and solubility might contribute to this biofilm formation induction. the use of more concentrated extracts is complicated by the low solubility of the components as the extract becomes viscous and difficult to dissolve at high concentrations.32 other studies added that the inhibitory power of the extracts against bacteria was not proportional to the increase in the concentration of the extract.36 the high concentration of ethanol extract causes an increase in its viscosity that affects the rate of diffusion of antibiofilm compounds on bacteria. an insoluble extract can promote the development of a thicker biofilm, as the biofilm thickness is influenced by the number of solid particles in the solution.37 the presence of bis-(2-ethylhexyl) phthalate, which is a plasticizer, in neem leaf extract is thought to cause the extract solution to be less soluble in high concentrations since di-(2-ethylhexyl) phthalate on bacterial biofilms increased the production of eps, providing functional and structural integrity to biofilms, which determine their physicochemical properties.38 overall, it can be concluded that the ethanolic extract of neem leaves has an effect on inhibiting the formation of the biofilms of e. faecalis bacteria at a concentration of 12.5%. phytochemical metabolite contained in the ethanolic extracts of neem leaves might be a promising agent in treating a biofilm-mediated root canal infection of e. faecalis. further studies addressing the potential of specific active compounds from the terpenoid and fatty acid groups in neem leaves for biofilm inhibition need to be conducted. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 103 suhartono et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 98–103 references 1. de castilho al, saraceni chc, díaz iec, paciencia mlb, suffredini ib. new trends in dentistry: plant extracts against enterococcus faecalis. the efficacy compared to chlorhexidine. braz oral res. 2013; 27(2): 109–15. 2. prasetyo ep, juniarti de, sampoerno g, wahjuningrum da, budi at, hasri d, tjendronegoro e. the antibacterial efficacy of calcium hydroxide–iodophors and calcium hydroxide–barium sulfate root canal dressings on enterococcus faecalis and porphyromonas gingivalis in vitro. dent j. 2022; 55(2): 62–6. 3. estrela c, holland r, estrela cr de a, alencar ahg, sousa-neto md, pécora jd. characterization of successful root canal treatment. braz dent j. 2014; 25(1): 3–11. 4. weckwerth ph, zapata ro, vivan rr, tanomaru filho m, maliza aga, duarte mah. in vitro alkaline ph resistance of enterococcus faecalis. braz dent j. 2013; 24(5): 474–6. 5. teja kv, janani k, srivastava kc, shrivastava d, jose j, marya a, karobari mi. comparison of herbal agents with sodium hypochlorite as root canal irrigant: a systematic review of in vitro studies. valadas lar, editor. evidence-based complement altern med. 2021; 2021: 8967219. 6. espíndola lcp, do nascimento mvmr, do souto rm, colombo ap v. antimicrobial susceptibility and virulence of enterococcus spp. isolated from periodontitis‐associated subgingival biofilm. j periodontol. 2021; 92(11): 1588–600. 7. kordmahaleh fa, shalke se. bacterial biofilms: microbial life on surfaces. j biol today’s world. 2013; 2(5): 242–8. 8. jakubovics ns, goodman sd, mashburn‐warren l, stafford gp, cieplik f. the dental plaque biofilm matrix. darveau rp, curtis ma, editors. periodontol 2000. 2021; 86(1): 32–56. 9. saber se-dm, el-hady sa. development of an intracanal mature enterococcus faecalis biofilm and its susceptibility to some antimicrobial intracanal medications; an in vitro study. eur j dent. 2012; 6(1): 43–50. 10. lekshmi ncjp, sowmia n, viveka s, brindha jr, jeeva s. the inhibiting effect of azadirachta indica against dental pathogens. asian j plant sci res. 2012; 2(1): 6–10. 11. jahan m, abuhena m, azad ak, karim mm. in vitro antibacterial and antibiofilm activity of selected medicinal plants and spices extracts against multidrug resistant pseudomonas aeruginosa. j pharmacogn phytochem. 2018; 7(3): 2114–21. 12. nimbulkar g, garacha v, shetty v, bhor k, srivastava k, shrivastava d, sghaireen m. microbiological and clinical evaluation of neem gel and chlorhexidine gel on dental plaque and gingivitis in 20-30 years old adults: a randomized parallel-armed, double-blinded controlled trial. j pharm bioallied sci. 2020; 12(5): s345–51. 13. suhartono s, ismail ys, muhayya sr, husnah m. ethanolic extracts of moringa oleifera leaves inhibit biofilm formation of vibrio alginolyticus in vitro. iop conf ser earth environ sci. 2019; 348(1): 012018. 14. suhendar u, sogandi s. identifikasi senyawa aktif ekstrak daun cengkeh (syzygium aromaticum) sebagai inhibitor streptococcus mutans. al-kauniyah j biol. 2019; 12(2): 229–39. 15. melo ldr, ferreira r, costa ar, oliveira h, azeredo j. efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model. sci rep. 2019; 9(1): 6643. 16. suhartono s, ismail ys, muhayya sr. the interference of moringa oleifera leaf extracts to modulate quorum sensing-facilitated virulence factors. biodiversitas j biol divers. 2019; 20(10): 3000–4. 17. hikaambo cn, kaacha l, mudenda s, nyambe mn, chabalenge b, phiri m, biete ll, akapelwa tm, mufwambi w, chulu m, kampamba m. phytochemical analysis and antibacterial activity of azadirachta indica leaf extracts against escherichia coli. pharmacol pharm. 2022; 13(1): 1–10. 18. fitriah r. uji aktivitas antibakteri ekstrak nheksana , etil asetat dan etanol daun mimba (azadirachta indica a. juss ) terhadap streptococcus mutans. borneo j pharmascientech. 2017; 1(2): 1–10. 19. sheneni vd, usman os, musa q. phytochemical constituent, percentage yield and phenolic content estimation of different solvent system of carica papaya leaves. korean j food heal converg. 2018; 4(2): 17–23. 20. kemit n, widarta iwr, nocianitri ka. pengaruh jenis pelarut dan waktu maserasi terhadap kandungan senyawa flavonoid dan aktivitas antioksidan ekstrak daun alpukat (persea americana mill ). itepa j ilmu teknol pangan. 2016; 5(2): 130–41. 21. nurnasari e, prabowo h. pengaruh ukuran sampel dan lama waktu destilasi terhadap rendemen minyak atsiri tembakau lokal indonesia. bul tanam tembakau, serat miny ind. 2020; 11(2): 47–57. 22. elaigwu ed, ogo oa, efiong ee, oche og. effects of ethanolic leaf extracts of neem (azadirachta indica) on oxidative stability of palm oil. res j phytochem. 2019; 13(1): 1–10. 23. pejin b, kartali t, stanimirovic b, karaman m. phytol may inspire new medicinal foods for the treatment of heart disease. asian j chem. 2014; 26(23): 8261–2. 24. srinivasan r, mohankumar r, kannappan a, karthick raja v, archunan g, karutha pandian s, ruckmani k, veera ravi a. exploring the anti-quorum sensing and antibiofilm efficacy of phytol against serratia marcescens associated acute pyelonephritis infection in wistar rats. front cell infect microbiol. 2017; 7: 498. 25. cheung lam ah, sandoval n, wadhwa r, gilkes j, do tq, ernst w, chiang s-m, kosina s, howard xu h, fujii g, porter e. assessment of free fatty acids and cholesteryl esters delivered in liposomes as novel class of antibiotic. bmc res notes. 2016; 9(1): 337. 26. devan k, peedikayil f, chandru t, kottayi s, dhanesh n, suresh kr. antimicrobial efficacy of medium chain fatty acids as root canal irrigants: an in vitro study. j indian soc pedod prev dent. 2019; 37(3): 258. 27. chava vr, manjunath s, rajanikanth a, sridevi n. the efficacy of neem extract on four microorganisms responsible for causing dental caries viz streptococcus mutans, streptococcus salivarius, streptococcus mitis and streptococcus sanguis: an in vitro study. j contemp dent pract. 2012; 13(6): 769–72. 28. harjai k, bala a, gupta rk, sharma r. leaf extract of azadirachta indica (neem): a potential antibiofilm agent for pseudomonas aeruginosa. pathog dis. 2013; 69(1): 62–5. 29. soviati n, widyarman as, binartha cto. the effect ant-nest plant (myrmecodia pendans) extract on streptococcus sanguinis and treponema denticola biofilms. j indones dent assoc. 2020; 3(1): 11–5. 30. lee j, kim y, khadke sk, lee j. antibiofilm and antifungal activities of medium‐chain fatty acids against candida albicans via mimicking of the quorum‐sensing molecule farnesol. microb biotechnol. 2021; 14(4): 1353–66. 31. jain a, parihar dk. antibacterial, biofilm dispersal and antibiofilm potential of alkaloids and flavonoids of curcuma. biocatal agric biotechnol. 2018; 16: 677–82. 32. kaverimanian v, heuertz rm. effects of neem extracts on formed biofilm of pseudomonas aeruginosa. faseb j. 2020; 34(s1): 1–1. 33. hemati s, kouhsari e, sadeghifard n, maleki a, omidi n, mahdavi z, pakzad i. sub-minimum inhibitory concentrations of biocides induced biofilm formation in pseudomonas aeruginosa. new microbes new infect. 2020; 38: 100794. 34. bernardi s, anderson a, macchiarelli g, hellwig e, cieplik f, vach k, al-ahmad a. subinhibitory antibiotic concentrations enhance biofilm formation of clinical enterococcus faecalis isolates. antibiotics. 2021; 10(7): 874. 35. martínez-hernández m, reda b, hannig m. chlorhexidine rinsing inhibits biofilm formation and causes biofilm disruption on dental enamel in situ. clin oral investig. 2020; 24(11): 3843–53. 36. sari er, nugraheni er. uji aktivitas antifungi ekstrak etanol daun cabai jawa (piper retrofractum) terhadap pertumbuhan candida albicans. biofarmasi j nat prod biochem. 2013; 11(2): 36–42. 37. sivadon p, barnier c, urios l, grimaud r. biofilm formation as a microbial strategy to assimilate particulate substrates. environ microbiol rep. 2019; 11(6): 749–64. 38. li y, zhang p, wang l, wang c, zhang w, zhang h, niu l, wang p, cai m, li w. microstructure, bacterial community and metabolic prediction of multi-species biofilms following exposure to di-(2ethylhexyl) phthalate (dehp). chemosphere. 2019; 237: 124382. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p98–103 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p98-103 �� vol. 42. no. 1 january–march 2009 inhibitory effect of n-hexane: ethyl acetate fraction from artemisia vulgaris l. on cell culture of oral epithelial carcinoma ira arundina department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: sudamala herb (artemisia vulgaris l.) is often used in society as an anti tumor for organs of digestive system including oral cavity. nevertheless, there are still no further scientific researches on active materials which can be used as anti carcinogen in oral cavity. most of anti carcinogens are actually obtained from the genus artemisia. moreover, in indonesia, the species of the genus artemisia that grows the most is artemisia vulgaris l. the problem of this research, however, is that the effect of n-hexane fraction: ethyl acetate from artemisia vulgaris l. towards the decreasing of oncogene in oral squamous cell carcinoma is still indefinite. purpose: the objective of this research is to explain the effect of giving n-hexane : ethyl acetate (3:7) fraction containing terpenoid from artemisia vulgaris l. towards the decreasing of oncogene in oral epithelial carcinoma cell line. methods: the method of this research is laboratory experimental research by using squamous cell carcinoma of oral cavity on cell culture. the inhibitory percentage test in vitro, furthermore, is taken during the analysis. the result then is analyzed by probit analysis with drawing relation curve between the inhibitory percentage and concentration. result: the result of n-hexane : ethyl acetate (3:7) fraction containing terpenoid from artemisia vulgaris l. has the smallest ic50, 3.902 μg/ml, less than 20 μg/ml suitable with nci criteria; thus, it can potentially be used as anti carcinogen. conclusion: there is the decreasing of oncogenes after being given n-hexane : ethyl acetate (3:7) fraction containing terpenoid from artemisia vulgaris l. towards oral epithelial carcinoma cell line. key words: n-hexane: ethyl acetate fraction, artemisia vulgaris l., oral squamous cells carcinoma correspondence: ira arundina, c/o: departmen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132. e-mail: arundinafkg@yahoo.com. introduction carcinoma in oral cavity is one of the tumor cases that often occur around the world. the tumor in oral cavity and oropharynx is found in about 3% of one million tumor cases detected in the united states during one year.1 in indonesia, on the other side, the rate of carcinoma cases in oral cavity is high enough which is on the sixth rank of all carcinoma cases that often occurrs and increases in every year. oral squamous cell carcinoma (oscc) in oral mucosa epitel is a kind of cancer often found in oral cavity about 90%.2 in addition, some herbal medicine discoveries showing pharmacology effects encourage researchers to exploit bioactive materials from herbs. the basic reason of this research is that herbs are often used by the society for curing cancer since they are safer, cheaper, and more available. sudamala herb (artemisia vulgaris l.), for instance, is often used in the society as anti tumor for organs of digestive system including oral cavity. nevertheless, there are still no further scientific researches on active materials which can be used as anti carcinogen in oral cavity. in indonesia the species of the genus artemisia that grows the most is artemisia vulgaris l. artemisia vulgaris l. called sudamala. this herb can be found growing wildly in fields, in forests, and in humid areas which are rich of humus. a lot of sudamala herbs used in the society is empirically useful as anti inflammation, analgesic, and anti cancer for digestive system and breast.3 in fact, there is no a lot of researches on artemisia vulgaris l. as anti carcinogen. however, with ethnopharmacology and chemotaxonomy approach it can be proved artemisia vulgaris l. as anti carcinogen. ethnopharmacology, moreover, is theoretical research report �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 37-40 approach using empiric indications about the use of herbal materials as medicine. on the other hand, chemotaxonomy is theoretical approach using another herb from the same genus proven that it contains active extracts. artemicinin, furthermore, is an isolate active material from artemisia annua l. used as anti carcinogen.4 artesunate derivative artemicinin from artemisia annua l. is also proved that it can prevent the growth of cancer cell in colon.5 the herbal extract of artemisia argyi l. containing terpenoid and flavonoid can also prevent cervix carcinoma and has citotoxic effects on he la cell culture.6 some natural compounds, moreover, are proven that they can prevent the interaction between bp-7,8-diol-9,10-oxide carcinogen and dna through some mechanisms. those compounds have polyphenol structure or are classified into flavonoid and terpenoid groups found a lot in some kinds of herbs.7 the purpose of this research, in addition, is to prove the inhibitory effect of n-hexane: ethyl acetate fraction from the n-hexane extract of artemisia vulgaris l. containing terpenoid towards oral ephitelial cell line carcinoma. in order to measure the inhibitory effect, the formulation of inhibitory percentage is used in analyzing the result. afterwards, probit analysis is used for measuring ic50 by drawing relation curve between inhibitory percentage and concentration. ic50 is a measure of the effectiveness of a compound which inhibitory percentage of cancer cell is 50%. the research result, finally, can be used as therapy development base using active materials of terpenoid compound from artemisia vulgaris l. in order to cure cancer, especially oral mucosa carcinoma. materials and methods the material of this research is artemisia vulgaris l. herb obtained and determined in balai konservasi of botanical garden purwodadi, pasuruan. this herb is taken from a plant about 24 months old and 50 cms tall. the herb, taken from the edge of the leaves to the tip of the leaves, is on 800 ms above the sea level. the herb then was cleaned from other herbs and sludge, washed, and dried in open air without being exposed to the sun directly. after being dried, the herb was milled by miller, and sifted by a powder sifter in order to make the active essence of the herb could easily react with solvent so that it could completely extracted. the powder of the herb then was saved into a closed vessel. the extract of artemisia vulgaris l. herb was made by maceration, a process of soaking artemisia vulgaris l. powder into n-hexane solvent in a closed vessel at ambient temperature for 2 x 24 hours, and stringing it at the same time. it then was sifted by buchner sifter, and its filtrate and pellet were macerated again for 6 times with new solvent. maceration would be stopped if after the orientation of terpenoid concentration with thin layer chromatography (tlc) using mobile phase (eluen) n-hexane : ethyl acetate = 1:2, the anisaldehyde stain of sulfate acid was not red purple on tlc pellet. the result of the maceration was collected and evaporated with rotavapor at low pressure until it could not be evaporated again. thus, the mass of condensed extract was obtained. the remains of solvent in the condensed extract then were evaporated in acid cabinet so that its result called as dried extract of n-hexane.8 the extract of n-hexane identified contains terpenoid, moreover, was fractionated by using column vacuum chromatography. the stationery phase of silica gel 60 (merck) was put into a dried sintered glass. the filling process was done until reaching 4–5 cm tall for each column with 2.5–3 cm diameter. hexane extract then was mixed with silica gel and poured on to sintered glass which had been watered by solvent. afterwards, it was closed again with silica gel 60, and then was eluted by mobile phase n-heksan-ethyl acetate with polarity increasing. mobile phase used was n-hexane : ethyl acetate (10:0, v/v), n hexane : ethyl acetate (9:1, v/v), n-hexane : ethyl acetate (8:2, v/v), n hexane : ethyl acetate (7:3,v/v), n-hexane : ethyl acetate (6:4,v/v), n-hexane : ethyl acetate (5:5,v/v), n-hexane : ethyl acetate (4:6,v/v), n-hexane : ethyl acetate (3:7, v/v), n-hexane : ethyl acetate (2:8,v/v), n-hexane : ethyl acetate (1:9,v/v), n-hexane : ethyl acetate (0:10,v/v). those 11 fractions produced then will be tested in vitro for their capabilities as anti carcinogen.8 furthermore, the inhibitory percentage of those 11 fractions was measured in vitro by using epithelial carcinoma cell line in oral cavity from american type culture collection (atcc) no ccl-17. this phase was done into laminar air flow cabinet (lafc) with three time replication. oral epithelial carcinoma cell line was cultured into dulbecco’s modification of eagles medium (dmem) 10% fetal bovine serum (fbs) 10 ml media, 2 ml penicillin streptomycin, and 0.5 ml fungizone in 100 ml dmem, and then was harvested by trypsine–edta 0.25%. afterwards, it was put into centrifuge tube and was centrifuged in five minutes, 1500 rpm. the result then was measured with hemocytometer, and was put into 20,000 cells/hole. the cells were put into 100 ml media in microwell plate. furthermore, 10 mg fraction of solution test (sample) was added and dissolved into 100µl methanol through the series of dilution until the concentration becomes 2.5, 5, 10, 20, and 40 mg/ml after the optimation.5 moreover, incubation process was done by using an incubator, 95% o2 and 5% co2, with temperature at 37° c for 24 hours. four hours before the incubation period was over, mtt (3-(4,5 dimethylthiazol-2yl)2-5 diphenyltetrazolin bromide) had been added about 5 µg/ml for each holes, and the incubation process then was continued again. after the incubation process had finished, centrifugation was taken. afterwards, 1 ml isopropil alcohol was added in order to destroy cell. vortex 30 rpm then was used for 5 minutes. in order to measure the absorbance of supernatant, colorimetry (elisa reader) with 550 nm long wave was used, and then the analysis of the result was taken by using the following formulation: ��arundina: inhibitory effect of n-hexane od control – od sample % inhibitory= × 100% od control note: od = optical density in order to determine the ic50, probit analysis was used by drawing the relation curve between inhibitory percentage and concentration. the ic50 was a measure of the effectiveness of a compound which inhibitory percentage of cancer cell is 50%.9 among 11 fractions that have been tested for their ability as anti-carcinogen in vitro, it was found that n-hexane : ethyl acetate (3:7,v/v) fraction has the lowest value of ic50 i.e. 3,902 mg/ml less than 20 mg/ml which is appropriate with criteria of national cancer institute (nci). therefore, it is potential to be used as anti-carcinogen. since the value of ic50 is 3,902 mg//ml, this ethyl acetate has the highest possibility as anti-carcinogen discussion researches of traditional medicines, which studied on herbal plants, are continuously done and recently the numbers of those researches are increased. in the contrary, only a few results of researches studying herbal plants are used as medicines in medical services. medicines consumed in society must meet some requirements; safe, valuable, and standardized. thus, pre-clinical and clinical experiments are undergone to examine the herbal plants. the pre-clinical experiment includes utility test based on experimental research that can be undergone either by in vivo or in vitro. in this research, experiment of in vitro using cell culture was applied to determine the fraction material which has the lowest value of ic50. the value of ic50 indicates the level of material experiment where the percentage of inhibitory towards cancer cell is 50%. in this experiment, radioactive material was not used but reagent that was reduced by metabolite of living cell was used, forming blue formazan called mtt or thiazolyl blue (3-(4,5-dimethylthiazol2yl)2-5-diphenyl tetrazolium bromide). process of mtt usage by redox reaction did not use radioactive material and it was more uncomplicated and harmless. furthermore, the result of measurement was the same as the result using radioactive material. the living cell would engulf the yellow mtt reagent and would be reduced by metabolite of cell which later formed blue crystal. the intensity of its color –blue– would be measured by calorimetry at the wave length 550 nanometer after the cell had been obliterated by isopropyl-alcohol solution. meanwhile, the intensity for yellow was equal to the number of living cell. the experiment used mtt method, which had to be completed cautiously especially when it came to the process of washing cell otherwise mtt on supernatant or there would be no cells in the fetched. these can cause error on reading elisa calorimetry.9 the experiment result in vitro of n-hexana: ethyl acetate fraction artemisia vulgaris l. is determined from the value of ic50 using probit analysis by drawing relation curve between the percentage of inhibitory and the concentration. from the analysis, n-hexane : ethyl acetate (3:7,v/v) fraction has the lowest value of ic50 of all fractions i.e. 3,902 mg/ml seen in the table 5.1. nci (national cancer institute) has determined criteria that a material has characteristics as anti-carcinogen if ic50 is less than 20 mg/ml for extract and fraction.10 of 10 fractions, the value of ic50 for n-hexane: ethyl acetate (3:7,v/v) fraction from n-hexane extract of artemisia vulgaris l. is the lowest so it has the highest ability as anti-carcinogen. sudamala herb is frequently used as anti tumor in organs of digestive system including in oral cavity but there has been no research studying active substance which has a role table 1. the average percentage of inhibitory of cancer cell in vitro and the value of ic50 after being given n-hexane : ethyl acetate fraction materials the average percentage of inhibitory value replication dosage dosage dosage dosage dosage ic50 2,5 mg/ml 5 mg/ml 10 mg/ml 20 mg/ml 40 mg/ml mg/ml n-hexane : ethyl acetate(10:0, v/v) 3 31,549 31,59090 35.95 36,046 35,960 54,462 n-hexane : ethyl acetate (9:1, v/v) 3 32,510 37,913 38,66 43,850 43,416 38,782 n-hexane : ethyl acetate (8:2, v/v) 3 34,980 39,316 40,297 45,030 44,487 37,160 n-hexane : ethyl acetate (7:3, v/v) 3 25,503 26,149 26,86 46,330 37,220 44,006 n-hexane : ethyl acetate (6:4, v/v) 3 39,800 40,070 47,87 51,050 49,300 29,324 n-hexane : ethyl acetate (5:5, v/v) 3 47,330 50,070 53,853 64,379 61,310 16,936 n-hexane : ethyl acetate (4:6, v/v) 3 42,3630 55,973 64,443 83,050 82,900 9,278 n-hexane : ethyl acetate (3:7, v/v) 3 59,290 64,155 81,675 85,486 83,429 3,902 n-hexane : ethyl acetate (2:8, v/v) 3 44,185 51,241 72,764 83,759 81,600 8,756 n-hexane : ethyl acetate (1:9, v/v) 3 40,792 54,496 60,918 83,143 74,743 10,837 n-hexane : ethyl acetate (0:10, v/v) 3 38,529 46,443 63,48 81,550 68,010 12,934 �0 dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 37-40 as anti-carcinogen in oral cavity. many active substances as anti-carcinogen are found in genus artemisia while the species that mostly grown in indonesia is artemisia vulgaris l. in this research, extraction process of artemisia vulgaris l applied meceration because the use equipment was simple and did not need any heat in order to avoid the compound being reduced by heat. extraction using n-hexane was applied in isolation of terpenoid compound found in artemisia vulgaris l that has a role as anti-carcinogen. general characteristic of non-polar terpenoid required non polar solution of n-hexane so it could take terpenoid compound.7 the fractination process using solution of n-hexane: ethyl acetate aimed to obtain terpenoid compound. terpenoid is a chemical compound derived from plant which has isoprena molecule (c5) and its carbon pattern is composed from connecting 2 or more (c5) units. terpenoid is classified into several kinds; isoprena (c5), monoterpenoid (10), sesquiterpenoid (c15), diterpenoid (c20), triterpenoid (c30), tetraterpenoid(c40), polyisoprene (cn). some terpenoid compounds from genus artemisia that have been studied have a function as anti-carcinogen for example artemisinin. artemisinin belongs to sesquiterpene lactone from artemisia annua l. that is able to inhibit breast cancer cell by in vitro by means of enhancing the activation of p53 wild.11 sesquiterpene lactone belongs to terpenoid compound that has potential effect of anticarcinogen and it can induce apoptosis on cancer cell in vitro. besides, it can metastasize various cancers in animal.7 terpenoid and saponin affect the permeability of cell membrane. the increase of its permeabilty can cause the liquid electrolite on the outside the cell will be easy to get into cells. as the result, the cell will be seperated into several fragments.7 this can explain one of mechanisms of terpenoid compounds in obliterating material. according to this research, it can be concluded that n-hexane: ethyl acetate (3:7,v/v) fraction which is derived from extract of n-hexane artemisia vulgaris l., has the lowest value of ic50 3,902 mg/ml of 10 fractions. consequently, it has the highest ability as anti carcinogen it is suggested that the findings of the research can be used as basis to compose a medicine containing n-hexane: ethyl acetate(3:7,v/v) fraction which later is used as anticarcinogen in oral cavity. references 1. greenberg ms, glick m. burket’s oral medicine diagnosis and treatment. hamilton bc decker inc; 2003. p. 194–201. 2. pindborg jj. kanker dan prakanker rongga mulut. jakarta: egc; 2000. p. 1–138. 3. dalimartha s. ramuan tradisional untuk pengobatan kanker. seriseri agrisehat. jakarta : penebar swadaya; 2003. p. 20–70. 4. kim dh, na hk, oh ty, kim wb, surh yj. eupatilin aeupatilin a pharmacologically active flavone derived from artemisia plants induces cell cycle arrest in ras transformed human mammary epithelial cells. biochemical pharmacology 2004; 68:1081–7. 5. efferth t, sauerbrey a, olbrich a, gebhart e. molecular modes of action of artesunate in tumor cell lines. molecular pharmacology 2003; 64(2):382–94. 6. dalimartha s. atlas tumbuhan obat indonesia. jilid 1. jakarta: trubus agriwidya; 2005. p. 7–12. 7. zhang s, won yk, ong cn, shen hm. anti cancer potential of sesquiterpene lactones bioactivity and molecular mechanisms. curr med chem anti canc agents 2005; 5(3): 239–49. 8. elferaly, farouk s, elsohly, hala n. method for the isolation of artemisinin from artemisia annua. j chrom 1990; 355:448–50. 9. freshney ri. culture of animal cells. a manual of basic technique. 4th ed. new wiley-liss inc; 2000. p. 329–43. 10. suffness m pezzuto jm. assays related to cancer drug discovery. methods in plant biochemistry assays for bioactivity. academic press; 1991 6:71–133. 11. singh np, lai h. selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells. life sciences 2001; 70:49–56. mkg vol 42 no 2 april 2009.indd 104 vol. 42. no. 2 april–june 2009 prognostic value of molecular markers of oral pre-malignant and malignant lesions peter agus department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract background: the representation of oral cancer and precancerous lesions is often undetected until at later stage and the survival rate of oral cancer has remained essentially unchanged over the past three decades. over 90% of these tumors are squamous cell carcinoma. the american cancer society estimates that among 28,900 new cases of oral diagnosis in 2002, nearly 7,400 people will die from this disease. oral pre-malignant and malignant lesions have multi-step process both at phenotype and genetic levels that influence tumor behavior and genetic mutations. purpose: the aim of this presentation was to review the current knowledge of prognostic value of tumor marker in order to achieve early detection, prognostic value, proper and accurate treatment of oral cancer. reviews: technological advances in molecular biology have greatly increased the number of new molecular markers that can be detected by molecular analysis such as immunohistochemistry (ihc), polymerase chain reaction (pcr) and surgical margin analysis that may increase prognosis and treatment of oral cancer. the result of most valuable tumor markers is twenty nine divided into four groups according to their function such as enhancement of tumor growth, tumor suppression and anti tumor defense, including immune response and apoptosis, angiogenesis, tumor invasion and metastatic potential, including adhesion molecules and matrix degradation. conclusion: in general the conclusion is that the location of markers within the tumor and not the quantitative assessment is as same as emphasized. especially, the analysis of new molecular markers have been used to be of great importance for early detection, surgical margin analysis, prognostication and treatment of oral pre-malignant and cancerous lesion. key words: tumor markers, oral pre-cancer, oral cancer, surgical margin, prognostic correspondence: peter agus, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: peteragus@yahoo.com review article introduction oral and oro-pharynx cancers are found in 28,900 new cases and 7400 victims were dead in 2002 in united states, which 3% found on men and 2% found on women among all malignancy of the body.1,2 it was reported that in the united states, one patient of oral cancer with oral squamous cell carcinoma dead every hour in every day, so the number of recurrences, morbidities, and mortalities tends to increase.3 if the oral cancer is detected in the early stage, the percentage of live expectation will be about 80–90% while if detected in the advanced stage the percentage will be about 50% within five years. unfortunately, there is still no current information about the prevalence of oral cancer from badan registrasi kanker indonesia. however, based on medical records of rsud dr. sutomo surabaya in poli kepala leher rs dr. sutomo from 1983 to 1992, there are 3.3% patients of head and neck tumor.4 the number of mortalities is relatively high in all parts of the world, there are 5.1% of oral cancer cases in early stage and 76.3% in advanced stage.4 the high rate of mortality, morbidity, and the worse prognosis of oral cancer are actually the world’s and clinician’s problem of cancer nowadays.4,5 unfortunately, the basic molecular pathogenesis of oral squamous cell carcinoma through multi-step process is still clinically not fully understood, so the early detection of oral cancer is only based on clinical diagnosis. the histopathology examination (hpa) also causes mistakes in diagnosis, recurrence, and mistakes in therapy.4-6 105agus: prognostic value of molecular markers the other problem is surgical margin analysis. in this approach, in order to obtain proper surgical margin of tissue that is free of tumor by using frozen section examination microscopically, the different results between clinical diagnosis and hpa diagnosis always occur. it indicates that it does not guarantee whether it will have no tumor recurrence or will worsen oral cancer prognosis. thus, it can increase the rate of morbidity, mortality and five years survival rate into 50%. it means that there has not been any significant progress in the last two decades.3,6 actually, the new approach of oral cancer treatment has radically changed in the last two decades since there were more understanding about molecular changing in oral carcinogens through multi-step process and advancement of technology in molecular biology so the new diagnosis can be obtained through immunohistochemistry (ihc), polymerase chain reaction (pcr) and surgical margin analysis. as a result, the target of molecular markers obtained is about 29 molecular markers in patients of either pre-malignant lesion or oral carcinoma. those molecular markers are classified into four based on the functions like: enhancement of tumor growth, including acceleration of proliferation and cell cycle; tumor suppression and anti tumor defense, including immune response and apoptosis; angiogenesis; tumor invasion and metastatic potential including adhesion molecules and matrix degradation. besides, nowadays the surgeons, especially oral and maxillofacial surgeons tend to use surgical margin analysis to make molecular diagnosis that is by using molecular marker, p53, so that the surgical margin that is free of tumor can be determined in order not only to eliminate tumor recurrence and the rate of morbidity and mortality, but also to use as prognostic indicator and therapy of pre-malignant lesion, especially for leukoplakia and oral cancer.3,7,8 epidemiology based on epidemiology study, leukoplakia in oral cavity, signed by white spots, is found in about 5–15%; dysplasia is found increasing about 31.4%. they are potentially considered to be oral cancer. meanwhile, oral cancer prevalence especially oral squamous cell carcinoma in some countries including indonesia is about 3–20% of all cancer cases.9 the etiology of leukoplakia pre-malignant lesion is related to tobacco use (severe smokers), betelchewing, marijuana use in young patients, severe alcohol consumption, and candida albicans.10 on the other hand, the etiology of oral cancer is still not identified clearly. nevertheless, the main risk factor is multi-factorials caused by external factors like tobacco, alcohol consumption, carcinogenic materials, radiation, and virus, especially hpv 16 (about 22%) and hpv 18 (about 14 %).11 dietary factors like low fruit and vegetable consumption can increase the risk factor of oral cancer.12 the internal factors like multi-steps and complex genetic change cause the variation of clinical type oral cancer. it depends on the high risk factor in patients such as age, topography location, and race.13,14 however, with the growth of molecular biology in the last three decades and supported by the techniques of genetic engineering, now cancer has obviously been proven that it has genetic basis. the etiologic factors of oral cancer is not only showed by the changing of molecular controlling through many lines, especially for g1-s phase of cell cycle, but also correlated with the description of phenotype, clinical description, and histopathology.15 diagnosis of pre-malignant lesions and oral cancer the description of pre-malignant lesions is related to erythroplakia and leukoplakia. erythroplakia is divided into granular and non-granular erythroplakia. however, erythroplakia are rarely found, so pre-malignant lesions is focused on leukoplakia which is divided into 2 types, homogenous and non homogenous. it is divided again into thin leukoplakia, thick leukoplakia, granular leukoplakia, verruciform leukoplakia, verrucous proliferative leukoplakia, and speckled leukoplakia.16 some researchers, furthermore, point out that the frequency of dysplasia lesion or cancerous changing leukoplakia lesion is about 15.6–39.2%, showing dysplasia degree started from mild, moderate, and severe. non homogenous type of leukoplakia like nodule, erythematous, and/or followed by verrucous components tends to be more cancerous than that from homogenous type of leukoplakia. meanwhile, verrucous proliferative leukoplakia shows type of aggressive lesion that almost tends to be cancerous.16 even though the risk of non homogenous type of leukoplakia is four to five times to be malignant compared to the risk of homogenous type of leukoplakia, only 5% of cases have just been reported. thus, leukoplakia cases considered to be malignant need more accurate alternative diagnosis than histopathology examination, an analysis of molecular marker detection either with immunohistochemical technique or with the most sophisticated molecules nowadays.16 the process of malignancy transformation takes a long time to detect premalignant tumor. besides, there is a multi-step theory under laying the malignancy process in oral epithelium either in phenotypic level or genetic level, so it can detect malignancy stage with different differentiation degree in pre-malignant lesion and the early stage of oral cancer that is difficult to detect by clinical observation.17 molecular changing in carcinogens cancer occurs by accumulation of genetic changing within a cell. the genetic changing shows a degree of genetic destruction which reflects a degree of tissue destruction because of the stimulus of carcinogenic materials in long term.17 carcinogen of oral cancer is a multi-step process involving many genetic events so that it can change normal function of oncogene and tumor suppressor gene. besides, this multi-step process can improve the production of growth factors or a number of nucleolus cell receptor, and then the increasing of intracellular messenger signs and production 106 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 104−108 of transcription factors. genetic events influenced by inactivity of tumor suppressor genes, moreover, shows a capability of cell phenotype that can cause the increasing of cell proliferation signed by the lost of cell formation, infiltration in local tissue, and tumor spread to the further location from its primer tumor.18 therefore, the study in other to obtain some potential molecular markers must be related with the malignancy growth like premalignant lesions and oral cancer, so it can predict the degree of recurrence, the cancer spreading to lymphoid gland and bone, and further metastases.19 molecular marker of patients with oral cancer identification of molecules that can potentially do malignancy transformation shows that the increased number of molecular marker has correlated with gradation, the severity of tumor, prognosis and cause of cancer. molecular marker for cell proliferation has been applied as molecular indicator for analyzing severity without clinically analyzing tumor description or behavior. the malignancy characters detected by the molecular marker hopefully can clinically improve the understanding of many variations of tumor description and behavior while can help the prognosis estimation of patients with oral cancer. then, the molecular marker relating with malignancy transformation hopefully can also show the possibility of conducting therapy without any surgery. molecular target of this treatment is achieved by conducting anti sense therapy or gene therapy. actually, the number of the studies concerning with molecular marker is very high, but they are still for the diagnosis and therapy of oral cancer, and there is also lack of knowledge about prognosis values. the number of molecular marker in the recent studies concerning with molecular marker is temporarily about 29 molecular markers, classified into 4 groups with different functions, a) enhancement of tumor growth; b) tumor suppression and anti tumor defense; including immune response and apoptosis; c) angiogenesis; d) tumor invasion and metastasis potential; adhesion molecules and matrix degradation.5,20 the group of molecular markers relating with enhancement of tumor growth consists of 9 molecular markers, which are a) epithelium growing factor (egf) and receptor of epithelium growing factor (egfr, c-erb-1 or her-2/neu); b) cyclin (cyclin a, b1,d1 e); c) proliferation cell nucleus antigen (pcna); d) ki 67/ mib; e) argyrophylic nucleolar organizer-region associated protein (agnor); f) skp2; g) bcl2/bag-1; h) heat shock protein (hsp27 and hsp70); and i) telomerase. the gene group relating with tumor suppression and anti tumor defense consists of 7 molecular markers, which are a) protein retinoblastoma (prb); b) cyclin dependent kinase inhibitors (cdkis)-p15, p16, p21, p27; c) p53; d) bax; e) fas/fasl; f) ζ–chain (zeta chains); and g) dendrite cells s 100/p55. next, the group relating with angiogenesis consists of 3 molecular markers, which are a) vascular endothelial factor/receptor (vegf/ vegf-r); b) nitric oxide synthase type 2 (nos2); c) platelet-derived endothelial cell growth factor (pd-ecgf). and, the group relating with tumor invasion and metastasis potential consists of 6 molecular markers, which are matrixmetallo-protease (mmps); cathepsines; integrins; cadherins and catenins; desmoplakin/plakoglobin; and ets-1.7,21 furthermore, the characterization of molecular markers in pre-malignant lesions and oral cancer, that are increasing related with the recurrence intensity, progression, and oral cancer prognosis that has not still finished yet and has many variations of molecular marker improvement, shows that carcinogenic process with genetic changing factors that are multi-steps and complex acquiring system control of activity and molecular function through the number of arrangements for cell behavior and cell coordination within a tissue or organ with cancer.7,21 nevertheless, some of other researchers tend to use surgical margin analysis with pcr technique in order to determine the surgical margin that is free from tumor and not detected by hpa examination using p53 as molecular marker detector since in that case there is recurrence in local and regional tissue. in other words, the use of molecular marker, p53, and other tumor markers is to detect whether there is gene mutation as the malignancy indicator or not. thus, the use of tumor marker p53 and other markers is can be useful as the marker for the surgical margin analysis of the pre-malignant lesions that is potential to be oral cancer. the application of molecular technique with molecular marker can also be used optimally in order to obtain the result as fast as frozen section analysis technique.22-24 procedure of immunohistochemistry technique (ihc), polymerase chain reaction (pcr), and surgical margin analysis on oral pre-malignant and malignant lesions immunohistochemistry technique (ihc) examination on proliferated cells using proliferating cell nuclear antigene (pcna) shows 100% cells of positive tumor. the increased pcna expression has correlation with other proliferation markers like the percentage of s. phase friction, ki 67, and mitotic count. however, some of researchers do not have the same opinion because they think that pcna is involved in the process of dna repair for some tumors. because of this, pcna remains controversial and it needs to be considered as proliferation marker in particular tumors.20,21 identification of immunohistochemistry on tumor marker of specimen is taken from oral mucosa swab. specifically it is gained from cytokeratin. profile of cytokeratin expression taken from cytology examination indicates that there is information on differentiation status of cell especially cytokeratin markers such as k8 and k19 which are useful for definitive indicators of undetected malignancy by other dna profiles.22 analysis of immunohistochemistry applies proliferation index of pcna and agnor on each cytology specimen to evaluate the presence of ki-67 expression before and after radiotherapy using dosage 24 gray on 43 patients of oral squamous cell carcinoma. the result of ki-67 expression shows that there are 10 cells of positive tumor and label index of proliferation varies from 0.1% to 0.01%. analysis toward the number of cells and nucleus 107agus: prognostic value of molecular markers activity (agnor) is applied on patients who have high risk to be infected oral cancer (severe smokers). besides, it is reported that agnor analysis on cytology specimen can be used as method of regular examination to diagnose oral cancer.23 findings of recent molecular markers by ihc analysis on saliva indicate that cyfra 21-1, tps and ca 125 are significantly increased on oral cancer.23 huang et al.24 applies pcr technique to amplify dna from samples of exfoliation cytology kssrm. furthermore restriction-fragment length polymorphism (rflps) analysis is applied. this analysis has found that the occurrence of loss of heterozygote (loh) is 66% in one position at sequence p53. meanwhile, another occurrence of loh is 55% in several places. analysis of pcr and rlfps has been applied as marker detection of microsatellite which is like short repetition of sequence p53. gene mutation using microsatellite and presence of loh is alteration of characteristic molecular from carcinoma of squamous cell in such areas as head and neck. in addition, the gene mutation can also be used as molecular marker of malignancy in oral cavity. nunes et al.25 applies micro satellite analysis on samples of carcinogen of oral cavity and samples of oro-pharynx which are taken by gargling and exfoliation cytology. since loh as much as 84% is found by various differentiations of cell, it can be used to diagnose or observe any malignancy as early as possible. recently surgical margin analysis has been used by some surgeons especially specialist of oral and maxillofacial surgeon to diagnose molecular using molecular marker p53. the molecular marker p53 was firstly used to detect the surgical margin in 1995 on patients of cancer. it should be noted that the cancer is located in the head and the neck. in 2002 specialist of oral and maxillofacial surgeon found that if the hpa examination was applied, 13 of 25 patients of oral cancer were diagnosed negative to have carcinogen. in contrary, if the molecular detection p53 was applied, the result was positive. five patients were positive toward lokoregional occurrence but they were negative if hpa examination was applied. the use of molecular marker p53 is very helpful to evaluate regular hpa examination, immunohistochemistry, and gene mutation on surgical margin analysis of patients of oral cancer and loh as molecular marker for oral pre-malignant lesions.26 discussion data of epidemiology for pre-malignant lesion and oral cancer are needed because the prevalence of pre-malignant lesion is increased from 5-15% to 31.4% in united states.9,10 meanwhile, for oral cancer and oro-pharynx cases, the number of patients were increased from 28,900 in 2002 to 34,000 in 2007. there are 481,000 new cases of oral cancer every year in the world. most of patients belong to oral squamous cell carcinoma. they die every hour and day in the united states.3,9 this leads to high mortality. however, the latest data of epidemiology on patients of pre-malignant lesion and oral cancer can not be gained from badan registrasi kanker indonesia. the data of epidemiology contain prevalence, sex, etiology, location, clinical picture of pre-malignant lesion, oral cancer, types of leukoplakia such as thin leukoplakia, thick leukoplakia, granular leukoplakia, verruciform leukoplakia, proliferative verrucous leukoplakia, speckled leukoplakia, diffuse leukoplakia, and erythroplakia including granular and non granular erythroplakia.16 these all are correlated with the presence of genetic factor which is multi steps and complex. the alteration of gene has caused clinical form of oral cancer vary depending on factors; age, topography, and race of patients.13,14 diagnosis of pre-malignant lesion and oral cancer is still applying examination of pre-malignant lesion and oral cancer by traditional technique that is false negative and its sensitivity is low. because the sample withdrawal is less adequate and interpretation diagnosis or procedure is subjective,18 more sophisticated technique is needed by applying immunohistochemistry and molecular biology examination such as polymerase chain reaction (pcr).19 on sample of cytology, particular examinations of immunohistochemistry technique are used like analysis of proliferation index, the number of keratin cell, nucleus activity with molecular marker ki-67. besides, these examinations are applied on patients of pre-malignant lesion and oral cancer. as consequence, they can be used as regular method of examination to diagnose cancer in oral cavity.20,21 identification of immunohistochemistry using marker tumor k18 and k19 on specimen from oral mucosa swab i.e. cytokeratin has been effective as definitive indicator of malignancy that can not be detected by other examinations like dna profile.22 the findings of recent molecular markers by ihc analysis on saliva indicate that cyfra 21-1, tps and ca 125 are significantly increased on oral cancer.23 there are 29 groups of the molecular marker. they are divided into four different groups based on their ability to control and arrange molecular. the first group is enhancement of tumor growth including the proliferation and the cell cycle. the second is tumor suppression and anti tumor defense: immune response and apoptosis. the third is angiogenesis. the fourth is tumor invasion and metastasis potential: adhesion molecules and matrix degradation.7 there are 29 tumor markers of oral cancer. they are egf, egfr (egfr, c-erb-1 or her-2/neu), cycline (cycline a, b1, d1, e); pcna,ki 67/mib, agnor, skp2, bcl2/bag-1, hsp (hsp27,70), telomerase, prb, cdkis (p15, p16, p21, p27), p53, bax, fas/fasl, ζ – chain (zeta chains), s 100/p55, vegf/vegf-r , nos2 , pd-ecgf, mmps, cathepsines; integrins, cadherins and catenins; desmoplakin/ plakoglobin dan ets-1. 7 the use of molecular marker has been very important since it could be used to detect the molecular alteration. this alteration can be seen before the morphological alteration of malignancy can be observed on clinical symptoms of pre-malignant lesion and oral cancer. in addition, it can be used to detect the 108 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 104−108 surgical margin of patients of oral cancer. if those patients are examined by hpa, the result will be negative. if they are examined by molecular detection p53, the result will be positive. the use of molecular marker p53 and other markers is effective to evaluate regular hpa examination. besides molecular marker p53, immunohistochemistry examination and gene mutation can be applied as molecular marker on patients of oral cancer and loh for surgical margin of pre-malignant lesion.3,7 it is expected that the use of all examinations will continuously be applied as markers for surgical margin of pre-malignant lesion. as a result, molecular technique can be optimized to gain rapid result as same as analysis technique of frozen section. the common molecular markers which are commonly applied are pcna, ki 67, genetic ploidy, oncogene cmyc, gene mutation of tumor supression p53 for pre-malignant lesion and oral cancer detection with immunohistochemistry and pcr technique. these markers are applied through aneuploidy cell, cell mutation, anaplasia, cell invasion, metastasis potential.2,3,7,8 in conclusion, there is an increase of molecular marker either for pre-malignant lesion or oral cancer. there are 29 molecular markers for surgical margin analysis with ihc and pcr technique. the surgeon especially oral and maxillofacial surgeon tend to apply pcna, ki 67, agnor, genetic ploidy, oncogene cmyc especially gene p53. it is recommended that there should be date base of molecular marker which can be used as treatment standard for premalignant lesion and oral cancer. later, it will be applied to reduce the prevalence, increase early detection, diagnosis, prognosis estimation with ihc and pcr technique, accurate therapy such as gene therapy. finally, it will decrease the morbidity and mortality rate on pre-malignant lesion and oral cancer. refferences 1. vokes ee, weichselbaum rr, lippman sm, hong wk. head and neck cancer. n eng j med 1993; 328 (3): 184–94. 2. lippman sm, hong wk. molecular marker of the risk of oral cancer. n engl j med 2001; 344 (17): 1323–6. 3. hill br. oral cancer foundation; histopathology, biology and markers. 2007. available at: http:// www. oralcancerfoundation.org/about/ index. htm. accessed january 24, 2008. 4. agus p. analisis molekuler patogenesis karsinoma sel skuamosa rongga mulut berdasarkan pola mutasi gen p53 dan p16. dissertation. surabaya: pasca sarjana universitas airlangga; 2004. p. 1–177. 5. ramli m. factors affect the treatment of oral cancer. warta ikabi ropanasuri 1999; 23(1): 61–70. 6. lippman sm, sudbo j, hong wk. oral cancer prevention and the evolution of molecular-targeted drug development. n engl j med 2001; 344(17): 1323. 7. schliephake h. prognostic relevance of molecular markers of oral cancer—a review. int j oral and maxillofac surg 2003; 32: 233–45. 8. natkunam y, mason yd. prognostic immunohistologic markers in human tumors: why are so few used in clinical practice? laboratory investigation 2006; 86: 742–7. 9. sudbo j. novel management of oral cancer: a paradigm of predictive oncology. clinical medicine & research 2004; 2(4): 233–42. 10. lee jj, hong wk, hittelman wn, mao l, lotan r, shin dm, et al. predicting cancer development in oral leukoplakia: ten years of translational research. clinical cancer research 2000; 6: 1702–10. 11. sugerman pb, shillitoe ej. the high risk human papillomaviruses and oral cancer. evidence for and against a causal relationship. oral dis 1997; 3: 145–7. 12. winn dm, ziegler rg, pickle lw. diet in the etiology of oral and pharyngeal among woman from the southern united states. cancer res 1984; 44: 1216–22. 13. chen gs, chen ch. a study on survival rates of oral squamous cell carcinoma. j kao-hsiung-i-hsueh-ko-hsueh-tsa-chih 1996; 12: 317–25. 14. todd r, donoff rb, wong dtw. the molecular biology of oral carcinogenesis. toward a tumor progression model. joms 1997; 55: 613. 15. suryohudoyo p. ilmu kedokteran molekuler. jakarta: kapita selekta, cv. sagung seto; 2000. p. 102–9. 16. reibel j. prognosis of oral pre-malignant lesions: significance of clinical, histopathological, and molecular biological characteristic. crit rev oral biol med 2003; 14(1): 47–62. 17. williams hk. molecular pathogenesis of oral squamous carcinoma. j clin pathol mol pathol 2000; 53:165–72. 18. neville bw, day ta. oral cancer and precancerous lesions. ca cancer j clin 2002; 52: 195–215. 19. bouquot je. oral precancer: dysplasia, molecular biology. microbiology 1994. available at: http:// www. maxillofacialcenter. co/precancerdysplasia.html. accessed june 1, 2009. 20. van diest pj, brugal g, baak jp. proliferation markers in tumours: interpretation and clinical value. j clin pathol 1998 october; 51(10): 716–24. 21. mehrotra r, gupta a, singh m, ibrahim r.. application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions, mol cancer 2006; 5: 11. 22. ogden gr, cowpe jg, chisholm dm, lane eb. dna and keratin analysis of oral exfoliative cytology in detection of oral cancer. oral oncol eur j cancer 1994; 30b: 405–824. 23. nagler rm, bahar g, shpitzer t, feinmesser r. concomitant analysis of salivary tumor markers—a new diagnostic tool for oral cancer, clinical cancer research, 2006; 12: 3976–84. 24. huang mf, chang yc, liao ps, huang th, tasy ch, chou my. loss of hetrozygosity of p53 gene of oral cancer detected by exfoliative cytology. oral oncol 1999; 35: 296–301. 25. nunes dn, kowalski lp, simpson aj. detection of oral and oropharyngeal cancer by microsatelite analysis in mouth washes and lesion brushings. oral oncol 2000; 36: 525–8. 26. eipstein bj, zhang l, rosin m. advances in the diagnosa of oral premalignant and malignant lesions. j can dent assoc 2002; 689(10): 617–21. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 160 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 160–165 original article the compressive strength and static biodegradation rate of chitosan-gelatin limestone-based carbonate hydroxyapatite composite scaffold devi rianti1, alqomariyah eka purnamasari1, rifayinqa ruyani putri1, noor zain salsabilla1, faradillah1, elly munadziroh1, titien hary agustantina1, asti meizarini1, anita yuliati1, ardiyansyah syahrom2 1department of dental materials, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2medical devices and technology centre (meditec), institute of human centered and engineering (ihumen), universiti teknologi malaysia (utm), johor bahru, malaysia abstract background: one of the main components in tissue engineering is the scaffold, which may serve as a medium to support cell and tissue growth. scaffolds must have good compressive strength and controlled biodegradability to show biological activities while treating bone defects. this study uses chitosan-gelatin (c–g) with good flexibility and elasticity and high-strength carbonate hydroxyapatite (cha), which may be the ideal scaffold for tissue engineering. purpose: to analyze the compressive strength and static biodegradation rate within various ratios of c–g and cha (c–g:cha) scaffold as a requirement for bone tissue engineering. methods: the scaffold is synthesized from c–g:cha with three ratio variations, which are 40:60, 30:70, and 20:80 (weight for weight [w/w]), made with a freeze-drying method. the compressive strengths are then tested. the biodegradation rate is tested by soaking the scaffold in simulated body fluid for 1, 3, 7, 14, and 21 days. data are analyzed with a one-way anova parametric test. results: the compressive strength of each ratio of c–g:cha scaffold 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w), consecutively, are 4.2 megapascals (mpa), 3.3 mpa, 2.2 mpa, and there are no significant differences with the p= 0.069 (p>0.05). the static biodegradation percentage after 21 days on each ratio variation of c–g:cha scaffold 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w) is 25.98%, 24.67%, and 20.64%. one-way anova welch test shows the result of the p-value as p<0.05. conclusion: the compressive strength and static biodegradation of the c–g:cha scaffold with ratio variations of 40:60 (w/w), 30:70 (w/w), and 20:80(w/w) fulfilled the requirements as a scaffold for bone tissue engineering. keywords: biodegradation; compressive strength; medicine; scaffold; tissue engineering article history: received 10 october 2022; revised 5 december 2022; accepted 4 april 2023; published 1 september 2023 correspondence: devi rianti, department of dental material, faculty dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47, surabaya, indonesia. email: devi-r@fkg.unair.ac.id introduction bone defects may occur in the maxillary and mandibular alveolar bone because of congenital anomaly, trauma, bone deficiency after tumor resection, periodontal diseases, and tooth loss.1–3 the most common treatment is the application of bone grafts using the concept of tissue engineering, which comprises three fundamental components: cells, scaffolds, and growth factors.4 tissue engineering aims to develop new biofunctional tissue to regenerate and repair damaged or diseased tissues.5,6 in tissue engineering, scaffolds are crucial as they provide support for cell and tissue growth and can imitate natural bone.7 the primary characteristics required in a scaffold for tissue engineering include biocompatibility, good mechanical properties, controlled biodegradability, osteoinductivity, osteoconductivity, and non-toxicity.8 the scaffold’s synthesization involves biomaterials consisting of a natural polymer from chitosan-gelatin (c–g) and a bio-ceramic from carbonate hydroxyapatite (cha), which display ideal scaffold characteristics.9 providing flexibility and elasticity, c– g is an organic material, while cha is high in crystals, which might contribute to the scaffold’s structural strength.10 chitosan is copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 mailto:devi-r@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 161rianti et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 160–165 a natural biopolymer derived from chitin with the desirable characteristics of biocompatibility and biodegradability, as well as being antibacterial and non-toxic.11 gelatin is a biocompatible, biodegradable, low-toxicity material derived from hydrolyzed and denaturalized collagen, one of the leading organic components in the natural bone.12,13 the combination of gelatin and chitosan may help improve the bone repair process.14 with greater homogeneity and fixation ability than hydroxyapatite, cha is an inorganic compound and is commonly used as a scaffold material for bone repair and replacement due to its bioactive, osteoconductive, and biocompatible characteristics.15 it is capable of activating cell adhesion differentiation and proliferation with good absorptivity for bone defects, which is essential for tissue engineering. 16,17 cha may also help increase the ion calcium and phosphate required for new bone formation.13 in this study, limestone-based cha from cirebon, west java, extracted by the indonesian center for ceramics (bbk indonesia), has potential application as a bio-ceramic material in the medical field for bone replacement in treating bone defects. this study uses freeze-drying to synthesize the c–g:cha scaffold. this method can produce porous 3-dimensional scaffolds with more than 90% porosity and a 20–400 micrometer (µm) pore diameter.18 combining several natural materials can improve each material’s properties to achieve the scaffold’s ideal characteristics, particularly good mechanical properties.19 chemical crosslinking between the polymer components of c–g and the addition of cha can affect the mechanical properties of a scaffold.20 scaffolds used as a bone replacement need a 60% to 90% porosity with an average pore size of 150 µm and compressive strength comparable to the cortical bone of 100mpa to 230 mpa or trabecular bone of 1mpa to 12mpa.21–23 the compressive strength of a scaffold material is mainly studied to determine its maximum load-bearing capacity.24 the ideal scaffold must have good mechanical properties, including compressive strength to withstand pressure from tissue and maintain space for cell and new bone growth.25 when a scaffold is implanted into the body, it must maintain its mechanical properties with enough structural integrity, determined by the biodegradability of the biomaterial that it can create space for new bone tissue to grow. thus, the research aims to determine the mechanical property requirements for a tissue engineering scaffold by testing the compressive strength of the c–g:cha scaffold composite with ratio variations of 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w) and biodegradation testing to determine how long it takes for the scaffold to degrade into the body completely.26 a scaffold’s controlled and stable degradation process may help regenerate new bone tissue.27 this study aims to analyze the compressive strength and static degradation rate of the c–g:cha scaffold composite with specific ratios as requirements for bone regeneration. materials and methods the materials used in this study were chitosan with a medium molecular weight (sigma aldrich 448877, usa), bovine gelatin (sigma aldrich g9391, usa), cha powder made from limestone produced by indonesian center for ceramics (bbk indonesia), natrium hydroxide (biomedicine), acetic acid (merck), distilled water (duta farma), and simulated body fluid (sbf merck). the c–g:cha 40:60 (w/w) scaffold is prepared by weighing 0.5 grams of chitosan powder, 0.5 grams of gelatin powder, and 1.5 grams of hydroxyapatite carbonate powder. up to 2% acetic acid is added to the weighed gelatin up to 2 milliliters (ml) and stirred with a magnetic agitator at 50°c until the gelatin powder is homogeneous. the weighed cha is mixed with 0.94 ml of distilled water and then incorporated with a metal spatula until homogeneous. then, the dilute cha is incorporated in the gelatin gel and then stirred until homogeneous, while chitosan powder is added gradually to form a c–g:cha gel. the c–g:cha gel was mixed with 0.5 ml of 0.1 molar naoh to neutralize the acid. the c–g:cha gel was measured using litmus paper until a ph of 7 was obtained. if a ph >7 (alkaline) was obtained, 0.1 ml of acetic acid solution is added, while if a ph <7 (acidic) is obtained, 0.1 ml of naoh solution is added. the ph measuring must be done simultaneously to ensure the scaffold’s ph is exactly 7. the ph 7 c–g:cha gel is placed into the 48-well plate using a glass spatula and then compacted using a cement stopper until no hollow spaces are left. the mixture is frozen at −40°c for 2x24 hours and freeze-dried for 2x24 hours.28 scaffolds with the ratios of 30:70 (w/w) and 20:80 (w/w) are processed the same way. on the 30:70 (w/w) ratio, 0.375 grams of chitosan powder, 0.375 grams of gelatin powder, and 1.75 grams of cha powder are used. for the 20:80 (w/w) ratio, 0.25 grams of chitosan powder, 0.25 grams of gelatin powder, and 2 grams of cha powder are applied. compressive strengths are tested using the mini autograph universal testing machine’s sensor load cell l ip3 class 0.02 with microcontroller software phyton 2.7 on the cylindrical-shaped scaffolds. the diameter and height of the scaffolds are measured using vernier calipers to measure their surface area. scaffold samples are placed in the middle of the pressing machine with their vertical axis perpendicular to the flat plane. activating the mini autograph tool, the suppressor crushes the sample slowly with a pressure load of 400 newtons and 2 mm/minute speed until the samples are distorted and break. the tool will be stopped when the graph in the monitor shows that there is an increase after a decrease. when this occurs, the load no longer pressures the scaffold but is distributed only onto the upper and lower suppressor. calculations will be made from the graph results, which show displacement and force accepted by the scaffold as the maximum amount of load divided by the surface area of the scaffold sample. the data are then put into the compressive strength formula to calculate the compressive strength value with units of mpa.29 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 162 rianti et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 160–165 static biodegradation testing is achieved by soaking the samples in 1.5 ml sbf in an eppendorf container at a temperature of 37°c. before soaking, the ph measurements of the sbf media are taken by weighing the sbf to determine the initial weight of the scaffold in its dry state (wo). the percentage of biodegradation is obtained after calculating the final weight (wt) as the scaffolds are dried after soaking for 1, 3, 7, 14, and 21 days. the chosen formula is used to calculate the biodegradation percentage from the scaffolds’ w and wt data.30 𝐵𝑖𝑜𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = ����� �� 𝑥 100% research data are then statistically analyzed using the kolmogorov–smirnov test to determine if the data distribution is normal, followed by homogeneity testing using the levene test. if the p > 0.05, one-way anova parametric tests are performed to identify the significance of every sample’s data results. results the compressive strength value of the c–g:cha scaffold is obtained after entering the strength test results from the mini autograph universal testing machine’s sensor load cell l ip3 class 0.02 with the python 2.7 microcontroller software. results and the standard deviation of the c–g:cha scaffolds’ compressive strength value are shown in table 1 and figure 1. the average compressive strength value of the c–g:cha scaffold appears to increase table 1. the compressive strengths of various c–g:cha scaffold ratios (mpa) sample n average of compressive strength value standard deviation c–g:cha scaffold 40:60 6 4.19 0.79 c–g:cha scaffold 30:70 6 3.29 0.22 c–g:cha scaffold 20:80 6 2.19 1.19 0 1 2 3 4 5 scaffold c-g:cha 40:60 scaffold c-g:cha 30:70 scaffold c-g:cha 20:80 = not significant average of compressive strength value figure 1. graph of the average compressive strength of c–g:cha scaffolds. table 2. static biodegradation test of c–g:cha scaffold ratios (%) c–g:cha scaffold ratios n day 1 day 3 day 7 day 14 day 21 x + sd x + sd x + sd x + sd x + sd 40:60 6 0.41±0.06 1.69±0.69 6.67±1.64 16.13±5.43 25.98±2.74 30:70 6 0.50±0.13 3.18±1.35 6.07±0.52 12.55±2.03 24.67±3.77 20:80 6 0.42±0.09 1.86±0.80 5.51±0.79 8.27±1.51 20.64±6.40 notes: x: average biodegradation percentage; sd: standard deviation 0 5 10 15 20 25 30 scaffold c-g:cha 40:60 scaffold c-g:cha 30:70 scaffold c-g:cha 20:80 = significant = not significant day 1 day 3 day 7 day 14 day 21 figure 2. the graph shows the average static biodegradation rate of the c–g:cha scaffolds on days 1, 3, 7, 14, and 21. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 163rianti et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 160–165 with an increase in the c–g ratio and a decrease in the cha ratio. initial and final weights are calculated on each sample at every time stamp during the research. time-stamp variations for the biodegradation tests are 1, 3, 7, 14, and 21 days. data from the initial and final weights are used to calculate the degradation percentage of the c–g:cha scaffolds with various ratios. results and the standard deviation of the c–g:cha scaffolds’ degradation are shown in table 2 and figure 2. discussion the scaffold’s mechanical properties are essential in the manufacturing process. developing a porous structured scaffold compatible with bone is a central issue in tissue engineering.31,32 the porous structure of the scaffold is to facilitate cell attachment and proliferation, but then it must also have the sufficient mechanical strength to enhance biostability.33 based on the results, the average compressive strength value increases when the cha ratio decreases, and the c–g ratio rises. a one-way anova statistical analysis showed no significant difference in the compressive strength values of the three variations of the c–g:cha scaffold ratios of 20:80 (w/w), 30:70 (w/w), and 40:60 (w/w). this result means that the average compressive strength value increase was insignificant. in previous studies, the c–g:cha scaffold had been tested by ftir, sem-edx, and xrd, and the results contained a phosphate group (po4 3-), a carbonate group (co3 2-), and a hydroxyl group (oh-). chitosan has several hydroxyl groups (-oh) and amine groups (-nh2) in its chain, while gelatin has active hydroxyl groups (-oh), carboxylic groups (-cooh), and amine groups (-nh2). the groups in chitosan and gelatin form hydrogen bonds between the amine and carboxylic groups or with phosphate and carbonate groups.33 bonds between carboxyl groups in gelatin and amine groups in chitosan also produce ionic bonds, which cause the formation of a scaffold with denser properties.34 bonds in chitosan and gelatin will form intermolecular hydrogen bonds. hydrogen bonds will be created due to bonds with –nh2 and carbonates hydroxyapatite or interactions between –cooh and carbonates apatite. combining these 3 materials will form crystalline particles, which are dominantly formed from cha material containing the elements o, ca, and p and has crystalline particles.33 during the freeze-drying process, the mixture of the three materials will produce crystals and amorphs, which will balance the crystallinity of the scaffold so that the addition of the c–g ratio can increase the bond to the 3 materials. the above confirms the opinion that chemical crosslinking between polymer components, namely c–g, can affect the mechanical properties of the scaffold.20 the compressive strength value in this study is still in the range of compressive strength values in trabecular bone of 0.1 to 16 mpa. engineering scaffold tissue must possess sufficient mechanical properties to support new bone tissue at the implantation site and maintain good integrity for cells in vitro and in vivo.35-38 thus, it is vital for a bone scaffold to have identical mechanical properties as trabecular bone.23 in the results of previous studies, the trabecular bone mechanical properties have a value of compressive strength of at least 1 mpa.39 a study by waletzko-hellwig showed that the compressive strength of trabecular bone is 2 to 48 mpa. in comparison, a study by mohaghegh suggested a compressive strength of 1.5 to 45 mpa, and research by gerhardt and boccaccini showed a compressive strength of 0.1 to 16 mpa.37,40,41 this indicates that trabecular bone has a highly anisotropic and heterogeneous structure whose mechanical properties depend highly on anatomical location.42 in addition, the stiffness level of the bone scaffold must not be too low to provide mechanical stability and not too high to prevent stress shielding, resulting in friction under continuous pressure and damage to the surrounding bones.43 this can affect bone remodeling.44 previous research on balai besar keramik’s hydroxyapatite (habbk) scaffold, combined with c–g, proved the habbk:c–g scaffold composite with a ratio of 60:40 (w/w) had the highest average compressive strength value, i.e. 0.81 mpa, less than the compressive strength value of this study using cha.29,45 this study shows that all ratios meet the requirements as a scaffold in tissue engineering: the compressive strength in the c–g:cha scaffold with the ratio of 40:60 (w/w) is 4.19 mpa; the c–g:cha 30:70 (w/w) is 3.29 mpa; and the c–g:cha scaffold 20:80 (w/w) is 2.19 mpa. all these values are still within the range of compressive strength values of the trabecular bone. developing chitosan, gelatin, and cha materials into a three-dimensional structural scaffold with good mechanical strength and biological function will increase the recovery of bone defects. it can be used as a good candidate for designing biomimetic bone scaffolds. table 2 and figure 2 show that the average value of the static biodegradation rate of the c–g:cha scaffold in each ratio variation increased during the study from day 1 to day 21. the test results for the highest average degradation rate value of the c–g:cha scaffold sample for each ratio variation were obtained on the 21st day. in the c–g:cha scaffold with a variation of the ratio of 40:60 (w/w), the static biodegradation rate is faster compared to the other ratio variations. one of the ideal properties of biomaterials in tissue engineering is biodegradability. biomaterials used for scaffolds have an essential role in the success of tissue engineering. tissue engineering in a scaffold must support the tissue growth process that acts as a temporary extracellular matrix during the cell attachment and adhesion processes. this matrix must be made of biodegradable materials capable of being metabolized by the body and eventually gradually degraded when cells begin to undergo a process of proliferation and differentiation.46,47 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 164 rianti et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 160–165 the static biodegradation rate of the 40:60, 30:70, and 20:80 scaffolds throughout 1, 3, 7, 14, and 21 days increased due to bonds between the scaffold components and calcium and phosphate ions from the sbf solution. this bond gradually damages the scaffold components and causes a decrease in the weight of the scaffold, increasing the degradation rate due to the interaction between the scaffold and the sbf media. in the degradation process of the c–g:cha scaffold in the sbf, the increasing weight loss was affected by the bond between the scaffold components with ca2+ ions and po4 3+ ions originating from the sbf solution. the interaction of the scaffold and the sbf also caused the degradation of chitosan in the form of an amide complex such as nh2 in the sbf. this process will cause the sbf to gradually damage the scaffold components and increase the percentage of scaffold weight loss as the static immersion time rises to 21 days.33,48 the static biodegradation test, the gold standard, is used in this study.49 according to previous research, the reduction in scaffold weight should not be too fast or slow, following the bone remodeling process of around 3–6 months.50,51 in another opinion, the minimum degradation process is between 1–2 weeks because this period is the bone repair stage, starting with the elimination of damaged cells and replacing the weak fibrin clot with a more mechanically stable structure called a callus.52 the biodegradation rate of the c–g:cha scaffold is 20% to 25.98% on day 21, the largest ratio variation in the study. based on previous research, the trabecular bone regeneration process takes place for about 2–3 months.53according to other studies, bone regeneration in the trabecular bone takes about 200 days (6 months).54 in this study, the degradation rate of c–g:cha scaffold of 20% to 25.98% for 21 days is expected to lead to complete degradation within 3–6 months, showing that the c–g:cha scaffold has a biodegradation rate suitable for the bone regeneration process. all of the c–g:cha scaffold ratios demonstrate biodegradable properties. over 1, 3, 7, 14, and 21 days, the scaffold underwent in vitro bone regeneration, or it could be said that the scaffold was degraded. therefore, the c–g:cha scaffold with the ratios of 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w) all have reasonable degradation rates for a scaffold in tissue engineering. this study has limitations because it has not been able to observe complete biodegradation, which will occur in the future. it is also necessary to undertake further research using dynamic degradation techniques by simulating the movement of bone marrow in the trabecular bone. in conclusion, the compressive strength value of the c–g:cha scaffold composite with ratio variations of 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w) met the requirements of a scaffold in tissue engineering. the variation of the ratio of the c–g:cha scaffold composite 40:60 (w/w), 30:70 (w/w), and 20:80 (w/w) increased throughout 1, 3, 7, and 14 days, with the highest percentage of biodegradation on day 21 of 25.98% on the c–g:cha scaffold with a ratio variation of 40:60 (w/w). with good flexibility and elasticity, c–g combined with high-strength cha, is an ideal scaffold for tissue engineering. references 1. prasadh s, wong rcw. unraveling the mechanical strength of biomaterials used as a bone scaffold in oral and maxillofacial defects. oral sci int. 2018; 15(2): 48–55. 2. i b r a h i m a. 3d biop r i nt i ng b one. i n: 3d biop r i nt i ng for reconstructive surgery. elsevier; 2018. p. 245–75. 3. shuk la s, chug a, ma hesh l, singh s, singh k. optimal management of intrabony defects: current insights. clin cosmet investig dent. 2019; 11: 19–25. 4. ghassemi t, shahroodi a, ebrahimzadeh mh, mousavian a, movaffagh j, moradi a. current concepts in scaffolding for bone tissue engineering. arch bone jt surg. 2018; 6(2): 90–9. 5. collins mn, ren g, young k, pina s, reis rl, oliveira jm. scaffold fabrication technologies and structure/function properties in bone tissue engineering. adv funct mater. 2021; 31(21): 2010609. 6. witzler m, ottensmeyer pf, gericke m, heinze t, tobiasch e, schulze m. non-cytotoxic agarose/hydroxyapatite composite scaffolds for drug release. int j mol sci. 2019; 20(14): 3565. 7. samadian h, farzamfar s, vaez a, ehterami a, bit a, alam m, goodarzi a, darya g, salehi m. a tailored polylactic acid/ polycaprolactone biodegradable and bioactive 3d porous scaffold containing gelatin nanofibers and taurine for bone regeneration. sci rep. 2020; 10(1): 13366. 8. darus f, jaafar m. enhancement of carbonate apatite scaffold properties with surface treatment and alginate and gelatine coating. j porous mater. 2020; 27(3): 831–42. 9. safarzadeh m, chee cf, ramesh s, fauzi mna. effect of sintering temperature on the morphology, crystallinity and mechanical properties of carbonated hydroxyapatite (cha). ceram int. 2020; 46(17): 26784–9. 10. li j, jansen ja, walboomers xf, van den beucken jj. mechanical aspects of dental implants and osseointegration: a narrative review. j mech behav biomed mater. 2020; 103: 103574. 11. ghiasi b, sefidbakht y, rezaei m. hydroxyapatite for biomedicine and drug delivery. in: nanomaterials for advanced biological applications. springer; 2019. p. 85–120. 12. zheng x, liu y, liu y, pan y, yao q. novel three‐dimensional bioglass functionalized gelatin nanofibrous scaffolds for bone regeneration. j biomed mater res part b appl biomater. 2021; 109(4): 517–26. 13. noor khairiyah hanisah h, muhammad ridhwan r, nurazreena a. synthesis of porous carbonate apatite/gelatin scaffolds via freeze drying method. j phys conf ser. 2018; 1082: 012004. 14. oryan a, alidadi s, bigham-sadegh a, moshiri a. comparative study on the role of gelatin, chitosan and their combination as tissue engineered scaffolds on healing and regeneration of critical sized bone defects: an in vivo study. j mater sci mater med. 2016; 27(10): 155. 15. iancu l, ion r-m, grigorescu rm, ghioca pn, spurcaciu b, david me, andrei re, ghiurea m, stirbescu rm, bucurica a. carbonated hydroxyapatite substituted with magnesium for stone consolidation. in: the 16th international symposium “priorities of chemistry for a sustainable development” priochem. basel switzerland: mdpi; 2020. p. 59. 16. he j, hu x, cao j, zhang y, xiao j, peng l, chen d, xiong c, zhang l. chitosan-coated hydroxyapatite and drug-loaded polytrimethylene carbonate/polylactic acid scaffold for enhancing bone regeneration. carbohydr polym. 2021; 253: 117198. 17. januariyasa ik, yusuf y. porous carbonated hydroxyapatite-based scaffold using simple gas foaming method. j asian ceram soc. 2020; 8(3): 634–41. 18. fereshteh z. freeze-drying technologies for 3d scaffold engineering. in: functional 3d tissue engineering scaffolds. elsevier; 2018. p. 151–74. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 165rianti et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 160–165 19. re f, sartore l, moulisova v, cantini m, almici c, bianchetti a, chinello c, dey k, agnelli s, manferdini c, bernardi s, lopomo nf, sardini e, borsani e, rodella lf, savoldi f, paganelli c, guizzi p, lisignoli g, magni f, salmeron-sanchez m, russo d. 3d gelatinchitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation. j tissue eng. 2019; 10: 204173141984585. 20. georgopoulou a, papadogiannis f, batsali a, marakis j, alpantaki k, eliopoulos ag, pontikoglou c, chatzinikolaidou m. chitosan/ gelatin scaffolds support bone regeneration. j mater sci mater med. 2018; 29(5): 59. 21. roohani-esfahani s-i, newman p, zreiqat h. design and fabrication of 3d printed scaffolds with a mechanical strength comparable to cortical bone to repair large bone defects. sci rep. 2016; 6(1): 19468. 22. sutthi r, kaewwinud n, chindaprasirt p, mutoh y, laonapakula t. effect of curing temperature and time on the mechanical properties of hydroxyapatite/calcined kaolin. scienceasia. 2018; 44(6): 397. 23. kim t-r, kim m-s, goh ts, lee js, kim yh, yoon s-y, lee c-s. evaluation of structural and mechanical properties of porous artificial bone scaffolds fabricated via advanced tba-based freezegel casting technique. appl sci. 2019; 9(9): 1965. 24. mondal s, nguyen tp, pham vh, hoang g, manivasagan p, kim mh, nam sy, oh j. hydroxyapatite nano bioceramics optimized 3d printed poly lactic acid scaffold for bone tissue engineering application. ceram int. 2020; 46(3): 3443–55. 25. chi h, song x, song c, zhao w, chen g, jiang a, wang x, yu t, zheng l, yan j. chitosan-gelatin scaffolds incorporating decellularized platelet-rich fibrin promote bone regeneration. acs biomater sci eng. 2019; 5(10): 5305–15. 26. bose s, roy m, bandyopadhyay a. recent advances in bone tissue engineering scaffolds. trends biotechnol. 2012; 30(10): 546–54. 27. maji k, dasgupta s, kundu b, bissoyi a. development of gelatinchitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength. j biomater sci polym ed. 2015; 26(16): 1190–209. 28. fanny g. uji karakteristik scaffold kitosan gelatin karbonat apatit batu kapur balai besar keramik. thesis. universitas airlangga. surabaya; 2020. p. 22–34. 29. karina ry. compressive strength scaffold komposit hidroksiapatit balai besar keramik dengan berbagai rasio. thesis. universitas airlangga. surabaya; 2020. p. 22–4. 30. wang x, yu t, chen g, zou j, li j, yan j. preparation and characterization of a chitosan/gelatin/extracellular matrix scaffold and its application in tissue engineering. tissue eng part c methods. 2017; 23(3): 169–79. 31. shahbazi s, zamanian a, pazouki m, jafari y. introducing an attractive method for total biomimetic creation of a synthetic biodeg radable bioactive bone sca ffold based on statistica l experimental design. mater sci eng c. 2018; 86: 109–20. 32. tamburaci s, tihminlioglu f. biosilica incorporated 3d porous scaffolds for bone tissue engineering applications. mater sci eng c. 2018; 91: 274–91. 33. rianti d, fanny g, nathania rv, purnamasari ae, putri rr, soekartono h, soebagio s, yuliati a, syahrom a. the characteristics, swelling ratio and water content percentage of chitosan-gelatin/ limestone-based carbonate hydroxyapatite composite scaffold. int j integr eng. 2022; 14(2): 13–23. 34. aufan mr. sintesis scaffold alginat-kitosan-karbonat apatit sebagai bone graft menggunakan metode freeze drying. j biofisika. 2012; 8(1): 16–24. 35. dressler m, dombrowski f, simon u, börnstein j, hodoroaba vd, feigl m, grunow s, gildenhaar r, neumann m. influence of gelatin coatings on compressive strength of porous hydroxyapatite ceramics. j eur ceram soc. 2011; 31(4): 523–9. 36. bello ab, kim d, kim d, park h, lee s-h. engineering and functionalization of gelatin biomater ials: from cell culture to medical applications. tissue eng part b rev. 2020; 26(2): 164–80. 37. gerhardt l-c, boccaccini ar. bioactive glass and glass-ceramic scaffolds for bone tissue engineering. materials (basel). 2010; 3(7): 3867–910. 38. escoba r-sier ra dm, ma r tins j, ossa-orozco cp. chitosan / hydroxyapatite scaffolds for tissue engineering manufacturing method effect comparison. rev fac ing univ antioquia. 2015; (75): 24–35. 39. levengood skl, zhang m. chitosan-based scaffolds for bone tissue engineering. j mater chem b. 2014; 2(21): 3161. 40. waletzko-hellwig j, saemann m, schulze m, frerich b, bader r, dau m. mechanical characterization of human trabecular and formed granulate bone cylinders processed by high hydrostatic pressure. materials (basel). 2021; 14(5): 1069. 41. mohaghegh s, hosseini sf, rad mr, khojasteh a. 3d printed composite scaffolds in bone tissue engineering: a systematic review. curr stem cell res ther. 2022; 17(7): 648–709. 42. oftadeh r, perez-viloria m, villa-camacho jc, vaziri a, nazarian a. biomechanics and mechanobiology of trabecular bone: a review. j biomech eng. 2015; 137(1): 010802. 43. pei p, wei d, zhu m, du x, zhu y. the effect of calcium sulfate incorporation on physiochemical and biological properties of 3dprinted mesoporous calcium silicate cement scaffolds. microporous mesoporous mater. 2017; 241: 11–20. 44. bahraminasab m. challenges on optimization of 3d-printed bone scaffolds. biomed eng online. 2020; 19(1): 69. 45. ghouse s, reznikov n, boughton or, babu s, ng kcg, blunn g, cobb jp, stevens mm, jeffers jrt. the design and in vivo testing of a locally stiffness-matched porous scaffold. appl mater today. 2019; 15: 377–88. 46. yuliati a, kartikasari n, munadziroh e, rianti d. the profile of crosslinked bovine hydroxyapatite gelatin chitosan scaffolds with 0.25% glutaraldehyde. j int dent med res. 2017; 10(1): 151–5. 47. widyamsari nks. biodegradasi komposit scaffold berbasis hidroksiapatit balai besar keramik. dissertation. universitas airlangga. surabaya; 2020. 48. shuai c, zhou y, yang y, feng p, liu l, he c, zhao m, yang s, gao c, wu p. biodegradation resistance and bioactivity of hydroxyapatite enhanced mg-zn composites via selective laser melting. materials (basel). 2017; 10(3): 307. 49. md. saad ap, jasmawati n, harun mn, abdul kadir mr, nur h, hermawan h, syahrom a. dynamic degradation of porous magnesium under a simulated environment of human cancellous bone. corros sci. 2016; 112: 495–506. 50. zhang k, fan y, dunne n, li x. effect of microporosity on scaffolds for bone tissue engineering. regen biomater. 2018; 5(2): 115–24. 51. yadav n, srivastava p. in vitro studies on gelatin/hydroxyapatite composite modified with osteoblast for bone bioengineering. heliyon. 2019; 5(5): e01633. 52. rodriguez i. tissue engineering composite biomimetic gelatin sponges for bone regeneration. thesis. virginia commonwelth university: virginia; 2013. p. 23–7, 35–56. 53. irish j, virdi as, sena k, mcnulty ma, sumner dr. implant placement increases bone remodeling transiently in a rat model. j orthop res. 2013; 31(5): 800–6. 54. eriksen ef. cellular mechanisms of bone remodeling. rev endocr metab disord. 2010; 11(4): 219–27. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p160–165 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p160-165 168 effectivity of blunt end with side hole irrigation needle to eliminate root canal bacteria laksmiari setyowati, sudarjani gunawan and achmad sudirman department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract the blunt end with side hole irrigation needles have some advantages. they can be placed close to irrigation area, produce turbulent motion of irrigation material, and not push debris to apical. there is no data about the effectiveness of blunt end with side hole irrigation needle to eliminate root canal bacteria in vivo, therefore the research about effectivity of this needle compare to conventional irrigation needle was permormed. in this study 12 samples were used and divided into two groups. the conventional irrigation needle in the first group was used as control and the blunt end with side hole needle was used in the second group. the bacteriological sampling and colony counting was conducted. the paired t-test analysis before and after irrigation showed significant difference on the first and second group. the result indicated that blunt end with side hole needle more effective in eliminating root canal bacteria than conventional needle. supporting to this study a software of fluent had been done in vitro. the result showed blunt end with side hole needle produced turbulent motion of irrigating liquid and the conventional needle produced laminar motion. key words: the conventional irrigation needle, the blunt end with side hole needle, root canal bacteria correspondence: laksmiari setyowati, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction the steps of root canal treatment are cleaning the debridement entire pulp tissue and inflamed fluid, root canal preparation, sterilization, and obturation the root canal. the successful of root canal treatment is depends on several factors, that is preparation, sterilization, and obturation of root canal.1,2,3 the purposes of root canal preparation are shaping, enlarging, smoothing the root canal wall, and cleaning necrotic tissue and bacteria. on vital teeth, preparation does not only for debridement pulp tissue and shaping root canal wall, but in order to make filling easier. it will be different if we do a preparation of necrotic teeth. necrotic tissue from root canal must clean until dentin debris which is grinded look white on preparation device. the root canal treatment will failure if we do not clean the debris perfectly.4 debris and bacteria in the one third apical root canal are the important factors in successful of the root canal treatment. preparation and irrigation of root canal treatment are methods for cleaning root canal. irrigation procedure will remove organic and anorganic debris.5 hydrogen peroxide (h2o2) in dentistry is used as irrigation solution since it is promoted by richardson in 1860. the concentration of h2o2 which is used for irrigation is 3%. the mechanism is by forming white foam from organic material oxidation. if contacts with blood, pus, serum, saliva, and other organic materials, the foam will bring out the root canal debris. the mechanical cleansing effect of h2o2 plays important function. h2o2 is a light antiseptic material and not irritate. after irigation with h2o2, it must be followed by irrigation using sterile aquadest. this step will neutralize o nasen.1 penetration of irrigation material depend on several variables like root canal configuration, volume of irrigation solution, the type of irrigation solution, and the most important is type of irrigation device. the irrigation device which is used in airlangga university dental hospital is irrigation needle that have sharp tip with hole on the tip. this needle design will produce laminar flow.6 this needle has disadvantage. this needle can push debris out to apical.7 another type of irrigation needle is blunt end with side hole irrigation needle. this type has some advantages. the end of this needle is blunt with many kinds of size that are suitable with file diameter, so the needle will close to apical foramen. the shape of blunt end with side hole irrigation needle is hopefully able to clean debris at apical area and not push debris out to apical. it also create turbulent motion irrigation fluid, so it is wished to clean debris effectively and eliminate bacteria in root canal.6,7 kahn7 performed an experiment that compared the effectivity of irrigation devices such as conventional needle, monoject endodontic, cavi endo ultrasonic micromega 1500 subsonic, and blunt end with side hole needle. the result showed that the blunt end with side hole irrigation needle is effective on maxillar and mandibular position. there is no research about the effectivity of blunt end with side hole irrigation needle to eliminate bacteria in vivo, so the aim of this research is to examine the effectivity of blunt end with side hole irrigation needle to eliminate root 169setyowati: effectivity of blunt end with side hole irrigation needle canal bacteria in vivo. the advantage of this research is to get the information about effectivity of irrigation needle to eliminate root canal bacteria for support the successful of root canal treatment. materials and methods the research was clinic experimental with pre-post controlled design and laboratories experimental. the mandibular anterior teeth from patients, age 18–45 years old who visited airlangga university dental hospital, were choose as the samples. the teeth were diagnosed as pulp necrosis due to dental caries with no periapical lesion, straight root canal, no obturation, diameter of root canal can be put the file no. 30 in, male or female, and teeth with close apices. the samples were divided into 2 groups. group i (control group) contained six irrigated samples with conventional irrigation needle (figure 1). group ii contained of six irrigated samples with blunt end with side hole irrigation needle (figure 2). needle. irrigation was done with light pressure, so that the irrigation procedure of 2.5 ml irrigation material could be completed in 32 seconds. the location of needle was 4 mm shorter than working length. roor canal was dried with five pieces sterile paper point. root canal bacteria was collected using sterile paper point and put into media (bhib) and sent to microbiology laboratory of faculty of dentistry, airlangga university for colony calculation. supporting to this study a software fluent had been done in vitro in laboratory of machine technique, its surabaya. results the research of the effectivity of blunt end with side hole irrigation needle to eliminate the number of bacteria had been done. normal distribution test with kolmogorovsmirnov test before and after irrigation, was done for group i and ii. it was indicated that the result of all groups was normally distributed with p > 0.05. paired t test was done to compared the difference of bacteria number between control group and treatment group. there was a significant difference (p < 0.05) of bacteria number in necrotic mandibular anterior teeth before and after irrigation between group i and ii (table 1). using independent t test, there was a significant difference (p < 0.05) between both groups on of bacteria number in root canal of necrotic mandibular anterior teeth (table 2). table 1. the mean, standard deviation and paired t test of bacteria number in mandibular anterior teeth after treatment table 2. statistical result with independent t test in different number of bacteria in necrotic mandibular anterior teeth root canal between group i and ii conventional irrigation needle produced fluid velocity direction into radial of anterior root canal (figure 3). the conventional irrigation needle which is entered to root canal with depth of ¾ root canal length, produced spraying pattern in axial direction with teeth axis. the velocity is 1.14–1.37 m/s (yellowish green color) (figure 3-a). then, the velocity decreased to 0.456–0.685 m/s (light blue color) until irrigation material contact with apical foramen and filled at the limit of irrigation needle end, so velocity figure 1. conventional irrigation needle. figure 2. blunt end with side hole irrigation needle. aseptic procedures were done for tools, materials and teeth before the treatment. the teeth were isolated using rubber dam and the cavity entrance was made using a high speed bur. the necrotic pulp tissue was removed with extirpation needle then root canal preparation was done with no.15 file. sterile paper point was put in root canal to take the bacteria,9,10 then put into media (bhib) and sent to microbiology laboratory for colony calculation. root canal preparation was continued with conventional technique until file no. 60 and irrigated using blunt end with side hole irrigation needle on six samples. another six samples were irrigated using conventional irrigation 170 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 168–171 becomes 0 (dark blue color) (figure 3-b). after that, the irrigation material flew out with bringing debris with bacteria composition, with velocity of 0.590–0.885 m/s (green to yellowish green color). blunt end with side hole irrigation needle produced the fluid velocity direction into radial anterior root canal and tangential velocity that contact the inner wall of anterior root canal (figure 4). the blunt end with side hole irrigation needle which was entered to root canal with depth of ¾ root canal length, produced spraying pattern shape 900 angle to teeth axis with velocity of 1.18–1.42 m/s (yellowish green color) (figure 4-a). then, velocity decreased to figure 3. spray pattern in axial direction (same direction with y axis) for y/h = 0,75. 0.237–0.473 m/s (light blue color) until irrigation material contacted with apical foramen and filled at the limit of irrigation needle end, so velocity becomes 0 (dark blue color) (figure 4-b). after that, the irrigation material flew out with bringing debris with bacteria composition, with velocity of 0.497–0.662 m/s (greenish blue color). discussion in this research, the combination of 3% h2o2 and sterile aquadest was used to neutralize on. 3% h2o2 is light numeric flow pattern of spraying by blunt end with side hole irrigation needle. figure 4. spraying pattern shapes 90° angle to y axis for y/h = 0.75. 171setyowati: effectivity of blunt end with side hole irrigation needle antiseptic,1 we used this irrigation material in order to removed confounding variable. the difference of bacteria number in this research was caused by the difference of irrigation needle, not by the effect of irrigation material. according to walton and torabinejad,5 irrigation material for root canal must has sterilization or disinfectant characteristic. this research was supported with in vitro research of software fluent process to examined the irrigation material flow that was sprayed by two kinds of irrigating needles. the result of software fluent process proved that the spray flow of blunt end with side hole irrigation needle was turbulent. this spray flow consists of tangential and radial flow. the spray flow which was resulted by the conventional irrigation needle was laminar. if we examined the numeric flow pattern of spraying irrigation needle, velocity from blunt end with side hole irrigation needle was less than conventional irrigation needle. it is mean, the blunt end with side hole irrigation needle clean the root canal wall more clearer than conventional irrigation needle. in vivo research indicated that blunt end with side hole irrigation needle decreased bacteria number significantly compared to conventional irrigation needle. blunt end with side hole irrigation needle produced flow direction velocity of irrigation material into radial direction and also tangential velocity that contact root canal wall. the fusion of this two kinds of velocity clean more debris and bacteria after root canal preparation. conventional irrigation needle has a bevel on end side and a hole located on the bevel. this kind of needle will produce flow velocity direction same with teeth axis (axial direction). whith this flow pattern, part of fluid that contacts with root canal wall and irrigation needle will produce zero velocity, so it can not clean debris and bacteria. this research indicated that the elimination of the number of bacteria in root canal mandibular teeth was not optimal, therefore it should be irrigated using combination of blunt end with side hole irrigation needle and conventional irrigation needle. by using blunt end with side hole irrigation needle, debris will be accumulate in apical area after irrigation. irrigation with conventional irrigation needle is for excretion of debris from root canal. this procedure is not valid for maxillary anterior teeth because earth gravitation will cause the debris and bacteria self-excreted after irrigation. in conclusion, blunt end with side hole irrigation needle is more effective to eliminate root canal bacteria than conventional irrigation needle. the blunt end with side hole irrigation needle is effective as irrigating device for mandibular teeth. for optimal result, we should use the combination between blunt end with side hole irrigation needle and conventional irrigation needle. references 1. grossman li, 0liet s, del rio ce. endodontic practice. 11st ed. philadelphia: lea and febiger; 1988. p. 179, 187–203. 2. ingle ji, taintor jf. endodontics. 3rd ed. philadelphia: lea and febiger; 2001. p. 166–86. 3. cohen c, burn rc. pathways of the pulp. 8th ed. st louis: mosby; 2002. p. 231–91. 4. weine fs. endodontic therapy. 3rd ed. st louis, toronto: mosby co; 2003. p. 317– 24. 5. walton re, torabinejad m. prinsip dan praktik ilmu endodonsi. edisi ke-2. jakarta: penerbit buku kedokteran egc; 1998. p. 277–78. 6. soedojo p. fisika dasar. cetakan ke 2. andi. yogyakarta: penerbit bhratara; 2000. p. 45–52. 7. kahn fh, rosenberg pa, gliksberg j. an in vitro evaluation of the irritating characteristics of ultrasonic and subsonic hand pieces and irrigating needles and probe. j endod 1995; 21:277–80. 8. widodo wa. pemodelan numerik soft ware fluent. its mesin surabaya: 2004. 9. pumarola-sune j, sola-vicens l, sentis-vilalta j, canalda-sahli c, brau-aguade e. absorbency properties of different brands of standardized endodontic paper point. j endod 1998; 24:796–98. 10. edwards ro, bandyopadhyay s. physical and mechanical properties 101 volume 46 number 2 june 2013 compressive strength resin komposit hybrid post curing dengan light emitting diode menggunakan tiga ukuran lightbox yang berbeda (compressive strength of hybrid composites resin with post curing light emitting diode using three different sizes of lightbox) mirza aryanto,1 milly armilia2 dan dudi aripin2 1bagian konservasi gigi, fakultas kedokteran gigi universitas prof. dr. moestopo (b), jakarta – indonesia 2bagian konservasi gigi, fakultas kedokteran gigi universitas padjadjaran, bandung – indonesia abstract background: the use of different polymerization methods may result in variation of mechanical properties of composite resin. polymerization increases the conversion rate of monomers reflecting in improvement of compressive strength. post-curing methods can be used to increase strength to the composite resin. purpose: to determine the difference of compressive strength of post cured hybrid composite resin by using three different size of lightbox. methods: this research was conducted in a true in vitro experiment. research carried out by making a tube-shaped cylinder hybrid with 3 mm diameter and 6 mm height composite resin samples post cured by using 3 different size of light box, 3 cm x 3 cm x 3 cm (a), 4 cm x 4 cm x 4 cm (b) 6 cm x 6 cm x 6 cm (c), and a non post-curing control. compressive strength test was then performed using a universal testing machine. each sample was tested and averaged to obtain values in order to be analyzed statistically using anova and multiple comparison. results: there is an increase in compressive strength of each group, namely group a (172.9460 mpa), b (154.821 mpa), c (154.0789 mpa) and control (123.3550 mpa), and a statistically significant difference (f<0.05). conclusion: the smaller size of the lightbox is used, the higher the compressive strength of composite resin. key words: compressive strength, post curing, lightbox, hybrid composite resin abstrak latar belakang: penggunaan berbagai metode polimerisasi dapat mengubah sifat mekanis resin komposit. proses polimerisasi dapat meningkatkan derajat konversi monomer, sehingga dapat meningkatkan compressive strength resin komposit. metode post curing dapat digunakan untuk menambah kekuatan resin komposit. tujuan: untuk mengetahui perbedaan compressive strength resin komposit hybrid yang dilakukan post curing menggunakan tiga ukuran lightbox yang berbeda. metode: jenis penelitian ini adalah eksperimental murni yang dilakukan secara in vitro. penelitian dilakukan dengan membuat sampel resin komposit hybrid berbentuk tabung silinder dengan diameter 3 mm dan tinggi 6 mm yang dilakukan post curing menggunakan lightbox ukuran 3 cm x 3 cm x 3 cm (a), lightbox ukuran 4 cm x 4 cm x 4 cm (b), lightbox ukuran 6 cm x 6 cm x 6 cm (c), dan kelompok kontrol yang tidak dilakukan post curing. kemudian dilakukan uji compressive strength dengan menggunakan alat uji universal testing machine. data dianalisis secara statistik menggunakan anova dan multiple comparison. hasil: terdapat peningkatan compressive strength pada tiap kelompok, yaitu kelompok a (172,9460 mpa), b (154,821 mpa), c (154,0789 mpa) dan kontrol (123,3550 mpa), dan secara statistik terdapat perbedaan yang signifikan (p<0,05). simpulan: semakin kecil ukuran lightbox yang digunakan, semakin tinggi tingkat compressive strength resin komposit hybrid. kata kunci: compressive strength, post curing, lightbox, resin komposit hybrid korespondensi (correspondence): mirza aryanto, bagian konservasi gigi, fakultas kedokteran gigi universitas prof. dr. moestopo (b). jl. bintaro permai raya no. 3, jakarta 12220, indonesia. e-mail: emyr2000@yahoo.com research report 102 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 101–106 pendahuluan resin komposit telah digunakan secara luas di bidang kedokteran gigi karena dapat memberikan hasil akhir restorasi yang baik, yaitu memiliki estetis yang memadai dan kekuatan yang cukup.1 resin komposit pertama kali diperkenalkan pada pertengahan tahun 60-an sebagai bahan restorasi pada gigi anterior, tetapi saat ini resin komposit digunakan juga pada gigi posterior.2 restorasi pada gigi harus dapat menerima beban kunyah, baik secara langsung maupun tidak langsung pada saat oklusi dan artikulasi. beban yang diterima oleh gigi posterior jauh lebih besar bila dibandingkan dengan gigi anterior, oleh karena itu jenis resin komposit yang digunakan harus memiliki compressive strength yang baik.3 compressive strength resin komposit salah satunya dipengaruhi oleh proses polimerisasi. proses polimerisasi akan menentukan persentase perubahan ikatan ganda monomer menjadi ikatan tunggal polimer dikenal sebagai derajat konversi. umumnya, resin komposit yang disinar hanya memiliki derajat konversi sebesar 55-75%. sisa monomer yang tidak berpolimerisasi akan menurunkan compressive strength resin komposit. hal ini sejalan dengan penelitian yang menyatakan terdapat hubungan langsung antara derajat konversi dengan compressive strength resin komposit, semakin besar derajat konversi, maka semakin tinggi nilai compressive strength resin komposit.4-7 compressive strength adalah kemampuan suatu bahan untuk menahan beban kekuatan tekan. compressive strength yang rendah dapat mengakibatkan kegagalan restorasi secara klinis, yaitu degradasi tepi pada daerah restorasi yang tipis dan terjadi patah serta retak pada restorasi.1,3 semakin banyak sisa monomer yang tersisa akibat proses polimerisasi yang tidak sempurna maka tingkat compressive strength semakin rendah dan menyebabkan terjadinya celah mikro yang dapat menyebabkan karies sekunder, dan sensitivitas gigi.6,8 akibat dari rendahnya compressive strength dan adanya celah mikro tersebut menyebabkan terjadi kegagalan restorasi resin komposit.9,10 teknik restorasi resin komposit indirek dengan post curing diperkenalkan untuk mengurangi kekurangan tersebut. post curing adalah suatu teknik polimerisasi tambahan yang dilakukan setelah penyinaran awal dengan tujuan agar meningkatkan derajat konversi untuk menyempurnakan polimerisasi. cara post curing ada beberapa, yaitu kombinasi penyinaran, pemanasan, dan pemanasan dengan tekanan.6,7,9 menurut penelitian, post curing dapat dilakukan menggunakan lightbox yang berupa kubus kaca yang terdiri dari cermin pantul. penelitian tersebut membuktikan bahwa post curing dengan lightbox memberikan kekerasan komposit yang lebih baik dibandingkan tanpa post curing. post curing dengan lightbox mampu menyempurnakan proses polimerisasi karena efek refleksi sinar yang lebih merata dan akhirnya akan meningkatkan kekerasan resin komposit.11,12 pembuatan lightbox pada penelitian sebelumnya dibuat sendiri dan menggunakan ukuran yang berbeda. hal ini disebabkan belum ada penelitian mengenai pengaruh ukuran lightbox terhadap compressive strength. tujuan penelitian untuk mengetahui perbedaan compressive strength resin komposit hybrid pada post curing dengan penyinaran light emitting diode menggunakan tiga ukuran lightbox yang berbeda. bahan dan metode jenis penelitian yang digunakan adalah eksperimental murni secara in vitro. populasi penelitian ini adalah bahan resin komposit jenis hybrid. sampel diambil dari populasi resin komposit secara acak. sampel berjumlah 40 buah, berbentuk silinder dengan ukuran diameter 3 mm dengan ketebalan 6 mm. pada penelitian ini dilakukan pembuatan sampel dengan cara menyiapkan cetakan resin komposit, masukkan selapis resin komposit (± 2 mm) ke dalam cetakan. tekan dan ratakan dengan stopper semen. resin komposit disinari dengan led selama 20 detik, kemudian ditambahkan lapisan resin komposit sampai cetakan penuh, pada permukaan atas lapisan terakhir dilapisi dengan celluloid strip kemudian disinar kembali. setelah resin komposit dilepaskan dari cetakan, sampel diukur kembali dengan jangka sorong. permukaan sampel diperiksa kembali menggunakan kaca karena harus berkontak tepat dengan bidang alat uji. sampel dibagi menjadi 4 kelompok yang masing-masing terdiri dari 10 sampel, yaitu kelompok a, b, c dan kelompok kontrol. pada kelompok a, resin komposit hybrid dilakukan post curing menggunakan lightbox ukuran 3 cm x 3 cm x 3 cm selama 60 detik. kelompok b, resin komposit hybrid dilakukan post curing menggunakan lightbox ukuran 4 cm x 4 cm x 4 cm selama 60 detik. kelompok c, resin komposit hybrid dilakukan post curing menggunakan lightbox ukuran 6 cm x 6 cm x 6 cm selama 60 detik. pada kelompok kontrol tidak dilakukan post curing. sampel diletakkan pada meja uji universal testing machine di bawah beban tekan hingga berkontak dengan sampel, sampai pusat beban mesin berada dalam arah vertikal dari tengah permukaan sampel. mesin uji diaktifkan lalu diberikan beban 1 n secara kontinu dengan kecepatan 1 mm/min, sampai terbentuk retakan atau patahan pada sampel. analisis data hasil penelitian menggunakan uji anova. jika hasil signifikan dilanjutkan dengan metode uji least square difference (lsd). hasil hasil pengujian menggunakan universal testing machine didapatkan rerata nilai compressive strength dari tiap sampel. rerata nilai compressive strength dianalisis menggunakan one way anova untuk menguji kesamaan rerata compressive strength resin komposit setelah diberi empat macam perlakuan dan didapatkan hasil yang sangat 103aryanto, et al.,: compressive strength resin komposit hybrid post curing bermakna (p<0,05). setelah dilakukan uji anova maka nilai data kembali dilakukan uji multiple comparison dengan least square different (lsd). hasil dari tabel 1 menunjukkan bahwa antara kontrol ketiga kelompok uji hasilnya adalah signifikan, artinya ada perbedaan antara ukuran lightbox yang digunakan dengan kontrol (p<0,05). kelompok uji b dibanding kelompok uji c hasil tidak ada perbedaan yang signifikan (p>0,05). gambar 1 memperlihatkan gambaran deskriptif rerata nilai compressive strength resin komposit hybrid yang dilakukan post curing dengan menggunakan ukuran lightbox yang berbeda. grafik tersebut terlihat rerata nilai compressive strength karena perlakuan post curing dengan menggunakan ukuran lightbox 3 cm x 3 cm x 3 cm paling tinggi dan rerata nilai compressive strength pada kontrol yang tidak dilakukan post curing paling rendah. pembahasan resin komposit sebagai bahan restorasi gigi telah banyak digunakan sebagai bahan restorasi gigi posterior yang menerima beban kunyah yang besar. kekuatan resin komposit dipengaruhi proses polimerisasi. sifat mekanis resin komposit dapat ditingkatkan dengan melakukan proses polimerisasi yang sempurna. pada kenyataannya, monomer yang terpolimerisasi hanya sekitar 48-60%. untuk menyempurnakan polimerisasi, dikembangkan teknik post curing yang bertujuan untuk menyempurnakan polimerisasi resin komposit dan meningkatkan sifat mekanis.3,13,14 beberapa faktor yang penting dipertimbangkan dalam sistem penyinaran pada saat proses polimerisasi adalah: waktu paparan, intensitas sinar, jarak dan sudut antara sinar dan resin komposit, ketebalan resin, warna resin, jenis filler, jumlah fotoinisiator15-20 tabel 2 menunjukkan terdapat perbedaan nilai compressive strength antara ke empat kelompok uji. hal ini sesuai dengan beberapa penelitian sebelumnya yang membuktikan bahwa post curing dapat meningkatkan derajat polimerisasi resin komposit yang diperiksa melalui uji compression test.7,14,32,33 polimerisasi resin komposit selalu meninggalkan monomer-monomer yang tidak terpolimerisasi sempurna. hal ini dipengaruhi oleh penetrasi sinar yang tidak sampai dengan sempurna ke seluruh matriks dan filler resin komposit, sehingga perlu dilakukan polimerisasi tambahan (post curing) untuk meningkatkan derajat konversi dan meningkatkan sifat mekanis restorasi resin komposit. teknik post curing yang dilakukan pada penelitian ini menggunakan lightbox dengan prinsip dasar pemantulan sinar pada cermin pantul. setiap sinar dari led dipantulkan secara merata dari permukaan cermin di dalam lightbox sehingga mampu menyempurnakan penetrasi sinar pada matrik resin dari segala arah.11,12 metode post curing dengan penyinaran muncul setelah beberapa penelitian memperlihatkan besarnya monomer yang tersisa tidak terpolimerisasi karena intensitas sinar tidak dapat mencapai keseluruhan fotoinisiator pada matrik resin komposit. jarak antara sinar dengan komposit sangat mempengaruhi penetrasi sinar. pada teknik restorasi indirek dilakukan penyinaran tambahan di luar mulut untuk meningkatkan penetrasi sinar, sehingga akan meningkatkan derajat polimerisasi.5,21,22 penyinaran tambahan dapat dilakukan dengan menggunakan alat bantu yaitu lightbox. lightbox adalah kotak kubus berukuran sama sisi, pada bagian dalam setiap sisinya adalah cermin pantul. ketika penyinaran dilakukan dari atap lightbox, sinar akan menyebar secara merata melalui pantulan cermin. penggunaan alat ini dapat menyebabkan penetrasi sinar lebih merata, sehingga derajat polimerisasi meningkat.11,12 konsep pemakaian lightbox dengan cermin pantul adalah dari teori bahwa sebuah sinar dengan panjang gelombang tertentu bila dikenakan pada cermin pantul akan mengalami pemantulan. pemantulan terjadi jika berkas sinar sejajar jatuh pada permukaan, maka sinar tersebut akan dipantulkan sejajar dan searah (gambar 2). sinar yang jatuh pada cermin akan mengalami pemantulan/refleksi sebesar 45%, penyerapan/absorpsi sebesar 17%, dan penerusan/transmisi sebesar 38%.23-25 penelitian membuktikan bahwa resin komposit hybrid memiliki compressive strength dan ketahanan fraktur yang tabel 2. rerata nilai compressive strength resin komposit hybrid post curing menggunakan lightbox dengan ukuran yang berbeda (mpa) no. kontrol kel a kel b kel c 1 2 3 4 5 6 7 8 9 10 98,513 121,02 119,07 116.99 122,56 105,66 123,11 112,78 128,20 125,65 146,90 204,82 142,85 145,36 191,32 233,16 194,25 176,18 185,25 109,37 158,26 169,58 124,89 168,41 211.54 133,88 176,93 81,101 74,949 248,67 179,86 182,01 259,27 106,87 165,22 163,30 120,96 121,13 81,749 160,31 rerata ± simpangan baku 123,3550 ± 24,306 172,9460 ± 36,487 154,821 ± 53,827 154,0789 ± 49,890 104 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 101–106 lebih besar dibandingkan jenis mikrofil. resin komposit hybrid merupakan bahan kombinasi ukuran partikel macrofilled dan microfilled dengan ukuran filler 0,4-1μ. bahan pengisi resin komposit hybrid terdiri dari silika koloida yang memiliki volume 75-80% dari keseluruhan volume. resin komposit jenis hybrid memiliki ekspansi termal yang setara dengan struktur gigi dan memiliki sifat fisik yang lebih kuat dibandingkan jenis lain.14 compressive strength adalah kemampuan suatu bahan untuk menahan beban kekuatan tekan. ketika batas nilai compressive strength terlewati, maka bahan tersebut akan mengalami kehancuran atau pecah.26-29 compressive strength resin komposit tergantung pada kandungan filler, tipe filler, ukuran filler dan derajat konversi.30,31 derajat konversi adalah perhitungan persentase ikatan ganda monomer karbon yang bergabung menjadi ikatan tunggal yang membentuk resin polimer. semakin tinggi derajat konversi bahan resin komposit, semakin tinggi pula tingkat kekerasan, ketahanan terhadap keausan dan sifat mekanis lainnya.14 derajat konversi sangat berpengaruh terhadap sifat mekanis restorasi resin komposit.32 beberapa penelitian menunjukkan bahwa polimerisasi dengan penyinaran dipengaruhi oleh beberapa faktor, misalnya kandungan resin komposit, warna resin komposit, kualitas unit sumber penyinaran, waktu paparan, serta ketebalan lapisan resin komposit.33 sistem polimerisasi tambahan dengan cara post curing dapat meningkatkan derajat konversi monomer sehingga dapat meningkatkan kekerasan permukaan, compressive strength dan fleksural.29 post curing dapat meningkatkan nilai konversi monomer dengan cara dilakukan polimerisasi sinar terhadap keseluruhan permukaan restorasi.7 banyak penelitian yang telah dilakukan pada post curing, menurut hasil penelitian terbukti bahwa post curing dapat meningkatkan sifat mekanis resin komposit.32,34 hal ini bertentangan dengan penelitian yang lain yang menunjukkan bahwa perlakuan post curing tidak meningkatkan ketahanan resin komposit terhadap keausan.34 salah satu metode post curing adalah penyinaran tambahan yang dilakukan dengan menggunakan lightbox yang berupa kubus kaca yang memiliki efek cermin sehingga dapat memantulkan sinar secara merata ke segala arah.11,12 menurut penelitian diketahui bahwa jarak dari sumber sinar mempengaruhi polimerisasi resin komposit. semakin jauh jarak sumber sinar dari resin komposit, maka polimerisasi resin komposit akan semakin menurun.35,36 hasil post curing dengan lightbox pada penelitian ini membuktikan bahwa terdapat perbedaan compressive strength resin komposit pada post curing dengan sinar tambahan menggunakan tiga ukuran lightbox yang berbeda. diagram hasil analisis seperti terlihat pada gambar 1 serta tabel 2 memperlihatkan bahwa rata-rata nilai compresssive strength pada ukuran lightbox 3 cm x 3 cm x 3 cm menunujukkan nilai paling tinggi dan telah terbukti paling signifikan secara statistik. hal ini membuktikan bahwa pada ukuran lightbox terkecil, terdapat pemantulan sinar yang paling baik sehingga proses polimerisasi lebih merata. proses polimerisasi yang merata dapat dilihat dari nilai compressive strength yang paling tinggi dibandingkan dengan pemakaian ukuran lightbox yang lain. nilai compressive strength resin komposit dipengaruhi oleh banyak faktor, antara lain komposisi matriks organik resin komposit, derajat konversi serta tipe dan jumlah partikel filler. faktor yang dapat dimodifikasi pada penelitian ini adalah intensitas sinar, panjang gelombang dan waktu paparan untuk mendapatkan derajat konversi yang lebih besar. hasil penelitian menunjukkan rerata nilai compressive strength pada lightbox ukuran 4 cm x 4 cm tabel 1. hasil analisis multiple comparison untuk menguji perbedaan rerata compressive strength resin komposit antara empat macam perlakuan (i) perlakuan (j) perlakuan sig. kontrol kelompok a .000** kelompok b .043* kelompok c .048* kelompok a kontrol .000* kelompok b .004* kelompok c .003* kelompok b kontrol .043* kelompok a .004* kelompok c .959 kelompok c kontrol .048* kelompok a .003* kelompok b .959 keterangan: * = signifikan gambar 1. gambaran deskriptif compressive strength resin komposit hybrid yang dilakukan post curing dengan ukuran lightbox yang berbeda. 105aryanto, et al.,: compressive strength resin komposit hybrid post curing x 4 cm terlihat lebih besar dari rerata nilai compresssive strength pada ukuran lightbox ukuran 6 cm x 6 cm x 6 cm tetapi tidak signifikan secara statistik. hal ini disebabkan pengurangan intensitas sinar pada jarak yang diperbesar. menurut penelitian penambahan jarak mempengaruhi pengurangan derajat konversi akibat pengurangan intensitas yang dipancarkan sinar. hal ini sesuai dengan penelitian bahwa penambahan jarak 10 mm dapat mengurangi intensitas sinar sekitar 10%.35,36 selain itu, saat sinar dipantulkan pada cermin pantul, terjadi pengurangan intensitas sinar. pada saat sinar dipancarkan pada cermin pantul, tidak semua intensitas sinar yang dipantulkan, karena sebagian intensitas sinar mengalami penerusan (transmisi) dan penyerapan (absorpsi).23-25 kombinasi antara jarak dan pengurangan intensitas setelah pemantulan mengakibatkan pengurangan derajat konversi yang mengakibatkan terjadinya pengurangan nilai compressive strength pada resin komposit. kesimpulan penelitian ini semakin kecil ukuran lightbox yang digunakan, semakin tinggi tingkat compressive strength resin komposit. daftar pustaka 1. roberson tm, heymann h, swift ej. sturdevant’s art and science of operative dentistry. 5th ed. st. louis: mosby inc; 2006. p. 505-15. 2. hervás-garcía a, martínez-lozano ma, cabanes-vila j, barjauescribano a, fos-galve p.. composite resins. a review of the materials and clinical indications. med oral patol oral cir bucal 2006; 11(2): e215-20. 3. craig rg, powers jm. restorative dental materials. 12th ed. st. louis: mosby; 2006. p. 191-208. 4. mohamad d, young rj, mann ab, watts dc. post-polymerization of dental resin composite evaluated with nanoindentation and microraman spectroscopy. arc of orofac sc 2007; 2: 26-31. 5. jain v. evaluation of second generation indirect composite resin. thesis. department of dental material. indiana university; 2008. p. 15-6. 6. aschheim kw, dale bg. esthetic dentistry. 2nd ed. st. louis: mosby; 2001. p. 97-101. 7. hargreaves km, cohen s, berman lh. cohen’s pathways of the pulp. 10th ed. st. louis: mosby; 2011. p. 303-9. 8. ciccone-nogueira jc, borsatto mc, souza-zaroniaro wc, ramos rp, palma-dibb rg. microhardness of composite resin at different depths varying the post-irradiation time. j appl oral sci 2007; 15(4): 305-9. 9. lombardo ghl, carvalho cf, galhano g, souza roa, nogueira jr l, pavanelli ca. influence of additional polymerization in the microhardness of direct composite resins. cienc odont bras 2007; 10(2): 10-5. 10. awan m. a study investigating the mechanical testing of a novel dental restorative material and its biocompatibility. thesis. birmingham: biomaterial unit school of dentistry st chad’s queensway; 2010. p. 13-4. 11. widyasari r. perbedaan compressive strength komposit pada post curing dengan pemanasan dan lightbox. tesis. bandung: fakultas kedokteran gigi universitas padjadjaran; 2010. p. 12-7. 12. rizany t. perbandingan daya tahan terhadap tekanan antara komposit hybrid dan komposit nannofilled setelah post curing menggunakan lightbox. tesis. bandung: fakultas kedokteran gigi universitas padjadjaran; 2010. p. 14-5. 13. van noort, r. introduction to dental materials. 3rd ed. london: mosby elsevier ltd; 2007. p. 43-50. 14. anusavice, kenneth. philip‘s science of dental material. 11th ed. st. louis: wb saunders co; 2003. p. 402-20. 15. pattel, v. study of the composite resin. thesis. chemistry education. university of pennsylvania; 2007. p. 23-5. 16. sankai sg, diwakar hs, babu r, krishna m, murthy vs. synopsis of dental materials. bangalore: paras med pub; 2002. p. 19-32. 17. nicholson jw. the chemistry of medical and dental materials. cambridge: mpg books ltd; 2002. p. 148-58. 18. gladwin m, bagby m. clinical aspects of dental materials: theory, practice and cases. 3rd ed. philadelphia: lippincott ltd; 2009. p. 55-72. 19. lindberg a. resin composites: curing techniques. disertation. sweden: department of dental education. umeå university; 2005. p. 27-9. 20. malhotra n, mala k. light-curing considerations. acailable at: http://cde.dentalaegis.com/courses/4466. diakses tanggal 15 juni 2011. 21. nandini s. indirect composite resin. j conserv dent 2010; 13(4): 184-94. gambar 2. gambaran pola pantulan sinar yang mengenai cermin pantul.24,25 (a) menunjukkan gambaran sinar jatuh akan sejajar dengan sinar pantul; (b) terlihat gambaran sinar dengan intensitas yang berkurang setelah dipantulkan pada cermin. a b 106 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 101–106 22. terry da, leinfelder kf. development of a processed composite resin restoration: preparation and laboratory fabrication. int dent sa 2003; 8(4): 12-20. 23. subadyo ta. impact of solar radiation reflection. disertasi. jakarta: universitas indonesia; 1997. p. 34-7. 24. beeson s, mayer jw. patterns of light. oxford: springer; 2008. p. 13-23. 25. fakhruddin h. measuring the refractive index of air using a vacuum chamber. american vacuum society international symposium; 1998. p. 21-2. 26. kilaru kr. comparative evaluation of compressive strength hybrid posterior composites: an in vitro study. thesis. bangalore: department of conservative dentistry and endodontics. rajiv gandhi university 2006. p. 23-5. 27. wang l, d’alpino php, lopes lg, pereira jc. mechanical properties of dental restorative materials. j appl oral sci 2003; 11(3): 162-7. 28. manolea h, degeratu s, deva v, coles e, draghici e. contributions on the study of the compressive strength of the light-cured composite resins. j chs. 2009; 36(4): 62-5. 29. klymus me, oshima hms. influence of the mechanical properties of composites for indirect dental restorations on pattern failure. baltic dent and maxillofacial j 2007; 9: 57-60. 30. mccabe jf, walls awg. applied dental materials. 9th ed. oxford: blackwell pub. 2008. p. 195-224. 31. elbishari h, silikas n, satterthwaite j. filler size of resin-composites, percentage of voids and fracture. dent mater j 2012; 31(4): 523–7. 32. xu, hhk. whisker-reinforced heat-cured dental resin composite: effect of filler level and heat-cure temperature and time. j dent res 2000; 79(6): 1392-7. 33. jong lcg, opdam njm, bronkhorst em, roeters jjm, wolke jgc, geitenbeek b. the effectiveness of different polymerization protocols for class ii composite resin restorations. journal of dentistry 2007; 35: 513-20. 34. martin jr lo, mota jm, vaz rr. evaluation of the mechanical properties of light-cure composite resins submitted to post-cure. rfo passo fundo j 2010; 15(3): 275-80. 35. sobrinho lc, lima aa, consani s, sinhoreti mac, knowles jc. influence of curing tip distance on composite knoop hardness values. braz dent j 2000; 11(1): 11-7. 36. aquiar ahb, lazzari cr, lima danl, ambrosano gmb, lovadino jr. effect of light curig tip distance and resin shade on microhardness of a hybrid resin composite. braz oral res 2005; 19(4): 302-6. issn 1978 3728volume 46 number 1 march 2013 editorial board of dental journal (majalah kedokteran gigi) sk: 166/h3.1.2/kd/2013 january 2nd– december 31st, 2013 patron: dean of faculty of dentistry universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dentistry – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 46 number 1 march 2013: dr. retno indrawati, drg., msi. (oral biology – universitas airlangga) dr. indah listiana, drg., m.kes. (oral biology – universitas airlangga) wisnu setyari, drg., m.kes. (oral biology – universitas airlangga) dr. ira arundina, drg., m.kes. (oral biology – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 205162250 -204225738 contents page printed by: airlangga university press. (148/11.13/aup-b5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 46 number 1 march 2013 issn 1978 3728 1. patient’s expectation on communication performances community of dental health services providers located in urban and rural area taufan bramantoro and ninuk hariyani ..................................................................................... 1–4 2. changes in setting time of alginate impression material with different water temperature decky j. indrani and niti matram ................................................................................................. 5–8 3. bactericidal and cytotoxic effects of erythrina fusca leaves aquadest extract janti sudiono, ferry sandra, nadya saputri halim, timotius andi kadrianto and melinia .. 9–13 4. the role of inducible nitric oxide synthase in teeth periapical lesions immunopathogenesis caused by enterococcus faecalis tamara yuanita, latief mooduto and kuntaman ........................................................................ 14–17 5. antitumor activity of antisense oligonucleotide p45skp2 in soft palate carcinoma cell squamous in vitro supriatno, sartari entin yuletnawati and iwa sutardjo rus sudarso ....................................... 18–22 6. evaluation of local muscle soreness treatment with anterior bite splint made of soft putty impression material harry laksono dan sherman salim ............................................................................................... 23–29 7. unidentified angular recurrent ulceration responsive to antiviral therapy rahmi amtha and siti aliyah pradono .......................................................................................... 30–34 8. the inhibition of malignant epithelial cells in mucosal injury in the oral cavity of strains by pomegranate fruit extract (punica granatum linn) through bcl-2 expression sri hernawati ................................................................................................................................... 35–38 9. shear strength of orthodontic bracket bonding with gic bonding agent after the application of cpp-acpf paste melisa budipramana, thalca hamid and sianiwati goenharto .................................................. 39–44 10. hemolysin activities as virulence factor of enterococcus faecalis isolated from saliva and periapical abscess (gene detection by pcr) dewa ayu n.p.a, sari dewiyani and dessy sulistya ashari ........................................................ 45–49 11. effect of citrus aurantifolia swingle essential oils on methyl mercaptan production of porphyromonas gingivalis anindya prima yusinta, ivan arie wahyudi and anne handrini dewi .................................... 50–54 �� volume 46 number 1 march 2013 research report shear strength of orthodontic bracket bonding with gic bonding agent after the application of cpp-acpf paste melisa budipramana, thalca hamid and sianiwati goenharto department of orthodontics faculty of dentistry, universitas airlangga surabaya indonesia abstract background: white spot lesion is a major problem during fixed orthodontic treatment. this problem can be solved by minimizing white spot lesion before the treatment and using a fluoride-releasing bonding agent. the application of casein phosphopeptidesamorphous calcium phospate fluoride (cpp-acpf) paste as remineralization agent before treatment and gic as orthodontic bonding agent is expected to overcome this problem as well as to strengthen gic bonding. purpose: to measure the shear strength of fix orthodontic appliance using gic bonding with cpp-acpf application prior treatment. methods: in this study, 50 extracted premolars were randomly divided into 2 groups: group 1 as treatment group and group ii as control group that was not given cppacpf pretreatment. after having been cut and put into acrylic device, the samples in group i were given pretreatment with cpp-acpf paste on enamel surface for 2 minutes twice a day as instructed in product label for 14 days. orthodontic brackets were bonded with gic bonding agent on all samples in both groups as instructed in product label. then, the shear strength was measured by autograph shimatzu with crosshead speed 0.5 mm/minute. the data was analyzed with independent t-test. results: the mean shear bond strength in treatment group was 19.22 ± 4.04 mpa and in control group was 12.97 ± 3.97 mpa. independent t-test analysis showed that there was a significant difference between treatment and control group (p<0.05). conclusion: cpp-acpf pretreatment could increase gic orthodontic bonding shear strength. key words: shear strength, gic bonding, cpp-acpf abstrak latar belakang: lesi putih karies merupakan masalah utama selama perawatan dengan peranti cekat ortodonti. hal ini dapat diatasi dengan cara mengurangi lesi putih sebelum perawatan dengan menggunakan bahan bonding yang mengandung fluorida. aplikasi pasta casein phosphopeptides-amorphous calcium phospate fluoride (cpp-acpf) sebagai bahan remineralisasi sebelum perawatan dan bahan bonding gic diharapkan dapat mengatasi masalah ini sekaligus menambah kekuatan cekat bahan bonding gic. tujuan: mengukur kekuatan geser piranti cekat ortodonti menggunakan bonding gic dengan aplikasi pasta cpp-acpf. metode: 50 gigi premolar dibagi menjadi 2 kelompok, yaitu kelompok i sebagai kelompok perlakuan dan kelompok ii sebagai kelompok kontrol. setelah gigi dipotong dan ditanam dalam tabung akrilik, diaplikasikan pasta cpp-acpf pada permukaan enamel sampel pada kelompok i selama 2 menit 2 kali sehari selama 14 hari. aplikasi pasta cpp-acpf tidak dilakukan pada kelompok kontrol. kemudian breket ortodonti direkatkan dengan bahan bonding gic pada semua sampel di kelompok i dan kelompok ii. kekuatan geser diukur dengan menggunakan alat autograph shimatzu dengan kecepatan cross head 0.5 mm/menit. data dianalisis dengan statistik independent t-test. hasil: rerata kekuatan geser pada kelompok perlakuan 19,22 ± 4,04 mpa dan pada kelompok kontrol 12,97 ± 3,97 mpa. analisis independent t-test menunjukkan bahwa terdapat perbedaan signifikan antara kelompok perlakuan dan kontrol (p<0.05). kesimpulan: kekuatan geser antara piranti cekat ortodonti dengan bahan bonding gic meningkat setelah aplikasi pasta cpp-acpf. kata kunci: kekuatan geser, bonding gic, cpp-acpf correspondence: melisa budipramana, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: bdprmn_melz@yahoo.com �0 dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 39–44 introduction recently, orthodontic treatment is a viable option to repair teeth arrangement and aesthetics, however, risks of caries will increase on patients using fixed orthodontic appliances. white spot lesions, which are an indication of early caries activity, usually occur after orthodontic treatment in varying degrees. in the first few weeks after the extrication of brackets, an exponential decrease of the white spot lesions was found remineralization, albeit only around half the initial lesions were remineralized after 6 months without any specific therapy. topical fluoride is often used by dentists to help remineralization.1 to prevent the worsening of occuring caries lesions, an oral hygiene improvement and a minimization of early caries lesions are needed before performing any orthodontic treatment. besides, bonding agents with cariostatic properties or those containing fluoride can also be used to deter this problem. fluoride may influence the strength of glass ionomer cement (gic) based orthodontic bracket adhesive, whereas the higher the fluoride content within the gic, the lower its shear strength.2,3 gic bonding agent has the lowest bonding strength and is also prone to debonding.4 however, it is also able to prevent caries better as compared with other bonding agents due to its fluoride-releasing property.5 moreover, having low bonding failure is a priority in fixed orthodontic treatments due to the frequent debonded bracket replacement which is inefficient, time-consuming and costly.6 because of this problem, an alternative remineralization agent is needed, one that does not lower the bonding strength of the orthodontic brackets. it is stated that casein phosphopeptides – amorphous calcium phospate (cpp-acp) paste can help remineralization.7 cpp-acp acts as a remineralization and anti-cariotic agent due to its plaque-preventing property and also plays the role of calcium and phosphate reservoir on dental surface.8 casein phosphopeptides-amorphous calcium phospate fluoride (cpp-acpf), the combination of cpp-acp with fluoride, is proven to have a stronger remineralization effect compared to cpp-acp.9 it was stated that 3% and 5% cpp-acp addition to the gic bonding agent attains a lower fluoride-releasing property than pure gic bonding agent, although there was an increased release of calcium ions and inorganic phosphates.10 demineralization lesions found on the enamel around the gic bonding agent are also fewer than those found in the gic bonding control group. however, the effect of using cpp-acpf paste before orthodontic treatment towards gic orthodontic bracket bonding agent is still unknown. one of the methods utilized to determine the bonding strength of orthodontic bracket bonding agents is performing shear strength tests. the shear strength value is the maximum force attained as the bracket shifted parallelly on dental surface.2 a low shear strength can mean that the bonding strength is also low. based on the idea, this study was done to measure the effect of cpp-acpf pretreatment to gic orthodontic bonding agent shear strength. this study was expected to widen the knowledge of dentists and dental students about the effect of remineralization prior to orthodontic treatment. figure 2. shear strength test aid appliance (a and b). a b figure 1. bracket attachment. ��budipramana, et al.,: shear strength of orthodontic bracket bonding materials and methods fifty samples of post extracted human maxilla and mandibular premolars with normal enamel. samples were obtained from the department of oral and maxillofacial surgery in faculty of dentistry, universitas airlangga. the teeth samples were divided randomly into 2 groups: the treatment group which received cpp-acpf paste (tooth mousse plus, gc) application, and the control group which did not receive cpp-acpf paste application. artificial saliva was made by mixing all following ingredients to attain a homogenic mixture. the ingredients are: na2hpo4 (0,426 grams), nahco3 (1,68 grams) cacl2 (0,147 grams), extraction h2o (800 ml), and hcl-1m (2,5 ml). 11 after extraction the teeth were cleaned in flowing water and stored in a moist condition by wrapping in a piece of cloth drabbled with distilled water, then placed in a closed container and frozen in the temperature of -4 oc until the experiment was done to preserve the condition and humidity the teeth as it would be within the mouth cavity.12 the tooth surface was cleaned using pumice on a low-speed handpiece brush to remove debris, and dried using cotton pellets. the teeth were cut on the crown with the same thickness as plastic tube i using a separating disc attached to a straight handpiece while holding the samples around the root by hand. the teeth were cut on the cervical section to separate the crown and the root. plastic tube i was placed on top a glass plate oiled with vaseline. then the acrylic admixture was filled into the plastic tube layer by layer up to the dough stage before placing the cut tooth inside the tube with its buccal surface facing upwards. mesio-distal and occlusogingival surfaces were arranged to be balanced to the most convex part at the same height as the top of the plastic tube. excessive acrylic was then cleaned using a modelling knife without changing the position of the attached tooth. the cpp-acpf paste was applied on the enamel surface of the tooth for 2 minutes. afterwards the tooth was then irrigated using 10 ml of distillated water twice to ensure that the tooth enamel is clean, then it was dried using sterile cotton pellet and stored in a glass bottle filled with artificial saliva. this treatment was given twice a day (once every 12 hours) for 14 days.13 samples were stored within glass bottles filled with artificial saliva when the samples do not receive any treatment. polyacrylate acid (20%) conditioner (fuji ortho lc, gc) was applied using cotton pellets on cleaned tooth surface. application was done on the area of the bracket bonding (20 mm2). conditioner was applied for 20 seconds, then flushed using water spray for 30 seconds. gic powder and liquid (fuji ortho lc, gc) were mixed according to manufacturer direction, which is 3:1 in weight (1 spoon of powder to 2 drops of the liquid). the powder was separated into 2 equal parts, one part was mixed with the whole liquid for 10 seconds, and the second part was mixed into the admixture in the following 10-15 seconds. gic adhesive was applied on bracket (ortho organizer) using an applicator stick, pressed on the bracket to allow mechanical tension on the back of the bracket. bracket was then attached onto the tooth to obtain the thinnest layer of adhesive as possible. excessive adhesive agent was removed using a probe. the bracket was then cured using a light curing unit from four directions, each for 10 seconds. all specimens were stored in normal saline for 3 days before performing the attachment into plastic tube ii using acrylic (vertex). area around the bracket was given blue wax (cavex) at the height of tube i to eliminate undercut areas before the application of acrylic in plastic tube ii. then, teeth attached in acrylic were removed from plastic tube i. the remaining height of plastic tube ii was filled with acrylic by inserting a layer of powder (3 mm), and then dropped with monomer. this was done repeatedly until the tube was full and then flattened using a glass plate. the excessive acrylic was removed using a modelling knife. after the acrylic hardens, the tooth and bracket attached in the acrylic were removed from plastic tube ii and soaked in distillated water for 24 hours before further testing was performed. the cast, tooth along with the bonding agent and bracket in a linear arrangement was then placed within a bronze cylinder opposite to the two halves of the bronze cylinder. to secure the movement that will occur, the two bronze halves were inserted into larger tube and fastened with a screw. the bronze aid appliance was placed on the autograph shimatzu from japan with its lower hook clasped on the autograph’s holder and the upper hook is attached to the autograph’s hook which will later move upwards. the speed of the cross head was set on 0.5 mm/minute on the autograph’s control panel and the load recorded on the monitor from the release of the bracket was figure 3. the diagram of shear strength test aid appliance. yellow and green area indicates the smaller two halves of bronze cylinder; each blue area indicates acrilic from plastic tube i and ii. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 39–44 then recorded. afterwards, obtained data were analysed using an independent t-test. results the shear strength (mpa) was obtained by dividing shear force value (kn) read on the shimatzu autograph device by the bracket surface area attached to tooth enamel, which was 12 mm2. the shear strength of the treatment group which received an application of cpp-acpf paste was greater than that of the control group. kolmogorov smirnov normality testing showed that data from both groups were normally distributed (p>0.05). homogenity testing using levene’s test showed that data from both groups were homogen (p>0.05). on a difference testing using independent t-test, it was found that there was a significant difference between shear strength of the treatment group and the control group (p < 0.05). discussion initial caries or white spot lesion is formed by the combination of carbohydrate diet and bacterial infection mediated by saliva. it is caused by the imbalance between enamel demineralization and remineralization. white spot lesion is a precursor to enamel caries. white discoloration of the early caries lesion is caused by an optical phenomenon due to the loss of minerals on the enamel surface or subsurface. the solubility of the enamel crystal started from demineralization at the enamel subsurface creating pores between enamel rods. this will cause alterations on enamel refraction index and coarseness.14 white spot lesion is a problem during orthodontic treatments, especially in fixed appliances treatments. the usage of fixed appliances will increase plaque retention. furthermore, in a bad oral hygiene condition, plaque and acidogenic bacteria accumulation will cause demineralization. a study stated that 38% of patients have white spot lesion within 6 months after the attachment of fixed appliances and 50% of patients has white spot lesion by the end of their orthodontic treatment.15 minimizing white spot lesions before fixed orthodontic treatment has become an important issue to clinicians. white spot lesion may exist on patients who have or have never received prior orthodontic treatment, or those who have received the first phase of orthodontic treatment before using fixed appliances, for instance: functional appliances, rapid maxillary expansion, or other treatments. clinicians can help patients to overcome white spot lesions using some pretreatment procedures on the enamel.16 besides, mineral content on enamel may also affect the attachment of orthodontic brackets. on demineralized enamel, for instance because of the presence of caries lesions or post bleaching, the bonding strength of the orthodontic bracket tends to be lower compared to the bracket bonding on normal enamel.17,18 therefore, a procedure is needed to remineralize the enamel to improve the bonding strength of the bonding agent. several mechanisms that can be applied to minimize the enamel demineralization process during orthodontic treatment are by using topical remineralization agent, maintaining oral hygiene and controlling diet. the use of cpp-acp as a remineralization agent showed a synergistic effect with topical fluoride, which has been widely known as a remineralization agent. topical anticariotic effect of milk is the initial idea of the production of cpp which has the ability to stabilize calcium and phosphate in amorphous form. cpp molecule contains residual phosphoseryl group which increases calcium phosphate solubility by stabilizing acp in neutral and alkaline condition. multiple phosphoseryl residue in cpp binds with acp nanoclusters in a supersaturated condition. this will hamper acp molecule growth towards critical size which will allow acp to change phase.14 in this study, the remineralization agent used is cppacpf paste, which is cpp-acp added with 0.2% of fluoride. the anticariotic activity of cpp-acpf has been proven in vitro and in vivo on humans and animals. cppacpf nanocomplexes binds with plaque and tooth surface, then acts as calcium and phosphate reservoir, and cause an increase in the concentration of calcium and phosphate ions in plaques. this will keep the supersaturated condition of acp on enamel mineral, hamper demineralization and increase enamel remineralization. an immunolocation study states that cpp-acpf fuses with supragingival plaque by binding to the bacteria’s cell surface, intercellular matrix components of plaque and adsorbed macromolecules of the tooth surface. this condition will reduce the formation of cariogenic plaque.19 recently, the most commonly used orthodontic bonding agent is the composite resin. composite resin possesses the highest bonding strength of other types of bonding agents.20 however, resin-based bonding agents have many disadvantages, such as the occurence of more demineralization lesions in areas adjacent to the bracket.21 acid etch is also required in the bonding procedure. the usage of phosphoric acid as acid etch can increase decalcification due to the loss of enamel minerals. this loss can also occur during debonding procedure. in addition, resin tags which penetrated the enamel surface during the bonding procedure will stay within the enamel even after debonding, which in turn causes the increase of staining on table 1. descriptive analytic mean and standard deviation shear strength (mpa) and shear force (kn) results on each group group shear force shear strength mean standard deviation mean standard deviation treatment 0.23 0.05 19.22 4.04 control 0.16 0.05 12.97 3.97 ��budipramana, et al.,: shear strength of orthodontic bracket bonding tooth surface after orthodontic treatment is completed.22 another alternative to resin-based bonding agent is matrix-based bonding agent, which is commonly known as gic bonding agent. gic has a number of advantages compared to resin-based ones. gic does not need absolute moisture control, which is hard to achieve because despite of isolation, the teeth in oral cavity will still be moistened by saliva.23 gic also possesses the ability to prevent enamel demineralization around the bracket because it contains fluoride that can be released to adjacent enamels in a relatively long period of time, other than capable to absorb fluoride from other sources, such as fluoride from toothpaste or mouthwash. in other words, this agent serves as a slowrelease and rechargeable fluoride media. it will cause a decrease in the incidence of decalcification and formation of early caries lesion adjacent to the orthodontic bracket. besides, gic does not require acid etch which damages enamel surface and increase vulnerability to caries.24 in spite of its advantages, gic orthodontic bonding agent still has a limited use due to its lower bonding strength than other bonding agents like composite resin.25 the bonding strength can be known by measuring the shear strength between gic-bonded orthodontic bracket and the enamel surface it is attached to. based on past studies, it has been known that the average shear strength of gic bonding agent after cpp-acpf paste treatment (19.22 mpa) was greater than that of control group which did not receive cpp-acpf paste treatment (12.97 mpa). this number far exceeds the required bonding strength for orthodontic treatment, which is 6-8 mpa.26,27 this is probably caused by an increase in the level of calcium ion on the enamel due to the cpp-acpf paste application. gic bonding agent adheres to the enamel through chemical bonding with the calcium ions. in this case, gic is capable to bond with more ion calcium on the enamel, resulting in a greater shear strength. this study was done with in vitro approach so that it cannot prove the remineralization influence of cpp-acpf paste towards the bonding strength of gic bonding agent in the oral cavity. there are a few limitations of an in vitro study which is not similar to the biological conditions of the oral cavity such as: the absence of caries bacteria which can cause demineralization and create biofilm or plaque on tooth surface.28 this plaque may inhibit cpp-acpf activity to caries bacteria or biofilm. to create an experimental condition as close to the condition within the oral cavity, freshly extracted teeth were used in this study. the teeth were stored and frozen right after extraction to avoid dehydration and loss of teeth minerals. the storage is intended so that all teeth have the same moisture during the experiment because the samples were not obtained at the same time. this moisture needs to be maintained because normal enamel in the oral cavity is comprised of 96 percent inorganic material, 1 percent organic material and 3 percent water29 and the loss of water molecules may cause enamel alteration which will influence the remineralization. artificial saliva was used in this study, however, it may never completely replace the function of saliva in the oral cavity. the composition of saliva in the oral cavity is different with those of the artificial saliva, thus the bonding of calcium and other molecules contained in the saliva to the tooth surface can not happen.28 the composition and flow of saliva in the oral cavity is also influenced by the diet and activity of the person, causing it to be different in each people.30 the volume of the artificial saliva also can not be the same to that of the saliva in the oral cavity. tooth surface in different position in the oral cavity, for instance on the maxilla or mandible, will be moistured by a different volume and source of saliva. an increase in remineralization agent concentration given also may not be equalized as the volume increase done in vivo, causing the results of this study to less represent the effect of remineralization in vivo.28 as a conclusion, the use of cpp-acpf paste pretreatment could significantly increase the shear strength of orthodontic bracket bonding using gic bonding agent. references 1. willmot d. white spot lesion after orthodontic treatment. seminars in orthodontics, 2008; 14(3): 209–19. 2. powers jm, messersmith ml. enamel etching and bond strength. in: brantley wa, eliades t, editors. orthodontic materials: scientific and clinical aspects. stuttgard: thieme; 2001. p. 111–3. 3. bishara se, soliman m, laffoon jf, warren j. shear bond strength of a new high fluorida release glass ionomer adhesive. angle orthod 2008; 78(1): 125–8. 4. valletta r, prisco d, de santis r, ambrosio l, martina r. evaluation of the debonding strength of orthodontic brackets using three different bonding systems. eur j orthod 2007; 29: 571–7. 5. watts dc. orthodontic adhesive resins and composites: principles of adhesion. in: brantley wa, eliades t, editors. orthodontic materials: scientific and clinical aspects. stuttgard: thieme; 2001. p. 190–200. 6. zachrisson bu, buyukyilmaz t. bonding in orthodontics. in: graber lw, vanarsdall rl, vig kwl, editors. orthodontics: current principles and techniques. 5th ed. philadelphia: elsevier; 2012. p. 727–37. 7. oshiro m, yamaguchi k, takamizawa t, inage h, watanabe t, irokawa a, ando s, miyazaki m. effect of cpp-acp paste on tooth mineralization: an fe-sem study. j oral sci 2007; 49(2): 115–20. 8. moule ca, angelis f, kim gh, malipaoil s, foo ms, burrow mf, thomas d. resin bonding using an all-etch or self-etch adhesive to enamel after carbamide peroxide and/or cpp-acp treatment. australian dent j 2007; 52: 2. 9. jayarajan j, janardhanam p, jayakumar p, deepika. efficacy of cpp-acp and cpp-acpf on enamel remineralization–an in vitro study using scanning electron microscope and diagnodent. indian j dent res 2011; 22(1): 77–82. 10. al zraikat h, palamara jea, messer hh, burrow mf, reynolds ec. the incorporation of casein phosphoptide-amorphous calcium phosphate into a glass ionomer cement. dent mater 2010; 27(3): 235–43. 11. jarpa p. medición del ph de 12 preparaciones distintas de pasta de tabaco de mascar, relacionándolas con la adición a la nicotina. revista de la facultad de farmacia. 2003; 45(2): 7–11. 12. b u d i p r a m a n a e s . p e n g a r u h a m i n e f l u o r i d a , s o d i u m monof luorophosphate, da n sodium f luor ida sebaga i ba ha n remineralisasi pada permukaan enamel gigi dengan karies buatan. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 39–44 thesis. surabaya: universitas airlangga; 1987. 13. rachmawati d. permeabilitas enamel gigi permanen muda setelah aplikasi pasta casein phosphopeptide amorphous calcium phosphate (cpp-acp). thesis. surabaya: universitas airlanggga; 2009. 14. sudjalim tr, woods mg, manton dj. prevention of white spot lesions in orthodontic practice: a contemporary review. australian dent j 2006; 51(4): 284–9. 15. tufekci e, dixon js, gunsolley jc, lindauer sj. prevalence of white spot lesions during orthodontic treatment with fixed appliances. angle orthod 2011; 81(2): 206–10. 16. baysa l a, uysa l t. do ena mel m ic roabra sion a nd ca sei n phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface?. angle orthod 2012; 82(1): 36–41. 17. gonzalez-lopes s, de madeiros clsg, defren ca, bolariosc a r m o n a m v, sa n ch ezsa n ch e s p, me n e n d ez-nu n ez m. demineralization effects of hydrogen peroxide on bovine enamel and relation of shear bond strength of brackets. j adhes dent 2009; 11(3): 1–7. 18. attin r, stawarczuk b, kecik d, knosel m, wiechmann d, attin t. shear bond strength of brackets to demineralize enamel after different pretreatment methods. angle orthod 2012; 82(1): 56–61. 19. ekizer a, zorba yo, uysal t, ayrikcil s. effects of demineralizationinhibiton procedures on the bond strength of brackets bonded to demineralized enamel surface. korean j orthod 2012; 42(1): 17–22. 20. mandall na, millett dt, mattic cr, hickman j, worthington hv, macfarlane tv. orthodontic adhesives: a systematic review. j orthod 2002; 29(3): 205–10. 21. benson pe, shah aa, millett dt, dyer f, parkin n, vine rs. fluoridas, orthodontics and demineralization: a systematic review. j orthod 2005; 32(2): 102–44. 22. komori a, ishikawa h. evaluation of a resin-reinforced glass ionomer cement for use as an orthodontic bonding agent. angle orthod 1997; 67(3): 189–96. 23. ali h, maroli s. glass ionomer cement as an orthodontic bonding agent. j contemp dent pract 2012; 13(3): 650–4. 24. godoy-bezerra j, vieira s, oliveira jhg, lara f. bond strength of resin-modified glass ionomer cement with saliva present and different enamel pretreatments. angle orthod 2006; 76(3): 470–4. 25. cook pa, luther f, youngson cc. an in vitro study of the bond strength of light-cured glass ionomer cement in the bonding of orthodontic brackets. european j orthod 1996; 18: 199–204. 26. reynolds ir. a review of direct orthodontic bonding. br j orthod 1975; 2: 171–8. 27. whitlock bo iii, elck jd, ackerman rj jr, glaros ag, chappell rp. shear strength of ceramic brackets bonded to porcelain. am j orthod dentofacial orthop 1994; 106(4): 358–64. 28. white dj. the application of in vitro models to research on demineralization and remineralization of the teeth. adv dent res 1995; 9(3): 175–97. 29. bath-balogh m, fehrenbach mj. illustrated dental embryology, histology, and anatomy. st. louis: elsevier saunders; 2006; p. 180. 30. almeida pdv, gregio amt, machado man, lima aas, azevedo lr. saliva composition and functions: a comprehensive review. j contemp dent pract 2008; 9(3): 1–11. vol 38 no 3 2005 130 pencegahan primer pada anak yang berisiko karies tinggi (primary prevention in children with high caries risk) ami angela departemen pedodonsia fakultas kedokteran gigi universitas sumatera utara medan indonesia abstract oral and dental health has got improvement tremendously over the last century but the prevalence of dental caries in children has remained a significant clinical problem. caries risk varies between individuals according to each subject’s balance between factors exposing to and protecting from caries attack. it is important to assess the risk of caries for all patients on a routine basis. caries risk is divided into three levels: high, moderate, and low. thereby, the dental professional is better to make a specific prevention and treatment recommendations to reduce a child risk and improve overall oral health. the goal of caries-risk assessment and preventive treatment is to eliminate caries or at least to reduce the high-caries occurrence down to the level of the remaining part of the age group. preventive treatment is divided into three parts: primary, secondary, and tertiary prevention. this paper describes the primary prevention in children with high caries risk by behavior modification and tooth protection. behavior modification is about dental health education, oral hygiene, diet and sugar consumption, sugar-free chewing gum and sugar-free medicines. tooth protection is about sealant, usage of flouride and chlorhexidine. key words: primary preventive, caries-risk assessment, high caries risk korespondensi (correspondence): ami angela, departemen pedodonsia, fakultas kedokteran gigi universitas sumatera utara. jln. alumni no. 2 kampus usu medan 20155, indonesia. email: ami harahap@yahoo.com pendahuluan kesehatan mulut dan gigi telah mengalami peningkatan pada abad terakhir tetapi prevalensi terjadinya karies gigi pada anak tetap merupakan masalah klinik yang signifikan. suwelo1 melaporkan prevalensi karies anak prasekolah di dki jakarta 89,16% dengan def-t rata-rata 7,02 ± 5,25 dan hasil survei di 10 provinsi (1984–1988) pada daerah kota, prevalensi karies anak umur 8 tahun 45,20% dengan dmf-t 0,94 serta menurut skrt 1995, indeks dmf-t anak umur 12 tahun menunjukkan rata-rata 2,21 dengan angka prevalensi sebesar 76,9%.2 hal ini menunjukkan suatu keadaan kerusakan gigi yang hampir tanpa penanganan. agar target pencapaian gigi sehat tahun 2010 menurut who bahwa angka dmf-t anak umur 12 tahun sebesar 1 dan didominasi oleh indikator f-t dapat tercapai maka diperlukan suatu tindakan pencegahan.3 seluruh tindakan pencegahan baik pencegahan primer, sekunder ataupun tersier harus berdasarkan pada pemeriksaan klinik dan radiografi, penilaian risiko karies, hasil perawatan terdahulu, kemajuan dari riwayat karies terdahulu, pilihan dan harapan orang tua dan dokter gigi akan perawatan serta penilaian kembali pada saat kunjungan berkala.4 penilaian tingkat risiko karies anak secara individu harus diketahui oleh dokter gigi karena semua anak pada umumnya mempunyai risiko terkena karies dan perawatannya juga berbeda pada setiap tingkatan. tingkat risiko karies anak terbagi atas tiga kategori yaitu risiko karies tinggi, sedang dan rendah. pembagian risiko karies ini berdasarkan pengalaman karies terdahulu, penemuan di klinik, kebiasaan diet, riwayat sosial, penggunaan fluor, kontrol plak, saliva dan riwayat kesehatan umum anak.4,5 anak yang berisiko karies tinggi harus mendapatkan perhatian khusus karena perawatan intensif dan ekstra harus segera dilakukan untuk menghilangkan karies atau setidaknya mengurangi risiko karies tinggi menjadi rendah pada tingkatan karies yang dapat diterima pada kelompok umur tertentu sehingga target pencapaian gigi sehat tahun 2010 menurut who dapat tercapai. oleh sebab itu makalah ini akan membahas mengenai pencegahan primer pada anak yang berisiko karies tinggi. karies karies gigi adalah penyakit infeksi dan merupakan suatu proses demineralisasi yang progresif pada jaringan keras permukaan mahkota dan akar gigi yang dapat dicegah. risiko karies adalah kemungkinan berkembangnya karies pada individu atau terjadinya perubahan status kesehatan yang mendukung terjadinya karies pada suatu periode tertentu. risiko karies bervariasi pada setiap individu tergantung pada keseimbangan faktor pencetus dan penghambat terjadinya karies.6 risiko karies dibagi menjadi tiga tingkatan yaitu risiko karies tinggi, sedang dan rendah. agar dapat mengidentifikasi risiko karies anak digunakan suatu penilaian risiko karies.4,6 131angela: pencegahan primer pada anak yang berisiko karies tinggi penilaian risiko karies ini merupakan suatu metode evaluasi klinik di mana dokter gigi nantinya dapat menyesuaikan tindakan pencegahan dan perawatan pada setiap anak. penilaian risiko karies ini harus dilakukan pada setiap anak sebagai suatu pemeriksaan dasar rutin.6 menurut american academy of pediatric dentistry, penilaian risiko karies pada anak berdasarkan atas tiga bagian besar indikator karies yaitu: kondisi klinik, karakteristik lingkungan, dan kondisi kesehatan umum (tabel 1).7 kondisi klinis yang merupakan indikator risiko karies tinggi pengalaman karies pengalaman karies sebelumnya merupakan suatu indikator yang kuat untuk menentukan terjadinya karies di masa yang akan datang.4-6 li and wang8 mengatakan bahwa anak yang mempunyai karies pada gigi sulung mempunyai kecenderungan tiga kali lebih besar untuk terjadinya karies pada gigi permanen. penemuan klinik yang dapat dilihat pada anak yang berisiko karies tinggi adalah terjadi karies yang baru pada setiap kunjungan berkala, ekstraksi yang prematur, terdapat lebih dari satu area demineralisasi enamel (white spot), adanya enamel hipoplasia, tingginya proporsi streptococcus mutans, penggunaan alat kedokteran gigi seperti alat ortodonti ataupun gigi palsu.5,7 alat yang dapat digunakan untuk mengetahui aktivitas karies adalah cariostat, dengan perubahan ph terlihat perubahan warna media sehingga diketahui urutan aktivitas karies, dari aktivitas karies tidak aktif sampai yang aktif berat.1 karies dini dapat dideteksi dengan menggunakan quantitative light fluorescence (qlf), infrared laser fluorescence (diagnodent) untuk permukaan oklusal dan permukaan halus dan digital imaging fiber optic trasillumination (difoti) untuk daerah approksimal.9 kontrol plak plak yang menempel erat di permukaan gigi dapat dipakai sebagai indikator kebersihan mulut. indikator kebersihan mulut pada anak yang lebih sederhana dapat digunakan oral hygiene index simplified (ohis) dari green dan vermillon. skor indeks ohis adalah skor 0,0–1,2 dikatakan kebersihan mulut baik, skor 1,3–3,0 kebersihan mulut sedang dan 3,1–6,0 kebersihan mulut buruk.10 anak yang berisiko karies tinggi mempunyai oral hygiene yang buruk ditandai dengan adanya plak pada gigi anterior disebabkan jarang melakukan kontrol plak.5,11,12 indikator risiko karies risiko rendah risiko sedang risiko tinggi kondisi-klinis − tidak ada gigi yang karies selama 24 bulan terakhir − ada karies selama 24 bulan terakhir − ada karies selama 12 bulan terakhir − tidak ada demineralisasi enamel (karies enamel white spot lesion) − terdapat satu area demineralisasi enamel (karies enamel white spot lesion) − terdapat satu area demineralisasi enamel (karies enamel white spot lesion) − tidak dijumpai plak, tidak ada gingivitis − gingivitis − secara radiografi dijumpai karies enamel − dijumpai plak pada gigi anterior − banyak jumlah s. mutans − menggunakan alat ortodonti karakteristik lingkungan − keadaan optimal dari penggunaan fluor secara sistemik dan topikal − keadaan yang suboptimal pengguna fluor secara sistemik dan optimal pada penggunaan topikal aplikasi − penggunaan topikal fluor yang suboptimal − mengkonsumsi sedikit gula atau makanan yang berkaitan erat dengan permulaan karies terutama pada saat makan − sekali-sekali (satu atau dua) di antara waktu makan terkena gula simpel atau makanan yang sangat berkaitan terjadinya karies − sering memakan gula atau makanan yang sangat berhubungan dengan karies di antara waktu makan − status sosial ekonomi yang tinggi − status sosial ekonomi menengah − status sosial ekonomi yang rendah − kunjungan berkala ke dokter gigi secara teratur − kunjungan berkala ke dokter gigi tidak teratur − karies aktif pada ibu − jarang ke dokter gigi keadaan kesehatan umum − anak-anak dengan membutuhkan pelayanan kesehatan khusus − kondisi yang mempengaruhi aliran saliva tabel 1. penilaian risiko karies menurut american academy of pediatrics dentistry7,9 guidelines on the use of pit and fissures sealants in paediatric dentistry: an eapd 132 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 130–134 saliva fungsi saliva adalah sebagai pelicin, pelindung, buffer, pembersih, anti pelarut dan antibakteri. faktor yang ada dalam saliva yang berhubungan dengan karies antara lain adalah aksi penyangga dari saliva, komposisi kimiawi, aliran (flow), viskositas dan faktor anti bakteri.1 anak yang berisiko karies tinggi memiliki aliran saliva yang rendah dimana tingkat unstimulated salivary flow (usf) < 0,1 ml per menit dan stimulated salivary flow (ssf) < 0,5 ml per menit. hal ini bisa disebabkan oleh penyakit sistemik maupun terapi sinar, kapasitas buffer yang rendah ditandai dengan test buffer yang menggunakan dentofuff strip didapat ph ≤ 4 dan tingginya s. mutans diukur dengan menggunakan teknik strip mutans (dentocult-sm) didapat koloni unit s. mutans > 1 × 106 per ml saliva dan lactobacilus diukur dengan menggunakan dentocult-lb pada saliva.13 karekteristik lingkungan yang merupakan indikator risiko karies tinggi penggunaan fluor tujuan penggunaan fluor adalah untuk melindungi gigi dari karies. fluor bekerja dengan cara menghambat metabolisma bakteri plak yang dapat memfermentasi karbohidrat melalui perubahan hidroksil apatit pada enamel menjadi fluor apatit. reaksi kimia: ca10(po4)6.(oh)2 + f ca10(po4)6.(ohf) menghasilkan enamel yang lebih tahan terhadap asam sehingga dapat menghambat proses demineralisasi dan meningkatkan remineralisasi yang merangsang perbaikan dan penghentian lesi karies.14 pada anak yang berisiko karies tinggi dilaporkan bahwa penggunaan fluor ini hampir tidak ada. riwayat sosial banyak penelitian menunjukkan bahwa prevalensi karies lebih tinggi pada anak yang berasal dari status sosial ekonomi rendah. hal ini dikarenakan anak dari status ini makan lebih banyak makanan yang bersifat kariogenik, rendahnya pengetahuan akan kesehatan gigi dapat dilihat dari kesehatan mulut yang buruk, karies tinggi pada keluarga (karies aktif pada ibu), jarang melakukan kunjungan ke dokter gigi sehingga banyak karies gigi yang tidak dirawat. 5,6,11 kebiasaan makan penelitian vipeholm (1945–1953) menyimpulkan bahwa konsumsi makanan dan minuman yang mengandung gula di antara jam makan dan pada saat makan berhubungan dengan peningkatan karies yang besar.5 faktor makanan yang dihubungkan dengan terjadinya karies adalah jumlah fermentasi, konsentrasi dan bentuk fisik (bentuk cair, tepung, padat) dari karbohidrat yang dikonsumsi, retensi di mulut, frekuensi makan dan snacks serta lamanya interval waktu makan.1,5 anak yang berisiko karies tinggi sering mengkonsumsi makanan minuman manis di antara jam makan.4,5,11 kondisi kesehatan umum yang merupakan indikator risiko karies tinggi kondisi kesehatan pada anak sangat berpengaruh pada risiko karies. anak dengan ketidakmampuan mental atau cacat fisik terutama cacat tangan memerlukan perhatian khusus secara terus menerus disebabkan anak ini mempunyai keterbatasan untuk melaksanakan prosedur membersihkan mulutnya dan membutuhkan bantuan dari orang lain. ketergantungan anak pada orang lain meningkatkan faktor predisposisi terjadi karies tinggi.3 demikian juga pada anak yang mempunyai penyakit sistemik yang tidak terkontrol dapat mengakibatkan perubahan pada rongga mulut dan kondisi saliva baik dari segi komposisi maupun aliran saliva. hal ini akan mengakibatkan tingkat karies anak menjadi lebih tinggi. tindakan pencegahan primer tindakan pencegahan primer adalah suatu bentuk prosedur pencegahan yang dilakukan sebelum gejala klinik dari suatu penyakit timbul dengan kata lain pencegahan sebelum terjadinya penyakit. tindakan pencegahan primer ini meliputi: modifikasi kebiasaan anak modifikasi kebiasaan anak bertujuan untuk merubah kebiasaan anak yang salah mengenai kesehatan gigi dan mulutnya sehingga dapat mendukung prosedur pemeliharaan dan pencegahan karies. pendidikan kesehatan gigi pendidikan kesehatan gigi mengenai kebersihan mulut, diet dan konsumsi gula dan kunjungan berkala ke dokter gigi lebih ditekankan pada anak yang berisiko karies tinggi. pemberian informasi ini sebaiknya bersifat individual dan dilakukan secara terus menerus kepada ibu dan anak. dalam pemberian informasi, latar belakang ibu baik tingkat ekonomi, sosial, budaya dan tingkat pendidikannya harus disesuaikan sedangkan pada anak yang menjadi pertimbangan adalah umur dan daya intelegensi serta kemampuan fisik anak. informasi ini harus menimbulkan motivasi dan tanggung jawab anak untuk memelihara kesehatan mulutnya.4–6 pendidikan kesehatan gigi ibu dan anak dapat dilakukan melalui puskesmas, rumah sakit maupun di praktek dokter gigi. kebersihan mulut penyikatan gigi, flossing dan profesional propilaksis disadari sebagai komponen dasar dalam menjaga kebersihan mulut. keterampilan penyikatan gigi harus diajarkan dan ditekankan pada anak di segala umur. anak di bawah umur 5 tahun tidak dapat menjaga kebersihan mulutnya secara benar dan efektif maka orang tua harus melakukan penyikatan gigi anak setidaknya sampai anak berumur 6 tahun kemudian mengawasi prosedur ini secara terus menerus.1,13 penyikatan gigi anak mulai dilakukan sejak erupsi gigi pertama anak dan tatacara penyikatan gigi harus ditetapkan ketika molar susu telah erupsi.15 133angela: pencegahan primer pada anak yang berisiko karies tinggi metode penyikatan gigi pada anak lebih ditekankan agar mampu membersihkan keseluruhan giginya bagaimanapun caranya namun dengan bertambahnya usia diharapkan metode bass dapat dilakukan. pemakaian sikat gigi elektrik lebih ditekankan pada anak yang mempunyai masalah khusus. pasta gigi yang mengandung 1000–2800 ppm menunjukkan hasil yang baik dalam pencegahan karies tinggi pada anak di antara umur 6–16 tahun.5 anak sebaiknya tiga kali sehari menyikat gigi segera sesudah makan dan sebelum tidur malam. telah terbukti bahwa asam plak gigi akan turun dari ph normal sampai mencapai ph 5 dalam waktu 3–5 menit sesudah makan makanan yang mengandung karbohidrat dan rider cit. suwelo1 mengatakan bahwa ph saliva sudah menjadi normal (6–7) 25 menit setelah makan atau minum. menyikat gigi dapat mempercepat proses kenaikan ph 5 menjadi normal (6–7) sehingga dapat mencegah proses pembentukan karies. pemakaian benang gigi dianjurkan pada anak yang berumur 12 tahun ke atas di mana selain penyakit periodontal meningkat pada umur ini, flossing juga sulit dilakukan dan memerlukan latihan yang lama sebelum benar-benar menguasainya. profesional profilaksis (skeling, apklikasi flour) dilakukan oleh dokter gigi atau tenaga kesehatan anak. pada anak cacat dan keterbelakangan mental, hal ini harus lebih ditekankan.5 diet dan konsumsi gula tindakan pencegahan pada karies tinggi lebih menekankan pada pengurangan konsumsi dan pengendalian frekuensi asupan gula yang tinggi. hal ini dapat dilaksanakan dengan cara nasehat diet dan bahan pengganti gula.4,5 nasehat diet yang dianjurkan adalah memakan makanan yang cukup jumlah protein dan fosfat yang dapat menambah sifat basa dari saliva, memperbanyak makan sayuran dan buah-buahan yang berserat dan berair yang akan bersifat membersihkan dan merangsang sekresi saliva, menghindari makanan yang manis dan lengket serta membatasi jumlah makan menjadi tiga kali sehari serta menekan keinginan untuk makan di antara jam makan.1,5 xylitol dan sorbitol merupakan bahan pengganti gula yang sering digunakan, berasal dari bahan alami serta mempunyai kalori yang sama dengan glukosa dan sukrosa. xylitol dan sorbitol dapat dijumpai dalam bentuk tablet, pastiles, permen karet, minuman ringan, farmasi dan lainlain. xylitol dan sorbitol mempunyai efek menstimulasi daya alir saliva dan menurunkan kolonisasi dari s. mutans. menurut penelitian, xylitol lebih efektif karena xylitol tidak dapat dimetabolisme oleh bakteri dalam pembentukan asam dan mempunyai efek anti bakteri.4,5,16 perlindungan terhadap gigi perlindungan terhadap gigi dapat dilakukan dengan cara, yaitu silen dan penggunaan fluor dan khlorheksidin.4–6 silen silen harus ditempatkan secara selektif pada pasien yang berisiko karies tinggi. prioritas tertinggi diberikan pada molar pertama permanen di antara usia 6–8 tahun, molar kedua permanen di antara usia 11–12 tahun, prioritas juga dapat diberikan pada gigi premolar permanen dan molar susu.17 bahan silen yang digunakan dapat berupa resin maupun glass ionomer. silen resin digunakan pada gigi yang telah erupsi sempurna sedangkan silen glass ionomer digunakan pada gigi yang belum erupsi sempurna sehingga silen ini merupakan pilihan yang tepat sebagai silen sementara sebelum digunakannya silen resin. keadaan dan kondisi silen harus terus menerus diperiksa pada setiap kunjugan berkala. bila dijumpai keadaan silen tidak baik lagi silen dapat diaplikasikan kembali.5 penggunaan fluor fluor telah digunakan secara luas untuk mencegah karies. penggunaan fluor dapat dilakukan dengan fluoridasi air minum, pasta gigi dan obat kumur mengandung fluor, pemberian tablet fluor, topikal varnis. fluoridasi air minum merupakan cara yang paling efektif untuk menurunkan masalah karies pada masyarakat secara umum. konsentrasi optimum fluorida yang dianjurkan dalam air minum adalah 0,7–1,2 ppm.18 menurut penelitian murray and rugg-gun cit. linanof4 bahwa fluoridasi air minum dapat menurunkan karies 40–50% pada gigi susu. bila air minum masyarakat tidak mengandung jumlah fluor yang optimal, maka dapat dilakukan pemberian tablet fluor pada anak terutama yang mempunyai risiko karies tinggi.5,6 pemberian tablet fluor disarankan pada anak yang berisiko karies tinggi dengan air minum yang tidak mempunyai konsentrasi fluor yang optimal (2,2 mg naf, yang akan menghasilkan fluor sebesar 1 mg per hari).5 jumlah fluor yang dianjurkan untuk anak di bawah umur 6 bulan–3 tahun adalah 0,25 mg, 3–6 tahun sebanyak 0,5 mg dan untuk anak umur 6 tahun ke atas diberikan dosis 0,5–1 mg.16,19 penyikatan gigi dua kali sehari dengan menggunakan pasta gigi yang mengandung fluor terbukti dapat menurunkan karies. obat kumur yang mengandung fluor dapat menurunkan karies sebanyak 20–50%. seminggu sekali berkumur dengan 0,2% naf dan setiap hari berkumur dengan 0,05% naf dipertimbangkan menjadi ukuran kesehatan masyarakat yang ideal. penggunaan obat kumur disarankan untuk anak yang berisiko karies tinggi atau selama terjadi kenaikan karies. obat kumur ini tidak disarankan untuk anak berumur di bawah 6 tahun.5 pemberian varnis fluor dianjurkan bila penggunaan pasta gigi mengandung fluor, tablet fluor dan obat kumur tidak cukup untuk mencegah atau menghambat perkembangan karies. pemberian varnis fluor diberikan setiap empat atau enam bulan sekali pada anak yang mempunyai risiko karies tinggi. salah satu varnis fluor adalah duraphat (colgate oral care) merupakan larutan 134 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 130–134 alkohol varnis alami yang berisi 50 mg naf/ml (2,5%– kira-kira 25.000 ppm fluor). varnis dilakukan pada anak umur 6 tahun ke atas karena anak di bawah umur 6 tahun belum dapat meludah dengan baik sehingga dikhawatirkan varnis dapat tertelan dan dapat menyebabkan fluorosis enamel.5,19 sediaan fluor lainnya adalah dalam bentuk gel dan larutan seperti larutan 2.2% naf, snf2 , gel apf. klorheksidin klorheksiden merupakan antimikroba yang digunakan sebagai obat kumur, pasta gigi, permen karet, varnis dan dalam bentuk gel. flossing empat kali setahun dengan gel klorheksidin yang dilakukan oleh dokter gigi menunjukkan penurunan karies approximal yang signifikan. demikian juga pada anak berisiko karies tinggi hal ini dapat digunakan untuk melengkapi penggunaan silen di bagian oklusal gigi.5 pembahasan pada umumnya anak mempunyai risiko terkena karies. penilaian risiko karies terbagi atas risiko karies tinggi, sedang dan rendah berdasarkan indikator yang meliputi kondisi klinis, karakteristik lingkungan dan kondisi kesehatan umum. penilaian ini harus dilakukan untuk setiap anak sebagai tindakan dasar rutin untuk menentukan tindakan pencegahan dan perawatan serta menentukan jadwal kunjungan berkala. tindakan pencegahan primer pada anak yang berisiko karies tinggi meliputi modifikasi kebiasaan anak (kebersihan mulut dan diet konsumsi gula) dan perlindungan gigi (penggunaan silen, fluor dan klorheksidin). pada anak di bawah umur 5 tahun, usaha untuk melakukan pencegahan primer diberikan kepada ibu seperti meningkatkan pengetahuan ibu tentang menjaga kebersihan mulut anak, pola makan anak yang baik dan benar serta tindakan perlindungan terhadap gigi anak yang dapat diberikan. hal ini berhubungan karena kemampuan anak terbatas dan anak lebih dekat kepada ibunya. pada anak 6 tahun ke atas, dokter gigi harus lebih menekankan kepada anak mengenai tanggung jawabnya untuk memelihara kesehatan mulut. tindakan pencegahan yang dilakukan harus melihat indikator mana sebagai penyebab utama. bila kontrol plak yang tidak baik sebagai penyebab utama, dokter gigi harus lebih menekankan pada modifikasi anak mengenai kebersihan mulut (menyikat gigi dua kali sehari dengan menggunakan pasta gigi mengandung fluor sedikitnya 1000 ppm), bila karena kebiasaan diet yang salah, maka pengaturan diet lebih ditekankan (pembatasan konsumsi makanan dan minuman yang mengandung gula, menggunakan bahan pengganti gula seperti xylitol atau sorbitol). bila morfologi gigi lebih rentan terhadap karies, seperti pit dan fissure yang dalam, enamel hipoplasia maka perlindungan terhadap gigi seperti penggunaan silen, fluor dan flossing klorheksidin lebih ditekankan. untuk mengevaluasi tingkat risiko anak dilakukan kunjungan berkala, 3 atau 4 bulan sekali untuk melihat keberhasilan tindakan pencegahan yang dilakukan serta penilaian tingkat risiko karies anak. berdasarkan kajian konsep pencegahan primer pada anak yang berisiko karies tinggi, maka dapat disimpulkan bahwa anak yang berisiko karies tinggi harus mendapatkan perhatian khusus karena perawatan intensif dan ekstra harus dilakukan untuk menghilangkan karies atau setidaknya mengurangi terjadinya karies tinggi menjadi rendah. tindakan pencegahan yang lebih baik dilakukan adalah pencegahan primer dengan cara modifikasi kebiasaan anak dan perlindungan terhadap gigi. daftar pustaka 1. suwelo is. karies gigi sulung dan urutan besar peranan faktor resiko terjadinya karies. tesis. yogyakarta: universitas gajah mada; 1988. h. 6–30. 2. octiara e, roesnawi y. karies gigi, oral higiene dan kebiasaan membersihkan gigi pada anak-anak panti karya pungai di binjai. dentika dental jurnal 2001; 6(1):18–23. 3. kristanti ch, rusiawati y. gigi sehat tahun 2000 dan tinjauan profil kesehatan gigi 1995. jurnal kedokteran gigi universitas indonesia 2002; 9(2):1–5. 4. tinanoff n. caries management in children: decision-making and therapies. compendium 2002; 23(12):9–13. 5. scottish intercollegiate guidelines network. sign guideline. preventing dental caries in children at high caries risk; targeted prevention of dental caries in the permanent teeth of 6–16 years olds presenting for dental care. edinburgh: sign publication 2000; 47:1–32. 6. varsio s. caries-preventive treatment approaches for child and youth at two extremes of dental health in helsinki, finland. academic dissertation. finland: university of helsinki; 1999. p. 1–63. 7. american academy of pediatric dentistry. policy on use of a caries-risk assessment tool (cat) for infants, children, and adolescent. oral health dental policies 2002; 18–20. 8. li y, wang w. predicting caries in permanent teeth from caries in primary teeth: an eight-year cohort study. j dent res 2002; 81(8):561–6. 9. mcdonald re, avery, dean. dentistry for the child and adolescent: dental caries in child and adolescent. 8th ed. united states of america: mosby co; 2004. p. 215–6, 225, 231. 10. suwelo is. petunjuk praktis sistem merawat gigi anak di klinik, diagnosis dan rencana perawatan. cetakan ii. jakarta: egc; 1991. h. 20–1. 11. curnow mmt, pine cm, burnside g, nicholson ja, et al. a randomised controlled trial of the efficacy of supervised toothbrushing in high-caries-risk children. caries research 2002; 36(4): 294–9. 12. vanobbergen j, martens l, lesaffre e, bogaerts k, et al. the value of a baseline caries risk assessment model in the primary dentition for the prediction of caries incidence in the permanent dentition. caries research 2001 nov/dec; 35(6):442–50. 13. koch g, poulsen s. pediatric dentistry; a clinical approach. 1st ed. denmark: blackwell munksgaard; 2003. p. 142–5. 14. featherstone jdb. the science and practice of caries prevention. jada 2000; 131:887–99. 15. andlaw rj, rock wp. perawatan gigi anak. edisi 2. jakarta: widya medika; 1994. h. 31–5. 16. welbury r, raadal m, lygiydakis. guidelines on the use of pit and fissures sealants in paediatric dentistry: an eapd policy document. available from: url:http://www.eapd.gr/guidelines/ guideliness_pitfissures.htm. accessed august 10, 2004. 17. lewis dw, ismail ai. periodic health examination. update: 2. prevention of dental caries. j can med assoc 1995;152:836–46. 18. oulis cj, raadal m, martens l. guidelines on the use of flouride in children: an eapd policy document. ejpd 2000; 1(1):7–12. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 131 vol. 43. no. 3 september 2010 cervical end preparation design on collarless metal ceramic crown to the decrease of bacterial colony edy machmud department of prosthodontics faculty of dentistry, hasanuddin university ujung pandang-indonesia abstract background: cervical end preparation design is an important procedure in fixed partial denture. if the cervical end preparation design is inadequate, dental plaque will easily be formed and this may indicate the beginning of periodontal disease. purpose: this study was aimed to analyze the effect of cervical end preparation design on collarless metal ceramic crown towards the decrease of bacterium colony number. methods: this study was quasi-experimental study applying pre and post test on a control group involving 48 subjects with shoulder, bevel shoulder, and deep chamfer cervical end preparation. the bacterium colonies were examined on the 1st, 7th, and 21st days after the insertion of collarless metal ceramic crown. results: the study showed that bacterium colony increased significantly in deep chamfer and bevel shoulder preparation design between the treatment group and the control group (p<0.05). in shoulder preparation there was not significant different between the treatment group and the control group (p>0.05). conclusion: compared to the bevel shoulder and deep chamfer, shoulder design is the best design for collarless metal ceramic crown restoration. key words: cervical end preparation design, bacterial colonization, collarless metal ceramic crown abstrak latar belakang: desain preparasi tepi servikal merupakan suatu tahap yang sangat menentukan dalam pembuatan gigi tiruan cekat. apabila desain preparasi tepi servikal tidak adekuat dapat menyebabkan pembentukan plak gigi pada daerah tersebut. keadaan ini merupakan tahap awal terjadinya penyakit periodontal. tujuan: untuk menganalisis pengaruh desain preparasi tepi servikal yang dibuat pada mahkota collarless metal ceramic untuk mengurangi jumlah bakterium koloni. metode: penelitian ini adalah penelitian eksperimental semu dengan metode pre and post test dan kelompok kontrol terhadap 48 subyek penelitian. dilakukan preparasi gigi dan pembuatan akhiran preparasi tepi servikal shoulder, bevel shoulder, dan deep chamfer pada subyek penelitian. pemeriksaan koloni bakteri dilakukan pada hari ke-1, 7, dan 21 setelah pemasangan mahkota collarless metal ceramic. hasil: menunjukkan bahwa terjadi, peningkatan jumlah koloni bakteri yang bermakna antara kelompok perlakuan dengan kelompok kontrol (p<0,05) pada desain preparasi deep chamfer dan bevel shoulder. sedangkan pada desain preparasi tepi servikal shoulder tidak ada perbedaan yang bermakna antara kelompok perlakuan dengan kelompok kontrol (p>0,05). kesimpulan: desain shoulder adalah desain tepi preparasi yang terbaik bagi mahkota collarless metal ceramic dibandingkan bevel shoulder dan deep chamfer. kata kunci: desain tepi servikal, koloni bakteri, mahkota collarless metal ceramic correspondence: edy machmud, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas hasanuddin. jl. perintis kemerdekaan km 10 kampus tamalanrea ujung pandang, indonesia. e-mail: edy.machmud@yahoo.co.id. research report introduction collarless metal ceramic restorations were the best esthetic quality of porcelain fused to metal crown. the conventional metal ceramic restoration is a metal collar on the buccal surface, that provides good fitting and gingival contour. however, it may esthetically be unacceptable and probably the patient become disappointed.1 the use of 132 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 131–135 porcelain fused to metal restorations has become unpopular because of the disadvantages on the buccal metal collar. the advantages of porcelain fused to metal restorations is on the esthetic aspect and on the excellent biocompatibility of the glazed porcelain, and also on the fabrication of this type of restoration.2 the disadvantages of collarless metal ceramic restorations are related to the sensitivity technique of the operator that required skilled and meticulous care of the operator with the risk of producing restoration on the poorer marginal adaptation compared to the metal restoration.1,2 bacteria associated with the periodontium exist in an extremely complex arrangement, and often involved sites mix bacteria from normal sites. plaque is having similar microbial compositions derived from pooled dental plaque and infectious plaque potential. supragingival plaque is usually more abundant and more easily removed than subgingival plaque. plaque bacteria are varied and modulated by host factors resulting in disease progression during period of exacerbation. disease activity periods may have caused contribution of pathogen bacteria to make a lesion. some bacteria have been identified as main pathogens bacteria in periodontal disease. many types of dental plaque bacteria deposit on tooth surface above and below gingival margin. the first stage of plaque forming is a form of flimsy coat on the teeth or on the denture that called acquired pellicle. the pellicle is a flimsy coat with 0.1–0.8 micrometer, translucency, soft, uncolored, and irregular shape. shortly after teeth brushing, acquired pellicle does formed again on the surface of the teeth. the pellicle is not only give a protection to the surface of the teeth, but also become a medium for supporting an early attachment of bacterium plaque.3,4 bacterium needs not the form of pellicle, but attaches and build a colony shortly after plaque is formed. the structure of the oral cavity, tongue, cheek, saliva, teeth, and gingival sulcus is an ideal places for bacterium colony. the bacteria are predominantly streptococcus mutants and its variants. bacterium in the saliva attach to the inorganic pellicle of the teeth. some bacteria will attach to the pit and fissure of the teeth and margin of the denture. others attach interactively to the pellicle surface.5 the bacterium attachment support pilli or fimbriae. adhesin is a group of protein molecule on the bacterium surface cell that can be recognize and relate to the pellicle glycoprotein. it will increase the attachment to the pellicle and eventually form a plaque. bacterium also attaches easily with the support of kation such as free calcium ion and saliva that connects negative content on the surface teeth with the bacterium cells.3,6 the plaque accumulation increases due to the saliva acidity and also increase during sleep while saliva flow decreasing. in the supragingival plaque, the anaerob bacterium form the inner part of the plaque, while the aerobic bacterium is found on the surface and facultative bacterium is disseminated in the whole plaque mass.3 final composition of the plaque depends on the bacterium, the environment, and the host factor. such factors as the place in oral cavity, time, saliva composition, systemic disease, oral hygiene, the use of control plaque agent, diet, as well as the patient’s age, gender, and ethnic background influence the pattern and degree of plaque formation.4,6 marginal adaptation is a critical factor for the success of dental restoration and marginal cervical preparation design. if the fitness restoration is inadequate or there is poor marginal adaptation, there will be marginal leakage and encourage the place of plaque and bacterium.7 the restored cervical end area is an important area of the fixed partial denture restoration and very susceptible for the attachment of bacterium plaque. accurate selection of preparation design for fixed partial denture restoration is very helpful for obtaining closed restoration in the cervical area and preventing gingivitis. if the restoration adaptation is not adequate there will be a gap in the cervical restoration.5 this study was aimed to analyze the effect of cervical end preparation design on collarless metal ceramic crown towards the decrease of bacterium colony number. materials and methods this study is a quasi experimental research applying the pre and post test with a control group, conducted at the faculty of dentistry, hasanuddin university, makassar, in 2009. this study compared the influence of three kinds marginal cervical end preparation of porcelain fused to metal crown on the incidence of gingivitis. there were 24 subject students who were willing to become volunteer of the faculty of dentistry, hasanuddin university. the students criteria were: a) subject must had healthy periodontal tissue, free from calculus, gingivitis, and periodontal disease, b) subject had caries on the proximal site of the upper central incisor and need crown treatment, c) subject was not smoking, d) the age of the subject were between 20 to 27 years of old, e) subjects were male or female students. the study involved 24 subjects divided in 3 kinds of marginal cervical end preparation design of deep chamfer, bevel shoulder, and shoulder (figure 1). each design consisted of 8 subjects. the treatment groups were central upper incisor preparation of shoulder, bevel shoulder, and deep chamfer. the control groups were the opposite side of the central upper incisor without caries and or treatment. those subjects were given dental health education and counseling, an explanation of the working procedures and signing informed consent letter. after that, gingival retraction, temporary crown was made, and permanent crown was inserted by using glass ionomer cement. obtaining bacterium colonies was done before treatment and on the 1st, 7th, and 21st days after fitting the collarless metal ceramic crowns. bacterium colonies were spread on the paper point from the marginal cervical edge of the collarless metal ceramic crowns and further drawn on the blood agar medium. it was stored on the instrument with 133machmud: cervical end preparation design the incubation temperature of 37° celcius for 24 hours, and then the number of bacterium colonies was counted. results this study of the marginal cervical end preparation design on collarless metal ceramic crown was aimed to choose which design has the less number of bacterium colony. the alteration number of bacterium colony after collarless metal ceramic crown was inserted at the 1st, 7th, and 21st days and the difference number of bacteria colony between the treatment groups and control groups can be seen on table 1. there was an alteration number of bacterium colony at the 1st, 7th, and 21st days in bevel shoulder and deep chamfer marginal cervical end preparation design, whereas the increasing number in shoulder design was not significant. there was a significant increase in the number of bacterium colony between the treatment groups with control groups in bevel shoulder and deep chamfer marginal cervical end preparation design. meanwhile the shoulder design had no significant increase in the number of bacterium colony. discussion fixed partial denture is one of the popular prosthodontic restoration. the reason of use the fixed partial denture as one of an alternative tooth restoration because it is smaller, simpler, more comfortable, and has more esthetic, moreover, it gives more conficence to the patient compared to removable denture. fixed denture restoration still have problem related to periodontal health. selected of preparation design and material will determine the success of fixed partial denture treatment.6,8 there are some choices of fixed restoration materials porcelain fused to metal (pfm) and all porcelain. the development of porcelain fused to metal restoration is the modification of collarless metal ceramic crown in order to obtain the esthetic aspect and the metal material biocompatible to the oral mucous. porcelain restorations require some thickness on the material to prevent a crack or fracture on the cervical end margin. the recommended marginal cervical end preparation are shoulder, bevel shoulder, and deep chamfer.2,8,9 choosing a wrong marginal cervical end preparation design will cause marginal crack, since the density of the marginal preparation lines with the material edge can not be tightly adaptive. many studies reported that the marginal gaps were suitable location for the growth and development of dental plaque bacteria which had an important role on inflammation of periodontal tissue. the designs on the marginal cervical end preparation such as feather edge, bevel, deep chamfer, shoulder, shoulder with bevel not all are suitable for collarless metal ceramic crown.8,9,10 in this study, the design of marginal cervical end preparation were shoulder, bevel shoulder and deep chamfer. they were selected because collarless metal ceramic crown for the marginal cervical end was made of porcelain which need material thickness due to the fragil nature of porcelain. porcelain material thickness was determined by the marginal cervical end preparation so that the marginal cervical line is suitable with porcelain material. shoulder marginal end preparation design enables operator to determine how much tooth tissue should be removed. this shoulder width is necessary to hold the incisor or occlusal force and an appropriate preparation for collarless metal ceramic crown restoration. on table 1, in shoulder preparation design, the total number of bacteria decreased on the 1st, 7th, and 21st day after collarless metal ceramic crown was inserted. the marginal cervical end preparation was well adapted with the cervical end finish line of collarless metal ceramic figure �. cervical end preparation design: a) deep chamfer, b) bevel shoulder, c) shoulder.3 table �. the number of bacterium colonies before and after insertion of collarless metal ceramic crown (cfu/ml) design days mean sd p deep chamfer 1 161.25 64.68 0.058 control 1 95.00 70.10 bevel shoulder 1 162.50 62.96 0.052 control 1 95.00 70.10 shoulder 1 145.00 59.76 0.054 control 81.25 65.34 deep chamfer 7 182.5 81.55 0.058 control 7 95.00 70.10 bevel shoulder 7 217.50 90.99 0.027 control 7 95.00 70.10 shoulder 7 160.00 71.51 0.059 control 7 81.25 65.34 deep chamfer 21 196.25 87.33 0.047 control 21 95.00 70.10 bevel shoulder 21 228.75 115.07 0.037 control 21 95.00 70.10 shoulder 21 178.75 87.41 0.055 control 21 81.25 65.34 sd: standard deviation, p: probability a b c 134 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 131–135 crown, and prevented plaque to adhere on that location. this could happen because the shoulder preparation design is sharp and firm on the cervical end line and had sufficient thickness. the design was different from the design on bevel shoulder and deep chamfer, the marginal cervical end were thinner. hence, it was not so clear which could cause a leak on the cervical end restoration, it could also be broken easily, and dental plaque has a better place to develop in the mouth.8,10,11 the marginal cervical end on deep chamfer had finish line design less confined tissue removal, and has more adequate cervical adaptation than the marginal cervical end on shoulder. bevel shoulder finish line preparation was made to prevent a poor cervical adaptation, but bevel placement which was made between 0.5–1.0 mm at the cervical area could cause debris entrapment and plaque formation. design and marginal cervical end preparation placement remarkably affected gingival tissue health. therefore, to obtain a good result and durable restoration in the mouth, despite of the esthetic and functional aspect, periodontal health aspect also supported treatment success. healthy periodontal tissue can make the restoration durable in the mouth. the edge proximity greatly affected the success of a fixed denture restoration. in adequate edge proximity may facilitate the accumulation of plaque that can generate dental caries and periodontal disease.6 edge proximity can be detected with scaning electron microscope (sem); however, in this research it was detected from the existence of dental plaque at cervical area restoration. the cervical edge deep chamfer preparation design also had the same thickness as the shoulder preparation design, yet with a slight inclination in the preparation. the inclination of the preparation in the cervical part would cause friction of cervical restoration if occlusal pressure resulted in marginal gap.10,11 dental plaque or bio film is a slack deposit containing a various group of micro-organism on the tooth or denture surface.10 dental plaque is a complex aggregate of microorganism attaching and multiplying on the surface of hard surface and soft tissue oral cavity, containing one or more species micro-organism that can stick with the help of glicocalics.11,12 the plaque formation process on the teeth or denture surface covers three phases: a) the absorption of saliva protein and glycoprotein form a thin layer on the teeth or denture surface known as pellicle (acquired pellicle); b) the colonization of bacteria in the pellicle attached on the denture or tooth enamel is an initial colonization of bacteria (streptococcus oralis, streptococcus mitis, streptococcus sanguis, actinomyces and naisseria); c) the secondary colonization resulting from the interaction between bacteria in the pellicle and other bacteria in the buccal cavity increases the bacterium species and eventually causes matriculation of the plaque on the tooth surface.13 denture attached near by the orifice will have a contact with the saliva, absorb a number of saliva molecules, and form a thin layer called pellicle. pellicle contain protein that can tie micro-organisms in the buccal cavity so that they attach on the denture surface, colonize with other microorganisms and multiply to form dental plaque.8,13 the bevel shoulder and deep chamfer preparation design in this research were the designs that could increase the number of bacterium colonies, since they had inadequate restoration edge adaptation. both design were compared to the shoulder preparation design whose edge proximity was not closely adapted to the periphery of the cervical edge restoration that enabled dental plaque to develop and live in these areas.14 the result of this research indicates that the shoulder preparation design has good edge proximity since this cervical edge shoulder preparation design did not show any significant increase in the number of bacteria colonies (p>0.05) from the 1st up to 21st day after the insertion of collarless metal ceramic crown. the bevel shoulder and deep chamfer preparation designs had unfavorable cervical edge proximity since there was a significant increase in the number bacterium colonies (p<0.05), as the effect of marginal gap, which was an ideal habitat for dental plaque.7,14 it is concluded that shoulder preparation design is the best design for crown porcelain restoration. it has sufficient thickness to prevent the cervical end restoration from being broken. shoulder preparation design has a clear and firm preparation edge line. the solidity of the restoration edge is more reliable compare to bevel shoulder and deep chamfer. acknowledgement i would like to express my deepest gratitude to the post graduate program of hasanuddin university makassar, the health laboratory of the south sulawesi province, prof. drg. dharmautama, ph.d., sp.pros (k), and prof. dr. drg. arifzan razak, m.sc., sp.pros (k) for grant the opportunity and support to conduct this research. references 1. toshio k. the effect of collarless metal ceramic crown on gingival tissue in the dog. j kyusu dent soc 2005; 41(1): 148–54. 2. matsumoto w, antunes rp, orsi ia, fernandes rm. collarless metalcollarless metal ceramic fixed partial denture: a clinical report. braz dent j 2001; 12(3): 215-23. 3. allen dl, jenzano j. periodontitis for the dental hygienist. 4th ed. philadelphia: lea and febiger; 1987. p. 30–7. 4. konradsson k. influence of dental ceramic and a calcium aluminate cement on dental biofilm formation and gingival inflammatory response. dissertation. sweden: umea university; 2007. p. 1–10. 5. nisengard rj, newman mg. oral microbiology and immunology. 2nd ed. philadelphia: wb saunders co; 1994. p. 320–33. 6. soekartono h, rachmadi p. pengaruh kombinasi pengenceran cairan bahan tanam phosphate bonded terhadap sela marginal mahkota tuang logam campur nicr. jurnal penelitian medika eksakta 2002; 3(1): 20–5. 135machmud: cervical end preparation design 7. devaki v. marginal fit of metal ceramic restorations with various finish lines. dissertation. madras: tamilnadu dr. m.g.r. medical university; 2005. p. 1–26. 8. beuer f, aggstaller h, edelhoff, gernet w. effect of preparation design on the fracture resistance of zirconia crown copings. j dent mat 2008; 27(3): 362–7. 9. rosenstiel s, land m, fujimoto j. contemporary fixed prosthodonthics. 4th ed. saint louis, missouri: mosby elsevier; 2006. p. 220. 10. yoshida a. kuramitsu hk. multiple streptococcus mutans genes are involved in biofilm formation. j appl environ microbial 2002; 68(12): 6283–91. 11. xie h, cook gs, costerton jw, bruce g, rose tm, lamon rj. intergeneric communication in dental plaque biofilm. j bacteriology 2000; 182(24): 7067–72. 12. merit j. mutation of luxs affects bio film formation in streptococcus mutans. j infect immun 2003; 71(4): 1972–9. 13. jefferson kk. what drives bacteria to produce a biofilm. j fed europ microbial soc 2004; 236(2): 163–73. 14. comlecoglu m, dindar m, ozcan m, gungor m, gokce b, artunec c. influence of finish line type on the marginal adaptation of zirconia ceramic crowns. j oper dent 2009; 34(5): 586–96.j oper dent 2009; 34(5): 586–96. 170 various polymerization temperature on dimensional accuracy of orthodontic acrylic base plate elly rusdiana department of orthodontic faculty of dentistry airlangga university surabaya indonesia abstract cold curing acrylic is more porous, having more residual monomer and dimensional change. physical property of cold curing acrylic shows to have very small dimensional change resulted from imperfect polymerization process and if dimensional change occurs, it would cause improper use of orthodontic appliance. orthodontic acrylic base plate which is made of cold curing acrylic, in order to achieve good result, it is suggested to use hydro flask or polyclav with water temperature 45°–50°c and polyclav and pressure in 2–3 atm during polymerization process. even though cold curing acrylic could polymerization at room temperature, the purpose to use hydro flask or polyclav is to reduce residual monomer and porosity. the purpose of the present study is to know the difference in dimensional accuracy of orthodontic acrylic at various polymerization temperature. the samples of the study were 18 acrylic base plate sized 65 × 10 × 25 mm classified into 3 control groups with polymerization temperature at 30° c, 40° c and 50° c. measurement was done using caliper in order to know dimensional accuracy of orthodontic acrylic. the measurement result was tabulated and one-way anova test one was done with significant level 95%, the probability result 0,0055 was found which means there was significant difference among each control group. lsd test showed that group i polymerization at 30° c, group ii polymerization at 40° c, and group iii polymerization at 50°c there was significant difference. the conclusion of this study is : dimensional difference of orthodontic acrylic base plate on polymerization at group i at 30° c, group ii at 40° c, and group iii at 50° c water temperature and could achieve better dimensional accuracy comparing with polymerization at 50° c. key words: cold curing acrylic, dimension, polymerization correspondence: elly rusdiana, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction removable orthodontic appliance consists of several components i.e. active, retention, anchoring and plate acrylic. in line with the advancement and development of dentistry technology, at first heat curing acrylic was applied in the making of removable orthodontic appliance, but at present cold curing acrylic is applied. if heat curing acrylic is used, it is true that it would produce solid plate acrylic, harder, nonporous, more stable color, but, the process is more complicated because wax model should be firstly made and the process of acrylic filling would take time.1 the use of cold curing acrylic is more advantageous because this material is more time saving, cheaper, easier working method, the possibility of distortion due to retention or springs could be eliminated as minimally as possible or it could be prevented. this material would also make the process easier if reparation or change should be done in removable orthodontic appliance1. the main disadvantage of cold curing acrylic is that: unstable color, and more porousity.2 acrylic base plate of removable orthodontic appliance is an attachment place of active and passive components. the functions of acrylic base plate are to sustain other components, to continue the strength of active component anchoring denture movement, to prevent undesired denture shift, to protect springs on the palate and also to be widened to make bite plane either anteriorly or posteriorly. acrylic base plate is made too thick, but strong enough so it will not disturb lingual, swallowing function and also well attached on the palate or lingual teeth.3 polymerization is repeated intermolecular reaction between polymer and monomer functionally unlimitedly.2,4 for dentistry material polymerization is usually through chemical addition reaction. polymerization process of cold curing acrylic would run in three stages i.e. initiation, propagation (change spreader) and termination. the free radical contains very reactive free electron and capable to break monomer multiple binding, so, the monomer would become free radical by itself. propagation (change spreader) would occur because the monomer which is activated has reaction with other monomers, the process would continue until it would reach termination stage. so, in this stage, lengthening change has occurred because the monomer which has been activated is interrelated. change of termination (stage of ended change ) appears because of reaction between two change of free radical which is in growing process, therefore, stable molecule is formed.2 imperfect polymerization of cold curing acrylic 171rusdiana: various polymerization temperature on dimensional accuracy shows the presence of higher residual monomer which resulting irritation in oral tissue and more porosity which could decrease transversal strength.4 physical property of cold curing acrylic shows very small dimensional change resulting from imperfect polymerization process. to obtain nonporous acrylic base plate, in order to achieve tooth strength, good aesthetic, hygienic, low residual monomer so it would not irritate oral tissue.5 houston and isaacson3 suggested that polymerization of acrylic base plate done in hydro flask or polyclav containing warm water 45–50° c with the pressure 2–3 atm and the purpose is to reduce residual monomer and porosity. american dental association6 also suggested that to achieve good result by increasing the temperature and giving pressure during polymerization process. because polymerization of cold curing acrylic was quick process, as a result dimensional change would happen.7,8 cold curing acrylic dimensional change would cause removable orthodontic appliance is not properly used. the purpose of this study is to know the difference in dimensional accuracy of orthodontic acrylic plate at various polymerization temperature. material and method the present study was done in experimental laboratory study and the materials were: cold curing acrylic resin, hard gypsum and devices were: model master die with diameter 65 × 10 × 2,5 mm metal cuvet, digital balance, polyclav, water thermometer, caliper, bowl, spatula, vibrator, glass plate. the process of sample making: firstly mould was made using 100 gram hard gypsum mixed 35 ml water (according to factory procedure) was stirred in bowl using spatula, then put in the cuvet and place on the vibrator. model master die, was put in the middle of the cuvet, the gypsum was flatten using glass plate, finally the gypsum was left hardening. after hardening the model was taken, the mould was soaked in the water for ten minutes, when it was almost dry it was polished by separator material, until it was dry. filling of acrylic resin into the mould with comparison polymer 3.5 gr and monomer 1.5 ml (according factory procedure) using layering i.e. monomer which was dropped on the mould, then, polymer was spread and monomer dropped again until polymer was absorb and the desired thickness was achieved. cuvet with acrylic resin was put into polyclav at 30° c, 40° c, 50° c, water temperature in 2 atmosphere for 10 minutes and next, sample was removed from the cuvet. the test of dimensional accuracy of acrylic base plate was done by measuring and using caliper on mould before acrylic filling, the different measurement was done on acrylic base plate which was already formed, data analysis using one-way anova test in order to know the significant difference among every group, lsd test was done. result the result of the study on accurate dimension of acrylic base plate at various polymerization temperature as shown on table 1. table 1 show that highest dimensional change occurred in group iii (50° c water temperature), while the lowest dimensional change in group ii (40° c water temperature). to know the presence of difference in variant polymerization temperature on the accuracy of orthodontic acrylic base plate dimension, the above data analyzed using one-way anova test variant analysis with significance level 95% was found probability < 0.05. it could be concluded that there is significant difference in accuracy of orthodontic acrylic base plate in every group. to be able to know significant difference among controlled groups, lsd test was done and it is shown on table 2. on the above table we could see the dimensional accuracy of orthodontic acrylic base plate in group i and group ii no significant difference was found, between group i and iii significant difference was found and between group ii and iii significant difference was also found. discussion to achieve the success of orthodontic removable appliance, some requirements which are needed are table 1. the mean difference of orthodontic acrylic base plate dimension with the working model of every group (mm) group number of sample mean deviation standard i. 30° c water temperature ii. 40° c water temperature iii. 50° c water temperature 6 6 6 0.0333 0.0200 0.0750 0.0258 0.0237 0.0274 table 2. lsd test among every group group i ii iii i ii iii – – – – – – + + – note: – : no significant difference; + : significant difference 172 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 170-172 appropriate acrylic base plate, nonporous, good aesthetic, hygienic, low residual content, preventing unmovable dental. the making of acrylic resin of removable appliance using cold curing acrylic with finer particles comparing with cold acrylic in orthodontia. various cold curing acrylics have the same size of particles. while particle of polymer used in removable orthodontic appliance is 75 mm. the smaller the size of a particle the quicker the monomer infiltrates. the properties of cold curing acrylic are in higher porosity. even though it is not easily seen in pigmented resin, it is because the air is dissolved monomer fluid which is not absorbed in polymer powder at room temperature, having satisfying color which is matched with the color of oral mucosa (pink in color), determined by transitional glass (tg). temperature glass transitional of cold curing is 90° c, so the possibility of distortion of hot water would occur, the glass temperature affects the average of molecular weight of a polymer. in general the average of molecular weight is lower and contains more than 80% of heat curing material, it is probably correlated with lower molecular weight. the rheology property of heat curing acrylic is better comparing with cold curing, because cold curing material shows higher distortion in implementation.2 the result of this study shows that acrylic resin in which the polymerization at 30° c water temperature and 40° c producing better accurate dimension comparing with polymerization at 50° c water temperature. it is possibly because physical property of cold curing acrylic shows every little dimensional change caused by less perfect polymerization process.4 on this study 30° c was used with consideration that room temperature was about 30° c, while 40° c was matched with polyclav which was used on this study. 50° c was according to statement given by ada6 suggested to increase temperature during polymerization process. in group i: polymerization at 30° c, there is not significant difference with polymerization at 40° c in groups ii. it is possible that cold curing acrylic could polymerize at room temperature due to the presence of chemical activator,7 i.e. amino group which is added in monomer fluid to activate benzoil peroxide to form free radical, so polymerization could run at room temperature in which amino tertier: dimetil-p-toludin (dmpt)7 is usually used, therefore, 30° c water temperature and 40° c is not too different with room temperature so there is no significant change found in dimensional acrylic change. in group ii polymerization at 40° c water temperature comparing with iii polymerization at 50° c water temperature, there is significant difference. it is possibly because the temperature is too high comparing to room temperature, so polymerization process would quickly occur. the result of quick polymerization process, dimensional change would occur.7,8 the conclusion is the accuracy of orthodontic acrylic base plate dimension in polymerization at 30° c and 40° c air temperature could produce better dimensional accuracy comparing with polymerization at 50° c room temperature. references 1. adams cp. the design and construction and use removable orthodontic appliance. 6th ed. london: butter worth haineman ltd; 1990. p. 209–11. 2. combe ec. notes on dental materials. 6th ed. new york: churchill livingstone; 1992. p. 79–120. 3. houston wjb, isaacson kg. orthodontic treatment with removable appliances. 2nd ed. bristol: john wright & sons ltd; 1992 p. 132–8. 4. anusavice kj. phillip’s science of dental materials. 11th ed. saint louis: wb saunders company; 2003. p. 207–10. 5. nuraini i. sitotoksisitas resin akrilik rapid heat polymerized terhadap kultur sel bhk. majalah kedokteran gigi (dental journal) 2004; 37(1):15–18. 6. american dental association. guide to dental material devices. 7th ed. chicago: american dental association 1974; p. 97–102. 7. anderson jn. applied dental materials. 5th ed. oxford: blackwell scientific publications; 1976. 284–95. 8. craig rg, obrien wj, powers jm. dental material properties and manipulation. 7th ed. st louis, missouri: mosby inc; 2000. p. 79–120. 75 volume 46 number 2 june 2013 case report peroxide alkaline for cleansing the baby bottle nipple to prevent oral thrush relaps maharani laillyza apriasari department of oral medicine study program of dentistry, faculty of medicine, universitas lambung mangkurat banjarmasinindonesia abstract background: oral candidiasis is the most prevalent opportunistic infection affecting the oral mucosa. a number of predisposing factors have the capacity to convert candida from the normal commensal flora to a pathogenic organism. oral candidiasis is divided into primary and secondary infection. the primary infections are restricted to the oral and perioral sites, where as secondary infections are accompanied by sistemic mucocutaneous manifestation. oral thrush is one of the candidiasis primary infection. some presdiposing factors of oral thrush are neonatal, old people, or where oral microflora is disturbed by the treatment with broad spectrum antibiotics. final diagnosis is determined by fungal culture examination, although through clinical examination oral thrush can be determined by swabbing the white pseudomembran. purpose: this case report presents about the importance of using the antiseptic cleanser for baby bottle nipple to prevent oral thrush relaps and shows about peroxide alkaline as the alternatif of antiseptic cleanser for baby bottle nipple that can substitute chlorhexidine gluconat 0.2%. case: a baby girl, 15 months old, when she was suffering influenza the pediatry gave amoxycillin 125 mg three times a day for ten days. then the white plaque appeared on her dorsum of tongue. the therapy was gentian violet 1% four times a day for ten days was applied on dorsum of the tongue. the patient was suspected to suffer alergy reaction after using nistatin oral suspension four times a day had applied for 1 day. the instruction was doing sterilization for the baby bottle nipple in boiling water. three days after the baby was cured, the white plaque was appeared on upper n lower lips mucous. case management: the diagnosis was oral thrush. the therapy was gentian violet 1% four times a day for ten days that applied on upper and lower lips mucous. the instruction was doing the sterilization for baby bottle nipple in denture cleanser contain peroxide alkaline for five minutes, then it was washed with antiseptic soap, and soaked it in boiling water. conclusion: the baby bottle nipple sterilization on the case of oral thrush can not only by boiling in the water, but it must be keep in the antiseptic solution before boiled in the water. it was for preventing oral thrush relaps. key words: baby bottle nipple, denture cleanser, gentian violet 1%, oral thrush, peroxide alkaline abstrak latar belakang: kandidiasis rongga mulut merupakan infeksi opurtunistik yang sering terjadi pada mukosa mulut. banyak faktor predisposisi yang menyebabkan candida yang awalnya merupakan flora normal dalam rongga mulut berubah menjadi organisme patogen. kandidiasis rongga mulut dibagi menjadi infeksi primer dan sekunder. infeksi primer terjadi pada rongga mulut dan sekitarnya, sedangkan infeksi sekunder akan diikuti oleh manifestasi mukokutan secara sistemik. oral thrush adalah salah satu dari infeksi primer candidasis. beberapa faktor predisposisinya adalah pada bayi, orang tua atau microflora rongga mulut terganggu karena pemakaian jenis antibiotik spektrum luas. diagnosis akhir didapatkan dari pemeriksaan kultur jamur, meskipun diagnosis dapat ditegakkan dengan pemeriksaan klinis melalui hapusan lesi pesudomembran putih. tujuan: laporan kasus ini tentang pentingnya penggunaan antiseptik untuk sterilisasi dot botol bayi untuk mencegah kekambuhan oral thrush dan menunjukkan alkalin peroxida sebagai alternatif bahan antiseptik untuk sterilisasi dot botol bayi, yang mampu mengganti klorheksidin glukonat 0,2%. kasus: pasien adalah bayi perempuan usia 15 bulan, selama sakit batuk pilek mendapat obat dari dokter spesialis anak amoxycillin 125 mg yang diberikan 3 kali sehari selama 10 hari. selanjutnya tampak plak putih pada lidah. terapi yang diberikan adalah gentian violet 1% yang oleskan 76 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 75–79 4 kali sehari selama 10 hari pada dorsum lidah. hal ini disebabkan pasien diduga mengalami alergi setelah diberi nistatin suspensi 4 kali sehari. instruksi yang dianjurkan adalah menyeteril dot botol bayi dalam rebusan air. setelah sembuh, 3 hari kemudian muncul lagi plak putih pada mukosa bibir atas dan bawahnya. tatalaksana kasus: pasien didiagnosis menderita oral thrush. terapi yang diberikan adalah gentian violet 1% diaplikasikan 4 x 1 selama 10 hari pada mukosa bibir atas dan bawah. instruksi yang diberikan adalah untuk menyeteril dot botol bayi dengan pembersih gigi tiruan yang mengandung alkalin peroksida selama 5 menit, kemudian dicuci dengan sabun mandi antiseptik, dan direbus dalam air. simpulan: sterilisasi pada bottle nipple pada pasien anak dengan oral thrush tidak dapat dilakukan dengan hanya merebus dalam air saja, melainkan harus merendam dengan cairan antiseptik terlebih dahulu sebelum direbus dalam air. hal ini untuk mencegah terjadinya kekambuhan pada oral thrush. kata kunci: dot botol bayi, pembersih gigi tiruan, gentian violet 1%, oral thrush, alkalin peroksida correspondence: maharani laillyza apriasari, c/o: program studi kedokteran gigi, fakultas kedokteran gigi universitas lambung mangkurat. jl. veteran 128 b banjarmasin, indonesia. e-mail: rany.rakey@gmail.com introduction oral candidiasis is the most prevalent opportunistic infection affecting the oral mucosa. in the vast majority of cases, the lesions are caused by the yeast candida albicans. the pathogenesis is not fully understood, but a number of predisposing factors have the capacity to convert candida from the normal commensal flora to a pathogenic organism. it is a change in the normal oral environment rather than actual exposure, that results in clinical candida infection.1,2 oral candidiasis is divided into primary and secondary infection. the primary infections are restricted to the oral and perioral sites, where as secondary infections are accompanied by sistemic mucocutaneous manifestation. oral thrush is one of the candidiasis primary infection. the clinical features is white or creamy plaques that can be wiped off to leave a red base. these can be easily wiped away with gauze leaving an erythematous base with minimal bleeding. lesions can be seen anywhe re but are frequently located on the dorsal surface of the tongue, buccal mucosa, and palate. the predisposing factors of oral thrush are neonatal, older people, disruption of oral microflora by consuming antibiotics or corticosteroids, xerostomia, immune defects, immunosuppressive management, diabetes mellitus, leukaemias and lymphomas.1-3 the prevalence of children who were fed with both breast milk and bottle milk or other fluids was 18.5%, while in children fed only with breast milk was 0%. the consumption of bottle milk may lead of retention of fluid in the mouth leading to acid production by the oral microflora creating an environment for growth of candida albicans.4 the most common form of oral thrush usually acute. it appears as creamy whitish spots or plaques, which usually can be detached. the lesions may be localized or generalized. the management of oral thrush are eliminiting the predisposing cause and giving the topical antifungal drugs such as nystatin oral suspension andmyconazole gel, or systemic antifungal drugs such as fluconazole, itraconazole, and ketoconazole.1,3 this case report presents about the importance of using the antiseptic cleanser for baby bottle nipple to prevent oral thrush relaps. the peroxide alkaline usually uses as a denture cleanser that contain antiseptic agent. in this case, peroxide alkaline could be as the alternatif of antiseptic cleanser for baby bottle nipple that can substitute chlorhexidine gluconat 0.2%. case a baby girl, 15 months old, her mother said that the thick white membran appeared on her baby dorsum tongue. she had suffered influenza, the pediatry gave her amoxycillin 125 mg that was consumed three times a day. then the thick white membran appeared on her tongue dorsum after consuming amoxycillin 125 mg three times a day for ten days. case management fist visit, the intra oral examination showed the thick white plaque on the dorsum of tongue, can be scrapped, it leaved erythematous area (figure 1). clinical diagnosis was oral thrush because of using broad spectrum antibiotic. the therapy was nistatin oral suspension four times a day that applied on dorsum of the tongue, then it was swallowed. then the patient should not eat and drink for thirty minutes. the instruction was doing sterilization for baby bottle nipple in boiling water. second visit, after using nystatin for three times in one day, there were erythematous lesion around lips and arms (figure 2 and 3). it were papula, multiple, erythemaous, and itching that showed the symptoms that similiar to allergic reaction after using nystatin for three times a day. it was suspect allergic reaction. the patient was given cetirizine 2.5 mg once time a day until the symptoms that similiar to alergy was gone. it had dissapeared for two days. nystatin was stopped, it was replaced with gentian violet 1% applied four times a day for ten days. it was left for 30 minutes in oral cavity, especially on dorsum of the tongue. the instruction was doing the baby bottle nipple sterilization in boiling water still continued. 77apriasari: peroxide alkaline for cleansing the baby bottle nipple third visit, after 14 days, the patient and her mother came again. on intra oral examination, there were white and thick plaque on her upper and lower lips mucous (figure 4). the lesion could be scrraped and left erythematous areas. it appeared after the white plaque on dorsum of the tongue had been healed for 3 days. the therapy was gentian violet 1% applied four times a day for ten days. it was left for 30 minutes in oral cavity, especially on upper and lower lips mucous. the instruction was doing the baby bottle nipple sterilization in chlorhexidine glukonat 0.2% for 15 minutes, then cleaned by antiseptic soap, and put into boiling water. in the evening, the patient’s mother called by phone. she said that was difficult to get chlorhexidine gluconate 0.2% at the drug strores, so that chlorhexidine gluconat 0.2% was replaced with denture cleanser contains peroxide alkaline. the baby bottle nipple was soaked into peroxide alkaline effervecent and 200ml water for 5 menit, then it was wash with antiseptic soap, and put into boiling water. fourth visit, after 24 days, the patient came to control. according to her mother explanation, her child was getting better. base on clinical examination, there were not any lesion, the patient was cured (figure 5 and 6). discussion on this case, the patient was a baby girl, 15 months old, consumed baby bottle milk. on first time, based on clinical examination, it was diagnosed as thrush. base on patient anamnesis, it appeared on dorsum of the tongue, after consuming the broad spectrum antibiotic. it changed the balance of oral commensal microorganisms in oral cavity and disturb the comensal bacterial growing that antagonistic against candida, so it made the population being increased.1 thrush appears because of some predisposing and a bad oral hygiene. the consumption of bottle milk may lead of retention of milk in the mouth leading to acid production by the oral microflora creating an environment for growth of candida albicans. some studies discuss about the interaction between candida and bacterial in oral cavity. the bacterial modulated the attachment and candida colonization. the chronical inflamation was happened because of some protein as phospolipase dan proteinase from candida that was supported by bacterial from baby bottle milk. the residual milk attached on tongue mucous. then candida albicans was more virulent.4-6 the differential diagnosis of oral thrush on dorsal surface of the tongue are furred tongue. clinical examination of furred tongue reveals a whitish lesion encompassing the entire dorsal surface of the tongue. there are multiple elongated projections within the body of the lesion. patients may have a bad taste in their mouth or discoloration of the tongue but usually have no pain or discomfort. it is different from thrush, the patient has burning sensation, dryness, and loss of taste.7,8 the management case were eliminiting the presdisposing factors, had a good oral hygiene and giving the topical antifungal drug. the oral cavity was cleaning with sterile gauze that had been given with gentian violet (gv) 1% for getting a good oral hygiene. gv 1% substituted nystatin oral suspension, because the patient suspected of allergic reaction. previous studies suggested mechanisms of action for gv 1% that production of the perhydroxy radicals may facilitate the penetration of gv 1% through the biofilm matrix leading to inhibition of candida cell wall synthesis. gv 1% activity against candida biofilms was demonstrated by a reduction in dry weight, disruption of biofilm architecture, and reduced biofilm thickness.9 so that in this case, gv 1% was as effective as nystatin oral suspension for killing candida albicans. the instruction were doing baby bottle sterilization in boiling water, but it was not effectif for killing candida. because the temperature used for sterilizing baby bottle nipple was not to high for preventing baby bottle nipple to be broken. after three days the patient was healed, the thrush appeared again. boiling water in fifteen minutes will kill most vegetatif bacteria and inactive viruses, but it is ineffective many bacterial and fungal spore, therefore boiling water is unsuitable for sterilization.10 for preventing thrush relaps, the sterilization instruction was given to soak the baby bottle nipple in peroxide alkaline for 5 minutes, then cleaned by antiseptic soap, and put into boiling water. chlorhexidine gluconate 0.2% was difficult to get at drug strore, so that chlorhexidine gluconat 0,2% can be replaced with denture cleanser contains peroxide alkaline. peroxide alkaline is the chemical denture cleanser as the effervescent tablet. it’s compotision are subtilisin, citric acid, sodium carbonate, potassium peroxymonosulfate dan sodium perbirate monohydrate. after it has dissolved in water, sodium perborate will unravel and make peroxide alkaline solution that release oxygen so that will be cleaning and eliminating the mikroorganisme mechanically. peroxide alkaline is denture cleanser that is better than other denture cleanser which contains acid. it will decrease the ability to kill candida. it is supported by the fact that adhesion plaques is through organic content. peroxide alkaline in water will form hydrogen peroxyde solution and release oxygen. oxygen bubbles will clean it mechanically. the acid is dissolving calcareous deposit. peroxide degradation would release oxygen bubbles that will clean the dentures mechanically if it contacts with debris. peroxide can also prevent the formation of stain and calculus. as desinfectan, it will kill candida through protein denaturation process.12-14 peroxyde alkaline is a denture cleanser that as desinfectan can kill candida. based on that theory, peroxyde alkaline can substitute chlorhexidine gluconat 0,2% that is desinfectan for cleansing the baby bottle nipple from candida. the baby bottle nipple sterilization with boiling in water is not effective that can cause thrush relaps. the using 78 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 75–79 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. 67apriasari: peroxide alkaline for cleansing the baby bottle nipple figure 1. the intra oral examination showed the thick white plaque on dorsum of tongue. figure 2. there were erythematous lesion around her lips. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 3. there were erythematous lesion around her arms. it were papula, multiple, erythemaous, and itching that suspected of allergic reaction after using nystatin. figure 4. there were white and thick plaque on her upper and lower lips mucous. the lesion could be scrraped and left erythematous areas. figure 5. base on clinical examination, there was not any lesion on upper lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. figure 6. base on clinical examination, there was not any lesion on lower lip mucous. the patient was cured. 79apriasari: peroxide alkaline for cleansing the baby bottle nipple of peroxyde alkaline can prevent thrush relaps, because it is a desinfectan with antifungal agent. it can be concluded that baby bottle nipple sterilization on the case of thrush can not only by boiling in the water, but it must be keep in the antiseptic solution before boiled in the water. it was for preventing thrush relaps. references 1. greenberg ms, glick m, ship ja. burket’s oral medicine. 8th ed. bc decker inc hamilton, ontario: 2008. p. 79. 2. bruch jm, treister ns. clinical oral medicine and pathology. springer, new york: humana press; 2010. doi 10.1007/978-160327-520-0 3. gandalfo s, scully c, carrozzo m. oral medicine. philadelphia, usa: churchill livingstone elsevier; 2006. p. 49. 4. kadir t, uygun b, akyu s. prevalence of candida species in turkish children: relationship between dietary intake and carriage. arch oral biol 2005; 50(1): 33-7. 5. lukisari c, setyaningtyas d, djamhari m. penatalaksanaan kandidiasis oral disebabkan candida tropicalis pada anak dengan gangguan sistemik. dentofasial 2010; 78(2): 9. 6. apriasari m.l, soebadi b. penatalaksanaan chronic atrophic candidiasis pada pasien gigi tiruan lepasan. dentofasial 2009; 8(2): 95. 7. laskaris g. treatment of oral diseases a concise textbook. newyork, usa: thieme stuttgart; 2005. p. 30-1. 8. rizzolo d, monroe j. “furry” lesion on a young woman’s tongue. j am academy of physician assistants 2007; 20(1): 61. 9. traboulsi rs, mukherjee pk, chandra j, salata ra, jurevic r, ghannoum ma. gentian violet exhibits activity against biofils formed by oral candida isolates obtained from hiv-infected patients. antimicrob agents chemother 2011; 55(6): 3043–5. 10. zadik y, peretz a. the effectiveness of glass bead sterilizer in the dental practise. j isr dent assoc 2002; 25 (2): 36-9. 11. ural c, şanal fa, seda c. effect of different denture cleansers on surface roughness of denture base materials. clinical dentistry and research 2011; 35(2): 15-7. 12. vieira ap, senna pm, silva wj, del bel cury aa. long-term efficacy of denture cleansers in preventing candida spp. biofilm recolonization on liner surface. braz oral res 2010; 24(3): 343. 13. kumar mn, thippeswamy hm, raghavendra swamy kn, gujjari ak. efficacy of commercial and household denture cleanser against candida albicans adherent to acrylic denture base resin: an in vitro study. indian j dent res 2012; 23(1): 39-42. 14. henrique m, francisco m, braun ko. in vitro antifungal action of different substances over microwaved-cured acrylic resins. j appl oral sci 2009; 17(5): 433. vol 50 no 4 desember 2017.indd 216 research report dental journal (majalah kedokteran gigi) 2017 december; 50(4): 216–219 comparison of salivary alpha-amylase levels in gingivitis and periodontitis dyah nindita carolina, yanti rusyanti, and agus susanto department of periodontics faculty of dentistry, university of padjajaran bandung – indonesia abstract background: the development of periodontal disease is influenced by bacteria-plaque, while there are also several factors modifying the host’s response, one of which is psychological stress. alpha-amylase as a biomarker is also associated with periodontal inflammatory disease. purpose: the purpose of this study was to examine the difference of alpha-amylase level between gingivitis and periodontitis. methods: this research constitutes a descriptive study involving 44 subjects, divided into two groups: one of 22 gingivitis subjects and the other of 22 periodontitis subjects. these individuals completed a pss-14 questionnaire before their levels of alpha salivary amylase were measured by cocorometer. data was analyzed by means of a paired t test and a mann whitney test with p < 0.05. results: there were significant differences between the alpha-amylase levels of gingivitis and periodontitis. however, no significant contrast existed in the pss-14 scores of the two periodontal disease groups. conclusion: in conclusion alpha-amylase levels in the periodontitis group were higher than those in the gingivitis group and could be used as marker indicators of stress. keywords: alpha-amylase level; gingivitis; periodontitis; saliva; stress correspondence: dyah nindita carolina, department of periodontics, universitas padjajaran, jl. sekeloa selatan i, bandung 40132, indonesia. e-mail: dyah.nindita@unpad.ac.id introduction the oral cavity can be regarded as one site of systemic diseases because several such ailments can be identified through its overall condition. the parameters of oral examination in diagnosing disease may comprise the clinical condition of the cavity supported by analysis of, for example, the blood, gingival crevicular fluids and saliva. blood and saliva can be used to monitor systemic conditions indicating the general state of oral health.1,2 saliva has achieved rapid adoption as a biomarker for more than a decade, especially in the fields of psychology and biomedical research.3 the analysis of saliva has several advantages including: rapid and easy collection of samples, the lack of need for special equipment and/or operators, non-invasive and painless processes, as well as the absence of stress caused to the patient. saliva contains several biomarkers such as cystatins, albumin, alpha-amylase, iga, and cortisol. certain studies argue that alpha-amylase as a biomarker is also associated with periodontal inflammatory disease.4,5 psychological stress is one of the important risk factors in periodontitis. one study highlighted the significant numbers of people suffering from psychosocial or occupational stress (17.05%) and the complex mechanisms through which such stress could affect their periodontal condition. it suggested that one possible cause involves modification of undesirable behavior such as smoking and poorly maintained oral hygiene.6 the measurement of psychological stress can be viewed subjectively based on individual feelings and thoughts regarding recent developments, one of the most commonly used measuring tools being perceived stress scale-14 (pss-14).7 stress is one factor evident in physiological changes manifested by an individual and an analysis of saliva can determine the associated level of stress.8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p216-219 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p216-219 mailto:dyah.nindita@unpad.ac.id 217217carolina, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 216–219 periodontal diseases, most notably gingivitis and periodontitis, are defined as inflammation caused by pathogenic microflora making up the biofilm surrounding the teeth. gingivitis is an inflammatory condition that affects the surface of the gingiva and is reversible in the absence of alveolar bone destruction, while periodontitis is a disease resulting in damage to the tooth-supporting tissues that can cause tooth loss.9,10 this study used alpha-amylase enzyme as a parameter of the severity of periodontal disease and a marker of psychological stress in the patient. within this study, the selection of alpha-amylase was based on the assertion of ackali et al. that it is one of the main saliva-based markers serving as a stress indicator that has been investigated in the hope of confirming the interrelationship between psychological stress and periodontal disease.9 it is also associated with other studies suggesting that alphaamylase levels in saliva tend to increase in individuals with periodontal disease and in others who are psychologically stressed.5,7,8 tanaka et al. identified a practical, japanesemade tool (the nipro cocorometer) for measuring stress based on alpha-amylase levels in saliva.11 using a cocorometer, the present study measured alpha-amylase levels in patients afflicted with gingivitis and periodontitis. furthermore, these levels are used as stress parameters when observing the relationship between periodontal disease and psychological stress. the authors were interested in conducting research into the measurement of salivary alpha-amylase levels in patients with gingivitis and periodontitis. the measurement results for alphaamylase levels were also linked since psychological stress parameters and psychological stress were often linked as a risk factor influencing the occurrence of periodontitis. materials and methods the research was conducted on 44 patients of the periodontic department, dental hospital, universitas padjadjaran, selected according to the inclusion criteria of there being 22 gingivitis patients and 22 chronic periodontitis patients. the inclusion criteria of subjects included the following: all were aged 17–55 years old, all examined teeth afflicted by gingivitis had probing depths of ≤ 3 mm, those with periodontitis had a probing depth of ≥ 4 mm and a loss of clinical attachment of ≥4 mm. as confirmed by screening conducted by an internist, no subjects suffered systemic disease: diabetes mellitus (dm), rheumatoid arthritis or kidney disease. none had undergone periodontal therapy or taken antibiotics or contraceptive pills during the preceding three months, nor were any pregnant or lactating. this study used a non-random technique, selecting samples by determining which subjects met the research criteria included in the study within a certain time period.5 the tools and research materials utilized included: a cocorometer, nipro co., japan,10 glass mouth, explorer, williams probe, gloves and mask, mouth rinse and a cotton roll. the research was descriptive in nature, comparing the level of alpha-amylase in the specific forms of periodontal disease, namely; gingivitis and periodontitis. the required ethical clearance was issued by the commission of dentistry ethics, faculty of dentistry, universitas padjadjaran. clinical examination was performed on the oral cavities of patients suffering from gingivitis and periodontitis who attended the department of periodontics, dental hospital, universitas padjadjaran. the objective of the pss-14 questionnaire completed by patients upon termination of the oral examination was to measure psychological stress based on individuals’ subjective thoughts and feelings about their ongoing situation. the stress level criteria relating to pss14 scores were as follows: low: 15–19, medium: 20–24, high: 25–29, very high: > 30.6 alpha-amylase measurement was conducted using a cocorometer (nipro, japan) tool to monitor an individual’s physiological changes, particularly by determining the associated stress level through the analysis of saliva. the end of a measuring stick was placed at the base of the tongue for one minute until it had become wet (figure 1a). it was then inserted into the cocorometer tool (figure 1b).11 the results of alpha-amylase levels were recorded on the cocorometer’s monitor which operates on the basis that a rise in norepinephrine will affect the level of alpha-amylase in human saliva (figure 1c). alphaamylase reacts faster than cortisol and norepinephrine to acute stress. therefore, the instant a person feels stressed, it will appear in the results. the criteria for stress levels based on the alpha amylase level (ku/l) are as follows:10 low: 0–30, medium: 30–45, height: 46–60, very high: > 61. figure 1. (a) measurement of alpha-amylase content using nipro japan cocorometer tool. (b) the sticks were included in the nipro japan cocorometer tool. (c) record the results of the alpha-amylase levels listed on the japanese nipro cocorometer monitor. a b c dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p216-219 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p216-219 218 carolina, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 216–219 the measurements of alpha-amylase levels in groups of individuals suffering from gingivitis were compared with those afflicted with periodontitis. the research data was processed by calculating the mean, standard deviation, median and range. the average difference in alpha amylase levels was analyzed by means of a mann whitney test, while the pss score was calculated using a t-test. results the research reported here was conducted on 44 subjects all of whom were patients of the department of periodontics, dental hospital, universitas padjadjaran. the inclusion criteria of 22 individuals suffering from gingivitis and 22 afflicted with chronic periodontitis were fully met. their demographic characteristics were shown in table 1. the alpha amylase levels and pss-14 score of sufferers of periodontal disease (gingivitis and periodontitis) can be seen in table 2. table 1 contains data on the general characteristics of the study sample with regard to gender and age. in terms of gender, the gingivitis group contained 11 males and 11 females, while in the periodontitis group there were 10 males and 12 females. when tested statistically, the latter did not show any significant differences. as for the age of the subjects, the mean and median of the periodontitis group were both higher than those of the gingivitis group. the alpha-amylase levels and pss-14 scores in those individuals suffering from periodontal disease (gingivitis and periodontitis) can be seen in table 2. the mean alphaamylase level of 82.4 ku/l in gingivitis patients compared to one of 147.6 ku/l in members of the periodontitis group. the median of the periodontitis group was greater than that of the gingivitis group. an examination of the scores on pss-14 revealed that those of the periodontitis group ranged from 15 to 30 with an average of 22.7, while in the gingivitis group they ranged from 18 to 31 with an average of 23.1. discussion the progress of periodontal disease remains unclear, although it is influenced by differences in the respective susceptibility of individuals. the progression of periodontal disease can be more rapid (aggressive) given the presence of systemic or environmental factors such as diabetes mellitus, smoking, or stress.2,3,12that influence the host’s response to plaque. stress reduces salivary flow and supports bacterial plaque formation, while emotional stress modifies ph levels and salivary composition.13 saliva contains several proteins that can be used as markers of stress, one such indicator being alpha amylase.9 the results of this study confirmed that there was an average difference in alphaamylase levels in gingivitis patients and periodontitis patients. in this study, the alpha-amylase levels present in cases of periodontitis were higher, compared to those in gingivitis patients. this finding was consistent with that of research conducted by sanchez et al.5 which suggested that periodontitis may stimulate the increased production of proteins in saliva, including mucin and amylase. the concentration of these two types of protein is increased in the sufferers of periodontitis moderate to severe in severity. the increasing alpha-amylase levels in periodontitis were associated with a study by papacosta which stated that salivary alpha-amylase is the first line of defense. according to rohleder, this enzyme prevents pathogens entering the body through the mucosal surface and could be considered as the best indicator of mucosal immunity in the oral cavity by inhibiting attachment of bacterial growth.14,15 the stress levels of patients suffering from gingivitis and periodontitis which were measured in this study by pss-14 confirmed there to be no difference in stress levels between members of either group. these were supposedly related to the level of subjectivity of each individual under stress recorded by the measuring tool which used a pss-14. measures the feelings and thoughts of individuals related to situations that occurred within the previous month. research subjects were asked to choose the statement that best suitable of his condition.7 the perceived stress scale (pss) was not a diagnostic tool, as there was no standard reference value that measures stress levels. however, table 1. characteristic of subjects characteristic groups gingivitis (n = 22) periodontitis (n = 22) gender male female 11 11 10 12 age x (sd) median range 23.9 (7.1) 21 17-47 40.9 (6.9) 39 32-55 table 2. examination of alpha-amylase levels and pss-14 score in the groups of gingivitis and periodontitis characteristic groups gingivitis (n = 22) periodontitis (n = 22) alpha-amylase x (sd) median range 82.4 (52.7) 74 7–193 147.6 (101.4) 118 10–436 score pss-14 x (sd) median range 23.1 (4.0) 22 18-31 22.7 (4.4) 23 15-31 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p216-219 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p216-219 219219carolina, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 216–219 from these measurements, comparative values in groups of research samples could be observed. stress was part of being human which occurs to varying degrees and produces different effects on one’s health.16 several questionnaires such as the perceived stress scale have been developed to better identify and classify stress, but this questionnaire had deficiencies in the standardization of psychological scales for the quantity and definition of stress.13 a more thorough questionnaire that includes lifestyles and other powerful influences such as gender, age and characteristic traits would be better at clarifying a person’s stress levels relating to the developments that occur in his or her life.9 there are certain factors, conditions or occurrences, that cause stress. these are often referred to as the stressor. the response or reaction to something that can cause stress and the processes that actively occur affect its impact on the individual. this phenomenon is called the locus of control and constitutes a measure of the extent to which an individual believes he has control over his life and where such control comes from.17,18 levels of alpha-amylase measured by means of a cocorometer were used as a marker of stress. when the levels were higher than normal, they were expressed as stress (> 60 ku/l).11 the group suffering from more severe periodontal disease (periodontitis) demonstrated a tendency towards higher levels of stress. such elevated levels were in accordance with the findings of research conducted by shende which suggested that psychosocial factors such as severe stress, personality type and individual coping strategies to deal with a stress environment can modify the immune response. these result in an individual being more sensitive to unhealthy conditions which also affects the state of periodontal tissue.19 patients suffering from psychiatric disorders demonstrated a level of susceptibility to periodontitis greater than that of individuals free of such conditions.20 this study shows that the objective measurement of alpha-amylase levels in the periodontitis group was higher compared to that of subjects with gingivitis. however, in the assessment of pss-14, there was no difference in stress levels between the gingivitis and periodontitis groups. this was influenced by individual subjectivity in completing the questionnaire. stress is a non-observable abstract condition that cannot be precisely measured by the completing of such a survey alone. other forms of measurements, including interviews and observations from the experts concerned, were required. their absence must, therefore, be regarded as representing a major limitation on the rigor of this study. in conclusion alpha-amylase levels in the periondontitis group were higher than those in the gingivitis group and could be used as marker indicators of stress. references 1. navalkar a, bhoweer a. alterations in whole saliva constituents in patients with diabetes mellitus and periodontal disease. j indian acad oral med radiol. 2011; 23(4): 498–501. 2. buduneli n, özçaka ö, nalbantsoy a. salivary and plasma levels of toll-like receptor 2 and toll-like receptor 4 in chronic periodontitis. j periodontol. 2011; 82(6): 878–84. 3. nater um, rohleder n, schlotz w, ehlert u, kirschbaum c. determinants of the diurnal course of salivary alpha-amylase. psychoneuroendocrinology. 2007; 32(4): 392–401. 4. baliga s, muglikar s, kale r. salivary ph: a diagnostic biomarker. j indian soc periodontol. 2013; 17(4): 461–5. 5. sánchez ga, miozza v, delgado a, busch l. determination of salivary levels of mucin and amylase in chronic periodontitis patients. j periodontal res. 2011; 46(2): 221–7. 6. mârţu s, solomon s, potârnichie o, ărin lpăs, mârţu a, nicolaiciuc o, rescu iursa. evaluation of the prevalence of the periodontal disease versus systemic and local risk factors. int j med dent. 2013; 3(3): 212–8. 7. reis rs, hino aaf, añez crr. perceived stress scale: reliability and validity study in brazil. j health psychol. 2010; 15(1): 107–14. 8. pani sc, al askar am, al mohrij si, al ohali ta. evaluation of stress in final-year saudi dental students using salivary cortisol as a biomarker. j dent educ. 2011; 75(3): 377–84. 9. akcali a, huck o, tenenbaum h, davideau jl, buduneli n. periodontal diseases and stress: a brief review. j oral rehabil. 2013; 40(1): 60–8. 10. bensley l, vaneenwyk j, ossiander em. associations of selfreported periodontal disease with metabolic syndrome and number of self-reported chronic conditions. prev chronic dis. 2011; 8(3): a50. 11. tanaka y, ishitobi y, maruyama y, kawano a, ando t, okamoto s, kanehisa m, higuma h, ninomiya t, tsuru j, hanada h, kodama k, isogawa k, akiyoshi j. salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in major depressive disorder patients. prog neuro-psychopharmacology biol psychiatry. 2012; 36(2): 220–4. 12. hinrichs je, novak mj. classification of diseases and conditions affecting the periodontium. in: newman m, takei h, klokkevold p, carranza f, editors. carranza’s clinical periodontology. 11th ed. st. louis: saunders elsevier; 2012. p. 34–55. 13. reners m, brecx m. stress and periodontal disease. int j dent hyg. 2007; 5(4): 199–204. 14. papacosta e, nassis gp. saliva as a tool for monitoring steroid, peptide and immune markers in sport and exercise science. j sci med sport. 2011; 14(5): 424–34. 15. rohleder n, nater um. determinants of salivary α-amylase in humans and methodological considerations. psychoneuroendocrinology. 2009; 34(4): 469–85. 16. iacopino a m. relationship between stress, depression and periodontal disease. j can dent assoc (tor). 2009; 75(5): 329–30. 17. calvete e, corral s, estévez a. cognitive and coping mechanisms in the interplay between intimate partner violence and depression. anxiety, stress & coping. 2007; 20(4): 369–82. 18. brouskeli v, markos a. the role of locus of control and perceived stress in dealing with unemployment during economic crisis. res humanit soc sci. 2013; 3(21): 95–102. 19. shende as, bhatsange ag, waghmare as, shiggaon lb, mehetre vn, meshram ep. determining the association between stress and periodontal disease: a pilot study. j int clin dent res organ. 2016; 8(2): 111–4. 20. rai b, kaur j, anand sc, jacobs r. salivary stress markers, stress, and periodontitis: a pilot study. j periodontol. 2011; 82(2): 287–92. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p216-219 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p216-219 138 research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 138–143 grafting effectiveness of anadara granosa shell combined with sardinella longiseps gel on the number of osteoblast-osteoclast cells eddy hermanto,1 rima parwati sari,2 asri cahyadita dwi imaniar,1 and kevin anggoro1 1department of oral surgery 2department of oral biology faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: bone grafts derived from anadara granosa shells contain calcium carbonate that possesses bone-healing properties. the combination of sardinella longiceps fish oil, containing epa and dha, and anadara granosa shells was assumed to regulate the number of osteoblasts-osteoclasts during the bone-healing process. purpose: this study aimed to determine the effectiveness of anadara granosa shell grafts, combined with sardinella longiceps fish oil, in the bone-healing process by observing the ratio of osteoblasts-osteoclasts in rattus novergicus rats. methods: the wistar rat subjects (n = 25) were divided into five groups, namely: one untreated group (control), one group treated with bone grafts derived from anadara granosa shells (p1), and the other three groups treated with a combination of anadara granosa shells and sardinella longiceps fish oil at concentrations of 10%, 20%, and 30% (p2, p3, and p4). then, a wound equivalent in size to half the diameter of a round bur (±1.5mm) was intentionally inflicted on the right femur of all the subjects. the rats were subsequently sacrificed on day 14, their femur in the transversal side being cut before he staining was completed. thereafter, the ratio of osteoblasts to osteoclasts was measured by means of a light microscopy. the data was subsequently analyzed using one-way anova. results: the average number of osteoblasts in all research groups increased, viz: 9.420±0.8044 for control group (k), 12.080±0.79811 for group p1, 20.020±0.7190 for group p2, 25.940±0.7197 for group p3, and 36.280±0.9985 for group p4. similarly, the number of osteoclasts in all groups subject to analysis also increased, namely: 1.73±0.098 for group k, 2.19±0.305 for group p1, 1.60±0.088 for group p2, 1.60±0.724 for group p3, and 1.80±1.302 for group p4. moreover, the results of the one-way anova test confirmed that there were no significant differences in osteoclasts between all research groups (p>0.05). the results of the one-way anova and lsd tests confirmed there to be significant differences (p <0.05) between group k and other treatment groups (p1, p2, p3, and p4). conclusion: the grafts derived from the combination of anadara granosa shells and sardinella longiceps gel can induce the production of osteoblasts, but not in the numbers necessary during the healing processin the femurs in rattus novergicus rats. keywords: bone graft; anadara granosa; sardinella longiceps; osteoblasts; osteoclasts correspondence: eddy hermanto, department of oral surgery, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: eddyhermanto_tarka@yahoo.com introduction in dentistry, the main causes of bone damage are predominantly those of tooth extraction (90%), trauma, and conditions, such as cysts or tumors affecting the jaw. postextraction, the alveolar bone will undergo an anatomical change in its shape involving several stages. this condition can, in turn, cause the jawbone to shrink and become thin and brittle, thus facilitating fractures, reducing the success of other dental treatments, and decreasing the function of mastication and food digestion.1,2 damage to the alveolar bone is a post-tooth extraction complication potentially impacting negatively on an individual’s health, while also having an undesirable dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 mailto:eddyhermanto_tarka@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 139139hermanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 138–143 aesthetic effect. the damage can, nevertheless, be corrected by bone grafts that serve to restore the contours of bones or stimulate the formation of new bone. bone grafting is a surgical procedure whereby missing bone is replaced with substitutes either taken from the patient’s own body or which are artificial, synthetic or naturally occurring. bone grafting is feasible since bone tissue demonstrates the ability to regenerate effectively, given the availability of space for bone growth. bone formation can then occur owing to several factors influencing the acceleration of healing. bone graft is one option to precipitate the bone healing process, incorporating the use of a material that promotes reconstruction, stabilizes the structure and bonding of bones, and stimulates the process of osteogenesis and the healing of large bone defects.3–5 the bone graft material normally utilised is derived from non-metallic synthetic materials obtainable from ceramic materials (potassium), composites, and polymers. this material must be biocompatible and osteoconductive and can also be fused with bone, thereby enhancing the bone regeneration process.4 in general, bone grafts can be classified into four common types, namely: autograft, allograft, xenograft, and alloplast. xenograft involves the use of bone derived from donors drawn from species other than that of the recipient, such as anadara granosa shells.5 anadara granosa shell is of a type commonly found in both east and southeast asia and contains red blood pigment/hemoglobin enabling the shell to exist in conditions with relatively low levels of oxygen.6,7 anadara granosa shell is also classified as a mineral since biopolymer composites consist of between 95% and 99% caco3 in the form of aragonite crystals. 8 unfortunately, anadara granosa shells are relatively little used, usually being thrown away and allowed to break up naturally. in addition, according to previous research, the presence of nano-calcium carbonate (caco3) crystals derived from shells can be used in bone tissue engineering since they possess the potential to mimic the original composition, structure, and bone properties.9 the investigation referred to also showed that an increase in osteogenic activity of alkaline phosphatase may accelerate the differentiation of mesenchymal stem cells. therefore, the activity of alkaline phosphatase is considered to be an indicator, or bone marker, that signifies the presence of mineralized bone and promotes the formation of ha in osteoblast matrix vesicles by releasing it into the extracellular matrix and increasing osteoblast cell differentiation. subsequently, calcium carbonate stimulates macrophages in areas containing defects, with the macrophages, together with inflammatory cells, strengthening angiogenesis processes.10 on the other hand, fish oil is rich in omega-3 polyunsaturated fatty acid (pufa), composed of eicosapentaenoid acid (epa) and docohexaenoic acid (dha), which plays an important role in maintaining human health.11 similarly, sardinella longiceps fish oil contains n-3 pufa, 13.70% (epa), and 8.91% dha.12 the high levels of epa and dha contained in sardinella longiceps fish oil possess the potential to regulate the formation and activity of osteoblasts and osteoclasts. meanwhile, omega-3 pufa has the ability to act as a vasoconstrictor and platelet aggregation secreting growth factors, such as vegf, that directly affect the formation of new blood vessels.13 previous research confirmed that omega-3 pufa is also able to increase mediators of bone and tooth formation.14 the study reported here aimed to evaluate the effectiveness of bone graft derived from a combination of anadara granosa shells and sardinella longiceps fish oil gel by measuring the number of osteoblasts and osteoclasts during the bone healing process in animal subjects. materials dan method the research was truly experimental in character with a completely randomized design. thus, animal subjects used in both the control and treatment groups were randomly selected and consisted of two-month old, male wistar rats weighing between 150 and 357 grams. the total number of animal subjects used as samples was 25, divided into five groups. the research was then conducted following approval from the ethical commission for animal subjects faculty of dentistry, universitas hang tuah, surabaya no.193/kepk/ix/2016. the bone graft was prepared by collecting, boiling and cleaning anadara granosa shells taken from seaweed waste.15 the shell powder was then sterilized by the application of gamma-ray radiation at batan’s jakarta laboratory. thereafter, 0.2 grams of sardinella longiceps fish oil was mixed with 2 ml of 10% gelatin to produce 10% sardinella longiceps gel. in addition to producing sardinella longiceps gel at concentrations of 20% and 30%, 0.4 grams and 0.6 grams of sardinella longiceps fish oil were mixed with 2 ml of 10% gelatin. the experimental procedure which the animal subjects underwent began with seven days of acclimatization. the wistar rats were then divided into five groups, namely: one group not receiving treatment (control/k), one group treated with bone grafts derived from anadara granosa shells (p1), and the other three groups administered a combination of anadara granosa shells and sardinella longiceps fish oil at concentrations of 10%, 20%, and 30% (p2, p3, and p4) respectively. following the acclimatization process, surgery was performed on the dextral side of the subjects’ femurs along the lines of the procedure performed by fleckhell.16 the wistar rats were anesthetized by the administering of ketamine and xylazine intramuscularly at a dose of 0.11 ml/100 gr bm.17 once unconscious, subjects’ fur in the area where the defect had been induced was shaved using a gillete razor. 10% povidine iodine was applied to the area for five minutes as an antiseptic.18 a two-centimetre long incision was made using a one med indonesia surgical dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 140 hermanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 138–143 knife, before the removal of soft tissue (skin and muscle) by means of an osung periosteal elevator. defects as deep as a half the diameter of the bur (1.5mm) were produced on the dextral and lateral areas of the femur using a germanmade, size 18, mcisinger® round bur, together with a straight hand piece. after defects in the femur had formed, all research groups received different types of treatment. then, the application of membrane was performed. each surgical procedure invariably culminated in suturing to close skin wounds and cover soft tissue.19 the subsequent administering of novalgin consisted of a dose 0.09 cc/200 gr bm, before the antibiotic interflox was given as a dose of 0.1 cc/100 gr bm for three days. in short, this procedure was usually required in order to control inflammation and pain.20 at this point, the rats were euthanized using ether before their femur was taken from the dextral side on day 14.17 the femur specimens collected were hulled in order to access the bone after grafting by cutting it with a separating disc, and then inserting it into 10% formalin buffer solution. this was intended to keep the tissue from decomposing and hardening, as well as to increase the affinity of the tissue against the paint.19 after the process of tissue fixation, a decalcification process was induced by means of administering ethylene diamine tetra acid (edta) over two months. the femur specimens collected were then subjected to sagittal piece preparations before haematoxylin eosin (he) staining was carried out. thereafter, the number of osteoblasts and osteoclasts in the defect areas was measured with a light microscope (olympus® cx21, japan) at 400x magnification. the data obtained was analyzed to obtain details of data distribution and data summary in order to clarify the results. the hypothesis was then tested using a parametric statistical test, a one-way anova test and, finally, a least significance difference (lsd) test. results clinically, the defects in the dextral and lateral sides of the femurs of group p1 subjects were slightly closed due to a less than optimal process of bone formation. there were also significant differences in the number of osteoblasts between group p1 and groups p2, p3, and p4. group p1 had fewer osteoblasts than groups p2, p3, and p4. the histological features indicated significant differences in the number of osteoblasts and osteoclasts located at the defect sites in all research groups (figures 1 and 2). the results also revealed that the average number of osteoblasts in all research groups increased, viz: 9.420 ± 0.8044 for group k, 12.080 ± 0.79811 for group p1, 20.020 ± 0.7190 for group p2, 25.940 ± 0.7197 for group p3 and 36.280 ± 0.9985 for group p4. similarly, the average number of osteoclasts in all research groups also increased, 1.73 ± 0.10 for group k, 2.19 ± 0.305 for group p1, 1.60 ± 0.09 for group p2, 1.60 ± 0.07 for group p3, and 1.80 ± 0.302 for group p4. moreover, the results illustrated the morphologies of osteoblasts and osteoclasts in the defect sites of all research groups. in figure 2, the lowest number of osteoblasts was found in group k, while the highest was that of group p3. similarly, the average number of osteoclasts showed almost the same results. furthermore, the statistical test results using spss 18.0 confirmed the data as being both normally distributed (p>0.05) and homogeneous (p=0.324) with a significance level of 0.05. furthermore, the one-way anova test results indicated a significant difference in the presence of osteoblast indicators, but not those of the osteoclast variety. another further statistical test, a lsd test, was then performed in order to compare osteoblast indicators between one group and another (table 1). the figure 1. the histologic features of osteoblasts in each research group. note: k (negative control), p1 (group with the administration of calcium powder derived from anadara granosa shells), p2 (group with the administration of ha powder derived from anadara granosa shells and 10% sardinella longiceps fish oil), p3 (group with the administration of ha powder derived from anadara granosa shells and oil 20% sardinella longiceps fish oil), and p4 (group with the administration of ha powder derived from anadara granosa shells and 30% sardinella longiceps fish oil). yellow arrows indicate the presence of osteoblasts, while osteoclasts are indicated by green arrows. figure 2. the average number of osteoblasts and osteoclasts in each research group (n=5). osteoblast osteoclast k p1 p3p2 p4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 141141hermanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 138–143 results of this lsd assay showed that there were significant differences between one group and another (p<0.05). discussion anadara granosa shells combined with sardinella longiceps gel at concentrations of 10% and 30% did not effectively induce osteoblast proliferation, although at these concentrations there was a tendency to increase osteoblast proliferation.20 therefore, this research used sardinella longiceps fish oil of 10% and 30% concentrations as a reference, while also employing the gel form to improve the attachment of the test materials to bone defects. the animal subjects used in this research were wistar rats since they not only possessed certain characteristics relatively similar to those of humans but also demonstrated similarities to the physiological aspects of human metabolism.21 this research specifically used wistar rat femurs since they possess a trabecular bone formation that is well developed in the cortical bone. this is identical to the alveolar bone in wistar rats, allowing it to be used as a model to study the regeneration of the jawbone.22 furthermore, male wistar rats were selected as samples for this research based on the consideration of their lacking or having relatively little estrogen, as well as their enjoying more stable hormonal conditions. stress levels in females are also known to be higher than those of males.23 besides, the effects of estrogen in suppressing osteoclastic activity are known to occur indirectly through their action on osteoblastic receptors. one of the cytokines produced by osteoblasts, transfoming growth factor-β (tgf-β), can even be suppressed by estrogen. in fact, tgf-ß plays a role in osteoclast differentiation.24 the normal bone healing process begins with inflammation since the cells of first defense, such as leukocytes, lymphocytes, monocytes, and macrophages are activated. macrophages are phagocytic cells produced in bone marrow that play an important role in inflammation, while they also remove cytokines consisting of proinflammatory, anti-inflammatory, and growth factors. the macrophages will then trigger the secretion of proinflammatory cytokines, such as tumor necrosis factor (tnf), interleukin-1 (il-1), and interleukin-6 (il-6) as inflammatory mediators to strengthen the immune response and increase metabolic processes.25 the presence of proinflammatory cytokines will, as a result, increase the production of preosteoclasts so that receptor activator of nuclear factor kappa (rank) are also widely produced. rank will subsequently bind to receptor activators of nuclear factor kappa ligand (rankl), thus inducing osteoclasts to increase bone damage.26 the proliferative or reparative phase begins when the inflammatory phase releases the cytokines and growth factors, resulting in the proliferation of fibroblasts to form extracellular matrix and calcium salts through an attachment, thus forming a woven bone.27 this can be seen in group k, a group with normal bone healing process, in which the osteoblasts remained visible, although without any treatment. osteoblasts are known not only to take various forms, from cuboid to cylindrical within the basophil cytoplasm, but also to be visible around the osteoidous layer where new bone is formed. meanwhile, the osteoclasts are known to have very large branched shapes with multiple cores.28 in group k, defects were not clinically closed since the process of bone formation was undetected. on the other hand, group p1 was a group given bone grafts derived from anadara granosa shells containing calcium carbonate. this material was expected to accelerate the bone healing process since calcium carbonate serves as a skeleton in bone formation, improves the wound healing process and acts as a mineral reservoir that helps in new bone formation.29 bone grafts derived from anadara granosa shells is beneficial in the treatment of bone defects.29 the structure of anadara granosa shells is generally similar to cancellous bone and demonstrates bone-like mechanical properties. the shells are also known to contain high concentrations of calcium carbonate which has biocompatible, osteoconductive, and biodegrable properties. moreover, the shells act as an adequate carrier for growth factors and enable cell attachment, cell growth, cell spread, and cell differentiation.30 however, anadara granosa shell powder can trigger an inflammatory response and increase the number of osteoclasts. therefore, group p1 had a higher number of osteoblasts compared to that of group k (table 1). nevertheless, the number of osteoclasts in group p1 tended to be descriptively higher than in group k, although there was no statistically significant difference (p>0.05). group p1 was treated only with bone graft derived from shells in the form of a brittle powder. this made it rather difficult for bone to be formed based on the required table 1. results of the post-hoc lsd test for osteoblast average mean difference (i-j) p valvue group (i) group (j) k p1 -2.3400* .048 p2 -11.2800* .000 p3 -16.2000* .000 p4 -26.5400* .000 p1 p2 -8.9400* .000 p3 -13.8600* .000 p4 -24.2000* .000 p2 p3 -4.9200* .000 p4 -15.2600* .000 p3 p4 -10.3400* .000 note: this table shows the comparison of the mean number of osteoblasts between groups. the average number of osteoblasts in group p> group k. the higher the concentration of group p, the greater the average number of osteoblasts. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 142 hermanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 138–143 substituting graft material. the powder used for the graft on the damaged bone usually suffers from weakness that does not promote stable grown as a graft.31 furthermore, groups p2, p3, and p4 were given bone grafts at concentrations of 10%, 20% and 30%, derived from a combination of anadara granosa shells containing calcium carbonate and sardinella longiceps gel containing omega-3. anadara granosa shells, can be selected as bone replacement biomaterials since they are known to contain calcium carbonate compounds.31 caco3 crystals derived from shellfish can facilitate osteoblast proliferation, differentiation and adhesion. the osteogenic activity of alkali phosphatase can also be enhanced by caco3 crystals. increased osteogenic activity of alkali phosphatase can then increase the differentiation of mesenchymal cells.9 sardinella longiceps fish oil contains 12.5% n-3 pufas. n-3 pufas consist of epa and dha and are capable of producing resolvin that can serve as an anti-inflammatory mediator.32 resolvin promotes the elimination of inflammation by creating a macrophage 2 (m2) phenotype which releases high levels of il-10 inhibiting tnfα, il-6, and il-1.32 these proinflammatory cytokines then trigger the formation of rankl in osteoblasts that can bind to rank in the pre-osteoclast to differentiate into osteoclasts.33 as proinflammatory cytokines decrease, the number of osteoclasts is also expected to decline, while that of osteoblasts is anticipated to increase. proinflammatory cytokines can directly stimulate osteoblast apoptosis and its precursors, or do so indirectly by stimulating fas expression of potential proapoptotic mediators.33 the bone graft used in this research was derived from a combination of anadara granosa shells and sardinella longiceps gel of three different concentrations in order to determine the most effective in the bone healing process. although within the investigation reported here the number of osteoclasts in all research groups was largely similar, the results of the oneway anova test highlighted a significant difference in the number of osteoblasts. this suggests that osteoclasts might be used as the only indicator. therefore, the osteoblastosteoclast ratio needs to be monitored during the healing process since it is considered an appropriate indicator of healing or bone formation.34 from a clinical perspective, in groups p2, p3, and p4 the defects were effectively, although not optimally, closed. closure defects were due to the administration of bone grafts derived from the combination of anadara granosa shells and sardinella longiceps fish oil gel. effective material attachment to the defects was usually expected to accelerate the bone healing process.35 thus, the provision of bone graft derived from the combination of anadara granosa shells and sardinella longiceps gel in this research was expected to accelerate the healing process since it could serve as a scaffold for new bone formation.35 the stimulation of bone grafts was then expected to improve the cellular biology activity by analyzing the osteoclast ratio (figure 2). during the bone healing process, bone graft usually stimulates and triggers osteoblast proliferation, before migrating to the defect site.33 it can be concluded that bone grafts derived from the combination of anadara granosa shells and sardinella longiceps gel can successfully generate osteoblasts and osteoclasts indicating a more effective healing process. the most effective concentration of sardinella longiceps gel used was 30%. references 1. hamzah z, kar tikasari n. pencabutan gigi yang ir rasional mempercepat penurunan struktur anatomis dan fungsi tulang alveolar. stomatognatic. 2015; 12(2): 61–6. 2. mezzomo la, shinkai rs, mardas n, donos n. alveolar ridge preservation after dental extraction and before implant placement: a literature review. rev odonto ciência. 2011; 26(1): 77–83. 3. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125-7. 4. ardhiyanto hb. peran hidroksiapatit sebagai bone graft dalam proses penyembuhan tulang. stomatognatic. 2011; 8(2): 118–21. 5. amini ar, laurencin ct, nukavarapu sp. bone tissue engineering: recent advances and challenges. crit rev biomed eng. 2012; 40(5): 363–408. 6. haslaniza h, maskat my, wan aida wm, mamot s, saadiah i. optimization of enzymatic hydrolysis of cockle (anadara granosa) meat wash water precipitate for the development of seafood flavor. int food res j. 2013; 20(6): 3053–9. 7. arita s, adipati as, sari dp. pembuatan katalis heterogen dari cangkang kerang darah (anadara granosa) dan diaplikasikan pada reaksi transesterifikasi dari crude palm oil. j teknik kimia. 2014; 20(3): 31–7. 8. saryati, giat s, handayani a, supardi, untoro p, sugeng b. hidroksiapatit berpori dari kulit kerang. j sains materi indonesia. 2012; edisi khus(desember): 31–5. 9. kamba as, zakaria zab. osteoblasts growth behaviour on biobased calcium carbonate aragonite nanocrystal. biomed res int. 2014; 2014: 215097. 10. mu r phy w l , si m mons ca, k a igler d, mooney dj. bone regeneration via a mineral substrate and induced angiogenesis. j dent res. 2004; 83(3): 204–10. 11. pike ih, jackson a. fish oil: production and use now and in the future. lipid technol. 2010; 22(3): 59–61. 12. udari ahgs, wickramasinghe i, attygalle mve. effects of cooking on omega 3 content of sardinella longiceps emphasizing an innovative method to elevate omega 3 intake. int j innov res technol. 2015; 2(3): 68–75. 13. wang w, zhu j, lyu f, panigrahy d, ferrara kw, hammock b, zhang g. ω-3 polyunsaturated fatty acids-derived lipid metabolites on angiogenesis, inflammation and cancer. prostaglandins other lipid mediat. 2014; 113–115: 13–20. 14. mcmahon ms. beneficial effect of omega-3 fatty acids on bone metabolism. orthopedics. 2012; 35(9): 735–6. 15. li y, chen s-k, li l, qin l, wang x-l, lai y-x. bone defect animal models for testing efficacy of bone substitute biomaterials. j orthop transl. 2015; 3(3): 95–104. 16. rokn ar, khodadoostan ma, reza rasouli ghahroudi aa, motahhary p, kharrazi fard mj, bruyn h de, afzalifar r, soolar e, soolari a. bone formation with two types of grafting materials: a histologic and histomorphometric study. open dent j. 2011; 5: 96–104. 17. kopschina mi, marinowic dr, klein cp, araujo ca, freitas ta, hoff g, da silva jb. effect of bone marrow mononuclear cells plus platelet-rich plasma in femoral bone repair model in rats. brazilian j vet res anim sci. 2012; 49(3): 179–84. 18. schmidlin pr, nicholls f, kruse a, zwahlen ra, weber fe. evaluation of moldable, in situ hardening calcium phosphate bone graft substitutes. clin oral implants res. 2013; 24(2): 149–57. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 143143hermanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 138–143 19. ridwan e. etika pemanfaatan hewan percobaan dalam penelitian kesehatan. j indones med assos. 2013; 63(3): 112–6. 20. sari rp, wahjuningsih e, soeweondo ik. modulation of fgf2 after topical application of stichopus hermanii gel on traumatic ulcer in wistar rats. dent j (maj ked gigi). 2014; 47(3): 126–9. 21. sihombing i, wangko s, kalangi sjr. peran estrogen pada remodeling tulang. j biomedik. 2012; 4(3): s18-28. 22. bronner f, farach-carson mc, rubin je. bone resorption. london: springer; 2005. p. 17. 23. sultani m, stringer am, bowen jm, gibson rj. anti-inflammatory cytokines: important immunoregulatory factors contributing to chemotherapy-induced gastrointestinal mucositis. chemother res pract. 2012; 2012: 490804. 24. sfeir c, ho l, doll ba, azari k, hollinger jo. fracture repair. in: lieberman jr, friedlaender ge, editors. bone regeneration and repair : biology and clinical applications. totowa: humana press; 2005. p. 21–30. 25. mao t, kamakshi v. bone grafts and bone substitutes. int j pharm pharm sci. 2014; 6(2): 88–91. 26. mescher al. histologi dasar junqueira: teks & atlas. 12th ed. jakarta: egc; 2012. p. 118-128. 27. bharatham h, zakaria mzab, perimal ek, yusof lm, hamid m. mineral and physiochemical evaluation of cockle shell (anadara granosa) and other selected molluscan shell as potential biomaterials. sains malaysiana. 2014; 43(7): 1023–9. 28. suhendi a, nurca hya nti, muhtadi, sutr isna em. a ktivitas antihiperurisemia ekstrak air jinten hitam (coleus ambonicus lour) pada mencit jantan galur balb-c dan standardisasinya. majalah farmasi indonesia. 2011; 22(2): 77–84. 29. hazmi aja, zuki abz, noordin mm, jalila a, norimah y. mineral composition of the cockle (anadara granosa) shells of west coast of peninsular malaysia and it’s potential as biomaterial for use in bone repair. j anim vet adv. 2007; 6(5): 591–4. 30. warastuti y, abbas b. sintesis dan karakterisasi pasta injectable bone substitute iradiasi berbasis hidroksiapatit. j ilmiah aplikasi isotop dan radiasi. 2011; 7(2): 73–95. 31. asakura t, koyanagi r, nishiyama n, kuboyama n, kiba h, abiko y. bone regeneration on the epicondyle of the femur supported by silk fibroin-based scaffold: a model system for dental surgery. j insect biotechnol sericology. 2011; 80: 25–30. 32. i nda hya n i de. minya k i ka n l emur u (sa rdinella longicep) menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (fish oil of lemuru (sardinella longicep) reduced the osteoblast apoptosis in wistar rat alveolar bone). dent j (maj ked gigi). 2013; 46(4): 185–8. 33. asami a, nakamura m, takeuchi m, nakayama a, nakamura h, yoshida t, nagasawa s, hiraoka by, ito m, udagawa n, miyazawa h. effects of heat treatment of hydroxyapatite on osteoblast differentiation. j hard tissue biol. 2008; 17(2): 37–46. 34. teti a. mechanisms of osteoclast-dependent bone formation. bonekey rep. 2013; 2: 1–6. 35. sari rp, hermanto e, divilia d, candra i, kuncoro w, liswanti t. effects of anadara granosa shell combined with sardinella longiceps oil on oesteoblast proliferation in bone defect healing process. dent j (maj ked gigi). 2016; 49(1): 27–31. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p138-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p138-143 173 vol. 44. no. 4 december 2011 research report in vivo characterization of polymer based dental cements widiyanti p1, 2 and siswanto1 1department of physics, faculty of science and technology, airlangga university 2institute of tropical disease, airlangga university surabaya indonesia abstract background: in vivo studies investigating the characterization of dental cements have been demonstrated. as few in vitro studies on this cement system have been performed. previous researches in dental material has been standardized dental cement which fulfilled the physical and mechanical characteristic such as shear strength but were on in vitro condition, the animal model and clinical study of dental cement from laboratory has not been done yet. this research examined physical and mechanical characteristic in vivo using rabbit by making the caries (class iii) in anterior teeth especially in mesial or distal incisive, fulfilled the cavity by dental cement and analyzed the compressive strength, tensile strength, and microstructure using scanning electron microscope (sem). purpose: this study is aimed to describe the in vivo characterization of dental cements based on polymer (zinc phosphate cement, polycarboxylate, glass ionomer cement and zinc oxide eugenol). methods: first, preparation was done on animal model’s teeth (6 rabbits, male, 5 months old). the cavity was made which involved the dentin. then the cavity was filled with dental cement. after the filling procedure, the animal model should be kept until 21 days and than the compressive test, tensile test and microstructure was characterized. compressive test and tensile test was analyzed using samples from extracted tooth and was measured with autograph. the microstructure test was measured using sem. results: the best compressive strength value was belongs to zinc phosphate cement which was 101.888 mpa and the best tensile strength value was belongs to glass ionomer cement which was 6.555 mpa. conclusion: in conclusion, comparing with 3 others type of dental cements which are zinc phosphate, polycarboxylate and glass ionomer cement, zinc oxide eugenol cement has the worst for both physical and mechanical properties. key words: zinc phosphate cement, polycarboxylate cement, glass ionomer cement, zinc oxide eugenol in vivo, characterization abstrak latar belakang: studi in vivo meneliti karakterisasi secara in vivo dari semen gigi. beberapa studi in vitro di bidang ini telah dilakukan. beberapa riset di bidang material gigi telah menghasilkan semen gigi yang memenuhi standart sifat fisik dan mekanik seperti regangan dan kekuatan secara in vitro, sedangkan uji in vivo dan uji klinis dari semen gigi dari laboratorium belum dilakukan. penelitian ini menguji karakteristik fisik dan mekanik semen gigi menggunakan hewan coba kelinci dengan membuat karies kelas iii di gigi anterior terutama di permukaan mesial atau distal insisif, mengisi kavitas dengan semen gigi dan menganalisa kekuatan tekan, kekuatan tarik dan struktur mikronya dengan menggunakan scanning electron microscope (sem). tujuan: studi ini bertujuan memberikan gambaran karakterisasi in vivo semen gigi berbahan dasar polimer (semen seng fosfat, polikarboksilat, ionomer kaca dan seng oksida eugenol). metode: pertama, kami melakukan preparasi pada gigi hewan coba (6 kelinci, jantan, usia 5 bulan). kemudian kita membuat kavitas yang melibatkan dentin. lalu kami menumpat kavitas dengan semen gigi. setelah prosedur penumpatan, hewan coba dipelihara selama 21 hari dan dikarakterisasi kekuatan tekan, kekuatan tarik dan struktur mikronya. kekuatan tekan dan kekuatan tarik dianalisa dari sampel uji gigi hewan coba yang diekstraksi dan diukur dengan autograf. struktur mikronya diuji dengan sem. hasil: hasil nilai kuat tekan terbaik diperoleh oleh semen seng fosfat (zinc phosphate cement) sebesar 101,888 mpa dan nilai kuat tarik semen gigi terbaik adalah semen gelas ionomer (glass ionomer cement) sebesar 6,555 mpa. kesimpulan: �apat disimpulkan, dari ketiga jenis bahan semen yaitu seng fosfat, polikarboksilat, dan ionomer kaca, yang mempunyai sifat fisik dan mekanikal terburuk adalah semen ionomer kaca. 174 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 1173–176 kata kunci: semen seng fosfat, polikarboksilat, ionomer kaca, seng oksida eugenol, karakteristik secara in vivo correspondence: widiyanti p, c/o: fakultas sains dan teknologi universitas airlangga. jl. mulyorejo surabaya, indonesia. e-mail: drwidiyanti@yahoo.com introduction technology of dental material has been started to develop since 50 years ago. nowadays, dentists have many choice to making restore caries lesion, fracture and missing teeth. one of the alternative sources of dental material is based on polymer. scientists keep on trying to develop polymer in order to get closer to characteristic and the performance of real tooth.1 polymer is the long chain-molecule which has many unit.2 polymer has been used in the domain of industry and medicine. one of the examples of medical usage of polymer is their role as dental cement. the type of dental cements are zinc phosphate , polycarboxylate, glass ionomer and zinc oxide eugenol cement.3 dental cement must be elastic (low strength material). this cement is made by mixing the powder with the liquid. the composition of this cement is varied in chemical composition, characteristic and function usage.6 the need of dental cement has been fulfilled by the overseas product but there are many candidate of dental material in indonesia. some previous researches in dental material resulted in standardized dental cements which fulfilled the physics and mechanical characteristic such as shear and strength but they still play in the in vitro condition, the animal model and clinical study of dental cement has not been done. the phenomena inspiring us to perform the clinical research (in vivo) which examined physics and mechanical characteristic of dental cement based on polimer using rabbit. the teeth has been perforated by the diamond bur to perform caries (class iii) in anterior teeth especially in mesial or distal incisive or caninus. then we analyze the compressive strength, tensile strength, and micro structure using sem. the result of this research would be beneficial as base theory for the development of dental material. materials and methods animal models (bunolagus monticularis) were prepared (6 rabbit, male, 5 month). they should be anesthesized based on the age and weight. the cavity preparation were done using round bur, fissure bur dan tappered bur. the preparation involved dentin. then the cavity were were cleaned by cotton and water spray. the cavity and the surrounding area should be isolated using cotton roll to prevent saliva contamination. the filling material should be prepared in the glass and mixed using spatula cement. the filling material should be place inside the cavity using plastic filling instrument. after 1-2 minutes, it was pressed using amalgam stopper and carved. the first rabbit was filled with zinc phosphate cement (group a). second rabbit was filled by polycarboxylate cement (group b). the third rabbit was filled with glass ionomer cement (group c) and the fourth rabbit was filled with zinc oxide and eugenol cement (group d). the fifth rabbit was filled zinc phosphate cement and the left cavity was filled with polycarboxylate cement. the sixth rabbit, the right cavity was filled by glass ionomer cement and left cavity should be filled by zinc oxide and eugenol cement. the whole sample are 12 samples. after the filling procedure, the animal model should be kept until 21 days and then the compressive test, tensile test and microtructure were characterized. results the result of compressive strength of zinc phosphate cement is 101,888 mpa and the tensile strength is 5,777 mpa. whether for polycarboxylate cement, the compressive strength is 56,555 mpa and the tensile strength 6,111 mpa. glass ionomer cement has the compressive strength value is 70,777 mpa and tensile strength is 6,5555 mpa. zinc oxide eugenol has compressive strength value around 46,111 mpa and tensile strength is 3,111 mpa. the detail data could be seen in the table 1. table 1. the compressive and tensile strength of various cement no. sample compressive strength (mpa) tensile strength (mpa) 1. a 101.888 5.777 2. b 56.555 6.111 3. c 70.777 6.555 4. d 46.111 3.111 175widiyanti and siswanto: in vivo characterization of polymer based dental cements the microstructure of tooth structure and dental cement are showed in figure 1. discussion the best compressive strength is 101.888 mpa for zinc phosphate cement and the best tensile strength is 6.555 mpa for glass ionomer cement. if done with the right manipulation, the compressive strength of zinc phosphate cement is 104 mpa and the tensile strength is 5.5 mpa.7 the compressive and the tensile strength is varied according to the ratio of powder and liquid. to increase the strength, much powder showed be added than it should be. the decrease of powder ratio could yield weak cement. the lost or the addition of water would decrease tensile and compressive strength of cement. zinc phosphate cement and glass ionomer cement could be easily ossified. after ossification, the excess cement could be throw out by gouging out the cement sheer off the edge of restoration. because of that, the edge of restoration should be prevented from early contact with the liquid.8 the compressive strength of polycarboxylate cement is lower than zinc phosphate cement, but the tensile strength is little higher than zinc phosphate cement. polycarboxylate cement is not as fragile as zinc phosphate cement so it is more difficult to loosing upon the excessive cement after the ossification. mechanical characteristic of zinc oxide and eugenol cement is lower than other cements. this type of cement is difficult to manipulate inside the mouth. the thickness of the layer is higher and the excessive cement is difficult to discard.9 figure 1. microstructure of tooth and dental cement examined by sem: for group a) zinc phosphate cement, b) policarboxylate cement, c) glass ionomer cement, and d) zinc oxide and eugenol cement. the difference of the compressive and tensile strength is caused by the mixing speed of powder and liquid, mixing plate and the temperature of the stirring tool. the fusion speed of powder and liquid could influence the hardness of dental cement because powder is mixed with liquid gradually in small sum would increase working time, the hardness and decrease the color and it give opportunity to add much powder in the mixture. mixing plate and temperature of stirring tool influence mechanical characteristic of dental cement. high temperature of stirring tool could accelerate the hardening of dental cement. the other site of the temperature of stirring tool is lower, then the hardening reaction could take longer time. the wrong mixing of powder and liquid could result in crack of the dental cement and can make the measurement of mechanical characteristic difficult. microstructure of zinc phosphate cement is showed that the dental cement could not fuse with the tooth correctly. when the powder is mixed with the liquid, phosphoric acid is contacted with the surface of particle and release zinc ion to the liquid. aluminium, which form the adhesion with phosphoric acid, then reacted with zinc produce zinc aluminophosphate gel in the surface of particle. the set cement is the main core structure including unreacted zinc oxide particle, covered with dense matrix which are not from aluminophosphate zinc. water plays important role in the acid-base reaction, so the composition of liquid should be arranged to make sure that the reaction is consistent. the alteration of composition and the speed of reaction could happen because of water evaporation of the liquid. this means, the composition alteration could influence the reaction. a b c d c d 176 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 1173–176 microstructure of polycarboxylate cement that the cement have strong bonding with the tooth. the hardening reaction of dental cement involved dissolving of surface particle of acid and zinc, magnesium, and tin release which fused to polymer chain through carboxyl cluster. ions are reacted with carboxyl cluster and surrounding poly acid chain to form salt from cross binding when the cement is set. the set cement including matrix gel without proper form including many unreacted scattered particles. microstructure of glass ionomer cement consist of unreacted powder particles. when the mixed powder and liquid in paste form, the surface of glass particle would dissolve in the acid. calcium ion, aluminium, natrium and fluorine are released to the based water medium. polyacrilate acid chain would form cross binding with calcium ion and dense mass. before 24 hours, it forms the new phase where aluminium ion bind in the cement mixture. it would make the cement rigid. natrium ion and fluorin are not interfering in the cross binding of cement. some natrium ion could replace hydrogen ion from carboxylic cluster, and the rest will join with fluorin ion to form fluoride natrium which is scattered inside the set cement. in the maturation process, cross binding phase is hydrated by water. unreacted part with glass particles will be covered by silica gel which formed during the release of surface cation. the hardening set, including the group of unreacted powder particle, surrounded by silica gel in the amorphous matrix of hydrate calcium and aluminium salt. microstructure of zinc oxide and eugenol cement showed the very hard. in proper condition, the reaction of zinc oxide and eugenol produce hard relative mass. mechanism of hardening zinc oxide eugenol including zinc oxide hydrolysis and reaction between hydroxide zinc and eugenol to form the cluster. setting reaction is accelerated by the presence of dehydrate acetate zinc, which is more dissolved than hydroxide zinc. high temperature could accelerate hardening reaction. the important characteristic of dental cement is the endurance of solubility and disintegration inside oral cavity.10 cement is regularly in contact with many acid produced by microorganism and mastication and swallowing. some acids has been brought by some food and beverages. ph and temperature inside the oral cavity is always changed. so, there would be no cement which could fulfill the ideal characteristic. one type of dental cement might suitable for certain condition than the others. every condition should be valued based on the environment, biological and mechanical factor. in conclusion, comparing with three others types of dental cements which are zinc phosphate, polycarboxylate and glass ionomer cement, zinc oxide eugenol cement has the worst for both physical and mechanical properties. references 1. lutviyah. pembuatan semen gigi zinc polikarboksilat dari bahan baku zinc oksida dan asam poliakrilat. skripsi. surabaya: fst unair; 2008. p. 31–8. 2. combe e, burke fjt, bernard dw. dental biomaterials. 1st ed. london: springer; 1999. p. 256–8. 3. noort rv. introduction to dental material. 3rd ed. london, china: mosby elsevier; 2007. p. 127–43. 4. arifudin af. pembuatan semen gigi seng fosfat berbahan dasar. pembuatan semen gigi seng fosfat berbahan dasar pembuatan semen gigi seng fosfat berbahan dasar seng oksida dan asam fosfat. sripsi. surabaya: fst unair; 2008.. sripsi. surabaya: fst unair; 2008. p. 28–30. 5. nugroho. pembuatan semen tambal gigi dengan bahan dasar polimer. bandung: lipi; 2007. p. 15–7. 6. van vlack lh. 1989. elemen-elemen ilmu dan rekayasa material. edisi ke-6. djaprie s, editor. jakarta: erlangga; 2004. p. 24–6. 7. anusavice kj. phillips' science of dental materials. 11th ed. philadelphia: wb saunders; 2011. p. 300–10. 8. powers jm, sakaguchi rl. craig’s restorative dental materials. 12th ed. london: mosby elsevier; 2006. p. 256. 9. hatrick cd, eakle ws, bird wf. dental materials: clinical application for dental assistant and dental hygienist. 2nd ed. philadelphia: saunders; 2010. p. 156. 10. atai z, atai m. side effects and complications of dental materials on oral cavity. am j of appl sci 2007; 4(11): 946–9. 213213 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 213–216 characterization of streptococcus sanguis molecular receptors for streptococcus mutans binding molecules deby kania tri putri,1 indah listiana kriswandini,2 and muhammad luthfi2 1department of oral biology, faculty of dentistry, universitas lambung mangkurat, banjarmasin indonesia 2department of oral biology, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: dental caries is a major problem in oral cavity. if dental caries causes cavity, the structure of dental hard tissue will not be reversible because of damage in the structure of the hard tissue. the early pathogenesis mechanism of dental caries is an adhesion interaction between cariogenic streptococcus mutans microorganisms and tooth surface pellicles. the attachment involves a specific molecular component interaction between the bacterial complement molecules and the surface of the host. streptococcus sanguis as a dominant ecology at the beginning of bacterial plaque aggregation will colonize the tooth surface earlier than s. mutans. the surface of bacterial cells can express some adesin. the bacteria also can express receptors for adhesins of other bacteria. specific receptors for adhesions of s. mutans bacteria are not only found in the pellicles, but also present in pioneer bacteria, such as s. sanguis. adhesion between those bacteria is called as coagregation. purpose: this study aimed to analyze the characterization of streptococcus sanguis molecular receptors for streptococcus mutans binding molecules. method: this study used a sonication method for protein isolation of s. mutans and s. sanguis bacterial biofilms, as well as electrophoresis method using 12 % sds-page gel and western blot analysis. result: results of the protein profile analysis of s. mutans biofilms using 12% sds-page showed that there were 17 bands, each of which molecular weights was 212, 140, 81, 65, 61, 48, 45, 44, 40, 39, 33 , 25, 23, 19, 17, 12, and 11 kda. on the other hand, results of the protein profile analysis of s. sanguis biofilms using 12% sds-page showed that there were 15 bands, each of which molecular weight was 130, 85, 65, 61, 48, 46, 40, 37, 29, 25, 23, 21, 17, 15, and 12 kda. and, results of the analysis of s. sanguis receptor molecules using western blot showed that there were three bands, each of which molecular weight was 130, 85, and 40 kda. conclusion: s. sanguis bacteria have specific receptor molecules for s. mutans bacteria with the molecular weight of 130, 85, and 40 kda. keywords: receptor; adherence; coagregation; biofilms correspondence: indah listiana kriswandini, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: indahkrisfkg@gmail.com introduction dental caries is a multifactorial phenomenon. this disease is a major problem in oral cavity. if dental caries cause cavity, the structure of dental hard tissue will not be reversible because of damage in the structure of the hard tissue.1 according to riskesdas 2013, the prevalence of active caries in the population of indonesia increased compared to the prevalence of active caries in 2007, ie from 43.4% (2007) to 53.2% (2013). to decrease the caries index as referenced by who guidelines, prevention of all aspects triggering dental caries is necessary.2 streptococcus mutans (s. mutans) is a normal flora of the mouth. however, s. mutans can frequently become pathogenic in acidic environment. cariogenic potential of these bacteria is manifested by its ability to ferment various carbohydrates, produce large amounts of acid, and to participate in the formation of tooth plaque.1 s. mutans as the main oral cariogenic bacteria, moreover, excrete glucosyltransferase (gtf) enzyme, which is useful to synthesize extracellular polysaccharides (glucans) from sucrose. glucan is an important virulence factor since it helps the attachment of bacteria to the pellicles of teeth, and also contributes to the integrity of the structure.3,4 biofilm dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p213-216 214 putri, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 213–216 is a community of microbes attached to a solid surface, embedded in a matrix of extracellular polymeric substance produced by microorganisms.5,6 biofilm community is a complex and dynamic structure that has accumulated over several oral bacterial colonizations.7 the early mechanism of pathogenesis of dental caries is preceded by an adhesion interaction between cariogenic microorganisms and salivary glycoprotein components (pellicles) of the tooth surface. the attachment then involves a specific molecular component interaction between bacterial complement molecules and the surface of the host, then the surface of bacterial cells express some adhesins. the bacteria also express receptors for other bacterial adhesins.8 in the initial stage of dental plaque formation, furthermore, tooth surface is coated with pellicles, followed with adhesion of several bacterial species (pioneer colonizers), such as streptococcus sanguis (s. sanguis).6,9 s. sanguis will provide a place of attachment for other bacteria (secondary colonizers) to stick to the pellicles and form biofilms.6 similarly, a research conducted by okahashi explains that pil b and pil c proteins on the fimbriae of s. sanguis are capable of binding salivary α-amylase to the tooth surface. this indicates a specific attachment by the pili of s. sanguis facilitating other organisms to adapt well in the oral cavity.12 alternative attempts to suppress the incidence of dental caries are by diagnosing the risk of dental caries early and using inhibitor compounds for proteins that play a role in the formation of biofilm. proteomic study involving determination of protein profiles composing biofilms and s. mutants adhesin molecules is a way to identify biomarker candidates of dental caries risk in humans. therefore, this research aimed to examine the molecular components of s. sanguis bacterial receptors pioneer bacteria involved in the initial attachment of s. mutans biofilm formation, triggering dental caries. material and method this research was a laboratory exploratory observational research. the bacteria used in this research were s. mutans from the central health laboratory surabaya, and isolates of s. sanguis from the laboratory of microbiology, faculty of medicine, universitas brawijaya, malang. experimental animals used in this research, moreover, were new zealand rabbits aged 3 months and weighed 1.5-2 kg. meanwhile, tools required for this research were micropipette, incubators, digital scales analytical balance (ohaus), shaker incubator (tungtec instruments, laminar flow cabinets kottermann 8580), electrophoresis tool (bio rad, usa) and western blot device (biorad, usa). this research was conducted in several stages, namely identification of bacteria, a tests on s. mutans and s. sanguis bacterial biofilm formations, isolation of bacterial protein biofilms, treatment in experimental animals, manufacture of s. mutans anti-biofilms, electrophoresis stage, and western blotting stage. identification of each bacteria was performed using tripticase yeast cysteine (tyc) media, then incubated at a temperature of 370 c for 24 hours. a test of bacterial biofilm formation was conducted using brain heart infusion broth (bhib) media, then incubated at 370 c for 24 hours in a candle jar. bhib as the bacterial culture media were put into vacutainer tubes, and then centrifuged for 15 minutes to separate the bacterial pellets from the medium. the supernatant was discarded. part of the s. mutans bacterial sediment was added with 2 ml of 0.05% nog, and then centrifuged at a speed of 12,000 rpm for 30 minutes. isolation of protein biofilms in each group was carried out using sonication method with a power of 7 x 30 sec at a frequency of 40hz in the tem buffer (10 mm of tris-hcl [ph 6.8], 1 mm of edta, 5 mg of mgso4).13,15 they then were stored at a temperature of -200. as a result, they were ready to be used as samples for analysis of crude protein of s. mutans and s. sanguis biofilms using 12% sodium dodecyl sulfate poly acrilamida gel electrophoresis (sds-page). the analysis of the crude protein of s. mutans and s. sanguis biofilms was performed using 12% sds-page (2.5 ml of acrylamide, 1.2 ml of tris hcl (ph 8.8), 1.2 ml of 0.5% sds, 1.1 ml of distilled water, 50 ml of temed, and 30 ml of 10% aps). staining then was conducted using silver stain and standard molecules of sigma low range marker. in the next stage, s. mutans anti-biofilms was made of protein derived from s. mutans biofilms mixed with adjuvant materials at a ratio 1 : 1. after that, they were vortexed to be homogeneous for 30 minutes, and then injected in the sub-cutaneous area of those rabbits to facilitate the absorption of the antigens. adjuvant materials used for the initial vaccination were complete freund’s adjuvant, while incomplete freund’s adjuvant was used as booster. booster was administrated until the 35th day of vaccination, and then the polyclonal antibodies were harvested.15 s. sanguis receptor molecules were analyzed by running the s. sanguis protein biofilms, and then transferring proteins in the nc membrane. they were incubated together with s. mutans biofilm suspensions and s. mutans polyclonal antibodies, and anti rabbit ig g as secondary antibodies. blotting process then was performed to generate bands, converted with bands on broad marker proteins from bio-rad. results analysis results of the protein profiles of s. mutans biofilms revealed seventeen bands, each of which molecular weight was 212, 140, 81, 65, 61, 48, 45, 44, 40, 39, 33, 25, 23, 19, 17, 12, and 11 kda. on the other hand, analysis results of the protein profiles of s. sanguis biofilms indicated fifteen bands, each of which molecular weight dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p213-216 215215putri, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 213–216 was 130, 85, 65, 61, 48, 46, 40, 37, 29, 25, 23, 21, 17, 15, and 12 kda. analysis results of s. sanguis receptor molecules using western blot showed three bands, each of which molecular weight was 130, 85, and 40 kda. discussion in the stage of the isolation of bacterial protein biofilms using the sonication method, tem buffer (10 mm of trishcl [ph 6.8], 1 mm of edta, and 5mm of mgso4) was added.13,14 this additional buffer aims to protect proteins from denaturation due to heat produced by ultrasonic sound vibrations.21 the analysis of the crude protein of s. mutans and s sanguis biofilms was performed to determine the protein profiles of s. mutans and s sanguis biofilms based on their molecular weight. the protein profiles of their biofilms depicted all the constituent proteins of s. mutans and s. sanguis biofilms. the protein profiles observed were molecular weight, existed protein bands, thick and thin protein bands, and total protein formed from the samples. the presence or absence of the bands at a certain migration distance indicates the presence or absence of migrated proteins that stop at such distances during electrophoresis process. the thickness of the bands can basically be divided into two, namely thick and thin bands. thick bands indicate high total protein or large protein concentration, while thin bands demonstrate low total protein.16,17 thick bands can be distinguished from the thin bands due to the number of molecules migrated. thick bands are formed from ixation of several bands. bands that have a greater ionic strength will migrate farther than the bands with small ionic strength.19 moreover, results of the analysis of the protein profile of s. mutans biofilms using 12% sds-page showed that there were 17 bands, each of which molecular weight was 212, 140, 81, 65.61, 48, 45, 44, 40, 39, 33, 25 , 23, 19, 17, 12, and 11 kda. according to kyle, there are 185,000 surface proteins that can be expressed by s. mutans serotype c. previous researches have established some s. mutans surface protein molecules that play a role in initial attachment process to other organisms on the tooth surface, which have multiple names or designations, namely pac molecules, antigen i/ ii, p1, sr and ms. antigen i/ ii in the cell walls of s. mutans has a molecular weight of 150 kda 215 kda.22 the molecular weights of other s. mutans protein molecules in this research were 81, 65, 61, 48, 45, 44, 40, 39, 33 , 25, 23, 19, 17, 12, and 11 kda. those protein molecules could be considered as fraction components of the fimbriae, as a degradation of other adhesins with greater molecular weights, or as expressions of other genes that have not known the role of proteins encoded. however, the protein molecular weights are not always in line with the protein molecular weights referenced since it can be affected by several factors, such as concentration of the gel, flow of supplied electricity, and effects of buffer, for instance, ph will affect the density of protein charge, consequently, affecting the level and direction of the movement.24 4 complete freund's adjuvant, while incomplete freund's adjuvant was used as booster. booster was administrated until the 35th day of vaccination, and then the polyclonal antibodies were harvested.15 afterwards, s. sanguis receptor molecules were analyzed by running the s. sanguis protein biofilms, and then transferring proteins in the nc membrane. next, they were incubated together with s. mutans biofilm suspensions and s. mutans polyclonal antibodies, and anti rabbit ig g as secondary antibodies. blotting process then was performed to generate bands, converted with bands on broad marker proteins from bio-rad. results analysis results of the protein profiles of s. mutans biofilms revealed seventeen bands, each of which molecular weight was 212, 140, 81, 65, 61, 48, 45, 44, 40, 39, 33, 25, 23, 19, 17, 12, and 11 kda. on the other hand, analysis results of the protein profiles of s. sanguis biofilms indicated fifteen bands, each of which molecular weight was 130, 85, 65, 61, 48, 46, 40, 37, 29, 25, 23, 21, 17, 15, and 12 kda. figure 1. bm protein profiles of s. mutans (1) and s. sanguis (2). m as a protein marker of 12% sdspage. analysis results of s. sanguis receptor molecules using western blot showed three bands, each of which molecular weight was 130, 85, and 40 kda. 260 10 15 25 35 40 50 7 0 100 140 40 kda 37 kda 23 kda 19 kda 1 2 m (kda) 11 12 17 19 23 25 33 39 40 244 245 48 65 61 81 140 212 12 15 23 25 29 48 61 85 130 1 m comment [hh8]: provide complete data along with the full name and location (city, province or state if usa/canada, and country) of the supplier such as a waterlase md dental laser (biolase technology inc., irvine, ca, usa) figure 1. bm protein profiles of s. mutans (1) and s. sanguis (2). m as a protein marker of 12% sds-page. 5 figure 2. bm receptor molecules of s. sanguis (2). m as a western blot marker of biorad. discussion in the stage of the isolation of bacterial protein biofilms using the sonication method, tem buffer (10 mm of tris-hcl [ph 6.8], 1 mm of edta, and 5mm of mgso4) was added.13,14 this additional buffer aims to protect proteins from denaturation due to heat produced by ultrasonic sound vibrations.21 next, the analysis of the crude protein of s. mutans and s sanguis biofilms was performed to determine the protein profiles of s. mutans and s sanguis biofilms based on their molecular weight. the protein profiles of their biofilms depicted all the constituent proteins of s. mutans and s. sanguis biofilms. the protein profiles observed were molecular weight, existed protein bands, thick and thin protein bands, and total protein formed from the samples. the presence or absence of the bands at a certain migration distance indicates the presence or absence of migrated proteins that stop at such distances during electrophoresis process. the thickness of the bands can basically be divided into two, namely thick and thin bands. thick bands indicate high total protein or large protein concentration, while thin bands demonstrate low total protein.16,17 thick bands can be distinguished from the thin bands due to the number of molecules migrated. thick bands are formed from ixation of several bands. bands that have a greater ionic strength will migrate farther than the bands with small ionic strength.19 12 kda 10 15 35 40 50 70 100 140 40 kda 85 kda 130 kda 260 figure 2. bm receptor molecules of s. sanguis (2). m as a western blot marker of biorad. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p213-216 216 putri, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 213–216 the addition of sodium dodecyl sulfate (sds) in the electrophoresis process, furthermore, serves as a electrophoresis buffer to make proteins become denatured, causing dissolved hydrophobic molecules, resulting in a negative charge on the entire structure of a protein by binding to the hydrophobic residues of each amino acid. as a result, protein molecules are separated based on their molecular weight only.18 on the other hand, western blot technique aims to determine the character of s. sanguis receptor molecules in recognizing s. mutans. this technique will detect a protein with a particular molecular weight as gene expression results on the nitrocellulose membrane by using a labeled antibody. in addition, the analysis results of the s. sanguis receptor molecules using western blot demonstrated that there were three bands, each of which molecular weight was 130, 85, and 40 kda. adhesin proteins in s. sanguis, according to some previous researches, are known as srta, fim a, abp a, abp b, pil b, and pil c. abp b in this research had a molecular weight of 85 kda. similarly, nikitkova argues that abp b has a molecular weight of 82 kda 87 kda.23 pil b and pil c, moreover, in this research had a molecular weight of 40 kda. like this research, pil b and pil c as proteins/ receptors/ adhesins in s. sanguis bacteria, according to the previous research, also have a molecular weight of 38 kda 45 kda.23 meanwhile, srta in this research had a molecular weight of 130 kda. similarly, in a previous research conducted by yamaguchi, srta molecules as s. sanguis surface antigens have molecular weights of 100 kda, 130 kda and 170 kda. yamaguchi states that srta involves in adhesion process to the tooth surface, dental restorative materials, and oral cavity epithelial cells.11 therefore, it can be said that the western blot results obtained in this research are in line with the previous researches. it can be concluded that s. mutans biofilms have an ability to recognize specific epitopes of the constituent proteins in s. sanguis biofilms. s. sanguis as pioneer bacteria on tooth surfaces have specific receptors against cariogenic s. mutans bacteria to facilitate s. mutans attach to the tooth surface receptors. references 1. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. policy and practice the global burden of oral diseases and risks to oral health. bulletin of the world health organization 2005; 83(05): 661–69. 2. sakti gm, rustandi k, putri np. rencana aksi nasional pelayanan kesehatan gigi dan mulut tahun 2015 -2019. available at: http:// perpustakaan.depkes.go.id:8180/handle/123456789/3294. accessed 20 januari, 2017. 3. koo h, xiao j, klein mi, jeon jg. exopolysaccharides produced by streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms. journal of bacteriology 2010; 192(12): 3024–32. 4. x iao j, ko o h. st r uct u r a l orga n i zat ion a nd dy na m ics of exopolysaccha r ide mat r ix a nd m icrocolon ies for mation by streptococcus mutans in biofilms. journal of applied microbiology 2010; 108: 2103–13. 5. sauer k, rickard ah, davies dg. biofilms and biocomplexity 2007; 2(7): 347–53. 6. ga r nett ja, matthews s. i nteractions in bacter ia l biof ilm development: a structural perspective. current protein and peptide science 2012; 13: 739-55. 7. hojo k, nagaoka s, ohshima t, maeda n. bacterial interactions in dental biofilm development. j dent res 2009; 88: 982-90. 8. ma rsh p, ma r tin vm. oral microbiology. 5th ed. churchill livingstone; 2009. p. 43, 65-7. 9. kreth j, merritt j, qi f. bacterial and host interactions of oral streptococci. dna cell biol 2009; 28(8): 397–403. 10. nagaya ma m, sato m, ya maguch i r, tokuda c, ta keuch i h. evaluation of co-aggregation among streptococcus mitis, fusobacterium nucleatum and porphyromonas gingivalis. the society for applied microbiology 2001; 33(71): 122-25. 11. yamaguchi m, terao y, ogawa t, takahashi t, hamada s, kawabata s. role of streptococcus sanguinis sortase a in bacterial colonization. microbes and infection 2006; 8: 2791–96. 12. okahashi n, nakata m, terao y, isoda r, sakurai a, kawabata s, ooshima t. microbial pathogenesis pili of oral streptococcus sanguinis bind to salivary amylase and promote the biofilm formation. microbial pathogenesis 2011; 50: 148-54. 13. welin j, welin jc, beighton d, swensater g. protein expression by streptococcus mutans during initial stage of biofilm formation. applied and environmental microbiology 2004; 70(6). 14. cury ja, koo h. extraction and purification of total rna from streptococcus mutans biofilms. analytical biochemistry 2007; 365(2): 208-14. 15. leenaars m, hendriksen cfm. critical steps in the production of p olyclona l a nd mono clona l a nt ib o d ies: eva luat ion a nd recommendations. ilar journal 2005; 46(3): 269-79. 16. aulanni’am. prinsib dan teknik analisis biomolekul. malang: fakultas pertanian universitas brawijaya press; 2004. 17. albert b, johnson a, lewis j, raff m, robert k, walter p. moleculer biology of the cell. 5th ed. new york: garland science; 2008. 18. ja nson jc, ryden l . p rotei n sepa rat ion a nd pu r i f icat ion. biotechnology science. 2nd ed. wiley. 2008. 19. cahyarini rd. identifikasi keragaman genetik beberapa varietas lokal kedelai di jawa berdasarkan analisis isozim. tesis. surakarta: program pasca sarjana universitas sebelas maret; 2004. 20. changyang p, mahmood t. western blot: technique, theory, and trouble shooting. north american journal of dental scienses 2012; 4(9): 429-34. 21. kang dc, zoe yh, cheng yp, xing lj. effects of power ultrasound on oxidation and structure of protein during curing processing. ultrasonics sonochemistry 2016; 33: 47-53. 22. kyle ph, ruby mas, paula jc, sofiane ekc, audrey b. identificaton of a supra moleculer functional architecture of streptococcus mutans adhesin p1 on the bacterial surface. j of biology chemistry 2015; 290(14): 9002-29. 23. nikitkova ae, haase em, scannapieco fa.. taking the starch out of oral biofilm formation: molecular basis. american society for microbiology. 2012. 24. orton dj, doucette aa, maksym gn, maclellan dl. proteomic analysis of rat proximal tubule cells following stretch-induced apoptosis in an in vitro model of kidney obstruction. journal of proteomics 2013; 100: 125-35. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p213-216 vol 49 no 3 juli-sept 2016.indd 120 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 120–124 correlation between p53 expressions and histopathological grading in oral cavity squamous cell carcinoma silvi kintawati department of oral biology faculty of dentistry, university of padjadjaran bandung – indonesia abstract background: squamous cell carcinoma is a malignancy of oral cavity mostly occurred and can also metastasize. p53 gene is a tumor suppressor gene that plays an important role in carcinogenesis. the role of wild-type p53 is very important in suppressing the formation of a malignancy. p53 also has many other important functions. p53 is a suppressor of tumor/ cancer progression through the response of cell cycle to dna damage and by giving time to repair dna prior to replication of genes. p53 mutation, mostly occurs in a malignancy, so earlier histopathological transformation can be detected by observing p53 mutation. the prognosis of squamous cell carcinoma in oral cavity, therefore, depends on histopathological grading and clinical staging of the tumor. to enforce the histopathological grading, in addition based on histopathology differentiation, the earlier histopathological transformation can also be assessed. purpose: this study aimed to determine the correlation of p53 expressions and histopathological grading in oral cavity squamous cell carcinoma. method: this study was a retrospective study on 20 cases of oral cavity squamous cell carcinoma examined at department of pathology anatomy in hasan sadikin hospital in bandung. immunohistochemical examination was then performed using p53 antibodies to determine the correlation of p53 expression and histopathological grading in oral cavity squamous cell carcinoma to predict prognosis. result: the overall results showed that there was no correlation between p53 expression and histopathological grading in oral cavity squamous cell carcinoma of the oral cavity although there was a very strong correlation between p53 expression and histopathological grading i (p<0.01). conclusion: it can be concluded that there was no correlation between p53 expression and histopathological grading in oral cavity squamous cell carcinoma. thus, p53 expression cannot be used to predict a prognosis. keywords: p-53; squamous cell carcinoma; immunohistochemical correspondence: silvi kintawati, department of oral biology, faculty of dentistry, university of padjadjaran. jl. raya jatinangor, cibeusi, jatinangor, kabupaten sumedang, jawa barat 45363, indonesia e-mail: silvikintawati@yahoo.com introduction squamous cell carcinoma is a tumor often found in head and neck, 90% of which is oral cavity malignancy. squamous cell carcinoma can also be considered as an aggressive disorder that can undergo metastasis and treatment failure, which can result in recurrence of the tumor.1 squamous cell carcinoma is a malignancy of the stratified squamous epithelium which can lead to local destruction and metastasis.2 unfortunately, etiology of squamous cell carcinoma in oral cavity as well as in other forms of carcinoma is still not known certainly. there are actually several stimulus factors playing a role in the incidence of oral cavity carcinoma, namely extrinsic and intrinsic factors. there is no specific factor that can be determined as a single carcinogen. extrinsic factors are composed of external resources, such as exposure to cigarette, tobacco, alcohol, bacteria, viruses, chemicals and ultra violet.2,3 clinically, squamous cell carcinoma has several variations, such as exophytic and endophytic. exophytic lesions typically have an irregular surface and papilla, as well as various color from normal to red with white dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p120-124 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p120-124 121121kintawati./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 120–124 patches depended on the amount of keratin produced. the surface sometimes is ulcerated, and on palpation the tumor mass is hard. endophytic lesions, on the other hand, are characterized by their curvature, irregular edges, ulceration, and elevated border (induration) with red mucosa and white patches. destruction of the underlying bone can occur with a radiology overview in the form of mouth-eaten radiolucent with unsmooth edges.1 histopathologically, broders (1920) classifies levels of carcinoma grading in the oral cavity into four levels, namely level i (well differentiated), level ii (moderately differentiated), level iii (poorly differentiated), and level iv (anaplastic). the most common incidences of squamous cell carcinoma in oral cavity are in lip inferior, lateral and ventral part of the tongue, and floor of the mouth, followed by the posterior part of the soft palate as well as tonsil area. to confirm the diagnosis of squamous cell carcinoma, histopathological examination is required. dysplasia rate will show the extent of cellular abnormalities, including increased mitotic figures, hyper-chromatics, and changes in normal cells as well as their maturation. mild, moderate, and severe dysplasia will show epithelial abnormalities with many different levels. if an abnormality has involved the entire thickness of the epithelium, it can be diagnosed as a insitu-carcinoma. if the basement membrane is damaged and the invasion into the underlying connective tissue occur, it can be diagnosed as a carcinoma. prognosis in oral cavity squamous cell carcinoma, further depends on histopathological grading and clinical staging of the tumor. to enforce the histopathological grading, in addition based on histopathology differentiation, the earlier histopathological transformation can also be assessed. p53 gene is a tumor suppressor gene that plays an important role in carcinogenesis. the role of wild-type p53 is very important in suppressing the formation of a malignancy. p53 also has many other important functions. p53 is a suppressor of tumor/ cancer progression through the response of cell cycle to dna damage and by giving time to repair dna prior to replication of genes, as well as the initiation of apoptosis in dna damage that cannot be repaired anymore. it basically can be considered as a central monitor directing cells to provide a response in the form of termination of the cell cycle and apoptosis.4,5 p53 mutation mostly occurs in a malignancy, so earlier histopathological transformation can be seen by observing p53 mutation. this study aimed to determine correlation of p53 expressions and histopathological grading in oral cavity squamous cell carcinoma that can be an indicator of the prognosis. materials and method this study was a retrospective study. samples used in this study were derived from paraffin blocks of oral squamous cell carcinoma examined at department of pathology in dr. hasan sadikin hospital. in total, there were 20 cases of oral cavity squamous cell carcinomas selected and were stained with hematoxylin eosin for enforcing diagnosis and histopathological grading according to broder’s modification (grading i, ii and iii). an immunohistochemical examination was performed using p53 monoclonal antibodies. immunohistochemical examination on p53 can indicate mutant p53/ p53 mutation that leads to transformation of malignancy.4,5 p53 mutation then will extend its half-life by increasing the stability of the protein, so the tumor cells with p53 mutation will show positive immunoreactive p53 in the nucleus/ cytoplasm of the tumor cells. immunohistochemical examination of the p53 protein in the tumor cells can also serve as an additional method to determine a correlation between the p53 genes and other tumor genes.6 p53 primary antibody do-7 clone, m7001 (dako, carpentaria, ca 93013. usa) can be used in immunohistochemical examination with streptavidin biotin peroxidase method. the results of the immunohistochemical staining can be indicated as positive ones if there is a brownish color expression in the nucleus and cytoplasm. the results of the staining then are compared to both of the positive control using a colon carcinoma that is known to be positive with p53 antibody and also the negative control. p53 expressions were rated quantitatively and intensively. p53 expressions were quantitatively calculated at 1000 tumor cells in 10 representative visual fields by using a light microscope cx-21 (olympus america inc. melville, ny 11747) with a magnification of 400x. in other words, p53 expressions were quantitatively categorized based on the percentage of positive tumor cells into score 1 if positive cells less than 25%, score 2 if positive cells between 25-50%, score 3 if positive cells between 51-75%, and score 4 if positive cells more than 75% with cut-off levels of <25% to >75%.3,7 on the other hand, the intensity of p53 expressions are categorized based on color into score 1 for weak intensity (light brown), score 2 for moderate intensity (chocolate between score 1 and score 3), and score 3 for stronger intensity (dark brown).8 based on the percentage and intensity of the p53 expressions, the values of p53 expressions were calculated by multiplying the percentage value of p53 expressions (score 1, 2, 3 or 4) with the intensity of the p53 expressions (score 1, 2 or 3). as a result, the values of p53 expressions obtained were in a range of 1-12, correlated later with histopathological grading for squamous cell carcinoma. data obtained then were statistically tested using correlation-regression test. results of the 20 sample cases of squamous cell carcinomas, there were eight (8) males and twelve (12) females with a mean age of 49 years old. the location of tumors was various, namely, 8 cases in tongue, 5 cases in gingival area, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p120-124 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p120-124 122 kintawati./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 120–124 2 cases in buccal area, 4 cases in the base of tongue, and 1 case in palate (table 1). grade i squamous cell carcinoma was found in 7 cases, grade ii in 5 cases, and grade iii in 8 cases. the number of tumor cells with immunoreactive p53 was also various from <25% to >75%, namely, <25% in 5 cases, 25-50% in 5 cases, 51-75% in 4 cases, and >75% in 6 cases. similarly, the color intensity of the p53 expressions was various, weak intensity in 4 cases, moderate intensity in 11 cases, and stronger intensity in 5 cases (table 2). based on the quantity and intensity of p53, the values of p53 expressions then were calculated by multiplying the quantity of p53 expressions with the intensity of p53 expressions. the values of p53 expressions obtained were between 1-12, consisted of score 1 in 4 cases, score 2 in 3 cases, score 4 in 4 cases, score 6 in 2 cases, score 8 in 2 cases, score 9 in 1 case, and score 12 in 4 cases. next, those scores were correlated with histopathological grading of oral squamous cell carcinoma (table 3) and (figure 1). based on the results of the statistical analysis using a correlation-regression test, there was no correlation between p53 expressions and histopathological grading i, ii, and iii as a whole. nevertheless, there was a very significant correlation between p53 expressions and histopathological grading i (p <0:01). discussion squamous cell carcinoma is a malignancy in oral cavity found mainly at the age of 45 years often associated with various risk factors, such as smoking and drinking alcohol in addition to other extrinsic factors. however, many cases of squamous cell carcinoma lately are also found in young adults associated with those risk factors.3 squamous cell carcinoma in oral cavity will have a poor prognosis when found in an advanced stage, in which most patients die within 5 years after the enforcement of diagnosis.3 prognosis of oral cavity squamous cell carcinoma actually depends on histopathological grading and clinical staging of table 1. characteristics of patients with oral squamous cell carcinoma variables n (%) median of age (years) <49 4 (20%) >49 16 (80%) sex males 8 (40%) females 12 (60%) location tongue 8 (40%) gingival area 5 (25%) buccal area 2 (10%) base of tongue 4 (20%) palate 1 (5%) table 2. expression and intensity of p53 in histopathological grading of squamous cell carcinoma histopathological grading expression intensity p n <25% 26-50% 51-75% >75% weak medium strong grade i 7 4 2 1 4 3 <0.01 grade ii 5 1 3 1 5 grade iii 8 2 6 3 5 table 3. the correlation between the scores of p53 expressions and histopathological grading of oral squamous cell carcinoma histopathological grading scores of p53 expressions total p 1 2 3 4 6 8 9 12 grade i 4 2 1 7 <0.01 grade ii 1 3 1 5 grade iii 1 2 1 4 8 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1 2 4 6 8 9 12 p-53 expression values n t um or grade i grade ii grade iii figure 1. graph of p53 expression values. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p120-124 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p120-124 123123kintawati./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 120–124 the tumor.1,2 to enforce the histopathological grading, in addition based on histopathological differentiation, histopathological transformation found early can be observed by using immunohistochemical examination. immunohistochemical examination has been widely used to evaluate and analyze the role of various markers in predicting prognosis of squamous cell carcinoma.9,10 p53 gene is a tumor suppressor gene that plays an important role in carcinogenesis. normally, p53 genes regulate cell cycle from g1 phase to s phase. increasing concentrations of wild-type p-53, consequently, will inhibit the cells in the g1 phase into the s phase during cell cycle by activating protein p-21 which will inhibit the function of cycling -dependent kinase enzyme, resulting in terminating the cell cycle, inhibiting cell growth, preventing duplication and replication of damaged dna, and allowing for dna repair. in other words, the roles of wild-type p53 are very important in suppressing the formation of a malignancy. wild-type p53 also has many other important functions, namely as a suppressor of tumor/ cancer progression through the response of cell cycle to dna damage and by giving time to repair dna prior to replication of genes, as well as the initiation of apoptosis in dna damage that cannot be repaired anymore, as a result, it basically can be considered as a central monitor that directs cells to provide a response in the form of termination of the cell cycle and apoptosis.4,5 if p53 mutation occurs, p53 will lose its function in regulating the cell cycle and apoptosis resulting in transformation and progression of malignancy. p53 mutation is one thing that most often occurs in a malignancy, so earlier histopathological transformation can be observed by looking at the p53 mutation. p53 mutation has been widely used as markers in a variety of malignancies, including in oral cavity squamous cell carcinoma.11 based on various researches that have been conducted, there were different correlation found between p53 expressions and histopathological grading in predicting prognosis.12,13 for instance, a research on 76 cases of squamous cell carcinoma in larynx conducted by luo et al. shows a significant correlation between p53 expressions and histopathological grading i, ii and iii. similarly, ashraf et al. also shows that p53 expressions can predict prognostic in squamous cell carcinoma in larynx.2 unlike the previous researches, the researches show that there is no significant correlation between p53 expressions and histopathological grading i, ii and iii.10,13-15 for those reasons, this study aimed to determine the role of p53 in immunohistochemistry as well as the correlation between p53 expressions and histopathological grading in oral cavity squamous cell carcinoma. in all 20 cases of squamous cell carcinoma classified by grading i, ii and iii, there was no correlation between p53 expressions and histopathological grading in those three groups as a whole. nevertheless, there was a highly significant correlation between p53 expressions and histopathological grading i (p <0.01). this finding is consistent with a research conducted by rodrigues et al. showing that there is no correlation between immunohistochemical p53 and histopathological grading in predicting prognosis for 30 cases of squamous cell carcinoma of larynx.13 similarly, another research conducted by motta et al.10 also shows that there is no significant correlation between p53 and histopathological grading in predicting prognosis for 28 cases of squamous cell carcinoma of oral cavity. therefore, it can be argued that p53 expressions possibly play a role at the beginning of the occurrence of malignant transformation. p53 mutation occurs as an initial change of a mutation to malignancy of oral cavity, such as dysplasia and leukoplakia.16,17 although the overall p53 expressions in this research were not statistically correlated with histopathological grading, there were greater distribution of high expression (6 cases) and strong intensity (5 cases) in histopathological grading iii. it means that p53 expressions in those squamous cell carcinoma cases of oral cavity were correlated with a poor prognosis (grade iii). this finding is in accordance with an opinion of motta et al.10 stating that there is a correlation between p53 and a poor prognosis in oral cavity squamous cell carcinoma. p53 expressions in grading i indicates a highly significant correlation (p<0.01) due to the low expression distribution (4 cases) and the weak intensity (4 cases) on histopathological grading i. this is possibly because of p53 mutation has a longer half-life, mostly found in a change/ occurrence of a malignancy. however, p53 mutation is not correlated with increased histopathological grading since p53 mutation emerges in the early stage of change into malignancy. this is in accordance with an opinion of khanna et al. stating that p53 mutation is considered as an initial change of a mutation to malignancy of oral cavity, such as dysplasia and leukoplakia.11,13,16,17 it can be concluded that there was no correlation between p53 expressions and histopathological grading in oral cavity squamous cell carcinoma. p53 cannot be used to predict prognosis, but p53 expressions in this research still can be considered as a part of the pathogenesis and development of a malignancy. references 1. ghali ge, larsen pe, waite pd. peterson’s principles of oral and maxilofacial surgery. 2nd ed. hamilton, london: bc decker inc; 2004. p. 617-71. 2. regezi ja, sciubba jj, jordan rck. oral pathology: clinical pathologic correlations. 5th ed. st louis, missouri: elsevier; 2008. p. 48-71. 3. chandra a, singh a, sebastian bt, agnihotri a, bali r, verma pk. oral squamous cell carcinomas in age distinct population: a comparison of p53 immunoexpression. j can res ther 2013; 9: 587-91. 4. maiuri mc, galluzzi l, morselli e, kepp o, malik sa, kroemer g. autophagy regulation by p53. j curr opin cell biol 2010; 22: 181-5. 5. levine b, abrams j. p53: the janus of autophagy? j nat cell biol 2008; 10: 637-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p120-124 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p120-124 124 kintawati./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 120–124 6. kasakov dv, grossmann p, supagnolo dv, vanecek t, vazmitel m, kacerovska d. expression of p53 and tp53 mutational analysis in malignant neoplasms arising in preexisting spiradenoma, cylindroma, and spiradenocylindroma, sporadic or associated with brooke-spiegler syndrome. am j dermatopathol 2010; 32: 21521. 7. barcones-martinez a, rodriguez-gutierrez c, rodriguez-gomez e, gilmontoya j.a., gomez-font r, gonzalez-moles ma. evaluation of p53, caspase-3, bcl-2, and ki-67 markers in oral squamous cell carcinoma and premalignant epithelium in a sample from alava province (spain). j med oral patol oral cir bucal 2013; 18(6): ne846-e850. 8. humayun s, ram prasad v. expression of p53 protein and ki67 antigen in oral premalignant lesions and oral squamous cell carcinoma: an immunohistochemical study. nanl. j maxillofac surg 2011; 2(1): 38-46. 9. li l, fukumoto m, liu d. p rognostic signif ica nce of p53 immunoexpression in the survival of oral squamous cell carcinoma patients treated with surgery and neoadjuvant chemotherapy. oncology letters 2013; 6(6): 1611-5. 10. motta rdr, zettler cg, cambruzzi e, jotz gp, berni rb. ki-67 and p53 correlation prognostic value in squamous cell carcinomas of the oral cavity and tongue. braz j otorhinolaryngol 2009; 75(4): 1-11. 11. sassi lm, loshii so, oliveira bv, pedruzzi pa, guerbur m, schussel jl, stramandinoli rt, ramos gh, orlandi d, fukuda e, cervantes o. second primary tumor: p53 and ki-67 expression in patients with oral squamous cell carcinoma. webmedcentral cancer 2011; 2(3): 2-8. 12. de oliveira lr, ribeiro-silva a, zucoloto s. prognostic impact of p53 and p63 immunoexpression in oral squamous cell carcinoma. j oral pathol med 2007; 36(4): 191-7. 13. rodrigues rb, da ros motta r, dos santos macbado sm. prognostic value of the immunohistochemistry correlation of ki-67 and p53 in squamous cell carcinomas of the larynx. bras otorrinolaringol 2008; 74(6): 855-9. 14. luo k, wang z, wang n, zhang x, yang j. effect of expression of p53 in squamous cell carcinoma of larynx and mucosa adjacent in tumor on the biological behavior. lin chuang er bi yan hou ke za zhi 2005; 19(9): 405-8. 15. micozkadioglu d, unal m, pata ys, basturk m, cinel l. prognostic value of expression of p53, proliferating cell nuclear antigen, and c-erbb-2 in laryngeal carcinoma. met sci monit 2008; 14(6): 299304. 16. k hanna r, vidhyarthi ak, k hanna s, singh u, singh uc. expression of p53 protein in leukoplakia and oral squamous cell carcinoma. world journal of surgical medical and radiation oncology 2012; 1: 16. 17. ashraf mj, maghbul m, azarpira n, khademi b. expression of ki67 and p53 in primary squamous cell carcinoma of the larynx. indian journal of pathology & microbiology 2010; 53(4 ): 661-5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p120-124 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p120-124 125 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 125–129 original article visualizing the velocity fields and fluid behavior of a solution using artificial intelligence during endoactivator activation harry huiz peeters1, elvira theola judith2, faber yosua silitonga3, lavi rizki zuhal3 1laser research center in dentistry, bandung, indonesia 2faculty of dentistry, universitas maranatha, bandung, indonesia 3faculty of mechanical and aerospace engineering, institut teknologi bandung, bandung, indonesia abstract background: electrical devices driven sonically have been found in several studies to be effective to clean root canals but the effect of the endoactivator irrigant activation flow behavior on cleaning efficacy is not completely understood. purpose: the study aimed to provide an initial understanding of flow behavior and velocity field generation during the irrigant activation process by endoactivator using artificial intelligence (ai). methods: a straight glass model was filled with a solution containing 17% edta. meanwhile, a medium activator tip with 22-mm polymer noncutting #25, 0.04 file driven by an electrical sonic hand-piece at 190 hz (highest level) was used to induce velocity field to produce micro-bubbles. the physical mechanisms involved were recorded using a miro 320s highspeed imaging system, the hydrodynamic responses were recorded, and analyzed using a motion estimation program supported by liteflownet (ai). results: the rapid fluid flow was visualized clearly in the model when it was activated by an endoactivator tip. it was also observed that the distal end of the endoactivator tip generated a near-wall high gradient velocity apically in all directions of the oscillation. conclusion: the analysis showed that the proposed motion estimation program, supported by liteflownet (ai), was able to capture velocity magnitude estimation of a non-piv experiment and visualize the bubbles generated in the solution. keywords: artificial intelligence; endoactivator; endodontics; natural frequency; shear stress correspondence: harry huiz peeters, laser research center in dentistry, cihampelas 41 bandung, west java, 40174, indonesia. email: h2huiz@cbn.net.id introduction a pre-requisite to the long-term success of root canal treatment is the thorough debridement and disinfection of the root canal system.1–4 meanwhile, the efficacy of an irrigation delivery or agitation system to debride depends on how the irrigant reaches the apical region, the uninstrumented areas, and the ability to create a strong current enough to carry the debris out of the root canal coronally.5–9 some examples of machine-assisted agitation are electrical ultrasonic and sonic devices10 that use a fine non-cutting polymer or metal tip vibrated within the root canal space at different frequencies based on the manufacturer’s instruction. it is important to note that sonically activated instruments utilize frequencies in the range of 1,000–6,000 hz and generate a single node near the point at which the file is attached and an antinode at the tip of the file.11–13 endoactivator (dentsply, tulsa dental) was introduced onto the market and has been developed with the intention of activating root canal irrigants by energizing the solution with sonic energy. several controversial results have been reported but sonically driven electrical devices have been found in different studies to be effective in cleaning root canals.12,14,15 powerful particle image velocimetry (piv) technology has also been used in several studies on dentistry to observe the natural flow behavior of hydrodynamic response during irrigation activation.16–19a deep learning approach for motion estimation currently showed promising results with higher accuracy and enhanced computational performance.20 moreover, the liteflownet network which is a state-of-theart deep learning model for motion estimation has also been developed by hui and colleagues.21 it is important to note that the reproduction of a deep learning model from dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p125–129 mailto:h2huiz@cbn.net.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p125-129 126peeters et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 125–129 estimator programs is not significantly different from measuring investigations using the piv image processing method. furthermore, an artificial intelligence model has been employed in dentistry to support the clinical decision-making process in certain disciplines22 such as radiology,23–25 endodontics,26 and surgery.27 a few studies have been conducted on the hydrodynamic response of endoactivator tip during the irrigation solution agitation but this present study investigates the solution dynamic behavior through the acquisition of real-time data using a transparent glass model of root canal in order to visualize the oscillation amplitude of the endoactivator tip during solution activation. this is necessary to provide more knowledge on the mechanism underlying the activation behavior of this device in the solution towards improving the outcomes of the root canal treatment. the main challenge is to develop a deep learning estimator for non-piv (absence of seeding particles) based on liteflownet21 to be applied to a relatively limited area such as a root canal space as a means of producing highresolution images. the program also has the ability to process a non-particle image pair input to produce a velocity field output with displacement vectors at every pixel. materials and methods a 22-mm polymer noncutting #25, 0.04 (dentsply) tip was mounted on an endoactivator hand-piece (dentsply tulsa dental specialties, tulsa, ok) set at a high mode of 190 hz to activate the irrigant. the handpiece was fixed in a holder to ensure the desired position was maintained. it is important to note that all the experiment was conducted by the same operator. the conditions within a straight root canal were simulated and visualized using a glass model with an artificial canal and pulp cavity that acted as the reservoir. the model was a glass root canal (kimia farma, bandung, west java, indonesia) with a canal inner diameter of 0.4 mm at the apex, the crown height of 8 mm, crown diameter of 6 mm, a taper of 0.06, and an overall length of 25 mm. the canal was filled with a solution containing 17% edta while the apex of the model was sealed with composite to allow the conditions within the root canal to be simulated. moreover, the tip of the sonic instrument was inserted into the solution around 6 mm from the interface and activated passively without any filing motion. a single transparent glass model was used to ensure uniformity in the width and size of the root canal. it is important to restate that the objective of this study is to observe the hydrodynamic response to endoactivator tip solution activation in the root canal model. the process was recorded using a phantom miro 320s high-speed digital imaging system (wayne, nj, usa) incorporated with a macro lens capable of producing 25,000 frames per second with 320x240 pixels per image (60 mm, f/2.8; nikon, tokyo, japan). the sample was illuminated by a fiber-lite figure 1. the deep learning motion estimator process in an optically accessible root canal model. lmi-6000 led continuous light source (dolan-jenner industries, boxborough, ma, usa). (see supplemental video s1 https://youtu.be/kbksxf-4nuw) the recordings of the hydrodynamic response were analyzed using in-house particle image velocimetry (piv) software developed by the aerodynamics laboratory at institut teknologi bandung (itb, west java, indonesia) to obtain estimation quantitative data images. the software used optical flow with a convolutional neural network for velocity field estimation rather than using the measured experimental piv settings with tracer particles. the data from non-piv experiments were extracted using a deep learning estimator incorporated with the piv software developed by the aerodynamics laboratory at itb (bandung, west java, indonesia). moreover, the user disregarded the last nete level and imposed the liteflownet trained weights to generate a liteflownet version. the flowchart of the stages involved in the data execution process is, therefore, presented in the following figure 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p125–129 https://youtu.be/kbksxf-4nuw https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p125-129 127 peeters et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 125–129 figure 2. the direction of the flow caused by an ea oscillating tip (indicated by solid arrows). (a) the red circle indicates the flow pattern occasionally occurring perpendicular to the axial of the tip and the oblique flow pattern in blue. meanwhile, (b) to (d) indicate the visualization of the instantaneous dense velocity fields within the small-scale flow structures near the wall with the orange to a red color indicating the location where a near-wall high-velocity gradient occurred. figure 3. (a) change of mean flow magnitude as indicated from 0 to a point showing accumulation of energy while the point to 2 sec depicts the decreased fluid flow velocity with the highest recorded at 0.09 sec. (b) the point with the highest magnitude at 824.75 mm/sec. figure 4. representative of instantaneous velocity fields with minimum flow magnitude (x). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p125–129 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p125-129 128peeters et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 125–129 to affect the cleaning mechanism. a pilot study was also conducted for confirmation and it showed that the ea tip did not separate when it was being forced to vibrate in the air at its applied frequency. this implies the ea tip did not achieve its resonant or natural frequency which can cause damage or tip separation due to excessive vibration at a higher frequency.28 bubbles regime stage: this stage lasted between approximately 0.09 and 2 seconds as indicated in figure 3a and the velocity was observed to reduce gradually to its lowest level reaching a constant speed of approximately 230 mm/sec during bubble formation as indicated in figure 3a and the near-wall velocity gradients were discovered not to occur. this phenomenon led to the recommendation of continuous replenishment of irrigation solution during activation to reduce the prolonged bubble regime (see supplemental video s3 https://youtu.be/hyxaspb-zzk). the up and down flow patterns were observed to hardly form during ea activation in these stages and this can lead to the development of a push-pull mechanism that enhances the removal of smear layer and debris.18,29 furthermore, it was very obvious during the activation that there were lots of areas where the velocity field had the lowest flow magnitude as indicated in figure 4. this implies the fluid flow was very limited in such areas, thereby, leading to minimal cleaning action and this was assumed to be due to the natural mode shape of the tip. this phenomena is in line with the study conducted by peeters et al.30 moreover, the node at the attachment and the antinode at the end of the tip vibrated freely as a cantilever beam model through a back-and-forth linear movement. this oscillation of the ea tip produced a near-wall high-velocity gradient which is proportional to wall-shear stresses but this was observed to be a rare occurrence. it is important to note that the areas of significant changes in the velocity over a short distance are indicated in red as presented in figure 2. furthermore, a near-wall high-velocity gradient and the bulk transports of solution are, potentially, the most important variables of the cleaning process within the root canal during activation from a fluid dynamic perspective.18 it was also observed from the experiment that high gradient velocity on the wall largely occurred more apically around the lower end of the tip and periodically in the same area and represented in red as indicated in figure 2. therefore, it is recommended based on the data relating to these stages that the tip should be moved up and down in order to distribute the shear stresses evenly along the canal while the pumping action of the tip induces additional shear stresses on the wall. these results simply showed that the proposed liteflownet-supported motion estimation program was able to conduct detailed flow estimation of a non-piv experiment and extract dense velocity fields. therefore, it is recommended that the tip should be moved up and down and continuous irrigation should be applied during ea activation. there is enormous potential for the development of related topics and this model facilitates further similar research in dentistry. results the results showed that the modified non-enhanced liteflownet program successfully extracted non-particle image pair input to produce velocity field output with displacement vectors in every pixel. the program also provided detailed high-resolution images which enabled smaller scale motion detection while generating a dense motion field for all images and rapidly completing the computer processing. the flow motion was highly unsteady and this observation was highlighted in this study by focusing only on a limited portion of the canal which was located around the distal end of the tip to enable velocity field visualization. moreover, the flow patterns perpendicular to the axial flow along the canal (red) and oblique flow patterns (blue) were periodically observed as indicated in figure 2a. the instantaneous image contained in figure 2 (b-d) also shows the flow pattern of the solution near the distal end of the tip where the highest amplitude occurs. furthermore, a periodic flow pattern was discovered at the velocity gradients recorded near the wall during activation. the findings also showed that the velocity fields within this region indicated in red were approximately three to six times higher than those in the surrounding area. the length of the arrows also indicates the magnitude of the velocity while bubbles were generated during activation. discussion human dentin structure makes it impossible to have a direct visualization of the root canal process and this is the reason in vitro models are usually made of transparent glass but previous studies showed that this method cannot reflect the actual activities in clinical settings. however, it is interesting to know how the fluid behavior and velocity field generation can be visualized in real-time. the experiment conducted showed that the ea tip activation process in the root canal model is in two stages which include the energy accumulation and bubbles regime stages. the energy accumulation stage: the vibrating ea tip transferred its energy to the solution and created waves which, in turn, caused surface waves to form at the interfaces at the 0-0.09 second range as indicated in figure 3a. moreover, the kinetic energy increased to the point that it was sufficiently high to disrupt the air-solution interface with an average fluid magnitude close to 824.75 mm/sec when the amplitude reached its peak (see supplemental video s2 https://youtu.be/6l24nueciy0). the highest peak of energy accumulation occurred at 0.085 second as shown in figure 3b where the bubble started to form for the first time. however, this point is not its natural frequency even though the amplitude of the ea tip seems to reach its peak depending on the frequency applied. therefore, the fluid flow was unable to reach its maximum needed dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p125–129 https://youtu.be/hyxaspb-zzk https://youtu.be/6l24nueciy0 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p125-129 129 peeters et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 125–129 acknowledgments the authors deny any conflict of interest related to this study and appreciate professor zainal abidin phd at the institut teknologi bandung (bandung, indonesia), mr. wowo watumas at the phantom company for providing helpful contributions to this study. references 1. moreno jo, alves frf, gonçalves ls, martinez am, rôças in, siqueira jf. periradicular status and quality of root canal fillings and coronal restorations in an urban colombian population. j endod. 2013; 39(5): 600–4. 2. gazzaneo i, vieira gcs, pérez ar, alves frf, gonçalves ls, mdala i, siqueira jf, rôças in. root canal disinfection by single and multiple-instrument systems: effects of sodium hypochlorite volume, concentration, and retention time. j endod. 2019; 45(6): 736–41. 3. souza ma, corralo dj, gabrielli es, figueiredo jap, cohen s, wolff m, steier l. oral bacterial decontamination using an innovative prototype for photocatalytic disinfection. clin oral investig. 2022; 26(3): 3005–10. 4. zhang d, shen y, de la fuente-núñez c, haapasalo m. in vitro evaluation by quantitative real-time pcr and culturing of the effectiveness of disinfection of multispecies biofilms in root canals by two irrigation systems. clin oral investig. 2019; 23(2): 913–20. 5. boutsioukis c, kastrinakis e, lambrianidis t, verhaagen b, versluis m, van der sluis lwm. formation and removal of apical vapor lock during syringe irrigation: a combined experimental and computational fluid dynamics approach. int endod j. 2014; 47(2): 191–201. 6. loroño g, zaldivar jr, arias a, cisneros r, dorado s, jimenezoctavio jr. positive and negative pressure irrigation in oval root canals with apical ramifications: a computational fluid dynamics evaluation in micro-ct scanned real teeth. int endod j. 2020; 53(5): 671–9. 7. boutsioukis c, verhaagen b, versluis m, kastrinakis e, van der sluis lwm. irrigant flow in the root canal: experimental validation of an unsteady computational fluid dynamics model using high-speed imaging. int endod j. 2010; 43(5): 393–403. 8. chen je, nurbakhsh b, layton g, bussmann m, kishen a. irrigation dynamics associated with positive pressure, apical negative pressure and passive ultrasonic irrigations: a computational fluid dynamics analysis. aust endod j. 2014; 40(2): 54–60. 9. layton g, wu w-i, selvaganapathy pr, friedman s, kishen a. fluid dynamics and biofilm removal generated by syringe-delivered and 2 ultrasonic-assisted irrigation methods: a novel experimental approach. j endod. 2015; 41(6): 884–9. 10. macedo r, verhaagen b, rivas df, versluis m, wesselink p, van der sluis l. cavitation measurement during sonic and ultrasonic activated irrigation. j endod. 2014; 40(4): 580–3. 11. arslan h, akcay m, capar id, ertas h, ok e, uysal b. efficacy of needle irrigation, endoactivator, and photon-initiated photoacoustic streaming technique on removal of double and triple antibiotic pastes. j endod. 2014; 40(9): 1439–42. 12. güven y, ali a, arslan h. efficiency of endosonic blue, eddy, ultra x and endoactivator in the removal of calcium hydroxide paste from root canals. aust endod j. 2022; 48(1): 32–6. 13. marques-da-silva b, alberton cs, tomazinho fsf, gabardo mcl, duarte mah, vivan rr, baratto-filho f. effectiveness of five instruments when removing calcium hydroxide paste from simulated internal root resorption cavities in extracted maxillary central incisors. int endod j. 2020; 53(3): 366–75. 14. swimberghe rcd, de clercq a, de moor rjg, meire ma. efficacy of sonically, ultrasonically and laser-activated irrigation in removing a biofilm-mimicking hydrogel from an isthmus model. int endod j. 2019; 52(4): 515–23. 15. jensen sa, walker tl, hutter jw, nicoll bk. comparison of the cleaning efficacy of passive sonic activation and passive ultrasonic activation after hand instrumentation in molar root canals. j endod. 1999; 25(11): 735–8. 16. jiang l-m, verhaagen b, versluis m, van der sluis lwm. evaluation of a sonic device designed to activate irrigant in the root canal. j endod. 2010; 36(1): 143–6. 17. koch jd, smith na, garces d, gao l, olsen fk. in vitro particle image velocity measurements in a model root canal: flow around a polymer rotary finishing file. j endod. 2014; 40(3): 412–6. 18. koch jd, jaramillo de, divito e, peters oa. irrigant f low during photon-induced photoacoustic streaming (pips) using particle image velocimetry (piv). clin oral investig. 2016; 20(2): 381–6. 19. robinson jp, macedo rg, verhaagen b, versluis m, cooper pr, van der sluis lwm, walmsley ad. cleaning lateral morphological features of the root canal: the role of streaming and cavitation. int endod j. 2018; 51(suppl 1): e55–64. 20. le cun y, matan o, boser b, denker js, henderson d, howard re, hubbard w, jacket ld, baird hs. handwritten zip code recognition with multilayer networks. in: proceedings 10th international conference on pattern recognition. atlantic city, nj, usa: ieee comput. soc. press; 1990. p. 35–40. 21. hui t-w, tang x, loy cc. liteflownet: a lightweight convolutional neural network for optical f low estimation. in: i eee/cv f conference on computer vision and pattern recognition. salt lake city, ut, usa: ieee; 2018. p. 8981–9. 22. shan t, tay fr, gu l. application of artificial intelligence in dentistry. j dent res. 2021; 100(3): 232–44. 23. orhan k, bayrakdar is, ezhov m, k ravtsov a, özyürek t. evaluation of artificial intelligence for detecting periapical pathosis on cone-beam computed tomography scans. int endod j. 2020; 53(5): 680–9. 24. z heng z , ya n h, setzer fc, sh i k j, muppa rapu m, li j. anatomically constrained deep learning for automating dental cbct segmentation and lesion detection. ieee trans autom sci eng. 2021; 18(2): 603–14. 25. hiraiwa t, ariji y, fukuda m, kise y, nakata k, katsumata a, fujita h, ariji e. a deep-learning artificial intelligence system for assessment of root morphology of the mandibular first molar on panoramic radiography. dentomaxillofacial radiol. 2019; 48(3): 20180218. 26. aminoshariae a, kulild j, nagendrababu v. artificial intelligence in endodontics: current applications and future directions. j endod. 2021; 47(9): 1352–7. 27. murphy m, killen c, burnham r, sarvari f, wu k, brown n. artificial intelligence accurately identifies total hip arthroplasty implants: a tool for revision surgery. hip int. 2021; : 1120700020987526. 28. thomson wt. theory of vibration with applications. 4th ed. london: crc press; 2018. p. 558. 29. jiang l-m, verhaagen b, versluis m, van der sluis lwm. influence of the oscillation direction of an ultrasonic file on the cleaning efficacy of passive ultrasonic irrigation. j endod. 2010; 36(8): 1372–6. 30. peeters hh, silitonga f, zuhal l. application of a r tificial intelligence in a visual-based fluid motion estimator surrounding a vibrating eddy® tip. g ital endod. 2022; 35. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p125–129 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p125-129 93 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 93–98 original article the effect of antimicrobial peptide gel rise-ap12 on decreasing neutrophil and enhancing macrophage in nicotine-periodontitis wistar rat model ika andriani1, ana medawati2, muhammad ihza humanindito3, maulida nurhasanah3 1department of periodontology, muhammadiyah university of yogyakarta, indonesia 2department of biomedic, muhammadiyah university of yogyakarta, indonesia 3school of dentistry, muhammadiyah university of yogyakarta, indonesia abstract background: periodontitis, an inflammation that causes alveolar bone destruction, is caused by bacteria and aggravated by nicotine exposure and is therefore a disease that many smokers have. antibacterial agents are essential for the rejuvenation process in periodontitis treatment; antimicrobial peptide (amp) gel is a broad-spectrum antibacterial agent that is hardly cause bacteria resistance. purpose: the objective of this study is to determine the effect of amp gel administration on neutrophil and macrophage counts on periodontitis regeneration in nicotine-exposed rats. methods: 24 wistar rats were separated into four groups: nicotineexposed, non-nicotine-exposed, treatment and control. rats with periodontitis were given amp in the gingival line on days 1, 3 and 7 after having their mandibular central incisors ligated for 14 days to induce periodontitis. after amp treatment, two groups of rats were collected randomly. each group were decapitated, followed by treatment and histological examination with hematoxylin-eosin staining in the pathology laboratory to view neutrophils and macrophages. the asymmetric kruskal wallis test was used to analyse the data. results: in mice treated with amp, neutrophil counts on day 3 were lower than in distilled water (aquadest) controls. the number of macrophages on day 3 was higher than that of the aquadest control. kruskal wallis test results for neutrophils were p = 0.017 and for macrophages p = 0.01, where both test results had p < 0.05, there were significant differences between the neutrophil and macrophage groups. conclusion: the administration of amp effects on decreasing the number of neutrophils and enhancing macrophages in the periodontitis regeneration. in nicotine-exposed rats. keywords: antimicrobial peptide; nicotine; neutrophil; macrophage; periodontitis correspondence: ika andriani, department of periodontology, muhammadiyah university of yogyakarta, jl. brawijaya, kasihan, bantul, yogyakarta 55183, indonesia. email: ika.andriani@umy.ac.id; ikaandriani@yahoo.com introduction periodontal disease is a chronic inflammatory condition caused by bacteria on the tooth’s surface and supporting tissues.1 smoking is associated with severe periodontal disease, as data show that smokers have higher rates of clinical attachment loss and chronic periodontal disease.2 one of the most harmful components of tobacco is nicotine. a more severe form of the disease is produced by nicotine, which disrupts the immune system and balances bacteria in the gingival biofilm.3,4 numerous studies have established that smoking contributes to periodontal disease, obstructs periodontal therapy and significantly affects the chance of effective periodontal treatment outcomes.5 an electronic database search comparing the outcomes of periodontal treatment in non-surgical and surgical patients including smokers and non-smokers, as well as supportive periodontal therapy, revealed that the healing response of smokers’ tissues to treatment was lower than that of non-smokers and that smokers had a higher risk of periodontal disease recurrence.2 periodontal therapy’s primary objective is to restore periodontal supporting tissue that has been lost or destroyed as a result of periodontitis. this happens because the healing of periodontitis accompanied by alveolar bone loss dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 mailto:ika.andriani@umy.ac.id mailto:ikaandriani@yahoo.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 94andriani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 93–98 is also influenced by the presence of large bone damage and is exacerbated by bacterial activity and smoking, so the treatment needs to be accompanied by therapy that involves an infection control process to obtain satisfactory tissue regeneration. until now, no satisfactory treatment for periodontitis has been found through conventional therapy, the use of antibacterials or with bone graft as an adjunct therapy.6 microbiological and biochemical analyses were performed in addition to clinical periodontal evaluations. it was shown that smoking and non-smoking patients received equivalent clinical results for non-surgical periodontal therapy. gram-negative bacteria repopulated far more quickly in smokers, presumably indicating a greater risk of recurrence.7 fusobacterium and porphyromonas gingivalis (p. gingivalis) bacteria are more commonly found in smokers than non-smokers, which the smokers group need potent antimicrobials.3 according to research by panpradit et al.7, nicotine and p. gingivalis had a negative effect on osteogenic differentiation. bacterial drug resistance is a severe health concern that affects people across the globe. long-term usage and misuse of antibiotics results in bacteria developing drug resistance.8 antimicrobial peptide (amp) is the first line of defence against invasion and infection caused by bacteria and other pathogens in the oral cavity.9 there are 45 different kinds of antimicrobial peptides released by the salivary glands, oral epithelial cells and neutrophils that are present in the mouth to inhibit the invasion and infection of microorganisms and bacteria. antimicrobial peptides’ inability to respond appropriately to inflammation results in the formation of periodontal disease.10 by delivering artificial antimicrobial peptides, various reactions and host circumstances that might result in decreased antimicrobial peptide synthesis can be resolved. artificial antimicrobial peptides, usually in the form of gels or creams, are natural molecules that do not cause side effects like other antibiotics in the form of gastrointestinal reactions, allergies or the production of antibiotic-resistant bacterial strains. jettingclean antimicrobial peptide rise-ap12, a periodontal gel biological agent, exhibits broad-spectrum, bactericidal effects and long-term antimicrobial activities, especially on periodontal inflammation caused by sensitive bacteria, such as p. gingivalis. this antimicrobial peptide plays a long-lasting inhibitor bacterium and promotes the repair of periodontal tissue.11 jettingclean as an antimicrobial peptide is naturally involved in apoptosis, wound healing and immunological regulation. it also possesses a broadspectrum bactericidal activity, capable of attacking grampositive and gram-negative bacteria, viruses, fungi and protozoa. thus, it can be an alternative to anti-infective therapy and immunomodulatory agents.12 many cells contribute to the regeneration of periodontal tissue, including neutrophils, monocytes, macrophages, lymphocytes, dendritic cells, fibroblasts and osteoblasts, each of which play a very important role from the beginning of the healing process, namely from the inflammatory stage to the final stage of readhesion.13 this is an inflammatory response starting from neutrophils moving from the gingival blood vessels to the junctional epithelial tissue and destroying the bacterial biofilm, turning it into apoptotic tissue. subsequently, macrophages play a critical role in cleaning apoptotic tissues by phagocytosis, activating lymphocytes that make il-17 and finally, producing fibroblasts which undertake the remodelling process.14 the novelty of this study is the use of peptide gel antimicrobial therapy in smokers with periodontitis. the aim of this study is to analyse the impact of the riseap12 antimicrobial peptide application on rats exposed to nicotine on the regulation of periodontitis in terms of the decreased number of neutrophils and enhanced number of macrophages. materials and methods the research was conducted at the integrated research and testing laboratory unit 4, gadjah mada university (lppt ugm unit 4). all the procedures of the in vivo experiment were approved by the ethical committee of the faculty of dentistry, muhammadiyah university of yogyakarta, 195/ep-fkik-umy/ix/2019. the laboratory experiment was done using jettingclean antimicrobial peptide periodontal gel containing antimicrobial peptide riseap12, which has bacteriostatic and bactericidal properties against p. gingivalis (rise biopharmaceuticals inc, beijing, china).10 this study used 24 healthy and active wistar male rats, weighing between 300 and 400 grams and aged 3 to 4 months. the experimental animals were adapted in a clean cage for 7 days. the rats were housed in separate cages and maintained under a 12-hour light/dark cycle at a temperature of 23 °c and relative humidity of 50%, with access to standard rat chow pellets and water ad libitum.15 the rats were then divided into two groups of nicotine (n = 12) and non-nicotine (n = 12), and each group was further divided into two, namely amp treatment (n = 6) and aqua dest control (n = 6) exposures. in the group of rats with nicotine exposure, nicotine (rts vapes, charlotte, usa) injection was provided at a dose of 16 µg/20 g of rat bodyweight for three consecutive days peritoneally on days 1, 2 and 3.16 furthermore, the experimental rats were locally anesthetized by using ketamine 10% (kepro bv, deventer, netherlands). a 3 mm silk ligature (b. braun, rubi, spain) was then attached as a ligation to the mandibular incisors for 14 days to generate periodontitis, with the description of the gingiva being red, swollen and moving apically away from the ligature.15 the ligation was released after 14 days. both nicotine and non-nicotine groups were then further divided into two subgroups with a total of four groups (n = 6) for anti-microbial peptide gel and control administration as dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 95 andriani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 93–98 follows: nicotine with amp (group 1); non-nicotine with amp (group 2); nicotine with aqua dest (group 3); and non-nicotine with aqua dest (group 4). all rats were given treatment on day 1, day 3, and day 7. for both group 1 and group 2, rats were given an application of a single spread of antimicrobial peptide periodontal gel consisting of rise ap-12 (rise biopharmaceuticals inc, beijing, china) using a cotton swab on the margin gingiva of mandibular anterior incisor that had undergone periodontitis. meanwhile, the negative control groups, group 3 and group 4, were given one spread of aqua dest. on days 3, 7, and 14 after the treatment, two rats were decapitated in each group of test animals. the wistar rats were anesthetized with ketamine (0.1 ml) and xylazine (0.1 ml) by intramuscular injection in the thigh (dose 6, 12 mg/kg).7,15 tissue that had periodontitis was then employed as a specimen to be made into preparations by the anatomical pathology laboratory of amc hospital using an automatic tissue processor (medimeas, haryana, india) and then placed in a paraffin block and cut using a finesse 325 microtome (thermo fisher scientific inc, massachusetts, united states). the slides were stained using an hematoxylin-eosin (he) stain and viewed using an olympus cx23 microscope (olympus corporation, tokyo, japan) with a magnification of 400x and captured using an optilab microscope camera (pt miconos, yogyakarta, indonesia) in the alveolar crest of the mandible incisor using six visual fields. the normality test was obtained from the observational data on the number of macrophages and neutrophils using the kolmogorov-smirnov test analytical method, and comparative tests were performed using kruskal wallis. the data analysis was done using spss statistics 23.0 (ibm corporation, new york, united states). results the data from the analysis of the number of neutrophils in each of the six fields of view are shown in table 1. this shows that there are significant differences in the number of neutrophils in the exposure and treatment groups with p = 0.017 (p < 0.05). the test results show the highest average treatment rating on nicotine on day 3 on amp and the lowest on day 7 on amp (figure 1). 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 day 3 day 7 day 14 a ve ra ge a m ou nt o f n eu tr op hi l time (day) nicotineamp non nicotine amp * * * nicotine-aquadest non nicotine-aquadest figure 1. the increasing pattern of the number of neutrophils between the nicotine exposure and treatment groups based on observation day 3, day 7 and day 14 between the neutrophils in nicotine + amp, nicotine + aqua dest, non-nicotine + amp and nonnicotine + aqua dest groups. *p < 0.05 table 1. the mean and standard deviation of the number of neutrophils in the kruskal wallis test in the nicotine + amp, non-nicotine + amp, nicotine + aqua dest and non-nicotine + aqua dest groups based on the day of observation amp aqua dest day 3 day 7 day 14 day 3 day 7 day 14 nicotine 4.17±1.72 1.83±0.75 2.50±0.84 3.00±2.00 3.00±1.79 3.00±1.26 non-nicotine 1.50±1.22 3.83±1.17 2.83±1.17 6.50±2.95 4.00±1.90 3.33±1.97 asymp. sig (p) 0.017* note: *p < 0.05 table 2. the mean and standard deviation of the number of macrophages in the kruskal wallis test in the nicotine + amp, nonnicotine + amp, nicotine + aqua dest and non-nicotine + aqua dest groups based on the day of observation amp aqua dest day 3 day 7 day 14 day 3 day 7 day 14 nicotine 5.50 ± 1.87 5.67 ± 4.08 8.50 ± 3.62 6.50 ±1.51 5.67±3.55 7.50±2.81 non-nicotine 5.50 ± 0.81 8.00 ± 5.02 8.50 ± 1.52 0.67±3.23 5.83±3.31 7.00±2.45 asymp. sig (p) 0.001* note: *p < 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 96andriani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 93–98 -2.00 0.00 2.00 4.00 6.00 8.00 10.00 day 3 day 7 day 14 a ve ra ge a m ou nt o f m ac ro ph ag e time (day) nicotineamp non nicotine amp * * nicotine-aquadest non nicotine-aquadest figure 2. the increasing pattern of the number of macrophages based on observation day 3, day 7 and day 14 between macrophage in nicotine + amp, nicotine + aqua dest, non-nicotine + amp and non-nicotine + aqua dest groups. *p < 0.05 the data in table 2 shows that there is a significant difference in the number of macrophages between the exposure and treatment groups p = 0.001 (p < 0.05). the test results showed that the average nicotine treatment was highest on day 14 on amp (figure 2). discussion based on the statistical results of the study a greater number of neutrophils and macrophages were observed in the periodontitis area in non-nicotine-exposed subjects compared to the nicotine-exposed. this shows that the inflammatory process is inhibited by nicotine through neutrophil activity and automatically reduces the function of macrophages in response to inflammation. based on ex vivo studies, it was stated that cigarette extract significantly (p < 0.001) resulted in minimal movement and a decreased neutrophil migration rate to where it could affect signal communication between neutrophil cells, which in turn could inhibit the production of neutrophils and other inflammatory cells. although neutrophils can still reach the infected area, it takes a longer time.17 the neutrophil count of the treatment of nicotine + amp on day 3 was lower than on day 7, with the possibility on the third day of new neutrophils slightly appearing and reaching their peak on days 5 and 6; unfortunately, on the fifth day of the study, neutrophil counts were not carried out. moreover, on the seventh day, the number of possible neutrophils started to decrease and was still decreasing on the fourteenth day. due to minimal movement and a decreased rate, this can cause more severe tissue damage mediated by neutrophils. in general, smoking can affect the success of neutrophils in eliminating pathogenic bacteria in periodontal disease.17 in their study, dhall et al. explained that nicotine can weaken monocytes to differentiate into macrophages, thereby interfering with the inflammatory process.18 likewise, research by ertugrul et al. stated that patients who smoked could reduce the bactericidal effect of antimicrobial proteins on periodontal tissue, which then caused damage to periodontal tissue.19 the number of neutrophils increased on day 3 compared to day 7, while neutrophil activity in the baseline periodontitis of subjects exposed to nicotine is inhibited because of the effects of nicotine that affect antimicrobial performance on neutrophils. neutrophils activity can be identified in the number of neutrophil in the subjects exposed to nicotine, which is lower than in the subjects without nicotine exposure, who were not inhibited by neutrophil activity in the inflammatory response. in a previous study, it was explained that one type of antimicrobial peptide, ll-37, which was found in neutrophils and gingival crevicular fluid, would be significantly higher in subjects with periodontitis than in healthy subjects.20 this is consistent with data on higher neutrophil counts in periodontitis subjects which were not exposed to nicotine. from an in vitro study, after 24 hours of administration of amp gel, it was identified that the metabolic activity of p. gingivalis bacteria decreased in the form of a loss of its ability to transfer resazurin to resorufin in the mitochondrial activity of bacterial cells.21 although different responses were seen in subjects without nicotine exposure, the number of neutrophils decreased in both the amp and control groups. this is because the high neutrophil count with no nicotine exposure is believed to have undergone apoptosis on day 3, leading to a decrease in the number of neutrophils. this is related to a previous study on neutrophils which stated that neutrophils in the tissue will undergo apoptosis after two days, which will then be phagocytized by macrophages.18 in contrast to the group of rats that were not exposed to nicotine on the third day, there was a decrease in the number of macrophages. it was explained in a study by reinke and sorg22 that on the third day after inflammation occurs, new macrophages move to the previous tissue that had experienced inflammation when neutrophils were found in the inflammatory tissue on day 2. on the third day of treatment, histological examination showed higher inflammation in macrophages and neutrophils in the amp treatment group than in the control group. according to the results, neutrophil and macrophage counts increased in nicotine-exposed subjects with amp dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 97 andriani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 93–98 therapy, indicating that neutrophil activity was restored to normal function in the inflammatory response after three days of topically applied artificial amp. on the seventh and fourteenth days of treatment, the amp treatment group had lower neutrophils than the control group, with or without nicotine exposure. this suggests that artificial amp improves in the acceleration of the periodontal healing process, which can be seen in the reduced inflammatory process in terms of the number of neutrophils. likewise, the number of macrophages on the seventh and fourteenth days of the amp treatment was higher than in the aqua dest control. this indicates that the inflammatory process returned to normal function on the seventh day of treatment. the effect of amp application was proven by lin et al., who researched wound-healing by employing an amp dressing mixed with alginate/ hyaluronic acid/collagen, wherein application of the amp dressing on day 7 had a significant effect in that it could reduce the number of s. aureus bacteria and e. coli on wound tissue properly and thereby accelerate the healing process.23 research by wang et al. also asserted that the application of amp can increase the ability of macrophage cells, as evidenced by the application of amp sublacin to inflammatory tissue, which can increase protein and mrna in il-1β, il-6 and tnf-α so that it can increase activity of macrophages in phagocytizing bacteria, dead cells or debris in inflammatory tissue.24 it is also evidenced by the research of wang et al. that one of the antimicrobial peptides, including salivary and gingival crevicular fluid (gcf) and known as ll-37, when administered to inflammatory tissue can increase the expression of cytokines in macrophages, which in turn increases the phagocytic ability of macrophages.25 in conclusion, this study has shown that the administration of amp, by examining the amount of neutrophils and macrophages, affects the regenerationsof. periodontitis in nicotine-exposed rats. acknowledgments the authors thank lp3m muhammadiyah university of yogyakarta for the support of the grants provided and the molecular medicine and therapy research laboratory, faculty of medicine and health sciences, muhammadiyah university of yogyakarta for their support in this research. references 1. loos bg, needleman i. endpoints of active periodontal therapy. j clin periodontol. 2020; 47(suppl 2): 61–71. 2. kanmaz m, kanmaz b, buduneli n. periodontal treatment outcomes in smokers: a narrative review. tob induc dis. 2021; 19: 77. 3. chrysanthopoulou a, mitroulis i, apostolidou e, arelaki s, mikroulis d, konstantinidis t, sivridis e, koffa m, giatromanolaki a, boumpas dt, ritis k, kambas k. neutrophil extracellular traps promote differentiation and function of fibroblasts. j pathol. 2014; 233(3): 294–307. 4. cor t és-viey ra r , rosa les c, ur ib e q uerol e . neut roph i l functions in periodontal homeostasis. j immunol res. 2016; 2016: 1396106. 5. kesim s, kılıc d, ozdamar s, liman n. effect of smoking o n a t t a c h m e n t of h u m a n p e r i o d o n t a l l ig a m e n t c e l l s t o periodontally involved root surfaces following enamel matrix der ivative application. biotechnol biotechnol equip. 2012; 26(5): 3215–9. 6. hajishengallis g. periodontitis: from microbial immune subversion to system ic inf la m mation. nat rev i m munol. 2015; 15(1): 30–44. 7. panpradit n, nilmoje t, kasetsuwan j, sangk hamanee ss, surarit r. effect of nicotine and porphyromonas gingivalis on the differentiation properties of periodontal ligament fibroblasts. eur j dent. 2021; 15(4): 727–32. 8. lei j, sun l, huang s, zhu c, li p, he j, mackey v, coy dh, he q. the antimicrobial peptides and their potential clinical applications. am j transl res. 2019; 11(7): 3919–31. 9. mahlapuu m, håkansson j, ringstad l, björn c. antimicrobial peptides: an emerging category of therapeutic agents. front cell infect microbiol. 2016; 6: 194. 10. gorr s-u, abdolhosseini m. antimicrobial peptides and periodontal disease. j clin periodontol. 2011; 38(suppl 1): 126–41. 11. xue c, li l. inhibitory effect of antimicrobial peptides rise-ap12® on porphyromonas gingivalis. chinese j pract stomatol. 2014; 7(4): 217–20. 12. mateescu m, baixe s, ga r nier t, jier r y l, ball v, haikel y, met z-bout ig ue m h , na r d i n m , sch a a f p, e t ien ne o, lavalle p. antibacterial peptide-based gel for prevention of medical implanted-device infection. plos one. 2015; 10(12): e0145143. 13. karatas o, balci yuce h, tulu f, taskan mm, gevrek f, toker h. evaluation of apoptosis and hypoxia-related factors in gingival tissues of smoker and non-smoker per iodontitis patients. j periodontal res. 2020; 55(3): 392–9. 14. krisanaprakornkit s, khongkhunthian s. the role of antimicrobial p ept ide s i n p e r io dont a l d ise a se ( pa r t i ): a n ove r v iew of human defensins and cathelicidin. thai j periodont. 2010; (1): 33–44. 15. ionel a, lucaciu o, moga m, buhatel d, ilea a, tabaran f, catoi c, berce c, toader s, campian rs. periodontal disease induced in wistar rats experimental study. hum vet med. 2015; 7(2): 90–5. 16. kubot a m, ya nag it a m, mor i k , hasegawa s, ya mash it a m, ya ma d a s, k it a mu r a m, mu r a k a m i s. t he ef fe ct s of c iga r et t e smoke c ond en sat e a nd n ic ot i ne on p e r io dont a l tissue in a periodontitis model mouse. plos one. 2016; 11(5): e0155594. 17. nicu ea, rijkschroeff p, wartewig e, nazmi k, loos bg. cha racter ization of oral polymor phonuclea r neutrophils in periodontitis patients: a case-control study. bmc oral health. 2018; 18(1): 149. 18. dhall s, alamat r, castro a, sarker ah, mao j-h, chan a, hang b, martins-green m. tobacco toxins deposited on surfaces (third hand smoke) impair wound healing. clin sci (lond). 2016; 130(14): 1269–84. 19. ertugrul as, sahin h, dikilitas a, alpaslan nz, bozoğlan a, tekin y. gingival crevicular fluid levels of human beta-defensin-2 and cathelicidin in smoker and non-smoker patients: a cross-sectional study. j periodontal res. 2014; 49(3): 282–9. 20. novak mj, novak kf, preshaw pm. smoking and periodontal disease. in: newman m, takei h, klokkevold p, carranza f, editors. carranza’s clinical periodontology. 11th ed. st. louis: saunders elsevier; 2012. p. 294–303. 21. papapanou pn, sanz m, buduneli n, dietrich t, feres m, fine dh, flemmig tf, garcia r, giannobile w v, graziani f, greenwell h, herrera d, kao rt, kebschull m, kinane df, kirkwood kl, kocher t, kornman ks, kumar ps, loos bg, machtei e, meng h, mombelli a, needleman i, offenbacher s, seymour gj, teles r, tonetti ms. periodontitis: consensus report of workgroup 2 of the 2017 world workshop on the classification of periodontal and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 98andriani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 93–98 peri-implant diseases and conditions. j periodontol. 2018; 89(suppl 1): s173–82. 22. reinke jm, sorg h. wound repair and regeneration. eur surg res. 2012; 49(1): 35–43. 23. lin z, wu t, wang w, li b, wang m, chen l, xia h, zhang t. biofunctions of antimicrobial peptide-conjugated alginate/ hyaluronic acid/collagen wound dressings promote wound healing of a mixed-bacteria-infected wound. int j biol macromol. 2019; 140: 330–42. 24. wang s, ye q, wang k, zeng x, huang s, yu h, ge q, qi d, q iao s. e n ha nc ement of m a c rophage f u nct ion by t he antimicrobial peptide sublancin protects mice from methicillinresistant staphylococcus aureus. j immunol res. 2019; 2019: 3979352. 25. wan m, van der does am, tang x, lindbom l, agerberth b, haeggström jz. antimicrobial peptide ll-37 promotes bacterial phagocytosis by human macrophages. j leukoc biol. 2014; 95(6): 971–81. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p93–98 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p93-98 185 vol. 42. no. 4 october–december 2009 detection of aggressive periodontitis by calprotectin expression desi sandra sari1 and suryono2 1department of periodontics, faculty of dentistry, university of jemberperiodontics, faculty of dentistry, university of jember faculty of dentistry, university of jember 2department of periodontics, faculty of dentistry, university of gadjah madaperiodontics, faculty of dentistry, university of gadjah mada, faculty of dentistry, university of gadjah mada abstract background: calprotectin is a calcium-binding protein expressed by neutrophil, monocytes, gingival keratinocytes, and oral epithelial cells. the concentrations of calprotectin increase in plasma, urine and synovial fluid of patients with inflammatory diseases. this protein is known as a marker for periodontal diseases and is detected in gingival crevicular fluids. purpose: this study was aimed to investigate the detection of inflammation on the aggressive periodontitis by calprotectin expression. method: the gingival crevicular fluids were taken from five aggressive periodontitis patients and five healthy subjects by using sterile paper points. calprotectin expression was analyzed by elisa technique. result: the results showed the significant difference in calprotectin expression between subject with aggressive periodontitis and healthy subjects p = 0.002 (p < 0.05). conclusion: it was concluded that the calprotectin expression on the aggressive periodontitis patients may be useful for evaluation the progression of inflammation in periodontitis. key words: calprotectin, aggressive periodontitis, gingival crevicular fluids correspondence: desi sandra sari, c/o: bagian periodonsia, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember.jl. kalimantan 37 jember. e-mail: desisandrasari@yahoo.com research report introduction aggressive periodontitis is a type of periodontitis attacking adult people in the age of 20 to 35 years old. the character of aggressive periodontitis is the fast and severe damage of periodontal tissue with gingival inflammation, bleeding, and exudation followed by the lost of alveolar bone in months, but it does not correspond with the amount of plague and calculus.1,2 aggressive periodontitis can occur locally or generally. the special mark of local aggressive periodontitis is started from the puberty period with the damage of the first insisive and molar alveolar bone. the damage of alveolar bone in general type occurs in almost all teeth. during the active phase, the development of aggressive periodontitis is marked by the inflammation of development gingival tissue with the proliferation in margin, and then is followed with the inactive phase, or even stops with or without gingivitis history.3 aggressive periodontitis is actually caused by the growth of negative periodontal pathogen in its gingival cycle followed by the inflammatory respond of immunity in the vulnerable host. thus, the patient of aggressive periodontitis has sensitive aspect in responding immune such as abnormality of neutrophile cells in the terms of chemotaxis, phagocytosis, adherence, and bactericide activities.1 neutrophile is very important during inflammation, especially in the mechanism of immunity towards the infection of periodontophatogenic bacteria. the function of neutrophile is to protect the integrity of periodontal tissue. however, in the severe periodontal disease the capability of neutrophile seems to decrease in controlling pathogenic bacteria.4 cytoplasm of neutrophile actually contains many anti-microbes inside its granules, myeloperoxides, defensin, elastase, proteinase, cathepsin g, azurosidin, laktoferin, lisosom, and calprotectin. the concentration of calprotectin in the cytoplasm of neutrophile is about 40–60%.5 calprotectin is protein binding with calcium contained in neutrophile, monocyte, keratinocyte cells, and epithelial cells with the molecular mass about 36.5 kda. calprotectin actually is also known as macrophage migration inhibitory factor-related protein 8 and 14 (mrp8 and mrp14), cystic 186 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 185-188 fibrosis antigen, calgranulin a and b, and s1000a8 and s1000a9. in healthy gingival epithelia, calprotectin is located in stratum spinosum cells, and during inflammation calprotectin can also be detected in stratum spinosum and granulosum cells.6,7 calprotectin is produced by neutrophile, monocyte, macrophag, keratinocyte cells and epithelial cells. the concentration of calprotectin increases in patients with infection, tumor, and allergic reaction. the concentration of calprotectin located in plasma, urine, and synovial fluids of patients with inflammatory diseases, such as pneumonia, meningitis, urine duct infection, rheumatoid arthritis, and cystic fibrosis. the concentration also increases compared with healthy patients.8 in addition, the in vitro study shows that calprotectin has anti-microbe function, bacteriastatics and fungistatics with minimal inhibitory concentration (mic) that is similar with some antibiotics. the mechanism of this action is actually caused by the increasing of bacteria binding zinc needed for proliferation.9 calprotectin, therefore, is not only found in calculus, but also in gingival cervicular fluid in which its concentration in the periodontal disease patients is higher than that in healthy patients.10 the fast damage occurred in patients with aggressive periodontitis is actually related more with the chemotaxis dysfunction of neutrophile and monocyte. since the normal role of neutrophile and monocyte in producing possibly has been changed, the chemotaxis dysfunction of neutrophile can get problem causing the susceptibility of immune system towards periodontophatic bacteria.7 calprotectin is secreted by neutrophile during the process of inflammation, and it also has an important role in the mechanism of body immune towards periodontal inflammation. the reason is because the concentration of calprotectin that detected in the gingival crevicular fluids can be potentially used as the maker of the clinical inflammation of periodontal disease. the concentration of calprotectin in the gingival crevicular fluids can also reflect the severity of the inflammation in patients with periodontal disease.7,8 thus, in order to analyze the mechanism or pathogenesis of this disease, the concentration of calprotectin showing the degree of the inflammation in those patients with aggressive periodontitis must be examined. the aim of the study was to detect the inflammation on patients with aggressive periodontitis by calprotectin expression in the gingival crevicular fluids. material and method the type of the study conducted was observational with laboratory approach. before the study was conducted, ethical clearance must be set up and approved by ethic commetee of health institusional. five patients with aggressive periodontitis and five healthy patients had also approved informed consent. the criteria of sample, moreover, were that they must be in the age of 20–35 years old; without any systemic abnormality; have no smoking habit; using no mouthwash or antibiotics minimally for about the last six months; not under periodontal treatment minimally for about the last six months; and that they must not have pregnancy or periods. in addition, the dental element of aggressive periodontitis patients suffering inflammation (diseased site) had pocket with  6 mm depth, while the healthy dental element (healthy site) of the healthy patients had light pocket (£ 3 mm). firstly, teeth were cleaned by sterile cotton rolls in order to clean supragingival plague. then, gingival crevicular fluids was taken. after that, the sterile paper point was put into the pocket and abandoned for about 30 seconds. the paper point then was put into 0.5 ml eppendorf tube, covered and sealed with paraffin tape, put into ice box, and stored in deep freezer –30o c in order to examine the concentration of calprotectin. afterwards, calprotectin in gingival crevicular fluids was examined with enzyme linked immunosorbent assay (elisa) method. the sample then was dissolved by using extraction solution and had got centrifugation with 2500 rpm for 15 minutes in the temperature of 4 degrees celsius. the sample then was diluted about 50–500 times. for optimization, it can also be done by using diluent solution, and by having centrifugation with 2500 rpm for 15 minutes in the temperature of 4 degrees celsius. the sample then was applied in 96 wells of microtiter plate coated by specific rabbit antibody for calprotectin. the application of the sample into each plate was about 50 μl. those plates then were covered by aluminium foil paper and incubated for 45 minutes in the room temperature. after that, the fluid was discharged by suction using washing buffer fluids. then, they were added by conjugated antibody enzyme, incubated again for 45 minutes at temperature of a dark room, and washed about 5 times. substract solution enzyme was added into each of those wells for about 100 ml in the room without light for about 20 minutes. the result of the average calprotectin concentration then was measured with optical densities of elisa reader 405 nm (biorad bench mark). one hundred ml stop solution (naoh) was also added into each of those wells. the data then was analyzed with t-test in order to find the difference of the calprotectin concentration between the patients with aggressive periodontitis and the healthy patients with the reliability degree 95%. result the respondents of the study were 10 people (21–35 years old) divided into 2 groups, with the average age of the subjects 29.3 years. the result of the study showed that the average and standard deviation of calprotectin concentration in the aggressive periodontitis patients was about 2.55 ± 0.44, meanwhile in the healthy patients was about 0.96 ± 0.6 (figure 1), so that concentration of 187sari and suryono: detection of aggressive periodontitis calprotectin in the aggressive periodontitis patients was higher than that in the healthy patients. the result of t-test analysis showed f = 0.002 and sig = 0.965 (> 0.05). it indicated that both groups have the same variant. the result of the analysis also showed that there was a significant difference of calprotectin expression in the aggressive periodontitis patients and the healthy patients with t = 4.73 and sig (2-tailed) = 0.002 (p < 0.05). 0 0.5 1 1.5 2 2.5 3 concentration of calprotectin aggressive periodontitis patients healthy patients 0.002* note* significance p < 0.05 figure 1. the average of calprotectin concentration based on the group. discussion the main cause of aggressive periodontitis was the infection of periodontophatogen bacteria in subgingival area. the species of bacteria colonizing in periodontal pocket area must adhere to the surface in order to prevent fluids from gingival crevicular fluids. the bacteria then might infuse into the main tissue through lesion of the gingival crevicular epithel or pocket, and infuse into the gingival tissue. another route of the bacteria in invading the tissue was by direct penetration into the main epithel or bound tissue. p. gingvalis, p. intermedia, a. actinomycetemcomitan, f. nucleatum, and t. denticola bacteria, could invade directly to the cells of the main tissue.11 the previous study actually has successfully i d e n t i f i e d e i g h t b a c t e r i a i n s u b g i n g i v a l p l a g u e , a. actinomycetemcomitan, t. denticola, f. nucleatum, p. intermedia, p. gingivalis, e. corrodens, t. forsythia, and c. rectus in the bleeding area, and only three of them are very patogen in aggressive periodontitis patients, which are a. actinomycetemcomitan, p. gingivalis, and p. intermedia.12 p. gingivalis and p. intermedia bacteria dominate periodontal pocket area of aggressive periodontitis patients, i.e. about almost 85%, compared with that of chronic aggressive periodontitis patients, about 65%. those anaerob negative gram bacteria are able to invade mucosa barrier and infuse into epithelial cells causing destruction in periodontal tissue because of the product of bacteria, lipopolysaccaride.13 the previous study conducted by kido et al.,14 also showed that lipopolysaccaride of periodontopathic bacteria can cause the in vitro discharging of calprotectin from neutrophile. however, the stimulation of lipopolysaccaride from p. gingivalis can also cause the discharging of calprotectin increasing about 15 times compared with the control one. the discharging of calprotectin is actually caused by lps and inflammatory cytokine, such as tnf-a, il-1b, and pge2. tnf-a and il-1b cause the significant production of calprotectin after 24 to 72 hours in monocyte. the concentration of calprotectin in gingival crevicular fluids relates with the clinical indicator, especially the depth of pocket marker biochemical marker, tnf-a, il-1b, and pge2 in the periodontal disease. calprotectin has chemotaxis, adhesion, regulation, and migration activities of neutrophile and monocyte, and also has important role in body immunity as natural immune system for periodontal disease.15 the concentration of calprotectin increases 3–4 times in periodontitis patients compared with that in the healthy patients. the concentration of calprotectin in the pocket with more than of 7 mm depth becomes 2400 ng/ml. it showed that there was significantly positive correlation between the concentration of calprotectin and the depth of the pocket.10 the height of the calprotectin concentration in gingival crevicular fluids of periodontitis aggressive patients is caused by the increasing number of periodontopathic bacteria in the periodontal pocket, so lipopolysaccaride of periodontopathic bacteria can stimulate the secretion of calprotectin from neutrophile cells.16 when calprotectin is discharged from neutrophile through the signal line of lipopolysaccaride, lipopolysaccaride interacts with some receptors, such as: cd14 and toll-like receptor (tlr) that activate the function of nuclear factor kb (nf-kb), so it can cause the process of transcription in the nucleus and the process of translation in the ribosom, which then it is synthesized into calprotectin.8,17 the concentration of calprotectin in gingival crevicular fluids increases about 12 times compared with that in chronic periodontitis patients and in the healthy patients in the longitudinal study of aggressive periodontitis patients.16 calprotectin is discharged through physiologic mechanism during the lifespan of neutrophile cells, in which there are more than 93.2% of cell viability after it is incubated with lps p. gingivalis.17 calprotectin can also be secreted by neutrophile in extracellular way as the result of the cell damage or death. the increasing concentration of calprotectin in extracellular fluids is considered as the result of the active or passive secretion when neutrophile is accumulated in local inflammation.18 calprotectin is actually considered as chemotaxis factor that has a role in early immune of an inflammation. with the stimulation of lps calprotectin will be discharged and then will cause migration from neutrophile to inflammatory area.19 the susceptibility of the immune system of periodontal tissue towards periodontophatic bacteria in the aggressive periodontitis patients is caused by the functional alteration of chemotaxis and phagocytosis from neutrophile. the functional damage of phagocytosis occurs at the phase in 188 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 185-188 which bacteria adhere on the surface of neutrophile and then are internalized.1 the concentration of calprotectin in gingival crevicular fluids also relates with clinical indicator, the depth of probing and bleeding on probing (bop) in which the concentration of calprotectin is higher in the positive bop than that in the negative one.14 based on the result of this study, it can be concluded that calprotectin expression in the aggressive periodontitis patients was higher than that the healthy patients. the infection of periodontophatic bacteria through lipopolysaccaride could also cause the producing of calprotectin as the factor of chemotaxis which has a role in margination and initiation of neutrophile cells into the infection area. the high calprotectin expression in gingival crevicular fluids then reflected the degree of gingival inflammation in the aggressive periodontitis patients. therefore, the concentration of calprotectin in gingival crevicular fluids could be used to detect the early symptom of an inflammation in periodontal disease. references 1. meng h, xu l, li q, han j, zhao y. determinants of host susceptibility in aggressive periodontitis. periodontology 2000, 2007; 43: 133–59. 2. gajardo m, silva n, gómez l, león r, parra b, contreras a, gamonal j. prevalence of periodontopathic bacteria in aggressiveprevalence of periodontopathic bacteria in aggressive periodontitis patients in a chilean population. j periodontol 2005; 76(2): 289–94. 3. novak kf, novak mj. aggressive periodontitis. in: newman mg,in: newman mg, takei hh, klokkevold pr, editors. clinical periodontology. 10th ed. philadelphia: wb saunders; 2006. p. 506–11. 4. nisengard rj, haake sk, newman mg, miyasaki kt. microbial interaction with the host in periodontal disease. in: newman mg, takei hh, klokkevold pr, editors. clinical periodontology. 10th ed. philadelphia: wb saunders; 2006. p. 236–43. 5. levy o. antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. journal of leukocyte biology 2004; 76(5): 909–25. 6. hayashi n, kido j, kido r, wada c, kataoka m, shinohara y, nagata t. regulation of calprotectin expression by interleukin-1a and transforming growth factor-b in human gingival keratinocytes. j periodont res 2007; 42(1): 1–7. 7. kido j, kido r, suryono, kataoka m, fagerhol mk, nagata t. induction of calprotectin release by porphyromonas gingivalis lipopolysaccharide in human neutrophils. oral microbiology immunology 2004; 19: 182–7. 8. suryono, kido j, hayashi n, kataoka m, nagata t. calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-b, and interleukin-1a . j periodontol 2005; 76: 437–42. 9. mørk g, schjerven h, mangschau l, soyland e, brandtzaeg p. proinflammatory cytokine upregulate expression of calprotectin (l1 protein, mrp-8/mrp-14) in cultured human keratinocytes. british journal of dermatology 2003; 149: 484–91. 10. suryono, kido j, hayashi n, kataoka m, nagata t. effect of porphyromonas gingivalis lipopolysaccharide, tumor necrosis factorb, and interleukin-1a on calprotectin release in human monocytes. j periodontol 2003; 74: 1719–24. 11. nisengard r, haake sk, newman mg, miyasaki kt. microbial interaction with the host in periodontal disease. in: newman mg, takei hh, klokkevold pr, editors. clinical periodontology. 10th ed. philadelphia: wb saunders; 2006. p. 236–43. 12. vinayak mj, vandana kl. the detection of eight putative periodontal pathogen in adult and rapidly progressive periodontitis patient. indian journal of dental research 2007; 18(1): 6–10. 13. botero je, contreras a, lafaurie g, jaramillo a, betancourt m, arce rg. occurence of periodontopathic and superinfecting bacteria in chronic and aggressive periodontitis subjects in a colombian population. j periodontol 2007; 78: 696–704. 14. kido j, kido r, suryono, kataoka m, fagerhol mk, nagata t. induction of calprotectin release by porphyromonas gingivalis lipopolysaccharide in human neutrophils. oral microbiology immunology 2004; 19: 182–7. 15. suryono, kido j, hayashi n, kataoka m, shinohara y, nagata t. norepinephrine stimulates calprotectin expression in human monocytes cells. j periodont res 2006; 41: 159–64. 16. kaner d, bernimoulin j, kleber b, heizman wr, friedmann a. gingival crevicular fluid levels of calprotectin and myeloperoxidase during therapy for generalized aggressive periodontitis. j periodont res 2006; 41: 132–9. 17. kido j, kido r, suryono, kataoka m, fagerhol mk, nagata t. calprotectin release from human neutrophils is induced by porphyromonas gingivlis lipopolysaccharide via the cd-14-tolllike receptor-nuclear factor b pathway. j periodont res 2003; 38: 557–63. 18. stríz i, trebichavský i. calprotectin a pleiotropic molecule in acute and chronic inflammation. physiol res 2004; 53(3): 245–53. 19. ryckman c, vandal k, rouleau p, talbot m, tessier pa. proinflamatory activities of s100a8, s100a9, and s100a8/a9 induce neutrophil chemotaxis and adhesion. the journal of immunologythe journal of immunology 2003; 170: 3233–42. vol 44 no 3 sept 2011.indd 145 vol. 44. no. 3 september 2011 threshold value of enamel mineral solubility and dental erosion after consuming acidic soft drinks muhammad ilyas department of dental public health faculty of dentistry, hasanuddin university makassar indonesia abstract background: dental erosion is irreversible and can caused by acidic soft drink consumption. dental erosion prevention had already been done, but it still has not been satisfying since the consumption of acidic soft drink is still high. there is still no explanation about the threshold value of enamel mineral solubility and the occurance of dental erosion after consuming acidic soft drink. purpose: this research is aimed to find the threshold value of enamel mineral solubility and dental erosion before and after consuming acidic soft drinks. methods: subjects of the research are saliva and enamel of 12 rabbits, which have some criteria such as age > 70 days, body weight > 600 grams, and teeth considered to be healthy. the sample devided equally into 4 groups. each of those marmooths was given a drink as much as 2.5 cc/consumption (there are 1, 2 and 3× per day) by using syringe without injection needle. salivary minerals then were examined by using atomic absorption spectrophotometric (ass), while dental erosion was examined using scanning electron microscop (sem). the data were analyzed by using paired t-test. results: it is known that the threshold value of enamel mineral solubility (k, na, fe, mg, cl, p, ca, f, c) has significant difference (p < 0.05) after being exposed to folic acid. meanwhile, fe did not have significant difference (p = 0.090) after being exposed to citric acid. similarly, c did not have significant difference (p = 0.063) after being exposed to bicarbonate acid. furthermore, it is also known that the threshold time value of dental erosion are on the 105th day for folic acid, on the 111th day for citric acid, and on the 117th day for bicarbonate acid. conclusion: threshold value of enamel mineral solubility before and after consuming soft drinks containing acid is different. based on the threshold value of dental erosion, it is known that folic acid is the most erosive acid. key words: soft drinks, threshold value, mineral solubility, dental erosion abstrak latar belakang: erosi gigi bersifat irreversible disebabkan oleh konsumsi minuman ringan yang mengandung asam. pencegahan erosi gigi telah dilakukan tetapi hasilnya tidak memuaskan karena masih banyak orang selalu mengkonsumsi minuman ringan yang berasam. tidak ada satupun yang menjelaskan lebih terperinci tentang perbedaan nilai ambang kelarutan email dan waktu erosi gigi setelah konsumsi minuman ringan yang berasam. tujuan: penelitian ini dilakukan untuk mengetahui perbedaan nilai ambang kelarutan mineral email dan erosi gigi sebelum dan setelah mengkonsumsi minuman ringan yang mengandung asam. metode: subjek dari penelitian ini adalah saliva dan enamel dari 12 ekor kelinci dengan criteria usia lebih dari 70 hari, berat lebih dari 600 gram, dan gigi dalam keadaan sehat. sampel dibagi menjadi 4 kelompok. masing-masing kelinci diberikan 2,5 ml minuman sekali konsumsi (1, 2, dan 3 kali sehari) menggunakan spite tanpa jarum. mineral saliva dianalisa menggunakan atomic absorption spectrophotometric (ass), sedangkan erosi gigi diperiksa dengan menggunakan scanning electron microscop (sem). data analisa dengan paired-t test. hasil: semua mineral email (k, na, fe, mg, cl, p, ca, f, c) nilai ambang kelarutannya berbeda secara bermakna sebelum dan setelah terpapar oleh asam folat (p < 0,05). sebelum dan setelah terpapar oleh asam sitrat nilai ambang kelarutan mineral fe tidak bermakna (p = 0,090), sebelum dan setelah terpapar oleh asam bikarbonat nilai ambang kelarutan mineral c tidak bermakna (p = 0,063). nilai ambang waktu erosi gigi didapatkan pada hari ke 105 untuk asam folat, hari ke 111 untuk asam sitrat dan hari ke 117 untuk asam bikarbonat. kesimpulan: nilai ambang kelarutan mineral email sebelum dan setelah konsumsi minuman ringan mengandung asam berbeda. berdasarkan nilai ambang erosi gigi, dapat diketahui bahwa asam folat merupakan asam yang paling erosif. kata kunci: minuman ringan, nilai ambang, kelarutan mineral, erosi gigi correspondence: muhammad ilyas, c/o: bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas hasanuddin. jl. perintis kemerdekaan km 10 kampus tamalanrea ujung pandang, indonesia. email: ilyasmils@yahoo.com research report 146 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 145–149 introduction there are 17% teens, more than 35% athletes in america, 20–30% athletes in australia, and 57% children aged 14 years in birminghan uk who have the prevalence of dental erosion due to the consumption of acidic soft drinks.1 another researcher also reported that consuming alcoholic beverages can cause regurgitation of stomach acid into the oral cavity so that ph in the mouth is decreasing and causes the solubility process of enamel and dentine minerals.7,10 the socio-economic conditions can also affect the occurrence of dental erosion. children with good socioeconomic condition usually are diligent to brush their teeth, but with wrong method, so they still can get dental abrasion. dental erosion will appear if the dental abrasion is not treated immediately and soft drinks containing acid are still consumed dental erosion.2–6 factors triggering the solubility of dental enamel minerals and dental erosion have actually been known, but none of researchers gives more details about the different threshold value of the dental enamel mineral solubility and the threshold time value of the dental erosion after consuming acidic soft drinks. the prevention of dental erosion has actually been done, but still not satisfied enough since acidic soft drinks are still consumed. mouth ph is above 5.5, through the process of salivary buffers it can cause supersaturation of ca+2 and po4-3 ions. in this situation, the dental hard tissues will pull minerals from saliva, called remineralization. on the other hand, if mouth ph below the critical point (≤ 5.5), it can cause subsaturation of ca+2 and po4-3 ions, the releasing of minerals into dental saliva, called demineralization. if this situation occurs repeatedly, it then can cause dental erosion.2–4 clinical symptoms of dental erosion are shiny enamel and dentin, as well as pain in incisal, palatal, labial, around cemento enamel junction of maxillary and mandibulary anterior and premolar tooth areas. the pain occurs with the short duration when the teeth are exposed to temperature, osmotics, chemical substances, both during brushing teeth and during consuming soft drinks.5–10 the purpose of this research was to find the threshold value of enamel mineral solubility and dental erosion before and after consuming acidic soft drinks. materials and methods this research is a quasi experimental research using pre-test and post-test designs at the laboratory gramik, medical faculy of airlangga university and bptp, maros regency in south sulawesi. research subjects; saliva and dental enamel of 12 rabbits (cavia aperea) divided into 4 groups (3 rabbits for bicarbonate acid, 3 rabbits for citric acid, 3 rabbits for folic acid, and 3 rabbits for mineral water) with age ≥ 70 days, weight ≥ 600 grams, and healty teeth. each of those rabbits then was given a drink as much as 2.5 cc per consumption (there are 1, 2 and 3× per day) by using a syringe without injection needles. the dental mineral in their saliva was examined before and after consuming soft drinks and mineral water by using atomic absorption spectrophotometric (ass). re-examination then was conducted with the first 5-minute intervals until the 35th minute after drinking, both in the treatment group and in the control group. the research was stopped after the raising of clinical symptoms of dental erosion. next, those marmmoths were slaughted and their teeth which got dental erosion or not were extracted. then, further examination was conducted. the dental crowns were separated from the dental roots by cutting the cervical area with high speed diomond burs, and then made dental enamel histopathological preparation with a size 4x6 mm for examining dental erosion under scanning electron microscop (sem). the data was analyzed by using paired t-test, in order to know the difference of the threshold value of enamel mineral solubility and dental erosion caused by acidic soft drinks. results based on the examination of sem with 750× magnfication, it is known that the dental enamel of the samples got erosion, in which their dental enamel surface became rough or full of big and small pores, while enamel surface that did not get erosion still smooth. based on sem result most of the dental enamel surface of the samples became rough marked by large and small pores indicating the occurence of dental erosion caused by soft drinks containing citric acid (figure 1). similarly, figure 2 shows some pores as big as those in figure 1. besides that, it also shows lines of cracks on the enamel surface of those marmmoths caused by the consumption of soft drinks containing bicarbonate acid. most of the dental enamel surface of the samples became rough marked by pores caused by the consumption of soft drinks containing folic acid (figure 3). figure 4 shows that most of the dental enamel surface of the samples became smooth indicating that there was no occurance of dental erosion caused by the consumption of mineral water. most of the the enamel minerals found in the saliva have significant difference of the solubility threshold values before and after consuming soft drinks containing citric acid, p < 0.05, except fe (p = 0.05) (table 1). in the other hand, most of the the enamel minerals found in the saliva have significant difference of the solubility threshold values before and after consuming soft drinks containing bicarbonate acid, p < 0.05, except c (p > 0.05) (table 2). 147ilyas: threshold value of enamel mineral table 1. the different threshold value of enamel mineral solubility in the saliva before and after consuming soft drinks containing citric acid mineral mean ± sd p before after k 157.00 ± 59.76 777.72 ± 707.26 0.001 na 820.00 ± 460.42 1144.68 ± 740.63 0.001 fe 40.01 ± 32.60 36.13 ± 28.21 0.090 mg 2.83 ± 1.22 10.18 ± 6.14 0.001 cl 36.66 ±14.61 48.72 ± 24.24 0.001 p 20.07 ± 7.44 71.05 ± 48.19 0.001 ca 74.82 ± 10.03 191.49 ± 114.79 0.001 f 163.16 ± 58.35 453.36 ± 245.13 0.001 c 205.66 ± 126.82 501.27 ± 169.67 0.001 sd: standard deviation, p: probability figure 1. the dental erosion occured (arrows) is caused of citric acid marked by the presence of pores. table 2. the different threshold value of enamel mineral solubility in the saliva before and after consuming soft drinks containing bicarbonate acid mineral mean ± sd p before after k 205,667 ± 67,236 718,682 ± 563,733 0.013 na 228,667 ± 45,386 542,409 ± 265,097 0.001 fe 5,167 ± 2,041 10,909 ± 3,294 0.001 mg 3,833 ± 1,169 11,500 ± 6,739 0.001 cl 66,667 ± 35,601 146,182 ± 83,211 0.001 p 248,003 ± 87,504 485,364 ± 202,732 0.001 ca 131,502 ± 38,501 320,591 ± 177,430 0.001 f 211,005 ± 52,014 493,591 ± 200,100 0.001 c 781,833 ± 287,482 615,818 ± 216,896 0.063 sd: standard deviation, p: probability figure 2. the dental erosion (arrow) occured is caused of bicarbonate acid marked by the presence of pores. table 3. the different threshold value of enamel mineral solubility in the saliva before and after consuming soft drinks containing folic acid mineral before mean ± sd after mean ± sd p k 241.23 ± 163.00 763.13 ± 522.48 0,013 na 450.66 ± 257.58 700.77 ± 411.98 0.001 fe 14.83 ± 4.23 22.03 ± 17.59 0.001 mg 6.66 ± 4.17 20.682 ± 14.90 0.001 cl 105.66 ± 72.15 345.40 ± 53.34 0.001 p 210.00 ± 49.32 283.81 ± 203.03 0.007 ca 197.66 ± 15.27 463.45 ± 274.47 0.001 f 177.16 ± 72.10 482.81 ± 200.35 0.001 c 395.83 ± 174.64 722.63 ± 255.33 0.001 sd: standard deviation, p: probability all of the enamel minerals found in the saliva can be indicated to have significant difference of the solubility threshold values before and after consuming soft drinks containing folic acid, (p < 0.05) (table 3). figure 3. the dental erosion (arrow) occured is caused of folic acid marked by the presence of pores. it indicated that soft drinks containing folic acid is the kind of soft drinks that can cause dental erosion more than those containing citric acid and bicarbonate acid, either on the frequency of consumption of 1 time, 2 times, or 3 times per day (table 4). figure 4. the smooth surface of the dental enamel (no erosion) after consuming mineral water on the examination (sem figure with magnification 750×). 148 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 145–149 discussion enamel consists of some minerals (k, na, fe, mg, cl, p, ca, f, c), and is also considered to be as a hard tissue. although considered as a hard tissue, enamel can still be dissolved by acidic chemicals. these acids are not only intrinsic (gastroesophageal reflux, vomiting), but also extrinsic (bicarbonate acid, citric acid or folic acid). thus, if mouth is always tasted acidic, it can indicate that its dental enamel gets mineral solubility, called demineralization. this demineralization of enamel actually occurs through a diffusion process, a process in which minerals are transfered from the dental enamel into the saliva due to the different acid concentration in the dental enamel and in the saliva. therefore, beverages with the high concentration of acid and the low ph will be able to diffuse into enamel through the tubuli of the enamel containing water and organic matrix or proteins. after the acid diffuses into the enamel, it is then ionized into h + and l-that can destruct calcium hydroxyapatite, and break them down into some ions, ca+2, oh –, po4 –3 and f-1. the ions formed then diffuses into the enamel and form complex compounds, ca (h2po4)2, cahpo4 and cacl2. these complex compounds with the high concentration diffuses out into the saliva. if this process always occurs, it can cause dental erosion. on the other side, if ph of mouth is high, the remineralization process will occur which can make the enamel pulls those ions from the saliva.9–11 there is significant difference of the threshold value of enamel mineral solubility in the saliva before and after consuming soft drinks containing citric acid, except fe. this is not only due to the lack of fe in dental enamel, as a result, it becomes difficult to be detected, but also due to the nature of the fe that is reactive causing reaction under acidic or basa condition.16 each mineral actually has a different solubility marked by the increasing of enamel mineral level in the saliva. this process is caused by citric acid obtained from soft drinks that have low ph, below the critical point (< 5.5).14,17 if it is found that enamel mineral generally has the significant difference of the threshold value of solubility before and after consuming soft drinks containing bicarbonate acid, except mineral c. this result is due to the nature of mineral c that can bind itself in small chains (cc, c = c and c ξ c). with this nature, mineral c can not be dissolved by bicarbonate acid which is weaker than and citric acid or folic acid.16 there is no significant difference in all of the enamel mineral before and after exposured to folic acid from soft drinks. citric acid and bicarbonate cannot dissolve all dental enamel minerals. this condition is actually caused by the differences of ph in all the three soft drinks, ph of folic acid is lower than that of citric acid and bicarbonate. this condition is actually in accordance with the opinion of earlier researchers who stated that most of soft drinks are acidic, but with different ph.14 it is also known that most of mineral k obtained is dissolved caused by the very quickly melting point of k.16 dental erosion occurs faster due to the consumption of soft drinks containing folic acid than that containg bicarbonate acid and citric acid. thus, it is proved that folic acid from soft drinks is more erosive. soft drinks containing folic acid are factor causing dental erosion, either on the frequency of consumption 1 time, 2 times, or 3 times per day. therefore, to avoid the exposure of soft drinks containing acid to teeth, it is better to use a pipette during consuming the soft drinks and then to gargle with water or mineral water in order to neutralize ph of the mouth before the solubility of dental mineral occurs. it is concluded treshold value of enamel mineral solubility before and after consuming soft drinks containing acid is defferent. based on the threshold value of dental erosion, it is known that folic acid is the most erosive acid. references 1. dlaigan yh, shaw l, smith aj. dental erosion in a group at british 14 year old, school children part iii influence of oral hygiene practises. british dent j 2002 11; 192(9): 2–3. 2. feathersone jd. diffusion phenomena during artificial erosion lesion formation. dent res j bristol 1997; 4: 8. 3. samuel smw, rubinstein c. micro hardness of enamel restored with fluoride and non fluoride releasing dental materials. braz dent j 2001; 12(1): 1–2. 4. schuurs. patologi gigi geligi. yogyakarta: gajah mada university pess; p. 164–73. 5. zandim dl, correa fob, sampaio jec, junior cr. influence of exposure of dentinal tubules: a sem evaluation. braz oral res 2004; 18(1): 63–8. 6. correa fob, sampaio jec, junior cr, orrico srp. influence of natural the smear layer from root surfaces an in vitro study. j canadian dent assoc 2004 nov; 70(10): 697–701. table 4. the description of the threshold value of dental erosion based on the time and frequency of the consumption of both soft drinks containing acid and mineral water (as a control group) consumption frequency of consumption teeth time of erosion (day) time of no erosion (day) bicarbonate acid 1× 140 2× 136 3× 117 citric acid 1× 135 2× 126 3× 111 1× 131 folic acid 2× 120 3× 105 1× 140 mineral water 2× 140 3× 140 149ilyas: threshold value of enamel mineral 7. bell ej, kaidonis j, townsend g, richard l. comparison of exposed dentinal surfaces resulting from abrasion and erosion. austr dent j 1998; 43(5); 362–4. 8. mok tb, mc. intyre, hunt d. dental erosion in vitro model of wine assessor’s erosion. austr dent j 2001; 46(14): 263–4. 9. brearly l, morgan mv. acidic diet and dental erosion among. austr dent j 2002; 20: 28. 10. linnet v, seow, connor f. oral health of children with gastro oesofageal reflux disease. a controlled study. austr dent j 2002; 47: 156–7. 11. ungehusak c, mongkolehaiarunya s, rattanarungsima k. risk factors on dental erosion among swimmers. fact sheet dental health 2003; 2(5): 2, 3. 12. sullivan eao. a new index for the measurement of erosion in children. european j paediatric dentistry 2000; 1: 69–70. 13. valena v, young wg. dental erosian patterns from intrinsic acid regurgitation and vomiting. australian dent j 2002; 47(2): 106–16. 14. simpson a, shaw l, smith aj. tooth surface ph during drinking at black tea. br dent j 2004; 190(7): 374–6. 15. smith jb, mangkoewidjojo s. pemeliharaan, pembiakan dan penggunaan hewan percobaan di daerah tropis. international development program of australian universities and colleges. jakarta: ui press; 1999. p. 65. 16. cotton fa, wilkinson g. 1976. kimia anorganik dasar. suharto s, editor. jakarta: ui-pres; 1999. p. 462–5. 17. winarno fg. kimia pangan dan gizi. jakarta: gramedia pustaka utama; 2002. p. 221. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 229229 case report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 229–233 management of herpes labialis triggered by emotional stress herlambang prehananto1 and kus harijanti2 1 department of oral medicine, institut ilmu kesehatan bhakti wiyata, kediri indonesia 2department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: herpes labialis is a form of secondary or recurrence of primary herpes simplex infection. herpes simplex virus is latent. it can reactivate due to reactivation of the virus induced by emotional stress, high fever, ultraviolet exposed, oral mucosal or nerve tissue trauma, immunosuppression condition, and hormonal disorders. purpose: the study aimed to report the management of patients with herpes labialis on the lower lip triggered by emotional stress. case: a 58 year-old woman complained of pain in her lower lip. the patient had suffered from the pain since one month ago. the patient had been treated with a lip ointment, triamcinolone acetonid 0.1% (kenalog®), for 2 weeks, but became thick, dry, and worse. she said that she got many calamities related to her family, leading to the increased busyness and psychologically distressed conditions. extra oral examination of the lower lip showed erythematous erosion sized 4x4 mm, yellowish red crusting sized 3x4 mm, and translucent multiple vesicles sized 1x1 mm with well circumscribed as well as irregular edges. on palpation submandibular lymph nodes, dextra and sinistra were palpable, rubbery, mobile and painless. based on intra-oral examination, however, there were no abnormalities. case management: the diagnosis was determined based on anamnesis, clinical examinations, and supporting examination of ig m and anti hsv-1 ig g. the patient then was prescribed systemic and topical acyclovir. conclusion: some laboratory tests are necessary to confirm the diagnosis and determine the accurate therapy of herpes labialis in addition to the history and clinical features. keywords: herpes labialis; the lower lip correspondence: kus harijanti, department of oral medicine, faculty of dental medicine, universitas airlangg. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: kus_oralmedair@yahoo.com introduction herpes labialis is a form of secondary or recurrence of herpes simplex infection primary caused by type 1 herpes simplex virus (hsv).1 hsv, categorized into the family herpesviridae that has no animal vector, but spreads among individuals, is enveloped double-stranded dna virus that has an ability to become latent in nerve cells of the host.2 the first infection usually occurs after the first contact with hsv contained in fluid secretion of the mucosa, skin, and eye on infected patients. hsv can also move to sensory nerve axons, then settle and transform into chronic latent one in the trigeminal nerve ganglion. hsv can also move to outside the nerve cell and become latent, such as epiteltium, primary leading to recurrences in lips.3 during the latent phase, hsv do not replicate, and hsv antigen will not be detected. hsv will be reactivated when there are trigger factors, such as sun, trauma, emotional stress, or menstruation,4 fever, and immunosuppressing.5 hsv replicates in the ganglion, and then centrifugally move along the axon to the skin or oral mucosa. hsv infects epithelial cells, then clinically triggering vesicles that are easily broken into ulcers.4 herpes labialis, occurs in 20-40% of the population in the united states, nearly 100 million times per year. the location of the occurrence is generally on lower lip, about one-third of the prevalence. herpes labialis sufferers often complain of cold sores or fever blisters. they also complain of prodromal symptoms, such as tingling, itching or burning sensations followed by the appearance of lesions in the form of vesicles that are easily broken into ulcers accompanied with crusts. 60% of patients who experience prodromal symptoms tend to have larger lesions. clinically, patients with herpes labialis will have vesicles on their lips, and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p229-233 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p229-233 230 prehananto and harijanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 229–233 then in a matter of hours they will transform into ulcers and crusts. in the first 24 hours, patients with positive hsv culture then will have lesions, 80% of which are in the form of vesicles and 34% of which are in the form of ulcers or crusts. in more severe conditions, the healing process requires longer time.4 generally, lesions are healed in 1-2 weeks without scars and bacterial secondary infection.1 furthermore, immunocompromised patients suffering from herpes labialis usually are given systemic acyclovir therapy. meanwhile, the use of topical antiviral medication aims to prevent replication, infectivity, pain, as well as wider size and longer duration of lesions. however, they cannot prevent the reoccurrence.1 topical antiviral medication, such as topical acyclovir cream 5%, penciclovir cream 3%, and docosanol cream 10%, can effectively be applied three to six times a day on the lesions. additionally, herpes labialis can be reduced by reducing the trigger factors, such as by using sunscreen cream.3 therefore, case report was aimed to analyze the case of herpes labialis on the lower lip triggered by emotional stress case on january 25, 2014, a 58-year-old female patient came to the dental and oral hospital, universitas airlangga, surabaya with complaints of pain in her lower lip from one month ago. the patient had ever treated her wounds with a lip ointment of acetonid triamcinolone 0.1% (kenalog®) for 2 weeks, but her lip became thick, dry and getting worse. the patient consulted to a dentist, and was given a topical aloclair® gel that was used for 2 weeks. unfortunately, her wounds were getting worse, and when the patient moved her lips, they were bleeding. one week before, the patient felt unwell after consuming miloxicam once a day for 2 days. the patient had a history of cancer sores when under stress. she also had a history of allergy to tetracycline, and her child also has a history of allergy to seafood. case management extra-oral examination of her lower lip, showed reddish erosion sized 4 x 4 mm with irregular edges, crusts sized 3x4 mm with irregular edges, and transparent multiple vesicles sized 1 x 1 mm with clear edges (figure 1a). on palpation of the submandibular lymph nodes, dextra and sinistra were palpable supple, mobile and painless. intraoral examination, showed no abnormalities. based on results of anamnesis and clinical examination, the patient then was diagnosed with herpes labialis, not pemphigus vulgaris. after that, the patient was referred to do a complete blood examination, such as sgot, sgpt, fasting blood sugar (fbs), 2-hour postprandial blood sugar (pp or pg), bun, creatinine, and total ige, and then given oxyfresh gel. on the second visit the patient still felt pain. the topical medications also were used according to the instructions. the patient came to submit results of the laboratory tests. results of the extra-oral examination showed that there were painful single erosion sized 5 x 3 cm with clear and irregular edges, reddish-yellow crusts, and vesicles on her lower lip. results of the intra-oral examination did not reveal any abnormalities. results of the complete blood laboratory test, moreover, showed that eosinophils was 1% (2-4%), lymphocytes was 38% (25-35%), erythrocyte sedimentation rate (esr) was 26 mm (1-20 mm), and the number of eosinophils was 70 (80-360). other laboratory 10 7. scully c. oral and maxillofacial medicine the basic of diagnosis and treatment. london, united koingdom: curchill livingstone elsevier; 2013. p. 311. 8. wilson dd. manual of laboratory and diagnostic test. united state; america: the mcgraw-hill companies; 2008. p. 619-20, 308, 341, 490, 564. 9. mitaart ah. infeksi herpes pada pasien imunokompeten. surabaya: prosiding seminar pendidikan kedokteran berkelanjutan new perspective of sexually transmitted infection problems; 2010. p. 83-93. 10. stoopler et, balasubramaniam r. topical and sistemic therapies for oral and perioral herpes simplex virus infections. cda journal 2013; 41: 259-62. 11. lokesh p, rooban t, elizabeth j, umadevi k, ranganathan k. allergic contact stomatitis; a case report and review of literature. indian journal of clinical practice 2012; 22(9): 458-62. 12. djajakusuma ts. the role of immunomodulator in the treatment of sexually transmitted infection. bandung: medical faculty of padjadjaran university; 2010. p. 147-8. 13. huber ma, terezhalmy gt. actinic cheilosis: etiology, epidemiology, clinical manifestations, diagnosis, and treatment. crest oral-b dental care continuing education course, december 9, 2015; p. 2. available at: https://www.dentalcare.com/enus/professional-education/ce-courses/ce400. 11 figure 1. on the first visit, erosion, crusts and vesicles appeared on the lower lip. figure 2. on the third visit, erosion and crusts still appeared on the lower lip. figure 3. on the fifth visit, lesions was getting improved, but there were multiple erosions on the lower lip. figure 4. on the sixth visit, the lesions on the lower lip was improved, but there were still multiple erosions with smaller areas. figure 5. on the seventh visit, erosions appeared again on the lower lip. figure 6. on the eighth visit, the erosions on the lower lip were getting smaller. 11 figure 1. on the first visit, erosion, crusts and vesicles appeared on the lower lip. figure 2. on the third visit, erosion and crusts still appeared on the lower lip. figure 3. on the fifth visit, lesions was getting improved, but there were multiple erosions on the lower lip. figure 4. on the sixth visit, the lesions on the lower lip was improved, but there were still multiple erosions with smaller areas. figure 5. on the seventh visit, erosions appeared again on the lower lip. figure 6. on the eighth visit, the erosions on the lower lip were getting smaller. 11 figure 1. on the first visit, erosion, crusts and vesicles appeared on the lower lip. figure 2. on the third visit, erosion and crusts still appeared on the lower lip. figure 3. on the fifth visit, lesions was getting improved, but there were multiple erosions on the lower lip. figure 4. on the sixth visit, the lesions on the lower lip was improved, but there were still multiple erosions with smaller areas. figure 5. on the seventh visit, erosions appeared again on the lower lip. figure 6. on the eighth visit, the erosions on the lower lip were getting smaller. 11 figure 1. on the first visit, erosion, crusts and vesicles appeared on the lower lip. figure 2. on the third visit, erosion and crusts still appeared on the lower lip. figure 3. on the fifth visit, lesions was getting improved, but there were multiple erosions on the lower lip. figure 4. on the sixth visit, the lesions on the lower lip was improved, but there were still multiple erosions with smaller areas. figure 5. on the seventh visit, erosions appeared again on the lower lip. figure 6. on the eighth visit, the erosions on the lower lip were getting smaller. 11 figure 1. on the first visit, erosion, crusts and vesicles appeared on the lower lip. figure 2. on the third visit, erosion and crusts still appeared on the lower lip. figure 3. on the fifth visit, lesions was getting improved, but there were multiple erosions on the lower lip. figure 4. on the sixth visit, the lesions on the lower lip was improved, but there were still multiple erosions with smaller areas. figure 5. on the seventh visit, erosions appeared again on the lower lip. figure 6. on the eighth visit, the erosions on the lower lip were getting smaller. 12 figure 7. on the ninth visit, multiple erosions still appeared on the lower lip. figure 8. on the tenth visit, lesions on the lower lip were healed. 12 figure 7. on the ninth visit, multiple erosions still appeared on the lower lip. figure 8. on the tenth visit, lesions on the lower lip were healed. figure 1. extra-oral examination of the patient lower lip on: a) first; b) third; c) fifth; d) sixth; e) seventh; f) eighth; g) ninth; h) tenth visit. a b c d e f g h dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p229-233 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p229-233 231231prehananto and harijanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 229–233 ash since at the time a natural volcanic disaster occurred (figure 1d). results of the extra-oral examination showed yellowish red multiple erosions sized 4 x 5 mm with clear and irregular edges on her lower lip. results of the intra-oral examination did not reveal any abnormalities. therefore, she was instructed to use a lip ointment, acyclovir cream, as well as immunomodulatory regularly. on the eighth visit the patient still felt the wounds on her lower lip. her lower lip was even still bleeding when used for talking and eating. the medication was used regularly according to the instructions (figure 1f). results of the extra-oral examination showed yellowish red multiple erosions sized 3 x 4 mm with clear and irregular edges on her lower lip. results of the intra-oral examination showed no abnormalities. as a result, she was instructed to stop applying the topical acyclovir cream. the patient then was given the following prescription: r / hydrocortisone 0.125 gr kemisitin 0.125 gr lanolin 0.25 gr vaseline ad 1 g m.f unguentum s 4 dd i smeared on the lips on the ninth visit the patient felt the wounds on her lips had been improved. her lips were not dried and bleeding anymore (figure 1g). the medication was used regularly according to the instructions. results of the extra-oral examination showed that there were multiple red erosions, sized 3 x 2 mm with clear and irregular edges on her lower lip. however, results of the intra-oral examination showed no abnormalities. on the tenth visits the patient felt the wounds on her lips had been improved. she even had no pain anymore (figure h). the medication was used regularly according to the instructions. results of the extra and intra oral examinations even did not reveal any abnormalities. thus, the oral topical medication was stopped. discussion herpes labialis is a recurrence or relapse of hsv-1 infection. one of factors triggering the recurrence of hsv is emotional stress. patients suffering from herpes labialis will typically have prodromal symptoms of tingling, burning, or pain in a place where a lesion appeares.1 a 58-years-old patient came with a complaint of discomfort started one month ago. prior to this complaint, she felt of dry and burn sensations on her lips, then vesicles appeared, and her lips became easily bleeding. based on her anamnesis, this patient lately had many problem related to her family. consequently, she was really busy, and her psychological condition was depressed. three days before lesions appeared, the patient felt unwell. these might underlie a recurrence in this case. results were still in normal limits. on the third visit the patient felt that the wound on her lower lip was getting improved. the topical lip medications were also still used regularly according to the instructions. results of the extra-oral examination showed red multiple erosions with clear and irregular edges, as well as crusts on her lower lip (figure 1b). results of the intra-oral examination showed no abnormalities. therefore, the patient was instructed to perform serologic anti hsv-1 ig m and ig g tests, and came back to show the results of the lab tests. on the fourth visit the pain on her lower lip was getting reduced, but sometimes the lower lip was still bleeding when used for eating and talking. the medications were still used regularly according to the instructions. she came to submit the results of the laboratory tests. results of the extra-oral examination showed multiple erosions sized 3x4 mm with clear and irregular edges on her lower lip. results of the intra-oral examination showed no abnormalities. in addition, the results of the laboratory results showed that anti hsv-1 ig m was non-reactive, about 5.12 (nonreactive if index <9 id u, reactive if index >11 en u), while anti hsv-1 ig g was reactive, about more than 50.08 (non-reactive if index <9 id u, reactive if index >11 en u). consequently, the patient was instructed to reduce excessive activity and to consume foods high in calories and protein. then, the patient was given the following prescription. r/ acyclovir 400 mg tab no. xxviii s 4 dd i r/ acyclovir cream tube no. i s 3 dd i lit or r/ immunomodulators (imbost force®) capl no. x s 1 dd i on the fifth visit the patient felt that the wounds on her lower lip were getting improved (figure 1c). the pain had also been reduced a lot. the medications were used regularly according to the instructions. results of the extra-oral examination showed red multiple erosions with clear and irregular edges on her lower lip. the results also showed no crusts. and, results of the intra-oral examination did not reveal any abnormalities. on the sixth visit the patient felt that the wounds on her lower lip were healed (figure 1d). the pain had also been reduced a lot. the medication was used regularly according to the instructions. results of the extra-oral examination still showed red multiple erosions with clear and irregular edges on her lower lip, and results of the intra-oral examination showed no abnormalities. thus, the patient was instructed to discontinue the use of systemic acyclovir, and then asked to use of acyclovir cream and immunomodulators. on the seventh visit the wounds appeared again on her lower lip. the patient said that a day before, she had outdoor activities without using any protection for her lip wounds. consequently, her lower lip was exposed to volcanic dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p229-233 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p229-233 232 prehananto and harijanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 229–233 stress actually can reactivate latent herpes virus through an increase in one or more stress-related hormones (neuropeptides) directly inducing latent virus. simultaneously, downregulation of cellular immune responses associated with stress can reduce an ability to control the cellular immune responses after reactivation of the latent virus. psychological stress in humans increases levels of corticotropin-releasing factor (crf) via the hypothalamus, then stimulating the pituitary gland to produce acth, which then stimulates the adrenal gland (hpa axis) to modulate immune interactions. many researches on a relationship between neuroendocrine peptides and immune function modulation have focused on neuropeptide derived from the polyprotein proopiomelanocortin (pomc), especially acth and betaendorphin. nevertheless, as already mentioned, other hormones, such as cortisol, growth hormone, prolactin, catecholamines, epinephrine, and norepinephrine also have been reported to modulate body immune functions.6 on the first visit, results of the clinical examination showed a red erosion sized ± 4 x 4 mm with an irregular edge, reddish-yellow crusts sized 3 x 4 mm with irregular edges, and transparent multiple vesicles sized 1 x 1 mm with clear edges on her lower lip. in herpes labialis, in a matter of hours, a few fragile and short-lived vesicles will appear, break easily, and form shallow ulcers, then forming a coalition.1 the differential diagnosis of these lesions was pemphigus vulgaris. herpes labialis and pemphigus vulgaris have similar clinical symptoms on lips, such as easily broken vesicles, triggering red erosions or ulcers on the lips, stimulating yellowish crusts to appear after a few days. however, there are some differences between herpes labialis and pemphigus vulgaris. for instance, in pemphigus vulgaris, vesicles can be easily moved by a sharing force, called a positive nikolsky sign. meanwhile, in herpes labialis, vesicles cannot be moved easily by a shearing force. in addition, in herpes labialis, lesions can be found only on lips. unlike in herpes labialis, lesions in pemphigus vulgaris can be found not only on lips, but also in oral mucosa.7 the diagnosis of herpes labialis sometimes can be confusing. to determine the diagnosis of herpes labialis, as a result, both further examination on complete blood and seroimmunological tests on anti hsv-1 ig m and ig g were conducted. complete blood examination showed that lymphocytes increased to 38 (25-35%), most likely a viral infection. erythrocyte sedimentation rate increased to 26 (1-20 mm) since the patient took aspilets drug, and lesions on her lower lip was already getting chronic. similarly, hematrokrit increased to 0.50 (0.35 to 0. 7) since the patient had a history of heart disease. moreover, eosinophils decreased to 1 (2-4%) since she was under stress. her -anti hsv-1 ig g was reactive, more than 50.08 reactive (nonreactive if index <9 id u, reactive if index >11 en u). it means that the patient was positive with hsv-1. igg is the most abundant immunoglobulin generated from gamma globulin, accounting for about 75% of the total amount of immunoglobulin in the body. igg provides protection against the virus. igg is very important in the secondary response of the immune system. when the immune system is faced with the antigen for the first time, the primary response is made by igm followed by igg level evaluation. igg retains antigen memory, so the immune system will already have a memory against the antigen next time. titers of anti-hsv igg antibody will usually have increased by 1 to 2 weeks after the primary infection, reaching a peak at 6 to 8 weeks after the infection. results of anti-hsv-1igm were non-reactive, about 5.12 (non-reactive if index <9 id u, reactive if index >11 en u). the increase in the titers of anti-hsv igm antibody occurrs a few days after the primary hsv infection8, and reached the peak after 2-4 weeks.9 igm and igg antibodies only give a picture of acute or chronic infections of simplex herpes disease.9 another additional examination is smear on the ulcer to see the characteristics of the virus infection in the form of multinucleated giant cell. nevertheless, this examination was not conducted since it could induce pain to the patient. in addition, results of the smear examination are still not specific to distinguish varicella zoster virus infection or other viral infection. direct fluorecent antibody (dfa) or immunoperoxidase antibody staining with smear preparation on vesicles actually will be more sensitive (7088%), but the examination is difficult because it requires intake vesicular stadium frequently resulting in negative false.2 the patient was given a therapy using acyclovir tablet 400 mg for 7 days with a dose of 1600 mg per day (4 x 1 tablet daily) and topical acyclovir cream 50 mg three times daily. acyclovir is an antiviral drug that is highly active against hsv-1 and hsv-2, as well as varicella zoster virus acting as inhibitors of the dna polymerase and preventing viral dna synthesis without affecting normal cellular processes. acyclovir is also active against herpes virus, but cannot kill the virus.10 the secondary herpes can be controlled with systemic acyclovir although reoccurrence cannot be prevented. systemic acyclovir prophylaxis is effective in cases of recurrent and stubborn problems and immunocompromised patients. the administration of topical acyclovir is recommended for the treatment of secondary herpes. acyclovir ointment 5% (or analog) is usually applied 5 times per day when the first symptoms appear in order to reduce the duration and severity of lesions.1 topical acyclovir is a topical medication in cream, a semisolid dosage in the form of a thick emulsion containing water not less than 60% and one or more medication ingredients in order to increase soft and flexible senses of the lips, resulting in a decrease of the discomfort on the lips of patients.11 consequently, a supportive therapy is needed for patients with viral infections since they often experience a decrease in endurance. in connection with the weak condition of the patient, the administration of immunomodulators dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p229-233 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p229-233 233233prehananto and harijanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 229–233 (imboost force®) supplements is expected to improve the general condition of the patient. these supplements contain echinacea, one type of herb. some species of echinacea plants are used to make a drug, extracted from leaves, flowers, and roots that pay a role as immunomodulators. 8 immunomodulator serves to enhance the immune system more active by increasing the body’s immune system, both natural and adaptive.12 as a result, this patient was instructed to have high protein/ high calorie diet, expected to increase the energy needed for the patient’s healing process. reducing her activities by taking a rest is also necessary to restore the patient’s condition. on the sixth visit, her clinical symptoms were getting improved. but, on the seventh visit, lesions appeared again on her lower lip. she said that she had done a lot of outdoor activities without protecting lesions on the lips (without using a mask). therefore, the lesions were exposed to a lot of flying dust. in addition, the patient was also exposed to direct sunlight in a long time. in herpes labialis, lesions usually can heal without any scar within 1 to 2 weeks and secondary infection. their exposure to direct sunlight constantly is suspected as an environmental trigger factor for the reoccurrence. exposure to ultraviolet radiation (uv) from the sun as uv-b (wavelengths 280-320 nm) and uv-a (320-400 nm wavelength) can usually cause damage to the affected tissue. exposure occurred continually then can cause loss of elasticity and tissue damage, especially exposure to uv-b.13 thus, the lesions possibly appeared on the seventh visit. finally, the patient was given a lip ointment concoction of hydrocortisone, kemisetin, lanolin, and petroleum jelly. hydrocortisone is a corticosteroid used as anti-allergen and anti-inflammatory. corticosteroid can prevent allergic reactions, and reduce inflammation. kemisetin is a broad spectrum anti-microbe that is effective against both grampositive bacteria and gram-negative bacteria by inhibiting protein synthesis of microbial cells. meanwhile, lanolin and petroleum jelly are the base materials for ointment, moisturizing stratum corneum, which can improve absorption and drug potential.10 on the tenth visit, after 10 days of the administration of the topical lip concoction (hydrocortisone, kemisitin, lanolin and petroleum jelly), lesions on her lower lip were healed. it can be concluded that further investigations are necessary to confirm diagnosis of herpes labialis, and also determine proper treatment in addition to anamnesis and clinical description. references 1. regezi ja, sciubba jj, jordan rck. vesiculobullous diseases at oral pathology clinical pathologic correlations. united states of america: elsevier saunders; 2012. p. 1-6. 2. costello m, sabatini l, yungbluth p. herpes simplex virus infections and current methods for laboratory detection. clinical microbiology newsletter 2006; 28(24): 185-92. 3. woo sb, greenberg m. ulcerative, vesicular, and bullous lesions in burkets oral medicine diagnosis & treatment. 12th ed. greenberg m, glik m, ship ja, editors. new jersey: bc decker inc; 2015. p. 58-62. 4. woo sb, challacombe sj. management of recurrent oral herpes simplex infections. oral surg oral med oral pathol oral radiol endod 2007;103(suppl): s12.e1-18. 5. lugito mdh, pradono sa. valacyclovir in the management of recurrent intraoral herpes infection. journal of dentistry indonesia 2014; 21(1): 27-31. 6. glaser r, glaser jk. stress-associated immune modulation and its implications for reactivation of latent herpesviruses. columbus, ohio: the ohio state university medical center; p. 245-70. 7. scully c. oral and maxillofacial medicine the basic of diagnosis and treatment. london, united koingdom: curchill livingstone elsevier; 2013. p. 311. 8. wilson dd. manual of laboratory and diagnostic test. united state; america: the mcgraw-hill companies; 2008. p. 619-20, 308, 341, 490, 564. 9. mitaart ah. infeksi herpes pada pasien imunokompeten. surabaya: prosiding seminar pendidikan kedokteran berkelanjutan new perspective of sexually transmitted infection problems; 2010. p. 83-93. 10. stoopler et, balasubramaniam r. topical and sistemic therapies for oral and perioral herpes simplex virus infections. cda journal 2013; 41: 259-62. 11. lokesh p, rooban t, elizabeth j, umadevi k, ranganathan k. allergic contact stomatitis; a case report and review of literature. indian journal of clinical practice 2012; 22(9): 458-62. 12. djajakusuma ts. the role of immunomodulator in the treatment of sexually transmitted infection. bandung: medical faculty of padjadjaran university; 2010. p. 147-8. 13. huber ma, terezhalmy gt. actinic cheilosis: etiology, epidemiology, clinical manifestations, diagnosis, and treatment. crest oral-b dental care continuing education course, december 9, 2015; p. 2. available at: https://www.dentalcare.com/en-us/professionaleducation/ce-courses/ce400. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p229-233 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p229-233 118 vol. 42. no. 3 july–september 2009 research report immunoglobulin-g level on aggressive periodontitis patients treated with clindamycin agung krismariono department of periodontics faculty of dentistry, airlangga university surabaya indonesia absctract background: aggressive periodontitis might occur as a result of complex interplay between bacteria and host defence, therefore, the host susceptibility plays important role. antimicrobial agents that could enhance host defence are required. clindamycin might influence host defence. purpose: the purpose of this study was to determine the influence of clindamycin on level of immunoglobulin-g (igg) patients with aggressive periodontitis, and its mechanism. methods: this study used the pre-test post-test control group design. eighteen aggressive periodontitis patients were divided into 2 groups at random. group 1 (treatment): 9 aggressive periodontitis patients were given with clindamycin of 150mg orally, 4 times a day, for 7 days. group 2 (control): 9 aggressive periodontitis patients were given with tetracycline of 250mg orally, 4 times a day for 12 days, and then metronidazole of 200mg orally, 3 times a day for 10 days. blood sample was collected from vena cubiti mediana. level of igg was measured at base line and day 28. data were analyzed statistically by using t-test (a = 0.05). result: examination for igg level showed there was significant difference between pre-test and post-test (p < 0.05). level of igg was significantly increased after therapy, both in treatment and control group. the increase of igg level in treatment group was not different significantly from control group (p>0.05). conclusions: this study shows that clindamycin clindamycin can be used as drug of choice for the treatment of aggressive periodontitis since clindamycin can improve the immunity status of aggressive periodontitis patients. key words: immunoglobulin-g, aggressive periodontitis, clindamycin correspondence: agung krismariono, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: agungkr@yahoo.com introduction the treatment of periodontal diseases still focused on local factors, the quality of hosts are still less concerned. therapy using antibiotic is usually concerned with old paradigm which aims to eliminate the bacteria. in fact, antibiotic certainly affects the immune response. some studies show that antibiotic is immunomodulatory becauseimmunomodulatory because because it has several characteristics; micro-organism eradicator, phagocytic, chemotactic and lymphocyte activity (effect by non-antibiotic of antibiotic).1–3 these characteristics of antibiotic are required to support the success of periodontal diseases treatment, specially for aggressive periodontitis. aggressive periodontitis is typical and it attacks the patients under 35 years old. its clinical symptoms are good oral hygiene, less accumulation of plague and calculus. in contrast, there is a rapid loss of alveolar bone. besides, much loss of attachment rapidly occurs, and it is followed by the occurrence of pocket deeper than 5mm. the amount of plague and calculus cannot be compared with the seriousness of disease. this disease appears because there is disturbance of the immune system,4 so in its treatment the efforts to increase the immunity are required. therefore, proper strategy of treatment needs to be seriously considered. this strategy can be applied by giving medicines effectively and efficiently which aims to eliminate the bacteria and increase the immune system. another alternative solution is by using the immunological effect of antibiotic. systemic antibiotic should be used in aggressive periodontitis treatment, because the micro-organisms which causing this disease is capable of invading the 119krismariono: immunoglobulin-g level on aggressive periodontitis pocket and leaving into sub epithelial gingival connective tissues. clindamycin is one of antibiotics used in aggressive periodontitis. it is effective against porphyromonas gingivalis and prevotella intermedia which are found in pocket of aggressive periodontitis.5,6 besides as antibiotic clindamycin influences the immune response of patients.7 by giving clindamycin to aggressive periodontitis patients, it is expected that the clindamycin can reduce pathogens owned by bacteria and increase the immunity. however, this statement needs further evidence and explanation. in such a chronic disease like aggressive periodontitis, the dominant immunoglobulin is igg.8 therefore, it is used as indicator in this study. the objective of the study was to identify the influence of giving clindamycin on igg level of aggressive periodontitis patients. the finding of the study is expected to be used as basis in determining proper strategy of treatment so that it can increase the success of treatment. clindamycin is a derivate of lincomycin antibiotic which is derived from fungus named streptomyces lincolnensis. it is bacteriostatic because it inhibits the protein blending of bacteria at sub unit ribosom 50s which functions as recipient. the clindamycin is tied at macrophage andthe clindamycin is tied at macrophage and leucocyte polymorphonuclear so it can increase the process of phagocytosis and intracellular killing.9,10 clindamycin is effective against anaerob bacteria either in positive gram or negative gram. it can be given per oral, intravenousvenous and topical. dosage per oral is 150mg–3g/day for adult,150mg–3g/day for adult, every 6 hours. for children, it is 10–20mg/kg/weight/day. concentration in serum is 2–3µg/ml that is obtained after one hour of intake.11 van winkelhoff et al.12 stated that in his study on refractory and adult periodontitis patients who were given clindamycin 150mg 4 times a day for 7 days, after a twentyfour month observation showed that there was activity decrease of disease from 10% to 0.5%, lengthening the emergence from 5 to 17 months. this study also concludesthis study also concludes that the given dosage can inhibit the growth of bacteria like porphyromonas gingivalis, prevotella intermedia and peptostrptococcus micros for 12 months. clindamycin is able to enhance the production of immunoglobulin-g in malignancy patients of epitel tumour with secondary infection. who studies the change of imunoglobulin-g level after being given clindamycin for 12 days on animals affected by paracite which is called babesia gibsoni. the disease characterized by cellular and humoral immune suppresion, with levels of igg antibody decline less rapidly in persistently infected patients. these results suggested that clindamycin damage paracites morphologically. clindamycin does not only destroy the paracite causing infection, but it also increases humoral and celluler immunity so that it can improve the clinical conditions.13–16 at first, the occurance of periodontitis caused by disturbance of local factors. one of the factors is the presence of bacteria. recently some researchers have believed that the growth of aggressive periodontitis depends on the interaction between periodontopathogen with immune response. aggressive periodontitis mayaggressive periodontitis may occurs because there is disturbance of immune system. aggressive periodontitis is closely related to the qualityggressive periodontitis is closely related to the quality and the susceptibility of the host, caused by abnormal immunocompetent cells that have disturbance of immune response. as consequence, the susceptibility of patients towards aggressive periodontitis is increased. one of abnormalities is phagocyte cells (neutrophils and monocytes).17–21 in aggressive periodontal treatment, the antibiotics which have clinically been studied and examined for the effectiveness are tetracycline and metronidazole on 30 aggressive periodontis patients, which are divided into two groups of treatment, one group of control given therapy with two kinds of antibiotics deliberately, concluded that tetracycline which is given to patients 250 mg 4 × a day for 12 days and metronidazole 200mg 3 × a day for 10 days give optimal treatment on aggressive periodontitis patients with clinical indicators; bleeding on probing (bop), probing depth (pd), gingival index (gi) and suppuration.22 the purpose of this study was to determine the influence of clindamycin on level of immunoglobulin-g (igg) patients with aggressive periodontitis, and its mechanism. the advantage of this study was to optimalize aggressive periodontitis treatment. materials and methods this study was designed and based on true experimental by using the post-test pre-test control group design. the population of this study was aggressive periodontitis patients who come to periodontic clinic at faculty offaculty of dentistry, airlangga university. the criteria of the samplesairlangga university. the criteria of the samples were as the following: male patients who are less than 35 years old; having clinically diagnosed of aggressive periodontitis, having no other infections either in oral cavity or other parts of the body, non-smokers, having no recent antibiotic therapy or other medicine. the samples of this study, moreover, were taken by using simple random sampling technique, which was divided into two groups; treatment group and control group. based on the trial method, each group on this study consisted of 9 the samples. first, the blood of the patients was taken from vena cubiti mediana of their arms for about 4 cc. then, the blood collected was put into venoject plain tubes for igg examination. afterwards, each patient was treated by scaling and root planing methods. for the treatment group, the patients were asked to take clindamycin of 150 mgs orally four times a day for seven days. meanwhile, for the control group, the patients were treated with active control method by asking them to take standard medicines for aggressive periodontitis orally, which were tetracycline and metronidazole. this treatment was then divided into two stages. in the first stage, the patients were asked to 120 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 118-122 table 1. mean and standard deviation of igg level igg treatment x ± sd control x ± sd pre-treatment post-treatment pre-treatment post-treatment 1392.3 ± 7.793 1488.4 ± 54.425 1420.8 ± 73.155 1506.4 ± 76.946 figure 1. level of igg pre and post therapy on the treatment and the control group. take tetracycline of 250 mgs orally 4 times a day for 12 days. and, in the second stage, the patients were asked to take metronidazole of 200mgs orally 3 times a day for 10 days.22 the igg level, furthermore, was measured on the twenty eighth day. the reason was because the period of igg formation took 28 days. thus, the blood of all patients either from the group of control or from the group of treatment was taken for about 4cc, which was then put into venoject plain tubes. afterwards, the tubes were examined based on standard laboratorial procedures for examining igg level. the method which was used, moreover, is “turbidimetry” with “immunoglobulin turbiquant automatic analysis tool. therefore, the blood sample in venoject plain tubes was stirred with centrifuge tool at a speed of 3000 rpm. 50 µl of serum was then taken and added with 1000 µl of nacl isotonic liquid. afterwards, 20 µl was taken and added with 500 µl of immunoglobulin reagent. the solution then was put into inhalator tubes which were set on “immunoglobulin turbiquant” tool. the results of the examinations finally could be read at the monitor screen. the result data were analyzed statistically by student’s t-test (a = 0.05). however, before being analyzed, all the data had to be examined first for their normality test by using “one-sample kolmogorov-smirnov test” with the level of significance at p > 0.05. result the level of igg in treatment group (clindamycin) and in the control group (tetracycline + metronidazole) are shown in table 1 and figure 1. kolmogorof -smirnov test shows p > 0.05 which mean the data have normal distribution, so the data can be examined by student’s t-test. in order to analyze the difference of igg levels in pretest and in post-test, either in the treatment group or in the control group, the data are examined statistically by paired t-test. the result showed that p = 0.000 (p < 0.05), which means that there was a significant difference. in other words, it indicated that giving either clindamycin or tetracycline+metronidazole could significantly increase the level of igg. in order to analyze the differences of pre-test data among both of groups, the data were examined by independent t-test. the results showed that p = 0.374 (p > 0.05), which means that there was no significant differences among them. in other words, it indicated that the level of igg in pre-test, either in the treatment group or in the control group, was considered the same. therefore, in order to determine whether there was a significant difference among the groups or not, independent t-test of the post-test data of both groups were done. the result shows that p = 0.576 (p > 0.05), which means there was no significant difference. in other words, it indicated that the level of igg in posttest, either in the treatment group or in the control group, is statistically not different. it meant that the change of igg level in the treatment group was the same as in the control group. therefore, the igg level of aggressive periodontitis patients who follow the therapy with clindamycin was the same as tetracycline + metronidazole. it might be suggested that the pattern of the change in both of groups tends to be the same, in which the increase of the igg level occurs after the therapy. however, if the increase of igg level in both of groups was compared to each other, it would show that the increase of igg level in the treatment group was bigger than in the control group. the increase of igg level in the treatment group was about 96.1 mg/dl; meanwhile, the increase of igg level in the control group was only about 85.6 mg/dl. discussion until now there are still many problems in aggressive periodontitis cases related to its treatment and prognosis. the reason is because the causes of aggressive periodontitis are not only from the local factors. the condition of the patients related to the abnormal immunity of the body also has an important role. therefore, immunoglobulin-g of the perifer of blood sample is used as a measurement of the immunity of the body in this study. based on the result data of aggressive periodontitis patients, the level of igg was affected by clindamycin. 121krismariono: immunoglobulin-g level on aggressive periodontitis the fact that clindamycin affects the immunity status of the body was proven by the increase of igg level. nevertheless, the effect caused by clindamycin has the same pattern as tetracycline + metronidazole in the control group. moreover, the data result slightly does not reflect the affectivity of clindamycin, but if it is analyzed further, it will show that the period of the therapy with clindamycin is shorter than that of the therapy with the other medicines in the control group. the therapy with clindamycin only needs 7 days, so the period of the treatment is shorter and the side effect of consuming the medicine can also be minimized. meanwhile, the therapy with the other medicines in the control group needs 22 days. igg actually is not only a main component of immunoglobulin serum, but also a kind of immunoglobulin which has most amount in blood. this antibody also has a role in preventing formation and colonization of bacteria in tissues, in improving the function of phagocytosis through opsonisation process, and in helping detoxification process of toxin produced by bacteria. for those reasons, igg has a potential role in body immunity.23 furthermore, even though the increase of igg level occurs in post therapy, the increase was still in normal level. therefore, the increase was not considered as the abnormal one which can cause the negative effect for the body. the highest level of igg after the therapy was 1574 mg/dl; meanwhile, the normal level of igg was between 800–1700 mg/dl. thus, the increase beyond the normal level must be prevented. in addition, there are some possibilities of the increase of igg level. first, the increase of igg level may be caused by the chemical bound between antibiotic and carrier protein, so they become immunogenic. immunogens can stimulate the immune responses. immunogens in b cell can cause proliferation and differentiation of b cell into plasma cell together with immunoglobulin result. this mechanism occurs since b cell acts as antigen presenting cell (apc), so immunogens can directly be bound by b cell. antibiotics can become immunogenic if there is the chemical bound between antibiotics and carrier protein. the immunogens recognized by b cell then pass proliferation and differentiation process, so it can stimulate the formation of antibodies.23 another possibility is that the increase of igg level after the therapy may be caused by the chemical bound between the medicines and macrophages. the immunogens which are from the kinds of medicines can be bound with macrophage which function is also as apc. as the explanation before, clindamycin is also able to distribute into mononuclear phagocyte cells. thus, with the chemical bound between the medicines and macrophage it then can stimulate the immunity responses. in addition, the increase of igg level may also be caused by the effect of inflammation mediator, prostaglandin-e2 (pge2). pge2 is arachidonate metabolic acid produced either by monocyte/macrophage cell or by fibroblasts.24 the macrophage cell which is activated will produce il-1, tnf and pge2. in this study, moreover, the inflammation causes the increase of pge2 level. the reason is because the condition of inflamed tissues can activate phagocyte cells which then cause the excretion of inflammatory mediator, pge2. moreover, according to harris, prostaglandin-e2 has double actions. in the high level, pge2 can decrease the level of igg; meanwhile, in the low level, pge2 can join il-4 which then can increase the level of igg deliberately. for those reasons, the low level of igg in this study may be caused by the high level of pge2. thus, it not only can inhibit the activation and proliferation of b lymphocyte by t lymphocyte (cd4+), but can also inhibit the differentiation of b cell into plasma cell, so it also causes the inhibition in producing antibodies.9 after the clindamycin therapy, the increase of the igg level occured. the reason was because of the ability of antibiotics, clindamycin, in eliminating bacteria causing infections, so it then could reduce the level of the inflammation. the decreasing of the inflammation level then will be followed by the decreasing of the inflammatory mediator production, including pge2. the low level of pge2, therefore, can reduce the inhibition of proliferation and differentiation of b lymphocyte into plasma cell, so the production of antibodies (immunoglobulin) by plasma cell will increase. the increase of the igg level is needed since igg has a role in opsonisation of antigen in phagocytes process, so the immunity mechanism can become more effective. the increase of the igg level in this study, thus, indicates that clindamycin has a role in increasing humoral immunity. in other words, it can cause aggressive periodontitis patients who follow the therapy with clindamycin can not be susceptible to diseases since there is the increase of the body immunity. based on the above explanation, it may be concluded that clindamycin can increase the level of igg since clindamycin has immunomodulatory character through the controlling mechanism of pro-inflammatory mediator. however, tetracycline with metronidazole can also role as immunomodulatory, but clindamycin more efficient than those because treatment with clindamycin needs single drug only so multiple drug side effects can be minimized. finally, clindamycin can be used as drug of choice for the treatment of aggressive periodontitis since clindamycin can improve the immunity status of aggressive periodontitis patients. references 1. omura m, satoh t. effects of antibiotics on chemotaxis of polymophonuclear leucocytes on experimental rabbit infection models. dentistry in japan 2001; 37:138–40. 2. hamilton-miller jm. immunopharmacology of antibiotics: direct and indirect immunomodulation of defence mechanisms. j chemother 2001; 13(2):107–11. 3. kitahara k, satoh t. effects of antibiotics on phagocytosis of polymophonuclear leucocytes on experimental rabbit infection models. dentistry in japan 2002; 38:146–9. 4. newman mg, takei h, carranza fa. clinical periodontology.clinical periodontology. 10th ed. philadelphia, london, new-york: wb saunders co; 2006.2006. p. 228–45, 506–11. 122 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 118-122 5. eickholz p, dannewitz b, kim ts. antibiotics in periodontal therapy. periodontology: clinical and research reports. 2005; 2(4):235–51. 6. kamma jj, nakou m, gmür r, baehni pc. microbiological profile of early onset/aggressive periodontitis patients. oral microbiol. immunol 2004; 19(5):314–21. 7. kuriyama t, karasawa t, nakagawa k, saiki y, yamamoto e, nakamura s. bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections. osomop 2000; 90(5):600–7. 8. guo s, takahashi k, kokeguchi s, takashiba s, kinane df, murayama y. antibody responses against porphyromonas gingivalis infection in patients with early-onset periodontitis. j clin periodontol 2000; 27(10):769–77. 9. katzung bg. basic and clinical pharmacology. 7th ed. california: med publ; 2001. p. 749–51. 10. eick s, pfister w, fiedler d, straube e. clindamycin promotes phagocytosis and intracellular killing of periodontopathogenic bacteria by crevicular granulocytes: an in vitro study. j antimicrob chemother 2000; 46(4):583–8. 11. goodman, gillman’s. the pharmacological basic of therapeutics. 10th ed. new york: mcgraw-hill; 2001. p. 1256–8. 12. van winkelhoff aj, rams te, slots j. systemic antibiotic therapy in periodontics. periodontology 2000. 1996; 10: 47, 51, 57, 60. 13. immunomodulating effect of antimicrobial agent on cytokine production by human polimorphonuclear neutrophlis. international journal of antimicrobial agent 2004; 23(2):150–4. 14. cerra r, castello g, abate g, dalla mora l, cremona f, parisi v, nisticò p. short-term antibiotic therapy with clindamycin phosphate in patients with malignant epithelial tumors: an immunological evaluation. j chemioterapia 1986; 5(1):33–6. 15. wulansari r, wijaya a, ano h, horri y, makimura s. lymphocytelymphocyte subsets and specific igg antibody levels in clindamycin-tretaed and treated dogs experimentally infected with babesia gibsoni. j vet med sci 2003; 65(5):579–84.2003; 65(5):579–84.65(5):579–84. 16. makimura s. canine babesia gibsoni infection–molecular biological diagnosis, phatopysiology of anemia and relapse, and chemotherapy with clindamycin. bulletin of the faculty of agriculture, university of miyazaki; 2006; 52(1.2):11–20. 17. d’aiuto f, graziani f, tete s, gabriele m, tonetti ms. periodontitis: from local infection to systemic diseases. int j immunopathol pharmacol 2005; 18(3):1–11. 18. mangini f, santacroce l, bottalico l. periodontitis and systemic diseases. clin ter 2006; 157 (6):541–8. 19. steinsvoll s, helgeland k, schenck k. mast cells-a role in periodontal diseases?. j clin periodontol 2004; 31(6):417. 20. guentsch a, puklo m, preshaw pm, glockmann e, pfister w, potempa j, eick s. neutrophils in chronic and aggressive periodontitis in interaction with porphyromonas gingivalis and aggregatibacter. journal of periodontal research 2006; 44(3):368–77. 21. lamster ib. antimicrobial mouthrinses and the management of periodontal diseases. j am dent assoc 2006; 137:5s–9s. 22. rubianto m. kombinasi pemberian tetrasiklin dengan metronidazole pada perawatan rapidly progressive periodontitis metode baru. jurnal kedokteran gigi univeristas indonesia 2001; p. 536–41. 23. baratawidjaja kg. imunologi dasar. edisi ke-6. jakarta: fakultasjakarta: fakultas kedokteran universitas indonesia; 2004. p. 73–84. 24. harris sg, padila j, koumas l, ray d, phipps rp. prostaglandinsprostaglandins as modulators of immunity. trends in immunology 2002;2002; 23(2):144–9. 217 volume 45 number 4 december 2012 research report antibacterial efficacy of salvadora persica as a cleansing teeth towards streptococcus mutans and lactobacilli colonies erlina sih mahanani,1 mohd fadhli khamis,2 erry mochamad arief,2 siti nabilah mat rippin,2 and zainul ahmad rajion2 1school of dentistry, faculty of medical and health science, universitas muhammadiyah, �ogyakarta indonesia 2school of dental sciences, universiti sains malaysia, 16150 kubang kerian, kelantan, malaysia abstract background: salvadora persica is a traditional chewing stick for cleaning teeth that it is known siwak. several studies have demonstrated the antimicrobial effects of salvadora persica. purpose: this study was aimed to examine the effectiveness of salvadora persica in several modified preparation against the salivary streptoccocus mutans and lactobacilli. methods: a single-blind, randomized clinical trial study with crossover design was used. the study comprised of 5 groups, per group consisted of 14 healthy dental students who had good oral hygiene. each participant was given 5 intervention to clean their teeth using, electric toothbrush modified with siwak, electric toothbrush with siwak toothpaste (colgate kayu sugi toothpaste), electric toothbrush with general toothpaste (colgate total toothpaste), original siwak chewing stick and normal saline. the wash out periode each intervention was 3 days. patients’ saliva was used to quantify the levels of streptococcus mutans and lactobacilli using caries risk test (crt) kit from vivadent. results: the results showed that there was a reduction in streptococcus mutans and lactobacilli risk score after cleansing different intervention except electric toothbrush modified with siwak. however, there was no significant difference for streptococcus mutans (p=0.158) and lactobacilli (p=0.396) risk score reduction when comparison was done between the groups. conclusion: the original siwak chewing stick has antimicrobial effects similar to toothbrushing with general toothpaste and salvadora persica toothpaste. however, electric toothbrush modified with siwak has no effect on microbial reduction. key words: salvadora persica, siwak, streptococcus mutans, lactobacilli, antibacteri abstrak latar belakang: salvadora persica adalah pembersih gigi tradisional yang lebih dikenal dengan sebutan siwak. beberapa penelitian menunjukkan bahwa salvadora persica memiliki daya antibakteri. tujuan: penelitian ini bertujuan untuk mengetahui efektivitas salvadora persica dalam berbagai bentuk sediaan untuk membersihkan gigi terhadap bakteri streptococus mutans dan lactobacilli dalam saliva. metode: desain penelitian yang digunakan adalah single-blind, randomized clinical trial study dengan crossover. lima kelompok perlakuan melakukan pembersihan gigi, tiap kelompok terdiri dari 14 mahasiswa kedokteran gigi, sehat dan memiliki kebersihan mulut yang baik. tiap subyek diberi 5 macam perlakuan untuk membersihkan gigi menggunakan sikat gigi elektrik dimodifikasi dengan siwak, sikat gigi elektrik dengan pasta gigi mengandung ekstrak siwak (colgate kayu sugi), sikat gigi elektrik dengan pasta gigi umum (colgate total), siwak asli, dan salin. saliva pasien digunakan untuk menganalisa pengurangan banyaknya bakteri streptococcus mutans dan lactobacilli sebelum dan sesudah perlakuan menggunakan caries risk test (crt) dari vivadent. hasil: hasil menunjukkan adanya penurunan skor streptococcus mutans dan lactobacilli setelah pembersihan gigi dibandingkan sebelumnya. sementara itu tidak ada perbedaan signifikan terhadap penurunan skor streptococcus mutans (p=0.158) dan lactobacilli (p=0.396) ketika dibandingkan antar kelompok. kesimpulan: mengunyah kayu siwak mempunyai efek antimikrobial yang hampir sama dengan menyikat gigi menggunakan kombinasi pasta gigi biasa dan pasta gigi yang mengandung siwak, akan tetapi meyikat gigi dengan sikat elektronik yang dimodifikasi dengan siwak dapat menurunkan jumlah bakteri. kata kunci: salvadora persica, siwak, streptococcus mutans, lactobacilli, antibacteria correspondence: erlina sih mahanani, c/o: prodi kedokteran gigi, fakultas kedokteran dan ilmu kesehatan, universitas muhammadiyah �ogyakarta. jl lingkar selatan, taman tirto, kasihan, bantul, �ogyakarta, indonesia. e-mail: erlinasihmahanani@yahoo.co.id 218 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 217–220 introduction plaque bacterial is solely responsible for the initiation and progression of caries and periodontal diseases. the mechanical and chemical methods are available for maintenance teeth. currently toothbrushes and dentifrices are mainly used for cleaning teeth. the traditional toothbrush or chewing stick (siwak), is used in many developing countries as the traditional means for oral hygiene.1 it is prepared from the roots, twigs and stem of salvadora persica. the stems and roots are spongy and can easily be crushed. pieces of the root are usually scented and become soft when soaked in water. it is chewed on one end until they become frayed into a brush. the brush-end is used to clean the teeth in a manner similar to the use of a toothbrush. the promotion of good oral health by siwak is mainly attributed mechanical cleansing, but may also be due in part to built-in antiseptics.2,3 in vitro studies have indicated that salvadora persica contain substances that possess plaque inhibiting and antibacterial properties against several types of cariogenic bacteria which are frequently found in the oral cavity. the growth and acid production of these bacteria is thus inhibited.4 various studies shows that streptococcus mutans were involved with the initiation of caries, whereas the lactobacillus were associated with the progression of the lesion.5,6 studies also have shown a positive correlation between the concentrations of streptococcus mutans in stimulated saliva and their level in dental plaque. salivary counts of streptococcus mutans and lactobacilli are also positively correlated with caries activity.7 good oral hygiene habits can prevent or retard the development of dental caries.8 the anti-microbial and cleaning effects of siwak have been attributed to various chemicals detectable in its extracts. these effects are believed to be due to its high content of vitamin c, salvadorine, salvadourea, alkaloids, trimethylamine, cyanogenic glycosides, tannins, saponins and salts mostly as chlorides in aqueous extract.1,9 the investigation has been done to assess the antimicrobial activity on salivary streptococcus mutans and lactobacilli.10 this study compared between cleaning teeth using siwak chewing stick and a toothbrush without toothpaste. however siwak consists of a chemical substantial that can reduce the bacteria, but it is not known the efficacy of siwak in several modified preparation for example as a toothpaste with siwak extract, customized in electric toothbrush to replace the brush. therefore, this study, was done to examine the effectiveness of salvadora persica (siwak) in several modified preparation against the salivary streptoccocus mutans and lactobacilli. materials and methods this was a randomized clinical trial study with crossover design, conducted for 3 weeks at school of dental sciences, university sains malaysia (usm) and ethical approved by human ethic committee usm (usmkk/ ppp/jepem [(206.3(4)]). this study was conducted in 5 groups, each group consist of 14 subjects. the subjects were randomly treated with 5 interventions. the interval period or wash out period between each intervention is 3 days.11 all the subjects were male and female dental students who had given their consents to involve in this study. the selected subjects were medically healthy with no systemic diseases and had not used any antibiotics or antiseptic mouthwash during the last two weeks. the subjects also had good oral hygiene (dmft less then 1) and had more than 20 natural teeth. stimulated whole saliva flow rate for each subjects were at ± 1.6 ml/sec. smokers, pitting (amelogenesis imperfecta), periodontitis and pregnant woman were not included in the study. the subjects were given a briefing on how to brush and use electric toothbrush, as well as how to use siwak chewing stick and scaling was done before interventions. the 5 intervention groups are: 1) electric toothbrush modified with siwak customized (figure 1); 2) electric toothbrush+siwak toothpaste (colgate kayu sugi); 3) electric toothbrush+general toothpaste (colgate total) as a positive control; 4) normal saline (0.9% nacl) mouthwash as a negative control; 5) original siwak chewing stick. saliva was collected for 5 minutes after one minutes of pre-stimulation by chewing paraffin wax. saliva was collected at 2 minutes interval for a total period of six minutes (5 minutes saliva collection+1 min pre-stimulated). saliva was collected before and after interventions to investigating the efficacy of intervention to againts streptococcus mutans and lactobacilli. the caries risk test (crt) vivadent (ivoclar, germany) was used to quantity bacterial colony. it was sufficiently figure 1. electric tootbrush modified with siwak customized. a) siwak, b) electric tootbrush. a b 219mahanani, et al.: antibacterial efficacy of salvadora persica as a cleansing teeth sensitive to provide a low, medium or high cariogenic bacterial challenge. the kit comes with two sided selective media sticks that asses streptococcus mutans on the blue side and lactobacilli on the green side. the samples were incubated at 37° c for 48 hours. growth density of the bacteria was evaluated under good lighting conditions by the naked eye and as per manufacturer’s instructions. bacterial growth was then scored by comparing with standards expressed in colony forming units (cfu) provided by the manufacturers as follows: streptococcus mutans scoring: 0 = very low colonies are detected; 1 = low, colonies growth are < 105 105 cfu; 2 = medium, colonies growth are > 105 105 but < 106 106 cfu; 3 = high, colonies growth are ≥ 106 107cfu. lactobacilli scoring: 0= very low colonies are detected; 1= low, colonies are ~ 105 cfu; 2= medium, colonies are ~ 104 104cfu; 3 = high, colonies are ~ 105 105 cf. the data were then be collected by single blind and spss version 12 for analysis. results the reduction of streptococcus mutans was presented in table 1 that showed in all group except modified siwak. the comparisons of the streptococcus mutant risks score within five groups using repeated measures anova revealed no significant effects. the values of comparisons were w=0.545, f (4, 10)=2.085, p=0.158, partial eta square=0.455. the descriptive statistics of mean and standard deviation for the lactobacillus risk score was presented in table 2. however the lactobacilli risk score after using different agents for cleansing teeth i.e siwak, colgate total, colgate kayu and saline sugi had reduction except for modified siwak as well. the comparison within five groups using repeated measures anova revealed no significant effects. the values of comparisons were w=0.689, f(4, 10)=1.130, p=0.396, partial eta square=0.311. it means that the modified siwak is not effective to reduce streptococcus mutans and lactobacilli. discussion modified siwak was prepared by cutting the end of siwak chewing stick for about 1 cm and we stick it to the electric toothbrush. study by hairudin shows that different length and size of siwak exert different effects,12 so the modified siwak released less chemical substance when compared to original siwak chewing stick. almas10 in his study reported that siwak had reduction streptococcus mutans better than toothbrushing without toothpaste. siwak clean the teeth by releasing chemical substance and mechanical cleansing action meanwhile tootbrusing without toothpaste has only mechanical cleansing action. the number of bacteria after cleansing teeth using original siwak, electric toothbrush with colgate total and colgate kayu sugi toothpaste had no significance difference in reduction of the streptococcus mutans and lactobacilli risk score. it mean that the siwak had same effect as tooth brushing with toothpaste in reducing the numbers of bacteria. many studies reported that toothpastes have antimicrobial activity both in vitro and in vivo.13 however salvadora persica contains antimicrobial anionic components such as sulphate (so42-), chloride(cl-) and thiocyanate (scn-). scnacts as a substrate for lactoperoxidase to generate hypothiocyanite (oscn-) in the presence of hydrogen peroxide. oscn has been demonstrated to react with sulfhydryl groups in bacterial enzymes which in turn lead to bacterial death. acid production in human dental plaque in vitro has been reported to be inversely proportional to the concentration of oscn in the test system, while supplementing saliva with hydrogen peroxide and scninhibited acid production.9 however, the use of miswak has also been reported to inhibit the formation of dental plaque chemically, and exert antimicrobial effect against many oral bacteria. in vitro studies have demonstrated that aqueous extracts of miswak have growth-inhibitory effects on several micro-organisms.14 using the checkerboard dna–dna hybridization (ckb) method, miswak may have a selective inhibitory effect on the levels of certain bacteria in saliva, particularly several oral streptococci species.15 the subjects that involved in this study had good oral hygiene (dmft 0) and this can influence to the result. previous studies have shown that subjects with low caries tended to have higher mean flow rates of unstimulated parotid saliva compared to those in the higher caries group.16 flow rates of saliva can influence the numbers of microorganism in the oral cavity. natural flow of saliva will detach microorganism not firmly attached to an oral surface. salivary components such as mucins can aggregate table 1. descriptive statistics for streptococcus mutans risk score n mean sd modified miswak 14 0.1 0.53 colgate kayu sugi 14 -0.1 0.66 colgate total 14 -0.2 0.70 saline 14 -0.3 0.73 miswak chewing stick 14 -0.4 0.93 table 2. descriptive statistics of lactobacillus risk score n mean sd modified miswak 14 0.1 0.73 colgate kayu sugi 14 -0.3 0.61 colgate total 14 -0.4 0.63 saline 14 -0.3 0.73 siwak chewing stick 14 -0.4 0.76 220 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 217–220 certain bacteria which facilitates their removal from the mouth. mucins are high molecular weight glycoprotein containing more than 40% of carbohydrate. other salivary components that act as antimicrobial are lysozme, lactoferrin, salivary peroxidase enzyme and histidinerich polypeptide.17 the other hand the continuous supply substance of salvadora persica can increase the salivary flow rate from the stem.14 the result proved that the cleansing teeth using original siwak chewing stick have similarity effectiveness with siwak toothpaste, general toothpaste to reducing streptococcus mutans and lactobacilli. suggestion for further research should be carried out in larger sample size with the high risk karies condition. controlled clinical trials are needed to find out the effect of salvadora persica on cariogenic microorganisms for a prolonged period of time to assess the substantively of the tested material. acknowledgment i would like to show my appreciations to universiti sains malaysia for short term research funding grant no.304/ppsg/6139033, school of dental sciences and dental health education hospital universiti sains malaysia for equipment and supporting staff and year 2, year 3, year 4 students who involved as volunteers in this research. references 1. al-otaibi m. the siwak (chewing stick) and oral health. studies on oral hygiene practices of urban saudi arabians. swed dent j suppl 2004; (167): 2-75. 2. ahmad h, rajagopal k. biological activities of salvadora persica l. (meswak). med aromat plants 2013; 2(4). 3. al-bayati fa, sulaiman kd. in vitro antimicrobial activity of salvadora persica l. extracts against some isolated oral pathogens in iraq. turk j biol 2008; 32: 57-62. 4. chentouf nc, meddah att, catherine m, aoues a, meddah b. in vitro and in vivo antimicrobial activity of algerian hoggar salvadora persica l. extracts against microbial strains from children’s oral cavity. j ethnopharmacol 2012; 144(1): 57-66. 5. almas k, al-zeid z. the immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study. j contemp dent pract 2004; 5(1): 105-14. 6. mortazavi s, akhlaghi n. salivary streptococcus mutans and lactobacilli levels following probiotic cheese consumption in adults: a double blind randomized clinical trial. j res med sci 2012; 17(1): 57-66. 7. van houte j. microbiological predictors of caries risk. adv dent res 1993; 7(2): 87–96. 8. axelsson p, nyström b, lindhe j. the long-term effect of a plaque control program on tooth mortality, caries and periodontal disease in adults. results after 30 years of maintenance. j clin periodontol 2004; 31(9): 749-57. 9. ismail ad, alfred ac, nils s, perk k e. identification and quantification of some potentially antimicrobial anionic components in miswak. indian j pharmacology 2000; 32: 11-4. 10. almas k, al-zeid z. the immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study. j contemp dent pract 2004; 5(1): 105-14. 11. turssi cp, faraoni jj, rodrigues jr al, serra mc. an in situ investigation into the abrasion of eroded dental hard tissues by a whitening dentifrice. caries res 2004; 38(5): 473-7. 12. hairuddin nm, jalil ra. the immediate term effect of chewing meswak (salvadora persica) on flow rate and ph of whole saliva. annal dent univ malaya 2000; 7: 6-10. 13. prasanth m. antimocrobial efficaccy of different toothpaste and mouthrinses: in vitro study. dent res j (isfahan) 2011; 8(2): 85-94. 14. sofrata a, lingström p, baljoon m, gustafsson a. the effect of miswak extract on plaque ph. caries res 2007; 41(6): 451–4. 15. darout ia, albandar jm, skaug n, ali rw. salivary microbiota levels in relation to periodontal status, experience of caries and miswak use in sudanes adults. j clin periodontol 2002; 29(5): 411–20. 16. gopinath vk, and arzreanne ar. saliva as a diagnostic tool for assessment of dental caries. arch orofac sci 2006; 1(1): 57-9. 17. philip m, and michael vm. oral microbiology. 5th ed. china: elsevier; 2009, 8-24. 221 volume 45 number 4 december 2012 literature reviews pomegranate juice (punica granatum) as an ideal mouthrinse for fixed orthodontic patients haryono utomo and kimberly clarissa oetomo faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: prevention of caries as well as periodontal disease is mandatory during orthodontic treatment. nevertheless, the use of antiseptic mouthrinse is contraindicated for prolonged use. pomegranate juice is a polyphenol-rich juice with high antioxidant capacity as well as antimicrobial properties. it has been shown to exert beneficial characteristics for orthodontic patients such as antioxidant and anti inflammatory effects. moreover, it contained fluoride and phosphorous which are cariostatic. previous study in fixed orthodontic patients revealed that rinsing with this juice showed reduced dental plaque and superior compared with chlorhexidine. if it has unwanted effect by reducing pro-inflammatory reaction that also needed in orthodontic movement is not clearly understood. purpose: the aim of the present review was to discuss the beneficial and unwanted effect of pomegranate juice mouthrinse towards orthodontic treatment. reviews: pomegranate has antimicrobial activity, its methanolic skin extract is the most potent followed by seed juice. nevertheless, seed juice is not only tastier, easier to make but also has mild antimicrobial potency which is beneficial for long-term use. healthy periodontal tissue is preferable for orthodontic movement since it resulted in less unwanted bone resorption. conclusion: regarding its beneficial effect and safety of pomegranate juice if use daily mouthrinse in fixed orthodontic patients, it could be proposed as an ideal long term use mouthrinse for fixed orthodontic patients. however, further researches should be done to verify this concept. key words: pomegranate juice, mouth rinse, fixed orthodontic abstrak latar belakang: pencegahan karies dan penyakit periodontal sangat penting dalam perawatan ortodontik. walaupun demikian, penggunaan obat kumur antiseptik jangka panjang merupakan kontraindikasi. jus buah delima sangat kaya akan polifenol dengan kemampuan antioksidan yang tinggi disertai kemampuan antimikroba. beberapa penelitian telah menunjukkan dampak menguntungkan pada pasien ortodonti, yaitu sebagai antioksidan dan anti radang. selain itu juga mengandung fluor dan fosfor yang bersifat kariostatik. penelitian terdahulu pada pasien ortodonti cekat menunjukkan penurunan plak gigi yang lebih banyak dibanding dengan klorheksidin. akan tetapi, apakah juga menyebabkan dampak merugikan yaitu mengurangi reaksi keradangan yang juga penting bagi pergerakan ortodonti masih belum jelas. tujuan: membahas dampak menguntungkan maupun merugikan jus buah delima pada perawatan ortodonti. tinjauan pustaka: buah delima merupakan antimikroba, ekstrak dari kulit yang mengandung metanol adalah paling kuat diikuti jus biji delima. akan tetapi, jus biji lebih enak rasanya, mudah dibuat dan merupakan antimikroba ringan sehingga menguntungkan untuk pemakaian jangka panjang. jaringan periodontal yang sehat diperlukan untuk pergerakan ortodonti karena dapat mengurangi resorpsi tulang. kesimpulan: berdasarkan dampak menguntungkan dan keamanan jus buah delima bila digunakan tiap hari, bahan ini dapat diusulkan sebagai obat kumur jangka panjang yang ideal untuk pasien ortodonti cekat. walaupun begitu, penelitian lebih lanjut harus dilakukan untuk verifikasi konsep ini. kata kunci: jus buah delima, obat kumur, ortodonti cekat correspondence: haryono utomo, c/o: fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id 222 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 221–227 introduction a frequent asked question: “is it safe to use mouthrinse when you have braces?”, it seems a simple question, nevertheless, to answer it many things should be considered. it is logical that wearing orthodontic appliances has been known to induce intraoral changes, such as increased plaque accumulation and elevated bacterial colonization along with potential enamel demineralization and a harmful effect on periodontal tissues such as gingivitis and periodontitis.1,2 fixed orthodontic appliances, such as orthodontic brackets, wires as well as elastomeric modules and chains etc., increase the number of retention places for dental plaque accumulation. all these factors make it even more difficult to maintain oral hygiene. self-cleaning is also more difficult because of the reduced effect of mechanical chewing and rinsing the food residues off by saliva. subsequent accumulation of plaque can causing to development of chronic periodontal inflammation and can progress to gingival enlargement. gingival enlargement inhibits hygiene measures, slows down orthodontic tooth movement and cause aesthetic and functional problems.2,3 even without brackets, for many people, especially children, it is difficult to adequately comply with recommended mechanical methods of plaque control and to achieve the necessary level of oral hygiene. tooth brushing removes up to 50% of plaque biofilm, and is even more effective when used in combination with flossing.2,4 such inadequate home care put these patients at a greater risk of developing gingivitis, which may progress to periodontitis in approximately 20% of patients.therefore, using mouth rinse is considered helpful.1-4 several requirements for long term use of oral rinse should be followed in orthodontic treatment such as minimal adverse effect towards the properties of brackets and wires5 as well as elastomeric modules and chains.6 the force degradation of elastomeric chain was related to water absorption and chemicals, the amount of loss in dry is less than in wet environments and especially in biofresh mouthrinse more than the neutral saliva.6 the role of ph fluctuation towards elastomeric chain force degradation.7 regarding the alcohol containing mouth rinse (acm), several opinions suggested that long term use may cause xerostomia and oral cancer which were not proven by studies. however, in orthodontics view, the use acm should be limited since a study by larrabee et al.,8 revealed that acm increase force degradation of elastomeric chain. the concept of mouthrinse is not new; they have been used for thousands of years, as mouthrinse ingredients containing salt, alum, and vinegar have been associated with chinese, indian, and greek and roman cultures. although some rinses were historically used to freshen breath, there is evidence that modern therapeutic rinses may improve health. therapeutic rinses may control oral biofilm, and biofilm is said to cause 70% to 80% of all human infections.9,10 the use of plants for treating diseases is as old as the human civilization. there are many plants which have been in use as traditional medicine, so they are called as medicinal plants. the use of plants for curing diseases was inevitable as is already proven by seeing the problems associated with synthetic antibiotics. peels of some plants such as punica granatum (having antibacterial properties) which are generally treated as wastes are true antibiotics as they are available for no cost, have no side effects and the most important benefit is that antibiotic resistant pathogens will be easily killed by these new and natural antimicrobials because they will take at least a few decades to get mutated and resistant to them.9 pomegranate juice as mouth rinse for fixed orthodontic patients is not a new idea. pomegranate mouth rinse was very efective to reduce dental plaque compared to chlorhexidine and distilled water in fixed orthodontic patients.11 however, the adverse effect towards fixed orthodontics accesories (wires, elastomeric modules, coil springs) are still unclear. the aim of the present review is to discuss the beneficial and unwanted effect of pomegranate juice towards orthodontic treatment. oral hygiene problem and maintenance in fixed orthodontic patients oral hygiene may be difficult to maintain during treatment, which may lead to plaque accumulation and gingival inflammation. it has been shown that orthodontic treatment induces changes in the oral environment, with an increase in the bacteria’s concentration, and alterations in buffer capacity, ph acidity and salivary flow rate.2,3 however, little is known about periodontal inflammation that results in diluted blood in saliva as well as the ph of dental plaque.12 plaque accumulation and subsequent gingivitis are common in orthodontic patients because of the challenge of controlling oral hygiene with the combination of brackets, bands, wires as well as elastomeric modules and chains present.2,3 the main goal of an at-home oral hygiene for fixed orthodontic patients is toothbrushing with a fluoride containing toothpaste. the role of fluoride as an inhibitor of demineralisation becomes important and it has been shown that the presence of fluoride in solution at the time of acidic attack on the enamel may considerably slow down the rates of decay. it has been suggested that if preventive measures (fluoride supplementation and good oral hygiene) are followed and maintained throughout the course of orthodontic treatment, then the number of white spot lesions may be reduced.12 mouthrinse mouthrinses or mouthwashes are generally formulated using antibacterial agents (fluoride, alcohol, and cetylpyridinium chloride), flavours (thymol, eucalyptol, menthol, and mint oils), humectants (sorbitol, glycerol, propylene glycol) and colorants in an aqueous/ alcoholic medium. the sweetener (sodium saccharin) may be used to reduce/ eliminate the base taste of the product. surfactants 223utomo and oetomo: pomegranate juice (punica granatum) as an ideal mouthrinse (sodium lauryl sulphate, cocoamidopropyl betaine) may also be used as foaming agents and water is the medium. most of the currently available mouthwash products in the market contain alcohol. though alcohol acts as an antiseptic and antibacterial, it induces dryness in the mouth and makes the environment more susceptible to the growth of bad breath producing bacteria. hence, the recent trend in the market is to move from the alcohol-containing products to alcohol-free mouthrinses.1,13 generally, some prescription of mouthrinse should be: (a) antimicrobial of oral pathogenic bacteria; (b) do not induce adverse effects (iritation, change taste buds perception, create oral flora imbalance, microbial resistance, decrease salivary fow and tooth or restoration staining); (c) contain fluorides.1 nevertheless, for fixed orthodontic patients several requirements should be added that does not affect the properties of elastomeric modules and chains14 as well as brackets and wires.15 in usa, mouthrinse should compared with ada and/or fda guidelines, two antiseptic mouthrinses (and their generic equivalents) have been awarded the ada seal for chemotherapeutic control of supragingival plaque and gingivitis: 0.12% chlorhexidine gluconate (chg) mouthrinse (peridex®) and essential oils (eo) mouthrinse (listerine®).1 some dental professionals may fear that antiseptic mouthrinses have a risk in killing or inhibiting normal flora with subsequent repopulation with opportunistic and/or more pathogenic or resistant organisms. the microbial shift would manifest as an overgrowth of opportunistic organisms, such as candida. fortunately, studies document no adverse effects on supragingival dental plaque microflora after 6 months of continued use with either chg or eo. evidence confirms that daily, long-term use (6 months or longer) of chg or eo does not adversely affect oral microbial flora, including no microbial overgrowth, opportunistic infection, or development of microbial resistance.1,16 benefits of herbal antimicrobials the widespread use of commercially available antimicrobials led to the consequence of emergence of antimicrobial resistant pathogens that ultimately led to the threat to global public health. since 1980 the introduction of new antimicrobials has declined due to the huge expense of developing and testing new drugs. all commercially available antibiotics with prolonged use may have negative effect on human and animal health because they kill gut flora, so they needs to take probiotics to replace the killed gut flora. all the above points make a clear way for herbal antimicrobials.14,15 the use of plants for treating diseases is as old as the human civilization. there are many plants which have been in use as traditional medicine, so they are called as medicinal plants. the use of plants for curing diseases was inevitable as is already proven by seeing the problems associated with synthetic antibiotics.15,17 peels of some plants as punica granatum (having antibacterial properties) which are generally treated as wastes are true antibiotics as they are available for no cost, have no side effects and the most important benefit is that antibiotic resistant pathogens will be easily killed by these new and natural antimicrobials because they will take at least a few decades to get mutated and resistant to them.17,18 pomegranate pomegranate (punica granatum l.) is native to the mediterranean region and has been extensively used in the folk medicine of many countries. pomegranate juice has potential anti-atherogenic effects in healthy humans and atherosclerotic effects in mice along with other nutritional and health advantages. as a result, pomegranate juice has become popular worldwide. numerous studies on antioxidant activity have shown that pomegranate juice contains higher levels of antioxidants than most fruit juice.14 pomegranate juice is an important source of anthocyanins (cyanidin, delphinidin, pelargonidin), which gives the fruit and aril its red color, and phenolics and tannins (punicalin, pedunculagin, punicalagin, ellagic acid).18 pomegranate fruit contains many different kinds of polyphenolic antioxidants and commercial pomegranate juice has been shown to posses antioxidant activity three times higher than those of red wine and green tea (table 1). the strongest antibacterial activity is the skin (rind, peel), followed by juice and the least is the seeds (red seeds are stronger than white). other constituent of pomegranate juice which is beneficial to dental and periodontal health are calcium and phosphorus15 as well as fluoride even only approximately 1/50th of green tea fluoride.19 table 1. phytochemical of pomegranate18 plant component constituents pomegranate juice anthocyanins, glucose, ascorbic acid, ellagic acid, gallic acid, caffeic acid, catechin, egcg, quercetin, rutin, numerous minerals, particularly iron, aminoacids pomegranate seed oil 95-percent punicic acid, other constituents, including ellagicacid, other fatty acids, sterols pomegranate pericarp (peel, rind) phenolic punicalagins, gallic acid and other fatty cids, catechin, egcg, quercetin, rutin and other flavonols, flavones, flavonones, anthocyanidins pomegranate leaves tannins (punicalin and punicafolin), and flavones glycosides, including luteolin and apgenin pomegranate flower gallic acid, ursolic acid, triterpenoids, including maslinic and asiatic acid, other unidentified constituents pomegranate roots and bark ellagitannins, including punicalin and punicalagin, numerous piperidine alkaloids 224 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 221–227 pomegranate’s mouth rinse and oral health investigators had been noted that pomegranate’s active components, including polyphenolic flavonoids (e.g., punicalagins and ellagic acid) (table 1), are believed to prevent gingivitis through a number of mechanisms including reduction of oxidative stress in the oral cavity, direct antioxidant activity; antiinflammatory effects; antibacterial activity; and direct removal of plaque from the teeth.10 changed of salivary measures relevant to oral health including gingivitis after rinsing with pomegranate extract.20 the changes were: (a) reduced total protein (which can correlate with plaque forming bacteria readings); (b) reduced activities of aspartate aminotransferase (an indicator of cell injury); (c) reduced alpha-glucosidase activity (a sucrose degrading enzyme); (d) increased activities of the antioxidant enzyme ceruloplasmin (which could give better protection against oral oxidant stress) and (e) increased radical scavenging capacity. a placebo of cornstarch in water did not affect these measures. pomegranate juice anti-iinflammatory and anti-oxidant effect.14 the effect of the hydroalcoholic extract (hae) from pomegranate fruits on dental plaque microorganisms.11 the results, expressed as the number of colony forming units per milliliter (cfu/ml), show that the hae was very effective against dental plaque microorganisms, decreasing the cfu/ml by 84% after mouthrinse. while similar values were observed with chlorhexidine, used as standard and positive control (79% inhibition), only an 11% inhibition of cfu/ml was demonstrated in the distilled water group, negative control. therefore, hae had an antibacterial activity against selected microorganisms, and may be a possible substitute for the treatment of dental plaque bacteria. moreover, studies conducted by bhandari et al.,10 and bhadbade sj et al.,21 indicate that the pomegranate mouth rinse has an antiplaque effect. it also states that pomegranate extract is succeeded against the aggregatibacter actinomycetemcomitans, porphyromonas gingivalis, and prevotella intermedia strains in vitro. these investigators suggest that pomegranate mouth rinse should be explored as a long-term antiplaque rinse, with prophylactic benefits. pomegranate extracts in especially fruit skin extract is highly effective on growth of streptococcus mutans in comparison with other extracts and various concentrations of tooth pastes, this fact may reflect efficiency of antibacterial activity of plant extracts, and ability of bacteria to resist other antibacterial agents such as pastes and antibiotics.22 pomegranate extract suppresses the ability of s. mutans, mitis and sanguis to adhere to the surface of the tooth.23 the trick is to inhibit a common species of streptococcus, preventing it from producing chemicals that create favorable conditions for fungi and other microorganisms to grow. inhibition of interbacterial adhesion or coaggregation which important in biofilm formation was verified.24 investigate the methanolic extract of punica granatum peel (mepgp) revealed its effective concentrations in mg/ml against microbes (table 2).17 effect of mouthrinse properties towards fixed orthodontic appliances there are several mouthrinse properties that may affect the characteristics of fixed orthodontics appliances and accesories for example: (a) fluoride; (b) alcohol; (c) ph (acidity). decay of elastomeric chain force in natrium fluoride 0.5% mouthwash plus saliva will degraded more rapidly than saliva only in higher force, that was 300 gram (p=0.020).2 nevertheless, in low force the difference was insignificant. meridol® mouthwash, which contains stannous fluoride, was the solution in which the nickel titanium (niti) wires coupled with the different brackets showed the highest corrosion risk, while in elmex® mouthwash, which contains sodium fluoride, the cuniti wires presented the highest corrosion risk.6 such corrosion has two consequences: deterioration in mechanical performance of the wire-bracket system, which would negatively affect the final aesthetic result, and the risk of local allergic reactions caused by released ni ions. the results suggest that mouthwashes should be prescribed table 2. antibacterial and antifungal properties of methanolic extract of punica granatum at three different concentrations17 microbials strains antimicrobial activity at p. value fconc. of 4 conc. of 8 conc. of 12 positive control**mean sd mean sd mean sd staphylococcus aureus 7.5 0.57 11.5 0.56 12.5 0.58 30 0.000 155.66 staphylococcus epidemis 11.5 0.57 13.5 0.59 13.5 0.58 29 0.000 20.00 lactobacillus acidophilus 6.5 0.57 10.0 0.00 10.0 0 14 0.000 227.00 actinomyces viscosus 6.0 0.00 6.5 0.57 6.5 0.57 25 0.168 2.00 streptococcus mutans 6.0 0.00 9.5 0.57 9.5 0.57 24 0.000 98.00 streptococcus sanguinis 6.5 0.57 10.0 0.00 11.5 0.58 25 0.000 172.00 streptococcus salivarius 6.5 0.58 8.5 0.59 9.5 0.60 26 0.000 43.66 candida albicans 6.0 0.00 6.5 0.57 6.5 0.57 40 0.168 2.00 *measured by the diameter of zone of inhibition in mm, conc= concentration, **ciprofloxacin and nystatin are the positive control group. 225utomo and oetomo: pomegranate juice (punica granatum) as an ideal mouthrinse according to the orthodontic materials used. a new type of mouthwash for use during orthodontic therapy could be an interesting development in this field.6 regarding to the safety of long term use of alcohol mouthrinse for oral cancer, it was still inconclusive.13 additionally, alcohol containing mouthwashes afford little or no advantage in terms of efficacy over the alcohol free competitors. alcohol causes a statistically significant (p=0.04) increase in the amount of force decay seen in elastomeric chains exposed to alcohol and commercial mouth rinse containing alcohol compared to those exposed only to water.8 ph of the mouthwash products are varying from 3.7 to 6.5. the mouthwashes containing ethyl alcohol in the range of 6–8% are having ph in the range of 6.0–6.5 whereas those containing alcohol higher than 20% are acidic in nature (ph below 4 also).26 there was chemical changes in ph and total ethanol of mouth rinse evaluated over time.27 therefore, it is recommend that mouthwash manufacturers routinely check the change of ph of the mouthwashes, and use preservatives that maintain desirable ph homeostasis in the products. elastomeric chain force degradation increase rapidly in neutral or alkaline ph,7 thus acidic environment facilitate higher performance. fruit juices including pomegranate (2.75-4.14) are acidic liquid.28 as the result it is considered beneficial to elastomeric chain properties. discussion based on this review, the prime question: “is it safe to use mouthwash when you have braces?”, should be answered: ”no problem”. until now the safety of alcoholcontaining mouthrinses has been called into question, since many proprietary mouthwashes contain alcohol (ethanol) and in some, the concentration of ethanol can be as high as 26% have fuelled the controversy.13 studies which appear to establish a relationship between the use of these rinses and oral cancer, are significantly flawed or imperfect.1,29 high risk predisposition (children, alcohol dependent persons, and persons with genetic deficiencies in acetaldehyde metabolism) should use alcohol-free mouthwashes for the maintenance of oral health.30 apart from these considerations for the individual, the risk of alcohol-containing mouthwash for public health appears very low compared to other routes of exposure to alcohol and acetaldehyde. nevertheless, in fixed orthodontic patients, especially in indonesia with high humidity lead to accelerate elastomers degradation, slowing down the force degradation by using an alcohol-free mouthwash is advisable. pomegranate juice is considered as acidic (ph 2.75– 4.1),7 it is beneficial for orthodontic movement by slowing down elastomeric degradation. even acidic, it is considered as a save mouthrinse, since ph of other mouthwash products are varying from 3.7 to 6.5. the mouthwashes containing ethyl alcohol in the range of 6–8% are having ph in the range of 6.0-6.5 whereas those containing alcohol higher than 20% are acidic in nature (ph below 4 also).26 another ingredients such as calcium and fluoride of pomegranate juice is beneficial for maintaining the integrity of tooth enamel.15 fluoride content of pomegranate juice is not as high as other fluoride containing mouthrinse. excess of fluoride content in daily mouthrinse is disadvantageous for brackets and cuniti wires since the are more prone to corrosion.6 other than its non-alcoholic contains, pomegranate as a phytoplants mouthrinse have been shown to be good alternatives to synthetic chemical antimicrobial agents and antibiotics because of the serious side effects, antimicrobial resistance and the emergence of previously uncommon infections that have been reported to be on the increase due to inappropriate or widespread overuse of antimicrobials. on the other hand, clinicians should remind the potential risk of urticaria, alteration in taste, increase of calculus staining of teeth and mucous membranes and more rarely, oral mucosa desquamation and parotid swelling before prescribing chlorhexidine mouth wash as an antimicrobial agent.14,18 extracts of punica granatum peel in different concentrations were effective against s. epidermidis, s. aureus, s. mutans, s. sanguinis and s. salivarius.24,31 antibacterial activity may be related to the presence of hydrolysable tannins and polyphenolics in the pomegranate extract specifically punicalagin and gallagic acid.32 it means that the antimicrobial effect of tannins is related to its toxicity and molecular structure. tannins may act on the cell wall and across the cell membrane because they can precipitate proteins. they may also suppress many enzymes such as gycosyltransferases. therefore, tannic acid has the highest antibacterial effect against tested sensitive strains even at low concentrations. p l a q u e m a y i n v o l v e f o u r o r m o r e d i f f e r e n t microorganisms combining forces to colonize the surface of the teeth. remarkably, nature’s own pomegranate fights the organisms’ ability to adhere by interfering with production of the very chemicals the bacteria use as “glue” in bacterial adhesion or coaggregation.24 the use of pomegranate as anti-adhesion of microbes. microbial adhesion is considered the first step in the sequence of events leading to colonization, is an important step leading to virulence and subsequent infection.33 since every part of the pomegranate plants and fruit has its own active ingredients, therefore in producing a pomegranate mouthrinse one should refer to a reference such as in table 1. hence, the antibacterial activity of punica granatum may be related to polyphenol structures because polyphenols may affect the bacterial cell wall, inhibit enzymes by oxidized agents, interact with proteins and disturb co-aggregation of microorganisms. therefore, if our intention is to make mouthrinse with the phenolic 226 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 221–227 compound, thus the peel and rind of pomegranate should be included. if only mild antimicrobial effect needed, just made it from white seed pomegranate.16,19 as an antioxidant, the effect of pomegrante mouthrinse towards orthodontic tooth movement (otm) is still unclear. a rat model of orthodontic tooth movement which consumed anti oxidants resveratrol or n-acetylcysteine.34 it suppressed the expression of proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in the periodontal ligament tissues compared to the vehicletreated group, thus inhibit bone resorption and retard otm. consumption of pomegranate extract accelerate bone formation.35 however, pomegranate consumption is different with mouthrinsing only, therefore the retardation of otm should be minimal. based on the literatures it is concluded that pomegranate is an ideal mouthrinse for fixed orthodontic patients since it has abundant benefits for long term use. the beneficial properties are: (a) antimicrobial of oral pathogenic bacteria; (b) prevent the interbacterial adhesion (coaggregation) as well as bacteria to tooth adherence by interfering adhesive production; (c) do not induce adverse effects (iritation, change taste buds perception, create oral flora imbalance, microbial resistance, decrease salivary fow and tooth or restoration staining); (d) non-alcoholic; (e) as fruit juice it has acceptable taste; (f) do not accelerate elastomeric chain force degradation since it has acidic ph; (g) non corrosive for orthodontic brackets and cuniti wires, cause of its mild fluoride content; (h) others are its antiinflammatory and antioxidant effect as well as calcium and phosphorus contents which beneficial towards dental and periodontal health. however, further researches should be done for its antiinflammatory and antioxidant effect towards bone remodelling which mandatory in orthodontic movement. references 1. goldie mp.the role of oral rinse technologies in a new daily oral healthcare regimen. compendium 2012; 32(3): 15–20. 2. perosa k, mestrovicb s, anic-milosevicc s, slaj m. salivary microbial and nonmicrobial parameters in children with fixed orthodontic appliances angle orthod 2011; 81(5): 901–6. 3. teixeira hs, kaulfuss smo, ribeiro js, do rosário pereira b, brancherja, elisa souza camargo es. calcium, amylase, glucose, total protein concentrations, flow rate, ph and buffering capacity of saliva in patients undergoing orthodontic treatment with fixed appliances. dental press j orthod 2012; 17(2): 157–61. 4. sanpei s, endo t, shimooka s. caries risk factors in children under treatment with sectional brackets. angle orthod 2010; 80(3): 509–14. 5. al-kassar ss. the force degradation of elastic chain in different environments and for different intervals (an in vitro study). al– rafidain dent j 2011; 11(2): 231–7. 6. schiff n, mickaël, boinet m, morgon l, lissac m, dalard f, grosgogeat b. galvanic corrosion between orthodontic wires and brackets in fluoride mouthwashes. eur j orthod 2006; 28(3): 298–304. 7. christnawati. pengaruh ph saliva terhadap sifat elastik rantai elastomerik. mi ked gigi 2007; 22(1): 16–20. 8. larrabee tm, liu ss�, torres-gorena a, soto-rojas a, george j, eckert gj, stewart kt. the effects of varying alcohol concentrations commonly found in mouth rinses on the force decay of elastomeric chain.angle orthod 2012; 82: 894–89. 9. ramesh ks, shamim sf. role of pomegranate in preventive dentistry. ijrap 2012; 3(5): 5–10. 10. bhandari pr. pomegranate (punica granatum l). ancient seeds for modern cure? review of potential therapeutic applications. int j nutr pharmacol neurol dis 2012; 2: 171–84. 11. menez es sm, cordei ro l n, via na gs. p u n ica g r a nat u m (pomegranate) extract is active against dental plaque. j herb pharmacother 2006; 6(2): 79–92. 12. lara-carrillo e, montiel-bastida nm, sánchez-pérez l, alanístavira j. effect of orthodontic treatment on saliva, plaque and the levels of streptococcus mutans and lactobacillus. med oral patol oral cir bucal 2010; 15(6): 924–9. 13. de a werner cw, seymour ra. are alcohol containing mouthwashes safe?. br dent j 2009; 207(10): 1–10. 14. kukreja bj, dodwad v. herbal mouthwashes – a gift of nature. int j pharm bio sci 2012; 3(2): 20–4. 15. eksi a, özhamamcı i̇. chemical composition and guide values of pomegranate juice gida 2009; 34(5): 265–70. 16. tufekci e, casagrande za, lindauer sj, fowler ce, williams kt. effectiveness of an essential oil mouthrinse in improving oral health in orthodontic patients. angle orthod 2008; 78(2): 294–8. 17. mousavinejad g, zahra emam-djomeh z, karamatollah k. identification and quantification of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight iranian cultivars. food chemistry 2009; 115: 1274–8. 18. abdollahzadeh s, mashouf r�, mortazavi h, roozbahani mn, vahedi m. antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens. j dentistry tehran 2011; 8(1): 1–6. 19. dahham ss, ali mn, tabassum h, khan m. studies on antibacterial and antifungal activity of pomegranate (punica granatum l .). american-eurasian j agric & environ sci 2010; 9(3): 273–81. 20. giljanović j, prkića, marija bralić m, brkljača m. determination of fluoride content in tea infusion by using fluoride ion-selective electrode. int j electroche sci 2012; 7: 2918–27. 21. disilvestro ra, disilvestro dj. pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk. phytother res 2009; 23(8): 1123–7. 22. bhadbhade sj, acharya ab, rodrigues sv, thakur sl. the antiplaque efficacy of pomegranate mouthrinse. quintessence int 2011; 42(1): 29–36. 23. alsaimary ie. efficacy of some antibacterial agents against streptococcus mutans associated with tooth decay. internet j microbiol 2009; 7(2). 24. vasconcelos lcs, sampaio fc, sampaio mcc, pereira mcv, higino js, peixoto m. minimum inhibitory concentration of adherence of punica granatum (pomegranate) gel against s. mutans, s. mitis and c. albicans. braz dent j 2006; 17(3): 223–7. 25. ramazanzadeh ba, jahanbin a, hasanzadeh n, eslami n. effect of sodium fluoride mouth rinse on elastic properties of elastomeric chains. j clin pediatr dent 2009; 34(2): 189–92. 26. teki k, bhat r. composition analysis of the oral care products available in indian market. part i: mouthwashes. ijarpb 2012; 1(3): 338–47. 27. lee ck. determination of ph, total acid, and total ethanol in oral health products: oxidation of ethanol and recommendations to mitigate its association with dental caries. j dent oral med dent ed 2009; 3(1): 1–4. 28. akbarpour v, hemmati k, sharifani m. ph physical and chemical properties of pomegranate (punica granatum l.) fruit in maturation stage. american-eurasian j agric & environ sci 2009; 6(4): 411–6. 29. iacopino am. use of alcohol-containing rinses to reduce oral microbial burden: safety and efficacy. cda 2009; 75(4): 160–4. 30. lachenmeier dw. alcohol-containing mouthwash and oral cancer– can epidemiology prove the absence of risk? an agric environ med 2012; 19(3): 609–10. 227utomo and oetomo: pomegranate juice (punica granatum) as an ideal mouthrinse 31. naz s, siddiqi r, ahmad s, rasool sa, sayeed sa. antibacterial activity directed isolation of compounds from punica granatum. j food sci 2007; 72(9): m341–5. 32. reddy mk, gupta sk, jacob mr, khan si, ferreira d. antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l. planta med 2007; 73(5): 461–7. 33. sharma s, sabnis s. study of anti adhesive properties of fruit juices and plant extracts on urinary tract pathogens. asian j exp biol sci spl 2010; 100–3. 34. chae hs, park hj, hwang hr, kwon a, lim wh, �i wj, han dh, kim �h, baek jh. the effect of antioxidants on the production of pro-inflammatory cytokines and orthodontic tooth movement. mol cells 2011; 32(2): 189–96. 35. monsefi m, parvin f, talaei-khozani t. effects of pomegranate extracts on cartilage, bone and mesenchymal cells of mouse fetuses. br j nutr 2012; 107(5): 683–90. vol 52 no 1 jan-mar 2019_new.indd 1313 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 13–17 research report effects of hydroxyapatite gypsum puger scaffold applied to rat alveolar bone sockets on osteoclasts, osteoblasts and the trabecular bone area amiyatun naini,1 i ketut sudiana,2 moh. rubianto,3 utari kresnoadi,4 and faurier dzar eljabbar latief5 1department of prosthodontics, faculty of dentistry, universitas jember, jember – indonesia 2department of electron microscopy, faculty of medicine, universitas airlangga, surabaya – indonesia 3department of periodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia 5micro-ct laboratory, faculty of mathematics and natural sciences, institut teknologi bandung, bandung – indonesia abstract background: damage to bone tissue resulting from tooth extraction will cause alveolar bone resorption. therefore, a material for preserving alveolar sockets capable of maintaining bone is required. hydroxyapatite gypsum puger (hagp) is a bio-ceramic material that can be used as an alternative material for alveolar socket preservation. the porous and rough surface of hagp renders it a good medium for osteoblast cells to penetrate and attach themselves to. in general, bone mass is regulated through a remodeling process consisting of two phases, namely; bone formation by osteoblasts and bone resorption by osteoclasts. purpose: this research aims to identify the effects of hagp scaffold application on the number of osteoblasts and osteoclasts, as well as on the width of trabecular bone area in the alveolar sockets of rats. methods: this research used posttest only control group design. there were three research groups, namely: a group with 2.5% hagp scaffold, a group with 5% hagp scaffold and a group with 10% hagp scaffold. the number of samples in each group was six. hagp scaffold at concentrations of 2.5%, 5% and 10% was then mixed with peg (polyethylene glycol). the wistar rats were anesthetized intra-muscularly with 100 mg/ml of ketamine and 20 mg/ml of xylazine base at a ratio of 1:1 with a dose of 0.08-0.2 ml/kgbb. extraction of the left mandibular incisor was performed before 0.1 ml preservation of hagp scaffold + peg material was introduced into the extraction sockets and suturing was performed. 7 days after preparation of the rat bone tissue, an hematoxilin eosin staining process was conducted in order that observation under a microscope could be performed. results: there were significant differences in both the number of osteoclasts and osteoblasts between the 2.5% hagp group, the 5% hagp group and the 10% hagp group (p = 0.000). similarly, significant differences in the width of the trabecular bone area existed between the 5% hagp group and the 10% hagp group, as well as between the 2.5% hagp group and the 10% hagp group (p=0.000). in contrast, there was no significant difference in the width of the trabecular bone area between the 2.5% hagp group and the 5% hagp group. conclusion: the application of hagp scaffold can reduce osteoclasts, increase osteoblasts and extend the trabecular area in the alveolar bone sockets of rats. keywords: alveolar bone; osteoblasts; osteoclasts; scaffold hydroxyapatite gypsum puger; sockets correspondence: amiyatun naini, department of prosthodontics, faculty of dentistry, universitas jember, jl. kalimantan 37, jember 68121, indonesia. e-mail: amiyatunnaini@yahoo.com introduction the post-extraction bone tissue healing process which begins in the alveolar socket area is characterized by bone remodeling, involving a cycle of bone resorption and bone formation.1 the bone structure that remains after the healing process will experience progressive resorption triggered by osteoclasts. resorption in the first six months after tooth extraction is usually extremely rapid, but subsequently decelerates and continues to a limited extent physiologically for the remainder of the life of the tissue.2 damage to bone tissue due to tooth extraction will cause alveolar bone resorption. in the field of dentistry, this process can influence the prognosis arrived at by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p13–17 mailto:amiyatunnaini@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p13-17 14 naini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1):13–17 prosthodontic treatment clinics. histologically, active bone formation occurs two weeks after tooth extraction, with the socket being filled with new bone within six months.3,4 furthermore, bone formation results from a complex cascade involving the proliferation of mesencymal stem cells and differentiation of osteoblast precursor cells, in addition to the maturing of osteoblasts, matrix formation and, finally, mineralization. osteoblasts move towards the base of the resorption cavity and then form osteoid which initiates mineralization lasting 13 days. osteoblasts are continuously formed and osteoid mineralization occurs until the cavity is filled. some osteoblasts differentiate into osteoid and attach to the matrix.5 it can be said that bone mass is regulated through a remodeling process involving two phases, namely; bone formation by osteoblasts and bone resorption by osteoclasts.6 in the field of prosthodontics, maintaining alveolar bone after tooth extraction should be prioritized to support the manufacture of conventional prostheses as well as the placement of implants.3 therefore, bone substitution material containing graph material is required for the preservation of alveolar sockets.7,8 one graph material developed as a synthetic biomaterial is hydroxyapatite with the chemical formula ca10 (po4)6 (oh)2. 9 hydroxyapatite also plays a role in the bone regeneration process, including osteointegration since it possesses osteoconductive properties that can stimulate mesenchymal cells to proliferate and differentiate during the bone regeneration process. the interconnected porous hydroxyapatite can even form a bond between extremely strong bones and accelerate the vascular procedure. nevertheless, the dimension and shape of the pore are also considered to be important factors in the osteointegration process. hydroxyapatite interconnects pores with rough surfaces and, therefore, facilitates penetration of osteoblast cells and becomes a supportive medium for osteoblast cells to attach to the surface of the bone graft matrix.10 previous research has indicated that hydroxyapatite can be synthesized from gypsum produced at gamping mountain in puger sub-district. consequently, hydroxyapatite gypsum puger (hagp), can be used as an alternative bioceramic material in the preservation of alveolar sockets.11 unfortunately, the effects of hagp scaffold as alveolar socket preservation material on the number of osteoblasts and osteoclasts as well as the width of trabecular area have not yet been comprehensively investigated. as a result, this research aimed to identify the effects of scaffold hagp application on the number of osteoblasts and osteoclasts as well as the width of the trabecular area in the alveolar bone remodeling process involving alveolar sockets in rats. materials and methods ethical approval for this research was obtained from the research ethics committee of the faculty of dental medicine, universitas airlangga (number: 247/kkepk. fkg/x/2016). this research was a pure experimental study with postest only control group design. the number of research samples totaled eighteen divided into three groups, each of which contained six members. this research employed hydroxyapatite gypsum puger (hagp) and gelatin to form a hydrogel for the manufacture of hagp scaffold. 10 g of solid gelatin was melted in hot water at a temperature of 600c to form 10% liquid gelatin. four grams of hydroxyapatite was subsequently mixed with 10ml of the liquid gelatin, before being frozen and dried using a sublimation/freeze dried system. thereafter, hagp particles were crushed, milled and sifted to a particle size of 150-355 μm at the tissue bank of dr. soetomo general hospital, surabaya. hagp scaffold at a concentration of 2.5% was prepared by mixing 0.05 grams of hagp scaffold and 0.45 grams of polyethylene glycol (peg). hagp scaffold at a concentration of 5% was then prepared by mixing 0.05 grams of hagp scaffold and 0.95 grams of peg. hagp scaffold at a concentration of 10% was prepared by mixing 0.05 grams of hagp scaffold and 1.95 grams of peg. peg was produced by mixing 3.92 grams of peg 400 (solid) with 0.98 grams of peg 4000 (liquid). all ingredients were then placed in a sterile container in preparation for application to the sockets of the rats. the wistar rats were anesthetized intra-muscularly with 100 mg/ml of ketamine and 20 mg/ml of xylazine base at a ratio of 1:1 and a dose of 0.08-0.2 ml/kgbm. after the subjects had been anesthetized, their left mandibular incisor was extracted with a needle holder. 0.1 ml of hagp scaffold + peg material was then applied to the extraction socket which was sutured with 75 cm of dr sella silk braided usp 3/0. seven days later, the subjects were sacrificed by means of a cotton swab moistened with ether and placed in a sealed glass box container for five minutes. their left lower jaw was carefully cut posteriorly from the anterior and then washed with pbs before the tissue was fixated in a 10% formalin buffer for 24 hours. alveolar bone demineralization was then carried out using 15% edta solution for 4-6 weeks (the solution being replaced once every three days). once the tissues had softened, they were processed into paraffin blocks. the soft tissues were washed with pbs at ph 7.4 three times for five minutes. dehydration using alcohol was performed at multilevel concentrations (70%, 80%, 96% and absolute) for 60 minutes in each case. clearing was then effected with xilol on two occasions times each of 60 minutes’ duration prior to infiltration with soft paraffin being conducted for 60 minutes at a temperature of 5860ºc. blocking was carried out for a day inside the hard paraffin in the molds. on the following day, the soft tissues were affixed to the holder and cut into pieces 4-6 microns wide with a rotary microtom. mounting on glass objects was subsequently performed using adhesive material. thereafter, they were sliced and put into a hot plate and then colored with hematoxilen-eosin (he). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p13–17 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p13-17 15naini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 13–17 0 2 4 6 8 10 12 14 16 2.5% hagp 5% hagp 10% hagp m ea n research groups osteoclasts osteoblasts trabecular bone area figure 1. the histogram of the mean number of osteoblasts and osteoclasts as well as the mean width of trabecular bone area in each research group. table 1. the mean and standard deviation of the number of osteoclasts and osteoblasts as well as the width of trabecular area in each research group. groups 10% hagp5% hagp2.5% hagp levene test anova x±sd, shapiro wilk x±sd, shapiro wilk x±sd, shapiro wilk p 9.67±0.235osteoclasts a 9.17±0.235. 0.167 a 4.00±0.359. 0.167 b 0.0000.727. 0.561 4.83±0.357osteoblasts a 6.33±0.592. 0.578 b 13.00±0.138. 0.399 c 0.0000.205. 0.683 13.88±0.242trabecular a 13.92±0.208. 0.198 a 14.86±0.189. 0.550 b 0.0000.908. 0.365 note: significance at α=0.05 the he staining was carried out to enable observation of osteoblasts and osteoclasts. the slides were washed with pbs at ph 7.4 three times for five minutes, before being colored with hematoxilen for ten minutes, soaked in tap water for the same period of time and rinsed with h2o. dehydration was conducted with 30% and 50% alcohol for five minutes respectively. the slides were colored with hematoxilen solution for 15 minutes, washed with running water and stained with eosin solution for three minutes. they were rinsed with 70%, 80%, 90% and 95% alcohol twice and with xylol three times. mounting was carried out with an entanglement and they were covered with a glass cover before being observed through a microscope. the number of osteoblasts and osteoclasts in the alveolar bone tissue incisions taken from the alveolar socket areas was measured using the he method and observed from 10 visual fields under a light microscope at 400x magnification. the trabecular area width was also measured by calculating the width of the trabecular bone formed in the alveolar socket area using he preparations using an optilab microscope camera and raster 3.0 image software. finally, statistical analysis was performed using a one-way anova test with a significance level of less than 0.05 (p < 0.05). results the mean number of osteoblasts and osteoclasts, as well as the mean trabecular area width in the 2.5% hagp group, the 5% hagp group and the 10% hagp group can be seen in table 1 and figure 1. the same superscript indicated no difference between groups when multiple lsd comparisons were made. the results of the anova test on the number of osteoclasts and osteoblasts as well as the width of the trabecular bone area indicated a p value of 0.000. this means that there was a significant difference between the 2.5% hagp group, the 5% hagp group and the 10% hagp group. consequently, an lsd multiple comparison test was conducted to identify any differences between two of the research groups. the lsd test results relating to the number of osteoclasts confirmed that there were significant differences between the 2.5% hagp group and the 5% hagp group with a p value of 0.034, between the 2.5% hagp group and the 10% hagp group with a p value of 0.000, as well as between the 5% hagp group and the 10% hagp group with a p value of 0.000. similarly, there were significant differences in the number of osteoblasts between the 2.5% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p13–17 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p13-17 16 naini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1):13–17 hagp group and the 5% hagp group with a p value of 0.001, between the 2.5% hagp group and the 10% hagp group with a p value of 0.000, as well as between the 5% hagp group and the 10% hagp group with a p value of 0.000. there were also significant differences in the width of trabecular bone area between the 2.5% hagp group and the 10% hagp group with a p value of 0.000 as well as between the 5% hagp group and the 10% hagp group with a p value of 0.000. histopathological anatomy descriptions showed osteoclast to be a multinucleated giant cell (figure 2), osteoblast to be a flat to round nucleated single nucleus (figure 3) and the trabecular bone area to be the hollow section of the bone (figure 4). discussion in this research, hagp scaffold at respective concentrations of 2.5%, 5% and 10% was employed to determine the appropriate and optimal concentration capable of increasing osteoblasts, extending the trabecular bone area and reducing the number of osteoclasts. it was demonstrated that, at the optimal concentration of 10%, hagp scaffold can increase osteoblasts, extend the trabecular bone area and decrease osteoclastsat concentrations higher than 5% and 2.5%. osteoblast and osteoclasts are the main components that play a role in bone remodeling. osteoblasts act as new bone formation, whereas osteoclasts are active in bone resorption processes.6,12 hydroxyapatite (ha), already known as a biomaterial in the biocompatible health field, constitutes a major constituent of bone material with specific properties such as the ability to promote chemical attachment to bone as well as that of reducing toxicity and inflammation. moreover, ha also possesses osteoconductive properties capable of stimulating mesenchymal cells to proliferate and differentiate as part of the bone regeneration process and osteoprogenitor cells which are considered active precursors of osteoblasts.10,13 a c b figure 2. accumulation of osteoclasts on the socket wall during microscopic observation at 400x magnification, in groups: (a) 2.5% hagp; (b) 5% hagp; and (c) 10% hagp. a c b figure 3. accumulation of osteoblasts on the socket wall during microscopic observation at 400x magnification, in groups: (a) 2.5% hagp; (b) 5% hagp; and (c) 10% hagp. a b c figure 4. accumulation of trabecular area on the socket wall during microscopic observation at 400x magnification, in groups: (a) 2.5% hagp; (b) 5%; hagp; and (c) 10% hagp. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p13–17 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p13-17 17naini, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 13–17 the contents of table 1 indicate that the higher the concentration of scaffold, the greater the increase in the number of osteoblasts and the more pronounced the reduction in the number of osteoclasts. in the initial phase, osteoblasts express rankl and stimulate the osteoclastogenic cascade. calcium released from the bone during resorption produces osteoclast apoptosis. as a result, osteoclastic differentiation is suppressed and bone formation is increased. osteoprotegrin produced by osteoblasts then prevents the interaction of rank with rank ligands. osteoprotegrin activity in osteoclast precursors (rank) inhibits osteoclastic formation.14–16 osteoblasts are commonly known as bone-forming cells derived from osteoprogenitor mesenchymal stem cells. osteoprogenitor cells, through calcium pathways or bone morphogenic protein (bmp) pathways, form and differentiate osteoblasts. the main function of osteoblasts is the formation of a bone matrix. osteoblasts also secrete certain products, such as collagen type i and type v, proteoglycans and non-collagen proteins (sialoprotein and osteopontin). on the other hand, osteoclasts, play a role both physiological and pathological in bone resorption. osteoclasts are derived from hematopoietic stem cells, their main function being in the resorption of bone matrix mineralized by the breakdown of hydroxyapatite crystals and organic matrix cleavage.14,17 in addition, the bone formation measurement results relating to post-extraction tooth sockets in rats indicated that hagp scaffold induction could have a positive effect on osteoblast response, thereby improving the physical properties of bone. similarly, research conducted by nishida et al. (2016), argues that graphene oxide scaffold can increase osteoblast proliferation.18 functionally, the surface of hagp is required for the interaction of cations and anions in order that calcium absorption can be increased. ca ions can actually stimulate bone marker expression in osteoblasts, stimulate alkaline activity and also adapt to the in-vivo environment for bone regeneration.19 accumulation of ca in hagp scaffold may even provide a favorable environment for bone tissue formation with the result that hagp scaffold is expected to have clinical applications in post-tooth extraction. these results indicate that hagp scaffold has high bone formation ability and is also expected to be useful for bone remodeling, especially for bone tissue engineering therapy. finally, it can be concluded that hagp scaffold at a concentration of 10% can increase osteoblasts, extend bone trabecular area and decrease osteoclasts in the alveolar bone of teeth sockets of rats more than that at concentrations of 5% and 2.5%. references 1. yang x, qin l, liang w, wang w, tan j, liang p, xu j, li s, cui s. new bone formation and microstructure assessed by combination of con foca l laser sca n n i ng m icroscopy a nd di f ferent ia l interference contrast microscopy. calcif tissue int. 2014; 94(3): 338–47. 2. sadr k, aghbali a, sadr m, abachizadeh h, azizi m, mesgari abbasi m. effect of beta-blockers on number of osteoblasts and osteoclasts in alveolar socket following tooth extraction in wistar rats. j dent (shiraz, iran). 2017; 18(1): 37–42. 3. d’souza d. residual ridge resorption– revisited. in: virdi m, editor. oral health care prosthodontics, periodontology, biology, research and systemic conditions. shanghai: intech; 2012. p. 15–24. 4. gupta a, tiwari b, goel h, shekhawat h. residual ridge resorbtion: a review. indian j dent sci. 2010; 2(2): 7–11. 5. kunert-keil c, gredes t, gedrange t. biomaterials applicable for alveolar sockets preservation: in vivo and in vitro studies. in: turkyilmaz i, editor. implant dentistry the most promising discipline of dentistry. shanghai: intech; 2011. p. 17–52. 6. vieira ae, repeke ce, ferreira junior s de b, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. 7. allegrini s, koening b, allegrini mrf, yoshimoto m, gedrange t, fanghaenel j, lipski m. alveolar ridge sockets preservation with bone grafting--review. ann acad med stetin. 2008; 54(1): 70–81. 8. kubilius m, kubilius r, gleiznys a. the preservation of alveolar bone ridge during tooth extraction. stomatologija. 2012; 14(1): 3–11. 9. ba lg ies, dewi su, da h la n k . si nt esis da n k a ra k t er isa si hidroksiapatit menggunakan analisis x ray diffraction. in: prosiding seminar nasional hamburan neutron dan sinar-x ke 8. tangerang: batan; 2011. p. 10–3. 10. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125–7. 11. naini a, ardhiyanto hb, yustisia y. proses sintesis dan karakterisasi hydoxyapatite menggunakan analisis xrd ftir dari gypsum puger kabupaten jember sebagai material augmentasi ridge alveolar. stomatognatic. 2014; 11(2): 32–7. 12. tanaka h, mine t, ogasa h, taguchi t, liang ct. expression of rankl/opg during bone remodeling in vivo. biochem biophys res commun. 2011; 411(4): 690–4. 13. pepla e, besharat lk, palaia g, tenore g, migliau g. nanohydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature. ann stomatol (roma). 2014; 5(3): 108–14. 14. veni mac, rajathi p. interaction between bone cells in bone remodelling. j acad dent educ. 2017; 2: 1–6. 15. sims na, martin tj. coupling signals between the osteoclast and osteoblast: how are messages transmitted between these temporary visitors to the bone surface? front endocrinol (lausanne). 2015; 6: 1–5. 16. tjoa sts, de vries tj, schoenmaker t, kelder a, loos bg, everts v. formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor. j clin periodontol. 2008; 35(7): 568– 75. 17. feng x, mcdonald jm. disorders of bone remodeling. annu rev pathol mech dis. 2011; 6: 121–45. 18. nishida e, miyaji h, kato a, takita h, iwanaga t, momose t, ogawa k, murakami s, sugaya t, kawanami m. graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket. int j nanomedicine. 2016; 11: 2265–77. 19. lü l-x, zhang x-f, wang y-y, ortiz l, mao x, jiang z-l, xiao z-d, huang n-p. effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo. acs appl mater interfaces. 2013; 5(2): 319–30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p13–17 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p13-17 189189 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 189–196 review article oral field cancerization: genetic profiling for a prevention strategy for oral potentially malignant disorders karlina puspasari1, togu andrie simon pasaribu1, meircurius dwi condro surboyo2, nurina febriyanti ayuningtyas2, arvind babu rajendra santosh2,3, diah savitri ernawati2 1oral medicine specialist program, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3school of dentistry, faculty of medical sciences, the university of the west indies, mona campus, kingston, jamaica abstract background: oral cancer therapy, such as radiation or surgical treatment, has pernicious long-term effects that patients suffer throughout their life, the disability being considerable with delayed diagnosis. it is well known that many oral cancers develop from oral potentially malignant disorders (opmds). patients diagnosed with opmds may have an increased risk of developing cancer anywhere in the oral cavity. early detection and intervention could be essential prevention strategies to inhibit oral cancer progression. opmds may not immediately develop into carcinoma. however, this condition provides a “field” of specific abnormalities wherein evolving altered genetic cells can be explained with the “field cancerization” concept. purpose: this review aims to describe the “field cancerization” concept in oral cancer and opmd, which is expected to contribute to a better clinical management strategy for oral cancer prevention. review: “oral field cancerization” describes oral cancers that develop in multifocal areas of pre-cancerous changes. it can be found as histologically abnormal tissue surrounding the tumor, suggesting that oral cancer often consists of multiple independent lesions. conclusion: the oral field cancerization concept should prompt healthcare professionals to remind their patients that frequent oral examination with histological studies and molecular testing is mandatory for those at high risk of developing malignancies. keywords: genetically altered field; oral cancer; oral field cancerization; oral potentially malignant disorders article history: received 30 november 2022; revised 5 january 2023; accepted 27 january 2023; published 1 september 2023 correspondence: diah savitri ernawati, department of oral medicine, faculty of dental medicine, universitas airlangga. jl mayjen prof. dr. moestopo no. 47, surabaya, 60132, indonesia. email: diah-s-e@fkg.unair.ac.id introduction oral cancer is one of the most common malignancies worldwide, with approximately 5% of those with diagnosed cancer coming from developing countries. the prevalence of head-and-neck squamous cell carcinoma (hnscc) worldwide is about 20 cases per 100,000 population per year.1 oral cancer, notably that induced by tobacco and alcohol consumption, may develop from an oral potentially malignant disorder (opmd) or pre-cancerization lesion.2–4 some opmds might disappear, while others result in oral cancer. the proportion of cases of opmd in oral cancers ranges from 3–50%.5 while the management of opmd might improve the outcome, standard therapy does not prevent cancer transformation from opmd.6 oral cancer therapies have pernicious long-term effects throughout the life of patients. these effects are substantial when the diagnosis is delayed.7 the hnscc 5-year survival rate is the lowest among aggressive cancers. the prognosis of oral squamous cell carcinoma (oscc) depends on the presence of new tumor growth.1 in 1953, slaughter published an article emphasizing the importance of examining and investigating the “field” surrounding an oral cancer lesion.8 this should be done at the risk-assessment stage and must be part of the comprehensive management of oral cancer. since then, many studies have used molecular techniques to explore this concept. the nature of oral cancer is genetically altered cells. during cancerization, several epithelial cells may undergo an altered genetic makeup called a field with a typical clinical appearance. these cells can provoke a process called “field cancerization.”9 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 mailto:diah-s-e@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 190 puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 some oral lesions display field cancerization and are classified as opmds or pre-cancerization lesions. common opmds are leukoplakia with a transformation possibility of 0.13–40.8%,10–12 erythroplakia with 33.1%,13 and oral lichen planus with 1.1–2.28%.14,15 the differences in transformation depend on the predisposing or risk factors.16 patients may develop cancer in the field of cancerization. unfortunately, this condition is only confirmed with invasive excisional surgery. there is no effective intervention for preventing transformation and cancerization.17 the purpose of this review is to discuss oral field cancerization, cancerization mechanisms, and deoxyribonucleic acid (dna), ribonucleic acid (rna), and tissue markers to be considered as cancerization biomarkers to provide recommendations for dentists, oral medicine specialists, and oral surgeons for better patient management. oral cancers the carcinogenesis process begins with a stem cell with one or more genetic or epigenetic alterations. then, a clone from the altered cells forms a patch or cluster. due to further genetic alteration, stem cells deviate from standard growth control patterns and enjoy advantages for the development of expanding clones. furthermore, lesions develop and become a field that replaces normal epithelium laterally. this field has genetically altered clonal units and advantages in proliferation activity, and finally dominates the overall process. additional genetic alterations occur along with enlargement of the lesion, creating various subclones within the field. because of divergence and clonal selection, clones are altered at different times and produce adequate modified stem cells. however, these cells share the same clonal origin. this process culminates in invasive cancer formation.18 histologically, it is considered a local recurrence when the distance between tumors is less than 2 cm; if it is more than 2 cm, it is regarded as a second primary tumor.19 even a single altered cell caused by tumorsuppressor gene inactivation and oncogene activation can overgrow and expand to form a clonal mass of tumor cells. clinically, this is a dynamic process. genetic alteration occurs from the accumulation of cell-growth phases and progresses from the benign to the pre-malignant and malignant stages.20 oral field cancerization the terms “field effect” and “field cancerization” are used when the pre-neoplastic process is in several locations.21 this was previously assumed to be multiple conditions developing independently. however, this was challenged due to the clinical diagnosis of a second primary tumor located distant from the original tumor, found on genetic analysis to be arising from the clonal spreading of the initial lesion.1 field pre-cancerization and its correlation with oral cancers are explained in figure 1. figure 1. field cancerization shows mucosal areas with normal cells (blue) and cells with cancer-associated genetic or epigenetic alterations (gray). a pre-neoplastic field is monoclonal in origin and does not show invasive growth or metastatic behavior, which are the hallmarks of invasive carcinoma (dark gray cells). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 191puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 oral cancers, especially osccs, develop in precancerous cells with clonal expansion of normal keratinocytes that have been altered genetically. the genetically unstable pre-cancerous keratinocyte manifests as aneuploidy, gaining or losing chromosomal material, or nucleotide sequence alteration. the instability of genomic support and further acquisition in genetic alteration leads to the growth superiority or inferiority of affected cells. genetically inherited cells eventually acquire a cancerous phenotype. although other oral cancers can develop from blood vessels or salivary glands, this mechanism underlies all cancer events. the probability of cancer development from genetically altered stem cells depends on the nature of the stem cells and the additional alteration. the proposed carcinogenesis model is based on a monoclonal origin and involves three stages.22,23 the first stage of patch formation is the conversion of a single stem cell (figure 2a) into a cell cluster with genetic alteration and without appropriate growth-pattern control. the second stage, or clonal expansion, is additional genetic alteration; the patch proliferates and forms a field that replaces normal epithelium (figure 2b). after exposure to another carcinogenic event, these cells turn to cancer cells with invasive growth or metastatic behavior, the third stage of tumor transition (figure 2c, 2d). surgical treatment is usually carried out at this stage (figure 2e). without proper molecular examination and prediction of field cancerization, cells with cancer-associated genetic or epigenetic alterations may be left behind (figure 2f). over time, with exposure to multiple carcinogenic events (unavoidable predisposing and risk factors), the remaining cells with cancer-associated genetics can develop into a second-field tumor, becoming overt carcinoma with invasive growth and metastases (figure 2g, 2h). markers of field cancerization carcinogenesis is a complex phenomenon with multiple genetic lesions and interactions.24 since every tumor has a figure 2. second field tumor model; a. a normal clonal unit. a stem cell (s) exposed to the carcinogenic event becomes a genetically altered cell. b. transit-amplifying cells (t) and daughter cells of the stem cell have the same genetic alteration. c. genetically altered cells with uncontrolled growth develop. d. cancer cells with invasive and metastatic behavior start to grow. e. the surgeon removes the carcinoma. f. post-surgery, without a proper genetic examination, the surgeon has left a field behind. g. a cell in the field turns into a cancer cell after another series of carcinogenic exposures. h. carcinoma develops in the same field: a second-field tumor.1 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 192 puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 unique alteration pattern, information about these markers can be used to measure the clonal correlation between lesions in a single patient. the presence of a field with genetically altered cells is a risk factor for cancer. many pre-cancerization cells within the proliferating area may increase cancer risk significantly. the early genetic event can lead to clonal expansion from pre-malignant daughter cells in specific tumor fields. subsequent genomic alteration in a few cells can induce a malignant phenotype.1 biomarkers can be used to monitor tumor progression, thus preventing invasive cancer transformation in precancerous lesions. the standard markers for identifying field cancerization are loss of heterozygosity, microsatellite alterations, chromosomal instability, and p53 gene mutations, generally detected by polymerase chain reaction, immunohistochemistry, and in situ hybridization.9 more specifically, some alteration or modification can detect both pre-cancerization and cancerization. in some instances of pre-cancerization, known as opmds, biomolecular markers have a significant role in detecting transformation to oral cancers. available data state that the intratumoral heterogeneity,25 proteome and lipidome profile,26 myofibroblasts,27 and cytokeratin markers like tissue polypeptide antigen (tpa) and tissue polypeptide specific antigen (tps),28 are the current biomarkers of pre-cancerization lesions. further, extensive analysis at the tissue and dna level has been developed. dna aneuploidy 29 and chromosome aberrations30 are commonly used to detect field cancerization at the dna level. several markers (p53, ki-67,31 cytokeratin fragments 21‐1,28 variations in nucleolar organizer regions,32 phosphatases and tensin homolog deleted on chromosome 10 allelic loss,33 dek overexpression,34 micro rna [hsa-mir-221, hsa-mir-21, hsa-mir-135b, and hsa-mir-29c] detection,35 atp-binding cassette subfamily g member 2,36 mutl protein homolog 1, methylguanine-methyltransferase methylation,37 interferonstimulated gene 15,38 aldehyde dehydrogenase, notch1,39 and bmi140) have been identified in pre-cancerization transformation into oral cancer, stimulating the cell cycle and promoting dna replication (figure 3). various protein expressions or markers have been revealed at the tissue level and may be easier to replicate in clinical settings than dna analysis. these include the expression of ki-67,38 kaiso, e-cadherin,41 stathmin,42 oct4+, sox2+,43 glut-3, glut 4,44 substance p, nk1r,45 podoplanin,36 matrix metalloproteinase 9 (mmp-9), tissue inhibitor of metalloproteinase 1 (timp-1), vimentin (vim),46 cornulin,38 transforming growth factor (tgfβ1) and interleukin 17-a (il-17a).47 these markers are essential in detecting oral cancer, especially oscc. all these markers have demonstrated strong reactivity, and detecting these markers increased the survival rate (figure 3). the specific techniques to obtain satisfactory results are whole-exome sequencing and targeted ultra-deep sequencing,48 dna high-resolution flow cytometry, arraycomparative genomic hybridization,30 mass spectrometry imaging based on matrix-assisted desorption-ionization,26 and liquid biopsy.49 discussion the world health organization has proposed the term opmd for classifying fifteen conditions, including leukoplakia, erythroplakia, proliferative verrucous leukoplakia, oral submucous fibrosis, and oral lichen figure 3. the markers of oscc, including dna analysis and tissue expression, are purposed for field cancerization to prevent malignant transformation or recurrence. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 193puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 planus.5 opmds are determined to be pre-cancerization lesions because some of the cells carry cancer-associated genetic or epigenetic alterations. some oral cancer specimens from the border of malignant lesions, showing a histologically normal appearance, have a genetic alteration, indicating that not all pre-cancerous fields can be identified histologically.50 genetic markers must be used to identify all potentially malignant areas.5 alteration can happen within the epithelium and/or the stroma.1 in the oral cavity, tobacco and alcohol work synergistically as primary carcinogens in developing osccs. environmental carcinogens reach a broader area simultaneously, destroy in more significant proportions and contribute to premalignant conditions within the exposed area and can manifest as micro-metastatic deposits.51 a pre-malignant field often needs a long time, about 67–96 months, to develop and become invasive carcinoma. an analysis of 783 patients by slaughter suggested that exposure to carcinogen-induced mucosal changes causes vulnerability of the surrounding area to multiple malignant foci.52 oral field cancerization is caused by either cell migration or from an independent cell. multiple tumors from the original primary cells and genetically altered cells from the primary cell are brought to their progenitor cells. investigating the development of primary lesions and their progression through cell expansion is crucial for measuring clonal markers based on the early identification of genetic events.18 clinically, oral cancer lesions may appear as white plaques, red plaques, ulcers, or verrucous forms, with a low degree of hyperplasia. however, the surrounding tissue may have a well-differentiated, verrucous hyperplasia, severe dysplasia, and even a carcinoma in situ.53 why the surrounding tissues transform into cancer is still not fully understood. the possible mechanism is that the adjacent tumor microenvironment and cancer occur through dynamic interactions by direct cell-to-cell communication or extracellular and intracellular agents. some hypotheses for cancer transformation are cell fusion, horizontal gene transfer, genetic instability, and microenvironment involvement.54 noncoding and micrornas represent the dynamic interaction between tumor and nontumor cells.55 this process may induce cancer-associated fibroblasts, the dominant cell type within the reactive stroma of many tumor types.56 other causal factors include cytokine involvement, growth factors, and reactive oxygen species (ros) as cell signaling molecules that aid cell-to-cell communication.57 field cancerization replaces the normal cell population with a histologically nondysplastic but pro-tumorigenic mutant cell clone.58 this mechanism is demonstrated in figure 2e; after surgical treatment, the surrounding tissue can progress to oral cancer because a single cell with cancer-associated genetic or epigenetic alterations can induce a neighboring cell to transform (figure 4). during the biopsy, it suggested that the sample should be larger than a single clonal unit, i.e., containing at least 200 cells in width and reaching 10 cm in diameter.19 this could identify pre-cancerization at the periphery of the incision, making the examination of possible areas of field cancerization more precise and adequate. however, it should be noted that some lesions and anatomical locations are a barrier to carrying out comprehensive biopsies. comprehensive biopsies aid in the adequate detection of field cancerization. clinical symptoms do not correlate with the pathogenesis and development of oral cancer in the early stages. thus, diagnostic biomarkers are crucial for determining histopathology grading and prognosis. when cancer has invaded, there are increased clinical symptoms, and the need for biomarker diagnostics is decreased. since figure 4. mechanism of field cancerization and cell transformation through noncoding rna, microrna, cytokines, growth factors, and reactive oxygen species. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 194 puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 the cancer is already in the final stage, the clinician focuses on the diagnosis and treatment plan with the possibility of a poor prognosis (figure 5). management strategies for opmd or pre-cancerization lesions should follow a strict protocol, including counseling emphasizing the patient’s commitment to discontinuing their bad habits. the clinician must provide a long-term follow-up and patient monitoring with an estimation of 67 – 96 months to detect invasive carcinoma transformation. based on the clinical characteristics, it is essential to examine and observe the entire oral area and not just the area with lesions. specific biomarkers must be used appropriately.17 these steps should contribute to improving prognosis.17 identifying molecular markers is essential in genetically transformed cells with normal histological appearance.59 thus, tumor-specific biomarker identification has an excellent role in monitoring tumor progression and, if possible, preventing invasive cancer transformation. early identification and management of field cancerization are critical for cancer mortality and morbidity prevention. in the clinical setting, oral field cancerization should prompt healthcare professionals to remind patients that frequent oral examination with histological studies and molecular testing is mandatory for those at high risk of developing malignancies. references 1. mallegowda h, theresa r, amberkar v. oral field cancerization: tracking the invisible. int j oral heal sci. 2019; 9(1): 28–35. 2. hecht ss, hatsukami dk. smokeless tobacco and cigarette smoking: chemical mechanisms and cancer prevention. nat rev cancer. 2022; 22(3): 143–55. 3. bhandari a, bhatta n. tobacco and its relationship with oral health. j nepal med assoc. 2021; 59(243): 1204–6. 4. vassoler t, dogenski lc, sartori vk, presotto js, cardoso mz, zandoná j, trentin ms, linden ms, palhano hs, vargas je, de carli jp. evaluation of the genotoxicity of tobacco and alcohol in oral mucosa cells: a pilot study. j contemp dent pract. 2021; 22(7): 745–50. 5. bouaoud j, bossi p, elkabets m, schmitz s, van kempen lc, martinez p, jagadeeshan s, breuskin i, puppels gj, hoffmann c, hunter kd, simon c, machiels j-p, grégoire v, bertolus c, brakenhoff rh, koljenović s, saintigny p. unmet needs and perspectives in oral cancer prevention. cancers (basel). 2022; 14(7): 1815. 6. abati s, bramati c, bondi s, lissoni a, trimarchi m. oral cancer and precancer: a narrative review on the relevance of early diagnosis. int j environ res public health. 2020; 17(24): 9160. 7. patil vm, noronha v, joshi a, abhyankar a, menon n, dhumal s, prabhash k. beyond conventional chemotherapy, targeted therapy and immunotherapy in squamous cell cancer of the oral cavity. oral oncol. 2020; 105: 104673. 8. desai rs, shirsat pm, bansal s, prasad p, satish arvandekar a. oral field cancerization: a critical appraisal. oral oncol. 2021; 118: 105304. 9. poh cf, zhang l, anderson dw, durham js, williams pm, priddy rw, berean kw, ng s, tseng ol, macaulay c, rosin mp. fluorescence visualization detection of field alterations in tumor margins of oral cancer patients. clin cancer res. 2006; 12(22): 6716–22. 10. warnakulasuriya s, ariyawardana a. malignant transformation of oral leukoplakia: a systematic review of observational studies. j oral pathol med. 2016; 45(3): 155–66. 11. aguirre‐urizar jm, lafuente‐ibáñez de mendoza i, warnakulasuriya s. malignant transformation of oral leukoplakia: systematic review and meta‐analysis of the last 5 years. oral dis. 2021; 27(8): 1881–95. 12. pinto ac, caramês j, francisco h, chen a, azul am, marques d. malignant transformation rate of oral leukoplakia—systematic review. oral surg oral med oral pathol oral radiol. 2020; 129(6): 600-611.e2. 13. brignardello-petersen r. proliferative verrucous leukoplakia and erythroplakia are probably the disorders with the highest rate of malignant transformation. j am dent assoc. 2020; 151(8): e62. figure 5. carcinogenesis model and clinical need for diagnostic biomarkers. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 195puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 14. aghbari smh, abushouk ai, attia a, elmaraezy a, menshawy a, ahmed ms, elsaadany ba, ahmed em. malignant transformation of oral lichen planus and oral lichenoid lesions: a meta-analysis of 20095 patient data. oral oncol. 2017; 68: 92–102. 15. gonzález‐moles má, ramos‐garcía p, war nakulasuriya s. an appraisal of highest quality studies reporting malignant transformation of oral lichen planus based on a systematic review. oral dis. 2021; 27(8): 1908–18. 16. offen e, allison jr. what is the malignant transformation potential of oral lichen planus? evid based dent. 2022; 23(1): 36–7. 17. sathiasekar a, mathew d, jaish lal m, arul prakash a, goma kumar k. oral field cancerization and its clinical implications in the management in potentially malignant disorders. j pharm bioallied sci. 2017; 9(5): 23. 18. mohan m, jagannathan n. oral field cancerization: an update on current concepts. oncol rev. 2014; 8(1): 244. 19. angadi p v., savitha jk, rao ss, sivaranjini y. oral field cancerization: current evidence and future perspectives. oral maxillofac surg. 2012; 16(2): 171–80. 20. ha pk, califano ja. the molecular biology of mucosal field cancerization of the head and neck. crit rev oral biol med. 2003; 14(5): 363–9. 21. curtius k, wright na, graham ta. an evolutionary perspective on field cancerization. nat rev cancer. 2018; 18(1): 19–32. 22. simple m, suresh a, das d, kuriakose ma. cancer stem cells and field cancerization of oral squamous cell carcinoma. oral oncol. 2015; 51(7): 643–51. 23. braakhuis bjm, tabor mp, rené leemans c, van der waal i, snow gb, brakenhoff rh. second primary tumors and field cancerization in oral and oropharyngeal cancer: molecular techniques provide new insights and definitions. head neck. 2002; 24(2): 198–206. 24. escher a, piotet e, waridel f, iggo r, monnier p. p53 mutation in histologically normal mucosa of the aero-digestive tract is not a marker of increased risk for second primary carcinoma in head and neck cancer patients. eur arch oto-rhino-laryngology. 2009; 266(4): 547–51. 25. gabusi a, gissi db, montebugnoli l, asioli s, tarsitano a, marchetti c, balbi t, helliwell tr, foschini mp, morandi l. prognostic impact of intra-field heterogeneity in oral squamous cell carcinoma. virchows arch. 2020; 476(4): 585–95. 26. bednarczyk k, gawin m, chekan m, kurczyk a, mrukwa g, pietrowska m, polanska j, widlak p. discrimination of normal oral mucosa from oral cancer by mass spectrometry imaging of proteins and lipids. j mol histol. 2019; 50(1): 1–10. 27. angadi p v., patil p v., kale ad, hallikerimath s, babji d. myofibroblast presence in apparently normal mucosa adjacent to oral squamous cell carcinoma associated with chronic tobacco/areca nut use: evidence for field cancerization. acta odontol scand. 2014; 72(7): 502–8. 28. amer hw, waguih hm, el-rouby dh. development of field cancerization in the clinically normal oral mucosa of shisha smokers. int j dent hyg. 2019; 17(1): 39–45. 29. giaretti w, pentenero m, gandolfo s, castagnola p. chromosomal instability, aneuploidy and routine high-resolution dna content analysis in oral cancer risk evaluation. futur oncol. 2012; 8(10): 1257–71. 30. pentenero m, donadini a, di nallo e, maffei m, marino r, familiari u, castagnola p, gandolfo s, giaretti w. field effect in oral precancer as assessed by dna flow cytometry and array-cgh. j oral pathol med. 2012; 41(2): 119–23. 31. peralta-mamani m, terrero-pérez á, tucunduva rma, rubira cmf, santos ps da s, honório hm, rubira-bullen irf. occurrence of field cancer ization in clinically nor mal oral mucosa: a systematic review and meta-analysis. arch oral biol. 2022; 143: 105544. 32. lópez-blanc sa, collet am, gandolfo ms, femopase f, hernández sl, tomasi vh, paparella ml, itoiz me. nucleolar organizer regions (agnor) and subepithelial vascularization as field cancerization markers in oral mucosa biopsies of alcoholic and smoking patients. oral surgery, oral med oral pathol oral radiol endodontology. 2009; 108(5): 747–53. 33. chaves fn, bezerra tmm, moraes dc, costa sf dos s, silva pgb, alves apnn, costa fwg, bernardes vf, pereira kma. loss of heterozygosity and immunoexpression of pten in oral epithelial dysplasia and squamous cell carcinoma. exp mol pathol. 2020; 112: 104341. 34. nakashima t, tomita h, hirata a, ishida k, hisamatsu k, hatano y, kanayama t, niwa a, noguchi k, kato k, miyazaki t, tanaka t, shibata t, hara a. promotion of cell proliferation by the protooncogene dek enhances oral squamous cell carcinogenesis through field cancerization. cancer med. 2017; 6(10): 2424–39. 35. lopes cb, magalhães ll, teófilo cr, alves apnn, montenegro rc, negrini m, ribeiro-dos-santos â. differential expression of hsa-mir-221, hsa-mir-21, hsa-mir-135b, and hsa-mir-29c suggests a field effect in oral cancer. bmc cancer. 2018; 18(1): 721. 36. yang x, shi l, zhou z, liu w. podoplanin and abcg2 expression in oral erythroplakia revisited: potential evidence for cancer stem cells driving the process of field cancerization. oral oncol. 2020; 101: 104368. 37. padin-iruegas e, chamorro-petronacci cm, sines-cajade i, lorenzo-pouso ai, blanco-carrión a, pérez-jardón a, gándara-vila p, pérez-sayans m. dna methylation by bisulfite next-generation sequencing for mlh1 and mgmt in oral squamous cell carcinomas and potentially malignant disorders: an integrative analysis towards field cancerization. medicina (b aires). 2022; 58(7): 878. 38. govindaraj pk, kallarakkal tg, mohd zain r, tilakaratne wm, lew hl. expression of ki-67, cornulin and isg15 in non-involved mucosal surgical margins as predictive markers for relapse in oral squamous cell carcinoma (oscc). ha p, editor. plos one. 2021; 16(12): e0261575. 39. de freitas filho saj, coutinho-camillo cm, oliveira kk, bettim bb, pinto cal, kowalski lp, oliveira dt. prognostic implications of aldh1 and notch1 in different subtypes of oral cancer. santhekadur pk, editor. j oncol. 2021; : 6663720. 40. feng j, zhou z, shi l, yang x, liu w. cancer stem cell markers aldh1 and bmi1 expression in oral erythroplakia revisited: implication for driving the process of field cancerization. j oral pathol med. 2020; 49(1): 96–9. 41. ahmed s, khan s, qureshi ma, bukhari u, anis m, mughal mn. expressional variations of kaiso: an association with pathological characteristics and field cancerization of oscc. bmc cancer. 2022; 22(1): 990. 42. vadla p, yeluri s, deepthi g, guttikonda vr, taneeru s, naramala s. stathmin! an immunohistochemical analysis of the novel marker in oral squamous cell carcinoma and oral leukoplakia. asian pacific j cancer prev. 2020; 21(11): 3317–23. 43. qiao b, he b, cai j, yang w. the expression profile of oct4 and sox2 in the carcinogenesis of oral mucosa. int j clin exp pathol. 2014; 7(1): 28–37. 44. feitosa sg, viana kf, luna ecm, costa fwg, cavalcante rb, chaves fn, chaves hv, pereira kma. immunohistochemical evaluation of glut-3 and glut-4 in oral epithelial dysplasia and oral squamous cell carcinoma. asian pac j cancer prev. 2018; 19(7): 1779–83. 45. gonzalez-moles. substance p and nk-1r expression in oral precancerous epithelium. oncol rep. 2009; 22(6): 1325–31. 46. m ig u e l a f p, e m b a l ó b, a lve s d i a s h b, r ive r o e rc . i m munoh istochem ica l expression of m mp-9, t i mp-1, a nd vimentin and its correlation with inflammatory reaction and clinical parameters in oral epithelial dysplasia. appl immunohistochem mol morphol. 2021; 29(5): 382–9. 47. yu t, tang q, chen x, fan w, zhou z, huang w, liang f. tgf‐β1 and il‐17a comediate the protumor phenotype of neutrophils to regulate the epithelial‐mesenchymal transition in oral squamous cell carcinoma. j oral pathol med. 2021; 50(4): 353–61. 48. tabatabaeifar s, larsen mj, larsen sr, kruse ta, thomassen m, sørensen ja. investigating a case of possible field cancerization in oral squamous cell carcinoma by the use of next-generation sequencing. oral oncol. 2017; 68: 74–80. 49. pérez-ruiz e, gutiérrez v, muñoz m, oliver j, sánchez m, gálvezcarvajal l, rueda-domínguez a, barragán i. liquid biopsy as a tool for the characterisation and early detection of the field cancerization copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 196 puspasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 189–196 effect in patients with oral cavity carcinoma. biomedicines. 2021; 9(10): 1478. 50. gabusi a, morandi l, asioli s, foschini mp. oral field cancerization: history and future perspectives. pathologica. 2017; 109(1): 60–5. 51. van oijen mg, slootweg pj. oral field cancerization: carcinogeninduced independent events or micrometastatic deposits? cancer epidemiol biomarkers prev. 2000; 9(3): 249–56. 52. nelem-colturato cb, cury pm, pereira tm, cosso is, pivato k, volpato ler, borges ah. sextuple tumors in head and neck area: evidence of field cancerization. case rep pathol. 2018; 2018: 8428395. 53. fortuna g, mignogna md. oral field cancerization. can med assoc j. 2011; 183(14): 1622–1622. 54. bansal r, nayak b, bhardwaj s, vanajakshi c, das p, somayaji n, sharma s. cancer stem cells and field cancerization of head and neck cancer an update. j fam med prim care. 2020; 9(7): 3178–82. 55. pirlog r, cismaru a, nutu a, berindan-neagoe i. field cancerization in nsclc: a new perspective on micrornas in macrophage polarization. int j mol sci. 2021; 22(2): 746. 56. liao z, tan zw, zhu p, tan ns. cancer-associated fibroblasts in tumor microenvironment – accomplices in tumor malignancy. cell immunol. 2019; 343: 103729. 57. liao z, chua d, tan ns. reactive oxygen species: a volatile driver of field cancerization and metastasis. mol cancer. 2019; 18(1): 65. 58. graham ta, mcdonald sa, wright na. field cancerization in the gi tract. futur oncol. 2011; 7(8): 981–93. 59. gadaleta e, thorn gj, ross‐adams h, jones lj, chelala c. field cancerization in breast cancer. j pathol. 2022; 257(4): 561–74. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p189–196 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p189-196 dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. � vol. 42. no. 1 january–march 2009 literature review the transformation of ordinal scale for parametric statistic analysis on dental health questionnaire adi hapsoro department of dental public health faculty of dentistry airlangga university surabaya indonesia abstract background: questioner measurement is very customize, it means that researchers make very individual based on the aim of the research. frequently, the results of questioner measurements are numeric rank and index or both of them. numeric rank and index are categorical data. beside of validity and reliability problems, they have analytical problems as well. so, they need transformation to scale type (ratio, interval) data in order to minimize their problems. purpose: this article reviews the effectiveness of two type data transformation in dental health research measurement. reviews: there are two type data transformations, i.e. interval equivalency and sum of rating transformation method. conclusion: interval equivalency transformation method is more effective for the data come from index, and sum of rating transformation method is more effective for the data come from numeric rank. key words: dental health, measurement, transformation method correspondence: adi hapsoro, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof dr. moestopo no. 47 surabaya 60132, indonesia. introduction there are many studies on dental health using questionnaire as measurement tools, especially in measuring either patients, clinic visitors’ respon, or society’s respons towards an object about dental health services. those responses can be used in measuring either opinion, reaction, interest, or level of knowledge. if the answers of the respondents are put into qualitative data/semi quantitative data (ordinal data) and analyzed with qualitative approach (non parametric), there would be no problem. however, it is often necessary to take qualitative data from a constructive phenomenon into quantitative one. in questionnaire, the measurement tools either on index or on scale can also be conducted. if the items of the questions and the options of the answers are not conducted into ranks, this measure is usually used in descriptive study.1–3 if the items of the questions and the options of the answers are not conducted into ranks, the result of this measurement on questionnaire is generally an ordinal data. this ordinal data only as codes instead of as values or scores. thus, the codes can not be put into quantitative data and can not describe parameter continuities. if the codes are analyzed with parametric statistics, the result will be bias and the conclusion will also be bias.4,5 based on those problems, two different methods will be discussed in this study as alternatives for researchers in conducting questionnaire in order to obtain quantitative data from an abstract phenomenon. it means that those two following methods can obtain values or scores not only on codes but on ordinal scales as well. conducting questionnaire with ordinal and interval scales the measurement on ordinal scale is shown with a qualitative grade or rank. if the options of the answers are scored with: 1, 2, and 3, the interval between 1 and 2 will not be the same as the interval between 2 and 3. nevertheless, it may be concluded that 2 is bigger than 1, or 3 is bigger than 2 and 1. the example of using ordinal scale, moreover, is in measuring the responses of the service quality with the options of the answers categorized into: good, average, and poor. good category is scored with 3, average category is � dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 1-7 scored with 2, and poor category is scored with 1. in other way, good category may also be scored with 9, average category may also be scored with 7, and poor category may also be scored with 3. it is indicated that the scores can be changed, but the ranks of the options must be the same. the result of measurement on interval scales, on the other side, is the same as the result of the measurement on ordinal scales, but it has the same intervals among the ranks. therefore, based on the example above, it may be concluded that the interval of 1–2 is the same as the interval of 2–3. interval scales, moreover, has no score, 0 as an absolute score. thus, if the results of the measurement are 1, 2, 3, and 4, score 4 can not be said as twice as score 2. for instance, if in an exam a gets 80 and b gets 40, it cannot be concluded that intelligence is as twice as the b. nevertheless, on interval scales the scores have already had their own values so that their average can statistically be for statistic parametric analysis purpose. in measuring an object with interval scales, the interval of the options of the answers in a questionnaire should also be measured. it means that the interval between the options good and average should be measured, and so the interval between the options average and poor. in the study of the concrete subject, moreover, the interval of two scores can be measured more easily than the interval of those in the study of the abstract subject. when the mass of two tennis balls is measured, for example, the mass of those balls can clearly measured; the ball a is 56 grams, and the ball b is 57 grams. it clearly indicate that the ball b is slightly heavier than the ball a. however, it may be difficult for measuring which ball is heavier only by holding those balls in our hands.1,6 the same problem, furthermore, also occurs in measuring abstract phenomenon or an activity or an object with a questionnaire as the measurement tool. for those reasons, in order to obtain continuous interval or score variations, the determined measurement tool is needed to be on a trial by using certain methods as the following. equal interval method the objective of the equal interval method is to assess the options of the answers. for example, respondent is asked to assess an object with criteria; good, average, and poor. each of criteria then will be on a trial with a 100 respondents even though 30 respondents. however, the sample for the trial can also be used later in analysis of the study so that it will not be useless.7 the first step is the respondents must determine criteria good, average, and poor into a continuum divided into nine or eleven intervals. a b c d e f g h i � � � � � � � � � the letter e is the central point and the letter a is the lowest score. thus, the quality of the score is getting high as to the right side. the quantitative interval among letters is still not known. however, the interval qualitatively increas as to letter i. it is indicated that the order of those letters is on ordinal scales. the interval among those letters then must be transformed on interval scales (equivalence). for instance, if the respondents choose the category good, this option must be divided more specifically into, good enough, good, or very good. therefore, box f to i can be choosed. moreover, if the respondents choose the option poor, box d to a can be choosed. if the respondents choose the option average, box d to f can be choosed. nevertheless, there will be possibilities of overlapping during . thus, the respondents must be clearly instructed that if they want to choose good, they must choose boxes the right side; if they want to choose poor, they must choose boxes the left side; and if they want to choose average, they must choose boxes on the center. moreover, in each form, the respondents must write down good, average, or poor on the top right of the form since the respondents are sometimes not consistent with their. therefore, it can be useful in classifying the data. finally, after all the respondents (100) have done their, for example, there are 35 respondents good, 35 respondents choosed average, and the rest respondents choosed poor, the data tabulate as the following. table 1 showed that out of 35 respondents, the letter f means frequency that means the number of respondents choosing ‘good’ category. for letter f the frequency is 10: it means that there are 10 respondents assessing the object with option good, or for letter g the frequency is 15 meaning that there are 15 respondents assessing the object with ‘really good’ option. moreover, p stand for proportion is a comparison between the frequency of each letter and the number of all respondents. thus, p = f/n. for example, for letter f, p = 10/35 = 0.286. the ‘pk’ symbol means cumulative proportion is proportion number on interval of certain interval or a table1. the result data of the respondents’ towards the category “good” a 1 b 2 c 3 d 4 e 5 f 6 g 7 h 8 i 9 f 0 0 0 0 0 10 15 6 4 p 0 0 0 0 0 .286 .428 .171 .114 pk 0 0 0 0 0 .286 .714 .885 1.0 �hapsoro: the transformation of ordinal scale certain number added with all the proportions less than that number. for instance, for number 7 or g letter the cumulative proportion is: 0.286 + 0.428 = 0.714. finally, the calculation of discriminal modal value as the final step is a process of evaluating a value representing rating or judgment of a measurement group towards an object. the value is estimated based on the median value, and symbolized with s. the formulation is as the following: s = bb + i [(0.50 – pkb)/p] bb = minimal limit of the number category with median value inside pkb = cumulative proportion below the number category with median value inside p = proportion of the number category with median value inside i = the width of interval (equal with 1) in statistics, median is a number that limits 0.50 of proportion or 50% of frequency – a number smaller than the median itself. in order to determine the position of median, could be seen in the column or the category in the table which there is 0.50% of cumulative proportion inside. for instance, in table 1 the cumulative proportion of the measurement result towards the criteria ”good” is in letter g’s column or in number 7. its minimal limit (bb), is about 6.5. this minimal limit is between the 6th and 7th box. the cumulative proportion below the number category with median inside (pkb) is about 0.286, and the proportion of the number 7 (p) is about 428. thus, the calculation of the score (on the scale) is as the following: s = 6.5 + 1 [(0.50 – 0.286)/0.428] s = 6.44 the total score of the criterion “good” is 7 the next example is the measurement towards the criterion “average”. before, the median of the data must be determined. based on table 2, the median is on column e with pk = 0.6, so that: bb = 4.50 pkb = 0.314 p = 0.286 thus, the total of the criterion “average” is: s = 4.50 + 1 [(0.50 – 0.314)/0.286] = 5.150 moreover, the result data of the towards the criterion “poor” is the following: the result of the is as the following: s = 2.5 + 1 [(0.50 – 0.263)/0.333] s = 3.21 the total of the criterion “poor” = 3.21 in conclusion, the total of the criterion “good”: 7 “average”: 5.15 “poor” : 3.21 the above result is an example of the towards an object. however, the result can also become the for a research about the index status of tooth cleaning (ohi’s) with 100 respondents. the problem, moreover, is that the from those 100 respondents is divided into three distributions. thus, if the scoring can be put in order as the example above, the continuum of the can be analyzed well. nevertheless, if the is coincided, the interval of the continuum can not be analyzed. therefore, in order to solve the problem, another method is needed for the ordinal data. rating summative method this method is used to solve overlapping results of distribution from and respondents since the of the respondents can be too homogenous or too heterogeneous. the respondents, thus, are put into one distribution and are asked to assess an object with 5 options. the options of the answer are usually totally disagree (sts), disagree (ts), table 2. the result data of the respondents’ towards the category “average” a 1 b 2 c 3 d 4 e 5 f 6 g 7 h 8 i 9 f 0 0 5 6 10 7 7 0 0 p 0 0 .142 .172 .286 .2 .2 0 0 pk 0 0 .142 .314 .6 .8 1.0 1.0 1.0 table 3. the result data of the respondents’ towards the category “poor” a 1 b 2 c 3 d 4 e 5 f 6 g 7 h 8 i 9 f 1 7 10 10 2 0 0 0 0 p .03 .233 .333 .333 .06 0 0 0 0 pk 03 .263 .596 .929 .989 1.0 1.0 1.0 1.0 � dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 1-7 abstain (tm), agree (s), and totally agree (ss). the category of those options of the answers, moreover, is clearly on ordinal scale, which then is assessed so that it can be changed onto interval scale (figure 3 & 4).8–10 the following table is an example of measuring the dental services at clinic of faculty of dentistry airlangga university from and respondents. the question is: was are dental services at faculty of dentistry airlangga university “satisfying”. table 4. respons distribution from or 100 respondents as a object (service) category of answercategory of answer sts ts tm s ss f 4 49 22 17 8 p = f/n 04 49 22 .17 .08 pk 04 53 75 .92 1.00 pk-t 02 285 640 .835 .96 z –2.054 –.568 .358 .974 1.751 sts = totally disagree ts = disagree tm = abstain s = agree ss = totally agree the first of the table is the frequencies of answers (f) for each responcategory. the total of all frequencies is the same as the total of respondents (n), in this case 100 respondents. proportion (p) is obtained by dividing each frequency with number of respondents. for instance, the proportion of tm responds is 22/100 = 0.22 cumulative proportion (pk) is a proportion of one respon category added to a proportion of all categories on the left side. for instance, pk for s category is obtained by adding (0.17 + 0.22 + 0.49 + 0.04) = 0.92. pk-t, is a median of cumulative proportion formulated as a half proportion of one category is added to cumulative proportion of another category on the left side, as the following formula: pk-t = ω p + pkb p = a proportion of one category pkb = cumulative proportion of another category on the left side. for example, pk-t for answer category ts is: 0.49 /2 + 0.04 = 0.285. the score of z is a median of each respon category for one continuum with interval scale. the interval among respond categories is stated by the interval of score z. deviation score for each pk-t is based on the table of normal deviation (appendix a). thus, the normal standard score in the curve can be determined by pk-t score. this process is ordinal data into an interval one or the semi quantitative data by using table z (appendix). for instance, ss category with pk-t = 0.96 has z score (see table) = 1.751 moreover, sts category with pk-t = .020 has z score= –2.054, and so do the other categories. if all z scores of each respon category are put into one continuum line, it will be as the following: -3 -2 -1 0 1 2 …...… ..,…...….,….…..,…….....,….……, sts ts tm s ss (–2.054) (–.568) (.358) (.974) (1.751) those respon scores are now in interval measurement ranks so that the lowest score can be changed into 0 by taking linier transformation, as the following: y = 2.054 + (1) x the result of the transformation is as the following: sts ts tm s ss x = –2.054 –.568 .358 . 974 1.751 y = 0 1.486 2.412 3.028 3.805 discussion transformation of ordinal scale as the measurement scale must be done before its validity and reliability are measured parametric statistics is required in analysis process. however, this scale transformation needs a long process. first, a trial must be taken in preparing scale transformation. second after the transformation, the validity and reliability process of measurement scale must be on another trial. in equal interval method the measurement is simple and easier. nevertheless, this method has some weaknesses. in measuring an object with scale, for example, there will be possibility of overlapping values since its categories are more than three categories (good, average, poor). if the respondents are homogenous, furthermore, there will also be possibility of coinciding values or even closely coinciding values among categories; good, average, poor. therefore, this equal interval method in measuring an object on index scales (withorank) so that the options of the answer; a, b, and c, can become b, a, and c after , or can also become another combination depending on the result of its equal interval. for example, in the measur of oral hygiene index simplified the mean score (code) of the data can not be estimated directly since the data still on ordinal scales. thus, in measuring this ohi’s data, a researcher must equal. in this case, scores 0, 1, 2, and 3 must be equal so that the exact interval can be obtained. for this reason, the ohi’s scores are not always 0, 1, 2, and 3. the scores can possibly become 0, 1.5, 2.3, and 2.9. for ohi’s tabulation, then, can be analyzed with parametric statistics by measuring its mean score. therefore, the score of debris index can not be the same as that of calculus index since the trial is separately taken in the equivalence of the measurement scales. for rating summative method, measuring approach, �hapsoro: the transformation of ordinal scale on the other side, is longer and uses table z (table of normal deviation) in making the ordinal data become interval one like equivalent interval method, this method an object. this method, thus, is more appropriate to be used in measuring an object with ranking scale and with unnecessary homogenous respondents. for instance, the questionnaire with likert scale usually has options of answers divided into five grades. however, if those grades must be transformed into interval scales, scales 1 to 5 must be equal a trial, not as a separate one like on index scales, since those grades are based on ranks. thus, as a unit, scale 1 can impossibly be changed into scale 2. nevertheless, only if the respondents are very homogenous, it will be possible that scale 1 and 2 will closely be coincided, so will scale 3, 4, and 5. as a conclusion, both of those methods can be applied as alternatives in conducting questionnaire, especially in determining values or scores from the options of the answers wh distribution approach is closed to normal distribution based on interval measurement scale. they, finally, can also be analyzed by parametric statistics. references 1. azwar, saifuddin. dasar-dasar psikometri. edisi ke-1. yogyakarta: pustaka pelajar offset; 1999. p. 112, 117. 2. ferguson ga. statistical analysis in psychology and education. auckland: mcgrawhill; 1981. p. 34. 3. howell dc. statistical methodes for psychology. boston: duxbury press; 1982. p. 23. 4. zimmerman dw. a simplified probability model of error measurement. psychological reports 1969; 25:175–86. 5. mccrae rr, costa pt. validation of the five factor model of personality across instruments and observers. journal of personality and social psychology1987; 52:81–90. 6. allen mj, yen wm. intoduction to measurement theory. monterey: brooks/cole publishing company; 1979. p. 7. 7. cliff n, keats ja. ordinal measurement in the behavioral sciences. mahwah: nj lawrence erlburn; 2003. p. 23. 8. michell j. measurement scales and statistics : a clash of para digsm psychological bulletin 1986; 3:398–407. 9. babbie e. the practice of social research. 10th edition. wadsworth: thomson learning inc; 2004. p. 14. 10. velleman pf, wilkinson l. nominal, ordinal, interval,and ratio typologies are misleading. the american statistician 1993; 47(1):65–72. available at: http://www.spss.com/ research/wilkinson/ publications/stevens.pdf. accessed january 9, 2009. �� vol. 42. no. 1 january–march 2009 antioxidant effect of minocycline in gingival epithelium induced by actinobacillus actinomycetemcomitans serotype b toxin ernie maduratna setiawati department of periodontology faculty of dentistry airlangga university surabaya indonesia abstract background: actinobacillus actinomycetemcomitans (aa) serotype b has been associated with aggressive periodontitis. gingival epithelial cell is exquisitely sensitive to the toxin and may lead to the epithel protective barrier disruption. experimental models show that minocycline is not related to it’s antimicrobial effect and protection against neuron cell apoptosis of a number experimental models of brain injury and parkinson’s disease. purpose: this study, examined antioxidant effect of minocycline to inhibit apoptosis of gingival epithelium induced crude toxin bacteria aa serotype b in mice. methods: thirty adult mice strain swiss webster (balb c) were divided randomly into three groups: control group (group a), toxin group (group b) and toxin and minocycline group (group c). the mice were taken at 24 hours after application, and then the tissue sections of gingival epithelium were stained with tunnel assay and immunohistochemistry. result: treatment with these toxin induced apoptosis of gingival epithelium and was associated with dna fragmentation and reduced gluthatione (gsh). minocycline 100 nm significantly increased gsh and reduced apoptosis (p < 0.05). minocycline provides antioxidant effect against citotoxicity of bacteria aa serotipe b. conclusion: nanomolar concentration of minocycline potential as new therapeutic agent to prevent progressivity of aggressiveness of periodontitis. key words: minocycline, gsh, apoptosis, aa serotype b crude toxin correspondence: ernie maduratna setiawati, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: setiawati_ernie@yahoo.co.id introduction aggressive periodontitis is a disease in dental supporting tissues which is characterized by rapid degeneration of periodontal ligament and alveolar bone in young patient. periodontitis leads to loss of periodontal attachment to the root surface and adjacent alveolar bone which ultimately results in tooth loss. recent paradigm shows the importance of balance between oxidant and antioxidant inside the cell.1 the imbalance between oxidant and antioxidant inside the cell will trigger the reaction of transcription factor which are nfkb, ap-1 and parp-1, which will cause apoptosis and inflammation reaction. toxin from actinobacillus actinomycetemcomitans (aa) serotype b bacteria will increase the apoptosis and reaction of parp-1 in gingival epithelium.2 aa serotype b crude toxin have cytotoxic effects that will trigger the increase in the amount of apoptosis in gingival epithelial cells, monocytes, lymphocytes, and macrophages.3,4 gingival epithelial cells is ten times more sensitive towards apoptosis than macrophage cells.5 the increase in the amount of apoptosis in host cell triggered by certain pathogenic bacteria is a new phenomenon in the pathogenesis of periodontal disease.6,7 minocycline is an antibiotic of choice for periodontitis treatment and often used topically.8 minocycline have different activities depending on the concentration. at the concentration above 10 mm minocycline is toxic towards epithelial cell, while above 100 mm it became toxic towards fibroblast cell. minocycline has an effect as antibacterial to aa bacteria at 4 mm concentration.9 minocycline on nanomolar concentration has bioactivity which is not connected to antibacterial, that is, as strong cytoprotection that protects the neuron cells from hypoxial trauma. therefore, it is often used in therapies for ischemic stroke, huntington’s disease, multiple sclerosis and parkinson’s disease.10–13 nevertheless, so far, the mechanism how minocyclin on research report �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 41-45 nanomolar concentration functions as antioxidant for gingival epithelial cell is still unclear. an antioxidant that is present in gingival sulcular fluid is reduced gluthatione (gsh) which has quite a high concentration. the existence of periodontopathogen bacteria can decrease in the concentration of gsh.1 one of the strategies for periodontal disease therapy is to increase the concentration of antioxidant in teeth support tissues cells. the objective of this research is to reveal the mechanism of minocycline as antioxidant caused by aa serotype b crude toxin in gingival epithelium through the apoptosis and gsh expression. materials and methods this research was categorized as experimental laboratory by using the experimental animal male mice (mus muculus) strain swiss webster (balb c). the design of this research was the completely randomized design. this research was done in the biomedical laboratory, faculty of medicine, brawijaya university and pathology anatomy, faculty of medicine, airlangga university. the unit analysis is the gingival epithelium in buccal anterior of the lower jaw of the mus musculus strain swiss webster balb/c male, chosen healthy physically, 2.5 months old with the weight of 25–35 grams obtained from pusat veterinaria farma surabaya (pusvetma). the material used is pure minocycline hydrochloride from sigma (155718), actinobacillus actinomycetemcomitans serotype b (atcc 43718), apoptag detection kit (chemicon-product s-7101). monoclonal antibody anti gluthatione (stressgen product, spa-542), streptavidine peroxidase from labvision co, ts-060-hr. the tools used are: incubator, centrifuge with cooler (eppendorf 5417r), vortex, water bath, micro pipette with sterilized points, eppendorf tube, binocular light microscope canon aplhaphot y5 connected to a monitor and camera. to ensure that all the procedure done in this research is ethically approved, before it was done, the proposal for this research was given to the ethical committee, faculty of dentistry, airlangga university for inspection. the procedure for this research includes the culturing of aa serotype b bacteria, the creation of crude aa serotype b toxin, the examination of immunohistochemistry using of assay tunnel and gsh. actinobacillus actinomycetemcomitans atcc 43718 (y4, serotype b), were cultured in dialysates todd hewitt broth (difco laboratories, detroit, mich) added with 1% of yeast extract at the temperature of 37° c, 5% co2 for 4 days.14 subsequently, the bacteria were centrifuges by 12.000 rpm, and had its supernatant extracted. to separate the non protein from crude toxin, rough toxin that comes from the supernatant was precipitated by adding thick ammonium sulphate 40%. the formed sediment is separated using centrifuges by 3000 rpm for 30 minutes at 4° c. the salt formed from the sediment was then separated by dialysis. the sample was then inserted into a membrane which had its top and bottom secured, after that, the cellophane bag was inserted into a beaker glass which contained buffer phosphate buffer saline (pbs) ph 7.2. the dialysis was stopped when it reached equilibrium.15 treatment on mice group 1: 10 mice is given sterile aquadest topically in the buccal gingival anterior lower jaw every 12 hours using disposable oral sponge swab (rynel inc, usa) which is inserted into the sterile aquadest until soaked, and applied by way of two double lateral strokes.16 after 24 hours, the mice's anterior gingival tissue of lower jaw was extracted as biopsy specimen.17 group 2: 10 mice were induced with bacterial toxin aa 100 mg/ml.14 after 24 hours, the mice's anterior gingival tissue of lower jaw was taken as biopsy specimen. group 3: 10 mice were induced with bacterial toxin aa 100 mg/ml on the anterior buccal gingiva of lower jaw by using hamilton syringe (reno, nev)18 continued with the administering of minociclyne 100 nanomolar topically on anterior buccal gingival of lower jaw every 12 hours using disposable oral sponge swab (rynel inc, usa) which are soaked in minocycline, and applied using two double lateral strokes. after 24 ours, the mice's anterior gingival tissue of lower jaw was taken as biopsy specimen. immunohistochemistry examination the immunohistochemistry examination with the method of streptavidin –biotin-complex and tunnel assay were used to get the expression of gsh and apoptosis. the counting of gingival epithelial cells which expressed apoptosis and gsh was done under light microscope with 400 times magnification. cells which proved to be positive gave brownish color between bluish/greenish epithelial cells. every reserves were examined at 4 different places clockwise 3, 6, 9, and 12. each field of vision are examined and counted at two places according to 6 and 12 needle using counting room and counter.19 the results were then averaged and data were then analized statistically one-way anova statistic analysis with 95% degree of significance (p < 0.05). result to prove the effect of antioxidant minocycline on gingival epithel cell on mice induced with crude bacterial toxin a. actinomycetemcomitans serotype b, the counting of cells which expressed apoptosis and gsh was done. the resulting data were then described and tested within 0.05 degree of significance. ��setiawati: antioxidant effect of minocycline figure 1. apoptosis expression appearance using tunnel assay with 400× magnification. a) control; b) bacterial toxin aa serotype b exposure; c) treatment with 100 nanomolar minocycline. red arrow indicates positive result which noticed by the presence brown spot in the nucleus of gingival epithelial cells, blue arrow indicates negative result which noticed by the absence of brown spot in the nucleus. a b c a b c figure 2. gluthatione expression appearance with 400× magnification. a) control; b) bacterial toxin aa serotype b exposure; c) treatment with 100 nanomolar minocycline. red arrow indicates positive result which noticed by the presence brown spot in the nucleus of gingival epithelial cells, blue arrow indicates negative result which noticed by the absence of brown spot in the nucleus. �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 41-45 table 1. mean, standard deviation, the significance of number of cells which express apoptosis in gingival epithelial cells that exposed to aa serotype b bacterial toxin after treated with 100 nanomolar solution of minocycline group n x sd significance control 10 5.17 0.27 0.00 toxin 10 45.17 7.68 0.00 toxin + minocycline 10 5.81 0.58 0.00 table 2. mean, standard deviation, the significance of number of cells which express glutathione in gingival epithelial cells that exposed to aa serotype b bacterial toxin after treated with 100 nanomolar solution of minocycline group n x sd significance control 10 60.51 1.12 0.00 toxin 10 29.11 2.30 0.00 toxin + minocycline 10 62.57 2.09 0.00 normality test is done using kolmogorov smirnov test. normal distribution group continued by parametric test of one-way anova and tukey hsd test to know the difference in the group with 5% significant rate. table 1 and table 2 shows that significant difference in number of cells which express apoptosis and gluthatione in gingival epithelial cell that exposed aa serotype b toxin, after treated with 100 nm solution minocycline and control (p < 0.05). in figure 1, it is showed the apoptosis examination using tunnel assay method on mice gingival epithelium biopsy with 400× magnification. in figure 2, it is showed the gsh expression on mice gingival epithelium biopsy with 400× magnification. discussion in an attempt to organize the appropriate treatment strategy, what is needed is the comprehensive understanding of etiopathogenesis mechanism of periodontitis, so that the progressivity process of the disease could be inhibited. in the past, aggressive periodontitis treatment is only centered on the local factor elimination and antibacterial administration. recent therapy progress is headed towards the increase of the capacity of tissues reparation by increasing cellular survival through the effect of cytoprotection by balancing oxidant and antioxidant.20 this research was an experimental research to reveal the effect of minocycline antioxidant on gingival epithelium on balb/c mice which were exposed to bacterial toxin actinobacillus actinomycetemcomitans serotype b. bacterial toxin aa serotype b is cytotoxic towards teeth support tissues, especially gingival epithelial cell 10 times more than macrophage.5,21 number of cells which express apoptosis in gingival epithelial cell that exposed aa serotype b toxin is higher than that were treated with 100 nm solution minocycline and control. aa serotype b toxin induce free radical increased in intracellular epithel. it will cause oxidative stress that can increased expression bax, caspase-3, enzyme poly adp ribose polymerase-1 (parp-1) and dna fragmentation. imbalance condition between free radical formation and antioxidant cause cell damaging with apoptosis pathway. gingival epithelium as the first defense for teeth support tissues need to be protected from the effect of free radical effect triggered by aa serotype b bacteria. the exposure to crude bacterial toxin aa serotype b caused the disturbance of energy metabolism in mitochondria and disturb the homeostasis of energy inside the cells through the increase of intracellular calcium.2 the result of this research showed that by giving minocycline 100 nanomolar, it can decrease figure 3. antioxidant mechanism of minocycline. ��setiawati: antioxidant effect of minocycline the apoptosis cells and increase the expression of gsh significantly. gsh is an important antioxidant cell needed for mitochondria to function. gsh is capable of giving protection towards the creation of ros.22 the increase in gsh showed the effect of cytoprotection to the cells. gsh is capable of disrupting chain reaction by suppressing dangerous radicals formed during chain reaction. gsh is also capable of preserving the rate of vitamin c in the body due to the ability of gsh in transforming radical ascorbic into ascorbic acid.1 the decrease in oxidative stress by the application of antioxidant could inhibit the progressivity of a disease. mitochondria is normally protected from oxidative damage by mitochondrial antioxidant systems, also, mitochondria have antioxidant with low-weight molecules such as alpha-tocopherol and ubiquinol, these molecule are effective for cleaning lipid peroxyl radical and preventing peroxidation of lipid. the defense reaction of cell towards the toxic effect of ros is the creation of antioxidant, such assuperoxide dismutase (sod), catalase, gluthatione peroxidase and gsh. catalase, gluthatione peroxidase enzymes are responsible for the detoxification of h2o2. gluthatione peroxidase lowers h2o2 by oxidizing gsh into gssg.23 some trials on humans by giving high dosage vitamin e didn’t show real improvement, this could be caused by the difficulty of getting through bloodbrain barrier (bbb). sod and catalase cannot enter cell membrane so it is less effective for intracellular ros. minocycline have the advantage of penetrating through cell membrane and work on mitochondria level, making it very effective as antioxidant.24 minocycline have the activity as antioxidant at the level of alpha tocopherol on neuron cell culture. minocycline works as antioxidant depending on the structure of phenol ring like that of alpha tocopherol (vitamin e).24 phenolic antioxidants are effective as antioxidant owing to the free radical chain reaction with phenol ring forming phenol-derived free radical which is relatively stable and non reactive. the main factor that minocycline has a potential effect as phenolic antioxidants are: 1) the level of resonation stabilization from phenol-derived radical; 2) amount and the size of phenol ring substituent that is able to inhibit the reaction with other molecules. kraus et al.,24 showed that minocycline was more effective as antioxidant 200–316 times more than tetracycline because the phenol ring on minocycline has dimethylamino substituent which is capable of increasing the resonation stabilization of phenol-derived free radical and had high steric stabilization. the conclusion of this research was that minocycline in 100 nanomolar proved to be an antioxidant through the lowering of the amount of apoptosis and the increase in expression of gsh. references 1. matthews j, chapple l. the role of rective oxygen and antioxidant species in periodontal tissue destruction. periodontol 2000 2007; 43:160–232. 2. setiawati em. efek sitoproteksi minosiklin pada epitel gingiva yang terpapar toksin bakteri actinobacillus actinomycetemcomitansserotype b. dissertation. surabaya: program studi ilmu kedokteran universitas airlangga; 2008. p. 84–102. 3. dirienzo jm, kang p, korostoff j, volgina a, grzesik w. differential effect of the cytolethal distending toxin of actinobacillus actinomycetemcomitans on co-cultures of human oral cells. j med microbiol 2005; 54:785–94. 4. shenker b, hoffmaster r, zekavat a, yamaguchi n. induction of apotosis in human t cells by actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of g2 arrest of the cell cycle. j immune 2001; 167:435–41. 5. paju s. virulence associated characteristics actinobacillus actinomycetemcomitans an oral and non oral pathogen. dissertation. finlandia: university of helsinki; 2003. p. 5–32. 6. bascones a, gamonal j, gomez m, silva`a, gonzalez ma. new knowledge of the pathogenesis of periodontal disease. quintessence int 2004; 35:706–16. 7. belibasakis gn, johansson a, wang y, chen c, kalfas s, lerner uh. the cytolethal discending toxin induces receptor activator of nf-{kappa}b ligand expression in human gingival fibroblasts and periodontal ligament cells. infect immun 2005; 73:342–51. 8. prajitno sw. periodontologi klinik. fondasi kedokteran gigi masa depan. jakarta: balai penerbit fakultas kedokteran ui. 2003. p. 12–32. 9. robert m, chopra i. tetracycline antibiotics: mode of action,appli cations,molecular biology and epidemiology of bacterial resistance. microbiol and molecular biology review 2001; 65: 232–60. 10. yansheng d, zhizhong m, lin s, gao f. minocycline prevents nigrostriatal dopaminergic neurodegeneration in the mptp model of parkinson disease. proceedings of the national academy of sciences 2002; 98:14669–74. 11. wang j, wei q, wang cy, hill d. minocycline up regulates and protects against cell death in mitochondria. j biol chem 2004; 279:19948–54. 12. stirling dp, khodarahmi k, liu j, phail m. minocycline treatment redces delayed oligodendrocyte death, attenuates axonal diebackand improves out come after spinal cord injury. j neurosci 2004; 24:2182–90. 13. swanson r, alano c, kauppinen t, valls v. minocycline inhibits poly(adp-ribose) polymerase-1 at nanomolar concentrations. proceedings of the national academy of sciences 2006; 103: 9685–90. 14. nishihara t, ohguchi m, ishisaki a, okohashi n, yamato k, noguchi t. actinobacillus actinomycetemcomitans toxin both cell cycle arrest in the g2/m phase and apoptosis. j infect and immun 1998; 66:5980–87. 15. aulanni’am. prinsip dan teknik analisis biomolekul. malang: fakultas pertanian universitas brawijaya press. 2004. p. 34–55. 16. logan ei. a model for evaluation of supragingival plaque and effects of mechanical and chemical paque control on gingivitis in the dog. dissertation. manhattan kansas: kansas state university; 1994. p. 49–52. 17. parwatisari r. pengaruhpemberian ekstrak biji jinten hitam terhadap jumlah sel makrofag, limfosit dan sel plasma pada jaringan ikat gingiva mencit yang diindukdi periodontopatogen. thesis. surabaya: program pascasarjana universitas airlangga; 2007. p. 47–56. 18. zubery y, dunstan cr, story bm, kesavalu l. bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin. infect and immun 1998; 66:4158–62. 19. pesik rn. ekspresi p53 pada apoptosis sel epitel mukosa lambung tikus putih wistar dengan indikasi indomethacin daya proteksi kombinasi vitamin e dan b karoten. thesis. surabaya: program pascasarjana universitas airlangga; 2002. p. 52–54. 20. bartold pm. periodontal tissues and health and disease. periodontol 2000.2006; 40: 7–70. 21. kato s, nakashima, sugimura, nishihara t, kowashi y. actinobacillus actinomycetemcomitans induces apoptosis in human monocytic thp-1 cells. med microbiol 2005; 54:293–8. 22. fernandes c, donovan d. mitochondrial gluthatione and oxidative stress: implication for pulmonary oxygen toxicity in premature infants. molecular, genetics and metabolism 2000; 71:352–8. 23. szeto hh. mitochondria-targeted peptide antioxidants: novel neuroprotective agents. american association of pharmaceutical scientists journal 2006; 8:3–21. 24. kraus r, pasieczny r, turner m, jiang a. antooxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity. j neurochem 2005; 94:819–27. 151 vol. 42. no. 3 july–september 2009 allergic asthma in children: inherited, transmitted or both? (the transmission of periodontopathic bacteria concept) seno pradopo1 and haryono utomo2 1 department of pediatric dentistry 2 dental clinic faculty of dentistry, airlangga university surabaya indonesia abstract background: in theory, allergic diseases including asthma, was the result of exposure to a transmissible agent and do not depend on early infection which is said to make children more allergy-resistant. this seems, to be a direct contradiction to the hygiene hypothesis, since epidemiologic evidence can be cited in this theory's support. the fact that nearly all children with asthma are allergic, but only a small proportion of allergic children have asthma, at least raises the possibility that some additional factor is involved. that this additional factor might be a transmissible agent is also suggested by the similarity between the gross epidemiologic patterns of children with paralytic poliomyelitis in the 1950s and children with asthma currently. purpose: the purpose of this study was to reveal the possible relationship between the transmissions of allergic asthma and periodontopathic bacteria. reviews: recent researches showed that periodontopathic bacteria are transmissible from mother and caregivers to infants. in addition, a collaborated research that was conducted by dental practitioners and pediatricians revealed that gram-negative bacteria were significantly predominant (p = 0.001) in uncontrolled allergic asthmatic children compared to well-controlled ones. nevertheless, how does these two phenomenon related was still uncertain. literatures showed that periodontopathic bacteria modulates host immune response and sometimes caused disadvantageous effect to allergic asthma. conclusion: according to the ability of periodontopathic bacteria and its components to stimulate immunocompetent cells, it is possible that they are able to modify host-immune response which tends to increase allergic asthma symptoms. key words: transmission, allergic asthma, periodontopathic bacteria, children correspondence: seno pradopo, c/o: departemen kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend.jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: pradopo_seno@yahoo.com. introduction the “hygiene hypothesis” proposes that the increase in allergic diseases in developing countries reflects a decrease in infections during childhood. cohort studies suggest, however, that the risks of asthma are increased in children who suffer severe illness from a viral respiratory infection in infancy. this apparent inconsistency can be reconciled through consideration of epidemiologic, clinical, and animal studies. the elements of this line of reasoning are that viral infections can predispose to organ-specific expression of allergic sensitization, and that the severity of illness is shaped by the maturity of immune function, which in turn is influenced by previous contact with bacteria and viruses, whether pathogenic or not.1 clinical studies of children and interventional studies of animals indeed suggest that the exposure to microbes through the gastrointestinal tract powerfully shaped immune function.1,2 in addition, the initial microbial exposure for children born by caesarean section is delayed compared with those born by vaginal delivery; epidemiological studies revealed that caesarian section3,4 and preterm5,6 infants have more asthma risk. coincidentally, the periodontopathic bacteria in dental plaque are also transmissible. some studies showed that if a child harbored a periodontal pathogen, then at least one of the parents will exhibit the same genotype of bacteria.7 tanner et al.8 found that various anaerobic species colonize the edentulous mouths of infants, and that maternal or caregivers saliva may act as a source of some review article 152 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 151-156 gram-negative anaerobes.7 regarding to allergic diseases which has t-helper2 (th2), or type 2 immune response, not as other pathogens, gram-negative periodontopathic bacteria, especially their lipopolysaccharides (lps) have a unique characteristics; in some instance enhance the type 1 immune response �i.e. �ps from actinobacillus actinomycetemcomitans and porphyromonas gingivalis (high dose)]; nevertheless, low dose of p. gingivalis lps enhance the type 2 immune response.9–12 a study by wiyarni et al.13 revealed that dental plaque control therapy improves respiratory quality and that the asymptomatic asthmatic children had significant lower gram-negative positive culture than uncontrolled asthma (wheezing and coughing) in one week study. evaluation of randomly selected subjects after two months revealed that their asthmatic symptoms and food allergy also diminished.14 therefore, there is a possibility that asthma is not merely inherited, but also transmissible, whether by transmission of virus, periodontopathic bacteria or both. the purpose of this review was to sketch the rationale for a new theory of asthma’s pathogenesis. the proposed theory is that asthma is caused, at least in part, by infection, especially in infancy, by a respiratory virus, most likely a human rhinovirus (hrv) only or concomitantly with periodontopathic bacteria such as p. gingivalis the understanding of this review may help dental practitioners, especially pediatric dentist to answer questions conducted by medical practitioners, especially pediatric allergy specialist regarding the possible connection between oral hygiene and allergic asthma. atopic and non-atopic asthma atopy is a personal and/or familial tendency to, usually in childhood or adolescence, become sensitized and produce immunoglobulin e (ige) antibodies in response to ordinary exposure to low doses of allergens, usually proteins.15 atopy can be detected by specific serum ige or skin-test reactivity to environmental allergens, is often associated with asthma. the prevalence of atopy has increased over time in some populations, whereas in others there has been a decrease or a plateau in prevalence since 1990. asthma and atopy can occur either independently or jointly in patients, in populations, and over time. in the united kingdom and australia, the prevalence of both asthma and skin-test reactivity has increased, whereas in hong kong, germany, and italy, the prevalence of atopy but not of asthma has increased.15 in some populations, the prevalence of asthma associated with allergies has increased more than that of non-atopic asthma, whereas in others the prevalence of the two types of asthma has increased to a similar degree. until today it is still not known what factors cause asthma in a person with atopy or what factors cause atopy in a person with asthma.1,15,16 several “new” factors also increase asthma risk, that are caesarian section3,4 and pre term birth.5,6 the “hygiene hypothesis” for the increase in prevalence of allergic disease the proportion of people affected by allergic diseases, including allergic rhinitis eczema, and asthma, has increased dramatically over the past 50 years. the increase has been most marked in children, and was first noted in developed, “westernized” countries. these diseases remain relatively uncommon in poor rural populations, but have increased sharply in such populations on migrating to urban areas or to regions of high prevalence. multiple different theories have been proposed to account for this phenomenon, with many focusing on differences in diet or in childhood exposure to allergens, like house dust mite, cockroach, and molds trapped in indoor air by western patterns of housing. the theory that has held up best so far, the “hygiene hypothesis,” was first put forward by strachan when he noted an inverse association between family size and the rate of allergic disease, with the greatest protection being associated with the number of older siblings.1,17 asthma as a transmissible disease a theory, seemingly in direct contradiction to the hygiene hypothesis, is that allergic diseases, including asthma, are the result of exposure to a transmissible agent, either virus (hrv) or microbes. as for the hygiene hypothesis, epidemiologic evidence can be cited in this theory’s support. on the simplest level, the fact that nearly all children with asthma are allergic, but only a small proportion of allergic children have asthma, at least raises the possibility that some additional factor is involved. that this additional factor might be a transmissible agent is also suggested by the similarity between the gross epidemiologic patterns of children with paralytic poliomyelitis in the 1950s and children with asthma currently. both are more common among children of small, well-off families and among children migrating to urban areas from rural ones.1 on a smaller scale, the pattern of asthma’s penetration into native populations also seems consistent with exposure to a transmissible agent. among the fore people of new guinea, asthma appeared first in adults returning to villages after working in a european influenced city. only thereafter did it appear in children; and another has found the rates of asthma to be nearly as high in adopted children of mothers with asthma as in natural children.1 microbial colonization and risk of atopy there are a number of unique features of immune responses during this early period. pregnancy is associated with complex interactions at the materno-fetal interface, which reduce cell-mediated tissue rejection and type 1 (ifn-g) immune responses. an increase in type 2 immune activity is among a number of mechanisms evolved to protect the fetus in this context. at birth, the cellular responses of the fetus continue to reflect this ‘type 2’ skewed pattern. whether due to immaturity or active type 1 regulation (or both), the neonatal capacity for ifn-g responses is significantly impaired compared with those of 153pradopo and utomo: allergic asthma in children adults, resulting in an increased vulnerability to infection during his period. in this context it is clear that intrauterine infection is associated with increased capacity for neonatal type 1 responses, confirming that antenatal exposures have the potential to influence maturation of type 1 function, which tends to be non-allergic. thus, it is possible that ‘cleaner environments’ could be having an effect even before birth.9,17 the effect of clean environment before birth had been studied by roduit et al.3 and thavagnanam et al.4 who reported that caesarean section may have contributed to the rise in asthma. the mode of delivery has been reported to influence the development of allergic diseases in childhood. the prevalence of allergies and asthma in childhood has increased dramatically over the past few decades, mostly in industrialized countries. in parallel, rates of caesarean delivery have risen in most of the developed countries, from about 5% in the 1970s to over 30% in 2000 in some regions of the world. the initial microbial exposure for children born by caesarean section is delayed compared to those born by vaginal delivery; thus the maturation of the immune systems might be different and delayed. especially their is a finding that the risk of asthma and allergy is lower in adults with serologic evidence of infection with microbes transmitted by the “orofecal” route. taken together, these observations suggest a protective effect not of viral infection, but of microbial exposure in a broader sense, including nonpathogenic microbes. this idea, that exposure to nonpathogenic microbes might play a role in preventing the development of asthma and other allergic diseases, was greatly advanced by studies of stool flora in infants from populations with different rates of allergic disease.1 role of bacteria in asthma toews18 review revealed that mycoplasma pneumoniae and chlamydophila. pneumoniae may be associated with asthma chronicity. the role of m. pneumoniae and c. pneumoniae infections with chronic asthma has been evaluated using pcr, culture and serology to detect m. pneumoniae, c. pneumoniae and viruses. altogether, 56% of asthmatic patients had a positive pcr for m. pneumoniae or c. pneumoniae. positive results for pcr were found in broncholaveolar lavage (bal) fluid or biopsy samples. cultures for these organisms were negative in all patients. asthma risk showed by significantly greater number of tissue mast cells were noted in the group of patients who were pcr positive. effect of dental plaque control therapy on respiratory quality wiyarni et al.13 study in 30 children (7-11 years old) with mild persistent asthma revealed significant predominance of gram-negative bacteria cultures in uncontrolled asthmatic children (i.e. wheezing, coughing at night) compared to controlled asthma (asymptomatic), and significant increase of respiratory quality based on forced expiratory volume in one second (fev1) (p = 0,001; ci 95%) in asthmatic children with dental plaque control therapy compared to without dental plaque control therapy after one week study. furthermore, randomly selected subjects did not manifest asthmatic symptoms after two months evaluation.14 transmission of periodontopathic bacteria despite the abundance of commensal bacteria present in the birth canal, none of these are able to successfully colonize the mouth of the infant suggesting that they do not have tropism for the oropharyngeal mucosa. it has been proposed that bacteria are transferred from the primary caregiver, external environment, and from other areas of the respiratory tract. successful colonization depends on the ability of the bacteria to circumvent host innate and acquired immunity in order that they can adhere to oral surfaces and avoid removal via the flushing action of saliva and mastication. neonatal saliva has been shown to contain secretory immunoglobulin a (siga) antibodies that react with these bacteria, but these antibodies appear insufficient to completely block adherence and subsequent colonization.19 it was unclear whether the initial colonization by periodontal pathogens occured in the oral cavity. mcclellan et al.20 reported the association between specific age groups and the time when the initial colonization by periodontal pathogens occurs in the oral cavity in such groups. according to this study and a study by tanner et al.8, p. gingivalis was detected in all age groups, even among children less than 1 year of age. the youngest child whom p. gingivalis was identified was 20 days old, and three of the six predentate infants in the sample population were positive for p. gingivalis. these data suggest that p. gingivalis was acquired rapidly upon exposure, even in the first days of life, and before what has been assumed to be its primary ecological niche, the gingival sulcus, has developed.8,20 the role of parents and caregivers in bacteria transmission studies showed that if a child harbored a periodontal pathogen, then at least one of the parents will exhibit the same genotype of bacteria found that various anaerobic species colonize the edentulous mouths of infants, and that maternal saliva may act as a source of some gram-negative anaerobes. a simple but reliable test is needed in routine clinical examinations to identify the presence of bacteria associated with periodontal diseases.7,21 bana test the bana (n-benzoyl-dl-arginine-2-naphthylamide) test detects a trypsin-like enzyme that is present in p. gingivalis, treponema denticola, and tannerella forsythensis. the bana test had 92% sensitivity and 70% specificity when compared with dna probes and polyclonal immunological reagents and children whose parents were colonized by bana-positive bacteria were 9.8 times more likely to be colonized by bana-positive species than were children whose parents were bana-test154 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 151-156 negative. children whose parents had clinical evidence of periodontitis were 12 times more likely to be colonized by bana-positive species. these data are compatible with the hypothesis that children may acquire the bana positive species from their parents, especially if the parent has periodontitis.7 a study revealed that 70 of the 140 caregivers tested bana-positive and/or weakly positive in one or more of the quadrants. eighty-four percent of children whose caregivers were bana-positive were also bana-positive, whereas only 7% of children whose caregivers were bana-negative were bana-positive. sixty-three percent of the children aged 3–5 yrs and 92% of the children aged 6–10 yrs, whose caregivers were bana-positive, were also bana-positive. if caregivers or family members had a history of periodontal disease, the children were significantly more likely to be bana-positive. forty-seven children (62%) whose caregivers were > 35 years old had bana-positive scores, while only 26% of children who had younger caregivers had bana-positive scores.7,21 bana test was done by taking plaque samples with toothpick then wiped onto the lower strip of the bana card. a separate toothpick was used for each plaque sample. after all tooth sites had been sampled, the upper strip was lightly moistened with distilled water by means of a cotton swab. the bana card was folded at the perforation mark, so that the lower and upper strips met and placed in an incubator at 55° c for 5 min. the bana card was removed, and the lower portion was discarded in a manner appropriate for contaminated material. the color on the upper strip was recorded by consensus of two different examiners, as 'no blue' (negative), a 'faint blue' (weakly positive), or a 'distinct blue' (positive). for statistical analysis, weakly positive and positive results were recorded as positive.7 the role of antimicrobial therapy in asthma infection has been thought to be responsible to asthma exacerbations, therefore there has been much progress in understanding the mechanisms of microbeinduced asthma exacerbations, and the development of new therapeutic agents as well as preventive strategies is needed. both antimicrobial and immune modulators could have therapeutic benefits in this respect. several different antibacterial agents, namely tetracyclines, macrolides, quinolones, azalides and the ketolide telithromycin have in vitro and in vivo activity against the common atypical bacteria c. pneumoniae and m. pneumoniae. they have shown some clinical benefit in patients with chronic stable asthma or acute exacerbations.22 discussion schroder and arditi23 reported that infectious diseases have a major impact on both the development and the severity of asthma. the rise of asthma in industrialized countries over the last decades has been attributed to increased hygiene standards as well as the concomitant use of antibiotics, which together lower the incidence of infection. although this point of view is supported by both clinical studies and experimental approaches in mice, an increasing body of evidence suggests that certain infectious diseases may predispose for the development of asthma, thus challenging the `hygiene hypothesis'. the effects of bacteria on immune development are likely to be the greatest in the postnatal period when the infant has more direct contact with environment. the first years of life through the gradual maturation of type 1 responses, although this appears not to consolidate until after 18 months of age so that responses during this early period are relatively skewed towards the type 2 which normally characterized allergic disease. however, despite this, the majority of infants do not go on to develop atopy. although it has not been confirmed, there has been longstanding speculation that an important contributing factor in the development of type 2 immune disease is delayed development of type 1 function by bacterial exposure in infancy. a reduction in the level and variety of early microbial burden is an obvious candidate in the search for culprits in the spiralling levels of allergic disease.1,18 this concept was supported by roduit et al.3 and thavagnanam et al.4 study that caesarean section may have contributed to the rise in asthma. it is well-known that the etiopathogenesis of allergy is multifactorial i.e. genetics, environmental and allergens factors.1 however, it is still unclear why oral focal infection may involve in allergic development and symptoms. it is interesting that preterm birth also increases asthma risk,5,6 and coincidentally, even still inconsistent, previous epidemiological studies revealed that periodontal disease increases preterm birth risk. nevertheless, a study by katz et al.24 in 2009 by using immunocytochemistry, identified the presence of p. gingivalis antigens in placental tissues. the antigens were detected in the placental syncytiotrophoblasts, chorionic trophoblasts, decidual cells, and amniotic epithelial cells, as well as the vascular cells. these results suggest that p. gingivalis may commonly colonize placental tissue, and that the presence of the organism may contribute to preterm delivery. thus periodontopathic bacteria may indirectly cause allergy, by inducing preterm birth. the role of infection in asthma etiopathogenesis is in accordance with pejcic et al., and paju et al.25 and scannapieco et al.26 who showed that oral focal infection can play a part in the creation of respiratory infections that manifest as sinusitis, tonsillitis, pneumonias, bronchial asthma. these diseases can be caused by microorganisms from the oral cavity, following a direct inhalation from saliva and dental plaque, or by blood dissemination.25 there have also been numerous other descriptions of the mechanism where oral bacteria have been included in the pathogenesis of respiratory infections, i.e. p. gingivalis and a. actinomycetemcomitans which can aspire into the lungs and cause infection (droplets infection); then the host`s and bacterial enzymes from the saliva can dissolve saliva pelicula on pathogens and allow them to adhere to the surface of mucous membrane; and also cytokines derived from the 155pradopo and utomo: allergic asthma in children periodontal tissue can damage the respiratory epithelium by causing an infection via respiratory pathogens. damage of respiratory epithelium may lead to increased sensitivity to respiratory allergens or stimulation.25,26 even though respiratory infection is proposed as one of the pathways that periodontopathic bacteria may involved in asthma transmission etiologies; nevertheless, the exact mechanism is still uncertain. it is logical that transmission of periodontopathic bacteria to infants may result from maternal or caregiver’s saliva, thus increases respiratory infection risk (via droplet infection) by touching with dirty hands, kissing, or via using the same spoon, glass or plate that are not sterilized.7,21,27 however it is interesting that according to guilbert et al.28 study on breast feeding by asthmatics may increase the risk of asthma in babies born to mothers with the respiratory disease; and leme et al.29 stated that mice pups born to normal mothers and breastfed by asthmatic foster mothers develop airway hyperresponsiveness and eosinophilic airway inflammation. in addition, perez et al.30 study revealed that breast milk is not always free of bacteria. it is also supported by the predominance of gramnegative bacteria in dental plaque of uncontrolled asthmatic children that had been verified in wiyarni et al.13 study. however, in toews’18 review, periodontopathic bacteria were not included in asthma pathogenesis; it was not surprising because according to yilmaz,31 these bacteria are able to internalized gingival epithelial cells, thus could not be detected easily. nevertheless, recently, not only the whole periodontopathic bacteria had to be confirmed for their presence, their enzymes could also detected with the bana test.21 therefore, the verification of the the hypothesis that periodontopathic bacteria may involved in asthma etiopathogenesis and exacerbation is possible. moreover, improving personal hygiene and treatment of periodontal diseases of mothers and caregivers is mandatory. several precautions were thorough cleaning or aseptic of breast area before breastfeeding; and sterilizing bottle or feeding set. thus also the bad habits of blowing for cooling of hot foods for the infants and children. for the concluding remarks, periodontopathic bacteria may involve in asthma transmission via several ways: direct transmission from parents and caregivers via inducing respiratory infection and type 2 immune response; indirectly via placenta and inducing preterm birth. additionally, for research needs, bana test for parents and caregivers is a beneficial diagnostic test for the possibility of periodontopathic bacteria transmission in asthmatic children with unknown etiology. however, further collaborated research of allergy specialists and dental researchers are mandatory to verify this concept. references 1. yoo j, tcheurekdjian h1, lynch sv, cabana m, boushey ah. microbial manipulation of immune function for asthma prevention inferences from clinical trials. proc am thorac soc 2007; 4: 277–82. 2. winkler p, ghadimi d, schrezenmeir j, kraehenbuhl jp. molecular and cellular basis of microflora-host interactions. j nutr 2007; 137: 756s–72s. 3. roduit c, scholtens s, de jongste jc, wijga ah, gerritsen j, postma ds, et al. asthma at 8 years of age in children born caesarean section. thorax 2008; 36(1):1–7. 4. thavagnanam s, fleming j, bromley a, shields md, cardwell cr. a meta-analysis of the association between caesarean section and childhood asthma. clin exp allergy 2008 38(4): 629–33. 5. jaakkola jjk, ahmed p, ieromnimon a, et al. preterm delivery and asthma: a systematic review and meta-analysis. j allergy clin immunol 2006 oct; 118: 823–30. 6. steffensen fh, sørensen ht, gillman mw, rothman kj, sabroe s, fischer p, et al. low birth weight and preterm delivery as risk factors for asthma and atopic dermatitis in young adult males. epidemiology 2000; 11(2): 185–8. 7. lee y, straffon lh, welch kb, loesche wj. the transmission of anaerobic periodontopathic organisms. j dent res 2006; 85(2):j dent res 2006; 85(2): 182–6. 8. tanner acr, milgrom pm, kent r, jr. mokeem sa, page rc, riedymokeem sa, page rc, riedy ca, et al. the microbiota of young children from tooth and tongue samples. j dent res 2002; 81(1): 53–57. 9. strachan dp. family size, infection and atopy: the first decade of the “hygiene hypothesis”. thorax 2000; 55: s2–10. 10. darveau rp, pham t thu-thao, leinley k, reife ra, brainbridge bw, et al. porphyromonas gingivalis lipopolysaccharide contains multiple lipid a species that functionally interact with both toll-like receptors 2 and 4. inf immun 2004; 72(9): 5041–51. 11. netea mg, van der meer jwm, sutmuller rp, adema gj, kullberg bj. from the th1/th2 paradigm towards a toll-like receptor/t–helper bias. antimicrob ag chemoth 2005; 49(10): 3991–6. 12. kato t, kimizuka r, okuda k. changes of immunoresponse in balb/c mice neonatally treated with periodontopathic bacterial endotoxin. fems immunol med microbiol 2006; 47: 420–4. 13. wiyarni, sudiatmika n, febiola i, utomo h, harsono a. pengaruh terapi pengendalian plak gigi pada kualitas respirasi anak asma alergi. penelitian akhir ppds kesehatan anak fakultas kedokteran universitas airlangga. 2007. 14. utomo h. reducing asthmatic symptoms through improving oral health: from imaginary to reality (case report). journal of indonesian dental association special edition for 23rd pdgi congress, march 2008. p. 28–33. 15. eder w, ege mj, von mutius e. the asthma epidemic. n engl j med 2006; 355: 2226–35. 16. world health organization. prevention of allergy and allergic asthma based on the who/wao meeting on the prevention of allergy and allergic asthma geneva, 8-9 january 2002. available online at url http://www.worldallergy.org/professional/who_paa2003.pdf. accessed october 1, 2009. 17. prescott s. bacteria and the allergy epidemic: the culprits and the cure? cur allerg clin immunol 2004; 17(3):108-14. 18. toews gb. impact of bacterial infections on airway diseases. eur respir rev 2005; 14: 95, 62–8. 19. kirchherr jl, bowden gh, richmond da, sheridan mj, wirth ka, cole mf. clonal diversity and turnover of streptococcus mitis bv on shedding and nonshedding oral surfaces of human infants during the first year of life. clin diag lab immunol 2005; 12 (10): 1184–90. 20. mcclellan dl, griffen al, ley ej. age and prevalence of porphyromonas gingivalis in children. j clin microbiol 1996; 34(8): 2017–19. 21. lee y, tchaou wc, welch kb, loesche wj. the transmission of bana-positive periodontal bacterial species from caregivers to children. jada 2006; 137(11): 1539–46. 22. papadopoulos ng, konstantinou gn antimicrobial strategies: an option to treat allergy? biomedicine & pharmacotherapy 2007; 61: 21–28. 23. schröder nwj, arditi m. ieiis meeting minireview: the role of innate immunity in the pathogenesis of asthma: evidence for the involvement of toll-like receptor signaling. j endotoxin res 2007; 13: 305–12. 156 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 151-156 24. katz j, chegini n, shiverick kt, lamont rj . localization of p. gingivalis in preterm delivery placenta. j dent res 2009; 88(6): 575–8. 25. pejcic a, pesevska s, grigorov i, bojovic m. periodontitis as a risk factor for general disorders. acta fac med naiss 2006; 23(2): 59–63. 26. scannapieco fa, bush rb, paju s. associations between periodontal disease and risk for nosocomial bacterial pneumonia and chronic obstructive pulmonary disease. a systematic review. ann periodontol 2003; 8(1): 54–69. 27. li y, ismail ai, ge y, tellez m, sohn w. similarity of bacterial populations in saliva from african-american mother-child dyads. j clin microbiol 2007; 45(9): 3082–85. 28. guilbert tw, stern da, morgan wj, martinez fd, wright al. effect of breastfeeding on lung function in childhood and modulation by maternal asthma and atopy. am j respir crit care med 2007; 176: 843–48. 29. leme as, hubeau c, xiang y, goldman a, hamada k, suzaki y, kobzik l. role of breast milk in a mouse model of maternal transmission of asthma susceptibility. j immunol 2006; 176: 762–9. 30. perez pf, dore jl, leclerc m, levenez f, benyacoub j, serrant p. bacterial imprinting of the neonatal immune system: lessons from maternal cells? pediatrics 2007; 119: e724–e732. 31. yilmaz o. the chronicles of porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay. microbiology 2008; 154: 2897–903. mkgs vol 45 no 2 april-juni 2012.indd 97 volume 45 number 2 june 2012 research report cytotoxicity of betel leaf (piper betel l.) against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells suprapto ma’at department of clinical pathology faculty of medicine, airlangga university surabaya indonesia abstract background: betel leaf (piper betel l.) has been used in modern and traditional medicine as antiseptic, antibacterial, and also prevention of plaque accumulation, but it still can stimulate cancer in lime-piper betel quid. betel leaf also has anti-inflammatory properties. purpose: the purpose of this study was examine the cytotoxicity of betel leaf extract (ble) against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by peripheral blood mononuclear cells (pbmc) stimulated with lps. methods: mtt assay was used to investigate the survival rate of the culture with the survival rate result of the given culture extract 4%, 2% and 1% about 82%, 83.4% and 85%. there was no significant difference between treatment with various concentrations of the extract and the control (p>0.05). to evaluate the effect of betel leaf extracts on the production of cytokines, proinflammatory was conducted by incubating the extracts of betel leaf with peripheral blood mononuclear cells stimulated with lipopolysaccharide. peripheral blood mononuclear cells were obtained from healthy volunteers isolated by density centrifugation method using ficoll-hypaque. once coupled with various concentrations of betel leaf extract and lipopolysaccharide, and then incubated for 24 hours, the culture supernatant was used to determine the level of ifn-γ and tnf-α by elisa method. results: it is known that the survival rates of ble 4%, 2% and 1% were 82%, 83.4% and 85%. there was no significant of difference between several concentrations of ble and those in the control group (p>0.05). the production of ifn-γ and tnf-α stimulated with lps was no significant difference between ble 4%, 2% and 1% and that in the control group (p>0.05). conclusion: it can be concluded that ble is not toxic against primary culture of chicken embryo fibroblast, and the production of ifn-γ and tnf-α by pbmc was not affected by ble. key words: betel leaf extract, mtt assay, proinflammatory cytokines abstrak latar belakang: daun sirih (piper betel l.) telah banyak digunakan dalam berbagai pengobatan tradisional maupun moderen sebagai antiseptik, antibakteri dan untuk pencegahan pembentukan plak, tetapi dapat juga menimbulkan kanker pada orang pengunyah sirih. daun sirih juga memiliki aktivitas sebagai anti-inflamasi. tujuan: penelitian ini dimaksudkan untuk mengevaluasi sitotoksisitas ekstrak daun sirih terhadap kultur primer sel fibroblas embrio ayam dan pengaruhnya terhadap produksi sitokin proinflamasi oleh sel mononuklear darah perifer yang distimulasi dengan lps. metode: uji mtt digunakan untuk menginvestigasi survival rate kultur, dengan hasil: survival rate dari kultur yang diberi ekstrak 4%, 2% dan 1% adalah 82%, 83,4% dan 85%. tidak terdapat perbedaan signifikan antara perlakuan dengan berbagai konsentrasi ekstrak terhadap kontrol (p>0,05). untuk mengevaluasi pengaruh ekstrak daun sirih terhadap produksi sitokin proinflamasi dikerjakan dengan menginkubasi ekstrak daun sirih bersama sel mononuklear darah perifer yang distimulasi dengan lipopolisakarida. sel mononuklear darah perifer diperoleh dari relawan sehat yang diisolasi dengan metode sentrifugasi densitas menggunakan ficoll-hypaque. setelah ditambah dengan berbagai konsentrasi ekstrak daun sirih dan lipopolisakarida, diinkubasi selama 24 jam, supernatan kultur digunakan untuk menentukan level ifn-γ dan tnf-α dengan metode elisa. hasil: tidak terdapat perbedaan signifikan produksi ifn-γ dan tnf-α antara kultur sel mononuklear darah perifer yang diinkubasi bersama ekstrak daun sirih dibandingkan dengan kontrol (p>0,05). kesimpulan: disimpulkan bahwa ekstrak daun sirih 98 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 97–101 tidak toksik terhadap kultur sel primer fibroblas embrio ayam dan tidak berpengaruh terhadap produksi sitokin proinflamasi ifn-γ dan tnf-α oleh sel mononuklear darah perifer manusia yang distimulasi dengan lps. kata kunci: ekstrak daun sirih, uji mtt, sitokin proinflamasi correspondence: suprapto ma’at, c/o: departemen patologi klinik, fakultas kedokteran universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya, indonesia. e-mail: adityo2wp@yahoo.com introduction betel leafe from piper betel l. is a traditional plant that is closely related to oral health. betel leaf contains a variety of compounds, such as chavicol compound with powerful antiseptic activity which is stated, five times stronger than hydroxychavicol, phenol, allylrocatechol, cinneol, caryiphyllene, menthone, eugenol and methyl ether compounds. 35% betel leaf extracts have greater antibacterial activity against streptococcous viridans than 10% povidone iodine.1 betel leaf is widely used for various traditional and modern treatments in dental health such as oral antiseptic mouthwash (gargle). the content of essential oil in a betel leaf is antibacterial, which is oftenly used as additional agent in toothpaste to replace the use of fluoride. the use of toothpaste with high fluoride concentration can cause side effects, such as enamel fluorosis. its antibacterial effect of fluoride only inhibit the differentiation rather than eliminate the bacteria.2 many experiments have been done to search better option. such as the use of betel leaf essential oil in toothpaste as antibacterial agents. toothpaste containing betel leaf essential oil is commercially available at the market recently. betel leaf extract is also used to prevent plaque accumulation.3 in some people chewing betel (limepiper betel quid), the incidence of cancer was found, but it is still unknown which one from the chewing material, such as betel leaf, lime, betel nut (areca nut), or tobacco that has carcinogenic effect.4-6 compared with extracts of betel nut (areca nut), betel leaf extract has a lower toxicity towards human gingival keratinocyte cells.7 it is also known that areca nut extract is considered as toxic towards cell line fibroblast culture (human buccal fibroblast culture),8 but few study has been conducted to examine the toxicity of betel leaf towards primary cell culture. therefore, clinical application of betel leaves in dentistry and others in the medical field is quite extensive and mostly applied topically (mouthwash, toothpaste, vagina antiseptics), which means more contacts with mucosa and epithelial cells, it is necessary to examine the toxicity of betel leaf against cells (cytotoxicity), and in this research cytotoxicity assay uses primary cell culture. betel leaves have anti-inflammatory activity that works by inhibiting lipoxygenase enzyme activity.9 the content of betel leaf is hydroxychavicol, an anti-inflammatory that works by inhibiting cyclooxygenase activity and platelet aggregation, and it is also expected to prevent atherosclerosis.10 one of various anti-inflammatory response is to inhibit proinflammatory cytokine.11 inflammation is one of the immune mechanisms to eliminate microbial pathogens, and classified in natural immunity. in the inflammatory process, proinflammatory cytokines, such as tnf, il-1 and il-12, would be secreted. furthermore, il-12 induces nk cells and lymphocytes t to secrete ifn-γ that can activate macrophage.12 for those reasons, this research is aimed to determine the effect of betel leaf on the production of proinflammatory cytokines after incubation with human peripheral blood mononuclear cells (pbmc) stimulated with lps. if proven that betel leaf can inhibit secretion of proinflammatory cytokines, particularly tnf, then it will likely be used in sepsis treatment.12 materials and methods betel leaf samples used in this research were obtained from traditional medicine industry, tradimun gresik, in the form of fine powder crude of dried leaves. the powder was extracted by maceration method using 70% alcohol. two hundred grams of powder was extracted with 70% alcohol for 24 hours at room temperature, and then filtered through whatmann filter paper. afterwards, the filtrate was dried in a vacuum evaporator at temperature of 40° c45° c. the final result obtained was in the form of 2.6% dried powder. cytotoxicity test was conducted on primary cell cultures of chicken embryo by preparing primary cell cultures made by standard methods of primary cell culture manufacturing.13 this test was performed in a laminar flow cabinet. the chicken embryos obtained from embryonated eggs or hatched eggs at the age of 8-9 days old. eggshells were disinfected with 70% alcohol. next, they were opened and the embryos were removed and placed on a sterile petri dish. afterwards, they were washed with sterile phosphate buffer, and its head, legs and wings were removed. the bodies were then cut with scissors into some pieces with the size of 2-3 mm. those pieces of embryos were placed in erlenmeyer 100 ml equipped with a magnetic bar, added with trypsin-versene solution, phosphate buffer solution containing 0.25% trypsin and 1 mm ethylenediamine tetra acetic acid (edta), and then stirred on the magnetic stirrer for 10 minutes at 200 rpm. afterwards, those were left for 3 minutes until those pieces of tissue settled, and then supernatants, cell suspension and tissue debris, were separated into another erlenmeyer. next, those were added with another trypsin99ma’at: cytotoxicity of betel leaf (piper betel l.) versene solution were added and then stirred until the pieces of embryos were unraveled. those cell suspensions were filtered by using three layers of sterile gauze to separate the remained tissue and debris. the filtrate containing fibroblasts was washed 3 times with phosphate buffer centrifugation, and then suspended in rpmi 1640 medium containing 10% fetal bovine serum (fbs), 100 units/ml penicillin, and 100 mg/ml streptomycin. cell density was calculated by using hemocytometer, made with the size of 4x105 cells/ml, and then ready for testing. moreover, preparation of the test was conducted by using modification of elution method (iso 10993-5).14 two grams of extract was then added to 25 ml rpmi 1640 culture medium + 10% fbs (concentration of extract 8%), and sterilized by filtration using millipore filter with the size of 0.22 μm. next, 10 ml of it was taken for dilution conducted 2 times (double dilution) with rpmi 1640 culture medium to obtain the concentration of the extract about 4% and 2%. afterwards, it was left at room temperature for 1 hour. the test was then conducted on cell culture plates with 96 wells. one hundred μl extract dilution was added into each well, 5 wells for each dilution, and then 100 μl fibroblast suspension was added, so the series of extract concentration was 4%, 2%, and 1%. each well then contained 2x105 fibroblast cells, and a row of wells containing 200 μl cell suspensions was used as control. the plates were incubated at 37° c in a 5% co2 incubator for 24 hours. cytotoxicity observation, was conducted on betel leaves by using mtt test method.15 one hour before the incubation period ended, 5 mg/ml of 20 μl mtt solution, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (sigma, m-5655), was added into all the wells. the incubation was continued for the next 1 hour. culture medium was replaced with isopropanol containing 0.01 m hcl to dissolve formasan crystals. optical density (od) of purple color of the formasan was measured by using multiwell spectrophotometer (elisa reader) at a wavelength of 570 nm. survival rate was then calculated as survival rate,% = od sample/od control 100%. the effects of betel leaves on the production of proinflammatory cytokines incubated in peripheral blood mononuclear cells were evaluated by preparing peripheral blood mononuclear cells. peripheral blood was obtained from healthy donor in department of clinical pathology in faculty of medicine airlangga university, and separated by gradient method using ficoll-hypaque 1.077 (ficoll hypaque density centrifugation). buffy coat containing mononuclear cells was then separated and washed 3 times with phosphate buffer. afterwards, the density of cells was measured by using hemocytometer, and adjusted to 5 105 cells/ml in rpmi-1640 medium + fbs 10%, penicillin, and streptomycin. the test was then prepared by using cell culture plates with 96 wells, and conducted as the same as cytotoxicity test. each well was filled with 80 ml suspension of mononuclear cells, and also added with 100 μl betel leaf extract dilution, each of which used 5 wells. afterwards, mononuclear cell density changed to 4x104 cells/ wells, and a series of wells containing 180 μl cell suspension, 4x104 cells/wells, was prepared as control. each well was added with 10 μg/ml of 20 μl lipopolysaccharide (lps) as a mitogen. the plates were then incubated at 37° c for 48 hours in co2 incubator. after incubated, the supernatant was used to analyze the content of ifn-γ and tnf-α by elisa method using quantikine colorimetic sandwich elisa kits. finally, the results obtained were analyzed by using anova, and then differences among treatment groups were analyzed with student-newman-keul’s test with p<0.05.13 results in the cytotoxicity assay of betel leaf extracts on primary cell cultures of chicken embryos, the average survival rates of betel leaf extracts 4%, 2%, and 1% in primary cell cultures of chicken embryo fibroblasts were 82%, 83.4%, and 85% respectively (the percentage of living cells), with control of 100% (figure 1). there was no significant difference between either the extract concentration of 4% (p = 0.5), 2% (p = 0.5), or 1% (p = 0.5) and the control group. it means that betel leaf extract had no effect on the growth of culture. in other words, it was not toxic on primary cell cultures of chicken embryo fibroblasts. production of ifn-γ and tnf-α from peripheral blood mononuclear cells after incubated with betel leaf extracts and stimulated with lps can be seen in table 1 and table 2. there was no significant difference in the secretion of ifn-γ in treatment group and controls group p = 0.285, 0.747 and 0.747 (table 1). there was also no significant difference in tnf-α secretion in treatment group and controls group had p = 0.873, 0.873 and 0.747 (table 2). 0 20 40 60 80 100 120 k 1 2 4 konsentrasi (%) su rv iv al r at e (% ) ** * concentration (%) control s u rv iv a l r a te ( % ) figure 1. survival rate (%) of primary cultures of chicken embryo fibroblasts with betel leaf extract treatment. k = control 1, 2, 4 = treatment by 4%, 2%, and 1% extract * = β>0.05 100 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 97–101 discussion primary cultures are cultures of cells isolated directly from tissues. their growth is limited compared to cell line culture and very easily inhibited by toxic materials. cytotoxicity test on betel leaves used primary cell cultures in order to determine the toxicity effects more sensitively. primary cell cultures of chicken embryo were considered as primary cell cultures with readily available raw materials and simple culture implementation. therefore, the implementation was conducted by using a modified elution method according to iso 10993-5 because betel extracts were soluble in water. according to iso 109935, moreover, materials that would be tested was added to the cell culture after the culture reached a 80% density of growth (confluent) cell line culture was used. in this research, if the primary cell culture was sub cultured, it might ruing its growth, so the tested betel leaf extracts were mixed with the cell suspension before incubation. there was no significant difference between the survival rates of the treatment group and the control groups. it was not considered as toxic to primary cell cultures of chicken embryos. although other researchers found that betel leaf extract was considered as toxic to human ginggival keratinocyte cell culture6 and human buccal fibroblast culture,8 chicken fibroblasts were more resistant to the toxicity of betel leaf extract possibly because human fibroblasts is different from avian fibroblasts. lipopolysaccharide (lps) is the largest component of the cell wall of gram-negative bacteria, and also known as endotoxin. lps activates a variety of mammalian cell types including monocytes/macrophages by activating nf-κb through intracellular signaling pathways and resulting in the synthesis and secretion of proinflammatory cytokines.16,17 in the buffy coat obtained by the gradient method using ficoll-hypaque 1.077 contained a population of lymphocytes t and lymphocytes b, as well as monocytes and nk cells. the giving of lipopolysaccharide mitogen, means that cells activated were b lymphocytes and monocyte cells. the activated monocyte cell would release il-1β proinflammatory cytokines, il-6, ifn-γ and tnf-α. monocytes have a central role in chronic inflammatory process,18 and its inflammatory reactions can be classified as the natural immune response againts pathogen invasion.12 materials or drugs that can affect the production of proinflammatory cytokines are called immunomodulator,19 including drug ingredients that can modulate proinflammatory cytokines work. several previous studies proved that betel leaf has antiinflammatory effect,20 and materials that can suppress the secretion of proinflammatory cytokines, such as ifn-γ and tnf-α, are categorized as antiinflammation.21 ingredients of medicine that are categorized anti-inflammation has several mechanism of action which are: 1) modulating inflammatory response with transcription factors as target, eg, cyclosporin a;22 2) inhibiting cyclo-oxygense activity, eg diclofenac, piroxicam, and ibuprofen;23 3) inhibiting lipoxygenase, eg mk-886 and a-63162;22 4) inhibiting complement system activity;24 and 5) inhibiting cytokine.25 anti-inflammation classified as cytokine inhibitors can be divided into two types. first, endogenous type, for example: 1) soluble cytokine receptors, such as tnf receptor (tnf-r) and il-1 receptor type ii (il-1r); 2) cytokine receptor antagonists, such as receptor antagonist il-1 (il-1ra); and 3) inhibitory cytokines, such as il-4, il-10, il-11, il-13, and tgf-β. second, exogenous type, such as antibodies against tnf-α (anti-tnf-α), includes ingredients that inhibit the synthesis and secretion of proinflammatory cytokines. in this research, there were no significant differences between the secretion of ifn-γ and tnf-α by either betel extract 1%, 2%, or 4% (p>0.05) compared with the control group. it means that the anti-inflammatory properties of betel leaves was not working through suppression of proinflammatory cytokines, ifn-γ and tnf-α, but through other channels in accordance with the research of pin et al.,9 which states that the anti-inflammatory properties of betel leaf works by suppressing the activity of lipoxygenase enzyme.8 a research conducted by ganguly et al.,26 revealed that the anti-inflammatory activity of betel leaf works by suppressing the expression of il-12 p40. meanwhile, by shalini t et al.,12 reported showed that the anti-inflammatory activity of betel leaf works by suppressing cyclooxygenase and thrombosis aggregation. however, this research has several weaknesses: first, embryonated chicken eggs was not taken from spf (specific table 1. the effect of betel leaf extract on ifn-γ secretion of peripheral blood mononuclear cells. ifn-γ concentrations in the picogram/ml control group pg/ml 1% extract pg/ml 2% extract pg/ml 4% extract pg/ml 112 113.5 112.5 109.5 11.5 112 111 109 112.5 114 113 111 113 113 111.5 110 111 112.5 112 110.5 p = 0.285 p = 0.747 p = 0.747 pg/ml = picogram/ml table 2. the effect of betel leaf extract on tnf-α secretion of peripheral blood mononuclear cells. tnf-α concentrations in the nanogram/ml control group ng/ml 1% extract ng/ml 2% extract ng/ml 4% extract ng/ml 4.8 4.5 4.7 4.8 4.7 4.6 4.65 4.9 4.9 4.55 4.75 5.0 4.75 4.65 4.65 4.95 4.85 4.7 4.75 4.85 p = 0.873 p = 0.873 p = 0.747 ng/ml = nanogram/ml 101ma’at: cytotoxicity of betel leaf (piper betel l.) pathogenic free) chickens, and second, mononuclear cells were isolated from only one donor. it would be better when it is done with cells from several donors. finally, it can be concluded that betel leaf extract (piper betel l) is not toxic to primary fibroblast culture of chicken embryo. betel leaf extract has no effect on the production of proinflammatory cytokines by human peripheral blood mononuclear cells stimulated with lipopolysaccharide. references 1. apriasari ml, soebadi b, hendarti ht. sensitivity difference of streptococcus viridans on 35% piper betle linn extract and 10% povidone iodine towards recurrunt apthous stomatitis. den j (maj ked gigi) 2011; 44(3): 159–63. 2. yeung ca. a systematic review of the efficacy and safety of fluoridation. evid based dent 2008; 9(2): 39–43. 3. mayasari ra, goenharto s, sjafei a. betel leaf toothpaste inhibit dental plaque formation on fixed orthodontic patients. den j (maj ked gigi) 2011; 44(4): 169–72. 4. secretan b. a review of human carcinogens. the lancet oncology 2009; 10(11): 1033–4. 5. amarasinghe hk, usgodaarrachchi us, johnson nw, lallo r, warnakulasuriva s. betel-quid chewing with or without tobacco is a major risk factor for oral potentially malignant disorders in sri lanka: a case-control study. oral oncology 2010; 46(4): 297–301. 6. lin mh, chou fp, huang hp, hsu jd, chou my, wang cj. the tumor promoting effect of lime-piper betel quid in jbl cells. food chem toxicol 2003; 41(11): 1463–71. 7. jeng jh, hahn lj, lin br, hsieh cc, chan cp, chang mc. effect of areca nut. inflorescence piper betel extracts and arecolin on cytotoxicity. total and unscheduled dna synthesis in cultured gingival keratinocytes. j oral pat & med 1999; 28(2): 64–71. 8. saraswati tr, sheeba t, nalinkumar s, ranganathan k. effect of glutathion on arecanut treated normal human buccal fibroblast culture. indian journal of dental research 2006; 17(3): 104–10. 9. pin ky, chuah al,rashih aa, mazura mp, fadzureena j, vimala s, rasadah ma. antioxidant and anti-inflammatory activities of extracts of betel leaves (piper betel) from solvent with different polarities. journal of tropical forest science 2010; 22(4): 448–55. 10. shalini t, verma nk, sigh dp, chaudary sk, asha r. piper betel: phytochemistry. tradisitional use & pharmacological activitya review. international journal of pharmaceutical research and development (ijprd) 2012; 4(04): 216–23. 11. feldmann m. cytokine inhibitors: biologics. in: gallin ji, snyderman r, editors. inflammation: basic and clinical correlates. 3rd ed. philadelphia: lipincott william & wilkins; 1999. p. 1207–11. 12. abbas ak, lichtman ah, pillai s. cellular and molecular immunology. 6th ed. philadelphia: saunders elsevier; 2010. p. 288–9. 13. ma’at s. teknik dasar kultur sel. surabaya: airlangga university press; 2011. p. 27–32. 14. international organization for standardisation. international standard iso 10993-5 biological evaluation of medical devices-part 5; tests for in vitro cytotoxicity. geneva: international organisation for standardisation, 1999. 15. mosmann t. rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. j immunol methods 1983; 65: 55–63. 16. ulevitch rj, tobias ps. recognition of gram-negative bacteria and endotoxin by the innate immune system. curr opin immunol 1999; 11: 19–22. 17. krappmann d, wegener e, sunami y, esen m, thiel a, mordmuller b, scheidereit c. the ikappab kinase complex and nf-kappab act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of ap-1. mol cell biol 2004; 24: 6488–500. 18. taylor pc, peters am, paleolog e; chapman pt, elliott mj, mccloskey r, feldmann m, maini rn. reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor α blockade in patients with rheumatoid arthritis. arthritis rheum 2000; 43: 38. 19. bessler h, bergman m, blumberger n, djaldetti m, salman h. coenzyme q10 decrease tnf-α and il-2 secretion by human peripheral blood mononuclear cells. j nutr sci vitaminol 2010; 56: 77–81. 20. guha p. betel leaf: the neglected green gold of india. j hum ecol 2006; 19(2): 87–93. 21. lee ch, butt ykcm wong msm lo scl. a lipid ekstract of perna canaliculus affects the expression of pro-inflammatory cytokines in a rat adjuvant-induced arthritis model. european annals of allergy and clinical immunology 2009; 40(4): 148–53. 22. manning am, rao a. agents targeting transcription factors. in: gallin ji, snyderman r, editors. inflammation: basic and clinical correlates. 3rd ed. philadelphia: lipincott william & wilkins; 1999. p. 1159–63. 23. showell hj, cooper k. inhibitors and antagonosits of cyclooxygenase. 5-lipoxygenase. and platelet activating factor. in: gallin ji, snyderman r, editors. inflammation: basic and clinical correlates. 3rd ed. philadelphia: lipincott william & wilkins; 1999. p. 1177–85. 24. platt jl. complement inhibitors. in: gallin ji, snyderman r. inflammation: basic and clinical correlates. 3rd ed. philadelphia: lipincott william & wilkins; 1999. p. 1195–204. 25. feldmann m. cytokine inhibitors: biologics. in: gallin ji, snyderman r, editors. inflammation: basic and clinical correlates. 3rd ed. philadelphia: lipincott william & wilkins; 1999. p. 1207–11. 26. ganguly s, mula s, chattopadhyay, chatterjee m. an ethanol extract of piper betel linn. mediates it regulation of nitric oxide. j pharmacy and pharmacology 2010; 59(5): 711–18. << 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkgs vol 45 no 2 april-juni 2012.indd 79 volume 45 number 2 june 2012 literature reviews the relation between salivary siga level and caries incidence in down syndrome children rosdiana1 and mochammad fahlevi rizal2 1resident at pediatric dentistry 2department of pediatric dentistry faculty of dentistry, indonesia university jakarta indonesia abstract background: down syndrome or trisomy 21 is a genetic disorder caused by extra chromosome on chromosome 21. down syndrome child, however, has good resistance against caries, and some of them even are caries-free. it is because the level of salivary siga in down syndrome children is equal or even higher than that in normal children. purpose: this review was aimed to review the relation between salivary siga level and caries incidence in down syndrome children. reviews: down syndrome is a collection of symptoms caused by chromosomal abnormality that has a number of physical and mental disorders. down syndrome children, nevertheless, have significantly lower incidence of caries than normal children. these conditions are thought to relate to characteristics of oral cavity and the level of salivary siga in down syndrome children. caries is a disease of dental hard tissues caused by the fermentation of sucrose into glucans by glucosyltransferase enzymes (gtf) of streptococcus mutans (s. mutans). one of proteins in saliva that acts as a defense mechanism is imunoglubulin. secretory immunoglobulin a (siga) inhibits the activity of s. mutans as bacteria causing caries forming glucan. this immunoglobulin, siga, is the most abundant immunoglobulin in saliva. the level of salivary siga in down syndrome children is significantly higher than that in normal children. conclusion: besides factors of tooth eruption delays, wide spaces among teeth, microdontia, ph, and high saliva contents (calcium, sodium, bicarbonate), the low incidence of caries in down syndrome children is also related with the higher level of salivary siga in down syndrome children than that in normal children. key words: down syndrome, siga, caries abstrak latar belakang: sindroma down atau trisomi 21 merupakan kelainan genetik yaitu adanya kromosom ekstra pada kromosom 21. anak sindroma down memiliki resistensi yang baik terhadap karies dan sebagian dari mereka bebas karies. kadar siga saliva anak sindroma down sama atau bahkan lebih tingi dari anak normal. tujuan: tujuan dari tulisan ini adalah mencari hubungan antara kadar siga di dalam saliva dengan kejadian karies pada anak sindroma down. tinjauan pustaka: sindroma down adalah suatu kumpulan gejala akibat abnormalitas kromosom yang memiliki sejumlah kelainan fisik dan mental. anak sindroma down secara signifikan memiliki prevalensi karies yang lebih rendah jika dibandingkan anak normal. kondisi ini diduga berhubungan dengan karakteristik rongga mulut dan kadar siga saliva anak sindroma down. karies merupakan penyakit jaringan keras gigi yang disebabkan oleh fermentasi sukrosa menjadi glukan oleh enzim glucosyltransferase (gtf) dari streptococcus mutans (s. mutans). salah satu protein di dalam saliva yang berperan sebagai mekanisme pertahanan adalah imunoglubulin. imunoglobulin a sekretori (siga) berperan menghambat aktivitas s. mutans sebagai kuman penyebab karies membentuk glukan. siga adalah imunoglobulin yang paling banyak terdapat pada saliva. kadar siga saliva sindroma down signifikan lebih tinggi dibandingkan anak normal. kesimpulan: rendahnya insiden karies anak sindroma down berhubungan dengan kadar siga di dalam saliva anak sindroma down yang lebih tinggi dibandingkan anak normal selain faktor keterlambatan erupsi gigi geligi, ruang antar gigi yang lebar, mikrodonsi, ph dan kandungan saliva (kalsium, sodium, bikarbonat) yang tinggi. kata kunci: sindroma down, siga, karies 80 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 79–83 introduction down syndrome or trisomy 21 is a collection of symptoms caused by abnormality of chromosome 21.1 in some reports, this disorder is closely related to mother’s age during her pregnancy.1,2 down syndrome children have abnormality characteristics which are delayed growth and development, both physically and mental.1,2 the incidence of down syndrome children in the world is about one of 800-10000 births.3 down syndrome children, however, have a good resistance against caries, and some of them even are cariesfree. some studies said that the incidence of caries in those down syndrome children is low.4–6 previous research even reported that 44% of down syndrome children are free of caries.3 these conditions are related to delay in eruption of permanent and deciduous teeth, hypodontia, microdontia, wide interdental space, and high concentration (ph) of saliva.5 saliva has protective function against microorganisms in oral. one of proteins in saliva that acts as a defense mechanism is imunoglubulin. the most abundant immunoglobulin in saliva is secretory immunoglobulin a (siga). as the most important specific defense, siga protect oral cavity from bacterial pathogens. the main function of the antibody, sig a is to limit penetration of microorganisms into oral. specifically, the role of siga is to inhibit adhesion of s. mutans adhesin on tooth surface.7 thus, several previous researches have been conducted to find the relation between the level of siga in the saliva and the incidence of caries in down syndrome children.5,6,8 the low incidence of caries is associated with the higher level of siga than that in normal children.6 therefore, this research is aimed to review the relation between salivary siga and caries incidence in down syndrome children. characteristics of systemic down syndrome down syndrome was first described by langdon down in 1865 based on physical findings in patients with down syndrome.5 down syndrome babies can be born from mothers with all ages, but the risk of down syndrome births increases as the increasing of maternal age during pregnancy.2 based on cytogenetic examination, down syndrome is generally divided into three types: trisomy 21, translocation, and mosaic. type of trisomy 21 occurs when there is an extra chromosome on chromosome 21 it has 47 chromosomes.9 type of translocation occurs when there is a segment of chromosome 21 attaching to another chromosome (usually chromosome 14), but the number of chromosomes is still 46.2 meanwhile, type of mosaic occurs when some cells have normal complement of 46 chromosomes and others have 47 chromosomes (an extra chromosome on chromosome 21).3 a number of physical abnormalities that can be found in down syndrome children are body with short and fat hand, round face with a flat profile, brachycefalics, pleated epicanthus, strabismus (crossed eyes), small maxilla, short hands and fingers, muscle hypotonus, and distance between the first and second fingers.1,2,10,11 moreover, bottom face of down syndrome children is more dominant, with slanted eyes and significant protruding forehead.9 the degree of retardation is determined by intelligence quotient (iq) and social quotient (sq). according to american association of mental deficiency (aamd), mental retardation is divided into four categories: mild retardation with a score of 55-69, moderate retardation with a score of 40-54, severe mental retardation with a score of 25-39, and profound mental retardation with score of less than 25.12 although there are some people with down syndrome have an iq above 69, but almost all people with down syndrome get mental retardation that varies from mild to profound.13 it can be found physical examination of down syndrome patients, leukemia, infections especially on respiratory track, hepatitis b, alzheimer, and congenital cardiac abnormalities can be found.1–3 gene expression of trisomy 21 in down syndrome, furthermore, causes various abnormalities of immune system. 1 down syndrome also has systemic immune system disorders, including immunodeficiency of mucosa humoral immune response.1,14 some researches even have found that high rate of infection in down syndrome patients is caused by immune system disorders.1,14 this can be shown by a defect in neutrophil chemotaxis of pmn leukocytes, antibody response damage against specific pathogens, decreasing number of t lymphocyte cells and immaturity of lymphocyte cells t.14 caries in down syndrome children down syndrome children have significantly lower prevalence of caries than normal children.4,5,15 caries lesion in down syndrome children is limited to the occlusal surface, while in smooth and proximal surface are rarely occurs.5 down syndrome children have good resistance against caries, and some of them are even free of caries.4 these conditions were related to delay in eruption of primary and permanent teeth, less contamination of cariogenic foods, hypodontia, microdontia, the interdental space, and high salivary ph.1,5 previous studies have reported that 29.4–53% of down syndrome children are free of caries, whereas only 0.5% of normal children are free of caries.4,6 microorganism that plays role in the caries process is s. mutans.1,12,16,17 previous studies have reported that the number of s. mutans in down syndrome children is lower than that in control group of normal children with caries. correspondence: rosdiana, c/o: program studi ilmu kesehatan gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta, indonesia. e-mail: rosdiana312@yahoo.co.id 81rosdiana and rizal: the relation between salivary siga level this is associated with a low prevalence of caries in down syndrome children.5,18 balance of ph and salivary buffering capacity is influenced by quantitative and qualitative of electrolytes in saliva. buffering capacity of saliva is an ability to maintain and neutralize low ph of saliva, its ability due to clusters of bicarbonate, phosphate, urea, protein, and mainly determined by the content of phosphate and bicarbonate. the composition of calcium, sodium, bicarbonate, ph and buffering capacity of saliva in down syndrome children was higher than that in normal children, so it can inhibit demineralization.8,14,18,19 the protective role of salivary siga in caries process saliva plays a role in oral health by both maintaining integrity of soft and hard tissues as well as protecting soft tissues of oral bacterial infections, fungi and viruses. saliva has a role to create a balance in oral cavity.7,20 a variety of compounds that plays a role in defense mechanisms found in saliva, one of which is siga.11 immunoglobulin is actually a substance classified as soluble proteins. immunoglobulin (ig) formed by plasma cells is derived from proliferation of cell b due to contact with antigen. immunoglobulin classified into five, namely igg, iga, igm, igd and ige based on antigenic differences in constant region of chain h. immunoglobulin a consists of two types, namely serum iga and mucosal iga. the amounts of iga in serum is small, whereas the higher level of iga is in the form of secretory iga (siga).21 moreover, siga is a dominant isotip antibody in human external secretions. secretory iga is mostly found in mucosal secretions, saliva, tracheobronchial, colostrum, breast milk, and urogenital.21 immunoglobulin a molecules secreted by plasma cells are found in salivary glands, whereas other protein components are produced in outer epithelial layer that covers the glands. 7 components of siga actually consist of four dimers consisted of two monomer molecules, a secretory, and a chain j. secretory components are produced by epithelial cells and connected to immunoglobulin a of crystallizable (fc) fragment by dimer chain j possibly passing mucosal epithelial cells.7 immunoglobulin a molecules are secreted by plasma cells found in the salivary glands, while other protein components are produced in outer epithelial layer that covers the glans. furthermore, siga is considered as the first defense mechanism at mucosal areas by inhibiting the development of local antigen, and it has also been known that it can inhibit virus to penetrate into mucosa.7,23–25 thus, siga is a product of mucosa immune system (mis) consisted of lymphocytes t and b. 20 in addition, siga in saliva is a sign of humoral immune response has been activated in oral cavity.19 humoral immune response in oral cavity actually has a relation to dental caries.7 dental caries infection can trigger salivary siga secretion.20 the level of salivary siga in caries-resistant group, therefore, is higher than that in caries-vulnerable group.5 the level of salivary siga in caries-resistant group is 17.88 ± 5.8 mg dl-1, whereas that in caries-vulnerable group is 11.78 ± 4.8 mg dl-1.7 discussion immune deficiency that occurs in down syndrome children is generally caused by excessive expression of superoxide dismutase (sod1) genes and low level of serum zinc.26 as a results, the number of t and b lymphocytes in down syndrome children become less.13 thus, the levels of iga, igg, and igm in down syndrome children have a tendency to be lower than those in normal children. previous research stated that the levels of iga and igg in the serum of down syndrome children who have lower respiratory tract infection are higher than those in normal ones, whereas the level of igm is lower than that in normal ones.26 these increasing levels of iga and igg are affected by slower elimination of infectious agents in down syndrome children caused by excessive stimulation of the immune system and increasing production of antibodies. meanwhile, the low level of igm is possibly affected by lower ability of antiinfection in down syndrome children. 26 the immunodeficiency in down syndrome children has caused the decreasing number of t cells, and if in immaturity condition, it can make the incidence of periodontal disease in those children increased, but not the incidence of dental caries.1,5,14 it is because the components of immune system in oral cavity in periodontal disorders include neutrophils, antibodies, lymphocytes, macrophages, lymphokine, secretory immune system. however, the level of siga in periodontal disorders is not increased since unlike dental caries process, in periodontal disease siga is considered as the most responsible immune component.7 though immunodeficiency occurs in down syndrome children, but the level of siga is higher than that in normal ones. there are unspecified causes for the higher level of siga in down syndrome children siga levels than normal children, but the levels of salivary siga itself is influenced by many factors, and salivary siga is also considered as the local mucosal immune system that does not really need to work together with other systemic immunities.14 factors that affect the production and concentration of salivary siga are antigen exposure, level of stress or emotional conditions, nutrition, history of consumption, power flow a  b  c  figure 1. a) structure of siga; b, c) structure of iga.21,22 82 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 79–83 of saliva, saliva stimulation, age, intensity of activity, hormonal and genetic background. 27,28 moreover, previous research has been conducted to find the cause of the low incidence of dental caries in down syndrome children.6 several previous researches have been conducted to assess the relationship between the overall levels of salivary siga and the incidence of dental caries in down syndrome children.6,29 it is known that the level of salivary siga in down syndrome is significantly higher compared to in normal children, as a result, this condition can protect their teeth from dental caries. 6,10,13 s. mutans, furthermore, is considered as the first bacteria colonizing to the tooth surface and initiating the formation of plague.1,29 s. mutans is also considered as a major pathogen microorganism involved in dental caries because it has virulence factors, such as abilities to produce adhesin, glucosyltransferase gtf, glucan-binding protein, acid, and tolerance to high concentration of acid.30,31 s. mutans secretes gtf enzyme that synthesize sucrose into soluble or non-soluble glucans (extracellular polysaccharide). extracellular polysaccharides produced then cause bacterial colonization and plaque formation on tooth surface.32,33 the roles of extracellular polysaccharides, mainly glucans, are strengthen the adhesion and accumulation of s. mutans and other streptococcus on tooth surface, strengthen the stability of the extracellular matrix that can increase the density of biofilm, protect microorganisms from microbial or other environmental influences, and to become a source of reservoir energy.34 the adhesin plays role in early colonization of s. mutans to pellicle on tooth surface through salivary receptor cells, and in co-aggregation with other bacteria.35,36 glukan-binding protein is a virulence factor of s. mutans that generate or glucan binding which produced by gtf.37 s. mutans is an antigen which will evoke an immune response in oral cavity. as the most important specific defense, siga plays a role to protect oral cavity from bacteria causing caries (s. mutans).7 secretory iga is able to control the colonization of s. mutans by reducing the initial adhesion of bacteria to tooth surface as well as neutralizing extracellular enzyme.34 the inhibition of s. mutans colonization by siga in vitro is presumably because siga can inhibit gtf work, so glucan is not formed, as a result, the attachment of bacteria does not occur on the mechanism of plaque formation.37 secretory iga has an ability not only to interfere either sucrose-dependent or sucroseindependent adhesion to the surface of hydroxyapatite, but also to inhibit the activity of metabolic adhesion.38 the inhibition of siga towards the sucrose-dependent adhesion of s. mutans is because siga can bind to bacteria and cover hydrophilic layer causing bacteria trapped in salivary causes mucus and eliminated from oral cavity.38,39 it is also known that the inhibition of siga towards the sucrose-dependent adhesion of s. mutans is because siga can inhibit the synthesis of glucan by gtf, principally caused by the ability of antibodies to bind to catalytic domain or glucan-binding domain which then would directly inhibit the function of enzymes.37,40 thus, it is known that glucosyltransferase gtf secreted by s. mutans play a role in the sucrose-dependent accumulation of streptococci. 40 in conclusion, children with down syndrome have a low incidence of caries. interaction of siga, a component of humoral immune system, against s. mutans, bacterial cariogenic antigens, in oral cavity possibly causes reduction in the incidence of dental caries. the higher level of siga in young down syndrome children than that of the normal ones makes the incidence of dental caries in those down syndrome children lower. the other reasons that make lower incidence of dental caries are delayed dental eruption, wide space among teeth, microdontia and higher level of ph and saliva contents (calcium, sodium, bicarbonate). references 1. mc donald re, avery dr, dean ja. dentistry for the child and adolescent. 8th ed. missouri: mosby inc; 2011. p. 540-2. 2. welbury rr, duggal ms, hosey mt. pediatric dentistry. 3rd ed. new york: oxford; 2005. p. 395. 3. koch g, sven p. pediatric dentistry a clinical approach. 2nd ed. uk: wiley blackwell; 2009. p. 338-9. 4. sharath a, muthu ms, sivakumar n. dental caries prevalence and needs of down syndrome children in chennai, india. indian j dent res 2008; 19(3): 224-9. 5. abou em, taha s, el shehaby f. relationsheep between salivary composition and dental caries among a group of egyptian down syndrome children. aus j basic and appl sciences 2009; 2: 720-30. 6. ranadheer e, vanugopal rn, arun pr, krisha k. the relationship of salivary immunoglobulin a with dental caries and oral hygiene status in down syndrome children. annal and essences j dentistry 2010; 2(2): 10-7. 7. roeslan bo. imunologi oral. kelainan di dalam rongga mulut. jakarta: fakultas kedokteran ui; 2002. p. 139-44. 8. bagherian a, jafarzadeh a, rezeian m. comparison of the salivary immunoglobulin concentration levels between children with early chilfhood caries and free of caries children. iran j immunol 2008; 5: 4. 9. ad k i nson lr, brown m d. elsevier’s i nteg rate d genet ics. philadelphia: cv mosby elsevier; 2007. p. 17-9. 10. suharsini m. pengaruh faktor genetik dan lingkungan terhadap bentuk fasial penderita sindroma down. indonesian journal of dentistry 2006; kppikg xiv: 124-7 11. fiske j, dickinson c, boyle c, rafique s, burke m. special care dentistry. quint essentials 2007; 16: 43-54. 12. lee sr, kwon hk, song kb, choi yh. dental caries and salivary immunoglobulin a in down’s syndrom children. j pediatric child health 2004; 40(9-10): 530-3. 13. suharsini m. perawatan gigi dan mulut pada anak retardasi mental. jurnal kedokteran gigi 2000; 7: 146-50. 14. chaushu s, yefenol e, becker a. severe impairment of secretory ig production in parotid saliva of down syndrome individual. j dent res 2002; 81(5): 308-12. 15. cogulu d, sabah e. evaluation of the relationship between caries indices and salivary secretory iga, salivary ph, buffering capacity and flow rate in children with down syndrom. archives of oral biology 2005; 51(1): 23-8. 16. cameron ac, widmer rp. handbook of pediatric dentistry. 3rd ed. toronto: mosby; 2008. p. 154. 17. pinkham jr, casamassimo ps, fields hw, mc tigue dj, novak aj. pediatric dentistry: infancy through adolescence. 4th ed. st louis: elsevier saunders; 2005. p. 64, 266-7,420. 83rosdiana and rizal: the relation between salivary siga level 18. dessai ss, fayeteville ny. down syndrome, a review of literature. j oral surg oral med oral path 1997; 84: 279-85. 19. siquera wl, nicolau j. stimulated whole saliva component in children with down syndrome. spec care dent 2002; 22(6): 22630. 20. handajani j. penggunaan pasta gigi ekstrak etanolik teh (caellia sinensis) dan pasta gigi epigallocatechin gallate ekstrak teh terhadap kadar siga saliva pasien penderita gingivitis. maj ked gigi 2009; 16(1): 25-30. 21. baratawidjaja kg. imunologi dasar. edisi tiga. jakarta: penerbit fakultas kedokteran universitas indonesia; 2004. p. 22-33. 22. mayer g. immunoglobulins structure and function. available from: http://pathmicro.med.sc.edu/mayer/igstruct2000.htm. accessed october 21, 2012. 23. iona ml. dental problems in people with down’s syndrome. available from: http://www.intellectualdisability.info/physicalhealth/dental-problems-in-people-with-downs-syndrome. accessed june 6, 2012. 24. vigna apd, gregio am, machado ma, azevedo lr. saliva composition and function. a comprehensive review. j contemp dent pract 2008; 9(3): 72-80. 25. cvetkovic a, ivonic m. the role of streptococcus mutans group and salivary immunoglobulins in etiology of early childhood caries. serbian dental j 2006; 53: 113-23. 26. deepa c, parkash chand, vishnu bhat b, negi vs, ramachandra rk. serum immunoglobulin levels and lower respiratory tract infectionsin children with down syndrome. curr pediatr res 2012; 16(1): 53-6. 27. jafarzadeh a, hassanshashi gh. comparison of salivary iga and ige levels in children with breast and formula feeding during infancy period. dent res j 2007; 4: 11-7. 28. timmons b. exercise and immune function in children. am j lifestyle med 2007; 1: 59-22. 29. mount gj, hume wr. preservation and restoration tooth structure. 2nd ed. queensland: knowledge books and software; 2005. p. 22-4, 111-8. 30. wang b, kuramitsu hk. a pleitropic regulator, effects polysaccharide synthesis, biofilm formation and competence development in s. mutans. infect and immune j 2006; 74(8): 4581-9. 31. noguiera rd, alves ac, napimoga mh. characterization of salivary immunoglobulin a responses in children heavily exposed to the oral bacterium streptococcus mutans: influence of specific antigen recognition in infection. infect immun 2005; 73: 5975-684. 32. c h ia js, l ien h t, h sueh pr. i nduct ion of cy tok i ne s by glikosyltransferases of streptococcus mutans. clin and diag lab immun 2002; 9(4): 892-7. 33. matsumoto m, fujita k. binding of glucan-binding protein c to gtfd-synthesized soluble glucan in sucrose-dependent adhesion os streptococcus mutans. j oral microbiol and immun 2006; 21: 42-6. 34. koo hyun, xiaou jin, klein mi. extracelluler polysacharides matrixan often forgotten virulence factors in oral biofilm research. in j oral sci 2009; 1(4): 229-34. 35. nakona k, nomura r. role of glucose side chains with serotypespecific polysaccharide in the cariogenicity of s. mutans. j caries res 2005; 39: 262-8. 36. pecarcki d, petersen c. involvement of antigen i/ii surface protein in streptococcus mutans and streptococcus intermedius biofilm formation. j oral microbiol and immune 2005; 20: 366-71. 37. smith dj. caries vaccines for the 21th century. j dent edu 2003; 67: 87-92. 38. sikorska m, mielnik-blaszczak m, kapec e. the relationship between the levels of siga, lactoferin and 1 proeteinase inhibitor in saliva and permanent dentition caries in 15-years-old. oral microbiol immunol 2002; 17: 272-6. 39. lundin ml, ericson d. salivary iga reaction to cell surface antigens of oral streptococci. oral microbiol immunol 2004; 19: 188-98. 40. walker dm. oral mucosa immunology: an overview. ann acad med sing 2004; 33: 27-30. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 121121 research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 121–126 fgf-2 expression and the amount of fibroblast in the incised wounds of rattus norvegicus rats induced with mauli banana (musa acuminata) stem extract didit aspriyanto,1 intan nirwana,2 and hendrik setia budi3 1department of oral biology, faculty of dentistry, universitas lambung mangkurat, banjarmasin indonesia 2department of dental material, faculty of dental medicine, universitas airlangga, surabaya-indonesia 3department of oral biology, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: traditional wound treatment using herbal medicine is thought to maintain the health of families and society in general economically, effectively, and efficiently without inducing side effects. one genus of plant that can be used as a traditional medicine is the mauli banana, indigenous to south borneo. mauli banana stem contains bioactive compounds, most of which are tannins along with ascorbic acid, saponin, β-carotene, flavonoids, lycopene, alkaloids, and flavonoids. tanin has antibacterial and antioxidant effects at low concentrations, as wells as antifungal ones at high concentrations. purpose: this study aimed to analyze the effects of mauli banana stem extract at concentrations of 25%, 37.5%, and 50% on the quality of incised wound healing in male rattus norvegicus rats by assessing fgf-2 expression and fibroblast concentration on days 3 and 7. methods: this research represented an experimental laboratory-based investigation involving 32 rats of the rattus norvegicus strain aged 2-2.5 months old. sampling was performed using a simple random sampling technique since the research population was considered homogeneous and divided into 8 treatment groups (c3, m3-25, m3-37.5, m3-50, c7, m7-25, m7-37.5, m7-50). the rats in each group were anesthetized before their back was incised with length and width of 15x15mm with a depth of 2mm. gel hydroxy propyl cellulose medium (hpmc) was applied to the incised wound of each rat in the control group, while stem mauli banana extract was applied to that of each rat in the treatment groups three times a day at an interval of 6-8 hours. on day 3, four rats from each group were sacrificed, while, in the remaining groups, the same procedure was performed until day 7, at which point they (8 groups) were sacrificed for he examination in order to assess the amount of fibroblast and for ihc examination to examine fgf-2 expression. data regarding fgf-2 expression and the amount of fibroblast were analysed by means of one-way anova and hsd. results: the results showed that the mauli banana stem extract could significantly improve the expression of fgf-2 and the amount of fibroblast cells compared to c3 and c7 groups. the highest increase in fgf-2 expression and fibroblast amount were found in all groups on days 3 and 7 treated with the mauli banana stem extract at a concentration of 50%. conclusion: there was an increase of fgf-2 expression and the amount of fibroblast cells in the incision wound healing process that induced with mauli banana stem extract. keywords: fgf-2; fibroblast cells; wound healing; mauli banana stem correspondence: intan nirwana, department of dental material, faculty of dental medicine, univesitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: intannirwana@gmail.com introduction the body responds to the presence of a foreign object. when the cells and tissues of the body experience damage due to an invasive agent, it will respond by repairing the tissue. the healing process represents tissue response to the wound which serves to restore the original structure. 1 wound healing constitutes a complex process of continuous interaction among cells as well as between cells and matrix summed up in three overlapping phases. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 mailto:intannirwana@gmail.com 122 aspriyanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 121–126 the three phases of the wound healing process are the inflammatory (0-3 days), proliferative and kult tissue formation (3-14 days), and remodeling phases.2 moreover, optimal wound healing will be achieved if complications in the form of deficiency or redundancy of wound healing components, especially collagen and epithelial cells, do not occur. the deficiency or redundancy of these components can be detected more clearly in the remodeling phase. another form of wound healing complication is that of excessive wound contraction.3–5 indonesia is rich in natural medicinal herbs that have traditionally been used to treat wounds. the long-standing treatment of wounds with medicinal plants is expected to be utilized by society to maintain the health of families and communities cheaply, effectively, and efficiently with minimal side effects. nevertheless, advances in knowledge and modern technology are expected to go hand-in-hand with the role of traditional medicine. as a result, in order to achieve those objectives, the government has recently promoted a return to natural treatment.6 one type of plant that can be used as in traditional medicine is the mauli banana, a typical banana from south kalimantan.7 evidence strongly suggests that mauli banana stems have historically been used as a form of wound healing medicine in the hulu sungai utara area of south kalimantan province.8 the mauli banana stem contains numerous bioactive compounds, the most common of which is tannin. tanin not only demonstrates antibacterial and antioxidant qualities at low concentrations, but also antifungal ones at high concentrations. other bioactive compounds contained include ascorbic acid, saponins, β-carotene, flavonoids, lycopene, alkaloids, and flavonoids. saponin serves as an antibacterial agent and painkiller, as well as stimulating the formation of new skin cells. meanwhile, flavonoids have antifungal, antioxidant, antiallergic, anti-inflammatory, anti-carcinogenic antithrombic, and hepatoprotective functions, while also stimulating fibroblast formation.9,10 materials and method this research constitutes an experimental laboratory study (truly experimental in character) using a post-test only control group design and was approved by the commission of health research ethics (kkepk) of the faculty of dental medicine, universitas airlangga, surabaya number: 203/kkepk.fkg/xi/2015. the 32 samples were male rattus norvegicus rats aged 2-2.5 months old and weighing 250-300 grams, which had never previously been used for research purposes, and were in a healthy condition without any anatomical abnormalities. the 25%, 37.5%, and 50% mauli banana stem extract was made into a gel using carbopol material, hydroxy propyl cellulose medium (hpmc) and propylenglycol. the carbopol material was mixed with water and then adjusted before propilenglikol was added. the hpmc was put into the first mixture. mauli banana stem extract was gradually added to the mixture until it reached a gel type-consistency. sampling was performed by means of a simple random sampling technique since the research population was considered to be homogeneous. after one week, certain rats were selected randomly and anesthetized, before their backs were incised using cardboard boxes to a length and width of 15 x 15 mm and a depth of 2 mm.5 thereafter, hpmc gel was applied to the incised wound of each rat in the control group on day 3 (c3) and the control group on day 7 (c7), while mauli banana stem extract gel was applied to the incised wound area of each rat in the treatment groups. the concentrations applied comprised: 25% mauli banana extract on day 3 (m3-25), 37.5% extract on day 3 (m3-37.5), 50% extract on day 3 (m3-50), 25% extract on day 7 (m7-25), 37.5% extract on day 7 (m7-37.5), 50% extract on day 7 (m7-50) three times a day at intervals of 6-8 hours. these rats were then placed into two cages which had respectively been labeled control group c3, c7 and treatment groups m3-25, m3-37.5, m3-50, m7-25, m7-37.5, m7-50. during the research, all the rats received equal treatment, being exposed to sunlight for 12 hours during the day, but not at night. they were also provided with sufficient food in the form of pellets. on day 3, four rats from each group c3, m3-25, m3-37.5, m3-50 were sacrificed. meanwhile, in the other treatment groups, c7, m7-25, m7-37.5, m7-50 the same process was performed up to day 7, when these rats were sacrificed. the tissue around the incision area was subsequently cut off, before each specimen was fixed with a formalin buffer. immuno histo chemistry (ihc) examination was then performed to assess the degree of fgf-2 expression and hematoksilin eosin (he) examination was conducted to assess the fibroblast count. results table 1. the mean and standard deviation of fgf-2 expression in the c3, m3-25, m3-37.5, m3-50 and c7, m7-25, m7-37.5, m7-50 groups n mean ± sd c3 4 3.25 ± 1.258 c7 4 6.00 ± 2.160 m3-25 4 8.50 ± 1.915 m7-25 4 15.25 ± 2.062 m3-37.5 4 8.50 ± 2.082 m7-37.5 4 15.50 ± 2.082 m3-50 4 14.75 ± 1.708 m7-50 4 18.50 ± 3.109 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 123123aspriyanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 121–126 table 2. the significance values of fgf-2 expression between treatment groups on days 3 and 7 group c3 c7 m3-25 m7-25 m3-37.5 m7-37.5 m3-50 m7-50 c3 0.077 0.002* 0.000* 0.002* 0.000* 0.000* 0.000* c7 0.106 0.000* 0.000* 0.000* 0.000* 0.000* m3-25 0.000* 1.000 0.000* 0.000* 0.000* m7-25 0.000* 0.868 0.740 0.039* m3-37.5 0.000* 0.000* 0.000* m7-37.5 0.619 0.055 m3-50 0.019* m7-50 description: * significant at α = 0.05 5 p1 k1 p2 p3 p3 p1 p2 k1 figure 1. fgf-2 expression in the control and treatment groups on day 3. note: c3 : fgf-2 expression in the control group on day 3 m3-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 3 m3-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 3 m3-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 3 figure 2. fgf-2 expression in the control and the treatment groups at day 7. note: c7 : fgf-2 expression in the control group on day 7 m7-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 7 c3 m3-25 m3-37.5 m3-50 c7 m7-25 m7-37.5 m7-50 figure 1. fgf-2 expression in the control and treatment groups on day 3. note: c3 : fgf-2 expression in the control group on day 3 m3-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 3 m3-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 3 m3-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 3 5 p1 k1 p2 p3 p3 p1 p2 k1 figure 1. fgf-2 expression in the control and treatment groups on day 3. note: c3 : fgf-2 expression in the control group on day 3 m3-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 3 m3-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 3 m3-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 3 figure 2. fgf-2 expression in the control and the treatment groups at day 7. note: c7 : fgf-2 expression in the control group on day 7 m7-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 7 c3 m3-25 m3-37.5 m3-50 c7 m7-25 m7-37.5 m7-50 figure 2. fgf-2 expression in the control and the treatment groups on day 7. note: c7 : fgf-2 expression in the control group on day 7 m7-25 : fgf-2 expression in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5: fgf-2 expression in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : fgf-2 expression in the treatment group with 50% mauli banana stem extract on day 7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 124 aspriyanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 121–126 discussion mauli banana (musa acuminata) stem extract contains the same substances as other bananas, such as terpenoid saponin. the ambon banana (musa paradisiaca var sapientum), for instance, also contains bioactive triterpenoid saponin which is known to accelerate the wound healing process in skin.11 terpenoid saponin contained in mauli banana stem extract is considered to be an immunostimulator that can increase activity and the number of macrophages. terpenoid saponins will also be captured by g protein receptors in macrophages. then, through a process of generating protein kinase c, it will activate nuclear factor kappa beta (nfkβ), thereby increasing the activity and number of macrophages.12 according to research conducted by apriasari in 2015, mauli banana extract gel table 4. the significance values of fibroblasts numbers between treatment groups on days 3 and 7 group c3 c7 m3-25 m7-25 m3-37.5 m7-37.5 m3-50 m7-50 c3 0.001* 0.002* 0.000* 0.000* 0.000* 0.000* 0.000* c7 0.887 0.016* 0.073* 0.000* 0.000* 0.000* m3-25 0.011* 0.055* 0.000* 0.000* 0.000* m7-25 0.478* 0.006* 0.008* 0.000* m3-37.5 0.001* 0.001* 0.000* m7-37.5 0.887 0.001* m3-50 0.001* m7-50 description: * significant at α = 0.05 table 3. the mean and standard deviation of fibroblast amount in the control and treatment groups on days 3 and 7 groups n mean ± sd c3 4 8.00 ± 2.582 c7 4 14.25 ± 2.217 m3-25 4 14.00 ± 2.582 m7-25 4 18.75 ± 1.708 m3-37.5 4 17.50 ± 3.000 m7-37.5 4 24.00 ± 2.944 m3-50 4 23.75 ± 2.217 m7-50 4 30.50 ± 2.082 7 k1 p1 p2 p3 figure 3. photographs of the he examination of the number of fibroblasts on day 3 (400x magnification). note: c3 : amount of fibroblasts cells in the control group on day 3. m3-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 3. m3-37.5: amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 3. m3-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 3. figure 4. photographs of the he examination of the number of fibroblasts on day 7 (400x magnification). note: c7 : amount of fibroblasts cells in the control group on day 7 m7-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5 : amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 7 k1 p1 p2 p3 c3 m3-25 m3-37.5 m3-50 c7 m7-25 m7-37.5 m7-50 figure 3. photographs of the he examination of the number of fibroblasts on day 3 (400x magnification). note: c3 : amount of fibroblasts cells in the control group on day 3. m3-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 3. m3-37.5: amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 3. m3-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 125125aspriyanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 121–126 at a concentration of 25% can accelerate the healing process in incised wounds on the oral mucosa of rats by increasing the number of macrophages on day 3, and subsequently decreasing them on the fifth day.13 macrophages play an important role in the wound healing process since they produce growth factors as well as trigger angiogenesis and fibro genesis. the excreted macrophages can even phagocytize bacteria and clean out tissue debris. during the transition from inflammatory processes to wound repair, macrophages can stimulate cell migration, proliferation, and tissue matrix formation. growth factors involved in angiogenesis are transforming growth factor-β (tgf-β), vascular endothelial growth factor (vegf), and fibroblast growth factors-2 (fgf-2).14,15 based on the contents of table 1, the highest mean number of fgf-2 expressions was found in the mauli banana stem extract of m3-50, namely 14.75 on the 3rd day and 18.5 on the 7th day. meanwhile, the lowest mean fgf-2 expressions were found in the control group, namely 3.25 on day 3 and 6.0 on day 7. according to the results of the research that was conducted on the third day, there was a significant increase in fgf-2 expression between the m3-25, m3-37.5, m3-50 groups treated with mauli banana stems compared to that of the c3 group. the result of the research conducted on the seventh day confirmed the increase in fgf-2 between the groups given m7-25, m7-37.5% and m7-50% extracts compared to that of c7 group. group extract of mauli banana m7-25 compared with group of extract of mauli banana m7-37.5 happened almost the same increase of fgf-2 whereas with group of extract of mauli banana m7-50 happened increase of fgf-2. group extract of mauli banana m7-37.5 showed result of increase of fgf-2 which almost equal to concentration of mauli banana m7-50 banana extract. for all treatment groups on the seven days that were compared there was an increase in fgf-2 compared to the control group. it can be said that giving mauli banana stem gelatin extract can increase fgf-2 expression compared with the control group on both the third and seventh days. the immunohistochemical results also showed that the highest increase in fgf-2 expressions was found in the treatment group administered mauli banana stem extract at a concentration of 50% since the higher the concentration of mauli banana stem extract, the greater the amount of tannin substances with anti-inflammatory properties contained. these include: saponin, alkaloids, flavonoids, lycopene, ascorbic acid, and β-carotene that work synergistically to suppress or decrease pro-inflammatory cytokines, thereby accelerating the wound healing process. the mauli banana stem extract containing terpenoid saponin has the same mechanism as the astragalus plant. the mauli banana is able to increase the proliferation and migration of fibroblasts and, similar to asiaticoside plants containing terpenoid saponins, can enhance collagen synthesis, granulation tissue formation, and wound contraction.16 previous research has also demonstrated that mauli banana extract gel has the potential to form high quality wound contractions in the oral mucosal of rats.9 the results of the research conducted on the third day confirmed an increase in the amount of fibroblast cells between the extracted groups m3-25, m3-37.5 and m3-50 compared to the c3 group. group of mauli banana extract m3-25 compared with mauli banana extract group m3-37.5 happened increase of amount of cells of fibroblas which almost same but still low when compared with mauli banana figure 4. photographs of the he examination of the number of fibroblasts on day 7 (400x magnification). note: c7 : amount of fibroblasts cells in the control group on day 7 m7-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5 : amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 7 7 k1 p1 p2 p3 figure 3. photographs of the he examination of the number of fibroblasts on day 3 (400x magnification). note: c3 : amount of fibroblasts cells in the control group on day 3. m3-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 3. m3-37.5: amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 3. m3-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 3. figure 4. photographs of the he examination of the number of fibroblasts on day 7 (400x magnification). note: c7 : amount of fibroblasts cells in the control group on day 7 m7-25 : amount of fibroblasts cells in the treatment group with 25% mauli banana stem extract on day 7 m7-37.5 : amount of fibroblasts cells in the treatment group with 37.5% mauli banana stem extract on day 7 m7-50 : amount of fibroblasts cells in the treatment group with 50% mauli banana stem extract on day 7 k1 p1 p2 p3 c3 m3-25 m3-37.5 m3-50 c7 m7-25 m7-37.5 m7-50 c7 m7-37.5 m7-50 m7-25 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 126 aspriyanto, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 121–126 extract m3-50. similar increase between concentration of m2-25 and m3-37.5 due on the third day is still not expected to show optimization of mauli banana extract the results of the research carried out on the seventh day confirmed an increase of the number of fibroblast cells between the extracted group m7-25, m7-37.5 and m7-50 compared to the control group. with regard to the m7-25 mauli banana extracts group, there was an increase in the amount of fibroblast cells which was similar to the m7-37.5 mauli banana extract, but still lower when compared with the m7-50 mauli banana extract group. the administration of mauli banana stem extract gel to the m7-25, m7-37.5, and m7-50 could significantly improve the amount of fibroblasts compared to that of c7. the results of this study indicated that the highest level of fibroblast occurred in the treatment group treated with mauli banana stem extract at a concentration of m7-50. previous research has even shown that plants containing anti-oxidants potentially play a role as immunomodulators, which produce immunostimulator and immunosuppressant effects triggered by the concentration level.17 in addition, the results of banana leaf extract study with he examination revealed that the highest amount of fibroblast occurred in the treatment group treated with mauli banana stem extract at a concentration of m7-50. in conclusion, there was an increase in fgf-2 expression and the number of fibroblast cells in the incision wound healing process that induced with mauli banana stem extract. references 1. kumar v, abbas ak, fausto n, robbins sl, cotran rs. robbins and cotran pathologic basis of disease. 7th ed. philadelphia: elsevier saunders; 2005. p. 1525. 2. reddy gak, priyanka b, saranya cs, kumar cka. wound healing potential of indian medicinal plants. int j pharm rev res. 2012; 2(2): 75–87. 3. peterson lj, ellis e, hupp jr, tucker mr. contemporary oral and maxillofacial surgery. 4th ed. st louis-missouri: mosby; 2003. p. 776. 4. nayak bs, sandiford s, maxwell a. evaluation of the wound-healing activity of ethanolic extract of morinda citrifolia l. leaf. evidencebased complement altern med. 2009; 6(3): 351–6. 5. granick ms, gamelli rl. surgical wound healing and management. new york: informa healthcare; 2007. p. 165. 6. wijayakusuma hmh. tanaman berkhasiat obat di indonesia. jakarta: pustaka kartini; 1996. p. 8-15. 7. sari sg, badruzsaufari b. hubungan kekerabatan fenetik beberapa varietas pisang lokal kalimantan selatan. j penelit sains. 2013; 16(1): 33–6. 8. apriasari ml, carabelly an, andini gt. ekstrak metanol batang pisang mauli (musa sp.) dosis 125-1000 mg/kg bb tidak menimbulkan efek toksik pada hati mencit (mus musculus). j dentofasial. 2013; 12(2): 81–5. 9. apriasari ml, iskandar i, suhartono e. bioactive compound and antioxidant activity of methanol extract mauli bananas (musa sp) stem. int j biosci biochem bioinforma. 2014; 4(2): 110–5. 10. apriasari ml, carabelly an, aprilia gf. efektivitas ekstrak metanol batang pisang mauli 100% pada penyembuhan luka punggung mencit (mus musculus) ditinjau dari jumlah sel radang. j dentofasial. 2014; 13(1): 33–7. 11. prasetyo bf, wientarsih i, pontjo b. aktifitas sediaan salep ekstrak batang pohon pisang ambon (musa paradisiaca var sapientum) dalam proses penyembuhan luka pada mencit (mus musculus albnus). majalah obat tradisional. 2010; 15(3): 121–37. 12. besung ink. pegagan (cantella asiatica) sebagai alter natif pencegahan penyakit infeksi pada ternak. bul vet udayana. 2009; 1(2): 61–7. 13. apriasari ml, endariantari a, oktaviyanti ik. the effect of 25% mauli banana stem extract gel to increase the epithel thickness of wound healing process in oral mucosa. dent j (maj ked gigi). 2015; 48(3): 150. 14. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219–29. 15. soni h, singhai ak. a recent update of botanicals for wound healing activity. int res j pharm. 2012; 3(7): 1–7. 16. tsala de, amadou d, habtemariam s. natural wound healing and bioactive natural products. phytopharmacology. 2013; 4(3): 532–60. 17. mukherjee pk, nema nk, bhadra s, mukherjee d, braga fc, matsabisa mg. immunomodulatory leads from medicinal plants. indian j tradit knowl. 2014; 13(2): 235–56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p121-126 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p121-126 77 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 7–12 case report interdisciplinary management of class iii malocclusion with cleft lip and palate retno iswati1, cendrawasih andusyana farmasyanti2, aulia ayub1,2, anne marie kuijpers-jagtman3,4,5, ananto ali alhasyimi2 1orthodontic resident, faculty of dentistry, universitas gadjah mada (ugm), yogyakarta, indonesia 2department of orthodontics, faculty of dentistry, universitas gadjah mada (ugm), yogyakarta, indonesia 3department of orthodontics, university medical centre groningen (umcg), groningen, the netherlands 4department of orthodontics and dentofacial orthopedics, school of dental medicine/medical faculty, university of bern, bern, switzerland 5faculty of dentistry, universitas indonesia, jakarta, indonesia abstract background: a cleft lip and palate (clp) is one of the most common birth defects of the face. individuals with clp often have a significant growth disturbance of the maxilla along three dimensions, resulting in skeletal class iii malocclusion and cross bite. oral rehabilitation can be complicated. purpose: the purpose of this case study was to highlight the necessity of sequential interdisciplinary management to improve facial esthetics and correct functional disturbances for a patient with clp. case: the patient was a 20-year-old woman complaining of the unpleasant appearance of her upper front teeth. she had a concave profile with class iii skeletal patterns (sna: 78o; snb: 82o; anb: -4o), cleft lip and palate, and an anterior and posterior crossbite. case management: a combined orthodontic, endodontic, conservative, periodontic, and prosthetic approach was proposed to achieve normal occlusion, function, and a harmonious profile. the combination of rapid maxillary expansion and fixed orthodontics (standard edgewise appliance) established good general alignment and a class i relationship. after 15 months of treatment, both the posterior and anterior crossbite had been completely corrected. in order to address the gingival margin differences, the patient was instructed to make another appointment with the periodontist and was referred to the restorative dentist for veneer restorations and the prosthodontist for fabrication of a removable retainer with obturator. conclusion: this interdisciplinary approach greatly improved both esthetics and function. the patient was satisfied with the results achieved. keywords: class iii skeletal malocclusion; cleft lip and palate; orthodontics article history: received 14 july 2022; revised 19 august 2022; accepted 22 september 2022 correspondence: ananto ali alhasyimi, department of orthodontic, faculty of dentistry, universitas gadjah mada. jl denta sekip utara, sinduadi, mlati, sleman, special region of yogyakarta 55281, indonesia. email: anantoali@ugm.ac.id introduction cleft lip and palate (clp) is a common type of congenital orofacial malformation which is characterized by varying degrees of deficient and displaced orofacial tissues, including soft tissue, musculature, bone, and cartilage.1 according to the world health organization (who), the prevalence of cleft lip and palate depends on the country, ranging anywhere between 3.4 and 22.9 out of every 10,000 births. it has been found that the rate of occurrence varies by ethnicity and geographical location, with the highest incidence seen in asian populations (0.84–4.04 per 1,000 live births), medium incidence in caucasians (0.9–2.69 per 1,000 live births), and the lowest incidence in african populations (0.18–1.67 per 1,000 live births).2 in indonesia, of all orofacial cleft types, cleft lip and palate is the most frequent (50.5%), followed by cleft lip (24.4%), and cleft palate (25.1%).3 there are multiple genes contributing to the etiology of clp. conte et al. reported 45 genes for deletions and 27 for duplications, including several known causative genes for orofacial clefts in humans, such as special at-rich sequence-binding protein 2 and meis homeobox 2, as well as 12 other genes that are associated with clefts. further, they identified a number of deletions and duplications in genes not previously reported. however, knowledge on the genetic background is still limited.4 individuals with clp are born with dentofacial deformity and may experience functional issues such as copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 mailto:anantoali@ugm.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 8 iswati et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 7–12 feeding difficulties, speech problems, respiration pattern alterations, and recurrent middle ear infections that affect their quality of life.5 a variety of professions are involved in the treatment. speech therapy is frequently needed to treat cleft-related muscle abnormalities at the time of speech development. disturbed facial growth and dental development leading to malocclusion necessitate dental and occasionally surgical care as the individual grows and matures.6 dental and cosmetic impairments such as midfacial deficiencies, crossbites, abnormalities, asymmetries of the soft tissues, and extraoral and intraoral soft-tissue scarring, as well as eating and communication difficulties are common in patients with these conditions. in many cases, lip and palate repair procedures have a deleterious impact on maxillary growth and development, resulting in a narrow maxillary arch and maxillary sagittal insufficiency. consequently, anterior and posterior crossbites and a reduction in the maxillary arch’s perimeter are usually noticed, and maxillary expansion is frequently necessary.7 comprehensive orthodontic treatment is needed for patients with clp to provide the best possible results in terms of dental occlusion and facial esthetics. it has also been noted that patients with cleft conditions are not only at a high risk of caries, but they also have a higher prevalence of caries than people who do not have cleft conditions.8 thus, the final treatment outcome for a clp patient is highly dependent on a multidisciplinary team approach.9 this case report highlights the importance of such an approach for the effective treatment of a class iii malocclusion in a clp patient in order to improve the patient’s overall functional, structural, and esthetic outcomes. case an indonesian female (aged 20 years 2 months) came to prof. soedomo at universitas gadjah mada dental hospital for orthodontic purposes. her chief complaint was her anterior crossbite which gives her face an unesthetic appearance. she was born with a nonsyndromic complete bilateral cleft lip, alveolus, and palate (cbclap). primary cheiloplasty and palatoplasty were performed when she was one year old. she did not undergo any additional orthodontic therapy or alveolar bone grafting procedure after this. both alveolar clefts were still open, the soft palate was partly closed, and there was an anterior palatal fistula present. the patient’s profile was concave, and her upper lip was retracted and lower lip everted. she had a mesocephalic head type and a hypereuriprosopic facial type. she showed an imbalance among the facial thirds (figure 1), and functional analysis revealed that the free-way space was normal (2.4 mm) with no evidence of temporomandibular disorder. intraoral examination revealed an angle class iii relationship bilaterally with a negative overjet up to –2 mm. due to a combination of factors, including the initial cleft and the previous surgical scarring in the palate, the a b c d figure 1. pre-treatment (a, b) and post-treatment (c, d) extraoral photographs. frontal photographs of spontaneous smile (upper) and profile view (lower). copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 9iswati et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 7–12 a b c figure 2. intraoral photographs: pre-treatment (a), during orthodontic treatment with fixed appliances (b), and after orthodontic treatment and prosthetic rehabilitation (c). a b c d figure 3. pre-treatment lateral cephalogram (a), post-treatment lateral cephalogram (b), pre-treatment panoramic radiograph (c), post-treatment panoramic radiograph (d). table 1. lateral cephalometric measurements parameters normal (mean ± sd) pre-treatment post-treatment horizontal skeletal sna (o) 82 ± 2 78 78 snb (o) 80 ± 2 82 82 anb (o) 2 ± 2 –4 –4 wits appraisal (mm) 1 ± 1 –2.35 –2.25 angle of convexity (o) 0 ± 5 –7 –4 vertical skeletal y-axis (o) 60 ± 4 58 59 sn-mandibular plane (o) 32 ± 3 29 29 mmpa (o) 27 ± 5 26 28 lafh (%) 55 ± 2 52 55 dental interincisal angle (o) 135 ± 10 128 133 u1-palatal plane (o) 109 ± 6 106 112 u1-na (mm) 4 ± 2 1 4 l1-mandibular plane (o) 90 ± 4 91 88 l1-nb (mm) 4 ± 2 6 4 soft tissue upper lip to e-line (mm) 1 ± 2 –6 –1 lower lip to e-line (mm) 0 ± 2 3 0 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 10 iswati et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 7–12 maxillary arch was unable to maintain its normal shape and had collapsed and narrowed. moderate crowding in the maxillary arch (arch length discrepancy −3.96 mm) was noted, whereas the mandibular arch showed slight anterior crowding (arch length discrepancy −0.92 mm). poor oral hygiene was present, as evidenced by bleeding in brushing, particularly in the posterior region (figure 2). the cephalometric findings revealed a skeletal class iii malocclusion (anb, –4o; wits appraisal, –2.35 mm) with a normal vertical facial growth pattern (sn-mp, 32 o). the maxillary incisors were extremely retroclined (u1-palatal plane, 106o; u1-na, 1 mm), whilst the mandibular incisors were slightly proclined but still within the normal range (l1-mp, 91o; l1-nb, 6 mm). the interincisal angle was 128º. rickett’s lip analysis indicated a retrusive upper lip and protrusive lower lip (table 1; figure 3). the panoramic radiograph indicated an absence of the maxillary left and right lateral incisors, an absence of the germ of teeth #38 and #48, radix second premolars (teeth #15 and #25), radix upper right canine (#13), gangrene in #37 and #46, and pulp necrosis in #11. all other permanent teeth were present (figure 3). case management the treatment’s objectives were to harmonize the facial profile through the expansion of the maxillary arch, which would correct the anterior and posterior crossbite as well as the maxillary transverse deficiency, levelling and aligning the dental arches and establishing a good interdigitation with enhanced intercuspation. a treatment plan was suggested as follows: (1) extraction of radix second premolars (teeth #15 and #25) and gangrenous teeth #37 and #46; (2) root canal treatment (rct) for #11; (3) restoration of all caries; (4) alignment of the upper and lower teeth with edgewise appliance, 0.022” slot; (5) retention using upper and lower a b c figure 4. occlusal view of the maxilla pre-treatment (a), during orthodontic treatment (b), and after orthodontic treatment and prosthetic rehabilitation (c). figure 5. superimposition of the pre(blue) and posttreatment (black) cephalometric tracings. note there were changes in maxillary and mandibular incisor inclination, and also in the lip position. removable retainers followed by prosthetic rehabilitation. the periodontist, general dentist, endodontist, restorative dentist, and prosthodontist were also involved in the planning. due to financial limitations, no additional surgical interventions were planned. after extraction of the upper radix premolars and gangrenous teeth, rct, and restoration, orthodontic treatment began with a slow maxillary expander. because rapid, heavy, intermittent forces created by a screw-type rapid palatal expansion device have the potential to cause tissue damage, a slow maxillary expansion was conducted to generate slow, suitable, continuous stresses. during the process of expansion, the maxilla, palatal mucosa, and dentition were tightly controlled for any difficulties that might arise, such as the enlargement of the palatal fistula or tipping of teeth. after a period of four months, an expansion of four millimeters was achieved. following expansion, a fixed standard edgewise appliance with a 0.022-inch slot (marquise, orthotech, usa) was bonded to the maxillary and mandibular teeth in order to begin the process of aligning the dental arches, while keeping the expansion appliance in place to maintain the width of the maxillary arch. the first step was processing alignment and leveling with 0.012”, 0.014”, 0.016” and 0.016 x 0.016-in stainless steel arch wires. the second stage, using a stainless steel 0.016 x 0.22-in arch wire, involved clockwise backward and downward rotation of the mandible while employing class iii intermaxillary elastics. this was done in order to enhance the maxillomandibular skeletal relationship in the sagittal dimension and to increase the lower anterior copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 11iswati et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 7–12 provide dental health education on the clp condition using motivational techniques as well as explain how the treatment plan will be carried out and how to deal with poor oral hygiene. the patient’s motivation and cooperation is essential in attaining these goals, and the patient must sign an informed consent acknowledging receipt of good information and agreeing to the course of treatment, in this case, the endodontist then performed treatment of tooth #11, and orthodontic treatment started using slow maxillary expansion (sme), which was performed until the interpremolar and intermolar width were acceptable. severe constriction in the posterior and anterior segments of the maxillary arch associated with the cleft lip and palate requires transverse expansion to achieve a better interocclusal relationship. the use of sme as a potential therapy modality for transverse deficiency in patients with clefts seems encouraging. here, a fixed palatal expander was used since there is a possibility that expansion would cause damage to the tissues in the mouth. this was also done in order to generate appropriate and continuous stresses.12 although there is little doubt that an expander appliance is effective in cleft patients, the question remains as to whether expansion is stable in the long term. patients with clp at a mean age of 30 years exhibited satisfactory stability in upper inter-canine and upper/lower inter-molar widths after expansion with an expander appliance for a period of 10 years.13 another study following 75 patients with a complete unilateral clp until five years posttreatment showed that the transverse relationship deteriorated further if maxillary expansion was performed during treatment than in patients without expansion.14 however, more long-term studies are needed to assess the stability of this approach. after the use of sme to achieve the ideal arch within four months, definitive orthodontic treatment can be initiated with fixed appliances. the aim of this phase is correction of the malrelationship and malposition of individual teeth and to align the dental arch to achieve good occlusion. the duration of definitive orthodontic treatment varies depending on the severity of the case and the degree of patient cooperation. in this case, the treatment was completed within 15 months, the posterior and anterior crossbite having been completely resolved. definitive orthodontic treatment should be carried out until all teeth have good interdigitation contact to improve stabilization, there is sufficient space for preparation of prosthetic placement, and a satisfactory patient profile has been achieved.15,16 in the final phase of treatment, in order to obtain optimal treatment results, patients are referred to various dental professionals such as a conservative dentistry specialist, periodontist, and prosthodontist. in this instance, the conservative dentistry specialist carried out dental veneer restoration of tooth #11 to achieve a more esthetic and stable result. the periodontist had performed a gingivectomy and gingivoplasty to correct the gingival margin discrepancies, and the prosthodontist had fabricated removable dentures to replace the upper lateral incisors. in this case, the face height. this rotation contributes significantly toward the development of an improved anterior overjet position. after 11 months of treatment, both the posterior and anterior crossbite had completely disappeared. the final step was finishing and settling of the occlusion using a 0.017 x 0.025-in stainless steel arch wire. subsequently, the patient was instructed to return to the periodontist in order to have the gingival margin inconsistencies corrected, to the conservative dentistry specialist for veneer restorations, and to the prosthodontist for fabrication of a removable maxillary denture. after 24 months of orthodontic treatment in total, the appliances were debonded. in the maxillary arch, a hawley retainer with two lateral incisors was used for esthetic reasons and to maintain tooth position and arch width. in the lower arch, a removable retainer was used to stabilize the position of the teeth and to replace tooth #46 (figure 4). soft tissue analysis indicated the position of the upper lip was more forward and upward while the lower lip was more backward by the end of treatment (figure 5). an improved profile, an ideal overjet and overbite, and class i relationship were established, a clockwise mandibular rotation was noted, and the inclination of the upper and lower incisors was within normal limits (figures 1c, 1d, 2c, and 5). discussion orthodontic management of clp patients requires a multidisciplinary approach. in the presented case, a multidisciplinary treatment involving orthodontic, endodontic, and prosthetic management was proposed to achieve a normal function, occlusion, and balanced profile. to enhance the patient’s quality of life, the treatment’s primary objective was to achieve a more functional and esthetically pleasing facial profile and dentition. the patient had a complex clp problem list. the width of the maxillary basal arch was exceptionally restricted. a deep curve of spee could be seen in the mandibular arch, and a class iii relationship was observed. the oral hygiene of the patient was poor, assessed by the presence of debris and calculus on the teeth, bleeding on probing of the gingiva, radix relicta, deep carious lesions, and pulp necrosis. the role of orthodontic treatment is important for the management of patients with dental arch discrepancy in clp conditions.4 there are several stages in this treatment that must be carried out. firstly, people with an orofacial cleft are more likely to have gingivitis and calculus than non-cleft patients. these issues may be caused by a lack of physical ability, which makes brushing teeth difficult, as well as a lack of knowledge of the necessity of oral health management, communication difficulties, and fear of oral health procedures.10 therefore, patients with an orofacial cleft need to take responsibility for maintaining proper dental hygiene and the long-term health of their teeth.11 at the beginning of treatment, the specialist must copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 12 iswati et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 7–12 partial denture in the maxilla was also able to serve as an orthodontic retainer to maintain the transverse dimensions. the choice of retainer used may vary depending on the situation.17 a recently published, evidence-based, clinical practice guideline on clp recommends maintaining the anterior teeth positions with a fixed retainer, and in addition, using a removable orthodontic retainer, such as a hawley retainer, to preserve the maxillary transverse dimensions.18 such a retainer must be worn on a nightly basis, lifelong.19 a retainer complete with denture and obturator was inserted. if the palate is severely scarred, the blood supply is often compromised, and a re-operation is not therefore advised. moreover, due to the long-term constricting effects of palatal scar tissue, the patient must wear a retainer anyway for the transverse dimensions. in this context, a retainer and denture with an obturator have a two-fold advantage: they retain the transverse dimensions and cover the remaining palatal defects to improve speech and feeding. following treatment, the patient was satisfied with the results, the facial esthetics and profile had improved significantly, and had a good occlusion while keeping a balanced profile. patient was educated to continue to use retainers regularly and to have regular check-ups with an orthodontist at least once every six months to assess the condition of the teeth so that good treatment results can be maintained.16,17 treatment for this patient with class iii malocclusion and cleft lip and palate was a challenge. the multidisciplinary approach resulted in significant improvements to facial and dental esthetics, and function was improved as well. the patient regarded the outcomes as satisfactory. acknowledgement this case report was partially supported by the cleft charity foundation ‘sumbing bibir’ (https://www.sumbingbibir. nl/english). references 1. kosowski tr, weathers wm, wolfswinkel em, ridgway eb. cleft palate. semin plast surg. 2012; 26(4): 164–9. 2. ahmed mk, bui ah, taioli e. epidemiology of cleft lip and palate. in: almasri ma, editor. designing strategies for ceft lip and palate care. london: intech; 2017. p. 1–7. 3. sjamsudin e, maifara d. epidemiology and characteristics of cleft lip and palate and the influence of consanguinity and socioeconomic in west java, indonesia: a five-year retrospective study. int j oral maxillofac surg. 2017; 46(suppl 1): 69. 4. conte f, oti m, dixon j, carels cel, rubini m, zhou h. systematic analysis of copy number variants of a large cohort of orofacial cleft patients identifies candidate genes for orofacial clefts. hum genet. 2016; 135(1): 41–59. 5. moreira hsb, machado ra, aquino sn de, rangel alca, martelli júnior h, della coletta r. epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana. brazilian j oral sci. 2016; 15(1): 39–44. 6. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10(1): 7. 7. pugliese f, palomo jm, calil lr, de medeiros alves a, lauris jrp, garib d. dental arch size and shape after maxillary expansion in bilateral complete cleft palate: a comparison of three expander designs. angle orthod. 2020; 90(2): 233–8. 8. grewcock re, innes npt, mossey pa, robertson md. caries in children with and without orofacial clefting: a systematic review and meta-analysis. oral dis. 2022; 28(5): 1400–11. 9. parsaei y, uribe f, steinbacher d. orthodontics for unilateral and bilateral cleft deformities. oral maxillofac surg clin north am. 2020; 32(2): 297–307. 10. shivakumar km, patil s, kadashetti v, raje v. oral health status and dental treatment needs of 5-12-year-old children with disabilities attending special schools in western maharashtra, india. int j appl basic med res. 2018; 8(1): 24–9. 11. nagappan n, john j. periodontal status among patients with cleft lip (cl), cleft palate (cp) and cleft lip, alveolus and palate (clap) in chennai, india. a comparative study. j clin diagn res. 2015; 9(3): zc53-5. 12. perillo l, vitale m, d’apuzzo f, isola g, nucera r, matarese g. interdisciplinary approach for a patient with unilateral cleft lip and palate. am j orthod dentofacial orthop. 2018; 153(6): 883–94. 13. jain s, shrivastav s, jain dn. maxillary expansion in cleft lip and palate casesa review. int j adv res. 2015; 3(9): 1455–61. 14. sumardi s, latief bs, kuijpers-jagtman am, ongkosuwito em, bronkhorst em, kuijpers mar. long-term follow-up of mandibular dental arch changes in patients with complete nonsyndromic unilateral cleft lip, alveolus, and palate. peerj. 2021; 9: e12643. 15. maeda-iino a, nakagawa s, nakamura n, miyawaki s. an adolescent patient with bilateral cleft lip and palate treated with late bone grafting and edgewise appliance for mesial movement of the maxillary molars and improvement of the retroclined maxillary central incisor. ajo-do clin companion. 2021; 1(1): 55–72. 16. veloso nc, mordente cm, de sousa aa, palomo jm, yatabe m, oliveira dd, souki bq, andrade i. three-dimensional nasal septum and maxillary changes following rapid maxillary expansion in patients with cleft lip and palate. angle orthod. 2020; 90(5): 672–9. 17. kim j-k, kim j-y, jung h-d, jung y-s. surgical-orthodontic treatment for severe malocclusion in a patient with osteopetrosis and bilateral cleft lip and palate. angle orthod. 2021; 91(4): 555–63. 18. elabbassy eh, sabet ne, hassan it, elghoul dh, elkassaby ma. bone-anchored maxillary protraction in patients with unilateral cleft lip and palate. angle orthod. 2020; 90(4): 539–47. 19. mink van der molen ab, van breugel jmm, janssen ng, admiraal rjc, van adrichem lna, bierenbroodspot f, bittermann d, van den boogaard m-jh, broos ph, dijkstra-putkamer jjm, van gemertschriks mcm, kortlever alj, mouës-vink cm, swanenburg de veye hfn, van tol-verbeek n, vermeij-keers c, de wilde h, kuijpers-jagtman am. clinical practice guidelines on the treatment of patients with cleft lip, alveolus, and palate: an executive summary. j clin med. 2021; 10(21): 4813. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p7–12 https://www.sumbingbibir https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p7-12 7676 management of chronic traumatic ulcer mimicking oral squamous cell carcinoma on the tongue revi nelonda and riani setiadhi department of oral medicine, faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: traumatic ulcers represent the most common oral mucosal lesions that can be differentiated from oral squamous cell carcinoma (oscc) by their clinical appearance. from a clinical perspective, oscc may resemble a chronic traumatic ulcer (ctu) because the base of the ctu that is healing is filled with reddish-pink granulated tissue, similar to that in oscc. purpose: the aim of this case report is to provide information about the oral management of a ctu case that imitates oscc. case: a 30-year old female presented with a major, painful, non-healing ulcer located on the right lateral of the tongue for the previous two months. approximately two years before, she had experienced a similar lesion on the tongue. intra oral examination showed a 10 mm x 5 mm yellowish ulcer with a fibrous center, erythematous irregular-induration margin and concave yellow base. the 15, 44 and 47 teeth were sharp and on occlusion caused trauma to the right lateral border of the tongue. case management: based on the clinical features, the lesion was imitating oscc. after a case history review, clinical examination and appropriate investigation, the patient was diagnosed as suffering from a chronic traumatic ulcer. the primary treatment of traumatic ulcers involves eliminating etiological factors. as pharmacological therapy, a mixture of triamcinolone acetonide and 1 mg dexamethasone tablet was administered in addition to folic acid and vitamin b12. conclusion: clinical presentation of traumatic lesions varies significantly and may, at times, be ambigous. it is important to immediately establish a correct diagnosis and implement prompt treatment of ctu lesions because they play a role at the oral carcinogenesis promotion stage. keywords: management; chronic traumatic ulcer; oral squamous cell carcinoma correspondence: revi nelonda, department of oral medicine, faculty of dentistry, universitas padjadjaran, jl. sekeloa selatan no.1, bandung 40132, indonesia. e-mail: revinelonda@gmail.com; riani.setiadhi@fkg.unpad.ac.id dental journal (majalah kedokteran gigi) 2018 june; 51(2): 76–80 case report introduction oral mucosa can feature numerous lesions resulting from chronic mechanical irritation caused by either teeth or dentures. the most common lesion is a chronic traumatic ulcer.1 a traumatic ulcer is usually a single lesion with erythematous, irregular margins and a clean base covered with a pseudo-membrane. usually painful, they occur due to a bite or trauma from sharp teeth or ill-fitting dentures.2 injury of the oral mucosa may result from physical, chemical and/or thermal injury possibly originating from an accidental bite to the inside of the cheek, sharp edged food, sharp edge of teeth or dentures, excessively hot food or overzealous brushing of the teeth.2 oral traumatic lesions are divided into acute and chronic varieties. clinical presentation of traumatic lesions varies significantly and the cause and the effect can usually be established by thorough case history compilation and clinical examination.3 a chronic traumatic ulcer (ctu) within the oral cavity is a relatively frequent lesion that usually develops in the posterior or middle third of the edge of the tongue or in the posterior third of the occlusal axis of the cheek mucosa.4 a chronic, yet painless, traumatic ulcer which sometimes develops with a hardened base and raised border is an etiologic agent in oral carcinogenesis, although this issue remains controversial to the present.5 moreover, ctu may clinically resemble an oral squamous cell carcinoma (oscc) because the base of the healing dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p76-80 77 nelonda and setiadhi/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 76–80 ctu may be filled with reddish-pink granulation tissue, similar to an oscc.6 oscc is the most common persistent ulcer affecting the oral cavity which encompasses 90-95% of oral cancers. usually painless, located on the tongue, especially on the lateral posterior border, early lesions are often asymptomatic6,7 – a fact that can cause the patient to delay seeking treatment.8 an early oscc lesion may be a shallow ulcer with a velvety red base and a firm, raised border. a healing chronic traumatic ulcer can resemble an early oscc lesion because its base may be filled with reddish-pink granulation tissue.6 a number of studies have revealed a connection between persistent inflammation and cancer through the overexpression of immune evasion and proliferation-regulating genes. for this reason, chronic mechanical irritation may also play a role in provoking a continuous inflammatory state.1 the management of ctu is largely based on eliminating all factors underlying the injury. needless to say, prompt diagnosis and elimination of the causative factors is expected to promote the healing process.9 this article reports the case of a tongue ulcer which was mimicking malignancy, but after extraction, followed by grinding the sharp teeth and pharmacological treatment, healed completely. case a 30-year old female attended the oral medicine departement of dr. hasan sadikin hospital with a painful, non-healing ulcer on the right lateral of the tongue that had been present for two months and gradually increasing in the size. the patient had experienced and recovered from the same medical condition two years before. she was not suffering from any systemic disease and demonstrated no undesirable habits such as chewing or smoking tobacco or consuming alcohol. this patient also complained about swelling of the right mandible gingiva causing difficulty when eating. on the first visit, (2nd october, 2017) the right submandibular lymph nodes were palpable, tender, firm and mobile during extra oral examination. this examination confirmed the presence of a 10 mm x 5 mm yellowish ulcer with a fibrous center, erythematous irregular-induration margin and yellow, concave base. the ulcer was on the lateral of the right tongue, parallel to the second molar mandibular. the 15 and 44 teeth were sharp and on occlusion causing trauma to the right lateral of the tongue. the base of the tongue and throat were normal and no other intra oral lesion was detected (figure 1). case management treatment for the patient included grinding the cups of the right maxillar second premolar and right mandibular first premolar teeth improving the patient’s nutritional intake by giving multivitamins, as well as antiseptic mouthwash and topical corticosteroid (a mixture of 0.1% triamcinolone acetonide in orabase and 1 mg dexamethasone tablet) which was prescribed for the inflammation. laboratory tests including an 8 parameter blood test, anti igg hsv-1, ig e and plasma glucose tests were conducted to eliminate the possible causes of ulcer due to viruses and allergens. the patient was referred to the oral surgery department for extraction of the right mandibular second molar in order to eliminate the predisposing factor. two weeks later (16th october 2017), these severity had been reduced. intra oral examination showed a yellowish ulcer with a fibrous center, an erythematous irregularinduration margin and a yellow concave base with slight figure 1. intra oral finding on the first visit : a 10x5 millimeter yellowish ulcer with a fibrous center, erythematous irregular-induration margin and concave yellow base (blue arrow). figure 2. slight improvement in the lesions found during the first visit at a 2-week-follow-up (blue arrow). figure 3. improvement in the lesions at the two-weekfollow-up (blue arrow). figure 1. intra oral finding on the first visit : a 10 x 5 millimeter yellowish ulcer with a fibrous center, erythematous irregular-induration margin and concave yellow base (blue arrow). figure 1. intra oral finding on the first visit : a 10x5 millimeter yellowish ulcer with a fibrous center, erythematous irregular-induration margin and concave yellow base (blue arrow). figure 2. slight improvement in the lesions found during the first visit at a 2-week-follow-up (blue arrow). figure 3. improvement in the lesions at the two-weekfollow-up (blue arrow). figure 2. slight improvement in the lesions found during the first visit at a 2-week-follow-up (blue arrow). figure 1. intra oral finding on the first visit : a 10x5 millimeter yellowish ulcer with a fibrous center, erythematous irregular-induration margin and concave yellow base (blue arrow). figure 2. slight improvement in the lesions found during the first visit at a 2-week-follow-up (blue arrow). figure 3. improvement in the lesions at the two-weekfollow-up (blue arrow). figure 3. improvement in the lesions at the two week followup (blue arrow). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p76-80 78nelonda and setiadhi/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 76–80 improvement (figure 2). laboratory investigation revealed a 38.10 (reactive) anti igg hsv-1. at this point, the patient was referred to the oncology department for a biopsy to eliminate suspected risk factors of the possibility of oscc, but she refused for psychological reasons. 15 days later, (20th november 2017) the lesion on the right lateral of the tongue gradually resolved (figure 3). there was no pain or inhibited chewing. at the time of writing, the patient is still under observation without any signs of relapse. discussion traumatic ulcers can result from physical, thermal or chemical injuries. injuries self-induced by the patient may be caused by accidental biting while talking, sleeping or chewing. accidental biting during mastication or rough food may cause acute traumatic ulceration. such ulcers generally heal within a few days without complications. however, chronic trauma caused by the sharp edges of teeth, restorations and appliances particularly ill-fitting dentures, may cause chronic ulcers. in newborns and infants trauma can be due to natal teeth (riga-fede syndrome)10 although the majority of such injuries are unintentional, self-inflicted injuries can also frequently occur.9,11–13 the aim of our case study is to provide information regarding the oral management of traumatic ulcer cases that mimicking oscc. the most common symptom of oscc is a non-healing sore or ulcer, while other potential symptoms including: pain, numbness, a persistent lump or thickened area, a persistent red or white patch, dysphagia, a sore throat or the sensation of something being "caught" in the throat. clinically, oscc appears as a mixed white or reddish proliferative growth-like lesion with raised margins and an ulcerated surface with yellowish greyish pseudomembranous tissue, particularly on the lateral and ventral of the tongue.14,15 oscc also demonstrates other clinical characteristics such as exophytic (outward growing) or endophytic (inward growing), leukoplakic, erythroplakic all of which show visible changes to the surface.3,7 oscc is characterized by firmness on palpation which can be a helpful diagnostic clue. upon identification and biopsy of a suspicious lesion, an oral or general pathologist will diagnose the oscc lesion by means of microscopic examination. a ctu appears as a single ulcer indicating loss of continuity of epithelial tissue with the base covered by yellowish-white pseudomembranous tissue which is firm on palpation. patients with ctu complain of tenderness or pain in the area of the lesion and the traumatic agent/factors can usually be readily identified.6,16 at the beginning, based on clinical features, the lesion in this case report was mimicking oscc (a white ulcer with yellowish greyish pseudomembranous tissue). however, after reviewing the case history (a painful ulcer for two months), clinical examination and appropriate investigation (the absence of induration during palpation, the ridges of the 15 and 44 teeth figure 4. diagram of the management of chronic traumatic ulcers in this case. the main management of ctu involves eliminating etiologic factor as non-pharmacological and using topical corticosteroid as pharmacological therapy. chronic traumatic ulcer: single ulcer with concave base with yellowish-white pseudomembranous cover, raised borders and firm on palpation dental factors prosthetic factors functional factors non-pharmacological: eliminating etiologic factor oral hygiene instruction follow up pharmacological: corticosteroid topical antiseptic mouthwash multivitamins non-healing, painless ulcer that does not respond to treatment induration-lack of inflammation around the ulcer with a rolled, thickened edge suspicion to a malignancy figure 4. diagram of the management of chronic traumatic ulcers in this case. the main management of ctu involves eliminating etiologic factor as non-pharmacological and using topical corticosteroid as pharmacological therapy. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p76-80 79 nelonda and setiadhi/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 76–80 being sharp), the patient was diagnosed as suffering from a chronic traumatic ulcer. the lesions showed significant improvement after the sharp teeth had been ground. the main treatment for chronic traumatic ulcer consists of eliminating the etiological factors. there are three contributory factors of chronic mechanical irritation that can cause ctu and oscc, namely: dental factors (dental malposition, diastema, sharp/jagged teeth and/or restoration, jagged teeth), prosthetic factors (sharp/rough dentures, denture retainers, overextended flanges, lack of retention and/or stability) and functional factors (tongue interposition, sucking, biting and parafunctional habits).1 from the anamnesa and clinical examination, the etiology factors were identified as sharp 15, 44 and 47 teeth as well as parafunctional factors such as the interposition of the tongue because of the missing 16, 14, 24, 26, 36, 45 and 46 teeth. these factors lead to the formation of ulcers due to their constant contact with the right lateral of the tongue. there are several stages of treatment that can be undertaken as planning such oral hygiene instruction (avoid coarse, hot and spicy food), correction of sharp cusps of the 15 and 44 teeth, multivitamins, topical corticosteroid application, antibacterial mouth wash and encouraging the patient to continue follow up.2 it was decided to grind the 15 and 44 teeth and to extract the 47 tooth in order to eliminate the etiology because continuous contact with the cause of trauma can cause delayed healing of the ulcer.17,18 topical corticosteroid (tc) plays a central role in the treatment of ulcerative oral mucosal lesions, but the evidence for efficacy of tc in oral medicine remains limited.19 the anti-inflammatory and immunosuppressive properties of topical corticosteroid is ideal for the management of specific immune mediated oral ulcerative conditions.20 the potency, frequency and vehicle of application of the topical steroid should be tailored to each individual case and subsequent response to treatment.21 0.1% triamcinolone acetonide was often used as the drug of choice. as pharmacological therapy, an unguentum mixture consisting of 0.1% triamcinolone acetonide in orabase and a 1mg dexamethasone tablet applied directly to the lesion three times a day in order to reduce both healing time and the size of lesions was employed. major challenges become more problematic as there are very few commercial products currently available for the topical treatment of the oral mucosal lesion. as the oral mucosal is constantly bathed in saliva, extremely mobile and highly permeable due to mostly non-keratinized epithelial tissue, adherent vehicles and aqueous solutions are among the most widely used.22 persistent ulcers such as ctu will heal in approximately two to four weeks after the removal of causative factors and topical corticosteroid therapy.6 0.1% triamcinolone acetonide in orabase that can adhere effectively to the dorsum of the tongue was mixed with a 1mg dexamethasone tablet because dexamethasone is a very powerful topical corticosteroid used in order to shorten healing time and reduce the size of the lesion. after 45 days, the ctu in this case resolved and healed on removal of the causative factor with scar formation dependent upon the extent of damage.16 persistent ulcers that did not respond to this therapy were candidates for biopsy. this patient was asked to undergo a biopsy in the oncology surgery department, but refused for psychological reasons. the ulcer showed significant improvement after the sharp teeth had been ground. malignancy should be suspected in the following cases: the ulcer is shallow with a velvety red base and firm raised border; a non-healing, painless ulcer has been present for more than three weeks after the elimination of predisposing factors or the ulcer has a rolled thickened edge and shows a lack of surrounding inflammation or local factors. the ulcer in this case showed significant improvement after the sharp teeth had been ground. therefore, malignancy was excluded. after the lesion on the tongue had healed, in order to avoid self-biting of the mucosa, the patient was referred to a prosthodontics department for a dental prosthesis. it is important to immediately establish a proper diagnosis and initiate prompt treatment of ctu lesions because they play a role at the stage of promotion of oral carcinogenesis. in this case, in order to facilitate interpretation, an algorithm of the management of chronic trumatic ulcers has been provided in figure 4. it can be concluded that clinical presentation of traumatic lesion varies significantly and may at times be ambigous. an accurate diagnosis will be obtained through appropriate review of the case history, clinical examination and investigation. if the ulcer persists two weeks after the eradicating of etiological factors, the conducting of anatomy histopatological examinations such as a punch biopsy, microbrush, and excision and/or incision biopsy are important in order to eliminate suspected potential risk factors of oscc. the main management of chronic traumatic ulcer imitating oral squamous cell carcinoma consists of eliminating etiological factors. as a pharmocological therapy, topical corticosteroid and multivitamins can reduce the size of lesions and shorten healing time. references 1. l a z o s j p, p iem o nt e e d, l a n f r a n ch i h e , br u no t t o m n. characterization of chronic mechanical irritation in oral cancer. int j dent. 2017; 2017: 1–7. 2. ishaquddin s, maya d, ghadage m. traumatic ulcer or squamous cell carcinoma of the t ongue? : case report. int j healthc biomed res. 2013; 2: 57–60. 3. nalin as, mary j, leukose t, sreedhar s, padiath s. traumatic ulcer – mimicking squamous cell carcinoma. iosr j dent med sci. 2016; 15(3): 83–6. 4. pérez ma, raimondi ar, itoiz me. an experimental model to demonstrate the carcinogenic action of oral chronic traumatic ulcer. j oral pathol med. 2005; 34: 17–22. 5. bombeccari gp, guzzi g, pallotti f, porrini m, giannì ab, spadari f. large oral ulcer of tongue related to dental trauma. stomatol balt dent maxillofac j. 2017; 19: 51–4. 6. nidarsh h, mithra h, aastha p, raksha b. differential diagnosis of long term tongue ulcers. int res j pharm. 2012; 3(8): 145–8. 7. hirota sk, migliari da, sugaya nn. oral squamous cell carcinoma dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p76-80 80nelonda and setiadhi/dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 76–80 in a young patient case report and literature review. an bras dermatol. 2006; 81(3): 251–4. 8. rao rs, patil s, ganavi bs. current updates on early detection and prevention of oral cancer. j med radiol pathol surg. 2015; 1: 1–2. 9. anura a. traumatic oral mucosal lesions: a mini review and clinical update. oral health dent manag. 2014; 13(2): 254–9. 10. parisay i, ghafournia m, shafagh m, mousavi sa. lingual traumatic ulceration (riga-fede disease): report of a case and review. j dent mater tech. 2013; 2(4): 142–7. 11. sunil a, kurien j, mukunda a, basheer a bin, deepthi d. common superficial tongue lesions. indian j clin pract. 2013; 23(9): 534– 42. 12. pereira t, shetty s, pereira s. sheep in wolf’s clothing – enigma unravelled. oral surg oral med oral pathol oral radiol. 2015; 3: 6–8. 13. bhavthankar jd, patil aa, singh p, mandale m, humbe j. malignant vs traumatic tongue ulcer : a clinical approach. j evol med dent sci. 2014; 3(14): 3758–63. 14. siu a, landon k, ramos dm. differential diagnosis and management of oral ulcers. semin cutan med surg. 2015; 34(4): 171–7. 15. apriasari ml. the management of chronic traumatic ulcer in oral cavity. dent j (maj ked gigi). 2012; 45(2): 68–72. 16. walsh t, liu lyj, brocklehurst p, glenny a, lingen m, kerr ar, ogden g, warnakulasuriya s, scully c. clinical assessment to screen for the detection of oral cavity cancer and potentially malignant disorders in apparently healthy adults. cochrane database syst rev. 2013; (11): 1–70. 17. ford pj, farah cs. early detection and diagnosis of oral cancer: strategies for improvement. j cancer policy. 2013; 1(1–2): e2–7. 18. tyagi n, tyagi r. squamous cell carcinoma (well differentiated): a case report. j dent oral hyg. 2013; 5(4): 31–4. 19. gonzález-moles má. the use of topical corticoids in oral pathology. med oral patol oral cir bucal. 2010; 15(6): 827–31. 20. savage nw, mccullough mj. topical corticosteroids in dental practice. aust dent j. 2005; 50(4 suppl 2): s40–4. 21. meddipour m, zenouz at. role of corticosteroids in oral lesion. intech. 2016; 6: 111–33. 22. gonzález-moles má, scully c. vesiculo-erosive oral mucosal disease — management with topical corticosteroids : (1) fundamental principles and specific agents available. j dent res. 2005; 84(4): 294–301. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p76-80 107 volume 46 number 2 june 2013 research report identifikasi bite marks dengan ekstraksi dna metode chelex (bite marks identification with chelex methods in dna extraction) imelda kristina sutrisno, ira arundina dan agung sosiawan departemen biologi oral fakultas kedokteran gigi universitas airlangga surabaya-indonesia abstract background: in the case of crime often encountered evidence in bite marks form that was found on the victim’s body. generally, bitemarks identification use standard techniques that compare the interpretation picture with the tooth model of suspected person. however, sometimes the techniques do not obtain accurate results. therefore another technique is needed to support the identification process,such as dna analysis that use the remaining epithelium attached in saliva to identify the dna of the suspected person. in this processes a limited dna material could be met, not only less in quantity but also less in quality. chelex known as one of an effective dna extraction method in dna forensic case is needed to overcome this problem. purpose: the study was aimed to examine the use of chelex as dna extraction method on a bitemarks sample models. methods: the blood and bitemarks of 5 persons with were taken. the dna of each subject was exctracted with chelex and quantified the quantity with uv spechtrophotometer. the dna results was amplified by pcr at locus vwa and th01 then vizualised by electrophoresis. results: the electrophoresis’s results showed band at locus vwa and th01 for blood sample and bite marks with no significant differences. conclusion: the study showed that chelex method could be use to extract dna from bitemarks. key words: bite marks, chelex, locus vwa and th01 abstrak latar belakang: dalam kasus kejahatan sering dijumpai bukti dalam bentuk bekas gigitan (bitemarks) yang ditemukan pada tubuh korban. umumnya, untuk mengidentifikasi bite marks menggunakan teknik standar yaitu membandingkan foto interpretasi dengan model gigi dari orang yang dicurigai. namun demikian teknik ini terkadang tidak mendapatkan hasil yang akurat, sehingga diperlukan teknik lain untuk menunjang keberhasilan proses identifikasi pelaku, yakni melalui analisis dna bitemarks, yang diperoleh dari saliva yang mengandung sisa epitel tersangka pelaku. sampel dna yang berasal dari bitemarks umumnya terbatas, tidak hanya terbatas dalam kuantitas tetapi juga terbatas dalam kualitas. hal ini seringkali menimbulkan kesulitan tersendiri dalam proses analisisnya. chelex yang dikenal sebagai salah satu metode ekstraksi yang efektif di bidang forensik, sangat diperlukan untuk mengatasi kendala tersebut . tujuan: penelitian ini bertujuan untuk meneliti penggunaan metode ekstraksi dna metode chelex pada sampel bite marks. metode: darah dan cetakan gigi dari 5 subjek diambil, dan dna di ekstraks dengan chelex dan kemudian diuji kuantitas dengan uv spechtrophotometer. setelah itu hasil diamplifikasi dengan pcr pada lokus vwa dan th01 kemudian divisualisasi dengan elektroforesis. hasil: hasil elektroforesis menunjukkan adanya band pada lokus vwa dan th01 untuk sampel darah dan cetakan gigi tanpa perbedaan yang signifikan secara statistika. simpulan: penelitian ini menunjukkan bahwa metode chelex dapat digunakan untuk mengekstraksi dna dari bite marks. kata kunci: bite marks, chelex, lokus vwa and th01 korespondensi (correspondence): imelda kristina sutrisno, departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: imel_lie92@hotmail.com 108 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 107–112 pendahuluan indonesia merupakan negara dengan tingkat kriminalitas yang cukup tinggi, berbagai macam tindakan kriminal sering terjadi seperti pembunuhan, pencurian, kekerasan, pemerkosaan dan lain sebagainya yang meninggalkan barang bukti berupa bite marks. proses identifikasi tersangka dengan bukti berupa bite marks umumnya dilakukan dengan membandingkan hasil foto interpretasi dengan model gigi dari tersangka yang dicurigai, meliputi analisa dan pengukuran ukuran, bentuk dan posisi dari masing-masing gigi.1 namun hasil identifikasi dengan teknik ini belum didapatkan hasil yang akurat. oleh karena itu perlu cara lain dalam mengidentifikasi bite marks yaitu dengan teknik irigasi pada bite marks kemudian dilakukan identifikasi dna. pada bite marks pemeriksaan dna dapat diambil dari saliva, stain yang menempel, sisa-sisa epitel mukosa pada saliva dan sebagainya.2 dna sebagai alat bantu identifikasi tidak lepas dari kelemahan misalnya saja kerusakan dna karena ada paparan dari lingkunngan seperti ph, temperatur dan lain sebagainya. oleh karena itu perlu penanganan yang tepat dan cepat dalam mengolah sampel salah satunya terkait pada proses ekstraksi. selain itu tidak jarang ditemui dna yang dijumlah dan kualitasnya terbatas sehingga perlu metode ekstraksi dna yang efektif dan efisien seperti metode chelex.3 kelebihan metode ini yaitu cepat, tahapan singkat serta mengurangi kemungkinan sample to sample contamination.3 larutan chelex yang digunakan terdiri dari styrene divinylbenzene copolymer yang berisi pasangan ion-ion iminodiacetate yang bertindak sebagai chelating group yang berikatan dengan ion mg2+, bila chelating resin ini terlarut pada hasil ekstraksi dna sehingga mengikat komponen mg 2+ sebagai kofaktor enzim dna polymerase, akibatnya tanpa ion ini enzim polymerase pada pcr tidak dapat bekerja.4 lokus pada dna yang digunakan dalam penilitan ini adalah tyrosine hidroxylase 1st intron (th01) dan von willebrand factor, 40th intron (vwa) dikarenakan dua jenis lokus ini termasuk lokus yamg memiliki tingkat mutasi yang rendah dari tiga belas lokus short tandem repeat (str) yang telah ditetapkan oleh federal bureau of investigation (fbi) sebagai sebuah sistem identifikasi dna forensik nasional.5 penelitian ini bertujuan untuk meneliti penggunaan metode ekstraksi dna metode chelex pada sampel bite marks. bahan dan metode jenis penelitian ini adalah observasional analitik dan dilaksanakan di laboratorium human genetic – institute of tropical disease (itd) universitas airlangga surabaya. penelitian ini telah mendapatkan ethical clearance dari komisi kelaikan etik penelitian kesehatan (kkepk) fakultas kedokteran gigi universitas airlangga (no. 45/kkepk.fkg/vi/2012). bahan yang digunakan adalah sampel bite marks dan sampel darah dari 5 orang relawan. pemilihan subyek penelitian dilakukan secara acak tanpa kriteria tertentu serta telah menandatangani inform concont. sampel bite marks didapatkan dengan mencetak gigi dengan alginat kemudian diirigasi dengan aquades steril (nuclease free water) menggunakan syringe 3 cc lalu dikumpulkan dalam tabung steril sampai 15 cc. sampel darah diambil pada masing-masing orang sebanyak 3 cc dari vena cubiti dan digunakan sebagai kontrol. sampel darah atau irigasi saliva dari bite marks diambil 500 μl kemudian ditambahkan 1000μl aqua bidestilata steril. setelah itu dilakukan centrifuge (himac scr 20b, hitachi) dengan kecepatan 1500-2000 rpm selama 10 menit. ambil bagian supernatant dari hasil centrifuge lalu dibuang kemudian ditambahkan 1000 μl aquades pada sisa pellet di tabung ependorf, kemudian dilakukan centrifuge dengan kecepatan 12000 rpm selama 10 menit, ambil bagian supernatant lalu dibuang setelah itu ditambahkan larutan chelex 20% sebanyak 500 μl seteleh itu di lakukan vortex, dipanaskan 560 c selama 20 menit setelah itu dilakukan vortex kemudian dipanaskan kembali dengan suhu 100° c selama 8 menit lalu ditunggu sampai dingin. setelah dengin dilakukan centrifuge lagi dengan kecepatan 12000 rpm selama 5 menit, kemudian dilakukan pengenceran dan dihitung kadar dna dengan uv specthrophotometer (uv-visible spectrophotometer, shimatzu). uji kuantitas dna hasil ekstraksi dilakukan dengan alat uv-spectrophotometer pada panjang gelombang 260 nm. menurut formula yang digunakan dalam perhitungan menggunakan alat rna/dna calculator menyatakan bahwa absorbansi λ260 1,0 sesuai untuk 50mg/ ml dna murni untai ganda, maka kadar dna sampel dapat dicari melalui perhitungan berikut:6 [dna] = å260 ×x 50 x faktor pengenceran keterangan: a260 : absorbansi sampel dna pada panjang gelombang 260 nm 50 : larutan dengan nilai absorbansi 1,0 sebanding dengan 50 ug untai ganda dna per ml (dsdna) setelah uji kadar kemudian dilanjutkan dengan optimasi pcr pada masing-masing lokus menggunakan pcr mix dari promega sebagai berikut, pcr mix dntp (atp,ctp,ttp,gtp), mgcl2, taq polimerase dan buffer sebanyak 12,5 μl kemudian ditambah lokus yang akan diperiksa sebanyak 2,5 μl vwa 1 kemudian ditambah 2,5 μl vwa 2 (5’-cctagtggatgataagaataat cagtatg3’5’ggacagatgataaatacatagga tggatgg-3’) ( promega primer, gen bank accession m25858) atau 2,5 μl th01 a kemudian ditambah 2,5 μl th01 b (5’-ctgggcacgtgagggcagcgtct-3’5’tgccggaagtccatcctcacagtc-3’) (promega primer, gen bank accession d00269) setelah itu ditambahkan nuclease free water sebanyak 6,5 μl dan terakhir ditambahkan hasil sampel dna sebanyak 1 μl. hasil akhir reaction volume sebesar 25μl dapat segera 109sutrisno, et al.,: identifikasi bitemark dengan ekstraksi dna metode chelex 10 gambar 1. hasil elektroforesis lokus vwa (sampel 1 dan 2). keterangan: m: marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada gambar terlihat semua band pada lokus vwa terdeteksi. gambar 2. hasil elektroforesis lokus vwa (sampel 3, 4 dan 5). keterangan: m : marker ladder 100 bp; c1: sampel 3 (darah); c2: sampel 3 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks); e1: sampel 5 (darah); e2: sampel 5 (bite marks). pada hasil terlihat band lokus vwa pada sampel bite marks no. 3 tidak terdekteksi. gambar 3. hasil elektroforesis lokus th01 pada sampel 1 dan sampel 4. keterangan: m : marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks). pada hasil terlihta semua band lokus th01 terdeteksi. m a1 a2 d1 d2 179-203 bp 131-171 bp c1 c2 d1 d2 m e1 e2 131-171 bp m a1 a2 b1 b2 m 500 bp 100 bp 500 bp 100 bp 500 bp 100 bp gambar 1. hasil elektroforesis lokus vwa (sampel 1 dan 2). keterangan: m: marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada gambar terlihat semua band pada lokus vwa terdeteksi. tabel 1. kadar dna sampel darah dan bitemark sampel kadar dna sampel darah (ng/μl) kadar dna sampel bitemark (ng/μl) sampel a 3496,5 98,2473 sampel b 1781,5 48,463 sampel c 1942,5 31,5835 sampel d 1204 45,2816 sampel e 1970,5 39,4686 rata-rata 2079 52,6088 dilakukan optimasi pcr. siklus optimasi dilakukan berdasarkan petunjuk dari promega dengan pengulangan siklus denaturation, annealing dan extension sampai kurang lebih 30 siklus.7 hasil amplifikasi pcr (gene amp, pcr system 2400, perkin elmer) selanjutnya divisualisasi dengan gel elektroforesis. proses elektroforesis diawali dengan pembuatan gel polyacrylamide (promega corp, 2010). pembuatan acrylamide 6% didapatkan dari rekasi antara acrylamide: bis acrylamide dengan perbandingan 19:1. setelah gel hampir mengeras diberikan cetakan/comb sebagai tempat sampel dna yang dimasukan pada bagian cetakan di gel sebesar masing-masing 10 μl, sedangkan marker ditempatkan sebanyak 3 μl. running gel elektroforesis dengan listrik 100 volt sampai rsb mencapai dasar. setelah itu dilakukan pengecatan pada gel dengan metode silver staining yang terdapat beberapa tahapan yaitu drying, fixation, staining, developing dan drying.6 hasil elektroforesis kemudian dilakukan visualisasi dengan adanya band di lokus th01 dan vwa pada sampel darah dan bite marks dengan menggunakan elektroforesis (promega corp, 2010). analisa dna dilakukan untuk menguji perbedaan kadar dna antara sampel bite marks dengan sampel darah dengan menggunakan paired t-test. pada hasil elektroforesis elektroforesis perbandingan band terdeteksi atau tidak terdeteksi akan diukur dengan fisher exact probability test.8 hasil kadar dna yang diperoleh dari hasil uji uv spectrophotometer menunjukkan rata-rata kadar dna pada sampel darah memiliki jumlah yang lebih banyak dibandingkan pada sampel bite marks (tabel 1). gambar 1, 2, dan 3 menunjukkan visualisasi hasil elektroforesis. hasil dari penelitian ini kemudian dilakukan analisa dengan statistika. pada perbedaan kadar dna antara sampel darah dengan sampel bite marks dilakukan paired t-test (tabel 2). dari tabel 2 didapatkan hasil p<0,05 sehingga dikatakan terdapat perbedaan yang signifikan antara kadar dna dari sampel darah dengan sampel bite marks. pada hasil elektroforesis di lokus vwa terdapat 1 sampel bite marks yang tidak terdeteksi (tabel 3). sampel yang tidak terdeteksi ini diuji perbedaan dengan menggunakan fisher exact probability test (tabel 4). hasil uji =1,000 lebih besar dari pada α = 0,05. hal ini menunjukkan bahwa tidak terdapat perbedaan yang signifikan antara dna bite marks yang terdeteksi dengan yang tidak terdeteksi. oleh karena itu metode chelex dinyatakan dapat digunakan untuk identifikasi pada sampel bite marks. pembahasan bukti berupa bite marks umumnya diidentifikasi dengan menggunakan teknik standar yaitu membandingkan hasil foto interpretasi dengan model gigi tersangka yang dicurigai meliputi analisa dan pengukuran ukuran, bentuk dan posisi masing-masing gigi,1 namun hasil identifikasi teknik ini tidak dapat dijadikan satu-satunya dasar identifikasi karena adanya kemiripan bentuk sehingga terjadi beberapa kasus kesalahan identifikasi. oleh karena itu digunakan cara lain 500 bp 100 bp 110 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 107–112 10 gambar 1. hasil elektroforesis lokus vwa (sampel 1 dan 2). keterangan: m: marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada gambar terlihat semua band pada lokus vwa terdeteksi. gambar 2. hasil elektroforesis lokus vwa (sampel 3, 4 dan 5). keterangan: m : marker ladder 100 bp; c1: sampel 3 (darah); c2: sampel 3 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks); e1: sampel 5 (darah); e2: sampel 5 (bite marks). pada hasil terlihat band lokus vwa pada sampel bite marks no. 3 tidak terdekteksi. gambar 3. hasil elektroforesis lokus th01 pada sampel 1 dan sampel 4. keterangan: m : marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks). pada hasil terlihta semua band lokus th01 terdeteksi. m a1 a2 d1 d2 179-203 bp 131-171 bp c1 c2 d1 d2 m e1 e2 131-171 bp m a1 a2 b1 b2 m 500 bp 100 bp 500 bp 100 bp 500 bp 100 bp gambar 2. hasil elektroforesis lokus vwa (sampel 3, 4 dan 5). keterangan: m : marker ladder 100 bp; c1: sampel 3 (darah); c2: sampel 3 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks); e1: sampel 5 (darah); e2: sampel 5 (bite marks). pada hasil terlihat band lokus vwa pada sampel bite marks no. 3 tidak terdekteksi. 10 gambar 1. hasil elektroforesis lokus vwa (sampel 1 dan 2). keterangan: m: marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada gambar terlihat semua band pada lokus vwa terdeteksi. gambar 2. hasil elektroforesis lokus vwa (sampel 3, 4 dan 5). keterangan: m : marker ladder 100 bp; c1: sampel 3 (darah); c2: sampel 3 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks); e1: sampel 5 (darah); e2: sampel 5 (bite marks). pada hasil terlihat band lokus vwa pada sampel bite marks no. 3 tidak terdekteksi. gambar 3. hasil elektroforesis lokus th01 pada sampel 1 dan sampel 4. keterangan: m : marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks). pada hasil terlihta semua band lokus th01 terdeteksi. m a1 a2 d1 d2 179-203 bp 131-171 bp c1 c2 d1 d2 m e1 e2 131-171 bp m a1 a2 b1 b2 m 500 bp 100 bp 500 bp 100 bp 500 bp 100 bp gambar 3. hasil elektroforesis lokus th01 pada sampel 1 dan sampel 4. keterangan: m : marker ladder 100 bp; a1: sampel 1 (darah); a2: sampel 1 (bite marks); d1: sampel 4 (darah); d2: sampel 4 (bite marks). pada hasil terlihta semua band lokus th01 terdeteksi. 11 gambar 4. hasil elektroforesis lokus th01 pada sampel 3. keterangan: m : marker ladder 100 bp, c1: sampel 3 (darah), c2 : sampel 3 (bite marks). pada hasil penelitian semua band pada lokus th01 terdeteksi. gambar 5. hasil elektroforesis lokus th01 pada sampel 5 dan sampel 2. keterangan: m : marker ladder 100 bp; e1: sampel 5 (darah); e2: sampel 5 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada hasil penelitian ini semua band pada lokus th01 terdeteksi. m c2 c1 179-203 bp e1 e2 b1 b2 m 179-203 bp 100 bp 500 bp 500 bp 100 bp gambar 4. hasil elektroforesis lokus th01 pada sampel 3. keterangan: m : marker ladder 100 bp, c1: sampel 3 (darah), c2 : sampel 3 (bite marks). pada hasil penelitian semua band pada lokus th01 terdeteksi. 500 bp 100 bp 500 bp 100 bp 500 bp 100 bp 111sutrisno, et al.,: identifikasi bitemark dengan ekstraksi dna metode chelex tabel 2. hasil uji perbedaan dengan paired t-test kelompok sig (2-tailed) dna darahbitemark .001 tabel 3. hasil ekstraksi dna metode chelex pada lokus vwa terdeteksi tidak terdeteksi total sampel darah 5 0 5 sampel bitemark 4 1 5 total 9 1 n = 10 tabel 4. hasil uji fisher exact probability test exact sig (2 sided) fisher’s exact test 1.000 untuk mengidentifikasi yaitu dengan mengekstraksi dna yang terdapat dalam bite marks dengan mengoleksi sampel pada bite marks yaitu dengan dilakukan irigasi pada bekas gigitan nuclease free water. pada bite marks pemeriksaan dna dapat diambil dari saliva, stain yang menempel, sisa-sisa epitel mukosa pada saliva dan sebagainya.2 hal ini dapat dibuktikan saat melakukan uji kuantifikasi dengan uv spechtrophotometer yakni terdapat kandungan dna dalam sampel bite marks yang berdasarkan hasil penelitian memiliki kadar dna rata-rata 52,61 ng/μl. selain sampel bite marks, sampel darah yang merupakan sampel yang umum digunakan untuk identifikasi dna dipilih oleh peneliti sebagai pembanding dari sampel bite marks. sampel darah dipilih menjadi sampel penelitian mengingat darah merupakan sumber pemeriksaan yang efektif. hal ini tidak terlepas dari kondisi darah yang mempunyai kadar dna rata-rata sebesar: 20.000-40.000 ng/μl, sehingga sangat efektif digunakan sebagai bahan atau spesimen pemeriksaan dna di bidang forensik.9 berdasarkan hasil dari penelitian bahwa ekstraksi dna dengan metode chelex ini dapat digunakan untuk ekstraksi dna dari sampel bite marks. hasil uji kuantitasi dna dari sampel darah menunjukkan angka rata-rata 2079 ng/μl sedangkan pada bite marks menunjukkan nilai rata-rata 52,61 ng/μl. hasil ekstraksi dari sampel darah menunjukkan kadar dna yang cukup tinggi sehingga dapat dikatakan ekstraksi chelex pada sampel darah menunjukkan hasil yang cukup bagus untuk selanjutnya diproses dalam amplifikasi dan dapat diidentifikasi dengan elektroforesis, sedangkan pada hasil bite marks menunjukkan angka yang lebih rendah. adanya perbedaan kadar dna pada sampel darah dan sampel bite marks ini tidak menghalangi proses pemeriksaan dna selanjutnya, mengingat teknik pcr masih dapat digunakan untuk menganalisis pada beberapa lokus dengan hasil ekstraksi dna yang berjumlah 1,0 ng, meskipun hasil akhir visualisasi sampel bite marks tidak sebagus dari sampel darah.10 metode yang di pasaran banyak dikenal sebagai chelex ®100 dari bio-rad laboratories ini merupakan suspensi dari sebuah chelating resin yang dapat ditambahkan secara langsung ke dalam sampel atau bahan pemeriksaan seperti halnya darah, bercak darah, atau sperma.11 hal ini mengingat bahwa chelex terdiri dari styrene divinylbenzene copolymer yang berisi pasangan ion-ion iminodiacetate yang bertindak sebagai chelating groups, yang dapat berikatan dengan ion-ion metal polyvalent seperti halnya ion mg2+ dan ca2+. dengan diikatnya ion magnesium dari bahan pemeriksaan yang diambil dnanya, maka enzim yang merusak dna sebagaimana halnya nuclease akan mengalami inaktivasi, sehingga molekul dna dapat terlindungi dan tidak sampai mengalami kerusakan yang berarti.3 metode chelex ini sering digunakan pada sampel yang berupa darah pada kasus forensik. berbagai keuntungan yang dapat diperoleh dari metode ekstraksi 11 gambar 4. hasil elektroforesis lokus th01 pada sampel 3. keterangan: m : marker ladder 100 bp, c1: sampel 3 (darah), c2 : sampel 3 (bite marks). pada hasil penelitian semua band pada lokus th01 terdeteksi. gambar 5. hasil elektroforesis lokus th01 pada sampel 5 dan sampel 2. keterangan: m : marker ladder 100 bp; e1: sampel 5 (darah); e2: sampel 5 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada hasil penelitian ini semua band pada lokus th01 terdeteksi. m c2 c1 179-203 bp e1 e2 b1 b2 m 179-203 bp 100 bp 500 bp 500 bp 100 bp gambar 5. hasil elektroforesis lokus th01 pada sampel 5 dan sampel 2. keterangan: m : marker ladder 100 bp; e1: sampel 5 (darah); e2: sampel 5 (bite marks); b1: sampel 2 (darah); b2: sampel 2 (bite marks). pada hasil penelitian ini semua band pada lokus th01 terdeteksi. 500 bp 100 bp 112 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 107–112 chelex antara lain prosesnya yang lebih cepat, tahapan yang dilakukan lebih sederhana sehingga resiko untuk terkontaminasi karena penggunaan banyak tabung (sample to sample contamination) dapat dihindari. meskipun demikian, metode ekstraksi ini juga tetap memiliki resiko untuk terlarutnya bahan chelating agent yang digunakan dalam metode chelex. bahan chelating agent bersifat mengikat ion-ion mg2+ yang berfungsi sebagai kofaktor dari enzim taq polymerase yang sangat diperlukan pada proses amplifikasi pcr. jika bahan chelating resin masih terdapat pada larutan dna, maka akan mengakibatkan kinerja taq polymerase tidak optimal. selain itu sifat chelating resin atau chelex adalah dapat merusak protein atau protein denaturant.12 padahal enzim taq polymerase itu sendiri adalah sejenis protein yang berfungsi sebagai enzim katalis dalam proses pcr. jika taq polymerase mengalami kerusakan yang disebabkan oleh chelating resin, maka dapat dipastikan bahwa proses pcr tidak akan dapat berlangsung atau berlangsung kurang optimal. hal ini ditandai dengan kegagalan proses elektroforesis, yakni tidak munculnya gambaran band atau pita dari dna yang telah digandakan tersebut. pada penelitian ini digunakan beberapa lokus str untuk pemeriksaan yaitu lokus th01 dan lokus vwa. dari hasil elektroforesis yang didapatkan pada kedua lokus ini tidak ada perbedaan yang berarti dari ketebalan band. kedua lokus ini termasuk lokus yang memiliki tingkat mutasi rendah sehingga hasil identifikasi dna juga lebih akurat. pada lokus th01 seluruh sampel terdeteksi, baik pada sampel dari darah maupun dari bite marks, sedangkan pada lokus vwa dari ekstraksi dna bite marks terdapat 4 sampel terdeteksi sedangkan 1 sampel tidak terdeteksi band pada elektroforesis yaitu pada sampel bite marks nomer 3. pada sampel ini didapatkan kadar dna dari ekstraksi bite marks sebesar 31,58 ng/μl yang menunjukkan bahwa pada bite marks tersebut terdapat dna, namun dna ini tidak terdeteksi pada elektroforesis. faktor yang dapat mempengaruhi terjadinya hal ini antara lain yaitu ikut terlarutnya bahan chelating resin pada saat mengambil supernatant hasil ekstraksi sehingga pcr tidak berjalan efektif akibatnya band yang muncul pada elektroforesis sangat tipis bahkan boleh dikatakan tidak terdeteksi. kualitas dari sampel bite marks sendiri yang telah mengalami penyimpanan selama 1 hari tidak berpengaruh pada tidak terdeteksinya band di elektroforesis. hal ini dapat diketahui berdasarkan hasil penelitian anthonappa et al yang menyatakan bahwa sampel saliva dapat disimpan dalam suhu 37°c sampai 18 bulan tanpa mengalami penurunan kualitas dan kemampuannya untuk dianalisa.13 adanya perbedaan hasil elektroforesis terdeteksi dengan tidak terdeteksi antara lokus th01 dan lokus vwa bukan merupakan suatu perbandingan antara lokus yang lebih baik dengan yang tidak karena keduanya merupakan alat untuk pemeriksaan. hasil penelitian ini menunjukkan bahwa ekstraksi dna chelex dapat digunakan pada sampel bite marks yang kadar dna nya lebih sedikit dibandingkan dengan darah. hal ini sesuai pada penelitian sebelumnya yang dilakukan oleh sweet et al.15 yang juga mendapatkan hasil dari ekstraksi pada sampel bite marks dengan metode chelex. hasil elektroforesis yang didapatkan dari 5 sampel pada lokus yang sama didapatkan 4 sampel yang terdeteksi baik dari sampel darah maupun sampel bite marks, dengan kata lain ekstraksi chelex ini dinyatakan berhasil untuk mendeteksi dna dari bite marks untuk proses identifikasi pada kasus forensik. daftar pustaka 1. van der velden a, spiessens m, willems g. bite mark analysis and comparison using image perception technology. j forensic odontostomatol 2006; 24(1): 14-7. 2. kennedy d. forensic dentistry and microbial analysis of bite marks. new zealand: university of otago; 2011. p. 6-15. 3. butler jm. short tandem repeat analysis for human identity testing. str typing current protocols in human genetic unit. new york: elsevier academic press; 2005. p. 1-37. 4. butler jm. forensic dna typing. united kingdom: elsevier academic press; 2001. p. 13-55. 5. butler jm. genetics and genomics of core short tandem repeat loci used in human identity testing. j forensic sci 2006; 51(2): 25365. 6. fatchiyah, esti la, sri w, sri r. biologi molekularprinsip dasar analisis. jakarta: erlangga; 2011. h. 34-55, 115-26. 7. promega corporation. technical manualgeneprint ® str systems (silver stain detection). us; 2010. p. 5-18. 8. budia r to e. biostatisti ka unt uk kedok tera n da n kesehata n masyarakat. jakarta: egc; 2002. h. 261. 9. butler jm. fundamentals of forensic dna typing. elsevier academic press; 2010. p. 63, 101. 10. frégeau cj, fourney rm. dna typing with fluorescently tagged short tandem repeats: a sensitive and accurate approach to human identification. biotechniques 1993; 15(1): 100-19. 11. walsh ps, metzger d, higuchi r. chelex100 as a medium for simple extrcation of dna for pcr based typing from forensic material. biotechniques 1991; 15: 506-13. 12. schiffner la, bajda ej, prinz m, sebestyen j, shaler r, caragine ta. optimization of a simple, automatable extraction method to recover sufficient dna from low copy number dna samples for generation of short tandem repeat profiles. croat med j 2005; 46(5): 847. 13. anthonappa rp, king nm, rabie ab. evaluation of the long-term storage stability of saliva as a source of human dna. clin oral investig 2013; 17(7): 1719-25. 14. sweet d, lorente m, lorente ja, valenzuela a, villanueva e. an improved method to recover saliva from human skin: the double swab technique. j forensic sci 1997; 42(2): 320-2. 172 roles of secretory leukocyte protease inhibitor amniotic membrane in oral wound healing elly munadziroh department of dental material and technology faculty of dentistry airlangga university surabaya – indonesia abstract secretory leukocyte protease inhibitor (slpi) is serine protease inhibitor. secretory leukocyte protease inhibitor is a protein found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, and cerebral spinal fluid, as in secretions from the nose and bronchi, amniotic fluid and amniotic membrane etc. these findings demonstrate that slpi function as a potent anti protease, anti inflammatory, bactericidal, antifungal, tissue repair, extra cellular synthesis. impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that slpi may have a role in the process. the objectives of this article are to investigate the role of slpi in oral inflammation and how it contributes to tissue repair in oral mucosa. the oral wound healing responses are impaired in the slpi sufficient mice and matrix synthesis and collagen deposition are delayed. this study indicated that slpi is a povital factor necessary for optimal wound healing. key words: secretory leukocyte protease inhibitor, amniotic membrane, oral wound healing correspondence: elly munadziroh, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction secretory leukocyte protease inhibitor (slpi) is a serine protease inhibitor has recently been shown to be capable of a range of immunological functions, including anti-inflammatory and anti-microbial activity. at tissue level, the ability of this ptotein to counteract the excessive degradation of functional and structural proteins such as collagen and fibronectin, as seen in impaired cutaneous wound has led to further inquiry into its degree of involvement within the wound healing process. moreover, since wound healing in the oral cavity occurs more rapidly and with minimal scarring compared to the skin, molecules in the oral cavity, such as slpi, may contribute to this differential healing response.1,2,3 the inflammatory process in wound healing includes the release of several mediators such as chemoattractants, cytokines and proteinases that regulate the adhesion of molecules, and the processes of cell migration, activation and degranulation. the characteristic destruction of tissue in inflammatory diseases is to a large extent mediated by an excess of neutral serine proteinases. states of impaired healing are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that slpi may also have a role in this process.4 human slpi is an 11.7–12 kda cationic protein and a member of the innate immunity-associated proteins.1,2,3 it is a nonglycosylated, highly basic, acid-stable, cysteinerich, 107-amino acid, single-chain polypeptide.5 the slpi gene, along with the elafin gene, is a member of the trappin gene family. the products of this family are characterized by an n-terminal transglutaminase domain substrate and a c-terminal four-disulfide core.6 slpi is produced by neutrophils, macrophages, betacells of pancreatic islets, epithelial cells investing the renal tubules, acinar cells of parotid and submandibular glands, acinar cells of submucosal glands, and epithelial cells lining mucous membranes of respiratory and alimentary tracts.7,8 slpi was originally isolated from parotid saliva and has been detected in a variety secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, and cerebral spinal fluid, as in secretions from the nose and bronchi, etc.5 the slpi gene was found to be expressed in lung, breast, oropharyngeal, bladder, endometrial, ovarian, and colorectal carcinomas, and slpi detection is correlated with poor prognosis. slpi is also found in neurons and astrocytes in the ischemic brain tissue.1 finally, slpi was found to play a pivotal role in apoptosis and wound healing.2,9 given that slpi is a ubiquitous protein, it has received many alternative names, including mucus protease inhibitor, antileukoprotease, bronchial secretory inhibitor, human seminal inhibitor i, cervix uteri secretion inhibitor, and secretory leukoprotease inhibitor.1 the objectives of this article are to investigate the role of slpi in oral inflammation and how it contributes to tissue repair in oral mucosa. amniotic membrane 173munadziroh: roles of secretory leukocyte amniotic membrane is the thin membrane that covers the placenta and baby before it is born. it has many properties that make it ideal for use as a transplant material. it was first used as a surgical dressing for skin burn. it is now utilized as a biological dressing for burned skin, skin wound and chronic ulcers of the leg as an adjunctive tissue in surgical reconstruction of artificial vagina and for repairing omphalocheles. it has also been used to prevent tissue addition in surgical procedures of the abdomen had and pelvis.10 amniotic membrane has been found to facilited epithelialization, maintain a normal epithelial phenotype, reduce inflammation, reduce scarring, reduce the addition of tissue, reduce vascularisation, a number cytokine, growth factors such as il-4, il-6, il-10, egf, fgf, tgf, hgf and 2-macrobulin. biological active protease inhibitors is slpi.11 secretory leukocyte protease inhibitor (slpi) the main function of slpi is to protect local tissue against the detrimental consequences of inflammation. indeed, a plethora of toxic (inflammatory) products, i.e., serine proteinases, is released from stimulated leukocytes during inflammation, and subsequent degradation of the tissues ensues. slpi protects the tissues by inhibiting the proteases, such as cathepsin g, elastase, and trypsin from neutrophils; chymotrypsin and trypsin from pancreatic acinar cells; and chymase and tryptase from mast cells.12 based on enzyme kinetic studies, its major physiologic function is probably the inhibition of neutrophil elastase.13 neutrophil elastase as well as mast cell proteolytic enzymes can cause extensive tissue degradation and has been shown to be involved in several diseases, such as cystic fibrosis, non-cystic fibrosis bronchectasis, emphysema, acute respiratory distress syndrome, chronic bronchitis, and bacterial pneumonia.12 it is generally postulated that the balance between proteinases and antiproteinases is a prerequisite for the maintenance of tissue integrity. indeed, it is shown that cleavage of slpi results in increased tissue damage.14 slpi also shields the tissues against inflammatory products by down-regulating the macrophage responses against bacterial lipopolysaccharides (lps). lps seem to induce slpi production by macrophages directly or by way of interleukin-1ß (il-1ß), tumor necrosis factor alpha, il-6, and il-10.15 secretory leukocyte protease inhibitor in turn inhibits the downstream portion of the nuclear factor b (nf-b) pathway by protecting i-ß (inhibiting factor of nf-b) from degradation by the ubiquitin-proteosome pathway. thus, slpi renders macrophages unable to release proinflammatory cytokines and nitric oxide.12 ding et al.16 point out that the inhibitory effect of slpi on macrophage responses may be due to its blockade of lps transfer to soluble cd14 (receptor of macrophages) and its interference with the uptake of lps from lps-soluble cd14 complexes by macrophages. taggart et al.17 suggest that slpi attenuates macrophages’ responsiveness by inhibiting the lps pathway through suppression of nf-b and activation of ccaat  enhancer-binding proteintranscription. thus, the accumulation of slpi in the local tissue environment may represent an intrinsic feedback inhibition mechanism. although there are only a few published studies pertinent to this field, recent scientific evidence suggests that slpi has broad-spectrum antibiotic activity that includes bactericidal and antifungal properties. in a recent study, fahey and wira18 examined the production of antibacterial factors by uterine epithelial cells from pre and postmenopausal women. apical rinse fluids from polarized epithelial cells recovered from women at the proliferative and secretory stages of the menstrual cycle were equally effective in killing staphylococcus aureus and escherichia coli, but those from postmenopausal women were not. slpi concentrations in apical wash fluids from premenopausal women were significantly higher than those in wash fluids obtained from postmenopausal women. slpi production correlated with bactericidal activity with respect to menstrual status and culture time. anti-slpi significantly decreased bactericidal activity of premenopausal epithelial cell rinse fluids. the endometrial epithelial cell line hec1a did not have a bactericidal effect, nor did it produce slpi. in contrast, hec-1b cells produced slpi and a factor that inhibited bacterial growth. it seems that the n-terminal domain is responsible for the dose-dependent bactericidal properties of slpi against both gram-positive (s. aureus) and gram-negative (e. coli) bacteria. hiemstra12 showed that the activity of this domain is not as efficient as the one of the intact molecule. hence, they speculated that a conformational change in the n-terminal domain is induced by the cleavage procedure of the native protein.12 in addition, suggested that the mechanism of the slpimediated bactericidal activity may include binding of the protease inhibitor to the bacterial mrna and dna, but hiemstra12 findings proved that this binding is not enough to explain the antibacterial activity of slpi. the antiprotease domain of slpi seems to play a crucial role in regulating host defense against infections by inhibiting the elastasemediated degradation of opsonins and receptors involved in phagocytosis and controlling the proteolytic processing of antimicrobial peptides, such as cathelicidins.12 tomee et al.20 showed that slpi has activity (50% fungicidal activity) against human isolates of the pathogenic fungi aspergillus fumigatus and candida albicans. they also found partial inhibition of fungal protease activity by recombinant slpi (rslpi), a putative virulence factor of a. fumigatus, and subsequent inhibition of the inductive proinflammatory cytokine response in cultured human airway epithelial cell lines. in a recent study showed that the increase of salivary slpi levels (to > 2.1 g/ml) along with other factors, such as low levels of cd4, antiretroviral monotherapy, and smoking, is a key predictor of oral candidiasis in human immunodeficiency virus type 1 (hiv1)-infected persons. a possible biological explanation for this association is that slpi is up-regulated in response to the infection in order to kill the pathogen and resolve the disease. an individual threshold limit to slpi production 174 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 172–176 and secretion may be reached. under this condition, the oral defenses are overwhelmed by the fungal insult and clinical disease ensues. in this scenario, an increase in salivary slpi is associated with greater odds of having oral candidiasis and thus may be a marker of oral fungal disease. slpi may also serve as an indicator of previous oropharyngeal candidiasis infection in the latter. as with the antibacterial-bactericidal activity, the antifungal activity was mainly localized in the nh2terminal domain. it is believed that killing of fungus protects the epithelia from the fungal proteases. probably the antibacterial and antifungal activities are related to the cationic nature of slpi.20 given its antimicrobial activity, slpi may provide a valuable therapeutic option in the future treatment or prevention of infectious diseases. wound healing and repair just after a surgical incision, a number of epithelial cells and connective tissue cells die and the basement membrane is disrupted. this clean and uninfected injury is enough to target an inflammatory response that will be absolutely necessary for the wound healing. immediately after the incision, the wounds covered with clotted blood containing fibrin and blood cells. this fibrin clots receives within 24 hours an amount of neutrophils, attracted by inflammatory factors locally released. at this time, we also have mitotic activity of the basal layer of the epidermis. by the day 3, macrophages are the most common cells in the tissue, instead of neutrophils. the main feature at this moment is the granulation tissue, that consists of fibroblasts and new capillary with amorphous substance all around. by the 5th day, granulation tissue and neovascularization are maximal. collagen fibrils are present and begin to bridge the incision, following the epithelial migration. after 1 week there is still connective tissue proliferation, but inflammatory features have virtually disappeared. at the end of the first month, the scar is completed within an intact epithelial layer, covering a new cellular connective tissue net, devoid of inflammation.21 in some instances, the wound (not surgical ones) has a large loss of cells and tissues, which makes the normal healing event impossible. in this case, we have the healing by second intention. this is characterized by a more complicated process with much more inflammation and granulation tissue. the original architecture is never attained and the main feature of the phenomemon is called wound contraction. the wound contraction is caused, at least in part, by the presence of myofibroblasts ¾ altered fibroblasts that have ultrastructural characteristics of smooth muscle cells. as noted, the disposition of connective tissue matrix, specially collagen, its remodeling into a scar and the acquisition of wound strength are the ultimate effects of the repair. the wound healing process is influenced by many systemic and local host factors. nutrition state of the patient is very important. protein deficiency and particularly ascorbic acid deficiency inhibits collagen synthesis and impairs healing. glucocorticoids therapy, by its antiinflammatory aspects, retards healing. patient’s age is also an systemic factor that plays a role. local infections are important causes of complicating and delaying healing process. hemorrhagic factors, such as ischemia, play a role and foreign bodies, such as sutures and/or other fragments constitute impediments to healing. the healing process may occur abnormally. there are many aberrations of growth, but the most common is called keloid. keloid is a tumoral scar resulted from accumulation os excessive amounts of collagen. the reasons for keloid formation still remain unknown, but is known that it’s more common in afro-caribbeans. the response to injury is a phylogenetically primitive, yet essential innate host immune response for restoration of tissue integrity. tissue disruption in higher vertebrates, unlike lower vertebrates, results not in tissue regeneration, but in a rapid repair process leading to a fibrotic scar. wound healing, whether initiated by trauma, microbes or foreign materials, proceeds via an overlapping pattern of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and tissue remodeling. the process of repair is mediated in large part by interacting molecular signals, primarily cytokines, that motivate and orchestrate the manifold cellular activities which underscore inflammation and healing. clearance of debris, foreign agents, and/or infectious organisms promotes resolution of inflammation, apoptosis, and the ensuing repair response that encompasses overlapping events involved in granulation tissue, angiogenesis, and re-epithelialization. within hours, epithelial cells begin to proliferate, migrate and cover the exposed area to restore the functional integrity of the tissue. re-epithelialization is critical to optimal wound healing not only because of reformation of a cutaneous barrier, but because of its role in wound contraction. immature keratinocytes produce matrix metalloproteases (mmps) and plasmin to dissociate from the basement membrane and facilitate their migration across the open wound bed in response to chemoattractants. the migration of epithelial cells occurs independently of proliferation, and depends upon a number of possible processes including growth factors, loss of contact with adjacent cells, and guidance by active contact. tgf-1 stimulates migration of keratinocytes in vitro,6,19 possibly by integrin regulation and/or provisional matrix deposition.20 behind the motile epidermal cells, basal cell keratinocyte proliferation is mediated by the local release of growth factors, with a parallel up-regulation of growth factor receptors including tnf-, heparin-binding epidermal growth factor (egf) and keratinocyte growth factor (kgf or fgf-7).21-23 such growth factors are released not only by keratinocytes themselves, acting in an autocrine fashion, but also by mesenchymal cells and macrophages, as paracrine mediators.24,25 numerous animal models in which cytokine genes have been deleted or over-expressed have provided 175munadziroh: roles of secretory leukocyte further evidence that such factors are involved in the process of epithelialization.23 tgf-1, and tgf-2 are potent inhibitors of keratinocyte proliferation, with the smad3 pathway implicated as the negative modulator. since epithelialization is significantly accelerated in mice null for the smad3 gene, with unchecked keratinocyte proliferation, but impaired migration in response to tgf-1, the implication is that the early proliferative event is critical to normal epithelialization.6 once contact is established with opposing keratinocytes, mitosis and migration stop, and in the skin, the cells differentiate into a stratified squamous epithelium above a newly generated basement membrane. other factors secreted by keratinocytes may exert paracrine effects on dermal fibroblasts and macrophages. one such factor is a keratinocyte-derived non-glycosylated protein termed slpi which inhibits elastase, mast cell chymase, nf-b and tgf-1 activation. in rodents, slpi is a macrophage-derived cytokine with autocrine and paracrine activities, but production by human macrophages has not yet been demonstrated. in mice, an absence of this mediator of innate host defense (slpi null) is associated with aberrant healing.12 discussion secretory leukocyte protease inhibitor exerts its antiprotease activity by means of its cooh-terminal domain (c-terminal domain), and the active center of which is formed by the leu72-met73 residues. 1 the nh2-terminal domain (n-terminal domain) has no such properties, but it may aid in stabilizing the protease-antiprotease complex and may mediate the enhancement of the antiproteinase activity of slpi by heparin. heparin augments the effectiveness of slpi as it induces a conformational change in the inhibitor.23 in addition, slpi increases glutathione levels, thereby reducing oxidant-mediated tissue injury, and prostaglandin e2 and matrix metalloproteinases are reduced. hiemstra12 hypothesized that slpi’s cysteine residues are utilized for the glutathione synthesis. slpi inhibits the pro-inflammatory activity of bacterial products such as lipopolysaccharide and regulates the activity of inflammatory cells. this has been suggested to be due to inhibition of activation of the transcription factor nuclear factor-kb (nf-b) by slpi, as a result of inhibition of the proteolytic degradation of ikb, the inhibitors of nfb, in unstimulated cells, nf-b is retained in the cytoplasm in complex with ikb proteins. upon cellular activation, ikb is degraded and nf-b is released, allowing it to move to the nucleus and influence gene expression. therefore slpimediated protection of ikb from proteolytic degradation may inhibit nf-b activity and its ability to increase the expression of pro-inflammatory genes.24 a role for slpi in tissue repair was suggested by the observation that the epithelial expression of slpi is increased upon cutaneous injury in humans. whereas these observations suggest an association between tissue repair and slpi expression, in that study it was shown that the absence of slpi resulted in delayed cutaneous wound healing, which was attributed to an increased and prolonged inflammatory response during the repair process and delayed matrix accumulation slpi stimulate the production of hepatocyte growth factor (hgf). hgf is a major cytokine product of mesenchymal cells and has been implicated in the regulation of mitogenesis, motogenesis and morphogenesis. in addition to regulating hgf production by fibroblast, slpi also affect other function of these cells, such as their ability to contract collagen gels in vitro. collagen gel contraction is thought to result from the ability of fibroblast to reorganize and compact collagen fibres, and the model is considered as in vitro model of wound healing and scar formation. analysis of the ability of conditioned medium from cultured human oral epithelial cells to contract collagen gels in vitro led to the identification of slpi as factors in this medium that inhibits fibroblast-mediated scar formation. t h e o r a l e p i t h e l i u m a l s o f o r m s p a r t o f a n intercommunicating network of immune system, in which signals are regularly exchanged in dynamic interactions. oral epithelial cells produce a range of cytokines including interlukin-1 beta (il-1), interlukin-6, tumor necrosis factor-alpha (tnf-), granulocyte-macrophage colony stimulating factor (gm-csf), transforming growth factorbeta (tgf-) and their receptors and il-8.25 three major function of slpi can be delineated: inhibition of local elastase, controlling of leukocyte activation and reduction of tgf- activation, leading to a reduced inflammatory response. aside from suppressing elastolytic release of tgf- complexed with elastin, however, slpi appears to control tgf- activity by regulating cellular activation.2 the local induction of slpi might be important to break the cycle of inflammation. however the mechanisms involved in the regulation of slpi expression and release still remain to be elucidated. it has been shown that slpi is up-regulated by proinflammatory stimuli including lps, tnf, il-6 and il-1, in vitro.26 in conclusion slpi has a multifaceted role in oral wound healing: indeed, slpi confers local protection against microbial, fungal and slpi is a pivopital endogenous factor necessary of optimal wound healing. reference 1. doumas s, kolokotronis a, stefanopoulos p. anti-inflammatory and antimicrobial roles of secretory leukocyte protease inhibitor. infection and immunity 2005. 2. ashcroft gs, lei k, jin w, longenecker g, kulkarni ab, greenwellwild t, hale-donze h, mcgrady g, song xy, wahl sm. secretory leukocyte protease inhibitor mediates non redundant functions necessary for normal wound healing. nat med 2000; 6(10):1147–53. 3. jana nk, gray lr, shugars dc. human immunodeficiency virus type 1 a stimulate the expression and production of secretory leukocyte protease inhibitor (slpi) in oral epithelial cells: a role for slpi in innate mucosal immunity. journal of virology 2005; 79:10. 4. hollander c, nyström m, janciauskiene s, westin u. human mast cells decrease slpi levels in type ii – like alveolar cell model, in 176 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 172–176 vitro. cancer cell international 2003, 3:14. 5. thompson rc, ohlsson k. isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. proc natl acad sci 1988; 83:6692–96. 6. schalkwijk j, wiedow o, hirose s. the trappin gene family: proteins defined by an n-terminal transglutaminase substrate domain and a c-terminal four-disulphide core. j biochem 1999; 340:569–77. 7. abe t, kobayasi n, yoshimura k. expression of the secretory leukoprotease inhibitor gene in epithelial cells. j clin investig 1991; 87:2207–15. 8. ashcroft gs, lei k, jin w. secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. nat med 2000; 6:1147–53. 9. chattopadhyay a, gray lr, patton ll, caplan dj. salivary secretory protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons. infect immun 2004; 72:1956–63. 10. dua hs, blanco aa. amniotic membrane transplantation. british journal opthalmology 1999; 83:748–52. 11. tseng scg, tsubota k. amniotic membrane transplantation for ocular surface reconstruction. in: ocular surface diseases: medical and surgical management. holland ej, mannis mj, editors. sringer, in press, 2001. 12. hiemstra ps. novel roles of protease inhibitors in infection and inflammation. biochemical society transactions 2002; 30(2): 116–20. 13. ying ql, simon sr. dna from bronchial secretions modulated elastase inhibition by proteinase inhibitor and oxidized secretory leukoprotease inhibitor. am j respir cell mol biol 2000; 23(4): 506–13. 14. greene c, taggart c, lowe g. local impairment of anti-neutrophil elastase capacity in community-acquired pneumonia. j infect dis 2003; 188:769–76. 15. jin f, nathan c, radzioch d, ding a. secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. 1997. 16. ding a, thieblemont n, zhu j. secretory leukocyte protease inhibitor interferes with uptake of lipopolysaccharide by macrophages. infect immun 1999; 67:4485–89. 17. taggart cc, greene cm, mcelvaney ng, o’neill s. secretory leukoprotease inhibitor prevents lipopolysaccharide-induced ikb degradation without affecting phosphorylation or ubiquitination. j biol chem 2002; 277. 18. fahey jv, wira cr. effect of menstrual status on antibacterial activity and secretory leukocyte protease inhibitor production by human uterine epithelial cells in culture. j infect dis 2002; 185: 1606–13. 19. grutter mg, frendrich g, huber r, bode w. the 2.5 å x-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymothrypsin. embo j. 1988; 7:345–51. 20. tomee jfc, kolter gh, hiemstra ps, kauffman hf. secretory leucoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic opinion? thorax 1998; 53:114–16. 21. braz fsv, loss ab, japiassu rm. wound healing and scarringsutures-surgery. 22. wahl sm, mcgrady g, song xy, wahl sm. cytokine modulation in the therapy of hepatic immunopathology and fibrosis in cytokines in liver injury and repair. kluwer academic publishers, lancaster, in press. 2001. 23. marone g, lichtenstein lm, galli sj. regulation by physiological inhibitors. in: marone g, lichtenstein lm, galli sj, editors. mast cells and basophils. san diego: academic press; 2000. p. 278–79. 24. taggart cc, cryan sa, weldon s, gibbons a, greene cm, kelly e, low tb, o’neill sj, mcelvaney ng. secretory leucoprotease inhibitor bind to nf-b binding sites in monocytes and inhibits p65 binding. jem 2005; 202:12. 25. walker dm. oral mucosalimmunology: an overview. ann acad med singapore 2004; suppl: 27s-30s. 114114 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 114–119 review article introduction based on the global burden of disease study (2016), the incidence of severe periodontal disease ranks the eleventh highest and most common with an average prevalence percentage of 25.9%, which accounts for around 20%–50% of the world’s population.1,2 periodontitis is a progressive periodontal disease that has the highest prevalence, at 10.5% to 12% globally, and is the most common chronic periodontitis.3,4 until now, the focus of treatment given to patients with periodontitis is to stop the progression of the disease and reduce the inflammation that occurs. non-surgical therapy is still the most recommended option, namely scaling root planning (srp), either without additional treatment or by administering a systemic antimicrobial dose of doxycycline.5 srp is a periodontal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 a mucoadhesive gingival patch with epigallocatechin-3-gallate green tea (camellia sinensis) as an alternative adjunct therapy for periodontal disease: a narrative review yeka ramadhani1, riski rahayu putri rahmasari1, kinanti nasywa prajnasari1, moh. malik alhakim1, mohammed aljunaid2, hesham mohammed al-sharani3,4, tantiana5, wisnu setyari juliastuti5, rini devijanti ridwan5, indeswati diyatri5 1undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2depatment of oral and dental medicine, faculty of medicine, university of taiz, taiz, yemen 3department of maxillofacial surgery, faculty of dentistry, ibb university, ibb, yemen 4department of maxillofacial surgery, school of stomatology, harbin, china 5department of oral biology, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: periodontitis is a progressive destructive periodontal disease. the prevalence of periodontal disease in indonesia reaches 74.1% and mostly occurs in the productive age group. most of the periodontopathogenic bacteria are gram-negative bacteria and have endotoxin in the form of lipopolysaccharide (lps), which can penetrate the periodontal tissue and induce an inflammatory response. in inflammatory conditions, osteoclastic activity is higher than osteoblastic activity, which causes bone destruction. this results in an imbalance between osteoclast-induced bone resorption and osteoblast-induced bone formation. the current preferred treatment for periodontitis is scaling root planning (srp), but this therapy cannot repair the damaged periodontal tissue caused by periodontitis. purpose: to describe the possibility of using a mucoadhesive gingival patch with epigallocatechin-3-gallate (egcg) green tea (camellia sinensis) as alternative adjunct therapy for periodontal disease. review: egcg is the main component of green tea catechins, which have antitumor, antioxidant, anti-inflammatory, anti-fibrotic, and pro-osteogenic effects. however, the weaknesses so far regarding the use of egcg as an alternative treatment is its low oral bioavailability and the concentration of egcg absorbed by the body decreasing when accompanied by food. egcg can be used with a mucoadhesive gingival patch to optimise bioavailability and absorption and increase local concentration and sustained release of egcg. egcg encourages bone development and braces mesenchymal stem cells (mscs) differentiation for osteoblast by enhancing the expression of bone morphogenic protein 2 (bmp2). egcg also has been proven to increase the expression of runx2 and alp activity that induces osteoblast differentiation and bone mineralisation. conclusion: a mucoadhesive gingival patch containing egcg green tea (c. sinensis) may potentially induce osteoblastic activity as an adjunct therapy to repair the periodontal tissue damage due to periodontal disease. keywords: dentistry; egcg; mucoadhesive gingival patch; osteoblast; periodontal disease correspondence: rini devijanti ridwan, department of biology oral, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: rini-d-r@fkg.unair.ac.id mailto:rini-d-r@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 115 ramadhani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 114–119 therapy in the form of a mechanical procedure to eliminate bacterial plaque and calculus on the tooth surface and is clinically able to reduce the clinical attachment loss (cal) and pocket depth (pd).6 the administration of srp therapy cannot restore the damaged periodontium and does not reduce the risk of a recurrence of periodontitis, so pharmacological therapy is needed as an accompanying therapy after srp.7,8 pharmacological therapy as a support for srp therapy can take the form of systemic or topical drug administration. the administration of supporting drugs after srp therapy showed a better reduction in cal and pd when compared to srp therapy alone, but only a few drugs were able to restore the structure of the damaged periodontal tissue.8,9 systemic administration of drugs is considered to be less effective and shows several shortcomings, so many studies are currently being carried out to develop a drug formulation for local periodontal therapy.10 the use of herbs in the health sector, including dentistry, has become widespread in recent years due to their medicinal and physicochemical properties that can provide additional therapeutic effects. one of the most effective and commonly used herbs for treatment is green tea.11–13 besides being rich in antioxidants, green tea also has many health properties, such as anti-cancer, anti-inflammatory, bone resorption, anti-diabetes, anti-hypertension, anti-tumour, anti-fibrosis, and pro-osteogenic.14–16 the catechins in green tea also exhibit antimicrobial and anti-inflammatory properties in the periodontium.17 furthermore, in this narrative review, the potential of (epigallocatechin-3-gallate) egcg topically administered via a mucoadhesive gingival patch to repair the periodontium damaged by periodontitis was described. periodontitis periodontitis is a destructive, multifactorial inflammatory disease of periodontal tissue, characterised by attachment and progressive loss of bone. the etiology of periodontal disease is influenced by the interaction of the microbial environment with the host’s immune response.4 the causative microbial biofilms are similar in aggressive and chronic periodontitis, so they cannot be distinguished based on certain periodontal pathogens.18 the global prevalence of periodontal disease ranks eleventh for severe periodontal diseases.1,2 destruction of the host’s immune and inflammatory responses by the dysbiotic microbiome is believed to be one of the leading causes of the initiation, formation, and progression of periodontitis and tissue destruction. cytokines and inflammatory mediators play important roles in the pathogenesis of periodontal disease. several inflammatory cytokines, such as tumour necrosis factor (tnf), interleukin (il)-1β, il-6, il-8, and il-17, enhance the inflammatory process of periodontal tissue. currently, there are several anti-inflammatory cytokines that reduce the regulation of periodontal inflammation, such as il-4 and il-10, and transform growth factor β (tgf-β).19,20 expressed pro-inflammatory mediators are able to stimulate osteoclastic activity and can cause damage to the periodontal tissue.4 treatment of periodontal disease requires a combination of mechanical treatments, such as debridement, scaling, and srp, to reduce stagnant bacteria. srp can effectively reduce the concentration of microbes present in the periodontal pocket and improve clinical parameters, such as bleeding and clinical adhesion levels, at probing and probing depth.4,21 porphyromonas gingivalis (p. gingivalis) porphyromonas gingivalis is a type of gram-negative anaerobic bacteria in the oral cavity that belongs to the group of black-pigment bacteroides. this group of bacteria form dark brown colonies on the blood agar plate. p. gingivalis is an important cause of periodontal disease. these bacteria produce many extracellular virulence factors and proteases that lead to the destruction of gingival tissue, including lipopolysaccharides (lps), pili, collagenase, hemolysin, endotoxins, fatty acids, ammonia, hydrogen sulphide, and indol. the various components on the surface of p. gingivalis enable the bacteria to interact easily with external media and support their growth, colonisation, nutrient absorption, and formation of a biofilm that protects it from the immune system.22,23 the pathogenesis of p. gingivalis has been observed in various animal models, such as mice, rabbits, drosophila, and cellular models, suggesting a complex mechanism of host interaction with p. gingivalis at the level of periodontal disease. the pathogenic mechanism is also influenced by genetic and environmental factors. the molecules involved in the pathogenesis of periodontitis can be divided into two major groups: those derived from the subgingival microbial flora (microbial virulence factor) of p. gingivalis and the host’s inflammatory immunity.4,22 green tea (camellia sinensis) green tea is a type of plant that has been used as a drink for 5,000 years. green tea is consumed because it can remove toxins, improve blood circulation, and increase resistance to illness. green tea beverages contain polyphenolic compounds, such as phenolic acids, flavanols, flavonoids, and flavandiols. most of the polyphenols in green tea are flavanols called catechins. catechins are also found in other plants, but these plants contain a lower quantity of catechins. the content of the tea rinse depends on the soil, climate, and general growing conditions.24,25 tea is an important product with economic and health benefits. as a result, the per capita consumption of tea in indonesia is about 0.35kg/person/year. in the field of health, green tea is known to have many benefits, including its effectiveness as an antifungal and immunomodulatory agent and for promoting of bone formation and bone resorption. green tea is a member of the genus camellia, which consists of shrubs and trees. the genus camellia is composed of more than 200 species, including camellia dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 116ramadhani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 114–119 sinensis (l.) kuntze.24 the following is the taxonomy of camellia sinensis (l.) kuntze: kingdom, plantae; super division, embryophyta; division, tracheophyta; subdivision, spermatophytina; class, magnoliopsida; order, ericales; family, theaceae; genus, camellia l; and species, camellia sinensis (l.) kuntze.25,26 egcg egcg is the most abundant catechin compound in green tea, accounting for about 59% of the total content of green tea. green tea contains 19% (-) epigallocatechin (egc), 13% (-) epicatechin gallate (ecg), and about 6% (-) epicatechin (ec). epigallocatechin-3-gallate (egcg) is poorly absorbed by the body, so egcg levels that enter the bloodstream are present only at low micromolar concentrations and disappear from plasma within hours. the bioavailability of egcg in humans ranges from 0.1–0.3%.27,28 the egcg has a higher content of superoxide and free lipid radicals and higher neutralisation activity of free radicals than egc and ec. egcg may alter biological activity and reduce the antioxidant capacity of the compartment.29 the main action of egcg is to suppress the expression of reactive oxygen species (ros) and to inhibit signal transduction during the inflammatory process. systemic dysfunction has decreased. egcg can inhibit osteoclast differentiation by inhibiting the transcriptional activity of the nuclear factor of activated t cell-cytoplasmic 1 (nfatc1) and nuclear factor kappa beta (nf-kb).30 catechins exhibit antioxidant activity through a variety of mechanisms: electron transfer, hydrogen atom transfer, and catalytic metal chelation. in egcg, the free radical inhibitory effect is due to the presence of defective groups at the 3-position of the trihydroxy bring structure and its chemical structure. egcg, which has eight hydroxyl groups mainly at positions 31, 41, and 51 and has a defect group at c3, is a better electron donor than other catechins and is therefore the best suppressor of free radical expression.31,32 egcg is widely used in the treatment of oral diseases, primarily due to its anti-inflammatory and antioxidant properties and its ability to inhibit bone resorption.33 egcg suppresses lps-induced alveolar bone resorption in vitro and suppresses lps-induced alveolar bone loss in vivo. the effective amount of egcg in vitro and in vivo is similar to the effective amount of polymethoxyflavonoids, such as nobiletin.16 catechins in green tea continue to be studied and developed as anti-virus and cancer therapy. according to kharisma et al.,34 tea catechin compounds may act as antiviral agents against hiv1 through apoptotic agonists and triple inhibitor mechanisms. apoptosis can occur during the interaction between the egcg and intracellular apoptosis-promoting proteins. as an anti-cancer, egcg inhibits angiogenesis, protects dna from carcinogens, and promotes apoptosis of cancer cells.26,34 mucoadhesive gingival patch mucoadhesives, either organic or synthetic, may be prescribed because of their ability to stick to organic tissue. generally, mucoadhesives are used without difficulty on available surfaces in the gingival, buccal, ocular, and nasal areas. mucoadhesive patches are long lasting, even on the floor of a membrane or mucosa, and might improve the absorption of the drug as it is not affected by metabolism, travelling first to the liver.35 mucosal adhesives provide direct contact between the surface and the adhesive. the american society for testing and materials defines mucosal adhesion as a condition in which two subjects are held together by interlocking interfacial forces. the word ‘muco’ refers to the mucous membrane. the mucous membrane is the moist surface that covers most of the body’s cavities, especially the inside of the oral cavity, and is responsible for lubrication and protection.36 the mucosal adhesion mechanism consists of two main stages: the contact stage and the consolidation stage. at the contact stage, there is strong contact between the adhesive and the surface of the periodontal tissue, which initiates the distribution of the target active ingredient. at the consolidation stage, the adhesive is activated by moisture, the system becomes plastic, the molecules break and open, and they bond to each other via weak van der waals forces and hydrogen bonds.37 the composition of mucosal adhesive plasters consists of active ingredients, polymers, plasticisers, and fortifiers. the polymer supplies the active ingredient and stays in contact with the mucosal surface longer. the active ingredient content of the patch is in the range of 5–25% by weight of the polymer. there are several polymers that can be used as gypsum materials, such as polyvinyl alcohol (pva), hydroxyethyl cellulose (hec), and hydroxypropyl methyl cellulose (hpmc). stucco plasticisers are used to prevent plaster damage if the plaster breaks or tears. glycerine, propylene glycol, and polyethylene glycol 400 (peg 400) can be used as plasticisers.38 enhancers work to increase the ability of the membrane to absorb drugs and active ingredients. enhancers that can be used include dimethyl sulfoxide (dmso), linoleic acid (la), isopropyl myristate (ipm), and oleic acid (oa).39 osteoblasts osteoblasts are bone cells with a single nucleus, located more peripherally. the cytoplasm is basophilic, cuboidal in shape, and are abundant on the surface of the bone matrix that makes up 4–6% of all bone cells. osteoblasts can be derived from differentiated mesenchymal stem cells and can secrete organic bone matrix proteins (osteoids) that are important for calcification and bone formation. morphologically, osteoblasts have the same organelles as other cells that can secrete proteins, such as rough endoplasmic reticulum, gorgi complexes, large mitochondria, and numerous secretory vesicles. osteoblasts can secrete molecules that can affect surrounding cells, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 117 ramadhani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 114–119 such as an osteoblast-derived vascular endothelial growth factor, which play a role in accelerating the healing process and bone formation. osteoblasts are also known to be able to secrete pro-collagenase enzymes that play a role in breaking down collagen fibres. the ability of osteoblasts to produce cytokines, such as receptor activator nuclear kappa beta ligand, osteoprotegrin, and macrophage colonystimulating factor, means these cells have an important role in regulating bone homeostasis.40,41 in periodontitis, bone destruction occurs progressively due to higher osteoclastic activity than osteoblastic activity. based on a study conducted on a rat calvaria model injected with p. gingivalis, troponema denticola, and tannerella forsythia bacteria, it showed that bone resorption occurred on days 3 to 5 after bacterial infection and on days 7 to 14. bone formation occurs as part of the bone healing process.42 discussion p. gingivalis is a normal flora in the oral cavity that has many virulence factors that cause periodontal tissue damage, such as lps. lpss are found in bacterial cell membranes and can interact with host cell components, toll-like receptor 2, and toll-like receptor 4.43 p. gingivalis was chosen by many researchers to trigger the periodontal process.44 periodontal disease is a chronic inflammation characterised by many reactions, including b. vasodilation and the recruitment of immune cells and plasma proteins to the site of infection or tissue damage. there are four main components to the inflammatory response: (1) intrinsic or extrinsic factors, such as pathogen-associated molecular patterns bacteria, viruses, fungi, parasites, and damageassociated molecular patterns derived from cell damage; (2) cellular receptors in the form of pattern recognition receptors, such as toll-like receptors; (3) inflammatory mediators, such as cytokines and chemokines; and (4) target cell or tissue.45 one of the drug delivery systems through the membrane of oral cavity is the buccal bioadhesive patch. one example is the mucoadhesive gingival patch, which creates a mucosal adhesion mechanism by forming an interaction between polymer and mucus. the mechanism of mucosal adhesion can be divided into two steps. the first is the contact step, and the second is the consolidation/integration step. in the first step, the mucous membrane comes into contact with the mucoadhesive, causing the formulation to swell and then spread over the mucous membranes. in the second, which is consolidation step, the moisture activates the mucosal adhesive material, which plasticises the system. this causes the separation of mucosal adherent molecules and allows them to connect to weak van der waals forces via hydrogen bonds.46 the theory of diffusion and dehydration explains the integration steps. the diffusion theory explains the interaction of mucosal adherent molecules with mucous glycoproteins and the formation of secondary bonds by the interpenetration of their chains. according to dehydration theory, the material turns into a gel when it comes into contact with mucus in an aqueous environment. this process increases the mucosal contact time between the formulation and the mixture of mucus. therefore, the movement of water, not the interpenetration of polymer chains, leads to the strengthening of the adhesive junction.46,47 the egcg content present in mucosal adherent gingival tissue will inhibit the induction of pro-inflammatory cytokine production from lps released by p. gingivalis. egcg is able to inhibit the expression of chemokines, such as il-8, monocyte chemoattractant protein-1 (mcp-1) and macrophage inflammatory protein-1 alpha (mip1α), by infected epithelial cells. the inhibited expression of il-8, mcp-1, and mip-1α resulted in the disruption of the chemotaxis process of inflammatory cells to areas of infection, such as macrophages, neutrophils, and lymphocytes. inflammatory cells have an important role in the severity of inflammation by expressing several inflammatory mediators, such as tumour necrosis factor alpha (tnf-α), il-1, il-6, il-17, prostaglandin e2, and ros metabolites. egcg was also able to inhibit the nf-κb inflammatory pathway activation by bacterial lps. inflammatory cell migration and inhibited nf-κb activation resulted in decreased expression of cytokines and inflammatory products. il-1, il-6, tnf-α, and ros are responsible for the apoptosis of osteoblast cells. the decreased expression of il-1, il-6, tnf-α, and ros will inhibit osteoblast cell death so that there is an obstacle in the decline of osteoblast cells.48–51 previous studies of egcg have demonstrated that egcg promotes differentiation of bone formation in bone marrow mesenchymal stem cells (bmscs) of mice. at certain doses, egcg can also promote differentiation of bmscs bone formation. the effect of egcg was found in a similar mouse bmsc: increased expression of bone-forming genes, such as bone morphogenetic protein-2 (bmp2), runt-related transcription factor 2 (runx2), alkaline phosphatase (alp), osteonectin, and osteocalcin; increased alp activity; and, finally, improved mineralisation.52 in another study, topical use of egcg in the femoral defect improved bone formation by increasing bone mass, which includes the bone’s maximum load, fracture point, stiffness, maximum load sub-curve area, area under the breakpoint curve, and ultimate stress. local egcg can be used to treat bone defects.53 bone formation will be induced by decreasing the osteoclast differentiation and increasing the differentiation of bone formation. green tea and its catechin compounds have been shown to suppress osteoclast differentiation. as shown in the result of research by nishioku et al.,30 egcg could inhibit osteoclastogenesis by suppressing the expression of nfatc1 in primary osteoclast cultures, a key regulator of osteoclast differentiation. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 118ramadhani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 114–119 the differentiation of osteoblast is important for bone formation, and osteoblast-specific gene products are involved in the differentiation process. runx2 is an important transcription factor and a central regulator of osteoblast-specific target genes such as osteocalcin during bone formation, transient activation, inhibition of cell proliferation, and osteoblast differentiation. runx2 regulates osteoblast progenitor cell proliferation and osteoblast differentiation through the mutual regulation of fgf, hedgehog, wnt, and pthlh signalling molecules with transcription factors, including sp7 and dlx5.54,55 this theory is supported by the results of a study conducted by byun et al.,56 which stated that the catechins in green tea can stimulate osteoblast differentiation with the help of a mediator in the form of runx2, the main regulator of transcription of osteoblast marker genes. furthermore, the egcg increases the transcriptional and post-transcriptional expression of the transcriptional coactivator with pdzbinding motif, a transcriptional coregulator involved in osteogenesis.57 although this mucoadhesive gingival patch containing egcg has the potential to be an alternative therapy for periodontitis, unfortunately, it is not known what the most effective dose and duration of application are to provide optimal results. in conclusion, a mucoadhesive gingival patch containing egcg green tea (camellia sinensis) can potentially repair damaged periodontal tissue by inducing osteoblastogenesis activity directly or through its anti-inflammation characteristic. for that, further research to find the right dose and duration of application of this mucoadhesive gingival patch with egcg is needed. references 1. vos t, abajobir aa, abate kh, abbafati c, abbas km, abd-allah f, et al. global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the global burden of disease study 2016. lancet. 2017; 390(10100): 1211–59. 2. nazir m, al-ansari a, al-khalifa k, alhareky m, gaffar b, almas k. global prevalence of periodontal disease and lack of its surveillance. sci world j. 2020; 2020: 2146160. 3. kumar s. evidence-based update on diagnosis and management of gingivitis and periodontitis. dent clin north am. 2019; 63(1): 69–81. 4. newman mg, takei hh, klokkevold pr, carranza fa. newman and carranza’s clinical periodontology. 13th ed. philadelphia: elsevier saunders; 2018. p. 30–1, 41–7, 101, 107, 164, 198–9, 484–5. 5. latif sa, vandana kl, thimmashetty j, dalvi pj. azithromycin buccal patch in treatment of chronic periodontitis. indian j pharmacol. 2016; 48(2): 208–13. 6. gross aj, paskett kt, cheever vj, lipsky ms. periodontitis: a global disease and the primary care provider’s role. postgrad med j. 2017; 93(1103): 560–5. 7. szulc m, zakrzewska a, zborowski j. local drug delivery in periodontitis treatment: a review of contemporary literature. dent med probl. 2018; 55(3): 333–42. 8. bao j, yang y, xia m, sun w, chen l. wnt signaling: an attractive target for periodontitis treatment. biomed pharmacother. 2021; 133: 110935. 9. lim sy, dafydd m, ong j, ord-mcdermott la, board-davies e, sands k, williams d, sloan aj, heard cm. mucoadhesive thin films for the simultaneous delivery of microbicide and anti-inflammatory drugs in the treatment of periodontal diseases. int j pharm. 2020; 573: 118860. 10. pagano c, giovagnoli s, perioli l, tiralti mc, ricci m. development and characterization of mucoadhesive-thermoresponsive gels for the treatment of oral mucosa diseases. eur j pharm sci. 2020; 142: 105125. 11. ningsih ds, idroes r, bachtiar bm, khairan. the potential of five therapeutic medicinal herbs for dental treatment : a review. iop conf ser mater sci eng. 2019; 523(1): 012009. 12. sakagami h, watanabe t, hoshino t, suda n, mori k, yasui t, yamauchi n, kashiwagi h, gomi t, oizumi t, nagai j, uesawa y, takao k, sugita y. recent progress of basic studies of natural products and their dental application. med (basel, switzerland). 2018; 6(1): 4. 13. moghadam et, yazdanian m, tahmasebi e, tebyanian h, ranjbar r, yazdanian a, seifalian a, tafazoli a. current herbal medicine as an alternative treatment in dentistry: in vitro, in vivo and clinical studies. eur j pharmacol. 2020; 889: 173665. 14. aboulwafa mm, youssef fs, gad ha, altyar ae, al-azizi mm, ashour ml. a comprehensive insight on the health benefits and phytoconstituents of camellia sinensis and recent approaches for its quality control. antioxidants (basel, switzerland). 2019; 8(10): 455. 15. musial c, kuban-jankowska a, gorska-ponikowska m. beneficial properties of green tea catechins. int j mol sci. 2020; 21(5): 1744. 16. tominari t, matsumoto c, watanabe k, hirata m, grundler fmw, miyaura c, inada m. epigallocatechin gallate (egcg) suppresses lipopolysaccharide-induced inflammatory bone resorption, and protects against alveolar bone loss in mice. febs open bio. 2015; 5: 522–7. 17. de almeida jm, marques bm, novaes vcn, de oliveira flp, matheus hr, fiorin lg, ervolino e. influence of adjuvant therapy with green tea extract in the treatment of experimental periodontitis. arch oral biol. 2019; 102: 65–73. 18. kinane df, stathopoulou pg, papapanou pn. periodontal diseases. nat rev dis prim. 2017; 3(1): 17038. 19. heidari z, moudi b, mahmoudzadeh-sagheb h. immunomodulatory factors gene polymorphisms in chronic periodontitis: an overview. bmc oral health. 2019; 19(1): 29. 20. könönen e, gursoy m, gursoy uk. periodontitis: a multifaceted disease of tooth-supporting tissues. j clin med. 2019; 8(8): 1135. 21. javadzadeh y, hamedeyaz s. floating drug delivery systems for eradication of helicobacter pylori in treatment of peptic ulcer disease. in: trends in helicobacter pylori infection. intech; 2014. p. 57353. 22. rafiei m, kiani f, sayehmiri f, sayehmiri k, sheikhi a, zamanian azodi m. study of porphyromonas gingivalis in periodontal diseases: a systematic review and meta-analysis. med j islam repub iran. 2017; 31: 62. 23. fiorillo l, cervino g, laino l, d’amico c, mauceri r, tozum tf, gaeta m, cicciù m. porphyromonas gingivalis, periodontal and systemic implications: a systematic review. dent j. 2019; 7(4): 114. 24. yang j-b, yang s-x, li h-t, yang j, li d-z. comparative chloroplast genomes of camellia species. unver t, editor. plos one. 2013; 8(8): e73053. 25. nugraha ap, narmada ib, sitasari pi, inayati f, wira r, triwardhani a, hamid t, ardani igaw, djaharu’ddin i, rahmawati d, iskandar rpd. high mobility group box 1 and heat shock protein-70 expression post (-)-epigallocatechin-3-gallate in east java green tea methanolic extract administration during orthodontic tooth movement in wistar rats. pesqui bras odontopediatria clin integr. 2020; 20: e5347. 26. narmada ib, sarasati a, wicaksono s, rezkita f, wibawa kgp, hayaza s, nugraha ap. phytochemical screening, antioxidant activity, functional groups and chemical element characterization analysis of (-)-epigallocatechin-3-gallate (egcg) in east javanese green tea methanolic extract: an experimental in vitro study. syst rev pharm. 2020; 11(5): 511–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 119 ramadhani et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 114–119 27. pastoriza s, mesías m, cabrera c, rufián-henares ja. healthy properties of green and white teas: an update. food funct. 2017; 8(8): 2650–62. 28. pervin m, unno k, takagaki a, isemura m, nakamura y. function of green tea catechins in the brain: epigallocatechin gallate and its metabolites. int j mol sci. 2019; 20(15): 3630. 29. chu ko, chan kp, yang yp, qin yj, li wy, chan so, wang cc, pang cp. effects of egcg content in green tea extract on pharmacokinetics, oxidative status and expression of inflammatory and apoptotic genes in the rat ocular tissues. j nutr biochem. 2015; 26(11): 1357–67. 30. nishioku t, kubo t, kamada t, okamoto k, tsukuba t, uto t, shoyama y. (−)-epigallocatechin-3-gallate inhibits ranklinduced osteoclastogenesis via downregulation of nfatc1 and suppression of ho-1–hmgb1–rage pathway. biomed res. 2020; 41(6): 269–77. 31. legeay s, rodier m, fillon l, faure s, clere n. epigallocatechin gallate: a review of its beneficial properties to prevent metabolic syndrome. nutrients. 2015; 7(7): 5443–68. 32. nikoo m, regenstein jm, ahmadi gavlighi h. antioxidant and antimicrobial activities of (-)-epigallocatechin-3-gallate (egcg) and its potential to preserve the quality and safety of foods. compr rev food sci food saf. 2018; 17(3): 732–53. 33. wu yr, choi hj, kang yg, kim jk, shin j-w. in vitro study on antiinflammatory effects of epigallocatechin-3-gallate-loaded nanoand microscale particles. int j nanomedicine. 2017; 12: 7007–13. 34. kharisma vd, widyananda mh, ansori anm, nege as, naw sw, nugraha ap. tea catechin as antiviral agent via apoptosis agonist and triple inhibitor mechanism against hiv-1 infection: a bioinformatics approach. j pharm pharmacogn res. 2021; 9(4): 435–45. 35. mishra m. concise encyclopedia of biomedical polymers and polymeric biomaterials. boca raton: crc press; 2017. p. 111– 220. 36. alawdi s, solanki ab. mucoadhesive drug delivery systems: a review of recent developments. j sci res med biol sci. 2021; 2(1): 50–64. 37. asati s, jain s, choubey a. bioadhesive or mucoadhesive drug delivery system: a potential alternative to conventional therapy. j drug deliv ther. 2019; 9(a): 858–67. 38. gales r, ghonaim hm, gardouh ar, ghorab mm, badawy ss. preparation and characterization of polymeric mucoadhesive film for buccal administration. br j pharm res. 2014; 4(4): 453–76. 39. prasanth v v, puratchikody a, mathew st, ashok kb. effect of permeation enhancers in the mucoadhesive buccal patches of salbutamol sulphate for unidirectional buccal drug delivery. res pharm sci. 2014; 9(4): 259–68. 40. florencio-silva r, sasso gr da s, sasso-cerri e, simões mj, cerri ps. biology of bone tissue: structure, function, and factors that influence bone cells. biomed res int. 2015; 2015: 421746. 41. henry jp, bordoni b. histology, osteoblasts. [updated 2021 may 10]. statpearls. treasure island (fl): statpearls publishing; p. 1–29. 42. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015; 2015: 1–10. 43. ramadhani nf, nugraha ap, ihsan is, agung ya, rantam fa, ernawati ds, ridwan rd, narmada ib, ansori anm, hayaza s, noor tnebta. gingival medicinal signaling cells conditioned medium effect on the osteoclast a nd osteoblast number in lipopolysaccharide-induced calvaria bone resorption in wistar rats’ (rattus novergicus). res j pharm technol. 2021; 14(10): 5232–7. 44. yesudhas d, gosu v, anwar ma, choi s. multiple roles of toll-like receptor 4 in colorectal cancer. front immunol. 2014; 5: 334. 45. muñoz-carrillo jl, hernández-reyes ve, garcía-huerta oe, chávez-ruvalcaba f, chávez-ruvalcaba mi, chávez-ruvalcaba km, díaz-alfaro l. pathogenesis of periodontal disease. in: yussif n, editor. periodontal disease diagnostic and adjunctive non-surgical considerations. london: intechopen; 2019. p. 86548. 46. singh j, deep p. a review article on mucoadhesive buccal delivery system. int j pharm sci res. 2013; 4(3): 916–27. 47. gilhotra rm, ikram m, srivastava s, gilhotra n. a clinical perspective on mucoadhesive buccal drug delivery systems. j biomed res. 2014; 28(2): 81–97. 48. khurshid z, zafar ms, zohaib s, najeeb s, naseem m. green tea (camellia sinensis): chemistry and oral health. open dent j. 2016; 10: 166–73. 49. kochman j, jakubczyk k, antoniewicz j, mruk h, janda k. health benefits and chemical composition of matcha green tea: a review. molecules. 2020; 26(1): 85. 50. reygaert wc. an update on the health benefits of green tea. beverages. 2017; 3(1): 6. 51. cai y, chen z, liu h, xuan y, wang x, luan q. green tea epigallocatechin-3-gallate alleviates porphyromonas gingivalisinduced periodontitis in mice. int immunopharmacol. 2015; 29(2): 839–45. 52. lin s-y, kang l, wang c-z, huang hh, cheng t-l, huang h-t, lee m-j, lin y-s, ho m-l, wang g-j, chen c-h. (-)-epigallocatechin-3gallate (egcg) enhances osteogenic differentiation of human bone marrow mesenchymal stem cells. molecules. 2018; 23(12): 3221. 53. lin s-y, kang l, chen j-c, wang c-z, huang hh, lee m-j, cheng t-l, chang c-f, lin y-s, chen c-h. (-)-epigallocatechin-3-gallate (egcg) enhances healing of femoral bone defect. phytomedicine. 2019; 55: 165–71. 54. komori t. regulation of proliferation, differentiation and functions of osteoblasts by runx2. int j mol sci. 2019; 20(7): 1694. 55. qin x, jiang q, miyazaki t, komori t. runx2 regulates cranial suture closure by inducing hedgehog, fgf, wnt and pthlh signaling pathway gene expressions in suture mesenchymal cells. hum mol genet. 2019; 28(6): 896–911. 56. byun mr, sung mk, kim ar, lee ch, jang ej, jeong mg, noh m, hwang es, hong j-h. (-)-epicatechin gallate (ecg) stimulates osteoblast differentiation via runt-related transcription factor 2 (runx2) and transcriptional coactivator with pdz-binding motif (taz)-mediated transcriptional activation. j biol chem. 2014; 289(14): 9926–35. 57. sitasari pi, narmada ib, hamid t, triwardhani a, nugraha ap, rahmawati d. east java green tea methanolic extract can enhance runx2 and osterix expression during orthodontic tooth movement in vivo. j pharm phyther res. 2020; 8(4): 290–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p114–119 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p114-119 � volume 46 number 1 march 2013 research report changes in setting time of alginate impression material with different water temperature decky j. indrani and niti matram department of dental material faculty of dentistry, universitas indonesia jakarta – indonesia abstract background: previous studies showed that setting process of alginates can be influenced by temperature. purpose: to determine the changes in setting time due to differences in water temperature and to determine the correlation between water temperature and the setting time. methods: seven groups of dough alginate were prepared by mixing alginate powder and water, each using a temperature between 13° c–28° c with a interval of 2.5° c. a sample mold ( = 30 mm, t = 16 mm) was placed on a flat plate and filled with doug alginate. immediately the flat end of a polished acrylic rod was placed in contact with the surface of dough alginate. setting time of alginat was measured from the starting of the mix to the time when the alginate does not adhere to the end of the rod. setting time alginate data were analyzed using one way anova, lsd and pearson. results: setting time of alginate with water temperature between 13° c–28° c were 87 to 119.4 seconds and were significantly different (p < 0.01). the setting time between group were also significantly different (p<0.01). there was an inverse correlation between water temperature and the setting time (r = -0.968). conclusion: water temperature between 13° c–28°c with a difference of 2.5° c produced significant differences in alginate setting time; the lower the water temperature being used the longer the setting time was produced key words: alginate, water temperature, setting time, polimerization abstrak latar belakang: penelitian-penelitian sebelumnya menunjukkan bahwa proses pengerasan alginat dapat dipengaruhi oleh suhu. tujuan: mengetahui perubahan waktu pengerasan alginat akibat perbedaan suhu air serta mengetahui hubungan antara suhu air dan waktu pengerasan. metode: tujuh kelompok adonan alginat yang dipersiapkan dengan mencampur bubuk alginat dan air, masingmasing menggunakan suhu antara 13° c–28° c dengan interval 2,5° c. pengukuran waktu pengerasan alginat dilakukan sesuai dengan spesifikasi ada no.18. sebuah cetakan sampel terbuat dari pralon berbentuk cincin (θ = 30 mm, t = 16 mm) ditempatkan di atas plat datar dan dipenuhi dengan adonan alginat. pengukuran waktu pengerasan dilakukan segera dengan menyentuhkan ujung datar batang akrilik yang telah dipoles di permukaan adonan alginat. waktu pengerasn alginat diukur dari awal pencampuran bubuk alginat dan air sampai dengan waktu awal ketika alginat tidak melekat di ujung batang. data waktu pengerasan yang diperoleh dianalisis menggunakan uji statistik one-way anova, lsd dan pearson. hasil: suhu air antara 13° c–28° c telah menghasilkan waktu-waktu pengerasan alginat 87 detik hingga 119,4 detik yang berbeda signifikan (p < 0,01). waktu pengerasan antar grup juga menunjukkan perbedaan signifikan (p<0,01). antara suhu air dan waktu pengerasan alginat terdapat hubungan terbalik (r=-0,968). kesimpulan. suhu air antara 13°c–28°c dengan interval 2,5° c menghasilkan perbedaan waktu pengerasan alginat; makin rendah suhu air yang digunakan untuk mencampur makin panjang waktu pengerasan alginat. kata kunci: alginat, suhu air, waktu pengerasan, polimerisasi correspondence: decky j. indrani, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no 5. jakarta 10430, indonesia. e-mail: decky@ui.ac.id � dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 5–8 introduction alginates are the most commonly used impression material to reproduce teeth and its surrounding tissue. popularity of alginate is related to its ability to reproduce detailed anatomical impression of teeth and its surrounding tissue, when it is manipulated well.1,2 internship students in the teaching hospital are not yet able to manipulate alginate well and thus not yet able to produce alginate dough with good consistency; whereas, students are expected to produce detailed and complete impression of teeth and other tissues. alginate dough produced by the students are often set too fast when used to make impression in the mouth, or even set far before it is used to make impression. a study showed that room temperature without air conditioning, ranging from 24º c–31º c, contributed to alginate’s setting time.3 in such condition, students often need to reduce the amount of alginate powder or add more water than what is recommended by the factory to delay alginate setting time. these methods will reduce mechanical strength of the alginate impression.4,5 alginates are salts from alginic acid that is obtained from brown algae. alginic acid is part of organic polymers from polysaccharide family, composed of two units b-lguluronate monomers (g unit) and a-d-manuronate (m unit) with combination of [gm-mg]n.6 processed alginates contain cation, e.g. k2+, na+, ca2+, which is used as primary components in alginate impression material. apart from that, alginate impression material also contains other components, i.e. calcium sulfate salts (caso4), monovalent retarder natrium triphosphate (na3po4) and potassium alginic (k2nalginic). all components take part in the setting process of alginates.7 the setting process of alginates contains of series of reaction. the first reaction series, polymerization and gelation, may be influenced by the size of alginates’ particles and/or retarder concentrations. manipulating retarder concentrations are the most reliable and controlled method to delay the setting time of alginates.7,8 such method can only be done during the fabrication of alginate powders. the easiest method to delay the setting time of alginates and prolong manipulating time is to use slow setting type of alginates; such type provides prolonged setting time than the normal one.7 if such type is not available, the normal one may still be used with water temperature lower than 80º f (± 28º c), as recommended by the manufacturer. the use of relatively low water temperature is important since it reduces collision among molecules in chemical reaction. the amount of the collision between substances’ molecules reacted in time unit determines the rate of chemical reaction.9 the less collision will slower the chemical reaction and thus the completion of the chemical reaction will have longer time. in such case, the application of lower water temperature will enable longer setting time. previous research has studied about the influence of temperature on the setting time of alginate. the research studied water with 10º c difference of temperature to mix alginates.10 water temperature during the mixing of alginates is highly sensitive; even small difference of temperature may change the rate of the setting process of alginates.9 the present study has used water temperature difference lower than 10º c, i.e. 2.5º c, in order to obtain detailed impression results. water temperature used were between 13º c to 28º c; this was because the laboratory temperature without air conditioning was approximately 28 (±1)º c and the lowest temperature achieved by adding ice cubes was 13 (±1)º c. therefore, the study aimed to determine the changes in setting time due to differences in water temperature between 13º c to 28º c and to determine the correlation between water temperature and the setting time. materials and methods firstly, alginate dough was prepared according to its manufacture direction. a part (equivalent to 6.5 gram) of alginate powders with normal setting type (new kromopan®, schultz science dental product, jerman, no. batch 015038011062) and water (equivalent to 17 ml) were put in a plastic bowl, then mixed and pushed with spatula to the wall of the bowl to get a homogenous dough. water temperature ranged between 13º–28º c with 2.5º c interval; there were 7 groups with different water temperature to make the dough. the test of alginate setting time followed ada specification no. 18.11 specimen molds were made from plastic cylinder (pipe) with 30 mm in diameter and 16 mm in height placed in glass plate. alginate dough in each group was poured into one specimen mold. the excess dough is removed using spatula until the surface of the dough reached the mold’s height. a stick made from resin acrylic with 10 cm in length and 6 mm in diameter was placed in contact to the surface of the specimen. alginate dough sticked to acrylic stick was cleaned and the contact was repeated with 10 seconds time interval. the setting time of alginate was counted from start of alginate mixing until no sticky dough found in the bar. the total number of specimens were 70. data of alginate setting time were analyzed with one-way anova test and continued with lsd test and pearsons test to find out the relationship between different temperature group and alginate setting time. results the setting time of alginate with water temperature between 13º–28º c was shown in the table 1. it showed that water temperature between 13º c–28º c produced various setting time of alginate, ranging from 87 seconds to 119.4 seconds. the longest difference of alginate setting time was 8.8 seconds, produced by temperature 25.5º c and 28º c, while the shortest time difference was 3.8 �matram and indrani: changes in setting time of alginate impression material with different water temperature seconds, i.e. between 15.5º c and 18º c. time difference in alginate setting time among seven groups ranged between 3–10%. the relationship between water temperature and alginate setting time was shown figure 1. it showed that the shortest alginate setting time was at 28º c, while the longest was at 13º c (figure 1). this showed that the relationship between water temperature and setting time was inversely proportional. one way anovatest showed that the setting time of alginates in different water temperature were significantly different (p<0.01). lsd test showed that the setting time between each temperature group were also significantly different (p<0.01). pearson’s test proved that there was significantly inverse correlation between water temperature and the setting time (r = -0.968). discussion longer set of alginate in the application of lower water temperature indicated that there was a delay in the setting time of alginate. prolonged set of alginate has already started since alginate powder is wet with water until the alginate turns into irreversible hydrocolloid gel. chemically, prolonged setting process may occur at the early stage of chemical reaction, polymerization stage and gelation stage. early reaction that takes place since the mixing of alginate powder with water triggers chemical reaction between caso4 with na3po4 retarder in the alginate powder, as reaction-1 below:7 3 caso4 + 2na3po4 ca3(po4)2 + 3na2so4 (retarder) ..................... (reaction-1) the reaction occurs continuously as long as there is retarder in reaction-1. retarder maintains [gm-mg]n polymers to stay in water soluble form. in such case, reaction-1 delays the next reaction to take place. after all of retarder (na3po4) reacts in the reaction-1, then reaction-2 takes place as follow:7 n caso4 + k2nalginat nk2so4 + can alginat ..................... (reaction-2) since there is no more retarder in reaction-2, caso4 reacts with k2nalginic to produce canalginic (calcium alginic/alginate). physically, reaction-2 is the alginate polymerization, which is the merging of [gm-mg]n polymers into alginate that is cross-linked.7 this process is illustrated in figure 2. cross-linked alginate occurs with the existence of caso4 as the source of calcium ions (ca +) to bind {gmmg]n alginate.12,13 ca+ ions make ether-oxygen bridge to unify carbon (c) at c1 position from m unit and c4 from g unit to become a cross-linked structure between [gmmg]n polymers. beside delaying reaction-1 and reaction-2, the application of lower water temperature may also delay alginate gelation. at the early gelation, the alginate formed is still water soluble. therefore, at the contact with acrylic bar, alginate still sticked to the bar. after passing the high table 1. setting time of alginate impression material with water temperature between 13º c-28º c water temperature (0c) n setting time (second) 28.0 10 87.0 ± 1.83 25.5 10 95.2 ± 2.82 23.0 10 100.7 ± 2.56 20.5 10 107.6 ± 2.42 18.0 10 111.9 ± 2.81 15.5 10 115.7 ± 2.85 13.0 10 119.0 ± 2.92 figure 1. the setting time of alginate was longer in the application of water with lower temperature. figure 2. scheme diagram (a) alginate expressed as guluronat acid (g unit) manuronat acid (m unit) forming (b) cross-linked at c1 and c4.12 � dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 5–8 viscosity solution stadium, alginate swells and eventually looks like gel-shaped thick paste.12,13 at the end of gelation stadium, alginat will no longer water soluble that can not be part. at this condition, alginate does not stick in the acrylic bar. during the setting process, water temperature influences the efficiency of the collision between molecules in chemical reaction through kinetic effectivity.9 the application of higher water temperature gives increasing effect on the efficiency of the collision between reacted molecules and thus produces an effective molecular collision kinetics. on the other hand, the application of lower water temperature produces less effective molecular collision kinetics. manipulating alginate using water temperature close to 13º c, produces kinetic effectivity of these collision and thus produces prolonged setting time of alginate. in other words, the lower water temperature used to mix the alginate powder, the longer alginate setting time produced. alginate setting time in this study could be compared to the previous studies. referring to ada,10 alginate setting time in normal setting type alginate (with water temperature ±18º c) is ranging from 120 to 390 seconds, other studies,9 102 to 282 seconds, while this study’s result is 87 to 119.4 seconds. even though the two studies using normal setting type impression material, it is likely that the time differences were contributed by room temperature and/or retarder concentration contained in the alginate. retarder concentration may vary in every alginate with different trade name, as what is used in the three studies above. alginate setting time obtained from this study may be useful for students during alginate manipulation. students may choose different water temperature based on their skill and competence to prolong setting time. therefore, less skilled students in impression technique with requirement to produce good alginate impression can still use normal setting alginate type by adjusting water temperature used to mix the alginate. this study showed that water temperature between 13° c to 28° c with a interval of 2.5° c produced significant differences in alginate setting time; the lower the water temperature used, the longer the setting time produced. references 1. nandini vv, venkatesh kv, nair c. alginat impression: a practical perspective. j conserv dent 2008; 11(1): 37–41. 2. ashley m, mccullagh a, sweet c. making a good impression: a ‘how to’ paper on dental alginate. dent update 2005; 32: 169–75. 3. irnawati d, sunarintyas s. functional relationship of room temperature and setting time of alginate impression material. dent j (majalah kedokteran gigi) 2009; 42(3): 137–40. 4. nallamuthu na, braden m, patel mp. some aspects of the formulation of alginate dental impression materials-setting characteristics and mechanical properties. dent mater 2012; 28(7): 756–62. 5. woortman r, kleverlaan cj, ippel d, feilzer aj, cavex holland bv. the effect of mixing method on the properties of alginate. scientific group programme. annual meeting of iadr continental european and israeli divisions 2007. 6. murillo-alvarez ji, hernandez-carmora g. monomer composition and sequence of sodium alginate extracted at pilot plant scale from three commercially important seaweeds from mexico j appl phycol 2007; 19: 545–8. 7. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231–48. 8. lemon jc, okay dj, powers jm, martin jw, chambers ms. facial moulage: the effect of a retarder on compressive strength and working and setting times of irreversible hydrocolloid impression material. j prosthet dent. 2003; 90(3): 276–81. 9. wright m. theories of chemical reaction-collision theory in an introduction to chemical kinnetics. john wiley & sons, ltd; 2004. p. 99–110. 10. rianti d. pengaruh variasi suhu air saat pencampuran bahan cetak alginat terhadap waktu pengerasan dan ketepatan model kerja yang dihasilkan. majajalah kedokteran gigi (dent j) 1998; 31 (4): 115–8. 11. barr jh, bowen r. ada specification no. 18 impression materials. in: guide to dental materials and device. 7th ed. chicago: american dental association; 1975. p. 219–23. 12. draget ki, smidsroed o, skjak-braek g. alginates from algae. in: steinbuchel a, rhoe sk, eds. polysaccharides, polyamides in the food industry properties, production and patents. wiley-vch verlag gmvh & co weinbeim; 2006. p. 379–82. 13. siew ck, williams p, young nw. new insights into the mechanism of gelation of alginate and pectin: charge annihilation and reversal mechanism. biomacromolecules. 2005; 6(2): 963–9. 136 vol. 43. no. 3 september 2010 treatment of sharp mandibular alveolar process with hybrid prosthesis sukaedi and eha djulaeha department of prosthodontic faculty of dentistry, airlangga university surabaya indonesia abstract background: losing posterior teeth for a long time would occasionally lead to the sharpening of alveolar process. the removable partial denture usually have problems when used during mastication, because of the pressure on the mucosa under the alveolar ridge. purpose: the purpose of this case report was to manage patients with sharp mandibular alveolar process by wearing hybrid prosthesis with extra coronal precision attachment retention and soft liner on the surface base beneath the removable partial denture. case: a 76 years old woman visited the prosthodontic clinic faculty of dentistry airlangga university. the patient had a long span bridge on the upper jaw and a free end acrylic removable partial denture on the lower jaw. she was having problems with mastication. the patient did not wear her lower denture because of the discomfort with it during mastication. hence, she would like to replace it with a new removable partial denture. case management: the patient was treated by wearing a hybrid prosthesis with extra coronal precision attachment on the lower jaw. soft liner was applied on the surface of the removable partial denture. hybrid prosthesis is a complex denture consisting of removable partial denture and fixed bridge. conclusion: it concluded that after restoration, the patient had no problems with sharp alveolar process with her new denture, and she was able to masticate well. key words: sharp alveolar process, hybrid prosthesis, precision attachment, soft liner abstrak latar belakang: kehilangan geligi posterior dapat menimbulkan processus alveolaris tajam. gigi tiruan sebagian lepasan mempunyai masalah selama pengunyahan karena adanya tekanan di mukosa di bawah alveolar ridge. tujuan: tujuan laporan kasus ini adalah untuk menjelaskan cara menangani pasien yang mempunyai prosesus alveolaris yang tajam di rahang bawah dengan dibuatkan protesis hybrid dengan daya tahan extra coronal precision attachment dan soft liner di permukaan bawah basis gigi tiruan sebagian lepasan. kasus: pasien wanita berumur 76 tahun datang di klinik prostodosia fakultas kedokteran gigi universitas airlangga. pasien memakai gigi tiruan lekat rentang panjang di rahang atas dan gigi tiruan sebagian lepasan akrilik free end di rahang bawah, pasien mengalami masalah waktu mengunyah. pasien tidak memakai gigitiruan lepasan rahang bawahnya karena tidak nyaman dipakai, dan pasien menginginkan pembuatan gigi tiruan lepasan rahang bawah yang baru. tatalaksana kasus: pada pasien ini dilakukan pembuatan hybrid prosthesis dengan daya tahan berupa extra coronal attachment di rahang bawah dan penggunaan bahan pelapis lunak yang diaplikasikan pada basis gigi tiruan lepasan nya. hybrid prosthesis adalah gigi tiruan himpunan yang terdiri dari gigi tiruan lepasan dan gigi tiruan lekat. kesimpulan: hasil perawatan menunjukkan setelah mengganti gigi tiruan dengan gigi tiruan sebagian lepasan yang baru, pasien tidak mempunyai masalah dengan gigi tiruan yang baru akibat processus alveolaris yang tajam dan pasien dapat mengunyah dengan baik. kata kunci: prosesus alveolaris yang tajam, hybrid prosthesis, precision attachment, bahan pelapis lunak correspondence: eha djulaeha, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ehadju@yahoo.com case report 137sukaedi and djulaeha: treatment of sharp mandibular processus alveolaris introduction the main function of removable partial denture is to support the mastication process of the patients. in order to improve the function, it must be designed for the convenience of the patient. unfortunately, in many cases, well designed removable partial denture still can not satisfy the patient’s need. the reason is because the success of the treatment for dental structural lost cases with removable partial denture is also determined by certain factors such as aesthetics, mastication, and phonetics. even though the removable partial denture is made according to the general clinical concepts, one of these three factors may still cause of failure.1 the most common dental structural cases suffered mostly by elderly patients are usually related to the loss of posterior teeth, pain of the alveolar bone, the thinness of soft tissue, and the sharpness of the alveolar process. the attachment of the removable partial denture, nevertheless, will be uncomfortable during mastication on the upper alveolar process. the pressure then continues to the mucosa at the edge of the alveolar process and the surface base of the removable partial denture. dentists should know and identify the detailed physical condition of the oral mucosa and structure according to the prosthodontic diagnostic index before designing a removable partial denture. precision attachment could be the right design to solve the problem of the posterior lower jaw. compared to the wire clasp, it also has better aesthetics. moreover, precision attachment can offer more biomechanical advantage than the wire clasp.2 another advantage of the precision attachment is that there are no buccal and lingual retention parts. since the retention does not depend on the crown contour, the stabilization becomes better and the pressure produced by the supporting teeth becomes minimal. eventually, patient will adapt more easily to the removable partial denture.3 however the use of precision attachment on the sharp alveolar process cases on the lower jaw still cannot solve the problem. the precision attachment should be combined with soft liner material applied on the bases of removable partial denture opposing the alveolar process. the function of soft liner is to cover the pain resulting from the mucosa pressure under the alveolar ridge during denture insertion.4 the aim of this case report was to improve mastication. considering that, the removable partial denture with the sharp alveolar process of the lower jaw may still cause pain. therefore, putting on the hybrid prosthesis with extra coronal precision attachment retention on the lower jaw and applying soft liner on the surface of the removable partial denture base will satisfy the patient’s need. case a 76 years old woman patient visited prosthodontic clinic, faculty of dentistry airlangga university. the patient has a long span bridge on the upper jaw (figure 1) and 34, 35, 36, 37, 38, 46, 47, 48 were lost. an acrylic removable partial denture on the lower jaw was made six months earlier. the patient could not masticate well, feel uncomfortable, and had not put on her denture for the past three months. there was no problem with the long span bridge on the upper jaw, yet she would like to have a new removable partial denture on the lower jaw. figure �. long span bridge on the upper jaw in the mouth. case management on clinical examination, the patient wore long span bridge on teeth number 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 25, 26 and acrylic removable partial denture on the lower jaw. patient has no problem with long span bridge on the upper jaw, the occlusion and radiographic photo were good and will be maintain as it is. anatomic duplication of her upper and lower jaws was conducted by using stock tray with irreversible hydrocolloid materials and was casted with type ii hard gypsum in order to make diagnostic model (figure 2). teeth number 33, 34 and 44, 45 were prepared for fixed splint before making the precision attachment on the lower jaw with extra-coronal attachment design. teeth number 33, 34 and 44, 45 were prepared with a shoulder-formed gingival margin for the making of porcelain fused to metal material figure �. diagnostic model of upper and lower jaws. 138 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 136–140 (figure 3). extra-coronal attachment was located outside the crown contour. generally, for female patient it is located on the removable partial metal denture of the lower jaw. figure �. fixed splint of lower jaw with extracoronal attachment in the mouth. figure 5. the removable partial metal denture of lower jaw. figure 6. the maxillary long-span bridge and the mandibular removable partial metal denture inside mouth. figure ��. lower jaw teeth preparation. the next stage was duplicating the lower jaw in order to make the fixed splint. by using tray, the duplicating process of the lower jaw was conducted by using putty materials. the result, especially in 33, 34 and 44, 45 areas, was swapped about 2 mm, filled with regular duplicating material or light body, and then the duplicating of the lower jaw was conducted for the second time. afterwards, the duplicating result was casted with type iii hard gypsum and used as working model. the making of the fixed splint was for teeth number 33, 34 and the 44, 45. the temporary cantilever bridge for teeth number 33, 34 and teeth number 44, 45 was made by duplicating diagnostic model with stock tray using putty materials. afterwards, the duplicating areas of 33, 34 and 44, 45 were filled with self curing acrylic materials. it was then reduplicated into the patient‘s mouth and the result was the temporary cantilever bridge. the temporary cantilever bridge was smoothened, polished, and inserted into the patient’s mouth. next, it was followed with bite registration by using bite rim. the working model of upper and lower jaw was put into the articulator, and sent to the laboratory to be prepared for the fixed splint with extra coronal attachment. after the fixed splint was copied, it was tested into the patient in order to check the occlusion. finally it was sent back to the laboratory for finishing. the fixed splint with extra coronal attachment was designed by using porcelain fused to metal, and tested into the patient in order to check the occlusion and the aesthetics (figure 4). the semi-permanent cementation was conducted after the fitting the aesthetic was good. the molding of the lower jaw was conducted for the second time by using putty material, teeth number 33, 34 and 44, 45 were swapped about 2 mm, and filled with regular molding material or light body, in which fixed splint was inserted in to the mold. the result of the molding then was filled with type iii hard gypsum and used as working model. then the bite registration was conducted on the working model of the lower jaw by using wax bite rim. the molding of the upper jaw was conducted with stock tray and irreversible hydrocolloid. the result then was poured with type iii hard gypsum and used as working model. the working models of the upper and lower jaws were placed on the articulator. after that, the removable partial metal denture was sent to the dental laboratory (figure 5), before it was ready to be inserted into the patient. in order to solve the problem with sharp alveolar process, the surface of the removable partial metal denture facing the anatomy surface was ragged and soft liner was applied on the surface of the removable partial metal denture base. the removable partial metal denture of the lower jaw then was inserted into patients (figure 6). during 139sukaedi and djulaeha: treatment of sharp mandibular processus alveolaris the attachment of the removable partial metal denture in the lower jaw, the occlusion was corrected by using articulating paper for adjustment in order to obtain the stable occlusion of the denture. discussion the structural loss with permanent deformity of the residual alveolar ridge occurred as the result of congenital defects, periodontal disease, and tooth extraction after surgical procedures. therefore, removable partial denture is needed to maintain the defective function of mastication. in other words, the function of the removable partial denture is to maintain the remaining structure without causing any lost of the supporting teeth or health problems of the temporo mandibular joint.5 furthermore, since the surface of the lower jaw was smaller than the upper jaw and the production of the removable partial denture was not good enough. the removable partial denture may loosen or break easily. the patients will also have problem with the distal extension on both sides of the lower jaw and with the sharp alveolar process. as a result, better mastication function was surely required.6 in fact the distal extension of the removable partial denture was supported by two different tissues: the hard tissue that was supporting the teeth and soft tissue that was covering the mucosal area. these differences will cause the removable partial denture to rotate through three planes if pressure exists on the removable partial denture. moreover, even a small pressure can still cause lifting power on the supporting tooth. if there was a pressure on the occlusal surface of the teeth, the removable partial denture would move and cause both rotating pressure on the teeth located at the most distal position and as well as trauma on the tissue. in other words, it was important to consider the supporting factors in choosing and deciding the design of the removable partial denture with distal extension.7,8 the removable partial denture with the distal extension in the posterior area also has tendency to lift up during mastication of sticky food. unfortunately, some types of wire clasp cannot prevent this movement, for example, when the removable partial denture rotates the edge of the wire clasp. the tendency of this rotation, however, can be prevented by combining structures known as indirect retainer.3 in addition, the indication of the use of extra coronal attachment can be determined by the condition of vertical height, mesio-distal space, and the distance between buccolingual teeth. it means that in order to fulfill the indication of using extra-coronal attachment, there must be enough distance for the vertical height, the wide mesiodistal space, and the wide bucco-lingual space on tooth number 33, 34, 44, and 45. furthermore, since the attachment does not make the removable partial denture to attached to the supporting teeth, but only to serve as a connection that enables movements between two components of the removable partial denture, it was better to use the extra coronal attachment on the lower jaw. thus, by using stress breaker, the pressure against the supporting teeth will be minimized. the use of precision attachment will not cause abrasion on the supporting teeth.2,3 the removable metal partial denture and the aspects of aesthetic and retention of the precision attachment were better than the wire clasp. nevertheless, the removable metal partial denture has some disadvantages that probably affect periodontal inflammation, abrasion on the abutment tooth, and make the wire clasp to move the abutment tooth when the removable metal partial denture was used in mastication.2,9 retention and stabilization of the removable partial denture depends on the maximum widening of the removable partial denture base covering the alveolar process, such as distolingual wing located physiologically on the retromylohyoid fossa. there will also be a problem on certain cases which the patients have sharp alveolar process. the pressure on sharp alveolar process during insertion period could be an anatomical problem. the base of the removable partial denture sometimes do not fit well and can reduce the retention and stabilization of the denture. therefore, it was better to use soft liner on the removable partial denture base since it can reduce the pain during mastication. moreover, since the characters of this soft liner material are elastic and viscoelastic, it can improve the convenience of the patients during mastication by protecting the mucosa and spreading the pressure evenly. as a result, optimal adaptation between the removable partial denture base and the procesus alveolaris will be acquired. however, since the attachment system of the soft liner was not conducted chemically, but physically, the rigidity on the layer of the removable partial denture base were loosened. the loosening of the removable partial denture base must be conducted during the application in order to improve the retention. the soft liner material has some weaknesses, soft liners could not resist longer, soft liner base must be replaced at least twice a year and then disinfection must also be conducted in order to prevent cross contamination.10 thus, regular cleaning of the removable partial denture must be performed by using cleaners such as polydent, chlorhexidine, sodium hypochlorite, or glutaraldehyde.4,11 it concluded that after restoration, the patient had no problems with sharp precessus alveolaris with her new denture, and she was able to masticate well. references 1. hickey jc, zarb ga, bolender cl. boucher‘s prosthodontic treatment of edentulous patients. 10th ed. st louis: the cv mosby co; 1990. p. 237–46. 2. ponsta. attachment more aesthetics than clasps. the dent liner journal 2004; 8(3): 1–4.3): 1–4. 3. preiskel hw. precision attachment in dentistry, 2nd ed. st louis, toronto, princeton: mosby company; 1973. p. 41–51. 140 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 136–140 4. hamada taizo, murata h, razak a. pelapisan gigi tiruan denture lining. surabaya: airlangga university press; 2003. p. 48–53.surabaya: airlangga university press; 2003. p. 48–53. 5. holmes jb. influence of impression procedure and occlusal loading on partial denture movement. j prosthet dent 2001; 11: 335–40. 6. carr ab, brown dt. mc cracken's removable partial denture prosthodontic. 11th ed st. louis, missouri: mosby co; 2011. p. 123–4. 7. habir e. prinsip-prinsip perawatan untuk kasus ekstensi distal. jitekgi. fkg ugm 2006; 101–4. 8. frenchette ar. the influence of partial denture design on distribution of force to abutment teeth. j prosthet dent 2001; 85: 527–58. 9. rosentiel sf, land mf, fujimot j. contemporary fixid prosthodontics. 4th ed. st. louis, missouri: mosby co; 2006. p. 666. 10. jorgenson eb. maternals and methods for deaning denture. j prosthet dent 1979; 42: 619–23. 11. abelson. denture plaque and denture cleaners. j prosthet dent 1981; 45: 376–9. vol 38-no4-2005-isi.pmd 169 elimination of oral focal infection: a new solution in chronic fatigue syndrome management? haryono utomo klinik vip rsgm fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract chronic fatigue syndrome (cfs) is an illness whose primary symptoms are debilitating fatigue and cognitive dysfunction. substantial symptom overlaping with fibromyalgia, depression, allergic diseases, and many other illnesses, and the absence of a universally applicable diagnostic test, makes the diagnosis of cfs complex and challenging. the pathophysiology of cfs is also complex, and not clearly understood. formerly, cfs was believed to be a variant form of depression, but due to an increasing body of evidence, cfs is now considered primarily as a biochemical derangement of the functioning of the neuroimmune and neuroendocrine systems. recently, most treatments still primarily emphasize analgesics, anti inflammatory and psychiatric treatment which correlates to psychosomatic disorders. one of the symptoms that is poorly understood is allergy, but according to the neurogenic switching hypothesis the correlation can be explained nowadays. the role of oral focal infection as one of the possible etiology has still rarely been discussed. the goal of this article is to explain the possible pathophysiology of cfs which could be elicited by oral focal infection, especially endotoxin (lipopolysaccharide) from gram negative bacteria. this case report discusses the history of illness, previous treatments, diagnosis, case management and treatment result. periodontal treatment of a patient with symptom mimicking cfss undergoing periodontal treatment has a remarkable result. the conclusion is that the elimination of oral focal infection could be a new solution in cfs management. key words: chronic fatigue syndrome, oral focal infection, neurogenic switching correspondence: haryono utomo, c/o: klinik vip spesialis terpadu, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo 47 surabaya 60132, indonesia. telp. (031) 5053195. introduction the proposed criteria for the diagnosis of chronic fatigue syndrome (cfs) are the presence of persistent and disabling fatigue for at least six months, coupled with an somatic and neuropsychological symptoms that are headache, myalgias, migrating arthralgia, sore throat, forgetfulness, difficulties in concentration and thinking, cervical or axillary lymphadenopathy, low-grade fever, irritability, and sleep disturbances.1 other symptoms are allergies, chest pain, rapid pulse, abdominal cramps, night sweats and rash.2 the pathophysiology and the etiology of cfs is still unclear, but the possibility of some obscure metabolic or immunologic derangement secondary to viral infection must be considered, but most of the cases lack such a history.1 one of the symptoms which are very disturbing in cfs is headache, that are tension headache and migraine.3 several headache that mimic the criteria of tension headache or migraine also accompanied by sinus pain, rhinorrhoe and nasal congestion and diagnosed as sinus headache, the problem are after consulting to an otolaryngologist (ent specialist) it were found that they had normal sinuses.4 however, cfs also manifest allergic symptoms that may confused with sinus headache symptoms and complicate the diagnosis.5 the pathophysiology of this phenomenon is still in controversy, but there is a hypothesis proposed by meggs so called neurogenic switching which can be considered as one that can satisfactory explain most of the mechanism of migraine with sinusitis-like symptoms.4–6 neurogenic switching explain that there is an interplay between neurogenic inflammation and immunogenic inflammation, for example the pathophysiology of allergic propagation, food allergy can elicit urticaria, rhinitis and asthma. ingestion of foods or drugs as well as cutaneous inoculation with vespid venom can trigger systemic anaphylaxis. however, in experimental models of anaphylaxis, ablation of neuronal pathways eliminates the anaphylactic response without blocking histamine release or antibody production. this switching of the site of inflammation in allergy and chemical sensitivity may be due to the same mechanism: there are neuronal pathways from the site of stimulation through the central nervous system to other peripheral locations. this mechanism of site switching has been termed neurogenic switching.6 the main chemical mediator involved in neurogenic switching are histamine and substance p (sp).4,6 other symptoms that may accompany are vestibular or cochlear disturbances and 170 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 169–172 it were caused by sp in the inner ear that stimulates expression of endothelium-leukocyte adhesion molecules from cochlea microvasculatures which decreases blood flow to cochlear sites resulting vestibular and hearing disorders.7 headache could be elicited by endotoxin (lipopolysaccharide) from gram negative bacteria that induced macrophages producing proinflammatory cytokines interleukin -1α (il-1β), il-6, tnf-α8–10 and other chemical mediators i.e. prostaglandin e2 (pge2) and nitric oxide (no). 8 prostaglandin e2 mediates vasodilatation, increases vascular permeability, enhances pain perception on nociceptor to bradykinin and histamine,8,9,11 and induce proliferation of t helper2 lymphocytes that are responsible for the development of allergy.12 excessive production of pge2 and no alter neurovascular condition which could elicit migraine.8,9 nervous system also contributes to the pathophysiology of peripheral inflammation including periodontal inflammation. inflammation that present in oral and periodontal tissue is mostly stimulated by endotoxin (lps), lps also stimulate nerve endings to release neuropeptides i.e sp which account for neurotransmitting and vasodilation. release of sp could be stimulated by hot temperature, capsaicin, bradykinin and tryptase (enzyme produced in mast cell degranulation). stress enhances the secretion of no and other inflammatory mediators in response to lps derived from porphyromonas gingivalis, thus providing accelerated periodontal destruction.13 mast cells which have an important role in immunologic inflammation are present in oral and periodontal tissue. degranulation of mast cells release histamine, arachidonic acid metabolites (i.e. leukotrienes, prostaglandins), enzymes (i.e. tryptase) and cytokines (i.e. il-1, il-6, tnf-α).14 it can be triggered by antigens, bacteria,13 neuropeptides (sp and cgrp), chemokines, calcium ionophores and physical factors (i.e cold temperature, exercise, trauma).14 substance p has also directly effect smooth muscle and indirectly induced vasodilatation of blood vessels.15 periodontal inflammation give rise to sp, neurokinin a (nka) and vasoactive intestinal peptide (vip) in gingival crevicular fluid.15 periodontium act as cytokine reservoir, the proinflammatory cytokines tnf-α, il-1β, and gamma interferon as well as pge2 reach high tissue concentrations in periodontitis. the periodontium can therefore serve as a renewing reservoir for spillover of these mediators, which can enter the circulation and induce and perpetuate systemic effects.16 oral focal infection, has a long history of controversy, however plenty of successful results of the evidence-based therapies reported. three mechanisms or pathways linking oral infections to systemic effects have been proposed that are 1) metastatic spread of infection from the oral cavity as a result of transient bacteremia, 2) metastatic injury from the effects of circulating oral microbial toxins (i.e lps from gram negative bacteria), and 3) metastatic inflammation caused by immunological injury induced by oral microorganisms.16 in this case, symptoms of the patient that mimicking and could be related to so called chronic fatigue syndrome are gone after scaling. the problem is how to explain pathophysiologically the successful management that happens and what is the main cause of the symptoms which are related to oral focal infection. case a male patient, 37 years, came to the clinic in the university of airlangga faculty of dentistry in august 2004 complained about continuous shoulder muscle pain, headache and runny nose. symptoms began in 2002, started with runny nose then followed by warm feeling in the ear. he had already suffered for 4 months then and had been examined and treated by a general practitioner but symptoms still exist. gradually the symptoms were getting worse everyday, and then he suffered from headache and warm feeling in his neck. the pain spread downward to the back and headache was felt in different places all over the head, the worse pain was in the neck. in 2004 backache went downward to the leg, the pain caused sleep disturbances, in the morning when he woke up the fatigue increased. patient consulted to an internist, the medicaments helped for a while, but symptoms arise after the medicaments were stopped. vertigo then arise and patient couldn't do daily work included driving and reading because everything seems to move and spinning. after consulting to a neurologist, patient was treated as vertigo patient but he still didn't feel well. patient already have a lot of treatment and medications, including massage. some of the medications were anti hypertensive, analgesic, antibiotic, anti vertigo, tranquilizer, anti depressant, muscle relaxant, cerebral and peripheral vascular vasodilator and anti inflammatory. in extra-oral examination, patient looked tired in the eyes and fatigue, red and hypertrophic gingiva but only a bit of calculus were seen in intra-oral examination because the patient also has visited a dental practitioner about 2 months ago, 36 was extracted because of mobile and painful. gingival bleed easily after probing. periodontal pockets were found in all regios with average depth 3–5 mm except in distal 46 was 7 mm and 47 were 6 mm. in orthopantomograph revealed the existence of horizontal resorption in all regios and vertical resorption in 46 (figure 1). figure 1. orthopantomograph. 171utomo: eliminasi of oral focal infection case management at first visit patient were asked about the medical and dental history, kinds of treatments that have already done, medications prescribed and the result of the treatments. superficial scaling were done with piezoelectric scaler and cleaning the debris and food impaction inside the pocket with half-moon explorer on the right side then the left side of the patient, after finishing on the right side patient feel more comfortable, headache and shoulder ache gradually disappear. then scaling were done to the left side, the result was the same. patient was prescribed chlorhexetidine 0,1% mouth wash and thiamfenicol 500 mg, he was told to take the antibiotics 2 hours before next visit three days later. the second visit patient was asked about the treatment result and the result was remarkable because patient felt very well, headache and rhinitis stopped no sleep disturbances, backache also disappeared, patient still felt uncomfortable in the leg but was getting better. intra oral examination revealed that the gingival was more healthy, pink colored and not easily bled. before deep scaling was performed, patient was local anesthetize with xylocaine adrenalin, during scaling periodontal pockets were flushed intermittently with hydrogen peroxide 3%. patient was then scheduled next visit in one week time. at the next visit pain in the legs was disappear completely and there was no complain about the recurrence of the symptoms, patient were told to check up every 6 months. on october 12nd, 2005 patient were evaluated and the symptoms did not recurrent. discussion the exact etiology and pathophysiology of chronic fatigue syndrome (cfs) is still unclear, some researchers propose the possibility of past viral infections but there are cases that the patients don't have the same medical history.1 some cases are accompanied with allergies such as rhinitis symptoms and sinus–headache, so the diagnosis and the treatment planning still confusing.4,5 drug of choice of cfs still symptomatic such as anti-inflammatory, anti depressant, corticosteroids and antibiotics.1,5 chronic fatigue syndrome (cfs) are also suspected as derangement of immune, neurologic and endocrine system,2,5 so it is still a dilemma: what is the treatment of choice, should we give all kind of medication, how about drug interactions and toxicity?. as proposed by meggs6 that is the neurogenic switching hypothesis, substance p (sp) produced by sensory neuron can elicit a neurogenic inflammation by degranulate mast cell thus combine neurogenic and immunologic inflammation. in this hypothesis the trigger of sp release by sensory nerves is chemical agents, lps can also indirectly triggers sp release by inducing macrophage to produce cytokines which stimulate the sensory nerves.15 reflected to the fact that immunecompetent cells and sp producing nerves are also present in oral tissue so it can be predicted that resolution of oral inflammation also diminished the symptoms which related to neurologic and immunologic inflammation. the explanation as follows: at site a, chemical irritants, ch interact with sensory nerve fibers to trigger release of substance p, sp, and other mediators of neurogenic inflammation. at site b, antigens, ag, are interacting with antibody on mast cells to release histamine, h, and other mediators of allergic inflammation. histamine interacts with nerve fibers to produce signal transmission to the central nervous system. at site c these mediators acting on effector cells to produce an inflammatory response. at site d there is an inflammation that being triggered at a site distant from the stimuli. signals from a or b are rerouted through the central nervous system to site d, where substance p, sp, is released from the nerve endings to initiate an inflammatory response (figure 2).6 mast cell mast cell effector cell effector cell figure 2. neurogenic switching.6 fibers innervating the periodontal tissues in human are immunoreactive to a number of neuropeptides, including sp, calcitonin gene-related peptide (cgrp), vasoactive intestinal peptides (vip) and neuropeptide y (npy). substance p has been located by immunohistochemistry in normal gingival tissue perivascularly and within the rete pegs. nerve fibers originating in the subepithelial connective tissue may also penetrate the junctional epithelium, junctional epithelium is extensively innervated by sp nerve fibers.13,15 in oral inflammation, neurogenic mechanism happened as follows, gram negative bacteria lps stimulate macrophage to produce cytokines. cytokines then stimulate the sensory nerves to release neuropeptides i.e sp and cgrp, sp could also interact with immune cells causing proliferation of t lymphocytes. there are several factors influencing the release of sp from sensory nerves, 1) cleavage of protease activated receptor-2 (par-2), 2) bradykinin binding to b2 receptors, 3) sensitization of cytokines produced by macrophages induced by lps and 4) nitric oxide (no) (figure 3).15 172 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 169–172 according to the past medical history, patient was prescribed anti-hypertensive, anti depressant, anti-vertigo, analgesics, muscle relaxant, cerebro-vascular vasodilator. the symptoms are relevant to chronic fatigue syndrome (cfs) criteria and still exist more than 6 months, we can conclude that this patient also suffered from cfs. in this patient, the symptoms initiates by rhinitis and warm feeling in the right side since 2002, was it the first inflammation which is the main source of the problem. considering the patient's age, oral and periodontal health and their effect to the alveolar resorption, the poor periodontal health initiate a long time before 2002. in this case neurogenic switching hypothesis might be began with the periodontal inflammation which also a source of sp, proinflammatory cytokines and chemical mediators. substance p then stimulates mast cell degranulation, histamine and sp then activate effector cells (i.e. lymphocytes) which also produced tnf-α. lymphocytic infiltrates are common to be seen in cfs patient. tumor necrosis factor-α make defect in tight junction integrity of epithelial cells, when the epithelial barrier is lost, neurogenic inflammation may be triggered at much lower doses. the result is ongoing inflammation, which in turn continues the damage to the epithelial barrier, if the distant inflammation occurs in the nose then symptoms of rhinitis appear, in another part of the body can cause muscle spasm and tenderness, tension headache and migraine.5,6 consistent pain in shoulder, neck and head could be triggered from the proinflammatory cytokines il-1β, il-6 and tnf-α mostly produced by macrophages induced by lps.10 stress which also the part of his complains also inducing the secretion of no and production of inflammatory that enhanced the periodontal destruction14 and in a systemic way could trigger head ache. 10 prostaglandin e2 that is arachidonic acid metabolites from the lipooxygenase pathway lowered the pain threshold and excitability of the sensory nerves and also an immunosuppressant that will cause the patient painful and fatigue in the muscles, joint and elsewhere in the body.9 systemic pge2 could also stimulate the humoral immunity (th2) which related to allergy diseases i.e. food allergy, allergic rhinitis and asthma, il-4 and il-13 induced b lymphocyte into ige specific producing plasma cells instead of igg specific (isotype switching mechanism).12 vertigo also suffered by the patient, it can be the effect of neuropeptide sp that affect the expression of endotheliumleukocyte adhesion molecules from cochlear microvasculatures which decreases blood flow to cochlear sites and cause vestibular and cochlear disorders.8 in the conclusion, oral focal infection an inflammation that includes neurogenic and immunologic response could also play an important role in the pathophysiology of cfs. elimination of oral focal infection is able to relief cfs symptoms, the first reason is because lps from oral infection can stimulates both neurogenic and immunologic inflammation, and the second reason is that oral tissue has sp producing sensory nerve fibers and immunocompetent cells such as mast cells and macrophages which needed for neurogenic switching mechanism. references 1. victor m, ropper ah. principles of neurology. 7th ed. new york: mc graw hill; 2001. p. 175–6. 2. kasper dl, braunwald e, fauci as, hauser sl, longo d. harrison's principles of internal medicine. 16th ed. new york: mcgraw-hill; 2005. p. 546. 3. american academy of otolaryngology. sinus headache facts. 2002; 20(1):10. available at:url http: //www.sinusnews.com. accessed september 5, 2005. 4. cady rk, schreiber cp. sinus headache or migraine? considerations in making a differential diagnosis. neurology 2002; 58:s10–s14. 5. gordon br. chronic fatigue syndrome: an allergic entity? current opinion in otolaryngology & head & neck surgery 2000; 8(3):253– 9. 6. meggs wj. neurogenic switching: a hypothesis for a mechanism for shifting the site of inflammation in allergy and chemical sensitivity. environ health perspect 1997; 105(2):54–6. 7. seelig m. review and hypothesis: might patients with the chronic fatigue syndrome have latent tetany of magnesium deficiency. j chr fatigue synd 1998; 4(2):1–41. 8. stirparo g, zicari a, favilla m, lipari m, martelletti p. linked activation of nitric oxide synthase and cyclooxygenase in peripheral monocytes of asymptomatic migraine without aura patients. cephalalgia 2000 mar; 20(2):100–6. 9. schaechter m, engleberg nc, eisenstein bi. microbial disease. 3rd ed. philadelphia: lippincott william wilkins; 1999. p. 123–5. 10. jeong hj, hong sh, nam yc. effect of acupuncture in inflammatory cytokine production in patients with chronic headache. am j chn med 2003; 31(6):945–54. 11. tsai cc, hong yc, chen cc, wu ym. measurement of prostaglandin e2 and leukotriene b4 in the gingival crevicular fluid. j dent 1998; 26(2):97–103. 12. mckay ab, rosen fs. allergy and allergic diseases. n eng j med 2001; (1):30–6. 13. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol med 2004; 15(2):82–98. 14. newman mg. carranza's clinical periodontology. 10th ed. philadelphia: saunders; 2002. p. 27, 113–47. 15. walsh lj. mast cells and oral inflammation. crit rev oral biol med 2003; 14(3):188–98. 16. lie xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection clinical. microbiology reviews 2000; 13(4):547– 58. figure 3. factors influencing the release of substance p from sensory nerves.15 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 185185 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 185–188 color stability of heat polymerized polymethyl methacrylate resin denture base after addition of high molecular nano chitosan ika devi adiana,1 trimurni abidin,2 and lasminda syafiar1 1department of dental material 2department of conservative dentistry faculty of dentistry, universitas sumatera utara medan indonesia abstract background: the addition of other ingredients to maintain color stability of heat polymerized polymethylmethacrylate is being developed. one of them is by adding high molecular nano chitosan. purpose: this study aimed to determine the color stability of heat polymerized polymethyl methacrylate denture base resin after an addition of high molecular nano chitosan. method: 30 sample pieces of acrylic plate (40x10x2 mm) were divied into 6 groups: control group and groups with the addition of chitosan nano gel percentages of 0.25, 0.50, 0.75, 1.0 and 1.50%. 2 ml chitosan nano gel was added into the mixture of acrylic resin with 23 g : 10 ml (p : l). after the mixture was inserted into a mold and then pressed and cured at 74oc for 120 minutes and then 100o c for 60 minutes. acrylic plates were then released from the mold and finished. color stability of acrylic resin were measured using uv-vis spectrophotometer and analyzed with a one way anova. result: the results showed significant differences in color stability after the addition of high molecular nano chitosan. the best color stability found in 1.0% the addition of chitosan nano gel group, the value was 0.07589 cm-1. conclusion: the chitosan nano gel can be used to maintain color stability of heat polymerized polymethyl methacrylate denture base resin. keywords: denture base; color stability; high molecular nano chitosan correspondence: ika devi adiana, department of dental material, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2 kampus usu padang bulan medan 20155, indonesia. e-mail: devi_ika27@rocketmail.com introduction the use of heat polymerized polymethyl methacrylate (acrylic) resin as denture base materials is becoming more common due to its esthetic, slightly transparent color.1-3 the ideal denture base materials should have the closest possibility to natural color. color stability is one of the clinical characteristics that is essential to denture base. color stability is the ability of a surface or color substance to avert degradation by environmental factors. for longterm usage, the color of heat polymerized acrylic resin will change due to degradation process in the material and disturb the esthetic of the denture.4 the discoloration of denture base might be due to both intrinsic and extrinsic factors. the intrinsic factors include chemical changes of the material due to addition of enforcing material/substance in the composition of acrylic resin, while extrinsic factors include stain caused by absorption of colored materials in exogenous sources, such as tea, coffee, soft drink, food, nicotine, mouthwash, denture cleanser and the interaction of other chemical material in the oral environment.4-7 the study showed significant discoloration occurred in heat polymerized polymethyl methacrylate and acrylic resin that were injected in coffee and nicotine intermittently (p<0,05). pmma showed discoloration value after 3 day soaking in several substances (coffee, saliva, tea and food colored materials).7 in order to maintain color stability of heat polymerized acrylic resin denture base to preserve esthetic function, substance binding with the pigment of heat polymerized acrylic resin is added to prevent its degradation. one of the materials with a good biodegradibility is chitosan. several other special properties of chitosan include good biocompatibility, good bioadhesion, non-toxic, non-allergic, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p185-188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p185-188 186 adiana, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 185–188 non-carcinogenic and safety for usage, hence its common use in biomedical application. chitosan is a broad spectrum agent and highly active in killing gram-positive and gramnegative bacteria as it is able to change the permeability of the bacteria cell that causes cell death. based on its viscosity, chitosan is classified into three molecular mass, i.e. low, moderate and high molecular chitosan. high molecular chitosan is obtained from horsecrab cells with deastilization degree of 84.20% and molecular mass of 893.000 mv. in its development, chitosan is modified in magnetic forms. nano chitosan particles sized 100-400 nm could increase its absorption ability. the use of chitosan in nanometer scale could increase its surface area, eventually increasing its affectivity in binding with other chemical clusters. previous studies on addition of high molecular nano chitosan in pmma showed several benefits, such as increases of flexural strengths, tensile strengths, and young’s modulus values. it is hoped that addition of nanochitosan would increase the color stability of heat polymerized polymethyl methacrylate, as it creates stronger and more stable bond (cross link). this study aimed to determine the color stability of heat polymerized polymethyl methacrylate after the addition of high molecular nano chitosan in various concentrations. material and methods t h e s t u d y i n v o l v e d h e a t p o l y m e r i z e d polymethylmetacrylate (qc 20 uk) and high molecular nano chitosan obtained from horsecrab to increase color stability of denture base material. chitosan gel is made by homogenously dissolving 0.25 gr, 0.50 gr, 0.75 gr, 1.0 gr and 1.50 gr of high molecular nano chitosan powder in becker glasses filled with magnetic stirrer and 100 ml of 1 % acetate acid on 200 rpm hot plate. the solution was added with 20 drops of tripolyphosphatepotassium (tpp) and stirred for 1 hour to form emulsion. it was then placed in ultrasonic bath for 20 minutes and centrifuged for 30 minutes at 3600 rpm to break the chitosan particle to form a high molecular nano chitosan gel. to ensure the nano size of chitosan particle, the gel was measured by particle size analysis (psa). the study used 30 pieces sample of heat polymerized polymethyl methacrylate plate (size 40 mm x 10 mm x 2 mm) as sample. the samples were divided into 6 treatment groups, i.e. control group (with no addition of high molecular nano chitosan), and groups that were added with 0.25%, 0.50%, 0.75%, 1.0% and 1.50% of high molecular nano chitosan gel. molds of heat polymerized polymethyl methacrylate plate were made by mixing hard gypsum in water (300 gr gyps in 90 ml water) to fill bottom half of the cuvette. metal plates were placed in the cuvette, their surface parallel to that of the gypsum. after the gypsum was set, the surface was applied with vaseline. the top and bottom halves of the cuvette were assembled and hard gypsum (300 gr gypsum in 90 ml water) was poured into it. the cuvette was then placed on a vibrator. after 45 minutes, the cuvette was disassembled, the plates were retrieved and the surface was applied with cold mold sealeant. polymer of polymethyl methacrylate was mixed with the monomer in acrylic pot (23 gr: 10 ml) and 2 ml of nano chitosan gel 0.25%, 0.50%, 0.75%, 1.0% and 1.5% were added separately with scaling pipe (control group received no addition of nano chitosan gel), the mixture was stirred with lecron to reach its dough-stage. the dough was then poured into the muld. the heat polymerized polymethyl methacrylate was covered with cellophane sheet and the top half of cuvette was assembled. the cuvette was then pressed with 1000 psi hydraulic press, then dissembled to rid excess acrylic resin, then re-pressed with 1200 psi press. afterwards, the cuvette was cured. first, the cuvette was put in a 74o c water bath for 120 minutes. then, the temperature was raised to 100oc for 60 minutes. the samples were taken out from the water bath. the samples were trimmed and polished to the desired size. 9 figure 4. uv-vis spectrophotometer. table 1. mean and deviation standard of color stability of heat polymerized polymethyl methacrylate sample color stability (cm-1) control chitosan 0.25% chitosan 0.50% chitosan 0.75% chitosan 1.0% chitosan 1.5% 1 2 3 4 5 0.07329 0.07327 0.07274 0.07410 0.07384 0.07391 0.07361 0.07422 0.07323 0.07271 0.07372 0.07401 0.07404 0.07375 0.07361 0.07492 0.07491 0.07492 0.07513 0.07509 0.07689 0.07533 0.07602 0.07608 0.07515 0.07535 0.07672 0.07516 0.07500 0.07547 mean + sd 0.07345+ 0.000533 0.07354+ 0.000589 0.07383+ 0.000189 0.07499+ 0.000107 0.07589+ 0.000692 0.07554+ 0.000684 figure 5. graphics of color stability value of heat polymerized polymethyl methacrylate with and without addition of nano chitosan gel 0.25%, 0.50%, 0.75%, 1.0% and 1.5%. figure 1. graphics of color stability value of heat polymerized polymethyl methacrylate with and without addition of nano chitosan gel 0.25%, 0.50%, 0.75%, 1.0% and 1.5%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p185-188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p185-188 187187adiana, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 185–188 the samples were then ground with carbide burrs, and pounded with mortar and stamper, then 0.3 gr of the samples were put into separate vials and each dissolved in 10 ml of xylene. color stability measurement was performed with a uv-vis spectrophotometer. the solution was put in tester cuvette, and the computer screen will measure its absorbance value with 552 nm wavelength. the values showed on screen were noted. the unit of measurement of this equipment was cm. results the color stability data from the six treatment groups were obtained in this study. graphic of the color stability value of heat polymerized polymethyl methacrylate resin denture base with and without addition of nanochitosan gel of 0.25 %, 0.50 %, 0.75%, 1.0% and 1.5% can be seen on figure 1. all treatment groups showed increase of values, but the group with 1% addition of nanochitosan gel presented significant increase of color stability compared to the other groups. in the study, the influence of 0.25%, 0.50%, 0.75%, 1.0% and 1,5% nanochitosan gel additions on color stability of heat polymerized polymethyl methacrylate was tested by the one way anova test, and show significance value of p = 0. to determine difference of color stability between the control group and other treatment groups with addition of 0.25%, 0.20%, 0.75% 1.0% and 1.5% nano chitosan gel, a least significance difference (lsd) test on absorption value of heat polymerized polymethylmetacrylate was performed and can be observed in table 1. discussion the differences in absorption values of heat polymerized polymethyl methacrylate resin denture base were shown in groups with and without nanochitosan gel addition. the highest color stability (0.07589 cm-1) was observed in group with addition of 1.0% nanochitosan gel, and lowest color stability (0.07345 cm-1) was found in group without addition of nanochitosan gel. if the forwarded light intensity is higher than the reflected one, the wavelength will increase. then, the results are in brighter and better stability of color. on the contrary, if forwarded light intensity is lower, wavelength will decrease which results in darker and worse stability of color.2 in the study, color stability of groups with addition of nano chitosal gel is higher than group without addition of nano chitosan gel. this shows a better color stability of group with nano chitosan gel. in modification process of chitosan to nanochitosan, crosslink agent (tripolyphosphatepotassium) is needed increase its stability in acid, due to its high solubility in organic acid. addition of this material (crosslink agent) will lower the adsorption ability of chitosan, leading to higher physical endurance.13 this will promote a better color stability of heat polymerized polymethyl methacrylate added with nanochitosan. several factors contributing to discoloration are chemical degradation, oxidation, oral hygiene, imperfect polymerization process, and water absorption.14 excessive water absorption in denture base material could cause discoloration.15 the liquid absorbed in the process of diffusion will fill the spaces in matrix, resulting in structural and physical changes of the resin. water absorption will dissolve some components, and will cause discoloration of a denture base materials.4 one of the factors that determine the color stability is the permeability of a material.16 absorption and desorption process are related to its environment. the material of polymer that absorbs air, both in air and water, could lead to expansion and mechanic strength changes in the material.17 in group with addition of nano chitosan gel, greater color stability could be observed due to lower water absorption and fewer residual monomer. this is due to the fact that the addition of nano chitosan gel to the polymer material as a filler will restore the properties of polymer material. the nano size and gel form of high molecular chitosan will affect the color stability of pmma. the smaller particle size will increase surface areas that leads to intensify the diffusion of chitosan to acrylic, eventually forming more bond of cluster –nh2 and color substance. the stronger bond formation will result in better color stability. in the process of dissolving heat polymerized acrylic resin added with nanochitosan gel in xylene solution, the resin will not dissolve due to chitosan’s insoluble properties. when this bond occurs hydrolysis reaction can not proceed and chitosan will bind the colored substances tabel 1. least significance difference (lsd) test on absorption value of color stability of heat polymerized polymethyl methacrylate (i) group (j) group mean difference (i-j) sig. control 0.25% 0.5% 0.75% 1.0% 1.5% -.000088 -.000378 -.001546* -.002446* -.002092* .791 .262 .000 .000 .000 0.25% 0.5% 0.75% 1.0% 1.5% -.000290 -.001458* -.002358* -.002004* 387 .000 .000 .000 0.5% 0.75% 1.0% 1.5% -.001168* -.002068* -.001714* .002 .000 .000 0.75% 1.0% 1.5% -.000900* -.000546 .012 .110 1% 1.5% .000354 .293 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p185-188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p185-188 188 adiana, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 185–188 of heat polymerized acrylic resin denture base to –nh2. this result is consistent with a study which stated that chitosan’s properties, if modified with several other polymers, such as polymethyl methacrylate, will bind colored substances to nh2. 9 low water absorption will result in lower dissolved component and higher color stability. the lower absorption value in group with additional 1.5% nanochitosan gel compared to the absorption value of group with addition of 1% nanochitosan gel is due to fewer bond of colored substances and –nh2 between chitosan and heat polymerized acrylic. this condition occurred because the concentration of coloring substances in heat polymerized acrylic resin added with 1.5% nanochitosan gel is lower compared to resin with addition of 1.0% nanochitosan gel, which is due to higher viscosity of 1.5% nano chitosan gel that inhibit its process of diffusion to bind with the pmma denture base material. the study concluded that addition of 1% nanochitosan gel in heat polymerized polymethyl methacrylate denture base material is suitable to maintain the stability of color. references 1. power s j m, wat a ha jc. dent a l mat er ia ls: prop er t ies a nd manipulation. 9th ed. missouri: mosby inc; 2008. p. 205-15, 28520. 2. anusavice kj, shen c, rawls hp. phillips’ science of dental materials. 12th ed. usa: elsevier saunders; 2013. p. 722-8, 735-4, 741-4. 3. noort rv. introduction to dental materials. 3rd ed. st. louis: mosby elvesier; 2008. p. 217-8, 221-2. 4. kortrakulkij k. effect of denture cleanser on color stability and f lexural strength of denture base materials. thesis. thailand: university of mahidol; 2008. 5. hipolito ac, barao va, faverani lp, ferreira mb, assuncao wg. color degradation of acrylic resin denture teeth as a function of liquid diet: ultraviolet-visible reflection analysis. j biomed opt 2013; 18(10): 1-9. 6. gujjari ak, bhatnagar vm, basavaraju rm. color stability and strength of polymethyl methacrylate) and bis-acrylic composite based provisional crown and bridge auto-polymerizing resins exposed to beverages and food dye: an in vitro study. indian j dent res 2013; 24(2): 172-5. 7. imirzalioglu p, karacaer, yilmaz b, ozmen i. color stability of denture acrylic resins and a soft lining material against tea, coffee, and nicotine. j prosthodont 2010; 19(2): 118-6. 8. ernani. studi perbandingan eksperimen dan simulsi ketahanan akar gigi pasca perawatan endodoontik dengan irigasi larutan kitosan molekul tinggi 0,2% setelah pemasangan pasak fiber prefabricated (penelitian in vitro). tesis. medan: fakultas kedokteran gigi universitas sumatera utara; 2014. 9. sugita p, wukisari t, sjahriza a, wahyono a. kitosan sumber biomaterial masa depan. bogor: ipb press; 2009. p. 27, 82-108, 125. 10. hen ny s. efek pena mba ha n k itosa n bla ngkas (tachypleus gigas) nanopartikel pada varian semen ionomer kaca terhadap mikrostruktur dentin dan komposisi kimia melelui sem_edx (in vitro). tesis. medan: fakultas kedokteran gigi universitas sumatera utara; 2014. 11. ningsih w. pengaruh viskositas larutan kitosan nanopartikel sebagai penyalut asam askorbat untuk menyerap asam lemak bebas (alb) dalam minyak goreng curah. tesis. medan: universitas sumatera utara; 2010. 12. amer zja, ahmed jk, abbas sf. chitosan/pmma bioblend for drug release applications. int j eng and tech 2014; 4(5): 1-6. 13. goiato mc, zulccolotti bcr, santos dm, moreno a, alves-rezende mcr. effects of thermocycling on mechanical properties of soft lining materials. acta odontol 2009; 22(3): 227-32. 14. saied hm. influence of dental cleansers on the color stability and surface roughness of three types of denture bases. j bagh college dentistry 2011; 23(3): 17-5. 15. ariyani. pengaruh penambahan fiber glass reinforced terhadap penyerapan air dan stabilitas warna bahan basis gigitiruan nilon termoplastik. tesis. medan: fakultas kedokteran gigi universitas sumatera utara; 2012. 16. al-hababeh r. water absorption and soluubility of propesional crown and bridge: the effect of the inclusion of polyethylene fibers. jrms; 14(1): 22-5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p185-188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p185-188 vol 44 no 3 sept 2011.indd 150 vol. 44. no. 3 september 2011 anterior makeover on fractured teeth by simple composite resin restoration eric priyo prasetyo department of conservative dentistry faculty of dentistry, airlangga university surabaya-indonesia abstract background: in daily practice dentists usually treat tooth fractures with more invasive treatments such as crown, veneer and bridges which preparation require more tooth structure removal. while currently there is trend toward minimal invasive dentistry which conserves more tooth structure. this is enhanced with the vast supply of dental materials and equipment in the market, including restorative materials. provided with these supporting materials and equipment and greater patient’s demand for esthetic treatment, dentists must aware of the esthetics and basic principle of conserving tooth which should retain tooth longevity. purpose: this article showed that a simple and less invasive composite resin restoration can successfully restore anterior esthetic and function of fractured teeth which generally treated with more invasive treatment options. case: a 19 year-old female patient came with fracture on 21 and 22. this patient had a previous history of dental trauma about nine years before and was brought to a local dentist for debridement and was given analgesic, the involved teeth were not given any restorative treatment. case management: the fractured 21 and 22 were conventionally restored with simple composite resin restoration. conclusion: fracture anterior teeth would certainly disturbs patient’s appearance, but these teeth could be managed conservatively and economically by simple composite resin restoration. key words: anterior makeover, fractured teeth, composite restoration abstrak latar belakang: dalam praktek sehari-hari pada umumnya dokter gigi merawat fraktur dengan restorasi invasif seperti mahkota, veneer dan jembatan yang semuanya memerlukan pengambilan jaringan gigi lebih banyak, sedangkan saat ini trend perawatan gigi lebih menuju kearah invasif minimal yang mempertahankan jaringan gigi sebanyak mungkin. keadaan ini ditunjang oleh tersedianya berbagai macam bahan dan peralatan kedokteran gigi di pasaran, termasuk bahan restorasi. dengan tersedianya bahan dan peralatan yang mendukung serta tingginya permintaan pasien akan perawatan estetik, dokter gigi harus mengetahui estetik dan prinsip dasar dari konservasi gigi yaitu mempertahankan gigi selama mungkin. tujuan: laporan kasus ini menunjukkan bahwa restorasi resin komposit sederhana yang tidak invasif dapat memperbaiki estetik dan fungsi geligi fraktur yang umumya dirawat dengan macam restorasi yang lebih invasif. kasus: seorang wanita berusia 19 tahun datang dengan fraktur pada gigi 21 dan 22. pasien tersebut memiliki riwayat trauma pada giginya sejak sembilan tahun sebelumnya dan telah dibawa ke dokter gigi untuk debridemen dan pemberian analgesik saja, tanpa perawatan restoratif. tatalaksana kasus: gigi fraktur pada 21 dan 22 diperbaiki dengan restorasi resin komposit sederhana. kesimpulan: fraktur pada gigi anterior akan sangat mengganggu penampilan pasien, akan tetapi fraktur tersebut dapat dirawat secara konservatif dan ekonomis dengan restorasi resin komposit sederhana. kata kunci: perbaikan anterior, fraktur gigi, restorasi resin komposit correspondence: eric priyo prasetyo, c/o departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ep_prasetyo@yahoo.com case report 151prasetyo: anterior makeover on fractured teeth introduction in the past twenty five years, the focus in dentistry has changed. years ago dentists were in the repair business, but now the demand for esthetic dentistry has grown vastly. nowadays it is not only on prevention and treatment of disease but also on meeting the demands for better esthetics. this fact presents great challenges for dentists in fulfilling the basic requirements of restoring teeth to form and function as well as creating the appearance of naturally pleasing teeth using appropriate esthetic based dental materials. currently various new dental materials and equipments have been introduced to support dentists with their esthetic works. there are many restorative materials, including varieties of specialized composite resins with different filler types and compositions as tooth colored dental materials with a wide range of shades to make successful color matching and easier manipulation techniques. this is also supported by improvements in adhesive technology through various bonding generations and equipment such as polishing instruments and led curing units for faster polymerization.1–3 in daily practice dentists usually treat tooth fractures with more invasive treatments such as crown, veneer and bridges which preparation require more tooth structure removal. while currently there is trend toward minimal invasive dentistry which conserves more tooth structure. this is enhanced with the vast supply of dental materials and equipment in the market, including restorative materials. provided with these supporting materials and equipment and greater demand for esthetic treatment, dentists must aware of the esthetics and basic principle of conserving tooth which should retain tooth longevity. when a makeover is planned for the esthetic appearance of a patient’s teeth, the dentist must have a logical diagnostic approach that results in appropriate treatment planning.4 although new esthetically pleasing materials and technology were introduced, a dentist should consider not only the principle of conserving tooth structure but also the patient’s financial capability and needs.5 this article presents a clinical case about anterior makeover on fractured teeth by simple composite restoration. case a 19 year old female patient came with fractured teeth on 21 and 22 (figure 1). this patient is an undergraduate student and starts to feel less confident with her fractured teeth since entering college. on anamnesis, the patient had a previous history of dental trauma about nine years ago and was brought to a local dentist for debridement and was given analgesic, the involved teeth were not given any treatment because based on the radiographic image, the apical were still in development and not constricted yet. the teeth were controlled by the previous dentist and no symptom was reported since then, but this patient never came back due to her parents’ job mutation to surabaya. on clinical examination, it was found that the patient was healthy. the overall general condition was good to receive dental treatment, no drug allergy and no previous history of transmittable diseases. posterior occlusion is within normal range with deep bite anterior relation. there were no carious lesions on other teeth. overall oral hygiene is good. there was no calculus, only a slight staining on palatal side of anterior teeth extended from 13 to 23. the gingival margins were normal. teeth 21 and 22 were fractured almost half cervicoincisally. these teeth were responsive to thermal and supported by healthy gingiva. there was no abnormal tooth movement. there were no pulp exposures on each tooth. after thorough explanations, the patient approved and consented about the single visit anterior makeover procedure by simple composite resin restoration. the restoration planning was determined using microhybrid composite and total etch technique. figure 1. pre-makeover anterior teeth condition. case management after anamnesis, clinical examination, thorough explanation, and patient’s consent about the makeover procedure, the involved teeth was cleaned and brushed with prophylaxis scrub (consepsis scrub, ultradent) and made ready for shade selection. shade selection was carried out on damp teeth with the provided composite shade guide from the manufacturer and shade a1 and pf were chosen for both teeth (figure 2). figure 2. shade selection was carried out to restore the teeth. 152 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 20111: 150–153 the teeth were prepared to form bevel surrounding the fracture part. this bevel was done by removing superficial enamel around the fracture part, including the proximal surfaces extending labially and lingually about 2 mm continuously. then the prepared areas were etched with 35% phosphoric acid solution (ultraetch, ultradent) for 15 seconds, suctioned, rinsed, and blot dried. the 21 and 22 teeth were isolated using cotton roll and celluloid matrix strip (svenska dentorama) was applied on interdental between 11–21, 21-22, and 22–23. a thin layer of bonding agent (pq1, ultradent) was applied using micro applicator (microbrush, sdi australia) and cured. the a1 body shade composite (vitlescence, ultradent) was applied layer by layer, while sculpting the mamelons until about one fifth incisally. pf enamel shade composite (vitlescence, ultradent) was applied over the surface and incisal of each tooth. this layering was finished by covering with a drop of t shade flowable composite resin (vitlescence, ultradent) on labial surface and disperse it with an artist’s brush to create a smooth surface. the restorations were then contoured and the excess composite resin was removed using fine finishing diamond bur (sharpcut, dentsply) and polished with diamond micro polisher (pogo, dentsply). the makeover showed better teeth hence changed the appearance and restore patient’s self confidence (figure 3). patient follow up was done six months after treatment, there were no complaint, the composite restorations were still in good condition and the patient was happily satisfied with the result. the patient came for regular dental check up about two years later and the restorations were still in good condition and functional (figure 4). figure 3. teeth condition after anterior makeover. figure 4. teeth condition on follow up (two years after makeover). discussion crown fractures account for the majority of all traumatic injuries on permanent dentition and some of these fractures are minor while others are severe which can cause the loss of affected tooth and require more extensive management.6 dental makeover may involve restoring the esthetic and functional characteristics of anterior teeth predictably and reliably.7 makeover doesn’t always come with complex and invasive treatments, but it can be done simply and predictably. it is surprising to hear of patients’ bad experience for leaving their previous dentist. this is happen because invasive care as seen by the patient is not limited to physical appearance but to treatment options given by the dentist who render care what the patient need and help them to feel trust and understanding. in daily practice, dentists face options of what treatment to recommend for a given clinical tooth condition. when those options are offered to patients with sufficient explanation, they usually prefer the simplest and least invasive treatments.8 any given restoration must has a natural appearance which is harmonized with the remaining teeth, the patient’s age and personality.9 based on this, composite restoration which includes the use of bonding agent was chosen in this case to minimize the amount of tooth structure sacrificed to achieve retention hence conservatively achieve good filling retention without significant loss of tooth structure.10 composite resins are the material of choice for the restoration of conservative defects in anterior teeth because of their adhesive and esthetic properties.11 discussing about composite restoration cannot be separated from dental adhesives or generally called bonding agents which was used in this case. bonding agents firstly introduced by dr. michael g. buonocore in mid 1950s are solutions used to assist in bonding fillings by two main functions: retention of the filling and sealing of the tooth-filling interface to prevent leakage.10 current dentine bonding systems are efficient to bear normal mastication forces. since application technique is critical for the success of the procedure; therefore manufacturer’s guide should be followed carefully. in this case, the teeth were prepared minimally to create a beveled labial and palatal area to facilitate acid etching therefore increasing the desirable bonding surfaces. the preparation technique and extent were relative to the enamel margins to assist the retention of composite restoration. this was coherent with minimal intervention dentistry, where conservation of tooth structure is of prime importance and to achieve this there needs to be a high level of visibility and an excellent tactile sense to avoid over-preparation and excess of tooth loss.12 flowable and regular microhybrid composite resins were used in this case in order to combine their fine properties, such as providing strong restorations that can be finished and polished well.13 microhybrid composite was chosen because of its acceptable strength, although other 153prasetyo: anterior makeover on fractured teeth types of composite resins may also be used as long as they are indicated to bear mastication forces and esthetically acceptable. flowable composite was used to create a smooth labial surface. in this case, the composite restorations’ surfaces were finished and polished to improve their esthetic quality and protect them against the aggressive oral environment and the colonization by micro-organisms.2 one step diamond micro polisher was used for several reasons; practical and capable to produce acceptable gloss finish on microhybrid composite restorations.14 even though the cervical gingival lining in 21 was not equal to 11 (figure 3), it was considerably accepted by the patient, realizing that the lip line could hardly reveal the cervical gingival lining when smiling (figure 4). however, the patient was explained about gingivoplasty as an optional procedure. patient’s cooperation is mandatory in retaining dental makeover result, this include the awareness to maintain basic oral health and daily hygiene. regular visit to dentist is also important to notice any changes on patient’s overall teeth condition which support the longevity of the established restoration. satisfactory esthetic makeover doesn’t always come with massive tooth structure removal during preparation or complicated advanced methods or expensive materials and equipments, but in contrary dentists as clinicians should try to do better on both simply and economically conserving the tooth structure while revealing the utmost esthetic, function and longevity out of it as well as appreciating what the patient wants and needs. in conclusion, fracture anterior teeth would certainly disturb patient's appearance, but there teeth could be managed conservatively and economically by simple composite resin restoration. references 1. prasetyo ep, samadi k, lunardhi cgj. the surface roughness difference between microhybrid and polycrystalline composites after polishing. maj ked gigi (dent j) 2008; 41(4):164–6. 2. koch jh. creating shiny composite surfaces without any detours. apdn 2009; 86: 27–8. 3. prasetyo ep. esthetic management for anterior teeth: a case report. apdc publishing; 2007. p. 123. 4. spear fm, kokich vg, mathews dp. interdisciplinary management of anterior dental esthetics. j of am dent assoc 2006; 137: 160–9. 5. lunardhi, cgj, prasetyo ep. esthetic rehabilitation of crowded and protruded anterior dentition. maj ked gigi (dent j) 2009; 42(1): 46–9. 6. wadhwani cp. a single visit, multidisciplinary approach to the management of traumatic tooth crown fracture. br dent j 2000; 188(11): 593–8. 7. felippe la, monteiro s jr, de andrada ca, ritter av. clinical strategies for success in proximoincisal composite restorations. j esthet restor dent 2005; 17: 11–21. 8. whitehouse ja. minimally invasive dentistry – clinical applications. j minim interv dent 2009; 2(1): 16–23. 9. price g. a perfect combination of material quality and professionals’ expertise for esthetic restorations. espertise 2008; 14: 8–9. 10. ritter av. dental adhesives. j esthet restor dent 2006; 18(6): 376–7. 11. macedo g, raj v, ritter av, swift ej jr. longevity of anterior composite restorations. j esthet restor dent 2006; 18(6): 310–1. 12. mount gj. minimal intervention dentistry: cavity classification and preparation. j minim interv dent 2009; 2(3): 150–62. 13. newsletter cra. do you want to use a nanofill composite resin? 2007, 31: 10. 14. prasetyo ep, lunardhi cgj, sukaton. the effectiveness of pogotm one step diamond micro polisher as a composite resin restoration polishing instrument. maj ked gigi (dent j) 2003; 36(4): 125–8. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 226226 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 226–230 original article the effect of giomer’s preheating on fluoride release muthiary nitzschia nur iswary winanto, irfan dwiandhono, setiadi warata logamarta, rinawati satrio, aris aji kurniawan department of conservative dentistry, faculty of medicine, jenderal soedirman university, purwokerto, indonesia abstract background: secondary caries occur due to imperfect plaque control. prolong the protective and therapeutic effects can be done with restorative materials that release fluoride. now composite resins have been developed a new restorative hybrid material with new matrix component, namely giomer. giomer composition containing surface pre reacted glass ionomer (s-prg) as a major source of fluoride production. increasing the mechanical strength and minimize microleakage to prevent secondary caries can be done with preheating treatment. purpose: this study is conducted to determine the effect of preheating temperature on the release of giomer’s fluoride. methods: this study used 9 cylindrical samples in 10 mm diameter and 2 mm thickness each group, divided into 3 groups and 3 subgroups. group 1: preheating at 37°c for 30 minutes. group 2: preheating at 60°c for 30 minutes. group 3: as a control group (without preheating treatment). each group divided into 3 subgroups immersion, on day 1, day 7 and day 14 with artificial saliva. fluoride release test was conducted by spectrophotometer. ibm's spss statistics used for the data analysis. results: the addition of preheating treatment decrease the amount of fluoride release. one-way anova test showed a significant difference (p < 0.05). a significant difference between groups and sub groups showed in lsd test (p < 0.05). conclusion: the group without preheating treatment has highest fluoride release and the 60°c preheating treatment group was the lowest. addition of preheating treatment may increase the mechanical strength and minimize microleakage, but also descrease the amount of fluoride release. keywords: fluoride release; giomer; preheating correspondence: muthiary nitzschia nur iswary winanto, department of conservative dentistry, faculty of medicine, jenderal soedirman university, jl. dr. soeparno, karangwangkal, purwokerto, 53123, indonesia. email: muthiarywinanto@gmail introduction secondary caries are the outcome of unsuccessful plaque control. the location of secondary caries usually occurs at the margin of the filling, and is most common on the gingival margin in class ii to v fillings, and is rarely seen in class i restorations.1 restorative materials that release fluoride could prolong the protective or therapeutic effect on tooth enamel, especially in areas prone to secondary caries.2 composite resin is a restorative material that is widely used for enamel abrasion, caries restoration, as well as for aesthetic needs, because it has a good fit with teeth.3 composite resin consists of three main components, namely matrix, filler, and coupling agent. composite resin matrix generally contains bis-gma, this matrix content is classified as conventional composite resin. the composite resin matrix is composed of monomers that have double-chain carbon bonds and have distances between the monomers.4 the disadvantage of composite resin is the shrinkage during polymerization (polymerization shrinkage), which can cause the formation of micro-leakages or gaps between the tooth surface and the composite resin.4 now composite resins have been developed a new restorative hybrid material with new matrix component, namely giomer.5 giomer is a new hybrid restorative material with a composition containing a derivative of glass ionomer cement called filler s-prg. surface pre reacted glass ionomer (s-prg) filler can release and recharge fluoride.2 restorative materials that release fluoride allow to extend the therapeutic effect or protection to the tooth enamel, especially in the approximal area that is contacted with the material. because the proximal area is an area with high plaque accumulation, it may be a cause for the formation of lesions of white dots when the surface is in contact with other surfaces with carious lesions.2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p226–230 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p226-230 227 winanto et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 226–230 saliva as a natural protective factor could prevent or inhibit caries formation, because of ca2+ and hpo4 2ion can replace the lost ions in the teeth, but if the ph in saliva is 5.5, which is a bad condition for hydroxyapatite, it will cause demineralization. giomer can reduce the risk of hydroxyapatite and fluorapatite’s breakdown. the acid neutralizing ion in the oral cavity released by giomer is strontium ion (sr2+).5 surface hardness can also be affected by ph in the oral cavity; according to research, there was an increase in hardness at a higher ph, about 125% after immersing giomer in artificial saliva ph 4.5 increased to 7.13±0.01 after soaking for 72 hours.5 preheating the composite resin before irradiation can reduce the occurrence of micro leakages and make it easier for application and manipulation.6 preheating is a method of heating the composite resin prior to irradiation. preheating can be done using a composite warmer or a conventional oven.7 preheating treatment can make the composite resin stronger, reduce viscosity so as to facilitate the adaptation and application of composite resin to the cavity, and can improve its mechanical properties.8,9 research on the giomer surface microhardness in previous studies, shows that preheating treatment at 60°c has a higher hardness than 37°c.10 another previous research, showed that preheating treatment at 60°c could lead to decreased microleakage and better marginal adaptation to composite resins.11 based on previous research that provides preheating treatment so as to increase mechanical strength and minimize microleakage in composites, it encourages researchers to conduct research that has never been done before to determine the effect of preheating treatment temperature on the release of giomer’s fluoride. materials and methods ethical approval which is managed as a condition for conducting research has been obtained from the research ethics commission of the faculty of medicine, jenderal soedirman university (no. 005.kepk.01.2021). this research is an experimental laboratory with a post-test only control group design. the data used primary data which were directly collected by the researcher. the bulkfill giomer beautifil ii from shofu.inc was used as a research sample for all groups and sub-groups. nine samples in each group were used in the study. this study divided these into three groups: preheating at 37°c group (group a), preheating at 60°c group (group b), and a control group without preheating treatment (group c). each group was divided into three sub-groups, immersion with artificial saliva on day 1, day 7 and day 14. each group and sub-group consisted of cylindrical samples with a diameter of 10 mm and a thickness of 2 mm.12 preheating was done using a conventional oven. sample mold was using a square shaped acrylic having a cylindrical hole in the center with diameter of 10 mm and height of 2 mm as a place to put the sample. the sample mold was given a prep glass which had been given vaseline previously using a microbrush. samples were molded after preheating treatment for each group to the mold. sample was condensed using cement stopper and coated with a celluloid strip. after that covered with another prep glass and then pressed gently so that the sample surface was flat. the sample was light cured for 20 seconds. after the sample was polimerized it was separate from the mold and polished using a rubber bur until the surface was smooth. all samples were immersed in 20 ml of artificial saliva solution in sterile bottles according to groups and subgroups. the immersion water of each group was collected on days 1, 7, and 14 to be tested for fluoride release. the sample test of fluoride release was carried out using a spectrophotometer (spectroquant pharo 300). figure 1. cylindrical sample. table 1. the result of giomer’s fluoride release group day 1 day 7 day 14 a (37°c) 0.87 0.81 0.71 b (60°c) 0.76 0.71 0.63 c (control) 1.03 0.91 0.72 table 2. one-way anova test results for the release of giomer’s fluoride group mean (ppm) standard deviation sig a (37°c) 0.79 0.04 b (60°c) 0.70 0.05 0.000* c (control) 0.88 0.04 note: * = significant difference (p<0.05). table 3. results of lsd further test for the release of giomer’s fluoride group a (37°c) b (60°c) c (control) a (37°c) 0.001* 0.002* b (60°c) 0.001* 0.000* c (control) 0.002* 0.000* note: * = significant difference (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p226–230 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p226-230 228winanto et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 226–230 results the test results are in the form of fluoride release data in ppm units. the results of the study on the effect of preheating on the release of giomer’s fluoride in direct restorations used a cylindrical sample measuring 10 mm x 2 mm. the results of the fluorine ion release test results are presented in table 1. data analysis was performed using ibm’s spss statistics version 20. data were found to be distributed normally (p>0.05) and homogeneous (p>0.05). anova test was then performed and a significant difference was found between the treatment groups (p<0.05) (table 2). the data were further tested for significant differences between treatments groups and sub-group using the lsd, and there were significant differences between treatments groups and sub-group (p<0.05) (table 3). discussion giomer is one of the restorative materials in dentistry that is capable of releasing fluoride.7 giomer has unique properties in the form of components that distinguish it from other composites, namely s-prg, which is coated with an ionomer layer within the resin matrix, and allows for the protection of the glass core from moisture, providing longterm aesthetics and durability of conventional composites with ion release and recharge.14 s-prg in giomer is contained in the resin matrix as a filler.14 s-prg filler can release and recharge fluoride, and can release five other ions such as sodium, strontium, aluminum, silicate, and borate. these ions have an important role in neutralizing the acidic state when exposed to lactic acid produced by bacteria in plaque. acid neutralization minimizes the possibility of secondary caries and makes restorations more durable.2 the advantage of using a fluoride-releasing restorative material is to protect the restored tooth surface during exposure to cariogenic agents, and has been widely studied with good results.15 previous research showed that a 13year-follow-up of in vitro study found a 66% retention rate and the secondary caries rate of only 3.27% with giomerbased restorative material. the research showed that giomer has a high amount of fluoride release and has the ability to be recharged, along with physical properties that could rival other composite system.14 preheating could lead to increase the degree of conversion, because polimerization involving free radicals will change the viscosity from high to low viscosity. this process will convert the c=c double bond into a covalent c–c single bond between the methacrylate monomers, which causes a change in the rate of free radical diffusion.16 the activation of free radicals is also influenced by high light intensity, so that more monomers will be converted. the degree of polymerization is also influenced by the mobility of free radicals and monomers so that polymer cross-links occur. polymer cross-links can form covalent bonds resulting from polymers adjacent to electrons. these cross-links act as a reaction bridge between linear macromolecules to form 3-dimensional working bonds that can change the strength, solubility, and water sorption of the composite resin, which could produce a material that is stronger than polymers that have single chains.9,17 additional polymerization can be carried out by adding preheating treatment to the composite resin before irradiation.17 the advantages of preheating treatment include a stronger composite resin, reducing viscosity, making it easier to manipulate and adapt the composite resin to cavities, reducing microleakage and increasing mechanical properties such as hardness, diametral tensile strength and compressive strength.8,9 the decrease of fluoride release pattern along with increased temperature is influenced by several factors, namely temperature, water sorption and giomer’s polymerization.16 the preheating treatment carried out in this study caused the carbon double chains breaking into single chains of the resin polymer contained in the giomer sample. the single polymer chain in the resin makes the polymer able to react with the monomer more evenly.16 the temperature increase of the giomer can cause a decrease in the rate of diffusion, which causes a delay in the rate of diffusion, reduced porosity and decreased surface roughness, and the opportunity for the s-prg filler to contact with water is also reduced, which results in a decrease of the amount of fluoride released.7,18 as seen in the sample group preheating treatment at 60°c (group a) on giomer showed the lowest fluoride ion release, with an average of 0.69 ppm and the group without preheating treatment (group c) had the highest fluoride ion release, with an average of 0.88 ppm. the polymerization phase in composite resins affects the quantity of fluoride release caused by segmental mobility of the polymer chain, so that when the preheating temperature is higher, the segmental polymer chains increases, and brings on more complete polimerization, which causes a decrease in fluoride release.19 in this study, it was seen that the quantity of fluoride ion released at 37°c (group a) had a higher average yield (0.79 ppm) than 60°c preheating. this is due to the lower degree of conversion at preheating temperature of 37°c which resulted in the final result of sample polymerization being less perfect when compared to preheating temperature of 60°c, which had more perfect polymerization.16 the average yield of fluoride ion release in the control group was higher (0.88 ppm) compared to the other treatment groups because, in the control group, giomer did not get a higher temperature change. the polymerization chain segmental formed on the composite without the treatment of higher temperature changes will be more so that it makes the surface have more gaps, and this causes the release of fluoride to increase.16 the results of fluoride release between groups that have been carried out in this study showed that the fluoride dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p226–230 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p226-230 229 winanto et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 226–230 release decreased along the increasing immersion time. this decreasing pattern of ion release is caused by the fluoride that has been liberated between the polymer chains; the amount will decrease if there is no intake or exposure to fluoride from the outside.20 in normal conditions of the oral cavity where individuals can seek fluoride exposure into the body (from food, drink, or additional supplements), then the amount of fluoride can be stable because the giomer is able to retake and release fluoride. however, in this study, artificial saliva that was not treated with added fluoride was used as the immersion medium, so that the amount of fluoride released would decrease over time. the number of ions released by the giomer is influenced by the amount of water absorbed and the porosity of the giomer. giomer’s low fluoride release caused by the limited fluoride content of the giomer filler, low water content, low solubility of ytterbium trifluoride in water, and permeability of the resin composite which causes the fluoride released to also decrease over time.19 the release of fluoride is less in artificial saliva, due to the possibility of calcium ions in the saliva immersion medium and the formation of caf2. 21 the immersion medium containing water also affects the release of fluorine ions in the giomer. in this study, the largest ion release occurred on the first day of immersion in each group, with an average result of group a of 0.87 ppm, group b of 0.76 ppm, and group c of 1.03. there was a greater release of ions on the first day of immersion, and in the first week to the fourth week there was no significant difference or had started to stagnate. the release of fluoride can occur due to mediation by the ability of the material to diffuse water.19 the absorption of liquid from resin-based materials occurs due to a combination of adsorption and absorption. adsorption can be seen from the ability of liquid molecules to reach the surface of a solid material. absorption involves the penetration of a liquid molecule into a solid structure primarily by diffusion. diffusion that occurs in resin-based restorations is a controlled diffusion process, and the most water absorption occurs in the matrix resin. giomer’s s-prg filler technology means the surface of the resin matrix containing s-prg reacts with polyacrylic acid during contact with water to form a thick silica hydrogel layer.19 the ion release mechanism occurs when the surface of the giomer which has gone through the light-curing process is in contact with water. silica gel is a material that is stable to high temperatures.22 the s-prg in contact with water then dissolves the ions that are not bound in the polymerization chain formed in the light cured giomer. the polymer contained in the giomer will react when given a light from the light cure to form a polymer chain. among the polymer chains, there are several ions that are not involved in the polymer chain so that they can dissolve in the immersion medium. ions that are not involved in this polymer chain include fluoride, calcium and aluminum.19 addition of preheating treatment may increase the mechanical strength and minimize microleakage, but the addition of preheating treatment also decreases the amount of fluoride release. to increase the mechanical strength and minimize microleakage, along with amount of fluoride release, according to this study, the best preheating temperature that can be used is 37°c. addition of preheating treatment may increase the mechanical strength and minimize microleakage, but also decreases the amount of fluoride release. references 1. fejerskov o, nyvad b, kidd e. dental caries: the disease and its clinical management. 3rd ed. oxford: wiley blackwell; 2015. p. 480. 2. guglielmi c de ab, calvo afb, tedesco tk, mendes fm, raggio dp. contact with fluoride-releasing restorative materials can arrest simulated approximal caries lesion. j nanomater. 2015; 2015: 1–7. 3. kafalia rf, firdausy md, nurhapsari a. pengaruh jus jeruk dan minuman berkarbonasi terhadap kekerasan permukaan resin komposit. odonto dent j. 2017; 4(1): 38–43. 4. permana dp, sujatmiko b, yulianti r. perbandingan tingkat kebocoran mikro resin komposit bulk-filldengan teknik penumpatan oblique incremental dan bulk. maj kedokt gigi indones. 2016; 2(3): 135. 5. ratna aa, triaminingsih s, eriwati yk. the effect of prolonged immersion of giomer bulk-fill composite resin on the ph value of artificial saliva and resin surface roughness. j phys conf ser. 2017; 884(1): 012011. 6. dionysopoulos d, tolidis k, gerasimou p, koliniotou-koumpia e. effect of preheating on the film thickness of contemporary composite restorative materials. j dent sci. 2014; 9(4): 313–9. 7. kashi tsj, fereidouni f, khoshroo k, heidari s, masaeli r, mohammadian m. effect of preheating on the microhardness of nanohybrid resinbased composites. front biomed technol. 2015; 2(1): 15–22. 8. didron pp, ellakwa a, swain m v. effect of preheat temperatures on me cha n ica l prop er t ies a nd polymer izat ion cont ract ion st r ess of dent a l comp osit es. mat e r sci appl. 2 013; 4 (6): 374–85. 9. nada k, el-mowafy o. effect of precuring warming on mechanical properties of restorative composites. int j dent. 2011; 2011: 1–5. 10. stoleriu s, iovan g, nica i, pancu g, sandu av, andrian s. the effect of heating on surface microhardness of resin-based mater ia ls for d i re ct restorat ion. mater plast. 2018; 55(4): 584–9. 11. taraboanta i, stoleriu s, iovan g, moldovanu a, georgescu a, negraia mr, andrian s. evaluation of pre-heating effects on marginal adaptation of resin-based materials. mater plast. 2018; 55(2): 238–42. 12. kurt a, tüzüner t, altıntepe i̇, aydınoğlu s, sökmen m. the effect of heat application on f luoride release in antibacterial added glass ionomer cement. cum hur dent j. 2019; 22(2): 218–25. 13. rusnac me, gasparik c, irimie ai, grecu ag, mesaroş aş, dudea d. giomers in dentistry – at the boundary between dental composites and glass-ionomers. med pharm reports. 2019; 92(2): 123–8. 14. griffin jr jd. unique characteristics of the giomer restorative system: a line of regenerative materials for anterior and posterior restorations. insid dent. 2014; 10(3): 4–5. 15. mickenautsch s, tyas mj, yengopal v, oliveira lb, bönecker m. absence of carious lesions at margins of glass-ionomer cement (gic) and resin-modified gic restorations: a systematic review. eur j prosthodont restor dent. 2010; 18(3): 139–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p226–230 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p226-230 230winanto et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 226–230 16. ribeiro bci, boaventura jmc, brito-gonçalves j de, rastelli an de s, bagnato vs, saad jrc. degree of conversion of nanofilled and microhybrid composite resins photo-activated by different generations of leds. j appl oral sci. 2012; 20(2): 212–7. 17. shen c, rawls hr, esquivel-upshaw j. phillips’ science of dental materials. 13th ed. philadelphia: elsevier saunders; 2021. p. 448. 18. soliman em, elgayar il, kamar a aa. effect of preheating on microleakage and microhardness of composite resin (an in vitro study). alexandria dent j. 2016; 41(1): 4–11. 19. harhash ay, elsayad ii, zaghloul ags. a comparative in vitro study on fluoride release and water sorption of different flowable esthetic restorative materials. eur j dent. 2019; 11(2): 174–9. 20. francois p, fouquet v, attal j, dursun e. commercially available fluoride-releasing restorative materials: a review and a proposal for classification. mater (basel, switzerland). 2020; 13(10): 1–28. 21. nik yusoff nna bt, ariffin z, hassan a, alam mk. fluoride release from dental restorations in de-ionized water and artificial saliva. int med j. 2013; 20(5): 635–8. 22. khonina tg, chupakhin on, shur vy, turygin ap, sadovsky v v., mandra y v., sementsova ea, kotikova ay, legkikh a v., nikitina ey, bogdanova ea, sabirzyanov na. siliconhydroxyapatite‒glycerohydrogel as a promising biomaterial for dental applications. colloids surf b biointerfaces. 2020; 189(february): 110851. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p226–230 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p226-230 107 vol. 43. no. 3 september 2010 research report computer-aided diagnosis for osteoporosis based on trabecular bone analysis using panoramic radiographs agus zainal arifin�, anny yuniarti�, lutfiani ratna dewi�, akira asano�, akira taguchi��, takashi nakamoto�, arifzan razak5, and hudan studiawan� 1 department of informatics, faculty of information technology, institut teknologi sepuluh nopember, surabaya – indonesia 2 graduate school of engineering, hiroshima university, hiroshima japan 3 department of oral and maxillofacial radiology, matsumoto dental university, nagano japan 4 hiroshima university hospital, hiroshima japan 5 faculty of dentistry, airlangga university, surabaya indonesia abstract background: mandibular bone on panoramic radiographs has been proven to be useful for identifying postmenopausal women with low skeletal bone mineral density. one of the important parts of mandibular bone is trabecular bone. trabecular bone architecture is one of the factors that governs bone strength and may be categorized as a contributor to bone quality. purpose: the purposes of this study were to develop a computer-aided system for measuring trabecular bone line strength on panoramic radiographs in identifying postmenopausal women with osteoporosis and to clarify the diagnostic efficacy of the system. methods: reduction and expansion of trabecular bone sample images using a two level gaussian pyramid for removing noises and small segments were first introduced. then, line strength at each pixel was calculated based on its existence on the trabecular bone with emphasizes line segment which has similar orientation with the root of tooth. the density was measured with respect to line strength of segment structure which has similar orientation with the root of tooth, either on the left and the right in the mandibular bone. number of pixels in the line segment area was compared with a threshold value to determine whether normal or osteoporosis. results: from experiment on 100 data, the accuracy of 88%, sensitivity of 92%, and specificity of 86.7% were achieved. conclusion: the computer-aided system of trabecular bone analysis may be useful for detecting osteoporosis using panoramic radiographs. key words: computer-aided system, line strength, trabecular bone, osteoporosis, panoramic radiographs abstrak latar belakang: tulang mandibula pada panoramik radiografi telah banyak diteliti dan terbukti mampu digunakan untuk mengidentifikasi wanita pasca menopause dengan menggunakan bone mineral density rendah. salah satu bagian tulang mandibula yang penting adalah tulang trabekula. arsitektur tulang trabekula merupakan salah satu dari faktor-faktor yang mempengaruhi kekuatan tulang dan dapat digolongkan sebagai kontributor bagi kualitas tulang. tujuan: penelitian ini bertujuan untuk membangun sebuah sistem dengan bantuan komputer untuk mengukur kekuatan garis pada tulang trabekula dan menggunakannya untuk mendeteksi osteoporosis pada wanita postmenopause. metode: dilakukan sampling pada sebagian tulang mandibular yang menghasilkan sebuah sampel citra. sampel citra ini selanjutnya diperbaiki dari derau (noise) dengan menggunakan piramida gaussian dua level. kekuatan garis pada tiap piksel dihitung berdasarkan orientasi segmen garis tulang trabekula yang sejajar dengan akar gigi. setelah dilakukan binerisasi, luasan segmen yang dihasilkan dihitung dan dibandingkan dengan sebuah nilai ambang. bila luasan melebihi nilai threshold maka dikategorikan sebagai normal. sebaliknya bila luasan dibawah nilai threshold, dikategorikan sebagai osteoporosis. hasil: berdasarkan eksperimen terhadap 100 data, sistem mampu mencapai akurasi identifikasi sebesar 88%, sensitivitas 92%, dan spesifisitas 86,7%. kesimpulan: sistem analisa trabecular bone dengan bantuan komputer ini dapat digunakan oleh para dokter gigi untuk mendeteksi osteoporosis menggunakan panoramik radiografi. kata kunci: sistem berbantukan komputer, kekuatan garis, tulang trabekula, osteoporosis, panoramik radiografi correspondence: agus zainal arifin, c/o: jurusan teknik informatika, fakultas teknologi informasi, institut teknologi sepuluh nopember. jln. raya its sukolilo surabaya. e-mail: agusza@cs.its.ac.id. tel.: +62 31 5922949; fax: +62 31 5939363. 108 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 107–112 introduction the number of hip fractures because of osteoporosis was rising from about 1.3 million in 1990 worldwide. moreover, it was estimated to be 4.5 million in 2050.1 the u.s. surgeon general reported that if there was no serious handling until 2020, half of americans were predicted to have osteoporosis.2 one parameter should be measured to determine whether someone has osteoporosis or not is called the bone mineral density (bmd), measured on lumbar spine and femoral neck. the commonly used scanner for measuring bone is dual-energy x-ray absorptiometry (dxa). however, dxa is very expensive and not every hospital has dxa even in developed countries.3 on the other hand, postmenopausal women rarely visit medical expert to diagnose osteoporosis. postmenopausal women would realize that they had osteoporosis after any bone fractures caused by an accident. postmenopausal women may have greater opportunity to visit dentists for treatment of dental caries and periodontal disease than to visit medical professionals for diagnosis of osteoporosis. a large number of panoramic radiographs were taken for diagnosis of teeth and jaws in general dental practice. trabecular bone is one of important parts of panoramic radiographs. the trabecular bone pattern may be analyzed visually by experts or with computer-aided methods to estimate the probability of having osteoporosis and predict the risk of future fractures.4 degrees of inter-examiner and intraexaminer agreement of visual assessment of the trabecular pattern are also expected to be relatively low because the trabecular pattern of the jaws is more diverse than that of the general skeleton, such as the vertebrae and proximal femur.5 however, other researchers analyzed trabecular bone pattern using customized image analysis software.6–9 this research proposed a method to analyze trabecular bone tissue using multiscale line operator on gaussian pyramid.10 the line strength of trabeculae that has similar orientation with the root of teeth was measured on both left and right sides of the mandible. high correlation between both experimental results and bmd assessed by dxa scanner proves the effectiveness of this method.10 the purposes of this study were to develop a computeraided system for measuring trabecular bone line strength on panoramic radiographs in identifying postmenopausal women with osteoporosis and to clarify the diagnostic efficacy of the system. the density was measured with respect to line strength of segment structure which has similar orientation with the root of tooth. this research also determined the threshold value of line strength considered as the osteoporosis sign. materials and methods there were 531 women for dxa measurement between 1996 and 2001, 100 postmenopausal women aged 50 years or older with no previous history on osteoporosis (mean 59.6 years; range 50–84 years) were randomly recruited for this study. none of the subjects had metabolic bone disease (hyperparathyroidism, hypoparathyroidism, paget’s disease, osteomalacia, renal osteodystrophy, or osteogenesis imperfecta), cancers with bone metastasis, or significant renal impairment or were taking medication that affect bone metabolism, such as estrogen. none had a history of smoking, and none had bone-destructive lesions in the mandible. no subject had menstruated for at least 1 year. panoramic radiography were taken for all subjects with informed consent at the time of dxa measurements of the lumbar spine (l2–l4). all panoramic radiographs were obtained with a az-3000 (asahi co., kyoto, japan) at 12 ma and 15 s; kvp varied between 70 and 80. screens of speed group 200 (hg-m, fuji photo film co., tokyo, japan) and film (ur2, fuji photo film co., tokyo, japan) were used. appearance of the mandibular inferior cortex was bilaterally clear in the radiographs. all radiographs were digitalized with the resolution of 300 dpi using a flat-bed scanner (es-8000, epson, japan). when using the definition of the japanese society for bone and mineral research,11 54 of the 100 women presented normal bmd (bmd more than 80% of japanese young adult), 21 osteopenia (70–80%), and 25 osteoporosis (less than 70%) in the lumbar spine. the rate of women with osteoporosis in the lumbar spine in our study was similar to that (26%) in 1,033 postmenopausal women aged 50 years or older in the adult health study (ahs) cohort in japan.12 region of interest (roi) was taken from four different areas of an image. two rois were taken from the left hand side and the right hand side. the location of trabecular bone samples were between root of the tooth and cortical bone. area where sample was taken on panoramic radiographs is illustrated in figure 1a. figure 1b and figure 1c show samples from left hand side, whereas figure 2d and 2e are samples from right hand side. in this experiment, each sample size which marked with white box is 128 × 128 pixels. multiscale line operator is one of line detection algorithm used for detecting linear structure on mammographic image together with other line detection methods.13 comparing with other methods, the line operator algorithm was proved to give good result from signal to noise aspect, line width accuracy, and localization. in the early implementation of multiscale line operator algorithm, the algorithm has been used to detect asbestos fiber.14 this algorithm was applied for detecting linear structure of iris blood vessel.15 multiscale line operator requires parameters of angle (q) and length (m).16 angle controls the number and size of analyzed rotation. angle size per rotation was sum of the current and previous angle size until the limit 180 degree. length parameter was needed to make moving window with length m. we used 12 rotations with angels of 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, and 165 degree. moving 109arifin et al.: computer-aided diagnosis figure �. input image. a) four samples from a dental panoramic radiographs, b) and c) two left hand side samples, d) and e) two right hand side samples. window size of 5 × 5 was applied to each pixel for analysis it with 24 neighbor pixels around. given a region of interest image, at each pixel (x, y), multiscale line operator algorithm measures the line strength s(x, y) by calculating the contribution of the foreground minus the contribution of the background. for each angle q, the foreground mask has a line of length m and width one pixel, oriented at the angle q. the foreground value, f(x, y, q), was the sum of pixel values multiplied by the corresponding foreground mask values. similarly, the background mask was a rectangle of size m × m, oriented at the angle q. the background value, b(x, y, q), was the sum of the image pixels multiplied by the corresponding background mask elements. the line-strength image value, s(x, y), was calculated by: gl(i,j) = s s w(m,n)gl–1(2i + m, 2j + n) m n linear strength detection detects structure of trabecular bone line segment which has similar orientation with root of tooth. detection of line strength in left hand side sample has orientation 0, 15, and 30 degrees. the right hand side has orientation 330, 345, and 360 degrees. angle size parameter, q, used in multiscale line operator algorithm was appropriate with angle to be detected. it was 0, 15, and 30 for left side sample and 330, 345, and 360 for right one. local linear structures which have good contrast and also match the foreground mask will have high values in the line strength map. bilinear interpolation was used to create the rotated image for non-zero angles to get better result. a b c d e figure �. output image. a) and b) result of linear strength detection applied to images in figure b) and c), respectively, c) and d) result of linear strength detection applied to images in figure d) and e), respectively. a b c d 110 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 107–112 each segment of the line structure has different width. to estimate the problem, we require multiscale analysis method. in this paper, we use gaussian pyramid to solve multiscale analysis problem. gaussian pyramid is generated by first smoothing the image with an appropriate smoothing filter and then subsampling the smoothed image as many as desired levels. this produces a set of gradually more smoothed images. nevertheless, the more smooth an image, the less sampling density. if we illustrate it graphically, this multiscale representation will look like a pyramid, from which the name has been obtained. images produced at the lower levels of the pyramid have higher resolutions, whereas those produced at the higher levels have lower resolutions. the levels of the pyramid obtained iteratively as follows. for 0 < l < n: gl(i,j) = s s w (m,n)gl–1 (2i + m, 2j + n) m n however it was convenient to refer to this process as a standard reduce operation and simply write: gl = reduce (gl+1) where w (m, n) was “generating kernel”. the structure of generating kernel is [0.25 a/2, 0.25, a, 0.25, 0.25 a/2] and a = 0.4. the equivalent weighting functions were particularly gaussian-like when a = 0.4, when a = 0.5 the shape was triangular, when a = 0.3 it was flatter and broader than a gaussian. with a = 0.6 the central positive mode was sharply peaked and was flanked by small negative loobs. after the line strength structure of the trabecular bone was detected, those images were transformed into binary images. this method changes the background into black with zero value and changed the image object (foreground) into white with one value. measurement of line strength in trabecular bone was applied to the binary image at each pixel based on its existence on the trabecular bone with prioritizes line segment which has similar orientation with the root of tooth. the total value of line strength was the mean of line strength in four samples on every panoramic radiographs. the number of the bone segment was used to decide whether the patient was affected osteoporosis or normal. results sample image from panoramic radiographs was processed using multiscale line operator algorithm and gaussian pyramid. figure 2a, 2b, 2c, and 2d show result images from detection of trabecular bone linear strength structure applied to images in figure 1b, 1c, 1d, and 1e, respectively. output for image in figure 1b and 1c from line strength structure detection using orientation 0, 15, and 30 degrees is shown in figure 2a and 2b. figure 2c and 2d show images from line strength detection using orientations 330, 345, and 360 degrees for image shown in figure 1d and 1e, respectively. after processing with multiscale line operator algorithm and gaussian pyramid, sample of trabecular bone image was converted to binary image. the total value of line strength from four black and white samples on every panoramic radiographs was added. after that, mean value of line strength on one panoramic radiographs was calculated. from 100 panoramic radiographs used in this experiment, line strengths were compared with threshold. the cut off threshold of trabecular bone line strength was selected at 3450 by 92% sensitivity. the result of line strength and its correlation with osteoporosis status was shown in table 1. the average pixel number of black and white image was used to diagnosis osteoporosis by comparing the number with threshold. this number was preferably 3400–3500, and more preferably 3425–3475, and 3450 was the best threshold value for the roi size of 128 × 128. the number of subjects identified by mandibular trabecular density less than and equal to the threshold were 10 and 23 for normal and osteoporotic subjects. there are 62 subjects for trabecular density higher that the treshold and 2 subjects for normal and osteoporotic, respectively. image that has a wide average of line strength of segment structure of trabecular bone means that the image was not affected by osteoporosis. image that has a small average of line strength of segment structure of trabecular bone means that the image was potentially affected by osteoporosis. diagnostic efficacy of manual and computeraided automatic measurements is presented in table 2. this method achieved the accuracy of 88%, sensitivity of 92%, and specificity of 86%. the correlation between bmd and trabecular bone line strength was 52%. discussion various diagnosis and treatment for osteoporosis has been discussed and some of them has been implemented.17 even though there have been several studies regarding osteoporosis on postmenopausal women in indonesia,18 however this study is the first demonstration comparing correlations between skeletal bmd and trabecular bone line strength measured by a computer-aided system on digitized panoramic radiographs for postmenopausal women. line strength measurement has been used for robust classification of anatomical types (vessels, spicules, ducts, etc) in a mammogram.13 in other study about trabecular bone for osteoporosis detection, it shows 92% sensitivity and 96% specificity.8 we have the same sensitivity but lower specificity. in our previous study,19 sensitivity and specificity for computeraided system were about 88.0% and about 58.7%, respectively. although it used cortical bone on mandible as subject, similar result can be got by using trabecular bone. with this image analysis system, line strength of trabecular bone can be measured and compared with 111arifin et al.: computer-aided diagnosis table �. mean of line strength and its bmd assessment result no. mean of line strength structure bmd assessment result no. mean of line strength structure bmd assessment result 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 3549.00 2653.50 3753.00 3239.50 3565.25 3619.50 3997.00 3266.75 2840.50 3849.75 4178.00 3302.50 3760.50 4025.50 2610.50 4086.00 3876.00 3913.00 3439.25 4145.25 3420.00 3150.75 3996.25 4306.25 3309.00 4022.25 3624.00 4002.50 4572.75 3364.00 3755.00 3565.00 4048.25 3793.75 3629.75 3515.75 3191.00 3623.00 3891.75 4077.50 4563.00 3673.75 4318.25 3599.00 3736.50 3891.25 3875.25 4557.25 2981.75 3493.75 normal osteoporosis normal osteoporosis normal normal normal osteoporosis osteoporosis normal normal osteoporosis normal normal osteoporosis normal normal normal osteoporosis normal osteoporosis normal normal normal osteoporosis normal normal normal normal normal normal normal normal normal normal normal osteoporosis normal normal normal normal normal normal normal normal normal normal normal normal normal 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 3031.00 3734.00 3947.25 4005.50 3522.25 3717.50 3688.50 3328.50 3820.75 3595.00 3039.75 4249.50 2754.25 3797.25 3527.75 3887.25 3967.50 2870.00 2995.50 3643.25 3863.50 3050.75 2977.50 3564.75 2885.00 3528.75 3287.50 3427.75 3524.75 3877.00 3121.75 3662.75 3185.50 3448.00 3050.00 4328.00 3662.25 3839.00 3195.00 4021.50 3040.75 3510.75 3691.75 3521.00 3665.25 3085.00 3153.75 3499.50 3267.50 3655.50 osteoporosis normal normal normal osteoporosis normal normal osteoporosis normal normal osteoporosis normal normal normal normal normal normal normal osteoporosis normal normal osteoporosis osteoporosis normal osteoporosis normal osteoporosis osteoporosis osteoporosis normal osteoporosis normal normal osteoporosis normal normal normal normal normal normal osteoporosis normal normal normal normal osteoporosis normal normal normal normal threshold value. if it was lower than threshold, the dentist could consider to advice further treatment for dual energy x-ray absorptiometry. it was also possible that general dental practitioners can identify women with low skeletal bmd by using digital panoramic radiographs with our computer-aided system. another advantage was due to low cost assessment, simply only with a file which scanned from panoramic 112 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 107–112 radiographs as the input file for the system. using conventional dexa scanner cost about idr900.000,for assessment on lumbar spine and femoral neck. while this system only need to have a panoramic radiograph and the assessment system which cost about idr100.000, and idr50.000,respectively for each assessment. panoramic radiograph can be easily taken in clinic and then scanned. considering these advantages, it is very possible to implement our proposed system to examine indonesian woman. this computer-aided system, however, has some limitations. dentists were asked to determine manually rois along trabecular bone area. error tends to occur with this determination due to the existence of root. automatic determination of the rois would be necessary to maintain good reproducibility around the world. the robustness of this system would also be necessary to overcome this system limitation. in conclusion, our approach of analyzing trabecular bone using panoramic radiographs has sensitivity and specificity of 92% and 86.7%, respectively. thus, we suggest that the computer-aided diagnosis system may be useful for detecting osteoporosis. acknowledgement this research was supported by directorate general of higher education (indonesia) under program hibah pascasarjana and by japan international cooperation agency (jica) under predict (project for research and education development in information and communication technology in its). references 1. gullberg b, johnell o, kanis ja. world-wide projections for hip fracture. osteoporos int 1997; 7(5): 407–13. 2. u.s. department of health and human services. bone health and osteoporosis: a report of the surgeon general. u.s. department of health and human services, public health service, office of the surgeon general, rockville, md. 2004. pp. 4. 3. kanis ja, johnell o. requirement for dxa for the management of osteoporosis in europe. osteoporos int. 2005; 16(3): 229–38. 4. majumdar s. current technologies in the evaluation of bone architecture. curr osteoporos rep 2003; 1(3): 105–9. 5. lindh c, horner k, jonasson g, olsson p, rohlin m, jacobs r, karayianni k, van der stelt p, adams j, marjanovic e, pavitt s, devlin h. the use of visual assessment of dental radiographs for identifying women at risk of having osteoporosis: the osteodent project. oral surg oral med oral pathol oral radiol endod 2008; 106(2): 285–93. 6. ruttimann ue, webber rl, hazelrig jb. fractal dimension from radiographs of peridental alveolar bone: a possible diagnostic indicator of osteoporosis. oral surg oral med oral pathol 1992; 74(1): 98–110. 7. law an, bollen am, chen sk. detecting osteoporosis using dental radiographs: a comparison of four methods. j am dent assoc 1996; 127(12): 1734–42. 8. faber td, yoon dc, service sk, white sc. fourier and wavelet analyses of dental radiographs detect trabecular changes in osteoporosis. bone 2004; 35(2): 403–11. 9. white sc, atchison ka, gornbein ja, nattiv a, paganini-hill a, service sk, yoon dc. change in mandibular trabecular pattern and hip fracture rate in elderly women. dentomaxillofac radiol 2005; 34(3): 168–74. 10. arifin az, asano a, taguchi a, nakamoto t, yuniarti a, dewi lr, studiawan h. line strength measurement for trabecular bone analysis of mandible on panoramic radiographs. proceeding of the international workshop on advanced image technology 2010, kuala lumpur, malaysia, 2010 january 11-12. p. 1–5. 11. orimo h, hayashi y, fukunaga m, sone t, fujiwara s, shiraki m, kushida k, miyamoto s, soen s, nishimura j, oh-hashi y, hosoi t, gorai i, tanaka h, igai t, kishimoto h. osteoporosis diagnostic criteria review committee: japanese society for bone and mineral research. diagnostic criteria for primary osteoporosis: year 2000 revision. j bone miner metab 2001; 19(6): 331–7. 12. fujiwara s, masunari n, suzuki g, ross pd. performance of osteoporosis risk indices in a japanese population. curr ther res clin exp. 2001; 62: 586–93. 13. zwiggelaar r, astley sm, boggis crm, taylor cj. linear structure in mammographic images: detection and classification. ieee trans on medical imaging. 2004; 23(9): 1077–86. 14. dixon rn, taylor cj. automated asbestos fiber counting. proc inst phys conf series 1979; 44: 178–85. 15. farnel djj, hatfield fn, knox p, reakes m, spencer s, parry d, harding sp. enhancement of blood vessels in digital fundus photographs via the application of multiscale line operators. journal of franklin institute 2008; 345: 748–65. 16. marti r, zwiggelaar r, rubin c. tracking mammographic structures over time 12th british machine vision conference. proceedings of the 12th british machine vision conference. 2001: 143–52. 17. kawiyana, iks. osteoporosis patogenesis diagnosis dan penanganan terkini. j peny dalam 2009; 10(2): 157–70. 18. kawiyana, iks. interleukin-6 yang tinggi sebagai faktor risiko terhadap kejadian osteoporosis pada wanita pascamenopause defisiensi estrogen. j peny dalam 2009; 10(1): 18–24. 19. arifin az, asano a, taguchi a, nakamoto t, ohtsuka m, tsuda m, kudo y, tanimoto k. computer-aided system for measuring the mandibular cortical width on panoramic radiographs in identifying postmenopausal women with low bone mineral density. osteoporos int 2006; 17(5): 753–9. table �. diagnostic efficacy of manual and computer-aided automatic measurements of mandibular cortical width in identifying women with low skeletal bone mineral densities computer-aided system sensitivity (95% ci) specificity (95% ci) positive predictive value (95% ci) negative predictive value (95% ci) accuracy (95% ci) likelihood ratio (+) (95% ci) automatic measurement 92.0 (81.4–100.0) 86.7 (79.0–94.4) 69.7 (54.0–85.4) 97.0 (92.9–100.0) 88.0 (81.6–94.4) 6.9 (3.8–12.4) ci: confidence interval 149149 9. premkumar s. orthodontics : preparatory manual for undergraduates. 2nd ed. new delhi: elsevier; 2008. p. 436-40. 10. cansunar ha, uysal t. comparison of orthodontic treatment outcomes in nonextraction, 2 maxillary premolar extraction, and 4 premolar extraction protocols with the american board of orthodontics objective grading system. am j orthod dentofac orthop. 2014; 145(5): 595–602. 11. anthopoulou c, konstantonis d, makou m. treatment outcomes after extraction and nonextraction treatment evaluated with the american board of orthodontics objective grading system. am j orthod dentofac orthop. 2014; 146(6): 717–23. 12. mavreas d, athanasiou ae. factors affecting the duration of orthodontic treatment: a systematic review. eur j orthod. 2008; 30(4): 386–95. d research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 149–153 rankl expressions in preservation of surgical tooh extraction treated with moringa (moringa oleifera) leaf extract and demineralized freeze-dried bovine bone xenograft s. soekobagiono, adrian alfiandy, and agus dahlan department of prosthodontics faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: preservation of sockets is a procedure aimed to reduce bone resorption after tooth extraction. one of the most commonly used xenograft materials is demineralized freeze-dried bone bovine xenograft (dfdbbx). meanwhile, one of the key regulations in osteoclast genesis process is rankl bond. a decrease in the number of rankl expressions can suppress the osteoclast genesis process so that bone resorption can be prevented. the combination of moringa leaf extract and dfdbbx, as a result, is expected to decrease the number of rankl. purpose: this study aimed to measure rankl expressions in tooth extraction socket treated with moringa leaf extract combined with dfdbbx. methods: fifty six cavia cobaya rats were divided into eight groups. the first group was a control group with peg administration onto their extraction sockets. the second group was a treatment group with dfdbbx administration. the third group was a treatment group with moringa leaf extract administration. the fourth group was a treatment group induced with a combination of dfdbbx and moringa leaf extract. examination then was performed on days 7 and 30. after 7 and 30 days, those cavia cobaya rats were executed and tested with immunohistochemical techniques. further research data collected then were tested with one-way anova. results: there were significant differences between the control group and the groups induced with the combination of moringa leaf extract and dfdbbx. on days 7 and 30, the groups induced with the combination of moringa leaf extract and dfdbbx had the lowest number of rankl expressions. conclusion: the combination of moringa leaf extract and dfdbbx can decrease the number of rankl expressions in cavia cobaya rats on the day 7 and day 30 after tooth extraction. keywords: dfdbbx; moringa leaf extract; socket preservation; rankl; alveolar bone correspondence: soekobagiono, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: soekobagiono@fkg.unair.ac.id introduction tooth extraction is an act of removing a tooth from an alveolar bone socket. tooth extraction may be performed due to caries, periodontal disease, impaction, cyst, tumor, and fracture. tooth extraction can also be conducted on healthy teeth with the aim of improving malocclusion and esthetics.1 tooth extraction may trigger an inflammatory response and alveolar bone resorption in the bucolingual and apicocoronal dimensions of the edentulous ridge region.2 therefore, extraction sockets are necessary to maintain in order to keep their original forms, so the volume of alveolar bone can be maintained. dental implants performed on poor alveolar bone conditions are at risk of poor osseointegration, thus increasing the risk of dental implant failure. the application of implant in edentulous ridge that has large resorption, as a result, requires intervention of augmentation procedure first.3 the process of alveolar bone resorption begins with a bond between the reactor activators of nuclear kapa-b ligand (rankl) in the reactor activator of nuclear kapa-b (rank) presented in preosteoclasts. rankl/rank is the key regulation in the osteoclastogenesis process.4 the formation of osteoclasts, nevertheless, is also influenced dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p149-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p149-153 mailto:soekobagiono@fkg.unair.ac.id 150 soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 149–153 by proinflammatory cytokines, such as tumor necrotizing factor-α (tnf-α), interleukin-1 (il-1), and interleukin -6 (il-6). the rankl/rank bond, consequently, will stimulate tnf receptor-associated factor 6 (traf6), nfκb, c-jun n-terminal kinase (jnk)/cjun/fos, and nuclear factor of activated t cells (nfat) initiating differentiation of precursor osteoclasts into preosteclast cells.5 the osteoclast mediation process for resorption usually takes about 1-4 weeks.6 one of procedures to prevent alveolar bone resorption is socket preservation. socket preservation is an act of preserving the alveolar bone through a surgical procedure that aims to maintain the bone and soft tissue maximally after tooth extraction.7 this action is essential for the preparation of dental implants. the most widely used material for regenerating bones for purposes of socket preservation is graft. the application of graft material can provide normal healing and bone-to-implant contact.8 autograft is a gold standard for bone regeneration. unfortunately, it has limited amounts and high morbidity risk. thus, it was abandoned. allograft in the form of demineralized freezedried bovine bone xenograft (dfdbbx), on the other hand, is a substitute material that is biocompatible, osteoinductive, and osteoconductive. dfdbbx can be used for preserving alveolar bone sockets.9 biomaterials that can decrease the post inflammatory response, therefore, are needed to prevent excessive resorption. one material that can be developed to reduce the inflammatory response after tooth extraction is moringa oleifera leaf.10 moringa oleifera leaf is composed of amino acid, fatty acid, beta carotene, minerals, vitamin e, and flavonoids.11 these flavonoids then can act as anti-inflammation, anticancer, antimicrobial, antiviral, immunomodulatory, antithrombotic, and osteoprotection.10,12 some previous researches have shown that moringa leaf extract may inhibit the inflammatory pathway by inhibiting carrageenan in rats induced with edema. barriers to the inflammatory pathway then will inhibit bone resorption.13 moringa leaf extract may also increase the proliferation and differentiation of osteoblast cells.14,15 an anti-inflammatory combination between moringa leaves and osteoconductive and osteoinductive properties of dfdbbx, thus, is expected to provide a good response to the body in minimizing the formation of osteoclasts. a previous research even showed that 2% moringa oleifera leaf extract and dfdbbx can generate osteoblasts, but decrease osteoclasts.10 this study aimed to examine rankl expressions in tooth extraction sockets treated with moringa leaf extract and dfdbbx. materials and method this research was an experimental research with a randomized factorial design. this research was conducted in october-november 2016. research subjects used were healthy and active male cavia cobaya (n = 56) rats weighed 300-350 grams and aged 3-3.5 months old. those rats also had to eat normally without any defects on their body, their skin, and their senses, so they could walk normally, not limping, as well as had normal body temperature. this research was also approved by the ethics committee of faculty of dental medicine, universitas airlangga no. 026/hrecc.fodm/iii/2017. moringa (moringa oleifera) leaves were extracted at balai penelitian dan konsultasi industri surabaya, while dfdbbx used was produced in batan (bonegraft®, size 10 mesh/2000 microns). the treatment of cavia cobaya rats then was performed in biochemistry laboratory of faculty of medicine, universitas airlangga, surabaya. cavia cobaya rats were divided into eight groups, and then the left incisive tooth of the cavia cobaya rats was extracted. the sockets of the tooth extraction in each research group were preserved differently. the sockets in group i (ki) and group v (kv) were filled with poly etyle glycol (peg) as the control groups, while the sockets in group ii (kii) and group vi (kvi) were filled with dfdbbx. moreover, the sockets in group iii (kiii) and group vii (kvii) were filled with moringa leaf extract, while the sockets in group iv (kiv) and group viii (kviii) were filled with moringa leaf extract and dfdbbx. afterwards, the post-retrieval wounds and tooth extraction sockets were stitched. rankl expressions on the extraction sockets in ki, kii, kiii, and kiv then were observed on day 7, while rankl expressions in kv, kvi, kvii, and kviii were observed on day 30. on the observation days, the rats were sacrificed, and their mandible was taken for decalcification using edta for 30 days to make paraffin blocks. the process of making preparation and reading preparatory reading was conducted at anatomical pathology laboratory of dr. soetomo surabaya. rankl expressions then were observed by imunohitochemical technique using anti rankl monoclonal antibody (biotech ®, santacruz). the observations of the preparation and the measurement of rankl expressions were performed by using a light microscope with a 1000x magnification. rankl expressions were calculated by measuring the cells that emitted brown chromogenic. the data of rankl expressions obtained were tested with one sample kolmogorov smirnov test to analyze the normality of the data. afterwards, levene’s test was performed to analyze the homogeneity of the data. one-way anova then was conducted to analyze differences between the research groups. results the expressions of rankl on the day 7 can be seen in figure 1. the ihc results indicated that the number of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p149-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p149-153 151151soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 149–153 10 figure 2. the mean number of rankl expression on days-7 and -30. table 1. results of tukey hsd test on the number of rankl expressions on day -7 groups ki kii k iii kiv ki 0.000* 0.000* 0.000* kii 0.000* 0.002* kiii 0.001* kiv *: a significant difference/ significance (p<α=0.05) table 2. results of tukey hsd test on the number of rankl expressions on day -30 groups group v group vi group vii group viii group v 0.881 0.000* 0.000* group vi 0.002* 0.000* group vii 0.540 group viii *: a significant difference/ significance (p<α=0.05) nu m be r o f r a n k l group group group group group days 7th days 30th table 1. results of tukey hsd test on the number of rankl expressions on day 7 groups ki kii k iii kiv ki 0.000* 0.000* 0.000* kii 0.000* 0.002* kiii 0.001* kiv *: a significant difference (p<α=0.05) table 2. results of tukey hsd test on the number of rankl expressions on day 30 groups group v group vi group vii group viii group v 0.881 0.000* 0.000* group vi 0.002* 0.000* group vii 0.540 group viii *: a significant difference (p<α=0.05) i & v iv & viiiiii & viiii & vii day 7 day 30 figure 1. rankl expressions during ihc examination on day 7 in ki (a), kii (b), kiii (c), and kiv (d) and on day 30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). 9 figure 1. rankl expressions during ihc examination on day -7 in ki (a), kii (b), kiii (c), and kiv (d) and on day -30 in kv (e), kvi (f), kvii (g), and kviii (h). a b c fed g h figure 2. the mean number of rankl expression on days 7 and day 30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p149-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p149-153 152 soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 149–153 rankl expressions on day 7 was higher than that on day 30 (figure 2). however, the lowest number of rankl expressions on days 7 and 30 was found in the treatment group induced with the combination of moringa leaf extract and dfdbbx. the number of rankl expressions in that treatment group even was lower than in the treatment groups only given moringa leaf extract or dfdbbx. meanwhile, the highest number of rankl expressions was found in the control group. the data of rankl expressions on the day 7 were statistically analyzed with a normality test, one-sample kolmogorov-smirnov test. results of the one-sample kolmogorov-smirnov test showed the data were normally distributed with a p value of 0.748 (p>0.05). the data then were analyzed with a homegeneous test, by using levene’s test then done and got value p = 0.123 which shows research data is homogeneous data (p>0.05). differences between groups were tested using tukey hsd one-way anova and can be seen in table 1. there were statistically significant differences between treatment groups on day 7 (p<0.05). the data of rankl expressions observed on day 30 were tested for their normality by using kolmogorovsmirnov one-sample test. the results of the kolmogorovsmirnov one-sample test showed the data were normally distributed (p>0.05). homogeneity test then was performed by using levene’s test. the results of the levene’s test revealed that the data were homogeneous (p>0.05). next, tukey hsd test and one-way anova test were conducted to evaluate differences between research groups. the results can be seen in table 2. on day 30, there were statistically significant differences between research groups (p <0.05), except between group vii and group viii (p>0.05). discussion these experimental animals were selected since they have metabolism as well as immunological responses similar to humans.16 dfdbbx is a type of xenograft derived from bovine. xenograft has osteoconduction properties with porous internal surface allowing for revascularization and osteoblast migration from the socket base to support osteogenesis.17 the structure and inorganic content of bone matrix from xenograft also have osteoconductive properties to facilitate bone formation.18 bone formation using bovine hydroxyapatite xenograft can lead to good results, namely an increase in osteoprotegrin (opg) expressions and a decrease in rankl expressions as indicators of bone formation.19 xenograft inserted into the extraction socket, as a result, can serve as a framework for new bone growth, derived from osteoblasts at the bottom of the socket.20 moringa oleifera contains benzyl isothiocyanate compounds, and based on the results of phytochemical studies, also contains secondary metabolite compounds such as flavonoids, alkaloids, and phenols which can inhibit bacterial activity.21 moringa oleifera leaf extract may also decrease the production of nitrous oxide in lps-induced macrophage cells (lipopolysaccharides).22 in addition, moringa leaves can decrease pro-inflammatory mediators, such as prostaglandins, il-1β, il-6, and tnfα. this is due to the inhibition of serotonin and histamine release as well as prostaglandin synthesis.23 inflammatory mediators are osteoclast activating factors that play a role in bone resorption.24 moringa oleifera extract on tooth extraction wounds, is expected to inhibit the inflammatory process so that macrophage infiltration can be reduced. the decrease in tnf α, il-1β, and il-6 then leads to a decrease in rankl production.3 moringa leaf extract is indirectly osteoinduced by suppressing nfκb activation.22 thus, nfκb products are reduced so that cytokines that act as inflammatory mediators, such as tnf α, il1β, and il-6 also serve to stimulate the formation of rankl produced by osteoblast cells leading to a decrease in rankl production. the results of the treatments given in all research groups on day 30, indicated a trend of decreasing in the number of rankl expressions when compared with the results on day 7. this suggests that on day 30, the number of osteoblasts was higher than that on day 7. simialrly, a research conducted by guskuma reveals that on day 7 bone defects are still in inflammation and enter the early stage of resorption, while on day 30, bone defects begin in the early stage of bone formation.24 like this previous research, a research conducted by irinakis suggests that in the 4th week, bone deposition begins to occur in the socket retraction.25 at that time, osteoblast and other osteogenic tissues begin to form with a significantly increased number. the results were also in line with a research conducted by kresnoadi arguing that the number of osteoblasts on the 30th day increase, while osteoclasts decrease significantly compared to the previous day.26 in addition, the results of this research showed that post-extraction socket preservation, a procedure to reduce bone loss after tooth extraction to maintain dental alveoli/ tooth socket in alveolar bone, can minimize alveolar bone resorption and accelerate bone formation in the area of damage. besides, selection of materials used in preservation of socket retraction also has an important role in the process of bone formation. like the results of this research, a research conducted by grover demonstrates that dfdbbx has osteoconduction properties that serve as a scaffold for new bone growth, derived from osteoblasts at the bottom of the socket.10 based on the results of this research, osteoconductive and osteoinductive properties of the two materials above can decrease the number of rankl expressions significantly. this is likely to enhance the success of socket preservation further, so bone dimensions and volume after dental extraction will be maintained.27 it can be concluded that the combination of moringa leaf extract and dfdbbx can decrease the number of rankl expressions in the tooth extraction sockets of the cavia cobaya rats on days 7 and 30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p149-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p149-153 153153soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 149–153 references 1. fragiskos fd. medical history. in: oral surgery. berlin, heidelberg: springer-verlag berlin heidelberg; 2007. p. 1–20. 2. hansson s, halldin a. alveolar ridge resorption after tooth extraction: a consequence of a fundamental principle of bone physiology. j dent biomech. 2012; 3: 1–8. 3. henderson b, ward jm, ready d. aggregatibacter (actinobacillus) act i nomycetemcom it a ns: a t r iple a* p er io dontopat hogen? periodontol 2000. 2010; 54(1): 78–105. 4. yustina ar, suardita k, agustin d. peningkatan jumlah osteoklas pada keradangan periapikal akibat induksi lipopolisakarida p o r p hy r omon a s g i ng iva l i s (su a t u p en el it ia n la b o r a t o r ie s menggunakan tikus). j biosains pascasarjana. 2012; 14(3): 140–4. 5. fernández-tresguerres-hernández-gil i, alobera-gracia ma, delcanto-pingarrón m, blanco-jerez l. physiological bases of bone regeneration ii. the remodeling process. med oral patol oral cir bucal. 2006; 11(2): e151-7. 6. kini u, nandeesh bn. physiology of bone formation, remodeling, and metabolism. in: fogelman i, gnanasegaran g, wall h van der, editors. radionuclide and hybrid bone imaging. berlin, heidelberg: springer berlin heidelberg; 2012. p. 29–57. 7. peck mt, marnewick j, stephen l. alveolar ridge preservation using leukocyte and platelet-rich fibrin: a report of a case. case rep dent. 2011; 2011: 345048. 8. barone a, aldini nn, fini m, giardino r, calvo guirado jl, covani u. xenograft versus extraction alone for ridge preservation after tooth removal: a clinical and histomorphometric study. j periodontol. 2008; 79(8): 1370–7. 9. weiss c, weiss a. principles and practice of implant dentistry. 1st ed. usa: mosby; 2001. p. 447. 10. grover v, malhotra r, kapoor a, sachdeva s. bone allografts: a review of safety and efficacy. indian j dent res. 2011; 22(3): 496. 11. burali sc, kangralkar v, sravani os, patil sl. the beneficial effect of ethanolic extract of moringa oleifera on osteoporosis. int j phamaccutical appl. 2010; 1(1): 50–8. 12. wihastuti ta, sargowo d, rohman ms. the effect of moringa oleifera leaf extract in inhibition of nfκb activation, tnf-α and icam-1 expression in oxydized ldl treated huvecs. j kadiologi indones. 2007; 28(3): 181–8. 13. coppin j. a study of the nutritional and medicinal values of moringa oleifera leaves from sub-saharan africa: ghana, rwanda, senegal and zambia. the state university of new jersey; 2008. p. 5-99. 14. garcía-lafuente a, guillamón e, villares a, rostagno ma, martínez ja. flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease. inflamm res. 2009; 58(9): 537–52. 15. patel c, rangrez a, parikh p. the anti-osteoporotic effect of moringa oliefera on osteoblastic cells : saos 2. iosr j pharm biol sci. 2013; 5(2): 10–7. 16. fox jg, anderson lc, otto gm, pritchett-corning kr, whary mt. laboratory animal medicine. 3rd ed. london: elsevier academic press; 2015. p. 427-9. 17. cohen jr. mm. the new bone biology: pathologic, molecular, and clinical correlates. am j med genet part a. 2006; 140a(23): 2646–706. 18. al-ghamdi h, mokeem sa, anil s. current concepts in alveolar bone augmentation : a critical appraisal. saudi dent j. 2007; 19(2): 74–90. 19. chaves md, de souza nunes ls, de oliveira rv, holgado la, filho hn, matsumoto ma, ribeiro da. bovine hydroxyapatite (bio-oss®) induces osteocalcin, rank-l and osteoprotegerin expression in sinus lift of rabbits. j cranio-maxillofacial surg. 2012; 40(8): e315–20. 20. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc. 2007; 73(6): 513. 21. coppin jp, xu y, chen h, pan mh, ho ct, juliani r, simon je, wu q. determination of flavonoids by lc/ms and anti-inflammatory activity in moringa oleifera. j funct foods. 2013; 5(4): 1892–9. 22. araújo lcc, aguiar js, napoleão th, mota fvb, barros als, moura mc, coriolano mc, coelho lcbb, silva tg, paiva pmg. evaluation of cytotoxic and anti-inflammatory activities of extracts and lectins from moringa oleifera seeds. plos one. 2013; 8(12): e81973. 23. mohd f, ahmad f, kumar a, yunus sm. an experimental evaluation of anti-inflammatory activity of moringa oleifera seeds. int j pharm pharm sci. 2013; 5(3): 1–5. 24. guskuma mh, hochuli-vieira e, pereira fp, rangel-garcia i, okamoto r, okamoto t, filho om. evaluation of the presence of vegf, bmp2 and cbfa1 proteins in autogenous bone graft: h istomet r ic a nd i m munoh istochem ica l a na lysis. j cra n io maxillofacial surg. 2014; 42(4): 333–9. 25. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc. 2006; 72(10): 917–22. 26. kresnoadi u, ariani m, djulaeha e, hendrijantini n. the potential of mangosteen (garcinia mangostana) peel extract, combined with demineralized freeze-dried bovine bone xenograft, to reduce ridge resorption and alveolar bone regeneration in preserving the tooth extraction socket. j indian prosthodont soc. 2017; 17(3): 282. 27. mezzomo la, shinkai rs, mardas n, donos n. alveolar ridge preservation after dental extraction and before implant placement: a literature review. rev odonto ciência. 2011; 26(1): 77–83. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p149-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p149-153 vol 38 no 3 2005 135 aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro) (in vitro antibacterial activity of flavonoids trigona sp propolis against streptococcus mutans) ardo sabir bagian konservasi gigi fakultas kedokteran gigi universitas hasanuddin makassar indonesia abstract a number of investigations have shown a positive correlation between the number of streptococcus mutans (s. mutans) in dental plaque and the prevalence of dental caries. consequently, this microorganism has been the prime target for the prevention of dental caries. propolis being a substance made by the honeybee, is a potent antibacterial agent. the main chemical class present in propolis is flavonoids. flavonoids are well-known plant compounds that have antibacterial property. because s. mutans is accepted to be one of the microorganisms responsible for dental caries and flavonoids in propolis are antibacterial, the purpose of this study was to evaluate in vitro the antibacterial activity of flavonoids trigona sp propolis against s. mutans as a first step in its possible use as an alternative anticaries agent. extract flavonoids was purified from ethanol extract of propolis which was obtained from bulukumba regency south sulawesi using thin layer chromatography. the purification of flavonoids was carried-out by uvradiation at λmax 254 nm and λmax 366 nm and treatment with ammonia. extract flavonoids was diluted in aquadest to 0.05%; 0.075%; 0.1%; 0.25%; 0.5%; 0.75% concentrations. aquadest and 10% povidone iodine were also used as control solution. s mutans were grown in medium glucose nutrient agar and incubated with flavonoids for 24 and 48 hours, at 37° c. antibacterial activity was reflected by the diameter of the inhibition zones around the stainless steel cylinder. the data were analyzed by using anova followed by lsd test with significance level of 5%. the results of this study showed that after being incubated for 24 and 48 hours, all flavonoid concentrations significantly (p < 0.05) inhibited the growth of s mutans. 0.1% flavonoid was the most effective concentration to inhibit the growth of s mutans after 24 hours of incubation and 0.5% flavonoid after 48 hours of incubator. key words: antibacterial activity, flavonoids, propolis, streptococcus mutans korespondensi (correspondence): ardo sabir, bagian konservasi gigi, fakultas kedokteran gigi universitas hasanuddin. jln. kandea 5 makassar, indonesia. pendahuluan seiring perkembangan zaman, maka masalah kesehatan khususnya kesehatan gigi dan mulut semakin lama makin meningkat pula. hal ini disebabkan timbulnya penyakit gigi dan mulut dipengaruhi oleh berbagai faktor yang saling berinteraksi satu dengan lainnya yakni faktor pendidikan, status sosial, penghasilan, pola makan, pekerjaan, bahkan budaya manusia itu sendiri.1 penyakit karies gigi dan penyakit periodontal merupakan dua penyakit gigi dan mulut yang paling sering ditemukan di klinik gigi dan merupakan penyebab utama hilangnya gigi di dalam rongga mulut.2 berdasarkan survei kesehatan gigi yang dilakukan oleh direktorat kesehatan gigi departemen kesehatan ri pada tahun 1994, ternyata selama pelita ke-v jumlah masyarakat yang berkunjung maupun pasien yang dirujuk ke rumah sakit karena menderita penyakit gigi dan mulut akibat karies gigi menduduki jumlah terbesar yaitu 53,05%, sedangkan penyakit periodontal menduduki tempat kedua yaitu sebanyak 28,32%.3 karies gigi merupakan suatu penyakit infeksi yang dapat menular dan terutama mengenai jaringan keras gigi, sehingga terjadi kerusakan jaringan keras setempat. proses terjadinya kerusakan pada jaringan keras gigi melalui suatu reaksi kimiawi oleh bakteri, dimulai dengan proses kerusakan pada bagian anorganik, kemudian berlanjut pada bagian organik.4 bakteri berperan penting pada proses terjadinya karies gigi, karena tanpa adanya bakteri maka karies gigi tidak dapat terjadi. terdapat berbagai spesies bakteri yang berkoloni di dalam rongga mulut khususnya pada plak gigi dan bakteri tersebut mampu menghasilkan asam sehingga terjadi proses demineralisasi jaringan keras gigi.5 salah satu spesies bakteri yang dominan dalam mulut yaitu bakteri streptococcus mutans (s. mutans). jenis bakteri ini diketahui merupakan bakteri penyebab utama timbulnya karies gigi.5 telah banyak penelitian yang membuktikan adanya korelasi positif antara jumlah bakteri s. mutans pada plak gigi dengan prevalensi karies gigi,4 hal ini disebabkan beberapa karakteristik dari bakteri s. mutans6 yaitu mampu mensintesis polisakarida 136 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 135–141 ekstraseluler glukan ikatan α (1–3) yang tidak larut dari sukrosa, dapat memproduksi asam laktat melalui proses homofermentasi, membentuk koloni yang melekat dengan erat pada permukaan gigi, dan lebih bersifat asidogenik dibanding spesies streptococcus lainnya. oleh karena itu bakteri ini telah menjadi target utama dalam upaya mencegah terjadinya karies gigi. telah banyak dilakukan penelitian dengan memanfaatkan bahan alam yang kesemuanya bertujuan untuk menghasilkan obat-obatan dalam upaya mendukung program pelayanan kesehatan gigi, khususnya untuk mencegah dan mengatasi penyakit karies gigi. kembalinya perhatian ke bahan alam yang dikenal dengan istilah back to nature ini dianggap sebagai hal yang sangat bermanfaat karena sejak dahulu kala masyarakat kita telah percaya bahwa bahan alam mampu mengobati berbagai macam penyakit. selain itu, pemanfaatan bahan alam yang digunakan sebagai obat jarang menimbulkan efek samping yang merugikan dibandingkan obat yang terbuat dari bahan sintetis.7 madu merupakan salah satu produk alam yang dihasilkan oleh lebah yang telah lama dikenal dan dimanfaatkan di indonesia karena khasiatnya dalam menyembuhkan berbagai macam penyakit. namun demikian, ternyata lebah juga menghasilkan produk lain seperti royal jelly, pollen, venom, dan propolis. setiap produk lebah tersebut mempunyai fungsi dan manfaat yang berbeda bagi kesehatan manusia.8 propolis atau lem lebah merupakan suatu bahan resin yang dikumpulkan oleh lebah madu dari berbagai macam jenis tumbuhan.9,10 salah satu jenis lebah yang mampu menghasilkan propolis dalam jumlah banyak yaitu jenis trigona sp. jenis lebah ini banyak dijumpai di propinsi sulawesi selatan baik didataran tinggi maupun dataran rendah, namun demikian propolis yang dihasilkan pemanfaatannya belum optimal oleh karena penelitian yang dilakukan masih terbatas.8 namun demikian, di luar negeri, penelitian terhadap propolis telah banyak dilakukan baik secara in vitro maupun in vivo dan hasilnya menunjukkan bahwa propolis memiliki beberapa aktivitas biologis dan farmakologis antara lain bersifat antibakteri baik terhadap bakteri gram positif11-13 maupun gram negatif.14 aktivitas antibakteri propolis yang sangat bervariasi ini lebih disebabkan komposisi dari propolis yang digunakan. komposisi propolis sendiri sangat dipengaruhi oleh jenis dan umur tumbuhan, iklim, dan waktu di mana propolis tersebut diperoleh.15,16 salah satu kandungan senyawa kimia yang penting pada propolis adalah senyawa flavonoid.9 flavonoid merupakan salah satu senyawa fenol alami yang tersebar luas pada tumbuhan, yang disintesis dalam jumlah sedikit (0,5–1,5%)17 dan dapat ditemukan pada hampir semua bagian tumbuhan.18 penelitian secara in vitro maupun in vivo menunjukkan aktivitas biologis dan farmakologis dari senyawa flavonoid sangat beragam, 19 salah satu diantaranya yakni memiliki aktivitas antibakteri.20,21 walau demikian, penelitian untuk mengetahui pengaruh flavonoid propolis trigona sp terhadap bakteri s. mutans belum pernah dilakukan. berdasarkan uraian di atas maka timbul suatu permasalahan yaitu bagaimana pengaruh flavonoid yang terdapat pada propolis trigona sp terhadap pertumbuhan bakteri s. mutans secara in vitro, sehingga tujuan penelitian ini adalah untuk melihat aktivitas antibakteri flavonoid yang terdapat pada propolis trigona sp terhadap pertumbuhan bakteri s. mutans secara in vitro. bahan dan metode jenis penelitian ini adalah eksperimental laboratoris, dan dilakukan di dua tempat yakni: laboratorium galenika, fakultas farmasi, universitas gadjah mada, yogyakarta untuk proses ekstraksi flavonoid dari propolis trigona sp dan laboratorium mikrobiologi farmasi, fakultas mipa, universitas hasanuddin, makassar untuk menentukan konsentrasi hambat minimal (khm) dan uji aktivitas antibakteri dari flavonoid terhadap bakteri s. mutans. proses ekstraksi senyawa flavonoid dari propolis trigona sp dilakukan dengan cara:22 ditimbang propolis sebanyak 1 kg, kemudian dimasukkan ke dalam maserator berpengaduk elektrik. lima liter etanol 95% ditambahkan sebagai pelarut. maserasi dilakukan dengan pengadukan sebanyak 12 kali selama 15 menit dengan tenggang waktu 5 menit antar pengadukan, dilanjutkan dengan perendaman selama 120 jam, selanjutnya dilakukan penyaringan dengan corong dan kertas saring untuk memisahkan filtrat dari ampas ke dalam labu erlenmeyer sehingga diperoleh filtrat ± 2,5 liter. filtrat diuapkan di atas cawan porselin sehingga kandungan etanolnya menguap dan diperoleh ekstrak yang konsistensinya kental (± 100 g). ekstrak kental tersebut kemudian dituang ke dalam labu erlenmeyer dan ditambahkan 500 ml larutan toluena, lalu diaduk sehingga semua ekstrak larut. larutan air : etanol = 2 : 1 (v/v) sebanyak 1,5 liter ditambahkan ke labu erlenmeyer yang berisi larutan ekstrak dalam toluena, kemudian diaduk hingga homogen dan didiamkan selama 24 jam. setelah 24 jam, larutan dipindahkan ke dalam corong pemisah dan didiamkan hingga terbentuk 2 lapisan. melalui corong pemisah, larutan bagian bawah (± 1,5 liter) dipisahkan dan diuapkan sehingga diperoleh ekstrak kental yang merupakan fraksi flavonoid polar (± 17 g). lapisan atas (± 500 ml) yang berada pada corong pemisah merupakan larutan toluena ditambah 1,5 liter larutan etanol : air = 2 : 1 (v/v), diaduk hingga homogen dan didiamkan hingga terbentuk 2 lapisan. lapisan bagian bawah (fraksi flavonoid semipolar) diuapkan sampai diperoleh ekstrak dengan konsistensi kental seberat ± 1 g, sedangkan lapisan bagian atas (fraksi non flavonoid) menghasilkan ekstrak yang konsistensinya kental seberat ± 100 g setelah diuapkan. tahap berikutnya adalah analisis kimia menggunakan teknik kromatografi lapis tipis (klt) dengan fase diam 137sabir: aktivitas antibakteri flavonoid propolis trigona sp lempeng silika gel gf 254 dan fase gerak larutan n-butanol : asam asetat : air = 3 : 1 : 1 (v/v) untuk mengetahui apakah kedua ekstrak flavonoid (polar dan semipolar) yang diperoleh tidak mengandung unsur lain, selain senyawa flavonoid. kedua ekstrak flavonoid yang telah dilarutkan dengan alkohol, diteteskan dengan pipet kapiler pada lempeng silika berukuran 10 cm × 4 cm dan dimasukkan ke bejana pengembang yang berisi fase gerak. setelah daya kapiler dari kedua ekstrak telah maksimal, lempeng dikeringkan, kemudian dilihat di bawah lampu ultraviolet sebelum dan setelah diuapi dengan amoniak. penentuan konsentrasi hambat minimal (khm) ekstrak flavonoid propolis trigona sp terhadap bakteri s. mutans diawali dengan membuat ekstrak flavonoid dalam beberapa konsentrasi yaitu 0,05%; 0,075%; 0,1%; 0,25%; 0,5%; dan 0,75%, dengan cara: untuk konsentrasi 0,75%, ekstrak flavonoid ditimbang seberat 0,075 g. setelah ditimbang ekstrak kemudian dilarutkan dengan aquades steril dalam lumpang hingga mencapai volume 10 ml, setelah itu dimasukkan ke dalam labu ukur, begitu pula untuk membuat konsentrasi lainnya. masing-masing sampel dimasukkan ke dalam botol yang berbeda, kemudian ditutup dengan menggunakan kapas dan aluminium foil, selanjutnya dibuat medium nutrient agar (na) yang komposisinya: ekstrak ragi 3 g, pepton 5 g, agar 15 g, dan aquades 1000 ml dibuat sebanyak 250 ml pada labu erlenmeyer. medium ini kemudian dipanaskan sampai seluruh bahan larut, selanjutnya dimasukkan ke dalam tabung reaksi dan disterilkan dalam autoklaf selama 15 menit pada temperatur 121° c. setelah steril, medium dimiringkan dan ditunggu sampai memadat. bakteri s. mutans diambil dengan menggunakan ose lalu digoreskan pada medium na miring dan diinkubasi selama 24 jam pada temperatur 37° c. medium na yang berisi bakteri s. mutans kemudian disuspensikan dengan menggunakan nacl 0,9 %. setelah itu dibuat medium nutrient broth (nb) dengan komposisi: ekstrak ragi 3 g, pepton 5 g, dan aquades steril 1000 ml dibuat sebanyak 250 ml di dalam gelas kimia. medium ini dimasukkan ke dalam 8 tabung reaksi masingmasing 5 ml, kemudian disterilkan dengan autoklaf selama 15 menit pada temperatur 121° c. tabung reaksi tersebut kemudian dibiarkan dingin. tahap selanjutnya adalah pada 6 dari 8 tabung reaksi dimasukkan biakan bakteri s. mutans sebanyak 0,02 ml dan diaduk hingga homogen kemudian ditambahkan 5 ml dari setiap konsentrasi flavonoid, sedangkan pada 2 tabung reaksi lainnya masing-masing diisi dengan flavonoid konsentrasi terendah (0,05%) dan konsentrasi tertinggi (0,75%) sebagai kontrol. semua tabung reaksi diinkubasi selama 24 jam pada temperatur 37° c, setelah masa inkubasi, dilakukan pemeriksaan ada/ tidaknya pertumbuhan bakteri s. mutans. metode yang digunakan untuk uji aktivitas antibakteri ekstrak flavonoid propolis trigona sp terhadap bakteri s. mutans adalah metode difusi agar. uji aktivitas antibakteri dilakukan dengan cara mengambil sampel satu konsentrasi di bawah khm, semua konsentrasi di atas khm, kontrol positif, dan kontrol negatif. adapun prosesnya adalah sebagai berikut, medium glucose nutrient agar (gna) dengan komposisi: glukosa 10 g, ekstrak ragi 5 g, pepton 10 g, nacl 2,5 g, agar 15 g, dan aquades steril 1000 ml dibuat sebanyak 300 ml di dalam labu erlenmeyer. medium ini kemudian dimasukkan ke dalam cawan petri steril dan dibuat menjadi 2 lapisan dengan ketebalan yang hampir sama (± 0,5 cm). lapisan pertama dibiarkan pada temperatur kamar ± 20 menit hingga mengeras, setelah itu dibuat lapisan kedua yang sebelumnya telah dicampurkan dengan biakan bakteri s. mutans sebanyak 1 ml dan dimasukkan dalam cawan petri. sebelum lapisan kedua mengeras, ditempatkan 7 silinder stainless steel (diameter luar 8 mm dan diameter dalam 6 mm) pada cawan petri, 5 untuk masing-masing sampel konsentrasi flavonoid, 1 untuk kontrol negatif (aquades steril), dan 1 untuk kontrol positif (povidone iodine 10%). pada silinder tersebut kemudian diisi dengan larutan sampel dan kontrol dengan menggunakan spuit, selanjutnya, cawan petri dimasukkan ke dalam inkubator selama 24 jam pada temperatur 37° c. pengukuran diameter dari setiap zone inhibisi pertumbuhan bakteri yang terjadi di sekeliling selinder dilakukan dengan menggunakan jangka sorong setelah 24 jam dan 48 jam masa inkubasi. prosedur ini dilakukan dengan replikasi sebanyak 3 kali terhadap bakteri s. mutans. zone inhibisi adalah jarak terdekat (mm) dari tepi luar selinder hingga mulai terjadinya pertumbuhan bakteri.23 data yang diperoleh merupakan hasil pengamatan secara laboratorium yang selanjutnya dianalisis dengan menggunakan statistik parametrik yaitu uji one-way anova. bila hasil uji anova tersebut menunjukkan hasil yang signifikan, maka dilanjutkan dengan uji least significant difference (lsd). sementara untuk mengetahui ada/tidaknya interaksi antara lama waktu kontak dengan konsentrasi flavonoid maka dilakukan uji two-way anova. hasil hasil proses ekstraksi senyawa flavonoid dari propolis trigona sp diperoleh 3 macam ekstrak kental yang masingmasing merupakan fraksi flavonoid polar, fraksi flavonoid semi polar, dan fraksi non flavonoid dengan berat berturutturut 17 g, 1 g, dan 100 g. hasil analisis dengan teknik kromatografi lapis tipis (klt) menunjukkan bahwa pengamatan di bawah sinar uv λmaks 254 nm ternyata pada fraksi flavonoid semi polar terdapat 3 bercak, sedangkan pada fraksi flavonoid polar 2 bercak (gambar 1). kelima bercak tersebut kemudian diberi tanda. pengamatan terhadap bercak di bawah sinar uv λmaks 366 nm, menunjukkan bahwa kelima bercak tersebut berfluoresensi (gambar 1). hal ini membuktikan bahwa tidak terdapat senyawa lain, selain senyawa flavonoid pada kedua ekstrak fraksi flavonoid tersebut. 138 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 135–141 a b penilaian uji konsentrasi hambat minimal (khm) ekstrak flavonoid propolis trigona sp terhadap bakteri s. mutans berdasarkan tingkat kekeruhan yang terjadi pada tabung reaksi yang berisi medium nb dan bakteri s. mutans. hasil uji khm menunjukkan bahwa khm ekstrak flavonoid propolis trigona sp terhadap bakteri s. mutans adalah 0,1%. hal ini disebabkan flavonoid 0,1% merupakan konsentrasi flavonoid terkecil yang masih dapat menghambat pertumbuhan s. mutans, yang ditandai dengan warna jernih pada tabung reaksi. setelah khm diperoleh, maka dilanjutkan dengan uji aktivitas antibakteri. hasil pengamatan aktivitas antibakteri flavonoid propolis trigona sp terhadap s. mutans setelah inkubasi selama 24 jam dan 48 jam pada temperatur 37° c dapat dilihat pada gambar 2. pada gambar 2 terlihat bahwa 24 jam dan 48 jam setelah inkubasi terlihat adanya zone inhibisi di sekitar selinder baik yang berisi berbagai konsentrasi ekstrak flavonoid propolis trigona sp maupun pada selinder yang berisi povidone iodine 10% (kontrol positif). rerata hasil pengukuran terhadap diameter zone inhibisi yang telah dilakukan pada setiap kelompok setelah inkubasi 24 jam dan 48 jam diperoleh bahwa semakin lama waktu kontak antara flavonoid dengan bakteri s. mutans maka diameter zone inhibisi yang terjadi juga semakin besar demikian pula dengan kontrol positif, kecuali kontrol negatif, sedangkan pada pengamatan 48 jam setelah masa inkubasi menunjukkan bahwa semakin tinggi konsentrasi flavonoid maka zone inhibisi yang terjadi semakin luas. untuk mengetahui ada/tidaknya perbedaan yang signifikan antarkelompok, maka dilakukan analisis statistik dengan menggunakan uji one-way anova yang hasilnya tampak pada tabel 1. gambar 2. hasil uji aktivitas antibakteri ekstrak flavonoid propolis trigona sp dengan konsentrasi 0,075%; 0,1%; 0,25%; 0,5%; 0,75%; dan kontrol positif terhadap pertumbuhan s. mutans pada temperatur 37° c setelah inkubasi; (a) 24 jam, (b) 48 jam. a b gambar 1. hasil kromatografi lapis tipis fraksi flavonoid polar dan semipolar dari propolis trigona sp. a: fraksi flavonoid semi polar; b: fraksi flavonoid polar; fase diam: silika gel gf254; fase gerak: n butanol: asam asetat : air = 3 : 1 : 1 (v/v); identifikasi bercak: sinar uvλmaks 366 nm; pereaksi: uap amoniak. 139sabir: aktivitas antibakteri flavonoid propolis trigona sp oleh karena hasil uji one-way anova di atas menunjukkan adanya perbedaan diameter zone hambat yang signifikan (p < 0,001) antara semua kelompok setelah masa inkubasi 24 jam dan 48 jam, maka dilakukan analisis lebih lanjut dengan menggunakan uji least significant difference (lsd) yang hasilnya tampak pada tabel 2. pada tabel 2 terlihat bahwa pada pengamatan 24 jam, terdapat perbedaan yang signifikan antara kontrol negatif dan flavonoid 0,075% dengan kelompok lainnya, sebailiknya tidak terdapat perbedaan yang signifikan antara flavonoid 0,1%; 0,25%; 0,5%; 0,75%, dan kontrol positif. sementara pada pengamatan 48 jam, selain terdapat perbedaan yang signifikan antara kontol negatif dan flavonoid 0,25% dengan kelompok lainnya, dan antara flavonoid 0,075% dengan kelompok flavonoid 0,25%; 0,5%; 0,75%; dan kontrol negatif, juga terdapat perbedaan yang signifikan antara flavonoid konsentrasi 0,5% dengan semua kelompok, kecuali dengan flavonoid 0,75%. untuk mengetahui ada/tidaknya interaksi antara lama waktu inkubasi dengan konsentrasi flavonoid, maka dilakukan analisis statistik dengan menggunakan uji two-way anova, yang hasilnya dapat dibaca pada tabel 3. pada tabel 3 tampak bahwa terdapat interaksi antara lama waktu kontak dengan konsentrasi flavonoid propolis trigona sp. tabel 3. hasil uji two-way anova mengenai interaksi antara lama waktu kontak dengan konsentrasi flavonoid jk db rk f sig f hari 157,567 1 157,567 1294,440 0,001* konsentrasi 831,109 6 138,518 1137,948 0,001* hari*konsentrasi 50,045 6 8,341 68,521 0,001* keterangan: jk = jumlah kuadrat; db = derajat bebas; rk = rerata kuadrat; f = f hitung; * = signifikan pada p < 0,001. pembahasan bahan uji yang digunakan pada penelitian ini berupa ekstrak hasil ekstraksi yang mengandung seluruh jenis senyawa flavonoid yang terdapat pada propolis trigona sp. propolis trigona sp dikumpulkan dari sarang lebah yang terdapat di kabupaten bulukumba propinsi sulawesi selatan. ekstrak flavonoid hasil ekstraksi selanjutnya dianalisis dengan menggunakan teknik kromatografi lapis tipis (klt) untuk mengetahui tingkat kemurnian atau purifikasi dari ekstrak. pemilihan teknik klt didasarkan atas beberapa alasan, yakni teknik ini hanya memerlukan cuplikan dalam jumlah sedikit, waktu yang dibutuhkan untuk menganalisis cuplikan relatif singkat, alat yang pergunakan cukup sederhana dan mudah diperoleh, biaya yang dibutuhkan relatif ekonomis, dan yang penting adalah memberikan hasil pemisahan yang memuaskan baik antara flavonoid itu sendiri, maupun antara flavonoid dengan senyawa lainnya.24,25 para peneliti menyatakan pendapat yang berbeda-beda sehubungan dengan mekanisme kerja dari flavonoid dalam flavonoid 24 jam 48 jam kontrol negatif 0,075% 0,1% 0,25% 0,5% 0,75% kontrol positif kontrol negatif − 0,001* 0,001* 0,001* 0,001* 0,001* 0,001* flavonoid 0,075% 0,001* − 0,001* 0,002* 0,004* 0,003* 0,001* flavonoid 0,1% 0,001* 0,315 − 0,148 0,060 0,082 0,324 flavonoid 0,25% 0,001* 0,001* 0,001* − 0,617 0,739 0,617 flavonoid 0,5 % 0,001* 0,001* 0,001* 0,023* − 0,867 0,324 flavonoid 0,75% 0,001* 0,001* 0,001* 0,012* 0,745 − 0,409 kontrol positif 0,001* 0,177 0,711 0,000* 0,001* 0,001* − keterangan: * = signifikan pada p < 0,05 tabel 2. hasil uji lsd mengenai diameter zone inhibisi antar kelompok setelah inkubasi 24 dan 48 jam tabel 1. hasil uji one-way anova jalur diameter zone inhibisi antara kelompok a jk db rk f sig f antar kelompok 271,832 6 45,305 787,920 0,001* dalam kelompok 0,805 14 5,750e-02 jumlah 272,637 20 b jk db rk f sig f antar kelompok 609,321 6 101,554 546,127 0,001* dalam kelompok 2,603 14 0,186 jumlah 611,925 20 keterangan: jk = jumlah kuadrat; db = derajat bebas; rk= rerata kuadrat; f = f hitung; * = singnifikan pada p < 0,001 (a) 24 jam, (b) 48 jam. 140 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 135–141 menghambat pertumbuhan bakteri, antara lain bahwa flavonoid menyebabkan terjadinya kerusakan permeabilitas dinding sel bakteri, mikrosom, dan lisosom sebagai hasil interaksi antara flavonoid dengan dna bakteri, 26,27 sementara mirzoeva et al.21 dalam penelitiannya mendapatkan bahwa flavonoid mampu melepaskan energi tranduksi terhadap membran sitoplasma bakteri selain itu juga menghambat motilitas bakteri. mekanisme yang berbeda dikemukakan oleh di carlo et al.28 dan estrela et al.29 yang menyatakan bahwa gugus hidroksil yang terdapat pada struktur senyawa flavonoid menyebabkan perubahan komponen organik dan transpor nutrisi yang akhirnya akan mengakibatkan timbulnya efek toksik terhadap bakteri. hasil dari uji konsentrasi hambat minimal memperlihatkan nilai khm yang diperoleh yaitu flavonoid 0,1%. kecilnya nilai khm ini mungkin disebabkan ekstrak flavonoid yang digunakan pada penelitian ini yang merupakan hasil proses ekstraksi dari propolis trigona sp sudah tidak mengandung senyawa lain yang mungkin tidak bersifat antibakteri yang dapat mengganggu daya antibakteri flavonoid, mengingat komposisi propolis sebagian besar berupa campuran resin dan getah (39–53%), serta lilin (wax) (19–35%).15,16 metode yang digunakan untuk mengevaluasi aktivitas antibakteri dari flavonoid terhadap bakteri s. mutans adalah metode difusi agar, oleh karena metode ini paling umum digunakan untuk menentukan suseptibilitas dari bakteri terhadap bahan yang diuji. 30,31hasilnya menunjukkan bahwa setelah 24 jam dan 48 jam, kecuali kontrol negatif, semua kelompok flavonoid dan kontrol positif mampu menghambat pertumbuhan bakteri s. mutans. setelah masa inkubasi 48 jam, ternyata semakin tinggi konsentrasi flavonoid maka rerata diameter zone inhibisi yang terjadi semakin luas pula. hal ini sesuai dengan pendapat pelzcar and chan32 bahwa semakin tinggi konsentrasi suatu bahan antibakteri maka aktivitas antibakterinya akan semakin kuat pula. hal yang berbeda terjadi pada hasil pengamatan 24 jam di mana flavonoid 0,1% menghasilkan rerata diameter zone inhibisi yang terluas dibanding flavonoid lainnya maupun kontrol positif. hal ini mungkin disebabkan karena diameter zone inhibisi yang terjadi sangat dipengaruhi oleh beberapa faktor antara lain toksisitas bahan uji, kemampuan difusi bahan uji pada media, interaksi antar komponen medium, dan kondisi lingkungan mikro in vitro.31 berdasarkan tabel 2 maka dapat disimpulkan bahwa setelah masa inkubasi 24 jam, semua konsentrasi flavonoid yang diuji mampu menghambat pertumbuhan s. mutans dan flavonoid dengan konsentrasi 0,1% merupakan konsentrasi yang paling efektif dibanding konsentrasi flavonoid lainnya. selain itu, pada periode waktu ini efektivitas daya antibakteri flavonoid 0,1% sama dengan povidone iodine 10% (kontrol positif). sementara setelah inkubasi 48 jam, semua konsentrasi flavonoid yang diuji mampu menghambat pertumbuhan s. mutans dan flavonoid dengan konsentrasi 0,5% merupakan konsentrasi yang paling efektif dibanding konsentrasi flavonoid lainnya, dan efektivitas daya antibakteri flavonoid 0,5% lebih baik dibanding povidone iodine 10% (kontrol positif). hasil uji two-way anova (tabel 3) memperlihatkan adanya interaksi antara lama waktu kontak dengan konsentrasi. hal ini tampak dengan terjadinya peningkatan konsentrasi flavonoid yang efektif dalam menghambat pertumbuhan s. mutans seiring dengan semakin lamanya waktu kontak atau dengan kata lain dengan bertambah lamanya waktu kontak maka terjadi penurunan aktivitas antibakteri dari flavonoid, hal ini mungkin disebabkan akibat terjadinya penurunan metabolisme flavonoid tersebut,33 walaupun pada penelitian ini terlihat bahwa semakin lama waktu kontak maka diameter zone inhibisi yang terjadi juga semakin luas. berdasarkan hal tersebut di atas, maka penulis berasumsi bahwa penggunaan flavonoid dengan konsentrasi rendah (0,1%) untuk periode waktu singkat (24 jam), sangat efektif dalam menghambat pertumbuhan s. mutans bila frekuensi aplikasinya dilakukan secara terus menerus atau kontinyu, sedangkan bila flavonoid digunakan untuk periode waktu yang lama (> 24 jam) dengan frekuensi aplikasi yang jarang, maka penggunaan flavonoid dengan konsentrasi yang tinggi (> 0,1%) sangat dianjurkan. berdasarkan hasil penelitian ini maka dapat disimpulkan beberapa hal, yakni setelah inkubasi selama 24 dan 48 jam, semua konsentrasi flavonoid yang diuji mampu menghambat pertumbuhan s. mutans. setelah masa inkubasi 24 jam, flavonoid 0,1% merupakan konsentrasi yang paling efektif dibanding konsentrasi flavonoid lainnya dan flavonoid 0,5% merupakan konsentrasi yang paling efektif dibanding konsentrasi flavonoid lainnya setelah masa inkubasi 48 jam, serta terdapat interaksi antara konsentrasi flavonoid dengan lama waktu kontak antara flavonoid dengan bakteri s. mutans. hasil penelitian ini menunjukkan bahwa flavonoid yang terdapat pada propolis trigona sp yang berasal dari kabupaten bulukumba, propinsi sulawesi selatan mampu menghambat pertumbuhan s. mutans secara in vitro. hasil ini merupakan langkah pertama kemungkinan pemanfaatan bahan alam ini sebagai salah satu bahan antikaries alternatif di bidang kedokteran gigi pencegahan, tentu saja masih diperlukan serangkaian uji lainnya, sehingga beberapa saran yang mungkin bermanfaat bagi penelitian mendatang, yaitu perlu dilakukan penelitian untuk mengetahui kemampuan antibakteri flavonoid propolis trigona sp secara in vivo, penting untuk dilakukan uji toksisitas dan uji biokompabilitas dari flavonoid propolis trigona sp, dan diperlukan adanya penelitian lebih lanjut mengenai kemampuan antibakteri flavonoid propolis trigona sp terhadap bakteri lain yang terdapat pada rongga mulut. 141sabir: aktivitas antibakteri flavonoid propolis trigona sp ucapan terima kasih penulis mengucapkan terima kasih kepada dr. suwidjiwo pramono, apt (fakultas farmasi, universitas gadjah mada, yogyakarta) atas segala bimbingannya selama pelaksanaan proses ekstraksi flavonoid. daftar pustaka 1. fajerkov o. concepts of dental caries and their consequences for understanding the disease. community dent oral epidemiol 1997; 25:5–12. 2. who. epidemiology: etiology and prevention of periodontal diseases. technical report series no 621. geneva: world health organization 1978; p. 1–7. 3. departemen kesehatan ri. profil kesehatan gigi dan mulut di indonesia pada pelita v. jakarta. 1994. h. 12–3. 4. lundeen tf, roberson tm. cariology: the lesion, etiology, prevention, and control. in: cm sturdevant, tm roberson, ho heymann, jr sturdevant, editors. the art and science of operative dentistry. 3th ed. st louis: mosby-year book inc; 1995. p. 62. 5. lavelle clb. applied oral physiology. 2nd ed. london: wright. 1988; p. 96–7. 6. roeslan ob. karakteristik streptococus mutans penyebab karies gigi. majalah ilmiah kedokteran gigi fakultas kedokteran gigi usakti. 1995; 29–30(10):112–5. 7. wiryowidagdo s. perkembangan dan masa depan mikrobiologi. kursus singkat pengontrolan kualitas bahan pangan secara mikrobiologi. ujung pandang: fakultas mipa universitas hasanuddin; 1996. h. 1–10. 8. sila m. madu tropis, gizi dan kesehatan masyarakat. ujung pandang: lembaga penelitian universitas hasanuddin;1998. h. 5-15. 9. ghisalberti el. propolis: a review. bee world1979; 60:59–84. 10. dadant cc. the hive and the honey bee. illinois: dadant and sons; 1984. p. 25–35. 11. dobrowolski jw, vohora sb, sharma k, shah sa, naqvi sah, dandiya pc. antibacterial, antifungal, antiamoebic, antiinflammatory and antipyretic studies on propolis bee products. j ethnopharmacol 1991; 35:77–82. 12. kujumgiev a, tsvetkova i, serkedjieva y, bankova v, cristov r, popov s. antibacterial, antifungal and antiviral activity of propolis of different geographic origin. j ethnopharmacol 1999; 64:235–40. 13. moreno min, isla mi, cudmani ng, vattuone ma, sampietro ar. screening of antibacterial activity of amaicha del valle (tucumán, argentina) propolis. j ethnopharmacol 1999; 69:97–102. 14. grange jm, davey rw. antibacterial properties of propolis (bee glue). j r soc med 1990; 83(3):159–60. 15. hill r. propolis: the natural antibiotic. 6th ed. wellingborough: thorsons publishers limited; 1981. p. 10-21. 16. chen y. apiculture in china. 1st ed. agricultural publishing house; 1993. p. 96–7. 17. havsteen b. flavonoids, a class of natural products of high pharmacological potency. biochem pharmacol 1983; 32 (7):1141–8. 18. markham kr. techniques of flavonoid identification. london: academic press inc ltd; 1982. p.1–20. 19. sabir a. pemanfaatan flavonoid di bidang kedokteran gigi. maj ked gigi (dent j) fkg unair 2003; (edisi khusus timnas iii): 81–7. 20. pepeljnjak s, jalenjak i, maysinger d. flavonoid content in propolis extracts and growth inhibition of bacillus subtilis. pharmazie 1985; 40:122-3. 21. mirzoeva ok, grishanin rn, calder pc. antimicrobial action of propolis and some of its components: the effects on growth, membrane potential, and motility of bacteria. microbiol res 1997; 152:239-46. 22. sabir a. identifikasi golongan flavonoid dalam propolis trigona sp dari kabupaten bulukumba sulawesi selatan yang digunakan pada perawatan kaping pulpa langsung. maj ked gigi (dent j) fkg unair 2003; (edisi khusus timnas iii):59–63. 23. gomes bpfa, ferraz ccr, garrido fd, et al. microbial susceptibility to calcium hydroxide pastes and their vehicles. j endod 2002; 28 (11):758–761. 24. sudjadi. metode pemisahan. yogyakarta: fakultas farmasi universitas gadjah mada; 1986. h. 11–5. 25. adnan m. teknik kromatografi untuk analisis bahan makanan. yogyakarta: penerbit andi; 1997. h. 9–23. 26. bryan le. bacterial resistance and suspectibility. sydney: mcgraw-hill co; 1982. p. 20–4. 27. wilson, gisvold. kimia farmasi dan medisinal organik. edisi ke8. achmad mustofa fatah. jakarta: dirjen dikti dan kebudayaan; 1982. h. 10–2. 28. di carlo g, mascolo n, izzo aa, capasso f. falvonoids: old and new aspects of a class of natural therapeutic drugs. life sci 1999; 65 (4):337–53. 29. estrela c, sydney gb, bammann ll, felippe jr o. mechanism of action calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. brazil dent j 1995; 6:85–90. 30. tobias rs. antibacterial properties of dental restorative materials: a review. int endod j 1988; 21:155–60. 31. mickel ak, sharma p, chogle s. effectiveness of stannous fluoride and calcium hydroxide against enterococcus faecalis. j endod 2003; 29 (4):259–60. 32. pelzcar mj, chan ecs. dasar-dasar mikrobiologi. jakarta: universitas indonesia press; 1977. h. 450–8. 33. havsteen bh. the biochemistry and medical significance of flavonoids. pharmacol ther 2002; 96:67–202. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 50 no 4 desember 2017.indd 205205 research report dental journal (majalah kedokteran gigi) 2017 december; 50(4): 205–210 analysis of ki-67 expression as clinicopathological parameters in predicting the prognosis of adenoid cystic carcinoma silvi kintawati, murnisari darjan, and winny yohana department of oral biology faculty of dentistry, universitas padjadjaran bandung – indonesia abstract background: adenoid cystic carcinoma is a malignant salivary gland tumor located in the head and neck region. although complete surgical resection and complementary radiotherapy have been shown to improve long-term survival rates, the prognosis of adenoid cystic carcinoma remains poor. ki-67 expression is considered a marker for the cellular proliferation rate, the detection of its expression usually being related to the aggressiveness and unfavorable prognosis of adenoid cystic carcinoma in the salivary gland. purpose: this study was conducted to quantify the expression of ki-67 in adenoid cystic carcinoma and to correlate the result with clinical parameters and histopathological grading in determining the prognosis. methods: twenty three cases of salivary gland adenoid cystic carcinoma were identified at the department of anatomical pathology, dr. hasan sadikin hospital between 2013 and 2015. clinical data such as age, gender, location of tumor and histopathological grading was also collected. the expression of ki-67 was assessed by immunohistochemical means to determine the correlation of ki-67 with clinical parameters and histopathological grading. results: there were no significant differences between the expression of ki-67 and clinical parameters, although a very strong correlation existed between the expression of ki-67 and histopathological grading (p < 0.01). conclusion: there were no correlation between the expression of ki-67 and clinical parameters, although a correlation existed between the expression of ki-67 and histopatological grading in salivary gland adenoid cystic carcinoma. thus, clinical parameters were unusable in determining the prognosis of adenoid cystic carcinoma, although ki-67 expression could be used for this purpose.. keywords: adenoid cystic carcinoma; histopathological grading; clinical parameters; immunohistochemistry; ki-67 correspondence: silvi kintawati, department of oral biology, faculty of dentistry, universitas padjadjaran. jl. raya jatinangor; cibeusi; jatinangor; kabupaten sumedang; jawa barat 45363, indonesia. e-mail: silvi.kintawati@fkg.unpad.ac.id introduction adenoid cystic carcinoma is a malignant tumor of the secretory glands often occurring in the major and minor salivary glands situated in the head and neck regions. adenoid cystic carcinoma presents certain clinical and histopathological properties, such as slow growth, perineural invasion and metastasis in isolated locations often leading to recurrence.1 nevertheless, the etiology and pathogenesis of adenoid cystic carcinoma remain unknown. up to the present, no research results exist confirming that environmental and genetic factors cause this tumor. however, they do indicate abnormalities in chromosomes 6q, 9p, and 17p of regions 12-13. there are also frequent genetic deletions in 12q, 13q, and 19q.2–4 clinically, adenoid cystic carcinoma develops slowly rendering detection of the tumor during examination difficult, while reducing patient survival rates. adenoid cystic carcinoma is also known to affect all age groups with its peak occurring in middle or old age. however, there is no specific higher incidence rate with a particular gender.2 histopathologically, adenoid cystic carcinoma demonstrates three different growth patterns, namely: 1) cribriform/glandular (classic); in the form of an epithelial cell nest with a cylindromatous cyst, 2) tubular; in the form of tumor cells forming ductal structures coating epithelial dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 mailto:silvi.kintawati@fkg.unpad.ac.id 206 kintawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 205–210 cells and 3) solid (basaloid); in the form of uniform, small, basofillic cells with hyperchromatic nuclei.2,3 although the solid type represents the most aggressive forms, the three types are often found to be present in a tumor, resulting in frequently unpredictable prognosis.5 adenoid cystic carcinoma can afflict either the parotid gland or the submandibularis gland. however, it is most commonly found in the minor salivary gland with the highest frequency being in the area of the durum palate, followed by the tongue, buccal mucosa, lips and mouth floor.2 in general, the prognosis of adenoid cystic carcinoma is influenced by several factors including: growth type, stage, anatomical location, tumor size and metastasis. although additional surgical therapy and radiotherapy are frequently administered, the survival rate of and prognosis for patients with adenoid cystic carcinoma remains poor.6 in other words, the prognosis of adenoid cystic carcinoma is clinically unpredictable.7 furthermore, a considerable body of research has linked ki-67 expression with the aggressiveness and prognosis of adenoid cystic carcinoma, although results remain inconclusive.8 consequently, it is important to predict the prognosis of the tumor. accurate identification of tumors by means of immunohistochemical examination is critical to reducing instances of misdiagnosis and establishing more appropriate therapies in order to avoid recurrence and metastasis and improve prognosis.9 ki-67, a protein located in chromosome 10q26.2 on the 16th exon, is a double band of polypeptide with a molecular weight of 345 and 395 kd. this protein lies within the cell nucleus and is expressed by cells undergoing proliferation with expression levels changing throughout the cell cycle. ki-67 will also be expressed in phase g1, phase s, phase g2 and then continually during phase m, but not the resting phase (phase g0).2,10,11 the occurrence of a malignancy fundamentally involves gene activity stimulating growth, in addition to gene mutation, which regulates apoptosis. in other words, a tumor can occur as the result of a higher rate of cell proliferation than cell death, resulting in the progression of the tumor.12,13 thus, in order to predict the aggressiveness of a tumor, any immunohistochemical examination conducted should employ ki-67 as an antibody marker. by utilising ki-67, cell proliferation will be detectable because this antibody will only be expressed under such conditions. the working principle of ki-67 antibodies is based on the form of antigen and antibody reaction. the ki-67 antibody will react positively to the tumor cell nucleus antigen, a process referred to as an immunoreactive condition.2 the proliferation rate of a tumor is closely related to its biological behavior. consequently, the higher and more rapid the rate of tumor proliferation, the greater its aggressiveness and the more serious the prognosis.14 therefore, this research aimed to investigate the relationship between ki-67 expression and the clinical parameters and histopathological grading of salivary adenoid cystic carcinoma. thus, ki-67 expression can be confidently utilised as an indicator underpinning a medical prognosis, leading to the implementation of an appropriate therapy to avoid recurrence. materials and methods this research constituted a retrospective study conducted between 2013 and 2015. the samples used consisted of twenty three paraffin blocks of adenoid cystic carcinoma derived from salivary glands examined at the department of anatomical pathology, dr. hasan sadikin hospital, bandung. data relating to these paraffin blocks in terms of the age, sex and tumor locations of patients was then recorded. with the help of two anatomical pathologists from fkup/rshs bandung the paraffin blocks were restained with eosin hematoxylin enabling diagnoses and histopathological types to be established. histopathological grading consisted of the following categories: grade i; if there was a tubular and cribriform pattern without a solid component, grade ii; when only cribriform pattern was present or mixed with a solid component comprising less than 30% and grade iii; when there was a tumor with a predominantly solid component.9 immunohistochemical preparations were made using a ki-67 antibody (clone sp6, biocare, usa) 1:50 dilution withjn a streptavidin-biotin peroxidase method (lsab kit k0492, dako, carpinteria, ca). such preparations were considered to be positive if a brownish immunoreactive tumor cell nucleus was present. the results were compared with both positive controls using lymphoid tissues and negative controls (performed on the same adenoid cystic carcinoma preparations using secondary/“non-immune serum” antibodies). thereafter, ki-67 immuno-expression was assessed according to its percentage and intensity. the percentage of ki-67 expression was calculated by means of a “hand tally counter” (vwr, no catalog 23609-102) on 1000 tumor cells in ten representative fields using a cx21 light microscope (olympus america inc. melville, ny 11747) at a magnification of 400x. in order to determine the relationship between ki-67 expression and clinical parameters (age, sex and location), ki-67 expression was categorized by the percentage of tumor cells falling within two categories, namely; one with a high proliferation index (pi ) where the positive cell percentage was greater than 40%, and another with a low proliferation index (pi ) with a positive cell percentage less than 40%.15 in addition, to determine the relationship between ki-67 immuno-expression and histopathological types, the percentage of ki-67 expressed by tumor cells was categorized as score 1 if positive cells accounted for less than 20%, as score 2 if positive cells represented between 21-50%, as score 3 if positive cells amounted to between 51-80% as and score 4 if positive cells totally over 80%. the intensity of ki-67 expression was also categorized into score 1 for weak intensity (light brown approaching dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 207207kintawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 205–210 negative control), score 2 for medium intensity (brown between score 1 and score 3) and score 3 for strong intensity (dark brown resembling positive control).16,17 based on the percentage and intensity values, the ki67 expression score was calculated by multiplying the percentage value (score 1, 2, 3 or 4) by the intensity value (score 1, 2 or 3). the results obtained ranged from 1 to 12 and were then associated with histopathological grading of salivary adenoid cystic carcinoma.9 thereafter, the data obtained were recorded for further statistical analysis using a chi-square test. results of the 23 cases of adenoid cystic carcinoma, 13 afflicted males and 10 females. the median of the average age was 48.44 years (with a range of 31-68 years). the ratio of male to female was 1: 0.77. there were four cases of tumors located in major salivary glands, three of which were located in the submandibular gland, with one being located in the parotid gland. on the other hand, the remaining nineteen cases were found in the minor salivary glands, thirteen of which were in the palate, two in the buccal mucosa, two in the tongue and two in the mouth (table 1). based on the examination results of ki-67 expression in the 23 cases of adenoid cystic carcinoma, four men had pi , while nine had pi . on the other hand, six women had pi , whereas four had pi . moreover, the results also showed that four people at the age of < 48 years had pi , while five at the age of < 48 years had pi . in contrast, six people aged > 48 had pi , whereas eight aged > 48 had pi . in addition, one case located in the major salivary glands had pi , while the other three had pi . then again, nine cases located in minor salivary glands had pi , whereas the other ten had pi . moreover, based on the statistical test results, there was no significant difference between the ki-67 expression percentage and the clinical parameters, such as sex (p = 0.16), age (p = 0.94), and location (p = 0.41) in the cases of adenoid cystic carcinoma (table 2, figure 1). based on the contents of table 3, of the 23 cases of adenoid cystic carcinoma studied, there were ten were of grade i, eight cases of grade ii and five of grade iii. the table also illustrated that the number of tumor cells expressed with ki-67 varied from < 20% to> 80%, i.e. three cases (< 20%), ten cases (21% -50%), six cases (51% -80%) and four cases (> 80%). furthermore, it was also known that the intensity of ki-67 indicated nine cases of weak intensity, eleven cases of moderate intensity, and three cases of strong intensity (table 3). based on the percentage value and intensity of ki-67, the ki-67 expression score was calculated by multiplying the percentage of ki-67 by its intensity, resulting in a score of 1-12. the results confirmed two cases with score 1, five cases with score 2, two cases with score 3, six cases with score 4, three cases with score 6, two cases with score 8, one case with score 9, and two cases with score 12. the data was then related to the histopathological grading of adenoid cystic carcinoma (table 4 and figure 2). in addition, based on the statistical test results, there was a significant difference between ki-67 expression and histopathological grading of adenoid cystic carcinoma (p < 0.01). it means that the higher the value of ki-67 expression, the higher the histopathological grading. table 1. characteristics of patients with adenoid cystic carcinoma variables n (%) median of age (years) < 48 > 48 sex male female location major salivary glands, 4 cases (17%): submandibular gland parotid gland minor salivary glands, 19 cases (83%): palate buccal mucosa tongue mouth base 9 (39) 14 (61) 13 (56.5) 10 (43.5) 3 (13) 1 (4) 13 (56) 2 (9) 2 (9) 2 (9) figure 1. the results of hematoxylin eosin staining in adenoid cystic carcinoma: (a) solid type, (b) cribriform type, and (c) tubular type. ki-67 immunoxpression in adenoid cystic carcinoma: (d) solid type, pi (> 40%), (e) cribriform type, and (f) tubular type, pi (< 40%). (100× magnification) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 208 kintawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 205–210 discussion adenoid cystic carcinoma is a rare tumor, 2% to 4% of such malignancies being found in head and neck.5 in general, adenoid cystic carcinoma is mostly found in the minor salivary gland and palate.2 similarly, the results of this research showed that 56% of adenoid cystic carcinoma cases occur in the palate. the results also confirmed that the ratio of men to women was 1: 0.77. the mean age of the patients was 48.44 years. as with the results of this research, a number of previous investigations found that adenoid cystic carcinoma may affect all age groups with its highest incidence at both middle and old age, but with no specific predominance of a particular sex.2,18 in recent years, molecular biology has been widely developed leading to invaluable results. various molecular markers have been identified and their association with oral cavity tumor development widely discussed.19 nowadays, considerable research has been focussed on ki-67 expression and malignant tumor prognosis.20 in mammary tumors, ki-67 expression has an independent and useful prognostic value in predicting recurrence, survival rate and therapeutic response.10 for example, since ki-67 expression increases in malignant oral tumors, it may be used as an indicator in predicting the prognosis of squamous cell carcinoma within the oral cavity.21 however, only limited research into ki-67 expression in salivary gland tumors exists. the basis of the occurrence of a malignancy actually lies in the fact that there is gene activation stimulating growth in addition to the gene mutation regulating apoptosis. as a result, when excessive cell proliferation and lack of apoptosis occurs, the tumor will develop.12,13 in tumors experiencing aggressive growth, there can also be an imbalance between cell production and cell death, in which the former exceeds the latter. the proliferation rate of a tumor is closely related to its biological behavior. thus, the faster and the higher the proliferation, the more aggressive the tumor will be and the greater the potential for a more serious prognosis. similarly, the higher the level of anti-apoptosis, the more progressive the tumor and the worse the prognosis might be.10,16 in this research, the ki-67 antibody marker was used to observe cell proliferation within a special table 2. the relation of the ki-67 expression percentage and the clinical parameters in acc characteristics ki-67 (%) p pi (< 40%) pi (>40%) sex male(13) female(10) 4 6 9 4 0.16 ns age (31–68 years old, mean 48,44 years old) < 48(9) >48(14) 4 6 5 8 0.94 ns location: major salivary glands (4) minor salivary glands (19) 1 9 3 10 0.41 ns ns = non significant table 3. the percentage and intensity of ki-67 in the histopathological grading of adenoid cystic carcinoma histopathological grading percentage (%) intensity total (n) < 20 21–50 51–80 > 80 weak moderate strong i 3 7 – – 7 3 – 10 ii – 3 4 1 2 6 – 8 iii – – 2 3 – 2 3 5 table 4. the relation of ki-67 expression and histopathological grading of adenoid cystic carcinoma histopathological grading ki-67 expression score total (n) sig p 1 2 3 4 6 8 9 12 i 2 5 – 3 – – – – 10 0.009* ii – – 2 3 2 1 – – 8 iii – – – – 1 1 1 2 5 *) significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 209209kintawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 205–210 immunohistochemical examination. ki-67 is a specific cell proliferation marker. by using the ki-67 antibody, cell proliferation will be detectable because the antibody will only be expressed in proliferative cells. the ki-67 antibody will be expressed in the cell nucleus during some cell proliferation phases, namely: phase g1, phase s, phase g2 and phase m, but not during the resting phase (phase g0) of the cell cycle. the more actively a cell divides, the more rapidly ki-67 antibodies will be expressed until phase m. then, it will decrease and disappear during the resting phase where there is no cell proliferation, so the ki-67 antibody cannot be expressed.2,10,11 this condition can also distinguish the types of ki-67 intensity, namely: weak, medium, and strong during the immunohistochemical staining process. therefore, the more actively tumor cells proliferate, the stronger the intensity of the color. meanwhile, the less actively tumor cells proliferate, the weaker the intensity of the color. moreover, many researchers have linked ki-67 expression as a marker with malignancy and a prognosis of adenoid cystic carcinoma, but the results are still confusing.8 consequently, this research linked ki-67 expression with clinical parameters (sex, age and location). however, the results of this research did not indicate any relation between ki-67 and clinical parameters. this suggests that adenoid cystic carcinoma can occur in both men and women, either in the major or minor salivary glands. this also suggests that both the < 48 years age group and that of > 48 years have the same chance of suffering adenoid cystic carcinoma, so it is ineffective in determining its aggressiveness and prognosis. there is a reference which suggests that the prognosis of adenoid cystic carcinoma is affected by growth type, stage, anatomical location, tumor size and metastasis. however, some references suggest that although additional surgical and radiotherapy therapies have been administered, the survival and prognosis rates of patients with adenoid cystic carcinoma remains poor. consequently, the prognosis of adenoid cystic carcinoma remains clinically unpredictable.6,7 furthermore, considerable previous research has linked ki-67 expression with the aggressiveness and prognosis of adenoid cystic carcinoma, although the results are still perplexing.8 for these reasons, the research reported here used previously unstudied samples of adenoid cystic carcinoma supplied by the department of anatomical pathology, dr. hasan sadikin hospital, bandung. similar to the results of this research, those of two previous investigations conducted by carlinfante et al. and amoueian et al. also showed there to be no relationship between ki-67 and the clinical parameters of adenoid cystic carcinoma.18 similarly, research conducted by jiang et al. found no relationship between bcl-2 expression and the clinical parameters in adenoid cystic carcinoma.9 since there was no relation between ki-67 expression and clinical parameters within this research, the clinical parameters cannot be used to determine the aggressiveness and prognosis of adenoid cystic carcinoma. in addition, this research linked ki-67 expression with the histopathological grading of adenoid cystic carcinoma which, according to szanto et al., is divided into three grades, namely: grade i, if there was a tubular and cribriform pattern without a solid component, grade ii, with the presence of a sole cribriform pattern or one mixed with a solid component of less than 30% and grade iii, when the presence of a tumor with solid component was the dominant one.18 in this research, 43.5% of tumors were classified as grade i, 34.8% as grade ii and 21.7% as grade iii. in general, the ki-67 expression in the 23 cases of salivary adenoid cystic carcinoma significantly augmented with the increase in histopathological grading. this result suggests that the higher the value of ki-67 expression, the higher the histopathological grading. similarly, research conducted by norberg et al. showed that the number of tumor cells positively expressing ki-67 correlates significantly with their tumor gradation. the same result was found in research conducted by triantafillidou et al. and suzzi et al.18 another previous piece of research conducted by faur et al.,10 also suggests that immunohistochemical examination can detect significant proliferation of tumor cells indicating an increasingly aggressive tumor. finally, it can be concluded that there is no relationship between ki-67 expression and clinical parameters, although there is correlation between ki-67 expression and the histopathological grading i, ii and iii of adenoid cystic carcinoma. as a result, clinical parameters are unusable in determining the prognosis of adenoid cystic carcinoma, although ki-67 expression can be used for this purpose. consequently, a more appropriate therapy can be implemented and recurrence avoided. acknowledgement the research reported here was supported by kemenristek dikti through drpmi universitas padjadjaran (grant # 718/un6.3.1/pl/2017). references 1. gondivkar sm, gadbail ar, chole r, parikh r v. adenoid cystic carcinoma: a rare clinical entity and literature review. oral oncol. 2011; 47(4): 231–6. 2. rosai j. rosai and ackerman’s surgical pathology. 10 th ed. philadelphia: mosby elsevier; 2011. p. 247, 248, 259-261, 873900. 3. kumar v, abbas ak, fausto n, aster jc. robbins and cotran pathologic basis of disease. 8th ed. philadelphia: saunders elsevier; 2010. p. 815-6. 4. se et ha la r r , cieply k , ba r nes e l , dacic s. p rog ressive genetic alterations of adenoid cystic carcinoma with high-grade transformation. arch pathol lab med. 2011; 135(1): 123–30. 5. zhao c, liu j-z, wang s-b, wang s-c. adenoid cystic carcinoma in the maxillary gingiva: a case report and immunohistochemical study. cancer biol med. 2013; 10(1): 52–4. 6. lloyd s, yu jb, wilson ld, decker rh. determinants and patterns of survival in adenoid cystic carcinoma of the head and neck, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 210 kintawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 205–210 including an analysis of adjuvant radiation therapy. am j clin oncol. 2011; 34(1): 76–81. 7. ko yh, lee ma, hong ys, lee ks, jung c-k, kim ys, sun d-i, kim bs, kim ms, kang jh. prognostic factors affecting the clinical outcome of adenoid cystic carcinoma of the head and neck. jpn j clin oncol. 2007; 37(11): 805–11. 8. al-ani ls, al-azzawi lm. evaluation of immunohistochemical expression of p53 and pcna in pleomorphic adenoma, mucoepidermoid and adenoid cystic carcinomas of salivary glands. tikrit j dent sci. 2013; 1: 1–8. 9. jiang lc, huang sy, zhang ds, zhang sh, li wg, zheng ph, chen zw. expression of beclin 1 in primary salivary adenoid cystic carcinoma and its relation to bcl-2 and p53 and prognosis. braz j med biol res. 2014; 47(3): 252–8. 10. faur ac, sas i, motoc agm, cornianu m, zamfir cl, lazăr dc, folescu r. ki-67 and p53 immunostaining assessment of proliferative activity in salivary tumors. rom j morphol embryol. 2015; 56(4): 1429–39. 11. ben-izhak o, laster z, araidy s, nagler rm. tunel – an efficient prognosis predictor of salivary malignancies. br j cancer. 2007; 96(7): 1101–6. 12. garewal j, garewal r, sircar k. expression of bcl-2 and mib-1 markers in oral squamous cell carcinoma (oscc)a comparative study. j clin diagn res. 2014; 8(7): qc01-4. 13. a r u l a s k j , s o l o m o n r d j , a r u l a s s j , s a n t h i v s . immunohistochemical evaluation of bcl-2 and ki-67 in varying grades of oral squamous cell carcinoma. j sci ind res (india). 2011; 70(11): 923–8. 14. li lt, jiang g, chen q, zheng jn. ki67 is a promising molecular target in the diagnosis of cancer (review). mol med rep. 2015; 11(3): 1566–72. 15. zeggai s, harir n, tou a, sellam f, mrabent mn, salah r. immunohistochemistry and scoring of ki-67 proliferative index and p53 expression in gastric b cell lymphoma from northern african population: a pilot study. j gastrointest oncol. 2016; 7(3): 462–8. 16. hornick jl. the prognostic role of immunohistochemistry in sarcomas. in: international society of bone and soft tissue pathology. washington: united state & canadian academy of pathology; 2010. p. 1–7. 17. humayun s, prasad vr. expression of p53 protein and ki-67 antigen in oral premalignant lesions and oral squamous cell carcinomas: an immunohistochemical study. natl j maxillofac surg. 2011; 2(1): 38–46. 18. a moueia n s, sag h a f i s, fa r h a d i f, toh id i e , sa d eg i l . immunohistochemical assessment of ki-67 expression in adenoid cystic carcinoma of the salivary glands. mashhad univ med sci. 2007; 10(2): 84–9. 19. da ros motta r, zettler cg, cambruzzi e, jotz gp, berni rb. ki-67 and p53 correlation prognostic value in squamous cell carcinomas of the oral cavity and tongue. braz j otorhinolaryngol. 2009; 75(4): 544–9. 20. sousa wat de, rod r igues lv, silva jr rg da, viei ra f l. immunohistochemical evaluation of p53 and ki-67 proteins in colorectal adenomas. arq gastroenterol. 2012; 49(1): 35–40. 21. kintawati s, darjan m. hubungan ekspresi ki-67 dengan gradasi histopatologi karsinoma sel squamous rongga mulut. bandung: universitas padjadjaran; 2015. p. 13–21. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p205–210 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p205-210 139 clinical application of the activity index to parameter for evaluation of electromyographic activity of the masticatory muscles takashi tanaka department of prosthetic dentistry, programs for applied biomedicine division of cervico-gnathostomatology, hiroshima university graduate school of biomedical sciences, hiroshima, japan. abstract the purpose of this study was to evaluate the relationship between the intended direction of clenching and changes in the applied activity index of the masticatory muscles. the subjects consisted of twelve male volunteers (average age of 26.3 years). the surface electromyographic activities of the anterior and posterior parts of the temporal muscles, the deep posterior part of the masseter muscle and the superficial central part of the masseter muscle were recorded during the intended clenching in vertical, anterior and posterior directions. the changes of the applied activity index (the relative different value between the examined muscle activity and the superficial central part of the masseter muscle activity) were evaluated. the applied activity indexes of the anterior and posterior parts of the temporal muscles and the deep posterior part of the masseter muscle decreased significantly during the intended clenching in the posterior direction. those of the anterior and posterior parts of the temporal muscles increased significantly during the intended clenching in the anterior direction. each applied activity index changed corresponding to the differences of the running directions in the sagittal plane between the superficial masseter muscle and these three muscles. the applied activity indexes of the anterior and posterior parts of the temporal muscles and the deep posterior part of the masseter muscle significantly changed during clenching in anteroposterior direction. therefore, it was suggested that the applied activity indexes of these three muscles could be used as a parameter to indicate the anteroposterior direction of force on the lower jaw. key words: electromyographic, activity index, direction, clenching correspondence: hitoshi abekura, department of prosthetic dentistry, programs for applied biomedicine division of cervicognathostomatology, hiroshima university graduate school of biomedical sciences, 1-2-3 kasumi, minami-ku, hiroshima, 734-8553, japan. tel 0081-82-257-5682, fax 0081-82-257-5684, e-mail: abekura@hiroshima-u.ac.jp introduction the temporo mandibular joint (tmj) has an anterior articular disk and is rich in blood vessels and nerves posteriorly.1 therefore, it is thought that force imparted on the posterior tissue of tmj by the mandibular condyle has an adverse.effect. therefore, the evaluation of the quantity and also the direction of force imparted around tmj are clinically important. previous studies have examined the tension applied to the tmj from such a viewpoint.2,3 some studies reported a strong relationship between the direction of the clenching force and the ratio of masticatory muscle activities.4–7 therefore, a change in the ratio of muscle activities is considered to be able to show a change in the direction of the clenching force, and could therefore become a meaningful parameter. the activity index advocated by naeije et al.8 was often adopted as parameters to objectively show the ratio of muscle activities. these parameters can overcome the influences of different conditions, such as variations in the thickness of the subcutaneous fat or electric resistance between electrodes and so on, which varies from patient to patient when the electromyography (emg) is used. naeije8 and visser et al9 insisted that these influences can be excluded by converting muscle activities to an index. surface electromyograms are clinically easy to record, and both sides of the masseter muscle and anterior parts of temporal muscles are often used as electrode attachment sites. these muscle bundles are broad, and their functions vary.10–12 there are other areas which can also be measured comparatively easily by the surface electromyogram,4,13 and it may also be useful to take electromyograms from each part. the ratio of muscle activities relates to the direction of the clenching force because the clenching force results from multiple muscle activities. the purpose of this study was to evaluate the relationship between the intended direction of clenching and changes in the applied activity index (parameter: the relative different value between the examined muscle activity and the superficial central part of the masseter muscle activity) for the anterior and posterior parts of the temporal muscles, and the deep posterior parts of the masseter muscles. materials and methods the subjects consisted of twelve male volunteers (average of 26.3 years with a standard deviation of 5.4 years) had normal occlusion, i.e. nearly a class 1 molar relation, and were without clinical evidence of symptoms of temporo mandibular disorder (tmd) or unusually 140 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 139–142 strong maxillofacial forms. none of the subjects had tooth defects excluding the presence or absence of wisdom teeth, dental caries and severe or moderate periodontal disease. each subject was informed about the aim and procedures of this study, and gave their informed consent prior to the start of the study. the emg activities were amplified and converted from analog to digital with a sample frequency of 2000 hz, and recorded on the hard disk of a personal computer with the biopac research system (mp100 ws biopac systems, biopac systems inc., santa barbara, usa ). the emg activities were recorded by attaching bipolar silver-silver chloride electrodes (el204s, biopac systems inc., santa barbara, usa) in parallel to the direction of the muscle fiber, and ground electrodes were attached to the central part of the frontal head area. when it was necessary to control the clenching level, subjects were able to control their emg level through visual feedback looking at an oscilloscope displaying the summated rectified emg signal of central part of masseter muscle. several parts of the muscles were selected, i.e. the left anterior part of the temporal muscle, the left posterior part of the temporal muscle, the left superficial central part of the masseter muscle and the left deep posterior part of the masseter muscle (figure 1). for the anterior part of the temporal muscle, the mid-point between the electrodes was placed 40 mm ahead of the anterior border of the outer ear canal and 40 mm above the frankfort plane. for the posterior part of the temporal muscle, a place superior to the auricle and outer ear canal was decided for one of the bipolar electrodes and a place 15mm behind that, for another bipolar electrode. electrodes were attached to these places along the general direction of the muscle fiber, after vigorous skin cleaning. the muscle bundle of the superficial part of the masseter muscle was confirmed by palpation. surface electrodes were placed 15 mm apart on the skin surface, in line with the general direction of the muscle fibers. the mid-point between the electrodes was located on the center of this muscle for the central part of the masseter muscle.the place for the left deep posterior part of the masseter muscle was decided as follows: part of the zygomatic arch, whose inferior border was confirmed by palpation, was decided on as the upper place to attach one of the bipolar electrodes. a place 15 mm downward of the upper electrode, reported as the area to palpate the deep part of the masseter muscle for examining tmd, was decided on as the lower place to attach another electrode. firstly, the influence of the clenching level on the applied activity index was examined. subjects were asked to clench at 25, 50, 75 and 100% of their maximal voluntary clenching level (mvc) for 5 seconds at the intercuspal position (icp) through a visual feedback. next, the influence of the intended clenching direction on the applied activity index was examined. emg activities were recorded during the intended clenching in vertical, anterior and posterior directions at the intercuspal position. subjects were asked to clench as strongly as possible and toward the intended direction as constantly as possible. each clenching was maintained for 3 seconds while measurements were performed, and this was carried out twice. at least one minute rest period was given between each clenching to avoid muscle fatigue. the whole sampled emg data were filtered through the range between 50 and 1000 hz and rectified. from each of the recorded emg activities, stable emg activities were selected respectively, and the integrated emg activities were calculated. finally, the four integrated emg activities in each clenching condition were averaged and used to calculate the next parameter. to evaluate the change of each muscle activity, the activity index, advocated by naeije, mccarroll and weijes8 was used and applied as figure 2 for present study. paired student’s t-tests were performed to determine difference between each condition. a bonferroni correction of the alpha-level for statistical analysis was performed to avoid type i error increased by multiplicity. statistical analyses were performed with significance set at the 0.05 figure 2. formula for the applied activity index.8 figure 1. location of electrodes. a: anterior part of temporal muscle, b: posterior part of temporal muscle, c: superficial central part of masseter muscle, d: deep posterior part of masseter muscle 141tanaka: clinical application of the activity index to parameter for evaluation probability level. results the differences in clenching level, i.e. 25%, 50%, 75%, and 100% mvc, did not influence the applied activity index for all muscles (figure 3, 4). the changes in the applied activity index for the anterior and posterior parts of the temporal muscles showed similar results to each other. the applied activity index for these muscles in the vertical direction was significantly lower than in the anterior direction, and the index in the posterior direction was significantly lower than in the vertical direction (figure 5). the applied activity index for the deep posterior part of masseter muscle in the posterior direction was significantly lower than in the vertical and anterior directions (figure 6). discussion the activity index, one of the proposed parameters, shows the relative value of the activities of the center of the masseter muscle against the activities of the anterior part of the temporal muscle, as an applicable muscle.8 the main direction of the clenching force aligns with the running of the masseter muscle fibers, and this muscle is considered to be one of the main working muscles in the generation of clenching force. other muscle activity increases the power and modifies the direction of the power generated by the masseter muscle. according to the formula of the applied activity index, this parameter is calculated by dividing the difference in the values between the masseter muscle and examining muscle activities by the total value of both muscle activities. the total value is in proportion to the clenching strength. therefore this parameter is expected to reflect the direction of the power regardless of the clenching strength. there are other muscles that modify the center of masseter muscle activity except for the anterior part of the figure 5. changes in the applied activity index for the temporal muscles caused by alteration in the clenching direction. figure 6. changes in the applied activity index for the deep posterior part of masseter muscles caused by alterations in clenching direction. figure 3. changes in the applied activity index for the temporal muscles caused by alteration in the clenching level. figure 4. changes in the applied activity index for deep posterior part of masseter muscle caused by alteration in the clenching level. 142 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 139–142 temporal muscle, and it is possible that similar parameters to the activity index are available for each muscle. the running of the posterior part of the temporal muscle does not identify that of the anterior part, so it is meaningful to examine both muscle activity patterns because the functions are different from each other.11 not only the running direction of the deep posterior masseter muscle but also origin and insertion are different from the superficial masseter muscle,14 and this muscle is also functionally different.13 therefore, to examine this muscle’s activity is beneficial. oppressive pain occurs at the area of the deep posterior masseter muscle in patients with tmd.15 this muscle is clinically important for these reasons but a few emg studies have been conducted on the deep masseter muscle.13,16 most of the deep masseter muscle is covered with superficial masseter muscle, but not the area just anterior to the temporomandibular joint.14 belser13 examined the electromyographic activity of this part as a target by means of surface and needle electrodes. a large muscle activity can be recorded during clenching in the posterior direction using both needle and surface electrodes. these four muscles, that is, the superficial central and deep posterior part of the masseter muscles and the anterior and posterior parts of the temporal muscles, are clinically important and easy to attach surface electrodes to.4 therefore, these muscles were selected for the present study. because the running of the muscles examined in this study varies in the sagittal plane, the clenching force direction was designated in the sagittal plane, that is, in the anterior and posterior directions. because the regulation of the clenching level makes it difficult for the subject to perform clenching in the designated direction, each subject was asked to clench in the intended direction at the maximal voluntary level. before recording emg during clenching, each subject practiced anterior and posterior mandibular sliding movements so that they could perform the intended direction of clenching at the intercuspal position. because the clenching level was not regulated during anterior and posterior intended clenching, changes in the applied activity indexes may have been influenced by the intensity of clenching. however, we did not observe significant influence of differences in clenching level intensity on the applied activity index. therefore, the results of figure 5 and 6 indicated the influence of the clenching direction. the power direction of each muscle activity depends on the running of the muscle fibers. when subjects clench in the posterior direction, it is considered that the deep posterior part of the masseter muscle and the anterior and posterior parts of the temporal muscle may become active and the applied activity indexes of these muscles decrease. these muscle activities decrease and the indexes increase when subjects clench in the anterior direction. the running directions of these muscles incline backward in the order of the posterior part of temporal muscle, the anterior part of temporal muscle, the deep posterior part of the masseter muscle, and the superficial central part of the masseter muscle in the sagittal plane.13 because the posterior part of the temporal muscle runs mostly in the posterior direction, this applied activity index changed most noticeably. the change in the numerical value of the deep part of the masseter muscle was similar to the temporal muscle, because the directions of both muscles are aligned with each other. if the running direction of these examined muscles is taken into consideration, the relationship between the direction of the clenching force and the change in the applied activity index could be explained well. it was suggested that the applied activity indexes of these three muscles, the deep posterior part of the masseter muscle, and the anterior and posterior parts of the temporal muscles, could be used as a parameter to indicate the anteroposterior direction of force on the lower jaw. references 1. scapino rp. the posterior attachment: its structure, function, and appearance in tmj imaging studies. part 1. j craniomandib disord facial oral pain 1991; 5:83–95. 2. ferrario vf, sforza c. biomechanical model of the human mandible in unilateral clench: distribution of temporomandibular joint reaction forces between working and balancing sides. j prosthet dent, 1994; 72:169–76. 3. osborn jw. biomechanical implications of lateral pterygoid contribution to biting and jaw opening in humans. arch oral biol 1995; 40:1099–108. 4. wood ww. a review of masticatory muscle functuion. j prosthet dent 1987; 57:222–32. 5. macdonald jw, hannam ag. relationship between occlusal contacts and jaw-closing muscle activity during tooth clenching: part ii. j prosthet dent 1984; 52:862–7. 6. mao j, osborn jw. direction of a bite force determines the pattern of activity in jaw-closing muscles. j dent res 1994; 73:1112–20. 7. van eijden tm. jaw muscle activity in relation to the direction and point of application of bite force. j dent res 1990; 69:901–5. 8. naeije m, mccarroll rs, weijes wa. electromyographic activity of the human masticatory muscles during submaximal clenching in the inter-cuspal position. j oral rehabil 1989; 16:63–70. 9. visser a, mccarroll rs, naeije m. masticatory muscle activity in different jaw relations during submaximal clenching efforts. j dent res 1992;71:372–9. 10. mcmillan as, hannam ag. task-related behavior of motor units in different regions of the human masseter muscle. arch oral biol 1992; 37:849–57. 11. blanksma ng, van eijden tm. electromyographic heterogeneity in the human temporalis muscle. j dent res 1990; 69:1686–90. 12. blanksma ng, van eijden tm, weijs wa. electromyographic heterogeneity in the human masseter muscle. j dent res 1992; 71: 47–52. 13. belser uc, hannam ag. the contribution of the deep fibers of the masseter muscle to selected tooth-clenching and chewing tasks. j prosthet dent 1986; 56:629–35. 14. eriksson po, thornell le. histochemical and morphological musclefibre characteristics of the human masseter,the medial pterygoid and the temporal muscles. archs oral biol, 1983; 28:781–95. 15. travell j, simons dg. myofascial pain and dysfunction: the trigger point manual. baltimore: williams and wilkins, 1983. p. 45–102, 16. santana u, mora mj. electromyographic analysis of the masticatory muscles of patients after complete rehabilitation of occlusion with protection by non-working side contacts. j oral rehabil 1995; 22:57–66. 154154 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 154–160 original article mapping of health care facilities, dental visits and oral health problems in indonesia to prevent covid-19 transmission ayu asri lestari1, melissa adiatman2, risqa rina darwita2 1master program of community dentistry, faculty of dentistry, universitas indonesia, jakarta, indonesia 2department of dental public health and preventive dentistry, faculty of dentistry, universitas indonesia, jakarta, indonesia abstract background: one of the goals of indonesia’s participation in sustainable development goals is to improve its health state. efforts to achieve health improvement are increasing the availability of health care facilities so people can easily access and get treatment for dental and oral health. as we know, the first case of covid-19 in indonesia was found in march 2020 and all cases were spread over 34 provinces. during this pandemic situation, health care facilities and some dental treatments generating aerosols are one of the environments that can potentially transmit covid-19 to the community. dentists have the riskiest job because they must be less than two meters from patients. purpose: this study aimed to evaluate the distribution of health care facilities, dental visits and oral health problems to prevent increased exposure to covid-19 by using a geographic information system to explore the distribution of regional data. methods: this study is a secondary data analysis and used data from indonesia basic health research 2018 and health facility research 2019. data of health care facilities, dental visits and oral health problems were tabulated using ms excel version 16.45. spatial mapping was done using quantum geographic information system desktop version 3.18.3 based on open-source software. results: java island has the highest distribution of health care facilities and receives treatment from the dentist. covid-19 reached a peak in june–july 2021, and java island became the region with the highest incidence rate. conclusion: the distribution of health care facilities and dental visits was in line with the spread of the covid-19 virus in 34 provinces. almost all treatments of oral health problems need a highspeed rotary instrument, which can be a source of transmission of this virus. keywords: health care facilities; dental visit; covid-19; teledentistry correspondence: ayu asri lestari, master program of community dentistry, faculty of dentistry, universitas indonesia. jl. salemba no. 4 jakarta, 10430, indonesia. email: ayyulestari@yahoo.com introduction coronavirus or severe acute respiratory syndrome coronavirus 2 was first discovered in wuhan, china in 2019. the increase in cases was due to human-to-human transmission, for which the origin is still unknown.1 hospitals and health facilities have challenges in dealing with pandemics, which showed in the handling of patients who needed treatment or referrals for covid-19 cases by controlling virus contamination. in addition, strict protocols were implemented, such as monitoring the movement of people and hospital staff or health care facilities who could infect themselves while in hospital.2 the study showed that there were 138 patients treated at the wuhan hospital and 40 of those patients (29%) were health care workers.3 the first case of covid-19 in indonesia was reported on march 2, 2020, and all cases are spread in 34 provinces. dki jakarta, di yogyakarta, west java, gorontalo, and north sulawesi are provinces with a high risk for covid19 infection.4 the total number of infected in may 2020 was 14.265 people and 214.746 people in september 2020. fast and high transmission occurring among humans increases the risk to health care workers because they interact with covid-19 patients daily and other patients without symptoms.4,5 several world health organisations, such as the center for disease control (cdc), the american dental association (ada) and the national health service (nhs), have made regulations to regulate and provide guidance for dental practices. new dental service protocols aim to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 mailto:ayyulestari@yahoo.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 155 lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 prevent the spread of this virus.6 dentists are at a high risk of being infected by covid-19 as they must work closely with the patient at a distance of fewer than two meters.7 various media, such as patients’ oral fluids, contamination of treatment tools and materials and some parts of the dental unit surface, are currently sources of increased transmission and contamination for dentists, assistants and patients.8 one instrument used in dentistry is a high-speed handpiece that can produce large amounts of aerosols and droplets mixed with the patient’s saliva or blood, and these particles will be airborne for a few moments before falling to the surface or being inhaled through the respiratory tract.3,7,9 basic knowledge regarding covid19 transmission is paramount in preventing transmission in dental practices.10 to minimise contact, several world health organization recommend postponing nonemergency procedures except for uncontrolled bleeding, cellulitis and trauma to facial bones causing airway disruption. to accomplish this, the ada advised dentists to use teledentistry to limit covid-19 spread and contact as the initial stage of screening patients during pandemic situations.11 epidemiology is the study of determining factors and distribution-related health states. there is a study regarding the development of technology and health information systems involving geographical conditions and epidemiology as health geographic information systems (gis). health gis is a tool with an integrated system for managing, analysing and presenting health data spatially. according to nykiforuk and flaman,12 one of primary uses of gis in health informatics is disease surveillance, which is the collecting and tracking of data incidence, prevalence and spread of disease. it is a key element in mapping and modelling disease using gis that helps us more easily understand where a disease is and how it can be minimised or stopped.13 this study aims to describe the distribution of health care facilities, dental visits to general dentists and specialists and oral health problems in 34 provinces to identify highrisk regions of covid-19 transmission using secondary data analysis and gis in presenting data. materials and methods this study aimed to evaluate the distribution of health care facilities, dental visits to general dentists and specialists and oral health problems using secondary data analysis. secondary data was obtained from july to august 2021 in indonesia and is available from the 2019 health facilities research and 2018 basic health research through the health research and development agency (https://www. litbang.kemkes.go.id/).14 internal and external data validity was performed by health facilities and basic health research teams. ethics approval was obtained from the dental research ethics committee, faculty of dentistry, universitas indonesia (protocol number: 030750921). the geographic map of indonesia was captured from indonesia geospasial (https://www.indonesia-geospasial. com/2020/04/download-shapefile-shp-batas.html).15 secondary data and maps of provinces in indonesia were mapped using quantum geographic information system (a free and open-source cross-platform desktop and worldwide association users and developers but legally constituted in swiss, https://www.qgis.org/) to identify the distribution of health care facilities, utilisation of dental health services and dental and oral health problems experienced by the indonesian community. figure 1. distribution map of health care facilities in indonesia. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://www.litbang.kemenkes.go.id/ https://www.indonesia-geospasial.com/2020/04/download-shapefile-shp-batas.html https://www.qgis.org/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 https://www.litbang.kemenkes.go.id/ https://www.indonesia-geospasial.com/2020/04/download-shapefile-shp-batas.html 156lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 figure 2. distribution map of dental visits to the dentist and dental specialist. results we can see the distribution of health care facilities, dental visits to general dentists and specialists and oral health problems in 34 provinces in figures 1, 2 and 3. there are 38,840 health care facilities based on the types of dentists who practise in each province in indonesia. from figure 1, the highest number of health facilities are on java island with 21,826, while the lowest is in the maluku islands with 490 facilities. figure 2 shows a map of the distribution of dental visits to general dentists and specialists. java island is an area that has the highest number of dental visits to general dentists and specialists. general dentists and specialists have a broader level of competence and authority in practise compared to other dental practitioners. west java has the highest number of visits to specialists (n=2,779) and general dentists (n=17,190), followed by east java province. the primary dental problem was tooth cavities with 45.3%. on the other hand, the two main oral health problems with the most complaints were abscesses (14%) and gingival bleeding (13.9%). gingival bleeding condition is closely related to periodontal tissue. sulawesi island shows the highest percentage of cavities, swollen gums or abscesses and bleeding gums among other provinces in indonesia (figures 3a, b and c). figure 3a. distribution of caries in indonesia. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 157 lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 figure 3b. distribution of swollen gums or abscesses in indonesia. discussion provinces in java island have more complete availability of health care facilities compared to other provinces. java island’s five provinces were identified as the most highrisk provinces due to the high number of infected people.4 health care facilities in indonesia are still not ready to face the pandemic regarding the availability of personal protective equipment (ppe), equipment and medicines.16 the incidence of covid-19 has increased in all countries; thus, lockdowns are an alternative to reduce the incidence by limiting people’s mobility and provide an opportunity for health facilities to prepare for covid-19 patients or prevent the viral spread to health care workers, inpatients and outpatients. infection prevention in health care facilities is crucial but challenging to achieve.17 health care facilities have contributed to the transmission of covid-19 due to several factors, such as high mobility in health care facilities as a referral place for infected patients and inadequate ppe and hospital facility and infrastructure readiness in the face of a pandemic.18 the potential for the spread of covid-19 in health care figure 3c. distribution of gingival bleeding in indonesia. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 158lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 facilities can come in various forms, such as surfaces of objects that have direct contact with covid-19 patients contaminated with droplets containing the virus and causing fomite transmission. a study showed that 202 surface samples in seven isolation rooms were positive. these samples were obtained from the patients’ personal belongings, such as towels, toothbrushes and the walls of the patient’s bedroom. another sample was collected from the surface of the public bathroom flushing toilet, and the test result was positive.19 the process of periodically spraying disinfectants on all rooms and surfaces in health care facilities must maintain a minimum distance of 1.5 meters, use appropriate masks and use hand sanitisers to reduce the risk of exposure.17,19 asymptomatic patients who visit health care facilities are difficult to detect and can increase the risk of transmission, so the rules for limiting visits to health care facilities need to be implemented.20,21 the health care facility zone is divided into three categories, namely the yellow zone, orange zone and blue zone, regarding the main principle that after the patient has carried out the screening process, the patient will not have direct contact with at-risk patients. the yellow zone (medium risk) is a gathering point where people enter through one door for initial screening, and this zone should be placed in an open room to provide good air circulation. the orange zone is for people confirmed positive, and the blue zone is for people confirmed negative.17 indonesia basic health research 2018 data shows that the number of visits to dentists on the island of java is very high, which is closely related to the availability of health care facilities (indonesia health facilities research 2019) centralised on java island.22,23 the total confirmed positives in indonesia on october 31, 2021, was around 4 million people, and mortality cases were 143.405.24 among the total number of deaths, health care workers who died during the pandemic were 2,032 cases. the peak total mortality amount was 485 people in july 2021. dentists rank seventh with 46 deaths. based on the number of deaths of health care workers in each province in indonesia, east java was ranked first, followed by west java, dki jakarta and central java.25 the death of health care workers is one of the main problems during the covid-19 pandemic. indonesia can learn from the outbreak of the ebola virus disease, which has caused a high number of deaths and workload level for health care workers.16 dentists have a crucial role in reducing the spread of covid-19 by ensuring adherence to practice activities that refer to the regulations set to protect themselves, patients and the nursing team.6 practicing dentists must ensure that all team members understand the forms of transmission and preventive measures from covid-19. dental procedures require air pressure to support the equipment used, which can produce aerosols and droplets from the patient’s saliva. the dental procedures that commonly produce aerosols are cavity preparation, the use of rotary instruments in root canal treatment, scaling and polishing dental implants and surgery with complicated cases.6,26 the significant impact of limitation in dental visit shows that only 38% of patients visiting the dentist with the most complaints were dental trauma and oral infections during this pandemic.26 a study showed that 131 of the 285 respondents (46%) visited the dentist during the pandemic, and the majority of complaints were toothaches (55.7%), ulcers (31.3%), bleeding gums (24.4%) and complaints of bad breath (16%). during this pandemic to minimize infectious spreading, only 6.1% who visited dentist, received treatment for their complaints and remaining 13.7% did online consultation. respondents who did not visit the dentist (93.9%) had personal reasons, namely not requiring treatment (34.1%), limited operational dental health services (27.6%) and fear of being exposed to covid-19 during dental treatment.27 in another study, there was a significant increase in emergency dentist visits during the pandemic from 51% to 71.9%. the most common emergency complaints were dental pulpal or periapical lesions and cellulitis. dental injury cases decreased from 14.2% to 10.5%, which was influenced by the reduced number of people doing activities due to the lockdown period.28 cavities are the most common dental problems in indonesia, and treatment requires a handpiece. currently, dentists have a high risk of exposure to the covid virus while practising as covid-19 can spread through the air as droplets, aerosols and airborne particles. the ada recommends postponing treatment if the availability of ppe is limited and only emergency dental treatments are allowed, such as uncontrolled bleeding, cellulitis or trauma to the face interfering with the airway.11 dental clinics, hospitals and other health care facilities treating dental problems can potentially transmit covid19 and require strict protocols. there are various operational procedures for dental services during this pandemic, starting from the patient’s arrival, while in health facilities and rooms, and after the procedure is complete.29 in the covid-19 situation, reducing contact with people is a preventive measure that can help to reduce exposure risk. tele means ‘far,’ so teledentistry is an attempt to fulfil longdistance communication needs in dentistry.30 teledentistry is the initial stage before patients come to the clinic that has used remote communication technology to avoid direct contact during the pandemic.11,29,30 teledentistry consists of several subunits, including teleconsultation, which is the most common form and the initial stage of teledentistry. patients consult through telecommunications media, which is considered very useful, especially for patients with physical and mental limitations, the elderly and prisoners who have limitations to visit health care services. studies have shown that teleconsultation can reduce the number of referrals from primary health services by more than 45%.30,31 telediagnosis is the initial stage of determining a diagnosis by using technology in sending clinical images or photos and supporting examination data. several studies show that smartphones are used in early diagnosis to detect dental caries and pre-malignant oral lesions.11,30 teletriage in teledentistry is commonly used to assess the needs dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 159 lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 of patients in receiving care in conditions with limited access to health services due to geographic location and socioeconomic conditions, such as the use of teleradiology in providing outcome information to the continuing health care level.30 telemonitoring can be used to evaluate treatment outcomes during a pandemic and reduce the frequency of visits, costs and the time required.30,31 the availability of the teledentistry system was well responded to by patients (97%), particularly in populations that have difficulty accessing dental health services.32,33 in addition to assessing and improving oral cavity health status, it can also monitor patients after dental surgery.34 in paediatric patients, this can reduce the anxiety felt by children when taking anamnesis. some orthodontic cases, except fixed appliances, can still be performed using e-dentistry.35 teledentistry can be implemented in a virtual clinic and telephone with users aged 10–70 and 8–88 years. the virtual clinic is more widely used than over the phone. paediatric patients are still accompanied by their parents during consultations.33 in its implementation as a supporting tool to limit faceto-face services, teledentistry has several limitations in the diagnostic accuracy level. the quality of clinical photos must have good resolution, and the target object must be taken properly.30 both methods require a supportive team and system in their implementation. dentists need to have the ability to use e-dentistry technology. in addition, infrastructure factors, such as good signal quality, limited hardware components and support from technicians and operational experts, both software and hardware, that are qualified for this system need to be considered.30,33 another obstacle is the regulation of prescriptions, particularly in the use of antibiotics, when handling cases of acute oral infections to prevent drug resistance.36 from secondary data and using gis as tools, we can visualise the highest number of health care facilities, dental visits and oral health problems easier than with graphics or tables on presenting data, and we can minimise and stop the spread of viruses based on pandemic distribution. the distribution of health care facilities and dental visits was in line with the spread of the covid-19 virus in 34 provinces in indonesia. java island had the highest distribution of health care facilities and dental visits and is also the covid-19 hotspot province in indonesia. as we know, almost all dental procedures need highspeed rotary instruments and produce droplets, aerosols and airborne particles, where these three things are a way of transmitting the covid-19 virus at health care facilities. teledentistry and postponed treatment are an effort to minimise contact with the patients for certain cases during the pandemic. acknowledgement we would like to thank the university of indonesia, the indonesian ministry of health and the indonesia medical council. references 1. munster vj, koopmans m, van doremalen n, van riel d, de wit e. a novel coronavirus emerging in china key questions for impact assessment. n engl j med. 2020; 382(8): 692–4. 2. capolongo s, gola m, brambilla a, morganti a, mosca ei, barach p. covid-19 and healthcare facilities: a decalogue of design strategies for resilient hospitals. acta biomed. 2020; 91(9-s): 50–60. 3. napimoga mh, de freitas arr. dentistry vs severe acute respiratory syndrome coronavirus 2: how to face this enemy. rgo rev gaúcha odontol. 2020; 68. 4. eryando t, sipahutar t, rahardiantoro s. the risk distribution of covid-19 in indonesia: a spatial analysis. asia pacific j public heal. 2020; 32(8): 450–2. 5. wiharsini w, sulistiadi w. determinant factors of covid-19 transmission among of health personnel: a systematic review. in: childhood stunting, wasting, and obesity, as the critical global health issues: forging cross-sectoral solutions. surakarta: masters program in public helath, universitas sebelas maret; 2020. p. 140–4. 6. odeh n-d, babkair h, abu-hammad s, borzangy s, abu-hammad a, abu-hammad o. covid-19: present and future challenges for dental practice. int j environ res public health. 2020; 17(9): 3151. 7. banakar m, bagheri lankarani k, jafarpour d, moayedi s, banakar mh, mohammadsadeghi a. covid-19 transmission risk and protective protocols in dentistry: a systematic review. bmc oral health. 2020; 20(1): 275. 8. villani fa, aiuto r, paglia l, re d. covid-19 and dentistry: prevention in dental practice, a literature review. int j environ res public health. 2020; 17(12): 4609. 9. silvestre f-j, martinez-herrera m, márquez-arrico c-f, silvestrerangil j. covid-19, a new challenge in the dental practice. j clin exp dent. 2021; 13(7): e709–16. 10. kochhar as, bhasin r, kochhar gk, dadlani h. covid-19 pandemic and dental practice. int j dent. 2020; 2020: 8894794. 11. abbas b, wajahat m, saleem z, imran e, sajjad m, khurshid z. role of teledentistry in covid-19 pandemic: a nationwide comparative analysis among dental professionals. eur j dent. 2020; 14(s 01): s116–22. 12. nykiforuk cij, flaman lm. geographic information systems (gis) for health promotion and public health: a review. health promot pract. 2011; 12(1): 63–73. 13. shaw n, mcguire s. understanding the use of geographical information systems (gis) in health informatics research: a review. j innov heal informatics. 2017; 24(2): 940. 14. badan penelitian dan pengembangan kesehatan kemenkes ri. laporan riset nasional. available from: https://www.litbang. kemkes.go.id/laporan-riset-nasional/. accessed 2022 aug 9. 15. indonesia geospasial. download shapefile batas administrasi provinsi, kabupaten/kota, kecamatan, desa tahun 2019. 2020. available from: https://www.indonesia-geospasial.com/2020/04/ download-shapefile-shp-batas.html. accessed 2022 may 21. 16. setiati s, azwar mk. covid-19 and indonesia. acta med indones. 2020; 52(1): 84–9. 17. wilkinson ls, moosa s, cooke r, muller am, boyles th. preparing healthcare facilities to operate safely and effectively during the covid-19 pandemic: the missing piece in the puzzle. s afr med j. 2020; 110(9): 835–6. 18. gordon cl, trubiano ja, holmes ne, chua kyl, feldman j, young g, sherry nl, grayson ml, kwong jc. staff to staff transmission as a driver of healthcare worker infections with covid-19. infect dis heal. 2021; 26(4): 276–83. 19. feng b, xu k, gu s, zheng s, zou q, xu y, yu l, lou f, yu f, jin t, li y, sheng j, yen h-l, zhong z, wei j, chen y. multi-route transmission potential of sars-cov-2 in healthcare facilities. j hazard mater. 2021; 402: 123771. 20. black jrm, bailey c, przewrocka j, dijkstra kk, swanton c. covid-19: the case for health-care worker screening to prevent hospital transmission. lancet (london, england). 2020; 395(10234): 1418–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://www.litbang.kemkes.go.id/laporan-riset-nasional/ https://www.indonesia-geospasial.com/2020/04/download-shapefile-shp-batas.html https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 https://www.litbang.kemkes.go.id/laporan-riset-nasional/ https://www.indonesia-geospasial.com/2020/04/download-shapefile-shp-batas.html 160lestari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 154–160 21. schneider s, piening b, nouri-pasovsky pa, krüger ac, gastmeier p, aghdassi sjs. sars-coronavirus-2 cases in healthcare workers may not regularly originate from patient care: lessons from a university hospital on the underestimated risk of healthcare worker to healthcare worker transmission. antimicrob resist infect control. 2020; 9(1): 192. 22. badan penelitian dan pengembangan kesehatan kemenkes ri. laporan hasil riset kesehatan dasar 2018. 2018. available from: https://www.litbang.kemkes.go.id/laporan-riset-kesehatan-dasarriskesdas/. accessed 2022 may 21. 23. badan penelitian dan pengembangan kesehatan. laporan riset fasilitas kesehatan (rifaskes) 2019. laporan dinas kesehatan rifaskes 2019. 2019. available from: https://www.litbang.kemkes. go.id/laporan-riset-fasilitas-kesehatan-rifaskes/. accessed 2022 may 21. 24. kawal covid-19. jumlah kasus di indonesia saat ini. kawal informasi seputar covid-19 secara tepat dan akurat. 2021. available from: https://kawalcovid19.id/. accessed 2021 nov 1. 25. lapor covid-19. tenaga kesehatan indonesia gugur melawan covid-19. statistik pusara digital tenaga kesehatan. 2021. available from: https://nakes.laporcovid19.org/statistik. accessed 2021 nov 1. 26. hudyono r, bramantoro t, benyamin b, dwiandhono i, soesilowati p, hudyono ap, irmalia wr, nor nam. during and post covid-19 pandemic: prevention of cross infection at dental practices in country with tropical climate. dent j (majalah kedokt gigi). 2020; 53(2): 81–7. 27. p a s i g a b d. r e l a t i o n s h i p k n o w l e d g e t r a n s m i s s i o n o f covid-19 and fear of dental care during pandemic in south sulawesi, indonesia. pesqui bras odontopediatria clin integr. 2021; 21. 28. baghizadeh fini m. what dentists need to know about covid-19. oral oncol. 2020; 105: 104741. 29. sosiawan a, wahjuningrum da, bhardwaj a, mishra k, khandelwal s, bhardwaj a, bhardwaj s. six commandments of treatment protocols during covid-19 pandemic in dentistry. biomol heal sci j. 2021; 4(1): 52–6. 30. ghai s. teledentistry during covid-19 pandemic. diabetes metab syndr. 2020; 14(5): 933–5. 31. telles-araujo g de t, caminha rdg, kallás ms, santos ps da s. teledentistry support in covid-19 oral care. clinics (sao paulo). 2020; 75: e2030. 32. achmad h, tanumihardja m, ramadhany yf. teledentistry as a solution in dentistry during the covid-19 pandemic period: a systematic review. int j pharm res. 2020; 12(sp2): 272–8. 33. rahman n, nathwani s, kandiah t. teledentistry from a patient perspective during the coronavirus pandemic. br dent j. 2020; : 1–4. 34. giudice a, barone s, muraca d, averta f, diodati f, antonelli a, fortunato l. can teledentistry improve the monitoring of patients during the covid-19 dissemination? a descriptive pilot study. int j environ res public health. 2020; 17(10): 3399. 35. crawford e, taylor n. the effective use of an e-dentistry service during the covid-19 crisis. j orthod. 2020; 47(4): 330–7. 36. dar-odeh n, babkair h, alnazzawi a, abu-hammad s, abuhammad a, abu-hammad o. utilization of teledentistry in antimicrobial prescribing and diagnosis of infectious diseases during covid-19 lockdown. eur j dent. 2020; 14(s 01): s20–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p154–160 https://www.litbang.kemkes.go.id/laporan-riset-kesehatan-dasar-riskesdas/ https://www.litbang.kemkes.go.id/laporan-riset-fasilitas-kesehatan-rifaskes/ https://kawalcovid19.id/ https://nakes.laporcovid19.org/statistik https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p154-160 https://www.litbang.kemkes.go.id/laporan-riset-fasilitas-kesehatan-rifaskes/ 144 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 144–153 original article ovalbumin’s potential as a wound-healing medicament in tooth extraction socket by induction of cell proliferation through the erk2 pathway in silico sri nabawiyati nurul makiyah1, sartika puspita2 1department of histology and cell biology, school of medicine, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta, yogyakarta, indonesia 2department of oral biology, school of dentistry, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta, yogyakarta, indonesia abstract background: the trend of studies on dental medicaments is increasing rapidly. antibacterial or anti-inflammatory activity is most frequently studied. ovalbumin is one of the proteins whose benefits have been studied, but these benefits are still limited because of ovalbumin’s potential for proliferative bioactivity. purpose: the aim of this study is to examine ovalbumin’s potential as a woundhealing medicament through molecular docking analysis on a protein related to the extracellular signal-regulated kinases/mitogenactivated protein kinase (erk/mapk) signaling pathway. methods: ovalbumin was hydrolyzed through biopep-uwm (the biopepuwm™ database of bioactive peptides). protein target and interaction were predicted using similarity ensemble approach target prediction webserver, superpred webserver, string webserver, and cytoscape version 3.9.1. selected fragments were docked using autodock vina in pyrx 0.8 with tukey’s multiple comparison test and biovia discovery studio version 19.1.0.18287 for visualization. results: this study found that ovalbumin has the potential to positively regulate cell proliferation, angiogenesis, and fibroblast growth factor production. six of the 131 fragments of ovalbumin could interact with 73 proteins, and the 20 proteins with the highest probability and score of betweenness centrality showed potential for bioactivity. five fragments and povidone-iodine interacted inside the adenosine triphosphate (atp) phosphorylation site of erk2, whereas fragment 1 (f1) and glycerin interacted outside the site. f1 could decrease the binding energy required for adenosine 5′-[,-methylene]triphosphate or an atp-analogue chemical compound to interact with erk2 compared to the control, with a score that was not significant. conclusion: ovalbumin has the potential to induce cell proliferation by affecting erk2-ligand interactions. keywords: angiogenesis; cell proliferation; erk2; mapk; ovalbumin article history: received 14 october 2022; revised 6 december 2022; accepted 14 february 2023; published 1 september 2023 correspondence: sri nabawiyati nurul makiyah, histology and cell biology department, school of medicine, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta, brawijaya street, tamantirto, kasihan, bantul, yogyakarta, 55183 indonesia. email: nurul.makiyah@umy.ac.id introduction the trend of studies on medicaments to prevent and treat dental problems has increased rapidly since 2010, based on data published in pubmed (https://pubmed.ncbi.nlm. nih.gov/). the antibacterial or anti-inflammatory activities of medicament ingredients have been widely examined in previous studies.1,2 medicament materials from antibiotics such as ciprofloxacin, metronidazole, and doxycycline,3 as well as other materials such as calcium hydroxide,1,3 odontopaste,3 and quaternary ammonium silane/k214 are known to have antibacterial activity. wound healing in the oral cavity, including in the tooth sockets, is affected by bacterial and inflammatory activity. other medicaments that have been studied for wound healing are povidone-iodine (pvp-i) and glycerin (glycerol; gly). pvp-i has been known to have anti-inflammatory activity, low toxicity, and good tolerability, making it popular in the use of medicaments even though it has been a decade since its first publication.5 in another study, pvp-i copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 mailto:nurul.makiyah@umy.ac.id https://pubmed.ncbi.nlm.nih.gov/ https://e-journal.unair.ac.id/mkg/index https://pubmed.ncbi.nlm.nih.gov/ https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 145makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 and gly were tested on 347 patients with acute otitis externa (aoe) to analyze their efficacy against the aoe. the clinical trial results showed that aoe treatment with both medicaments has good efficacy and can relieve canal edema and pain in the patient’s tragus.6 wound healing is a very complex process involving multiple bioactivities and molecular signaling. several important bioactivities in wound healing are related to the induction/suppression of inflammation, proliferation, and cell differentiation. the mitogen-activated protein kinase (mapk) pathway is one of the important signaling pathways associated with these bioactivities.7 the woundhealing process after a tooth extraction is the same as any other tissue-healing process; that is, it is complex and dynamic. restoration of damaged tissue integrity involves cellular components and an extracellular matrix (ecm).8 however, research on medicaments for molecular wound healing is still rare, necessitating the current study. ovalbumin, the main component in chicken eggs (gallus domesticus), makes up more than 50% of all protein components in eggs. molecularly, ovalbumin (serpinb14) is a protein with a molecular weight of 45 kda with 385 amino acids and is a part of the large serpin group (the serpin superfamily). based on the amino acid sequence (aa), there is 1 disulfide bond linking cys74 and cys121, with half of the residues being hydrophobic and one-third being acidic.9,10 ovalbumin is known to play a role as a carrier protein that can increase the antioxidant effect and solubility of curcumin.11 however, there is still limited information related to its proliferative activity. therefore, this study aims to examine the potential of ovalbumin as a wound-healing medicament in tooth extraction sockets by induction of cell proliferation through the erk/mapk signaling compared to pvp-i and gly in silico. materials and methods protein structures of ovalbumin (uniprot id: p01012) and extracellular signal-regulated kinase2 (erk2) (pdb id: 5v60) were downloaded from the uniprot database (https://www.uniprot.org/) in fasta format for ovalbumin and from the rcsb pbd (https://www.rcsb.org/) in pdb format for erk2. the molecules used were pvp-i (cid: 11989721), gly (cid: 753), and atp-analogue (adenosine 5’-[β,γ-methylene]triphosphate [amp-pcp]) (cid: 91532), which were downloaded in .sdf format from pubchem (https://pubchem.ncbi.nlm.nih.gov/). prediction of ovalbumin peptide bioactivity was carried out via the biopep-uwm web server (http://www.uwm.edu.pl/ biochemia/index.php/en/biopep).12 then, ovalbumin was hydrolyzed by the extracellular protease enzymes chymotrypsin (ec 3.4.21.1), trypsin (ec 3.4.21.4), and pepsin, ph 1.3 (ec 3.4.23.1) using the biopep-uwm web server, which resulted in 141 fragments.8 the fragments were selected based on how many active peptides are in the fragment sequence and the bioactivity of those peptides based on the biopep-uwm database. after that, six fragments were selected that had peptides with anti-inflammatory-related bioactivity, including anti-inflammatory and antioxidant activity. six of the selected fragments were converted to simplified molecular input line entry system (smiles) via novoprolabs (https://www.novoprolabs.com/tools/ convert-peptide-to-smiles-string). next, each smiles fragment was loaded onto the similarity ensemble approach (sea) (https://sea.bkslab.org/) and superpred (https://prediction.charite.de/subpages/target_prediction. php) webservers to obtain protein prediction targets.13,14 the cut off chosen from the sea results was max tc > 0.5, and superpred had a probability > 90%. then, from the prediction results, ≥ 70 target proteins were obtained. the target protein was inputted into string (https://stringdb.org/) to see the bioactivity of the target protein.15 the setting used was a “physical” type network that showed “confidence” through the thickness of the line with high confidence criteria (0.700). the target protein without interaction was eliminated so that 29 target proteins remained. the selected target proteins (29 proteins) were downloaded from string and analyzed using cytoscape ver. 3.9.1 (cytoscape consortium) and the golorize plugin.16,17 first, the protein network was analyzed using the networkanalyzer to obtain data betweenness and closeness centrality and degree.18 then, the network was analyzed by gene ontology (go) using the golorize plugin, which generated go data related to proteins in the tissue and colored protein nodes in the tissue according to their bioactivity. furthermore, bioactivities related to wound healing including regulation of proliferation, angiogenesis, growth factor production, and anti-inflammatory19 were selected for further study. six fragments (fragment 1 to fragment 6), pvp-i (cid: 11989721), gly (cid: 753), and amp-pcp (atpanalogue) (cid: 91532), were used for molecular docking analysis with the predicted protein. the selected target protein had average value betweenness centrality (bc) and the highest predictive value of all fragments with pvp-i (cid: 11989721) and gly (cid: 753) as controls. furthermore, the second docking was carried out to examine the effect of the fragments on the interaction of atp with mapk1. the second docking was carried out between amp-pcp (cid: 91532) and mapk1 (erk2) (pdb id: 5v60) with the previous six ovalbumin fragment ligands. molecular docking was carried out using autodock vina on pyrx 0.9.7 with ligands that were minimized in energy through the open babel plugin and proteins that were removed by water molecules and ligands using biovia discovery studio ver 19.1.0.18287.20 finally, visualization was carried out with biovia discovery studio ver 19.1.0.18287 to visualize the interaction between fragments and the amp-pcp (atp-analogue) to erk2. statistical analysis was conducted to validate the results of the docking analysis, which presented standard copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://www.uniprot.org/ https://www.rcsb.org/ https://pubchem.ncbi.nlm.nih.gov/ http://www.uwm.edu.pl/biochemia/index.php/en/biopep https://www.novoprolabs.com/tools/convert-peptide-to-smiles-string https://sea.bkslab.org/ https://prediction.charite.de/subpages/target_prediction.php https://string-db.org/ https://e-journal.unair.ac.id/mkg/index https://www.novoprolabs.com/tools/convert-peptide-to-smiles-string http://www.uwm.edu.pl/biochemia/index.php/en/biopep https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 146 makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 deviations. the analysis was conducted using graphpad prism9 with a one-way anova followed by tukey’s multiple comparisons test. results the ovalbumin peptide fragments obtained were 142 fragments from 141 sites cut by the selected enzymes. this hydrolysis model used independent variables in the form of the number and type of enzymes, where the following fragments were the result of the activity of chymotrypsin (ec 3.4.21.1); trypsin (ec 3.4.21.4); and pepsin, ph 1.3 (ec 3.4.23.1), which acted at the same time. there were 50 fragments with one amino acid (phenylalanine, histidine, lysine, methionine, asparagine, arginine, tryptophan, and tyrosine), 38 and 20 fragments with two and three amino acids, 18 and 3 fragments with four and five amino acids, and 13 fragments with more than 5 amino acids, one of those was composed of 19 amino acids (table 1). furthermore, to analyze the fragment’s potential bioactivity related to cell proliferation, we used ovalbumin’s active peptide database, which has antioxidant and anti-inflammatory bioactivity. the active peptide was then used to determine which fragments had the same peptide composition. next, 142 fragments were reduced to six active fragments: aah (fragment 1/f1), el (f2), gsigaasm (f3), giir (f4), tsvl (f5), and vy (f6) (table 2). the six fragments were predicted to interact with a total of 74 proteins, and five proteins—capn1 (calpain-1 catalytic subunit), cfb (complement factor b), fohl1 (folate hydrolase 1), human leukocyte antigens class i histocompatibility antigen (hla-a), and n-acetylated-alpha-linked acidic dipeptidase 2 (naalad2)—were predicted to interact with two fragments. meanwhile, pvp-i and gly were predicted not to form interactions with any proteins based on the sea target prediction. the next protein prediction was conducted using superpred to obtain additional prediction results from other table 1. fragment peptide ovalbumin (uniprot id: p01012) after hydrolysis through biopep-uwm sequence location sequence location sequence location sequence location m [1-1] sl [101-102] vteq esk [201-207] eek [289-291] gsigaasm [2-9] asr [103-105] pvqm [208-211] y [292-292] ef [10-11] l [106-106] m [212-212] n [293-293] cf [12-13] y [107-107] y [213-213] l [294-294] dvf [14-16] aeer [108-111] qigl [214-217] tsvl [295-298] k [17-17] y [112-112] f [218-218] m [299-299] el [18-19] pil [113-115] r [219-219] am [300-301] k [20-20] pey [116-118] vasm [220-223] gitdvf [302-307] vh [21-22] l [119-119] asek [224-227] sssan [308-312] h [23-23] qcvk [120-123] m [228-228] l [313-313] an [24-25] el [124-125] k [229-229] sgiss aesl [314-322] en [26-27] y [126-126] il [230-231] k [323-323] if [28-29] r [127-127] el [232-233] isqavh [324-329] y [30-30] ggl [128-130] pf [234-235] aah [330-332] cpiaim [31-36] epin [131-134] asgtm [236-240] aein [333-336] sal [37-39] f [135-135] sm [241-242] eagr [337-340] am [40-41] qtaad qar [136-143] l [243-243] evvgsa eagvda asvseef [341-359] vy [42-43] el [144-145] vl [244-245] r [360-360] l [44-44] in [146-147] l [246-246] adh [361-363] gak [45-47] sw [148-149] pdev sgl [247-253] pf [364-365] dstr [48-51] vesqtn [150-155] eql [254-256] l [366-366] tqin [52-55] giir [156-159] esiin [257-261] f [367-367] k [56-56] n [160-160] f [262-262] cik [368-370] vvr [57-59] vl [161-162] ek [263-264] h [371-371] f [60-60] qpssv dsqtam [163-173] l [265-265] iatn [372-375] dk [61-62] vl [174-175] tew [266-268] avl [376-378] l [63-63] vn [176-177] tssn [269-272] f [379-379] pgf [64-66] aivf [178-181] vm [273-274] f [380-380] gdsieaq cgtsvn [67-79] k [182-182] eer [275-277] gr [381-382] vh [80-81] gl [183-184] k [278-278] cvsp [383-386] ssl [82-84] w [185-185] ik [279-280] r [85-85] ek [186-187] vy [281-282] dil [86-88] af [188-189] l [283-283] n [89-89] k [190-190] pr [284-285] qitk [90-93] dedtqam [191-197] m [286-286] pn [94-95] pf [198-199] k [287-287] dvy [96-98] r [200-200] m [288-288] sf [99-100] copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 147makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 table 2. prediction of active peptide ovalbumin (uniprot id: p01012) with anti-inflammatory and antioxidant bioactivity active peptides sequence location active peptides sequence location active fragment sequence location aeeryp [110-115] lw [190-191] aah [340-342] ah [341-342] lwe [190-192] el [18-19], [124-125], [144-145] dedtqamp [197-204] ly [108-109], [129-130] gsigaasm [2-9] el [18-19], [128-129], [148-149], [238-239] mm [217-218] giir [160-163] fc [11-12], [379-380] my [218-219] tsvl [303-306] gaa [5-7] nen [25-27] vy [42-43], [281-282] hh [22-23] ry [113-114] ir [162-163] salam [37-41] kd [47-48], [196-197] svl [304-306] kglwe [188-192] vhh [21-23] kp [95-96], [213-214] vhhanen [21-27] lfc1 [378-380] vy [42-43], [99-100], [289-290] lk [19-20], [332-333] ylg [43-45] lpf [239-241] ynl [300-302] note: (aa)1: amino acid with anti-inflammatory bioactivity. the bold print indicates the amino acids are predicted to be active peptides. sea target superpred figure 1. prediction of target protein for each fragment from sea target prediction and superpred prediction. the target proteins displayed are only those that had a probability value exceeding the specified cutoff (sea target prediction, cutoff maxtc ≥ 0.5; superpred, cutoff probability ≥ 90%). the black color of the squares corresponds to the increase in the probability score. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 148 makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 table 3. betweenness centrality score in the highest ppi and probability name betweenness centrality score probability mean (%) app 0.26 0 mapk1 0.14 72.19 mme 0.13 0 egfr 0.11 15.16 casp8 0.10 23.01 pik3ca 0.08 11.25 tp53 0.08 0 il1b 0.08 0 nfkb1 0.07 82.91 nfkbia 0.06 0 mapk14 0.05 0 cxcr4 0.04 11.31 ep300 0.04 41.34 ptgs2 0.04 0 traf2 0.04 0 note: the proteins in bold are among the 29 proteins selected from the previous analysis (figure 1). some proteins have an average probability of 0% because they are not predicted to interact with any fragments during target prediction analysis. figure 2. protein network associated with previously predicted proteins. a total of 59 proteins (29 predicted proteins [red label] and 30 enrichment proteins) were obtained. yellow-colored proteins indicate positive regulation of cell proliferation (go-id: 8284) bioactivity, blue-colored proteins indicate positive regulation of angiogenesis (go-id: 45766) bioactivity, and green-colored proteins indicate positive regulation of fibroblast growth factor production (go-id: 90271) bioactivity. table 4. prediction of bioactivity of 59 proteins based on functional annotation gene ontology analysis go-id description p-value corrected p-value gene 8284 positive regulation of cell proliferation 1.6103e-5 1.6163e-4 capns1 mme ripk2 il1b mapk1 s1pr3 kras capn1 ptgs2 egfr 45766 positive regulation of angiogenesis 7.3175e-4 3.7681e-3 il1b ptgs2 tnfrsf1a 90271 positive regulation of fibroblast growth factor production 4.1250e-3 1.4003e-2 ptgs2 databases and increase the probability of approaching the real condition. a total of 34 proteins were predicted to interact with six fragments and control drugs. pvp-i was predicted to interact with ctsd, erab, and nfkb1, whereas gly was predicted to interact with lamin a/c (lmna; figure 1). next, 30 proteins from the enrichment process were obtained and arranged according to the value of bc. bc was obtained from the protein interaction network. the higher the bc score, the more important the role of the protein in the network.18 the target protein for docking analysis was selected from the previous 29 proteins with the highest bc and maxtc/probability scores (figure 2; table 3). erk2 was chosen as a protein target for molecular docking analysis because it is known to interact with all fragments based on superpred predictions, and it has a bc score of 0.14 with a mean probability of 72.19% to interact with all ligands. then, based on go analysis, erk2 had a role in the processes of proliferation and cell survival. it would be interesting to study further the effect of the ovalbumin fragment in influencing the interaction of erk2 with its downstream protein. hereafter, bioactivity analysis based on the interactions of 59 proteins demonstrates the presence of bioactivity, and table 4 shows associated proteins. the three biological activities of the selected ontology genes had different confidence and strength scores. the confidence score used was the p-value score and bonferroni’s corrected p-value to increase the credibility of the results. based on bonferroni’s p-value and corrected p-value scores,21 the three bioactivities had a score of < 0.05, meaning they had a probability of occurring. positive regulation of cell proliferation was the most potent bioactivity, followed by copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 149makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 a b in di ng e ne rg y (k ca l/m ol ) ligand binding energy (kcal/mol) f1 -7.10 + 0.00 f2 -5.90 + 0.17 f3 -6.67 + 0.32 f4 -6.67 + 0.55 f5 -6.63 + 0.30 f6 -7.20 + 0.00 gly -3.83 + 0.11 pvp-i -4.60 + 0.10 b b in di ng e ne rg y (k ca l/m ol ) erk2 + ligand binding energy (kcal/mol) c -8.73 + 0.06 f1 -8.87 + 0.06 f2 -7.83 + 0.31 f3 -7.13 + 0.64 f4 -7.37 + 0.21 f5 -7.20 + 0.20 f6 -7.30 + 0.26 gly -8.70 + 0.00 pvp-i -8.57 + 0.21 figure 4. comparison of the binding energy scores of fragments and compounds. (a) comparison of binding energy scores of all ligands against f6, which had the lowest binding energy score. one-way anova (p < .05); ***p =.0004, ****p < .0001. (b) the amp-pcp binding energy score against erk2, which interacted with all ligands, was compared with the amp-pcp binding energy score against the control erk2 (c). one-way anova (p < .05); *p = .0216, ***p < .001, ****p < .0001. a fragment 1 fragment 5 fragment 2 fragment 6 b fragment 3 pvp i fragment 4 glycerin figure 3. the location of the six fragments interacting with erk2 at the atp phosphorylation site. (a) the location of the six fragments, f1 = blue, f2 = green, f3 = yellow, f4 = purple, f5 = pink, f6 = turquoise, gly = orange, and pvp-i = brown. (b) location of atp phosphorylation at the erk2 phosphorylation site. comparison of amp-pcp (cid: 91532) against original (native) amp-pcp. the two ligands were juxtaposed to justify the similarity of the positions and interactions formed. (blue) amp-pcp (atp-analogue) interacts at the phosphorylation site of erk2 according to the study of lechtenberg et al.22 (gdp id: 5v60). (red) amp-pcp control (cid: 91532). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 150 makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 native amp-pcp control (c) fragment 1 (f1) figure 5. the interaction between amp-pcp and erk2 with f1, gln, and pvp-i. positive regulation of angiogenesis and fibroblast growth factor (fgf) production. next are the results related to molecular docking analysis. the atp-binding site or phosphorylation site of erk2 (pdb id: 5v60) was found in amino acids 3139 and 54, including ile31, gly32, glu33, gly34, ala35, tyr36, gly37, met38, val39, and lys54.22 the six fragments were docked blindly to find out where the optimal location of interaction would occur in each ligand. docking was carried out in three replications with different coordinates. replication coordinates 1, center: x: -4.591, y: 8,733, z: 47,759; dimension (angstrom): x: 79,999, y: 49,579, z: 63,872. replication 2, center: x: -2.857, y: 6.812, z: 46.304; dimension (angstrom): x: 64,739, y: 48,945, z: 70,444. replication 3, center: x: -3,270, y: 6,527, z: 46,102; dimension (angstrom): x: 66,329, y: 50,285, z: 70,678. f6 required the least amount of binding energy (interaction energy), followed by f1. from these results, it can be concluded that the interaction between f1 and gly, which is outside the atp phosphorylation site, may affect the molecular bioactivity of erk2 without having to compete with atp. at the same time, the interaction between f2, f3, f4, f5, f6 and pvp-i may act as an inhibitor of atp phosphorylation on erk2 (figure 3). the second docking was carried out by interacting amp-pcp (atp analogue) as a native experimental ligand with erk2 to evaluate the interaction docked with the previous ligand. the docking procedure was the same as in the previous step in triplicate, with the original amp-pcp ligand anchored to erk2 (amp-pcp original) (pdb id: 5v60) substituted with amp-pcp (cid: 91532) anchored to erk2 (pdb id: 5v60), which was prepared as a control. replication coordinates 1, center: x: 14,533, y: 13,085, z: 15,442; dimension (angstrom): x: 25,607, y: 29.3, z: 36,223. replication 2, center: x: 13,998, y: 10,744, z: 15,947; dimension (angstrom): x: 30,065, y: 25,309, z: 28,456. replication 3, center: x: 13,658, y: 11,993, z: 15,166; dimension (angstrom): x: 27,266, y: 25,481, z: 25,573. according to the second docking result, f1 interaction can reduce amp-pcp binding energy to erk2 by 0.01% compared to the control (figure 4). a visualization was carried out to understand in detail the interactions that occurred between the ligand and erk2. the visualization of amp-pcp control and original amppcp in the erk2 ligand (pdb id: 5v60) aims to validate the amp-pcp pose after re-docking (figure 5). table 5 is the list of all residues that interacted with the ligand. glycerin (gly) povidone-iodine (pvp-i) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 151makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 discussion tooth extraction is an activity that causes injury to the tooth socket because of the presence of parts of the gums and nerves that interact directly with the environment (the oral cavity). pathogenic microorganisms can enter the wound, causing the body to automatically respond with inflammation and the wound-healing process.23 wound healing is a very complex process in the human body. it involves various types of cells and ranges from repair and arrangement of specialized structures, such as collagen, migration, proliferation, and differentiation of cells.19,24 in this study, we report potential bioactivity that positively regulates cell proliferation (go-id: 8284), angiogenesis (go-id: 45766), and fgf production (goid: 90271) by proteins targeted by an ovalbumin fragment. these three bioactivities are known to play a crucial role in the proliferative phase compared to the inflammatory phase or the maturation (remodeling) phase in the woundhealing process. in the inflammatory phase, the process of vascular vessel contraction and blood clotting is followed by an increase in the number of leukocytes in the wound tissue, including an increase in the number of neutrophils due to an increase in pro-inflammatory cytokines such as interleukin-1, tumor necrosis factor-alpha (tnf-α), and interferon-gamma and chemotactic agents such as pathogen-specific associated molecular pattern, damageassociated molecular pattern, complement, histamine, prostaglandins, and leukotrienes. in addition to neutrophils, there is also an increase in the macrophage population due table 5. residues that form interactions with amp-pcp native erk2/amppcp erk2 control erk2/f1 – amppcp erk2/gly – amp-pcp erk2/pvp-i – amp-pcp hydrogen bond lys54 lys54 asp106 met108 met108 asp111 lys114 ser153 ser153 ala35 tyr36 arg67 gln105 asp106 met108 asp111 ala35 tyr36 gly37 lys54 gln105 met108 ser153 ala35 tyr36 lys54 gln105 asp106 met108 asp111 lys54 gln105 asp111 asp111 asp149 lys151 lys151 ser153 cys166 cys166 electrostatic bond lys54 lys54 arg67 lys151 tyr36 asp167 asp167 asp167 tyr36 asp167 asp167 tyr36 asp167 asp167 asp167 asp149 asp167 asp167 asp167 hydrophobic bond ile31 val39 ala52 leu156 leu156 ile31 val39 val39 ala52 ala52 lys54 arg67 leu156 leu156 ile31 val39 val39 val39 ala52 ala52 ala52 leu156 leu156 leu156 ile31 ile31 val39 val39 val39 ala52 ala52 ala52 leu156 leu156 val39 val39 ala52 lys54 leu156 van der waals bond gly32 glu33 gly34 ala35 ile84 gln105 leu107 asp149 asn154 asp167 gly32 glu33 gly34 gly37 ile84 leu107 lys114 ser153 asn154 gly169 gly32 glu33 gly34 lys55 ile56 ile84 asp106 leu107 lys114 asn154 cys166 gly32 glu33 gly34 gly37 ile84 leu107 asn154 cys166 gly169 ile31 gly32 glu33 ile84 lys114 asn154 thr190 unfavorable bond lys54 lys54 arg67 asp111 lys54 lys54 arg67 arg67 asp111 lys114 lys54 lys54 arg67 asp111 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 152 makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 to the chemotactic compounds tumor growth factor-beta (tgf-β) and monocyte chemoattractant protein-1.19,24 furthermore, in the proliferative phase, there are reepithelialization, neovascularization (angiogenesis), and immunomodulators aimed at repairing and restructuring damaged tissue. in this phase, tissue granulation occurs by fibroblasts, which play an important role in inducing the formation of a new ecm and blood vessels. another process that is no less important is angiogenesis and vasculogenesis (neovascularization), which aims to form new blood vessels that will supply nutrients and maintain oxygen homeostasis in the healing process in injured tissues. this process is inseparable from the presence of pro-angiogenic signals such as vascular endothelial growth factor (vegf), fgf, platelet-derived growth factor beta, tgf-β, and angiopoietins. the last involves the formation of pericytes, which play a role in microvascular stability, regulation of blood flow, and the formation of vascular protection from bacteria.25 the last phase is maturation (remodeling), which causes contraction of the wound and replacement of type-iii collagen with type-i collagen.24 the three bioactivities are obtained from a network of 59 proteins where amyloid-β precursor protein (app), mapk1 (erk2), mme, epithelial growth factor receptor (egfr), and caspase-8 (casp8) are important links in the network. in wound healing, app plays an important role in the proliferation, migration, and adhesion of endothelial cells. endothelial cells require app as a mediator of the scr/fak pathway in vegf signaling.26 furthermore, in regard to erk2, upregulation, and phosphorylation of erk1/2 and akt are known to be consistent with increased proliferation and migration of human skin fibroblasts and human umbilical vein endothelial cells in vitro due to sea cucumber peptide treatment.27 other studies have also shown that the activation of the egfr/mek/erk signaling pathway by the sox2 gene is known to accelerate wound healing through the induction of keratinocyte cell migration and proliferation.28 meanwhile, the inhibition of corneal mme is known to improve corneal epithelial wound healing in mice.29 eliminating casp8 is known to increase the proliferation and migration of human epidermal keratinocytes, which can promote wound healing in mice.30 molecular docking analysis results show that peptide fragments vy (f6), aah (f1), gsigaasm (f3) and require the lowest binding energy. interestingly, the molecular docking results blindly show two interaction sites outside and inside the atp phosphorylation site. then, according to the molecular docking results of the amp-pcp interaction with erk2, f1 has the potential to reduce the binding energy of the amp-pcp interaction with erk2 in the phosphorylation domain, although the difference is not significant. the interaction of f1 outside the phosphorylation site minimizes the probability that f1 will compete with atp. additionally, the interaction of gly and pvp-i on erk2 increases the binding energy of amp-pcp when it interacts with erk2, although the difference is again not significant. what is interesting about the interaction between the two is the different interaction sites, where gly interacts outside the atp phosphorylation site and pvp-i acts inside the phosphorylation site. based on its location and required binding energy, pvp-i has the potential to be an inhibitor of atp phosphorylation on erk2 when it has interacted with erk2 first. however, the binding energy required for gly and pvp-i to interact with erk2 is high, so further research is needed to obtain more comprehensive conclusions. furthermore, pvp-i is known to have anti-inflammatory activity through suppression of tnfexpression in human neutrophil cells in vitro31 and decreased galactosidase activity in e. coli cultures32 so that it can reduce the level of interleukin-6, tnf-α, and rheumatoid factor in the serum of rheumatoid arthritis patients.33 gly is more often used as a viscous mixing agent with a heavy molecular weight, such as a ca2+ and ohmixer for intracanal ca(oh)2 medicaments.34 furthermore, the sodium alginate (naalg)pvp-i complex is known to have a wound-healing effect by accelerating the closure process.35 structurally, pvp-i is composed of a polyvinyl pyrrolidone (povidone) polymer complex with elemental iodine, which is intended for health practitioners. various studies on pvp-i have shown anti-inflammatory, anti-bacterial, anti-biofilm, anti-edema, and hemostatic activity; low toxicity; and good tolerability. thus, it is still used as a medicament even though it has been more than a decade since its development.5,31 ovalbumin was found to affect the interaction of erk2 and amppcp in this study. ovalbumin is also predicted to have the potential to be an additional medicament/component complex for better wound healing than pvp-i and gly. in conclusion, ovalbumin has the potential to induce cell proliferation by decreasing the binding energy required for amp-pcp to interact with erk2 compared to gly and pvp-i, which slightly increase the binding energy required for amp-pcp to interact with erk2. further in vitro/in vivo development and testing are needed to validate and develop ovalbumin as a pro-proliferative medicament. references 1. athanassiadis b, walsh lj. aspects of solvent chemistry for calcium hydroxide medicaments. materials (basel). 2017; 10(10): 1219. 2. manohar m, sharma s. a survey of the knowledge, attitude, and awareness about the principal choice of intracanal medicaments among the general dental practitioners and nonendodontic specialists. indian j dent res. 2018; 29(6): 716–20. 3. govinda raju l , jena r tha na n s, subra ma nya m d, ajitha p. antibacterial activity of various intracanal medicament against enterococcus faecalis, streptococcus mutans and staphylococcus aureus: an in vitro study. j pharm bioallied sci. 2021; 13(5): 157–61. 4. kok esk, lim xj, chew sx, ong sf, see ly, lim sh, wong la, davamani f, nagendrababu v, fawzy a, daood u. quaternary ammonium silane (k21) based intracanal medicament triggers biofilm destruction. bmc oral health. 2021; 21(1): 116. 5. bigliardi pl, alsagoff sal, el-kafrawi hy, pyon j-k, wa ctc, villa ma. povidone iodine in wound healing: a review of current concepts and practices. int j surg. 2017; 44: 260–8. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 153makiyah and puspita. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 144–153 6. patel m, dehadaray a. povidone-iodine and glycerine for treatment of acute otitis externa. saudi j heal sci. 2018; 7(3): 178–82. 7. sun y, liu w-z, liu t, feng x, yang n, zhou h-f. signaling pathway of mapk/erk in cell proliferation, differentiation, migration, senescence and apoptosis. j recept signal transduct. 2015; 35(6): 600–4. 8. velnar t, bailey t, smrkolj v. the wound healing process: an overview of the cellular and molecular mechanisms. j int med res. 2009; 37(5): 1528–42. 9. fatoki th, aluko re, udenigwe cc. in silico investigation of molecular targets, pharmacokinetics, and biological activities of chicken egg ovalbumin protein hydrolysates. j food bioact. 2022; 17: 34–48. 10. chay pak ting bp, pouliot y, gauthier sf, mine y. fractionation of egg proteins and peptides for nutraceutical applications. in: separation, extraction and concentration processes in the food, beverage and nutraceutical industries. elsevier; 2013. p. 595– 618. 11. liu y, ying d, cai y, le x. improved antioxidant activity and physicochemical properties of curcumin by adding ovalbumin and its structural characterization. food hydrocoll. 2017; 72: 304–11. 12. minkiewicz, iwaniak, darewicz. biopep-uwm database of bioactive peptides: current opportunities. int j mol sci. 2019; 20(23): 5978. 13. keiser mj, roth bl, armbruster bn, ernsberger p, irwin jj, shoichet bk. relating protein pharmacology by ligand chemistry. nat biotechnol. 2007; 25(2): 197–206. 14. nickel j, gohlke b-o, erehman j, banerjee p, rong ww, goede a, dunkel m, preissner r. superpred: update on drug classification and target prediction. nucleic acids res. 2014; 42(w1): w26–31. 15. szklarczyk d, gable al, lyon d, junge a, wyder s, huerta-cepas j, simonovic m, doncheva nt, morris jh, bork p, jensen lj, mering c von. string v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. nucleic acids res. 2019; 47(d1): d607–13. 16. garcia o, saveanu c, cline m, fromont-racine m, jacquier a, schwikowski b, aittokallio t. golorize: a cytoscape plug-in for network visualization with gene ontology-based layout and coloring. bioinformatics. 2007; 23(3): 394–6. 17. shannon p, markiel a, ozier o, baliga ns, wang jt, ramage d, amin n, schwikowski b, ideker t. cytoscape: a software environment for integrated models of biomolecular interaction networks. genome res. 2003; 13(11): 2498–504. 18. xia j, benner mj, hancock rew. networkanalyst integrative approaches for protein–protein interaction network analysis and visual exploration. nucleic acids res. 2014; 42(w1): w167–74. 19. gonzalez ac de o, costa tf, andrade z de a, medrado arap. wound healing a literature review. an bras dermatol. 2016; 91(5): 614–20. 20. dallakyan s, olson aj. small-molecule library screening by docking with pyrx. in: hempe je, williams ch, hong cc, editors. chemical biology methods and protocols. new york: humana press; 2015. p. 243–50. 21. jafari m, ansari-pour n. why, when and how to adjust your p values? cell j. 2019; 20(4): 604–7. 22. lechtenberg bc, mace pd, sessions eh, williamson r, stalder r, wallez y, roth gp, riedl sj, pasquale eb. structure-guided strategy for the development of potent bivalent erk inhibitors. acs med chem lett. 2017; 8(7): 726–31. 23. ningsih jr, haniastuti t, handajani j. re-epitelisasi luka soket pasca pencabutan gigi setelah pemberian gel getah pisang raja (musa sapientum l) kajian histologis pada marmut (cavia cobaya). jikg (jurnal ilmu kedokt gigi). 2019; 2(1): 1–6. 24. primadina n, basori a, perdanakusuma ds. proses penyembuhan luka ditinjau dari aspek mekanisme seluler dan molekuler. qanun med med j fac med muhammadiyah surabaya. 2019; 3(1): 31–43. 25. rodrigues m, kosaric n, bonham ca, gurtner gc. wound healing: a cellular perspective. physiol rev. 2019; 99(1): 665–706. 26. ristori e, cicaloni v, salvini l, tinti l, tinti c, simons m, corti f, donnini s, ziche m. amyloid-β precursor protein app down-regulation alters actin cytoskeleton-interacting proteins in endothelial cells. cells. 2020; 9(11): 2506. 27. zheng z, li m, jiang p, sun n, lin s. peptides derived from sea cucumber accelerate cells proliferation and migration for wound healing by promoting energy metabolism and upregulating the erk/ akt pathway. eur j pharmacol. 2022; 921: 174885. 28. uchiyama a, nayak s, graf r, cross m, hasneen k, gutkind js, brooks sr, morasso mi. sox2 epidermal overexpression promotes cutaneous wound healing via activation of egfr/mek/erk signaling mediated by egfr ligands. j invest dermatol. 2019; 139(8): 1809-1820.e8. 29. genova rm, meyer kj, anderson mg, harper mm, pieper aa. neprilysin inhibition promotes corneal wound healing. sci rep. 2018; 8(1): 14385. 30. liu y, xiong w, wang c-w, shi j-p, shi z-q, zhou j-d. resveratrol promotes skin wound healing by regulating the mir-212/casp8 axis. lab investig. 2021; 101(10): 1363–70. 31. amtha r, kanagalingam j. povidone-iodine in dental and oral health: a narrative review. j int oral heal. 2020; 12(5): 407. 32. lachapelle j-m, castel o, casado af, leroy b, micali g, tennstedt d, lambert j. antiseptics in the era of bacterial resistance: a focus on povidone iodine. clin pract. 2013; 10(5): 579–92. 33. su z, gao j, xie q, wang y, li y. possible role of β-galactosidase in rheumatoid arthritis. mod rheumatol. 2020; 30(4): 671–80. 34. putri kusuma ar. pengaruh lama aplikasi dan jenis bahan pencampur serbuk kalsium hidroksida terhadap kekerasan mikro dentin saluran akar. odonto dent j. 2016; 3(1): 48–54. 35. summa m, russo d, penna i, margaroli n, bayer is, bandiera t, athanassiou a, bertorelli r. a biocompatible sodium alginate/ povidone iodine film enhances wound healing. eur j pharm biopharm. 2018; 122: 17–24. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p144–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p144-153 123 vol. 42. no. 3 july–september 2009 research report color stability of visible light cured composite resin after soft drink immersion alizatul khairani hasan, siti sunarintyas, and dyah irnawati department of dental biomaterials faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: composite resin is a tooth-colored filling material containing bis-gma which exhibits water sorption properties. people tend to consume soft drink with various colors. water sorption properties can alter the color stability of composite resin purpose. purpose: this study was to determine the influence of immersion durations of composite resin in soft drink on color stability. methods: the visible-light cured hybrid composite resin and soft drink were used. ten disk specimens (2.5 mm thickness and 15 mm diameter) of composite resin were prepared and light cured for 20 seconds, then stored in distilled water for 24 hours at 37° c. the initial color of specimens were measured by chromameter. after that, each specimen was immersed in 30 ml of soft drink up to 48, 72, and 96 hours at 37° c. the specimens’ color were measured again after each immersion. the color changes were calculated by cie l*a*b* system formula. the data was analyzed by one-way anova and lsd (a = 0.05). result: the anova showed that the immersion durations of composite resin in soft drinks had significant influence on the color stability (p < 0.05). the lsd0.05 tests showed significant differences among all groups. the least color change was detected from the group of 48 hours immersion, while the greatest color change was from the group of 96 hours immersion. conclusions: the immersion of composite resin in soft drinks influenced the color stability (began after 48 hours immersion). key words: composite resin, soft drink, immersion durations, color stability correspondence: siti sunarintyas, c/o: bagian biomaterial kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara, yogyakarta 55281, indonesia. e-mail: sunarintyassiti@yahoo.com, phone/fax no. 062-274-515307. introduction dental caries is a disease of the calcified tissues of the teeth caused by the action of microorganisms on fermentable carbohydrates. it is characterized by disintegration of organic materials of the teeth. dental caries occur in the presence of four factors; dental plaque, carbohydrate, susceptible tooth surface, and time.1 tooth decay is generally repaired by removing the carious tissue and replacing it with an appropriate restoration.2 a study revealed that there appears to be a fairly enthusiastic adoption of tooth-colored restorative materials, influenced by clinical indications and patient’s demands.3 it showed that tooth-colored restorative materials such as composite resins are much more preferred compared to amalgams. in the era of esthetic composite restorations, the demand for overall good color stability is increasing. most anterior restorations are replaced because of unacceptable color match.4 the color stability of dental composites is due to exogenous and endogenous reasons. exogenous influences are staining food or even mouthrinses. the examples of staining food are coffee and red wine.5 tea and soft drinks are also classified as strong staining agents. endogenous or instrinsic color change of a visible-light activated composite may caused by the decomposition of the camphorquinone.6 a high water sorption value for a composite resin may indicate that the material has a high soluble fraction.7 it has been shown that materials exhibiting high water sorption values are more easily stained by hydrophilic colorings in aqueous solutions. the water presumably acts as a penetration vehicle.8 modern lifestyles have changed the behavior of people all around the world to be more practical. people with time constraints would be more inclined to choose food requiring little manipulation before consumption. for example, most 124 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 123-125 north americans under 25 years of age readily accept convenience food and beverages. this is because they became familiar with the food early in their lives and are knowledgeable about the time-saving qualities.9 one of the most popular convenience food and beverages available for consumption is soft drink. soft drink is a flavored, nonalcoholic beverage prepared with carbonated water.10 although soft drink is water based, much of the taste and appeal comes from the addition of significant amounts of sugar, sugar substitutes and other chemicals that are harmful to health.11 the flavor of soft drink sometimes comes from a mixture of vanilla, cinnamon and citrus flavorings. soft drink may be sweetened with sugar, corn syrup or an artificial sweetener.12 most soft drinks also contain coloring.10 people learn to associate certain colors with certain flavors, and this causes the color of food to influence the perceived flavor. for this reason, food manufacturers add dyes or colorings to their products.13 wide selections of soft drinks with different colors such as orange, blue, red, yellow, and dark brown to black are available in the market. cola, an example of soft drink, contains caramel color iv. caramel color iv is also known as sulfite-ammonia, soft drinks caramel or acid proof caramel. caramel colors are dark brown to black liquids or solids having an odor of burnt sugar and pleasant, somewhat bitter taste. caramel colors are prepared by controlled heat treatment of carbohydrates.14 in clinical studies, a change in color was observed in anterior composite restorations over a 3-year period.15 most of water sorption takes place during the first week. specimen discolorations tend to follow the evolution of water sorption.8 the hydrophilic characteristic of composite resin may affect the color stability of the restoration after the consumption of soft drinks which contain food colorings in certain times. this condition can be very displeasing especially when the anterior restoration is involved. the present study was aimed to determine the influence of immersion durations of composite resins in soft drink on the color stability. materials and methods ten disk specimens were prepared using two glass slides and a fiberglass mold with an internal thickness of 2.5 mm and a diameter of 15 mm (to fit diameter of chromameter tip). one glass slide was placed on a glass plate and the fiberglass mold was placed on the top of the glass slide. visible-light-cured hybrid composite resin (solare a2, gc corporation, japan) was put into the mold by a plastic spatula. the mold was then covered by glass slide and glass plate. the glass plate was removed and the specimen was cured by visible-light-curing unit (litex 600 dentamerica, usa) for 20 seconds on the top surface of the specimen. after that, the specimen was removed from the mold. all specimens were stored in distilled water for 24 hours at 37° c. the initial colors of specimens in commision internationale del’eclairage system (cie l*a*b*) were measured by chromameter (cr-200 b minolta co. ltd., japan). the chromameter was calibrated to a standard white plate. after that, the specimens were put on a table that was covered by a piece of paper and the measuring tip was placed flat against the surface of the specimens. after the ready lamp had lit, the measuring button on the meter body was pressed and the measured value appeared in the display. ten plastic jars were filled with 30 ml of coca-cola soft drink (pt coca-cola bottling, indonesia). the specimens were immersed in the coca cola soft drink up to 48 hours (group a), 72 hours (group b), and 96 hours (group c). immersion duration of 48, 72, and 96 hours are equivalent to soft drink consumption in 2, 3, and 4 years. the soft drink used for immersions were replaced every 24 hours. the jars were put into the incubator (mir 162 sanyo, japan) at the 37° c temperature. after each immersion, the specimens’ colors were measured again. the effect of the immersion durations on the color stability of composite resins were detected by calculating the color changes (the differences between initial and after immersion measurements) using the formula:16 δ��� ������������ �� ��δ�������� ������������ �� ��δ�����δ�����l*)2 + �δ �����δ ����� a*)2 + �δ�����δ�����b*)2 ]½ notes: e* = color change l* = brightness a* = amount of red (positive values) and green (negative value) b* = amount of yellow positive value) and blue ( negative value) the data was analyzed by one-way anova and least significant difference tests (a = 0.05). result the means and standard deviations of color changes �δ����� of the composite resins �fter immersion in soft drink are presented in table 1. the results showed trend of color changes increasing by the immersion duration. test of normality (kolmogorov-smirnov test) showed that the probability is greater than 0.05. test of homogeneity of variances showed that the probability is also greater than 0.05. the result of one-way anova showed that the immersion durations of composite resin in soft drinks significantly influenced the color stability (p < 0.05). the lsd tests showed significant differences between the means of all groups (table 2). composite resin consists of organic polymer matrix, inorganic filler particles, coupling agent, and the initiatoraccelerator system.16 most filled resin systems use bisgma as the organic polymer matrix. bis-gma showed high water sorption value because of the diluents added to reduce the viscosity of the paste.17 the study to compare 125hasan, et al.: color stability of visible light cured water sorption and solubility of hybrid and microfilled composite resin proved that microfilled composite resin exhibits higher water sorption than hybrid composite resin, because the microfilled composite has greater matrix content than hybrid composite.18 however, the hybrid composites are the ones widely used for anterior restorations because of its smooth surface and good strength.17 this is why the hybrid composite resin was used in the present study instead of microfilled composite resin. results of this study showed that the color of composite specimens tend to become darker after each immersion in soft drink. soft drink used in this experiment contains dark brown caramel coloring. the results were consistent with previous study where food red 3 solution was used as one of the staining agent. in the study, the colors of composite specimens become darker after each immersion in staining agent for 10, 20, and 30 days.19 water soluble food coloring agent can be absorbed by hydrophilic basic resin. the water will act as a penetration vehicle.8 the interfaces between the filler particles and the matrix of composite will accommodate the water.18 if the composite can absorb water, then it can also absorb other fluids.7 this means, the composite resin is able to absorb caramel coloring from the soft drink and leads to the color change of the material. composite resin will become darker over time. one of the explanations of this phenomenon is because the surface staining caused by food coloring.20 in clinical studies, a change in color was observed in anterior composite restorations over a 3-year period.15 in the previous study, the immersion durations of 48 hours, 72 hours, and 96 hours represented soft drink consumption for 2 years, 3 years, and 4 years respectively. the results of this study showed th�t the δ��� of the composite specimens were higher �s the immersion duration were increased. color differences of esthetic restorations presenting a higher color change than 1 are considered as acceptable up to color change of 3.3.8 in the research of this study, color change of hybrid composite resin for both 48 hours and 72 hours immersion were clinically acceptable. meanwhile, the color change after 96 hours immersion was clinically unacceptable. this means, soft drink consumptions for 4 years will results in clinically unacceptable color change of hybrid composite resin. however, the color change was already statistically detected from the 48 hours immersion. this means, the color stability of the hybrid composite resin will already be influenced after 2 years consumption of soft drink. according to the results of the research, it can be concluded that the immersion duration of composite resin in soft drink influenced the color stability (began after 48 hours immersion). references 1. kidd eam, smith bgm, pickard hm. pickard’s manual of operative dentistry. 6th ed. new york: oxford medical publications; 1990. p. 3. 2. nicholson jw. the chemistry of medical and dental materials. cambridge: rsc; 2002. p. 15–7, 148–58. 3. burke fjt, mchugh s, randall rc, meyers ia, pitt j, hall ac. direct restorative materials use in australia in 2002. aus dent j 2004; 49: 185–91. 4. schulze ka, marshall sj, gansky sa, marshall gw. color stability and hardness in dental composites after accelerated aging. dent mat 2003; 19:612–9. 5. kolbeck c, rosentritt m, lang r, handel g. discoloration of facing and restorative composites by uv-irradiation and staining food. dent mat 2006; 22:63–8. 6. mount gj, hume wr. preservation and restoration of tooth structure. sydney: mosby; 1998. p. 97–8, 196–7. 7. van noort r. introduction to dental materials. london: mosby; 2007. p. 99–126. 8. dietschi d, campanile g, holz j, meyer j. comparison of the color stability of ten new-generation composites: an in vitro study. dent mat 1994; 10:353–62. 9. lau d, krondl m, coleman p. psychological factors affecting food selection. in: galler jr, editor. human nutrition: a comprehensive treatise. new york: plenum press; 1984. p. 397–411. 10. chamblee ts. soft drink. in world book 2005. chicago: world book inc; 2005. p. 1. 11. stout m. water quality and carbonated soft drinks article from element h2o. 2006. available from http://www.elementh2o.com/ articles/ customlabelbottlearticles.aspx?pageid=397. accessed: may 19, 2007. 12. anonymous a. cola. wikipedia the free encyclopedia. 2006. available from: http://en.wikipedia.org/wiki/cola. accessed october 15, 2006. 13. anonymous b. food coloring. wikipedia the free encyclopedia. 2006. available from: http://en.wikipedia.org/wiki/food_coloring. accessed: october 15, 2006. 14. aacc. caramel. 2003. available from: http://www.aaccnet. org/cerealfoodsworld/pdfs/w03020501f.pdf#search=’caramel’. accessed october 15, 2006. 15. craig rg. restorative dental materials. 9th ed. st. louis: mosby-year book, inc; 1993. p. 264. 16. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby; 2006. p. 28–30, 189–212. 17. manappallil jj. basic dental materials. 2nd ed. new delhi: jaypee; 2003. p. 146–73. 18. el-hejazi aa. water sorption and solubility of hybrid and microfine resin-composite filling materials. saudi dent j 2001; 13:139–42. 19. satou n, khan am, matsumae i, satou j, shintani h. in vitro color change of composite-based resin. dent mater 1989; 5:384–7. 20. de souza fb, guimaraes rp, silva chv. a clinical evaluation ofa clinical evaluation of packable and microhybrid resin composite restorations: one-year report. quintessence int 2005; 36: 41–8. table 1. means and standard deviations of color changes of the composite resins after immersion in soft drinks �δ����� immersion durations (hours) means ± standard deviations 48 1.0607 ± 0.3098 72 1.8433 ± 0.3784 96 4.9793 ± 0.7526 table 2. statistical results of lsd tests from the color changes �δ����� of composite resins �fter immersion in soft drink group mean difference a (48 hours) b (72 hours) c (96 hours) a (48 hours) b (72 hours) 0.7827* c (96 hours) 3.9187* 3.1360* * the mean difference is significant at the 0.05 level discussions issn 1978 3728volume 45 number 4 december 2012 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm (oral and maxillofacial surgery – airlangga university); prof. dr. m. rubianto, drg., ms., sp.perio (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy's dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic university of hiroshima, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontica – airlangga university); prof. dr. latief mooduto, drg., m.s., sp.kg (conservative dentistry – airlangga university); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort (orthodontic – airlangga university); prof. dr. istiati soehardjo, drg., ms (oral biology – airlangga university); prof. dr. anita yuliati, drg., m.kes (dental material – airlangga university); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – airlangga university); prof. dr. diah savitri ernawati, drg., m.si (oral medicine – airlangga university); thalca i. agusni, drg., mhped., ph.d., sp.ort (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes (dental public health – airlangga university); dr. elly munadziroh, drg., ms (dental material – airlangga university); priyawan rachmadi, drg., ph.d (dental material – airlangga university); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes (dental radiology – airlangga university); bagus soebadi, drg., mhped (oral medicine – airlangga university); endang pudjirochani, drg., ms., sp.pros (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes (oral biology – airlangga university); susy kristiani, drg., m.kes (oral biology – airlangga university); ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms (orthodontic – airlangga university); devi rianti, drg., m.kes (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros (prosthodontic – airlangga university); an'nissa chusida, drg., m.kes (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg (conservative dentistry – airlangga university); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university); hendrik setiabudi, drg., m.kes (oral biology – airlangga university); otty ratna wahyuni, drg., m.kes (dental radiology – airlangga university); anis irmawati, drg., m.kes (oral biology – airlangga university); yuliati, drg., m.kes (oral biology – airlangga university); retno palupi, drg., m.kes (dental public health – airlangga university); eka augustina, drg., sp.perio (periodontica – airlangga university); febriastuti, drg., sp.kg (conservative dentistry – airlangga university); mega m. puteri, drg., sp.kga (pediatric dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 45 number 4 december 2012: prof. dr. regina titi christinawati, drg., m.sc (oral biology – universitas gadjah mada) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) dr. ida bagus narmada, drg., sp.ort(k) (orthodontics – universitas airlangga) dr. retno indrawati, drg., msi. (oral biology – universitas airlangga) dr. theresia indah budhy, drg., m.kes. (oral pathology & maxillofacial – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com; website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 205162250 -204225738 contents page printed by: airlangga university press. (147/11.13/aup-b5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 45 number 4 december 2012 1. the role of actinobacillus actinomycetemcomitans fimbrial adhesin on mmp-8 activity in aggressive periodontitis pathogenesis rini devijanti ridwan .................................................................................................................... 181–186 2. aesthetic treatment on anterior teeth crown fracture caused by dental trauma nanik zubaidah ............................................................................................................................... 187–191 3. molecular detection of interleukin-1a +4845gt gene in aggresive periodontitis patients chiquita prahasanti and harianto notopuro ................................................................................ 192–196 4. antitumor activity of intratumoral injection of pcdna3.1-p27kip1mt followed by in vivo electroporation in a malignant burkitt’s lymphoma cell xenograft supriatno and sartari entin yuletnawati ...................................................................................... 197–201 5. the effect of nickel as a nickel chromium restoration corrosion product on gingival fibroblast through analysis of bcl-2 fx ady soesetijo and mandojo rukmo ......................................................................................... 202–207 6. inhibition of dental plaque formation by toothpaste containing propolis nurin aisyiyah listyasari and oedijani-santoso .......................................................................... 208–211 7. inhibition effect of cashew stem bark extract (anacardium occidentale l.) on biofilm formation of streptococcus sanguinis rizni amaliah, sri larnani and ivan arie wahyudi .................................................................... 212–216 8. antibacterial efficacy of salvadora persica as a cleansing teeth towards streptococcus mutans and lactobacilli colonies erlina sih mahanani, mohd fadhli khamis, erry mochamad arief, siti nabilah mat rippin, and zainul ahmad rajion ............................................................................................................... 217–220 9. pomegranate juice (punica granatum) as an ideal mouthrinse for fixed orthodontic patients haryono utomo and kimberly clarissa oetomo ......................................................................... 221–227 10. the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast due to the induction of the combination of aloe vera and 2% xenograft concelous bovine utari kresnoadi ................................................................................................................................ 228–233 11. the ph changes of artificial saliva after interaction with oral micropathogen basri a. gani, cut soraya, sunnati, abdillah imron nasution, nurfal zikri and rina rahadianur ....................................................................................................................... 234–238 228 volume 45 number 4 december 2012 research report the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast due to the induction of the combination of aloe vera and 2% xenograft concelous bovine utari kresnoadi department of prosthodontics faculty of dentistry, universitas airlangga surabaya indonesia abstract background: to make a successfull denture prominent ridge is needed, preservation on tooth extraction socket is needed in order to prevent alveol bone resorption caused by revocation trauma. an innovative modification of the material empirically suspected to be able reduce inflammation caused by the revocation trauma is a combination of aloe vera and xenograft concelous bovine (xcb) and aloe vera is a biogenic stimulator and accelerating the growth of alveolar ridge bone after tooth extraction. purpose: the research was aimed to determine of the increasing alveol bone formation by inducing the combination of aloe vera and 2% xenograft concelous bovine. methods: to address the problems, the combination of aloe vera and xenograft concelous bovine was induced into the tooth extraction sockets of cavia cabayas which devided on 8 groups. groups control, filled with xcb, aloe vera and aloe vera and xcb combination, at 7 days and 30 days after extraction. afterwards, immunohistochemical examination was conducted to examine the expressions of fgf-2 and osteocalcin, as the product of the growth of osteoblasts. results: there were significantly increases expression of fgf-2 and osteocalcyn on group which filled with xcb, aloe vera and combined aloe vera and xcb. conclusion: it may be concluded that the induction of the combination of aloe vera and xenograft concelous bovine into the tooth sockets can enhance the growth expressions of fgf-2 and osteocalcin as the product of osteoblasts, thus, the growth of alveolar bone was increased. key words: fibroblast growth factor 2, osteocalcin, osteoblast, alveolar bone, combination of aloe vera and graft abstrak latar belakang: untuk keberhasilan pembuatan gigitiruan diperlukan ridge yang prominent, maka diperlukan suatu preservasi soket pencabutan gigi untuk mencegah terjadinya resopsi tulang alveolar akibat trauma pencabutan. suatu inovasi modifikasi bahan yang diduga secara empiris dapat mengurangi keradangan karena trauma pencabutan adalah berupa kombinasi aloe vera dan xenograft concelous bovine (xcb). aloe vera yang merupakan biogenik stimulator untuk merangsang dan mempercepat pertumbuhan tulang alveolar setelah pencabutan gigi. tujuan: untuk membuktikan bahwa induksi kombinasi aloe vera dan xenograft concelous bovine 2% pada soket pencabutan gigi, dapat meningkatkan pembentukan tulang alveolar. metode: pengisian kombinasi aloe vera dan xcb pada soket pencabutan gigi cavia cabaya yang dibagi dalam 8 kelompok. kelompok: kontrol, aloe vera, xcb, kombinasi aloe vera dan xcb, masing-masing dalam pemeriksaan 7 dan 30 hari. kemudian dilakukan pemeriksaan imunohistokimia ekspresi fgf-2 dan osteocalcyn sebagai produk pertumbuhan sel osteoblas. hasil: terdapat peningkatan ekspresi fgf-2 dan osteocalcyn pada kelompok yang diisi xcb, aloe vera dan kombinasi aloe vera + xcb dibanding dengan kelompok kontrol. kesimpulan: induksi kombinasi aloe vera dan xenograft concelous bovine 2% pada soket pencabutan gigi dapat, meningkatkan ekspresi fgf-2 dan osteocalcyn, sehingga dapat meningkatkan pertumbuhan tulang alveol. kata kunci: fibroblast growth factor 2, osteocalcin, osteoblas, tulang alveolar, kombinasi aloe vera dan graft correspondence: utari kresnoadi, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: ut.kres@yahoo.com 229kresnoadi: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblastthe increasing of fibroblast growth factor 2, osteocalcin, and osteoblast introduction the success of denture manufacture requires supporting anatomical condition of oral cavity, especially prominent ridge. to achieve the good condition of ridge, after tooth extraction, the ridge must be prevented from resorption first. some users of fixed denture and partially removable denture feel uncomfortable with the dentures due to sustained resorption of residual ridge after tooth extraction, and this situation may cause structural changes in jaw bone. level of residual ridge resorption in each person, however, is not the same, so we need to conduct the prevention of residual ridge resorption. 1 after tooth extraction, the alveolar ridge usually decreases about 50%, and this condition occurs in the period of 6-12 months. the decreasing even occurs several times due to residual ridge resorption that physiologically occurs after tooth extraction.2 residual ridge resorption is a biomechanical disease caused by multi-factors, such as anatomic, metabolic, and mechanical determinants.3 after tooth extraction, there will usually be inflammation as rapid reaction in the area of extraction, and the sockets of the extraction will be filled with blood clot. later, the epithelial tissue will be proliferated and migrated in one week, and the tissue will fix the sockets of the extraction quickly. the use of bovine graft to improve either damaged bone or augmentation is actually often conducted in the field of general surgery and oral surgery. however, the use of bovine graft still can not give satisfactory results. therefore, we need an innovative material, biogenic stimulator, to stimulate graft that can accelerate bone growth. aloe vera has been considered as biogenic stimulator and hormonal activator in healing wound. aloe vera extract can be used to prevent scar tissue during incision, and when aloe vera gel is used after surgery, the incision will heal quickly.4 significant changes of the amount of collagen of the control and treatment groups indicate how aloe vera can stimulate fibroblast cell growth and wound healing process.5 thus, this research was aimed to prove whether the induction of the combination of aloe vera and 2% xenograft concelous bovine (xcb) into tooth extraction sockets can increase the expression of fgf2 and osteocalcin, which then may cause the increasing of alveolar bone growth. materials and methods this research was an experimental research with randomized post-test control group design with the combination of aloe vera and 2% xcb. the combination of aloe vera and 2% xcb can indicate that each active substance contained is about 2%. xcb is the product of bank of tissue of dr. soetomo hospital surabaya. other materials used in this research consist of aloe vera extracts, sterile distilled water, xcb, absolute alcohol, 70% alcohol, anti-fgf2 monoclonal antibodies, anti osteokalcin monoclonal antibodies, and reagents. those materials were used for immunohistochemical examination, and reagents used for staining was hematoxilin-eosin (he). instruments used in this research, moreover, were a set of tools for both immunohistochemical examination and preparation manufacture, such as micropipette, tip (yellow, white, blue), light microscopy, object glass, and cover glass. animals used in this research, furthermore, were healthy and active male cavia cobaya (marmots) with 300 -350 grams of weight and in the age of 3 to 3.5 months. those animals were then divided into 8 groups. next, lower right incisor extraction was then conducted in all those groups. firstly, cavia cobaya were taken from their care center, and then anaesthetized with intra venous ketamine 0.2 cc/300g bm.6 the tooth extraction process was conduced by using special pliers (needle holder). after the tooth extraction, the sockets were filled with materials as much as 0.1 cc, and then stitched. the materials were loaded as follows: group i: the sockets of tooth extraction were filled with polyetilene glycol (peg) as the control group, and then examined after 7 days; group ii: the sockets of tooth extraction were filled with peg (as the control group), and then examined after 30 days; group iii: the sockets of tooth extraction were filled with xenograft concelous bovine (xcb) and peg, and then examined after 7 days; group iv: the sockets of tooth extraction were filled with xcb and peg, and then examined after 30 days; group v: the sockets of tooth extraction were filled with aloe vera and peg, and then examined after 7 days; group vi: the sockets of tooth extraction were filled with aloe vera and peg, and then examined after 30 days; group vii: the sockets of tooth extraction were filled with aloe vera, xcb, and peg, and then examined after 7 days; and group viii: the sockets of tooth extraction were filled with aloe vera, xcb, and peg, and then examined after 30 days. after 7 days and 30 days, after being decalcified with 2% nitric acid, the jaws of those tested animals were cut to make preparations of block paraffin. afterward, slides were made for staining with imunnohistochemistry and hematoxilin eosin (he). next, the calculation was conducted on fgf-2, osteocalcin, and immunohistochemical staining by using microscopic examination, and then the amount of osteoblasts and osteoclasts was calculated on he staining. the tested animals were taken care at biochemical laboratory of medical faculty, universitas airlangga, while the manufacture of tissue preparation was conducted at histology laboratory and pathology anatomi laboratory of dr. soetomo hospital. moreover, the manufacture of aloe vera extract was conducted at physical chemistry laboratory of pharmacy faculty, universitas airlangga, while the manufacture of aloe vera frezze dryed was conducted at biology laboratory of science and technology faculty, universitas airlangga. and, immunohistochemical examination was conducted at immunohistochemistry laboratory, lpt tropical disease center (tdc) of universitas airlangga, and at biomedical laboratory of 230 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 228–233 universitas brawijaya. the calculation results were then recorded and tabulated. afterwards, the result data were analyzed by using anova, and then multiple comparison of the data of those four groups was conducted. results the increasing of the expressions of fgf-2 and osteocalcin in the control group and in the treatment groups induced with xcb, aloe vera, and xcb and aloe vera on day 7 and day 30 can be seen in figure 1. based on figure 1, it can be indicated that the expressions of fgf2 and osteocalcin in those three treatment groups induced with xcb, aloe vera, and aloe vera + xcb were higher than those in the control group. however, the highest expressions of fgf-2 and osteocalcin were found in the group induced with aloe vera + xcb on day 30. the expression of fgf-2 in those four groups can be seen in figure 2. based on the figures 2, it is known that the expressions of fgf-2 in the treatment groups induced with xcb (2b), aloe vera (2c), and aloe vera and xcb (2d), increased more as pointed by black arrows than those in the control group (2a). meanwhile, the expressions of osteocalcin on day 30 of immunohistochemical examination can be seen in figures 3a, b, c, d. based on figures 3, it is known that the expressions of osteocalcin in the treatment groups induced with xcb (3b), aloe vera (3c), and aloe vera + xcb (3d) increased more as pointed by black arrows than those in the control group (3a). based on anova analysis, moreover, it is also known that there was significant difference of the expressions of fgf2, p <0.05, between the control group and the treatment groups induced with xcb, aloe vera, and aloe vera + xcb. similarly, there was also significant difference of the expressions of osteocalcin, p<0.05, between the control group and the treatment groups induced with xcb, aloe vera, and aloe vera + xcb. figure 1. the average expressions of fgf-2 and osteocalcin in the control group, in the treatment groups induced with xcb+peg, aloe vera + peg, and aloe vera + xcb + peg on day 7 and on day 30. figure 2. a) the expression of fgf-2 on day 30 in the control group; b) the expression of fgf-2 on day 30 in the group induced with xcb; c) the expression of fgf-2 on day 30 in the group induced with aloe vera; d) the expression of fgf-2 on day 30 in the group induced with aloe vera dan xcb. black arrows indicate the expressions of fgf-2. fgf-2 osteocalcyn a b c d 231kresnoadi: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblastthe increasing of fibroblast growth factor 2, osteocalcin, and osteoblast furthermore, based on tukey hsd analysis, there was significant difference of the expressions of fgf-2, p<0.05, between the control group and the groups induced with xcb, aloe vera, and aloe vera + xcb. there was also significant difference of the expression of osteocalcin between the control group and the treatment groups induced with xcb, aloe vera, and aloe vera + xcb. however, there was no significant difference between the group induced with aloe vera and the group induced with xcb. next, the expressions of fgf-2 and osteocalcin on day 7 and on day 30 were analyzed by using t-test. the results then showed that there was significant difference of the expressions of fgf-2 and osteocalcin, t<0.05, started from day 7 to day 30. finally, how the increasing of the expressions of fgf-2 and osteocalcin stimulated the growth of osteoblast can be seen in figure 4. based on figure 4, it is known that the growth of osteoblasts in the control group and in the treatment groups induced with xcb, aloe vera, and aloe vera + xcb increased from day 7 to day 30 (see red bar). and, the highest growth of osteoblasts occurred in the group induced with aloe vera+ xcb on day 30. discussion based on figure 1, it is known that that the expressions of fgf2 and osteocalcin in the control group and in the treatment groups induced with xcb, aloe vera, and aloe vera + xcb increased started from day 7 to day 30 of the examination. the highest fgf-2 expression and osteocalcin were found in the group induced with aloe vera + xcb started from day 7 to day 30 of the examination. it indicates that aloe vera + xenograft concelous bovine can stimulate the growth of alveolar bone in the socket of tooth extraction. healing process with graft induction can prevent alveolar bone from resorption prior to the formation of new alveolar bone.7 thus, graft material used must be biocompatible, so it can inhibit bone resorption and stimulate osteogenesis. figure 3. a) the expression of osteocalcin on day 30 in the control group; b) the expression of osteocalcin on day 30 in the group induced with xcb; c) the expression of osteocalcin on day 30 in the group induced with aloe vera; d) the expression of osteocalcin on day 30 in the group induced with aloe vera and xcb. black arrows indicates the expression of osteocalcin figure 4. the average and standard deviation of osteoblast cells in the control group and the treatment groups induced with xcb, aloe vera, and aloe vera + xcb after he on day 7 and on day 30. a b c d co ntr ol 7 co ntr ol 30 osteoblast osteoblast 232 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 228–233 this opinion is also supported by some experts stating that graft induced into tooth extraction sockets can cause osteoinduction, stimulating bone growth.8-11 the increasing of osteocalcin expression can also be caused by osteocalcin mostly produced by osteoblasts. there are actually collagen and non-collagen proteins in osteoblasts, which usually synthesize non-collagen protein in bone matrix, namely osteocalcin and osteonektin as much as 40–50% in bone.12 fibroblast growth factor 2, furthermore, is an important modulator for both the growth of bone as well as the differentiation, expression and regulation of osteoblast.11 in addition, fgf-2 is also an important signaling molecule for the regulation of bone development stages, thus, if fgf-2 increases, the amount of osteoblasts will also increase.13,14 the expressions of fgf-2, osteocalcin, and osteoblast in the group induced with aloe vera were higher than those in the group induced with xcb. it indicates that aloe vera is an anti-inflammatory, antibacterial, anti-virus because it contains not only antthraquinones components, namely aloin, aloe emodin, and barbaloin, but also pure mannan carbohydrate, acetyl mannan glukomannan, as well as alkaline phosphatase and bradikinase enzymes. there are also healing hormones, auxin, gibberellin, and saponins, as well as proteins in aloe vera that can enhance tissue and bone healing process so that aloe vera can reduce inflammation caused by tooth extraction trauma, then can induce the healing process of the wound, and can finally stimulate osteoblast, as consequence, the growth of new bone will increase.4,15-22 this increasing process is also stimulated by aloeride as polysaccharides derived from aloe vera that has a high molecular weight and strong immunostimulator activity.23 thus, the group induced with aloe vera and xcb had the highest expressions of fgf2, osteocalcin, osteoblasts, and fibroblasts started from day 7 to day 30 of examination. it indicates that aloe vera and xcb in tooth extraction cases can accelerate wound healing process, can prevent inflammation, and then can induce new bone growth. it is because when aloe vera is combined with xcb which is osteoinduction, it will accelerate healing process and new bone growth.2,8,9,10 the increasing of osteoblast will not occur continuously, but at a certain time it will decrease. in a previous study, it is known that the growth of osteoblasts in tooth extraction socket filled with aloe vera and xcb with 0.5% concentration of the active substance increased on day 30, but on histologic examination it is known that the growth of osteoblasts decreased on day 60.24 on day 12 after the tooth extraction, the formation of new bone occurred along the socket wall as well as in trabecular spaces around the tooth extraction area, and trabeculae bone was also woven at the periphery socket, osteoprogenitor cells, preosteoblasts, and osteoblasts. periodontal ligament also moved into the middle of the socket, but not attached to the socket wall, and then got apoptosis. collagen density, as a result, increased, and then was gradually replaced by bone. after this phase of bone resorption, freezing, granulation, and collagenase phases of the healing process can be bypassed, and then the socket of tooth extraction may regenerate.7 finally, it may be concluded that the induction of the combination aloe vera and 2% xenograft concelous bovine into the socket of tooth extraction can increase the expressions of fgf2 and osteocalcin, so the growth of the alveolar bone can increase. references 1. nishimura i, damiani pj, atwood da. resorption of residual ridge (rrr) in rat. j dent rest 1987; 66(12): 1753–7. 2. pelegrine aa, sorgi da costa ce, pizzigati correa me, marques jr jfc. clinical and histomorphometric evaluation of extraction socket treted withan autologous bone marrow graft. clinical oral implant rest 2010; 21: 535–42. 3. gupta a, tiwari b, goel h, shekawat h. residual ridge resorption: a review. indian journal of dental science 2010; 2 issue: 207–11. 4. roostita & tim redaksi qanita. lidah buaya. cetakan i. bandung: penerbit qanita, pt. mizan pustaka; 2008. h. 19–38. 5. ariyani e, khoswanto c. the use 90 % aloe vera frezze dryiing as modulator of collagen density in exctraction socket of incisivus cavia cabaya. dent j (maj. ked. gigi) 2008; 41(2): 74–6. 6. kusumawati d. bersahabat dengan hewan coba, cetakan pertama. jogjakarta: gajah mada university press; 2004. p. 67. 7. steiner gg, francis w, burrell r, kallet mp, steiner dm, macias r. the healing socket and socket regeneration. j compendium 2008; 29(2): 1–11. 8. khan, sn, cammisa fp, sandhu, hs, diwan ad, girardi fp, lane jm. the biology of bone grafting. j am acad orthop surg 2005; 13(1): 77–86. 9. de boix pw, gauthier o, guicheux, bouler sm, pilet p, daculsi g, grimandi g. injectable bone substitute to prever alveolar ridge resorption after toothextraction, a study in dog. j mater sci mater med 2006; 17(11): 1145–52. 10. fickl s, zuhr o, wachtel h, kebschull m, hurzeler mb. hard tissue alteration after socket preservation with additional buccal overbuilding: a study in beagle dog. j clin periodontol. 2009; 36(10): 898–904. 11. montero a, okada �, tomita m, ito m, tsurukamu h, nakamura t, doetschman t, coffin jd, hurley mm. disruption of the fibroblast growth factor-2 gene result in decreased bone mass and bone formation. j clin invest 2000; 105(8): 1085–93. 12. k hudhany ls. penentuan indeks densitas tulang mandibula perempuan pasca menopause dengan memperhatikan beberapa fa k tor r isi ko ter jad i nya osteoporosis. diser t at ion ja ka r t a: pascasarjana universitas indonesia; 2003. p. 34, 52. 13. schmid gj, kobayashi c, sandell lj, ornitz dm. fibroblast growth factor expressing during skeletal fracture healing in mice. dev dyn 2009; 238(3): 766–74. 14. su n, du x, chen l. fgf signaling: its role in bone development and human skeleton, front biosci 2008; 13: 2842–65. 15. vasquez b, avila g, segura d, escalante b. antiimflammatory activity of extract from aloe vera gel. j ethnopharmacol 1996; 55(1): 69–75. 16. kurniawati r, marminah mt. efek anti inflamasi gel lidah buaya (aloe vera linn) terhadap tikus putih. solo: prosiding seminar nasional tumbuhan obat indonesia; 2006. xxix: 82–9. 17. chun-shu �u, fu-shun �u, jack kai-sheng chan, te-mao li, song-shei lin, ssu-ching chen, te-chun hsia, �ung-hsien chang, jing-gung chung. aloe-emodin affect the level of cytokines and functions of leukocytes from sprague-dawley rat. in vivo 2006; 20(4): 505–9. 18. hamman jh. composition and application of aloe vera leaf gel. molecules 2008; 3(8): 1599–616. 233kresnoadi: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast: the increasing of fibroblast growth factor 2, osteocalcin, and osteoblastthe increasing of fibroblast growth factor 2, osteocalcin, and osteoblast 19. park m�, kwon hj, sung mk. evaluation of aloin and aloe-emodin as anti inflammatory agents in aloe by using murine macrophages. biosci biotechnol biochem 2009; 73(4): 828–32. 20. lawrence r, tripathi p, jeyakumar e. isolation, purification and evaluation of antibacterial agents from aloe vera. braz j microbiol; 40(4): 906–15. 21. moghadasi sm, verma sk. aloe vera their chemicals composition and applications: a review. int j biol med res 2011; 2(1): 466–71. 22. pachanon c. a study the element in aloe vera powder by neutron activation analysis. thesis. faculty of graduates studies, mahidol university; 2005; p. 11. 23. pugh nd, tamta h, balachandram p, xiangmei wu, howell jl, dayan fe, pasco ds. the majority of in vitro macrophage activation exhibited by extract of some immune enhancing botanicals is due to bacterial lipoproteins and lipopoly saccharides. int immunopharmacol. 2008; 8(7): 1023–32. 24. kresnoadi u, rahayu rp. stimulation of osteoblast activity by induction aloe vera and xenograft combination. dent j (maj. ked. gigi) 2011; 44(4): 200–4. 165 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 165–173 review article the effect of herbal medicine in reducing the severity of oral lichen planus: a systematic review and meta-analysis kharissa kemala vychaktami1, rahmi amtha2, indrayadi gunardi2, rosnah binti zain3 1dental student, faculty of dentistry, universitas trisakti, jakarta, indonesia 2department of oral medicine, faculty of dentistry, universitas trisakti, jakarta, indonesia 3faculty of dentistry, mahsa university, bandar saujana putera, jenjarom, selangor, malaysia abstract background: oral lichen planus (olp) is a chronic autoimmune mucocutaneous disease of unknown aetiology. the reported use of herbal medicines may promote the healing of olp lesions. purpose: we aim to determine the effectiveness of herbal medicine to reduce the clinical and pain severity of olp. methods: pubmed, cochrane library and wiley online library were reviewed according to the inclusion criteria. risk of bias was performed for the randomised control trial (rct) and cohort studies to assess the effectiveness of herbal medicines for olp treatment. outcomes were recorded based on pain severity and the quality of life of patients with olp. the mean difference and effect size of studies were pooled. reviews: out of 1,034 papers, six publications were selected and reviewed. the most common types of olp lesions were erosive and atrophic and were mainly found at the buccal site. olp was common in the range of 27–74 years, especially in females. the herbal medicines used in the publication were curcumin, lycopene, purslane, aloe vera and quercetin. improvement in quality of life or olp severity was recorded in the intervention group treated with purslane, curcumin and lycopene (p<0.05) but not in the control group. the total effect of herbal medicine in reducing pain severity (measured with the visual analogue scale [vas]) in olp patients was not significant (mean difference 0.13; 95% ci -0.202 to 0.463; p=0.442). conclusions: herbal medicine cannot be used as a single regime to reduce pain severity. further research is recommended to evaluate cohort design studies to observe the prolonged effect of herbal medicine in olp lesions. prospero registration number: crd42021262282 (2021) keywords: oral lichen planus; herbal medicine; meta-analysis correspondence: indrayadi gunardi, department of oral medicine, faculty of dentistry, universitas trisakti, jl. kyai tapa no. 260, jakarta, 11440, indonesia. email: indrayadi@trisakti.ac.id introduction oral lichen planus (olp) is a chronic mucocutaneous autoimmune disease with unknown aetiology. predisposing factors for this disease could be genetics, infection, stress, malnutrition, endocrine gland disorders and a poor immune system.1 the prevalence of olp in indonesia is more than 10%, and it is mainly found in female patients over 40 years of age. 1,2 other lesions that resemble olp clinically and/ or histologically have been termed oral lichenoid lesions (olls) and oral lichenoid reactions (olrs). 3,4 in oral mucosa, olp is commonly found on the buccal mucosa, but the tongue and gingiva may also be involved.5 clinically, olp is categorised into two types: non-erosive (reticular, papular and plaque) and erosive (erosion, atrophic and bullous).6 patients with asymptomatic olp (non-erosive type) usually do not require any treatment.7 in symptomatic patients (erosive), topical treatments, such as corticosteroids, calcineurin inhibitors, cyclosporine, retinoids and rapamycin, may relieve pain.7 other olp treatments, such as phototherapy, laser therapy, photodynamic therapy and ultraviolet therapy, have also been reported.8 corticosteroids are the main treatment for olp, but due to their local (burning sensation and irritation) and systemic (immunosuppression) side effects, various studies have been carried out to find alternative treatments, such as herbal medicines,9 which have been widely used because they are safe, easy to find and low cost.10 thus far, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 mailto:indrayadi@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 166vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 many studies have shown that herbal medicines may reduce the severity of olp; for example, liu wei di huang,9 tripterygium glycosides,9 zeng sheng ping,9 liquorice,11 purslane,12 lycopene,13 raspberry,14 propolis,15 green tea,16 ignatia amara,17 aloe vera,18 quercetin19 and curcuma longa.20 different types of herbs have had varied results; for example, curcumin may reduce the inflammatory cytokine response caused by the activity of t lymphocytes in olp. as olp treatment originally used corticosteroids, resulting in patients experiencing many side effects, many countries sought alternative approaches using herbal medicine. until now, there have been limited systematic reviews on the usefulness of these herbs for olp treatment, although a meta-analysis was conducted on pain severity between intervention and control groups. this study aims to conduct a systematic review and meta-analysis on the epidemiology and effectiveness of herbal medicine for treating olp. this review may contribute to the benefits of applying herbal medicine to reduce clinical symptoms in olp patients. methods this meta-analysis was conducted using the preferred reporting items for systematic reviews and metaanalyses (prisma) guidelines, and it was registered in the prospero database (crd42021262282). selection criteria a patient/population, intervention, comparison, outcomes and time (pico(t)) strategy was used to define the following eligibility criteria: (p) patients with olp and/or olr/oll with no histological dysplastic changes; (i) herbal medicine (aloe vera, curcumin, liquorice, green tea, purslane, liu wei di huang, zeng sheng ping, tripterygium glycosides, lycopene, raspberries, propolis, quercetin and ignatia amara); (c) population who received the placebo; (o) epidemiological data of clinical olp (severity, quality of life and side effects); (t) all literature published up until july 2021. inclusion and exclusion criteria the inclusion criteria were patients older than 18 (as paediatric olp is very rare) who had been diagnosed clinically and histologically as olp/oll/olr with or without skin lesions, english literature with an rct study design and a cohort from three database sources (pubmed, cochrane literature and wiley online literature) and olp patients who experienced dysplastic changes to squamous cell carcinoma and irrelevant conditions/lesions such as olp with a systemic condition. search strategies each keyword was determined and the following boolean words were applied: (“aloe vera” or (curcumin or “curcuma longa” or curcuminoids) or (liquorice or “glycyrrhiza glabra” or glycyrrhizin) or (“green tea” or “camellia sinensis” or epicatechin or epigallocatechin or “epicatechin 3 gallate” or “epigallocatechin 3 gallate”) or (purslane or “portulaca oleracea”) or (“liuwei dihuang” or “liu wei di huang” or “six flavor rehmanni” or “cornus officinalis” or “rehmannia glutinosa” or “rhizoma dioscoreae” or “cortex moutan radicis” or “poria cocos” or “alisma plantago aquatica”) or (“zeng sheng ping” or zsp or “sophora tonkinensis” or “polygonum bistorta” or “prunella vulgaris” or “sonchus brachyotus” or “dictamnus dasycarpus” or “dioscorea bulbifera”) or (“tripterygium glycosida” or “tripterygium wilfordii”) or lycopene or (raspberry or “rubus idaeus”) or propolis or (quercetin or “ginkgo biloba” or “hypericum perforatum” or “sambucus canadensis”) or (ignatia or “ignatia amara” or “strychnos ignatii” or strychnine) or “natural ingredients”) and (“oral lichenoid reaction” or “olp” or olr or “oral lichen planus” or oll or “oral lichenoid lesion” or “oral lichenoid contact lesion” or olcl or “oral lichenoid drug reactions” or oldr) and prevention. study selection after eliminating duplicate studies, the remaining studies were screened based on their titles and abstracts. then, the studies that were not available in full-text form were excluded. the studies with full texts were evaluated to comply with the inclusion criteria for this review. one investigator (kkv) worked independently to screen the studies using boolean words and retrieved reports using microsoft excel office 2019. the result was reviewed by all investigators before conducting the risk-of-bias assessment. risk of bias assessment three reviewers (kkv, ig, ra) independently assessed the risk of bias in the final six studies. the joanna briggs institute (jbi) critical appraisal tools for systematic reviews were used to assess the cohort study, and the cochrane risk-of-bias tool was used to assess studies with an rct design. data extraction three investigators (kkv, ig, ra) extracted data from six studies according to subjects, type of olp, the severity of olp, quality of life, types of herbal medicine and side effects. data analysis the outcome variables, such as pain severity taken from the vas score, were collected and calculated. the mean differences (mds) were calculated for continuous data. as there was low heterogeneity between the included studies, the fixed-effects model was used to pool the data. the heterogeneity levels of the eligible rcts were assessed using i2 statistics. subgroup analysis was not performed dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 167 vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 because only four studies were included. the study’s md and effect size were pooled using openmetaanalyst for windows 10 64-bit (cebm® brown university). sensitivity analysis could not be performed as the number of studies included for meta-analysis was low. results study selection one independent reviewer selected the studies. the pubmed database provided 234 studies, seven studies were found in cochrane literature and 796 studies were from wiley online literature. after removing the duplicates based on their titles, 1,034 studies were obtained. the abstract screening was carried out, and 925 studies were excluded because the abstracts were unavailable and did not discuss olp, oll or olr. only 91 studies remained out of 109 after full texts were screened. a total of 85 studies were excluded for irrelevant topics, incomplete data (epidemiological data of clinical olp severity and quality of life) and different study designs. finally, six studies met the inclusion criteria and were included for the review (figure 1). meanwhile, only four studies provided mean vas scores between the groups, so these studies were used for meta-analysis. risk-of-bias assessment quality assessment was carried out on the final six studies, five of which were rcts (salazar-sánchez et al.;18 amirchaghmaghi et al.;19 amirchaghmaghi et al.;20 kia et al.;21 agha-hosseini et al.22), and only one study was a cohort design (prasad kushwaha et al.23). studies conducted by salazar-sánchez et al.,18 amirchaghmaghi et al.,19 amirchaghmaghi et al.,20 kia et al.21 and prasad kushwaha et al.23 had a low risk of bias (figure 2 and table 1). in contrast, the study by agha-hosseini et al.22 had a high risk of bias due to unclear allocation concealment and selective reporting, in addition to other sources of bias, including blinding of participants and personnel, blinding outcome assessment and incomplete outcome data (figure 2). this study was included despite its high risk of bias because of the completeness of data. none of the studies described selective reporting except for amirchaghmaghi et al.19 data extraction this research included 212 patients aged 27–74. olp was found predominantly in females with various types of olp (erosive, atrophic and reticular). the research results from the six selected reports were based on the severity of olp and quality of life. the severity of olp in three reports was measured using the thongprasom scale and records identified through pubmed (n = 234) records after duplicates removed (n = 1034) records excluded (n = 925) abstract not available (n = 14) irrelevant topic (n = 911) records screened (n = 109) no full-text availability (n = 18) full-text articles assessed for eligibility (n = 91) full-text articles excluded, with reasons (n = 85) irrelevant topic (n = 54) incomplete data (n = 18) different study design (n = 13) systematic reviews included in the overview (n = 6) id en tif ic at io n sc re en in g in cl ud ed records identified through cochrane (n = 7) records identified through wiley (n = 796) e lig ib ili ty systematic reviews analysed for meta (n = 4) figure 1. prisma flow diagram. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 168vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 table 1. risk-of-bias assessment for cohort study (y=yes; n=no) author design score based on appropriate jbi appraisal* overall appraisal 1 2 3 4 5 6 7 8 9 10 prasad kushwaha et al. (2019) cohort y y y n y n y n y y included table 2. studies of herbal medicine for olp treatment (m=male; f=female; iv=intervention group; c=control group; na=not available) studies study design subjects olp type / location olp severity quality of life natural agent result aghahosseini et al. (2010) rct purslane (iv) = 20 placebo (c) = 17 16 m, 21 f age (mean) = 47.4 ± 10.8 erosive, atrophic, reticular tool: thongprasom scale iv group 4 deg worse = 0 3 deg worse = 0 no change n = 17% 1 deg improvement = 29% 2 deg improvement = 29% 3 deg improvement = 13% 4 deg improvement = 12% c group 4 deg worse = 5% 3 deg worse = 5% no change n = 73% 1 deg improvement = 17% 2 deg improvement = 0 3 deg improvement n = 0 4 deg improvement n = 0 tool: vas iv group 1 deg worse = 0 no change n = 0 1 deg improvement = 43% 2 deg improvement = 43% 3 deg improvement = 14% c group 1 deg worse n = 14% no change n = 15% 1 deg improvement n = 71% 2 deg improvement n = 0 3 deg improvement n = 0 purslane capsule dosage: 235 mg capsules for 6 months (1x1) significant differences in olp severity and pain score between the purslane group and control group (p<0.001) r an do m s eq ue nc e ge ne ra tio n a llo ca tio n co nc ea lm en t se le ct iv e re po rt in g o th er s ou rc es o f b ia s b lin di ng o f p ar tic ip an ts a nd p er so nn el b lin di ng o f o ut co m e as se ss m en t in co m pl et e ou tc om e da ta amirchaghmaghi et al., 2015 amirchaghmaghi et al., 2016 salazar-sánchez et al., 2010 agha-hosseini et al., 2010 + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? _ _ _ _ _ _ __ kia et al., 2020 figure 2. risk-of-bias assessment for rct studies. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 169 vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 salazarsánchez et al. (2010) rct aloe vera (iv) = 31 (3 m, 28 f) placebo (c) = 24 (1 m, 23 f) age (mean) iv = 62.19 ± 10.45 c = 60.71 ± 12.23 erosive, atrophic buccal (n = 52), tongue (n = 38), lip (n = 7), gingival (n = 33), palate (n = 5) na tool: vas, ohip-49, had iv group had-d baseline = 6.32 ± 5.77 3 months = 6.19 ± 5.90 had-a baseline = 8.90 ± 4.54 3 months = 7.84 ± 5.10 ohip-49 baseline = 40.26 ± 24.60 3 months = 20.35 ± 17.61 vas baseline = 5.5 ± 2 12 weeks = 2.5 ± 3.0 c group had-d baseline = 5.83 ± 3.38 3 months = 6.08 ± 3.43 had-a baseline = 10.08 ± 4.03 3 months = 9.42 ± 3.52 ohip-49 baseline = 40.75 ± 19.88 3 months = 29.50 ± 20.82 vas baseline = 5.8 ± 1.8 12 weeks = 3.7 ± 3.3 aloe barbadensis gel dosage: 60ml for 12 weeks (0.4 ml keeping it within the oral cavity for 1 minute) natural ingredients: aloe vera there was no significant difference in pain, depression, anxiety or ohip score between the aloe vera group and the control group; except on ohip49 domain psychological disability (p = 0.007) and total score ohip-49 (p = 0.046) amirchaghmaghi et al. (2015) rct quercetin (iv) = 15 placebo (c) = 15 8 m, 22 f age (mean) iv = 48.26 ± 16.28 c = 44.6 ± 10.22 erosive, atrophic tool: individual severity index baseline iv = 9.40 ± 3.16 c = 9.63 ± 3.83 1 week iv = 5.93 ± 3.15 c = 4.63 ± 2.6 2 weeks iv = 4.73 ± 3.23 c = 3.70 ± 2.35 3 weeks iv = 3.70 ± 3.30 c = 2.50 ± 2.45 4 weeks iv = 3.23 ± 3.47 c = 1.33 ± 1.87 8 weeks iv = 2.23 ± 2.93 c = 1.10 ± 2.35 tool: vas baseline iv = 1.92 ± 0.86 c = 1.80 ± 0.77 1 week iv = 1.07 ± 0.95 c = 0.8 ± 0.86 2 weeks iv = 0.53 ± 0.66 c = 0.86 ± 0.91 3 weeks iv = 0.33 ± 0.48 c = 0.66 ± 0.89 4 weeks iv = 0.23 ± 0.43 c = 0.46 ± 0.83 8 weeks iv = 0.46 ± 0.51 c = 0.53 ± 0.91 quercetin hydrate dosage: 250 mg capsules 2 times a day there was no significant difference in clinical and pain severity between systemically administered quercetin and the placebo (p>0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 170vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 studies year intervention control weight (%) estimate 95% c.i. n mean sd n mean sd salazar-sánchez et al 2010 31 2.5 3 24 3.7 3.3 3.874 -1.2 (-2.891, 0.491) amirchaghmaghi et al 2015 15 1.92 0.86 15 1.8 0.77 32.448 0.12 (-0.464, 0.704) amirchaghmaghi et al 2016 12 0.33 0.65 8 0.13 0.35 57.055 0.2 (-0.241, 0.641) kia et al 2020 29 2.69 2.89 28 2.33 2.03 6.624 0.36 (-0.933, 1.653) overall p = 0.442 100 0.13 (-0.202, 0.463) heterogeneity chi-square = 2.597, df = 3, p = 0.458, i2 = 0%, fixed standardised mean difference intervention control -2 -1 0 1 figure 3. forest plot of visual analogue scale for pain between intervention group and control group. amirchaghmaghi et al. (2016) rct curcumin (iv) = 12 placebo (c) = 8 7 m, 13 f age (mean) iv = 49.42 ± 11.22 c = 52.75 ± 9.43 erosive, atrophic buccal (n = 18), gingival (n = 5), tongue (n = 10), lips (n = 2) tool: thongprasom scale baseline iv = 3.17 ± 1.03 c = 3 ± 1.30 after 4 weeks iv = 1.08 ± 0.66 c = 1.5 ± 1.06 tool: vas baseline iv = 6.5 ± 2.15 c = 4.63 ± 3.20 after 4 weeks iv = 0.33 ± 0.65 c = 0.13 ± 0.35 curcumin (tablets) dosage: 500 mg 2x1 for 4 weeks there was no significant difference in clinical and pain severity between the curcumin group and the control group (p = 0.77) prasad kushwaha et al. (2019) cohort iv = 13 7 m, 6 f age iv = 27–74 years atrophic = 6 erosive = 4 reticular= 3 buccal (n = 25), gingival (n = 6), tongue (n = 3), lips (n = 2), hard palate (n = 1) tool: thongprasom scale baseline = 2.77 ± 1.74 2 weeks = 2.69±1.65 4 weeks = 2.62±1.66 6 weeks = 1.54±1.19 8 weeks = 0.85±0.37 na lycopene (lycored 2mg capsules) dosage: lycopene capsules 4mg/day for 8 weeks lycopene significantly reduced olp severity based on the thongprasom index (p = 0.005) adverse effects nausea mild abdominal pain/cramps increased appetite diarrhoea headaches dizziness dry mouth flatulence kia et al. (2020) rct curcumin (iv) = 29 (4 m, 25 f) prednisolone (c) = 28 (5 m, 23 f) age (mean) iv = 51.86 ± 9.94 c = 52.67 ±8.9 na tool: thongprasom scale baseline iv = 3.83 ± 1.17 c = 3.83 ± 1.18 4 weeks iv = 2.34 ± 1.14 c = 1.83 ± 0.92 tool: vas baseline iv = 4.65 ± 3.39 c = 4.89 ± 3.34 1 week iv = 4.38 ± 3.03 c = 4.67 ± 3.45 2 weeks iv = 3.41 ± 2.74 c = 3.28 ± 2.74 4 weeks iv = 2.69 ± 2.89 c = 2.33 ± 2.03 curcumin (nanocurcumin) micellar soft gel capsule dosage: 80 mg once daily after breakfast curcumin can be an alternative drug for olp lesions compared to the control group. in the curcumin group, pain decreased significantly at 2 weeks. (p<0.001) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 171 vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 the vas (amirchagmaghi et al.;20 kia et al.;21 aghahosseini et al.22). the study by salazar-sánchez et al.18 measured quality of life using the vas, the oral health impact profile (ohip-49) and hospital anxiety-depression (had) instruments; however, the study did not measure the severity of olp. another report by amirchagmaghi et al.19 measured the severity of olp using the individual severity index and measured quality of life using the vas. prasad kushwaha et al.23 measured the severity of olp using the thongprasom scale and did not measure the quality of life (table 2). meta-analysis figure 3 depicts the total effect of the vas in the form of a forest plot, based on the findings of four published studies (salazar-sánchez et al.;18 amirchagmaghi et al.;19 amirchagmaghi et al.;20 kia et al.21). the forest plot reveals that the heterogeneity of studies was low (chisquared=2.597, p=0.458, i2=0%); therefore, a fixed-effect model was utilized for the analysis. the overall pooled effect of the md vas score between the intervention and control groups was not significantly different (p=0.442, 95% ci -0.202 to -0.463). the study by prasad kushwaha et al.23 could not be compared because it only had one group, and the researchers did not provide quality-of-life data. agha-hosseini et al.22 could not be compared because the data were presented as a proportion. discussion this systematic review of five rcts and one cohort study on herbal medicines for olp treatment found that the general risk of bias was low in all studies, but the resulting meta-analysis performed well. based on the results of the fixed-effects analysis of four publications,18–21 herbal medicines may be less effective in reducing the severity of olp pain, although the results varied between the types of herbal medicine used for treatment. in the study by salazar-sánchez et al.,18 lycopene significantly reduced the severity of olp pain, but there were some adverse effects. additionally, agha-hosseini et al.22 stated that purslane had been used as an alternative medicine for treating olp patients without any side effects. in this systematic review, olp was more likely to occur in females (n=161) than males (n=51), as it may be influenced by hormonal cycles, especially during pregnancy, menstruation and menopause. in the perimenopause or menopause phase, the decrease in oestrogen levels can cause physical and emotional symptoms such as depression, irritability, insomnia and fatigue.24 oestrogen is a hormone that plays a role in controlling the menstrual cycle, and it can modulate t cells, including cd4+ (th1, th2, th17 and tregs) and cd8+, which play an important role in the pathogenesis of olp.25 based on six studies, olp was found to occur from 27–74 years of age. this lesion may be related to decreased immunological reactivity, impaired dna repair and the atrophy of oral tissues, especially oral epithelium and the salivary glands. furthermore, at an advanced age, other factors could include systemic diseases, nutritional disorders, the side effects of drugs and the use of ill-fitting dentures.26 the most studied types of olp (salazar-sánchez et al.;18 amirchagmaghi et al.;19 amirchagmaghi et al.;20 agha-hosseini et al.;22 kushwaha et al.23) are erosive and atrophic types because patients often complain about pain, and there have been reports on its potential to become malignant.27 olp was most commonly found in the buccal mucosa area, which could be because this area is most susceptible to trauma.28 until now, the aetiology of olp remains unknown, but several factors can trigger its occurrence, including autoimmune disorders, stress, trauma, malnutrition, systemic diseases, and endocrine and salivary gland disorders.2 in some cases, genetic and viral infections could also trigger the development of olp.29 however, none of the six selected studies discussed the predisposing factors of olp. curcumin could decrease the inflammatory response by suppressing the activity of cox-2, lipoxygenase, inducible nitric oxide synthase enzymes (inos) as well as inhibiting the production of cytokines that cause inflammation, such as il-1, il-2, il-6, il-8 and il-12. therefore, curcumin was found to effectively reduce the signs and symptoms of olp such as a burning sensation and the occurrence of erythema and ulceration. however, in its topical use, there were some complaints, such as xerostomia, itching, burning, mild inflammation and the yellowish colour of the gingiva and the surrounding area.30 aloe vera is known to inhibit the inflammatory process by interfering with the activity of the arachidonic acid pathway through cox and reducing leukocyte adhesion and tumour necrosis factor (tnf) levels.31 due to its properties, aloe vera was found to be effective in reducing the burning sensation and decreasing the healing duration, but the side effects of its topical use may cause allergic reactions.31,32 quercetin has anti-inflammatory properties that inhibit cytokines such as il-12, ifn-γ, ifn-α, il-8, cox-2 and prostaglandin e. at the same time, its antioxidant content could inhibit free radicals and nitric oxide. therefore, quercetin was found to reduce pain in olp patients.33 purslane contains melatonin, which is known as an antioxidant agent, and omega-3 fatty acids, which are rich in fatty acids and have anti-cancer and anti-inflammatory effects that have been shown to reduce il-6 levels. because of its ingredients, the use of purslane leads to clinical improvements in olp patients.9 lycopene is known to have antioxidants, and immunomodulatory and free radical scavenging properties.13 it has been shown to reduce the burning sensation in olp patients and may be used to treat atrophic and erosive types of olp.34 based on the six journal reports, curcumin is the most studied herbal medicine to reduce the severity of olp. curcumin is widely used as the main ingredient in cooking, food colouring and traditional medicine. this natural ingredient originates from india and is widely cultivated in southern china, taiwan, japan, burma and indonesia.35 to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 172vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 reduce the severity of olp, herbal medicines have several ingredients that contain anti-inflammatory, antioxidant and anti-cancer effects.36 thomas et al.33 found that a topical 1% curcuminoid gel reduced signs and symptoms of olp, although it was not as effective as 0.1% triamcinolone acetonide. chainani-wu et al.37 found that 5% curcumin paste reduced the severity of olp lesions. no serious effects of herbal medicine were reported, except for lycopene. curcumin, aloe vera, purslane, quercetin and lycopene were effective in reducing the clinical severity of olp. the results of the study by prasad kushwaha et al.,23 found that lycopene can significantly reduce the severity of olp based on the thongprasom index (p=0.005), but it had some side effects such as nausea, cramps, diarrhoea, headaches, etc. at the same time, agha-hosseini et al.22 stated that purslane could be used as alternative medicine or a supplement for treatment in olp patients without side effects. based on the meta-analysis, a low heterogeneity (p=0.458, i2=0%) was calculated in four studies with an overall fixed-effect analysis, and herbal medicine had a more negligible effect in reducing pain severity (vas) against olp (md=0.13, p=0.442, 95% ci -0.202 to -0.463). it has been demonstrated that these natural ingredients reduce the inflammatory symptoms of olp. since olp is not only a disease with a chronic inflammatory background, but also an autoimmune disorder, the anti-inflammatory and antioxidant properties of the five natural ingredients are insufficient and have little effect on reducing the severity of olp. the limitations and proposed recommendations of this study are: 1) the trend of herbal medicine research reports on a variety of herbs, which may lead to difficulty in comparing the same herbs that have the potential of reducing the severity of olp, as each study uses different tools to measure lesion severity and the quality of life of olp patients; 2) most of the studies that examined herbal medicine were conducted over a short period; 3) some studies found improvements in olp only among baseline data in the intervention group but did not make comparisons with the control group due to the study design. it was sometimes concluded that herbal medicine was effective in treating olp. in conclusion, herbal medicine cannot be used as a single regime but might be used as a supplement or additional medicine to reduce the symptoms and severity of olp lesions. further research is recommended to evaluate larger cohort design studies to observe the prolonged use of herbal medicine in treating olp lesions. acknowledgement authors contributions kkv, ig and ra were responsible for conceptualisation, writing the original draft, and reviewing and editing the manuscript. kkv and ig were responsible for data acquisition and investigation. kkv was also the administrator for projects, resources and the visualisation of charts and tables. ig and ra were responsible for the methodology, supervision and validation of collected data. rbz was responsible for critically editing the project and manuscript. funding this research did not receive any specific grant from funding agencies in the public, commercial or not-forprofit sectors. competing interests no competing interests in this study. references 1. nosratzehi t. oral lichen planus: an overview of potential risk factors, biomarkers and treatments. asian pac j cancer prev. 2018; 19(5): 1161–7. 2. gonzález-moles má, warnakulasuriya s, gonzález-ruiz i, gonzález-ruiz l, ayén á, lenouvel d, ruiz-ávila i, ramos-garcía p. worldwide prevalence of oral lichen planus: a systematic review and meta-analysis. oral dis. 2021; 27(4): 813–28. 3. fortuna g, aria m, schiavo jh. drug-induced oral lichenoid reactions: a real clinical entity? a systematic review. eur j clin pharmacol. 2017; 73(12): 1523–37. 4. dudhia bb, dudhia sb, patel ps, jani y v. oral lichen planus to oral lichenoid lesions: evolution or revolution. j oral maxillofac pathol. 2015; 19(3): 364–70. 5. madalli v, basavaraddi sm. lichen planus –a review. iosr j dent med sci. 2013; 12(1): 61–9. 6. alrashdan ms, cirillo n, mccullough m. oral lichen planus: a literature review and update. arch dermatol res. 2016; 308(8): 539–51. 7. olson ma, rogers rs, bruce aj. oral lichen planus. clin dermatol. 2016; 34(4): 495–504. 8. yang h, wu y, ma h, jiang l, zeng x, dan h, zhou y, chen q. possible alternative therapies for oral lichen planus cases refractory to steroid therapies. oral surg oral med oral pathol oral radiol. 2016; 121(5): 496–509. 9. ghahremanlo a, boroumand n, ghazvini k, hashemy si. herbal medicine in oral lichen planus. phytother res. 2019; 33(2): 288– 93. 10. kalaskar ar, bhowate rr, kalaskar rr, walde sr, ramteke rd, banode pp. efficacy of herbal interventions in oral lichen planus: a systematic review. contemp clin dent. 2020; 11(4): 311–9. 11. sharma v, katiyar a, agrawal rc. glycyrrhiza glabra: chemistry and pharmacological activity. in: mérillon jm, ramawat k, editors. sweeteners reference series in phytochemistry. springer, cham; 2018. p. 87–100. 12. thanya k, lakshmi t. ethnobotanical approach for oral lichen planus a review. int j drug dev res. 2013; 5(4): 54–7. 13. pratibha, shekhawat ks, deepak ta, srivastava c. assessment of lycopene and levamisole in management of oral lichen planus a comparative study. j oral med oral surgery, oral pathol oral radiol. 2016; 2(1): 4–10. 14. vickers er, woodcock k l. raspber r y lea f herba l ext ract significantly reduces pain and inflammation in oral lichen planus patients – a case series analysis. open j dent oral med. 2015; 3(3): 73–81. 15. vagish kumar ls. propolis in dentistry and oral cancer management. n am j med sci. 2014; 6(6): 250–9. 16. zhang j, zhou g. green tea consumption: an alternative approach to managing oral lichen planus. inf lamm res. 2012; 61(6): 535–9. 17. thongprasom k, prapinjumrune c, carrozzo m. novel therapies for oral lichen planus. j oral pathol med. 2013; 42(10): 721–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 173 vychaktami et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 165–173 18. salazar-sánchez n, lópez-jornet p, camacho-alonso f, sánchezsiles m. efficacy of topical aloe vera in patients with oral lichen planus: a randomized double-blind study. j oral pathol med. 2010; 39(10): 735–40. 19. amirchaghmaghi m, delavarian z, iranshahi m, shakeri mt, mosannen mozafari p, mohammadpour ah, farazi f, iranshahy m. a randomized placebo-controlled double blind clinical trial of quercetin for treatment of oral lichen planus. j dent res dent clin dent prospects. 2015; 9(1): 23–8. 20. amirchaghmaghi m, pakfetrat a, delavarian z, ghalavani h, ghazi a. evaluation of the efficacy of curcumin in the treatment of oral lichen planus: a randomized controlled trial. j clin diagn res. 2016; 10(5): zc134-7. 21. kia sj, basirat m, mortezaie t, moosavi m-s. comparison of oral nano-curcumin with oral prednisolone on oral lichen planus: a randomized double-blinded clinical trial. bmc complement med ther. 2020; 20(1): 328. 22. agha-hosseini f, borhan-mojabi k, monsef-esfahani h-r, mirzaiidizgah i, etemad-moghadam s, karagah a. efficacy of purslane in the treatment of oral lichen planus. phytother res. 2010; 24(2): 240–4. 23. prasad kushwaha r, prasad rauniar g, rimal j. clinical assessment of the effects of lycopene in the management of oral lichen planus. kuga mc, editor. int dent med j adv res vol 2015. 2019; 5(1): 1–5. 24. dalal pk, agarwal m. postmenopausal syndrome. indian j psychiatry. 2015; 57(suppl 2): s222-32. 25. mohan rps, gupta a, kamarthi n, malik s, goel s, gupta s. incidence of oral lichen planus in perimenopausal women: a crosssectional study in western uttar pradesh population. j midlife health. 2017; 8(2): 70–4. 26. bozdemir e, yilmaz hh, orhan h. oral mucosal lesions and risk factors in elderly dental patients. j dent res dent clin dent prospects. 2019; 13(1): 24–30. 27. ramos-garcía p, gonzález-moles má, warnakulasuriya s. oral cancer development in lichen planus and related conditions-3.0 evidence level: a systematic review of systematic reviews. oral dis. 2021; 27(8): 1919–35. 28. panta p, andhavarapu a, sarode sc, sarode g, patil s. reverse koebnerization in a linear oral lichenoid lesion: a case report. clin pract. 2019; 9(2): 1144. 29. nogueira pa, carneiro s, ramos-e-silva m. oral lichen planus: an update on its pathogenesis. int j dermatol. 2015; 54(9): 1005–10. 30. kia sj, shirazian s, mansourian a, khodadadi fard l, ashnagar s. comparative efficacy of topical curcumin and triamcinolone for oral lichen planus: a randomized, controlled clinical trial. j dent (tehran). 2015; 12(11): 789–96. 31. reddy rl, reddy rs, ramesh t, singh tr, swapna la, laxmi nv. randomized trial of aloe vera gel vs triamcinolone acetonide ointment in the treatment of oral lichen planus. quintessence int. 2012; 43(9): 793–800. 32. rajeswari r, umadevi m, rahale cs, pushpa r, selvavenkadesh s, kumar kps, bhowmik d. aloe vera: the miracle plant its medicinal and traditional uses in india. j pharmacogn phytochem. 2012; 1(4): 118–24. 33. thomas ae, varma b, kurup s, jose r, chandy ml, kumar sp, aravind ms, ramadas aa. evaluation of efficacy of 1% curcuminoids as local application in management of oral lichen planus interventional study. j clin diagn res. 2017; 11(4): zc89– 93. 34. gupta s, jawanda mk. oral lichen planus: an update on etiology, pathogenesis, clinical presentation, diagnosis and management. indian j dermatol. 2015; 60(3): 222–9. 35. nisar t, iqbal m, raza a, safdar m, iftikhar f, waheed m. turmeric: a promising spice for phytochemical and antimicrobial activities. j agric environ sci. 2015; 15(7): 1278–88. 36. singh v, pal m, gupta s, tiwari sk, malkunje l, das s. turmeric a new treatment option for lichen planus: a pilot study. natl j maxillofac surg. 2013; 4(2): 198–201. 37. chainani-wu n, madden e, lozada-nur f, silverman s. high-dose curcuminoids are efficacious in the reduction in symptoms and signs of oral lichen planus. j am acad dermatol. 2012; 66(5): 752–60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p165–173 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p165-173 208 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 208–212 case report the early detection of tongue cancer with the etiology of mechanical trauma aris setyawan, zhavira dwiyanti anang, rizki nurida rahmawati, winda kumalasari, alfi dian uly noor, rima talitha yulianti department of dentistry, faculty of medicine, diponegoro university, semarang, indonesia abstract background: oral squamous cell carcinoma (oscc) occurs in approximately 95% of people older than 40 years; usually, it is diagnosed at the age of 60 years. the etiology of oscc is multifactorial. chronic mechanical trauma due to sharp teeth has also been suggested as an etiology of oral squamous of the tongue. purpose: this case report aimed to describe the etiology of oscc resulting from mechanical trauma and to prevent further metastasis by correctly diagnosing the lesion earlier. case: a 49-year-old female patient presented with painful and swollen stomatitis two months ago. no submandibular lymph nodes were palpable. an indurated ulceration and sharp teeth on the right region of the jaw were obtained from the intraoral examination. malignancy was concluded from histopathology examination (hpe) and magnetic resonance imaging (mri) radiography results. case management: multiple extractions were chosen to eliminate the etiology of mechanical trauma due to sharp teeth, and the patient underwent hemiglossectomy with the keyhole method. postoperative, the histopathology examination revealed an oscc of the tongue. the patient is currently in the second cycle of chemotherapy by hematologist-oncologists. conclusion: the early appropriate diagnosis of a tongue ulcer can prevent metastasis, decrease morbidity, and increase quality of life. keywords: carcinoma; chronic trauma; oral cancer; sharp teeth; tongue article history: received 30 november 2022; revised 15 february 2023; accepted 2 march 2023; published 1 september 2023 correspondence: aris setyawan, department of dentistry, faculty of medicine, diponegoro university, jl. prof soedarto, semarang, 50275, indonesia. email: arisomfsundip@gmail.com introduction ninety-five percent of oral squamous cell carcinoma (oscc) cases occurs in people who are older than 40 years; it is usually diagnosed at the age of 60 years.1 oscc is more common in men than women; this is due to the high risk of tobacco and alcohol consumption. the etiology of oscc is multifactorial, including tobacco, alcohol, and nutritional factors. chronic mechanical trauma due to sharp teeth has also been suggested as a possible etiology of tongue-oscc.2 treatment of oscc is determined by the metastatic and degree of tissue dysplasia. the first-choice treatment for tongue oscc is surgery that is followed by radiotherapy or radiochemotherapy.3 eliminating local factors, such as sharp teeth, is especially important because this condition is an etiological factor of oscc development. the patient’s general condition and age become important during the management of oscc, especially in a patient with underlying diseases. the early diagnosis of this condition and proper multidisciplinary management of oscc must be made to improve the prognosis and the patient’s quality of life.4 this case report presented the management of oscc in a young patient with mechanical trauma due to sharp teeth. case a 49-year-old female patient was presented to the dental department of mardi rahayu hospital in kudus, central java, with slightly painful and swollen stomatitis on the right side of the tongue on april 14, 2022. intraoral examination showed a major white ulceration more than 10 mm in diameter, with a diffuse and irregular margin, indurated and slightly raised in the dextral lateral tongue. the cusp of teeth (44) was observed sharp and in close contact with the ulcer (figure 1). with an extraoral examination, no submandibular lymph nodes were palpable. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p208–212 mailto:arisomfsundip@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p208-212 209setyawan et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 208–212 figure 1. the intraoral examination showed an indurated ulceration in the lateral border of the tongue (a); tooth 44 close to the ulcer and in contact during tongue movement while eating and talking (b). the patient had no history of smoking, drinking alcohol, or hypertension, but had under-controlled diabetes mellitus that required routinely taking oral medication. there is no history of family-related cancer. case management due to the indurated ulceration, the lesion was suspected to be malignant. an mri was performed on the patient. the mri showed a mass in the anterior-mid-posterior lateral of the tongue [tr: 26.2 mm x ap: 19.3 mm x cc: 22.1 mm] (figure 2a) and classified as t1-2 n0 m0 (figure 2b and 2c). malignancy was concluded from mri radiography results, but no definitive diagnosis was decided yet. multiple extractions were performed to eliminate the etiology of mechanical trauma due to sharp teeth, and the patient underwent a hemiglossectomy with the keyhole method under general anesthesia. the excision was made 2 cm from the affected site, and the malignant mass and figure 2. mri radiographs showed a malignancy mass in the lateral border of the tongue (red arrow) (a), and the mass expansion that classifies as t1-2 n0 m0 (yellow arrow) (b-c). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p208–212 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p208-212 210 setyawan et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 208–212 healthy surrounding tissue were removed (figure 3a-b). the defect was repaired and sutured (figure 3c). after performing the surgery, the excised tissue was taken for histopathology examination. analysis showed the tissue was covered with keratinized stratified squamous epithelium, partially with mild to moderate pleomorphic nuclei, hyperchromatic, coarse chromatin, mitoses present, cytoplasm eosinophilic, with partially clear intercellular bridging and keratin pearl infiltration between groups of moderate striated muscle cells lymphocytes, and lymph angioinvasion. the description above supports the diagnosis of well-differentiated squamous cell carcinoma accompanied by lymph angioinvasion. after a postoperative histopathology examination, the patient was referred to a hematologist-oncologist for the first cycle of chemotherapy, which consisted of six visits. the patient is currently undergoing the second cycle. a long-term clinical evaluation is needed to prevent the recurrence. discussion the appearance of oscc is usually linear to aging because it is related to the length of the exposure and causes genetic changes, including chemical or physical irritants, viruses, or hormonal changes. of oral cancers, tongue cancer commonly occurs in 20to 44-year-olds, while cancers of other anatomical features like lips, gingiva, palate, and dorsal tongue appear less commonly. recent data showed that 9% of younger patients, under 40 years old and nonsmokers, suffered from oral cancer.5 the reason behind this is due to the impaired immune system,5 but further explanation needs to be explored.6 in the present case, a 49-year-old female patient presented an oscc due to mechanical trauma caused by sharp teeth. in the literature, it is mentioned that tongue cancer can be seen in young patients, under 45 years old, mainly in females who abstain from tobacco and alcohol.7 mechanical trauma, like sharp teeth and other etiologies such as fractured fillings and illfitting dentures, can induce the development of oscc.8,9 the pathogenesis is debatable, but the characteristic of the trauma must be low intensity and persistent.10 the lateral of the tongue is the highest area close to trauma, because, during the physiological function of a normal swallowing pattern, this area is trapped between dental arches approximately three times per minute.10 this trauma can promote epithelial cell transformation,11 alone or in association with other risk factors, and has also been reported to be implicated in the development of oscc.12 the persistent trauma can induce the disruption of the normal physical architecture of the extracellular matrix, promoting oncogene expression, increasing the hyperproliferative status, and creating an inflammatory microenvironment.12 however, the exact pathogenesis is questionable and the traumatic event alone is unable to develop an oscc13 without the presence of genetic alteration such as loss of heterozygosity.12 although the mouth is visually accessible for examination, the diagnosis of oscc is frequently delayed because it may be difficult to distinguish clinically from other diseases. over 50% of oscc patients have metastases both local or distant during the diagnosis.14 this patient figure 3. the partial hemiglossectomy. pre-operation (a), during operation (b), and post-operation (c). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p208–212 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p208-212 211setyawan et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 208–212 was identified by a general practitioner who suspected a malignant lesion on the lateral side of the tongue because of the clinical appearance, from the induration around the ulceration, raised border and asymptomatic.15,16 the general practitioner directly referred the patient to an oral and maxillofacial surgeon to receive the correct diagnosis and treatment. no regional lymph nodes were palpable. oscc usually involves the submandibular, submaxillary, submental, digastric, and upper cervical nodes, and has metastatic lung involvement.17 but in this case, thorax radiography or chest x-ray (cxr) was performed on the patient and showed no metastatic involvement to the lungs or any lymphatic spread. the need for rapid diagnosis of oscc is critical because early diagnosis can reduce mortality, and general practitioners have a better chance of detecting this disease at the early stage.18 the world health organization (who) proposed that general dental practitioners could perform oral cancer screenings based on inspection and palpation as part of an essential oral examination. oral cancers may or may not cause pain, whereas a common symptom of ulceration is pain that usually resolves within 7–14 days. the classical features of an oral cancer lesion a persistent ulceration with hardening and peripheral infiltration and may be associated with red or whitish staining. in the present case, persistent ulceration with peripheral induration was noticed. the ulceration was located on the lateral border of the tongue.19 the predominant location is the lateral border of the tongue or oral floor because this area is often in continuous contact with teeth.10 after enlarging, oscc may become a raised nodule and develop into ulceration with induration resulting from fibrosis, infiltration, and inflamed tissue. in several months, oscc may form into an indurated ulcer with a rolled border.20 mri radiograph results showed a malignant mass, but the definitive diagnosis was not concluded yet. treatment of oscc remains mainly surgical, depending on the anatomic site, with adjuvant radiotherapy added for advancedstage disease.21 the patient, in this case, underwent hemiglossectomy surgery on the right side of the tongue with the keyhole method followed by chemotherapy; it should be the optimal surgery for the early cancer stage. the selection of appropriate treatment modalities depends on tumor factors such as size (t-stage), location and multiplicity, proximity to the bone, pathological features, histology grade, and depth of invasion.22 h e m i g l o s s e c t o m y o r g l o s s e c t o m y t y p e i i i b (compartmental) is the primary treatment option for oral cancer patients with one-sided tongue involvement.3,23,24 the specimen includes intrinsic and extrinsic muscles ipsilateral to the lesion, resected up to healthy tissue with appropriate safety margins (at least 1.5 cm), and the lingual artery must be ligated and removed en bloc with the lingual and hypoglossal nerves. the ipsilateral base and the tip of the tongue are preserved.23 the surgery was performed under general anesthesia. the hole left by the excision of the cancer was small and repaired by suturing the tongue immediately. pain and discomfort after the procedure were managed with medication; this patient was given antibiotics, anti-inflammatory, and analgesic drugs. a long-term clinical evaluation is needed to determine oscc recurrence. the prognosis of oscc in the oral cavity depends on several factors, including size, location, histopathology, metastasis, and the patient’s age.25 lymph node involvement and tumor size are the most important prognostic factors. this case had no lymph node involvement or further metastasis to the lungs, so the stage was: t1-2 n0 m0, which means the oscc was expected to have a better prognosis. generally, oral cancer has a poor prognosis; when carcinoma has metastasized to the lymphatic gland, the survival rate will decrease. in this case, the general practitioners were able to diagnose quickly. the patient received appropriate treatment by direct referral to an oral maxillofacial specialist and continued by chemotherapy so that metastasis could be prevented, and poor diagnosis could be avoided. in conclusion, oscc is usually diagnosed at an advanced stage, which has a poor prognosis. mechanical trauma, like sharp teeth, could also be the main etiology of developing oscc. early diagnosis of oscc is helpful to increase the survival rate of the patient. references 1. glick m, greenberg ms, lockhart pb, challacombe sj. introduction to oral medicine and oral diagnosis. in: burket’s oral medicine. 13th ed. usa: wiley; 2021. p. 1–18. 2. odell ew. cawson’s essentials of oral pathology and oral medicine. 9th ed. elsevier; 2017. p. 317–22. 3. mannelli g, arcuri f, agostini t, innocenti m, raffaini m, spinelli g. classification of tongue cancer resection and treatment algorithm. j surg oncol. 2018; 117(5): 1092–9. 4. riskayanti np, riyanto d, winias s. manajemen multidisiplin oral squamous cell carcinoma (oscc): laporan kasus. intisari sains medis. 2021; 12(2): 621–6. 5. valero c, yuan a, zanoni dk, lei e, dogan s, shah jp, morris lgt, wong rj, mizrachi a, patel sg, ganly i. young non-smokers with oral cancer: what are we missing and why? oral oncol. 2022; 127: 105803. 6. yu c, zhou z. relationship between young non-smokers and oral cancer: what can we learn? oral oncol. 2022; 133: 106064. 7. farquhar dr, tanner am, masood mm, patel sr, hackman tg, olshan af, mazul al, zevallos jp. oral tongue carcinoma among young patients: an analysis of risk factors and survival. oral oncol. 2018; 84: 7–11. 8. gilligan g, piemonte e, lazos j, simancas mc, panico r, warnakulasuriya s. oral squamous cell carcinoma arising from chronic traumatic ulcers. clin oral investig. 2022; 27(1): 193–201. 9. ra h ma n k h, su rboyo m dc, rad it h ia d, pa r mad iat i a e , wihandono a, ernawati ds. oral squamous cell carcinoma with essential thrombocythemia and positive jak2 (v617f) mutation. j taibah univ med sci. 2022; 17(2): 326–31. 10. l a z o s j p, p iem o nt e e d, l a n f r a n ch i h e , br u no t t o m n. characterization of chronic mechanical irritation in oral cancer. int j dent. 2017; 2017: 6784526. 11. shetty sr, al-bayati saaf, hamed ms, abdemagyd hae, elsayed ws. carcinoma of tongue in a 40-year-old male: a case report. albanian med j. 2017; 3: 59–64. 12. pentenero m, azzi l, lodi g, manfredi m, varoni e. chronic mechanical trauma/irritation and oral carcinoma: a systematic copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p208–212 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p208-212 212 setyawan et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 208–212 review showing low evidence to support an association. oral dis. 2022; 28(8): 2110–8. 13. piemonte ed, lazos jp, gilligan gm, panico rl. association between chronic mechanical irritation and oral cancer needs more original research. oral dis. 2022; 28(8): 2304–6. 14. xu t, wang dc, shan xf, cai zg. [delayed diagnosis of oral squamous cell neoplasms at different sites]. beijing da xue xue bao. 2019; 51(4): 748–52. 15. kremer j. tongue cancer in children on the rise. j oral maxillofac surg. 2021; 79(6): 1385–6. 16. hudyono r, bramantoro t, benyamin b, dwiandhono i, soesilowati p, hudyono ap, irmalia wr, nor nam. during and post covid-19 pandemic: prevention of cross infection at dental practices in country with tropical climate. dent j (majalah kedokt gigi). 2020; 53(2): 81–7. 17. mendenhall wm, foote rl, sandow pl, fernandes rp. oral cavity. in: gunderson ll, tepper je, editors. clinical radiation oncology. 4th ed. elsevier; 2016. p. 570-596.e3. 18. wimardhani ys, warnakulasuriya s, wardhany ii, syahzaman s, agustina y, maharani da. knowledge and practice regarding oral cancer: a study among dentists in jakarta, indonesia. int dent j. 2021; 71(4): 309–15. 19. morikawa t, shibahara t, nomura t, katakura a, takano m. non-invasive early detection of oral cancers using fluorescence visualization with optical instruments. cancers (basel). 2020; 12(10): 2771. 20. paderno a, morello r, piazza c. tongue carcinoma in young adults: a review of the literature. acta otorhinolaryngol ital. 2018; 38(3): 175–80. 21. riemenschnitter ce, morand gb, schouten cs, rupp nj, balermpas p, gander t, broglie däppen ma. need for adjuvant radiotherapy in oral cancer: depth of invasion rather than tumor diameter. eur arch oto-rhino-laryngology. 2023; 280(1): 339–46. 22. arrangoiz r, cordera f, caba d, moreno e, luque de leon e, munoz m. oral tongue cancer: literature review and current management. cancer reports rev. 2018; 2(3): 1–9. 23. ansarin m, bruschini r, navach v, giugliano g, calabrese l, chiesa f, medina je, kowalski lp, shah jp. classification of glossectomies: proposal for tongue cancer resections. head neck. 2019; 41(3): 821–7. 24. de berardinis r, tagliabue m, belloni p, gandini s, scaglione d, maffini f, margherini s, riccio s, giugliano g, bruschini r, chu f, ansarin m. tongue cancer treatment and oncological outcomes: the role of glossectomy classification. surg oncol. 2022; 42: 101751. 25. tagliabue m, belloni p, de berardinis r, gandini s, chu f, zorzi s, fumagalli c, santoro l, chiocca s, ansarin m. a systematic review and meta‐analysis of the prognostic role of age in oral tongue cancer. cancer med. 2021; 10(8): 2566–78. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p208–212 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p208-212 189 vol. 42. no. 4 october–december 2009 the ability of igy to recognize surface proteins of streptococcus mutans basri a. gani1, santi chismirina1, zinatul hayati2, endang winiati b3, boy m. bachtiar3, and i. wayan t. wibawan4 1 department of oral biology, faculty of dentistry, syiah kuala university, banda aceh 2 department of microbiology, faculty of dentistry, syiah kuala university, banda aceh 3 department of oral biology, faculty of dentistry, indonesia university, jakarta 4 department of bacteriology and immunology, vertirinary faculty, pertanian bogor institute, bogor abstract background: streptococcus mutans are gram positive bacteria classified into viridians group, and have a role in pathogenesis of dental caries. it’s adhesion to the tooth surface is mediated by cell surface proteins, which interact with specific receptor located in tooth pellicle. glucan binding protein, glukosyltransferase, and antigen i/ii are basic proteins of s. mutans, which have a role in initiating the interaction. a previous study showed that chicken’s igy can interfere the interaction. purpose: the objective of this study was to assess the ability of igy in recognizing the surface molecule of streptococcus mutans expressed by various serotypes (c, d, e, f) and a strain derived from ipb, bogor. method: western blot was used as a method to determine such capability. result: the result showed that igy has a potency to recognize antigen i/ii, but not the other proteins on the cell surface of all bacteria tested. conclusion: the ability of igy to bind the surface protein, antigen i/ii, indicates that this avian antibody could be used as a candidate for anti-adhesion in preventing dental caries. key words: igy, streptococcus mutans, adhesion, surface proteins, and dental caries correspondence: basri a. gani, c/o: prodi kedokteran gigi, fakultas kedokteran, universitas syiah kuala darussalam banda aceh 23111. e-mail: basriunoe@yahoo.com research report introduction streptococcus mutans (s. mutans) are bacteria that have an important role in the pathogenesis of dental caries. among bacterium species in oral cavity, s. mutans are known as one of bacteria that have recently been discussed because of its ability in forming extracellular polysaccaride and plaque. these bacteria were isolated from dental plaque for the first time by clarke in 1924.1 these bacteria are classified into the monera kingdom, firmicutes division, bacilli class, lactobacillus ordo, streptococcaceae family, streptococcus genus, and streptococcus mutant species.2 in 1890, miller formulated chemo-parasitic theory of dental caries, known as hypothesis of non-specific plaque describing a process from decalcification process of enamel to the forming of dental caries as the cumulative effect of acid produced by bacteria of dental plaque.3 molecularly, the surface proteins of s. mutans that are commonly involved in the process of dental caries are glucan binding protein (gbp) and antigen i/ii (ag i/ii).4,5 besides that, s. mutans also express molecules as enzyme in the process of carbohydrate fermentation that has a role in the activity of s. mutans, such as glucosyltransferase (gtf), dextranase (dex), and fruktosiltranferase (ftf). each of those three enzymes then breaks sucrose in order to form glukan, dextran, and fruktan. actually, there are other proteins that have a role in supplying energy reverse of s. mutans in order to be able in oral cavity, such as dextranase a (dexa), dextranase b (dexb), fruktanase, and dlt1-4 (protein intracellular cells of s. mutans).6,7 furthermore, there are two approaches for preventing caries, which are host aspects like diet control, clean control of oral cavity, fluorides, and agent aspects obtained from both active and passive immunizations. the use of mammalian immunoglobulin g (igg) in active and passive immunizations can prevent the adhesion of s. mutans on dental pellicle by recognizing cell surface proteins of s. mutans, especially gbp, gtf, and agi/ii, so the antibody 190 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 189-193 can be used for preventing dental caries.8 besides mammalian antibody like igg, immunoglobulin y (igy) can also be used as immune-prophylaxis matter for preventing dental caries through its use as complement materials of tooth paste used for decreasing the percentage of s. mutans in human saliva.9 tests for the igg in passive immunization actually has been done, but the producing process of igg has many technique limitations, is not economics, and can hurt animal that produce it during blood taking process. based on the phenomenon, passive immunization with antibody (igy) specifically located in egg yolk was used to substitute the role of igg in preventing the interaction of adhesive molecule of s. mutans on dental surface.10,11 chicken’s egg is the source of igy that potentially can be used as immunotherapy materials since it is more resistant to temperature (60–65° c) and ph alteration, and does not cause cross-reaction with structural component of tissue and protein in mammalians compared with igg. these characters indicate that igy can be applied to diagnose and prevent disease.12 based on some aspects in dentistry, igy anti s. mutans actually can be used to decrease the frequency of caries in the experiments on caries.13 for instance, based on the result of the experiment in which rats are used as testing animal, it was known that igy has a potency in preventing the activity of gtf and gbpb, so it can present colonization of s. mutans in dental pellicle, but the serotype is not known.14,15 besides that, the reference about the ability of igy relating with antigen i/ii of s. mutans with many serotypes is still not known since protein has an important role in the adhesion of s. mutans to dental pellicle.3 therefore, this research was aimed to analyze the potency of igy anti s. mutans in recognizing cell surface proteins of s. mutans with serotype c, d, e, and f. the significance of this experiment is to inform both the potency of igy located in egg yolk of chicken as anti-s. mutans, especially for the researchers and pharmacy companies, and the possibility of igy as passive immunization materials. material and method this research was an experimental laboratory conducted in molecular laboratory in faculty of dentistry-ui and faculty of veterinary-ipb. specific igy anti s. mutans produced by faculty of veterinary, pertanian bogor institute (fkh-ipb) was used as detector of bacteria from 4 serotypes, s. mutans serotype c (mutans xc), s. mutans serotype d (sobrinus omz176), s. mutans serotype e (mutans lm7), and s. mutans serotype f (mutans omz175). those four serotypes of s. mutan were laboratory strain, obtained from professor yamashita, department of preventive dentistry, faculty of dentistry, kyushu university, japan. s. mutans strain derived from ipb was used as positive control. in order to obtain antigen, each serotype of s. mutans was cultured in the selective solid media of trypticase soy with sucrose and bacitracin (tys20b) and incubated for 12–72 hours at the temperature of 37° c. next, one colony was taken usingh oase after being cultivated in the water media of trypticase soy broth (tsb), and incubated for 24–72 hours at the temperature of 37° c in microaerophilic setting. afterwards, whole cell s. mutans were centrifuged at 3000 rpm for 10 minutes. sediment obtained was added with 200 ml lisozim, and incubated at ice temperature for about 5 minutes before centrifuged again at 3000 rpm for 5 minutes. next, the sediment, as s. mutans antigen, was re-suspended in 500 ml phosphate buffer saline (pbs) and added with 12.5% sodium dodecyl sulphate (sds) for about 65 ml. before the profile of cell proteins of s. mutans with serotype c, d, e, f, and a strain derived from ipb with western blot method was detected, the level of protein was determined by using bradford (bio-rad) method in order to make the number of cell proteins of s. mutans that was analyzed have the same level of protein for each. s. mutans cells that have already been prepared with lisozim then were put into elisa plate wells for about 160 ml (10 ml sample + 150 ml pbs). next, other same elisa plate wells were given with bovine serum antibody (bsa) as standard proteins about 160 ml (10 ml bsa + 150 ml pbs). either sample or bsa was added with 40 ml protein assay (bradford), and then re-suspended by using multi-chanel pipet and incubated at the room temperature for 1 hour. the concentration of proteins was measured by using elisa reader based on optical density with 655 nm wave length. western blot technique was used in order to detect the reactivity of igy. based on the method conducted by yera et al.,16 the method will be as the following; sds page. the prepared collector gel was put into sds tank that has already been filled with reservoir running buffer stock (biorad). next, separator gel was put into 20 ml sample and 5 ml standard protein (invitrogen) in different wells as the mass indicator of the sample protein molecule. then, protein was separated through 2 phases. first, the sample and marker proteins in the wells were collected near separator gel by setting up the electricity vertically at 100 ma, 100 volt, and 16 watt for 30 minutes. second, the proteins in separator gel were separated by setting up the electricity at 100 ma, 150 voltage, 25 watt, for 80 minutes. the next step was electrotransfer of antigen protein. first, gel containing antigen protein was put onto blotting nitrocellulose paper, and then was immersed in buffer transfer blot for about 2 minutes inside glass container. at the same time, transferring cassette that has already been halved was prepared. second, each of them was put in foam pad (sponge), and then filter papers of mini trans-blot were put on them. third, gel was put on one of them with blotting nitrocellulose on it. afterward, the surface part was leveled up in order not to cause any bubble. then, the transferring cassette was girded again and put into electrophoresis tank. finally, electro-transfer was used with 100 voltage of electricity for 1 hour. 191gani, et al.: the ability of igy to recognize surface proteins detection of antigen protein with igywas done. first, blotting nitrocellulose (bio-rad) paper containing antigen was incubated in 20 ml 5% milk on sheker for about 1 hour at the temperature of 30° c. next, it was washed 3 times with pbs (sigma) for about 5 minutes. igy in 10 ml 5% non-fat milk with the concentration 1:2000 was poured into the surface of blotting papers that have already been washed and incubated on shaker for about 1 hour at 30° c. then, they were washed 4 times with pbs for 5 minutes. in order to visualize the interaction between igy and targeted antigen protein, those blotting papers must be added with anti igy antibody labeling peroxides (horse rabbit peroxide anti-chicken/turkey igg) with 1:2000 concentration that have already diluted with 10 ml 5% milk. next, they were incubated on shaker for 1 hour at the temperature of 30° c. after that, those blotting papers were washed 4 times with pbs for 5 minutes. to visualize the binding between igy and targeted antigen, finally, developing reagent was used. result in this research, the series of working procedures for determining the profile and reactivity of igy towards cell proteins of s. mutans with serotype c, d, e, and f, were conducted by using western blot technique. streptococus mutans that have already been extrated with lisozim was analyzed by sds page method and colored with commasie blue in order to analyze the profile of s. mutans proteins visualised in figure 1. paralelly, blotting of igy anti s. mutans was conducted by using western blot technique in order to analyze cell proteins of s. mutans binding with igy (figure 2). discussion chicken’s egg yolk is considered as the food component which also contain specific antibody towards antigen inducing the respond of the chicken’s immune system. the production of igy from chicken’s egg actually can give more advantages than the production of imunoglobulin from other animals, like mammals.14 igy, moreover, can be used to detect some viral brands through elisa test, immunodiffusion, and immunofluorescence since the score of its isoelectrics is lower than human’s igg. 17 igy can also be used to detect immunoglobulin in animal serum.14 the technology for producing igy as an alternative antibody substituting antibody produced by mammalian animals, moreover, is considered as one of reasons for using it in immunotherapy and immunopropilaxis.18 in dentistry, igy can actually be used as antibody of anti s. mutans.15,19 thus, igy, as anti adhesion, is expected to have potency to recognize the surface proteins causing the dental caries. in this case, the surface proteins of s. mutans commonly reported as the initiator of the adhesion of bacteria to dental pellicle are gbp, gtf, and ag i/ii.20 the choosing of whole cell s. mutans as the vaccine materials used for producing chicken’s antibody was aimed to expose many kinds of surface epitope used for producing many kinds of paratop with many binding sites in hypervariabel regio, thus, igy produced can interact with epitop of the surface proteins of s. mutans with high aviditas.21 it means that the binding of the surface proteins of s. mutans by igy antibody can decrease the character of figure 2. the surface proteins of s. mutans recognized by igy anti s. mutans, using western blot method (figure 2), showed similar protein ribbon with molecule mass (mm) about 188 kda for all of those five isolates of s. mutans. number 1–5 show that the well with 20 ml antigen extracted with lisozim from whole cels of s. mutans. line 1 (s. mutans isolate fkh ipb), line 2–4, each of which shows s. mutans with serotype f, serotype e, serotype d, and serotype c. m = prestaining protein marker, kda = kilo dalton, and the number under it showed the molecule mass of proteins. figure 1. the protein profile of s. mutans colored with commassie blue using sds-page method s. mutans in line 1, 2, and 3 showed similar protein ribbon profile, while, s. mutans in line 4 and 5 shows the different protein ribbon from the previous one. 192 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 189-193 s. mutans pathogens since they can restrain the adhesion of those bacteria in the host dental surface, so it can possibly restrain the process of caries.22 the result from this research can give important information about the potency of igy as anti surface proteins of s. mutans. this research may oven answer whether igy produced by fkh ipb can interact with high avidities towards the surface proteins of s. mutans. therefore, in order to answer the question, western blot technique then was used in this research. based on the analysis result of protein profile with commasie blue and the use of igy anti s. mutans with western blot technique (figure 1 and 2), the surface proteins of s. mutans that can be recognized by igy were proteins with molecule mass (bm) about 188 kda, while other proteins was not detected by western blot technique. idone7 and matos-graner23 report that proteins, about 180-190 kda, are considered as ag i/ii proteins of s. mutans, which means that igy used in this research can specifically recognize antigens i/ii considered as the surface proteins expressed by those serotypes of s. mutans, meanwhile gtf and gbp can not be detected. the reason of this igy potency is because fragment antigen binding (fab) of igy can recognize the surface proteins of s. mutans.24 thus, this result supports the assumption that chicken immunized with whole cell s. mutans can produce specific igy towards ag i/ii expressed by those four serotypes of s. mutans. the potency of igy is possibly related with the higher concentration of antibody in egg yolk.25 however, this hypothesis still needs further researches. one of them conducted by nikki,26 shows that besides ag i/ii has 190 kda molecule mass, ag i/ii also locates in the position that is relatively higher than the cell surface of s. mutans compared with gtf and gbp. therefore, ag i/ii become the surface proteins that are more dominant as the initiator of adhering to dental pellicle. by using of igy with water soluble fraction (wsf) and immune-blotting technique, wibawan14 and smith24 reports that igy wsf can detect 59 kda proteins considered as the surface proteins of s. mutans (gbpa), nevertheless, igy only shows the weak reactivation towards pac proteins classified into antigen i/ii family.10 method used in this research, therefore, supports and is relevant with the aim of igy anti s. mutans in recognizing both the surface proteins of s. mutans with serotype c, d, e, and f, and affinity of its interaction. thus, the potency of igy in recognizing ag i/ii specifically shows that igy used in this research is specific and can be used to restrain ag i/ii of s. mutans expressed by all those serotypes. petersen27 reports that ag i/ii has a good role as adhesin of s. mutans, strong tendency to bind saliva component, and a good role as the main initiator in the adhesion and colonization of s. mutans in dental pellicle. the potency of igy in restraining the surface proteins of s. mutans in in-vitro way, thus, can be assumed that igy can prevent the synthesis of glucan from sucrose caused by s. mutans, and can decrease the colonization and carcinogenicity of s. mutans in the dental surface. furthermore, kruger28 reports that the use of igy through passive immunization is very effective in protecting caries in rats as the testing animals, which means that igy can restrain the colonization of s. mutans and s. sobrinus in in vivo experiment. not only in s. mutans, the use of igy anti salmonella enteritis can also show the good reactivation through elisa test,29 similarly, rawendra11 reports that igy can interact well with enterophatogenic escherichia coli and restrain the colonization of bacteria in the surface of intestine. moreover, the research conducted by smith 6 showed that the use of siga antibody of s. mutans with serotype c, showed that the reactivation of siga in the caries free group was higher than that in the caries sensitive group. it means that the antibody can prevent the adhesion of s. mutans to hydroxiapatite layers. in other words, the content of siga in saliva can prevent the process of caries by restraining the adhesion of s. mutans.30 like siga, the result of this in-vitro research shows that igy could be able to recognize ag i/ii considered as the surface proteins of s. mutans. chismirina31 in her research also reports that the use of polymerase chain reactions (pcr) method successfully determines the serotype of s. mutans strain derived from ipb with serotype d (s. mutans used as the positive control of antigen produced by igy). thus, it may be concluded that igy anti s. mutans with serotype d can recognize proteins of ag i/ii cell surface of s. mutans with serotypes c, d, e, and f similar with s. mutans strain derived from ipb used as the positive control in this research. however, in other to strengthen the result of this research, it needs the further researches about clinical isolate s. mutans strain, especially concerning with the allergic test and cross reaction with some bacteria and fungi of oral cavity, both of which facilitate the adhesion of s. mutans to dental pellicle. therefore, igy is expected to be used as biology material in the prevention program of dental caries through passive immunization. acknowledgement this project was funded by university of syiah kuala, higher education of indonesia national education. national research strategies, contact number 096/h11p2t/a.01.2009, february, 27 2009 a well a bbnad and department of dentistry, medical faculty, syiah kuala university also molecular laboratory of fkh ipb and fkg ui. references 1. basri ag, antonia t, soeherwin t. molecular aspect of virulence streptococcus mutans. indonesia journal of dentistry 2006; 13(2): 107–14. 2. samaranayake l. essential microbiology for dentistry. 3rd ed. churchill livingstone elsevier; 2006. p. 255–93. 193gani, et al.: the ability of igy to recognize surface proteins 3. banas ja. virulence properties of streptococcus mutans. frontiers in bioscience; journal and virtual library 2004; 9: 1267–77. 4. deepan s, syah h, roy r, russell rb. a novel glucan-binding protein with lipase activity from the oral pathogen streptococcus mutans. microbiology 2004; 150: 1947–56. 5. banas ja, vickerman mm. glucan binding proteins of the oral streptococci. crit rev oral biomed2003; 14(2): 89–99. 6. smith dj. caries vaccines for the twenty-first century. journal of dental education 2003; 67(10): 1130–7. 7. idone v, brendtro s, gillespie r, kocaj s, peterson e, rendi m, warren w, michalek s, krastel k, cvitkovitch d, spatafora g. effect on orphan respon regulator on streptococcus sucrose-dependent adherence and cariogenesis. infect immune 2003; 8(71): 4351–60. 8. biswas s, biswas i. role of htra in surface protein expression and biofilm formation by streptococcus mutans. infection and immunity 2005; 73 (10): 6923–34. 9. kruger c. passive immunization against oral phatogens. division of clinical immunology at the departement of laboratory medicine and center for oral biology at novum, institute of odontology, karolinska university hospital, huddinge, stockholm, sweden. karolinska university press; 2004. p. 14–29. 10. crowley pj, trevor bs, isoda r, van tilburg m, monika wo, rebekah ar, william pm, arnold sb, brady jl. requirements for surface expression and function of adhesin p1 from streptococcus mutans. infection and immunity 2008; 76(6): 2456-68. 11. rawendra r. immunoglobulin y (igy) water soluble fraction (wsf) kuning telur kering beku anti enterophatogenic escherichia coli (epec). disertation. sekolah pascasarjana institut pertanian bogor; 2005. p. 41-100. 12. mitoma m, oho t, michibata n, okano k, nakano y, fukuyama m, koga t. passive immunization with bovine milk containing antibody to a cell surface protein antigen-glucosyltransferase fusion protein protects rats against dental caries. infect immune 2002; 70(5): 2721–4. 13. chi zb, gao yx, pan y, zhang b, feng xp. the inhibitive effect of igy toothpaste against oral streptococcus mutans. article in chinese. department of preventive & pediatric dentistry, ninth people's hospital, school of stomatology, shanghai second medical university. shanghai 200011. china: shanghai kou qiang yi xue; 2004; 13(4): 256–8. 14. wibawan iwt. pemanfaatan telur ayam sebagai pabrik biologis: produski “yolk immunoglobulin” (igy) anti plaque dan diare dengan titik berat pada anti streptococcus mutans, escherichia coli dan salmonella enteritidis. laporan penelitan. lembaga penelitian dan pemberdayaan masyarakat ipb, fkh-ipb; 2005. p. 1–20. 15. basri ag, boy mb, dewi f. surface proteins of streptococcus mutans ccell that can be recognized by immunoglobulin y. dentika dental journal 2006; 11(2): 188–93. 16. yera h, andiva s, perret c, limonne d, boireau p, dupovy canet. development and evaluation of a western blot kit for diagnosis of human trichinellosis. american society for microbiology; 2003. 10(5): 793–6. 17. carlander d. avian igy antibody in vitro and in vivo. disertation for the degree of doctor of philosophy (faculty of medicine) in clinical chemistry presented at uppsala university; 2002. p. 3–53. 18. zhou z, zhou r, tang z. effects of topical application ofeffects of topical application of immunoglobulin yolk on mutans streptococci in dental plaque. hua xi kou qiang yi xue za zhi 2003; 21(4): 295–7. 19. wen b, zhou rj, yang j, tang z, zhou z. the inhibitory effect of anti-streptococcus mutans immunoglobulin of yolk on glucan synthesis of streptococcus mutans. shanghai kou qiang yi xue 2002; 11(2): 141–2. 20. sato y, okamoto k, kagami a, yamamoto y, igarashi t, kizaki, h. streptococcus mutans strains harboring collagen-binding adhesion. research reports. j dent res 2004; 87(7): 534–9. 21. west ap jr, herr ab, bjorkman pj. the chicken yolk sac igy receptor, a functional equivalent of the mammalian mhc-related fc receptor, is a phospholipase a2 receptor homolog. immunity 2004; 20(5): 507–8. 22. matsumura m, izumi t, matsumoto m, tsuji m, fujiwara t, ooshima t. the role of glucan-binding protein in the cariogenecity of streptococcus mutans. j microbiol immunol 2003; 47(3): 213–15. 23. mattos-graner ro, smith dj. the vaccination approach tothe vaccination approach to control infections leading to dental caries. bra j oral sci 2004; 3(11): 595–608. 24. smith dj, king wf, godiska r. passive transfer of immunoglobulin y antibody to streptococcus mutans glucan-binding protein b can confer protection against experimental dental caries. infect immun 2001; 69(5): 135–42. 25. mine y, kovacs-nolan j. chicken egg yolk antibody as therapeutics in enteric infectous disease: a review. j med food 2002; 5(3): 159–69. 26. nikki r, jenny mc, knneth bt, william pm, brady jl. william p, mcarthur. characterization of the streptococcus mutans p1 epitope recognized by immunomodulatory monoclonal antibody 6-11a. infection and immunity 2004; 72(8): 4680–88. 27. petersen fc, assev s, van der mei hc, busscher hj, scheie aa. functional variation of the antigen i/ii surface protein in streptococcus mutans and streptococcus intermedius. infect immun 2002; 70: 249–56. 28. kruger c, pearson sk, kodama y, vacca smith a, bowen wh, hammarstrom l. the effects of egg-derived antibody to glucosyltransferases on dental caries in rats. caries res 2004; 8(1): 9–14. 29. lee en. anti-bacterial activities of chicken egg yolk antibody (igy) against enteric pathogens. a thesis master of science submitted to the faculty of graduate studies and research in partial fulfillment of the requirements for the degree of in food science and technology university of alberta, canada; 2000. p. 93–106. 30. smith dj. dental caries vaccines: prospects and concerns. crit revcrit rev oral biol med 2002; 13(4): 335–49. 31. chismirina s. efek imunoglobulin y (igy) sebagai anti adhesin pada pembentukan biofilm oleh streptococcus mutans (serotype c, e, f) dan streptococcus sobrinus (serotype d) secara in-vitro. thesis. jakarta: fakultas kedokeran gigi universitas indonesia; 2006. p. 10–20.2006. p. 10–20. vol 51 no 4 okt-des 2018.indd 185 dental journal (majalah kedokteran gigi) 2018 december; 51(4): 185–188 considerations in performing odontectomy under general anesthesia: case series anindita zahratur rasyida and andra rizqiawan department of oral and maxillofacial surgery faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: an odontectomy constitutes a common surgical procedure performed by oral and maxillofacial surgeons on a daily basis. the anesthesia procedure required during this form of operation may involve the administering of a general anesthesia which, while a safe procedure when performed by an anesthesiologist, still involves an element of risk and should only be undertaken with appropriate safeguards. various measures, not only anxiety control-based, are suggested in this article. purpose: the purpose of this study was to report considerations factors, other than anxiety, as indications in performing odontectomy conducted under a general anesthesia at universitas airlangga hospital, surabaya, indonesia. cases: four cases of patients who had undergone an odontectomy under a general anesthetic are reported here. case management: the four cases of management involved odontectomies conducted under a general anesthetic for a variety of reasons with contrasting outcomes. the treatment of the four patients was based on an anatomical approach and previously ineffective pain control due to greater trauma. one of the patients also suffered from schizophrenia that produced comorbidity requiring holistic observation. this individual required intricate surgery whose performance was challenging under a local anesthetic. none of our other patients suffered from serious complications related either to surgery or the administering of a general anesthetic. conclusion: in conclusion, important factors relating to an odontectomy performed under a general anesthetic on four patients in universitas airlangga hospital, surabaya included: anxiety, anatomical approach, adequate pain control, comorbidity of systemic medical conditions and the potential need for surgical procedures difficult to perform under a local anesthetic. keywords: consideration; general anesthesia; odontectomy; oral and maxillofacial surgeon correspondence: andra rizqiawan, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: andra-r@fkg.unair.ac.id case report introduction an impacted tooth is one that fails to erupt into the dental arch within the anticipated time period.1 a study of 392 patients in the dental and oral hospital, universitas padjadjaran, bandung, indonesia indicated that 76.8% of these individuals had third molar impactions.2 as a general rule, unless removal is contraindicated, all impacted teeth should be extracted by means of a process known as an odontectomy. the performance of an odontectomy is common in oral and maxillofacial surgery (omfs) during which pain control is usually achieved through the administering of a local anesthetic or, less frequently, a general anesthetic (ga). the first application of the latter for the purposes of removing impacted teeth in december 1884 is widely credited to horace wells.3 various considerations have been highlighted regarding the use of a ga during the performance of an odontectomy. a ga is generally required in cases of major surgery when a local anesthetic produces an inadequate level of sedation, when patient cooperation or compliance is unnecessary, when muscle relaxation in apprehensive patients is required for stabilization or when the patient is allergic to local anesthetics.4 several other reviews of the application of a ga in dentistry highlight issues such as the lack of patient cooperation due to anxiety, mental disability or medical conditions (for example, extreme gag reflexes or an inability dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p185–188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p185-188 186rasyida, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 185–188 to keep the mouth open) which render surgical intervention while he/she is conscious difficult.5,6 the administering of a ga under the supervision of an anesthesiologist constitutes a relatively safe procedure, but still carries a certain degree of risk and should not be undertaken merely as a first-line means of anxiety control. such risk is associated with dental-soft tissue trauma and potentially fatal cardiopulmonary dysfunction. a ga should be strictly limited to those patients and clinical situations in which the administering of a local anaesthetic (with or without sedation) is not an option.5 as surgeons, omfs doctors are required to analyze multiple factors in order to decide on the use, or otherwise, of a ga. this article describes odontectomic procedures performed on patients with symptoms other than anxiety under a ga at universitas airlangga hospital, surabaya, indonesia. these case reports may be useful for dentists as a means of educating patients prior to surgery. cases the four cases reported here relate to patients undergoing an odontectomy under a ga at the in-patient section of the oral and maxillofacial surgery department, universitas airlangga hospital. case 1: a 45-year old male, with no history of previous illness or infection, complained of a partly erupted lower left third molar he wished to have extracted due to the discomfort caused by impacted food near the tooth. clinical examination confirmed the partial eruption of teeth 38 and 48, while orthopantomographic evaluation (figure 1) revealed bilateral horizontal impaction of the distomolar diagnosed as a potential odontoma adjacent to the impacted third molar on the left and right side of the lower jaw. the positions were deep and overlapped with the mandible canals. the left impacted teeth showed enlargement of the dental follicle which was provisionally diagnosed as a dentigerous cyst. case 2: a 53 year-old male referred by a prosthodontist for multiple extractions prior to dental prosthesis treatment. this patient also suffered from schizophrenia, for which he was taking clozapine medication, in addition to allergies to antibiotics, plastics and metal. clinical evaluation indicated partial eruption of 48 and multiple chronic apical periodontitis of 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 26, 36, 38, 45, 46 gangrene radix. the orthopantomographic view is shown in figure 2. case 3: a 25-year old female complained of a dull non-specific pain radiating from the right side of the lower jaw and extending to the neck and also swelling in the pericoronal of 38. the subject wanted to have four of her third molars removed. no previous medical illness was recorded. clinical examination showed impaction of 48 and partial impaction of 18, 28, 38. the results of an orthopantomography indicated that the apical of 48 overlapped with the mandible canal and the distance to the inferior border of the mandible was too short. the patient had already been informed that the deep position of 48 figure 1. orthopantomograph view of first patient show deep tooth impaction and odontoma. figure 2. orthopantomograph of a second patient showing multiple gangrenous radix. figure 3. orthopantomograph of third patient showing deep impaction of 48. figure 4. orthopantomograph view of fourth patient showing the unsual position of supernumerary teeth in the lower left jaw. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p185–188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p185-188 187 rasyida, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 185–188 increased the risk of a fracture and she agreed to undergo the appropriate surgical procedure. case 4: a 28-year old male complained of frequent tenderness and tooth decay in his upper and lower molars and expressed the desire to have all of his wisdom teeth extracted. an orthopantomograph (figure 4) revealed unusual invertion of the distomolars (kissing molars) located in the ascending ramus of the mandible in the left lower jaw. a mesiodens was also visible between the upper incisors. however, the patient refused to have it extracted due to its being asymptomatic. case managements the therapy applied to the four cases constituted an odontectomy under a ga. each patient remained in hospital for three days: one day for pre-op, one day for surgery and one day for post-surgical observation prior to discharge. further follow-up dreing which the stitches were removed occurred one week after surgery. none of the patients above suffered any serious complications resulting from either their operation or the general anesthetic administered. they showed minimal edema, no wound dehiscence, infection, nausea or systemic complications resulting from the administering of a ga. discussion ga induces a loss of consciousness by blocking brain function which, consequently, renders a specific operative area insensitive to pain. it is usually desirable to keep the patient in a state of consciousness during dental treatment by means of a local anesthetic.7 however, in particular cases, such as an odontectomy, there are various reasons for administering a ga which needs to take account of the balance between risk versus benefit.5 the cases presented here had various motives for their decision to undergo an odontectomy performed under ga. the first and fourth patients did so because of the need for adequate pain control during the relatively protracted duration of the procedure and its traumatic impact. the administering of a ga also helped to support the surgeon. the condition of the second patient, who suffered from specific allergies and schizophrenia, was managed through consultation with a psychiatrist and internist. he underwent examination and psychoeducation to prepare him for dental treatment. the administering of a ga was decided upon because the surgeon could remove not only the impacted 48 but also the overall focal infections during a single surgical procedure. the patient would, consequently, remain in his comfort zone without the need for longterm clinical observation. evaluation and observation of his medical condition and medication used to treat his schizophrenia before, during, and after surgery could also be performed holistically together with an anesthesiologist. an odontectomy under a ga was performed on the third patient to not only adequately control pain, but also to prepare mandibular plating should it be required in order to stabilize the back-up of any potential fracture. thus, a ga helps to facilitate surgery that would prove too extensive and/or challenging on a conscious patient.5 during surgery, the intact condition of the mandible was reviewed and the need for further stabilization by means of a plate evaluated immediately on removal of the 48. the final decisions in this regard were not taken by the surgeon alone since the patients had to be kept fully informed and their consent obtained regarding the administering of a ga. therefore, an odontectomy conducted under a ga involves a clinical decision on the part of the surgeon meeting with the support of the patient. pre-operative assessment of the patient, during which he/she is made aware of the potential risks of the proposed procedure and provides informed consent, is a prerequisite.7 further additional considerations have been cited as influencing the decision to treat patients either under sedation or a ga, including their overall state of health, their own preferences as well as those of their carers or family, the specific surgical procedures involved and operator or facility-related factors.6 instances of odontectomies performed under a ga at universitas airlangga hospital are conducted solely due to patient anxiety. this is because, in most cases, the administering of a ga allows the dentist to complete the treatment promptly rather than delay care because of the anxiety experienced by a patient rendering him/her uncooperative with the surgical procedure. it is possible for orthodontists to offer significantly improved dental care since omfs will become increasingly straightforward. the administering of a ga results in total relaxation, while patient recall of the procedure is minimized, facilitating successful treatment of even the most dental-phobic of individuals. nevertheless, both pharmacological and non-pharmacological behavior guidance techniques can be applied to alleviate anxiety.8 ultimately, the capability of health care professionals and the surgical facilities available will decide the best option regarding anesthetic procedures. one study reported that comprehensive dental treatment was simpler, the general condition of the patient population more stable and the risk of postoperative complications lower when compared to the results of general surgery. in 2017, chen et.al investigated post-operative complications associated with comprehensive dental treatment under ga at the taipei veterans general hospital in august 20112012 found that the three most common were: lip swelling (69.2%), nausea (59.6%) and oral ulceration (46.1%). however, most of the above complications gradually self-eliminated post-operatively under appropriate medical care.8 a major factor that has to be considered with an odontectomy under ga is that of its disadvantages which comprise a higher cost than procedures conducted under a dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p185–188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p185-188 188rasyida, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 185–188 local anesthetic and the need for laboratory tests and chest x rays. pain control during procedure is more effective under a ga. however, precisely because the patient feels no pain, the surgeon may apply excessive force to extract the tooth and/or drill so deeply into the bone or tooth that alveolar nerve injury ensues. the relationship of the mandibular third molar roots to the inferior alveolar nerve must be considered when surgical removal is contemplated. surgical planning and proper informed consent depend on detailed knowledge of the positional relationships in this area.9,10 to avoid such damage, a split technique constitutes the best odontectomy approach to avoid unnecessary trauma and reduce complications.11,12 in conclusion, in addition to anxiety, the considerations underpinning the decision to conduct an odontectomy under a ga on patients at univeritas airlangga hospital, surabaya included: an anatomical approach, adequate pain control, systemic medical illness as a comorbid and the requirements of surgical procedures that are difficult to perform under a local anesthetic. acknowledgement we would like to express our gratitude to universitas airlangga hospital for providing necessary data relating to the cases described here. references 1. hupp jr, ellis e, tucker mr. contemporary oral and maxillofacial surgery. 6th ed. st louis missouri: mosby elsevier; 2013. p. 703. 2. singh s, sam b, sitam s. prevalence third molar agenesis and impaction among indonesian people. in: indonesia 10th asian congress oral and maxillofacial radiology. bandung: faculty of dentistry, universitas padjadjaran; 2014. p. 1–20. 3. malhotra n. general anaesthesia for dentistry. indian j anaesth. 2008; 52(suppl 5): 725–37. 4. malik na. textbook of oral and maxillofacial surgery. 3rd ed. new delhi: jaypee brothers medical publishers; 2012. p. 145. 5. hutchinson s. general anaesthesia for dentistry. anaesth intensive care med. 2011; 12(8): 347–50. 6. borle rm. textbook of oral and maxillofacial surgery. new delhi: jaypee brothers medical publishers; 2014. p. 813. 7. chitre ap. manual of local anesthesia in dentistry. 2nd ed. new delhi: jaypee brothers medical publishers; 2010. p. 357. 8. chen yp, hsieh cy, hsu wt, wu fy, shih wy. a 10-year trend of dental treatments under general anesthesia of children in taipei veterans general hospital. j chinese med assoc. 2017; 80(4): 262–8. 9. deliverska eg, petkova m. complications after extraction of impacted third molars literature review. j imab annu proceeding (scientific pap. 2016; 22(3): 1202–11. 10. azenha mr, kato rb, bueno rbl, neto pjo, ribeiro mc. accidents and complications associated to third molar surgeries performed by dentistry students. oral maxillofac surg. 2014; 18(4): 459–64. 11. farish se, bouloux gf. general technique of third molar removal. oral maxillofac surg clin north am. 2007; 19: 23–43. 12. singh v, alex k, pradhan r, mohammad s, singh n. techniques in the removal of impacted mandibular third molar: a comparative study. eur j gen dent. 2013; 2(1): 25–30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p185–188 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p185-188 mkgs vol 45 no 2 april-juni 2012.indd 102 volume 45 number 2 june 2012 research report deoxypyridinoline level in gingival crevicular fluid as alveolar bone loss biomarker in periodontal disease agustin wulan suci dharmayanti department of biomedical faculty of dentistry, jember university jember indonesia abstract background: periodontal diseases have high prevalence in indonesia. they are caused by bacteria plaque that induced host response to release pro inflammatory mediator. pro inflammatory mediators and bacteria product cause degradation of collagen fibers in periodontal tissue. deoxypyridinoline is one of pyridinoline cross-link of collagen type i that can be used as biomarker in bone metabolic diseases, however, their contribution to detect alveolar bone loss in periodontal diseases remains unclear. purpose: this study was to evaluate deoxypyridinoline level in gingival crevicular fluid as alveolar bone loss biomarker on periodontal disease. methods: this study used 24 subjects with periodontal diseases and 6 healthy subjects. dividing of periodontal disease was based on index periodontal. gingival crevicular fluid was taken at mesial site of maxillary posterior tooth by paper point and deoxypyridinoline be measured by elisa technique. results: we found increasing of deoxypyridinoline level following of the severity of periodontal diseases. there was also significant difference between healthy subjects and periodontal diseases subjects (p<0.05). conclusion: deoxypyridinoline level in gingiva crevicular fluid can be used as alveolar bone loss biomarker in periodontal disease subjects. key words: deoxypyridinoline, gingival crevicular fluid, alveolar bone loss abstrak latar belakang: prevalensi penyakit periodontal di indonesia cukup tinggi. ini disebabkan oleh bakteri plak yang merangsang respon tubuh untuk mengeluarkan mediator keradangan. mediator keradangan dan produk bakteri menyebabkan degradasi serat kolagen jaringan periodontal. deoksipiridinolin merupakan salah satu ikatan piridinium dari kolagen tipe i yang dapat digunakan sebagai biomarker penyakit metabolisme tubuh. akan tetapi, penggunaan deoksipiridinolin untuk mendeteksi kehilangan tulang alveolar pada penyakit periodontal masih belum jelas. tujuan: tujuan penelitian ini untuk mengetahui bahwa kadar deoksipiridinolin pada cairan krevikular gingival dapat digunakan sebagai biomarker kehilangan tulang alveolar pada penyakit periodontal. metode: penelitian ini menggunakan 24 subyek penelitian yaitu 24 orang dengan penyakit periodontal dan 6 orang tidak menderita penyakit periodontal. pembagian penyakit periodontal berdasarkan indeks periodontal. cairan krevikular gingival diambil dari bagian mesial gigi posterior atas dengan menggunakan paper point dan diukur kadar deoksipiridinolin dengan menggunakan teknik elisa. hasil: hasil penelitian menunjukkan ada peningkatan kadar deoksipiridinolin seiring dengan tingkat keparahan penyakit periodontal. hasil statistik juga menunjukkan ada perbedaan rata-rata antara subyek penelitian yang tidak menderita penyakit dengan subyek yang menderita penyakit periodontal (p<0,05). kesimpulan: kadar deoksipiridinolin pada cairan krevikular gingival dapat digunakan sebagai biomarker kehilangan tulang alveolar pada penderita penyakit periodontal. kata kunci: deoksipiridinolin, cairan krevikular gingiva, kehilangan tulang alveolar correspondence: agustin wulan suci dharmayanti, departemen biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember. e-mail. agustinwulan.fkgunej@gmail.com, telp. +6281336455748. fax. +62331331991. 103dharmayanti: deoxypyridinoline level in gingival crevicular fluid introduction periodontal disease and dental caries are the primary causes of permanent tooth loss. in indonesia, periodontal diseases prevalence was higher than dental caries.1 survey kesehatan rumah tangga (skrt) of indonesia healthy department in 2008 showed that 46% indonesia’s population affected periodontal disease and the prevalence increased as followed the age.2 periodontal diseases are inflammation and degeneration of soft and hard tooth supporting tissue that caused by dental plaque bacteria. progressivity of periodontal diseases involves some factors, such as local, systemic and environment factor. these factors will influence host and bacteria interaction. bacteria of oral cavity can cause inflammation by host cell activation to produce pro-inflammatory mediator.3 pro-inflammatory mediator causes collagen fibers of periodontal tissue degradation, including collagen crosslink of alveolar bone.3 collagen cross-link reinforces collagen fibers of tissue. however, the presence of inflammation in supporting tissue causes collagen fibers degradation and also collagen cross-link destruction. the product of collagen degradation cannot be re-metabolized in body and will be released into bloodstream and excreted in urine.4 in mature tissue, collagen type i cross-link is formed by pyridinium cross-link, such as pyridinoline and deoxypyridinoline. pyridinoline is the most collagen cross-link in cartilage and soft tissue, while deoxypyridinoline is the most collagen cross-link in bone and ligament.5 deoxypyridinoline has specificity for bone loss. deoxypyridinoline can be used to know bone loss in osteoporosis and bone metabolic diseases, such as hyperthiroid, hyperparathyroid, and paget’s disease.6 shibutani showed that deoxypyridinoline level in gingival crevicular fluid, urine, and serum can be used to detect periodontitis in beagle dog.7 dentists need information and examination for determining diagnostic of periodontal disease. they used clinical examination such as probing depth, bleeding on probing, loss attachment, plaque index, and radiographic examination. the advantage of this method is easy, cheap and non invasive.7 however, this examination only can detect alveolar bone destruction in late period or the destruction is more than 3 mm.8 recent it is being developed procedures for a more practical examination. it uses biology indicator or biomarker. indicator or biomarker is more specific because it is related with host resistance to local and systemic factor. indicator use samples from biofilm plaque, gingival crevicular fluid (gcf) and saliva.3 periodontal diseases indicators are usually related to collagen fiber destruction, such as deoxypyridinoline, but it is still unclear and has never been proven on human. we measure deoxypyridinoline level in gcf of healthy and subjects with periodontal diseases to indicate alveolar bone loss, as a biomarker. materials and methods this research was admitted and approved by agreement from ethical commission of dentistry faculty, gadjah mada university. thirty patients consecutively recruited for the study at the periodontic department of prof. soedomo dental hospital gadjah mada university. all of patient must signed inform concern as agreement legally of research subject. there were 24 patients with periodontal disease and 6 healthy subjects. inclusion criteria: man or woman 30–50 years old, had 20 teeth minimally in oral cavity, did not have systemic disease, non smoker, did not use oral rinse, antibiotic, or drug that had calcium metabolic effect for 6 months, did not get periodontal treatment for 6 month, and were not pregnant, menstruation, or menopause. all patients also were examined loss attachment degree, probing depth, and bleeding on probing. subjects were divided into 5 groups: patients with gingivitis, mild, moderate, and severe periodontitis, and healthy subject (as control). clinical criteria of periodontal index used russel’s modification for determining diagnostic of periodontal diseases. determination of tooth sample that was taken the gcf was depended on probing depth and loss attachment. healthy subject (control) showed no loss attachment, pocket and bleeding on probing. gingivitis showed no loss attachment and pocket, but there is bleeding on probing. in mild periodontitis, there is loss attachment less than 3 mm, periodontal pocket 3–4 mm, and bleeding on probing. in moderate periodontitis, there is loss attachment more than 3 mm, periodontal pocket 4–5 mm, and bleeding on probing. in severe periodontitis, there is loss attachment more than 3 mm, periodontal pocket more than 5 mm, and bleeding on probing.7,9 having taken gcf from teeth was based on clinical examination and radiographic, particularly first molar of maxilla. if first molar was missing, it could be substituted by second molar or second premolar. however, the site of collecting sample must not be near residual ridge. gcf samples were collected using a paper point. paper point #25 was inserted into pocket periodontal for 30 seconds gently. previously selected tooth was isolated with sterile cotton rolls, and the supragingival plaque was removed with sterile cotton pellets.10,11 paper point was removed into 0.5 ml eppendorf tube and closed by paraffin tape. then, eppendorf tube was inserted into ice box and kept in deep freezer -20 °c until deoxypyridinoline test. when paper point inserted into periodontal pocket, paper point must not make injury in gingival sulcus, because it made bleeding and influenced the result. that eppendorf tube was holed under the tip of tube by sterile needle and given 50 μl 0.02 m phosphate buffer solutions (pbs) (ph 7.0–7.2) as solvent. then, that eppendorf tube was put in a new 1.5 ml eppendorf tube and centrifuged in 1000xg for 20 minutes at room temperature 18–25 °c. its procedure was for getting 104 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 102–106 gcf+pbs solutions. eppendorf tube was centrifuged again in 1000xg for 20 minutes. deoxypyridinoline test used elisa technique (uscn life, china).12 the result were analyzed by kruskal-walis-h test and followed by mann-whitney-u test with 5 % (p<0.05) significant degree. results there was increasing of deoxypyridinoline level following the severity of periodontal diseases. the increasing of deoxypyridinoline level in periodontal diseases subjects was 4–60 times from healthy subjects (table 1). based on kruskal–wallis–h test, there was significant different in deoxypyridinoline level of subject with and without periodontal disease (p<0.05). then, the result was analyzed by mann–whitney–u test to know mean differences between groups. it showed there is significant different of deoxypyridinoline level between healthy, gingivitis, mild, moderate and severe periodontitis subjects (p<0.05) (table 2). discussion based on research result, there was increasing of deoxypyridinoline level following severity of periodontal disease. the highest of deoxypyridinoline level was in severe periodontitis subjects. it showed that there was alveolar bone destruction in severe periodontitis subject more than the other groups. research result also showed there was significant different of deoxypyridinoline level in subject with and without periodontal diseases (p<0.05). periodontal diseases are inflammation disease that causes connective tissue and bone surrounding tooth destruction. the inflammation is started from gingival then spread into periodontal tissue. there was different pattern of tissue destruction between gingivitis and periodontitis. in gingivitis, there was just gingival inflammation surrounding the tooth and without loss attachment. in periodontitis, there were loss attachment and alveolar bone loss.13 in gingivitis subject, there is significant different of deoxypyridinoline level. because gingival is formed by collagen fibers of type i, iii, and v. although, the composition of type i collagen fibers is lesser than type iii and v. alveolar bone and ligament periodontal are formed by collagen fibers of type i and iii. the type i collagen fibers is higher than type iii.14 gingival inflammation in gingivitis subjects caused type iii collagen fibers degradation. de coster research showed on immunofluoressence test, the most type iii collagen fibers and some type i and v collagen fibers of gingivitis subjects was lost. there was no type iii collagen fibers and less of type i collagen fibers in gingivitis.7 deoxpyridinoline was type i collagen crosslink and the most in ligament and bone. deoxypiridinolin cross link is support link of type i collagen fiber.3 it caused significant different of deoxypyridinoline level between gingivitis and peridontitis subjects. based on research result, deoxypyridinoline level changing was found in gcf of periodontal disease patients. gcf is inflammatory exudates fluid that seeps out into the gingival crevicular or periodontal pockets around teeth with inflammation gingival. gcf and serum contains local materials, such as tissue breakdown products, inflammatory mediators, antibodies to kill bacteria of dental plaque.15 in bone loss, deoxypyridinoline will be released into bloodstream and excreted into urine. initial events are triggered by lipopolysaccharides (lps). lps from gram negative plaque biofilms on the periodontal tissues. as a first line of defense, pmns are recruited to the site. monocytes and activated macrophages respond to endotoxin by releasing cytokines (tnf and il-1) that direct further destruction processes. mmps, which can act as powerful collagen-destroying enzymes, are produced by fibroblasts and pmns. tnf, il-1, and receptor activator of nuclear factor-kappa b ligand (rankl) are elevated in active sites and mediate osteoclastogenesis and bone breakdown. bone-specific markers, such as i-carboxytelopeptide pyridinoline (ictp) and deoxypyridinoline, table 2. result of mann–whitney–u deoxypyridinoline level control gingivitis mild periodontitis moderate periodontitis severe periodontitis control 0.001* gingivitis 0.001* mild periodontitis 0.001* moderate periodontitis 0.001* severe periodontitis 0.001* explanation: *: there is significant different between groups (p<0.05) table 1. mean and standard deviation (sd) of deoxypyridinoline level in healthy and periodontal diseases subjects (nmol/ l) variable n mean sd control 6 6.38 1.29 gingivitis 6 25.43 3.92 mild periodontitis 6 142.71 27.31 moderate periodontitis 6 270.13 53.99 severe periodontitis 6 388.61 21.39 105dharmayanti: deoxypyridinoline level in gingival crevicular fluid are released into the surrounding area and transported by way of gcf into the sulcus or pocket and serve as potential biomarkers for periodontal disease detection. when deoxypyridinoline is into bloodstream, it will penetrate and go out from blood vessel to gingival tissue through gcf (figure 1). pyridinium cross link such as deoxypyridinoline and ictp was found in gcf because they were collagen cross-link that released when collagen of periodontal tissue in periodontal diseases was degraded.16 deoxypyridinoline levels in gcf can be used as early marker of alveolar bone destruction in periodontal disease. several reasons deoxypyridinoline levels can be used as early marker of alveolar bone destruction in periodontal disease are a) deoxypyridinoline is the result of type i collagen degradation and alveolar bone constituent; b) deoxypyridinoline will be released during the bone loss and will not be re-metabolized; and c) deoxypyridinoline on gcf is a safe, non-invasive and efficient biological sample to see inflammation or alveolar bone loss.17 gcf also has good value for diagnosis of periodontal disease. gcf contains protein as periodontal tissue destruction product, one of them is deoxypyridinoline.10 deoxypyridinoline level in periodontal disease patients is higher than control (table 1). this indicates that since the beginning of periodontal disease is gingivitis to chronic periodontal disease, there are damage collagen fibers, which continues on dental alveolar bone destruction. results of animal studies showed that deoxypyridinoline level increased in gcf and serum of experimental animals suffering from periondontitis.7 deoxypyridinoline level was different significantly between mild, moderate and severe periodontitis subjects. there was loss attachment ad alveolar bone destruction, but the severity of destruction is different. in mild periodontitis, alveolar bone destruction just was in interdental septum of alveolar bone. in moderate periodontitis, alveolar bone destruction was less a third of bone of tooth support, and in severe periontitis, alveolar bone destruction was more than third of bone of tooth support.17 severity of alveolar bone destruction was related with collagen degradation of alveolar bone that was manifested in deoxypyridinoline level changing. deoxypyridinoline is potential agent for periodontal diseases biomarker, although, it need further research. it was caused deoxypyridinoline is pyridinium crosslink that formed between type i collagen molecules and will be released into circulation when collagen is catabolized or degradation.17 furthermore, deoxypyridinoline could detect progressivity of periodontal disease, since early stage (gingivitis) to advanced stage (severe periodontitis), so it could be called as biomarker. although it need validation and qualification test, before it used widely. the tests are functioned to be accurate and good standardization and have clinical end point.19 this research could be concluded that deoxypyridinoline level in gcf could be used as alveolar bone loss biomarker in periodontal disease. besides, it needed further research about the changing of deoxypyridinoline level longitudinally, so it could be used as biomarker or alveolar bone loss. references 1. a lbayaty h f, a l-fatlawi zm. mirza k b, abdulla ma. prevalence and severity of periodontal disease among iraqi twin population. scientific research and essays 2011; 6(5): 1034–38. 2. riset kesehatan dasar. laporan nasional 2008. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan; 2008. p. 131–2. 3. taba m jr, janet k, amy sk, william vg. diagnostic biomarker for oral and periodontal disease. dental clinical north america 2005; 49(3): 551–6. 4. eyre rd, jiann-jiu wu. collagen cross-links. top curr chem 2005; 247: 207–29. 5. arican mö, köylu a, uyaroglu m, erol kn, çalim. diagnostic importance of deoxypyridinoline and osteocalcine in equine osteoarthritis. acta veterinary brno 2004; 73: 491–6. 6. kraenzelin em, claude ak, christian m, cecilia g, beat s. automated hplc assay for urinary collagen cross-link: effect of age menopause and metabolic bone diseases. clinical chemistry 2008; 54(9): 1546–53. figure 1. schematic overview of pyridinium cross link such as ictp and deoxypyridinoline was released into gingival crevicular fluid.14 106 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 102–106 7. dye ba, gina te. a brief history of national surveillance efforts for periodontal disease in the united states. j periodontol 2007; 78(suppl): 1373–9. 8. kinney js, christoph ar, wv. giannobile. oral f luid–based biomarkers of alveolar bone loss in periodontitis. ann ny acad sci 2007 1098; 230–51. 9. ciancio sg. taking oral health to heart: an overview. jada 2002; 133: 4s–6s. 10. reimseier ca, janet sk, amy eh, thomas b, james vs, charlie as, lindsay ar, huu mt, anup ks. identification of pathogen and host response markers correlated with periodontal disease. j periodontol 2009; 80: 436–46. 11. masunaga h, wataru t, hyun o, naoki s, noriyoshi k, yorimasa o. use of quantitative pcr to evaluate methods of bacteria sampling in periodontal patients. j oral sci 2010; 52(4): 615–21. 12. wulan a, widjijono, suryono. c-telopeptide pyridinoline level in gingival crevicular fluid as indicator of alveolar bone loss. dentika dent j (maj. ked. gigi) 2011; 16(1): 1–3. 13. herr ae, anson vh, william vg, daniel jt, huu mt, james sb, anup ks. integrated microfluid platform for oral diagnostics. ann ny acad sci 2007; 1098: 367–74. 14. ahuja t, dhakray v, mittal m, khanna p, yadav b, jain m. role of collagen in the periodontal ligament a review. the internet journal of microbiology 2012; 10(1). 15. armitage gc. analysis of gingival crevice f luid and risk of progession of periodontitis. periodontology 2000, 2004; 34: 109–19. 16. giannobile wv. host-response therapeutics for periodontal diseases. j periodontol 2008; 79: 1592-600. 17. reinhardt ra, julia as, lorne mg, hsi_ming l, pirkka vn, timo s, jeffery bp. association of gingival creivular fluid biomarkers during periodontal maintenance with subsequent progressive periodontitis. j periodontol 2010; 81: 251–9. 18. dreyer p, jose gilberto h v. bone turnover assessment: a good surrogate marker?. arq bras endocrinol metab 2010; 54(2): 99–105. 19. furness p, zimmern r, wright c, adam m. the evaluation of diagnostic laboratory test and complex biomarkers. summary of diagnostic summit 14–15 january 2008. available at: www.rcpath. org. accessed on august, 25, 2009. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 206 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 206–212 perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome siti fitria ulfah,1 darmawan setijanto,2 and taufan bramantoro2 1dental nursing program study, poltekkes kemenkes surabaya, surabaya indonesia 2departement of dental public health, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: the number of children with down syndrome in surabaya has reached 924 children. prevalence of gingivitis and dental caries (91% and 93.8%, respectively) occurs in children with down syndrome aged 6 to 20 years. oral and dental health problems are found in children with down syndrome because they have physical and motoric limitation in maintaining oral and dental hygiene, thus require parental care from mother. perceived parenting style includes responsiveness and demandingness. perceived parenting is crucial for mother whose children have down syndrome in order to guide their health behavior, particularly to maintain oral and dental health. purpose: the study aimed to analyze correlation between perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome. method: this cross sectional analytical study involved 40 mothers of children aged 7-13 years with down syndrome enrolled in special education elementary schools surabaya and association of parents of children with down syndrome surabaya. data of perceived parenting style (responsiveness and demandingness) and mother’s behavior in maintaining dental health were obtained by questionnaire. composition of each item in questionnaire of perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome was passed through validity and reliability test. data analysis was carried out using multiple linear regression correlation test. result: this present study showed that perceived parenting style is significantly correlated with mother’s behavior in maintaining dental health of children with down syndrome (r = 0.630, p = 0.000), with perceived parental responsiveness as a strong predictor. mean score and standard deviation of perceived parental responsiveness and demandingness were 33.00±2.99 and 15.55±1.99, respectively. conclusion: perceived maternal parenting style in children with down syndrome is closely related to mother’s behavior in maintaining dental health of children with down syndrome. keywords: perceived parental responsiveness; perceived parental demandingness; dental health maintenance behavior; children with down syndrome correspondence: taufan bramantoro, department of dental public health, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: taufan-b@fkg.unair.ac.id introduction down syndrome or trisomy-21 is congenital disorder characterized by an extra chromosome 21 on the chromosome 21 pair.1 this fenotype of triplicate expression is observed on 95% of trisomy 21, the remaining 5% shows a close correlation with other chromosomal disorders. in general, chromosome 21 is related to partial trisomy disorder/anomaly, mosaicism, and translocation.1 the number of down syndrome case in the world remains unknown, but it is estimated up to 8.000.000 cases. in indonesia, the number of people with down syndrome is about more than 300.000 (3.75%).2 according to statistic indonesia, the number of children with down syndrome in surabaya is about up to 924 children.2 people with down syndrome have different oral health problems compared to general population.3 approximately 96% of adults with down syndrome commonly have considerably high prevalence of periodontal disease.4 gingivitis affects 91% of children aged 6-20 years with down syndrome.3 in riyadh, children aged 11 years on average with down syndrome had more gingivitis (46.9%) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 207207ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 than control group/normal children (34%) in all sextants of the mouth except the mandibular middle sextants.5 prevalence of poor oral and dental hygiene in children aged 7 to 12 years with down syndrome was significantly higher (87.5%) than normal children.6 individuals with down syndrome require assistance from parents and caregivers in order to maintain their daily oral and dental health.7 parents and caregivers usually face oral health care problems in children with down syndrome. comprehensively oral health care is indispensable in order to obtain an optimal care.8 mother’s perception on children with down syndrome provides significant impact on their oral and dental hygiene practice. mother’s perception concerning impact of oral and dental health in children with down syndrome plays an important role in their social contact.9,10 global perceptions of parenting children with down syndrome were less positive than those of parenting normal children.11 korean mothers of children with down syndrome experienced two perceived parenting aspects, positive and negative, in raising their children. in addition, mothers of children with down syndrome in some regions of korea showed more attention to their children. mothers tend to be the primary caregivers for children with down syndrome in korea.12 parenting children with down syndrome is considered less favorable and more expensive (i.e, financial, emotional, social) than parenting children with no disabilities.11 perceived parenting style has two dimensions, that are, responsiveness and demandingness.13 parental care of children with down syndrome is more focused on parental responsiveness.14 responsiveness is the families’ ability to respond to the demands and needs of children in an accepting and supportive way by supporting the individuality and assertiveness of the child.14 perceived parental responsiveness is perceived parenting style that includes warmth, open-communication, and care. warmth is the family’s ability to assert the love they feel towards their children sentimentally, emotionally and empathically within the family.15 parental responsiveness is shown through motivation on children in order to support children’s development of internalized moral orientation.15 in addition to demonstrate parental responsiveness, parents of children with down syndrome also need to apply parenting style in such discipline, firm, and controlling way.16 parents who give contribution of perception in discipline, firm, and controlling way are called perceived parental demandingness. perceived parental demandingness is perceived parenting style that highlights monitoring, demandingness, and control of the children’s behavior.13 parental care given by parents to their children reflects an behavior, strategy applied by parents themselves to achieve goal in any certain situations.17 parents’ behavior in maintaining oral health of children with special needs plays an important role in oral health of their children.18 parents and caregivers particularly mother of children with down syndrome should guide their children how to practice tooth brushing skill and use of flouride-containing toothpaste to improve oral hygiene of children with down syndrome.7 according to the existing phenomena and supported by study results, we need to analyze the correlation between perceived maternal parenting style and mother’s behavior in maintaining dental health of children with down syndrome in special education elementary schools surabaya and association of parents of children with down syndrome surabaya. materials and methods this study was observational analytical quantitative study with cross sectional study design which investigates dynamic of correlation between risk factors and effects. this present study involved mothers of children aged 7-13 years with down syndrome enrolled in special education elementary schools surabaya and association of parents of children with down syndrome surabaya. total respondents involved in this study were 40 mothers of children with down syndrome. sampling technique was performed as simple random sampling. inclusion criteria in this study as follows: 1) mother of children with mild and moderate down syndrome; 2) mother of children aged 7-13 years with down syndrome; 3) education level of mother of children with down syndrome is minimum graduated from senior high school; 4) mothers who are full-time in taking care of their down syndrome children. this study used questionnaire as instrument to measure perceived maternal responsiveness and demandingness, and mother’s behavior in maintaining dental health of children aged 7-13 years with down syndrome. assessment of perceived maternal responsiveness was based on four dimensions included communication, support promotion, expressing affection and harmony in the motherchild relationship.12,13,19 meanwhile assessment of maternal demandingness was based on three dimension included monitoring, demandingness, and control of children’s behavior. assessment of mother’s behavior in maintaining dental health of children aged 7-13 years with down syndrome was based on questionnaire instrument of chand et al.20 answers for each statement item about mother’s perception of children with down syndrome shown by options “strongly agree, agree, disagree, strongly disagree”. scoring for each answer option was 4 for strongly agree; 3 for agree; 2 for disagree; and 1 for strongly disagree. answer options for measurement of mother’s behavior were “very often, fairly often, sometimes, and never”. scoring for each answer option was 4 for very often; 3 fairly often; 2 for sometimes; and 1 for never. composition of each item in questionnaire of perceived parental responsiveness and demandingness was adopted from perception of parenting questionnaire;13 perceived maternal styles;15 malaysian (sarawak) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 208 ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 mothers’perspectives;19 maternal perceptions to openended questions about life with down syndrome in korea;12 parenting a child with down syndrome;21 impact of mothers’ oral hygiene knowledge and practice on oral hygiene status of their 12-year-old children.20 before questionnaire instrument passed through realibility and validity test, researcher had involved some experts to compose item of questionnaire, such as public health dentist, pedodontist, and child psychologist. involvement of these experts aimed to obtain explisitness, sequence of relevance, and comprehensiveness on all items in questionnaire. after all items in questionnaire had been composed, researchers performed validity and reliability test. in those tests, researchers involved 30 mothers of children with down syndrome to fill out the questionnaire. those respondents were mothers of children with down syndrome, members in association of parents of children with down syndrome surabaya. location of study was at special education elementary schools surabaya and association of parents of children with down syndrome surabaya. study period started from may-juny 2016. this study had been approved by ethics committe of faculty of dentistry, airlangga university. the analysis used distribution table which confirmation shown as precentage.22 data analysis in this study used multiple linear regression correlation test (table 5). results results obtained from questionnaire of perceived maternal parenting style in children with down syndrome consisted of parental responsiveness and demandingness. there were 4 dimensions in perceived parental responsiveness which consisted of 9 items of questionnaire. whereas perceived parenting demandingness had 3 dimensions which consisted of 5 items of questionnaire. according to validity and reliability test, cronbach’s alpha of perceived parental responsiveness, demandingness, and mother’s behavior in maintaining dental health of children with down syndrome were 0.858, 0.754 and 0.813, respectively. the result of reliability test showed that each instrument had higher value of cronbach’s alpha than critical value for two tailed correlation coefficient with the number of sample as 30 subjects, r=0.374. assessment of questionnaire items in perceived maternal responsiveness of children with down syndrome after passed through the reliability and vaidity test consisted of several items, included: “i reassure my child when he/she is afraid, i cheer him/her up when he/she is sad”, “i treat him/her as a friend, i pay attention to what he/she should eat and drink”, “i pay attention to his/her need of self-care, such as bathing, defecation, urination, and menstruation”, “i hug and kiss my child, i am happy when my child could speak a few words that are understandable by anyone”, “i spend my strength and time to take care of my child”, “i spend a lot of time to assist my child in learning (for example: speaking, writing, reading, etc)”. assessment of questionnaire items in perceived maternal demandingness of children with down syndrome after passed through the reliability and validity test consisted of several items, included: “i threaten my child with scary things when he/she is fussy, stubborn, disobedient, very demanding”, “i ignore for a while if he/she keeps on fussy, stubborn, disobedient, very demanding”, “i yell loudly when my child keep crying, very demanding”. “i watch my child when he/she is in public areas”, “i watch my child because i worry about things that could possibly harm him/her”. assessment of questionnaire items of mother’s behavior in maintaining dental health of children with down syndrome after passed through reliability and validity test consisted of several items, included: “i clean up my child’s teeth”, “i watch my child when he/she clean up his/her teeth and mouth”, “i clean up my child’s teeth using toothbrush and toothpaste”, “i clean up my child’s teeth twice daily”, “i clean up my child’s teeth only at night before go to bed”, “i clean up my child’s teeth in the morning after breakfast and at night before go to bed”. in the present study, characteristics of children with down syndrome and their mothers were presented. majority of mothers of children with down syndrome, i.e 26 of them (65%), aged 40-54 years. majority of educational level of respondents (n=30; 75%) was graduated from senior high school. employment status of all respondents (n=40; 100%) was homemaker. majority of gender of children with down syndrome was female (n=23; 57.5%). ten children with down syndrome aged 7 years (n=10; 25%). there were 36 children had moderate level of down syndrome (n=36; 90%). in subscale of reassuring children when they are afraid, majority of the respondents stated strongly agree (n=29; 72.5%), and only one of them stated strongly disagree (n=1; 2.5%). in subscale of cheering up the children when they are sad, majority of the respondents stated strongly agree (n=30; 75%), and only one of them stated strongly disagree (n=1; 2.5%). in subscale of treating the children as friend, approximately 70% or 28 respondents stated strongly agree, and there were still few respondents stated disagree to treat their children as friend. in subscale of paying attention to what their children should eat and drink, approximately 77.5% or 31 respondents stated strongly agree, only one of them or 2.5% stated disagree to pay attention to what their children should eat and drink. table 5.3 shows subscale of children’s need of self-care such as bathing, defecation, urination, and menstruation. those who stated strongly agree were 67.5% or 27 respondents, and agree were 32.5% or 13 respondents. in subscale of hugging and kissing the children with down syndrome, there were 67.5% or 27 respondents stated strongly agree, 32.5% or 13 respondents stated agree. approximately 87.5% of respondents stated strongly agree that they are happy when their children dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 209209ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 could speak a few words that are understandable by anyone. approximately 21% of respondents stated agree to spend strength and time to take care of their children. approximately 60% or 24 respondents stated agree to spend a lot of time to assist their children in learning (for example: speaking, writing, reading, etc) (table 1). there were 45% or 18 mothers of children with down syndrome who stated agree to threaten their children with scary things when children are fussy, stubborn, disobedient, and very demanding, but there was also one of them or 2.5% stated disagree. many respondents, approximately 60% or 24 of them stated disagree to ignore their children for a while when the children keep on fussy, stubborn, disobedient, and very demanding. many mothers of children with down syndrome stated agree, approximately 60% or 24 respondents, to yell loudly when the children keep crying and very stubborn. but there were 12.5% or 5 respondents stated disagree to yell loudly when the children keep crying and very stubborn. many respondents, approximately 52.5% of them stated strongly agree to watch their children when going in public areas. respondents who stated strongly agree to watch their children because they worry about things that could possibly harm the children were 50% or 20 respondents (table 2). there were 62.5% or 25 mothers of children with down syndrome who often clean up their children’s teeth, even there were 12.5% or 5 mothers of children with down syndrome who never clean up their children’s teeth. mothers of children with down syndrome who often assist their children to clean up the teeth were 57.5% or 23 respondents, but there were also 12.5% or 5 respondents who never assist their children to clean up the teeth. mothers of children with down syndrome who often watch their children cleaning up the teeth and mouth were 70% or 28 respondents, and also there were 10% or 4 respondents who never watch their children cleaning up the teeth and mouth. there were 62.5% or 25 mothers of children with down syndrome who often clean up their children’s teeth using toothbrush and toothpaste, even there were 10% or 4 respondents who never use toothbrush and toothpaste table 1. frequency distribution of perceived maternal parenting responsiveness for children with down syndrome item strongly agree agree disagree strongly disagree n % n % n % n % reassuring my child when he/she is afraid 29 72.5 10 25 1 2.5 0 0 cheer up my child when he/she is sad 30 75 9 22.5 1 2.5 0 0 treating my child as friend 28 70 10 25 2 5 0 0 paying attention to what my child should eat and drink 31 77.5 8 20 1 2.5 0 0 paying attention to self-care needs of my child such as bathing, defecation, urination, menstruation 27 67.5 13 32.5 0 0 0 hugging and kissing my child 27 67.5 13 32.5 0 0 0 0 feeling happy when my child could speak a few words that are understandable by anyone 35 87.5 5 12.5 0 0 0 0 spending strength and time to take care of my child 18 45 21 52.5 0 0 1 2.5 spending a lot of time to assist my child in learning (for example: speaking, writing, reading, etc) 24 60 15 40 0 0 1 2.5 table 2. frequency distribution of perceived maternal parenting demandingness for children with down syndrome item strongly agree agree disagree strongly disagree n % n % n % n % threatening my child with scary things when he/she is fussy, stubborn, disobedient, and very demanding 13 32.5 18 45 8 20 1 2.5 ignoring for a while if he/she keeps on fussy, stubborn, disobedient, very demanding 3 7.5 11 27.5 24 60 2 5 yelling loudly when my child keep crying, and very demanding 10 25 24 60 5 12.5 1 2.5 watching my child when he/she is in public areas 21 52.5 19 47.5 0 0 0 0 watching my child because i worry about things that could possibly harm him/her 20 50 20 50 0 0 0 0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 210 ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 to clean up their children’s teeth. in addition, there were 55% or 22 mothers of children with down syndrome who often clean up their children’s teeth twice daily. tehre were 45% or 18 mothers of children with down syndrome who never clean up their children’s teeth at night before go to bed. total of mothers of children with down syndrome who often clean up their children’s teeth in the morning after breakfast and at night before go to bed were 37.5% or 15 respondents, and those who never were 17.5% or 7 respondents (table 3). perceived maternal responsiveness in children with down syndrome reached high score, as it approached the highest score of 36, with mean score of 33.00 and standard deviation of 2.99. perceived maternal demandingness in children with down syndrome reached high score, as it approached the highest score of 20 with the mean score of table 3. frequency distribution of mother’s behavior in maintaining dental health of children with down syndrome item very often fairly often sometimes never n % n % n % n % i clean up my child’s teeth 4 10 25 62.5 6 15 5 12.5 i assist my child to clean up his/her teeth 5 12.5 23 57.5 7 17.5 5 12.5 i watch my child when he/she clean up his/her teeth and mouth 6 15 28 70 2 5 4 10 i clean up my child’s teeth using toothbrush and toothpaste 10 25 25 62.5 1 2.5 4 10 i clean up my child’s teeth twice daily 7 17.5 22 55 6 15 5 12.5 i clean up my child’s teeth only at night before go to bed 2 5 8 20 12 30 18 45 i clean up my child’s teeth in the morning after breakfast and at night before go to bed 8 20 15 37.5 10 25 7 17.5 table 4. description of results of mean score and standard deviation of mother’s perception and behavior in maintaining dental health of children with down syndrome variable n mean standard deviation perceived parental responsiveness 40 33.00 2.99 perceived parental demandingness 40 15.55 1.99 behavior 40 18.55 4.74 table 5. correlation between perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome perception n behavior p value perceived parental responsiveness 40 0.000** perceived parental demandingness 40 0.642 15.55 and standard deviation of 1.99. mother’s behavior in maintaining dental health of children with down syndrome showed low score, far from the highest score of 28, with mean score of 18.55 and standard deviation of 4.74 (table 4). in addition, result of data analysis using multiple linear regression showed that there was strong correlation between perceived maternal parenting style and mother’s behavior in maintaining dental health of children with down syndrome (r=0.630, p=0.000). strong predictor that influence perceived maternal parenting style in children with down syndrome and mother’s behavior in maintaining dental health of children with down syndrome was perceived maternal responsiveness. discussion perceived parenting style defined as an opinion of children about styles of parental behaviors during their childhood.23,24 perceived parenting style is related to parental behaviors that parents use to socialize their children.25 in this study we discuss about perceived maternal responsiveness and demandingness in children with down syndrome in relation to mother’s behavior in maintaining dental health of children with down syndrome. according to the study result, there is strong correlation between perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome, wherein the strongest predictor is perceived parental responsiveness. parents with responsiveness style are those who always show affection, often smile, give appreciation, and encourage their children.13 respondents always reassure when their children are afraid, cheer up their children when they are sad, treat them as friend, hug, kiss and pay attention to what their children should eat and drink. in addition, respondents strongly agree to pay attention to self-care of their children such as bathing, defecation, urination, and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 211211ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 menstruation. the most challenging part in self-care of children with down syndrome experienced by respondents is when their daughter get menstruation. girls with down syndrome could not use pads at all, they could only stare at blood stains on pants.19 respondents strongly agree that they feel happy when children with down syndrome could speak a few words that are understandable by anyone. many mothers of children with down syndrome feel very happy when their children could speak a few words that are understandable by anyone.26 in perceived maternal responsiveness of children with down syndrome, total of mothers who agree to spend strength and time to take care of their children were approximately 21%. in korea, this is considered as family responsibility, which means that family is responsible for additional needs related to care for them.27 korean mothers of children born with down syndrome experienced any difficulties in caring for their children.27 respondents agree to spend a lot of time to assist their children in learning (for example: speaking, writing, reading, etc). in a qualitative study, it had been found that there was a chinese mother trying to increase learning potential of her child that born with down syndrome, as her child experienced delay in learning.19 in addition to perceived maternal responsiveness style in children with down syndrome, it was also found in respondents answers which still applied perceived parenting demandingness. perceived parenting demandingness is a perception that describes standards set by parents are related to parental control of child’s behavior.13 in the present study we found that many mothers of children with down syndrome stated agree to yell loudly when their children keep crying and very stubborn. it is possible that when parents are more hostile, critical, irritable, and harsh, children do not feel like they are important or competent.28 further, when parents express a dislike or aversion to their children, the parents might be less likely to be actively engaged in their children’s development that lead to increased negative behavioral problems. whereas in this study, mothers of children with down syndrome were homemakers that should pay attention to their children’s needs. although many mothers of children with down syndrome stated agree to yell loudly when their children keep crying, very stubborn, but they keep watching and monitoring their children. it is shown by their answers which stated strongly agree to watch their children when going in public areas, as they worry about things that could possibly harm their children. mothers of children with down syndrome consider their children are susceptible to the danger, because there are barriers of social contact thus mothers try to watch their children intensively.29 scoring for mother’s behavior in maintaining dental health of children with down syndrome in the present study appears low. this is due to many mothers of children with down syndrome did not assist their children to clean up their teeth at night before go to bed. whereas the optimal time to brush teeth is after breakfast and before go to bed.30 the reasons of someone apply health behavior or not are thoughts and feelings that reflect knowledge, perception, attitude, belief and assessment regarding health.31 in this term, perceived maternal parenting style of children with down syndrome is crucial to influence mother’s behavior in maintaining dental health of their children born with down syndrome. perceived parenting style is closely related to parental behavior in caring, raising, and educating children.32 parenting comprises care practices used by parents of children up to three years old, as well as beliefs they hold and that guide their behavior.13 the more mothers engaged in good dental health-related behavior for themselves, the more likely they were to know more about promoting their children’s oral health.33 it could be concluded that perceived maternal parenting style in children with down syndrome that mostly applied is perceived parental responsiveness compared to demandingness. perceived parental responsiveness applied by mothers of children with down syndrome has shown correlation with mother’s behavior in maintaining dental health of their children. the results of this study are expected to be a basis of development of promotive and preventive approach for dental health of children with down syndrome, either by dentists or other dental health professionals. references 1. galley, r. medical management of the adult patient with down syndrome. jaapa 2005; 18(4): 45-6, 48, 51-2. 2. mulia a, kristi e. fasilities of kids therapy down syndrome in surabaya. jurnal edimensi arsitektur 2012; 1: 1-6. 3. amaral loureiro ac, oliveira costa f, eustáquio da costa j. the impact of periodontal disease on the quality of life of individuals with down syndrome. downs syndr res pract 2007; 12(1): 50-4. 4. shore s, lightfoot t, ansell p. oral disease in children with down syndrome: causes and prevention. community pract 2010; 83(2): 18-21. 5. oredugba f. oral health condition and treatment needs of a group of nigerian individuals with down syndrome. down’s syndrome. research and practice 2007; 12(1): 72-6. 6. al sarheed. comparative study of oral health among trisomy 21 children living in riyadh, saudi arabia: part 2, gingival condition. the saudi dental journal 2015; 27: 224–7. 7. haliza t, normastura ar, azizah y. dental anomalies and oral hygiene status of down syndrome children. international journal of public health and clinical sciences 2015; 2(3): 33-41. 8. al-maweri s, al-sufyani g. dental caries and treatment needs of yemeni children with down syndrome. dent res j (isfahan) 2014; 11(6): 631–5. 9. oliveira ac, pordeus ia, luz cl. mothers’ perceptions concerning oral health of children and adolescents with down syndrome: a qualitative approach. european journal of paediatric dentistry 2010; 11(1): 27-9. 10. bracksley s, dickson-swift v, anderson k, gussy m. an exploration of mothers’ perceptions about dental health. journal of theory and practice of dental public health 2013; 1(1): 9-14. 11. lawson k l. expectations of the pa renting exper ience and willingness to consider selective termination for down syndrome. journal of reproductive and infant psychology 2006; 24(issue 1): 43-59. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 212 ulfah, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 206–212 12. choi h, riper, mv. maternal perceptions to open-ended questions about life with down syndrome in korea. the qualitative report 2016; 21(2): 288-98. 13. pasquali l, gouvea vv, santos ws, fonseca pn, andrade jm, lima tjs. perceptions of parents questionnaire: evidence for a measure of parenting styles. paidéia 2012; 22 (52): 155-64. 14. mahoney fp. the relationship between parenting stress and maternal responsiveness among mothers of childreen with developmental problems. dissertation. mandel school of applied social sciences. case western reserve university. 2009. 15. calik var e, kilic s, kumandas h. investigating opinions of mothers on different socioeconomic status in terms of perceived maternal styles. eurasian journal of educational research 2015; 61: 81-98. 16. goleman d. emotional intelligence (10th anniversary re-issue edn.). new york: bantam books; 2006. 17. stewart sm, bond mh, kennard bd, ho ln, zaman rm. does the chinese construct of guan export to the west?. international journal of psychology 2002; 37(2): 74–82. 18. smith ls, ree m, leonard h. oral health and children with an intellectual disability: a focus group study of parent issues and perceptions. journal of disability and oral health 2010; 11(4): 171-7. 19. chan kg, lim ka, ling hk. care demands on mothers caring for a child with down syndrome: malaysian (sarawak) mothers’ perspectives. international journal of nursing practice 2014; 1-10. 20. chand s, chand s, dhanker k, chaudhary a. impact of mothers’ oral hygiene knowledge and practice on oral hygiene status of their 12-year-old children: a cross-sectional study. journal of indian association of public health dentistry 2016; 12(4): 323-6. 21. joosa e, berthelsen d. parenting a child with down syndrome: a phenomenographic study. school of early childhood. australia: queensland university of technology.victoria park road; 2006. p. 49-54. 22. arikunto s. research procedure, a practical approach. edition revision. jakarta: rineka cipta; 2010. 23. fonte ba. relationship between parenting style, emotional intelligence and self esteem. 2009. 24. lopes pn, brackett ma, nezlek jb, schütz a, sellin i, salovey p. emotional intelligence and social interaction. personality and social psychology bulletin 2004; 30(8): 1018-34. 25. kobarg apr, vieira v, vieira ml. validação da escala de lembranças sobre práticas parentais (embu). avaliação psicológica 2010; 9(1): 77-85. 26. choi h, riper, mv. maternal perceptions to open-ended questions about life with down syndrome in korea. the qualitative report 2016; 21(2): 288-98. 27. choi ek, lee yj, yoo iy. factors associated with emotional response of parents at the time of diagnosis of down syndrome. journal for specialists in pediatric nursing 2011; 16(2): 113-20. 28. phillip ab. a comparison of parenting dimensions between mothers of childreen with down syndrome and mothers of typically developing children. dissertation. tuscaloosa: department of psychology in the graduate school of the university of alabama; 2016. 29. lupton d, schmied v. the right way of doing it all’: first-time australian mothers’ decisions about paid employment. women’s studies international forum 2002; 25: 97–107. 30. wong dl, hockenberry m, wilson d, winkelstein ml, schwartz p. textbook of pediatric nursing. 6th ed. jakarta: egc; 2009. 31. budiharto. introduction health behavior and dental health education. jakarta: egc; 2010. 32. doinita ne, maria nd. attachment and parenting style. procediasocial and behavioral sciences 2015; 203: 199-204. 33. akpabio a, klausner cp, inglehart mr. mothers’/guardians’ knowledge about promoting children’s oral health. journal of dental hygiene 2008; 82(1): 8-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p206-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p206-212 p-issn: 1978-3728 e-issn: 2442-9740 volume 48, number 2, june 2015 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2015 january 2nd – december 31st, 2015 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga) editorial boards: prof. tri murni abidin, drg., m.kes., sp.kg(k) (department of conservative dentistry – faculty of dentistry, universitas sumatera); prof. dr. melanie s. djamil, drg., m.biomed (department of oral biology – faculty of dentistry, universitas trisakti); prof. dr. h. boedi oetomo roeslan, drg., m.biomed (department of biochemistry – faculty of dentistry, universitas trisakti); prof. dr. anita yuliati, drg., m.kes (department of dental material – faculty of dental medicine, universitas airlangga); prof. dr. istiati, drg., su (department of oral pathology and maxillofacial – faculty of dental medicine, universitas airlangga); prof. dr. adioro soetojo, drg., ms., sp.kg(k) (department of conservative dentistry – faculty of dental medicine, universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (department of periodontics – faculty of dental medicine, universitas airlangga); dr. retno pudji rahayu, drg., m.kes (department of oral pathology and maxillofacial – faculty of dental medicine, universitas airlangga); dr. theresia indah budhy, drg., m.kes. (department of oral pathology and maxillofacial – faculty of dental medicine, universitas airlangga); dr. indah listiana kriswandini, drg., m.kes. (department of oral biology – faculty of dental medicine, universitas airlangga); dr. retno indrawati, drg., msi. (department of oral biology – faculty of dental medicine, universitas airlangga); dr. rini devijanti ridwan, drg., m.kes. (department of oral biology – faculty of dental medicine, universitas airlangga); wisnu setyari, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); dr. hendrik setiabudi, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga). managing editors: dr. ira widjiastuti, drg., m.kes., sp.kg (department of conservative dentistry – faculty of dental medicine, universitas airlangga); udijanto tedjosasongko, drg., ph.d., sp.kga (department of pediatric dentistry – faculty of dental medicine, universitas airlangga); markus budi rahardjo, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); sianiwati goenharto, drg., ms (department of orthodontics – faculty of dental medicine, universitas airlangga); an’nisaa chusida, drg., m.kes. (department of odontology forensic – faculty of dental medicine, universitas airlangga); anis irmawati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); yuliati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); eric priyo prasetyo, drg., m.kes., sp.kg (department of conservative dentistry – faculty of dental medicine, universitas airlangga). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 contents page printed by: airlangga university press. (rk 163/06.15/aup-b3e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 48, number 2, june 2015 p-issn: 1978-3728 e-issn: 2442-9740 1. expression of bone morphogenetic protein-2 after using chitosan gel with different molecular weight on wound healing process of dental extraction sularsih and endah wahjuningsih ................................................................................................. 53–58 2. the role of ubiquinone supplementation on osteogenesis of non-vascularized autogenous bone graft irham taufiqurrahman, achmad harijadi, roberto m. simanjuntak, coen pramono d, and istiati ........................................................................................................................................... 59–63 3. expression of cd133 in various premalignant and proliferative lesions rahmi amtha, indrayadi gunardi, ferry sandra, and diah savitri ernawati ......................... 64–68 4. the effects of curcuma zedoaria oil on high blood sugar level and gingivitis juni handajani and dhinintya hyta narissi ................................................................................ 69–73 5. micronucleus frequency in exfoliated buccal cells from hairdresser who expose to hair products koh hui yee, alma linggar jonarta, and regina tc. tandelilin ............................................. 74–79 6. the level’s changing of transforming growth factor β2 during canine retraction in non-growing age patient adianti and ameta primasari ........................................................................................................ 80–83 7. fungal inhibitory effect of citrus limon peel essential oil on candida albicans iwan hernawan, desiana radithia, priyo hadi, and diah savitri ernawati ............................. 84–88 8. expression analysis of cd63 in salivary neutrophils and the increased level of streptococcus mutans in severe early childhood caries muhammad luthfi ........................................................................................................................... 89–93 9. the decrease of fibroblasts and fibroblast growth factor-2 expressions as a result of x-ray irradiation on the tooth extraction socket in rattus novergicus fatma yasmin mahdani, intan nirwana, and jenny sunariani .................................................. 94–99 10. the effects of golden sea cucumber extract (stichopus hermanii) on the number of lymphocytes during the healing process of traumatic ulcer on wistar rat’s oral mucous ira arundina, yuliati, pratiwi soesilawati, dian w damaiyanti, and dania maharani ........... 100–103 11. the cleanliness differences of root canal irrigated with 0.002% saponin of mangosteen peel extract and 2.5% naocl anis sakinah, laksmiari setyowati, and devi eka j .................................................................... 104–107 181 volume 45 number 4 december 2012 research report the role of actinobacillus actinomycetemcomitans fimbrial adhesin on mmp-8 activity in aggressive periodontitis pathogenesis rini devijanti ridwan department of oral biology faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: actinobacillus actinomycetemcomitans (a. actinomycetemcomitans) is gram negative and a major bacterial agent associated with aggressive periodontitis in young adult, this bacteria was an important factor in pathogenesis of aggressive periodontitis. a. actinomycetemcomitans possesses fimbriae with an adhesin protein that was the first bacterial molecules to make physical contact with host. purpose: the objective of this research was to analyzed the influence of a. actinomycetemcomitans fimbrial adhesin protein induction on mmp-8 activity. methods: the research was an experimental laboratory study, the step in this study were isolation and identification a. actinomycetemcomitans, characterize a. actinomycetemcomitans adhesin and study the role of a. actinomycetemcomitans adhesin in wistar rats. results: the result of this research on the role of adhesin in wistar rats after analysis with analysis of variance (anova) showed significant differences in the control group with group induction with a. actinomycetemcomitans, a. actinomycetemcomitans plus adhesin and adhesin. mmp-8 activity increased with induction a. actinomycetemcomitans and 24 kda a. actinomycetemcomitans adhesin. this fimbrial adhesin protein showed that a. actinomycetemcomitans has the ability to adhesion, colonization and invasion for host in aggressive periodontitis pathogenesis. conclusion: a. actinomycetemcomitans fimbrial adhesin protein induction increasing mmp-8 activity for aggressive periodontitis pathogenesis. key words: a. actinomycetemcomitans, adhesin, mmp-8, aggressive periodontitis abstrak latar belakang: a. actinomycetemcomitans merupakan salah satu bakteri gram negatif yang terkait dengan periodontitis agresif yang menyerang penderita usia muda dan merupakan faktor penting dalam patogenesis periodontitis agresif. a. actimycetemcomitans mempunyai fimbriae dengan protein adhesin yang merupakan molekul pertama dari bakteri untuk melakukan kontak fisik dengan host. tujuan: tujuan penelitian ini adalah menganalisis pengaruh induksi adhesin a. actinomycetemcomitans terhadap aktivitas mmp8. metode: penelitian ini merupakan studi eksperimental laboratori, langkah dari penelitian ini adalah isolasi dan identifikasi a.isolasi dan identifikasi a.identifikasi a. actinomycetemcomitans, isolasi dan karakterisasi adhesin a. actinomycetemcomitans dan uji peran adhesin a. actinomycetemcomitans di tikus wistar. hasil: hasil analisis menunjukkan adanya aktivitas mmp-8 yang meningkat bermakna di kelompok kontrol dibandingkan dengan kelompok dengan induksi a. actinomycetemcomitans, a. actinomycetemcomitans + adhesin dan adhesin. hal ini menunjukkan bahwa a. actinomycetemcomitans berkemampuan melakukan adesi, kolonisasi dan invasi pada host dalam patogenesis periodontitis agresif. kesimpulan: induksi protein adhesin a. actinomycetemcomitans dengan berat molekul 24 kda meningkatkan aktivitas mmp8 pada patogenesis periodontitis agresif. kata kunci: a. actinomycetemcomitans, adhesin, mmp-8, periodontitis agresif correspondence: rini devijanti ridwan, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya 60132, indonesia. email: devi.rini@yahoo.co.id 182 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 181–186 introduction aggressive periodontitis is a disease of the tooth supporting tissues characterized by rapid destruction of periodontal ligament and alveolar bone in young patients, usually occurs at the age under 30 years. in the process of aggressive periodontitis tissue attachment loss and gingival recession four times faster than chronic periodontitis.1,2 the national survey results (riskesdas data 2007) showed that 90% of indonesia population suffers from periodontal disease are quite aggressive, since 20.3% of east java residents had problems of the teeth and mouth.3 the pathogenesis of aggressive periodontitis are caused by the interaction between host and bacterial factors. major bacterial in aggressive periodontitis are dominated by actinobacillus actinomycetemcomitans and now better known as aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans). the presence of these bacteria in dental plaque associated with aggressive periodontal tissue damage and aggravated by the presence of genetic and environmental factors.4 matrix metalloproteinase (mmp) levels of saliva increased in patient with aggressive periodontitis.5 mmp is an endopeptidase which is an important mediator of the inflammatory tissue damage that breaks most of the extracellular matrix and basement membrane proteins at physiological ph and temperature, especially as tissue damage in periodontitis. among mmps, collagenase (ie: mmp-1, mmp-8, and mmp-13) are the largest matrix metalloproteinases and have the intertitial capacity on collagen damage. mmp-8 is secreted by neutrophils in inactive form and become active when the periodontal tissues inflamed. activation of mmp-8 can be induced by inflammatory mediators such as il-1β, tnfα and derivatives microbial proteases, and reactive oxygen species (ros) produced by neutrophils induced.6 in chronic periodontitis collagenase activity of mmp-8 about 90-95% of the gingival crevicular fluid.2 at the time of being aggressive periodontitis, the activity of mmp8 in gingival crevicular fluid (gcf) and significantly increased mmp-8 causes damage to periodontal tissues and alveolar bone. it is not yet known the role of adhesin a. actinomycetemcomitas in the pathogenesis of aggressive periodontitis, so it is necessary to investigate the role of a. actinomycetemcomitans adhesin against mmp-8 activity. materials and methods the bacterial strain used in this study was a. actinomycetemcomitans clinical isolate. bacteria were grown in actinobacillus actinomycetemcomitans growth medium (aagm) and incubated at 37 ºc anaerobically for 24 hours. identification of a. actinomycetemcomitans on aagm plates based on gross morphology such as adherence to the medium surface, a starlike inner structure and positive catalase. the identification of a. actinomycetemcomitans confirmed using the microbact system and pcr. from the cellular bacteria pellet, genomic dna was extracted using pcr which was performed by adding 1 μl dna to a reaction mixture (50 μl final volume) containing 20 nmol of each primer, 40 nmol of deoxynucleotide triphosphates and 1u of taq polymerase. the following cycling conditions were used: denaturation at 94 ºc for 1 minutes, annealing at 42 ºc for 2 minutes, and elongation at 72 ºc for 3 minutes. finally, 10 minutes elongation at 72 ºc followed 22 cycles of amplification. the pcr products were purified with the qiaquick pcr purification kit (qiagen, valencia, ca). for clinical evaluation, subgingival plaque samples from patients with periodontitis were obtained by inserting a sterile endodontic paperpoint into the subgingival site for 10 seconds. the paper point was transferred into 200 ml of phosphate buffer saline (pbs) and centrifuged at 15.000 rpm at 4 ºc for 5 minutes. after denaturation at 96 °c for 2 minutes, a total of 25 pcr cycles were performed; each cycle consisted of 15 seconds of denaturation at 94 °c, 30 seconds of annealing at 54 °c, and 60 seconds of extension at 72 °c. amplification products were loaded into 1.8% (wt/vol) agarose gels by electrophoresis, stained with ethidium bromide (0.5 mg/ml), and photographed under uv light.7 a. actinomycetemcomitans culture in 250 ml aagm was added with 10 ml of 3% tricholoro acetic acid (tca) and allowed to stand for 30–60 minutes, then centrifuged at 6,000 rpm at 4 ºc for 15 minutes. supernatant was discarded and the sediment suspended in 50 ml pbs ph = 7.4 and fimbriae was cut using omnimixer cutting tools. when cutting, the suspension of bacterial fimbriae was placed in cooled vessel. cutting was performed for 1 minute and resting for 30 seconds (for cooling). this process was done for 5 times. each of these pieces were centrifuged at 12.000 rpm for 15 minutes and the supernatant (pieces fimbriae) were stored. figure 1. pcr profiles of a. actinomycetemcomitans clinical isolates. 12 lane 1-8 : s1 8: sampel 1 – 8 a.a : a. actinomycetemcomitans m : marker: fermentas 100 . pcr profiles of a. actinomycetemcomitans clinical isolates. 12 lane 1-8 : s1 8 : sampel 1 – 8 a.a : a. actinomycetemcomitans s1 s2 s3 s4 s5 s6 s7 s8 a.a m 327 pb 183ridwan: the role of actinobacillus actinomycetemcomitans fimbrial adhesin a 20 ml sample plus 20 μl reducing sample buffer (rsb) included in eppendorf was heated for 5 minutes in boiling water. enter the sample on the electrophoresis gel wells running the sample at 120 v for 90 minutes. lift the gel, perform staining with coomassie brilliant blue r 250 on shaker for 20-30 minutes. after that, gel was transfered into the destaining solution overnight in a shaker until the gel looks clean and then calculated the protein molecular weight in protein bands that appear on the gel. the result of sds-page in the form of the protein bands was performed hemagglutination test and continued with elution of mice erythrocytes to demonstrate the presence of protein haemagglutinin. several studies have shown that bacterial adhesin played by hemagglutinin protein. the hemagglutination test was done to found the hemagglutinin protein on a.actinomycetemcomitans bacteria with fimbrial adhesin proteins from sds-page results. a.actinomycetemcomitans fimbrial adhesin protein was reacted with the erythrocytes of mice then seen the hemaglutination titers. at first step, the mice erythrocytes were washed 3 times with pbs ph 7.4 and then made into 0.5% suspension and included 50 ml pbs. into the first well was added 50 ml protein fimbriae, subsequent serial dilution was made into the next wells, except wells-12 is used as a control (without protein sample). then into each well was added 50 ml of erythrocyte suspension, shaken in 15 minutes and then left in the room temperature until visible results was obtained. hemagglutination assay results for the sample is read when the control wells had visible results. proteins with the highest titer used for subsequent studies. to determine the presence of certain protein in the gel used western blotting method with antibody anti adhesin. a. actinomycetemcomitans bacteria was cultured in aagm medium, at 37 °c for 4-5 days. liquid culture was centrifuged at 6000 rpm, at 4 °c for 15 min. the precipitate was suspended in pbs containing bsa 1%. hemaglutin fimbriae protein dose were divided respectively into 0 mg (control), 25 mg, 50 mg, 100 mg, 200 mg and 400 mg. furthermore, for each dose protein fimbriae added enterocytes suspension of 300 ml and shaken on water bath at 37 °c for 30 minutes. then the mixture was added to the bacterial suspension (108/ml) of 300 μl. the mixture was incubated on the ‘shaking incubator’ for 30 min at 37 °c. furthermore centrifuged 1500 rpm, at 4 °c for 3 minutes, then washed sediment using pbs twice. the precipitate was taken, and stain with gram staining. preparations were observed under a microscope 1000x magnification, and the number of bacteria that attach to the enterocytes were counted. adhesion index was the average number of bacteria that attach to hela cells and was calculated until on 100 hela cells for every observation.8 ten weeks old male wistar rats with 120–150 grams weight were divided into 4 groups, each group consists of 10 rats. in group one was the control group, induced with 0.9% nacl, the group 2 induced with adhesin, group 3 induced with adhesin + a. actinomycetemcomitans and group 4 induce with a. actinomycetemcomitans whole cell.9 before the treatment, it was examined a. actinomycetemcomitans in the rat oral cavity. adhesin was induced in rat by giving 200 ml a. actinomycetemcomitans adhesin with a protein content of 200 ug/ml at 108 a. actinomycetemcomitans density and given at least 7 days to get real aggressive periodontitis symptoms.10 induction done on the upper right of first molar gingival sulcus of wistar rats.11 then examination to determine the severity of periodontal tissue destruction and alveolar bone through mmp-8 activity with zymogram analysis. anova are used to data analysis for mmp-8 activity. results the profile result of a. actinomycetemcomitans identification in this study using polymerase chain reaction (pcr) (figure 1). pcr examination for a. actinomycetemcomitans was done after identification tests using microbiology, biochemistry and performed with scanning electron microscopy (sem). three from eight samples of a.actinomycetemcomitans dna from pcr profiles showed positive reaction, i.e, sample number two (s2), three (s3) and five (s5). we used sample number 2 (s2) to isolated and identified as a. actinomycetemcomitans adhesin since this bacteria has rough colony and fimbriae. table 1. haemaglutination test result of fimbriae adhesin protein a. actinomycetemcomitans fraction fimbriae wells 1 (1/2) 2 (1/4) 3 (1/8) 4 (1/16) 5 (1/32) 6 (1/64) 7 (1/128) 8 (1/256) 9 (1/512) 10 (1/1024) 11 (1/2048) 12 (k) 60 kda + – – – – – – – – – – – 53 kda – – – – – – – – – – – – 42 kda – – – – – – – – – – – – 28 kda – – – – – – – – – – – – 24 kda + + + + + + – – – – – – 184 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 181–186 in this study, there were five fimbriae protein adhesin, at 60 kda, 53 kda, 42 kda, 28 kda and 24 kda. protein profile on sds-page of five pieces a. actinomycetemcomitans fimbriae protein using a stratified omnimixer showed a picture of the most prominent protein bands i.e. the protein with a molecular weight of 60 kda, 53 kda, 42 kda, 28 kda and 24 kda. the five proteins that exhibit prominent picture was collected and purification for electroelution then performed to obtain the protein solution. the protein with highest titer results from electroelusion test used to fimbriae adhesin protein hemagglutination test. table 1 showed the result of hemagglutination test performed to find proteins hemagglutinin (ha) from a. actinomycetemcomitans fimbriae after bacterial culture was cut by using a modification omnimixer for 5 (five) times. for the hemagglutinin protein of fimbriae fraction preceded by hemagglutination assay using rat erythrocytes, ½ titer obtained from fimbriae protein fraction with 60 kda molecular weight, 1/128 from the 24 kda fraction fimbriae protein and negative from 53 kda, 42 kda and 28 kda fractions fimbriae protein. further, analysis of the result from haemaglutination test for fimbriae adhesin protein with 60 kda and 24 kda done using western blotting test. western blotting assay was the specific method to determine the presence of certain proteins in the gel by using anti adhesin antibodies that was obtained from 24 kda fimbriae protein. the 24 kda fimbriae protein was a specific protein of fimbriae a. actinomycetemcomitans because on western blotting assay results obtained fimbriae protein bands at 24 kda. this band showed fimbriae protein 24 kda can be detected its existence because they are specific and have a high sensitivity. at 6.7 kda protein fimbriae there was no band, it shows that the 6.7 kda fimbriae protein did not have a strong ability to bind to anti adhesin antibodies because could not be reacted with the substrate. this condition also showed there was no cross-reaction between the protein fimbriae with 24 kda and 6.7 kda molecular weight because western blotting assay use polyclonal antibody from protein fimbriae with 24 kda and 6.7 kda molecular weight. result of adhesion test in hela cell culture in some dose i.e 400 μg/ml, 200 μg/ml, 100 μg/ml, 50 μg/ml and 0 μg/ml showed the decrease of a. actinomycetemcomitans amount that was attached to the surface of hela cells and obtained results were a significant reduction of the a. actinomycetemcomitans amount on hela cells with the increasing dose of fimbrial adhesin protein. the activity of mmp-8 in zymogram was aimed to identify mmp-8 through the degradation of the substrate and by molecular weight. the mmp-8 activity was performed by measuring the density of bands on sdspage by silver staining. the higher band density in zymogram showed the higher of mmp-8 activity. the result of zymogram assay with silver staining to measure mmp-8 activity is showed in figure 2. it was shown that mean of mmp-8 activity in group with a.actinomycetemcomitans induction was higher (60.4) than the group with adhesin + a. actinomycetemcomitans induction (43.7), adhesin induction (33.2) and control group (16.8) (table 2). one way anova test results showed the value of the activities of mmp-8 in the control group and the group with adhesin, adhesin + a. actinomycetemcomitans and a. actinomycetemcomitans induction were significantly different, p=0.001 (p<0.005) and then analyzed by tukey hsd test. analysis of tukey hsd found a significant differences from the activity of mmp-8 in the control group and the group treated with induction adhesin, adhesin + a. actinomycetemcomitans and a. actinomycetemcomitans and between the treatment groups. discussion five fimbriae adhesin protein have been identified from clinical isolates of a. actinomycetemcomitans by molecularby molecular mass at sds-page. the fimbrial adhesin were 60 kda, 53 kda, 42 kda, 28 kda and 24 kda, after haemaglutination table 2. the mean and standart deviations number of mmp-8 activity group x sd min max anova control 16.8a 0.5 16.5 17.1 f=490.56 adhesin 33.2b 0 33.2 33.2 p=0.001 adhesin+a. actinomycetemcomitans 43.7c 1.3 42.9 44.6 a. actinomycetemcomitans 60.4d 1.9 59 71.7 note: there different superscript indicates that there are significantly differences between groups (p <0.05) figure 2. mmp-8 activity of the group induction with a.actinomycetemcomitans showed the highest activity m adhesin+a.a c a.a adhesin 200 kda 140 kda 100 kda 70 kda 50 kda 40 kda 185ridwan: the role of actinobacillus actinomycetemcomitans fimbrial adhesin test we identified two fimbrial adhesin with positive test, that were 60 kda and 24 kda and the titre were ½ and 1/128. in western blotting assay result obtained fimbrial adhesin band at 24 kda molecular weight, this suggests that fimbrial adhesin protein could be detected its existence because it is specific and have a high sensitivity. adhesion index was calculated by counting the number of a. actinomycetemcomitans that was attached to the surface of hela cells. the results of the adhesion index showed significant reduction of the number of a. actinomycetemcomitans in hela cells with the increasing dose of fimbrial adhesin protein. the more protein that causes the point saturated on hela cell receptors will caused decrease ability a. actinomycetemcomitans to attach in hela cell. this condition could be said that the fimbrial adhesin protein of a.actinomycetemcomitans could prevent the attachment of a. actinomycetemcomitans on hela cells with methode to binding the 24 kda a. actinomycetemcomitans fimbrial adhesin protein to hela cell receptors. the result of haemaglutination, westernblotting and adhesion assay indicating that fimbrial adhesin protein at 24 kda molecular weight detected as a fimbrial adhesin protein for a. actinomycetemcomitans clinical isolate. mmp-8 activity was processed with zymogram to identify mmp-8 through the degradation of the substrate and by molecular weight. mmp-8 activities performed by measuring the density of bands on sds-page by silver staining, band density in zymogram results indicate a bond between the enzyme and the substrate to form a bond of enzyme-substrate complex (es complex). at es complex bonding, substrate bound to the active side region depicting the activity of enzymes that examined mmp-8. mmp8 activity can be measured when it is done by looking further reaction products produced or residual undigested substrate. the higher density obtained band showed that the activity of mmp-8 were higher. the results showed there was an mmp-8 activity increase in zymogram was significantly differed compared a. actinomycetemcomitans, a. actinomycetemcomitans + adhesin, adhesin induction and control groups. this suggests that the colonization and invasion of a. actinomycetemcomitans role in the stimulation of proinflammatory cytokine il-8 is secreted by monocytes, keratinocytes, endothelial cells and fibroblasts, this spending will stimulate mmp-8 by neutrophils. mmp-8 as a collagenase-2 which is a potential and important role in the degradation of connective tissue in the area of inflammation. mmp-8 is secreted in the form of not glycosylated one with 55 kda molecular weight or glycosylated secreted by 75 kda molecular weight and after activation will decrease with 10–20 kda of the molecular weight.12,13 mmp-8 is secreted in a latent form in the 75–80 kda and 55 kda molecular weight, become an active form in the 65 kda and 45 kda molecular weight.14 mmp-8 is released from neutrophils in a latent, inactive proform and becomes activated during periodontal inflammation by independent and/or combined actions of host-derived inflammatory mediators, such as tnfα and il-1β, and microbial-derived proteases and reactive oxygen species (ros) produced by triggered neutrophils. the molecular mass of mmp-8 varies in different publications between 50 and 85 kda, and forms as small as 20 kda have been reported reflecting different degrees of mmp8 glycosylation and/or whether the enzyme is in latent or activated/truncated form. naturally activated mmp-8 obtained from peripheral neutrophils can be detected by immunoblotting at 65–70 kda, and the mmp-8 in gingival crevicular fluid migrates primarily as a 60 kda form with smaller amounts of 78 kda species, corresponding to active and latent forms of the enzyme, respectively. mmp-8 is also the major collagenase present in inflamed human gingival tissue. extracts of periodontitis patients untreated gingival tissue in contrast to healthy subjects gingiva contain pathologically elevated levels of mmp-8 in a catalytically active form. mmp-8 is also the major mmp present in human mature dental plaque.15 mmp-8 is released in a latent form in periodontal inflammation as a result of stimulation by host derived inflammatory mediators such as il-1β, tnf-α, various periopathogenic bacteria and their virulence factors. the molecular weight of mmp-8 differs a lot according to cell source varying from 85 kda (sometimes even >100 kda), to smaller than 20 kda sizes. the proform of pmn typed mmp-8 can be detected in 75-80 kda and converted to 65 kda active form, whereas non-pmn type mmp-8 is detected in 55 kda and 45 kda for latent and active forms, respectively. activation can be proteolytic (e.g. by mmp-3) or non-proteolytic (initial activation by oxygen radicals).16 in conclusion, 24 kda fimbrial adhesin protein a. actinomycetemcomitans has a role in the increased of mmp8 activity in aggressive periodontitis pathogenesis. acknowledgment this research is supported by universitas airlangga (dipa universitas airlangga, penelitian unggulan perguruan tinggi tahun anggaran 2012). references 1. velden v, abbas f, armand s, loos bg, timmerman mf, weijden v. java project on periodontal diseases. the natural development of periodontitis: risk factor, risk predictors and risk determinants. j clin periodontol 2006; 33(8): 540–9. 2. newman mg, takei n, klokkevold p, carranza f. carranza’scarranza’s clinical periodontology. 10th ed. philadelphia, new �ork, london:philadelphia, new �ork, london: wb saunders co; 2006. p. 168–81, 409–14, 675–88. 3. laporan riset kesehatan dasar propinsi jawa timur. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia; 2007. p. 143. 4. stabholz a, soskolne wa, saphira l. genetic and environmental risk factors for chronic and aggressive periodontitis. periodontology 2000, 2010; (53): 138–53. 186 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 181–186 5. levine l, baev v, lev r, stabholz a, ashkenazi m. aggressiveaggressive periodontitis among young israeli army personennel. j periodontol 2006; 77(8): 1392–6. 6. sorsa t, tjdcrhanc l, salo t. matrix metalloproteinases (mmps) in oral apoptosis in cancer. am j pathology 2004; 153(4): 1041–8. 7. suzuki n, nakano �, �oshida �, ikeda d, koga t. identification of actinobacillus actinomycetemcomitans serotypes by multiplex pcr. j clin microbiol 2001; 39(5): 2002–5. 8. santoso s. protein adhesin salmonella typhii sebagai faktor virulensi berpotensi imunogenik pada produksi s-iga protektif. dissertation. surabaya: program pascasarjana universitas airlangga; 2002. p. 85–107. 9. schreiner h, markowitz k, miryalkar m, moore d, diehl s, fine dh. aggregatibacter actinomycetemcomitans induced bone loss and antibody response in three rat strain. j periodontol 2011; 82(1): 142–50. 10. zhou q, desta t, fenton m, graves dt, amar s. lps cytokineslps cytokines profiling of macrophage exposed to porphyromonas gingivalis, its lipopolysachcharide, or its fim a protein. infect immun 2005; 73(2):2005; 73(2):73(2): 935–43. 11. du m ist rescu a l . h istolog ica l compa r ison of p er iodont a l inflammatory changes in two models of experimental periodontitis the rat: a pilot study. tmj 2006; 56(2): 211–7. 12. moilanen m, pirila e, grenman r, sorsa t, salo t. expression and regulation of collagenase-2 (mmp-8) in head and neck squamous cell carcinomas. j pathol 2002; 197(1): 72–81. 13. moilanen m, sorsa t, stenman m, nyberg p, lindy o, vesterinen j, paju a, konttinen �t, stenman uh, sato t. tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (mmp-1, -8, -13) and stromelysin -1 (mmp-3) and degrades type i collagen. biochemistry 2003; 42(18): 5414–20. 14. korpi j. collagenase-2 (matriks metalloproteinase-8) in tongue squamous cell carcinoma, bone osteosarcoma, and wound repair. dissertation. acta university, 2010. p. 31–41. 15. mantyla p. the scientific basis and development of a matrix metalloproteinase (mmp) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid. dissertation. university of helsinki, 2006. p. 25–33. 16. tanzer ab. characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction. dissertation. university of helsinki, 2010. p. 13–39. � vol. 42. no. 1 january–march 2009 chronic gingivitis and aphthous stomatitis relationship hypothesis: a neuroimmunobiological approach chiquita prahasanti1, nita margaretha2, and haryono utomo3 1department of periodontics, faculty of dentistry, airlangga university, surabaya 2department of oral medicine, dentistry study program, brawidjaja university, malang 3dental clinic, faculty of dentistry, airlangga university, surabaya abstract background: traumatic injuries to the oral mucosa in fixed orthodontic patients are common, especially in the first week of bracket placement, and occasionally lead to the development of aphthous stomatitis or ulcers. nevertheless, these lesions are selflimiting. purpose: the objective of this study is to reveal the connection between chronic gingivitis and aphthous stomatitis which is still unclear. case: a patient with a persistent lesion for more than six months. case management: ras was treated with scaling procedure, the gingival inflammation was healed. however, in this case report, despite the appropriate management procedures had been done, the lesion still worsen and became more painful. moreover, the symptoms did not heal for more than two weeks. actually, they had been undergone orthodontic treatment more than six months and rarely suffered from aphthous stomatitis. coincidentally, at that time they also suffered from chronic gingivitis. it was interesting that after scaling procedures, the ulcer subsides in two days. conclusion: recently, the neuroimmunobiological researches which involved neurotransmitters and cytokines on cell-nerve signaling, and heat shock proteins in gingivitis and stomatitis are in progress. nevertheless, they were done separately, thus do not explain the interrelationship. this proposed new concept which based on an integrated neuroimmunobiological approach could explain the benefit of periodontal treatment, especially scaling procedures, for avoiding prolonged painful episodes and unnecessary medications in aphthous stomatitis. however, for widely acceptance of the chronic gingivitis and aphthous stomatitis relationship, further clinical and laboratory study should be done. regarding to the relatively fast healing after scaling procedures in this case report; it was concluded that the connection between chronic gingivitis and aphthous stomatitis is possible. key words: chronic gingivitis, aphthous stomatitis, orthodontic treatment, neuroimmunobiological approach correspondence: chiquita prahasanti, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132. e-mail: chiquita_prahasanti@yahoo.com. introduction hipocrates (460-370 ad) was the one who, for the first time, used the word “aphtha” (aphtodea), came from the greek word aptein (arteiv), translated as “set fire”. until today, the etiology of recurrent aphthous stomatitis (ras) remains unknown, although hints of its etiological basis lay on genetic susceptibility, infectious agents and alterations in immune mechanics.1 others are deficiencies of iron, vitamin b, or folate had been reported in patients with ras, but the data are conflicting.2 infection with various microorganisms had been suggested but not proven to be a contributing factor, although cross-reactivity between a microbial antigen and a homologous peptide (the heat shock protein theory) within the oral epithelium may play a role.2 various factors have been suggested to precipitate outbreaks of ras in predisposed persons, including oral trauma,, anxiety or stress, sensitivities to food (e.g., to preservatives), and hormonal changes related to the menstrual cycle. nevertheless, evidence to support the causative role of these factors is rarely.3 in addition, ras management does not change radically throughout time. recent management protocols according to scully in 2008 are still conventional medication such as topical corticosteroids, topical anti-inflammatory, topical antibiotics, and antiseptic mouthwash. if they failed, immunomodulators such as thalidomide could be used.4 case report �prahasanti, et al.: chronic gingivitis and aphthous stomatitis because of the uncertain etiology of ras, it may lead to inappropriate medication, a condition that may provide adverse effects after prolonged use; thus, other concept which related to oral homeostasis should be considered. it is also a common sense that in fixed orthodontic treatment, brackets and wires are to be blamed as a prominent causative factor of ras. nevertheless, the role of fixed orthodontic accessories (i.e. brackets, modules, chains) as bacterial adherence media which may provoke ras is often overlooked.5,6 we report two cases in which, ras was found in orthodontic patients who rarely experienced the disease before. at that time they also suffered from chronic gingivitis adjacent to the lesion. even though maintaining good oral hygiene is suggested in ras management, scaling procedure is not mentioned specifically in ras management protocol. in our cases the procedure resulted quicker healing compared to previous conventional treatment. however, the mechanism of the prompt healing of ras symptoms after scaling procedure is still unclear. current literatures discussed about the role of cytokines, neuropeptides and heat shock proteins (hsps) which have an important role in health and disease. the balance of these substances could be modified after exposure to stressors such as physiological (i.e. hormones), physical (i.e. trauma), pathophysiological (i.e. bacterial infection) and psychogenic.7 additionally, oral inflammation such as chronic gingivitis is able to stimulate interactions of immunogenic and neurogenic inflammation, so called the “neurogenic switching” mechanism.8 periodontal treatments reduce either local or systemic inflammation; i.e. by decreasing pro-inflammatory cytokines, neuropeptides8, 9 and hsps; furthermore, they also increase antiinflammatory mediators i.e. hsp10.10 pro-inflammatory hsps 60 and 65 are abundantly found in chronic periodontal disease.10 interestingly, ras is also related with increase response to these hsps.2, 11 however, until now, the relationship between chronic gingivitis and ras is still unclear. moreover, even though scaling is regarded as a simple and routine procedure, the relationship with the etiopathogenesis and the healing process of the ras lesion should be elucidated. hopefully, after further researches, scaling procedure could be explicitely included in ras management protocol. the objective of this case report is to discuss evidence– based cases which may explain a new hypothesis concerning the possible relationship between chronic gingivitis and ras. successful researches verifying this hypothesis may include scaling procedure in ras management protocol, therefore preventing from unnecessary medications. for this reason, since interaction of the neural, immune and biological system of the oral cavity is possible; this hypothesis will be elucidated through a neuroimmunobiological approach. case case 1: a 14-year-old male orthodontic patient suffered from recurrent ulceration on the lower labial oral mucosa. the orthodontic treatment began one and a half year before the ulcer occurred, when the teeth had already aligned. actually, he seldom complained about traumatic ulcer, if existed, it healed spontaneously. nevertheless, despite antiseptic mouthwash and topical corticosteroid treatment, within the last one month the ulceration persisted for more than 2 weeks. they were multiple ulcers on the lower labial mucosa, about 3–4 mm in diameter, and painful. coincidentally, at that time he also missed several regular appointments, thus oral hygiene could not be controlled as usual. intra oral examination revealed that the gingiva was bright red and severely inflamed, especially in the lower labial gingiva. at this region, the labial surface of the teeth were half covered with hyperthropic papillae and supragingival plaque. moreover, the lingual aspects of lower anterior teeth were covered with calculus. case 2: a 13-year-old female orthodontic patient complained of recurrent ulceration on the oral mucosa. the orthodontic treatment was started a year before, she had dental protrusion without crowding. after brackets placement, ulcerations developed and usually healed within 1–2 weeks. recently, ulcerations recurred and required more than 2 weeks to heal. ulcerations developed on the various site on the non-keratinized mucosa. the last ulcer was found on the lower right labial mucosa, adjacent to the bracket placed on 42, round in shape, shallow, about 4 mm in diameter, grayish white, surrounded by erythematous halo, and painful. self medication history for the oral ulceration included chinese herbs and commercial mouthwash (listerine®). the medication failed to promote healing. intra oral examination revealed that supra gingival calculus was found on the lingual and labial sides of the lower gingiva, accompanied by inflammation on the marginal gingiva. the diagnosis in these cases are ras and chronic marginal gingivitis. it is suggested that the gingival inflammation resulted from dental plaque and calculus is playing a role in the etiopathogenesis of both cases. case management the male patient initially came for regular orthodontic control when the persistent ulceration was reported. the last visit was two months before. since there was chronic gingivitis on the lower anterior gingiva, the first attempt was to reduce this lesion by scaling and not intentionally done for the ras. ras symptoms still treated by previous topical corticosteroid which actually was not very helpful in this case, and only acts for tissue protector. he was scheduled � dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 6-11 for another visit one week later and was instructed to maintain good oral hygiene. at the second visit, the result of scaling procedure was remarkable. the gingival inflammation was healed. interestingly, the ulcer also subsided. although it was still present but the diameter was smaller, only one mm, pink colored, and not painful anymore the lesion still present but only one mm in diameter and pink colored. the lesion did not elicit pain anymore. additionally, his oral hygiene was good. anamnesis revealed that the pain subsides significantly after two days, so did the lesion diameter. the patient was told to come to the dental office two weeks later for regular orthodontic control. after experiencing similar cases in several ras patients that responded well to scaling procedures, we became more convinced with this phenomenon. therefore scaling procedure was also done as the treatment of ras in the second case. chronological pictures of the lesions were also documented. these figures were shown: pre-treatment lesion (figure 1-a); 2 days after (figure 1-b); 5 days after (figure 1-c); one week after (figure 1-d). discussion chronic gingivitis is an inflammatory lesion of the soft tissues surrounding the tooth and a consequence of the local accumulation of dental plaque. it is a multifactorial disease, principally associated with infection by specific pathogenic organisms. stimulation by bacteria, cell surface molecules like lipopolysaccharides (lps) and bacterial metabolites to immunocompetent cells are responsible for the initiation and early development of gingivitis. these immunocompetent cells release an array of pro-inflammatory mediators i.e. interleukin 6 (il-6), prostaglandins.9 recurrent aphthous stomatitis is a common condition, restricted to the mouth. in these patients the lesions were diagnosed as minor aphthous ulcers (2 to 8 mm in diameter) and usually heal spontaneously in 10 to14 days.2 however, the lesions were still painful and had insignificant healing after more than 2 weeks. even though the exact immunopathogenesis of ras remains unclear, it probably involves cell-mediated mechanisms. macrophages, mast cells and t cells probably aid in the destruction of oral epithelium that is directed and sustained by local cytokine release.2,3 compared with control subjects, individuals with ras have raised serum levels of cytokines such as il–6 and il-2r. crossreactivity between a streptococcal hsps60 and 65, and the oral mucosa has been demonstrated, and significantly elevated levels of serum antibodies to hsps are found in patients with ras. it was predicted that ras may be a t cell–mediated response to antigens of streptococcus i.e. s sanguis, which cross-react with self-hsp and induce oral mucosal damage.3,11 interestingly, the human hsps 60 and 65 also implicated in the pathogenesis of periodontal disease and the involvement of t-cells response. albeit ras may be caused by the cross-reactivity between self-hsp and hsps from periodontopathic bacteria i.e. phorphyromonas a b dc figure 1. chronological intra oral photographs of apthous stomatitis resolution. �prahasanti, et al.: chronic gingivitis and aphthous stomatitis gingivalis;10 nevertheless, this interactions scarcely discussed. in chronic gingivitis, the inflammatory reaction is not purely immunogenic, since according lundy and linden, the role of nervous system, especially the primary afferent neurons should be taken into account. this neurogenic inflammation involved neurotransmitter, the neuropeptides. the pro-inflammatory neuropeptides are substance p (sp) and calcitonin gene-related peptide (cgrp); and the anti-inflammatory is vasoactive intestinal peptide (vip).8 studies have shown that most neuron expressing sp are nociceptors (related to pain), whereas those expressing cgrp may belong to either nociceptive or non nociceptive afferents.12 these neuropeptides increased expression in local nerve in the inflamed periodontium. in their animal study, saleem et al.,12 concluded that the up-regulated cgrp in periodontal disease may be principally associated with neuroimmune interaction rather than nociceptive spinal inputs. it is agreed by wadachi and hargreaves13 that chronic periodontal disease did not cause pain. saleem, et al,12 study revealed that the increase of cgrp after stimulation in one branch of the trigeminal nerve also propagate to the other branch; increase of cgrp also stimulates vip secretions to counteract.8, 14 moreover, there is a special characteristic of this anti-inflammatory neuropeptide which acts as a long-lasting vasodilator.14 if vip present persistently in the buccal or labial mucosa there should be a persistent mucosal inflammation that is prone to trauma from biting or brackets. several studies by kemmpainen et al.,15 support this proposed mechanism. kempainnen et al.,16 showed that pulpal stimulation which considered as neurogenic inflammation was able to induce lip vasodilation, nevertheless, the exact mechanism was still unclear. another study by kemmpainen et al.,16 also revealed that non-keratinized mucosal tissue is prone to nerve stimulation which leads to neurogenic inflammation. interestingly, either sulcular epithelium or labial mucosa are non-keratinized mucosa. therefore pseudopockets in chronic gingivitis facilitate the initiation of the neurogenic inflammation in inflammatory reaction, prostaglandin e2 (pge2) increases the sensitivity of primary afferent nerve fibers to stimuli.17 interestingly, that according to burt epidemiological study, chronic gingivitis had the higher pge2 level in gingival crevicular fluid than periodontitisonly.18 therefore, nerve stimulation in chronic gingivitis is considered easier than periodontitis-only or healthy periodontium. other literatures revealed that local inflammation could be propagated to distant organ via the nervous system, this mechanism often called as “neurogenic switching mechanism”.19 moreover, according to boyd, stimulation of the maxillary nerve in the nasal cavity also stimulates branches of the maxillary nerve in the temporal area via neurogenic inflammation.20 does it also happen in the mandibular nerve innervation area as in this case report?. periodontopathic bacteria and their products are able to stimulate immunocompetent cells which then initiate the neurogenic switching mechanism. lipopolysaharides (lps) from gram negative bacteria is able to stimulate these cells which then release mediators to activate primary afferent nerve endings.8 this mechanism also verified in saleem et al.,12 study, since lps injection in the mandibular gingiva also increase cgrp expression in the trigeminal ganglion, began with the mandibular followed by maxillary and ophthalmic branch. heat shock proteins (hsps) are normally intracellular proteins that have functions involved in protein folding and maintenance of protein integrity under both normal and stress conditions, yet if they are to act signals in response to environmental stresses (i.e. infection, temperature, ph, redox potential, physiological stress), they should be present in extra cellular environment.7 infection or other environmental stress stimulates self-hsps including the self-hsp60 (pro-inflammatory hsp) to appear, it is beneficial for stimulating macrophages to produce proinflammatory cytokines in an inflammatory process. unfortunately, hsp60 also act as lipopolycsaccharides binding protein (lbp) which increase lps immunogenicity, thus exaggerate the inflammatory process, which is an unwanted effect.21 local and systemic hsps are increase in periodontal disease. yamazaki et al.,22 revealed that in chronic gingivitis, serum antibody to self-hsp60 was increased. in addition, according to ueki et al.,23 self-hsp60 expressed abundantly in periodontal lesion and similar to bacterial lps, thus it is able to stimulate tnf-a production by macrophages. periodontal treatment is able to decrease inflammation by increasing anti-inflammatory hsps (i.e. hsp10) that inhibit pro-inflammatory hsps.10 it also reduce pro-inflammatory cytokines; neuropeptides sp, cgrp and vip.9,14 it is common that initial bracket placement will cause trauma to buccal or mucosal tissue which may cause ras. nevertheless the frequency will reduce after adaptation. in orthodontic patients, orthodontic braces, wires and accessories made them more susceptible to food impaction and difficulties in maintaining optimal oral hygiene. therefore, supragingival and subgingival plaque which also called biofilm accumulated. the developing pseudopockets in chronic gingivitis made the subgingival plaque elimination became more difficult. subsequently, there were increase of infectious agents which also increase either self-hsp60 or bacterial-hsp60. this condition also facilitates activation of immunocompetent cells by lps to generate the neurogenic switching mechanism. several literatures explained the cross-reactivity between microbes and oral epithelial cells, which are active components of the innate immunity. porphyromonas gingivalis components: i.e. fimbriae, gingipains and lps, are able to elicit gingival epithelial cells (gecs) reaction via molecular pathway. other current finding revealed that p. gingivalis are able to invade epithel cells (intra-epithelial) in vivo (figure 2).24,25 �0 dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 6-11 moreover, p. gingivalis also able to degrade epithelial cell junctional complex and the infection can be transmitted cell to cell without passing through the extra cellular space.25 this mechanism of spreading may allow p. gingivalis to colonize oral tissues without exposure to the humoral immune response.26 it is also interesting that p. gingivalis, which commonly colonized in the subgingival plaque, a study by suzuki et al.,26 found their existence in saliva; and hafajeel et al.,27 in the supragingival plaque. these literatures support the possible involvement of periodontopathic bacteria such as p. gingivalis in ras. p. gingivalis could act as intraepithelial bacteria, thus is more resistant to antiseptic mouthwash as in this case report. recent hypotheses postulate ras are a consequence of an autoimmune reaction against oral epithelium. it has been suggested that this autoimmune reaction could be a cross-reaction immune response, activated by hsps released by oral bacteria and targeting similar peptides in the oral epithelium. therefore, in our opinion there should be a possible bacteria similarity between chronic gingivitis and ras; nevertheless, it was contradictory with marchini et al.,28 study which found that among periodontopathic bacteria only prevotella sp which appear in 16% ras sufferers, porphyromonas sp was not significantly detected in samples. however, this result was based on bacterial study of the buccal mucosa swab samples only, and did not investigate the intraepithelial bacteria. the role of intraepithelial bacteria such as p. gingivalis in ras is supported by a recent clinical study; the application of topical minocycline which usually indicated for periodontal disease was successful for ras treatment. minocycline, besides its microbial effect, it also a host response modulator and able to give effect intraepithelially.29 it is likely that elastomeric modules and chain act as media for plaque accumulation. nevertheless, a study by ahn et al.,5 showed that despite its “smooth:” surface, bacteria also adhere to brackets, especially on metal.6 therefore, optimal oral hygiene maintenance and regular changing of the elastomeric accessories should be conducted. these orthodontic patients wear metal brackets and elastomeric modules, with poor oral hygiene; thus more susceptible to bacterial adhesion and facilitate contacting bacterial colony to the buccal or lip mucosa. based on these literatures, the chronic gingivitis and ras relationship hypothesis could be explained by neuroimmunobiological approach as follows: 1) pge2 in chronic gingivitis lowered activation threshold of afferent primary nerve fibers which become more sensitive to stimuli; 2) activation of immunocompetent cells by lps release mediators that able to activate nerve endings which then produced neuropeptides; especially in the same innervation area (in this case was the lower lip); 3) prolonged sensitization increase vip level which not easily degraded that also lead to long lasting vasodilatation of tissue vessels, including the buccal or lip mucosa which become more inflamed; 4) the presence of intraepithelial bacteria and the cross-reactivity of hsps exaggerate the ongoing mucosal inflammation. therefore, the affected lip or buccal mucosa became more susceptible to trauma from brackets or unintentional biting. the disrupted epithelial junction by tnf-a worsen the effect. for the concluding remarks, since the interesting phenomenon, ras lesion which relief after scaling procedures could be explained by neuroimmunobiological perspectives, thus our hypothesis is possible. moreover, this discussion could explain the uncertain etiopathogenesis of ras, and in our opinion, the cause of ras is rather locally, but could be exaggerated by systemic condition (i.e. stress, immunocompromise). nevertheless, more evidence based cases and randomized controlled trials should be done and supported by immunopathological and microbiological examination to verify this hypothesis. references 1. de oliveira martinez k, lúcio l mendes ll, alves jb. secretory asecretory a immunoglobulin, total proteins and salivary flow in recurrent aphthous ulceration. rev bras otorrinolaringol 2007; 73(3):323–8. 2. challacombe sj, shirlaw p. immunology of diseases of the oral cavity. in: mestecky j, et al. editors. mucosal immunology. 3rd ed. burlington. elsevier academic press; 2005. p. 1530–3. 3. scully c. aphthous ulceration. n engl j med 2006; 355:165–72. 4. scully c. aphthous ulcers. available online at url http://www. emedicine.com/ent/ entoral_medicine.htm. accessed september 20, 2008. 5. ahn sj, kho hs, lee sw, nahm ds. 1 roles of salivary proteins in the adherence of oral streptococci to various orthodontic brackets. j dent res 2002; 81(6):411–5. 6. benson pe, shah aa, campbell if. fluoridated elastomers: effect on disclosed plaque. j orthod 2004; 31(1):41–6. figure 2. epithelial internalizing by porphyromonas gingivalis.29 ��prahasanti, et al.: chronic gingivitis and aphthous stomatitis 7. westerheide sd, morimoto ri. heat shock response modulators as therapeutic tools for diseases of protein conformation. j biol chem 2005; 280(39):33097–100. 8. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 9. d’aiuto f, nibali l, parkar m, suvan j, tonetti ms. short-term effects of intensive periodontal therapy on serum inflammatory markers and cholesterol. j dent res 2005; 84(3):269–73. 10. shamaei-tousi a, d’aiuto f, nibali l, steptoe a, coates arm, et al. differential regulation of circulating levels of molecular chaperones in patients undergoing treatment for periodontal disease. plos one 2007; 2(11):1–7. 11. hasan a, shinnick t, mizushima y, van der zee r, lehner t. defining a t-cell epitope within hsp 65 in recurrent aphthous stomatitis. clin exp immunol 2002; 128:318–25. 12. saleem a, abd el-aleem, begonia m, aza m, donaldson lf. sensory neuropeptide mrna up-regulation is bilateral in periodontitis in the rat: a possible neurogenic component to symmetrical periodontal disease. eur j neurosci 2004; 19:650–9. 13. wadachi r, hargreaves km. trigeminal nociceptors express tlr-4 and cd14: a mechanism for pain due to infection. j dent res 2006;j dent res 2006; 85(1):49–53. 14. foster n, lea sr, preshaw pm, taylor jj. pivotal advance: vasoactivepivotal advance: vasoactive intestinal peptide inhibits up-regulation of human monocyte tlr2 and tlr4 by lps and differentiation of monocytes to macrophages. j leu biol 2007; 81:893–903. 15. kemppainen p, forster c, koppert w, handwerker ho. blood flow increase in the human lip after high-intensity tooth stimulation is not based on cholinergic mechanisms. neurosci lett 2001; 23:109–11. 16. kemppainen p, avellan nl, handwerker ho, forster c. differences between tooth stimulation and capsaicin-induced neurogenic vasodilatation in human gingiva. j dent res 2003; 82:303–7. 17. kidd bl, urban la. mechanisms of inflammatory pain. brit j anaesth. 2001; 87(1):3–11. 18. burt b. epidemiology of periodontal disease. j periodontol 2005; 76:1406–19. 19. cady rk, schreiber cp. sinus headache or migraine. neurology 2002; 58:s10–s14. 20. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http://www. migraineprevention.com/index/html. accessed february 15, 2006. 21. habich c, kempe k, van der zee r, rümenapf r, akiyama h, et al. heat shock protein 60: specific binding of lipopolysaccharide. j immunol 2005; 174:1298–305. 22. yamazaki k, ohsawa y, tabeta k, ito h, ueki k, oda t, et al. accumulation of human heat shock protein 60-reactive t cells in the gingival tissues of periodontitis patients. infect immun 2002; 70:2492–501. 23. ueki k, tabeta k, yoshie h, yamazaki k. self-heat shock protein 60 induces tumour necrosis factor-alpha in monocyte-derived macrophage: possible role in chronic inflammatory periodontal disease. clin exp immunol 2002; 127:72–7. 2 4 . r u d n e y j d , c h e n r , s e d g e w i c k g j . a c t i n o b a c i l l u s actinomycetemcomitans, porphyromonas gingivalis, and tannerella forsythensis are components of a polymicrobial intracellular flora within human buccal cells. j dent res 2005; 84(1):59–63 25. yilmaz o. the chronicles of porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay. microbiology 2008; 154:2897–903. 26. suzuki n, yoshida a, nakano y. quantitative analysis of multispecies oral biofilms by taqman real-time pcr. clin med res 2005; 3(3):176–85. 27. haffajee ad, socransky ss, patel mr, song x. microbial complexes in supragingival plaque. oral microbiol immunol 2008; 23:196–205. 28. marchini l, campos ms, silva am, paulino lc, nobrega fg. bacterial diversity in aphthous ulcers. oral microbiol immunol 2007; 22:225–31. 29. gorsky m, epstein j, raviv a, yaniv r, truelove e. topical minocycline for managing symptoms of recurrent aphthous stomatitis. spec care dentist 2008; 28(1):27–31. mkgs vol 45 no 2 april-juni 2012.indd 84 volume 45 number 2 june 2012 inhibition of 10% alpinia galanga and alpinia purpurata rhizome extract on candida albicans growth fakhrurrazi, rachmi fanani hakim, and cut cahya study program of dentistry, faculty of medicine, syiah kuala university aceh indonesia abstract background: one of normal oral flora that found in human oral cavity is candida albicans (c. albicans). the overgrowth of this species can lead to opportunistic infection known as candidiasis. two natural plants, alpinia galanga rhizome and alpinia purpurata rhizome, are natural remedies containing flavonoid, saponin, tannin, and triterpenoid used as antifungal component. purpose: this experimental laboratory study is aimed to determine the inhibition of alpinia galanga rhizome and apinia purpurata rhizome on the growth of c. albicans. methods: alpinia galanga rhizome and alpinia purpurata rhizome were extracted in ethanol solvent using soxhletation method. the ratio test was conducted on those two extracts at the concentration of 10% toward the growth of c. albicans through agar diffusion method. results: the results showed that 10% alpinia galanga rhizome extract and 10% alpinia purpurata rhizome extract were able to inhibit the growth of c. albicans, about 7.33 mm for alpinia galanga rhizome extract and 6 mm for alpinia purpurata rhizome extract. the results of statistical tests using independent samples t-test showed that there was no significant difference between the inhibition of 10% alpinia galanga rhizome extract and that of 10% alpinia purpurata rhizome extract. conclusion: in conclusion 10% alpinia galanga rhizome extract and 10% alpinia purpurata rhizome extract have weak inhibition on c. albicans growth. key words: candida albicans, candidiasis, alpinia galanga rhizome, alpinia purpurata rhizome abstrak latar belakang: candida albicans (c. albicans) merupakan flora normal yang terdapat dalam rongga mulut, jika keseimbangannya terganggu maka jamur tersebut akan menjadi patogen dan dapat menyebabkan infeksi dalam rongga mulut yaitu kandidiasis. lengkuas rimpang putih maupun lengkuas rimpang merah merupakan tanaman yang mengandung senyawa antijamur berupa flavonoid, saponin, tanin, dan triterpenoid. tujuan: penelitian eksperimental laboratoris ini dilakukan untuk mengetahui perbandingan daya hambat ekstrak lengkuas rimpang putih (alpinia galanga) dengan ekstrak lengkuas rimpang merah (alpinia purpurata) terhadap pertumbuhan c. albicans. metode: ekstrak lengkuas rimpang putih maupun lengkuas rimpang merah diperoleh dengan metode soxhletasi. dilakukan pengujian perbandingan kedua ekstrak pada konsentrasi 10% terhadap pertumbuhan c. albicans dengan menggunakan metode difusi agar. hasil: ekstrak lengkuas rimpang putih 10% dan ekstrak rimpang lengkuas merah 10% mampu menghambat pertumbuhan c. albicans dengan daya hambat rata 7,33 mm untuk ekstrak lengkuas rimpang putih dan 6 mm untuk ekstrak lengkuas rimpang merah 10%. hasil uji statistik menggunakan independent sampel t test menunjukkan tidak ada perbedaan bermakna antara respon hambat ekstrak lengkuas rimpang putih dan ekstrak lengkuas rimpang merah. kesimpulan: dari hasil penelitian ini dapat disimpulkan bahwa ekstrak lengkuas rimpang putih 10% dan ekstrak lengkuas rimpang merah 10% memiliki daya hambat yang lemah terhadap pertumbuhan c. albicans. kata kunci: candida albicans, kandidiasis, lengkuas rimpang putih, lengkuas rimpang merah correspondence: rachmi fanani hakim, c/o: program studi kedokteran gigi, fakultas kedokteran universitas syiah kuala aceh, indonesia. e-mail: abunidafahiza@gmail.com research report 85fakhrurrazi, et al.: inhibition of 10% alpinia galanga introduction unlike bacteria which considered as prokaryotic microorganisms, fungi are considered as eukaryotic microorganisms. one of the most commonly found fungi in oral cavity is candida. candida is a normal fungus found in oral cavity, gastrointestinal tract, genital tract, and sometimes in skin.1 candida is also known to present approximately in 40-60% of human population.1,2 there are actually 200 different species of candida, including candida albicans (c. albicans), candida glabrata (c. glabrata), candida crusei (c. crusei), candida tropicalis (c. tropicalis), but c. albicans in oral cavity is the most common species that may cause disease. as much as 75-90% of fungal infections in humans are triggered by c. albicans.1-3 another report also shows that 40-60% of the oral cavity in healthy adult population contains c. albicans with small concentration (200-500 cells/ml saliva).3,4 c. albicans, as a normal flora in oral cavity will become pathogenic and cause candidiasis when a person has risk factors of the excessive growth of c. albicans. those risk factors are triggered not only by local factors such as xerostomia, but also by the use of topical corticosteroids, prostheses, and smoking habit. in addition, there are also systemic factors considered as risk factors, such as either the use of antibiotics and systemic corticosteroids or hormonal changes caused by pregnancy or diabetes mellitus. candidiasis is often found in people infected with human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome (aids) triggered by weak immunity.2,3 nowadays a lot of medicine can be used to overcome infections caused by candida. generally, infections caused by candida can be solved by the following three groups of agents, namely polyenes, azole and dna analogues. those agents can be used based on the type and severity of infections.1 however, these drugs tend to be expensive and have more side effects in long-term use, such as being resistant to fungus and being harmful when used in patients with hepatitis (azole class).2 therefore, it is necessary to look for alternative drugs that is cheap, relatively safe to use, and easy to get, such as alpinia. alpinia is herbal medicine known as anti-fungal derived from zingeberaceace family. ginger rhizome is traditionally used as a drug to treat skin diseases, especially those caused by fungi.5 there are two kinds of galanga, namely red ginger (alpinia purpurata rhizome) and white ginger (alpinia galanga rhizome). white ginger is widely used as spices or herbs, while red ginger is widely used as herbs.5,6 white ginger, furthermore, contains 1% greenish yellow volatile oil and some other compounds. one of the researchs on exposed white ginger shows that some other compounds were succesfully isolated from white ginger, such as acetoxychavicol acetate considered as antifungal.7,8 another compound that also has antifungal activity is a diterpene isolated from white ginger. further studies also show that diterpene works by altering the lipid membrane of c. albicans then resulting in the changes of the permeability of its membrane.9 a test conducted on alpinia galanga rhizome extract with concentration of 10% even shows that there were antifungal activities against c. albicans in vaginalis candidiasis.8 on the other hand, red ginger (alpinia purpurata) contains flavonoids, saponins and tannins. one of the functions of flavonoids is as antimicrobial and antifungal activities.10 for those reasons, this study is aimed not only to determine the inhibition response of white ginger extract (alpinia galanga) and red ginger extract (alpinia purpurata) toward the growth of c. albicans, but also to determine the ratio of the inhibition response of 10% white ginger extract (alpinia galanga) and 10% red ginger extract (alpinia purpurata) to the growth of c. albicans. materials and methods this study is considered as an experimental laboratory study conducted at biological chemistry laboratory of mathematic and natural science faculty and at microbiology laboratory of veterinary faculty, university of syiah kuala, in banda aceh. the unit of analysis in this study was the dosage form of c. albicans atcc 10231 obtained from microbiology laboratory of medical faculty, university of indonesia. samples used in this study were white ginger (alpinia galanga) and red ginger (alpinia purpurata) obtained from peunayong market in banda aceh. at the first stage, c. albicans was obtained by using a sterile loop, and then was grown in sabouraud dextose agar (sda) media. next, it was incubated in incubator at 37 ˚c for 24 hours until its growth occured. afterwards, c. albicans grown on sda media was identified by gram by using staining method, and then was observed under a microscope with a magnification 1000. the preparation of suspension was conducted to produce c. albicans more. this process was conducted by inoculating c. albicans into 10 ml peptone, and then compared with the turbidity level of mc. farland solution about 0.5 (1.5 x 106 cfu/ml). at the next stage, white ginger (alpinia galanga) and red ginger (alpinia purpurata) obtained was extracted. fresh white ginger (alpinia galanga) and red ginger (alpinia purpurata) obtained as much as 1 kg were washed, cut crosswise, and dried at 60 ˚c for 15 minutes before they were then mashed. next, the powder of white ginger (alpinia galanga) and red ginger (alpinia purpurata) obtained was wrapped in filter paper, and then soaked separately in 600 ml of 96% ethanol in soxhletasi tool until the solvent droplets were colorless. afterwards, the filtrate mix with the solvent was evaporated with rotary evaporator at 40 °c to obtain pure extract. since the amount of the extracts obtained was not sufficient, then the gingers 86 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 84–88 were needed to be added.10, 11 the pure extracts obtained was then diluted by using sterile aquadest water to obtain specific concentration about 10%. the effectiveness of those extracts of 10% white ginger (alpinia galanga) and red 10% ginger (alpinia purpurata) obtained toward the growth of c. albicans was tested by using agar diffusion method with sterile sda media. afterwards, the suspension of c. albicans that had been measured was poured about 0.1 ml by using eppendorf pipette into the media surface, and then was smoothed by using a sterile spreader bar (hocky sterile). the next step is to put disc papers, each of which had been soaked in white ginger extract (alpinia galanga) 10% and red ginger extract (alpinia purpurata) 10%, while disc papers soaked in aquadest were used as negative control. those three media were then incubated at 37 °c for 24 hours. the zone of inhibition toward the growth of fungi could be observed by measuring and recording the diameter of inhibition zone using long slide in millimeters. the observation was conducted three times. the parameters that would be observed was the diameter of inhibition zone of c. albicans growth. the results obtained way interpreted using table 1.12 data obtained were tested with shapiro-wilk test and were normally distributed, later analyzed with parametric independent sample t-test with 95% confidence level. the test showed a significant result (p<0.05). results c. albicans that had been cultured on sda media for 48 hours and stored in the incubator had a spherical colony shape with slightly convex, creamy colour, and soft surface as well as yeast aroma. confirmatory test was conducted using gram staining test. the results on c. albicans using gram staining test microscope observation with 1000 magnification showed that there were round cells, pseudohyphae, and purple smooth tube hyphae indicating that the strain was c. albicans (figure 1). test of the inhibition response was conducted on alpinia galanga rhizome extract and alpinia purpurata rhizome extract using agar diffusion method and disc papers soaked in each of those solution extracts. the test was repeated three times. statistical test used was independent sample t-test. there was no significant differences between alpinia galanga rhizome and alpinia purpurata rhizome in inhibiting the growth of c. albicans with p = 0.057. discussion the results of c. albicans cultured on sda media showed that colonies formed were slightly convex, creamy, and soft surface as well as yeast aroma smell. the morphology was is similar to the statement of jawetz,13 kayser15 and rippon14. sda is considered as a standard medium for culturing c. albicans since it contains dextrose and peptone to support the growth of c. albicans, however, several other fungi can also grow on this medium because sda is not a selective medium only for c. albicans therefore a confirmation test is required to ensure the process of culturing is not contaminated with other fungi.16 c. albicans have a thick structure as composed of chitin, mannan, and glucan which causes stiffness and low permeability so that when moistened with alcohol, the cell walls cannot be penetrated and the violet gentian dye is still remained in the cell and will make cells looks purple.14 the 10% alpinia galanga rhizome extract and 10% alpinia purpurata rhizome extract were capable of inhibiting the growth of c. albicans with the average of the inhibition response about 7.33 mm for alpinia galanga rhizome extract and 6 mm for alpinia purpurata rhizome figure 1. the result of gram staining test. a) cell, b) pseudohyphae, c) hyphae. tabel 2. the independent sample t-test results diameter of the inhibition zone of the growth of c. albicans of the extracts of alpina galangal rhizome and alpina purpurata rhizome (mm) extract mean of inhibition diameter p 10% alpinia galanga rhizome 7.33 0.057 10% alpinia purpurata rhizome 6 table 1. the classification of the inhibition response12 diameter of light zone inhibition response toward the growth 20–30 mm +++ (strong) 11–20 mm ++ (moderate) 6–10 mm + (weak) 0 – 87fakhrurrazi, et al.: inhibition of 10% alpinia galanga extract. both of alpinia galanga rhizome and alpinia purpurata rhizome are actually two plants derived from the family of zingeberacea known as one of the herbs that have known benefits as antifungal.5,10,17 alpinia galanga rhizome extract and alpinia purpurata rhizome extract could inhibit the growth of c. albicans.8-10 the inhibition response of alpinia galanga rhizome extract toward the growth of c. albicans can happen because the extract contains both diterpene compounds, such as acetoxychavicol acetate, and flavonoid compounds, such as kaempferol, kaempferide, galangin, alpinin and essential oils which are antifungal compounds.6,7 the diterpene compounds work by altering the lipid membrane of c. albicans, so the changes of their membrane permeability occur.9 meanwhile, flavonoids work by inhibiting the synthesis of nucleic acids of fungi and destabilizing the cell membranes due to the change of the nature of the fungal cell membrane that may cause the exchange of fluid in the cell. on the other hand, the inhibition response of alpinia purpurata rhizome is caused by the fact that it contains saponins, tannins, flavonoids, essential oils and diterpen compounds.18,19 saponin works as an antifungal agent by interfering with the permeability of the fungal cell wall. the antifungal activity of saponin, however, is related to the composition of aglycone and the structure of monosaccharide unit in their sugar chain group.20,21 the results of the study conducted by silvina,8 also showed that among 30 samples of sda containing 10% alpinia galanga rhizome extract in candidiasis vaginalis patients there was no c. albicans grown. however, the result is different from the result in this study due to the different test method conducted. solid dilution method was used to test the effectiveness of 10% galanga extracts, thus, it made the extract distributed, and make the contact with the fungus more effective.8, 21 unlike that previous study, in this study agar diffusion method was chosen since the process is relatively simpler, more practical and thorough. nevertheless, this method still has weaknesses one of which causes the limitation of the average size of the inhibition zone formed. other disadvantages of this method include the results of the testing of several samples with different antimicrobial potency cannot always be compared among them because each sample has different physical properties, such as solubility, volatility, and diffusion characteristics. thus, the content of the extract which has good diffusion coefficient, but weak antifungal activity can diffuse into the agar well. meanwhile, although the content of the extract has good antifungal activity, but without having good diffusion coefficient, it will affect the inhibition zones formed. in addition, another weakness of this method will also arise when comparing the inhibition zone formed from different samples. in the disc paper method, the inhibition zone will also be affected by concentrations given in the disc paper.22 alpinia purpurata rhizome extract is more effective than alpinia galanga rhizome extract in inhibiting the growth of streptococcus mutans.23 similarly, it is also known that alpinia purpurata rhizome extract is more effective in inhibiting trichopyton ajelloi than alpinia galanga one.24 unlike those studies, in this study it is known that there was no significant difference between alpinia galanga rhizome extract and alpinia purpurata rhizome extract. this is due to the differences of the structures of both of fungi.25–28 based on the classification of the inhibition response according to morales,12 it is also known that the inhibition response of alpinia galanga rhizome extract 10% and alpinia purpurata rhizome extract 10% toward the growth of c. albicans is in the weak category (+). the cause of the weak inhibitition zone was affected by the quality of the rhizome extract obtained for test material. the quality of simplicia and natural materials that will be used as drugs can be standardized based on the method of making simplicia issued by ministry of health of the republic of indonesia. the poor quality of extract is caused by the amount of the active substances contained in the rhizome.29 the active substances is actually affected by temperature and humidity. alpinia have different levels of maturity. the different levels of maturity can lead to the differences of the texture and color of substances costituted.30 the weak inhibition response, can also be caused by the extraction method using soxhletasi method. this method has some disadvantages, the solvent used must be volatile and can only be used for the extraction of heat-resistant compounds. this negative side then may affect the amount and quality of antifungal compounds contained in the extracts of both alpinia galanga rhizome and alpinia purpurata rhizome contained will be damaged at high temperature. if the extracts are broken, it will affect the ability of these substances to inhibit the growth of c. albicans. one of the effects is that volatile oil, such as sineol is unstable and sensitive to high temperature. during the drying and withering process, plant cell membrane gradually breaks, as a result, water penetrates freely from one cell to another to form a volatile compound and the amount of water in plant cells will diffuse into the top surface.31 the minimal inhibitory zone in 10% alpinia galanga rhizome extract and 10% alpinia purpurata rhizome extract may occur due to the heating process at soxhletasi period. c. albicans grow in an environment that has a specific concentration and is still able to survive from minimum concentartion to maximum. c. albicans can optimally grow from at ph 4.5 to at ph 6.5.32 thus, the small inhibition zone of both of 10% alpinia galanga rhizome extract and 10% alpinia purpurata rhizome extract may be caused by the value of those extract ph that ranged in ph optimum for the growth of c. albicans. similarly, according to a study conducted by wahyuni,24 it is also known that based on the measurement of the acidity of both of n-hexane extract and alpinia galanga rhizome extract, the ph obtained was ranging from 5% to 50% that was equal to 5. this result indicates that the ph of alpinia galanga rhizome extract can become a factor triggering the growth of c. albicans around disc papers since they are in the range of ph for 88 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 84–88 their optimum growth. it can finally be concluded that both 10% alpinia galanga rhizome extracts and 10% alpinia purpurata rhizome extract have weak inhibition response toward the growth of c. albicans. acknowledgement we would like to deliver our gratitude and appreciation to research institute of university of syiah kuala, which has facilitated this study to be funded by unsyiah, ministry of national education. references 1. samaranayake lp. essential microbiology for dentistry. 2nd ed. london uk: churchill, livingstone. elsevier; 2002. p. 177. 2. silverman sj, eversole lr, edmond lt. essential of oral med. london: bc. decker inc, hamilton; 2001. p. 170–7. 3. lamont rj, jenkinson hf. oral microbiology at a glance. united stated: wiley-blackwell press; 2010. p. 66–7. 4. greenberg ms, glick. burket’s oral medicine diagnosis and treatment. 10th ed. new york: bc decker inc; 2003. p. 95–101. 5. sinaga e. lengkuas. jakarta: pusat penelitian dan pengembangan tumbuhan obat universitas nasional jakarta; 1999. p. 1–3. 6. siddiq j. rahasia, khasiat dan manfaat bumbu dapur, rempah-rempah dan sayuran. jogjakarta: penerbit surya media; 2010. p. 54. 7. chudiwl ak, jain dp, somani rs. alpina galanga wild.-an overview on phyto-pharmacological properties. india: singhad collage of pharmacy; 2009. p. 144–6. 8. silvina. uji banding efektivitas rimpang lengkuas (alpinia galanga) 10% terhadap ketokonazol 2% secara invitro terhadap pertumbuhan candida albicans pada candidiasis vaginalis. skripsi. semarang: fakultas kedokteran universitas diponegoro; 2006. 9. haraguchi h, kuwata y, inada k, shingu k, miyahara k, nagao m, yagi a. antijamur activity of from alpina galanga and the competition for incoporation in unsaturrated fatty acid in cell growth. planta med 1996; 62(4): 308–13. 10. kochutheressia kp, britto sj, jaseentha, raj lr, senthilkumar sr. antimicrobial efficacy of extract from alpina purpurata (viell.) k. schum. against human phatogenic bacteria and jamur. agriculture and biology journal of north america 2010; 1(6): 1249–52. 11. yenni. pengaruh perbedaan kadar cairan penyari (etanol 10%, 40%, 70%, 96%) rimpang lengkuas merah terhadap pertumbuhan candida albicans. skripsi. surabaya: fakultas farmasi universitas; 2001. 12. morales g, sierra p, mancillia a, paredes a, loyola la, galardo o, jorge b. secondary metabolites from northen chile: antimicrobial activity and biotoxicity againts artemia salina. journal of the chilean chemical society chile 2003; 48(2): 1–6. 13. geo fb, janet sb, stephen am. jawetz melnic adelberg’s medical microbiology. 24th ed. newyork: mcgraw-hill companies, inc; 2007; p. 277–9. 14. rippon jw. medical mycology. philadelphia: wb saunders co; 1998. p. 532–75. 15. kayser fh, kurt ab, johannes e, ralph mz. medical microbiology. 10th ed. stuggart: thieme; 2005. p. 173. 16. bhavan ps. rajkumar r, radhakrishnan s, seenivasan c, kannan s. culture and identification of candida albicans from vaginal ulcers and separation of enolase on sds-page. international journal of biology 2010; 2(1): 84–93. 17. kusumawardani nf. formulasi salep minyak atsiri rimpang lengkuas (alpina galanga) basis lemak dan peg 4000 dengan uji sifat fisik dan uji anti jamur candida albicans. skripsi. surakarta: fa k u lt a s fa r ma si un iver sit a s mu ha m ma d iya h su r a k a r t a; 2009. 18. permadi a. membuat kebun tanaman obat. jakarta: pustaka bunda; 2008. p. 39–40. 19. sirait mh, liamen mr. chemical constituents of alpina purpurata. pertanika journal science and technology 1995; 3(1): 67–71. 20. utami wd. perbedaan daya hambat ekstrak dan perasan rimpang lengkuas (alpinia galanga) terhadap pertumbuhan candida albicans. skripsi. jember: universitas jember; 2010. p. 27–38. 21. maryati, fauzia rs, rahayu t. uji aktivitas antibakteri minyak atsiri daun kemangi (ocimun basilicum l.) terhadap staphylococcus aureus dan escherichia coli. jurnal penelitian sains & tekhnologi 2007; 8(1): 30–8. 22. liliana s, tatiane b, ana mfa, dulce hss, vanderland sb, maria jsmg. the use of standard methodology for determination of antijamur activity of natural products againts medical yeast candida sp and cryptococcus sp. brazilian journal of microbiology 2007; 392–3.i. 23. tiurlina s, ferdinan sd, anna f. pertumbuhan streptococus mutans pada bioaktivitas ekstrak rimpang lengkuas secara in vitro dan pemamfaatannya sebagai zat aktif pada pasta gigi. jurnal kimia universitas udaya e-journal 2011; 5(1): 9–23. 24. wahyuni s. perbandingan daya antijamur ekstrak rimpang lengkuas putih dan lengkuas merah terhadap tricophyton ajelloi. penelitian tanaman obat di beberapa perguruan tinggi di indonesia. jakarta: balai penelitian dan pengembangan kesehatan republik indonesia; 1995. p. 83–4. 25. segal b. phatogenic yeast and yeast infection. tokyo: crc press inc; 1994. p. 14. 26. kokare cr. pharmaceutical microbiology principles and application. mumbai: nirali prakashan; 2008. p. 148. 27. mukoma fs. dermathopytes: their taxonomy, ecology and pathogenecity. revista iberoamericana de micología. bilbao: spain; 2000. p. 3. 28. limyati, ariani d, artawan, halim i.g.k, junita. daya antimikroba ekstrak brotowali terhadap staphylococcus aureus, escherichia coli, candida albicans dan trichophyton ajelloi. jakarta: warta tumbuhan obat indonesia; 1998. p. 16–7. 29. trisnamurti rh, basuki t. funtcional food industri: trend and chalange. jakarta: lipi press; 2005. p. 59–77. 30. siswanto yw. penanganan hasil panen tanaman obat komersial. jakarta: penerbit penerbar swadaya; 2004. p. 99. 31. sastrohamidjojo h. kimia minyak atsiri. yogyakarta: gadjah mada university press; 2004. p. 30. 32. michael jp, chan ecs. dasar-dasar mikrobiologi. jilid 2. jakarta: ui-press; 2008. p. 456. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice � volume 46 number 1 march 2013 research report patient’s expectation on communication performances community of dental health services providers located in urban and rural area taufan bramantoro and ninuk hariyani department of dental public health faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: the quality of dentist’s communication skills is considered as one of important aspects on the quality of dental health services assessment. during the initial interview conducted at ketabang, dupak, and kepadangan community dental health services at surabaya and sidoarjo, indonesia, it appeared that eighty percent of initial respondents were not satisfied with the communication aspect. community dental health services (cdhs) need to assess the communication performances based on community characteristics in effort to promote the quality and effectiveness of the dental health services. purpose: the objective of this study was to analyze patient’s expectation values priorities on dentists’ communication performances in cdhs that located in urban and rural area. methods: the study was conducted in ketabang surabaya, dupak surabaya and kepadangan sidoarjo cdhss. the participants were 400 patients above 18 years old. participants were assessed their expectation value using the communication performances of dental health services questionnaire. results: patients in urban cdhs appeared that there were two priority aspects which had high values, namely the clarity of instructions and the dentist’s ability of active listening to the patient, while patients in rural cdhs revealed that the clarity of instructions and dentist-patient relationship were the aspects with high values. conclusion: patients in cdhs that located in rural area expect more dentist-patient interpersonal relationship performance than patients in cdhs located in urban area. this finding becomes a valuable information for cdhs to develop communication strategies based on community characteristics. key words: communication performances, patients expectation, dental health services abstrak latar belakang: kualitas komunikasi dari dokter gigi merupakan salah satu aspek penting dalam penilaian kualitas layanan suatu sarana pelayanan kesehatan. pada wawancara pendahuluan yang dilaksanakan di puskesmas ketabang, dupak dan kepadangan di surabaya dan sidoarjo, indonesia, diperoleh hasil bahwa delapan puluh persen responden merasa tidak puas dengan aspek komunikasi dari tenaga kesehatan gigi. penyedia layanan kesehatan gigi perlu untuk menilai penampilan komunikasi dari tenaga kesehatan gigi sesuai dengan karakteristik masyarakat sebagai usaha untuk meningkatkan kualitas dan efektivitas layanan kesehatan gigi. tujuan: tujuan penelitian ini adalah untuk menganalisis prioritas harapan pasien terhadap penampilan komunikasi dokter gigi di puskesmas yang berlokasi di daerah perkotaan dan pedesaan. metode: penelitian dilakukan di puskesmas ketabang surabaya, dupak surabaya dan kepadangan sidoarjo. responden berjumlah 400 pasien, berusia diatas 18 tahun. penilaian harapan pasien akan aspek komunikasi dari tenaga kesehatan akan diukur dengan menggunakan kuesioner kualitas layanan terkait penampilan komunikasi. hasil: pasien pada puskesmas di daerah perkotaan menyampaikan bahwa ada dua aspek prioritas yang mereka anggap lebih bernilai, yaitu kejelasan instruksi dokter gigi dan kemampuan dokter gigi untuk mendengarkan mereka secara aktif, sementara pasien di puskesmas pedesaan lebih memberikan nilai prioritas yang tinggi pada aspek kejelasan instruksi dokter dan hubungan kedekatan antara dokter–pasien. kesimpulan: pasien pada puskesmas yang terletak di daerah pedesaan � dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 1–4 introduction communication performances become the successful factor to build the effective relationship between dentist and patient in dental health services.1 moreover, effective communication performances in dental health services do not only have an important role with patient satisfaction and patient compliance, but also with patient anxiety and the risk of malpractice claims. previous research also showed that communication performances relate with dental health service quality.2,3 dupak surabaya and ketabang surabaya community dental health services (cdhss) as community dental health services that located in urban area and kepadangan sidoarjo cdhs as community dental health services that located in rural area need to be assessed the communication performances based on community characteristics in order to promote the quality and effectiveness of dental health services. thus, patient’s assessment on cdhs performances, including the communication performances become an important information due to dental health service quality improvement. as one of the government health service programmes is having responsibility to improve its service quality. during the initial interview conducted at ketabang, dupak, and kepadangan cdhs, it was examined that 80% of initial respondents were not satisfied with the communication aspect of cdhs providers. currently, patients are more concerned regarding the quality of communication skill that is shown by their dentist. patients assess how the dentist communicating effectively with patients, gathering information effectively through active listening skills, and demonstrating her/his professionalism.4,5 the differences of patients perspective can affect the consumer behavior on fulfilling their needs.6 that differences explain that the service providers must be aware of the service approaches. as a service provider, dental health services need to understand their community characteristics for improving their dental treatment approaches. the approaches are not only related with dental knowledge and technical skills, but also the dentist’s ability on communicating with patients. the objective of this study was to analyze patient’s expectation value on the communication performances of cdhs that located in urban and rural area. materials and methods this was an observational analistical study and applied cross sectional design. the participants were 400 patients above 18 years old selected from patients that had dental treatment consist of 245 patients in ketabang surabaya and dupak surabaya cdhss and 155 patients in kepadangan sidoarjo cdhs indonesia. participants were assessed their expectation value using the communication performances of dental health services questionnaire. research data were collected for two months. t h i s s t u d y u s e d f o u r i t e m s q u e s t i o n n a i r e o f communication performances in dental health services derived from the service quality instrument. 7 the questionnaire consists of four aspects including dentistpatient relationship, ability to listen to the patient, adequacy of explanations to the patient, and clarity of instructions. patients were asked to respond the expectation value questionnaire using likert scale, from one until five. mean value between patients in urban and rural cdhs were calculated to obtain expectation value of the communication performances. the higher expectation value of communication performances aspect, the more important the aspect compared to the others. results the majority of participants were female (67.5 percent). the age range of the participants between 18 and 65 years old. total of 245 patients in ketabang surabaya and dupak surabaya cdhss to represented patients from urban area while 155 patients in kepadangan sidoarjo cdhs represented patients from rural site. the results showed that there were differences priority expectation assessment on communication performances between patients in cdhs that located in urban and rural area. patients in urban community dental health service revealed two priority aspects with had high expectation values, namely the clarity of instructions and the ability of active listening to the patient. the lowest priority aspect was dentist-patient relationship. in contrast, patients in rural community dental health service assessed that the clarity of instructions and dentist-patient relationship had high values, while the ability of active listening to the patient aspect had lebih mengharapkan upaya membangun hubungan interpersonal dokter gigi dengan pasien dibandingkan dengan pasien pada puskesmas terletak di daerah perkotaan. temuan ini merupakan informasi yang berharga bagi penyedia layanan kesehatan gigi untuk membangun strategi komunikasi berdasarkan karakteristik masyarakat sasaran. kata kunci: penampilan komunikasi, harapan pasien, pelayanan kesehatan gigi correspondence: taufan bramantoro, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: tbramantoro@yahoo.com �bramantoro and hariyani: patient’s expectation on communication performances community the lowest expectation value (table 1). the priority order of communication performance in urban and rural community dental health services was different (table 2). discussion cdhs as a health service provider has identical characteristic with other service provider. it has intangible aspects that shape the interaction between health service provider and service users or the patients.8,9 the patients characteristics have significant influence on the services of the health care. patients as an individual influence health care provided that their management improvement strategy depend on customers with identical needs.10 patients’ perspective have an important role in monitoring, evaluation, and improving quality of dental health care services. dental health quality ensure their patients’ oral health and provide optimal solutions regarding their oral health function,11 the dental health service quality also was determined by good relations between doctor and patient, availability of basic infrastructure, time spent for consultation and information given during consultation, and waiting time.12 other perspective that have important role in dental health service quality is the dental health services provide appropriate service in a sociocultural view.13 the interaction between provider and patients make health service providers aware to always improve their service quality. the effort of quality improvement needs to be emphasized not only in the improvement of physical performances, but also in the awareness of the importance of community aspects, as it can establish personal and humanistic approach in a dental health service. the improvement efforts should be focused in fulfilling patient’s needs and expectation of health service.8 it will affect in improving the utilization of services, health service outcomes, and clinical decision making.11,14 understanding patient’s perspective in service quality is a basic strategy to achieve the quality improvement. patient’s perpective are shaped by their expectation which they set as their standards as well as their experiences.9 thus the assessment of patients’ expectation value on communication performances can be used as a supporting material in institutional programs planning process. this research illustrates that there were similarity and difference of expectation value on communication performances between patients in urban and rural cdhss. this research showed that both patients in urban and rural cdhss have a high expectation value on the clarity of instruction. however, urban and rural patient’s expectation values on communication performances differ with regard to dentist-patient relationship aspect and ability active listening to the patient aspect. both urban and rural cdhss patients showed the highest expectation value on the clarity of instruction given by the dentist. it represents that patients need a clear instruction and they value it as the most important thing in the dentist’s communication performances. in contrast, urban and rural patients have different perspective related with dentist-patient building relationship aspect. patients in urban community dental health service assess that dentist-patient building relationship aspect was not the main priority aspect. meanwhile, patients in rural area consider that dentist-patient building relationship aspect was the important aspect on dental health service process. this phenomenon was caused by the differences of social living characteristic. people in rural community have low variation of social life, culture and economic condition. on the other hand, urban community has high variation of social, culture and economic condition, as well as the high density of living area. the differences between urban and rural social characteristic have significant role in building people perspective.15 people in urban and rural area also differ regarding the social life perspective. people in urban table 2. priority order of communication performances in urban and rural community dental health services variables priority series in cdhs urban rural dentist-patient relationship 4 2 ability active listening to the patient 2 4 adequacy of explanations to the patient 3 3 clarity of instructions 1 1 table 1. expectation value of communication performances in urban and rural community dental health service variables urban rural mean standard deviation mean standard deviation dentist-patient relationship 4.44 0.736 4.88 0.329 ability active listening to the patient 4.53 0.675 4.79 0.411 adequacy of explanations to the patient 4.51 0.716 4.86 0.343 clarity of instructions 4.54 0.610 4.90 0.305 � dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 1–4 area have more self-individual focus and pragmatism perspective than in rural area. as a consequence, people in urban area tend to be more sensitive in expecting pragmatic approach and in assessing their needs of fulfillment.15 urban’s people have different characteristics than rural’s that can affect the dental treatment approaches in the two areas. the approaches are not only related with dental knowledge and technical skills, but also the dentist’s ability on communicate with patients. the differences of patients perspective in regard to their social living environment affect their buying and consuming consideration that shaping their decision in selecting service providers.6,16 patients perceive the quality of communication performances as an important health service quality assessment factor.16 therefore, the service approaches that related with patients concerns in regard to communication performances become important issues for dental health service providers. dental health service as a dental treatment providers must understand the characteristics of their targeted community for promoting their effectiveness and efficiency of dental health services. this study suggested that located in rural area expect more dentist-patient interpersonal relationship performance than patients in cdhs located in urban area. this study gives valuable information for community dental health service to develop communication strategies based on the community characteristics. references 1. soelarso h, soebekti rh, mufid a. peran komunikasi interpersonal dalam pelayanan kesehatan gigi. maj ked gigi (dent j) 2005; 38(3): 124–9. 2. kent gg, blinkhorn as. 1991. pengelolaan tingkah laku pasien pada praktik dokter gigi. budiman ja, lilian juwono, editor. jakarta: egc; 2005. p. 155–81. 3. yoshida t, milgrom p, coldwell s. how do u.s. and canadian dental schools teach interpersonal communication skills. j dent educ 2002; 66(11): 1281–8. 4. hannah a, millichamp c, ayers m. a communication skills course for undergraduate dental students. j dent educ 2004; 68(9): 970–7. 5. elizabeth ar, margaret m, hinrichs mm, beth al. a model for communication skills assessment across the undergraduate curriculum. medical teacher 2006; 28(5): e127–e134. 6. suryani t. perilaku konsumen: implikasi pada strategi pemasaran. yogyakarta: graha ilmu; 2008. p. 55–125. 7. tjiptono f. pemasaran jasa. malang: bayumedia publishing; 2006. p. 25–84. 8. sabarguna b. pemasaran rumah sakit. diy: konsorsium rsi jateng; 2004. p. 88–93. 9. gurdal p, cankaya h, onem e, dincer s, yilmaz t. factors of patient satisfaction/dissatisfaction in a dental faculty outpatient clinic in turkey. comm dent oral epid 2000; 28: 461–9. 10. cooper br, monson al. patient satisfaction in a restorative functions dental hygiene clinic. j dent educ 2008; 72(12): 1510–5. 11. perneger tv. adjustment for patient characteristics in satisfaction surveys. int j qual health care 2004; 16: 433–5. 12. karydis a, komboli-kodovazeniti m, hatzigeorgiou d, panis v. expectations and perceptions of greek patients regarding the quality of dental health care. int j qual health care 2001; 13: 409–16. 13. bankauskaite v, saarelma o. why are people dissatisfied with medical care services in lithuania? a qualitative study using responses to open-ended questions. int j qual health care 2003; 15: 23–29. 14. van der velden t, ping c. the introduction of norplant in cambodia through the private sector. asia pac j public health 2002; 14: 69–74. 15. yunus hs. dinamika wilayah peri-urban: determinan masa depan kota. yogyakarta: pustaka pelajar; 2008. p. 185–210. 16. supriyanto s, ernawaty. pemasaran jasa industri kesehatan. surabaya: fkm universitas airlangga; 2009. p. 174–93. mkg vol 42 no 2 april 2009.indd 55 vol. 42. no. 2 april–june 2009 case report diagnosis and management of crohn’s disease in retarded child rahmi amtha department of oral medicine faculty of dentistry, trisakti university jakarta indonesia abstract background: crohn’s disease is an uncommon condition characterized by granulomatous lesions. it is a rare disease and affects mainly in the terminal ileum. it may also manifest in the oral cavity as an unhealed, painful chronic apthous-like ulcer and may be undiganosed, so that it leads to the mismanagement of the disease. purpose: to overview the establisment and managment of a retarded child with chronic painfull ulcers and gastrointestinal problems. case: an 11-year-old retarded child who complained of ulcers since 6 months ago on the both side of the cheek and the lateral border of the tongue. large irregular lesions were found accompanied by lips swelling, gingival hyperplasia, mucosal tags, hypersalivation. the blood test showed that the patient was suffering from anemia and haematinic (fe, folat and vitamin b12) deficiencies. weight loss occurred for last 6 months, abdominal pain and constipation were also identified. daily food pattern showed imbalance food intake. histopathology features showed granolumatous lesions and was confirmed as a crohn’s disease. case management: reassurance and team work with gastroenterologist were performed. systemic sulfasalazin combined with corticosteroid and multivitamin were administered. oral hygiene was maintained with hyaluronic acid mouthwash. reduced in size of ulcer, pain and swelling were shown gradually. oral ulcers and gastrointestinal symptoms disappeared after 2 months treatment. conclusion: diagnosis of crohn’s disease needs a comprehensive clinical examination and histopathological test are mandatory to be able to manage the disease thoroughly. key words: crohn’s disease, diagnosis, management, child correspondence: rahmi amtha, c/o: bagian penyakit mulut, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa, grogol jakarta, indonesia. e-mail: rahmi_amtha@yahoo.com introduction crohn’s disease is a chronic inflammatory condition which may affect any part of the whole gastrointestinal tract, from mouth to anus.1 most commonly, it affects the terminal ileum and the colon. cronh’s disease is rare and may influence oral health because it causes malabsorption and vitamin deficiencies which predispose to oral lesions. crohn’s disease occurs throughout the world, but primarily in western developed population. the annual incidence and prevalence of crohn’s disease has been steadily rising not only in united states and northern europe, but lately also increases in asian countries such as china, japan and thailand.2 it affects mainly from young childhood to advanced age, but the peak ages are second and third decades.2 previously, epidemiology studies showed that both sexes can be affected equally,1 however currently females are affected slightly more than males.2 crohn’s disease is part of inflammatory bowel disease which is characterized by granulomatous lesions.1-3 the etiology is still unknown, with very little pathognomonic feature of the disease. the diagnosis may be delayed for months or even years.4 crohn’s disease appears as to be heterogeneous group of disorders probably cause by commensal bacteria in person with a genetically determined dysregulation of mucosal t-lymphocytes, the inflammatory response being mediated by various factors such as tumour necrosis alpha. susceptibility appears to be related to a locus 56 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 55−59 on chromosome 16. the microorganism incriminated is mycobacterium paratuberculosis, but it appears unlikely that this is of major importance.5 the manifestations of crohn’s disease depend on its severity and the affected site. general symptoms are produced from gastrointestinal problem such as abdominal pain, diarrhea, constipation, anemia and weight loss.3,6 this comes from the inflammation process in the bowel which causes swelling, redness and ulceration. the disease process may also involve the orofacial tissues. the mucosa may have areas of serpiginous ulceration with adjacent oedematous, hyperplastic mucosa, creating a “cobblestoned” appearance. the ulcer may be superficial and shallow or deep and fissuring.1,2 other orofacial features concurrently found are facial/lips swelling, angular stomatitis/cracked lips, apthous-like ulcers, gingival hyperplasia and mucosal tag appearance.2 granulomas may also be seen in other conditions, especially sarcoidosis and in relation to foreign bodies. the term orofacial granulomatosis has been introduced for oral granulomatous reactions which are unassociated with any detectable systemic disease or with foreign bodies. in some patients the granulomatous reaction appears to be due to common additives to food and drink such as benzoates, cinnamon, or tartrazine. in some patients develop the condition after drinking carbonated drinks.3 the diagnosis of crohn’s disease depends on the demonstration of typical clinical, radiology, or histopathologic findings.3,6 the similar general symptoms of crohn’s disease and ulcerative colitis produce difficulty in making straight diagnosis, so that it needs the diagnostic histopathology.4 however, the findings of orofacial granulomatosis in these patients will be very useful, and the non-caseating granuloma from oral lesion biopsy is highly suggestive (65–85%) of cronh’s diasease.7 this article reported a case of crohn’s disease in retarded child, which one of the manifestation was orofacial granulomatosis. the diagnostic approach to and the treatment are also reviewed. case an 11-year-old retarded child was referred from pediatrician (asthma and allergy clinic) in jakarta of having persistent, painful ulcers on both side of the cheek and lateral border of the tongue since 6 months ago. the patient suffered from frequent diarrhea, constipation, weight loss and has been treated with medications with minimal improvement. there was no history of asthma, allergy and tuberculosis (to exclude sarcoidosis) reported. patient and also family member admitted of daily food pattern/food intake imbalance of patients. the extra oral examination revealed lymphadenopathy on both submandibular nodes. the lower lip was markedly edematous (figure 1). the lip was firm to palpation. intraoral examination of the right buccal mucosa revealed a wide, deep and yellowish ulcer, with irregular margins, slightly indurated (figure 2). on the left buccal mucosa revealed mucosal tag and similar ulcer, but slightly smaller than the right side (figure 3). on the left lateral of the tongue was found an irregular yellowish ulcer, as well as depapilated areas on the dorsal and extended to ventral of the tongue, as it showed healed ulcers (figure 4 and 5). both buccal mucosa were fibrous, caused limitation of mouth opening. hypersalivation and halitosis were noticed. figure 2. wide, fissured, irregular yellowish ulcers with softtissue swelling on the right buccal mucosa (first visit). figure 3. irregular yellowish ulcers with mucosal tag (arrow) on the left mucosa (first visit). figure 1. lip swelling was shown on the first visit. 57amtha: diagnosis and management of crohn's disease there were no appreciable changes on the palatum and hyperplasia gingival were slightly noticed although prominent. the provisional diagnosis based on clinical manifestations was established as crohn’s disease. differential diagnosis included sarcoidosis and orofacial granulomatosis with background of allergic reaction was necessary to be ruled out. case management under consent (represented by family member, as the patient was a retarded child), treatment planning was made. chest radiography and a series of full blood tests (including level of vitamin b12, ferrum and folic acid) were requested to rule out anemia and deficiencies. referral letter to gastroenterologist was made to obtain optimum management for patients. on the first visit, biopsy was not performed as patient needed to be handled psychologically. biopsy was planned to be done after laboratory results and gastroenterologist opinion. a mouth wash contain of hyaluronic acid was prescribed to relieve ulcer pain. gastroenterologist recommended patient to do gastrointestinal tract endoscopy, and patch test to rule out allergic reaction. however, patient refused to do the endoscopy. second visit, laboratory findings showed patient was suffering from anemia, followed by deficiency of a haematinic (fe, folat, and vitamin b12) which mean malabsorption as the effect of crohn’s disease. to confirm the diagnosis, punch biopsy was done on the right buccal ulcer with sutures and was sent for histopathological evaluation. patient was instructed to continue the gargle and clobetasol propionate 0.5% was applied topically three times daily for 2 weeks. third visit, the histopathology result showed some non-caseating epitheloid granulomatous with infiltration of perivascular lymphocyte and no malignant cells detected. the patch test was also negative. therefore, a final diagnosis of crohn’s disease was made. fourth visit, under coordination with gastroenterologist, systemic sulfasalazine 40 mg/kg body weight/day, 4 times daily for 2 weeks, corticosteroid therapy (dexamethasone 45 mg per day, 3 times daily, tapered dose for 2 week days) and multi vitamin were started and were well tolerated. the lip swelling decreased, and there was an obvious reduction in the ulcer size and pain. treatment was maintained until next 3 visits. the ulcers were gradually healed and disappeared eventually (figure 6, 7, 8), except the fibrosis of mucosa and cobblestoning, as the effect of disease (figure 9, 10, 11). patients were instructed to routine 6 months checked up to maintain oral hygiene and consume a proper nutrition. figure 5. irreguler ulcers extended to ventral of the tongue (first visit). figure 4. irregular ulcers on the lateral border and dorsal of the tongue were also shown on the first visit. figure 7. left buccal ulcer showed gradually healing (fourth visit). figure 6. ulcers on the right buccal mucosa showed healing (fourth visit). 58 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 55−59 discussion the diagnosis of crohn’s disease depends on the demonstration of typical clinical, radiology and histopathologic findings.3 differential diagnosis of lip swelling and ulcers which is called apthous-like ulcers in this patient includes sarcoidosis, ulcerative colitis, orofacial ganulomatosis (ofg), meischer’s cheilitis, and melkersonrosenthal syndrome.6 all of these conditions must be taken into account during investigation/anamnesis. the medical history and the results of the clinical examination showed that this patient had gastrointestinal problems, no history of tuberculosis, asthma, and demonstrated typical common oral lesions (cobbletoning and mucosal tag). it excluded the possibility of sarcoidosis (which usually present with background of tuberculosis). besides that it also ruled out ofg, which is presenting a swelling and ulcers without any gastrointestinal symptoms. meischer’s cheilitis is a condition where the swelling of the lips is isolated, without ulcers in the oral cavity.3 melkerson-rosenthal syndrome shows a lip and facial swelling in combination with fissured tongue and facial palsy.3 that clinical appearance was not found in this patient. ulcerative colitis was excluded by histological findings that found non-caseating granulomas from oral lesion biopsy. establishing the diagnosis of crohn’s disease in this patient followed the guideline of diagnosis of crohn’s disease in children and adults which has been published in 2007.8 it stated that crohn’s disease was established if the patient fulfills at least 2 of these criteria: a) clinical history shows abdominal pain, weight loss, fatigue; b) endoscopic findings: cobblestoning, linear ulceration, skip areas, perianal disease; c) radiological findings: fistula, mucosal cobblestoning, or ulceration; 4) macroscopic appearance: patchy penetrating lesions, cobblestoning, discrete ulcerations, fissuring, strictures); 5) histological finding of transmural inflammation or granulomas. hence, based on those criteria, this patient has fulfilled the criteria of cronh’s disease. in addition, ancillary test (complete blood count) has performed. it comprised of erythrocyte level, serum levels of folic acid, iron and vitamin b12 figure 8. ulcers on the dorsal of the tongue healed gradually. figure 10. healed ulcers (left buccal mucosa). figure 9. after 2 months of treatment, chronic ulcers on the right buccal mucosa have disappeared, and left a cobblestoned and mucosal fissures (mucosal tags). figure 11. complete healing of ulcers on dorsum of the tongue. 59amtha: diagnosis and management of crohn's disease for crohn’s disease to assess whether a systemic disease was responsible for the granulomatous inflammation. the assessment was essential, especially in the presence of signs of anemia and intestinal malabsorption, as one of the major effect of crohn’s disease, where it caused by the damaged of villi of ileum.1,2 oral lesions present vary 6-11%.4 in this case, the major ulcers present as cobblestoning appearance of oral mucosa and apthous-like ulcer, as most commonly reported.7 according to several authors9,10 a linear ulceration in the buccal vestibule surrounded by hyperplastic mucous folds is highly suggestive of crohn’s disease, however in this case, linear ulcer was not present. research on children who suffered crohn’s disease found that labial swelling and mucosal tags were the most frequent findings and majority of the children may have more than one type of oral mucosa.4 it is similar with the clinical appearance of this patient. analysis on 79 cases of crohn’s patient indicated that granulomas formation was more presented on oral lesions (67-85%) biopsy than on intestinal lesions (50%).11 biopsy on intestinal was not carried out as the patient refused to do that. however, finding of non-caseating granulomas from the oral biopsy was adequate enough to establish the diagnosis, based on previous study above. in this case, diagnosis of crohn’s disease was slightly difficult as patient was a retarded child, so that the information of gastrointestinal symptoms was firstly inadequate. in addition, patient also refused to undergo endoscope evaluation. patient was handled patiently and involved teamwork (with gastroenterologist and family member). patient refused to apply topical ointment (clobetasol propionate). it was due to the limitation of mouth opening because of the mucosal thickening, and rigidity of buccal mucosa. it was also because of hypersalivation that disturbed the attachment of ointment to oral lesions. hence, the treatment was changed into systemic corticosteroid and sufasalazine as a corticosteroid-sparing agent to reduce systemic side effect.12 the steroid was used in managing acute phases of the disease. sulfasalazine was used mainly for maintenance between active episodes6 and was given under cooperation with patient’s gastroenterologist. other therapeutic measures have been reported in the literature, including hydroxychloroquine, methotrexate, clofazimine, metronidazole, minocycline alone or in combination with oral prednisone, thalidomide and dapsone.13-15 oral hygiene maintenance was improved by dental health education every visit and using mouthwash. ulcers in the mouth and symptoms of gastrointestinal improved gradually. it helped patient a lot in taking food, so that the haematinic deficiency was corrected automatically. crohn’s disease is a rare disease with major oral manifestations of chronic apthous-like ulcer. in this case, crohn’s disease was established on retarded child, based on medical history, clinical appearance (general and oral manifestations), histological findings and laboratory assessment. the establishment of diagnosis of crohn’s disease can not be based on single manifestation. besides typical oral lesions, granulomas formations are the major essential hints to distinguish other similar gastrointestinal diseases and orofacial granulomatosis. due to a lack of understanding of etiology, relapses are common, and longterm treatment may be required. therefore, a comprehensive clinical evaluation and histopathological test are mandatory to diagnose and manage crohn’s disease. references 1. mcmahon rft, sloan p. diseases of gastrointestinal tract. in: essentials of pathology for dentistry. edinburg: churchil livingstone; 2000. p. 154–5. 2. liu c, crawford jm. the gastrointestinal tract. in: robbins, cotran. editors. pathologic basis of diseases. 7th ed. vinay kumar, abul k abbas, nelson fausto, editors. china: elsevier saunders; 2005. p. 846–9. 3. scully c, porter s. orofacial disease: update for dental clinical team: 2. ulcers, erosions and other causes of sore mouth. part ii. dental update 1999; 26:31–9. 4. pittock s, drumm b, fleming p, mcdermott m, imrie c, flint s, bourke b. the oral cavity in crohn’s disease. j pediatr 2001; 138:767–71. 5. zheng j, shi x, chu x, jia l, wang f. clinical features and management of crohn’s disease in chinese patients. chinese med j 2004; 117(2):183–8. 6. scully c, cawson ra. gastrintestinal disorders. in: medical problems in dentistry. oxford: wright; 2000. p. 189–91. 7. kauzman a, quesnel-mercier a, lalonde b. orofacial granulomatosis: 2 case reports and literature review. j can dent assoc 2006; 72(4):325–9. 8. ccfa working group (crohn’s and colitis foundation of america working group). differentiating ulcerative colitis from crohn disease in children and young adults: report of a working group of the north american society for pediatric gastroenterology, hepatology, and nutrition and the crohn’s and colitis foundation of america. journal of pediatric gastroenterology and nutrition.2007; 44:653–74. 9. dupuy a, cosnes j, revuz j, delchier jc, gendre jp, cosnes a. oral crohn’s disease: clinical characteristics and long-term follow-up of 9 cases. arch dermatol 1999; 135(4):439–42. 10. kalmar jr. crohn’s disease: orofacial considerations and disease pathogenesis. periodontol 2000 1994; 6:101–15. 11. plauth m, jenss h, meyle j. oral manifestations of crohn’s disease. an analysis of 79 cases. j clin gastroenterol 1991; 13(1):29–37. 12. scully c, felix dh. oral medicine-update for dental practicioner. aphtous and other common ulcers. british dent j 2005; 199(5): 259-64. 13. van der waal ri, schulten ea, van der meij eh, van de scheur mr, starink tm, van der waal i. cheilitis granulomatosa: overview of 13 patients with longterm follow-up results of management. int j dermatol 2002; 41(4):225–9. 14. hegarty a, hodgson t, porter s. thalidomide for the treatment of recalcitrant oral crohn’s disease and orofacial granulomatosis. oral surg oral med oral pathol oral radiol endod 2003; 95(5): 576–85. 15. lloyd da, payton kb, guenther l, frydman w. melkerssonrosenthal syndrome and crohn’s disease: one disease or two? report of a case and discussion of the literature. j clin gastroenterol 1994; 18(3):213–7. << /ascii85encodepages false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� volume 46 number 1 march 2013 research report hemolysin activities as virulence factor of enterococcus faecalis isolated from saliva and periapical abscess (gene detection by pcr) dewa ayu n.p.a,1 sari dewiyani 2 and dessy sulistya ashari3 1department of conservative dentistry, faculty of dentistry, universitas indonesia 2department of conservative dentistry, faculty of dentistry, universitas prof. dr. moestopo 3department of oral biology, faculty of dentistry, universitas indonesia jakarta – indonesia abstract background: enterococcus faecalis is a normal flora of the oral cavity, commonly detected in saliva and persistence in endodontic infections. these bacteria have diverse survival and virulence factors. hemolysin is one of the factor and still had unclear role as a virulence factor of the enterococcus faecalis to survive in the root canal. purpose: the purpose of this research was to analyze the presence and activity of hemolysin gene and its activity as a virulence factor isolated from saliva and root canals with periapical abscess. yet by understanding one of the phenotypes characters which is hemolysin, it is expected a successful endodontic treatment can be provided with the persistent of enterococcus faecalis bacteria. methods: method of the research starting with the identification of enterococcus faecalis bacteria in isolated saliva and periapical abscess was done in the first part of the study. then the phenotypes character of enterococcus faecalis such as gene detection and expression of hemolysin in blood agar cultures of the 60 colonies samples were performed in the later part. results: not all of the colonies cultured were identified as enterococcus faecalis. all positive detection on hemolysin gene showed hemolysin expresion in both isolated samples. however, there were samples with hemolysin expression eventough no hemolysin gene detected. hemolysin expression detection in saliva was higher due to different activation phase of hemolysin in saliva. the study with just one primer could lead to the possibility of undetected hemolysin gene, eventough there were samples that did not have hemolysin gene. the proportion of hemolysin expression in root canals were less than saliva, this could be influenced by environmental factors. however, hemolysin was considered as important virulence factor, particularly for disease therapy. conclusion: the conclusion of this research was hemolysin gene discovered in clinical isolated saliva and root canals samples as virulence factor of the enterococcus faecalis, and hemolysin expression occured from both sources. key words: hemolysin, virulence, enterococcus faecalis, saliva, root canal, primer abstrak latar belakang: bakteri enterococcus faecalis adalah flora normal rongga mulut dan merupakan mikroorganisme yang umum dideteksi dalam saliva dan infeksi endodontik persistensi. bakteri ini memiliki berbagai faktor survival dan virulensi. hemolysin adalah salah satunya tetapi masih merupakan faktor virulensi yang belum terlalu jelas mekanismenya. tujuan: tujuan riset ini adalah untuk menganalisis keberadaan gen hemolysin dan aktifitas hemolysin enterococcus faecalis sebagai faktor virulen yang diisolasi dari saliva dan saluran akar gigi dengan abses periapikal. mengetahui salah satu karakter fenotip enterococcus faecalis yaitu hemolysin diharapkan berguna untuk kesuksesan perawatan endodontik akibat pesistensinya bakteri tersebut. metode: penelitian diawali pada bagian pertama penelitian adalah identifikasi bakteri enterococcus faecalis isolat saliva dan abses periapikal. bagian kedua melihat karakter fenotip enterococcus faecalis berupa deteksi gen hemolysin serta ekspresi hemolysin di kultur agar darah dari 60 sampel koloni. hasil: tidak semua kuman yang tumbuh dikultur teridentifikasi sebagai enterococcus faecalis. pada deteksi gen hemolysin positif menunjukkan seluruhannya terekspresi hemolysin di kedua sumber isolat klinik. namun, terdapat sampel yang menunjukkan terekspresi hemolysin meskipun gen hemolysin tidak ada dan itu lebih banyak di saliva, walaupun tidak bermakna. hal ini dapat disebabkan perbedaan tahap aktivasi hemolysin di saliva. pengujian hanya dengan satu primer dapat menyebabkan kemungkinan �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 45–49 introduction enterococcus faecalis is a normal commensal flora, normally found in gastrointestinal and oral cavity.1-3 this bacteria could become pathogenic and caused of many infections including root canal infection.4-13 the characteristics of these microbes allow it to survive in conditions that are not common to other microbes, because it has some virulence factors which hold important role in the pathogenesis such as lipoteichoic acid, aggregation substance (as), hemolysin, gelatinase (gel e) and extracellular surface protein (esp).7-10,14 hemolysin has been reported as an imprtant virulene factor especially for disease therapy. this virulence factor can be use for guard antibiotic and corticosteroid combinations of treatment in hemolysin or non-hemolysin strain cases.15 on the other hand, even though hemolysin has been researched in many aspects but still had unclear role as a virulence factor of the enterococcus faecalis to survive in the root canal, and induce inflammation in the periapical tissues.16 enterococcus faecalis bacteria is a normal flora of the oral cavity, but often found in cases of periapical abscess and endodontic treatment failure cases, therefore it is assumed that there are differences in phenotype activity of hemolysin in saliva and root canals with periapical abscess.6 the involvement of enterococcus faecalis in periapical tissue infections was not fully understood from literatures. thus, more detailed data is needed to explain the behavior of the bacterial population in the root canal of the tooth, whether as the pathogen causing the infection of periapical tissues or opportunistic species that become pathogenic because of the conducive micro-environment to their survival. a question needs to be answered through this research because these bacteria are normal flora of the mouth and digestive tract. the purpose of this research was to analyze the presence and activity of hemolysin gene and its activity as a virulence factor isolated from saliva and root canals with periapical abscess. yet by understanding one of the phenotypes characters which is hemolysin, it is expected a successful endodontic treatment can be provided with the persistent of enterococcus faecalis bacteria. materials and methods the first part of the study was the identification of enterococcus faecalis bacteria in isolated clinical saliva and periapical abscess. then the phenotypes character of enterococcus faecalis such as gene detection and expression of hemolysin in blood agar cultures were performed in the later part. samples were taken from six patients with periapical abscess. the samples taken from saliva and root canals were then cultured in chromatogenic agar. sixty greenish blue color colonies were taken. extraction of dna proceeded for pcr preparation. dna was extracted using the real genomic hi-yield dna mini kit. sequence primers use for 16srna, forward: tggc ataa gagt gaaa ggcgc, revers: gggg acgt tcag ttac taac gt. sequence primers use for hemolysin gene forward: gact cggg gatt gata ggc, revers: gctg ctaa agct gcgc ttac.17-19 two pcr reactions were performed. the first one was using 16srna to ensure enterococcus faecalis dna analyzed, while the second reaction was to confirm the extracted dna contains hemolysin genes. pcr reaction performed in a total volume of 25µl containing pcr mix dream tag fermentation (real biotech co. usa), 3 µl primer 16srna forward 10 µm, 3 µl primer 16srna revers 10 µm, 3 µl nuclease free water. a total of 3 µl of extracted dna was added to the reaction mixture. pcr was also performed using a positive control (dna extracted from the american type culture collection [atcc] species). for the first amplification, samples were subjected to 22 denaturation cycles at 95° c for 15 min, heating at 94° c for 20 seconds. for the second amplification, pcr reaction conditions were 40 cycles of 67° c for 45 seconds, 72° c for 15 seconds, 72° c for 7 minutes and then 4° c. hemolysin gene pcr reaction was performed in the same way. the first temperature was 95° c during 15 minutes as the stage of activation of the dna denaturation enzyme. followed with 35 cycles at a temperature of 94° c for 20 seconds, 56° c for 45 seconds and 72° c for 60 seconds. pcr products were analyzed by 1.5% agarose gel electrophoresis, stained with gelred nucleic acid gel stain (biotium inc. usa) in tae electrophoresis buffer ii, ada gen hemolysin tetapi tidak terdeteksi. walaupun memang ada sampel yang tidak memiliki gen hemolysin. proporsi keberadaan ekspresi hemolysin pada saluran akar lebih sedikit dari saliva karena ekspresi hemolysin dipengaruhi faktor lingkungan. namun demikian, hemolysin adalah faktor viruensi yang penting khususnya untuk terapi penyakit. kesimpulan: kesimpulan penelitian ini adalah ditemukan gen hemolysin pada sampel isolat klinik saliva dan saluran akar sebagai faktor virulen bakteri enterococcus faecalis serta terjadi ekspresi hemolysin dari kedua sumber tersebut. kata kunci: hemolysin, virulen, enterococcus faecalis, saliva, saluran akar, primer correspondence: dewa ayu n.p.a, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta 10430, indonesia. e-mail: dewaayunpa@yahoo.co.id ��ayu, et al.,: hemolysin activities as virulence factor of enterococcus faecalis and viewed under ultraviolet translumination. positive or negative identification was done based on the presence of clear bands of the expected molecular size using a 100bp dna ladder (generuler, ca). then the enterococcus faecalis phenotypic characters test was done to confirm the hemolysin expression of isolated saliva and root canal samples in blood agar plates. blood agar medium was made by mixing 40 ml fresh lamb blood into 1 l of blood agar solution. colony dilution was done (10-5) in order to distribute colonies on blood agar. hemolysin expression activity will be seen in the form of clear circular nodes (halo) around the bacterial colonies. results about 15% of 70 colonies samples were not enterococcus faecalis colonies. this suggests the possibility of other types of bacteria isolated in the sample and were not studied further. a total of 60 enterococcus faecalis colony samples were taken for the next stage in this research (figure 1). the proportion of hemolysin gene in the isolated saliva samples were higher compared to root canal but no significant differences (table 1). the proportion of hemolysin expression in root canal sample is less than in saliva samples, but no significant differences (table 2 and figure 2). table 1 and 2 showed proportion of hemolysin gene and hemolysin expression not compare each other. figure 2. bacterial colonies showed no halo (left) and positive halo (right). table 2. hemolysin expression of clinical isolated samples clinical isolated samples hemolysin expression total p*yes no n % n % n root canal saliva 20 21 66.7 69.0 10 9 33.3 31.0 30 30 1.000 total 41 67.8 19 32.2 60 * chi square test figure 1. pcr 16srn result from 60 samples using a 100 bp dna ladder (generuler, ca). table 1. hemolysin gene distribution of clinical isolated samples clinical isolated samples hemolysin gene total p*yes no n % n % n root canal saliva 17 19 56.7 63.3 13 11 43.3 36.7 30 30 0.792 total 36 60.0 24 40.0 60 * chi square test on positive hemolysin gene detection showed that all of them had hemolysin expression in both clinical isolated samples. however, there were samples with hemolysin expression eventough no hemolysin gene detected (table 3). the proportion of hemolysin expression without hemolysin gene was 23.1% in root canal samples and 18.2% in saliva samples. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 45–49 discussion the identification of enterococcus faecalis bacteria was performed using chromatogenic agar as medium. this medium was used for positive gram bacteria. a more distinctive color display showed bacteria colonies in chromatogenic agar, greenish blue color colonies suspected as enterococcus faecalis or enterococcus faecium. pcr 16srna was used to confirm the enterococcus faecium colonies.20 the results showed that 85% of the samples were enterococcus faecalis colonies and the remaining 15% were not. this suggests the possibility of other types of bacteria isolated in the samples and were not studied further. the proportion of hemolysin gene in isolated root canal was 56.7%, smaller than the proportion of saliva which was 63.3%, there were no significant difference (p>0.05). in this study, cytolisin a primers were used to detect the presence of hemolysin gene, while hemolysin gene can detect using cytolisin a, b or m primers. the production and activation of hemolysin has several stages. lysis precursor factors (cyll) synthesized in ribosome, modified and undergo a process of translation by cylm, secreted from the cell by cylb, then activated by cyla.15 cyla primers selection was based on the final stage. hemolysin gene was dicovered more in saliva than in the root canal, although not significant, due to the activation of extracellular stage of hemolysin in saliva, while in root canal was still in the initial stage where bacteria grow. one primers was used in this test (cyla), with only cyla might cause other types of hemolysin gene undetected. the results of this study found that not all of the samples had hemolysin gene. a total of 43% of root canal samples and 36.7% saliva samples showed negative hemolysin gene. the literature showed that most of the strains of enterococcus faecalis were non-haemolytic.7,9 several studies supported the finding.18 nevertheless, primers of all hemolysin phases should be used to ensure complete detection of all stages in the formation of hemolysin gene. hemolysin expression of root canal samples was less than saliva samples, this could be caused by silent cyl genes; hemolytic activity on blood agar was not likely to happen because of environmental factors. the infected surrounding environment would be activated during the expression. it found that 6 of the 31 samples had cyla enterococcus faecalis gene but none expressed hemolysin activity.17 because of the small percentage, the role of this protein as a virulence factor considered very small. however, recent findings indicated that negative phenotype profile could be activated due to environmental factors to finally express hemolysin. researches using animal models demonstrated that hemolysin as an important virulence factor. antibiotic and corticosteroid combinations of treatment showed effective results in non-hemolysin strain cases, and has the effect to decrease tissue damage in endophthalmitis cases, while not useful in the case of strains that produce hemolysin.15 it was found that positive hemolysin gene indicates expression of hemolysin (100%) in both isolated clinical sources (root canal and saliva). however, there were samples that showed hemolysin expression without hemolysin gene existed. in root canal samples, the proportion of hemolysin expression with negative hemolysin gene was 23.1%, and 18.2% in the saliva samples. only cyla was used in this study, this might showed negative result but perhaps other types of hemolysin gene was involved. this research showed that hemolysin is one of the virulence factor of enterococcus faecalis isolated from saliva and root canal. all samples detected hemolysin gene indicates expression of hemolysin. on the other hand, there were samples that had hemolysin expression without hemolysin gene. it can occur because the research used only one primers, in fact, there are three stage of formation hemolysin gene. highly suggestion to use all of the kind of primers to detect all of the hemolysin gene for the further researches. acknowledgments this research was funded by the research fund drpm ui budget year 2012. directorate of research and community service, university of indonesia. table 3. hemolysin expression presence distribution with hemolysin gene of clinical isolated samples expression total yes no n % n % n root canal positive hemolysin gene negative hemolysin gene saliva positive hemolysin gene negative hemolysin gene 17 3 19 2 100.0 23.1 100.0 18.2 0 10 0 9 0.0 76.9 0.0 81.8 17 13 19 11 ��ayu, et al.,: hemolysin activities as virulence factor of enterococcus faecalis references 1. portenier i, waltisno tm, haapsalo m. enterococcus faecalis the root canal survival and star in post treatment disease. endodontic topics 2006; 135–59. 2. sunqvist g, fidgor d. life as an endodontic pathogen: ecological difference between untreated and filled root canal. endodontic topics 2003; 6: 3–28. 3. abdullah m, yuan-ling, gulabivala k, moles dr, spratt da. susceptibilties of two enterococcus faecalis phenotypes to root canal medications. j endod 2005; 31(1): 30–6. 4. vianna me, gomes bpfa, berber vb, zaia aa, ferraz ccr, souza-filho fj. in vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. oral surg oral med oral pathol 2004; 97(1): 70–84. 5. distel jw, hatton jf, gillerspie mj. biofilm formation in medicated root canals. j endod 2002; 28(10): 689–93. 6. dunuvant tr, regan jd, glickman gn, solomon es, honeyman an. comparative evaluation of endodontic irrigants against enterococcus faecalis biofilm. j endod 2006; 32(6): 527–31. 7. gutmann j. problem solving in endodontics. 4th ed. st. louis: elsevier mosby co; 2006. p. 91–123. 8. siqueira jf, rocas in. endodontic microbiology. in: torabinejad m, walton re, eds. endodontics principles and practice. 4th ed. st. louis: saunders elsevier; 2009. p. 38–48. 9. cohen s, burn r. pathways of the pulp. 8th ed. st. louis: by mosby inc; 2002. p. 231–92. 10. ingle, backland, et al. endodontic. 5th ed. california: elsevier; 2002. p. 179–83, 871. 11. carver k, nusstein j, reader a, beck m. in vivo antibacterial efficacy of ultrasound after hand and rotary instrumentation in human mandibular molars. j endod 2007; 33(9): 1038–44. 12. ercan e, ozekinci t, atakul f, gul k. antibacterial activity of 2% chlorhexidine gluconate and 5.25% sodium hypoclorite in infected root canal: in vivo study. j endod 2004; 30(2): 84–7. 13. shuping gb, orstavik d, sigurdsson a, trope m. reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. j endod 2000; 26(12): 751–5. 14. george s, kisben a, song kp. the role environment changes on monospesies biofilm formation on root canal wall by enterococcus faecalis. j endod 2005; 12(2): 867–71. 15. kayaoglu g, orstavik d. virulence factors of enterococcus faecalis: relationship to endodontic disease. crit rev oral biol med. 2004; 15(5): 308–20. 16. day am, cove jh, phillips-jones mk. cytolisin gene expression in enteroccus faecalis is regulated in response to aerobiosis condition. mol genet genomics 2003; 269(1): 31–9. 17. sedgley cm, molander a, flannagen se, nagel ac, appelbe ok, clewell db, dahlen g. virulence, phenotype and genotype characteristics of endodontic enterococcus spp. oral microbiol immunol 2005; 20(1): 10–9. 18. sedgley cm, lennan sl, clewell db. prevalence, phenotype and genotype of oral enterococci. oral microbiol immunol 2004; 19(2): 95–101. 19. bittencourt e, suzart s. occurrence of virulence-associated genes in clinical enterococcus faecalis strains isolated in londrina, brazil. j med microbiol 2004; 53(pt 11): 1069–73. 20. rocas in, siqueira jf. comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study. j endod 2011; 37(2): 143–50. 177 the potential application of stem cell in dentistry ketut suardita department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract stem cells are generally defined as cells that have the capacity to self-renewal and differentiate to specialize cell. there are two kinds of stem cell, embryonic stem cell and adult stem cells. stem cell therapy has been used to treat diseases including parkinson’s and alzheimer’s diseases, spinal cord injury, stroke, burns, heart diseases, diabetes, osteoarthritis, and rheumatoid arthritis. stem cells were found in dental pulp, periodontal ligament, and alveolar bone marrow. because of their potential in medical therapy, stem cells were used to regenerate lost or damage teeth and periodontal structures. this article discusses the potential application of stem cells for dental field. key words: embryonic stem cell, adult stem cells, dentistry correspondence: ketut suardita, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the inability of most tissues and organs to repair and regenerate after damage is a problem in medical and dentistry that should be solve. to repair or regenerate damage tissues and organs, many materials and devices were used, but the results are not good. many complications including infection, inflammation, impaired function, and loosening were happen. these conditions make opportunities to scientist improved therapies. in medical therapy, stem cells have been used for engineering many tissues and organs. stem cell researches are interesting knowledge about how to regenerate healthy cells, tissues, and organs from a single cell. stem cell is a multipotent cell, which can proliferate and differentiate to specific cell. these cells have the capacity to form many different tissue types. stem cell therapy has been used to treat diseases including parkinson’s and alzheimer’s diseases, spinal cord injury, stroke, burns, heart diseases, diabetes, osteoarthritis, and rheumatoid arthritis.1 regeneration of damage periodontal tissue, bone, pulp, and dentin are problems that the dentist should solve. at present, some traditional approaches are used to repair damage dental tissues. direct pulp capping using calcium hydroxide is a conventional technique to repair the damage of tooth pulp. demineralized bone graft was used in order to repair fractured bone. furthermore, guided tissue regeneration (gtr) and growth factor, for example bone morphogenic proteins (bmps) were used to regenerate new periodontal tissues. in fact, it is difficult to predict the result of treatments stated above.2 since, stem cells were used to regenerate damage tissue in medical therapy successfully; it is possible that the dentist use stem cell to regenerate lost or damage dental and periodontal structures. the purpose of this article is to describe and discuss stem cell potential application in dentistry. in future, stem cell therapy will enable new dental treatments for caries, endodontic, periodontal and oral-maxillo facial surgery, alveolar ridge augmentation, and cartilage in the temporomandibular joint. what is stem cell? stem cell can be described as an immature or undifferentiated cell that is capable of producing an identical daughter cell. stem cell has two important characteristics that distinguish them from other types of cells. first, they are unspecialized cells that renew themselves for long periods through cell division. the second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions. stem cell self-renewal may be perpetuated over many generations, resulting in considerable amplification of stem cell numbers. a stem cell is able to produce at least one type of highly differentiated cell. in traditional thinking, stem cells have been generally recognized as undifferentiated cells with varying degrees of potency. there are three basic measures of stem cell potency i.e. totipotent, pluripotent, and multipotent.3 stem cell has been identified in two kinds of tissues that are in adult tissues, so-called adult stem cell and in embryo, called embryonic stem cell. in a blastocyst of a developing embryo, stem cells differentiate into all of the specialized embryonic tissues. in adult organisms, stem cells act as a repair system for the body; replenishing specialized cells.4 generally adult stem cells present a more limited range of differentiated lineages. compared to embryonic stem cells, adult stem cell are preferable for therapeutic purposes since they are considered safer for implantation, with lesser 178 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 177–180 proliferation capacity and tumorogenecity. adult stem cells are also easier to differentiate to specific lineages.5 source of stem cell embryonic stem cells are isolated from the inner cell mass of the preimplantation blastocyst and have been derived from mice, non-human primates, and human. they are pluripotent cells, retaining the capacity to generate any and all fetal and adult cell types in vivo and in vitro. by manipulating the culture conditions under which embryonic stem cell differentiate, it has been possible to control and restrict the differentiation pathways. embryonic stem cell, especially mouse embryonic stem cell have been used to generate a range of distinct phenotypes including haematopoietic precursors, neural cells, adipocytes, muscle cells, myocytes, chondrocytes, pancreatic islet, and osteoblasts in vitro. because of their capability to differentiate into many different cell types, embryonic stem cell have been recognized as a valuable model system for studying the mechanisms underlying lineage specification during the early stages of mammalian development.6,7 stem cell can be identified in many adult mammalian tissues. in some tissues, such as epithelia, blood, and germ line, stem cells contribute to replenishment of cells lost through normal cellular senescence or injury. stem cells may also be present in other adult organs, such as the brain and pancreas, which normally undergo very limited cellular regeneration or turnover. adult stem cells are found in specific niches or tissue compartment including skin, liver, intestine, brain, skeletal muscle, myocardium, fatty tissue, and bone marrow.8 bone marrow contains hematopoetic stem cells, which differentiate into every type of mature blood cell; endothelial cell progenitors; and marrow stromal cells, also called mesenchymal stem cells (msc). msc can fabricate a spectrum of specializes mesenchymal tissues including bone, cartilage, muscle, marrow stroma, tendon, ligament, fat, and variety other connective tissues.9,10,11 in oral environment, stem cells were isolated from adult dental pulp tissues, periodontal ligament and alveolar bone marrow.12,13,14,15 there was an evidence that remnant dental pulp derived from exfoliated deciduous teeth contains a multipotent stem-cell population.16 application of stem cell for medical and dental therapies because of their abilities of unlimited expansion and pluripotency, embryonic stem cells are a potential source for regenerative medicine and tissue replacement after injury or disease. to date, no approved medical treatments have been derived from embryonic stem cell research. controversies surrounding the legal and moral status of human embryos and the use of embryonic stem cells encompass fundamental issues such as contraception, abortion, the definition of human life, and the rights and legal status of an embryo. the use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does require the destruction of an embryo. adult stem cells have been proven clinically useful because they can be isolated, transplanted, and effectively reconstitute the damage tissues. using autologous mscs dispersed in a collagen-type i gel, wakitani et al.17 succeeded in repairing full-thickness defects on the weight-bearing surface of medial femoral condyles. furthermore, treatment of msc with synthetic glucocorticoid dexamethasone stimulates msc proliferation and support osteogenic lineage differentiated.18 adult stem cells have been used to repairing or regenerating tissues because the limitation of adult cell for tissue regeneration. grande et al.19 reported that autologous chondrocyte cultures could be utilized to repair articular cartilage defects in the rabbit knee. subsequently, this technique has been applied to the clinical treatment of articular cartilage defects. although repairing the defect with chondrocytes is attractive, there are limitations related to the harvesting of chondrocytes and expanded these cells. we need large amount of biopsy to get enough cells for transplantations. in addition to the well-established bone and cartilage lineages, the induction of msc differentiation into other connective tissues, such as muscle, tendons, and ligaments is also being investigated. for a tissue-engineering approach, marrow-derived mscs have been used for achilles tendon repair. mscs seeded onto a collagen-type i construct incorporated into healing tendons. these mscs-loaded scaffolds had better alignment of cells and collagen fibers and were more similar to the native tendon than unloaded controls.20 recently, some groups have been examined the treatment of myocardial infarction by application of autologous mscs in the pig model, and these studies show engraftment, differentiation, and improved function in animals treated with autologous marrow mscs.21 in periodontal treatment, bone marrow-derived mesenchymal stem cells were transplanted to experimental class iii periodontal defects. the aim of this research was to elucidate the behavior of transplanted mscs in periodontal defects. four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue.22 discussion caries, pulpitis, apical periodontitis and another craniofacial diseases increase health costs and attendant loss of economic productivity. they ultimately result in premature tooth loss and therefore diminishing the quality of life. within the next few decades, changes in the methods and materials used to treat dental disease will take place. tissue engineering is a new concept that might be solves the problem in craniofacial regeneration. tissue engineering is the science of design and manufacture of new tissues to replace damage tissues because of diseases and trauma. the three key elements of tissue engineering are signal for morphogenesis, stem cells for responding to morphogens, and the scaffold of extra cellular matrix. stem cells are generally defined as cells that have the 179suardita: the potential application of stem cell capacity to self-renewal and differentiate to specialize cell. stem cells are present in small numbers in many vertebrate adult and fetal tissues. they are responsible for tissue renewal and for regeneration of damaged tissues. during wound healing, dental pulp stem cells have the potential to proliferate and to differentiate into odontoblasts to form dentin.23 in the other hand, stem cell derived from periodontal ligament may migrate into periodontal defect, proliferate, and differentiate.24 the ability of high expansion and multipotent of differentiation make stem cells are the cell sources for potential therapeutic use and tissue engineering in dentistry. several studies indicated that stem cells are present in dental pulp, alveolar bone and in the periodontal ligament.12,13,14 in endodontic and conservative dentistry, to restore and regenerate the dentin-pulp complex is a problem that very difficult to solve. direct pulp capping using calcium hydroxide cannot induce new dentin regeneration when there are no odontoblasts remain in dental pulp. stemcell based tissue engineering may be a new technique to regenerate dentin-pulp complex. using methodology developed to isolate and characterize mesenchymal stem cells, clonogenic and highly proliferative dental pulp stem cells (dpscs) have been isolated from adult human teeth. these stem cells maintained their high rate of proliferation even after extensive sub culturing and generated s dentin/ pulp-like complex. furthermore, it is noteworthy that the amount of dentin and pulp-like tissue formed in transplant far exceeds the amount that would be generated in situ during the lifetime of an organism. consequently, there is a great potential for the isolation of a large number of dpscs from a single tooth that could be used for dentinal repair of a number of teeth.12 in addition, iohara et al.25 in their research stated that the autogenous transplantation of bmp2-treated dental pulp stem cell pellet culture onto the amputated pulp stimulated reparative dentin formation. these results prove that stem cell therapy has considerable promise in dentin regeneration. in near future, stem cells in combination with appropriate scaffolds and growth factors are materials, which may use for direct pulp capping. in addition, multipotent stem cells were isolated from the remnant pulp of exfoliated deciduous teeth. it found that these stem cells are distinct from dpscs with respect to their high proliferation rate, increases cell-population doublings, sphere-like cell-cluster formation, osteoinductive capacity in vivo, and failure to reconstitute a dentin-pulp like complex. it is indicate that deciduous teeth may be an ideal resource of stem cells to repair damage tooth structure, induce bone regeneration, and possibly to treat neural injury or degenerative diseases. as we know, periodontal diseases can destroy the periodontal ligament, bone, and cementum. destruction of this tissue is a cause of tooth loss. recently, seo et al.26 discovered stem cells from human periodontal ligament. these stem cells have the potential to generate periodontal ligament and cementum. in another research, mesenchymal stem cells have been used for periodontal defect treatment. hasegawa et al.22 was transplanted bone marrow-derived mesenchymal stem cell to experimental class iii periodontal defects. four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue. cementoblasts, osteoblasts, osteocytes, and fibroblasts of the regenerated periodontal tissues were detected. as a dentist, we frequently encounter defects in alveolar bone caused by trauma or inflammatory processes. due to the high healing capacity of oral tissues, small defects frequently heal without major problems. however, if the affected area is large or complex tissues are involved, regeneration is generally incomplete. to regenerate the deteriorated tissues biologically based technique are required. stem cell based-tissue engineering is a good alternative therapy. several studies have already detailed the ability of mscs transduced with bmp7 to elicit periodontal bone formation.27 stem cell has a clinical potential for bone defect therapy. until now, bone graft technique have been used for bone graft therapy. autogenous bone graft from iliac bone make good healing, but this therapy is too expensive and morbidity. furthermore, 8% of iliac graft make infection, nerve injury, blood loss, short and long-term pain and functional deficit. recently, ueda 28 were able to demonstrate the transplantibility and therapeutic effects of msc in bone defect. he formed mscs transplantation in combination with biodegradable scaffold (beta tcp). the result of this research was the increased of bone regeneration in the defect. stem cells are also found in alveolar bone marrow. isolated stem cells from alveolar bone marrow were cultured and expanded. these stem cells had potent osteogenic potential in vitro and in vivo. based on the results, the researchers hope that transplantation of alveolar bone marrow stem cell can promote regeneration of alveolar bone in patients with periodontal diseases.15 in conclusion, stem cell present in dental pulp, periodontal ligament and alveolar bone marrow, and has a potential to repair and regenerate tooth and periodontal structures. stem cells can be harvested from dental pulp, periodontal ligament, alveolar bone marrow, expanded, embedded in a appropriate scaffold, and transplanted back into defect to regenerate bone and tooth structures. references 1. bonassar lj, vacanti ca. tissue engineering: the first decade and beyond. j cell biochem supp 1998; 30(31): 297–303. 2. nakashima m, reddi h. the application of bone morphogenetic proteins to dental tissue engineering. nature biotechnology 2003; 21:1025–32. 3. ballas cb, zielske sp, gerson s. adult bone marrow stem cell and gene therapies: implications for greater use. j cell biochemistry supp 2002; 38:20–8. 4. caplan ai, bruder sp. mesenchymal stem cells: building blocks for molecular medicine in the 21st century. trends in mol med 2001; 7:259–64. 5. pelled g, turgeman g, aslan h, gazit z, gazit d. mesenchymal stem cells for bone gene therapy and tissue engineering. current pharmaceutical design, 2002; 8:1917–28. 180 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 177–180 6. rathjen pd, lake j, whyatt lm, bettes md, ratjhen j. properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. reprod fertil dev 1998; 10:31–47. 7. odorico js, kaufman ds, thomson ja. multilineage differentiation from human embryonic stem cell lines. stem cell 2001; 19:193–204. 8. kuehnle i, goodell m. the therapeutic potential of stem cells from adults. bmj 2002; 325:372–6. 9. caplan ai. the mesengenic process. clin plastic surg 1994; 21:42935. 10. makino s, fukuda k, miyoshi s, kinoshi f, kodama h, pan j, sano m, takahashi t, hori s, abe h, hata j, umezawa a, ogawa s. cardiomyocytes can be generated from marrow stromal cells in vitro. j clin invest 1999; 103:697–705. 11. bianco p, robey pg. marrow stromal stem cells. j clin invest 2000; 105:1663–8. 12. gronthos s, mankani m, brahimj, robey pg, shi s. postnatal human dental pulp stem cells (dpscs) in vitro and in vivo. proc natl acad sci usa 2000; 97:13625–30. 13. gronthos s, brahim j, li w, fisher lw, cherman n, boyde a, denbesten p, robey pg, shi s. stem cell properties of human dental pulp stem cells. j dent rest 2002; 81:531–5. 14. batouli s, miura m, brahim j, tsutsui tw, fisher lw, gronthos s, robey pg, shi s. comparison of stem-cell-medicated osteogenesis and dentinogenesis. j dent res 2003; 82:976–81. 15. matsubara t, suardita k, ishii m, sugiyama m, igarashi a, oda r, nishimura m, saito m, nakagawa k, yamanaka k, miyazaki k, shimizu m, bhawal u, tsuji k, nakamura k, kato y. alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells. j bone and min. res. 2005; 20: 399–409. 16. miura m, gronthos s, zhao m, lu b, fisher lw, robey pg, shi s. shed: stem cells from human exfoliated deciduous teeth. proccedings of the national academy of science. 2003; 100: 5807–12. 17. wakitani s, gotot, pineda sj, young rg, mansour jm, caplan ai, goldberg vm. mesenchymal cell-based repair of large, fullthicknesss defects of articular cartilage. j bone joint surg am 1994; 76:579–92. 18. liu f, aubin, je, malaval l. expression of leukimia inhibitory factor (lif)/interleukin-6 family cytokines and receptors during in vitro osteogenesis: differential regulation by dexamethasone and lif. bone 2002; 31:212–9. 19. grande, da, pitman mi, peterson j, menche d, klein m. the repair of experimentally produced defect in rabbit articularcartilage by autologous chondrocyte transplantation. j orthop res 1989; 7:208–18. 20. shake jg, gruber pj, baumgartner wa, senechal g, mryers j, redmont jm, pittenger mf, martin bj. mesenchymal stem cell implantation i a swine myocardial infarct model: engraftment and functional effects. ann thorac surg 2002; 73: 1919–26 21. young rg, butler dl, weber w, caplan ai, gordon sl, fink dj. use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. j orthop res 1998; 16: 406–13. 22. hasegawa n, kawaguchi h, hirachi a, takeda k, mizuno n, nishimura m, koike c, tsuji k, iba h, katoy, kurihara h. behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects. j periodontol 2006; 77(6):1003–7. 23. tziafas d, smith aj, lesot h. designing new treatment strategies in vital pulp therapy. j dent 2000; 28: 77–92. 24. cho mi, garant pr. development and general structure of the periodontium. periodontol 2000; 24: 9–27. 25. iohara k, nakashima m, ito m, ishikawa m, nakashima a, akamine a. dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. j dent res. 2004; 83(8): 590–5 26. seo bm, miura m, gronthos s, bartold pm, batouli s, brahim j, young m, robey pg, wang cy, shi s. investigation of multipotent postnatal stem cells from human periodontal ligament. the lancet 2004; 364:149–155. 27. jin qm, anusaksathien o, webb sa, rutherford rb, giannobile wv. gene therapy of bone morphogenic protein for periodontal tissue engineering. j periodontol 2003; 74:202–13. 28. ueda m. maxillofacial bone regeneration using tissue engineering concepts. dentistry in japan 2003. 39:199–205 � volume 46 number 1 march 2013 bactericidal and cytotoxic effects of erythrina fusca leaves aquadest extract janti sudiono,1 ferry sandra,2 nadya saputri halim,3 timotius andi kadrianto3 and melinia3 1department of oral pathology, faculty of dentistry, universitas trisakti 2department of biochemistry, faculty of dentistry, universitas trisakti 3dental practitioner jakarta – indonesia abstract background: empirically, erythrina fusca has been used as traditional herb for its antibacterial and antiinflammation properties. periodontal disease is one of the most oral infectious diseases with microorganism predominated as the contributing factors. porphyromonas gingivalis (p. gingivalis) is one of the main bacteria pathogen found in periodontal diseases. purpose: the purpose of this study was to examine the bactericidal effect of erythrina fusca leaves aquadest extract (eflae) at various concentrations on p. gingivalis and cytotoxic effect on fibroblast. methods: pure p. gingivalis was cultured in brain heart infusion (bhi) medium for 24 hours with or without various concentrations of treatment of eflae. calculation and statistical analysis of remaining bacteria were performed by inhibitory zone method to evaluate the eflae bactericidal effect and compared to chlorhexidine as positive control. to evaluate the cytotoxic effect, nih 3t3 cells were cultured in dulbecco’s modification of eagle’s medium (dmem) containing of 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin, ph 7.2, in 5% co2, and stored in humidified incubator under temperature 370 c. cells were treated with/without various concentrations of eflae for 48 hours. the viable cells were then counted using 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazodium bromide (mtt) method. results: eflae have bactericidal effect on p. gingivalis in a concentration dependent manner starting from 78%. the concentration of 90% eflae had stronger bactericidal effect (35.004 ± 1.546) than those of chlorhexidine as positive control (32.313 ± 1.619). one-way anova showed significant bactericidal effect differences among concentrations of eflae and chlorhexidine (p<0.05) while tuckey hsd test showed significant difference only between lower concentration of eflae (78%, 79%) and chlorhexidine. with the highest concentration of eflae (100%) applied in the bactericidal test, no cytotoxic effect of eflae on nih 3t3 cells was detected. conclusion: eflae could inhibit the growth of p. gingivalis in a concentration dependent manner, starting from 78%. there was no evidence of eflae’s cytotoxic effect on fibroblast. key words: eflae, bactericidal, citotoxicity abstrak latar belakang: erythrina fusca telah digunakan secara empiris sebagai tanaman obat tradisional untuk khasiat antibakteri dan antiradang. penyakit periodontal merupakan salah satu penyakit infeksi mulut terbanyak dengan mikroorganisme sebagai faktor kontributor utama. porphyromonas gingivalis (p. gingivalis) merupakan salah satu bakteri patogen utama yang ditemukan pada penyakit periodontal. tujuan: tujuan penelitian ini untuk mengamati efek bakterisid terhadap p. gingivalis dan efek sitotoksik terhadap sel fibroblast dari beberapa konsentrasi ekstrak akuades daun erythrina fusca (eflae). metode: p. gingivalis murni dikultur pada medium brain heart infusion (bhi) selama 24 jam dengan atau tanpa pemberian beberapa konsentrasi eflae. perhitungan dan analisis statistik terhadap bakteri yang masih hidup dilakukan dengan metode zona hambat untuk mengevaluasi efek bakterisid eflae dibandingkan dengan chlorhexidine sebagai kontrol positif. untuk mengevaluasi efek sitotoksik, digunakan kultur sel nih 3t3 pada medium dulbecco’s modification of eagle’s medium (dmem) yang berisi fetal bovine serum (fbs) 10% dan penicillin-streptomycin 1%, ph 7.2, dalam co2 5%, dan diinkubasi pada suhu 37° c. sel diberi perlakuan dengan atau tanpa beberapa konsentrasi eflae selama 48 jam, kemudian sel yang masih hidup dihitung menggunakan metode 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazodium bromide (mtt). hasil: eflae mempunyai efek bakterisid terhadap p. gingivalis mengikuti kenaikan konsentrasinya dimulai dari 78%. pada konsentrasi 90%, eflae menunjukkan efek bakterisid lebih kuat (35.004 ± 1.546) dibandingkan dengan chlorhexidine (32.313 ± research report �0 dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 9–13 introduction erythrina fusca (e. fusca) is the most widespread species in the genus available wild in both the old and new world tropics. in asia and oceania it occurs along coasts and rivers planted throughout the humid tropic. e. fusca is found from sea level up to 200 m altitude, within a wide range of rainfall pattern, from 1,200 mm to over 3,000 mm annually, with or without seasonal distribution. e. fusca has many functions and been used by several countries; as in indonesia, the scraped inner bark is used for poulticing fresh wounds.1 prior study of ethanol extract of e.fusca showed inhibitory effect of cyclooxygenase 2 (cox2).2 in vietnam, the bark is used to treat toothache. the young leaves are eaten as a vegetable in java, bali, and guatemala.1 the first compounds isolated from erythrina were alkaloids. subsequently, homoerythrina alkaloids were investigated for their anti-cancer activity. recently, research involving erythrina has focused on other chemical effects, primarily the antimicrobial action of erythrina lectins and the enzymology of proteinase inhibitors isolated from erythrina. however there was no research about its effect on periodontal disease as one of the most prevalent oral diseases in indonesia therefore this research was conducted. the incidence of periodontal disease reached 70% in entire population of the world, including indonesia, especially in elderly.3 periodontal disease is an infectioustyped disease which can be caused by local factor as well as systemic factor. commonly, the main cause of periodontal disease is local factor, which is caused by bacteria and afterwards is aggravated with the existence of systemic factor. the main pathogenic bacteria which cause the periodontal disease is pophyromonas gingivalis (p. gingivalis), this bacteria has the ability to infect the periodontal ligament; which in early stage starts with infection of the gum (gingivitis) and continue to chronic infection which involve all the periodontal ligament (periodontitis).4,5 phytochemistry test on e. fusca leaves aquadest extract (eflae) in balai penelitian tanaman obat dan aromatik (balittro), bogor (2010), showed that eflae contains alkaloid, glycoside, saponin, tanin, triterphenoid and steroid. the strongest compounds found in eflae are alkaloid and glycoside. alkaloids have the ability as anti-bacterial agent. tanin has also shown potential as antibacterial agent.6,7 and other previous research concluded that triterphenoid and saponin worked as antibacterial agent.8 these knowledge become the foundation for the implementation of this scientific research about the bactericidal effect of eflae on p. gingivalis. concerning to its bactericidal potency, the cytotoxic effect of this natural biomaterial need to be evaluated to know whether eflae biocompatible to be applied on oral mucosa. cytotoxic test was conducted on nih3t3 cells as fibroblast is one of the important structures of oral mucosa.9 therefore the purpose of this study was to examine the bactericidal effect on p. gingivalis and cytotoxic effect on fibroblast of eflae at various concentration. materials and methods this study is an experimental laboratory research. extraction of e. fusca leaves were performed by maceration tehnique using aquadest to find out gel form extract.10 e. fusca leaves (50 mg) were dried for 5 days, grinded, diluted in aquadest (500 ml) for 24 hours, refined, then evaporated with rotary evaporator 40° c up to gel formation of eflae. dimethyl sulfoxide (dmso) used in this study as polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. this study used serial dilution method to get various concentration of eflae. pure p. gingivalis was cultured in brain heart infusion (bhi) medium for 24 hours in 37° c humidified incubator, with/without various concentrations treatment. observation and calculation of remaining bacteria were performed by inhibitory zone method to evaluate eflae bactericidal effect and compared to chlorhexidine as positive control since up to know chlorhexidine is accepted as gold standard of periodontal treatment.11 the results were then analyzed using one way anova with α = 0.05. the bactericidal test showed that bactericidal effect occured up to concentration 80% of eflae while the concentration of 70% showed negative result therefore the concentration treatment diluted into lower concentration gradually which were 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% in order to find out the minimum bactericidal effect concentration. 1.619) sebagai kontrol positif anova-1 jalan menunjukkan perbedaan efek bakterisid yang bermakna di antara beberapa konsentrasi eflae dan chlorhexidine (p<0.05) sedangkan uji tuckey hsd menunjukkan perbedaan bermakna hanya ditemukan antara konsentrasi eflae yang lebih rendah (78%, 79%) dengan chlorhexidine. efek sitotoksik terhadap sel nih 3t3 tidak terdeteksi pada pemberian konsentrasi tertinggi eflae (100%) yang telah diaplikasikan pada uji bakterisid. kesimpulan: eflae dapat menghambat pertumbuhan p. gingivalis sesuai dengan konsentrasinya dimulai dari 78%. tidak ada efek sitotoksik eflae terhadap sel fibroblast. kata kunci: eflae, bakterisid, sitotoksisitas correspondence: janti sudiono, c/o: departemen patologi mulut, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa no. 260 grogol-jakarta 11440, indonesia. e-mail: jantish@hotmail.com ��sudiono, et al.,: bactericidal and cytotoxic effects of erythrina fusca leaves aquadest extract nih3t3 cells were cultured in 100 µl dulbecco’s modification of eagle’s medium (dmem) containing 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin using the 96 wells-plate, ph 7.2, in 5% co2 37° c humidified incubator.14 hemacytometer with trypan blue staining was used to count the number of viable cells. the viable cells is unstaining cells. viable cell per ml is equal to average viable cell count per square into dilution factor into 104. dilution factor is total volume sample and diluting liquid divided by volume of sample. percentage cell viability is total viable cells (unstain cells) divided by total cells (viable and dead cells) into 100. total viable cell/ sampel is viable cell per ml into the original volume of fluid from which the cell sample was removed. volume of media needed is number of cells needed divided by total number of viable cells into 1.000. the number of cells used in this cytotoxicity test was 2.000 cells. cells were treated with/ without various concentrations of eflae (0%, 10%, 100%) for 48 hours. the viable cells were then counted by 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazodium bromide (mtt) assay method using the standard curve formula. this assay based on the changes of tetrazodium salt. mtt will transmute formazan in mitochondria. the formazan’s concentration, purple in colour can be determined by spectrophotometry. formazan crystal which was formed will be dissolved by the addition of acid isopropanol. the absorbance was then evaluated using elisa plate reader with wave length (λ) of 570nm.12 the cells absorbance was in liniar with viability. results eflae used in this study was in gel form, brownishgreen in colour and solid consistency. bactericidal effect of eflae occured starting from concentration 78% of eflae and having tendencies to increase and reached its peak on 80% (figure 1). eflae has bactericidal effect on p. gingivalis in a concentration dependent manner starting from 78%. the concentration of 90% eflae had stronger bactericidal effect (35.004±1.546) than those of chlorhexidine as positive control (32.313±1.619). no significant difference between the concentration of 100% and 80% eflae with those of chlorhexidine as positive control. one-way anova showed significant bactericidal effect differences among concentrations of eflae and chlorhexidine (p<0.05) while tuckey hsd test showed significant difference only between low concentration of eflae (78%, 79%) and chlorhexidine (figure 1). various quantitities of nih3t3 cells were cultured in dmem using 96 wells-plate for 24 hours to find out standard curve. the test was conducted in three consecutive weeks using elisa plate reader, with λ = 570 nm to find out the absorbance of the cells; in which a formula of standard curve acquired. nih3t3 cells with the same quantity (2.000 cells) were treated with various concentrations (0%, 10%, 100%) of eflae. the test was done in three consecutive weeks (week 1, 2, and 3). afterward, the result of the cell’s absorbance was substituted to the formula of standard curve to calculate the number of viable cells. number of viable nih3t3 cells which were cultured in dmem with/without various concentrations of eflae showed that eflae did not induce cytotoxicity on nih3t3 cells (figure 2). on day 3, the number of nih3t3 cells with 10% and 100% of eflae were slightly higher than those in control (0%). anova test showed significant difference between groups (p=0.00<0.05). however, tuckey hsd test showed that on day 3, there was no significant difference number of viable cells between control and 100% (p=0.256>0.05) and between 10% and 100% (p=0.080>0.05), while there was significant difference of viable cells number between control and 10% of eflae group (p=0.005<0.05) (figure 3). figure 1. diameter of bactericidal effect of eflae in various concentrations (blue); aquadest (orange); and chlorhexidine (red). this line diagram showed that bactericidal effect of eflae (blue dot) tends to increase following increase concentration while aquadest showed no bactericidal effect and chlorhexidine showed bactericidal effect equal as 100% eflae. figure 3. number of nih3t3 cells with various concentrations of eflae at the time of seeding (d0) and day 3 (d3). on day 3, the number of nih3t3 cells with 10% and 100% of eflae were slightly higher than control (0%). �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 9–13 discussion this study used aquadest to find out erythrina fusca leaves extract (eflae) with consideration that aquadest is not influenced in phytochemical compound of erythrina fusca leaves compared to those of other solvent such as ethanol, chloroform or methanol. beside the result of phytochemical test of eflae consisted of potential compounds as also found by other studies such as alkaloid, glycoside, saponin, tannins, triterpenoids, and steroids. another reason, the exploration of this aquadest extract can be used orally at the future as its simply soluble in gastrointestinal tract that be absorbed fastly. moreover, this type of extract can be directly applied on the oral mucosa. in indonesia, there is not much scientific research about eflae as phytotherapeutic agents especially against p. gingivalis as one of the most pathogen bacteriae of periodontal disease. outside of indonesia such as in sub-saharan africa, erythrina species is sources of lead compounds or new class of phytotherapeutic agents for fighting against major public health (mdr infections, cancer, diabetes, obesity). some phytochemicals (vogelin b, vogelin c, isowightcone, abyssinin ii, derrone) were demonstrated as the active principles as antibacterials, antifungals, antiplasmodials and inhibitors of enzyme borne diseases (protein tyrosine phosphatase (ptp) inhibitor/ ptp1b, hiv protease).13 in japan and thailand, there was also some studies about the phytotherapeutic agents of erythrina.14–16 this study found that 80%, 90% and 100% of eflae has bactericidal effects on p. gingivalis as strong as those of chlorhexidine and the 90% concentration of eflae had stronger bactericidal effect than those of chlorhexidine which was in general accepted as commercial antibacterial dental medicine. however, the optimum concentration of eflae was at 80% because no significant increase in bactericidal effect after this concentration. indeed, the 80% of eflae can be explored to be used as dental medication in the future. bactericidal effect of eflae were suspected from some of the components found in eflae such as alkaloid, tannins, and saponin.6–8 phytochemistry test done in this study showed alkaloid is a highest percentage component of eflae. the result of this study assumed that alkaloid play a role in resulting the bactericidal effect on p. gingivalis as stated in previous research that alkaloid act as antibacterial agent, by means of disrupting the compiler’s component of peptidoglycan bacterial cell therefore the cell wall is not fully formed, resulting in apoptosis of the cell.8 beside that, alkaloid is known as the compound to be a dna intercalator and an inhibitor of dna synthesis through topoisomerase inhibition.17–19 in order to ensure this mechanism, further research need to be conducted. there were several cytotoxicity studies on erythrina species. the cytotoxicity study of innok et al.20 about flavanoids and pterocarpans compound in the bark of erythrini fusca (which was also found in the leaves at this phytochemistry study) revealed that three new isomeric flavanones, fuscaflavanones a; six known flavanones, lupinifolin; lonchocarpol a; phaseollidin showed moderate to weak activity against kb, bc and ncih187 cells, whereas fuscaflavanones a(2) exhibited only weak activity against kb cells. another cytotoxicity study of erythrina stricta roots and erythrina subumbrans stems extracts by rukachaisirikul et al.16 stated that erybraedin a (2) showed the highest activity against the nci-h187 and bc cells (ic50 2.1 and 2.9 microg/ml, respectively), whereas erysubin f exhibited the highest activity against the kb cells (ic50 4.5 microg/ml). cytotoxic test in this study was done to examine eflae’s cytotoxic effect on fibroblast cells. the result of this test showed that the highest concentration of eflae (100%) did not induce cytotoxicity of cells. however the lower concentration (10%) of eflae showed increase viability of cells compared to those of control. a b c a b c a b c figure 2. eflae did not induce cytotoxicity on nih3t3 cells. nih3t3 cells which were cultured in 96 wells-plate for 24 h then treated by 0% (a), 10% (b) and 100% (c) eflae for 48 h. pictures were captured under light microscope on day three. black bar = 100 mm. the viable cells determine based on the microscopic feature refer to no cytopathic effect (cpe). maginification (40x10). ��sudiono, et al.,: bactericidal and cytotoxic effects of erythrina fusca leaves aquadest extract based on the bactericidal test and citotoxicity test of eflae, this study suggested to develop 80% eflae as a traditional herbs in gel state for periodontal disease since this concentration does not show cytotoxic effect but urges the growth of cells result in recuperation and proliferation of cells beside its optimum bactericidal property. in conclusion, eflae could inhibit the growth of p. gingivalis in a concentration dependent manner starting from 78%. there was no evidence of eflae’s cytotoxic effect on fibroblast. references 1. valkenburg jlch, bunyapraphatsara n. plant resources of southeast asia: medicinal and poisonous plants 2. leiden: backhuys publishers; 2001. p. 252-53. 2. perera p, ringbom t, huss u, vasage m, bohlin. search for natural product which affect cyclooxygenase-2. in: tringali c, editor. bioactive compounds from natural sources: isolation, characterisation, and biological properties. london: taylor and francis; 2001; 434-65. 3. situmorang n. profil penyakit periodontal (periodontal disease profile). dentika dental journal 2004; 9: 71-7. 4. carranza fa, newman mg, takei hh. clinical periodontology. 9th ed. philadelphia: saunders; 2002. p. 64-9. 5. nield js, willman de. foundation of periodontics for dental hygienist. philadelphia: lippincott; 2003. p. 54-5. 6. akiyama h, fujii k, yamasaki o, oono t, iwatsuki k. antibacterial action of several ta nnins aga inst staphylococcus aureus. j antimicrob chemother 2001; 48(4): 487–91. 7. funatogawa k, hayashi s, shimomura h. antibacterial activity of hydrolysable tannins derived from medicinal plants against helicobacter pylori. microbiol immunol 2004; 48(4): 251–61. 8. yadava rn, jharbade j. new antibacterial triterpenoid saponin from lactuca scariola. fitoterapia 2008; 79(4): 245-9. 9. oie y, hayashi r, takagi r, yamato m, takayami h, tanu y, mishida k. a novel method of culturing human oral mucosal epithelial cell sheet using post-mitotic human dermal fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction. br j ophthalmol 2010; 94(9): 1244–50. 10. saefudin a, rahayu v, teruna hy. standarisasi bahan obat alam. edisi 1. yogyakarta: graha ilmu; 2011. p. 1-75. 11. marsh pd, martin mv. oral microbiology. 4th ed. edinburgh: elsevier; 2009. p. 74-100. 12. freshney ri. culture of animal cells. 4th ed. new york: wiley-liss; 2000. p. 153-63, 364. 13. karou d, savadogo a, canini a, yameogo s, montesano c, simpore j, colizzi v. traore as. antibacterial activity of alkaloids from sida acuta. african j of biotechnology 2005; 4(12): 1452-7. 14. sato m, tanaka h, oh-uchi t, fukai t, etoh h, yamaguchi r. antibacterial activity of phytochemicals isolated from erythrina zeyher i against vancomycin-resistant enterococci and their combinations with vancomycin. phytother res 2004; 18(11): 906-10. 15. rukachaisirikul t, innok p, suksamrarn a. erythrina alkaloids and a pterocarpan from the bark of erythrina subumbrans. j nat prod 2008; 71(1): 156-8. 16. rukachaisir ikul t, saekee a, tha r ibun c, watkuolham s, suksamrarn a. biological activities of the chemical constituents of erythrina stricta and erythrina subumbrans. arch pharm res. 2007; 30(11): 1398-403. 17. innok p, rukachaisirikul t, suksam rarn a. flavanoids and pterocarpans from the bark of erythrina fusca. chem pharm bull. 2009; 57(9): 993-6. 18. guittat l, alberti p, rosu f, van miert s, thetiot e, pieters l, gabelica v, de pauw e, ottaviani a, roiu jf, mergny jl. interaction of cryptolepine and neocryptolepine with unusual dna structures. bioch 2003; 85: 535-41. 19. lisgarten jn, coll m, portugal j, wright cw, aymami j. the antimalarial and cytotoxic drug cryptolepine intercalates into dna at cytosine-cytosine sites. nature structural biol 2002; 9: 57-60. 20. kone wm, solange k n, dosso mj. assessing sub-saharan erythrina for efficacy: traditional uses, biological activities and phytochemistry. pak j biol sci 2011; 14(10): 560-71. 127 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 127–131 original article post-tooth extraction induction effect of moringa oleifera leaf extract and demineralized freeze-dried bovine bone xenograft treatment on alveolar bone trabecula area utari kresnoadi1, najla salsabila2, primanda nur rahmania1, phara aster chandra adventia2, bima subiakto rahmani2, nobuhiro yoda3 1department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3division of advanced prosthetic dentistry, tohoku university graduate school of dentistry, sendai, miyagi, japan abstract background: after tooth extraction, alveolar bone resorption occurs naturally, followed by alveolar bone remodeling. alveolar bone formation is characterized by an increase in density and expansion of the trabecular bone. socket preservation using a combination of moringa oleifera leaf extract and demineralized freeze-dried bovine bone xenograft (dfdbbx) is expected to increase the area of the alveolar bone trabeculae and thus accelerate the process of alveolar bone formation. purpose: this study aimed to determine if a combination of moringa oleifera leaf extract and dfdbbx could increase the area of the alveolar bone trabeculae in tooth extraction sockets. methods: with their lower left incisors extracted, the 56 cavia cobayas were divided into eight treatment groups according to the material given: polyethylene glycol (peg), dfdbbx and peg, moringa oleifera leaf extract and peg, and a combination of moringa oleifera leaf extract, dfdbbx, and peg. on the seventh and thirtieth days, the cavia cobayas were sacrificed and examined. histopathological samples were stained with hematoxylin-eosin (he) to evaluate the trabecula area, and data were analyzed using one-way anova and tukey hsd. results: on the thirtieth day, the group that received a combination of moringa oleifera leaf extract and dfdbbx had the greatest area of alveolar bone trabeculae. conclusion: a combination of moringa oleifera leaf extract and dfdbbx induced in the tooth extraction socket can increase the area of the alveolar bone trabeculae. keywords: alveolar bone; dfdbbx; moringa oleifera leaf extract; socket preservation; trabecular area article history: received 29 november 2021; revised 21 november 2022; accepted 12 december 2022 correspondence: utari kresnoadi, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo 47, surabaya 60132, indonesia. email: utari-k@fkg.unair.ac.id introduction tooth extraction will result in the formation of a socket. in the post-extraction socket, bleeding will occur as an early marker of the socket healing process, followed by coagulation, inflammation, proliferation, and remodeling.1 after tooth extraction, alveolar bone resorption happens, causing changes in the morphology and dimensions of the alveolar bone. after six months, the alveolar bone will resorb by 29–63% in the horizontal plane and 11–22% in the vertical plane.2 the bone remodeling process will involve concurrent bone resorption and deposition. the balance of osteoclasts and osteoblasts influences this process. preosteoclasts will differentiate into osteoclasts, causing increased bone resorption. the activation of osteoclasts stimulates the differentiation and maturation of osteoblast precursor cells, resulting in bone matrix mineralization, which is an indicator of the bone formation process and osteoclast apoptosis.3 on the seventh day after tooth extraction, bone regeneration will begin at the periphery and extend to the socket’s middle area toward the bone trabeculae. on the twelfth day, woven bone trabeculae have formed at the socket’s periphery and are surrounded by preosteoblasts, osteoblasts, and osteoprogenitor cells.4 by the fourteenth day, trabecular bone is actively forming and will cover most of the bone graft’s surface. on the twenty-eighth day, the trabecular bone will fill most of the alveolar sockets.5 an increase in trabecular bone density and expansion also indicates the formation of new alveolar bone.6 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p127–131 mailto:utari-k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p127-131 128kresnoadi et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 127–131 alveolar bone resorption following a tooth extraction is a physiological process, but it can be minimized. socket preservation is the process of minimizing alveolar bone resorption by inserting regenerative material into the postextraction socket. also, socket preservation is expected to preserve the dimensions of the alveolar ridge after tooth extraction. bone graft is a regenerative material commonly used in dentistry, one of which is demineralized freezedried bovine bone xenograft (dfdbbx). dfdbbx is a xenograft graft material derived from bovine bone, which is biocompatible, osteoinductive, and osteoconductive.5 dfdbbx promotes the growth of new bone.6 several studies have reported that moringa oleifera leaves can aid in bone formation and prevent bone resorption. moringa oleifera in combination with xenograft is effective in generating tgf-β1 and osteocalcin expression in alveolar bone, as well as accelerating alveolar new bone formation and decreasing bone resorption.7,8 based on pharmacological studies, flavonoids and tannins have an anti-inflammatory effect by reducing tnf-α, il-1β, and il-6, all of which play a role in bone resorption.6 a combination of moringa leaf extract and dfdbbx with an effective dose of 2% can increase the number of osteoblasts, thereby accelerating the process of post-extraction alveolar bone formation on the experimental subject cavia cobaya.9 this study was conducted to determine if inducing a combination of moringa leaf extract (moringa oleifera) and dfdbbx in the tooth extraction socket increased the area of the alveolar bone trabeculae. materials and methods this research began once the airlangga university faculty of dental medicine committee approved it with the number 533/hrcee.fodm/ix/2021. this was an experimental laboratory study with randomized post-test-only control group design samples using healthy and active cavia cobaya males, weighing about 300–350 grams and at 3–3.5 months in age. moringa leaf extract was made at the surabaya industrial consultation and research institute. moringa leaves were soaked in water before being heated and filtered. the filtrate was thickened and concentrated to a volume of 1:1 with water. the extract was treated with silicate to make it nonhygroscopic and then dried. this study used four different solutions as treatments for each group: (i) only polyethylene glycol (peg), (ii) a combination of dfdbbx and peg, (iii) a combination of moringa leaf extract and peg, and (iv) a combination of moringa leaf extract, dfdbbx, and peg, with each concentration of the active substance being 2%. to obtain a 2% concentration of moringa leaf extract and dfdbbx, 0.5 grams of moringa leaf extract were combined with 0.5 grams of dfdbbx and 24 grams of peg (peg 400 + peg 4000, in a 1:1 ratio). peg was used as a carrier to turn the mixture into a gel, making it easier to fill into the socket. cavia cobayas were anesthetized intramuscularly (im) with ketamine at a 20mg/300 mg body weight (bw) dose. the same motion, direction, and force were used to extract the left lower incisor. the socket was then irrigated with sterile distilled water. the 56 cavia cobayas with their lower left incisors extracted were divided into eight groups of seven. as a control, groups 1 and 2 were given 25 grams of peg. on the other hand, groups 3 and 4 received 0.5 grams of dfdbbx and 24.5 grams of peg. groups 5 and 6 were both given 0.5 grams of moringa leaf extract and 24.5 grams of peg. for groups 7 and 8, 0.5 grams of moringa leaf extract, 0.5 grams of dfdbbx, and 24 grams of peg were administered. the combination of moringa leaf extract, dfdbbx, and peg was injected into the socket (± 0.1 ml) using a syringe. then, the post-retraction wound area was sutured with polyamide monofilament, ds 12 3/8 c, 12 mm, 6/10 meters, 0.7 sterile braun aesculap. the cavia cobayas from groups 1, 3, 5, and 7 were terminated on the seventh day, and groups 2, 4, 6, and 8 on the thirtieth day. their mandibular bones were collected for a 30-day decalcification using ethylenediaminetetraacetic acid (edta). the tissue was then prepared for histopathological examination using paraffin blocks. moreover, histopathological samples were prepared using hematoxylineosin (he) staining. the area of the alveolar bone trabeculae was calculated using a light microscope with 400x magnification and ten visual fields equipped with raster 4.1 image software (optilab). the measurement results were analyzed using the shapiro–wilk, levene’s, one-way analysis of variance (anova), and tukey honestly significant difference (hsd) tests on the statistical package for the social sciences (spss) version 21 software. results the results of the mean value of the alveolar bone trabeculae area and the standard deviation in the histological observations of five visual fields during the seventhand thirtieth-day examinations are shown in figure 1. the highest mean value of the alveolar bone trabeculae was found in the combined leaf extract treatment group. the moringa + dfdbbx + peg group had the highest value on the thirtieth day, while the control group had the lowest value on the seventh day. furthermore, the mean alveolar bone trabecular area in the treatment group was greater compared to the control group on both the seventh and thirtieth days. histopathological views of cavia cobaya tooth sockets showing the trabecular area on the seventh and thirtieth days are depicted in figures 2 and 3, respectively. the research samples were normally distributed and homogeneous based on the shapiro–wilk test and levene’s test. the one-way anova test showed that on both the seventh and thirtieth day, there were significant differences copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p127–131 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p127-131 129 kresnoadi et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 127–131 a b c d figure 2. histopathological view of cavia cobaya tooth sockets in each group on day-7. (a) control group (peg); (b) dfdbbx treatment group; (c) moringa leaf extract treatment group; (d) combined dfdbbx + moringa leaf extract treatment group. 0 50 100 150 200 250 300 control dfdbbx moringa extract moringa extract + dfdbbx day-7 day-30 figure 1. the mean and standard deviation of an alveolar bone trabecular area on day-7 and -30 in the control group and treatment groups: dfdbbx, moringa leaf extract, and combined moringa leaf extract and dfdbbx. figure 3. histopathological view of cavia cobaya tooth sockets in each group on day-30. (a) control group (peg); (b) dfdbbx treatment group; (c) moringa leaf extract treatment group; (d) combined dfdbbx + moringa leaf extract treatment group. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p127–131 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p127-131 130kresnoadi et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 127–131 between the treatment groups (p < 0.001). the tukey hsd test also revealed a significant difference on the seventh day between the control group and the moringa leaf extract group (p = 0.026), between the control group and the moringa leaf extract and dfdbbx combination group (p = 0.001), and between the dfdbbx group and the moringa leaf extract and dfdbbx combination group (p = 0.034). on the thirtieth day, however, a significant difference was noted between all groups (p = 0.001) except for the moringa leaf extract group and the dfdbbx group (p = 0.913). discussion alveolar bone consists of trabecular and cortical bone but is mainly composed of the former.10 cortical bone has a mechanical function, while trabecular bone plays a role in metabolic processes and physiological responses.11 alveolar bone has biological properties in the form of plasticity, which allows it to adapt to physiological or pathological changes in the oral cavity.12 the resorption process is characterized by a decrease in alveolar bone, which is influenced by osteoclasts.13 alveolar bone deposition is characterized by an ossification process that begins with the mineralization of the bone matrix and continues with osteoblast proliferation and differentiation into osteocytes. ossification changes osteocytes, which leads to the formation of bone trabeculae. therefore, increased trabecular bone expansion is a marker for the formation and mineralization of new alveolar bone.5 the results of this study prove that inducting a combination of moringa leaf extract and dfdbbx can increase the area of the alveolar bone trabeculae. dfdbbx is a bovine xenograft with a structure and inorganic content similar to humans, which gives it osteoconductive properties. also, dfdbbx will act as a scaffold to support cell adhesion and proliferation, as well as stabilize blood clotting to prevent tissue formation from being disturbed.6 its osteoconductive properties will stimulate osteoblast migration from the socket base, thereby supporting osteogenesis. moreover, dfdbbx will decrease receptor activator of nuclear factor kappa β-ligand (rankl) expression while increasing osteoprotegerin (opg) expression, which leads to a decrease in osteoclasts, the main factor in bone resorption.7 moringa oleifera is rich in flavonoids, tannins, terpenoids, alkaloids, saponins, ascorbic acid, phenolics, carotenoids, potassium, calcium, β carotene, protein, and vitamin c.9 the flavonoid compounds in moringa leaves are phytochemical compounds with high osteoinductive and anti-inflammatory properties. flavonoids stimulate osteoblast proliferation and differentiation, as well as prevent bone resorption by inhibiting the cyclooxygenase-2 (cox-2) enzyme, which hinders prostaglandin synthesis and decreases peg-2 and macrophage infiltration.14 reduced macrophage infiltration results in fewer inflammatory mediators and proinflammatory cytokines (tnf-α, il-1β, and il-6). furthermore, rankl production is disrupted, which inhibits osteoclast formation and stimulates osteoclast growth for apoptosis, reducing bone resorption and activating osteoblastogenesis, which plays a role in the formation of new bone.8 flavonoids can also regulate cell function by producing tgf-β, which induces osteoblast proliferation and migration while inhibiting osteoblast apoptosis.15 flavonoids also increase the enzyme alkaline phosphatase (alp), which is an indicator of osteoblast proliferation. alp participates in the mineralization process that facilitates calcium storage in tissues and reflects the activation of osteoblast cells during bone formation.7,16 phytochemical compounds of tannins play a role in inhibiting osteoclast differentiation by preventing receptor activator of nuclear factor kappa-β (rank) activity.15 in comparison, phytoestrogens stimulate increased osteoblast activity, because they have bioactivity similar to estrogen produced by the body. estrogen is involved in bone growth and bone homeostasis. estrogen also increases osteoblast activity by inhibiting osteoclast activity, preventing osteoblast and osteocyte apoptosis, and stimulating osteoblast activity. phytoestrogens stimulate new bone formation by binding to estrogen on target cells and increasing osteoblast activity. hence, estrogen mediates the activity of phytoestrogens in the bone formation process.17 flavonoids have the potential to stimulate osteoblasts, while saponins in moringa leaf have an osteogenic effect, aiding in osteoblast proliferation and differentiation.18 flavonoids can also regulate cell function by stimulating tgfβ production, which induces osteoblast proliferation and migration.19 kaempferol and quercetin in moringa can also directly inhibit rankl and tnfα activity by inhibiting the inflammatory pathway. it is expected that there will be a reduction in macrophage infiltration, followed by a decrease in proinflammatory cytokines (tnfα, il1β, and il6), which will then reduce rankl production and decrease alveolar bone resorption.20 the moringa leaf extract and dfdbbx treatment group having the highest mean area of alveolar bone trabeculae is a result that is in line with a study conducted by rostiny et al. (2016), which found that when given a moringa leaf extract and dfdbbx combination, the number of osteoblasts increased, accelerating the formation of alveolar bone post-extraction.9 this is because the combination of osteoconductive and osteoinductive properties found in dfdbbx and moringa leaf extract can significantly increase the area of alveolar bone trabeculae. dfdbbx, which acts as a scaffold, will facilitate new bone formation and stabilize blood clots, as well as be a source of minerals.6 moreover, the moringa leaf extract’s flavonoids, tannins, and phytoestrogens have anti-inflammatory osteoinductive properties that will reduce bone resorption and stimulate osteoblast proliferation and differentiation. increased migration, proliferation, and differentiation of osteoblasts will accelerate new bone growth along the socket and trabecular space in the extraction area, resulting in woven copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p127–131 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p127-131 131 kresnoadi et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 127–131 bone trabeculae and an increase in the area of the alveolar bone trabeculae.7 this study’s results established that a combination of moringa leaf extract and dfdbbx induced in the tooth extraction socket can increase the alveolar bone trabeculae’s area on the seventh and thirtieth day. references 1. de sousa gomes p, daugela p, poskevicius l, mariano l, fernandes mh. molecular and cellular aspects of socket healing in the absence and presence of graft materials and autologous platelet concentrates: a focused review. j oral maxillofac res. 2019; 10(3): e2. 2. pagni g, pellegrini g, giannobile w v, rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 151030. 3. kresnoadi u, rahayu rp, rubianto m, sudarmo sm, budi hs. tlr2 signaling pathway in alveolar bone osteogenesis induced by aloe vera and xenograft (xcb). braz dent j. 2017; 28(3): 281–6. 4. kresnoadi u, lunardhi lc, agustono b. propolis extract and bovine bone graft combination in the expression of vegf and fgf2 on the preservation of post extraction socket. j indian prosthodont soc. 2020; 20(4): 417–23. 5. hassumi js, mulinari-santos g, fabris al da s, jacob rgm, gonçalves a, rossi ac, freire ar, faverani lp, okamoto r. alveolar bone healing in rats: micro-ct, immunohistochemical and molecular analysis. j appl oral sci. 2018; 26: e20170326. 6. naini a, sudiana ik, rubianto m, kresnoadi u, latief fde. effects of hydroxyapatite gypsum puger scaffold applied to rat alveolar bone sockets on osteoclasts, osteoblasts and the trabecular bone area. dent j. 2019; 52(1): 13–7. 7. kresnoadi u, rahmania p, caesar h, djulaeha e, agustono b, ari ma. the role of the combination of moringa oleifera lea f ext r a ct a nd dem i ner a l i z e d f re ez e d r ie d b ov i ne b one xenograft (xenograft) as tooth extraction socket preservation materials on osteocalcin and transforming growth factor-beta 1 expressions in alveolar bo. j indian prosthodont soc. 2019; 19(2): 120–5. 8. rahmania pn, kresnoadi u, mundiratri k, ari mda. macrophage analysis of the combination of moringa leaf extract and dfdbbx in cavia cobaya tooth extraction sockets. biochem cell arch. 2020; 20(1): 3113–7. 9. rostiny r, djulaeha e, hendrijantini n, pudijanto a. the effect of combined moringa oleifera and demineralized freeze-dried bovine bone xenograft on the amount of osteoblast and osteoclast in the healing of tooth extraction socket of cavia cobaya. dent j. 2016; 49(1): 37–42. 10. berawi kn, wahyudo r, pratama aa. potensi terapi moringa oleifera (kelor) pada penyakit degeneratif. j kedokt univ lampung. 2019; 3(1): 210–4. 11. jiang n, guo w, chen m, zheng y, zhou j, kim sg, embree mc, songhee song k, marao hf, mao jj. periodontal ligament and alveolar bone in health and adaptation: tooth movement. front oral biol. 2015; 18: 1–8. 12. yuliati, sari gm, setyawan s, hendromartana s. pemberian tambahan kalsium pada masa pertumbuhan terhadap tebal tulang kortikal dan trabekular. maj ilmu faal indones. 2007; 6(3): 169–74. 13. berkovitz b, moxham b, lindern r, sloan a. master dentistry volume 3 oral biology. edinburgh: churchill livingstone; 2010. p. 221–25. 14. naini a. the comparative micro-ct analysis on trabecular bone density between hydroxyapatite gypsum puger scaffold application and bovine hydroxyapatite scaffold application. dent j. 2021; 54(1): 11–5. 15. guskuma mh, hochuli-vieira e, pereira fp, rangel-garcia i, okamoto r, okamoto t, filho om. evaluation of the presence of vegf, bmp2 and cbfa1 proteins in autogenous bone graft: h istomet r ic a nd i m munoh istochem ica l a na lysis. j cra n io maxillofacial surg. 2014; 42(4): 333–9. 16. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc. 2006; 72(10): 917–22. 17. kresnoadi u, rahayu rp. stimulation of osteoblast activity by induction of aloe vera and xenograft combination. dent j. 2011; 44(4): 200–4. 18. djais ai, salam f, jubhari eh, rahma s, bachtiar r. ridge and socket preservation management for prevention of bone resorption as a preparation for the placement of implant and denture. makassar dent j. 2021; 10(2): 125–8. 19. syarif rd, kusumaningsih t, arundina i. changes in osteoblast and osteoclast cell count after moringa oleifera leaf extract administration during orthodontic tooth movement. j dentomaxillofacial sci. 2020; 5(2): 98–102. 20. soekobagiono s, salim s, hidayati he, mundiratri k. effects of moringa oleifera leaf extract combined with dfbbx on type-1 collagen expressed by osteoblasts in the tooth extraction sockets of cavia cobaya. dent j. 2018; 51(2): 86–90. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p127–131 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p127-131 vol 49 no 3 juli-sept 2016.indd 143143 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 143–147 effects of robusta coffee (coffea canephora) brewing on levels of rankl and tgfβ1 in orthodontic tooth movement herniyati,1 ida bagus narmada,2 and soetjipto 3 1department of orthodontic, faculty of dentistry, universitas jember, jember-indonesia 2department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya-indonesia 3department of biochemistry, faculty of medicine, universitas airlangga, surabaya-indonesia abstract background: orthodontic tooth movement will be followed by periodontal ligament and alveolar bone remodeling. orthodontic mechanical force (omf) will be distributed through the teeth to periodontal ligament and alveolar bone and then will generate local pressure resulting in bone resorption and tension areas that will form new bone. robusta coffee contains caffeine, chlorogenic acid and caffeic acid. caffeine may increase osteoclastogenesis, and caffeic acid has antioxidant effects that may reduce oxidative stress in osteoblasts. purpose: this study conducted to analyze the effect robusta coffee steeping on levels of rankl and tgf-β1 in orthodontic tooth movement. method: 16 male rats were divided into 2 groups. group c: rats given omf, group t: given omf and coffee brew at 20 mg/ 100 g bw. omf in rats was conducted by applying ligature wire on the molar-1 (m-1) and both incisivus of right maxilla. subsequently, m-1 of right maxilla was moved to mesial with a niti closed coil spring. observations were made on days 15 and 22 by taking the gcf by putting paper point on the gingival sulcus of mesioand disto-palatal areas of m-1 of right maxilla to determine the levels of rankl and tgf-β1 using elisa method. result: the administration of coffee brew was effective to increase levels of rankl and tgf-β1 in the compression and tension areas (p <0.05). rankl levels in compression area were higher than in the tension area (p <0.05), while the levels of tgf-β1 in the tension area were higher than in the compression area (p <0.05). conclusion: the administration of coffee brew was effective to increase the levels of rankl and tgf-β, therefore it might improve alveolar bone remodeling process. keywords: rankl; tgf-β1; robusta coffee; orthodontic tooth movement; alveolar bone remodeling correspondence: herniyati, department of orthodontic, faculty of dentistry universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: herny_is@yahoo.com; tel: +081358681200. introduction the prevalence of malocclusion in indonesia is still very high, approximately about 80% of the population. malocclusion, consequently, is considered as the biggest dental and oral health problem. this condition is triggered by low dental care awareness and bad habits in society, such as sucking thumb or something else. since the number and severity level of malocclusion will continually increase, so malocclusion must be prevented or treated.1 orthodontic treatment aims to adjust the position of teeth to the right tooth curve. thus, chewing function efficiency, face harmony, oral health, dentofacial aesthetics, and tooth position stability can be improved. orthodontic treatment usually takes 2-3 years.2 orthodontic tooth movement, will be followed by remodeling of alveolar bone and periodontal ligament. 3 orthodontic mechanical force will be distributed from the teeth to the periodontal ligament and the alveolar bone, resulting in bone resorption at pressure site and new bone formation at tension site during tooth movement.4 application of orthodontic mechanical force on teeth is marked with inflammation activating macrophages, then releasing cytokines and growth factors. 5 those grow factors are receptor activator of nuclear factor κβ ligand (rankl) and transforming growth factor β (tgf-β1).6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p143-147 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p143-147 144 herniyati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 143–147 rankl is a regulator of bone remodeling during the orthodontic movement process.4 rankl is expressed on osteoblasts and stromal cells as a respond to parathyroid hormone (pth) and stimulation triggered by active 1,25-dihydroxyvitamin d (1,25 vit d3).7 rankl binds to receptor activator of nuclear factor κβ (rank) on osteoclast precursors, triggering osteoclast differentiation and proliferation. as a result, osteoclasts then become active. next, the active osteoclast will trigger bone resorption.8 on the other hand, osteoblasts also express osteoprotegerin (opg) as a receptor inhibiting rankl-rank interaction, resulting in prevention of osteoclastogenesis. pressure force will mechanically induce osteoclastogenesis in vitro through an increase in rankl expression and a decrease in opg expression.9 the ratio of rankl and opg expressions is necessary to determine in inflammation inducing bone resorption. bone resorption will occur if rankl expression is higher than opg expression. in contrary, bone formation will occur if opg expression is higher than rankl expression.10 there is also tgf-β1 as a growth factor also considered as a periodontal homeostasis biomarker, promoting cell migration, cell differentiation, cell proliferation, as well as extracellular matrix synthesis. tgf-β1 is also known as osteogenic protein, needed in bone mineralization.11 many efforts have been undertaken to accelerate orthodontic movement, such as medicines, surgical methods, as well as physical and mechanical stimulation methods.12 one of materials used in those efforts are coffee. coffee has recently been a popular drink consumed in the world. one of kinds of coffee consumed is robusta coffee. robusta coffee contains certain substance, known as caffeine (1, 3, 7 trimetilxantin).13 robusta coffee also contains chlorogenic acid and caffeic acid generating antioxidant effects.14 a research on rats given orthodontic mechanical force shows that the administration of caffeine at a high dose (10 mg/ 100 g bb) on them can improve osteoclasts and bone resorption at tension site on day 15.15 caffeine increases osteoclastogenesis by improving rankl.16 caffeic acid has antioxidant effects that can reduce oxidative stress on osteoblasts.17 another research also illustrates that chlorogenic acid promotes osteogenesis on human adipose tissue derived from mesenchymal stem cells (hamscs), indicated by an increase in bone mineralization.18 therefore, this research aimed to analyze the effects of robusta coffee brew on rankl and tgf-β1 levels during orthodontic tooth movement. the results of this research then were expected to reveal whether coffee could be used as a therapy for accelerating bone remodeling process and orthodontic tooth movement or not. as a result, orthodontic treatment could be conducted more easily, cheaper, and faster since coffee is easy to obtain and relatively cheap with minimal side effects. materials and method this research was a laboratory experimental study conducted on sixteen (16) healthy male rats (spraque dauwley) aged 3-4 months and weighed 250-300 grams. those rats were selected since they had complete dental structure as well as good oral cavity and periodontal tissue conditions. those rats were divided randomly into two groups, namely control group (c), given orthodontic mechanical force and 2 ml of distilled water, and treatment group (t) given orthodontic mechanical force and drip of coffee brew at a concentration of 20 mg/ 100 g bw (equivalent to a cup of coffee for an adult man), dissolved into 2 ml of distilled water. the administration of orthodontic mechanical force was conducted after those rats were anesthezing with ketamine. a ligature wire with a diameter of 0.20 mm was installed from their molar-1 (m-1) on the upper right jaw (uj) to their two insivus. m-1 ra was moved into mesial by using tension gauce to generate a force of 10 g/ cm2 with a nickel titanium orthodontic closed coil spring sized 6 mm length.19 observation was conducted on days 15 and 22 to take gingival crevicular fluid (gcf) by putting paper point on mesio and disto-palatal areas of m-1 uj for 30 seconds, and then put it into eppendorf tube.20 rankl and tgf-β1 levels then were measured by using elisa method. installation of the closed coil spring on those rats can be seen in figure 1. data obtained were analyzed using student’t-test, paired t-test, and wilcoxon signed ranks test at a confidence level of 95% (α=0.05). this research was approved by the research ethics committee of the faculty of dental medicine, universitas airlangga with a letter no. 18/ kkepk.fkg/ ii/ 2015. results the results of the research indicated that there were some effects of coffee brew on rankl and tgf-β1 levels figure 1 installation of closed coil spring on the rats. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p143-147 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p143-147 145145herniyati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 143–147 as shown in table 1, table 2, table, 3 and table 4. table 1 shows the mean and standard deviations of rankl levels at the pressure and tension sites on days 15 and 22. the results of the wilcoxon signed ranks test on the pressure site and t test on the tension site on day 15 indicated that the rankl levels in the treatment group were significantly higher than those in the control group (p<0.05). similarly, the results of t test on the pressure and tension sites on day 22 showed the rankl levels in group t were significantly greater than in group c (p<0.05). those rankl levels in groups c and t indicated that the pressure sites were larger table 1. mean and standard deviation of rankl levels at the pressure and tension sites in between the research groups groups n rankl (pg/ ml) (mean ± standard deviation) on day -15 on day -22 pressure tension p pressure tension p c 8 17.30 ± 5.93 15.33 ± 4.40 0.514** 10.95 ± 4.16 5.98 ± 1.71 0.014* t 8 41.82 ± 4.22 40.50 ± 3.85 0.484** 38.91 ± 4.95 32.72 ± 6.07 0.026* 0.000* 0.000* 0.000* 0.000* note: *: significantly different; **: insignificantly different table 2. results of the difference test on rankl levels at the pressure and tension sites between on day 15 and on day 22 in each group research group n rankl (pg/ ml) (mean ± standard deviation) pressure tension on day-15 on day -22 p on day-15 on day-22 p k 8 17.30 ± 5.93 10.95 ± 4.16 0.101** 15.33 ± 4.40 5.98 ± 1.71 0.002* p 8 41.82 ± 4.22 38.91 ± 4.95 0.208** 40.50 ± 3.85 32.72 ± 6.07 0.014* note: *: significantly different; **: insignificantly different table 3. mean and standard deviations of tgf-β1 levels at the pressure and tension sites in between the research groups group n tgf-β1 mean ± standard deviation) on day-15 on day-22 pressure tension p pressure tension p c 8 3.83 ± 0.70 4.11 ± 0.65 0.271** 3.59 ± 0.91 3.76 ± 0.49 0.542** t 8 24.26 ± 2.52 32.46 ± 4.95 0.006* 12.09 ± 1.54 15.75 ± 2.39 0.001* t 0.000* 0.000* 0.000* 0.000* note: *: significantly different; **: insignificantly different table 4. results of the difference test on tgf-β1 levels at the pressure and traction areas between on day 15 and on day 22 in each group research group n tgf-β1 (pg/ ml) (mean ± standard deviation) pressure traction on day-15 on day-22 p on day-15 on day-22 p c 8 3.83 ± 0.70 3.59 ± 0.91 0.577** 4.11 ± 0.65 3.76 ± 0.49 0.313** t 8 24.26 ± 2.52 12.09 ± 1.54 0.000* 32.46 ± 4.95 15.75 ± 2.39 0.000* note: *: significantly different; **: insignificantly different than the tension sites on day 15, but it was not statistically significant (p> 0.05). meanwhile, the rankl levels in groups c and t on day 22 indicated the pressure sites were significantly different from the tension sites (p<0.05). table 2 illustrates the results of paired t-test on group c and wilcoxon signed ranks test on group t. the results showed that the rankl levels at the pressure sites of the research groups significantly decreased on day 22 compared to those on day 15, but it was not statistically significant (p>0.05). on the other hand, the rankl levels at the tension areas of the research groups decreased significantly on day 22 compared to those on day 15 (p<0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p143-147 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p143-147 146 herniyati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 143–147 table 3 shows the mean and standard deviations of tgf-β1 levels at the pressure and tension sites on days 15 and 22. the results of the t test on the pressure and tension sites on day 15 indicated that the tgf-β1 levels in the treatment group were significantly higher than those in the control group (p <0.05). the tgf-β1 levels in group c indicated that the tension site was insignificantly larger than the pressure site on both days 15 and 22 (p>0.05). meanwhile, the tgf-β1 levels in group t indicated that the tension site was significantly larger than the pressure site on both days 15 and 22 (p<0.05). table 4 illustrates the results of paired t-test on group c and group t. the results showed that the tgf-β1 levels at both of the pressure and tension sites in group c decreased on day 22 compared to those on day 15, but it was not statistically significant (p>0.05). meanwhile, tgf-β1 levels at both of the pressure and tension areas in group t significantly decreased on day 22 compared to those on day 15. discussion the results of this research showed that the administration of coffee brew triggered an increase in rankl levels at pressure and tension sites on days 15 and 22. this is due to caffeine contained in coffee binding to adenosine receptors and modulating several other receptors, including glucocorticoid receptor, insulin, estrogen, androgen, vitamin d, cannabinoid, glutamate, and adrenergic receptors, expressed in osteoblasts or osteoprogenitor cells which have important functions during osteoblast differentiation.21 an in vitro research shows that caffeine at a low concentration can also trigger cyclooxygenase-2 (cox-2)/ prostaglandin e2 (pge2), which then activate rankl levels on osteoblasts, resulting in increased osteoclast formation, as well as reduce opg expression on osteoblasts. 22 meanwhile, an in vivo research illustrates that caffeine can reduce bone mineral density (bmd) in rats and increase osteoclastogenesis. previous research also shows that caffeine can improve an osteoclastogenic ability of periodontal ligament cells under stress, and increase tooth movement through pge2-rankl.16 thus, a decrease in opg expression may probably be caused by an increase in proinflamatory cytokine triggered by orthodontic pressure, will inhibit opg expression.6 results of this research also indicated the increased levels of rankl at the pressure sites after the administration of coffee brew was larger than at the tension sites, especially on day 22. this is consistent with a research showing that the application of orthodontic force on pressure site can trigger osteoblasts to generate more rankl expression, resulting in enhancement of osteoclastogenesis and then improvement of bone resorption.23.24 in rankl levels on day 22 decreased compared to those on day 15 either after the administration of coffee brew or not. this is because the strength of the mechanical orthodontic force decreased on day 22, therefore osteoblast activity also decreased. the decreased levels of rankl then could inhibit osteoclastogenesis and bone remodeling. as a result, it can be said that the administration of coffee brew can effectively increase the levels of rankl on day 15. the increased levels of tgf-β1 at the pressure and tension sites on days 15 and 22 in this research is due to caffeic acid, phenolic acid classified into non acds phenolic flavonoids, contained in coffee, that can give an antioxidant effect in reducing oxidative stress on osteoblasts. 17 several in vitro and in vivo researches on experimental animals also show that oxidative stress can reduce the rate of bone formation by decreasing osteoblast differentiation and survival. a report even shows that reactive oxigen species (ros) can activate osteoclasts, resulting in an increase in bone resorption. in other words, antioxidant activity is important in stimulating osteoblastic activity through specific receptors to support bone growth.25 on day 22, the results of the research showed that the levels of tgf-β1 at the pressure and tension sites after the administration of coffee brew significantly decreased compared to those on day 15. it is due to the reduced orthodontic pressure, so bone formation also decreased. the results of this research also indicated that tgf-β1 levels were greater at the tension sites than those at the pressure sites since the tension sites always requires more bone formation than the pressure sites. tgf-β-1, produced by various cells, including osteoblasts and fibroblasts stimulated mechanically, has a highly osteogenic properties that can enhance osteoblast activity and inhibit osteoclast activity.26 a research also shows that tgf-β1 can be associated with tissue remodeling in periodontal ligament during orthodontic tooth movement, and the mechanical loads of the tension strength can regulate tgf-β1 expression in osteoblasts and periodontal ligament cells in vitro.9 it can be concluded that the administration of coffee brew can increase rankl and tgf-β1 levels in order to improve alveolar bone remodeling process. acknowledgement the researches would like to gratitude to the managers of biomedical laboratory in faculty of dentistry, universitas jember for services provided in the process of giving treatment on animal as well as to the managers of biomedical laboratory in faculty of medicine, universitas brawijaya for services provided in the process of gcf analysis using elisa. references 1. laguhi va, anindita ps, gunawan pn. gambaran maloklusi dengan menggunakan hmar pada pasien di rumah sakit gigi dan mulut universitas sam ratulangi manado. j e-gigi 2014; 2(2): 1-7. 2. profitt wr, field hw, safer dm. contemporary orthodontics. 4th ed. toronto: cv mosby co; 2007. p. 331-41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p143-147 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p143-147 147147herniyati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 143–147 3. krishnan v, davidovitch z. cellular, molecular and tissue-level reaction to orthodontics force. am j orthod dentofacial orthop 2006; 129(4): 469.e1-32. 4. henneman s, von den hoff jw, maltha jc. microbiology of tooth movement. eur j orthod 2008; 30(3): 299-306. 5. nimeri g, kau ch, aboe-kheir ns, corona r. acceleration of tooth movement during orthodontic treatment – a frontier in orthodontic. progress in orthodontics 2013; 14(42): 1-8. 6. d’apuzzo f, cappablanca s, clavarella d, monsurro a, biavati as, perillo l. biomarkers of periodontal tissue remodelling during orthodontic tooth movement in mice and men: overview and clinical relevance. sci world j 2013; 41: 342-53. 7. leibbrandt a, penninger jm. rank/rankl: regulators of immune responses and bone morphology. ann ny acad sci 2008; 1143: 123-50. 8. meikle cm. the tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after carl sandstedt. eur j orthod 2006 j; 28(3): 221-40. 9. andrade ji, taddei sra, souza pea. inflammation and tooth movement: the role of cytokines, chemokines, and growth factors. seminar in orthodontics 2012; 18(4): 25769. 10. fili s, karalaki m, schaller b. therapeutic implication of osteo protogerin. cancer cell international 2009; 9(26): 1-8. 11. alves aca. the impact of orthodontic treatment on periodontal support loss. dental press j orthod 2012; 17(1): 18-20. 12. shenava s, naya k kus, bhaska r v, naya k a. accelerated orthodontics-a review. international journal of scientific study 2014; 1(5): 35-9. 13. redaksi health secret. khasiat bombastis kopi. jakarta: pt alex media komputindo; 2012. p. 37-42. 14. yashin a, yashin y, wang jy, nemzer bs. antioxidant and antiradical activity of coffee. antioxidants 2013; 2: 230-45. 15. sun p, he yc. effect of caffeine on alveolar bone remodeling during orthodontic tooth movement in rats. journal of tongji university (medical science) 2011; 03. 16. yi j, yan b, li m, wang y, zheng w, li y, zhao z. caffeine may enhance orthodontic tooth movement through increasing osteoclastogenesis induced by periodontal ligament cells under compression. j archoral bio 2016; 64: 51-60. 17. baek kh, oh kw, lee wy, lee ss, kim mk, kwon hs. association of oxidative stress with postmenopausal osteoporosis and effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. calcif tissue internat 2010; 87(3): 226-35. 18. bi n hs, je ong j h, c hoi u k . c h lorogen ic acid promot es osteoblastogenesis in human adipose tissue-derived mesenchymal stem cells. j food sci biotechnol 2013; 22(supplement 1): 107-12. 19. sella rc, de mendonça mr, osma r a, cuoghi oa, li a. histomorphic evaulation of periodontal compresion and tensien sides during orthodontic tooth movement in rats. j orthod 2012; 17(3): 108-17. 20. zia a, khan s, bey a, gupta nd, mukhtar s. oral biomarker in the diagnosis and progression of periodontal diseases. biology and medicine 2011; 3(2): 45-52. 21. reis ams, ribeiro lgr, ocarino nm, goes am, serakides r. osteogenic potential of osteoblasts from neonatal rats born to mothers treated with caffeine throughout pregnancy. bmc musculoskelet disord 2015; 16(1): 10. 22. liu sh, chen c, yang rs, yuan py, yang yt, tsai c. caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats. j orthop res 2011; 29: 954–60. 23. seifi m, badiee mr, abdolazimi z, amdjadi p. effect of basic fibroblast growth factor on orthodontic tooth movement in rats. cell j autumn 2013; 15(3): 230-7. 24. yamaguchi m. rank/ rankl/opg during orthodontic tooth movement. orthod craniofal res 2009; 12: 113-9. 25. banfi g, iorio el, corsi mm. oxidative stress, free radicals and bone remodeling. clin chem lab med 2008; 46: 1550–5. 26. van sa, sloten jv, geris l. a mechanobiological model on orthodontic tooth movement. j biomechan model mechanobiol 2013; 12(1): 244-65. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p143-147 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p143-147 6 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 6–9 the cleanliness differences of root canal walls after irrigated with east java propolis extract and sodium hypoclorite solutions tamara yuanita department of conservative dentistry, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: root canal instrumentation produces smear layer that covers dentine tubules of the root canal surface. smear layer is organic and inorganic particles that have to be removed. east java propolis extracts contais of saponin components used as a natural surfactant. 2.5% naocl and 5% naocl solutions have been widely used for irrigation in root canal treatment. purpose: the purpose of this study was to analyze the cleanliness of the root canal walls, irrigated with aquadest, 8% east java propolis extract, 2.5% naocl and 5% naocl. method: forty extracted teeth with straight single root canals were randomly divided into four groups (n=10). the specimens were prepared with protaper. during instrumentation, the root canals were irrigated with different solutions: control group irrigated with aquadest; group 1 irrigated with 8% east java propolis extract; group 2 irrigated with 2.5% naocl and group 3 irrigated with 5% naocl. the root canals were cut at apical third and sem scores were tested by using mann-whitney test at the significance level of p=0.05 and median control test. result: the results of mann-whitney test, there were significant differences between control group with group 1, 2 and 3 (p<0.05). based on the median control test, the value of 8% east java propolis extracts was 1,000, which was the best value compared to 2.5% naocl, 5% naocl and aquadest. conclusion: it can be concluded that 8% east java propolis extract is the most effective solution for cleaning root canal walls compared with 2.5% naocl and 5% naocl. keywords: propolis extracts; naocl; root canal walls correspondence: tamara yuanita, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tamara25jun@yahoo.com; hp: 081703490329/08155130747 introduction root canal treatment is one type of dental conservation treatments that aims to maintain teeth function in oral cavity. root canal treatment consists of several stages, namely root canal preparation including cleaning and shaping (biomechanical preparation), disinfection, and root canal filling. the main principle of root canal cleaning is the preparation process should reach and clean the entire surface of the root canal walls.1 however, root canal preparation has some risks, one of which is that instrumentation can cause the formation of smear layers composed of organic and inorganic materials, such as dentin powders and necrotic pulp tissue remnants. the smear layers formed would make colonizes of bacteria form biofilms on the root canal walls.2 smear layers can also reduce the adaptation of filler materials with canal walls that can cause leakage between the obturation materials and the root canal walls leading to treatment failure.3 the key role of root canal irrigants is to clean the canal during the enlarging and shaping process. consequently, one or more irrigants must be used for the complete elimination of smear layer and debris from the root canal system. the ideal irrigation materials are materials which have antimicrobial properties, ability to dissolve soft tissue or smear layers, low surface tension, and low toxicity. various materials are commonly used for irrigation, such as citric acid, edta, chlorhexidine, and sodium hypochlorite.4 sodium hypochlorite (naocl) can be classified as halogenated groups that are oxygenating. chemical reactions in naocl showed that naocl acts as an organic dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p6-9 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p6-9 77yuanita/dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 6–9 solvent and degrades fats into fatty acids and glycerine which serve to reduce the surface tension of the liquid.5 naocl in solution form hypochlorus acid (hocl) and oxychloride (ocl). these disinfectants contain chlorinebased solution (cl 2). these solutions are considered as high level disinfectants because they are very active in all bacteria, viruses, fungi, parasites, and some spores. the ingredients work fast or fast acting. naocl has specific properties when in contact with vital tissue since these substances can be cytotoxic and destructive.6 naocl nevertheless, is not able to eliminate smear layers because it can dissolve inorganic materials perfectly and smells unpleasant.7 propolis is a natural product, which is interesting in pharmaceutical application, a mixture of resin and wax bees collected from important parts of plants.8 antimicrobial activities of propolis against a variety of bacteria, fungi and viruses have been studied since the late 1940s, and have showed different variable activities from microorganisms.9 the antimicrobial effects of propolis can resist more than 100 types of bacteria, fungi and viruses, including agents causing tuberculosis, syphilis, diphtheria, and influenza.10 saponins in propolis, moreover, act as surfactants, which resemble the properties of detergent. saponins, thus, are often referred to as ‘natural detergents’, a foaming solution classified by aglykon complex structure into triterpenoid and steroid saponins. saponins are characterized by their ability as surfactants that can reduce surface tension to wet root canal walls optimally 11 this study used 8% east java propolis extract based on minimal concentration of east java propolis to inhibit enterococcus faecalis (e. faecalis) bacteria.12 the study aimed to compare the effectiveness among 8% east java propolis extract, 2.5% naocl and 5% naocl on the cleanliness of root canal wall. materials and methods east java propolis extraction was conducted by maceration method in balai penelitian dan konsultasi industri, surabaya, east java. 350 grams of raw east java propolis was macerated with 650 grams of 70% ethanol in a sealed container. propolis and ethanol were shaken by using a shaker at a speed of 80 rpm in balai penelitian dan konsultasi industri, surabaya, east java, indonesia. after 7 days, the maceration process was stopped and filtered. the maceration process was repeated for 7 days until the color of ethanol was stable. it was evaporated until the substance free from ethanol and then was diluted with aquadest to obtain 8% propolis extracts. each first permanent mandibular premolar with matured apices in 21 mm length (n=40) was placed in an acrylic container that has a hole resembling a tooth socket for having a treatment. root canal preparation was conducted using pro tapper with hand instrumentation and crown down technique. during instrumentation, the canal of each samples were irigated with 25 gauge open ended needle just 2 mm before working length. to remove the smear layer, the root canals in every each sample were irrigated with aquadest as control group and group i: irrigated with 8% propolis extract, in group ii : irrigated with 2.5% naocl (kimia farma, surabaya, indonesia) and group iii: irrigated with 5% naocl (kimia farma, surabaya, indonesia) and aquadest as final rinse in every each sample irrigation was conducted by using the same pressure (1 atm). irrigation solution in each group was used every change of instrument as much as 3 ml for 30 sec, and aquadest as final rinse then dried with sterile paper points 3 times. the samples were stored in a desiccator to keep them dry. those samples were cut with a low speed diamond disc in a horizontal plane along the apical third of 4 mm from the apex tip then cut longitudinally in bucco-lingual. samples that have been cut were attached to the coated holders. after coating with paladium and aurum, one by one of the sample was inserted into scanning electron microscope (sem) and photographed with a magnification of 1000x. cleanliness assessment then was carried out by using a transparent plastic tool (13.5x8.5 cm). it then was divided into nine squares of the same size. the observation was conducted by three observers. finally, assessment was conducted by placing a transparent plastic on each photo, then a score for each box was taken as follows : score 0: 95-100% of dentin tubulesare open; score 1: 50-95% of dentin tubules, score 2: less than 50% of dentin tubules are open, score 3: dentin tubules are not open. 13 results this study was conducted to know the effectiveness of 8% east java propolis extract, 2,5% naocl and 5% naocl in cleaning root canals. this study had four treatment groups, the group irrigated with aquadest as a control group and three treatment group irrigated with 8% east java propolis extracts, group irrigated with 2,5% naocl, and group irrigated with 5% naocl. (figure 1). the assessment of sem results was conducted by three dentist as observers, and then its validity was tested by using friedmann test. the data showed that there was no difference among the three observers as shown in table 1. the data was tested by using a non-parametric test, kruskal-wallis test, to know the difference among all groups. the results then showed that the significance value obtained was 0.001, smaller than 0.05 (p<0.05). it means that there was a significant difference among all treatment groups. mann-whitney test was conducted to know the differences in each treatment group (table 2). the results showed that the treatment group irrigated with 8% propolis extract compared with the group irrigated with aquadest had 0.001 score, while the treatment group irrigated with 2,5% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p6-9 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p6-9 8 yuanita/dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 6–9 naocl compared with the group irrigated with aquadest had 0.007 less than 0.05 (p<0.05). it means that there was a significant difference between the treatment group and the control group. furthermore, the treatment group irrigated with 8% propolis extract compared with the treatment group irrigated with 2.5% naocl had 0.015 and 5% naocl had score less than 0.05 (p<0.05). it indicates that there was a significant difference between the treatment group irrigated with 8% propolis extract and the treatment group irrigated with 2.5% naocl and 5% naocl. the median value of each group was measured. based on the results, it is known that 8% propolis extract had the smallest value compared to the other groups. discussion during root canal preparation, endodontic instruments usually cause friction leading to smear layer formation. 7 10. remanuskiene k, inkeniene am, savickas a. analysis of the antimicrobial activity of propolis and lisozyme in semisolid emulsion system. acta poloniae pharm and drug research 2009; 66(6): 681-8. 11. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of coronal obturation a systematic review and meta analysis. j endod 2007; 33(2): 96-105. 12. yuanita t, hutagalung j, widjiastuti i, rulianto m, mooduto l. minimum bacterial concentration of east java propolis to biofilm of enterococcus faecalis. e journal apimondia kiev, ukraina. 2013. 13. perard m, goff a. study of rins endo action on the smear layer and debris removal by scanning electron microscopy. j endod 2013; 7(1): 15-21. 14. halackova z, martina k. rinsing of the root canal. smfm 2003; 76 (1): 49-54. 15. schaudinn c, carr g, gorur a, jaramillo d, costerton jw, webster p. imaging of endodontic biofilm by combined microscopy. j microsc 2009; 235(2): 124-7. aquadest naocl 2,5% figure 1. the results of the cleanliness irigating with aquadest, 8% propolis extract, 2.5% naocl and 5% naocl with 1000x magnification. table 1. friedman test treatment groups friedman test naocl 2,5% aquadest aquadest 7 10. remanuskiene k, inkeniene am, savickas a. analysis of the antimicrobial activity of propolis and lisozyme in semisolid emulsion system. acta poloniae pharm and drug research 2009; 66(6): 681-8. 11. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of coronal obturation a systematic review and meta analysis. j endod 2007; 33(2): 96-105. 12. yuanita t, hutagalung j, widjiastuti i, rulianto m, mooduto l. minimum bacterial concentration of east java propolis to biofilm of enterococcus faecalis. e journal apimondia kiev, ukraina. 2013. 13. perard m, goff a. study of rins endo action on the smear layer and debris removal by scanning electron microscopy. j endod 2013; 7(1): 15-21. 14. halackova z, martina k. rinsing of the root canal. smfm 2003; 76 (1): 49-54. 15. schaudinn c, carr g, gorur a, jaramillo d, costerton jw, webster p. imaging of endodontic biofilm by combined microscopy. j microsc 2009; 235(2): 124-7. aquadest naocl 2,5% figure 1. the results of the cleanliness irigating with aquadest, 8% propolis extract, 2.5% naocl and 5% naocl with 1000x magnification. table 1. friedman test treatment groups friedman test naocl 2,5% aquadest 7 10. remanuskiene k, inkeniene am, savickas a. analysis of the antimicrobial activity of propolis and lisozyme in semisolid emulsion system. acta poloniae pharm and drug research 2009; 66(6): 681-8. 11. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of coronal obturation a systematic review and meta analysis. j endod 2007; 33(2): 96-105. 12. yuanita t, hutagalung j, widjiastuti i, rulianto m, mooduto l. minimum bacterial concentration of east java propolis to biofilm of enterococcus faecalis. e journal apimondia kiev, ukraina. 2013. 13. perard m, goff a. study of rins endo action on the smear layer and debris removal by scanning electron microscopy. j endod 2013; 7(1): 15-21. 14. halackova z, martina k. rinsing of the root canal. smfm 2003; 76 (1): 49-54. 15. schaudinn c, carr g, gorur a, jaramillo d, costerton jw, webster p. imaging of endodontic biofilm by combined microscopy. j microsc 2009; 235(2): 124-7. aquadest naocl 2,5% figure 1. the results of the cleanliness irigating with aquadest, 8% propolis extract, 2.5% naocl and 5% naocl with 1000x magnification. table 1. friedman test treatment groups friedman test naocl 2,5% aquadest 7 10. remanuskiene k, inkeniene am, savickas a. analysis of the antimicrobial activity of propolis and lisozyme in semisolid emulsion system. acta poloniae pharm and drug research 2009; 66(6): 681-8. 11. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of coronal obturation a systematic review and meta analysis. j endod 2007; 33(2): 96-105. 12. yuanita t, hutagalung j, widjiastuti i, rulianto m, mooduto l. minimum bacterial concentration of east java propolis to biofilm of enterococcus faecalis. e journal apimondia kiev, ukraina. 2013. 13. perard m, goff a. study of rins endo action on the smear layer and debris removal by scanning electron microscopy. j endod 2013; 7(1): 15-21. 14. halackova z, martina k. rinsing of the root canal. smfm 2003; 76 (1): 49-54. 15. schaudinn c, carr g, gorur a, jaramillo d, costerton jw, webster p. imaging of endodontic biofilm by combined microscopy. j microsc 2009; 235(2): 124-7. aquadest naocl 2,5% figure 1. the results of the cleanliness irigating with aquadest, 8% propolis extract, 2.5% naocl and 5% naocl with 1000x magnification. table 1. friedman test treatment groups friedman test naocl 2,5% aquadest 2,5% naocl 5% naocl 8% east java propolis figure 1. the results of the cleanliness irigating with aquadest, 8% propolis extract, 2.5% naocl and 5% naocl with 1000x magnification. table 1. friedman test treatment groups friedman test aquadest p = 0.368 8% east java propolis p = 0.060 2,5% naocl p = 0.091 5% naocl p = 0.083 table 2. the result of man-whitney test treatment groups aquadest 8%east java propolis 2,5%naocl 5%naocl aquadest p= 0,001 0,007 0,009 8% east java propolis p = 0,001 0,015 0,025 2,5% naocl p = 0,007 p= 0,015 0.003 5% naocl p = 0,009 p= 0,025 0,003 table 3. median value treatment group median aquadest 3 2,5% naocl 2 5% naocl 2 8% east java propolis 1 smear layer is defined as a surface film of debris that is retained on the dentin or other surfaces after instrumentation with either rotary instruments or endodontic files, which composed of organic and inorganic particles of calcified tissue, necrotic tissue and microorganisms.14 this study was conducted to determine the effectiveness of 8% east java propolis extract, 2.5% naocl and 5% naocl as irrigation materials in the cleanliness of root canal walls. the indicators of the cleanliness can be observed from the covered areas of smear layer on the surface of root canal as seen on photo. the effectiveness of 8% east java propolis extract, 2.5% naocl, and 5% naocl, moreover, can be determined by using sem. sem can display images of the cleanliness of root canal surface because it can show the topography of the surface of the root canal walls with high resolution. thus, it can be said that the less smear layers cover the dentinal tubules, the cleaner the root canals are.15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p6-9 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p6-9 99yuanita/dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 6–9 based on the sem images assessment, the covered areas of smear layer in the group irrigated with 8% east java propolis extract was less and almost none. it was indicted by the dentine tubules were opened and clean. it means that saponins contained in 8% east java propolis extract were effective in cleaning the root canals from smear layers. meanwhile, there were still smear layers covering the dentinal tubules in the groups irrigated with 2.5% naocl and 5% naocl. naocl furthermore, is able to dissolve organic tissues through several stages, namely saponification reaction, neutralization reaction of amino acids and chlorination reaction. saponification process of naocl acts as an organic solvent that can degrade fats into glycerin (alcohol) and fatty acids, containing -oh which makes fatty acids polar (hydrophilic), as a result, it can reduce the surface tension of the liquid.15 naocl does not have an ability to clean smear layers, one of which consists of inorganic dentin debris. naocl can only clean organic materials and does not have an ability to clean inorganic materials so that its power to clean smear layers cannot be optimal.1 8% east java propolis extract, moreover, contains active substances, such as saponins. saponins have the same characteristics as detergent, often referred to “natural detergents”. saponins can also be considered as glycosides found in many plants, characterized as surfactants. surfactants serve as active compounds that can be used to lower energy barrier limiting two nonmutually soluble liquids. surfactants will lower cohesion force (cohesion force will make two substances not stick together when mixed). on the other hand, surfactants can improve adhesion force (adhesion force will make two substances stick together when mixed) so they can reduce the surface tension. this ability is due to the hydrophilic and hydrophobic groups owned by surfactants.11 saponins actually have a long hydrocarbon chain with the tip of the ion group consisting of non-polar (hydrophobic) and polar (hydrophilic). non-polar groups interact with grease/ oil/ dirt (in this study, the dirt in the form of smear layer). saponin molecules move around smear layers and then form a ring called a micelle or micelles. the tip containing hydrophilic group will attract water molecules, while the other tip containing hydrophobic group will bind dirt. cleaning process will occur, in which smear layers will be absorbed into the center of micelles making them change into substances easily dispersed and dissolved in water. meanwhile, the polar group will dissolve in water to form foam and bind smear layer particles to form an emulsion. when there is maxi probe turbulence movement, smear layers will be carried out. therefore, saponins can be characterized as surfactants because they can lower surface tension so that smear layers can be dissolved and carried out as irrigation repeated at every protaper/file substitution.11,14 in addition, the smear layers in the control group irrigated with aquadest covered almost the entire dentin tubules. this is because aquadest only serves to moisten the root canal alone and does not have an ability as a surfactant to be able to dissolve smear layers.13 based on the results of kruskal-wallis test followed by mann-whitney test, there were significant differences among the groups. the median value also showed that aquadest had the biggest value. it indicates that aquadest was the least effective. meanwhile, 8% east java propolis extract had the smallest value that indicates propolis extract as the most effective. it means that the group irrigated with 8% propolis extract showed the cleanliness of the root canals. this is because an active substance, saponin, contained in propolis extract acts as a surfactant that can reduce the surface tension of the root canals, so smear layers can be dissolved.13,14 finally, it can be concluded that 8% east java propolis extract is the most effective material for cleaning smear layers in root canal compared to 2.5% naocl and 5% naocl. references 1. torabinejad m, walton re. endodontics, principles and practice. 4th ed. st louis, missouri: sanders, elseviers inc; 2009. p. 258-86. 2. zehnder m. root canal irigants. j endod 2006; 32(5): 389-98. 3. cohen s, hargreaves km. cohen’s pathway of the pulp. 10th ed. st louis missouri: mosby inc; 2011. p. 529-58. 4. van frounhover a. dental materials at a glance. oxford: blackwell; 2010. p. 46-7. 5. mohammdi z. sodium hypoclorite in endodontics: an update review. int dent j 2008; 58(6): 329-41. 6. farren st, sadoff rs, penna kj. sodium hypoclorite chemical burns. sdj 2008; 74(1): 61-71. 7. ingle ji, bakland lk, baumgartner jc. endodontics. 6th ed. shelton usa: mc graw-hill; 2008. p. 992-1018. 8. surendra ns, bhushanam m, raikumar h. antimicrobial activity of propolis trigona sp and apis mellifera of kamataka india. pjmr 2012; 2(2): 80-85. 9. temiz a, şener a, tüylü aö, sorkun k, salih b. antibacterial activity of bee propolis samples from different geographical regions of turkey against to two food-borne pathogens, salmonela enteritidis and listeria monocytogenes. turk j biol 2011; 503-11. 10. remanuskiene k, inkeniene am, savickas a. analysis of the antimicrobial activity of propolis and lisozyme in semisolid emulsion system. acta poloniae pharm and drug research 2009; 66(6): 681-8. 11. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of coronal obturation a systematic review and meta analysis. j endod 2007; 33(2): 96-105. 12. yuanita t, hutagalung j, widjiastuti i, rulianto m, mooduto l. minimum bacterial concentration of east java propolis to biofilm of enterococcus faecalis. e journal apimondia kiev, ukraina. 2013. 13. perard m, goff a. study of rins endo action on the smear layer and debris removal by scanning electron microscopy. j endod 2013; 7(1): 15-21. 14. halackova z, martina k. rinsing of the root canal. smfm 2003; 76 (1): 49-54. 15. schaudinn c, carr g, gorur a, jaramillo d, costerton jw, webster p. imaging of endodontic biofilm by combined microscopy. j microsc 2009; 235(2): 124-7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p6-9 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p6-9 143 the periodontal pain paradox: difficulty on pain assesment in dental patients (the periodontal pain paradox hypothesis) haryono utomo,* indah listiana kriswandini* and diah savitri ernawati** ** department of oral biology ** department of oral medicine faculty of dentistry airlangga university surabaya indonesia abstract in daily dental practice, the majority of patients’ main complaints are related to pain. most patients assume that all pains inside the oral cavity originated from the tooth. one particular case is thermal sensitivity; sometimes patients were being able to point the site of pain, although there is neither visible caries nor secondary caries in dental radiograph. in this case, gingival recession and dentin hypersensitivity are first to be treated to eliminate the pain. if these treatments failed, pain may misdiagnose as pulpal inflammation and lead to unnecessary root canal treatment. study in pain during periodontal instrumentation of plaque-related periodontitis revealed that the majority of patients feel pain and discomfort during probing and scaling. it seems obvious because an inflammation, either acute or chronic is related to a lowered pain threshold. however, in contrast, in this case report, patient suffered from chronic gingivitis and thermal sensitivity experienced a relative pain-free sensation during probing and scaling. lowered pain threshold which accompanied by a blunted pain perception upon periodontal instrumentation is proposed to be termed as the periodontal pain paradox. the objective of this study is to reveal the possibility of certain factors in periodontal inflammation which may involved in the periodontal pain paradox hypothesis. patient with thermal hypersensitivity who was conducted probing and scaling, after the relative pain-free instrumentation, thermal hypersensitivity rapidly disappeared. based on the successful periodontal treatment, it is concluded that chronic gingivitis may modulate periodontal pain perception which termed as periodontal pain paradox key words: periodontal pain paradox, dental pain assessment correspondence: haryono utomo, c/o: bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. telp. 62 (31) 5053195. e-mail: dhoetomo@indo.net.id introduction pain is categorized as physiological and clinical. in addition, clinical pain consisted of inflammatory and neuropathic pains.1 modulation of clinical pain perception in individuals could be from central nervous system, that is suppressed transmission and facilitated transmission. hyperalgesia refers to an increase in sensitivity to stimulation at the site of pain. primary hyperalgesia occurs as the result of a lowered pain threshold or sensitization in the peripheral structures presumably due to the presence of algogenic substances or pro-inflammatory mediators.2 periodontal pockets are source of subgingival biofilm and function as reservoir of periopathogenic gram negative bacteria, including prevotella intermedia, porphyromonas gingivalis and actinobacillus actinomycetecomitans. they are also the source of pro-inflammatory mediators in the periodontal tissues, which may lower the pain threshold of nociceptors.2,3 concerning pain during periodontal treatment, there is a study in probing and scaling procedures to patients who treated for plaque-related periodontitis. the study revealed that subgingival instrumentation causes in pain and discomfort.4 in some cases, gingivitis or chronic periodontitis patients may experience a strange pain sensation which proposed as a periodontal pain paradox. it could be explained as a contradiction in pain perception; the noxious stimulation such as periodontal probing may not elicit pain; in contrary, non-noxious stimulation such as rinsing with tap water resulting in the opposite. despite the literatures discussed about pain and inflammation, the possible mechanism of the proposed periodontal pain paradox was rarely discussed. presumably because it was thought to be a dentinal hypersensitivity and treated successfully with particular toothpaste which also improve periodontal health thus eliminating the pain symptoms. the objective of this case report is to reveal the possible mechanism that occurs in the periodontal pain paradox and substances which involved in pain modulation. case a 53 years female came to a private dental practice, her main complaint was pain upon hot and cold stimulation in the lower right region, in which the cold stimulus gave more painful sensation. rinsing with room temperature tap water also elicit pain even though it had a lower intensity than cold stimulation. she was able to point the exact location of pain, that was the lower right region. nevertheless, 144 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 143–146 previous dental treatment that was done on the right lower molar tooth made no improvement. extra oral inspection seemed normal. intraorally, there was a temporary filling in mesial of 46, the suspected source of pain. the interdental space between 46 and 45 was dark red and bleed easily upon probing. rinsing with room temperature tap water elicit sudden and diffuse pain. however, pulpal thermal test with ethyl chloride on 46 and 45 did not show any pain. in other regions there were also mild periodontitis without gingivitis. periapical radiograph in 46 45 did not reveal significant alveolar resorption and an overhanging filling on 45. the overhanging filling was suspected to be the cause of the symptoms. case management at first visit, the first thing to be done was reshaping the overhanged filling, followed by periodontal instrumentation. preceding the periodontal instrumentation, that were scaling and probing procedures, the suspected area was irrigated with hexetidine 0.1%, after 30 second, the procedures started. as pain sensation could also be felt by the patient. anticipation was conducted by telling her to raise her left hand if sharp pain appeared. deep scaling along the subgingival area, especially in the interdental space between 46 and 45 resulted in bleeding. despite the bleeding, it seemed to be a paradox since the patient still comfortable and did not feel pain during this procedure. after the periodontal instrumentation had finished, the patient was told to rinse with room temperature tap water; at that time she did not feel pain anymore. in order to give more confidence to the patient for the successful result, she was given ice water for rinsing; surprisingly it did not elicit pain either. patient was scheduled for the next visit one week later second visit, the patient said that the pain symptom was completely disappeared. intraorally, the gingival area which previously inflamed was pink-colored and not easily bleeds. scaling procedures and probing which were done at the second visit felt more painful than the first visit. in order to maintain her periodontal health, she was told to floss her teeth regularly and had a routine dental check-up. discussion there are three kinds of pain receptors or nociceptors: a) mechanical nociceptor, b) thermal nociceptor, and c) polymodal nociceptor. mechanical and thermal nociceptors transmitted the impulse created by stimulation faster (12–18 m/sec) which dominates by the afibers than polymodal nociceptors which are the c-fibers (0, 5 m/sec). pricking or sharp sensation is mediated by the a-, whereas burning sensation which is slightly delayed, by the c-fibers. it is known, however, that a- fibers also conduct touch, warm and cold, whereas c-fibers also conduct itch, warm, and cold.5,6 nevertheless, the a-fibers dominate the cold sensation which is quickly felt as a sharp pain. the c-fibers also responsible for the diffuse and dull pain which experience shortly after the sharp pain. polymodal nociceptors respond equally to all kinds of damaging stimuli, including irritating chemicals released from injured tissues. despite their special characteristics, nociceptors do not have specialized receptor structures; they are all free nerve endings.5,7 each sensory receptor is attached to a first order or primary afferent neuron that carries impulses to the cns. the axon of these first order neuron are found to have varying thickness, thicker fibers have faster conduction velocities. pain receptors are also called nociceptors.5–7 pain that are conducted by a and c-fibers nociceptors can be sensitized by the presence of pro-inflammatory mediators and algogenic substances such as prostaglandin e2 (pge2), bradykinin, nitric oxide (no), histamine, serotonin etc. which cause primary hyperalgesia; or by repeated noxious stimulation (figure 1).2,9–11 figure 1. pain receptors in free nerve ending.10 145utomo: the periodontal pain paradox the a-fibers, the thinly-myelinated fiber is rapidly activated and associated with acute pain. this is a good pain because it warns the host to take care of the problems, e.g. touching a hot saucepan. it also related with the glutamate neurotransmitter. the c-fibers are unmyelinated, conduct impulses slowly; and associated with diffuse, dull, chronic pain. this is a bad pain because it cannot be alleviated simply by removing the stimulus. this pain is generated by such things as damaged tissue and cancer, and also related to the substance p neurotransmitter.2,8 sensitized or stimulated afferent nerve fiber release neuropeptides that are substance p (sp) and calcitonin generelated peptide (cgrp) from their terminals. substance p is slow to build up at the synapse and also slow to be destroyed, it is proposed as the result of the interplay between sp and cgrp.6,9 substance p also able to enhance nociception, and this neurotransmitter can be released into the extracellular space. in addition, it is thought to be the major neurotransmitter in slow pain (e.g. deep, dull or aching sensation) which primarily carried by c-fibers. glycine is a neurotransmitter which acts as the suppressor of pain transmission in the dorsal root ganglion, however, in inflammation prostaglandin potentiates the pain by blocking its action. stimulation will be perceived as pain if exceeds a certain threshold of the receptors, which then transmit signals that the brain interprets as pain.2,6,7 thermal sensation, pain elicited by heat or cold stimulation is perceived by different nerve fibers, cold perception is by the a- fibers which quickly response to cold stimulation. on the other hand, heat stimulation is perceived by the c – polymodal fibers which have the slower response.5 there are several detectors or receptors of heat, cold and which are transmembrane proteins, embedded in the plasma membrane of the endings of sensory neurons. there are four heat receptors: 1) transient receptor potential vanilloid4 (trpv4) -warm (> 25 °c); 2) trpv3 -warmer (> 31 °c); 3) trpv1 (also known as vr1) hot (> 43 °c), also activated by capsaicin, the active ingredient of hot chili peppers, and by acids (protons); 4) trpv2 (also called vrl-1) painfully hot (> 50 °c) (figure 2). in addition, there are two cold receptors: 1) trpm8 (or cmr1), is a channel that admits ca2+ and na+ in response to moderate cold (< 28 °c) or menthol (the ingredient that gives mint its cool touch and taste) and 2) trpa1 (or anktm1), responds to lower temperatures (< 18 °c) and elicit signals that the brain interprets at pain.5,8,12 according to a cross-sectional study, in patients suffered from chronic gingivitis only, the prostaglandin e2 has a greater quantity than gingivitis plus untreated periodontitis.13 the greater quantity of pge2 may lower the pain threshold of the nociceptors to thermal changes which coincidence to this case report. sensitization of nociceptors also occur from activation of kinases in the signaling pathway of the cytokines which present in abundant quantity in periodontal inflammation.2,11,13 other pain modulators which involved in periodontal inflammation is bradykinin, which is a product of the kallikrein-kinin system. this system can be activated by mast cell tryptases and microbial proteinases, including gingipains. gingipains, is a cysteine proteinase from porphyromonas gingivalis, the major pathogen of periodontal diseases, activates the kallikrein-kinin system through alternative cleavage of kininogens. this process subsequently produced bradykinin and kallidin.14,15 prostaglandin e2 and bradykinin work in concert in lowering pain threshold of nociceptors. bradykinin triggers an inflammatory positive feedback cycle, stimulating the release of prostaglandins and cytokines which in turn amplify the responsiveness of afferent nociceptors. in figure 2. nociceptive stimuli, nociceptive receptors, and subpopulation of type c neurons.12 146 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 143–146 the presence of nitric oxide (no) which is a product of macrophages during an inflammatory process, the lowered pain threshold is maintained.2 since the patient complained about pain upon eating or drinking hot and cold water, it was suggested that rinsing with room temperature tap water which was a non-noxious (unharmful) stimuli and do not elicit pain. however, in this patient it caused a pricking pain; the sudden pain might be related to the lowered threshold of a- fibers which had faster impulse velocity than c-fibers.5 while previous treatment was intended to relieve pulpal inflammation, it seemed plausible that several visits of pulpal treatments were failed to alleviate the pain, because the main problem in this patient was related to periodontal inflammation, that was chronic gingivitis. after deep scaling which surprisingly was a painless procedure in this patient, that also caused the inflamed gingiva to bleed; subsequently, the pain perception was back to normal. in order to evaluate the result of the treatment, the patient was told to rinse with tap water with room temperature, and followed by rinsing with ice water. rinsing with the tap water did not elicit pain, and surprisingly, rinsing with ice water also gave the same result. the possible explanation was that periodontal instrumentation removed the pro-inflammatory mediators (i.e. pge2, bradykinin and no), which responsible for lowering pain threshold were drained out from the inflamed gingiva. the exudates which came out accompanied by blood that oozed after the periodontal instrumentation also reduced the severity of chronic inflammation, and the gingival environment gradually returned to its normal condition. lipo polisaccharides were able to elicit pain in trigeminal nociceptor via the toll-like receptor-4 (tlr-4) and cd14 in dental pulp.16 logically, in chronic gingivitis which involved the lps and produces an array of proinflammatory mediators that may lower pain threshold should have a painful sensation. in contrary, in marginal periodontitis, a process associated with lps is not generally reported as painful. several hypothesis might explained this mechanism: 1) periodontal pathogenenic bacteria in marginal periodontitis might inactivate tlr/cd14 via released peptidases that are known to cleave receptors; 2) chronic lps may induce a down-regulation in tlr4 expression, and in fact, a nine-fold reduction in tlr4 has been reported in marginal periodontitis; and 3) nociceptor innervations or function might differ in differing target tissues, such as dental pocket vs. periodontal pockets.16,17 periodontal instrumentation, including scaling in probing may elicit pain and discomfort in patients, although it is also operator dependent.4 nevertheless, in this case, the patient did not show any evidence of pain. the possibility was something blunted the pain stimulation to nociceptor which suspected to be the cleave of pain receptors by peptidases of the periodontal pathogenic bacteria.14 a possible explanation in this case is that peptidases from periodontal pathogenic bacteria cleave mechanoreceptor but not thermoreceptor, which resulted in blunted pain reception upon scaling and probing, whereas hot and cold sensation which are non-noxious become painful (compared to the other side of the mouth). in this case, the periodontal pain paradox suspected etiology was the lowered pain threshold which caused by the higher pge2 level in chronic gingivitis compared to periodontitis. sudden relief of pain symptoms occurred after scaling and drainage of pro-inflammatory mediators of the chronic gingivitis resulted from the normalizing pain threshold. since the rapid resolution of inflammation in the gingiva also normalize the pain threshold, it concluded that chronic gingivitis, especially which bleed easily upon periodontal instrumentation is the main etiology of the periodontal pain paradox. however, further researches are needed to verify this hypothesis. references 1. meliala. terapi rasional nyeri: tujuan khusus nyeri neuropatik. 1st ed. jokyakarta: aditya media; 2004. p. 3. 2. kidd bl, urband la. mechanisms of inflammatory pain. brit j anaesth 2001; 87(1):3–11. 3. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microb rev 2000; 13(4):547–58. 4. steenberghe d, garmyn p, geers l, hendrickx e, marechal m, huizar k, et al. patients’ experience of pain and discomfort during instrumentation in the diagnosis and nonsurgical treatment of periodontitis. j periodontol 2004; 75(11): 1465–70. 5. squire lr, bloom fe, mcconnell sk, roberts jl, spitzer nc, zigmond mj. the somatosensory sytem. in: fundamental neuroscience. 2nd ed. orlando: academic press-elsevier science; 2003. p. 669–76. 6. okeson jp. bell’s orofacial pain. 6th ed. carol stream: quintessence pub; 2005. p. 262. 7. sherwood l. fundamentals of physiology. 1st ed. belmont: thomson/ brooks-cole; 2006. p. 88, 147–8. 8. kimball j. heat, cold and pain. available on line at: url http:// users. rcn. com/ jkimball. ma. ultranet/biology/ pages/p/pain. html. accessed october 12, 2006. 9. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2): 82–98. 10. millan mj. the induction of pain: an integrative review. neurobiol 1999; 57:1–16. 11. moriyama t, higashi t, togashi k, iida t, et al. sensitization of trpv1 by ep1 and ip reveals peripheral nociceptive mechanism of prostaglandins. molecular pain 2005; 1(3):1–13. 12. chih-feng t, baraniuk jn. upper airway neurogenic mechanisms. cur al clin immunol 2002; 2(1):11–9. 13. burt b. epidemiology of periodontal disease. j periodontol 2005; 76:1406–19. 14. deschner j, singhal a, long p, liu cc, piesco n, agarwal s. cleavage of cd14 and and lbp by a protease from prevotella intermedia. arch microbiol 2003; 179:430–6. 15. imamura t, potempa j, travis j. activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases. biol chem 2004; 385:989–96. 16. wadachi r, hargreaves km. trigeminal nociceptors express tlr-4 and cd14: a mechanism for pain due to infection. j dent res 2006; 85(1):49–53. 17. muthukuru m, jotwani r, cutler cw. oral mucosal endotoxin tolerance induction in chronic periodontitis. infect immune 2005; 73:687–94. mkg vol 42 no 2 april 2009.indd 82 vol. 42. no. 2 april–june 2009 research report the effect of watermelon frost on prostaglandin e2 (pge2) in inflamed pulp tissue (in vitro study) dennis and trimurni abidin department of conservative dentistry faculty of dentistry, university of north sumatera medan indonesia abstract background: pulp inflammation can be marked by the increase of prostaglandin e2 (pge2) level compared to normal pulp. the increase of pge2 may lead to vasodilatation, increase of vascular permeability, pain and bone resorption. watermelon frost has been well known in chinese society for pain relief and inflammation in oral cavity and teeth. purpose: the aim of this study was to investigate that watermelon frost can be used to decrease the pge2 level. method: 27 samples of pulp tissues used in this in-vitro study, were extirpated from the patients’ teeth with symptomatic irreversible pulpitis referred to clinic of conservative dentistry, rspgm faculty of dentistry, usu. trial materials were applied to 27 samples i.e. watermelon frost as a trial material and commercial watermelon frost and eugenol to observe their effect on pge2. pge2 level of each material was detected through elisa method by measuring and comparing the absorbance reading of the wells of the samples against standards with a micro plate reader at w1 = 650 nm and w2 = 490 nm. result: the result showed the biggest effect was found in the third group (eugenol), mean 4.6933, followed by the first group (watermelon frost as a trial material), mean 18,1578 then the second group (commercial watermelon frost), mean 82,2689. oneway anova revealed that there were significant differences among all trial materials (p < 0.001) on pge2 level. conclusion: this study demonstrated that watermelon frost can be used to decrease the pge2 level in inflamed pulp tissue and led to the acceptance of traditional medicine and natural products as an alternative form of dental care. key words: watermelon frost, prostaglandin-e2, inflammation, pain correspondence: dennis, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas sumatera utara. jl. alumni no.2, kampus usu medan 20155, indonesia. e-mail: dennis_dionisius@yahoo.co.uk introduction pulp inflammation is a case which is often found in dental practice. this inflammation has caused pain to the patients. the inflamed pulp will cause pain that is obviously seen by the increase of prostaglandin e 2 (pge2) if it is compared with normal pulp. the increase of pge2 will trigger pain on teeth, vein vasodilatation, vascular permeability, and bone resorption.1-7 hospitals and dental clinics with patients of symptomatic irreversible pulpitis and periodontitis need intradental pain relief to decrease inflammation. generally, the pain relief, which is often used, is eugenol (clove oil) as sedative. apparently, most of indonesians have used clove oil as traditional medicine to relieve intradental pain for a long time. they unconsciously have known eugenol as local sedative but they have not precisely figured out how much eugenol should be consumed.8 a study by olsson et al.,9 showed that any materials containing eugenol had more serious reaction of tissues than those without eugenol.10 another pain relief commonly used is pulperyl containing creosate, procaine, phenol and chloroform. several corticosteroids can be used to reduce intradental pain for acute pulpitis patients. practically their use is combined with polyantibiotic. because polyantibiotic is imported, its market price is expensive and it is not widely sold. watermelon frost, a traditional medicine for therapy, has been widely known by the chinese community for ages. clinical experiment has proven that watermelon frost affects inflammation treatment of oral cavity, tonsilitis, laryngitis, 83dennis: the effect of watermelon frost pharyngitis, dental pain caused by high fever, inflamed gums, scald, etc.11,12 this indicates that inflammation can be reduced or healed after being given watermelon frost as traditional medicine. research dealing with watermelon frost which will be used as pain relief should be increased more because indonesia is enriched by tropical fruit like watermelon. in endodontic treatment the materials used should be biocompatible (nontoxic in human tissues). however, concerning the use of watermelon frost as pain relief, a question arises as the following: can watermelon frost that has been widely known for medical treatments of oral cavity, pharyngitis, laryngitis, gingivitis be used as intradental pain relief? the objective of the study was to figure out how watermelon frost affects pge2 which is one of inflammatory mediators. it is expected that the study can be seen as one consideration to develop the use of watermelon frost as traditional medicine for the inflammation and the pain relief besides eugenol, miswak and corticosteroid.13,14 furthermore, the study can familiarize the use of watermelon frost so that people can utilize watermelon which is not expensive. in addition, the study will encourage primary health services to provide economical material used as pain relief which is nontoxic and biocompatible enough in dental tissues. materials and methods type of this study was comparative experimental research. it involved watermelon frost as the material. the steps to make watermelon frost were: chose a watermelon weighing 2.5 kg, sliced its top, and part of the flesh was removed. put 500 grams of glauber’s salt (sodium sulfate decahydrate) on it (figure 1). then a small stick of bamboo to retained it and put the top back so the hole was covered. afterwards the watermelon was hung in fridge (not a part of freezer) approximately 7–10 days or until frost (white powder) was formed in outer part of watermelon rind. this frost has a therapeutic effect in reducing pain and inflammation.15 watermelon as a trial material produced white powder (frost) by adding glauber’s salt (na2so4.10h2o) for several days and frost was dissolved in aquadest (figure 2). figure 2. watermelon frost found in the rind surface of watermelon. sample was divided based on the materials: group i (9 samples) was given watermelon frost with concentration 50% (dissolved with aquadest), group ii (9 samples) was given watermelon frost with commercial 50% (content based on the supply of factory) and group iii (9 samples) was given eugenol 50% (dissolved with dimethyl sulfoxide/ dmso). pulp tissues were obtained from the teeth which were chosen by opening the cavity and extirpating pulp with extirpating needle (barbed-broach). the teeth rooted with more than one pulp were gained from the largest root canal. then, the pulp was placed in eppendorf containing 0.5 ml of phosphate buffer saline (pbs) and 10–3 m of nimesulide; centrifuge was applied at 2400 rpm for 10 minutes; after that, it was restored at temperature –23° c. each eppendorf was given watermelon frost as trial material, commercial watermelon frost and eugenol. analysis of pge2 quantitative level was seen by using enzyme-linked immunosorbent assay kit (neogen, usa) through an equipment called ”elisa reader” that showed the absorbance level of color at the wave length w1 = 650 nm and w2 = 490 nm. result the result from reading the absorbance level of color on each sample by elisa reader showed the largest decrease of pge2 level in eugenol group then followed by watermelon as trial material. meanwhile, the group of figure 1. watermelon which is given glauber’s salt. 84 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 82−85 commercial watermelon frost showed the smallest decrease of pge2 level (table 1). table 1. the pge2 level from watermelon frost as trial material, commercial watermelon frost and eugenol (ng/ml) number sample concentration of pge2 (ng/ml) group i (watermelon frost as trial material) group ii (commercial watermelon frost) group iii (eugenol) 1. 17.87 86.62 4.30 2. 14.96 98.95 6.51 3. 28.35 68.99 12.27 4. 17.87 108.05 5.68 5. 15.95 68.65 3.76 6. 15.80 71.25 2.92 7. 13.59 83.50 3.80 8. 26.15 92.78 1.95 9. 12.88 61.63 1.05 table 2. the average level and standard of deviation from the decrease of pge2 level (ng/ml) type of materials n x sd watermelon frost as trial material 9 18.16 5.45 commercial watermelon frost 9 82.27 15.74 eugenol 9 4.69 3.31 total 27 35.04 35.75 table 2 indicated that the average level and standard of deviation at group of watermelon frost as trial material were 18.16 ± 5.45 ng/ml. at commercial watermelon frost group, the average level and standard of deviation were 82.23 ± 15.74 ng/ml. at eugenol group, they were 4.69 ± 3.31 ng/ml. these implied that effect of eugenol in reducing the pge2 level is higher than that in watermelon frost (as trial material and commercial). in the meantime, the effect of watermelon as trial material in reducing the pge2 level is higher than that of commercial watermelon frost. to see the difference of pge2 level in watermelon frost as trial material, commercial watermelon frost and eugenol, one-way anova was applied with α = 0.001 (table 3). the result of anova showed that there was statistically significant difference (p < 0.001) among groups of watermelon frost as trial material, commercial watermelon frost and eugenol in the decrease of pge2 level. discussion in this study, the effect of watermelon frost as trial material was compared to the effect of commercial watermelon frost and eugenol. of those three materials, it was eugenol that had the greatest effect on pge2. this was followed by watermelon frost as trial material and commercial watermelon frost. however, the decrease of pge2 in commercial watermelon frost was lower than in watermelon frost as trial material. this was probably caused by the content of pure watermelon frost in commercial product as much as 50% and the mixture with other materials; rhizoma belamcandae (5%), bulbus fritillariae (15%), radix sophorae tonkinensis (10%), mentholum (5%). indigo naturalis (5%) and borneolum (10%). the content of watermelon frost as much as 50% has caused the decrease of pge2 lower than the watermelon frost as trial material where the content is pure. besides, other contents found in commercial watermelon frost can influence the effectiveness of watermelon frost in reducing the pge2 level. according to cohen et al. cit hashimoto et al.,8 the pge2 level on the tissue of symptomatic inflamed pulp was significantly higher than that of asymptomatic inflamed pulp. isett j et al.17 in his study stated that higher pge2 level is found in irreversible pulpitis and it leads to vein vasodilatation, the increase of vascular permeability, chemotaxis, pain, and bone resorption.16 the tables in this study implied that each group had several samples showing higher pge2 level (out layer) even though it was given trial material. in table 1, it was seen that even though the pulp tissue was taken from symptomic irreversible pulpitis, the pge2 level in each case could be different. in severe inflammation, the pge2 level would be high. consequently, in group i the pge2 level of the third sample was 28.55 ng/ml; in group ii the fourth sample was 108.05 ng/ml; in group iii the third sample was 12.27 ng/ml. these cases are possibly caused by the pge2 level that was higher than that found on other samples. eugenol has the greatest capability to reduce the pge2 level. it was shown from a study by dewhirst and hirafuji cit. hashimoto et al.,8 where eugenol can inhibit biosynthesis of pge2 through in vitro. in contrary, a study by prashar et al.18 showed that eugenol is very cytotoxic in fibroblast and endothelium cells. the toxicity effect has caused the cell inactive through apoptosis and necrosis. finally, if the inflamed cells in the pulp tissue are inactive, the pge2 level will be significantly decreased. table 3. analysis of variant for the groups: watermelon frost as trial material, commercial watermelon frost, and eugenol the pge2 level (ng/ml) df f p. between groups 2 160.821 .000* within groups 24 total 26 explanation : p: anova test * : significance f : frequency df : degree of freedom 85dennis: the effect of watermelon frost according to the study by ho et al.,10 it was mentioned that eugenol can inhibit the growth and proliferation of osteoblastic cell line u2os. therefore, eugenol has significant role on periapical toxicity and destroys the cell in the pulp tissue. watermelon frost is a traditional/herbal medicine which has been empirically known by the chinese community as one of therapies against inflammation. the use of watermelon frost has been approved by chinese drugs administration department. watermelon frost is obtained from watermelon by using glauber’s salt. since watermelon frost is naturally gained from watermelon, it is biocompatible enough in oral tissues. as consequence, the cells in the pulp tissue including the inflamed cells after being given watermelon frost are probably vital.19 from the data analysis and the discussion, it can be concluded that there were differences among the decrease of pge2 level found in commercial watermelon frost, watermelon frost as trial material, and eugenol. based on the analysis above, it can be seen that eugenol has bigger effect of the pge2 decrease 4 times than watermelon frost as trial material and it is bigger 17 times than commercial watermelon frost. meanwhile the effect of the pge2 decrease is bigger 5 times than commercial watermelon frost. eugenol is very cytotoxic in fibroblast and endothelium cells. the effect of cytotoxicity can lead to the death of cells through apoptosis and necrosis. if the inflamed cells in the pulp tissue are inactive, the pge2 level will be significantly decreased. because watermelon frost is naturally gained from the watermelon, watermelon frost is biocompatible and nontoxic in oral tissues, so the cells in the pulp tissue including the inflamed cells after being given watermelon frost are vital. to identify the active substance which functions as anti pge2 in watermelon frost, fraction and characterization tests need to be carried out. the tests refer to the physical and chemical characteristics of watermelon frost. besides, cell culture needs to be applied to observe the condition of cells in the pulp tissue after being given watermelon frost. references 1. waterhouse pj, nunn jh, whitworth jm. prostaglandin e2 and treatment outcome in pulp therapy of primary molars with carious exposures. international journal of paediatric dentistry 2002; 12:116–23. 2. waterhouse pj, whitworth jm, nunn jh. development of a method to detect and quantify prostaglandin e2 in pulpal blood from cariously exposed, vital primary molar teeth. int endodon j 1999; 32:381–7. 3. nakano k, ohishi m, ogawa y, ohba t, kido j, miyake y, nagata t. prostaglandin e2 inhibits alveolar bone resorption in experimental periodontitis in hamster. dentistry in japan 1998; 34:108–11. 4. trowbridge ho. history of pulpal inflammation. in: seltzer, bender, editors. dental pulp. il: quintessance publishing co, inc; 2002. p. 227–45. 5. fouad af. molecular mediators of pulpal inflammation. in: seltzer and bender. dental pulp. il: quintessance publishing co, inc; 2002. p. 247–79. 6. isett j, reader a, gallatin e, beck m, padgett d. effect of an intraosseous injection of depo-medrol on pulpal concentrations of pge2 and il-8 in untreated irreversible pulpitis. j endodon 2003; 29:268–71. 7. ohnishi t, suwa m, oyama t, et al. prostaglandin e2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation. j dent res 2000; 79:748–55. 8. hashimoto s, uchigama k, maeda m, et al. in vivo and in vitro effects of zinc oxide eugenol (zoe) on biosynthesis of cyclooxygenase products in rat dental pulp. j dent res 1988; 67:1092–109. 9. ollson b, sliwkoarski a, largeland k. subcutaneous implantation for the biological evaluation of endodontic materials. j endodon 1981; 7:355–65. 10. ho yc, huang fm, chang yc. mechanisms of cytotoxicity of eugenol in human osteoblastic cells in vitro. int endodon j 2006; 39:389–93. 11. fang yc, huang hc, chen hh, juan hf, tcm gene dit: a database for associated traditional chinese medicine, gene and disease information using text mining biomed central ltd 2008; 8(58): 1–11. 12. zhang, yi fang. comprehensive methods for preventing and treating influenza. j chinese medicine 2001; 65:6–10. 13. sulaiman mi, al-khateeb tl. the analgesic effects of miswak. the saudi dent j 1996; 8:140–4. 14. al-samh da, al-nazhan s. in vitro study of the cytotoxixity of the miswak ethanolic extract. the saudi dent j 1997; 9:125–32. 15. yin-fang, dai, cheng-jun, liu. terapi buah. adi loka sujono, editor. jakarta: prestasi pustaka publisher; 2002. p. 32–4. 16. nakano k, ohishi m, ogawa y, et al. prostaglandin e2 inhibits alveolar bone resorption in experimental periodontitis in hamster. dentistry in japan 1998; 34:108–11. 17. isett j, reader a, gallatin e, beck m, padgett d. effect of an intraosseous injection of depo-medrol on pulpal concentrations of pge2 and il-8 in untreated irreversible pulpitis. j endodon 2003; 29:268–71. 18. prashar a, locke ic, evans cs. cytotoxicity of clove (syzygium aromaticum) oil and its major components to human skin cells. cell prolif 2006; 39:241–8. 19. zong-chang, xiu. traditional chinese medical therapies: melons, fruits and vegetables as medicine. selangor darul ehsan malaysia: pelanduk publication; 2003. p. 15–28. << /ascii85encodepages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 141 vol. 43. no. 3 september 2010 the combination of miacalcic, calcium lactate, and vitamin c as postextracted alveolar bone resorption inhibitor sri kentjananingsih department of biology, surabaya state university surabaya indonesia abstract background: tooth extraction can cause alveolar resorption, and will reduce the denture retention. the process of bone resorption looks like the process of osteoporosis. calcium and vitamin d supplementation is the rational therapy for minimizing bone loss. miacalcic is the drug of choice for osteporotic patient. purpose: this study is aimed to know whether the combination of miacalcic, calcium lactate, and vitamin c are effective in inhibiting post extracted alveolar resorption. methods: thirty three healthy postmenopausal women were chosen as samples and they were classified randomly into control group (without treatment), 1st experiment group (treatment was started 3 months post extraction), and 2nd experiment group (treatment was started at the 2nd day post extraction). the treatment was done by giving miacalcic nasal spray, calcium lactate 500 mg and vitamin c 100 mg tablets every morning in 10 days every month for 3 months. x-ray photo of the post extracted area were taken an hour, 3 months, and 6 months post-extraction. results: after 6 month, there was significant difference in buccolingual thickness decreasing among three groups (p<0.05). the maximum mean difference of buccolingual thickness decreasing was 0.72 mm, between control and 2nd experiment groups. there was no significant difference about decreasing bone density among them (p>0.10). the maximum difference of the mean of density decreasing was 1,906 g/cm2/mm between control and 2nd experiment groups. the increasing density mostly occurred in the 2nd experiment group. conclusion: the combination of miacalcic, calcium lactate, and vitamin c are effective for inhibiting alveolar resorption, although statistically there was no significant difference about bone density decreasing. the sooner this treatment is given the better result will be achieved. key words: miacalcic, calcium lactate, vitamin c, alveolar resorption abstrak latar belakang: pencabutan gigi menyebabkan resorpsi tulang alveolaris, dan akan mengurangi retensi geligi tiruan. proses resorpsi tulang alveol pada osteoporosis mirip dengan proses resorpsi tulang pada penyembuhan luka bekas pencabutan. miacalcic adalah obat utama untuk penderita osteoporosis. kalsium dan vitamin d merupakan terapi yang rasional untuk meminimalkan resorpsi tulang. tujuan: membuktikan apakah kombinasi miacalcic, kalsium laktat, and vitamin c juga efektif menghambat resorpsi tulang alveol pasca pencabutan. metode: sampel 33 wanita postmenopause yang sehat, terbagi secara acak ke dalam kelompok kontrol (tanpa perlakuan), kelompok eksperimen 1 (perlakuan mulai 3 bulan pasca pencabutan) dan kelompok eksperimen 2 (perlakuan mulai hari kedua pasca pencabutan). perlakuannya yaitu: pemberian miacalcic semprot hidung, tablet kalsium laktat 500 mg dan vitamin c 100 mg setiap pagi, 10 hari dalam sebulan, selama tiga bulan. foto sinar-x dari regio pasca pencabutan dibuat satu jam, 3 bulan, dan 6 bulan pasca pencabutan. hasil: 6 bulan pasca-cabut, ada beda bermakna perihal selisih tebal bukolingual tulang alveol antar ketiga kelompok (p<0,05). rerata penurunan ketebalan ini maksimal sebanyak 0.72 mm, antara kelompok kontrol dan kelompok eksperimen 2. penurunan kepadatan tulang antar ketiga kelompok tidak bermakna (p>0,10). beda maksimum rerata kepadatan tulang antara kelompok kontrol dan kelompok eksperimen 2 sebesar 1,906 g/cm2/mm. peningkatan kepadatan terbanyak dialami anggota kelompok eksperimen 2. kesimpulan: kombinasi miacalcic, kalsium laktat, vitamin c efektif menghambat resorpsi tulang alveolaris, walaupun secara statistik beda penurunan kepadatan tidak bermakna. makin awal pemberian perlakuan, hasilnya akan lebih baik. kata kunci: miacalcic, kalsium laktat, vitamin c, resorpsi tulang alveolaris correspondence: sri kentjananingsih, jurusan biologi universitas negeri surabaya. e-mail: sri_kentjananingsih@yahoo.co.id research report 142 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 141–145 introduction tooth extraction is often chosen by patients because of strong pain or the unwillingness to visit their dentist several times, although dental conservative science and technology are continually developing, the consequence of tooth extraction is alveolar resorption. it occurs fast in the first three months in mesiodistal, apico-occlusal, bucco lingual directions and it influences its density. after that it occurs slowly but progressively during our life until the alveolar processus lost. this condition is related to minimum physiological pressure during wound healing.1 there are some other factors influence the resorption rate, such as general health, local condition, the quality of food intake, medicine usage, and some hormones concentration such as parathyroid hormone (pth), blood calcium concentration, sex hormone, and calcitonin.2 if most of the factors can be controlled, the influence of each factor can be proved. due to this reason, healthy postmenopausal women, who have the lowest concentration of sex hormone and calcitonin were chosen as samples. in this condition, the osteoclasts’ activity are 20–40 times more efficient then osteoblasts, so the density of the bone is fastly reduced.2 the food’s quality in unstarvation person is considered normal, because in adult, the more protein intake, the faster bone resorption will occur.2 hypocalcemia will stimulate pth secretion and further high pth concentration will stimulate calcium deposition from our bone. calcium and vitamin d supplementation is rational therapy for minimizing bone loss.3 during post extraction wound healing, vitamin c as the cofactor for prolilhydroxylation is needed in hidroxyprolin forming to get better bone quality.1 miacalcic is a salmon calcitonin, a calcictropic hormon, which inhibits bone resorption by a direct inhibiting action on osteoclast activity via its receptors. it is the physiological treatment of choice for osteoporotic patients, because it inhibits the bone resorption process and its minimum side effect as compared to estrogen.4,5 its nasal spray makes it easily used. it still works several days after being exposured in several minutes. it increases calcium excretion, which needed in osteoclasts’ activities, so that the bone resorption is reduced;6 even it can increase the bone’s mass and density6–8 and enhance the healing process.7,9 in two weeks administration, it had effectively inhibited the reducing bone mass,9 but with 15 iu/kg dose, calcitonin can not prevent the reducing calcium incorporation that happened in immobilized bone.10 administration more than one year caused the lost of calcitonin receptors.6 it could reduce pain disturbance.11 conversely, duarte et al.,12 found that it could not prevent bone loss caused by estrogen deficient. thamsboro et al.,13 got significant bone mass density (bmd) increasing, but no significant bmd difference. gurkan et al.,14 found that giving miacalcic without calcium would increase the bone resorption process. this research will prove whether miacalcic, calciumlactate and vitamin c treatment was effective in inhibiting post extracted alveolar resorption. it will help to overcome the decreasing denture retention. materials and methods the samples were found in puskesmas tambakrejo with the inclusion criteria: normal in finger articulation; p o s t u r e ; b o d y m a s s i n d e x ; r a n g e o f c o m p l e t e l y hematology and urine laboratory test; glucose, thyroid stimulating hormone (tsh), t4 and alkalin phosphatase concentration, and normal backbone photos. they have their mandibular’s second premolar, first molar, or second molar been extracted. they would not wear the denture. the exclusion criteria were: irregular period during one year; malignant process; hypertension, asthma, prolong using glucocorticosteroid, prostaglandin, and analgesic, tetracycline and chloramphenicol; unwillingness to be observed for at least 6–12 months. for this, each of them should sign an inform consent. this research was a clinical trial using completely randomized design, with three groups. they were control group, without treatment; 1st experiment group, who received treatment 3 months post extracted; and 2nd experiment group, who received treatment at the following day post extracted. after being extracted she was taken her alveolar x-ray photos. at that day she should fast from 7 pm to got a series laboratory test at following day. at the same day her body weight and body height were measured; her backbone x-ray photo was taken. the impression result was held in a plastic sac, which can be tightly bound for inhibiting the impression shrinkage. in the afternoon it was filled with gypsum. if her laboratory result was good, she became the sample of one of the three groups randomly. depended on which experiment group’s member she was, at that day or three month later or never, she was given one calcium-lactate 500 mg (kimia farma) and one vitamin c 100 mg tablets (kimia farma) to be swallowed, then one spray of miacalcic (sandoz) for each nostril, every morning, 10 days continually in a month for three months at her home. at the end of the 3rd month x-ray photo at her post extracted area and mandible impression were taken again and those was repeated three monthly until one year. alveolar x-ray photo, was taken when the patient sat vertically, lean on the back of the chair, with straight vision to front direction, the cone was arranged horizontally as high as the root part of the posterior teeth, and the duration had been already arranged. the patients were not using an apron protection while their x-ray photos were taken, because each of them was given x-ray exposure each of kind three months. before processing the film, both of the poured processing solution were into two small bowls. the film was opened in dark room, shaken 23 seconds in the developer solution and 30 seconds in fresh water, then it was shaken 5 minutes in the fixation solution. finally the film was washed in flowing fresh water for several minutes until it cleaned from the fixation solution. the film was dried in the air. the target regions were circled not less than 1 mm2 wide by ink pen. the darkness of the target area in the film was measured with calibrated densitometer type 07–424 (figure 1). for this, turn on the switch for at least 10 minutes, then close 143kentjananingsih: the combination of miacalcic, calcium lactate, and vitamin c the arm and the digital should show 0, by arranging the small wheel at the right side. the target area in the film should be located on the window of densitometer and the stable number showed on the digital was recorded. this was repeated three times and then the mean was counted. the mean number was conversed to the bone density, through the formula.15 the density difference of the target area could then be counted. the buccal and lingual parts of the plaster model was signed as in figure 2. the buccolingual thickness of this model was measured by a caliper, as the distance of the buccal’s and lingual’s signs. this was repeated in 6 month’s model. the alveolar resorption was counted as the width difference. results the data of the alveolar thickness difference and the alveolar density difference of the 33 sample were provided in table 1 and table 2. table �. the alveolar thickness differences (mm) of the three groups after six months atd�c�d�c� atd�1st e� atd�2nd e� 2�27 2�26 0�98 2�09 2�29 2�34 2�40 3�38 3�06 2�70 3�34 1�24 0�99 2�19 1�25 0�71 1�69 3�47 2�82 1�25 0�15 0�75 0�73 1�01 0�65 2�31 2�04 2�40 1�35 2�30 2�30 1�50 2�40 mean: 2�446 var� coef�: 27�72% 1�501 65�29% 1�726 40�38% atd.cg : alveolar thickness difference of the control group atd.1st eg : alveolar thickness difference of the 1st experiment group atd.2nd eg : alveolar thickness difference of the 2nd experiment group table �. the alveolar density difference (g/cm3/mm) of the three groups after 6 months add.cg add.1st eg add.2nd eg 1.134 3.755 1.991 3.730 9.601 1.109 7.661 0.806 8.871 2.999 -0.554 1.810 3.856 0.328 5.141 6.048 7.686 3.024 1.033 2.591 -3.553 0.690 6.351 -2.167 3.325 -1.965 1.814 -1.159 2.344 5.544 4.058 -1.984 3.982 mean: 3.737 var. coef.: 92.86% 2.605 118.77% 1.831 172.86% add.cg : alveolar density difference of the control group add.1st eg : alveolar density difference of the 1st experiment group add.2nd eg : alveolar density difference of the 2nd experiment group the anova of this result showed a significant decreasing thickness difference among those groups (p<0.05), while the difference between both experiment groups was non significant (p=0.519). it means that the significant difference is only happens between control group and both experiment groups. the samples of the control group got 0.72 mm reduction more than the two others. this means that the treatment can inhibit the alveolar resorption. the anova of this result showed that there wasn’t significant bone density difference among those groups (p>0.05), although the mean value of both experiment group was lesser than the control group. the bone density of some patients in those three groups increase, four members of the 2nd experiment group and one member in each of two other groups. discussion the exclusion factors have been fulfilled here. one thing that contrary to public idea is, the more protein 1st eg.0 1st eg.3 1st eg.6 figure �. target areas in one member’s of 1st experiment group (1st eg) x-ray photoes. 1st eg.0 was taken soon after tooth extraction; 1st eg.3 was taken 3 months after tooth extraction, and 1st eg.6 was taken 6 months after tooth extraction. the cervical points of the neighboring teeth were related and the center of the circle line was put 3 mm below the line. the circle line above the edentulous was used as the guidance in measuring the blackened degree; while the circle between two other teeth was made to know whether this treatment had influenced another area of alveolar bone. 144 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 141–145 intake, especially animal protein in adult will cause loss of calcium from the bone, because it will increase blood uric acid and urea concentration.2,16,17 they store in the body as toxins and fats.18 for excreting them in urine the water will much loss and our bones tend to release calcium.19 excess protein may be associated with negative calcium balance.17 our body needs only about 35–50 grams protein per day, either from animal, plant, or their combination.17as far as someone is not at starvation condition, he was in normal food quality, because our body needs balance nutritions, consists of carbohydrate, protein, fat, minerals and vitamin.17,20 postmenopausal women were chosen, so that the calcitonin could be controlled at the lowest concentration. through this treatment, their calcitonin concentration would enable the precipitation of calcium and phosphate on the bone,2 but this was unproved by tuukkanen et al.10 although bone wound healing usually has completed in 3 months,2 even in young rats, it needed only 4 weeks,14 but it’s calcification can progressively occur until 6 months.1 it was the reason that the 1st experimental group was treated 3 months after extracted. there are two reasons of choosing miacalcic in this research. the first reason is, there are only two medications which currently approved by federation dental association (fda) for osteoporosis treatment, they are estrogen and salmon calcitonin.5 estrogen tends to raise breast cancer,5 in contrary calcitonin has slight side effect.6 the second reason is, calcitonin is a first-line choice in several bone diseases treatment.4 placebo was unneeded in this research, because each sample was treated individually at her home. calcium-lactate was given because calcitonin without calcium will increase the bone resorption process.14 this condition is related to the decreasing blood calcium concentration soon after calcitonin administration, while calcium is needed for spouting chemical and electric signal from cellular membrane into the cell.6 vitamin c is a cofactor of prolilhydroxylation, it helps the hydroxyprolin forming.2 for getting better new bone quality, it is important to give it during wound healing process. if this vitamin is given after the wound healed, it will not give much effect to the new bone quality.1 alveolar resorption can happen in mesiodistal, buccolingual, and occlusoapical directions, besides the reduction of bone density.1 in this research the data were only the buccolingual thickness and the bone density reduction differences, because the occlusoapical resorption made irregular occlusal surface of the alveolar bone, so it could not be measured; while mesiodistal distance wasn’t completely gotten, because some of the extracted teeth were the most distal teeth. there was only a slight thickness-decreasing difference (mean difference was 0.72 mm). this was supporting the research findings of canavero et al.,7 that there was no larger amount of new bone at the end of 21 days observation and there was no significant clinical size.6 this slight difference was worthy enough for denture retention, especially if it occurs at the whole maxilla or mandible. there was not significant alveolar density difference, although the maximum difference of the mean densitydecreasing difference was 1.906 g/cm2/mm. actually this slight increase is also worthy, because it will better maintain the physiological resorption. from these data, some members of the experimental group got slight increased their alveolar density. it supports the previous study, that although there is significant increase in cortical bone mass density with calcitonin treatment, but calcium and vitamin d administration is more effective in preventing bone loss.11 furthermore, calcitonin could not prevent the effect of estrogen deficiency,12 and nasal salmon calcitonin 200 iu daily produces only a minor increase in bone mass.13 alveolar density was increased more on the samples who begin the treatment at the following day after extracted, which the wound healing starts. this was easily understood, because miacalcic enhanced the wound healing in early stage,10 although gurkan et al.,14 found no significant effect on it. it may be caused by the inability to hinder the food intake activity of the rat that may cause slight infection, so the healing was postponed. miacalcic will help much more calcium precipitating in the bone matrix.10 salmon calcitonin had no significant effect on mineralization of the alveolar bone.14 furthermore, two weeks immobilization will cause bone mass reduction, so that the postponed miacalcic treatment will cause their bone density lower than the ones who directly got the treatment.10 about the increasing bone density in one member of the control group, the most possible reason is they have got enough calcium from their food, so that the higher calcium blood concentration will prevent the bone calcium figure �. the way to sign the points to be measured in plaster model of mandibular. a) mandibular model of a patient who has been extracted her 1st molar, b) mandibular model of a patient has been extracted her 1st molar which was the most distal tooth. ba 145kentjananingsih: the combination of miacalcic, calcium lactate, and vitamin c release.12 it might also be caused by the difference of gut epithelial capability in calcium and phosphorus absorption, the difference of personal vitamin d concentration, or the difference of their body mass index. because there were many exclusion criterias to be fulfilled, the samples were not so easy to get. it needed almost four years to get 33 samples to fulfill the sample size. during this time there are many patients failed to be the samples, although they had signed the inform consent; because each member had her own activity and business. this research needs special social skill in holding the good relationship to the samples, and to get their data in the exact schedule. a good schedule should be arranged for the researcher’s daily activity. this means that besides the main result, the researcher got another advantage. the conclusion of this research was the combination of miacalcic calcium lactate, and vitamin c are effective for inhibiting alveolar resorption, although statistically there was no significant difference about bone density decreasing. the sooner this treatment is given the better result will be achieved. references 1. peterson lj, indresano at, marciani rd, roser sm. principles of oral and maxillofacial surgery. vol. 2. philadelphia: jb lippincott co; 1992. p. 1173. 2. guyton ac. textbook of medical physiology. 7th ed. philadelphia, london, toronto, mexico city-rio de janeiro, sydney, tokyo, hongkong: wb saunders company, 1996. p. 732, 782–6, 861, 868–83, 891–2, 905, 910. 3. buckley lm, leib es, kathryn s, cartularo rn, vacek pm, cooper sm. calcium and vitamin d3 supplementation prevents bone loss in the spine secondary to low-dose corticosteroids in patients with rheumatoid arthritis. article. available at: http://www.annals.org/ content/125/12/961.full. accessed december 31, 2010. 4. reginster jy. calcitonins: newer routes of delivery. osteoporosis int 1993; 3(suppl 2): 53–6. discussion s6–7. 5. cosman f, nieves j, walliser j, lindsay r. postmenopausal osteoporosis: patient choices and outcomes. maturitas 1995; 22(2): 137–43. 6. vered m, shohat i, buchner a, dayan d, taicher s. calcitonin nasal spray for treatment of central giant cell granuloma: clinical, radiological, and histological findings, and immunohistochemicall expression of calcitonin and glucocorticoid receptors. netter oral surgery, oral medicine, oral pathology, oral radiology, and endodontology 2007; 104(2): 226–39. 7. canavero e, januario al, sallum ea, novaes pd, nociti fh jr. histometric evaluation of local action of salmon calcitonin on bone repair: a study in rats. pesqui odontol bras 2000; 14(2): 183–7. 8. kaskani e, lyritis gp, kosmidis c, galanos a, andypas g, chorianopoulos k, giagiosis a, iliadou k, karagianis a, katsimichas k, koskinas a, matsouka k. effect of intermittent administration of 200 iu intranasal salmon calcitonin and low doses of 1alpha(oh) vitamin d3 on bone mineral density of the lumbar spine and hip region and biochemical bone markers in women with postmenopausal osteoporosis: a pilot study. clin rheumatol 2005; 24(3): 232–8. 9. dogan h, ozcelik b, gediko g, senel s. the effect of calcitonin on osse-ous healing in guinea pig mandible. j endod 2001; 27(3):j endod 2001; 27(3): 160–3. 10. tuukkanen j, jalovaara p, vaananen k. calcitonin treatment of immobil-ization osteoporosis in rats. acta physiologica scandinavica 1991; 141(91): 119–24. 11. kopaliani m. effectiveness of intranasal salmon calcitonin treatment in post-menopausal osteoporosis. georgian med news 2005; 121: 38–42. 12. duarte pm, goncalves p, sallum aw, sallum ea, casati mz, nociti jrfh. effect of an estrogen-deficient state and its therapy on bone loss. j period 2004; 39(2): 107–10. 13. thamsboro g, jensen jeb, kollerup g, hauqe em, melsen f, sarensen oh. effect of nasal salmon calcitonin on bone remodelling and bone mass in postmenopausal osteoporosis. bone 1996; 18(2): 207–12. 14. gurkan l, ekeland a, langeland n, ronningen h, solheim lf. effect of salmon-calcitonin on growth of teeth and on extraction socket healing in young rat. wiley online library oct 1wiley online library oct 1st 2007. availableavailable at: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0722.1983. tb00841.x/abs-tract. accessed aug 16, 2010. 15. kentjananingsih s. penghambatan penyusutan tulang alveol daerah bekas cabut oleh kalsitonin, ditambah kalsium dan vitamin c. disertasi. surabaya: pascasarjana universitas airlangga; 1997. 16. preventing osteoporosis using natural methods. p 1–4. available at: http://www.healingdaily.com/ conditions/osteoporosis,htm. accessed december 28, 2010. 17. stevemark. health problems and side effects that has been associated` with too much high protein. p. 1–7. available at: http://hubpages. com/hub/health-concerns-regarding-high-proteins-diets. accessed december 28, 2010. 18. hannan mt, tucker kl, dawson-hughes b, cupples la, felson dt, kiel dp. effect of dietary protein on bone loss in elderly men and women: the framingham osteoporosis study. j bone miner res 2000 dec; 15(12): 2504–12. 19. hopkins s. could high protein diets prove harmful for people with liver or kidney disease. article. p. 1–3. available at:article. p. 1–3. available at: http://www. diethealthclub.com/articles/42/diet-and-wellness/could-high-proteindiets-pro. accessed december 28, 2010.accessed december 28, 2010. 20. heaney r. the protein and calcium paradox in osteoporosis. am j clin nutrit 2002; 75(4): 609–10. vol 38-no4-2005-isi.pmd 176 the effect of different concentrations of neem (azadiractha indica) leaves extract on the inhibition of streptococcus mutans (in vitro) sri kavi subramaniam, widowati siswomihardjo, and siti sunarintyas department of biomaterials faculty of dentistry gadjah mada university yogyakarta indonesia abstract the neem plant has a history for treating gum and teeth problems and this plant is used for oral care in india. the active component (azadirachta indica) has been proven to exhibit antibacterial properties. the objective of this study was to determine the influence of different concentrations of neem leaves extract on the inhibition of streptococcus mutans. neem leaves extract at concentrations of 10%, 20%, 40%, 60%, 80%, and 100% was prepared. fifty milliliters of each concentration were dropped into holes of 6 millimeters in diameter on a mha agar that has been inoculated with streptococcus mutans. distilled water was used as a control. after 24 hours of incubation, the inhibition diameters were measured and analyzed. the statistical results of the one-way analysis of variance (anova) illustrated that the different concentrations of neem extract had a significant influence on the inhibition of streptococcus mutans. this was followed with the least significant difference (lsd) which implied that there were significant differences between all the concentrations of neem leaves extract used in this experiment. the conclusion of this study was that neem leaves extract exhibited antibacterial effect towards streptococcus mutans and different concentration of neem leaves extract influenced the inhibition of streptococcus mutans. key words: azadirachta indica, streptococcus mutans, antibacterial correspondence: sri kavi subramaniam, department of biomaterials, faculty of dentistry gadjah mada university. denta ii, sekip utara street yogyakarta, indonesia. introduction dental caries is one of the most common human diseases that affect the vast majority of individuals. samaranayake1 defines caries as the localized destruction of tooth tissue by bacterial fermentation of dietary carbohydrates. among the types present in the oral cavity, the cariogenic bacteria are the ones responsible for dental caries.1 the most common and most destructive bacteria are the streptococcus mutans.2 cross sectional and longitudinal epidemiology surveys have implicated streptococcus mutans in the etiology of human dental caries.3 streptococcus mutans can rapidly metabolize sugars to lactic acid and other organic acids, reducing ph and initializing enamel demineralization.4 inhibiting the growth of the streptococcus mutans in the oral cavity would lead to healthier teeth and gums.1 manson and aley4 also states that with the current soft civilized diet, teeth wear is slight or absent, thus encouraging bacterial deposition.2 as the public awareness for oral hygiene increases, many people are now turning to traditional medicine for a solution. the neem tree originates from northeast india.5 it is also known as margosa or the persian lilac.6 in indonesia, this plant is referred to as mimba.7 over centuries, this plant has provided leaves, seed oil and barks as a range of healing properties.8 in india this plant is referred to as the village pharmacy because of its ability to cure many disorders ranging from bad teeth and bed bugs to ulcers and malaria.9 the neem is of particular interest to the field of dentistry for it has a long history treating teeth and gum problems.9 in rural areas of india, the twigs are used as toothbrush to prevent gingivitis.10 in a study to determine the most effective method for reducing plaque formation and the level of bacteria on tooth surface, it was found that micro-organisms in inflamed gums are resistant to penicillin (44%) and tetracycline (30%) but were not resistant to antibacterial plant extracts like the neem.11 in another report by the ucla school of dentistry, it was found that neem could reduce the ability of streptococcal bacteria to colonize on the surface of teeth, thus providing an explanation for neem's long-standing reputation as a cavity fighter.12 from the above, it is deduced that although the anti-bacterial effect of neem has been proven, there is still a grey area concerning its effective concentration against the streptococcus mutans specifically. the objective of this study is to determine the effectiveness of the neem leaves extract as an anti-bacterial against the growth of streptococcus mutans. materials and method this was a laboratory experimental study. the extractions of neem leaves extract were carried out at the traditional medicine research center, gadjah mada university. the materials used in this experiment are neem 177subramaniam, et al.: the influence of different concentration of neem leaves aqueous extract (10%, 20%, 40%, 80%, and 100%), distilled water, brain heart infusion (bhi) as a culture medium, muller hinton agar (mha) and cultured streptococcus mutans. among the instruments used are petri dishes (10 cm in diameter), test tubes, test tube rack, and water bath, sliding calipers, sterile loop, incubator, autoclave and micropipettes. one kilogram of matured and complete neem leaves is selected. one litre of distilled water was added to the leaves and then it was blended in the blender. the mixture was put through a buchner filter to separate the filtrate and the residue. the filtrate was then evaporated over a water-bath at 60° c to obtain concentrated extract. the extract was then ready for dilution.13 based on previous experiments carried out by satya14 and pramularsih,15 in this study the concentrations of neem leaves extract used are 10%, 20%, 40%, 80%, and 100%. distilled water was used to dilute the neem leaves extract and the various concentration of neem leaves were prepared by the mass of extract per volume. the dilution details are presented below: group a: for the control only 100 ml of distilled water; group b: for 10%, 10 mg of neem leaves extract and distilled water added little by little to make the volume 100 ml; group c: for 20%, 20 mg of neem leaves extract and distilled water added little by little to make the volume 100 ml; group d: for 40%, 40 mg of neem leaves extract and distilled water added little by little to make the volume 100 ml; group e: for 80%, 80 mg of neem leaves extract and distilled water added little by little to make the volume 100 ml; group f: for 100%, 100 mg of neem leaves extract and distilled water added little by little to make the volume 100 ml. the bacterial sensitivity test was carried out to determine the antibacterial effect of the neem leaves extract. in this experiment the bacterial sensitivity test was carried out using the diffusion techniques.16 this technique was used by most laboratories to test routinely for bacterial sensitivity. the sample of streptococcus mutans strain for this experiment was obtained from the microbiology department of the veterinary faculty, gadjah mada university. the streptococcus mutans was cultured on mueller hinton agar. after 24 hours of incubation at 37° c, 5 colonies were transferred into 2 ml of brain heart infusion (bhi). its turbidity was compared to the standard brown iii solution. using a sterile loop of about 4 mm in diameter, the suspension was inoculated three times on the centre of a mha plate. a sterile cotton wool pad is then used to spread the inoculum evenly over the plate. six holes were then punctured on the mha each six milimetres in diameter. fifty microlitres extract of each concentration was dripped into holes. for the control, fifty microlitres of distilled water was used. similarly, two other petri dishes were prepared as well. after incubation for 24 hours at 37° c, the inhibition zones are measured with a sliding caliper. after 24 hours of incubation, the petri dishes were observed for inhibition zones around each hole. this translucent area around the hole where there was no streptococcus mutans growth is also referred to as the radical zone. the required area was from the edge of the hole to the outer border of bacterial inhibition. the diameter is measured using a sliding caliper with a precision of 0.01 mm. each measurement was taken three times to ensure higher accuracy. the horizontal diameter of the inhibition zone was measured, followed by the vertical diameter and finally the diameter between the first two lines at an angle of 45°. the average of the three measurements for each zone was recorded. the procedure was repeated for all three petri dishes. the results obtained were then systematically documented in a form of a table. statistical analysis of the data was carried out using the one-way analysis of variance (anova). in this test, the calculated f ratio was compared to the value from the f distribution table at a confidence level of 95%.17 anova is to see if the different concentrations and control have a significant influence on the inhibition of streptococcus mutans. the anova was then followed by a post-hoc comparison, in this case, the least significant difference (lsd). lsd was used to further speculate if there was a significant difference between the various concentrations.17 results a line diagram is plotted to see the relationship between the inhibition diameters and extract concentrations. the chart shows that the increase of streptococcus mutans inhibition diameter corresponds with the increase of neem leaves extract concentration. the diagram representing these results is presented in figure 1. the data is first analyzed with the normality tests. the results of the test show that the probability is 0.85. this value is greater then 0.05, thus implying that the acquired data is normally distributed (table 1). 0 2 4 6 8 10 12 14 10 20 40 80 100 consentration of neem leaves extract (%) d ia m et er o f in hi bi to n zo ne (m m ) figure 1. relationship between the inhibition diameters of streptococcus mutans and neem leaves extract. 178 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 176–179 table 1. statistical results of the normality test kolmogorov-smirnov df sig radical zone diameters 0.190 54 0.850 the normality test is then followed by the anova. in this test, the calculated f ratio is compared to the value from the f distribution table at a confidence level of 95%. the anova speculates if the different concentrations of neem leaves extract have a significant influence on the inhibition of streptococcus mutans. the results of this analysis are presented in table 2. table 2. statistical results of the one-way analysis of varience (anova) source ss df ms f p between groups 1255.568 5 251.114 6495.874 0.001 within groups 3328.267 48 0.038 – – total 68983.000 53 – – – the results of the anova show that the probability is 0.001; this value is less than the 0.05 confidence level. the interpretations of the anova illustrates that the different concentration of neem leaves extract has a significant influence on the inhibition of streptococcus mutans. to further speculate if there is a significant difference between the various concentrations, the anova test is then followed by a post-hoc comparison, the least significant difference (lsd). these results imply that there are significant differences between all the concentrations of neem leaves extract used in this experiment (table 3). table 3. least significant difference (lsd) statistical results between concentration mean difference sig 10% and 20% 10% and 40% 10% and 80% 10% and 100% 20% and 40% 20% and 80% 20% and 100% 40% and 80% 40% and 100% 80% and 100% 1.7556 5.2000 9.0667 9.3111 3.4444 7.3111 7.5556 3.8667 4.1111 0.2444 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.011 discussion the results of this study exemplifies that the neem leaves extract has antibacterial activity towards streptococcus mutans. the inhibition area that is formed is larger as the concentration of neem leaves extract is increased. the statistical analysis carried out using the anova proves that the different concentrations have significant influence on the growth of streptococcus mutans. these results also coincide with the results in the research of satya14 and pramularsih.15 satya14 documented a study using neem leaves to inhibit the growth of bacillus subtilis and e. coli. pranularsih15 on the other hand studied the influence of neem leaves extract in the inhibition of staphylococcus aureus and salmonella typhi. although the bacteria used in the both the experiment are different, all the results correspond with the theory that azadiachtin the active component of neem is of the phenol group and that phenols substances can destroy bacterial cell walls which will inevitably inhibit the growth of bacteria.18 the breakdown of cell wall disturbs osmotic pressure and leads to cell death.19 the statistical analysis summarized in table 2 proves that the different concentration of neem leaves extract have a significant influence on the growth of streptococcus mutans. in table 3, the post hoc test shows that the probability between the concentration of 80% and 100% is 0.011. although this value is statistically significant, it is higher that the probability of all the other concentration. the different value confirms that there is less statistical difference between the concentration of 80% and 100%. this gives evidence that the effectiveness of neem leaves extract on the streptococcus mutans at concentration 80% and 100% is clinically almost the same. nevertheless, it is still not viable to determine the effective concentration of the neem leaves extract to inhibit the growth of streptococcus mutans. the effective concentration is the concentration where beyond that value the inhibition diameter is constant or decreases. in this experiment it can only be approximated that the effective concentration is above 80%. the value is indistinct hence, future experiments with a narrower range are necessary to determine the effective concentration of neem leaves extract to inhibit the growth of streptococcus mutans. in this experiment, the neem leaves are extracted using distilled water as the dissolvent, therefore the active component that is extracted is less. the use of organic dissolvent such as ethanol is more effective because theoretically the active component belongs to the phenol group and phenols would dissolve better in organic dissolvent.11 ethanol extractions may also be able to show the effective concentration of neem leaves extract in the inhibition of streptococcus mutans which could not be determined in this experiment. however, ethanol extractions are more complicated and expensive, therefore in this study, distilled water is used as it is more economical. from this study it can be concluded that the neem leaves extract has antibacterial properties towards the streptococcus mutans. this could be further developed as an alternative method to prevent caries. in this experiment, the extracting method has been kept simple without the use of ethanol and sophisticated methods. the experiment 179subramaniam, et al.: the influence of different concentration of neem is performed keeping in mind that the ultimate aim is to find a cheaper and safe method to curb caries in developing countries. references 1. samaranayake lp. essential microbiology for dentistry. london: churchill livingstone; 2002. p. 121–6. 2. marsh p, martin mv. oral microbiology. 4th ed. usa: reed educational and professional, ltd; 1999. p. 82–100. 3. willet pn, white rr, rosen s. essential dental microbiology. new jersey: appleton and lange: prentice hall inter inc; 1991. p. 157–9. 4. manson jd, eley bm. outline of periodontics. 4th ed. usa: a division of the reed educational and professional, ltd; 2000. p. 318–9. 5. puri hs. neem the divine tree (azadiracthta indica). netherlands: harwood academic publishers; 1999. p. 182. 6. backer ca, bakhuizen, brink rc. flora of java. volume 2, nvp nordhoff, gronigen the netherlands: nvp nordhoff. 1965. p. 120. 7. anonym. medicinal herbs index in indonesia. 2nd ed. jakarta: pt eisai indonesia; 1995. p. 168. 8. duke ja. chemicals and the biological activities in azadirachta. 1992. available at: http: //www.ars-grin.gov/cgi_bin/duke/ pharmacy/scroll.3.pl. accessed december 15, 2004. 9. narula as. neem: the tree of 1000 uses, alpha omega labs. 1997. available at: http: //www.altcancer.com./neem.1000.htm. accessed january 10, 2005. 10. mccaleb r. neem the ancient herb from india.1986. available at: http: //www.altcancer.com./images/. accessed december 20, 2004. 11. wolinsky le, mania s, nachnani s, ling s. the inhibiting effect of aqueous azadirachta indica (neem) extract upon bacterial properties influencing in-vitro plaque formation. j dent res 1996; 75:816–22. 12. vanka a, tandon s, rao sr, udupa n, ramkumar p. the effect of indigenous neem (azadirachta indica) mouth wash on streptococcus mutans and lactobacilli growth. indian j dent res 2001; 12(3):133–44. 13. wibowo s. efektivitas antimalaria ekstrak biji mimba (azadirachta indica a.juss) pada mencit (swiss mice). skripsi. yogyakarta: fakultas farmasi universitas gadjah mada; 1990. h. 7–17. 14. satya rcd. daya antibakteri terhadap bacillus subtilis dan e. colii daun dan kulit ranting tanaman mimbo (azadirachtin indica) serta profil kromatrografi lapis tipis dari fraksi aktif. skripsi. yogyakarta: fakultas farmasi, universitas gadjah mada; 2000. h. 12–16 15. pramularsih ed. uji aktivitas antibakteri daun mimba (azadirachtin indica) terhadap staphlylococcus aureus and salmonella typhi berserta profil kromatografi lapis tipisnya. skripsi. yogyakarta: fakultas farmasi, universitas gadjah mada. 2001. h. 9–15. 16. cheesbrough m. medical laboratory manual for tropical countries. volume ii. england: tropical health technology; 1984. p. 128. 17. dawson b, saunders, trapp rg. basic and clinical biostatistics. 2nd ed. norwalk, connecticut: appleton and lange; 1994. p. 97. 18. trewari dn. monograph on neem (azadirachta indica). dehra dun, india: int. book distributors; 1992. p. 179. 19. robinson t. the organic constituens of higher plants. 6th ed. bandung: penerbit itb; 1995. p. 191–2. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 120120 saliva analysis in children with active caries before and after dental treatment ebru akleyin1, cansu osmanoğulları sarıyıldız2, i̇zzet yavuz3, i̇smet rezani toptancı1 1 department of pediatric dentistry, faculty of dentistry, dicle university, diyarbakır, turkey 2 diyarbakır oral dental health center, diyarbakır, turkey 3 department of pediatric dentistry, faculty of dentistry, harran university, urfa, turkey abstract background: the amount and quality of saliva play important roles in maintaining an intraoral bacterial balance. the quality of saliva is defined by its buffering capacity, viscosity, ph and protein content. the amount of saliva is usually related to the flow rate. purpose: this study aimed to compare the flow rate, ph, viscosity and buffering capacity of saliva as well as plaque formation in children before and after dental treatment. methods: saliva samples were taken from paediatric patients before their treatments and one month after their dental treatments had ended, and these saliva samples were then analysed. for each sample analysis, the gc saliva-check buffer kit (gc corporation, tokyo, japan) was used to evaluate buffering capacity, ph and flow rate, and the gc saliva-check mutans kit (gc corporation, tokyo, japan) was used for the determination of streptococcus mutans. gc tri plaque id gel (gc corporation, tokyo, japan) was applied to evaluate plaque maturation. results: the pre-treatment buffering capacity, ph and viscosity sample values were found to be significantly lower than the post-treatment values (p<0.05). no statistically significant difference was determined in the amount of saliva preand post-treatment (p>0.05). when examining plaque maturation, it was determined that all of the post-treatment plaque was pink. conclusion: this study showed that the ph, viscosity and buffering capacity of saliva had increased significantly post-treatment and that the formation of plaque had decreased in children with active caries after all their dental treatments had been completed. keywords: child; dental treatment; saliva analysis; gc saliva-check buffer correspondence: ebru akleyin, department of pediatric dentistry, dicle university, sur, diyarbakır, 21010, turkey. e-mail: dt.eakleyin@gmail.com introduction fighting tooth decay, a common disease for dental, is vital as it impacts people’s social lives.1 dental decay is an infectious and multifactorial disease that develops through the proliferation and colonisation of bacteria inside the mouth and through the interaction of diet and host factors over time.2 saliva enables oral functions such as chewing, swallowing and talking by wetting the tissues inside the mouth.3 saliva has a high diagnostic potential for the study of human pathologies. the ph, circulation rate, calcium content and microbial profile of saliva can be used to predict the risk of developing dental caries.4,5 high saliva quantity and quality are essential in balancing demineralisation and remineralisation of the enamel in a cariogenic environment. specific changes, such as increases in ph, buffering capacity and flow rate, can contribute to decreased sensitivity to dental decay.6 after the intake of sugar-containing foods, cariogenic bacteria decrease the ph in dental plaque, causing demineralisation of the teeth. the neutralising effect of salivary flow, the buffering capacity of bicarbonate and the impact of salivary proteins on microorganisms’ movements on dental plaque are essential for the prevention of dental caries.7 bacterial species associated with dental caries have been detected in higher rates in the saliva of children with severe dental caries.8,9 kits such as the gc saliva-check buffer kit (gc corporation, tokyo, japan) can be used to determine the saliva buffering capacity to evaluate the risk of decay. dental journal (majalah kedokteran gigi) 2022 september; 55(3): 120–124 original article dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p120–124 mailto:dt.eakleyin@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p120-124 121 akleyin et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 120–124 this kit works on the principle of reverse titration.10 monoclonal antibodies specific to streptococcus mutans (s. mutans) have recently been used to determine the number of s. mutans in saliva, and by using the gc salivacheck buffer kit, the antigen–antibody reaction can be evaluated in only 15 minutes.11 this study analyses and compares saliva samples taken before and after ten paediatric patients with dental complaints had completed their dental procedures (fillings, extractions, root canal treatments, fissures and fluoride applications). in contrast to studies on the effect of caries on saliva, the aim of this study is to evaluate whether improved oral hygiene and health in children affect saliva flow, ph, buffering capacity and plaque maturation. materials and methods children between the ages of seven and eight who had presented to the dicle university, faculty of dentistry, department of pediatric dentistry were included in this study. specifically, paediatric patients who did not have any systemic diseases that may have affected saliva flow and who had not used any medication in the last four weeks were included in the study. information was given to each child and their parents about the study, and a consent form was obtained. the patient’s age, gender, brushing frequency and dental caries index values (dmft) were recorded in the information form prepared for the patients. in the first session, the correct method of brushing teeth was shown. the world health organization recommends using the dmft index (total number of decayed, extracted and filled teeth due to caries) to assess the status of caries in permanent teeth. the dmft index was used to assess the children’s primary teeth. pre-treatment saliva samples were obtained from fifty children with active caries with a dmft index of five and above. dmtf indexing and all treatments were performed by two paediatric dentists. the treatments could only be completed in ten (10) patients. saliva samples were retaken one month after the completion of treatment, which had consisted of fillings, root canal treatments, extractions, periodontic treatments and protective applications. the patients were instructed not to eat, drink, brush their teeth or chew gum for at least one hour before the examination during which the saliva sample was to be collected. the saliva samples were collected between 09.00 and 12.00. each patient rinsed their mouth with distilled water and was then instructed to lean forward and spit into a saliva collection tube for five to seven minutes. then, to stimulate saliva flow, each child chewed a paraffin tablet for five minutes, and the stimulated saliva was then collected. the gc saliva-check buffer kit was used for each saliva analysis to evaluate buffering capacity, ph, viscosity and flow rate. to determine s. mutans in the saliva, the gc saliva-check mutans kit (gc corporation, tokyo, japan) was used. gc tri plaque id gel (gc corporation, tokyo, japan) was applied to evaluate plaque maturation. the applications were made as per the manufacturer’s instructions. a drop of saliva taken from the tube was dropped onto each of the three pads on the test strip in the gc salivacheck buffer kit, and the saliva was spread onto the absorbent surface. the wait time was two minutes for an accurate result. the result was then scored by evaluating the colour of each pad: 0–5 points indicated very low (red), 6–9 points indicated low (yellow) and 10–12 points indicated normal/high (green) (figure 1). a saliva sample was collected from each patient when the patient was in a resting state, and the ph level was measured using the ph strips in the gc saliva-check buffer kit. the colour shown on the strip was checked against the colour control chart in the kit. the ph values were evaluated with 5.0–5.8 indicating low, 6.0–6.6 indicating moderate and 6.8–7.8 indicating healthy (figure 1). the amount of saliva was measured in ml as marked on the cup in which the stimulated saliva had been collected. according to the data of lund university faculty of odontology department of cariology, sweden,7 the amount of saliva collected in five mins was evaluated with <3.5 ml indicating very low, 3.5–5.0 ml indicating low and >5.0 ml indicating normal. stimulated saliva at the rate of 1–6 ml is seen as normal in healthy individuals. plaque maturation was evaluated with the application of gc tri plaque id gel. after application, the gel was removed from the surface of figure 1. application of the gc saliva-check buffer kit. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p120–124 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p120-124 122akleyin et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 120–124 the teeth with a small swab. the colouration of the teeth was evaluated with pink or red indicating newly formed plaque, blue or purple indicating plaque of at least 48 hours maturity and light blue indicating mature plaque producing acid (figure 2). the measuring of saliva viscosity was made visually while the patient was resting. sticky foaming saliva was evaluated as high viscosity (red), foaming saliva with bubbles as increased viscosity (yellow) and watery clean saliva as normal viscosity (green). the gc saliva check mutans kit measured the level of s. mutans in saliva within 15 minutes without the need for a special device and bacteria culture (figure 3). one drop of reagent #1 was dropped into the collection cup of stimulated saliva; after ten seconds, four drops of reagent #2 were added, and the mixture was shaken. using a graded pipette, saliva that had turned light green was placed in the window of the test device and left for 15 minutes. the s. mutans level was evaluated as high (>5 x 105 cfu/ml saliva) with the determination of a red line in the control (c) window and as low (<5 x× 105 cfu/ml saliva) if no red line was formed. approval for the study was granted by the ethics committee of dicle university dental faculty (decision no: 2021-26). data obtained in the study were statistically analysed using ibm spss version 21 software. due to unit numbers, the shapiro wilk test was used in the assessment of conformity of the variables to normal distribution. to examine the differences between two dependent categorical variables, the marginal homogeneity test was applied according to the levels. when examining the difference between two dependent variables not showing normal distribution, the wilcoxon test was applied. a value of p<0.05 was accepted as statistically significant in all tests. results saliva buffering capacity was significantly lower after treatment than before treatment (p<0.05) (table 1). no statistically significant difference was determined between the pre-treatment and post-treatment amounts of saliva (flow rate) (p>0.05) (table 2). the salivary ph value was determined to be statistically significantly lower before table 1. results of the analysis of the difference between the pre-treatment and post-treatment saliva buffering capacity values saliva buffering capacity n mean ± sd median (min–max) p pre-treatment 10 7.60 ± 2.27 6 (6–12) 0.024* post-treatment 10 9.40 ± 2.12 9 (6–12) *wilcoxon test table 2. results of the analysis of the difference between the pre-treatment and post-treatment saliva amount values [n (%)] saliva amount pre-treatment <3.5 ml 3.5–5 ml >5 ml post-treatment <3.5 ml 0 (0) 0 (0) 0 (0) 3.5–5 ml 1 (50) 5 (71.43) 0 (0) >5 ml 1 (50) 2 (28.57) 1 (100) figure 2. intraoral application of gc tri plaque id gel. figure 3. gc saliva-check mutans kit. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p120–124 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p120-124 123 akleyin et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 120–124 treatment compared with the post-treatment value (p<0.05) (table 3). when examining plaque maturation, it was determined that 100% of patients with pink, purple and blue plaque pretreatment had pink plaque after treatment (table 4). all the cases with increased viscosity of saliva pre-treatment were seen to have returned to normal viscosity post-treatment (p<0.05). the presence of s. mutans in the saliva was determined to be positive in all of the patients before and after treatment. discussion saliva is a complex secretion that has a significant protective effect against tooth decay through its buffering capacity, cleansing ability, antibacterial effect and its ability to preserve calcium and phosphate levels. it can also be used as a diagnostic fluid.12 many studies in the literature have analysed and compared saliva samples taken from children who have been categorised into two groups, with and without caries.7,12 as a result, our study evaluated salivary flow rate, ph, buffering capacity and viscosity as well as plaque maturation and s. mutans levels in children with active caries (dmtf ≥ 5) before and after dental treatment. the main limitation of this study was that as it was conducted during the covid-19 pandemic, only a small number of patients (10 patients) who had completed treatment could be reached. however, the results the study obtained were invaluable and established that there is a need for further studies with greater numbers of patients in order to obtain more data for more precise results. the amount of saliva flowing into the mouth in one minute is known as the saliva flow rate.13 pyati et al.7 reported in their study that the salivary flow rate had been shown to be significantly reduced (p<0.05) in children with active caries. however, some studies have reported no relationship between caries activity and salivary flow rates.12,14 similar to results reported in published study findings, no statistically significant difference was found regarding the saliva flow rate. however, this result could be attributed to the study group not including patients with systemic salivary gland disease that can cause hyposalivation or xerostomia, but only healthy children who had not used any drugs such as antidepressants, antihistamines, diuretics or narcotics, which can reduce saliva flow, for at least four months. the buffer capacity of saliva is a key factor in caries prevention. when the ph level in the mouth falls below the critical ph value of 5.5, inorganic tooth matter may dissolve. the presence of bicarbonate in saliva neutralises acid formation in the mouth and dissolves it into dental plaque.15 therefore, in this study, it was predicted that the ph and buffer capacity of saliva that were found to be low in children with active caries would have an important roles in the formation of caries in children’s teeth, which can be detected by the saliva-check buffer kit method. the results of this study determined that salivary ph and buffering capacity in children with dental caries increased significantly after treatment compared with pre-treatment values. these findings were similar to many previous studies.16–18 bagherian and asadikaram19 concluded that children who had not had early childhood caries (ecc) had higher salivary ph levels and better buffer capacity than children with ecc and children without current ecc. pyati et al.7 reported that in 50 children with active caries (dmfs/ dfs ≥ 5) and 50 children without caries (dmfs/dfs=0) aged between six and twelve, buffering capacity, salivary flow rates and ph levels were significantly lower (p<0.05) in children with active caries. in light of these results, it is clear how important the results of our study are. plaque accumulation increases the risk of dental caries. plaque is a thin layer on the tooth surface that contains a bacterial community. utami20 reported that dental plaque is a risk factor for the severity of dental caries in preschool students. the risk of dental caries is 3.3 times higher in children with high dental plaque than in children with low dental plaque. in this study, the finding that 100% of the plaque scores improved after dental treatment was thought to be due to improved oral hygiene post-treatment. karabekiroğlu et al.21 reported in their study that although no significant correlation was found between saliva viscosity and the risk of decay, they were observed that patients with less intense saliva viscosity had lower table 4. results of the analysis of the difference between the pre-treatment and post-treatment plaque scores [n (%)] plaque score value pre-treatment pink purple blue purple-blue post-treatment 2 (100) 5 (100) 1 (100) 2 (100) 2 (100) 5 (100) 1 (100) 2 (100) table 3. results of the analysis of the difference between the pre-treatment and post-treatment saliva ph values saliva ph n mean ± sd median (min–max) p pre-treatment 10 6.89 ± 0.44 6.8 (6.4–7.6) 0.007* post-treatment 10 7.56 ± 0.23 7.6 (7.2–7.8) * wilcoxon test dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p120–124 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p120-124 124akleyin et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 120–124 dmtf-dmfs values than patients with more intense saliva viscosity. voelker et al.22 found no significant relationship between saliva viscosity and decay in the saliva analyses of 53 patients, but reported that dense saliva consistency did not help in the removal of plaque from the teeth. the results of this study demonstrated that saliva viscosity returned to normal after dental treatment. further largerscale studies would be needed in order to better understand the relationship between saliva viscosity and decay. s. mutans is one of the most important bacteria in the formation of cavities in children. recent studies have shown that a colony variation of s. mutans can form in the oral cavity at the age of 3-6 months with the horizontal transmission. the basic defence against s. mutans is provided by immunoglobulin a in the saliva, serum and gingival groove fluid.23 dogra et al.24 reported that the number of s. mutans was significantly higher in children with active decay in the 7–14 age. in this study, all the patients had s. mutans positivity before and after treatment. the advantage of using the s. mutans kit is that after adding the samples to the device, the results are obtained in 15 minutes, and the kit can therefore be easily utilised in daily practice without having to use a laboratory. however, that the same result emerges for moderate risk progressing to high risk seems to be a disadvantage of the s. mutans kit. the ready availability and positive correlations between the components of saliva are the most important advantages to using saliva as a diagnostic tool. in this study, after dental treatment and oral hygiene education, it was observed that the ph, viscosity and buffering capacity levels of the saliva in all ten children with active caries had increased significantly and that the formation of plaque had decreased. references 1. seredin p, goloshchapov d, ippolitov y, vongsvivut p. pathologyspecific molecular profiles of saliva in patients with multiple dental caries-potential application for predictive, preventive and personalised medical services. epma j. 2018; 9(2): 195–203. 2. kılınç g, çetin m, ellidokuz h. the relationship of salivary flow rate and salivary ph on dental caries in children. j pediatr res. 2015; 2(2): 87–91. 3. anu v, madan kumar pd, shivakumar m. salivary flow rate, ph and buffering capacity in patients undergoing fixed orthodontic treatment a prospective study. indian j dent res. 2019; 30(4): 527–30. 4. gao x, jiang s, koh d, hsu c-ys. salivary biomarkers for dental caries. periodontol 2000. 2016; 70(1): 128–41. 5. guo l, shi w. salivary biomarkers for caries risk assessment. j calif dent assoc. 2013; 41(2): 107–9, 112–8. 6. schipper rg, silletti e, vingerhoeds mh. saliva as research material: biochemical, physicochemical and practical aspects. arch oral biol. 2007; 52(12): 1114–35. 7. pyati sa, naveen kumar r, kumar v, praveen kumar nh, parveen reddy km. salivary flow rate, ph, buffering capacity, total protein, oxidative stress and antioxidant capacity in children with and without dental caries. j clin pediatr dent. 2018; 42(6): 445–9. 8. belstrøm d, sembler-møller ml, grande ma, kirkby n, cotton sl, paster bj, twetman s, holmstrup p. impact of oral hygiene discontinuation on supragingival and salivary microbiomes. jdr clin transl res. 2018; 3(1): 57–64. 9. skelly e, johnson nw, kapellas k, kroon j, lalloo r, weyrich l. response of salivary microbiota to caries preventive treatment in aboriginal and torres strait islander children. j oral microbiol. 2020; 12(1): 1830623. 10. cheaib z, ganss c, lamanda a, turgut md, lussi a. comparison of three strip-type tests and two laboratory methods for salivary buffering analysis. odontology. 2012; 100(1): 67–75. 11. wennerholm k, emilson c-g. comparison of saliva-check mutans and saliva-check iga mutans with the cariogram for caries risk assessment. eur j oral sci. 2013; 121(5): 389–93. 12. jamal abbas m, khairi al-hadithi h, abdul-kareem mahmood m, mueen hussein h. comparison of some salivary characteristics in iraqi children with early childhood caries (ecc) and children without early childhood caries. clin cosmet investig dent. 2020; 12: 541–50. 13. battino m, ferreiro ms, gallardo i, newman hn, bullon p. the antioxidant capacity of saliva. j clin periodontol. 2002; 29(3): 189–94. 14. bilyschuk l, keniuk a, goncharuk-khomyn m, yavuz i. association between saliva quantity and content parameters with caries intensity levels: a cross-sectional study among subcarpathian children. pesqui bras odontopediatria clin integr. 2019; 19(1): 1–10. 15. guy s. general practice | children’s caries history predicts future tooth decay | medwirenews.com. 2012. available from: https://www. medwirenews.com/general-practice/family-medicine/children-scaries-history-predicts-future-tooth-decay/112758. accessed 2022 may 17. 16. sarode g, shelar a, sarode s, bagul n. association between dental caries and lipid peroxidation in saliva. int j oral maxillofac pathol. 2012; 3(2): 2–4. 17. animireddy d, reddy bekkem vt, vallala p, kotha sb, ankireddy s, mohammad n. evaluation of ph, buffering capacity, viscosity and flow rate levels of saliva in caries-free, minimal caries and nursing caries children: an in vivo study. contemp clin dent. 2014; 5(3): 324–8. 18. pop as, campian rs, jiman p, ionescu e, milicescu s, teodorescu e, pacurar m, bechir es, mola fc, tarmure v. correlations between ph values of oral fluid and dental caries epidemiologic indicators in children aged within 6-12 years. rev chim. 2018; 69(2): 484–7. 19. bagherian a, asadikaram g. comparison of some salivary characteristics between children with and without early childhood caries. indian j dent res. 2012; 23(5): 628–32. 20. utami s. the relationship between dental plaque and the severity of dental caries among preschool children. ind dent j. 2013; 2: 9–15. 21. karabekiroğlu s, gönder hy, çayır i, ünlü n. the effect of different etiologic factors on caries experience in young adults with high caries risk. necmettin erbakan univ dent j (neu dent j). 2020; 2(3): 103–12. 22. voelker ma, simmer-beck m, cole m, keeven e, tira d. preliminary findings on the correlation of saliva ph, buffering capacity, flow, consistency and streptococcus mutans in relation to cigarette smoking. j dent hyg jdh. 2013; 87(1): 30–7. 23. baltacı e, baygın ö, korkmaz fm. early childhood caries: a literature review. turkiye klin j dent sci. 2017; 23(3): 191–202. 24. dogra s, bhayya d, arora r, singh d, thakur d. evaluation of physio-chemical properties of saliva and comparison of its relation with dental caries. j indian soc pedod prev dent. 2013; 31(4): 221–4. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p120–124 https://www https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p120-124 126 vol. 42. no. 3 july–september 2009 storage duration effect on deformation recovery of repacked alginates siti sunarintyas and dyah irnawati department of dental biomaterials faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: manufacturers supply alginate impression materials as a powder that is packaged in bulk and in individual container. some indonesian dental suppliers often repackage the bulk alginate into individual plastic packages which are not tied tightly and stored in the display room without air conditioner. it is known that critical factors to the shelf life of alginate includer avoidance of moisture contamination which may lead to premature setting of the alginate and avoidance of high temperature which may cause depolymerization of the alginate. purpose: the aim of this study was to determine storage duration effect of repacked alginates on deformation recovery. methods: two brands of alginates (tulip®tu, and aroma fine df iii®af) were repacked into 120 plastic containers. the samples were stored in room condition (temperature 29° c ± 1° c, relative humidity 60% ± 10%) for 1, 2, 3, 4 and 5 weeks. the alginates setting time and recovery from deformation were measured according to the ansi/ada specification number 18 (iso 1563). result: the results revealed that there was decreased setting time during 5 weeks but there was slight decreased in deformation recovery after 3 weeks storage. the anova showed there was no significant difference of alginates deformation recovery among the storage times (p > 0.05). conclusion: storage duration of repacked alginates in plastic containers during 5 weeks in room condition do not influence the alginate deformation recovery. key words: storage time, alginates, deformation recovery correspondence: siti sunarintyas, c/o: bagian biomaterial kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara, yogyakarta 55281, indonesia. e-mail: sunarintyassiti@yahoo.com, phone/fax no. 062-274-515307. research report introduction alginate is the most widely used dental impression material. the wide use of alginates relates to the ease of mixing and manipulating the material, the flexibility, the accuracy, and the relatively inexpensive comparing to other impression materials.1 the disadvantages of alginates are that they do not transfer as much surface details to gypsum dies as agar or rubber impressions do, and the storage stability is a critical characteristic for them.2 the physical, mechanical, and chemical properties of alginates may be affected by the period of time they are stored before use and the conditions under which they are stored.3 alginates are supplied by manufacturers as powder that are packaged in bulk or in individual container. the bulk materials are packaged in a sealed screw-topped plastic container or in a hermetically sealed metal can, such as one used to package coffee. the pre weighed packages are constructed of plastic and metal foil and contain enough material for a single full-arch impression.4,5 the bulk alginate is cheaper than the individual manufacturer package. the dental suppliers often repackage the bulk one into individual plastic packages in order to be able to sell more alginates. the consumers tend to buy the repacked one because the cost is lower than the bulk one or the manufacturer pre weighed one. the repacked alginates are supplied in plastic bags which are tied in their one end. these repacked are not legibly marked with the information of manufacturer’s name, initial setting time, storage conditions, expiry date, minimum net mass of the contents, and batch number as the ansi/ada specification number 18 recommendation.6 these repacked were often stored by the dental supplier in their display case for one up to five weeks until all the 127sunarintyas and irnawati: storage duration effect of repackaging alginates are sold. these plastic repacked alginate do not seem to have the property of being tightly closed container to protect the alginate from moisture contamination. critical factors to the shelf life of alginate include avoidance of moisture contamination, which may lead to premature setting of the material in the container, and avoidance of high temperature, which may cause depolymerization of the alginate.7 alginate powder is unstable in a storage with presence of moisture or in warm temperatures over 23° c. storaging alginate powder in room temperature of 65° c for 1 month could make the alginate deteriorate and could not be used as impression material as the setting time was faster than usual.4 storage of alginate in a tropical country with temperature around 27–30° c and relative humidity of 50–70% without air conditioner for a long time is risky.8 storage-dependent deterioration may have detrimental effects on the clinical usefulness of alginate. since the set alginate is held between the impression tray and the tissue, it is important to know the extent of any recovery from deformation during the removal of the impression from the mouth. the purpose of this study was to determine storage duration effect of repacked alginates on recovery from deformation. materials and methods the materials used in this study were two brands of bulk dental impression material (tulip® tu, and aroma fine df iii® af), plastic bag, and tap water. the equipments used were analytical balance, measurement glass, rubber bowl and spatula, stopwatch, room thermometer, hygrometer, setting time and recovery from deformation apparatus (as indicated in the ansi/ada specification number 18),6 water bath, and flat glass plate. bulk dental impression materials were repacked in plastic bags. the alginates which were packaged in a sealed screw-topped plastic container were opened and weighed of 6.5 gram to be stored in 120 plastic bags. the air in the plastic bag was removed by pressing both of the plastic end sides outward, and then the plastic end was tied. the packages were placed in an opened plastic box and stored in the display case. the samples were stored in room condition (temperature 29° c ± 1° c, relative humidity 60% ± 10%) for 1, 2, 3, 4, and 5 weeks. the alginates recovery from deformation were measured according to the ansi/ada specification number 18 (iso 1563). the control group was alginate without storage.6 the deformation measurement needed the initial setting time data of the repacked alginates. the setting time measurement was done by a poly (methyl methacrylate) cylindrical test rod of 10 cm long and 6.35 mm in diameter. the sample ring mould was overfilled with repacked alginate powder and water mixed, then stroke off even with the top of the mould. immediately thereafter, the end of the test rod was placed into momentary contact with the unset material. the test rod was withdrawn and cleared it off any material left from the contract. the contact and withdrawal steps were repeated at 10 second intervals until the rod was separated cleanly from the material. the recovery from deformation measurement needed the deformation apparatus: split mould with fixation ring, flat glass plates, water bath and c-clamps. there were two steps in measuring the deformation: the preparation of the test specimen and the measurement procedure. in preparation of the test specimen, the fixation ring was placed on a glass plate and filled it slightly more than one-half full with alginate material mixed in accordance with the manufacturer’s instructions. the split mould was pressed into the fixation ring until the bottom of the mould touched the glass plate and alginate extruded above the top of the mould. the second plate was clamped over the mould to force away the excess alginate and to form the upper surface of the specimen. thirty seconds after the end of mixing, the split mould assembly and its accompanying plates were placed and fixed by a c-clamp in the water bath maintained at 35±1° c. at the initial setting time that had been measured, the assembly was removed from the water bath. after removing the flask, the specimen was separated from the split mould assembly. the flat plate was centered on the top of the specimen. the specimen was placed on the table of the deformation apparatus. the deformation measurement was done following the criteria in table 1 (t was the initial setting time obtained). the obtained alginates recovery from deformation data were calculated as a percentage using the following formula:6 100 (1a-b/20) where 20 was the length of the mould in millimeters. the data were analyzed by two ways analysis of variance (anova). table 1. the alginate recovery from deformation measurement time test procedure t + 45 s t + 55 s t + 60 s t + 90 s t + 100 s the spindle of the dial indicator was gently lowered so that it came into contact with the plate on the specimen. the dial indicator was read, the value was recorded as reading a, and the spindle was fixed in the up position. the specimen was deformed to height of 16 mm±0.1 mm within 1 s and maintained this deformation for 5 s ± 0.5 s, then the deforming force was released. the spindle of the dial indicator was gently lowered so that it came into contact with the plate on the specimen. the dial indicator was read and the value was recorded as reading b. 128 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 126-129 result the average of setting time and recovery from deformation of repacked alginates in various storage duration were shown at table 2. the results revealed the decreasing in setting time during 5 weeks but slight decreasing in recovery from deformation after 3 weeks storage. the two-ways anova (table 3) showed there was not any significant difference of alginates recovery from deformation among the storage duration (p > 0.05). there was a significant difference between brands and also among brand and storage duration (p < 0.05). discussion storage stability or shelf life was a critical property of perishable dental materials. two major factors that affected the shelf life of alginate impression materials were storage temperature and moisture contamination from ambient air.9,10 table 2 revealed that during 5 weeks storages there was a trend toward a shorter setting time in both of the alginate brands and decreasing in recovery from deformation during 3 weeks storage. those phenomena may be because of partial spontaneous polymerization of the alginate material in the plastic bags, perhaps prompted by moisture contamination from relative humidity during storage. repacked bulk alginates by the plastic bags which were tied in their one end did not seem to protect the alginate powder from the environmental humidity. the presence of water in the alginate powder caused a chemical reaction that cross linked the polymer chain, so formed a three-dimensional polymer network structure. as some of the repacked alginate powder had changed into the polymer networks, the setting time measurement of the material became shorter. research on material containers11 revealed that aluminum foil package became the best container followed by plastic and paper. plastic container was recommended as the alternative of aluminum container regarding on its thickness. the slight decrease in recovery from deformation of the repacked alginates after 3 weeks storage up to 5 weeks storage may be explained by premature polymerization because fewer sites may be available for calcium crosslinking. as the polymer network formed, viscosity increased, resulting in less elasticity and decreasing in recovery from deformation.12 however, the anova showed that there was not any significant influence of storage duration up to 5 weeks of repacked alginates on recovery from deformation. the recovery from deformation of the alginate was still within the ada limit of 95%.6 this finding may indicate that for most clinical cases, this repacked alginate in plastic container with one end tied (not sealed tightly enough) which was stored by dental supplier for 5 weeks remained efficacious to be used, assuming of course, that the ada limit was not excessively lenient. shelf life study on the physical and mechanical properties of an alginate impression material on exposure to various environmental conditions for more than 78 months revealed that there was an increase in strength and working time and a decrease in recovery from deformation at 30 to 50 months: strength and recovery from deformation then remained constant past 6 years, whereas working time and creep compliance decreased. only the most stressful environmental conditions (heat and humidity) caused spontaneous failure of the material to set.13 another shelf study on the storage effect of non aqueous elastomeric impression materials revealed that there were changes in viscosity, working and setting time, elastic recovery, and creep compliance over 72 months storage period.14 table 2. average of setting time and recovery from deformation of repackaging alginates in various storage duration storage duration tu af setting time (second) recovery from deformation (%) setting time (second) recovery from deformation (%) control 1 week 2 weeks 3 weeks 4 weeks 5 weeks 66.00 ± 2.23 62.80 ± 0.84 59.80 ± 2.28 48.60 ± 0.55 46.60 ± 0.54 45.60 ± 0.52 97.93 ± 0.09 97.95 ± 0.47 97.57 ± 0.72 97.96 ± 0.23 97.73 ± 0.13 97.56 ± 0.19 78.80 ± 1.09 76.40 ± 0.55 76.20 ± 4.15 75.20 ± 3.56 73.60 ± 2.07 67.20 ± 0.83 98.56 ± 0.26 98.27 ± 0.16 98.60 ± 0.39 98.62 ± 1.09 98.55 ± 0.49 98.29 ± 0.24 tu: tulip®; af: aroma fine df iii® table 3. result of two-ways anova of recovery from deformation of repackaging alginates in various storage duration source sum of squares df mean square f sig brand storage duration brand*storage duration error total 4.320 1.402 2.979 10.347 576866.810 1 5 5 48 60 4.320 0.280 0.596 0.216 20.039 1.301 2.764 0.001 0.279 0.028 129sunarintyas and irnawati: storage duration effect of repackaging this recent finding strengthen the above studies as it was proven that 5 weeks storage of repacked alginate in a tropical country did not alter the alginate physical property significantly. combination of elevated temperature and moisture has an adverse effect upon the shelf life of the material.15 tulip® and aroma fine df iii® guaranteed that the alginates were in good quality for 3 years, period provided that the packs were unopened and stored in a cool and dry place.16,17 storage of such a perishable material in tropical country with high humidity without air conditioner needed special attention especially in its container and storage periods. further research on the shelf life of repacked alginates was needed especially on the molecular weight determination to prove the contribution of water from the environment humidity on alginates. the conclusion of the study revealed that the storage duration of repacked alginates in plastic container during 5 weeks in room condition did not significantly influence alginate recovery from deformation. the alginates recovery from deformation remained within the ada specification limit. references 1. craig rg, powers jm, wataha jc. dental materials properties and manipulation. 7th ed. st louis: mosby co; 2000. p. 138–41. 2. gladwin m, bagby m. clinical aspect of dental materials. philadelphia: lippincot williams & wilkins; 2000. p. 100–1. 3. craig rg, powers jm. restorative dental materials. 11th ed. st louis: mosby co; 2002. p. 330–40. 4. combe ec. notes on dental materials. 6th ed. edinburgh: churchill livingstone; 1992. p. 115–25. 5. mccabe jf. anderson’s applied dental materials. 6th ed. new york: blackwell scientific publication; 1987. p. 113–5. 6. council on dental materials, instruments and equipment, american national standard/american dental association (ansi/ada specification no. 18, alginate impression materials); 1992. p. 1–8. 7. noort rv. introduction to dental materials. 1st ed. london: mosby co; 1994. p. 162–5. 8. badan meteorologi dan geofisika. prakiraan cuaca kota propinsi indonesia. available at: http://www.meteo.bmg.go.id/cuacaindo. jsp. accessed january 27, 2007. 9. vanable de, lopresti lr. using dental materials. new jersey: pearson prentice hall; 2004. p. 87–91. 10. klettke t, kuppermann b, ranftl d, hampe r. temperature effect on setting of dental impression materials. iadr/aadr/cadr 85th general session and exhibition 2007 march; p. 110. 11. robi’in. perbedaan bahan kemasan dan periode simpan dan pengaruhnya terhadap kadar air benih jagung dalam ruang simpan terbuka. buletin teknik pertanian 2007; 1: 7–9. 12. ellis b, lamb dj. the setting characteristics of alginate impression materials. brit dent j 1981; 151: 343–6. 13. hondrum so, fernandez jr. r. effect of long-term storage on properties of an alginate impression material. j prosthet dent 1997; 77: 601–6. 14. hondrum so. changes in properties of non aqueous elastomeric impression materials after storage of components. j prosthet dent 2001; 85: 73–81. 15. ferracane jl. materials in dentistry principles and applications. 2nd ed. philadelphia: lippincot williams & wilkins; 2001. p. 173–201. 16. tulip alginate. product information on tulip color switch dust free alginate impression material with color change. available at: http:// www.cavex.nl/3/3_imp_alg_tul_pic.html. accessed december 10, 2006. 17. aroma. aroma fine df iii normal set. available at: http://www. gceurope.com/pid/iii/ifu/g.caroma_fine_dfiii_normal/pdf. accessed january 18, 2007. vol 50 no 4 desember 2017.indd 183183 research report dental journal (majalah kedokteran gigi) 2017 desember; 50(4): 183–187 doi: 10.20473/j.djmkg.v50.i4.p183-187 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg effects of filler volume of nanosisal in compressive strength of composite resin dwi aji nugroho,1 w. widjijono,2 n. nuryono,3 widya asmara,4 wijayanti dwi astuti,5 and dana ardianata1 1dental school, faculty of medical and health science, universitas muhammadiyah yogyakarta 2department of biomaterial, faculty of dentistry, universitas gadjah mada 3department of chemistry, faculty of mathematics and natural science, universitas gadjah mada 4department of microbiology, faculty of veterinary, universitas gadjah mada 5department of electrical engineering and informatics, vocational college, universitas gadjah mada yogyakarta indonesia abstract background: one of the composite resin composition is inorganic filler. the production of inorganic filler materials was highly dependent on non-degradable, and nonrenewable fossil fuels. therefore, natural fibers can be used as substitute for inorganic fillers. one that can be developed is sisal. purpose: this study aimed to determine the effects of nanosisal filler volume on compressive strength of composite resin. methods: in this study, composite resins with nano-sized sisal as filler were manufactured and labeled as nanosisal composites. this research processed sisal fibers into nano size and mixed them with bis-gma, udma, tegdma, champhorquinone (sigma aldrich). nanofiller composite (z350 xt, 3m, espe) was utilized as a control. the 20 samples utilized were divided into 4 groups (each group containing five samples): group a contained nanosisal composite of 60% filler volume, group b, nanosisal composite of 65% filler volume, group c, nanosisal composite of 70% filler volume and group d, nanofiller composite (z350 xt, 3m, espe). samples were 2 mm in diameter and 6 mm in height. the sample was tested for compressive strength using a universal testing machine (utm). data was analyzed by means of a kruskal wallis procedure. results: the mean of the compressive strength of the nanosisal composite 60% was 16.80 mpa; the nanosisal composite 65% was 10.80 mpa, the nanosisal composite 70% was 7.20 mpa and the nanofiller composite was 7.40 mpa. there was a significant difference in data analysis (p = 0.033; p < 0.05). conclusion: in this study, the filler volume of nanosisal influenced the compressive strength of a composite resin and the nanosisal filler volume was recomended at 60%. keywords: nanosisal; composite resin; compressive strength; nanofiller correspondence: dwi aji nugroho, dental school, faculty of medical and health science, universitas muhammadiyah yogyakarta. jl. lingkar selatan, tamantirto, kasihan, bantul, yogyakarta 55184, indonesia. e-mail: dwiajinugrohodrg@gmail.com. introduction composite resin is one of the most commonly used dental fill materials since it has high aesthetic attractiveness compared to other dental fill materials.1 the mechanical properties of composite resin are known to be influenced by filler volume. therefore, the higher the volume of filler, the greater the hardness, stiffness, strength, and resistance against fracture.2 based on the amount of fill material used, composite resin can be classified into traditional composite resin, micro filter composite resin, hybrid composite resin, and nanofil composite resin. nanofil composite resin possesses high aesthetic value since it is easy to polish and produces a shiny dental filling.3,4 composite resin consists of matrix, inorganic filler and a coupling agent. the matrix contained in composite resins plays a role in forming its physiology. inorganic filler is a reinforcing material dispersed within the matrix. http://dx.doi.org/10.20473/j.djmkg.v50.i4.p183-187 184 nugroho, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 183–187 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p183–187 a coupling agent plays a role in combining the matrix and inorganic filler. in addition to these three components, there are others present, namely: activator, pigment, initiator and ultraviolet absorbent.3 the greater the filler volume used in the composite resin, the greater the mechanical strength.5 the volume of filler in the composite resin material is usually between 60–70%.6 filler used in composite resin is derived from inorganic materials such as glass, quartz, and silica since these materials are strong, hard and stable.7 nevertheless, inorganic materials, still demonstrate several weaknesses. first, inorganic materials are produced through energy process heavily dependent on fossil fuels. as a result, the emulsion of pollutants produced by the production of inorganic materials is so high that it is prejudicial to the environment and, by extension, health. second, inorganic materials are also non-degradable, non-renewable and non-recyclable.8,9 therefore, natural fibers are expected to become substitutes for inorganic materials due to their high compressive strength, low weight, and environmentalfriendly relation.10 one such natural fiber that can be developed is nanosisal since its hard fibers are produced from sisal plants (agave sisalana) which are easily cultivated. however, the use of sisal fibers is still limited to marine and agricultural fields, usually being used as rope, yarn, carpets and handicrafts.11 the research reported here focused on the use of nanosisal fibers in manufacturing composite resin by mixing the resin matrix with the nanosisal filler without the use of a coupling agent due to their being organic materials. thus, both can bind without coupling agent. the bond between the nano-sisal and the resin matrix can be considered to be a chemical bond of oh group.12 this study aimed to analyze the effects of the volume of nanosisal fibers used as filler in composite resin on its compressive strength. materials and methods sisal fibers used in this research were obtained from the indonesian crops and fiber research institute (balittas), malang, indonesia (figure 1). they were cut into pieces weighing up to three grams. the fibers were then scoured (alkalized) by soaking in naoh solution at 100ºc for two hours while being agitated with a magnetic stirrer. this treatment was repeated three times. the fibers were then filtered and washed in aquadest, before being bleached using a mixture solution of naoh, h2o2 and aquadest. the bleaching process was carried out at 80ºc for two hours while agitated with a magnetic stirrer. the bleaching process then was repeated four times. after each stage of the bleaching process, the fibers were filtered and washed in aquadest. after the bleaching process, the sisal fibers were processed by means of an ultrasonic machine (cole-parmer ultrasonic processor, model cp 505). the sisal fibers obtained were filtered with fritted glass filter no 1 to remove residual aggregate and then dried in a freeze drier (flex-drytm μpmicroprocessor control, fts systems, inc., usa) in order to obtain solid nanosisal fibers (figure 2). the solid sisal fibers were subsequently observed by means of a scanning electron microscopy (sem) to determine its size. sem analysis showed that the size of the sisal fibers was 143.4–260.3 nm. (figure 2). solid nanosisal fibers was obtained. at the next stage, the solid nanosisal fibers were weighed by a digital balance and separated into samples weighing 0.003 grams (60% filler volume), 0.005 grams (65% filler volume), and 0.007 grams (70% filler volume). each sample was mixed with 0.5 grams of bis-gma (bisphenol a glycerolate dimethacrylate, sigma aldrich), 0.02 ml of tegdma (triethylene glycol dimethacrylate, sigma aldrich), 0.02 grams of udma (diurethane dimethacrylate, sigma aldrich) and 0.09 grams of champorquinone (sigma aldrich) to obtain a nanosisal composite dough. this was then placed into a cylindrical-shaped mold 6 mm in height and 2 mm in diameter in accordance with iso 4049 guidelines.13 the materials were mixed on a glass plate using a stainless steel spatula. thereafter, the samples were figure 1. sisal fibers figure 2. sem analysis of the nanosisal fiber (the marked image) with a magnification of 50.000. http://dx.doi.org/10.20473/j.djmkg.v50.i4.p183-187 185185nugroho, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 183–187 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p183–187 radiated with a visible light cure (led curing unit, lyb200, s&d dental international co., ltd, shanghai, china) for 40 seconds until they set. the nanosisal composite resin samples were then divided into three groups of varying concentrations, namely: a (60%), b (65%), and c (70%). on the other hand, the nanofiller composite (z350 xt, 3m espe) which was to act as the control (group d) was extracted from the tube with plastic instruments, inserted into a mould, and radiated with a visible light cure for 40 seconds until setting. the four groups were then marked at the center of the samples to be tested for their compressive strength by using a universal testing machine (mettler toledo al 204). each mould was placed in the centre of the utm with the 1000 n load being applied on top of the material with the vertical printout position at 1 mm/min. the data obtained was in the form of n unit which was then converted into mpa using the following formula:14 rc = f a note: rc : compressive strength (mpa) f : maximum force (n) a : width of sample base area (πr2) (mm2) the score of the compressive strength obtained then was analyzed by non-parametric tests, namely; kruskal wallis and mann-whitney tests. results the results showed that the average value of compressive strength in the nanosisal composite resins group a was 16.80 mpa, in group b 10.80 mpa, in group c 7.20 mpa and in group d 7.40 mpa. the normality of the resulting data was then tested by means of a saphiro wilk test, the results of which demonstrated the distribution of the data to be normal. the homogenity of the data was then tested using a levene test whose results indicated that the variance of the data was not homogeneous. as a result, a kruskal wallis procedure was carried out. table 1 show that the largest mean value of compressive strength was found in the group a nanosisal composite resins. therefore, the optimum nanosisal filler volume recommended by this research was one of 60%. furthermore, table 2 demonstrates a p value of < 0.033 (p < 0.05). this confirms that there was a filler volume effect on the compressive strength of the nanosisal composite resins. in addition, table 3 shows the nanosisal composite resins group a have significant differences in compressive strength compared of the nanosisal composite resins c and d groups with p value (p < 0.05). however, there were no significant differences between the nano-sisal composite resins when comparing group a with group b, group b with group c and group d and group c with group d. discussion the nanosisal composite resin in group a had greater compressive strength than the nanofiller composite group d. this is because the nanosisal composite resin forms a stronger bond between the nanosisal fiber and resin matrix due to a chemical bond of the oh group.12 based on the observation results of the chemical structures of the resin matrix and nanosisal fibers, the h atoms present in the resin matrix bind to the o atoms present in nanosisal fiber. as a result, a new oh group is formed. moreover, mechanical bonds are also formed between the resin matrix and nanosisal fiber due to the roughness of the surface of the sisal fibers. the initial process of nanosisal composite manufacture greatly influences the mechanical properties of the composite resins.15 in this research, scouring including alkalization treatment was conducted. the sisal fibers themselves contain a group of hydroxyl groups that can form hydrogen bonds which can affect the dimensional stability of natural table 1. the mean value of compressive strength of the nanosisal composite resins group n mean (mpa) compressive strength a 5 16.80 b 5 10.80 c 5 7.20 d 5 7.40 total 20 group a: nanosisal composite 60% filler group b: nanosisal composite 65% filler group c: nanosisal composite 70% filler group d (control): nanofiller composite z350 xt table 2. results of the kruskal wallis test compressive strength test chi-square 8.707 df 3 asymp. sig. .033 table 3 results of the mann-whitney test variable variable a b c d a – 0.076 0.009 0.028 b – – 0.295 0.347 c – – – 0.917 d – – – – group a: nanosisal composite 60% filler group b: nanosisal composite 65% filler group c: nanosisal composite 70% filler group d (control): nanofiller composite z350 xt http://dx.doi.org/10.20473/j.djmkg.v50.i4.p183-187 186 nugroho, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 183–187 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p183–187 fibers. this will then lead to a weak bond between the resin and the matrix.15 according to previous research, alkalization can break the hydrogen bonds in the tissue structure, thereby increasing the surface roughness of the sisal fibers. this, in turn, results in the stronger bonds of the resin matrix and the sisal fiber. in other words, alkalization treatment has been shown to increase the mechanical strength of natural fibers.16 the imbalance of the coupling agents on the z350 xt composite resins may also lower the compressive strength of the composite resins. the coupling agent used in z350 xt composite resins is silane which is an adhesion promoter containing two different reactive functions that can react and combine with variations of organic and inorganic materials. silane is used to strengthen the bonds of different materials. the hydrolysable functional group will react with the hydroxyl group on the surface of the inorganic substrate to form a siloxane (si-o-si) bond. organic groups that cannot be hydrolyzed together with a double bond c = c can polymerize with a double-bonded composite resin monomer.17 a balance between the number of inorganic substrate hydroxyl groups and the hydrolysable functional groups in the silane must exist. if the amount of silane contained in the z350 xt composite resin is imbalanced, the compressive strength distribution from the matrix to the filler will not proceed properly and composite resin will then be easily broken.18 nanosisal composite resins used in this research were prepared without the use of coupling agents because the resin matrixes and nanosisal fibers are organic materials. thus, both can bond chemically without the presence of a coupling agent.12 in the pre-eleminary study, resin matrix, silane (coupling agent), photoionization, and nano-sisal fibers were mixed. unfortunately, the composite resins did not harden after being radiated with a visible light cure for 40 seconds. based on the data obtained, the nanosisal composite resins at group a demonstrated the highest compressive strength. previous research comparing hybrid composite and filler composite consisting of areca fibers at concentrations of 50% and 60% showed that the filler composite at a concentration of 60% had a lower water absorption capacity than the filler composite at a concentration of 50%.19 this may be due to the higher compatibility between hydrophilic fibers and composite matrixes at a concentration of 60%.20 in contrast, this research indicates that the higher the volume of the nanosisal volume used in composite resins, the smaller the compressive strength. the results of this research were supported by that of a previous investigation examining palm fibers as composite fillers at volumes varying from 30% to 70%. this showed that the compressive strength of the palm fibers increased as the filler volume expanded to 60% and decreased when the filler volume was 70%.21 this could be explained by the compressive strength decreasing as filler volume increases since, at the higher volume, many fiber ends will experience fibrous discohesion within the resin matrix leading to initial cracking on the composite resins.22 increasing the concentration of sisal/palm hybrid fibers resulted in a reduction of tensile strength and tear strength. palm fibers can, therefore, be seen to have similar characteristics as sisal fibers.23 table 3 showed no significant difference in the compressive strength of nanosisal composite of groups a and b since the structure of nanosisal composite resins began to crack if the filler volume was slightly higher than 60%. this was the finding of a previous study on palm fibers.21 similarly, there was also no significant difference in the compressive strength of nano-sisal composite resins at the volumes of b and c groups since the probability of the number of cracks occurring did not vary much.21 these results may be caused by the fibrous discohesion in the number of fiber ends of both nanosisal composites not differing greatly.22 finally, it can be concluded that nanosisal filler volume has an effect on the compressive strength of the nanosisal composite resins. the recommended filler volume of the nanosisal composite resin is 60%. acknowledgement we would like to express our graitude to the institute for research and community service and human resources bureau of universitas muhammadiyah, yogyakarta. references 1. mccabe jf, walls awg. applied dental materials. 9th ed. oxford: blackwell publishing ltd.; 2008. p. 197–8. 2. sintawati j, soemartono sh, suharsini m. pengaruh durasi aplikasi asam fosfat 37% terhadap kekuatan geser restorasi resin komposit pada email gigi tetap. j dent indonesia. 2008; 15(2): 97–103. 3. anusavice kj, shen c, rawls hr. phillips’ science of dental materials. 12th ed. st. louis: elsevier saunders; 2012. p. 275–6. 4. khaled an. physical properties of dental resin nanocomposites. thesis. manchester: university of manchester; 2011. p. 26–7. 5. thomaidis s, kakaboura a, mueller wd, zinelis s. mechanical properties of contemporary composite resins and their interrelations. dent mater. 2013; 29: e132–41. 6. masouras k, silikas n, watts dc. correlation of filler content and elastic properties of resin-composites. dent mater. 2008; 24: 932–9. 7. gladwin ma, bagby md. clinical aspects of dental materials: theory, practice, and cases. 3rd ed. philadelphia: lippincott williams & wilkins; 2009. p. 121–2. 8. wambua p, ivens j, verpoest i. natural fibres: can they replace glass in fibre reinforced plastics?. compos sci technol. 2003; 63: 1259–64. 9. joshi sv, drzal lt, mohanty ak, arora s. are natural fiber composites environmentally superior to glass fiber reinforced composites?. compos part a appl sci manuf. 2004; 35: 371–6. 10. natarajan n, bharathidhasan s, thanigaivelan r, suresh p. sisal fiber/glass fiber hybrid nano composite: the tensile and compressive properties. in: 5th international & 26th all india manufacturing technology, design and research conference. assam; 2014. p. 1–6. 11. kusumastuti a. aplikasi serat sisal sebagai komposit polimer. j kompetensi teknik. 2009; 1(1): 27–32. http://dx.doi.org/10.20473/j.djmkg.v50.i4.p183-187 187187nugroho, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 183–187 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p183–187 12. ahmad eem. the influence of microand nanosisal fibers on the morphology and properties of different polymers. thesis. qwaqwa: university of the free state; 2011. p. 3–60. 13. international organization of standardization (iso). dentistry -polymer-based restorative materials. 4th ed. iso 4049: 2009. p. 1–28. 14. bresciani e, barata tje, fagundes tc, adachi a, terrin mm, navarro mfl. compressive and diametral tensile strength of glass ionomer cements. j appl oral sci. 2004; 12(4): 34–8. 15. ilomäki km. adhesion between natural fibers and thermosets. thesis. tampere: tampere university of technology; 2011. p. 36–7. 16. li x, tabil lg, panigrahi s. chemical treatments of natural fiber for use in natural fiber-reinforced composites: a review. j polym environ. 2007; 15: 25–33. 17. zaghloul h, elkassas dw, haridy mf. effect of incorporation of silane in the bonding agent on the repair potential of machinable esthetic blocks. eur j dent. 2014; 8(1): 44–52. 18. lung cyk, matinlinna jp. aspects of silane coupling agents and surface conditioning in dentistry: an overview. dent mater. 2012; 28: 467–77. 19. venkateshappa sc, bennehalli b, kenchappa mg, ranganagowda rpg. flexural behaviour of areca fibers composites. bioresources. 2010; 5(3): 1846–58. 20. betan ad, soenoko r, sonief aa. pengaruh persentase alkali pada serat pangkal pelepah daun pinang (areca catechu) terhadap sifat mekanis komposit polimer. j rekayasa mesin. 2014; 5(2): 119–26. 21. velmurugan r, manikandan v. mechanical properties of glass/ palmyra fiber waste sandwich composites. indian j eng mater sci. 2005; 12: 563–70. 22. nutt sr, needleman a. void nucleation at fiber ends in al-sic composites. scr metall. 1987; 21: 705–10. 23. jacob m, thomas s, varughese kt. mechanical properties of sisal/palm hybrid fiber reinforced natural composites. compos sci technol. 2004; 64(7): 955–65. http://dx.doi.org/10.20473/j.djmkg.v50.i4.p183-187 vol 44 no 3 sept 2011.indd 154 vol. 44. no. 3 september 2011 management of horizontal crown fracture caused by traumatic injury with endorestoration treatment nanik zubaidah department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: traumatic injuries of teeth are the main cause of emergency treatment in dental practice. the horizontal crown fracture more frequently observed usually occurs in maxillary anterior region and young male patients. the most common type of coronal fracture is in the middle third, followed by root and apical part. purpose: the aim of this case report is to present the management of crown fracture of teeth with pulp exposure caused by dental trauma with endorestoration treatment in order to reconstruct the shape and the function of the teeth. case: a 22 years old male with horizontal crown fracture of anterior teeth. the patient asked for aesthetic dental treatment both for its form and function. case management: this horizontal crown fracture of anterior teeth with pulp exposure caused by dental trauma still could be reconstructed, mainly by endorestoration treatment. the endodontic treatment with post and core insertion in the root canal then would increase its retention. later, the porcelain crown would aesthetically recover its original form and function, therefore, it would improve the patient’s confidence and teeth function. conclusion: endorestoration treatment on anterior teeth with harizontal crown fractures and pulp exposure is able to recover the normal function, aesthetic, and self-confidence. key words: dental trauma, horizontal crown fracture, endorestoration treatment abstrak latar belakang: trauma pada gigi merupakan penyebab utama perawatan darurat dalam praktek dokter gigi. fraktur mahkota horisontal pada umunya terjadi pada gigi anterior rahang atas dan terjadi pada penderita pria muda. jenis yang paling sering dari fraktur mahkota adalah pada sepertiga tengah, daerah akar dan apical. tujuan: laporan kasus ini menjelaskan penatalaksanaan fraktur mahkota gigi dengan pulpa terbuka akibat trauma dengan perawatan endorestorasi untuk mengembalikan bentuk dan fungsi gigi. kasus: penderita pria umur 22 tahun dengan fraktur mahkota horizontal pada gigi anterior. penderita tersebut menginginkan perawatan estetik untuk mengembalikan bentuk dan fungsi giginya. tatalaksana kasus: fraktur akar horisontal gigi anterior dengan pulpa terbuka oleh karena trauma gigi dapat direstorasi dengan perawatan endorestorasi. perawatan endodontic dengan pasak dan inti dimasukkan ke dalam saluran akar dapat meningkatkan retensi. kemudian mahkota porselen dapat mengembalikan bentuk dan fungsinya, karena itu dapat meningkatkan percaya diri pasien dan fungsi giginya. kesimpulan: perawatan endorestorasi pada gigi anterior dengan fraktur mahkota harizontal dan pulpa terbuka dapat mengembalikan fungsi estetik dan percaya diri pasien. kata kunci: trauma gigi, fraktur mahkota horizontal, perawatan endorestorasi correspondence: nanik zubaidah, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132. indonesia. e-mail: nanikzubaidah@yahoo.com case report 155zubaidah: management of traumatic injury introduction dental traumatic injuries are the main cause of emergency treatment in dental practice. it occurs in young patients with varying severity from enamel fractures to avulsion.1 dental trauma can cause damage to pulp, with or without either crown or root damage. the percentage of maxillary of anterior tooth fracture is high, and 90% of them are caused by protrusive teeth which are not covered by lips stand out, so they can not quite be covered by the lips.2 the dental trauma followed by anterior tooth fractures, especially permanent incisors can cause a strong psychological impact on young patients. dental trauma involving extensive loss of tooth structure can cause pain, discomfort and bad appearance. with recent advances in esthetic dentistry, restoration treatment has caused high expectations for the patients to be able to smile with confidence.3,4 crown fracture is a fracture that involves enamel and dentin. tooth fracture accompanied by open pulp is considered as complicated. the number of complicated crown fracture cases is about 2–13% of all trauma cases, and most of them involves first maxillary incisors, which are frequent in children, according to chan,5 it is about 5-20%. horizontal fracture cases, occur more frequently than the vertical ones. various kind of fractures usually occur because of severe trauma, 3% of which are caused by traffic accidents and trauma obtained at the time of exercise, while others are caused by trauma obtained in crime and rape cases.6,7 in addition, strong frontal and horizontal force can produce fracture line from some point on crown to mesial or distal subgingival regions with or without the involvement of pulp. according to ellis, the classification of dental fractures consists of six basic categories, namely: enamel fractures, dentin fractures without pulp exposure, crown fractures with pulp exposure, root fractures, luxation teeth, and intrusion. crown fractures can be classified into three classes.2 it is because the degree of pulp exposure can vary from small open pulp to fully open coronal pulp.6,8,9 there are four kinds of possible treatments for horizontal crown fracture with pulp exposure: pulpotomy (vital pulp), apexification (necrotic pulp), pulpectomy (endodontic treatment), and root resection.10 in dentistry, especially in esthetic restoration, horizontally fractured crown with pulp exposure needs to obtain endorestoration treatment involving endodontic therapy using retention, such post core and jacket porcelain crown.1,7 the purpose of this case report is to inform that crown fracture with pulp exposure due to trauma can be managed through endorestoration treatment to restore the form, function and esthetics. the success of treatment, however, depends on the careful selection of cases based on clinical and radiographic examination. in other words, the treatment must be planned carefully to avoid accident, such as root fracture or extensive periodontal ligament damage. case the patient was a 22 year old man who came to the dental and oral medical vip hospital of the faculty of dentistry, airlangga university, surabaya. the patient come three days after he got a traffic accident in which he fell off from the bike with fractured and cracked of teeth 12, 11 and 21, and swelling around the vestibule of the teeth 12, 11 and 21 and the upper lip. the patient wanted aesthetic improvement for his anterior teeth and hoped that the original esthetic and function can be restored. based on intra-oral examination it was known then there was horizontal complicated crown fracture tooth 11. it was also known that there was horizontal complicated crown fracture on tooth 12, but the condition of the fracture was crown still in place, in the center of the crown, involving enamel, dentin, and the pulp, while there was a minor crack only in the center of the crown of tooth 21 involving the enamel and dentin (figure 1). for the purposes of diagnosis and treatment planning, local x-ray and panoramic photographs were taken. based on the results of the radiographic photos, it was known that there was radiolucency on the periapical region of tooth 11, and there was also crack on tooth 12 involving dental pulp. it was also known that the diagnosis of teeth 12 and 11 was irreversible pulpitis, while the diagnosis of his tooth 21 was reversible pulpitis. therefore, the dental treatment plan for tooth 21 was by conducting composite veneer restoration (direct veener), while the dental treatment for teeth 12 and 11 were endodontic treatment (pulpectomy vital treatment) accompanied with post and porcelain jacket crown made of zirconia materials using dental cad/cam method. case management when the patient came at the first time, emergency treatment conducted consisted of cleaning the soft tissues arround his mouth and lips by using saline and hydrogen peroxide, inducing antibiotics, analgesics and antiinflammatory, using chlorhexidine mouthwash, giving instructions to maintain oral hygiene and soft diet that are recommended. it is because this treatment could help the patient avoid anything more severe than soft-tissue edema.6 next, capturing printed images on his teeth 11, 12 and 21 which were traumatized was conducted before further treatment (figure 1). then, the maxillary and mandibular tooth anatomy was printed both for the study model and dental record, and also for diagnosing occlusion and relation that might occur as well as for preparing jacket crowns on teeth 11 and 12 in order not to reduce the esthetics of those teeth during the treatment. afterwards, pulpectomy treatment was conducted on the teeth 11 and 12 in one visit by using both crown down pressurelless technique with preparation tools, such 156 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 154–158 as file protaper, and single cone filling technique with endomethason sealer pasta materials. next, the preparation of post canal was conducted by taking guta percha point with gates gliden drill, and then peeso reamer leaved approximately 4–5 mm area from the apex of the root canal. after that, the insertion of post with tapered serrated type (unimetric) cemented by using type i of glass ionomer cement (luting cement). and then, the core post was made by using composite resin (figure 2). figure 2. the insertion of post on teeth 12 and 11. the preparation of the teeth 11 and 12 with cervical line and chamfer shape was conducted by using tapered fissure bur and wheel-shaped diamond bur (figure 3). after that, those teeth that had already been prepared were impressed by using double impression technique using elastomer impression materials with injection and putty types. the next stage was setting temporary crowns on those teeth, and sending the cast to a dental laboratory for making all porcelain jacket crowns made of zirconia materials. and then, all porcelain jacket crowns were cemented on teeth 11 and 12 using adhesive bonding technique (figure 4). after that, the restoration of the direct veneer composite was conducted on teeth 21 by using light cure microfill composite material (figure 4). first, the teeth were cleaned, and the proper shade that matched with the original shade of next teeth was chosen. second, those teeth were isolated with cotton rolls and retraction cords. third, preparation was conducted with a coarse and round end diamond instrument after the insertion of all porcelain jacket crowns in order to get similar composite shade to the shade of the porcelain crowns for esthetic purpose. fourth, window preparation was conducted with a depth of 0.5 mm and tapered shape towards the gingival margin with a depth of 0.2 mm, and then it was etched, washed, dried. bonding agent was applied and cured for 20 seconds, and then was coated with micropfilled composite resin and cured for 20 seconds. the composite was placed a little too much to get contouring, particularly along the gingival margin, to reduce the effects of polymerization contraction. the polishing was conducted by using fine finishing diamond bur and silicone rubber with polishing pasta for the resin composite. and, the patient was finally asked for control 1 week, 1 month, 6 months and 1 year after the treatment. discussion various conditions of trauma can cause crown fractures although many literatures suggest several predominant causes, such as trauma while playing and running or during sports activities, traffic accidents, and hit on face. anterior teeth are more susceptible to trauma, approximately 80% of maxillary incisors followed by maxillary lateral incisors and central mandibular incisisors.1 in this case, it is known that tooth 11 suffered complicated horizontal crown fracture, while tooth 12 suffered with complicated horizontal crown fracture in the middle of the crown involving enamel, dentin, and pulp, but the condition of the crowns had already attached each other. it is also known that tooth 21 suffered with fracture only figure 1. the condition of teeth 12, 11, 21, and 22 before the treatment. figure 3. the preparation of teeth 12 and 11. figure 4. the condition of the teeth 12, 11 and 21 after treatment. 157zubaidah: management of traumatic injury at enamel layer and thin dentin layer, so composite veneer restoration was conducted on the tooth 21. meanwhile, pulpectomy endorestoration treatment was conducted on teeth 11 and 12 in which the retention of prefabricated post and the core of the composite were inserted, and all porcelain jacket crown was set. if an open fracture of crown with pulp is reported to the dentist after 72 hours or more, the option will only be endodontic treatment by removing the entire pulp tissue; but if the fracture is more than ½ of coronal lost, post-core and crown will be required. the post-core restorations are used to reshape the structure of the lost crown.3,7 the principle of dental care for those who have experienced endodontic treatment is actually to restore dental root and crown by using retentive and stable post crown, so it will not easily separated and can be used as long as possible in the oral cavity as same as the original ones. tooth that has got endodontic treatment, moreover, is relatively more brittle and prone to fracture than the vital one because of tooth internal moisture that is reduced during endodontic treatment and can weaken the remaining tooth structure, furthermore, and a non-vital tooth often discloved.5 endodontic treatment was conducted on teeth 11 and 12 in one visit because the form of the root canals is normal, mild periapical abnormality with no clinical symptoms. this effecient time for the treatment was suitable with the activities of the patient as a student who was so bussy in collage. this one visit endodontic treatment aimed to prevent the spread of diseases of the pulp to the periapical tissues or if it has occurred, it will aim to restore the periapical tissues. it also gives the advantage to reduce the risk of infection that may occur between visit periods, and to save time.2 the preparation of root canals 11 and 12 was then conducted by using crown down pressureless technique with protaper instrument conducted with coronal-apical approach. this technique is advantageous since most of the microorganisms located in 1/3 coronal and 1/3 center had been cleaned before getting into the apical regions, and the irrigation is more perfect in 1/3 apical.11 the intracanal retention was conducted in order to restore the crown shape of the post used as the retention of the restoration. post used in this this case was prefabricated post since the post has several advantages, namely more effective and efficient as it can be completed only in one visit, more variety in design as a result, it can meet the needs of patients concerning with the condition of their treated teeth, as well as stiffer and stronger as it is made of metal. this kind of post then can be classified as the passive one. during cementation, this passive post, moreover, requires no pressure on the post canal, so it can reduce the risk of fractures. the taper of the post even is almost like the natural shape of the root canal. the cementation process of this manufactured post in root canals then was followed by the formation of the core with composite resin because it has advantages in terms of aesthetics, compressive strength, and rapid hardening so that it can be readily prepared, easily manipulated, and dimensionally stabilized with minimal edge leakage.12,13 the selection of the final restoration involving allporcelain jacket crowns made of zirconia materials by using cad/cam method is actually considered as the best choice since zirconia material is metal-free restorative material with high-quality ceramics in esthetic factors as well as high-tech ceramic material that has characters of stability, biocompatibility, higher strength than other ceramic materials, and higher fracture roughness than other restorative materials.14 similarly, the results of qualthrough’s research15 also states that among 956 patients, only 63% of whom felt satisfied when one of their anterior teeth was crowned, while 79% of whom were satisfied when 4 or more of their anterior teeth were crown with porcelain. hume,16 also declares that the use of porcelain jacket crown is the best way to restore the first incisive teeth with optimal esthetics. according to bulem,17 porcelain is actually considered as the most satisfying material for patients due to its natural color and aesthetics. furthermore, cementation of porcelain jacket crown towards post and core by using resin cement (dual cure bonding system) actually depends on the increasing of the retention of passive post retention that is linier with that of active post since resin cement has both better tensile bond strength than glass ionomer cement, and also better shear bond strength than phosphate zinc cement.12 the composite veneers was conducted on tooth 21 since the tooth was suffered from minor cracks located in the center of the crown only involving enamel layer and thin dentin layer with reversible pulpitis diagnosis. the cost of the treatment was cheaper than that with veneer ceramic (porcelain) since there was no laboratory cost and it needed only one visit. the making of dental restoration on the fracture depended on the width of the coronal structure of the dental fracture. thus, if the fracture only involved enamel and dentin, it then can be restored with composite or porcelain laminated veneers.18 it can be concluded that horizontal crown fractures due to trauma can be treated with endorestoration treatment in order to restore the form, function and esthetics of the teeth in accordance with their original ones. references 1. mutan ha, erdal o, yahya oz, muzaffer a. treatment of traumatized maxillary permanent lateral and central horizontal root fractures. a case report. indian j dent res 2008; 19(4): 354–6. 2. grossman li. oliet s, del rio ce. 1988. ilmu endodontik dalam praktik. 1st ed. jakarta: penerbit buku kedokteran ecg; 1995. p. 196–380. 3. jain v, gupta r, duggal r, parkash h. restoration of traumatized anterior teeth by interdisciplinary approach: report of three cases. j indian soc pedod prev dent 2000; 20(1): 193–6. 4. heda cb, heda aa, kulkarni ss. a multi-disciplinary approach in the management of traumatized tooth with complicated crown-root fracture: a case report. j indian soc pedod prev dent 2006; 24(4): 197. 158 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 154–158 5. chan dcn, myers ml, chipped, fracture, or endodonticcally treated teeth. in: goldstein re, editor. esthetics in dentistry. 2nd ed. hamilton, london: bc decker inc; 2002. p. 537–9. 6. aggarwal v, logami a, shah n. complicated crown fracturesmanagement and treatment option. int endodontic j 2009; 42: 740–53. 7. baritcigil c, harorli ot, yildiz m. restoration of crown fracture with a fiber post, polyethylene and composit resin. rev clin pesq odontol curitiba 2009; 5(1): 73–7. 8. andreassen jo, andreassen fm. texbook and color atlas of traumatic injuries to teeth. 3rd ed. copenhagen: munksgaard; p. 219–56. 9. nandlal b, daneswari v. restoring biological width in crown–root fracture: a periodontal concern. a case report. j indian soc pedod prevent dent 2007; supplement: s20–s23. 10. schulze a. dental traumatic injuries in sports. clinical sports medicine international (csmi) 2008; 1(8): 13–5. 11. garg n, garg a. texbook of endodontic. 1st ed. jaype brothers medical publisher; 2007. p. 196. 12. arianti n, untara te. perawatan saluran akar molar mandibula teknik crown down disertai mahkota porcelain fusi metal pasak tappered serrated. majalah ilmiah kedokteran gigi 2007; 14(1): 29–34. 13. christina sd, sri ds. mahkota all porcelain dengan penguat pasak fiber pada gigi insisivus maksila pasca perawatan saluran akar. majalah ilmiah kedokteran gigi 2007; 14(1): 17–22. 14. lava im. crown and bridges. available at: http://www.nilabsolution. com/lava htm. 2004. accessed april 16, 2009. 15. qualthrough aje, burke fjt. a look at dental esthetics. j quintessence international 1994; 25(1): 7–9. 16. hume wr. preservational restoration of tooth structure. london: the cv mosby co; 1998. p. 185–90. 17. murali s. unconventional prosthodontics: post, core and crown technique. j indian prosthodontic society 2007; 7(4): 191–4. 18. summit jb, robbin jw, hilton tj, schartz rs. fundamentals of operative dentistry. 3rd ed. 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 113 vol. 43. no. 3 september 2010 research report anti-inflammation effects of sardinella longicep oil against paw oedema on rattus novergicus induced by 1% carrageenan rima parwati sari and yenny sugiharto department of oral biology faculty of dentistry, university of hang tuah surabaya indonesia abstract background: people usually used non steroid anti-inflammation drugs (nsaid) such as aspirin in chronic inflammation treatment. however, using nsaid at long term therapy will cause many effects such as nausea and vomiting. sardinella longiceps oil, on the other side, is reported as an alternative treatment for anti-inflammation since it is natural and also contained eicosapentaenoid acid (epa) and decohexaenoic acid (dha). thus, it may reduce paw oedema. purpose: the aim of this study was to know anti-inflammation effects of sardinella longiceps oil against paw oedema of wistar rats induced by 1% carrageenan. methods: the samples of this research were 32 wistar rats which were divided into four groups, in group 1, the rats were given aquadest; in group 2, the rats were given aspirin; in group 3, the rats were given 1 ml sardinella longiceps oil; in group 4, the rats were given 1.5 ml sardinella longiceps oil. all of the rats, nevertheless, were given intraplantar induction of 1% carrageenan into the paw of rats to induce the inflammation condition. results: all data were tested with normality test. the normal data were then analyzed with homogenity of variances and also anova test which result showed significant differences. the data which showed significant differences were tested again with lsd test. result then showed that group given 1 ml sardinella longiceps oil and group given 1.5 ml sardinella longiceps oil had no significant differences from group given aspirin, but there were significant differences between group given 1 ml sardinella longiceps oil and group given 1.5 ml sardinella longiceps oil, and also between group given 1.5 ml sardinella longiceps oil and group given aquadest. conclusion: sardinella longiceps oil could reduce paw oedema in wistar rats induced with 1% carrageenan. key words: sardinella longiceps oil, aspirin, 1% carrageenan, anti-inflammation abstrak latar belakang: penggunaan obat anti-inflamasi non steroid (oains) seperti aspirin sering digunakan dalam pengobatan inflamasi kronis. namun penggunaan oains dalam jangka waktu panjang akan menyebabkan efek samping seperti mual dan muntah. di sisi lain, minyak sardinella longiceps, dilaporkan dapat digunakan sebagai alternatif anti-inflamasi karena selain alami, juga terdapat kandungan eicosapentaenoid acid (epa) and decohexaenoic acid (dha) yang dapat mengurangi edema pada telapak kaki. tujuan: tujuan dari penelitian ini adalah mengetahui efek anti-inflamasi minyak sardinella longiceps pada edema telapak kaki tikus wistar yang diinduksi karagenan 1%. metode: sampel penelitian ini adalah 32 tikus yang dibagi ke dalam empat kelompok. kelompok 1, tikus diberi akuades; kelompok 2, tikus diberi aspirin; kelompok 3, tikus diberi minyak sardinella longiceps 1 ml; kelompok 4, tikus diberi minyak sardinella longiceps 1,5 ml. semua tikus tidak terkecuali, diinduksi intraplantar karagenan 1% secara intraplantar pada telapak kaki untuk membuat kondisi inflamasi. hasil: semua data dilakukan uji normalitas. selanjutnya data yang berdistribusi normal dilakukan uji homogenitas dan juga uji anova yanng hasilnya menunjukkan adanya perbedaan bermakna. data yang menunjukkan perbedaan bermakna diuji lagi dengan lsd. pada uji lsd menunjukkan bahwa tidak ada perbedaan bermakna antara kelompok minyak sardinella longiceps 1 ml dengan kelompok aspirin dan kelompok minyak sardinella longiceps 1,5 ml dengan kelompok aspirin, tetapi ada perbedaan bermakna antara kelompok minyak sardinella longiceps 1 ml dan kelompok akuades dengan kelompok minyak 114 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 113–116 sardinella longiceps 1.5 ml group given aquadest. kesimpulan: sardinella longiceps dapat mengurangi edema pada telapak kaki tikus wistar yang diinduksi karagenan 1%. kata kunci: minyak sardinella longiceps, aspirin, karagenan 1%, anti-inflamasi correspondence: rima parwati sari, c/o: bagian biologi oral, fakultas kedokteran gigi universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: rima.sari@yahoo.com introduction inflammation is a living tissue reaction to invasion of pathogenic microorganisms, traumas, burns, or chemical materials.1 inflammation is often found in almost every case in dentistry. but, there are certain cases that are difficult to be treated since the inflammation is considered to be chronic and persistent, such as periodontitis. synthetic or chemical drugs have already become alternative medicines for those particular cases, especially acute inflammation. in chronic inflammation, those drugs, unfortunately, are not recommended since in the long-term treatment they can cause nausea, vomiting, and gastric bleeding since they can inhibit the cyclooxygenase-1 (cox-1) more than the cyclooxygenase-2 (cox-2).2,3 cox-1 is useful in physiological processes such as for maintaining and protecting stomach, kidneys and other organs, whereas cox-2 contributes to the production of pro-inflammatory prostaglandins and other inflammatory mediators in inflammatory process.3 for those reasons, in the treatment of chronic inflammation it is better to select drugs that have the fewest side effects since the treatment process requires long time. under these circumstances, it is necessary to choose another alternative treatment which use traditional medicines. traditional medicine is a concoction of natural ingredients derived from plants, animals, minerals, whole preparation, or mixture of those materials which have traditionally been used for treatment based on empiric experience.4 another advantage is that the traditional ingredients are easily available, and their benefit on certain diseases is quite large with low risk ratio and useful benefit for patients.4 in health sector, traditional medicines, have already been researched widely for their benefits, including traditional medicines that have anti-inflammatory effects. the result of recent researches even shows that fish oil containing omega-3 has anti-inflammatory effects that are good for long-term use. omega-3 fatty acids are actually rich of eicosapentaenoid acid (epa) and decohexaenoic acid (dha) that can inhibit the synthesis of arachidonic acid. the reason is because epa and dha can inhibit cox line and lypoxygenase (lox) line.5 the inhibition of lox line evan can be more effective for treating chronic inflammation cases than nsaids that can only inhibit enzyme cox.6 in addition, fish mostly found in many of indonesia's marine waters are sardinella longiceps. the distribution of sardinella longiceps is mostly found around muncar near banyuwangi (east java).7 actually, sardinella longiceps is a type of fish that has a significant economic value. this fish is one of the resources which has a large potential and good prospects. for instance, they can be processed to become canned food, cue, and salted fish, and their waste can also be processed to become fish meal. sardinella longiceps can also be used as bait to catch bigger fish.8 the use of sardinella longiceps as medicine is widely studied. it is also known that sardinella longiceps contain much omega3 that is good for health. the reason is because omega-3 widely contained in sardinella longiceps is good for brain and chronic inflammatory medication.9 therefore, the aim of this study was to investigate anti-inflammatory effects of sardinella longiceps against the induction of 1% carrageenan suspension in rats’ paw, compared to those induced with aspirin as anti-inflammatory drug. as a consequence, from this research, sardinella longiceps can be widely cultivated in indonesia. materials and methods this research was an experimental laboratory using completely randomized design.10 the parameter of this research was the size of edema (mmhg) of rat paw that had been induced with 1% carrageenan suspension.11 the samples of this research were 32 rats then randomly divided into 4 groups.12 the criteria of those samples were male white wistar rats (rattus novergicus) in the age of 2–3 months old and with the weight of 150–200 grams. before the rats were treated, they had to adapt with the environment for one week under supervised condition. the research then was conducted in the biochemistry laboratory of faculty of medicine, airlangga university surabaya for 6 months. sardinella longiceps oil was created through intake process, cooking process, and separator process. intake process was conducted to check the freshness of meat and oil of fish in order to obtain the expected good results. in the cooking process, raw fish was heated at the temperature of 90–95° c. the heating process was aimed to sterilize the fish, so their protein could be frozen and their cell membrane could be disrupted, as a consequence, the fat depot (thick and stiff) would be separated and the oil would be released. in the separator process, the liquid containing water and mostly oil was seperated from fish, and then the protein and salt were dissolved in it. afterwards, it was put in a decanter, and send to drying to be mixed with press 115sari and sugiharto: anti-inflammation effects of sardinella longicep oil cake. the liquid from the decanter, finally, was separated and stored.13 before this research was conducted, nevertheless, those white rats fasted approximately for 18 hours, but they were still given water to drink. if there were any of them sick, those would be excluded from this research. those white rats then were divided into four groups: group 1 was a control group orally given only 1 ml aquadest, group 2 was the treatment group orally given aspirin at the dose of 360 mg/kg, group 3 was orally given 1 ml sardinella longiceps oil, while group 4 was orally given 1.5 ml sardinella longiceps oil.14 onset of action in each of those anti-inflammatory drugs was estimated around 30 minutes, so those drugs had already demonstrated anti-inflammatory effects before the induction of 1 % carrageenan. after 30 minutes, the left paw of each of those white rats was dipped into mercury (pletismometer) to be measured. each left foot of those white rats was given intraplatar induction of 1 % carrageenan suspension, 0.05 ml, as illustrated by vinegar et al.11 next, every 15 minutes, the volume of those white rats' left paw was measured again for 3 hours. all data obtained were then recorded, and the results of each group were also averaged. data obtained from the measurement of those white wistar rats’ paw every 15 minutes for 3 hours in each of those groups were tabulated. to analyze the effects of antiinflammatory, the difference of the volume of inflammation of the rat paw between that at t minutes and that at initial volume (t=0) was calculated. based on the calculation of the difference of the volume of inflammation in each group, the statistical calculations with anova test followed by least significant difference test (lsd) was then conducted for the significance analysis in each group. results figure 1 shows the results of the mean volume of oedema in each group of treatment groups, starting from the largest to the smallest one; aquadest, 1 ml sardinella longiceps oil, aspirin, and 1.5 ml sardinella longiceps oil. based on anova test, it is known that there were the mean differences of oedema volume for 180 minutes after the injection of 1% carrageenan (p < 0.05). based on lsd test, it is also known that there was no significant difference (p > 0.05) not only between group given aquadest and group given 1 ml sardinella longiceps oil (p = 0.229), between group given aspirin and group given 1 ml sardinella longiceps oil (p = 0.544), but also between group given aspirin and group given 1.5 ml sardinella longiceps oil (p = 0.102). meanwhile, there were significant differences (sig < 0.05) not only between group given aquadest and group given aspirin (p = 0.007), between group given aquadest and group given 1.5 ml sardinella longiceps oil table �. lsd test results dependent variable groups of treatment aquadest aspirin 1 ml sardinella longiceps oil 1.5 ml sardinella longiceps oil the mean reduction of oedema volume aquadest 0.007* 0.229 0.001* aspirin 0.544 0.102 1ml sardinella longiceps oil 0.029* 1.5 ml sardinella longiceps oil figure �. the mean differences of oedema volume for 180 minutes after the injection of 1% carrageenan. figure �. classical mechanism of anti-inflammatory activity of omega-3 fatty acids.5 0.004 0.0035 0.003 0.0025 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045 control 1 ml aspirin 1.5 ml groups of treatment mean of inflamatory difference sardinella longiceps oil sardinella longiceps oil 116 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 113–116 (p = 0.001), but also between group given 1 ml sardinella longiceps oil and group given 1.5 ml sardinella longiceps oil (p = 0.029) (table 1). discussion inflammation is body's defense mechanism due to tissue response to the damaging effects that can be local or induced into the body.12 inflammatory reaction can be observed from clinical symptoms, such as oedema. oedema is due to the release of various mediators of pain so that the liquid of blood plasma come out from blood vessels and come into the wound, as a result, tissue oedema then clinically occurs and make the condition of the acidosis area.15 aspirin, on the other side, known as nsaids, has already been proven to have anti-inflammatory effects, but it has also side effects such as nausea, vomiting, and so on.16 in this study, therefore, anti-inflammatory effect test was conducted on white rats based on the ability of drugs to reduce the volume of oedema in the feet of white rats caused by the intraplatar induction of 1% carrageenan. in the analysis of the test then it is known that group given aspirin and group given 1.5 ml sardinella longiceps oil had anti-inflammatory effects against carrageenan indicated by the decrease of the volume of oedema compared to group with aquadest and group with 1 ml sardinella longiceps oil. this result, can also be seen in figure 1 and table 1 describing there was no difference between group given 1.5 ml sardinella longiceps oil and group given aspirin. this indicates that group given 1.5 ml sardinella longiceps oil and group given aspirin had similar effects, so the potential of 1.5 ml sardinella longiceps oil was the same as that of aspirin. however, group given 1.5 ml sardinella longiceps oil had significant differences from group given 1 ml sardinella longiceps oil, which means that the effects of 1 ml sardinella longiceps oil were not as effective as those of 1.5 ml sardinella longiceps oil. similarly, group given aspirin also same at the group given aquadest. it indicates that group given 1.5 ml sardinella longiceps oil had the strongest anti-inflammatory effects compared group given aspirin and 1 ml sardinella longiceps oil. this could be due to omega 3 contained in 1 ml sardinella longiceps oil is less than that in 1.5 ml sardinella longiceps oil. sardinella longiceps is actually one of the most important fish resources because of its great potency and benefits. sardinella longiceps, contains high omega-3 considered to be useful as anti-inflammatory since it contains epa and dha.9 epa could inhibit 5-lox line and cox-2 line. meanwhile, dha could only inhibit cox-2 line.5 epa and dha from fish or fish oil could cause the decreasing of the metabolite pge2, the decreasing of thromboxane a2 (txa2), the increasing of thromboxane a3 (txa3) (weak platelet aggregator and weak vasoconstrictor), and the increasing of prostaglandine i3 (pgi3) triggering the increasing of the total of prostacyclin by increasing pgi3 without reducing pgi2. meanwhile, lipoxigenase (lox) line could not only decrease the formation of leucotriene b4 (ltb4), an inflammatory inductor and a strong leukocytes chemotaxis agent, but can also increase leucotriene b5 (ltb5), an inflammatory inductor and a weak chemotaxis agent.17 the content of epa and dha in 1 ml sardinella longiceps oil, however, is not as much as in 1.5 ml sardinella longiceps oil. this is what causes barriers against cox and lox not as big as on 1.5 ml sardinella longiceps oil. from these results, it can be concluded that sardinella longiceps oil could reduce oedema paw in wistar rats induced with 1% carrageenan. references 1. guyton ac, hall je. buku ajar fisiologi kedokteran. irawati dkk, editor. edisi 11. jakarta: egc; 2008. p. 455. 2. wilmana pf, gunawan sg. farmakologi dan terapi. edisi 5. gunawan sg, editor. jakarta: gaya baru; 2007. p. 262, 248, 407.. 3. hartoyo b. inflammere. dental horison. 2002; iii(9): 24. 4. wiryowidagdo, sudjaswadi, dan sitanggang m. tanaman obat untuk penyakit jantung, darah tinggi, dan kolesterol. cetakan 11. jakarta:cetakan 11. jakarta: agromedia pustaka; 2007. p. 24–5. 5. calder pc. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. am j clin nutr 2006; 83(suppl): 1505s–19s. 6. helmy m, munasir z. pemakaian cetirizine dan kortikosteroid pada penyakit alergi anak. dexa media. 2007; 20(2): 26–9. 7. myers p, espinosa r, parr cs, jones t, hammond gs, dewey ta. sardinella longiceps. the animal diversity web (online). 2008. available from http://animaldiversity.ummz.umich.edu/site/accounts/ classification/path/sardinella_longiceps.html#sardinella longiceps. accessed november 10, 2007. 8. wahyuni s. pengaruh substitusi berbagai kadar minyak ikan lemuru dalam diet terhadap profil lipid serum serta pengaruhnya terhadap peroksidasi lipid tanpa maupun dengan suplementasi vitamin e pada tikus. tesis. pasca sarjana universitas airlangga. 1999. p. 37–45. 9. fadilah s. pengaruh diet minyak ikan lemuru terhadap kadar lipid plasma dan agregasi platelet pada orang sehat. tesis. jakarta: fakultastesis. jakarta: fakultas kedokteran universitas indonesia; 1987. p. 74–81. 10. hanafiah ka. rancangan percobaan, teori dan aplikasi. jakarta: pt raja grafindo persada; 2003. p. 56–60. 11. vinegar r, truax jf, selph jl, johnston pr, venable al, mckenzie kk. pathway to carrageenan-induced inflammation in the hind limb of the rat. fed proc 2007; 46:fed proc 2007; 46: 118–26. 12. higgins je, ap klinbaum. determining sample size in introduction to randomized clinical trials. usa: family health international; 1985. p. 24–5. 13. sr–mjöl. the production process. 2007. available from http://www. srmjol.is/srmjol/content/view/19/34/. accessed november 15, 2007. 14. tangka j. pengaruh pemberian minyak ikan tuna (thunus albhacares) terhadap kadar kolesterol total, kolesterol ldl, kolesterol hdl dan kadar triasilgliserol pada darah tikus rattus norvegicus dengan hiperkolesterolemia. skripsi. surabaya: universitas airlangga; 2004. p. 28–32. 15. soekanto a. the rationale of use non steroid anti-inflammation drug in dentistry. j ked gigi. ed khusus kppikg xii. 2000 nov; 7: 83–6. 16. katzung bg. farmakologi dasar dan klinik. edisi 8. bagian farmakologi fakultas kedokteran universitas indonesia, editor. jakarta: salemba medika; 2008. p. 449–60. 17. simopoulos, artemis p. omega-3 fatty acids in inflammation and autoimmune diseases. journal of the american college of nutrition. 2002; 21(6): 495–505. 10 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 10–13 effect of electrolyzed reduced water on wistar rats with chronic periodontitis on malondialdehyde levels rini devijanti ridwan,1 wisnu setyari juliastuti,1 and r. darmawan setijanto2 1department of oral biology 2department of dental public health faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: periodontal disease is a progressive destructive change that causes loss of bone and periodontal ligaments around the teeth that can eventually lead to its loss. the main bacteria in chronic periodontitis is porphyromonas gingivalis. aggregatibacter actinomycetemcomitans, a pathogen associated with aggressive periodontitis, initiates a proinflammatory response that causes tissue destruction of periodontal, alveolar bone resorption and subsequent tooth loss. electrolyzed reduced water (erw) is an alkaline water, erw not only has a high ph and low oxidation reduction potential (orp), but also contains several magnesium ions. magnesium ions proven effective for the prevention of various diseases. purpose: to analyze the level of malondialdehyde (mda) in wistar rats with cases of chronic and aggressive periodontitis that consumed erw. method: wistar rats were divided into four groups, each group with 10 rats. the first and second group were wistar rat with chronic periodontitis and consume drinking water and erw. the third and fourth group were wistar rat with aggressive periodontitis and consume drinking water and erw. this experiment is done by calculating the levels of mda. the calculation of the levels of mda is done with spectrophotometric assay for mda. result: the results of this experiment show that the level of mda in serum in group that consume erw had decreased significantly different with thegroup that consume drinking water with the statistical test. conclusion: it can be concluded that erw can decrease the mda level in wistar rat with chronic and aggressive periodontitis case. keywords: chronic periodontitis; aggressive periodontitis; electrolyzed reduced water; malondialdehyde level; wistar rat correspondence: rini devijanti ridwan, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: devi.rini@yahoo.co.id introduction electrolysis of water produces reduced water at the cathode and oxidized water at the anode. electrolyzedreduced water (erw) has a very negative oxidationreduction potential. erw is also called alkali electrolysis water, alkaline-ionic water, alkaline cathode water, and alkaline ionized water, based on its physicochemical and physiological aspects. erw indicates an alkaline ph, rich in hydrogen molecules, and has a negative oxidation reduction potential (orp) scavenging activity and reactive oxygen species (ros) scavenging1. erw with high ph and significant negative redox potential (rp) was shown to have superoxide dismutase (sod)-like activity and catalase-like activity, and thus, scavenge active oxygen species and protect dna from damage by oxygen radicals in vitro.2 bioactivity of erw is its antioxidant activity. erw mimics the activity of antioxidant enzymes, such as sod and catalase (cat) by scavenging ros. cellular oxidative damage to dna, rna, and protein molecules caused by ros can be markedly opposed by erw. additionally, erw has a therapeutic effect on various diseases, including diabetes, tumors, and renal disease. reduced water shows high ph, low dissolved oxygen (do), very high molecular dissolved hydrogen (dh), and highly redox potential (rp) values. reduce water, as well as catalase and ascorbic acid, can directly scavenge h2o2. reducing water suppresses the damage of single stranded dna by the active oxygen dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p10-13 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p10-13 1111ridwan, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 10–13 species produced by the oxidation of ascorbic acid cu (ii) by dose dependent, indicating that reduced water can scavenge not only o2 and h2o2, but also o2 and oh. 1,2 periodontitis can be further classified as chronic or aggressive. chronic periodontitis usually has a slow to moderate rate of progression, with local and systemic factors such as plaque, calculus, smoking, and diabetes often contributing to the disease. porphyromonas gingivalis, a gram-negative anaerobe bacteria, has been frequently isolated from lesions in chronic periodontitis patients and is considered an etiological agent of the disease.3 aggressive periodontitis is a type of periodontitis in which periodontal ligament destruction and rapid alveolar bone occur in healthy systemic individuals generally in younger age groups but patients may be older.4 although the prevalence of aggressive periodontitis is much lower than for chronic periodontitis, management of aggressive periodontitis is more challenging than chronic periodontitis due to its strong genetic predisposition as an unmodifiable risk factor. although the prevalence has been reported to be much smaller than for chronic periodontitis, this may result in loss of teeth in affected people if not diagnosed at an early stage and treated appropriately.5 aggressive periodontitis can be distinguished from chronic periodontitis by age onset, rapid disease progression rate, and related subgingival microflora composition, changes in host immune response, and family aggregation of diseased individuals.6 the presence of inflammation of chronic periodontitis have resulted in an influx of immune cells use a lot of oxygen, causing excess reactive oxygen species (ros) production.7 oxidative stress causes oxidative damage to lipids that can be detected by elevated levels of malondialdehyde (mda) in the cells.8 in normal circumstances in cell there is a balance between ros generation and antioxidant activity.9,10 if there is interference on the balance it will cause oxidative stress that can damage cell components. this research aims to study the role of erw to mda level which is one biomarker of their oksidative stress on wistar rats with chronic periodontitis. materials and methods this study using wistar rats as animals model. fourty wistar rats divided into four groups, ten wistar rat as the first group was wistar rats with chronic periodontitis that induced with porphyromonas gingivalis bacteria and consume erw (ph8.5). second group was ten wistar rats with chronic periodontitis and consume with drinking water. the other group were wistar rat that induced with aggregatibacter actinomycetemcomitans as a aggressive periodontitis models. twenty wistar rat with aggressive periodontitis consume erw and drinking water. mda samples for examination were taken from wistar rat blood. as much as 5 cc of blood drawn by using a syringe inserted into tubes that had contained edta, then centrifuged at 3500 rpm for 5 minutes. liquid blood plasma that has been separated from the solid part of blood was transferred to mda microplate for examination. mda level measurement is done using elisa method with a kit mda586 bioxytech on spectrophotometry. result in chronic periodontitis case, kolmogorof smirnov test showed that the data were normally distributed (p<0.05) and the value levene test shows that the data homogeneous (p>0.05) so that it can proceed with different test one-way anova. one-way anova test results show the value of p <0.05 thus concluded that there are significant differences between all groups. then followed by tukey hsd post hoc test to see differences in each group. there are significant differences (p<0.05) between the control group 7 days with treatment group seven days, as well as the control group and the treatment group 14 days to 14 days. the results showed a decrease in mda levels of a group of wistar rats with chronic periodontitis by erw for 7 and 14 days than in the group given drinking water (table 1). in table 1 shows 7 16. itoh t, fujita y, ito m, masuda a, ohno k, ichihara m, kojima t, nozawa y, ito m. molecular hydrogen suppresses fcepsilonri-mediated signal transduction and prevents degranulation of mast cells. biochem biophys res commun 2009; 389(4): 651-6. table 1. mean and standart deviation of mda levels in wistar rat with chronic periodontitis that consume erw and drinking water group level of mda (µm) sd kolmogorof smirnov anova p control group (7 days) 0.591 0.032 0.644 0.193 0.000 erw (7 days) 0.304 0.026 0.991 control group (14 days) 0.564 0.057 0.991 erw (14 days) 0.311 0.033 0.999 figure 1. mean number of mda levels in wistar rat. table 2. mean and standart deviation of mda levels in wistar rat with aggressive periodontitis that consume erw and drinking water group level of mda (µm) sd kolmogorof smirnov anova p control group (7 days) 0.646 0.041 0.995 0.001 0.000 erw (7 days) 0.357 0.118 0.749 0.591 0.304 0.564 0.311 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 le ve l o f m d a group control erw control erw 7 days 7 days 14 days 14 days figure 1. mean number of mda levels in wistar rat. table 1. mean and standart deviation of mda levels in wistar rat with chronic periodontitis that consume erw and drinking water group level of mda (µm) sd kolmogorof smirnov anova p control group (7 days) 0.591 0.032 0.644 0.193 0.000 erw (7 days) 0.304 0.026 0.991 control group (14 days) 0.564 0.057 0.991 erw (14 days) 0.311 0.033 0.999 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p10-13 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p10-13 12 ridwan, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 10–13 significantly differences between the groups of wistar rats with chronic periodontitis with erw administration for 7 days and 14 days compared to the group with drinking water same at figure 1. kolmogorof smirnov test in aggressive periodontitis showed that the data were normally distributed (p<0.05) and the value levene test shows that the data are not homogeneous (p<0.05) and so can not proceed with different test one-way anova. different test done using kruskal wallis test. kruskal wallis test results show the value of p<0.05 thus concluded that there are significant differences between all groups. then proceed with the test mann-witney to see the difference in the two groups. there are significant differences (p<0.05) between the control group 7 days with treatment group and control 7 days to 14 days; the control group and the treatment group 14 days to 14 days; as well as the treatment group and control 7 days to 14 days (table 2). the results showed a decrease in mda levels of a group of wistar rats with aggressive periodontitis by erw for 7 and 14 days than in the group given drinking water (table 2). in table 2 shows significantly differences between the groups of wistar rats with aggressive periodontitis with erw administration for 7 days and 14 days compared to the group that consume drinking water. discussion in the group of wistar rats with chronic and aggressive periodontitis that administration by erw decreased levels of mda, it will show a decrease in ros levels due in normal circumstances there is a balance between ros and antioxidant activity in cell. if the balance is disrupted will cause oxidative stress which can causing damage to the cell components. one of the damages caused by the condition oxidative5-7 produce a number of compounds such as epoxides, hydrocarbons and aldehydes. between aldehyde compounds produced were mda. some antioxidants endogenous that acts to prevent the occurrence of oxidative damage is the mnsod, catalase and reduced glutathione (gsh). the decrease of mda in groups of wistar rats with chronic and aggressive periodontitis that administration by erw at 7 and 14 days showed the role of erw that have the potential of oxidation and reduction of low and high ph capable of eliminating reactive oxygen in cells and were able to cause damage to the plasmid dna of bacteria, this situation is consistent with research on the park et al. in 2012.1,11 erw produces reduced water at the cathode and oxidized water at the anode. erw has an extremely negative oxidation-reduction potential. erw scavenges cellular reactive oxygen species (ros) and suppresses single-strand breaks of plasmid dna in bacteria.11 molecular hydrogen has ability as an effective antioxidant treatment,12 based on its free radical scavenger properties, and has been successfully used in a variety of pathological conditions involving acute oxidative stress. 13,14 the main molecular target of molecular hydrogen is not clearly understood. the main mechanism of action is advised to rinse hydroxyl radicals (ho) and peroxynitrite (onoo) in particular, thereby reducing oxidative damage to membrane lipids and dna.9 in addition, recent reports show a consistent effect on gene and protein expression and phosphorylation.15,16 based from these research it can be concluded that the erw administration for 7 days and 14 days resulted in decreased levels of mda in blood of wistar rats with chronic and aggressive periodontitis. acknowledgement this research was supported by the director general of higher education which has provided funding for this research through dana hibah penugasan penelitian unggulan perguruan tinggi baru in 2016. references 1. park sk, park sk. electrolyzed-reduced water increases resistance to oxidative stress, fertility, and lifespan via insulin/igf-1-like signal in c. elegans. biol res 2013; 46(2): 147-52. 2. shirahata s, hamasaki t, teruya k. advanced research on the health benefit of reduced water. trends in food science & technology 2012; 23: 124-31. 3. kobayasi t, kaneko s, tahara t, hayakawa m, abiko y, yoshie h. antibody responses to porphyromonas gingivalis hemagglutinin a and outer membrane protein in chronic periodontitis. j periodontol 2006; 77(3): 364-9. 4. american academy of periodontology. parameter on aggressive periodontitis. j periodontol 2000; 71(5 suppl): 867-9. 5. demmer rt, papapanou pn. epidemiologic patterns of chronic and aggressive periodontitis. periodontol 2000, 2010; 53: 28-44. table 2. mean and standart deviation of mda levels in wistar rat with aggressive periodontitis that consume erw and drinking water group level of mda (µm) sd kolmogorof smirnov anova p control group (7 days) 0.646 0.041 0.995 0.001 0.000 erw (7 days) 0.357 0.118 0.749 control group (14 days) 0.842 0.066 0.930 erw (14 days) 0.212 0.053 0.991 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p10-13 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p10-13 1313ridwan, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 10–13 6. armitage gc, cullinan mp. comparison of the clinical features of chronic and aggressive periodontitis. periodontol 2000, 2010; 53: 12-27. 7. hendler a, mulli tk, hughes fj, perrett d, bombardieri m, hourihaddad y, weiss ei, nissim a. involvement of autoimmunity in the pathogenesis of aggressive periodontitis. j dent res 2010; 89(12): 1389-94 8. zainuri m, wanandi si. aktivitas spesifik manganese superoxide dismutase (mnsod) dan katalase pada hati tikus yang diinduksi hipoksia sistemik: hubungannya dengan kerusakan oksidatif. media litbang kesehatan 2012; 22(2): 87-92 9. bag a, bag n. target sequence polymorphism of human manganese superoxide dismutasegene and its association with cancer risk: a review. cancer epidemiol biomarkers prev 2008; 17(12): 3298305. 10. harju t, kaarteenaho-wiik r, sirviö r, pääkkö p, crapo jd, oury td, soini y, kinnula vl. manganese superoxide dismutase is incresed in the airways of smokers’ lungs. eur respir j 2004; 24(5): 765-71. 11. park sk, kim jj, yu ar, leemy, park sk. electrolyzed reduced water confers increased resistance to environmental stresses. mol cell toxicol 2012; 8(3): 241-7. 12. ohsawa i, ishikawa m, takahashi k, watanabe m, nishimaki k, yamagata k, katsura k, katayama y, asoh s, ohta s. hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. nat med 2007; 13(6): 688-94. 13. ohta s. molecular hydrogen is a novel antioxidant to efficiently reduce oxidative stress with potential for the improvement of mitochondrial diseases. biochim biophys acta 2012; 1820(5): 58694. 14. huang c, kawamura t, toyoda y, nakao a. recent advances in hydrogen research as a therapeutic medical gas. free radic res 2010; 44(9): 971-82. 15. itoh t, hamada n, terazawa r, ito m, ohno k, ichihara m, nozawa y, ito m. molecular hydrogen inhibits lipopolysaccharide/interferon c-induced nitric oxide production through modulation of signal transduction in macrophages. biochem biophys res commun 2011; 411(1): 143-9. 16. itoh t, fujita y, ito m, masuda a, ohno k, ichihara m, kojima t, nozawa y, ito m. molecular hydrogen suppresses fcepsilonrimediated signal transduction and prevents degranulation of mast cells. biochem biophys res commun 2009; 389(4): 651-6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p10-13 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p10-13 137 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 137–141 original article immunohistochemical differential expression of p16 proteins in follicular type and plexiform type ameloblastoma haris budi widodo1, anung saptiwulan2, helmi hirawan3, christiana cahyani prihastuti1, tirta wardana4,5 1department of oral biology, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 2undergraduate student, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 3department of oral surgery, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 4department of biomedicine, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 5integrative laboratory, faculty of medicine, jenderal soedirman university, purwokerto, indonesia abstract background: differences in histopathological features that describe the growth mechanism and biological behaviour of follicular and plexiform ameloblastomas are associated with benign, aggressive and destructive tumour markers. p16 has inhibitory interactions between cyclin d and cdk 4/6 to block the cell cycle and alterations related to severity. purpose: this study intends to evaluate and determine differential expressions of p16 protein in follicular and plexiform ameloblastomas. methods: this is a descriptive analytics study. a total of 21 specimens consisting of follicular and plexiform ameloblastomas and healthy gingiva tissues as the negative control were examined using the immunohistochemistry assay. the analysis of p16 protein expression was interpreted by immunoreactive scoring. statistical analysis was conducted using spss software with the mann–whitney test. a p-value <0.05 shows the significance of the change in expression. results: an increased expression of p16 protein was found in the follicular ameloblastoma type (2.13 ± 1.808) and the plexiform type (4.44 ± 2.506) in comparison to the negative control group (0 ± 0). the increase of p16 expression in the follicular and plexiform ameloblastomas was significant compared to the negative control group (p-value <0.05); however, there was no significant difference between either type of ameloblastoma (p-value >0.05). conclusion: the highest intensity of p16 protein expression was found in the plexiform type, even though it was not significantly different from the follicular type ameloblastoma. keywords: ameloblastomas; follicular; immunohistochemistry; plexiform; p16 protein expression correspondence: tirta wardana, department of biomedicine, dental medicine study programme, faculty of medicine, jenderal soedirman university, purwokerto, indonesia. email: tirta.wardana@unsoed.ac.id introduction the most common types of ameloblastomas are follicular and plexiform, whose clinical findings and specific clinical behaviours are associated with histopathological appearance. given that appearance, this tumour shows signs of being a benign tumour, although clinically, it is aggressive and destructive.1 ameloblastoma is an odontogenic tumour in tooth-forming tissue that grows slowly and locally invasive. usually, the patient is unaware until the inflammation enlarges. its recurrence is high, and its spread is expansive and infiltrative, giving the impression of malignancy.2 ameloblastoma is commonly found in the mandibular and maxillary areas, with swelling resulting in facial deformity. on clinical examination, ameloblastoma does not have a specific feature because the stain of the tumour tissue is the same as the surrounding tissue.3 in addition, the consistency can be soft or hard, with no pain and paraesthesia and no ulceration of the mucosa around the tumour tissue.4 the growth of ameloblastoma is influenced by oral infection, tooth extraction, trauma to the teeth or jaws and genetic factors, such as tumour suppressor genes (tsg) and oncogenes (c-myc gene and ras gene). the loss of function of tsg, which plays a role in controlling cell proliferation and preventing cells from becoming malignant, causes tumour formation.5–7 p16 is a group of tsg called mts1 (multiple tumour suppressor 1), cdkn2 (cyclin-dependent kinase inhibitor 2) and p16ink4a, which functions as an inhibitor of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p137–141 mailto:tirta.wardana@unsoed.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p137-141 138widodo et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 137–141 the interaction between cyclin d and cyclin-dependent kinase (cdk) 4 and 6; it blocks the cell division cycle in g1 phase-control points.8,9 high throughput technology analyses using microarray gene expression profiling offer a technology to classify the tumour subtypes, such as breast cancer,10 bladder cancer11 and pancreatic adenocarcinoma.12 however, because of the high cost and complexity of laboratory procedures, alternate immunochemistry (ihc) assay is used to identify subtypes of tumour classification. the identification of the subtype and the clinical impact of the tumour can be used to determine the success of treatment due to tumour biological properties and behaviour.13 based on its role, p16 can be used as a marker of the cellcycle phase to study pathophysiological conditions, such as abnormal cell differentiation and tumour prognosis.14,15 this study aims to determine the differential expression of p16 mutant proteins in the formation of follicular and plexiform ameloblastomas. this may inform future investigations into the molecular mechanism and increase potential therapeutics for indonesian ameloblastoma. materials and methods this descriptive analytics study uses 21 paraffin blockstained specimens from the department of anatomical pathology laboratory of prof. dr. margono soekarjo hospital, purwokerto and the asri medical centre (amc), yogyakarta. the paraffin blocks were cut with a microtome, deparaffinised, rehydrated and then subjected to ihc staining to observe the p16 expression using the anti-cdkn2a/p16ink4a antibody (abcam ab108349, usa) with a 30-minute incubation using 1:100 dilution. counterstaining using mayer’s hemalum was performed to determine the differences in three groups: follicular and plexiform types of ameloblastomas as well as healthy gingiva tissue as a negative control.16,17 all procedures followed the manufacturer’s recommendations. observation of the p16 protein expression was performed by 400x magnification in five viewpoints using a light microscope camera with optilab® (motic® b2-series, usa) and software raster image (us national institutes of health, usa). observation with a grading picture of cells recorded as positive and the reaction intensity were as follows:13,18 grade 0 (no cells recorded) and negative reaction intensity; grade 1 (>10%–50% of recorded cells) and weak staining intensity; grade 2 (>10%–50% of recorded cells) and medium staining intensity (2); grade 3 (>50%–80% of recorded cells) and strong staining intensity; and grade 4 (>80% of recorded cells) and powerful staining intensity. based on a previous study, immunohistochemistry p16 expression analysis was carried out based on grading status and intensity scores, with expression scores ranging from 0 to 12.18 immunoreactive scores are categorised as 1–4 positive scores (+), weak definition; 5–8 positive scores (++), moderate definition; and 8–12 positive scores (+++), strong definition. data analysis was carried out using spss software version 22 (ibm corp version 23, chicago, il). cohen’s kappa coefficient was used to test the validity of the examination by two observers. the analysis of differences in p16 expression was carried out using the kruskal–wallis non-parametric test. the mann–whitney test was performed to determine significant differences between groups; a p-value <0.05 indicates a significant difference. results in this study, the specimen sample consisted of follicular and plexiform ameloblastomas (table 1). immunohistochemical staining was conducted to evaluate the expression of p16 by discolouration. the brown and dark brown discolouration in follicular and plexiform ameloblastoma specimens showed mutant p16 expressions (figure 1). the differential in colour intensity indicated weak positive, table 1. mean of p16 mutant protein expression tissue specimens total sample average std. deviation std. error mean plexiform ameloblastoma 9 4.44 2.506 0.835 follicular ameloblastoma 8 2.13 1.808 0.639 healthy gingiva epithelial tissue control group 4 0 0 0 a b c figure 1. positive p16 mutant protein expression by immunohistochemical staining in study groups (a) plexiform type ameloblastoma (x400); (b) follicular type ameloblastoma (x400); and (c) healthy gingival epithelium tissue (x40). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p137–141 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p137-141 139 widodo et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 137–141 moderately positive and strong positive differences in immunosuppression scores (figure 1). the mean value of the difference in the expression of mutant p16 from the results of immunohistochemical examinations in the sample group are shown in table 1. two observers analysed mutant p16 expression by multiplying the positive cell grading and the reaction intensity from five fields of view observations. the kappa coefficient test analysis showed a significance value of p = 0.000 with an ideal value of 0.598. in our study, the mean p16 expression for the follicular ameloblastoma group was 2.13 ± 1.808, for the plexiform ameloblastoma group 4.44 ± 2.506 and for the healthy gingival epithelium control group 0 ± 0. the analysis of differences in the expression of p16 showed a significant difference (p-value <0.05) in the follicular ameloblastoma, the plexiform ameloblastoma and the control groups (can be seen in table 2). the expression of p16 mutant proteins showed differences between the control group compared with the follicular type ameloblastoma (p-value <0.05) and the control group compared with the plexiform type ameloblastoma (p-value <0.05), whereas the follicular type ameloblastoma compared with the plexiform type ameloblastoma group showed no significant difference (p-value = 0.071; p-value >0.05) (figure 2). discussion ameloblastoma is a tumour with a high incidence, unrelated to age and gender and with no specific clinical symptoms. histopathological and radiographic examinations are the gold standard for diagnosis in the incidence of ameloblastoma, with several types often found, namely follicular, plexiform and adenomatous. different types of ameloblastomas can represent characteristics such as aggressiveness, recurrence and severity. increased p16 expression in the incidence of ameloblastoma can provide an overview of the severity of its role as a tumour-suppressor gene in inhibiting the uncontrolled proliferation process.19,20 this study found a significant increase of p16 expression in both types of ameloblastomas compared to the healthy gingival tissue, with the highest expression of p16 shown in the plexiform type ameloblastoma. increased expression of the p16 mutant ameloblastoma indicated the incidence of a malignancy. on the other hand, the wild-type p16 protein is difficult to detect in normal conditions because it has a short half-life.21 increased expression of mutant p16 causes failure of cell proliferation in the g1 phase so that it is often found in follicular and plexiform types of ameloblastomas.22,23 the imbalance of cell cycle regulatory pathways involving p16-rb can impair cell proliferation, ultimately leading to unrestricted proliferation and tumourigenesis.22,24,25 mutant p16 expression was not found in the regular gingival epithelial control group in normal cell proliferation, implying there were no malignant changes in cells. the p16 protein expressed in the g1 phase is a product of the cdkn2a gene, a tumour suppressor gene (antioncogene) that can prevent the overgrowth of cells in the g1 phase.26 the p16 protein acts as a negative regulator of cell proliferation. in normal cells, wild-type p16 is expressed and binds to cdk4 and cdk6 so that free cyclin d and protein kinase complexes are inactive.27 decrease or inactivation of p16 causes cdk4/6 to bind to cyclin d, causing an active protein kinase complex. the protein kinase complex triggers the phosphorylation of prb so that prb is inactive. inactivation of prb causes the release of the transcription factor e2f so that the cell enters the s phase. continuous e2f transcription will cause normal cells to become ameloblastoma.28–30 the results showed an increase in mutant p16 expression in follicular and plexiform ameloblastoma types. these results are supported by the research of kumamoto et al.(2001), who demonstrated over-expression of p16 in most neoplastic cells from ameloblastoma so that odontogenic epithelium would be found to be under the control of this oncoprotein.31 another study also showed the immunohistochemical expression of p16 in odontogenic tumours, including ameloblastomas, finding a particularly positive trend in tumour cell nuclei for tumours with low recurrence risk and a similar reaction for the nucleus and cytoplasm of tumours with high recurrence rates.32 l ev el e xp re ss io n fo lli cu la r pl ex ifo rm co nt ro l figure 2. immunohistochemical expressions of p16 mutant proteins on follicular and plexiform ameloblastomas and healthy gingiva control group. table 2. different expressions of p16 mutant proteins variable i variable ii significance control follicular ameloblastoma 0.028 control plexiform ameloblastoma 0.005 follicular ameloblastoma plexiform ameloblastoma 0.071 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p137–141 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p137-141 140widodo et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 137–141 the difference in p16 expression in follicular type ameloblastoma was not significantly different from the plexiform type ameloblastoma group. this shows that the two groups have similar characteristics of mutant p16 expression. the results of this study are supported by a previous study that showed that there was no statistically significant difference in the expression of positive p16 in the central cells of low-risk and high-risk odontogenic tumours; in both groups, the results were equally high.32 another study showed that the expression of the tumour suppressor p16 was not significantly different in odontogenic keratoses and unicystic ameloblastomas.13 this suggests that the invasive growth of odontogenic keratosis and the cystic behaviour of unicystic ameloblastoma are closely related to the state of p16 expression in the lesional epithelium. a candidate tumour marker can be used to analyse mutant p16 protein expression changes in follicular and plexiform ameloblastomas. however, it cannot be used as a progression marker between follicular and plexiform ameloblastoma groups. this study has several limitations: the limited number of specimens involved and the clinical data that may have risk factors associated with p16 protein expression. in addition, we believe that our findings impact the understanding of p16 protein expression in different types of ameloblastomas. there are significant differences in p16 protein expression using immunohistochemical analysis between the follicular and plexiform types of ameloblastomas compared to healthy tissue. in addition, the highest increase in the expression of the p16 protein is shown in the plexiform type’s ameloblastoma. acknowledgments the authors would like to thank the educators and teaching staff of the faculty of medicine, jenderal soedirman university; the anatomical pathology laboratory of the regional general hospital prof. dr margono soekarjo purwokerto; the anatomical pathology laboratory of faculty of medicine, gadjah mada university yogyakarta; and the anatomical pathology laboratory of dr sardjito general hospital yogyakarta for their help. all authors declare there is no conflict of interest in this study. references 1. gomes cc, de sousa sf, gomez rs. craniopharyngiomas and odontogenic tumors mimic normal odontogenesis and share genetic mutations, histopathologic features, and molecular pathways activation. oral surg oral med oral pathol oral radiol. 2019; 127(3): 231–6. 2. staines ks, crighton a. benign oral and dental disease. in: watkinson jc, clarke rw, editors. scott-brown’s otorhinolaryngology and head and neck surgery. 8th ed. boca raton: crc press; 2018. p. 699–718. 3. speight pm, takata t. new tumour entities in the 4th edition of the world health organization classification of head and neck tumours: odontogenic and maxillofacial bone tumours. virchows arch. 2018; 472(3): 331–9. 4. doroy gar, gelbolingo n. primary intraosseous carcinoma of the mandible: a case report. philipp j otolaryngol head neck surg. 2020; 35(1): 56–9. 5. dean ke. a radiologist’s guide to teeth: an imaging review of dental anatomy, nomenclature, trauma, infection, and tumors. neurographics. 2020; 10(5): 302–18. 6. schwartz jl, sroussi h. genomic foundation for medical and oral disease translation to clinical assessment. in: sonis st, villa a, editors. translational systems medicine and oral disease. st. louis: elsevier; 2020. p. 17–92. 7. du y, cheng y, su g. the essential role of tumor suppressor gene ing4 in various human cancers and non-neoplastic disorders. biosci rep. 2019; 39(1): bsr20180773. 8. sánchez-martínez c, gelbert lm, lallena mj, de dios a. cyclin dependent kinase (cdk) inhibitors as anticancer drugs. bioorg med chem lett. 2015; 25(17): 3420–35. 9. tripathy d, bardia a, sellers wr. ribociclib (lee011): mechanism of action and clinical impact of this selective cyclin-dependent kinase 4/6 inhibitor in various solid tumors. clin cancer res. 2017; 23(13): 3251–62. 10. shan m, zhang x, liu x, qin y, liu t, liu y, wang j, zhong z, zhang y, geng j, pang d. p16 and p53 play distinct roles in different subtypes of breast cancer. plos one. 2013; 8(10): e76408. 11. dadhania v, zhang m, zhang l, bondaruk j, majewski t, siefkerradtke a, guo cc, dinney c, cogdell de, zhang s, lee s, lee jg, weinstein jn, baggerly k, mcconkey d, czerniak b. metaanalysis of the luminal and basal subtypes of bladder cancer and the identification of signature immunohistochemical markers for clinical use. ebiomedicine. 2016; 12: 105–17. 12. moffitt ra, marayati r, flate el, volmar ke, loeza sgh, hoadley ka, rashid nu, williams la, eaton sc, chung ah, smyla jk, anderson jm, kim hj, bentrem dj, talamonti ms, iacobuziodonahue ca, hollingsworth ma, yeh jj. virtual microdissection identif ies d istinct t umora nd st roma-specif ic subt ypes of pancreatic ductal adenocarcinoma. nat genet. 2015; 47(10): 1168–78. 13. razavi sm, poursadeghi h, aminzadeh a. immunohistochemical comparison of cyclin d1 and p16 in odontogenic keratocyst and unicystic ameloblastoma. dent res j (isfahan). 2013; 10(2): 180–3. 14. ogrodnik m, salmonowicz h, jurk d, passos jf. expansion and cell-cycle arrest: common denominators of cellular senescence. trends biochem sci. 2019; 44(12): 996–1008. 15. canaud g, bonventre j v. cell cycle arrest and the evolution of chronic kidney disease from acute kidney injury. nephrol dial transplant. 2015; 30(4): 575–83. 16. cadavid amh, araujo jp, coutinho-camillo cm, bologna s, junior cal, lourenço sv. ameloblastomas: current aspects of the new who classification in an analysis of 136 cases. surg exp pathol. 2019; 2(1): 17. 17. verneuil a, sapp p, huang c, abemayor e. malignant ameloblastoma: classification, diagnostic, and therapeutic challenges. am j otolaryngol. 2002; 23(1): 44–8. 18. surowiak p, materna v, maciejczyk a, pudelko m, suchocki s, kedzia w, nowak-markwitz e, dumanska m, spaczynski m, zabel m, dietel m, lage h. decreased expression of p16 in ovarian cancers represents an unfavourable prognostic factor. histol histopathol. 2008; 23(5): 531–8. 19. wang y, chen s, yan z, pei m. a prospect of cell immortalization combined with matrix microenvironmental optimization strategy for tissue engineering and regeneration. cell biosci. 2019; 9(1): 7. 20. lazăr cs, şovrea as, georgiu c, crişan d, mirescu şc, cosgarea m. di f ferent pat ter ns of p16i n k4a i m mu noh istochem ica l expression a nd t hei r biolog ica l i mpl icat ions i n la r y ngea l squamous cell carcinoma. rom j morphol embryol. 2020; 61(3): 697–706. 21. li m, yang j, liu k, yang j, zhan x, wang l, shen x, chen j, mao z. p16 promotes proliferation in cervical carcinoma cells through cdk6-hur-il1a axis. j cancer. 2020; 11(6): 1457–67. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p137–141 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p137-141 141 widodo et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 137–141 22. lee sk, kim ys. current concepts and occurrence of epithelial odontogenic tumors: i. ameloblastoma and adenomatoid odontogenic tumor. korean j pathol. 2013; 47(3): 191–202. 23. diniz mg, guimarães bva, pereira nb, de menezes ghf, gomes cc, gomez rs. dna damage response activation and cell cycle dysregulation in infiltrative ameloblastomas: a proposed model for ameloblastoma tumor evolution. exp mol pathol. 2017; 102(3): 391–5. 24. boscolo-rizzo p, da mosto mc, rampazzo e, giunco s, del mistro a, menegaldo a, baboci l, mantovani m, tirelli g, de rossi a. telomeres and telomerase in head and neck squamous cell carcinoma: from pathogenesis to clinical implications. cancer metastasis rev. 2016; 35(3): 457–74. 25. merlin jpj, rupasinghe hpv, dellaire g, murphy k. role of dietary antioxidants in p53-mediated cancer chemoprevention and tumor suppression. oxid med cell longev. 2021; 2021: 9924328. 26. lapak km, burd ce. the molecular balancing act of p16(ink4a) in cancer and aging. mol cancer res. 2014; 12(2): 167–83. 27. pack lr, daigh lh, chung m, meyer t. clinical cdk4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin d-cdk4 to inhibit cdk2. nat commun. 2021; 12(1): 3356. 28. ombiro em, kwena a, melly e, kamau t, maiyoh gk. genotypes a nd preva lence of h igh-r isk huma n papillomavi r us a mong patients diagnosed with head and neck cancer at alexandria ca ncer cent re. jco glob o ncol. 2020; 6(supplement 1): 30–30. 29. mahale s, bharate sb, manda s, joshi p, bharate ss, jenkins pr, vishwakarma ra, chaudhuri b. biphenyl-4-carboxylic acid [2-(1hindol-3-yl)-ethyl]-methylamide (ca224), a nonplanar analogue of fascaplysin, inhibits cdk4 and tubulin polymerization: evaluation of in vitro and in vivo anticancer activity. j med chem. 2014; 57(22): 9658–72. 30. salari fanoodi t, motalleb g, yegane moghadam a, talaee r. p21 gene expression evaluation in esophageal cancer patients. gastrointest tumors. 2015; 2(3): 144–64. 31. kumamoto h, kimi k, ooya k. detection of cell cycle-related factors in ameloblastomas. j oral pathol med. 2001; 30(5): 309–15. 32. artese l, piattelli a, rubini c, goteri g, perrotti v, iezzi g, piccirilli m, carinci f. p16 expression in odontogenic tumors. tumori. 2008; 94(5): 718–23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p137–141 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p137-141 subject index volume 45 38% hydrogen peroxide, 89 a. actinomycetemcomitans, 181 acetic acid, 156 actinobacillus actinomycetemcomitans, 22 adhesin, 181 aggregatibacter actinomycetemcomitans, 234 aggressive periodontitis, 181, 192 alcohol, 216 alpinia galanga rhizome, 84 purpurata rhizome, 84 alveolar bone, 228 bone loss, 102 analgesic effect, 156 anchorage, 121 antibacteri, 217 bcl-2, 203 betel leaf extract, 97 biopsy, 68 bite deepening, 121 mark, 73 bleaching agent, 43 bone tissue engineering, 150 brown algae, 177 caffeic acid, 52 calcium, 93 candida albicans, 28, 84, 234 candidiasis, 84 carbonate apatite, 150 caries, 79, 133 casein phosphopeptide-amorphous calcium phosphate, 93 cashew stem bark, 212 cd4, 114 cephalometrics, 107 children, 127 chitosan, 17, 150 chlorine dioxide, 22 chlorogenic acid, 52 chronic, 68 periodontitis, 192 class ii major histocompatibility complex, 133 combination of aloe vera and graft, 228 complete unilateral cleft lip and palate, 107 complicated crown fracture, 187 composite resins, 43 craniofacial morphology, 107 crude toxin of aggregatibacter actinomycetemcomitans serotype-b, 39 deacetylation degree, 17 dental care service, 48 pulp, 133 plaque, 208 deoxypyridinoline, 102 diabetes mellitus, 22 differential moment, 121 digital photography, 73 discoloration, 43 dmf-t index, 35 dmt-1, 202 dna damage, 202 fragmentation , 39 down syndrome, 6, 79 elderly, 138 electroporation, 197 enamel, 93 endodontic, 1 endorestoration, 187 endotoxin, 144 fetal growth restriction, 144 fibroblast growth factor 2, 228 fixed orthodontic, 221 fluor concentration, 35 forensic, 73 gene polymorphisms, 192 gingival crevicular fluid, 102 hemimaxillectomy, 172 henoch-schönlein purpura, 127 hiv, 114 hiv/aids, 28 hsv, 114 human malignant burkitt's lymphoma, 197 hyposalivation, 138 igg, 114 inhibition of biofilm formation, 212 immediate overdenture, 1 infectious foci, 127 inflammation, 89 infrared, 73 insulin, 22 interleukin-1a +4845g→t, 192 irreversible hydrocolloid, 177 lactobacilli, 217 liquid smoke of coconut shell, 156 madura strait, 177 malocclusion, 6 management, 68 masticatory functional analysis, 59 maxilla reconstruction, 172 medication, 138 mmp-8, 181 mouth rinse, 221 mutant type p27kip1, 197 mtt assay, 97 ni2+, 202 omplicated crown fracture, 187 oral candidosis, 28 orthodontic, 6 ossifying fibroma, 172 osteoblast, 228 osteocalcin, 228 parp-1, 39 pcr-rflp, 192 periodontitis, 22, 144 pge2, 161 physical characteristic, 177 pmn, 161 polypropylene mesh, 172 porphyromonas gingivalis, 144 pregnancy, 144 proinflammatory cytokines, 97 pomegranate juice, 221 prpopolis, 208 prosthodontic rehabilitation, 59 rapd, 28 red betel leaf infusion (piper crocatum), 12 relining , 1 robusta coffee bean extract, 52 saliva, 35 salivary ph, 234 salvadora persica, 217 saponin, 12 scaffolds, 150 scrubbing technique, 167 segmented arch, 121 self-adhering flowable composite, 167 severe attrition, 59 shear bond strength, 167 shrimp shell waste, 17 siga, 79 siwak, 217 smear layer, 12 streptococcus mutans, 217, 230 sanguinis, 212 surfactant, 12 the assessments of patients, 48 the expectations of patients, 48 the healing process, 52 the satisfactory of patients, 48 tooth movement , 161 paste, 208, 212 traumatic dental injury, 187 ulcer, 68 ultrasonic cleaning bath, 216 vital tooth bleaching, 89 xerostomia, 138 authors index volume 45 amaliah, rizni, 212 andriani, ardiny, 89 apriasari, maharani laillyza, 68 arifin, rafinus, 161 bramantoro, taufan, 48 dharmayanti, agustin wulan suci, 102 dwi ariani, maretaningtias, 150 dwi cs, meircurius, 156 ermawati, tantin, 22 fakhrurrazi, 84 gani, basri a., 234 goenharto, sianiwati, 6 haniastuti, tetiana, 133 jaya, ferry, 167 kenisa, �orinta putri, 52 kresnoadi, utari, 228 kusumawardani, banun, 144 laksono, harry, 59 listyasari, nurin aisyiyah, 208 ma’at, suprapto, 97 mahanani, erlina sih, 217 pangabdian, fani, 12 pertiwi, arlette suzy puspa, 127 prahasanti, chiquita, 192 prananingrum, widyasri, 93 pratiwi, vidyana, 35 r. soesanto, 172 rahayu, retno puji, 28 ridwan, rini devijanti, 181 rosdiana, 79 sampoerno, galih, 43 setiawatie, ernie maduratna, 39 soesetijo, fx. ady, 1, 202 sufiawati, irna, 114 sularsih, 17 supriatno, 198 utomo, haryono, 73, 221 utomo, sigit handoko, 107 widiyanti, prihartini, 177 wijaya, harryanto, 121 wimardhani, �uniardini septorini, 138 zubaidah, nanik, 187 thanks to editor dental journal (majalah kedokteran gigi) volume 45 number 1 march 2012: 1. sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) volume 45 number 2 june 2012: 1. prof. dr. regina titi christinawati, drg., m.sc.(oral biology – gadjah mada university) 2. endrajana, drg., ms., sp.bm (oral and maxillofacial surgery – airlangga university) 3. kus harijanti, drg., ms., sp.pm(oral medicine – airlangga university) 4. dr. retno indrawati, drg., m.si (oral biology – airlangga university) 5. dr. theresia indah budhy, drg., m.kes.(oral biology – airlangga university) 6. dr. indah listiana kriswandini, drg., m.kes. (oral biology – airlangga university) 7. david buntoro kamadjaja, drg., mds., sp.bm.(oral and maxillofacial surgery – airlangga university) 8. agung krismariono, drg., m.kes., sp.perio (periodontic – airlangga university) volume 45 number 3 september 2012: 1. prof. dr. drg. iwa sutardjo rus sudarso, su., sp.kga(k) (pediatrics dentistry – universitas gadjah mada) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentstry – universitas airlangga) 3. kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) 4. dr. indah listiana kriswandini, drg., m.kes. (oral biology – universitas airlangga) 5. indeswati diyatri, drg., ms (oral biology – universitas airlangga) 6. wisnu setyari, drg., m.kes.(oral biology – universitas airlangga) 7. david buntoro kamadjaja, drg., mds., sp.bm. (oral and maxillofacial surgery – universitas airlangga) volume 45 number 4 december 2012: 1. prof. dr. regina titi christinawati, drg., m.sc.(oral biology – gadjah mada university) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) 3. prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) 4. dr. retno indrawati, drg., msi. (oral biology – universitas airlangga) 5. dr. theresia indah budhy, drg., m.kes. (oral pathology & maxillofacial – universitas airlangga) 6. aster arjani, drg., ms (oral biology – universitas airlangga) 7. wisnu setyari, drg., m.kes (oral biology – universitas airlangga) dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and 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reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. �u f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new �ork, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; �ork, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. �ogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. �amagishi h, hiroe a, nishio h, miki k, tawada �. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 235 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 235–239 case report management of impacted maxillary canine with surgical exposure and alignment by orthodontic treatment meralda rossy syahdinda1,2, alexander patera nugraha2, ari triwardhani2, tengku natasha eleena binti tengku ahmad noor3,4 1orthodontic department, faculty of dentistry, hang tuah university, surabaya, indonesia 2orthodontic department, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3membership of faculty of dental surgery, royal college of surgeon, edinburgh university, united kingdom 4malaysian armed forces dental officer, 609 armed forces dental clinic, kem semenggo, kuching, sarawak, malaysia abstract background: maxillary canines play a critical point in creating an aesthetic smile as they support the part of facial muscle. canines are also the second most frequently impacted teeth after the third molars. in some cases, it is possible to retract canines into their correct position by orthodontics treatment. purpose: this article highlighted a treatment option with surgical exposure, in which a maxillary canine was impacted. it presented gradual steps from pre-surgical to post-surgical orthodontic treatment. case: a 16 years old female patient referred to orthodontic department by oral surgeon for pre-surgical orthodontic treatment of her impacted maxillary canine. case management: after pre-surgical orthodontic treatment, the impacted canine was surgically exposed by closed technique since open surgical exposure might need excessive removal of the surrounding bone. traction was given through a gold chain which attached to the palatal surface of the impacted canine. the tooth was ideally positioned with fixed orthodontic appliances. the permanent right maxillary canine was successfully positioned into proper alignment with the remaining teeth. aesthetic smile was improved. conclusion: malocclusion with impacted canine was successfully treated with mbt orthodontic prescription combined with gold chain that can retract the impacted canine into its physiological position and the patient was satisfied with the aesthetic result. keywords: tooth impacted; orthodontic appliance; medicine; dentistry; tooth movement technique correspondence: ari triwardhani, orthodontic department, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya, 60132 indonesia. email: ari-t@fkg.unair.ac.id introduction canine impaction is a condition in which the canine is embedded in the surrounding tissues so that the eruption is prevented. impactions are twice as common in females as in males with palatal impactions being twice as common as labial impactions. impacted maxillary canines are a rather frequent condition. when the third molar is excluded, the maxillary canine is the most often impacted tooth. the prevalence of impacted maxillary canines has been observed to range between 0.9 and 3.3%. the maxillary impacted canine is more commonly seen palatally (85% of the time) than labially (15%). root dilaceration has been recorded in up to 59.5% of cases.1 the prevalence of palatally impacted canine of the worldwide population ranges from 0.27% to 2.4%.2 there are several options to treat impacted canine: no treatment—leaving the tooth where it is, removal of the tooth if orthodontic treatment is not possible, and surgical exposure followed by orthodontic treatment.3 after surgical exposure, the impacted tooth may erupt naturally during early to late mixed dentition period or moved orthodontically after bonding an attachment on the tooth.4 the goal of this research was to provide a strategy for surgically exposing an impacted maxillary canine and orthodontically placing it. the cosmetic and functional care of impacted canines is critical. the effective alignment of impacted canines requires careful surgical and orthodontic procedure selection. even though there are very many published-articles about management of impacted maxillary canine with surgical exposure, this study describes the mbt orthodontic prescription combined with gold chain to treat the malocclusion with canine impaction. this article dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p235–239 mailto:ari-t@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p235-239 236syahdinda et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 235–239 highlighted a treatment option with surgical exposure, in which a maxillary canine was impacted. it presented gradual steps from pre-surgical to post-surgical orthodontic treatment. case this case report was granted permission from the patient and patient’s guardian willing to fill the written informed consent for the scientific publication. a 16 years old female patient presented to orthodontic department as referral from department of oral surgery. the patient wanted to have her upper right decayed tooth extracted. the tooth which she referred was a deciduous canine. the periapical radiograph showed the permanent maxillary canine which should have replaced the primary canine was impacted. it was palatally placed and borne in semi-vertical position, classified as class i case. the radiograph also showed the agenesis of permanent right maxillary lateral incisor. intra-oral examination showed angle class i malocclusion with mild crowding in lower anterior and upper posterior, scissor bite of 25/35, and 2 mm upper mid-line shift (figure 1 a-e). the patient had a negative arch length discrepancy as much as 1.5 mm in both the upper and lower arches. extra-oral examination showed straight facial profile. cephalometric radiograph showed ∠ sna 87.5º, ∠ snb 85º, ∠ anb 2.5º, ∠ i-na 27º, ∠ i-nb 28º, ∠ fh-np 90º, ∠ nap 3º, ∠ y-axis 57.5º, interincisal angle 117º. the upper and lower lips were in the normal range based on ricketts’ and steiner’s analysis (figure 2a). in addition, the impacted canine was examined with periapical radiography analysis, as can be seen in figure 2b and 2c. a e c d b figure 1. pre-treatment intra oral view, (a) maxillary occlusal, (b) mandibular occlusal, (c) sagital right side, (d) anterior view, (e) sagital left. a b c figure 2. pre-treatment radiograph of patient case. (a) cephalometry digital analysis, (b) canine impaction examined with periapical radiography, (c) canine impaction radiography occlusal view. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p235–239 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p235-239 237 syahdinda et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 235–239 case management the main treatment goals for the patient were to improve the patient’s cosmetics as well as the functional occlusion by aligning and occluding the permanent canine. surgical exposure of the impacted maxillary canine was planned, followed by directed pressures to correct the canine’s position. correction of posterior crowding and a 25/35 scissor bite in the maxillary arch were planned, as were correction of anterior crowding and a mid-line shift in the mandibular arch. pre-surgical orthodontics consist of maxillary and mandibular arch alignment start from initial niti to 0.016” x 0.022” stainless steel arch wires. niti open coil spring was inserted between deciduous lateral incisor and first premolar in upper right region in every figure 3. traction of maxillary canine using gold chain. a b c d e figure 4. post-treatment intra oral view. (a) maxillary occlusal, (b) mandibular occlusal, (c) sagital right side, (d) anterior view, (e) sagital left. before treatment figure 5. superimposed cephalometric radiograph before (black line) and after treatment (red line). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p235–239 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p235-239 238syahdinda et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 235–239 stage. adequate anchorage was planned before canine exposure by using bands on first molars and tubes on second molars in upper arches. after the space for canine had been obtained, exposure was performed by a close window procedure. the mbt orthodontic prescription was conducted in this study. the exposed crown area was covered in gold chain. in addition to the scissor bite correction, a glass ionomer cement bite block was placed on the mandibular posterior teeth to prevent obstruction and promote mobility of the maxillary canine. light orthodontic forces were applied by placing the end of the chain into the 0.016” x 0.022” stainless steel wire which inserted to the bracket slots. the chain was activated every two weeks. the force was increased by using ligature wire. after the canine was close enough to the wire, the chain was replaced by bracket attachment on labial surface of the crown. double wires technique was used to pull the canine into the arch. after completion of the traction, a series of stainless-steel wires (up to 0.019” x 0.025”) were ligated. triangle elastics were used. final alignment and leveling accomplished, followed by passive and retention phase using hawley retainer. leveling-aligning and canine space regaining for surgical preparation took six months. after the exposal treatment, there were no complications observed at the surgical site, and the gingiva remained healthy. the canine was pulled out using gold chain (figure 3). in the first two weeks, the chain remained passive, then the forces were increased by using ligature wire. this process took 10 months until the canine was close enough to the wire and the chain could be replaced by bracket. re-leveling to finishing and detailing needed four more months before the whole arches could be ligated passively for two months before debonding. after the whole treatment process, the permanent upper right canine was positioned into proper alignment with the remaining teeth. the upper mid-line shift and 25/35 scissor bite were corrected. the upper posterior and lower anterior crowding was aligned (figure 4a-e). post-treatment cephalometric radiograph showed ∠ sna 86.15º, ∠ snb 83.8º, ∠ anb 2.35º, ∠ i-na 23.89º, ∠ i-nb 24.75º, ∠ fh-np 90º, ∠ nap 3º, ∠ y-axis 57.5º, ∠ interincisal 115º. upper and lower lips were in normal range based on ricketts’s and steiner’s analysis. superimposed cephalometric radiograph showed no changes in any skeletal aspects (figure 5). as for the dentition, the interincisal angle were slightly increased and brought the lips forward, but it did not have any impact to the patient’s profile. the overall result was quite acceptable and fulfilled patient expectation. discussion permanent canine teeth are essential for functional occlusion, dental aesthetic and a well-balanced grin. canines also give the cheeks a lot of support. the lack of canines leads to a flattened upper lip.5,6 impaction in dogs has been linked to an increased risk of infection and cyst formation.2 canine impaction can be caused by various factors. localization, such as tooth size and arch length discrepancy, prolonged retention or early loss of primary canine, ankylosis of permanent canine; systemic condition; and genetics.2 in this case, what probably caused the impaction of canine could be the absence of permanent lateral incisor. according to the guidance theory, the canine erupts along the root of the lateral incisor, acting as a guide. the canine will not erupt if the root of the lateral incisor is missing or deformed.6,7 the deciduous lateral incisor is still maintained in the end of treatment. it is not replaced. otherwise, it is restored with veneer due to the proficient condition of the root. there are some options to treat impacted canine. in this case, surgical exposure was chosen because of several considerations—the canine position, where the tip was at the cervical third of the adjacent tooth; severity of impaction, which the canine lied at less than 45º angle; patient age, and patient consent. an apical third with 3045º angle of impacted canine position is the most favorable for orthodontic traction.8,9 before the oral surgeon perform the maxillary impacted canine treatment, cone-beam computed tomography (cbct) examination may helpful to determining maxillary bone density.6,10,11 the most common method used in surgical exposure is to allow the tooth to erupt naturally during early or late mixed dentition. but some factors e.g denser palatal bone, thicker palatal mucosa and a more horizontal position causes palatally displaced cuspids to be impacted and they rarely erupted without requiring complex biomechanical intervention. so, a closed flap technique can be employed in which the impacted tooth is surgically exposed and gold chain is bonded. then orthodontic forces are used to move the tooth into oral cavity.12 in the present case, a closed flap technique was used which usually produces best gingival aesthetics and increased ease of tooth movement.13 at the end of treatment, the inclination of upper incisive angle was slightly increased. it happened because of the occurring thrust due to the canine appearance in the arch. the space regaining for the impacted canine using niti coil spring in the initial phase was also beneficial for mid-line shift correction. the mid-line shift appeared because of the non-existence of the permanent canine, where the primary canine also failed to maintain the space due to caries.14 whereas in lower arch, the incisive angle was increased because of the anterior crowding correction. it normally happens in non-extraction cases, leading the soft tissue to move forward.15 from this case report, it can be concluded that the treatment goal has been achieved to treat the malocclusion with impacted canine. the impacted canine could be retracted with gold chain combined with mbt orthodontic prescription into its physiological position and the patient was satisfied with the aesthetic result and stomatognathic function. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p235–239 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p235-239 239 syahdinda et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 235–239 references 1. grisar k, piccart f, al-rimawi as, basso i, politis c, jacobs r. three-dimensional position of impacted maxillary canines: prevalence, associated pathology and introduction to a new classification system. clin exp dent res. 2019; 5(1): 19–25. 2. manne r, gandikota c, juvvadi sr, rama hrm, anche s. impacted canines: etiology, diagnosis, and orthodontic management. j pharm bioallied sci. 2012; 4(suppl 2): s234-8. 3. becker a, chaushu s. palatally impacted canines: the case for closed surgical exposure and immediate orthodontic traction. am j orthod dentofacial orthop. 2013; 143(4): 451–9. 4. bedoya mm, park jh. a review of the diagnosis and management of impacted maxillary canines. j am dent assoc. 2009; 140(12): 1485–93. 5. chrystinasari na, narmada ib, triwardhani a. position of unilateral / bilateral permanent canine impaction on the prognosis of treatment with kpg index: 3d cone beam computed tomography analysis. j int dent med res. 2021; 14(4): 1523–30. 6. gunardi oj, danudiningrat cp, rizqiawan a, mulyawan i, amir ms, kamadjaja db, sumarta npm, anugraha g, fessi r al, barus l, ono s. decision-making criteria of odontectomy or surgical exposure in impacted maxillary canine based on treatment difficulty index modification. eur j dent. 2022; . 7. de ca r valho ab, motta r hl, de ca r valho emd. relation between agenesis and shape anomaly of maxillary lateral incisors and canine impaction. dental p ress j or thod. 2012; 17(6): 83–8. 8. dubovská i, špidlen m, krejčí p, borbèly p, voborná i, harvan ľ, kotas m. palatally impacted canines – factors affecting treatment duration. iosr j dent med sci. 2015; 14(2): 16–21. 9. pitt s, hamdan a, rock p. a treatment difficulty index for unerupted maxillary canines. eur j orthod. 2006; 28(2): 141–4. 10. adiwinarno b, narmada ib, hamid ta. comparison of trabecular bone in impacted and normal erupted unilateral maxillary canine teeth using cone-beam computed tomography in patients scheduled for orthodontic treatment at the universitas airlangga dental and oral hospital. acta med philipp. 2022; . 11. tallo fr, narmada ib, ardani igaw. maxillary anterior root resorption in class ii/i malocclusion patients post fixed orthodontic treatment. dent j (majalah kedokt gigi). 2020; 53(4): 201–5. 12. hassan gs, rubby mg, babu mri, sultana n, hasan mn. management of an unerupted maxillary canine: a case report. city dent coll j. 2012; 9(2): 22–4. 13. brézulier d, sorel o. impacted canines-literature review. j dentofac anomalies orthod. 2017; 20: 1–10. 14. christensen rt, fields hw, christensen jr, beck fm, casamassimo ps, mctigue dj. the effects of primary canine loss on permanent lower dental midline stability. pediatr dent. 2018; 40(4): 279–84. 15. machado gb. treating dental crowding with mandibular incisor extraction in an angle class i patient. dental press j orthod. 2015; 20(3): 101–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p235–239 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p235-239 166 dental journal (majalah kedokteran gigi) 2017 september; 50(3): 166–170 research report potency of garcinia mangostana l peel extract combined with demineralized freeze-dried bovine bone xenograft on il-1β expression, osteoblasts, and osteoclasts in alveolar bone imam safari azhar,1 utari kresnoadi,1 and retno pudji rahayu2 1department of prosthodontics 2department of oral pathology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: alveolar bone plays an important role in supporting dentures. one socket preservation procedure is the application of bone graft to the tooth socket immediately after extraction. the administration of garcinia mangostana l peel extract combined with demineralized freeze-dried bovine bone xenograft (dfdbbx) is assumed to decrease the number of osteoclast cells and il-1β expressions, while also increasing that of osteoblast cells. purpose: this study aimed to establish the potency of garcinia mangostana l peel extract combined with dfdbbx on interleukin 1β expressions, osteoclast cells, and osteoblast cells during the preservation of tooth extraction sockets. methods: this research constituted experimental laboratory-based research using 56 cavia cobayas as specimens with randomized factorial design (true experimental design). these subjects were divided into eight groups, given peg, dfdbbx, mangosteen peel extract or a combination of mangosteen peel extract and dfdbbx, and subsequently anesthesized before their left mandibular incisors were extracted. thereafter, the post-extraction sockets were treated with peg, dfdbbx, mangosteen peel extract or a combination of mangosteen peel extract and dfdbbx. the sockets were examined by means of he and immunohistochemical staining on days 7 and 30 after extraction. the data obtained was analyzed with a one-way anova test. results: the results of the one-way anova test showed that the average number of osteoblasts, osteoclasts, and il-1β expressions varied significantly between the groups. conclusion: the combination of mangosteen peel extract and dfdbbx can potentially increase osteoblasts, while also decreasing osteoclasts and il-1β expressions in the alveolar bones of cavia cobaya. keywords: alveolar bone; dfdbbx; mangosteen peel extract; il-1β expression; osteoblasts, osteoclasts correspondence: imam safari azhar, department of prosthodontics, faculty of dental medicine universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132. e-mail: imam-safari-azhar@fkg.unair.ac.id introduction interleukin 1 beta (il-1β) is a mediator in infection and injuries which is is activated by monocytes and macrophages that are cells generally active in the inflammatory process.1 the increased level of il-1β in tissues can trigger inhibition of osteoblast differentiation resulting in a decrease in the number of osteoblasts leading, in turn, to the inhibition of bone formation. such inhibition will then impede further treatment such as the prescribing of dentures. as a result, the suppression of il-1β level in tissues is required in order to promote effective wound healing.2 the development of treatment evolves into the use of natural materials, one of which that has been studied and proven to be effective as an anti-inflammatory and antioxidant in the wound healing process being garcinia mangostana l or mangosteen.3 previous research has shown that mouse cells with 5 mg of gamma-mangostin can inhibit the production of such cyclooxygenase-2 enzymes that reduce inflammation. therefore, gamma-mangostin is demonstrated to have a superior anti-inflammatory effect.4 another previous piece of laboratory-based research analyzing the toxicity of mangosteen peel extract also dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p166-170 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p166-170 mailto:imam-safari-azhar@fkg.unair.ac.id 167167azhar, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 166–171 found that mangosteen extract at concentrations of 200 µg/ ml, 300 µg/ml, 400 µg/ml, 500 µg/ml, 600 µg/ml, 700 µg/ ml, and 800 µg/ml cannot cause toxicity to human gingival fibroblast cell cultures.5 on the other hand, preservation of tooth extraction sockets constitutes a surgical procedure intended to preserve the alveolar bone through the application of bone graft on sockets immediately after teeth have been extracted. this procedure aims to optimally maintain the bone and soft tissue post-tooth extraction.6,7 in general, bone graft is used to support bone regeneration, severe bone defects caused by trauma or surgical procedures, repair of bone damage resulting from dental problems, post-extraction socket filling to maintain the height and width of the alveolar ridge, and immediate post-extraction alveolar ridge reconstruction.8 one of the bone graft materials used is demineralized freeze-dried bovine bone xenograft (dfdbbx). dfdbbx is one bone graft xenograft type derived from cows that is osteoconductive. therefore, dfdbbx can play a role as a scaffold in new bone growth triggered by osteoblasts derived from the base of the post-extraction socket.9 moreover, dfdbbx is often used because of its osteoconduced inorganic matrix components which serve to provide scaffold for bone regeneration without being involved in bone formation itself. dfdbbx can also affect protein-inducing exposed bone during the demineralization process. consequently, dfdbbx can be said to be both osteoinductive and osteoconductive.10 for these reasons, an innovative material that can induce graft activity to accelerate bone formation is required. this study aimed to analyze the effects of mangosteen peel extract at a concentration of 2% as an active substance combined with dfdbbx in il-1β expressions, osteoclast cells and osteoblast cells in extraction sockets. results of this study are expected to be used as a reference for the development of mangosteen peel extract as an alternative treatment combined with dfdbbx in accelerating aveolar bone formation. materials and methods the research reported here was approved with ethical clearance certificate number 067/hrecc.fodm/ vi/2017 and constituted an experimental laboratory-based investigation featuring randomized factorial design (true experimental design). 56 cavia cobayas were used as research subjects divided into eight sample groups each of which contained seven members. the inclusion criteria applied comprised the following: healthy, active male cavia cobayas weighing 300-350 grams, aged 3-3.5 months with a normal appetite and skin and limb injury-free. furthermore, the subjects were required to enjoy full use of their faculties, while exhibiting a normal gait and movement as well as a standard body temperature. they were subsequently kept in one location and placed on identical diets. mangosteen peel extract was produced through a process involving several stages. mangosteen fruit was washed before being separated according to whether their peel was hard or soft. as the primary raw material, soft mangosteen peel was mashed in order to facilitate the extraction process carried out using a mixture of ethanol and water at a ratio of 1:2 as a solvent. meanwhile, the ratio of materials extracted to solvent used was 1:4. the products of extraction were immersed for 24 hours and filtered to separate them from their dregs. to produce a combination of mangosteen peel extract and dfdbbx at a concentration of 2%, 0.5 gr of mangosteen peel extract was added to 0.5 gr of dfdbbx and 24 gr of peg. thereafter, the cavia cobaya subjects were anesthesized with a 20 mg/ 300 mg bb dose of ketamine administered intramuscularly.11 their left mandibular incisors were then cleansed of food scraps with a water spray and dried before being fully extracted (no root fracture) using a sterile needle holder. extraction was performed by means of the same movement, direction and strength. the sockets were then irrigated with sterile aquade solution. post-extraction, the mangosteen peel extract + dfdbbx, mangosteen peel extract, dfdbbx, and peg were introduced into the selected sockets with a syringe until they were full, at approximately +0.1 ml. all wound sites were then stitched with polyamide monofilament sewing thread, ds 12 3/8 c, 12 mm, 6/10 meth, 0.7 sterile braun aesculap.12 at the next stage, the members of groups i, ii, iii, and iv were sacrificed on day 7, while those in groups v, vi, vii, and viii were sacrificed on day 30 since deposition and bone resorption were assumed to occur in the sockets within four weeks. the subjects were sacrificed using ketamine in 100 mg/ml (pfizer) at a dose of 0.2 ml. their mandibula was then removed before burial. subsequent steps performed included dehydrating, clearing, infiltrating, and embedding. the most commonly used reagent for fixating histological specimens consisted of 10% neutral buffered formaldehyde. each tissue was then cut to a thickness of 2-4 mm. the size of the tissues did not exceed the embedding of the cassette, so the reagent could flow around the tissues. the next stage is that of dehydrating aimed at removing water from the tissue and replacing it with paraffin. the dehydrating process was affected by washing the tissues with 70% ethanol for 15 minutes, 80% ethanol for 1 hour, 95% ethanol for 1 hour and, finally, ethanol (absolute) for one hour. this process was repeated twice. clearing was subsequently conducted by immersing the tissues in xylol solution for one hour and then xylol for a further two hours. this process was then repeated. after clearing had been completed, infiltration was carried out on the tissues. a piece of tissue was placed in the middle of the embedding cassette and closed. impregnation was then carried out by reducing the tissues in paraffin at a temperature of 56-580 c for two hours, a process repeated three times. thereafter, the tissues were planted in paraffin blocks. liquid paraffin at a boiling point between 56 and 600 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p166-170 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p166-170 168 azhar, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 166–171 c was poured into a metal moulding device. an embedding cassette was then placed into the metal moulds with the surface of the tissue to be cut facing downwards. before the liquid paraffin was poured into the moulds, the upper part was labeled. after the paraffin had hardened, the resulting blocks were removed from their metal molds. at the next stage, the paraffin blocks were cut using a shear microtome of 4µm thickness. the results were collected using a brush, and then placed carefully on the surface of the waterbath at a fixed temperature of 56-580 c. the incision results were placed on a poly-llysine (preparat) microscope slide. the tissue-containing preparations were placed on a host plate at a minimum temperature of 30-350 c for 12 hours. the preparations were then ready for continuous immunohostical staining to facilitate observation of il-1β expressions and he staining to enable examination of osteoblast and osteoclast cells. the number of il-1β expressions, osteoblasts, and osteoclasts was observed and calculated using a light microscope at 400x magnification. the observed portion was calculated manually through 20 viewing fields. based on the calculation results, a levene’s test was conducted followed by a one-way anova test intended to analyze any differences between groups. a tukey hsd test was subsequently performed to establish the respective difference between each group. results the results of this research indicated that the average number of il-1β expressions was significantly different across the groups. there were differences in the average number of il-1β expressions between the mangosteen peel extract, group, the dfbbx group, and the peg group. the highest average number of il-1β expressions was found in the control group on day 7, while the lowest was in the group with mangosteen peel extract + dfdbbx on day 30 (figure 1). figure 2 illustrates macrophage cells expressing il-1β that observed using a light microscope. 26(1): 77–83. 14. allegrini s, koening b, allegrini mrf, yoshimoto m, gedrange t, fanghaenel j, lipski m. alveolar ridge sockets preservation with bone grafting--review. ann acad med stetin. 2008; 54(1): 70–81. 15. reynolds ma, aichelmann-reidy me, branch-mays gl. regeneration of periodontal tissue: bone replacement grafts. dent clin north am. 2010; 54(1): 55–71. 16. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc. 2007; 73(6): 513. 17. chaverri jp, rodríguez nc, ibarra mo, rojas jmp. medicinal properties of mangosteen (garcinia mangostana). food chem toxicol. 2008; 46(10): 3227–39. 18. mardiana l. ramuan & khasiat kulit manggis. jakarta: penebar swadaya; 2011. p. 11-20. 19. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc. 2006; 72(10): 917–22. figure 1. mean and standard deviation of the number of il-1β expressions on days 7 and 30. day 7 day 30 control dfdbbx mangosteen peel extraxt + dfdbbx mangosteen peel extraxt figure 1. mean and standard deviation of the number of il-1β expressions on days 7 and 30. figure 2. microscopic pictures of il-1β expressions in each treatment group during cpi examination, arrows indicating macrophage cells expressing il 1β viewed through a light microscope at a magnification of 400x. figure 3. the graph of the mean and standard deviation of osteoblasts on days 7 and 30. control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx day 7 day 30 control dfdbbx mangosteen peel extraxt + dfdbbx mangosteen peel extraxt figure 2. microscopic pictures of il-1β expressions in each treatment group during cpi examination, arrows indicating macrophage cells expressing il 1β viewed through a light microscope at a magnification of 400x. figure 3. the graph of the mean and standard deviation of osteoblasts on days 7 and 30. control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx day 7 day 30 control dfdbbx mangosteen peel extraxt + dfdbbx mangosteen peel extraxt figure 2. microscopic pictures of il-1β expressions in each treatment group during cpi examination, arrows indicating macrophage cells expressing il 1β viewed through a light microscope at a magnification of 400x. figure 2. microscopic pictures of il-1β expressions in each treatment group during cpi examination, arrows indicating macrophage cells expressing il 1β viewed through a light microscope at a magnification of 400x. figure 3. the graph of the mean and standard deviation of osteoblasts on days 7 and 30. control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx day 7 day 30 control dfdbbx mangosteen peel extraxt + dfdbbx mangosteen peel extraxt figure 3. the graph of the mean and standard deviation of osteoblasts on days 7 and 30. figure 3 illustrates that there were differences in the average number of osteoblasts between the group with mangosteen peel extract, the group with dfbbx, and the group with peg. the highest number of osteoblasts was found in the group with mangosteen peel extract + dfdbbx on day 30, while the lowest one was in the control group with peg on day 7. figure 4 illustrates osteoblast cell seen with a light microscope on day 30. figure 5 demonstrates that there were differences in the average number of osteoclasts between the group with mangosteen peel extract, the group with dfbbx, and the group with peg. the highest number of osteoclasts was found in the control group with peg on day 7, while the lowest one was in the group with mangosteen peel extract + dfdbbx on day 30. figure 6 indicating osteoclast cells that observed using a light microscope on day 30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p166-170 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p166-170 169169azhar, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 166–171 discussion alveolar bone becomes atrophied after tooth extraction.13 if it becomes severely atrophied, fitting dentures effectively becomes difficult. as a result, an attempt to recover the lost alveolar bone is required. several types of materials such as autograft, allographs, xenografs, synthetic biomaterials, and osteoactive agents have been used widely as bone replacement.14 furthermore, the application of bone graft to posttooth extraction sockets is intended to stimulate new bone growth. in other words, the bone graft functions as a skeleton (scaffolds) and a matrix for the attachment and proliferation of osteoblasts. the bone graft was also expected to provide a skeletal structure for the development, maturation, and remodeling of the clot supporting bone formation. therefore, bone graft materials should have biocompatibility and osteoconductivity properties for cellular attachment (proliferation and migration) as well as osteogenesis and osteoinduction.15 morever, bone graft materials should be more osteoinductive, stimulating the osteoprogenitor to differentiate into osteoblasts and form new bone.16 certain previous research has shown that the administration of dfdbbx and peg can decrease the number of il-1β expressions compared to those of the control group. the number of il-1β expressions in the treatment group with the administration of dfdbbx was seen to differ significantly compared to the control group on the 7th and 30th days. dfdbbx is a kind of xenograft derived from cows. xenograft has osteoconductive properties with porous internal surfaces to enable revascularization and osteoblast migration from the socket base supportive of osteogenesis.9 consequently, dfdbbx with its osteoconductive properties can play a role as a skeleton (scaffold) in new bone growth triggered by osteoblasts derived from the base of the post-tooth extraction socket.17 mangosteen peel contains a number of pigments derived from two metabolites, namely α-mangistin and β-mangostin. mangosteen peel is also very beneficial to health since it contains numerous xanton compounds. in fact, such compounds contained there are 27 times greater than those contained in mangosteen flesh. for the human body, xanton compounds play a role as a strong antioxidant, anti-proliferation, anti-inflammatory, and anti microbacterial.18 similarly, within this research, the group treated with mangosteen peel extract had a significantly different number of il-1β expressions on days 7 and 30 compared to that in the control group and the group treated with dfdbbx. this is because mangosteen peel extract is rich in xanton compounds, especially α-mangostin and γ-mangostin, that can decrease tnf α, il-1β, il-6, il-8, mcp-1, and tlr-2 expressions. however, γ-mangostin control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx figure 4. microscopic pictures of osteoblast cells in each treatment group during he examination, arrows indicating osteoblast cells seen with a light microscope with a magnification of 400x on day 30. figure 5. the graph of the mean and standard deviation of osteoclasts on days 7 and 30. a b c d day 7 day 30 control dfdbbx mangosteen peel extraxt + dfdbbx mangosteen peel extraxt figure 5. the graph of the mean and standard deviation of osteoclasts on days 7 and 30. control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx figure 6. microscopic pictures of osteoclast cells in each treatment group during he examination, arrows indicating osteoblast cells seen with a light microscope with a magnification of 400x on day 30. control group group with dfdbbx group with mangosteen peel extract group with mangosteen peel extract + dfdbbx figure 6. microscopic pictures of osteoclast cells in each treatment group during he examination, arrows indicating osteoblast cells seen with a light microscope with a magnification of 400x on day 30. figure 6. microscopic pictures of osteoclast cells in each treatment group during he examination, arrows indicating osteoblast cells seen with a light microscope with a magnification of 400x on day 30. figure 4. microscopic pictures of osteoblast cells in each treatment group during he examination, arrows indicating osteoblast cells seen with a light microscope with a magnification of 400x on day 30. group with mangosteen peel extract control group group with dfdbbx group with mangosteen peel extract + dfdbbx dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p166-170 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p166-170 170 azhar, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 166–171 dfdbbx has greater anti-inflammatory properties than α-mangostin. moreover, γ-mangostin also can inhibit lipopolysaccharide (lps) inducing activation of ikk, nfκb, and cox-2 transcriptions.17 the inhibition of cox-2 can then decrease proinflammatory (il-1, tnf α) cytokines directly or indirectly leading to the inhibition of osteoclast formation through rankl. consequently, there is no differentiation and formation of osteoclasts resulting in their decrease. nevertheless, within this research, the group receiving an administration of mangosteen peel extract combined with dfdbbx produced better results than either the group treated with mangosteen peel extract alone or the group treated with dfdbbx. it means that a combination of mangosteen peel extract and dfdbbx can execute a scaffold function as well as demonstrate anti-inflammatory properties. the results in the group treated with mangosteen peel extract combined with dfdbbx on day 7 were also different from those on day 30. the number of osteoblast cells on day 30 was higher than that on day 7. in contrast, osteoclast cells and il-1β expressions on day 30 were fewer than those on day 7. similarly, previous research showed that during weeks 4-6, alveolar bone is mostly filled with woven bone, followed by a bone maturation process in the next phase.19 it can be argued that the post-extraction socket preservation conducted in this research could reduce the occurrence of alveolar bone resorption, while accelerating the process of bone formation in the defective bone since the additional ingredients of mangosteen peel extract are indirectly osteoinductive. as a result, mangosteen peel extract can suppress nfkb activities leading to a proliferation of osteoblast progenitor cells. consequently, it can be said that the combination of osteoconductive and osteoinductive properties of mangosteen peel extract mixed with dfdbbx can significantly reduce the number of il-1β expressions. this combination can potentially be expected to further increase the success of socket preservation with the result that the dimension and volume of post-tooth extraction bone can be maintained. it can be concluded that mangosteen peel extract at a concentration of 2% as an active substance combined with dfdbbx demonstrates the potential to increase the number of osteoblast cells as well to decrease that of osteoclast cells and il-1β expressions in the alveolar bone of cavia cobaya. references 1. dinarello ca. immunological and inflammatory functions of the interleukin-1 family. annu rev immunol. 2009; 27(1): 519–50. 2. lin fh, chang jb, mcguire mh, yee ja, brigman be. biphasic effects of interleukin-1β on osteoblast differentiation in vitro. j orthop res. 2010; 28(7): 958–64. 3. yatman e. kulit buah manggis mengandung xanton yang berkhasiat tinggi. majalah ilmiah widya. 2012; 29(324): 1–9. 4. nakatani k, nakahata n, arakawa t, yasuda h, ohizumi y. inhibition of cyclooxygenase and prostaglandin e2 synthesis by γ-mangostin, a xanthone derivative in mangosteen, in c6 rat glioma cells1. biochem pharmacol. 2002; 63(1): 73–9. 5. hayyu ns. sitotoksisitas ekstrak kulit garcinia mangostana linn terhadap sel fibroblas gingiva manusia (penelitian semi experimental laboratoris). thesis. surabaya: universitas airlangga; 2013. p. 43. 6. peck mt, marnewick j, stephen l. alveolar ridge preservation using leukocyte and platelet-rich fibrin: a report of a case. case rep dent. 2011; 2011: 1–5. 7. kotsakis g, markou n, chrepa v, krompa v, kotsakis a. alveolar ridge preservation utilizing the “socket-plug” technique. int j oral implantol clin res. 2012; 3(1): 24–30. 8. albanese a, licata me, polizzi b, campisi g. platelet-rich plasma (prp) in dental and oral surgery: from the wound healing to bone regeneration. immun ageing. 2013; 10: 1–10. 9. cohen re, alsuwaiyan a, wang b. xenografts and periodontal regeneration. j orthod endod. 2015; 1(1): 1–6. 10. al-ghamdi h, mokeem sa, anil s. current concepts in alveolar bone augmentation : a critical appraisal. saudi dent j. 2007; 19(2): 74–90. 11. kusumawati d. bersahabat dengan hewan coba. yogyakarta: gajdah mada press; 2004. p. 14, 26-28, 114. 12. kresnoadi u. toll like receptor 2 sebagai signaling pathway osteogenesis tulang alveoli yang diinduksi kombinasi aloe vera dan graft. disertation. surabaya: universitas airlangga; 2012. p. 49-50. 13. mezzomo la, shinkai rs, mardas n, donos n. alveolar ridge preservation after dental extraction and before implant placement: a literature review. rev odonto ciência. 2011; 26(1): 77–83. 14. allegrini s, koening b, allegrini mrf, yoshimoto m, gedrange t, fanghaenel j, lipski m. alveolar ridge sockets preservation with bone grafting--review. ann acad med stetin. 2008; 54(1): 70–81. 15. reynolds ma, a ichelmann-reidy m e, branch-mays gl. regeneration of periodontal tissue: bone replacement grafts. dent clin north am. 2010; 54(1): 55–71. 16. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc. 2007; 73(6): 513. 17. chaverri jp, rodríguez nc, ibarra mo, rojas jmp. medicinal properties of mangosteen (garcinia mangostana). food chem toxicol. 2008; 46(10): 3227–39. 18. mardiana l. ramuan & khasiat kulit manggis. jakarta: penebar swadaya; 2011. p. 11-20. 19. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc. 2006; 72(10): 917–22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p166-170 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p166-170 165 the effect of gender differences on dentist’s performance (a study in health centers in east java) titiek berniyanti1 and kromodiharjo sudiyono2 1 departement of dental public health, faculty of dentistry airlangga university 2 departement of manufacture, faculty of tecnology industry, its surabaya – indonesia abstract gender is a social relationship concept that differentiates role and function between man and women. equality of rights and obligations between man and woman have been normatively guaranteed and the constitution does not explicitly discriminate the rights and obligations of woman in law and government, in occupation and decent life, in politics, in religion and beliefs, and in national defend. related with gender perspective, the need of health worker especially dentist worker has to be determined and planned properly. the productivity is one of the factor on dentist worker, that affect on the quality of health delivary. some companies try to increase efficiency in performing their activities and try to measure activities they do. in this case, time & motion study method is one of the solutions to help the company measuring their activity. using these techniques, company can measure the productivity of resources used for every activity. in order to get better performance in cost reduction, the company should assign their cost to the product resulted. determination of the standart time to work is one of the method that contain high value of efeciency to gain the productivity. the aim of this study was to learn how gender has an effect on dentist’s work performance. the time of completing dentist’s task i.e: teeth extraction were measured and the standard time were determined. in addition the patient’s respond were measured through questionair. this study was conducted in east java. the conclusion of this study revealed that there was no difference of work performance between male and female dentist’s. key words: gender, work performance correspondence: titiek berniyanti, c/o: bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction the study investigated the effect of gender on the dentist’s performance, which also implied to the performance of dentists in health centers in east java. gender is a social relations concept differentiating the role between man and woman. the differentiation is not due to biological or natural differences. instead, it is differentiated or divided according to each position, function, and role in various life and development areas.1 natural difference between man and woman comprises four conditions, i.e., menstruation, pregnancy, delivery, and breastfeeding. equality of rights and obligations between man and woman have been normatively guaranteed by the constitution 1945. the constitution does not explicitly discriminate the rights and obligations of woman in law and government, in occupation and decent life, in politics, in religion and beliefs, and in national defend.2 health development is a sector that involving much women. its percentage, which is slightly higher than man, 50.2% of indonesian total population, represents a potentiality to actively take part in health development together with their male counterparts.3 according to ichromi4 gender is a social concept. however, different view holds by abdullah5 who regarded that gender is actually a cultural product built on the idea that functionally there are two categories of man and woman. as a cultural product, gender has a hierarchical system that produces operational groups. these groups depend on each other or even compete each other to defend their own power. the efforts to find solution of gender problems in indonesia had not provided maximum results due to heterogeneous and unclear perception on gender problem in this country. until now the studies on women in gender perspective is limited only to find aspiration developing among female community. in fact, there were many criteria to evaluate the role of women, i.e.: 1) the extent to which they play a role as participant in development, 2) the extent to which they receive benefit from development, 3) the extent to which they have access to society resources, 4) the extent to which they have control to human resources, including their social facilities. hence, it is interesting to know whether or not the gender perspective would effect male or female dentist and give the different result in their productivity. ergonomics is the study of the interaction between people and machines an the factor that affect the interaction. its purpose is to improve the performance (work accelerate, accuracy, work safety, reduce work exaggerate and fatigue) of system by improving human machine interaction.6 according to kroemer7 success is measured by improved productivity, 166 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 165-169 efficiency, safety, acceptance of the resultant system design, and last, but truly not least, improved quality of human life. the concept of individual performance, according to wignjosubroto,8 is the outcome appearance of efforts by an individual. the concept of performance is related with productivity and has a strong correlation with the surrounding systems. there are two ways to accelerate production. the one of them is work simplification. work simplification or time motion studies is term freely used by engineers who are concerned with improving the efficiency work performed. time and motion studies were introduced in the usa by frederick taylor at the beginning of the 20th century. since then, the practice has spread throughout the industrialized world. it is a method to increased the productivity, so that production is increased but speed with is accompanying fatigue is reduced. this word in dentistry means shorter and easier way to practice dentistry. process of analysis applied to a job or number of jobs to check the efficiency of the work method, equipment used, and the worker. its findings are used to improve performance.9 in production theory, system comprises factors providing contribution to the results of production. system in the gender concept refers to social and cultural behavior contributing to gender meaning. data in health office, east java 1998 showed that since 1988 a community health center has a dentist and dental nurse serving averagely 35 patients a day.10 result of study by berniyanti10 showed that performance produced by a dentist, measured from the capability in diagnosis, aenesthesis and extraction skill in rvu (relative value unit, 1 rvu equal to 5 minutes), indicated that the average work of a dentist was 3 rvu in extracting molar-1. the purpose of this research is to know whether or not gender differences (male and female) have an effect on work performance to raise the productivity. basically we will be dealing with a synthesis of the two: the application of certain measurement systems for the purpose of analyzing, classifying, and quantifying, that are, work measurement. work systems consist of integrated activities of people and machines, engaged in the production of goods and services, or in those activities which support that production. measurement systems are somewhat more abstract. they are integrated activities of people and instrument, use to analyze, classify, and quantify certain attributes of those entities to which they are applied.12 work measurement is an indispensable part of planning and control of an organization. it provides information which is the basis for almost all management decision making. material and method this study used combination of review and quasiexperimental method using the following frame of thinking (figure 1). variables observed was gender perspective, the viewpoint regarding working division according to sex, and performance, the outcome of work measured by working time standard and working quality, including injection precision, extraction skill and working method. population in this study was divided into two groups, the dentists who carried out tooth extraction and the patients whose tooth was extracted, in community health centers in east java. figure 1. frame of thinking dentist work performance in central health care indonesia. no method gender issue dentist working inefficiency predominant factor reduced performance increased performance equal partner elimination of predominant factor culture & perspective standard method method appraisal 167berniyanti and sudiyono: the effect of gender on dentist's performance using purposive sampling and simple random sampling we obtained 26 dentists and 64 patients as samples in 8 community health centers. data collecting was carried out using questionnaire and direct observation to the dentist’s working procedure during patient care, based on prevailing standard criteria in international health care, with the following criteria (table 1, 2, 3, and 4).13 this standard is performance rating table that contains score according to the level for each factor. to normalize the time would be carry on by calculating the time acquired from the work measurement, times the sum of four factor’s rating chosen according to the performance shown by the operator.7 the table of the performance rating can be seen below: table 1. standard criteria observation westinghouse skill ratings system for performance level13 score or rating value quality + 0.15 + 0.13 + 0.11 + 0.08 + 0.06 + 0.03 0.00 – 0.05 – 0.10 – 0.16 – 0.22 a1 a2 b1 b2 c1 c2 d1 e1 e2 f1 f2 super-skill super-skill exellent exellent good good average fair fair poor poor table 2. standard criteria observation westinghouse effort ratings system for performance level13 score or rating value quality + 0.13 + 0.12 + 0.10 + 0.08 + 0.05 + 0.02 0.00 – 0.04 – 0.08 – 0.12 – 0.17 a1 a2 b1 b2 c1 c2 d1 e1 e2 f1 f2 excessive excessive exellent exellent good good average fair fair poor poor table 3. standard criteria observation westinghouse system condition ratings for performance level13 score or rating value quality + 0.06 + 0.04 + 0.02 0.00 – 0.03 – 0.07 a b c d e f ideal exellent good average fair poor table 4. standard criteria observation westinghouse system consistency ratings for performance level13 score or rating value quality + 0.04 + 0.03 + 0.01 0.00 – 0.02 – 0.04 a b c d e f perfect exellent good average fair poor data analysis was done using multivariate (t. hotteling) test and independent two sample t test. data were interpreted using quantitative and qualitative approach. results measurement was conducted for performance variable, comprising diagnosing time and aenesthetic time for the parameter of preparation and the duration of act, as well as extraction time. measurement was undertaken to the time needed for molar extraction activity. measurement was also carried out for gender perspective responded by the patient. results of measurement presented in table 5 were analyzed for the difference of extraction time of male and female dentists. the analysis was divided into two forms, partial analysis for each measured parameter and simultaneous analysis for performance. the result of statistical analysis in each parameter was as follows. statistical analysis for each variable was independent two-sample average discriminant test, that was for the variable of diagnosing time, aenesthetic preparation time, and extraction time with the probability value for each 168 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 165-169 statistical test was t > 0.05. its is mean that the affect on dependent variable not significant. the summary of multivariate analysis is presented as follows. performance observed in this study consisted of skill, effort, condition and consistency with criteria as mentioned in methodology. the following table present performance in both observed groups. it can be explained that there is no difference in the performance in both gender groups, either in skill, effort, condition or consistency. the result of statistical analysis showed that there is no difference in the performance in both gender groups, either in skill, effort, condition or consistency. table 5. description of dentist work performance measurement in central health care east java dentist variable n male female mean sd mean sd diagnosing time preparation time extraction time 26 26 26 32.869 2.802 3.021 3.101 0.434 0.635 31.969 2.919 3.307 2.996 0.473 0.635 source: result of primary data processing table 6. result of two-sample discriminant test of dentist work performance measurement in central health care east java variable n db tcount prob diagnosing time preparation time extraction time 26 26 26 50 50 50 1.065 –0.927 –1.450 0.292 0.358 0.153 source: result of primary data processing table 7. result of two-sample discriminant test of dentist work performance measurement in central health care east java statistical value db fcount prob hotteling's trace 0.073 3 1.164 0.333 resource: result of primary data processing table 8. result of performance tabulation of dentist work performance measurement in central health care east java dentist groups rating quality skill effort condi consy skill effort condi consy male female 0.104 0.103 0.103 0.099 0.024 0.025 0.013 0.001 good good good good good good good good table 9. result of two-sample discriminant test of dentist work performance measurement in central health care statistical value db fcount prob hotteling's trace 0.039 4 0.457 0.676 resource: result of primary data processing 169berniyanti and sudiyono: the effect of gender on dentist's performance discussion the result of the above analysis revealed that there was no difference in the variable of diagnosing time, extraction preparation time, and extraction time in both compared male dentist and female dentist groups. thus, it can be explained that there was no difference in the performance between male dentists and female dentists in terms of their time in completing the task of diagnosing patient, extraction preparation, and extraction. analysis was also done using multivariate approach, whose results showed no difference in extraction time for measured variables (diagnosis, extraction preparation, aenesthesis, and extraction) for male and female dentist groups. the gender division of dentist in term of their physical feature is apparent and may affect the predilection of patient to them. however, after being analyzed, there was no significant difference in professional context between male and female dentist. gender concepts that divide male and female in their role and function apparently does not involve in professional dentistry field. based on the respondents experience, which was affected by their cognitive and cultural perspective, several substantial questions were delivered in order to describe the trend of dentist preference for dental care. from 6 items of question responded by the patients representing the community it was found that their perspective on gender was relatively homogeneous. there was no perception difference from the respondents on the male and female dentist performance. these responses indirectly provided justification that gender perspective had actually no effect on working activity, although some experts wrote that in the society there is a set of social relations, which also includes the aspect of power in gender relations. when the gender relations is unbalanced, in which women remains subordinated, other unjust relations may be present in that social relations. results obtained in this study, particularly in regard with the patient’s response that represents the user of health care, revealed that gender perspective does not affect the dentist performance. two conclusions can be drawn from this study: no effect of gender difference on dentist activity in community health centers, and no performance difference in gender perspective (male and female dentists). acknowledgment this article is a part of junior lecturers research funded by human resource development project, directorate general of higher education. in addition to this institution, we also thank to airlangga university research center and all parties who had supported this study. references 1. wijaya hr. mewujudkan kemitraan sejajar laki-laki dan perempuan. 1995. p. 25–27. 2. gadner o, wagemmann. m, sulaeman e, sulastri. perempuanperempuan indonesia dulu dan kini. jakarta: gramedia pustaka utama; 1996.jakarta: gramedia pustaka utama; 1996. p. 13–25. 3. wiek w. identitas dan peran gender. makalah seminar. ikip malang.makalah seminar. ikip malang. 1995. 4. ichromi. analisisa gender dan transformasi sosial. jakarta: gramedia pustaka utama; 1995. 5. abdullah i. sangkan paran gender. jogjakarta: pustaka pelajar offset gajah mada; 1997. p. 14–18. 6. bridger rs. intoduction to ergonomic. london, new york: taylorergonomic. london, new york: taylor & francis group; 2003. p. 15–17.2003. p. 15–17. 7. kroemer k, kroemer h, kroemer-elbert k. ergonomic how to design for ease and efficiency. 2nd ed. international series in industrial & system engineering. 2001. p. 1–6. 8. wignjosubroto s. teknik tata cara dan pengukuran kerja. surabaya: its; 1993. p. 115–123. 9. dinas kesehatan jawa timur. pedoman pelaksanaan pelayanan kesehatan gigi dan mulut. dinas kesehatan jawa timur. 1998. p. 20. 10. berniyanti. penetapan waktu baku dari pencabutan satu gigi dengan metode time motion study di puskesmas kodya surabaya. 1992. p. 1–7. 11. smith, geoege l. work measurement, system approach. columbus, ohio: grid publishing, inc; 1978. p. 7–10. 12. barnes rm. motion and time study: design and measuremment of work. new york, london: john wiley and sons, inc; 1980. p. 12. 13. stielle hb. motion and time study. new york, london: john wiley and sons, inc; 1997. p. 233. 194 vol. 42. no. 4 october–december 2009 the management of over closured anterior teeth due to attrition eha djulaeha and sukaedi department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: tooth is the hardest tissue in human body, that can be injured because of attrition process. for old people, denture attrition process is caused by psysiological process relating with the mastication function which also supported by some bad habits such an bruxism, premature contact, and consuming habit of abrasive food. attrition or abrasion can also be happened with patien’t dentition who does not have teeth subtutition for long time due the lost of their maxillary as well as mandibulary. the pasient will loose their vertical dimension of occlusion, injure, and the lower jaw becomes over closed which is called over closure. purpose: this article reported the management of over closured anterior teeth due to attrition. case: a seventy six year old woman patient came to prosthodontic clinic in faculty of dentistry, airlangga university, to rehabilitate her upper and lower severe attrited anterior teeth and her posterior teeth. the patient has experienced of wearing acrylic removable mandibular partial denture ten years ago. unfortunaly, the denture was uncomfortable, and she did not wear it anymore since five years ago. case management: the severe attrition of anterior teeth with the lost of occlusal vertical dimension can be treated by improving the occlusal vertical dimension gradually. the treatment is then followed by the increasing of the height of the anterior teeth by lengthening the crown teeth of upper jaw with 12 units of span bridge and the acrylic removable partial denture of lower jaw. conclusion: the severe attrition of anterior teeth with the lost of occlusal vertical dimension can be treated by improving the occlusal vertical dimension gradually, using long span bridge and acrylic removable partial denture. key words: attrition, over closure correspondene: eha djulaeha, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: eha_dj@yahoo.com introduction tooth is the hardest tissue in human body; however, this tissue can be injured because of attrition process. for old people, the dental attrition process is caused by physiological dan pathological processes. physiologically, it is related with the mastication function which supported by any bad habits like bruxism, premature contact, and consuming abrasive food, while pathologically is caused by endogenous or exogenous factors as well as by para function. the causes of attrition itself can be mechanically and chemically classified, mechanically in the form of attrition and abrasion, while chemically in the form of erosion.1,2 attrition or abrasion can also be happened with patien’t dentition who does not have teeth subtutition for long time due the lost of their maxillary as well as mandibulary. the pasient will loose their vertical dimension of occlusion, injure, and the lower jaw becomes over closed which is called over closure. under this condition, the patients tend to move the mandible forward and used their anterior teeth for mastication resulting attrition and abrasion on the anterior teeth.2 over closure is an occluding vertical dimension that results in excessive inter occlusal distance when the mandible is in the rest position. it results in reduced interidge distance when the teeth are in contact.2 over closure is signed by a deep fold at the corner of the mouth. in this condition the patients normally get easily tired while masticating food, having problem with clicking of the temporomandibular joint, and having their lower faces on third shorter.1 there are two kinds of vertical relation, first is when the teeth contact in centric occlusion. the second one case report 195djulaeha and sukaedi: the management of over closured anterior teeth is when during rest position. the rest position is neutral position of mandible when the muscles of the mouth are opening and closing in balance (minimal muscle tonus). the difference between the two vertical dimensions is called free way space which usually about 2–4 mm. if the free way space is more than 2–4 mm, it will indicate that over closure occurs.4,5 several methods could be applied to determine the occlusal vertical dimension. the first method is niswonger, the occlusal vertical dimension is obtained from jaw vertical dimension during the rest position subtracted by free way space (2–4 mm).5 second method is willis, the distance between nasal base to the point below the chin when the teeth or bite wall contacts.4 third method is silverman using phonetic method, in which the patient is asked to say such as “yesss” (words containing ‘s’ letter), and then the distance between it is noted from incisal edge to the most maximum line of occlusal contact when ‘s’ sound is produced.6 in this case, incisal gap is about 2–4 mm similar to free way space. the lost occlusal vertical dimension in long term can influence the appearance of the face, so it will look older, and in severe condition it may cause angular cheilitis.7 when the occlusal vertical dimension is lost, a therapy should be carried out to improve the vertical dimension. some kinds of therapies can be conducted, for example by lengthening the crown, doing the orthodontical tooth movement, repositioning teeth by surgical procedure, or making substitute of removable dentures.8 maryono3 did the over closure therapy by heightening the occlusal vertical dimension gradually. the result of her therapy was the disappearing of the over closure symptoms in two weeks until one month. the patient are also satisfied since they can get mastication comfort and aesthetical face again.3 the purpose of this case report is showing the treatment of a patient who had severe anterior teeth attrition with over closure and temporo mandibular joint problems. case a seventy six year old woman patient came to prosthodontic clinic in faculty of dentistry, university of airlangga, to rehabilitate her upper and lower severe attrited anterior teeth and her posterior teeth. the patient has experienced of wearing acrylic removable partial denture of lower jaw ten years ago. unfortunaly, the denture was uncomfortable, and she did not wear it anymore since five years ago. figure 1. panoramic photo of the patient showed severe teeth attrition and several posterior teeth lost. case management during the clinical examination of temporomandibular joint, it was found a clicking in the left and right temporomandibular joint, that made the patient felt uncomfortable. the intra oral examination, found the lost of 12, 14, 15, 23, 26, 27,34, 35, 36, 37,46, 47, and affected by attrition 11, 13,21, 22, 31, 32, 33, 41,42, 43, 44, and deep bite in anterior relation (over closure). morover after taking panoramic photo of the patient’s dentition (figure 1), it was showed that there were lost teeth and post endodontic treatment on 11, 21, and 22. anatomic duplicate of her upper and lower jaws impression was conducted by using stock tray with irreversible hydrocolloid materials and was casted with type ii of hard gyps in order to make diagnostic model or study model (figure 2). figure 2. diagnostic model of upper and lower jaws. the examination of occlusal vertical dimension with niswonger’s and willis’s ways were conducted by concerning with the appearance of the patient. the measuring result of vertical dimension of occlusion was 61 mm, and the rest position was 69 mm. it means that the occlusal vertical dimension is lost or reduced. in conclusion, the rest position was subtracted by the occlusion position, 69 mm – 61 mm = 8 mm, and then was subtracted by free way space. the result of the lost vertical dimension of occlusion was 8 mm – 4 mm = 4 mm. then, the diagnostic wax up for upper jaw model was taken by heightening the bite, about 2 mm (figure 3). figure 3. diagnostic wax up of maxillary model. 196 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 194-198 figure 4. the lengthening of anterior teeth and lower jaws with composite. diagnostic wax up were a long span maxillary bridge and heightening crown of mandibular anterior teeth which was for her lower jaw. both diagnostic waxed up were made by using wax material. the improving process of the lost occlusal vertical dimension was taken gradually conducted. the first stage, the restoration of the 33, 32, 31, 41, 42, and 43 were conducted by lengthening incisal, 2 mm, with of composite restoration a3 and a1 to improve the aesthetics and heighten the occlusal vertical dimension (figure 4). then the patient was evaluated for two weeks. the patient had no problems with her temporomandibular joint. the second stage was the preparation process of these teeth mention 11, 13, 21, and 22 (figure 5). figure 5. upper jaw teeth preparation. figure 6. the temporary bridge of upper anterior teeth.anterior teeth. the making process of temporary bridge of upper jaw for 11, 12, 13, 21, and 22 was conducted followed by the heightening process of occlusion about 2 mm of the temporary bridge in order not only to maintain the aesthetics and to improve the vertical dimention of occlusion, but also to treat the over closure condition of the patient. temporary bridge was by self curing acrylic (figure 6). the teeth should get anesthetic treatment 16, 24, and 25 as the preparation for making the long span bridge with 12 units made of porcelain materials fuced to metal materials. therefore, the final formation after the preparation of gingival margin area was in ridge form, meanwhile pontic form facing the gingival for anterior teeth was ridge lap, and for posterior teeth was sanitary. during the important preparation, the parallel position of axial areas of all supporting teeth must be concerned by using an aid tool, paralelometer, in order to make the insertion process of the long span bridge more easier. after the preparation process, the duplicating process with irreversible hydrocolloid materials and cast with the type iii of hard gyps was conducted. the next step was having correction of the parallel position among the prepared teeth with paralelometer tool that has parallel axis must be conducted on the model. the making of the long span bridge for 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 25, and 26, and the cantilever design for 26 with porcelain fuced to metal materials were conducted in order to find out the occlusal area of upper jaw appropriated with curve of spee (figure 7-a). after the making of the long span bridge is finished,it was inserted into the patient. the next stage was the making of acrylic removable partial denture for lower jaw. the 34 was given two fingerwrought wire with rest mesial, meanwhile 43 was given gilet clamer (figure 7-b). figure 7. a) the long span bridge of upper jaw; b) the lower jaw acrylic removable partial denture. figure 8. the upper jaw long span bridge and the lower removable partial denture inside the mouth. aa b 197djulaeha and sukaedi: the management of over closured anterior teeth during insertion the acrylic removable partial denture of lower jaw, the correction of occlusion must be conducted by articulating paper in order to obtain stable occlusion. after being polished, the acrylic removable partial denture was inserted into the mouth of the patient (figure 8). controls are conducted of the first and the seventh day after the insertion. there is no problem during the usage of acrylic removable partial denture. discussion the most common problem occurred is concerning with the severe problems of dental and periodontal damage treated by tooth extraction. therefore, in order not to reduce further problems of mastication process caused by the tooth extraction, the use of the removable partial denture or the fixed bridge should be done. the substituting of the lost teeth in upper and lower jaws by using the fixed bridge and the removable partial denture is not only to improve the function of mastication, talking, and aesthetics, but also to maintain the health of tissue in the mouth. the abnormality of the occlusion contact will occur when there is failure in the mastication system. it can disturb the movement that is supposed to be smooth among the teeth of upper and lower jaws. the failure of the mastication system then can cause the irregular movement pattern of lower jaw compared to what usually occurs in the normal condition since everyone will always tend to try to find a new movement pattern considered to be more comfort.9 during preparation of teeth, the spring line must be concerned in order not to obtain difficulties during the insertion process of the long span bridge. moreover the colar of long span bridge must also be appropriated with the color of the original color of the patient’s teeth in lower jaw. in order to obtain the treatment result of the fixed denture that meet the aesthetic aspects and the health requirements, it must be concerned with some factors like the relation between the teeth and the curve of the jaw, the health of the periodontium tissue, gingival retraction, the formation of dental anatomy, the pontic form like ridge lap, and the color of the facing fixed denture that must be appropriated with the original color of the teeth. the restoration of the dentures in this case involves regio anterior and regio posterior of upper jaw. this condition is also followed by the decreasing of occlusal vertical dimension. the treatment for this case is the long span bridge. the reason is because of its rigid characters that can avoid the fracture during the restoration, and therefore the apportionment of load can have the occlusion balanced for all the jaw curves.10 in other words, the restoration of the long span bridge that has rigid character can distribute the load more balance on the teeth of the antagonist jaw. besides, the long span bridge that exceeds the median line connecting the right and left sides of the upper jaw can hopefully have the balanced occlusion. in order to obtain the balance occlusion and stable denture, furthermore, there must be occlusal contact on working side. the contact must occur contemporaneously in working side of the fixed denture of upper jaw and the acrylic removable partial denture of lower jaw in order to spread the accepted load. it was conducted by doing occlusal adjustment to the patient’s denture.11 moreover, 31, 32, 33, 41, 42, 43, 44 of lower jaw must be treated by the heightening of the bite with the increment of initial part of those teeth in order to improve the occlusal vertical dimension and over closure. the heightening of the occlusal vertical dimension must be done gradually in order to let the muscles of the mastication adapt to the new occlusal vertical dimension. the long span bridge with cantilever design for 26 of upper jaw was chosen. the design was possibly chosen since the antagonist tooth using the acrylic removable partial denture which has smaller mastication ability than the original one. to find out the occlusal area of the long span bridge of upper jaw, it must be appropriated with the curve of spee of upper jaw, meanwhile the curve of the teeth must be appropriated with the curve of the jaw. according to hickey et al.,5 over closure is a problem of tempororo mandibular joint with the symptoms like sharp pain in temporo mandibular joint, discomfort, clicking, dizzy and neuralgia. the problem of ear function is caused by the decreasing distance between upper jaw and lower jaw, so the tongue will move to the backside caused the near tissue pushed and therefore closed the eustachian hole caused the problem of ear function. the occlusal vertical dimension that is too much low can cause condili move forward and push the front part of fossa articularis. the continual pressure can cause sharp pain around jaw joint that sometimes can cause dizzy. by improving the occlusal vertical dimension gradually the condili must gradually be repositioned into the original position in articularist fossa. because of the movement of condili gradually to the original position, then the pressure on fossa wall can be elimanated.5 problems with temporo mandibular joint can also cause headache and tinnitus. over closure caused by the lost of teeth can also cause costen’s syndrome with initial symptoms like tinitus, vertigo and dull pain around the ears.11 actually, all of the over closure therapy taken for any cases are followed by the improving of occlusal vertical dimension. though the improving of occlusal vertical dimension can change the position of lower jaw that then causes the elongation of the muscles of mastication, it still can not cause any symptoms in patients as long as it is not more than the appropriate vertical dimension of occlusion.12 meanwhile, much increasing of occlusion vertical dimension can cause the lost of free way space, inflammation on the tissues under the removable denture, pain in muscles, reabsorb of residual alveolar bone, horse 198 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 194-198 sound producing, sounds p,b, and m becomes unclear since the mouth can not close completely in which symptom of temporo mandibular joint syndrome then becomes increasing due to the load relating with joint.13 the improvement of occlusal vertical dimension is generally to reduce occlusal trauma of all chewing system, headache, by increasing functional comfort, improving aesthetics and stability, reducing temporo mandibular joint syndrome, and improving the occlusal damage because of the use of dentures.12 in conclusion, the severe attrition of anterior teeth with the lost of occlusal vertical dimension can be treated by improving the occlusal vertical dimension gradually, using long span bridge and acrylic removable partial denture. references 1. xhonga fa. bruxism and its effect on the teeth. j oral rehabil 1997; 4: 65–7. 2. glossary of prosthodontic terms. j prosthet dent 1999; 81: 39–110. 3. maryono r. overclosure rahang pada pemakai gigitiruan lengkap (hasil penelitian). perpustakaan unair. surabaya: fkg unair. 1992. p. 3. 4. sharry jj. complete denture prosthodontics. 3rd ed. new york: mcgraw-hill book co; 1974. p. 21–3. 5. hickey jc, zarb ga, bolender cl. boucher‘s prosthodontic treatment of edentulous patients. 10th ed. st louis: the cv mosby co; 1990. p. 237–76. 6. dawson pe. evaluation, diagnosis and treatment of occlusal problems. saint louis: the cv mosby co; 1974. p. 275–85. 7. rostiny. the correction of occlusal vertical dimention of tooth wear. dent j 2007; 40(4): 161–4. 8. turner ka, missirlian dm. restoration of extremely worn dentition. j prosthet dent 1984; 52: 467–74. 9. gunadi ha, burhan lk, surya tf. buku ajar ilmu gigi tiruan sebagianbuku ajar ilmu gigi tiruan sebagian lepasan. 1st ed. penerbit hipocrates, 1991. p. 52–55. 10. vena, hedge. significance of the frankfort mandibular planesignificance of the frankfort mandibular plane angle in prosthetic management partially of completely edentulous patients with class ii malocclutions. j indian prosthodontic soc 2005 ; 5(4): 175–9. 11. ash mm, ramfjod s. occlussion. 4th ed. philadelphia: wb saunders co; 1995. p. 1–75. 12. endang p. overclosure dan permasalahannya di bidang prostodonsia. maj. ked. gigi (dent j) 2001; 34(4): 161–3. 13. grant aa, johnson w. removable denture prosthodontics. 2nd ed. london, madrid, melbourne, new york, tokyo: churchill, livingstone; 1992. p. 71–7. 234 volume 45 number 4 december 2012 the p�� changes of artificial saliva after interaction with oralp�� changes of artificial saliva after interaction with oralchanges of artificial saliva after interaction with oral micropathogen basri a. gani,1 cut soraya,2 sunnati,3 abdillah imron nasution,1 nurfal zikri1 and rina rahadianur1 1 department of oral biology 2 department of conservative dentistry 3 department of periodontics 4 dentistry program study, medical faculty, universitas syiah kuala banda aceh indonesia abstract backgorund: saliva contains several protein elements, exocrine proteins and antibodies, such as lactoferrin, siga, peroxidase, albumin, polypeptides, and oligopeptides that contribute to the defense of oral mucosa and dental pellicle to prevent infection caused by oral micropathogen, such as candida albicans, streptococcus mutans and aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans). those micropathogens have a role to change salivary ph as an indicator of oral disease activities. purpose: this study was aimed to analyze the changes of artificial saliva ph after interaction with s. mutans, c. albicans, and a. actinomycetemcpmitans. methods: the materials used in this study consist of s. mutans (atcc 31987), c. albicans (atcc 10231), a. actinomycetemcomitans (attc 702 358), and artificial saliva. to examine the ph changes of artificial saliva, those three microbiotas were cultured and incubated for 24 hours. results: the results showed that the interactions of s. mutans, c. albicans, and a. actinomycetemcomitans in the artificial saliva can change the salivary on neutral. there were no significant difference with the control treatment salivary ph 4, 5, 6, 8, and 9 (p>0.05). similarly, there was also no significant difference when those three microorganism interacted each other in the artificial saliva (p<0.05). conclusion: it can be concluded that the biological activity of s. mutans, c. albicans, and a. actinomycetemcomitans in artificial saliva can change the salivary ph into neutral. it indicates that those microbiotas mutually supported and cooperated in influencing the biological cycle of the oral cavity with salivary ph as an indicator. key words: salivary ph, candida albicans, streptococcus mutans, aggregatibacter actinomycetemcomitans abstrak latar belakang: saliva merupakan cairan eksokrin yang mengandung unsur protein dan antibodi seperti siga laktoferin peroksidase, albumin, polipeptida dan oligopeptida yang berperan pada pertahanan mukosa rongga mulut dan gigi guna mencegah infeksi oral mikropatogen seperti c. albicans, s. mutans, dan a. actinomycetemcpmitans. patogenesis ketiga oral mikropatogen tersebut diawali dengan mempengaruhi perubahan ph saliva sebagai langkah invasi dan infeksi pada mukosa oral dan pelikel gigi. tujuan: penelitian ini bertujuan untuk untuk mengetahui perubahan ph saliva buatan setelah diinteraksikan dengan s. mutans, c. albicans, dan a. actinomycetemcpmitans. metode: materi penelitian ini berupa streptococcus mutans strain atcc 31987, candida albicans strain atcc 10231, aggregatibacter actinomycetemcomitans strain attc 702358, dan saliva buatan. untuk mengetahui perubahan ph saliva, maka ketiga mikrobiota tersebut dikultur dan untuk menguji perubahan ph saliva dilakukan uji interaksi ketiga mikroorganisme tersebut dalam saliva buatan selama 24 jam dengan pengaturan ph saliva sebagai indikator hasil penelitian. hasil: hasil penelitian menunjukkan interaksi s. mutans, c. albicans, dan a. actinomycetemcomitans dalam saliva buatan mampu mereduksi perubahan ph saliva mengarah ke ph netral dengan kontrol perlakuan ph saliva 4, 5, 6, 8, dan ph 9 secara statistik tidak tidak menunjukkan perbedaan bermakna (p>0,05), begitu juga ketika dilakukan interakasi diantara masing-masing mikroorganisme tersebut dalam saliva buatan menunjukkan adanya perbedaan bermakna (p<0,05). kesimpulan: dapat disimpulkan bahwa aktivitas biologi s. research report 235gani, et al.: the p�� changes of artificial saliva after interaction with oral micropathogenp�� changes of artificial saliva after interaction with oral micropathogenchanges of artificial saliva after interaction with oral micropathogen mutans, c. albicans, dan a. actinomycetemcomitans dalam saliva buatan mampu merubah ph saliva sekaligus mempertahankan ph netral. hal ini menggambarkan bahwa mikrobiota tersebut saling mendukung dan bekerjasama dalam mempengaruhi siklus biologi rongga mulut dengan ph saliva sebagai indikator. kata kunci: ph saliva, candida albicans, streptococcus mutans, aggregatibacter actinomycetemcomitans correspondence: basri a. gani: c/o: bagian biologi oral, program studi kedokteran gigi, fakultas kedokteran universitas syiah kuala darussalam-banda aceh. 23111. email: basriunoe@yahoo.com introduction as pathogens in oral cavity, streptococcus mutans (s. mutans) and aggregatibacter actinomycetemcomitans (a. actinomycetemcomitans) are considered as the cause of dental caries and periodontitis.1,2 although infection caused by them is different, but it is known that both of these bacteria support each other in pathogenic process, especially in biofilm formation of dental caries infection and periodontitis.3 these two micropathogens also play an important role on the activity of moleculer fungus, candida albicans (c. albicans), as an agent of oral candidiasis infection. in alkaline condition, those two bacteria can facilitate c. albicans to form biofilms on oral mucosa,4,5 on the other hand, in acidic condition, c. albicans is precisely able to adapt to changes in the acidic environment of the oral cavity, that is affected by salivary ph.6 it means that the salivary ph can determine the pathogenicity of those micropathogens. saliva is exocrine fluid containing water, approximately about 99%. other supporting elements consist of an organic component involving sodium, calcium, potassium, magnesium, bicarbonate, chloride, rodanida and thiocynate (cns), phosphate, potassium, and nitrate; meanwhile, inorganic components consist of amylase, peroxidase, maltase, albumin protein, kretinin, mucin, vitamin c, amino acids, lysozyme, lactic acid, and hormones, such as testosterone and salivary cortisol; besides, there are also siga antibodies, lactoferrin, polypeptides and oligopeptides that contribute to oral mucosal defense and dental pellicle.7, 8 the degree of acidity (ph) of saliva is affected by diet, stimulation of salivary secretion, and activity of oral microorganisms. diet with high carbohydrate can either lead to a decrease in salivary ph, accelerate the demineralization of tooth enamel, or produce acid through glycolysis process that can lower the salivary ph into the critical one (5.5 to 5.2).9 in contrast, the alkaline properties of saliva can either neutralize the acidity of the mouth, reduce tooth decay, prevent the formation of plaque and calculus, or reduce the risk of periodontitis. furthermore, calcium contained in saliva may play a role in tooth enamel remineralization.10 the changes of the biological properties of the saliva that are likely to affect oral biological abnormalities, such as xerostomia are caused by the imbalance of salivary ph regulation.11 in addition, the critical ph of saliva is also worsened by carbohydrate fermentation activities triggered by a number of pathogenic oral microorganisms, such as c. albicans, s. mutans, and a. actinomycetemcomitans.12 the changes of the salivary ph in oral soft tissues are often associated with dental caries, periodontitis, and oral candidiasis. the changes of the ph of acidic and alkaline saliva can trigger saliva viscosity and facilitate the fermentation of carbohydrates and salivary proteins that can cause the imbalance of the growth of those three micropathogens11 since s. mutans can survive only at the critical ph of 4.5-5.0,1 c. albicans can grow only at the ph of 4.5-6.5,13 while a. actinomycetemcomitans can only grow better at the ph of 7-8.5.2 in addition to being considered as pathogens in the oral cavity infections, those three microorganisms also contribute to endocarditis infection.14 antibacterial medicines can either accelerate the growth of fungi or provide the threat of chronic candidiasis oral infections. thus, the ph of the oral cavity must be adjusted with salivary ph as an indicator and supporter of biological activity in oral cavity preventing the virulence factors of those three micropathogens from biofilm formation, colonization, invasion and infection of the host. therefore, saliva specifically becomes the controler of pathogenic microorganism activities in oral cavity through salivary ph regulation in order to maintain the ph balance of the oral cavity. as a result, this study was aimed to analyze the changes of salivary ph (acidic and alkaline) after interaction with c. albicans, s. mutans, and a. actinomycetemcomitans as well as after those interaction with each other. finally, the results of this study are expected to be able to prevent invasion and infection caused by oral micropathogens, mainly on dental caries, periodontitis, and oral candidiasis through salivary ph control in order to regulate oral biological and ecological cycles. materials and methods this study is an in-vitro laboratory experiment conducted in the laboratory of microbiology and immunology, faculty of veterinary, syiah kuala university in banda aceh. materials used in this study were a laboratory strain of s. mutans, atcc 31 987, and a laboratory strain of c. albicans, atcc 10231, obtained from the laboratory of microbiology, faculty of veterinary, syiah kuala university, as well as a laboratory strain of a. actinomycetemcomitans, attc 702 358, 236 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 234–238 obtained from the laboratory of oral biology, faculty of dentistry, universitas indonesia. those three pathogenic microorganisms were used as biological control or balance of acid and alkaline environment changes. other materials used as artificial saliva were 0.702 g nacl, 0.221 g kcn, 1.495 g nahco3, 1.153 g kcl, 1.100 g h2nconh2, 0.213 g na2hpo4, and 0.204 g kh2po4 obtained from the laboratory of biochemistry, faculty of medicine, universitas indonesia. the artificial saliva was used as a reference for the interactions of those micropathogens, especially in acidic and alkaline environments. the various components of these materials were then analyzed with various forms of analysis approaches. the laboratory strains of a. actinomicetemcomitans and s. mutans were cultured by using treak late. the laboratory strain of s. mutans was cultured on muller hilton agaragargar (mha) media, while a. actinomicetemcomitans using nutrient agar (na) media. each of those two bacteria wasutrient agar (na) media. each of those two bacteria wasagar (na) media. each of those two bacteria wasgar (na) media. each of those two bacteria was then put into anerobic jar and incubated at 370 c for 48 hours. for confirmatory test, those bacteria were stained. afterwards, they were cultured in tsb liquid medium for 48 hours in order to become samples for analysis. on the other hand, the laboratory strain of c. albicans was taken about 1 ose, and then cultured on selective sabouraud dextrose agar (sda) media using t scratchingdextrose agar (sda) media using t scratchingextrose agar (sda) media using t scratchingagar (sda) media using t scratchinggar (sda) media using t scratching technique. next, it was incubated in an incubator for 2448 hours at 370 c. colonies of c. albicans grown on sda media were suspended by taking a colony of c. albicans from the sda media, which was then put into peptone solution. the level of turbidity of c. albicans in the peptone solution was compared with mc farland solution (1.5 x 108 cfu/ml). s. mutans and a. actinomicetemcomitans, that have been cultured from tsb liquid medium, and c. albicans, that has been cultured in peptone, were respectively centrifuged at 2000 rpm for 5 min. their residue was then collected and added with 15 ml pbs. afterwards, 15 ml pbs was also added to the bacteria and c. albicans was added with 15 ml peptone solution. they were then vortexed and resuspended. next, each of these microorganisms was taken and put into 2 ml of the artificial saliva prepared with ph of 4, 5, 6, 8, and 9. afterwards, they were incubated for 72 hours, and every 24 hours the changes of the salivary ph were measured by using a ph meter, and the artificial saliva with normal ph was used as control. finally, data obtained from the interaction between s. mutans, c. albicans and a. actinomycetemcpmitans in the artificial saliva were statistically analyzed with a normality test, kolmogorov-smirnov test, followed with repeatedrepeatedepeated measures and anova test using spss software.easures and anova test using spss software. results the results of this study were then reported in two forms of analysis, the analysis of the changes of the salivary ph after interacted with oral micropathogens (table 1) and the analysis of the changes of the ph of the artificial saliva after interacted with oral micropathogen (table 2). the ph of the saliva that has been interacted with s. mutans, c. albicans, and a. actinomycetemcomitans was measured 3 times during 24 hours. each of the ph values presented in the table was the average value derived from the examintaions conducted three times. discussion many researches on s. mutans which cause dental caries, a. actinomycetemcomitans which cause periodontitis, and c. albicans which cause oral candidiasis, have showed that they can cause oral health problems, one of which is related to saliva ph imbalance.15 this is due to the salivary ph that has an important role to regulate both metabolic activities of normal flora microbiotas and biological balance of oral cavity. thus, the changes of the saliva ph can cause the table 1. the changes of the ph of the artificial saliva after interacted with s. mutans, c. albicans, and a. actinomycetemcomitans types of microorganism in the artificial saliva the changes of the salivary ph ph 4 ph 5 ph 6 ph 8 ph 9 s. mutans 5.79 5.25 6.38 7.38 7.53 c. albicans 4.71 5.59 6.42 7.16 7.29 a. actinomycetemcomitans 5.84 6.75 7.91 8.49 8.43 table 2. the changes of the ph of the artificial saliva after s. mutans, c. albicans, and a. actinomycetemcomitans interacted each other types of microorganism in the artificial saliva the changes of the salivary ph ph 4 ph 5 ph 6 ph 8 ph 9 s. mutans and c. albicans 6.76 6.93 7.07 8.18 8.19 s. mutans and a. actinomycetemcomitans 6.47 6.53 7.04 7.95 7.82 a. actinomycetemcomitans and c. albicans 7.32 7.23 7.25 7.27 7.34 237gani, et al.: the p�� changes of artificial saliva after interaction with oral micropathogenp�� changes of artificial saliva after interaction with oral micropathogenchanges of artificial saliva after interaction with oral micropathogen normal flora microorganisms of the oral cavity evolved into a pathogen accelerating invasion, inflammation and infection of the host.16 the changes of the ph of the artificial saliva after interacted with s. mutans, c. albicans, and a. actinomycetemcomitans for 24 hours (table 1) indicate that those three microorganisms have the properties of adaptation to its ph growth. besides, it is also known that the saliva could also serve as a biological control for the activities of the oral microbiotas contained in it although in general the changes were not significantly different (p>0.05) from the control ph, so it is suspected that there was adjustment phase for the properties of each of these microorganisms. this nature will change as there is a change in the ecology and biology of the oral cavity caused by an imbalance triggered by various factors, such as disorders of hormonal system and oral cavity ph.17 temperature factors, chemical factors, and psychological factors can also become the main determinant of the pathogenic properties of oral microbiota, such as s. mutans, c. albicans, and a. actinomycetemcomitans.18 this shows that the virulence properties of the three oral microbiotas will become active and pathogenic when influenced by those factors.19 it is known that the interactions between s. mutans and a. actinomycetemcomitans, and between c. albicans and a. actinomycetemcomitans as shown in table 2 were significant (p<0.05). it means that those oral micropathogens could lower the control ph 8 and 9 to neutral one (7.2 to 7.9). it is related with the bioactive components contained in the artificial saliva, that is possible to affect the changes of the ph caused by bacteria and fungi, while in the normal condition, siga and lactoferrin proteins have a role to inhibit the biological activity of the oral microbiota associated with the changes of the salivary ph.17 interaction of s. mutans and c. albicans in the artificial saliva can actually make the ph more alkaline, specifically the control ph 4, 5, and 6. it can be assumed that the microorganisms, in addition to making the ph stable in an acidic environment, is also able to adapt to an alkaline environment, called the instability of acid.19 s. mutans and c. albicans are two microorganisms that always express virulence properties when the instability of acid occurs in the oral cavity, and their growth is still stable despite the critical acidic ph (3 to 4.5).1 the instability of acid can trigger and stimulate the virulence properties of the microorganisms to become more pathogenic, as well as can affect the biological properties of saliva.20 s. mutans not only have both aciduric and acidogenic properties by producing a dextran, which play a role in the formation of biofilm on the tooth surface before causing colonization, invasion and infection of the tooth enamel and chronic caries.21 meanwhile, c. albicans can live in various acidic and alkaline ph as well as in neutral ph although at 4.56.5 ph. the activity and expression of manoprotein of c. albicans are affected by changes in temperature and ph that can inhibit protein synthesis and reduce manan activity (cell wall protein) that affect the growth of c. albicans.22 as bacteria which are stable in alkaline environments, a. actinomycetemcomitans and c. albicans were able to make the ph of the artificial saliva neutral or alkaline (table 2). this is because a. actinomycetemcomitans has strong adhesion properties and can colonize well in saliva, so the condition of the salivary ph can be controlled by the bacteria in order to balance the ph and the growth of c. albicans.23,24 in molecular level, s. mutans (39-864) molecules, played a role in attachment to the salivary protein, so the localization of acid products occured with a high concentration on the surface of tooth enamel. this acid will lower the ph of the oral cavity, so it can cause demineralization of the enamel.14 in cellular level, two proteins of the cell wall were hydrolyzed, such as fructose hydrolyzed by fructosyltransferase and dextran hydrolyzed by glucosyltransferase (gtf). this process aims to maintain the instability of the critical ph of gtf and glucan binding protein (gbp) as trigger protein to produce lactic acid either by s. mutans or by other bacteria that mediates colonization on tooth enamel, so glucosedextran can lower saliva properties either as a protector or as antibacteria on tooth surface.25 interactions among microbiotas in the oral cavity were either facilitated by components of salivary proteins or caused by interactions of molecule bondings, such as interactions between proteins, hydrophobicity bonding on cell surface, electrostatic bonding, and biofilm matrix protein molecule bonding that interacts with either c. albicans, s. mutans or a. actinomycetemcomitans. the bonding and interaction among those protein molecules then can form coaggregasion and cause adhesion among microorganisms.15 one function of the molecular interaction among the microbiotas is to maintain the ph balance of oral cavity, including salivary ph, as biology and ecology control of the oral cavity. the acidity of the saliva is also dependent on hydrogen ions contained in the solution. at alkaline ph, c. albicans unable to adapt because the increasing of cell wall polysaccharide reactiviting and components of salivary proteins, thereby affecting cell wall resistance and instability of the absorption of hydrogen ions caused by cytoplasma that can interfere energy supply as a results of cell lysis. 24 the research of oral candidiasis in denture users indicates that the interaction of c. albicans, streptococcus sp, and actinomyces sp in the saliva may decrease the population of c. albicans. in addition, this condition occurs as a result of the activity of lipopolysaccharide molecules produced by bacteria that are able to inhibit the formation of hyphae of c. albicans, so this condition then can facilitate the penetration of protein molecules of the bacterial surface into the cells of c. albicans.12, 2626 therefore, this research showed that there was a close relation between the influence of oral activity of microphatogen and the changes of the ph of the artificial saliva since the results of this research provide information 238 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 234–238 that in normal conditions, c. albicans, s. mutans, and a. actinomycetemcomitans can consistently maintain ph of the oral cavity in order to maintain their stable life as normal floras, but they will change into pathogens when the salivary ph changes. this research also provides information that the salivary ph will change when there is biological imbalance of oral cavity, thus, it is necessary to balance the salivary ph as an attempt to control the growth of oral pathogenic microbiotas and to support ecological and biological activities in oral cavity. in other words, it can also be considered as an attempt to maintain the integrity of pathogenic bacteria as normal flora microorganisms, so it can prevent infection and oral diseases, such as dental caries, periodontitis and oral candidiasis. it can be concluded that biological activities of s. mutans, c. albicans, and a. actinomycetemcomitans in artificial saliva can change salivary ph as well as maintainsalivary ph as well as maintainlivary ph as well as maintain its neutral ph. this condition indicates that the microbiotas can support and co-operate each other in influencing the biological cycle of the oral cavity with saliva ph as an indicator. acknowledgments this study was financially supported by universitas syiah kuala, ministry of education, in accordance with the agreement of research assignment for �oung lecturer, number: 2159/h11/lk-pnbp/2011, dated may 18, 2011. references 1. gani ba. acidogenic and aciduric properties of streptococcus mutans as the bacteriostatic against oral microbiota pathogen. cakradonya dental journal 2010; 2(1): 128–36. 2. ardila cm, alzate j, guzmán ic. relationship between gram negative enteric rods, aggregatibacter actinomycetemcomitans, and clinical parameters in periodontal disease. j indian soc periodontol 2012; 16(1): 65–9. 3. loesche w. dental caries and periodontitis: contrasting two infections that have medical implications. infect dis clin north am. 2007; 21(2): 471–502. 4. filoche s, wong l, sissons ch. oral biofilms: emerging concepts in microbial ecology. j dent res 2010; 89(1): 8–18. 5. vílchez r, lemme a, ballhausen b, thiel v, schulz s, jansen r, sztajer h, wagner-döbler, i. streptococcus mutans inhibits candida albicans hyphal formation by the fatty acid signaling molecule trans2-decenoic acid (sdsf). chembiochem 2010; 11: 1552–62. 6. puy cl. the rôle of saliva in maintaining oral health and as an aid to diagnosis. med oral patol oral cir bucal 2006; 11: 449–55. 7. dawes c. salivary flow patten and the health of hard and soft oral tissues. j am dent assoc 2008; 139: 185–245. 8. damle sg, vidya i, �adav r, bhattal h, loomba a. quantitative deter m ination of inorganic constituents in saliva and their relationship with dental caries experience in children. dentistry journal 2012; 2(3): 131–5. 9. ilie o, van loosdrecht mcm, picioreanu c. mathematical modelling of tooth demineralization and ph profiles in dental plaque. journal of theoretical biology 2012; 309: 159–75. 10. vijayaprasad ke, ravichandra ks, vasa aa, suzan s. relation of salivary calcium, phosphorus and alkaline phosphatase with the incidence of dental caries in children. j indian soc pedod prev dent 2010; 28: 156–61. 11. lončar b, stipetić mm, baričević m, risović d. the effect of lowlevel laser therapy on salivary glands in patients with xerostomia. photomedicine and laser surgery 2011; 29(3): 171–5. 12. morales dk, hogan da. candida albicans interaction with bacteria in the context of human health and disease. microbiol mol biol rev 2010; 21: 245–345. 13. gani ba. diversity of candida albicans virulence factor as the infectious recognizer. cakradonya dental journal 2011, 3(1): 323–31. 14. basri ag, tanzil a, mangundjaja s. molecular aspect of the streptococcus mutans virulence properties. indonesian journal of dentistry 2006; 13(2): 107–14. 15. taubman ma, genco rj, hillman jd. the specific pathogen-free human: a new frontier in oral infectious disease research. adv dent res 1989; 3(l): 58–68. 16. hasturk h, kantarci a, thomas e, dyke v. oral inflammatory diseases and systemic inflammation: role of the macrophage. front immunol 2012; 3(118): 1–17. 17. marcotte h, lavoie mc. oral microbial ecology and the role of salivary immunoglobulin a. microbiol mol biol rev 1998; 62(1): 71–109. 18. fernanda gb, camila fo. amanda f, cristina k, vanderlei sb, denise m p, spolidório, josimeri h, carlos adsc. in vitro effect of low-level laser therapy on typical oral microbial biofilms. braz dent j 2011; 22(6): 502–10. 19. saha s, tomaro-duchesneau c, malhotra m, tabrizian m, prakash s. suppression of streptococcus mutans and candida albicans by probiotics: an in vitro study. dentistry journal 2012; 2(6): 141. 20. niemi lz. host ligands and oral bacterial adhesion: studies on phosphorylated polypeptides and gp-340 in saliva and milk. dissertation. umeå university: umeå department of odontology; 2010. p. 11–26. 21. arthur ra, cury aadc, mattos-graner ro, rosalen pl, vale gc, leme afp, cury ja, tabchoury cpm. genotypic and phenotypic analysis of s. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions. braz dent j 2011; 22(4): 267–74. 22. kruppa m, rachel r. greene, noss i, douglas w. lowman, williams dl. candida albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature. oxford: oxford university press; 2011. p. 1–28. 23. ouhara k, komatsuzawa h, shiba h, uchida �, kawai t, sayama k, hashimoto k, taubman ma, kurihara h, sugai m. actinobacillus actinomycetemcomitans outer membrane protein 100 triggers innate immunity and production of β-defensin and the 18-kilodalton cationic antimicrobial protein through the fibronectin-integrin pathway in human gingival epithelial cells. infection and immunity 2006; 74(9): 5211–20. 24. lima ll, farias ff, carvalho mar, alviano cs, farias lm. influence of abiotic factors on the bacteriocinogenic activity of actinobacillus actinomycetemcomitans. microbiology 2002; 153(4): 249–52. 25. bikandi j, moragues, md, quindos, g, polonelli l, ponton j. influence enviromental ph on reactivity of candida albicans with salivary iga. j dent rest 2000; 79: 1439. 26. mayocchi k, restelli, alfredo m. ultra structural study of the effect of candida albicans on the surface of the temporary enamel acta microspora. american dental association 2009; 18: 453–4. �� vol. 42. no. 1 january–march 2009 the role of microendodontics in treating mandibular second molar with five canals harry huiz peeters private practitioner abstract background: finding the incidence of anatomical variation in daily practice becomes more frequent due to the development of diagnostic tools such as microscopes and ultrasonic devices. therefore, a thorough understanding of the normal anatomy of canals and its variations in root canal treatments would be helpful in achieving the desired result. the usual root configuration of a mandibular second molar is two separate roots, distal and mesial, of which the distal root usually has one canal and the mesial root two canals. the incidence of 4 distinct root canals in the distal root of c-shaped orifices is a rare phenomenon that is reported in literature. purpose: to understand the role of microendodontics in trating mandibulary second molar with five canals. case: this case shows how to locate and manage a rare anatomical variation of mandibular second molar with 4 distinct root canals in the distal aspect of a c-shaped orifice successfully. case management: the anatomical variation was overcome with the help of diagnostic tools such as microscopes and ultrasonic tips. conclusion: the successful treatment of this case demonstrates the necessity for awareness of the presence of additional canals and abnormal morphology of the canals. besides that the operator’s perseverance and patience as well as sophisticated diagnostic tools, such as the dental operating microscopes and ultrasonic tips, have an important role in treating unusual configurations of root canals. key words: c-shaped orifices, dental operating microscope, ultrasonic tips correspondence: harry huiz peeters, c/o: private practitioner, cihampelas 41 bandung, indonesia. e-mail: h2huiz@cbn.net.id introduction the basic precondition for successful root canal treatment is to seal the root canal system completely through a thorough mechanical and chemical debridement of the entire root canal followed by 3 dimensional obturation with an inert filling material and a final coronal restoration, thereby preventing from reinfection of microorganisms.1 one of the root canal treatment failures is the presence of microorganism in the root canal system, because of insufficient debridement of the entire root canal system especially in the complex root canal system such as c shaped canal.2 the complexity of the root canal system has both technical and microbiological objectives that should be overcome. the most frequent reason for failure is oversight of their existence. therefore, a thorough understanding of canal anatomy in treating root canals which have anatomical variations and racial characteristic groups is an important figure 1. intraoperative photograph of the pulp chamber floor revealing 4 separate orifices in distal aspect. (db, dm, dl1, dl2). case report ��peeters, et al.: the role of microendodontics in treating mandibular prerequisite in negotiating and determining the location of a canal as well as its subsequent management.3 finding the incidence of anatomical variations in daily practice becomes more frequent due to the development of diagnostic tools. this case study shows how to locate and manage a rare anatomical variation successfully, especially in treating mandibular second molar with 4 distinct canals in the distal aspect of a c-shaped orifice. case a 65-year-old female patient with a chief complaint of intermittent pain in relation to tooth 47 (mandibular right second molar), especially when she closed her mouth. that particular pain had lasted over a 6 months period, and had increased in intensity over the previous 2 days. medical history was noncontributory. intraorally, the patient had moderate oral hygiene. tooth 47 was non-responsive to cold and electric pulp test. further examination showed that tooth 47 was tender to percussion and non-responsive to palpation, and there was an intact big composite restoration. there was no evidence of either swelling or sinus tract. the periodontal condition was excellent, with no gingivitis and absence of pocket. radiographic evaluation of the involved tooth showed no evidence of radiolucency and has already been treated endodontically. the tooth was diagnosed as a pulpless tooth with an infected root canal system. root canal treatment needed be done as a treatment of choice, followed by restoration and crowning. the patient signed an informed consent form as a medicolegal procedure. the patient would be reviewed after 6 months and then annually. at the first visit, the tooth was isolated under rubber dam, accessed with local anesthesia and sterilized by iodine solution. under microscope inspection, composite restoration and carious dentine were removed prior to accessing the pulp chamber by fissure round bur and straight line access was achieved. it was discovered that tooth 47 had a c-shaped orifice. using a size 0. 8 c+ file a canal was identified in each of the mesial and distal roots. further microscopic investigation (under magnification 1x) of the distal root orifice revealed possible other canals in the distal aspect. when explored with size 0.8 c+ file, there were 4 canals in distal aspect. all canals were confirmed radiographically. it was concluded that tooth 47 had 5 canals. an apex locator was used to verify the working length in all five root canals. an additional operative radiograph was taken to confirm the independent presence of the four canals in the distal root. individual canal instrumentation was performed sequentially with rotary ni-ti protaper universal files nos: 15, 20, 25, 30 (6%) using a crown-down pressureless technique to a master apical size 30. copious chemical irrigation was performed with 2.5% sodium hypochlorite. after completion of chemomechanical preparation, ultrasound was used to activate the irrigants in the entire canals, this has been proved as an valuable action in penetrating small spaces. the root canals were dried with sterile paper points, the pulp chamber was examined again under the microscope for any additional canals and photographs were taken under magnification 1.6× (figure 1) and an intracanal dressing of ultracal paste was used before placing temporary filling. during the second visit the patient had no symptoms. obturation was performed at this second appointment using warm vertical compaction of gutta-percha and ah26 as a root canal sealer. prior to obturation, edta (17%) followed to remove the smear layer and a final irrigation of 2.5% sodium hypochlorite solution was used. the excess of gutta-percha cones were cut at the level of the root canal orifices and vertical compaction was applied immediately with iso size 40 finger plugger after being heated by system-b. this action will ensure better homogeneity of gutta-percha and the gutta-percha will fill canal irregularties as well as accessory canals. a coronal permanent restoration of composite was placed and a postoperative radiograph was taken in order to assess the quality of obturation in all canals (figure 2). the patient was requested to return postoperatively after 6 months (figure 3) and again after one year. at the recall appointment, the patient was symptom free. figure 2. postoperative radiograph showing the canals were filled with gutta-percha. figure 3. after 6 months recall with no symptoms. �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 12-14 discussion success in root canal therapy was originally based on cleaning, shaping, and filling the entire root canal system effectively. in achieving a successful result, a thorough knowledge of the morphology of the root canal system is an essential prerequisite. many methods are used to investigate the root canal anatomy in vitro and in vivo, however, the specimens are destroyed by grinding or splitting. based on these concerns, radiography becomes the most practical and often used method to predict the root canal anatomy in both laboratory and clinical studies. careful assessment of the preoperative radiograph is a key step for subsequent root canal treatment, such as root canal preparation and obturation. preoperative radiographs may aid in visualizing and observing the anatomy of various root canal systems, especially the complicated ones, such as a c-shaped canal configuration. a ccanal system is a canal variant mostly seen in mandibular second molars.4 the incidence of c-shaped molars in the general population is approximately 8 percent, but can be as high as 31 percent in asian ethnic groups.5 anatomically, the teeth do not always have a normal shape. a great number of variations may occur in formation, number of roots, and their shape. however, dentists must have an awareness of the presence of rare anatomical variation; even though abnormalities are rare.6 many studies have been performed over the years to investigate tooth morphology, including mandibular second molars.1 the major variant in this group is the madibular first molar with 3 roots. the additional root is usually located on the lingual aspect.7 whereas the most prominent prevalence of c-shaped root canals is reported in the mandibular second molar.8 yang et al. claimed that there was a high prevalence of c-shaped roots in mandibular second molars (31.5%), in which 68.3% of the teeth had c-shaped canal orifices. the usual root configuration of a mandibular second molar is two separated roots, mesial and distal. the distal root usually has one canal and the mesial root has two canals that often converge in the apical area.10 in general, two-rooted mandibular second molar have a single distal canal (about 90%) and two mesial canals (more than 70%).11 c-shaped molar configuration develops when hertwig’s epithelial root sheath fails to fuse either on the buccal or lingual surface. that fusion failure on both the buccal and lingual aspect would results in a groove on the opposite side of the root that is present coronoapically.11 the canal configuration was significantly related to race, with more asian having c-shaped canals.11 recognizing the anatomical variation of c-shaped canal configuration would be helpful in determining and locating canal orifices to prevent catastrophic perforations when instrumenting the thin isthmuses. because of this complex morphology of second mandibular molar more over with the presence of a high incidence of transverse anatomoses, lateral canals and apical deltas make it difficult to clean and seal the canal system adequately. in this case report to minimize the failure of sealing the canal, the obturation was done with warm vertical compaction that allows the guttapercha to flow into small spaces, deltas, anastomoses and lateral canals effectively and will ensure better homogeneity of gutta-percha and a hermetical sealing can be achieved. the ultrasonic device that was used is believed to be very effective to provide optimal cleaning. the main goal of this case study is to report a successful treatment of 4 distinct root canals in the distal of the cshaped orifices. the success of nonsurgical root treatment of mandibular second molar with 5 root canals is a result of the combination of anatomical knowledge and the variations thereof, together with experience as well as the support of sophisticated diagnostic tools such as dental operation microscope and ultrasonic device. references 1. vertucci fj. root canal morphology and its relationship to endodontic procedures. endodontic topics 2005; 10:3–29. 2. nair pn. on the causes of persistent apical periodontitis: a review. int endod j 2006; 39:249–81. 3. plotino g. a mandibular third molar with three mesial roots: a case report. j endod 2008; 34:224–6. 4. bing f, yuan g, wei fj. gutmann. identification of a cshaped canalgutmann. identification of a cshaped canal system in mandibular second molars-part ii: the effect of bone image superimposition and intraradicular contrast medium on radiograph interpretation. j endod 2008; 34:160–5. 5. cooke hg, cox fl. c-shaped canal configurations in madibular molar. j am dent assoc 1979; 99:836–9. 6. jamileh g, neda n, mina z, ehsan r. mandibular first molar with four distal canals. j endod 2007; 33:1481–3. 7. speber gh, moreau jl. study of the number of roots and canals in senegalese first permanent mandibular molars. int endod j 1998; 31:117–22. 8. weine fs. the c-shaped mandibular second molar: incidence and other considerations. j endod 1998; 24:372–5 9. yang zp, yang sf, lin yc, shay c, chi cy. c-shaped root canals in mandibular second molar in a chinese population. endod dent traumatol1988; 4:160–3. 10. yi min, bing fan, gary sp, cheung, gutmann jl, mingwen f. c-shaped canal system in madibular second molars. part iii: the morphology of the pulp chamber floor j endod 2006; 32:1155–9. 11. manning sa. root canal anatomy of mandibular second molars. part ii: c-shaped canals.int endod j 1990; 23:40–5. 173 the ability of 5% tamarindus indica extract as cleaner of the root canal wall smear layer erawati wulandari1, latief mooduto2, and theresia indah budhy s3 1 department of conservative dentistry, faculty of dentistry, jember university, jember indonesia 2 department of conservative dentistry, faculty of dentistry airlangga university, surabaya indonesia 3 depatment of oral biology faculty of dentistry, airlangga university, surabaya indonesia abstract tamarindus indica is one of traditional medicines. pulpa tamaridorum consist of organic acid that is usually used as irrigant and to remove root canal wall smear layer. the aimed of this study was to elucidate the ability of 5% tamarindus indica extract as a root canal irrigant to remove root canal wall smear layer. eighteen tooth samples were cut on cervical line and divided into 2 then groups were prepared with k file and irrigated. group 1 was irrigated by sterile aquabidest and group 2 was irrigated by 5% tamarindus indica extract. samples were cut longitudinally and formed 7 × 2× 2 mm specimen. each specimen was photographed by scanning electron microscope, scored and summed. the total score obtained is used as the hygiene value of root canal wall. the collected data were statistically analyzed by using independent t test at 0.05 level. the result of the study showed there was a significant difference between 5% tamarindus indica extract and sterile aquabidest (p < 0.05), the hygiene value of 5% tamarindus indica extract was higher than sterile aquabidest. the conclusion of this investigation showed that 5% tamarindus indica extract remove root canal wall smear layer. key words: tamarindus indica, smear layer, root canal wall correspondence: erawati wulandari, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. introduction tamanridus indica has been known as traditional herbal medicine. the pulpa tamarindorum has an efficacy for mouth sore, injury and fever.1 the chemical content of pulpa tamarindorum consist of invert glucose, tartaric acid, citric acid, 1-malic acid, pipecolic acid, serine, beta-alanine, proline, phenylalanine, leucine.1 tjitrosoepomo2 suggested that pulpa tamarindorum contains various organic acid such as tartrate, lactate, malonate acid, 12–15% citric acid, to k-bitartra, pectine, tanine, invert glucose. in dentistry, citric acid and could be used as irrigant material.3 tidmash et al. cit. ingle and bakland3 suggested that 50% citrate acid as material of root canal irrigant could only be removed by demineralization material such as citric acid, edta, tetracycline hydrochloride.4 scelza et al.5 suggested that 10% citric acid could be used as dentinal decalcification material. smear layer is a layer which covers dentinal tubule and root canal wall. this layer is formed when dentine was cut by using hand instrument or rotary instrument and during root canal preparation.6 smear layer consist of organic and inorganic tissue such as fragment of odontoblast, microorganism and necrotic tissue. smear layer cleaning would decrease micro flora as well as toxin and would increase dentinal permeability. this condition might cause diffusion and action intracanal irrigation and medication so it would increase effectiveness of root canal obturation.5,6,7 in performing root canal preparation is always followed by irrigation using irrigant which is capable to remove smear layer with minimal toxicity. the function of root canal irrigant is to dissolve the content of root canal in unreachable region by an instrument.8,9 complex anatomical internal dentine such as the presence of apical delta, lateral root canal and additional root canal causing root canal debridement could not be done by instrument only but requiring irrigant during root canal treatment. preparation of root canal which could be done without irrigant cause debris are leftover inside root canal so root canal treatment would failed.8,9 to achieve clean result of root canal preparation, it is necessary to choose adequate irrigant material of root canal by considering nontoxic, being able to remove smear layer, anti bacteria, easily obtained, low cost, so alternative irrigant which could fulfill the requirement is needed. on the previous study on toxicity of 5% tamarindus indica extract was nontoxic against cell line bhk-21. based on the above explanation, it is necessary to perform a study on 5% tamarindus indica extract to remove the smear layer of root canal. the aim of this study was to examined the capability of 5% tamarindus indica extract as irrigant to remove smear layer of root canal wall using scanning electron microscope (sem). the study result is expected to add scientific perception of dentistry field especially on technology development of root canal irrigant. 174 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 173-175 material and method the material of this study were 5% tamarindus indica extract, sterile aquabidest, gold of 24 carat. red wax, araldyte glue, extracted tooth with criteria i.e. having single root, unobstructive root canal, milipore 0.45 mm. the procedure of 5% tamarindus indica extract preparation, is the shell of tamarindus indica was opened, the pulpa tamarindorum was weighed 5 gr added by 100 cc sterile aquabidest and mixed using magnetic stirring until the pulpa was well and homogeneously dissolved and mixed. to separate the supernatant and precipitation, it was centrifuged at 250 rpm for 15 minutes, the tamarindus indica supernatant was filtered using milipore 0,45 mm. the instrument were used in this study: disposable syringe, high speed drilling with water cooler, sem, coating tool: joel, fissure bur, file type k no. 15–40. sample was divided into 2 groups, the crown was cut on cervical line and measured according to the length of the root reduced 1 mm. sample was fixated on metal ring, red wax and put on the balance then prepared. the preparation produce was file type k no. 15 was inserted into root canal as far as working length. file was pushed to the wall of root canal, by push and pull force to scrape the wall of root canal until it was spacious, smooth and adequate working length. root canal was irrigated using sterile aquabidest (group1). tamarindus indica extract (group ii). each irrigation used 0.5 cc, sprayed without pressure in 6 seconds then irrigant was sucked out and it was done repeatedly (four times). until the volume of irrigant was 2 cc. the next, root canal was dried with paper point. preparation was continued in the same way until it reached file no. 40. the total of contact period between irrigant and root canal wall for 150 seconds (2.5 minutes). in group ii, every time after irrigation with test material, root canal was irrigated using sterile aquabidest with the aim to remove root canal wall from debris of irrigant. the samples were cut longitudinally into two parts on third of the part was formed 7 × 2 × 2 mm specimen. the specimen was cleaned by spraying aquadest and dried in incubator (at 30° c for 2 × 24 hours). the specimen was observed and attached on the holder (stub) using araldyte glue in which the surface was observed facing upward. it was left to dry for about one day, then polished by pure gold. the specimen one was put into sem, photograph was done on the targeted part and magnified 5000 times. to evaluate the result of hygiene value using transparent sheet divided into 10 boxes then attached on the result of sem. every box was scored i.e. 0 = dentinal tubule orifis invisible, the surface was completely covered by smear layer 5 = dentinal tubule orifis unequally spread, a part of the surface free from smear layer 10 = dentinal tubule orifis opens and spreads equally, the whole part free from smear layer. to achieve the result of evaluation is to sum the score of the 10 boxes. the whole sum is the hygiene value of root canal wall. the higher the value means the more hygiene the root canal. result in group irrigated with sterile aquabidest (control) the surface of root canal wall was dirty and covered completely by smear layer and dentinal tubule orifis was really invisible (figure 1), while the groups irrigated with 5% tamarindus indica extract, the surface of root canal wall was clean, dentinal tubule orifis was opened and equally spread (figure 2) while the mean of hygiene value of root canal could be seen on table 1. the data is achieved by performing normality test using kolmogorov smirnov test and data of normal distribution is obtained so test of difference was done using parametric statistical analysis. homogenous data was found based on homogenesity test (levene’s test) and independent t test showed significant difference between group irrigated with sterile aquabidest and 5% tamarindus indica extract on hygiene of root canal wall (p < 0.05) with hygiene value of root canal wall group irrigated with 5% eti higher than group irrigated with sterile aquabidest. discussion on this study sem was used to investigate the hygiene of root canal wall due to the capability showing rapid and accurate investigation result of the surface of root figure 1. the observation of root canal surface using sem after irrigation with sterile aquabidest (5000×). figure 2. the observation of root canal surface with sem after irrigation with 5%eti (5000×). 175wulandari et al.: the ability of 5% tamarindus indica extract canal wall. it is more important because specimen surface could be directly observed, the sharpness of investigation appears to be more distinctive and the capability producing better analysis compared with microscope with common beam.10 based on the present study shows that sample irrigated by 5% tamarindus indica extract the root canal wall is cleaner compared with sterile aquabidest due to acid character 5% tamarindus indica extract (ph = 2). a material with acid nature could contribute demineralization. acid dissolved in water would be ionized to be carboxylate ion, and h+ ion, if having contact with root canal wall it would dissociate into hydroxyapatite it would release ca2 + ion and hpo4 2– and demineralization occurs. in addition, one of tamarindus indica extract contents is citric acid which can dissolve hydroxyapatite. nightingale and sheridan cit. arief11 suggested that the reaction between citric acid and hydroxyapatite by releasing hydrogen ion and binding calcium (cathode). citric ion (anode) would replace phosphate ion (anode) in hydroxyapatite structure until structure of dentine crystal demineralized. ingle and bakland3 and walton and torabinejad12 suggested that citric acid could solve saline mineral in dentin and it is chelating agent therefore it can remove smear layer during root canal treatment and it can vanish bacterial endotoxin. citric acid can decrease the number of bacteria in root canal so that the surface of root canal would be clean. wulandari14 reported that citric acid has capability of anti bacteria against streptococcus viridans. the effect of anti bacteria of citric acid is obtained from its low ph so that the high concentration of hydrogen ion can denaturate component on microorganism.15 dharmayati16 reported on the efficacy of anti bacterial solution of tamarindus indica extract has anti bacterial effect against root canal bacteria streptococcus viridans. it is concluded that 5% tamarindus indica as root canal irrigant is capable to remove smear layer of root canal wall. references 1. wijayakusuma. tanaman berkhasiat obat di indonesia. jilid 3. pustaka kartini; 1997. p. 26–9. 2. tjitrosoepomo g. taksonomi tumbuhan obat-obatan. jogjakarta: gadjah mada university press; 1994. p. 205. 3. ingle j, bakland l. endodontics. 4nd l. endodontics. 4th ed. lea & febiger book, waverly co; 1994. p. 180–2.p. 180–2. 4. maduratna e. biokompatibilitas tetrasiklin pada kultur sel fibroblas4. maduratna e. biokompatibilitas tetrasiklin pada kultur sel fibroblasibilitas tetrasiklin pada kultur sel fibroblas dan pengaruhnya terhadap pelepasan lapisan smir. tesis. surabaya: program pascasarjana universitas airlangga; 1999.rjana universitas airlangga; 1999.1999. 5. scelza mfz, teixeira am, scelza p. decalcifying effect of edta-t, 10% citric acid, and 17% edta on root canal dentin. oral surg oral med oral pathol oral radiol endod 2003; 95:234–6. 6. torabinejad m, cho y, khademi aa, bakland lk, shabahang s. the effect of various concentrations of sodium hypochlorite on ability of mtad to remove the smear layer. j endod 2003; 29(4):233–9. 7. beltz re, torabinejad m, pouresmail m. quantitative analysis of the solubilizing action of mtad, sodium hypochloride, and edta on bovine pulp and dentine. j endod 2003; 29(5):334–7.j endod 2003; 29(5):334–7. 8. grossman li, oliet s, del rio ced. ilmu endodontik dalam praktek. rafia a, editor. endodontic practice. 11th ed. philadelphia: lea & febiger; 1995. p.196, 205. 9. vianna me, gomes bpfa, berber vb, zaia aa, ferraz ccr, de souza-filho fj. in vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. oral surg oral med oraloral surg oral med oral pathol oral radiol endod 2004; 97:79–84. 10. samadi k. efektivitas edtac sebagai bahan irigasi pada preparasi saluran akar. tesis. surabaya: program pascasarjana universitas airlangga; 1986. 11. arief em. pengaruh demineralisasi sementum pada periodontitis marginalis kronis terhadap respon sel epitel dan jaringan ikat tikus dengan menggunakan asam sitrat. tesis. surabaya: program pascasarjana universitas airlangga; 1991. 12. walton rem, torabinejad m. prinsip dan praktek ilmu endodonsi. narlan sumawinata, editor. principle and practice of endodontics. 2nd ed. 1998. p. 277–80. 13. chan cp, jeng jh, hsieh cc, lin cl, lei d, chang mc. morphology alterations associated with the cytotoxic and cytostatic effects of citric acid on cultured human dental pulp cells. j endod 1999; 25(5):354–8. 14. wulandari e. perbedaan khasiat antibakteri bahan irigasi hidrogen peroksida 3% dan asam sitrat 6% terhadap streptococcus viridans. majalah kedokteran gigi (dental journal) 2000; 33(1):14–6. 15. nizar m. daya antibakteri perasan buah nanas muda (ananas comosus) terhadap streptococcus viridans. skripsi. jember: program sarjana kedokteran gigi universitas jember; 2003. 16. dharmayanti aw. kemampuan larutan buah asam jawa (tamarindus indica l) dalam menghambat pertumbuhan streptococcus viridans. skripsi. jember: program sarjana kedokteran gigi universitas jember; 2003. table 1. mean, standard deviation, the significance of hygiene value control group 3% h2o2 and 5% tamarindus indica extract with independent test group n x significance control 5% eti 9 9 0 83.33 ± 3.54 0.001 0.001 �� volume 46 number 1 march 2013 the role of inducible nitric oxide synthase in teeth periapical lesions immunopathogenesis caused by enterococcus faecalis tamara yuanita,1 latief mooduto1 and kuntaman2 1department of conservative dentistry, faculty of dentistry, universitas airlangga 2faculty of medicine, universitas airlangga surabaya – indonesia abstract background: periapical lesions, are characterized by an immune response to the invading bacteria consequences periapical bone destruction. in root canal treatment failure was found enterococcus faecalis (e. faecalis) as most species. inos found an important role in protection against infection, plays vital roles in fighting pathogens and contributing to disease pathology. purpose: this study was to observed the role of inos in teeth periapical lessions immunopathogenesis caused by e. faecalis. methods: the randomized post-only control group design used in this study, this study used 24 wistar rats, were divided into three groups (each group consisted of 8 rats), as negative controls group is a normal teeth, in the positive controls group was made by drilling the upper right first molar to penetrate the dental pulp and was induced with 10µl bhi-b then filled with glass ionomer cement (gic) and the treatment group, after drilling the teeth, then inoculated with e. faecalis atcc 29212 106 cfu into 10µl bhi-b then filled with gic to prevent contamination. it takes 21 days to get periapical lesions and rat were sacrificed, and then the expression of inos was measured. results: statistical analysis using anova found a significant differenced between control and treatment groups (p<0.05). conclusion: this study concluded that inos role in teeth periapical lesions immunopathogenesis caused by e. faecalis. key words: enterococcus faecalis, inducible nitric oxide synthase, periapical lesions abstrak latar belakang: lesi periapikal merupakan hasil suatu respon imun untuk melawan invasi bakteri yang mengakibatkan destruksi tulang periapikal. pada perawatan saluran akar yang mengalami kegagalan ditemukan enterococcus faecalis sebagai spesies terbanyak. inos berperan penting untuk proteksi terhadap bakteri, mempunyai peran yang vital untuk melawan patogen dan berkonstribusi secara patologik untuk menyebabkan suatu penyakit. tujuan: penelitian ini bertujuan untuk mengobservasi peran inos secara imunohistokimia pada lesi periapikal tikus wistar. metode: penelitian ini menggunakan disain randomized post-only control group, digunakan 24 ekor tikus wistar yang dibagi menjadi tiga kelompok yang masing-masing terdiri dari 8 ekor tikus, sebagai kelompok kontrol negatif adalah gigi normal, pada kelompok kontrol positif dilakukan pengeboran pada gigi molar pertama rahang atas sampai menembus pulpa kemudian diinduksi 10µl bhi-b kemudian ditumpat (glass ionomer cement) gic dan pada kelompok perlakuan, setelah dilakukan pengeboran dilakukan induksi e. faecalis atcc 29212 sebanyak 106 cfu ke dalam 10 µl bhi-b kemudian ditumpat gic untuk mencegah kontaminasi. diperlukan waktu 21 hari untuk mendapatkan lesi periapikal pasca perlakuan kemudian tikus dikorbankan lalu dihitung sel-sel yang mengekspresikan inos. hasil: analisis menggunakan anava membuktikan bahwa ada perbedaan yang bermakna antara kelompok kontrol dan kelompok perlakuan (p<0,05). kesimpulan: inos berperan pada imunopatogenesis lesi periapikal gigi akibat e. faecalis. kata kunci: enterococcus faecalis, inducible nitric oxide synthase, lesi periapikal correspondence: tamara yuanita, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: tamara25jun@yahoo.com research report ��yuanita, et al.,: the role of inducible nitric oxide synthase introduction periapical lesions are formed due to an infection in the root canal by the bacteria that live and breed in the apical part of root canal. a persistent infection after the preparation of root canal is a principal etiology in the failure of root canal treatment. enterococci exist in the gastrointestinal tract and oral cavity in humans as normal comensals. they can cause a wide variety of diseases in humans. enterococci can withstand hars enviromental conditions, they grow at 10° c to 45° c at ph 9,6; in 6,5% naocl and survive at 60° c for 30 minutes. (e. faecalis) can adapt to adverse conditions. periapical lesions as the indicator of canal root treatment failure sometimes without clinical symptoms so the radiographic is the only way to check the presence of periapical lesions.1 periapical lesions in chronic state is usually without symptoms which is classified as apical periodontitis (ap) may also be the result of a secondary infection in the root canal treatment procedure. infection in the periapical region is caused by the lack of control at the time of intracanal endodontic treatment resulting in re-infection of root canal system caused by non hermetic obturation or inadequate manufacturing of dental restorations resulting in a leakage that can be entered by bacteria.2 e. faecalis bacteria is found in the root canal that has been obturated and has undergone a 77.2% periapical disorders.3 e. faecalis bacteria can survive in post endodontic treatment condition because of its ability of forming biofilm to defend themselves in harsh conditions, able to increase calcium ion (ca2+) in biofilm structure in anaerobic environments. the ability of this bacteria to enter and survive in the root canal system in a long term remains unclear, its ability to survive in stressful conditions including intracellular survival in macrophages and can survive for 4 months in urban water (tap water). e. faecalis is identified as a species which is able to survive during the root canal treatment process and persistent as pathogens on dentin tubuli because it has a broad spectrum of genetic polymorphisms and can hold a bond to dentin because it has serine protease, gelatinase, and gelatin binding protein.4 nitric oxide (no) is a short lived free radicals known to cause several different cellular processes. no is an important messenger molecule involved in many physiological and pathological processes in mammal’s body that can be beneficial or harmful.4 no is gaseous molecule that plays in nervous, cardiovascular and immune systems. no acts as a regulators that also serves as an effector when there is inflammation and infection. one of the functions of this effector is to yield toxicity effect on bacteria found in the inflamed tissue. the appropriate level of no production is protective but excessive no will induce nf-κb which can lead to toxicity in tissues. chronic expression of no is associated with various carcinomas and inflammatory conditions.5 no is produced from amino acid l-arginin by the nitric oxide synthase (nos) enzymatic reaction.6 human and rat have three genomes containing three different genes that encode different synthesis nos, they are neuronal nos (nnos or nos-1), cytokine-inducible nos (inos or nos-2) and endothelial nos (enos or nos-3). no products from inos synthesize are very different from enos and nnos. inos enzyme produces very much products and can last a long time which can lead to toxicity. no was obtained from inos plays an important role on host defense.6 no in bone loss induced apical periodontitis with deficiency of inos produce inflammatory cells and increase osteolytic lesions.7 the objective of this study was to observe increase of inos expressions in the immunopathogenesis of periapical lesions of wistar rat by induction of e. faecalis. materials and methods type of the research was laboratory experimental, post only control group design. twenty four wistar rats aged 12 weeks was divided by random technique into 3 groups, each groups consisted of 8 rats. negative control group was the normal teeth, positive control group was the maxillary first molar tooth which was drilled until penetrating the pulp. ten ml of bhi-b was put in then filled with gic and the treatment group, after drilling then 106 cfu of e. faecalis atcc 29212 was inoculated into the 10 ml bhi-b then filled with gic to prevent contamination. it takes 21 days to get periapical lesions after pulp infections. rats were sacrificed then immunohistochemical study was done to measure the inos expression by counting the number of cells that gave positive reactions to anti inos monoclonal antibody in the specimen under a microscope with 400 times magnification by 10 times the field of view then averaged.8 statistical analysis done with anova to discover the differences among each group. results inos expression data obtained from observation of the number of cells on the periapical tissue that gave positive reactions to anti-inos monoclonal antibody using immunohistochemical method in the negative control group, positive control, and treatment group as shown in figure 1. statistical test was done using anova to discover the differences among each group (table 1). tukey hsd test was done to discover the differences of inos among each research group as shown in table 2. the result showed that there ware significant differences among the negative control group, positive control group, and treatment group as shown in figure 2. data analysis result showed that there were significant differences for inos variable among three research groups. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 14–17 macrophages. no plays an important role on protection against bacteria that causes infection but also cause tissue damage. production of no is thought to have an important role in the development of periapical lesions. in periapical lesions, macrophages and polymorphonuclear leukocytes are the major source of no.9 no is produced by phagocytes (monocyte, macrophage, and neutrophil) which is part of the immunity response. no is synthesized inside the cell by nos enzyme. no product by inos synthesis is very different from enos and nnos because inos produces so much and can last long which will lead to tissue toxicity and plays an important role in host defense. inos is activated by interferon-gamma (ifnγ) as a single signal or by tumor necrosis factor (tnf) as a secondary signal. no can activate nf-κb which is an important transcription factor on inos gene expression in the reaction against inflammation. inos enzyme produces so much and can last for a long time which will lead to toxicity.10 the role of no generated by inos is very complex. no has an anti microbial effect and macro molecule nitrolyzation. in a few seconds no will be oxidized into nitrite or nitrate produced by anion superoxide (o2-) which can form peroxinitrite (onoo-) which has cytotoxic effect. allegedly, an increase of inos is due to the increasing of nf-κb transcription factor resulting in an increase of the secreted genes which is inos that releases no which can induce apoptosis in osteoblasts that are likely to increase the development of periapical lesions.11 in this study, it is proved that there is an increase of inos expression in the treatment group compared to the control groups (p<0.05) (table 1 and 2). large amount of increase in inos will lead to cell toxicity.13 increasing in inos is a response of ifnγ-activated macrophage as a single signal secreted through th-1 cytokine.12,13 inos shows an opposite effect on the physiology of osteoclasts. no has a different function during the figure 1. inos expression on wistar rat periapical tissue with 400 times magnification. a: negative control, b: positive control, c: treatment. positive inos expressions are marked with arrows (d, e, f). table 1. anova test result for inos variable treatment group n σ inos positive cells average sd p negative control 8 14.5 1.19 0.001*positive control 8 18.50 4.37 treatment 8 32.62 1.40 explanation: * : significant (p < 0.05); n: amount of sample; sd: standard deviation table 2. tukey hsd test result negative control positive control negative control – p = 0.021* positive control p = 0.021* – treatment p = 0.001* p = 0.001* * = significant (p < 0.05) figure 2. three groups of inos expression on wistar rat periapical tissue. group positive controlnegative control bacteria e. faecalis 9 5 % c i in o s 35 30 30 20 15 10 discussion periapical lesions is a chronic infectious disease, many kind of bacteria in the oral cavity are involved. nitric oxide (no) is a key molecule for fighting pathogens, is a short lived free radicals produced by inflammatory ��yuanita, et al.,: the role of inducible nitric oxide synthase development and activation of osteoclasts. expression inos for no generation is stimulated by ifn or lipopolysaccharide inos expression and no releasing cause an increasing of receptor activator of nf-kb ligand (rankl), and that response depends on the duration and dosage. needs activation of nf-κb and protein synthesize that are specifically inhibited by osteoprotegerin (opg) which is an angler receptor. this causes inhibition of noinduced rankl to increase the formation of osteoclast, this indicates that normal no controls the osteoclastogenesis mediated by rankl. inos deficiency accelerates the formation of osteoclast and resorption through in vivo and in vitro. rankl induced in inos derived no function is a negative signal to limit osteoclastogenesis which were jointly stimulated by rankl.14 no donor will increase the production of opg and inhibit osteoclastogenesis activities in stromal bone marrow cells on ovariectomy rat.15 increasing in bone resorption on rats with inos deficiency is correlated to the increasing of rank expression and opg reduction, so that can be concluded the no deficiency causes disproportion of bone resorption modulation factor that stimulates bone loss.6 no and superoxide are likely to react in vivo to produce peroxynitrite which molecularly will increase tissue damage. production of extracellular superoxide and release of lytic enzymes gelatinase and hyaluronidase and the toxin cytolisin by e. faecalis can cause direct damage in dentinal as well as in periapical tissues. the role of no in bone loss caused by bacteria infection which induces apical periodontitis, inos produce more inflammatory cells and osteolytic lesions than control rats. tartrate resistant acid phosphatase positive (trap+) osteoclasts significantly have a greater amount, this is correlated to the expression increasing of receptor activator nf-κb (rank) stromal cell and decreasing in osteoprotegerin (opg) expression. no deficiency will result in disproportion of bone resorption modulation factor and aggravating bone loss.7 no and superoxide are likely to react in vivo to produce peroxynitrite which molecularly will increase tissue damage. the role of inos and no to control bone resorption progress in experimental rats in apical periodontitis.10 inos deficiency is associated with the inbalance state in cytokine proinflammatory il-1β and tnf-α, bone resorption modulator (rank and rankl) and mp1 chemokin. interestingly, ros production showed no involvement in the progress of periapical lesions that may mediate osteoclast differentiation. the conclusion of this study inos role in teeth periapical lesions immunopathogenesis caused by enterococcus faecalis. references 1. kayaoglu g, orstavik d. virulence factors of enterococcus faecalis: relationship to endodontic disease. crit rev oral biol med 2004; 15(5): 308–20. 2. cohen s, hargreaues km. cohen’s pathways of the pulp. 10th ed. st. louis, missouri: mosby inc; 2011. p. 529–58. 3. rocas in, siqueira jf jr, santos kr. association of enterococcus faecalis with different form of periradicular diseases. j endod 2004; 30(5): 315–20. 4. stuart c h, schwartz s a, beeson t j, owatz cb. enterococcus faecalis: it’s role in root canal treatment failure and current concepts in retreatment. j endod 2006; 32(2): 93–8. 5. hou yc, yancuk a, wang pg. current trends in the development of nitric oxide donors. curr pharm des 1999; 5(6): 417–41. 6. hof rj, ralston sh. cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity. j bone miner res 1997; 12: 1797–1804. 7. fukada sy, silua ta, socanato lf, garlet gp. inos-derived nitric oxide modulates infection-stimulated bone loss. jdr 2009; 87(12): 1155–60. 8. pizem j, cor a. detection of apoptosis cells in tumour paraffin section, radio oncol 2003; 37(4): 225–32. 9. hama s, takichi s,saito i, ito k. involvement of inducible nitric oxide synthase and receptor for advanced glycation end products in periapical granuloma. j endod 2007; 33(2): 137–41. 10. silva mjb, souza lma, lara upl, cardosa fp. the role of inos and phox in periapical bone resorption. j dent res 2011; 90(4): 495–500. 11. lin sk, kok sh, lin ld, wang cc. nitric oxide promotes the progression of periapical lessions via inducing macrophage and osteoblast apoptosis. oral microbial immunol 2007; 22(1): 24–9. 12. gorzyniski g, stanley a. clinical immunology landes bioscience 2002; austin tx. isbn 1570596255. 13. guzik tj, korbut r, adamek-guzik t. nitric oxide and superoxide in inflamamtion and immune regulation. available from: www. mifhorbut @cyf-kv.edu.pi. accessed at november 20, 2011. 14. zheng h, yo x, osdoby pc, osdoby p. rankl stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclast. an autocrine negative feedback mechanism trigered by rankl-induced interferon γ via nf-kb that restrain osteoclastogenesis and bone resorption. j biol chemistry 2006; 281(3): 15809–20. 15. wang fs, wang cj, chen yj. nitric oxide donor increases osteoprotegerin production and osteoclastogenesis inhibitory activuty in bone marrow stromal cells from ovariectomized rats. j endocrinology 2004; 145(5): 2148–61. mkgs vol 45 no 2 april-juni 2012.indd 89 volume 45 number 2 june 2012 research report pulpal inflammation after vital tooth bleaching with 38% hydrogen peroxide ardiny andriani1, juni handajani2, and tetiana haniastuti2 1 dental student, faculty of dentistry, gadjah mada university 2 department of oral biology, faculty of dentistry, gadjah mada university yogyakarta – indonesia abstract background: in-office vital tooth bleaching is a treatment to remove tooth stains. tooth sensitivity is one of side effect commonly complained by patients receiving this treatment. purpose: the aim of this study was to examine histological inflammatory cells infiltration of dental pulp after application of 38% h2o2 as a vital tooth bleaching agent. methods: under informed consent, a total of 15 premolars from 8 healthy subjects scheduled for orthodontic extraction were used in this study. thirty eight percent h2o2 was applied on the buccal surface of the treated group. the treated teeth were extracted after 1 hour, 5, 8, and 15 days. all specimens were embedded in paraffin wax, sectioned serially and stained with hematoxyllin eosin. histological specimens were then observed under a light microscope. results: all treated groups showed a slight disorganization of odontoblasts layer and slight inflammation in the pulp tissue adjacent to the 38% h2o2 application site. the number of polymorphonuclear leukocytes (pmn) had increased significantly 1 hour after application of 38% h2o2 (p<0.05), while macrophages had significantly increased 5 days after the application (p<0.05). the most intense pmn and macrophages infiltration was found 5 days after the application and gradually decreased 8 days after application of38% h2o2. conclusion: application of 38% h2o2 as a vital tooth bleaching agent induces acute inflammation in human dental pulp; however, the inflammation will decrease 8 days after the application. key words: vital tooth bleaching, 38% hydrogen peroxide, inflammation abstrak latar belakang: perawatan pemutihan gigi vital metode in-office merupakan tindakan untuk menghilangkan pewarnaan pada gigi. salah satu efek samping yang sering dikeluhkan oleh pasien yang menjalani perawatan ini adalah sensitivitas gigi. tujuan: penelitian ini bertujuan untuk mengamati infiltrasi sel inflamasi pada pulpa gigi setelah aplikasi h2o2 38% sebagai bahan pemutih gigi. metode: sampel penelitian ini berupa 15 gigi premolar yang berasal dari 8 subjek sehat yang akan melakukan pencabutan gigi untuk perawatan ortodontik. seluruh subjek telah menandatangani informed consent. hidrogen peroksida 38% diaplikasikan pada permukaan bukal gigi kelompok perlakuan. gigi kemudian dicabut 1 jam, 5, 8, dan 15 hari setelah aplikasi h2o2 38%. seluruh spesimen kemudian ditanam dalam parafin, dipotong secara serial dan diwarnai dengan hematoxillin eosin. pengamatan preparat histologis dilakukan dengan menggunakan mikroskop cahaya. hasil: hasil penelitian ini menunjukkan gangguan pada lapisan odontoblas dan peradangan pada jaringan pulpa di bawah daerah aplikasi h2o2. jumlah pmn meningkat secara signifikan (p<0,05) 1 jam setelah aplikasi h2o2 38% sedangkan jumlah makrofag meningkat secara signifikan 5 hari setelah aplikasi hidrogen peroksida 38%. infiltrasi pmn dan makrofag paling banyak ditemukan 5 hari setelah aplikasi dan menurun secara bertahap 5 dan 8 hari setelah aplikasi h2o2 38%. kesimpulan: aplikasi h2o2 38% sebagai bahan pemutih gigi vital dapat menginduksi inflamasi akut pada pulpa gigi manusia, namun, inflamasi akan mereda 8 hari setelah aplikasi. kata kunci: perawatan pemutihan gigi vital, hidrogen peroksida 38%, inflamasi correspondence: tetiana haniastuti, bagian biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: haniastuti@yahoo.com 90 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 89–92 introduction many patients nowadays are interested in aesthetic dental treatments for a variety of reasons, from personal satisfaction to work-related needs. the goal of dental aesthetics is determined by various factors and one of the greatest causes of dissatisfaction is tooth discoloration that does not meet patient’s expectations.1 vital tooth bleaching is a therapeutic procedure that enables dental discoloration to be removed, thus giving the shade that meet the patient’s expectation. the treatment is therefore, presented as non-invasive and conservative procedures that do not alter the natural shape of the teeth. this procedure is suitable for the vital teeth affected by shade problems which present no other dental or periodontal pathology and retain a correct anatomy and appropriate position in the arch.1,2 vital tooth bleaching techniques include night guard and in-office techniques. these techniques may be used separately or in combination with one another. night guard technique can be performed by the patients using low concentration of hydrogen peroxide all night for 1–2 weeks.2 the in-office bleaching technique is performed by dentist by using high concentration of hydrogen peroxide. this treatment needs 1–2 visits. although it has more potential for causing gingival irritation, this technique is often chosen because of the shorter treatment time.3 the active agent for the vital tooth bleaching commonly used today is hydrogen peroxide. this agent has to be applied directly on the tooth surface.4 hydrogen peroxide acts as a strong oxidizing agent through the formation of free radicals. the reactive molecule attacks the long-chained, dark-colored chromophore molecules and split them into smaller, lighter color, and more diffusible molecules.5 the common side effect of vital tooth bleaching using high concentration of hydrogen peroxide is tooth sensitivity.6,7 this side effect normally persists for up to 4 days after the cessation of bleaching treatment.7 in vitro and in vivo experiments have shown that the peroxide had ability to penetrate enamel and dentinal tubule and therefore, enter the pulp chamber.8-10 little is known about the effects of hydrogen peroxide on dental pulp tissues. many controversies arise related to the safety of vital tooth bleaching procedure although peroxide-based products had been accepted by the american dental association (ada) as save and effective agents.11 the aim of the study was to examine inflammatory cells infiltration of dental pulp after application of 38% h2o2 as a vital tooth bleaching agent. materials and methods this research was approved by ethical commission of the faculty of dentistry universitas gadjah mada. all patients who agreed to be a part of the study signed a consent form. the consent form for the patients under 18 years of age were signed by the parents. patients were recruited from dental clinics in sleman and makassar. all of them were healthy, age 12–26 years, and have never done tooth bleaching treatment before. the subject of this study consisted of 15 premolars which were scheduled for extraction for orthodontic treatment. subject were divided into 2 group, control and treated group. treated group were then divided into 4 subtreated group based on the extraction time, which were 1 hour, 5, 8, and 15 days after the application of 38% hydrogen peroxide (h2o2). each subtreated group and control group were consisted of 3 premolars. the application of opalescence xtra boost (ultradent, utah) which consisted of 38% hydrogen peroxide was performed according to the manufacturer's protocol. in brief, the tooth was drained from saliva and buccal retractor was mounted on the patient’s mouth. gingival barrier (opaldam, ultradent, utah) was applied on interdental gingiva and gingiva around the treated tooth and activated by using a light curing unit. opalescence xtra boost was mixed with activator (ultradent, utah) and applied 0.5–1 mm thick layer on the buccal surface of the treated tooth. after 45 minutes, 38% h2o2 was cleaned from the tooth surface by using the suction. patients were then asked to rinse and the gingival barrier were cleaned by using an explorer. the treated teeth were extracted 1 hour, 5, 8, and 15 days after the application of 38% h2o2. in control group, the teeth were extracted without application of 38% h2o2. immediately after extraction, the most apical 4 mm of the root was sectioned off by using fissure bur, and fixed with 10% buffered formalin. the teeth were then decalcified in morse solution (mixture of 50% formic acid and 20% sodium citrate with the same ratio) for 45 days. after decalcification completed, teeth were embedded in paraffin, serially sectioned and stained with hematoxylin and eosin. the whole condition of the pulp tissue in the pulp chamber was examined. the number of polymorphonuclear leukocytes (pmn) and macrophages at the area of the pulp which encompassed the dentinal tubules corresponding to h2o2 application site were counted at 3 different fields using a light microscope at 400√ magnification. results of each number of pmn and macrophages are presented as mean ± standard deviation. difference among means of pmn and macrophages cells number were then analyzed separately using analysis of variance (anova) and followed by least significant difference (lsd) test at 5% level of significance. results all teeth in control group showed a normal dental pulp tissue organization. odontoblast cell layer, cell free zone, cell rich zone, and pulp core were observed in all specimens (figure 1a). a small number of inflammatory cells (pmn and macrophages) were seen in cell rich zone. 91andriani, et al.: pulpal inflammation after vital tooth bleaching the result of anova test showed a statistical significant difference of inflammatory cells number among groups, indicating that application of 38% h2o2 increased the number of pmn and macrophages. in all specimens of the treated groups, cell rich zone could not be observed because infiltration of the inflammatory cells. vacuolization and inflammatory cells infiltration were also found in odontoblast cell layer (figure 1b). lymphocytes were not found in both treated and control group. the number of pmn was increased significantly immediately (1 hour) after application of 38% h2o2 (figure 2). the most intense pmn infiltration were found in the specimens 5 days after application and gradually decreased 8 and 15 days after application of 38% h2o2. the number of macrophages was significantly increased 5 days after application and decreased gradually 8 days after application (figure 3). on extended time observation (15 days) macrophages cells number became fewer and had no significant differences compared to the control group. discussion present study showed that application of 38% h2o2 as a vital tooth bleaching agent induced inflammatory cells infiltration in human dental pulp. this finding supported previous study by costa et al.12 they showed that vital tooth bleaching with 38% h2o2 induced pulp inflammation, but they only observed the pulp 2 days after the treatment. h2o2 is a strong oxidizing agent that produces free radicals with unpaired electron, such as perhydroxyl radical and superoxyde anion. free radicals break down large pigmented molecules in enamel and dentin into smaller and less pigmented molecules.13,14 in order to promote the tooth lightening effect, h2o2 have to penetrate into enamel and dentin.15,16 14% h2o2 have the ability to penetrate into enamel and dentin; therefore, it can enter the pulp chamber through the dentinal tubules. the higher h2o2 concentration of bleaching agent results in the higher pulpal peroxide penetration.16 enamel and dentin have a high permeability to h2o2 and free radical.17 penetration of h2o2 and free radical through the tooth structure occur mainly because of their low molecular weight which increases the ion movement.18 dental enamel contains 0.6% of organic material. h2o2 increases the porosity and loss of substances of enamel matrix as a result of free radical oxidation, thus increases figure 2. the mean ratio of pmn cells in dental pulp of control group and treated groups (*p < 0.05). figure 3. the mean ratio of macrophages in dental pulp of control group and treated groups (*p < 0.05). a b figure 1. dental pulp tissue in control group (a) and after application of 38% h2o2 (b). four zones of the dental pulp tissue can be observed in the control group, while in the treated group, cell free zone can not be observed since it is occupied by pmns and macrophages (black arrows). 1 = odontoblast layer, 2 = cell free zone, 3 = cell rich zone, 4 = pulp core. 92 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 89–92 h2o2 penetration. 19 h2o2 also has ability to increase enamel porosity by denaturation of enamel’s protein.20 dentinal tubules connect dentin with pulp chamber. this morphology encourages physical passage of h2o2 and free radical to enter pulp chamber.15 odontoblasts are cells located in the peripheral of the pulp chamber, being the first defense against irritation of the pulp. penetration of h2o2 into the pulp chamber causes changes in the odontoblast cell layer.21 the present study revealed vacuolization of odontoblast cell layer in all treated groups. vacuolization of odontoblast cell layer on the pulp tissue is the first response of odontoblast cells to injury22 and usually occurs before the pulp is inflamed.23 h2o2 and perhydroxyl radicals induced the expression of interleukin-8 (il-8).24 h2o2 and radical perhidroksil which penetrate into the pulp may induce the odontoblast to produce il-8. interleukin-8 is a proinflammatory cytokine which has ability to stimulate chemotaxis of pmn and macrophages into the exposed area.21,25 in this study, the number of pmn increased immediately (1 hour) after application of 38% h2o2, while the number of macrophages were started to increase 5 days after the application of 38% h2o2. inflammation is a local protective response generated by tissue damage, which serves to eliminate the irritating material or damaged tissue. acute inflammation occurs a few minutes or hours after tissue damage and usually lasts for 1–2 weeks.26 pmn and macrophages play an important role in the inflammation. they migrate from blood vessels into the tissue at the beginning of inflammation. pmn migration into inflamed areas occurs only in a few minutes after inflammation.27 inflammation continues unless the irritating material can be successfully removed.28 eight days after the application, the number of pmn and macrophages were decreased, indicating that the irritating materials were begun to be eliminated from the pulp chamber. dental pulp has defense mechanisms to eliminate h2o2 from the pulp chamber, by producing catalase and peroxidase enzymes. catalase breaks down hydrogen peroxide into water and oxygen; while peroxidase uses h2o2 to oxidize some other substrates.17 in conclusion, application of 38% h2o2 as a vital tooth bleaching agent induces acute inflammation in human dental pulp; however, the inflammation will decrease 8 days after the application. references 1. el askary aes. fundamentals of esthetic implant dentistry. 2nd ed. iowa: blackwell publishing; 2007. p. 3–12. 2. roberson tm, heymann ho, swift ej. sturdevant’s art and science of operative dentistry. 4th ed. missouri: mosby; 2002. p. 609–11. 3. patil r. esthetic dentistry: an artist’s science. india: pr publication; 2002. p. 84–91. 4. dahl je, pallesen, u. tooth bleaching: a critical review of the biological aspects. crit rev oral biol med 2003; 14 (4): 292–304. 5. paravina rd, powers jm. esthetic color training in dentistry. texas: elsevier mosby; 2004. p. 91–3. 6. nathoo s, santana e, zhang yp, lin n, collins m, klimpel k. comparative seven-day clinical evaluation of two tooth whitening products,. compend contin educ dent 2001; 22: 599–604. 7. tredwin cj, naik s, lewis nj, scully cbe. hydrogen peroxide tooth-whitening (bleaching) products: review of adverse effects and safety issues. brit dent j 2006; 200: 371–6. 8. pugh g. high levels of hydrogen peroxide in overnight toothwhitening formulas: effects on enamel and pulp. j esthet restor dent 2006; 17(1): 40–5. 9. markovic m, sieck ba, takagi s, chow lc, majeti s. diffusion of hydrogen peroxide through sound tooth enamel. j dent res 2000; 79: 305. 10. camargo s, valera m, camargo c, mancini mg, menezes m. penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique. j endod 2007; 33(9): 1074–7. 11. fugaro jo, nordahl i, fugaro oj, matis ba, mjor ia. pulp reaction to vital bleaching. oper dent 2004; 29(4): 363–8. 12. costa ca, riehl h, kina jf, sacono nt, hebling j. human pulp responses to in-office tooth bleaching. oral surg oral med oral pathol oral radiol endod 2010; 109(4): e59–64. 13. kashima-tanaka m, tsujimoto y, kawamoto k, senda n, ito k, yamazaki m. generation of free radicals and/or active oxygen by light or laser irradiation of hydrogen peroxide or sodium hypochlorite. j endod 2003; 29: 141–3. 14. walsh lj. safety issues relating to the use of hydrogen peroxide in dentistry. aus dent j 2000; 45(4): 257–69. 15. gokay o, yilmaz f, akin s, tuncbilek m, ertan r. penetration of the pulp chamber by bleaching agents in teeth restored with various restorative materials. j endod 2000; 26(2): 92–4. 16. gokay o, mujdeci a, algin e. peroxide penetration into the pulp from whitening strips. j endod 2004; 30(12): 887–9. 17. jain a, bahuguna r. bleaching effectiveness of 35% carbamide peroxide and superoxol in fluorosis case study. ind j dent sci 2010; 2(6): 28–30. 18. chapple ilc, matthews jb. the role of reactive oxygen and antioxidant spesies in periodontal tissue destruction. periodontol 2000, 2007; 43: 160–232. 19. benetti ar, valera mc, mancini mn, miranda cb, balducci i. in vitro penetration of bleaching agents into the pulp chamber. int endod j 2004; 37: 120–4. 20. greenwall l. bleaching techniques in restorative dentistry. 1st ed. new york: martin dunitz; 2001. p. 106. 21. hargreaves km, goodis he. seltzer and bender’s dental pulp. chicago: quintessence publishing co; 2002. p. 99–115. 22. bjorndal l. dentin and pulp reactions to caries and operative treatment: biological variabels affecting treatment outcome. endod top 2002; 3(123): 36. 23. saraf s. textbook of oral pathology. india: jaypee brothers publishers; 2006. p. 179. 24. lee ys, bak ej, kim m, park w, seo jt, yoo yj. induction of il8 in periodontal ligament cells by h2o2. j microbiol 2008; 46(5): 579–84. 25. gomes ac, gomes-filho je, oliveira shp. mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of il-1, mip-2 and ltb4. oral surg oral med oral pathol oral radiol endod 2010; 109(3): e135–42. 26. kumar v, cotran rs, robbins sl. robbins basic pathology. 7th ed. philadelphia: saunders; 2003. p. 330–45. 27. mohan h. essential pathology for dental student. 3rd ed. india: jaypee brothers publishers; 2005. p. 105. 28. guyton ac, hall je. textbook of medical physiology. 11th ed. philadelphia: elsevier; 2006. p. 432. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 197197 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 197–201 case report closed reduction and drainage incision for the treatment of neglected and infected mandibular fractures eddy hermanto1, fanny m. laihad1, amelia elizabeth pranoto1, monika elidasari1, ivan tantra1, sarianoferni2, dian widya damaiyanti3 1department of oral and maxillofacial surgery, faculty of dentistry, universitas hang tuah, surabaya, indonesia 2department of oral radiology, faculty of dentistry, universitas hang tuah, surabaya, indonesia 3department of oral biology, faculty of dentistry, universitas hang tuah, surabaya, indonesia abstract background: jaw fracture is the most common facial fracture in oral and maxillofacial bone and is usually caused by trauma. the fracture itself could lead to infection due to bone and tissue damage, which is the port of entry for microorganisms. fracture-related infection (fri) in the patient discussed in this study manifested as a submandibular abscess. the goals of fracture treatment were achieving the anatomic reduction of the fracture line and regaining acceptable occlusion. there are two methods for treating mandibular fractures: the closed method, also called conservative treatment, and the open method, which requires advanced surgery. closed method treatment uses a maxillomandibular fixation (mmf) device in order to reduce and immobilize fracture fragments. treatment of fri should use a multidisciplinary approach to achieve an outstanding result, such as wound debridement, antimicrobial therapy, and implant retention. purpose: the purpose of this article is to report a case of neglected mandibular fracture with a submandibular abscess, which was treated with a combination of the closed reduction method and incision drainage. case: a 25-year-old female visited nala husada hospital because of a submandibular abscess on the neglected mandibular fracture of the right parasymphysis and left corpus. case management: the case was managed using an arch bar in the mandible and an eyelet in the maxilla while continuing with mmf and an extra oral drainage incision. conclusion: combination therapy (mmf and incision drainage) was needed to treat this case because of the occurrence of a submandibular abscess due to a neglected mandibular fracture. keywords: closed reduction; drainage incision; mandibular fracture; neglected fracture; submandibular abscess article history: received 12 september 2022; revised 5 january 2023; accepted 25 january 2023; published 1 september 2023 correspondence: eddy hermanto, department of oral and maxillofacial surgery, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150, surabaya, indonesia. email: eddy.hermanto@hangtuah.ac.id introduction jaw fracture is the most common facial fracture in the oral and maxillofacial bone, which can be caused by direct or indirect trauma and pathological conditions such as degenerative bone disorders/osteoporosis. the external wound involves the skin, mucosa, or periodontal membrane associated with the fracture site.1 mandibular fractures may develop several complications such as malocclusion, infection (abscesses and osteomyelitis), and delayed wound healing (malunion and non-union fractures as well as wound dehiscence).2 the fracture itself could lead to infection due to bone and tissue damage, which is the port of entry for microorganisms.3 bone and tissue damage, if not treated properly in time, are also one of the causes of infection.4 microorganisms in fracture-related infections (fri) are staphylococcus aureus (30–42%), coagulase-negative staphylococci (20–39%), enterobacteriaceae (14–27%), anaerobes (16%), and streptococci (11%).5 the fri in this patient manifested as a submandibular abscess because of pus formation in the submandibular space and occurs due to infection.3 the submandibular space is the most common site for deep neck space infections. the usual symptoms are fever and neck pain accompanied by swelling under the mandible and/or under the tongue and possibly trismus.6 evacuation of the abscess can be performed under local anesthesia for shallow and localized abscesses or under general anesthesia if the abscess is deep and wide. early copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p197–201 mailto:eddy.hermanto@hangtuah.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p197-201 198 hermanto et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 197–201 incision and drainage should always be considered for the patient, even in seemingly noncritical cases. drainage can be performed through either intraoral or extraoral incisions, depending on the site of infection.7 among all maxillofacial fractures, mandibular fractures are thought to have the greatest infection rates.8 the systemic health of the patient, type of injury, timing of medical treatment, and type of treatment employed are a few examples of factors that can raise the risk of infection. the pharynx, submandibular lymph nodes, floor of the mouth, and teeth are the most common sources of infection in the submandibular area.9 in addition to being caused by dental infection, infection in the submandibular space can be caused by lymphadenitis, trauma, or surgery and can also be a continuation of deep neck space infection. in addition to bacteria, infection in mandibular fractures can result from inadequate interfragmentary stability, foreign bodies, loose screws from an open reduction internal fixation system, a tooth or a part of a tooth in the fracture line, and necrotic bone fragments.10 management of mandibular fractures requires a comprehensive understanding of anatomical, biomechanical, and occlusion factors. the goal of fracture treatment is achieving an anatomical reduction of the fracture line and regaining acceptable occlusion. depending on whether direct visual access to the fracture site is available, reduction procedures used to treat mandibular fractures can be characterized as either open or closed. in contrast to open reduction, which requires direct visual access to the fracture site through a surgical incision, closed reduction enables manipulation of the fracture segment with guided tooth occlusion.11,12 closed reduction and maxillomandibular fixation (mmf) can be performed using splints such as bonded orthodontic brackets, arch bars, or eyelet wire. the closed reduction method of treating mandibular fractures is referred to as non-surgical treatment, since it involves manually realigning the fractured pieces, gradually realigning the teeth, and immobilizing the teeth and jaws with mmf.8,11 in the following case, we will discuss the treatment of a neglected mandibular fracture with a submandibular abscess due to fri. the purpose of this case report is to understand how to manage fri. case a 25-year-old indonesian female was referred from a private dental clinic to the oral and maxillofacial surgery department at nala husada hospital, surabaya, indonesia, in june 2022. she was diagnosed with a left submandibular abscess due to a neglected mandibular fracture. her record revealed that the patient had been involved in a fight last month and had been given painkillers. one week after treatment, the patient’s face was swollen and painful, which did not decrease even though she used the painkillers as prescribed. later, the patient returned to the private dental clinic and was given two different drugs to reduce the swelling. the patient did not know the name of the medicines. one week after this treatment, the swelling and pain still existed, so the treating doctor suggested a radiographic examination; however, the patient had had a radiographic examination only two weeks ago. the patient consulted another private dental clinic and was referred to nala husada hospital. extra-oral clinical examination showed swelling of the left submandibular region, approximately 3x2x1 cm in size, more erythematous than the surrounding tissue, clear boundaries, painful palpation, and fluctuations (figure 1). there is no intraoral picture because the patient was not able to open her mouth widely due to submandibular swelling. the radiograph shows a radiolucent line from the alveolar bone distal at tooth 37 and 43 to the inferior border of the mandible (figure 2). based on the history, clinical examination, and panoramic figure 1. clinical photo of the patient on her first day at the hospital. (a) large swelling over her left submandibular and angle of the mandible showing limited mouth open; (b) with her mouth in centric occlusion, no malocclusion detected. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p197–201 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p197-201 199hermanto et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 197–201 figure 2. panoramic radiograph shows a radiolucent line (white arrows) from the crest of the alveolar bone at distal tooth 37 to the mandibular border and from the crest of the alveolar bone at distal tooth 43 to the mandibular border. gfigure 3. (a) an extraoral incision has been made, and a rubber drain has been placed in the submandibular space to drain pus and decompress abscess space. (b) the patient has been treated with mmf, with eyelet wire on the maxilla and erich bar on the mandibula. she showed normal centric occlusion while intermaxillary wiring was being applied. figure 4. panoramic radiograph shows a reduction of the radiolucent line at the right parasymphysis and right corpus mandibula; meanwhile, the left body mandible has not been manipulated due to infection. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p197–201 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p197-201 200 hermanto et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 197–201 radiographic examination, the patient was diagnosed with a left submandibular abscess due to either a neglected mandibular fracture (left body and right parasymphysis mandibular region) or a lower left wisdom tooth infection, which occurred before her injury. case management the management of this case was carried out in combination, namely closed reduction and a drainage incision with minor surgery. the patient was pre-medicated before the procedure, and both procedures were performed simultaneously. the first time, the submandibular abscess was incised and drained with a rubber drain in the left submandibular space. the wound was closed with sterile gauze and adhesive flexible tape (figure 3a). after draining out the pus in the left submandibular space, the mandibular fractures were able to be treated by closed reduction and cross-jaw ties mmf. in the lower jaw, an erich bar was placed on teeth 37 to 46, and an eyelet wire was placed in the upper jaw on teeth 15–16, 11–21, and 25–26. the mmf was installed by joining the wire through the eyelet loop attached to the upper jaw and the erich bar on the lower jaw (figure 3b). after the procedure, the patient was given antibiotics (amoxicillin tablet 500 mg and metronidazole tablet 500 mg) every eight hours (three times a day) and pain-reliever medication (mefenamic acid caplet 500mg) three times a day. the patient was instructed to maintain oral hygiene, take medication as prescribed, and change extra oral gauze every day or every time the gauze became wet with pus. she was instructed to come for check-ins every two to three days until there was no pus production or if the rubber drain came off. the patient came on the second day to change the gauze and have the intraoral region cleaned, whereas the drain was changed every two days or if the drain was detached with saline irrigation. two weeks later, the mmf was removed temporarily, the patient was trained to open her mouth, the intraoral area was irrigated with 0.9% nacl, and then the mmf wire ligature was reattached. the mmf was permanently removed after eight weeks, and radiographs showed a reduction of the radiolucent line (figure 4). discusion open fractures can generally be regarded as contaminated. since fractures in the dentate area have contact with the oral cavity, these are considered open fractures.13 in this case, the patient developed a left submandibular abscess from injuries untreated on her lower jaw. moreover, in her panoramic radiograph, there was a partial eruption of the lower left wisdom tooth, and radiolucent imaging surrounded the distal crown of m3 (figure 4). the prediction was that her impacted tooth around the fracture line became the port of entry for fri (submandibular abscess). the impacted tooth on the left side of the mandible also increased mandibular fracture possibilities.14,15 the hypothesis that the m3 level of impaction further increases the risk of angle fractures originated with the work of reitzik et al.16 the reasoning behind this hypothesis is that when m3 occupies more osseous space, it weakens the mandible against outside stresses.14 fri in this patient manifested as a submandibular abscess. fracture consolidation, soft tissue envelope restoration, functional recovery, prevention of persistent chronic infection, and infection eradication are all essential components of effective fri care. debridement, antimicrobial treatment, and implant retention are the main ideas of surgical management of fri.3 the success of this case’s treatment is due to the use of appropriate techniques for closed reduction and drainage incision as well as the operative patient. the gold standard for the treatment of mandibular fractures is repositioning/ reduction, fixation, and immobilization, using either the open reduction and rigid internal fixation method or the closed reduction method, depending on the circumstances of each individual case.17 due to the non-displacement of the fracture fragments and the normal occlusion of the patient’s teeth, the mandibular fracture in this case was treated with the closed reduction method. fixing the fracture fragments in this case is done primarily to make them anatomically fit together.17,18 to better immobilize the fracture fragments, the operator used closed reduction fixation with an arch bar on the lower jaw and eyelet wire on the upper jaw, followed by mmf for two weeks. the benefit of closed reduction is that it does not necessitate surgery, so there is no risk of scar tissue or infection after the procedure. additionally, this procedure has fewer complications and is less expensive.9,19 the arch bar was used to reduce and repair fracture fragments in the mandible due to its rigid metal crosssection and function as an adjustable splint for the jaw arch. the arch bar’s length is also adjustable to the work area, and its installation is relatively simple.20 placing eyelets is a wire splinting technique that requires 0.4 mm-diameter wire placed on the left and right maxillary posterior teeth. the placement of eyelets on the maxilla serves as an instrument for mmf. eyelets were chosen because it is a relatively simple wiring technique, so little food debris gets left on the wire, and the patient can manage her oral hygiene compare. in this instance, mmf was performed by attaching the wire to the arch bar and loop eyelets for additional immobilization. mmf and eyelets wire can be removed after two weeks because a callus has formed on the fracture fragment, thereby eliminating the need for additional intermaxillary immobilization.21 the arch bar is kept in place for up to eight weeks because it accommodates for new bone formation between fracture fragments.21,22 a subjective examination of the patient revealed that the mandibular fracture was caused by a fight that occurred one month prior to the patient’s visit to the oral surgery clinic of copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p197–201 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p197-201 201hermanto et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 197–201 nala husada hospital. the patient’s fracture fragment did not heal because she was not immediately treated for her injury and was only given painkillers (neglected fracture). a neglected fracture is a fracture with or without dislocation that is improperly treated, resulting in a deterioration of the condition and/or disability.6 in this instance, the patient developed a left submandibular abscess as a result of a neglected fracture. the left submandibular abscess was treated with an incision for drainage, which is done so that the inflammatory products in the submandibular space can be excreted properly, and tissue oxygenation can be increased, allowing anaerobic bacteria to be eradicated and promoting faster healing. according to a study conducted by the university of witwatersrand, streptococcus and staphylococcus bacteria were the predominant flora found in pus cultures of submandibular abscess cases.23 administration of antibiotics, empirically amoxicillin 500 mg tablets and metronidazole 500 mg tablets, are considered to be in accordance with the literature, though culture and antibiotic sensitivity tests are required for more definitive treatment.17 following the drainage incision, the physician also prescribed 500 mg of mefenamic acid every eight hours to reduce the patient’s pain after treatment. fri such as this case should be treated immediately and appropriately. incision draining of abscesses followed with closed reduction fracture treatment and antimicrobial prescription will decrease the patient’s morbidity and lead to the bone healing quickly. this patient’s fracture was resolved with a single arch bar on the mandible and eyelet wiring for the upper jaw coupled with mmf. this indicated that the treatment for this case was adequate to reduce, fixate, and immobilize fragment fractures. in conclusion, fri often happens from open fractures in dentate areas, with untreated fractures triggering a more serious infection. in this case, the patient had an impacted tooth around the fracture line on the left lower jaw, which increases fri possibility. for treatment, an incision was made, and the abscess drained in her left submandibular space. her jaws were then fixated using an arch bar in the mandible and eyelets in the maxilla with mmf, in accordance with the main concepts of surgical management for fri. references 1. hirani nn, pujara n. comparison of open reduction and internal fixation in case of symphysis and parasymphysis mandible fracture. int j sci res. 2015; 4(6): 2129–31. 2. sjamsudin e, adiantoro s, saragih ga, rausyanfikr ya, simarmata ra, kadrianto ta. combination of open and closed reduction methods in the treatment of multiple mandible fractures. int j sci res. 2020; 9(7): 914–8. 3. depypere m, morgenstern m, kuehl r, senneville e, moriarty tf, obremskey wt, zimmerli w, trampuz a, lagrou k, metsemakers w-j. pathogenesis and management of fracture-related infection. clin microbiol infect. 2020; 26(5): 572–8. 4. kuehl r, tschudin-sutter s, morgenstern m, dangel m, egli a, nowakowski a, suhm n, theilacker c, widmer af. time-dependent differences in management and microbiology of orthopaedic internal fixation-associated infections: an observational prospective study with 229 patients. clin microbiol infect. 2019; 25(1): 76–81. 5. ma x, han s, ma j, chen x, bai w, yan w, wang k. epidemiology, microbiology and therapeutic consequences of chronic osteomyelitis in northern china: a retrospective analysis of 255 patients. sci rep. 2018; 8(1): 14895. 6. isya wahdini s, dachlan i, seswandhana r, hutagalung mr, putri il, afandy d. neglected orbitozygomaticomaxillary fractures with complications: a case report. int j surg case rep. 2019; 62: 35–9. 7. metsemakers w-j, fragomen at, moriarty tf, morgenstern m, egol ka, zalavras c, obremskey wt, raschke m, mcnally ma. evidence-based recommendations for local antimicrobial strategies and dead space management in fracture-related infection. j orthop trauma. 2020; 34(1): 18–29. 8. nasser m, pandis n, fleming ps, fedorowicz z, ellis e, ali k. interventions for the management of mandibular fractures. cochrane database syst rev. 2013; : cd006087. 9. abdelfadil e, salem as, mourad si, al-belasy fa. infected mandibular fractures: risk factors and management. j oral hyg heal. 2013; 1: 102. 10. perez d, ellis e. complications of mandibular fracture repair and secondary reconstruction. semin plast surg. 2020; 34(4): 225–31. 11. omeje ku, rana m, adebola ar, efunkoya aa, olasoji ho, purcz n, gellrich n-c, rana m. quality of life in treatment of mandibular fractures using closed reduction and maxillomandibular fixation in comparison with open reduction and internal fixation – a randomized prospective study. j cranio-maxillofacial surg. 2014; 42(8): 1821–6. 12. yudianto c, sylvyana m, yuza at, sjamsudin e. management of multiple fractures of the maxilla and mandible with closed reduction: a case report. int j med biomed stud. 2022; 6(9): 27–34. 13. kumar g, narayan b. prevention of infection in the treatment of one thousand and twenty-five open fractures of long bones. retrospective and prospective analyses. in: banaszkiewicz p, kader d, editors. classic papers in orthopaedics. london: springer; 2014. p. 527–30. 14. metin m, sener i, tek m. impacted teeth and mandibular fracture. eur j dent. 2007; 1(1): 18–20. 15. farhadi f, emamverdizadeh p, hadilou m, jalali p. evaluation of infection and effective factors in impacted mandibular third molar surgeries: a cross-sectional study. scribante a, editor. int j dent. 2022; 2022: 8934184. 16. reitzik m, lownie jf, cleaton-jones p, austin j. experimental fractures of monkey mandibles. int j oral surg. 1978; 7(2): 100–3. 17. pickrell b, serebrakian a, maricevich r. mandible fractures. semin plast surg. 2017; 31(2): 100–7. 18. van den bergh b, heymans mw, duvekot f, forouzanfar t. treatment and complications of mandibular fractures: a 10-year analysis. j cranio-maxillofacial surg. 2012; 40(4): e108–11. 19. gunardi oj, diana r, kamadjaja db, sumarta npm. closed reduction in the treatment of neglected mandibular fractures at the department of oral and maxillofacial surgery, universitas airlangga. dent j (majalah kedokt gigi). 2019; 52(3): 147–53. 20. kumar m, hussain shah sf, kumar panjabi s, abdullah s, shams s. mandibular fracture management; comparison of efficacy of maxillomandibular fixation of screws versus erich arch bar. prof med j. 2019; 26(4): 615–9. 21. dergin g, emes y, aybar b. evaluation and management of mandibular fracture. in: gözler s, editor. trauma in dentistry. london: intechopen; 2019. 22. romero h, guifarro j, díaz f, umanzor v, pineda m, cruz c, gabrie m. management of mandibular fractures: report of three cases. dent res manag. 2021; 5(1): 17–22. 23. maharaj s, ahmed s, pillay p. deep neck space infections: a case series and review of the literature. clin med insights ear, nose throat. 2019; 12: 117955061987127. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p197–201 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p197-201 mkg vol 42 no 2 april 2009.indd 60 vol. 42. no. 2 april–june 2009 case report pseudomembranous candidiasis in patient wearing full denture nurdiana1 and m. jusri2 1resident of oral medicine 2staff of oral medicine faculty of dentistry airlangga university surabaya indonesia abstract background: oral candidiasis is a common opportunistic infection of the oral cavity caused by an overgrowth of candida species, the commonest being candida albicans. candida albicans is a harmless commensal organism inhabiting the mouths but it can change into pathogen and invade tissue and cause acute and chronic disease. dentures predispose to infection with candida in as many as 65% of elderly people wearing full upper dentures. purpose: the purpose of this case report is to discuss thrush in patient wearing full denture which rapidly developed. case: this paper report a case of 57 year-old man who came to the oral medicine clinic faculty of dentistry airlangga university with clinical appearance of pseudomembranous candidiasis (thrush). case management: diagnosis of this case is confirmed with microbiology examination. patient was wearing full upper dentures, and from anamnesis known that patient wearing denture for 24 hours and he had poor oral hygiene. patient was treated with topical (nystatin oral suspension and miconazole oral gel) and systemic (ketoconazole) antifungal. patient also instructed not to wear his denture and cleaned white pseudomembrane on his mouth with soft toothbrush. conclusion: denture, habit of wearing denture for 24 hours, and poor oral hygiene are predisposing factors of thrush and it can healed completely after treated with topical and systemic antifungal. key words: thrush, candida albicans, denture, poor oral hygiene correspondence: nurdiana, c/o: departemen oral medicine, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: nurdiana_drg@yahoo.com introduction candidiasis is a common opportunistic oral candida infection that develops in the presence of one of several predisposing conditions.1 candidiasis is caused by an overgrowth of the superficial fungus candida albicans (c. albicans).2 c. albicans is a commensal organism residing in the oral cavity in a majority of healthy persons.1 oral candidiasis (oc) has been arranged into two classification categories. primary oc is confined to oral or perioral tissues. these are sub classified as: acute: pseudomembranous and erythematous; chronic: pseudomembranous, erythematous and hyperplastic; and candida-associated lesion (denture-induced stomatitis, angular cheilitis, and median rhomboid glossitis.3–4 secondary oc is an oral manifestation of a generalized systemic mucocutaneous candidal infection, subdivided based on various immunological disease etiologies includes chronic mucocutaneous and candida-endocrinopathy syndrome.3–5 pseudomembranous candidiasis (thrush) is the most common form of candidiasis.1 thrush forms soft, friable, and creamy plaques on the mucosa that can be wiped off, leaving a red, raw or bleeding, and painful surface. the buccal mucosa, palate, and tongue are common location.2 the lesions may involve the entire oral mucosa or relatively localized areas where normal cleansing mechanisms are poor.6 the pseudomembrane consists of a network of candidal hyphae containing desquamated cells, microorganisms, fibrin, inflammatory cells, and debris.3 diagnosis of thrush is usually based on clinical criteria. direct smear microscopic examination with potassium hydroxide and culture are helpful.5 61nurdiana: pseudomembranous acadidiasis in patien wearing figure 1. visit i: white pseudomembrane on almost all over oral mucosa. thrush predominantly occurs in middle-aged or older persons.7 candida infection is also found commonly in denture wearer.8 one study found 63.1% of adults were asymptomatic carriers of c. albicans, with the occurrence of thrush at 64.8% in adults and 66.7% in adults with dentures. non-retentive denture, poor oral hygiene, constant irritation by a prosthesis, higher salivary yeast counts, and adherence of c. albicans to denture base may explain the higher occurrence of thrush in patient who wear denture.4 oral candidiasis (oc) that usually occurred in denture wearer is chronic atrophic candidiasis or denture stomatitis which characterized by chronic erythema and edema of the mucosa that contacts the fitting surface of the denture.7 however, in this case report, a patient whose wearing denture had thrush that rapidly developed on almost all over his oral mucosa. it is thought that thrush is caused by several predisposing factors of oc, such as wearing full denture, wearing denture for 24 hours, and poor oral hygiene. the purpose of this case report is to discuss thrush in patient wearing full denture which rapidly developed. case on april 1st 2008, a 57-years-old male patient came to oral medicine department, faculty of dentistry, airlangga university with almost all over his oral mucosa covered by white patches. according to anamnesis known on march 26th 2008, the patient came to prosthodontics department to make lower denture. in intra oral examination was found white lesion on lower edentulous ridge with clinical diagnosis was chronic hyperplastic candidiasis, the patient then referred to oral medicine department. since 3 days ago this patch developed in almost all over his oral mucosa and painful. patient then came to community health service and was given several drugs including amoxicillin, methampyrone, and vitamin b, but the patient did not take the drugs. patient has been wearing full upper denture since 4 months ago. since the patient wear denture, he used to wear this denture for 24 hours. patient cleaned his denture with toothbrush. since 1 month ago, patient had sore throat. patient visited tambak rejo hospital and was given several drugs, including ambroxol. patient’s general condition was thin and weak because since he had sore throat about a month ago he only drunk milk and not ate other food. in dental history known that patient wear full upper denture since 4 month ago. patient had smoking habit. there’s no known disorder in patient’s medical history and family history. extra oral examination showed chronic lymphadenitis on the right and left submandibular glands. in intra oral appeared white, elevated, pseudomembrane that can be wiped off on almost all over oral mucosa (figure 1). 62 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 60−64 figure 2. visit ii (3rd day), white pseudomembranous only seen on several part of oral mucosa and erythematous patch on hard palate (arrow). figure 3. white spots on filliform papilla (arrow). all pseudomembrane had healed (8th day). case management from anamnesis and clinical examination at first visit (april 1st, 2008), clinical diagnosis of this case was thrush. the patient refused to do complete blood examination, because he just had complete blood examination for hemorrhoid operation which is scheduled on april 3rd 2008, and the result of the complete blood examination was normal. the patient was treated with chlorhexidine mouthwash 2 times/day, nystatin oral suspension 2 times/ day, miconazole oral gel 2 times/day, ketoconazole tablet 200 mg 2 times/day, vitamin c 100 mg 2 times/day, and vitamin b complex 100 mg 2 times/day, for 14 days. patient instructed not to wear his denture and cleaned white pseudomembrane on his mouth with soft toothbrush. according to anamnesis two days later (april 3rd, 2008), the pain is greatly reduced and patient already ate solid food. right and left submandibular glands are normal. in intra oral appeared thin and discrete white pseudomembrane on several part of oral mucosa (figure 2a–g). on hard palate appeared erythematous patch (figure 2h). swab was done on lateral surface of the tongue and lower labial fold for microbiology examination. patient was instructed to continue the treatment. from anamnesis 5 days later (april 8th, 2008) known that there’s no more pain. intra orally, all white pseudomembrane was gone, except on lateral surface of the tongue there was white spots on filiform papilla (figure 3). microbiology examination result found candida colony. patient was instructed to continue the treatment, but ketoconazole dose was reduced into 200 mg 1 time/day. one week later (april 15th 2008), there was no pain anymore. intra oral examination showed white spot on filiform papilla that can not be wiped off. the lesion was clinically diagnosed as hairy tongue (figure 4). the treatment was stopped. patient was instructed to maintain the oral and denture hygiene, and take off the denture at night. 63nurdiana: pseudomembranous acadidiasis in patien wearing figure 4. white spot on filiform papilla (arrow) was clinically diagnosed as hairy tongue. discussion prevalence, characteristic appearance, and ease of removal of the lesions makes thrush easily recognized, and a diagnosis of thrush is frequently made based on the appearance of the lesion.6 taking a history followed by a thorough examination of the mouth, at the soft and hard palate, and examining the buccal mucosa in those wearing dentures after they have been removed are usually good starting points.9 diagnosis can be confirmed microbiologically either by staining a smear from the affected area with periodic acidschiff (pas) stain, gridley stain, or gomori methenamine silver (gms) stain or by culturing a swab from an oral rinse.7,9 culture candida using a sabouraud's agar slant was done to aid in the definitive identification of the fungal organism.10 this case was diagnosed based on clinical features, which characterized of thrush that is white creamy pseudomembrane (patch) that can be wiped off and leaving erythematous base. diagnosis of this case was confirmed with microbiology examination through swab and culture using sabouraud’s agar. candida colony was found in microbiology examination. predisposing factors is so important in the etiology of candidiasis that it is extremely rare to find a case of oc in which one or more of these factors cannot be identified. a diagnosis of thrush should always be followed by a search for a possible undiagnosed medical disorder, a review of patient’s medications, and some locally acting predisposing factor such as denture.6 according to neville, et al. cit. firriolo10 there are three general factors that may lead to clinically evident oc. these factors are: immune status of the host; oral mucosal environment; and particular strain of c. albicans (the hyphal form is usually associated with pathogenic infection). factors that alter immune status of the host are blood dyscrasias or advanced malignancy, old age or infancy, radiation therapy or chemotherapy, and hiv infection or other immunodeficiency disorders, endocrine abnormalities (such as diabetes mellitus, hypothyroidism, hypoparathyroidism, pregnancy, corticosteroid therapy or hypoadrenalism). factors that alter oral mucosal environment are xerostomia, antibiotic therapy, poor oral or denture hygiene, malnutrition or gastrointestinal malabsorption, iron, folic acid, or vitamin deficiencies, acidic saliva or carbohydrate-rich diets, heavy smoking, and oral epithelial dysplasia. the yeast form of c. albicans is believed to be relatively innocuous, this is associated with the fact that candida is poorly equipped to invade and destroy tissue, but the hyphal form is usually associated with invasion of host tissue.6 few candida hyphae are associated with the atrophic epithelium in erythematous candidiasis, whereas numerous organisms are found invading the prickle cell layer of oral epithelium in pseudomembranous candidiasis. c. albicans is a polymorphic organism which undergoes morphological transition among yeast, pseudohyphal, and hyphal forms. all three morphogenetic forms of c. albicans are frequently encountered in the oral mucosa, and, in most oral infections, both yeast and filamentous organisms can be found in the infected tissues. however, clinicopathologic findings have correlated the presence of filamentous forms with localized tissue invasion in oral candidiasis. the transition from commensalism to infection in the oral mucosa is dictated by changes in the local oral microenvironment (breach of mucosal integrity, qualitative or quantitative shifts in oral microbial flora), or by an inadequate host defense, which results in overgrowth of the organism. in oral mucosal infections, c. albicans organisms colonize the outermost layers of epithelium, rarely invading past the spinous cell layer. it is well recognized that the epithelial cells is an infection barrier against candida.11 a major pathogenicity mechanism of candida is its adherence capacity to the host cell. adherence capacity depends on several factors, such as the hydrophobic state of the fungal cell wall and the characteristics of the substrate surface. the expression of cellular surface hydrophobicity of c. albicans is a dynamic process on which the culture conditions have a fundamental influence.12 in this patient found several factors that can contribute for oc, which is denture, poor oral hygiene, and smoking habit. denture wearing, poor oral hygiene, smoking habit, and habit of wearing denture for 24 hours makes thrush develop rapidly. candida-associated denture stomatitis is a recalcitrant disease in some 60% of otherwise healthy denture wearers.13 denture wearers are predisposed to the development of candida colonization, candidiasis, and presence of candida. it is observed that denture base composition influences significantly in the adhesion of candida to denture.14 the surface irregularities of acrylic resin is a factor in the entrapment of microorganisms, especially c. albicans.8 microbial plaque accumulation on the base surface of removable dentures plays a critical role, promoting a switch from a commensal to a pathogenic oral flora. the denture-palatal interface offers a unique ecological niche for microorganism colonization because of the relatively anaerobic and acidic environment favoring yeast proliferation.15 the isolated candida associated with dentures are related to the poor hygienic condition of the 64 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 60−64 dentures, to the long time of the usage, wearing dentures at night and to the modifications of the hard supporting tissues.8 some studies have reported that smoking significantly increased carriage from 30–70%.7 oral hygiene and topical antifungals are usually adequate for uncomplicated oc. oral hygiene involves cleaning the teeth, buccal cavity, tongue, and dentures, if present, daily.9 denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals.13 this case was treated with topical and systemic antifungals. topical antifungals that were used in this case were nystatin oral suspension and miconazole oral gel. majority of candidiasis maybe simply treated with topical applications of nystatin oral suspension.1 nystatin oral suspension 100.000 units/ml was used for 14 days after meals and at bedtime.10 miconazole, an imidazole, can be used as a local application in the mouth.9 systemic antifungal are usually indicated in cases of disseminated disease and/or in immunocompromised patients.10 systemic antifungal that was used in this case was ketoconazole. oral ketoconazole can be effective for treatment of severe oral and esophageal candidiasis, but patient’s compliance often is poor because of taste of drug.8–9 ketoconazole tablets, 200 mg twice daily can be used for 14 days.10 chlorhexidine mouthwash was given to improve oral hygiene, and vitamin c and b complex was given to improve patient’s general condition. denture should be cleaned and disinfected daily and left out overnight or for at least six hours daily.9 patient was instructed to remove the denture at night and the denture should always be cleaned. patient was advised to reduce the smoking habits. the conclusion of this case is thrush developed due to 3 factors are reduced of host’s immune status that was affected by patient’s age (57 years old); oral mucosa environment that contributed to candidiasis i.e. poor oral hygiene, denture wearing, and smoking habits; and the present of c. albicans which is normal oral flora. in this case, thrush was diagnosed through clinical feature which was confirmed with microbiology examination and treated with topical antifungals (nystatin oral suspension and miconazole oral gel) which were combined with systemic antifungal (ketoconazole). references 1. regezi ja, sciubba jj, jordan rck. oral pathology clinical pathologic correlations. 4th ed. st. louis: saunders; 2003. p. 100–4. 2. langlais rp, miller cs. color atlas of common oral diseases. 3rd ed. philadelphia: lippincott williams and wilkins; 2003. p. 130–1. 3. field a, longman l. tyldesley’s oral medicine. 5th ed. oxford: university press; 2003 p. 35–40. 4. wiler jl. diagnosis: oral candidiasis / thrush. available from: http://www.health.am/ab/more/ diagnosis-oral-candidiasis-thrush. htm. accessed april 4, 2008. 5. laskaris g. treatment of oral diseases. a concise textbook. 1st ed. stuttgart: thieme; 2005. p. 30–2. 6. bhattacharyya i, cohen dm, silverman jr. s. red and white lesions of the oral mucosa. in: greenberg ms, glick m. burket’s oral medicine diagnosis and treatment. 10th ed. hamilton: bc decker; 2003. p. 94–101. 7. scully c. candidiasis, mucosal. available from: http://www. emedicine.com/derm/topic68.htm. accessed april 4, 2008. 8. daniluk t, tokajuk g, stokowska w, fiedoruk k, sciepuk m, zaremba ml, et al. occurrence rate of oral candida albicans in denture wearer patients. advances in medical sciences 2006; 51(suppl 1):77–80. 9. akpan a, morgan r. oral candidiasis. available from: http://pmj. bmj.com/cgi/content/full/78/ 922/455.htm. accessed april 4, 2008. 10. firriolo fj. oral candidiasis. available from: http://www.dentalcare. com/soap/intermed/ oralcan.htm. accessed april 4, 2008. 11. dongari-bagtzoglou a, fidel pl. the host cytokine responses and protective immunity in oropharyngeal candidiasis. j dent res 2005; 84(11):966–77. 12. blanco mt, morales jj, lucio l, pérez-giraldo c, hurtado c, gómez-garcía ac. modification of adherence to plastic and to human buccal cells of candida albicans and candida dubliniensis by subinhibitory concentration of itraconazole. oral microbiology immunology 2006; 21(1):69–72. 13. samaranayake yh, cheung bp, parahitiyawa n, seneviratne cj, yau jy, yeung kw, samaranayake lp. synergistic activity of lysozyme and antifungal agents against candida albicans on denture acrylic surfaces. archives of oral biology 2009; 54(2):115–26. 14. marcos-arias c, vicente jl, sahand ih, eguia a, de-juan a, madariaga l, aguirre jm, eraso e, quindós g. isolation of candida dubliniensis in denture stomatitis. archives of oral biology 2009; 54(2):127–31. 15. avon sl, goulet jp, deslauriers n. removable acrylic resin disk as a sampling system for the study of denture biofilms in vivo. j prosthet dent 2007; 97(1):32–8. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 99 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 99–104 original article the correlation between the knowledge level related to practice protocols and dentists’ anxiety levels in practice during the covid-19 pandemic merlya balbeid, yuanita lely rachmawati, marchella anestya wibowo department of preventive and public health dentistry, faculty of dentistry, universitas brawijaya, malang, indonesia abstract background: the global epidemic of covid-19 has reached an emergency status in the health system, including dentistry. the dentist profession is inseparable from the possibility of direct or indirect contact with microorganisms in the patient’s blood or saliva. national and international dental associations, such as persatuan dokter gigi indonesia and the american dental association, have published practice protocols that must be applied by dentists who choose to continue practicing during the covid-19 pandemic. dentists’ knowledge of practice protocols in the current situation is very important, as it enables dentists to take infection control measures against virus transmission in the dental practice environment. strong knowledge can have a positive impact on the psychological state of dentists, such as by reducing the anxiety level of dentists when treating patients during the pandemic. purpose: to determine the correlation between the level of knowledge of dentists regarding practice protocols and the level of anxiety that they face regarding practicing during the covid-19 pandemic in indonesia. methods: the research design is a correlation analysis, namely research with a cross-sectional approach and purposive sampling, with a total sample of 170 respondents. data were collected through google form and univariate analysis was carried out then bivariate analysis with kendall’s tau correlation test. results: this study found that as many as 166 respondents (97.6%) had a good level of knowledge and as many as 87 respondents (51.2%) had a minimum level of anxiety. the results of the analysis were obtained and found to be 0.031, which means p <0.05 so that it shows a relationship between the two variables. conclusion: there is a correlation between the level of knowledge and the level of anxiety of dentists in practice during the covid-19 pandemic. keywords: anxiety; covid-19; dentist; knowledge correspondence: merlya balbeid, department of preventive and public health, faculty of dentistry, universitas brawijaya. jl. veteran, malang 65145, indonesia. email: merlya.fk@ub.ac.id introduction at the beginning of the new decade, on 30 january 2020, the world health organization (who) declared a global public health emergency against the coronavirus disease outbreak, which is known as corona virus disease 2019 (covid-19). 1it started with the discovery of a new pathogen that spread across china to europe2 and has since rapidly reached pandemic status.1 covid-19 has created a state of emergency in the health system, including dentistry.2 health professionals, especially dentists, are at a higher risk of exposure to infection due to having close contact with infected patients.3 transmission of covid19 during dental procedures can occur through droplet or aerosol inhalation from an infected patient or direct contact with oral fluids, mucous membranes and contaminated instruments and surfaces.2 knowledge plays an important role in human life because it reflects how an individual understands the situation in the surrounding world, which can later determine how a person acts.4 factors that influence disease prevention include a person’s knowledge, attitude and actions towards the disease. someone who is knowledgeable about something tends to make more appropriate decisions regarding the problem.5 meanwhile, anxiety is an unpleasant emotional state that is experienced by individuals when thinking about something unpleasant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 mailto:merlya.fk@ub.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 100balbeid et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 99–104 that will happen and can cause feelings of fear, caution and vigilance. anxiety is an unpleasant fear or a sign that something bad will happen.6 especially in the current covid-19 pandemic situation, fear increases symptoms of anxiety and stress in healthy people.7 therefore, anxiety is used as an indicator in research. anxiety causes a mixture of emotions that are felt by an individual, where fear is the dominant emotion. anxiety is more appropriate to describe a person’s emotional condition during the current covid19 pandemic because the outbreak of this virus can cause a person to worry about the unexpected that might happen in the future.8 in the current era of the covid-19 pandemic, dentists are vulnerable to anxiety when dealing with patients because effective drugs against covid-19 are still in the research and development process. moreover, vaccinations have been used all over the world. as stated by the who, the virus is transmissible through droplets, and this poses a risk to dentists when performing dental procedures. anxiety and fear are strong emotions that may be related to the overreporting of the pandemic being disseminated through social media, electronic or print, not all of which contain scientific evidence and well-structured knowledge about covid-19. this is because screening and diagnostics take time, and there is inadequate personal protective equipment (ppe) and unclear treatment and immunisations.7 the level of anxiety that is experienced in a covid-19 pandemic situation can affect dentists’ performance and decision-making.9 in their research, lai et al.10 conducted a survey on health workers who were working in hospitals in wuhan and hubei. most of the respondents had symptoms of insomnia (34%), anxiety (44.6%), depression (50.4%) and distress (71.5%).10 kinariwala et al.11 conducted a survey on dentists in india. in the survey, 45.9% of 403 respondents were worried about the risk of contracting covid-19 through patients.11 an anonymous online survey was sent to dentists who were practicing in modena and reggio emilia, which are some of the regions in italy that were most affected by covid-19. it found that almost 85% of dentists reported that they were worried about exposure to infection during clinical activities. the results of general anxiety disorder-7 (gad-7) showed that 9% of respondents reported experiencing severe anxiety. in conclusion, the covid-19 emergency had a negative impact on the activities of dentists who were practicing in the modena and reggio emilia areas. this negative impact perception is accompanied by feelings of fear (42.4%), anxiety (46.4%) and worry (70.2%).2 national and international dental associations, such as persatuan dokter gigi indonesia (pdgi)12 and the american dental association (ada), have published practice protocols that must be applied by dentists who choose to continue practicing during the covid-19 pandemic. although the ada has published prevention guidelines, most dentists are reluctant and afraid to perform treatment during the covid-19 pandemic. because indonesia is the fourth most populous country in the world, it is likely to suffer greatly over a longer period compared with countries with a small population. this will affect various aspects of society, such as work, health and psychological factors, and clinical dentists, who will be more susceptible to transmission from covid-19. in this study, the researchers wanted to investigate the correlation between the level of knowledge of dentists regarding practice protocols and the level of anxiety that they face regarding practicing during the covid-19 pandemic in indonesia. materials and methods this research has been approved by the health research ethics commission of the health polytechnic of malang, with protocol number 965/kepk-polkesma/2020. this study is a cross-sectional study that was conducted on both general dentists and specialists in indonesia, who practiced during the covid-19 pandemic. the sampling method was purposive sampling because the researchers wanted to provide a more representative value regarding the knowledge and anxiety levels of dentists who actively practiced in this situation from several provinces in indonesia, such as east java, central java, west java, dki jakarta, north sumatra, riau, east nusa tenggara, bali, east kalimantan, and south sulawesi. the participants had to have whatsapp social media and be willing to participate in this research. the sample size estimation was carried out by referring to the number of dentists in several provinces of indonesia who had different transmission risks. the type i error (α) was set at 5%, which is a common range in health or social research. the minimum sample size that was calculated was 164. the type of research used is correlation analysis to determine the correlation between two groups of variables in a situation or group of subjects. the variables that were measured were the level of knowledge related to practice protocol and the level of anxiety of the dentist, and measured using a research instrument in the form of a closed questionnaire. the questionnaire was created and packaged using a google form that can be accessed for free, and links to online surveys were sent via whatsapp social media. the knowledge questionnaire in this study focused on assessing respondents’ understanding of the practice protocol when dealing with patients during the covid19 pandemic. the anxiety questionnaire consisted of question items that were related to anxiety symptoms, such as fear, worry and anxiety, which affect the respondent’s psychology regarding the future of their practice. to avoid research bias, the researcher studied the research on several previous surveys to investigate the question items that were relevant to the research. therefore, the question items in this study have been adapted from english-language surveys in modena and reggio emilia2 which were then translated into valid and appropriate indonesian, as well as practice guidelines issued by pdgi. in addition, to assess dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 101 balbeid et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 99–104 the psychological impact on dentists when practicing in the current situation, such as fear, anxiety, worry, sadness and anger, an anxiety-measuring instrument in the form of the gad-7 scale was used to determine the presence or absence of anxiety dysfunction in the respondents.13 the primary data was obtained directly from respondents and analysed using statistical product and service solution (spss) statistics 21. researchers carried out content and face validity by identifying 22 questions from knowledge and anxiety questions. the reliability test was carried out by testing the questionnaire on 14 general dentists and specialist dentists, who were spread across the cities of malang and blitar, twice with an interval of 7–14 days. it was analysed using cronbach’s alpha (α) with a value of 0.787 for the variable knowledge and 0.889 for the anxiety variable so that the items in this questionnaire would be reliable. after that, the questionnaire was distributed to the research sample. data collection was carried out from 15 november 2020 to 27 april 2021, with a total sample of 170 respondents. the respondent data that was collected was then analysed using univariate and bivariate analyses. in the bivariate analysis, kendall's tau correlation test was used to measure the correlation between the level of knowledge and the anxiety level of the dentist. data on the characteristics of the respondents and the categorisation of each variable, namely the level of knowledge related to practice protocol and the level of anxiety in the respondents, was described. results a total of 170 dentists completed the questionnaire in full. of the respondents, 19.4% were men, and 80.6% were women. most of the respondents were general dentists (150, 88.2%) and aged under 35 years (48.2%). therefore, most had practiced for less than five years (42.9%). a total of 55 (32.4%) participating dentists reported that they worked in private practices, while 22.9% worked in public health centres, 18.2% in hospitals and 26.5% in clinics. moreover, 100 (58.8%) of the respondents came from the east java province, as illustrated in table 1. approximately 97.6% of respondents had a good level of knowledge, as they understood the practice guidelines that were issued by pdgi. almost all dentists (161, 94.7%) feared being infected with covid-19 by both their patients and co-workers, and 91.8% of them felt anxious when treating patients who showed symptoms of a cough or were suspected of being infected with covid-19. approximately 51.8% of dentists wanted to close their practice until the number of confirmed cases started to decline (table 2). regarding covid-19, only 34.7% reported experiencing anxiety, approximately 17.2% experienced fear, 7.1% felt sad and most (41.2%) felt concerned about the covid19 situation. the average gad-7 score was 5,211, which indicates an overall mild generalised anxiety level. more precisely, 51.2% of respondents showed minimal anxiety (score 0–4), 35.3% showed mild anxiety (score 5–9), 8.8% table 1. demographic information of dental practitioners (n=170) characteristic frequency percentage (%) gender female 137 80.6 male 33 19.4 age (years) less than 35 82 48.2 35–55 74 43.5 above 55 14 8.2 last education general dentist 150 88.2 specialist dentist 20 11.8 dental practice public health centre 39 22.9 hospital 31 18.2 clinic 45 26.5 private 55 32.4 province east java 100 58.8 central java 16 9.4 west java 5 2.9 dki jakarta 3 1.8 bali 13 7.6 north sumatera 4 2.4 riau 12 7.1 east nusa tenggara 6 3.5 east kalimantan 7 4.1 south sulawesi 4 2.4 professional experience (years) less than 5 73 42.9 5 – 10 31 18.2 10 – 15 23 13.5 above 15 43 25.3 table 2. assessment of dentists’ fear and anxiety when p r a c t i c i n g d u r i n g t h e c o v i d 1 9 p a n d e m i c (n = 170) questions yes n (%) no n (%) are you afraid of getting infected with covid-19 from a patient and co-worker? 161 (94.7) 9 (5.3) do you feel anxious when providing treatment to a patient who is coughing or showing suspicious symptoms? 158 (91.8) 12 (8.2) do you want to close your dental practice until the number of covid-19 cases starts declining? 88 (51.8) 82 (48.2) table 3. respondents’ concerns about the future of practice questions: how worried are you about your professional future? frequency (n=170) percentage (%) extremely 6 3.5 a lot 12 7.1 quite 56 32.9 a little 70 41.2 not at all 26 15.3 total 170 100 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 102balbeid et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 99–104 showed moderate anxiety (score 10–14) and 4.7% showed severe anxiety (score 15–21). to the question, “how worried are you about the future of your practice?”, most respondents (41.2%) felt slightly worried, as illustrated in table 3. for the last question, “what worries you the most?”, several answers could be selected by the respondent. most of the respondents in this study stated that “they do not know when this emergency situation will end”, and 142 respondents (83.5%) and the second highest percentage answered that “new procedures and new devices are needed for safety and infection prevention” namely 60% of respondents. the percentage with a lower preference on this question resulted in respondents’ answers, as follows the “difficult situation in dental practice will get worse” (29.4%), “patients will have less money to spend” (18.2%) and the “opportunity to lose their job or have to lay off employees” received the smallest indicated preference (11.8%). based on the analysis of this study, it was found that kendall’s tau correlation was 0.031, which means p < 0.05, with a correlation coefficient (r) of 0.158. this means that there is a significant correlation between the level of knowledge and the level of anxiety of dentists when practicing during the covid-19 pandemic, with a weak correlation strength. this shows that the research hypothesis is rejected and that the level of knowledge that is possessed by dentists affects their level of anxiety when practicing. discussion in a pandemic situation, the levels of stress, fear and anxiety increase. correspondingly, the level of difficulty that is experienced among healthcare staff is higher than that experienced by the general population, as they have a higher risk of infection.7 since the sars-cov-2 pandemic, other surveys have been proposed by other international agencies, with the purpose of measuring the impact of this epidemic in the dentists’ environment.2 the older group of dentists were less likely to develop anxiety than younger dentists, which has also been observed among the public during the covid-19 outbreak. in addition, personal protective measures can reduce anxiety among dentists, as these measures have the potential to reduce the fear of covid-19.14 this survey was conducted by taking samples from several provinces in indonesia with different levels of risk representing the covid-19 situation in indonesia so that various data were obtained. this survey reached 170 respondents, who comprised general and specialist dentists in indonesia. many female respondents who participated in this study showed that female dentists were more dominant in the field of dentistry. this is related to data obtained from the central statistics agency of malang city, which of the 72 number of dentists in malang city spread across several health centers and hospitals in 2019 it was found that 58 of them were female, and the remaining 14 were male. the results of this study also showed that respondents who were aged under 35 years comprised the majority (82 respondents, 48.2%). according to the data reported by the ada, among 201,117 dentists working in the united states in 2020, 17% were under 35 years old, 24% were 35–44 years old, 21.6% were 45–54 years old, 21.1% were aged 55–64 years and 16% were aged 65 years and over. this shows that most dentists actively practice in adulthood, which is under 35 years of age. most of the respondents were general dentists, namely 150 respondents (88.2%) which from the pb pdgi statistics in 2021 also showed that of the number of dentists in indonesia as many as 40,380 people, 35,979 of them were general dentists, and the rest were divided into several specialties. based on the results of the research above, most of the respondents have a private practice (55 respondents, 32.4%). this is followed by respondents who work in clinics (45 respondents, 26.5%) and health centres (39 respondents, 22.9%), and the rest work in hospitals (31 people, 18.2%). most respondents came from east java because the researchers optimised the distribution of questionnaires to dentists who were working in the east java area. respondents who had practiced for <5 years (73 respondents, 42.9%) were the majority in this study. this correlates with the data held by the indonesian medical council, which shows that the number of dentist graduates is 1,000–1,500 per year. this means that the population of dentists in the field is currently dominated by new dentist graduates with practical experience of less than five years. the knowledge of dentists in this study was seen from their understanding of practice guidelines that must be applied during the covid-19 pandemic. as with other infectious infections, this practice guideline covers personal protective equipment, hand washing, detailed patient evaluation, isolation using a rubber dam, anti-retraction handpieces, mouth rinses before dental procedures and clinical disinfection.2 based on relevant practice and research guidelines, dentists should implement strict personal protective measures and avoid or minimise operations that can generate aerosols or droplets.2 the results that were obtained in this study were very similar to the findings in previous studies, in that most of the respondents (97.6%) had a high level of knowledge.3 in previous studies, the researchers believed that dental professionals can play an important role in suppressing the transmission of covid-19. the infection control guidelines that are adopted in dentistry were last provided during the human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome (aids) pandemic, and similar guidelines may have prevented the spread of the epidemic of severe acute respiratory syndrome (sars) which is a viral respiratory disease caused by the sars-associated coronavirus in 2003 in dentistry.15 this is not in accordance with the situation in indonesia at the time of the initial outbreak of the covid-19 outbreak, transmission rates were found in several dental practices, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 103 balbeid et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 99–104 although they only represented 4% of the total population of dentists in indonesia. the gad-7 scale assessment found that 51.2% of respondents had a minimum level of anxiety. the anxietymeasuring instrument only assessed the frequency of respondents’ anxiety and concerns among dentists about the inability to prevent the end of the pandemic, followed by the need for new procedures and devices to prevent the spread of sars-cov-2 in dental practice. in similar studies that were conducted previously, symptoms of anxiety, such as fear, worry, sadness and anger, showed almost similar results among dentists regarding the covid-19 pandemic.3,14 the correlation results that were obtained in this study are not in line with the research by koçak et al.16 entitled “knowledge and anxiety levels of dentists about the covid-19 pandemic”, which found no significant correlation. the differences in the variables that were studied also influenced the results in this study. the results of the study by koçak et al.16 showed that most respondents experienced somatic symptoms, such as discomfort, fatigue and sleep disturbances, while in this study, no data was collected regarding the somatic symptoms of anxiety.16 the research data was obtained over a short period, as there will always be changes or impacts from the covid19 pandemic on the psychological condition of dentists, which will always be balanced by increasing the application of infection control guidelines during the covid-19 pandemic. therefore, the knowledge and mental conditions of dentists, especially anxiety, can change according to how the pandemic develops. zhao et al.’s14 research stated that there are several factors that could potentially be related to the anxiety state of workers in the dental practice environment, including the following: i) number of working hours per day; ii) number of working days per week; iii) number of working hours between breaks; iv) whether aerosolisation procedures are performed frequently; v) whether they have ever treated confirmed or suspected cases of covid-19; and vi) whether their skin or wounds were exposed to saliva, blood or other materials from the patient’s body fluids. however, in this study, these were not analysed. as the data collection was related to these potential factors, it became a limitation in the assessment of the respondent’s level of anxiety. in addition, the respondents who participated in this study did not cover the entire population of dentists in indonesia. therefore, generalisation of the study needed to be done. there is a possibility that the results that were obtained will be more varied, as the general conditions of each province in indonesia have a different risk of transmission. this affected the sample size in the study, as it was small. another problem in this study is the selection bias and sampling limitations because this research is an online survey, and the respondents were only obtained using social media in the form of whatsapp. this study shows that during the covid-19 pandemic, most dentists in indonesia could overcome their psychological state when deciding whether to continue practicing. researchers found that the anxiety status of dentists in indonesia was at a minimal level. this is because most dentists have a good knowledge of the practice guidelines that were issued by the local dentist association, namely pdgi, for cross-infection control during the covid-19 pandemic. therefore, it can be concluded that having a good level of knowledge correlates with anxiety management among dentists when practicing during the covid-19 pandemic. acknowledgments the authors would like to thank all dentists who participated in this study. references 1. kamate s, sharma s, thakar s, srivastava d, sengupta k, hadi aj, chaudhary a, joshi r, dhanker k. assessing knowledge, attitudes and practices of dental practitioners regarding the covid-19 pandemic: a multinational study. dent med probl. 2020; 57(1): 11–7. 2. con solo u, bel l i n i p, benciven n i d, i a n i c , c he c ch i v. epidemiological aspects and psychological reactions to covid-19 of dental practitioners in the northern italy districts of modena and reggio emilia. int j environ res public health. 2020; 17(10): 3459. 3. ahmed ma, jouhar r, ahmed n, adnan s, aftab m, zafar ms, khurshid z. fear and practice modifications among dentists to combat novel coronavirus disease (covid-19) outbreak. int j environ res public health. 2020; 17(8): 2821. 4. niedderer k. mapping the meaning of knowledge in design research. des res q. 2007; 2(2): 1, 5–13. 5. utami f, putri ks, hidayati h. hubungan pengetahuan dan sikap dengan tindakan mahasiswa program profesi dokter gigi rsgmp universitas andalas terhadap pengendalian infeksi. andalas dent j. 2017; 5(2): 88–98. 6. julistia r, sari k, sulistyani a. perbedaan tingkat kecemasan pada dokter gigi muda dan perawat gigi muda saat menghadapi pasien. j psikogenes. 2018; 4(1): 73–84. 7. aly mm, elchaghaby ma. impact of novel coronavirus disease (covid-19) on egyptian dentists’ fear and dental practice (a crosssectional survey). bdj open. 2020; 6(1): 19. 8. annisa df, ifdil i. konsep kecemasan (anxiety) pada lanjut usia (lansia). konselor. 2016; 5(2): 93. 9. olivieri jg, de españa c, encinas m, ruiz x-f, miró q, ortegamartinez j, durán-sindreu f. general anxiety in dental staff and hemodynamic changes over endodontists’ workday during the coronavirus disease 2019 pandemic: a prospective longitudinal study. j endod. 2021; 47(2): 196–203. 10. lai j, ma s, wang y, cai z, hu j, wei n, wu j, du h, chen t, li r, tan h, kang l, yao l, huang m, wang h, wang g, liu z, hu s. factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019. jama netw open. 2020; 3(3): e203976. 11. kinariwala n, samaranayake lp, perera i, patel z. concerns and fears of indian dentists on professional practice during the coronavirus disease 2019 (covid-19) pandemic. oral dis. 2021; 27(s3): 730–2. 12. persatuan dokter gigi indonesia. surat edaran no.2776/pb pdgi/ iii-3/2020 tentang pedoman pelayanan kedokteran gigi selama pandemi virus covid-19. 2020. p. 1–13. available from: http://pdgi. or.id/artikel/pedoman-pelayanan-kedokteran-gigi-selama-pandemivirus-covid-19. accessed 2020 sep 22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 http://pdgi https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 104balbeid et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 99–104 13. baker a, simon n, keshaviah a, farabaugh a, deckersbach t, worthington jj, hoge e, fava m, pollack mp. anxiety symptoms questionnaire (asq): development and validation. gen psychiatry. 2019; 32(6): e100144. 14. zhao s, cao j, sun r, zhang l, liu b. analysis of anxiety-related factors amongst frontline dental staff during the covid-19 pandemic in yichang, china. bmc oral health. 2020; 20(1): 342. 15. bakaeen lg, masri r, altarawneh s, garcia lt, alhadidi a, khamis ah, hamdan am, baqain zh. dentists’ knowledge, attitudes, and professional behavior toward the covid-19 pandemic. j am dent assoc. 2021; 152(1): 16–24. 16. koçak s, saġlam bc, özdemir o, hazar e, koçak mm. knowledge and anxiety level of dentists about covid-19 pandemic. j oral heal community dent. 2021; 14(3): 104–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p99–104 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p99-104 147 prevalence of hypodontia in chinese orthodontic patients pambudi rahardjo department of orthodontics faculty of dentistry airlangga university surabaya indonesia abstract hypodontia is a phenomenon of congenitally missing teeth in human. a thousand and twelve panoramic radiographs of chinese orthodontic patients were examined for agenesis of permanent teeth including third molars. the patients with missing teeth were divided into four group: the patients with missing less than 4 molars, missing all third molars, hypodontia and oligodontia. the result revealed, 210 patients affected one or more teeth agenesis with a total of 455 teeth missing. the prevalence of tooth agenesis was maxillary third molar 259 (56.9%), mandibular third molar 143 (31.4%), mandibular second premolar 15 (3.3%), mandibular lateral incisors 13 (2.8%), maxillary lateral incisors 8 (1.7%), maxillary second premolars 7 (1.5%), other teeth 10 (2.2%) respectively. of the mesial mandibular first permanent molar, mandibular second premolars were the most frequent missing teeth. although hypodontia did not represent a public health problem, from orthodontic point of view it might cause esthetic and masticatory function disorders as well as more complex mechanotherapy of a patient. key words: hypodontia, chinese, mandibular second premolar correspondence: pambudi rahardjo, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction some people born, having problem with unable to develop a full set of teeth, due to disturbance occurred during the development of the tooth. this condition commonly called hypodontia, a congenitally absence of one or a few deciduous or permanent teeth. hypodontia could occur as an isolated sporadic or a familial trait. the most common permanent teeth missing are the third molars (20%), second premolars (3.4%) and maxillary lateral incisors (2.2%).1 some authors excluded the third molars in their studies.2-8 an investigation in two countries in norway reported that the most often missing teeth were mandibular second premolars (47% of all missing teeth) followed by maxillary second premolars and lateral incisors (both 20% of all missing teeth). the prevalence was higher in females (5.1%) than in males (4.0%).2 steffen et al.3 after examined 968 panoramic radiographs of orthodontic patients (463 females, 505 males) reported that hypodontia of at least one missing tooth was recorded in 89 patients. one hundred and thirty one tooth buds were missing. the most frequent effected tooth was the lower second premolars (52%), second upper premolar (27%) and upper lateral incisors (21%) respectively. an examination of 739 healthy caucasian 7 year-olds proved the prevalence of hypodontia excluding third molars in girls was 8.4%, in boys 6.5% and in both sexes combined 7.4%. of the children with hypodontia, the majority (90.9%) lacked one or two teeth. lower second premolar were the teeth most frequently missing.4 another study of 111 orthodontic patients (46 males and 65 females) revealed the fact that agenesis was found in 8.1% of the patients (2 males and 7 females) and the mandibular second premolars was the most commonly affected teeth, excluding the third molars.5 a survey in korea which performed on 721 school children found the most common congenitally missing teeth were mandibular second premolars (32.7%) followed by the mandibular incisors (28.7%), the second maxillary premolars (16.7%) and the maxillary lateral incisors (10.2%). the prevalence of congenitally missing teeth was 6.7% in boys and 9.5% in girls and 8.0% for both sexes combined.6 a study conducted in hong kong to determine the prevalence of hypodontia and hyperdontia of permanent teeth amongst southern chinese children. the results was as follows: the prevalence of congenitally missing teeth (third molar excluded) 6.1% in boys and 7.7% in girls and 6.9% for both sexes combined; the most commonly absent tooth was the mandibular incisors, affecting 58.7% of the children with hypodontia.7 an investigation was performed to examine the effects of advanced hypodontia on craniofacial morphology in japanese patients. the most frequently missing teeth were the mandibular and maxillary second premolar, followed by the maxillary first premolars and the maxillary first molar, in that order.8 the aim of this survey was to examine the prevalence of hypodontia including third molars in chinese orthodontic patients treated in private orthodontic practice and ranked the missing teeth according to the prevalence. 148 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 147–150 material and methods the pre-treatment panoramic radiographs of 1012 chinese orthodontic patients (734 females and 278 males) treated at private practice were evaluated retrospectively for agenesis of permanent teeth including third molars. the patients age range from 8 to 38 years. none of the patients has undergone extraction of permanent teeth and orthodontic treatment as well. the patients with missing teeth were grouped into 4: the patients with missing less than 4 molars, the patients with missing all third molars, the patients with hypodontia (missing 1 to 6 teeth) and patients with oligodontia (missing more than 6 teeth). the data was analyzed descriptively and the prevalence of missing teeth was then ranked. results the distribution of the patients according to age, the number of patients with agenesis and the number of teeth missing can be seen in table 1–3. table 1. the distribution of the patients according to age table 2. the number of patients with agenesis table 3. the number of teeth missing the patients under 10 years of age and over 30 years were almost the same in number. the patients between 10 to 19 years of age was 832 (82.2%) and the patients between 20 to 29 years of age was 160 (15.8%). the total number of teeth missing was 455 and upper third molars was the most missing teeth (56,9%). the third mandibular molars was in the second rank, followed by mandibular second premolars. discussion all permanent tooth crowns except the third molars have begun their mineralization by age of six. usually at the age of 8 to 10 years the first sign of the third molar appeared on the radiograph, but occasionally on older age. the formation of dentition continues many years and differences exist in mineralization depending on race, gender, family and individual. therefore, diagnosis of tooth agenesis in permanent dentition should be made after the age of 6 (excluding third molars), and by 10 years of age if third molars were also studied.9 the youngest patient in this study was 8 years of age, and the number of the patients under 10 years were only 8 (0.7%). the distribution of patients by age was dominated by patients whose age between 10 to 19 years and followed by patients of 20 to 29 years of age. all the patients were suitable as sample in this study according to age. in general, the patients in group of 10 to 19 years was the most having benefit of orthodontic treatment. in this survey the third molar was included even though some authors 2–8 excluded third molar in their investigation. the reason for including third molar in this study was the presence of third molar was still in controversy as an etiologic factor of mandibular anterior crowding in later age. as a tendency of human evolution it was believed that most of the people would not have third molar in their dentition. the total number of patients with missing third molars, in a variation from 1 to 4, was 185 (18.2%), smaller than what was reported by da silva.1 unfortunately, most of the study did not include third molars, especially the study with the sample of mongoloid race,6–8 so it was difficult to compare the prevalence of missing molars. the prevalence of patients who has four molars was still much higher than those who did not. it means that the patients who will have the probability of facing sequelae of the third molars were still high. thirty nine (3.8%) patients missed all four molars, a very small portion of patients who would not suffer from negative effects from the presence of third molars. the prevalence of patients with hypodontia was 24 (2.37%) and oligodontia was found in only 1 patient (0.09%). this patient with oligodontia missed 14 teeth (including third molars) in symmetrical order (right and left side). he lost both the maxillary third molars, second molars, second premolars, canines and mandibular third molars, second premolars and lateral incisors. the number of teeth missing was 455 and the most 149rahardjo: prevalence of hypodontia frequent teeth missing were maxillary third molars (259 teeth), followed by mandibular third molars (143). fifteen mandibular second premolars were missing (3.3%), mandibular lateral incisors 13 (2.8%), maxillary lateral incisors 8 (1.7%) and maxillary second premolars 7 (1.5%). of the mesial first permanent teeth, mandibular second premolars were the most frequent missing teeth and this finding was in accordance with other studies.1–6,8 the term hypodontia was most frequently used when describing the phenomenon of congenitally missing teeth in general. many other terms appeared in literature to describe a reduction in number of teeth: oligodontia, anodontia, aplasia of teeth, congenitally missing teeth, absence of teeth, agenesis of teeth and lack of teeth. but two of this terms (oligodontia and anodontia) had a specific meaning; oligodontia was defined as missing a large number of teeth (more than 6 teeth), while anodontia was an extreme condition denoting complete absence of teeth. an author proposed a term advance hypodontia, for agenesis more than 4 teeth excluding third molars.8 even though hypodontia could occur in primary teeth but the term hypodontia was used for permanent dentition in this study. hypodontia could be easily found by examining panoramic or periapical radiograph but panoramic was recommended. the advantage of using panoramic radiograph in assessing agenesis was the broad coverage of the area to be studied. a horizontally maxillary canine located high in palatal vault was invisible in the periapical radiograph, and might be misinterpreted as agenesis of maxillary canine. tooth agenesis in human can be understood as an evolutionary trend, and lef1 is one of the molecules that played a leading role during the evolution of dentition patterning.1 the underlying causes of this phenomenon were mostly unknown. mutation of the msx1 and pax9 gene were identified as the cause of selective tooth agenesis in human, and played a role in early tooth development.9–13 it affected both primary and permanent teeth, but predominantly involving premolars. the hypodontia of mandibular lateral incisors was the second most frequently found in this survey. in case of hypodontia it was some times difficult to clinically differentiate mandibular lateral incisors and the central which was missing. but it was believed and proved in many studies that mandibular lateral incisors was more frequently missing than the central. as a general rule, if only one or a few teeth were missing, the absence of tooth would be the most distal tooth of any given type. if a molar tooth was congenitally missing, it was almost always the third molar; if an incisor was missing, it was nearly always the lateral; if a premolar was missing, it almost always the second rather than the first.14 the occurrence of missing second premolars can cause difficulty in treatment planning of orthodontic problem. if extraction of first premolar was indicated in the treatment plan but the second premolar was congenitally missing there would be difficulty in carrying the treatment on, because the operator should retract more teeth to correct anterior crowding. extracting second premolar would have a risk of loosing of anchorage. the extraction of more distal teeth might alter the treatment planning and modified the mechanotherapy as well. the amount of space available to correct crowding was greater when a first premolar was extracted. if a more distal tooth was selected for extraction, space would inevitably be lost if molars slipped forward rather than canine or premolars moving distally. in this case, a maximum anchorage was needed to prevent anchorage loss and it means a longer treatment time due to preparing the good anchorage before correcting anterior malalignment. another clinical association of missing premolars was infraocclusion of deciduous first molars due to ankylosis.15–17 the exact mechanism for initiation of ankylosis was not known. infraoccluded deciduous molars were believed to remain static while the adjacent teeth move vertically with growth and development of the alveolar process. this left the deciduous molar teeth in a progressively inferior position in relation to the occlusal plane, giving the visual impression that it was submerging. a restorative crown some times needed for this case. the conclusion of this survey is the rank of hypodontia in chinese orthodontic patients is as follows: maxillary third molars, mandibular third molars, mandibular second premolars, mandibular lateral incisors, maxillary lateral incisors and maxillary second premolars. although hypodontia did not represent a public health problem, from orthodontic point of view it might cause esthetic and masticatory function disorders as well as more complex mechanotherapy of a patient. acknowledgements the author was very much obliged to late prof. dr. soekotjo djokosalamoen, msc. drg. sp. ort. whose permission to examine his orthodontic patients’ records made possible this survey. references 1. da silva er, peres rcr, scarel-caminaga rm, deconto f, line srp. absence of mutation in the promoter region of the lef1 gene in patients with hypodontia. braz j oral sci. 2003; 2(4):144–6. 2. nordgarden h, jensen jl, storhaug k. reported prevalence of congenitally missing teeth in two norwegian countries. comm dent health 2002; 19:258–61. 3. steffen w, rohling j, bauss o. prevalence of symptoms of disturbed development of the dentition. j dent oral med 2006; 8(1): 307. 4. backman b, wahlin yb. variation in number and morphology of permanent teeth in 7-year-old swedish children. int j pediatric dent 2000; 10:11–7. 5. thongudomporn u, freer tj. prevalence of dental anomalies in orthodontic patients. aust dent j 1998; 43(6):395–8. 6. lee t, moon h. genetic linkage analysis for the pedigree data of hypodontia of permanent teeth. available at: www.isi.cbs.nl/ iamamember/cdi/abtracts/papers/2182pdf. accessed october 26, 2006. 7. davis pj. hypodontia and hyperdontia of permanent teeth in hong kong schoolchildren. com dent oral epidemiol 1987; 15:218–21. 8. endo t, ozoe r, yoshino s, shimooka s. hypodontia pattern and 150 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 147–150 variation in craniofacial morphology in japanese orthodontic patients. angle orthod 2006; 76(6): 996–1003. 9. arte s. phenotypic and genotypic features of familial hypodontia. academic dissertation, helsinky university 2001. available at: www. ethesis,helsinky.fi/julkaisut/laa/hamma/vk/arte. accessed october 27, 2006. 10. klein ml, nieminen p, lammi l, niebuhr e, kreiborg s. novel mutation of the initiation of pax9 causes oligodontia. j dent res 2005; 84(1):43–7. 11. frazier-bowers sa, pham ky, le ev, cavender ac, kapadia h, king tm, et al. a unique form of hypodontia seen in vietnamese patients: clinical and molecular analysis. j med genet 2003; 40: 79–82. 12. vastardis h, karimbou n, guthua sw, seidman jg, seidman ce. a human msx1 homeodomain missence mutation causes selective tooth agenesis. nat genet 1996; 13:417–21. 13. graber tm, vanarsdall jr. rl, vig kwl. orthodontics, current principles and techniques. 4th ed. st louis missouri, toronto: mosby, an imprint of elsevier; 2005. p. 109. 14. proffit wr, field jr hw. contemporary orthodontics. 3rd ed. mosby, an imprint of elsevier; 2000. p. 118, 228–9. 15. kurol j, thilander b. infraocclusion of primary molars with aplasia of the permanent successor, a longitudinal study. angle orthod 1984; 54:285–94. 16. sidhu hk, ali a. hypodontia, ankylosis and infraocclusion: report of a case restored with a fibre-reinforced ceromeric bridge. br dent j 2001; 191(11):613–6. 17. kokich vg, kokich vo. congenitally missing mandibular second premolars: clinical options. am j orthod dentofac orthop 2006; 130(4): 437–44. isi vol 39 no 3 juli-september 2006.pmd 112 the effect of humidity on peak value of hema carbonyl absorbance band adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya – indonesia abstract bond strength between 2-hydroxyethyl methacrylate hema-based dentin bonding agent and dentin collagen had proved by presence of the chemical interaction. this union involved the carbonyl group on hema resin with amino group on dentin collagen following produce an amide chain. however, this bond strength influence by humidity condition of dentin collagen and hema resin. the aim of this study is to measure the effect of humidity on peak value of the hema carbonyl absorbance band. the bond strength between by hema bonding agent and dentine collagen was analyzed in different humidity, e.g. in 60%, 70%, 80%, and 90% humidity. control experiment was done in ambient room humidity that is 65%. chemical bond that formed between hema and dentine collagen was carried out by mixing pure hema with bovine type-i collagen which had been stored in different humidity. the esther carbonyl group of hema will react with the amino group of collagen, produced an amide bonding, which is observed by measuring the ir spectrum absorbance of the esther carbonyl group of hema in kbr method. the decrease of the carbonyl group absorbance indicates the more chemical bonds formed. by measuring the absorbance of c=o esther in different humidity, it showed that the greatest number of chemical bonds resulted when the experiment was done in 70% humidity condition. key words: humidity, ftir, hema, carbonyl group, type-i dentine collagen correspondence: adioro soetojo, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. dentin collagen network carefully. so we need an optimal condition (optimal humidity) on dentin surface to obtain a maximal bonding between dentin bonding materials and dentin collagen. besnault and attal8 expressing that at 50% and 60% humidity, bond strength of bonding materials at dentine is higher than at 80% and 90% humidity. at the minimum humidity, the amount of water around dentine is optimal. water molecules can form hydrogen bond with collagen peptide, and then break hydrogen bond between molecules of fibril collagen. at drying procedure, fibrils will re-expansion again after collapse. kemp9 reported that hema carbonyl when reacting with amino collagen group, peak reduction of the mixture esther carbonyl and hema with collagen will happened at infra red spectrum. it can be assumed that there are many groups of carbonyl esther bonding with amino if there is a high peak reduction of hema carbonyl absorbance band. this means chemical bond between hema and collagen become stronger. the purpose of this research is to know the effect of humidity on peak value of the hema carbonyl absorbance band. in knowing, the smallest value of hema carbonyl absorbance band will also know the chemical bond strength between hema and dentine collagen maximally at optimal humidity. introduction the 2-hydroxyethyl methacrylate (hema) is often used as liquid base of dentine bonding.1,2,3 several reports claims that hema has a good physical and chemical characteristic.4-7 the 2-hydroxyethyl methacrylate bind to dentine surface by chemical or mechanical bond. chemical bond of hema to dentine collagen is covalent bond or primary bond between atoms. covalent bond between nitrogen atom of collagen and carbon atom of resin aldehyde will form in chemical interaction.3 xu et al.4 reported that by f-t raman spectroscopy, if there are new bonding, for example c-n of c=o at esther group of hema, hence will yield product of amide [c(o)nh]. clinically, one of total etched technique step is wash and dries the dentin surface. the purpose of washing step is to discard salt formed as reaction of acid etched materials and dentin minerals. the drying procedure will remove excess water that are used for washing dentin surface after etching process.1,2,3 many researchers said that at the time of drying procedure, the dentin surface should neither too dry nor wet.4,7 if the surface is too dry, the dentin collagen will collapse. however, if the surface is too wet, there are many water molecules around collagen hence yield hydrogen bond between water and amino collagen. with this phenomenon, dentin bonding agents will not bond to 113soetojo: the effect of humidity on peak value of hema carbonyl absorbance band materials and methods this research is an experimental laboratory research.10,11 bovine incisive were obtained from animal slaughtering house at jl. pegirian surabaya. after cleaning, tooth was soaked in physiological salt solution and kept in refrigerator (temperature 40 c). pure bovine type i collagen and hema pure liquid were purchased from sigma chemical, st. louis, usa. appliances that were used: desiccators with vacuum faucet (china), hygrometer (haar. synth. hygro, germany), air suction (schuco, usa), and ftir (jasco ft / ir 5300, japan). to arrange 90 % humidity, 150 cc of water were putted at the bottom part of desiccators then put hygrometer that have been calibrated. at this condition humidity which seen at hygrometer was range from 94 to 95%. hereafter from faucet located above desiccators, air pumped out by air suction until reach humidity 90% and closed the faucet. for humidity 80%, air inside the desiccators were pumped out until reach humidity 80%. to quicken the process, put silica gel that activated before in the desiccators. same process was done for 70 % and 60 % humidity. to make kbr pellets, 300 mg kbr powder (bromide kalium) was prepared, then 10 μl (10,7 mg weight) of hema (1,07 g/ml) were dropped using micro-pipette. kbr powder and hema (310,7 mg ) were crushed with mortal and pestle. some part of this mixture (50 mg) were put into kbr die appliance then were compressed and vacuuming by compressor until 10 ton. this vacuuming and compressing process was done in 5-10 minutes. the obtained result was a transparent pellet (form like thin tablet). the pellets were observed into fourier transform infrared spectroscopy (ftir) appliance. these samples were control samples. room humidity was 65% and room temperature was 250 c ± 20 c. to make a mixture of collagen sample and hema, 10 μl (10,7 mg) hema was dropped to collagen (2 mg), then kbr powder were added till the weight of the mixtures were 310,7 mg. the mixture of this materials were crushed, 50 mg of them were put into kbr die, then compressed and vacuumed until 10 ton. the pellets were observed into ftir appliance. to make sample for the group of treatment by different humidity, 2 mg collagen fibers were packed into desiccators with hygrometer appliance that have been calibrated. then desiccator’s humidity was arranged to 60%, 70%, 80% and 90%. as soon as released from desiccators, hema and kbr powder were added on collagen until 310,7 mg in weight. the mixtures were crushed, 50 mg of them were, compressed and vacuumed until 10 ton and pellets were made in the same method, which has been explained above. in this study, each sample group was conducted until three times research (n = 3 to each group). the measuring was conducted at peak value of carbonyl absorbance band (c=o) of treatment group which was compared to control group. the method to calculate peak value of carbonyl absorbance band (p) as follows:9 figure 2 and 3 shows peak value of carbonyl absorbance band at various humidity: figure 2. infrared spectrum of pure hema (kbr pellet) peak value of carbonyl absorbance band is 46.1 (number 11) wave number (c=o) at 1720 cm-1, t value: ± 80%. 0.00 4.000.0 3.000.0 2.000.0 wave number 1.000.0 100.00 %t 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 h.3 figure 1. peak of the hema carbonyl absorbance band. description: p = (bc / ab) x 100 ; ab, bc measured in centimeters. this calculation is accurate enough and can be trusted if absorbance band intensity at t: 30-60%. transmittance = t (%) 100 0 a b c absorbance band c = 0 (carbonyl wave number) figure 3. infrared spectrum of hema + collagen (kbr pellet) peak value of carbonyl absorbance band is 9.8 (number 9) wave number (c=o) at 1720 cm-1, t value: ± 50% 4.000.0 3.000.0 2.000.0 wave number 1.000.0 0.00 100.00 %t h.2 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 9 114 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:112–115 results principally, the peak value of hema carbonyl absorbance band will decreased if many groups of hema carbonyl bind with groups of amino at dentin collagen,. this matter designate that many groups of carbonyl are bonding with groups of amino, thus it means chemical bond between hema and collagen is strong. mean value of the hema carbonyl absorbance band and standard deviation were shown at table 1. hema and group at 70%, 80%, 90%, and 65%. there was no significant difference between group at 60% and 65%, and 80% humidity (p > 0.05). there was a significant difference between group at 70% humidity and sample group of 60%, 80%, and 90% humidity (p < 0.05), but if compared with group at 65% humidity (control), there was no significant difference. furthermore, there was a significant difference between group at 80% humidity and 70% humidity, but there was no difference significance between group at 80% humidity and group of humidity 60%, 90%, and 65% (control). group at 90% humidity showed a significant difference if compared with group at 60% and 65%. discussion some expert expressed that chemical interactions between hema and dentine collagen is through reaction between hema carbonyl group and amino dentine collagen group. bonding between amino collagen (nh2) group and hema carbonyl group is only some of all chemical bond that possibly happened. this reaction form the new formation that are an amide bonding and also the side effect in the form of etilen-glikol.4 renzo and ellis12 stated chemical bond between dentin and hema happened as result of reaction between resin and collagen function group, for example carboxylate, hydroxyl, amine group, or amide. could also happened complex reaction with ion of ca++ of hydroxy-apatite. wang and spencer13 that investigating chemical interaction between hema and bis-gma at dentin surface, obtained that penetrating of hema into dentin surface which forming hybrid layer can reach until 10 μm. at this depth, penetration of hema is decreased until remain 8.85%. they used spectroscopy of micro-raman and absorbance band which are observed was carbonyl absorbance band (1720 cm–1), c=c phenyl (1609 cm–1), group of ch2 (1453 cm–1) and others. bonding of hema resin at dentine collagen also depend on to the number of hydrogen bonds.6 if there are any resistance of carboxylic acid dissociation or amino collagen group hence will improve hydrogen bond between hema resin and dentine collagen. previously, they studied the absorption of hema resin at dentine collagen characteristically by c-nmr.12 the reduction of t1 value of hema carbonyl esther carbon is bigger than at other carbon, which estimated will form hydrogen bond between hema carbonyl ester and dentine collagen. the amount of hema concentrations in solution and hema adsorption into collagen has correlation with tensile bond strength of resin at dentine. in present chemically research, pure hema is used to directly knowing the role of hema to chemical bond with pure collagen. mean average peak value of pure hema carbonyl absorbance band was equal to 47.5 ± 5.6. thus at 60%, 65%, and 70% humidity, the peak value decreased table 1. mean value of peak of hema carbonyl absorbance band at various humidity pure hema have peak carbonyl absorbance band equal to 47.5 + 5.6, then the peak value decreased at 60%, 65%, and 70% humidity, the value was 21.2 + 8.8; 19.7 + 6.6 and 9.5 + 2.9 respectively. this result mean at the lowest humidity, the bonding between hema carbonyl group and amino collagen group will increase. in the other hand, bonding between hema carbonyl group and amino collagen decreased at 80% and 90% humidity, where the peak of absorbance band was at 29.0 ± 6.3 and 32.3 ± 3.2. to know that the measurement data chemically is normal, normal distribution test was done. p value of entire group treatment of 60–90% humidity shows the higher number than 0.05 (p > 0.05). this means group data attempt of chemical bond at 60–90% humidity is normal distribution. next, homogeneity test was conducted to prove whether the treatment chemical sample group is homogenous, hence from calculation obtained by value of p = 0.460 (p > 0.05) meaning chemical sample group at 60–90% humidity was homogeneous. by anova test, effect of 60–90% humidity to chemical bond between hema and dentine collagen shows the significant difference for each sample group (p < 0.05). the significance of each chemical sample group at 60–90% humidity was calculated with lsd test. the result has shown a significant difference between peak value of pure hema carbonyl absorbance band and hema carbonyl absorbance band at 60% humidity (p < 0.05). there is a significant difference between pure humidity n x (mean) sd hema 60% 70% 80% 90% 65% 3 3 3 3 3 3 47.5667a 21.2000b 9.5000c 29.0000bdf 32.2667de 19.7333bcdf 5.6448 8.8606 2.9614 6.3553 3.2083 6.6214 description: n = amount of sample x = mean value of peak hema carbonyl absorbance band sd = standard deviation anova f = 14.257, p > 0.001 superscript with different letter showed there significances at = 0.05 115soetojo: the effect of humidity on peak value of hema carbonyl absorbance band significantly to 21.2 ± 8.8, 19.7 ± 6.6, and 9.5 ± 2.9. it means that the amount of hema carbonyl group, which is bonding with amino collagen group, is increase at 60%, 65%, and 70% humidity. the tensile bond strength of hema with collagen also increase and the highest tensile bond strength obtained at 70 % humidity. at 70% humidity the water condition around fibril is very optimal in causing chemical interaction between hema and collagen. water could make collagen to re-expansion, thus the fibril are more active and permeable to hema resin. at 80% and 90% humidity, the chemical bond between hema resin and collagen was decreased significantly (peak value of carbonyl absorbance band at 80 % humidity: 29.0 ± 6.3; 90 % humidity: 32.3 ± 3.2). the increasing amount of water molecules around collagen will block the penetration of hema materials to collagen fibril. the increasing of water amount around collagens will also increase hydrogen bonding between water and hema carbonyl group. the increase of this hydrogen bonding will block the bonding between hema and collagen, so it will decrease the amount of hema carbonyl group which is bonding to amino collagen and also decreasing the tensile bond strength. this situation is according to statement of nakabayashi and pashley.7 the difficulty to obtain good sample was also explained by wang and spencer.13 they said that absolute intensity of raman absorbance band depend on some factors: softness of sample surface, focusing position, detection depth, fluorescence biological component, equipments stability, and the power of laser-ray. for each sample, the result of absorbance band intensity will differ at the time of measurement, even for the same area of spot at one sample. this research demonstrated that 70% humidity is the most optimal humidity to get maximal chemical bond between hema carbonyl group and dentine collagen amino. references 1. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 2. noort rv. introduction to dental materials. 2nd ed. edinburgh, london, new york, oxford: cv mosby co; 2002. p. 11–78. 3. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia, london, toronto: wb saunders co; 2003. p. 21–395. 4. xu j, stangel i, butler is, gilson dfr. an ft raman spectroscopy investigation of dentin and collagen surfaces modified by hema. j dent res 1997; 76:596–601. 5. finger wj, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4:277–82. 6. nishiyama n, suzuki k, nagatsuka a, nemoto k. dissociation states of collagen functional groups and their effects on priming efficacy of hema bonded to collagen. j dent res 2003; 82:257–61. 7. nakabayashi np, pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 8. besnault c, attal jp. influence of a simulated oral environmental on dentin bond strength of two adhesive systems. am j dent 2001; 14:367–72. 9. kemp w. organic spectroscopy. 2nd ed. edinburgh: elbs/macmillan ltd; 1988. p. 12–81. 10. pratiknya aw. metodologi penelitian kedokteran dan kesehatan. edisi ke-4. jakarta: pt raja–gratindo persada; 2001. h. 19–174. 11. notoatmodjo s. metodologi penelitian kesehatan. edisi ke-2. jakarta: pt rineka cipta; 2002. h. 36–202. 12. renzo md, ellis th. chemical reactions between dentin and bonding agents. j adhes dent 1994; 47:115–21. 13. wang y, spencer p. hybridization efficiency of the adhesive/dentin interface with wet bonding. j dent res 2003; 82:141–45. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 162 volume 46, number 3, september 2013 restorasi mahkota logam dengan pasak fiber komposit pada molar permanen muda (metal crown restoration with fiber composite post in young permanent molar) theresia dhearine pratiwi dan mochamad fahlevi rizal departemen ilmu kedokteran gigi anak fakultas kedokteran gigi, universitas indonesia jakarta – indonesia abstract background: the first permanent molar has a high prevalence of caries with the most rapid progression in the first two years after eruption. the destruction can extend to the pulp and require endodontic treatment. after endodontic treatment the teeth should have a final restoration due to the possibilities of fracture. the teeth with a few remaining tissue need a restoration such as crown with post and core support. fiber composite post is widely used today because it has a similar modulus elasticity as dentin. purpose: the case report was aimed to share the endodontic treatment which was followed by fiber composite post and metal crown insertion on young permanent molar. case: an 11 years old girls was referred to pediatric dentistry clinic at universitas indonesia dental hospital due to caries #36 that extend to the pulp. case management: endodontic treatment with metal crown supported by fiber composite post and composite core was done as final restoration. one month after procedure there was no subjective complaints or inflammation. conclusion: the case report showed that endodontic treatment followed by fiber composite post and metal crown insertion could be done succesfully on young permanent molar of 11 years old patient. key words: fiber composite post, metal crown, young permanent molar abstrak latar belakang: gigi molar pertama permanen muda (m1) merupakan gigi dengan angka kejadian karies yang tinggi dengan kerusakan paling cepat terjadi pada dua tahun pertama setelah gigi tersebut erupsi. kerusakan tersebut dapat mencapai pulpa sehingga diperlukan perawatan endodontik. gigi yang sudah dirawat memerlukan restorasi akhir yang baik, karena kemungkinan terjadi fraktur. sisa jaringan gigi yang sedikit membutuhkan restorasi akhir berupa mahkota tiruan dengan dukungan pasak dan inti. pasak fiber komposit merupakan pasak yang saat ini sering digunakan karena memiliki keunggulan modulus elastisitas yang menyerupai dentin. tujuan: tujuan penulisan laporan kasus ini adalah untuk melaporkan perawatan endodontik yang diikuti dengan pemasangan pasak fiber komposit dan mahkota logam pada molar pertama permanen muda. kasus: anak perempuan usia 11 tahun dirujuk ke klinik kedokteran gigi anak rumah sakit gigi dan mulut universitas indonesia dengan kerusakan gigi #36 mencapai pulpa. tatalaksana kasus: perawatan endodontik dengan restorasi akhir mahkota tiruan tuang logam dengan dukungan pasak fiber komposit dan inti resin komposit. pada kontrol setelah 1 bulan tidak didapatkan keluhan subjektif serta kondisi peradangan simpulan: laporan kasus ini menunjukkan bahwa perawatan endodontik yang diikuti dengan pemasangan pasak fiber komposit dan mahkota logam dapat dilakukan dengan baik pada molar pertama permanen muda dari pasien berusia 11 tahun. kata kunci: pasak fiber komposit, mahkota logam, molar permanen muda korespondensi (correspondence): theresia dhearine pratiwi, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta 10430, indonesia. e-mail: dhearine@yahoo.com case report 163pratiwi dan rizal: restorasi mahkota logam dengan pasak fiber komposit pendahuluan tingkat kejadian karies pada anak dan remaja sering terjadi pada gigi molar pertama tetap (m1). di amerika, asia, dan brazil angka kejadian karies pada gigi m1 dapat mencapai 40-50% dari populasi yang ada, dengan adanya kavitas pada gigi, restorasi, atau tanggalnya gigi m1. perkembangan karies pada gigi m1 paling cepat terjadi pada dua tahun pertama setelah gigi m1 erupsi dengan adanya kemungkinan gigi tersebut mengalami kerusakan hingga mencapai pulpa.1 perawatan yang harus dilakukan adalah perawatan endodontik dengan memperhatikan beberapa faktor seperti sisa jaringan mahkota, vitalitas pulpa, penutupan daerah apikal, dan tingkat kepatuhan pasien.2 gigi yang telah dirawat endodontik membutuhkan suatu restorasi akhir yang baik agar dapat berfungsi optimal dalam rongga mulut. gigi tersebut lebih rapuh daripada gigi vital sehingga meningkatkan kemungkinan fraktur selama berfungsi.3,4 pasak merupakan retensi tambahan yang diletakkan di dalam saluran akar gigi yang telah dirawat saluran akar. pasak bertujuan sebagai pemegang inti dan mahkota serta membantu melindungi penutupan apikal dari kontaminasi bakteri yang disebabkan karena kebocoran mahkota.5 fungsi dari pasak dan inti adalah untuk meningkatkan daya tahan struktur jaringan gigi terhadap tekanan lateral dan mendistribusikan ke seluruh jaringan gigi yang ada.3 beberapa syarat konstruksi inti dan pasak adalah panjang pasak yang harus seimbang atau panjangnya sesuai perkiraan mahkota klinis idealnya 2/3 panjang akar. memiliki apikal stop untuk mencegah pergerakan ke arah apikal, konstruksi yang dapat menahan gaya normal dalam mulut, ketebalan yang cukup untuk mencegah pergerakan dan stabil, sisa gutta-percha yang tertinggal minimal 3-5 mm. diameter pasak sebaiknya tidak lebih dari 1/3 diameter akar, dengan ketebalan minimum 1 mm.3 pada tahun 1990 pasak fiber mulai sering digunakan. pasak fiber mengandung kumpulan serat-serat karbon yang dikelilingi matriks resin, sehingga pasak menjadi kuat, tetapi kurang kaku dan kuat jika dibandingkan dengan pasak keramik dan metal.3 modulus elastisitas menyerupai dentin berkisar antara 13-47 gpa sehingga dapat mencegah terjadinya fraktur akar.3,4,6,7 berbeda dengan pasak logam yang isotropik di mana modulus elastisitasnya sama pada semua sudut, sedangkan pasak fiber bersifat anisotropik yaitu modulus elastiknya menurun dari 0 sampai 90 derajat.7 pasak fiber memiliki nilai estetik yang baik karena sewarna dengan gigi atau bersifat translusen, sehingga tidak membutuhkan opaquer dan cocok untuk semua bahan restorasi termasuk mahkota komposit dan all-ceramic. biokompatibilitas baik, tidak toksik, tidak korosif, tidak galvanic, tidak terlalu invasif dibandingkan pasak logam, mudah diperbaiki atau dikeluarkan, dan tidak memberi efek sensitif terhadap gigi. apabila dilakukan sementasi dengan baik, maka dapat mencegah terjadinya kebocoran mikro.3,4 penggunaan pasak fiber diperlukan pembuatan inti untuk meggantikan struktur mahkota yang hilang dan dikombinasikan dengan sisa jaringan mahkota. inti pada pasak prefabricated terkadang sudah menjadi satu dengan pasak, namun ada juga beberapa pasak yang memerlukan pembuatan inti sebelum pembuatan mahkota tiruan.3 bahan yang dapat digunakan untuk mahkota tiruan antara lain mahkota logam, metal porselen, atau, full porselen. pada gigi molar tetap muda dengan masih mengalami pertumbuhan erupsi dan perubahan oklusi, mahkota logam stainless steel dapat dipilih sebagai restorasi sementara hingga gigi mendapat restorasi permanen.10 namun, pada kondisi yang sudah stabil, mahkota tiruan dapat menggunakan mahkota tiruan tuang logam, mahkota tiruan metal porselen, mahkota tiruan seluruhnya porselen, mahkota tiruan metal akrilik, dan mahkota tiruan seluruhnya akrilik. pembuatan mahkota tiruan logam dari base metal alloy atau high noble metal alloy dapat digunakan karena memiliki beberapa keuntungan seperti retensi dan resistensi yang baik serta kekuatan yang baik terutama di daerah posterior.3,5 namun kerugiannya antara lain secara estetik tidak menguntungkan, test vitalitas yang sulit dilakukan pada gigi yang vital, dan pembuangan struktur gigi yang cukup besar.3 tujuan penulisan laporan kasus ini adalah untuk melaporkan perawatan endodontik yang diikuti dengan pemasangan pasak fiber komposit dan mahkota logam pada molar pertama permanen muda. kasus pasien anak perempuan berusia 11 tahun datang ke klinik kedokteran gigi anak rumah sakit gigi dan mulut universitas indonesia dengan keluhan gigi molar pertama kiri bawah berlubang, keadaan umum baik. hasil pemeriksaan pada ekstra oral tidak ditemukan asimetris wajah dan kelenjar getah bening submandibularis kiri teraba, keras, dan sakit dan sebelah kanan teraba, lunak, dan tidak sakit. pemeriksaan intraoral, gingiva regio #36 terdapat kemerahan dan oedem. hubungan vertikal molar satu permanen kiri dan kanan kelas i, susunan gigi berjejal, dan terdapat gigitan silang pada anterior antara gigi #11 dengan #41 dan #42. pada pemeriksaan gigi geligi ditemukan gigi #36 karies mencapai pulpa (kmp), test perkusi menunjukkan adanya keluhan sedang pada test tekanan tidak ada keluhan (gambar 1). pada gigi m1 tetap lain mengalami karies enamel baik pada gigi #16, #26, dan #46. pemeriksaan radiografik pada gigi #36 menunjukkan radiolusensi di oklusal mencapai kamar pulpa, radiolusensi di periapikal baik mesial dan distal, dan pembentukkan akar yang sudah sempurna. diagnosis gigi #36 adalah nekrosis pula dengan abses periapikal. rencana perawatan untuk kasus ini adalah perawatan saluran akar dengan restorasi akhir mahkota tiruan dengan pasak (dowel crown). 164 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 162–166 tatalaksana kasus kunjungan pertama dilakukan ekskavasi karies pada gigi #36 dan ditumpat sementara. kunjungan kedua dilakukan open akses pada gigi #36 lalu penjajakan saluran akar menggunakan k-file #15. preparasi dilakukan dengan protaper sampai ukuran f2 sesuai panjang kerja dengan irigasi naocl konsentrasi 2,5% setiap kali dilakukan penggantian alat lalu dikeringkan dengan paper point. setelah itu, dilakukan sterilisasi dengan medikamen chkm dan ditumpat sementara. kunjungan berikutnya sudah tidak ditemukan keluhan subyektif dan dari pemeriksaan obyektif edema sudah tidak ada dan perkusi serta palpasi tidak ada keluhan dan kondisi gigi tertinggal dua dinding saja. kemudian dilakukan pengisian saluran akar dengan gutta percha protaper serta sealer endomethasone sesuai dengan panjang kerja sebelumnya, diberi basis gic dan ditumpat sementara (gambar 2). setelah satu minggu pengisian saluran akar dilakukan pemeriksaan gigi #36, perkusi, palpasi, keluhan subjektif, serta fistula pada gigi #36 dan tidak didapatkan keluhan. setelah itu, dilakukan pengambilan guttap sepanjang 2/3 saluran akar distobukal dan mesiolingual menggunakan gates gliden drill (ggd) mulai dari ukuran terkecil hingga saluran akar bersih. kemudian dilakukan sementasi pasak fiber ukuran 1.25 dengan menggunakan semen resin dual cured. ukuran disesuaikan dari foto radiograf pengisian yang ada pada akar distobukal dan mesiolingual dan dilakukan pembuatan inti dengan resin komposit (gambar 3a dan 3b). setelah itu, dilakukan preparasi metal crown dan pencetakan dengan rubber base. kemudian dilakukan sementasi mahkota logam dengan gic luting. pada kontrol 1 bulan setelah sementasi mahkota logam didapatkan hasil pemeriksaan perkusi dan palpasi tidak ada keluhan, tidak ada hiperemi dan edema, serta tidak ada keluhan subyektif. hasil kontrol melalui foto radiograf didapatkan perbaikan lesi periapikal pada akar distal dan mesial namun penyembuhan di periapikal akar mesial masih belum sempurna meski sudah mulai terjadi penulangan (gambar 4a dan 4b). gambar 1. kondisi klinis awal rongga mulut dengan gigi #36 karies mencapai pulpa. gambar 2. foto pengisian saluran akar gigi #36. gambar 3. pemasangan pasak fiber. a) kondisi klinis saat pemasangan pasak fiber pada saluran akar mesiolingual dan distobukal; b) radiografis pasak fiber dalam saluran akar. a b 165pratiwi dan rizal: restorasi mahkota logam dengan pasak fiber komposit pembahasan gigi m1 merupakan gigi yang paling sering mengalami karies pada anak, terutama m1 rahang bawah.10 pasien ini berusia 11 tahun yang merupakan usia sekolah dan gigi m1 erupsi dengan pembentukkan akar yang sudah sempurna. hal ini sesuai dengan penelitian ahmed dkk.,10 di mana pada pasien ini didapatkan karies pada seluruh gigi m1 dengan kondisi terparah terjadi pada gigi m1 kiri bawah dengan karies yang sudah mencapai pulpa. gigi #36 pada pasien ini mengalami karies hingga kondisi pulpa yang sudah terbuka dan sudah nekrosis. pada kasus kerusakan gigi m1 dapat dilakukan perawatan endodontik atau ekstraksi. pada keadaan gigi #36 ini, sisa gigi yang tersisa masih dapat dilakukan perawatan endodontik. selain itu, kondisi gigi #37 dari foto rontgen terlihat sudah menembus tulang dan pembentukkan akar mencapai 1/3 tengah akar. oleh karena itu, diperlukan perawatan endodontik dan restorasi pasca endodontik yang sesuai untuk dapat menjaga ruangan yang ada dan mempertahankan gigi di dalam lengkung rahang karena hilangnya gigi m1 dapat berakibat masalah lain yang lebih kompleks seperti hilangnya kunci oklusi, migrasi gigi tetangga, ekstrusi gigi lawan dan sebagainya, kecuali jika pasien yang menginginkan dilakukan ekstraksi.11 rencana perawatan pada gigi #36 ini adalah perawatan endodontik dengan restorasi akhir berupa mahkota tiruan dengan pasak dan inti karena sisa gigi yang ada hanya dua dinding mahkota yaitu bukal dan mesial. hal ini sesuai dengan pernyataan bahwa di mana indikasi dari pembuatan mahkota tiruan dengan pasak dan inti jika kerusakan mahkota cukup luas kurang dari tiga dinding.6 pembuatan restorasi mahkota tiruan pasak dan inti diawali dengan pengambilan 2/3 bahan pengisi untuk penempatan pasak. jenis pasak yang digunakan pada pasien ini adalah pasak prefabricated dengan bahan fiber komposit. pasak ini dipilih karena pasak ini cukup kuat meskipun tidak sekuat pasak keramik atau logam, tetapi pasak ini memiliki ketahanan terhadap fraktur karena modulus elastisitasnya yang sama dengan dentin.3 sebuah penelitian juga menyatakan keuntungan lain dari pasak prefabricated adalah lebih efisien secara waktu dan memberikan hasil yang cukup memuaskan, dibandingkan dengan pasak custom made yang memiliki kerugian utama berupa tingginya resiko terjadinya fraktur akar.2,12 posisi gigi yang berada di posterior juga menjadi salah satu pertimbangan karena adanya beban kunyah yang besar di daerah posterior. pengambilan isi gutta point dilakukan dengan menggunakan gates gliden drill. pengambilan gutta percha dapat menggunakan endodontik plugger yang dipanaskan atau dengan menggunakan rotary instrument seperti gates gliden drill. pengambilan ini dilakukan perlahan sehingga meninggalkan bahan pengisi sepanjang 4 mm dari periapikal.3 pada kasus ini, dua saluran akar dipakai untuk penggunaan pasak yaitu akar distobukal dan mesiolingual. pada gigi berakar ganda (lebih dari 1 saluran akar) diperlukan retensi pasak lebih dari 1 saluran akar. saluran akar yang dipilih untuk peletakkan pasak adalah saluran akar terbesar, dimana pada gigi m1 saluran akar terbesar terdapat pada saluran akar distal, sedangkan dua saluran akar diletakkan pasak agar distribusi gaya yang terjadi pada gigi tersebut dapat semakin merata.3 sementasi pasak fiber komposit dilakukan dengan semen resin dual cured untuk memastikan terjadinya polimerisasi yang sempurna. adanya photo initiator dan chemical initiator pada semen resin dual cured sehingga polimerisasi dapat terjadi dengan sempurna dan menghindari kebocoran mikro yang dapat menyebabkan inflamasi berulang.8 pasak fiber terletak pasif dalam saluran akar dan luting agent berbahan dasar resin (resin semen) merupakan bahan yang diindikasikan untuk menambah retensi.4,8,9 hal yang perlu diperhatikan ketika melakukan sementasi pasak adalah memastikan semen pengisian saluran akar tidak tertinggal dalam saluran akar karena dapat mengakibatkan terjadi kebocoron mikro.4 a b c gambar 4. gambaran klinis dan radiograf setelah 1 bulan pasca pemasangan restorasi pasak fiber dengan mahkota logam. a) mahkota logam pada gigi #36 dalam rongga mulut; b) radiograf awal gigi #36 sebelum perawatan c) radiograf setelah 1 bulan paska insersi mahkota logam, terlihat terjadi penyembuhan lesi periapikal. 166 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 162–166 pembuatan inti pada pasien ini menggunakan bahan resin komposit sebelum dilakukan pencetakkan untuk pembuatan mahkota tiruan. hal ini dilakukan karena cukup menguntungkan yaitu ikatan yang baik dengan pasak fiber, jumlah kunjungan yang lebih sedikit, prosedur laboratorium yang lebih sedikit, dapat mempertahankan sisa jaringan gigi, dan cukup kuat karena dapat beradaptasi dengan sisa jaringan gigi.3 restorasi akhir yang dipilih adalah restorasi tuang logam. restorasi ini dapat menjadi restorasi akhir untuk gigi tersebut karena gigi tersebut sudah gigi tetap, sudah dirawat saluran akar dan sudah diberi pasak, usia pasien 11 tahun, dan pembentukkan akar yang sudah sempurna. ada beberapa alternatif mahkota tiruan yang sesuai untuk gigi tersebut yaitu mahkota tiruan metal porselen dan mahkota tiruan logam (all metal). restorasi mahkota logam dipilih karena kuat menahan beban kunyah pada daerah posterior, membentuk kembali anatomi gigi, dan mempertahankan gigi dalam lengkung rahang.3 pada kasus ini jarak oklusogingival rendah karena itu digunakan mahkota tiruan logam tuang.5 posisi gigi yang berada di posterior juga tidak memerlukan sifat estetik seperti gigi anterior. dari segi biaya, mahkota tiruan all metal lebih terjangkau dibandingkan dengan mahktoa tiruan metal porselen, sehingga pasien memilih jenis restorasi ini. pada pasien ini kondisi oklusi pasien daerah posterior juga berat sehingga mahkota logam dipilih menjadi restorasi akhir. laporan kasus ini menunjukkan bahwa perawatan endodontik yang diikuti dengan pemasangan pasak fiber komposit dan mahkota logam dapat dilakukan dengan baik pada molar pertama permanen muda dari pasien berusia 11 tahun. daftar pustaka 1. chen jw, varner ll. pulp treatment for young first permanent molars: to treat or to extract. endodontic topics 2012; 23: 34-40. 2. raducanu am, victor f, claudiu h, mihai ar. prevalence of loss of permanent first molars in a group of romanian children and adolescents. ohdmbsc 2009; 3: 3-11. 3. rosenstiel sf, land mf, fujimoto j. restoration of the endodontically treated tooth. contemporary fixed prosthodontics. 4th ed. china: mosby elsevier; 2006. p. 336-74. 4. schwart rs, robbins jw. post placement and restoration of endodontically treated teeth: a literature review. j endod 2004; 30(5): 289-301. 5. cheung dmd. a review management of endodontocally treated teeth post, core and final restoration. j am dent assoc 2006; 136(5): 611-9. 6. cohen s, hargreaves km. pathways of the pulp. 9th ed. st louis, missouri: mosby inc; 2006. p. 786-810. 7. hicks n. esthetic fiber reinforced composite posts. smile j 2008; issue 9: 1-4. 8. goracci c, corciolani g, vichi a, ferrari m. light-transmitting ability of marketed fiber posts. j dent res 2008; 87: 1122. 9. teixeira ec, teixeira fb, piasick r, thompson jy. an in vitro assessment of prefabricated fiber post systems. j am dent assoc 2006; 137: 1006-12. 10. ahmed na, astram an, skaug n, petersen pe. dental caries prevalence and risk factors among 12-year old schoolchildren from baghdad, iraq: a post-war survey. int dent j 2007; 57(1): 36-44. 11. carrotte p. endodontic treatment for children. british dent j 2005; 198: 9-15. 12. kaur j, verma pr, archana n. fracture resistance of endodontically treated teeth restored with different post systems: a comparative study. indian j dent sci 2011; 3: 5-9. 2323 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 23–27 socioeconomic characteristics of the parents and the risk prediction of early childhood caries wahyu aji wibowo,1 retno indrawati,1 and retno pudji rahayu2 1department of oral biology 2department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: the high prevalence of early childhood caries still becomes a major health problem. it is because children prefer to consume sweet foods, which are also cariogenic. the oral mucosa is a mirror of general health or any systemic disease which usually shows visible symptoms in the oral cavity. dental caries in children is often related to the socioeconomic characteristics of the parents. some references suggest that there is a relationship between the socioeconomic status with the incidence of caries. purpose: the purpose of this study was to examine the relationship of socioeconomic characteristics of the parents with the risk prediction of early childhood caries by using cariogram approach to the elementary school students in wonosobo. method: the research is an observational analytic study with cross-sectional approach survey method. the research sampling uses proportional random sampling with 201 respondents of fifth graders. the socioeconomic status is measured through questionnaires, while the risk prediction of early childhood caries is measured by using cariogram. the research data analysis uses spearman rank. result: the results of the study show that the socioeconomic characteristics of the respondents’ parents mostly belong in the middle category as many as 145 respondents (72.1%), lower category as many as 31 respondents (15.4%) and in the upper category as many as 25 respondents (12.4%). prediction of the respondents avoiding early childhood caries is 55%, while the prediction of the respondents being risked of having early childhood caries is 45%. the results of data analysis show that the relationship between the socioeconomic characteristics of the respondents’ parents and the risk of having early childhood caries for the respondents is p<0.05. conclusion: there is a relationship between the socioeconomic characteristics of the respondents’ parents with the early childhood caries, which shows that the higher the socioeconomic status of the parents, the lower the risk of the respondents from having early childhood caries. keywords: socioeconomic; the risk prediction of early childhood caries; cariogram correspondence: retno indrawati, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: retno_in2007@yahoo.co.id. introduction dental caries is caused by the demineralization of enamel and dentin, which is highly related to the excessive consumption of cariogenic foods and the bacterial activity of mutant streptococcus.1 the high prevalence of early childhood caries still becomes a major health problem. the world health organization (who) has determined that the prevalence of early childhood caries is 60-90%. the data from household health survey (skrt / survey kesehatan rumah tangga) in 2007 states that the prevalence of dental caries is 76.92%. basic health research (riskesdas / riset kesehatan dasar) in 2007 by the ministry of health republic of indonesia states that the dmf-t score in indonesia shows four dental caries, with the prevalence of active caries in central java is 43.1% and the incidence of dental caries is 67.8%. the average index of dmft in wonosobo is 6.5, with the prevalence of active caries in is 44.9% and the incidence of dental caries is 78.6%.2 dental caries is a common case in children because they prefer to consume sweet foods, which are also cariogenic. oral cavity is an important part of the body and is a mirror dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p23-27 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p23-27 24 wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 23–27 of general health or any systemic disease which usually shows visible symptoms in the oral cavity. children tend to have early childhood because of they are likely to have bad oral hygiene of children and they also prefer to eat sweet foods.3 generally, school-age children have a high-risk of early childhood caries, because the habit of consuming snack foods and drinks usually grow the highest around this age. the five graders are selected because their average age are 10-13 years old when they start to have the habit of consuming sweet foods and drinks, that are also cariogenic, both at school and at home.1 there should be any precaution taken since the beginning to minimize the cost of their healthcare treatment. one of the precaution strategies of early childhood caries is by identifying the risk factors. it should be necessary to acknowledge the level of risk factors of early childhood caries and distinguish the precaution strategies.4 some references state that there is a relationship between socioeconomic status with incidence of early childhood caries, one of them is the study by tulangow.5 the result of that study shows that children coming from the lower socioeconomic status have the higher index of dmf-t, compared to children from the higher socioeconomic status. cariogram is a computer application for predicting the incidence of dental caries and describing the relationship between the interrelated risk factors of dental caries. there has been developed the education program on cariogram to achieve the better understanding on the multifactorial aspects of dental caries and to estimate the risk factors of dental caries. this program can be used in the clinic or a variety of educational purposes. the computer application for cariogram has several advantages, such as to give recommendations for preventive care and to encourage the motivation of the patient. there has been a research using the computer application for cariogram conducted in wonosobo. this study aims to examine the relationship between the socioeconomic characteristics of the respondents’ parents with the risk prediction of early childhood caries of the elementary school students in wonosobo and the use of cariogram to find out the order of risk prediction of early childhood caries in wonosobo.6 materials and methods this study uses an analytic observational with cross sectional approach. the research subjects are 201 students from 31 elementary schools in 15 subdistricts in wonosobo which are taken randomly proportional. the socioeconomic characteristics of the respondents’ parents is measured by using questionnaire consisting of nine questions from the previous validated researches. cariogram is used to find the risk prediction of early childhood caries. the cariogram is equipped with 10 assessment criteria, in which the risk prediction of the dental caries can be found out once the 7 components of the assessment have been filled in. in this study, the eight assessment criteria are caries experience (dmft), the affecting disease, the food contents (measured by using dietary survey), eating frequency, plaque score, fluoride program, salivary ph and volume of saliva. the research data is analyzed statistically by using spearman rank, if p <0.05, then there is a significant relationship. results by using linkert scale, it is found that 72.1% of the research subjects (145 respondents) are in the middle category, 15.4% of the research subjects are in the lower table 1. overview of the socioeconomic characteristics of the respondents’ parents socioeconomic amount percentage (%) higher 25 12.4 middle 145 72.1 lower 31 15.4 table 2. the risk predictions of early childhood caries of the respondents the risk predictions of early childhood caries amount percentage (%) very high 2 1 high 36 17.9 medium 84 41.8 low 64 31.8 very low 15 7.5 9 table 1. overview of the socioeconomic characteristics of the respondents' parents socioeconomic amount percentage (%) higher 25 12.4 middle 145 72.1 lower 31 15.4 table 2. the risk predictions of early childhood caries of the respondents the risk predictions of early childhood caries amount percentage (%) very high 2 1 high 36 17.9 medium 84 41.8 low 64 31.8 very low 15 7.5 figure 1. the diagram of risk prediction of early childhood caries of the respondents. informations: : the chance in avoiding dental caries : dietary : bacteria figure 1. the diagram of risk prediction of early childhood caries of the respondents. ( : the chance in avoiding dental caries; : dietary; : bacteria; : susceptibility ; : the influential condition) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p23-27 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p23-27 2525wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 23–27 category as many as 31 respondents (15.4%) and in the high category by 25 respondents (12.4%) (table 1). table 2 shows that most of the research subjects are in the medium category, as many as 84 respondents (41.8%), 2 respondents are in the very high category (1%), 36 respondents are in the high category (17.9%), 64 respondents are in the low category (31.8%) , while 15 repondents are in the very low category (7.5%). figure 1 shows the prediction of the respondents to avoid the early childhood caries as many as 55% and the risk prediction of early childhood caries as many as 45%, with the highest risk factor is susceptibility, bacteria, dietary, and the influential condition. table 3 shows the cross tabulation between the socioeconomic characteristics with the risk predictions of early childhood caries. table 4 shows the fluorine content in the spring and public water supply table 5 shows the significance score between the socioeconomic characteristics with the risk predictions of the early childhood caries, which is measured by using spearman rank test, is 0.043. it shows that there is a significant relationship between the socioeconomic characteristics with the risk predictions of early childhood caries. discussion vulnerability is one of the risk factors gained from the combinations of the research data obtained from the salivary ph, volume of saliva and fluoride program. there are several functions of saliva, including to lubricate the tissues in oral cavity, to protect the tissues in the oral cavity so that there will be no abrasion during the mastication process, to help metabolizing carbohydrates, as an antibacterial activity against the pathogenic bacteria in the oral cavity, to clean up debris and food scraps left in the oral cavity, and to maintain the stability of the buffer system in the oral cavity. the low salivary secretion may cause such a disturbance in the salivary function.7 the results of the study show that most of the respondents only use fluoride containing toothpaste so that they don’t get the maximum intake of fluoride program. most of respondents only brush their teeth once a day during the morning shower. the research laboratory data shows that the water used by the respondent, either it is from the public water supply or springs, contain fluoride as much as 0.18 and 0.08 ml per gram, respectively. fluoride is the primary anticaries agent. fluoride has the potentials to inhibit dental demineralization, enhance remineralization and inhibit cariogenic bacteria. fluoride may enhance remineralization of enamel or crystals by partially dissolving dentin by combining with calcium and phosphate in saliva. remineralization is the natural repair process against caries lesions that have not formed any cavity. fluoride may help accelerating the process of remineralization and forming layers, like the new layer of fluoroapatite on top of the remineralization crystals which are left under caries lesions. hence, it may decrease the solubility of the aforementioned crystals.8 caries prevention can be done by increasing the resistance of email by using flour either locally or systemically. it can also be obtained from toothpaste. brushing teeth twice a day with a toothpaste containing fluoride is proven to reduce caries.4 most respondents have used toothpaste for tooth brushing, although it is only once a day. there should be any counseling and supervision from the parents to increase the frequency of tooth brushing to twice a day, after having breakfast and before going to bed. table 3. cross tabulation between the socioeconomic characteristics with the risk predictions of early childhood caries the risk predictions of early childhood caries very low low medium high very high socioeconomy lower 1 8 16 5 1 middle 14 48 55 27 1 higher 0 8 13 4 0 table 4. the fluorine content in water parameter public water supply (ml) springs (ml) fluor 0.18 0.08 table 5. the analysis of the relationship between the socioeconomic characteristics with the risk predictions of early childhood caries free variable bound variable p spearman rank socioeconomic characters the risk predictions of early childhood caries 0.043 there is a relationship dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p23-27 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p23-27 26 wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 23–27 fluoridation of drinking water is the most effective way to reduce the of caries problem in the community generally.5 fluoridation of drinking water can only be done if there is a centralized public water supply. it is quite expensive though, which is why it cannot be done yet here in indonesia. the second higher risk factor of early childhood the bacteria. it is obtained from the plaque score. the average of the plaque score of the respondents is categorized as quite well. plaque is a thin layer of microorganisms, food waste and organic material that can be formed on the teeth, and sometimes can also be found on the gums and tongue. plaque is the aggregate of a large numbers and a wide varieties of microorganisms on the surface of the teeth which begins erupted so quickly that would be protected by the thin layer of a glycoprotein called the acquired pellicle. glycoproteins in saliva will be specifically absorbed on hydroxiapatite and firmly attach to the surface of the teeth plaque.9 one of the preventive methods is teeth brushing. a routine dental care check-up is one way to prevent dental and oral health care problems. teeth brushing is a common way that is recommended to clean any debris that is attached to the surface of the teeth and gums. in order to obtain the optimum results in tooth brushing, try to acknowledge the influential factors, including the teeth brushing technique and the frequency of teeth brushing (twice a day, at the very least, in the morning after having breakfast and at night before bedtime). ideally, teeth brushing should be done after having breakfast every me morning, but the most important time is right before bedtime.10 oral health treatment should be done from early age. primary school period is the ideal age to train the motor skills of a child, including teeth brushing. teaching teeth brushing to young children should be done by using models and simple techniques, with is accompanied with interesting and attractive methods without changing the content, such as live demonstrations of teeth brushing, audio-visual programs, or through a controlled program of mass teeth brushing activity.11 the third higher risk prediction of early childhood caries is dietary. dietary is the risk factor obtained from the content of foods (carbohydrates), and eating frequency. substrate (carbohydrates) also has an important role in the occurrence of dental caries, since the substrate is the source of energy for bacteria and helps in forming plaque. not all types of carbohydrates have the important role in the formation of caries. sucrose (disaccharide) and glucose (monosaccharide) are the two types of substrates that are highly cariogenic, while the other types of disaccharide have no strong cariogenic properties.12 some of the references state that there is a relationship between socioeconomic status with the incidence of dental caries, one of the references is the study of tulangow on the overview of caries status of elementary school students in sdn 48 manado based on the socioeconomic status of their parents. the results of the study show that the children with lower socioeconomic status have a higher dmf-t index than the children from the higher status. there are several variables that are often used as the indicator to measure the elements of socioeconomic status, including the job, regular income, and level of education.13 gerungan states that the criteria of either low or high socioeconomic status depends on the type and location of the house, the regular income of the household, and several other criteria in regards with the welfare of the family. the socioeconomic status of the household is associated with oral and dental hygiene, which is why the economic status highly affects (even restrains, in some cases) the dental health care of the respondents.14 the result of the statistical test shows that p <0.05, with the significance value of 0.043, which means that there is a significant relationship between the socioeconomic characteristics with the early childhood caries. there is a similar research shows that the prevalence of early childhood caries is higher in children who come from low socioeconomic status, because they eat less fiber foods. they also have low levels of education, which lead to the lack of knowledge about the importance of oral health. hence, it increases the rate of dental healthcare problems.15 the social and economic factors are some of the factors that influence the number of dental caries problems. the low socioeconomic status, which are measured by the level of education and regular income, is associated with the lack of fiber consumption in individuals who live in areas with bad socioeconomic surroundings.16 it can be concluded that there is a relationship between the socioeconomic characteristics with the risk predictions of the early childhood caries measured by using cariogram. the higher the socioeconomic status is, the lower the risk prediction is. references 1. worotitjan i, mintjelungan cn, gunawan p. pengalaman karies gigi serta pola makan dan minum pada anak sekolah dasar di desa kiawa kecamatan kawangkoan utara. jurnal e-gigi 2013; 59-68. 2. riset kesehatan dasar. laporan hasil riset kesehatan dasar. jakarta.: departemen kesehatan ri; 2007. 3. machfoedz i, zein ay. menjaga kesehatan gigi dan mulut anak-anak dan ibu hamil. yogakarta: tramaya; 2005. p. 59-68. 4. angela a. pencegahan primer pada anak yang beresiko karies tinggi (primary prevention in children with high caries risk). dental journal (majalah kedokteran gigi) 2005; 38(3): 130-4. 5. tulangow jt, mariati nw, mintjelungan c. gambaran status karies murid sekolah dasar negeri 48 manado berdasarkan status sosial ekonomi orang tua. jurnal e-gigi 2013; 1(2): 85-93. 6. bratthall d, petersson gh. cariogram-a multifactorial risk assessment model for a multifactorial disease. community dent oral epidemiol 2005; 33(4): 256-64. 7. roland sm. dental health advisor oral action. london: general dental practitioners st. john wood; 2005. p. 553-4. 8. featherstone jdb. delivery challenges for fluoride, chlorhexidine and xylitol. bmc oral health 2006; 6(1): s8. 9. roeslan bo. imunologi oral: kelainan di dalam rongga mulut. jakarta: fakultas kedokteran ui; 2002. p. 139-41. 10. liliwati as. pengaruh frekuensi menyikat gigi terhadap tingkat kebersihan gigi dan mulut. dentika dent j 2005; 88–90. 11. pratiwi d. gigi sehat dan cantik. jakarta: media nusantara; 2009. p. 17-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p23-27 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p23-27 2727wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 23–27 12. limeback h. comperehensive preventive dentistry. iowa: john wiley & son ltd; 2012. p. 11-4. 13. suryani t. perilaku konsumen. yogyakarta: graha ilmu; 2008. p. 269. 14. reilly b. socioeconomic status and oral health. austr dent assoc 2006; 4(1): 91–100. 15. budisuari ma, oktarina, mikrajab ma. hubungan pola makan dan kebiasaan menyikat gigi dengan kesehatan gigi dan mulut (karies) di indonesia. buletin penelitian sistem kesehatan 2010; 13(1): 8391. 16. adi r. metodologi penelitian sosial dan hukum. jakarta: granit; 2004. p. 39. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p23-27 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p23-27 editorial team of dental journal (majalah kedokteran gigi) sk: 15/un3.1.2/2022 january 4 – december 31, 2022 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga editor in chief: muhammad dimas aditya ari department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200578006] editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478, 5030255. fax. +62 31 5039478, 5026288 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg/index accredited no. 158/e/kpt/2021 cover photo purchased from: https://stock.adobe.com order number: ae00770304146cid volume 55, issue 4, december 2022 p-issn: 1978-3728 e-issn: 2442-9740 editorial boards roeland jozef gentil de moor, department of restorative dentistry and endodontology, dental school, ghent university, belgium [scopus id: 7005928380] cortino sukotjo, department of restorative dentistry, university of illinois at chicago college of dentistry, united states [scopus id: 6508194317] guang hong, liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan [scopus id: 7203031334] kenji yoshida, department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, japan [scopus id: 57080640700] miguel rodrigues martins, co-worker aachen dental laser center, rwth aachen university, germany [scopus id: 55993479000] sajee sattayut, department of oral surgery, faculty of dentistry, khon kaen university, thailand [scopus id: 55431381300] samir nammour, department of dental science, faculty of medicine, university of liege, belgium [scopus id: 6602922393] reza fekrazad, laser reseach center in medical science, dental faculty, aja university of medical science, iran [scopus id: 22952665700] hong sai loh, department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore [scopus id: 7202491277] hamid nurrohman, missouri school of dentistry & oral health, a.t. still university, united states [scopus id: 52564067000] harry huiz peeters, laser research center, bandung, indonesia [scopus id: 51864447300] rahmi amtha, department of oral medicine, faculty of dentistry, universitas trisakti, indonesia [scopus id: 26031894400] elza ibrahim auerkari, department of oral biology, faculty of dentistry, universitas indonesia, indonesia [scopus id: 10139113000] r. darmawan setijanto, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 55212583700] anita yuliati, department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 43462222100] udijanto tedjosasongko, department of pediatric dentistry, faculty of dental medicine, universitas airlangga [scopus id: 6508026751] associate editors ketut suardita, department of conservative dentistry, faculty of dentistry, iik bhakti wiyata, indonesia [scopus id: 6506788956] alexander patera nugraha, department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57194112535] astari puteri, department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200385443] nastiti faradilla ramadhani, department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57191881659] managing editors beshlina fitri widayanti roosyanto prakoeswa, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57467259800] saka winias, department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57211330310] aulia ramadhani, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57205630113] beta novia rizqy, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57214805206] editorial assistant novi dian prastiwi, faculty of dental medicine, universitas airlangga; abdullah mas’udy, faculty of dental medicine, universitas airlangga. printed by: airlangga university press. campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 55, issue 4, december 2022 p-issn: 1978-3728 e-issn: 2442-9740 1. oral microbiota in oral cancer patients and healthy individuals: a scoping review irna sufiawati, alamsyah piliang, vatchala rani ramamoorthy .............................................. 186–193 contents review article page case reports 2. role of cytotoxic t-lymphocyte antigen 4 (ctla-4) expression in the pathogenesis of warthin’s tumor growth alvionika nadyah qotrunnada, tecky indriana, jane kosasih, meiske margaretha, mei syafriadi .................................................................................................................................... 194–199 3. gender differences in cephalometric angular measurements between boys and girls helsa alyayuan, johan arief budiman ......................................................................................... 200–203 4. effectiveness of telemedicine approach as a treatment to reduce severity of temporomandibular disorders ricca chairunnisa, siti dyah fadilla ............................................................................................ 204–208 5. il-17 plasma levels and erythrocyte sedimentation rate on oral candidiasis animal model erna sulistyani, iin eliana triwahyuni, happy harmono, lisa miftakhul janna, saikha adila azzah, muchamad ziyad afif, ainunnusak ayuningtyas .................................... 209–214 6. strategy for improving the quality of school dental health efforts at tabanan public health center i gusti ayu ari agung, i nyoman panji triadnya palgunadi .................................................... 215–220 7. cleft lip and palate based on birth order and family history at mitra sejati general hospital, indonesia hendry rusdy, isnandar, indra basar siregar, veronica ........................................................... 221–225 8. the effect of giomer’s preheating on fluoride release muthiary nitzschia nur iswary winanto, irfan dwiandhono, setiadi warata logamarta, rinawati satrio, aris aji kurniawan ........................................................................................... 226–230 original articles 9. role of supportive periodontal management in patient with metastatic cancer kevin chee pheng neo, nurul syahirah mohamad, avita rath, melissa li zheng wong, myint wai, bennete fernandes ...................................................................................................... 231–234 10. management of impacted maxillary canine with surgical exposure and alignment by orthodontic treatment meralda rossy syahdinda, alexander patera nugraha, ari triwardhani, tengku natasha eleena binti tengku ahmad noor .................................................................... 235–239 11. successful traction of a mesially 90° dilacerated root of impacted maxillary canine: a case report fani tuti handayani, ida ayu evangelina ................................................................................... 240–245 3333 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 33–37 original article addition of gourami (osphronemus goramy) fish scale powder on porosity of glass ionomer cement erawati wulandari1, farah rachmah aulia wardani2, nadie fatimattuzahro3, i dewa ayu ratna dewanti3 1department of conservative dentistry, faculty of dentistry, universitas jember, jember, indonesia 2student of faculty of dentistry, universitas jember, jember, indonesia 3department of biomedical dentistry, faculty of dentistry, universitas jember, jember, indonesia abstract background: porosity is one of the disadvantages of glass ionomer cement (gic) restorative materials, as it causes a reduction in strength and durability; the greater the porosity, the lower the strength of the restorative material and vice versa. as gourami fish scales contain calcium and phosphate, they have the potential to reduce the porosity of gic. purpose: this study aimed to analyse the effect of adding gourami fish scale powder (gfsp) on the pore size and porosity level of the gic. methods: this experimental research included a post-test-only control. the gfsp was fabricated using the freeze-drying method. sixteen fuji ix extra sample cylinders with a diameter of 5 mm and a height of 3 mm were divided into four groups: k0, which comprised gic without the addition of gfsp; k1, which contained gic powder + 2.5% gfsp (by weight); k2, which comprised gic powder + 5% gfsp (by weight), and k3, which contained gic powder + 10% gfsp (by weight). the samples were observed using scanning electron microscopy and measured using imagej software. data were analysed using a one-way analysis of variance (anova) test. results: the addition of 2.5% gfsp (by weight) produced the smallest pore size and lowest porosity, while the one-way anova test results were significant among all groups at p = 0.000. there was no significant difference in pore sizes between k0 and k1 (p = 0.359), but a significant difference was found in the level of porosity (p = 0.024). conclusion: the addition of gfsp affected the porosity of the gic; the pore size and porosity level of the gic were reduced by the addition of 2.5% gfsp. keywords: glass ionomer cement; gourami fish scale powder; porosity correspondence: erawati wulandari, department of conservative dentistry, faculty of dentistry, universitas jember. jl. kalimantan no. 37, jember, 68121, indonesia. e-mail: era.fkg@unej.ac.id introduction glass ionomer cement (gic) is a restorative material with several advantages, including anti-cariogenic properties (due to fluoride release), biocompatibility, has a natural tooth colour and low toxicity. however, it also has disadvantages, namely, low fracture and wear resistance, brittleness and porosity, which lead to poor polishing results.1 porosity refers to the presence of an open cavity,2 and pores act as a source of stress concentration, increasing the brittleness of specimens.3 the greater the porosity, the lower the strength and resistance of a material; this affects its compressive strength and allows it to change colour easily.4,5 the appearance of pores in gic may also facilitate the increased adhesion of microorganisms on the surface of restorations due to increased roughness.6 a previous study found the porosity of a conventional self-cured gic to be 7.27%–7.81%; the value for resinmodified glass ionomer cement was 5.42%–5.96%, while it was 1.01%–1.41% for composite resin.7 this shows that the porosity of conventional gic is greater than resin-modified or composite resin. furthermore, a study examined several types of gic and found the total number of pores in light-cured fuji ix gic to be 13, while there were 295 in conventional fuji ix material. 8 based on these studies, the porosity of conventional fuji ix gic is high. pore sizes and porosity levels can be reduced or increased using materials containing calcium and phosphate,9 and the physical and mechanical properties of gics can be improved by modification with hydroxyapatite.10,11 one substance that contains calcium, phosphate and hydroxyapatite is gourami (osphronemus goramy) fish dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p33–37 mailto:era.fkg@unej.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p33-37 34 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 33–337 scales.12 gourami fish scales contain 5%–7.5% calcium and 5% phosphate, while other freshwater fish scales contain only 2%. in addition, the hydroxyapatite content is similar to that found in bone and dentin. the addition of 2.5%, 5%, and 10% powdered gourami scales to gic materials tends to reduce tool-like receptors 2 (tlr2) and tlr4 in rats.12 the addition of 2.5% gourami fish scale powder (gfsp) can decrease the width of the marginal gap (a gap at the tooth-material junction) and increase the compressive strength and inhibition zone of streptococcus mutans and lactobacillus acidophilus.13 gourami fish scales are reported to contain phosphate and calcium, which are the main materials used in teeth restoration. however, the use of these scales in dentistry has not been optimised, and their application to reduce the porosity of gic is limited. as a restoration material, it is suspected that gourami fish scales have the potential to reduce the pore size and porosity and improve the mechanical properties of gics. therefore, this study aimed to analyse the effect of gfsp addition on the pore size and porosity level of conventional gic. materials and methods this experimental research study included a post-test-only control group design and was conducted using a random sampling technique. first, fish body scales were cleaned of fat and dirt using a cleaning brush under running water. then, they were placed on a tray and allowed to dry at room temperature (28-33°c) for 48 hours. next, the samples were placed in a freeze dryer for 24 hours (zirbus technology vaco 5-ii-d serial no. 11/3184, bad grund, germany).14 the dried fish scales were ground using a blender (miyako, jakarta, indonesia) and refined with a test sieve analys mesh 200 to produce gfsp (74 µm) (abm jakarta, indonesia), which was stored in a dry, airtight glass bottle.15 this study used 16 gic sample cylinders with a diameter of 5 mm and a height of 2 mm,16 which were divided into four groups. sixteen gic samples (fuji ix extra gc gold label hs posterior, gc corporation, tokyo, japan) were prepared at a 1:1 ratio of one spoonful of solid powder to one drop of liquid, respectively, based on the manufacturer’s instructions. one spoonful of powder weighed 0.23 grams. the samples were divided into four groups, as follows: the k0 (control) group consisted of gic without added gfsp. the k1 group contained gic powder and 2.5% gfsp (by weight), where the weights of the gic powder and gfsp were 0.224 and 0.006 g, respectively. the k2 group contained gic powder and 5% gfsp (by weight), where the weights of the gic powder and gfsp were 0.218 and 0.012 g, respectively. group k3 contained gic powder and 10% gfsp (by weight), where the weights of the gic powder and gfsp were 0.207 and 0.023 g, respectively. the samples were prepared by mixing gic powder with gfsp on a paper pad. then, the gic liquid was added according to the manufacturer’s instructions. it was stirred with an agate spatula until homogeneous and placed into a cylindrical mould with a plastic filling instrument (onemed, jakarta, indonesia). subsequently, the gic was compacted with stopper cement (schwert ss, cologne, germany), and the surface was covered with a celluloid strip. the top of the mould is loaded with 0.5 kg to obtain a similar density after setting, the gic was removed from the mould and stored in a closed container. the porosity (which appeared as dark round or irregular shapes) was observed using scanning electron microscopy (sem) (hitachi tm3030 plus, tokyo, japan) under 500-x magnification. the pore size and porosity levels on the surface of the samples were calculated for all five fields of view using imagej software (maryland, us). the research data were tested for normality using shapiro–wilk and levene homogeneity tests. a statistical test was performed using a one-way analysis of variance (anova) and continued with a least-significant difference (lsd) test using spss version 22 software (ibm, us). * ** * ** * * 100 90 80 70 60 50 40 30 20 10 0 k0 k1 k2 k3 64 36 76 88 k0: gic k1: gic + 2.5% gfsp k1: gic + 5% gfsp k1: gic + 10% gfsp figure 1. average total porosity level and least-significant difference test results between groups. *p < 0.05, ** p > 0.05. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p33–37 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p33-37 35wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 33–37 results the smallest pore size and the lowest level of porosity were found in the k1 group, as shown in figures 1 and 2. the pore size in the k2 and k3 groups was larger than in the k0 and k1 groups. the average value from the lowest to highest was k1, k0, k2 and k3, which indicates that the result was directly proportional to the degree of porosity. the one-way anova test results on the level of porosity showed a significant difference between all groups (p = 0.003). additionally, the lsd test results indicated significant differences between k0 and k1, k0 and k3, k1 and k2, and k1 and k3, as shown in figure 1. the one-way anova test for pore size revealed a significant difference between all groups (p = 0.000), while the lsd test between groups showed significant differences between k0 and k3, k1 and k2, k1 and k3, and k2 and k3 (figure 2). the sem image results revealed that the smallest pore size and lowest level of porosity were found in k1. moreover, visible crack lines in the form of porosity-related fractures were observed in all groups. based on the results, the largest cracks were found in the k3 group (figure 3). 14 12 10 8 6 4 2 0 k0 k1 k2 k3 7.161 6.093 9.39 12.659 k0: gic k1: gic + 2.5% gfsp k1: gic + 5% gfsp k1: gic + 10% gfsp * ** * * ** * figure 2. average pore size (µm) and least-significant difference test results between groups. *p < 0.05, ** p > 0.05. a b c d figure 3. microscopic characterization of the pore size and level of porosity under a 500-x scanning electron microscope. (a) k0 (glass ionomer cement [gic] without the addition of gourami fish scale powder [gfsp]). (b) k1 (gic + 2.5% gfsp). (c) k2 (gic + 5% gfsp). (d) k3 (gic + 10% gfsp). porosity is indicated by the white arrows. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p33–37 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p33-37 36 wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 33–337 discussion the results showed that the pore size and level of porosity in the gic with 2.5% added gfsp were smaller and lower, respectively, than the control. it is suspected that the hydroxyapatite in gfsp binds strongly to gic and plays a role in the chemical changes that occur during the initial setting reaction of the cement. moreover, hydroxyapatite dissolves rapidly below ph 2.05 when mixed with gic liquid with a ph of 1.23.17 after the reaction, hydroxyapatite from the gfsp adsorbs in the gic matrix and fills the vacancies (distances) between the glass particles, thereby increasing the density of the cement and reducing its porosity.17 when gic powder and gfsp containing hydroxyapatite are mixed with liquid, calcium ions are released; they initiate an acid–base reaction against metal ions, such as al3+ and sr2+, on the surface of the gic powder, forming more salt bridges and crosslinking structures.18,19 the pore size and porosity level increased with higher concentrations of gfsp (5% and 10%), which is probably due to the absence of a bond between the gic and gfsp. an increase in the amount of added gfsp did not lead to the formation of optimal crosslinking bridges. the addition of an extremely large amount of gfsp presumably caused an ineffective reaction in the gic with no formation of bonds between the particles, thereby increasing the porosity.20 the supplementation of other materials to a gic powder affects its mechanical properties. previous studies have confirmed that a smaller amount of gic powder caused inadequate crosslinking, thereby reducing the matrix formed.21 it is assumed that the higher porosity at concentrations of 5% and 10% was caused by differences in the size of the powder particles. the largest particle size in the gic was 50 µm, while the particle size of the gfsp reached 74 µm. groups k2 and k3 revealed a larger pore size and a higher level of porosity due to the addition of more gfsp than in k1. the addition of larger-sized particles with smaller surface areas in the gic/gfsp mixtures reduced the adhesion force between the powder mixtures.22 in this study, higher viscosity was obtained at concentrations of 5% and 10% due to the difference in particle size and the extremely large amount of added gfsp. a high viscosity causes the inhomogeneous mixing of samples and increases air trapping, thereby causing the pore size and level of porosity to increase.23 air trapped during mixing reduced the polymer conversion rate by inhibiting the setting reaction and causing an inadequate acid–base reaction, thereby reducing polymer crosslinking.24,25 porosities were observed in the k0 group (gic without the addition of gfsp). this involves an acid–base reaction between polyacrylic acid as a proton donor and aluminosilicate glass as a proton acceptor. the polyacrylic acid further destroys the bonds in the aluminosilicate glass, while h+ ions from polyacids and tartaric acid cause the release of al3+, ca2+, na+ and fluorine cations from the surface of the gic powder, leading to the formation of porosity.17 furthermore, porosity occurs during the hardening process, where na+ and fluorine ions are unable to bond completely, leading to the release of fluorine ions and the development of empty cavities (or porosity) in the particle structure of the gic. the release of cations from the gic’s surface causes the release of glass particles, leading to porosity.26,27 moreover, the technique of placing the material in a dental cavity or impression also causes air to enter the material.7 in conclusion, the addition of gfsp affected the porosity of the gic; the addition of 2.5% gfsp reduced both the pore size and the level of porosity. further research is needed regarding the appropriate mass weight gain of gfsp for improving the mechanical and physical properties of gic. in addition, it is necessary to investigate obtaining a gfsp particle size identical to that of gic powder. acknowledgements the authors would like to thank the rector and lembaga penelitian dan pengabdian kepada masyarakat (lp2m) of the university of jember for their support with a funding grant for this research. references 1. chen s, cai y, engqvist h, xia w. enhanced bioactivity of glass ionomer cement by incorporating calcium silicates. biomatter. 2016; 6: e1123842. 2. dorland. kamus saku kedokteran dorland. 30th ed. siangapore: elsevier; 2020. p. 900. 3. al-kadhim aha, abdullah h, mahmood a. effect of porosity on compressive strength of glass ionomer cements. malays dent j. 2012; 34(1): 23–9. 4. wardhani wp, meizarini a, yuliati a, apsari r. perubahan warna semen ionomer kaca setelah direndam dalam larutan teh hitam. dentofasial. 2010; 9(2): 123–9. 5. anusavice k, shen c, rawls hr. phillips’ science of dental materials. 12th ed. philadelphia: saunders; 2013. p. 592. 6. cabello malagón i, cánovas hernández b, martínez hernández e, serna-muñoz c, pérez-silva a, ortiz-ruiz aj. analysis of the porosity and microhardness of glass ionomer cements. mater sci. 2021; (early access). 7. rodrigues ds, buciumeanu m, martinelli ae, nascimento rm, henriques b, silva fs, souza jcm. mechanical strength and wear of dental glass-ionomer and resin composites affected by porosity and chemical composition. j biotribo-corrosion. 2015; 1(3): 24. 8. ok e, kamalak h, canbay ca, yiğit o. the evaluation of thermal conductivity of different restorative glass ionomer cements. int j appl dent sci. 2018; 4(3): 360–4. 9. setyawati a, waladiyah f. porositas email gigi sebelum dan sesudah aplikasi pasta cangkang telur ayam negeri. j kedokt gigi univ padjadjaran. 2019; 31(3): 221–7. 10. khaghani m, doostmohammadi a, golniya z, monshi a, arefpour ar. preparation, physicochemical characterization, and bioactivity evaluation of strontium-containing glass ionomer cement. int sch res not. 2013; 2013: 583989. 11. kutuk zb, vural uk, cakir fy, miletic i, gurgan s. mechanical properties and water sorption of two experimental glass ionomer cements with hydroxyapatite or calcium fluorapatite formulation. dent mater j. 2019; 38(3): 471–9. 12. dewanti idar, lestari pe, purwanto p, wulandari e, yani rwe, wibisono s, burlakovs j. bones, scales of upeneus sulphureus and osphronemus gouramy increase adhesion and decrease il-1β dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p33–37 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p33-37 37wulandari et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 33–37 expression on monocytes against streptococcus mutans. ann trop med public heal. 2020; 23(8): 1253–8. 13. ayu id, dewanti r, firdausya da, mantika si, amalia af, rifqoh t, wulandari e. marginal gap , compressive strength and bacterial inhibition of gurami fish (osphronemus gouramy) scales powder. iosr j dent med sci. 2022; 21(1): 53–9. 14. puspitasari e, umayah e, hidayat ma. instruksi kerja freeze drier zirbus vaco 5-ii-d serial no.11/3184. jember: universitas jember; 2013. 15. yudhit a, harahap k, nasution sc. effect of hydroxyapatite from nile tilapia (oreochromisniloticus) scale on surface hardness of conventional and resin modified glass ionomer cement (in vitro study). in: oral health international conference on art, nature and material science development. 2019. p. 5–10. 16. rahman iab, masudi sm, luddin n, shiekh ra. one-pot synthesis of hydroxyapatite-silica nanopowder composite for hardness enhancement of glass ionomer cement (gic). bull mater sci. 2014; 37(2): 213–9. 17. khurshid z, zafar m, qasim s, shahab s, naseem m, abureqaiba a. advances in nanotechnology for restorative dentistry. mater (basel, switzerland). 2015; 8(2): 717–31. 18. mawa d a r a pa , moza r t ha m, k t. penga r u h p ena mba ha n hidroksiapatit dari cangkang telur ayam terhadap kekerasan permukaan gic. j mater kedokt gigi. 2016; 5(2): 8–14. 19. choudhary k, nandlal b. comparative evaluation of shear bond strength of nano-hydroxyapatite incorporated glass ionomer cement and conventional glass ionomer cement on dense synthetic hydroxyapatite disk: an in vitro study. indian j dent res. 2015; 26(2): 170–5. 20. barandehfard f, kianpour rad m, hosseinnia a, khoshroo k, tahriri m, jazayeri he, moharamzadeh k, tayebi l. the addition of synthesized hydroxyapatite and fluorapatite nanoparticles to a glassionomer cement for dental restoration and its effects on mechanical properties. ceram int. 2016; 42(15): 17866–75. 21. saran r, upadhya np, ginjupalli k, amalan a, rao b, kumar s. effect on physical and mechanical properties of conventional glass ionomer luting cements by incorporation of all-ceramic additives: an in vitro study. abdalla ai, editor. int j dent. 2020; 2020: 1–9. 22. alobiedy an, al-helli ah, al-hamaoy ar. effect of adding micro and nano-carbon particles on conventional glass ionomer cement mechanical properties. ain shams eng j. 2019; 10(4): 785–9. 23. girinath reddy m, dinesh pa, sandeep n. effects of variable viscosity and porosity of fluid, soret and dufour mixed double diffusive convective flow over an accelerating surface. iop conf ser mater sci eng. 2017; 263(6): 062012. 24. panpisut p, monmaturapoj n, srion a, angkananuwat c, krajangta n, panthumvanit p. the effect of powder to liquid ratio on physical properties and fluoride release of glass ionomer cements containing pre-reacted spherical glass fillers. dent mater j. 2020; 39(4): 563–70. 25. poorzandpoush k, omrani l-r, jafarnia sh, golkar p, atai m. effect of addition of nano hydroxyapatite particles on wear of resin modified glass ionomer by tooth brushing simulation. j clin exp dent. 2017; 9(3): e372–6. 26. van noort r. introduction to dental materials. 4th ed. sheffield: elsevier health sciences; 2013. p. 264. 27. sidhu sk, nicholson jw. a review of glass-ionomer cements for clinical dentistry. j funct biomater. 2016; 7(3): 16. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p33–37 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p33-37 �0 volume 46 number 1 march 2013 research report effect of citrus aurantifolia swingle essential oils on methyl mercaptan production of porphyromonas gingivalis anindya prima yusinta, ivan arie wahyudi and anne handrini dewi department of dental biomedical sciences faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: halitosis is a term used to describe an unpleasant odors emanating timely from oral cavity. the unpleasant smell of breath most common caused from volatile sulphure compound (vsc). methyl mercaptan is the major component of vsc. p. gingivalis produced large amount of methyl mercaptan. the essential oils of citrus aurantifolia swingle contain antibacterial component. purpose: the purpose of this study was to determine the effect of essential oil of citrus aurantifolia swingle on the production of methyl mercaptan compounds in p. gingivalis. methods: bacterial suspension of p. gingivalis in tsb medium with 108 cfu/ml concentration cultured in a microplate and added by the essential oils of citrus aurantifolia swingle with 1%, 2%, 3% and 4% concentration. distilled water was used as negative control and 0.2% chlorhexidine mouthwash was used as a positive control. microplate was incubated anaerobically for 48 hours. after the periode of incubation, 0.6% methionine as the exogenous substrate and 0.06% dtnb as a reagen for determining methyl mercaptan concentration were added to each wells. the microplate was futher incubated for 12 hours. concentration of methyl mercaptan produced by the p. gingivalis was measured spectrophotometrically using microplate reader at 415 nm. results: one-way anova showed that the essential oil of citrus aurantifolia swingle take effect on the concentration of methyl mercaptan produced by p. gingivalis. lsd test results indicated that there was a significant difference of methyl mercaptan concentration between treatment groups of the essential oils of citrus aurantifolia swingle and distilled water that used as negative control. conclusion: the essential oil of citrus aurantifolia swingle has decreased the production of methyl mercaptan produced by p. gingivalis. key words: citrus aurantifolia swingle, porphyromonas gingivalis, methyl mercaptan, halitosis abstrak latar belakang: halitosis adalah istilah yang digunakan untuk menggambarkan bau tidak sedap yang berasal dari rongga mulut. penyebab utama halitosis adalah senyawa volatile sulphur compound (vsc) dan metil merkaptan merupakan komponen vsc yang paling dominan menyebabkan halitosis. p. gingivalis dapat memproduksi metil merkaptan dalam jumlah banyak. minyak atsiri kulit jeruk nipis (citrus aurantifolia swingle) memiliki kandungan antibakteri di dalamnya. tujuan: untuk mengetahui pengaruh minyak atsiri kulit jeruk nipis terhadap produksi senyawa metil merkaptan pada bakteri p. gingivalis. metode: suspensi bakteri p. gingivalis dalam media tsb dengan konsentrasi 108 cfu/ml dibiakkan dalam microplate. selanjutnya dilakukan penambahan minyak atsiri kulit jeruk nipis konsentrasi 1%, 2%, 3%, dan 4%. obat kumur chlorhexidine 0,2% digunakan sebagai kontrol positif dan akuades sebagai kontrol negatif. microplate diinkubasi selama 48 jam untuk selanjutnya dilakukan penambahan metionin 0,6% dan dtnb 0,06% dan diinkubasi kembali selama 12 jam. konsentrasi senyawa metil merkaptan yang diproduksi oleh bakteri p. gingivalis dihitung dengan menggunakan microplate reader pada panjang gelombang 415 nm. hasil: anova satu jalur menunjukkan bahwa minyak atsiri kulit jeruk nipis berpengaruh terhadap konsentrasi metil merkaptan yang diproduksi oleh bakteri p. gingivalis. hasil uji lsd menunjukkan adanya perbedaan yang signifikan antara kelompok perlakuan minyak atsiri kulit jeruk nipis dengan akuades sebagai ��listyasari: inhibition of dental plaque formation by toothpaste containing propolis kontrol negatif. kesimpulan: minyak atsiri kulit jeruk nipis dapat menurunkan produksi senyawa metil merkaptan yang dihasilkan oleh bakteri penyebab halitosis p. gingivalis. kata kunci: kulit jeruk nipis (citrus aurantifolia swingle), porphyromonas gingivalis, metil merkaptan, halitosis correspondence: ivan arie wahyudi, c/o: departemen ilmu biomedik kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: ivanocovic@yahoo.com introduction halitosis is one of oral health problems that many people complain after caries and periodontal diseases.1 halitosis is a general term used to describe the bad smell of breath coming from the oral cavity.2 detection upon the presence of halitosis can be a manifestation of a systemic illness and is a good indicator of oral diseases or certain systemic diseases.3 halitosis is mainly (85-90%) initiated by the bacteria that live in the oral cavity.4 the oral cavity is home to hundreds of bacteria species that produce some badsmelling substances as a result of protein degradation.5 microbial degradation products which primarily cause halitosis are volatile sulphur compound (vsc).6 vsc compounds are mainly produced by negative gram anaerobic oral bacteria through a process of sulfurcontaining amino acid degradation derived from peptides and proteins.7 these proteins are obtained from organic substrates derived from saliva, crevicular fluid, epithelial cells that exfoliate from the oral mucous membranes, and food debris.8 bacteria that produce vsc is negative gram anaerobic.7 volatile sulphur compound components, among of them, include hydrogen sulfide (h2s), methyl mercaptan (ch3sh), and dimethyl sulfide [(ch3)2s] 3. ch3sh is believed to be the major component of the vsc causing halitosis.2 ch3sh compound could cause halitosis three times more halitosis than vsc h2s. 7 porphyromonas gingivalis (p. gingivalis) is a negative gram anaerobic bacterium, has no spores, and is nonmotile. these bacteria has fimbriae which is important as adhesion molecule when these bacteria interact with oral epithelial cell, fibroblast ligament periodontal, endothelial cell, extracellular matrix protein, saliva protein, and others bacteria.9 p. gingivalis has strong proteolysis’ activities that can degrade proteins,10 so that this bacterium can produce large amounts of ch3sh compound derived from the enzymatic metase reaction upon amino acid l-methionine.11 citrus aurantifolia swingle is known for long as a plant with many benefits. as a natural herb, limes are efficacious to relieve sore throat and cough, mucus laxative (mucolytics), and urine laxative (diuretics).12 citrus aurantifolia swingle belong to the citrus genus, which is one of essential oil producers.13 the essential oil content in citrus is concentrated in the peel and leaves of the fruits.14 the fresh citrus aurantifolia swingle contains about 1.25% of essential oil with limonene as its main components.15 limonene acts as an anti-bacterial agent by expanding the cell membranes, increasing the membrane voltage and penetrating cell membranes of bacteria in order to inhibit bacterial respiration enzyme which is important as the energy system of the cell.16 this study aims to examine the effect of citrus aurantifolia swingle (lime peel essential oil) on the production of ch3sh (methyl mercaptan) compound on halitosis causing bacteria p. gingivalis. materials and methods the material used in this study was lime peel from purworejo, breeding suspension of porphyromonas gingivalis in the liquid medium of trypticase soy broth (tsb), 5% of peg organic solvent to dilute the essential oil into the required concentration, reagent 5, 5 ‘-dithiobis (2-nitrobenzoic acid) (dtnb) 0.06% (w/v), methionine 0.6% (w/v) as the source of sulfur-containing amino acids, 1 µm edta and 0.1 m naoh as dtnb reagent solvent, distilled water as a negative control, and chlorhexidine 0.2% (minosep®) were used as a positive control. citrus aurantifolia swingle used first were identified to ensure that the materials used in this study were the right limes in question. this study used 10 kg of citrus aurantifolia swingle and yielded as much as 2.5 kg of citrus aurantifolia swingle. furthermore, a small piece of citrus aurantifolia swingle was put in distillation container that has been assembled with a cooler (condenser), and then they were heated. the distillation process carried out in this study was a distillation with steam and water (water and steam distillation). the distillation process resulted the essential oil with 100% concentration in which dilution were then performed using an organic solvent of 5% peg to obtain the required concentrations (1%, 2%, 3% and 4%). the next step was to prepare the bacterial suspension of p. gingivalis. the bacteria used were pure-cultured bacteria derived from balai laboratorium kesehatan ngadinegaran, yogyakarta. before used, the bacteria were inoculated and optimized first in blood gelatin plate media for 1 week. after the optimization process for one week, a few bacterial colonies were taken using a sterile loop and suspended into a liquid medium of trypticase soy broth (tsb) and then the bacterial concentration was calculated �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 50–53 using densichek ® to obtain bacterial concentration 108 cfu/ml which is equivalent to mcfarland standard 0.5 and brown iii standard solutions. this study uses dtnb (ellman reagent). dtnb is a reagent which is used to measure the concentration of thiol compounds in a sample. thiol compound is a compound that contains the functional group consisting of sulfur and hydrogen atom (-sh). methyl mercaptan is a thiol compound. the chemical reaction occurring between the thiol group and 5.5’-dithiobis (2-nitrobenzoic acid) (dtnb) will form yellow 2-nitro-5-thiobenzoate compound (ntb-). suspension of p. gingivalis was cultured in 96-well microtiter plates, each was 30 µl. the concentration of p. gingivalis bacteria of each well was 108 cfu/ml. then, citrus aurantifolia swingle essential oil with concentration 1%, 2%, 3%, and 4%, was added, as the positive control 0.2% chlorhexidine mouthwash was used and as the negative control was distilled water, 30 µl for each well and was incubated in a co2 incubator of thermo scientific barnstead lab-line® for 48 hours. after the 48 hoursincubation, 30 µl of methionine (0.6% w/v) and 30 µl of dtnb (0.06% w/v) were added at each pitting. then, they were incubated once more for 12 hours. the concentration of the ch3sh compound produced by bacteria p. gingivalis was calculated using the bio-rad microplate reader benchmark model 680xr® at a wavelength of 415 nm carried out at the laboratory of integrated research and testing (lppt) unit iii, universitas gadjah mada, yogyakarta. the data obtained in the form of absorbance values were equal to the concentration of methyl mercaptan produced by the p. gingivalis bacteria. the overall data obtained were ratio-scaled data which were then grouped according to the respective treatment to be calculated for the mean. results research on the effect of essential oil of citrus aurantifolia swingle upon the production of ch3sh (methyl mercaptan) compound in vitro toward halitosis-causing bacteria, porphyromonas gingivalis, has been carried out in the microbiology laboratory of balai laboratorium kesehatan ngadinegaran and lppt iii universitas gadjah mada. the data obtained were quantitative in the form of absorbance values which were equivalent to the concentration of methyl mercaptan produced. the research findings suggest that the concentration of methyl mercaptan compound produced varies in each treatment group. highest production of methyl mercaptan compounds were found in the negative control. based on the test results using one way anova, their significance was 0.001 (p<0.05), there was a significant difference in the concentration of methyl mercaptan production between group of treatments. it means that the essential oil of citrus aurantifolia swingle take effect on the concentration of methyl mercaptan produced by p. gingivalis. afterward, advanced analysis test of post-hoc least significant difference is conducted to know which group of treatment that had a significant mean difference. in this test, a significant differences in average (p<0.05) was found between the negative control group treatment and essential oils of citrus aurantifolia swingle skin concentration of 1%, 2%, 3% and 4%. lsd test results indicated that there was a significant difference of methyl mercaptan concentration between treatment groups of the essential oils of citrus aurantifolia swingle and distilled water that used as negative control. discussion this study occupied ellman reagent or dtnb (5.5’dithiobis 2-nitrobenzoic acid) to detect the concentration of methyl mercaptan compounds produced by the bacteria p. gingivalis in microplate wells. dtnb is a reagent which is used to measure the concentration of thiol compounds within a sample. thiol compounds are a compound that contains the functional group consisting of sulfur and hydrogen atom (-sh). methyl mercaptan is a thiol compound which, when bonded with dtnb, will produce yellow tnb compounds (2-nitro-5-thiobenzoic acid). the yellow color results from the formation of tnb (2nitro-5-thiobenzoic acid) then its absorbance values were calculated using the bio-rad microplate reader benchmark model 680xr ® at a wavelength of 415 nm.17 the chemical reaction between the reagent dtnb and thiol compound (figure 1). the research findings in table 1 showed that the average concentration of methyl mercaptan compounds produced by p. gingivalis in the negative control treatment had the highest values compared with the other treatment groups which can be seen qualitatively from the most concentrated yellow color resulted than other treatment groups. this was because in the negative control treatment, the bacterial suspensions within the wells were added only by distilled water which is neutral and has no antibacterial power so that p. gingivalis living in the wells remains alive and able to produce methyl mercaptan in large quantities derived from the amino acid methionine. based on previous theory regarding the action mechanism of dtnb reagent that is the more thiol compounds are produced, the more thiol compounds bounding with dtnb and subsequently form a yellow tnb compound so that reading using a microplate absorbance reader produces a high value.18 the average value of methyl mercaptan production in the treatment group of the positive control and the citrus aurantifolia swingle essential oil of concentration 1%, 2%, 3% and 4% had a lower value than the negative control. this was because 0.2% chlorhexidine and citrus aurantifolia swingle essential oil have antibacterial activities so that the generated methyl mercaptan compound will decrease. these results are in accordance with the previous statement ��listyasari: inhibition of dental plaque formation by toothpaste containing propolis figure 1. chemical reaction between dtnb and thiol compound.17 table 1. means and standard deviations of methyl mercaptan concentrations after treatment with the essential oil of various concentrations, positive controls, and negative controls no. treatment n x ± sd 1. negative control 5 2.244 ± 0.092 2. positive control 5 0.322 ± 0.065 3. citrus aurantifolia swingle essential oil 1% 5 0.327 ± 0.128 4. citrus aurantifolia swingle essential oil 2% 5 0.246 ± 0.067 5. citrus aurantifolia swingle essential oil 3% 5 0.241 ± 0.039 6. citrus aurantifolia swingle essential oil 4% 5 0.262 ± 0.050 that the use of mouthwash with antibacterial ingredients contained within them can reduce halitosis by reducing the amount of bacteria and inhibit bacterial activities so that the productions of compounds that cause halitosis including methyl mercaptan will decline.3 citrus aurantifolia swingle essential oil was used to be one of the treatment groups in this study because citrus aurantifolia swingle essential oil has antibacterial power. the ability as an antibacterial agent is suspected due to of the existence of limonene content as the main compounds. limonene is antibacterial in nature by expanding the cell membranes, increasing the voltage across the membranes and inhibiting various enzymes in the membranes. these compounds can penetrate the cell membranes of bacteria so that it can inhibit the respiratory enzyme of the bacterial cell and cause death of the bacteria.16 essential oil is not a single compound, but rather a combination of various compounds and therefore limonene is not the only component contained in the essential oil of citrus aurantifolia swingle. in citrus genus, the essential oil content is concentrated in the peel and leaves of fruits.14 fresh citrus aurantifolia swingle contains about 1.25% of essential oils with the main component is liomonene.15 the other components of the citrus aurantifolia swingle essential oil, geranial (α-citral) and neral (β-citral) can inhibit the growth of bacteria, both negative gram and positive gram.19 based on the results of lsd test, there was a significant difference in term of the average between the negative control and all other treatment groups. this was supported by the significant value which was less than 0.05 (p <0.05) which was 0.001. it means that the essential oil of lemon peel and 0.2% chlorhexidine may affect the concentration of methyl mercaptan produced by p. gingivalis. lsd analysis also showed there is no significant difference on the average concentrations of methyl mercaptan among citrus aurantifolia swingle essential oil used in the study, hence it can be interpreted that citrus aurantifolia swingle essential oil concentration of 1%, 2%, 3% and 4% influence relatively similar to the decline in the production of methyl mercaptan in p. gingivalis. the insignificant difference in term of their average may be resulted from the essential oil used in this study which concentration interval was too short that the effect upon the decreased production of methyl mercaptan was insignificant. but, when viewed descriptively according to the table on the average concentration of methyl mercaptan compound in table 1, it can be concluded that the concentration of methyl mercaptan produced tended to decrease with the increase of citrus aurantifolia swingle essential oil concentration of 1%, 2%, and 3%. this is consistent with the statement pelczar and chan20 said that the higher the concentration of a certain antibacterial agent, the higher the capability to inhibit or to kill so that in this study the production of methyl mercaptan compound would decrease along with the increasing concentration of essential oil used. according to the data presented in table 1, the production of methyl mercaptan in the treatment of essential oil concentration 4% increased. however, based on the lsd test analysis, such increase in the concentration of methyl mercaptan taking place was not significant to the treatment of essential oil concentration of 3%. based on the findings showed in the data in table 1, it is known that the average value of methyl mercaptan concentration in the positive control was 0.322; it is lower than that in the negative control treatment. chlorhexidine is an antibacterial active ingredient belonging to the gold standard contained in mouthwash.21 chlorhexidine has a broad-spectrum antibacterial activity both against the gram positive and gram negative.22 antimicrobial activities of chlorhexidine are performed by damaging the cytoplasmic membrane. bacterial cells characteristically contain negative loads, while the �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 50–53 chlorhexidine molecule is a cation that will be quickly attracted to the surface of the negative load bacterial cell. this process will change the integrity of the bacterial cell membrane. chlorhexidine will engage to the phospholipids in the inner membrane/cytoplasmic membrane resulting in increased cytoplasmic membrane permeability and leakage in components with low molecular weight such as potassium ions. then, cytoplasm coagulation resulting in irreversible cell damage happens.23 however, besides being antibacterial in nature, chlorhexidine also has local side effects when used as a mouthwash that is it can cause extrinsic staining on the teeth so that the teeth become dark yellow brown.21 based on these research, it can be concluded that the essential oil of lemon peel (citrus aurantifolia swingle) of concentration 1%, 2%, 3% and 4% can reduce the production of ch3sh (methyl mercaptan) compound on halitosis-causing bacteria, porphyromonas gingivalis. references 1. pintauli s. masalah halitosis dan pelaksanaannya. dentika dent j 2008; 13(1): 74-9. 2. ongole r, shenoy n. halitosis: much beyond oral malodor. kathmandu univ med j 2010; 8(2): 269-75. 3. djaya a. halitosis: nafas tak sedap. jakarta: pt. dental lintas mediatama; 2000. p. 1-9. 4. anwar ai. penyebab dan penanggulangan halitosis. jitekgi 2007; 4(1): 1-6 5. krespi yp, shrime mg, kacker a. the relationship between oral malodor and volatile sulfur compound–producing bacteria. otolaryngol head neck surg 2006; 135(5): 671-6. 6. loesche wj, kazor c. microbiology and treatment of halitosis. periodontol 2000, 2002; 28: 256-79. 7. ada council on scientific affairs. oral malodor. j am dent assoc 2003; 134(2): 209-14. 8. xu x, zhou xd, wu cd. tea catechin egcg suppresses the mgl gene associated with halitosis. j dent res 2010; 89(11):1304-8. 9. lamont rj, burne ra, lantz ms, lebanc dj. oral microbiology and immunology. washington dc: asm press; 2006. p. 263. 10. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical peridontology. 10th ed. st. louise: mosby elsevier; 2006. p. 161. 11. yoshimura m, nakano y, yamashita y, oho t, saito t, koga t. for mat ion of met hyl mercapt a n f rom l -met h ion i ne by porphyromonas gingivalis. infect immun 2000; 68(12): 6912-6. 12. nuraini dn. aneka manfaat kulit buah dan sayuran; manfaat dan cara pemakaian. yogyakarta: penerbit andi; 2011. p. 58-59. 13. chutia m, bhuyan dp, pathak mg, sarma tc, boruah p. antifungal activity and chemical composition of citrus reticulata blanco essential oil against phytopathogens from north east india. j food sci and technol 2009; 42: 777-80. 14. gunawan d, mulyani s. ilmu obat alam, farmakognasi. depok: penebar swadaya; 2004. 106-12. 15. agusta a. minyak atsiri tumbuhan tropika indonesia. bandung: penerbit itb; 2000. p. 1-9, 89. 16. mizrahi b, shapira l, domb aj, houri-haddad y. citrus oil and mgcl2 as antibacterial and anti-inflammatory agents. j periodontal 2006; 7(6): 963-8. 17. ellman gl. tissue sulfhydryl groups. arch biochem biophys 1959; 82(1): 70-7. 18. onawunmi go, yisak w, ogunlana eo. antibacterial constituentsin the essensial oil of cymbopogon citratus (dc.) stapf. j ethnopharmacol 1984; 12(3): 279-86. 19. peng h, chen w, cheng y, hakuna l, strongin r, wang b. thiol reactive probes and chemosensor. sensors (basel) 2012; 12(11): 15907-46. 20. pelczar mj, chan ecs. 1986. dasar-dasar mikrobiologi 2. jakarta: ui press; 2009. p. 655. 21. pires jr, junior cr, pizzolitto. in vitro antimicrobial efficiency of a mouthwash containing triclosan/gantrez dan sodium bicarbonate. brazilian oral research 2007; 21(4): 1-7. 22. mathur s, mathur t, srivastava r, khatri r. chlorhexidine: the gold standard in chemical plaque control. national journal of physiology, pharmacy and pharmacology 2011; 1(issue 2): 45–50. 23. denton gw. chlorhexidine in block ss, disinfection, sterilization and preservation. 4th ed. philadelphia: lea and febiger; 1991. p. 279-89. vol 38-no4-2005-isi.pmd 180 the molecular changing mechanism of ampicillin-sulbactam resistant staphylococcus aureus towards methicillin resistant staphylococcus aureus mieke hemiawati satari department oral biology faculty of dentistry padjadjaran university bandung – indonesia abstract the aim of this study was to determine the molecular changing of s.aureus, which is resistant to ampicillin-sulbactam and then become resistant to methicillin as a result of improper dosage. the study was conducted by isolating ampicillin-sulbactam resistant and methicillin resistant s.aureus (mrsa), afterwards an amplification process was performed by pcr (polymerase chain reaction.) to isolate the betalactamase enzyme regulator and pbp 2a genes. the result of this research showed that there were a deletion of few amino acids from the regulator gene, and a suspicion that the dna sequence had been substituted from pbp 2 gene into pbp 2a (gen mec). this process had formed mrsa. key words: s.aureus, betalactamase gene, pbp 2a gene correspondence: mieke hemiawati satari, c/o: bagian oral biologi, fakultas kedokteran gigi universitas padjadjaran. jln. sekeloa selatan i bandung, indonesia. introduction until now, infection is still a prevalent problem especially in developing countries. one of dental caused infections is periapical abscess. prolonged infection will cause jaw osteomyelitis with staphylococcus aureus (s. areus) as the main bacteria. in constant efforts to cope with infection caused by s. aureus, several antibiotics had been used, among others the betalactam group. however, the present condition revealed many bacteria are resistant to betalactam. to overcome the resistance, betalactam is mixed with betalactamase inhibitor, in this case ampicillin with sulbactam complying to antibiotic-consumption-strict-rules of correct doses, correct indication, and correct bacteria. should this rules be violated, therapy becomes difficult and expensive, and also escalating resistance. based on a research by satari1 from 94 clinical isolates, 79% was ampicillin resistant, 53% was ampicillinsulbactam resistant and out of this 53% resistance, 38% was resistant towards methicillin causing bacteria resistance to many antibiotics. from an empirical 7 years study (1986-1993) conducted at the microbiology department of school of medicine, university of indonesia, it was revealed that s.aureus pattern of resistance had changed to multiresistance.2 lyon and skuray3 had proven that s.aureus was resistant to 20 antibiotics. according to on brooks,4 since 1961 the multiresistant s. aureus has caused health problems particularly at health centers that can be fatal. the multiresistant s. aureus was later known as resistant to methicillin s. aureus (mrsa). in molecular process, the forming of mrsa was initiated by a mutation of pbp 2 which was a transpeptidase enzyme functioning in forming a murein bag of peptidoglican, to become pbp 2a which had low affinity towards betalactam antibiotic group. the aim of this research was to molecularly analyze the influence of s.aureus resistance pattern against ampicillin-sulbactam to form mrsa. this research gave scientific contribution to understand the resistibility process at molecular stage. materials and method the sample was taken from isolate s. aureus which was resistant against ampicillin-sulbactam and methicillin, complying with the criteria as follows: 1) producing betalactamase enzyme; 2) producing total dna isolation; 3) pcr fragment gene regulator technique (blai and b l a r i • using 2 primary pairs) and fragment (pbp 2a (gen mec) • using 1 primary pair) could be well isolated. the primary which was used to isolate betalactamase enzyme regulator gene (blari and blai) was designed by okamoto, okubuta and inoue.5 181satari: mechanism of staphylococcus aureus forward bla z: primary sense p1 : 5’actctttggcatgtgaactg 3’ reverse blari: primary antisense p3 : 5’ ggacaaatctatcggcttct 3’ p4 : 5’ tgagttgagtcgcagtatag 3’ blai: p5 : 5’ cataacatcccattcagcca 3’ p6 : 5’ aacttttcaatgttcccctcc 3’ the primary which was used to isolate gen mec was designed by murakami, minamide.6 forward : 5’ aaaatcgatgttaaaggttggc 3’ reverse : 5’ agttctgcagtaccggatttgc 3’ the research material was isolate s. aureus which was resistant to ampicillin-sulbactam and methicillin. it was obtained from the microbiology clinic of hasan sadikin general hospital/school of medicine padjadjaran university and school of medicine diponegoro university. the chemical substance to totally isolate dna: tris hcl 50mm containing edta 5mm, nacl 50mm, lysostapin 20µg/ml and rnase 200µg/ml. the total dna isolation was performed with tokue and shoji7 technique. the chemical substance for pcr technique were: buffer lysis, pcr kit core system (promega), primary (p1-p6) and primary mec. blari and blai isolation with pcr technique using primary (p1-p6) under pcr condition i.e.: 1) denaturation process 94º c for 4 minutes, 2) 94º c denaturation cycle stage in 30 seconds, 3) 50º c primary attachment stage in 30 seconds, and 4) 72º polymerisation stage in 2 minutes. polymerase chain reaction (pcr) was done in 40 cycles with 72º stabilizing stage. the chemical substance for sequensing technique was abi prims dye terminator cycle sequensing ready kit (perkin elmer corporation). the equipments for microbiologic examinations were eppendorf tube, 25ºc and -4ºc ultra centrifugation, vortex mixter, e-c minicell, pcr machine, transilluminator with ultraviolet, polaroid, dna sequensing abi prims 377. results amplification result of blaz, blari and blai genes using 4 primary pairs can be seen on figure 1. figure 1. electrophoresis pcr product utilizing p1-p6 to amplify blaz, bla ri and blai gene fragments. 182 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 180–182 hinfl (puc19) was the marker used to give restriction results sized 1419,517,396,214 and 65 pb. figure a: isolate using primary p1 p3 → 878pb figure b: isolate using primary p1 p4 → 1426pb figure c: isolate using primary p1 p5 → 1579pb figure d: isolate using primary p1 p6 → 1686pb primary p1–p6 with primary internal p3, p4, p5 were used in this research. p1–p3 had 878pb, amplifying blaz which was a betalactamase enzyme structure gene. p1–p4 had 1426pb, amplifying blaz and part of blari. p1–p5 had 1579pb, amplifying blaz, blari and part of blai. p1–p6 had 1686pb, amplifying blaz, bla ri and blai. to study the deletion of regulator gene, which later on was suspected being fusion with gen mec, a sensitivity test was performed towards s. aureus ampicillin-sulbactam resistant with methicillin. thirty eight percent of samples were resistant to methicillin. after amplifying gen mec a, the result was an amplification with 517pb length. figure 2. gen mec detection of mrsa column 1. marker puc19/hinfl 5. pcr isolate c3 product 2. pcr isolate a5 product 6. pcr isolate 88 product 3. pcr isolate a5 product 7. pcr isolate 88 product 4. pcr isolate c3 product 8. negative control discussion using p1-p3, the output of blaz gene fragment amplificaton could be isolated in accord to the length of the structured gene which was 878pb due to the relatively stable blaz, and from a homology output with s. aureus tn 552, where no mutation occurred. all these outputs showed that to the resistance of s. aureus against ampicillinsulbactam was not caused by the structured gene mutation. an amplification output of blari and blai genes using two primary pairs of p1-p5 was 1579pb from the expected 1960pb. while with p1-p6, the output was 1686pb from the expected 2223pb. this was assumed due to a deletion of some nucleotides from both genes resulting in disturbed function of the regulator gene for betalactamase enzyme production i.e. a hyperproduction. this hyperproduction was the cause of the resistance of s. aureus against ampicillin-sulbactam. hyperproduction took place evoked by nucleotides changes on regulator genes where they transmitted (blari) and received signal (blai). wiederman and peter8 stated that should a regulator gene be inactive as a result of several nucleotides deletion, then the production of betalactamase enzyme multiplied enormously. according to okamoto5 and murray9 a deletion of 150 nucleotides on the tip of the regulator gene could vanish its regulating function, a change of production characteristic occurred, that was from inductive to constitutive production manifolding betalactamase output in 50–100 times. while lewis, curnok and dyke10 clarified that mrsa was formed due to the fusion between the regulator gene and the structural pbp2 gene which afterward expressed a pbp 2a gene with a low affinity towards ampicillin-sulbactam. the shaping of pbp 2a was the reason for the resistibility of s.aureus against several antibiotics, this was later known on as mrsa. the conclusion of the research revealed that the usage of antibiotics in particular of ampicillin-sulbactam mixture, should stick to certain regulations to avoid resistance. the resistance against ampicillin-sulbactam, phenotypically was caused by a hyperproduction of betalactamase enzyme. genotypically, the hyperproduction was due to the deletion of several nucleotides of regulator genes. it was assumed that the vanished nucleotide fusioned with pbp 2 becoming pbp 2a. the fusion happened because the regulator gene with pbp 2 gene had formed a tertiary dna structure kernodle.11 references 1. satari mh. the molecular phenomenon of betalactamse enzyme hyperproduction on resistant staphylococcus aureus against ampicillin sulbactam. desertation. bandung: postgraduate program padjadjaran university; may 2002. p. 1. 2. sudarmono p, radji m. features of typhoid fever in indonesia, singapore. world scientific 1994; 11-6. 3. lyon bk, skuray r. antimicrobial resistance of s. aureus genetic basis. microbiology reviews, dept of microbiology monash university, victoriaaustralia march 1987; 12(3):94-8. 4. brooks gf, butel js, morse sa. staphylococcus. in: jawetz, melnick, adelberg, editors. medical microbiology. 21th ed. connecticut: appleton & lange; 2001. p. 197-202. 5. okamoto r, okubuta y, inoue r. detection of gene regulating betalactamase productionin s. aureus. antimicrobial agents and chemotherapy 1996 november; 40(11):2550-4. 6. murakami k, minamide w. pcr detection of methicillin–resistant s. aureus. diagnostic moleculer microbiology, bios scientific pub co; 1994. p. 76-82. 7. tokue y, shoji s. comparison of polimerase chain reaction assay. antimicrobial agents and chemotherapy 1992 january; 44(6):6-9. 8. wiederman b, peter gk. induction of betalactamase in gram positif bacteria. diag microbial & infectious disease1989; 12:131-7. 9. murray be. betalactamase producing s. aureus. j antimicrobial agents & chemistry 1992 april; 873-9. 10. gregory pd, lewis ra, dyke kgh, curnock sp. study represor (blai) betalactamase synthesis of staphylococcus aureus. moleculer microbiology 1997; 24 (5):1025-37. 11. kernodle dj. mechanism of resistence to betalactam antibiotic in gram positive pathogens. american society for microbiology 2000; 609–23 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 44 no 3 sept 2011.indd 122 vol. 44. no. 3 september 2011 dental measurements of deuteromalayid javanese students of the faculty of dentistry airlangga university myrtati dyah artaria1 and bambang soegeng herijadi1 1department of anthropology, airlangga university 2department of oral biology, faculty of dentistry, airlangga university surabaya indonesia abstract background: dental anthropology is a new field of study in indonesia, hence there are few numbers of research that can be found in this field. knowledge in this field is needed due to the large area and diversity of the people. moreover, knowledge regarding the possibility to differentiate the sexes of skeletons is still needed for the purpose of identification. purpose: this research intended to study the differences in mesio-distal measurements of the teeth of deuteromalayid javanese males and females studying in the faculty of dentistry in airlangga university. methods: this study used mesiodistal metric dental data, using dental caliper, to test the existence of sexual dimorphism. sample were teeth of freshman students of the airlangga university, from 52 individuals comprising 26 males and 26 females deuteromalayid originated from java (indonesia) studying in the faculty of dentistry airlangga university. measurements were not performed on damaged casts due to caries or other reasons. differences between males and females were tested using independent t-test. results: the mean of mesiodistal measurements in males and females differs, where the males have greater size of mesiodistal measurements. the results showed that there were significant differences between sexes in the sample, in all field of teeth except the second upper and lower premolars. reverse sexual dimorphism-female teeth measurement is larger than those of males-has not been found in these samples. the result of this study revealed that the range of mesiodistal measurements of every tooth in males and females overlapped. conclusion: it is concluded that teeth measurement of males is bigger than females, except maxillary and mandibular second premolars. key words: teeth measurement, sex, mesio-distal, odontometry, sexual dimorphism abstrak latar belakang: antropologi dental adalah bidang studi baru di indonesia, dan karenanya penelitian di bidang ini masih sedikit dijumpai di indonesia. apalagi masih dibutuhkan pengetahuan mengenai apakah jenis kelamin dapat dilihat dari geligi tengkorak manusia, untuk keperluan identifikasi. tujuan: tujuan penelitian ini adalah untuk mengetahui perbedaan ukuran mesiodistal gigi antara laki-laki dan perempuan deuteromalayid dari jawa. metode: penelitian ini menggunakan data pengukuran mesio-distal gigi untuk mengetahui apakah ada perbedaan antar jenis kelamin pada ukuran gigi mahasiswa fakultas kedokteran gigi universitas airlangga keturunan deuteromalayid dari jawa. penelitian ini merupakan penelitian tahap awal dengan jumlah sampel yang masih terbatas. sampel diambil dari 52 orang individu yang bersedia dicetak giginya dengan jumlah 26 laki-laki dan 26 perempuan. pengukuran dilakukan dari mesial ke distal gigi. pengukuran tidak dilakukan mesial gigi yang mengalami kerusakan seperti misalnya karena karies atau aus yang parah. signifikansi perbedaan antar jenis kelamin dianalisis menggunakan independent t-test. hasil: hasil penelitian menunjukkan bahwa terdapat perbedaan yang signifikan antar laki-laki dan perempuan di semua jenis gigi, kecuali pada premolar ke dua atas dan bawah. ”reverse sexual dimorphism” di mana rata-rata ukuran gigi perempuan lebih besar dari lakilaki, tidak ditemukan pada sampel ini. pada penelitian ini meskipun dijumpai ”overlap” pada ukuran gigi laki-laki dan perempuan, tetapi sebagian besar rata-rata ukuran gigi berbeda secara bermakna antar kedua jenis kelamin pada sampel mahasiswa fakultas research report 123artaria: dental measurements of deuteromalayid introduction dental anthropology is a new field of study in indonesia, hence there are few numbers of research can be found in this field. knowledge in this field is needed due to the large area and diversity of the people. anthropometrical research has been done several times,1-8 but research in sexual dimorphism of the groups of people dwelling in this area based on dental research has not been found. indonesia is a large country, occupied by more than 200 million of people who live in chains of islands. some of the islands are isolated from each other, but some other islands can be reached by the neighboring islands inhabitors easily. the isolations and the bridging between the islands that changed from time to time may cause the similarity and dissimilarity of the morphology of the people, as well as different pattern of dental traits variation, and dental measurements. species other than human primates showed so much of differences between the sexes.9 however, different developmental processes can produce traits that appear similar at the enamel surface, suggesting caution in intra and intertaxonomic comparisons,10 so that comparison of sexual dimorphism with other species may not be possible. sexual dimorphism in teeth size has been found in several studies, but the pattern is less consistent. if dental measurements in javanese sample showed an indication of sexual dimorphism, it might be used as a tool for identifying individuals such as in forensics cases. materials and methods a number of 52 javanese subjects were observed in 2009. the sample consisted of 26 males and 26 females aged 18 to 20 years of age. the sample came from freshman students of the faculty of dentistry airlangga university so that the age may not exceed 20 years of age. the casts were made out of the teeth of subjects who were willing to participate for this study. subjects were assigned to javanese only if both parents belonged to that ethnic group. observations were written down on a form and subsequently transferred to excel spreadsheets. observations were not made on damaged casts due to caries or other reasons. tooth diameters were recorded with a calliper accurate to 0.5 mm. the maximum lengths of left and right antimeres of all permanent teeth were measured, and the measurements of the tooth in question taken as the average of both sides. where only one of the antimeres could be measured, its diameters were used, and where neither of the antimeres could be accurately measured-such as carries, incomplete or non-eruption, excessive wear, or cast damage--no value was recorded. although a study reported that heavily worn teeth may be measured because of the strong correlation between the crown measurements and cervical measurements,11 it is decided to use only teeth that are not heavily worn. the measurements were using callipers instead of scanning the objects with computed tomography, and measurements were made digitally with a 3-dimensionalbased dental measurements program (3dd, biodent, cairo, egypt). the reason was simply because the researchers did not have the device to do the computed tomography. besides, according to el zanatya et al.,12 there was no significant differences between the 3dd and conventional measurements. differences between males and females were tested using independent t-test. the differences were significant when the value were 0.05 and below. results it shows that the mean of mesiodistal measurements in males and females, where the males have greater size of mesiodistal measurements (table 1). the larger size of the male teeth of the males, compared to their female counterparts, is usually in evidence, as expected.13,14 however the result if this research revealed that the differences between the sexes were significant in all field except in upper and lower second premolars. the insignificant differences between the males and females in upper and lower premolar were caused by the diversity of the measurements of those teeth, both in males and females. therefore, the overlapping of measurements was also great, in those two teeth, between the males and the females. in the upper canine, a measurement between 5.8 mm and 6.4 mm would almost be certain to be a female, and in the lower canine, a measurement between 4 mm and 5.9 mm would almost be certain to be a female. meanwhile, measurements of the upper and lower canines between 8.1 mm and 9 mm would be on a male subject. in the upper second incisor, a measurement between 7.6 mm and 9 mm would almost be certain to be a male, kedokteran gigi universitas airlangga yang berasal dari jawa keturunan deuteromalayid. kesimpulan: dapat disimpulkan bahwa pengukuran gigi laki-laki lebih besar daripada perempuan, kecuali premolar kedua maksila dan mandibula. kata kunci: ukuran gigi, jenis kelamin, morfometri, mesio-distal, odontometri, dimorfisme seksual correspondence: m. d. artaria, c/o: departemen anthropology, fakultas ilmu sosial dan ilmu politik universitas airlangga. jl. airlangga 4–6 surabaya 60286, indonesia. e-mail: myrtati@gmail.com, phone: +62 31 5011 744. 124 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 122–126 while in the lower second incisor, a measurement between 6.1 mm and 7 mm would almost be certain to be a male. measurements below 7.6 in the upper second incisor and below 6.1 in the lower second incisor would be difficult to decide whether it belongs to a male or a female. a measurement between 9.1 mm and 9.5 mm of the upper first incisor would certainly grouped into male, while the lower first incisors could be differentiated more into males and females. a measurement between 4 mm and 4.7 mm belonged to a female, and between 5.9 mm and 8 mm belonged to a male. the upper second premolar overlapped greatly so that it was difficult to differentiate. meanwhile, the lower second premolar that had a measurement between 3.5 mm and 4.9 mm belonged to a female. an upper first premolar that had a measurement between 7.1 mm and 8 mm belonged to a male. a measurement of the lower first premolar between 7.6 mm and 8 mm belonged to a male. the measurement of upper second molar that fell between 10.1 mm and 11 mm would be grouped into a male. the lower second molar could be grouped further. a measurement fell between 5 mm to 5.9 mm belonged to a female, and a measurement between 11.1 mm to 13 mm belonged to a male. when the measurements of the upper first molar fell between 10.4 mm and 12 mm, it would be grouped into a male. lower first molar measurement that fell between 11.1 mm and 13 mm was grouped into a female. discussion according to barrett et al.,15 based on the coefficients of variation, the third molars and the maxillary lateral incisors varied most in size and the first molars least. in the samples of the present study in both males and females the mandibular second molar showed relatively large size variability. the least varied in tooth size was found in lower first incisors (li1) in females, and lower first premolar (lp1) in males. the mesiodistal molar crown diameters of these teeth ranged from 6 mm to 13 mm in males, and from 6 mm to 11 mm in the females. barrett et al.,15 found somewhat larger teeth sizes in his study, between 10.50 mm to 13.60 mm. the mandibular first molars is larger than mandibular second molars, so is the case of the upper molars. these findings were found in both males and females. several authors16–19 had drawn attention to the fact that in general tooth size and morphology were more stable in the mesial teeth than in the distal teeth within each tooth group, but here the reverse was true for the maxillary molars of males, and maxillary upper incisors of females. reverse sexual dimorphism-female teeth measurement is larger than those of males-has not been found in this sample such as found in prabhu and acharya.20 this is not surprising because comparisons of sexual dimorphism in teeth between different populations showed that it differed among different groups.21 table 1. the number of sample, minimum, maximum, mean, standard deviation, and the significance of differences between males and females males females sig. (2-tailed) n min max mean s n min max mean s uc 26 6.5 9.0 7.7 0.68 26 5.8 8.0 7.1 0.53 0.001 ui2 26 5.5 9.0 6.9 0.74 26 5.5 7.5 6.3 0.46 0.001 ui1 26 7.0 9.5 8.3 0.73 26 7.0 9.0 7.8 0.48 0.004 up2 26 4.0 9.0 6.6 0.96 25 5.0 10.0 6.4 0.95 0.467 up1 26 6.0 8.0 7.2 0.58 26 6.0 7.0 6.5 0.54 0.001 um2 26 8.0 11.0 9.6 0.75 25 8.0 10.0 8.6 0.67 0.001 um1 26 5.5 12.0 10.4 1.26 26 8.5 10.3 9.7 0.44 0.007 lc 26 6.0 9.0 6.9 0.60 26 4.0 8.0 6.0 0.70 0.001 li2 26 5.0 7.0 6.2 0.64 26 5.0 6.0 5.5 0.49 0.001 li1 26 4.8 8.0 5.9 0.76 26 4.0 5.8 5.0 0.31 0.001 lp2 26 5.0 8.5 7.0 0.77 26 3.5 10.0 6.6 1.18 0.229 lp1 26 6.0 8.0 7.0 0.54 26 6.0 7.5 6.6 0.46 0.002 lm2 26 6.0 13.0 10.3 1.41 26 5.0 11.0 8.9 1.37 0.001 lm1 26 6.0 13.0 10.9 1.23 26 6.0 11.0 9.8 1.03 0.002 notes: uc: upper canine, ui2: upper second incisor, ui1: upper first incisor, up2: upper second premolar, up1: upper first premolar, um2: upper second molar, um1: upper first molar, lc: lower canine, li2: lower second incisor, li1: lower first incisor, lp2: lower second premolar, lp1: lower first premolar, lm2: lower second molar, lm1: lower first molar 125artaria: dental measurements of deuteromalayid the degree of dimorphism varies within different populations although usually males have bigger tooth crowns than females even in recent human populations.22 although modern human did not emerge as significantly dimorphic as fossilized hominids, it is well-known that sexual dimorphism in canine exists.23 it was suggested that canine dimorphism is not developmentally homologous across primates.24 furthermore those researchers argued that there were growth rate differences between males and females resulting in canine dimorphism in primates, including human primate. recent studies have shown that dimorphism is the product of changes in both male and female traits, and developmental studies demonstrate the variety of ontogenetic pathways that can lead to dimorphism.25 similar degree of sexual dimorphism such as in recent human seems to have existed in australopithecus anamensis (living approximately 4 million years ago) from estimated canine crown heights, suggesting a low degree of sexual dimorphism.26 the result of this study revealed that the range of mesiodistal measurements of every tooth in males and females overlapped. this is similar to the size of body of males and females. this will make it more difficult to assign the sex of a single tooth. however, someone can always be stated that overall teeth size of females is significantly smaller than those of males. the size of the permanent human canine is one of the few sexually dimorphic features to be present in childhood and as such offers the opportunity to assist in the identification of sex in remains where no other appropriate criteria exist, such as in subadults.27 this sexual dimorphism in mesiodistal measurements canines of this sample showed highly significant differences-0.000 in lower canine and 0.001 in upper canine-which can be used as one of the teeth to aid sex identification. upper canine had significant mean differences in all measurements in swedish population.28 in this research, however, the most dimorphic mesiodistal measurements were found in upper first premolar, upper second molar, lower first and second incisors, and lower canine. it would be easier to assign the sex of a single tooth in cases where the tooth falls at the lowest end of range, or at the highest end of range. for example, an upper canine which has the mesiodistal measurement of 8 mm or above would be a male, and an upper canine which has the mesiodistal measurement of 6 mm or smaller would be a female. when the measurement of one tooth falls into the overlapping area, it is best to measure the complete set of teeth, as stated by prabhu and acharya20 that “the teeth from both jaws taken together were able to determine sex to higher levels”. when compared to other population, according to kuswandari and nishino,8 the mean of javanese mesiodistal crown measurements fell between hong kong chinese and australian aboriginal. it is suggested that the mean varied between populations. it should be noted that indonesia has a wide diversity of population, ranging from deuteromalayid at the western part to australomelanesoid at the eastern part. it is therefore exceptional to assume that there is a significant difference between the western population and the eastern population in indonesia, regarding the mean of mesiodistal measurements of teeth. assessment of variation in dental size gives a clue about the behavior of a population.29 the next step after measurement is considering the correlation of size to shape of teeth, which was not done in this research. this information can be obtained by calculating the correlation of two measurements within the same tooth. this should be done in a bigger size of sample. it then could be compared to other populations whether the sexual dimorphism is greater or less than those of other populations. according to ate et al.,29 the sexual dimorphism of populations could be different between one to another. it is concluded that teeth measurement of males is bigger than females, except maxillary and mandibular second premolars. larger sample size will be useful to find more evidence of differences in the tooth size in each field. further research involving other dental traits for sexual dimorphism studies would be useful. references 1. artaria md. growth of javanese. in: henneberg m, kilgariff j, eds. causes and effects of human variation. adelaide: australian society for human biology; 2001. p. 139–56. 2. artaria md. growth of adolescence. lambert academic publ 2009; isbn 3-83832879-5. p. 1–52. 3. glinka j. anthropological research on newborn infants of the middle flores in indonesia. przegl antropol 1969; 35: 249–60. 4. glinka j. growth of head and face in 7–17-year-old children and adolescents on palue island (lesser sunda islands). z morphol anthropol 1972; 64(1): 20–8. 5. glinka j. body-weight increase of newborn infants from middleflores from birth to the 12th month. zeitschrift für morphologie und anthropologie 1973; 65(2): 186–91. 6. kristiani s, koesbardiati t, glinka j. stature and weight of indonesian children compared to nchs-reference. folia medica indonesiana 2003; 39(2): 122–6. 7. rayner drt. the dental morphology of the indigenous people of the malay peninsula. thesis. anu, canberra, australia, 2000. 8. kuswandari s, nishino m. the mesiodistal crown diameters of primary dentition in indonesian javanese children. archives of oral biology 2004; 49(3): 217. 9. scott r, turner cg. the anthropology of modern human teeth. cambridge: cambridge university press; 2000. p. 105–8. 10. skinner mm, wood ba, boesch c, olejniczak aj, rosas a, smith tm, hublin jj. dental trait expression at the enamel-dentine junction of lower molars in extant and fossil hominoids. j hum evol 2008; 54(2): 173–86. 11. hilson s, fitzgerald c, flinn h. alternative dental measurements. am j phys anthr 2005; 126: 413–26. 12. el-zanatya hm, el-beialyb ar, el-ezzc ama, attiad kh, elbialye ar, mostafaf ya. three-dimensional dental measurements: an alternative to plaster models. am j orthod and dentofacial orthopedics 2010; 137(2): 259–65. 13. jain ak, garq n, singh j, anshari a, sangamesh b. mesiodistal crown diamension of the permanent dentition of the north indian population. indian journal of dentistry 2011; 2(2): 16–20. 14. singh sp, goyal a. mesiodistal crown dimensions of the permanent dentition in north indian children. j indian soc pedod prev dent 2006; 24: 192–6. 126 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 122–126 15. barrett mj, brown t, macdonald mr. dental observations on australian aborigines: mesiodistal crown diameters of permanent teeth. austr dent j 1963; 8(2): 150–6. 16. butler pm. studies of the mammalian dentition – differentiation of the post-canine dentition. journal of zoology 1939; b109(1): 1–36. 17. dahlberg aa. the dentition of american indians. in: laughlin ws, editor. the physical anthropology of the american indian. new york: viking fund; 1949. p. 138–76. 18. dahlberg aa. the paramolar tubercle (bolk). american journal of physical anthropology 1945; 3(1): 97–103. 19. moorrees cfa. the aleut dentition. cambridge: harvard university press; 1957. p. 5. 20. prabhu s, acharya ab. odontometric sex assessment in indians. forensic sci int. 2009; 192(1–3): 129. 21. zorba e, moraitis k, manolis sk. sexual dimorphism in permanent teeth of modern greeks forensic sci int. 2011; 210(1-3): 74–81. 22. schwartz gt, dean mc. sexual dimorphism in modern human permanent teeth. am j phys anthropol. 2005; 128(2): 312–7. 23. schwartz gt, dean mc. ontogeny of canine dimorphism in extant hominoids. am j phys anthropol. 2001; 115(3): 269–83. 24. schwartz gt, miller er, gunnell gf. developmental processes and canine dimorphism in primate evolution. j hum evol. 2005; 48(1): 97–103. 25. plavcan jm. sexual dimorphism in primate evolution. am j phys anthropol. 2001; suppl 33: 25–53. 26. plavcan jm, ward cv, paulus fl. estimating canine tooth crown height in early australopithecus. j hum evol. 2009; 57(1): 2–10. 27. hasset b. technical note: estimating sex using cervical canine odontometrics: a test using a known sex sample. am j phys anthropol. 2011; 146(3): 486–9. 28. lund h, mörnstad h. gender determination by odontometrics in a swedish population. j forensic odontostomatol. 1999; 17(2): 30–4. 29. ate mm, karaman ff, ican my, erdem tl. sexual differences in turkish dentition. legal medicine 2006; 8(5): 288–92. << 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 187 volume 45 number 4 december 2012 case report aesthetic treatment on anterior teeth crown fracture caused by dental trauma nanik zubaidah department of conservative dentistry faculty of dentistry, universitas airlangga surabaya indonesia abstract background: complicated crown fracture is a tooth fracture that involve enamel, dentine and pulp. the incidence of complicated crown fracture ranges from 2% to 13% of all dental injuries and the most commonly involved teeth are the maxillary central incisors. various treatment modalities are available depending on the clinical, physiological and radiographic examination of the involved teeth. purpose: the aim of this case report is to present the management of crown fractures with pulpal exposure caused by traumatic injury, through endorestoration approach to reconstruct the shape and function of the teeth. case: a 17 years old male with complicated crown fractures of anterior teeth #11 #21 and #22. the patient wish for aesthetic dental treatment in both of its form and function. case management: crown fractures of anterior teeth with exposed pulp caused by traumatic injury were reconstructed by endorestoration approach. the endodontic treatment with post and core insertion in the root canal which will increase its retention and porcelain fused to metal crown which will aesthetically recover its original form and function. after restoration the patient feel very glad and confident with the result. conclusion: endorestoration treatment on anterior teeth with complicated crown fractures and exposed pulp is able to recover the normal form, function and dental aesthetic in accordance with stomatognatic system and self confidence. key words: traumatic dental injury, complicated crown fracture, endorestoration abstrak latar belakang: fraktur mahkota kompleks (complicated) adalah fraktur pada mahkota gigi yang melibatkan enamel, dentin dan pulpa. kejadian dari fraktur mahkota kompleks bervariasi antara 2-13% dari semua trauma gigi dan sebagian besar gigi yang terkena adalah gigi insisif pertama rahang atas. berbagai macam cara perawatan yang dilakukan tergantung pada hasil pemeriksaan klinis, psikologis dan radiografis dari gigi yang terkena. tujuan: laporan kasus ini menjelaskan penatalaksanaan fraktur mahkota gigi dengan pulpa terbuka akibat trauma dengan perawatan endorestorasi untuk mengembalikan bentuk dan fungsi gigi. kasus: penderita pria umur 17 tahun dengan fraktur mahkota pada gigi anterior #11, #21 dan #22. penderita tersebut menginginkan perawatan estetik untuk mengembalikan estetik baik bentuk maupun fungsi giginya. tatalaksana kasus: fraktur mahkota gigi anterior dengan pulpa terbuka akibat trauma gigi dikembalikan melalui pendekatan perawatan endorestorasi. perawatan endodontik dengan pasak tuang dan inti yang dimasukkan ke dalam saluran akar akan meningkatkan retensi dan kemudian ditutup dengan mahkota porselen fused to metal dapat mengembalikan bentuk maupun fungsinya. selesai perawatan, pasien merasa senang dengan hasil perawatan tersebut dan hal ini menunjukkan peningkatan kepercayaan diri pasien. kesimpulan: perawatan endorestorasi gigi anterior dengan fraktur mahkota kompleks gigi anterior dengan pulpa terbuka dapat mengembalikan bentuk, fungsi dan estetik yang normal sesuai dengan sistem stomatognatik dan meningkatkan kepercayaan diri. kata kunci: jejas gigi traumatik, fraktur mahkota kompleks, endorestorasi correspondence: nanik zubaidah, c/o departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: nanikzubaidah@yahoo.com 188 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 187–191 introduction dental traumatic injury treatment is an important aspect of dentistry. dental traumatic injury occurs mostly in young patients with varying degrees of severity ranging from enamel fracture to avulsion that will affect psychologically to parents and children, especially if the injury affects permanent teeth and involves extensive loss of tooth structure.1-3 the trauma, moreover, can also cause either damage to the pulp of teeth, with or without crown or root damage, or removal of teeth from the socket. sweet stated that a high percentage of the anterior teeth in the upper jaw suffer from fractures, and 90% of which really stand out, so the lip can not adequately cover them.1,4 teeth can suffer from trauma caused by activities, such as falling off a bike, car accidents, crashes, domestic violence, sports or other causes that may lead to disability wound in individuals as well as various forms of fractures, including crown fracture and tooth root fracture. crown fracture with open pulp involving enamel, dentin, and pulp known as complicated crown fracture.5 complicated crown fracture occurs about 2-13% of all dental injuries, most of which involve maxillary first incisors.2,6,7 according to chan,8 the frequency of fracture in permanent incisors occurs in children about 5-20%. the degree of the open exposured pulp can vary from a hole with needle size to a hole opened on coronal pulp. if the surface is left open, there will be necrosis process caused by bacterial contamination. the result of treatment depends on the extent of injury, the quality and timeliness of initial care, and the technique of treatment. trauma followed with fracture of anterior teeth, especially permanent incisor, is considered not only as a tragic experience for young people, but also as the loss of anterior teeth that have a strong psychological impact on children and parents. if trauma involves both extensive loss of tooth structure and pain and discomfort sensation, it will change the appearance of the child and become the subject of bully by his or her peers. however, with recent advances in the field of esthetic in dentistry, restoration treatment is considered as the best solution to meet the high expectations of patients to be able to smile confidently.9,10 a violent blow from frontal and horizontal direction can cause fracture line from several points on the crown, and then can longitudinally extend, with or without the involvement of the pulp, to mesial or distal subgingival area. fractures of teeth can be classified into six classes: class 1: enamel fracture; class 2: dentin fracture without opening the pulp; class 3: crown fracture with opening the pulp; class 4: root fracture; class 5: subluxation teeth; and grade 6: intrusion teeth. meanwhile, crown fracture can be classified into fracture class 3.4 crown fracture with pulp exposed due to trauma can be treated with four kinds of treatment: pulpotomy, apexification, pulpectomy and root resection.11 the appropriate treatment for the pulp and its required restoration, nevertheless, is determined with the extent of the fracture. extensive fracture also requires root canal treatment using post and core that support the crown, but it still depends on the age of patients. in dentistry, especially in aesthetic dental conservation, fracture is a kind of tooth decay requiring an aesthetic care. a tooth suffering from complicated crown fracture with open pulp can be treated with endorestoration treatment including endodontic treatment using post-core retention and porcelain fused to metal crown.1,5,12 this case report is aimed to report the esthetic improvement of the anterior teeth the anterior teeth suffering from complicated crown fracture with open pulp due to trauma by conducting endorestoration treatment in order to improve its forms, functions, and aesthetics as normal as the original ones with stomatognatic system. a case of trauma to the maxillary incisors suffering from complicated crown fracture and treated with endorestoration treatment, including endodontic treatment, followed by insertion of casting post-core and of porcelain fused to metal crown will be reported. case a 17 years old male came to universitas airlangga dental hospital. he had motorcycle accident four days ago. the crown of his anterior teeth #11, #21 and #22 fractured around the cervical areas. torn ragged wound and swelling figure 1. a) the condition of upper lip with torn ragged wound; b) the initial condition of complicated crown fracture on teeth #11, #21 and #22. a b 189zubaidah: aesthetic on anterior teeth crown fracture caused by dental trauma was found around the vestibule areas of the teeth #11, #21, #22 and the upper lip (figure 1a). the patient wanted to have his anterior teeth aesthetically improved to normalized its form and function. intra oral examination was conducted on the teeth #11, #21 and #22 suffering from complicated crown fracture causing an fully opened pulp around tooth cervical areas (figure 1b). panoramic and local x-rays were conducted (figure 2). based on the results, there was radiolucency appeared on the periodontal ligament of the teeth #11, #21 and #22. the teeth #11, #21 and #22 were then diagnosed as pulpitis irreversible. case management at the first visit, to dental hospital emergency treatment that consisted of soft tissue cleansing around the mouth and lips with saline and hydrogen peroxide and giving antibiotics, analgesics, and anti-inflammatory were conducted. the patient was also suggested to maintain his oral hygiene by using chlorhexidine mouthwash and having soft food diet. the emergency care should immediately be taken to prevent the patient from soft tissue.2 the crowns of fractured teeth #11, #21 and #22 were extracted until the cervical areas and gingivectomy was done in the palatal rugae frenulum areas to improve the shape of excessive gingival contour lines that corresponded to palatal and labial cervical portion of the anterior teeth (figure 3). after wound healed, an impression were taken on maxillary and mandibula for model study. dental records figure 2. panoramic x-ray image. figure 3. the condition of teeth after the releasing the crowns of #11, #21, #22 prior to the treatment. figure 4. temporary tooth crown on teeth #11, #21 and #22. were then also conducted in order to record occlusion and relationship, and to be used as temporary jacket crowns on teeth #11, #21 and #22 to prevent bad appearance during the dental treatment. moreover, pulpectomy treatment was conducted for one visit on the teeth #11, #21 and #22 with crown down pressurelless technique by using file pro taper preparation tools and single cone filling technique by using ultracal pasta sealer material. in the next stage, the preparation of post channel was conducted by taking gutapercha using gates gliden drill, and then followed with conducting peeso reamer leaving the gutta-percha about 4-5 mm from the apex of the root canal. afterwards, the root canal was printed by using elastomer print materials to make post and core. the temporary crown was then inserted for maintaining the aesthetics of the teeth during manufacturing process of casting post and core in dental laboratoium (figure 4). few days later cast posts and cores were inserted into the root canal of teeth #11, #21 and #22 by using glass ionomer cement type i luting cement (figure 5a). local x-ray were conducted (figure 5b). impression was done on teeth #11, #21 and #22 by double impression technique. in the next stage, temporary crown was inserted and the print result was sent to the dental lab for making porcelain fused to metal crowns. then, the porcelain fused to metal crowns were inserted on the teeth #11, #21 and #22 by using glass ionomer cement type i luting cement (figure 6). evaluation was followed 3 and 6 months after treatment in which the patient got a good result (figure 7). discussion various conditions of trauma can generally cause crown fractures although many literatures suggest several predominant causes, such as falling either while playing and running or during sports activities, as well as having car accidents and a blow on the face. anterior teeth are actually more susceptible to trauma, approximately 80% it the central maxillary incisors followed by lateral maxillary incisors and central mandibular incisors.1 generally dental trauma can involve pulp both directly and indirectly, so endodontics is considered to play an important role in the evaluation and treatment of dental 190 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 187–191 injuries. fractures that involving the edge of gingiva area need an endodontic treatment followed with crown restorations using post and core but it depend on the age of patient.5 in this case, teeth #11, #21 and #22 suffering from complicated crown fracture were treated by endorestoration treatment, which was pulpectomy care followed by the installation of retention, such as casting post and core, and finished with the insertion of porcelain fused to metal crown. if patient with an open pulp tooth fracture case came to be treated after 72 hours or more, the treatment option is endodontic treatment by removing all affected pulp tissue; but if more than half the coronal missing, it is necessary for post and core to be inserted restored with core crown. the post and core restorations are used to reshape the structure of the lost crown.9,12 restoration of a fractured tooth after endodontic treatment depends on the structure lost of tooth crown. if the fracture is only on the enamel and dentin, it can be restored with a simple composite or porcelain veneers, but, if it is more than half of the crown missing, the tooth will require post and core crowns.9 the dental care after endodontic treatment is restore the remaining root and crown of tooth by using retentive and stable post crown, so it will not easily broken and can be used as long as possible in the oral cavity to replace the original tooth. characteristic of tooth is relatively more brittle and prone to fracture than vital one due to tooth internal moisture decrease and it weaken after endodontic treatment are weaken the remaining tooth structure and non-vital tooth often get color shifts.8.21 therefore, one visit endodontic treatment was conducted on teeth #11, #21 and #22 because the shape of the root canals was normal and single-rooted with the diagnosis of pulpitis irreversible with periodontal ligament abnormalities without clinical symptoms. thus, this one visit endodontic treatment aimed not only to prevent the spread of the disease from pulp into periapical tissues, but also to restore the periapical tissues. it also gives the advantage to reduce the risk of infection between visits.4 preparation of the root canal #11, #21 and #22 was then conducted by using crown down pressureless technique with a protaper tool through the coronal-apical approach. this technique is very beneficial because 1/3 of microorganisms are located in coronal, another 1/3 that has drawn first before going into the apical area is located in center, and the other 1/3 that had more perfect irrigation are located in apical.13 afterwards, intracanal retention was conducted to restore the crown by using post as retention on its restoration. post used in this case was casting post (artificial post) and core also used was aimed to increase the retention of teeth and position of porcelain fused to metal crown restoration. position of teeth with crowns that have been depleted or with heavy occlusal forces can be indicated by the crown with the casting post and core. teeth both with short clinical crowns or without clinical crowns, but still having adequate roots in terms of length, thickness and embedded in the alveolar bone, and with the ratio between the root and the crown of the teeth can qualify for post length inserted into the root canal length at least equal to the crown length, thus, casting post and core are the best choices.15,16 figure 5. a) the inserted of casting post and core on teeth #11, #21 and #22; b) casting post and core insertion in the root canal. figure 7. control after 3 month.figure 6. the insertion of porcelain fused to metal crown on teeth #11, #21 and #22. a b 191zubaidah: aesthetic on anterior teeth crown fracture caused by dental trauma moreover, the selection of the post and core designs as the retention in the root canal is depend on to the rest of the crown, the occlusal pressure force (chewing power), the diameter of the root canal, the location of the teeth, and the health of the periodontal tissue as post crown supporter.8 in determining the type of post crown, it can not be separated from efforts to design the post. the procedure of selection of design and preparation of root canal should be done in such a choice that does not further debilitating the remaining tooth tissues and cause a risk of post disposition.17 furthermore, the teeth #11, #21 and #22 used post and core since it generally had several advantages such as: post and core become one unit, and could accurately adapt the shape of root canal preparation, as a result, it become retentive and stable, it does not require additional retention form pin, and could easily adjust to the irregular shape of root canal that result strong and effective post and core. the setting of post and core on the teeth #11, #21, and #22 was prepared one by one at the same time, to get the shape and size similar to the normal size of each tooth and anterior dental arch with normal overbite and overjet to achieve a good and harmonious aesthetics.15 the principle of dental care that has already obtained endodontic treatment is actually to restore tooth root and crown by using retentive and stable post and core crowns, so they are not easily malpositioned and can be used as long as possible in the oral cavity like the original tooth. moreover, it should be noted that tooth having endodontic treatment is relatively more brittle and fragile (easily fracture) than healthy tooth due to organic and biological changes caused pulp necrose, the reduction of the tooth internal tissue, as well as the lack of the linkage between the tooth enamel and dentin due to the grinding of dentin tissue during preparation of root canal, that can caused discoloration. therefore, comprehensive protection is necessary by strengthening post and core as well as porcelain fused to metal crown restoration in order to prevent the tooth from fracture.8,15 porcelain fused to metal crown restoration is the best option to restore tooth aesthetics, especially the shape and color of the tooth anatomy in accordance with natural teeth, and to be able to function naturally. similarly, hume18 also said that the porcelain jacket crown is the best choice to restore the aesthetics of the central incisors optimally. furthermore, among 956 patients, only 63% of them were satisfied with the appearance of the anterior tooth using a crown veil, and 79% of them were satisfied with 4 or more anterior teeth using porcelain fused to metal crowns.19 porcelain is also considered to be the most satisfiying treatment because of its natural color and aesthetics. the cementation of the porcelain fused to metal crown towards post-core uses glass ionomer cement type i (luting sement).20 the success of a restoration, however, is determined by retention, stability, aesthetics (especially anterior teeth) as well as biological aspects. cooperation and understanding between patient and dentist is also the basis of success. improvement on aesthetic appearance becomes increasingly important in today’s modern dental practice.21 this case showed that that tooth with complicated crown fracture due to trauma can be treated with endorestoration care not only to maintain the tooth as long as possible in the oral cavity, but also to restore the form, function and aesthetic of the tooth in accordance with stomatognatic system. references 1. mutan ha, erdal o, �ahya oz, muzaffer a. treatment of traumatized maxillary permanent lateral and central horizontal root fractures. a case repord. indian j dent res 2008; 19(4): 354–6. 2. aggarwal v, logami a, shah n. complicated crown fracturesmanagement and treatment option. int endod j 2009; 42(8): 740–53. 3. biner s, dang l, gomes, j, mc murran t, schneiders. whats is the best treatment of horizontal root fracture teeth. an evidance based report. toronto: faculty of dentistry toronto on canada; 2010. p. 1–23. 4. grossman li. oliet s, del rio ce. 1988 ilmu endodontik dalam praktek. 1st ed. jakarta: penerbit buku kedokteran ecg; 1995. p. 196–380. 5. torabinejad m, walton re. endodontics principles and practice. 4th ed. st louis: missouri; 2009. p. 165–7. 6. parirokh m, kakoel s, aeskandizadeh a. maturogenesis of complicated crown fracture: case report with 8 years follow up. iranian endod j 2007; 2: 32–6. 7. altinok b, kargul b. case report. use of mineral trioxide agregate in permanen incisor with horizontal root fractures: a five �ear follow up. 2010, december (ix), 4: 2–6. 8. chan dcn, myers ml, chipped. fracture, or endodonticcally treated teeth. in: goldstein re, editor. esthetics in dentistry. 2nd ed. hamilton, london: bc decker inc; 2002. p. 537–9. 9. jain v, gupta r, duggal r, parkash h. restoration of traumatized anterior teeth by interdisciplinary approach: report of three cases. j indian soc pedod prev dent-december 2000; 20(1): 193–6. 10. heda cb, heda aa, kulkarni ss. a multi-disciplinary approach in the management of traumatized tooth with complicated crown-root fracture: a case report. j indian soc pedod prev dent 2006; 24(4): 197–200. 11. schulze a. dental traumatic injuries in sport accident. clinic sports medicine international (csmi) 2008; 1(8): 13–5. 12. catagay b, osman th, mehmet �. restoration of crown fracture with a fiber post, polyethylene and composit resin. rev clin pesq odontol curitiba 2009; 5(1):73–7. 13. nesha g, amit g. texbook of endodontic. 1st ed. jaype brothers medical publishers; 2007. p. 196. 14. shillingburg ht, kessler w. restoration of endodontically treated tooth. chicago, quintessence publishing co. inc; 1982. p. 17–8. 15. tohiroh dj, rahardo tbw. retensi mahkota pasak berdasarkan desain pasak. kumpulan naskah temu ilmiah nasional i (timnas i) peringatan 70 tahun pendidikan dokter gigi indonesia, 1998; p. 145–6. 16. kamizar. etiologi dan pencegahan kasus-kasus iatrogenic dalam restorasi pasca endodontic. jkgui 2000; (edisi khusus): 470–4. 17. ziebert gj. restoration of endodonticcally treated teeth. in: malone wfp, koth dl, kaiser da, morgano sm, eds. tylman’s theory and practice of fixed prosthodontic. 8th ed. st louis, tokyo: ishiyaku euro america inc; 2009. p. 407–17. 18. hume wr. preservationand restoration of tooth structure. london: the cv mosby co; 1998. p. 185–90. 19. qualthrough aje, burke fjt. a look at dental esthetics. j quintessence international 1994; 25(1): 7–9. 20. �uzugullu b, canay s. metal–ceramic dowel crown restoration for severely damaged teeth: a clinical report. indiana j dent res 2009; 20(1): 110–2. 21. zubaidah n. the management of horizontal crown fracture caused by traumatic injury with endorestoration treatment. dent j (majalah kedokteran gigi) 2011; 44(3): 154–8. 130 vol. 42. no. 3 july–september 2009 research report calprotectin mrna (mrp8/mrp14) expression in neutrophils of periodontitis patients with type 2 diabetes mellitus ahmad syaify1, marsetyawan hnes2, sudibyo1, and suryono1 1 departement of periodontology, faculty of dentistry gadjah mada university 2 department of immunology, faculty of medicine gadjah mada university yogyakarta indonesia abstract background: calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils and monocytes/machrophages. this protein is known to be a marker for several inflammatory diseases including periodontitis. in type 2 diabetes mellitus patients, the severity of periodontitis was strongly thought to be caused by decreasing of leukocytes function such as neutrophils. previous research found that the calprotectin level in serum of periodontitis patients with type 2 dm is higher than periodontits patients non dm. purpose: the aim of this study was to determine calprotectin mrna (mrp8/mrp14) expression in human neutrophils of periodontitis patients with type 2 diabetes mellitus. methods: neutrophils were isolated from the peripheral blood of periodontitis patients with uncontrolled type 2 dm, controlled type 2 dm, and non dm. the expression of calprotectin mrna (mrp8 and mrp14) were detected by rtpcr. result: the result showed that the value of mrna calprotectin expression in dm patients were higher than non dm, and the highest expression was on the uncontrolled type 2 dm. conclusion: the basal level of calprotectin mrna mrp8/mrp14 expression increased in neutrophil of periodontitis patient with type 2 dm compared non diabetic subjects. it was suggested that high basal level of calprotectin mrna has a role in the regulation of periodontitis severity with diabetes mellitus patients. key words: calprotectin mrna, periodontitis, type2 diabetes mellitus correspondence: ahmad syaify, c/o: bagian periodonsia, fakultas kedokteran gigi universitas gadjah mada. jl. bulaksumur yogyakarta 55281, indonesia. e-mail: ahmad_syaify@yahoo.com introduction calprotectin is a calcium binding protein which has a molecular mass of 36.5 kda, belongs to the s-100 protein family which can be detected in neutrophils, monocytes, and epithelial cells, being composed of two subunits macrophage migration inhibitory factor-related protetin 8 and 14 (mrp8 and mrp14).1,2 it is known that calprotectin plays an important role in the innate immunity, and its level is markedly increased in plasma, feces, and synovial fluid from patients with infections and inflammatory diseases.3 calprotectin level in gingival crevicular fluid (gcf) of periodontitis patients was significantly higher than healthy subjects and it was detected in gingival tissue only from periodontitis patients.4,5 diabetes mellitus (dm) is one of health problems found in the world and about 90 percent were type 2 non insulin dependent diabetes mellitus (niddm). in indonesia, the incidence rate among those who are above 15 years old in indonesia was 1.2–2.3% and tends to increase.4 among the late complications associated to the diabetes mellitus, periodontal disease has been highlighted, and it can be more severe and refractory to treatment than in healthy subjects.5 the incidence of periodontitis increases, more frequent and severe in diabetic patients with more advanced systemic complications, and the increased susceptibility does not correlate with increased levels of dental plaque or calculus.6 the severity of periodontitis in diabetic patient was strongly thought caused by decreasing of leukocytes function such as neutrophils. periodontitis, as one of very frequent complication of diabetes mellitus, was known as caused by immune response disturbaces such as; decrease of chemotactic, adherence, and phagocytosis of neutrophils.7 it is known that calprotectin (mrp8 and mrp14), forms about 60% protein in neutrophil, plays a role for neutrophils 131syaify, et al.: calprotein mrna (mrp8/mrp14) expression in neutrophils function. however, the exact role of calprotectin in periodontitis patient with diabetes mellitus is unclear. in previous research, it was found that the calprotectin level in serum of periodontitis patients with type 2 dm was higher than periodontitis patients non dm.8 the aim of this study was to determine calprotectin expression in human neutrophils of periodontitis patients with type 2 diabetes mellitus. materials and methods peripheral venous blood from 10 periodontitis patients with type 2 diabetes mellitus (consisted of 5 uncontrolled dm, 5 controlled dm), and 5 subjects non dm were collected into heparinized tubes to avoid blood aglutination before next procedure. all patients who visited dr. sardjito teaching hospital yogyakarta gave their informed consent to participate in this research. neutrophiles were separated from heparinized blood by density gradient centrifugation using histopaque®-1077 (sigma-aldrich), and this cells were collected in eppendorf tube as samples for rna determination neutrophils from uncontrolled dm patients, controlled dm, and non dm were isolated from its rna using trizol® reagent (invitrogen) according to the manufacturer’s protocol. trizol reagent 1 ml was added into pellet cell, suspensed with injection spuit and incubated in room temperature for 5 minutes. chloroform 20 ml was added and mixed by hands, then centrifugated 12.000 g for 15 minutes in 4° c temperature. the aqueous phase was transfered by mixing with fresh tube and precipitated the rna from the aqueous phase by mixing with 500 ml isoprophyl alcohol. rna samples were incubated at room temperature for 10 minutes and then centrifuged at 12.000 × g for 10 minutes at 4° c. rna pellet was washed with 1 ml ethanol 75% and mixed by vortex and centrifugedml ethanol 75% and mixed by vortex and centrifuged at 7.500 × g for 5 minutes at 4° c. then rna pellet was dried with vacuum dry for 3 minutes and redisolved in 30 ml rnase free water. calprotectin mrna (mrp8 and mrp14) expression was determined by reverse transcriptase polymerase chain reaction (rt-pcr) according to manufacturer’s procedure. we used two steps rt-pcr procedure, the first step was cdna synthesis from rna samples and continued with pcr procedure as the second step. master mix for cdna syntesis were ; 10 x reaction buffer, 25mm mgcl2, deoxy nucleotid mix, primer pd (t)6, rnase inhibitor, and amv rt. to determined mrna calprotectin, cdna samples and pcr primers for calprotectin (table 1) were amplified by polymerase chain reaction (pcr). pcr products then were checked by electrophoresis to measured band intesity of the mrp8 and mrp14 calprotectin mrna. the intensity of each band was normal if compared to the gapdh band. the expression of calprotectin (mrp18 and mrp14) mrna was represented as the intensity of bands that were checked by thin layer chromatography. table 1. pcr primers oligonucleotide sequence product mrp8 sense 5’-gctggagaaagccttgaactc-3’ 232 bp mrp8 antisense 5’-ccacgcccatctttatcacca-3’ mrp14 sense 5’-tcgcagctggaacgcaacata-3’ 213 bp mrp14 antisense 5’-agctcagctgc ttgtctgcat-3’ gadph sense 5’-tccacacc ctgttgctgta-3’ 558 bp gapdh antisense 5’-accacagtccatgccatcac-3’ result polymerase chain reaction (pcr) product of calprotectin mrna (mrp8 and mrp14) and also gapdh were checked by electrophoresis to know each band position. in this study, band position of all oligonucleotides were suitable with base pairs (bp) values of mrp8, mrp14, and gapdh as mentioned by the manufacturer’s protocol. compared with the 100 bp dna ladder, these bands were in the correct position as described on figure 1. to investigate whether diabetes mellitus affects the mrp8 and mrp14 expression in human neutrophils, the expression of mrp8/14 mrna was examined. when neutrophils were isolated from diabetic and non diabetic subjects, the expression of mrp8 mrna significantly higher than mrp14 mrna. the intensity of bands markedly increased in uncontrolled dm patient (figure 2). mrp8 mrp14 dna ladder 232 bp 213 bp 558 bp dna ladder gapdh figure 1. band position of mrp8, mrp14, gapdh primers comparing with 100bp dna ladder. all primers were in the correct positions where the values of base pairs (bp) were 558 bp for gapdh, 213 bp for mrp14, and 232 bp for mrp8. both mrp8 and mpr14 mrna expression in periodontitis patients with type 2 dm were higher than non dm patients, while the highest expression of mrna mrp8/mrp14 was in uncontrolled dm (figure 3); suggesting that mrp8/mrp14 mrna has important role on severity of periodontitis in diabetic patient. 132 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 130-133 in blood of uncontrolled diabetes mellitus patient. pro inflammatory cytokines such as tnf-alfa and il1-beta were present in large amount in blood circulating diabetic patients, and it was reported that the level was higher in uncontrolled dm than controlled dm.12,13 these cytokines, both tnf-alpha and il-1beta, are found in the circulating peripheral blood and their level were increased in several inflammatory diseases, including periodontitis. in previous study, it was reported that expression of mrp8/mrp14 was increased in monocyte by several factors and compounds including tnf-alpha, and it was also known that this cytokine caned stimulate calprotectin expression in human neutrophils.14,15 mrp8/mrp14 is found predominantly in a cytosolic location in both neutrophils and monocytes, it represents about 45–60% of the total neutrophils cytosolic protein.1,16 large amounts of mrp8/mrp14 is necessary in cell such as the neutrophil which must make quick responses to environmental signals.10 after activations of neutrophils, mrp8 and mrp14 are released into the compartment extracellular via tubulin dependent pathway, where they are known to promote the adhesion of neutrophlis on endothelium.16 previously, high basal level concentration of calprotectin intracellular in monocyte and neutrophils that was determined by elisa kit was identified (unpublished data). increasing basal level of calprotectin in serum of periodontitis patients with type 2 diabetes mellitus was also found.8 in this study, the same pattern of increasing calprotectin mrna expression in uncontrolled type 2 diabetic patients was found. it can be understood, because uncontrolled diabetic patients have persistently high concentration of tnf-alpha in their blood circulation, whereas tnf-alpha potentially stimulate neutrophils to increase mrp8/mrp14 mrna expression and calprotectin production. in conclusion, the calprotectin mrna mrp8/mrp14 expression was increased in neutrophil of periodontitis patient with type 2 dm compared to non diabetic subjects. it was suggested that high expression of calprotectin mrna has a role in the regulation of periodontitis with severity in diabetes mellitus. acknowledgment the authors were very grateful to lembaga ejikman jakarta which gave us an opportunity to conduct the research. this study was supported by grant from research and development of health department republic indonesia (litbang depkes ri). references 1. nisopakultorn k, ross kf, herzberg mc. calprotectin expression inhibits bacterial binding to mucosal epithelial cells. infection and immunity2001; 69(6):3692–6. 2. kerkhoff c, klempt m, sorg c. novel insights into structure and function of mpr8 (s199a8) and mpr14 (s100a9). biochem biophsy acta 1998; 1448:200–11. figure 2. calprotectin mrp8/mrp14 mrna expression of neutrophils from diabetic and non diabetic subjects. the expression of mrna was determined in 1ug rna from resting neutrophils by rt-pcr using mrp8, mrp14, and gapdh primers. discussion this study identified that calprotectin mrna (mrp8/ mrp14) expression in neutrophils of periodontitis with type 2 dm was different from periodontitis patients without dm, and the highest calprotectin expression was on uncontrolled type 2 dm patients compared with controlled dm and non dm (figure 3). the difference expression of calprotectin from neutrophils on diabetic and non diabetic patients were strongly suspected to be correlated with impairment of immune cell function, especially innate immunity cells such as neutrophils and monocytes. some authors mentioned that this impairment of function including chemotaxis, diapedesis, and phagocytosis of neutrophils,9 but which one of those functions that was very dominant to cause the severity of diabetic periodontitis is unclear. our result showed that calprotectin mrp8/14 mrna expression in periodontitis patients with diabetes mellitus was different from non diabetic subjects, while calprotectin has been well known as a chemotactic factor.10,11 the previous study demonstrated that diabetic patients with severe periodontitis had depressed pmn (neutrophils) chemotaxis compared to those with periodontitis on non diabetic subjects with severe or mild periodontitis.6 the result also identified that the highest calprotectin mrp8/14 mrna expression were in periodontitis patients with uncontrolled type 2 diabetes mellitus. it maybe caused by pro inflammatory cytokines that markedly increased figure 3. expression of mrp8 and mrp14 mrna in neutrophils of uncontrolled type 2 dm, controlled dm, and non dm subjects. 133syaify, et al.: calprotein mrna (mrp8/mrp14) expression in neutrophils 3. fagerhol mk. calportectin, a faecal marker of organic gastrointestinal abnormality. lancet 2000; 356:1783–4.lancet 2000; 356:1783–4. 4. perkeni. konsensus pengelolaan diabetes mellitus tipe 2 di indonesia.perkeni. konsensus pengelolaan diabetes mellitus tipe 2 di indonesia. jakarta. 2001. p. 1, 42. 5. arrieate-blanco jj, bartolome-villar b, jimenez-martinez e, saavedravallejo p. dental problems in patients with dm (ii): gingival index and periodontal disease. med oral. 2003; 8:233–47.med oral. 2003; 8:233–47. 6. mealey b. diabetes and periodontal disease: position paper. j periodontol 2000; 70:935–49. 7. iacopino am, cuttler cw. pathophysiological relationship between periodontitis and systemic disease: recent concept involving serum lipids in periodontitis and systemic disease. j periodontol 2000; 71(8): 1375–84. 8. syaify a, marsetyawan, suryono. level of calprotectin in serum of periodontitis patient with type 2 diabetes mellitus. journal of pdgi 2008 march; xxii(special edition in congress):1–5. 9. little jw, falace da. dental management of the medically compromised patient. 3rd ed. st. louis: mosby co; 1988. p. 291–307. 10. hessian pa, edgeworth j, hogg n. mrp-8 and mrp-14, two abundant ca –binding proteins of neutrophil and monocytes. j leukocyte biol 1993; 53:197–203. 11. miyasaki kt, bodeua am, shafer wm, pohl j, murthy rk, lehrer ri. new ideas about neutrophil antimicrobial mechanisme: antibiotic peptides, postphagocytic protein processing, and cytosolic defense factors. in: molecular pathogenesis of periodontal disease. washington dc: american society for microbiol; 1994. p. 321–35. 12. marhamah. pathomechanism of periodontal tissue damage in patients with type 2 diebets mellitus. dissertation. makasar: hasanuddin university; 2004. p. 79–82. 13. graves dt, liu r, alikhani m, al-mashat h, trackman pc. diabetes-enhanced inflammation and apoptosis: impact on periodontal pathology. j dent res 2006; 85(1):15–21. 14. suryono, kido j, hayashi n, kataoka m, nagata t. effect of porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alfa and interleukin 1-beta on calprotectin release in human monocytes. j periodontol 2003; 74:1719–24.j periodontol 2003; 74:1719–24. 15. kido j, kido r, suryono, kataoka m, fagerhol mk, nagata t. calprotectin release from human neutrophils is induced by porphyromonas gingivalis lipopolysaccharide via the cd-14-toll like receptor pathway. j periodontol res 2003; 38:557–63. 16. ehlermann p, eggers k, bierhaus a, most p, weuchenhan d, greten j, nawroth. pp, katus ha, remppis a. increased proinflamatory endothelial respons to s100a8/9 after preactivation through advanced glycation end products. cardiovascular diabetology 2006; 5(6):1–9. mkgs vol 45 no 2 april-juni 2012.indd 107 volume 45 number 2 june 2012 craniofacial morphology of children with complete unilateral cleft lip and palate following labioplasty and palatoplasty sigit handoko utomo1, krisnawati2, and benny m. soegiharto2 1 dental practitioner 2 department of orthodontics, faculty of dentistry, universitas indonesia jakarta indonesia abstract background: a complete unilateral cleft lip and palate generally results in asymmetry of the midface. the lack of continuity in the perilabial musculature through the midline contributes to a malpositioning of the underlying osseus structures which are often underdeveloped. purpose: the purpose of this study was to determine whether there are differences in the craniofacial morphology among children with complete unilateral cleft lip and palate following labioplasty and palatoplasty as compared with children without cleft lip and palate at the same pubertal age. methods: a series of 14 consecutively treated subjects with complete unilateral cleft lip and palate following labioplasty and palatoplasty were compared with 14 pubertal stage-matched controls with normal craniofacial structure. pubertal stage was determined with cervical vertebral maturation (cvm) method improved by baccetti et al, 2002. lateral cephalograms were used for comparison. an unpaired t-test was run for 14 subjects with complete unilateral cleft lip and palate and 14 normal subjects. results: there were significant cephalometric differences in anterior cranial base length (p = .002), cranial base length (p = .001), maxillary length (p = .000), mandibular length (p = .000), mandibular ramus height (p = .000), mandibular body length (p = .002), and upper anterior face height (p = .004). there was no significant cephalometric difference in posterior cranial base length (p = .051), lower anterior face height (p = .206), posterior face height (p = .865), growth pattern/ facial type (p = .202). conclusion: there were craniofacial morphology differences between children with complete unilateral cleft lip and palate post labioplasty and palatoplasty and children without cleft lip and palate at the age of pubertal. children with complete unilateral cleft lip and palate post labioplasty and palatoplasty had shorter length of the anterior cranial base, cranial base, maxilla, mandible, mandibular ramus height, mandibular body, and upper anterior face height as compared with children without cleft lip and palate at the age of pubertal. key words: cephalometrics, complete unilateral cleft lip and palate, craniofacial morphology abstrak latar belakang: celah bibir dan langit-langit unilateral komplit umumnya menghasilkan asimetri wajah bagian tengah. berkurangnya kontinuitas otot di sekitar bibir yang melewati garis tengah wajah mengakibatkan malposisi struktur tulang di bawahnya yang seringkali kurang berkembang. tujuan: tujuan penelitian ini adalah untuk menentukan apakah terdapat perbedaan morfologi kraniofasial anak dengan celah bibir dan langit-langit unilateral komplit pasca labioplasti dan palatoplasti dibandingkan anak tanpa celah bibir dan langit-langit pada usia pubertal. metode penelitian: sejumlah subyek penelitian berupa 14 orang anak penderita celah bibir dan langit-langit pasca labioplasti dan palatoplasti dibandingkan dengan 14 orang anak yang normal pada masa pubertal yang sama. masa pubertal ditentukan menggunakan metode cervical vertebral maturation (cvm) yang dikembangkan oleh baccetti dkk, 2002. dilakukan perbandingan hasil pengukuran sefalogram lateral dari kedua kelompok. uji-t tidak berpasangan dilakukan untuk mengetahui perbedaan gambaran kraniofasial antara kelompok anak dengan celah bibir dan langit-langit unilateral komplit pasca labioplasti dan palatoplasti dan kelompok anak normal. hasil: terdapat perbedaan bermakna pada panjang basis kranium anterior (p = .002), panjang keseluruhan basis kranium (p = .001), panjang maksila (p = .000), panjang mandibula (p = .000), tinggi ramus mandibula (p = .000), panjang badan mandibula (p = .002), tinggi wajah anterior atas (p = .004). tidak terdapat perbedaan bermakna pada panjang basis kranium posterior (p = .051), tinggi wajah anterior bawah (p = .206), tinggi wajah posterior (p = .865), pola pertumbuhan/tipe wajah (p = .202). kesimpulan: terdapat perbedaan morfologi kraniofasial antara anak dengan celah bibir dan langit-langit unilateral komplit pasca labioplasti dan palatoplasti dibandingkan anak tanpa celah bibir dan langit-langit research report 108 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 107–113 introduction cleft lip and/or palate is a common congenital malformation, with incidence between 1/500 to 1/1000 births worldwide and is associated with a high incidence of geographic origin, racial background/ethnicity, and social economic status.1 these inherited disorders occur because of disruption to normal mechanisms during early development in the embryonic face. cleft can vary from a small base on the edge of the lip vermilion to a complete separation of the alveolar ridge, to the bottom of the lip and nose.2 cleft is due to hypoplasia of the mesenchyme layer resulting in a failure of the unification of the medial nasal and maxillary processes, whereas cleft palate occurs due to failure of palatal shelves to fuse.3 etiology of congenital abnormalities is multifactorial with genetic and environmental factors play an important role through a complex mechanism at the molecular stage during embriogenesis.4 patients with cleft lip and/or palate have many risk factors including inadequate nutrition, pulmonary aspiration, impaired masticatory function, aesthetic problems, hearing loss, speech impairment, and psychosocial, and growth disorders.2 shapira found an incidence of 74% for missing maxillary lateral incisors and 18% for missing second premolars in children with cleft lip, cleft palate, or both.5 when not absent, the maxillary lateral incisor on the cleft site is nearly always abnormal in size and shape, and the second premolars show delay in development and eruption.5,6 root development was significantly delayed, especially on the side of the cleft.7 intrauterine growth and development of the nasomaxillary complex in patients with clefts often show anterior and posterior cross bite and midfacial deficiencies with class iii malocclusion tendency.8 growth pattern in patients with cleft can be influenced by the type of cleft, post-surgical scar tissue, orthodontic/orthopedic, and alveolar bone graft.9 lack of information about craniofacial morphology of children with complete unilateral cleft lip and palate (culp) during pubertal encourage writers to do research on craniofacial morphology of children with complete unilateral cleft lip and palate during pubertal who had undergone labioplasty and palatoplasty using cephalometric analysis. determination of pubertal age in this study was done by the method of cervical vertebral maturation (cvm) developed by baccetti et al.10 the purpose of this study was to determine whether there are differences in the craniofacial morphology among children with complete unilateral cleft lip and palate post labioplasty and palatoplasty as compared with children without cleft lip and palate at the same pubertal age. material and methods this study was a cross-sectional study using lateral cephalometric radiographs from children with complete unilateral cleft lip and palate and normal children. the radiographs were taken during routine orthodontic examination. the study was conducted in cleft lip and palate unit rsab harapan kita-jakarta and rsgm faculty of dentistry universitas indonesia-jakarta during apriljune 2012. patients were included if they fulfilled the following criteria: 1) patients who were diagnosed with non-syndromic complete unilateral cleft lip and palate corresponding visual inspection by oral surgeon listed on the medical record; 2) birth weight 2500-3500 g;11 3) has a lateral cephalometric radiographs with good clarity and contrast; 4) in the pubertal growth period (cvms ii and iii according to the cvm index developed by baccetti et al.;10 5) has had labioplasty and palatoplasty with the same surgical protocol (cronin technique labioplasty and pushback partial split flap palatoplasty). the exclusion criteria of subjects: 1) being/ have been treated orthodontically; 2) has alveolar bone grafting performed previously. criteria for inclusion of control: 1) class i jaw relationship, determined by cephalometric analysis (anb pada masa pubertal. anak dengan celah bibir dan langit-langit unilateral komplit pasca labioplasti dan palatoplasti memiliki panjang basis kranium anterior, panjang keseluruhan basis kranium, panjang maksila, panjang mandibula, tinggi ramus mandibula, panjang badan mandibula, dan tinggi wajah anterior atas yang lebih pendek dibandingkan anak tanpa celah bibir dan langit-langit pada masa pubertal. kata kunci: sefalometri, celah bibir dan langit-langit unilateral komplit, morfologi kraniofasial correspondence: sigit handoko utomo, c/o: praktisi kedokteran kedokteran gigi. perum persada depok blok b2/7 tapos-depok 16959, jakarta, indonesia. e-mail: sigithandokoutomo@yahoo.com figure 1. the newly improved cervical vertebral maturation (cvm) method.10 109utomo, et al.: craniofacial morphology of children with complete unilateral cleft lip 2°±2°); 2) class i incisor relationship (insisif classification of the british standards institute). using unpaired numerical analytic formulas with a 5% error, as many as 14 of the samples are obtained in the group of children with complete unilateral cleft lip and palate and 14 samples in the control group. a conventional cephalometric approach was used to examine the data. the parameters evaluated on the lateral cephalogram are listed in table 1. lateral cephalometric radiographs of children with complete unilateral cleft lip and palate and normal children were observed in a darkened room, and a black surround was used on the light box to eliminate excess light and facilitate landmark identification. each radiograph was then classified according to the cervical vertebral maturation (cvm) method improved by baccetti et al.10 the outlines of cervical vertebrae and cephalometric landmarks were traced on cephalometric tracing paper (ortho organizers) with a 3h lead pencil. digital caliper (mitutoyo, japan) was used to measure the distance between the landmark, cephalometric protractor (ortho organizers) was used to measure the angle. to establish intraexaminer repeatability between the first and the second measurement 6 sefalogram were randomly selected (consisting of 3 subjects with cleft lip and palate and 3 normal subjects) performed with 1 week interval. because cephalograms demonstrated different magnification, adjustment for enlargement factor was made. data processing and statistical analysis performed on the data from each study variable. statistical tests performed by univariat analysis to obtain the mean, the maximum and minimum, and standard deviation of each group. data analysis was performed with unpaired t-test to examine craniofacial morphology differences between children with complete unilateral cleft lip and palate post labioplasty and palatoplasty and the normal children as controls. table 1. operational definition of variables12 variables (cephalometric measurement) definition anterior cranial base length posterior cranial base length cranial base length maxillary length mandibular length mandibular ramus height mandibular body length upper anterior facial height lower anterior face height posterior face height y-axis distance from point s to point n (mm) distance from point s to point ba (mm) distance from point n to point ba (mm) distance from ans to pns (mm) distance from point ar to point pog (mm) distance from point ar to point go (mm) distance from point go to point pog (mm) distance from point n to point ans (mm) distance from point ans to point me (mm) distance from point pns to point go(mm) angle from fhp and s-gn line (degree) table 2. cephalometric landmark on hard tissues13 cephalometric landmark definition s sella: mid point of the fossa hipophysealis/sella turcica n nasion: anterior point at the frontonasal suture ba basion: most posteroinferior point of the clivus ans anterior nasal spine: most anterior point of anteroposterior profile of the upper jaw pns posterior nasal spine: most posterior point of the bony palate defined by the junction of the hard palate, the soft palate, and the extension of the pterygomaxillary fissure ar articulare: the intersection of a line along the posterior border of the mandible and the inferior border of the basilar occipital bone pog pogonion: most anterior point of the mandibular profile in the mental region go gonion: point of intersection between the line bisecting the posterior and inferior border of the mandible and the contour of the chin me menton: most inferior point of the mandibular symphysis frankfort horizontal plane fhp: po-or gn gnathion: most anteroinferior point on mandibular symphysis po porion: most superior point of the external auditory meatus or orbitale: the deepest point on the infraorbital margin 110 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 107–113 8 9 1 2 3 11 4 5 6 7 10 figure 2. measurement used in the cephalometric analysis12 linear measurements: 1) anterior cranial base length (s-n); 2) posterior cranial base length (s-ba); 3) cranial base length (n-ba); 4) maxillary length (ans-pns); 5) mandibular length (ar-pog); 6) ramus height (ar-go); 7) mandibular body length (go-pog); 8) upper anterior facial height (n-ans); 9) lower anterior facial height (ans-me); 10) posterior facial height (pns-go); angular measurement: 11) y-axis (fhp-s-gn).12 results t w e n t y e i g h t s u b j e c t s w e r e f i n a l l y s e l e c t e d . characteristics of the groups are presented in table 3 with 14 subjects with complete unilateral cleft lip and palate which consist of 9 male subjects and 5 female subjects, whereas of the 14 subjects without cleft lip and palate which consist of 10 female subjects and 4 male subjects. the measurement errors were calculated using dahlberg formula. the error of measurement did not exceed 1.2 mm for linear measurement and 0.87 º for angular measurement. the normality test (levene tests) was performed to asses differences in variance of each measurement. normal distribution of data obtained thus followed by paired t-test to test the intergroup differences of measurement. the differences was considered significant for p≤0.05. the results of intra-observer suitability test shows no statistically significant difference between the first and second measurements for linear and angular measurements (p = .157 in linear measurements and p = .076 in angular measurement). shapiro-wilk test results show that the overall measurement data has a normal distribution and levene test results showed homogeneous data. with a normal distribution of data, unpaired t-test was performed to test the research hypothesis. the p ≤05 value considered as statistically significant differences between the two groups with 95% confidence intervals were calculated for the difference of means for each variable. there were statistically significant differences in the length of the anterior cranial base (sn, p = .02) and overall length of cranial base (n-ba, p = 0.001) between patients with complete unilateral cleft lip and palate compared with control patients, but there was no statistically significant difference in the length of the posterior cranial base (s-ba, p = .051). there were statistically significant differences in the length of the maxilla (ans-pns) among patients with table 3. distribution of study subjects by sex and agethe cleft group and the normal group cleft normal n % mean sd n % mean sd male 9 64.28 12.22 .83 4 28.57 12.75 1.71 female 5 35.72 10.60 1.34 10 71.43 12.60 1.43 14 14 table 4. intergroup differences in measured varibles detected by unpaired t-test; 95% confidence interval (ci) calculated for the difference between means variable cleft group normal group difference p mean sd mean sd s-n 64.42 3.82 68.95 3.29 -4.53 .002* s-ba 45.70 4.74 48.65 2.59 -2.95 .051 n-ba 99.12 5.88 106.53 5.12 -7.41 .001* ans-pns 48.73 4.42 58.70 5.60 -9.98 .000* ar-pog 96.42 6.14 105.78 5.71 -9.36 .000* ar-go 38.96 3.84 44.11 2.86 -5.15 .000* go-pog 71.07 5.53 78.73 5.99 -7.65 .002* n-ans 49.94 4.49 54.80 3.67 -4.86 .004* ans-me 60.02 3.99 61.99 4.02 -1.97 .206 pns-go 40.70 6.70 40.34 4.26 0.36 .865 y axis 63.50 3.06 61.71 4.08 1.79 .202 * p≤05 statistically significant difference 111utomo, et al.: craniofacial morphology of children with complete unilateral cleft lip complete unilateral cleft lip and palate with a control group (p = .000). there were statistically significant differences in the length of the mandibular ramus (ar-go, p = .000), length of mandibular body (go-pog, p = .002), and overall mandibular length (ar-pog, p = .000) between group of complete unilateral cleft lip and palate patients with a control group. anterior facial height was divided into upper anterior facial height (n-ans) and the lower anterior facial height (ans-me). there is a statistically significant difference (p = .004) in the upper anterior facial height (n-ans) between the groups but there was no statistically significant difference (p = .206) in the lower anterior facial height (ans-me). posterior facial height (pns-go) did not show a statistically significant difference (p = .189) between the groups of patients with cleft lip and palate in the control group. there was no difference in the pattern of growth and the facial type, measured as the acute angle formed by the fhp plane and s-gn line (y-axis) between the groups of patients with complete unilateral cleft lip and palate with a control group (p = .202). discussion this study was conducted with a cross-sectional design, the researchers conducted the measurement variable at a given moment. all subjects were observed only once and the subject variable measurement performed during the inspection. the research was conducted by utilizing lateral cephalometric radiograph made for the purposes of orthodontic treatment in patients with cleft lip and palate (secondary data) so as to prevent the patient from additional radiation. cross-sectional design has the main advantage that is relatively easy, inexpensive, and the results are obtained faster. it can be used to investigate many variables at once, and can be used as a basis for further research that is more conclusive.14 although the selection and determination of the subject of the research were conducted in a cleft center but there were constraints in choosing subjects that meet the criteria: 1) the limited number of pubertal age patients that owned lateral cephalographic radiograph because they were in the phase of evaluating the need to do alveolar bone grafting to prepare canine eruption in the area of the gap so most of the pubertal age patients only have orthopantomogram (opg); 2) social-economic factor limiting orthodontist to refer patients to make a lateral cephalometric radiograph because it would add to the burden of the cost of care; 3) there were many variation of severity in cleft lip and palate so treatment needs may also differ between individual patients thus causing differences in treatment stages although patients were in the same age ranges. chronological age, dental development, sexual maturation, voice changes, and height are all ways that have long been used to assess skeletal maturation, but can not be used to determine the peak of the pubertal growth.15,16 hand-wrist radiographs can be used to assess the skeletal maturation.15 however, the complexity in identification of landmarks can lead to less accurate predictions and the child will be exposed to additional radiation.17 to overcome these shortcomings cvm used as a method of assessment of skeletal maturation stages by observing changes in the cervical spinal form and the cvm method is a reliable indicator for assesing skeletal maturity.16 the peak of the pubertal growth occurred between cvms ii and iii.10 this method eliminates the need for additional radiographic exposure because it can be observed on routine lateral cephalometric radiographs.16,18 lateral cephalometric radiographs used in this study to see the craniofacial morphology differences between children with of complete unilateral cleft lip and palate post labioplasty and palatoplasty with children without cleft lip and palate as it is a standardized method of measuring.12 cephalometric analysis is the most commonly used method to determine the dentocraniofacial morphology.19 cephalometric analysis is also a diagnostic support to determine the type and pattern of growth in the face so that the clinician can determine facial disharmony.20 lateral cephalometric shows craniofacial morphology and anatomical structures on the lateral direction. the intraexaminer reliability were tested using paired t-test. intraexaminer measurements test performed on the data showed that there was no significant difference between the results of the first measurement and the second measurement. this findings showed good intraoperator reliability. levene tests showed homogeneous data (p >.05), this may be due to the inclusion criteria determining which sets the level of skeletal maturation as a guide in determining pubertal age in both groups and this result increased internal validity of the research result. the results of this study showed a significant difference in the length of the anterior cranial base (sn) and overall cranial base length (n-ba) between groups of children with cleft lip and palate compared with normal children. the length of the anterior cranial base (sn) and overall cranial base length (n-ba) in the group of children with complete unilateral cleft lip and palate shorter than normal children. maxillary complex is the most affected part of the craniofacial structures because the existence of a gap, but growth disorders in children with complete unilateral cleft lip and palate are not limited only at the maxilla. it can be shown from the results of this research that there is a significant difference in both the length of the maxilla (ans-pns) and the dimensions of the mandible; mandibular length (ar-pog), mandibular ramus height (ar-go), and mandibular body length (gopog) between groups of children with complete unilateral cleft lip and palate compared with the children without cleft lip and palate at pubertal age. the growth of the maxilla in the horizontal direction as measured by the distance between 112 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 107–113 the ans-pns in groups of children with cleft lip and palate that was shorter than the control group. it can not be determined for certain whether this was due to the surgical procedure but the growth of the maxilla in patients with cleft lip and palate who had surgery often restricted in the 3-dimensional direction.21 mandibular length (ar-pog), mandibular ramus height (ar-go), and mandibular body length (go-pog) in children with complete unilateral cleft lip and palate significantly different compared with children without cleft lip and palate at the age of pubertal. the dimensions of the mandible in children with complete unilateral cleft lip and palate at pubertal age was relatively shorter than those of children without cleft. this is interesting because the mandible is not affected by either a gap or surgical procedures such as labioplasty and palatoplasty, however this is in accordance with the results of da silva et al, who stated that patient with cleft lip and palate have a shorter mandibular length than individual without cleft lip and palate.21 mandibular length that is shorter in children with complete unilateral cleft lip and palate balanced with a cranial base length that is also shorter, which implies the existence of an equivalent growth in the mandible with anterior cranial base in the anteroposterior direction. although both the upper anterior facial height (n-ans) and the lower anterior facial height (ans-me) in groups of children with complete unilateral cleft lip and palate shorter than normal children, but significant differences occur only in the upper anterior facial height (n -ans) and there is no significant difference in the lower anterior facial height (ans-me) between the two groups. significant differences in upper anterior facial height can be understood as a consequence of the existence of a gap in the maxilla. maxillary growth affects not only in the anteroposterior and transverse direction, but also has an effect in the vertical direction. posterior facial height (pns-go) showed no significant difference between the two groups as well as the pattern of growth and the facial type (y-axis). in a study conducted in adult patients with complete unilateral cleft lip and palate found a reduction in posterior facial height and the relationship between the rotation of the mandible in the cleft palate (downward and backward rotation) were also marked by the addition of gonial angle which also resulted in the addition of anterior facial height.21 in this study there was no significant difference between the two groups in both the posterior facial height and y-axis, but the y-axis value in the groups of children with cleft lip and palate has a greater value than normal children (table 4). this lateral cephalometric studies only examine the craniofacial structure in the anteroposterior and vertical direction, posteroanterior cephalometric or 3-d ct studies of the cleft are necessary to further examine the craniofacial morphology difference between the two groups in the transverse direction. based on this study it can be concluded that there were craniofacial morphology differences between children with complete unilateral cleft lip and palate post labioplasty and palatoplasty and children without cleft lip and palate at the age of pubertal. children with complete unilateral cleft lip and palate post labioplasty and palatoplasty had shorter length of the anterior cranial base, cranial base, maxilla, mandible, mandibular ramus height, mandibular body, and upper anterior face height as compared with children without cleft lip and palate at the age of pubertal. the maxillary complex was most affected by cleft lip and palate but growth disturbance in chidren with complete unilateral cleft lip and palate were not restricted only at the maxilla. acknowledgement thanks and appreciation go to the board of directors and staff of rsab harapan kita-jakarta, particularly to drg. syafrudin hak sp.bm (k) and drg. enny tyasandarwati sp.ort from cleft lip and palate unit for their help during this study. references 1. meng l, bian z, torensma r,von den hoff j w. biological mechanisms in patogenesis and cleft palate. j dent res 2009; 88: 22–33. 2. tighe d, petrick l, cobourne mt, rabe h. cleft lip and palate: effects on neonatal care. neo reviews 2011; 12: 315–24. 3. berhman re, kliegman rm, jenson hb. nelson textbook of pediatrics. 16th ed. philadelphia: wb saunders; 2000. p. 1111. 4. cobourne mt. the complex genetics of cleft lip and palate. eur j orthod 2004; 26: 7–16. 5. shapira y, lubit e, kuftinec mm. hypodontia in children with various types of clefts. angle orthod 2000; 70: 16–21. 6. baek sh, kim ny. congenital missing permanent teeth in korean unilateral cleft lip and alveolus and unilateral cleft lip and palate patients. angle orthod 2007; 77: 88–92. 7. solis a, figueroa aa, cohen m, polley jw, evans ca. maxillary dental development in complete unilateral alveolar clefts. cleft palate craniofac j 1998; 35: 321–8. 8. shetye pr, evans ca. midfacial morphology in adult unoperated complete unilateral cleft lip and palate patients. angle orthod 2006; 76: 810–6. 9. seo yj, park jw, kim yh, baek sh. initial growth pattern of children with cleft before alveolar bone graft stage according to cleft type-unilateral cleft lip and alveolus, unilateral cleft lip and palate, and cleft palate. angle orthod 2011; 1–8 10. bacetti t, franchi l, mcnamara ja. an improved version of the cervical vertebral maturation (cvm) method for the assesment of mandibular growth. angle orthod 2002; 72: 316–23. 11. pa ndu a n pelaya na n kesehat a n bay i ba r u l a h i r berba sis perlindungan anak. direktorat kesehatan anak khusus 2010. available from: http://www.gizikia.depkes.go.id/wp-content/ uploa d s /dow n loa d s / 2 011/ 01/ pa n dua n ya n k e s bbl berbasis-perlindungan anak.pdf. 12. jacobson a, jacobson rl. radiographic cephalometry from basic to 3-d imaging. 2nd ed. hanover: quintessence; 2006: 79–87. 13. meazzini mc, glussani g, morabito a, semb g, gamatini g, brusati r. a cephalometric inter center comparison of patients with unilateral cleft lip and palate: analysis at 5 and 10 years of age and long term. cleft palate craniofac j 2008; 45: 654–60. 113utomo, et al.: craniofacial morphology of children with complete unilateral cleft lip 14. sastroasmoro s, ismael. s. dasar-dasar metodologi penelitian klinis. 3rd ed. jakarta: sagung seto; 2010: 124. 15. mito t, sato k, mitani h. cervical vertebral bone age in girls. am j orthod dentofac orthop 2002; 122: 380–5. 16. ozer t, kama jd, ozer sy. a practical method for determining pubertal growth spurt. am j orthod dentofac orthop 2006; 130: 131.e1–.e6. 17. soegiharto bm, moles dr, cunningham sj. discriminatory ability of the skeletal maturation index and the cervical vertebrae maturation index in detecting peak pubertal growth in indonesian and white subjects with receiver operating characteristics analysis. am j orthod dentofac orthop 2008; 134: 227–37. 18. tancan u, ramoglu si, basciftci fa, sari z. chronologic age and skeletal maturation of the cervical vertebrae and hand wrist: is there a relationship?. am j orthod dentofac orthop 2006; 130: 622–8. 19. wu j y, hagg u, wong rw, mcg r at h c. comp r ehen sive cephalometric analysis of 10 to 14 year old southern chinese. the open anthrop j 2010; 3: 85–91. 20. ku r a m a e m, mag na n i m, bo e ck e , lucat o a. ja r aba k’s cephalometric analysis of brazilian black patients. braz dent j 2007; 18: 258–62. 21. filho og, normando adc, filho lc. mandibular growth in patients with cleft lip and/or cleft palate-the influence of cleft type. am j orthod dentofac orthop 1993; 104: 269–275. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 42 no 2 april 2009.indd 86 vol. 42. no. 2 april–june 2009 research report shear bond strength between porcelain and nano filler composite resin with or without 9% hydrofluoric acid etching kun ismiyatin department of conservative dentistry faculty of dentistry, airlangga university surabaya-indonesia abstract background: reparation technique on restorations with broken or damaged porcelain which are still attached with the teeth are difficult, because it is very hard to remove the porcelain restoration without damaging it, and it needs a long time. various ways have been developed to repair the broken porcelain, one of them is the use of composite resin as the material for the restoration of fractured porcelain. repairing porcelain inside the mouth without removing the restoration of the damaged porcelain using light cured composite resins material seems to be an advantageous option because it is relatively simple, has low risks, good esthetically and cheap. purpose: the objective of this study was to find out the difference of shear bond strength in porcelain reparation using nano filler composite resin with or without 9% hydrofluoric acid etching by using autograph measuring device. methods: twenty pieces of the porcelain samples devided into 2 groups. group i: etching process using 9% hydrofluoric acid, and group ii : without etching process. result: the data was analyzed using t test in a p value of 0.0001 (p≤0.05), which means there is a significant different of shear bond strength between treated group i and ii. the biggest shear bond strength was in treatment group i. conclusion: the use of 9% hydrofluoric acid on the surface of porcelain can increase the shear bond strength between porcelain and nano filler composite resin. key words: shear bond strength, porcelain, nano filler composite, 9% hydrofluoric acid, etching correspondence: kun ismiyatin, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: kunismiyatin@gmail.com introduction porcelain restoration material has advantages in its strength which resembles email, translucent, not easily worn out, stable and esthetically close to the tooth color, while in dentistry, porcelain material is used for inlay, onlay, laminate veneer, and crown.1,2 however, porcelain has disadvantages in its fragile character, thus porcelain has fracture potential, even in light pressure.1 some factors that may cause porcelain restoration undergo defect are the heavy pressure of occlusion, excessive chewing, micro defect of the material it self, inappropriate design, trauma and its fragile character.3 reparation treatment towards restorations of broken or damaged porcelain which are still well attached to teeth is difficult to do, because it is very hard to remove porcelain restoration without damaging it, and it needs a long time. various ways have been developed to repair the broken porcelain, one of them is what is now being developed, that is the use of composite resin as the material for the restoration of fractured porcelain. composite resins has become the chosen material because it is esthetically good, strong enough, not easily abraded, easy to manipulate, able to stick to porcelain surface, provided in various colors so that one may choose the color matching to porcelain color.4–6 repairing porcelain inside the mouth without removing the restoration of the damaged porcelain using light cured composite resins material seems to be an advantageous option because it is relatively simple, has low risks, esthetically good and relatively cheap.7,8 composite resins containing very small particles are nano filler composite resins, whose particle diameter ranges from 20–27 nm. the compressive strength and fracture resistance of this nano composite is the same as, or a little higher than other kinds of composites, in addition, nano composite shows better polishing result than that of micro 87ismiyatin: shear bond strength between porcelain hybrid composite.9 the shear ability between porcelain and composite resins becomes significant when reparation on porcelain restoration is going to be done, therefore a lot of studies to increase the shear bond strength of composite resins and porcelain have been done. the shear bond strength of the material is determined by the nature of the physical shear, the mechanism and the chemistry of the surface of the material.2 one of the efforts to obtain the characteristic of chemical shear between composite resins and porcelain is the use of silane solution. some researchers have proved that composite resins is able to adhere well on porcelain with silane solution.4 silane is the combination of organic-inorganic materials which function as a mediator, a coupling agent to obtain shear between different organic and inorganic materials.8 the silane mostly used in dentistry is γ-methacryloxypropyltrimethoxysilane (γ-mpts), and this material functions as the mediator between composite shear and porcelain because the methacryloxypropyl clusters of γ-mpts match with the ones of dimethacrylate used in resin technology.10 besides, this silane has alcoxy clusters which can react on hydroxyl clusters of inorganic silicone substrate of the porcelain,8 and this will enable silane to become a chemical bond mediator between porcelain and composite resins. for the first time some researchers did on porcelain etching, they used hydrofluoric acid, then the glass/silica molecules in the porcelain reacted on the hydrofluoric acid was following this reaction: sio2(s) + 6hf(aq) → h2[sif6](aq) + 2h2o(l) the result of the reaction, which is h2[sif6](aq), dissolves, resulting a micro porous porcelain surface.11 the study on the etching of porcelain surface using 9% hydrofluoric acid for one minute produces as wide as 50 μm pore diameter of porcelain surface,12 so that the resins applied to the surface of the porcelain will penetrate into those pores and form resin tag, producing micro mechanical interlocking between porcelain and composite resin. the hydrofluoric acid used for intra oral is a buffer solution in the shape of gel, which in general has 9% content and 1.57 ph. this solution will produce constant hydrogen fluoride, but during the etching process it will not do any harm. repairing porcelain reparation using composite resins needs to result in good bond strength. therefore a test to asses the shear strength of composite resins to the porcelain which undergoes treatment needs to be done. nowadays, different opinions whether porcelain surface treatment will develop a better bond strength as well as the use of 9% hydrofluoric acid due to its irritating characteristic to oral tissues still exist. the purpose of this study was to know shear bond strength between porcelain and nano filler composite resin with or without 9% hydrofluoric acid etching. materials and methods experiment samples are 20 pieces of porcelain, metal kind porcelain of ivoclar made according to the factory instruction, 4 mm in diameter, 4 mm height, no cracking part, having flat surface, soft, and no glazing has been done.5 the porcelain samples are made more than 20 pieces. then, the 20 pieces which match with the criteria are taken randomly, 10 each for one group of treatment. the composite resin used is nano filler composite resins polimerated with the help of visible light curing unit. group i: the porcelain’s surfaces are made rough using no. 100 taiyo sand paper with medium pressure for one minute, then etching process using 9% hydrofluoric acid is done for one minute to the porcelain’s surfaces, and then they are washed with aquadest spray using 2,5 cc syringe, next they are dried using air blow. after that, the porcelain’s surfaces were smeared with silane solution twice, and let them dry in one minute. group ii: the surfaces of the porcelains are made rough, first by means of number 100 taiyo sand paper, using medium pressure for one minute then they are washed with aquadest spray using 2.5 cc syringe for two times, and after that they are dried using air blower, next the surfaces of the porcelains smeared twice with silane solution, and then they are let dry in one minute after having been treated, each porcelain samples group is placed on a plastic tray. in order to prevent scraping during composite aplication, the treated surfaces must face the composite molds. the composite mould ring was inserted and applied approximately 2 mm thick composite, and cured it with blue light for 40 seconds. next, approximately 2 mm thick composite was applied for the second time, and then celluloid strip and thin glass were put on it, weighted with one kilogram of weight for one minute, then the excessive composite was removed using scalpel, and cured with blue light of visible light cure unit for 40 seconds. after the composite application was finished, the specimen was taken out from the composite laying aid and the mold ring was removed, at last the finished samples were put into a plastic tube containing aquadest for 24 hours prior to the shearing test. the test of shear bond strength for each sample group was measured with autograph ag-10 te (shimadzu, japan) with a cross head speed of 1/10 mm/second and load cell capacity of 5 kn/500 kgf (1 kgf = 9.81 n). result from the result of the experiment to inquire the effect of 9% hydfluoric acid on the porcelain surface to shear bond strength using composite resins, the data as depicted in table 1 are gotten: 88 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 86−89 table 1. mean and standar deviation as the results of shear bond strength measurement between composite resins materials and the porcelains undergo acid etching and without acid etching (n/mm2) treatment n x sd group i (acid etching) 10 245.77 ± 24.28 group ii (without acid etching) 10 148.42 ± 13.12 notes: x : mean, n: numbers of samples, sd: standard deviation the result of the experiment of treated group i gave a mean value of 245.77 and the deviation standard is ± 24.28; the treated group ii gave a mean value of 148.42 and the deviation standard is ± 13.12. before the deviation standard test between both treated groups was done, first, normal distribution test using kolmogorov smirnov test has been given to each group to see the data distribution in each group. from the kolmogorov smirnov test done to all groups, a value of p > 0.05 was got, this result showed that all groups distribute normally. next, t test was done to see the difference of the shear strength in treated group i and treated group ii. t-test results in a p value of 0.0001 (p ≥ 0.05), which means there was a significant different of shear bond strength between treated group i and ii. discussion this experiment was done to find out if 9% hydrofluoric acid etching on porcelain surfaces would cause an increase in the composite resin’s shear bond strength towards the porcelains without acid etching. after having been acid etched or without acid etching the porcelain surfaces were smeared two times using silane solution that will increase the shear bond strength, and this is an important step which cannot be passed up in the procedure of fusing composite resins to porcelain. some test can be used to evaluate the shear bond strength between porcelain and composite resins, such as flexural test, shear test, tensile test. each way of assessments has its own advantages and disadvantages and some writers state that shear test is the most adequate one to measure shear bond strength between two materials.4–6,8,9,13 this kind of test is done through the potentials which directly develop on the surfaces of two materials which were going to be tested. the test on the shear bond strength with flat surface may direct most of the potential pressure to the fusing surface, without any influences of elasticity modules of the tested material as what happens in flexural test.5 in this kind of test, the testing point has directed precisely to the surface of the material so that the oblique strength generated during the test could be minimized. it was appropriate with the research which stated that parallel directing of the strength to the unification field between two materials tested may minimize cohesive failure and the test result tends to result in adhesive failure.13 from the result of this experiment we get statistically significant differences on 95% trust in group i and ii. this fact showed that the kind of surface treatment on porcelain determine the value of composite resin shear bond strength that will be got. the highest shear bond strength was gotten in treatment group i, which was suitable with the result of the experiment from another reseacher,13 who did the experiment on micro shear bond strength, it might be because group i had two kinds of bonds between porcelain and composite resin were micromechanical bond and chemical bond, while in group ii the bond was just chemically. another researcher said that hydrofluoric acid which is smeared to the surface of the porcelain may react with the glass/silica phase which form the porcelain. the result of the reaction which is h2[sif6](aq) becomes dissolved and generating micro pores porcelain surface.11 there is a research which promotes the micro pores of the material surface will cause the widening of the surface of the contact and it will cause a micromechanical interlocking between porcelain and composite resin.16 many kinds of acid solution can be used to etch a porcelain, but according to other researcher that comparison between the effect of the kinds of acid etching on porcelain surface, hydrofluoric acid showed the most effective result.16 the application of silane solution on the porcelain surface will result in chemical bond between porcelain and composite resin. a previous research said that silane solution functions as a promoter bonding which triggers chemical bonding between organic and inorganic surfaces. the fusion of composite resin on porcelain happens because of polymerization reaction between dimethacrylate cluster on resin matrix and the cluster methacryloxypropyl silane solution during composite resin curing process and through condensation reaction between hydroxyl (si-oh) on the molecules of porcelain silica, with alcoxy cluster on hydrolysed cylanol molecule. this reaction generates cyloxane cluster (si-o-si) and water molecule (h2o) as the last product.16,17 the clean and dry surface are needed in forming optimum fusion because after the surface of the material is cleaned, the surface energy will increase and result in the surface of the material will be easier to absorb adhesive material,2 and that is why the surface of the porcelain needs to be washed with aquadest and needs to be dried using air blow. another researcher also said that silane solution also helps the bonding of porcelain and composite resin because there is an increase of the wetting of the porcelain surface.16,17 the wetting on porcelain is proved with the small size of the contact angle of the surface and that the silane through out all part of the porcelain surface, including to the micro pores which has been formed because of acid etching.2 the use of 9% hydrofluoric acid is proved to be able to increase the shear bond strength between nano filler composite resin with porcelain, but because of the 89ismiyatin: shear bond strength between porcelain irritating characteristic of hydrofluoric acid, when it is used intra orally, optimal isolation method must be done to protect soft tissues,3 and air vacuum needs to be used to lessen steam which might be inhaled during the etching process. if an open dentin is found, using silane solution without hydrofluoric acid etching is enough, because 9% hydrofluoric acid has an irritating effect on dentin.17 the conclusion of this study is the use of 9% hydrofluoric acid on the surface of porcelain can increase the shear bond strength between porcelain and nano filler composite resin. references 1. anusavice kj. philips science of dental material. 10th ed. philadelphia: wb saunders co; 2003. p. 69–298. 2. craig rc. restorative dental materials. 7th ed. st. louis: the cv mosby co; 2002. p. 260–4. 3. jordan re. esthetic composite bonding techniques and materials. 2nd ed. st louis: mosby co; 1993. p. 318–38. 4. dent rj. repair porcelain fused to metal restoration. j prosthet dent 1979; 41:663. 5. beck da, janus ce, douglas h.b. shear bond strength of composite resin porcelain repair material. j prosthet dent 1990; 64(5): 529–33. 6. gregory wa, moss sm. effect of heterogenous layers of composite and time on composite repair of porcelain. j oper dent 1990; 15:18–22. 7. wood l. visible light-cured composite resins: an alternative for anterior provisional restoration. j prosthet dent 1984; 51(2): 192-4. 8. özcan m. evaluation of alternative intraoral repair techniques for fractured ceramic-fused-to-metal restorations. j oral rehab 2003; 30(2): 194–203. 9. sumita bm, dong w, brian nh. an application of nanotechnology in advanced dental materials. j am dent assoc 2003; 134(10): 1382–90. 10. hooshman t, noort, keshvad. storage of pre-activated silane on resin to ceramic bond. j dent mat 2000; 20: 635–42. 11. nelson e, barghi. effect of apf etching time on resin bonded porcelain. j dent rest 1989; 68:271. 12. gonzaga, cc. micro structure of dental porcelain. iadr tech program, san diego. 2002 available from: url:http//iadr.confex. com/iadr/2002sandiego/techprogram/abstract.9029 accessed october 12, 2006. 13. pretti m. evaluation of the shear bond strength of the union between two cocr-alloys and a dental ceramic. j appl oral sci 2004; 12(4): 19–24. 14. cheung ppl. method for etching dental porcelain. available from: url:http://www. sacnewsmonthly.com. accessed november 4, 2006. 15. hydrofluoric acid. retrieved from http://en.wikipedia.org/wiki/ hydrofluoric_acid. accessed december 22, 2006. 16. filho am. effect of different ceramic surface treatments on resin micro tensile bond strength. j prosthet dent 2004; 13(1): 28–35. 17. goyal s. silanes: chemistry and applications. j indian prost soc 2006; 6: 14–8. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 177 vol. 44. no. 4 december 2011 research report oral rinse as a potential method to culture candida isolate from aids patients desiana radithia, hening t. hendarti, and bagus soebadi department of oral medicine faculty of dentistry, airlangga university surabaya indonesia abstract background: candida isolate is easily sampled from oral cavity by swabbing directly on the candidiasis lesion, to be smeared onto slides for direct examination or cultured in a growth medium. this method is by far the gold standard for defining candidiasis diagnosis. however it is difficult to apply on sensitive patients and almost impossible on patients showing no clinical appearance of oral candidiasis. ai�s patients are very prone to candida infection and have a tendency of repetitive infection involving mixed species. as many candida species show different susceptibility to anti-fungal agents, it is necessary to identify the species causing the infection in the management of oral candidiasis. oral rinse is a suggested method to obtain candida isolate to be cultured for further analysis such as species identification. this method is simple and less risky on infection transmission as less tools are required in the procedure. purpose: this study aimed to assess the application of oral rinse as an alternative method to culture candida isolate from ai�s patients. methods: a cross-sectional observative study was conducted in hiv/ai�s in-patient facility of intermediate care unit for infection �isease, �r. soetomo hospital surabaya. fourteen stadium 4 ai�s patients matching criteria were swabbed on 1/3posterior of the tongue, and then given 10 ml phosphate buffer saline to rinse vigorously for 15 seconds. both specimens were cultured on sabouraud’s dextrose agar and colony growth was observed. results: candida colonies were able to grow from all 14 isolates (100%) by both methods. qualitatively, cultures from oral rinse specimens were more populated than cultures from swab specimens. conclusion: oral rinse is an applicable technique to obtain candida species isolate. this technique is safe, easy, non-invasive, and needs less tools therefore less risky for hiv transmission. key words: hiv/ai�s, oral candidiasis, candida species, methods for obtaining isolate abstrak latar belakang: isolat candida mudah diambil dengan cara mengusap lesi candidiasis, baik untuk dioleskan pada kaca preparat untuk pemeriksaan langsung maupun dikultur. hingga kini, metode tersebut dinyatakan sebagai “standar emas” untuk menentukan diagnosis. namun kekurangan metode ini yaitu berisiko merangsang muntah pada pasien sensitif, dan hanya bisa dilakukan bila tampak jelas ada lesi. candidiasis adalah penyakit nosokomial yang sering terjadi. pasien ai�s sangat rentan terhadap infeksi oportunis ini secara rekuren dan persisten. identifikasi spesies penyebab harus dilakukan karena berbagai spesies candida memiliki kerentanan yang berbeda terhadap berbagai jenis antifungal. oral rinse adalah metode pengambilan isolat candida yang non-invasif. isolat yang didapat bisa dikultur dan diidentifikasi, selain itu bisa dilakukan pada pasien yang belum menunjukkan adanya lesi candidiasis untuk menentukan besar risiko pasien terkena candidiasis, sehingga dapat ditentukan perlu tidaknya pemberian profilaksis antifungal. tujuan: penelitian ini bertujuan mengamati efektivitas metode oral rinse untuk mengisolasi candida dari rongga mulut pasien ai�s. metode: penelitian observasional dilakukan di bagian rawat inap unit perawatan intermediate penyakit infeksi rsu� �r. soetomo. swab pada 1/3 posterior lidah dilakukan pada 14 pasien yang memenuhi kriteria, kemudia pasien diberi 10 ml phosphate buffer saline untuk berkumur kuat-kuat selama 15 detik. spesimen yang didapat melalui kedua metode dikultur pada medium sabouraud untuk diamati. hasil: koloni candida berhasil dikultur dari 14 spesimen (100 %) melalui kedua metode isolasi. secara kualitiatif tampak bahwa hasil kultur dari oral rinse tampak lebih subur. kesimpulan: oral rinse adalah metode yang dapat diaplikasikan untuk 178 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 177–180 mengisolasi candida dari rongga mulut pasien. tekniknya mudah, aman, non-onvasif, dan tidak memerlukan peralatan dan ketrampilan khusus, sehingga mengurangi risiko transmisi hiv. kata kunci: hiv/ai�s, oral candidiasis, candida species, metode isolasi spesimen correspondence: desiana radithia, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas airlangga. jl. myjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: deisy_radithia@yahoo.com introduction candida species (candida spp.) is likely the most familiar cause of fungal infections to clinicians and patients.1 it is reported to cause over 70% of invasive fungal infection in hospitalized patients. beginning with contracting mucosal candidiasis, certain hospitalized patients are at risk of contracting nosocomial candidemia because of their underlying medical conditions, while medical interventions such as antibiotic use, the presence of central venous catheter, and hemodialysis further increase the risk of contracting candidemia.2 patients infected with hiv and aids are at the highest risk of candida infection. oropharyngeal candidiasis may be seen even in patients with cluster of differentiation 4+ (cd4+) counts over 200/ mm3, and becomes more common when the cd4+ count falls below 300/mm3.3,4 opportunistic infections are major factors contributing the progressivity of hiv infection as inflammation induces viral replication and eventually leads to cd4+ depletion.5 oral candidiasis in hiv and aids patients must be managed urgently and thoroughly to delay the progresivity of aids. preventive measures may be achieved by detecting the infection as early as possible in hospitalized hiv positive individuals. management of oral candidiasis consists of correct diagnosis and proper choice of treatment. defining the species causing the infection is very important, because every species of candida has different sensitivity to antifungal. the specificity might not be as specific as bacteria against antibiotics, and most fungal species are sensitive to azoles. however, resistence to azole treatment has recently become widely reported. it is very important for clinicians to identify the microorganism causing the infection to be able to prescribe the most potent anti-fungal agent.4 the gold standart to obtain candida isolate from any mucosal site is by swabbing the creamy-white pseudomembran directly from the oral candidiasis lesion. this allows the isolate to later be observed by direct microscopy and cultured in growth medium.6 however this method is hardly applicable when obvious lesion is not present. swab sticks, although simple and considered non-invasive, may provoke vomitting when swabbed in posterior areas, especially in sensitive patients. therefore, an alternative method must be developed to obtain candida isolate from patients without clear clinical presentation of oral candidiasis and to determine the possibility for the patient to contract oral candidiasis during his hospitalization. collecting oral rinse, or oral lavage, is relatively easy to apply, without any special skill from the clinician and caretakers needed. less tools are also needed when applying this method, and this might be the most beneficial point while working with hiv positive individuals. this study is aimed to assess the application of oral rinse as an alternative method to obtain candida isolate from hiv/aids patients, in the hope of setting up a better and safer protocols in the management of oral candidiasis in hiv/aids patients. materials and methods this cross-sectional observative study was conducted in in-patient facility of intermediate care unit for infectious disease (hiv and aids facility) in dr. soetomo hospital surabaya. all stadium 4 aids patients in the facility were clinically examined. fourteen met the sampling criteria, which included receiving no treatment for fungal infection. swab specimen was taken from 1/3-posterior of tongue dorsum, then the swab stick was immersed in sabouraud’s broth and incubated for 24 hours in 37° c. from the same patient, oral rinse specimen was taken by telling the patient to rinse vigorously with 10 ml phosphate buffer saline (pbs) for 15 seconds. oral rinse specimen was then centrifugated, and the harvested pellet was also immersed in the sabouraud’s broth and incubated for 24 hours in 37° c. cotton swab stick was then used to stir the liquid medium, then spreaded onto sabouraud’s dextrose agar, and incubated for another 24 hours in 37° c. agar mediums were then observed visually and qualitatively for colony growth comparison. results out of 14 patients involved in the study, 12 were males and 2 were females. all of them diagnosed with stadium 4 aids. oral examination showed that none of the patients had good oral health status. they all had one or overlapping oral mucosal lesions. oral pseudomembranous candidiasis was clinically suspected in 6 patients. atophic erythematous condition on tongue and buccal mucosa was present in 8 patients. exfoliative cheilitis is generally common as patients tended to have dry mouth, but only 3 severe cases were noted. two patients presented angular cheilitis and one oral hairy leukoplakia. 179radithia, et al.,: oral rinse as a potential method to culture candida isolate as shown in figure 1 and 2 candida spp from 14 isolates (100%) taken by both methods were able to grow on the sabouraud medium. qualitatively, cultures from oral rinse specimens were more populated than cultures from swab specimens. discussion hiv-infected individuals are at the highest risk of candida infection. oral candidiasis is associated to 90% of hiv/aids cases.7 opportunistic infections are the most contributing factor in the progressivity of hiv infection. inflammation induces hiv replication and eventually leads to cd4+ count depletion.5 the severity of cd4+ depletion is not the sole determinant of risk, however, as recent data suggest an independent, inverse relationship between plasma hiv viral load and the prevalence of oropharyngeal and oesophageal candidiasis.4 since the introduction of highly active antiretroviral therapy (haart), the incidence and prevalence of opportunistic infections on hiv positive individuals has declined. however, in countries with limited settings where haart is not readily available, candidiasis still constitute a large disease burden.3 the most common species identified in human infections at any mucosal sites is candida albicans (c. albicans), however additional species are increasing in frequency and include c. dubliniensis, c. glabrata, c. krusei, c. lusitaniae, c. parapsilosis and c. tropicalis. c. albicans is isolated from the oropharynx of over 40% of normal individuals and is a standard commensal of the gastrointestinal tract , therefore the strain causing disease is derived from the patient’s own flora.1,4 candida species are generally sensitive to broad spectrum antifungal agents eventhough reports suggest resistance to fluconazole by non-albicans species and a certain strain of c. albicans. this is the reason for clinicians to identify correctly the microorganism causing the disease.8–10 eighteen patients recently admitted to the intermediate care unit for infectious diseases were included in the study, however 4 of them must be excluded because 2 were barely conscious while the other 2 were not able to sit up. all 14, 12 males and 2 females, were diagnosed with stadium 4 aids, indicating cd4+ counts below 200/mm3. most of them were totally unaware of their oral health condition and could not decide if they have ever contracted candidiasis on any mucosal part or if they have ever been given treatment for candidiasis before. oral examination on those patients revealed that none of them had a good oral health condition. all of them had at least one or overlapping mucosal lesions which are closely-associated to hiv infection such as oral pseudomembranous candidiasis, oral hairy leukoplakia and linear gingival erythema, or lesions which are less associated with hiv infection such as mucosal erythema, angular cheilitis and exfoliative cheilitis. figure 1. colony growth from swab specimen. figure 2. colony growth from oral rinse specimen. 180 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 177–180 diagnosis of oral candidiasis is made based on clinical presentation and laboratory confirmation. there are three types of clinical presentations of oral candidiasis and the pseudomembranous type is probably the easiest one to recognize clinically.4,6 swabbing directly on the lesion is the easiest way to obtain the isolate for further laboratory examination. the isolate can then be smeared on a glass slide and stained with koh or gram preparation. appearance of blastophores, pseudohyphae and true hyphae would confirm the diagnosis of oral candidiasis. the rest of the isolate can also be grown on to standard growth medium for candida species.3,6,10 however, the gold method does not cover taking isolates from patients presenting less obvious form of candidiasis, such as the erythematous type. patients with risk of contracting candidiasis, such as hospitalized patients and especially hiv positive ones, should receive early management for this debilitating opportunistic infection.3,4,6 as swabbing can only be done on obvious lesion, a different approach should be developed to take isolates from patients who do not present clinical candidiasis yet but at risk of contracting one. oral rinse is very potential for these conditions. rinsing movement allows dettachment of salivary mucin containing microorganisms. phosphat buffer saline ensures the cells obtained are well preserved during transfer time to the laboratory.8,10 by telling the patient to rinse his mouth, we also gather information about the patient’s motoric and sensoric reflex. this will also be a consideration for clinician to prescribe topical antifungal and antiseptic in the form of rinse or gargle. the most beneficial point in this method is that it takes less tool and it does not require a special skill from the clinician. swabbing method, eventhough considered non-invasive, requires a skillful dexterity to swab the cotton swab stick on the mucosal surface. if the lesion is situated in the posterior region, the movement would easily trigger the patient to vomit, and this will increase the risk of infection transmission. in conclusion, as long as the patient is able to perform oral rinsing and has good gag reflex, oral rinse might stand as an applicable technique to obtain candida species isolate for further laboratory assessment. this technique is safe, easy, non-invasive, and needs less tools therefore less risky in the sense of hiv transmission. acknowledgement this research is supported by the indonesian ministry of national education (direktorat jendral pedidikan tinggi, dp2m, hibah strategis nasional tahun anggaran 2010–2011). references 1. cramer ra, perfect jr. recent advances in understanding human opportunistic fungal pethogenesis mechanisms. in: anaissie ej, mcginnis mr, pfaller ma. clinical mycology. 2nd ed. edinburg: churchill livingstone, elsevier, inc; 2009. p. 15–28. 2. lockhart sr, diekema dj, pfaller ma. the epidemiology of fungal infections. in: anaissie ej, mcginnis mr, pfaller ma. clinical mycology. 2nd ed. churchill livingstone, elsevier, inc; 2009. p. 1–12. 3. devitt e, powderly wg. candida in hiv infection. in: volberding pa, sande ma, greene wc, lange jma, gallant je, walsh cc, eds. global hiv/aids medicine. philadelphia: saunders elsevier; 2008. p. 365–72. 4. saccente m. fungal infections in the patient with human immunodeficiency virus infection. in: volberding pa, sande ma, greene wc, lange jma, gallant je, walsh cc, eds. global hiv/ aids medicine. philadelphia: saunders elsevier; 2009. p. 417–9. 5. abbas ak, lichtman ah, pilai s. cellular and molecular immunology. 6th ed. philadelphia: saunders, elsevier inc; 2007. p. 475–88. 6. anaissie ej, solomkinm js, dignani mc. candida. in: anaissie ej, mcginnis mr, pfaller ma. clinical mycology. 2nd ed. edinburg: churchill livingstone, elsevier, inc; 2009. p. 197–218. 7. repentigny l, lewandowski d, jolicoeur p. immunopathogenesis of oropharyngeal candidiasis in human immunodeficiency virus infection. clinical microbiology reviews 2004; 17(4): 729–59. 8. shahid m, malik a, rizvi mw, singhai m. protein profile of a fluconazole-resistant candida albicans isolated from hiv-1 infected patient: evaluation of protein extraction methods and development of a simple procedure. global j biotech & biochem 2006; 1(1): 1–6. 9. matsuki m, kanatsu h, watanabe t, ogasawara a, mikami t, matsumoto t. effects of antifungal drugs on proliferation signals in candida albicans. biol pharm bull 2006; 29(5): 919–22. 10. oliveira gs, ribeiro et, baroni fa. an evaluation of manual and mechanical methods to identify candida spp from human and animal sources. rev inst med trop s. paulo 2006; 48(6): 311–5. vol 50 no 4 desember 2017.indd 211211 dental journal (majalah kedokteran gigi) 2017 december; 50(4): 211–215 research report dentoalveolar changes in post-twin block appliance orthodontic treatment class ii dentoskeletal malocclusion y. yoana, eka chemiawan and arlette suzy setiawan department of paediatric dentistry faculty of dentistry, universitas padjadjaran bandung – indonesia abstract background: the analysis of cephalometric radiographs provides information about facial skeletal structures, jaw bone-base relationships, incisive-axial inclination relationships, soft tissue morphology, growth direction and pattern, malocclusion classification and the limitations of orthodontic treatments. in class ii malocclusion, the mesiobuccal cusp of the permanent maxillary first molar rests between the first mandibular molar and the second premolar. a twin block appliance is recommended to treat class ii dentoskeletal malocclusion with retrognathic mandible characteristics. purpose: the aim of this study was to analyze the dentoalveolar alterations in class ii dentoskeletal malocclusion with retrognathic mandible characteristics after orthodontic treatment with twin block appliance based on a steiner analysis. methods: this research constitutes a retrospective study using secondary data derived from the lateral cephalometric radiographs of patients with class ii malocclusion treated with twin block appliance at the pediatric dentistry department of the oral and dental hospital, universitas padjajaran, bandung. the data was analyzed using a t-test for normally distributed paired data. in cases where data was not normally distributed, a wilcoxon test was employed. results: the average measurements showed statistically significant dentoalveolar changes among class ii malocclusion patients after twin block appliance treatment when analyzed using the paired t-test based on steiner method cephalometric radiograph analysis (p < 0.05). conclusion: it is concluded that a twin block appliance is effective in treating class ii dentoskeletal malocclusion with a retrognathic mandible based on dentoalveolar changes resulting from steiner analysis keywords: cephalometric; dentoalveolar alterations; dentoskeletal malocclusion; retrognathic mandible; twin block correspondence: arlette suzy setiawan, department of paediatric dentistry, faculty of dentistry, universitas padjadjaran. jl. sekeloa selatan 1 bandung 40132, indonesia. e-mail: arlettesuzy@yahoo.com; arlette.puspa@fkg.unpad.ac.id introduction numerous clinical studies have been conducted to reveal the skeletal and dentoalveolar changes related to the treatment of class ii malocclusion. however, the implications of the resulting scientific data are still debatable.1 several studies have argued that the skeletal effects induced by the functional appliances significantly influenced mandibular growth, while others indicated that they might have little effect on skeletal changes. major modifications identified following orthodontic treatment with functional appliances include those dentoalveolar in nature, i.e. distalization of the buccal part and retroclination of maxillary anterior teeth, along with mesial changes in the buccal section of mandibular teeth and proclination of their labial sections. the availability of a diagnostic report supported by study models, radiographs and images showing the pre-treatment conditions necessary to select the form of malocclusion treatment and to assess its effectiveness was essential.2 the analysis of cephalometric radiographs provided information regarding facial skeletal structures, jaw bone-base relationships, incisive-axial inclination relationships, soft tissue morphology, growth direction and pattern, malocclusion classification, and limitations on orthodontic treatments.3 the purpose of cephalometric dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p211-215 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p211-215 mailto:arlettesuzy@yahoo.com mailto:arlette.puspa@fkg.unpad.ac.id 212 yoana, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 211–215 radiograph examination is to evaluate the face type, jawcranial base relationship, growth pattern, dentoalveolar relationship, the position of malocclusion, soft tissues and their relationship with regard to etiology and prognostics, functional relationship, and therapeutic possibilities.4 the method frequently used to analyze cephalometric radiographs as part of dentofacial relationship evaluation is that of a steiner analysis. this form of analysis is the most widely employed because it is simple, easy to understand and produces maximum clinical information with minimal methods that include calculation of the position and inclination of the teeth in relation to the jaw and the position of the jaw vis-a-vis the cranium base.4 the outcome of incompatible treatment may be due to incorrect line points in preand post-treatment cephalometric radiograph analysis. this creates difficulties in assessing the real involvement of the skeletal and dentoalveolar components in the changes evident in the treatment outcome.5 cephalometric radiographs are frequently used to identify and evaluate changes during malocclusion treatment.4 the world health organization stated that malocclusion is a dentofacial abnormality leading to defects in both appearance and function which affect a person both physically and mentally. malocclusion is a deviation from the ideal occlusion that may cause discomfort and, specifically, poor esthetics. this is due to the imbalance in size and position of teeth, facial bones and soft tissues (lips, cheeks, and tongue).6,7 edward angle stated that class ii dental malocclusion is defined as a condition where the first mandibular molar is more distal than under normal occlusion in relation to the first maxillary molar. the british dental institute defined class ii malocclusion as a condition where the mandibular incisor edge is in a posterior position in relation to the palatal cingulum of the maxillary incisor in a proclined inclination with increased overjet.8 an epidemiological study at an elementary school in jakarta confirmed the prevalence of the class ii malocclusion as one of 31.6%.9 several mandibular proclination methods applied to correct class ii malocclusion include: functional and extraoral appliances, camouflage treatment and surgical jaw repositioning.5,10 the functional appliances comprise: bionators, fr-2 or frankel, fixed and removable herbst functional appliances and the twin block appliance introduced by william j. clark.11 the twin block appliance consists of maxillary and mandibular acrylic plates with bite blocks that guide the mandible to a forward position during closure of the mouth.10 the indication of the twin block appliance is to correct class ii dentoskeletal malocclusion with mandibular retrognathy.10–13 a comparative study between patients treated with twin block appliances and a control group showed a significant increase in mandible length, but with the frequent occurrence of overjet correction as a result of dentoalveolar compensation.12 meanwhile, a comparative study of twin blocks and other functional appliances confirmed the former to be the best functional appliance for producing sagittal modification; including: mandibular skeletal changes, dentoalveolar changes and normal growth pattern changes.13 this study aimed to analyze post-orthodontic treatment dentoalveolar modifications using twin block appliances in class ii dentoskeletal malocclusion with retrognathic mandibles based on a steiner analysis. materials and methods the study reported here was retrospective analytical in nature incorporating a purposive sampling method based on the secondary data derived from a lateral cephalometric radiograph of class ii dentoskeletal malocclusion with retrognathic mandible patients. during the period 2010–2017, these individuals were treated using twin block appliances at the pediatric dentistry installation of the oral and dental hospital, universitas padjadjaran, bandung. dentoalveolar alterations were assessed using steiner cephalometric radiograph analysis (i-na angle, i-na distance, i-nb angle, i-nb distance, and interincisal angle). steiner analysis was also used to recognize the relationship between the position of the jaw and cranial base, the position of the mandible against the maxilla, in addition to that of teeth in the arch. the dentoalveolar alteration indicators employed in the steiner analysis include: the maxillary first incisor inclination angle (i-na angle), the anteroposterior position of the maxillary first incisor against the maxilla (i-na distance), the mandibular first incisor inclination angle (i–nb angle), the anteroposterior position of the mandibular first incisor against the mandible (i–nb distance), the axial inclination angle between the maxillary and mandibular first incisors (interincisal angle). these measurements were taken preand post-treatment by means of a twin block appliance. data was evaluated using a shapiro-wilk test on a small sample (≤50) before analysis. normally distributed paired data was analyzed using a t-test. if data was not normally distributed, a wilcoxon test would be used. results this study was performed during the period marchapril 2017. twenty-one lateral cephalometric radiographs taken preand post-treatment using a twin block appliance were collected. the age of the samples ranged from 8 to 16 years, with and the majority being 11 years old. the number of male and female samples was almost equal, at 10 and 11 respectively. measurement of preand post-treatment dentoalveolar modification with the twin block conducted using a steiner analysis are listed in table 1. it was apparent that the i-na angle, i-na distance and interincisal angle demonstrated very significant statistical (p < 0.01) variation. the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p211–215 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p211-215 213213yoana, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 211–215 i-nb distance also demonstrated a significant variation (p < 0.05). the i-nb angle did not show any significant change (p≥0.05). the percentage reduction in dentoalveolar changes are listed in table 2. the (–) mark on the interincisal angles in the table represents an increase in angle percentage which is inversely proportional to that of other angles. the greatest decrease occurred in i-na distance (32.1%). discussion a dentoalveolar relationship is one between the maxillary and mandibulary dentoalveolar processes (apical base) and respective skeletal base and the craniofacial bone.1 this dentoalveolar relationship may experience changes. a distal step dentoalveolar relationship in primary teeth will become a class ii molar relationship in permanent ones.14 the terminal planes during primary and mixed dentition may develop into an unpleasant relationship (class ii molar relation) in permanent teeth. in this case, observation should be undertaken carefully to ensure that orthodontic treatment can be performed as early as possible. several factors are involved in the development of the molar dentoalveolar relationship, including flush terminal plane relationship, leeway space, mandibular growth and environmental factors affecting the dental arch pattern. in class ii malocclusion, the mesiodistal cusp of the maxillary first permanent molar occludes mesially to the buccal groove of the mandibular first permanent molar. this creates a disharmony between the incisor teeth and the facial profile.4,15–17 the main objective of orthodontic treatment using a twin block functional appliance is to stimulate mandibular lengthening by stimulating the growth of cartilage and condylar cartilage, as well as inhibiting the growth of the maxilla. growth modification treatment for class ii malocclusion is recommended at an early age to prevent unfavorable growth patterns. treatment performed during the primary and mixed dentition periods produces better results compared to that started on conclusion of the mixed dentition period.17,18 early treatment produced superior results, evidenced by greater efficiency in moving the mandibulla forward and inhibiting maxillar growth, while simultaneously correcting the occlusal relation.19 the samples analyzed were lateral cephalometric radiographs from class ii dentoskeletal malocclusion with retrognathic mandible patients taken before and after orthodontic treatment using a twin block appliance. this study consisted of 21 samples taken from patients aged 8 to 16 years due to the fact that functional appliances table 1. average comparative testing on dentoalveolar change measurement before and after treatment with the twin block steiner analysis variable measurement before treatment after treatment nilai p* i-na (0) < 0.01* mean (sd) 31.21 (7.38) 26.59 (8.31) range 14 – 44.5 6.5 – 40.5 ina (mm) < 0.01** mean (sd) 7.61 (2.13) 4.98 (1.38) range 3.5 – 11,0 1.5 – 7.5 i-nb (0) 0.931 mean (sd) 32,46 (6,66) 32.36 (6.51) range 20 – 43.5 17.5 – 43 i-nb (mm) 0.011* mean (sd) 6.86 (2,32) 5.69 (2.20) range 3.5 – 11.5 1.2 – 11.5 interincisal angle (0) < 0.01** mean (sd) 108.09 (8.66) 115.60 (8.09) range 94.5 – 127.0 100.0 – 129.5 description: data was analyzed using paired t-test with a significance score of p < 0.05 (significant) (*) and p < 0,01 (very significant) (**) table 2. the percentages of reduction in dentoalveolar changes based on cephalometric radiographs in class ii dentoskeletal malocclusion retrognathic mandible treatment using twin block in the oral and dental hospital, universitas padjadjaran no. variable % decrease 1 i-na angle 15.0 2 i-na distance 32.1 3. i-nb angle 3.2* 4 i-nb distance 14.1 5 interincisal angle -7.3 description: *data used the median score due to not being normally distributed. (–) increase dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p211–215 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p211-215 214 yoana, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 211–215 appliance reduces overjet through the combination of dentoalveolar and skeletal changes. the anterior teeth are significantly tipped on the upper arch, but insignificantly so on the lower arch. the construction of the twin block explains the tipping result of the upper incisors. functional appliances used in class ii malocclusion treatment are designed to alter the mandible position both sagittally and vertically.2 therefore, the functional appliance is used in sagittal and vertical malocclusion treatment for patients in a growth and developmental period. the functional appliance works through back and forth mandibular movements, culminating in a stretching of soft orofacial and muscle tissues and myotatic reflexes resulting from muscle extension.22 the myotatic reflexes combined with the viscoelastic characteristics of the muscle exert stresses on both teeth and bone structure during the treatment. this muscle action is an important factor in generating of anticipated orthodontic and orthopedic forces. these forces are transmitted both directly and indirectly to the dentoskeletal tissues in order to correct malocclusion by increasing the sagittal intermaxillary relationship, i.e. changes in the molar relationship and decreased overjet.21 this condition can also be seen in the results of this study. the correction of malocclusion is achieved through dentoalveolar modifications reflected in the cephalometric radiographs of class ii dentoskeletal malocclusion with the retrognathic mandible treated by twin block appliances. treatment involving the use of functional appliances is intended to improve the functional relationship of dentofacial structures by eliminating poor growth factors and improving the muscle condition around the developing occlusion area. the amended dental position and supporting tissue will achieve the new functional pattern and be able to support the new position equally.2,22 the success of orthodontic treatment in class ii dentoskeletal malocclusion patients depends on the growth and development of each individual and sufficient treatment time. the success of treatment with twin block appliances also depends on these factors, as well as being profoundly affected by patient cooperation, meaning that the outcome of the treatment will vary. other factors include the age and maturity of the patient, growth pattern, etiology and initial severity of the malocclusion, duration of treatment, soft tissue characteristics and the force applied by the appliances. there are several factors affecting the stability of the treatment, i.e. mandibular growth rotation direction, respiratory tract obstruction, effective appliance manipulation, duration of treatment and sufficient retention period.2 twin block appliances may also prove able to correct transversal discrepancy in class ii malocclusion by activating the screw in the palatal section. however, the twin block appliance usage can also cause a posterior open bite, resulting in the need for further fixed orthodontic treatment to correct the open bite.22 it is concluded that a twin block appliance is effective in treating class ii dentoskeletal malocclusion with retrognathic mandible were indicated for patients with a relatively good arch, mild or moderate class ii skeletal pattern and who were experiencing a period of growth.15 within a steiner analysis, several angles are used as the reference for measuring the relationships between the maxillary first incisor and the maxilla, the mandibular first incisor and the mandible, and the maxillary and mandibular teeth.20 according to the steiner analysis, the results of measuring the average change in the dentoalveolar before and after treatment by means of a twin block appliance showed that there was a statistically significant change in i-na angle, i-na distance, i-nb distance, and interincisal angle. the greatest reduction was seen in i-na distance. within this study, the primary change observable in functional treatment consisted of dentoalveolar changes comprising buccal distalization and maxillary anterior teeth retroclination as well as mesial-shifting of the buccal part of mandibular teeth and proclination of their labial section. such findings are in line with a study performed by sharma et al.20 that indicated a significant increase in the interincisal angle in cases of orthodontic treatment utilising twin block appliances. within this study, a contrasting result was observed in the i-nb angle which confirmed there to be no statistically significant difference in this variable. this finding is similar to that of a study conducted by tarvade et al.21 that showed no proclination of the lower anterior teeth, while another piece of research revealed lower incisor proclination of 3.20.2 after treatment with twin block appliances, the reduction of proclination in the upper incisors was statistically significant. the contact between the labial bow in upper incisors and labial muscles produced a palatal retroclination of incisors. this result is to be expected from a treatment involving the use of a functional appliance due to the class ii traction effect.21 within this study, the lowest reduction was found in the i-na distance and in the degree of upper incisor protrusion. this indicates that the anterior inclination of the upper incisor was reduced, due to palatal tipping movement of the upper incisor and forward movement of the pogonion. the position of the lower mandibular incisor in class ii is important in orthodontic treatment with functional appliances. excessive labial tipping must be restricted to reduce the posibility of orthopedic alteration. after orthodontic treatment with twin block appliances, a proclination of the mandibular incisor in relation to the mandibular plane (impa) can be observed, even though it is statistically insignificant. several studies reported significant proclination of mandibular incisors after treatment with twin block appliances, while the mandibular incisors remain stable in their position.20 this result may be explained by the design of the twin block appliance. the use of an acrylic cover on mandibular incisors and the eyelet clasps produce rigid retention on the labial lower jaw, as well as the use of sounthend clasps by tarvade.21 this study indicates that the twin block dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p211–215 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p211-215 215215yoana, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 211–215 based on dentoalveolar alterations resulting from a steiner analysis. references 1. naini fb. facial aesthetics: concepts and clinical diagnosis. london: wiley-blackwell; 2011. p. 105–7. 2. clark wj. twin block functional therapy: applications in dentofacial orthopedics. 3rd ed. philadelphia: jp medical; 2014. p. 7–24, 85–118. 3. rakosi t, graber tm, alexander rg. orthodontic and dentofacial orthopedic treatment. new delhi: thieme; 2010. p. 179. 4. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st louis-missouri: mosby; 2012. p. 178. 5. sidlauskas a. the effects of the twin-block appliance treatment on the skeletal and dentolaveolar changes in class ii division 1 malocclusion. medicina. 2005; 41(5): 392–400. 6. sim jm, finn sb. clinical pedodontics. 4th ed. philadelphia: w. b. saunders; 2003. p. 209–24. 7. hassan r, rahimah ak. occlusion, malocclusion and method of measurements-an overview. arch orofac sci. 2007; 2: 3–9. 8. almustaseb e, jing m, hong h, bader r. the recent about growth modification using headgear and functional appliances in treatment of class ii malocclusion: a contemporary review. iosr-jdms. 2014; 13(4): 39–54. 9. wijayanti p, krisnawati k, ismah n. gambaran maloklusi dan kebutuhan perawatan ortodonti pada anak usia 9-11 tahun (studi pendahuluan di sd at-taufiq, cempaka putih, jakarta). j pdgi. 2014; 63(1): 25–9. 10. saptarini r, gartika m, runkat j. penggunaan twin block pada perawatan maloklusi kelas ii angle anak-anak. j dent indonesia. 2005; 12(2): 50–4. 11. baccetti t, franchi l, toth lr, mcnamara ja. treatment timing for twin-block therapy. am j orthod dentofac orthop. 2000; 118(2): 159–70. 12. ireland aj, mcdonald f. the orthodontic patient: treatment and biomechanics. london: oxford university press; 2003. p. 300–1. 13. ghislanzoni lth, baccetti t, toll d, defraia e, mcnamara ja, franchi l. treatment timing of mara and fixed appliance therapy of class ii malocclusion. eur j orthod. 2013; 35(3): 394–400. 14. bishara se. textbook of orthodontics. philadelphia: w. b. saunders; 2001. p. 354–9. 15. staley rn, reske nt. essentials of orthodontics: diagnosis and treatment. iowa: wiley-blackwell; 2011. p. 338. 16. mitchell l. an introduction to orthodontics. 4th ed. london: oxford university press; 2013. p. 336. 17. moyers re. handbook of orthodontics. michigan: year book medical publishers; 1988. p. 577. 18. oltramari-navarro pvp, de almeida rr, conti ac, navarro rl, de almeida mr, fernandes ls. early treatment protocol for skeletal class iii malocclusion. braz dent j. 2013; 24(2): 167–73. 19. soemantri es. sefalometri. bandung: universitas padjajaran; 1999. p. 1–58. 20. sharma a, sachdev v, k irtaniya b, singla a. skeletal and dentoalveolar changes concurrent to use of twin block appliance in class ii division i cases with a deficient mandible: a cephalometric study. j indian soc pedod prev dent. 2012; 30(3): 218–26. 21. ta r vade s, yamya r s, choudha r i c, biday s. skeletal and dentoalveolar changes seen in class ii div 1 mal-occlusion cases treated with twin block appliance: a cephalometric study. iosrjdms. 2014; 13(1): 5–9. 22. susilowati s, sulastry s. korelasi antara lebar mesiodistal gigi dengan kecembungan profil jaringan lunak wajah orang bugismakassar. j dentomaxillofacial sci. 2007; 6(2): 11–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p211–215 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p211-215 issn 1978 3728volume 45 number 3 september 2012 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm (oral and maxillofacial surgery – airlangga university); prof. dr. m. rubianto, drg., ms., sp.perio (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy's dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic university of hiroshima, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontica – airlangga university); prof. dr. latief mooduto, drg., m.s., sp.kg (conservative dentistry – airlangga university); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort (orthodontic – airlangga university); prof. dr. istiati soehardjo, drg., ms (oral biology – airlangga university); prof. dr. anita yuliati, drg., m.kes (dental material – airlangga university); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – airlangga university); prof. dr. diah savitri ernawati, drg., m.si (oral medicine – airlangga university); thalca i. agusni, drg., mhped., ph.d., sp.ort (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes (dental public health – airlangga university); dr. elly munadziroh, drg., ms (dental material – airlangga university); priyawan rachmadi, drg., ph.d (dental material – airlangga university); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes (dental radiology – airlangga university); bagus soebadi, drg., mhped (oral medicine – airlangga university); endang pudjirochani, drg., ms., sp.pros (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes (oral biology – airlangga university); susy kristiani, drg., m.kes (oral biology – airlangga university); ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms (orthodontic – airlangga university); devi rianti, drg., m.kes (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros (prosthodontic – airlangga university); an'nissa chusida, drg., m.kes (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg (conservative dentistry – airlangga university); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university); hendrik setiabudi, drg., m.kes (oral biology – airlangga university); otty ratna wahyuni, drg., m.kes (dental radiology – airlangga university); anis irmawati, drg., m.kes (oral biology – airlangga university); yuliati, drg., m.kes (oral biology – airlangga university); retno palupi, drg., m.kes (dental public health – airlangga university); eka augustina, drg., sp.perio (periodontica – airlangga university); febriastuti, drg., sp.kg (conservative dentistry – airlangga university); mega m. puteri, drg., sp.kga (pediatric dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 45 number 3 september 2012: prof. dr. drg. iwa sutardjo rus sudarso, s.u., sp.kga(k) (pediatrics dentistry – universitas gadjah mada) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentstry – universitas airlangga) kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) dr. indah listiana kriswandini, drg., m.kes. (oral biology – universitas airlangga) indeswati diyatri, drg., ms (oral biology – universitas airlangga) wisnu setyari, drg., m.kes. (oral biology – universitas airlangga) david buntoro kamadjaja, drg., mds., sp.bm. (oral and maxillofacial surgery – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 contents page printed by: airlangga university press. (054/04.13/aup-b5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 45 number 3 september 2012 issn 1978 3728 1. the benefit of differential moment concept in managing posterior anchorage and avoiding bite deepening harryanto wijaya and joko kusnoto ............................................................................................ 121–126 2. henoch-schönlein purpura in children: its relation to oral and dental health arlette suzy puspa pertiwi ............................................................................................................. 127–132 3. distribution of class ii major histocompatibility complex antigen-expressing cells in human dental pulp with carious lesions tetiana haniastuti ........................................................................................................................... 133–137 4. medication intake and its influence on salivary profile of geriatric outpatients in cipto mangunkusumo hospital yuniardini septorini wimardhani, winanda annisa and febrina rahmayanti ....................... 138–143 5. maternal endotoxin-induced fetal growth restriction in rats: fetal responses in toll-like receptor banun kusumawardani, marsetyawan hne. soesatyo, djaswadi dasuki and widya asmara .... 144–149 6. novel development of carbonate apatite-chitosan scaffolds based on lyophilization technique for bone tissue engineering maretaningtias dwi ariani ............................................................................................................. 150–155 7. analgesic effect of coconut shell (cocos nucifera l) liquid smoke on mice meircurius dwi c.s, tantiana and ira arundina ......................................................................... 156–160 8. penetration effect of prostaglandin e2 gel on oral mucosa of rats rafinus arifin, retno widayati, erni h purwaningsih and dewi fatma s .............................. 161–166 9. shear bond strength of self-adhering flowable composite on dentin surface as a result of scrubbing pressure and duration ferry jaya, siti triaminingsih, andi soufyan s and yosi kusuma eriwati .............................. 167–171 10. facial reconstruction using polypropylene mesh after resection of maxillary ossifying fibroma r. soesanto ....................................................................................................................................... 172–176 11. physical characteristic of brown algae (phaeophyta) from madura strait as irreversible hydrocolloid impression material prihartini widiyanti and siswanto ................................................................................................. 177–179 146 vol. 43. no. 3 september 2010 the molecular phenomena of the blaz genes forming betalactamase enzymes structure in staphylococcus aureus resistant to beta-lactam antibiotics (ampicillin) mieke satari department of oral biology faculty of dentistry, padjadjaran university bandung indonesia abstract background: nowadays, infectious disease still an important problem. one of the bacteria causing infectious diseases is staphylococcus aureus (s. aureus). in the effort to deal with infections caused by s. aureus, beta-lactam antibiotics, such as ampicillin, are used. in fact, it is unfortunately known that many of s. aureus bacteria are resistant to this group of antibiotics. because of nucleotide base changes in the structure of the genes blaz which encode beta-lactamase enzymes in s. aureus. purpose: the objective of this study was to analyze the nucleotide base changes in the structure of the genes blaz forming beta-lactamase enzymes in s. aureus resistant to ampicillin based on molecular point of view. methods: molecular examinations was conducted by isolating the genes, forming beta-lactamase enzyme, which length was 845bp, from 7 isolates of s. aureus resistant to ampicillin by using pcr technique. the results of blaz amplification were then subjected to homology by using tn 552 of s. aureus obtained from bank of genes. results: based on the result of the homology, it was found that there was a change in purine base tg, which was a pyrimidine base at the -37 position of the initial codon of blaz. this change, however, did not affect the strength of the promoter since the number of a and t is still more than the number of g and c. in the structure of the blaz gene there was even no mutation or deletion or nucleotide base substitution found, so it would not affect the effectiveness of beta-lactamase enzyme. conclusion: it can be concluded that the resistance of s. aureus towards ampicillin was not caused by nucleotide base deletion/substation. it is suspected that there were other causes leading to the resistance, including the overproduction of beta-lactamase enzyme of the blaz gene, causing the degradation of beta-lactam antibiotics. key words: staphylococcus aureus, ampicillin, blaz abstrak latar belakang: penyakit infeksi sampai saat ini masih merupakan masalah. salah satu bakteri penyebab infeksi yaitu staphylococcus aureus (s. aureus). upaya menangani infeksi yang disebabkan s. aureus dapat menggunakan antibiotik golongan betalaktam, salah satunya ampisilin. pada kenyataannya banyak s. aureus resisten terhadap antibiotik ini. salah satu penyebab timbulnya resistensi ampisilin terhadap s. aureus yaitu adanya dugaan perubahan basa nukleotida dari gen struktur (blaz) yang mengkode enzim betalaktamase. tujuan: untuk menganalisis perubahan basa nukleotida gen struktur pembentuk enzim betalaktamase pada s. aureus yang resisten ampisilin ditinjau secara molekuler. metode: pemeriksaan enzim betalaktamase secara molekuler dilakukan dengan mengisolasi gen pembentuk ensim betalaktamase (blaz) yang memiliki panjang 845 pb terhadap 7 isolat s. aureus hasil isolasi yang berasal dari abses yang resisten terhadap ampisilin dengan mengunakan pcr . hasil amplifikasi blaz kemudian dilakukan homologi dengan tn 552 s. aureus yang diperoleh dari bank gen. hasil: hasil homologi ditemukan adanya perubahan basa purin t  g yang merupakan basa pirimidin pada posisi –37 dari kodon awal blaz. perubahan ini tidak mempengaruhi kekuatan promoter karena jumlahperubahan ini tidak mempengaruhi kekuatan promoter karena jumlah a dan t masih lebih banyak dari g dan c. pada gen struktur blaz ini tidak terdapat adanya mutasi ataupun delesi maupun subsitusi basa nukleotida hingga tidak akan mempengaruhi efektifitas kerja enzim betalaktamase. kesimpulan: terjadinya resisten s. aureus terhadap ampisilin bukan disebabkan adanya mutasi maupun delesi/ subsitusi basa nukleotida dari blaz namun diduga adanya sebab lain yaitu produksi berlebih enzim betalaktamase hingga semua antibiotik betalaktam akan didegradasi oleh enzim betalaktamase. kata kunci: stafilokokus. aureus, ampisilin, blaz correspondence: mieke satari, c/o: bagian biologi oral, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: mieke_satari@yahoo.com research report 147satari: the molecular phenomena introduction nowadays, infectious disease still an important issue, especially in developing countries. one of the bacteria causing infectious diseases is staphylococcus aureus (s. aureus), which is a major bacterium causing osteomyelitis with the average percentage about 83% of 589 clinical isolates.1,2 antibiotics are commonly used to treat the infectious diseases caused by s. aureus. one of them is beta-lactam antibiotics, namely ampicillin. unfortunately, the use of antibiotics often does not follow the right rules, such as unapproriate dosage and usage procedure, and misuse (not in accordance with the illness or indications), as a consequence, it can make the resistance of the bacterium towards ampicillin increased.3 s. aureus, for instance, is often resistant to beta-lactam antibiotics, one of which is ampicillin, not only because of the activity of beta-lactamase enzymes in inactivating betalactam antibiotics, but also because of the characteristics changes of beta-lactamase enzymes encoded by blaz gene, from inductive into constitutive.4 the use of ampicillin related with beta-lactamase enzyme, is to split the active site of serine until this enzyme is not able to break the beta-lactam ring. then, ampicillin will bind to proteins binding to penicillin (transpeptidase). this activity occurs since it has a structure similar to the peptidoglycan precursor, which is d-alanin type-d-alanin penta-peptide of n-acetyl muramics. ampicillin then disrupts the formation of inter-peptide, so the final stage of the formation of peptidoglycan is disrupted since instead trans-peptidase cuts penta-peptide (d-ala), trans-peptidase bind to ampicillin, as a consequence, the spliting of d-ala is disrupted, and the formation of murrain sac becomes imperfect.5 the use of ampicillin currently still generates a lot of problems as s. aureus is known to be resistant. the mechanism of the resistance is not only due to the inability of the antibiotics to split serine, considered as the active site of beta-lactamase enzyme, but also due to the amount of beta-lactamase enzymes produced is so high that ampicillin cannot inactivate beta-lactamase enzymes produced. the reason is because of the disruption of genes that regulate the production of beta-lactamase enzymes which change to be constitutive.6 some researchers even say that the character of resistance is suspected to be encoded in plasmids that have genes resistant to ampicillin, as a result, these plasmids can conjugatively be transferred. actually, ampicillin is molecularly resistant to s. aureus allegedly either because of the mutation of the binding area or the active site of serine in the structure of blaz genes forming beta-lactamase enzyme, or because of the deletion of the structure of the genes forming betalactamase enzymes or the signal interference from the regulator genes in order to suppress the production of beta-lactamase enzyme.6–8 beta-lactamase enzyme, on the other side, is enzyme produced extracellularly. in gram-positive bacteria, this enzyme will be produced if there is the induction of betalactam antibiotics. as a consequence, in gram-positive bacteria, the formation of beta-lactamase enzymes is considered to be inductive. this formation even is 10 to 100 times higher than that in gram-negative bateria constitutively forming beta-lactamase enzyme. it means that if there is overproduction, it is supposed to be caused by the disruption of the regulation in the formation of beta-lactamase enzyme, thus, the amount of beta-lactamase enzymes will get higher. in other words, the disruption of the regulation in the production of beta-lactamase enzymes is caused by the loss of protein function regulation.9–11 another reason that causes the increasing of betalactamase enzymes is the changing of the nucleotide sequence in the structure of the genes encoding betalactamase enzyme, blaz, which is phenotypically increased 2–3 times. there was also some researchers stating that if there is a change of nucleotide base, blaz, it will then cause the effectiveness of this enzyme decreased.9,10 therefore, the purpose of this study is to analyze the nucleotide base changes of the structure of the blaz gene in forming beta-lactamase enzymes in s. aureus resistant to ampicillin. materials and methods the selection of samples then was conducted with consecutive sampling method based on the following inclusion criteria: a) s. aureus produces beta-lactamase enzymes tested by savinase disc; b) s. aureus produces total dna isolations, chromosomes and plasmids; and c) blaz gene fragments are isolated well. the study consists of two stages: conducting a laboratory examination for selecting s. aureus resistant to ampicillin by using 10 mg ampicillin disc, and determining minimal inhibitor concentration (mic) of s. aureus resistant to ampicillin. besides that, savinase disc is also used to determine whether s. aureus produces beta-lactamase enzyme. next, the molecular examination of total dna isolation in which 5 ml bacterial culture is washed with 2 ml of 50 mm tris-hcl and 200 mg/ml of rnase.11–12 afterwards, the suspension is incubated while shaken at 50rpm at the temperature of 37° c for about 30 minutes, and then is added with 400 ul of 50 mm 0.5% tris sodium dodecyl sulfate, 0.4 m edta, and 1 mg proteinase k, and after that it is incubated in 500 c water bath for about one hour. the result of dna isolation, finally, is precipitated with ethanol, and then is resuspended in 50–100 ml of 10 mm tris-hcl containing 1 mm edta.13 moreover, blaz amplification used primer pairs and pcr. primer pairs used are as the following:13 forward: 5’ tacaactgtaatatcggaggg 3’ reverse: 5’ cattacactcttggcggtttc 3’ the sequence of pcr condition at blaz amplification is: initial denaturation process occured at the temperature of 94° c for about 4 minutes; the cycle of denaturation process 148 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 146–150 occured at the temperature of 94° c for about 30 seconds; the attachment of primer occured at the temperature of 50° c for about 30 seconds; polymerization process occured at the temperature of 72° c for 2 minutes; and pcr performs in the 35th cycle and the stabilization phase occured at the temperature of 72° c. for one cycle, it takes 7 minutes with the last storage phase occured at the temperature of 4° c. if blaz fragments are amplified, sequencing then must be conducted to obtain nucleotide sequences, and finally homology process with tn552 derived from the gen-bank is conducted by using the dna star program. results from this laboratory examination, 102 samples of s. aureus isolates derived from various types of abscess were obtained. based on the result of the sensitivity test, it is then known that 69 isolates of them were resistant to ampicillin. to prove that these bacteria produce betalactamase enzyme, savinase discs then were used by applying s. aureus colonies on the savinase discs. if the color alters into pink, it will indicate that the bacteria produce beta-lactamase enzyme. molecular examination was also conducted to analyze the nucleotide changes that occur in the structure of the genes (blaz fragment) by isolating the total dna of 8 isolates that were resistant to ampicillin first. the results of the isolation observed through the analysis of agarose gel electrophoresis 1% then can be seen in the following figure 1. as shown with a thin band, it is also known that the result of the isolated total dna in obtaining plasmid was less satisfied since it migrated faster than the chromosomes did. the reason is because plasmid carriying genes encoding beta-lactamase enzymes in s. aureus are relatively small, which is about 6,7 kb, and has 4–10 copies, as a consequence, it can be concluded that the plasmid is classified into a kind of plasmid with small number of copies. besides that, unlike in e. coli, in which beta-lactamase enzymes are more greatly encoded in the plasmid, in s. aureus, beta-lactamase enzymes are more greatly encoded in the chromosome than in the plasmid.9,10 the electrophoresis results of amplified blaz gene fragments of some isolates of s. aureus in agarose gel 1% is considered to be pcr products with 845 bp length. in amplification process, there were only 7 isolates of 8 ones amplified by blaz. the results of the amplification process then were sequenced. afterward, the nucleotide base sequence of the sequencing results were homologed, and the amino acid was deduced based on the sequence of s. aureus, tn 552, obtained from the gene bank. this analysis used dna star program in order to determine the homology of the nucleotide sequence and the deduction of amino acid sequence. the homology result of blaz isolates of s. s. aureus and tn 552 of s. aureus then can be seen in the following figure 3. discussion the amplification process of blaz gene created dna with 845 bp length, which was in line with the size of the base surrounded by a pair of primers located at nucleotide position 5372–5392 as primer 1 (forward) and at nucleotide positions 6192–6212 as primer 2 (reverse). it means that the amplification of blaz was in line with the expected figure 1� the isolated total dna of 8 isolates of s. aureus resistant to ampicillin� figure 2� the results of blaz isolation with 845 bp length� figure 3� the homology result of s. aureus isolates resistant to ampicillin and s. aureus obtained from the bank of �enes in promoter area showed that there were nucleotide changes at –37 (tg) position� chromosome plasmid 149satari: the molecular phenomena length of base pairs since blaz was relatively stable and even at the nucleotide sequence there would be not any secondary structure of dna in blaz fragment until blaz could be amplified well. this condition actually has already been predicted that the blaz does not form any secondary structures meanwhile genes that regulate the production of beta-lactamase enzymes either as repressor genes or as regulator genes are located in areas where secondary dna was found, as a consequence, it is difficult to amplify those two genes. the analysis results of sequencing blaz isolates of s. aureus then was conducted by using the same pair of primers used during the blaz amplification process. after that, the homology analysis of nucleotide sequence and s. aureus tn 552 was conducted by using dna star program. next, the result was deduced into amino acids. based on the result of the sequencing analysis of the nucleotide sequence of the promoter, ribosome binding side (rbs) of blaz then was conducted to determine whether possesing strong promoter and rbs. the result of the homology of blaz of s. aureus that is resistant to ampicillin has the same nucleotide composition with s. aureus tn 552. based on the homology analysis of sequencing result of blaz fragments, it is then known that there was mutation in the –37 position of the initial codon of t-g purine base of pyrimidine bases. however, this alteration did not affect the strength of the promoter since the number of a and t was still more than that of c and g. similarly, another study also states that the alteration of the nucleotide sequence around the promoter consensus area with the number of a-t more than that of g-c still makes the promoter so strong that the transcription process can run well.14 generally, the forming process of beta-lactamase enzymes in gram-positive bacteria is actually considered to be inductive, as a consequence, strong promoter is needed. gram-positive bacteria has a strong promoter by generating beta-lactamase that is 8-18 times higher than gram-negative ones.16 beta-lactamase enzymes generated by grampossitive bacteria is 30–60 times higher than that generated by gram-negative ones which production is considered to be constitutive.14 the reason is because gram-positive bacteria have strong promoter, as a consequence, if the induction characteristics alters into the constitutive one with the strong promoter, the amount of beta-lactamase enzymes generated will be increasing. besides that, the analysis of rbs homologed with th 552 of s. aureus indicates that there was no mutation, as a result, the number of a-t (6) (purine base) was still more than that of g-c (1) (pyrimidine bases). therefore, it can be concluded that s. aureus that has strong rbs will have strong translation process which can increase the initiation complex on the 30s ribosomal subunit that still has the higher amount of purine base than the pyrimidines. the alteration of the nucleotide sequence about +1 in the consensus area caused by the addition of base a or t is very important for the strength of the promoter.17 the mutation in this area then led to the increasing of the production of beta-lactamase enzyme. the observation conducted by rowland and dyke19 even gives an illustration that the promoter of the blaz structure genes overlaps with the promoter of regulator gene. in addition, in gram-positive bacteria, beta-lactamase enzymes will only be produced if there is the induction of beta-lactam antibiotics, and then the production betalactamase enzymes will be higher than that in gramnegative bacteria which beta-lactamase enzymes are produced as much as their base products.9,11 this condition makes strong promoter needed for a strong transcription process in order to produce large amounts of beta-lactamase enzyme. the results of nucleotide sequence analysis of the 7 isolates of s. aureus resistant to ampicillin at blaz coding areas, called coding sequences. it indicates that there was no mutation, deletion, or substitution of nucleotide bases. betalactamase will become ineffective if there is mutation in the blaz structure genes, particularly in the active site, serine.18 however, in this study, the resistance in s. aureus was not caused by the alteration of the active site of serine, but it was caused by another factor, which was the mutation of genes regulating the product of beta-lactamase enzyme. s. aureus resistant to ampicillin was suspectedly caused by the excessive product of beta-lactamase, as a result, an ampicillin molecule could only be degraded by a molecule of beta-lactamase enzymes by breaking beta-lactam ring. thus, ampicillin could bind to pbp 1–3 until the bacteria could stay alive or become resistant. to find out how many doses were needed to kill bacteria, a test of mic then was also conducted in order to determine the inhibition dose of ampicillin. according to some researchers, the value of mic is supposed to be 32u g/ml,4,15 while the results of this study showed that the value of mic was about 39–312 ug/ ml. the high mic volume then caused s. aureus become resistant because of the high mic. the treatment of infection still becomes problem until nowadays since there are still many bacteria, such as s. aureus, resistant to antibiotics, one of which is ampicillin. this resistance occurs because of the interference of betalactamase enzymes produced by s. aureus. besides that, based on several studies, it is known that the resistance occurs because of mutations occured either in the binding areas or in serine active site in the structure genes of betalactamase enzyme, namely blaz. however, in this study it is found that the resistance occured was not caused by the mutations in blaz, but it was caused by another reason, which was supposed to be caused by interference in the regulator genes that caused the overproduction of betalactamase enzyme.20 this condition is based on the test which result showed that the value of mic obtained was high, about 39–312 ug/ml. it then can be concluded that to kill s. aureus a high amount of ampicillin is required since one molecule of ampicillin can be degraded by one molecule of beta-lactamase enzyme. in conclusion, the resistance of s. aureus towards ampicillin is not caused by nucleotide base deletion/ 150 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 146–150 substation. it is suspected that there are other causes leading to the resistance, including the overproduction of betalactamase enzyme of the blaz gene, causing the degradation of beta-lactam antibiotics. references 1. peterson lj. principles of management and prevention of odontogenic infection. 3rd ed st. louis: mosby year book inc; 1998. p. 65–9. 2. dojosugito a, supardi i. infeksi nosokomial (hasil penelitian). jakarta. 2001. p. 10–5. 3. nordmann ay, yamaguchi a. mechanism of beta -lactamasee inhibition. diag microbial infect 1998; 12: 1215–95. 4. richard a, lewis k, dyke kgh. meci represess synthesis from betalactamase operon s. aureus. j antibacterial and chemotherapy 2000; 45: 139–44. 5. lees l, melson ja, kneusch ak. sulbactam plus ampicillin, interimsulbactam plus ampicillin, interim review of efficacy and safety for therapeutic and prophylactic use. review of infectious diseases 1998 nov-dec; 18(5): 3078–90. 6. jeshina j, surekha k. moleculer characterization of methicillin resistant s. aureus strain isoloated in kerala south india. current research in bacteriol 2008; 2: 1–6. 7. tetsuo s, akihito y. mechanism of betalactamase inhibitor; differences between sulbactam and other betalactamase inhibitor. antibacterial agents and chemotherapy 2000; 13(6): 78–82. 8. girlich d, naas t, nordmann p. regulation of betalactamase gene expression in s. aureus. j microbiology 2006; 152(9): 2661–72. 9. bennet pn, chopra j. moleculer basis of betalactamase induction in bacteria. antimicrobial agents and chemotherapy 2003; (9): 153–8. 10. murphy jt, walsche r, devocelle m. computational model of antibiotic resistance in mrsa. j theoritical biology 2008; 25(4): 284–93. 11. wiedemann b, peter s. induction of betalactamase in gram negative. diagnoses microbial and infectious diseases 1998; 12: 131–7. 12. gordon la, posato ae, krusuwith b, craig wa, eisner w, clino mw. mec ablaz corepresor in clinical s. aureus isolates. antimicrobial agents and chemotherapy 2003; (4): 146-51. 13. tomayko y, zchek s, murray bs. sequence analysis of betalactamase from s. aureus. antimicrobial agent and chemotherapy 1996; (10): 2265–9. 14. kernodle dj. mechanism of resistance to betalactam antibiotics in gram positif. american society for microbiology 2000; 609-23. 15. dipalma jr, gregorio gj, ferco ap. basic pharmacology in medicine.basic pharmacology in medicine. 4th ed. weschester: medical surveillance inc; 1994. p. 697–703. 16. nelson ec, elisha bg. clasification based on promoters strength. j gene 1996; 86: 319–25. 17. neidhart fc, ingraham jl, schachter m. physiology bacterial cell: a moleculler approach. sundeland, massachuestts: sinuer assiciates inc. pub; 1990. p. 321–2. 18. salerno aj, lsampen o. transcriptional analysis of betalactamase regulation in s. aureus j bacteriol 1989; 166(3): 769–78. 19. rowland sj, dyke kgh. tn 552 a novel transposable element from s. aureus. molecul microbiology 1990; 4: 961–75. 20. cha j, vakulenko s, mobaschery s. characterization of betalactam antibiotic sensor domain of the mec r1 signal sensor from mrsa. j biochemestry 2007; 46(26): 7822–31. 52 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 52–56 original article streptococcus mutans detection on mother-child pairs using matrix-assisted laser desorption ionization – time of flight mass spectrometry and polymerase chain reaction udijanto tedjosasongko, dwi mulia ramadhaniati and seno pradopo department of pediatric dentistry, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: streptococcus mutans (s. mutans) bacteria mainly cause dental caries in children. these bacteria are not considered oral indigenous bacteria since they are transmitted from people around children during their deciduous teeth eruption. the detection of these bacteria can be used for dental caries prevention in children. purpose: to determine the strain and serotype of s. mutans by using matrix assisted laser desorption ionization – time of flight mass spectrometry (maldi-tof ms) and polymerase chain reaction (pcr) on dental plaque samples taken from mother-child pairs. methods: sixteen dental plaque samples of mother-child pairs were cultured on brain heart infusion broth (bhib) and mitis salivarius bacitracin (msb) media until s. mutans colony isolates were obtained. next, the isolates of s. mutans colony were introduced into the target plates of maldi-tof ms, and then ionized to become peptide mass fingerprint (pmf). afterwards, the colony isolates were detected by database software. the detected s. mutans dna then was extracted by using conventional 727 bp pcr (serotype c). results: six strains of s. mutans were detected by maldi-tof ms method. five samples were classified into ua159, two samples were 3sn1, two samples were nfsm1, two samples were 11a1, two samples were u138, two samples were 4sm1, and one sample was classified into another bacterium. five out of 16 samples were detected by pcr as serotype c (ua159). conclusion: six strains of s. mutans were detected, namely ua159, 3sn1, nfsm1, 11a1, u138, and 4sm1, one of them (ua159) was detected as serotype c. keywords: maldi-tof ms; mother-child pairs; pcr; streptococcus mutans correspondence: udijanto tedjosasongko, department of pediatric dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: udijanto@fkg.unair.ac.id introduction dental caries is an infectious disease that can transmit from one to another.1,2 children have high dental caries prevalence.3,4 dental caries is considered a significant unsolved problem. moreover, it is a multifactorial disease since it is triggered by several interrelated factors including streptococcus mutans (s. mutans) bacteria which are the most influential microorganisms in dental caries formation with a percentage of 45% in dental plaque.5,6 the polysaccharide composition of s. mutans’ extracellular layer enhances their survival rate in a very low plaque ph as to why s. mutans bacteria have high virulence besides being in high colonies that promote their environmental transmission.5 caufield et al.7 described the concept “window of infectivity” as an early vulnerable period during which infants acquired s. mutans at the age between 19 and 31 months. furthermore, another research argued that s. mutans colonization in the child’s mouth starts with first tooth eruption and continues to grow with age.7 however, this is closely related to the mother’s oral cavity state since the mother is considered the primary child’s caregiver with the highest contact frequency.8,9 strain is a single colony progeny or subculture that has been isolated in pure culture. most individuals have one s. mutans strain but some may have 1-4 s. mutans strains.10 however, since s. mutans strains have similarities and differences, they may be used to identify the kinship.11 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p52–56 mailto:udijanto@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p52-56 53tedjosasongko et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 52–56 on the other hand, subspecies microorganism marking based on its antigenic component is called serotype (serovar). s. mutans is classified into serotypes c, e, f, and k. serotype c s. mutans is the most abundant in the human oral cavity with a complex rhamnose-glucose-polymer (rgp) structure which is responsible for s. mutans survival, attachment, and colonization in the oral cavity.2,12,13 matrix assisted laser desorption ionization – time of flight mass spectrometry (maldi-tof ms) is an alternative detecting microorganisms’ method with relative higher speed, more sensitivity and specificity, simpler work procedure, less laboratory equipment requirement, and lower cost compared to other molecular and immunological based methods.14–16 this method has been widely used by microbiologists for several purposes including identification of strains and taxonomic microorganisms (bacteria, viruses, and fungi), and epidemiological studies in addition to detection of bioterrorism, water and food pathogens, and antibiotic resistance, etc.15,16 maldi-tof ms method’s principles of strains and taxonomic microorganisms’ identification are based on the generated peptide mass fingerprint (pmf) by samples. the unknown organisms can be identified whether by comparing their pmf with existed database pmf or by matching their biomarker mass with proteomic databases.16–18 recently, microorganism detection is conducted mainly by molecular methods such as polymerase chain reaction (pcr) followed by dna sequencing which is considered the gold standard method in detecting and identifying microorganisms up to chromosome dna level.19,20 furthermore, this method has the highest sensitivity and specificity, which is fast and accurate (100%). therefore, this research aimed to determine the strain and serotype of s. mutans using maldi-tof ms and pcr methods on dental plaque samples taken from mother-child pairs. materials and methods the research samples were dental plaque taken from 16 subjects in jagiran tambaksari area in surabaya. the subjects were eight mother-child pairs; the children were younger than 2 years old, the pairs were healthy and did not consume antibiotics and corticosteroid drugs while the mothers had dmf-t index of more than 2.7, and were willing to participate in the research signing the informed consents. the ethical clearance certificate was submitted by the ethical clearance committee of faculty of dental medicine, universitas airlangga (no. 77/kkepk.fkg/vi/2016). the research was conducted at the institute of tropical disease (itd), universitas airlangga. the plaque samples were taken by brushing method for maxillary and mandibular teeth surfaces, including the tongue, using sterile toothbrushes.8 after that, the plaque was added to the brain heart infusion broth (bhib) liquid media and incubated at 37°c for 48 hours. thereafter, the plaque samples were cultured on mitis salivarius bacitracin (msb) media and incubated anaerobically at 37°c for 48 hours using gas-pack anaerobic jars. subsequently, a solitary colony from the incubated samples was taken carefully using a stick and cultured on the second bhib media tubes. all tubes with s. mutans colonies then were incubated again at 37°c for 48 hours. after the incubation process, the s. mutans colonies were re-cultured by diffusing them in a zigzag pattern on the second msb media, in which each petri dish was divided into 4 parts for 4 samples. the second msb media then were incubated anaerobically at 37°c for 48 hours using gaspack anaerobic jars. next, the morphology of each sample on the second msb media was examined under an inverted microscope (olympus ck 128, tokyo, japan) to ascertain whether the emerged bacterial colonies were s. mutans or not. afterward, a solitary colony was taken carefully using a stick to transfer it to the third bhib media tubes and then was incubated at 37°c for 48 hours. after that, the tubes were vortexed to be homogeneous, and each sample was then transferred into two different sterile eppendorf tubes with a size of 2 ml micropipette. one eppendorf tube was used as a sample for the maldi-tof ms process,17 while the other eppendorf tube was used as a sample for the pcr process. in the maldi-tof ms process, the eppendorf tubes samples were diluted with 3 ml of 0.45% nacl, then introduced to the target plates, and mixed with reagents including a matrix (mixture of water and organic solvent) of acetonitrile and strong acid (trifluoroacetate tfa), as well as a matrix of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). next, they were dried. after that, the target plates were inserted into the maldi-tof ms machine (vitex, biomérieux s.a, marcy l’étoile, france) to match the microorganisms with the software existing data. then, the results were printed.18 in the pcr process, the eppendorf tube samples containing dna were isolated with the dna isolation purification kit wizard (wizard® genomic dna purification kit, promega corporation, singapore) to obtain the dna extract of each sample. next, each dna extract was processed by the conventional pcr process that performed in 25 cycles under initial denaturation at 96°c for 2 minutes. in each cycle, they exposed to denaturation at 96°c for 15 seconds, annealing at 61°c for 30 seconds, extension at 72°c for 1 minute, and post extension at 72°c for 10 minutes using sc-f primer pairs (cgg agt gct ttt tac aag tgc tgg) and sc r (aac cac ggc cag caa acc ctt tat) at site 727 bp.21 after that, dna samples were processed by bio-rad t100 pcr machine, california, usa. subsequently, the pcr product was confirmed by 1% agarose gel electrophoresis (mupid2plus) on a voltage of 90 volts for 30 minutes. after the electrophoresis process was completed, the gel was soaked into a 2% ethidium bromide immersion solution for 30 minutes, and the pcr product then was visualized using a translucent uv to observe whether the band was in the predetermined location or not. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p52–56 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p52-56 54 tedjosasongko et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 52–56 results there were 16 dental plaque samples taken from motherchild pairs. after samples’ culturing on bhib and msb media, their morphology was tested using an inverted microscope (olympus ck 128, tokyo, japan). the results showed round or ovoid single cubes with a 1-2 μm diameter arranged in chains. the pure s. mutans isolation was processed on the vitex machine (biomérieux s.a, marcy l’étoile, france) using maldi-tof ms method to detect s. mutans strains. the results of the detection of s. mutans strain with malditof ms method are presented in table 1. during the pcr process, serotype c s. mutans was detected since serotype c is the most dominant serotype in the human oral cavity. the results of serotype c s. mutans detection with pcr method were shown in figure 1. based on pcr process results at the 727 bp amplification site, three samples which are line 1 (an 8), line 2 (ib1), and line 3 (ib 4) samples were detected as serotype c s. mutans, while the other13 samples were not. discussion the results of the s. mutans detection on the dental plaque samples of the mother-child pairs with maldi-tof ms approach showed six different strains of s. mutans with some samples had the same s. mutans strains. gibbons et al.11 stated that the microorganism strains show similarities and differences among individuals, that is why the strain can be used to identify the individuals’ kinship. according to klein et al.10 humans can have 1-4 strains of s. mutans in their oral cavity; at least one strain in each. the strains are varied among individuals according to their geographical locations, bacterial culture conditions (not significantly different), and the sample preparation methods. the national center for biotechnology information (ncbi) in 2016 recognized 172 strains of s. mutans.22 these strains have been detected from complete previous studies. for instance, s. mutans ua 159 with id taxonomy 637000288 and id ncbi 21007 was officially announced on 1st december 2006 as a common facultative and pathogenic gram-positive cocci in the oral cavity of children with active caries , including serotype c s. mutans which is the sequencing status has already been studied. strain, according to dijkshoorn et al,’s statement cited in bergey’s manual of systemic bacteriology, is a progeny or subculture of a solitary colony isolated in pure culture.23 furthermore, strain is divided into several types according to certain basic properties. first of all, biotype strain, in which the strain is classified based on the biochemical or physiological structure of species. although biotype strain is often used to describe the species’ characteristics, it has not the capability to demonstrate the whole species properties. therefore, other classifications, based on other criteria, were conducted such as morphotype strain (morphovar) in which the strain is grouped based on their morphology and serotype strain (serovars) in which the strain is classified based on antigenic structures, as well as patotype strain (patovars), or phagotype strain (fagovars) which are used sometimes to denote certain properties of strain variation.24,25 s. mutans is classified based on its antigenic structure (cell wall carbohydrate specificity, h2o2 production, bacitracin sensitivity, fermented substrate, and dna content) into four serotypes which are: serotype c, serotype e, serotype f, and serotype k.21,26,27 serotype c s. mutans is the most common type in the human oral cavity, especially in dental plaque with a prevalence of 75-90%, since serotype c s. mutans has a complex rgp structure that enhances its survival, attachment, and colonization in the oral cavity.28 therefore, s. mutans serotype c was selected in this research as s. mutans determinant. substantially, bacterial identification methods include the phenotypic method in which bacteria are classified based on their profile, metabolic properties, and chemical composition and the genotypic method in which bacteria are categorized according to their genetic material (dna). in the phenotypic method, bacteria are taken from various 727 bp 500 bp m an8 ib1 ib4 figure 1. the results of the pcr process. three samples were detected as serotype c s. mutans, i.e. an8, ib 1, and ib 4, while the other 13 samples were not. table 1. the results of s. mutans strain detection with malditof ms method samples taxon ids name of the organism mother 1 child 1 637000288 637000288 s. mutans ua159 s. mutans ua159 mother 2 child 2 2558860343 2558860343 s. mutans 3sn1 s. mutans 3sn1 mother 3 child 3 2558860323 2558860323 s. mutans nfsm1 s. mutans nfsm1 mother 4 child 4 637000288 2639762775 s. mutans ua159 s. gordonii ie35 mother 5 child 5 2558860348 2558860348 s. mutans 11a1 s. mutans 11a1 mother 6 child 6 2558860328 2558860328 s. mutans u138 s. mutans u138 mother 7 child 7 2558860342 2558860342 s. mutans 4sm1 s. mutans 4sm1 mother 8 child 8 637000288 637000288 s. mutans ua159 s. mutans ua159 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p52–56 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p52-56 55tedjosasongko et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 52–56 specimens to be cultured, isolated, and then detected based on taxonomic principles. the microscopic observation, in which bacteria is detected based on shape, size, group, gram staining reaction, and motility, should be combined with the natural environment data during the bacterial identification method. bergey’s manual of determinative bacteriology is an example of a guidebook that detects bacteria based on their microscopic and physiological characters. however, the phenotypic method can only distinguish genus from the same family members and cover some bacterial species, while the genotypic method has the capability to recognize the microorganism subtypes as to why modern taxonomy tend to use more complex detection method including molecular analysis.29,30 on the other hand, maldi-tof ms is considered an efficient analytical technique for detecting chemical structures in which chemical compounds are ionized into molecules based on the mass calculation of the molecule and its fragmentation pattern (m / z). this method is considered an alternative method in bacterial identification because of its advantageous characteristics including simplicity, fast result, high specificity and sensitivity, lower cost compared to the molecular and immunological method, and no laboratory requirements. furthermore, this method is used for various purposes including identification of strain type, microbial taxonomy, bioterrorism, water and food pathogens, blood and urinary tract pathogens, and antibiotic resistance, in addition to supporting the bacterial, fungal, and viral diseases diagnosis, etc.15,17,18 maldi-tof ms is based on pmf generated by samples to detect the strain type or the microbial taxonomy. target samples that have been trapped and dissolved in the matrix solution would be exposed to laser spectrometry to undergo an ionization process turning them into pmf. pmf is used in the detection process by comparing unknown organisms pmf with the existing database pmf or by matching the unknown microorganism biomarkers mass with proteome databases. furthermore, matching the sample pmf pattern with the ribosome protein pmf is required to detect some particular species’ microorganisms and their strain type. however, the pmf pattern matching in this method still has some limitations since the new isolates can be detected if the software database matches the sample pmf pattern. that is why the database of this method should be locally prepared for a specific taxonomy (e.g. streptococcus or staphylococcus) as to why geographical variations can occur among the genotype and phenotype of a microorganism.14,17,18 recently, microorganisms identification tends to be more dependant on dna sequencing approaches. for example, pcr and dna sequencing methods become the “gold standard” in microorganism identification in modern taxonomy, defining phylogenies, and analyzing epidemiological studies ecosystem. furthermore, this method is used for revealing bacterial evolution, constructing phylogenetic trees, tracing species diversity, and detecting new species without isolating the microorganisms. this method has many superior properties including the fast procedure, accurate result, high specificity and sensitivity reach to 100%, low vulnerability to contamination, and simultaneous detection for several microorganisms. however, this approach requires high cost, specific primary determinations, appropriate thermal cycles, and specialized expertise.29 serotype c s. mutans, in particular, can be detected through serotype c primers wherein a specific primary pcr process will be encoded according to the nucleotide base sequence of a dna sample. thus, dna samples that do not match with the nucleotide base sequence on the pcr primers will not be recognized or appeared in the amplified band. in this research, serotype c s. mutans were detected with sc-forward primers (cgg agt gct ttt tac aag tgc tgg) and sc-reverse primers (aac cac ggc cag caa acc ctt tat) at the amplification region of 727 bp19. from the 16 s. mutans dna samples, three samples were detected as serotype c s. mutans as follows: in line 1 (child 8), line 2 (mother 1), and line 3 (mother 4), while the other 13 samples were not identified as serotype c s. mutans since serotype c primers were the only used primers. the other 13 samples may follow other serotypes, such as serotype e s. mutans, serotype f s. mutans, etc., or genetic polymorphism of previously studied s. mutans.19,31 genetic polymorphism is a variation in the microorganism dna structure in which a change in the nucleotide base sequence is occurred due to the insertion, addition, or subtraction of a particular base.32 genetic polymorphism is caused whether by the spontaneous gene mutations triggered by the normal cell function changes or by interaction with the environment. if the changes occur in only one base, referred to as point mutation, it can be inferred that the microorganism has been genetically polymorphed. most point mutations occur as substitutions of g c (guanin cytosine) or a t (adenine thymine). in conclusion, there were six strains of s. mutans detected by maldi-tof ms method follows five samples of s. mutans ua159, two samples of s. mutans 3sn1, two samples of s. mutans nfsm1, two samples of s. mutans 11a1, two samples of s. mutans u138, and two samples of s. mutans 4sm1. however, ua159 was the only strain that was detected as serotype c by pcr method. it is expected to use these results as a basis for further researches related to early detection of dental caries, identification of new s. mutans isolates, and epidemiological studies. references 1. mattos-graner ro, li y, caufield pw, duncan m, smith dj. genotypic diversity of mutans streptococci in brazilian nursery children suggests horizontal transmission. j clin microbiol. 2001; 39(6): 2313–6. 2. baca p, castillo a-m, liébana m-j, castillo f, martín-platero a, liébana j. horizontal transmission of streptococcus mutans in schoolchildren. med oral patol oral cir bucal. 2012; 17(3): e495500. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p52–56 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p52-56 56 tedjosasongko et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 52–56 3. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10(1): 7. 4. pierce a, singh s, lee j, grant c, cruz de jesus v, schroth rj. the burden of early childhood caries in canadian children and associated risk factors. front public heal. 2019; 7: 328. 5. okada m, soda y, hayashi f, doi t, suzuki j. pcr detection of streptococcus mutans and s. sobrinus in dental plaque samples from japanese pre-school children. j med micro. 2002; 51(5): 443–7. 6. jiang q, yu m, min z, yi a, chen d, zhang q. ap-pcr detection of streptococcus mutans and streptococcus sobrinus in caries-free and caries-active subjects. mol cell biochem. 2012; 365(1–2): 159–64. 7. caufield pw, cutter gr, dasanayake ap. initial acquisition of mutans streptococci by infants: evidence for a discrete window of infectivity. j dent res. 1993; 72(1): 37–45. 8. tedjosasongko u, kozai k. initial acquisition and transmission of mutans streptococci in children at day nursery. asdc j dent child. 2002; 69(3): 284–8, 234–5. 9. hameş-kocabaş ee, uçar f, kocataş ersin n, uzel a, alpöz ar. colonization and vertical transmission of streptococcus mutans in turkish children. microbiol res. 2008; 163(2): 168–72. 10. klein mi, flório fm, pereira ac, höfling jf, gonçalves rb. longitudinal study of transmission, diversity, and stability of streptococcus mutans and streptococcus sobrinus genotypes in brazilian nursery children. j clin microbiol. 2004; 42(10): 4620–6. 11. gibbons rj, cohen l, hay di. strains of streptococcus mutans and streptococcus sobrinus attach to different pellicle receptors. infect immun. 1986; 52(2): 555–61. 12. pieralisi fjs, rodrigues mr, segura vg, maciel sm, ferreira fba, garcia je, poli-frederico rc. genotypic diversity of streptococcus mutans in caries-free and caries-active preschool children. int j dent. 2010; 2010: 1–5. 13. damé-teixeira n, arthur ra, parolo ccf, maltz m. genotypic diversity and virulence traits of streptococcus mutans isolated from carious dentin after partial caries removal and sealing. sci world j. 2014; 2014: 1–6. 14. murray pr. what is new in clinical microbiology-microbial identification by maldi-tof mass spectrometry: a paper from the 2011 william beaumont hospital symposium on molecular pathology. j mol diagn. 2012; 14(5): 419–23. 15. singhal n, kumar m, kanaujia pk, virdi js. maldi-tof mass spectrometry: an emerging technology for microbial identification and diagnosis. front microbiol. 2015; 6(aug): 791. 16. croxatto a, prod’hom g, greub g. applications of maldi-tof mass spectrometry in clinical diagnostic microbiology. fems microbiol rev. 2012; 36(2): 380–407. 17. cla rk a e, kaleta ej, a rora a, wolk dm. matr ix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. clin microbiol rev. 2013; 26(3): 547–603. 18. bilecen k, yaman g, ciftci u, laleli yr. performances and reliability of bruker microf lex lt and vitek ms malditof mass spectrometry systems for the identification of clinical microorganisms. biomed res int. 2015; 2015: 516410. 19. childers nk, osgood rc, hsu k-l, manmontri c, momeni ss, mahtani hk, cutter gr, ruby jd. real-time quantitative polymerase chain reaction for enumeration of streptococcus mutans from oral samples. eur j oral sci. 2011; 119(6): 447–54. 20. gilbert k, joseph r, vo a, patel t, chaudhry s, nguyen u, trevor a, robinson e, campbell m, mclennan j, houran f, wong t, flann k, wages m, palmer ea, peterson j, engle j, maier t, machida ca. children with severe early childhood caries: streptococci genetic strains within carious and white spot lesions. j oral microbiol. 2014; 6(1): 1–11. 21. shibata y, ozaki k, seki m, kawato t, tanaka h, nakano y, yamashita y. analysis of loci required for determination of serotype antigenicity in streptococcus mutans and its clinical utilization. j clin microbiol. 2003; 41(9): 4107–12. 22. schoch cl, ciufo s, domrachev m, hotton cl, kannan s, khovanskaya r, leipe d, mcveigh r, o’neill k, robbertse b, sharma s, soussov v, sullivan jp, sun l, turner s, karsch-mizrachi i. ncbi taxonomy: a comprehensive update on curation, resources and tools. database (oxford). 2020; 2020(2): 1–21. 23. dijkshoorn l, ursing bm, ursing jb. strain, clone and species: comments on three basic concepts of bacteriology. j med microbiol. 2000; 49(5): 397–401. 24. prescott lm, harley jp, klein da. microbial taxonomy. in: microbiology. 5th ed. new york: mcgraw-hill; 2002. p. 422– 49. 25. maki y, sakayori t, hirata s, ishii t, tachino a. monitoring caries risks before the window of infection and later caries increment: a caries prediction study on rapid detection of streptococcus mutans using monoclonal antibodies. bull tokyo dent coll. 2014; 55(1): 19–23. 26. nakano k, lapirattanakul j, nomura r, nemoto h, alaluusua s, grönroos l, vaara m, hamada s, ooshima t, nakagawa i. streptococcus mutans clonal variation revealed by multilocus sequence typing. j clin microbiol. 2007; 45(8): 2616–25. 27. nakano k, nemoto h, nomura r, homma h, yoshioka h, shudo y, hata h, toda k, taniguchi k, amano a, ooshima t. serotype distribution of streptococcus mutans a pathogen of dental caries in cardiovascular specimens from japanese patients. j med microbiol. 2007; 56(4): 551–6. 28. tabchoury cpm, sousa mck, arthur ra, mattos-graner ro, del bel cury aa, cury ja. evaluation of genotypic diversity of streptococcus mutans using distinct arbitrary primers. j appl oral sci. 2008; 16(6): 403–7. 29. franco-duarte r, černáková l, kadam s, s. kaushik k, salehi b, bevilacqua a, corbo mr, antolak h, dybka-stępień k, leszczewicz m, relison tintino s, alexandrino de souza vc, sharifi-rad j, melo coutinho hd, martins n, rodrigues cf. advances in chemical and biological methods to identify microorganisms—from past to present. microorganisms. 2019; 7(5): 130. 30. sato t, matsuyama j, kumagai t, mayanagi g, yamaura m, washio j, takahashi n. nested pcr for detection of mutans streptococci in dental plaque. lett appl microbiol. 2003; 37(1): 66–9. 31. kazemtabrizi a, haddadi a, shavandi m, harzandi n. metagenomic investigation of bacteria associated with dental lesions: a crosssectional study. med oral patol oral y cir bucal. 2020; 25(2): e240–51. 32. williams jgk, kubelik ar, livak kj, rafalski ja, tingey s v. dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acids res. 1990; 18(22): 6531–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p52–56 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p52-56 231 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 231–234 case report introduction oral cancer can be mistakenly diagnosed as periodontitis, particularly when the patient’s signs and symptoms present localised periodontitis or acute periodontal infections such as a periodontal or periapical abscess. neoplasms such as oral squamous cell carcinoma (oscc), odontogenic tumours, other primary neoplasms of periodontal tissue and secondary metastatic neoplasms of periodontal tissue have a moderate association with the destruction of periodontal tissue, thus causing progressive loss of the alveolar bone and subsequently, mobility of teeth.1 however, little information on salivary gland neoplasm can be obtained, particularly adenoid cystic carcinoma (acc) and its relationship with periodontitis. acc is a rare malignant neoplasm of the salivary gland in the head and neck malignancies, with a prevalence of 10% among salivary gland disorders.2–4 it commonly affects women at the average age of 50 years and occurs in the parotid glands, minor salivary glands and, rarely, in the buccal mucosa.3,5 clinically, acc presents as an indolent and slow-growing lesion.3,6–8 it is locally invasive and can invade peripheral nerves and blood vessels at an early stage. around 40% of acc cases have the potential to metastasise to other distant organs.3,4,7–9 its high propensity for perineural invasion affects the trigeminal and facial nerves of the patient.3 risk of distant metastasis is influenced by the difficulty of surgical removal at the primary site and stages of the acc.10 treatment modalities for acc can range from conservative to radical surgical removal followed by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p231–234 role of supportive periodontal management in patient with metastatic cancer kevin chee pheng neo1,3, nurul syahirah mohamad2,3, avita rath3, melissa li zheng wong3, myint wai3, bennete fernandes3 1klinik pergigian tawau, ministry of health, sabah, malaysia 2klinik pergigian bandar tun razak, ministry of health, pahang, malaysia 3faculty of dentistry, segi university, selangor, malaysia abstract background: treatment of head and neck cancers may cause sequelae affecting patients’ quality of life during and after treatment. as a result, periodontal management of a patient with parotid gland adenoid cystic carcinoma, particularly in advanced stage 4, can be challenging for dental practitioners, especially if the patient is on active oral molecular therapy and undergoing long-term radiotherapy and chemotherapy. purpose: this report was intended to describe the conservative non-surgical management of tooth 27 with poor prognosis owing to grade ii mobility in a patient on active lenvatinib therapy – where the extraction was not advisable to lessen the risk of osteonecrosis. case: a 52-year-old female patient was referred by an oncologist to our dental clinic for non carious toothache. five years ago, the patient was diagnosed with adenoid cystic carcinoma (acc) and had a treatment history of radiotherapy, chemotherapy and surgical removal of the left parotid gland to manage the malignancy. oral examination revealed characteristic findings of periodontitis. case management: ultrasonic scaling, antimicrobial mouth rinses and reinforcement in oral hygiene instructions manage the tooth 27 conservatively. conclusion: a cautious approach by the dental surgeon, together with the multidisciplinary team caring for cancer patients, is fundamental and helps with the palliative periodontal management of this patient to enhance oral health-related quality of life. with a lack of reports on conservative periodontal therapy in acc patients, this report highlights the combination of smoking cessation, reinforcement of oral hygiene instructions and conservative periodontal treatment. keywords: adenoid cystic carcinoma; periodontitis; periodontal maintenance therapy; periodontal management correspondence: dr. kevin chee pheng neo, klinik pergigian tawau, ministry of health. klinik pergigian tawau, klinik kesihatan 2, jalan chong thien vun, jalan sin on, 91008 tawau, sabah, malaysia. email: kevincpneo@gmail.com mailto:kevincpneo@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p231-234 232neo et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 231–234 radiotherapy, chemotherapy or molecular therapy.3,4,9,10 however, these treatment modalities do not improve the prognosis of the disease due to its metastasis ability and responsiveness to treatment, especially the chemotherapeutic agents due to their low sensitivity and responsiveness toward acc.3,11 oral molecular drugs such as lenvatinib (molecule anti-angiogenic drug) are used for the treatment of acc and have a low risk of causing osteonecrosis of the jaw (onj). however, when combined with other antiresorptive drugs, such as bisphosphonate, these may increase onj incidents.12 moreover, previous literature reports the occurrence of oral molecule medications and could act as a trigger for the occurrence of medicationsrelated mronj.13 hence, the main clinical objective is to relieve the patient’s pain and prevent tooth loss, as extraction is not advisable during this period as the patient is at risk of onj. this case report was intended to describe the conservative non-surgical management of tooth 27 with poor prognosis owning to grade ii mobility in a patient – on active lenvatinib therapy – where extraction was contraindicated to ameliorate the risk of osteonecrosis. case an oncologist referred a 52-year-old female patient due to localised pain concerning tooth 26. her primary complaint was localised pain on tooth 26 and difficulty in eating and swallowing in the last three weeks before visiting our dental clinic. it was described as non-radiating, chronic and dull pain in the region of tooth 26. the patient’s medical history revealed stage 4 metastatic acc involving the left parotid gland since 2017. previously, the patient had undergone radiotherapy until june 2020, including chemotherapy during the last cycle of oral-targeted therapy in the middle of 2019. currently, she is on the second cycle of oral targeted therapy with lenvatinib for her cancer. the patient had been smoking since the age of 16, with an average of 25 cigarettes per day (45 packs per year). the patient also admitted to drinking alcohol occasionally. the patient visited a general dental practitioner three weeks prior, and the dentist suggested root canal treatment at the dental specialist clinic to relieve the pain. upon clinical examination, the patient presented with an asymmetrical face and left-side facial paralysis during centric relation, as shown in figure 1. the facial paralysis was due to surgical removal of acc, which includes the complete removal of the left parotid gland. in addition, tooth 26 showed poor periodontal status with miller grade ii mobility indicated for extraction. f u r t h e r e x a m i n a t i o n o f t h e p a t i e n t u s i n g a n orthopantomogram (opg) is shown in figure 2, revealing that the patient has trismus, as shown in figure 3. the opg revealed that the bone level of tooth 26 was at the middle third of the root. generalised horizontal bone loss can be observed in both upper and lower jaws, with localised figure 1. the inability of the patient to close the left eyelid due to facial paralysis. figure 2. the orthopantomogram (opg) of the patient. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p231–234 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p231-234 233 neo et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 231–234 vertical bone loss seen in relation to tooth 35 and tooth 46. the patient was diagnosed with periodontitis as a manifestation of systemic disease. case management following a clinical and radiographic examination, the patient was prescribed paracetamol 500 mg (prn), chlorhexidine and saline mouth rinse for two weeks, and she was referred back to her oncologist to acquire a clearance letter for the necessary dental procedures. after two weeks, the patient visited with a letter from her oncologist urging that we pursue conservative management options. however, if tooth extraction is inevitable, the oncologist advised withholding her oral lenvatinib medication a week before the surgery to avoid osteonecrosis. full mouth ultrasonic scaling was carried out, and the patient was referred to the oral surgery department for extraction of tooth 26. an oral maxillofacial surgeon educated the patient and guardian before extraction regarding the post-extraction complications when the patient is on oral lenvatinib. the oral maxillofacial surgeon recommended that the patient be handled conservatively for another week, such as rinsing with chlorhexidine mouthwash and saline mouth rinse to relieve the non-carious pain. an extensive oral hygiene education was given to enhance the patient’s existing gingival health. during a follow-up phone call, the patient reported improved pain control after using chlorhexidine mouthwash and saline mouth rinse in conjunction with the reinforced oral hygiene instructions. discussion while oral cancer, such as oscc, has been intensively investigated for its relationship with periodontitis, acc has received less attention due to its rarity, accounting for just 1% of all head and neck cancers.14 many studies reported intraosseous acc mimicking apical periodontitis,15–17 but no results associating acc to periodontitis were discovered. the patient had both metastatic acc and typical periodontitis symptoms. the clinical characteristics are consistent with periodontitis, but for this patient to be diagnosed with a systemic illness affecting the periodontium, a biopsy and histological testing were required to determine if the radiolucency is of metastatic or inflammatory origin.1 since a biopsy was not performed, this posed a challenge for comprehensive management. the benefits of conservative management include the patient’s ability to retain their teeth for mastication, enhancing their quality of life. it also aids in the prevention of extraction, which is contraindicated in our case. extraction may cause the patient to develop onj due to the medication, whereas lenvatinib makes future care more complex. however, the disadvantages of this management include that it only gives short relief to the patient if the patient maintains good dental hygiene. the periodontitis and tooth discomfort will return if the patient does not cooperate. the patient also needed to be evaluated regularly to monitor and manage the condition closely. resultantly, in light of the diagnostic inadequacies, periodontal management was carried out as conservatively as feasible to avoid difficulties for the patient. in the present study, irradiated patients’ preferred periodontal treatment includes scaling and root planing with antimicrobial therapy.18 increased incidence of opportunistic pathogens after radiotherapy to the head and neck region – with or without chemotherapy – renders plaque control extremely vital to prevent disease progression and periodontal pocket colonisation in cancer patients.17,19 additionally, meticulous oral hygiene instruction should be given to the patient, emphasising avoiding alcohol consumption and smoking.20 patients should be instructed on the proper tooth brushing technique, preferably the modified bass technique, and interdental cleaning using floss or an interdental brush.18,21 the usage of mouthwash containing chlorhexidine and fluoride is encouraged to reduce the side effects of radiotherapy by preventing a decrease in the tensile strength of irradiated dentin and enamel.22 periodontal maintenance is crucial in determining the outcome of the periodontal treatment and long-term care of teeth.23 tooth loss rate for patient with high periodontitis risk is significantly higher than low-risk patients.24 in this case, the patient is under the high-risk category due to acc and smoking habits.23,25 it is important to start tobacco cessation programmes for the patient to achieve a better outcome of the periodontal disease and reduce the risk of tooth loss during the maintenance period.23,26 studies showed that smokers respond less than non-smokers to non-surgical periodontal therapies in terms of healing and clinical parameters.27,28 chlorhexidine mouthwash effectively improves plaque and gingival index among cancer patients as an oral hygiene regimen before and during cancer treatment.29 figure 3. the mandibular arch and the maximum mouth opening of the patient. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p231–234 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p231-234 234neo et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 231–234 using anti-angiogenic agents such as lenvatinib causes side effects such as dry mouth and fatigue.6 dry mouth increases the risk of caries in patients. patients with high caries risk require good oral hygiene maintenance to prevent tooth decay and further management. certain patients without motivation to maintain oral hygiene might choose to extract the tooth when it is decaying or mobile. this action might further complicate the medical condition of patients with the occurrence of onj – common among patients under cancer therapy that opted for dental extraction. the coincidence of onj among patients under lenvatinib is significantly low, but the risk of onj is still present as dental extraction acts as a common risk for the occurrence of onj.12 hence, we must consider the balance between risk and reward when such a scenario occurs. to summarise, periodontal care of acc patients throughout the cancer therapy phase may be challenging. a cautious approach by the dental surgeon, together with the multidisciplinary team caring for cancer patients, is fundamental and helps with the palliative periodontal management of this patient to enhance oral health-related quality of life. due to the lack of reports on conservative periodontal therapy in acc patients, the current case report highlights the combination of smoking cessation and reinforcement of oral hygiene instructions in addition to conservative periodontal treatment. references 1. albandar jm, susin c, hughes fj. manifestations of systemic diseases and conditions that affect the periodontal attachment apparatus: case definitions and diagnostic considerations. j clin periodontol. 2018; 45 suppl 2: s171–89. 2. young a, okuyemi ot. malignant salivary gland tumors. [updated 2022 feb 16]. statpearls [internet], editor. treasure island (fl): statpearls publishing; 2022. 3. karimi a, parhiz a, eslamiamirabadi n, moradzadeh khiavi m, derakhshan s. adenoid cystic carcinoma of buccal mucosa: a report of two rare cases and review of literature. clin case reports. 2021; 9(1): 23–30. 4. dillon pm, chakraborty s, moskaluk ca, joshi pj, thomas cy. adenoid cystic carcinoma: a review of recent advances, molecular targets, and clinical trials. head neck. 2016; 38(4): 620–7. 5. waechter j, xavier cb, correa g, gomes e de f, fernandes filho rb. oral and maxillofacial rehabilitation of a patient suffering from intraosseous adenoid cystic carcinoma. rgo rev gaúcha odontol. 2017; 65(2): 168–73. 6. locati ld, galbiati d, calareso g, alfieri s, singer s, cavalieri s, bergamini c, bossi p, orlandi e, resteghini c, platini f, granata r, quattrone p, mancinelli m, mariani l, lo vullo s, licitra lf. patients with adenoid cystic carcinomas of the salivary glands treated with lenvatinib: activity and quality of life. cancer. 2020; 126(9): 1888–94. 7. liu j, shao c, tan ml, mu d, ferris rl, ha pk. molecular biology of adenoid cystic carcinoma. head neck. 2012; 34(11): 1665–77. 8. andreasen s. molecular features of adenoid cystic carcinoma with an emphasis on microrna expression. apmis. 2018; 126 suppl: 7–57. 9. jaso j, malhotra r. adenoid cystic carcinoma. arch pathol lab med. 2011; 135(4): 511–5. 10. gao m, hao y, huang mx, ma dq, luo hy, gao y, peng x, yu gy. clinicopathological study of distant metastases of salivary adenoid cystic carcinoma. int j oral maxillofac surg. 2013; 42(8): 923–8. 11. sahara s, herzog ae, nör je. systemic therapies for salivary gland adenoid cystic carcinoma. am j cancer res. 2021; 11(9): 4092–110. 12. lu w, guo q, ma z, liu l, zhao z. lenvatinib and osteonecrosis of the jaw: a pharmacovigilance study. eur j cancer. 2021; 150: 211–3. 13. mauceri r, panzarella v, morreale i, campisi g. medication-related osteonecrosis of the jaw in a cancer patient receiving lenvatinib. int j oral maxillofac surg. 2019; 48(12): 1530–2. 14. beaumont s, bhatia n, mcdowell l, fua t, mccullough m, celentano a, yap t. timing of dental extractions in patients undergoing radiotherapy and the incidence of osteoradionecrosis: a systematic review and meta-analysis. br j oral maxillofac surg. 2021; 59(5): 511–23. 15. schuurhuis jm, stokman ma, witjes mjh, langendijk ja, van winkelhoff aj, vissink a, spijkervet fkl. head and neck intensity modulated radiation therapy leads to an increase of opportunistic oral pathogens. oral oncol. 2016; 58: 32–40. 16. ammajan rr, joseph r, rajeev r, choudhary k, vidhyadharan k. assessment of periodontal changes in patients undergoing radiotherapy for head and neck malignancy: a hospital-based study. j cancer res ther. 2013; 9(4): 630–7. 17. lajolo c, rupe c, gioco g, troiano g, patini r, petruzzi m, micciche’ f, giuliani m. osteoradionecrosis of the jaws due to teeth extractions during and after radiotherapy: a systematic review. cancers (basel). 2021; 13(22): 5798. 18. irie m-s, mendes e-m, borges j-s, osuna l-g-g, rabelo g-d, soares p-b-f. periodontal therapy for patients before and after radiotherapy: a review of the literature and topics of interest for clinicians. med oral patol oral cir bucal. 2018; 23(5): e524–30. 19. sroussi hy, epstein jb, bensadoun r-j, saunders dp, lalla r v, migliorati ca, heaivilin n, zumsteg zs. common oral complications of head and neck cancer radiation therapy: mucositis, infections, saliva change, fibrosis, sensory dysfunctions, dental caries, periodontal disease, and osteoradionecrosis. cancer med. 2017; 6(12): 2918–31. 20. lanzós i, herrera d, lanzós e, sanz m. a critical assessment of oral care protocols for patients under radiation therapy in the regional university hospital network of madrid (spain). j clin exp dent. 2015; 7(5): e613-21. 21. rapone b, nardi gm, di venere d, pettini f, grassi fr, corsalini m. oral hygiene in patients with oral cancer undergoing chemotherapy and/or radiotherapy after prosthesis rehabilitation: protocol proposal. oral implantol (rome). 2017; 9(suppl 1/2016 to n 4/2016): 90–7. 22. abdalla r, niazy ma, jamil we, hazzaa ha, elbatouti aa. the role of fluoride and chlorhexidine in preserving hardness and mineralization of enamel and cementum after gamma irradiation. radiat environ biophys. 2017; 56(2): 187–92. 23. vieira tr, martins cc, cyrino rm, azevedo amo, cota lom, costa fo. effects of smoking on tooth loss among individuals under periodontal maintenance therapy: a systematic review and metaanalysis. cad saude publica. 2018; 34(9): e00024918. 24. farina r, simonelli a, baraldi a, pramstraller m, minenna l, toselli l, maietti e, trombelli l. tooth loss in complying and non-complying periodontitis patients with different periodontal risk levels during supportive periodontal care. clin oral investig. 2021; 25(10): 5897–906. 25. lang np, tonetti ms. periodontal risk assessment (pra) for patients in supportive periodontal therapy (spt). oral health prev dent. 2003; 1(1): 7–16. 26. ba r told pm. lifestyle and periodontitis: the emergence of personalized periodontics. periodontol 2000. 2018; 78(1): 7–11. 27. bunaes df, lie sa, enersen m, aastrøm an, mustafa k, leknes kn. site-specific treatment outcome in smokers following nonsurgical and surgical periodontal therapy. j clin periodontol. 2015; 42(10): 933–42. 28. kanmaz m, kanmaz b, buduneli n. periodontal treatment outcomes in smokers: a narrative review. tob induc dis. 2021; 19: 77. 29. hong chl, hu s, haverman t, stokman m, napeñas jj, braber jb, gerber e, geuke m, vardas e, waltimo t, jensen sb, saunders dp. a systematic review of dental disease management in cancer patients. support care cancer. 2018; 26(1): 155–74. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p231–234 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p231-234 199 vol. 42. no. 4 october–december 2009 glucosyltransferase b/c expression in streptococcus mutans of rampant and caries-free children yetty herdiati h. nonong department of pediatric dentistry faculty of dentistry, padjajaran university bandung indonesia abstract background: streptococcus mutans (s. mutans) as specific bacteria causing dental caries have cariogenic characteristic related to glucosyltransferase (gtf) b/c that can change sucrose into insoluble glucan. insoluble glucan functions as an attachment media and bacteria colonization, and also as a source of extracellular polysaccharide which is needed for the bacteria and may lead to caries formation. purpose: the aim of this study was to find out the gtf b/c expression in isolated s. mutans from dental plaque of rampant and caries-free children. methods: an observational study was done on 96 isolated bacteria grown in sucrose and bacitracin containing media, which include s. mutans ina 99, s. mutans eu3, s.mutans eu7, s.eu10a, and s.mutans 10b. pcr technique was used as amplification technique for gtf b/c. result: this study showed that gtf b/c gene was found in s. mutans, s. constellatus, s. bovis, s. anginosus, l. fermentum, l. salivarius, and kleibsiella oxytoca. the presence of gtf b/c gene was found in 9 of 10 samples identified in the sample of rampant caries children. conclusion: the gtf b/c enzyme was found not only in s. mutans, but also in other bacteria. key words: cariogenic bacteria, s. mutans, glucosyltransferase correspondence: yetty herdiati h. nonong, c/o: bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: yettynonong@yahoo.com introduction dental caries was not a new disease and has become an important problem in all around the world. it has been the focus of researchers for decades. the researchers tried to identify the bacteria which cause dental caries at the end of the nineteenth century. they found that l. acidophilus and s. mutans are specific cariogenes which trigger dental caries. these cariogenes called as lactic acid bacteria are the main causes.1 they are considered as specific agents that produce primary acid for dental caries.2,3 to ensure the influence of lactobacilllus toward dental caries, a study by byun et al.,4 showed that the increased amount of lactobacillus has correlation with the increase of carbohydrate on teeth. the average amount of carbohydrate found on l.gaseri and l.ultunens was higher than that on other lactobacillus. another observation indicates that s. mutans was the main factor causing dental caries because of its characteristics which can change sucrose into glucan, produce lactic acid by homofermentation, form colony on teeth surfaces, be more aciduric than other streptococcus. as consequence, s.mutans was considered as the main etiology organism because it was potential to be virulent in triggering dental caries.5 munson et al.,6 divided the role of s. mutans and lactobacilus in the process of dental caries. their emphasized that s. mutans was responsible in the initiation of caries lesion which was attached on dental surfaces by glucan production or retention of pit and fissure. based on above statements, all oral bacteria may cause caries, but the level of their potential needs to be researched. if the previous researchers have already identified that s. mutans was cariogenic, the presence of characteristics found in other bacteria should also be observed. as a result, researchers can prove that the most cariogenic bacteria are those which have the highest potential. the purpose of this study was to find out the gtf b/c that was expressed due to the exceeded gtf b/c gene in bacteria isolated from dental plaque of rampant and caries-free children. research report 200 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 199-203 material and methods samples of plaque were taken from student clinic in department of dentistry, padjadjaran university, kindergarten and elementary school of bale indah, bandung regency, elementary school of sukasari i and elementary school of sukasari ii, bandung regency. samples of s. mutans were gained from laboratory in faculty of dentistry, trisakti university, jakarta, and laboratory in faculty of dentistry, airlangga university, surabaya. ninety six strained bacteria that were separated from the material in the form of plaque and 5 strained bacteria which have been identified as s. mutans which were s. mutans ina 99, s.mutans ue3, s.mutans eu7, s.mutans eu10a, and s.mutans eu10b. the separation was identified by applying a test on msa, tycsb, the coloring of gram, and biochemistry. the characteristic was taken based on streptococcus morphology, positive gram, positive mannitol, positive sorbitol, positive aesculin, negative arginin, positive melibiose, positive raffinose which was taken by fermentation test. mitis salivarius agar plate was used after incubated anaerobically for 2 × 24 hours. the strained bacteria generally identified by the coloring of gram forming colony which turned into blue. then, their morphology was observed by microscope. by gram coloring it can be seen from microscope that streptococcus was purple. if the coloring of gram and morphological identification are appropriate with the characteristic owned by streptococcus, the positive gram will be in the form of chained or paired coccus. after applying incubation on tycsb by 15 g/l bactocasitone (difco), 5 g/l yeast extract (difco), 0.2 g/l l-cystine, 0.1 g/l na2so3, 1 g/l nacl, 2 g/l na2hpo4. 12 aq, 2 g/l nahco3, 20 g/l na-acetate, 15 g/l bacto agar, the colony will turn into white and the size was 0.5–1.0 mm. further identification of biochemistry found in the bacteria will be done by adding proliferated bacteria on the tube which contains arginin solution, aesculin, sucrose, lactose, mannitol, sorbitol, raffinose, melibiose 1%. to add the proliferated bacteria on the tube, an ose which has been heated was used. dna extraction and pcr dna of anaerob bacteria was isolated by applying wizard dna isolation purification kit with a half composition of reaction. cell was taken 10 ml from culture and dissolved with 240 ml, 50 mm of edta, and 60 ml of lysozyme 10 mg/ml. after that, it was incubated for 30–60 minutes at 37° c and spun for 2 minutes with 13,000 rpm rotation. then, 300 ml of nuclei lysis solution was added. the supernatant was removed and incubated at 80° c. next, it was stored at room temperature and added with 1.5 ml of rnase, and incubated at 37° c for 30–60 minutes. then, it was added with 100 ml of vascor protein, and stored for 5 minutes. next, it was taken with 13,000 rpm for 5 minutes. after that, supernatant was put in the eppendorf containing 300 ml of isopropanol. it was spun back and forth with 13,000 rpm rotation for 2 minutes. then, the supernatant was removed and the pellet was washed by ethanol 70%. later, the same process was repeated while dna was dried by concentrator. when the dna was completely dried, it was dissolved with 50 ml of dna rehydration. amplification using primer universal gene 16s rdna was applied in: double denaturation at 94° c for 2 minutes, annealing at 48° c for 1 minute, elongation at 72° c for 1 minute, and post-elongation at 72° c for 10 minutes, and amplification as many as 30 cycles. the genes which were used include: forward: 5’ agagtttgatc(a/ c)tggctac3’ (19 pairs of alkali); reverse: 5’ ggttc(g/ c)ttgttacgactt3’ (18 pairs of alkali) amplification was applied by using primer gene gtf b/c. forward: 5’ agattt ccgt ccctt actg 3’; reverse: 5’ atca tatttgt cgccat cata 3’ and tegenerated primer.6 both primer genes were used in early denaturation at 94° c for 2 minutes. the cycle consists of denaturation at 94° c for 1 minute, primer attachment at 50° c for 1 minute, and extension process at 72° c for 1 minute as many as 35 cycles. at the last cycle, the extension was applied at 72° c for 10 minutes. optimization of pcr was completed by setting the template of pcr, concentration of magnesium chloride, primer concentration, temperature of primer attachment, and concentration of dntp. based on optimization, the proper compositions of pcr were 40 printings, 20 genes of forward, 20 genes of reverse, 9 ml of magnesium chloride 25 mm, 5 ml of tag polymerase dapar, 1 ml of tag polymerase enzyme, and 1 ml of dntp. at last step, sterile aquabidest was added as much as 50 ml. pcr product was confirmed with electrophoresis of agarose gel 1% (w/v) by comparing dna token, positive control, and negative control. agarose gel 1% was made by dissolving 400 mg of agarose and 40 ml of tae 1x dapar (trwas-base, edta 0.5 m ph 8.0, glacial acetic acid and natrium hydroxide. electrophoresis was applied in 90 volt for 45 minutes. after the electrophoresis was completed, pcr product was observed by using transluminator uv. nucleotide was arranged based on dideoxy method of sanger. the compound to set nucleotide in order was pcr product that was purified by 10 ng/3 ml of primer forward, tag polymerase dapar 10×, tag polymerase enzyme, dnpt, ddtp, and stop solution. result based on the research through pcr technique using primer universal gene 16s rdna by token of dna puc19/ hinfi, it was known that 18 samples can be identified to produce dna with band sized 1,500 pb (figure 1) and 1,400s pb (figure 2). 201nonong: glucosyltransferase b/c expression table 1. result of bacteria identification by 16s rdna approach num sample sample category name of bacteria homology (%) 1 k12 rampant caries streptococcus mutans 93% 2 k17 lactobacillus fermentum 94% 3 k18 klebsiella oxytoca 96% 4 k19 streptococcus anginosus 97% 5 k20 streptococcus constellatus 85% 6 k21 streptococcus bovis 96% 7 k39 streptococcus bovis 85% 8 k40 streptococcus anginosus 96% 9 k41 lactobacillus salivarius 99% 10 7eu streptococcus mutans 100% 11 bk46 free caries kleibsiella oxitoca 94% 12 bk54 streptococcus anginosus 96% 13 bk55 streptococcus constellatus 97% 14 bk42 streptococcus anginosus 99% 15 bk43 lactobacillus salivariussubsp. 99% 16 bk44 lactobacillus salivarius-subsp. 95% 17 bk45 lactobacillus salivarius-subsp. 95% 18 3eu streptococcus mutans 92% description: identified bacteria are homologized based on the data in gene bank, and percentage (%) shows the homology level. the result showed that the bacteria which were isolated include: s. mutans, s. anginosus, s. constellatus, s. bovis, l. salivarius, l. fermentum, dan k. oxytoca (table 1). in this study the fragment amplification result of gene gtf b/c using specific primer showed that the band was 600 pb (figure 4), and less than 600 pb (figure 5). complete amplification result of the band length of gtf b/c on various bacteries can be seen on table 2. figure 1. amplification result of gene 16s rdna with band length 1,500s pb. 1) puchinfi; 2) isolate bk48; 3) isolate bk47; 4) isolate k24; 5) isolate k32; 6) isolate bk42; 7) isolate bk45; 8) isolate bk43; 9) isolate bk46; 10) isolate k21; 11) isolate k19; 12) isolate k18; 13) isolate bk44; 14) isolate k20; 15) isolate k17; 16) isolate k16. figure 2. amplification result of gene 16r sdna with band length 1,400s pb. 1) token of molecule weight1) token of molecule weight puc-hinfi; 2) isolate k31; 3) isolate k12; 4) isolate 10aeu; 5) isolate bk22; 6) isolate bk25; 7) isolate 7eu; 8) isolate 3eu; 9) isolate k39; 10) isolate bk55; 11) isolate k40; 12) isolate k41; 13) isolate bk54. 202 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 199-203 table 2. fragment amplification result of gtf b/c on various bacteria num sample 16s rdna band length amplification sample category 1 k21 streptococcus mutans 600 pb rampant caries 2 k17 lactobacillus fermentum 700 pb 3 k18 klebsiella oxytoca 600 pb 4 k19 streptococcus anginosus 500 pb 5 k20 streptococcus constellatus 700 pb 6 k21 streptococcus bovis 500 pb 7 k39 streptococcus bovis 600 pb 8 k40 streptococcus anginosus 600 pb 9 k41 lactobacillus salivarius 600 pb 10 7eu streptococcus mutans no band 11 bk46 kleibsiella oxitoca 450 pb caries-free 12 bk54 streptococcus anginosus 600 pb 13 bk55 streptococcus constellatus no band 14 bk42 streptococcus anginosus no band 15 bk43 lactobacillus salivarius subsp. no band 16 bk44 lactobacillus salivarius subsp. no band 17 bk45 lactobacillus salivarius subsp. no band 18 3eu streptococcus mutans no band description: the band length that can be identified shows amplification of gtf b/c, if there was no band, there will be no amplification of gtf b/c figure 3. colonies of s.mutans on tycsb media. the coloniesthe colonies of s.mutans are white, glossy, not flat, cauliflower like, in the form of crystal, and sticky on media. figure 4. fragment amplification of gtf b/c with band length 600 pb. 1) pcr product: isolate k12, s. mutans; 2) pcr product isolate k18, k. oxitoca; 3) pcr product isolate k39, s. bovis; 4) molecule token puc-hinfi. figure 5. fragment amplification of gtf b/c with band length less than 600 pb. 1) isolate bk42 (no amplification); 2) isolate bk55 (no amplification); 3) pcr product isolate k21 (s. bovias); 4) pcr product isolate k40 (s. angunisus); 5) pcr product isolate k46 (k. oxitoca); 6) pcr product isolate k46 (k. oxitoca); 7) isolate 7eu (no amplification); 8) molecule token puc-hinfi. 203nonong: glucosyltransferase b/c expression discussion several s.mutans which can be identified are k12 and 7eu (taken from dental caries plaque) and 3eu (taken from free dental caries) (table 1). in the process of proliferation all samples have s.mutan characteristics: which has blue color on msa, glossy, sticky on proliferation media, has diameter of 0.1–1 m or could reach 1–1.5 mm. on tycsb media, their color was white and their surface was not flat. their form was crystal like cauliflower and attached on media (figure 3). these findings have reconstructed the previous opinions which stated that msa and tyscb are selective media for s.mutans. the reason was that after pcr was applied with primer gene 16s rdna, some colonies besides s.mutans have similar characteristics. on this basis, it was predicted that msa and tycsb media are not selective for s.mutans proliferation and those characteristics appeared because the bacteria used sucrose as substrate to produce glucan. however, further research needs to be conducted to study these findings. a little amount of s.mutans which can be identified can contribute to the development of microbiology study focusing on characteristics of biochemical bacteria. some bacteria have particular characteristics; forming ammonia from arginin, fermenting mannitol, sorbitol, aesculin, melibiose, and raffinose. these characteristics are similar to the biochemistry of s.mutans. enzyme that forms caries was gtf b/c. insoluble glucan is indicated by gft b by while gtf c indicates the soluble and insoluble glucan.7,8 gtf b and c enzyme has high homology when forming operon. the operon has strong promoter at the top of gtf b. while for grf c, its operon was at the bottom of gtf c.9 s.mutansgtf b/c enzyme creates pathogenic insoluble glucan because polimer glucose produced by these two enzymes was aggregation mediator for bacteria on dental surface.10,11 this can contribute to the intensity of thickness and structure integrity of dental plaque which later can form caries. the presence of gtf b/c enzyme was expressed by the presence of gtf b/c coding the two enzymes. gene gtf b/f can be amplified by pcr techniques using primer gene gtf b/c. the presence of gtf b/c enzyme was one characteristic owned by cariogenic bacteria.13,14 bacteria which are isolated from the plaque of dental caries have gtf b/c. based on the result of the study and the discussion. it is concluded that the presence of caries was closely related to the presence of gtf b/c gene, since it was one of characteristics owned by cariogenic bacteria. this implies that the presence of glucosyltransferase enzyme was expressed by the presence of gtf b/c. this study indicates that bacteria with gtf b/c have one of the characteristics owned by cariogenic bacteria. besides s. mutans, other oral bacteria which indicate glucosyltransferase enzyme can trigger caries. bacteria like s. mutans, s. anginosus, s. constellatus, l. salivarius, and k. oxytoca are not only found in rampant caries children, but also in free-caries children. all bacteria (s. mutans, s. bovis, s. anginosus, s. constellatus, l. salivarius, l. fermentum, and k. oxytoca) found in rampant caries children contain gene gtf b/c. s. mutans which does not contain gene gtf b/c was found either in rampant caries or caries-free children. if gene gtf b/c was found in caries-free children, it was suggested that prevention to eradicate the bacteria causing caries should be applied as soon as possible. s. mutans was classifed as cariogenic bacteria although it has no gtf b/c, acidogenic bacteria funtion as cariogenic. therefore, further studies need to be conducted to identify other causes. references 1. thylstrup a, fejerkov o. clinical and pathological features of dental caries. in: thylstrup a, fajerkov o, editors. textbook of clinical pediatric. copenhagen: munkgaard, 1996. p. 111–15. 2. vanhoute j, lopman j, kent r. the predominant cultivable flora of sound and carious human root surfaces. j dent res 1994; 73(11): 1727–17. 3. klein b. a mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to s. mutans and the specific-plaque hypothesis. crit rec. oral biol med 2002; 13(2): 108–12. 4. byun r, nadkarni ma, chhour kl, martin fe. quantitative analysis of diverse lactobacillus species present in advanced dental caries. j clin microbiol 2004; 42(7): 3128–31. 5. beighton d, brailsford s, samarayanake, lp, brown jp, ping fx, mils dg, et al. a multi country comparison of caries associated microflora in demographically diverse children. community dental health 2004; 21(suppl): 96–1. 6. munson ma, banerjee a, watson tf, wade wg. molecular analysis of the microflora associated with dental caries. j clin microbiol 2004: 3023–30. 7. kralj s. glucansucrase of lactobacilli: characterization of genes, enzymes, and products synthesized. netherlands: ponsen & looijen bv, 2004: 466–89. 8. burne ra, chen yy, penders. analysis of gene expression in s. mutans in biofilms in vitro. adv dent res 1997; 11(1): 100–1. 9. kopec lk, smith amv, ng-evans l, bowen wh. influence of antibody on the structure of glucans. caries res 2002; 36: 108–11. 10. hanada n, kuramitsu hk. isolation and characterization of the streptococcus mutans gtf d gene, coding for primer-dependent soluble glucan synthesis. infect immun 1989; 57: 2079–208. 11. yamashita y, bowen wh, kuramitsu hk. molecular analysis of a streptococcus mutans strain exhibitng polymorphism in the tandem gtf bang? gtf b/c genes. infect immun 1992; 60(4): 1618–16. 12. jespersgaard c, hajwashengallwas g, russel mw, michalek s. identification and characterization of a nonimmunogbulin factor in human saliva that inhibits streptococcus mutans glucosyltransferase. infect immun 2002; 70(3): 1136–11. 13. newman mg, nwasengard r. oral microbiology and immunology. philadelphia: wb saunders co, 1988: p. 117–25. 11 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 1–5 rinsing effect of 60% bay leaf (syzygium polyanthum wight) aqueous decoction on the accumulation of dental plaque during fixed orthodontic treatment fitria avriliyanti, sri suparwitri, and ananto ali alhasyimi department of orthodontics faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: fixed orthodontic appliance patients have a high risk to increasing plaque accumulation in tooth surface due to the presence of fixed appliance components. using mouthwash with antibacterial agent from herbal material can control dental plaque accumulation. bay leaf (bl) contains active compounds such as essential oil, tannin and flavonoid that have been known as an antibacterial agent. purpose: the purpose of this study was to determine the effect of rinsing with 60% of bl aqueous decoction to the accumulation of dental plaque in fixed orthodontic appliance patients. method: this research was an experimental clinical research with pretest and post-test control group design conducted on 20 subjects with age ranged between 18-25 years old. all subjects were undergoing the last stage (finishing) of fixed active orthodontic treatment. the subjects were instructed to rinse with 60% of bl aqueous decoction and 0.2% chlorhexidine as a control. wash out period that needed between rinsing with 60% of bl aqueous decoction and rinsing with 0.2% chlorhexidine was 7 days. each mouthwash used routinely for 7 days with same duration and intensity. plaque scoring was measured before and after each treatment using orthodontic plaque index (opi). result: one-way anova test showed that there was a significant difference in the plaque index before and after treatment between the group of rinsing with 60% of bl aqueous decoction and group of rinsing with chlorhexidine (p<0.05). conclusion: rinsing with 60% bl aqueous decoction can decrease the accumulation of dental plaque in fixed orthodontic appliance patients. keywords: fixed ortodontic appliance; plaque accumulation; 60% bay leaf aqueous decoction correspondence: ananto ali alhasyimi, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: anantoali@ugm.ac.id introduction orthodontic treatment used to be considered cosmetic, but nowadays, dentists and patients alike realize that orthodontic treatment may be a necessity. there are two kinds of orthodontic appliances; removable and fixed appliances. fixed orthodontic appliance has more complicated design compared to removable appliance which makes cleaning procedure more difficult and increasing the risk of developing plaque retention.1,2 accumulation of plaque can be found on fixed orthodontic appliance such as the bracket, wire, interface between bonding material and bracket. an orthodontic bracket may create difficulties to maintain good oral hygiene, leading to plaque accumulation and increase of enamel demineralization. wire that is used for a long time during orthodontic treatment tends to plaque accumulation which can increase the level of microorganism in oral cavity. the roughness of composite surface as bonding material for orthodontic bracket also can be a predisposition factor for the attachment and growth of intraoral microorganisms.3,4 plaque accumulation that is not properly cleaned during fixed orthodontic treatment may result a bad oral hygiene which leads to various oral diseases and failure of orthodontic treatment. negligence in maintaining good oral hygiene will result in several negative effects, such as the destruction of periodontal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p1-5 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p1-5 2 avriliyanti, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 1–5 tissue (gingivitis, periodontitis), caries, halitosis and affecting the length of orthodontic treatment time.5 around 5-10% of patients failure in fixed orthodontic treatments are caused by poor oral hygiene.6 one of the indicators for dental and mouth hygiene is dental plaque. plaque control is an attempt to prevent the formation of plaque on the tooth surface. plaque control can be performed mechanically or chemically. mechanical plaque control includes tooth brushing and flossing, while the use of mouthwash is an example of chemical plaque control.7 mouthwash is a type of plaque control that is easily accessible dan practical to use. this solution is a necessity for most people because it is believed to prevent the formation of dental plaque and occurrence of gingivitis mechanically and chemically. rinsing using mouthwash can help the solution to reach more areas of teeth and intraoral surfaces, especially the interproximal areas which are not accessible mechanically.8 the example of mouthwash that is easily accessible in the market is chlorhexidine (chx). chx has been proven to decrease the accumulation of plaque due to its broad spectrum antimicrobial, bactericidal, and bacteriostatic properties to any kind of microorganisms, including bacteria, fungi, and virus.9 chx is still the gold standard for its antimicrobial action and high substantiveness, but side effects in longterm used such as tooth and restoration staining, soft tissue staining, increased calculus deposition, unpleasant taste, taste alteration, burning sensation, desquamation and mucosal irritation limit its continued use. chx can also lead oral discomfort in some of patients with chemotherapyinduced mucositis, xerostomia or ulcerative oral mucosal conditions.8,10 nowadays, natural materials are being used and developed as more safe and cheaper alternatives with lesser side effects compared to chemical based products.11 a type of traditional herbal plant that is commonly seen in indonesia is bay leaf (syzygium polyanthum weight). bay leaf (bl) that is also used as cooking spice, has many pharmacological activities that are useful in the field of dentistry. the chemical properties of bl consist of tannins, flavonoid and essential oils (0.05%), including citric acid and eugenol.12 tannin is an active compound that has antibacterial activity. tannin can inhibit the growth of bacteria by reacting with protein due to solidification on cell protein of bacterium (occurrence of protein denaturation). flavonoid has biological and pharmacologic activities, including antibacterial activity.13 essential oil also can inhibit the growth of some bacteria. bl is very effective against several bacteria such as salmonella enterica and e. coli.14,15 the antibacterial activity of bl can be also due compounds non-flavonoid origin. the high contents of eugenol, methyl eugenol and fatty acid methyl esters together with other active components could contribute to its overall antibacterial activity.16,17 this research was carried out to investigate the effect of rinsing with 60% of bl aqueous decoction to the decreasing accumulation of dental plaque in fixed orthodontic appliance patients. materials and methods this research was a clinical experimental with pretest and post-test control group design conducted on 20 subjects with age ranged between 18-25 years old. the study was approved by the ethics committee of faculty of dentistry, universitas gadjah mada with number 00789/kkep/fkgugm/ec/2016. a total of 100 grams of fresh bay leaves were minced and boiled in 200 ml of boiling water until 100 ml of decoction was left. the concentration of the decoction was 100%. it was separated from the minced leaves and left to chill in room temperature (25o c). the decoction was filtered afterward with a 0.45 µ pore (millipore) diameter filter. sixty percent of bl decoction was obtained by adding 40 ml of aquades into 60 ml of 100% bl decoction. all eligible subjects were given oral and written informations about the products and the purpose of the study and were asked to sign an informed consent prior to the studied procedures. all subjects were in the last stage (finishing) of fixed active orthodontic treatment. exclusion criteria were considered: pregnancy, systemic disorders, patients under preventive treatment, and absence of studied evaluated teeth. subjects were instructed to rinse their mouths with 60% bl aqueous decoction and 0.2% chx mouth rinse as a control. the time interval between mouth rinsing with 60% bl aqueous decoction and 0.2% chx mouth rinse was 7 days. each mouthwash was routinely used for 7 days with the intensity of 2 times a day (after tooth brushing in the morning and at night) for 30 seconds strongly, using 10 ml of the solution in each rinsing. after a week of washout period, each group was instructed to use the opposite mouthwash for 7 days. the subjects in the study were still instructed to brushing (with bass technique) using pumice toothpaste for seven days of treatment. plaque measurement was performed before and after application each mouth rinse using the orthodontic plaque index (opi) method. the opi developed by declerk in 1989, is a special index for patients with fixed orthodontic appliances. opi has a higher diagnosis performance and accuracy compared to quigley and hein index, and modified navy plaque index.18,19 in this study, we used disclosing agents that were applied on vestibular surfaces of teeth with orthodontic brackets and then the presence of dyed plaque was evaluated with the yes-no system in 3 areas of vestibular surfaces of a tooth (figure 1). all measurements were carried out under the same conditions and were performed by two calibrated examiners who were blinded to the applied regimen. the examiners performed their analysis with a satisfactory intraexaminer and interexaminer reliability kappa index value (0.75). each area has its own level of difficulty depending on the accessibility for cleaning (occlusal area = 1 = easily accessible; cervical area = 2 = accessible with certain difficulty; central area = 3 = poorly accessible). the values found are entered into a work table (table 1).20 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p1-5 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p1-5 33avriliyanti, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 1–5 resulting index is obtained by means of the formula:20 opi % = sum of values from dyed areas x 100% total number of teeth x 6 the condition of oral hygiene then evaluated according to the following scheme: 0-30%= excellent hygiene; 30-50%= average hygiene; over 50% = insufficient hygiene.20 4 decoction and 0.2% of chx mouth rinse as a control. the time interval between mouth rinsing with 60% bl aqueous decoction and 0.2% chx mouth rinse was 7 days. each mouthwash was routinely used for 7 days with the intensity of 2 times a day (after tooth brushing in the morning and at night) for 30 seconds strongly, using 10 ml of the solution in each rinsing. after a week of washout period, each group was instructed to use the opposite mouthwash for 7 days. the subjects in the study were still instructed to brushing (with bass technique) using pumice toothpaste for seven days of treatment. plaque measurement was performed before and after application each mouth rinse using the orthodontic plaque index (opi) method. the orthodontic plaque index developed by declerk in 1989, is a special index for patients with fixed orthodontic appliances. orthodontic plaque index has a higher diagnosis performance and accuracy compared to quigley and hein index, and modified navy plaque index.18,19 in this study, we used disclosing agents that were applied on vestibular surfaces of teeth with orthodontic brackets and then the presence of dyed plaque was evaluated with the yes-no system in 3 areas of vestibular surfaces of a tooth (figure 1). all measurements were carried out under the same conditions and were performed by two calibrated examiners who were blinded to the applied regimen. the examiners performed their analysis with a satisfactory intraexaminer and interexaminer reliability kappa index value (0.75). each area has its own level of difficulty depending on the accessibility for cleaning (occlusal area = 1 = easily accessible; cervical area = 2 = accessible with certain difficulty; central area = 3 = poorly accessible). the values found are entered into a work table (table 1).20 figure 1. scheme distribution of vestibular surface for evaluation of opi: i = occlusal area=easily accessible; cervical area = 2 = accessible with certain difficulty; central area = 3 = poorly accessible21 figure 1. scheme distribution of vestibular surface for evaluation of opi: i = occlusal area=easily accessible; cervical area = 2 = accessible with certain difficulty; central area = 3 = poorly accessible21 table 1. orthodontic plaque index: recorded dental plaque in individual areas multiplied by the relevant factor of cleaning difficulty20 cervical 2× total central 3× total occlusal 1× total teeth 5 4 3 2 1 1 2 3 4 5 occlusal 1× total central 2× total cervical 3× total 5 table 1. orthodontic plaque index: recorded dental plaque in individual areas multiplied by the relevant factor of cleaning difficulty20 resulting index is obtained by means of the formula:20 the condition of oral hygiene then evaluated according to the following scheme: 0-30%= excellent hygiene; 30-50%= average hygiene; over 50% = insufficient hygiene.20 results the result showed decreasing of plaque index before and after rinsing with 60% of bl aqueous decoction as well as rinsing with chlorhexidine. decreasing of plaque index on group using bl aqueous decoction 60% amounts 43.1% ± 4.02%, while other group rinsing with cl amounts 42.1% ± 4.3%. figure 2 shows the coloured dental plaque after application of disclosing agent solution in patients rising with 60% of bl aqueous decoction and rinsing with 0.2% cl. figure 2. coloured dental plaque after application of disclosing agent solution in patients rinsing with 60% of bay leaf aqueous decoction (a) and rinsing with 0.2% chx (b). 5 table 1. orthodontic plaque index: recorded dental plaque in individual areas multiplied by the relevant factor of cleaning difficulty20 resulting index is obtained by means of the formula:20 the condition of oral hygiene then evaluated according to the following scheme: 0-30%= excellent hygiene; 30-50%= average hygiene; over 50% = insufficient hygiene.20 results the result showed decreasing of plaque index before and after rinsing with 60% of bl aqueous decoction as well as rinsing with chlorhexidine. decreasing of plaque index on group using bl aqueous decoction 60% amounts 43.1% ± 4.02%, while other group rinsing with cl amounts 42.1% ± 4.3%. figure 2 shows the coloured dental plaque after application of disclosing agent solution in patients rising with 60% of bl aqueous decoction and rinsing with 0.2% cl. figure 2. coloured dental plaque after application of disclosing agent solution in patients rinsing with 60% of bay leaf aqueous decoction (a) and rinsing with 0.2% chx (b). figure 2. coloured dental plaque after application of disclosing agent solution in patients rinsing with 60% of bay leaf aqueous decoction (a) and rinsing with 0.2% chx (b). results the result showed decreasing of plaque index before and after rinsing with 60% bl aqueous decoction as well as rinsing with chx. decreasing of plaque index on group using 60% bl aqueous decoction 60% amounts 43.1% ± 4.02%, while other group rinsing with chx was 42.1% ± 4.3%. figure 2 shows the coloured dental plaque after application of disclosing agent solution in patients rising with 60% of bl aqueous decoction and rinsing with 0.2% chx. normality test result by shapiro-wilk showed normal distribution of data (p>0.05), while homogeneity test by levene’s test showed all the data have a homogeneous variance (p>0.05). the result of normality test and homogeneity test showed analysis could be done by parametric test using one-way anova. table 2 showed the result of one-way anova analysis obtained p-value p<0.05. the result showed that there were significant differences in plaque indexes before and after rinsing between treatment groups. differences between treatment groups could be determined by post-hoc test using least significant difference (lsd). the results of lsd test in table 2 showed that there were significant differences in group before and after rinsing with bl, group after rinsing with bl and before rinsing with chx, group before and a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p1-5 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p1-5 4 avriliyanti, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 1–5 after group rinsing with chx (p<0.05). whereas in the group before rinsing with bl and before rinsing with chx, group after rinsing with bl and after rinsing with chx had no significant difference (p>0.05). discussion based on the study there were decreasing of plaque indexes in groups rinsing with 60% bl aqueous decoction as well as groups rinsing with chx in fixed orthodontic appliance patients. percentage of plaque index reduction in groups gargling by 60% bl aqueous decoction was 43.1% ± 4.02%, while other group rinsing with chx was 42.1% ± 4,3%. both of groups belongs to average hygiene category (31-50%).20 the study used pumice as a toothpaste thus anti-bacterial agent only came from mouthwash (bl and chx) so reduction of plaque index was not optimal and only achieved moderate level. the used of pumice toothpaste without detergent and abrasive materials aimed to minimalize a bias observation. results of the study in each group showed rinsing with 60% bl aqueous decoction as well as rinsing with 0.2% chx could decrease accumulation of dental plaque in fixed orthodontic appliance patients (p<0.05). another study showed that chlorhexidine was a gold standard of mouthwash and proven to be broad-spectrum of anti-bacterial agent and also having bactericidal and bacteriostatic towards all of the type microbes, fungi and virus.22 in this study, bl potentially proved on reduction plaque index in fixed orthodontic appliance patients. it is supported by a previous study conducted by sumono and wulan12 that rinsing with bl aqueous decoction could decrease the colony number of streptococcus sp. bl are rich in essential oils, flavonoids and tannins which have been known as antibacterial agent.23 the activity of essential oils can affect both the external envelope of the cell and the cytoplasm of bacteria. the specific mechanisms action of essential oils in decreasing bacterial accumulation is via hydrophobicity. the typical of essential oils allows them to partition in the lipids of the bacterial cell membrane and mitochondria and leading to leakage of its cell contents and responsible for the disruption of bacterial structures.24,25 tannin also has antibacterial activity and it is related to the ability to inactivated adhesin bacterial cell, enzyme and interfered protein transport on cell layer so that bacterial metabolism becomes impaired.26 tannin have the ability to reduce the attachment of bacteria by binding to proline-rich protein of the salivary pellicle or to the cell-surface lipoteichoic acid.27 tannin can also inhibit growth and kill bacteria by reacting with the cell membrane, resulting in leakage of essential metabolites that inactivate the bacterial enzyme system.28 bl also contained flavonoid besides essential oil and tannin. flavonoids are well-known plant compounds that have antibacterial property.29 action mechanism of flavonoid as an antibacterial is to interfere motility of bacteria, synthesis of nucleic acid, damage fluidity of membrane therefore membrane fluidity of outer and inner layer will decrease and interfere energy metabolism of bacteria.30,31 moreover, their mode of antimicrobial action may be related to their ability to inactivated microbial adhesion, enzymes and cell envelope transport proteins.32 the antibacterial activity of bl can be also due to compounds non-flavonoid origin. the high contents of eugenol, methyl eugenol and fatty acid methyl esters together with other active components could contribute to its overall antibacterial activity.16,17 plaque index score in group before rinsing using bl with group before rinsing with chx showed no significant differences (p>0.05), while plaque index score in group after rinsing using bl and group before rinsing with chx showed significant differences (p<0.05). the result showed washout process period in this study was successful. washout period aimed to ensure the effect of prior exposure of first mouthwash had been stopped before the second mouthwash applied.33 plaque index score in group after rinsing with bl and after rinsing with chx showed no significant difference (p>0.05), the means rinsing used 60% bl aqueous decoction have the same effect with chlorhexidine as gold standard mouthwash to decrease the accumulation of dental plaque. one of excellences rinsing with bl was no alteration in taste sensation, while rinsing with chx raised bitter tastes, caused change of temporary sensation and burning sensation and also long-term use of chx can be associated with local side effects such as impaired sense of taste, tooth staining and occasional irritation and desquamation of mucous membranes.8,10,34,35 table 2. descriptive statistics and results of the anova and lsd tests comparing the plaque index in the 4 groups tested group n plaque index (%) significance* p-value after bl before chx after chx before bl 10 56.4±3.05 p=0.001 0.001* 0.621 0.001* after bl 10 43.1±4.02 0.,001* 0.741 before chx 10 57.0±3.78 0.001* after chx 10 42.7±4.30 values are presented as mean ± standard deviation or p-value only. *by anova, *significant differences between groups (p < 0.05). anova: analysis of variance; bl: 60% bay leaf aqueous decoction, chx: 0.2% chlorhexidine dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p1-5 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p1-5 55avriliyanti, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 1–5 finally, it can be concluded that rinsing with 60% bay leaf (syzygium polyanthum wight) aqueous decoction can reduce the accumulation of dental plaque in fixed orthodontic appliance patients. references 1. singh g. fixed orthodontic appliances. text book of ortodontics. 1st ed. new delhi: jaypee publishers; 2007. p. 449. 2. karadas m, cantekin k, celikoglu m. effects of orthodontic treatment with a fixed appliance on the caries experience of patients with high and low risk of caries. j of dental sciences 2011; 6: 19599. 3. lee hj, park hs, kim kh, kwon ty, honge sh. effect of garlic on bacterial biofilm formation on orthodontic wire. angle orthod 2011; 81(5): 895–900. 4. sukontapatipark w, el-agraudi ma, seliseth nj, thunold k, selvig ka. bacterial colonization associated with fixed orthodontic appliances. a scanning electron microscopy study. eur j of orthod 2001; 23(5): 475-84. 5. al-jewaira t, suri s, tompson bd. predictors of adolescent compliance with oral hygiene instr uctions dur ing two-a rch multibracket fixed orthodontic treatment. angle orthod 2011; 81(3): 525-31. 6. mehra t, nanda rs, sinha pk. orthodontists’ assessment and management of patient compliance. angle orthod 1998; 68: 115–22. 7. carranza fa, newman mg. clinical periodontology. 9th ed. philadelphia: wb saunders; 2006. p. 76. 8. camile sf, mcintosh l, mccullough mj. mouthwash. aust prescr 2009; 32: 162-4. 9. fajriani, andriani jn. reduction of salivary streptococcus mutans colonies in children after rinsing with 2.5% green tea solution. jdi 2014; 21(3): 79-84. 10. eley bm, manson jd. periodontic. 5th edition. saunders: elsevier; 2004. p. 21-7, 209. 11. alam mt, parvez n, sharma pk. fda-approved natural polymers for fast dissolving tablets. journal of pharmaceutics 2014; 952970: 1-6. 12. sumono a, wulan sda. the use of bay leaf (eugenia polyanthum weight) in dentistry. dent j (majalah kedokteran gigi) 2008; 41(3): 147-50. 13. lestari ika, nazip k, estuningsih s. test of effectiveness of antibacterial of ethanol extract of loranthus of tea (scurulla atropurpurea bl danser) on the growth of enterobacter sakazakii. international conference on food, biological and medical sciences, bangkok, thailand: 2014. p. 10-15. 14. santos fa, rao vsn . antiinflammatory and antinociceptive effects of 1.8-cineole a terpenoid oxide present in many plant essential oils. phytotherapy research 2000; 14: 240-4. 15. friedman m, henika pr and mandrell re. bactericidal activities of plant essential oils and some of their isolated constituents against campylobacter jejuni, escherichia coli, listeria monocytogenes, and salmonella enterica. j food prot 2002; 65: 1545-60. 16. marzouki h, piras a, marongiu b, assunta r, assunta m. extraction and separation of volatile and fixed oils from berries of laurus nobilis l. by supercritical co2 dessì 3. molecules 2008; 13: 170211. 17. ivanović j, mišić d, ristić m, pešić o, žižović i. supercritical co2 extract and essential oil of bay (laurus nobilis l.) – chemical composition and antibacterial activity. j serb chem soc 2010; 75(3): 395–404. 18. paschos e, bucher k, huth ck, crispin a, wichelhaus a, dietel t. is there a need for orthodontic plaque indices? diagnostic accuracy of four plaque indices. clin oral invest 2014; 18(4): 1351-8. 19. beberhold k, sachse-kulp a, schwestka-polly r, hornecker e, ziebolz d. the orthodontic plaque index: an oral hygiene index for patients with multibracket appliances. orthodontics 2012; 13(1): 94-9. 20. ticha r, bohmova h. influence of fixed orthodontic appliance on the level of patient’s oral hygiene. orthodoncie 2005;14(4): 29-34. 21. heintze sd, jost-brinkmann pg, finke c, miethke rr. oral health for the orthodontic patient. illinois: quintessenz publishing co, inc; 1999. p. 39. 22. fajriani, andriani jn. reduction of salivary streptococcus mutans colonies in children after rinsing with 2,5% green tea solution. jdi2014; 21(3): 79-84. 23. said cm, hussein k. determination of the chemical and genetic differences of laurus collected from three different geographic and climatic areas in lebanon. european scientific j 2014; 2: 412-9. 24. nazzaro, fratianni f, martino ld, coppola r, feo vd. effect of essential oils on pathogenic bacteria filomena. pharmaceuticals 2013; 6: 1451-74. 25. burt s. essential oils: their antibacterial properties and potential applications in foods – a review. int j food microbiol 2004; 94(3): 223–53. 26. akiyama h, fujii k, yamasaki o, oono t, iwatsuki k. antibacterial action of several ta nnins aga inst staphylococcus aureus. j antimicrob chemother. 2011; 48(4): 487-91. 27. islam b, khan an, khan au. dental caries: from infection to prevention. med sci monit 2007; 13 (11): 196-203. 28. mailoa mn, mahendradatta m, laga a, djide n. antimicrobial activities of tannins extract from guava leaves (psidium guajava l) on pathogens microbial. int j sci technol 2014; 3(1): 236-41. 29. sabir a. in vitro antibacterial activity of flavonoids trigona sp propolis against streptococcus mutans. dent j (majalah kedokteran gigi) 2005; 38(3): 135–41. 30. mirzoeva ok, grishanin rn, calder pc. antimicrobial action of propolis and some of its components: the effects on growth, membrane potential, and motility of bacteria. microbiol res 1997; 152: 239-46. 31. vasconcelos lcs, sampaio fc, sampaio mcc, pereira msv, higino js, peixoto mhp. minimum inhibitory concentration of adherence of punica granatum linn (pomegranate) gel againts s. mutans, s. mitis, and c albicans. braz dent j 2006; 17(13): 223-7. 32. alghazeer r, elmansori a, sidati m, gammoudi f, azwai s, naas h, garbaj a, eldaghayes i. in vitro antibacterial activity of flavonoid extracts of two selected libyan algae against multi-drug resistant bacteria isolated from food products. jbm 2017; 5: 26-48. 33. roberts aw, dusetzina sb, farley jf. revisiting the washout period in the incident user study design: why 6–12 months may not be sufficient. j comp eff res 2015; 4(1): 27–35. 34. charles ch, sharma nc, galustians hj, qaqish j, mcguire ja, vincent jw.comparative efficacy of an antiseptic mouthrinse and an antiplaque or antigingivitis dentifrice. a six-month clinical trial. j am dent assoc 2001;132: 670-5. 35. graziani f, gabriele m, d’aiuto f, suvan j, tonelli m, cei s. dental plaque, gingival inflammation and tooth discolouration with different commercial–formulations of 0.2% chlorhexidine rinse: a double-blind randomized controlled clinical trial. oral health prev dent 2015; 13: 101-11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p1-5 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p1-5 8888 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 88–92 original article the relationship between maxillary and mandibular lengths of ethnic bataks of chronological age 9–15 years hilda fitria lubis, nurul ulfa simanjuntak department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: maxillary and mandibular growth have an important role in determining diagnosis and treatment plans. knowledge of the growth of the maxilla and mandible becomes very important in designing a proper treatment plan and knowing the mean maxillary and mandibular lengths from the ages of 9–15 means malocclusion can be treated at the appropriate age. purpose: the aim of this study was to determine the relationship between 9–15-year-old males and females and the length of the maxilla and mandible. methods: this study used a cross-sectional design. the subjects consisted of 35 male and 45 females aged 9–15 years and 80 cephalometric radiograms were collected using a purposive sampling method from universitas sumatera utara (usu) oral and dental hospital based on inclusion and exclusion criteria. data were collected by tracing the lateral cephalogram, the maxillary length and mandible lengths being measured on the cephalogram based on the mcnamara method through a computer program, coreldraw. pearson’s correlation coefficient was used for statistical analysis. results: the average maxillary length for 9–15-year-olds was 96.35 ± 7.56 mm. the mean mandibular length for 9–15-year-olds was 122.29 ± 10.43 mm. based on assessment and result, using the pearson correlation coefficient test between maxillary length and mandibular length and chronological age, a maxillary length of p=0.003 and mandibular length of p=0.00 were obtained. conclusion: there was a significant positive relationship between chronological age and maxillary length and mandibular length in 9–15-year-olds of batak ethnicity. keywords: chronological age of 9-15 years; mandibular; maxillary correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2 medan, 20155, indonesia. email: hilda.fitria@usu.ac.id; hildadrgusu@gmail.com introduction the process of growth and development of the craniofacial area is one area of knowledge that must be possessed by dentists, especially orthodontists. this knowledge has an important role in establishing a diagnosis and treatment plan, especially in cases that require modification of facial bones in patients, such as the maxilla and mandible.1,2 malocclusion is a dental and oral problem that ranks third, after caries and periodontal disease, with a rate of prevalence of 80% of the population of indonesia.3 treatment of malocclusion needs to be done early in order to achieve maximum treatment results, as it have not yet reached maturity. bone growth in the craniofacial area is more significant before reaching maturity as this bone growth will provide space for the malocclusion repair process. research by enikawati, et al.4 displays results indicating that the greatest increase in maxillary length in males occurs at 14–15 years of age. the greatest increase in maxillary and mandibular length in girls, and mandibular length in boys, occurs between the ages of 13 and 14 years.4 this period of accelerated growth is called adolescence, or pubertal growth spurt, and always shows variations in growth rates, onset, intensity and duration in each child.2,5 research conducted by hsiao, et al.6 over the range of 7–12 year-old school children shows results indicating that the maxillary length experienced significant growth in group 3, namely aged 11–12-years-old, compared to the age group of 7–10 years; also, in respect to mandibular length, there was a significant difference with age. various studies were conducted to assess the relationship between peak growth period and indicators of child development, such as chronological age, physiological dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p88–92 mailto:hilda.fitria@usu.ac.id mailto:hildadrgusu@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p88-92 89 lubis and simanjuntak/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 88–92 age with height and weight, dental age, skeletal maturation and secondary sex characteristics.2 djoeana, et al.7 argue that different racial groups will display different patterns of craniofacial growth. therefore, every ethnic group in indonesia has different maxillary and mandibular growth from each other.7 most of indonesia’s population is dominated by ethnic malays, who are then divided into proto-malays and deutro-malays. the batak ethnic group is part of the proto-malay ethnic group that occupies the island of sumatra and dominates north sumatra.8 research on maxillary and mandibular length in ethnic bataks has not been well researched and is still limited, especially in medan city. based on statistics from the sumatera utara agency (bps), the bataks are the largest ethnic group in north sumatra with a percentage of 44.75% of the total population there.9 so we chose and were interested in conducting a study of maxillary and mandibular length in children aged 9–15 years of batak ethnicity, which is the largest ethnic in north sumatra. material and methods this descriptive cross-sectional study was carried out at the universitas sumatera utara (usu) oral and dental hospital, medan, indonesia. the research sample consisted of 80 lateral cephalograms consisting of 35 boys and 45 girls aged 9–15 years batak ethnicity, collected using a purposive sampling method based on the inclusion and exclusion criteria. the inclusion criteria were lateral cephalograms of patients aged 9–15 years, skeletal class i (patients who have not received orthodontic treatment), and lateral cephalograms with good quality. the exclusion criteria included a history of craniofacial trauma and fractures, incomplete patient medical records and craniofacial disease, and symptoms or anomalies. this study had permission from the research ethics committee of universitas sumatera utara (number 132/ kep/usu/2021). after collecting the samples of cephalograms that matched the inclusion criteria, then tracing manually using a pencil, ruler, tape, tracing paper, and a tracing box, the cephalometric anatomical landmarks at the anterior nasal spine (ans) – spinous process of the maxilla forming the most anterior projection of the floor of the nasal cavity – were marked points: a (the deepest point on the curved, bony outline between the ans and prosthion [pr]), pog (the most prominent point on the anterior aspect of symphysis of the mandible), me (the most inferior point on the symphysis of the mandible), gn (the intersection of facial plane and mandibular plane), co (the highest point of superior curvature of the condyle of the mandible).10 tracing was done in a systemic manner. the major references, landmarks, and line measurement of the mcnamara analysis were traced and are shown in figures 1a and 1b. an analysis of the growth length of the maxillary and mandible was carried out by determining the points on the cephalogram using coreldraw x7 (canada) on a computer. after tracing and defining landmarks, the paper is then scanned and transferred to a computer by using a printer (figure 1a). entering the scanned file into the coreldraw software application then gives the patient’s name and age. the maxillary length was measured using the mcnamara method with the coreldraw software, with the help of a mouse, by the line from the reference point of the condyle to point a (drawing a line from point a to the point of the condyle), and the length of the mandible was measured by the co-gn reference line from condyle point to gnathion (figure 1b).11 the data obtained was then processed and this data fed to the computer and analysed using statistical testing. figure 1. the results of tracing mcnamara’s measurement using the ‘parallel dimension tool’ in coreldraw x7 (a). cephalometric tracing of mcnamara’s measurement (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p88–92 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p88-92 90lubis and simanjuntak/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 88–92 after all the data had been collected, to prove the data distribution is normal the kolmogorov-smirnov normality test was performed. the pearson correlation coefficient (r) test was performed to assess the correlation of maxillary and mandibular lengths associated with chronological age 9–15 years of different sexes. results based on the data of table 1, it could be seen that the mean maxillary length of males was highest at the age of 15 years with a length of 111.79 ± 7.97 mm and the mean maxillary length of females was highest at the age of 15 years with a length of 101.98 ± 2.30 mm. the lowest mean maxillary length for males was at age 12 with a length of 91.97 ± 8,14 mm and the lowest mean maxillary length for females was at age 13 years with a length of 91.65 ± 7.06 mm. table 1 shows the average maxillary length of 9–15year-olds from the sample group of the usu oral and dental hospital. the results of this study are in line with the results of the study by fouda, et al.10 of 60 male and female egyptian patients, where the results determined that the mean maxillary length of males was 76.80 ± 5.15, higher than that of females, which was 73.55 ± 5.90 mm, and the study by enikawati, et al.4 of 10–16-year-olds using a different maxillary length measurement point (namely from the ans-pns points), showing the mean maxillary length for males was 45.91 ± 3.34 mm, higher than that for females, which was 43.96 ± 3.24 mm. based on table 2 data, it can be seen that the highest mean mandibular length for males was at the age of 15 years with a length of 146.93 ± 10.76 mm and the highest mean mandibular length for females was at the age of 15 years with a length of 127.29 ± 2.87 mm. the lowest mean mandibular length for males was at the age of 12 years with a length of 115.49 ± 9.85 mm and the lowest mean mandibular length for females was at the age of 9 years with a length of 113.01 ± 3.04 mm. table 2 shows the average length of the mandible at the age of 9–15-year-olds from the sample group of the usu oral and dental hospital. the results of this study are in line with the results of the study by fouda, et al.10 of 60 male and female egyptian patients, where the results determined that the mean mandibular length for males was 100.15 ± 7.14 mm, which was higher than in females, which was 96.18 ± 6.94 mm and the study by enikawati, et al.4 using different mandibular length measurement points (namely the measurement between the gonion and menton points), where the mean mandibular length for males was 62.01 ± 3.24 mm, which was higher than that for females, being 60.52 ± 4.20mm. table 3 shows the results of the correlation test between chronological age and maxillary length (r count > r table), obtaining the value of r = 0.329 and an r table value of 0.220. it can be concluded that chronological age is positively correlated with maxillary length, where a positive correlation value indicates a directional relationship between chronological age and maxillary length. mandibular correlation value obtained r = 0.370, which means that chronological age is positively correlated with the mandible with a directional relationship between chronological age and mandibular length. there is an increase in maxillary and mandibular length growth at different ages. the level of correlation is included in the category of sufficient correlation because it is in the class interval 0.25–0.5. the significance value was obtained (p < 0.05), which means that the length of the maxilla and mandible has a correlation with chronological age. table 1. the mean maxillary length from 9–15-year-olds in the sample group from the usu oral and dental hospital age (years) males females n mean ± sd (mm) n mean ± sd (mm) 9 5 95.89 ± 7.64 6 91.71 ± 1.28 10 11 93.54 ± 8.88 10 97.68 ± 7.64 11 9 96.68 ± 6.14 14 94.01 ± 5.01 12 3 91.97 ± 8.14 6 97.97 ± 4.80 13 2 101.88 ± 6.56 4 91.65 ± 7.06 14 2 109.04 ± 9.03 2 96.46 ± 3.20 15 3 111.79 ± 7.97 3 101.98 ± 2.30 total 35 97.47 ± 9.25 45 95.48 ± 5.90 table 2. the mean mandibular lengths at the age of 9–15-year-olds in the sample group from the usu oral and dental hospital age (years) males females n mean ± sd (mm) n mean ± sd (mm) 9 5 125.03 ± 8.89 6 113.01 ± 3.04 10 11 118.48 ± 8.65 10 127.04 ± 9.94 11 9 123.51 ± 7.97 14 117.51 ± 7.73 12 3 115.49 ± 9.85 6 119.25 ± 7.60 13 2 124.31 ± 4.07 4 121.72 ± 10.83 14 2 141.58 ± 9.71 2 122.18 ± 2.02 15 3 146.93 ± 10.76 3 127.29 ± 2.87 total 35 124.54 ± 11.9 45 120.49 ± 8.82 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p88–92 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p88-92 91 lubis and simanjuntak/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 88–92 discussion knowledge of the growth of the skull and jaw, especially the maxilla and mandible, is very important during adolescence when the growth spurt occurs.1 according to evälahti’s11 study, the male mandibles begin to grow more rapidly, with a total average increase of 36.5 mm, between the ages of 4 and 25, while in females at the same age the average increase was 28.2 mm. peak growth is a period of dynamic development characterised by rapid changes in size, shape, and body, with sexual dimorphism.12 from this study we found that the maxillary length growth of ethnic bataks was higher in males than females. this is in accordance to a previous study by laowansiri, et al. 13 that states a significant difference in the maxillary size of males and females where the maxillary size of males is larger than that of females. differences in growth that occur in males and females are caused by either natural factors or disruptive factors. natural factors include genetic variation, and pressure/the biomechanical theory and disruptive factors include malnutrition, hormones, and habits.14 a natural factor that can control the growth of the maxilla and mandible is the presence of genetic variation. based on the biomechanical theory, the main factor in controlling bone growth is pressure. mechanical stress represents one of the many signals involved in the activation of osteogenic connective tissue. however, what regulates the complex balance of genic activity among the various cells and tissues that play a role is not known.15,16 also, from this study we also found that the mandibular length growth was higher in males than females. the mandible in males is 9.3 mm longer on average than in females.11 there are differences in men and women because the pattern of bone remodelling is not the same and can be influenced by genes, hormones, and the environment.12,13,15 other types of factors that affect the growth of the maxilla and mandible are disruptive factors, one of which is malnutrition.15,16 poor nutrition during childhood growth can affect the normal pattern of craniofacial development. nutritional deficiencies can lead to a reduction in maxillomandibular length and lower facial height. arifin stated that girls who consumed more animal protein than vegetable protein, and fat from ages 6 to 8 experienced an earlier peak of growth.17 nutrients that are essential for normal postnatal growth such as calcium, magnesium, phosphorus, fluoride, vitamin a, and vitamin d are needed for bone growth.5 good nutrition can provide normal bone growth. calcium, phosphorus, magnesium, manganese, and fluoride are essential for the growth of good bones and teeth.4,18 vitamin a controls the activity of osteoblasts and osteoclasts. deficiency of essential amino acids, essential fatty acids, vitamins, or minerals also affects skeletal maturation. vitamin d is a good nutrient for bone growth because it contains calcium that bones need. poor nutritional intake will cause interference with growth in height, age, and bone structure.4,18 we found in this study the test results of maxillary length and mandibular length to be statistically positive significant with chronological age (9–15 years). the maxilla and mandible are bones that can provide an overview of gender differences because males and females have morphological differences in each of these bones.14 based on the research of azhari ,et al.19 in men and women aged 9–25 (and also the study of astuti, et al.14), the maxillary and mandibular growth was higher in men than in women aged 15–25. the study also outlined the functioning of the different types of hormones between the sexes, such as the difference in testosterone levels between men and women, where men are heavily influenced by the hormone testosterone and women are heavily influenced by the hormones estrogen and progesterone. regarding the size and mass of muscle and bone, as well as changes in facial shape, the hormone estrogen plays an important role in bone metabolism, in this case affecting the regulation of osteoblast and osteoclast activity by paying attention to the speed of resorption and bone formation taking place at the same rate (under normal conditions) so that bone mass remains constant.4,12 litsas20 states that somatotropin (growth hormone or, gh) is an important factor in craniofacial and skeletal growth during childhood and adulthood. gh can increase bone elongation by stimulating maturation and cell division of chondrocytes in the epiphyseal plate; thus, there is a continuous widening of the disc and production of more cartilage for bone formation.20 another factor that can affect the growth of the maxilla and mandible is the environment. one of the environmental factors that can influence is habit. abnormal habits affect facial growth patterns, which have an important influence on craniofacial growth and occlusal physiology. abnormal habits or bad habits can affect or inhibit bone growth, cause malposition of teeth, breathing difficulties and speech disorders, disrupt facial muscle balance, and create psychological problems. examples of these bad habits are thumb sucking and finger sucking, sticking out of the tongue, sucking and biting lips, poor posture, and biting nails, among others.21 the research conducted is in line with the research of enikawati, et al.4,that the increase in maxillary length in males is greater than in females and the mandibular length in males is greater than in females aged 10–16, which is influenced by genetic, hormonal and nutritional factors. table 3. results of the r-test (correlation) of maxillary and mandibular lengths associated with chronological age age (years) maxillary length mandibular length r p r p 9-15 0.329 0.003* 0.370 0.001* *significant p < 0.05 r test description: 0: there is no correlation between the two variables 0.01 – 0.25: weak correlation 0.26 – 0.5: sufficient correlation 0.51 – 0.75: strong correlation 0.76 – 0.99: very strong correlation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p88–92 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p88-92 92lubis and simanjuntak/dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 88–92 to conclude, there was a significant positive relationship between chronological age and maxillary and mandibular lengths of ethnic bataks aged 9–15. the small but statistically significant gender differences in mandibular and maxillary lengths may not be clinically significant. taking into consideration the ethnic features, age and gender of the patients, plays a critical role in setting objectives for successful orthodontic treatment. thereby, a single set of batak norms from the mcnamara analysis may be advisable and practical in orthodontic diagnosis. references 1. proffit wr. malocclusion and dentofacial deformity in contemporary society. in: proffit wr, fields hw, larson be, sarver dm, editors. contemporary orthodontics. 6th ed. st. louis: mosby; 2018. p. 1–6. 2. velisya v, wijaya h. profil perubahan dimensi mandibula selama fase-fase pubertas. j kedokt gigi terpadu. 2019; 1(1): 58–62. 3. citra nabila r, sapta r ini p r ima r ti r, a hmad i. hubungan pengetahuan orang tua dengan kondisi maloklusi pada anak yang memiliki kebiasaan buruk oral. j syiah kuala dent soc. 2017; 2(1): 12–8. 4. enikawati m, soenawan h, suharsini m, budihardjo sb, sutadi h, rizal mf, fauziah e, wahano na, indriati is. maxillary and mandibular lengths in 10 to 16-year-old children (lateral cephalometry study). j phys conf ser. 2018; 1073: 022015. 5. nahhas rw, valiathan m, sherwood rj. variation in timing, duration, intensity, and direction of adolescent growth in the mandible, maxilla, and cranial base: the fels longitudinal study. anat rec (hoboken). 2014; 297(7): 1195–207. 6. hsiao s-y, cheng j-h, tseng y-c, chen c-m, hsu k-j. nasomaxillary and mandibular bone growth in primary school girls aged 7 to 12 years. j dent sci. 2020; 15(2): 147–52. 7. djoeana hk, nasution fh, trenggono bs. antropologi untuk mahasiwa kedokteran gigi. jakarta: universitas trisakti; 2005. p. 40–9. 8. rieuwpassa ie, hamrun n, riksavianti f. ukuran mesiodistal dan servikoinsisal gigi insisivus sentralis suku bugis, makassar, dan toraja tidak menunjukkan perbedaan yang bermakna. (size of mesiodistal and cervicoincisal maxillary central incisors between buginese, makassarese, and torajanese showe). j dentomaxillofacial sci. 2013; 12(1): 1–4. 9. badan pusat statistik (bps) provinsi sumatera utara. sosial dan kependudukan. 2020. available from: https://sumut.bps.go.id. accessed 2021 sep 27. 10. fouda a, nassar e, hammad y. mcnamara’s cephalometric norms of egyptian children. egypt dent j. 2017; 63(4): 2923–9. 11. evälahti m. craniofacial growth and development of finnish children a longitudinal study. faculty of medicine doctoral programme in oral sciences. dissertation. helsinki: university of helsinki; 2020. p. 12–3, 40, 77–9. 12. soliman a, de sanctis v, elalaily r, bedair s. advances in pubertal growth and factors influencing it: can we increase pubertal growth? indian j endocrinol metab. 2014; 18(suppl 1): s53-62. 13. laowansiri u, behrents rg, araujo e, oliver dr, buschang ph. maxillary growth and maturation during infancy and early childhood. angle orthod. 2013; 83(4): 563–71. 14. astuti er, iskandar hb, nasutianto h, pramatika b, saputra d, putra rh. radiomorphometric of the jaw for gender prediction: a digital panoramic study. acta med philipp. 2022; 56(3): 113–21. 15. ardani igaw. dasar pertumbuhan kraniofasial setelah kelahiran. surabaya: airlangga university press; 2021. p. 24–8. 16. proffit wr. concepts of growth and development. in: proffit wr, fields hw, larson b, sarver dm, editors. contemporary orthodontics. 6th ed. st. louis: mosby; 2018. p. 27–58. 17. arifin r, noviyandri pr, shatia ls. hubungan usia skeletal dengan puncak pertumbuhan pada pasien usia 10-14 tahun di rsgm unsyiah. cakradonya dent j. 2017; 9(1): 44–9. 18. achmad mh, natsir m, samad r, setijanto d. maloklusi pada anak dan penanganannya. jakarta: sagung seto; 2016. p. 4–20. 19. azhari a, pramatika b, epsilawati l. differences between male and female mandibular length growth according to panoramic radiograph. maj kedokt gigi indones. 2019; 5(1): 43–9. 20. litsas g. growth hormone and craniofacial tissues. an update. open dent j. 2015; 9: 1–8. 21. sulandjari h. buku ajar ortodonsia i kgo i. yogyakarta: fakultas kedokteran gigi, universitas gadjah mada; 2008. p. 9, 35–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p88–92 https://sumut.bps.go.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p88-92 121 volume 45 number 3 september 2012 case report the benefit of differential moment concept in managing posterior anchorage and avoiding bite deepening harryanto wijaya and joko kusnoto department of orthodontics faculty of dentistry, universitas trisakti jakarta indonesia abstract background: anchorage is one of the major concerns in orthodontic space closure. various methods have been proposed to enhance posterior anchorage in space closure such as headgear, nance holding appliance, and micro implant as temporary anchorage devices. however, several issues such as patient's compliance, appliance effectiveness, and cost of the device become many clinicians concern. the differential moment concept in segmented arch is a technique that requires no patient compliance but can effectively manage posterior anchorage and avoid bite deepening by careful application of forces and moments. purpose: the purpose of this case report is to show the use of differential moment concept in segmented arch technique to manage posterior anchorage and to avoid bite deepening. case: a 21 years old female patient with protrusive teeth as her chief complaint was treated using fixed orthodontic appliance. case management: the treatment included four first bicuspid extraction and space closure utilizing differential moment concept in segmented arch. conclusion: it can be concluded that application of differential moment concept in segmented arch technique is a non invasive, compliance independent, effective, and cost efficient method to manage posterior anchorage and to avoid bite deepening. key words: anchorage, bite deepening, differential moment, segmented arch abstrak latar belakang: penjangkaran merupakan salah satu aspek yang sering kali menjadi masalah dalam penutupan ruang pada perawatan ortodonti. berbagai metode disarankan untuk memperkuat penjangkaran posterior dalam penutupan ruang seperti headgear, piranti penahan nance, dan implan mikro sebagai alat penjangkar sementara. namun demikian, beberapa hal seperti kerjasama pasien, efektivitas piranti, dan biaya dari alat-alat tersebut sering menjadi perhatian/pertimbangan bagi klinisi. konsep momen diferensial pada segmented arch adalah suatu cara yang efektif untuk memperkuat penjangkaran dan menghindari pendalaman gigitan tanpa memerlukan kerjasama pasien. tujuan: laporan kasus ini bertujuan untuk menunjukkan penggunaan konsep momen diferensial pada segmented arch untuk mengatasi masalah penjangkaran posterior dan pendalaman gigitan. kasus: seorang perempuan usia 21 tahun dengan keluhan utama gigi-gigi anterior protrusif dilakukan perawatan dengan alat ortodonti cekat. tatalaksana kasus: perawatan yang dilakukan meliputi pencabutan 4 premolar pertama dan penutupan ruang dengan konsep momen diferensial pada segmented arch. kesimpulan: dapat disimpulkan bahwa penggunaan konsep momen diferensial pada segmented arch efektif untuk mengatasi masalah penjangkaran posterior dan pendalaman gigitan. kata kunci: penjangkaran, pendalaman gigitan, momen diferensial, segmented arch correspondence: harryanto wijaya, c/o: departemen ortodonsia, fakultas kedokteran gigi trisakti. jl. kyai tapa no. 260, grogol, jakarta barat 11440, indonesia. e-mail: harryantowijaya@yahoo.com. introduction orthodontic anchorage is the resistance to force provided by other teeth or by structures outside the mouth. controlling anchorage is one of the most critical elements of orthodontic treatment.1 generally, there are two types of anchorage used in orthodontic: tooth anchorage and 122 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 121–126 auxiliary anchorage. tooth anchorage may be defined as resistance to movement by using teeth as anchorage. auxiliary anchorage are those adjunctive procedures or appliances that increase anchorage by incorporating adjacent soft and hard tissue components, i.e., headgear, nance holding appliance, and temporary anchorage device.2 headgear, which used to control anchorage, is a device that has been used in orthodontic for at least 100 years. unfortunately, the use of headgear depends on patient compliance for success. in recent years, with the introduction of temporary anchorage devices, a paradigm shift has occurred in the overall perspective toward patient compliance, preservation of anchorage and facilitation of treatment for various difficult malocclusions. however, temporary anchorage devices are invasive as well as expensive and are best reserved for problems that cannot be effectively managed with conventional mechanics.3 the differential moment concept is a mean of anchorage management that increases anchorage by a careful application of forces and moments. differential moment offer many advantages in orthodontic treatment, including simultaneous correction overbite and overjet, arch length, and class ii malocclusion.4 the application of differential moment induces differential tooth movement due to differential stress in the periodontal ligament, which can aid the anchorage control. moments applied to the anterior unit (alpha moment) must be sizeable enough to prevent labial movement of the roots (uncontrolled tipping movement); on the other hand, moment on the posterior unit (beta moment) must be of enough magnitude to induce bodily movement or even root movement. the clinical expression of tipping movements regularly occurs faster than root movement, so that the anterior teeth retract distally into the space before any mesial molar movement is seen.5,6 the purpose of this case report is to demonstrate the ability of differential moment concept in segmented arch technique to manage posterior anchorage and to avoid bite deepening. case patient was a 21 years 10 months old female who presented a protrusive teeth and crowding on lower front teeth as her chief complain. past and present medical history was negligible. the conditions of dentition and other intra oral structure were unerupted lower right third molar, partially erupted lower left third molar, calculus on lower teeth and no other past dental history. the etiology was probably combination of genetic and environment factors. patient had symmetrical dolichofacial face and convex facial profile. lips were strain upon closure. when smiling, the wide buccal corridors due to narrow dental arch became evidence and there was excessive gingival display. facial and upper dental midlines were coincided. patient was in permanent dentition stage. molar along with canine relationships were bilaterally 25% and 50% class ii, respectively. overjet was 4 mm and overbite was 1.5 mm. the upper and lower dental midlines were almost coincided. the lower arch was moderately crowded (figure 1). figure 1. pre-treatment facial and intraoral photographs: a) frontal; b) frontal smiling; c) lateral; d) upper arch; e) lower arch; f) right; g) front; h) left. 123wijaya and kusnoto: the benefit of differential moment concept the cephalometric analysis revealed that patient had class ii skeletal relationship and high mandibular plane angle. lower incisors were protruded with normal inclination, upper incisors were protruded and proclined. interincisal and nasolabial angle were acute. lower and upper lips were protruded relative to e-line. the panoramic confirmed that there was unerupted lower right third molar and no other pathology existed (figure 2). this patient was diagnosed as class ii skeletal malocclusion with bimaxillary dental protrusion. case management the treatment objectives were established to masked skeletal discrepancy via dental movement, reduce upper incisors protrusion and proclination, reduce lower incisors protrusion, achieve adequate overbite and overjet, achieve class i occlusion of buccal segment, and improve facial profile. the treatment plan was determined as follows: (1) extraction of upper and lower first bicuspids; (2) leveling and aligning upper and lower dental arch; (3) closing the extraction space with group a and b anchorage for upper and lower arch, respectively; (4) retraction of upper and lower anterior teeth with controlled tipping and bodily movement, respectively; (5) retention to maintain the treatment result. the fixed appliances used were 0.018 slot roth prescription brackets, molar band with auxiliary tube, and transpalatal arch. after extraction of first bicuspids, leveling and aligning were started using 0.014 and 0.016 superelastic nickel titanium archwire, respectively. once alignment was achieved in the upper arch, three-piece segmented arches (0.016√0.022 ss) were put on posterior and anterior segment, and then the upper canines were retracted using module chain. intrusion arch (0.016√0.022 cna) was included to prevent bite deepening and increase posterior anchorage by tipping molar distally (figure 3). in lower teeth, canines were retracted and posterior teeth were slightly protracted to achieve class i occlusion on continuous arch (0.016√0.022 ss). once the canine retraction had been completed, the anterior segment was retracted. differential moment figure 2. pre-treatment panoramic radiograph. figure 3. three-piece segmented arches and intrusion arch were used on the upper arch to simultaneously retract canines, prevent bite deepening and enhance posterior anchorage by tipping molar distally. on the lower arch, canine retraction and posterior protraction were done to achieve class i occlusion. a) upper arch; b) lower arch; c) right; d) front; e) left. 124 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 121–126 mechanic was used to retract the upper and lower incisors. in order to do this mechanic, t-loops were pre-activated, off-centered gable bend were placed on distal legs. preactivation of the t-loops achieve the necessary moment to force ratio. this pre-activation started by carefully separating the legs of both t-loops by approximately 3 mm. because of the posterior anchorage was the objective, the loops were offset to the posterior and additional gable bends were placed distal to the t-loops to increase anchorage moment. to take the advantage of the positional effects, the loops were directly engaged to the molar auxiliary tube bypassing the second premolars. the archwire was ready to be activated approximately 4 mm; 3 mm of pre activation plus 1 mm of additional activation (figure 4). it is not necessary to be very far off center to obtain an adequate moment differential, with most cases requiring only 1–2 mm off-centering. when the space closed, the finishing phase could be started. this phase of treatment involved the use of coordinated 0.017√0.025 cna wire. minor bends were placed in these beta titanium wires for finishing details. retention consisted of an upper and lower circumferential retainer. figure 4. t-loop was used to retract four incisors on upper and lower arch. pre-activated, off-centered position and gable bend on distal of tloop would create differential moment. the differential moment concept would preserve posterior anchorage through differential tooth movement. a) upper arch; b) lower arch; c) right; d) front; e) left. figure 5. post-treatment facial and intraoral photographs. a) frontal; b) frontal smiling; c) lateral; d) upper arch; e) lower arch; f) right; g) front; h) left. 125wijaya and kusnoto: the benefit of differential moment concept facial profile and lips strain were improved. patient had better smile appearance with broader dental arch. the bilateral class i molar and canine relationship were achieved as well as good overjet and overbite. the upper and lower dental midlines coincided with the facial midline. the buccal segments had good interdigitation. the upper and lower arch form were ovoid and symmetric (figure 5). the superimposed cephalometric tracing confirms the changes achieved with treatment. overall superimposition showed backward reposition of point a and point b. lips were retracted significantly. maxillary superimposition showed upper incisors were retracted in control tipping movement approximately 14.5 degrees. maxillary first molars moved mesially less than 0.5 mm in crown level. maxilla was rotated in counter clockwise direction about 2 degrees relative to sella-nasion plane. mandibular superimposition showed that mandibular incisors were retracted in bodily movement about 3 mm and intruded approximately 1 mm. mandibular molars moved forward 2 mm in bodily fashion. mandible was rotated in counter clockwise direction about 3 degree relative to sella-nasion plane. the post-treatment panoramic showed adequate root parallelism (figure 6). table 1 shows the comparison of cephalometric measurement between pre and post treatment. discussion few studies have investigated the effectiveness of differential moment strategies for anchorage control. well controlled clinical studies of orthodontic treatment strategies are difficult because of the great number of confounding figure 6. post-treatment panoramic radiograph. table 1. preand post-treatment cephalometric measurement analysis variables mean tracing 1 tracing 2 skeletal sna 84.6 83.5 82 snb 81 77.5 77 facial angle (fhp n pog) 87.8 86 87.5 convexity (a n pog) 3.8 8 6.5 mandibular plane angle (fhp mp) 25 36.0 35 dento-skeletal lower incisors a pog (mm) 3 12 9 lower incisors a pog (degree) 23.2 30 27 upper incisors a pog (mm) 5.5 17 13 upper incisors a pog (degree) 34.8 45 32.5 dental molar relationship -3 -1 -3 overjet 2.5 4 3 overbite 2.5 1.5 2 interincisal angle 121.9 106 121 soft tissue nasolabial angle 99.6 77 90 lower lip e line 1.6 9 5 upper lip e line 2 5 2 126 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 121–126 variables associated with orthodontic treatment. the differences among patients and the specific objectives of their treatment complicate the analysis of the effectiveness of particular treatment mechanisms. however, the studies that have been completed provide support for a differential moment concept for anchorage control.7 the conventional canine retraction on light continuous wire will generate extrusive effect on incisors and bite deepening due to change in canines inclination.8 to counteract this tendency, segmented arches and intrusion arch were used. the essence of the segmented arch is the establishment of well-defined units of teeth, so that anchorage and segments movement are clearly defined. the other advantage of segmented arch technique is force system can be defined as statically determinate instead of indeterminate. the meaning of statically determinate force systems is the moments and forces can readily be discerned, measured and evaluate.1 the intrusion arch not only created vertical forces but also delivered a distal crown tip back moments on the molars to effectively control the anchorage loss often associated with sliding mechanic.9 a frequently overlooked consideration in anchorage control is the first order side effect of space closure. the mesially directed, buccally located force on molar will tend to produce a mesially inward rotation. a transpalatal arch provides an excellent means for preventing this side effect.10 many methods have been proposed to increase the anchorage. those methods are ranged from traditional headgear to contemporary tads (temporary anchorage devices) but those methods have several shortcomings such as invasive procedures; patient compliance dependent; and additional treatment cost. however, differential moment concept is the method of choice because it is non invasive, independent of patient compliance, effective, and cost efficient. differential moment concept is not without side effects. according to the principle of static of equilibrium, the unequal moments must be balanced by a third moment or couple. this couple is represented as a pair of vertical forces, intrusive to the anterior teeth and extrusive to the posterior teeth. the magnitudes of the forces are proportional to the moment differential. fortunately, these vertical forces may be beneficial to correct of excessive overbite during space closure.11,12 the overall superimposition of cephalometric tracings showed that lips were retracted significantly due to maximum retraction of upper incisors and optimum retraction of lower incisors. maxillary superimposition demonstrated minimum mesial movement of the upper molars and upper incisors were retracted in control tipping movement due to the proper application of alpha and beta moment. mandibular superimposition showed that lower molars were protracted in bodily movement and lower incisors were bodily retracted to meet the treatment objective. it can be concluded that application of differential moment concept in segmented arch is a non invasive, compliance independent, effective, and cost efficient method in managing posterior anchorage and avoiding bite deepening. it is recommended to provide more evidence in the efficacy of this treatment approach via well controlled clinical trials. acknowledgement the authors thank irawati gandadinata, drg., sp.ort for reviewing the manuscript. references 1. proffit wr, fields hw, sarver dm. contemporary orthodontics. 4th ed. st. louis: mosby co; 2007. p. 343, 383–92. 2. langberg jb, todd a. treatment of a class i malocclusion with severe bimaxillary protrusion. am j orthod dentofac orthop 2004; 126(6): 739–46 3. nanda r, uribe fa. temporary anchorage devices in orthodontics. st. louis: mosby co; 2009. p. 3. 4. mulligan tf. the advantages of differential moments. j clin orthod 2009; 43: 379–86. 5. viecilli rf. self-corrective t-loop design for differential space closure. am j orthod dentofac orthop 2006; 129(1): 48–53. 6. martins rp, buschang ph, gandini lg. group a t-loop for differential moment mechanics: an implant study. am j orthod dentofac orthop 2009; 135(2): 182–9. 7. andrew jk, priebe dn. space closure and anchorage control. semin orthod 2001; 7(1): 42–9. 8. uribe f, nanda r. treatment of class ii, division 2 malocclusion in adult: biomechanical considerations. j clin orthod 2003; 37: 599–606. 9. şenişik en, türkkahraman h. treatment effects of intrusion arches and mini-implant systems in deepbite patients. am j orthod dentofac orthop 2012; 141(6): 723–33. 10. kuhlberg a. segmented arch mechanics. in: rakosi t, graber tm †, editors. orthodontic and dentofacial orthopedic treatment. stuttgart: thieme 2010. p. 222–34. 11. nanda r, andrew jk, uribe f. biomechanic basis of extraction space closure. in: nanda r, editor. biomechanics and esthetic strategies in clinical orthodontics. st. louis: elsevier saunders 2005. p. 194–210. 12. choi yj, chung jc, choy kc, kim kh. absolute anchorage with universal t-loop mechanics for severe deepbite and maxillary anterior protrusion and its 10-year stability. angle orthod 2010; 80(4): 771–82. editorial team of dental journal (majalah kedokteran gigi) sk: 17/un3.1.2/2021 january 4 – december 31, 2021 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga chief editor: muhammad dimas aditya ari, drg., m.kes department of prosthodontics, faculty of dental medicine, universitas airlangga editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (department of restorative dentistry, university of illinois at chicago college of dentistry, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health, a.t. still university, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); beshlina fitri widayanti (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); aulia ramadhani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers ida ayu evangelina (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); arlette setiawan (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); siti sunarintyas (deparment of dental biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); niswati fathmah rosyida (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); ananto ali alhasyimi (deparment of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); mei syafriadi (deparment of biomedical sciences, faculty of dentistry, universitas jember, indonesia); sianiwati goenharto (vocational faculty, universitas airlangga, indonesia); diah savitri ernawati (deparment of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); eha renwi astuti (department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia). tania saskianti (department of pediatric dentistry, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); priyawan rachmadi (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia) an'nisaa chusida (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478, 5030255. fax. +62 31 5039478, 5026288 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg/index accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 volume 54, issue 4, december 2021 p-issn: 1978-3728 e-issn: 2442-9740 printed by: airlangga university press. campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 54, issue 4, december 2021 p-issn: 1978-3728 e-issn: 2442-9740 1. management of pericoronitis for partial eruption of second permanent molar in a pediatric patient tengku natasha eleena binti tengku ahmad noor, james lian yoon chen, mohd safwani affan alli and mohd hosni bin mahmood .......................................................... 169–173 2. management of a complete 180° rotation of bilateral maxillary canines putri intan sitasari, niken merrystia and ida bagus narmada ................................................. 174–180 contents case reports page 3. inhibitory effect of calcium hydroxide combined with nigella sativa against enterococcus faecalis myrna nurlatifah zakaria, yusfien shabrina putri, asih rahaju, sri fatmawati and arief cahyanto .......................................................................................................................... 181–185 4. an effective concentration of propolis extract to inhibit the activity of streptococcus mutans glucosyltransferase enzyme riyan iman marsetyo, sagita putri andyningtyas, chonny salsabilla zamrutizahra, ivan nur fadela, agus subiwahjudi and ira widjiastuti ............................................................ 186–189 5. the effect of persea americana mill. seed extract on inflammatory cells and fibroblast formation in tooth extraction socket healing yessy ariesanti, irvan septrian syah putra rasad, maylan nimas and nadira syabilla ........ 190–194 6. the effects of mixing slurry water with type iii gypsum on setting time, compressive strength and dimensional stability chindy fransiska br nainggolan and dwi tjahyaning putranti ............................................... 195–199 7. comparison of maxillary sinus on radiograph among males and females rona aulianisa, rini widyaningrum, isti rahayu suryani, rurie ratna shantiningsih and munakhir mudjosemedi .......................................................................................................... 200–204 8. comparison of the occlusal feature index (ofi) and dental aesthetic index (dai) in 10–14-yearold children at the universitas sumatera utara dental hospital hilda fitria lubis and arfah azriana ........................................................................................... 205–209 9. effectiveness of capsaicin nanoparticle gel of capsicum frutescens l. on oral squamous cell carcinoma in rattus norvegicus fitri aniowati, cantika nadrotan naim, nova dwi anggraeni and pratiwi nur widyaningsih ....................................................................................................... 210–215 10. runx2 rs59983488 polymorphism in class ii malocclusion in the indonesian subpopulation fadli jazaldi, benny m. soegiharto, astrid dinda hutabarat, noertami soedarsono and elza ibrahim auerkari ............................................................................................................. 216–220 11. bioinformatic approach of propolis as an inhibitor of peptidoglycan glycosyltransferase to improve antibacterial agent: an in-silico study imelia arifatus sani, siska maulidina cahyani, safira fariha, oliresianela and diah ........... 221–226 original articles vol 50 no 4 desember 2017.indd 220 dental journal (majalah kedokteran gigi) 2017 desember; 50(4): 220–225 research report the role of cervical vertebrae maturation in defining the chronological age of down syndrome children anggiani dewi rahmawati, iwan ahmad, and arlette suzy setiawan department of paediatric dentistry, faculty of dentistry, universitas padjadjaran bandung – indonesia abstract background: the difficulty of determining chronological age is increased in individuals with conditions that may affect normal development. some systemic conditions in children, for example down syndrome, may cause abnormal physiological maturation. skeletal and dental age are considered the most apt physiological age indicators in determining chronological age. purpose: this study aimed to compare and analyze the relationship between two developmental parameters (dental history and skeletal age) as indicators of the chronological age of children with down syndrome. methods: the study design was cross-sectional with a paired t-test to analyze the differences in chronological and dental age of the samples. the radiograph selection was based on purposive sampling. the study material consisted of 30 panoramic radiographs and lateral cephalometrics of 6-14 years old children with down syndrome and those experiencing normal development (control group) divided into two groups of 15 subjects who attended the pediatric dentistry polyclinic, rsgm, universitas padjadjaran. statistical analysis employed a t-test to determine the difference between chronological and dental age, while a spearman rank correlation was used to evaluate the correlation between dental and skeletal age. results: the results showed there to be no statistical difference between chronological and dental age, where p > 0.05, but a significant relationship between dental and skeletal age in children diagnosed with down syndrome, where p = 0.05. conclusion: it is concluded that dental age identified by means of the nolla method is closer to chronological age than skeletal age using the cervical vertebrae maturation method. keywords: down syndrome; dental age; skeletal age; cvm; chronological age correspondence: arlette suzy setiawan, department of paediatric dentistry, faculty of dentistry, universitas padjadjaran. jl. sekeloa selatan 1 bandung 40132, indonesia. e-mail: arlettesuzy@yahoo.com; arlette.puspa@fkg.unpad.ac.id introduction chronological age has many applications in areas as varied as: pediatrics, orthopedics, orthodontics, forensic medicine, anthropological, social and legal contexts such as criminal cases, kidnapping, employment, marriage, premature birth, adoption, illegal immigration, lack of a birth certificate or document-based fraud.1-4 chronological age is defined as the length of time that has elapsed since an individual’s birth5,6 and can be used to identify the specific stage of child development. however, it is a weak predictor of growth, rendering physiological age more reliable for evaluating the developmental period. physiological age is determined by the level of functional development (maturation) of various body tissue systems measured by a range of parameters including: somatic maturation, sexual, skeletal and dental age.3,7-9 somatic maturation can be assessed by analyzing a child’s increasing height and weight during the growth period, while sexual maturation is associated with secondary sexual characteristics in boys and girls. quantification of age by skeletal maturation can be undertaken through reference to changes in bone ossification, one of which is cervical vertebrae maturation (cvm), which are monitored by radiograph. meanwhile, dental age is established based on the timing of the emergence of teeth and the stage of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220–225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 mailto:arlettesuzy@yahoo.com mailto:arlette.puspa@fkg.unpad.ac.id 221221rahmawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 220–225 dental formation (calcification) which can also be observed radiographically.1,3,7,8,10 the assessment of dental age based on tooth calcification using the nolla method is more reliable than results obtained by other means, such as the demirjian method. this is because the latter method uses two decimal fractions in the calcification stage, more than 90% researchers approved this method due to the more accurate result.11 skeletal age can be determined through hand and wrist analysis by means of cvm method, of which the most widely-used variety is cervical vertebrae maturation stage (cvms) analysis. various studies on cvm have been performed.12-14 in 1972, lamparski et al. as cited in bacetti et al.,13 suggested a method for the evaluation of skeletal maturation based on morphological changes in the cervical vertebrae. the original lamparski method was modified by baccetti et al.,8,13 and has proven to be sufficiently reliable and valid to replace palm and wrist analysis. certain systemic conditions may cause abnormal physiological maturation with the result that skeletal age is more delayed than dental age.1 systemic delayed eruption is associated with numerous genetic conditions including: cleidocranial dysplasia, hemifacial atrophy, mucopolysaccharidosis, turner syndrome and down syndrome.15 the latter is a genetic disorder afflicting more than 5.8 million people that has become the most common genetic disorder resulting in intellectual disability. the clinical condition of down syndrome, first diagnosed by john langdon down in the mid-nineteenth century (1866), is characterized by a central growth deficiency resulting in impaired mental development that ranges in severity from mild to moderate. the growth of individuals suffering from the condition is delayed in almost every aspect, including the development of teeth.16–19 de moraes et al.20 studied the chronology of dental mineralization in down syndrome children and found the dental age of two-thirds of subjects of either gender to be generally more advanced, while that of one-third was delayed, meaning that the majority of patients had normal development. other studies by de moraes et al.18 into the chronological analysis of mineralization using nolla’s method concluded that the dental age of down syndrome individuals is similar to that in normal individuals. the correlation between the stage of dental calcification and skeletal maturation that has been reported found there to be a correlation between dental age and skeletal analysis of the palms and wrists.10 research by carinhena et al.,21 into skeletal age comparing the cvm method and analysis of the palms and wrists in the pubertal growth spurt curve of down’s individual syndrome confirmed that both analyzes produce similar results. few studies are known to link dental age assessment through the stage of dental calcification with skeletal age determined by cvm. therefore, the authors were interested in conducting research that aimed to compare dental and skeletal age, while linking both with chronological age. taking the fact that both the panoramic radiograph and lateral cephalometry constitute dental diagnostics frequently performed by the dentist into account, the authors wished to examine further the comparison between dental age established by the nolla method and skeletal age through cvm in down syndrome children who were based in bandung. materials and methods the research material of this study was secondary data obtained from the results of the panoramic radiographic images and lateral sefalometri of 6–14 years old children with down syndrome who attended the oral and dental hospital, universitas padjadjaran. the control group was composed of healthy children of the same age and the sampling undertaken was purposive in nature. the study sample was divided into two groups, namely: down syndrome children as the test group using primary data, and normal children as the control group using secondary data. group membership satisfied the following inclusion criteria: the test group included down syndrome children of all types aged 6–14 years, while membership of the control group was based on the panoramic radiographs and lateral cephalometric tests of normal children aged 6–14 years who underwent dental and mouth care at oral and dental hospital, universitas padjadjaran. the results of a panoramic radiographic and lateral cephalometry test confirmed well-defiined detail, contrast and density and did not experience any distortion resulting from a radiologist’s assessment. the research samples meeting the inclusion criteria consisted of 15 children with down syndrome as the test group and 15 normal children as the control/comparison group. the grouping of samples according to age shows the results of chronological, dental, and skeletal age calculations for those subjects with down syndrome and their normal/control counterparts. the research was conducted using a cross-sectional study design with parametric and non-parametric statistical tests in order to compare two developmental parameters (dental and skeletal age) as chronological age images in down syndrome children and to assess the relationship between dental and skeletal ages based on chronological age in children with this condition. the study was conducted by assessing the results of the panoramic radiographs and lateral cephalometry tests in the form of analysis of ten radiographs completed on one day and repeated once at an interval of one week. assessment and calculation of dental age based on the results of the panoramic radiographs followed the nolla method. assessment of skeletal age based on the radiographic results of lateral cephalometry in the determination of cvm level adhered to the methodology recommended by baccetti et al.22 assessment and calculation of dental age using the results of the panoramic radiograph was based on the nolla method, including the following dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220-225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 222 rahmawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 220–225 steps: identification of ten stages of maxillary tooth and mandible verification, the first column (right) assesses the growth stage of the central and lateral incisors, the second column the canine teeth, the third column the premolars and the fourth column the molars (figure 1). each stage was scored, with the score of each stage then being totaled. if one-third of the crown had been formed when the observation was conducted a value of 3.0 was assigned, whereas if one third of the roots had been formed a value of 7.0 was recorded. if the tooth was between the appropriate class of illustrations, the decimal fractional values of 0.2, 0.5 or 0.7 were registered as recommended by nolla. if the radiographs were seen between two stages, a value of 0.5 was assigned. for example, if the radiograph reading was between one-third and two-thirds of the roots that had been formed, it was rated 7.5. if the radiographs showed a slightly larger step than the illustration class, but not as much as halfway between that stage and the next, a value of 0.2 was recorded. for example, if a radiographic reading was taken indicating that slightly more than twothirds of the crown had been formed, it was rated 4.2. if the radiographs confirmed fewer stages than the illustrated class indication, a value of 0.7 was alloted. for example, if the stage is in the class of crowns that are two-thirds completed, the value was 3.7. the number of scores obtained for each tooth (maxillary and mandible) was recorded. this figure was then matched with the corresponding one within the age table of maxillary and mandibular teeth of both genders to translate the developmental value into a dental age. assessment of skeletal age by radiographic results of lateral cephalometry in the determination of cvm level was based on the findings of baccetti et al. the stages of cvm classification according to the method of baccetti et al. (figure 2) are as follows: cvm 1 the lower limit of the entire cervical vertebrae (c2-c4) was flat, the cervical vertebrae 3 and 4 (c3 and c4) bodies exhibited a trapezoidal shape (the superior vertebrae of the vertebrae body decreased from posterior to anterior/tappered). cvm 2 – the presence of concavity at the lower limit of cervical vertebrae 2 (c2) (depth of basin equal to 0.8 mm), lower limit of cervical vertebrae 3 and 4 (c3 and c4) flat and body c3 and c4 still trapezoidal. cvm 3 – the presence of concavity at the lower limit of cervical vertebrae 2 and 3 (c2 and c3), the cervical vertebrae bodies 3 and 4 (c3 and c4) display a shape between the trapezoid or rectangle without substantial changes. cvm 4 presence of concavity at the lower limit of cervical vertebrae 2, 3 and 4 (c2, c3 and c4). the cervical vertebrae bodies 3 and 4 (c3 and c4) show a horizontal rectangular shape. cvm 5 presence of concavity at all lower boundaries of cervical vertebrae 2, 3 and 4 (c2, c3 and c4). at least one of the cervical vertebrae 3 and 4 (c3 and c4) shows a square shape, or one of which is still a horizontal rectangle. cvm 6 the presence of clear concavity on the entire lower border of cervical vertebrae 2, 3 and 4 (c2, c3 and c4). at least one of the cervical vertebrae 3 and 4 (c3 and c4) is a vertical rectangle (vertical border becomes longer than the horizontal) or one of them is still square. the statistical tests used in this study constituted the following: a paired t-test to analyze chronological age difference and dental age in down syndrome children, a rank spearman correlation test to analyze the correlation between dental age with skeletal age, a wilcoxon analysis test to analyze comparison of chronological age to childhood down syndrome skeletal age, an unpaired t-test to know the difference in dental age between down syndrome and control children and a wilcoxon’s analysis test to determine the skeletal age difference between down syndrome and control children. results down syndrome children constituted a test group of 15 people, with a highest chronological age of 178 months and a lowest of 85 months. the highest dental age was 144 months, while the lowest was 84. the mean age of skeletal childhood down syndrome was in cvs2. the normal children acting as a control group amounted to 15 individuals, with a highest age of 164 months and a lowest of 98 months. the highest dental age was one of 156 months, while the lowest was 84 months. the average skeletal age of a normal child was in cvs2. figure 1. stages of tooth classification.4 figure 2. stages of cervical vertebrae development according to baccetti.22 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220-225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 223223rahmawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 220–225 grouping of chronological age based on skeletal age in down syndrome children was completed in order to compare chronological age with skeletal age as shown in table 1. this shows a comparison of chronological age at cvs 2 and cvs 3 levels. a wilcoxon analysis test obtained a p-value of 0.0050 < 0.05 and it can be concluded that there is a statistically significant chronological age difference between the cvs 2 and cvs 3 groups. table 1 shows a comparison of chronological age at cvs 2 and cvs 3 levels. a wilcoxon analysis test obtained a p-value of 0.0050 < 0.05, thereby supporting the conclusion that there is a statistically significant chronological age difference between the cvs 2 and cvs 3 groups. table 1 descriptively shows that the chronological age ratio at cvs 2 and cvs 3 levels is different. the wilcoxon analysis test obtained a p-value of 0.0050 < 0.05, supporting the conclusion that there is a statistically significant chronological age difference between the cvs 2 and cvs 3 groups. the cvs 4 group (n = 1) was not included in the wilcoxon analysis test because only one sample was found in patients with a chronological age of 178 months. the dental age of children with down syndrome was then compared with their chronological age to enable a comparison of the two ages as shown in table 2, which descriptively shows that the mean of the chronological age in down syndrome children is higher than that of their dental age. the difference between the mean chronological and dental age was 8.2 months, indicating that the dental age of a down syndrome child was 8.2 months lower than his/her chronological age. statistical analysis of paired t-tests was applied to identify the chronological and dental age differences in children with down syndrome. this was not statistically significant (p-value = 0.2377 > 0.05). therefore, it can be concluded that there is no chronological and dental age difference among down syndrome children. an insignificant result is possible because of the small number of samples in order to identify the relationship between dental and skeletal age in children with down syndrome, a spearman correlation analysis was completed and confirmed the existence of a statistically significant relationship between dental and skeletal age in children with down syndrome with a value of 0.783. the test results obtained t value = 4.539. the coefficient of determination of the calculation results obtained was 61.3%. the unpaired t-test was used to establish whether any difference in dental age between the children with down syndrome and those in the control group existed. the result of the analysis confirmed a difference in dental age between those children with down syndrome and the control group, although a significant test result (p-value = 0.0636 > 0.05) was absent. consequently, it can be concluded that there is no difference in dental age between down children and normal/control children. the average dental age of the down syndrome children was 108.80 months, whereas in the control group it was 121.60 months. this suggests that the dental age of the down syndrome child in the 6-14 years chronological age range was 12.80 months or 1 year 8 months lower than in the control group. the wilcoxon analysis test for skeletal age differences between the children with down syndrome and the control group members shows that the majority of individuals with down syndrome and normal children have a skeletal age in cvs 2. down syndrome sufferers register a higher percentage (73.4%) when compared with that of normal children (46.7%). wilcoxon test results obtained a p-value of 0.0763 > 0.05 indicating that there is no significant difference in skeletal age between children with down syndrome and those in the control group based on chronological age. discussion within this study, the chronological age of subjects was calculated from differences between radiographic exposure and date of birth, such as in the study conducted by leonelli de moraes et al.18 the research sample consisted of a down syndrome children group and control group of children in the 6-14 years age range. this age-based retrieval is based on the cervical vertebrae ossification process commencing when the fetus is in the womb and continuing until childhood. therefore, changes in cervical table 1. results of chronological age comparison test for skeletal age of down syndrome children variable n sum of ranks sd z p-value chronological age-cvs 2 11 66 6.41 -2.57 0.0050*)chronological age-cvs 3 3 39 total 14 105 description: p-value = (< 0.05); *) = significant table 2. differences test of chronological and dental age in children with down syndrome chronological age average (months) dental age average (month) differential average between chronological and dental age (month) n sd t-test p-value 115.8 108.8 8.2 15 9.49 0.723 0.2377 description: n = sample, sd = standard deviation, p-value = (< 0,05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220-225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 224 rahmawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 220–225 vertebrae maturation can be observed during the growth period,37 in addition to the time of permanent dental eruption (excluding the third molar) between the ages of 6–13 years. calcification of the permanent teeth begins at the end of the gestation period and continues to average until around the age of 16.23 this represents a good time for dental age assessment based on tooth formation (dental calcification) and radiographic features. children with down syndrome are at the cvs2 level, confirming that the majority experience delays in development when compared to their chronological age. this result is consistent with the finding of leonelli de moraes et al. that the skeletal age of children with down syndrome is delayed during the ages of 7 to 15. generally, the growth and development of children with down syndrome experiences delays. excess chromosomes in children with down syndrome will alter the genetic balance of the body and result in changes in physical characteristics and intellectual abilities, as well as impairments to the body’s physiological functions.24 the difference between chronological and dental age in down syndrome children falling within the 6–14 years age range indicates that there is a delay in the growth and development of teeth in such individuals. the dental age of down syndrome children lags 8.2 months behind their chronological age – a difference falling within the medium category. the division of dental age categories into “slightly, “moderately”, or “notably” in terms of their contrast with chronological age is a classification previously used by other authors who consider a difference of approximately three months between dental and chronological ages as falling within the “normal” category.2,20 sachan et al. evaluated the relationship between skeletal and palmar skeletal maturation indicators and cvm indicators with canine calcification based on the nolla method in a lucknow-india population. they concluded that there was a strong correlation in both male (r = 0.849) and female (r = 0.932) subjects and that canine calcification stages can be used in assessing skeletal maturation. however, regardless of the substantial correlations reported in the study, clinical significance may be limited to the individual level.7 the same results in this study showed there to be a statistically significant relationship between dental and skeletal age in down syndrome children, with a coefficient of determination amounting to 61.3%. this provides insight into the fact that skeletal age is influenced by dental age in 61.3% of cases, while the remaining 38.7% represents the impact of variables other than dental age, such as: genetic, hormonal, nutritional, socio-economic, climatic, seasonal and factors including pharmacological biochemicals which can delay or accelerate aging because of certain abnormalities. down syndrome is one of the most common causes of skeletal retardation.21 in their research, abouhala et al.1 show that there is a closer value and a smaller difference between dental and chronological age compared to skeletal and chronological age in down syndrome individuals. it can therefore be said that establishing age by means of the nolla method is more accurate than using the greulich and pyle method in determining skeletal and chronological age. abouhala et al.1 support these results. in this study, there was a statistically significant correlation between dental age and skeletal age with a t-count of 4,539. this correlation showed that establishing age by means of the nolla method produces a result closer to a subject’s chronological age than does skeletal age using the cvm method. this is because the nolla method advocates analysis of two degrees of mineralization from the crown, thereby providing a more detailed value for the stage of dental calcification. it may be that dental age correlates more with chronological age. it is concluded that the dental age as determined by the nolla method is closer to the chronological age than the skeletal age established by means of the cvm method. references 1. hala la, de moraes mel, carvalho mflv, lopes slpdc, gamba tdo. comparison of accuracy between dental and skeletal age in the estimation of chronological age of down syndrome individuals. forensic sci int. 2016; 266: 578.e1–10. 2. diz p, limeres j, salgado afp, tomás i, delgado lf, vázquez e, feijoo jf. correlation between dental maturation and chronological age in patients with cerebral palsy, mental retardation, and down syndrome. res devel disabil. 2011; 32(2): 808–17. 3. lund e, tømmervold t. relationship between dental age, skeletal maturity and chronological age in young orthodontic patients. thesis. northern norway: uit the arctic university of norway; 2014. p. 8–21. 4. altunsoy m, nur bg, akkemik o, ok e, evcil ms. dental age assessment: validity of the nolla method in a group of western turkish children. marmara dental journal. 2013; 2: 49–52. 5. dorland w. kamus kedokteran dorland. jakarta: egc; 2002. p. 201. 6. tandon s, bhat m, deshpande a. textbook of pedodontics. 2nd ed. new delhi: paras medical publisher; 2009. p. 917. 7. sachan k, sharma vp, tandon p. a correlative study of dental age and skeletal maturation. indian j dent res. 2011; 22(6): 882. 8. valizadeh s, eil n, ehsani s, bakhshandeh h. correlation between dental and cervical vertebral maturation in iranian females. iran j radiol. 2012; 10: 1–7. 9. ogodescu ae, bratu e, tudor a, ogodescu a. estimation of a child’s biological age based on tooth development. rom j leg med. 2011; 19(2): 115–24. 10. saranya b, ahmed j, shenoy n, ongole r. comparison of skeletal maturity and dental maturity a radiographic assessment. scholars j appl med sci. 2013; 1(5): 427–31. 11. miloglu o, celikoglu m, dane a, cantekin k, yilmaz ab. is the assessment of dental age by the nolla method valid for eastern turkish children? j forensic sci. 2011; 56(4): 1025–8. 12. rakosi t, jonas i, graber tm. color atlas of dental medicine: orthodontic-diagnosis. new york: thieme medical publisher; 1993. p. 34. 13. baccetti t, franchi l, mcnamara ja. an improved version of the cervical vertebral maturation (cvm) method for the assessment of mandibular growth. angle orthod. 2002; 72(4): 316–23. 14. alhadlaq am, al-shayea ei. new method for evaluation of cervical vertebral maturation based on angular measurements. saudi med j. 2013; 34(4): 388–94. 15. friedrich re, habib s, scheuer ha. eruption times of permanent teeth in children and adolescents in latakia. arch kriminol. 2009; 223(3-4): 84–97. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220-225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 225225rahmawati, et al./dent. j. (majalah kedokteran gigi) 2017 december; 50(4): 220–225 16. welbury r, duggal ms, hosey mt. paediatric dentistry. oxford: oxford university press; 2005. p. 234–61. 17. cheng rhw, yiu cky, leung wk. oral health in individuals with down syndrome. in: dey s, editor. prenatal diagnosis and screening for down syndrome. hongkong: intech; 2011. p. 59–76 18. de moraes mel, de moraes lc, cardoso m, ursi w, lopes slpdc. age assessment based on dental calcification in individuals with down syndrome. res dev disabil. 2013; 34(11): 4274–9. 19. hennequin m, faulks d, veyrune jl, bourdiol p. significance of oral health in persons with down syndrome: a literature review. dev med child neurol. 2009; 41(4):275–83. 20. de moraes mel, bastos ms, dos santos lrda, castilho jcdm, de moraes lc, filho em. dental age in patients with down syndrome. braz oral res 2007; 21(3): 259–64. 21. carinhena g, siqueira df, sannomiya ek. skeletal maturation in individuals with down’s syndrome: comparison between pgs curve, cervical vertebrae and bones of the hand and wrist. dental press j orthod. 2014; 19(4): 58–65. 22. baccetti t, franchi l, mcnamara ja. the cervical vertebral maturation (cvm) method for the assessment of optimal treatment timing in dentofacial orthopedics. semin orthod. 2005; 11(3): 119–29. 23. beunen gp, rogol ad, malina rm. indicators of biological maturation and secular changes in biological maturation. food nutr bull. 2006; 27(4): s244–56. 24. de moraes me, tanaka jlo, de moraes lc, filho em, castilho jcdm. skeletal age of individuals with down syndrome. spec care dentist. 2008; 28(3): 101–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i4.p220-225 http://dx.doi.org/10.20473/j.djmkg.v50.i4.p220-225 �� volume 46 number 1 march 2013 antitumor activity of antisense oligonucleotide p��skp� in soft palate carcinoma cell squamous in vitro supriatno,1 sartari entin yuletnawati1 and iwa sutardjo rus sudarso2 1department of oral medicine 2department of pediatric dentistry faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: human soft palate cancers are characterized by a high degree of local invasion and metastasis to the regional lymph nodes. treatment options for this cancer are limited. however, a new strategy for refractory cancer, gene therapy is watched with keen interest. p45skp2 gene as a tumor promoter gene is one of target of the oral cancer therapy. to inhibit the activity of p45skp2 gene is carried-out the genetic engineering via antisense technique. purpose: to examine the antitumor activity of p45skp2 antisense (p45skp2 as) gene therapy in human soft palate [hamakawa-inoue (hi)] cancer cells. methods: pure laboratory experimental study with post test only control group design was conducted as a research design. to investigate the apoptosis induction of p45skp2 astransfected cell was evaluated by colorimetric caspase-3 assay and flow cytometry. furthermore, to detect the suppression of in vitro hi cell invasion and cell growth of p45skp2 as-treatment cell was examined by boyden chamber kit and mtt assay, respectively. results: the cell number of p45skp2 as-treated hi cell was significant decreased when compared with that of p45skp2 sense (p45skp2 s) cells (p<0.05). p45skp2 as-treated cell induced apoptosis characterized by an increase in the early and late apoptosis, and activation of caspase-3 (p<0.05). therefore, suppression of hi cell invasion and cell growth were markedly increased by p45skp2 as treatment (p<0.05). conclusion: antisense oligonucleotide p45skp2 has a high antitumor activity in human soft palate cancer cell, targeting this molecule could represent a promising new therapeutics approach for this type of cancer. key words: antisense p45skp2 gene, apoptosis, invasion, soft palate cancer cell abstrak latar belakang: kanker palatum lunak mempunyai karakteristik invasi dan metastasis ke limfonodi regional yang tinggi. pilihan perawatan kanker tersebut masih sangat terbatas. walaupun demikian, strategi baru untuk penanganan kanker yaitu terapi gen menjadi pilihan utama. gen p45skp2 sebagai gen pemacu tumor merupakan salah satu target terapi kanker oral. untuk menghambat aktivitas gen p45skp2 tersebut dilakukan rekayasa genetik melalui teknik antisense. tujuan: menguji aktivitas antitumor gen p45skp2 antisense (p45skp2 as) terhadap sel kanker palatum lunak (sel hi). metode: jenis penelitian yang digunakan adalah eksperimen laboratorik murni dengan rancangan posttest only control group design. induksi apoptosis sel yang ditransfeksi p45skp2 as dievaluasi menggunakan uji caspase-3 kolorimetrik dan flow cytometry. untuk mendeteksi hambatan invasi dan pertumbuhan sel hi yang ditransfeksi p45skp2 as dilakukan uji boyden chamber dan uji mtt. hasil: pertumbuhan sel hi yang ditransfeksi p45skp2 as menurun secara signifikan dibandingkan dengan p45skp2 sense (s) (p<0,05). sel hi transfeksi p45skp2 as menginduksi apoptosis dengan meningkatkan aktivitas proteolitik caspase-3 dan early and late apoptosis (p<0,05). hambatan invasi dan pertumbuhan sel hi secara signifikan meningkat pada sel yang diperlakukan dengan p45skp2 as (p<0,05). kesimpulan: p45skp2 as oligonukleotida mempunyai aktivitas antitumor yang kuat pada sel kanker palatum lunak. target dari molekul tersebut dapat menjanjikan suatu terapeutik baru untuk jenis kanker palatum tersebut. kata kunci: gen p45skp2 as, apoptosis, invasi, sel kanker palatum lunak research report ��supriatno, et al.,: antitumor activity of antisense oligonucleotide p45skp2 soft palate cancer is a rare cancerous growth of the mouth. cancer of the soft palate accounts for approximately 2% of head and neck mucosal malignancies and most cancer of the palates are squamous cell carcinoma (scc).1 human soft palate cancers are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes. moreover, human soft palate cancer frequently shows local recurrence after initial treatment, probably due to micro invasion and/or metastasis of tumor cells at the primary site.2 despite advanced in surgery, radiotherapy and chemotherapy, the survival of patients with oral cancers include soft palate cancer has not significantly improved over the past several decades. also, treatment options for recurrent or refractory soft palate cancers are limited.3 furthermore, the ratio of mortality in 1980 and 1990 was 48% and 47% respectively,4 and the oral cancer prognosis has not changed during the past 10 years. however, as a new strategy for refractory cancer, gene therapy is watched with keen interest. p45 s-phase kinase associated protein 2 (p45skp2 or skp2), a member of the f-box family, is the substraterecognition subunit of the scfskp2 ubiquitin ligase complex.5 skp2 has been implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase (cdk) inhibitor p27kip1, and positively regulates the g1/s transition. 6 the targeted disruption of skp2 leads to the accumulation of p27 and cell cycle arrest in g1. over expression of skp2 has been observed in various types of human tumors. elevated expression of skp2 indicates poor prognoses for patients with colorectal,7 lymphoma,8 gastric,9 and lung cancers.10 therefore, skp2 knock-out mice grow more slowly and have smaller organs than littermate controls, and they show a reduced growth rate and increased apoptosis.11 however, the role of skp2 over expression in cancer cells or the nature of its contribution to the malignant phenotype is little known. in the present study, p45skp2 was combined with antisense oligonucleotide. this antisense is designed to hybridize to their specific mrna target. the hybrid formation causes a steric block or conformational obstacle for protein translasion. antisense oligonucleotides are used to knock down protein expression by inhibiting the translation of the mrna of a desired target gene.12 the main purpose of the study was to examine the antitumor activity of p45skp2 antisense (p45skp2 as) gene therapy in human soft palate [hamakawa-inoue (hi)] cancer cells. materials and methods derivation of the human soft palate cancer cell line (hi) has been described.13 hi cell line was cultured in dulbecco’s modified eagle medium (dmem, sigma-aldrich, usa) supplemented with 10% fetal calf serum (fcs, moregate biotech, bulimba, australia), and 100 mg/ml streptomycin, 100 u/ml penicillin (invitrogen corp., carlsbad, ca, usa). the cultures were incubated in humidified atmosphere of 95% air and 5% c02 at 37° c. furthermore, two oligonucleotides (antisense and sense) containing phosphorothioate backbone were treated into a soft palate cancer (hi) cell line that had exhibited overexpression of the protein. antisense experiments were performed as described previously.10 two oligonucleotides containing phosphorothioate backbones were synthesized as follows: as, 5’-tcctgtgcatagcgtccgcaggccc– 3’ (as direction of human skp2 cdna nucleotides 15 to 40); s, 5’-cccggacgcctgcgatacgtgtcct-3’ (s for as). the oligonucleotides were delivered into hi cell line directly according to the manufacturer’s instructions (biognostik’s antisense, germany). the amount of replication was performed in quartet. moreover, hi cell line was seeded on 100 mm dish (falcon, becton dickinson labware, lincoln park, nj, usa) at 2x105 cells/well in dmem containing 10% fcs. after 72 hours, the sticked and floating cells were collected in conical tube (falcon). then, the cells were incubated with 5 ml fitc and propidium iodine (pi) in 500 ml binding buffer. flow cytometry was analysed by a digital flow cytometry system epics (coulter, miami, fl, usa). next, caspase-3 activities were measured using the colorimetric assay kit. this test is based on the addition of a caspase-specific peptide conjugated to a color reporter molecule p-nitroanilide (p-na). the cleavage of the peptide by caspase releases the chromophore p-na, which is quantitated spectrophotometrically at 405 nm. briefly, equal amounts of cell extracts prepared from hi cell line treated with as or s were incubated with the substrate (dved-pna) in the assay buffer for 2 hours at 37° c. absorbance was measured at 405 nm using a microplate reader (bio-rad laboratories, hercules, ca, usa). each determination was conducted in triplicate. furthermore, hi cell line was seeded on 96-well plates (falcon, nj, usa) at 5 x 103 cells per well in dmem containing 10% fcs, the day before treatment. cell line was treated with oligonucleotides at final concentration 100 mm. after 24 and 48 hours, the number of cells was quantitated by an assay in which mtt; 3-(4,5-dimethylthiazol-2corespondence: supriatno, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: pridentagama_oncolog@yahoo.com introduction �0 dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 18–22 yl)-2,5-diphenytetrazolium bromide (sigma-aldrich) was used. therefore, hi cell line (5 x 105 cell/kit) was seeded and placed in the upper compartment (each well was 50 ml) and was allowed to migrate through the pores of the membrane into the lower compartment, in which chemotactic agents (medium + p45skp2 as or s) are present. after an appropriate incubation time (24 hours), the membrane between the two compartments is fixed and stained, and the number of cells that have migrated to the lower side of the membrane is determined by light microscope with 40x magnitude. statistical differences between the mean for the different groups were evaluated with stat view 4.5 (abacus concepts, berkeley, ca) using post-hoc t-test. the significance level was set at 5% for each analysis. results to determine whether down regulation of p45skp2 protein can induce apoptosis, flow cytometry analysis was performed on each transfectant. flow cytometry analysis demonstrated that high percentage of early apoptosis was detected in hi-skp2 as (33.5%) compared with that of hi-skp2 s (18.8%). however, slightly high percentage of late apoptosis was detected in hi-skp2 as (17.6%) (figure 1). it means hi-skp2 as induced apoptosis in both early and late level of apoptosis. the activity of caspase-3 in hi cells treated with oligonucleotide (as and s) for 48 hours were examined. as seen in figure 2, hi-skp2 as increased caspase-3 proteolytic activity (> 1.75 fold) compared with that of hi-skp2 s (p<0.01). it means skp2 as was significantly induced the proteolitic activity or apoptosis of hi transfectant cell in external pathway at least 1.75 times compared that of skp2 s. relative cell number was evaluated by comparing the absorbance in each cell using mtt assay at 24 and 48 hours. relative number of cell hi-skp2 as decreased significantly when compared to cell treated by skp2 s (p<0.05) (figure 3). it suggests that skp2 as was markedly suppressed the hi cell growth effectively compared that of skp2 s. cell invasion was examined by boyden chamber kit for 24 hours incubation. as seen in figure 4, hi cell transfected by skp2-as was markedly decreased the cell invasion compared with that of hi-skp2 s (p<0.05). it means skp2-as has strong enough inhibiting the hi cell invasion compared with that of skp2-s. 18.8% 10.9% 33.5% 17.6% hi-skp2 s hi-skp2 as figure 1. p e r c e n t a g e s o f a p o p t o t i c c e l l s i n h i s k p 2 oligonucleotide-treated cell. 0 0.5 1 1.5 2 2.5 skp2 s skp2 as hi cells ** fold increase in caspase-3 activity figure 2. the proteolytic activity of caspase-3 in hi transfectant cells. 0 0.3 0.6 0.9 1.2 24 hours 48 hours skp2 sc skp2 as * ** hi cell relative cell number (od 540) figure 3. growth of hi cell transfectants in vitro at 24 and 48 hours. 0 20 40 60 80 100 120 skp2 s skp2 as hi cell * number of cell invasion figure 4. suppression of hi-skp2 cells invasion at 24 hours incubation. ��supriatno, et al.,: antitumor activity of antisense oligonucleotide p45skp2 discussion the strategy of therapy with skp2 as in human head and neck cancer includes human soft palate cancer becomes the focus of attention in this decade. antisense oligonucleotides p45skp2 contained phosphorothioate backbone was used to knock down protein expression by inhibiting the translation of the mrna of a desired target gene.12 antisense oligonucleotides are synthesized in the hope that they can be used as therapeutic agents-blocking disease processes by suppressing the synthesis of a particular protein. this would be achieved by the binding of the antisense oligonucleotide to the mrna from which that protein is normally synthesized. binding of both may physically block the ability of ribosomes to move along the mrna or simply hasten the rate at which the mrna is degraded within the cytosol.14 in order to be useful in human therapy, antisense oligonucleotides must be able to enter the target cells; avoid digestion by nucleases, and not cause dangerous sideeffects. to achieve these goals, antisense oligonucleotides are generally chemically modified to resist digestion by nucleases, attached to a targeting device such as the ligand for the type of receptors found on desired target cells, antibodies directed against molecules on the surface of the desired target cells. several commercial factories are presently examining antisense oligonucleotides as weapons against: hiv/aids, human cytomegalovirus (hcmv), asthma, several cancers include chronic myelogenous leukemia, and inflammation caused by cell-mediated immune reactions.15 in the present study, an antisense strategy to investigate the effect of skp2 on growth of human soft palate cancer that was overexpressing this gene was employed. transfection of an antisense oligonucleotide skp2 into hi cell induced a decrease in growth inhibition followed by invasion cell suppression. these circumstances, together with observations of cell death in the skp2-antisense treated cells, prompted us to investigate possible involvement of apoptotic mechanism in the inhibition of cell growth following antisense treatment. an increase in early and late apoptosis percentage and activation of caspase-3 in as-treated cells strongly suggested that apoptosis had occurred in those cultures. activation of caspase-3 (an executioner caspase in the apoptosis pathway) leads to the cleavage of poly (adp-ribose) polymerase (parp) and dna fragmentation, indicating that caspase-3 targets cellular proteins for proteolytic cleavage that results in cell death. caspase-3 can be activated by either an extrinsic apoptosis pathway, by the activation of caspase-8, or an intrinsic apoptosis pathway (via release of cytochrome-c from mitochondria). activated caspase-8 can directly cleave and activate the executioner caspases, such as caspase-3. alternatively, it can cleave one of the bcl-2 family members (such as bid) to induce the release of mitochondrial cytochrome c, which also leads to activation of caspase-3 via formation of apoptosome (consisting of apaf-1 and caspase-9).16 in the current study, increased caspase-3 activation in as-treated cells revealed that apoptosis ensured extrinsic pathway. in fact, apoptosis was originally described as a mechanism of controlled or physiological cell death. it is associated with the regulation of cellular homeostasis in organs and the elimination of damaged cells or cells with deleterious reactivities from the host. apoptosis is very common in organs with high proliferation activity and in tissue with intense hematopoietic activity. additionally, apoptosis has been implicated in the progression of a number of pathological conditions, including cancer and autoimmune diseases.17 as expected from its stronger growth inhibition and apoptosis induction, a significant suppression of tumor growth was detected in hi-skp2 as when compared with that of hi-skp2 s in vivo animal tumor model. several investigators have already shown a relationship between expression of skp2 and apoptosis. s-phase induction by adenovirus-vector mediated expression of skp2 in quiescent cells was followed by apoptosis.6 mouse embryonic fibroblast (mef) in skp2-deficient mice showed an increased tendency toward spontaneous apoptosis.11 antisense skp2 and jab1 induce apoptosis in human oral tongue cancer cells through induction of p27kip1 protein.13 since components of apoptotic programs represent promising targets for anticancer therapy, down-regulation of skp2 by the antisense oligonucleotide approach could be a useful apoptosis-modulating strategy for treatment of human head and neck cancers including human soft palate cancers. in conclusion, antisense oligonucleotide p45skp2 has a high antitumor activity in human soft palate cancer, targeting this molecule could represent a promising new therapeutics approach for this type of cancer. acknowledgments the author heart fully thanks and appreciates prof. mitsunobu sato dds., ph.d; dr. koji harada, dds., ph.d; dr. hamakawa dds., ph.d and teams, department of oral maxillofacial surgery and oncology, school of dentistry, tokushima university, japan, for providing some materials and discussion. references 1. cohan dm, popat s, kaplan se, rigual n, loree t, hicks wl jr. oropharyngeal cancer: current understanding and management. curr opin otolaryngol head neck surg 2009; 17(2): 88–94. 2. ammar a, uchida d, begum nm, tomizuka y, iga h, yoshida h, sato m. the clinicopathological significance of the expression of cxcr4 protein in oral squamous cell carcinoma. int j oncol 2004; 25(1): 65–71. 3. inagi k, takahashi h, okamoto m, nakayama m, makoshi t, nagai h. treatment effects in patients with squamous cell carcinoma of the oral cavity. acta otolaryngol suppl 2002; 547: 25–9. 4. bray f, sankila r, ferlay j, parkin dm. estimates of cancer incidence and mortality in europe in 1995. europ j cancer 2002; 38(1): 99–166. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 18–22 5. wu l, grigoryan av, li y, hao b, pagano m, cardozo tj. specific small molecule inhibitors of skp2-mediated p27 degradation. chemi biol 2012; 19(12): 1515–24. 6. zheng wq, zheng jm, ma r, meng ff, ni cr. relationship between levels of skp2 and p27 in breast carcinomas and possible role of skp2 as a target therapy. steroids 2005; 70(11): 770–4. 7. hershko d, bornstein g, ben-izhak o, carrano a, pagano m, krausz mm, hershko a. inverse relation between levels of p27kip1 and of its ubiquitin ligase subunit skp2 in colorectal carcinoma. cancer 2001; 91(9): 1745–51. 8. chiarle r, fan y, piva r, boggino h, skolnik j, novero d, palestro g, de wolf-peeters c, chilosi m, pagano m, inghirami g. s-phase kinase-associated protein 2 expression in non-hodgkin’s lymphoma inversely correlates with p27 expression and defines cells in s phase. am j pathol 2002; 160(4): 1457–66. 9. masuda ta, inoue h, sonoda h, mine s, yoshikawa y, nakayama k, mori m. clinical and biological significance of s-phase kinaseassociated protein 2 (skp2) gene expression in gastric carcinoma: modulation of malignant phenotype by skp2 overexpression, possibly via p27 proteolysis. cancer res. 2002; 62: 3819–25. 10. yokoi s, yasui k, lizasa t, takahashi t, fujisawa t, inazawa j. down-regulation of skp2 induces apoptosis in lung-cancer cells. cancer sci 2003; 94(4): 344–9. 11. nakayama k, nagahama h, minamishima ya, matsumoto m, nakamichi i, kitagawa k. targeted disruption of skp2 results in accumulation of cyclin e and p27kip1, polyploidy and centrosome overduplication. embo j 2000; 19(9): 2069–81. 12. supriatno. oligonukleotid s-phase kinase associated protein-2 (skp2) antisense induces the growth inhibition and increases apoptosis activity on head and neck cancer cell lines. proceeding gadjah mada university of inter-cluster research. 57th dies natalis gmu, november 28, yogyakarta; 2006. p. 1–13. 13. supriatno, harada k, yoshida h, sato m. basic investigation on the development of molecular targeting therapy against cyclin-dependent kinase inhibitor p27kip1 in head and neck cancer cells. int j oncol 2005; 27(3): 627–35. 14. wacheck v, weiss ujw. antisense molecules for targeted cancer therapy. crit rev oncol hematol 2006; 59(1): 65–73. 15. pastorino f, stuart d, ponzoni m, allen tm. targeted delivery of antisense oligonucleotides in cancer. j contr rel 2001; 74(1): 69–75. 16. azuma m, harada k, supriatno, tamatani t, motegi k, ashida y, sato m. potentiation of induction of apoptosis by sequential treatment with cisplatin followed by 5-fluorouracil in human oral cancer cells. int j oncol 2004; 24(6): 1449–55. 17. qiao l, wong bcw. targeting apoptosis as an approach for gastrointestinal cancer therapy. drug resist 2009; 12(3): 55–64. �� vol. 42. no. 1 january–march 2009 toxicity testing of chitosan from tiger prawn shell waste on cell culture maretaningtias dwi ariani1, anita yuliati2, and tokok adiarto3 1 department of prosthodontic, faculty of dentistry airlangga university 2 department of dental material, faculty of dentistry airlangga university 3 department of chemistry, faculty of sience & technology airlangga university surabaya indonesia abstract background: a biomaterial used in oral cavity should not become toxic, irritant, carcinogenic, and allergenic. chitosan represents a new biomaterial in dentistry. purpose: to examine the toxicity of chitosan from tiger prawn shell waste on cell culture with mtt assay. methods: chitosan with concentration of 0.25%, 0.5%, 0.75% and 1% was used in this experiment. each sample was immersed on eppendorf microtubes containing media culture. after 24 hours, the immersion of media culture was used to examine the toxicity effects on bhk-21 cell based on mtt assay method. the density of optic formazan indicates the number of living cells. all data were then statistically analyzed by one-way anava. results: the number of living cells in chitosan from tiger prawn shell waste was 93.16%; 85.07%; 78.48%; 75.66%. thus, there was no significant difference among groups. conclusion: chitosan with 0.25%, 0.5%, 0.75% and 1% concentrations from tiger prawn shell waste were not toxic for bhk-21 cell culture when using parameter cd50. key words: toxicity, chitosan, mtt assay correspondence: maretaningtias dwi ariani, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: etaprosto@yahoo.com introduction chitosan is a distillation product of chitin. nowadays, chitosan is still considered as harmful waste for the environment. more than 109–1010 tons of chitosan are predictably produced every year. according to statistical data, countries which have shell producing industries produce this waste about 5,200 tons. however, product of chitin derivatives, chitosan oligomer, is the most expensive product in world market today.1 for instance, the use of chitosan in medical fields known as biomaterial nowadays can be used in many clinical applications. the reason is because chitosan has some special characters such as good biocompatibility, nontoxic and non bioactive, biodegradable, non allergenic and non carcinogenic.2,3 the result of medical research on chitosan, moreover, shows that its commercial products can be used as skin regeneration for burn wounds or necrotic ulcers, as wound bandage, as surgical thread, and as membrane barrier for preventing abnormal ligament formation. the combination of chitosan and other materials cannot only be used as drug delivery system, oral vaccine carrier, and scaffold for tissue engineering, but can also react with living tissues during implementation procedure.3 furthermore, in dentistry, the other use of chitosan, especially 0.5% phosphorylated chitosan, as mouthwash can significantly can reduce the early plague formation.4 the combination of chitosan and chlorhexidine in reducing the plague formation is better than chlorhexidine without chitosan.5 in endodontic field, chitosan can also be used as dressing material which has anti-inflammation effect for root path in periapical lesion since chitosan can stimulate fibroblastic cell to release chemotatic inflammatory cytokines, especially interleukin 8 (il-8).6,7 chitin and chitosan can actually be distinguished based on their nitrogen content. the polymer can be known as chitin if the nitrogen content is less than 7%, meanwhile, the polymer can be known as chitosan if the nitrogen content is more than 7%. even though both polymers are found in nature, the terminology of chitosan nowadays is research report �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 15-20 referred to chitin which acetyl structure can artificially be discharged.8 chitin in tiger prawn shells, moreover, is produced as mucopolysacarida which relates to inorganic salt, especially calcium carbonate (caco3), protein and fat including pigment. therefore, deproteination process (protein separation), demineralization process (mineral separation), and depigmentation process (fat and color essence discharge) must be involved in order to obtain or produce chitin in tiger prawn shell.10 figure 2. distillation process of chitin into chitosan.9 chitosan in some specification has actually been examined and produced by chemistry department of science and technology faculty of airlangga university. thus, the chitosan is expected to be used later as biomaterial in dentistry. in dentistry, however, if chitosan would be used orally, it must be biocompatible, which means that it could be accepted by human body, non toxic, non irritant, non carcinogenic, and also save without causing allergic reaction.11 therefore, in order to reduce the bad effects of using chitosan, toxicity testing is needed. the toxicity testing can use animal testing in vivo or cell culture in vitro (cytotoxicity). the examination in vivo can give complete description about the response of the animal testing, but it can be difficult to comprehend the biologic response of the animal testing towards the tested material since the biologic response actually involves many stimulant reactions. nevertheless, cell culture method is often used in examining biologic effects for several reasons such as: to reduce pressure from society towards the using of animal testing, to control the environment factors of cell culture (temperature, ph, and osmotic pressure), to obtain the result faster, and to expose the cell culture directly with the tested materials. cell culture, moreover, is also very sensitive with toxic materials. thus, not only can toxicity be measured quantitatively, but its response towards living cell can also be examined. cell culture used in this experiment is bhk-21 cell from fibroblast of baby hamster’s kidney. fibroblast is often used by the researchers for toxicity testing of dentistry materials since it is the most important cell in the components of pulp, ligament periodontal and gingiva.12 this toxicity testing, furthermore, is done in some ways which are by measuring the number of cells and their development after being exposed with the tested materials, by examining cell status after the changing of membrane permeability, and by measuring toxic response based on enzymatic activities. nevertheless, toxicity testing based on enzymatic cell activities is often done since this method can monitor specific function of cell metabolism, it needs only short duration (4 hours), gives quantitative results, has high sensitivity towards toxic materials, and has potentials for standardization of testing method.12 one of methods used for toxicity testing by monitoring enzyme activities is mtt assay. mtt is a yellow soluble molecule, which can be used to analyze cellular enzymatic activities. if the cell can reduce mtt, formazan produced will be blue-purple, insoluble, and precipitated in cell. the amount of formazan formed is proportional to enzymatic activities. this testing, furthermore, is measuring cellular dehydrogenizing activities, and changing the chemical material, mtt, through the number of cellular reductive materials into blue and insoluble formazan compound. mtt assay actually is based on the capability of living cells in reducing mtt salt. the principals of this assay are to break tetrazolium mtt ring (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) by the existence of dehydrogenase in active mitochondria, and then to produce insoluble blue-purple formazan product. the mechanism is that the yellow tetrazolium salt will be reduced in cell which has metabolic activities. mitochondria of living cell has important role in producing dehydrogenase. if the dehidrogenase is not active because of sitotoxic effects, formazan will not be produced. formazan production can be measured by diluting it and measuring the optic density of the solution produced. there are actually many protocols in using mtt assay, but the concentration of mtt used must be the same as to dilute 5mg/ml yellow mtt powder in pbs. the reaction of blue-purple color is used as the measurement of the number of living cells. the number of living cells can be measured as the product result of mtt by using spectrophotometer with 570–690 nm wave length.13 the objective of this experiment, finally, is to analyze the concentration of chitosan from tiger prawn shell waste which is produced by chemistry department, faculty of science and technology, airlangga university and does not figure 1. chemical structure of chitin, chitosan, and cellulose.9 ��ariani, et al.: toxicity testing of chitosan have toxic effects on bhk-21 cell culture testing. the result of this experiment is then expected to give information about the concentration of chitosan which does not give toxic effects on bhk-21 cell culture testing so that later it cannot only become references for next experiments on animal and patch test for humans, but can also add kinds of data specification about quality control of chitosan isolated from tiger prawn shell waste produced. materials and methods this laboratory experimental research designs post test-only control group experiment. the subject of this experiment is chitosan isolated from tiger prawn shell waste. the location of this experiment is at pusat veterinaria farma (pusvetma–centre of pharmaceutical veterinary) surabaya and chemistry laboratory of chemistry department of science and technology faculty of airlangga university. the materials used in this experiment were chitosan isolated from tiger prawn shell waste, composing of 100 g tiger prawn shells, hcl 1n, 3.5% naoh, 50% naoh, acetone, 1% acetate acid, sterile aquadest, bhk-21 cell, culture media containing eagle’s minimum essential medium (mem), rpmi-1640, 1% penstrep, 10% foetal bovine serum (fbs), 100 unit/ml fungizone, mtt reactor (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) lot. 042k5313 (sigma-usa), phosphate buffer saline (pbs), and dimethylsulfoxide analar (dmso) lot 208ki6687088 (bdh-england). the tools used in this experiment, moreover, were 0.20 mm filter millipore minisart lot. 16534040736 (sartorius), flask (nunc), microplate 96 well nunc batch. 04644 (nunclondenmark), pipette pasteur, shaker vari shaker (dynatech), autoclave (foundry), 5 cc syringe injection (terumo), 1 cc syringe tuberculin (terumo), eppendorf micropipette (titertek, england), incubator memmert w. germany 37° c 5% co2, laminar flow oliphant australia, and 630 nm elisa reader opsysmr denmark. in addition, there were some steps in preparing sample of tiger prawn shell (penaeus monnodon). first, the shells of tiger prawn were washed cleanly and boiled in boiling water (± 100° c) for 15 minutes. second, the shells were sun dried, then dissolved and filtered with 50 mesh size. third, the phase of chitin extraction from the tiger prawn shells consisted of some processes, deproteination, demineralization, depigmentation, and distillation. in deproteination process the filtered powder of the tiger prawn shells was put into a beker glass, and then added with 3.5% naoh solution with the ratio 1:10 (b/v). this process consisted of stirring process for 2 hours with temperature at 65° c. afterwards, the solution was filtered by using filtering paper to obtain its residue. the residue is then washed by aquadest until the water ph became neutral, and dried in oven with temperature at 80° c until it was dried. the result of this process was known as crude chitin. in demineralization process: crude chitin was put into beker glass, and then added with hcl 1n solution with the ratio 1: 15 (b/v). this process covered stirring process for 30 minutes at ambient temperature. afterwards, the solution was filtered by using filtering paper to obtain its residue. the residue was then washed by aquadest until the water ph became neutral, and dried in oven with temperature at 80o c until it was dried. the result of this process was known as chitin powder. in depigmentation process: the chitin powder was immersed in acetone for about 20 hours. the immersion result was then washed cleanly by aquadest and filtered with using filtering paper. after being dried in oven with temperature at 80° c, white chitin powder (cleaner) was obtained. in distillation process: the chitin powder was immersed in 50% naoh solution with the ratio 1:10 (b/v) for 6 hours with temperature at 90° c. the result was then washed with aquadest and filtered with using filtering paper. after being dried in oven with temperature at 80° c, chitosan was obtained.9 the sample of this research was made of one gram chitosan powder diluted in 100 ml 1% acetate acid so that it became chitosan gel. based on the standard procedure, 1% chitosan liquid was made of 1 gram chitosan gel mixed with 100 ml sterile aquadest (% b/v = 1 g/100 ml). thus, chitosan solution with 0.25%; 0.5%; 0.75% and 1% concentration was made based on the procedure explained before. the sample was then sterilized in autoclave for 15 minutes. the sample was then ready to be tested with cell culture. bhk-21 cell culture in cell-line form was cultivated inside a bottle. after confluent, the cell culture was harvested by using trypsine versene solution. the harvesting result was taken out little bit and then cultivated again into rosewell park memorial institute (rpmi-1640) media containing with 10% bovine serum albumin incubated for 24 hours with temperature at 37° c. those cells, afterwards, were put into small bottles with 2 × 105 cell/ml density as the testing samples. this toxicity testing used 96 well plates of cell culture with flat base (figure 3). the testing was done based on standard protocol required for mtt assay. each plate containing cells with 2 × 105 densities into 100 µl culture media. before being tested, those samples had to be sterilized by ultra violet rays for 15 minutes. the samples were then put into well plates about 50 µl. control cell and control media had to be prepared also. the control cell was that each well plate had to be contained only with cells and culture media. in this experiment, the testing was done in duplo. those well plates were then incubated for 20 hours with temperature at 37° c. after that, each plate was contained with 5 mg/ml mtt which had been diluted in 25 ml pbs, and those well plates were then incubated for 4 hours with temperature at 37° c. the next step, samples were taken from each well plate which was added with 50 ml dmso and piped up and down in order to dilute crystals formed. those well plates were then incubated for 5 minutes with temperature at 37° c. those well plates, afterwards, was monitored by elisa reader with 630 nm wave length.12 the result was �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 15-20 then shown in optical density (absorbents). the amount of absorbents in each well plate showed the number of viability cell in media culture. in order to analyze the percentage of living cell, the following formulation could be used: treatment + media % living cell = × 100% cell + media the measuring result was then tabulated based on each groups, and tested by one-way anova with 5% level of significance. if there was significant difference, lsd test would be done. result the research result of toxicity testing on chitosan from tiger prawn shell waste on cell culture, in which chitosan toxicity parameter in bhk-21 cell culture monitored based on the number of living cells/cell viability shown as optical density (absorbent), can be shown in table 1. based on table 1, the formazan optic density values are decreasing as the concentration is increasing. kolmogorofsmirnof test shows that all groups have probability values more than 0.05 (p > 0.05). it means that all groups have normal distribution. in homogeneity test, however, the figure 3. toxicity testing on 96 well plates. note: concentration of chitosan a: 1%, b: 0.75%, c: 0.5%, d: 0.25%, e: control cell, f: control media. a b c d e f table 1. average values, standart deviation, and percentage of chitosan (od) concentration optic density values pn n x sd % control cell 0.56650 0.17254 100.00 0.554 4 0.25% 0.53875 0.06698 93.16 0.447 4 0.50% 0.52625 0.06009 85.07 0.437 4 0.75% 0.42600 0.14615 78.48 0.609 4 1% 0.39725 0.11723 75.66 0.416 4 note: x: average of formazan optic density values; sd: standart deviation; %: percentage average of formazan optic density; pn: normal probability; n: number of samples. table 2. the result of one-way anova test of formazan optic density values on 0.25%, 0.5%, 0.75%, 1% chitosan and control cell variation source quadrate total db quadrate average f p among treatments 0.089 4 0.022 1.524 0.246 under treatment 0.219 15 0.015 total 0.308 19 note: db: free degree; f: f count; p: probability ��ariani, et al.: toxicity testing of chitosan value obtained is about 0.233 (p > 0.05) which means that all groups are homogeny. thus, one way anava test is needed to be done with the result as in table 2. the result of one-way anova test shows that there is no significant difference of optic density values of each chitosan concentrations with probability value = 0.246 (p > 0.05). in other words, chitosan with concentration from 0.25% to 1% can not influence formazan optic density values. discussion in recent years, biomaterial has been known to have many special characteristics that can be used for many clinic applications. chitosan is one of biomaterials which is continuously improved for many clinical applications. chitosan with chemical formulation of 2-amino-2-deoxyd-glucan, produced by distillation process of chitin, is chitin derivative polysaccharide which can be used widely for biomedical applications.15 the use of chitosan is generally including biochemistry, enzymology, microbiology, drugs/pharmacy, food and nutrition, agriculture, waste management, paper industry, textile, membrane /film, cosmetics, etc. in the last decade, chitosan, moreover, can be used for many clinical applications.8 chitin and chitosan can give effects on fibroblast cell proliferation of human skin and keratinocyte in vitro. chitosan chloride cl 313a has stimulant effects on fibroblast cell proliferation depending on high distillation degree. the combination of chitosan and arginine can be used as biomaterial for anticoagulant.10 in this research bhk-21 cell culture is used for toxicity testing of chitosan which is isolated from tiger prawn shell waste. the chitosan with the concentration of 0.25%; 0.5%; 0.75% and 1% is used based on the previous research that minimum inhibitory concentration (mic) of chitosan in s. mutans is about 1%. based on this research, the estimated density values of formazan optic is decreasing as the concentration is increasing from 0.25% to 1%. the percentage of living cell of all groups is up to 50% if using parameter cd50. statistic measurement using one-way anova with level of significance 5% shows that the increasing of chitosan concentration from 0.25% to 1% can not influence optic density values of formazan. it means that there is no toxicity on bhk-21 cell culture since chitosan does not have function cluster and structure which can make chitosan becomes toxic. a material usually can produce toxic especially because of its toxic cluster and structure. the process of chitosan production, moreover, has passed deproteination phase which is a phase of separating or breaking bond between protein and chitin so that chitosan will not become toxic. since chitosan is not toxic, the combination of chitosan and other materials can be used as drug delivery system and oral vaccine carrier. chitosan, furthermore, cannot only be used as scaffold for tissue engineering, but can also interact with living tissue in implantation procedures.3 some researches show that chitosan can be used as substitute materials for bone. the combination of phosphorchitosan and calcium phosphate cements, for example, can produce compression power and modulus young which is enough for bone cement material. the combination of chitosan-citrate acid and calcium phosphate cements, moreover, can become bone cement materials with good concentration.16 the research on the difference of chitosan concentration on cell culture with mtt assay is done in order to analyze the percentage of living cell with different concentration of chitosan. mtt assay is done based on the ability of living cell in reducing mtt salt. the principal of this assay is to break tetrazolium mtt ring, (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide), which has yellow color because of dehydrogenesis in active mitochondria, and to produce insoluble blue-purple formazan product. the mechanism of the yellow tetrazolium salt will be reduced in cell which has metabolic activities. in this mechanism, mitochondria of living cell have an important role in producing dehydrogenises. if the dehydrogenises are not active because of cytotoxic effects, the formazan is not produced. the formazan production can be measured by dissolving it and measuring optic density of the solution produced. the lower the percentage of optic density is, the fewer the number of active metabolic cells that can reduce mtt. the number of living cell detected by spectrophotometer or elisa reader is the result of mtt product. the purpler the color is, the higher the absorbent values are and the more the number of living cells.14 spectrophotometer is used in this toxicity testing since it can make the testing not only faster and easier than plat measuring method, but also more sensitive than visual reader method. measuring with spectrophotometer is the best method which can give the best result since chitosan is highly viscous so that the visual monitoring can hardly be done on generating result of bhk-21 cell. based on the laboratory experimental research on toxicity testing of chitosan from tiger prawn shell waste on bhk-21 cell culture with mtt assay, it can be concluded that chitosan with concentration 0.25% to 1 % in tiger prawn shell waste does not have toxic effect on bhk-21 cell culture since chitosan does not have any structure which can cause toxic reaction. based on this research result, finally, the next phases of biocompability test (secondary test and tertiary test) are needed to be done. references 1. meidina, sugiyono, jenie sl, suhartono. aktivitas antibakteri oligomer kitosan yang diproduksi menggunakan kitonase dari isolat b.lincheniformis mb-2. institut pertanian bogor. 2006. p. 288–93.institut pertanian bogor. 2006. p. 288–93. 2. zhu ap, zhang z, shen j.preparation and characterization of novrl silica butyrylchitosan hybrid biomateial. j mater sci mater med 2003;j mater sci mater med 2003; 14:27–31. 3. howling gi, dettmar pw, goddard pa, hampson fc, dornish m, wood ej. the effect of chitin and chitosan on proliferation of human skin fibroblast and keratinocytes invitro biomaterials 2001; 22:2959–66. �0 dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 15-20 4. sano h, shibasaki k, matsukubo t, takaesu y. effect of rinsing with phosphorilated chitosan on four day plaque regrowth. bull tokyo dent coll 2001; 42(4):251–6. 5. decker em, weiger r, wiech i, heide pe, brecx m. a synergistic chlorhexidine/chitosan combination for improved antiplaque strategies. j periodontal res 2005; 40(5):373–7. 6. mori t, okumura m, matsumura m, ueno k, tokuro s, okamoto y, minami s, fujinaga t. effect of chitin and its derivates on the proliferation and cytokine production of fibroblast in vitro. biomaterials 1997; 18:947–51. 7. ikeda t, yanagiguchi k, viloria ii, hayashi y. relationship between lysozyme activity and clinical symptoms following the application of chitin/chitosan in endodontic treatment. in: muzarelli raa, editor. chitosan per os : from dietary supplement to drug carrier. crottammare : atec edizioni; 2000. p. 275–92. 8. suhardi. khitin dan kitosan. yogyakarta: universitas gajah mada; 1993. p. 11–20, 120–40. 9. patel ss. pharmaceutical significance of chitosan: a review. 2006; 4(6). available at: http:// www.pharmainfo.net/reviews/ pharmaceutical-significance-chitosan-review. accessed decemberaccessed december 18, 2006. 10. muzzarelli raa, mattioli-belmonte m, pugnaloni a, biagini g. biochemistry, histology and clinical uses of chitins and chitosans in wound healing. in: jolles p, muzzarelli raa, editors. chitin and chitinases. based : birkhauser verlag; 1999. p. 285–93. 11. anussavice kj. phillips science of dental material. 11st ed. usa: elsevier science, saunders; 2003. p. 172–94. 12. fazwishni s, hadijono bs. uji sitotoksisitas dengan esei mtt. jkgui 2000; 7:28–32.2000; 7:28–32.; 7:28–32. 13. zubaidah n. the citotoxicity of calcium hydroxide intracanal dressing by mtt assay. dental journal 2007; 40(4):157–58. 14. telli c, serper a, dogan al, guc d. evaluation of the cytotoxicity of calcium phosphate root canal sealers by mtt assay. j endodon 1999; 25:811–3. 15. irawan b. chitosan dan aplikasi klinisnya sebagai biomaterial. indonesian journal of dentistry 2005; 12(3):146–51. 16. yokoyama a, yamamoto s, kawasaki t, kohgo t, nakasu m. development of calcium phosphate cement using chitosan and citric acid for bone substitute materials. biomaterials 2002; 23(4): 1091–101. 184 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 184–188 original article the effect of nanoparticle tooth grafts on osteoblast stimulation in the first stages of the bone healing process in wistar rats compared to the micro-tooth graft technique ega lucida chandra kumala,1 malianawati fauzia,1 hana salsabila junivianti2 1department of periodontics, faculty of dentistry, university of brawijaya, malang, indonesia 2bachelor of dentistry study program, faculty of dentistry, university of brawijaya, malang, indonesia abstract background: the use of a bone graft in bone regeneration is challenging. tooth graft material has been used as a bone graft alternative due to its similar composition of organic and inorganic materials close to the bone. recently, nanotechnology has been used to improve bone graft quality. the osteoconduction rate in the defect area represents the bone graft quality. purpose: this study aimed to compare the number of osteoblasts using nano-tooth grafts and micro-tooth grafts in wistar rats. methods: wistar rats were divided into six groups: the negative control groups (examined on days 7 and 14), the micro-tooth graft groups (examined on days 7 and 14), and the nano-tooth graft groups (examined on days 7 and 14). the control group received nothing, the micro-tooth group received a micro-size tooth graft, and the nano-tooth graft group received a nano-size tooth graft on the injured femur. histological observations of osteoblasts were carried out using a light microscope with 1000x magnification. data were analyzed using one-way analysis of variance and least significant difference tests. results: on day 7, the nano-tooth graft group showed a higher osteoblast number (11.75) than the micro-tooth graft group (7.5) (p = 0.039). there was no significant difference in the micro-tooth graft group compared to the control (p > 0.05). on day 14, the nano-tooth graft group showed a decrease in osteoblast number close to normal (control) (p > 0.05), while the micro-tooth graft group still experienced significant elevation. conclusion: nano-tooth grafts accelerate the stimulation of osteoblasts in the first stages of the healing process compared to micro-tooth grafts. keywords: micro hydroxyapatite; nanohydroxyapatite; osteoblast; tooth graft article history: received 19 september 2022; revised 23 november 2022; accepted 12 december 2022; published 1 september 2023 correspondence: ega lucida chandra kumala, department of periodontics, faculty of dentistry, university of brawijaya. jl. veteran, malang, 65145 indonesia. email: egalucidachk@gmail.com introduction the maxillofacial bone defect is common and can be caused by trauma, neoplasms, infections, and congenital abnormalities. reconstruction of bone defects is still challenging, as the healing process is often impaired or even fails. bone graft is often used in bone reconstruction, as it is expected to help accelerate the healing process.1 bone grafting is a surgical procedure to replace missing or damaged bone using materials from the patient’s body: artificial, synthetic, or natural.2 a bone graft can be taken from a donor and substituted into the damaged bone tissue. bone grafts should have three basic functions: osteogenesis, osteoinduction, and osteoconduction.1 osteogenesis is the graft’s ability to produce new bone.2 osteoinduction is the differentiation induction of polyvalent stem cells from the surrounding tissue to an osteoblastic phenotype to form bone. osteoconduction is the ability to carry out and promote bone growth by penetration of capillaries and bone-forming cells into the graft material.3 bone grafts should be biocompatible, have good mechanical properties, and be easy to manipulate. there are four kinds of bone grafts: autograft, xenograft, allograft, and alloplastic or alloimplant synthetic material. autografts, using bone graft from the patient’s body, are still the primary option for bone defect reconstruction. allografts use tissue from other donors within the same species, while xenografts use tissue from other species. materials of tissue rehabilitation should have the same characteristics as natural bone.1 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p184–188 mailto:egalucidachk@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p184-188 185kumala et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 184–188 teeth are composed of both organic and inorganic mineral components which include calcium phosphate, collagen, and other organic elements. several teeth components, such as hydroxyapatite (ha) and betatricalcium phosphate, are known to be osteoconductive and biocompatible to bone.4 kaur5 found recycled dentin roots should be used in bone grafts since they induce new bone formation in the periodontium. dental cementum and its progenitor cells also have several growth factors, including transforming growth factor (tgf)-beta and insulin-like growth factor (igf)-i.6 among them, ha is one of the ceramics with good biocompatibility properties, as its mineral content is chemically and physically the same as human bones and teeth.1 in recent years, nano-sized ha, with a particle size of 100 nm in at least one direction, is believed to have high osteoconduction and possibly can be used in bone grafting.7 research has shown that micro-sized ha grafts have lower mechanical strength, so they cannot be used in areas that bear high loads. nano-ha has biocompatibility, superior bioactivity, and osteointegration ability and causes a lower inflammatory reaction compared to micro-ha.8,9 osteogenesis involves osteoblasts, osteoclasts, and osteocytes.10 osteoblasts contribute to bone matrix mineralization through the secretion of type i collagen and release calcium, magnesium, and phosphate ions. in both the embryonic and fetal stages, osteoprogenitor cells are active precursors to osteoblasts. however, when mature, these cells are resting and require stimulation to proliferate and differentiate into osteoblasts. the osteoconductive properties of ha promote the proliferation and differentiation of osteoblasts and accelerate vascularization.11 ha particle size is considered to affect the substance absorption in the bone graft. therefore, this study aims to determine the number of osteoblasts at the bone healing stage after implantation of nanoand micro-sized tooth graft materials in the femurs of wistar rats. materials and methods this research used 30 wistar rats (rattus norvegicus). the inclusion criteria were that they were male, three months old, and had a mass of 250–300 g. the rats were then divided into six groups. they were kept in the cage for seven days to adapt and fed with 50 g pellet each day. these study methods were approved by the brawijaya university research ethics commission (certificate number 008-kepub-2021). tooth grafts were made by selecting post-extraction human teeth that had no filling. these were then washed and disinfected using 70% ethyl alcohol. any soft tissue that still adhered was removed.12 the teeth were then stored and sent to the national nuclear energy agency of indonesia (badan tenaga nuklir nasional) for powder production by ball-milling with a ratio of 1:5 (ball:powder) at 250 rpm for five hours. next, the powder was filtered and dried (100°c, 24 hours), and then sieved until the microparticles formed. to convert the particles into nanoparticles, the sample was prepared, then micro-sized tooth graft powder was dried for 1.5 hours at 60°c. wet milling was carried out with a ball-to-powder ratio parameter of 1:5 for 5 hours. after that, the suspension was filtered using filter paper and redried at 60°c for 1.5 hours. the powder was then ground with a mortar and sieved. x-ray diffraction was used to determine the chemical composition, phase identification, and crystallization size. the rats were divided into six groups: the day 7 groups (control, nano-tooth graft, and micro-tooth graft) and the day 14 groups (control, micro-tooth graft, and nano-tooth graft). the bone defect in rats was made by applying 2.5 ml of ketamine and then antiseptic on the surgical area using 10% povidone-iodine. an incision was made horizontally on the femur of the wistar rats. the full-thickness mucoperiosteal flap was opened with a rasparatorium. a circular defect was made in the femur with a diameter of 2 mm using a round bur on the right lateral femoral condyle and irrigated with 0.9% nacl. on days 7 and 14, the defects in the control group were allowed to fill with blood; in the micro-tooth graft group, they were filled with tooth grafts measuring 250–710 µm, and in the nano-tooth graft groups, they were filled with tooth grafts with an average size of 484 nm. the tooth graft was applied while the rats were still under anesthesia. after being treated, the flap defect was closed and sutured using a simple interrupted technique. the rats were injected with 0.2 ml of amoxicillin and 0.2 ml of novalgin for three days. on days 7 and 14, after the administration of the tooth grafts, all rats were injected with ketamine and decapitated. the femur of the rat was removed and immersed in 10% formalin. the femur was then prepared for histological analysis of the osteoblast count using hematoxylin-eosin staining. observations were carried out using a light microscope with 1000x magnification. data were analyzed using one-way analysis of variance (anova) and least significant difference (lsd) tests using the spss version 19 software (ibm statistic, usa). statistical significance was determined when p < 0.05. results in this study, we counted the number of osteoblasts in the bone healing process in the femur of wistar rats. the nanotooth group showed the highest osteoblast mean number (11.75) on day 7 compared to the micro-tooth (7.5) and control (7.0) groups. meanwhile, on day 14, the micro-tooth graft group showed a higher osteoblast number (11.75). on the other hand, the nano and control groups showed a lower osteoblast number (figures 1 and 2). the one-way anova test showed there were significant differences in the osteoblast counts on both the day 7 and day 14 observations (p < 0.05). the lsd test showed that on day 7, there was a significant difference in copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p184–188 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p184-188 186 kumala et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 184–188 table 1. the post hoc least significant difference test between groups observation time between groups p-value day 7 control micro-tooth graft group >0.05 control nano-tooth graft group >0.05 micro-tooth graft group nano-tooth graft group 0.039 day 14 control micro-tooth graft group 0.028 control nano-tooth graft group >0.05 micro-tooth graft group nano-tooth graft group >0.05 (a) (b) (c) (d) (e) (f) figure 1. osteoblasts (arrows) with 1000x magnification in the wound area of femurs of wistar rats with hematoxylin-eosin staining: (a) control group day 7, (b) micro-tooth graft group day 7, (c) nano-tooth graft group day 7, (d) control group day 14, (e) micro-tooth graft group day 14, (f) nano-tooth graft group day 14. 0 7 7.75 0 7.5 11.75 0 11.75 9.25 0 2 4 6 8 10 12 14 0 7 14 a ve ra ge n um be r of o st eo bl as ts days control micro nano figure 2. the average number of osteoblasts. the number of osteoblasts between the control group and the nano-tooth graft group (p = 0.024). on day 7, the microtooth graft group significantly differed from the nano-tooth graft group (p = 0.039). the control group had no significant difference from the micro-tooth graft group (p > 0.05). on day 14, the number of osteoblasts in the micro-tooth graft group was significantly higher than that in the control group (p = 0.028), while the control group and nano-tooth graft group had no significant difference (table 1). discussion this study showed that the osteoblast count on the defective femur receiving the nano-tooth graft was higher than in the control and micro-tooth graft groups. this study supports the previous study that revealed that nano-ha could increase proliferation, differentiation, adhesion strength, osteoblast scattering, bone morphogenic protein (bmp) expression, and bone re-conduction properties.13 therefore, copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p184–188 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p184-188 187kumala et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 184–188 using nanotechnology in tooth graft materials can improve the osteoconductive properties of bone grafts. the dimensions and shape of the pores are critical in the process of osteointegration. nano-ha interconnected between pores with a rough surface will facilitate osteoblast cells’ penetration and attachment. the surface of bone mineral crystals with nano-sized particles is large, and absorption by osteoclasts can occur evenly because these crystals present in the organic matrix have very loose connections to each other.3 in vitro and in vivo studies of calcium ion release from ha powder using nanoparticles have shown similar results to bone apatite and are much faster than ha powder with microparticle size. the size reduction of enamel and dentin to the nanoparticle in nanotechnology applications can produce the crystalline degree and properties of the ha content close to natural sources of ha in bone. compared to nano-ha, microcrystalline ha has a low level of solubility, so it is not easily absorbed. based on a study conducted by pascawinata et al.14, when implanted in bone defects, this ha can be well covered and even partially replaced by new bone. however, the resorption process takes a long time and is still seen in the experimental animal after nine months. nanocrystalline ha (10–100 nm) resembles the natural form of ha found in bone more than microcrystalline ha. moreover, it has tighter contact with the surrounding tissue, and many molecules on its surface are more quickly absorbed. the ideal implant material should have a resorption capacity equal to the time of new bone formation. osteoblasts and osteoclasts that adhere more to the nanocrystalline ha accelerate the healing process of bone injuries.14 as well as the particle size, tooth graft powder also contains some beneficial components for cell proliferation. ninety percent of the organic components of teeth are type i collagen and contain growth factors such as igf-ii, bmp2, and bone-like tgf-beta. bmp2 is present in the enamel and is potentially osteoinductive.14 cementum and dentin have other growth factors, including plateletderived growth factor, igf, and fibroblast growth factor. according to research, autogenous demineralized dentin matrix is chemotactic for osteoprogenitor cells and osteoblasts, encouraging the faster process of bone repair in bone defects.15 in this study, the number of osteoblasts in the nanotooth graft group increased on day 7 compared to day 14, while the micro-tooth graft group found that the number of osteoblasts on day 7 was lower than in the nano-tooth graft. this shows that there is a faster maturation process of osteoblast precursors in nanocompared to microtooth grafts. research conducted by li et al.16 showed that nanoparticles can accelerate surrounding stem cells to differentiate into osteoblasts. research conducted by mahmoud et al.17 also proves that surface modification of titanium using nanotubes can increase and accelerate the differentiation of osteoblast cells. furthermore, research conducted by wu et al.18 found a significant osteoblast proliferation acceleration by using titanium nanomaterials. on day 14, there is a difference in osteoblast number between the nano-tooth graft and micro-tooth graft groups. even though the nano-tooth graft group had the highest osteoblast number on day 7 compared to the other groups, it shows a depletion on day 14. however, the osteoblast number is still close to control. the micro-tooth graft still promotes osteoblast proliferation. the decrease of osteoblasts in the nano-tooth graft group may be explained by brannigan et al.19, who stated that in the bone healing process, the first two weeks are the most critical period, and the differentiation of mesenchymal cells into osteoclasts and osteoblasts occurs around the second week.22 in the second week after injury, mature osteoblasts turn into osteocytes, while others remain on the surface of the periosteum and endosteum.20 according to a study conducted by griffin et al.21, nanoparticle grafts increase the adhesion and proliferation of osteoblasts compared to microparticle grafts. nanostructure scaffold surfaces have increased surface area and improved protein attachment, which can stimulate cell attachment, compared to micro-size scaffolds.21 liang et al.22 also demonstrated that the use of nano-ha increases the induction of osteoblasts. this study also shows that nano-tooth grafts promote faster osteoblast proliferation in the first week after bone injury. a previous study showed cell proliferation in osteoblast cell cultures after seven days.13 the activity of osteoblasts in bone remodeling then begins in the second week.23 micro-tooth grafts provide slower osteoblast proliferation in the first week after bone injury. nanotooth graft groups need one week to accelerate osteoblast proliferation, while the micro-tooth graft group needs two weeks. in conclusion, nano-tooth grafts accelerate osteoblast stimulation in the bone healing process more so than micro-tooth grafts. nano-tooth grafts accelerate and shorten the healing process because osteoblasts are stimulated faster, i.e., on the 7th day, while the peak of osteoblast proliferation in micro-tooth grafts happens on the 14th day. even though nano-tooth grafts have the potential to induce osteoblasts, further experiments are needed to evaluate toxicity and other negative effects. acknowledgment this research is supported by grants from the dentistry faculty, brawijaya university (decree number: 20/2021). references 1. anita lett j, sagadevan s, fatimah i, hoque me, lokanathan y, léonard e, alshahateet sf, schirhagl r, oh wc. recent advances in natural polymer-based hydroxyapatite scaffolds: properties and applications. eur polym j. 2021; 148: 110360. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p184–188 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p184-188 188 kumala et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 184–188 2. anggaraeni pi. alloplastic bone graft for pocket reduction after third molar surgery. universitas udayana; 2018. p. 1–19. 3. widhia r tini i a a, wirasuta mag, suk rama dm, rai i bn. t herapeutic dr ug monitor ing of r ifa mpicin, isoniazid, a nd pyra zi na m ide i n newlyd iagnosed pul mona r y t uberculosis outpatients in denpasar area. bali med j. 2019; 8(1): 107–13. 4. khanijou m, seriwatanachai d, boonsiriseth k, suphangul s, pairuchvej v, srisatjaluk rl, wongsirichat n. bone graft material derived from extracted tooth: a review literature. j oral maxillofac surgery, med pathol. 2019; 31(1): 1–7. 5. kaur n. natural teeth-a novel biomaterial for bone regenration. online j dent oral heal. 2021; 4(2): 1–3. 6. dilip bhalla n, r patil s, a belludi s, prabhu a, hr v, dani s, birla a. autogenous tooth bone graft a biomimetic promise for regenerative dentistry. acta sci dent scienecs. 2019; 3(9): 56–62. 7. mozartha m. hidroksiapatit dan aplikasinya di bidang kedokteran gigi. cakradonya dent j. 2015; 7(2): 835–41. 8. du m, chen j, liu k, xing h, song c. recent advances in biomedical engineering of nano-hydroxyapatite including dentistry, cancer treatment and bone repair. compos part b eng. 2021; 215: 108790. 9. rajula mpb, narayanan v, venkatasubbu gd, mani r, sujana a. nano-hydroxyapatite: a driving force for bone tissue engineering. j pharm bioallied sci. 2021; 13(5): s11–4. 10. ramadhani t, sari rp, w w. efektivitas kombinasi pemberian minyak ikan lemuru (sardinella longiceps) dan aplikasi hidroksiapatit terhadap ekspresi fgf-2 pada proses bone healing. dent j kedokt gigi. 2016; 10(1): 20–30. 11. ardhiyanto hb. stimulasi osteoblas oleh hidroksiapatit sebagai material bone graft pada proses penyembuhan tulang. stomatognatic (j k g unej). 2012; 9(3): 162–4. 12. fatemeh mirjalili, navabazam a, samanizadeh n. preparation of hydroxyapatite nanoparticles from natural teeth. russ j nondestruct test. 2021; 57(2): 152–62. 13. pilloni a, pompa g, saccucci m, di carlo g, rimondini l, brama m, zeza b, wannenes f, migliaccio s. analysis of human alveolar osteoblast behavior on a nano-hydroxyapatite substrate: an in vitro study. bmc oral health. 2014; 14(1): 22. 14. pascawinata a, prihartiningsih p, dwirahardjo b. perbandingan proses penyembuhan tulang antara implantasi hidroksiapatit nanokristalin dan hidroksiapatit mikrokristalin (kajian pada tulang tibia kelinci). b-dent, j kedokt gigi univ baiturrahmah. 2018; 1(1): 1–10. 15. kim y-k, lee j, um i-w, kim k-w, murata m, akazawa t, mitsugi m. tooth-derived bone graft material. j korean assoc oral maxillofac surg. 2013; 39(3): 103–11. 16. li jj, kawazoe n, chen g. gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis. biomaterials. 2015; 54: 226–36. 17. mahmoud ns, mohamed mr, ali mam, aglan ha, amr ks, a hmed hh. nanomater ial-induced mesenchymal stem cell differentiation into osteoblast for counteracting bone resorption in the osteoporotic rats. tissue cell. 2021; 73: 101645. 18. wu y, liu c, gao m, liang q, jiang y. effect of titanium nanoparticles on osteoblast proliferation. nanosci nanotechnol lett. 2020; 12(4): 455–60. 19. brannigan k, griffin m. an update into the application of nanotechnology in bone healing. open orthop j. 2016; 10(1): 808–23. 20. kumar s, dewi ah, listyarifah d, ana id. remodeling capacity of femoral bone defect by pop-cha bone substitute: a study in rats’ osteoclast (first series of pop-based bone graft improvement). indones j dent res. 2015; 1(2): 116. 21. griffin m, kalaskar d, seifalian a, butler p. an update on the application of nanotechnology in bone tissue engineering. open orthop j. 2016; 10(1): 836–48. 22. liang w, ding p, li g, lu e, zhao z. hydroxyapatite nanoparticles facilitate osteoblast differentiation and bone formation within sagittal suture during expansion in rats. drug des devel ther. 2021; 15: 905–17. 23. vidyahayati il, dewi ah, ana id. pengaruh substitusi tulang dengan hidroksiapatit (hap) terhadap proses remodeling tulang. media med muda. 2016; 1(3): 157–64. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p184–188 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p184-188 vol 52 no 1 jan-mar 2019_new.indd 11 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 1–7 research report evaluation of orthodontic tooth movement by 3d micro-computed tomography (μ-ct) following caffeine administration h. herniyati,1 happy harmono,2 leliana sandra devi,1 and sri hernawati3 1 department of orthodontics 2 department of dental biomedicine 3 department of oral medicine faculty of dentistry, universitas jember, jember – indonesia abstract background: the compressive strength of orthodontic tooth movement will be distributed throughout the periodontal ligament and alveolar bone, resulting in bone resorption on the pressure side and new bone formation on the tension side. caffeine, a member of the methyl xanthine family, represents a widely-consumed psychoactive substance that can stimulate osteoclastogenesis through an increase in rankl. a 3d micro-computed tomography (μ-ct) x-ray device can be used to measure orthodontic tooth movement and changes in periodontal ligament width. purpose: the purpose of this research was to analyze the effects of caffeine on the distal movement distance of two mandibular incisors using 3d μ-ct. methods: this research constituted an experimental study with post test control group design. the research subjects (guinea pigs) were randomly divided into four groups. of the two control groups created, one received two weeks of treatment and the other three weeks. the members of these two control groups were subjected to orthodontic movement but received no caffeine. meanwhile, the other two groups were treatment groups whose members also received either two or three weeks of treatment. in these two treatment groups, the subjects were subjected to orthodontic movement and received a 6 mg/500 bm dose of caffeine. the orthodontic movement of the subjects was induced by installing a band matrix and orthodontic bracket on each mandibular incisor to move distally by means of an open coil spring. observations were then conducted on days 15 and 22 with μ-ct x-rays to measure the distal movement distance of the two mandibular incisors and the width of the periodontal ligament. results: the administration of caffeine increased the tooth movement on day 15 (p<0.05) and day 22 (p<0.05). the increase in the tooth movement on day 22 was greater than that on day 15 (p<0.05). the width of the periodontal ligament on the pressure side of the treatment groups experienced greater narrowing than that of the control groups (p<0.05). meanwhile, the width of periodontal ligament on the tension side of the treatment groups widened more than that of the control groups (p<0.05). conclusion: μ-ct x-ray can be used to evaluate the extent of orthodontic movement in addition to the width of the mandibular incisor periodontal ligament during orthodontic tooth movement. moreover, it has been established that the administering of caffeine can improve orthodontic tooth movement. keywords: caffeine; micro-computed tomography; orthodontic tooth movement correspondence: herniyati, department of orthodontics, faculty of dentistry, universitas jember, jl. kalimantan 37, jember 68121, indonesia. e-mail: herny_is@yahoo.com introduction the success of orthodontic treatment depends on periodontal tissue health, oral hygiene and orthodontic strength.1 the latter causes the periodontal tissue to be divided histologically into pressure and tension sides.2 bone resorption occurs in the periodontal ligament subject to pressure, while bone formation occurs in the periodontal ligament experiencing tension.3 osteoclast activities will increase on the pressure side, whereas osteoblasts will start to proliferate and extracellular matrix mineralization, resulting in bone remodeling, occurs in the tension side.4 osteoclasts play a significant role in bone resorption5, while the alveolar bone marrow plays a role in osteoclast formation during orthodontic tooth movement.6 when orthodontic mechanical forces are applied, changes that dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 mailto:herny_is@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 2 herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 occur to the bone are accepted by mechanoreceptors regulated by osteocytes. these will then stimulate osteoclast proliferation and differentiation. the differentiation of osteoclasts is regulated by two important cytokines, namely; macrophage colonystimulating factor (m-csf) and receptor activator of nuclear factor-κβ ligand (rankl). m-csf is an important factor responsible for the survival and proliferation of osteoclast precursors which also triggers expression of receptor activator of nuclear factor-ββ (rank) in osteoclast precursor cells to generate an efficient response to the rankl-rank signaling pathway.6 the binding of rankl-rank receptors to osteoclast precursors subsequently stimulates osteoclast differentiation and proliferation rendering osteoclasts active. these active osteoclasts will then induce bone resorption.7 previous research on male wistar rats posited that rankl expression on day 21 increases significantly followed by bone resorption caused by orthodontic tooth movement. in addition, the results of the polymerase chain reaction (pcr) examination featured in that research also indicated that rankl increases significantly on the pressure side.8 orthodontic treatment is of relatively lengthy duration. consequently, numerous efforts have been made to accelerate it, including drugs, surgical methods and physical/mechanical stimulation methods. at present, no drug exists capable of safely accelerating orthodontic tooth movement.9 caffeine, on the other hand, is an alkaloid compound (purine base) that is white and psychoactive. it is usually consumed in the forms of coffee, tea and carbonated drinks such as cola, but is also used as a central nervous system stimulant (in this case, diuretic) which accelerates metabolism. moreover, consumption of caffeine is beneficial since it increases alertness, eliminates drowsiness and enhances mood.10 prior research conducted on mice indicated that during orthodontic tooth movement low doses of caffeine (2.5 mg/100 g bm) can increase the number of osteoclasts, while simultaneously accelerating bone resorption on the pressure side of the alveolar bone on day 14.11 a method has been developed to enable the viewing of three-dimensional (3d) images of dentoskeletal and craniofacial relationships before and after orthodontic treatment. a 3-dimensional picture can show the results of treatment on both hard and soft tissues, such as the teeth, face and bones, while also being used to determine the diagnosis, prognosis and treatment plan.12 unfortunately, it is not possible to use histology, a scanning electron microscope (sem), a transmission electron microscope (tem) or 3-dimensional imaging as means of observing periodontal tissue responses to orthodontic tooth movement with any degree of precision. consequently, 3d micro-computed tomography (μ-ct) x-ray method has been developed to observe tooth movement and periodontal ligament width during this process.13 as a result, the research aims to observe and analyze the effect of caffeine on the distal movement distance of two mandibular incisors using the 3d μ-ct method. materials and methods this research constituted an experimental study with posttest control group design. ethical approval was granted by the research ethics committee of the faculty of medicine, universitas jember, no: 1150/h25.1.11/ ke/2017. the research was conducted at the biomedical laboratory of the faculty of dentistry, universitas jember and included the administering of treatment of subjects through to tissue sampling. the subjects consisted of 20 male guinea pigs aged 10-12 months and weighing 500 grams which were randomly divided into four groups of five members.14 the tissue samples were observed and measured to detect any increase in tooth movement among the subjects at the micro-ct laboratory of the faculty of mathematics and natural science (fmipa) at institut teknologi bandung (itb). of the four groups to which the subjects had been randomly assigned, one was a 2-week control group and another a 3-week control group. the members of these two control groups, received orthodontic movement and 3ml of distilled water. meanwhile, the other two groups constituted those receiving 2 and 3 weeks of treatment respectively. the members the two treatment groups were subjected to orthodontic movement and received a daily 6 mg/500 bm dose of caffeine (equivalent to that contained in one cup of coffee containing 100 grams of coffee powder in 150ml of water) dissolved in 3ml of distilled water. at the next stage, the subjects were anesthetized with ketamine and orthodontic movement was set by installing the band matrix and orthodontic bracket on each mandibular incisor to enable it to move distally using an open coil spring (ortho-tech, america)15 with a power of 0.0525 n or 52.5 grams (figure 1). observations were then made after the subjects had been sacrificed on days 15 and 22 by extracting both their right and left mandibular incisors as well as periodic tissue and placing these in the fixative solution. observations were subsequently performed by scanning the periodontal tissue samples with x-ray devices using the 3d method μ-ct bruker skyscan 1173 high energy micro-ct at the micro-ct laboratory of the itb faculty of mathematics and natural science (fmipa). scanning was performed at 65 kv and 30 μa with a 1mm-sized aluminum filter for 250 minutes. 20 samples were scanned at standard resolution, producing output in the form of a 560560 pixel-sized projection image in 16-bit tiff format which was recorded using an average of ten frames to minimize the noise produced. this device uses a silent source method and a rotating object detector which, for the purposes of this scan, operated with a rotation hose of 0.4° dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 3herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 figure 1. installation of orthodontic devices in guinea pigs. the orthodontic bracket attached to the band matrix (yellow arrow) was affixed to the two right and left mandibular incisors of the guinea pigs, their two right and left mandibular incisors subsequently being moved distally using an open coil spring (green arrow). figure 3. histograms of distal movement distances for the two mandibular incisors (the width between the two mandibular incisors) (a) and the width of periodontal ligament in the resorption and apposition (b) areas in the research groups for two weeks and three weeks marked in green and the caffeine treatment group marked in blue. the control groups the treatment groups sagital sagital transversal transversal figure 2. μ-ct figures of the distal movements of the two mandibular incisors and the widths of periodontal ligaments on the sagittal pieces (a and b for 2 weeks; c and d for 3 weeks) and on transverse pieces (a1 and b1 for 2 weeks; c1 and d1 for 3 weeks); tooth root (a); periodontal ligament (b); alveolar bone (c); yellow arrow: the distal movement distance of incisor; green arrow: the width of the periodontal ligament in the apposition area; red arrow: the width of the periodontal ligament in the resorption area. and a total rotation of 240°. the spatial resolution of the resulting image was ~50 micrometers/pixel. the complete image in the form of a 3d map of object density was then obtained by reconstructing the projection image using the feldamp backpropagation method. after the reconstruction process, 539 pieces with a 2d image were produced in an 8-bit bitmap image format. initial processing in the form of repositioning objects in 3d space and analyzing objects in the form of long measurements using dataviewer software (bruker micro-ct, belgium) were subsequently carried out. observations of sagittal pieces were conducted from the crown to the root of the mandibular incisors in order to perform two functions. firstly, to quantify the increased tooth movement in the subjects by measuring the distance dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 4 herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 from the mesial section of the right mandibular incisor through the alveolar bone peak between the two teeth to the mesial section of the left mandibular incisor. secondly, to measure the width of the periodontal ligament both in the mesial part of the left and right mandibular incisors representing the tension side or bone apposition area as well as in the distal part representing the pressure side or bone resorption area in the right and left first mandibular incisors (figure 2). thereafter, the mean width of the periodontal ligament on both pressure and tension sides of the two incisors was measured. the research data were analyzed using an independent t test with a confidence level of 95% (α=0.05). results the results of this research into the effects of caffeine on the distal movement of mandibular incisors using the 3d μ-ct method are illustrated in tables 1, 2, 3; as well as figures 2 and 3. the distal movement of mandibular incisors was indicated by changes in the width or thickness of the periodontal ligaments in the cervical region measured from the mesial side of the right mandibular incisor through the alveolar bone to the mesial side of the left mandibular incisor (figure 2 and 3) . table 1 shows the mean and standard deviation of the distal movements of two mandibular incisors in the treatment and control groups on days 15 and 22. the distal movements of two mandibular incisors in the treatment groups were greater than those in the control groups. based on the t-test results, it is evident that significant differences in the distal movements of mandibular incisors existed between those groups on day 15 (p<0.05) and day 22 (p<0.05). this indicates that the administration of caffeine can elevate the distal movement of mandibular incisors to a greater extent than in cases where caffeine is not administered. table 2. the mean and standard deviation of the widths of periodontal ligaments in the resorption and apposition areas of each research group on day 15 and day 22. ngroups width of periodontal ligaments (mm) (mean ± standard deviation) day-22day-15 pappositionresorptionpappositionresorption control group 0.000*0.897 ± 0.0010.147 ± 0.0010.000*0.797 ± 0,0010.349 ± 0.0015 treatment group 0.000*0.180 ± 0.0010.113 ± 0.0030.000*1.577 ± 0.0015 0.147 ± 0.001 0.000*0.000*0.000*p 0.000* note: *based on the independent t-test results table 3. the results of the comparison test on the width of periodontal ligament in the resorption and apposition areas between day 15 and day 22 in each research group. ngroups width of periodontal ligaments (pg/ml) (mean ± standard deviation) appositionresorption pday-22day-15pday-22day-15 control group (-) 0.000*0.897± .0010.797 ± .0010.000*0.147± .0010.349± .0015 treatment group (p) 0.000*0.180± .0011.577±0.0010.000*0.113± .0030.147 ± .0015 note: *based on the independent t-test results table 1. the mean and standard deviation of the distances or widths between the two mandibular incisors, as well as the results of a comparison test between the research groups on day 15 and day 22. ngroups tooth movement (mm) (mean ± standard deviation) p day-22day-15 control group 0.000*3.273±0.0032.987±0.0045 treatment group 0.000*5 4.472±0.002 4.572±0.002 p 0.000* 0.000* note: *based on the independent t-test results dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 5herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 table 2 depicts the mean and standard deviation of the periodontal ligament width of the resorption and apposition areas in each research group on days 15 and 22. based on the t-test results, the width of the periodontal ligament in the resorption area in the treatment groups were smaller than those in the control groups on both days 15 (p<0.05) and 22 (p<0.05). meanwhile, the widths of the periodontal ligament in the apposition areas of the treatment groups were greater than those in the control groups on both days 15 (p<0.05) and 22 (p<0.05). this means that the provision of caffeine can increase the distal movement of the mandibular incisor. table 3 shows that the results of the comparison test on periodontal ligament widths in the resorption and apposition areas in each research group on days 15 and 22. according to the t-test results, the width of the periodontal ligament in the resorption area on day 22 was smaller than that on day 15 (p<0.05). meanwhile, the width of the periodontal ligament in the apposition area on day 22 was wider than that on day 15 (p<0.05). this means that the longer the duration of caffeine administration, the greater the distal movement of mandibular incisor (figure 2). discussion the force of orthodontic pressure will be distributed through the teeth into the periodontal ligament and alveolar bone. this will result in bone resorption in the pressure side during tooth movement while, on the other hand, new bone formation will be triggered in the tension side.16 such movement caused by orthodontic treatment can actually cause sequential reactions involving periodontal tissue and alveolar bone, resulting in the release of various substances from dental tissues and surrounding structures. the initial response of the periodontal tissue to mechanical stress involves metabolic changes that enable tooth displacement. minor changes in the thickness of the periodontal ligament occur one hour after the administration of orthodontic strength, while significant changes occur after a period of six hours.17 during orthodontic tooth movement, the tension side of the periodontal ligament will widen due to initial movement and fibroblast proliferation.18 complex molecular signals produce cellular responses to resorb alveolar bone with the result that the tooth moves. thus, orthodontic tooth movement will be followed by alveolar bone and periodontal ligament remodeling processes.7 a previous study of mice revealed that during molar teeth movement the presence of rankl and rank in periodontal tissues, in addition to mechanically stressed periodontal ligament cells, induces osteoclastogenesis through increased regulation of rankl expression,8 followed by bone resorption on the pressure side.19 during orthodontic treatment, the application of optimal strength is important for adequate biological response in the periodontal system. however, the force applied during orthodontic treatment should not exceed capillary blood pressure (20-25g/cm)7 since, if it does surpass that level, tissue necrosis can ensue.16 large forces can exert excessive pressure on the periodontal ligament and cause hyalinization which can, in turn, inhibit alveolar bone surface resorption.20 another earlier study into the response of periodontal tissue after orthodontic tooth movement using tem and sem was carried out, but initial changes in the thickness of the periodontal ligament have yet to be identified. contrastingly, in histological studies the thickness of periodontal ligaments is easily influenced by tissue preparation, such as decalcification and tissue dehydration. moreover, the limited size of tissue sample preparation slides render it impossible to obtain an overall 3-dimensional picture of orthodontic tooth movement.12,13 3d μ-ct constitutes a new image examination with high-resolution and non-intrusive analysis techniques that have developed rapidly in recent years. therefore, 3d μ-ct is expected to provide qualitative and quantitative data with three-dimensional images of specimens tested in order to learn about and better understand the breakdown of alveolar bone microstructure.21,22 the 3dμ-ct technique has also been employed to measure tooth movement and changes in the width of the periodontal ligament.13 previous research on periodontal tissue, especially in the trabecular bone microstructure, applies histological techniques capable of observing microstructure, but the parameters of alveolar bone microstructure remain challenging to obtain and describe.23 the results of evaluation using 3d μ-ct techniques on sagittal and transverse pieces obtained during this research showed that the provision of caffeine increased the distal movements of the two mandibular incisors on days 15 and 22 indicated by an increase in the intervening distance. the results also confirmed that the width of the periodontal ligament, indicated by the radiolucent area between the teeth and the alveolar bone on the pressure side, demonstrated narrowing resorption. in contrast, that on the tension side showed an apposition area that was increasing in width. in the caffeine treatment groups, the pressure side narrowed to a greater extent than that in the control groups, whereas the tension side of the caffeine treatment groups had greater periodontal ligament width. this indicates that tooth movements were more pronounced in the treatment groups supplied with caffeine. the increasing distal movement of mandibular incisors is caused by caffeine generating osteoclastogenesis through an increase in rankl.24,25 the results of this research are in accordance with those of several previous investigations which posited that a low dose of caffeine can elevate osteoclasts and bone resorption on the pressure side of alveolar bone on day 14.11 research conducted by yamaguchi (2009), indicated that rankl expression increases significantly followed by bone resorption caused by orthodontic tooth movement on day 21.8 rankl, a member of the tumor receptor necrosis factor (tnf) family, mediates signals that lead to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 6 herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 osteoclastogenesis.26 rankl plays a role in bone resorption and will interact with rank on osteoclast precursors, thereby triggering osteoclast differentiation and proliferation resulting in osteoclasts becoming active and leading to bone resorption.7,27 this will, in turn, cause orthodontic tooth movement28 followed by remodeling of periodontal ligament and alveolar bone.7 caffeine binds to adenosine receptors and modulates several other receptors including: glucocorticoid, insulin, estrogen, androgens, vitamin d, cannabinoids, glutamate and adrenergic receptors, all of which are expressed in osteoblasts or osteoprogenitor cells and have important functions during osteoblast differentiation.29 moreover, vitamin d will promote greater transcription of rankl and limit the production of osteoprotegerin (opg).30 previous research has argued that, while low caffeine concentration (0.005-0.01 mm) cannot affect cell survival and osteoblast differentiation from bone marrow mesenchymal stem cells (msc), it can significantly increase both osteoclast differentiation from bone marrow hematopoietic stem cells (hscs) and bone resorption activity. such research has also indicated that lower caffeine concentration can increase rankl protein expression in osteoblast cell cultures and reduce opg expression in osteoblasts. meanwhile, in vitro research has confirmed the findings of the aforementioned investigations that low caffeine concentration triggers cyclooxygenase (cox-2)/ prostaglandin (pg) e2 and increases rankl in osteoblasts, thereby promoting osteoclast formation.31 the absence of mononuclear osteoclast precursors from the normal periodontal ligaments of mice and the presence of active osteoclasts there are caused by the pressure exerted by the orthodontic appliance. mononuclear precursors in differentiated bone marrow will become mononuclear osteoclast precursors, before migrating to the periodontal ligaments on the pressure side and differentiating into active multinuclear osteoclasts. multinuclear osteoclasts, subsequently, induce bone resorption.32 in addition, the orthodontic tooth movement on day 22, following the provision of caffeine, was significantly greater than that on day 15. it means that the longer the duration of caffeine provision, the greater the subsequent orthodontic tooth movement. as a result, caffeine is expected to be an alternative in accelerating orthodontic treatment. finally, it can be concluded that 3d μ-ct method can observe changes in tooth movement and periodontal ligament width during orthodontic tooth movement both in the resorption and apposition areas. furthermore, it is also known that caffeine provision can accelerate orthodontic tooth movement. references 1. cardaropoli d, gaveglio l. the influence of orthodontic movement on periodontal tissues level. semin orthod. 2007; 13(4): 234–45. 2. ariffin shz, yamamoto z, abidin izz, wahab rma, ariffin zz. cellular and molecular changes in orthodontic tooth movement. sci world j. 2011; 11: 1788–803. 3. roberts-harry d, sandy j. orthodontics. part 11: orthodontic tooth movement. br dent j. 2004; 196(7): 391–4. 4. sprogar š, vaupotic t, cör a, drevenšek m, drevenšek g. the endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats. bone. 2008; 43(4): 740–7. 5. kominsky sl, abdelmagid sm, doucet m, brady k, weber kl. macrophage inflammatory protein-1: a novel osteoclast stimulating factor secreted by renal cell carcinoma bone metastasis. cancer res. 2008; 68(5): 1261–6. 6. kim jh, kim n. regulation of nfatc1 in osteoclast differentiation. j bone metab. 2014; 21(4): 233–41. 7. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofac orthop. 2006; 129(4): 469.e1-32. 8. yamaguchi m. rank/rankl/opg during orthodontic tooth movement. orthod craniofac res. 2009; 12(2): 113–9. 9. shenava s, nayak sk, bhaskar v, nayak a. accelerated orthodontics – a review. int j sci c study. 2014; 1(5): 35–9. 10. ennis d. the effects of caffeine on health: the benefits outweigh the risks. perspectives (montclair). 2014; 6: 1–5. 11. peng s, yong-chun h. effect of caffeine on alveolar bone remodeling during orthodontic tooth movement in rats. j tongji univ (medical sci. 2011; 3. 12. karadeniz ei, gonzales c, elekdag-turk s, isci d, sahin-saglam am, alkis h, turk t, darendeliler ma. the effect of fluoride on orthodontic tooth movement in humans. a twoand threedimensional evaluation. aust orthod j. 2011; 27(2): 94–101. 13. gonzales c, hotokezaka h, arai y, ninomiya t, tominaga j, jang i, hotokezaka y, tanaka m, yoshida n. an in vivo 3d micro-ct evaluation of tooth movement after the application of different force magnitudes in rat molar. angle orthod. 2009; 79(4): 703–14. 14. daniel ww. biostatistics: a foundation for analysis in the health sciences. 6th ed. new york: john wiley & sons; 1995. p. 224. 15. suparwitri s, pudyani ps, haryana sm, agustina d. effects of soy isoflavone genistein on orthodontic tooth movement in guinea pigs. dent j (maj ked gigi). 2016; 49(3): 168–74. 16. henneman s, von den hoff jw, maltha jc. mechanobiology of tooth movement. eur j orthod. 2008; 30(3): 299–306. 17. nakamura y, noda k, shimoda s, oikawa t, arai c, nomura y, kawasaki k. time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography. eur j orthod. 2008; 30(3): 320–6. 18. isaacson kg, muir jd, reed rt. removable orthodontic appliances. new delhi: wright; 2002. p. 1–8. 19. wise ge, king gj. mechanisms of tooth eruption and orthodontic tooth movement. j dent res. 2008; 87(5): 414–34. 20. martín-badosa e, amblard d, nuzzo s, elmoutaouakkil a, vico l, peyrin f. excised bone structures in mice: imaging at threedimensional synchrotron radiation micro ct. radiology. 2003; 229(3): 921–8. 21. waarsing jh, day js, van der linden jc, ederveen ag, spanjers c, de clerck n, sasov a, verhaar jan, weinans h. detecting and tracking local changes in the tibiae of individual rats: a novel method to analyse longitudinal in vivo micro-ct data. bone. 2004; 34(1): 163–9. 22. salazar m, hernandes l, ramos al, micheletti kr, albino cc, nakamura cuman rk. effect of teriparatide on induced tooth displacement in ovariectomized rats: a histomorphometric analysis. am j orthod dentofac orthop. 2011; 139(4): e337–44. 23. masoud s, jesri m. correlation of bone resorption induced by orthodontic tooth movement and expression of rankl in rats. vol. 26. j dent sch shahid beheshti univ med sci. 2009; 26(4): 369–74. 24. yi j, yan b, li m, wang y, zheng w, li y, zhao z. caffeine may enhance orthodontic tooth movement through increasing osteoclastogenesis induced by periodontal ligament cells under compression. arch oral biol. 2016; 64: 51–60. 25. herniyati h. pengaruh kafein terhadap ekspresi rankl dan jumlah osteoklas pada pergerakan gigi ortodonti. denta. 2016; 10(1): 62. 26. nakagawa n, kinosaki m, yamaguchi k, shima n, yasuda h, yano k, morinaga t, higashio k. rank is the essential signaling dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 7herniyati, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 1–7 receptor for osteoclast differentiation factor in osteoclastogenesis. biochem biophys res commun. 1998; 253(2): 395–400. 27. bilezikian jp, raisz lg, rodan ga. principles of bone biology. 2nd ed. london: elsevier; 2002. p. 109–26. 28. yamaguchi m, kasai k. inflammation in periodontal tissues in response to mechanical forces. arch immunol ther exp (warsz). 2005; 53(5): 388–98. 29. reis ams, ribeiro lgr, ocarino n de m, goes am, serakides r. osteogenic potential of osteoblasts from neonatal rats born to mothers treated with caffeine throughout pregnancy. bmc musculoskelet disord. 2015; 16: 1–11. 30. purroy j, spurr nk. molecular genetics of calcium sensing in bone cells. hum mol genet. 2002; 11(20): 2377–84. 31. liu sh, chen c, yang r sen, yen yp, yang yt, tsai c. caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats. j orthop res. 2011; 29(6): 954–60. 32. xie r, kuijpers-jagtman am, maltha jc. osteoclast differentiation dur ing exper imental tooth movement by a shor t-ter m force application: an immunohistochemical study in rats. acta odontol scand. 2008; 66(5): 314–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p1–7 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p1-7 134 vol. 42. no. 3 july–september 2009 research report the influence of xylitol containing toothpaste on plaque formation inhibition on fixed bridge hamim fithrony, eha djulaeha and michel soedjono department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: plaque is the main cause of teeth and periodontal tissue damage, which usually accumulates on crown surfaces. to avoid this, plaque control is the best way that not only has a close connection to oral hygiene but also become important element in dental practice. previously, xylitol was used as alternative sweetener for diabetic patients, but later it is used to maintain healthy teeth. xylitol is capable to inhibit streptococcus mutans growth which changes sugar and other carbohydrate into acid, because xylitol cannot be fermented. purpose: this study was aimed to understand the inhibition capability of toothpaste containing xylitol to plaque formation on fixed bridge. methods: this clinical experiment study was carried out in fifteen patients wearing fixed bridge at prosthodontics department, faculty of dentistry, airlangga university in surabaya from 2005 to 2008. samples were based on selective random sampling technique. plaque index was analyzed by mann whitney test. result: this study showed that there was significant difference of plaque scores in patients who brush their teeth using xylitol containing toothpaste compared to the control group (placebo). conclusion: xylitol was capable to inhibit plaque formation on fixed bridge. key words: xylitol, fixed bridge, plaque correspondence: hamim fithrony, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: hamimf@unair.ac.id. introduction tooth lost due to caries or other periodontal diseases must be replaced to maintain good oral health. one of many dental treatments to handle is the making of fixed bridge. this treatment is needed to improve aesthetic, mastication function, and maintain oral health after tooth loss. the success of fixed bridge is determined from treatment planning, pontic design, abutment teeth preparation, cement choice which doesn’t destroy tooth and gingiva, harmonious occlusion, aesthetic terms, and the ability of patient to maintain oral hygiene to avoid teeth and periodontal damage.1 restoration with fixed bridge must be able to reduce the plaque adherence while support periodontal tissue health.2 plaque accumulation under fixed denture pontic could inflame the underlying mucosa.3 therefore the success and failure of fixed denture solely depend on the health of underlying tooth and bone structure. the main cause of tooth and periodontal damage are plaque on tooth crowns. plaque control is the best way and closely related to oral health as the main element in dental practice, because every patient is responsible for his or her own oral health. plaque consists of organic and inorganic material in solid form and about 20% of plaque is water. almost 70% bacteria live in the solid form and the rest in intercellular matrix. organic solid matter contains complex polysaccharide protein with main component of carbohydrate and protein 30%, lipid 15%, and the rest is still not known.3 according to manson,4 there are two methods to eradicate dental plaque, chemically and mechanically. from both ways, the mechanical through brushing with tooth brush or other devices is more important. mechanical cleaning with toothbrush and other devices is the most effective method in plaque control and calculus inhibition. tooth brushing only will not inhibit new plaque 135fithrony, et al.: the influence of xylitol in toothpaste accumulation.4 previous researches have been done on chemical substances which inhibit or reduce plaque and calculus formation.3 at first xylitol was used as alternative sweetener for diabetic patients.5 presently this sweetener is used as alternative to maintain healthy teeth. xylitol could inhibit streptococcus mutans’ growth while processing other sugar and other carbohydrates into acid. this could happen because xylitol is not fermented by the bacteria.6 some superiority of xylitol compared to other sweetener are that xylitol is unable to be fermented by oral bacteria, capable of reducing caries, plaque, increasing saliva production, replacing fluoride in toothpaste, has acceptable taste without leaving unwanted aftertaste, and producing lower calories compared to sucrose or cane sugar. one gram of xylitol produce 2.4 calories. this number is lower than sucrose which produce 4 calories/gram energy. consuming 5–10 grams of xylitol per day is still considered save.7 xylitol inhibition could reach 90%. xylitol effectiveness is optimal if contained more than 50% in a product. in indonesia, xylitol is produced from agricultural waste like rice, corn, or cane waste.7 the purpose of this research was to know the influence of xylitol in toothpaste as fixed bridge plaque inhibitor. materials and methods this research was clinical experimental study on fixed bridge patients who were treated in prosthodontics department clinic, faculty of dentistry, airlangga university, surabaya, during 2005–2008 period, with selective random sampling method. the materials used in this research were xylitol containing toothpaste (ciptadent) and placebo toothpaste. this xylitol toothpaste contains calcium carbonate, dicalcium phosphate, silica, sorbitol, propylene glycol, xanthan gum, sodium laurylsulphate, sodium saccharine, sodium monofluorophosphate, sodium fluoride, methyl paraben, flavor, vitamin a, vitamin c, vitamin e, xylitol, and aqua. placebo toothpaste contains cellulose 2%, sorbitol 40%, saccharine 0,2%, flavor 1%, surfact 2%, preservatives 0,2%, carbonate 15%, precipitate silicone 20%, titanium dioxide 0,2% and water ad 100%. other materials were disclosing solution, polishing material, and cotton. on the first day, fixed bridge were polished with rubber and brush therefore the plaque score = 0. at home, subjects were instructed to brush their teeth with placebo toothpaste 1 cm on toothbrush with roll method for 2 minutes, followed by 30 seconds gargle. subjects were instructed not to eat but allowed to drink plain water after polishing and tooth brushing until after 4 hours (in prosthodontics department clinic, faculty of dentistry, airlangga university). the fixed bridges were given disclosing solution by gargling for 30 seconds, and then the plaques were scored. subjects were not given any treatment for 1 week to condition the oral cavity to normal. after that, teeth brushing were done with xylitol toothpaste according to above step and the plaques were scored. result sample’s plaque score on 15 patients with 3 units fixed bridges in prosthodontics department clinic, faculty of dentistry, airlangga university, surabaya during 2005– 2008, according to sample category and after teeth brushing with placebo toothpaste and xylitol toothpaste was shown on table 1. the plaque were scored using plaque index from turesky-gilmore-glickman. table 1. means and standard deviation of sample’s plaque score after teeth brushing with placebo toothpaste and xylitol toothpaste after 4 hours mean sd placebo 0.80 0.13 xylitol 0.22 0.18 samples’ plaque score after brushing with xylitol toothpaste is lower than with placebo toothpaste. normality test with kolmogorov-smirnov test showed that all group has normal distribution (p > 0.05). to know the difference of patients’ plaque score between patients who brushed their teeth with placebo toothpaste and xylitol toothpaste, mann whitney test was performed, the result was shown on table 2. table 2. mann whitney test result on patients’ plaque score between brushing with placebo toothpaste and xylitol toothpaste toothpaste mean sd p > 0.05 plaque score placebo 0.80 0.13 0.000 xylitol 0.22 0.18 there was significant difference on patients’ plaque score between brushing with placebo toothpaste and xylitol toothpaste. hypothesis can be tested using hypothesis test. on hypothesis test, first the placebo and xylitol result must be counted and put into formula, finding z = 4.60. since counted z (4.60) > z from table (1.96), ho was rejected. ho is zero hypothesis which means there was no difference between patients’ plaque score which brushed with placebo toothpaste and xylitol toothpaste. hypothesis test showed significant difference on patients’ plaque score between brushing with placebo toothpaste and xylitol toothpaste. discussion in this research, the plaque was checked four hours after teeth brushing, because bacteria colony was formed on tooth surfaces within four hours.3 before treatment, 136 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 134-136 the fixed bridges were polished to get plaque score = 0. subjects were given the same toothpaste and were told the same brushing method (roll technique). these were done to reduce heterogenity. subjects were asked not to consume food for four hours after brushing till examination. this research showed difference in plaque formation between patients who brushed their teeth with placebo toothpaste and xylitol toothpaste. plaque formation was less in patients who brushed their teeth with xylitol toothpaste. statistic analysis using mann whitney test showed p = 0.000. this result is smaller than half a = 0.025. this is true according to edgar6 who stated that xylitol was not fermented by many microorganisms, which cause plaque former microorganisms cannot live. beside that, xylitol is artificial sweetener and grouped into sugar alcohol. xylitol is more stable in chemical structure and enzymatic compared to other sweeteners. this made xylitol hard to be fermented by bacteria, therefore oral ph could be maintained.6 this research proved the use of xylitol containing toothpaste has effect on plaque decrease of patients with fixed bridge, this can also help avoid caries and plaque formation. xylitol can also increase saliva production hence could remineralize tooth decay from bacterial demineralization. xylitol cannot be fermented into cariogenic acid and showed caries decrease by inhibiting streptococcus mutans’ growth.8 other positive value of xylitol is the use as sucrose replacement sweetener since 1983 because xylitol has low calories, therefore it is save for diabetic mellitus patient consumption.7 there are some weaknesses in this research which can reduce research validity, such as how to make sure that the subject did not drink or eat during research, which must be controlled well. if patients eat or drink, they will influence the result. during plaque scoring, there might be different results between individuals, even though generally bacteria colony formed after four hours, still there were variations. from the observation after brushing with experiment toothpaste or placebo, it was seen that plaques were found most under pontic and metal part near gingival, while there are less found on the porcelain part. this is according to hanoem9 which stated that plaque stick easier on metal than on porcelain. through observing this research and data analysis, it can be concluded that the use of xylitol containing toothpaste was effective to reduce the plaque index on fixed bridge. references 1. horn hr. practical consideration for successful crown and bridge therapy. 1st ed. philadelphia, toronto, london: mosby co; 1976. p. 257–59. 2. pameijer hn. periodontal and occlusal factors in crown and bridge procedures. usa: dental center for postgraduate courses; 1985. p. 17, 22, 148–50. 3. carranza fa, newman mg. clinical periodontology. 8th ed philadelphia: wb saunders company; 1996. p. 68, 84–9, 493–6, 500–2. 4. manson jd. periodontics. 4th ed. london: hendry kimpton publisher; 1980. p. 116–38. 5. xylitol (who food additives series 12). tolerance studies in diabetes. joint fao/ who expert committee on food additives. geneva, 1977 april; p. 18-27. available from: http: //www.in chem.. org/documents /jecfa/jecmono/v12 je 22.htm. accessed january 19, 2009. 6. edgar wm. the journal of clinical dentistry. london: the university of liverpool; 2007. x(2):563–6. 7. dadan r, arista b. sekilas tentang xylitol. pikiran rakyat bandung,pikiran rakyat bandung, pusat data redaksi; 2006. 8. kibbe, arthur h. handbook of pharmaceutical excipients. london united kingdom. 2000. p. 4, 28, 111, 143, 427, 455–7, 478, 487, 515, 602. 9. hanoem eh. pengaruh pugaran porcelain dan logam paduan nikelkrom terhadap terjadinya keradangan gingival. majalah kedokteran gigi surabaya 1993; (edisi khusus, peringatan 65 tahun pendidikan dokter gigi di surabaya): 178–83. mkgs vol 45 no 2 april-juni 2012.indd 93 volume 45 number 2 june 2012 the increasing of enamel calcium level after casein phosphopeptideamorphous calcium phosphate covering widyasri prananingrum and puguh bayu prabowo department of dental material faculty of dentistry, hang tuah university surabaya – indonesia abstract background: caries process is characterized by the presence of demineralization. demineralization is caused by organic acids as a result of carbohydrate substrate fermentation. remineralization is a natural repair process for non-cavitated lesions. remineralization occurs if there are ca2+ and po4 3ions in sufficient quantities. casein-amorphous calcium phosphate phosphopeptide (cpp-acp) is a paste material containing milk protein (casein), that actually contains minerals, such as calcium and phosphate. the casein ability to stabilize calcium phosphate and enhance mineral solubility and bioavailability confers upon cpp potential to be biological delivery vehicles for calcium and phosphate. purpose: the aim of this study was to determine the calcium levels in tooth enamel after being covered with cpp-acp 2 times a day for 3, 14 and 28 days. methods: sample were bovine incisors of 3 year old cows divided into 4 groups, namely group i as control group, group ii, iii and iv as treatment groups covered with cpp-acp 2 times a day. all of those teeth were then immersed in artificial saliva. group ii was immersed for 3 days, while group iii was immersed for 14 days, and group iv was immersed for 28 days. one drop of cpp-acp was used to cover the entire labial surface of teeth. the measurement of the calcium levels was then conducted by using titration method. all data were analyzed by oneway anova test with 5% degree of confidence. results: the results showed significant difference of the calcium levels in tooth enamel of those groups after covered with cpp-acp 2 times a day for 3, 14 and 28 days (p = 0.001). there is also significant difference of the calcium levels in tooth enamel of those treatment groups and the control group (p = 0.001). conclusion: the calcium levels of tooth enamel are increased after covered with cpp-acp 2 times a day for 3, 14 and 28 days. key words: calcium, enamel, casein phosphopeptide-amorphous calcium phosphate abstrak latar belakang: proses terjadinya karies gigi ditandai oleh adanya demineralisasi. demineralisasi terjadi oleh asam organik sebagai hasil fermentasi substrat karbohidrat oleh bakteri. remineralisasi adalah proses perbaikan alami untuk lesi non cavitated. remineralisasi terjadi jika terdapat ion ca2+ dan po4 3dalam jumlah cukup. casein phosphopeptide-amorphous calcium phosphate (cpp-acp) adalah bahan berbentuk pasta berisi suatu protein susu (kasein). pada kasein terkandung mineral kalsium dan fosfat. kemampuan kasein untuk menstabilkan kalsium fosfat dan meningkatkan kelarutan dan bioavailabilitas sehingga cpp memiliki potensi menghantarkan kalsium dan fosfat. tujuan: tujuan penelitian ini untuk mengetahui kadar kalsium enamel setelah pengulasan cpp-acp pada permukaan enamel 2 kali sehari selama 3,14 dan 28 hari. metode: sampel adalah gigi insisif sapi, usia 3 tahun. pada penelitian ini sampel (n = 24) dibagi menjadi 4 kelompok yaitu kelompok i sebagai kelompok kontrol, dan kelompok ii, iii, iv sebagai kelompok perlakuan dengan pengulasan cpp-acp 2 kali sehari. semua kelompok direndam dalam saliva buatan. kelompok ii direndam 3 hari, kelompok iii direndam 14 hari, kelompok iv direndam 28 hari. pengulasan cpp-acp sejumlah 1 tetes diratakan pada seluruh permukaan labial gigi. pengukuran kadar kalsium dilakukan dengan metode titrasi. semua data dianalisa dengan uji one-way anova dengan taraf kemaknaan 5%. hasil: hasil penelitian menunjukkan perbedaan yang signifikan kadar kalsium enamel gigi di antara kelompok (p = 0,001). hasil penelitian menunjukkan perbedaan yang signifikan kadar kalsium gigi pada kelompok research report 94 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 93–96 kontrol dan perlakuan antara lama waktu pengulasan 3, 14, dan 28 hari (p = 0,001). kesimpulan: pengulasan cpp-acp selama 3, 14 dan 28 hari mampu meningkatkan kadar kalsium pada enamel. kata kunci: kalsium, enamel, casein phosphopeptide-amorphous calcium phosphate correspondence: widyasri prananingrum, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas hang tuah. jl. arif rahman hakim no. 150 surabaya. e-mail: wprananingrum@gmail.com introduction there are four important factors that could cause caries, namely bacteria, carbohydrates, host, and time. some kinds of bacteria have ability to perform fermentation of carbohydrate substrate in food (e.g glucose and sucrose) in order to form acid and to lower ph to below 5 or 4.5 within 1–3 minutes. to return to normal ph around 6-7, it takes about 30–60 seconds.1 mineral component of enamel is hydroxyapatite with chemical formula ca10(po4)6(oh)2. in normal condition, hydroxyapatite is balanced with saliva containing ca2+ and po4 3ions. hydroxyapatite is also reactive with hydrogen ions at ph <5.5 called as critical ph. thus, h+ then reacts with po4 3to hpo4 2-. it is known as ca10(po4)6(oh)2(s) + 8h+(aq) 10ca2-(aq) + 6hpo4 2-(aq) + 2h2o(l) ]. 2 the process of dental caries is characterized by the presence of demineralization. demineralization is caused by fermentation of organic acids from carbohydrate substrates caused by bacteria. the demineralization process of carbohydrates caused by bacterial colonies is often called as plaque that produces acids which damage hydroxyapatite minerals of teeth, therefore the releasing process of mineral in enamel is called demineralization.1 remineralization is a natural process for repairing dental tissue suffering from demineralization. remineralization occurs if there are ca2+ and po4 3ions in sufficient quantities generally obtained from saliva.3 casein phosphopeptide-amorphous calcium phosphate (cpp-acp) is material with the form of paste containing milk protein (casein). casein phosphopeptide (cpp), has ability not only to bind and stabilize calcium and phosphate in solution, but also to bind dental plaque and tooth enamel. calcium phosphate, which is in the form of the crystal structure is usually insoluble at neutral ph, but cpp keeps calcium and phosphate in the form of an amorphous form. in the amorphous form, calcium and phosphate ions can diffuse into tooth enamel. thus, casein contains calcium and phosphate minerals. casein even can improve the quality of the enamel surface to be more resistant to organic acids as a result of the fermentation of carbohydrate substrates caused by bacteria. enamel that gets demineralization, therefore, can be improved by the provision of calcium and phosphate ions contained in cpp-acp inserted to the inside of enamel in order to replace the dissolved minerals, so remineralization can occur.4 casein actually contains minerals, such as calcium and phosphate. the casein ability to stabilize calcium phosphate and enhance mineral solubility and bioavailability confers upon cpp potential to be biological delivery vehicles for calcium and phosphate.5 for those reasons, the purpose of this study was to determine the level of calcium in tooth enamel on which cpp-acp is covered 2 times a day with a 12-hour interval for 3, 14 and 28 days. materials and methods this study was considered as an experimental research with completely randomized design by using bovine incisors of ± 3 year old cows as samples. those teeth were cleaned and stored in normal saline for ± 1 week with several criteria of eruption crown, without abrasion, without fracture/crack, and without caries.7 the study was conducted at the laboratory of chemistry university of hang tuah surabaya. samples (n=24) were divided into 4 groups. group i was control group, each sample was not covered with cpp-acp. group ii, iii and iv were treatment groups, each sample was covered with cpp-acp. the surfaces of the tooth enamel then were covered with cpp-acp, and left for 15 seconds. next, they were washed with normal saline thoroughly and dried with a blower until they became white. afterwards, those tooth enamel surfaces were covered with cpp-acp 2 times a day with 12 hour interval, and then were immersed in 90 ml of artificial saliva. artificial saliva used was made of 36.00 g nacl, 1.69 g kcl, 0.96 g cacl 2, 0.80 g nahco3 and added with 400 ml aquadestilata. this mixture can actually produce neutral ph (ph 7).7 group ii was immersed for 3 days, while group iii was immersed for 14 days, and group iv was immersed for 28 days. cpp-acp was used for about 1 drop, and then it was applied at the entire labial surface of the teeth after the teeth were washed with normal saline and dried for 10 minutes. afterwards, the artificial saliva was replaced every time it was used. the measurement of calcium levels was then conducted by titration method. samples were boiled with hydrochloride acid which would extract minerals and solve sample protein, and then were filtered. the calcium level of the filtrate was analyzed by using permanganometry titration. the procedures included the preparation of the ash, the preparation of ash solution/sample preparation, and the examination of dental calcium levels of wistar rats’ teeth. data results were then tested by using statistical test, one-way anova. 95prananingrum and prabowo: the increasing of enamel calcium level results the mean and standard deviations of calcium levels in enamel among the treatment groups covered with cpp-acp were higher than the control group at the time of observation 3, 14 and 28 days (table 1). the mean level of calcium in enamel of the group covered with cpp-acp for 28 days was the highest compared to that in groups covered with cppacp for 3 and 14 days. the mean and standard deviations of enamel calcium levels obtained were analyzed with kolmogorov smirnov test. the results indicate that all data were normally distributed (p>0.05), thus, parametric test can be used. levene test was conducted on the observation of enamel calcium level covered with cpp-acp. the results show that the probability value was >0.05. it means that the homogeneous assumption was met, so parametric test could be conducted. the results of one-way anova showed that there were significant differences of the enamel calcium levels (p<0.05), about 0.001 (table 2), between the treatment groups covered with cpp-acp for 3, 14, and 28 days and the control group. it was also known that there was significant difference of the calcium level between the treatment groups covered with cpp-acp for 3 and 14 days with p about 0.001 (p<0.05). similarly, there was significant difference of the calcium level between the treatment groups covered with cpp-acp for 3 and 28 days with p about 0.001 (p<0.05). there was significant difference of the calcium level between the treatment groups covered with cpp-acp for 14 and 28 days with p about 0.003 (p<0.05) (table 3). discussion bovine fresh extracted teeth were used referring a previous study because bovine teeth are easily obtained in large quantities and in good condition (rarely getting any caries).8 in this study, samples of the study were covered with 37% phosphoric acid in order to obtain surface roughness of enamel. the samples consisted of 4 groups: 3 treatment groups covered with cpp-acp for 3 days, 14 days, 28 days, and control group. each group then consisted of 6 samples. each sample was then covered with acids made of h+ ion reacting with po4 3and altering into hpo4 2-. this hpo4 2compound, however, did not play a role in the balance of hydroxyapatite since hydroxyapatite contained more po4 3than hpo4 2-, so hydroxyapatite crystals would dissolve and cause demineralization2. acid, dissolved ± 10 μm of the enamel surface (enamel rods), so porosity was formed about 5-50 μm.9 this study took 3 days, 14 days, and 28 days to compare dentin tubule porosities formed since according to the result of the research conducted by oshiro et al.,13 using sem with those cover times was effective enough to determine the effect of the use of cpp-acp. besides, according to the factory rules the use of cpp-acp was supposed to be 2 times a day. therefore, the total of the use of cpp-acp was 6 times for 3 days, 28 times for 14 days, and 56 times for 28 days. the average level of calcium in enamel of the treatment groups covered with cpp-acp for 28 days was the highest of all. the levels of calcium were different between the treatment groups and the control group, and there was significant difference of the calcium levels among those treatment groups with different cover times. this is because in cpp-acp there is a complex amalgamation of nano, amorphous calcium phosphate (acp), diffusion into dental plaque and tooth surface. cpp has an important role as carrier of acp to tooth surface location where the solubility level of calcium phosphate is very high. this localization then will maintain the high concentration gradient of calcium and phosphate ions below the surface of the enamel, so remineralization can easily occur.10 cpp-acp is a material with pasta form shape derived from casein, a milk protein containing calcium and phosphate minerals. in the study conducted by reynolds table 1. the mean and standard deviation of the calcium levels of tooth enamel based on cover times of cpp-acp, namely for 3, 14 and 28 days 3 days 14 days 28 days mean sd mean sd mean sd cpp-acp 40.1867 0.97558 44. 8567 1.44750 47.3650 0.88090 control 36.0217 0.75056 36.0217 0.75056 36.0217 0.75056 table 2. the differences of the significant level of the calcium level of tooth enamel in treatment groups and control group based on cover times of cpp-acp, namely for 3, 14 and 28 days variable pvalues control – cpp-acp 3 days 14 days 28 days level of calcium 0.001* 0.001* 0.001* note: * = significance of difference table 3. the significant level of the calcium level of tooth enamel in treatment groups and control group based on cover times of cpp-acp, namely for 3, 14 and 28 days variable cover time pvalues control cpp-acp level of calcium 3 days – 14 days 0.001* 0.001* 3 days – 28 days 0.001* 0.001* 14 days – 28 days 0.001* 0.003* 96 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 93–96 and johnson proved that the use of milk containing casein may play a role in preventing caries in oral environment. it is because cpp-acp can bind to plaques, provide large amounts of calcium, and then slow diffusion of free calcium. thus, it can restrict mineral loss during cariogenic episode and provide calcium to be used to remineralization process later.11 cpp can keep calcium and phosphate in the form of anamorphous non-crystalic. in amorphous form, calcium and phosphate ions can diffuse into tooth enamel.11 the use of cpp-acp on the surface of tooth enamel can accelerate the process of remineralization compared to that without the use of the cpp-acp. it occurs because cpp-acp does not only contains calcium and phosphate minerals, but also creates both physical-chemical bond between ca2+ and po4 3 ions, and complex compound, cahpo4, decomposed in demineralization process of tooth enamel and then forming strong bond with calcium, phosphate, and fluoride ions which later forms fluoroapatite crystals [ca10(po4)6(oh)f]. these crystals are more resistant to acid ion with ph above 4.5 compared to pure hydroxyapatite [ca10(po4)6(oh)2] with critical ph about 5.5.2 once calcium and phosphate ions diffused into the carious lesion through dissociation, the activity of calcium and phosphate ions will be increased, so the adhesion of bacteria on the surface of the enamel can be reduced and remineralization can be increased.12 similar study conducted by kumar4 showing that there was not only a high efficiency of cpp-acp in remineralization of early lesions in enamel, but also a high potential of cpp-acp in remineralization when applied topically after tooth brushing with toothpaste containing fluoride. some studies even suggest that cpp-acp will be more effective when mixed with saliva in mouth.13 in conclusion the use of cpp-acp for 3, 14 and 28 days can increase the levels of calcium in tooth enamel. the longer the use of cpp-acp, the higher the level of calcium in tooth enamel. references 1. fejerskov o, kidd e. dental caries: the disease and its clinical management. 2nd ed. australia: blackwell munksgaard; 2008. p. 101–10. 2. mount gj, hume wr. preservation and restoration of tooth structure. 2nd ed. australia: knowledge book and software; 2005. p 2, 25, 39, 87, 212. 3. featherstone jd. dental caries: a dynamic disease process. aust dent j 2008; 53(3): 286–91. 4. kumar vln, itthagar un a, k ing nm. the effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: an in vitro study. aust dent j 2008; 82(3): 34–40. 5. cross k j, huq nl, palamara je, perich jw, reynolds ec. physicochemical characterization of casein phosphopeptide amorphous calcium phosphate nanocomplexes. the journal of biological chemistry 2005; 280(15): 15362–9. 6. soetojo a. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada berbagai kelembaban. disertation. surabaya: pascasarjana fakultas kedokteran gigi universitas airlangga; 2006. 7. setyawan kb. perbedaan jumlah pelepasan fluorida pada kompomer setelah perendaman dalam saliva buatan dengan ph 7 dan ph 4,5. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2004. 8. edmunds dh, whittaker dk, green rm. suitability of human, bovine, equinine, and bovine tooth enamel for studies of artificial bacterial carious lesions. caries res 1988; 22: 327–36. 9. anusavice kj. philips: buku ajar ilmu bahan kedokteran gigi. edisi 10. jakarta: ecg; 2003. p. 251–3. 10. ola b. the clinical applications of tooth moussetm and other cppacp products in caries prevention: evidence-based recommendation. smile dental journal 2009; 4(1): 8–12. 11. rose rk. effects of an anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaques. arch oral biol j 2000; 45(7): 569–75. 12. afanty a. penga r uh aplikasi pasta casein phosphopeptideamorphous calcium phosphate pada white spot gigi desidui. tesis. yogyakarta: universitas gajah mada; 2009. 13. oshiro m, yamaguchi k, takamizawa t, inage h, watanabe t, irokawa a. effect of cpp-acp paste on tooth mineralization: an fe-sem study. oral sci 2007; 49(2): 115–20. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 51 no 4 okt-des 2018.indd 194194 cytotoxicity test and characteristics of demineralized dentin matrix scaffolds in adipose-derived mesenchymal stem cells of rats desi sandra sari,1 ernie maduratna,2 f. ferdiansyah,3,5 i ketut sudiana,4 and fedik abdul rantam5,6 1department of periodontics, faculty of dentistry, universitas jember, jember – indonesia 2department of periodontology, faculty of dental medicine, universitas airlangga, surabaya – indonesia 3department of orthopedics & traumatology, dr. soetomo general hospital, surabaya – indonesia 4department of electron microscopy, faculty of medicine, universitas airlangga, surabaya – indonesia 5regenerative medicine and stem cell centre, universitas airlangga and dr. soetomo general hospital, surabaya – indonesia 6stem cell laboratory, stem cell research and development center, universitas airlangga, surabaya – indonesia abstract background: demineralized dentin matrix (ddm) scaffold is a substitute material for the bone contained in human teeth. ddm is a scaffold-derived tooth dentine containing type i collagen and bone morphogenetic protein (bmp). while ddm possesses the ability to perform osteoinductive and osteoconductive roles, a cytotoxicity test of ddm scaffold remains extremely important in evaluating the level of toxicity of a material if cultured in cells. adipose-derived mesenchymal stem cells (admscs) are multipotent in nature because they contain progenitor cells and have the potential for differentiation via adipogenic, osteogenic and chondrogenic pathways. admscs are also known to have high biocompatibility and the ability to combine with other bone material. purpose: the purpose of this study was to determine the cytotoxicity and characteristics of ddm scaffolds derived from bovine teeth in the admscs of rats cultured in vitro. methods: this research constituted an experimental study. admscs were isolated from the inguinal fat of rats. thereafter, ddm was extracted from bovine teeth and formed 355-710 μm-sized particles. ddm scaffolds were assessed using sem and the effects of ddm scaffolds on the cell viability of admscs at concentrations of 10%, 50%, and 100% analyzed by means of 3-4,5’dimethylihiazol-2-yl,2.5-di-phenyl-tetrazolium bromide (mtt) assay. the results obtained were then analyzed by an anova to establish the difference between the groups. results: sem results showed the diameter sizes of the dental tubulis ddm scaffolds to be approximately 4.429 μm and 7.519 μm. the highest cell viability (97.08%) was found by means of an mtt test to be in admscs at a concentration of 10% compared to those at concentrations of 50% and 100%. conclusion: in conclusion, ddm scaffold derived from bovine teeth with a particle size of 355-710 μm produces a low cytotoxicity effect on admscs. keywords: adipose-derived mesenchymal stem cells; cytotoxicity test; demineralized dentin matrix; scaffold correspondence: desi sandra sari, department of periodontics, faculty of dentistry, universitas jember, jl. kalimantan 37 jember 68121, indonesia. e-mail: desi_sari.fkg@unej.ac.id research report introduction demineralized dentin matrix (ddm) derived from bovine teeth is a material used as a substitute for human teeth. in recent years, ddm has widely been used in dental research as it is easily obtained in large quantities of high quality with a more uniform composition than that of human teeth.1,2 bovine dentin is similar in composition to human dentin which consists of 70% inorganic material, 20% organic material and 10% water. dentine microstructure consists of collagen fiber tissue3 and contains various growth factors, such as insulin growth factor-2 (igf-2), bone morphogenetic protein (bmp), tumor growth factor-ß (tgf-ß), platelet-derived growth factor (pdgf) and fibroblast growth factor (fgf).2,4,5 ddm can play an important role as scaffold because it contains both organic and inorganic components in addition to being microporous.6 ideally, scaffold must be biocompatible, biodegradable and non-toxic. moreover, dental journal (majalah kedokteran gigi) 2018 december; 51(4): 194–199 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 195 sari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 194–199 ddm also possesses the ability to proliferate and differentiate between cells and is, therefore, considered a scaffold deployable as a bone substitute.7,8 according to the findings of several studies, ddm is not only biocompatible, but also executes osteoinductive and osteoconductive roles.9,10 nevertheless, demineralized bone and dentin can decrease its antigenic properties.11 the osteoconductive activities of scaffolds can provide a microenvironment supportive of new bone growth. on the other hand, its osteoinductive activities involve growth factors as well as adhesion of exogenous and endogenous progenitor cells to proliferate, differentiate and produce bone cells.12,13 adipose-derived mesenchymal stem cells (admscs) demonstrate a multi-differentiational ability because they contain numerous multipotent progenitor cells and possess differentiation abilities by means of adipogenetic, osteogenetic and condrogenetic pathways.14 therefore, when combined with the appropriate scaffold admscs are expected to improve the healing process of bone damage in the maxilla, mandible and calvarium. the combination of admscs and inorganic bovine bone can even stimulate proliferation and differentiation within osteogenics.15 the viability of hydroxyapatite (ha) derived from tooth bone indicates that this material is non-toxic when cultured in bone marrow mesenchymal stem cells and can be used as an alternative bone graph material.16 hence, this study aimed to determine the cytotoxic characteristics and effects of 355-710 μm-sized ddm scaffolds derived from tooth bone on the admscs of rats using an in vitro technique. materials and methods this research was approved by the research ethics committee, faculty of veterinary medicine, universitas airlangga (number 637-ke). three 4-week old, male, wistar rats were sacrificed to obtain fat tissue from the perirenal region. in order to isolate the admscs, their adipose tissue was washed with phosphat-buffer saline (pbs) containing 10% penicillin-streptomycin mixture (sigma-aldrich, usa). meanwhile, their fat tissue was chopped into small pieces, soaked in 0.2% type i collagenase (worthington, usa), added to pbs and agitated slowly for 40 minutes at 370c. it was then filtered with a 10 μm mesh (spl, korea) before being centrifuged at 1,250 rpm for five minutes to remove the supernatant.17,18 the admscs were cultured with eagle alpha-modified minimum essential medium (α-mem) (gibco, usa) mixed with 15% fetal bovine serum (biowest, usa), 2 mm of l-glutamine (sigma-aldrich, usa), 100 iu/ml of penicillin (sigma-aldrich, usa), 100 mg/ml streptomycin (sigma, usa), and 2.5 μg/ml of fungizone (sigma-aldrich, usa) and subsequently incubated at 370c with 5% co2. the cells were grown in six wells on tissue culture plates (iwaki, japan) at a concentration of 107 in each well. observations were then completed with an inverted microscope at 80x magnification. in cases of nucleus cells that were similar to fibroblast cells, their morphology was evaluated, while their attachment to the plastic culture plates was passaged 4-5 times.19,20 characterization of admscs in the culture media was carried out by means of an immunocytochemical staining technique. single cells derived from the trypsinization process were centrifuged. the pellets and medium were then re-suspended and placed on glass objects, before being incubated at 370 c for one hour. they were subsequently fixated with formaldehyde (bioworld, usa), and washed with pbs. samples plus anti-rat cd 105 fitc (biolegend, usa) and anti-rat cd 45 fitc (biolegend, usa) were incubated 370c for 45 minutes.17,18 the production and processing of scaffolds derived from bovine teeth were conducted at the tissue bank of dr. soetomo general hospital. bovine teeth were removed from the jaws of the subjects with osteotomes, hammers and saws. the teeth were cleaned by immersion in 3% peroxidase for one week and their crowns and roots were subsequently separated using a bone cutter and knable pliers. the process of producing a powder commenced with smashing the tooth roots, composed of dentine tissue and cementum, by inserted them into a bone miller. the resulting powder was sifted to produce particles of the desired size. the demineralization process, incorporating a bone mineral release method, was performed during which ddm particles were soaked in 1% hydrochloric acid (hcl) for one day, then washed thoroughly and dried.21,22 freezedrying of ddm particles was conducted during which they were sublimated into a dry condition in the form of 355710 μm-sized particles and sterilized at batan (jakarta, indonesia). the form of bovine tooth particles was tested by sem (central laboratory, universitas negeri malang). a ddm toxicity test was carried out on admsc cell cultures during which ddm was immersed in x-mem for 24 hours. the admscs were centrifuged and the pellets cultured in 96 wells at a maximum of 5 x 104 cells per well, before being incubated for 24 hours at 370c with 5% co2. after 80% of the cells had developed, they were incubated for 20 hours at 370 c with 5% co2 before being added to 200 μl of the ddm supernatant. this had been immersed in x-mem. 25 μl of 3-(4.5-dimethylthiazol-2yl)-2,5diphenyltetrazolium bromide (mtt) (bioassay system, usa) added to each well and incubated for four hours at 370 c. the results were subsequently observed under an inverted microscope. color changes in the wells were then read by an elisa reader at a wavelength of 595 nm.16,23 all data were expressed at the mean and sd the statistical analysis were performed using one-way anova. p value <0.05 were considered statistically significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 196sari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 194–199 results observation conducted on the first day confirmed the appearance of prospective cells. after three days, the cell growth reached 80% confluence leading to the medium being changed. on the 15th day after phase 4, the cells filling the plates possessed a fibroblast-like shape. the admscs culture results are contained in figure 1. the characteristics of the admscs were observed through immunocytochemical examination to confirm whether those that had been cultured represented mesenchymal stem cells. two markers of mesenchymal stem cells (mscs) were obtained, namely; cd 105 as a positive marker and cd 45 as a negative marker. the immunocytochemical examination results showed a higher number of positive cd 105 markers than the negative cd 45 markers in passage 4 (figure 2). ddm scaffolds with a diameter of between 355 μm and 710 μm were produced at the dr. soetomo hospital tissue bank in surabaya. the results of micro-computed tomography (μ-ct) indicated that the differing diameters of the ddm scaffold particles rendered them heterogeneous. the distribution of particles was then measured from the medial axis. the results confirmed that the average size of ddm scaffold particles was within the range of 355-710 μm (figure 3). sem images of ddm scaffolds indicated the various diameter sizes of the dentinal tubular pores. the smallest diameter was approximately 4.429 μm, while the largest was in the region of 7.519 μm (figure 4). the mtt assay performed produced significantly contrasting results between ddm scaffolds at concentrations of 10%, 50% and 100%. in ddm scaffold at 10% concentration, the average concentration of living admscs was 97.08% with a mean ± sd value of 97.08 + 12.67. meanwhile, in ddm scaffold at a concentration of 50%, the average number of living admscs cells 88.58% with a mean ± sd value of 88.58 ± 12.38. finally, in ddm scaffold at a concentration of 100%, the average number of living admscs cells was 76.64% with a mean ± sd value of 76.64 + 5.76 as shown in figure 5. in addition, the formation of purple formazan crystals indicated that mtt would be reduced by the mitochondria in admscs present in purple formazan compounds and insoluble in water. hence, the higher the intensity of purple, the greater the number of cells that survive (figure 6). discussion in this study, admscs were derived from the adipose tissue of wistar rats that had been passaged four times. during this process, the cells were attached to the plates and increased by approximately 80% with fibroblast-like morphologies (figure 1). the growth of adipose mscs can be sub-cultured up to 9-10 times before the cells degenerate. research conducted by yang et al. (2018) revealed that in vitro mscs derived from bone marrow used for osteogenic purposes are supposed to be extracted in the fourth passage, rather than the eighth, since the former is considered to be the initial passage where cell communication is optimal and cell proliferation maximal. therefore, the most widely used passages of admscs culture in regeneration therapy are passages 1-5.24,25 based on the results of this research, the particle sizes of ddm scaffolds obtained were between 355 μm and 710 μm (figure 3), while the ideal particle size for bone graph material is 500 μm. this means that those obtained in this research were still recommended since resorption will continue for a significant period if the particle size is excessively large, while the particles will be absorbed before they can function as graft material if they are too small. in other words, the material particle size of bone graph can affect bone formation. hence, small scaffold pore size can facilitate osteoblast cell proliferation, while low porosity can help in vitro osteogenic differentiation. newly-formed bone structure is related to the pore size of the scaffolds in that smaller pores can support more trabecular formation.26–28 based on the sem analysis results, the ddm scaffold surface which was 4,429 μm and 7,519 μm in diameter with exposed dentinal tubules, dentine bundle bonds and intermittent peritubular capillaries provided a channel to release proteins and growth factors. therefore, interconnecting porosity is the predominant factor in osteoconduction. however, a number of alternative arguments have been put forward stating that the pore size required for bone growth in implants is 100 to 500 μm.29,30 several factors, such as the size of scaffold pores, must be considered when designing scaffolds since this will affect nutrient diffusion and cell migration throughout the scaffold, inhibit the formation of vascular tissue in the scaffold and facilitate integration with the surrounding vascular tissue. previous research has gone as far as stating that collagen combined with demineralized bone powder with a particle size of 250-500 μm may prove suitable for osteoblast differentiation and, possibly, bone tissue engineering.31,32 the mtt assay indicate that ddm scaffolds at a concentration of 10% are non-toxic to admscs cultures. the mtt assay is used to evaluate material cytotoxicity in tissue engineering as well as to provide an indication of cell growth and proliferation. the results showed that 10% ddm scaffold caused a lower incidence of admsc cell death than ddm scaffolds at concentrations of 50% and 100% (p<0.05) (figure 5). this means that ddm scaffold is non-toxic and can be attached to mesenchymal stem cells derived from the adipose tissue of wistar rats.33 it can be argued that a relationship exists between the cells and scaffold. scaffolds can release a chemical messenger that binds to membrane receptors, subsequently affecting intracellular communication. thereafter, growth factors will bind not only to cell membrane receptors dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 197 sari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 194–199 b a figure 1. admscs culture (a) cells began to grow on the first day after the isolation, and cell colonies attached to the passage plate 1; (b) cells became monolayer with fibroblast-like morphologies in passage 4 (with inverted microscope at 200x magnification). a b figure 2. the immunocytochemical images of admscs at the 4th passage (a) the expression of cd 105 markers looked strong with the presence of green fluorescent cells; (b) the expression of cd 45 markers looked weak marked with the absence of green fluorescent cells (with immunofluorence microscope at 200x magnification). figure 3. the 3d images of μ-ct showing the shape of ddm scaffold particles. figure 4. sem images of ddm scaffolds depicting the presence of pores on the surface of the scaffolds (red arrows indicate dentinal tubular pores at 5000x magnification). 20.000 40.000 60.000 80.000 100.000 120.000 10% 50% 100% cy to to xi ci ty te st s ( % ) cons e ntration (%) ** * figure 5. results of the cytotoxicity tests on ddm scaffolds at different concentrations in admscs cells (significance * p≤ 0.05). a b d c figure 6. the formation of formazan crystals in which the cells became purple (see red arrows). (a) the control group; (b) mtt assay at a concentration of 10%; (c) mtt assay at a concentration of 50%; (d) mtt assay at a concentration of 100% (inverted microscope, 200x magnification). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 198sari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 194–199 initiating the intercellular cascade that affects gene expression and molecular signals released from scaffolds, but also to cell receptors triggering cell intracellular communication. the link between ddm scaffolds and cells occurs through integrin molecules on the cell surface, where cell attachment to scaffold through integrins is very important for the migration, proliferation and differentiation of various cell types.34,35 in conclusion, ddm scaffolds with a pore size of 355-710 μm derived from bone teeth will not produce cytotoxicity effects in admscs. acknowledgement the researchers would like to express their gratitude to the following bodies: the doctoral research section, directorate general of research and developmental enhancement, indonesian ministry of research and technology; the stem cell research and development center, universitas airlangga; the laboratory of the experimental animal stem cell research and development center, universitas airlangga; the postgraduate school of the medical faculty, universitas airlangga; dr. fourier dzar eljabbar latief, micro ct laboratory (bruker microct skyscan 1173), faculty of mathematics and science, bandung institute of technology. references 1. teruel j de d, alcolea a, hernández a, ruiz ajo. comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth. arch oral biol. 2015; 60(5): 768–75. 2. yassen gh, platt ja, hara at. bovine teeth as substitute for human teeth in dental research: a review of literature. j oral sci. 2011; 53(3): 273–82. 3. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125–7. 4. hussain i, moharamzadeh k, brook im, josé de oliveira neto p, salata la. evaluation of osteoconductive and osteogenic potential of a dentin-based bone substitute using a calvarial defect model. int j dent. 2012; 2012: 1–7. 5. tollemar v, collier zj, mohammed mk, lee mj, ameer ga, reid rr. stem cells, growth factors and scaffolds in craniofacial regenerative medicine. genes dis. 2016; 3: 56–71. 6. ahn k-j, kim y-k, yun p-y, lee b-k. effectiveness of autogenous tooth bone graft combined with growth factor: prospective cohort study. j korean dent sci. 2013; 6(2): 50–7. 7. saebe m, suttapreyasri s. dentin as bone graft substitution. songklanakarin dent j. 2014; 2(1): 39–47. 8. dhandayuthapani b, yoshida y, maekawa t, kumar ds. polymeric scaffolds in tissue engineering application: a review. int j polym sci. 2011; 2011: 1–19. 9. kim y-k. bone graft material using teeth. j korean assoc oral maxillofac surg. 2012; 38(3): 134–8. 10. kim y-k, keun j, kim k-w, um i-w, murat m. healing mechanism and clinical application of autogenous tooth bone graft material. in: advances in biomaterials science and biomedical applications. intech; 2013. p. 405–35. 11. murata m, akazawa t, mitsugi m, um i-w, kim k-w, kim y-k. human dentin as novel biomaterial for bone regeneration. in: biomaterials physics and chemistry. intech; 2011. p. 127–40. 12. miron rj, zhang yf. osteoinduction: a review of old concepts with new standards. j dent res. 2012; 91(8): 736–44. 13. gokul k, arunachalam d, balasundaram s, balasundaram a. validation of bone grafts in periodontal therapy a review. int j curr res rev. 2014; 6(14): 7–16. 14. dai r, wang z, samanipour r, koo k, kim k. adipose-derived stem cells for tissue engineering and regenerative medicine applications. stem cells int. 2016; 2016: 1–19. 15. liang l, song y, li l, li d, qin m, zhao j, xie c, sun d, liu y, jiao t, liu n, zou g. adipose-derived stem cells combined with inorganic bovine bone in calvarial bone healing in rats with type 2 diabetes. j periodontol. 2014; 85(4): 601–9. 16. setiawatie em, ulfah n, wahjuningrum da, sari ds, rubianto m. viability bovine tooth hydroxiapatite on bone marrow mesenchymal stem cells. in: international medical device and technology conference. johor bahru: universiti teknologi malaysia; 2017. p. 100–4. 17. lotfy a, salama m, zahran f, jones e, badawy a, sobh m. characterization of mesenchymal stem cells derived from rat bone marrow and adipose tissue: a comparative study. int j stem cells. 2014; 7(2): 135–42. 18. bunnell b, flaat m, gagliardi c, patel b, ripoll c. adipose-derived stem cells: isolation, expansion and differentiation. methods. 2008; 45(2): 115–20. 19. strioga m, viswanathan s, darinskas a, slaby o, michalek j. same or not the same? comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. stem cells dev. 2012; 21(14): 2724–52. 20. kamal a, iskandriati d, dilogo i, siregar n, hutagalung e, yusuf a, mariya s, husodo k. comparison of cultured mesenchymal stem cells derived from bone marrow or peripheral blood of rats. j exp integr med. 2014; 4: 17–22. 21. malinin ti, temple ht, garg ak. bone allografts in dentistry: a review. dentistry. 2014; 4(2): 1–8. 22. um i-w, cho w-j, kim y-k. experimental study on human demineralized dentin matrix as rhbmp-2 carrier in vivo. j dent appl. 2015; 2(7): 269–73. 23. abdelfattah mi, nasry sa, mostafa aa. characterization and cytotoxicity analysis of a ciprofloxacin loaded chitosan/bioglass scaffold on cultured human periodontal ligament stem cells: a preliminary report. open access maced j med sci. 2016; 4(3): 461–7. 24. yang y-hk, ogando cr, wang see c, chang t-y, barabino ga. changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. stem cell res ther. 2018; 9: 131. 25. ikebe c, suzuki k. mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols. biomed res int. 2014; 2014: 1–11. 26. koga t, minamizato t, kawai y, miura k, i t, nakatani y, sumita y, asahina i. bone regeneration using dentin matrix depends on the degree of demineralization and particle size. papaccio g, editor. plos one. 2016; 11: 1–12. 27. yu j, xia h, ni q-q. a three-dimensional porous hydroxyapatite nanocomposite scaffold with shape memory effect for bone tissue engineering. j mater sci. 2018; 53(7): 4734–44. 28. sundelacruz s, kaplan dl. stem celland scaffold-based tissue engineering approaches to osteochondral regenerative medicine. semin cell dev biol. 2009; 20(6): 646–55. 29. nassif l, el sabban m. mesenchymal stem cells in combination with scaffolds for bone tissue engineering. materials (basel). 2011; 4(10): 1793–804. 30. de oliveira g, miziara m, silva e da, ferreira e, biulchi a, alves j. enhanced bone formation during healing process of tooth sockets dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 199 sari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 194–199 filled with demineralized human dentine matrix. aust dent j. 2013; 58(3): 326–32. 31. shimauchi h, nemoto e, ishihata h, shimomura m. possible functional scaffolds for periodontal regeneration. jpn dent sci rev. 2013; 49(4): 118–30. 32. thitiset t, damrongsakkul s, bunaprasert t, leeanansaksiri w, honsawek s. development of collagen/demineralized bone powder scaffolds and periosteum-derived cells for bone tissue engineering application. int j mol sci. 2013; 14: 2056–71. 33. chen q, shou p, zheng c, jiang m, cao g, yang q, cao j, xie n, velletri t, zhang x, xu c, zhang l, yang h, hou j, wang y, shi y. fate decision of mesenchymal stem cells: adipocytes or osteoblasts? cell death differ. 2016; 23(7): 1128–39. 34. steward aj, liu y, wagner dr. engineering cell attachments to scaffolds in cartilage tissue engineering. jom. 2011; 63(4): 74–82. 35. sanz ar, carrión fs, chaparro ap. mesenchymal stem cells from the oral cavity and their potential value in tissue engineering. periodontol 2000. 2015; 67: 251–67. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p194–199 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p194-199 vol 38-no4-2005-isi.pmd 183 deciduous teeth eruption in full and mosaic type of down’s syndrome patient willyanti sjarif department of pediatric dentistry faculty of dentistry padjadjaran university bandung indonesia abstract the purpose of this study was to examined the correlation of deciduous teeth eruption with the karyotipe of) down’s syndrome patient. full and mosaic karyotype in down’s syndrome (ds) patients have different prognostics. a total of 33 ds patients constituted of 23 full and 10 mosaics were enrolled in this cross sectional study. the chi-square statistical test was utilized to analyze the data. the result revealed that a full trisomy ds patients had their deciduous teeth erupted in 13-18 months old while a mosaic ds patients in 8-12 months old. the conclusion affirmed that the deciduous teeth eruption in mosaic ds patients is earlier than full ds patients (p = 0.002). key words: down’s syndrome, mosaic, full, karyotype, deciduous teeth, eruption correspondence: willyanti sjarif, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jln. sekeloa selatan no. 1 bandung, indonesia. introduction the terminology down’s syndrome was named after a british physician, langdon down who in 18661,2 described patient’s clinical features of the syndrome. the trisomy 21 down’s syndrome was initially called mongolism, but many people thought that it was too racialistic. the term down’s syndrome is more often used among researchers.[, it is known as down’s syndrome (ds).] 1,2,3 down’s syndrome (ds) incidence is quite high. in mothers under 30 years old, the incidence is one in every 1500 birth and the incidence increase to one every 65 birth if the mothers older than 45 years old. the incidence of down’s syndrome tends to increase along with mother’s age.1,2 the older the mothers, the higher the risk to have a down’s syndrome child. down’s syndrome is a syndrome caused by an extra chromosome in chromosome 21. the etiology of ds is a non-disjunction during meiosis resulting in one extra chromosome in chromosome 21. important factors influencing the non-disjunction process is mother’s age during pregnancy and labor. the incidence of ds increase in the mothers older than 35 years of age during labor.1-4 down’s syndrome (ds) clinical features are low birth weight, short, microcephaly, flat head, flat face, low set ear, straight soft hair, up slanting eyes, syndactyly and clinodactyly on metacarpal and phalanges, simian crease with a sandal gap between the first and second toes.1,4-6 the prominent features are macroglossia, fissured and geographic tongue, high palatum and hypotonia. several missing teeth, delayed deciduous teeth eruption and exfoliation are also observed. the teeth are smaller and conus. the hypotonia causes stick-out tongue, openedmouth and drooling. down’s syndrome (ds) patients have delayed body and dental growth and development. their deciduous and permanent teeth have delay eruption, and dental anomaly in) structure and morphology e.g. hypoplatia, conus. a normal person has 46 chromosomes 23 pairs consisted of 22 pairs autosom al chromosom and 1 pair of sex-chromosome in each body cell. every pair of autosom al chromosom is numbered from one to twenty-two. according to its etiology, ds is divided in to 3 types, i.e.: mosaic, full and translocated. down’s syndrome (ds) or trisomy 21 is an anomaly caused by chromosome’s disturbance with varied clinical manifestations. the majority shows a full trisomy (full karyotype) which demonstrates an extra chromosome in chromosome 21, with 47 chromosomes in every cell. the mosaic type ds has normal chromosomes (46) in several cells, thus it shows lighter physical characteristics and a better mental condition. while the full type ds shows more severe clinical features. the mosaic type has two cell populations in his body, some cells contain normal chromosome complements with 2 chromosome 21, whereas other cells have 3 chromosome 21 (a joint cell with chromosome 46 and 47).6 the mosaic ds is a type with a variation of ds and normal condition. whenever there is only few amount of cells undertake trisomy, the clinical signs are very difficult to find, thus it is hard to diagnose ds. the bigger the cell proportion with normal chromosome, the higher possibility of normal appearance.2 184 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 183–184 the possibility of mosaic occurrence is due to a deviation of the 2nd and the 3rd zygote meiosis, resulting in 46 chromosomes and 47 chromosomes with an extra chromosome in chromosome 21. the purpose of this study was to examined the correlation of deciduous teeth eruption with the karyotipe of down’s syndrome patient mosaic and full type. materials and method the study was a cross-sectional research. the subject was patients with pediatrician’s diagnosed ds, in range of age between 7 to 18 months. the ds was confirmed based on clinical and chromosome examinations. karyotyping was used to analize the chromosome, and trisomy 21 defined if an extra chromosome in chromosome 21 was found. the mosaic ds type was determined if there are more than 40% normal cell (cell with 46 chromosomes), while the full ds type was determined if the number of normal cells are less than 40%. the deciduous teeth eruption was defined if the white sign appear in the oral mucosa and the number of erupted deciduous teeth. chi-square was used to analize the data. results table 1. the duration of healing ras' ulcer in the female students with and without history of cp deciduous teeth eruption time (month) trisomy n 8-12 month 13-18 month mosaic 10 10 full 23 12 11 chi square p = 0.002 table 1 showed the deciduous eruption time of the samples. eleven out of 23 full type patients, had teeth eruption between 13 to 18 months old (p = 0.002). the results showed that the full type ds had a delayed deciduous teeth eruption, while the mosaic type showed the eruption of deciduous teeth occurred in the age of 8 to 12 months. discussion normally, deciduous teeth eruption begins from the mandibular central incisor at the age of 4– 6 months.7,8 delay of deciduous teeth eruption is often found in ds patients. the ds patients showed a disturbance in eruption and [the] eruption order. the first erupted teeth is mandibular central incisor and the latest eruption is second primary molar. the primary central incisor in trisomy 21 erupted in 12 to 13 months of age. it could erupted in 24 months old, with (the completition of primary tooth eruption) in 4 to 5 years of age.5 many ds patients had no teeth until the age of 2 years old.7 primary teeth eruption is influenced by gene and the environment. the presence of three chromosome 21 can caused the disturbance of growth and development in children, including dental growth and development in particularly the primary teeth eruption.6,7 the mosaic type has a combination of normal karyotypes and ds karyotypes. the mosaic ds has many normal cells (46 chromosomes) than the full type (47 chromosomes). it explained why the ds mosaic type has lighter physical signs than the full type. the same experience happened in deciduous teeth eruption. this research revealed that the primary teeth in mosaic ds erupted earlier than the full ds type. there was a significant difference between the primary teeth eruption of the full type ds than the mosaic type.) the finding was in accord ance with the statement that heavier physical evidences in full type ds1,2 due to the presence of more abnormal cells 47 chromosomes. the delayed eruption of deciduous teeth correlated with a delayed development in ds patients, including delayed physical and mental development.4-6,9 the conclusion are the deciduous teeth eruption of full type ds was more delayed than of the mosaic type ds. the eruption in the full type ds occurred in 13 months of age and in the mosaic type occurred less than 11 months of age. references 1. smith, wilson: the child with down syndrome. philadelphia: wb saunders co; 1973. p. 20-5. 2. smith’s. recognizable pattern of human malformation. 5th ed. philadelphia: wb saunders; 1997. p. 10–2. 3. nowak aj. dentistry for the handicapped patient joint lower. st louis: the cv mosby; 1976. p. 46–8, 302–14. 4. jones kl. morphogenesis and dysmorfogenesis: smith recognizable pattern of human malformation. 5th ed. philadelphia: wb saunders; 1997. p. 695–7. 5. limbrock g, fischer h, avalle c, castillo m. orofacial therapy treatment of 67 children with down syndrome. dev, med chilled new york 1991; 33:196–303. 6. welburry r. pediatric dentistry. 2nd ed. new york: oxford university; 2001. p. 294, 394– 5. 7. stewart re. pediatric dentistry. st louis: cv mosby; 1982. p. 835–7. 8. sindoor sd. down syndrome a review of the literature. oral surgery, oral medicine, oral pathology and pedodonties 1997 september; 84(3):279–85. 9. sjarif w, oewen r. sindroma down mosaik, sudut pandang bidang kedokteran gigi anak. buku ilmiah piikga iv idgai jabar, 2003; h. 159-64. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice subjects index volume 55 400mcg, 76 acute pseudomembranous candidiasis, 105 adenoid cystic carcinoma, 231 adherence, 21 ageing population, 16 alveolar bone thickness, 148 osteitis, 7 ameloblastic carcinoma, 174 ameloblastoma, 137, 174 angular measurement, 200 animal model, 209 annona, 130 anterior teeth, 44 temporalis muscle activity, 38 antimicrobial peptide, 93 antineoplastic agent, 130 anxiety, 99 aphthous-like ulcer, 49 aplastic anaemia, 49 apoptosis, 56 artificial intelligence, 125 bilateral ramus mandibulectomy, 174 biofilm, 21 biological age, 13 bubbles formation, 71 c.albicans, 21, 209 calcium hydroxide, 62 cancer, 186 candida albicans, 105 ch–barium sulfate, 62 child, 120 children, 105 chronological age of 9-15 years, 88 cleft lip, 221 and palate, 76 cleft palate, 221 cocoa bean, 7 complete denture, 179 covid-19, 99, 154 cvm, 13 cytotoxic t-lymphocyte antigen-4 (ctla-4), 194 cytotoxicity, 130 demirjian, 161 demirjian’s method, 13 dental age estimation, 161 health education, 215 students, 142 treatment, 120 visit, 154 dentist, 99 dentistry, 114 , 161, 215, 235 diastema closure, 44 direct composite, 44 effective suction method, 179 egcg, 114 endoactivator, 71, 125 endodontics, 71, 125 enterococcus faecalis, 62 exercise, 56 fibroblasts, 81 first trimester,76 fissure sealant, 67 fluoride release, 226 folic acid, 76 follicular, 137 fonseca anamnestic index, 204 forensic odontology, 161 gc saliva-check buffer, 120 gender, 200 genetic, 221 giomer, 226 glass ionomer cement, 33 gourami fish scale powder, 33 health care facilities, 154 herbal medicine, 165 il-17, 209 immune escape, 194 immunohistochemistry, 137 impacted dilacerated canine, 240 infectious disease, 209 interdisciplinary, 109 intrusion, 109 iodophors, 62 jaw exercises, 204 kawamura, 142 knowledge, 99 lateral cephalogram, 200 light force, 240 lime (citrus aurantifolia swingle) peel extract, 81 macrophage, 93 malocclusion, 13 mandibular, 88 length growth, 13 maxillary, 88 medicine, 209, 235 meta-analysis, 165 microbiota, 186 microleakage, 67 moderate-intensity exercise, 56 mucoadhesive gingival patch, 114 natural frequency, 125 neutrophil, 93 nicotine, 93 oral cancer, 56 cavity, 186 hygiene, 142 lichen planus, 165 rating index (ori), 142 squamous cell carcinoma, 56 ulcer, 49 orthodontic, 109 appliance, 235 traction, 240 treatment, 240 osteoblast, 114 osterix, 26 p16 protein expression, 137 pathogenesis; 1 pathologic tooth migration, 109 patient satisfaction, 62 periodontal disease, 114 dressing, 81 maintenance therapy, 231 management, 231 periodontitis, 16, 93, 109, 231 physical activity, 56 plate reconstruction, 174 pleomorphic adenoma, 1 plexiform, 137 porosity, 33 porphyromonas gingivalis, 62 portunus pelagicus, 26 pregnancy, 76 preheating; 226 preparation technique, 67 progressivity, 1 retraction, 148 risk factors, 221 saliva analysis, 120 scaffold, 26 school dental health efforts, 215 semi-adjustable articulator, 179 severity, 16 shear stress, 125 smartphone apps, 204 socket healing, 7 preservation, 26 sonic, 71 squamous cell carcinoma of head and neck, 130 superficial masseter muscle activity, 38 surface electromyography, 38 teledentistry, 49, 154 telemedicine, 204 tinospora crispa l, 21 tipping, 148 tooth extraction, 7 impacted, 235 movement technique, 235 torque, 148 transform cells, 56 tumor necrosis factor-α, 26 unilateral posterior crossbite, 38 warthin’s tumor, 194 wild p53, 56 willems, 161 wound healing, 81 authors index volume 55 agung, i gusti ayu ari, 215 akleyin, ebru, 120 alyayuan, helsa, 200 andriani, ika, 93 ari, muhammad dimas aditya, 179 balbeid, merlya, 99 bhanuwati, areta vania, 130 chairunnisa, ricca, 204 dewi, alfira putriana, 13 fauzia, malianawati, 81 femilian, afryla, 105 handayani, fani tuti, 240 hutomo, suryani, 21 irmawati, anis, 56 isnandar, 7 izach, agnes imelda, 38 lay, eunike, 174 lestari, ayu asri, 154 louisa, marie, 109 lubis, hilda fitria, 88 neo, kevin chee pheng, 231 peeters, harry huiz, 71, 125 prasetyo, eric priyo, 62 pribadi, nirawati, 44 qotrunnada, alvionika nadyah, 194 ramadhani, yeka, 114 rusdy, hendry, 76, 221 salim, irvan, 26 sari, gesti kartiko, 67 sari, suci purnama, 148 setiabudi, tirza oktarina, 142 sosiawan, agung, 161 sufiawati, irna, 186 sulistyani, erna, 209 syafriadi, mei, 1 syahdinda, meralda rossy, 235 talahatu, lani berlina, 49 vychaktami, kharissa kemala, 165 widodo, haris budi, 137 winanto, muthiary nitzschia nur iswary, 226 wulandari, erawati, 33 wulandari, pitu, 16 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. all manuscripts submitted to the journal must be written in english. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak1, matsuo hamada2, ninuk hartati3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur, malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima, japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne, australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in original articles should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in review articles should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (original articles, case reports, or review articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission  the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair. ac.id/mkg/about/submissions#onlinesubmissions  the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id 192 volume 45 number 4 december 2012 research report molecular detection of interleukin-1a +4845gt gene in aggresive periodontitis patients chiquita prahasanti1 and harianto notopuro2 1 department of periodontics, faculty of dentistry, universitas airlangga 2 department of biochemistry, faculty of medicine, universitas airlangga surabaya indonesia abstract background: abundant researches had been conducted based on the clinical and histopathological pathogenesis of aggresive periodontitis. nevertheless, there were still few researches which based on molecular biology, and especially related to gene polymorphism. this study was done based on il-1a +4845gt gene polymorphism in aggressive periodontitis patients. purpose: the purpose of this tudy was to characterized the generic variation of il-1a +4845gt as a risk factor aggressive periodontitis and chronic periodontitis. methods: dna from patients with aggressive periodontitis and chronic periodontitis was taken determination of il-1a +4845gt polimorphism was conducted with pcr-rflp technique. results: homozygous allele tt polymorphism was not found in all samples, only allele gg (wild type) and allele gt (heterozygous mutant) were not affect aggressive periodontitis and chronic periodontitis. conclusion: the study showed there was no significant association between il-1a +4845gt gene polymorphism and aggressive periodontitis and chronic periodontitis. key words: interleukin-1a +4845gt, gene polymorphisms, aggressive periodontitis, chronic periodontitis, pcr-rflp abstrak latar belakang: penelitian tentang patogenesa periodontitis agresif berdasar klinis dan histopatologi telah banyak dilakukan, akan tetapi penelitian berdasar biologimolekuler terutama polimorfisme gen masih sangat jarang dilakukan. penelitian ini dilakukan berdasarkan pada polimorfisme gen il-1a +4845gt pada penderita periodontitis agresif. tujuan: tujuan dari penelitian ini adalah untuk mengetahui variasi genetik dari il-1a +4845gt yang merupakan faktor risiko periodontitis agresif dan periodontitis kronis. metode: dna dari penderita periodontitis agresif dan periodontitis kronis diisolasi, selanjutnya dilakukan determinasi dari polimorfisme gen il-1a +4845gt dengan menggunakan teknik pcr-rflp. hasil: pada seluruh sampel penelitian ini tidak dijumpai polimorfisme allel tt (homosigot mutan), yang didapat adalah jenis allel gg (wild type) dan allel gt (heterosigot mutan) yang tidak berpengaruh terhadap periodontitis agresif dan periodontitis kronis. kesimpulan: polimorfisme gen il-1a +4845gt tidak mempunyai hubungan terhadap kejadian periodontitis agresif dan periodontitis kronis. kata kunci: interleukin -1a +4845gt, polimorfisme gen, periodontitis agresif, periodontitis kronis, pcr-rflp correspondence: chiquita prahasanti, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132. e-mail: chiquita_prahasanti@yahoo.com 193prahasanti and notopuro: molecular detection of interleukin-1a+4845gt introduction periodontitis is commonly found in society, such as cardiovascular disorders, diabetes, and other complex diseases due to various factors. periodontitis, however, is an infectious disease commonly found in periodontal tissues in oral cavity caused by various factors and later can cause periodontal tissue damage. it is known that the periodontitis was triggered by bacterial plaque and defense mechanisms of host, so the understanding of the relationship between host and oral bacteria is the basis understanding of the pathogenesis of periodontal disorder.1 many studies have reported that smoking, diabetes, and genetic factors can increase the severity risk of periodontitis. determination of the etiology of severe of periodontal disease continues to be discussed by researchers, who stated that environmental and genetic are factors contributing to the occurrence of periodontitis.2 many studies showed that genetic factors influence the occurrence of periodontitis. genetic polymorphism is closely related to the variety of clinical conditions of periodontitis. based on research conducted, it is known that the variation of the host immune response is associated with genetic factors, and plays an important role in the occurrence of aggressive periodontitis and chronic periodontitis.1,3 in the united states of america (usa), it is also known that race gives strong influence on the occurrence of periodontitis. several studies have shown that the occurrence of aggressive periodontitis in africanamericans is higher than in the white race (caucasoid), and this condition shows that the african-americans are vulnerable to aggressive periodontitis, about 10%.4,5 cytokines, interleukin-1, is a pro-inflammatory protein with cytokine functions, such as as chemotactic factors playing a role in the onset of inflammatory, and as a mediator and regulator of inflammatory responses in the host innate immune system which plays in a number of biological activities, including proliferation, homeostasis, regeneration, reparation and inflammation.6,7 biological effects of il-1 depend on the number of cytokines that are released at low levels with their main function as local inflammatory mediators. meanwhile, il-1 at high levels moves into circulation, and invited endocrine effects. there are three genes that play a role to regulate the production of il-1: il-1a, il-1b, and il-1 rn. il-1a gene associated with the production of il-1α cytokines is associated with the occurrence of inflammation.8 in in vitro study, it is known that there is a direct relationship between il-1 genotype and the amount of cytokine secreted in the culture macrophages.9,10 il-1a polymorphism is associated with the severity of periodontitis.11 based on it, it was necessary to characterize the genetic variation of il-1a +4845gt as a risk factor for aggressive periodontitis and chronic periodontitis as a control group, especially in societies in surabaya because until now there are no data about the characterization of the il1a gene. this research, therefore, was expected to reveal the basic pathogenesis of aggressive periodontitis and chronic periodontitis as well as to be able to be used in determining the basic treatments of patients with aggressive periodontitis and chronic periodontitis. genetic polymorphism of il-1a +4845gt was a point mutation at nucleotide number +4845 from guanine becomes thymine. materials and methods this study was considered as an observational analytic study with a case-control study design in patients who suffer from aggressive periodontitis and chronic periodontitis. further gene variant test was also conducted on il-1a +4845gt with pcr-rflp. the subjects of study were patients with aggressive periodontitis and chronic periodontitis who came to periodontics clinics dental hospital universitas airlangga have gained ethical clearance. the venous blood of all patients with aggressive periodontitis and chronic periodontitis were taken for about 3 ml by an analyst, and then extracted for its dna. dna amplification was conducted by using pcr for about 32 cycles. primer specific sequences of il-1a +4845gt used were f: 5’-atg gtt tta gaa atc atc aag cct agg gca-3’ and r: 5’-aat gaa agg agg gga gga tga cag aaa tgt-3’. afterwards, visualization of pcr il-1a product was conducted by 3% agarose gel and added with 1μl ethidium bromide, then it placed in submarine gel agarose electrophoresis apparatus at 100v, 70 mamp, for 40 minutes, and recorded by using gel doc system. determination of il-1a +4845gt polymorphisms was conducted with pcr-rflp technique by using restriction endonuclease enzymes; fnu4hi. criteria for il-1a +4845gt polymorphism were: (a) allele gg: 124 bp + 29 bp (wild type); (b) allele tt: 153 bp (homozygous mutant); (c) allele gt: 153 bp and 124 bp + 29 bp (heterozygous mutant). restriction endonuclease enzyme incubated at 37°c for overnight in waterbath incubator. then, digestion fragments of dna by rflp were detected by electrophoresis using 3% high resolution of agarose mixed with 1 μl ethidium bromide and then run on gel electrophoresis at 100 volts for 40 minutes. finally, the results were read and recorded by using gel doc system. results patients who have abnormalities in this study were majority women with both aggressive periodontitis and chronic periodontitis. among patients with aggressive periodontitis, 26 subjects were female, and 11 subjects were male. meanwhile, among patients with chronic periodontitis, 22 subjects were female, and 12 subjects were male (figure 1), that there was no significant difference 194 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 192–196 between male patients and female by statistical test (chi square p = 0.821). the role of interleukin-1 in periodontal disease, based on in vitro and in vivo studies, can be mentioned as a factor causing degradation of extracellular matrix as well as damage to alveolar bone. it has been mentioned that table 2. distribution alleles of interleukin–1a +4845gt gene on aggressive periodontitis genotype n frequency % allele 1 g allele 2 t 62 12 83.8 16.2 total 74 100.0 table 3. distribution genotype polymorphism of interleukin– 1a +4845gt gene on chronic periodontitis genotype n frequency % allele 1 gg allele 1.2 gt allele 2 tt 21 13 0 61.7 38.3 0.0 total 34 100.0 table 4. distribution alleles of interleukin-1a +4845gt gene on chronic periodontitis genotype n frequency % allele 1 g allele 2 t 55 13 80.9 19.1 total 68 100.0 table 1. distribution genotype polymorphism of interleukin– 1a +4845gt gene on aggressive periodontitis genotype n frequency % allele 1 gg allele 1.2 gt allele 2 tt 25 12 0 67.5 32.5 0.5 total 37 100.5 0 5 10 15 20 25 30 p agresif p kronis perempuan laki-laki women female aggressive periodontitis chronic periodontitis figure 1. distribution of patients based on gender. polymorphism more influenced by racial factors, thus, the results of a study conducted on different races is likely to give different results. genotype frequency of il-1a +4845gt on aggressive periodontitis was about 67.5% allele gg (wild type), 32.5% allele gt (heterozygote mutant), and no one (0%) allele tt (homozygous mutant). meanwhile, the frequency of the mutant alleles t in aggressive periodontitis was about 16.2% (table 1 and 2). genotype frequency of il-1a +4845gt in chronic periodontitis was 61.7% allele gg (wild type), 38.3% allele gt (heterozygote mutant), and no one (0%) allele tt (homozygous mutant). while the frequency of the mutant allele t in chronic periodontitis was about 19.1% (table 3 and 4). in this study, we found that allele tt (homozygous mutant) is not found in all samples, but allele gt is the most polymorphism appeared (heterozygous mutant). based on figure 2, it shown that there were difference in the distribution of gene polymorphism in aggressive periodontitis and that in chronic periodontitis. incidence of alleles gt in patients with aggressive periodontitis was the same as that in patients with chronic periodontitis, nearly about 32.5% and 38.2%. by using fisher’s exact test, the significant value obtained was about 0.629. thus, if α used is about 5%, there will be no different between gene polymorphisms of il-1a gene +4845gt in aggressive periodontitis and that in chronic periodontitis. discussion the results of this study showed that based on gender there was no difference between women and men, which means that every individual has the same risk for getting the disease. based on the number of people, the number of women was higher than that of men probably due to the fact that more women concern with their aesthetic and appearance than men do so that defects in women’s oral cavity will soon be detected earlier. moreover, periodontal figure 2. cross tabulation between the aggressive and chronic periodontitis samples and the polymorphism types. male female 0 5 10 15 20 25 30 p agresif p kronis perempuan laki-laki women female aggressive periodontitis chronic periodontitis 195prahasanti and notopuro: molecular detection of interleukin-1a+4845gt disorder is known as a disease causing no complaint to the sufferer, and the other possibilities, such as puberty in early age, hormonal changes during menstruation, and pregnancy even can also worsen the clinical condition of periodontitis patients. another researchers found that the majority of the sample was female, and also found men and women have the same representation in these disorders.7,13 many studies conducted to examine the role of gene polymorphisms on the host response of patients with periodontitis. gene polymorphisms that occur in patients with periodontitis are likely to affect protein expression they produce, and will ultimately change the cell morphology and function affecting innate and adaptive immune responses, and possibly manifested in the clinical condition of the patients. gene polymorphism of il-1a +4845gt in this study was type of heterozygous mutant affected on aggressive periodontitis later also affected protein expression. based on the results of this study, it is also known that gene polymorphisms of il-1a +4845gt, allele gt were mostly found in patients with aggressive and chronic periodontitis as disease risk markers. gonzales et al.,14 did not find gene polymorphisms of il-1a +4845gt, but gulzerdemir et al.,7 they found few gene polymorphisms of il-1a +4845gt allele gt. these results indicate that race factor can influence the characteristics occurence of gene polymorphisms. some studies found that single nucleotide polymorphisms (snps) can affect genes encoding pro-inflammatory cytokines, such as il-1a genes that play a role in chronic and aggressive periodontitis pathogenesis. number of genotypes and alleles may differ in sick people and healthy ones. thus, since detected allele can be associated with the disease a research can be conducted to understand role of genes as etiology or a risk factor.15 patients with positive mutant genotypes showed statistically lower igg antibody than those with negative mutant genotype. it indicates that body response to periodontal bacterial pathogens also reduced.16 it is also known that patients with polymorphisms will have higher bacterial pathogen, and a low antibody titer will against periodontal bacteria.17,18 the response of il-1a gene polymorphisms with periodontal treatment then reported that patients with an allele tt polymorphism did not show a good response to the treatment using guided tissue regeneration.19 on the other hand, patients who obtained non-surgical treatment after being evaluated for two years were not associated with genotype polymorphism.20 therefore, more information and research are needed to understand the biological influences on the clinical effects of il-1a gene polymorphisms in order to set a more accurate treatment to get better results. in addition, since periodontitis is considered as multifactorial disease, it requires a lot of data and research to get a clear association between genetic factors, pathogenic process, and clinical condition. however, in this study it can be concluded that there is no difference found between polymorphism in aggressive periodontitis and that in chronic periodontitis, and also no specific genetic biomarkers for aggressive periodontitis and chronic periodontitis. further studies with larger samples are needed. studies about other genes are needed since periodontics is a polygenic disease that needs control group from a healthy persons (normal group). acknowledgments this research can be carried out in cooperation between department of periodontics, faculty of dentistry, universitas airlangga and department of biochemistry, faculty of medicine, universitas airlangga, as well as the funding of dp2m of national higher education directorate references 1. newman mg, takei n, klokkevold p, carranza f. carranza’ clinical periodontology. 11th ed. wb saunders; 2011. p. 16881, 409-14. 2. sakellari d, koukoudetsos s, arsenakis m, kontantinidis a. a prevalence of il-1a and il-1b polymorphisms in a greek population. j clin periodontol 2003; 30: 35–41. 3. hart tc, atkinson jc. mendelian forms of periodontitis. periodontol 2000, 2007; 45: 95–112. 4. levin l, baev v, lev r, stabholz a, ashkenazi m. aggressive periodontitis among young israeli army personnel. j periodontol 2006; 77: 1392–6. 5. meng h, xu l, li q, han j, zhao �. determinants of host susceptibility in aggressive periodontitis. periodontology 2000, 2007; 43: 133–59. 6. chen h, wilkins lm, aziz n, cannings c, wyllie dh, bingle c, rogus j, beck jd, offenbacher s, cork mj, kolpin mr, hsieh cm, kornman ks, duff gw. single nucleotide polymorphisms in the human interleukin-1b gene affect transcription according to haplotype context. human molecular genetic 2006; 15(4): 519–29. 7. guzeldemir e, gunhan m, ozcelik o, tastan h. interleukin-1 and tumor necrosis factor-α gene polymorphisms in turkish patients with localized aggressive periodontitis. j of oral science 2008; 50(2): 151–9. 8. arab hr, afshari jt, radvar m, moeintaghavi a, sargolzaei n, rigi a, shirkhani m. il-1b +3954 genetik polimorfisme association with generalized aggressive periodontitis in khorasaniran province. j med sci 2007; 7(2): 222–7. 9. agrawal aa, kapley a, �eltiwar rk, purohit hj. assessmen of single nucleotide polymorphism at il-1a+4845 and il-1b+3954 as genetic susceptibility test for chronic periodontitis in maharashtrian ethnicity. j periodontol 2006; 77(9): 1515–21. 10. kowalski j, gorska r, dragan m, and kozak i. clinical state of the patients with periodontitis, il-1 polymorphism dan pathogens in periodontal pocket – is there a link? (an introductory report). advances in medical sciences 2006; 51: 9–12. 11. poulsen ah, sorensen lk, bendtzen k, holmstrup p. polymorphisms within the il-1 gene cluster: effects on cytokine profiles in peripheral blood and whole blood cell cultures of patients with aggressive periodontitis, juvenile idiopathic aethritis, and rheumatoid arthritis. j periodontal 2007; 78: 475–92. 12. nikolopoulos gk, dimou nl, hamodrakas sj, bagos pg. cytokine gene polymorphisms in periodontal disease: a meta-analysis of 53 studies including 4178 cases and 4590 controls. j clin periodontol 2008; 35: 754–67. 196 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 192–196 13. brett pm, zygogianni p, griffiths gs, tomaz m, parkar m, d’aiuto f, tonetti m. functional gene polymorphisms in aggressive and chronic periodontitis. j dent res 2005; 84(12): 1149–53. 14. gonzales jr, michel j, rodrıguez el, herrmann jm, bodeker rh, meyle j. comparison of interleukin-1 genotypes in two populations with aggressive periodontitis. eur j oral sci 2003; 111: 395–9. 15. loos bg, john rp, laine ml. identification of genetik risk factors for periodontitis and possible mechanisms of action. j clin periodontol 2005; 32(suppl. 6): 159–79. 16. papapanou pn, neiderud am, sandros j, dahlẻn g. interleukin-1 gene polymorphism and periodontal status. a case-control study. j clin periodontol 2001; 28: 389–96. 17. agerbaek mr, lang np, persson gr. microbiological composition associated with interleukin-1 gene polymorphism in subjects undergoing supportive periodontal therapy. j periodontol 2006; 77(8): 1397–402. 18. wagner j, kaminski we, aslanidis c, moder d, hiller k-a, christgau m, schmitz g, schmalz g. prevalence of opg and il-1 gene polymorphisms in chronic periodontitis. j clin periodontol 2007; 34: 823–27. 19. de sanctis m, zucchelli g. interleukin-1 gene polymorphisms and long-term stability following guided tissue regeneration therapy. j periodontol 2000; 71: 606–13. 20. lang np, tonetti ms, suter j, sorrell j, duff gw, kornman ks. effect of interleukin-1 gene 1 polymorphism on gingival inflammation assessed by bleeding on probing in a periodontal maintenance population. j periodontal res 2000; 35: 102–7. 167 volume 46, number 3, september 2013 ukuran kranial dan indeks sefalik pada anak retardasi mental (cranial size and cephalic index of mentally retarded children) dewi elianora,1 iwa sutardjo2 dan bambang udji rianto3 1bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas baiturrahmah padang – indonesia 2bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas gadjah mada yogjakarta – indonesia 3fakultas kedokteran universitas gadjah mada yogjakarta – indonesia abstract background: mental retardation is imperfect condition of mental development which resulted in delay of motoric development, speech and in adaption with the environment. the common symptoms is brain growth disorder, which affects the cranial size and the intelectual function lower than average (<70). purpose: this study was aimed to determine the difference of cranial size and cephalic index of mentally retarded children compared with normal chilren based on antropometry and cephalometric measurement. methods: this research was epidemiology analytic observational with case control design. the cranial size and cephalic index measurements were carried out on 168 children in range of age 7-12 years old (84 were moderate mental retarded children and 84 were normal children). data was statistically analyzed with t-test. results: the size of cranial and cephalic on index on mentally retarded children were smaller than normal children. s-n and g-op size were shorter than normal children, the results of s-n differences (-4.4), s-ar (-2.38) and g-op (-5.5), eu-eu (-8.24). the results analysis of linear and angle component cranial base (s-n, s-ar and <nsar) was significantly smaller than normal children. based on age and genders, and the normal child was normocephalic. odds ratio value of the head profile based on age groups 2.10 times (ci 0.290-3.390) compared to normal children 0.99 (ci 0.025-0.375). based on gender the odds ratio value 1.469 times (ci 0.429-5.035) compared normal child 0.562 (ci 0.19-1.65). the head length and cranial base had related with gender. cranial size on boys were bigger than girls. conclusion: it is concluded that the cephalic index and the size of cranial base of mentally retarded children were smaller than normal children. key words: antropometry, lateral cephalometric, mental retardation, children abstrak latar belakang: retardasi mental merupakan ketidaksempurnaan perkembangan mental yang mengakibatkan keterlambatan perkembangan motorik, bicara dan penyesuaian diri dengan lingkungan. gejala umum adalah gangguan pertumbuhan otak yang berpengaruh terhadap ukuran kranial dan fungsi intelektual (quetient intelegence) rendah (kurang dari 70). tujuan: penelitian ini bertujuan mengukur perbedaan ukuran kranial dan indeks cephalik anak retardasi mental umur 7-12 tahun dibandingkan anak normal berdasarkan pengukuran antropometri dan sefalometri. metode: penelitian epidemiologi analitik observasional dengan rancang penelitian case control. pengukuran ukuran kepala dan indeks sefalik dilakukan pada 168 anak umur 7-12 tahun (84 anak retardasi mental sedang dan 84 anak normal). data dianalisis dengan uji-t. hasil: ukuran kranial dan indeks sefalik anak retardasi mental lebih kecil, terlihat dari ukuran s-n dan g-op lebih pendek pada anak retardasi mental dibanding anak normal. rerata selisih perbedaan -4.4 (s-n), -2.38 (s-ar) dan g-op (-5.05 mm), eu-eu (-8.24). hasil komponen garis dan sudut ukuran basis kranium (s-n, s-ar dan sudut n-s-ar) lebih pendek secara bermakna pada anak retardasi mental dibanding anak normal. berdasarkan kelompok umur dan jenis kelamin pada anak retardasi mental lebih banyak ditemukan bentuk kepala brakisefalik, dan anak normal normosefalik. nilai odds ratio bentuk kepala berdasarkan kelompok umur 2.10 kali (95% ci 0.025-0.375) dibanding anak normal 0.99 (ci 0.290-3.390). berdasarkan jenis kelamin nilai odds ratio 1.469 kali (ci 0.429-5.035) dibanding normal 0.562 (ci 0.19-1.65). panjang kepala dan research report 168 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 167–172 pendahuluan ilmu kedokteran gigi anak pada saat ini semakin maju dan berkembang.1-3 permintaan akan perawatan gigi dan mulut anak retardasi mental semakin meningkat.1,4 orangtua yang mempunyai anak cacat mental pada umumnya menginginkan peningkatan status kesehatan gigi dan mulut anaknya. kendala serta masalah perawatan gigi dan mulut anak retardasi mental lebih kompleks dibanding anak normal.5,6 keadaan fisik dan mental anak yang tidak memungkinkan terjadinya kerja sama yang baik, kurangnya pengetahuan dan keterampilan dokter gigi dalam penanganan serta banyak kendala lain dapat menghambat perawatan kesehatan gigi dan mulut pada anak retardasi mental ini. masalah kesehatan gigi dan mulut pada anak retardasi mental relatif banyak, salah satunya adalah maloklusi.7 anak retardasi mental mempunyai quetient intelegence (iq) yang rendah di bawah rata-rata (kurang dari 70)8,9 akibat gangguan pertumbuhan otak.10 selain berpengaruh terhadap iq anak kemungkinan besar dapat berpengaruh pada pertumbuhan tulang kranial. retardasi mental sering dikaitkan dengan deformitas dan penurunan ukuran kranial.10,11 secara fenotip anak retardasi mental tipe ringan dan sedang sering terlihat seperti anak normal, terdiagnosis setelah dilakukan pemeriksaan intelligence quetiont (iq) dan social quotient (sq).7,12 tujuan penelitian ini untuk mendapatkan gambaran ukuran kranial dan indeks sefalik anak retardasi mental pada anak umur 7-12 tahun. umur 7 tahun diambil karena pada umur ini pertumbuhan otak maksimal terjadi.13,14 hasil penelitian ini diharapkan dapat memberikan pencegahan awal terhadap kemungkinan lebih lanjut terjadinya maloklusi yang cenderung menimbulkan gangguan psikologis dan fungsional sistem pengunyahan.5,15 bahan dan metode penelitian ini merupakan penelitian epidemiologi analitik observasional dengan desain case control dan telah mendapatkan ethical clearence dari komite etik fakultas kedokteran gigi gadjah mada. subyek penelitian terdiri dari 168 anak (84 anak retardasi mental sedang dan 84 anak normal) usia 7-12 tahun. rerata iq anak retardasi mental sedang antara 36-51. diambil secara random dari 102 anak retardasi mental sedang yang ada di slbn tipe c yogyakarta dan 204 anak normal yang masuk kriteria inklusi. belum pernah melakukan perawatan ortodonsi, belum pernah mengalami trauma yang dapat berpengaruh pada struktur kraniofasial. identifikasi kepala berdasarkan pengukuran antropometri dan sefalometri. pengukuran indeks kepala (ci) mengunakan rumus lebar kepala maksimal/panjang kepala maksimal 100.8 panjang kepala diukur dari titik glabella (titik tengah alis) dan opisthocranion (external occipital protuberance). lebar kepala diukur dari euryon-euryon (gambar 1-a) dan panjang kepala diukur dari glabella-opisthocranion (gambar 1-b). diklasifikasikan menjadi bentuk kepala dolichocephalic apabila ci < 74,9, mesocephalic bila ci < 75-79,9 dan brachycephalic bila ci < 84,9. masingmasing pengukuran dilakukan 3 kali, dan hasilnya diambil reratanya. pengukuran variabel skeletal meliputi s-n, s-ar dan < nsar yang dikelompokkan menjadi kelompok ukuran kranial (gambar 2). nilai odds ratio diperoleh dengan membagi kelompok umur menjadi 2 yaitu umur 7–9 tahun dan 10–12 tahun. umur 6–9 tahun merupakan periode transisi pertama, umur 9–11 tahun disebut periode transisi kedua di mana terjadi pergantian gigi posterior dan gigi molar kedua permanen erupsi.16 pengukuran dilakukan setelah pengisian informed consent. pengukuran yang sama juga dilakukan pada anak normal. untuk menentukan perbedaan ukuran kranial antara anak retardasi mental dan anak normal dilakukan uji-t. data dianalisa mengunakan program spss 18. basis kranial ada kaitannya dengan jenis kelamin, anak laki-laki ukuran kranialnya lebih besar dari anak perempuan. smpulan: ukuran kepala dan basis kranial anak retardasi mental lebih kecil dari anak normal. kata kunci: antropometri, sefalometri lateral, retardasi mental sedang, anak korespondensi (correspondence): dewi elianora, bagian kedokteran gigi anak, fakultas kedokteran gigi universitas baiturrahmah. jl. raya by. pass km. 15 aie pacah, padang, indonesia. e-mail: dewi.eliaonora12345@gmail.com gambar 1. a) euryon-euryon (lebar kepala); b) glabellaopisthocranion (panjang kepala).7 169elianora, dkk.: ukuran kranial dan indeks sefalik pada anak retardasi mental gambar 2. variabel ukuran kranial.2 keterangan: 1. s-n = jarak titik sella tursica dan titik nasion (panjang basis kranium anterior); 2. s-ar = jarak titik sella tursica dan titik articulare (panjang basis kranium posterior); 3. < nsar = sudut yang dibentuk perpotongan garis sn dan sar. hasil hasil uji normalitas kolmogorov-smirnov menunjukkan indeks kepala p>0,05 dan q-q plot menunjukkan data berdistribusi normal (gambar 3). dari 168 subyek yang diteliti, 63% anak retardasi mental ditemukan dengan bentuk kepala brakisefalik, anak normal dengan bentuk kepala normosefalik (45%) (gambar 4). untuk melihat perbedaan ukuran kranial antara kelompok anak retardasi mental dan anak normal dilakukan uji-t. hasil rerata indeks kepala (euryon-euryon dan glabellaopisthocranion) anak retardasi mental 81,25 ± 11,71 dan rerata indeks kepala anak normal 76,10 ± 5,49. hasil uji-t menunjukkan perbedaan signifikan (p = 0,001). bentuk kepala berdasarkan kelompok umur ditunjukkan pada tabel 1. nilai odds ratio diperoleh dengan membagi kelompok umur menjadi dua kelompok yaitu umur 7–9 tahun dan 10–12 tahun. hasil uji statistik menunjukkan signifikan (p = 0,001). pada kelompok umur 7–12 tahun, anak retardasi mental lebih banyak ditemukan bentuk kepala brakisefalik dengan nilai odds ratio 2,10 dibanding anak normal (0,99). berdasarkan jenis kelamin bentuk kepala pada anak laki-laki dan perempuan retardasi mental lebih banyak ditemukan brakisefalik (64% dan 57%), sedangkan anak normal lebih banyak bentuk kepala normosefalik (43% dan 48%). laki-laki lebih banyak dibanding perempuan.gambar 3. grafik sebaran indeks kepala. 170 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 167–172 gambar 4. bentuk kepala anak retardasi mental dan anak normal. uji statistik menunjukkan nilai odds ratio 1.469 kali dibanding normal (0,562) dengan nilai ci pada anak retardasi mental 0,429-5,035 dan 0,19-1,65 pada anak normal, ditunjukkan pada tabel 2. pengukuran antropometri kepala berdasarkan jenis kelamin didapatkan signifikan (p<0,05). sebelum uji beda dilakukan antara kelompok anak retardasi mental dan kelompok anak normal, terlebih dahulu dilakukan pengelompokkan variabel menjadi kelompok ukuran kranial terdiri dari eu-eu (euryon-euryon), g-op (glabella-opisthocranion), s-n (sella-nasion), s-ar (sellaarticulare), <nsar (<nasion-sella-articulare). hasil uji beda antara kelompok anak retardasi dan anak normal dengan variabel kranial dianalisis dengan uji-t dapat dilihat pada tabel 3. hasil uji-t ukuran eu-eu (lebar kepala) dan g-op (panjang kepala) menunjukkan perbedaan yang bermakna (p=0,001) dengan rerata ukuran eu-eu lebih besar pada anak retardasi mental disbanding anak normal. rerata ukuran panjang kepala (g-op) lebih pendek pada anak retardasi mental dari anak normal. selisih perbedaan rerata eu-eu (-8,24) dan g-op (-5,05). rerata indeks kepala (eu-eu/g-op) pada anak retardasi mental 81,29 dan anak normal 76,12, ini berarti bahwa rerata ukuran kranial anak tabel 1. nilai odds ratio bentuk kepala berdasarkan kelompok umur umur brakisefalik n (%) dolikosefalik n (%) p or 95% ci retardasi mental 7-9 35 (92,11) 3 (7,89) .001 2,10 10-12 18 (52,94) 16 (47,1) (0,290-3,390) normal 7-9 6 (30,00) 17 (68,0) .051 0,99 10-12 7 (33,33) 14 (66,7) (0,025-0,0375) *p < 0,05 gambar 5. distribusi sebaran bentuk kepala berdasarkan jenis kelamin (a) laki-laki retardasi mental, (b) laki-laki normal, (c) perempuan retardasi mental, (d) perempuan normal. 23% 14% 63% retardasi mental profil kepala dolikosefali profil kepala normosefali profil kepala brakisefali 37% 45% 18% normal profil kepala dolikosefali profil kepala normosefali profil kepala brakisefali a bretardasi mental normal dolikosefalik normosefalik brakisefalik dolikosefalik normosefalik brakisefalik a laki-laki retardasi mental dolikosefalik brakisefalik normosefalik b laki-laki normal dolikosefalik brakisefalik normosefalik d perempuan normal dolikosefalik brakisefalik normosefalik c perempuan retardasi mental dolikosefalik brakisefalik normosefalik retardasi mental menunjukkan profil kepala brakisefalik, sedangkan anak normal normosefalik. hasil pengukuran basis kranium menunjukkan perbedaan yang lebih bermakna (p=0,001) pada s-n daripada s-ar (p=0,01), dimana ukuran basis kranium anak 171elianora, dkk.: ukuran kranial dan indeks sefalik pada anak retardasi mental retardasi mental lebih pendek dari anak normal, dengan selisih perbedaan -4,4 (s-n) dan -2,38 (s-ar). walaupun sudut n-s-ar tidak menunjukkan perbedaan (p=0,53), namun ukuran kranial retardasi mental didapatkan lebih kecil dari anak normal. ini menunjukkan bahwa basis kranium anterior dan posterior pada anak retardasi mental lebih pendek dari anak normal. tabel 3. hasil uji-t ukuran kranial anak retardasi mental dan 95% ci ukuran kranial retardasi mental normal selisih p 95% cin = 84 x ± sd n = 84 x ± sd eu-eu 126,52 ± 11,17 118,28 ± 4,95 8,24 .001 5,60 ± 10,87 g-op 150,55 ± 12,24 155,60 ± 6,03 -5,05 .001 -2,99 ± 2,88 s-n 64,33 ± 5,35 66,71 ± 6,50 -4,44 .001 -4,19 ± -.057 s-ar 32,30 ± 4,61 36,74 ± 5,63 -2,38 .01 -6,01 ± -2,87 < nsar 124,46 ± 7,03 125,05 ± 4,77 -0,58 .53 -2,41 ± 11,25 keterangan: eu-eu (euryon-euryon); g-op (glabella-opisthocranion); s-n (sella-nasion); s-ar (sella-articulare); nsar (nasion -sella-articulare) tabel 2. nilai odds ratio bentuk kepala berdasarkan jenis kelamin sex brakisefalik n (%) dolikosefalik n (%) p or 95% ci retardasi mental laki-laki 27 (79,41) 7 (20,59) .029 1,479 perempuan 24 (70,59) 12 (31,58) (0,429-5,035) normal laki-laki 7 (20,59) 17 (72,00) .039 0,562 perempuan 10 (29,41) 14 (63,64) (0,19-1,65) *p < 0,05 perbedaan ukuran kranial berdasarkan jenis kelamin, hasilnya ditunjukkan pada tabel 4. hasil rerata ukuran kranial anak retardasi mental lebih pendek dari anak normal, laki-laki didapatkan ukurannya lebih besar daripada perempuan. terlihat dari ukuran eu-eu, g-op, s-n, s-ar, dan <nsar. perbedaan yang lebih bermakna ditunjukkan pada ukuran kranial g-op (p=0,001) dan s-n (p=0,051), ini menunjukkan bahwa ada hubungan retardasi mental dengan ukuran kranial. pembahasan ukuran basis kranium dan indeks sefalik anak retardasi mental lebih pendek secara bermakna dari anak normal. selisih perbedaannya dengan anak normal adalah basis kranium anterior (s-n) 4,4 mm dan basis kranium posterior (s-ar) 2,38 mm. hal ini menunjukkan ada hubungan retardasi mental dengan ukuran kranial. hasil penelitian ini sesuai dengan penelitian fink et al.,17 pada penderita sindroma down umur 14 tahun yang mengalami defisiensi pertumbuhan signifikan (p<0,001) pada daerah tabel 4. ukuran kranial anak retardasi mental dan anak normal berdasarkan jenis kelamin dan hasil uji-t ukuran kranial laki-laki perempuan selisih p 95% ci x ± sd x ± sd eu-eu retardasi mental 118,54 ± 4,31 118,01 ± 5,56 1,66 0,535 -1,62 -2,69 normal 127,29 ± 11,77 125,75 ± 10,63 1,54 0,195 -3,33 -6,41 g-op retardasi mental 155,66 ± 12,74 153,44 ± 12,74 2,21 0,001 -5,13 – 5,56 normal 156,61 ± 5,40 154,59 ± 6,51 1,88 2,021 -.57 – 4,62 s-n retardasi mental 66,54 ± 4,90 64,12 ± 4,27 2,43 0,001 11,91 – 2,76 normal 67,55 ± 7,56 65,88 ± 5,20 1,67 6,604 -1,15 – 4,48 s-ar retardasi mental 32,76 ± 4,93 30,83 ± 4,28 2,10 0,005 -2,93 – 1,08 normal 37,90 ± 5,72 35,57 ± 5,71 2,33 0,002 -4,74 0,07 < nsar retardasi mental 126,86 ± 7,76 123,24 ± 6,31 3,48 0,484 -5,54 – 1,69 normal 124,86 ± 4,87 124,67 ± 3,94 0,40 0,495 -3,47 – 2,67 keterangan: eu-eu (euryon-euryon); g-op (glabella-opiscranion); s-n (sella-nasion); s-ar (sella-articulare); nsar (nasionsella-articulare) 172 dent. j. (maj. ked. gigi), volume 46, number 3, september 2013: 167–172 endokranial yaitu basis kranium anterior lebih pendek 8-9 mm, basis kranium posterior lebih pendek 3-4 mm dibanding anak normal. pendapat ini didukung allanson et al.15 yang meneliti variabel ukuran kraniofasial pada sindroma down usia 6-61 tahun. menurut hendarto,9 ukuran kranial dipengaruhi oleh pertumbuhan otak dan berkorelasi signifikan dengan fungsi intelektual. ukuran kranial anak laki-laki lebih besar dari anak perempuan. hal ini berarti panjang kepala dan basis kranium ada kaitannya dengan jenis kelamin. fakta ini menunjukkan bahwa panjang kepala berkaitan dengan pertumbuhan posterior otak dan perkembangan struktur yang lebih pada laki-laki.18,19 sexual dismorphism menunjukkan perbedaan indeks kepala antara laki-laki dan perempuan20 dan volume kepala laki-laki (1500 cm3) lebih besar dari perempuan (1300 cm3).10 lingkungan prenatal dan postnatal mungkin berpengaruh pada perbedaan panjang kepala laki-laki dan perempuan.20,21 hasil komponen garis dan sudut pada ukuran basis kranium (s-n, s-ar dan sudut n-s-ar) lebih pendek secara bermakna pada kelompok anak retardasi mental daripada kelompok anak normal. hal ini menunjukkan pertumbuhan basis kranial anak retardasi mental kurang berkembang. kurang berkembangnya basis kranial pada anak retardasi mental, kemungkinan disebabkan gangguan pada pertumbuhan otak dan proses pertumbuhan tulang kranial, ditunjukkan pada jarak s-n yang pendek pada anak retardasi mental. sidiarto,12 dalam penelitiannya menemukan pengurangan berat otak 67%-76% pada hitung sel otak penderita retardasi mental secara mikroskopik dibanding dengan berat otak orang normal. hal ini menunjukkan bahwa otak penderita retardasi mental, khususnya cerebellum dan batang otak kurang berkembang. pada anak retardasi mental euryon-euryon (eu-eu) dan glabella-opisthocranion (g-op) didapatkan ukurannya lebih pendek dari kelompok anak normal. hal ini mungkin dapat menjadi dasar pemikiran pada anak retardasi mental terjadi gangguan pertumbuhan tulang kranial akibat gangguan pertumbuhan otak. pertumbuhan kranial dipengaruhi pertumbuhan kapsula otak dan pertumbuhan kapsula otak dipengaruhi pertumbuhan otak.22 pertumbuhan kubah tulang kepala (cranial vault) dipicu bagian dalam tulang kepala, pertumbuhan dalam kepala tulang kepala dipengaruhi oleh pertumbuhan otak.13 bentuk otak akan mengatur struktur dasar tengkorak yang berada di bawahnya yang pada akhirnya akan mempengaruhi struktur kepala dan wajah.13,19 berdasarkan kelompok umur dan jenis kelamin bentuk kepala pada anak retardasi mental lebih banyak brakisefalik. hasil penelitian ini hampir sama dengan yang ditemukan beberapa peneliti sebelumnya.23-25 disimpulkan bahwa ukuran kepala dan basis kranium anak retardasi mental ukurannya lebih kecil dari anak normal. hal ini menunjukkan ada hubungan retardasi mental dengan ukuran kepala. ukuran kepala anak retardasi mental kurang berkembang disebabkan gangguan pertumbuhan otak dan pertumbuhan tulang kranial. daftar pustaka 1. camassimo ps, hendry wf, dennis jm, arthur jn. pediatric dentistry infancy through adolescence. baltimore: elseiver; 2013. p. 149-99. 2. mcdonald re, avery dr. dentistry for child and adolescent. 9th ed. st louis: mosby co; 2011. p. 32-6. 3. richmond s, chesnutt i, shennan j, brown r. the relationship of medical and dental factors to perceived general and dental health. community dent oral epidemiol 2007; 35(2): 89-97. 4. kementrian kesehatan ri tahun 2012. perencanaan program pelayanan kesehatan gigi dan mulut 2011-2025. perpustakaan. depkes.go.id. 5. orelant a, heijbel j, jagell s, persson m. oral function in the physically handicapped with or without severe mental retardation. j dent child 1989; 56(1): 17-25. 6. dubrul el. oral anatomy. 7th ed. st. louis: cv mosby co; 1980. p. 93-132, 142-73, 200-209. 7. heijbel j, oreland a, jagell s. malocclusions in physically and/ or mentally handiccapped children. j swed dent 1987; 11(3): 103-10. 8. lumbantobing sm. anak dengan mental terbelakang. jakarta: balai penerbit fkui; 1997. h. 4-19. 9. hendarto sk. retardasi mental. simposisum perkembangan genetik kedokteran. kumpulan naskah neurologi anak idai, ukk. tumbuh kembang pediatri sosial idai, 30 april-2 mei 1991, fkui. jakarta. 10. manjunath ky, narayanan hs, thomas im. cranial volume of mental retardates. karnataka pediatric j 2002; 16(2):7-10. 11. oewen rr. facial antropometric deviation in mental retardation relation to syndromes. scientific papers the 40th anniversary celebration of the ida, 1990. p. 192-200. 12. sidiarto ld. aspek neurologic sindroma down & penangganannya. dalam kumpulan naskah neurologi anak idai, ukk. tumbuh kembang pediatri sosial idai, 30 april-2 mei 1991, fkui. jakarta. 13. langman j. medical embryology. 4th ed. baltimore: williams and wilkins co; 1988. p. 268-306. 14. esomunu ug, badamasi mi. cephalic anthropometry of ndi igho of abia state of nigeria. asian j scientific research 2012; 10(5): 178-84. 15. allanson je, o’hara p, farkas lg, nair rc. anthropometric craniofacial pattern profiles in down syndrome. am j med genet 1993; 47(5): 748-52. 16. van der linden fpgm. facial growth and facial orthopedics. chicago: quintessence, publishing; 1986. p. 184 -94. 17. fink gb, madus wk, walker gf. a quantitative study of the face in down syndrome. am j orthod 1975; 67(5): 540-53. 18. ilayperuma i. evaluation of cephalic indices: a clue for racial and sex diversity. int j morphol 2011; 29(1): 112-7. 19. lux cj, concradt c, burden d, komposch g. tra nsverse development of the craniofacial skeleton and dentition between 7 and 15 years of age-a longitudinal postero-antero cephalometric study. eur j orthod 2004; 26(1): 31-42. 20. funatsu m, sato k, mitani h. effects of the growth hormone on craniofacial growth. angle orthod 2006; 76(6): 970-7. 21. garba sh, numan ai, mishara ig. craniofacial classification of normal newborns in maiduguri metropolis nigeria. int j morphol 2008; 26(2): 407-10. 22. enlow dh. growth of the craniofacial skeleton. in: moyers re. handbook of orthodontics 4th ed. chicago: year book medical publisher, inc; 1988. p. 51-117. 23. golalipour mj. the effect of ethnic factor on cephalic index in 1720 years old females of north of iran. int j morphol 2006; 24(3): 319-322. 24. oladipo gs, okoh pd, akande pa, oyakhire mo. anthropometric study of some craniofacial parameters: head circumference, nasal height, nasal width and nasal index of adult omoku indigenes of nigeria. am j sci ind res 2010; 2(1): 54-7. 25. vojdani z, bahmanpour s, momeni s, vasaghi a, yazdizadeh a, karamifar a, najafifar a, setoodehmaram s, mokhtar a. cephalometry in 14-18 years old girls and boys of shiraz-iran high school. int j morphol 2009; 27(1): 101-4. 80 volume 46 number 2 june 2013 research report treatment of non-vital primary molar using lesion sterilization and tissue repair (lstr 3mix-mp) tania saskianti, udijanto tedjosasongko and irmawati department of pediatric dentistry faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: root canal preparation and anatomic variations of deciduous teeth often cause the child patient uncooperative and sometimes the treatment failure. the non-threatening treatment and non-invasive approaches is needed to obtain a good cooperation from child patient. purpose: the study was aimed to clinically evaluate the use of 3mix-mpa combination of antibacterial drugs, i.e. metronidazole, minocycline and ciprofloxacin (3mix), and macrogol and propylene glycol (mp) as pulp medicament on a necroses primary molar. methods: subject were the children patients of pediatric dental clinic universitas airlangga dental hospital. eight primary molars with pulp necroses due to dental caries were selected as samples. the treatment was done based on the concept of lesion sterilization and tissue repair (lstr) therapy. a slice of 3 mix-mp pastes was placed in the cavity and then sealed with glassionomer cement. subjects were asked for recall visit in 1, 3 and 6 months post treatment, for clinical and radiographic evaluation. the antibacterial effect of 3 mix-mp was compared with tempophore on mixed bacteria of pulp cavity which was isolated prior to therapy. the antibacterial effect was determined by measuring the inhibition zone after 24 hours anaerobe incubation. results: seven out of 8 subjects on recall visit showed no acute or chronic clinical symptoms, such as fistulae, abscess, purulent exudates, swelling or feel any pain during mastication. microbiological test result showed lstr 3mix-mp had antibacterial effect higher than tempophore (p<0.001). conclusion: the study revealed that 3mix-mp treatment showed clinical and radiographic positive response on necrose primary molar. key words: lstr 3mix-mp, pulp necroses, primary teeth abstrak latar belakang: preparasi saluran akar dan variasi anatomi gigi sulung seringkali menyebabkan pasien anak tidak kooperatif dan kadang menyebabkan kegagalan perawatan. perawatan yang tidak menakutkan dan non-invasif diperlukan untuk mendapatkan kerjasama yang baik dari pasien anak. tujuan: penelitian ini bertujuan untuk evaluasi klinis penggunaan 3mix mpkombinasi obat antibakteri, yaitu metronidazole, minocycline dan ciprofloxacin (3mix), dan makrogol dan propilen glikol (mp) – sebagai pengobatan pulpa pada gigi molar sulung yang nekrose. metode: subjek adalah pasien anak dari klinik kedokteran gigi anak rumah sakit gigi & mulut universitas airlangga. delapan gigi molar sulung dengan nekrosis pulpa akibat karies gigi dipilih sebagai sampel. pengobatan dilakukan berdasarkan konsep lesion sterilization and tissue repair (lstr). selapis pasta 3 mix mp ditempatkan di kavitas gigi dan kemudian ditumpat dengan semen glass-ionomer. subjek diminta untuk kontrol untuk evaluasi klinis dan radiografi 1, 3 dan 6 bulan pasca perawatan. efek antibakteri 3 mix mp terhadap bakteri campuran rongga pulpa yang diisolasi sebelum terapi dibandingkan dengan tempophore . efek antibakteri ditentukan dengan mengukur zona hambat setelah 24 jam inkubasi anaerob. hasil: tujuh dari 8 subjek pada saat kontrol tidak menunjukkan gejala klinis akut atau kronis, seperti fistula, abses, eksudat purulen, bengkak atau 81saskianti, et al.,: treatment of non-vital primary molar using lesion sterilization merasa sakit selama pengunyahan. hasil uji mikrobiologi menunjukkan lstr 3mix mp memiliki efek antibakteri yang lebih tinggi daripada tempophore (p<0,001). simpulan: hasil penelitian menunjukkan bahwa penggunaan 3mix mp memberikan respon positif pada perawatan molar sulung yang nekrotik secara klinis dan radiografis. kata kunci: lstr 3mix-mp, nekrose pulpa, gigi sulung correspondence: tania saskianti, c/o: departemen kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: taniapedo@yahoo.com introduction approximately 75% of the teeth with deep caries have been found from clinical observation to have pulp exposure. over 90% of the asymptomatic teeth with deep carious lesions could be successfully treated without pulp exposure using indirect pulp therapy techniques, so the premature extraction can be prevented. 1 primary teeth play an integral role in the development of occlusion. premature loss of a primary tooth through infection has the potential to destabilize the developing occlusion with space loss, arch collapse, and premature, delayed or ectopic eruption of the permanent successor teeth. effective pulp therapy in the primary dentition must not only stabilize the affected primary tooth, but also create a favorable environment for normal exfoliation of the primary tooth. it does not harm the developing enamel or disturb normal eruption of its permanent successor. the primary teeth preserved, are consider to be the best space maintenance treatment until natural exfoliation.2 however, before treatment plan of primary molar with deep caries was made, it is important to consider some factors, e.g. age of children, the general health, the period of permanent dental eruption, the severity of dental caries, the cooperation of children, and the cost of treatment.3 these examples, in any combination, illustrate many treatment considerations that could be important in an individual patient with pulp necrose.1 the general treatment for primary molar teeth with diagnose of necroses pulp actually consists of some root canal treatment techniques. however, complete elimination of bacteria is not always achieved in clinical practice due to the anatomical complexities of root canals and consequent limitations of access by instruments and irrigators. 4 furthermore, root canal treatment procedure in children is more challenging and difficult than in adult due to the following reasons: behavior management problems in children, limited mouth opening, complexities of the root canal system, and danger of injuring the permanent tooth bud. the variation and eventual alterations in the number and size of the primary root canals made it inaccessible even to the smallest endodontic instruments.1 in general, the least invasive intervention that is predictably associated with a healthy, adaptive healing response in the affected primary or permanent tooth, therefore, is needed to be done.2 all inflammatory periapical lesions should be initially treated with conservative nonsurgical procedures.5 thus, in order to anticipate those problems, nowadays the cariology research unit of niigata university school of dentistry in japan has developed the concept of lesion sterilization and tissue repair (lstr) by using combination of metronidazole, ciprofloxacin, and minocycline (3mix), and combination of carrier materials, macrogol and propylene glycol (mp) for disinfecting the lesion in oral mouth, including dentinal, pulp, and periapical lesion. the recovery of the infected tissue then is expected to occur if the lesion is disinfected.6-8 in addition, in vitro and in situ researches are done extensively for analyzing the affectivity of mixing medicines (3mix) towards bacteria in oral mouth, including endodontic lesion in permanent teeth. the research of lstr 3mix-mp on permanent dental actually has been done before by hoshino,9 kuswandari,10 and khalil,11 nevertheless, other supporting researches are still needed in different places in order to examine whether this treatment is also effective for primary molar teeth considering with the morphological difference between the crown of primary molar teeth and that of permanent teeth with many difficulties in maintaining root canal. the wider dentin tubules of primary molar teeth and the thinner enamel of primary molar teeth than those of the permanent teeth can make the spreading of bacteria into root canal easier. furthermore, in child patients treatment sometimes is difficult to be achieved since the flat anatomy of root canal in the primary molar teeth, and since it also have many branches that can make the conventional root canal treatment difficult. due to the polymicrobial nature of the infected root canal, combination of antibacterial drug is required. the treatment with lstr is considered to be useful to cure the pulp necroses since it can exterminate bacteria in root canal. clinical procedures of lstr therapy is also simple and does not need long and continual visiting, thus it makes dentists easier to treat pulp necroses of primary molar teeth.6 the aim of study was to evaluate the success of lstr 3mix-mp treatment subjectively, clinically, and radiographically in primary molar teeth with diagnosis of pulp necroses in 1, 3, and 6 months after the treatment. the study could give an alternative treatment of pulp necroses in primary molar teeth that is easily, simple, effective, cheap acceptable for children. 82 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 80–84 material and methods this study was a clinical research study with the pre test-post test control group design. the locations of study were clinic of pediatric dentistry universitas airlangga dental hospital and ikhlas kindergarten at jalan sekolahan 22, surabaya. the targeted population in this research were the students of ikhlas kindergarten (51 children) who meet the criteria of sample. the samples were the maxillary or mandibulary primary molar teeth with diagnosis of pulp necroses due to dental caries, based on the clinical and radiologic examination. the teeth must also met criteria of the following root canal treatment indication: the tooth should be restorable after the treatment, the root was in full length or 1/3 of root length resorbed based on periapical radiographic. patients were cooperative, had no any systemic abnormality or not under medical therapy relating with their systemic condition, had no allergic history with any chemical materials and antibiotics, must be permitted by their parents who then signs informed consent. finally, the method of sample collecting in the study was random sampling. the criteria of treatment success were subjective, clinical and radiographic evaluations.12 the treatment was subjectively considered to be success if the early symptoms or spontaneous pain in percussion decreased or disappeared in period of observation (1, 3, and 6 months). the treatment was clinically considered to be success if the patients with or without early symptoms or spontaneous pain in percussion and fistula, inflammation of intra oral, and subjective complains at the first visit had decreased, or had no pain at all during observation period. the treatment was considered to be radiographically success if the area of periapical radiolucency decreased and had no new periapical radiolucency at the end of observation period (6 months). clinical examination conducted on primary molar teeth with pulp necroses. this clinical examination was aimed to examine the presence of gingival inflammation; abscess or fistula; abnormal dental mobility; pain after light pressure on tooth; and any spontaneous pain.2 moreover, radiographic examination before and after the treatment was also needed to obtain some following information; the existence of external root resorbtion of treated primary molar tooth, the existence of internal root resorbtion of treated primary molar tooth, the existence of bifurcation involvement, and the existence of periapical radiolucency.2 for children who met the criteria of subject, their parents were explained about the aims, the expected results, the advantages and disadvantages of lstr 3mix-mp treatment, and the stages of the research as well as the schedule of recall visit. after the parents approved it, they signed the informed consent. tools and hand instruments were sterilized prior the examination. enteric coating and capsule covering the medicine were opened. each of the medicines then was smoothened by using porcelain mortar and pestle. the powder was stored in covered container, and saved in refrigerator at the temperature of 16o c. next, the powder was mixed with the ratio of metronidazole 500 mg: ciprofloxacin 500 mg: minocycline 500 mg (3mix) = 1:3:3. and, the carrier materials then were mixed with macrogol and propylene glycol (mp) = 1:1. finally, those mixing medical powder were mixed with the carrier materials for forming pasta 3 mix-mp. the isolation of working area must be conducted in children by using cotton roll. the wall of dental cavity was cleaned chemically by using 35% of phosphate acid. the cavity then was half-filled with 3mix-mp and covered with glassionomer cement fuji ix with liquid consistency (the ratio 1 powder: 2 liquid) without pressure. at recall visit (1, 3, and 6 months posttreatment) aanamnesis was conducted towards the patients’ parents. in intra oral examination on treated teeth of the children, moreover, palpation, percussion, and pressure examinations were conducted. then, radiology examination was conducted. the obtained data, finally, were noted in the prepared form. sterile paper point was put into the dental root canal for 1 minute to obtain mixed bacteria in the necrose pulp. paper point then was put into the medium tube containing bhi broth, and aerobically incubated for 24 hours. the blood agar was divided into 4 zones. bacteria culture in the bhi broth was taken by micropipette. each of zones of blood agar was given one drop (10 μliter), and leveled with spreader. afterwards, lstr 3mix-mp and tempophore, about half of measuring spoon, were applied in each of absorbed papers. those absorbed papers then were put on blood agar and anaerobically incubated in for 48 hours. the antibacterial effect determined by the inhibition zone. the bigger zone of inhibition the stronger its antibacterial effect. results based on the observation of lstr 3mix-mp treatment on the 4-6 year old children of tk ikhlas in surabaya, 7 of 8 children were successfully treated with lstr 3mixmp. there were 5 children who were successfully treated without any complains and 2 children were successfully treated though they got pain in the first and third months, but they recovered in the sixth month. there was one child who was not successfully treated. moreover, during the examination of subjective symptom one month after lstr 3mix-mp treatment, there were 3 students got pain. one student reliefed the pain at the third month, yet the pain emerged again at the sixth month. the two children recovered their pain from the third month to the sixth month. however, there was one student who had pain during mastication at the sixth month. meanwhile, five children had no pain from the start and had stable condition until the end of the treatment. the radiographic examination result, however, was difficult to measured since from the beginning of treatment 83saskianti, et al.,: treatment of non-vital primary molar using lesion sterilization there was no periapical radiolucency or resorbtion of alveolar bone in seven of eight children. in general, lstr 3mix-mp treatment radiographically did not show differences between before and after the treatment. nevertheless, there was permanent radiolucency in one child and physiologic resorbtion in one of the primary molar root (data not shown). based on the result, it showed that average of the inhibition zone of lstr 3mix-mp was bigger than that of tempophore on bacteria culture of root canal (table 1). before taking the test for measuring the difference of inhibition zone between lstr 3mix-mp group and tempophore group, statistic test had been taken on each group for analyzing the distribution of the data. kolmogorov smirnov test on lstr 3mix-mp group and tempophore groups were more than 0.05 which means that either lstr 3mix-mp group or tempophore group has normal distribution of the data. moreover, in order to determined the difference of resistance zone between lstr 3mix-mp and tempophore independent t-test conducted. based on the result of the statistic test, it was known that p=0.001 (p<0.05). it means that the difference of the inhibition zone was significant between lstr 3mix-mp and tempophore on bacteria culture of root canal. discussion based on the result it was known that the treatment was successfully conducted in seven of eight children. similarly, the research conducted by takushige6 also showed successfull lstr 3mix-mp treatment, about 79% of his cases. besides that, the research conducted by kuswandari10 and khalil11 also pointed out that lstr 3mix-mp treatment on primary molar teeth with diagnosis of pulp necrose is considered to be a successful procedure, which the level of success reached 100% in two months of treatment. furthermore, kuswandari10 also stated that the early condition of primary molar teeth before lstr 3mixmp treatment determined the level of clinical recovery. it means that primary molar teeth without any clinical symptoms in initial condition will recover faster that those with periapical inflammation. the failure in one child was due to broken restoration of gic at the first month and at the sixth month of the treatment, so that the elimination of bacteria could not be reached, and the child had pain during mastication. according to chugal et al.,14 eradication of microorganisms from an infected root canal system is the key to successful root canal treatment since there are millions of bacteria in root canal with pulp necrose. thus many bacteria infected root canal, especially anaerob bacteria. the virulency of endodontic flora is generally low, but the pathogenic character and intraradicular defense are affected by the combination of many factors, e.g. interaction with other microorganisms in root canal, the ability of bacteria in disturbing the defense system of the host, the secretion of lipopolysakarida (lps) and other bacterial products, and the enzyme synthesis breaking the tissue of the host. it is also supported by narayanan & vaishnavi,15 that bacteria can infect through many pathways, such as 1) through dental crown or root after the broken pulp due to trauma; 2) through periodontal tissue into broken dentin tubuli, lateral cavity, and accessory cavity or apical and lateral foramen; 3) through lymphatic route or hematogenus (anakoresis). thus, in order to eliminate those bacteria more than one antibiotic is needed. lstr 3mix-mp is considered as the combination of metronidazole, minocycline, and ciprofloxacin (3mix), and those three antibiotics mixed with macrogol and propylene glycol (mp) were proven to be able to penetrate efficiently through root canal and to disinfect the lesion of caries. laboratory test showed that lstr 3 mix-mp could kill mixed bacteria in root canal more potent than that of tempohore. this result inline with the clinical result. since lstr 3mix-mp was bactericidal in aerob bacteria and in obligate anaerob bacteria that are resistant, lstr 3mixmp could eliminate bacteria from dental tissue infected in primary or permanent molar teeth. this assumption is that by eliminating bacteria, the infection, inflammation, and pain are also eliminated.6 the success of lstr 3 mix-mp treatment on those 5 samples was marked by the fact that it did not cause any pain, or any clinical and radiological abnormality that indicates the successful disinfection process of bacteria. it is evident that in primary endodontic cases root canal environment provides better nutritional supply, rich with peptides and amino acids for bacterial inhabitants of root canal system, so that elimination of microorganisms from infected root canals is a difficult task.16 due to the polymicrobial nature of infected root canal, single empirical antibiotic is insufficient in disinfection of the root canal. non specific antibiotic suppress most of the microbial flora and allow residual virulent micro-organisms to repopulate the root canal. therefore it is essential to use combination of antibiotics to act against all endodontic pathogens and to prevent resistance.17 besides the main root canal considering as capillary blood vessel that can be cleaned, there are also many additional root canal in primary molar teeth that cannot be cleaned with mechanic tools.17 thus, good material are needed to deliver three-antibiotic mixture (3mix) and create asepsis condition either in the main or in the additional root canal. propylene glycol is solvent commonly used in table 1. the average of inhibition zone (mm) group n x sd lstr 3mix-mp 10 38.0250 3.2789 tempophore 10 19.9300 3.3067 note: n = the number of research sample, sd = standard deviation 84 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 80–84 industries, food, and daily goods. the penetration effect of propylene glycol into root dentin was investigated by cruz et al.18 the area and the depth of penetration of safranin-o dye in propylene glycol were reported to be significantly greater than dye with distilled water into root dentine. propylene glycol delivered dye through the root canal system rapidly and more effectively indicating its potential use in delivering intracanal medicaments. lesion sterilization and tissue repair (lstr), involves the use of antibacterials combined with a mixture of macrogol and propylene glycol (mp) as the efficient vehicle to carry the antibacterials within the tooth. like the procedure of indirect pulp capping, the application of 3mix-mp topically on dental lesion caries in vivo could also be done by taking the tissue that cannot be re-mineralized, and by leaving the tissue of caries in the deepest area of cavity. studies have shown that dentin matrices contain reservoirs of bioactive molecules and odontoblast capable of directing tissue repair. by sterilizing the infection and not taking all dentin in pulp, it means that dentin matrix is maintained and preserved as the reservoir source of growth factor secreted by functional odontoblast and pulp fibroblast.9,10,19 growth factor then gives molecular signal for the surviving post-mitotic odontoblast cells to proliferate and differentiate to stimulates the secretion of reactionary type of tertiary dentin matrix.21 lstr treatment consisting of three-antibiotic mixture could eliminate bacteria taken from carious lesions, pulp necrose, infected root dentine and endodontic lesions of permanent teeth20 and deciduous teeth6 that disturbing the process of odontoblast proliferation and differentiation. by using propylene glycol and macrogol considering as carrier of materials (solvent), the growth factor in dentin matrix was possibly penetrated into dentin tubuli, so it could stimulate the form of tertiary dentin matrix. based on this advantage, the treatment of root canal with 3mix-mp on primary molar teeth with pulp infection could give positive effect in this treatment. finally, lstr 3mix-mp treatment could be considered to be successful if the tooth did not have any clinical symptoms until that tooth was physiologically extracted and then substituted by the permanent one. therefore, the evaluation was supposed to be done continually until the tooth is physiologically extracted with the various numbers of samples. the result of this research clinicaly and radiographically showed that lstr 3mix-mp could be used to treat primary molar teeth with pulp necrose diagnosis. lstr 3mix-mp treatment can be used as an alternative therapy for conventional root canal treatment in primary molar teeth with pulp necrose diagnosis. nevertheless, in order to ensure the success rate and to examine other factors determining the success of this treatment, it is needed to take further research with the bigger number of samples in the longer period, and by comparing with the conventional treatment of root canal. references 1. mcdonald re, avery dr. dentistry for the child and adolescent. 9th ed. missouri: mosby; 2011. p. 344, 346, 354. 2. cameron ac, widmer rp. handbook of pediatric dentistry. 3rd ed. toronto: elsevier; 2008. p. 95-6, 347. 3. ounsi hf, debaybo d, salameh z, chebaro a, bassam h. endodontic consideration in pediatric dentistry: a clinical perspective. dent hor 2009; 1: 5-10. 4. kovak j, kovak d. effect of irrigating solution. bratisl lek listy 2011; 112(7): 410-5. 5. lin lm, huang gt, rosenberg pa. proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing. j endod 2007; 33(8): 908-16. 6. takushige t, cruz ev, asgor moral a, hoshino e. endodontic treatment of primary teeth using a combination of antibacterial drugs. int endod j 2004; 37(2): 132-8. 7. vijayaraghavan r, mathian vm, sundaram am, karunakaran r, vinodh s. triple antibiotic paste in root canal therapy. j pharm bioallied sci 2012; 4(suppl 2): 230–3. 8. fernandes m, ataide id. nonsurgical management of periapical lesions. j conserv dent 2010; 13(4): 240-5. 9. hoshino e. sterilization of carious lesion by drugs. j jap asc for dent sc 1990; 9:32-37 10. kuswandari s, chrisnawati r, lukito e. endodontic treatment on deciduous molars with 3mix-mp. jurnal kedokteran gigi indonesia 2007; edisi khusus pin ikga ii: 11. 11. khalil i, islam km, zahid md, shah ak, badruddoza a, moral mdaa. lesion sterilization and tissue repair (lstr)-3mix mp therapy showed reliable efficacy against the most resistant endodontic bacteria enterococcus faecalis. city dental college j 2012; 9(2): 1-4. 12. mortazavi m, mesbahi m. comparison of zinc oxide and eugenol, and vitapex for root canal treatment of necrotic primary teeth. j clin pediatr dent 2004; 14(6): 417-24. 13. tkachenko o, karas ja. standardizing an in vitro procedure for the evaluation of the antimicrobial activity of wound dressings and the assessment of three wound dressings. j antimicrob chemother 2012; 67(7):1697-700. 14. chugal nm, clive jm, spangberg lsw. endodontic treatment outcome: effect of the permanent restoration. oral surg oral med oral pathol oral radiol endod 2007; 104(4): 576-82. 15. narayanan l, vaishnavi. endodontic microbiology. j conserv dent 2010; 13(4): 233–9. 16. peciuliene v. maneliene r, balcikonyte e, drukteinis s, rutkunas v. microorganisms in root canal infection: a review. stomatologija 2008; 10(1): 4-9. 17. parasuraman vr, muljibhai bs. 3mix-mp in endodontics–an overview. j dent and medical sci 2012; 3(1): 36-45. 18. cruz ev, kota k, huque j, iwaku m, hoshino e. penetration of propylene glycol into dentine. int endod j 2002; 35(4): 330-6. 19. handajani j, haniastuti t, ohshima h, hoshino e. survival of root canal pulp tissue after pulpitis. j lstr therapy (international web version) 2010; 9: 1-6. 20. takushige t, hoshino e. clinical evaluation of 3mix-mp methodtakushige t, hoshino e. clinical evaluation of 3mix-mp method in endodontic treatment. japan j conserv dent 1998; 41: 970-4.japan j conserv dent 1998; 41: 970-4.1998; 41: 970-4.; 41: 970-4.-4. 21. smith aj, cassidy n, perry h, kirn cb, ruch jv, lesot h.smith aj, cassidy n, perry h, kirn cb, ruch jv, lesot h. reactionary dentinogenesis. int j dev biol 1995; 39(1): 273-80. mkg vol 42 no 2 april 2009.indd 65 vol. 42. no. 2 april–june 2009 research report cooperation of patient as key factor to overcome oral habits magdalena lesmana department of orthodontic faculty of dentistry, university prof. dr. moestopo (b) jakarta indonesia abstract introduction: orthodontic treatments have developed rapidly on the last decade even though; the sophisticated treatment has been limited by patient’s behavior. orthodontist should acknowledge that the success of treatment not only depends on the knowledge and skill of the operator, but on the patient’s behavior as well, which is very critical to overcome the oral habit, including anterior tongue thrust swallow (atts). patient’s cooperation is the key factor to set prognosis of oral habit correction and have to be proven through analysis of questioners. methods: patient cooperation scale (pcs) was made for this research involving 68 orthodontic patients for correction of atts from faculty of dentistry indonesia university and ladokgi as respondents. pcs assessment was based on data sets of: patient awareness of atts (paw), patient acceptance to receive treatment (pac), and patient comfortable for using appliances (pc). the evaluation was obtained by calculation the total score of 10 items from each component, which gave 0–30 range of scale and would be 0–90 range of cooperation scale as the final result. result: pcs assessment can be based on data sets of paw, pac, and pc. it is valid and reliable. the score for the cooperative patients range between 0–25. the higher the score from this questionnaire shows less cooperation from respondent. on the other hand, the lower the score showed better cooperation. conclusion: pcs can be used for the prognosis of the successful oral habits correction and has a significant relation with the length of successful treatment. key words: anterior tongue thrust swallow (atts), patient cooperation, patient awareness, patient acceptance, patient comfortable correspondence: magdalena lesmana, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas prof. dr. moestopo (b). jl. bintaro permai raya 3 jakarta 12330, indonesia. e-mail: gandangr@cbn.net.id introduction the success of orthodontic treatment depends not only on the skill and knowledge of the operator, but also understanding the psychological aspect of the treatment. various treatments and appliances have been done and developed to overcome anterior tongue thrust swallow (atts), but have failed to fix atts permanently, it may be lead the relapse of the malocclusion.1 the difficulties to overcome the oral bad habit, might be due to the treatment that only focused on mechanical aspects but ignoring the psycho-physiological aspects. the psychological aspect of orthodontic treatment needs to be taken care as it often creates a problem in the future, for example the length and discomfort caused by the treatment. therefore for the success of the orthodontic treatment, psychological approach needs to be done such as educating the patient, building the expected behavior, guiding to observe important thing, changing the environment, bringing back self respect and showing the attitude to support the success of the treatment.2,3 it is recognized that encouraging individuals to change their behavior with respect to health is a considerable challenge. behavioral psychologists have developed an approach to therapy for changing patient’s behavior, known as behavior modification therapy. the usefulness has been realized in dental treatment. behavior modification technique is required to change diverged behavior into structured behavior by using learning principle. the behavior is “shaped” in a systematic step-by-step manner until it conforms to the desired pattern. researcher has been encouraged to use the behavior modification technique to overcome atts.4,5 the key success for stopping oral habit is a good cooperation of patient through psychological approach.6,7 cooperation of patient is needed to obey the rules of 66 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 65−69 treatment, such as regularity visiting the doctor, using and taking care of the appliances, doing exercise as instructed and maintaining oral hygiene.6 cooperation towards treatment is an interaction between patient’s character, treatment, and the operator.3 those characteristics can be measured using questioners,8,9 and for this research were used questionnaires of: patient awareness of atts (paw), patient acceptance to receive treatment (pac), and patient comfortable for using appliances (pc). these questionnaires were designed as a modification of ngan’s questionnaire.9 degrees of patient cooperation scale assessment are important in determining the prognosis of the treatment,10 and for this research was based on data sets analysis of paw, pc and pac as the evaluation of patient’s behaviors (awareness, acceptance and comfort ably). the advantage of using patient cooperation scale(pcs) is to predict the effectiveness of a correcting device and to know which component will be the risk factor for the successful of treatment. the validity of pcs value, needs to be done to proof that pcp can be used as prognosis assessment of atts treatment. to asses the validity of pcp in this research need measurement in construction validity, that confirms every item in pcp can represent what is measured. for the reliabilities, the cronbach coefficient alpha was used to asses the internal consistency. materials and methods sixtyeight patients were collected from orthodontic clinic faculty of dentistry indonesia university and ladokgi and would be the tongue guidance (tg) appliance users for correcting retained atts. tg is a modification of hawley retainer. patients were in 0-2 months retention period during september 2004 to may 2005, with completed anterior teeth and had fully erupted. these patients would be the subjects of clinical test for atts correcting device effectiveness. subjects were informed about atts existence and problem caused by atts, what to do or treatment would be given, and consequences of the treatment. at the beginning of treatment subjects were instructed to be the respondents in a questionnaire research (preliminary research) to determine the prognosis of atts treatment based on their cooperation value as key factor for successful treatment. for that purpose, the subjects need to fill in respondent data sheet, assignment sheet and signing medical informed consent. they were given questionnaires and report card, must be filled everyday, for the recording of total hours using the appliance and intensity of tongue exercise. to assess patient cooperation scale (pcs), three components data set were needed: data set of atts existence awareness component (paw) consists of 10 items, data set of comfort component (pc) consists of 10 items; data set of willingness to accept treatment component (pac) consists of 10 items. from those components measurement were done: for paw 10 items that indicated patient‘s awareness of atts as a problem that needed to be observed and corrected; for pc 10 items that indicated functional and social comfort using tg appliance, which would be sensed during treatment, and for pac that indicated patient’s willingness to accept the atts treatment. scoring criteria for these questionnaires were excellent – sufficient – insufficient – nil with the scale 0/1/2/3. the evaluation was obtained by calculation the total score of 10 items from each component, which gave 0-30 range of scale and would be 0-90 range of cooperation scale as the final result during the treatment. the higher the score from this questionnaire shows less cooperation from respondent. on the other hand, the lower the score showed better cooperation. the score for the cooperative patients range between 0–25. a good cooperation behavior during treatment is required for behavioral change to the establishment of a new swallowing pattern. the explanation for questionnaire completion was given on the 1st meeting day between researcher and respondent; that was after the respondent had signed the informed consent. it took one month to complete questionnaires of comfortable component as it had to recorded on the 1st day, 1st week, and 1st month. respondents had to complete the questionnaires by their self (respondent’s self report). there were no difficulties to complete these questionnaires. questionnaires to assess pcs consists of: questionnaires of atts existence as problem awareness (paw); questionnaires of tg appliance comfortable (pc); questionnaires of treatment acceptance (pac). the following items of paw should be noted: awareness to use retention appliance for the stabilization result in malocclusion treatment; awareness that atts can create relapse after malocclusion treatment; awareness that relapse can cause disturbances in orofacial function; awareness that relapse can make treatment and all sacrifice become useless; awareness that atts needs to be corrected; awareness that atts correction needs the usage of the appliance; awareness that atts correction appliance is also retention appliance; awareness the importance of tongue exercise according to sop; awareness the importance of regular visit to the doctor; awareness that the success of the treatment really depends on self willingness to change tongue behavior. comfortable using atts correcting appliance would be noticed from 10 items of pc : comfortable speaking; comfortable swallowing; comfortable appearance; comfortable socializing; comfortable doing daily activity; comfortable sleeping; comfortable to follow treatment rules; comfort for the teeth; comfort for the soft tissue; comfortable doing tongue exercising. the following 10 items of treatment acceptances (pac) should be noted as willingness feeling discomfort during the treatment; willingness facing difficulties; 67lesmana: cooperation of patient as key factor table 1. cronbach coefficient alpha value total observation total item cronbach alpha coefficient range component item correlation power level 68 30 0.9668 0.0149 1.000 1.000 1.000 1.000 1.000 1.000 pc-pc pac-pac pac-pac paw-paw paw-paw paw-paw pac-pac 13 & 19 27 & 25 21 & 22 5 & 2 6 & 7 10 & 8 27 & 25 weak strong strong strong strong strong strong willingness adhering to treatment rules; willingness adhering to visiting schedule; willingness adhering to use appliance regularly; willingness adhering to oral hygiene instruction; willingness doing tongue exercises; willingness filling report correctly; willingness participating actively; willingness communicating openly. questionnaire to measure respondent’s awareness towards atts (paw) the purpose of paw questionnaire is to know respondent’s response towards atts treatment, which will be taken later. the positive or negative attitude towards treatment is one of the important factors, which determine the success of treatment.11 questionnaire is completed after the explanation of what is atts and how it can cause malocclusion relapse with interrelated problems in the future. by using the mirror and mouth mirror, atts existence is shown to the respondents. they are also given explanation how important it is to correct the atts using a correcting device and have to combined with tongue exercise according to the rules (sop) has been given. questionnaire of patients comfortable (pc) in order to correct atts, wearing an appliance in the mouth can cause physical (functional) and psychic (social) discomfort.12-14 this will have significant effect on the attitude through treatment, which will impact the success of the treatment.15 as the time goes, comfort will become more after using the appliance for 1 day, 1 week, and 1 month. questionnaire of patient willingness to accept treatment (pac) this questionnaire shows patient motivation for the success of atts correction treatment. the completion of report card by the patient needs to be checked by operator on every visit. at the end of the treatment, after atts has been corrected or examination time period was over, and the evaluation will be conducted for the average of visiting frequency per month, average appliance usage (hours) and exercise frequency every day. cooperation applied as willingness to communicate openly with the researcher.3 result table 1 showed the range of correlation for the 30 items of pcp: 0.0149 – 1 pcp analysis was done with stata 7. the weakest correlation (0.0149) was the correlation value between item no 19 with item no. 13. this correlation was in the same component:pc. the strongest correlation (1.000) was correlation value between item no. 6 (awareness) with item no. 7 (awareness), item no. 22 (willingness) with item no. 21 (willingness), between item no 5 (awareness) and item no 2 (awareness), and between item no. 10 (awareness) with item 8 awareness (8). each of these 8 items shows the compatibility in 1 component. the cronbach coefficient alpha value of 0.9668 shows that pcs is reliable, so that it can be used for the clinical research of tongue guidance appliance effectiveness comparing with the tongue crib appliance as a correcting device of atts for all this time. in table 2, the value of cronbach coefficient alpha for all items in pc were strong, not only in the component but also to total component. it means that the items were in the right place and could be an indicator of patient’s cooperation. in this research the test for validity and reliability of pcp was being done. the pcp validity testing was based on thinking if pcp can be functioning as a measure to predict cooperation in atts treatment. for pcp validity testing only conducted through construction validity measurement to confirm that every item in pcp represents what will be measured. the test result of 30 items shows correlation range between all items divided in 3 components (comfort, awareness, willingness). table 2 shows if every item is coincidentally for the component. the values 0.9643, 0.9512, and 0.9299 respectively for pc, paw and pac from data analysis with cronbach alpha, means that these questionnaires can be used for treatment prognosis assessment based on patient motivation and cooperation which are imperative for the successful of changing the tongue behavior. subject characteristics that will take part in the success of atts correction for this research was shown in table 3 through cox regression test. 68 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 65−69 table 2. pcp coefficient correlation item to total total respondent component total item item no. correlation to total in component (pcp) respondent = tg users subject (68) paw 10 1 0.9279/strong ; 0.6059/strong 2 0.8840/strong ; 0.4495/weak 3 0.8374/strong ; 0.1437/weak 4 0.9279/strong ; 0.2646/weak 5 0.8809/strong ; 0.6680/strong 6 0.9776/strong ; 0.7056/strong 7 0.8809/strong ; 0.6680/strong 8 0.9776/strong; 0.7056/strong 9 0.9776/strong ; 0.7056/strong 10 0.8020/strong ; 0.6626/strong pc 10 11 0.9427/strong ; 0.8310/strong 12 0.9874/strong ; 0.8790/strong 13 0.9855/strong ; 0.9072/strong 14 0.9848/strong ; 0.8076/strong 15 0.9475/strong ; 0.8790/strong 16 0.9817/strong ; 0.9144/strong 17 0.9817/strong ; 0.9144/strong 18 0.9415/strong ; 0.6087/strong 19 0.9800/strong ; 0.7882/strong 20 0.9415/strong ; 0.6087/strong pac 10 21 0.9337/strong ; 0.8592/strong 22 0.9337/strong ; 0.8592/strong 23 0.9498/strong ; 0.8528/strong 24 0.8018/strong ; 0.6476/strong 25 0.9230/strong ; 0.8697/strong 26 0.9558/strong ; 0.9265/strong 27 0.9230/strong ; 0.8697/strong 28 0.9863/strong ; 0.8986/strong 29 0.9144/strong ; 0.8565/strong 30 0.9558/strong ; 0.9265/strong table 3. characteristic factors that influence the tg accomplishment characteristic haz. ratio p > |z| [95% conf. interval] comfortable 0.6472874 0.014 0.4571853 0.916436 acceptance 1.006794 0.913 0.8916583 1.136797 awareness 1.04062 0.728 0.8316845 1.302045 the result of this analysis has shown that “comfortable” was the most influence factors for the treatment result. discussion comfort data of a device can be a cooperation predictor between subjects and operators.6,9,14,16 this comfort feeling include speech function, swallowing, and appearance,8 as items in pc. according to nanda,8 comfort/discomfort feeling influences cooperation during treatment. the value of pcs, describes subjects characteristics that contribute to the prognosis of treatment and was obtained through analysis of paw, pc, and pac questionnaires. according to rich,4 cooperation is an absolute condition for the successful of treatment. cooperation prediction is required before applying the correcting appliance. as cooperation predictor was comfortable,9,10,14,16 awareness of atts as a problem, and willingness of undergoing treatment, that can be measured with pc, paw and pac. with ficher’s exact calculation result and t test, the p value was 0,0000 for tg comfort, willingness to accept treatment and awareness of atts. this showed that the factors that contribute to the achievement of atts treatment through behavior modification were willingness, comfortable, and awareness. cooperation applied at the time doing behavior 69lesmana: cooperation of patient as key factor modification program and needs to be observed during treatment, so that it can be done consistently, preventing the relapse of atts. patients cooperation scale can be used for the prognosis of oral habits therapy so that operator can do the anticipation for the successful outcome. references 1. barrett rh, hanson ml. oral myofunctional disorders. saint louis: the cv mosby company; 1974. p. 146–61. 2. eli i. oral psychophysiology: stress, pain, and behavior in dental care. crc press, inc; 1992. p. 25–35, 133–6. 3. klages u, sergl hg, burucker i. relationships between verbal behaviour of the orthodontist and communicative cooperation of the patient in regular orthodontic visits. am j orthod dentofoc orthop 1992; 102(3):265–9. 4. rich sk. behavior modification for orthodontic patients: an exploratory approach to patient education. am j orthod 1980; 78(4):426–37. 5. martin g, pear j. behavior modification: what it is and how to do it. 3rd ed. prentice hall, englewood cliffs, nj. 1988. p. 6–13. 6. el-mangoury nh. orthodontic cooperation. am j orthod 1981; 80(6):604–22. 7. albino je, lawrence sd, lopes ce, nash lb, tedesco la.. cooperation of adolescents in orthodontic treatment. j behav med 1991; 14:53–70. 8. nanda rs, kierl mj. prediction of cooperation in orthodontic treatment. am j orthod dentofac orthop 1992; 102(1):15–21. 9. ngan p, kess b, wilson s. perception of discomfort by patients undergoing orthodontic treatment. am j orthod dentofac orthop 1989; 96(1):47–53. 10. slakter mj, albino je, fox rn, lewis ea. reliability and stability of the orthodontic patient cooperation scale. am j orthod 1980; 78(5):559–63. 11. tedesco l, keffer ma, fleck-kandath c. self-efficacy, reasoned action and oral health behaviour reports: a social-cognitive approach to compliance. j behav med 1991; 14:341–55. 12. lewis hg, brown wab. the attitude of patients to the wearing of a removable orthodontic appliance. br dent j 1973; 134:87–90. 13. sergl hg, klages u, zentner a. pain and discomfort during orthodontic treatment: causative factors and effects on compliance. am j orthod dentofac orthop 1998; 114(6):684–91. 14. sergl hg, klages u, zentner a. functional and social discomfort during orthodontic treatment-effects on compliance and prediction of patient’s adaptation by personality variables. eur j orthod 2000; 22:307–15. 15. jones m, chan c. the pain and discomfort experienced during orthodontic treatment: a randomized controlled clinical trial of two initial aligning arch wires. am j orthod dentofac orthop 1992; 102(4):371–81. 16. bos a, hoogstraten j, prahl-andersen b. expectations of treatment and satisfaction with dentofacial appearance in orthodontic patients. am j orthod dentofac orthop 2003; 123(2):127–32. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 181 vol. 44. no. 4 december 2011 in vitro effect of q-switched nd:yag laser exposure on morphology, hydroxyapatite composition and microhardness properties of human dentin retna apsari1, siswanto1, anita yuliati2, and noriah bidin3 1 department of physics, faculty of sciences and technology, airlangga university, surabaya indonesia 2 department of dental material, faculty of dentistry, airlangga university, surabaya indonesia 3 department of physics, faculty of sciences, universiti teknologi malaysia abstract background: a q-switched nd:yag laser was employed as a source of ablation. the fundamental wavelength of the laser is 1064 nm, with pulse duration of 8 nanosecond operates with uniphase mode of tem00. in the following experiments, dentin samples (without caries and plaque) are exposed to pulse laser with q-switching effect at various energy dose. purpose: the aim of this study was to investigate the effect of laser ablation on dentin samples using q-switched nd:yag laser exposure. methods: the laser was operated in repetitive mode with frequency of 10 hz. the energy dose of the laser was ranging from 13.9 j/cm2, 21.2 j/cm2 and 41.7 j/cm2. the target material comprised of human dentin. the laser was exposed in one mode with q-switched nd:yag laser. energy delivered to the target through free beam technique. the exposed human dentin was examined by using x-ray diffraction (xr�) and fluoresence scanning electron microscopy for energy dispersive (fesem-e�ax). microhardness of human dentin were examined by using microhardness vickers test (mvt). results: the result obtained showed that the composition of hydroxyapatite of the dentin after exposed by q-switched nd:yag laser are 75.02% to 78.21%, with microhardness of 38.7 kgf/mm2 to 86.6 kgf/mm2. this indicated that exposed pulsed nd:yag laser on the human dentin attributed to the phototermal effect. the power density created by the q-switched nd:yag laser enables the heat to produce optical breakdown (melting and hole) associated with plasma formation and shock wave propagation, from energy dose of 21.2 j/cm2. from xr� analysis showed that the exposure of nd:yag laser did not involve in changing the crystal structure of the dentin, but due to photoablation effect. conclusion: in conclusion, the application of q-switched nd:yag laser as contactless drills in dentistry should be regarded as an alternative to the classical mechanical technique to improve the quality of the dentin treatment. key words: plasma, laser ablation, crystal structure, microhardness, hydroxyapatite, nd:yag laser abstrak latar belakang: mode q-switch pada laser nd:yag dapat menghasilkan fenomena ablasi pada dentin. laser nd:yag yang digunakan mempunyai panjang gelombang 1064 nm, durasi pulsa 8 ns beroperasi dengan mode tem00. sampel dentin yang digunakan tanpa karies dan plak, yang dipapari laser dengan mode q-switch dalam berbagai variasi dosis energi. tujuan: tujuan penelitian ini adalah mengamati efek ablasi dentin secara in vitro akibat paparan laser nd:yag q-switch dengan pengamatan morfologi permukaan, komposisi hidroksiapatit, dan uji kekerasan mikro. metode: laser nd:yag q-switch dengan frekuensi 10 hz dan variasi dosis energi 13,9 j/cm2, 21,2 j/cm2 dan 41,7 j/cm2 ditembakkan pada sampel dentin manusia dengan teknik penyinaran bebas tanpa dilewatkan serat optik. �entin yang terbuka diamati menggunakan x-ray diffraction (xr�) dan fluoresence scanning electron microscopy for energy dispersive (fesem-e�ax). kekerasan mikro dari dentin juga diamati menggunakan microhardness vickers test (mvt). hasil: hasil penelitian menunjukkan bahwa komposisi hidroksiapatit dari dentin setelah paparan laser nd:yag q-switch menunjukkan peningkatan berkisar 75,02% sampai 78,21% dibandingkan normal, dengan kekerasan mikro berkisar 38,7 kgf/mm2 sampai 86,6 kgf/mm2. perubahan pada struktur mikro tersebut disebakan karena adanya efek fototermal. kerapatan daya yang bervariasi berdasarkan variasi dosis energi menyebabkan efek panas pada dentin yang menyebabkan adanya fenomena optical breakdown, yang ditandai dengan munculnya efek leleh dan lubang pada research report 182 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 181–186 sampel karena produksi plasma dan adanya gelombang kejut, mulai dosis 21,2 j/cm2. berdasarkan uji xr�, efek yang muncul pada dentin tidak menyebabkan terjadinya perubahan struktur kristal hidroksiapatit, tetapi menyebabkan perubahan komposisi hidroksiapatit yang disebut dengan fotoablasi. kesimpulan: �apat disimpulkan bahwa penggunaan mode q-switched pada laser nd:yag sebagai alat dengan kontak minimal dapat dijadikan teknik alternatif untuk meningkatkan kualitas perawatan dental. kata kunci: plasma, ablasi laser, struktur kristal, kekerasan mikro, hidroksiapatit, laser nd:yag correspondence: retna apsari, c/o: departemen fisika, fakultas sains dan teknologi universitas airlangga. jl. mulyorejo surabaya, indonesia. e-mail: retnoapsari@unair.ac.id introduction nd:yag laser technology has continuously developed during the last years partly driven by the medical demand or adapted from technical application and transferred to the medical use, especially in dentistry. laser has firmly established itself as an indispensable tool for various applications such as in micromachining and medical surgery.1–5 dental laser based on nd:yag laser is used in dentistry area for therapy supporting on soft tissues in 1980. recently, laser development is used to save application for dental hard tissues therapy. in dentistry, laser is used for supporting therapy on soft tissues and evaluating dental enamel, caries morphology, dentine resistance, and dental plaque composition.1,4–6 among the most used laser systems in dentistry relies one nd:yag laser with a wavelength of 1064 nm assuring the specific characteristic of presenting affinity with pigmented tissues, which particularly makes it selective for carious tissues.1,2 nd:yag laser (1064 nm) is also used in cosmetic application,3 photomedicine and laser surgery.4,5 nd:yag laser irradiated was used to evaluate the dental enamel caries morphology through sem images,1 dental plaque composition of human enamel,4 surface dentin modification,8,9 phototermal and optical breakdown production.10 the aim of this in vitro study was to investigate the effect of a q-switched nd:yag laser irradiated to healthy human dentin (without caries and plaque) by regulating the voltage oscillator ranging from 660 to 740 volt, with the variataion of power density are 13.9 j/cm2, 21.2 j/cm2 and 41.7 j/cm2. there are three effects of human dentin that will be measured: surface morphology through fluorescence scanning electron microscopy for energy dispersive x-ray (fesem-edax) images, hydroxyapatite composition through x-ray diffraction (xrd) analysis, and microhardness properties. materials and methods a q-switched nd:yag laser with fundamental wavelength of 1064 nm manufactured by lumonics model hy 200, was employed as a source of ablation. the pulse duration of the beam is 8 nanoseconds and operates with uniphase mode of tem00. the laser was conducted in repetitive mode with frequency of 10 hz. the various energy dose of the laser are 13.9 j/cm2, 21.2 j/cm2 and 41.7 j/cm2. the energy of nd:yag laser was verified by regulating the capacitor voltage in xenon flashlamp driver ranging from 640 to 740 volt. the specimens comprised of healthy human dentin (upper premolar) without caries and plaque. the sampels provided by oral surgery clinic, faculty of dentistry, airlangga university. the teeth were extracted from consenting patients (ages of 13 until 19 years old) who undergoing extractions for orthodontic treatment. prior to teeth collection, approvals for using human hard tissue samples were obtained from the human ethics committees of all participating institutes involved in this study. the samples were cleaned using ultrasonicator branson 5210 at normal temperature within 10 minutes duration. the cleaned teeth were then stored in distilled water at constant temperature of 4° c before ready to cut. the purposed of cutting is to obtained a flat dentin surface. machine model of edenta with diamond disk was employed to cut the sample at low speed. the dentin surfaces were polished using abrasive paper grading from 400 to 2000 with diameter ranging of 1.5 to 2 mm. after polishing, the specimens were randomly divided into five groups according to the class of delivering energy. the nd:yag laser was conducted in one modes with q-switched. the laser was focused using converging lens of 28 mm focal length. protective googles specific for this wavelength for eye safety and also double masks were used to avoid vapor aspiration during the irradiation procedure. the exposed human dentin was examined by metallurgical method. in this case xrd (siemens diffractometer d 5000) and fesem-edax (zeiss supra 35 vp) machines were conducted. microhardness properties of human dentin were examined by using mvt (future tech fm 7). the surface of specimen was stressed by 2 kgf using piramide diamond in this system which inclined at an angle of 136°. the optical alignment of this experimental set-up is shown in figure 1. distance between human enamel and beam splitter is 50 cm, and exposure time was set around 9–11 second. he-ne laser was also coaxial with the nd: yag laser for an ease of alignment, because nd:yag laser produced invisible light. couple charge device video camera also, utilized for an accurate and precisely location of the sample. 183apsari, et al.,: in vitro effect of q-switched nd:yag laser exposure figure 1. experimental set up to expose human dentin with q-switched nd:yag laser. results the exposed and unexposed dentin specimens were observed under fesem. the typical results are shown in figure 2. initially the unexposed of dentin surface was observed. the morphological of normal dentin is depicted in figure 2a. the exposed dentin with q-switched nd:yag laser using energy dose of 13.9 j/cm2, operated pumping voltage at 660 v, and repetition rate of 10 hz is depicted in figure 2b. entirely different structure of damage occurred when the dentin surface was focused by a q-switched nd:yag laser such as shown in figure 2c using energy dose of 21.2 j/cm2, operated pumping voltage at 700 v, and repetition rate of 10 hz, and figure 2d using energy dose 41.7 j/cm2, operated pumping voltage at 740 v, and repetition rate of 10 hz. the typical of xrd results are emerging in figure 3-a for normal human dentin, figure 3b for q-switched nd:yag laser operated pumping voltage at 660 v with energy dose and repetitive rate of 10 hz, and figure 3c for q-switched nd:yag laser using pumping voltage of 700 v, energy dose of 21.2 j/cm2 and frequency of 10 hz, and figure 3d for q-switched nd:yag laser using pumping voltage of 740 v, energy dose of 41.7 j/cm2 and frequency of 10 hz . the peaks in each figure represented the hydroxyapatite compound [ca10(po4)6](oh)2 of the tested human dentin sample. the percentage of the composition for each tested sample are calculated based on this figure 3. the computed results are tabulated in table 1. the lattice parameters for each tested sample are also shown in the this table. the results of microhardness measurement of the human dentin after the exposure to the pulsed laser with and without q-switching are tabulated in table 2. at a maximum energy dose of 41.7 j/cm2, the microhardness of the dentin is measured to be 86.6 kgf/mm2 with q-switched nd:yag laser. figure 2. enamel surface morphology using fesem and repetition rate of 10 hz. a) without exposure (normal human dentin); b) with exposure to energy dose of 13.9 j/cm2, operated pumping voltage at 660 v); c) with exposure to energy dose of 21.2 j/cm2, operated pumping voltage at 700 v); d) with exposure to energy dose of 41.7 j/cm2, operated pumping voltage at 740 v). a b c d 184 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 181–186 table 1. percentage of hydroxyapatite compound [ca10(po4)6] (oh)2 using q-switched nd:yag laser with repetition rate of 10 hz no energy dose (j/cm2) lattice parameter (å) percentage of hydroxyapatite (%) 1 0 a=b=9.416; c=6.884 73.42 2 13.9 a=b=9.399; c=6.883 75.02 3 21.2 a=b=9.463; c=6.856 76.03 4 41.9 a=b=9.447; c=6.832 78.21 table 2. microhardness properties of human dentin samples with q-switched nd:yag laser exposure no energy dose (j/cm2) hardness value (kgf/mm2) 1 0 32.3 2 13.9 38.7 3 21.2 46.9 4 41.9 86.6 discussion there were many microcracking observed on the dentin surface exposed to laser with the q-switching on energy dose of 13.9 j/cm2. the surface comprised of microcrack all over the surface. the focused of nd:yag pulsed laser acts like hammering. the impact results rough surface associated with microcrack. this means the infrared of nd:yag laser produced thermal effect which absorbed the surface and diffused the heat laterally. the multiple impacts of the focused pulsed laser cause the surface dehydrated and crack. this is attributed to excess heat, which causes surface dehydration and crack. in this case, the multiple impacts of the focused pulsed laser cause rough heat diffusion, which affects the interaction between light and hard tissue. however, an entirely different structural damage was observed when the dentin surface was exposed to a q-switched nd:yag laser, as shown in figure 2c on energy dose of 21.2 j/cm2 and figure 2d on energy dose of 41.7 j/cm2, that occured melting and variation of deep hole. although the dentin exposed by multiple impacts of pulse laser but only one pit is appeared on the dentin surface. the pit like a deep hole been drilled by the focused laser. power density created by the q-switched nd:yag laser enable to produce breakdown associated with plasma formation and shock wave propagation. the high temperature from the plasma and high pressure from the shock wave responsible to melt and vaporize the enamel surface which result the formation of deep hole, depending on variation energy dose. furthermore, damage observed under fesem analysis due to q-switched exposed is distributed more uniformly as a resulted of mechanical mechanism when sample preparation. rather than only single deep pit due to the effect of melted and vaporization after interaction with figure 3. xrd spectra of the dentin specimens at energy dose various and fixed repetition rate of 10 hz a) xrd output from normal dentin; b) xrd output of dentin exposed to q-switched nd:yag laser (energy dose of 13.9 j/cm2); c) xrd output of dentin exposed to q-switched nd:yag laser (energy dose of 21.2 j/cm2); d) xrd output of dentin exposed to q-switched nd:yag laser (energy dose of 41.7 j/cm2). 185apsari, et al.,: in vitro effect of q-switched nd:yag laser exposure microplasma induced by focused of q-switched nd: yag laser, associated with pressure wave induced by the propagation of acoustic shock wave. non-switched is more energetic in this interaction subjected to higher energy carried by every single pulse, compared to q-switched laser. furthermore, the longer duration and the integration of pulsed energy delivered by multipulses on the same spot, subject to greater and longer exposure to the surface of human dentin. in contrast with q-switched the peak power deliver to the target is much higher but the duration within nanosecond time will not allow the thermal conductivity on surface of human dentin. this result is more localized, damage the pits, and may cause some particle removal due to the melted and vaporization effect. in this experiment, the q-switched nd:yag lasers operate at energi dose of 13.9 j/cm2, 21.2 j/cm2, and 41.7 j/cm2 with repetitive rates of 10 hz. hydroxyapatite (ca10(po4)6](oh)2) is found having high percentage after exposed by q-switched nd:yag laser. these results show that human dentin consists of more than 73% of hydroxyapatite, in which the ablation threshold fluence is expected to be. it is lower than that of monocrystalline fluoroapatite. the composition of hydroxyapatite of the dentin after exposure to q-switched nd:yag laser varies from 75.02% to 78.21%. this results indicate that exposing pulsed nd:yag laser on the human dentin induced greater composition of hydroxyapatite. the chemical bonding between the atoms in molecules are affected due to the vibration or rotational results from the absorption of infrared laser radiation. this effect of chemical composition is also indicated by the shifted on the lattice parameter of the crystal structure of the dentin material. after the dentin specimen exposed by q-switched infrared laser, the lattice parameters of the hydroxyapatite is shifted greater than compared to the exposure by normal dentin unexposed as long as no new appearance of peaks of loss of peaks meaning that the crystal structure of the dentin material is remain the same. the results also proved by the xrd spectrum analysis (figure 3). this indicated that exposed by pulsed nd:yag laser on the human dentin induced greater composition of hydroxyapatite and higher microhardness in comparison to normal unexposed dentin. the absorption of infrared laser radiation also affects the chemical bonding between the atoms in molecules due to the vibration or rotational effect. this resulted in a shift of lattice parameter of the crystal structure of the dentin material. it is also observed that the q-switched pulses exposure shifts the lattice parameters of the hydroxyapatite greater than that of the non-q-switched laser. since no new appearance of peaks of loss is observed in the xrd spectra the crystal structure of the dentin materials assumed unchanged. exposure of pulsed nd:yag laser on the human dentin caused photoablation effect. exposed the dentin by infrared laser radiation is similar as laser annealing process. the longer the time taken to anneal the surface the more the harder the surface due to the loss of water and carbon content of the dentin material. the results of the microhardness properties of the dentin after anneal with infrared material is shown in table 2. this means the pulse nd:yag laser exposed almost 15000 times longer than nanosecond signal from q-switched nd:yag laser. furthermore the power received after 10 second exposure for example by the dentin surface is almost 166 w for 10 hz repetition rate. such power radiation will be enough to generate high temperature within 100 to 650° c since the area of the enamel just around 4 to 9 mm in diameter. the photothermal effect is possible to decrease the water and carbon content from the dentin surface. the evaporation of water and carbon content is subjected to increase the microhardness property of the dentin. this result is in good agreement with other previous researchers.1,4,6–9 these results show that nanoseconds q-switched nd: yag laser can be used for tooth ablation applications. the material removal remains localized on the area of the laser spot. almost no signs of collateral damage and cracks are observed with a sharply defined cavity edges. this study suggests that the application of short-pulse lasers as contactless drills in dentistry can be considered as an alternative to the classical mechanical technique to improve the quality of the dentin treatment. ablation of the dentin is demonstrated at localized area and no signs of collateral damage and cracks are observed using an 8 ns pulses at wavelength of 1064 nm, energy dose of 13.9 j/cm2 and 10 hz repetition rate from a q-switched nd:yag laser, while the energy dose of 21.2 j/cm2 and 41.7 j/cm2 occured melting and deep hole. this exposure from q-switched nd:yag laser increases the microhardness property and hydroapatite percentage of the dentin, which produces a side effect of surface dehydration and crack, melting, and deep hole. this side effect can use to dentin treatment in dentistry. however, the crystal structure of dentin is unchanged since the shifted crystal lattice is small with exposure to q-switched nd:yag laser, so safe for application to dentin treatment. in conclusion the application of q-switched nd:yag laser as contactless drills in dentistry should be regarded as an alternative to the classical mechanical technique to improve the quality of the dentin treatment. acknowledgement the authors would like to thanks the government of indonesia through directorate general of higher education and faculty of sciences and technologi airlangga university for financial support in this research. the author would also than university technology malaysia (utm) for the collaboration work and ms. yuni trisnawati for her cooperation in analysis work. 186 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 181–186 references 1. de andrade ak, lizarelli r de f, pelino, bagnato vs, de oliveire ob. enamel caries resistance accidentaly irradiated by nd:yagenamel caries resistance accidentaly irradiated by nd:yag laser. laser phys lett 2007; 4(6): 457–63. laser phys lett 2007; 4(6): 457–63.lett 2007; 4(6): 457–63. 2. walsh lj. the current status applications in dentistry. aust dent j 2003; 48(3): 146–55. 3. steiner w. new laser technology and future application. medical laser appl 2006; 21(2): 131–40. 4. korytnicki d, mayer mp, daronch m, singer j da m, grande rh. effects of nd:yag laser on enamel microhardness and dental plaque composition: an in situ study. photomedicine and laser surgery 2006; 24(1): 59–63. 5. villa gep, catirse abceb, lia rcc, lizarelli rfz. in vivo analysis of low-power laser effects irradiation at stimulation of reactive dentin. laser phys lett 2007; 4: 690–5. 6. rohanizadeh r, jean a, daculsi g. effect of q-switch nd:yag laser on calsified tissues. laser in medical science 1999; 14(3): 221–7. 7. magalhaes ac, rios d. effect of nd:yag irradiation and fluoride application on dentine resistance to erosion in vitro. photomedicinephotomedicine and laser surgery 2008; 26(6): 559–63. 8. lizarelli rfz, costa mm, carvalho-filho e, nunes fd, bagnato vs. selective ablation of dental enamel and dentine using femtosecond laser pulses. laser pyhs lett 2008; 5(1): 63–9. 9. arianto. study of microstructure characterization and thermal properties of human dentin as surface dentin modification. thesis. jakarta: indonesia university; 2003. 10. neimz mh. laser-tissue interactions, fundamental and applications. 3rd ed. germany: springer; 2007. p. 45–200. vol 44 no 3 sept 2011.indd 127 vol. 44. no. 3 september 2011 literature review recent pharmacological management of oral bleeding in hemophilic patient monica widyawati setiawan faculty of pharmacy, widya mandala university surabaya indonesia abstract background: hemophilia is a hereditary bleeding disorder that can increase the risk of disease in oral cavity. sometimes hemophilia is not always established already in a patient. the lack of awareness of hemophilia presence can cause serious problem. purpose: the purpose of this review is to explain about dental bleeding manifestation and management in hemophilic patient. reviews: hemophilia can be manifested as dental bleeding that cannot stop spontaneously. it should be treated with factor viiii either by giving whole blood, fresh plasma, fresh frozen plasma, cryoprecipitate, and factor viii concentrate. factor viii dose for hemophilia treatment can be calculated based on factor viii present in hemophilia patient’s body. factor viii can also be given as prophylaxis to prevent bleeding. complications that can be caused by factor viii replacement therapy are the presence of factor viii inhibitor and transfusion related diseases. treatment of dental bleeding due to hemophilia consists of factor replacement therapy and supportive therapy. conclusion: treatment of dental bleeding due to hemophilia consists of factor replacement therapy and supportive therapy. there are complications that can happen due to factor viii replacement therapy that should be considered and anticipated. key words: hemophilia patient, oral bleeding, management abstrak latar belakang: hemofilia adalah kelainan pembekuan darah yang diturunkan. hemophilia dapat meningkatkan resiko penyakit rongga mulut. hemofilia tidak selalu sudah terdiagnosa saat penderita melakukan kunjungan ke dokter gigi. kurangnya kewaspadaan akan adanya hemofilia dapat menyebabkan masalah serius. tujuan: tujuan dari kajian pustaka ini adalah memaparkan tentang manifestasi dan penanganan perdarahan gigi pada penderita hemofilia. tinjauan pustaka: hemofilia dapat bermanifestasi sebagai perdarahan gigi yang tidak dapat berhenti secara spontan. pada keadaan perdarahan tersebut, pemberian faktor viii yang diberikan sebagai whole blood, fresh plasma, fresh frozen plasma, cryoprecipitate, dan konsentrat faktor viii. dosis faktor viii sebagai terapi hemofilia dapat dihitung berdasarkan kadar faktor viii yang terdapat dalam tubuh penderita hemofilia. faktor viii juga dapat diberikan sebagai terapi profilaksis untuk mencegah perdarahan. komplikasi yang dapat terjadi pada pemberian factor viii replacement therapy adalah timbulnya inhibitor faktor viii dan penyakit yang terkait transfusi. terapi perdarahan gigi pada penderita hemofilia terdiri dari factor replacement therapy dan terapi suportif. kesimpulan: terapi perdarahan gigi pada penderita hemofilia terdiri dari factor replacement therapy dan terapi suportif. komplikasi factor viii replacement therapy harus diwaspadai dan ditatalaksana dengan baik. kata kunci: pasien hemofilia, perdarahan mulut, tatalaksana correspondence: monica widyawati setiawan, c/o: fakultas farmasi, universitas widya mandala surabaya, indonesia. e-mail: monicawidya@yahoo.co.id introduction spontaneous bleeding in oral cavity can easily happen, due to gingival bleeding. the risk of gingival bleeding during brushing teeth causes poor oral hygiene in most hemophilic patient. the poor oral hygiene will increase the risk of disease in oral cavity, due to dental caries or periodontal disease. morbidity and death are primarily the 128 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 127–131 result of hemorrhage, although infectious diseases (eg, hiv, hepatitis) became prominent.1 hemophilia is an x-linked chromosome disorder and will be manifested as bleeding due to clotting factor deficiency. hemophilia must be suspected in patients with abnormal bleeding tendency. deficiencies of factor viii and ix are the most common severe inherited bleeding disorder.1 the classification of the severity of hemophilia has been based on either clinical bleeding symptoms or on plasma procoagulant levels; the latter are the most widely used criteria. persons with less than 1% normal factor (< 0.01 iu/ml) are considered to have severe hemophilia. persons with 1–5% normal factor (0.01–0.05 iu/ml) are considered to have moderately severe hemophilia. persons with more than 5% but less than 40% normal factor (> 0.05 to < 0.40 iu/ml) are considered to have mild hemophilia.1,2 the treatment of hemophilia may involve management of hemostasis, management of bleeding episodes, use of factor replacement products and medications, treatment of patients with factor inhibitors, and treatment and rehabilitation of patients with hemophilia synovitis. treatment of patients with hemophilia ideally should be provided through a comprehensive hemophilia care center.1 to stop the bleeding, factor that is deficient should be given. several options for factor viii replacement therapy are: whole blood, fresh plasma, fresh frozen plasma, cryoprecipitate, and factor viii concentrate. although factor viii concentrate has been given, bleeding can still happen due to inhibitor factor viii. inhibitor factor viii should be managed well to prevent prolonged bleeding.2 etiology, patophysiology, and laboratory examination of hemophilia hemophilia is a hereditary bleeding disorder. hemophilia a is caused by an inherited or acquired genetic mutation or an acquired factor viii inhibitor, while hemophilia b is factor ix deficiency. the defect results in the insufficient generation of thrombin by the fixa and fviiia complex by means of the intrinsic pathway of the coagulation cascade. it creates an extraordinary tendency for spontaneous bleeding.1,3,4 hemophilia was stated in 1820 as bleeder’s disease transmitted by unaffected females to their son. this disorder is inherited in an x-linked recessive pattern. prolonged clotting time was found in hemophilia patient. hemophilia is caused by decrease of factor viii levels.4,5 factor viii deficiency, dysfunctional factor viii, or factor viii inhibitors lead to the disruption of the normal intrinsic coagulation cascade, resulting in spontaneous hemorrhage and/or excessive hemorrhage in response to trauma. hemorrhage sites include joints (eg, knee, elbow), muscles, cns, gi system, genitourinary system, pulmonary system, and cardiovascular system.4 platelet count, activated partial thromboplastic and prothrombin test are screening tests if there is suspicion of hemophilia. specific test needed to diagnose hemophilia is factor viii assay.6 in patients with hemophilia, there will be prolong activated partial thromboplastin time (aptt), normal platelet count, and normal prothrombin time (pt). specific assay for factor viii is needed to know which factor is deficient.2,7 clinical manifestation of hemophilia bleeding symptoms may be present from birth or may occur in the fetus.1,8 like other parts of the body, hemophilia will also give effect to oral cavity such as dental caries or gingivitis. hemophilia patients will be afraid to brush their teeth and afraid to receive dental treatment because they fear about bleeding that may occur. every dental management must be done very carefully to prevent bleeding.1 suspicion should always be raised in the presence of abnormal bleeding. although there can be considerable overlap, in general, platelet problems result in ptechiae, especially on dependent parts of the body and mucosal surfaces. ecchymoses are suspicious for coagulation factor deficiencies or platelet problems when they occur in unusual areas, are out of proportion with the extent of described trauma, or are present in different stages of healing.9 management of hemophilia to stop the bleeding, factor that is deficient should be given. in hemophilia a patients, factor viii should be given. there are several options for factor viii replacement therapy, such as: whole blood, fresh plasma, fresh frozen plasma, cryoprecipitate, and factor viii concentrate. whole blood contains the least factor viii. cryoprecipitate or fresh frozen plasma is considerably less effective and less safe. the best treatment is factor viii concentrate.2,10–13 the in vivo percent elevation in factor viii level can be estimated by multiplying the dose of ahf per kilogram of body weight (iu/kg) by 2%. we will know the factor viii increment needed by measuring the factor viii the patient had and increased it to the normal level. factor viii needed to stop the bleeding is calculated by multiplying expected % factor viii increase with body weight and divided it with 2%/iu/kg.14,15 in oral bleeding, topical thrombin can be applied especially if bleeding is minimal or has been for only a few hours.16 prophylaxis factor viii can be given every 2–-3 days to maintain the normal level of factor viii.17,18 supportive management that can be done to stop the bleeding in hemophilia patient are rest, ice, compression, and elevation (rice).9 in addition, chlorhexidine gluconate mouthwash can be used to control periodontal problems. blood loss of all kinds can be controlled locally with direct pressure or periodontal dressings with or without topical antifibrinolytic agents.19 complications that can be caused by factor viii replacement therapy are the presence of inhibitor and transfusion related diseases.20 transfusion related diseases above all are hiv, hepatitis, cytomegalovirus, epstein-barr virus, syphilis, malaria, etc. the most prevalent transfusion related diseases are hiv and hepatitis.21. 129setiawan: recent pharmacological management discussion hemophilia was called royal disease because it spread to the royal families of europe through victoria’s descendants. it was untreatable and only a few hemophiliacs survived to reproductive age because any small cut or internal hemorrhaging after even a minor bruise were fatal, until recently it is treated with factor replacement therapy.22 positive family history is not always available, and it complicates the diagnosis of hemophilia. a retrospective descriptive study done from january 2007–december 2010 done in rd kandou hospital manado found only 5 of 21 patients (23.8%) had positive family history.23 the major signs and symptoms of hemophilia are excessive bleeding and easy bruising. the extent of bleeding depends on the type and severity of the hemophilia. children who have mild hemophilia may not have symptoms unless they have excessive bleeding from a dental procedure, an accident, or surgery. bleeding can occur on the body's surface (external bleeding) or inside the body (internal bleeding). signs of excessive external bleeding include: bleeding in the mouth from a cut or bite or from cutting or losing a tooth, nosebleeds for no obvious reason, heavy bleeding from a minor cut, and bleeding from a cut that resumes after stopping for a short time.24 platelet count must be included among the basic screening tests for patients exhibiting a bleeding diathesis. prothrombin test is the time taken by a recalcified citrated platelet poor plasma to clot in the presence of tissue thromboplastin & phospholipids. it is a very good screening tests for coagulation factors involved in the extrinsic and common pathway of coagulation–namely vii, x, v, ii and i. activated partial thromboplastic is the time taken by a recalcified citrated platelet poor plasma to clot in the presence of a surface activator (silica, koalin or ellagic acid) and phospholipid (partial thromboplastin). it is good screening test of coagulation factors involved in the contact activation and common pathway namely xii, xi, ix, viii, x, v, ii and i. specific test needed to diagnose hemophilia is factor viii assay.6 factor viii is involved in intrinsic pathway of haemostatic process. factor viii is needed to convert factor x to be factor xa, which in turns will convert prothrombin to be thrombin. thrombin is needed to convert fibrinogen to be fibrin which needed to stop bleeding (figure 1). that is why in patients with positive bleeding history or active bleeding should have platelet count, pt and ppt test. in patients with hemophilia, there will be prolong ppt, normal platelet count, and normal pt. specific assay for factor viii is needed to know which factor is deficient.2,7 management of hemophilia consists of supportive and specific therapy. specific therapy is factor replacement therapy, which available in several forms that should be given according to the availability of the agent. supportive therapy for dental bleeding consist of avoidance of giving antifibrinolytic agent systematically, evaluate the presence of anemia, topical agent supplementation (thrombin/fibrin sealant, ice, pressure), and soft diet. dental care should be done very carefully. before any dental procedure that may cause bleeding, factor replacement therapy should be given first.25 desmopressin acetate can also be given intranasal with 0.2–0.4 mcg/kg/dose, 2 hours before procedure.26 there are several things to remember in dental check up for hemophilia patient: antibiotics should be taken before all invasive procedures, factor viii replacement therapy should be given if there is prolonged bleeding after teeth cleaning work, pretreatment with factor concentrate or short term hospitalization may be required for oral surgery and periodontal treatment in hemophilia patient, pretreatment with an antifibrinolytic agent and possibly infusion of factor concentrate if there is likelihood of bleeding difficulties after dental treatment, and if the patient had regular prophylaxis regimen, dental treatment should be done in the same day with the prophylaxis treatment day.27 in hemophilia patient, accidents involving the mouth may happen, especially during childhood. when bumps, falls, and collisions occur, there are several things that can be done: we should pick up the tooth by the crown, avoiding the roots, rinse it off, and place in milk, if possible so that the tooth may be reinserted. while waiting for the process, apply firm pressure to the bleeding site with a piece of clean gauze. patient must go directly to the emergency room if there are bleeding on the tongue, cheek, or floor of the mouth doesn’t stop; the tongue, throat, or neck is swollen or bruised; or there is trouble to breath or swallow. we should prevent the clot from breaking away from the wound site after an injury or extraction with treatment that can include an antifibrolytic agent, factor concentrate, or desmopressin nasal. a word to the wise about preventing emergencies: children with hemophilia should always wear mouth guards when they play sports.27 it is essential to prevent accidental damage to the oral mucosa when carrying out any procedure in the mouth. injury can be avoided by: using saliva ejectors carefully; removal of impressions carefully; extra care in the placement of x-ray films, particularly in the sublingual region; giving figure 1. factor viii action in haemostatic function.2 130 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 127–131 protection to the soft tissues during restorative treatment by using a rubber dam or applying yellow soft paraffin (vaseline®).27 previously, when factor viii concentrate is still not available, blood product such as whole blood and cryoprecipitate are used to replace the need of factor viii concentrate. lately, factor viii concentrate is used to replace the need of factor viii concentrate. in 1970, the factor viii available is acquired from many donors by separating factor viii from plasma. this way, the treatment can be given right away and it can be given at home. unfortunately, at 1980, it is reported that many virus can be transmitted from factor viii concentrate replacement therapy. in 1984, recombinant factor viii technology is developed. today, recombinant factor viii therapy is available.28–30 factor viii replacement therapy can have several complications i.e. the presence of inhibitor and transfusion related diseases.29,31 the most prevalent transfusion related diseases are hiv and hepatitis.32 if factor viii concentrate has been given, but no significant improvement is present, factor viii inhibitor should be considered. to confirm the present of factor viii inhibitor, blood analysis should be done. it is reported that factor viii inhibitor is common in patients treated with factor viii recombinant replacement therapy.33 to overcome the presence of factor viii inhibitor, the amount of factor viii concentrate given to the patient should be increased or a by passing agent can be given. by passing agents are prothrombin complex concentrates and activated prothrombin complex concentrates. in 1990, recombinant activated factor vii was used to overcome the factor viii inhibitor.34 it is concluded that treatment of dental bleeding due to hemophilia consists of factor replacement therapy and supportive therapy. there are complications that can happen due to factor viii replacement therapy that should be considered and anticipated. references 1. pradopo s. perawatan dasar di kedokteran gigi bagi pasien hemophilia. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 91–6. 2. monroe dm, hoffman m, roberts hr. molecular biology and biochemistry of the coagulation factors and pathways of hemostatis. in: kaushansky k, lichtman m, beutler e, kipps t, prchal j, seligsohn u, eds. 8th ed. new york: williams hematology; 2010. p. 1978–85. 3. susanah s. pertimbangan pemilihan terapi kriopresipitat, konsentrat factor atau rekombinan pada hemophilia. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 18–25. 4. mosher df. disorders of blood coagulation. in: wyngaarden jb, smith lh, bennet jc, eds. 19th ed. philadelphia: cecil textbook of medicine; 1992. p. 1004–7. 5. sutaryo, ardianto b. history of hemophilia. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 44–50. 6. peyvandi f. carrier detection and prenatal diagnosis of hemophilia in developing countries. seminar thromb hemost 2005; 31(5): 544–54. 7. scott jp, montgomery rr. hemostasis. in: kliegman rm, behrman re, jenson hb, stanton bf, eds. nelson textbook of pediatrics. 18th ed. philadelphia: elsevier; 2007. p. 2060–6. 8. scott jp, montgomery rr. hereditary clotting factor deficiencies. in: kliegman rm, behrman re, jenson hb, stanton bf, eds. nelson textbook of pediatrics. 18th ed. philadelphia: elsevier; 2007. p. 2066–70. 9. mckenzie se. coagulation disorders. in: polin ra, ditmar mf, eds. pediatric secrets. 4th ed. philadelphia: elsevier; 2005. p. 272–4. 10. widjajanto ph. mengenal hemophilia. cetakan kedua. yogyakarta: sumber aksara; 2011. p. 25–37. 11. permono b. clinical perspective in understanding von willebrand complex for hemophilia a therapy. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 84–90. 12. clarke a. factor viii concentrate factor ix complex (coagulation factors ii, vii, ix, x) concentrate application for retention on the who model list from: european plasma fractionation association (epfa). 2004. available from: http://factorviii&ix_epfa_ submission_29oct.pdf. accessed may 6, 2011. 13. philipp c. the aging patient with hemophilia: complications, comorbidities and management issues. in: gewirtz am, mikhael jr, schwartz bs, crowther ma, eds. hematology. 30th ed. florida: american society hematology; 2010. p. 191–6. 14. permono b, ugrasena idg, ratwita m. pedoman diagnosis dan terapi ilmu kesehatan anak buku satu. edisi 3. surabaya: rsud dr. soetomo; 2008. p. 139–42. 15. duncan c, heeney mm. hematologic disorders. in: graef jw, wolsdorf ji, greenes ds, eds. manual of pediatric therapeutics. 7th ed. philadelphia: lippincott williams & wilkins; 2008. p. 482–4. 16. lee ca, kessler cm, varon d, martinowitz u, heim m, gringeri a. treatment protocol of haemophilia and other congenital bleeding disorders in italy. haemophilia 1998; 4(4): 423–4. 17. gatot d. tatalaksana profilaksis terkini pada penderita hemophilia. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 80–3. 18. lubis b, windiastuti e, amali p, amelia n. pedoman pelayanan medis ikatan dokter anak indonesia. edisi 1. jakarta: idai; 2010. p. 92–7. 19. brewer a, correa me. guidelines for dental treatment of patients with inherited bleeding disorders. treatment of hemophilia. quebec: world federation of hemophilia; 2006. p. 40. 20. widjajanto ph. hemofilia: etiologi, komplikasi dan terapi. kongres nasional iii himpunan masyarakat hemofilia indonesia: penanganan terpadu hemophilia menuju masa depan yang lebih cerah, surabaya; 2011. p. 6–8. 21. contreras m. abc for transfusion. new york: wiley-blackwell; 4th ed. 2008. p. 80–9. 22. aronova-tiuntseva y, herreid cf. hemophilia: “the royal disease”. available from: http://www.sciencecases.org/hemo/hemo.asp. accessed may 6, 2011. 23. salim j, gunawan s. profile of hemophilia in manado. pediatrica indonesiana 2011; 51(4): 179. 24. roberts hr, key ns, escobar ma. hemophilia a and hemophilia b. in: kaushansky k, lichtman m, beutler e, kipps t, prchal j, seligsohn u, eds. 8th ed. new york: williams hematology; 2010. p. 2000–8. 25. world federation of hemophilia. guidelines for the management of hemophilia. available from: http:// www.wfh.org. accessed may 6, 2011. 26. barone ma. the harriet lane handbook. 17th ed. philadelphia: mosby; 2008. p. 518–9. 131setiawan: recent pharmacological management 27. peterson d. hemophilia & dental care. available from: http://www. dentalgentlecare.com/hemophilia_dental_care.htm. accessed may 6, 2011. 28. montgomery rr, gill jc, paola jd. hemophilia and von willebrand disease. in: nathan dg, orkin sh, ginsburg d, look at, eds. nathan and oski’s hematology of infancy and childhood. 7th ed. philadelphia: saunders; 2009. p. 1487–524. 29. stachnik j. hemophilia: etiology, complications, and current options in management. formulary 2010; 45: 218–27. 30. manucci pm. back to the future: a recent history of hemophilia treatment. hemophilia 2008; 14(3): 10-8. 31. abdel-messih iy, habashy dmm, moftah sg, el-alfy m. persistent factor viii inhibitors and orthopaedic complications in children with severe haemophilia a. haemophilia 2011; 17(3): 490–3. 32. harmening dm. harmening: modern blood banking and transfusion practices. 5th ed. pennsylvania: fa davis company; 2005. p. 207–14 33. wight j, paisley s. the epidemiology of inhibitors in hemophilia a: a systematic review. hemophilia 2003; 9: 418-35. 34. tjonnfjord ge, holme pa. factor viii inhibitor bypass activity in the management of bleeds in hemophilia patients with 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 32 research report dental journal (majalah kedokteran gigi) 2017 march; 50(1): 32–35 correlation between working position of dentists and malondialdehyde concentration with musculoskeletal complaints hari wibowo,1 titiek berniyanti,1 and jenny sunariani2 1department of dental public health 2department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: musculoskeletal complainstof dentists have become common issues in dentistry since the awkward positions during treating the patients; such as position of a dentist who bends towards the patient, moving abruptly, twisting the body from a side to another side. all those movements are done several times in long term. such high level of activity without sufficient recovery time may lead to an oxidative stress, so it will affect on the musculoskeletal and concentration of malondialdehyde (mda) in blood. purpose: the purpose of this study was to identify the correlation between mda concentration on the risk of musculoskeletal complaint on dentist with working position of maxilla dental patching at community health center(puskesmas) in surabaya. method: this study was observational analytics using cross sectional approach with cluster random sampling technique. the total samples were 19. musculoskeletal complains assesment is conducted using a nordic body map questionaire that devided into 4 scores. working position of samples were asseses using ovako working posture analysis (owas). bloos sampling was conducted to examine the concentration of mda. result: from data analysis result using spearman correlation test, it was found that there was a significant correlation between working position and musculoskeletal complaint using spearman correlation test and the p value obtained was<0.05. this research also found that there was a significant correlation between malondialdehyde concentration and musculoskeletal complaint using pearson correlation test and the p value obtained was <0.05. conclusion: there was a correlation between working positions of dentists and musculoskeletal complaints. there was a correlation between working position and mda concentration with the musculoskeletal complaints. keywords: malondialdehyde; musculoskeletal complaint; working position correspondence: titiek berniyanti, department of dental public health, faculty of dental medicine, universitasairlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: berniyanti@gmail.com introduction musculosceletaldisorders (msds) is a chronic disorder on muscles, tendons and nerves caused by repetitive use of energy, rapid movement, enormous use of energy, contact with pressure, awkward or extreme posture, vibration, and low temperature.1 dentist is a profession with frequent mdss due to the works. from the literatures, it can be identified that prevalence of msds on dentists in saudi arabia is 82.9%. in australia, musculoskeletal disorder reaches 87.2%, in india it is 78%, lithuania 86.5%, and turki reaches 94%. from those data, it can be seen that the high prevalence of musculoskeletal prevalence on dentist in other countries.2 in indonesia, the number of incidents is unidentified since there has been no data provided about the prevalence of musculoskeletal complained by dentists in indonesia according to the result of screening conducted at faculty of dentistry, universitas indonesia using body discomfort map and brief survey as instruments, which found that 80% of dentists who work in the clinic experience musculoskeletal disorder, particularly on neck, shoulders, forearms, upper arms, hands and back.2 one of the causes of musculoskeletal syndrome experienced by dentists is because the dentists assume that dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p32-35 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p32-35 3333wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 32–35 they are the one who should make moves to get close to the patients rather than they should arrange patient’s sitting position on the dental chair. most musculoskeletal disorders among dentists occur since the dentists are positioning their body unconsciously that do not support their movement during treating the patients. when preparing for dental or pulling of the teeth, for example, sometimes the dentist frequently bends towards the patient, move abruptly, twist the body from a side to another side. all those movements are done several times for prolonged period.3 musculoskeletal disorder is also proven to contribute in the decrease of productivity and even finish their professional career earlier. obviously, this risk is a serious matter so that it requires a preventive treatment and education starting from the time when the dentists are still in their learning period at faculty of dentistry.4 the activities of dentists and a lack of attention for their comfort when treating the patient could be a workload for the dentists. activities and the workload may cause oxidative stress. oxidative stress is a condition where there is an imbalance between free radicals production or reactive oxygen species (ros) with the antioxidant, where the level of free radical is higher compared to the antioxidant.5 several results of the research exhibits that the level of free radical increases after performing activities or exercises signified with the occurring of increasein lipid hydroperoxide.5 in other words, oxidative stress is a condition where there is an imbalance between free radical and antioxidant. oxidative stress is a component on the mechanism of tissue damage in human. oxidative stress can be identified by having a higher level of malondialdehyde (mda) serum or tissue.6 the main indicator utilized to identify the presence of lipid peroxide and the parameter of incurring oxidative stress is mda. mda is one of the substances whose molecule weight is quite light which is generated as the final product of lipid peroxide within the body due to free radical reaction.7 mda is formed from lipid peroxyde (lipid peroxidation) on cell’s membrane namely free radical reaction (hydroxyl radical) with polyunsaturated fatty acid (pufa). while mda level increase, it shows that there is a lipid peroxidation process, which is highly potential causing complication whether it is microvascular or macrovascular.6 the activities conducted by the dentists without sufficient recovery time will lead to oxidative stress so it may affect on musculoskeletal complaints and concentration of mda. from the previous backgroundsof the study, this study aimed to identify whether there was a correlation between concentration of mdain blood and musculoskeletal complaint on the subject of dentists. materials and methods this research is categorized into observational analytic research using cross sectional approach. sampling technique used in this research was cluster random sampling using 19 partisipants. all partisipants were dentists. partisipants criteria in this research are: maximum age of 50 years old, not yet menopause and are not in the middle of period (during blood sampling) for female, maximum tenure of 5 years, physically and mentally healthy, and were willingly to act as research subjects. musculoskeletal complaint assessment is conducted using a nordic body map questionnaire. this questionnaire provides picture of human body which has been categorized into 9 main parts, namely: neck, shoulders, upper back, elbows, lower back, wrists/hands, hip/bottom, knees, heels/ feet. a nordic body map questionnaire is divided into 4 scores, namely low between score of 0-20, medium between score of 21-41, high between score of 42-62, and very high between score of 63-84. this research also assessed the working position of dentist using an ovako working posture analysis (owas) method. owas method is a simple method and is utilized to assess body posture during working. working position assessment using this method is by providing risk score on the back parts of the body (4 positions), arms (3 positions), feet (7 position), and loading (3 intervals).8 according to the observation on the posture which has been conducted, it may identify the urgency of remedial action on such posture through the classification of the 4 action categories from scale 1 to 4. combination of the four digit numbers then will provide us an overview of which action categories of the posture that we observe is located at. manually, we may identify the score of action category by looking at the combination of those four numbers. 8 blood sampling was conducted to examine the concentration of mda, which was conducted using syringe to take the vein blood samples for 2cc. after that, the blood samples were insertedinto red tubes and delivered to the laboratory for centrifuge and to take 0.5 ml the serum. the serum was processed by adding cold phosphatebuffered saline (pbs) solution, 15% trichloroacetic acid (tca) and 0.37% 2-thiobarbituric acid (tba) in 0.25 n hydrochloric acid (hcl). after it was cooled down, serum was centrifuged and a measurement on mda concentration according to mda supernatan absorbent score was conducted using microprocessor controlled uv/visible range spectrophotometer (boeco, hamburg, germany). results in this research, working position of respondent was identified using an owas method. table 1 shows that the total number of respondents with the highest frequency were 10 respondents (52.6) of which the score was 2. meanwhile, the total number of those with the lowest frequency was 3 respondents (15.8%) of which the score was 1. most respondents in this research had medium muscoskeletal complaint risk level, which means that their working position (posture) had several effects, which may harm the musculoskeletal system. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p32-35 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p32-35 34 wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 32–35 in this research, musculoskeletal complaint was assessed using a nordic body map questionnaire. from the classification, the risk level was divided into four levels such as low, medium, high, and very high levels. table 1 shows that among 19 respondents, there were only low, medium, and high risk levels. 11 respondents (57.9%) experienced low level of complaints, 5 respondents (26.3%) experienced medium level of complaints and 3 respondents (15.8%) experiencedhigh levelcomplaints. the statistical test result using spearman correlation test shows a significant correlation between working position and musculokseletal complaints. the statistical test result using pearson correlation test shows a significant association between mda concentration in blood and musculokseletal complaints (table 2). discussion assessment of dentist’s working position in this research utilized an ovako working posture analysis (owas) method, which is an ergonomical evaluation method to observe working posture at back, arms, and limbs parts objectively. an owas identifies several postures, which commonly occur on an occupation (particularly table 1. frequency of working position of respondents and respondents’ musculoskeletal complaints among dentists at community health center (puskesmas) in surabaya in 2016 risk categories (owas scores) frequency (people) percentage (%) low (1) average (2) high (3) very high (4) 3 10 6 0 15.8 52.6 31.6 0 total 19 100 musculoskeletal complaints low average high 11 5 3 57.9 26.3 15.8 total 19 100 table 2. analysis of association between working position and mda concentration in blood and respondents’ musculoskeletal complaints among dentists at community health center (puskesmas) in surabaya in 2016 independent variable dependent variable p explanation working position (owas) mda musculoskeletal complaints musculoskeletal complaints 0.011 0.048 related related manufacture). body posture that includes back, arms, and limbs has particular codes so that a result of owas evaluation may provide a series of 7 digit numbers. the first digit of the code represents back posture, the second digit represents arms posture, the third digit for feet limbs posture, and the fourth digit is the code for the load handled by the worker. the next two digits describe the sectional code of work set whose posture is being observed.9 results of this research showed that there was a significant correlation between working position and musculoskeletal complaints, which means that there was an association between the incidents of musculoskeletal complaints and working position of dentists. these results showed the working position of dentists with long duration and frequency, which may lead to a risk incurring musculoskeletal complaints. when administering a treatment, a dentist perform the treatment in the position of bowing the body and bowing the neck in long duration, so that it may lead to the possibility of vertebral injury. data on results of this research is shown in table 1, which says that if the dentists do not have sufficient break, it may harm the dentists. working positions of the subjects in treatment action are mostly conducted in standing, bowing position in long duration repetitively and the neck, which tends to go upfront. this is in line with the research conducted by ikrimah10 who stated that the complaints experienced by the workers due to bowing working position and twisting movement on hip area, bowing neck and repetitive movements without any interval of sufficient rest or break. career in dentistry is shown by the presence of statical and stiff body position in executing treatments for the patients. the patients who are treated on dental chair will force the dentists to sit or standing while bowing in long duration. this kind of body position may cause the dentists experience sort of pain during their clinical practice or discomfort on neck, shoulders and backbone areas so that it may cause, among other things, musculoskeletal disorder in the form of low back pain.3 standing position in long period is actually the body is only able to tolerate the standing-still pose in one position only for 20 minutes. if it is more than that period, tissue elasticity will gradually decrease and muscle pressure will increase so that there will be discomfort on back area. if such back muscles receiving static loads when the person is standing for such a long time, it may cause complaints in the form of damage on joints, ligaments and tendons. these complaints and damages which is commonly called as musculoskeletal or injury on musculoskeletal system.8,11 the works conducted in sitting position shows that the body parts being complained for are hip, back and neck area. sitting position at musculoskeletal and vertebral should be retained by backrest so that it may prevent the body from back pain and fatigue. in addition, when sitting, the feet should be on footwear and on the sitting pose which allows for a bit movement and relaxation. working position in sitting needs a smaller amount of energy compared to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p32-35 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p32-35 3535wibowo, et al./dent. j. (majalah kedokteran gigi) 2017 march; 50(1): 32–35 standing, therefore, it may decrease the amount of static muscle load at feet. the wrong sitting position may cause back problems such as hip and neck pain. pressure on vertebral will increase during sitting, compared to when standing or lying down. straining sitting position needs more muscle or back nerves activities. therefore, sitting position may affect the performance of a worker. 11 dentists, in their clinical practice, often perform multiple extreme statical postures such as bowing their heads, bowing their bodies down, leaning their bodies, putting their hands up and rising their shoulders up. such extreme positions may cause muscle fatigue and mechanical pain on neck, shoulders and lower hip. during working, the needs of blood circulation may increase from ten to twenty times. the increase of blood circulation on the working muscles enforces the heart to pump more blood. when the body is used to stand for a long duration, the muscles tend to work statically, of which this static muscle work is signified by muscle contraction for a long time, which is commonly according to body position. it is not recommended to continue the work of static muscle for long time since it may cause pain.12arda stated that one of the triggers of back pain is sitting or standing in such position a long period, or the same repetitive movement, which makes the muscles become stiff (spasme).13 a musculoskeletal complaint occurred among dentists is not only caused by position factor during treatment, but the workload also provides several roles on musculoskeletal complaints. too many patients, long period of treatment and business hours per day is the workload that should be done by the dentists. excessive workloads may cause oxidative stress which lead to an increase on lipid peroxidation. lipid peroxidation is a mechanism of cell’s trauma, whether it is on plants or animals, therefore, lipid peroxidation is used as an indicator of oxidative stress on cells and tissues. mdais a dialdehyde compound, which is the final product of lipid peroxidation within the body. mda shows unsaturated lipid acid oxidation product by free radical. an increase of free radical will cause oxidative stress. an increase of oxidative stress is according to the increase of mda forming.14,15 in this research, we found an association between mda and musculoskeletal complaints on the subjects of dentists. these data explained the correlation between workloads and musculoskeletal complaints. these musculoskeletal complaints will increase free radical production and if it exceeds antioxidant resistance capacity, it may cause oxidative stress which is reflected by an increase in mda concentration within the serum.16 in this research, mda concentration in group of normal body mass index (>25 bmi) was higher than normal (18.5 bmi). this was probably signified by several things such as an increase of lipoprotein oxy-disability or antioxidant decreases. in a study, body weight loss can be specifically taking role in reducing mda concentration as an indicator of change on oxidative stress and lipid profiles. mda concentration significantly can be reduced when reducing body weight. mda acts as peroxidase lipid indicator because this molecule is the main product of oxidative stress. 17 the activities done by the dentists without sufficient recovery time will be a burden for the dentists. excessive workloads may cause oxidative stress. the bmi is one of dentists’ workloads, which significantly affects mda concentration. it can be concluded that there was a correlation between working position of dentist and musculoskeletal compaint. there was a correlation between working position and mda concentration with the risk of musculoskeletal complaint. references 1. anonymous. tlvs and beis. american conference of governmental industrial hygienists. united states: signature publication. 2010. 2. wijaya at, darwita rr, bahar a. the relation between risk factors and musculoskeletal impairment in dental students: a preliminary study. journal of dentistry indonesia 2011; 18(2): 33-7. 3. andayasari l, anorital. gangguan muskuloskeletal pada praktik dokter gigi dan upaya pencegahannya. media litbang kesehatan 2012; 22(2): 70-6. 4. leggat pa. occupational health problems in modern dentistry. industrial health 2007; 45: 611–21. 5. kürkçür. the effects of short-term exercise on the parameters of oxidant and antioxidant system in handball players. african journal of pharmacy and pharmacology 2010; 4(7): 448-52. 6. marjani a. lipid peroxidation alterations in type 2 diabetic patients. pak j biol sci 2010; 13(15): 723-30. 7. nielsen f, mikkelsen bb, nielsen jb, andersen hr, grandjean p. plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. clin chem 1997; 43(7): 1209-14. 8. tarwaka. ergonomi industri: dasar-dasar pengetahuan ergonomic dan aplikasi di tempat kerja. surakarta: harapan press; 2015. 9. helander m. a guide to human factors and ergonomics. second edition. danvers: crc press; 2006. 10. ikrimah n. faktor faktor yang berhubungan dengan keluhan musculoskeletal disorders pada pekerjakon veksisektor usaha informal di wilayah ketapang cipondoh tanggerang 2009. uin 2009. p. 85-89. 11. susanti n, hartiyah, kuntowatod. hubungan berdiri lama dengan keluhan nyeri punggung bawah miogenik pada pekerja kasir di surakarta. jurnal pena medika 2015; 5(1): 60–70. 12. effendi f. ergonomi bagi pekerja sektor informal. cermin dunia kedokteran 2007; 34: 1-154. 13. susanti n. hubungan berdiri lama dengan keluhan nyeri punggung bawah miogenik pada pekerja kasir di surakarta. jurnal pena medika 2015; 5(1): 60–70. 14. jeyabalan a, ca r itissn. antioxidant and the prevention of preeklapmsia-unresolved issues. new england j med 2006; 354(17): 1841-3. 15. winarsi h. antioksidan alami dan radikal bebas. yogyakarta: kanisius; 2007. p. 50-5. 16. peake jm, suzuki k, coombes js. the influence of antioxidant supplementation on markers of inflammation and the relationship to oxidative stress after exercise. j nutrbiochem 2007; 18(6): 35771. 17. siswonoto s. hubungan kadar malondialdehid plasma dengan keluaran k linis stroke iskemik akut. jur nal ilmu biomedik universitas diponegoro. 2008. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i1.p32-35 http://dx.doi.org/10.20473/j.djmkg.v50.i1.p32-35 mkg vol 42 no 2 april 2009.indd 90 vol. 42. no. 2 april–june 2009 research report screening of oral premalignant lesions in smokers using toluidine blue yanti leosari, sri hadiati and dewi agustina faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: a smoker is associated with the risk of developing oral premalignant lesions due to the cacinogenic contents in cigarette. toluidine blue is a basic chromatic dye used in screening the presence of premalignant lesions due to its ability to detect acidic components in cells and tissues. purpose: this study was purposed to observe the outcomes of toluidine blue staining on oral mucosa of smokers and non smokers and to find out whether quantity and duration of smoking affect the final results of toluidine blue staining. methods: forty male subjects, aged 20-60 years old were involved in this study, consisted of 10 heavy smokers, 10 moderate smokers, 10 light smokers and 10 non smokers. subjects were instructed to rinse their mouths with mineral water for 20 seconds followed by acetic acid 1% for another 20 seconds. toluidine blue stain was applied in excess and left on site for 1 minute. subjects were instructed to rinse with acetic acid 1% and sufficient water consecutively for 20 seconds each. the areas of oral mucosa that stained blue were captured with intraoral camera and transferred to the computer unit. the staining procedure was repeated after 14 days. results: chi-square test showed that toluidine blue positive staining dominates the smokers group. regression and correlation test indicate that toluidine blue staining is more obvious in subjects who consume more cigarettes. conclusion: it was concluded that oral mucosa of smokers absorbed more toluidine blue than that of non smokers and retention of toluidine blue is affected by quantity and duration of smoking. key words: cigarette, oral mucosa, toluidine blue, smokers correspondence: dewi agustina, c/o: fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara yogyakarta 55281. e-mail: dewiagustina2004@yahoo.com introduction tobacco usage has become a major health problem worldwide since it causes many harmful effects, such as causing serious systemic effects and damaging tissues of the mouth.1,2 in many places, smoking is practiced mostly in the form of cigarette, however, other forms of tobacco, including smokeless tobacco, cigars, and pipes, are also used. every single bar of cigarette contains more than 4000 chemical substances, including more than 60 carcinogens. some of them are nicotine, tar, carbon monoxide and hydrogen cyanide.3,4 there is good evidence that tobacco in all forms is carcinogenic in the upper aerodigestive tract, including the mouth.5,6 the disruption of normal cellular and molecular mechanisms would appear to be the target of tobacco ingredients.7 the strong association between cancers of the oral cavity and pharynx with tobacco use is well established. epidemiological studies show that the risk of developing oral cancer is five to nine times greater for smokers than for non smokers.8 the duration of smoking habits and the number of cigarettes consumed daily were significantly associated with the presence of premalignant lesions.9 in spite of its lower incidence as compared to other cancers, oral cancer has a low survival rate, largely because the disease is often not diagnosed until it is advanced. public awareness of oral cancer is also low and this contributes to delay in diagnosis.10,11 however, the diagnosis of oral cancer is challenging due to its multitude of ill-defined, variable appearing, controversial and poorly understood lesions that appear in the mouth.12 91leosari, et al.: screening of oral premalignant lesions toluidine blue (tolonium chloride), an acidophilic, metachromatic dye belonging to the thiazine group, is a vital tissue dye which exhibits differential uptake into tissue, resulting in metabolically active areas of lesions being stained a deep blue.13 when reacting with the tissues, toluidine blue selectively stains acid tissue components (sulfate, carboxylate and phosphate radicals) such as dna and rna. there is no evidence of chemical reaction with any component of the cell within the reactions.14 oral premalignant lesions that stained with toluidine blue consistently contained loss of chromosomal genetic information, termed loss of heterozygosity.15 previous studies showed that the sensitivity and specificity of toluidine blue were quite reliable in revealing early oral premalignant lesions and monitoring recurrences of oral carcinoma.16,17 toluidine blue-positive staining correlated with clinicopathologic risk factors and high-risk molecular patterns. oral premalignant lesions which stained positively with toluidine blue showed a consistently higher frequency of loss of heterozygosity for chromosome arms.15 a question needs to be answered is whether toluidine blue can be used to screen premalignant lesions which would likely occurred in smokers. the objectives of this study were to screen the premalignant lesions using toluidine blue staining on oral mucosa of smokers and non smokers and to find out whether quantity and duration of smoking would effect the screening results. this study is expected to provide sufficient scientific information about toluidine blue staining as a screening tool to detect the presence of oral premalignant lesions in smokers and non smokers, with attention to quantity and duration of smoking. materials and methods this study involved 40 men whose ages range from 20 to 60 years. subjects were divided into 4 groups: group i consisted of 10 heavy smokers (consume more than 15 cigarettes daily), group ii consisted of 10 moderate smokers (consume 10-15 cigarettes daily), group iii consisted of 10 light smokers (consume less than 10 cigarettes daily) and group iv consisted of 10 non smokers. all smokers have smoked at least 5 years prior to the study. the sampling technique is purposive sampling. subjects were asked for their consents as the study samples and to answer the standardized smoking questionnaires. then, the anamnesis was accomplished by reviewing the medical and dental histories, oral and systemic health, lifestyles and habits. the staining procedure was preceded by a complete and thorough clinical intraoral examination. subjects accepted brief elaborations about the process before rinsing their mouths with mineral water for 20 seconds to clean up the debris and followed by acetic acid 1% as pre-rinse solution for another 20 seconds. the oral mucosa surface was then dried with gauze. toluidine blue stain was applied in excess and left on site for 1 minute. in order to eliminate excess stain, the oral mucosa was again rinsed with acetic acid 1% and sufficient water consecutively for 20 seconds each.19 the areas of oral mucosa that stained blue were captured with intraoral camera and transferred to the computer unit. inflammation, irritation and ulcers would take up the stain. because the test carried a risk of false positives, a second test was required to minimize the risk. the recommended protocol, thus included another 14 days later to give inflammatory lesions an opportunity to heal.21 results if one or more regions in the mouth were persistently stained blue during the first and the second staining, the results were considered positive, otherwise, the staining that did not meet the criteria were considered negative. the pictures of positive and negative staining were displayed in figure 1 and 2, respectively. figure 1. photographs of the toluidine blue positive staining. the same region was persistently stained blue during the first (a) and second (b) staining. 92 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 90−93 the numbers of subjects with positive or negative outcomes were calculated with the number of cigarette smoked daily and the duration of smoking habit. table 1 showed that positive staining dominates the smoker group. the fisher’s exact statistic (alternative test of chi-square test) analyzed the data and gave a result that retention of toluidine blue were significantly higher in smokers than that in non smokers. it was demonstrated by fisher's exact test with the significance of 0.003. table 1. result of toluidine blue staining categories smokers non smokers total positive staining 27 4 31 negative staining 3 6 9 total 30 10 40 the total lifetime cigarette consumption was calculated by the following formula: total cigarette use = bars/day × 365 days/year × years of smoking bb aa the regression and correlation analysis were completed to check the influence of total lifetime cigarette consumption. the regression test showed that the quantity and duration of smoking affected the final result with a significance of 0.026. the spearman correlation test also proved significant relation between total cigarette use and staining results (sig. = 0.001) with correlation coefficient = 0.510, therefore, interpreted as moderate relation. the clinical appearance of the toluidine blue staining was not restricted to positive and negative only. in most clinical situations, the probability of staining results lay somewhere between these two extremes. the pattern of dye retention was assessed by the intensity as strong, equivocal, weak, and no staining. strong staining was rarely found in this study. weak and equivocal staining were common, and thus might not be ignored. in this study, they were categorized as positive due to an interesting finding that there were no significant differences in the molecular profiles of lesions with strong staining and those with weak staining.18 further evaluation of every toluidine blue stain is important, even when the staining is faint. figure 2. photographs of the toluidine blue negative staining. no areas was stained during the first (a) and the second (b) staining. no staining in the first (c) and weak staining in the second (d). equivocal in the first (e) and no staining in the second (f). 93leosari, et al.: screening of oral premalignant lesions discussion nearly all smokers included in this study were positively stained, meanwhile, the rate of negative staining was higher among the non smokers group. statistically, the fisher’s exact test also proven that oral mucosa of non smokers absorbed less stain than that of smokers. the results confirmed that the content of acidic components in oral mucosa of smokers are probably higher than that of non smokers.22 referring to the high sensitivity rates of toluidine blue,16,21 the results of this study indicated that smokers are more susceptible to develop oral premalignancies. this is supported by the fact that cigarettes contain carcinogenic agents such as tar, carbon monoxide and hydrogen cyanide.3 intraoral regions that stained positively, when confirmed with biopsy, expressed various histologic changing from mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and invasive cancer.21 toluidine blue is useful in raising or confirming clinical suspicion. even after having the test, subjects might still not having the disease. they only have a probability of the disease. toluidine blue is not more than a screening tool used for early diagnosis of presymptomatic diseases, thus should never be used as a single determinant. toluidine blue positive lesion may not be ascertained as premalignancy unless confirmed by biopsy. histopathologic assessment is the most reliable and valid measure of disease, and therefore it is used as the gold standard. another supportive finding was that the risk of positive staining increased with the increasing number of lifetime cigarette use. the relative risk of developing oral premalignancy was associated with a positive doseresponse relationship.9 although quantity and duration of smoking markedly affect the retention of toluidine blue dye, a lot of factors still need to be considered. many variables were not under control in this study, such as the oral health, systemic health and lifestyle. these features are likely to contribute more to the staining results. the occurrence of oral cancer is apparently an interaction of many factors, such as genetics, alcohol consumption, smokeless tobacco use, hpv, hiv seropositive, narcotics abuse, sunlight exposure and dietary intake.13 in conclusion, the results of this study demonstrate that toluidine blue retention is more noticeable on oral mucosa of smokers than that of non smokers. the quantity and duration of smoking also affect the retention of toluidine blue on oral mucosa. therefore, it is suggested that toluidine blue staining can be used as a screening tool to detect the presence of oral premalignant lesions in smokers. it is recommended that the next study may include individuals with higher lifetime cigarette consumption as subjects to produce stronger intensity of toluidine blue staining. acknowledgement the authors would like to express their special gratitude to community fund of faculty of dentistry, gadjah mada university that has funded this research. references 1. siddiqui ia, farooq mu, siddiqui ra, rafi smt. role of toluidine blue in early detection of oral cancer. pak j med sci 2006; 22(2): 184–7. 2. wood nk, goaz pw. differential diagnosis of oral and maxillofacial lesions. missouri: mosby, inc; 1997. p. 587–9. 3. sukendro s. filosofi rokok: sehat tanpa berhenti merokok. yogyakarta: pinus book publisher; 2007. p. 83–4. 4. weinberg ma. oral cancer risk factors and the pharmacist’s role in intervention. us pharm 2006; 8: 79–84. 5. zakrzewska jm. fortnightly review: oral cancer. bmj 1999; 318(7190): 1051–4. 6. regezi ja, sciubba j. oral pathology: clinical-pathologic correlations. 2nd ed. philadelphia: wb saunders company; 2003. p. 52–3. 7. hecht ss. tobacco carcinogens, their biomarkers and tobacco-induced cancer. nature reviews cancer 2004; 3(10): 733–44. 8. neville bw, day ta. oral cancer and precancerous lesions. cancer j clin 2002; 52: 195–215. 9. bokor-brati m, vukovi n. cigarette smoking as a risk factor associated with oral leukoplakia. archive of oncology 2002; 10(2): 67–70. 10. agustina d. kewaspadaan yang perlu diberikan di balik kasus kanker mulut. maj. ked. gigi (dent j) 2006; 13(2): 203–7. 11. kujan o, glenny am, oliver rj, thakker n, sloan p. screening programmes for the early detection and prevention of oral cancer. cochrane database of systematic reviews, issue 3, page number cd004150, 2006. 12. carnelio s, rodrigues g. oral cancer at a glance. the internet journal of dental science 2004; 1(2). 13. lynch dm. oral cancer risk and detection: the importance of screening technology. adts peer-reviewed publication. 2007. p. 3–7. 14. rajmohan m. assessment of oral mucosa in normal, precancer and cancer using chemiluminescent illumination, toluidine blue supravital staining and oral exfoliative cytology. dissertation. 2005. p. 5–27. 15. epstein jb, zhang l, poh c, nakamura h, berean k, rosin m. increased allelic loss in toluidine blue-positive oral premalignant lesions. oral surg oral med oral pathol oral radiol endod 2003; 95: 45–50. 16. onofre ma, sposto mr, navarro cm, paolo s. reliability of toluidine blue application in the detection of oral epithelial dysplasia and in situ and invasive squamous cell carcinomas. oral surg oral med oral pathol oral radiol endod 2001; 91(5): 535–40. 17. zhang l, williams m, poh cf, laronde d, epstein jb, durham s, et al. toluidine blue staining identifies high-risk primary oral premaligant lesions with poor outcome. cancer research 2005; 65(17): 8017–21. 18. gandolfo s, scully c, carrozzo m. oral medicine. philadelphia: churchill livingstone elsevier; 2006. p. 187–9. 19. craig g, newell j. opportunistic oral cancer screening. bda occasional paper issue 6, 2000. p. 26–7. 20. canto mi. staining in gastrointestinal endoscopy: the basics. endoscopy 1999; 31(6): 479–86. 21. epstein jb, oakley c, millner a, emerton s, van der meij e, le n. the utility of toluidine blue application as a diagnostic aid in patients previously treated for upper oropharyngeal carcinoma. oral surg oral med oral pathol oral radiol endod 1997; 83: 537–47. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isi vol 39 no 3 juli-september 2006.pmd 116 facial, upper facial, and orbital index in batak, klaten, and flores students of jember university masniari novita biomedical department – dental forensic faculty of dentistry jember university jember indonesia abstract forensic anthropology is a neglected branch of physical anthropology in indonesia. the role of anthropology in forensics including medical and dental forensic is identification. anthropology could be used in identify skeleton including sex, age, height and race. the aim of this study was to know the facial index, the upper facial index and the orbital index among three different students race population of jember university used posteroanterior radiography. the subjects of this study were batak, klaten and flores students of jember university. the craniometric indices in this study according to the el-najjar classification. the result showed that all the subjects facial index classified as hypereuryprosopic with the mean between 78.05–79.184. batak population upper facial index classified as hyperueryene, while flores and klaten population were euryene. all the population orbital index classified as hypsiconch. key words: anthropology forensic, craniometric index, population, pa radiography correspondence: masniari novita, c/o: bagian biomedik, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. in the forensic context, the approach to race must be a pragmatic one. the identification of unknown individuals is one of the most important justification for maintaining biologically based racial typologies. the racial classification is also essential to facial reconstruction, where recognizability is a major aim. the skull is the best part of the skeleton to use for determination of racial affinity, both morphologically and osteometrically.3 in forensic anthropology, only three ‘major’ group of racial affiliation are normally encounter (at least outside the pacific rim and australia): caucasian (including europeans, asians from the indian subcontinent, mediterraneans and americans with similar ancestry); mongoloid (asiatics and native americans); and negroid (africans and african americans).4 indonesia belongs to mongoloid, and glinka cit. herniyati5 divided indonesia into four races and nine cluster: deuteromelayid in the west and north side, dayakid in the celebes; protomalayid in south east, and melanesid in irian jaya (papua). physical characterize of deuteromalayid is brachycephal head with wide face and projection of zygomatic arch as well as dayakid, while protomelayid and melanesid are meso-to dolicocephal head.5 distinction between the racial subgroups is best made from feature of the skull and, secondarily, from the postcranial skeleton. traits useful in the assessment of racial affiliation include the overall morphology of the skull–its length, breadth and height–the shape of the face, the width of the zygomatic arches, the shape of the orbits, the interorbital breadth, and the size, shape and degree of guttering of the nasal aperture.4 introduction forensic anthropology is a neglected branch of physical anthropology in indonesia. the role of anthropology in forensics including medical and dental forensic is identification. in the police opinion, identification is an individual identification but in anthropology forensic the process to identify an individual is called identification, such as racial identification, sex and age. anthropology could be used in identify skeleton including sex, age, height and race, or in individual identification by physiognomic reconstruction, lip print and dermatoglyphy. the role of anthropology was very strict in paternity disputed, because using the anthropometric, anthroposcopy and dermatoglyphy beside serology the reliability was very high.1 sometimes in disasters, whether natural, technological or man-made, there were lots of dead bodies which already mutilated or incineration. the ultimate aim of all disaster victim identification operation must invariably be to establish the identity of every victim by comparing and matching the accurate ante-mortem (am) and post-mortem (pm) data.2 in those cases the role of an anthropologist is a must. the three most vital determinations that must be made when dealing with skeletal remains are age, sex, and racial affinity. it would be nearly impossible to attempt to identify, much less reconstruct, the face of an individual without this information. there is no question that all of these factors have a significant bearing on appearance and also serve to narrow the range of possible matches.3 117novita: facial, upper facial, and orbital index it cannot be emphasized strongly that correct determination of age, sex, and race are prerequisites to all aspects of craniofacial reconstruction, superimposition, and identification. a significant error in age assignment could eliminate the actual individual in question from consideration. diagnosing race or sex incorrectly makes identification absolutely impossible. therefore, an experienced forensic anthropologist or skeletal biologist should always be consulted when skeletal remains are found.3 anthropometric studies are an integral part of craniofacial surgery and syndromology. for these reasons, standards based on ethnic or racial data are desirable because these standard reflect the potentially different patterns of craniofacial growth resulting from racial, ethnic, and sexual differences.6 face width is the maximum distance between directly opposite on the malar or cheek bone. face length is measured from the nasion to the lowest point in the center line on the lower jaw (gnathion). this relation, the facial index, is calculated by dividing the length of the face by its width and multiplying the quotient by 100.6 the upper facial index is calculated by dividing the distance from nasion to prosthion (aleolare) by its width and multiplying the quotient by 100. and the orbital index is calculated by dividing the maximum orbital breadth by its maximum orbital length. these craniometric indices are the measurement of the skull, included in osteometry.7 many disaster cases leaved several dead bodies unidentified because of the mutilation of the bodies or the incineration. the jw marriot bombing 2003 leaved only a head of the dead body, the situbondo tragedy 2003 killed 55 incineration students, and the kuta bombing 2005 leaved three head without body. in all these cases identification of the body was very difficult because there was no am data. the role of an anthropologists in those cases was determining sex, age and race from the skull. that’s why we need some method and measurement of the skull. fortunately, there is only little information of the measurement of indonesian. the aim of this study is to know the facial, upper facial, and orbital index among three different students population of jember university used pa radiography and we hope that this measurement could be used in identification the skull in incineration and mutilation cases, or in superimpose technique and facial reconstruction. materials and methods the subjects of this cross sectional study were students from jember university that belongs to three different population according from their hometown: batak, klaten and flores. using purposive sampling technique with sample criteria: male, age between 18-27, there is no mixmarriage to other population up to two generation, no malocclusion, no dental restoration and prosthetic, and good key of occlusion; we found 10 samples for klaten, 10 samples for flores, and 11 samples for batak population. all the samples used pa radiography and we calculated the facial, upper facial, and orbital index according to the craniometric indices from el-najjar7 as follows: facial index = nasion gnathion height 100× bizygomatic breadth hypereuryprosopic x –79,9 euryprosopic 80,0–84,9 mesoprosopic 85,0–89,9 leptoprosopic 90,0–94,9 hyperleptoprosopic 95,0–x upper facial index = nasion prosthion height 100× bizygomatic breadth hypereuryene x–44,9 euryene 45,0–49,9 mesene 50,0–54,9 leptene 55,0–59,9 hyperleptene 60,0–x orbital index = max. orbital breadth 100× max. orbital length chamaeconch x–75,9 mesoconch 76,0–84,9 hypsiconch 85,0–x results we studied 31 students of jember university that belongs to three different population according to their hometown. both flores and klaten population classified as hyperueryprosopoic (50%), euryprosopic (40%) and mesoprosopic (10%); while batak population classified as hypereuryprosopic (64%), mesoprosopic (27%) and euryprosopic (9%) as shown in table 1. the mean of facial index of these population between 78.76–79.184 and classified as hyperueryprosopic (table 4). table 1. facial index classification on flores, batak and klaten population of jember university facial index classification population n a b c d e flores batak klaten 10 11 10 5 (50%) 7 (64%) 5 (50%) 4 (40%) 1 (9%) 4 (40%) 1 (10%) 3 (27%) 1 (10%) 0 0 0 0 0 0 a. hypereuryprosopic, b. euryprosopic, c. mesoprosopic, d. leptoprosopic, e. hyperleproprosopic 118 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:116–119 upper facial index of flores population was so variety as shown in table 2. 50% of this population classified as euryene, 20% classified as hypereuryene and mesene, while 10% classified as lepene. 64% of batak population classified as hypereuryene, and 18% classified as euryene and mesene. 50% of klaten population classified as euryene, 30% as mesene and 20% as hypereuryene. the mean of upper facial index of these population between 43.46–47.8 (table 4) and classified as hypereuryene and euryene. the frontal bone is a saucer-shaped bone which forms the forehead and the upper part of the orbital cavities. the frontal bone articulates with the two parietal bones at the coronal suture, laterally with the great wing of the sphenoid and the frontal process of the zygomatic bone, and below with the ethmoid, lacrimal, maxillary and nasal bones posteroanteriorly.7 the zygomatic or malar bone supports the cheek. it articulates medially with the maxilla, above with the frontal bone, laterally with the temporal bone, and behind with the greater wing of the sphenoid. the posterior projection forms the anterior portion of the zygomatic arch, and the upward projection completes the outer wall of the orbit.7 anatomically modern the human show considerable geographical variation in the form of the facial skeleton. during growth the facial skeleton changes dramatically n shape as well as in size. it comprises several interdependent bones that grow and develop under the influence of various local and systemic factors. although different bones and/or different parts of the same bone may grow independently to some degree under the influence of localized factors, the facial skeleton remains a functional whole throughout the course of development. it well known that adult modern human populations show significant differences in both facial size and shape.8 klaten (central java) belongs to deuteromalayid while batak and flores are protomalayid, and in the dendogram of indonesian population by glinka cit. herniyati5 they all at the d group.5 it supported this present study that the facial index of the three population were similar classified as hypereuryprosopic (table 1) with the mean of the facial index between 78.05–79.184 (table 4). in contrast, cakirer et al.9 showed that leptoprosopic facial types were more often associated with dolicocephalic head forms, and euryprosopic facial forms were more often associated with brachycephalic head forms. the different classification of the upper facial index, batak was hypereuryene (mean 43.46) but klaten and flores were euryene (table 2) with the mean of the upper facial index respectively 47.8; 47.43 caused by batak and tapanuli were in the same group (d), while east flores at the c group and bama (east flores) at the c group. some of east java are in the d group but some of them in the f group. the different result could be made by the different aspect of view. we analyzed the indices through the pa radiography and compared it by the classification of physical anthropology because pa radiography is the most useful tools in identification the skull of death body. in the superimpose technique where we compared the victim photograph and skull found in the scene, the used of the pa radiography is a must. radiography studies play an important part in the forensic autopsy and are particularly relevant in the cases where identity of the deceased is unknown. comparison of the post mortem radiographs with clinical radiographs taken in life can result in identity being established with a very high degree of certainty.10 ante mortem x-ray table 2. upper facial index classification on flores, batak and klaten population of jember university all of these population orbital index classified as hypsiconch (100%) as shown in table 3 with the mean of orbital index between 99.26 – 106.63 (table 4). table 4. mean of facial, upper facial, and orbital index on flores, batak and klaten population of jember university table 3. orbital index classification on flores, batak and klaten population of jember university population n mean of facial index mean of upper facial index mean of orbital index flores batak klaten 10 11 10 79.184 ± 4.96 78,05 ± 7.04 78.76 ± 4.4 47.43 ± 4.75 43.46 ± 5.43 47.8 ± 3.03 106.63 ± 7.63 99.26 ± 6.99 102.73 ± 5.77 a. chamaconch, b. mesoconch, c. hypsiconch orbital facial index classification population n a b c flores batak klaten 10 11 10 0 0 0 0 0 0 10 (100%) 10 (100%) 10 (100%) a. chamaconch, b. mesoconch, c. hypsiconch upper facial index classification population n a b c d e flores batak klaten 10 11 10 2 (20%) 7 (64%) 2 (20%) 5 (50%) 2 (18%) 5 (50%) 2 (20%) 2 (18%) 3 (30%) 1 (10%) 0 0 0 0 0 a. hypereuryene, b. euryene, c. mesene, d. lepene, e. hyperlepene discussion the skull is the bony structure of the head which includes the cranium, skeleton of the face, and the mandible or lower jaw. skull bones vary in thickness, size, and shape in relation to each other and between different individuals. they may be flat (parietal and temporal), irregular (sphenoid), or curved (frontal).7 119novita: facial, upper facial, and orbital index photographs of the head and oro-dental region are excellent evidence for identification purposes. the size, shape and dimensions of many structures are very stable in the skeleton of the head. post mortem radiographs of skulls can, however, be taken under conditions identical to those which were employed during life, allowing superimposition comparisons to be made.11 the orbital index of all population classified as hypsiconch (table 3). it was hard to measure the orbital breadth and length due to the quality of the pa radiogram. various factors have been proposed to influence the adult form of the facial skeleton. although the basic structure is determined in accordance with genetically regulated blueprint while in utero this is modified preand postnatally through functional matrices responding to environmental and epigenetic influence such as climate, activity patterns and masticatory function.8 metric traits are continuous morphological variables dealing with the size and dimension of the skull and postcranial skeleton. the inheritance of these traits depends on the combined influence of many gene.7 there are certain universal traits of the skull that influence appearance, regardless of geographic or racial differences.3 many traits in radiographs, which suggest that a familial study may be possible using films now stored in hospitals and other repositories.7 selection, gene flow, and genetic drift may have acted singly or in combination at the same time or at different time levels to produce the observed similarities or differences in the skeletal material. similar diets and environment conditions under which the groups lived may have also been responsible for the observed similarities or differences in their skull.7 the similar classification of facial and orbital index and the different classification of the upper facial index in those three population maybe caused by the economic and transportation factors that lead to the same condition on diet. in conclusion, this study showed that there was no different of facial and orbital index on batak, klaten and flores population of jember university, but there was different classification of upper facial index among these populations. we haven’t found any relation of facial and upper facial index yet, maybe because the variety of the result and the little number of samples used in this study. we suggest more samples in the future study and more study of craniometric indices on indonesian population because indonesia has lots of island with different kind of population in order to make a special indonesian facial measurement. references 1. jacob t, indriati e. buku bacaan antropologi biologis. yogyakarta: dirjen dikti departemen pendidikan nasional; 1999/2000. h. 3–159. 2. anonim. disaster victim identification guide. new york: interpol; 2002. p. 1–21. 3. vladimir n, iscan my, loth sr. morphologic and osteometric assessment of age, sex and race from the skull. in: iscan my, helmer rp, editor. forensic analysis of the skull. new york: wiley-liss inc; 1993. p. 71–88. 4. briggs ca. anthropological assessment. in: clement jg, ranson dl, editor. craniofacial identification in forensic medicine. great britain: arnold; 1998. p. 49–61. 5. herniyati. bentuk embilang insisivus pertama permanent dan tonjol carabeli molar pertama permanen rahang atas pada populasi jawa, cina di jember dan populasi tengger di proboinggo. tesis. surabaya: pascasarjana universitas airlangga; 1999. h. 5–17. 6. evereklioglu c, doganay s, er h, gunduz a, tercan m, balat a, cumurcu t. craniofacial anthropometry in a turkish population. the clef palate-craniofacial journal 2002; 39(2):208–18. avaiable from: url: http:///cpcj.allenpress.com/cpcjonline/? request = get-toc & issn = 1545–67 & volume = 39 & issue = 2. accessed march 6, 2006. 7. el-najjar my, mcwilliams kr. forensic anthropology. illinois usa: charles c thomas publ; 1978. p. 15–143. 8. vidarsdotir us, o’higgins p. stinger c. a geometric morphometric study of regional differences in the ontogeny of the modern human facial skeleton. journal of anatomy 2002; 201(3):211. available from: url: http://www.blackwell-synergy.com/doi/full/10.1046/ j.1469. accessed july 16, 2006. 9. cakirer b, hans mg, graham g, aylor j, tishler pv, redline s. the relationship between craniofacial morphology and obstructive sleep apnea in whites and in african-americans. am.j.respir.crit.care.med 2001; 163(4):947-950. available from: url: http://ajrccm.atsjournals.org/cgi/content/full/163/4/947. accessed july 16, 2006. 10. raymond ma, ashley wj. management of the scene and forensic evidence. in: clement jg, ranson dl, editor. craniofacial identification in forensic medicine. great britain: arnold; 1998. p. 9–24. 11. clement jg. dental identification. in: clement jg, ranson dl, editor. craniofacial identification in forensic medicine. great britain: arnold; 1998. p. 63–81. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 49 no 3 juli-sept 2016.indd 148 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 148–152 inhibitory effect of jengkol leaf (pithecellobium jiringa) extract to inhibit candida albicans biofilm muhammad luthfi, ira arundina, and nizamiar hanmi department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: candida albicans (c. albicans) are dimorphic fungi in oral cavity, considered not only as normal flora, but also as pathogens. c. albicans have an ability to grow biofilm, which has a thick layer of outer skin structure, called as extracellular matrix. jengkol leaves (pithecellobium jiringa) contain alkaloids, flavonoids, terpenoids, and lectins, which have an ability as antifungal agent purpose: this study aimed to analyze the optimum dose of jengkol leaf extract as antibiofilm against c. albicans biofilms. method: c. albicans were cultured on yeast peptone dextrosa (ypd) media in 96-well microtiter plate flat bottom plates. there were one control group (without treatment) and three treatment groups. the first treatment group was given jengkol leaf extract at a dose of 100 mg/ ml. the second treatment group was given jengkol leaf extract at a dose of 200 mg/ ml. and, the third treatment group was given jengkol leaf extract at a dose of 400 mg/ ml. semi quantitative method was applied to determine c. albicans biofilmsis using crystal violet staining technique. the absorbance of the cells then was calculated using a spectrophotometer with a wavelength of 570 nm. result: the mean value of optical density in the control group was 1.23. the mean value of optical density in the treatment group with a dose of 100 mg/ ml was 0.2. meanwhile, the mean value of optical density in the treatment group with a dose of 200 mg/ ml was 0.2, and 0.21 in the treatment group with a dose of 400 mg/ ml. the results also showed that there were significant differences between the control group and all of the treatment groups (p<0.05), but there was no significant difference between the treatment groups (p>0.05). conclusion: the optimum dose of jengkol leaf extract used as antibiofilm against c. albicans biofilms is 100 mg / ml with an inhibitory percentage of 83.7%. keywords: jengkol leaf extract; antibiofilm; candida albicans correspondence: muhammad luthfi, department of oral biology, faculty of dental medicine, universitas airlangga. jl mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: m.luthfi7@yahoo.com introduction candida albicans (c. albicans) are dimorphic fungi in oral cavity, considered not only as normal flora, but also as pathogens. if the growth of c. albicans in the oral cavity is excessive, it can trigger certain diseases.1 those diseases are usually such fungal infections that are known as oral candidiasis.2 according to a research conducted by parman, the prevalence of fungal infections caused by c. albicans is high, ie 55 cases out of 100 patients. similarly, based on data from the directorate general of disease control and environmental health of the ministry of health of the republic of indonesia, there were 21,591 cases in 2010, 21,031 cases in 2011, and 21,511 cases in 2012 related to human immunodefisiency virus (hiv) in indonesia, 10,689 of which are also classified as oral candidiasis caused by c. albicans.3,4 candida is a type of fungus that has a high prevalence in the formation of biofilms, particularly c. albicans. c. albicans biofilms have the outermost layer structure, called as extracellular matrix. extracellular matrix is formed by polysaccharide that serves to maintain stability of c. albicans while growing and developing.5 oral candidiasis, can be classified based on clinical symptoms. classic type commonly occurs is pseudomembranous candidiasis. pseudomembranous candidiasis patients dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p148-152 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p148-152 149149luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 148–152 have certain clinical symptoms, such as disturbing pain in the area of oral infections. bleeding then can occurs when pseudomembranous open. this disease even occurs potentially in patients who have been infected with hiv.2 actually, there have been many choices for antifungal drugs on markets today, one of which widely used is azole class with certain side effects when consumed in long term. one of the side effects is in the form of hepatotoxicity. a research using experimental animals given fluconazole at a dose of 12.26 mg (the therapeutic dose in humans) per animal per day for 20 weeks, liver tumors even can emerge at week 10 after the administration.6 jengkol (pithecellobium jiringa) is a typical plant of southeast asia. jengkol leaves contain flavonoids, tannins, alkaloids, steroids, glycosides, lectins, and steroids/ terpenoid.7,8 alkaloids are able to impair mitochondrial function and consistency of the cell wall. lectin has an ability to attach to mannose and hyphae. lectins and flavonoids, furthermore, have an ability to disrupt cell interaction process.9,10 terpenoid has an active role in inhibiting cell cycle process of c. albicans.11 similarly, a research conducted by bakar12 shows that the methanol extract of jengkol leaves (pithecellobium jiringa) has antifungal power against c. albicans cells with minimum inhibitory concentration (mic) of 100 mg/ ml. unfortunately, there have not many researches on the effects of antibiofilm derived from jengkol leaf extract, especially against c. albicans biofilms. jengkol leaves have some natural chemical compounds considered to be able to work synergistically as antibiofilm. thus, the results of this research can be considered as a scientific study for the development of traditional medicine expected to be applied as a candidate of antifungal drug selection. materials and method this research was an in vitro laboratory experimental research. c. albicans were cultured at the laboratory of microbiology in faculty of dentistry, universitas airlangga. meanwhile, jengkol leaves were extracted at the laboratory of pharmacognosy in faculty of pharmacy, universitas airlangga. and, antibiofim test on c. albicans were conducted at the laboratory of microbiology in faculty of medicine, university of brawijaya from june to july 2015. jengkol leaf extract at various concentrations were carried out in several stages. jengkol leaves were taken from a cultivating garden in ngantang, malang. jengkol leaves then were identified in the technical implementation unit of plant conservation center in purwodadi (unit pelaksana teknis balai konservasi tumbuhan kebun raya purwodadi lembaga ilmu pengetahuan indonesia) as a part of indonesian institute of sciences in pasuruan. sampling of jengkol leaves was conducted with certain criteria, such as fresh, green, and planted at least for 5 years. jengkol leaves were separated from the stems and stalks. those leaves then were dried up through wind, and crushed with a blender. 50 grams of jengkol leaf powder was put in an erlenmeyer flask sized 250 ml, added with 150 ml of methanol, and then macerated for 24 hours using a shaker. the results of this maceration were filtered with filter paper to obtain a filtrate. jengkol leaf powder residue was added to the methanol and then re-macerated for 24 hours. steps 4 and 5 were repeated until there was no color (clear) in thin layer chromatography (tlc) check. the solution derived from the filtration process was evaporated with rotary vacuum evaporator. the extract resulted was in the form of condensed extract weighed 5 grams. it was diluted at doses of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml. c. albicans used this research were cultured on yeast peptone dextrosa (ypd) media in a 96-well microtiter plate flat bottom plate. ca. albicans biofilms then were made in several stages. 100 l suspension of c. albicans as the test concentration and sucrose 2% were added using a multichannel pipette to induce biofilm formation into 96well microtiter flat bottom plate. the entire microtiter plates were covered, wrapped with cling wrap, and then incubated for 24 hours at a temperature of 370 c. multichannel pipette was used to rinse the plates repeatedly 3 times with sterile pbs (200 ml per well). alternatively, automated microtitter plate washer can be used. during flushing and after the last rinsing, the plates were placed upside down and then closed with a paper towel to remove any residual pbs. in these circumstances, biofilms formed could be seen by inverted microscope. biofilms then were ready to proceed to the antibiofilm testing process. afterwards, the samples were divided into four groups, consisted of one control group (no treatment) and three treatment groups. those three treatment groups were given jengkol leaf extract at doses of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml. replication was performed in each treatment five times. jengkol leaf extract with doses of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml were applied to c. albicans biofilms that had been cultured in a 96-well microtiter flat bottom plate. by using multichannel pipette, jengkol leaf extract at a dose of 100 mg/ ml was added to column 1, a dose of 200 mg/ ml to column 2, and a dose of 400 mg/ ml to column 3. column 4 as a control was not given the extract. the plates then were covered and incubated for 48 hours at a temperature of 370 c. after that, the plates were rinsed three times with sterile pbs (200 ml per well). staining then was performed using crystal violet, and incubated for 45 minutes. the whole wells were rinsed four times with sterile distilled water, and immediately given 200 ml of 95% methanol. 100 μl of the methanol was taken using pipette to be replaced to a new well. afterwards, absorbance of each well was measured using a spectrophotometer with a wavelength 570 nm.13 data of the mean value of optical density then were derived from spectrophotometric readings. optical density (od) is a unit used to look turbidity of c. albicans biofilms dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p148-152 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p148-152 150 luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 148–152 on microtiter plates. turbidity illustrates the number of c. albicans biofilms attached to microtiter plates.14 the mean optical density value of each treatment group then was converted into an inhibitory percentage using the following formula: inhibitory percentage = (od control at 570 nm od treatment at 570 nm) × 100 od control at 570 nm results in this research, there were four groups, one control group (no treatment) and three treatment groups. replication of each group was performed 6 times. table 1 shows the values of optical density in each group, the control group and the treatment groups. in table 1, column with control information indicates the optical density value of the control group. column with a caption 1 shows the optical density value of the jengkol leaf extract at a dose of 100 mg/ ml. meanwhile, column with a caption 2 shows the optical density value of the jengkol leaf extract at a dose of 200 mg/ ml. column with a caption 3 shows the optical density value of the jengkol leaf extract at a dose of 400 mg/ ml. table 2 shows the mean optical density values of the research groups. in the control group, the mean od value was 1.23 with a standard deviation of 0.53. in the group with jengkol leaf extract at a dose of 100 mg/ ml, the mean od value was 0.2 with a standard deviation of 0.01. meanwhile, in the group with jengkol leaf extract at a dose of 200 mg/ ml od, the mean od value was 0.2 with a standard deviation of 0.02. in the group with jengkol leaf extract at a dose of 400 mg/ ml, the mean od value was 0.21 with a standard deviation of 0.03. normality test was performed using one-sample kolmogorov-smirnov test. p value obtained for each sample was more than 0.05. it means that the data had normal distribution. furthermore, after the homogeneity test (levene test) was conducted, p value obtained was 0.00 (p <0.05). it indicates that the data were homogeneous. afterwards, to know the differences between the groups, kruskal test was carried out. the significance value obtained was 0.003 (p<0.05). it means that there was a difference between the groups. mann whitney test then was performed to find significant differences between two groups. the results of kruskal wallis test also showed that there was significant differences between groups since the significance value obtained was less than 0.05 (0.003) as shown in table 3. based on the results of mann whitney test, there was a significant difference between the control group and the treatment group with a dose of 100 mg/ ml (0.004). there was also a significant difference between the control group and the treatment group with a dose of 200 mg/ ml (0.004). similarly, there was a significant difference between the control group and the treatment group with a dose of 400 mg/ ml (0.004). however, there was no significant difference between the treatment group with a dose of 100 mg/ ml and the treatment group with a dose of 200 mg/ ml (0.371). similarly, there was no significant difference between the treatment group with a dose of 100 mg/ ml and the treatment group with a dose of 400 mg/ ml (0.421). there was also no significant difference between the treatment group with table 1. od values of candida albicans biofilms after the administration of jengkol leaf extract (pithecellobium jiringa) at a dose of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml in the treatment groups as well as in the control group (without the administration) description doses of jengkol leaf extract (mg/ ml) replication i ii iii iv v vi control control 1.972 1.012 0.887 1.731 0.556 1.249 1 100 0.196 0.184 0.181 0.190 0.214 0.220 2 200 0.195 0.177 0.183 0.172 0.201 0.235 3 400 0.248 0.206 0.175 0.239 0.175 0.230 table 2. the mean and standard deviation of od values of candida albicans biofilms after the administration of jengkol leaf extract (pithecellobium jiringa) at a dose of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml in the treatment groups as well as in the control group (without the administration) data n x sd control 8 1.23 0.53 jengkol leaf extract (pithecellobium jiringa) (100 mg/ml) 8 0.2 0.01 jengkol leaf extract (pithecellobium jiringa) (200 mg/ml) 8 0.2 0.02 jengkol leaf extract (pithecellobium jiringa) (400 mg/ml) 8 0.21 0.03 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p148-152 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p148-152 151151luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 148–152 a dose of 200mg/ ml and the treatment group with a dose of 400 mg/ ml. in addition, the diagram below (figure 1) shows the inhibitory percentages of the whole groups against c. albicans biofilms. in the groups with doses of 100 mg/ ml and 200 mg/ ml, the inhibitory percentage for each was 83.7%. meanwhile, in the group with a dose of 400 mg/ ml, the inhibitory percentage was 81.3%. it means that there was a decrease of 2.4% in the inhibitory percentage of the treatment groups, from a dose of 400 mg/ ml to a dose of 200 mg/ ml. discussion in this study, jengkol leaf extract used was at doses of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml. the determination of these doses was based on a preliminary study stating that jengkol leaf extract containing antifungal materials that has a minimum inhibition concentration (mic) of 100 mg/ ml against c. albicans.12 in table 2, the mean value of the optical density (od) in the control group (untreated group) was 1.23, higher than the groups treated with doses of 100 mg/ ml and 200 mg/ ml generating 0.2 for each, and a dose of 400 mg/ ml deriving 0.21. it means that the number of c. albicans biofilms in the treatment groups was fewer than in the control group (untreated group). it also indicates that there was no decline in the mean value of od as the dose increased. there was an increase in the mean value of od in the treatment group with a dose of 400 mg/ ml compared to the treatment group with a dose of 200 mg/ ml. the increase in the mean value of od at a dose of 400 mg/ ml is likely to be caused by the resistance of c. albicans biofilms against jengkol leaves (pithecellobium jiringa). the resistance of c. albicans biofilms to antifungal drugs is due to complex and multifactorial causes. efflux pump is one of factors causing the resistance of c. albicans biofilms.15 efflux pump is a form of fungal cell’s defense against agent/ antifungal drugs. efflux pump occurs as a result of huge pressure from jengkol leaf extract at a dose of 400 mg/ ml, which affects osmolarity of the fungal cells. efflux pump mechanism occurs by pumping out or secreting the antifungal agents through the fungal cell membrane using adenosine triphosphatase of binding cassette transporter (atp-binding cassette transporter). as a result, the antifungal drugs that are already in the cells pumped out without making any contact with the drug targets.16 the inhibitory percentage of jengkol leaf extract against c. albicans biofilms (figure 1) at a dose of 100 mg/ ml was 83.7%. meanwhile, at a dose of 200 mg/ ml, the inhibitory percentage was 83.7%, and at a dose of 400 mg/ ml the inhibitory percentage was 81.3%. it indicates that jengkol leaf extract had inhibitory effects on c. albicans biofilm formation. similarly, an initial research claims that jengkol leaf extract is antifungal against c. albicans cells.12 based on phytochemical examination in the laboratory of the faculty of pharmacy, universitas airlangga, jengkol leaf extract positively contains alkaloids, flavonoids, terpenoids, and polyphenols. jengkol also contains lectin compounds.17,18 alkaloids are antifungal derived from organic plants. in a research conducted by dhamgaye, alkaloid undermines the integrity of the cell walls of c. albicans as well as interferes calcineurin pathway system. such damage then causes mitochondrial dysfunction, and c. albicans ultimately will die.19 lectin, on the other hand, has an ability to bind to hyphae and mannose. hyphae in c. albicans is a structure that serves as a major tool in the absorption of nutrients. therefore, impairing absorption of nutrients can cause disruption of spore germination of c. albicans. the binding of lectin mannose that is elements of the extracellular matrix of the biofilm, furthermore, 83.7 83.7 81.3 80 81 82 83 84 figure 1. diagram of the inhibitory percentages in each treatment group against candida albicans biofilms. table 3. differences between groups based on the results of mann whitney test control 100mg/ml 200mg/ml 400mg/ml control 0.004* 0.004* 0.004* 100 mg/ml 0.371 0.421 200 mg/ml 0.257 400 mg/ml in hi bi to ry ( % ) dose (mg/ml) 100 200 400 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p148-152 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p148-152 152 luthfi, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 148–152 will trigger antibiofilm activities.9 meanwhile, lectin and flavonoids have an ability to disrupt the interaction of cells (cell signaling pathway). the interaction of cells is a process of interaction between c. albicans and host cells that occurs in the attachment stage. the interaction stage, consequently, can be considered as an important stage in the colonization and penetration of c. albicans.9,10 terpenoid has an ability to damage cell cycle system of c. albicans by binding proteins on the cell membrane of c. albicans, resulting in disruption of cell cycle leading to the death of c. albicans.11 in other words, jengkol leaves (pitheecellobium jiringa) contain alkaloids, flavonoids, terpenoids, and lectin that work synergistically as antibiofilm of c. albicans, ultimately degrading c. albicans biofilms. the degradation of c. albicans biofilms is described by a decrease in the mean value of od at doses of 100 mg/ ml, 200 mg/ ml, and 400 mg/ ml (tabel 2). it may be concluded that the optimum dose of jengkol leaf extract (pithecellobium jiringa) in inhibiting c. albicans bioilms is 100 mg/ ml with an inhibitory percentage of 83.7%. references 1. staniszewska m, bondaryk m, swoboda-kopec e, siennicka k, sygitowicz g, kurzatkowski w. candida albicans morphologies revealed by scanning electron microscopy analysis. braz j microbiol 2013; 44(3): 813-21. 2. burket lw. burket’s oral medicine. hamilton: bc decker inc; 2008. p. 79–80. 3. aditama ty. p rofil pengendalian penya k it dan penyehatan lingkungan tahun 2013. jakarta: direktorat jendral pengendalian penyakit dan penyehatan lingkungan; 2013. p. 75. 4. parmar r, sharma v, thakkar c, chaudhary a, pateliya u, ninama g, mistry k, goswami y, kavathia gu, dan rajat r. prevalence of opportunistic fungal infections in hiv positive patients in tertiary care hospital in rajkot. natl j med res 2102; 2(4): 463-5. 5. chandra j, kuhn dm, mukherjee pk, hoyer ll, mccormick t, ghannoum ma. biofilm formation by the fungal pathogen candida albicans: development, architecture, and drug resistance. j bacteriol 2001; 183(18): 5386-94. 6. e ssaw y a e , hela l sf, e lz ohei r y a h , e lba rd a n e m. hepatotoxicity induced by antifungal drug fluconazole in the toads (bufo regularis). j drug metab toxicol 2010; 1: 106. 7. warintek. pithecellobium lobatum benth. available from : (http:// www.warintek.ristek.go.id). diakses pada 12 november 2014. 8. muslim ns, nassar zd, aisha af, shafaei a, idris n, majid am, ismail z. antiangiogenesis and antioxidant activity of ethanol extracts of pithecellobium jiringa. bmc complement altern med 2012; 12: 210. 9. paiva pmg, gomes fs, napoleão th, sá ra, correia mts, coelho lcbb. antimicrobial activity of secondary metabolites and lectins from plants. current research. brazil: formatex; 2010. p. 399400. 10. onsare jg, arora ds. antibiofilm potential of flavonoids extracted from moringa oleifera seed coat against staphylococcus aureus, pseudomonas aeruginosa and candida albicans j appl microbiol 2015; 118(2): 313-25. 11. zore gb, thakre ad, jadhav s, karuppayil sm. terpenoids inhibit candida albicans growth by affecting membrane integrity and arrest of cell cycle. phytomedicine 2011; 18(13): 1181-90. 12. bakar ra, ahmad i, sulaiman sf. effect of pithecellobium jiringa as antimicrobial agent. bangladesh j pharmacol 2012; 7(2): 131-4. 13. pierce cg, uppuluri p, tummala s, lopez-ribot jl. 96 well microtiter plate based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilm. j vis exp 2010; (44). pii: 2287. 14. mer rit jh, kadouri de, toolkomakomae ga. growing and analyzing static biofilms. new york: john wileey & sos inc; 2011. p. 2. 15. mukherjee pk, chandra j, duncan mk, ghannoum ma. mechanism of fluconazole resistance in candida albicans biofilm: phase–specific role of efflux pumps and membrane sterlos. infection and immunity 2003; 71(8): 4335-6. 16. gordon r, bachman s, patterson tf, brian l, wickes jl. lopezribot. investigation of multidrug eff lux pumps in relation to fluconazole resistance in candida albicans biofilms. j of antimicrob chemotherapy 2002; 49: 976-7. 17. charungchitrak s, petsom a, sangvanich p, karnchanatat a. antifungal and antibacterial activities of lectin from the seeds of archidendron jiringa nielsen. j food chem 2011; 126: 1028–32. 18. panadda v, karnchanatat a, sangvanich p. an alpha-glucosida inhibitory activity of thermostable lectin protein from archindendron jiringa nielsen seeds. journal of biotechnology 2012; 11(42): 10030. 19. dhamgaye s, devaux f, vandeputte p, khandelwal nk, sanglard d. molecular mechanisms of action of herbal antifungal alkaloid berberine in candida albicans. plos one 2014; 9(8): 6-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p148-152 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p148-152 151 the fractographic analysis of three dentin bonding agents on tooth surface adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract the dentin bonding agent is hydrophilic resin that can strongly bind to dentin surface, both in chemical and physical-mechanical ways. the dentin surface is good for the resin bonding when the surface is in moist condition. three types of dentin bonding agents: voco, prime & bond nt and excite were used in this research and their application methods are called as total-etched technique. the objective of this research is to examine the difference of tensile bond strength of the three bonding agents on the moist dentin surface. bovine incisivus teeth were cut and sharpened using diamond bur then smoothened with sandpaper. dentin surfaces were etched with 37% phosphoric acid, washed with 20 cc aquadest, and dried with blot-dry technique. the preparation teeth were inserted into desiccator with minimum humidity 60% and maximum 90% for one hour. after removed from the desiccator, the voco agent was applied on the teeth in first group, and then followed by the prime & bond nt and excite agents, respectively. the resulting sample was stored within the room temperature. after 24 hours, the tensile bond strength was tested using autograph instrument. the results indicated that the tensile bond strength of voco and prime & bond nt agents were higher than excite both at humidity 60% and 90% (p  0.05). in conclusion, the dentin bonding agents with acetone solvents have a higher tensile bond strength compared with those with alcohol solvents. key words: dentin bonding agents, dentin collagen, blot-dry technique, fractographic analysis correspondence: adioro soetojo, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the dentin bonding agents are widely used in the operative dentistry, particularly as materials for treating the class v cavity. this is because gingival tissues with the increasing ages will physiologically experience a retraction so that their dentins and cementum will be fairly open. one proper option to restore this condition is using a composite resin restoration.1,2,3 as the combined bonding between the dentin and composite resin, the dentin bonding agents, notably the hydrophilic resins that can strongly bind to the moist dentin tissues will be used here.4,5 contrary to the enamel, the wet environment in the dentin is mainly caused by a presence of the fluids in dentin tubule. the deeper cavities in the dentin produce an elevated number of dentin tubules making this region wetter. in some areas next to the pulp, the number of tubules decrease from ± 45,000/mm2 to ± 20,000/mm2 in several regions close to dentin-enamel junction.2,6 previous researches suggested that chemical compounds which could be used as the dentin bonding agents were including hema (2-hydroxyethyl methacrylate), bpdm (bisphenyl dimethacrylate), 4-meta (methacryloyloxyethyl trimellitate anhydride) and other agents. however, hema is the most common used agent because it has advantageous chemical-physical properties, stable enough since preservative agents are added, such as hydroquinone, and its construction is inexpensive.7–11 the hema-based dentin bonding agents on the dentin surface may take the form of either chemical or mechanical bonding. in the first place, the chemical bonding occurs when hema carbonyl groups interact with the dentin collagen amino groups which in turn generate amide or peptide bonds.12,13 conversely, the mechanical bonding can be explained below. in three-dimensional sense, the dentin collagen represents a network or braid of the fibril collagen. there are nano cavities between the fibrils into which the hema agents will penetrate and undergo polymerization. the hema solidifying within the interfibrillar cavities constitute a mechanical anchoring/retention of the dentin bonding agents.14,15 in general, the success of the hema-based dentin bonding on the collagen is generally dependent on several factors, such as low monomer viscosity, type and concentration of monomer, the conditioning acid application, temperature and humidity around the collagen.6,8,16according to craig et al.4 and swift et al.,17 some dentin bonding agents contain multi-functional monomers (primer and adhesives) where hydrophilic groups are useful for reaching adequate wetting and penetrative properties. the hydrophobic properties will polymerize with and bind to composite resin on them. the solvent agents usually used are acetone, alcohol and 152 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 151–155 water. furthermore, collagen tissues existing in the dentin are type i collagen.18 collagen usually used as material in the research is a sequence of amino acids, proline, proline, glycine {hn-(pro-pro-gly)5-cooh}.19,20 according to breschi et al.,16 the fibril collagen length is about ± 0.5–1,0 mm, mayor fibril diameter 60-80 nm, minor branch diameter 10–25 nm and interfibrillar cavity 15-20 nm. in this research, the moist dentin surface was made consistent with the previous researches.21-24 the researchers argued that the moist surface was correlated with a water content or humidity surrounding the dentin. hence, our analysis was carried out based on the minimum humidity 60% and maximum humidity 90% at temperature 25 °c. the humidity is defined as a condition related to water content existing around the material.22,23 moreover, the fractographic analysis put into execution in the research represented “a tensile strength test” imposed on composite resin/dentin bonding agents until they were dissociated from the dental surface and some researchers called this technique as microtensile bond test.25,26 the purpose of this research is to analyze fractographically the three types of the hema-based dentin bonding agents using moist-milieu approach. materials and methods the materials used in the research are bovine incisivus teeth (obtained from the slaughterhouse in pegirian surabaya); three types of the hema-based dentin bonding agents: voco (germany), prime & bond nt (dentsplycaulk, germany) and excite (ivoclar-vivadent, schaan/ liechtenstein); acid etching solution (ivoclar-vivadent), self-curing acrylic: vertex (dentimex, holland). the ingredients of the three dentin bonding agents are shown in table 1. table 1. ingredients of the dentin bonding agents instruments used in the research are diamond disk, diamond bur, sandpaper no. 400 and 1000 (fuji star, japan), desiccator with vacuum-tap, united thermometer and hygrometer (haar-synth, hygro, germany), compressor/air suction (schuco, usa), autograph ag-10 te (shimadzu, japan). the methods in this research is similar to the previous researches.21 the preparation teeth were cleaned carefully and gently by removing debris present on the teeth surface using brush, while for soft or hard tissues using sharp scalpel. during the cleaning process, the teeth should always be in wet condition. then the teeth were cut using diamond disk and embedded firmly on the dental stone cylinder block. the dentin should face forward. for preparation up to the dentin part, diamond bur was required. the dentin surface was smoothen using silicone sandpaper no. 400 and proceeded with no.1000. the dentin was covered with adhesive tape (isolation) 3 mm in diameter and attached just in the middle of the surface. in the next stage, the dentin specimens were primed or smeared with 37% phosphoric acid etching agent using cotton pellets for 15 seconds, then washed gently with 20 cc aquadest from injection spuit and dried by wiping gently using cotton pellets. in subsequent stage, the preparation teeth were put into desiccator for one hour under minimum humidity 60% and maximum humidity 90%. when removed immediately from the desiccator, the teeth were primed with primary solution and bonding agents that have been thoroughly mixed (voco) as described shortly below. firstly, the solution was dropped slightly on disposable brush and primed/smeared on the dentin surface and stand for 30 seconds and irradiated with light curing unit for 20 seconds (the method was carried out in accordance with the manufacturer’s direction). the cylinder block was inserted into plunger. the opposite plunger was filled with self-cured acrylic as the solidifying material on the dentin bonding. for solidification with dentin bonding agents of prime & bond nt and excite, their application was the same as voco. all resulting samples were kept in the room at ± 28 °c for 24 hours. after 24 hours, the tensile bond strength test was carried out using autograph instrument (at airlangga joint laboratory). when in use, the instrument was operated with following provisions: cross-head speed = 10 mm/minute, operational (range) level: 5, load cell capacity = 5 kn/500 kgf. the results as seen on the display have kgf unit. the test dentin surface area is about 7.1 mm2. subsequently, the data collected were then analyzed using one-way anova test at  = 0.05, and proceeded with turkey hsd test. results the tensile bond strength of three dentin bonding agents, means and standard deviation are showed in table 2. from table 2 it appeared that the tensile strength of voco at humidity 60% was higher than at 90%. in addition, the t test results indicated that p value = 0.001 (p < 0.05), indicating that there was a significant difference between 153soetojo: the fractographic analysis the voco’s tensile strengths at humidity 60% and 90%. to know whether the voco’s tensile strength was normal or not, the tensile strength was tested using kolmogorovsmirnov technique. the test results showed that at humidity 60%, p value = 0.916 (p > 0.05) and at humidity 90%, p value = 0.560 (p < 0.05). this indicates that the two trial groups are normal. table 2. the tensile bond strength of three dentin bonding agents on the dentin surface at minimum and maximum humidity (mpa) along similar lines, the tensile bond strength of prime and bond nt at humidity 60% was higher than at humidity 90%. the t test was carried out to know whether there was a difference between the two trial groups. the test results demonstrated that there was a significant difference between the two trial groups in associated with the tensile bond strength (p < 0.05). likewise, the results of the kolmogorov-smirnov test suggested that prime & bond nt trial group possessed normal data distribution (at humidity 60%, p value = 0.988 > 0.05, while for humidity 90%, p value = 0.539 > 0.05). similarly, the tensile bond strength of excite at humidity 60% was statistically significant higher compared with that at humidity 90% (p = 0.001 < 0.05). the data distribution in the two trial groups both at humidity 60% and 90% were normal (p value for humidity 60% = 0.746 and p value for humidity 90% = 0.540). to prove that the research had homogenous data, the statistical analysis was carried out using levene test. the test results showed that at humidity 60% the dentin bonding agents with voco, prime & bond nt and excite brands possessed p value = 0.686 (p > 0.05), meaning that the three agents were homogenous. similarly, at humidity 90% those agents were also homogenous with p value = 0.921 (p > 0.05). to know whether there was a difference between overall trial groups the statistical analysis using anova test was carried out. at humidity 60% the significance level of the three dentin bonding agents was 0.001 (p < 0.05), suggesting that significant differences existed among all trial groups at humidity 60%. conversely, at humidity 90% the significance level was 0.004 (p < 0.05), indicating that at humidity 90% all trial groups possessed significant differences. moreover, to see a difference in each trial group the tukey hsd test was carried out and the results could be seen in table 3. table 3. the difference in tensile bond strength of three bonding agents at humidity 60% when tensile strengths of voco and prime & bond nt were compared, there was no statistically significant difference at humidity 60% (p > 0.05). on the other side, there was statistically significant difference between voco and excite groups in line with their tensile strengths (p < 0.05). in comparison between prime & bond nt and excite groups, there was statistically significant difference (p < 0.05). table 4. the difference in tensile bond strength of three bonding agents at humidity 90% the difference in tensile strengths at humidity 90% can be seen in table 4. the tensile bond strength between the trial groups was the same at humidity 60% and 90%. there were statistically significant differences (p < 0.05) between voco and prime & bond nt and excite groups when they were compared. on the other hand, there was no significant difference (p > 0.05) between voco and prime & bond nt. discussion it has been commonly argued that optimal tensile bond strength of the hema-based bonding agents on the dentin surface was reached in moist condition.2,3 summitt et al.2 argue that the dentin bonding agents (hema) can bind sufficiently to the fibril collagen when the dentin surface 154 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 151–155 is in moist condition instead of the dry or wet surface. this may be true since when the dentin surface is dry, the fibril collagen will collapse. consequently, the dentin bonding agents have difficulty in binding to those collapsed collagen. on the contrary, when the surface is too wet, more water molecules exist around the fibrils thereby inhibiting the bonding agents penetrative power to bind to the fibril collagen. therefore, the moist environment in the dentin surface plays a considerable role in the bonding or the environment of the dentin surface must have an optimal and proper humidity.6 the results of this research have proved that humidity 60% is an optimal humidity since the tensile strengths in voco, prime & bond nt and excite agents are significantly higher than the trial groups at humidity 90%. this is because fibril collagen tissues are greatly permeable and active at humidity 60% thereby making it easier for them to bind to the dentin bonding agents both chemically and mechanically.2,6 in chemical bonds, a bonds between resin agents and collagen will generate strong amide bonds, namely interatomic primer bond/covalent bond.12,14 in addition, there were hydrogen bonds, interfunctional group bonds between both compounds, including hydroxyl group, carboxylate, amino and carbonyl, and a complex reactions happened between dentin calcium ions and resin bonding agents. on the other hand, the mechanical bonds may occur because the bonding agents penetrate into dentin tubules, on irregularly dentin surfaces (microscopically forming slit, pore, crack and undercut). the mechanical bonds may also occur due to a penetration of the bonding agents into nano interfribrillar cavities of the collagen network. these exposed fibrils are not merely collagen tissues but also enclosed by several proteins, such as noncollagen protein and proteoglycan. both proteins have good wetting properties so that when adhesive monomers will make a contact with fibrils, they must compete with water molecules on the protein surfaces. the results showed that tensile strengths in the voco and prime & bond nt agents were significantly higher than excite agents both at humidity 60% and 90% (p  0.05). this could be explained by the fact that the voco and prime & bond nt were equipped with acetone solvent while excite with alcohol solvent. as we have known that acetone possesses higher vaporousness and water chasing effect compared with alcohol. thus, when primed or smeared on the dentin the acetone will quickly vaporize and prevent water molecules from dentin surface and allow many resin bonding molecules to bind to the fibril collagen. it is also noted that acetone is able to dilute resin bonding solution which in turn reduces resin viscosity. the low resin viscosity produces a good wetting on the dentin surface, leading to an increased surface energy and ultimately enhances resin tensile strength on the dentin.27 however, the researcher said that too high acetone concentration could cause hybrid layers to undergo a crack and the strain strength of the resin would decline. the maximum acetone concentration is about 37% of the weight. summitt et al.2 and anusavice3 proposed that an adequate tensile bond strength between the dentin bonding agents and the dentin surface was much dependent on adequate wetting properties of the bonding agents that brought about a small contact angles between the two agents. thus, in order to wet the dentin surfaces evenly and completely, the resin viscosity should be low. in addition, the bonding agent’s capability to wet the surface (wettability) is mainly influenced by several factors. for example, a cleanliness of the agent surface to which the dentin surface is attached and oxyde layers in the surface may inhibit the bonding, including organic fluids. however, the acid etching on the dentin could increase the wetting and surface roughness, and may cause the opening of the dentin tubule.28 the proper wetting procedure will result in a good joining between the resin agents and fibril collagen. but until today there is a scant knowledge about the bonding monomer affinity against the dry or wet fibril collagen. therefore, it was widely stated that the resin agents could bind to the dentin surface even though its affinity was fairly low.6 anusavice3 and craig et al.4 explained that when a contact angle between the adhesive fluids and the solid surface at an interface was small, the adhesive molecules could adhere strongly to the agent molecules. this means that the wettability of the adhesive material is good enough. on the contrary, if the contact angle is large the wetting capability will decrease accordingly. additionally, both interface surfaces must be able to attract one another to allow the adhesion occurrence. this condition can occur without considering the second phase of the substance whether it is solid, liquid or gas. the energy on surface of the substance is usually larger than inside. this is because of the geometrical lattice pattern for its molecules. that is, the molecule lattices in all atoms attract one another in balanced fashion inside the substance. the increased energy for each area unit on the surface is intimately correlated with the surface energy or surface tension.2,3,5 regarding the tensile bond strength on the dentin surface, some factors we should consider are those which may cause a failure in bonding, including, firstly, whether a fluoride has ever been applied in the teeth. this can reduce the wetting properties of the resin agents. second, the presence of smear layer on the tooth surface is likely to decrease the bond strength. third, the tooth composition may be not homogenous; and finally, the tooth surface may be contaminated by saliva or blood. again, these factors are likely to produce a bonding failure. similarly, the acidity degree or ph of the resin agent solution generally also affected the bond strength on the dentin surface.13 it was commonly argued that when a dissociation of carboxylic acid or amino groups were inhibited, the hydrogen bond between resin and collagen would improve. in fact, this condition can boost the bond strength. this usually occurs at ph 2. however, when ph increases from 6.6 to 9.0, the tensile strength will decline sharply. this is due to the deformation in hydrogen bond 155soetojo: the fractographic analysis and functional groups from carboxylic acid undergoing dissociation. the voco solution used in the research has ph 2, while prime & bond nt 1.5 and excite 1.5. thus, there is no significant difference in ph of the three bonding agents. the water content within the dentin is very vital for the presence of the physical and chemical properties of collagen.6,29 according to them, the presence of water molecules around the collagen generates hydrogen bonds on the fibril collagen or between the fibril collagen. furthermore, these hydrogen bonds will produce an optimal physical appearance in fibril collagen, making it easier for the fibril to bind to the resin bonding agents. on the other side, if the water molecules are too small, the hydrogen bonds will dissociate, leading to the fibril collapse, and close contact between the fibrils may happen. consequently, the bond between peptides is weaken; collagen matrix will wrinkle and being hard so that the collagen is not permeable any longer for the hemabased resin bonding agents. with respect to the secondary structure of the collapsed collagen, the amino groups are masked or hidden. then, the hema carbonyl groups have difficulty in binding to the collagen amino groups. in conclusion, the tensile bond strength of the dentin bonding agents voco and prime & bond nt were significantly higher than excite. this could be explained by the fact that voco and prime & bond nt were equipped with acetone solvent while excite with alcohol solvent. references 1. peutzfeldt a, vigild m. a survey of the use of dentin bonding systems in denmark. dent mat 2001; 17:211–16. 2. summitt jb, robbins jw, hilton tj, schwartz r. fundamentals of operative dentistry. 3rd ed. chicago: quintess publ.co, inc; 2006. p. 183–242. 3. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia: wb saunders co; 2003. p. 21–395. 4. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 5. noort rv. introduction to dental materials. 2nd ed. edinburgh, london, new york, oxford: cv mosby co; 2002. p. 11–78. 6. nakabayashi np, pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 7. tay fr, pashley dh. aggresiveness of contemporary self-etching systems. dent mat 2001; 17:296–308. 8. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32:19–26. 9. frankenberger r, tay fr. self etch vs etch-and-rinse adhesive: effect of thermo-mechanical fatique loading on marginal quality of bonded resin compositerestorations. dent mat 2005; 21:397–412. 10. zohairy aa, de gee aj, mohsen m. effect of conditioning time of self-etching primers on dentin bond strength of three adhesive resin cements. dent mat 2005; 21:83–93. 11. brackett mg, brackett ww, haish ld. microleakage of class v resin composites placed using self-etching resins. quintess int 2006; 37:109–13. 12. renzo md, ellis th. chemical reactions between dentin and bonding agents. j adhesion 1994; 47:115–21. 13. nishiyama n, suzuki k, nagatsuka a, nemoto k. dissociation states of collagen functional groups and their effects on priming efficacy of hema bonded to collagen. j dent res 2003; 82:257–61. 14. jacques p, hebling j. effect of dentin conditioners on the micro tensile bond strength of a conventional and a self-etching primer adhesive systems. dent mat 2005; 21:103–9. 15. carrilho mr, tay fr, pashley dh. mechanical stability of resindentin bond components. dent mat 2005; 21:232–41. 16. breschi l, gobbi p, marzotti g, falconi m. high resolution sem evaluation of dentin etched with maleic and citric acid. dent mat 2002; 18:26–35. 17. swift ej, wilder ad, may kn, waddell sl. shear bond strength of one-bottle dentin adhesives using multiple applications. operative dent 1997; 22:194–9. 18. cohen s, burns rc. pathways of the pulp. 8th ed. st louis, london, philadelphia: mosby inc; 2002. p. 411–54. 19. nishiyama n, asakura t, suzuki k. adhesion mechanism of resin to etched dentin primed with n-mgly studied by 13c nmr. j biomed mater res 1998; 40:458–63. 20. nishiyama n, asakura t, suzuki k, komatsu k. bond strength of resin to acid-etched dentin studied by 13nmr. j dent res 2000; 79:806–11. 21. soetojo a. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada berbagai kelembaban. disertation. surabaya: airlangga university; 2006. p. 66–9. 22. finger wj, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4:277–82. 23. besnault c, attal jp. influence of a simulated oral environmental on dentin bond strength of two adhesive systems. am j dent 2001; 14:367–72. 24. chiba y, miyasaki m, rikuta a. moore bk. influence of environmental conditions on dentin bond strengths of one application adhesive systems. oper dent 2004; 29:554– 59. 25. hashimoto m, ohno h, kaga m, sano h. fractured surface characterization: wet versus dry bonding. dent mat 2002; 18:95–102. 26. fuentes v, ceballos r, osorio r, toledano m. tensile strength and microhardness of treated human dentin. j dent mater 2004; 20:522–9. 27. cho bh, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the microtensile bond strength. dent mat 2004; 20:107–15. 28. rosales ji, marshall gw, watanabe lg. acid etching and hydration influence on dentin roughness and wettability. j dent res 1999; 78:1554–9. 151 vol. 43. no. 3 september 2010 management of idiopathic alveolar bone necrosis associated with oroantral fistula after upper left first molar extraction ni putu mira sumarta department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia absract background: complications such as alveolar osteonecrosis and oroantral fistula can occure in maxillary molar extraction. the management of such complication is done by treating to treat any persisting maxillary sinusitis if present, prevent further antral contamination, wound bed preparation, and oroantral fistula closure with appropriate method. purpose: this case report presents a treatment stage of an idiopathic upper alveolar bone necrosis and oroantral fistula that occurred 4 months after left upper first molar extraction. case: a case of an idiopathic upper alveolar bone necrosis associated with oroantral fistula that occurred 4 months after left upper first molar extraction is presented. patient suffered from pain and swelling at left upper jaw since 2 month before admission. there was a history of complicated tooth extraction 4 months earlier. patient also complained pus and blood discharge from post extraction socket. patient occasionally choked when drinking and fluids escaped through the nostril. there was a diffuse swelling in the left maxillary region; there was no hyperemia, with soft consistency and no pain on palpation. in the 26, 27 region there was a defect in the post extraction area, fragile bone exposed, granulation tissue, there was pain and pus occasionally escaped on palpation. patient had already taken antibiotics. case management: the treatment performed was treatment of persisting maxillary sinusitis with saline irrigation through fistula, surgical acrylic splint to reduce further contamination to the wound bed and antral cavity, and also inflammation reduction. necrotomy on the necrotic bone and residual tooth roots extraction were done to prepare the wound bed. after there was no sign of infection in the wound bed, two stages of surgical closure were performed. closure with buccal fat pad flap and buccal advancement flap was done after there was small wound dehiscence. conclusion: management of bone necrosis and oroantral fistula is to treat persisting maxillary sinusitis, preparation of wound bed using, necrotomy, infection source removal, and closure of the fistula with appropriate method. the best and the easiet way to manage such complication is to prevent it from happening through a thorough pre operative assessment. key words: alveolar bone necrosis, oroantral fistula, oroantral fistula closure abstrak latar belakang: beberapa komplikasi seperti osteonekrosis alveolar dan oroantral fistula dapat timbul pada ekstraksi gigi molar atas. penatalaksanaan komplikasi tersebut adalah merawat sinusitis maksilaris bila ada, mencegah kontaminasi pada antrum, mempersiapkan bed luka, dan penutupan fistula oronatral dengan metode yang tepat.tujuan: untuk melaporkan tahapan perawatan pada kasus nekrosis tulang alveolar rahang atas idiopatik yang menyebabkan fistula oroantral yang terjadi 4 bulan setelah ekstraksi gigi molar pertama rahang atas kiri. kasus: nekrosis tulang alveolar rahang atas yang menyebabkan fistula oroantral, yang timbul 4 bulan setelah ekstraksi gigi molar rahang atas kiri. pasien mengeluh nyeri dan bengkak pada rahang atas kiri sejak 2 bulan sebelum datang ke klinik bedah mulut dan maksilofasial fakultas kedokteran gigi universitas airlangga. didapatkan riwayat pencabutandidapatkan riwayat pencabutan gigi atas kiri yang sulit dan lama. pasien juga mengeluh keluarnya nanah dan darah dari bekas ekstraksi gigi, pasien kadang-kadang merasa tersedak dan cairan keluar melalui lubang hidung pasien bila minum. terdapat pembengkakan dengan batas difus pada regio maksila kiri, tidak terdapat hiperemia, pada palpasi didapatkan konsistensi lunak. pada regio 26, 27 terdapat defek post ekstraksi gigi, tulang yang terekspos dan rapuh, terdapat jaringan granulasi, pada palpasi didapatkan rasa nyeri dan pus. pasien sudah minum antibiotika yang diresepkan dari klinik sebelumnya. tatalaksana kasus: penatalaksanaan pada kasus ini terdiri dari perawatan sinusitis maksilaris yang terjadi dengan irigasi dengan larutan salin melalui fistula, splint akrilik untuk mencegah kontaminasi kembali case report 152 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 151–156 pada bed luka dan rongga sinus, sehingga menurunkan inflamasi. nekrotomi tulang nekrotik dan ekstraksi sisa-sisa akar dilakukan untuk mempersiapkan bed luka. setelah bed luka tidak lagi menunjukkan tanda-tanda infeksi, dilakukan 2 tahap penutupan dengan pembedahan, yaitu penutupan dengan buccal fat pad flap dan buccal advancement flap yang dilakukan setelah didapatkan wound dehiscene kecil. kesimpulan: penatalaksanaan osteonekrosis dan fistula oroantral adalah perawatan pada sinusitis maksilaris yang ada, mempersiapkan bed luka, nekrotomi, dan menghilangkan sumber infeksi, serta nekrotomi pada tulang nekrotik dan menghilangkan sumber infeksi seperti sisa akar yang lain, serta penutupan fistula dengan metode yang sesuai. cara terbaik dan termudah untuk mengatasi komplikasi seperti ini adalah dengan pencegahan agar tidak terjadi melalui pemeriksaan preoperatif yang meyeluruh. kata kunci: nekrosis tulang alveol, fistula oroantral, penutupan fistula oroantral correspondence: ni putu mira sumarta, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: putumira_omfs@yahoo.co.idjl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: putumira_omfs@yahoo.co.id introduction necrosis of the jaw bones or osteonecrosis of the jaw refers to exposure of dead bone, regardless of the cause. the necrosis may result from ischemia, regardless of its cause that can be divided into. the use of systemic medications, radiation, infection, direct chemical toxicity, trauma, idiopathy, and other etiologies.1 clinically; it appear as an exposed yellow-white, hard bone in mandible or maxilla. the patient may or may not be symptomatic.2 oroantral fistula can be defined as a fistular canal covered with epithelia which may or may not be filled with granulation tissue or polyposis of the signal mucous membrane that communicate maxillary sinus and oral cavity.3,4 frequently occur because of iatrogenic oroantral communication after tooth extraction, infection, inflammation, cyst, neoplasma, and trauma.4–6 the most common cause of oroantral fistula is tooth extraction, mostly from extraction of upper lateral teeth or teeth posterior to the maxillary canines.3,7 oroantral fistula formed from communication between oral cavity and maxillary sinus, which do not healed by means of blood clot, and there is granulation tissue formation and migration of gingival epithelial cells that partially grow into the canal. during expiration there is air current passes the alveoli into the oral cavity facilitating a fistular canal formation. the presence of a fistula makes the sinus permanently open, which enable micro flora passage from oral cavity into the maxillary sinus causing sinusitis. symptoms of oroantral fistula are similar to the symptoms of oroantral communication. when drinking, patient feels a part of liquid entering the nose and runs into the nostril on the same side and when the patient is asked to blow through the nose while the nostrils are closed with fingers, air hisses from the fistula into the mouth.3 the study conducted by güven from 1983 to 1997 demonstrated that oroantral fistula usually occurs after third decade of life, the frequency of oroantral fistula was nearly the same in both sexes.6 the incidence of oro-maxillary perforation after maxillary tooth extraction was 3.8%.8 the purpose of this case report is to present a case and proper treatment stages of alveolar bone necrosis and oroantral fistula, and also closure of the oroantral fistula. thus general practioner can perform management before refering the patient to oral and maxillofacial surgeon. figure �. clinical appearance, a) diffuse swelling on the left maxilla; b) post extraction defect and oroantral fistula. 153sumarta: management of idiopathic alveolar bone figure �. a) panoramic view showing alveolar defect; b) water’s view showing radioapaque mass on basal area of the left antrum. figure ��. obturator placement. case sixty five years old woman was referred to oral and maxillofacial surgery clinic faculty of dentistry airlangga university with a diagnosis of a sinus perforation after tooth extraction, complaining of pain and swelling at left upper jaw since 2 months before admission. there was a history of complicated tooth extraction 4 months earlier. patient still suffer from pain after tooth extraction, followed by pus and blood discharge from post extraction socket, patient occasionally choked when drinking and fluids escaped through the nostril. patient had already taken antibiotics (oral clindamycin 3×300 mg) prescribed from the previous clinic. patient had no history of underlying systemic disease. patient presented with a good general condition and normal vital signs. extraorally there was a diffuse swelling with no hyperemia on the left maxillary region (figure 1-a), which was soft and nontender on palpation. from intra oral examination in the left molar region there was a post extraction defect, measuring 1.5 cm with a yellowish exposed bone covered with debris (figure 1b) and there was a mass measuring 1 cm which was soft and pain on palpation and hyperemia surrounding the exposed bone. there was no pus discharged on palpation. from clinical examination, the patient was diagnosed with alveolar bone necrosis and oroantral fistula after left upper first molar extraction. panoramic radiograph showed alveolar defect at upper left first molar region with radiopaque margin, there appeared retained roots appearance in mesial and distal side of the defect (figure 2-a). water’s view showed radiopaque thickening on basal area of the left antrum (figure 2-b). management performed in this patient was the treatment of persisting left maxillary sinusitis with saline irrigation through the fistula until clear fluid were obtained along a b 154 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 151–156 figure 5. first oroantral fistula closure with buccal fat pad flap. a) immediate view post closure; b) wound dehiscence 9 days post closure; c) wound dehiscence 2 months post closure. a b c figure �. clinical examination (a) day 1 post necrotomy; (b) tooth roots remnants showing anomaly; (c) resolution of swelling in the left maxillary region 2 weeks after; (d) intraoral view showing a good wound bed without signs of infection. with oral antibiotic administration (oral clindamycin 3×300 mg), acrylic surgical splint that entirely covered edentulous area and the fistula were worn by the patient to eliminate further contamination to the wound bed and antral cavity and to reduce inflammation (figure 3). necrotomy and residual tooth roots extraction were performed to prepare the wound bed, after the obturator was continuously worn by the patient. two weeks after necrotomy, wound bed was good and there was no sign of infection. then two stages of surgical closure were performed. closure with buccal fat pad flap was performed first (figure 4a), nine days after closure patient complaining of fluid escape from nostril when dinking and there was a small wound dehiscence on the flap 2 months later, second closure with buccal d a b c 155sumarta: management of idiopathic alveolar bone advancement flap were performed. after second closure patient did not complain of escaped fluid from nostril when drinking and healing occurred. discussion osteonecrosis of the jaw can be caused by various etiologies such as use of systemic medications primarily bisphosphonate, systemic steroids, antiangiogenic agents; radiation; infections like noma, necrotizing ulcerative periodontitis, herpes zoster, deep fungal infection; direct chemical toxicity; trauma such as tooth extraction, direct chemical toxicity from agents used in dental treatment that was a caustic chemicals such as arsenic paste and formocresol that are still in use as root canals obturator in some developing countries; idiopathic benign sequestration of the lingual plate of the mandible consists of spontaneous sequestration on the lingual mandibular bone usually in the mylohyoid ridge in patients with no significant underlying systemic condition. other etiologies such as avascular necrosis which includes aseptic necrosis, ischemic necrosis, osteochondritis dissecans which maybe caused by compromise of the blood supply leading to ischemia and necrosis; osteomyelitis associated with sclerotic disease and malignancies, and also other trauma such as difficult intubation causing exposure of the mylohyoid ridge, orthognathic surgery, temporomandibular joint puncture. non-traumatic causes such as hemoglobinopathies, fat embolism, alcoholism, and systemic lupus erythematosus.1 alveolar bone necrosis in this patient may be caused by buccal plate fracture occurred after complicated left first molar extraction that can be observed from intra oral loss of buccal plate (figure 1-b, c). from the extracted retained root remnant we can see that there was dilacerated root (figure 4-b), this correlate with literature review that odontogenic anomalies can make oral surgery complicated, such as maxillary sinus exposures, tuberosity fractures, and buccal plate fractures.9 dilacerated root also may be easily fractured during tooth extraction.10,11 clinically the lesion appear as exposed bone which is painful in 60 to 69 percent of cases.1 this patient complained of swelling and pain at post extraction site for 2 months before escaping fluid when drinking, showing that oroantral fistula develop after the presence of osteonecrosis. the maxillary sinus reaches its greatest dimension during third decade of life, so the incidence of oroantral communications should be higher after that age,4,12 study conducted by hernando et al 12 showed that the average age incidence of oroantral communications is 47.5. oroantral communication commonly caused by: close anatomic relationship between root apices of the premolar and molar tooth and maxillary sinus base (80%), maxillary cyst (10–15%), benign or malignant tumor (5–10%), trauma (2–5%), and other causes.4,6,12 if the oroantral communication is maintained open to the oral cavity for more than 48 hours or if there is infection, chronic inflammation of the sinus membrane and permanent epithelization of the fistula will increase the risk of sinusitis.5 management of oroantral fistula closure consists of nonsurgical and surgical management. if fistulas are small (less than 5 mm) and sinusitis is absent or eliminated, spontanues healing may occur. it is also important to remove epithelial lining of the fistula in order to facilitate healing. acrylic surgical splint were used to cover the entire edentulous area to prevent further contamination to the antrum. patient must wear the acrylic splint continuously, removing it only when cleaning after meal. patient also instructed to avoid or minimize any activity increasing intraoral or intranasal pressure, such as smoking, drinking through straws and blowing nose. various surgical techniques for oroantral fistula closure have been reported, including local flaps (palatal rotation advancement flaps, buccal advancement flaps, buccal fat pad flaps), distant flaps (tongue flaps), and grafts (bone, fascia lata, dura, hydroxiapatite blocks).2,4,7 it is important to establish wether or not infection of sinus had occurred during the existence of the fistula, duration and width of the fistular canal lumen contributes to the sinus infection.2 this patient presented with signs of sinus infection, and was treated with acrylic surgical splint and maxillary sinus irrigation with saline until clear fluid were obtained combined with oral clyndamycin administration 3×300 mg to treat maxillary sinus pathology. this was coherent with literature review which stated that the correction of maxillary sinus pathology is essential to get a successful therapy with drainage, adequate aeration and antibiotic administration.4,7 two weeks after, clear fluid were obtained, then necrotomy and retained tooth root extraction were done. acrylic surgical splint was maintained for 2 weeks until good wound bed were obtained. first closure with buccal fat pad flap 9 days after closure, patient complained about escaped fluid through nostril when drinking, second closure with buccal advancement flap. there is no superior method for oroantral fistula closure. when choosing surgical method for oroantral closure, location, size, height of the alveolar ridge, its relation to neighbouring teeth, existence and figure 6. intra oral view showing healing after second oroantral fistula closure with buccal advancement flap. 156 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 151–156 duration of sinus inflammation, and patient’s general health should be taken into consideration.2 alveolar bone necrosis in this case caused alveolar vertical height reducing, wider buccal-palatal dimension, and loss of buccal plate of the alveolar bone that complicate surgical closure, causing wound dehiscence after closure with buccal fat pad flap and there was still an oroantral fistula. the patient need a longer treatment time because she need second closure of the oroantral fistula that was done with buccal advancement flap. dehiscence after closure with buccal fat pad flap occurred also maybe because of a large and deep defect, a little fat pedicle obtained during closure, and the patient was a 65 years old woman. healing after second closure with buccal advancement flap occurred with shallowing of the buccal vestibulum which was a disavantage of this method.2 it is concluded that oroantral fistula most commonly caused by communication after tooth extraction on maxillary posterior teeth. in this case maybe caused by alveolar necrosis after complicated extraction of first molar with a dilacerated root. alveolar necrosis caused loss of alveolar vertical height, wider buccal-palatal dimension, and loss of buccal plate of the alveolar bone that caused difficulty in oroantral fistula closure. management of oroantral fistual consist of non-surgical and surgical management. references 1. a l m a z r o o a s a , w o o s o o k b i n . b i s p h o s p h o n a t e a n db i s p h o s p h o n a t e a n d nonbisphosphonate-associated osteonecrosis of the jaw. a review. jada 2009; 140(7): 864–75. 2. sokler k, vuksan v, lauc t. treatment of oroantral fistula. acta stomat croat 2002; 135–40. 3. kim hkw. introduction to osteonecrosis of the femoral head (ofh) and osteonecrosis of the jaw (onj). j musculoscelet meuronal interact 2007; 7(4): 350–3. 4. yilmaz t, suslu ae, gursel b. treatment of oroantral fistula: experience with 27 cases. am j otolaryngol 2003; 24: 221–3. 5. adeyemo wl, ogunlewe mo, ladeinde al, james o. closure of oro-antral fistula with pedicled buccal fat pad. a case report and review of literature. afr j oral health 2004; 1: 42–6. 6. güven o. a clinical study on oroantral fistulae. j cranio-maxillofac surg 1998; 26: 267–71. 7. logan rm, coates ea. non-surgical management of oro-antral fistula in a patient with hiv infection. aus dent j 2003; 48(4): 255–8. 8. hirata y, nagaoka s, miyamoto r, yoshimasu h, amagasa t. a clinical investigation of oro-maxillary sinus-perforation due to tooth extraction. kokubyo gakkai zasshi 2001; 68(3): 249–53. 9. gernhover kj. conscrescence of a maxillary second and third molar. cda journal 2009; 27(7): 479–81. 10. miloglu o, cakic f, cglayan f, yilmaz ab, demirkaya f. the prevalence of root dilacerations in a turkish population. med oral patol oral cir bucal 2010; 15(3): 441–4. 11. thongudomporn u, freer tj. prevalence of dental anomalies in orthodontic patients. aus dent j 1998; 43(6): 395–8. 12. hernando j, gallego l, junquera l, villareal p. oroantral communications. a retrospective analysis. med oral patol oral cir bucal 2010; 15(3): 499–503. 2121 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 21–25 original article the effect of brotowali (tinospora crispa l.) stem ethanolic extract on the inhibition of candida albicans biofilm formation suryani hutomo1, christiane marlene sooai2, maria silvia merry1, ceny gloria larope3, haryo dimasto kristiyanto1 1department of microbiology, faculty of medicine, duta wacana christian university, yogyakarta, indonesia 2department of parasitology, faculty of medicine, duta wacana christian university, yogyakarta, indonesia 3undergraduate student, faculty of medicine, duta wacana christian university, yogyakarta, indonesia abstract background: candida albicans (c. albicans) is an opportunistic pathogen that can be found in the oral cavity and other parts of the body. this species is the main cause of oral candidiasis and forms a biofilm as its virulence factor. due to increasing cases of antifungal resistance, research is needed on methods to control candida biofilm formation. brotowali (tinospora crispa l.) is known to be antifungal, antiseptic and antiparasitic. purpose: the purpose of this study is to analyse the ability of brotowali stem extract to inhibit candida biofilm formation. methods: the susceptibility of c. albicans to this extract was examined by a minimum inhibitory concentration (mic) test using the broth microdilution method. a bacterial adherence assay was performed by similar methods of the mic assay. a brotowali stem extract of various concentrations were incubated in a yeast peptone dextrose broth medium and stimulated with a c. albicans suspension. the 0.1% crystal violet was used to stain the adherent fungi and measured using a microplate reader at 595 nm. scanning electron microscopy (sem) was performed to provide a general overview of the biofilm formation. results: the mic value for the brotowali stem extract was at a concentration of 5,000 μg/ml. moreover, this extract inhibited fungal adherence starting at a concentration of 250 μg/ml. observation using sem confirmed these results. statistical analysis using one-way analysis of variance demonstrated a significant difference of c. albicans adherence following stimulation with brotowali extract (p < 0.005). conclusion: brotowali stem extract can inhibit c. albicans biofilm formation at an optimal concentration of 1,000 μg/ml. keywords: adherence; biofilm; c. albicans; tinospora crispa l. correspondence: suryani hutomo, department of microbiology, faculty of medicine, duta wacana christian university. jl. dr. wahidin sudirohusodo no. 5-25 yogyakarta, 55224, indonesia. email: suryanihutomo_drg@yahoo.com introduction candida albicans (c. albicans), the most common type of candida spp., is a normal flora of the human body. these fungi can to turn into opportunistic pathogens that are responsible for mucosal infections, including oral and vaginal.1,2 candida albicans is the main cause of 95% of oral candidiasis infections.3 oral candidiasis can be found on the buccal mucosa, oropharynx and tongue.4 this species can change into opportunistic pathogens in immunocompromised hosts, patients with catheters, dental prostheses, and those who are taking long-term antibiotics causing superficial candidiasis and systemic candidiasis.5 the main predisposing factors for developing oral candidiasis are salivary dysfunction, poor denture hygiene, long-term prostheses, topical corticosteroid therapy and smoking.3 candida albicans have distinctive characteristics that include morphological changes and biofilm formation. its ability to grow into three different morphologies (yeast, pseudohyphae and true hyphae) contributes to biofilm formation,6 which makes the treatment of c. albicans challenging. the national institute of health reports that biofilms are responsible for more than 80% of microbial infections.7 these microbial infections are due to resistance to antifungal agents and increased pathogenicity of c. albicans.8 the biofilm’s extracellular matrix plays an essential role in the development of c. albicans resistance, as it serves as a physical barrier against drug penetration.9 in addition, it protects fungi from the innate immune system dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p21–25 mailto:suryanihutomo_drg@yahoo.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p21-25 22 hutomo et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 21–25 by killing neutrophils and monocytes.10 previous studies have reported multiple cases of antifungal resistance, including candida resistance to echinocandin drugs, such as micafungin, anidulafungin and caspofungin, and the azole, including fluconazole, itraconazole and isavuconazole.11,12 therefore, the utilisation of antifungal agents should be minimised, and methods to control biofilm formation need to be developed. indonesia has around 30,000–50,000 species of plants, 7,500 of which have been used as medicinal plants. a well-known medicinal plant, brotowali (tinospora crispa l.), is a wild vine that grows in asian countries, including indonesia. extracts of its stem, root and leaves have been shown to have antifungal properties.13 more than 65 compounds were identified specifically from brotowali stems, such as flavones, alkaloids, berberine and diterpenes. the antifungal content of brotowali stems is derived from berberine alkaloids and flavonoids.14 in addition to its antifungal properties, it is analgesic, antipyretic, antiparasitic and antiseptic.15 this study aims to determine the ability of brotowali stem ethanolic extract to inhibit c. albicans biofilm formation. materials and methods candida albicans was obtained from a patient with candidiasis from bethesda hospital, in yogyakarta, indonesia. the fungi were isolated and identified using a chromagar candida medium (becton dickinson, germany). the colonies of c. albicans appeared to be light to medium green (figure 1). after identification, two colonies of c. albicans were grown in a yeast peptone dextrose (ypd) broth medium (sigma, missouri, usa) for 24 h at 37°c. the concentration of stock suspension was prepared as a 0.5 mcfarland. the brotowali stem simplicia was obtained and confirmed by a botanical expert at the herbal manufacturing company, cv merapi farma, yogyakarta, indonesia (ref. 122/mfh-simp/iv/2021). the plants used in this study were grown in the village of hargobinangun, pakem, sleman, yogyakarta. the plants were cultivated in appropriate conditions for three months and their brownblack–coloured stems were obtained. the simplicia was processed into an ethanolic extract via a maceration technique (a modification of our previous study16) in the biology laboratory of the faculty of pharmacy, universitas gadjah mada yogyakarta, indonesia. the 1,000 g of simplicia was mixed with 1,000 ml ethanol 96% and stored at 4°c for 24 hours. the macerate was separated by filtration three times and concentrated by a vacuum rotary evaporator at 70°c to obtain an ethanolic extract of brotowali stem. the extract, in paste form, was stored in a refrigerator at 4°c until used. the paste was dissolved in a dimethyl sulfoxide 1% solution (dmso, merck, germany) as a stock in a concentration of 10,000 μg/ml for the analysis of its fungal susceptibility and a concentration of 4,000 μg/ ml for the adherence assay. the stock of the extract was filtered using a 0.45 μm syringe filter (sartorius, germany) before being diluted with the ypd broth. the susceptibility of c. albicans to the brotowali stem extract was determined via a minimum inhibitory concentration (mic) test using the broth microdilution method. ten μl of 0.5 mcfarland fungal culture was inoculated into 100 μl of ypd broth containing the brotowali stem ethanolic extract at a concentration ranging from 312.5 to 10,000 μg/ml in a 96-well culture plate (iwaki, japan). the experiments were performed by using three well replicates. fluconazole (dexa medica indonesia) was used as the standard antifungal agent at a concentration of 2,000 μg/ml. the plates were incubated for 24 h at 37°c and observed for the visual absence of turbidity. the mic of the extract was defined as the lowest concentration that showed no turbidity.16 the method used for fungal adherence assay was similar to the susceptibility assay.17 a 10 μl brotowali stem ethanolic extract at a concentration ranging from 250 to 4,000 μg/ml were added to the 100 μl ypd medium and incubated for 30 min at 37°c in a 96-well culture plate. the culture was stimulated with 10 μl of the 0.5 mcfarland c. albicans suspension and incubated at 37°c for 24 hours. following incubation, the ypd medium was removed from each well via washing with a phosphate buffer saline solution (pbs, sigma-aldrich, germany) and fixed with 150 μl absolute methanol for 15 min. the adherent fungi on the wells were stained with 0.1% (wt/vol) crystal violet for 10 min at room temperature and rinsed with pbs twice. the stained, adherent fungi were extracted from the wells using 200 μl of 96% ethanol and transferred to a fresh 96well plate. the absorbance of the stained adherent bacteria was measured at 595 nm using a microplate reader (thermo scientific, usa). the data were analysed via one-way analysis of variance (anova) at a significance level of 0.05 with p < 0.05 considered significant. data analysis was performed via graphpad prism (la jolla, ca, usa). a general overview of the bacterial adherence to the hydroxyapatite (ha) disks were performed using figure 1. candida albicans culture in chromagar medium. a scanning electron microscope (sem). the ha discs dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p21–25 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p21-25 23hutomo et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 21–25 (10mm in diameter and 1.2mm in thickness) were created by placing 500 mg of hydroxyapatite powder into a mould and compressed at 120 mpa. finally, the discs were sintered for two hours at 1,300°c and placed in the autoclave for 15 minutes at 100°c to achieve sterilisation.18 the ha discs in the 500 μl ypd medium were incubated with 100 μl of the brotowali stem ethanolic extract at a concentration of 0 and 2,000 μg/ml for 30 min at 37°c in a 24-well culture plate (iwaki, japan). the culture was inoculated with 100 μl of the 0.5 mcfarland c. albicans suspension and incubated at 37°c for 24 hours. thereafter, the discs were rinsed twice with sterile pbs and placed in a primary fixative solution (glutaraldehyde 0.15 m 2.5% [vol/vol] in pbs) for 12 h at 4°c. the discs were rinsed with sterile pbs and treated with the secondary fixative (osmium tetroxide [oso4 1% w/v]) for 1 hr. the discs were subsequently rinsed with distilled water, dehydrated in an ethanol series (70% for 10 min, 95% for 10 min, and 100% for 20 min) and air dried overnight in a desiccator. the discs were coated twice with platinum vanadium using a sputter ion (bal-tec scd 005; bal-tec, balzers, liechtenstein) and bonded to carbon double-sided tape for examination via sem (hitachi su3500, japan).19 results the mic value for the brotowali stem extract was at a concentration of 5,000 μg/ml. it was indicated by no turbidity in the ypd medium just as the fluconazole group as a standard of antibacterial agent (figure 2). the data were representative of three times experiments. the fungal adherence assay demonstrated that inhibition occurred at concentrations of 500, 1,000, 2,000 and 4,000 μg/ml and were characterised by a decreased optical density (od) value compared with the control group. the od value decreased proportionally with increasing extract concentrations, but at a concentration of 4,000 μg/ml, the od values had values similar to those at a concentration of 2,000 μg/ml. there was no significant difference of the od value between concentrations of 4,000 μg/ml and 2,000 μg/ ml, as seen in figure 3. the experiment was repeated three times to confirm the data. statistical analysis via one-way anova showed that there was a significant difference between groups (p = 0.000). the aim of this study was to inhibit c. albicans biofilm formation, and the optimum concentration of brotowali stem deletedextract was found to be 1,000 μg/ml. a b ic d e f g h figure 2. the fungal susceptibility. no treatment (i); the concentration ranging from 10,000 μg/ml (b) to 156.25 μg/ml (h). the mic was at a concentration of 5,000 μg/ml (c, blue box); fluconazole used as a standard of antifungal agent (a). 4000200010005002500 ns 1.5 1.0 0.5 0.0 2.0 o pt ic al d en si ty v al ue dose figure 3. optical density values of c. albicans adherence at various concentrations. notes: ns: not significant; ***: p < 0.001. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p21–25 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p21-25 24 hutomo et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 21–25 observations via sem confirmed these results as representative of the experimental groups. yeast cells can be observed on both the untreated and extract-treated disks. the number of c. albicans colonies were higher in the untreated disk compared with the 1,000 μg/ml extracttreated disk. the extracellular matrix of c. albicans biofilm was also detected in a higher intensity on the untreated group (figure 4). discussion the ethanolic extract of brotowali stem (tinospora crispa l.) showed antifungal activity against c. albicans starting at a concentration of 500 μg/ml. the mic value for the brotowali stem extract was at a concentration of 5,000 μg/ml. the antifungal activity of the brotowali stem (tinospora crispa l.) occurs due to its flavonoids and berberine alkaloids. so far, there has been little research on brotowali stem extract’s antifungal properties. a previous study conducted by warsinah et al.20 using a disk diffusion method showed antifungal activity of this extract against c. albicans and also reported that the ethanol fraction had the best inhibition due to the ethanol’s ability to extract plant antifungal compounds, such as phenols, fatty acid groups and terpenoids compared with the chloroform, ethyl acetate, n-hexane and distilled water fractions.20 therefore, in this study, ethanol was chosen as the solvent. many factors contribute to the candida biofilm formation process. the initial step involves the attachment of the hyphae to a surface. the hyphal cells then proliferate to form microcolonies, forming the biofilm base layer. morphological changes occur, followed by the secretion of extracellular polymeric substances and the formation of an extracellular matrix.10,21,22 the attachment of c. albicans to the buccal epithelium and mucosa is mediated by various protein molecules, such as als and hwp1, which are dominantly expressed.23,24 the antifungal effect of brotowali stem (tinospora crispa l.) on c. albicans biofilms is influenced by berberine alkaloids and flavonoids. berberine is a natural isoquinoline alkaloid found in herbal plants and distributed in the roots, bark and stem of the plant.25 this substance has a yellow color and bitter taste. berberine and its derivatives have antifungal, antibacterial and anti-inflammatory potential.24 berberine affects biofilm formation by reducing the thickness of the biofilm and destroying its structure. berberine is able to suppress the dimorphic changes of c. albicans, thus inhibiting the development of biofilms.23 the mechanism of berberine as an antifungal agent occurs via the reduction of regulatory gene expression in biofilm formation and proliferation of hyphae. berberine causes changes in the mitochondrial membrane potential (δψm). the role of mitochondria in fungi is essential in signaling the metabolic pathway during virulence and infection in the host and defense against oxidative stress.23 changes in the mitochondrial membrane potential opens pores in the membrane, triggering the release of proapoptotic factors and resulting in fungal apoptosis.26 in addition, the combination of berberine and fluconazole accumulates mainly in the nucleus of c. albicans and cause dna damage.27 flavonoids are secondary metabolites and the largest class of polyphenols widely found in plants.26 the antifungal activities of flavonoid occurs via several mechanisms, such as plasma membrane disruption, induction of mitochondrial dysfunction, inhibition of cell formation, protein synthesis and efflux pumps.28 flavonoids such as quercetin can increase the production of farnesol, which suppresses biofilm formation and hyphae development in c. albicans.22 the combination of the flavonoid quercetin with fluconazole has the ability to prevent cell-to-cell communication, thereby disrupting gene expression in biofilm formation. in this study, it was hypothesised that alkaloid, berberine and flavonoid compounds in the brotowali stems inhibited fungal growth and the biofilm formation. in this study, an ha disk was used as a medium for c. albicans adherence. however, these results had not yet represented the c. albicans adherence to mucosal surface. future studies are recommended to analyse the ability of this extract to inhibit c. albicans adherence on mucosal figure 4. scanning electron microscopy of ha surface. the number of c. albicans colonies (1) were higher in the untreated disk (a) compared with the 1,000 μg/ml extract-treated disk (b). the extracellular matrix (2) of the c. albicans biofilm was detected in a higher intensity on the ha crystal (3) of the untreated disk (magnification 3,000×). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p21–25 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p21-25 25hutomo et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 21–25 epithelium. in conclusion, brotowali (tinospora crispa l.) stems were found to have antifungal activity against c. albicans. we suggest that this extract may serve as a potential natural product for oral care medicaments to control candida biofilm formation, particularly in immunocompromised hosts. acknowledgement the authors would like to thank the faculty of medicine, universitas kristen duta wacana, yogyakarta, indonesia for the grant that funded this research. references 1. moyes dl, naglik jr. mucosal immunity and candida albicans infection. clin dev immunol. 2011; 2011: 346307. 2. kadosh d. regulatory mechanisms controlling morphology and pathogenesis in candida albicans. curr opin microbiol. 2019; 52: 27–34. 3. vila t, sultan as, montelongo-jauregui d, jabra-rizk ma. oral candidiasis: a disease of opportunity. j fungi (basel, switzerland). 2020; 6(1): 15. 4. singh a, verma r, murari a, agrawal a. oral candidiasis: an overview. j oral maxillofac pathol. 2014; 18(suppl 1): s81-5. 5. kabir ma, hussain ma, ahmad z. candida albicans: a model organism for studying fungal pathogens. isrn microbiol. 2012; 2012: 538694. 6. kim j, sudbery p. candida albicans, a major human fungal pathogen. j microbiol. 2011; 49(2): 171–7. 7. nobile cj, johnson ad. candida albicans biofilms and human disease. annu rev microbiol. 2015; 69: 71–92. 8. lestari pe. peran faktor virulensi pada patogenesis infeksi candida albicans. stomatognaic j kedokt gigi. 2010; 7(2): 113–7. 9. pereira r, dos santos fontenelle ro, de brito ehs, de morais sm. biofilm of candida albicans: formation, regulation and resistance. j appl microbiol. 2021; 131(1): 11–22. 10. ponde no, lortal l, ramage g, naglik jr, richardson jp. candida albicans biofilms and polymicrobial interactions. crit rev microbiol. 2021; 47(1): 91–111. 11. perlin ds. echinocandin resistance in candida. clin infect dis. 2015; 61(suppl 6): s612-7. 12. a rend r up mc, patterson t f. multid r ug-resista nt ca ndida: epidemiology, molecular mechanisms, and treatment. j infect dis. 2017; 216(suppl_3): s445–51. 13. aminul h, m ias, mohammad s. antimicrobial, cytotoxicity and antioxidant activity of tinospora crispa. j pharm biomed sci. 2011; 13(12): 1–4. 14. ahmad w, jantan i, bukhari sna. tinospora crispa (l.) hook. f. & thomson: a review of its ethnobotanical, phytochemical, and pharmacological aspects. front pharmacol. 2016; 7: 59. 15. zuza-alves dl, silva-rocha wp, chaves gm. an update on candida tropicalis based on basic and clinical approaches. front microbiol. 2017; 8: 1927. 16. hutomo s, putri du, welviyanda bc, susilowati h. inhibition effect of garlic (allium sativum) extract on streptococcus sanguinis biofilm formation involving bacterial motility mechanism. malaysian j med heal sci. 2021; 17(2): 169–74. 17. raja af, ali f, khan ia, shawl as, arora ds. acetyl-11-keto-βboswellic acid (akba); targeting oral cavity pathogens. bmc res notes. 2011; 4: 406. 18. siswomihardjo w, sunarintyas s, tontowi ae. the effect of zirconia in hydroxyapatite on staphylococcus epidermidis growth. int j biomater. 2012; 2012: 432372. 19. setiawati s, nuryastuti t, ngatidjan n, mustofa m, jumina j, fitriastuti d. in vitro antifungal activity of (1)-n-2-methoxybenzyl1,10-phenanthrolinium bromide against candida albicans and its effects on membrane integrity. mycobiology. 2017; 45(1): 25–30. 20. warsinah, harwoko, nuryanti. screening of volatile compounds of brotowali (tinospora crispa) and antifungal activity against candida albicans. int j pharmacogn phytochem res. 2015; 7(1): 132–6. 21. inci m, atalay ma, özer b, evirgen ö, duran n, köksaldi motor v, koç an, önlen y, kilinç ç, durmaz s. investigations of als1 and hwp1 genes in clinical isolates of candida albicans. turkish j med sci. 2013; 43(1): 125–30. 22. tits j, cammue bpa, thevissen k. combination therapy to treat fungal biofilm-based infections. int j mol sci. 2020; 21(22): 8873. 23. huang x, zheng m, yi y, patel a, song z, li y. inhibition of berberine hydrochloride on candida albicans biofilm formation. biotechnol lett. 2020; 42(11): 2263–9. 24. burhannuddin b, karta iw, tresnanda b, putra ignd, darmada ipa, pradnyadhita, iida, gunawan iwba, ariawan imb. daya hambat virgin coconut oil terhadap pertumbuhan jamur candida albicans yang diisolasi dari sampel swab vagina. jst (jurnal sains dan teknol). 2017; 6(2): 209–19. 25. gan r. bioactivities of berberine : an update. int j mod biol med. 2012; 1(1): 48–81. 26. da silva ar, de andrade neto jb, da silva cr, campos r de s, costa silva ra, freitas dd, do nascimento fbsa, de andrade lnd, sampaio ls, grangeiro tb, magalhães hif, cavalcanti bc, de moraes mo, nobre júnior hv. berberine antifungal activity in f luconazole-resistant pathogenic yeasts: action mechanism evaluated by f low cytometry and biofilm growth inhibition in candida spp. antimicrob agents chemother. 2016; 60(6): 3551–7. 27. li d-d, xu y, zhang d-z, quan h, mylonakis e, hu d-d, li m-b, zhao l-x, zhu l-h, wang y, jiang y-y. fluconazole assists berberine to kill fluconazole-resistant candida albicans. antimicrob agents chemother. 2013; 57(12): 6016–27. 28. a l aboody ms, m ick yma ray s. a nt i-f u nga l ef f icacy a nd mechanisms of flavonoids. antibiot (basel, switzerland). 2020; 9(2): 45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p21–25 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p21-25 editorial team of dental journal (majalah kedokteran gigi) sk: 15/un3.1.2/2022 january 4 – december 31, 2022 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga editor in chief: muhammad dimas aditya ari department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200578006] editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478, 5030255. fax. +62 31 5039478, 5026288 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg/index accredited no. 158/e/kpt/2021 cover photo purchased from: https://stock.adobe.com order number: ae00770304146cid volume 55, issue 3, september 2022 p-issn: 1978-3728 e-issn: 2442-9740 editorial boards roeland jozef gentil de moor, department of restorative dentistry and endodontology, dental school, ghent university, belgium [scopus id: 7005928380] cortino sukotjo, department of restorative dentistry, university of illinois at chicago college of dentistry, united states [scopus id: 6508194317] guang hong, liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan [scopus id: 7203031334] kenji yoshida, department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, japan [scopus id: 57080640700] miguel rodrigues martins, co-worker aachen dental laser center, rwth aachen university, germany [scopus id: 55993479000] sajee sattayut, department of oral surgery, faculty of dentistry, khon kaen university, thailand [scopus id: 55431381300] samir nammour, department of dental science, faculty of medicine, university of liege, belgium [scopus id: 6602922393] reza fekrazad, laser reseach center in medical science, dental faculty, aja university of medical science, iran [scopus id: 22952665700] hong sai loh, department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore [scopus id: 7202491277] hamid nurrohman, missouri school of dentistry & oral health, a.t. still university, united states [scopus id: 52564067000] harry huiz peeters, laser research center, bandung, indonesia [scopus id: 51864447300] rahmi amtha, department of oral medicine, faculty of dentistry, universitas trisakti, indonesia [scopus id: 26031894400] elza ibrahim auerkari, department of oral biology, faculty of dentistry, universitas indonesia, indonesia [scopus id: 10139113000] r. darmawan setijanto, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 55212583700] anita yuliati, department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 43462222100] udijanto tedjosasongko, department of pediatric dentistry, faculty of dental medicine, universitas airlangga [scopus id: 6508026751] associate editors ketut suardita, department of conservative dentistry, faculty of dentistry, iik bhakti wiyata, indonesia [scopus id: 6506788956] alexander patera nugraha, department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57194112535] astari puteri, department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200385443] nastiti faradilla ramadhani, department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57191881659] managing editors beshlina fitri widayanti roosyanto prakoeswa, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57467259800] saka winias, department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57211330310] aulia ramadhani, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57205630113] beta novia rizqy, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57214805206] editorial assistant novi dian prastiwi, faculty of dental medicine, universitas airlangga; abdullah mas’udy, faculty of dental medicine, universitas airlangga. printed by: airlangga university press. campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 55, issue 3, september 2022 p-issn: 1978-3728 e-issn: 2442-9740 1. saliva analysis in children with active caries before and after dental treatment ebru akleyin, cansu osmanoğulları sarıyıldız, i̇zzet yavuz, i̇smet rezani toptancı ............ 120–124 2. visualizing the velocity fields and fluid behavior of a solution using artificial intelligence during endoactivator activation harry huiz peeters, elvira theola judith, faber yosua silitonga, lavi rizki zuhal .............. 125–129 3. cytotoxic test of different solvents of soursop (annona muricata) leaf extract against hsc-3 cell line areta vania bhanuwati, alfred pakpahan ................................................................................... 130–136 4. immunohistochemical differential expression of p16 proteins in follicular type and plexiform type ameloblastoma haris budi widodo, anung saptiwulan, helmi hirawan, christiana cahyani prihastuti, tirta wardana ................................................................................................................................ 137–141 5. oral hygiene assessment of dental students using the oral rating index (ori) tirza oktarina setiabudi, fajar hamonangan nasution ........................................................... 142–147 6. labial and palatal alveolar bone changes during maxillary incisor retraction at the universitas sumatera utara dental hospital suci purnama sari, mimi marina lubis, muslim yusuf ............................................................ 148–153 7. mapping of health care facilities, dental visits and oral health problems in indonesia to prevent covid-19 transmission ayu asri lestari, melissa adiatman, risqa rina darwita ......................................................... 154–160 8. estimation of children’s age based on dentition via panoramic radiography in surabaya, indonesia agung sosiawan, an’nisaa chusida, beshlina fitri widayanti roosyanto prakoeswa, arofi kurniawan, maria istiqomah marini, beta novia rizky, tito krisna gianosa, najminoor ramadhani ridlo, mumtaz ramadhani putra pesat gatra, aspalilah alias ....... 161–164 contents original articles page case reports 9. the effect of herbal medicine in reducing the severity of oral lichen planus: a systematic review and meta-analysis kharissa kemala vychaktami, rahmi amtha, indrayadi gunardi, rosnah binti zain .......... 165–173 review article 10. bilateral ramus mandibulectomy with plate reconstruction in ameloblastic carcinoma patient eunike lay, widodo ario kentjono ............................................................................................. 174–178 11. management of a complete denture in the flat mandibular ridge using a semi-adjustable articulator along with an effective suction method muhammad dimas aditya ari, harry laksono, valerian laksono, real akbar aucky sanjaya, tasya regita pramesti, ratri maya sitalaksmi ........................... 179–185 240240 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 240–245 case report successful traction of a mesially 90° dilacerated root of impacted maxillary canine: a case report fani tuti handayani1, ida ayu evangelina2 1school of dentistry, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 2department of orthodontics, faculty of dentistry, padjadjaran university, bandung, indonesia abstract background: impacted maxillary anterior teeth are a problem that has a significant impact on the aesthetics of the smile, dental arches, and occlusion. teeth that have higher tendencies to get impacted are third molars, maxillary central incisors, maxillary canines, and mandibular premolars. impacted teeth with severe root dilacerations are usually extracted surgically, then the space will be closed by orthodontic treatment or using a prosthesis. purpose: this study aims to report the successful orthodontic traction of mesially 90° dilacerated root of impacted maxillary canine. case: a 15-year-old female patient with the chief complaint of an unerupted left maxillary canine. intraoral examination showed a class i molar relationship on both sides, a unilateral crossbite on the right side, 8 mm spacing between the maxillary left lateral incisor and the premolar. also, mild crowding was found on both upper and lower anterior segments. cbct results showed a left maxillary canine was palatally semi-vertical impacted and had a mesially 90° dilacerated root. a significant stress concentration occurred at the middle and apical of the dilacerated root apex when exposed to orthodontic force; this tends to be a higher potential for resorption. case management: the impacted dilacerated canine was successfully moved to the proper position by combining crown exposure surgery, orthodontic traction using continuous light force, and gingivectomy. furthermore, traction was held using a gold chain combined with an elastic thread tied initially to a modified stainless-steel main archwire; this was followed by piggyback tandem wire and a vertical 3/16” light elastic traction. conclusion: the patient showed successful traction of the canine in less than six months. in addition, the unilateral posterior crossbite was corrected and functional occlusion was achieved. that canine showed good orthodontic and periodontal stability at one-year follow-up, without any evidence of root resorption. keywords: impacted dilacerated canine; orthodontic treatment; light force; orthodontic traction correspondence: fani tuti handayani, school of dentistry, faculty of medicine, jenderal soedirman university, jl. dr. soeparno, karangwangkal, purwokerto, 53123, indonesia. email: fanitutihandayani@gmail.com introduction impaction has been well-defined as total or partial lack of eruption of a tooth well after the normal age of eruption.1 the tooth impaction is caused by multiple factors which are divided into local and systemic factors.1,2 the impacted tooth can be caused by lack of space, lack of eruptive force, and sometimes a physical barrier such as mucosa, supernumerary tooth, bone or even retained deciduous tooth can prevent a tooth from erupting.1,3 teeth that have higher tendencies to get impacted are third molars, maxillary central incisors, maxillary canines, and mandibular premolars.1 in treatment plans, surgical and orthodontic management, those teeth are usually harder to treat and more challenging. achieving the ideal result in the treatment of teeth impaction usually depends on several factors such as biomechanical and clinical considerations, parent’s and patient’s commitment to their decision regarding the therapeutic limitation and possible complications of tooth traction like tooth ankylosis, periodontal support’s loss of attachment and the resorption of root apical external area.1,4 impacted maxillary anterior teeth are a problem that has a significant impact on the aesthetics of the smile, dental arches, and occlusion.5 they can disturb the tooth function in patients and have to be repaired as soon as possible so the greater deviation like alveolar height loss, the inclination of adjacent teeth to an incorrect space and the deviation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 mailto:fanitutihandayani@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 241 handayani and evangelina/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 240–245 of midline will not occur. canines are the most impacted anterior teeth and this can be caused by narrowing of the eruption space, wrong tooth placement, root dilaceration, and ankylosis.5,6 additionally, this can be alternated with surgical extraction of the tooth, or traction of the impacted tooth with orthodontic treatment.6 the success rate of eruption and the procedure’s risk are essential factors to consider in treating of impacted teeth.5,6 dilaceration refers to abnormal angulation or curvature formed in the root or crown of a tooth.7 root dilaceration is a shape abnormally in the root of a tooth and the deviation of the longitudinal axis is usually seen. the curve is usually seen in labiolingual or mesiodistal.7,8 moreover, the roots’ deviation from the axis of the tooth crown makes it difficult for the teeth to erupt according to the normal eruptive pathway.9,10 root dilaceration is usually caused by idiopathic developmental disorders or trauma and has also been associated with advanced root canal infection. other etiologies that can cause root dilacerations are cysts, developmental of dental germ that cause a change in anatomic structures, lack of space that cause ectopic tooth development, and facial clefts.8 an impacted tooth with dilacerated tooth can be treated with several treatments like tooth’s extraction or orthodontic traction. it all depends on the prognosis of periodontal and biomechanical considerations. impacted teeth with severe root dilacerations are usually extracted surgically and the space can be closed by orthodontic treatment or using a prosthesis, but usually a tooth with the such condition can be challenging to be treated by a surgical-orthodontic approach.5,6 this case report describes the interdisciplinary treatments of impacted maxillary left canine with root dilaceration in the apical third portion at 90° mesially. the treatment included orthodontic, oral surgery and periodontal approach. this study aims to report the successful orthodontic traction of mesially 90° dilacerated root of impacted maxillary canine. case a 15-year-old girl came in for treatment for an unerupted permanent left maxillary canine. there was no history of serious health problems, allergies, or trauma in her medical history. the patient had a convex profile and a class i skeletal base-jaw relationship. except for the left maxillary canine, intraoral examination revealed that all permanent teeth completely erupted. she demonstrated a class i molar relationship with 4.5 mm overjet and 1 mm overbite. there was mild crowding in the maxillary arch (ald +6 mm; 2.5 mm anterior crowding; 0.5 mm central diastema and 8 mm spacing between the left maxillary lateral incisor and the first premolar); mild crowding in the mandibular arch (ald -2 mm) with a unilateral crossbite on the right side (figure 1). the panoramic radiograph demonstrated an impacted left maxillary canine in a semi-vertical position with root laceration (figure 2a). additionally, the threedimensional cone-beam computerized tomographic (cbct) reconstructed images showed that the tooth was semi-vertical with the crown of the tooth palatally and the root of the tooth buccally. meanwhile, 1/3 of the root tip was lacerated 90° mesially (figure 2b). as shown in figure 2, the root formation was completed. cbct confirmed that the left impacted maxillary canine was located close to the alveolar ridge with only a thin layer of bone covering the crown. the crown was in the palatal with the buccal side facing the dental arch which is favorable to orthodontic traction using one-step approach, in which the attachment is placed on the tooth at the time of surgical exposure. it is considered as a factor leading to a good prognosis. this case can be treated using several options such as surgical exposure and followed by prosthesis or surgical exposure with orthodontic traction. it was all discussed with the patient and parents. the difficulty during extraction was expected because the root of the impacted canine was already severely dilacerated. the patient and her family had a strong desire to retain the tooth. as a result, the decision to expose the tooth surgically followed by orthodontic traction was taken. we would also maintain the integrity of periodontal tissue supporting the tooth at the same time. since this treatment plan was approved, informed consent was signed. case management fixed orthodontic appliances (roth .022 3m unitek, monrovia, ca usa) were bonded from the second molar forward on both sides in the maxillary and mandibular arches, except the left maxillary canine. initial alignment and leveling were achieved with super-elastic nickel-titanium (niti) wire followed by 0.016x0.022-in stainless steel (ss) wire. after that, the surgical exposure of the impacted left maxillary canine with dilacerated root was done using the open window method. under local anesthesia, the surgeon created a window to expose the crown of the impacted tooth (figure 3a). the gold chain was attached to the buccal part of the crown in the palatal. the use of gold chain was considered because it can support good adhesive strength and almost no one has an allergic reaction to it. subsequently, the chain was tied with an elastic thread using light orthodontic force (≈ 20g measured by tension gauge) on a modified 0.016x0.022-in ss wire; this was performed with an omega-shaped hook facing occlusal in the distal third wire length area spacing (figure 3b). every three weeks, the elastic thread was replaced. after nine weeks, the crown of the tooth was getting labially closer to the arch. as a result, the force was applied differently using the piggyback tandem wire technique with full arch anchorage; this was performed with a 0.012-in niti wire over a 0.016x0.022-in ss wire as the main archwire while still using light orthodontic force (≈ 30-40g measured by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 242handayani and evangelina/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 240–245 figure 1. pretreatment intraoral photographs. a b c d e f a b figure 2. a. pretreatment panoramic; b. cbct. figure 3. treatment progress. a. surgical exposure, b. traction with gold chain and elastic thread, c.gingivectomy, d. bracket placement, e. re-alignment and re-leveling, f. traction result. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 243 handayani and evangelina/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 240–245 tension gauge). after six weeks, the tooth appeared more extruded (the height of the visible crown was about 6 mm) and more labial in position; gingivectomy and bracket placement of the tooth were performed (figure 3c). the force was then applied using the piggyback tandem wire technique as before. in addition, the patient was instructed to use a light force 3/16 inch vertical-triangular elastic band, replaced twice a day for three weeks by anchoring all the mandibular teeth that had been aligned and leveled. after the next control period, the canine position did not crossbite with a distance of about 2 mm from the buccal surface of the tooth to the main archwire. this stage of using piggyback tandem wire was completed. therefore, bracket repositioning, realignment, and re-leveling were performed using an initial super-elastic 0.014 niti wire with the other tooth positions having figure-eight ligation to add anchors (figure 3d). in less than six months, the left maxillary canine was in a good arch of occlusion (figure 3e); this happened in may to november 2019. occlusion setting and detailing was started in january 2020, but unfortunately, since march 2020 the patient had difficulty coming to visit for routine control because of the covid-19 pandemic, so the fixed appliances were debonded in october 2020 according to the patient’s request related to the uncertain situation at that time. hawley retainers were placed in maxillary and mandibular arches after debonding. in less than six months, surgical exposure and orthodontic traction of mesially 90° dilacerated root of impacted maxillary canine was successfully performed. the roots of the teeth showed no resorption. additionally, no abnormalities were found in the alveolar bone or the surrounding tissues since the traction or after the recall one year following the completion of orthodontic treatment (figure 4). treatment limitations occurred due to the pandemic situation in 2020. root parallelism did not achieve because the setting and detailing phases were disrupted during the pandemic. post-treatment photographs could not be taken because the procedure for handling patients during the pandemic at that time did not allow cameras. panoramic after one-year follow-up (figure 4b) has shown a slight decrease in alveolar bone height in the anterior region which could be related to various factors, including the lack of monitoring of the patient’s oral hygiene since 2020 and during the use of the hawley retainer. discussion impacted and dilacerated are two different types of conditions that can complicate dental care, especially orthodontic treatment. this case is quite rare, and because the success rate is still rarely reported, the treatment is a a b figure 4. panoramic a. after orthodontic traction b. after one-year follow-up. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 244handayani and evangelina/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 240–245 big challenge. the impact of tooth’s position and direction, the extent of root formation, direction and angulation of dilacerations, and the amount of space available for aligning the impacted tooth are all factors affecting the successful alignment of impacted dilacerated teeth. meanwhile, the prognosis of the orthodontic traction treatment can be seen from the low position of the alveolar ridge, incomplete formation of the root and fan obtuse inclination angle.9–11 this case had a semi-vertical impacted tooth crown palatally and root buccally positioned, mesially 90° root laceration. furthermore, this position causes stress concentrated at the middle and apical regions of the root during intrusive, extrusive, tipping, and rotational force application. therefore, the potential for root resorption is increased.9,12 root resorption causes many dental problems and complications. in orthodontics, root resorption is referred to as resorption caused by inflammation. it is a pathological condition because the orthodontic force will be focused on the tooth and the area will be hyalinized. this will cause the loss of periodontal tissue.13 the root resorption can be caused by a complex multifactorial etiology like the combination of genetic, mechanical and biological factors and also some factors that are related to the orthodontic treatment.13–15 the application of force in orthodontic treatment is essential for successful treatment and is an iatrogenic factor that harms the teeth and surrounding tissues.16 the mean optimal force on teeth with normal roots should not be applied to teeth with dilacerated roots. applying the same magnitude of force to the two different root states resulted in higher stress apical to the dilacerated root state. this difference could be up to 20 times higher.12 also, the first force applied in this case was tipping; this was accomplished by applying a light force from a continuous elastic thread of about 20 grams, resulting in a tooth shift of 4 mm for nine weeks. a twisting effect delivered by the direction of the elastic thread knot was made oblique to the distal 10°. since the initial position of the labial teeth was facing anterior, the elastic thread’s pull direction would transmit tensile forces and rotate the labial teeth laterally. abnormal root morphology such as dilacerated root is a high enough risk factor for root resorption. similarly, orthodontic treatment is also a mechanical risk factor for root resorption.16 the application of orthodontic force to the dilacerated root can be a bad scheme for the tooth condition. therefore, consideration of the magnitude and direction of the force is needed to minimize adverse effects.17 however, no references specifically describe applying the optimum orthodontic force to perform tooth traction with dilacerated roots. orthodontic traction on impacted teeth is performed in many ways, including cantilever springs such as kilroy springs, ballista springs by jacoby, buccal auxiliary springs by kornhauser, and other spring innovations.18–21 the design and dimensions of the wire are used to influence the force generated by the spring. making a spring to produce a continuous light force is not simple, mainly if applied to a tooth with a root dilaceration.12,20 due to the relatively easy application, tooth traction using an elastomeric chain is also a preferred option.22,23 the principle of treatment performed refers to two principles: oppenheim’s principle (1911) regarding light forces and martin schwarz’s (1932) principle that the limit of orthodontic forces should not exceed capillary blood pressure, 20 g/cm2 for tipping and 40-50 g/cm2 for bodily movement.24,25 in addition, the first application of force was performed using an elastic thread, with the pulling distance adjusted based on the force measured with an orthodontic force gauge or tension gauge; this makes it easy to control the magnitude of the force and determine the direction of the pull. the increase in styling is performed gradually. using only the piggyback technique with super-elastic 0.012 niti wire, the force application was performed. after that, a combination of the piggyback technique was performed using the super-elastic 0.012 niti wire with elastic vertical light force. this combination divides the force into two directions, buccal and occlusal, without increasing the magnitude of the previous force. since the distance of the tooth’s crown is getting closer to the labial arch, the buccal force of the piggyback technique is smaller than the previous one. this significant difference in force is applied to pull the tooth occlusally with a vertical elastic. the anchorage was obtained from ligation of all other teeth, both from the maxillary and mandibular dental arches aligned and leveled. the success of orthodontic treatment with the traction of impacted teeth lies in pulling the teeth into the normal arch, maintaining the surrounding tissue, and functional occlusion. orthodontic traction of mesially 90° dilacerated root of impacted maxillary canine has been successfully performed by applying light force from an elastic thread, tandem wire and vertical elastic. after the traction was successful, the alignment and leveling process was repeated, with the total duration of orthodontic treatment lasting two years. the smile aesthetics and tooth occlusion of the patient were improved and the patient was delighted by the result. in a one-year follow-up, the treated canine showed good stability both orthodontically and periodontally without resorption of the root. however, this treatment showed some limitations. the pandemic covid-19 occurred when approaching the end of treatment. this became a significant obstacle to achieving the ideal finishing, such as root parallelism, occlusal adjustment and periodontal monitoring. the clinical implications of this condition can lead to periodontal problems, so the patient was informed for having a routine dental checkup when the pandemic situation improved. some suggestions for clinical management are to plan and carry out the orthodontic treatment, especially in cases of traction always using light force, and also to establish professional communication with patients, which is very important so that, when unexpected things happen, they remain under supervision and patients are willing to cooperate well. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 245 handayani and evangelina/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 240–245 acknowledgment the researchers would like to thank the patient for her willingness to publish her case and treatment. references 1. singh n, bagga d, tripathi t, gupta p, singh r. orthodontic management of impacted teeth: an overview. indian j orthod dentofac res. 2017; 3(2): 59–63. 2. singh n, tripathi t, rai p, kalra s, neha. impacted central incisor with dilacerated root-treat with ease. j clin diagn res. 2016; 10(3): zj07-8. 3. becker a. orthodontic treatment of impacted teeth. 3rd ed. wileyblackwell publishing ltd; 2012. p. 456. 4. chaushu s, becker t, becker a. impacted central incisors: factors affecting prognosis and treatment duration. am j orthod dentofacial orthop. 2015; 147(3): 355–62. 5. kaczor-urbanowicz k, zadurska m, czochrowska e. impacted teeth: an interdisciplinary perspective. adv clin exp med. 25(3): 575–85. 6. manne r, gandikota c, juvvadi sr, rama hrm, anche s. impacted canines: etiology, diagnosis, and orthodontic management. j pharm bioallied sci. 2012; 4(suppl 2): s234-8. 7. regezi ja, sciubba jj, jordan rck. abnormalities of teeth. in: oral pathology: clinical pathologic correlations. 7th ed. st. louis: saunders; 2017. p. 374–88. 8. neville b, damm d, allen c, chi a. oral and maxillofacial pathology. 4th ed. missouri: wb saunders, elsevier; 2016. p. 449–450. 9. refai wm, hassan ms. effect of root curvature of anterior teeth. egypt dent j. 2012; 58(2): 53–61. 10. tausche e, ha rzer w. treatment of a patient with class i i malocclusion, impacted maxillary canine with a dilacerated root, and peg-shaped lateral incisors. am j orthod dentofacial orthop. 2008; 133(5): 762–70. 11. pavlidis d, daratsianos n, jäger a. treatment of an impacted dilacerated maxillary central incisor. am j orthod dentofacial orthop. 2011; 139(3): 378–87. 12. kamble rh, lohkare s, hararey p v, mundada rd. stress distribution pattern in a root of maxillary central incisor having various root morphologies: a finite element study. angle orthod. 2012; 82(5): 799–805. 13. teja kv, ramesh s. comprehensive narrative review. drug invent today. 2020; 13(2).58–63 14. bansal p, nikhil v, kapur s. multiple idiopathic external apical root resorption: a rare case report. j conserv dent. 2015; 18(1): 70–2. 15. adaki drv, adaki dsr, agrwal djm, nanjannawar dlg. external apical root resorption – an unusual case report. iosr j dent med sci. 2014; 13(1): 60–2. 16. jacob a, m.v a, shetty s, nambiar s, philip jose n. a literature review on orthodontically induced root resorption: the aftermath of the pursuit of an attractive smile. eur j mol clin med . 2020; 7(3): 941–57. 17. hsu y-c, kao c-t, chou c-c, tai w-k, yang p-y. diagnosis and management of impacted maxillary canines. taiwan j orthod. 2019; 5(4): 741–9. 18. bowman sj, carano a. the kilroy spring for impacted teeth. j clin orthod. 2003; 37(12): 683–8. 19. raghav p, singh k, munish reddy c, joshi d, jain s. treatment of m a x i l la r y i mpa ct e d ca n i ne usi ng ba l l ist a sp r i ng a nd orthodontic wire traction. int j clin pediatr dent. 2017; 10(3): 313–7. 20. kachoei m, ghanizadeh m, nastarin p. a novel spring for impacted canine traction: a mew method presentation. adv biosci clin med. 2019; 7(2): 25. 21. cruz rm. orthodontic traction of impacted canines: concepts and clinical application. dental press j orthod. 2019; 24(1): 74–87. 22. alqahtani h. management of maxillary impacted canines: a prospective study of orthodontists’ preferences. saudi pharm j. 2021; 29(5): 384–90. 23. grisar k, luyten j, preda f, martin c, hoppenreijs t, politis c, jacobs r. interventions for impacted maxillary canines: a systematic review of the relationship between initial canine position and treatment outcome. orthod craniofac res. 2021; 24(2): 180–93. 24. ren y, maltha jc, kuijpers-jagtman am. optimum force magnitude for orthodontic tooth movement: a systematic literature review. angle orthod. 2003; 73(1): 86–92. 25. asiry ma. biological aspects of orthodontic tooth movement: a review of literature. saudi j biol sci. 2018; 25(6): 1027–32. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p240–245 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p240-245 p-issn: 1978-3728 e-issn: 2442-9740 volume 49, number 4, december 2016 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2015 january 2nd – december 31st, 2016 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga editorial boards: roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); harry huiz peeters (laser research center, bandung, indonesia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); fajar hamonangan nasution (department of orthodontics faculty of dentistry, universitas trisakti, indonesia); pinandi sri pudyani (department of orthodontics faculty of dentistry, universitas gadjah mada, indonesia); boedi oetomo roeslan (department of biochemistry faculty of dentistry, universitas trisakti); rahmi amtha (department of oral medicine faculty of dentistry, universitas trisakti, indonesia); anita yuliati (department of dental material faculty of dental medicine, universitas airlangga, indonesia); darmawan setijanto (department of dental public health faculty of dental medicine, universitas airlangga, indonesia); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); boy muchlis bachtiar (department of oral biology faculty of dentistry, universitas indonesia, indonesia) managing editors: priyawan rachmadi (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); markus budi rahardjo (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty vocational, universitas airlangga, indonesia); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia) assistant editors saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia) peer-reviewers ismet danial nasution (department of prosthodontics, faculty of dentistry, universitas sumatera utara); melanie sadono djamil (department of biomedic, faculty of dentistry, universitas trisakti, indonesia); al. supartinah santoso (department of pediatric dentistry, faculty of dentistry, universitas padjajaran, indonesia); muhammad rubianto (department of periodontic dentistry, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); mei syafriadi (oral pathology, faculty of dentistry, universitas jember, indonesia); rasmidar samad (department of epidemiology, faculty of dentistry, universitas hasanuddin, indonesia); titiek berniyanti (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); haryono utomo (dental clinic, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health faculty of dental medicine, universitas airlangga, indonesia) administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 086/01.17/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 49, number 4, december 2016 p-issn: 1978-3728 e-issn: 2442-9740 1. effects of sarang semut (myrmecodia pendens merr. & perry) extracts on enterococcus faecalis sensitivity cut soraya, hendra dian adhyta dharsono, dudi aripin, mieke h. satari, dikdik kurnia, and danny hilmanto ............................................................................................. 175–180 2. contemporary guided bone regeneration therapy for unaesthetic anterior peri-implantitis case benso sulijaya, sandra olivia kuswandani, and yuniarti soeroso ............................................ 181–184 3. color stability of heat polymerized polymethyl methacrylate resin denture base after addition of high molecular nano chitosan ika devi adiana, trimurni abidin, and lasminda syafiar ......................................................... 185–188 4. effects of silane application on the shear bond strength of ceramic orthodontic brackets to enamel surface pinandi sri pudyani and setiarini widiarsanti ............................................................................ 189–194 5. aggregatibacter actinomycetemcomitans sensitivity towards chlorophyll of moringa leaf after activated by diode laser i gde bagus yatna wibawa, suryani dyah astuti, and ernie maduratna setiawati ............... 195–200 6. correlation between working positions and lactic acid levels with musculoskeletal complaints among dentists fiory dioptis putriwijaya, titiek berniyanti, and indeswati diyatri .......................................... 201–205 7. perceived parenting style and mother’s behavior in maintaining dental health of children with down syndrome siti fitria ulfah, darmawan setijanto, and taufan bramantoro ................................................ 206–212 8. characterization of streptococcus sanguis molecular receptors for streptococcus mutans binding molecules deby kania tri putri, indah listiana kriswandini, and muhammad luthfi .................................... 213–216 9. the effect of peer support education on dental caries prevention behavior in school age children at age 10-11 years old debby syahru romadlon, taufan bramantoro, and muhammad luthfi .................................. 217–222 10. effectiveness of line communication application as a social media on changes in tooth brushing behavior of junior high school students in banjarmasin w. widodo, r. darmawan setijanto, and agung sosiawan ......................................................... 223–228 11. management of herpes labialis triggered by emotional stress herlambang prehananto and kus harijanti ................................................................................. 229–233 215 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 215–220 original article strategy for improving the quality of school dental health efforts at tabanan public health center i gusti ayu ari agung, i nyoman panji triadnya palgunadi department of dental public health and preventive dentistry, faculty of dentistry, universitas mahasaraswati denpasar, denpasar, bali, indonesia abstract background: the school dental health efforts or usaha kesehatan gigi sekolah (ukgs) is a public health effort to maintain and improve the dental and oral health of elementary school students. ukgs is the strategy and the flagship of dental health in schools. the implementation of ukgs involves three elements, namely the public health centre or pusat kesehatan masyarakat (puskesmas), schools, and parents. the three elements in ukgs are the driving force and restraining force, which greatly affect the quality of ukgs services. purpose: this study aimed to analyze a strategy that can improve the quality of ukgs at tabanan puskesmas. methods: this research used evaluative research and was analyzed by kurt lewin’s force field analysis. results: the driving forces that have the highest score are good knowledge, the skill of personnel, and guidelines for implementing the ukgs at puskesmas; whereas the restraining forces that have the highest score are the unavailability of guidebooks and health teacher skills, as well as lack of budget for the ukgs activities. conclusion: strategies that can improve the quality of ukgs at tabanan puskesmas can be done by utilizing the facilities at the puskesmas for the ukgs activities in schools, transmitting knowledge and skills from puskesmas officers to the ukgs staff in schools, and increasing the budget by means of self-help. keywords: dental health education; dentistry; school dental health efforts correspondence: i gusti ayu ari agung, department of dental public health and preventive dentistry, faculty of dentistry, universitas mahasaraswati denpasar, jl. kamboja no.11a denpasar, bali, 80233 indonesia. email: ayuariagung@unmas.ac.id introduction the global burden of disease study 2019 estimated that dental diseases affect close to 3.5 billion people worldwide, with caries of permanent teeth being the most common condition. globally, it is estimated that 2 billion people suffer from caries of permanent teeth and 520 million children suffer from caries of primary teeth.1 in most lowincome and middle-income countries, the prevalence of dental disease continues to increase as urbanization grows and living conditions change. this idea is mainly due to inadequate exposure to fluoride (in water supplies and dental hygiene products such as toothpaste), the availability and affordability of high-sugar foods, and poor access to dental healthcare services in the community.2 the world health assembly approved a resolution on dental health in 2021 at the 74th world health assembly. the resolution recommends a shift from the traditional curative approach toward a preventive approach that includes the promotion of dental health within the family, schools, and workplaces, which includes timely, comprehensive, and inclusive care within the primary healthcare system.3 dental diseases in indonesia are at the top of the list of the 10 most common diseases in indonesia. the perception and the behavior of the indonesian people toward dental health are still poor. it can be seen from a large number of dental caries diseases in indonesia, which tend to increase, so dental health problems in indonesia still need attention. the prevalence of caries and periodontal disease is still relatively high.4 the dental and oral problems of the people in bali are higher than the national average, which is 58.4%. one of the reasons is that 95.7% of balinese people have never visited a dental medical facility. furthermore, only 5.3% of balinese people brush their teeth at the right time, which should be twice a day, in the morning after breakfast, and at night before going to bed. it allows for other factors that influence the high level of dental and oral problems in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 mailto:ayuariagung@unmas.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 216agung and palgunadi/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 215–220 province of bali, one of which is the behavior of brushing teeth that is not good and correct, and dental and oral health services are not evenly distributed.5 to control dental and oral diseases by the public health center or pusat kesehatan masyarakat (puskesmas) through the actualization of the local school dental health efforts or usaha kesehatan gigi sekolah (ukgs) for both examination and treatment of dental and oral diseases is still low. public awareness of dental and oral health is also still low, so it is necessary to develop a health improvement system through counseling and improving the quality of services.5,6 the puskesmas program on ukgs is not yet optimal. this shows that the implementation of ukgs has not succeeded.7 the efforts to improve dental and oral health services, especially in elementary schools, face several obstacles which certainly require solutions. obstacles faced include the limited number of dental health workers in elementary schools. the health law of the republic of indonesia number 36 the year 2009 about health states that improvements in health status can be realized by increasing integrated health facilities and public health services carried out by health workers according to their area of expertise. therefore, there is a need to strengthen cross-program and cross-sectoral integration, the development of which is the responsibility of health workers, teachers, and parents. the puskesmas play an important role as the ukgs’s development team at the district level, particularly in dental and oral health services such as screening for dental and oral health problems, regular dental health check-ups, and consultations. the role of teachers in schools is also crucial, that is, to monitor student behavior daily.8 tabanan regency in bali province is one of the regencies in indonesia that has a prevalence of dental caries experience that is higher than the national prevalence of 68.2%. based on interviews with school principals, it was found that most ukgs in the tabanan district were not active. this is in accordance with the results of a study in denpasar which stated that more than 95% of ukgs were inactive,9 and when the research was conducted, it turned out that, during the covid-19 pandemic, the ukgs program could not be implemented. student learning is conducted online which worsens the condition of students’ consumption of sweet snacks during the online learning process. this will increase the occurrence of caries in students. therefore, dental and oral health education counseling through the ukgs online program is highly needed. elementary school students (ages 6-12 years) are often referred to as a vulnerable period because the baby teeth begin to fall out one by one, and the first permanent growth begins. new teeth are immature and susceptible to decay.5 ukgs is a technical strategy for implementing dental and oral health for elementary school students. the scope of its activities is to carry out dental health checks, routine dental care, and dental and oral health counseling for school children.3 however, at the time of the study, the covid-19 pandemic occurred, so the ukgs program in schools was suspended. it is necessary to have a strategy to improve services and human resources quality through the ukgs program. therefore, the purpose of this study is to investigate the driving and restraining force in developing efforts to improve the quality of ukgs services materials and methods this qualitative research used evaluative research and was analyzed by kurt lewin’s force field analysis,10 which assesses and measures results with standard indicators. the population in this study was 40 ukgs officers at tabanan puskesmas. the research sample consisted of 15 ukgs officers who had signed an informed concern. the method of determining and selecting samples is by purposive sampling, of which one sample of ukgs officers was taken from each puskesmas with the support of one elementary school. the population in this study were all puskesmas in tabanan district, while the target population was all puskesmas in tabanan district that foster ukgs. the research sample was ukgs officers at the tabanan health center, while the intended sample was a sample that met the inclusion and exclusion criteria. the sample that is actually researched is the sample that really follows the research to completion. inclusion criteria are ukgs puskesmas tabanan officers who are willing to be investigated by signing the informed consent. the exclusion criteria were ukgs puskesmas tabanan officers who were not willing to be investigated. the drop-out criteria are the research sample who for some reason cannot continue the research. the researcher absolutely guarantees that the identity of the research participants will be kept confidential and fully protected. validity assessment is through cross-checking of information sources.11 ethics approval was obtained from the ethics review board of the faculty of dentistry at mahasaraswati denpasar university (no.356/a.17.01/ fkg-unmas/iii/2022). the technique used is a force field analysis (ffa) approach by analyzing inputs, including the condition of health centers and elementary schools, the availability of ukgs program tools and materials, ukgs program implementation guidelines, ukgs program planning, and ukgs program budget. in addition, the process analysis includes planning discussions, counseling to teachers, parents, and students, screening, plenary treatment, mass toothbrushing, recording and reporting, monitoring, evaluation, and feedback.12 the results of the input and process analysis are expected to provide input that can produce efforts to improve the quality of ukgs services. the data obtained were analyzed using ffa to determine the key restraining force (rf) and driving force (df) of the study results. a qualitative method study was carried out using the ffa regarding improving the quality of ukgs in puskesmas tabanan, bali, in 2020. implementation of the ffa using transparency overhead let’s all participants see dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 217 agung and palgunadi/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 215–220 the ongoing discussion process. this study included six steps for improving the quality of ukgs: first, discover df and rf from references; second, select key df and rf through focus group discussion; third, df and rf assessment by the first group (ukgs officers); fourth, determine key df and rf from head school perspective; fifth, determining the score of the strength of the influence on each of the agreed strength; and sixth, the largest factor value from the driving factors and restraining factors, is then used as a key success factor in formulating a strategy to improve the quality of ukgs in tabanan puskesmas. results tabanan regency is divided into 10 sub-districts and consists of 133 villages. according to the results of the population registration carried out by the central statistics agency (bps) at the end of 2015, the population of tabanan regency reached 448,033 inhabitants. the population is spread over ten sub-districts in tabanan regency. most of the tabanan regency area is a rural/mountainous area. tabanan regency has 20 puskesmas, which covers ukgs in 310 elementary schools. the 15 puskesmas where the research was carried out based on regional characteristics is shown in table 1. questionnaires, field observations, suggestions, and expectations of ukgs staff (puskesmas and school) are expected to improve the quality of ukgs program services at tabanan puskesmas. the procedures, efforts, and actions for improving the quality of ukgs program services based on the key driving and restraining forces are presented in table 2. ffa efforts to improve the quality of ukgs program services in tabanan puskesmas are presented in figure 1. table 1. number of puskesmas based on regional characteristics of research location, from puskesmas to elementary school puskesmas n % in easily accessible locations: tabanan i, ii, iii 3 20 in locations that are difficult to reach: marga i, ii; kerambitan ii; pupuan i, ii; selemadeg timur i, ii; selemadeg barat; baturiti i, ii; penebel i, ii 12 80 table 2. efforts to improve the quality of ukgs program services based on the key driving forces and restraining forces key driving and restraining forces efforts and activities made good knowledge and the skills of ukgs personnel at the puskesmas providing skills and understanding of the meaning, objectives, targets of activities, and targets of the ukgs program for teachers and elementary school students, especially in the implementation of dental and oral health counseling and mass toothbrushing there are guidelines for implementing the ukgs program at the puskesmas distributing and explaining the ukgs implementation guidelines for school health teachers there is a program planning at the puskesmas utilizing the ukgs program planning at the puskesmas as a reference for implementing ukgs in schools and making ukgs program planning together there is no ukgs guidebook at school organizing ukgs guidebooks for students and teachers lack of budget for ukgs program in schools implementing a healthy fund program through the student parents committee board lack of training for ukgs personnel in schools, and puskesmas staff rarely go to school providing training and counseling on how to brush teeth properly and correctly, as well as practicing it to teachers and elementary school students; improving the discipline of ukgs puskesmas officers force strength driving forces (positives) +5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5 restraining forces (negatives) (df1) good knowledge and skill of ukgs personnel at puskesmas (rf1) there is no ukgs guidebook at school; and health teachers are not skilled (df2) there are guidelines for implementing the ukgs program at the puskesmas (rf2) lack of budget for ukgs program in schools (df3) there is a program planning at the puskesmas (rf3) lack of training for ukgs personnel in the school; and puskesmas staff rarely go to school total scores: +12.5 total scores: -11 figure 1. ffa strategy for improving the quality of ukgs at tabanan puskesmas.10 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 218agung and palgunadi/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 215–220 the results showed that the driving factors were the availability of skilled and knowledgeable health workers and ukgs personnel who were willing to carry out the ukgs program. in addition, there were also restraining factors in the form of infrequent visits by officers to schools, lack of equipment and materials facilities, and lack of budget for ukgs activities. efforts to improve the quality of ukgs program services based on the key driving forces are utilizing the guidelines, program planning, knowledge, and skills of ukgs personnel puskesmas. efforts to improve the quality of ukgs program services based on the key restraining forces are increasing the budget for the ukgs program by holding health funds. based on figure 1, the total score for the force strength of the driving forces (+12.5) is higher than that of the restraining forces (-11). so the ukgs program is feasible to be developed. the driving forces that have the greatest value are df1 and df2, whereas the restraining forces that have the greatest value are rf1 and rf2. discussion dental and oral health services in the province of bali begin with basic dental health activities at the puskesmas. the research report says the effectiveness of the utilization of the dental health department of puskesmas tabanan is very low.13 this is in accordance with the results of research at the karangasem puskesmas. it is due to the road conditions which are difficult to reach.14 the results of this study are shown in table 1, which shows that 80% of the road conditions are very difficult for ukgs puskesmas officers to go to elementary schools. moreover, since each puskesmas only has two dentists, it does not meet the standards for services for the number of residents that must be handled. the rate of addition of puskesmas in the tabanan district is not proportional to the rate of population growth, so the ukgs program is difficult to implement due to the lack of existing facilities.15 this is consistent with the results of an evaluation of the ukgs implementation in the districts of denpasar and karangasem, bali province, which found around 95% of ukgs were inactive. this can be overcome by multiplying the ukgs guidebook at the puskesmas, distributing them to students and teachers, and training school health teachers through the implementation of the student parent committee board’s health fund program (table 2 and figure 1).16 the active ukgs was found to be around 5%, significantly improving the dental and oral health of elementary school students.9,14 some aspects of the service can influence the utilization of the health services in puskesmas tabanan, including the activities being done for health such as factors of the health personnel doing the health service, facility, and factor of the services users. puskesmas’ flagship program to prevent dental health problems in elementary school students is the ukgs program. it is conducted to maintain and improve the dental and oral health of all students in school. it is carried out through health education, dental health services, and fostering a healthy school environment. the impact of the ukgs is that, hopefully, there will be changes in students’ attitudes and behavior. in addition, students will understand when and how they should brush their teeth properly and correctly, and they can take advantage of available dental and oral health service facilities to improve their oral and dental health.17 the lack of budget for the ukgs program in schools is the restraining power with the highest score (figure 1). this is in accordance with the results of research at the halmahera health center, semarang, which stated that the ukgs activities were not successful because they were influenced by the lack of operational funds for ukgs activities.3 this is confirmed by the results of research in saudi arabia on increasing oral health literacy, as well as reducing organizational and financial barriers, resulting in better oral and dental health in school children.18 therefore, the strategy for the success of the ukgs program is to implement it in an integrated manner, across programs and sectors that are targeted and sustainable.12 ffa analysis found the restraining force with the highest score is health teachers in schools are not skilled (figure 1). several research reports recommend that it is necessary to regularly conduct training for school health teachers to motivate teachers to actively take the initiative to develop various activities related to the promotion of dental health in schools.19–21 this activity is in the form of counseling for teachers on how to assess oral hygiene or oral condition, including dental caries and gum disease, as well as explanations about efforts or programs that need to be carried out.22,23 ukgs services for students are training for school health teachers on integrated dental and oral health knowledge.24–26 dental health education and counseling are carried out by school health teachers by following the curriculum applicable to all students in grades i-vi. the teacher guides daily tooth brushing activities, at least for grades i, ii, and iii, by using toothpaste containing fluoride.27 other ukgs program activities are duplicating, distributing, and training ukgs implementation guidelines in the puskesmas to school ukgs teachers (table 2). the ukgs implementation guidelines can be used as guidelines for implementing the ukgs program in elementary schools. elementary school-age children are vulnerable to dental and oral health due to a lack of knowledge. the level of teachers’ knowledge about oral health affects students’ attitudes and behavior towards dental and oral health.19 applying the latest science, technology, and motivation to stimulate student participation and break the caries chain by preventing and protecting teeth. primary prevention and protection technologies include the latest caries theory, such as demineralization versus remuneration, and minimum intervention, such as protecting caries-prone teeth. principles of treatment and minimum intervention from an early age are proven to add value to be more effective and measurable. this is in accordance with the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 219 agung and palgunadi/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 215–220 research results that the current ukgs development is the emergence of innovative ukgs, which aims to increase students’ awareness of caries risk factors by teaching them how to overcome caries.12 united nations educational, scientific, and cultural organization (unesco) and the world health organization (who) call on schools to play an important role in maintaining dental health education and equip students with skills for their future health and well-being. the results showed that dental health education improved the students’ knowledge, attitudes, and oral hygiene status.28 there is a significant correlation between knowledge, attitude, source of information, and teacher support with oral treatment behavior of oral hygiene in children. source of information is the dominant factor that affects the behavior of oral hygiene in children aged 12 years old in puskesmas i baturiti, tabanan.29 the results of the study indicate that it is important for educators to incorporate education on medicinal plants with nutrition for oral health into the school curriculum. this is reinforced by a reminder sticker book, which can increase knowledge of oral health. the results of the study indicate that it is important for educators to incorporate dental and oral health nutrition, as well as medicinal plant garden and dental health nutrition education, into the school curriculum.30,31 reminder sticker books can increase oral health knowledge and reduce ohi-s scores in seven-to eight-year-old children.32 unesco proposes to prepare students to understand the reality to build individual student empowerment through improving curriculum content (focus on related to health, nutrition, and well-being) with collaboration between various sectors and actors to achieve an integrated system focused on students and teachers.28,33 good dental health contributes considerably to the health and quality of life of the population.34 providing elementary school educators with web-based resource materials improves their attitudes, increases their knowledge, and leads to positive behavioral intentions concerning educating their students about dental health.32 based on research, it is stated that the acquisition of learning outcomes through a combination of the senses of sight (visual) and hearing (audio) becomes higher.35 based on the description above, it can be concluded that the strategy that can improve the quality of ukgs at the tabanan puskesmas can be done by utilizing the facilities at the puskesmas for ukgs activities in schools, transmitting knowledge and skills from puskesmas ukgs officers to school ukgs officers, and increasing the budget by the implementation of the student parent committee board’s health fund program. this research was carried out during the covid-19 pandemic, which is the main limitation of this research, so respondents can only be taken from 15 ukgs puskesmas officers. the findings of this study have important implications that the ukgs program is very important to be immediately noticed by parents, the health office, the government, research and community service institutions, and funders, considering that only about 5% of the ukgs program can be active. on the other hand, the condition of students’ dental health is getting worse. it is recommended for the development of an innovative ukgs program, namely the development of a few dentists with a garden of nutritious medicinal plants, which is the main focus of the physical education and health curriculum, and appears on the website. the findings of this study have very important implications, both for readers, students, parents of students, ukgs officers (schools and puskesmas), principals, heads of health centers, leaders of the puskesmas, as information that the ukgs program is more than 95% unable to be active because of constraints, such as lack of funds and lack of awareness to dental health for elementary school students. references 1. global burden of disease collaborative network. global burden of disease study 2019 (gbd 2019). 2019. available from: http://ghdx. healthdata.org/gbd-results-tool. accessed 2021 nov 9. 2. world health organization. oral health. 2020. available from: https://www.who.int/news-room/fact-sheets/detail/oral-health. accessed 2021 aug 12. 3. taftazani rz, rismayani l, santoso b, wiyatini t. analisis program kegiatan usaha kesehatan gigi sekolah (ukgs) di puskesmas halmahera. j kesehat gigi. 2015; 2(1): 32–7. 4. suanda iw. gerakan masyarakat hidup sehat dalam mencegah terjadinya penyakit gigi dan mulut. j kesehat gigi. 2018; 6(1): 29–34. 5. badan penelitian dan pengembangan kesehatan. laporan provinsi bali riskesdas 2018. kementerian kesehatan republik indonesia; 2018. available from: https://ejournal2.litbang.kemkes.go.id/index. php/lpb/article/view/3751. accessed 2021 nov 9. 6. pemerintah republik indonesia. peraturan pemerintah republik indonesia nomor 47 tahun 2016 tentang fasilitas pelayanan kesehatan. 2016. available from: https://peraturan.bpk.go.id/home/ details/4820. accessed 2021 nov 9. 7. santoso b, gejir n, fatmasari d. information system monitoring model implemented in school health dental unit. arc j dent sci. 2017; 2(4): 8–11. 8. galuh a, supriyana, rasipin, sunarjo l, fatmasari d. management model of school dental health effort (sdhe) of website-based for improving quality of information system at elementary school. int j nurs heal serv. 2021; 4(2): 112–9. 9. wirata in. perbedaan derajat kesehatan gigi dan mulut pada siswa sd dengan program ukgs aktif dan tidak aktif di wilayah kerja puskesmas denpasar utara ii tahun 2015. j ilmu dan teknol kesehat. 2016; 3(2): 124–36. 10. shrivastava s, shrivastava p, ramasamy j. force field analysis: an effective tool in qualitative research. j curr res sci med. 2017; 3(2): 139–40. 11. sastroasmoro s. dasar-dasar metodologi penelitian klinis. 5th ed. jakarta: sagung seto; 2018. p. 89–298. 12. kementrian kesehatan ri. pedoman usaha kesehatan gigi sekolah (ukgs). jakarta: kementerian kesehatan republik indonesia; 2012. p. 11–14. 13. dwiastuti sap. hubungan status kesehatan gigi dan mutu layanan dengan pemanfaatan balai pengobatan gigi puskesmas kabupaten tabanan. j kesehat gigi. 2013; 1(1): 10–5. 14. su r ya n i n w, ta mba i m, ag ung iga a. eva luat ion of t he implementation of school dental health program in karangasem regency, year 2017. sci reasearch j. 2018; 6(5): 31–4. 15. dinas kesehatan provinsi bali. profil kesehatan provinsi bali. 2021. available from: https://diskes.baliprov.go.id/profil-kesehatanprovinsi-bali/. accessed 2021 nov 9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 http://ghdx https://www.who.int/news-room/fact-sheets/detail/oral-health https://ejournal2.litbang.kemkes.go.id/index https://peraturan.bpk.go.id/home/ https://diskes.baliprov.go.id/profil-kesehatan-provinsi-bali/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 220agung and palgunadi/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 215–220 16. kementrian kesehatan ri. puskesmas berperan penting wujudkan sekolah sehat. 2013. available from: https://www.kemkes.go.id/ article/view/2411/puskesmas-berperan-penting-wujudkan-sekolahsehat.html. accessed 2021 nov 9. 17. lestari dr, indarjo s. evaluasi penerapan manajemen ukgs dalam perilaku perawatan gigi dan mulut siswa sekolah dasar. j heal educ. 2016; 1(2): 8–11. 18. al agili de, farsi nj. need for dental care drives utilisation of dental services among children in saudi arabia. int dent j. 2020; 70(3): 183–92. 19. nugraheni h, sunarjo l, wiyatini t. teacher’s role on oral health promoting school. j kesehat gigi. 2018; 5(2): 13–21. 20. siswanto sh, abraham jf, ‘aini nq, damayanti m, wulansari aa, aprilia v, guna idaw, sary hp, nabella ti, jatiatmaja na, setijanto rd. the effect of identification and management of dental health problems on kindergarten and elementary school teachers knowledge levels in keputih public health center (puskesmas). indones j dent med. 2020; 2(1): 16–8. 21. bramantoro t, santoso cma, hariyani n, setyowati d, zulfiana a a, nor na m, nag y a, p rat a mawa r i dn p, i r ma l ia w r. effectiveness of the school-based oral health promotion programmes from preschool to high school: a systematic review. plos one. 2021; 16(8): e0256007. 22. raiyanti iga, ratmini nk, nyoman n, supariani d. perawat gigi dalam pelaksanaan program ukgs di puskesmas kabupaten badung tahun 2015. j kesehat gigi. 2017; 5(2): 42–51. 23. pradnyadani iga. revitalisasi usaha kesehatan gigi sekolah (ukgs) dalam meningkatkan kesehatan gigi dan mulut anak usia sekolah. j kesehat gigi. 2014; 2(1): 190–4. 24. g e et ha p r iya pr, asoka n s, ja na n i rg, k a nd aswa my d. effectiveness of school dental health education on the oral health status and knowledge of children: a systematic review. indian j dent res. 2019; 30(3): 437–49. 25. geetha priya pr, asokan s, kandaswamy d, shyam s. impact of different modes of school dental health education on oral healthrelated knowledge, attitude and practice behaviour: an interventional study. eur arch paediatr dent. 2020; 21(3): 347–54. 26. marliny, hasnita e, silvia. analisis pelaksanaan pelayanan usaha kesehatan gigi sekolah (ukgs) di masa pandemi covid-19. j hum care. 2021; 6(3): 541–50. 27. ari agung iga, wedagama dm, hervina. dokter gigi cilik dengan taman sirih (implementasi konsep tri hita karana). denpasar: unmas press; 2021. p. 9. 28. world health organization. unesco and who urge countries to make every school a health-promoting school. 2021. available from: https://www.who.int/news/item/22-06-2021-unesco-and-whourge-countries-to-make-every-school-a-health-promoting-school. accessed 2021 nov 19. 29. wulandari nnf, handoko sa, kurniati dpy. determinan perilaku perawatan kesehatan gigi dan mulut pada anak usia 12 tahun di wilayah kerja puskesmas i baturiti. intisari sains medis. 2018; 9(3): 55–8. 30. nicksic ne, massie aw, byrd-williams ce, kelder sh, sharma s v, butte nf, hoelscher dm. dietary intake, attitudes toward healthy food, and dental pain in low-income youth. jdr clin transl res. 2018; 3(3): 279–87. 31. stein c, santos nml, hilgert jb, hugo fn. effectiveness of oral health education on oral hygiene and dental caries in schoolchildren: systematic review and meta-analysis. community dent oral epidemiol. 2018; 46(1): 30–7. 32. inglehart mr, zuzo ga, wilson jj. kindergarten/elementary school teachers and web-based oral health-related resources: an exploration. oral health prev dent. 2017; 15(3): 229–36. 33. thomson s, cylus j, evetovits t. can people afford to pay for health care? new evidence on financial protection in europe. switzerland: world health organization. regional office for europe; 2019. p. 119. 34. bernabé e, masood m, vujicic m. the impact of out-of-pocket payments for dental care on household finances in low and middle income countries. bmc public health. 2017; 17(1): 109. 35. anwar ai. buku ajar ilmu kesehatan gigi masyarakat: teori dan praktik penyuluhan. ester m, editor. jakarta: egc; 2019. p. 20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p215–220 https://www.kemkes.go.id/ https://www.who.int/news/item/22-06-2021-unesco-and-who-urge-countries-to-make-every-school-a-health-promoting-school https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p215-220 200200 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 200–203 original article gender differences in cephalometric angular measurements between boys and girls helsa alyayuan, johan arief budiman department of orthodontics, faculty of dentistry, universitas trisakti, jakarta, indonesia abstract background: gender determination is an important aspect of human biologic profile identification. the human skull is part of the body that has many gender indicators. lateral cephalogram is used for human skull analysis because of its morphological biologic details, including gender. purpose: the objective of this study was to determine the difference of angular measurements, those are sella-nasion-point a (sna), sella-nasion-point b (snb), point a-nasion-point b (anb), gonial, mandibular plane, glabella-metopion and sella-nasion (gm-sn), glabella-metopion and frankfort horizontal plane (gm-fhp), and glabella-metopion and basion-nasion (gm-ban) angles measurement’s results between boys and girls aged 8-12 years. methods: this study was an observational analytic on cephalometric radiographs in children aged 8-12 years from july-december 2018 using 54 samples from the faculty of dentistry universitas trisakti’s oral and dental hospital radiology installation. landmarks determination and angular measurement were digitized. the data were analyzed to a univariate test followed by a statistical test using the independent t-test. results: the independent t-test showed there are no differences between boys’ and girls’ angular measurement results (p > 0.05). conclusion: there are no differences in the angular measurements results between boys and girls aged 8-12 years. keywords: angular measurement; gender; lateral cephalogram correspondence: johan arief budiman, department of orthodontics, faculty of dentistry, universitas trisakti. jl. kyai tapa no. 260, jakarta, 11440, indonesia. email: johanarief@trisakti.ac.id introduction gender determination is the most important aspect of medico-legal cases and anthropological research. identification techniques such as facial reconstruction will be difficult to perform if gender determination is not carried out correctly.1 therefore, separating and assessing gender manifestations is an integral part of identifying human skeletons. in forensic and physical anthropology, elements of the human skeleton play an important role in gender determination. the human skull has many excellent indicators of gender although the pelvis is considered a gender indicator for craniofacial growth.2 lateral cephalometrics are one of the tools used for the human skull analysis because it can provide information on anatomical points in one radiography and show morphological details for evaluation including various anatomical points and morphological details for gender determination.3,4 lateral cephalometrics are becoming popular in orthodontics and are used for craniofacial assessment which assists in the determination of diagnosis and treatment planning.5 in determining the gender of an incomplete skull, the lateral cephalometric has an important role because it can provide details of skull morphology and thus aid in identifying the characteristics of the skull.1 since broadbent and hofrath’s introduction in 1931, the cephalometric has become an important tool in assisting diagnosis in orthodontic treatment planning cases. its main advantage is that it can provide facial and tooth relationships measurements, identification and classification of bone and tooth disorders, and assessment of facial growth.6 lateral cefalometrics were used in many growth studies before being recognized for their role in determining the diagnosis.7 one analysis that has an important role in diagnostics the orthodontic routine is an angular measurement.8 angular measurements on the face have more informative than linear measurements. faces vary in size from person to person and it is difficult to assign specific directions to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p200–203 mailto:johanarief@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p200-203 201 alyayuan and budiman/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 200–203 linear measurements.7 angular measurement of faces can provide information about the variability in facial profiles concerning biological profiles including gender.9 one of the angles that can be measured from the cephalogram is the gonial angle. several studies have shown that the gonial angle is a parameter with acceptable accuracy and precision for determining gender. however, in several studies found that there was no significant difference in the measurement of the gonial angle between men and women with a mean of 122.17o for men and 124.99o for women.10 apart from the gonial angle, several other angles, sellanasion-point a (sna), sella-nasion-point b (snb), point a-nasion-point b (anb), and the mandibular plane angle are also used in several studies such as research conducted by mathur et al.1 the results of this study indicate that there is a difference in the gonial angle (124.766o in women and 119.96o in men) and the mandibular plane angle (28.133o in women and 24.533o in men) with women being greater than men. other measurements show that there is no significant difference between men and women, namely sna with a mean of 81.316o for men and 80.4o for women, snb with a mean of 77.35o for men and 77o for women, and anb with 3.9o in men and 3.6o in women. although, in general, the skeleton does not manifest gender characteristics until puberty, in establishing the gender identity from a defleshed skull, lateral cephalometrics and posteroanterior view radiographs assume a predominant role, as they can provide architectural and morphological details of the skull, thereby revealing additional characteristics and multiple points for comparison. various researchers have claimed that the gender identification by skull radiographs is a reliable method with up to 80–100% accuracy.1 in a study conducted on the kuruba caste in india. the angle of the intersection of the glabella-metopion (gm) and sella-nasion (sn) lines obtained an average angle measurement of 96.5o in men and 92.7o in women.2 while in the measurement of the intersection angle of the glabellametopion (gm) and frankfort horizontal plane (fhp) lines, it was found an average of 77.8o in men and 75.8o in women. then, at the intersection angle of the glabella-metopion (gm) and basion-nasion (ban) lines, it was obtained an average of 104.6o in men and 102.2o in women.2 therefore, the objective of this research was to find out the differences in cephalometric angular measurements, sna, snb, anb, gonial, mandibular plane, gm-sn, gm-fhp, and gm-ban angles between boys and girls aged 8-12 years at our oral and dental hospital. materials and methods this is an observational analytic study on the lateral cephalometrics of patients. the population in this study were all lateral cephalometrics which were produced from the veraviewepocs 2d panoramic machine (morita®japan) for patients aged 8-12 years at our oral and dental hospital. which was conducted at the radiology installation of our oral and dental hospital from july-december 2018. the population in this study were all lateral cephalometrics which were produced from the veraviewepocs 2d panoramic machine (morita®-japan) at our oral and dental hospital. the sample size was determined using the lemeshow formula. based on the formula, the minimum number of samples is 26. the taken samples are 27 for each gender. the total lateral cephalometrics samples were digitized using i-dixel 2.0 software (morita®, japan) on a pc hp® i7 (8700) gtx1060 with an hp® 23f monitor with 1920 x 1080 resolution. the anatomical reference point was determined on the lateral cephalometrics. those were sna, snb, anb, gonial, mandibular plane, gm-sn, gm-fhp, and gm-ban angles. a line was drawn (figure 1), then the angles in the study were measured. anatomical landmark determination, tracing, and angular measurements were carried out twice at different time intervals to avoid fatigue. all measurement results are analyzed and tabulated according to the name and angular measurement and gender. the data were analyzed using shapiro-wilk normality test, the cronbach’s alpha reliability test and a univariate test using the independent t-test (p<0.05). results the study was conducted on 54 samples of lateral cephalometrics of children aged 8-12 years old. information was obtained by collecting the lateral cephalometrics angular measurements and gender. the sample distribution can be seen in table 1. landmarks determination and angular measurement (sna, snb, anb, gonial, mandibular plane, gm-sn, gm-fhp, and gm-ban angles) were carried out twice at different times to minimize errors. the two assessment results are carried out by a normality test to find out whether the data are distributed normally. from figure 1. landmarks and lines for angular measurements. the normality test, it can be seen that the two assessment dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p200–203 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p200-203 202alyayuan and budiman/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 200–203 results are normally distributed (p>0.05). the normality test can be seen in table 2 then an internal reliability test was carried out on both assessments using the cronbach’s alpha method to determine the consistency of the assessment for each angular measurement according to gender. the reliability test can be seen in table 3. furthermore, the homogeneity test was carried out on the two assessments. the results of the homogeneity test showed that in the first measurement there was one angle where the assessment was not homogeneous, the glabella-metopion (gm) and sellanasion (sn) lines (p<0.05) while in the second assessment all angular measurements showed homogeneous results (p>0.05). the homogeneity test can be seen in table 4. table 2. shapiro-wilk normality test on 1st and 2nd assessment angular measurement gender degree of freedom p* 1 2 sna boys 27 0.387 0.685girls 27 0.559 0.839 snb boys 27 0.880 0.938girls 27 0.167 0.299 anb boys 27 0.607 0.123girls 27 0.376 0.880 gonial boys 27 0.861 0.623girls 27 0.161 0.996 mandibular plane boys 27 0.863 0.148girls 27 0.951 0.66 gm-sn boys 27 0.121 0.697girls 27 0.71 0.662 gm-fhp boys 27 0.237 0.760girls 27 0.65 0.613 gm-ban boys 27 0.179 0.394girls 27 0.088 0.737 *p=0.05 table 1. sample distribution gender age total8 9 10 11 12 boys 0 8 9 5 5 27 girls 0 9 10 7 1 27 total 0 17 19 12 6 54 table 3. reliability test on 1st and 2nd assessment assessment gender cronbach’s alpha* 1 boys 0.445girls 0.502 2 boys 0.512girls 0.704 *p=0.05 table 4. homogeneity test on 1st and 2nd assessment angular measurement p* 1 2 sna 0.768 0.279 snb 0.741 0.938 anb 0.806 0.248 gonial 0.985 0.554 mandibular plane 0.844 0.113 gm-sn 0.022 0.66 gm-fhp 0.078 0.326 gm-ban 0.060 0.284 *p=0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p200–203 the test results showed that the first assessment data was not homogeneous at the gm-sn angle (p=0.022), whereas, in the second assessment, all of the angular measurements showed homogeneous data (p>0.05). from the results of the three univariate tests carried out, only the second measurement result data can be continued for a different test with an independent t-test to determine whether there is a difference in the results of angular measurement between genders. discussion digital analysis is slowly replacing the manual method because it uses less time, can modify the size and contrast as needed, and solves the problem of film damage over time, which reduces the information on a radiograph.11,12 the landmarks determination followed by angular measurement was carried out twice at different times to minimize errors. after the second assessment, the normality test was carried out to determine whether the data were normally distributed. the normality test used the shapiro-wilk method because of the small number of samples for each man and woman (27 samples). the test results showed that in the 1st and 2nd data assessment, all angular measurements for each gender were normally distributed (p>0.05).13 after that, the internal reliability test, using the cronbach’s alpha method, was assessed on the two-assessment data. the result showed that both assessments are statistically reliable for each gender, with no significant difference (p>0.05). gender determination of an unknown human skeleton is important information needed in the development of the identification of a human biological profile.14 the skull is the second-best accurate skeleton after the pelvis in gender determination because it consists of hard tissue so that the skull is the most widely available part for forensic examination.15 lateral cephalometrics is one of the tools used in identifying gender. the lateral cephalometrics shows morphological details of the skull architecture on a radiograph.16 in previous research conducted by mathur et al.1 among young adults in nashik, india, there was no significant difference in the measurement results of sna (p = 0.369), snb (p = 0.71), and anb (p = 0.6) measurement results. the men are bigger than the women. meanwhile, in the gonial and mandibular plane angles, there were significant differences (p <0.05) with women being greater than men.1 another study concluded that the gonial angle of the digital lateral cephalometrics has the same result.17 according to previous study, in cases where an intact skull is not found, gender can be predicted by analyzing the mandible because it is the most dimorphic skull bone.1 one that can be analyzed on the mandibular is the gonial angle. several studies in south africa, europe, america, and egypt prove that the gonial angle is gender dimorphic.1,17 the results of this research conducted whose race was unknown showed similarities at the sna (p = 0.563), https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p200-203 203 alyayuan and budiman/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 200–203 nashik: a lateral cephalometric study. j adv med dent sci. 2014; 21–5. 2. devang divakar d, john j, al k heraif aa, mavinapalla s, ramakrishnaiah r, vellappally s, hashem mi, dalati mhn, durgesh bh, sa fadi r a, a n il s. sex deter m ination using discriminant function analysis in indigenous (kurubas) children and adolescents of coorg, karnataka, india: a lateral cephalometric study. saudi j biol sci. 2016; 23(6): 782–8. 3. datta a, chandrappa siddappa s, karibasappa gowda v, revapla channabasappa s, babu banagere shivalingappa s, dey d. a study of sex determination from human mandible using various morphometrical parameters. indian j forensic community med. 2015; 2(3): 158–66. 4. farhidnia n, soltani s, aghakhani k, salehi s, khloosy l, chehreii s, fallah f, memarian a. the value of lateral cephalometric variables measured by cephalogram in sex determining among iranians. glob j health sci. 2016; 9(6): 214. 5. qamruddin i, alam mk, shahid f, tanveer s, mukhtiar m, asim z. assessment of gender dimorphism on sagittal cephalometry in pakistani population. j coll physicians surg pak. 2016; 26(5): 390–3. 6. mehta p, sagarkar rm, mathew s. photographic assessment of cephalometric measurements in skeletal class ii cases: a comparative study. j clin diagn res. 2017; 11(6): zc60–4. 7. premkumar s. textbook of craniofacial growth. jaypee brothers medical publishers (p) ltd.; 2011. p. 396. 8. heil a, lazo gonzalez e, hilgenfeld t, kickingereder p, bendszus m, heiland s, ozga a-k, sommer a, lux cj, zingler s. lateral cephalometric analysis for treatment planning in orthodontics based on mri compared with radiographs: a feasibility study in children and adolescents. kleinschnitz c, editor. plos one. 2017; 12(3): e0174524. 9. adamu lh, ojo sa, danborno b, adebisi ss, taura mg. sex determination using facial linear dimensions and angles among hausa population of kano state, nigeria. egypt j forensic sci. 2016; 6(4): 459–67. 10. leversha j, mckeough g, myrteza a, skjellrup-wakefiled h, welsh j, sholapurkar a. age and gender correlation of gonial angle, ramus height and bigonial width in dentate subjects in a dental school in far north queensland. j clin exp dent. 2016; 8(1): e49-54. 11. kamath m, arun a. comparison of cephalometric readings between manual tracing and digital software tracing: a pilot study. int j orthod rehabil. 2016; 7(4): 135–8. 12. nava r ro r de l, oltrama r i-nava r ro pvp, fer nandes t mf, oliveira gf de, conti ac de cf, almeida mr de, almeida rr de. comparison of manual, digital and lateral cbct cephalometric analyses. j appl oral sci. 2013; 21(2): 167–76. 13. ghasemi a, zahediasl s. normality tests for statistical analysis: a guide for non-statisticians. int j endocrinol metab. 2012; 10(2): 486–9. 14. thomas rm, parks cl, richard ah. accuracy rates of ancestry estimation by forensic anthropologists using identified forensic cases. j forensic sci. 2017; 62(4): 971–4. 15. badam rk, manjunath m, rani m. determination of sex by discriminant function analysis of lateral radiographic cephalometry. kailasam s, editor. j indian acad oral med radiol. 2011; 23(3): 179–83. 16. binnal a, yashoda devi b. identification of sex using lateral cephalogram: role of cephalofacial parameters. j indian acad oral med radiol. 2012; 24: 280–3. 17. belaldavar c, acharya ab, angadi p. sex estimation in indians by digital analysis of the gonial angle on lateral cephalographs. j forensic odontostomatol. 2019; 37(2): 45–50. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p200–203 snb (p = 0.651), and anb (p = 0.797). there was a significant difference between the mean of the lateral cephalometrics angular measurement between genders. the gonial and mandibular planes showed different results. in the average measurement of the gonial angle, showed that there is no significant difference (p = 0.789), and the mean measurement of women is greater than that of men. while, the results of the mandibular plane angle measurement showed very different results, because the mean measurement of men was greater than that of women with no significant difference (p = 0.272). previous research conducted on a population of children and adolescents of the kurubas caste in india showed a significant difference (p <0.05) between the results of the angular measurement between gender.2 the angular intersection of the glabella-metopion (gm) and sella nasion (sn) lines, glabella-metopion (gm), and frankfort horizontal plane (fhp), as well as glabella-metopion (gm) and basion-nasion (ban), have the average measurement of men being greater than the women. meanwhile, the other research showed that the measurement results of women’s angular measurement were greater than men’s at the three angles of gm-sn, gm-fhp, and gm-ban with significant differences (p< 0.05).15 the results of this study were in line with research on the kurubas caste in india, the gm-sn angle (p = 0.484) and gm-fhp (p = 0.637), with the average measurement result for men, is greater than that for women, but statistically insignificant.2 however, the gm-ban angle (p = 0.582) showed different results, with women greater than men with no statistically significant difference. the differences in the results of previous studies with this study are due to differences in various races.1 gender differentiation in one human group may not be the same in another group. factors such as genetics, environmental conditions, socioeconomic status, daily diet, and physical activity allow for differences among populations.1,2 those factors are not the part of this study. this research can be concluded that there is no difference in the angular measurement of sna, snb anb, gonial, mandibular plane, gm-sn, gm-fhp, and gm-ban between boys and girls in children aged 8-12 years old. this study can be developed with more specific techniques and methods to assess gender differentiation on angular lateral cephalometrics measurements. references 1. m athur ru, mahajan am, dandekar rc, patil rb. determination of sex using discriminant function analysis in young adults of https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p200-203 mkgs vol 45 no 2 april-juni 2012.indd 114 volume 45 number 2 june 2012 research report seroprevalence of herpes simplex virus types 1 and 2 and their association with cd4 count among hiv-positive patients irna sufiawati1, sunardhi widyaputra2, and tony s. djajakusumah3 1department of oral medicine, faculty of dentistry, university of padjadjaran 2department of oral pathology, faculty of dentistry, university of padjadjaran 3department of dermatology & venereology, faculty of medicine, university of padjadjaran bandung indonesia abstract background: herpes simplex virus (hsv) is a common cause of viral opportunistic infections among hiv-positive patients. frequent, more severe and prolonged episodes of recurrent hsv infection can be a source of significant morbidity and mortality among hiv-positive patients with advanced immunosuppression, reflected by low cd4 count. however, conflicting results have also been reported. purpose: the aim of this study was to investigate the seroprevalence of hsv type 1 (hsv-1) and type 2 (hsv-2) in hiv-positive patients compare with the rate in hiv-negative patients, and to evaluate their association with cd4 count. methods: a cross sectional study was conducted among 145 subjects consisting of 80 hiv-positive and 65 hiv-negative patients attending the top referral hospital in bandung, west java, indonesia. the serum obtained was assayed for the presence of hsv-1 and hsv-2 igg antibodies using elisa kits. data were analyzed using a chi-square test, t-tests and analysis of variance (anova). results: there were no significant differences in hsv-1 seroprevalence between hiv-positive patients (71%) and hiv-negative patients (66%). hsv2 seroprevalence was significantly higher in hiv-positive patients (30%) than hiv-negative patients (5%). the titers of hsv-1 igg antibodies in hiv-positive patients (mean 24.63 ± 19.06 idu) were significantly lower than those of hiv-negative patients (mean 44.62 ± 33.22 idu). in contrast, hsv-2 igg antibody titers in hiv-positive patients (mean 13.31 ± 20.28 idu) were significantly higher than hiv-negative patients (mean 4.42 ± 10.99 idu). there was no significant correlation between hsv-1 and hsv-2 seropositivity and cd4 count among hiv-positive patients. however, most of hsv-2 seropositive patients had cd4 count < 200 cells/mm3. conclusion: seroprevalence of hsv-1 and hsv-2 among hiv-positive patients was high with no correlation with cd4 count. key words: hsv, igg, hiv, cd4 abstrak latar belakang: herpes simplex virus (hsv) adalah penyebab infeksi virus oportunistik yang paling umum pada pasien hivpositif. infeksi hsv rekuren yang sering terjadi, lebih berat, dan episode yang berkepanjangan dapat menjadi penyebab morbiditas dan mortalitas yang signifikan pada pasien hiv-positif dengan imunosupresi lanjut, ditandai dengan jumlah cd4 yang rendah. namun, hasil yang bertentangan juga telah dilaporkan. tujuan: penelitian ini bertujuan untuk mengetahui seroprevalensi hsv tipe 1 (hsv-1) dan tipe 2 (hsv-2) pada pasien hiv-positif dibandingkan dengan pasien hiv-negatif, dan untuk mengevaluasi hubungannya dengan jumlah cd4. metode: penelitian potong lintang ini dilakukan pada 145 subjek yang terdiri dari 80 pasien hiv-positif dan 65 pasien hiv-negatif yang berkunjung ke rumah sakit pusat rujukan di bandung, jawa barat, indonesia. antibodi igg hsv-1 dan hsv2 di dalam serum diperiksa dengan menggunakan elisa. data dianalisis dengan uji chi-square, t-test dan anova, nilai p < 0.05 dianggap signifikan secara statistik. hasil: seroprevalensi antibodi igg hsv-1 pada pasien hiv-positif (71%) tidak berbeda secara signifikan dengan pasien hiv-negatif (66%). namun, seroprevalensi hsv-2 secara signifikan lebih tinggi pada pasien hiv-positif (30%) dibandingkan dengan pasien hiv-negatif (5%). titer antibodi igg hsv-1 pada pasien hiv-positif (mean 24.63 ± 19.06 idu) secara signifikan lebih rendah dibandingkan pasien hv-negatif (mean 44.62 ± 33.22 idu). sedangkan, titer antibodi igg hsv-2 pada pasien hiv-positif (mean 13.31 ± 20.28 idu) secara signifikan lebih tinggi dibandingkan pasien hiv-negatif (mean 4.42 ± 10.99 idu). tidak ada hubungan yang signifikan antara seropositivitas hsv-1 dan -2 dengan jumlah cd4. namun, sebagian besar pasien 115sufiawati, et al.: seroprevalence of herpes simplex virus types 1 and 2 and their association introduction the increase in the immunocompromised populations due to hiv infection is a factor that can contribute to the change in epidemiology of herpesviruses-associated disease. the hallmark of herpesviruses infection is the ability to establish latent, life-lasting, and periodically reactivating infections in the host. the immunosuppressive state induced by hiv-1 may facilitate herpes viruses’ reactivation or re-infection.1 on the other hand, it is growing evidence that herpevirus infection may increase an pasienal’s susceptibility to hiv infection,2 and could interact with hiv to accelerate disease progression.3,4 to date, there are eight known human herpes viruses, they are herpes simplex virus type 1 (hsv-1), hsv-2, varicella-zoster virus (vzv), and cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpesvirus-6, human herpesvirus-7, and hhv-8 (kaposi’s sarcoma herpes virus).5 among herpesviruses family members, hsv is the most common co-infection and pathogenic in hiv-1-positive patients.6 herpes simplex viruses (hsv), an alpha-herpesvirus, are categorized into two types, herpes type 1 (hsv-1) and herpes type 2 (hsv-2). for each virus, the primary mode of transmission is different and there is a tendency to infect different anatomical sites, causing a wide variety of mucocutaneous infections. hsv-1 is mainly localized around the oral region and hsv-2 around the genital region, however it is quite possible to transmit the virus to either region. the prevalence of hsv-1 infection in general populations is high in most geographic areas worldwide ranges from roughly 65% to 90% and has been found to be higher than hsv-2 infection.7,8 studies from different parts of the world demonstrated that rates of hsv-1 infection were much higher in hiv-positive patients or with a high risk of hiv than in the general population. the prevalence rates of hsv type 1 (hsv-1) among hiv-1 infected people ranging from 90% to 100%.9,10 hsv-2 prevalence is highly variable and depends on many factors, including country and region of residence, population subgroup, sex, and age. prevalence of hsv-2 infection in the general population in developing asian countries appears to be lower (10–30%) than developed regions.11 in the united states, hsv-2 seroprevalence was 16.2%.12 hsv-2 seroprevalence in central and south america are estimated at 20% to 60%.7,11 in europe, hsv-2 seropositivity varies by region ranging from 4.2% to 23.9%.13 sub-saharan africa has the highest hsv-2 seroprevalence in the world, reaching 80% in adult population.7 hsv type 2 affects 50–90% of hiv1infected people higher than in the general population.9,11,14 hsv-2 infection reported as a major risk factor for hiv acquisition.15 a meta-analysis of the association between hsv-2 infection and risk of hiv-1 acquisition reviewed 31 studies have demonstrated that prevalent hsv-2 is associated with a 2to 4-fold increased risk of hiv-1 acquisition.16,17 these epidemiological evidence indicated a strong relationship exist between hsv-2 and hiv. previous studies have demonstrated that hsv infections are associated with a compromised immune system in hiv-positive patients. hoots et al.5 reported that there was a statistically significant association between hsv seropositivity and the degree of immunosuppression, as reflected by cluster difference 4 (cd4) count. other studies showed that in hiv-positive patients, asymptomatic hsv shedding increases with lower cd4 count.36,37 another study also confirmed that risk factors for increased hsv shedding among hiv-positive men were low cd4 cell count.38 however other studies have shown conflicting results, santos et al.39 reported a weak and statistically nonsignificant association of hsv and cd4 count. patients with hsv infection can present with severe manifestations even after their cd4 count increases to > 500 cells/mm3.40 it has been suggested that immune reconstitution inflammatory syndrome (iris), usually occurs in individuals with a rapidly rising cd4 count, associated with severe hsv lesions after haart initiation. due to the apparent evolving epidemiological trends of herpesviruses infection in hiv-positive people, this study was conducted to assess the seroprevalence of hsv-1 and hsv-2, and their correlation with cd4 count among hiv-positive patients in bandung, west java, indonesia. since more herpesviruses infections are asymptomatic, the seroepidemiological studies are critical in understanding the pattern and distribution of infection, which have not been previously investigated among hiv-positive patients in west java, indonesia. materials and methods data were collected in a cross-sectional study from january until march 2012 in a referral hospital in bandung, west java. we recruited 80 patients who were diagnosed as hiv-positive patients. we also enrolled 65 sex and agematched healthy volunteers as controls. ethical clearance was obtained from the institutional review board of the seropositif hsv-2 memiliki jumlah cd4 < 200 sel/mm3. kesimpulan: seroprevalensi hsv-1 dan hsv-2 pada pasien hiv-positif adalah tinggi, tetapi tidak berkorelasi dengan jumlah cd4. kata kunci: hsv, igg, hiv, cd4 correspondence: irna sufiawati, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan no.1 bandung 40132. e-mail: irnasufiawati@yahoo.com. fax: +62222532805. 116 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 114–120 ethical committee hasan sadikin hospital and patients gave written informed consent for participation. samples of venous blood (5 ml) was drawn from all the enrolled subjects into edta blood collection tubes and immediately kept at +4° c. hiv status was confirmed by enzyme-linked immunosorbent assay (elisa). cd4 testing was done using a bd facscount™ cytometer. the sera were obtained on the same day and stored at 20° c freezer in aliquots until tested. the presence of igg antibodies against hsv-1 and hsv-2 were examined using elisa kits (indec diagnostic, indonesia), in accordance with the manufacturer’s instructions. positive and negative standard sera, accompanying the kit were included in each assay. data were entered and analyzed in spss 11.0 for windows. differences in hsv-1 and -2 seropositivity rates among different groups were evaluated using the chi-square test. mean titer levels were compared between hiv-positive patients and hiv-negative controls using two sample t-tests. the statistical significance of correlation between hsv-1 and hsv-2 igg titers with cd4 count was obtained using anova. p-values < 0.05 were considered statistically significant. the mean, median, mode and standard deviation has also been done by using the same software. results of all 145 subjects included in the analysis, the hiv group comprised 80 hiv-positive patients (42 male and 38 female), the mean age was 30.8 + 8.3 years (median 31.5 years, range 1-55). the control group consisted of 65 hivnegative patients (34 male and 31 female), the mean age was 29.1 + 12.1 years (median 28 years, range 1-56 years). there was no statistically significant different between hiv-positive patients and hiv-negative control in age (p > 0.05) and gender distributions (p > 0.05). the majority of hiv-positive patients were adequately controlled as determined by cd4 count ranging from (mean 393.4 + 210.8 cells/mm3) (table 1). the results showed that seroprevalence of hsv-1 igg was found slightly higher in hiv-positive patients (71%) than in hiv-negative patients (63%), however the different was no statistically significant (p > 0.05). while, we found that hsv-2 seroprevalence was significantly higher in hiv-positive than hiv-negative patients (30% vs. 5%, respectively; p < 0.05). hsv-1 and-2 igg antibodies were not found in 34% hiv-negative patients and in 20% hiv-positive patients. further, we identified that of all subjects there were a number of subjects gave a positive test for both hsv-1 and-2 igg antibodies. out of 80 hivpositive patients, 21% of them have both hsv-1 and-2 igg antibodies, significantly higher (p < 0.05) than those of hiv-negative patients (3%) (figure 1). immunoglobulin g antibodies against to hsv-1 are more frequently found in hiv-positive patients with cd4 count > 500 cells/mm3. in contrast, many hiv-positive patients who were hsv-2 seropositive had cd4 count < 200 cells/mm3. in detail, hsv-1 igg antibodies were found in 13% of hiv-positive patients with cd4 count < 200 cells/mm3, 22% of them have cd4 count ranging from 200 to 349 cells/mm3, 28% of them have cd4 count 350-499 cells/mm3, and many of them (37%) have cd4 count more than 500 cells/mm3. in contrast, only 17% of hiv-positive patients with cd4 count > 500 cells/mm3 had hsv-2 igg antibodies, 28% of them have cd4 count table 1. characteristics of study participants characteristics hiv-positive patients (n = 80) hiv-negative patients (n = 65) p age (yeard old) mean ± sd (range) 29 ± 13 (1-58) 31 ± 8 (1-55) 0.24 median 28 32 gender (%) female 38 31 0.37 male 42 34 0.42 cd4 counts (cell/mm3) mean ± sd (range) 394 ± 209 nd – median 393 note: n: number of subjects; nd: not determined; *p < 0.05, statistically significant figure 1. seroprevalence of igg antibody against hsv-1 and hsv-2 among hiv-positive and hiv-negative patients. 117sufiawati, et al.: seroprevalence of herpes simplex virus types 1 and 2 and their association < 200 cells/mm3, 22% of them have cd4 count 200-349 cells/mm3, and 33% of them have cd4 count 350-499 cells/mm3 (figure 2). there were no significant correlation between hsv-1 and hsv-2 seropositivity and cd4 count (p > 0.05). the mean titer of igg antibodies against both two herpes simplex viruses were statistically significantly different compared to hiv-negative control group. the titer of hsv-2 igg antibodies were detected significantly higher (p < 0.05) in hiv-positive patients compared with hivnegative patients. in contrast, the titers of hsv-1 igg antibodies in hiv-positive patients were significantly lower (p < 0.05) than in hiv-negative patients. in addition, table 2 showed that there were no significant correlation between the titer of both hsv-1 and hsv-2 igg antibodies and cd4 count (p > 0.05). however, when we used post hoc analysis (2-tail p-values for pairwise independent groups t-tests), we found a significant different in the titer of hsv2 between hiv-positive patients with cd4 t-cell count < 200 cells/mm3 and those patients with cd4 count > 500 cells/mm3 (p = 0.0233). discussion to our knowledge, this is the first study to compare the seroprevalence of hsv type 1 and 2 antibodies in hiv-positive and hiv-negative patients in a large public referral hospital serving the urban and surrounding rural area in bandung, west-java, the province with the highest burden of hiv in indonesia. we were particularly interested in determining the seroprevalence of these herpes viruses in hiv-positive patients and their associations with immune status as measured by cd4 count, since there is little known about this. our study demonstrated that overall igg antibodies againts hsv-1 and hsv-2 were more prevalent in both hiv-positive than hiv-negative patients (tabel 1). however, result from statistical analysis showed that only hsv-2 seroprevalence rates were significantly higher in hiv-positive than in hiv negative patients. in this study, seroprevalence of hsv-1 igg was found slightly higher in hiv-positive than in hiv negative patients. this results were not much different with other studies that showed the prevalence of hsv-1 infection in general populations worldwide is high ranges from roughly 65% to 90%.7,8 the high prevalence of hsv-1 clearly shows that most people are infected with some type of infection at least once in their lifetime. most of them are asymptomatic during the initial stage, they remain undiagnosed for long periods of time or even throughout the life. however, the seroprevalence rates of hsv-1 in hiv-positive patients in our study lower than in the other figure 2. distribution of hsv-1 and hsv-2 seropositive patients by the rate of cd4 count. table 2. the titers of igg antibody against hsv-1 and hsv-2 and their correlation with cd4 count virus type virus titers (idu) p hiv-positive patients hiv-negative patients hsv-1 mean ± sd 24.63 ± 19.06 44.62 ± 33.22 0.0000118 median 21.8 52.6 hsv-2 mean ± sd 13.31 ± 20.28 4.42 ± 10.99 0.0019 median 2.1 2 cd4 t-cell counts (cells/mm3) p < 200 200 – 349 350 – 499 ≥ 500 hsv-1 mean ± sd 23.14 ± 22.789 20.38 ± 17.734 21.09 ± 17.734 25.69 ± 17.774 0.8013 median 13.85 17.45 22 24.1 hsv-2 mean ± sd 22.76 ± 27.392 11.18 ± 18.647 13.43 ± 21.351 6.12 ± 11.675 0.1665 median 7.25 1.5 2 1.8 note: sd: standard deviation; id u (indec units); *p < 0.05, statistically significant 118 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 114–120 epidemiological studies carried out in various countries within the range of 90–100%.9,10,18 the high prevalence of hsv-1 igg antibody in hiv-positive patients than in general population is possibly because many of them were acquired hsv-1 through sexual activities. it is well known that hsv-1 is usually acquired orally during childhood via non-sexual contacts, however the virus can be transmitted also through sexual contacts. hsv-2 is the main causative agent of genital herpes worldwide.19 however, recent data reported that hsv-1 has also emerged as a major causative agent of genital herpes in some developed countries,20,21 and the increase in the frequency of genital herpes caused by hsv-1 compared with genital hsv-2 infection.22,23 the results of this study also showed that hsv-1 igg seroprevalence was higher than hsv-2 in both groups which is in agreement with the reported results from different regions of the world where the prevalence of hsv-1 prevalence is almost always greater than hsv2 prevalence.7 a recent study also reported a higher prevalence rate of igg antibody against hsv-1 than hsv-2 in both groups.24 hsv-1 infections usually occur earlier in life. it has been suggested that a high percentage of hsv-2 antibodies because of prior hsv-1 protected from subsequent hsv-2 infection. in developed countries, while childhood acquisition of hsv-1 has decreased, hsv-2 seroprevalence has increased, suggesting the possible protective effect of hsv-1 against hsv-2 infection.24 a prior infection with hsv-1 has an acquired immune response that confers moderate protection against getting hsv-2, and reduces its severity.25 a study reported that previous hsv type 1 infection appeared to reduce the risk for acquisition of hsv type 2 infection by 40%.26 it is not known whether previous genital hsv-1 infection modifies the risk of hsv-2 acquisition more substantially than previous oral hsv-1 infection.27 however, hsv-1 infections are still at risk of hsv-2 acquisition. there are conflicting results from studies on the risk of hsv-1 positive patients of acquiring hsv-2 that are reported that previous hsv-1 infection does not reduce susceptibility to subsequent genital hsv-2.28 furthermore, our results indicate statistically significant a higher prevalence of hsv-2 igg in hiv-positive patients when compared to 5% hiv-negative patients. this finding is consistent with most previous studies in other countries. hsv-2 infection is present in 30 to 70% of those in europe and 50 to 90% of those in africa among patients with hiv infection.16 a study reported that the hiv-positive men shed hsv-2 orally more frequently than did the hiv-negative men.29 however, other studies have identified that genital shedding of hsv-2 is higher in hiv-positive patients.30 the higher rate of hsv-2 seroprevalence of hsv-2 among hiv-aids patients because hsv-2 is transmitted via sexual contact with an hiv-positiveperson. several studies demonstrated that behavioral and sexually transmitted infection (stis) as predictors of hsv-2 acquisition.16,17,31 primary genital hsv-2 occurring in an hiv-1-infected person is a marker for unsafe sexual practices. genital ulcer caused by hsv-2 provides a site for hiv entry on hiv negative patients and the associated inflammation increases the number of activated cells that can be targeted by hiv. in contrast, many hiv-1-infected patients are already infected with hsv-2 at the time of hiv-1 acquisition and having herpes doubled the risk of subsequently catching hiv. epidemiological studies found that at least a 2to 4-fold increased risk of acquiring hiv among patients infected with hsv-2,16,32 and may account for 40–60% of new hiv infections in high hsv-2 prevalence populations.2,31-33 susceptibility to hiv is higher among patients who have recently acquired hsv-2. nonetheless, it also found an increased risk of hiv infection even when herpes infection appeared dormant or was causing no symptoms.34 the high prevalence and incidence of hsv-2 infection among hiv-positive patients compared with the general population suggests a critical need for screening and preventive programs among this targeted group. this would be of help in prevention of hiv infection. it has been stated that more frequently virus infections are associated with a compromised immune system in hiv-positive patients. prior study confirmed that immunesuppressed patients are more vulnerable to common virus infections.4 our findings showed many hiv-positive patients who were hsv-1 seropositive had cd4 count more than 500 cells/mm3. in contrast to hsv-1, many hiv-positive patients who were hsv-2 seropositive had cd4 count < 200 cells/mm3. some studies also reported that there were an association between hsv-2 seropositivity and cd4 count,35 but others have shown conflicting results.39 interestingly, when we analysis of a comparison of igg antibody titers, the results showed significantly higher titers of igg antibody against both hsv-1 and hsv-2 in hiv-positive patients compared to hiv-negative patients. we also found a significant different in the titer of hsv2 between hiv-positive patients with cd4 cell count < 200 cells/mm3 and those patients with cd4 count > 500 cells/mm3. however, we did not find significant correlation between the titer of both herpes viruses and cd4 count. there are some possibilities could explain this finding. first, hsv infection did not modulate the relationship of hiv-1 to cd4+ t cell count suggests that the effect of hsv-2 infection on cd4+ t cell count manifests prior to acquisition of hiv-1.41 second, it is suggested that cd8+ t-cells are a critical component of the response to hsv infection but not cd4+ t-cells. the level of anti-hsv antibody did not have any impact on the percentage or absolute number of late-differentiated cd4+ t-cells.42 in contrast, hsv-1 infection reduced the number of infiltrating cd8+ t cells.43 a study also confirmed that among hivpositive patients, the frequency of hsv-2 -specific cd-8 t cells is inversely related to hsv-2 severity.44 in conclusion, seroprevalence of hsv-1 and hsv-2 among hiv-positive patients was high with no correlation with cd4 count. this study may increase the understanding about the spread of herpes simplex virus and may be valuable for guiding prevention efforts of recurrent herpes 119sufiawati, et al.: seroprevalence of herpes simplex virus types 1 and 2 and their association simplex virus disease among hiv-positive patients. in addition, the detection of igg antibodies against herpes simplex virus may help seropositive people identify symptoms and protect their partners from acquiring hiv, or vice versa, protect hiv-positive patients from acquiring the most common viral opportunistic infection. acknowledgement we are grateful to sharaf m. tugizof, ph.d., dsc., at the university of california, san francisco (ucsf), usa, for his kind support and great expertise, and valuable suggestion in this study. we would like also to thank the staff of the clinical pathology laboratory, hasan sadikin hospital, bandung, indonesia, for their laboratory help. this work was supported by the directorate general of higher education, ministry of national education indonesia. references 1. fauci as. immunopathogenesis of hiv infection. j acquir immune defic syndr. 1993; 6(6): 655–62. 2. serwadda d, gray rh, sewankambo nk, wabwire-mangen f, chen mz, quinn tc, lutalo t, k iwanuka n, k igozi g, nalugoda f, meehan mp, ashley morrow r, wawer mj. human immunodeficiency vir us acquisition associated with genital ulcer disease and herpes simplex virus type 2 infection: a nested case-control study in rakai, uganda. j infect dis 2003; 188(10): 1492-7. 3. suligoi b, dorrucci m, uccella i, andreoni m, rezza g. effect of multiple herpesvirus infections on the progression of hiv disease in a cohort of hiv seroconverters. j med virol 2003; 69(2): 182–7. 4. shieh b, chang mj, ko wc, chen ej, wu jc, lee cf, shieh b, chang tt, li c. effects of multiple virus coinfections on disease progression in hiv-positive patients. intervirology. 2003; 46(2): 105–13. 5. straus se. introduction to herpesviridae. in: mandell gl, bennett je, dolin r, eds. principles and practice of infectious diseases. 6th ed. philadelphia: elsevier; 2005. p. 1756–62. 6. stewart ja, reef se, pellet pe. herpesvirus infections in persons infected with human immunodeficiency virus. clin infect dis 1995; 21 (suppl 1): s114–20. 7. smith js, robinson nj. age-specific prevalence of infection with herpes simplex virus types 2 and 1: a global review. j infect dis 2002; 186 (suppl 1): s3–28. 8. chayavichitsilp p, buckwalter jv, krakowski ac, friedlander sf. herpes simplex. pediatr rev 2009; 30(4): 119–29. 9. hill c, mckinney e, lowndes cm, munro h, murphy g, parry jv, gill on; gum anon network. epidemiology of herpes simplex virus types 2 and 1 amongst men who have sex with men attending sexual health clinics in england and wales: implications for hiv prevention and management. euro surveill 2009; 14(47): 19418. 10. romanowski b, myziuk ln, walmsley sl, trottier s, singh ae, houston s, joffe m, chiu i. seroprevalence and risk factors for herpes simplex virus infection in a population of hiv-positive patients in canada. sex transm dis 2009; 36(3): 165–9. 11. weiss h. epidemiology of herpes simplex virus type 2 infection in the developing world. herpes. 2004; 11 (suppl 1): 24a-35a. 12. xu f, sternberg mr, gottlieb sl, berman sm, markowitz le, forhan se, et al. seroprevalence of herpes simplex virus type 2 among persons aged 14-49 years united states, 2005-2008. mmwr 2010; 59(15): 456–59. 13. pebody rg, andrews n, brown d, gopal r, de melker h, françois g, gatcheva n, hellenbrand w, jokinen s, klavs i, kojouharova m, kortbeek t, kriz b, prosenc k, roubalova k, teocharov p, thierfelder w, valle m, van damme p, vranckx r. the seroepidemiology of herpes simplex virus type 1 and 2 in europe. sex transm infect. 2004; 80(3): 185–91. 14. bohl dd, katz ka, bernstein k, wong e, raymond hf, klausner jd, mcfarland w. prevalence and correlates of herpes simplex virus type-2 infection among men who have sex with men, san francisco, 2008. sex transm dis. 2011; 38(7): 617–21. 15. barnabas rv, wasserheit jn, huang y, janes h, morrow r, fuchs j, mark ke, casapia m, mehrotra dv, buchbinder sp, corey l; niaid hiv vaccine trials network. impact of herpes simplex virus type 2 on hiv-1 acquisition and progression in an hiv vaccine trial (the step study). j acquir immune defic syndr. 2011; 57(3): 238–44. 16. corey l, wald a, celum cl, quinn tc. the effects of herpes simplex virus-2 on hiv-1 acquisition and transmission: a review of two overlapping epidemics. j acquir immune defic syndr 2004; 35: 435–45. 17. wald a. herpes simplex virus type 2 transmission: risk factors and virus shedding. herpes 2004; 11 (suppl 3): 130a–137a. 18. luchsinger v, luzoro a, martínez mj. high seroprevalence of cytomegalovirus, herpes simplex type 1 virus and epstein barr virus infection among human immunodeficiency virus-infected adults. rev med chil. 2010; 138(7): 809–14. 19. tao g, kassler wj, rein db. medical care expenditures for genital herpes in the united states. sex transm dis. 2000; 27(1): 32–8. 20. nieuwenhuis rf, van doornum gj, mulder pg, neumann ha, van der meijden wi. importance of herpes simplex virus type-1 (hsv1) in primary genital herpes. acta derm venereol. 2006; 86(2): 129–34. 21. xu f, sternberg mr, kottiri bj, mcquillan gm, lee fk, nahmias aj, berman sm, markowitz le. trends in herpes simplex virus type 1 and type 2 seroprevalence in the united states. jama 2006; 296(8): 964–73. 22. roberts cm, pfister jr, spear sj. increasing proportion of herpes simplex virus type 1 as a cause of genital herpes infection in college students. sex transm dis 2003; 30: 797–800. 23. haddow lj, dave b, mindel a, mcphie ka, chung c, marks c, dwyer de. increase in rates of herpes simplex virus type 1 as a cause of anogenital herpes in western sydney, australia, between 1979 and 2003. sex transm infect 2006; 82: 255–9. 24. venkateshwaran sp, murugesan k, sivaraj r. seroprevalence of igg and igm antibodies in patients with herpes simplex virus -1 & 2 infection in hiv positive and negative patients of south indian population. j app pharm sci 2011; 01(10): 154–8. 25. nahmias aj, lee fk, beckman-nahmias s. sero-epidemiological and-sociological patterns of herpes simplex virus infection in the world. scand j infect dis suppl. 1990; 69: 19–36. 26. mertz gj, benedetti j, ashley r, selke sa, corey l. risk factors for the sexual transmission of genital herpes. ann intern med 1992; 116(3): 197–202. 27. sucato g, wald a, wakabayashi e, vieira j, corey l. evidence of latency and reactivation of both herpes simplex virus (hsv-1) and hsv-2 in the genital region. j infect dis 1998; 177: 1069–72. 28. roest rw, van der meijden wi, van dijk g, groen j, mulder pg, verjans gm, osterhaus ad. prevalence and association between herpes simplex virus types 1 and 2-specific antibodies in attendees at a sexually transmitted disease clinic. int j epidemiol. 2001; 30(3): 580–8. 29. kim hn, meier a, huang ml, kuntz s, selke s, celum c, corey l, wald a. oral herpes simplex virus type 2 reactivation in hivpositiveand -negative men. j infect dis. 2006; 194(4): 420–7. 30. mbopi-kéou fx, gresenguet g, mayaud p, weiss ha, gopal r, matta m, paul jl, brown dw, hayes rj, mabey dc, bélec l. interactions between herpes simplex virus type 2 and human immunodeficiency virus type 1 infection in african women: opportunities for intervention. j infect dis. 2000; 182(4): 1090–6. 31. freeman ee, weiss ha, glynn jr, cross pl, whitworth ja, hayes rj. herpes simplex virus 2 infection increases hiv infection in men and women: systematic review and meta-analysis of longitudinal studies. aids 2006; 20(1): 73–83. 120 dent. j. (maj. ked. gigi), volume 45 number 2 june 2012: 114–120 32. wald a, link k. risk of human immunodeficiency virus infection in herpes simplex virus type 2–seropositive persons: a meta-analysis. j infect dis 2002; 185: 45–52. 33. celum c, levine r, weaver m, wald a. genital herpes and human immunodeficiency virus: double trouble. bull world health organ 2004; 82: 447–53. 34. reynolds sj, risbud ar, shepherd me, zenilman jm, brookmeyer rs, paranjape rs, divekar ad, gangakhedkar rr, ghate mv, bollinger rc, mehendale sm. recent herpes simplex virus type 2 infection and the risk of human immunodeficiency virus type 1 acquisition in india. j infect dis. 2003; 187: 1513–21. 35. hoots be, hudgens mg, cole sr, king cc, klein rs, mayer kh, rompalo am, sobel jd, jamieson dj, smith js. lack of association of herpes simplex virus type 2 seropositivity with the progression of hiv infection in the hers cohort. am j epidemiol. 2011; 173(7): 837–44. 36. augenbraun m, feldman j, chirgwin k, zenilman j, clarke l, dehovitz j, dehovitz j, landesman s, minkoff h. increased genital shedding of herpes simplex virus type 2 in hiv-seropositive women. ann intern med 1995; 123: 845–7. 37. wright pw, hoesley cj, squires ke, croom-rivers a, weiss hl, gnann jw, jr. a prospective study of genital herpes simplex virus type 2 infection in human immunodeficiency virus type 1 (hiv1)–seropositive women: correlations with cd4 cell count and plasma hiv-1 rna level. clin infect dis 2003; 36: 207–11. 38. schacker tw, zeh je, hu hl, hill e, corey l. frequency of symptomatic and asymptomatic herpes simplex virus type 2 reactivations among human immunodeficiency virus-infected men. j infect dis 1998; 178: 1616–22. 39. santos fc, de oliveira sa, setúbal s, camacho la, faillace t, leite jp, santos fcvelarde lg. seroepidemiological study of herpes simplex virus type 2 in patients with the acquired immunodeficiency syndrome in the city of niterói, rio de janeiro, brazil. mem inst oswaldo cruz. 2006; 101(3): 315–9. 40. lanzafame m, mazzi r, di pace c, trevenzoli m, concia e, vento s. unusual, rapidly growing ulcerative genital mass due to herpes simplex virus in human immunodeficiency virus–infected women. br j dermatol 2003; 149: 216–7. 41. barbour jd. hiv-1/hsv-2 co-infected adults in early hiv-1 infection have elevated cd4+ t cell count. plos one 2007(2): e1080. 42. derhovanessian e, maier ab, hähnel k, beck r, de craen aj, slagboom ep, westendorp rg, pawelec g. infection with cy tomega lovi r us but not her p essi mplex vi r us i nduces t he accumulation of latedifferentiated cd4+ and cd8+ t-cells in humans. j gen virol. 2011; 92: 2746–56. 43. himmelein s, st leger aj, knickelbein je, rowe a, freeman ml, hendricks rl. circulating herpes simplex type 1 (hsv-1)-specific cd8+ t cells do not access hsv-1 latently infected trigeminal ganglia. herpesviridae. 2011; 2(1): 5. 44. posavad cm, koelle dm, shaughnessy mf, corey l. severe genital herpes infections in hiv-positive patients with 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 117 vol. 43. no. 3 september 2010 research report the correlation between immunoexpression of estrogen receptor and the severity of periodontal disease yuliana mahdiyah da’at arina�, s. sunardhi widyaputra�, and koeswadji�� 1 departement of periodontology, faculty of dentistry, jember university, jember-indonesia 2 departement of oral biology, faculty of destistry and departement of pathology anatomi, faculty of medicine padjadjaran university, bandung-indonesia 3 department of biochemistry, faculty of medicine, padjadjaran university, bandung-indonesia abstract background: the decreased level of estrogen during menopause may be one of the risk factors of periodontal disease. the influence of estrogen to periodontal tissue disturbance is mediated by the presence of estrogen receptor on tissue. the precise mechanism how the estrogens mediate this effect is still unclear. purpose: the aim of this study was to determine the correlation between estrogen receptor a and b on the periodontal pocket of women who had severe chronic periodontitis measured based on the periodontal pocket depth. methods: twenty four periodontitis patients from menopausal and productive women according to the criteria were examined upon her periodontal status and immunoexpression of estrogen receptor a and b on their periodontal pocket wall. results: the result showed that in the menopausal and productive women, immunoexpression of estrogen receptor a and b was not correlated with the periodontal pocket depth (p>0.05). however, the pocket depth seemed to show higher correlation with immunoexpression of estrogen receptor a than that with estrogen receptor b, r=0.37 vs. r=0.12 for menopausal women, and r=41 vs. r=0.11 for productive women. conclusion: it was concluded that no significant correlation was found between the estrogen receptor and periodontal pocket depth both on menopausal and productive women, presumed that estrogen has little role in the severity of periodontitis based on periodontal pocket depth. however, the estrogen receptor a has valuable effect on the severity of periodontal disease more than the estrogen receptor ß. key words: estrogen, estrogen receptor a and b, periodontal disease/severity, periodontal pocket abstrak latar belakang: berkurangnya kadar estrogen pada masa menopause merupakan salah satu faktor resiko penyakit periodontal. peran estrogen dalam kerusakan jaringan periodontal dimediatori oleh reseptor estrogen a dan b yang terdapat dalam jaringan. akan tetapi, mekanisme estrogen mempengaruhi efek ini sampai saat ini masih belum jelas. tujuan: penelitian ini bertujuan untuk menentukan korelasi antara reseptor estrogen pada poket periodontal wanita menopause penderita periodontitis kronis dengan keparahan periodontitis yang ditentukan berdasarkan kedalaman poket. metode: dilakukan pemeriksaan status periodontal dan immunoekspresi reseptor estrogen a dan b pada dinding poket periodontal dari 24 wanita menopause dan belum menopause penderita periodontitis yang sesuai dengan kriteria yang telah ditetapkan. hasil: hasil mendapatkan bahwa immunoekspresi reseptor estrogen a dan b tidak berkorelasi dengan kedalaman poket periodontal wanita menopause dan belum menopause (p>0,05). meskipun demikian, kedalaman poket periodontal tampak lebih berkorelasi dengan reseptor estrogen a daripada dengan reseptor estrogen b, dengan nilai r=0,37 vs r=0,12 pada wanita menopause, dan r=0,41 vs r=0,11 pada wanita belum menopause. kesimpulan: dapat disimpulkan bahwa tidak terdapat korelasi yang signifikan antara reseptor estrogen dan kedalaman poket periodontal wanita menopause dan belum menopause sehingga diduga bahwa estrogen mempunyai sedikit pengaruh pada keparahan periodontitis. meskipun demikian, reseptor estrogen a tampak lebih berperan terhadap keparahan penyakit periodontal dibandingkan reseptor estrogen b. kata kunci: estrogen, reseptor estrogen a dan b, keparahan penyakit periodontal, poket periodontal correspondence: yuliana md arina, bagian periodonsia, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: yuliana_mahdiyah@yahoo.com 118 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 117–121 introduction in women, hormonal changes throughout lifetime influence their periodontal tissue health. estrogen can modulate some cytokines that regulate the host response to infection.1 a study previously has reported that the periodontal disease of menopausal women is more severe than reproductive women.2 estrogen deficiency at menopausal women could be suspected to be an aggravating factor in periodontal disease.3 estrogen deficiency is related with the increase of alveolar bone resorption,3 and periodontal loss attachment leading to severity of periodontal disease that can cause dental loose.4,5 the influence of estrogen to periodontal tissue disturbance is mediated by the presence of estrogen receptor on tissue. estrogen receptor (er) is a protein mediating estrogen biological effect on cells.6 two kinds of er are identified in human being, er a and er b.7 distribution of er a and b is different, although, it is occasionally overlapped.8 in oral cavity, the immunoexpression of er a had been detected on odontoblast, endothelial, schwan cells of pulp tissue and show no difference based on the age and sex.9 the periodontal ligament cells showed the immunoexpression of er b.10 leimola-virtanen et al.11 had demonstrated that there was no significant difference of mrna expression of er in the oral mucosa of menopausal women and nineteen year old women. presently, the presence of er a and b in periodontitis patients has not been discussed. the precise mechanism by which estrogens mediate these effects is still unclear. it was hypothesized that the decreased of estrogen on menopausal women will decrease the immunoexpression of estrogen receptor so that the severity of periodontitis will increase. the specific of er which mediate the estrogen effect in periodontitis is unknown. the purpose of this study is to determine the immunoexpression of estrogen receptor a and b on the periodontal pocket of menopausal women which had chronic periodontitis and to know the correlation between er a and b with the severity of periodontitis measured based on the periodontal pocket depth. materials and methods this study was a cross-sectional observation. the material used was the result of scrapping periodontal pocket in the menopausal women and productive women with periodontitis coming to the periodontal division in the oral and dental clinic dr. hasan sadikin hospital bandung. the patients within the inclusion criteria were provided informed consent. the inclusion criteria of the sample were a) menopausal women, women having had no menstruation for 12 months respectively, getting no estrogen replacement therapy and having no diabetes mellitus history. b) productive women (as a control), above 30 years old, getting routine menstruation monthly, not pregnant, not using oral contraception and having no diabetes mellitus history. subsequently, the patients were examined upon their periodontal tissue including clinical gingival examination, bleeding on probing, and probing depth. probing was conducted using periodontal probe inserted in the gingival sulcus in direction with tooth axis. periodontal pocket depth was determined from the distance to which a tip of periodontal probe penetrates into the pocket. periodontitis was marked by gingival inflammation, probing depth more than 3 mm and clinical attachment loss. the periodontal lateral pocket wall was scrapped using tooth pick made of smooth bamboo split into two parts. periodontal pocket scrapping was conducted twice for each examination of er a and b. the result of periodontal pocket scrapping was subsequently smeared on the object glass and fixed in 95% alcohol solution. furthermore, immunocytochemical staining was conducted using antibody of er a and b in the laboratory of pathology anatomi dr. hasan sadikin hospital bandung. briefly, the rabbit polyclonal antibody mc20 from santa cruz biotechnology (santa cruz, usa) was used for detection of er a, and the chicken polyclonal er b 503 immunoglobulin y for er b. the slides were immersed in pbs, incubated in 0.3% h2o2 to quench endogenous peroxidase. to block unspecific binding of secondary antibodies, slides were incubated in 100μl serum blocking reagent. primary antibody of er a antibody mc20 (santa cruz biotechnology, 1:100 dilution in blocking solution, incubated one hour at room temperature) was added. the polyclonal er b 503 immunoglobulin y was diluted 1:100. after several washes, slides were incubated with a secondary biotinylated antibody (sc-2040, santa cruz biotechnology, usa). finally, streptavidin conjugated to horseradish peroxidase (dako, lsab2 kit) was applied and visualized by incubated in 0.5% diaminobenzidine (dako). all slides were slightly counterstained with mayer’s hematoxylin, mounted and examined with light microscope. as the positive control of estrogen receptor a, invasive ductal carcinoma of breast was used, and hyperplasia prostate for positive control of estrogen receptor b, while for negative control, slides was incubated with normal serum. the immunostaining was assessed in a quantitative manner by dividing the number of cells which immunoreactivity with the number of total cells.12 statistical analysis was carried out using the regression analysis test to determine the correlation between immunoexpression of estrogen receptor and probing depth. results a total of 24 periodontitis patients from menopausal and productive women were examined. menopausal group consisted of 13 women aged 58.08 years old in average (the age range was 50–70 years old) and menopausal duration was 5.85 years in average (the range was 1–16 years). the examination of periodontal pocket depth showed 4.62 mm in average with the deepest was 7 mm. the productive group consisted of 11 women aged 40.73 years old in 119arina et al.: the correlation between immunoexpression average (the range was 30–47 years old). the result of periodontal pocket depth examination was 3-6 mm with 4.27 mm in average. the result of this study is presented in the table 1. the study showed the presence of immunoexpression of er with brown-colored in nucleus and or cytoplasm. the intensity of nuclear brown-colored was stronger than that in the cytoplasm. likewise, the result of immunocytochemical smear using antibody of er b showed stronger intensity of nuclear brown color than that in the cytoplasm (figure 1). the result of regression analysis test between the immunoexpression of er and periodontal pocket depth on the menopausal and productive group was presented in table 2. it showed that the immunoexpression of er a and b was not correlated with periodontal pocket depth (p > 0.05) both on the menopausal and productive women. discussion periodontitis is a multi-factorial infectious disease. although the presence of bacteria on the plaque is considered as the main factor of periodontitis, the systemic factors may play an important role in its initiation and progression. there are some factors that may influence the table �. periodontal pocket depth and immunoexpression of estrogen receptor of the menopausal women and productive women sample groups n age periodontal pocket menopause duration immuno-expression er a immuno-expression er b (years) (mm) (years) (%) (%) menopausal women 13 58.08 (sd=6.06) 4.62 (sd=1.19) 5.85 (sd=4.3) 27.16 (sd=15.69) 14.72 (sd=16.78) productive women 11 40.73 (sd=7.04) 4.27 (sd=1.01) ------32.11 (sd=18.22) 13.51 (sd=10.48) table �. regression analysis test between the periodontal pocket depth and immunoexpression of estrogen receptor probing depth menopausal women productive women er a r = 0,37 r = 0,41 er b r = 0,12 r = 0,11 figure �. immunoexpression of estrogen receptor a and b on the periodontal pocket, a) immunoexpression of estrogen receptor a in the nucleus and or in the cytoplasm (black arrow) and negative cells of estrogen receptor a (blank arrow), b) positive cells of estrogen receptor b (black arrow) and negative estrogen receptor b, c) positive control of estrogen receptor a in the breast invasive ductal carcinoma, d) positive control of estrogen receptor b in the hyperplasia prostate. a b c d 120 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 117–121 extent and severity of periodontitis, including hormonal factors, genetic factors, smoking, emotional stress, nutrition, and degradation of immune response on hiv.13 in menopausal women, the decrease of estrogen is correlated with the increase of loss attachment leading to the increase of periodontal disease severity.3,5 this study demonstrated that the periodontal disease of menopausal women is more severe than reproductive women3 and it correlated with the menopausal periode.14 the result of this study showed that cells from periodontal pocket smear expressed the er a and b both on the menopausal and productive women through immunocytochemical method. immunocytochemical method has been found favorable since many years ago for routine examination of er. it was a simple, quick and moderately reliable technique to evaluate er status.15 even though the number of cells examined is small but immunocytochemistry showed high sensitivity and specificity, short procedure time and did not need retrieval antigenic procedure.16 furthermore, the samples are simply collected, not invasive nor painful. the presence of er a and b in the periodontal pocket cells in this study suggests that estrogen has important role in the periodontal pocket. this result is in agreement with study conducted by xie and shu17 that have found the expression of er in the gingival tissues that might have relation to periodontitis of females. periodontal pocket is a main clinical sign of the periodontal tissue destruction on the periodontitis due to specific periodontopathogen bacteria in the gingival sulcular area. these bacteria have ability to invade the gingival through sulcular epithelium and bring out their products into deeper periodontal tissue. this may induce the host response through bringing out inflammatory mediator that initiating inflammatory process on the periodontal tissue. inflammatory mediator is produced by inflammatory cells, epithelial cells and gingival fibroblast. these mediators may stimulate fibroblast and macrophage to produce matrix metalloproteinase, collagenase and prostaglandin e2 that cause breakage on collagen, glycoaminoglican and alveolar bone resulted in a periodontal pocket, clinical attachment loss and alveolar bone resorption.18,19 estrogen play role in managing periodontal response to periodontopathogen bacteria.1 the role of estrogen is to accelerate the wound healing process through increasing matrix deposition, accelerating epithelization, and reducing inflammatory response.20 estrogen also has been reported can induce cellular proliferation in gingival fibroblasts.21 estrogen increases cellular proliferation, differentiation and sulcular epithelial keratinization.22 sulcular epithelial has a role as gingival defense toward bacterial invasion through proliferation, keratinization degree and continuous epithelial substitution. the continuous substitution on gingival epithelial cells may cause bacteria released from cellular surface of gingival epithelium. mitotic activity of gingival epithelial cells is once in 24 hours periodically.23 estrogen also inhibits the release of inflammatory mediators by decelerating gene transcription through estrogen receptor a and b.24 several studies had reported that estrogen inhibits the release of cytokine proinflammatory il1,24 il-i ß, il-6,25 pge2, mmp-926 and tnf-a.27 consequently, the periodontal pocket formation, clinical attachment loss and alveolar bone resorption can be inhibited. a l t h o u g h t h e s e s t u d y h a v e d e m o n s t r a t e d t h e immunoexpression of estrogen receptor a and b on the periodontal pocket, but the correlation between periodontal pocket depth and immunoexpression of er a and b on the menopausal and productive women exhibited the presence of value, but statistically not significant. this result suggested that the estrogen is not the main factor on periodontal pocket. the effect of estrogen depends on the number of complex binding between estrogen and estrogen receptor on the sulcular epithelial cells. the more complex binding, activity of estrogen is greater. besides acting indirectly on the gingival tissues, estrogen could affect the gingival tissues directly by er.17 this study is not asses the serum estrogen level so it cannot extensively explain the result. the explanation of these result is may be the estrogen status is more influential to the alteration of alveolar bone density.4 osteoporosis on the menopausal women is also a risk indicator for periodontal disease in postmenopausal caucasian women.28 a theory reveals that the decrease of mineral in the alveolar bone due to osteoporosis is risk factor of attachment loss during periodontitis.29 bone mineral density (bmd) is related to resorption of alveolar crest, and clinical attachment loss on post-menopausal women.28 study on the post-menopausal women of asianamerican race showed the relation among bone mineral density, edentulous, and clinical attachment loss, however, there was no correlation between bmd and periodontal pocket depth. bmd is a predictive factor of edentulous and clinical attachment loss.29 among postmenopausal women, there were significant trends for increasing prevalence of moderate or severe periodontal disease and tooth lost as the level of bmd decreased.5 however, in the study conducted by lopes et al.,30 that in postmenopausal women, there is no significant correlation between bmd and the increase of periodontitis risk measured based on the gingival index, plaque index and the clinical attachment level. a study in two groups of pre-menopausal women, the group with severe generalized periodontitis and periodontally healthy group, showed that there was no significant correlation between the clinical attachment level and bmd value.31 the presence of these differences needs a further study to determine the influence of estrogen on the periodontal tissue either through er a or estrogen b on the menopausal and productive women with periodontitis. however, this study showed that in the menopausal and productive women, the immunoexpression of er a seemed to have higher correlation with periodontal pocket depth than that with er b (r= 0.37 vs. r = 0.12 for menopausal women, and r = 41 vs. r = 0.11 for productive women). this confirms the result of previous study that in the gingival tissues of female with periodontitis, the positive rate of er in the 121arina et al.: the correlation between immunoexpression gingival tissues increased significantly.17 nevertheless, detection frequency of estrogen receptor a genotypes in female chinese population was not statistically different among the aggressive periodontitis, chronic periodontitis and healthy control.32 it concluded that the immunoexpression of er had no correlation with the severity of periodontal disease determined based on the periodontal pocket depth both on menopausal and productive women which presumed that estrogen has little role in the severity of periodontitis based on periodontal pocket depth. however, the er a was more correlated with the periodontal pocket depth than the er b. to get better evaluation of the estrogen role on the pathogenesis and severity periodontitis, additional prospective longitudinal studies with further analysis of possible confounding factors for menopause status, estrogen receptor, osteoporosis and periodontal status in larger cohorts of post-menopausal women are needed. the results of the studies will have a practical significance in the diagnosis, prevention and approach for the treatment of periodontal disease in menopausal women. acknowledgement the author thanks prof. jan-ake gustafsson, md, phd, the head division of biotechnology center of karolinska novum institute, sweden for providing estrogen receptor antibody b igy used in this investigation. references 1. mascarenhas p, gapski r, al-shammari k, wang hl. influence of sex hormones on the periodontium. j clin periodontol 2003; 30(8): 671–81. 2. arina ymd. perbedaan status kesehatan jaringan periodontal wanita menopause dan belum menopause. spirulina 2008; 3(1): 39–46. 3. duarte pm, goncalves pf, sallum aw, sallum ea, casati mz, notici fh. effect of an estrogen-deficient state and its therapy on bone loss resulting from an experimental periodontitis in rats. j perio res 2004; 39: 107–10. 4. johnson rb, gilbert ja, cooper rc, parsell de, stewart ba, nick tg, streckfus cf, butler ra, boring jg.effect of estrogen deficiency on skeletal and alveolar bone density in sheep. j periodontol 2002; 73(4): 383–91. 5. inagaki k, kurosu y, kamiya t, kondo f, yoshinari n, noguchi t, krall ea, garcia ri. low metacarpal bone density, tooth loss and periodontal disease in japanese women. j dent res 2001; 80(9): 1818–22. 6. nilsson s, makela s, treuter e, tujague m, thomsen j, andersson g, enmark e, pettersson k, warner m, gustafsson ja. mechanism of estrogen action. physiol rev 2001; 81(4): 1535–65. 7. matthews j, gustafsson ja. estrogen signaling: a subtle balance between era and erb. mol interv 2003; 3(5): 281–92. 8. gruber cj, tschugguel w, schneebrger c, huber jc. production and actions of estrogens. n engl j med 2002; 346(5): 340–52. 9. jukic s, prpic-mehicic g, talan-hranilovc j, miletic i, egovic s, anic i. estrogen receptor in human pulp tissue. oral surg oral med oral pathol oral radiol endo 2003; 95(3): 340–4. 10. jonsson d, andersson g, ekblad e, liang m, bratthall g, nilsson bo. immunocytochemical demonstration of estrogen receptor in human periodontal ligament cells. arch oral biol 2004; 49(1): 85–8. 11. leimola-virtanen r, salo t, toikkanen s, pulkkinen j, syrjanen s. expression of estrogen receptor (er) in oral mucosa and salivary glands. maturitas 2000; 36(2): 131–7. 12. saji s, jensen ev, nilsson s, rylander t, warner m, gustafsson ja. estrogen receptors a and b in the rodent mammary gland. proc natl acad sci usa 2000; 97(1): 337–42. 13. nagy rj, novak mj. chronic periodontitis in carranza’s clinical periodontology. 10th ed. philadelphia: wb saunders co; 2006. p. 494–9. 14. arina ymd, sari ds, yuniar nh. hubungan antara status jaringan periodontal wanita menopause dengan lama menopause. spirulina 2006; 1(1): 43–52. 15. jarayam g, elsayed em. cytologic evaluation of prognostic marker in breast carcinoma. acta cytol 2005; 49(6): 605–10. 16. cano g, milanezi f, leitao d, ricardo s, brito mj, schmitt fc. estimation of hormon receptor status in fine-needle aspirates and paraffin-embedded sections from breast cancer using the novel rabbit monoclonal antibodies sp1 and sp2. diagn cytopathol 2003; 29(4): 207–11. 17. xie yf, shu r. expression of estrogen and progesterone receptors in the gingival tissues of female patients with moderate and advanced periodontitis. shanghai kou qiang yi xue 2003; 12(5): 366–9. 18. carranza fa, camargo pm. the periodontal pocket in carranza’s clinical periodontology. 10th ed. philadelphia: wb saunders co; 2006. p. 434–51. 19. eley bm, manson jd. periodontics. 5th ed. london: wright; 2004. p. 55–82. 20. ashcroft gs, mills sj, lei k, gibbons l, jeong m, taniguchi m, burow m, horan ma, wahl sm, nakayama t. estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor. j clin invest 2003; 111(9): 1309–18. 21. mariotti aj. estrogen and extracellular matrix influence human gingival fibroblast proliferation and protein production. j periodontol 2005; 76(8): 1391–7. 22. corgel jo. periodontal therapy in the female patient in carranza’s clinical periodontology. 10th ed. philadelphia: wb saunders co; 2006. p. 636–49. 23. bulkacz j, carranza fa. defense mechanisms of the gingiva in carranza’s clinical periodontology. 10th ed. philadelphia: wb saunders co; 2006. p. 344–54. 24. pfeilschitefter j, koditz r, pfohl m, schatz h. changes in proinflammatory cytokine activity after menopause. endocr rev 2002; 23(1): 90–119. 25. cuzzocrea s, mazzon l, dugo l, genovese t, paola rd, ruggeri z, vegeto e, caputi ap, van de loo fa, puzzolo d, maggi a. inducible nitric oxide synthase mediates bone loss in ovariectomized mice. endocrinology 2003; 144(3): 1098–107. 26. vegeto e, bonincontro c, pollio g, sala a, viappiani s, nardi f, brusadelli a, viviani b, ciana p, maggi a. estrogen prevents the lipopolysaccharide-induced inflammatory response in microglia. j neurosci 2001; 21(6): 1809–18. 27. cenci s, toraldo g, weitzmann mn. estrogen deficiency induces bone loss by increasing t cell proliferation and lifespan through ifnγ-induced class ii tran activator. proc natl acad sci 2003; 100(18): 10405–10. 28. tezal m, wactawski-wende j, grossi sg, ho aw, dunford r, genco rj. the relationship between bone mineral density and periodontitis in postmenopausal women. j periodontol 2000; 71(9): 1492–8. 29. mohammad ar, hooper da, vermilyea sg, mariotti a, preshaw pm. an investigation of relationship between systemic bone density and clinical periodontal status in postmenopausal women. int dent j 2003; 53(3): 121–5. 30. lopes ff, loureiro fh, alves cm, pereira ade f, oliveira ae. systemic bone mineral density versus clinical periodontal condition: cross-sectional study in postmenopausal women. rev assoc med bras 2008; 54(5): 411–4. 31. sonmez eh, ozcakar l, kutsal yg, karaagaoglu e, demiralp b, erverdi hn. no alteration in bone mineral density in patient with periodontitis. j dent res 2008; 87(1): 79–83. 32. zhang l, meng h, zhao h, li q, xu l, chen z, shi d, feng x. estrogen receptor-alpha gene polymorphisms in patients with periodontitis. j periodontal res. 2004; 39(5): 362–6. �� vol. 42. no. 1 january–march 2009 the effect of toothpaste containing kayu sugi extract on plaque formation widowati w1, rar awang2, nh ismail2, and sh othman2 1kulliyyah of dentistry, international islamic university, malaysia 2school of dental sciences, universiti sains malaysia, kelantan, malaysia abstract background: although many researches had revealed the beneficial effect of kayu sugi as a chewing stick, study on the effectiveness of its extract in toothpaste is still inadequate. purpose: the objective of this study was to compare the effect of toothpaste, with and without kayu sugi extract on preventing plaque formation. methods: the study consists of two sessions which was separated by three days washout period. the subjects were given two types of toothpaste, with and without kayu sugi extract to be used in the first and second session separately. the subjects were polished and plaque score were measured after one hour for the first quadrant, two hours later for the second quadrant and after four hours for the third/fourth quadrant. subjects were not allowed to eat, drink or rinse during this four hours period. the procedures were repeated for the second session after three days washout period. the plaque score were recorded as absent (code 0) and present (code 1), and only labial and palatal/lingual surfaces of each tooth were used for plaque scoring. result: the study showed that there was no significant difference of the amount of plaque formed after polishing using two different toothpastes, with and without kayu sugi extract. conclusion: we concluded that toothpaste with or without kayu sugi extract give similar level in preventing plaque formation. key words: kayu sugi, plaque formation, toothpaste correspondence: widowati witjaksono, kulliyyah of dentistry, international islamic university malaysia. bandar indera mahkota,bandar indera mahkota, 25200 kuantan, pahang darul makmur, malaysia.e-mail:drwidowati@iiv.edu.my. telephone: +609-5716441/6444 introduction kayu sugi, known as miswak or chewing stick is used as teeth cleaning tool in many regions in the world especially in muslim predominant areas. it is made from the roots or twigs of salvadora persica tree. the world health organization1 has recommended and encourages the use of kayu sugi as an effective alternative tool for oral hygiene. the use of kayu sugi, when preceded by professional instruction has been shown to be more effective than toothbrush in reducing plaque formation and gingivitis.2 the observational study on sudanese volunteers3 showed that kayu sugi users demonstrated better periodontal status as compared to toothbrush user. besides, the kayu sugi users were also found to present with lower salivary level of streptococci species4 and actinobacillus actinomycetemcomitans5 in comparison to the toothbrush user. due to its long history of usage as a natural tooth brush and many positive findings were reported on the kayu sugi users especially concerning its effects in reducing gingivitis and some oral pathogens,2-5 many researches were carried out the extract of kayu sugi. a numbers of in vitro studies had reported on the antibacterial properties of the kayu sugi extract on some cariogenic bacteria and periodontal pathogens,5-7 thus suggesting another potential antibacterial agent suitable to be added in the oral health products. based on its antibacterial properties, kayu sugi extract was included in many oral health products such as toothpaste and mouthwash in an attempt to inhibit bacterial growth and thus slowing down the process of plaque formation. although in vitro studies had shown the antibacterial properties of the kayu sugi extract, there were very few studies, if any that look at the effectiveness of kayu sugi extract added in oral health products especially toothpaste in hindering plaque formation. hence, the objective of the study was to compare the effect of the toothpaste, research report �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 21-24 with and without kayu sugi extract in preventing plaque formation materials and methods this was a community trial study performed at school of dental sciences, universiti sains malaysia (usm) to compare the efficiency of two different toothpastes, with and without kayu sugi extract in hindering plaque accumulation. the ethical clearance was obtained from the research and ethics committee usm. thirdty six 36 subjects who were the students of school of dental sciences usm volunteered for the study. their ages were ranged from 20to 25-year-old and comprised of 31 ladies and 5 gentlemen. the inclusion criteria were as follows: a) subjects with intact periodontal tissues which means free from calculus, gingivitis and periodontal diseases; b) subjects without any prosthodontics or orthodontics appliance; and c) subjects were non smokers. two types of toothpastes, with and without kayu sugi extract were provided. double blinding method was employed in the study where both operator and subjects did not know the type of toothpaste used in each visit. data collection procedures were carried out in two sessions where three days washout period applied in between the sessions. the washout period was where the subjects were instructed to use their normal oral hygiene method. in the first session, disclosing agent was applied to the entire teeth and the subjects were polished using the toothpaste until the disclosing agent was not visible. plaque score measurement were carried out at the following scheduled periods: a) after one hour (measurement on first quadrant); b) after two hours (measurement on second quadrant); and c) after four hours (measurement on third or fourth quadrant). the subjects were not allowed to eat, drink or rinse during this four hours period. the procedures were repeated in the second session with the other provided toothpaste. plaque score measurements were carried out by single operator using mouth mirror and periodontal probe. five teeth from each quadrant were selected which were central incisor, lateral incisor, canine, first premolar and second premolar. the plaque score measurements were carried out on facial and lingual/palatal surfaces of the tooth by running the probe on the cervical areas. the plaque score were recorded as absent (code 0) and present (code 1). the overall teeth surfaces evaluated in each variable were 360 surfaces. the data analysis was carried out using spss version 12.0 (spss inc., chicago, il), where pearson’s chi-square test was used to assess the significant of an association between proportion of plaque accumulation after polishing using toothpaste, with and without kayu sugi extract. result from the 36 selected subjects, the overall of 720 teeth surfaces were observed for plaque formation at each plaque evaluation time namely one hour, two hours and four hours. details of the descriptive statistics of the study are shown in table 1, and the frequency of plaque for tooth paste are shown in table 2. the proportions of plaque formation after polishing between toothpastes, with and without kayu sugi extract was not significantly different at one hour (p = 0.110), two hours (p = 0.328) and four hours (p = 0.823). therefore, there was no significant association between polishing using toothpaste, with and without kayu sugi extract and plaque formation. table 1. demographic data of 36 subjects with plaque evaluation on 720 surfaces in each time variable variable mean (sd) frequency (%) age (year) 22.75 (1.131) gender male 5 (14) female 31 (86) proportion of plaque formation on teeth surfaces (n = 720) one hour 230 (31.9) extract kayu sugi 105 (45.7) without kayu sugi 125 (54.3) two hours 311 (43.2) extract kayu sugi 149 (47.9) without kayu sugi 162 (52.1) four hours 353 (49.0) extract kayu sugi 178 (50.4) without kayu sugi 175 (49.6) ��widowati, et al.: the effect of tootpaste comtaining kayu sugi extract discussion the use of toothpaste as a vehicle in carrying chemical adjunct is widely established.8 a wide range of chemicals especially antibacterial agents9 including kayu sugi extract have been added in toothpaste as an attempt to produce a direct inhibitory effect on oral pathogens and thus preventing plaque accumulation. however, in the present study, when we compared the effect of polishing using toothpaste, with and without kayu sugi extract for their effect on plaque formation, we found that the difference was not significant. since there was no similar study regarding the effect of toothpaste containing kayu sugi extract on plaque formation, the comparison of the result cannot be made. however, many other studies were carried out regarding the effect of toothpaste containing other chemical adjunct on their effect on the plaque formation. the study on tooth paste and extract containing herbal,10–13 amine fluorides,14 sodium fluoride or stannous fluoride15 and triclosan16,17 were failed to prove the superiority of the tested toothpaste as compared to the control. on the other hand, there were also studies indicated the toothpaste containing stannous fluoride,18 salivary substitutes19 and chlorhexidine mouth rinses20 had better prevention in the plaque formation in comparison to the control materials. although many in vitro studies had proved the antibacterial capability of the kayu sugi extract6,9 its capability, especially when carried in toothpaste for inhibiting oral pathogen is questionable/doubtful. as in our study, we found that the plaque formation was equal in the subjects who polish using toothpaste either with or without kayu sugi extract, where the plaque formation should be less in the subjects who used toothpaste with kayu sugi extract, since its has the antibacterial properties. the possible reason for the ineffectiveness of the adjunct added in toothpaste in inhibiting bacterial or plaque formation is may be due to the clearance effect of the saliva. the dawes model of oral clearance stated that the extraneous component of saliva such glucose can be cleared by half after 2.2 minutes if the unstimulated flow rate is 0.3 ml/minute.21 with the high efficiency of the oral clearance of the saliva, the only possible way for chemical adjunct from toothpaste to stay in the mouth is by their ability to bind to oral tissues. however some chemical adjuncts such as stannous fluoride and chlorhexidine somehow had the ability to bind with oral tissue.22 hence, may be explained the reason why some study showed that certain toothpaste (containing fluoride or chlorhexidine) showed superiority in preventing plaque formation. from the present study, we concluded that when polishing with toothpaste containing kayu sugi extract, the effect of preventing plaque formation of this material is not significantly better than the one without the extract. the study of kayu sugi extract into the reduction of dental plaque have been studied from many point of views using modern scientific methods such as local and clinical,2,3,10,12,23 microbial4–7,23 and chemical effects.8,9, 22,23 however, so far, there is no study have been done in the mechanisms of the reduction of dental plaque by molecular point of view. based on this fact we suggest to further study in the relation of the mechanism of plaque reduction from the point of molecular perceptions acknowledgement we would like to express our thanks to all the volunteers that had participated in this study and all staff in hospital usm dental clinic, school of dental sciences, universiti sains malaysia for their help and support. this elective study is granted by the short term grant of universiti sains malaysia. references 1. world health organization. national policy on traditional medicine and regulation of herbal medicines. geneva: report of a who global survey; may 2005. p. 11, 22. table 2. proportion of plaque formations after polishing using toothpaste, with and without kayu sugi extract in four hours variable n with extract freq (%) without extract freq (%) x2 statistica (df) p-value one hour present 230 105 (45.7) 125 (54.3) 2.555 0.110 absent 490 255 (52.0) 235 (48.0) two hours present 311 149 (47.9) 162 (52.1) 0.957 0.328 absent 409 211 (51.6) 198 (48.4) four hours present 353 178 (50.4) 175 (49.6) 0.050 0.823 absent 367 182 (49.6) 185 (50.4) a chi-square test for independence; significant level was set at p = 0.05 �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 21-24 2. al-otaibi m, al-harthy m, soder b, gustafsson a, angmar-mansson b. comparative effect of chewing sticks and toothbrushing on plaque removal and gingival health. oral health prev dent 2003; 1(4):301–7. 3. darout ia, albandar jm, skaug n. periodontal status of adult sudanese habitual users of miswak chewing sticks or toothbrushes. acta odontol scand 2000; 58(1):25–30. 4. darout ia, albandar jm, skaug n, ali rw. salivary microbiota levelssalivary microbiota levels in relation to periodontal status, experience of caries and miswak use in sudanese adults. j clin periodontol 2002; 29(5):411–20. 5. al-otaibi m, al-harthy m, gustafsson a, johansson a, claesson r, angmar-mansson b. subgingival plaque microbiota in saudi arabians after use of miswak chewing stick and toothbrush. j clinj clin periodontol 2004; 31(12):1048–53. 6. almas k, al-zeid z. the immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study. j contemp dent pract 2004 february; 1(5):105–14. 7. almas k. the antimicrobial effects of seven different types of asian chewing sticks. odontostomatol trop 2001; 24(96):17–20. 8. institutt for klinisk odontologi, det odonto logiske fakultet, universitetet i oslo. dentifrices and mouthwashes ingredients anddentifrices and mouthwashes ingredients and their use. in components of toothpastes and mouthwashes. 2003. p. 5–8. 9. the ubiquitous triclosan. a common antibacterial agent exposed pesticides and you beyond pesticides/national coalition against the misuse of pesticides 2004; 24(3):12–7. 10. poureslami hr, makarem a, mojab f. paraclinical effects of miswak extract on dental plaque. dent res j 2007; 4(2):106–10. 11. darmani h, nusayr t, al-hiyasat as. effects of extracts of miswak and derum on proliferation of balb/c 3t3 fibroblasts and viability of cariogenic bacteria. int j dent hyg 2006; 4(2):62–6. 12. pannuti cm, mattos jp, ranoya pn, jesus am, lotufo rf, romito ga. clinical effect of a herbal dentifrice on the control of plaque and gingivitis: a double-blind study. pesqui odontol bras 2003; 17(4):314–8. 13. almas k, skaug n, ahmad i. an in vitro antimicrobial comparison of miswak extract with commercially available non-alcohol mouth rinses. int j dent hyg 2005; 3(1):18–24. 14. auschill tm, deimling d, hellwig e, arweiler nb. antibacterialantibacterial effect of two toothpastes following a single brushing. oral health prev dent 2007; 5(1):25–32. 15. white dj. effect of a stannous fluoride dentifrice on plaque formation and removal: a digital plaque imaging study. j clin dent 2007;j clin dent 2007; 18(1):21–4. 16. mcclanahan sf, bollmer bw, court lk, mcclary jm, majeti s, crisanti mm, beiswanger bb, mau ms. plaque regrowth effects ofplaque regrowth effects of a triclosan/pyrophosphate dentifrice in a 4-day non-brushing model. j clin dent 2000; 11(4):107–13. 17. moran j, newcombe rg, wright p, haywood j, marlow i, addy m. a study into the plaque-inhibitory activity of experimental toothpaste formulations containing antimicrobial agents. j clin periodontol 2005; 32(8):841–5. 18. willumsen t, solemdal k, wenaasen m, ogaard b. stannous fluoride in dentifrice: an effective anti-plaque agent in the elderly. gerodontology 2007; 24(4):239–43. 19. hatti s, ravindra s, satpathy a, kulkarni rd, parande mv. biofilm inhibition and antimicrobial activity of a dentifrice containing salivary substitutes. int j dent hyg 2007; 5(4):218–24. 20. sheen s, addy m. an in vitro evaluation of the availability of cetylpyridinium chloride and chlorhexidine in some commercially available mouthrinse products. british dental journal 2003; 194(4):207–10. 21. dawes c. salivary clearance and its effects on oral health. in: edgar m, dawes c, o’mullane d, eds. saliva and oral health. 3rd ed. london: british dental association; 2004. p. 71–85. 22. dawes c. salivary flow patterns and the health of hard and soft oral tissues. j am dent assoc 2008; 139(suppl):18s–24s. 23. darout ia, skaug nils. comparative oral health status of an adult sudanese population using miswak or toothbrush regularly. saudi dental journal 2004; 16(1):29–38. 204 vol. 42. no. 4 october–december 2009 treatment results evaluation using the index of orthodontic treatment need thalca hamid department of orthodontics faculty of dentistry, university of airlangga surabaya indonesia abstract background: the use of orthodontic indices were increasingly popular in the last few years. index of orthodontic treatment need (iotn) usually was used to assess the needs and demand of orthodontic treatment, eventhough, indices can be used for more than one purpose. purpose: to determine whether the index of orthodontic treatment need (iotn) could be able to evaluate the treatment results as well. method: data was obtained by evaluating each of 202 study models from 17 orthodontic postgraduate students. the ‘before’ and ‘after’ treatment models were assessed, using the index of orthodontic treatment need. result: using the wilcoxon signed-rank test, the assessment of dental health component (dhc) and aesthetic component (ac), before and after orthodontic treatment showed significantly differences from each others (p : 0,000 < a : 0.05). the null hypothesis were rejected. the grade of dhc and ac were decreasing to a better score. conclusion: the index of orthodontic treatment need (iotn) could be used to assess the orthodontic treatment outcomes, to evaluate before and after orthodontic treatment of the patients. the results of the treatment showed good improvements of dentofacial appearance of the patients which means the successful achievement of the clinical works programs. key words: index of orthodontic treatment need, dental health component, aesthetic component correspondence : thalca hamid, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: thalcaia@gmail.com research report introduction the use of orthodontic indices were increasingly popular in the last few years. an index usually used ton index usually used to consider the needs and demands of orthodontic treatment, or to evaluate the severity of malocclusion.1–3 in fact, indices is not only used for screening and measuring the need for treatment, but it may be used for many other purposes. one of the index as an example is the index of orthodontic treatment need (iotn), which usually was used to assess the needs and demand of orthodontic treatment. nevertheless, this index could be used for more than one purpose.1,2 the uses of orthodontic indices are: first, screening for handicapping malocclusion, diagnostic classification and describe severity or treatment complexity then, describe and ranking subjects for priority treatment or measure the treatment need and finally, for epidemiological surveys of malocclusion.3–5 the index of orthodontic treatment need (iotn) was developed by a team in the university of manchester and has two components which rank malocclusion in terms of the significance of various occlusal traits for the individual's dental health and perceived aesthetic impairment. the iotn incorporates a dental health component (dhc) and aesthetic component (ac). the dental health component was developed by brook and shaw6 and the aesthetic component of the index was developed by evans and shaw.7 the main aim of this study is to determine whether the index of orthodontic treatment need (iotn) could be able to evaluate the treatment results as well. the subsidiary aim is to study whether the output of the clinical orthodontic teaching at the specialist training program is having a successful results. 205hamid: treatment results evaluation material and method to evaluate the orthodontic treatment outcome, the index of orthodontic treatment need (iotn) was used in this study. this index aims to rank malocclusion in terms of the significance of various occlusal traits for an individual's dental health and perceived aesthetic impairment, with the intention of identifying those individuals who would be most likely to benefits from orthodontic treatment. the index corporate a dental health and an aesthetic component. the dental health component (dhc) will be considered first, then the aesthetic component (ac). the dental health component was roughly modelled on the index of the swedish dental board.8 the swedish index was meant as a basic guide, and its practical implementation called for a “good sense of judgement”. the dhc was developed to reduce this subjectivity in measurement, with well defined cut off points. the dhc records the various occlusal traits of malocclusion which would increase the morbidity of the dentition and surrounding structures.6 it has five grades, grade 1 represents a small or negligible need for treatment while grade 5 indicates a great need for treatment. in use, various features or traits of malocclusion are recorded. cleft palate, severe overjets greater than 9mm would fall into grade 5. displacements between contact points less than 1mm would fall into grade 1. however, only the highest scoring traits need to be recorded (table 1). the grade of dhc describes the priority for treatment, as for example: grade 1–2 means no or little need for treatment; grade 3 means borderline treatment need; while grade 4–5 which usually in this group including severe malocclusion thus it is grade need for treatment. figure 1. the dental health component ruler. table 1. dental health component of iotn (treatment need from a dental health perspective)1,6 206 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 204-209 the dental health component of the iotn has five categories ranging from 1 (no need for treatment) to 5 (great need) which may be applied clinically or to patients' study casts. the most severe occlusal trait is identified for any particular patient and the patient is then categorised according to this most severe trait. patients in grade 5 would include patients with cleft lip and palate, multiple missing teeth or a destructive malocclusion, which would include those with minor tooth displacements where there is little need for treatment. the dental health component uses a simple ruler (figure 1 & 2) and an acronym mocdo to guide the observer to the single worst feature of the malocclusion. mocdo represents missing teeth; overjets; crossbites; displacement of contact points; overbites. there are 5 categories, from 1 representing no need for treatment to 5 representing a great need for treatment. thus a patient who has an impacted upper incisor is immediately categorised as falling into the highest group iotn 5 and no further assessment of the dhc is required. a ruler has been designed containing all the information necessary to record the dental health component. the ruler has been developed for the clinical setting by which information is collected regarding competence of the lips, displacement on closure and masticary or speech problems. where there are no anomalies of tooth number or position, the ruler will figure 3. aesthetic component photographs for study model.7 figure 2. ruler used to measure the dental health component: (a) measuring over-jet; (b) measuring open bite. rules used to measure oj – 4.a rules used to measure contact point displacement 3.doj – 4.a a b 207hamid: treatment results evaluation be useful to measure the overjet (positive or negative), to see where this will place the patient. thus, an increased overjet in the range 6–9mm will be iotn 4. there are two ways of recording the dhc. the first is to record the grade only, and in the second the initiating feature would be recorded, for example, an overjet greater than 9 mm. would be graded as 5a (the grade being 5 and the overjet signified by letter a). the second method provides more information regarding the prevalence of the individual occlusal traits. the aesthetic component consists of ten scaled colour photographs showing different levels of dental attractiveness (figure 3).7 the dental attractiveness of prospective patients can be rated with reference to this scale. black and white photographs are used for dental cast assessments. grade 1 represents the most attractive tooth arrangement with grade 10 the least attractive arrangements of teeth. the score reflects the aesthetic impairment. monochrome photographs and dental casts have an advantages in that raters are not influenced by oral hygiene, gingival conditions or poor colour matches in restorations affecting anterior teeth.9 the grade of ac describes the aesthetic matters and priority for treatment, that is: grade 1–4 means no or slight need for treatment; grade 5–7 means borderline treatment need; while grade 8–10 which usually in this group including severe malocclusion thus it is grade need for treatment. the aesthetic component of the iotn consists of a ten-point scale illustrated by a series of photographs which were rated for attractiveness by a lay panel and selected as being equidistantly spaced through the range of grades.7 a rating is allocated for overall dental attractiveness rather than specific similarities to the photographs. the final value reflects the treatment need on the grounds of aesthetic impairment and by implication the sociopsychological need for orthodontic treatment. both parents and patients find this easy to apply and there is a high level of agreement between the scores obtained by dentists, parents and children. there seems to be a general agreement that a dhc of less than 4 and an ac score of below 7 do not justify treatment by a hospital based consultancy except for teaching or research purposes. to use this index for the survey, the examiner had been calibrated in the university dental hospital of manchester, in the united kingdom with some of others dentist. the samples for this study included of 202 study models from 17 orthodontic postgraduate students of the specialist orthodontic program at the faculty of dentistry, university of airlangga who had agreed to be evaluated. the study models taken were the ‘before’ and ‘after’ treatment study casts impression of the patients. the study casts which included in this study, should have good details of ‘before’ and ‘after’ treatment alignment and have no broken tooth in the casting model impressions (figure 4). the data were collected by using the index of orthodontic treatment need (iotn). firstly the study casts were measured using the dental health component ruler of the iotn for the dental health component (dhc) score. the aesthetic component (ac) of the iotn is also measured and scored by reference to 10 scaled standard black and white photographs for study model showing levels of attractivenes, from grade 1 (most) to grade 10 (least attractive). the scores are said to reflect the aesthetic impairment and need for treatment. the iotn assessment took only 1 minutes per pair of study cast. the data were measured and collected at the clinic of orthodontic postgraduate student at the university of airlangga dental faculty. the measurement of the dhc and the ac were recorded and calculated for the statistical test for the results of the calculation of dhc preand posttreatment as well as the ac pre-and post treatment to reinforce the hypothesis of this study. figure 4. example of the study model, “before” and “after” treatment. (a–d) study model before treatment –dhc: 3c : ac : 4, (e–g) study model after treatment –dhc: 2g : ac : 2 a b c d e f g 208 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 204-209 result it was observed that when the data analysed by statistical calculation using one sample kolmogorof-smirnov test showed the abnormal distribution as p : 0.00 < a : 0.05, then regarding this result, the statistical non-parametric was used for this study. the index of orthodontic treatment need (iotn) was separated into two components, i.e. the dental health component (dhc) and the aesthetic component (ac) to assist evaluation. in fact originally, the index of orthodontic treatment need was used as an objective measurement of need and demand for orthodontic treatment. the two component of the iotn usually inter-related to each other. to prove whether this index could be used for other purposes, thus some statistical tests were used for this study. the spearman‘s ranks correlation between dhc pre and posttreatment showed non-significant correlation since r = 0.118 with p : 0.096 > a : 0.05; it means the dhc pre treatment is not correlate to dhc after treatment while when tested the correlation between the two components before treatment, the correlation between dhc and ac before treatment according spearman’s rank correlation test, with r : 0.684 and p : 0.00 (< a : 0.05) showed a significance correlation, it means the dhc pre treatment is correlating to ac before treatment. if the dhc have a high score in its assessments, the ac tends to follow the grade of dhc as mentioned by some researchers previously.6,7 the correlation between dhc and ac after treatment according spearman’s rank correlation test, with r : 0.614 and p : 0.00 (< a : 0.05) showed a significance correlation, the dhc after orthodontic treatment correlating to ac after treatment, it means if the dhc have a lower score after treatment assessments, thus the ac follows the grade of dhc after treatment consecutively. as seen in the results after treatment for example, the dhc from 3c decreases to dhc 2g while the ac decreases from ac grade 4 to ac score 1 (figure 4). the same indicator to prove that the scores for each orthodontic cases, evaluated by using the two components of iotn could be used as assessment for evaluating the treatment results, the wilcoxon signed rank test, was performed for the dhc beforeand after treatment, the result of this test, with p : 0.000, (< a : 0.05) showed a significance difference between the dhc beforeand after treatment. it means the dhc before treatment have higher scores compared to dhc after treatment assessments. the same pattern showed similar results as ac before and aftertreatment according the wilcoxon signed rank test; with p: 0.000, (< a : 0.05) showed a significance difference. the ac before treatment have higher scores compared to ac scores after treatment assessments. the dhc beforeand aftertreatment according the wilcoxon signed rank test, with p : 0.000, (< a : 0.05) showed a significance difference and the aesthetic component beforeand after-treatment according the wilcoxon signed rank test, with p : 0.000, (< a : 0.05) showed a significance difference as well. discussion the examination of each 202 pairs study models using the index of orthodontic treatment need showed good results, table 1 showed the abnormal distribution. thus, a non parametric statistical test was applied for this study. the collecting of data was done fast, only taken two days of examination for the study models to measure the dhc and ac scores. this is due to the fact that the examiner was familiar to use the index of orthodontic treatment need. moreover, the index proved to be simple, efficient, easy to use and to learn . eventhough, the index of orthodontic treatment need may not an ideal one, but this index could be considered as good malocclusion index, sice it has fulfil some of criteria of goo index as stated by young and striffler.3 the examination only takes less than one minute if the malocclusion is not too complicated. the spearman‘s ranks correlation between dhc pre and posttreatment showed non-significant correlation since r = 0.118 with p : 0.096 (> a : 0.05); means the dhc pre treatment is not correlate to dhc after treatment. on the other hands, the correlation between dhc and ac after treatment according spearman’s rank corelation test, with r : 0.614 and p : 0.00 (< a : 0.05) showed a significance correlation. in this case it is obvious if the dhc and ac showed positive correlation because the position of teeth after treatment is almost always in a better or good alignment, then the appearance of the 10 scale photographs which is represent most of anterior teeth would be improved as well. accordingly, if the dhc is having high score, the ac will followed. the dhc before and after treatment according the wilcoxon signed rank test, with p : 0.000, (< a : 0.05) showed a significance difference which means there were improvement of dhc scores after treatment. if the dhc grade improves, for example from dhc 3c to dhc 2g means there is improvement of treatment, the treatment showed a good results. there is also a consistency with the aesthetic component. the aesthetic component before and after treatment according the wilcoxon signed rank test, with p : 0.000, (< a : 0.05) showed a significance difference, the ac grade improves, for example from ac 4 to ac 1 means there is improvement of treatment, the treatment showed a good results. the dhc and the ac are always corelate to each other. all results after treatment having a significant differrence, it could be concluded that the treatment results in dhc and ac as well showed a good improvement. the ac grade is decreasing to a smaller scores, since the appearance of the aesthetic improved means the result of treatment is good. the dhc and ac before treatment according the wilcoxon signed rank test, with p: 0.000, (< a : 0.05) 209hamid: treatment results evaluation showed a significance difference. the dhc and ac after treatment according the wilcoxon signed rank test, with p : 0.000, (< a : 0.05) showed a significance difference. the comparison between the dhc and ac before and after treatment showing much different. it means the treatment delivered by the orthodontics postgraduate students showed a good improvement of the facial aesthetic of the patients. the output of the clinical orthodontic teaching at the specialist training program showed a good successful training and fulfil the expectation of the patients. all the grades of dhc and ac were decreasing to a better score. the null hypothesis were rejected. the use of this index, the index of orthodontic treatment need (iotn) was very good that it could be able not only to evaluate the treatment, but it has been used used for other purpose such as the main purpose is to measure the treatment need in a population. it is concluded that the index of orthodontic treatment need (iotn) could be used to assess the orthodontic treatment outcomes, to evaluate before and after orthodontic treatment of the patients treated by the students of the orthodontic specialist at the dental school of the university of airlangga. the results of the treatment showed good improvements of dentofacial appearance of the patients which means the successful achievement of the clinical works programs. references 1. agusni t, barnard pd. assessment of dental-facial appearance and malocclusion of urban and rural schoolchildren in surabaya, indonesia. j dent res 1995; 74: 763. 2. cons nc, jenny j, kahout fj dai. the dental aesthetic index. iowa city: university of iowa; 1986. p. 10–16. 3. young wo, striffler df. the dentist, his practice and the community. 2nd ed. philadelphia, london, toronto: wb saunders; 1969. p. 15–29. 4. carlos. evaluation of indices of malocclusion. int dent j 1970; 20(4): 606–17. 5. angle eh. classification of malocclusion. dental cosmos 1899; 41: 248–64. 6. brook ph, shaw wc. the development of an orthodontic treatment priority index. eur j orthod 1989; 11: 309–20. 7. evans r, shaw wc. preliminary evaluation of an illustrated scale for rating dental attractiveness. eur j orthod 1987; 9: 314–18. 8. linder-aronson s. orthodontics in the swedish public dental health system. transactions of the european orthodontic society 1974; 4: 233–40. 9. woolass kf, shaw wc. validity and reproducibility of rating dental attractiveness from study casts. br j orthod 1987; 14: 187–90. subjects index volume 54 3% binahong extract gel, 57 aesthetic zone, 160 aloe vera 90% gel, 124 angle’s classification of malocclusion, 96 anthropometry, 96 anticancer, 210 antifungal, 82 antioxidant, 87 asymmetry, 21 bag, 165 binahong, 57 biofilm, 63 bitewing radiographs, 35 bleaching, 87 bone graft, 11 tissue engineering, 68 bovine hydroxyapatite, 11 calcium hydroxide, 174, 181 candida albicans, 82 canine rotation, 174 capsaicin, 210 carbohydrate intake, 46 carbonated hydroxyapatite, 16 caries, 165 cephalometric, 200 x-ray, 128 children, 205 chinese student, 132 chitosan-hydroxyapatite scaffold, 68 class ii, 216 cleft lip with or without cleft palate (cl/p), 108 cobb’s angle, 74 compressive strength, 195 couple force, 174 covid tongue, 155 covid-19, 155 cpp–acp, 165 cultivation, 39 dai, 205 demineralisation, 78 dental caries prevention, 113 implant, 160 trauma, 1 derotation, 174 deutero-malayid, 96 diabetes, 137 digital radiograph, 21 dimensional stability, 195 dmf-t index, 46 edentulous, 160 educational game, 5 effectiveness, 25 elderly, 31 electrosurgery, 169 enamel, 165 hardness, 78 roughness, 78 endodontic infection, 174, 181 enterococcus faecalis, 181 eruption, 137 expansion, 39 facial height proportion, 96 family history, 108 ffq, 46 fibroblast, 190 fluoride, 165 formula milk, 113 g330t, 216 gingival-derived mesenchymal stem cells, 39 glucosyltransferase enzyme, 186 height, 200 hiv/aids, 82 home visits, 25 hsc-3 cell, 150 human & health, 160 amniotic mesenchymal stem cells, 68 hydroxyapatite, 119 gypsum puger, 11 hypoxia, 68 idiopathic scoliosis, 74 immediate placement, 160 imperata cylindrica, 150 indonesia, 216 inflammatory cells, 190 intracanal medicament, 181 lactoferrin, 113 lateral photograph, 132 maldi-tof ms, 52 malocclusion, 143, 205 mandible deviation, 74 mandibular, 21 masseter muscle, 143 maxillary sinus, 200 medicine, 186 micro-computed tomography, 11 micronucleus, 128 mild disability, 25 milk formula, 78 moringa oleifera, 63 mother-child pairs, 52 mtt assay, 150 multifactorial, 108 nance appliance, 174 n-hap, 165 nigella sativa, 181 nsf, 165 obesity, 46 occlusal characteristics, 92 occlusion, 92 ofi, 205 operculectomy, 169 oral cancer, 150 health care, 155 hygiene index simplified, 5 hygiene, 221 orthodontic indices, 205 oscc, 210 osteoblasts, 119 osteoclasts, 119 osteocytes, 119 p. gingivalis, 63 paediatric, 102 panoramic x-ray, 128 parents/caregivers, 25 pcr, 52 pediatric, 169 peptidoglycan glycosyltransferase, 221 pericoronitis, 169 periodontal ligament stem cells, 39 periodontitis, 16 persea americana mill.,190 pmn count, 124 polychromatic erythrocyte cell, 128 polymorphism, 216 pomegranate extract, 87 portunus pelagicus, 119 posterior crossbite unilateral, 143 premature loss, 102 prevalence, 1 preventive, 102 primary dentition, 92 proliferation rate, 39 promotion of oral health, 25 propolis, 16, 63, 221 extract, 186 quality images, 35 recurrence, 108 reminder sticker, 5 remineralisation, 78, 165 residual socket volume, 57 rs59983488, 216 runx2, 216 salivary volume; 31 zinc, 31 school, 25 students, 1 scratch assay, 150 second molar, 169 semg, 143 setting time, 195 sex estimation, 200 shear bond strength, 87 silicone, 35 slurry water, 195 socket healing, 57 soft tissue profile analysis, 132 space maintainer, 102 management, 102 sterilized milk, 113 streptococcus mutans, 52, 186 susceptibility and resistance, 82 taste disorder, 31 tcp, 165 temporal muscle, 143 terminal plane relationships, 92 tetracycline, 63 thymoquinone, 137 tooth extraction, 57, 124 socket healing, 190 tooth, 137 type 1 collagen, 16 iii gypsum, 195 vertical, 21 whey protein, 113 width, 200 wound healing, 124 authors index volume 54 aisy, aulia rohadatul, 96 anggraini, laelia dwi, 102 aniowati, fitri, 210 ariesanti, yessy, 190 astuti, eha renwi, 128 aulianisa, rona, 200 effendi, muhammad chair, 5 hanafiah, olivia avriyanti , 57 irianti, amaliyah nur, 78 jazaldi, fadli, 216 kamadjaja, michael josef kridanto, 68, 119 kunarti, sri, 165 kurniawan, hansen, 63 kusumawardani, banun, 39 kusumawati, indi, 16 lubis, hilda fitria, 132, 205 luthfiani, l., 113 marsetyo, riyan iman, 186 moorthy, kirubanandan sathya, 21 nainggolan, chindy fransiska br, 195 naini, amiyatun, 11 noor, tengku natasha eleena binti tengku ahmad, 169 oetomo, kimberly clarissa, 74 padmanabhan, vivek, 92 permatasari, dewi kania intan, 31 prativi, shinta amini, 35 qotrunnada, salsabila, 137 raisah, putri, 25 rezeki, sri, 82 roeslan, moehamad orliando, 150 sani, imelia arifatus, 221 sari, nila, 160 setianingtyas, dwi, 155 setiawan, ignatius, 46 siregar, yona pricilia anggi, 143 sitasari, putri intan, 174 soesilawati, pratiwi, 124 sosiawan, agung, 108 suratno, indes rosmalisa, 87 tedjosasongko, udijanto, 52 younus, mohamed salim, 1 zakaria, myrna nurlatifah, 181 zubaidah, nanik, 71 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. all manuscripts submitted to the journal must be written in english. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) 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articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content 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no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission  the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair. ac.id/mkg/about/submissions#onlinesubmissions  the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id 127 volume 45 number 3 september 2012 henoch-schönlein purpura in children: its relation to oral andits relation to oral and relation to oral andrelation to oral and oral and dental health arlette suzy puspa pertiwi department of pediatric dentistry faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: henoch-schönlein purpura (hsp) is a rare systemic small vessel vasculitis, which commonly occur in children between 2 and 10 years of age. the course of the disease is often self-limiting, although may manifest long-term renal morbidity. the severity of renal involvement decides about the prognosis of this disease. many factors can trigger the disease attack, which is the most common is bacterial invasion. since the oral cavity is often refer as infectious foci to other part of the body, it seemed rationally to be part that contribute the course of disease, thus management of these infectious foci, if possible, gives rise to an astoundingly good prognosis. purpose: this paper will describe a review on hsp and the possible association with oral and dental health since it might be related to the prognosis of hsp. reviews: rashes in children are common; they may develop a rash after prescription of antibiotics. nevertheless there are some childhood diseases that may manifest a rash presentation, such as hsp. it is important for pediatric dentist to have knowledge about hsp and consider the possibility of dental treatment or disease as potential triggers. conclusion: oral and dental condition may be the trigger cause of hsp attack. therefore, it is important for pediatric dental practitioner to be aware of the course of the disease in order to limit the expanding complications. key words: henoch-schönlein purpura, infectious foci, children abstrak latar belakang: henoch-schönlein purpura (hsp) merupakan vaskulitis pembuluh darah kecil sistemik yang jarang terjadi dan biasanya menyerang anak usia 2 hingga 10 tahun. penyakit tersebut seringkali dapat sembuh sendiri, tetapi pada jangka panjang dapat bermanifestasi dengan morbiditas ginjal. keparahan keterlibatan ginjal menentukan prognosis penyakit. banyak faktor yang dapat memicu serangan penyakit, tersering adalah invasi bakteri. karena rongga mulut sering kali merupakan fokus infeksi terhadap bagian lain dari tubuh, maka mempunyai peluang sebagai faktor pemicu timbulnya penyakit, sehingga penatalaksanaan fokus infeksi dalam rongga mulut, jika ada, dapat memberikan prognosis yang baik pada pasien. tujuan: makalah ini akan menggambarkan tinjauan mengenai hsp dan hubungannya dengan kesehatan gigi dan mulut berkaitan pengaruhnya terhadap prognosis hsp. tinjauan pustaka: ruam sering terjadi pada anak; pasien anak dapat memperlihatkan gejala ruam setelah pemberian antibiotika. selain itu, beberapa penyakit dapat bermanifestasi sebagai ruam, misalnya hsp. merupakan hal yang penting bagi dokter gigi anak untuk memiliki pengetahuan mengenai hsp dan mempertimbangkan perawatan atau penyakit gigi sebagai pemicu potensial. kesimpulan: keadaan gigi dan mulut dapat merupakan pencetus serangan hsp, oleh karena itu penting bagi dokter gigi anak untuk memahami perjalanan penyakit sehingga dapat membatasi komplikasi yang terjadi. kata kunci: henoch-schönlein purpura, fokus infeksi, anak correspondence: arlette suzy puspa pertiwi, c/o: departemen kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran, jl. sekeloa selatan 1 bandung, indonesia. e-mail: arlettesuzy@yahoo.com literature reviews 128 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 127–132 introduction henoch-schonlein purpura (hsp) is the most common type of systemic vasculitis in childhood and diagnosed when palpable purpura is present plus one of the following: diffuse abdominal pain, any biopsy showing predominant immunoglobulin a (iga) deposition, arthritis or arthralgia, and renal involvement.1 symptoms can begin in children, most commonly between the ages of 4 and 7 years, soon after an upper respiratory tract infection or streptococcal pharyngitis. children may develop arthritis, leading to pain. a rash may start as urticaria or erythematous maculopapules on the legs and buttocks. eventually these spots blend to form purpura in the skin. abdominal pain that can be quite severe may also present in some cases. children younger than 2 years with hsp are more likely to develop edema of various areas of their bodies, which is a result of leaky small blood vessels in the skin. kidney involvement can also cause edema, hematuria or proteinuria.2 although its cause is unknown, it is often associated with infectious agents such as group a streptococci3 and also as immune complex disease since iga, which is caused by mucosal infections, is known to play an important role in its immunepathogenesis.4a high prevalence of infectious foci in oral as well as ear, nose, and throat diseases was revealed in children with hsp.5 hsp is known to have a high probability of being spontaneously cured if supportive treatment is the primary intervention. however, it sometimes develops into severe conditions with a high rate of reoccurrence. no form of therapy has ever been shown to decrease the duration of the disease or prevent recurrences. nephritis is the most serious long-term complication of hsp. although early aggressive therapy has been recommended for children with such severe renal involvement, there is little evidence to indicate the best treatment for it.6 since the oral cavity is often refer as infectious foci to other part of the body, and also structured by mucous tissue, it seemed rationally to be related to hsp. management of these infectious foci, if possible, gives rise to an astoundingly good prognosis in reducing the recurrent attacks of hsp. tahmassebi3 in 2007 first reported a case of hsp following endodontic treatment. inoue, et al.7 in 2008, reported that dental caries (70%) along with apical periodontitis (53%) was found in hsp cases and conclude that early and extensive treatment for these lesions may prevent the complication of hsp. this paper will discuss an overview of hsp and the possible relation of oral focal infection, which will be valuable to pediatric dentists, since, although hsp is generally a benign, self-limited condition, oral and dental health of the patient might be related to the prognosis of hsp. epidemiology and etiology henoch-schönlein purpura (hsp) is an inflammatory disorder characterized by a generalized vasculitis involving the small vessels of the skin, gastrointestinal (gi) tract, kidneys, joints, and, rarely, the lungs and central nerve system. the syndrome takes its name from two german physicians. in 1837, johan schönlein first described several cases of peliosis rheumatica or purpura associated with arthritis. thirty years later, edouard henoch described the gi manifestations, including vomiting, abdominal pain, and melena. henoch-schönlein purpura has also been referred to as rheumatic purpura, leukocytoclastic vasculitis, and allergic vasculitis.8 although the exact cause of hsp is unknown, exposure to various infective pathogens, drugs, vaccines, food allergens and insect bites may be possible immunological triggers.9 an upper respiratory tract infection (urti) preceding presentation with hsp by some days or weeks has been reported in up to 50% of cases and the occurrence of hsp in children particularly in the autumn and winter months suggests an infectious etiology.9–11 in patients with hsp, immunoglobulin a (iga) immune complexes are deposited in small vessels, as a result of exposure to an antigen from an infection, medication, or other environmental factors. group a streptococci, which can cause an upper respiratory tract infection, are the most common pathogenic microorganisms that cause hsp.12 several dermatological or autoimmune diseases are thought to correlate with odontogenic infectious diseases. for example, burger's disease has been linked to periodontitis, or palmoplantar pustulosis and chronic pigmented purpura have also been reported to have an association with an oral focal infection.6 hsp is considered to be associated with odontogenic infectious diseases as well. there are a few reports that mention the correlation between hsp and odontogenic infectious diseases. jinous et al.3 have reported a case of hsp that had developed after endodontic treatment. this report suggested that root canal treatment could be a trigger for hsp, as it assumed that trepanation of the apex may cause a streptococcal bacteremia. environment and microbiological flora changes in the root canal may also cause a bacteremia. inoue et al.,7 have reported on the efficacy of dental treatment in preventing nephropathy in pediatric hsp. igawa et al.,13 reported that an oral focal infection could be a precipitating factor for adult hsp, as improvements in the skin lesions were observed after patients being treated for the oral infection. hsp preferentially affects children aged 2–11 years. the median age is 5 years and occurs a slight predominance in males, which is twice as females. the condition has been reported to have an incidence of 10–20 cases per 100 000 school-aged children each year.3,11,14 more than 90 percent of patients are children younger than 10 years, with a peak incidence at six years of age. however, it is also seen in infants, adolescents, and adults. hsp is milder in infants and children younger than two years. disease is more severe in older children and adults, especially with regards to renal involvement.12 the true prevalence may be underestimated because cases are often not reported. as seen in indonesia, the exact data about the prevalence hsp is still unclear. 129pertiwi: henoch-schönlein purpura in children according to secondary data in harapan kita hospital jakarta during 6 years period (2004–2010), it was reported 70 cases of hsp in 2–16 years old children with the rate for boys are higher (55.7%) than girls (44.3%).15,16 based to ethnic groups, hsp has a higher prevalence in caucasians and asians than in those of african descent.17 the disease occurs more often in the colder months and is usually preceded by an upper respiratory infection, particularly streptococcal, but a recent study suggests that an occult malignancy may be the cause.12 despite the exact etiology of hsp remains unknown, histologically hsp exhibits an immune mediated leukocytoclastic vasculitis, with deposits of immunoglobulin a (iga) and its immune complexes within the walls of involved vessels and organs. patients have elevated serum levels of iga, iga immune complexes, iga anticardiolipin antibodies, and transforming growth factor-ß, as well as altered iga glycosylation.18 antigen and antibody complexes, mostly iga, form as a result of bacterial and viral infections, vaccinations, drugs, and autoimmune mechanisms. these antigen antibody complexes deposit in the small vessel walls and activate the alternate complement pathway that leads to neutrophil accumulation resulting in inflammation and vasculitis without a granulomatous reaction. this can involve multiple systems including skin, gastrointestinal tract, kidney, and joints but it can involve any organ system. vasculitis causes extravasation of blood and its components into the interstitial spaces resulting in edema and hemorrhage.19 clinical feature the major clinical manifestations of hsp are purpura, arthritis, abdominal pain, gastrointestinal bleeding, and henoch-schönlein purpura nephritis (hspn).7 the most common clinical manifestations are illustrated in figure 1. these can develop over days to weeks and may vary in the order that they present. it can masquerade as many different conditions, depending on the symptoms. palpable purpura and joint pain are the most common and consistent presenting symptoms; initial diagnosis of hsp in the absence of these symptoms may not be obvious.18 the classic rash (figure 2) of hsp begins as erythematous, urticarial and macular wheals. it then coalesces and develops into the typical ecchymoses, petechiae, and palpable purpura. the rash occurs in 96% cases, often manifests in a symmetrical pattern at pressure dependent areas, such as the lower extremities and the figure 1. the four classic features of henoch-schönlein purpura.18 figure 3. typical distribution of palpable purpura in henochschönlein purpura.20 figure 4. henoch-schönlein purpura of the upper limb with swollen elbow joint.21 figure 2. closer look of skin rashes.20 130 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 127–132 buttocks (figure 3). in no ambulatory children the face, trunk, and upper extremities may be more affected. may be urticarial and cause edema, particularly in children.18 joints are involved in the majority of cases, involving lower limbs (ankles and knees) more commonly.21 arthralgia occurs in 84% of hsp patients and often coexists with other symptoms. the large joints of the lower extremities are most commonly affected. it is none destructive arthritis. transient oligoarticular arthritis and periarticular swelling may cause pain, tenderness and restricted movement (figure 3).18 gi pain is often the most debilitating of the hsp symptoms, and can be further complicated by git hemorrhage (14–38%), intussusception, obstruction or perforation.21,22 gi problems started as cramping abdominal pain, often with vomiting, appears about a week or more after the rash begins. although there are cases where gi problems occur without a rash. 25% have gib and 50% have occult blood loss. on endoscopy you see pupuric lesions +/– edema, ulceration, or bowel spasm (figure 5).8 renal symptoms have a wide range of severity, from asymptomatic microscopic hematuria, to full-blown nephritic syndrome or nephritis. most renal involvement occurs early, 85% within the first month, although it can develop later; follow-up to 6 months is recommended.21 begins few days to weeks after other symptoms. urine analysis can show proteinuria (mild), red blood cells, and cellular casts. many patients will be asymptomatic, but others can develop nephritic syndrome.8 other symptoms are rare and usually involve the central nervous system or lungs, from pulmonary hemorrhage through to convulsions. age does play a role in the symptomatology, with children younger than two years showing predominantly cutaneous symptoms and signs, as well as a much lower incidence of renal and gastrointestinal involvement. the peak incidence is in the 4–6 year-old age group with figures around 70/100 000 population, with a very slight male predominance. recurrence is relatively common and 30% of patients will have one or more recurrences of acute vasculitis. the average duration of disease is 4 weeks and while steroids will shorten this period, current data suggest there is no correlation between steroid use and increased frequency of relapse.21 diagnosis, differential diagnosis and prognosis diagnosis of hsp depends on clinical findings and history. it is usually not difficult if the classic triad of rash, gastrointestinal complaints or hematuria, and arthritis is present. when symptoms are not typical, however, the differential diagnosis can become extensive. there is not a specific laboratory test for the disorder, although an elevated serum iga level is suggestive. some laboratory studies can also help in excluding other diagnoses and in evaluating renal function, including urinalysis.8 hsp is a clinically obvious condition in the majority of cases, but laboratory investigation would include: full blood count, to exclude thrombocytopenia; most often thrombocytosis is found in hsp. anemia may be present but is usually an indicator of git hemorrhage or severe hematuria. renal function is obviously very important and assists in identifying some with a rapidly progressive glomerular disease. erythrocyte sedimentation rate (esr) is elevated in approximately 60%, but is a nonspecific inflammatory marker. iga levels are elevated in 25–50% of patients. albumen levels are diminished in cases of nephritic syndrome and/or protein-losing enteropathy, which may occur. occult fecal blood is seen in 25%. factor xiii plasma levels can be measured in atypical cases and are decreased in the majority, even prior to purpura formation. skin biopsy is a useful diagnostic tool in atypical cases, and reveals a typical leucocytoclastic vasculitis with necrosis of the vascular wall and inflammatory cell infiltrate, accompanying iga dermal deposition.21 vasculitis is not a common childhood condition and hsp is difficult to confuse with other small-vessel vasculitides, but a relatively short list of alternative possibilities, which includes kawasaki disease. most of these conditions can be excluded or diagnosed clinically, but immune serological markers and a full blood count will distinguish doubtful cases.21 generally the prognosis is good, with the exception of those with significant renal involvement.23 hsp is only fatal in the most rare of cases. initial attacks of hsp can last several months, and relapses are possible. kidney damage related to henoch-schoenlein purpura is the primary cause of morbidity and mortality. overall, an estimated 2% of cases progress to renal failure; as many as 20% of children who have hsp and are treated in specialized centers require hemodialysis. the renal prognosis appears to be worse in adults than in children.24 management management is mainly supportive and symptomatic. most patients can be managed as outpatients with treatment being directed at adequate oral hydration and pain relief. figure 5. e n d o s c o p i c f i n d i n g o n e s o p h a g u s s h o w i n g inflammation, sub mucosal hemorrhage, and small ulceration.19 131pertiwi: henoch-schönlein purpura in children edema of the lower extremities, buttocks, and genital area are improved with bed rest and elevating the affected area.18 non-steroid anti inflammations drug also may reduce the inflammation.25 no effective therapeutic protocol to reduce recurrent attacks of hsp or prevent the complication of nephropathy has been established.21 discussion although hsp is a rare inflammatory disorder of childhood, it has clinical significance. the most serious sequel of hsp is renal involvement. this complication occurs in 50% of older children, but only 25% of children younger than 2 years of age. less than 1% of cases progress to end-stage renal disease. patients who develop renal involvement generally do so within three months of the onset of rash.3,26,27 hsp is a self-healing benign disorder, but renal and gastrointestinal involvement can lead to poor prognoses. odontogenic focus infection (ofi) as one of several trigger cause to hsp attack need to be consider as one of the risk factors that determine the prognosis of hsp. ofi is a bacterial infection that tends to be overlooked by dermatologists. dental screening of hsp patients could help to decrease the risk of renal and/or abdominal complications and facilitate treatment.13 the concept that focal infection may produce chronic systemic diseases has now been generally accepted. local, septic, or mucosal infections foci anywhere in the body can be sources of systemic diseases. to date, foci of specific infections of the gums and the presence of abscesses around the roots of the teeth, often unsuspected, have not received attention in the treatment of pediatric diseases.7 the etiological role of chronic oral infection in hsp is supported by several other studies. the antigens of the outer membranes of haemophilus parainfluenzae, a common bacterium within apical periodontitis, and antibodies against these have been identified in the glomerular mesangium and sera of hsp and iga nephropathy patients.28 in one case, endodontic dental therapy coincidentally induced hsp.3 given that iga nephropathy and hspn are pathologically identical diseases, all of these data suggest that chronic infections in the oral cavity may play pathogenic roles in hsp.7 the high caries levels of hsp children may support this view. dental caries in premature teeth easily invade through infected root canals into surrounding bony tissues, forming apical periodontitis.29 the most commonly identified ofi in hsp patients was apical periodontitis in association with dental caries. although both are infectious diseases by nature, apical periodontitis, which is mostly initiated from dental caries by oral bacteria invading through infected root canals, is a much more complex disorder in regard to infection as well as inflammation. a thousand billion bacteria colonize a single lesion, and more than 300 species of aerobic and anaerobic bacteria can be isolated. within the associated lesions, various inflammatory cytokines are produced by cellular components of the periapical lesion, resulting in the persistence of active immune reactions.7 many degraded bacterial products and the decomposition products of pulp tissue stagnate there. meanwhile, bacteria and their toxic derivatives and destroyed peripheral tissues may egress through the apical foramen and be captured continuously within tonsils through their surface epithelium.30 however, the innate secretory iga-mediated oral mucosal defense system may fail to eliminate bacterial antigens owing to the presence of a tremendous amount of bacteria. on the other hand, bacterial pathogens may enter the blood stream during transient bacteremia, damaging the inside smooth lining of the blood vessel walls. collectively, chronic and long-standing apical periodontitis have the potential to trigger hsp.7 it is concluded that hsp is the most common systemic vasculitis primarily affecting children aged 3–15 years. hsp is characterized by palpable purpura without thrombocytopenia or coagulopathy, arthritis or arthralgia, abdominal pain, and renal disease. diagnosis depends on clinical manifestations and no single diagnostic test can confirm the disease. management is mainly supportive and symptomatic and can usually occur in the ambulatory setting. oral and dental condition may be the trigger cause of hsp attack. therefore, it is important for dental practitioner to be aware of the course of the disease in order to limit the expanding complications. references 1. ozen s, ruperto n, dillon m. eulars/pres endorsed consensus criteria for the classification of childhood vasculities. ann rheum dis 2006; 65(7): 936–41. 2. punnoose a, lynm c, gollub r. henoch-scho ̈nlein purpura. jama 2012; 307(7): 742. 3. tahmassebi jf, paterson sa. development of acute henoch– schönlein purpura subsequent to endodontic treatment. int j paed dent 2007; 17: 217–22. 4. kilis ́-pstr usin ́ska k. paediatric henoch-schönlein pur pura– immunological and clinical aspects for a family doctor. int rev allergol clin immunol family med 2012; 18(1): 41–5. 5. nakaseko h, osamu u, takuhito n, satoshi y, yoshiko h, yasuhito y, yamamoto m. high prevalence of sinusitis in children with henoch-scho ̈nlein purpura. int j paed 2011; 15:1–3. 6. abe m, mori y, saijo h, hoshi k, kazumi o, takato t. the efficacy of dental therapy for an adult case of henoch–schönlein purpura. oral sci int 2012; 9: 59–62. 7. inoue c, nagasaka t, matsutani s, masako i, rikako h, yasushi c. efficacy of early dental and ent therapy in preventing nephropathy in pediatric henoch-schönlein purpura. clin rheumatol 2008; 27: 1489–96. 8. tarvin se, ballinger s. henoch schonlein purpura. curr. ped 2006; 16: 259–63. 9. saulsburry f. henoch-schönlein purpura in children: report of 100 patients and review of the literature. medicine 1999; 78(6): 395–406. 10. trapani s, micheli a, grisolia f, resti m, chiappini e, falcini f, de mar tino m. henoch schonlein pur pura in childhood: epidemiological and clinical analysis of 150 cases over a 5-year period and review of literature. semin arthritis rheum 2005; 35(3): 143–53. 132 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 127–132 11. yang y, hung c, hsu c, wang l, chuang y, lin y, chiang bl. a nationwide survey on epidemiological characteristics of childhood henoch-schonlein purpura in taiwan. rheumatol 2005; 44(5): 618–22. 12. reamy b, williams p, lindsay t. henoch-schönlein purpura. am fam psychians 2009; 80(7): 698–704. 13. igawa k, satoh t, yokozeki h. possible association of henoch– schönlein purpura in adults with odontogenic focal infection. int j dermatol 2011; 50(3): 277–9. 14. dolezalova p, telekesova p, nemcova d, hoza j. incidence of vasculitis in children in the czech republic: 2-year prospective epidemiology survey. j rhematol 2004; 31(11): 2295–9. 15. widjayanti m. manifestasi dan komplikasi gastrointestinal pada purpura henoch schonlein. sari pediatri 2012; 13(2): 334–9. 16. lestari e. manifestasi renal pada anak dengan purpura henochschoenlein. sari pediatri 2012; 14(1): 36–9. 17. gardner-medwin j, dolezalova p, cummins c, southwood t. incidence of henoch-schonlein purpura, kawasaki disease, and rare vasculitides in children of different ethnic origins. lancet 2002; 360: 1197. 18. lim d, cheng l, wong f. could it be henoch-schönlein purpura?. australian family physician 2009; 38(5): 321–4. 19. sohagia a, gunturu s, tong t, hertan h. henoch-schonlein purpura—a case report and review of the literature. gastroenterology research and practice 2010: 1–7. 20. bain s. physical signs for the general dental practitioner. case 62. henoch-schonlein purpura. dental update 2009; 36(2): 124. 21. sinclair p. henoch-schönlein purpura – a review. current allergy & clinical immunology 2010; 23(3): 116–20. 22. setiabudiawan b, ghrani r, sapartini g, sari n, garna h. bula hemoragik dengan komplikasi perforasi gaster sebagai manifestasi klinis purpura henoch-schonlein yang tidak biasa pada anak. sari pediatri 2011; 13(4): 257–64. 23. watson l, richardson a, holt r, jones c, beresford m. henoch schonlein pur pura – a 5-year review and proposed pathway. plosone 2012; 7(1): 1–7. 24. chartapisak w, opastiraku s, willis ns, craig jc, hodson em. prevention and treatment of renal disease in henoch-schonlein purpura: a systematic review. arch dis child 2009; 94(2): 132–7. 25. syafri m, kurniati n, munazir z. pemberian steroid pada purpura henochschonlein serta pola perbaikan klinis di departemen ilmu kesehatan anak fkui/rscm jakarta. sari pediatri 2008; 10(4): 268–71. 26. tendean s, siregar s. henoch-schonlein purpura: laporan kasus. sari pediatri 2005; 7(1): 45–9. 27. sugiyama h, watanabe n, onoda t, kikumoto y, yamamoto m, maeta m, ohara n. successful treatment of progressive henochschönlein purpura nephritis with tonsillectomy and steroid pulse therapy. internal medicine 2005; 44(6): 611–5. 28. ogura y, suzuki s, shirakawa t. haemophilus parainfluenzae antigen and antibody in children with iga nephropathy and henochschönlein nephritis. am j kidney dis 2000; 36(1): 47–52. 29. inouea c, matsutanib s, ishidoyab m, hommab r, chibaa y, nagasakac t. periodontal and ent therapy in the treatment of pediatric henoch-schönlein purpura and iga nephropathy. adv otorhinolaryngol 2011; 72: 53–6. 30. jouhula o. henoch schonlein purpura in children. dissertation. acta universitatis ouluensis, medical; 2012. �� vol. 45. no. 1 march 2012 effectiveness of bleaching agent on composite resin discoloration galih sampoerno department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: the discoloration of teeth, especially anterior teeth, is one of aesthetic problems. the use of tooth bleaching agents for discolored natural teeth is becoming increasingly popular. many dentists, however, get many problems when they conduct bleaching process since there is much composite filling on patient’s anterior teeth. although many research have focused on the discoloration of composite resin after bleaching process, the problem still becomes debatable. purpose: the purpose of this study was to investigate the difference of the discoloration between hybrid composite and nano composite before and after the application of tooth bleaching agent, 38% hydrogen peroxide. methods: eighteen disk-shaped specimens (5 mm) of each of two composite resins, hybrid and nano filler, were prepared. the each group was treated 3 times and the specimens were divided into two groups consisted of 9 specimens for each, and then immersed in black tea solutions for 72 hours. next, after having staining and bleaching processes, the color of the specimens was measured with a optic spectrophotometer by using photo with type bpy-47 and digital microvolt. the differences of the light intensity among three measurements were then calculated. afterwards, glm manova repeated measure and parametric analysis (independent t-test and paired t-test) were then used to analyze the data. results: after staining process, it is then known that the nano composite had more discoloration and more affected by the black tea solution than the hybrid one. conclusion: after bleaching, the discoloration was finally removed completely from both hybride and nano filler composite resins and became brighter from the baseline color. key words: discoloration, composite resins, bleaching agent abstrak latar belakang: salah satu problem estetik adalah adanya perubahan warna pada gigi anterior. peningkatan pemakaian bahan bleaching semakin popular. banyak dokter gigi mempunyai problem ketika mereka akan melakukan proses bleaching dan ditemukan banyak tumpatan komposit pada gigi anteriornya. meskipun telah banyak penelitiahan yang dilakukan tetapi masalah ini masih menjadi pro dan kontra. tujuan: melihat perbedaan perubahan warna antara komposit hybrid dan komposit nano sebelum dan sesudah terpapar bahan pemutih gigi hidrogen peroksida 38%. metode: tiap kelompok mendapatkan perlakuan 3 kali. dipersiapka 18 spesimen berbentuk tabung (5 mm) dan dibagi menjadi 2 kelompok masing-masing 9 untuk resin komposite hybrid dan komposit nano. direndam larutan teh hitam selama 72 jam. perubahan warna diukur dengan spektrofotometer tipe bpy-47 dan mikrovolt digital, baik sebelum, setelah perendaman larutan teh hitam dan setelah proses bleaching. perbedaan intensitas cahaya dihitung dengan glm manova repeated measuse dan analisa parametrik. hasil: setelah perendaman teh hitam, komposit nano mempunyai perubahan warna yang lebih gelap dibandingkan komposit hybrid. setelah bleaching, baik komposite hybrid dan komposit nano kembali ke warna aslinya. tetapi perubahan warna komposit nano lebih besar dari pada komposit hybrid. kesimpulan: setelah bleaching, baik resin komposit hybrid dan nano menjadi lebih terang dari warna aslinya. kata kunci: perubahan warna, resin komposit, bahan bleaching correspondence: galih sampoerno, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: sampoernog@gmail.com research report �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 43–47 introduction the main concern in the dental profession is currently not only the prevention and treatment of disease, but also with the aesthetic factor.1 the discoloration of teeth, especially front teeth, is one of the aesthetic problem that can be experienced by everyone, especially those whose profession is directly related to public service, such as television broadcasters, artists, teachers, secretaries, etc. thus, it can lead to psychological disturbances, such as lack of confidence, embarrassment, and low self-esteem. the aesthetic factors are mostly influenced by the nature of smile, the shape of a symmetrical face, the neat row of teeth, and the color of teeth.2 the discoloration of teeth can also occur in composite restoration that comes from intrinsic and extrinsic factors. intrinsic factor occurs due to the changes of the composite resin matrix itself, the separation of matrix and filler materials, and the oxidation or hydrolysis of the composite resin matrix. extrinsic factors are caused by the absorption of the dye as a result of the contamination of the various exogenous sources, for instance, the habit of consuming foods and beverages contributes to the formation of stain or stain on the tooth surface, such as black tea, coffee, red wine, and soft drink.3 the colored beverage most widely consumed in the world is actually tea.4 tea mostly sold in markets is black tea, about 98%. and, since tea contains tannin and polyphenol molecules absorbed by the surface of enamel, it can cause the discoloration of the teeth.5 tooth bleaching can also be considered as one of the aesthetic treatments that is often conducted since it is easy to be conducted, far more conservative than the method of restoration, not necessary to have laboratory work, relatively simpler and cheaper to be implemented, and not necessary to have much tissue removed. therefore, tooth bleaching becomes more popular and attractive to everyone, from young people to adult ones.6-7 nowadays, many new products of tooth bleaching have recently emerged in markets.8 however, if materials of those products contact with tooth structure for long periods of time, it will also bleach tooth materials.9 as a results, many dentists have difficulties to whiten teeth if there are so many composite casts in anterior teeth, especially if the composite restorations are still in good shape, either in terms of color and anatomy. thus, since the discoloration of the composite resin becomes a problem, many studies were then conducted to determine the effect of tooth bleaching on composite resin.10 tooth bleaching conducted on vital teeth, for instance, can affect the restoration of composite resin which dark color will change into lighter one.11 the discoloration of composite resin veneer can be eliminated partly through in-office tooth bleaching and repolishing procedures.12 the use of 10% carbamide peroxide or 10% hydrogen peroxide can lead to the discoloration of composite resin into lighter one.13 but, for teeth with the discoloration of composite restoration class iv, tooth bleaching must be conducted with external in office procedures by using 35% hydrogen peroxide and heat that then will make the teeth and the restoration lighter.14 actually, there are many studies focusing on the discoloration problem of composite resin caused by tooth bleaching process, but the problem still become debatable nowadays, in terms of advantages and disadvantages. a research states that if the composite resin is exposed to tooth bleaching agents, it will cause the leakage of the edge of the restoration, reduce the strength and surface roughness of the composite. on the other side, another study states that tooth bleaching causes few or almost no negative effects on all restorative materials. thus, if composite resin is exposed to tooth bleaching agents, it will only cause less prone to surface roughness, and make compressive strength increased, although it has significant meaning. in other words, although tooth bleaching cause a lot of oxygen released into teeth, the strength of the existing attachment of the restoration will not be reduced or become weak. however, after being exposed to tooth bleaching agents, tooth structure that contains lots of oxygen do not provide a good surface condition for binding new restoration.15 for these reasons, it is necessary to study the effects of tooth bleaching agent, 38% hydrogen peroxide, on the different discoloration problem of composite resins with hybrid type and nano filler type. thus, this study is aimed to determine the difference of the discoloration of hybrid composite resin and nano filler composite before and after the application of tooth bleaching agents, 38% hydrogen peroxide. materials and methods each group was treated 3 times and the sample groups were then distinguished based on the type of composite resin and the application of tooth bleaching agent. group 1 was composite resin with hybrid type applied with tooth bleaching agent, 38% hydrogen peroxide. group 2 was composite resin with nano filler type applied with tooth bleaching agent, 38% hydrogen peroxide. to create the samples, plastic rings with a diameter of 5 mm and with a height of 2 mm were fixated with modeling wax on the glass slab. two mm composite resin was removed from the tube with plastics filling and then put on the plastic rings. those rings were closed by celluloid strip, and glass slab was put on the top of them. the excess resin was then cleaned. both sides of them were light cured with the distance of 0 cm between the surface of the tools and materials, and the tip of light curing unit was perpendicular to the surface of the rings. light curing was conducted for 20 seconds on both sides with the intensity of 450 mw/cm2. polishing was conducted for 10 minutes. after the first light cured, polishing was conducted with superfine diamond burs for 3 times with one way shear direction of movement followed by polishing with urethane dimethacrylate resin (pogo) for 3 times with one way shear direction of movement without water. polishing was ��sampoerno: effectiveness of bleaching agent then conducted by using low speed micrometer (10,000 rpm) with light pressure. after polishing, all samples were then washed with water as much as 10 ml. next, steeping tea was made by dipping a black tea bag in 150 ml of boiling water for 4 minutes, and then the tea bag was removed. afterwards, the whole parts of the samples of the composite resin were immersed in black tea for 72 hours. the dye was slowly inserted into the resin composites after the immersion for 3 days,16 because of the inhibition process. every 24 hours, the solution of black tea was replaced in order not to make fungus aspergillus formed.17 the composite resin was then washed with distilled water, dried, and stored in artificial saliva. the composite resin, as a result, had discolored due to the immersion in black tea, and then exposed to tooth bleaching agent, 38% hydrogen peroxide. prior to the bleaching process, composite resin was polished with pumice and then washed with distilled water. the surface of the composite resin was then bleached and cured as much as 15 rounds (10 minutes). one rotation consisted of both the exposure duration of 30 seconds and the rest duration of 10 seconds. after the tooth bleaching process was completed, the composite resin was washed with running water for 1 minute, dried with suction paper, and stored in artificial saliva. before measurement was conducted, the samples were cleaned with water. the measurement was conducted through laser from helium neon gas laser with uniphase brand which size of the light was reduced by using gap of optical spectrophotometer. the light was dropped on the samples, and then the intensity of light was emitted from the samples. the measurement was conducted by using photo with type bpy-47 and digital microvolt, with the unit of lux (lumen/m2) and the scale of 102. it then could indicate the size of the intensity of the light absorbed by the sample by reducing the intensity light intensity that came from one that was reflected.18 results the results of kolmogorov-smirnov test on the hybrid composite resin and the nano filler composite resin showed the value of p>0.05. this indicates that either the hybrid composite resin groups or the nano filler composite resin groups had a normal distribution of data. to know the difference of the light intensity of the hybrid composite resin and the nano filler composite resin among all groups including before the treatment, after the immersion in black tea, and after the application of tooth bleaching agent, glm manova repeated measures was conducted. the results showed that p-value was about 0.001. this indicated that there were significant differences of the light intensity of both hybrid composite resin and nano filler composite resin including before the treatment, after the immersion in tea, and after the application of tooth bleaching agent without distinguishing the type of composite resin. furthermore, to know which treatment groups of the hybrid composite resin have the different light intensity, paired t-test was conducted. the difference of the light intensity among all treatment groups of the hybrid composite resin was about 0.001 (p<0.05). it indicates that there were significant differences of the light intensity before and after the immersion in tea, after the immersion in tea and after the application of tooth bleaching agent, and before the treatment and after the application of tooth bleaching agent. moreover, to know the difference of the light intensity in the nano filler composite resin, paired t-test was conducted. the difference of the light intensity among all treatment groups of the nano filler composite resin was about 0.001 (p<0.05). it indicated that there were significant differences of the light intensity before and after the immersion in tea, after the immersion in tea, after the immersion in tea and after the application of tooth bleaching agent, and before and after the application of tooth bleaching agent. then, to know both the differences of the light intensity between the hybrid composite resin and the nano filler composite resin before and after the immersion in tea and the differences of the light intensity after the immersion in tea and after the application of tooth bleaching agent, independent t-test was conducted. the difference of the light intensity between the hybrid composite resin and the nano filler composite resin before the treatment was about 0.102 (p>0.05). it indicated that there was no significant difference of the intensity of the light between the hybrid composite resin and the nano filler composite resin before the treatment (table 1). but, there table 1. the results of independent t-test on the light intensity of the hybrid composite resin and that of the nano filler composite resin treatment composite p value of independent ttest before hybrid p = 0.102 nano filler the difference before and after the immersion in tea hybrid p = 0.006 nano filler the difference after the immersion in tea and after the application of tooth bleaching agent hybrid p = 0.004 nano filler �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 43–47 was the difference of the light intensity differences before and after the immersion in tea, p-value = 0.006 (p<0.05). this suggests that there were significant differences of the light intensity before and after the immersion in tea. and, it is also known the differences of the light intensity between after the immersion in tea and after the application of tooth bleaching agent, p-value = 0.004 (p<0.05). this suggests that there was a significant difference of the light intensity after the immersion in tea after the application of tooth bleaching agent. discussion in the study, the results showed that both of the hybrid composite resin and the nano filler composite resin had got discoloration. they even had different light intensity after being exposed to tooth bleaching agent, hydrogen peroxide 38%. the change of the light intensity into darker one after the immersion in black tea was because tannin dye in black tea could cause the formation of brown spots on the surface of the composite. tannin in tea is known to have polar structure that can make the chemical bonds stronger physically, and also make the absorption occur. moreover, tannin can easily oxidize oxygen out and in water causing the easier change of the color into darker one.19 the study used optic spectrophotometer so that the infrared light emitted from the laser was absorbed more by the samples that had been immersed in black tea. this indicates that the color of the samples that had been immersed became more reddish or brownish. in the bleaching process, the samples were bleached by using hydrogen peroxide 38%. the results showed that the light intensity of the hybrid composite resin and the nano filler composite resin had changed into brighter color. this increasing of the light intensity was caused by hydrogen peroxide that can remove any spot attached to the composite resin by breaking h2o2 derived from tooth bleaching agents into h2o+oµ which not only have the ability to generate free radicals that are highly reactive, but also have unpaired electrons.20 unpaired electrons or highly reactive o-, therefore, would oxidize tannin from black tea which attached to the composite resin matrix resulting the change of the color of the composite resins into brighter one. immersion in black tea can cause the decreasing of the light intensity of both of the hybrid composite resin and the nano filler composite resin. however, the magnitude of the decreasing of the light intensity was significantly different between the hybrid composite resin and the nano filler composite resin. it means that the decreasing of the light intensity of the nano filler composite resin was greater than that of hybrid composite resin. thus, it can be indicated that the application of hydrogen peroxide 38% can cause the increasing of the light intensity of both of the hybrid composite resin and the nano filler composite resin. nevertheless, there was the significant difference of the increasing of the light intensity between the hybrid composite resin and the nano filler composite resin. the light intensity of hybrid composite was smaller than that of nano filler composite resin. this suggests that if the nano filler composite resin was exposed to tooth bleaching agent, the light intensity of the nano-filler composite resin would be greater than that of hybrid composite resin. it is because the volume fraction in the hybrid composite resin was bigger, about 60%, than that in the nano filler composite resin, only about 57%59.5%.21 as a result, the low volume fraction can cause the increasing size of the gap between the composite resin matrix and the filler material, so it cause more absorption of tooth bleaching. the amount of the nano filler composite resin absorbing tooth bleaching agent makes the oxidation of tannin derived from a black tea attaching to the composite resin matrix by the obe more effective. in addition to the hybrid composite resin, the composition of tegdma has actually been replaced by using a combination of udma and bisphenol a polyethylene glycol diether dimethacrylate (bis-ema). both of these resins have higher molecular weight, and more double bonds per weight unit, so shrinkage can be reduced and it modulus of elasticity can also be higher than the nano filler composite resin using a combination of bisgma/bis-ema/udma and small amount of tegdma. free radicals released by tooth bleaching agent then can destroy the composite resin matrix through a process of oxidation and degradation that is by destroying the bonds of monomers contained in the composite matrix. the degradation will occur by breaking the chain of low molecular weight, namely tegdma considered as diluent in the organic resin matrix with low viscosity. the hybrid composite has a composition of resin matrix with higher molecular weight and more bonds than the nano filler one. therefore, if the nano filler composite is exposed to tooth bleaching agent, the process of degradation will be larger. the degradation process then can cause the decreasing of physical and mechanical properties of the composite resins. later, the condition will lead to the capability of oin reaching the color pigment of the composite resin to be oxidized into the lighter one. however, this condition more often occurs in the nano filler composite resin than in the hybrid composite resin.22-26 based on the above discussion, it can be concluded that the application of tooth bleaching agent, hydrogen peroxide 38%, can lighter the color of both hybrid and nano filler composite resin which had darker color after the immersion in black tea. the color of nano filler composite resin even became brighter than that of hybrid composite resin. thus, it can be said that the application of tooth bleaching agent, hydrogen peroxide 38%, can cause the color of both hybrid composite resin and nano filler composite resin became lighter than the original one. ��sampoerno: effectiveness of bleaching agent references 1. ratna dp. esthetic dentistry. an artist’s science. 1st ed. mumbai: pr publications; 2002. p. 13. 2. fauzi a. pengaruh berbagai konsentrasi h2o2 sebagai bahan bleaching dan lamanya waktu terhadap kebocoran tepi restorasi resin komposit. karya tulis akhir. surabaya: program studi pendidikan dokter gigi spesialis konservasi gigi fakultas kedokteran gigi universitas airlangga; 2005. 3. gupta r, parkash h, shah n, jain v. a spectrophotometric evaluation of color changes of various tooth colored veneering materials after exposure to commonly consumed beverages. j indian prosthodontic society new delhi 2005; 5(2): 72–8. 4. spiller ga. caffeine. new york: crc press; 1998. p. 35–55, 97–150. 5. mercola j. shedding some light on teeth whiteners. mercola.com. 2003. p. 1–2. 6. tse cs, lynoh e, blake dr, william dm. is home tooth bleaching gel cytotoxic. j esthet dent 1991; 3(5): 162–8. 7. goldstein re, garber da. bleaching: a new role for restorative dentistr y. chicago: quintessence p ublishing co, inc; 1995. p. 1–24. 8. auschill tm, hellwing e, schmidale s, sculean a, arweiler nb. efficacy, side-effects and patients’ acceptance of different bleaching techniques (otc, in-office, at-home). oper dent 2005; 30(2): 156–63. 9. schemehorn b, gonzalez-cabezas c, joiner a. a sem evaluation of a 6% hydrogen peroxide tooth bleaching gel on dental materials in vitro. j dent 2004; 32(suppl 1): 35–9. 10. kim jh, lee yk, lim bs, rhee sh, yang hc. effect of toothwhitening strips and films on changes in color and surface roughness of resin composites. clin oral investig 2004; 8(3): 118–22. 11. turkun ls, turkun m. effect of bleaching and repolishing procedures on coffee and tea stain removal from three anterior composite veneering materials. j esthet restor dent. 2004; 16(5): 290–301. 12. canay s, cehreli mc. the effect of current bleaching agents on the color of light-polymerized composites in vitro. j prosthet dent. 2003; 89(5): 474–8. 13. patricia villalta, huan lu, zeynep okte, franklin garcia godoy, john m. powers. effects of staining and bleaching on color change of dental composite resins. j prosthet dent 2006; 92(2): 137–42. 14. dahl je, pallesen u. tooth bleaching. a critical review of the biological aspects. crit rev oral biol med 2003; 14(4): 292–304. 15. summitt jb, robbins jw, schwartz rs. operative dentistry. a contemporary approach. 2nd ed. illinois: quintessence publishing co, inc; 2001. p. 420. 16. erdrich a. discoloration face-off fine hybrid composites versus nanofilled. dental products net 2004; p. 1–11. 17. bugno a. occurrence of toxigenic fungi in herbal drugs. braz j microbiology 2006; 1: 1–13. 18. musanje l, darvell bw. aspect of water sorption from the air, water and artificial saliva in resin composite restorative materials. dent mater 2003; 19(5): 414–22. 19. kresnoadi u, widjoseno tm. the polyester ebp 2421 denture base endurance to the penetration of drinking liquid color. maj ked gigi (dent j) 2001; 34(2): 81–4. 20. fessenden rj. kimia organik. edisi ke 3. jakarta: erlangga; 1991. p. 223–41. 21. sarrett dc. professional product review. am dent assoc j 2006; 1(1): 4. 22. z 250 technical product profile. 3m filtektm z 250 universal restorative system. st paul. mn. usa. 1998. p. 7–10, 26, 29. 23. z 350 technical product profile. 3m filtektm z 350 universal restorative system. st paul. mn. usa. 2005. p. 5–9, 18. 24. taher nm. the effect of bleaching agents on the surface hardness of tooth colored restoratif materials. journal of contemporary dental practice 2005; 6(2): 1–7. 25. lodhi ta. surface hardness of different shades and types of resin composite cured with a high power led light curing unit. a mini thesis for the degree of master of science in dental sciences in restorative dentistry at the faculty of dentistry university of the western cape, south africa. 2006. p. 1–10, 21–6. 26. robinson fg, haywood vb, myers m. effect of ten percent carbamide peroxide on color of provisional restoration materials. j am dent assoc (jada) 1997; 128: 727–31. �� volume 46 number 1 march 2013 evaluation of local muscle soreness treatment with anterior bite splint made of soft putty impression material harry laksono dan sherman salim department of prosthodontics faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: local muscle soreness is the most common temporomandibular disorders complaint of patients seeking treatment in the dental clinics. the emergency treatment that can be done in the clinics to manage this disorder is by making anterior bite splint. anterior bite splint is usually made of acrylic, but currently there is a soft putty impression material that can also be used for making anterior bite splint. the effectiveness of soft putty anterior bite splint in local muscle soreness treatment still has not clear. purpose: to determine the effectiveness of the soft putty impression material as a material used for making anterior bite splint in the treatment of local muscle soreness. case: six patients was reported five female patients aged 20-40 years old and one male patient aged 37 years old with local muscle soreness. four female patients with a “click” sound on tmj. case management: make differential diagnosis with screening history (anamnesis), clinical examination consists of extra oral examination such as muscle and temporomandibular joint palpation, measure the mandibular movement, end-feel, load test, intra oral examination and radiographic evaluation. record the results and make the diagnosis. make a soft putty anterior bite splint, adjusted and inserted in the maxillary anterior teeth. record the results based on signs and symptoms. conclusion: it can be concluded that anterior bite splint made of soft putty impression material is effective for treatment the local muscle soreness. key words: soft putty, anterior bite splint, local muscle soreness abstrak latar belakang: salah satu tipe temporomandibular disorders yang paling sering dijumpai di klinik dokter gigi adalah local muscle soreness. perawatan yang dapat dengan segera dilakukan di klinik untuk mengelola gangguan tersebut adalah dengan pembuatan anterior bite splint. biasanya anterior bite splint terbuat dari akrilik, namun saat ini telah ada bahan cetak soft putty yang memungkinkan untuk dipakai sebagai bahan pembuatan anterior bite splint. efektivitas pemakaian anterior bite splint dari bahan putty untuk perawatan local muscle soreness sampai saat ini masih belum jelas. tujuan: mengetahui efektivitas pemakaian bahan cetak soft putty sebagai bahan anterior bite splint pada perawatan local muscle soreness. kasus: dilaporkan enam pasien terdiri dari lima pasien wanita usia 24-40 tahun dan satu pasien laki-laki usia 37 tahun dengan diagnosis local muscle soreness. empat pasien wanita disertai suara “klik” pada sendi. tatalaksana kasus: membuat diagnosis banding dengan anamnesis, pemeriksaan klinis terdiri dari pemeriksaan di luar rongga mulut yang meliputi palpasi otot pengunyahan dan sendi temporomandibular, mengukur pergerakan rahang bawah, end-feel, uji beban, pemeriksaan di dalam rongga mulut dan radiologis. mencatat hasil pemeriksaannya dan membuat diagnosis. setelah itu membuat soft putty anterior bite splint dan melakukan penyesuaian dan pemasangan. mencatat hasilnya berdasarkan keluhan-keluhan dan tanda-tanda. kesimpulan: anterior bite splint yang terbuat dari bahan cetak soft putty efektif untuk perawatan local muscle soreness. kata kunci: soft putty material, anterior bite splint, local muscle soreness correspondence: harry laksono. c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: harrylaksono@yahoo.com case report �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 23–29 introduction temporomandibular disorders (tmds) includes a variety of clinical problems, such as pain in facial area, limited mouth opening, joint sounds, and others involving temporomandibular joint (tmj) as well as its supporting structure and/ or masticatory muscles.1-3 it is extremely important that they be differentiated because their treatments are quite different.2,4,5 epidemiological studies have proven that tmds is more commonly found in the population between the ages of 20-40 years old.1,2,6 in general, tmds is more commonly in women than in men.1,7 in a study conducted through interviews on asian and caucasian populations, the prevalence shows 6%-30% of joint sounds, 5%-33% of joint pain, and 4%-16% of any abnormality or limitation in mouth opening. another studies shows that 2,2 out of every 100 suffer with tmds, especially on women.8 in general, the prevalence is 4%-6%.1 local muscle soreness (lms) is the most common tmds complaint of patients seeking treatment in the dental clinics.2,9 lms is a non inflammatory, myogenous pain disorder, and usually occurs after a few hours or one day after an event with the clinical signs, such as pain to palpation, and pain will increase as the jaws function.2 the main goal of lms treatment is to eliminate pain and improve the function of masticatory muscles.2 nowadays, many international studies have shown the success of tmds treatment with various types of splint.1,2 one of the splint types that can be used is anterior bite splint (abs).1,2,10,11 anterior bite splint is a partial coverage splint worn over 2-4 maxillary anterior teeth, and few anterior symmetrical contact with opposing mandibular anterior teeth.2,7 this splint used as a tool for emergencies treatment that is immediately fabricated by dentist without the help of an articulator and can easily be adjusted in dental office.11 however, studies about the effectiveness of abs in decreasing pain are still limited either in their reports or in the number of patients involved.7 treatment using abs can provide the same results with that using stabilization splint.10 nevertheless, some studies do not recommend it since it is still considered to be less effective, increasing of pressure on the tmj, excessive eruption on the back teeth, as well as being easily swallowed.1-3,7,12,13 some studies even stated that the use of abs can only be used for the treatment of acute muscle pain in short period of time, about no more than 2 weeks.1-3, 11 the use of hard splint is more effective to reduce hyperactivity of masticatory muscle than the use of soft splint.14 meanwhile, the use of soft splint is more effective in protecting the antagonist teeth from pressure than the use of hard splint, as well as better patient compliance, and fewer side effect.15 the use of soft splint is more cost-effective than pharmacotherapy in long period of time.16 however, the deficiencies of wearing the soft splint are always be very limited in time,17 and need for close monitoring in their use.18 nowadays, there was a soft putty material as an addition (vinyl) silicone impression materials.19 these materials may be use as an abs because of the intermediate hardness. nevertheless, the effectiveness of the use of abs made of soft putty materials for lms treatment is still not clear. the aim of this case report is to determine the effectiveness of the use of soft putty abs in lms treatment. case in this case report, data sources were obtained from the universitas airlangga dental hospital. the number of cases used was 6 cases, in which there were 5 female patients aged 20-40 years old and 1 male patient aged 37 years old with lms diagnosis as well as 4 female patients with a “click” sound on tmj. those diagnosis were made based on the classification system of american academy of orofacial pain (aaop).2 case management in this case report, the standard measurement of lms diagnosis was made by using anamnesis (comprehensive history), clinical examination, muscle and tmj palpation, and millimeter ruler to measure the movement of the lower jaw when the mouth opens, moves to the right and the left, and protrutes.2 anamnesis was conducted with the operator sits on the right beside the patient who sits in upright position.6 the initial evaluation involves interviewing and recording about his or her symptoms consists of several question about duration of pain, pain character, activities of lower jaw which can cause the increasing and decreasing of the pain, and the effects of pain on jaw activities like chewing food, talking, and swallowing.2,20 afterwards, type of treatment that will be conducted as well as possible treatment outcomes was clearly explained to the patient prior to the treatment. after anamnesis was completely, followed by recording the extra and intra oral examination. in extra oral examination, range of motion (rom) examination measured prior to palpating the masticatory muscles. rom examination consist of interincisal maximum distance measurements (figure 1), left and right lateral movement, and protrusion.1,2,20,21 palpation examination was done by manual palpation in the temporalis and masseter muscles, and tmj by using bilateral techniques with the middle finger and the index finger.20 the examination was done by pressing slowly, but strong enough with rotating movement for 1-2 seconds, and each pain emerged in each muscle was recorded on examination form with four categories, which are “a0” if ��salim: evaluation of local muscle soreness treatment with anterior bite splint made the patient does not feel pain during muscle palpation, “a1” if the patient feels uncomfortable (tenderness or soreness), “a2” if the patient feels pain, “a3” if the patient feels hard pain signed with closing his or her eyes or moving the body.2,22 afterwards, end-feel or passive stretch of the mandible evaluation was done by placing the thumb finger on maxillary incisive incisors and the index finger on mandibular incisive incisors, and then slowly press down for 10-15 seconds. if the lower jaw slightly pushed down, it will be called “soft end feel”, while if the lower jaw can not be pushed down, it will be called “hard end feel”.2,8,20 in intra oral examination, the lateral pterygoid muscle evaluate by pressing the vestibule area of the maxillary alveolar ridge as the most posterior by using the index finger (figure 2), and the medial pterygoid muscle by pressing the inferior alveolar injection area by using the index finger,1,2,20 teeth and surrounding tissue examinations and the occlusion. these evaluation followed by radiological evaluation, then cotton-roll clench test by asking the patient to bite the cotton roll placed on the area of the canines and premolars on the right side, and then moved to the left side (figure 3). if the pain occured on the ipsilateral, the disorder could be considered as muscle disorders, whereas if the pain occured on the contralateral, the disorder could be considered as tmj disorders.4,5 after all of the examinations were completely, a diagnosis was then made based on the classification system of aaop. patients were indicated with lms if the history reported by the patient reveals that the pain complaint began several hours or one day following an event. the clinical characteristics were structural dysfunction, minimum pain at rest, increased pain to function, actual muscle weakness, and increased tenderness and pain to palpation on local muscle.2,7 after a diagnosis is achieved by careful evaluation of information derived through the history and examination procedures, the treatment was immediately done by making soft putty abs. the process was done by mixing base and catalyst soft putty, and then it was placed on a stock tray for partial edentulous, followed with impression the maxillary incisors regions (figure. 4), and after the soft putty hardened, the stock tray removed. afterwards, the abs was trimmed with a scalpel, and smoothened by using a frazer. the next step is to adjust the abs occlusion with the help of articulating paper and frazer (figure 5) in order to make a symmetrical contact only with opposing 2 anterior mandibular teeth (figure 6), and disocclusion on posterior teeth with interocclusal distance of 2 mm (figure 7 and 8). on lateral movement, abs must be made no contact with the mandibular canines. once completed, the abs inserted in anterior maxillary teeth, and the patient was instructed to wear it all day long, except when eating and sleeping, to eat soft foods, not to open the mouth too wide, to put a cold compress on the sore area, and to have exercise therapies, such as assisted muscular stretching and stretching against resistance. evaluation process was conducted after 1, 3, 7, and 14 days of the use of abs.4 in evaluation process, the same examination process was done as the initial examination step, and the results of the examination were then recorded and graphed to evaluate pain caused by the treatment. results the results of lms treatment with soft putty abs showed the reducing of pain. t1 showed that the pain was reduced from a3 to a2 (day 1), a1 (day 14), and a0 (day 24); t2 showed that the pain was reduced from a3 to a1 (day1), and a0 (day 7); t3 showed that the pain was reduced from a3 to a2 (day 1), a1 (day 3), and a0 (day 7); t4 showed that the pain a2 is still on day 1 and was reduced to a1 (day 7), and a0 (day 14); t5 showed that the pain a3 is still on day 1 and was reduced to a2 (day 3), and a0 (day 7); t6 showed that the pain was reduced from a2 to a1 (day 1), and a0 (day 14) (figure 9). figure 1. interincisal maximum distance. figure 2. the palpation of lateral pterygoid muscle. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 23–29 figure 3. cotton roll clench test. figure 4. impression the maxillary incisors. figure 5. adjusting with articulating paper. figure 6. symmetrical contact with opposing teeth. figure 8. disoclusion of right posterior teeth. figure 7. disoclusion of left posterior teeth. discussion the evaluation of the effectiveness of the use of abs made of soft putty in lms treatment as a conservative treatment has been conducted. treatment with soft putty abs was selected for the treatment because of its simple construction technique, and also because it can easily be repaired, non-invasive, and relatively cheaper than other treatments. this appliance serves as a stop for the incisal mandibular teeth and the surface must be flat and parallel to the long axis of the mandibular anterior teeth in order to make the position of the lower jaw does not deflect when there is a pressure on maximal intercuspation position (mi). this situation leads to two condyles sitting musculoskeletaly stable position (ms) by elevator muscles.2 lms is a condition characterized by local changes of muscle tissue. these changes are characterized by the release of certain algogenic substances, such as bradykinin, substance prostaglandins, and histamine that produce pain. the changes were caused by ischemia occured in lms.9 ��salim: evaluation of local muscle soreness treatment with anterior bite splint made according to manfredini1, pain in lms is caused by ischemia along with muscle contraction. the mechanism involves the activation of acid-sensing ion channel (asic) and transient receptor potential vanilloid 1 receptor (trpv1) that play a role in physiological pain that releases ions h+, from low ph due to ischemia. lms presents clinically with muscles that are tender to palpation and reveal increased pain with function. structural dysfunction is common, and when the elevator muscles are involved, limited mouth opening and weak muscle results.2,9 in this case report, magnetic resonance imaging (mri) was not conducted because the use of manual measurement standards for tmds diagnosis with anamnesis, clinical examination, manual palpation of the muscles and tmj, millimeter ruler to measure mandibular movement are still credible (reliable, valid, sensitive, and specific), relatively inexpensive.22 in addition, muscle palpation is still considered as a diagnostic gold standard which is very useful to diagnose the masticatory muscle disorders. this is because the main complaint of musculoskeletal disorders is pain.1,2,4,13,23 although until now how occlusal splints reduce pain still does not clear, there are some concepts that still can be used to explain several things, such as the concept of distribution of forces, relaxing the muscles, allowing condyles to seat in the centric relation (cr), normalizing periodontal ligament proprioception, cognitive awareness theory, placebo effects, and increase in the vertical dimension of occlusion (vdo). however, all of these concepts are overlapping.24 the concept of relaxing the muscles is often used to explain how occlusal splint reduce pain. this concept explained about the occlusal disorders during centric relation will cause hyperactivity of lateral pterygoid muscle, and in which posterior teeth disruption during the lower jaw movement will cause hyperactivity of mouth muscles when the mouth closes; thus, muscle fatigue can then cause hyperactivity later causing pain complaints. as a result, when hyperactive muscles are terminated, the pain will be eliminated.1,2,4,20,24 treatment of pain caused by masticatory muscle disorders with abs has still been debated recently. some studies even do not recommend its use because of many deficiencies1-3,15,16 and was not as effective as a stabilization splint (ss).7,25 another risk of abs is its small dimensions, which can lead to swallowing or aspiration. medical emergency due to swallowed abs has been reported in norway.26 abs has the same effectiveness with ss for reducing muscle pain.27 in addition, another research showed the use of abs does not lead to the changes of condyles position with maximum bite force.28 some of literatures and researches also still recommend the use of abs, but not more than 2 weeks with closely monitored.4,11,24,29 for occlusal bite registration, this case report used mi position, because patients have complete teeth and stable occlusion. position of mi can be used as a guide for making figure 9. the result of pain evaluation after using soft putty abs in 6 patients with lms. note: t1 is the result of pain evaluation for patient 1; t2 for patient 2; t3 for patient 3; t4 for patient 4; t5 for patient 5; and t6 for patient 6. a0: 0–1 cm; a1: 1–2 cm; a2: 2–3 cm; a3: 3–3.5 cm. �� dent. j. (maj. ked. gigi), volume 46 number 1 march 2013: 23–29 occlusal splints in patients with stable occlusion without large differences between cr and mi.30 this technique is relatively simple and inexpensive. in intra-oral examination, showed that pain occured during the palpation of the lateral pterygoid muscle so that they were able to close their mouth in mi position. based on some previous studies, it can be caused by muscle fatigue or inability to stretch as the normal one during resting position. in patients 1, 2, 4, and 6, the pain was also accompanied with “click” sound on tmj. it could be caused by both lateral pterygoid muscles that hold the discs and condyles impaired so that the disk position would be pushed over to the anterior, especially when the condyles sliding. this condition known as anterior disc displacement with reduction.2,20 in patient 4 and 5, showed that one day after the used of the anterior bite splint the pain was still not decreased and they also still felt less comfortable. it may be due to the increasing of the masticatory muscle activities as stated in a research with emg showing that masseter muscle activities were immediately increased after the used of a soft splint for maximum clenching.31 the result of study showed that in 5 of 6 patients (t2-t6), the pain was eliminated (from a3 to a0) from 1-3 weeks after the used of abs. this finding is in accordance with okeson2 that lms treatment can be recovered in 1-3 weeks. however, it is also showed that in patient 1, the pain just could disappeare in 24 days. this is because once the pain was reduced and she was able to open his mouth on day 18, the patient has odontectomy of her left third maxillary molar. thus, the pain in the area of the extraction possibily was spread to his face, especially in front of his ear, and then perceived by the patient as tmds pain symptom. in the evaluation phase, it is showed that the decreasing of pain could significantly improve the ability to chew, maximum mouth opening and optimally after 1-3 weeks after the used of soft putty abs as well as to reduce click sounds on tmj. it is seems that the condition of the masticatory muscles was back to normal again after the used of the abs. in terms of the eliminating of masticatory muscle pain and ability to chew, the result in according withprevious studies. they stated that pain in the masticatory muscles play an important role in the decreasing of the bite force so that patients are difficult to chew food, the decreasing of the pain will directly improve the patient’s ability to chew32-34 due to the recovery of the muscle strength, and the pain can reduce the bite force, approximately about 35% to 50%.9 based on the evaluation report, it can be concluded that soft putty impression material can be used as abs material. however, to determine whether soft putty abs is effective for treating lms still requires more samples. nevertheless, based on this case report, soft putty abs can be used to reduce pain in emergency cases caused by lms. if the pain does not reduce within 2 weeks, it is advisable to make a new diagnosis or replace the abs with ss appliances. references 1. manfredini d. current concept on temporomandibular disorders. london, berlin, chicago, tokyo, barcelona, istanbul, milan, moscow, new delhi, paris, beijing, praque, sao paulo, seoul and warsaw: quintessence publ; 2010. p. 25-31, 72-7, 171-90, 373. 2. okeson jp. management of temporomandibular disorders and occlusion. 6th ed. st louis, missouri: mosby elsevier; 2008. p. 252, 300-5, 468, 486-8, 491–2. 3. wassell r, naru a, steele j, nohl f. applied occlusion. london, berlin, chicago, paris, milan, barcelona, istanbul, sao paulo, tokyo, new delhi, moscow, praque, warsaw: quintessence publ co, ltd; 2008. p. 73–80, 146. 4. dos santos j jr. occlusion. principles and treatment. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque, and warsaw: quintessence publ co, inc; 2007. p. 108–11, 135–42. 5. dawson pe. functional occlusion. from tmj to smile design. st louis, missouri: mosby elsevier; 2007. p. 265–75. 6. al-jundi ma, john mt, setz jm, szentpetery a, kuss o. metaanalysis of treatment need for temporomandibular disorders in adult non patients. j orofac pain 2008; 22(2): 97–107. 7. de leeuw r. orofacial pain. guidelines for assessment, diagnosis, and management. 4th ed. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque, and warsaw: quintessence publ co, inc; 2008. p. 129–33, 153–4, 158, 169. 8. laney wr, salinas tj, carr ab, koka s, eckert se. diagnosis and treatment in prosthodontics. 2nd ed. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, new delhi, moscow, praque, and warsaw: quintessence publ co, inc; 2011. p. 83–9. 9. okeson jp. bell’s orofacial pains. the clinical management of orofacial pain. 6th ed. chicago, berlin, tokyo, copenhagen, london, paris, milan, barcelona, istanbul, sao paulo, new delhi, moscow, praque, and warsaw: quintessence publ co, inc; 2005. p. 161–70, 287–4, 316–7. 10. jokstad a, mo a, krogstad bs. clinical comparison between two different splint designs for temporomandibular disorders therapy. acta odontol scand 2005; 63(4): 218–26. 11. re jp, perez c, darmouni l, carlier jf, orthlieb jd. the occlusal splint therapy. j stomat occ med 2009; 2: 82–6. 12. becker im. comprehensive occlusal concepts in clinical practice. west sussex, uk: blackwell publ. ltd; 2011. p. 177–80. 13. murphy e. managing orofacial pain in practice. london, berlin, chicago, paris, milan, barcelona, istanbul, sao paulo, tokyo, new delhi, moscow, praque, warsaw: quintessence publ co, ltd; 2008. p. 64–7, 76. 14. turp jc, komine f, hugger a. efficacy of stabilization splints for the management of patient with masticatory muscle pain: a qualitative systematic review. clin oral investig 2004; 8(4): 179–95. 15. halachmi m, gavish a, gazit e, winocur e, brosh t. splints and stress transmission to teeth: an in vitro experiment. j dent 2000; 28(7): 475–80. 16. naikmasur v, bhargava p, guttal k, burde k. soft occlusal splint therapy in the management of myofascial pain dysfunction syndrome: a follow-up study. ijdr 2008; 19(3): 196–203. 17. truelove e, huggins kh, manci l, dworkin sf. the efficacy of traditional, low cost and non splint theraphies for temporomandibular disorder: a randomized controlled trials. j am dent assoc 2006; 137(8): 1099–1107. 18. fricton j, look jo, wright e, alencar fgp, chen h, lang m, ouyang w and velly am. systematic review and meta-analysis of randomized controlled trials evaluating intra oral orthopedic appliances for temporomandibular disorders. j orofac pain 2010; 24(3): 237–54. 19. o’brian wj. dental materials an their selection. 4th ed. chicago, berlin, tokyo, london, paris, milan, barcelona, istanbul, sao paulo, mumbai, moscow, praque, and warsaw: quintessence publ co, inc; 2008. p. 102. ��salim: evaluation of local muscle soreness treatment with anterior bite splint made 20. wright ef. manual of temporomandibular disorders. iowa usa: blackwell publ co; 2005. p. 27–53, 84–7. 21. gray r, al-ani z. temporomandibular disorder. a problem-based approach. west sussex. uk: blackwell publ; 2011. p. 29–38. 22. okeson jp. the classification of orofacial pain. oral maxillofac surg clin north am 2008; 20(2): 133–44. 23. joh n m t, dwork i n sf, ma ncl la. rel iabi l it y of cl i n ica l temporomandibular disorder diagnoses. pain 2005; 118(1): 61–9. 24. yadav s and karani jt. the essentials of occlusal splint therapy. int j prosthet dent 2011; 2(1): 12–21. 25. magnusson t, adiels am, nilsson hl, hakim m. treatment effect on signs and symptomps of temporomandibular disorders. comparison between stabilization splint and a new type of splint (nti). a pilot study. seved dent j 2004; 28(1): 11–20. 26. fleten a, gjerdet nr. accidental swallowing of an incisal splint. nor tannlegeforen tid 2004; 114: 638–9. 27. al-quran fa, kamal ms. anterior midline point stop device (amps) in the treatment of myogenous tmds: comparison with the stabilization splint and control group. oral surg oral med oral pathol oral radiol endol 2006; 101(6): 741–7. 28. santosa re, azizi m, whittle t, wanigaratne k, klineberg ij. the influence of the leaf gauge and anterior jig on jaw muscle electromyography and condylar head displacement: a pilot study. austral dent j 2006; 51(1): 33–41. 29. klasser gd, green ss. oral appliances in the management of temporomandibular disorders. oral surg oral med oral pathol oral radiol endol 2009; 107(2): 212–23. 30. hamata mm, zuim prj, garcia ar. comparative evaluation of the efficacy of occlusal splints fabricated in centric relation or maximum intercuspation in temporomandibular disorders patients. j appl oral sci 2009; 17(1): 32–8. 31. savahi o, nejatidanesh f, khosravi s. effect of occlusal splints on the electromyographic activities of masseter and temporal muscles during maximum clenching. quintessence int 2007; 38(2): e129–32. 32. kogawa em, calderon ps, laurus jrp, araujo crp, conti pcr. evaluation of maximal bite force in temporomandibular disorders patients. j oral rehabil 2006; 33(8): 559–65. 33. pereira lj, gaviao mbd, bonjardin lr, castelo pm, van der bilt a. muscle thickness, bite force and craniofacial dimensions in adolescents with signs and symptoms of temporomandibular dysfunction. eur j orthod 2007; 29(1): 72–8. 34. pizolato ra, gaviao mbd, berretin-felix g, sampaio acm, junior ast. maximal bite force in young adults temporomandibular disorders and bruxism. braz oral res 2007; 21(3): 278–83. 142142 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 142–147 original article introduction oral health problems have a high rate of occurrence in many countries worldwide and affect various aspects of an individual’s life, often causing pain and discomfort.1 according to the data and information center of the indonesia ministry (2007 and 2013) and baseline health research (2018), oral health problems in indonesia continue to increase every year.2 however, most people with oral health problems have minimal knowledge, and are not concerned, about their situations.3 people who should understand and care about dental and oral problems are dental students. dental students are future dentists responsible for maintaining the oral health and hygiene of the community. consequently, they are expected to maintain their oral hygiene to be good examples for the community.4 research on dental students’ oral hygiene levels conducted in several countries showed varying results. a study in sudan showed a low score on dental hygiene behaviour, and another study in saudi arabia showed a high level in oral health attitudes, but this was not reflected in the oral hygiene and gingival statuses. research conducted on trisakti university dental students using the hu-dbi (hiroshima university dental behavioural inventory) questionnaire, which describes oral health perception and behaviour, showed good results. thus, further studies using intraoral assessments are needed to confirm the findings of the prior studies.5–8 oral hygiene assessments are usually carried out in dental practices with various oral hygiene indexes, but these assessments are not possible to perform during the covid-19 pandemic.9 the covid-19 pandemic began in early 2020 and caused a lockdown condition that impacted dental practices, mainly routine dental check-ups.10 research conducted on the brazilian population during the pandemic reported that oral hygiene behaviour decreased, and the anxiety level associated with visiting dental clinics increased because of the risk of virus transmission, meaning that oral hygiene could not be monitored appropriately, and eventually, this oral hygiene assessment of dental students using the oral rating index (ori) tirza oktarina setiabudi1, fajar hamonangan nasution2 1dental student, faculty of dentistry, trisakti university, jakarta, indonesia 2department of orthodontics, faculty of dentistry, trisakti university, jakarta, indonesia abstract background: oral hygiene screening should be done on a regular basis, notably during the covid-19 outbreak, during which lifestyle changes and government lockdown policies lower the oral hygiene level. the oral rating index (ori), established by kawamura, is the suitable oral hygiene screening index during the covid-19 pandemic. dental students are supposed to be role models for the community by maintaining good dental hygiene. purpose: to study the use of online oral hygiene screening using the ori and to determine the oral hygiene of dentistry students at trisakti university. methods: this research is a cross-sectional descriptive observational study. a total of 100 preclinical dental students from trisakti university took part in the study. intraoral photos were taken and sent to the researcher via the internet. the ori was used to evaluate the data. the reliability of the results was determined using a per cent agreement test and cohen’s kappa coefficient. results: the average oral hygiene score of dental students at trisakti university was 0.58±0.88. the per cent agreement was 88%, and cohen’s kappa coefficient was κ = 0.79, indicating excellent reliability. conclusion: based on their ori score, it can be stated that dentistry students at trisakti university have good oral hygiene and that online evaluation using the ori is a useful tool for routine oral hygiene screening. keywords: dental students; kawamura; oral hygiene; oral rating index (ori) correspondence: fajar hamonangan nasution, department of orthodontics, faculty of dentistry, trisakti university. jl. kyai tapa, no. 260, jakarta, 11440, indonesia. email: fajar@yahoo.co.jp dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 mailto:fajar@yahoo.co.jp https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 143 setiabudi and nasution/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 142–147 led to dental and oral health problems.11,12 particularly in dental students, the switch in activity to online learning may affect their oral health; however, there is no data yet about this. dental health during the pandemic can be monitored by using an oral hygiene screening method that can be applied under lockdown conditions. oral hygiene screening methods that are suitable during the pandemic are assessments that do not require face-to-face contact between dentists and patients and can be done online while still providing reliable results.13 an example of an oral hygiene assessment that can be done during the pandemic is the oral rating index (ori), which was developed by kawamura and will be modified in this research by using remote examination through intraoral photos to adapt to the pandemic situation.14,15 the urgency of this research is based on the current conditions of the pandemic, during which oral hygiene cannot be monitored as usual, and there is no data yet regarding dental students’ oral hygiene. therefore, the researchers are interested in conducting this study, which aims to obtain an overview of oral hygiene among dental students in the faculty of dentistry, trisakti university – the most accessible population for testing the new screening method during the pandemic. this study will contribute to current knowledge as it reflects the community oral hygiene condition and reveals whether we will have good future dentists. furthermore, the purpose of this research is to assess the reliability of online oral hygiene screening using the ori to monitor oral health as an alternative reliable option during the pandemic. materials and methods this research employed a descriptive observational method and a cross-sectional method. this research was conducted online from september to october 2021 and received ethical clearance from the faculty of dentistry ethics commission at trisakti university on 23 july 2021, with letter number 471/s1/kepk/fkg/7/2021. the population in this study comprised preclinical dental students from the faculty of dentistry, trisakti university, jakarta. the research sample was taken by consecutive sampling. the minimum sample size required was 97, which was obtained based on the lemeshow formula, but 100 samples were taken to prevent data shortages.16 the inclusion criteria in this study included preclinical students of the faculty of dentistry, trisakti university, who were willing to be research subjects and use the same brand of smartphone to take the intraoral photos according to the instructions given. the exclusion criteria for this study were those using fixed orthodontic appliances and students who had performed scaling after march 2020. preclinical dental students were chosen because they had the same education and knowledge background, they were of a similar age range and they were the most accessible population on which to conduct the research during the pandemic. the variables in this study were the dental and oral hygiene of preclinical students who were assessed based on the ori. the ori is an index of dental and oral hygiene assessment, which is based on gingival condition and plaque and calculus accumulation by visual examination as mentioned and established by kawamura. the area examined included the labial surfaces of the upper and lower anterior teeth and the lingual surfaces of the upper and lower right posterior teeth. the ori rating uses an ordinal scale from +2 to –2. a score of +2 (very good) was assigned if the gingivae were healthy and no plaque and calculus were detected. a score of +1 (good) was given if there was a slight gingival inflammation but oral hygiene was generally good. a score of 0 (questionable) was given if the researcher found it difficult to determine a positive or negative score. a score of –1 (poor) was assigned when the gingival inflammation was visible and there was a lot of plaque and calculus. a score of –2 (very poor) was given if the gingival inflammation was severe and the oral hygiene was very bad.14 the data collection was carried out online through google forms, which included examples and instructions for taking intraoral photos. there were four intraoral photos that should have been taken: the labial surface of the anterior upper teeth and lower teeth and the lingual surface of the posterior upper and lower right teeth. the instructions for taking intraoral photos included using the same brand of smartphone with the specification of a 12-megapixel rear camera and activating the camera flash. the distance should have been adjusted accordingly so that the result would be focused and not blurry. the angle and the intraoral photo results should have followed the example from the researchers (figure 1). the data were collected and assessed based on the parameters. by use of microsoft excel, the mean score of the data was measured and analysed based on the gender group and the academic year’s group. the differences among the gender groups and the academic year groups were analysed using the mann–whitney u test and the kruskal–wallis test, respectively, as well as statistical package for the social sciences (spss) statistics 25 software. the intra-rater reliability test was carried out by one researcher who reassessed the data within two weeks of the first assessment by blinding the respondent's identity to prove that the assessment carried out had reliable results. reliability analysis was carried out using cohen’s kappa coefficients and spss statistics 25 software. the results of the cohen’s kappa assessment can be categorised as poor agreement (κ < 0.40), good agreement (0.40 < κ < 0.75) or excellent agreement (κ > 0.75). the data obtained in this study were then calculated using microsoft excel software to find the mean of oral hygiene and per cent agreement. the results of per cent agreement can be categorised as none (0%–4%), minimal (4%–15%), weak (15%–35%), moderate (35%–63%), strong (64%–81%) and almost perfect (82%–100%). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 144setiabudi and nasution/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 142–147 figure 1. example for taking intraoral photos and the desired results. +2 (very good) normal gingival condition and no detectable plaque or calculus +1 (good) normal gingival condition; small amount of plaque and calculus accumulation detected; fairly good oral hygiene 0 (questionable) localised gingival inflammation but small amount of plaque and calculus accumulation detected; questionable appearance –1 (poor) gingival inflammation; notable amount of plaque and calculus accumulation detected; poor oral hygiene –2 (very poor) gingival inflammation; large amount of plaque and calculus accumulation detected; very poor oral hygiene figure 2. examples of data collected and oral rating index (ori) assessment, in order from top to bottom: +2 to –2. table 1. respondents’ characteristics distribution (n = 100) year male female total first 6 21 27 second 13 21 34 third 3 15 18 fourth 2 19 21 table 2. results distribution of oral rating index (ori) examination based on gender (n = 100) gender ori score (+2) (+1) (0) (–1) (–2) male 2 7 10 2 3 female 5 53 11 6 1 (n = 100, mann–whitney test = 0.003 (p < 0.05)) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 145 setiabudi and nasution/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 142–147 results data were collected online via google forms from 100 preclinical dental students attending trisakti university. of the 100 respondents, 76 were female and 24 were male; they were distributed from first year to fourth year (table 1). the collected intraoral photos were then compiled and assessed for each ori score based on the gingival condition and the plaque and calculus that were visible in the intraoral photos. the acceptable intraoral photos were those that showed the examination parameters clearly. the researcher marked the inflammation signs of the gingivae and the accumulation of plaque and calculus (figure 2). the majority of the students (60%) got a +1 score, which indicates good oral hygiene and good gingival healthcare levels, while only 4% of the total respondents registered the lowest score of –2 (very poor) (table 2). the data were then analysed using microsoft excel, and the average ori score was 0.58 ± 0.88, which could be categorised as good oral hygiene. the average ori score can be reviewed based on gender; the oral hygiene in female dental students was 0.72 ± 0.76, while in the male students, it was lower, at 0.13 ± 1.12. there is a significant difference between the oral hygiene scores of males and females (p < 0.05). in addition to the gender distribution, the results of the ori examinations can be assessed by year of study. the average ori score of third-year students is the highest, at 0.72 ± 0.57, followed by first-year students at 0.55 ± 0.89, second-year students at 0.55 ± 1.49, and lastly, fourth-year students, whose average was 0.52 ± 0.87 (table 3). there is no significant difference between oral hygiene scores among students in different academic years (p < 0.05). the intra-rater reliability test was then carried out by conducting a second examination using the same intraoral photos from respondents with a blinding method, which uses code numbers to replace respondents’ initials and randomises the examination order. the results of the first and second examinations were combined in a crosstabulation, which was then processed with the per cent agreement using microsoft excel and cohen’s kappa coefficient through spss statistics (table 4). in this research, the per cent agreement was 88%, which can be deemed almost perfect reliability. in this study, the cohen’s kappa results obtained were κ = 0.79, with a significance value of 0.00 (0.00 < 0.05), which means the reliability was excellent. discussion oral hygiene screening using the ori on preclinical dental students was conducted online through intraoral photos. instructions were given for standardising the photo results for the ori examination as the respondents took photos of their oral cavity by themselves. hitherto, there had been no available reference for the standardisation of intraoral photos for ori assessment. however, the overall results of the intraoral photos obtained showed the parameters needed for the assessment, so they were considered adequate for ori examination. therefore, this method can be an alternative for conducting similar research remotely during the pandemic or after the pandemic ends, as long as the participants are able to take the photos according to the instructions. the average score in the oral hygiene of dental students at the faculty of dentistry at trisakti university was considered good. the research results aligned with the dental and oral hygiene behaviour assessment using the hiroshima university dental behavioural inventory (hu-dbi) questionnaire, previously conducted in 2020 with similar respondents.6 the ori and the hu-dbi are complementary tools; data from the two studies conducted at trisakti university showed a direct, unidirectional relationship between the ori and the hu-dbi. specifically, the higher the hu-dbi score, the higher the ori score. table 4. intra-rater agreement for oral rating index examination first examination second examination total very good (+2) good (+1) questionable (0) poor (–1) very poor (–2) +2 7 0 0 0 0 7 +1 1 57 2 0 0 60 0 0 4 13 4 0 21 –1 0 1 0 7 0 8 –2 0 0 0 0 4 4 total 8 62 15 11 4 100 (n = 100, % agreement = 88%, κ = 0.79 (sd = 0.05)) table 3. results distribution of oral rating index examination based on year of study (n = 100) year ori score (+2) (+1) (0) (–1) (–2) first 2 15 7 2 1 second 4 18 8 1 3 third 0 14 3 1 0 fourth 1 14 3 4 0 (n = 100, kruskal–wallis test = 0.928 (p < 0.05)) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 146setiabudi and nasution/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 142–147 good oral and dental hygiene can also be found in dental faculty students in korea who were examined with the ori index and in dental faculty students in india and romania, where other intraoral clinical examinations, such as the gingival index, the plaque index and the oral hygiene index were used.17–19 dental students at the faculty of dentistry at trisakti university have been learning subjects related to oral health and hygiene since the first year; hence, good oral hygiene may indicate that the students have implemented dental and oral hygiene knowledge, yet further analysis and assessment are required to confirm this conclusion. the ori examination was given according to the condition of the gingivae and the plaque and calculus accumulation on a scale of +2 (very good) to –2 (very poor). the higher the ori value, the better the level of dental and oral hygiene. the score of +2 (very good) was expected to appear the most in the respondents of this research as they are dental students. however, from the results data, only a few respondents (7%) who met the assessment criteria were very good, while the majority of respondents (60%) got a score of +1 (good). healthy gingival conditions and the presence of minimal or even no calculus during the pandemic are signs that oral hygiene behaviour has been applied routinely and adequately considering that calculus is formed from mineralised plaque, which can only be prevented by daily plaque control, and if calculus has formed, it can be cleaned only by scaling at the dental clinic.20 scores of –1 (poor) and –2 (very poor) were still found in a small proportion of respondents (12%). although only slight, these results were worrying because, as prospective dentists who should provide awareness about the importance of maintaining oral and dental hygiene, these respondents had not yet taken care of their oral hygiene properly. research conducted on students of the dental faculty in denpasar also showed that there was still a small proportion of students from the faculty who had poor levels of oral and dental hygiene.21 a score of 0 (questionable) is a condition in which the researcher finds it difficult to determine a positive or negative value in the ori assessment. in this study, a score of 0 was given to respondents who had asynchronous conditions, such as gingivae that looked healthy alongside quite a lot of plaque and calculus accumulation (or vice versa) seen on the photo. although online assessment of the ori using photographs is recommended, there are still limitations that cannot be avoided; in some cases, the researchers were hesitant to determine the extent of gingival inflammation and plaque and calculus accumulation. therefore, it is necessary to conduct another study by faceto-face ori examination with the respondent to confirm the results. if necessary, another dental and oral hygiene index examination can be added using an instrument to confirm and facilitate the determination of the level of oral hygiene. the results obtained in this research had a refractive factor because the research was carried out during a pandemic and a government-mandated lockdown policy under which conditions were not normal, so the existing results could not be used as baseline data. after the pandemic, oral hygiene examinations can be carried out again to obtain baseline data on the oral hygiene level of dental students and to be used as a basis of comparison between oral hygiene during a pandemic and oral hygiene in everyday situations. ori assessment can be viewed from the gender perspective, where the average score in this study was higher among women than it was among men and where women got scores of 0.72 ± 0.76 while men got 0.13 ± 1.12. consequently, it can be stated that oral hygiene among women is better than it is among men. women have better oral and dental hygiene behaviour than men in terms of brushing teeth, using dental floss and visiting the dentist. women tend to care more about their bodies and their appearance, and they form habits that support dental and oral hygiene even before getting lessons about dentistry.22 reliability tests were carried out to prove that the assessments that have been conducted have consistent and reliable results. the reliability test of this research used the intra-rater test-retest method, in which one researcher performed two assessments on the same data so that agreement was obtained between the results of the first and second assessments. the ori assessment reliability test results in this research were processed with per cent agreement and cohen’s kappa coefficient. excellent results were obtained (% agreement = 88%, κ = 0.79), meaning that the ori examination index has a clear definition and is understood by researchers so that it gives the same results when repeated assessments are carried out.23,24 the limitations of this study are that dental and oral hygiene examinations were conducted remotely. therefore, it was very dependent on the respondent because the researcher could not observe directly when the respondent took the intraoral photos. the accuracy of the intraoral photos was influenced by many factors, so it was not easy to standardise the results of these photos. ideally, however, the intraoral photos collected in this research can still be assessed.25 the blinding that was carried out in this study was done by only one researcher, so there could still be an examination bias. efforts that can be taken to standardise the photos include using the same smartphone brand, giving exact instructions to all respondents and providing examples of intraoral photos and how to take the photos. above all the shortcomings and limitations, it can be stated that online assessment using the ori is reliable and safe, and it can be used as an initial dental screening tool and routine oral hygiene examinations, providing an alternative reliable option during the pandemic. from this research, it can be concluded that the oral hygiene level of dental students from the faculty of dentistry at trisakti university is classified as good based on ori examination (0.58 ± 0.88), and online oral hygiene screening using the ori can be implemented well. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 147 setiabudi and nasution/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 142–147 references 1. baiju rm, peter e, varghese no, sivaram r. oral health and quality of life: current concepts. j clin diagn res. 2017; 11(6): ze21–6. 2. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar (riskesdas). jakarta: kementerian kesehatan republik indonesia; 2018. 3. soesanto s, octarina, kusnoto j. peningkatan kesadaran warga rt 014/rw 008 jatipulo, jakarta barat mengenai kesehatan gigi pada masa pandemi covid-19. akal abdimas dan kearifan lokal. 2021; 2(1): 22–9. 4. mekhemar m, ebeid k, attia s, dörfer c, conrad j. oral health attitudes among preclinical and clinical dental students: a pilot study and self-assessment in an egyptian state-funded university. int j environ res public health. 2020; 18(1): 1–13. 5. rahman b, al kawas s. the relationship between dental health behavior, oral hygiene and gingival status of dental students in the united arab emirates. eur j dent. 2013; 7(1): 22–7. 6. saptari vs. perilaku kesehatan gigi dan mulut mahasiswa fakultas kedokteran gigi universitas trisakti : kajian dengan menggunakan hiroshima university dental behavioral inventory. thesis: jakarta, universitas trisakti; 2021. p. 1–35. 7. kawamura m, ikeda-nakaoka y, sasahara h. an assessment of oral self-care level among japanese dental hygiene students and general nursing students using the hiroshima university--dental behavioural inventory (hu-dbi): surveys in 1990/1999. eur j dent educ. 2000; 4(2): 82–8. 8. al-shiekh l, muhammed me-d, muhammed ae-r, el-huda ma, hashim nt. evaluation of dental students’ oral hygiene attitude and behavior using hu-dbi in sudan. sci postprint. 2015; 1(2): 2–6. 9. marya c. a practical manual of public health dentistry. new delhi: jaypee brothers medical publishers; 2012. p. 146–148, 156–160, 231. 10. sinjari b, rexhepi i, santilli m, d addazio g, chiacchiaretta p, di carlo p, caputi s. the impact of covid-19 related lockdown on dental practice in central italy-outcomes of a survey. int j environ res public health. 2020; 17(16): 1–14. 11. pinzan-vercelino c-r-m, freitas k-m-s, girão v-m-p, da silva do, peloso r-m, pinzan a. does the use of face masks during the covid-19 pandemic impact on oral hygiene habits, oral conditions, reasons to seek dental care and esthetic concerns? j clin exp dent. 2021; 13(4): e369–75. 12. peloso rm, pini nip, neto ds, mori aa, de oliveira rcg, valarelli fp, freitas kms. how does the quarantine resulting from covid-19 impact dental appointments and patient anxiety levels? braz oral res. 2020; 34: 1–11. 13. estai m, kanagasingam y, huang b, checker h, steele l, kruger e, tennant m. the efficacy of remote screening for dental caries by mid-level dental providers using a mobile teledentistry model. community dent oral epidemiol. 2016; 44(5): 435–41. 14. kawamura m, fukuda s, inoue c, sasahara h, iwamoto y. the validity and reproducibility of an oral rating index as a measurement of gingival health care and oral hygiene level in adults. j clin periodontol. 2000; 27(6): 411–6. 15. camgoz m, gurgan ca, akkaya m. turkish dental students’ and dentists’ ability to assess gingival health status with daags software. j dent educ. 2011; 75(8): 1127–32. 16. arifin wn. introduction to sample size calculation. educ med j. 2013; 5(2): 89–96. 17. kim kj, komabayashi t, moon se, goo km, okada m, kawamura m. oral health attitudes/behavior and gingival self-care level of korean dental hygiene students. j oral sci. 2001; 43(1): 49–53. 18. lalani a, dasar pl, sandesh n, mishra p, kumar s, balsaraf s. assessment of relationship between oral health behavior, oral hygiene and gingival status of dental students. indian j dent res. 2015; 26(6): 592–7. 19. bobu li, saveanu ci, ciceu o, balcos c, armencia a, murariu a. association between oral health perceptions and oral health status of dental students in iasi, romania. rom j med dent educ. 2020; 9(5): 84–90. 20. perry da, beemsterboer pl, essex g. periodontology for the dental hygienist. 4th ed. missouri: elsevier; 2014. p. 54. 21. astini nwrs, susanti dna, handoko sa. hubungan antara pengetahuan dan perilaku menjaga kesehatan gigi dengan oral hygiene pada mahasiswa program studi pendidikan dokter gigi fakultas kedokteran universitas udayana. bali dent j. 2019; 3(2): 70–3. 22. mamai-homata e, koletsi-kounari h, margaritis v. gender differences in oral health status and behavior of greek dental students: a meta-analysis of 1981, 2000, and 2010 data. j int soc prev community dent. 2016; 6(1): 60–8. 23. gwet kl. handbook of inter-rater reliability. 4th ed. gaithersburg: advanced analytics, llc; 2014. p. 16,74-75,100. 24. mchugh ml. interrater reliability: the kappa statistic. biochem medica. 2012; 22(3): 276–82. 25. ahmad i. essentials of dental photography. wiley; 2019. p. 5, 18. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p142–147 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p142-147 vol 51 no 4 okt-des 2018.indd 179 a study of cytotoxicity and proliferation of cosmos caudatus kunth leaf extract in human gingival fibroblast culture zhafira nur shabrina, ni putu mira sumarta, and coen pramono department of oral and maxillofacial surgery faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: post-extraction dental sockets clinically resolve within a period of 3-4 weeks. however, complete healing and bundling of gingival fibers may require several months. medication is therefore required to accelerate the healing process. cosmos caudatus (c. caudatus), a local plant with antioxidant properties and high calcium content, has the potential to promote wound healing while also reportedly capable of strengthening bone. previous studies have demonstrated the effectiveness of c. caudatus as an alternative treatment for post-menopausal osteoporosis by investigating the dynamic and cellular parameters of bone histomorphometry. purpose: the study aimed to examine the citotoxicity and proliferation of human gingival fibroblast cells culture after the application of c. caudatus extract. methods: cultures of human gingival fibroblast cells with 5x104 cell density were divided into two groups and placed in a 30-well culture dish. the control group contained human gingival fibroblast cell culture without extract, while the experimental group consisted of human gingival fibroblast cells culture with extract. the concentrations of extract were 1200 μg/ml, 600 μg/ml, 300 μg/ml, 150 μg/ml, and 75 μg/ml. a toxicity test was conducted and the optimum concentration evaluated using an mtt assay, while fibroblast numbers on were calculated days 1 and 2 by means of a hemocytometer. research data was analyzed using a one-way anova test. results: no toxicity was found. the optimum concentration was 600 μg/ml and fibroblast proliferation was significantly higher in the experimental group compared to the control group, p=0.002 (p<0.05). conclusion: c. caudatus leaf extract is non-toxic and increases the proliferation of human gingival fibroblast culture at an optimum concentration of 600 μg/ml. keywords: cosmos caudatus leaf extract; human gingival fibroblasts culture; wound healing correspondence: ni putu mira sumarta, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 60132, surabaya, indonesia. e-mail: niputu.mira@fkg.unair.ac.id; zhafiranurshabrina@yahoo.com dental journal (majalah kedokteran gigi) 2018 december; 51(4): 179–184 introduction a wound is considered healed if tissue has regenerated to its original anatomic structure, function and within a reasonable period. most wounds result from minor injuries, but some do not heal fully in a timely fashion. several local and systemic factors can impede wound repair by disrupting the process of balance improvement, resulting in chronic wounds failing to healing.1 fibroblasts, cells in the connective tissue that affect the wound healing process, enter the wound area three days after extraction and became dominant after 6-7 days.2 fibroblasts will experience certain changes to the phenotype and become myofibroblasts that serve to retract the wound. fibroblasts produce extracellular matrix, collagen primer and fibronectin which promote cell migration and proliferation. fibroblasts derived from the undifferentiated mesenchymal cells produce mucopolysaccharides, amino acid glycine and proline, a basic ingredient of collagen fibers, that binds wound edges. however, in cases of significant bone damage, the natural repair processes within the body cannot restore its function or promote clinical improvement.3 cosmos caudatus (c. caudatus), known as kenikir in indonesia, had been used in eastern regions of the country to reduce high blood pressure. as well as acting as an antiresearch report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 mailto:zhafiranurshabrina@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 180shabrina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 179–184 hypertensive, it possesses anti-diabetic, anti-inflammatory, bone protective, anti-microbial and anti-fungal properties.4 the polyphenol content of c. caudatus is high, as is the level of vitamin e, vitamin c, folic acid, β-carotene and polyphenols in the c. caudatus lycopene.5 c. caudatus also promotes oral health, especially as an antibacterial, as it contains polyphenol derivates such as flavones, flavonols, flavonones, catechins and isoflavones. the catechin derivate has antioxidant and anti-inflammatory properties. other substances contained in c. caudatus include phytochemical flavonoids. previous studies have shown flavonoids to have anti-inflammatory and antioxidant properties, the capacity to accelerate tissue regeneration and to act as a natural antioxidant that enhances the wound healing process.6 another study stated that c. caudatus leaves contain terpenoids (essential oils), alkaloids and saponin.7 c. caudatus was studied in this experiment because it is readily available, widely consumed and used in the manufacture of drugs to enhance the process of postextraction healing. tooth extraction socket healing occurs through a secondary healing process that involves the formation of granulated tissue and subsequent covering by epithelium. c. caudatus leaf properties have proved capable of balancing the production of reactive oxygen species (ros) and promoting cell growth, such as fibroblasts.8 this research was conducted as a preliminary study of the development of drugs applied to promote fibroblast proliferation. it followed the successive stages of drug development, namely: in vitro examination, experimentation on live subjects (in vivo), clinical trials and a market launch.9 the initial step of this study prior to analyzing the proliferation of human gingival fibroblast (hgf) culture was that of conducting a citotoxicity assay/ study.10 the study aimed to quantify the citotoxicity and proliferation of human gingival fibroblast cell culture after the application of c. caudatus extract. c. caudatus leaf properties were able to balance the production of ros and promote cell growth such as fibroblast so that tooth extraction socket healing occurred through a secondary healing process that increased the proliferation of granulated tissue and the epithelium. materials and methods this was an experimental laboratory study using a post-test control group design. the experiment involved c. caudatus leaf extract application to primary cell cultures of human gingival fibroblasts. preliminary cytotoxicity and optimum concentration tests were conducted using a colorimetric assay that measured the reduction of yellow 3-(4.5-dimethythiazol2-yl) -2.5-diphenyl tetrazolium bromide (mtt) assay by mitochondrial succinate dehydrogenase. five concentrations of extract, consisting of 1200 μg/ml, 600 μg/ml, 300 μg/ml, 150 μg/ml, and 75 μg/ml based on the ic50, were tested. the optimum concentration for cell growth was found to be approximately 504.840 μg/ml.11 the mtt results did not demonstrate toxicity after 24 hours with an elisa reader used to calculate cells spectrophotometrically with a wavelength of 620-650 nm based on formazan crystal absorbance values. extract concentrations of 600 μg/ml were selected because, at this concentration, viable cell numbers of hgf were optimal with no bias in spectrophotometric absorbance. these concentrations correspond to the proliferation of fibroblasts compared to ones below 600 μg/ ml because lower extract concentrations will not provide maximum results. two groups, a control group and an experiment group each containing five samples placed in 30-well culture disk, were used in this study, together with a monolayer hgf culture of 5x104 cell density. a control group was hgfcultured without extract application, while the experiment group was hgf-cultured with the application of 600 μg/ml extract. c. caudatus leaf extract was applied by means of a 1 ml pipette with the fibroblast numbers being counted after 24 hours and 48 hours. fibroblast cell counts were conducted using a hemocytometer after trypan blue staining. cells were taken at the ratio 1:1 and resuspended with trypan blue stain and observed under a light microscope at 100x magnification. cells were considered viable if they were single, clear and round. an overlap between two or more cells was counted as two cells. non-viable cells were defined as those absorbing the color of trypan blue. the measurement results of the observations were added together and calculation values obtained for the subjects of research using the formula:12 number of cells = (a+b+c+d) 4 x x 2 x sample dilution note: count the number of cells – both viable (unstained) and nonviable (stained) in each of the four corner quadrants (a, b, c and d). data were then analised statistically using one-way anova. a p-value less than 0.05 was considered statistically significant. results all concentrations increased the number of fibroblasts. there were differences in formazan crystal formation between each concentration. this was due to the difference in the number of living cells in each extract concentration as presented in figure 1. the optimum concentration was 600 μg/ml which produced a high level of hgf proliferation as shown in table 1. extract concentrations of 1200 μg/ ml produced a false positive result due to the dark color as shown in figure 1a. the proliferation of hgf culture in both the control group and experiment group at 600 μg/ml extract applications showed a higher density under light microscope dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 181 shabrina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 179–184 examination in the experiment group on both the first and second days, as shown in figures 2 and 3. the number of fibroblast in experiment groups were higher compared to control groups. anova statistical analysis showed that there were significant differences in average fibroblast numbers between groups, p=0.002 (p<0.05) as presented in table 2. table 1. toxicity test results of c. caudatus leaf extract no. control well with medium control well with hgf well hgf + 1200 μg/ml extract well hgf + 600 μg/ml extract well hgf + 300 μg/ml extract well hgf + 150 μg/ml extract well hgf + 75 μg/ml extract 0.7240.7860.8941.0711.4170.660.0561 0.7250.7890.8741.0371.4130.6410.0572 0.7260.7680.8471.0581.3910.6670.0563 0.7280.7680.8561.0681.4130.6610.0564 0.7250.7690.8581.0711.4140.650.0575 0.7260.7710.8571.0691.4060.6620.0576 4.3544.6415.1866.3748.4543.9410.339total 0.7260.7740.8641.0621.4090.657mean 0.057 111119135168225.3total living cell % figure 1. figure fibroblasts after application of c. caudatus extracts for 24 hours under light microscope at 10x magnification. (a) concentration of 1200 μg/ml. (b) concentration of 600 μg/ml. (c) concentration of 300 μg/ml. (d) concentration of 150 μg/ml. (e) concentration of 75μg/ml. figure 2. microscopic picture of fibroblasts on the first day (40x magnification). (a) control fibroblasts on the first day. (b) treatment of fibroblast cells. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 182shabrina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 179–184 discussion this was an experimental in vitro study with an application of c. caudatus leaf extract on hgf cell culture. the study was carried out following a drug development sequence which comprised of in vitro examination, animal experiments (in vivo), clinical trials and preparation for marketing.9 cell culture was obtained from human gingival cells because these could be cultured into primary cell line culture demonstrating a similar pattern of behavior to in vivo reactions compared to cell lines.13 cell lines have been used for decades to produce culture, resulting in stronger cells often genetically and phenotypically different from their original networks which underwent morphological changes. unlike cell lines, primary cells are isolated directly from the network, have a limited lifespan and expansion capacity. on the positive side, primary cells demonstrate normal cell morphology and retain many important observable markers and functions, and the data obtained from primary cells is more relevant for interpretation and can be generalized into an in vivo setting.14 this finding was in line with that produced by the research conducted by wong et al15 indicating that primary cells are more sensitive to drugs than cell lines and tend to survive at higher concentrations and proliferation. in contrast, the lifespan of primary cells was shorter at 16-24 hours after the administering of drugs at lower concentrations. the administering of 20 μm (6 μg/ml) cisplatin to primary cells resulted in a viability range of 64.0% cells, while the cell line with 33 μm (6.6 μg/ ml) cisplatin concentration produced a viability level of 71.6% cells.15 extract concentrations were set at 1200 μg/ml, 600 μg/ ml, 300 μg/ml, 150 μg/ml and 75 μg/ml. dose reduction was based on a geometric series of measurements to evaluate the dose-cell response relationship to the exposure of the extract.11 all concentrations of c. caudatus extract were non-toxic as shown in table 1. the concentration of 600 μg/ml was selected because it showed the highest number of proliferated living cells. moreover, the absorbance value of formazan crystals at a concentration of 600 μg/ml could be read spectrophotometrically without bias caused by the extremely dark color. this concentration was suitable for fibroblast cell proliferation compared to those below 600 μg/ml because, according to the dose-response relationship, weaker extract concentrations will produce a lower number of living fibroblasts or will fail to produce optimum results. the concentration of 1200 μg/ml was rejected because it was found that significantly elevated proliferation rates would induce dense cell growth resulting in greater competition for nutrients in the media. as a result, the cells did not mature rapidly and experienced high mortality rates culminating in a false negative result. the possibility of such a result occurring also existed because the concentrated color of dense cells caused the spectrophotometric reading to indicate a higher level of absorbance. fibroblast proliferation was significantly higher in the experiment group compared to that in the control group, p = 0.002 (p <0.05). this was due to the active ingredients in c. caudatus leaf extract containing catechins, polyphenols, flavonoids, vitamins c and e and saponins.9 schuck et al16, citing weisburg et al (2004), stated that catechins do not induce oxidative stress in normal human cells originating in the oral cavity because catechins in extracts can reduce ros table 2. statistical analysis results of fibroblast numbers control and experiment group standard deviationaverage kolmogorovsmirnov anovalevenne 0.29,082.9582,000k1 0.0020.782 0.28,366.6088,000p1 0.27,905.6995,000k2 0.26,519.20106,000p2 figure 3. microscopic picture of fibroblasts on the second day (40x magnification). (a) control fibroblasts on the second day. (b) treatment of fibroblast cells. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 183 shabrina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 179–184 levels in normal cells, while causing intracellular oxidative stress in cancer cells. this indicates that the cathechin effect on normal and cancer cells worked in different biochemical pathways that induce normal cell proliferation. polyphenol content promotes both anti-microbial and antifungal activities that play a role in maintaining the growth of cell cultures. the catechins present in polyphenols can reduce ros levels in normal cells, while flavonoids stimulate fibroblast synthesis and are able to balance the production of ros with antioxidant capacity that promotes fibroblast growth.8 other ingredients found in c. caudatus include high levels of vitamin c and e. flavonoids increase the effectiveness of vitamin c conducive to the formation of fibroblasts.14 ascorbic acid, also known as vitamin c, is necessary for normal responses to physiological stressors and its requirement increases during injury or stress. studies have shown that physiological stress produces excessive ros leading to vitamin c playing the role of activating intracellular signaling which regulates fibroblast proliferation.17 terpenoids or essential oils, as well as polyphenol, promote antibacterial and antifungal activity. fibroblasts are able to proliferate effectively because of the eugenol content of essential oils that damage the cell walls of bacteria causing damage to the cells themselves for example those of gram-positive bacteria, especially streptococcus aureus. phenolic compounds present in essential oils cause protein denaturation and bacterial cell death.8 saponins, active substances that act as anti-microbes, increase cell membrane permeability and have antioxidant propoerties.18 saponins can stimulate the formation of a cellextracellular matrix by stimulating fibronectin synthesis in fibroblasts capable of promoting the wound healing process.19 the saponin content stimulating transforming growth factor-beta 1 (tgf-β1) can affect fibroblast growth factor (fgf) in fibroblast cells thereby promoting the formation of collagen fibers. when fgf is stimulated by tgf-β1 fibroblast proliferation will be increased.20 alkaloids represent one of the other active components in c. caudatus leaves with toxic properties that significantly affect physiological activity. most alkaloids that have been isolated from plants are crystalline solids with a certain melting point.21 the toxic content of alkaloids in c. caudatus leaf extract was at small levels, caused no effect on cell culture and does not increase ros but induced oxidative stress in normal cells. in this study, cell death occurred but there were also fibroblast proliferation that did not effect the results.22 the wound healing process is evident from several indicators, including an increase in the number of fibroblasts that will stimulate the collagen forming extra-cellular matrices. the proliferation of fibroblasts promoted by transforming growth factor-β (tgf-β) will increase collagen and fibronectin synthesis and promote extracellular matrix deposition.20 in the next stage, there is a decrease in endothelial cell proliferation and the number of fibroblast cells, but fibroblasts become more progressive in synthesizing collagen and fibronectin involving changes in the composition of extracellular matrix (ecm).19 the ecm provides a sub-layer for cell attachment and effectively regulates cell growth, movement and differentiation propagated by growth factors and cytokines namely platelet-derived growth factor, fgf, tgf-β and interleukin-1, interleukin-4, immunoglobulin-g produced by leukocytes and lymphocytes during collagen synthesis. remodeling will commence after an extracellular matrix is formed.20 an inherent weakness of this study was the fact that dense fibroblasts in the culture plate can perish because of an over-proliferation of their cells. further research needs to be undertaken by reducing the number of cells to less than 5x104. further study will be conducted to develop a herbal drug which promotes post-extraction tooth socket healing. it can be concluded that c. caudatus leaf extract is non-toxic to human gingival fibroblast culture resulting in a significant increase in fibroblast numbers. the optimum concentration of c. caudatus leaf extract to increase fibroblast proliferation is 600 μg/ml. references 1. velnar t, bailey t, smrkolj v. the wound healing process: an overview of the cellular and molecular mechanisms. j int med res. 2009; 37(5): 1528–42. 2. thiruvoth fm, mohapatra dp, sivakumar dk, chittoria rk, nandhagopal v. current concepts in the physiology of adult wound healing. plast aesthetic res. 2015; 2(5): 250–6. 3. li b, wang jh-c. fibroblasts and myofibroblasts in wound healing: force generation and measurement. j tissue viability. 2011; 20(4): 108–20. 4. amalia l, anggadired k, sukrasno, fidrianny i, inggriani r. antihypertensive potency of wild cosmos (cosmos caudatus kunth, asteraceae) leaf extract. j pharmacol toxicol. 2012; 7(8): 359–68. 5. cheng s-h, barakatun-nisak my, anthony j, ismail a. potential medicinal benefits of cosmos caudatus (ulam raja): a scoping review. j res med sci. 2015; 20(10): 1000–6. 6. kurnia pa, ardhiyanto hb, suhartini. potensi ekstrak teh hijau (camellia sinensis) terhadap peningkatan jumlah sel fibroblas soket pasca pencabutan gigi pada tikus wistar. e-jurnal pustaka kesehat. 2015; 3: 122–7. 7. liliwirianis n, musa nlw, zain wzwm, kassim j, karim sa. premilinary studies on phytochemical screening of ulam and fruit from malaysia. e-journal chem. 2011; 8(s1): s285–8. 8. siagian wm. efektivitas pemberian kenikir (cosmos caudatus kunth) terhadap performa, organ limfoid dan profil darah ayam kampung (gallus gallus domesticus). thesis. bogor: institut pertanian bogor; 2012. p. 285-8. 9. turner jr, hoofwijk tj. clinical trials in new drug development. j clin hypertens. 2013; 15(5): 306–9. 10. ratra m, gupta r. future prospects and aspects of herbal drug discovery in herbal medicines. columbia j pharm sci. 2015; 2(2): 16–21. 11. fauzia a, any u, wati t, rizkita cg, wahyuni e, qori ’ah n, arifin i. the effect of marigold leaves and doxorubicin toward cell cycle and apoptosis of t47d cells. indones j cancer chemoprevention. 2016; 7(3): 79–86. 12. louis ks, siegel ac. cell viability analysis using trypan blue: ma nua l a nd automated met hods. met hods mol biol. 2011; 740: 7–12. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 184shabrina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 179–184 13. sabaliauskas v, juciute r, bukelskiene v, rutkunas v, trumpaitevanagiene r, puriene a. in vitro evaluation of cytotoxicity of permanent prosthetic materials. stomatol balt dent maxillofac j. 2011; 13(3): 75–80. 14. pan c, kumar c, bohl s, klingmueller u, mann m. comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. mol cell proteomics. 2009; 8(3): 443–50. 15. wong ah-h, li h, jia y, mak p-i, martins rp da s, liu y, vong cm, wong hc, wong pk, wang h, sun h, deng c-x. drug screening of cancer cell lines and human primary tumors using droplet microfluidics. sci rep. 2017; 7: 1–15. 16. schuck a, weisburg j, esan h, robin e, bersson a, weitschner j, lahasky t, zuckerbraun h, babich h. cytotoxic and proapoptotic activities of gallic acid to human oral cancer hsc-2 cells. oxid antioxid med sci. 2013; 2(4): 265–74. 17. kurutas eb. the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. nutr j. 2015; 15: 1–22. 18. desai sd, desai dg, kaur h. saponins and their biological activities. pharma times. 2009; 41(3): 13–6. 19. park ks, park dh. the effect of korean red ginseng on full-thickness skin wound healing in rats. j ginseng res. 2018;: 1–10. 20. mei h, gonzalez s, deng s. extracellular matrix is an important component of limbal stem cell niche. j funct biomater. 2012; 3(4): 879–94. 21. krishnaiah d, devi t, bono a, sarbatly r. studies on phytochemical constituents of six malaysian medicinal plants. j med plants res. 2009; 3(2): 067–72. 22. mckenna t. oxidative stress on mammalian cell cultures during recombinant protein expression. thesis. linköping: linköping university; 2009. p. 1-66. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p179–184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p179-184 137 vol. 42. no. 3 july–september 2009 functional relationship of room temperature and setting time of alginate impression material dyah irnawati and siti sunarintyas department of dental biomaterials faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: indonesia is a tropical country with temperature variation. a lot of dental clinics do not use air conditioner. the room temperature influences water temperature for mixing alginate impression materials. purpose: the aim of this study was to investigate the functional relationship of room temperature and initial setting time of alginate impression materials. methods: the new kromopan® alginate (normal and fast sets) were used. the initial setting time were tested at 23 (control), 24, 25, 26, 27, 28, 29, 30 and 31 degrees celcius room temperatures (n = 5). the initial setting time was tested based on ansi/ada specification no. 18 (iso 1563). the alginate powder was mixed with distilled water (23/50 ratio), put in the metal ring mould, and the initial setting time was measured by test rod. data were statistically analyzed by linear regression (a = 0.05). result: the initial setting times were 149.60 ± 0.55 (control) and 96.40 ± 0.89 (31° c) seconds for normal set, and 122.00 ± 1.00 (control) and 69.60 ± 0.55 (31° c) seconds for fast set. the coefficient of determination of room temperature to initial setting time of alginate were r2 = 0.74 (normal set) and r2 = 0.88 (fast set). the regression equation for normal set was y = 257.6 – 5.5 x (p < 0.01) and fast set was y = 237.7 – 5.6 x (p < 0.01). conclusions: the room temperature gave high contribution and became a strength predictor for initial setting time of alginates. the share contribution to the setting time was 0.74% for normal set and 0.88% for fast set alginates. key words: alginates, room temperature, initial setting time correspondence: dyah irnawati, c/o: bagian biomaterial kedokteran gigi, fakultas kedokteran gigi universitas gadjah mada. jl. denta, sekip utara, yogyakarta 55281, indonesia. e-mail: ninnad38@yahoo.com, phone/fax no. 062-274-515307 research report introduction the function of an impression material is to record accurately the dimensions of oral tissues and their spatial relationships.1 impression material should fulfill certain requirements to obtain an accurate impression, such as: adaptive to the oral tissues, viscous enough to be loaded in the tray, set into rubbery or rigid solid in a reasonable amount of time, dimensionally stable, biocompatible, and low cost.2 the alginate hydrocolloid, agar hydrocolloid, and synthetic elastomeric impression materials are the most widely used today.3 the general use of alginate exceeds than the other impression materials.2 the widely used of alginates results from: the ease of mixing and manipulation, the minimum equipment necessary, the flexibility of the set impression, accuracy on proper handling, and low cost.1 alginate impression are used to form study casts to plan treatment, monitor changes, and fabricate crown, bridges, and removable prostheses.3 alginate impression materials is an irreversible hydrocolloids. the main active ingredient of this materials is soluble alginates such as sodium, potassium or thiethanolamine alginate. the other components are reactor (calcium sulfate), filler particles (zinc oxide, diatomaceous earth), accelerator (potassium titanium fluoride), and retarder (sodium phosphate).2 when the alginate powder is mixed with water, the setting reaction is occurred and the gel alginate is set. the typical sol-gel is a reaction of soluble alginate with calcium sulfate and the formation of an insoluble calcium alginate gel. structurally calcium ion replaces the sodium or potassium ions of two adjacent molecules to produce a cross-linked complex or polymer network. the production 138 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 137-140 of the calcium alginate is so rapid that it does not allow sufficient working time. the retarder (trisodium phosphate) is added to prolong the working time. the calcium sulfate will react with this salt in preference to the soluble alginate, so the rapid reaction between calcium sulfate and the soluble alginate is deferred as long as there is unreacted trisodium phosphate.2 alginate powder is mixed with water in a flexible bowl to a soft paste consistency, loaded into a tray to carry it to the mouth, then placed over the patient’s teeth and allowed to set.4 ideally the total setting time should be less than 7 minutes.2 setting time of alginate is ranged from 1 to 5 minutes.3 the setting reaction of alginate is a typical chemical reaction.3 this reaction is a temperaturedependence reaction.5 to fulfill the critical requirements of a dental impression material, the reaction must be controlled to attain the desirable properties of consistency, working time, setting time, strength, elastic quality, and smooth hard surface on gypsum cast. these requirements are achieved by adding agents to control the rate of reaction, develop strength and elasticity in the gel, and counteract the delaying effect of alginate on the setting of gypsum products.3 the setting time of alginate is best regulated by the amount of retarder added during the manufacturing process.2 environmental conditions and the characteristics of the tissue often influence the choice of materials, the quality of impression, and the quality of cast.2 the setting time of alginate depends on the water and room temperatures of mixing.6 the standard room temperature is 23 ± 2° c.7 alginate is normally mixed with water at room temperature.4 the water temperature is between 20° c and 22.2° c.8 in such a case, the temperature of mixing water should be controlled carefully within a degree or two of standard temperature, usually 20° c, so that a constant and reliable setting time can be obtained.2 indonesia is a tropical country with dry and rainy seasons. many cities in indonesia have different elevation. these factors influence air temperature of the cities in a certain time. in dry season, the temperature is varied from 18° c to 26° c (minimum) and 27° c to 34° c (maximum).9 the air temperature will influence room temperature. in dry season, room temperature in yogyakarta (300 m above sea level) is 27° c to 29° c and in cianjur (1000 m above sea level) is 20° c to 23° c.10 some public health centers, dental private clinics, and laboratory in faculty of dentistry do not use air conditioner. consequently, it is difficult to maintain the room in a certain temperature. also, it is important to know the room temperature influences and the strength of its relationship on alginate setting time. the aim of this study was to investigate the functional relationship of room temperature and initial setting time of alginate impression materials. materials and methods two types (normal set and fast set) of the new kromopan® alginate impression materials (lascod, germany) were examined. the analytical balance, measurements glass, rubber bowl, spatula, stopwatch, glass plate, metal ring mould (16 mm in height and 33 mm in diameter),7 and poly methylmethacrylate cylindrical test rod (10 cm long and 6.35 mm in diameter)7 were used. the initial setting time were tested in air conditioned laboratory room with temperatures of 23 (control group), 24, 25, 26, 27, 28, 29, 30, and 31 degrees celcius. five specimens were tested for each group. before testing, the alginate powder, distilled water, and test equipments were conditioned for 6 hours in these certain temperature and 60 ± 10% relative humidity. measurement was done from the beginning of water and alginate powder mixing until the rod is cleanly separated from the mixed material.7 the proportion of alginate powder and water was 23:50 (w/v). the metal ring mould was put on the glass plate. the water was poured into the rubber bowl, and then the alginate powder was added. the water and alginate powder was stirred by spatula until a smooth creamy mix results. the mixing time was 30 seconds. the ring mould was overfilled with the mixed material and the top of the mould was strike off with spatula to get a smooth and parallel surface. immediately, end of the test rod was placed into momentary contact with the unset material, then withdrawn and cleaned with tissue. the contact/withdrawal steps were repeated every 10 seconds until the rod was separated cleanly from the material. the setting time of alginate was recorded. data were statistically analyzed by linear regression (a = 0.05). result the mean and standard deviation of initial setting time of normal set and fast set alginate impressions was shown in table 1. the regression analysis results of the relationship between room temperature and initial setting time can be seen in table 2. discussion the setting time of alginate is best regulated by the amount of retarder added during the manufacturing process.2 normal set materials gel in 3 to 4 minutes and fast set materials gel in 1 to 2 minutes.11 normally, the manufacturer make both fast and normal setting alginate to provide clinicians the opportunity to choose the materials 139irnawati and sunarintyas: functional relationship of room temperature that best suit their working style.2 the standard setting time is achieved when the alginate is manipulated in 23° c room temperature and mixed with water in this room temperature. the setting time of alginate is influenced by many factors, such as: the w/p ratio, mixing time, water temperature,2 composition (trisodium phosphate content), temperature of mixing,6 second alginate layer added, retarder added, and certain liquid products added.12 local environment conditions, such as: air temperature, surface temperature, humidity, and wind also have an effect on the final setting time of alginate impression materials.13 the setting time of alginate is affected by the room temperature. the higher the temperature the less time is required.14 room temperature primarily has an effect on the setting time of the outside surface of the material. on a very hot day, the air temperature can accelerate the set of alginate.13 the results of this study showed that there was a significant share contribution of room temperature to the value of alginate initial setting time (0.74% for normal set alginate and 0.88% for fast set alginate). the room temperature influences the temperature of materials (alginate powder and water) and instruments (rubber bowl, spatula, and impression tray) which are put in that room for some hours. consequently, the setting time of alginate impression materials will be affected. the setting reaction of alginate impression materials is a chemical reaction.1,3 the temperature dependence of the alginate reaction process was of the arrhenius type.5,15 the arrhenius law is the dependency of chemical reaction rate on the temperature.16 the chemical reaction rate speed up with increasing temperature. the rate of reaction doubles with a 10° c rise in temperature.3,17 the setting time of calcium alginate is very temperature dependent.18 the temperature of the water controls the rate of setting reaction.11 the reaction of alginate is faster at higher temperatures.2,6,11,12 an increase in the temperature of the water used to prepare the mix shortens the setting time.1 the warm water reduce the setting time by accelerating the rate at which sodium phosphate is consumed and by subsequently increasing the rate of cross–linking reaction.19 the results of this study showed the functional relationship of room temperature and initial setting time of alginate impression materials. for every 1° c room temperature increased, 5.5 seconds (normal set) or 5.6 seconds (fast set) initial setting time reduction of alginate impression materials was occurred. this results is accordance with previous study that examined the rheological properties of alginate impression materials at various temperatures (37.4, 20.5, 15.5, 11.5, and 7.0 degrees celcius). the setting reaction of alginates was faster at higher temperature and so the alginates in contact with tissues.20 the relationship of room temperature and alginate setting time in this study was relatively similar with the relationship of water temperature and alginate setting time. in general, 1 minute reduction in setting time occurs for every 10° c of temperature increase.2 the setting time of one alginate impression materials product has a 10 seconds water temperature dependence for each 2° c departure from 23° c.13 in a hot weather, the standard setting time of alginate impression materials can be achieved by using water that is cooler than room temperature.2 although, alginate mix made with water at room temperature is the most comfortable for the patient.8 it may even be necessary to pre cool the mixing bowl and spatula, to avoid premature gelation of alginate. because the materials exhibit different degrees of sensitivity to temperature, it is better to select a product with the desired setting time and less sensitivity table 1. mean and standard deviation of initial setting time of alginates (seconds) room temperature (° c) normal set alginate fast set alginate 23 (control) 24 25 26 27 28 29 30 31 149.60 ± 0.55 122.40 ± 0.55 112.20 ± 0.45 106.20 ± 0.84 102.20 ± 0.83 99.60 ± 0.55 98.40 ± 0.55 97.20 ± 0.45 94.60 ± 0.89 122.00 ± 1.00 97.60 ± 0.89 95.00 ± 0.71 90.60 ± 1.14 85.20 ± 1.09 81.20 ± 0.84 76.0076.00 ± 1.41 72.20 ± 0.84 69.60 ± 0.55 table 2. regression analysis of the relationship between room temperature and initial setting time of alginate impression normal set alginate fast set alginate the coefficient of determination (r2) the regression equation significancy 0.74 y = 257.6 – 5.5 x p < 0.01 0.88 y = 237.7 – 5.6 x p < 0.01 140 dent. j. (maj. ked. gigi), vol. 42. no. 3 july–september 2009: 137-140 to temperature change.2 in the future, it is necessary to produce alginate impression materials which has standard setting time in hot weather with room temperature higher than 23° c. it concluded that the room temperature gave high contribution and became a strength predictor for initial setting time of alginates. the room temperature has significant share contribution to the value of alginate initial setting time (0.74% for normal set alginate and 0.88% for fast set alginate). for every 1° c room temperature increase, 5.5 seconds (normal set) or 5.6 seconds (fast set) initial setting time reduction of alginate impression materials was occurred. references 1. craig rg, powers jm, wataha, jc. dental materials properties and manipulations. 7th ed. st louis: mosby co; 2000. p. 138–41, 145–56. 2. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205–9, 231–48. 3. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330–40. 4. vanable de, lopresti lr. using dental materials. new jersey: pearson prentice-hall; 2004. p. 87–91. 5. craig rg. review of dental impression materials. adv dent res 1988; 2(1): 51–64. 6. combe ec. notes on dental materials. 6th ed. edinburgh: churchil livingstone; 1992. p. 115–6, 121–5. 7. council on dental materials, instruments and equipment, american national standard/american dental association (ansi./ada specification no. 18, alginate impression materials, 1992). 8. ferracane jl. materials in dentistry principles and applications. 2nd ed. philadelphia: lippincot williams & wilkins; 2001. p. 173–201. 9. badan meteorologi dan geofisika, prakiraan cuaca kota propinsi indonesia, 2007, http://meteo.bmg.go.id/cuacaindo.jsp. accessed january 18, 2007. 10. purwanti s. kajian suhu ruang simpan terhadap kualitas benih. journal online universitas sriwijaya, 2004. available at: http://journal.unsri. ac.id/detail.php?no=15. accessed august 16, 2007. 11. gladwin m, bagby m. clinical aspects of dental materials. philadelphia: lippincot williams & wilkins; 2000. p. 100–1. 12. parvin d, how to extend the setting time of alginate. contemporary art media 2003; 1–3. 13. accu-cast, life casting instruction for accu-cast products. 2006, available at: http://www.accucast.us/prod_instruct.html. accessed july 7, 2007. 14. hollins c. nvqs for dental nurses. oxford: blackwell pub professional; 2003. p. 136–7. 15. ellis b, lamb dj. the setting characteristics of alginate impression materials. brit dent j 1981; 151: 343–6. 16. bird t. kimia fisik untuk universitas. cetakan ke-2. jakarta:cetakan ke-2. jakarta:jakarta: pt gramedia; 1993. p. 282. 17. anonymous. the encyclopedia americana international edition.the encyclopedia americana international edition. vol. 2. americana corporation. 1973. p. 377. 18. doubleday b. orthodontic products update impression materials. bjo 1998; 25(2): 133–40. 19. mccabe jf, walls awg. applied dental materials. 8th ed. cambridge: blackwell science ltd; 1998. p. 118–26, 133–40. 20. fish sf, braden m. characterization of the setting process in alginate impression materials. j dent res 1964; 43(1): 107–17. 172 vol. 42. no. 4 october–december 2009 case report fibrous epulis associated with impacted lower right third molar ni putu mira sumarta and david b kamadjaja department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia absract background: epulis or epulides are lesions associated with gingival tissues. fibrous epulis is a type of hyperplastic fibrous tissue mass located at the gingival which is slow growing, painless, having same color as the oral mucosa and firm on palpation. anterior regions of the oral cavity are the frequently affected sites as these areas are more prone to be affected by calculus deposition and poor plaque control due to frequent teeth malposition. removal of any irritating factors and excision of the lesion are the usual treatments. purpose: this case report presents a rare case of fibrous epulis which occurred in the posterior region of the oral cavity and associated with impacted lower third molar. case: a case of fibrous epulis at the lower right third molar area of three months duration is presented. the mass was slow growing, painless and on examination it was a pedunculated mass overlying the unerupted lower right third molar, having same color with the oral mucosa and firm on palpation. clinically, the lesion was diagnosed as fibrous epulis associated with impacted lower right third molar. case management: the treatment were surgical excision of the epulis and removal of the lower right third molar. the histopathology result showed tissue with squamous epithelial lining, achanthotic fibrous connective tissue, mononuclear inflammatory cells and few capillaries without signs of malignancy. this is consistent with the diagnosis of fibrous epulis. conclusion: fibrous epulis, although frequently occurred at the anterior region of the oral cavity, may rarely grow at the area of lower third molar. this phenomenon supports the theory that epulis can grow on any surface of oral mucous membrane as long as local irritants are present. key words: epulis, fibrous epulis, excision, impacted molar correspondence: ni putu mira sumarta, c/o: ppdgs departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: putumira_omfs@yahoo.co.id introduction epulis or epulides means “on the gums” are lesions associated with the gingival tissues.1 other literature states that when a reactive focal connective tissue proliferation is confined to the gingiva and its exact histologic nature is unknown, it is clinically designated as an epulis. the most common lesions referred to as epulis are peripheral fibroma, peripheral ossifying fibroma, pyogenic granuloma, and peripheral giant cell granuloma.2 traumatic and inflammatory factors constitute favorable conditions for epulis development. their size varies from 2.5 to 3 cm, in most cases they are pedunculated, and their color varies depending on the tissue structure. depending on the histopathological image and clinical picture there are several types of epulis: fibrous epulis, granulomatous epulis, and gigantocellulare epulis.3 fibrous epulis is another hyperplastic fibrous tissue mass located at the gingiva. it may represent resolving pyogenic granulomas. usually the fibrous epulis has the same color as the oral mucosa which is light pink, firm on palpation, and has a fibrous texture although sometimes hard if bone is present within the lesion.1,3 the majority of inflammatory reactive hyperplasias occur on the surface of the oral mucous membrane where irritants are quite common.4 fibrous epulis in adults occurs due to chronic irritation or trauma, due to factors such as dentures, a carious tooth, faulty restorations, and 173sumarta and kamadjaja: fibrous epulis associated subgingival calculus.5,6 removal of any irritating factors and excision is the usual treatment. these lesion, however may recur if the lesion is not excised totally.1,5 most reports showed that the anterior region of oral cavity is most frequently affected than the posterior region.7 a case of fibrous epulis at the posterior region of oral cavity which is considered rare in terms of site of occurrence, is presented here. case a 33 year old female patient presented with main complaint of a mass at lower right third molar area since 3 months previously. the mass was slow growing and there had been no pain. the growth was felt by the patient to be preceded by swelling and pain in that area. clinically the patient was in good general condition. intra orally, there was a pinkish lobulated mass, with 3 cm in diameter about 3 cm, distal to the lower right second molar overlying the unerupted lower right third molar (figure 1). it was found to be pedunculated, firm on palpation, and there was no pain on palpation. the lesion was clinically diagnosed as fibrous epulis. orthopantomogram showed impacted lower third molar with widened periodontal space of the lower right third molar tooth. there was bone destruction distal to the lower right third molar, indicating there was a chronic inflammation in the bone, distal of the impacted tooth crown. the bone surrounding the crown of the impacted left lower third molar was within normal limit (figure 2). case management the patient was treated with complete excision of the epulis and removal of the impacted lower right third molar. on excision, the mass was found to be pedunculated distal to the crown of lower third molar, measuring about 3 cm in diameter, firm in consistency and there was subgingival calculus on the lower right second and third molar tooth. histopathologic result was fibrous epulis. microscopic examination revealed tissue with squamous epithelial lining, achanthotic fibrous connective tissue, mononuclear inflammatory cells and few capillaries. there was no sign of malignancy (figure 3). figure 1. the pinkish lobulated mass located distal to the right lower second molar overlying the unerupted lower right third molar. figure 2. orthopantomogram showing impacted lower third molar with radiolucencies along periapical of the lower right third molar tooth and bone destruction distal to the lower right third molar tooth. figure 3. histopathological examination was fibrous epulis. microscopic examination showed tissue with squamous epithelial lining, achanthotic fibrous connective tissue, mononuclear inflammation cells and few capillaries. there was no sign of malignancy. (he, ×200). 174 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 172-174 discussion a variety of swellings located on or near the gums is clinically included under the heading of epulis. epulis usually occur as a result of gingival hyperplasia due to local irritation of the gums.6 fibrous inflammatory hyperplasias, if located on the gingiva often referred as epulis.4 epulises are lesions which do not give pain, however, their presence causes difficulties in chewing and eating.3 the patient presented was with sign and symptom in accordance to that of fibrous epulis, which is a slow growing, asymptomatic, pedunculated mass with firm consistency. within the oral cavity most local irritants are physical and stimulate the submucosal connective tissue, periodontal ligament, or the periosteum.2 fibrous epulis arises in response to local irritation from sharp margins of a carious tooth or the presence of subgingival calculus. the common variety of epulis often arises from the interdental papilla.6 the etiology of the fibrous epulis in this case may be the subgingival calculus, as the chronic irritating factor, at the distal part of the crown. as suggested by peralles et al.,7 other possible etiology of this lesion is local tissue irritation by bacterial agents and cellular debris at the distal aspect of the lower right third molar which induced hyperplasia of the pericoronal tissue. this is supported by the orthopantomogram which showed bone destruction distal to the crown of lower right third molar. the majority of inflammatory reactive hyperplasia occurred on the surface of the oral mucous membrane where irritants occured presented are quite common.4 these are in accordance with the reported case that the epulis grew on the mucous membrane of the pericoronal tissue of the lower right third molar. however, most reports showed that the anterior regions of the oral cavity were affected more frequently, varying from 57% to 71% of the cases, and this can be explained by the fact that these regions are drier than the posterior regions, are prone to be affected by calculus deposition on the inferior region, and the frequent teeth malposition also in this area, making hygiene and plaque control difficult.7-9 those are in contrast to the case in this patient where the epulis grew in the posterior regions of oral cavity, which is at the lower third molar area. in the author’s opinion this is considered a rare site for fibrous epulis as no such cases have been documented neither in our department nor in the literature so far. the fibrous epulis in the current case occurred in a 33 year-old female patient. this is in line with the majority of reports that epulis occur more frequently in women. it is probably caused by high estrogen concentration that is considered to be a factor favourable to their formation and influencing their growth.3 study by bataineh and al-dwairi4 showed that fibrous lesion frequently affected people between age 21 and 60 years old. this study also found that females were more commonly affected than males. other study also showed that inflammatory gingival hyperplasia affect adults on their third or fourth decades of life, specially females.7 treatment of epulis involves excision with gingival recontouring and removal of the source of irritation is done to prevent recurrence.6 this patient was treated with surgical excision of the epulis and removal of the impacted lower third molar because its position predispose to calculus formation, which may be the most irritating factor in this case, and the increased concentrations of bacterial agents can cause chronic irritation inducing hyperplasia of the pericoronal tissue. in conclusion, epulis can grow on any surface of oral mucous membrane as long as local chronic irritants are present. references 1. reichart pa. surgical management of nonmalignant lesions of the mouth. in: booth pw, schendel sa, hausamen je, editors. maxillofacial surgery. 2nd ed. st louis: churcill livingstone; 2007. p. 1457. 2. sapp jp, eversole lr, wysocki gp. salivary gland disorders. in: sapp jp, eversole lr, wysocki gp, editors. contemporary oral and maxillofacial pathology. 2nd ed. st louis: mosby co; 2004. p. 337–44. 3. hopkala jk, szykowska am, hopkala m, koszel uo, czajkowsi. observations on epulises based on clinical material with a focus on histopathological diagnosis. ann. univ. marie curie-sklodowska lublin-polonia 2007; lxii (1,27): 127–30. 4. bataineh a, al-dwairi zn. a survey of localized lesions of orala survey of localized lesions of oral tissues: a clinicopathological study. j contemp dent pract 2005; 6(3): 30–9. 5. inan m, yalçin o, pul m. congenital fibrous epulis in the infant. yonsei med j 2002; 43(5): 675–7. 6. dabholkar jp, vora kr, sikdar a. giant fibrous epulis. indian j otolaryngol head and neck surg. 2008; 60: 69–71. 7. peralles pg, viana apb, azedevo alr, pires fr. gingival and alveolar hyperplastic reactive lesions: clinicopathological study of 90 cases. brazilian j oral sci 2006; 5(18): 1085–9. 8. cuisia zes, brannon rb. peripheral ossifying fibroma-a clinical evaluation of 134 pediatric cases. pediatr dent 2001; 23: 245–8. 9. al-khateeb t, ababneh k. oral pyogenic granuloma in jordanians: a retrospective analysis of 108 cases. j oral maxillofac surg 2003; 61: 1285–8. 133 volume 45 number 3 september 2012 distribution of class ii major histocompatibility complex antigenexpressing cells in human dental pulp with carious lesions tetiana haniastuti department of oral biology faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: dental caries is a bacterial infection which causes destruction of the hard tissues of the tooth. exposure of the dentin to the oral environment as a result of caries inevitably results in a cellular response in the pulp. the major histocompatibility complex (mhc) is a group of genes that code for cell-surface histocompatibility antigens. cells expressing class ii mhc molecules participate in the initial recognition and the processing of antigenic substances to serve as antigen-presenting cells. purpose: the aim of the study was to elucidate the alteration in the distribution of class ii mhc antigen-expressing cells in human dental pulp as carious lesions progressed toward the pulp. methods: fifteen third molars with caries at the occlusal site at various stages of decay and 5 intact third molars were extracted and used in this study. before decalcifying with 10% edta solution (ph 7.4), all the samples were observed by micro-computed tomography to confirm the lesion condition three-dimensionally. the specimens were then processed for cryosection and immunohistochemistry using an anti-mhc class ii monoclonal antibody. results: class ii mhc antigen-expressing cells were found both in normal and carious specimens. in normal tooth, the class ii mhc-immunopositive cells were observed mainly at the periphery of the pulp tissue. in teeth with caries, class ii mhc-immunopositive cells were located predominantly subjacent to the carious lesions. as the caries progressed, the number of class ii mhc antigen-expressing cells was increased. conclusion: the depth of carious lesions affects the distribution of class ii mhc antigen-expressing cells in the dental pulp. key words: class ii major histocompatibility complex, dental pulp, caries abstrak latar belakang: karies merupakan penyakit infeksi bakteri yang mengakibatkan destruksi jaringan keras gigi. dentin yang terbuka akibat karies akan menginduksi respon imun seluler pada pulpa. kompleks histokompatibilitas utama (mhc) merupakan sekumpulan gen yang mengkode histokompatibilitas antigen-antigen permukaan sel. sel-sel yang mengekspresikan molekul-molekul ini berpartisipasi dalam pengenalan awal substansi-substansi antigenik untuk selanjutnya diproses dan dipresentasikan pada permukaan sel. tujuan: penelitian ini bertujuan untuk mengetahui perubahan distribusi sel-sel yang mengekspresikan molekul mhc kelas ii pada pulpa gigi manusia dengan meningkatnya keparahan karies. metode: penelitian ini menggunakan 15 gigi molar ketiga yang mengalami karies pada permukaan oklusal dengan berbagai tingkat kedalaman dan 5 gigi molar ketiga normal (tidak mengalami karies). sebelum didekalsifikasi dengan larutan edta 10% (ph 7,4), seluruh sampel diamati dengan menggunakan micro-computed tomography untuk mengetahui kedalaman lesi karies secara tiga dimensi. spesimen kemudian diproses untuk dilakukan cryosection dan dilakukan immunohistokimia dengan menggunakan monoklonal antibodi anti mhc kelas ii. hasil: ekspresi mhc kelas ii oleh sel-sel pada ruang pulpa dijumpai disemua spesimen baik pada kondisi normal maupun karies. pada gigi normal, sel-sel yang mengekspresikan mhc kelas ii terletak terutama pada tepi pulpa. pada gigi-geligi yang mengalami karies, agregasi sel-sel yang mengekspresikan mhc kelas ii terutama terletak di bawah lesi karies. semakin dalam lesi karies, jumlah sel-sel yang mengekspresikan mhc kelas ii semakin meningkat. kesimpulan: kedalaman lesi karies berpengaruh terhadap distribusi sel-sel yang mengekspresikan mhc kelas ii pada pulpa. kata kunci: kompleks histokompatibilitas utama, pulpa gigi, karies correspondence: tetiana haniastuti, c/o: departemen biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. email: haniastuti@yahoo.com case report 134 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 133–137 introduction dental caries is a bacterial infection which causes destruction of the hard tissues of the tooth. this disease remains the most prevalent infectious disease in the world. among the oral bacteria, streptococcus mutans and streptococcus sobrinus are strongly associated with human dental caries. the ability of these bacteria to produce caries is directly related to their production of acid and their ability to tolerate large quantities of lactic acid. the acid will lead to the dissolution of the mineralized matrix of the teeth.1,2 dental pulp is a connective tissue resides in a rigid encasement of the mineralized dentin. when enamel is lost for any reason, the exposed dentinal tubules provide diffusion channels from the surface of the tooth to the pulp. it is known, however, that the pulp contains a variety of indigenous and recruited immunocompetent cells which have ability to initiate and maintain immune responses against pathogenic challenges that permeate the dentinal tubules. these cells seem to form a network that surveys invasion of noxious stimuli and, upon injury, establishes reaction pathways that characterize specifity of pulpal diseases. exposure of the dentin to the oral environment as a result of caries inevitably results in a cellular response in the pulpo-dentinal organ.3,4 major histocompatibility complex (mhc) is a group of genes that code for cell-surface histocompatibility antigens. the function of the mhc molecules is to bind peptide fragments derived from the pathogens and display them on the cell surface for recognition by the appropriate t cells. the consequences are almost always deleterious of the pathogen.5 all cells in the body except erythrocytes express class i molecules, while only some cells such as dendritic cells and macrophages express class ii molecules. cells expressing class ii molecules are called antigen presenting cell. these cells participate in the initial recognition and the processing of antigenic substances to serve as antigen-presenting cells. they migrate to lymphoid tissues via afferent lymph to induce t-cell proliferation and activation.6 previous studies have revealed the presence of class ii mhc antigen-expressing cells in normal dental pulp, both in permanent7 and deciduos teeth.8 however, the distribution of the class ii mhc-expressing cells in the dental pulp remains obscure, and is not sufficiently understood. changes in the distribution of class ii mhc antigen-expressing cells have been shown during tooth development as well as cavity preparations.9 the present study aimed to elucidate the alteration in the distribution of class ii mhc antigen-expressing cells in human dental pulp as carious lesions progressed toward the pulp. materials and methods the protocol of this study was approved by ethical committee of faculty of medicine, universitas gadjah mada. twenty third molars from 20 to 40-years-old patients were used in this study. informed consent was obtained from all patients who were enrolled in this study. fifteen third molars with caries at the occlusal site at various stages of decay and 5 intact third molars were extracted for orthodontic or therapeutic reasons. the depth of the cavity judged by clinical examination was recorded. under local anesthesia, the teeth were extracted and then immersed in 10% buffered formalin for 3 days. before decalcifying with 10% edta solution (ph 7.4), all the samples were observed by micro-computed tomography to confirm the lesion condition three-dimensionally. the specimens were processed for cryosection. the tissues for cryosection were equilibrated in a 30% sucrose solution for cryoprotection. the specimens were then cut at thickness of 50 μm on a freezing microtome, and processed for the avidin-biotin peroxidase complex (abc) method using an anti-mhc class ii (hla-dr)-monoclonal antibody (lab vision, usa). following incubation with the primary antibodies for 3 days in 4oc, the sections were reacted by two consecutive incubations with biotinylated anti-mouse igg and abc complex (vector lab. inc., burlingame, usa) for 2 hours each at room temperature. the sites of antigen-antibody reaction were then visualized by placing sections in 0.05 m tris buffer (ph 7.6) containing 0.04% 3–3'-diaminobenzidine tetrachloride and 0.002% h2o2. the immunostained sections were counterstained with methylene blue. immunohistochemical controls were performed by: replacing the primary antibody with nonimmune serum or pbs and omitting the anti-mouse igg or the abc complex. these immunocontrol sections did not show any specific immunoreactions. results an intense immunoreactive for class ii mhc molecule was observed in the pulp tissue of the tooth in all cases. class ii mhc-immunopositive cells were distributed widely throughout the dental pulp. some of them showed spindle-like or dendritic profiles, while some of them revealed macrophage morphology. in normal intact tooth, the class ii mhc-immunopositive dendritic cells were situated mainly at the cell-free zone and along the pulp-dentin border (figure 1). some of their cell processes were situated in the predentin and showed a close relationship to the odontoblast processes. they extended their thick cytoplasmic processes into the dentinal 135haniastuti: distribution of class ii major histocompatibility tubules. class ii mhc-immunopositive macrophages were dispersed sparsely in the pulp tissue. the number of the class ii mhc-immunopositive macrophages observed in normal pulps was fewer than the class ii mhc-immunopositive dendritic cells. in the cases of pulp affected by early caries, all specimens showed caries confined to the enamel. all specimens revealed small cluster of class ii mhc-immunopositive cells in the pulpal restricted area beneath the pulpal ends of dentinal tubules communicating with the carious lesion (figure 2). numerous class ii mhc-immunopositive dendritic cells extended their cytoplasmic processes into the affected dentinal tubules in the odontoblastic layer, while class ii mhc-macrophage cells were seen distibuted sparsely in pulp chamber. compare to the intact teeth, the density of class ii mhc-immunopositive cells in this case was higher. in the cases of pulp affected by advanced caries, all teeth showed caries confined to the dentin. the aggregation of class ii mhc-immunopositive cells was more densely gathered subjacent to the carious lesion than elsewhere. the specimens with advanced caries exhibited an expansion of the area of increased class ii mhc-immunopositive cells; thus, the dense network of class ii mhc-immunopositive cells was widely seen in the pulp chamber. the density of mhc-immunopositive cells was higher in the cases of carious lesion confined to dentin than enamel. their expression became highest in the cases of carious lesion confined to dentin with pulp exposure. in advanced caries, lymphocytes and endothelial cells were also expressed class ii mhc molecules in addition to dendritic cells and macrophages. tertiary dentin was observed in all specimens with advanced caries. in the area beneath the tertiary dentin, limited presence of class ii mhc-immunopositive cells were observed. discussion this study showed the alteration of class ii mhc antigen-expressing cells distribution in human dental pulp as caries progressed toward the pulp. in normal tooth, the class ii mhc-immunopositive cells were distributed sparsely throughout the dental pulp, and located predominantly at the periphery of the pulp tissue. as the caries progressed, the number of class ii mhc-immunopositive cells was increased and distributed more widely in the pulp. they located mainly subjacent to the carious lesions. the presence of class ii mhc antigen-presenting cells in the normal dental pulp implied important roles played by these cells in maintaining physiological conditions of the pulp tissue. in the present study, the normal dental pulps were shown to contain two types of class ii mhcimmunopositive cells: dendritic cells and macrophages. dendritic cells are widely accepted to be the most potent and versatile antigen presenting cells in the immune system. these cells are believed to possess a more potent antigenpresenting capacity than macrophages. in peripheral tissues, dendritic cells are highly motile, possess superior capacity for acquiring and processing antigens for presentation to t lymphocytes, and within secondary lymphoid organs have the potential to express high levels of the co-stimulatory molecules that direct and fine-tune t cell activation.10 dendritic cells are extremely efficient in stimulating naive t-lymphocytes.5 this study revealed that the dendritic cells were strategically positioned in the periphery of the pulp where foreign antigens are most likely to enter the tissue. their primary function may be to monitor invasion of antigens. numerous dendritic cells were situated in the predentin and extended their cytoplasmic processes into the affected dentinal tubules. these finding implying their high motility and support the concept that the dendritic cells play a critical figure 1. a specimen of normal intact tooth. the class ii mhcimmunopositive dendritic cells are situated mainly at the cell-free zone (red arrows) and along the pulpdentin border (black arrows). figure 2. a specimen with early karies. a cluster of class ii mhc-immunopositive cells are seen in the limited area subjacent to the pulpal ends of dentinal tubules communicating with the carious lesion (red arrows). 136 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 133–137 role in the initial immune defense of the dental pulp against trans-dentinal antigenic stimuli.10 macrophages function as a first-line defense to detect and eliminate invading microbes and toxic macromolecules.11 this study showed that macrophages were distributed sparsely in the pulp and seemed to form a network together with pulpal dendritic cells. there are three major biological functions of macrophages: phagocytosis, antigen processing and presentation, and cytokine secretion. in addition to its importance in defense against pathogenic bacteria, phagocytosis is an essential step in antigen processing. following endocytosis, digestion, and degradation, peptide fragments from phagocytosed proteins are able to associate with class ii mhc peptide within organelles of the endosomal pathway. the class ii mhc-peptide complexes are then transported to the cell surface, where they interact with t lymphocytes. at the same time, the macrophages secrete interleukin (il) 1, a necessary signal in antigen presentation which stimulates proliferation of the lymphocytes.12 in carious teeth, cluster of class ii mhc-immunopositive cells were observed mainly beneath the lesion. the density of these cells in the area subjacent to the lesions was highest compare to other area in the pulp chamber. the dendritic cells which are class ii molecule-expressing cells carry receptors on their surface that recognize common features of many pathogens. cariogenic bacterial components will bind to these receptors, then stimulate the dendritic cells to engulf the cariogenic bacteria and degrade them intracellularly.5 after capture protein antigens, dendritic cells will migrate to the regional lymph nodes and present the peptide fragments in conjunction with class ii mhc molecules to antigen-specific t lymphocytes (naive t lymphocytes). helper t lymphocytes will subsequently clonally expand and differentiate into effector t cells or memory t cells. effector t cells and some memory t cells then enter into the vasculature and migrate to the pulp. resident antigen-presenting cells such as macrophages will interact with and present antigen directly to the t cells, which are locally activated and trigger the effector phase of the immune response against bacterial antigens.13 macrophages have a variety of receptors that recognize microbial surface components which are involved in the ingestion of bacteria by phagocytosis and in signaling for the secretion of proinflammatory cytokines, which then recruit and activate more phagocytes.11 as the caries progressed, the number of the bacteria invaded the dentinal tubules increase.14 this study demonstrated that the number of class ii mhcimmunopositive cells increased as the caries advanced and the lesions progressed toward the pulp. after capture the antigen, dendritic cells secrete certain chemokine namely il-8, which then attract the inflammatory cells to the site of infection.15 thus, as caries progressed, the inflammatory cells infiltration may increase as well. this finding was supported by the previous study16 which proved that the number of inflammatory cells infiltration increased as the caries progressed toward the pulp. when a carious lesion has invaded dentin, the pulp responds by depositing a layer of tertiary dentin over the dentinal tubules of the secondary dentin that communicate with the carious lesion. compare to secondary dentin, tertiary dentin differs morphologically and less permeable to externally derived matter.17 this study revealed only few number of class ii mhc-immunopositive cells presence beneath the tertiary dentin, implying its low permeability. the dental pulp is known to have a high potential for participation in both defense against foreign stimuli and tooth repair. the class ii mhc-immunopositive cells in the pulp-dentin border was assumed to have some unknown functions other than immunosurveillance. kannari et al.,8 proposed an inductive role for these cells in the differentiation, migration and/or activation of odontoclasts and cementoblast-like cells during physiological root resorption. thus, the predentinal class ii mhc-immunopositive cells as shown in the present study might also exert some effects on the odontoblasts under physiological conditions. however, this hypothesis is needed to be tested in future studies. previous report18 has demonstrated a rich supply of pulpal nerve fibers beneath the odontoblast layer to form the subodontoblastic nerve plexus in the coronal dental pulp. the distribution pattern of the class ii mhc antigen-expressing cells in the dental pulp of the teeth with carious lesions in this study was similar to the distribution pattern of the nerve fibers which have been shown in the previous study. this similarity in distribution patterns between the class ii mhc antigen-expressing cells and neural elements in the tooth with carious lesions revealed an intimate functional correlation between them. indeed, the author suggested the involvement of neural elements in the functional capacities of the class ii mhc antigen-expressing cells. in conclusion, the depth of carious lesions affects the distribution of class ii mhc antigen-expressing cells in the dental pulp. references 1. marsh pd, nyvad b. the oral microflora and biofilms on teeth. in: fejerskov o, kidd e, editors. dental caries: the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p. 179–80. 2. chaussain-miller c, fioretti f, goldberg m, menashi s. the role of matrix metalloproteinases (mmps) in human caries. j dent res 2006; 85(1): 22–32. 3. pashley dh, liewehr fr. structure and functions of dentin-pulp complex. in: cohen s, hargreaves km, editors. pathways of the pulp. 9th ed. missouri: mosby elsevier; 2006. p. 461. 4. yu c, abbot pv. an overview of the dental pulp: its functions and responses to injury. aus dent j 2007; 52(1 suppl): s4–16. 5. murphy k, travers p, walport m. janeway's immunobiology. 7th ed. new york: garland science; 2008. p. 331. 6. hahn c, liewehr fr. update on the adaptive immune responses of the dental pulp. j endod 2007; 33(7): 773–81. 137haniastuti: distribution of class ii major histocompatibility 7. olgart l, bergenholtz g. the dentine-pulp complex: responses to adverse influences. in: bergenholtz g, horsted-bindslev p, reit c, editors. textbook of endodontology. oxford: blackwell munksgaard; 2003. p. 26–7. 8. angelova a, takagia y, ok iji t, kanekoc t, yamashita y. immunocompetent cells in the pulp of human deciduous teeth. arch oral biol 2004; 49(1): 29–36. 9. kawagishi e, nakakura-ohshima k, nomura s, ohshima h. pulpal responses to cavity preparation in aged rat molars. cell tissue res 2006; 326(1): 111–22. 10. ricart bg, john b, lee d, hunter ca, hammer da. dendritic cells distinguish individual chemokine signals through ccr7 and cxcr4. j immunol 2011; 186(1): 53–61. 11. taylor pr, martinez-pomares l, stacey m, lin hh, brown gd, gordon s. macrophage receptors and immune recognition. annu rev immunol 2005; 23: 901–44. 12. vega ma, corbi al. human macrophage activation: too many functions and phenotypes for a single cell type. immunol 2006; 25(4): 248–72. 13. iwasaki a. mucosal dendritic cells. annu rev immunol 2007; 25: 381–418. 14. love rm. invasion of dentinal tubules by root canal bacteria. endod top 2004; 9: 52–65. 15. haniastuti t. histological evaluation of human pulp tissue in response to caries progression. dentika dental j 2010; 15(2): 165–9. 16. oz-arslan d, ruscher w, myrtek d, ziemer m, jin y, damaj bb, soricter s, idzko m, norgauer j, maghazachi aa. il-6 and il-8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids. j leukoc biol 2006; 80(2): 287–97. 17. haniastuti t, nunez p, djais aa. the role of transforming growth factor beta in tertiary dentinogenesis. dent j (majalah kedokteran gigi) 2008; 41(1): 15–20. 18. haniastuti t. pulp nerve fibers distribution of human carious teeth: an immunohistochemical study. dent j (majalah kedokteran gigi) 2010; 43(4): 186–9. 197 volume 45 number 4 december 2012 antitumor activity of intratumoral injection of pcdna3.1-p27kip1mt followed by in vivo electroporation in a malignant burkitt’s lymphoma cell xenograft supriatno and sartari entin yuletnawati department of oral medicine faculty of dentistry, universitas gadjah mada �ogyakarta – indonesia abstract background: human malignant burkitt’s lymphomas are an uncommon type of non-hodgkin lymphoma commonly affects in children. it is a highly aggressive type of b-cell lymphoma. treatment for this malignant are still limited. however, a new strategy for refractory cancer, gene therapy is watched with keen interest. recently, a novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electroporation and plasmid cdna. in the present study, a non-viral gene transfer system, in vivo electroporation in human malignant burkitt’s lymphoma (raji) cell xenograft was investigated. purpose: the purpose of this study was to evaluate p27kip1 gene therapy in raji cell xenografts using pcdna3.1-p27kip1 mutant type (mt) and pcdna3.1 empty vector (neo) with the local application of electric pulses. methods: true experimental study using post-intervention with control group design was performed in this study. material sample was obtained from integrated research laboratory at faculty of dentistry, universitas gadjah mada, yogyakarta. the efficiency of transfection of exogenous p27kip1 gene by electroporation was confirmed by western bloting analysis. to evaluate the reduction of malignant burkitt’s lymphoma cell xenografts by this method, the volume of raji cell xenografts in mice after electroporation with p27kip1 mt or neo gene was measured. results: up-regulation of p27kip1 protein was detected in pcdna3.1-p27kip1 mt. furthermore, the growth of tumors was markedly suppressed by p27kip1 mt gene transfection compared with transfection of neo. conclusion: injection of pcdna3.1-p27kip1 mt gene followed by in vivo electroporation has a high-potentially to suppress the growth of malignant burkitt’s lymphoma cells. furthermore, combination system of pcdna3.1-p27kip1 mt-injected tumor and electroporation might be used for human oral cancer. key words: mutant type p27kip1, human malignant burkitt’s lymphoma, electroporation abstrak latar belakang: limfoma burkitt’s maligna banyak terjadi pada anak-anak dan merupakan jenis yang langka dari limfoma nonhodgkin (nhl). limfoma burkitt’s maligna adalah tipe yang sangat agresif dari limfoma sel b. perawatan penyakit ini masih sangat terbatas, walaupun demikian strategi baru perawatan kanker menggunakan terapi gen menjadi pusat perhatian. suatu metode baru transfer gen untuk meningkatkan efisiensi dan kontrol area telah dikembangkan dengan mengkombinasi elektroporasi in vivo dan plasmid cdna. pada penelitian ini, telah diteliti sistim transfer gen non-virus dengan elektroporasi in vivo terhadap xenograft sel limfoma burkitt’s maligna (sel raji). tujuan: tujuan dari penelitian ini adalah untuk mengevaluasi terapi gen p27kip1 terhadap xenograft sel raji menggunakan pcdna3.1-p27kip1 mutant type (mt) dan pcdna3.1 empty vector (neo) dengan aplikasi lokal elektroporasi. metode: jenis penelitian yang digunakan adalah eksperimen murni memakai rancangan pasca intervensi dengan kelompok kontrol. sampel dan bahan penelitian didapat dari laboratorium riset terpadu, fakultas kedokteran gigi, universitas gadjah mada, yogyakarta. efisiensi transfeksi gen p27kip1eksogen dengan elektroporasi dilakukan dengan analisis western bloting. untuk mengevaluasi hambatan xenograft sel limfoma burkitt’s maligna dengan metode elektroporasi, dilakukan pengukuran volume xenograft sel raji pada tikus pasca elektroporasi dan injeksi gen p27kip1 mt atau neo. hasil: peningkatan regulasi protein p27kip1 terdeteksi pada gen pcdna3.1-p27kip1 mt. selanjutnya, pertumbuhan tumor secara signifikan terhambat oleh transfeksi gen p27kip1 mt dibandingkan dengan transfeksi neo. kesimpulan: injeksi gen pcdna3.1-p27kip1 mt disertai elektroporasi in vivo mempunyai potensi yang kuat menghambat pertumbuhan research report 198 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 197–201 sel limfoma burkitt’s maligna. kombinasi sistim injeksi tumor menggunakan gen pcdna3.1-p27kip1 mt dan elektroporasi kemungkinan dapat digunakan untuk terapi kanker oral. kata kunci: p27kip1 mt, limfoma burkitt’s maligna, elektroporasi correspondence: supriatno, c/o: bagian penyakit mulut, fakultas kedokteran gigi universitas gadjah mada. jl. denta 1, sekip utara, �ogyakarta 55281, indonesia. email: pridentagama_oncolog@yahoo.com atau supriatno-fkg@ugm.ac.id introduction electroporation (electro-gene therapy or electric pulse) has been developed for the purpose of achieving highly efficient in vitro gene or drug transfer.1 this system provides markedly higher efficiency transfer compared with other non-viral transfer system, including cationic liposome.2 electroporation has been applied to in vivo drug transfer for cancer treatment and clinical trial has been started.3 electroporation has become more and more popular as an effective technique for introduction of foreign dna into cells of various kinds of mammalian cells,4 for investigation of gene regulation,5 and has been demonstrated to be highly useful in transfecting human hematopoietic stem cells for gene therapy.6 however, the transfection efficiency in mammalian cells using in vivo electroporation has received little attention7 and usually is still low, typically about 0.01-1%.8 because electroporation is a physical method, it has a little biological side effect and is free of chemical toxicity.5 many types of methods and techniques for in vivo gene transfer have been developed, and some of them have already been applied in clinical trials.1 non-viral gene transfer, “naked” plasmid dna is an ideal system for gene transfer. a plasmid mediated method would be economical and easy because use of this system obviates the necessity to construct viral vectors, establish clones of producer cells, assessed viral titers and presence of replication-competent helper virus, which has been known to activate passive oncogenes. the transfer procedure could be easily repeated because “naked” plasmid dna has little antigenicity to the host body.9 burkitt’s lymphoma (bl) is a rare, highly aggressive lymphoma. it is a tumour of the immune system with variable range of incidence depending on age, geographic location, race and epstein–barr virus (ebv) exposure. bl is classified as a type of non-hodgkin’s lymphoma (nhl) of monoclonal small, non-cleaved b-cell lymphocytes, which is subclassified as either endemic or non-endemic.10 this is one of the most rapidly growing paediatric tumours that require prompt diagnosis before initiation of a specific treatment.11 although the clinical features of endemic and non-endemic bl are dissimilar, the characteristics of the tumour cells and prognostic factors are similar.12 according to their clinical and cytological characteristics, burkitt’s lymphoma is classified into low, medium and high grades of malignancy. high-grade burkitt’s lymphoma affects mainly young people, while low-grade malignancies are more frequent in advanced age.10 the majority of lymphomas arise in lymphoid tissue, especially the cervical nodes (80% of all childhood neoplasm), and only 20% arise at extranodal sites.12 the commonest tumour was seen in children 3–10 years of age,11 and epstein-barr virus has been implicated in its etiology. a number of studies have assessed clinical and treatments of bl and contradictory results have been reported.13 p27kip1 is an universal cyclin-dependent kinase inhibitor that directly inhibits the enzymatic activity of cyclin-cdk complexes, resulting in cell cycle arrest at g1. 14 p27kip1 has an important prognostic factor in various malignancies. recently, decreased expression of p27kip1 has been frequently detected in human cancer.15-17 in addition, loss of p27kip1 has been associated with disease progression and an unfavorable outcome in several malignancies.18 furthermore, mice lacking the p27kip1 gene show an increase in body weight, thymic hypertrophy and hyperplasia of pituitary intermediate lobe adrenocorticotropic hormon cells, adrenal glands and gonadal organ.19 also, malignant human oral cancer cells transfection with p27kip1 gene leads to inhibition of proliferation, invasion and metastasis.20,21 in the present study, the antitumor activity of p27kip1 gene therapy in human malignant burkitt’s lymphoma (raji) cell xenografts using pcdna3.1-p27kip1 mutant type (mt) and pcdna3.1 empty vector (neo) with the local application of electric pulses was evaluated. materials and methods raji cells were obtained from the integrated research laboratory, faculty of dentistry, gadjah mada university, yogyakarta. cells were maintained in dulbecco’s modified eagle medium (dmem, sigma, st louis, mo, usa) supplemented with 10% fetal calf serum (fcs, moregate biotech, bulimba, australia), 100 µg/ml streptomycin, and 100 units/ml penicillin (invitrogen corp., carlsbad, ca, usa). the mammalian expression vectors pcdna3.1-p27kip1 mt containing sense oriented human mutant type p27kip1 cdna was constructed. briefly, pcdna3.1 (+) was digested with age1 (takara biomedicals, kusatsu, japan) and nhe1 (takara), and dephosphorylated by calf intestinal alkaline phosphate (roche diagnostics, mannheim, germany). the human mutant type p27kip1 cdna fragment (0.59 kb agei and nhe1 fragment) was obtained as a generous gift from dr. j massague (howard hughes medical institute, 199supriatno and yuletnawati: antitumor activity of intratumoral injection of pcdna3.1-p27kip1mt memorial sloan-kettering cancer center, ny) and dr. k harada (department of therapeutic regulation for oral tumor, institute of health bioscience, tokushima university, japan). this fragment containing the human mutant type p27kip1 open reading frame was ligated to the prepared cloning site of pcdna3.1 (+) by t4 dna ligase (takara). the direction of the ligated fragmen was confirmed by sequencing analysis with a spesific primer (p27kip1-sqp: 5’-atgtcaaacgtggcgagtgtc3’) for human p27kip1 cdna the dna sequence was determined by the dideoxy chain termination method, using fluorescene-labeled primers and a thermo sequenase cycle sequencing kit (amersham pharmacia biotech, sweden). electrophoresis and scanning were performed with a shimadzu dsq-500 dna sequencer (shimadzu, kyoto, japan). however, sequencing data is not shown in this article. cell lysates were prepared from the xenograft tumor tissue. briefly, samples containing equal amounts of protein (50 µg) were electrophoresed on a sds-polyacrylamide gel and transferred to a nitrocellulose filter (pvdf membrane: biorad, hercules, ca, usa). the filters were blocked in tbs containing 5% nonfat milk powder at 370c for 1 hour and then incubated with a 1: 500 dilution of the monoclonal antibody against p27 protein (clone 1b4, monoclonal antibody, novocastra laboratories, new castle, uk) and an amersham ecl kit (amersham pharmacia biotech) as the primary antibody. mouse antibody igm was used as the secondary antibody. anti-α tubulin monoclonal antibody (zymed laboratories, san fransisco, ca, usa) was used for normalization of western blot analysis. the raji cells were trypsinized, washed with pbs, and suspended in saline solution at 1 x 106 cells in 0.1 ml. cell suspension (0.1 ml) was injected into each male wistar mouse with balb/ca genetic background (lppt ugm, yogyakarta, indonesia) subcutan in the flank area. a pair of 1 cm diameter of disc-shaped electrodes (pinsettes-type electrode 449-10 prg, meiwa shoji, tokyo, japan) was used to nip the tumor nodule through the skin. a series of eight electrical pulses with pulse length of 1 msec was delivered with a standar square wave electroporator btx t820 (btx, inc, san diego, ca). the voltage of 80 v/1.0 cm diameters of xenografts was used. then, it delivered an appropriate pulse length and frequency of pulses according to previous report.4 immediately after electrical pulsing, 20 µg of pcdna-neo or pcdna3.1-p27kip1 mt dissolved in 50 µl of tris edta buffer was directly injected into the tumor nodule. this electroporation and injection were performed a total of three times at 3-day intervals. tumor volume and body weight were measured every 3 days from the time electroporation started until the mice were sacrificed. the tumor volume was determined by measuring length (l) and width (w) diameters of the tumor and calculated as v = 0.4 x l x w2. statistical analysis was performed with a stat work program for macintosh computers (cricket software, philadelphia, pa, usa). in vivo tumor volume data in tumorigenesis assay were analyzed for statistical significance of 95% with two way anova followed by post-hoc lsd. results to evaluate the efficiency of transfection of p27kip1 gene, the expression of p27kip1 protein by western blotting was evaluated. equal amounts of each transfected cell protein (50 µg) were electrophoresed on a sds-polyacrylamide gel and transferred to a nitrocellulose filter. up-regulated of p27kip1 protein in pcdna3.1-p27kip1 mt-injected tumors was detected when compared with that in pcdna3.1 empty vector (neo)-injected tumors. however, the expression of α-tubulin as an internal control was approximately the same in all of the tumors (figure 1). the mean relative volume for raji xenografts treated with an injection of pcdna3.1-p27kip1 mt or pcdna3.1 empty vector was shown in figure 2. pcdna3.1-p27kip1 mt-injected became much smaller than pcdna3.1 empty vector-injected tumors, and p27kip1-up-regulated tumors revealed significantly suppressed the tumor volume compared with that of neo (p<0.01). interestingly, during the experimental period, no loss of body weight was observed in each treatment group, and that no skin region including a burn also was observed. discussion lack of detectable expression of p27kip1 cyclin dependent kinase inhibitor has previously been correlated with high degree of malignancy in human cancers include breast, colorectal, gastric and small cell lung carcinomas. furthermore, we were demonstrated that an inverse correlation between p27kip1 expression and tumor malignancy in oral cancer.20,21 in the present study, antitumor activity of intratumoral injection of pcdna3.1p27kip1 mutant type followed by electroporation in a human malignant burkitt’s lymphoma cell xenograft was examined. the mutant type p27kip1 gene was used as a transfection gene and was evaluated its antitumor activity in human malignant burkitt’s lymphoma cell (raji cell) xenograft. the results of study demonstrated the transfection of mutant type p27kip1 gene by electroporation could induce the expression of p27kip1 protein (figure 1), which has the negative regulator function in the cell cycle. accumulation of p27kip1 protein in human malignant burkitt’s lymphoma cell was marked the good prognosis. the same result was reported by barnouin et al.,22 that p27kip1 has the antiproliferative function in burkitt’s lymphoma cell in vitro. therefore, mutant type p27kip1 gene was markedly suppressed the growth of raji cancer xenografts through tumorigenesis analysis (figure 2a). also, during the 200 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 197–201 achieved using disk-shaped electrodes. suggesting that clinical application using this electroporation system for oral cancer may be possible in the future. on the other hands, some disadvantages of this method should be considered. although transfection by electroporation inhibited the growth of raji cell xenografts, the target area was limited to local tumors and the growth of multiple metastatic lesions cannot be target for efficient suppression. for that reason, with a view to obtaining more effective gene therapy using electroporation for head and neck cancer and oral cancer, we plan to attempt gene transfer with several other genes and to use various anticancer agents in combination with gene transfection by this electroporation system. in conclusion, injection of pcdna3.1-p27kip1 mt gene following in vivo electroporation has a highly antitumor activity in human malignant burkitt’s lymphoma cell xenografts. it might be possible to transfer pcdna3.1p27kip1 mt gene into human malignant burkitt’s lymphoma cell xenograft. in vivo gene transfer method is a simple procedure and can solve some of the critical drawbacks of the present gene transfer techniques, thus providing a new strategy for gene therapy. acknowledgment i thank dr. koji harada, dds., ph.d, second department of oral maxillofacial surgery and oncology, school of dentistry, tokushima university, japan, for their valuable advices and providing materials. also, thank our staff in oral medicine and co-asst faculty of dentistry universitas gadjah mada for finishing this research. references 1. supriatno, �uletnawati, widiasto. effect of intratumoral injection of mutant type p27kip1 followed by in vivo electroporation on radiotherapy-resistant human oral tongue cancer xenografts. mol med report 2011; 4(1): 41–6. 2. kanduser m, miklavcic d, pavlin m. mechanisms involved in gene electrotransfer using highand lowvoltage pulsesan in vitro study. bioelectrochemistry 2009; 74(2): 265-71. 3. mali b, miklavcic d, campana lg, cemazar m, sersa g, snoj m, jarm t. tumor size and effectiveness of electrochemotherapy. radiol oncol 2013; 47(1):32–41. 4. sato m, akasaka e, saitoh i, ohtsuka m, watanabe s. development of a technique for efficient gene transfer to antral follicular cell in the mouse ovary. syst biol reprod med 2012; 58(3): 136-41. 5. sharma s, sun x, agarwal s, rafikov r, dasarathy s, kumar s, black sm. role of carnitine acetyl transferase in regulation of nitric oxide signaling in pulmonary arterial endothelial cells. int j mol sci 2012; 14(1): 255–72. 6. nakamura h, funahashi j. electroporation: past, present and future. dev growth differ 2013; 55(1): 15–9. 7. shah k, connolly rj, chapman t, jaroszeski mj, ugen ke. electrogenetherapy of b16.f10 murine melanoma tumors with an interleukin-28 expressing.dna plasmid. hum vaccin immunother 2012; 1: 8–11. 8. guo h, hao r, wei �, sun d, sun s, zhang z. optimization of electrotransfection conditions of mammalian cells with different biological features. j membr biol 2012; 245(12): 789–95. p27kip1 α-tubulin neo mt figure 1. western blotting analysis. expression of p27kip1 and α-tubulin protein in human malignant burkitt’s lymphoma cell transfected with pcdna3.1-p27kip1 mt or empty vector. experimental period, no loss of body weight was observed in each treatment group, and that no skin region including a burn also was observed (figure 2b). supriatno et al.,23 reported that degradation of p27kip1 can be promoted by phosphorylation of skp2 and thr-187. inversely, mutant type p27kip1 was originally from mutation on thr-187/ pro-188 (acgccc) to met-187/ile-188 (atgatc) is not influenced by ubiquitin-mediated degradation. met187/ile-188 (atgatc) is resistance to degradation.23 increasing p27kip1 protein in cancer tissues is associated with low aggressive of cancer cell and good prognosis.23 next, electro-transfer of plasmid cdna p27kip1 mt into burkitt’s lymphoma cell xenograft can be successfully a 0 1000 2000 3000 4000 5000 0 3 6 9 12 15 day m ea n of tu m or v ol um e (m m 3) p27 neo p27 mt * * electroporation b 21.2 21.6 22 22.4 22.8 23.2 0 3 6 9 12 15 day b od y w ei gh t ( g ra m ) p27 neo p27 mt figure 2. a) tumorigenesis suppression of human malignant burkitt’s lymphoma (raji) cell after injection of pcdna3.1-p27kip1 mt or empty vector (neo) followed by in vivo electroporation. * p < 0.05; b) change of body weight. *p < 0.05. 201supriatno and yuletnawati: antitumor activity of intratumoral injection of pcdna3.1-p27kip1mt 9. kimura s, ikezawa m, cao b, kanda �, pruchnic r, cummins j, huard j, miike t, suzuki s. ex vivo gene transfer to mature skeletal muscle by using adenovirus helper cells. j gene med 2004; 6(2): 155–65. 10. muralee mc, thakral a, bhat sk. primary extranodal nonhodgkin’s lymphoma involving masseter and buccinator muscles. int j oral maxillofac surg 2012; 41(11): 1393–6. 11. stárek i, mihál v, novák z, pospísilová d, vomácka j, vokurka j. pediatric tumors of the parapharyngeal space. three case reports and a literature review. int j pediatr otorhinolaryngol 2004; 68(5): 601–6. 12. �amaguchi m. hodgkin lymphoma and non-hodgkin lymphoma. rinsho ketsueki 2013 ; 54(1): 81–8. 13. marques h, catarino r, domingues n, barros e, portela c, almeida mi, costa s, reis rm, medeiros r, longatto-filho a. detection of the epstein-barr virus in blood and bone marrow mononuclear cells of patients with aggressive b-cellnon-hodgkin’s lymphoma is not associated with prognosis. oncol lett 2012; 4(6): 1285–89. 14. jain sk, bharate sb, vishwakarma ra. cyclin-dependent.kinase inhibition by flavoalkaloids. mini rev med chem 2012; 12(7): 632–49. 15. wang x, gao p, long m, lin f, wei jx, ren jh, �an l, he t, han �, zhang hz. essential role of cell cycle regulatory genes p21 and p27 expression in inhibition of breast cancer cells by arsenic trioxide. med oncol 2011; 28(4): 1225–54. 16. akli s, zhang xq, bondaruk j, tucker sl, czerniak pb, benedict wf, keyomarsi k. low molecular weight cyclin e is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer. cell cycle 2012; 11(7): 1468–76. 17. kudo �, kitajima s, ogawa i, miyauchi m, takata t. downregulation of cdk inhibitor.p27.in.oral.squamous cell carcinoma. oral.oncol 2005; 41(2): 105–16. 18. guan x, wang �, xie r, chen l, bai j, lu j, kuo mt. p27(kip1) as a prognostic factor in breast cancer: a systematic review and metaanalysis. j cell mol med 2010; 14(4): 944–53. 19. taguchi r, �amada m, horiguchi k, tomaru t, ozawa a, shibusawa n, hashimoto k, okada s, satoh t, mori m. haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (men1)-related genes, mll,.p27kip1.and p18ink4c in endocrine organs. biochem biophys res commun 2011; 415(2): 378–83. 20. supriatno, harada k, kawaguchi s, �oshida h, sato m. effect of p27kip1 on the ability of invasion and metastasis of an oral cancer cell line. oncol rep 2003; 10: 527–32. 21. supriatno, harada k, kawaguchi s, onoue t, �oshida h, sato m. characteristics of antitumor activity of mutant type p27kip1 gene in an oral cancer cell line. oral oncol. 2004; 40(7): 679–87. 22. roorda bd, ter elst a, scherpen fj, meeuwsen-de boer tg, kamps wa, de bont es. vegf-a promotes.lymphoma.tumour growth by activation of stat proteins and inhibition of p27(kip1) via paracrine mechanisms. eur j cancer 2010; 46(5): 974–82. 23. supriatno, harada k, �oshida h, sato m. basic investigation on the development of molecular targeting therapy against cyclin-dependent kinase inhibitor p27kip1 in head and neck cancer cells. int j oncol 2005; 27(3): 627–35. vol 44 no 3 sept 2011.indd 132 vol. 44. no. 3 september 2011 treatment of lingual traumatic ulcer accompanied with fungal infections sella1 and mochamad fahlevi rizal2 1resident at pediatric dentistry 2departement of pediatric dentistry faculty of dentistry, indonesia university jakarta indonesia abstract background: traumatic ulcer is a common form of ulceration occured in oral cavity caused by mechanical trauma, either acute or chronic, resulting in loss of the entire epithelium. traumatic ulcer often occurs in children that are usually found on buccal mucosa, labial mucosa of upper and lower lip, lateral tongue, and a variety of areas that may be bitten. to properly diagnose the ulcer, dentists should evaluate the history and clinical description in detail. if the lesion is allegedly accompanied by other infections, such as fungal, bacterial or viral infections, microbiological or serological tests will be required. one of the initial therapy given for fungal infection is nystatin which aimed to support the recovery and repair processes of epithelial tissue in traumatic ulcer case. purpose: this case report is aimed to emphasize the importance of microbiological examination in suspected cases of ulcer accompanied with traumatic fungal infection. case: a 12-year-old girl came to the clinic of pediatric dentistry, faculty of dentistry, university of indonesia on june 9, 2011 accompanied with her mother. the patient who had a history of geographic tongue came with complaints of injury found in the middle of the tongue. the main diagnosis was ulcer accompanied with traumatic fungal infection based on the results of swab examination. case management: this traumatic ulcer case was treated with dental health education, oral prophylaxis, as well as prescribing and usage instructions of nystatin. the recovery and repair processes of mucosal epithelium of the tongue then occured after the use of nystatin. conclusion: it can be concluded that microbiological examination is important to diagnose suspected cases of ulcer accompanied with traumatic fungal infection. the appropriate treatment such as nystatin can be given for traumatic fungal infection. key words: traumatic ulcers, fungal infections, nystatin abstrak latar belakang: ulkus traumatic merupakan bentuk umum dari ulserasi rongga mulut yang terjadi akibat trauma mekanis baik akut maupun kronis yang mengakibatkan hilangnya seluruh epitel. ulkus traumatic sering terjadi pada anak-anak, biasanya ditemukan pada mukosa bukal, mukosa labial bibir atas dan bawah, lateral lidah, dan berbagai daerah yang mungkin dapat tergigit. untuk mendiagnosis ulkus dengan tepat, dokter gigi harus mengevaluasi riwayat dan gambaran klinis secara detil dan jika lesi tersebut diduga disertai infeksi lainnya seperti fungal, bakteri atau virus maka diperlukan tes mikrobiologi atau serologi. salah satu terapi awal jika diketahui adanya keterlibatan fungal dapat digunakan nystatin untuk mendukung pemulihan dan perbaikan jaringan epitel pada ulkus traumatic tersebut. tujuan: laporan kasus ini bertujuan untuk menekankan pentingnya pemeriksaan mikrobiologi pada kasus ulkus traumatic yang diduga disertai infeksi fungal. kasus: seorang anak perempuan usia 12 tahun datang ke klinik ilmu kedokteran gigi anak fakultas kedokteran gigi universitas indonesia pada tanggal 9 juni 2011 diantar ibunya. pasien dengan riwayat geographic tongue datang dengan keluhan terdapat luka di bagian tengah lidah. diagnosis utama adalah ulkus traumatic yang disertai infeksi fungal. penegakan diagnosis ditetapkan dari hasil pemeriksaan swab. tatalaksana kasus: kasus ulcus traumatic ini diatasi dengan dental health education, oral profilaksis, pemberian resep dan instruksi pemakaian nystatin. terjadi pemulihan dan perbaikan epitel mukosa lidah setelah penggunaan nystatin. kesimpulan: pemeriksaan mikrobiologi penting dilakukan untuk menegakkan diagnosa case report 133sella: treatment of lingual traumatic ulcer ulkus traumatic yang diduga disertai infeksi fungal. pengobatan yang tepat seperti pemberian nystatin dapat diberikan pada kasus ulkus traumatic yang disertai infeksi fungal. kata kunci: ulkus traumatic, infeksi fungal, nystatin correspondence: sella, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no.4 jakarta pusat 10430, indonesia. e-mail: jacksell_chanel@yahoo.com introduction lesion occured on tongue is associated with various oral mucosal diseases, both in general and specific conditions. normal variations of the lesions are fissured tongue, hairy tongue, and geographic tongue.1,2 geographic tongue is oftenly associated with various systemic factors and/or psychological conditions, including gastrointestinal disorders, anemia, reiter's syndrome, psoriasis, emotional stress, allergies, diabetes, and hormonal disorders.3 numbness and burning pain derived from geographic tongue accompanied with high mobility of the tongue can become a predisposing factor of trauma causing traumatic ulcers.1 traumatic ulcers are considered as a form of mechanical injury, either acute or chronic, resulting in loss of the entire epithelial layer.2 the clinical features of ulcer are usually single trauma, painful, having a smooth surface and erythematous with a yellowish base and red edge, and without any induration.2,4 the size of the lesion is various ranging from several millimeters to centimeters. in general, large traumatic ulcer is caused by biting or trauma.2 chronic one is usually solitary and covered by yellowish white fibrin clot located on the site of trauma. ulcer pain caused by trauma, usually has spinning and hard edges as in palpation. it indicates that the improvement of consistency is caused by the formation of scar tissue and chronic inflammatory infiltration.4 thus, the diagnosis of traumatic ulcers involves aphtous stomatitis, necrotizing ulcerative gingivitis, eosinophilic ulcer, as well as carsinoma cell squamosa.2 to properly diagnose the ulcer, dentists should evaluate the history and clinical description in detail. the number (single or multiple), location, shape, edge characteristics of the ulcer, the basic description of ulcer, induration (hardness on palpation), and the condition of mucosa (white, red with vesicobullae) should be examined carefully.2,3 in this situation, microorganisms, such as bacteria, fungi, and virus can easily attached.5 fungi, for instance, are considered to be opportunistic and form pseudohyphae. in pathological conditions, the pseudohyphae seems to penetrate into epithelium.6 the symptoms of fungal infection involves burning and sensitive sensations. if it involves the pharyngeal or esophageal, dysphagia may occur. intraoral clinical description of the fungal infection is various from reddish (erythematous) to white pseudomembranous (thrush), with or without angular cheilitis. erythematous appearance is usually found on the dorsal surface of tongue marked with the loss of filliform papillae. meanwhile, pseudomembranous form is usually found in organism colonies attached to surfaces that can be lost if polished, but it usually leaves redness surface and bleeding. the diagnose of fungal infection, can be established through swabs examination processed with potassium hydroxide or gram-negative bacteria before microscopic examination or/by cultural examination to determine the quantity and quality of the number and species of candida.6 fungal infection can be treated with both topical and systemic antifungal drugs. the choice depends on several factors, such as the level of pain, the ability to use topical agents, and the condition of patients. nowadays, topical antifungal drugs are available in mouthwash, suspense, creams, tablets, and lozenges. one of topical antifungal drugs is nystatin.6,7 on the other hand, systemic antifungal drugs provide comfort care even though their prices and the medical conditions need to be considered in determining the therapy. therefore, this case report was focussed on the importance of the microbiological examination in the treatment of ulcers occured on tongue accompanied with traumatic fungal infection in a 12 year old child. case a 12-year-old girl came to the clinic of pediatric dentistry, faculty of dentistry, university of indonesia accompanied by her mother. the patient came with complaints of injuries found in the middle of tongue since five days before (figure 1a and b). the examination showed that the patient often feels numbness in her tongue, with sufferent locations. it is also known that the patient felt sick when eating spicy and hot foods because of her numb tongue, as a result, the child began to bite her tongue to suppress the discomfort. two days ago, the patient then consumed herbal medicines available at home, and then got sick. currently, the patient complained of pain when eating, drinking and talking. the patient had just completed a general test, during her menstrual period and had biting nails habit. in extraoral examination, facial asymmetry was not found, and her right and left submandibular lymph nodes was palpable, soft, and painless. in intraoral examination on her tongue, it is known that there was red oval-shaped ulcer with a diameter of ±7 mm. there was also a pseudomembrane 134 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 132–136 in the middle of the lesion with a yellowish base and red edge without induration (figure 2). her oral hygiene status was good with plaque index of 0.5. the primary diagnosis is suspected traumatic ulcer with fungal infection. the differential diagnoses were aphtous stomatitis, necrotizing ulcerative gingivitis, eosinophilic ulcer and squamosis cell carcinoma. case management the treatment plan involved dental health education (dhe) and oral prophylaxis (op); referral for culture and swab examinations of the lesion area; as well as topical fluoride applications. the treatment conducted was dhe to the mother and the patient in the form of instructions on how to maintain oral hygiene, including tongue and oral prophylaxis. the patient was also given knowledge not to bite her tongue if there was bad taste on the tongue. then, the mother and the patient were given an understanding of geographic tongue that this condition is a benign condition and not a malignancy. the patient was also given a letter of introduction to both lesion swab examination and culture examination in the laboratory of parasitology, faculty of medicine, university of indonesia. based on direct swab examination result in the laboratory, it was then known that there was positive yeast cells. based on the results of such examination, furthermore, the patient was prescribed nystatin, about 100 000 units/ml. the usage instruction of nystatin was 5 ml of nystatin every 6 hours (or 4 times in a day) or at least 10 days or 48 hours after the lesion was disappeard. the patient was asked to come back 7 days later. from of anamnesis, it was known that nystatin was used as recommended. as a result, the patient reported there was no pain at the lesion. based on clinical examination, it was also known that the ulcer on tongue began to dissapear (figure 3). it indicated that there was a regrowth of filiform papilla, so the color around the lesion is the same as the previous color. the patient was instructed to maintain the cleanliness of her oral cavity and tongue and to continue to consume nystatin for the next 10 days. on this visit, topical fluoride application was also conducted. finally, on the second control, 2 weeks later, it was known that there was no sign of inflammation, but there was still visible presence of scar tissue in former lesion area (figure 4). figure 2. the red oval ulcer with diameter of ± 7 mm, and pseudomembrane in the middle of the lesion. figure 1. early lesion with erythematous area on the tongue with white edge and atrophy from filiform papilla filiformis as the description of geographic tongue. aa bb figure 3. the clinical examination on day 7. a) few hours later after nystatin was given, pseudomembrane was dissapeared leaving reddish area; b) one day after nystatin was given, there was clot on the lesion area. a b 135sella: treatment of lingual traumatic ulcer discussion in this case report, a child came with injury in the middle of her tongue since 5 days before. there was a visible lesion, erythematous area of tongue with a white edge that appeared more prominent than the surrounding area. the patient complained of both numbness in accordance with the description of geographic tongue, and irregular reddish spots (erythematous) clearly bordered by white or cream arising keratotic lines which seemed more prominent than the surrounding area and occured at several different locations.3,8,9 in the erythematous, her tongue surface looked red, smooth, and shiny because of atrophy or the loss of filliform papilla.7,10 the white boundary also consisted of a regenerating filliform papilla and a mixture of keratin and neutrophil.2,3 based on the results of anamnesis, it was known that this child had just completed a general test during by her menstrual period so that stress occurrance could trigger exacerbations of geographic tongue, especially when the patient was in the hormonal changes.3,8,11 in clinical examination on her intraoral, it was known that there was a red oval ulcer with a diameter of ±7 mm on the surface of tongue. at the lesion, it was known that there was pseudomembrane layer with yellowish base and red edge as same as the diagnosis of traumatic ulcer.4 the diagnose of traumatic ulcer was also supported by the anamnesis which was confirmed by the patient that she liked biting her tongue due to bad taste on her tongue. she also admitted that she consumed herbal drug on the area so that other diagnosis were excluded. the patient also complained of burning sensation and pain when eating hot and spicy foods. the lesion was also accompanied by other infections, which is fungal infection because of the description of the lesion, pseudomembranous layer. thus, to ensure the diagnose, the patient was asked to get swab examination of the lesion area. this examination was aimed to avoid the contamination of the other side of the lesion.3,4 results of the swab showed that there was fungal infection that may be related to her daily habit of nail biting. the patient was given nystatin, about 100 000 units/ml which usage instruction was 5 ml of nystatin for every 6 hours (or 4 times in a day) or at least 10 days or 48 hours after the lesions were disappeard.7,12,13 there are actually various opinions on the method of nystatin use.6, 12,13 nystatin application must follow the instructions recommended by the manufacturer. nystatin was chosen because it could inhibit the growth of fungi and yeast, but not active against bacteria, protozoa, and virus.14 nystatin can also be used as initial therapy for patients with fungal infections.4,5 nevertheless, it can not be absorbed by skin, mucous membranes, gastrointestinal tract, and vagina, thus, it is excreted through faeces.13,14 it is because nystatin will only be bound by sensitive fungi or yeast.14 based on the pictures taken a few hours after nystatin was given, it was known that pseudomembrane layer was dissapeared, but left a reddish. one day later, clot was performed, indicating the repair process of epithelials and the formation of new epithelial layers. clot is very important in epithelial repair and must endure the initial healing process for the formation of the new epithelial layers.5 when the patient came back to control 7 days later, the lesion was disappeared with repopulation of filliform papillae on her tongue surface, but the ulcer was still obvious. two weeks later, the former lesion was still visible. this area indicated the scar tissue. in general, oral mucosal epithelial recovery is faster than skin. it was known that oral mucosal epithelial recovery only takes 14 days, while skin takes 27 days.5 therefore, biopsy examination, blood tests, and imaging was not conducted since a single lesion can recover less than 2 weeks, so no further investigation was needed. the patient was warned not to bite her tongue if there are geographic tongue lesions in the future, so there will be no more trauma on her tongue surface and to keep her tongue clean to prevent from opportunist fungal infections. it can be concluded that microbiological examination is important to diagnose suspected cases of ulcer accompanied with traumatic fungal infection. the appropriate treatment such as nystatin can be given for traumatic fungal infection. figure 4. the clinical examination of the patient tongue on day 10. a) on the seventh day after the treatment, the lesion on the oral dorsal was dissapeared, but there was still visible presence of scar ulcer; b) in two week control later, there was still visible presence of scar tissue in the area of the former lesion. ba 136 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 132–136 references 1. field a, longman l, tyldesley wr. tyldesley’s oral medicine. 5th ed. new york: oxford university press; 2003. p. 35–40, 51, 67–73. 2. laskaris g. color atlas of oral disease in children and adolescents. new york: thieme; 2000. p. 46–52, 80–2, 128–31. 3. jainkittivong a, langlais rp. geographic tongue: clinical characteristics of 188 cases. j contemp dent pract 2005; 6: 123–35. 4. compilato d, cirillo n,termine n, kerr ar, paderni c, ciavarella d, campisi g. long-standing oral ulcer: proposal for a new ‘s-c-d classification system’. j oral pathol med 2009: 38: 241–53. 5. balogh mb, fehrenbach mj. dental embryology, histology, and anatomy. 2nd ed. st.louis: elsevier saunders; 2006; p. 127–50. 6. epstein jb, silverman s, fleischmann j. oral fungal infections. in: eversole lr, truelove el. essentials of oral medicine. ontario: bc decker inc; 2001. p. 170–9. 7. meechan j. oral pathology and oral surgery. in: welbury r, editor. paediatric dentistry. 2nd ed. new york: oxford; 2001. p. 337–46. 8. greenberg ms, glick m, ship ja. burket’s oral medicine. 11th ed. ontario: bc decker inc; 2008. p. 103–4. 9. aldred m, cameron a, hall r. paediatric oral medicine and pathology. in: cameron a, widmer r, editors. handbook of paediatric dentistry. edinburgh: mosby; 2003. p. 161. 10. mcdonald re, avery dr, dean ja. dentistry for the child and adolescent. 8th ed. st. louis: mosby elsevier; 2004. p. 139–40, 282. 11. cawson ra, odell ew, porter s. oral pathology and oral medicine. edinburgh: churchill livingstone; 2002. p. 216–7. 12. requa-clark b. applied pharmacology for the dental hygienist. 4th ed. st. louis: mosby; 2000. p. 200–1. 13. park nh, kang mk. antifungal and antiviral agents. in: yagiela ja, dowd fj, neidle ea, editors. pharmacology and therapeutics for dentistry. 5th ed. st louis: mosby; 2004. p. 660–2. 14. bahri b, setiabudy r. obat jamur. in: ganiswarna sg, editor. farmakologi dan terapi. 4th ed. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 42 no 2 april 2009.indd 94 vol. 42. no. 2 april–june 2009 research report biocompatibility of acrylic resin after being soaked in sodium hypochlorite nike hendrijatini department of prosthodontic faculty of dentistry, airlangga university surabaya indonesia abstract background: acrylic resin as basic material for denture will stay on oral mucosa for a very long time. the polymerization of acrylic resin can be performed by conventional method and microwave, both produce different residual monomer at different toxicity. acrylic resin can absorb solution, porous and possibly absorb disinfectantt as well, that may have toxic reaction with the tissue. sodium hypochlorite as removable denture disinfectant can be expected to be biocompatible to human body. the problem is how biocompatible acrylic resin which has been processed by conventional method and microwave method after being soaked in sodium hypochlorite solution. purpose: the aim of this study was to understand in vitro biocompatibility of acrylic resin which has polimerated by conventional method and microwave after being soaked in sodium hypochlorite using tissue culture. methods: four groups of acrylic resin plate were produced, the first group was acrylic resin plate with microwave polymeration and soaked in sodium hypochlorite, the second group was acrylic resin plate with microwave polymeration but not soaked, the thirdwas one with conventional method and soaked and the last group was one with conventional method but not soaked, and in 1 control group. each group consists of 7 plates. biocompatibility test was performed in-vitro on each material using fibroblast tissue culture (bhk-21 cell-line). result: the percentage between living cells and dead cells from materials which was given acrylic plate was wounted. the data was analyzed statistically with t test. conclusion: the average value of living cells is higher in acrylic resin poimerization using microwave method compared to conventional method, in both soaked and non soaked (by sodium hypochlorite) group. this means that sodium hypochlorite 0.5% was biocompatible to the mouth mucosa as removable denture disinfectant for 10 minutes soaking and washing afterwards. key words: biocompatibility, cell culture, disinfectant, acrylic resin, polimerization correspondence: nike hendrijatini, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: hendrijantininike@yahoo.com introduction biocompatibility is a harmonius condition without any toxic effect on biological function, which is measured according to local cytotoxity, systematical response, and carcinogen characteristic.1 the treatment of a denture is by taking the denture off and soaking it at night, beside the reservation and cleansing action. the cleansing method of denture generally can be done in two ways, either mechanically or chemically. the process of chemical cleansing is done by putting the denture on the cleanser which contains disinfectant.2 in dentistry, disinfectant material play an important role to decontaminate the disposable or reusable tools which are being used. disinfectant materials can also be used as cleanser for acrylic resin denture. one of the disinfectant materials is sodium hypochlorite (naocl). as a decontamination material, the use of naocl is to prevent infection from patient to the medical health personnel or to other patients, especially in this era in which the number of infection caused by virus is increasing, such as hepatitis and hiv. sodium hypochlorite which is a chemical based material consists of chlorine, is a high level disinfectant and very effective for all bacteria, virus, yeast, parasite and spora.3 disinfectant is aimed to prevent cross-contamination that occurs between denture’s user who suffered from infection, which involved the dentist, dental technician and the 95hendrijatini: biocompatibility of acrylic resin on bhk-21 surrounding people. in daily use, denture’s user can also use denture cleanser with disinfectant by soaking the denture, and rinse it with water, and put it back on. since most part of a denture is acrylic resin which has characteristics of absorbing liquid material,4 it is feared that the cleansing material will eventually make in contact with the oral mucosa. naocl as disinfectant is toxic, especially in high concentration.4 it is necessary to do toxicity test of acrylic resin by using fibroblast tissue culture (bhk-21 cell-line) after being soaked in naocl, which is being used as disinfectant material on removable denture acrylic resin. beside the acrylic resin polymerization process which has been commonly used in indonesia, microwave polymerization process becomes the latest and more efficient process which is more hygienic and resulting a better physical characteristic of acrylic resin with less residual monomer. it is important to do toxicity test of acrylic resin which polymerized by conventional and microwave method which is being soaked in naocl solution. the objective of this study was to observe the in vitro biocompatibility of acrylic resin material which was polymerized by the conventional method and microwave method after being soaked in sodium hypochlorite as denture cleanser material by using tissue culture. the result of this study can give information for dentist in choosing acrylic resin denture disinfectant materials and polymerization method which is more biocompatible, so it can be guaranteed that the material which is being used is safe and more biocompatible to the oral mucosa. materials and methods this study was done by using laboratory experimental method. the samples were twenty eight round shape plates of acrylic resin of heat cured type qc-20 (with diameter of 12 mm, and thickness of 1 mm),5 which were divided into four groups, with 7 plates in each group. this research was conducted at faculty of dentistry laboratory of airlangga university and surabaya pusvetma laboratory. to make the sample, mixtures of gypsum and water, was prepared with a ratio based on the manufacture’s dosage, which was put into the low cuvet. master model was put on the mixtures’s surface. each of it was 10 pieces in one cuvet, and then let it stay for 15 minutes. after the gypsum has been set, apply all the surface of gypsum with vaseline, put the top cuvet, and filled it with gypsum. the gypsum is idled until it harden. the cuvet was opened, and the master model was taken out. for conventional method of polymerization, the brass cuvet was being used, and the plastic cuvet was used for polymerization by microwave. the mold which has been formed was filled with acrylic resin with w/p ratio according to manufacture’s dosage. the cuvet was closed and then pressed it by using hydraulic bench with the pressure of 22 kg/cm 2 hg.6 polymerization by conventional method was performed with japane industrial standard (jis) procedure, where the cuvet was placed into the pan with the temperature of 70° c for 90 minutes, and then continued for 30 minutes in the temperature of 100° c, the cuvet was opened and let it cool down for 24 hours, and taken out afterward.7 for polymerization by microwave method, special cuvet was placed right in the centre of turn table microwave oven. polymerization by microwave was very short. it only took 5 minutes in 500 watt microwave. the cuvet was opened when it’s already cold and the plate was taken out.8 nodules and excess of acrylic on the plate was cut by straight hand piece, and then were rubbed with abrasive paper, number 300 and 600, under flowing water. heat-cured acrylic resin plate which was processed by conventional and microwave polymerization method, was round shape with a diameter of 12 mm, and thickness of 1 mm. all plates are soaked in aquadest for 48 hours. then the two groups were soaked in naocl 0.5% for 10 minutes, and the two other groups was not soaked in naocl solution. concentration of naocl 5.25% being diluted to 0.5% (1:10), was used for soaking. the first group was plate acrylic resin which was processed by conventional method. the second group was processed by conventional method and being soaked in sodium hypochlorite 0.5% for 10 minutes. the third group was processed by the micro wave. the forth group was processed by the microwave and being soaked in naocl 0.5% for 10 minutes. the fifth group was a control group, petri dish containing media and cell culture of bhk-21, without acrylic resin plate which naocl 0.5 % was used as soaking material. acrylic resin plates were rinsed 3 times with aquadest, and sterilized with uv lights for 1 hour inside laminar flow. then the plate was sticked on the base of small petri dish with silicone grease, each of one plate on one petri dish. in every petri dish was added by a media, which was incubated for 48 hours, at 37° c, co2 5%. for the control group, in seven petri dish as which only contained of media without any acrylic resin plate. in the roux tube, cell culture line bhk-21 was added with 20ml eagle media serum (ems) 10%, and then being incubated for 48 hours at 37° c, co2 5%. cell line was tested under the microscope, and when it had been already full (confluent), the ems solution was removed, and was then rinsed twice, with pbs 10%. adding 1 ml of versin trypsin after the cell was detached, and then adding media culture which had bovine serum 10% to stop the versin trypsin reaction, and then making cell with the density of 2 × 105. the cell was ready for the test (figure 1). the media solution was removed and rinsed it twice with pbs 10%, 1ml of versin trypsin 0.25%, 2 ml of ems 10%. 0.1 ml of cell was added 0.9 ml of tryphan blue, and mixed until homogenized. it was dropped on hemositometer, the number of living cells and dead cells was calculated under the microscope, using the bird and forrester method (figure 2).9 96 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 94−98 results table 1. means and deviation standard of living cell percentage in toxicity test of acrylic resin which was soaked and not soaked in naocl to tissue culture number of sample living cells percentage means deviation standard k 7 96.45 0.73 k–n 7 95.43 0.72 m 7 97.86 0.11 m–n 7 96.48 0.52 control group 7 98.04 0.20 note: k : acrylic resin processed by conventional method k–n : acrylic resin processed by conventional method and soaked in naocl m : acrylic resin processed by microwave m–n : acrylic resin processed/by microwave and soaked in naocl the highest mean and standard deviation of living cell percentage in toxicity test of acrylic resin which was processed by microwave, which was not soaked in naocl (97.86 ± 0.11) compared to the one which was not soaked in naocl (96.48 ± 0.52). and for acrylic resin with conventional method which was not soaked in naocl (96.45 ± 0.73) is higher than acrylic resin which was not soaked in naocl (95.43 ± 0.72) (table 1). to observe the level of significance of different means and deviation standard of living cell percentage in toxicity test of acrylic resin which was processed by conventional either microwave method after soaked in naocl, the data then was analyzed statistically by t-test (table 2). table 2. the result of t test of living cell percentage of acrylic resin which is processed by conventional and microwave method and being soaked and not soaked in naocl k k-n m m-n control group k 0.022 * 0.002 * 0.000 * k-n 0.009 * 0.001 * m 0.001 * 0.064 m-n 0.001 * control group note: k : acrylic resin processed by conventional method k–n : acrylic resin processed by conventional method and soaked in naocl m : acrylic resin processed by microwave m–n : acrylic resin processed /by microwave and soaked in naocl t-test was done to observe the toxicity of dental material being used to the tissue culture. from the calculation of living cell percentage was acquired p = 0.001 smaller from α = 0.05 which means there was a quite significant difference between acrylic resin being soaked with or without naocl, either processed by conventional method or microwave. discussion sodium hypochlorite (naocl) is a high level disinfectant material which is broad spectrum, effective to bacteria, spora, yeast, hiv and hepatitis. the substance which has concentration of 0.5% can be used as disinfectant of acrylic resin denture.9 because the majority of removable denture material is acrylic resin which can absorb fluid, it is feared that naocl will be absorbed by acrylic resin. to make a prove, toxicity test in vitro was performed by using cell culture method. in this research the number of cell was calculated by direct counting on the number of living cell and death cell by hemositometer. living cells do not absorb stain of tryphan blue, while dead cell absorbed the stain. after the calculation was done, the average number of living cells from all of the 5 groups was acquired. from all those four group, resulting the average mean of living cells between (95.43 ± 0.72) to (95.86 ± 0.11). figure 1. microscopic appearance of bhk-21 cells which was magnified 200x, the cells is forming a firm tie and creating monolayer cell around the sample. figure 2. microscopic appearance of the living cells (white color) and the dead cells (blue color which has absorbing tryphan blue color). 97hendrijatini: biocompatibility of acrylic resin on bhk-21 the percentage of living cells of all the group of acrylic resin which was soaked by naocl compared to the control group showed significant difference, except for group number 4 which was processed by microwave method. it happened because microwave polymerization produced less residual monomer compared to conventional method.10 on conventional polymerization, the heat energy derived from outside, and it caused monomer molecules outside which the accepted heat and continued the heat to the monomer molecules inside. the monomer molecules moved passively because of the heat from outside, therefore the process of polymerization started from outside to inside. this process caused the residual monomer trapped inside the mixture, which caused the residual monomer on the acrylic resin.11 on microwave polymerization, the heat resulted as the effect of a very fast movement of the monomer molecules from a high frequency electromagnetic, where the crashes of inter molecular occurs and creating heat from inside to outside the direction of the energy reduced the possibility of the residual monomer being trapped inside acrylic resin. residual monomer is the monomer which is not reacted with the polymer, which eventually have the potention to irritate the mucous tissue of the mouth,12,14 which determine that acrylic resin with microwave polymerization is more compatible compared to the conventional method. acrylic resin which was processed either by microwave or conventional method which was then soaked in naocl 0.5 % show significant difference. it was shown that by the soaking process, the percentage of living cells was reduced, which explain that naocl is a toxic disinfectant for the tissue. naocl is toxic and could destroy the cellular tissue. the content of chlorine in naocl solution acted fast and very effective to hbv and hiv. chlorine can cause irritation to the skin or mucosa, because chlorine is able to release the free oxygen which will enter the protoplasmic cells which will destroy cells. the combination of chlorine with the membrane cells will form n-chloro compound which will disturb the metabolism of the cells. the changing of membrane cells, will cause diffusion that make the cell content come out, beside it can also destroy the membrane cell mechanically.5 the death of cells also caused by chlorine oxidation process in sh group and important enzyme, and it can cause the dysfunction of enzymatic process.3 this research used 0.5% naocl concentration. neidle15 argued that the concentration can be used as antiseptic for membrane mucosa. contradiction with neidle, mehra5 determined that the limit of toxic for tissue is 0.25%, but disinfectant of naocl with 0.5% concentration is very effective against hepatitis virus and hiv beside yeast, parasite and sporas. according to mehra 5 the concentration (0.5%) has reached toxic limit, and there is a possibility of residual solution of naocl which can cause tissue toxicity, so the percentage of living cells on tissue culture would be reduced. the percentage of acrylic resin living cells which was processed by microwave method and soaked in naocl is higher than acrylic resin which was soaked in naocl and processed by conventional method. even though the effect of soaking process decreased the percentage of living cells, acrylic resin which was processed by microwave resulting higher percentage of living cells compared to acrylic resin which was processed by conventional method, because at the beginning the residual monomer of acrylic resin is lower. it showed that the decrease of the number of living cells was because of the effect of naocl solution with chlorine. the result of all toxicity test showed that the average mean of living cells which was processed by two methods of polymerization and being soaked in naocl solution resulting the mean more than (95.43 ± 0.72) which was closer to 100%, so it could be said that resin which was processed by both ways either being soaked or not resulting good biocompatibility. material which has good biocompatibility has to be closer or equal to 100% the average mean of living cells percentage, or 92.3–100%.14,17 the result of this research was acrylic resin denture which either being soaked or not by naocl fulfilled the condition. it is important to remember that the procedure of naocl 0,5% usage as disinfectant is by soaking the denture for 10 minutes and then rinsed it off. so the naocl will not make a contact with mucosa tissue directly, then naocl might cause less toxicity reaction to the oral mucosa. it concluded that acrylic resin which was processed with microwave and soaked in naocl was more biocompatible compared to the conventional method which was soaked in naocl. it means that naocl 0.5% is biocompatible as removable denture disinfectant by means of soaking for 10 minutes and then rinsed. the usage of naocl 0.5% as cleanser for denture acrylic resin which was processed by microwave either by conventional method has to be done in a careful instruction, guaranteed that it is rinsed as clean as possible. it was found out in the research of culture cell media that the decrease of the number of living cells still occurred, although it was compatible. references 1. anusavice kj. philip’s science of dental materials. 10th ed. philadelphia: wb saunders co; 1996. 237–72. 2. budtz-jorgensen e. materials and methods for cleaning dentures. j prothest dent 1979; 42:619–22. 3. kinyon tj, schwartz rs, burgess jo, bradley dv. the use of warm solutions for more rapid disinfection of prostheses. int j prosthodont 1989; 2:518–23. 4. combe ec. notes on dental materials. 6th ed. edinburg, london, melbourne, new york: churchill livingstone; 1992. p. 1233–5. 5. mehra p, clancy c, wu j. formation of a facial hematoma during endodontic therapy. jada 2000; 131:67–72. 6. sheridan pj, koka s, ewoldsen no, lefebve ca, lavin mt. cytotoxicity of denture base resins. int j prosthodont 1997; 10:73–7. 7. melani wg. hubungan antara suhu dan waktu proses curing dengan porositas dan sisa monomer pada polimerisasi resin aklirik heat cured. thesis. surabaya: universitas airlangga; 1981. p. 19–22. 98 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 94−98 8. salim s. pengaruh humiditas dan lama penyimpanan serta cara kuring terhadap sifat fisik, kimia dan mekanik resin aklirik basis gigi tiruan. dissertation. program surabaya: pascasarjana universitas airlangga; 1995. p. 20–59 9. bird br, forrester ft. basic laboratory technique in cell culture. us: department of health and human services, public health service. centre of disease control; 1981. p. 33–43. 10. nike h. pengaruh konsentrasi larutan sodium hipoklorid terhadap kekuatan transversa plat resin akrilik. ceramah ilmiah lustrum viii fkg ugm, majalah ilmiah dies natalis fkg-ugm ke-40 2001; 262–5. 11. haslinda zt. pengaruh ketebalan dan jenis resin akrilik heat-cured gigi tiruan terhadap jumlah monomer sisa, porositas dan kekuatan transversa. thesis. surabaya: pascasarjana universitas airlangga; 1996. p. 60–2. 12. sanders jl, levin b, reitz pv. porosity in denture acrylic resins cured by microwave energy. quintessence international 1987; 18(18):453–6. 13. zografakis ma, harrison a, hugget r. measurement of residual monomer in denture base materials: studies on variations in methodology using gas-liquid chromatography. j prosthodont restor dent 1994; 2(3):101–7. 14. muslita i. biokompatibilitas bahan basis gigi tiruan resin akrilik. majalah ilmiah kedokteran gigi 2002; 50:133–8. 15. neidle ea, kroeger dc, yagiela ja. pharmacology and therapeutics for dentistry. st. louis, toronto, london: the cv mosby; 1980. p. 662–3. 16. neyt lf. augmentasi tulang alveolaris dan sinus lift dengan tehnik mutakhir. ceramah sehari di fakultas kedokteran gigi universitas airlangga, 19 februari 1998; p. 8. 17. rubianto m. biokompabilitas bahan allograft (human bone mineral powder) dibandingkan dengan bahan alloplast (hydroxyapatite). kumpulan naskah temu ilmiah nasional i 1998; 507–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 156 the transverse strength of acrylic resin after coleus amboinicus, lour extract solution immersion devi rianti department of dental material and technology faculty of dentistry airlangga university surabaya indonesia abstract a laboratoric experimental study was conducted on the transverse strength of acrylic resin after coleus amboinicus, lour extract solution immersion. the aim of this study is to know the difference of acrylic resin transverse strengths caused by immersion time variations in a concentrate solution. the study was carried out on unpolished acrylic resin plates with 65 × 10 × 2,5 mm dimension; solution with 15% coleus amboinicus, lour extract, and 30, 60, 90 days immersion times to measure the transverse strength and sterilized aquadest was used as control. acrylic resin plates transverse strength was measured using autograph ag-10 te. the data was analyzed using one-way anova and lsd with 5% degree of significance. the result showed that longer immersion time will decrease the transverse strength of the acrylic resin plates. after 90 days immersion time, the transverse strength decrease is still above the recommended standard transverse strength. key words: coleus amboinicus, lour, candida albicans, transverse strength, denture cleanser, resin acrylic correspondence: devi rianti, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction up to now acrylic resin is still used as denture base due to the advantage of having good physical and esthetic character, small dimension change and being easily repaired. in addition to the advantage, acrylic resin also has the disadvantage such as: residual monomer, porous, water absorption and less resistant to abrasion.1,2 acrylic resin in oral cavity is covered by saliva with high protein content as a result pellicle formation occurs. this pellicle is able in having adherence to microorganism such as candida albicans.3 pellicle, in two-hour period, would change into plaque which consist of a cluster of microorganism, glycoprotein matrix as well as polysaccharide which adheres to the teeth surface. the process of plaque formation is similar to the process occurs in denture surface.4 stomatitis on a patient wearing removable denture is called denture stomatitis in which the prevalence is quite high in indonesia and characterized by the present of candida albicans cluster.5 the statement is supported by a study which reported that the present of candida albicans detected in 64% of 50 patients using acrylic resin denture base.6 some researchers stated that the character of denture material, pellicle as well as candida albicans give the most contribution to the occurrence of denture stomatitis.3,7 denture stomatitis can be prevented by taking care, cleansing and taking off the denture at night.8 to clean the denture can be done in two-ways by either mechanically or chemically. mechanical cleaning can be done by using tooth brush or ultra sonic device, meanwhile chemical cleaning can be done by immersing the denture in cleansing solution in 15 minutes, 30 minutes, one hour or the whole night depends on the cleansing material being used.9 furthermore, other researcher stated with fungicide effect should be done minimally 30 minutes and more effectively done in two hours.10 denture cleanser which is available in the market is frequently imported but currently the indonesian government is actively promoting traditional herb as an alternative medication, and as a matter of fact, indonesia is really reach or herbal plant. therefore, based on national health system emphasizing on effective and efficient traditional medication, in which it should be guided and done through / for experiment and scientific research on herbal plant. one of family herbal medicines which is easily planted and grown in the garden is daun jinten with the latin name: coleus amboinicus, lour or plectranthi amboinicus folium. the plant is efficacious medication for stomatitis and fungicidal.11 it has kalium content and essential oil containing carvacrol, isopropyl o cresol, phenol and cineol. additionally explained that 120 kg of fresh coleus amboinicus, lour could obtain 25 ml of essential oil. this is equivalent to 0.2% of essential oil consisting of phenol derivation that is isopropyl-o-cresol with high antiseptic capability.12 the previous study reported that 15% concentration of extract coleus amboinicus, lour effectively killed candida albicans in acrylic resin after a two-hour of immersion time.13 cineol chemical substance has anti microbial capability towards candida albicans, trichiphyton metagrophytes 157rianti: the transverse strength of acrylic resin and cryptococcus neoformans.14 phenol and cresol could kill vegetative cell, fungi and spore configuring bacteria by creating protein denaturizing and decreasing surface strain, so bacterial permeability would increase.15 meanwhile according to merck index16 carvacrol has fungicidal. in addition to its fungicidal, phenol which is one of the essential oil element, in case of having contact to acrylic resin, it would cause chemical damage to the surface of acrylic plate.17 coleus amboinicus, lour had fungicidal and phenol content which could destroy acrylic resin. other researcher stated that acrylic resin immersion in cleansing solution or anti bacterial solution could change the basic physical and mechanical character of acrylic resin denture such as decreasing the transverse strength.18 based on the above analysis, the problem arose whether there might be differences in acrylic resin transverse strength caused by various duration of immersion in extract solution of coleus amboinicus, lour. how long is the effective immersion time in extract solution of coleus amboinicus, lour without decreasing the transverse strength of acrylic resin. the purpose of the study was to know the transverse strengths of acrylic resin caused by various duration of immersion in extract solution of coleus amboinicus, lour and to determine the effective duration of immersion without decreasing the transverse strength. the advantage of the study outcome would contribute scientific information on the effective duration of immersion of acrylic resin in extract solution of coleus amboinicus, lour without decreasing the transverse strength. this shall be used as the basic determination to the use of this solution as an alternative of cleansing material for denture treatment. materials and methods experimental laboratory study was conducted with the subject of (65 × 10 × 2.5) mm unpolished acrylic resin plate, using free variable of which the immersing period were 30, 60, and 90 days, the variable depended on the strength of transverse acrylic resin. control variable were: the type of acrylic resin, method of making samples, sample size, polymerization method, similarity of soil for planting coleus amboinicus, lour, harvest time, drying time, method of making coleus amboinicus, lour extract using ethanol solution, concentration of extract solution, device and method of measuring transverse strength. the study was initiated by making the samples as well as preparing extract solution of coleus amboinicus, lour, diluting the extract of solution coleus amboinicus, lour, preparing profile chromatogram with thin layer extract as done by former researchers to know the active content in the extract, and also the transverse strength test.13 the extract solution of coleus amboinicus, lour preparation was done in the fitochemical laboratory of faculty of pharmacy of airlangga university. the extract was prepared as follows: 3–4 months aged of coleus amboinicus, lour leaves freshly cropped at 07.00 a.m. at the traditional medicine research center experimental plantation, then washed and dried in a room with 24°c of temperature. after being dried, they were grinded. the powder weighted as of 1000 gram then solved in 3000 ml of ethanol for 72 hours and afterwards it was filtered using a buchner quill. the filtrated solution evaporated using vacuum evaporator for 5 hours, and it yielded 100 grams of pure coleus amboinicus, lour extract.19 afterwards 1.5 grams of the extract solved in 10 ml of sterile aquadest and vibrated in an ultrasonic vibrator for 15 minutes to obtain a 15% concentrated solution. the transverse strength test was carried out by using autograph a6-10 te with cross head speed 1/10 mm/sec. the distance between two supporting parts was 50 mm.20 at the beginning all of the acrylic resin plates were immersed in aquadest for 48 hours.21,22 then they were immersed in coleus amboinicus, lour extract solution and classified into 3 groups based on the duration of immersion i.e. 30, 60, and 90 days. replacement of immersion solution was done in every 24 hours. all immersion processes were performed in room temperature 27 ± 1 °c. immersion for control is specially done in aquadest. after immersions performed in individual period, the acrylic resin plates were cleansed with aquadest, dried, and finally transverse strength tested. the result of the test was calculated based on the following formula:18 s: transverse strength, b: specimen width (mm), i: support distance (mm), d: specimen thickness, p: load weight (n) the result of measurement was tabulated according to the individual group, followed by statistical test by using one-way anova with = 0,05. if the outcome was significantly different, the test would be continued using lsd test. result after the acrylic resin plates immersion processes in 15% concentration of extract solution of coleus amboinicus, lour in the period of 30, 60, 90 days, and using aquadest as control were completed, the transverse strength was measured by using autograph ag-10 te. the outcome of this is shown on table 1. prior to do the parametric test, a kolmogorov-smirnof test as a normality test was performed to get the significance difference. the result of the three treated group as well as the control showed normal distribution (p > 0.005) and followed by comparation test for the four groups and control through one-way anova test. the result of one-way anova test showed significant difference among the 158 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 156–160 table 1. mean and standard deviation of transverse strength of acrylic resin after immersion in 15% extract solution of coleus amboinicus, lour (mpa) figure 1. the surface structure of acrylic resin plate immersed in aquadest for 90 days (light microscope, magnified 100×). treated groups with p = 0.001 (p < 0.05), in order to know further the difference, the analysis was continued by using the least significant difference (lsd) test illustrates on table 2. table 2 shows that there is significant difference in every sample group except control group meaning lsd test on table 2 shows significant difference among every immersing group in a period of 30, 60, 90 days in other words that the increase of immersion period in extract solution of coleus amboinicus, lour will decrease infl the transverse strength of acrylic resin plate. discussion the result of the study showed that mean of transverse strength of acrylic resin immersed in extract solution of coleus amboinicus, lour for 30, 60, 90 days. decreased of the transverse strength of acrylic resin (table 1). one-way anova statistical analysis was used and continued by lsd test, the result showed of the longer the immersion duration is, the transverse strength of acrylic resin will significantly decreases. the above outcome might be caused by extract table 2. least significant difference test of transverse strength of acrylic resin plates immersed in extract solution of coleus amboinicus, lour figure 2. the surface structure of acrylic resin plate immersed in extract solution of coleus amboinicus, lour in 15% concentration for 90 days (light microscope magnified 100×). 159rianti: the transverse strength of acrylic resin of coleus amboinicus, lour through chromatography thin layer test, consist of phenol 5.15% and cineole content in the extract solution of coleus amboinicus, lour would influence the transverse strength of acrylic resin. acrylic resin is polymer in the form of long polyester which consists of repeated methyl methacrylate with low polarity.1 meanwhile phenol is acid in nature with high polarity. in acid condition would be hydrolyzed in case the hydrolysis occurs, polymer will experience degradation and as a result it will probably decrease the transverse strength. degradation process on structural surface of acrylic resin could be seen on figure 1 and 2 supported by previous researchers. shen et al.17 suggested that the transverse strength dependend on the alteration of morphological surface in which it is strongly influenced by the duration of immersion and the type of disinfectant. the polyphenol group is responsible for the cause of crazing to polymer.23 the main character of acrylic resin is to absorb liquid, therefore, phenol in extract solution will be absorbed into acrylic resin, so that crazing will occur not only in the surface but also will continue to penetrate into acrylic resin plate and finally it would contribute to the decrease of transverse strength. modern plastic encyclopedia17 also supported that pure phenol in 5% concentration would destroy structural surface of acrylic resin. othmer24 also proved that chemical resistant of acrylic resin could be influenced by phenol. in this study the duration of acrylic resin plate immersion was 30, 60, 90 days. it is identical to the duration of acrylic resin denture use for 1, 2, 3 years, if it is 2 hours/day; for 2, 4, 6 years it was immersed in one hour/day immersion duration. additional duration of immersion shows the significant of transverse strength. anderson25 reported that due to phenol absorption, acrylic resin molecular bind will be easily broken and eventually the bind among the molecules will be cut off resulting in the decrease of transverse strength. based on the report acrylic resin immersion for 30, 60, 90 days might have weakened molecular bind and decreased transverse. the lowest mean transverse strength in 72.70 mpa originated from acrylic resin plate immersed in extract solution of coleus amboinicus, lour with 15% concentration in 90 days. the transverse strength is still above the recommended value as minimal acceptable transverse strength for acrylic resin denture base that is not allowed to be less than 55 n/mm2 or 66.04 mpa.18 this was due to the use of cross linked type of heat cured acrylic utilized in this study. according to ada no.12,21 the cross linked type of neat cured acrylic resin consists of cross linked material in the monomer liquid with 1-2%. adding this material will make the possibility of the connection between two long polymer molecules, so that it create a stronger, harder and more resistant to scratch, crack and to bear the action of solution. a study by asad et al.18 reported that the decrease of transverse strength occurs in heat cured acrylic resin either cross linked or non cross linked after immersion in disinfectant solution with alcohol base material for 7 days, however transverse strength of acrylic resin for non cross linked is higher than cross linked. this study is a preliminary study, because the extract processing is more accurate and measurable. extract material availability is more difficult to obtain, is it is, medicinal herbs processing alternatives is required in order to allow the public easier to utilize it. based on a study, medicinal herbs processing by infusum technique is apparently better than boiling. the infusum advantages, besides of easier to make and more socialized, it is cheaper because it does not need special treatment and sophisticated equipments as compared to extract.26 the outcome of this experimental laboratory study observing transverse strength of acrylic resin immersed in extract solution of coleus amboinicus, lour 15% concentration, it can be concluded that the longer duration of immersion 30, 60, 90 days will cause the decrease of transverse strength of acrylic resin plate. after 90 days duration of immersion the decrease of immersion strength still above the recommended standard transverse strength. it is suggested that extract solution of coleus amboinicus, lour can be used as an alternative cleansing solution for acrylic resin denture and also, it is necessary to conduct further study on the color alteration of acrylic resin after immersion in extract solution of coleus amboinicus, lour considering color alteration will influence denture esthetic. references 1. combe ec. notes on dental materials. 6th ed. edinburg: churchill livingstone; 1992. p. 79–120. 2. anusavice kj. phillips’ science of dental materials. 11th ed. usa: saunders; 2003. p. 89–90, 144–6, 723–4, 726–7, 733, 741–4. 3. edgerton m, levine mj. characterization of acquired denture pellicle from healthy and stomatitis patients. j prosthet dent 1992; 68: 683–91. 4. abelson dg. denture plaque and denture cleansers. j prosthet dent 1981; 45: 376–9. 5. soenartyo h. prevalensi candida albicans rongga mulut orang dewasa serta hubungannya dengan faktor-faktor lokal dan sistemik. disertation. surabaya: universitas airlangga; 1987. p. 113–4. 6. elizabeth m. prevalensi candida species di daerah tissue surface dari basis gigi tiruan penuh rahang atas. rimbawan1996; 1b: 1217–26. 7. radford dr, challacombe sj, walter jd. denture plaque and adherence of candida albicans to denture base materials in vivo and in vitro. crit rev oral biol med 1999; 10:99–110. 8. devenport jc. the oral distribution of candida in denture stomatitis. brit dent j 1970; 129:151–6. 9. jorgensen be. material and method for cleaning dentures. j prosthet dent 1979; 42:619–22. 10. nikawa h, hamada t. efficacy of commercial denture cleansers. dent j 1998; 31:77–82. 11. wijayakusuma h, dalimartha s, wirian ag. tanaman obat berkhasiat indonesia. jilid iv. edisi 1. jakarta: pustaka kartini; 1996. p. 38–41. 12. heyne k. tumbuhan berguna indonesia. jilid iii. jakarta: badan litbang kehutanan. yayasan sarana wana jaya; 1987. p. 1698. 13. rianti d. efektivitas lama perendaman resin akrilik dalam ekstrak 160 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 156–160 daun coleus amboinicus, lour terhadap keberadaan candida albicans. majalah kedokteran gigi surabaya 2003; 46(4):129–33. 14. hammerschmidt fj, clark am, soliman fm, el-kashoury es, kawy mm, fishawy am. chemical composition and antimicrobial activity of essential oil of jasonia candicans and jasonia montana. planta med 1993; 59:68–70. 15. rahardjo mb. perbedaan daya antibakteri allium sativum linn dan kaempferia galanga terhadap streptococcus mutans dan bermacammacam bakteri yang berasal dari saluran akar gigi gangraena pulpae. thesis. surabaya: universitas airlangga; 1993. p. 13. 16. windholz m. the merck index. 11st ed. usa: merck & co., inc; 1989. p. 34–5. 17. shen c, javid ns, colaizzi fa. the effect of glutaraldehyde base disinfectants on denture base resins. j prosthet dent 1989; 61:583–9. 18. asad t, watkinson ac, huggett r. the effect of disinfection procedures on flexural properties of denture base acrylic resins. j prosthet dent 1992; 68:191–5. 19. dep kes ri. farmakope indonesia. edisi 3. jakarta: dep kes ri; 1979. p. 12–13. 20. gy szabo, stafford gd, huggett r. some mechanical properties of denture base polymers treated with an ultraviolet light activated coating material. j dent 1987; 15:261–65. 21. american dental association (ada). guide to dental materials and devices. 7th ed. chicago: american dental dental association; 1974. p. 97–102, 203–8. 22. beyli ms, fraunhofer ja. repaired of fracture acrylic resin. j prosthet dent 1980; 44:497–503. 23. craig rg, powers jm. restorative dental materials. 6th ed. london: mosby co; 2002. p. 135–40. 24. othmer k. encyclopedia of chemical technology. 3rd ed. new york: new york wiley interscience publication. john wiley & son; 1982. p. 382. 25. anderson jn. aplied dental materials. 5th ed. oxford blackwell scientific publication; 1976. p. 245–84. 26. eha d. khasiat obat kumur infusa daun kacapiring terhadap perubahan mikroorganisme rongga mulut pemakai gigi tiruan lepasan. majalah ilmiah kedokteran gigi fakultas kedokteran gigi universitas trisakti 1999; edisi khusus foril vi: 497–501. 176 a new concept in orthodontics: faster and healthier tooth movement by regularly consuming xyilitol chewing gum haryono utomo department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract xylitol was first discovered in the 19th century, it wasn’t until the 1960’s that commercial production was first implemented. recent studies showed that xylitol chewing gum is beneficial for preventing caries and periodontal disease. therefore, it is also advantageous for orthodontic treatment, especially the fixed orthodontics patients who have difficulties in acquiring optimal oral health, particularly periodontal health which important in remodeling. however, how consuming xylitol chewing gum may stimulate tooth movement and preventing root resorption is still unclear. it is suggested that chewing activities may stimulate tooth movement, since jaw hypofunction leads to lower mineral apposition and bone function; and narrow periodontal ligament (pdl). these conditions may lead to impaired remodeling process, and increases the susceptibility of root resorption during orthodontic tooth movement. moreover, since stimulation of the pdl could be mechanoreceptive (i.e. chewing action) or nociceptive (i.e. painful stimulation), periodontal nerve fibers are supposed to play an important role in bone remodeling. it is supported by a study which revealed that during tooth movement, the galanin-containing immunoreactive nerve fibers, a part of primary sensory neurons in the pdl is increasing. galanin is able to induce osteoclast differentiation that needed for bone resorption in orthodontic treatment. the objective of this study is to elucidate a new concept in using xylitol chewing gum as an excellent media to have a faster and healthier orthodontic movement. since continuous chewing stimulates the pdl which enhances tooth movement, improves oral health, and prevents root resorption; it is concluded that this concept is possible. key words: xylitol, chewing gum, orthodontic treatment correspondence: haryono utomo, c/o: bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof.jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id, telp. (031) 5053195 introduction successful orthodontic treatment should include rapid tooth movement concomitantly with healthy periodontal tissues, and minimal root resorption. it is a common problem that plaque removal is difficult in individuals carrying orthodontic brackets and ligatures. in 1970, the dental importance of xylitol for dental plaque inhibition was discovered in turku, finland, followed by the launching of first commercial xyilitol chewing gums in 1975.1 after more than 30 years of xylitol dental use, unfortunately, lack of study had been investigated the advantageous effect of xylitol products to orthodontic treatment. there were studies which found that xylitol products were able to increase salivary ph2 and inhibit lactic acid formation3 in fixed orthodontic patients; therefore, reduces demineralization. however, the idea of consuming xylitol chewing gums to improve the quality of orthodontic treatment by promoting both periodontal health and rapid tooth movement; and also preventing root resorption is considered unusual. the rate of tooth movement depends on the remodeling process. it could be affected by biological, mechanical or other factors i.e. force magnitude, age, sexual hormone, bone density, genetic variability, activation interval etc.5 there were some recent ideas to accelerate tooth movement rate such as “accelerated osteogenic orthodontics, aoo”, also called wilckodontics; nevertheless, it was complicated and still expensive (us$ 10.000–15.000).6 others were drugs that favor osteoclast recruitment and genetic engineering of cells involved in tooth movement, but it should take years and million of dollars of researches for commercial use in orthodontic treatment.5 therefore, this new concept is regarded easier and more economical. owing to the remodeling process, a simple matter such as continuous chewing that may act as tooth movement stimulator was rarely discussed; thus it is considered as a new concept in orthodontic treatment. some literatures supported the possibility of this concept. during tooth movement, it was found that the galanin-immunoreactive nerve fibers in periodontal ligament were increasing; galanin was able to stimulate osteoclast activity. 7 in addition, the application of continuous force to the tooth also triggers addition bone volume, which enhances remodeling.8 other theory is the hypofunctional periodontium concept which stated that in the absence or reduced occlusal function, the periodontium narrows, and its cushioning effect decreases which lead to increase vulnerability to root resorption during orthodontic movement.9 177utomo: a new concept in orthodontics: faster and healthier tooth movement the objective of this review is to elucidate the possibility of using xylitol chewing gum as an appropriate media to stimulate tooth movement, optimizing dental and periodontal health, and simultaneously prevent root resorption during orthodontic treatment. hopefully, this new concept could be included as a routine procedure during orthodontic treatment. xylitol xylitol is a 5-carbon sugar that was discovered by fischer and stahel in germany, and bertrand in france around 1890. since the 1960s it was used for medical purposes such as parenteral nutrition and special dietary purposes. in 1970, the dental importance of xylitol for dental plaque inhibition was discovered in turku, finland, followed by the launching of first commercial xyilitol chewing gums in 1975, then usa a few months later.1 until now, the use of xylitol chewing gums are widespread worldwide, that also supported by the launching of ”xylitol oral health program” by the academy of dental resources in 2006.10,11 however, researches of xylitol in orthodontics, especially in fixed orthodontics treatment were still unusual. one of the investigations was related to the effect of xylitol lozenges for increasing dental plaque ph2 and the other was the effect of xylitol tablets to streptococcus mutans and the formation of saliva lactic acid.3 studies showed that 4 to 12 grams of xylitol per day are enough. the “all xylitol” mints and gums contain about one gram of xylitol in each piece. you could begin with as little as one piece four times a day for a total of four grams. it is not necessary to use more than 15 grams per day as higher intakes yield diminishing dental benefits.11,12 if used only occasionally or even as often as once a day, xylitol may not be effective, regardless of the amount. use xylitol at least three, and preferably 5 times every day. use immediately after eating and clearing the mouth by swishing water, if possible. between meals, replace ordinary chewing gum, breath mints, or breath spray with comparable xylitol products.12 mastication and malocclusion mastication is a complex task with numerous anatomical, physiological, and psychological components. given the remarkable ability of individuals to adapt and compensate, a deficiency in one of the componenets can be compensated for by other components. for example, poor masticatory biomechanics might be compensated for by increased muscle strength. masticatory ability, efficiency, and performance are three ways of measuring the capability of individuals to breakdown foods. masticatory ability is a subjective measure or a perception, how well subjects think they break down foods. on the contrary, masticatory efficiency and performance are objective measures.13 efficiency pertains to the number of masticatory cycles (i.e. number of chews) required to reduce foods to a certain size.13 malocclusion negatively affects subjects’ ability to process and break down foods. compared to normal occlusion, the median particle sizes for class i, class ii, and class iii malocclusions were approximately 9%, 15%, and 34% larger, respectively. individuals with normal occlusion also produced a wider distribution of particles, which indicates better masticatory performance. in addition, malocclusion affects an individual’s perception of how well they can chew. compared with normal occlusion, individuals with class iii malocclusions reported the greatest difficulty, followed by class ii malocclusions and class i malocclusions.14 biologic basis of tooth movement orthodontic tooth movement is induced by mechanical stimuli and facilitated by remodeling of the periodontal ligament (pdl) and alveolar bone. a precondition for those remodeling activities, and ultimately tooth displacement, is the occurrence of an inflammatory process. vascular and cellular changes were the first events to be recognized and described, and a number of inflammatory mediators, growth factors, and neuropeptides have been demonstrated in periodontal supporting tissues. their increased levels during orthodontic tooth movement have led to the assumption that interactions between cells producing these substances, such as nerve, immune, and endocrine system cells, regulate the biological responses that occur following the application of orthodontic forces.15 application of force to the tooth, could either increasing or decreasing the loading of the pdl. this strain is relayed to the osteoblasts in the bony socket lining. one of proteins in the membrane of the osteoblast is called an integrin. integrins translate mechanical strain into a signal within the cell that can turn stimulate a gene to make the cell develop ligands. this “talk” between osteoblasts and osteoclasts is accomplished through a receptor activator of nuclear factor k b (rank) and rank ligand. rank ligand on the osteoblast membrane which can interact with developing monocytes to cause them to differentiate into osteoclasts. ligands allow intracellular communication, which stimulates undermining resorption, allowing tooth movement. interestingly, other ligands can develop to block the rank ligand and prevent osteoclast formation.4,5,15 however, this simple mechanism is modified by many other factors i.e. force magnitude, age, sexual hormone, bone density, genetic variability, activation interval etc.4,15 according literatures and practical experience, teeth move at different rates and there are differences between individuals. these differences can be experimentally demonstrated in dogs, where two to three times the amount of movement occurs in different dogs using the same force levels. the reason is beginning to be elucidated.5 the role of periodontal ligament nerve fibers in remodeling the pdl is partly innervated by primary sensory neurons in the trigeminal ganglion. trigeminal ganglion neurons are considered to transmit nociceptive and mechanoreceptive information from the periodontal ligament to the brainstem. 178 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 176-180 experimental tooth movement causes several changes in periodontal tissues such as the blood vessels, bone, and periodontal ligament.7,16 in the pdl, nerve fibers which contain calcitonin generelated peptide (cgrp) also increase in number during orthodontic tooth movement; numerous free nerve endings and perivascular endings appear in the apical region of the periodontal ligament. these nerve fibers are considered to be associated with the pain and discomfort that patients experience during orthodontic tooth movement. therefore, it is possible that periodontal nerve fibers play a role in remodeling of the periodontal tissues during orthodontic tooth movement.7 calcitonin gene-related peptide (cgrp) and galanin are known to have nociceptive functions in the spinal cord). cgrp can enhance nociceptive inputs to secondary neurons, while galanin has a suppressive effect on nociceptive transmission in the cord. therefore, the increased galanin-immunoreactive neurons and coexpression of galanin and cgrp suggest that galanin is associated with the modulation of nociceptive information. cgrp has been suggested to affect bone remodeling during experimental tooth movement. this peptide is known to suppress differentiation of osteoclasts, and may promote bone formation.7,16 bone remodeling bone remodeling is stimulated in a hierarchy. the chemical stimulation and interaction of osteoblasts and osteoclasts controls tooth movement. factors such as prostaglandins like prostaglandin e2 (pge2) and proinflammatory cytokines (i.e. tumor necrosis factor-a, tnf-a; interleukin-1b, il-1b) can all be “up or down regulated” by hormones such as estrogen and are activated by environmental stimulation to remodel bone. postmenopausal women tend to have a problem with increased bone resorption because estrogen levels are decreased in the body.5,15 factors that inhibit and increase tooth movement chemical factors were identified that favor osteoclast recruitment so teeth can move faster. some factors inhibit osteoclast activity and block inhibitors of osteoclast activation (i.e. rank ligand inhibitor). still other factors are known to promote osteoblastic activity and may help someday with increased anchorage for moving teeth (i.e. bone morphogenic protein, bmp). however, before these chemical applied to human, problems such as an appropriate delivery system need to be worked out and would involve questions of systemic vs. local delivery, dosage, effectiveness of the drug based on the delivery route chosen effect, side effects of the drugs etc.5 “accelerated osteogenic orthodontics” also called wilckodontics, has been done to move teeth faster. a slow speed bur is used to place dimples in the bone exposed by a flap procedure. this selective decortication is done and then the bone is covered with demineralized freeze dried bone and bovine xenograft. this resulted in faster tooth movement and completion of an 18-month case in six months.6 root resorption root resorption is another unwanted effect or orthodontic treatment. researches had been identified factors that predispose to root resorption. these are the following: a) root shape, longer and tapered roots are predisposed to more root resorption; b) amount of movement of the root apex; c) forces such as class ii elastics on upper canines; d) longer treatment times; e) individual variation and genetic predisposition.5 additionallly, periodontal hypofunction which manifestates as narrower periodontal ligament (pdl) has the propensity to increase root resorption by diminished cushioning effect.9 discussion studies by sengun et al. and steckesen-blicks et al. supported the use of xylitol chewing gums to optimize dental and periodontal health during orthodontic treatment.2,3 clinical studies have shown that chewing sugarless gum for 20 minutes following meals can help prevent tooth decay, i.e. by increase salivary ph.17 in the future, look for chewing gum that delivers a variety of therapeutic agents that could provide additional benefits to those provided by the ability of gum to mechanically stimulate salivary flow. for instance, some gum might contain active agents that could enhance the gum’s ability to remineralize teeth and reduce decay, or enable gum to help reduce plaque and gingivitis.18,19 based on literatures, it had been found that chewing performance of young adults with excellent buccal segment occlusion is 40% better than that of “less than ideal” buccal segment relationship, and these differences increase with increasing number of chews. moreover, masticatory performance also related to the classification of malocclusion, the greatest difficulty in chewing was class iii, followed by class ii and class i respectively.14 therefore, it is plausible that the majority of orthodontic patient already have masticatory hypofunction which has deleterious effects during orthodontic treatment such as the predisposition to root resorption, and poorer bone turnover or remodeling than normal occlusion. as the result, enhancing masticatory performance or occlusal function should be beneficial for a healthier root and periodontal tissues during tooth movement. according to deguchi et al. (2003)16 and deguchi et al. (2006),(2006),7 cgrp in the periodontal nerve fibers has been suggested to affect bone remodeling during experimental tooth movement. this peptide is known to suppress differentiation of osteoclasts, and may promote bone formation; this action is counter-regulated by galanin. it is the peripherally transported galanin that plays a role in periodontal ligament remodeling.7,16 consequently, 179utomo: a new concept in orthodontics: faster and healthier tooth movement continuous stimulation-inhibition impulses by consuming chewing gum, even though is not a nociceptive stimulus, could enhance pdl remodeling by up-regulate galanin synthesis in the trigeminal primary neurons innervating the periodontal ligament. referred to these studies, it is possible that the pdl stimulation may accelerate tooth movement. it is the chewing action that stimulates mechanoreceptor during gum chewing. however, the possibility of nociceptor stimulation could also be expected. during tooth movement there is an increase of pro-inflammatory mediators (i.e. prostaglandin e2, pge2) which able to sensitize nociceptor and decrease pain threshold, and modulate pain perception during orthodontic treatment.15 therefore, it is common that even light touch may elicit pain in moved teeth. thus, occasionally, continuous chewing action such as gum chewing may act both as mechanoceptive and nociceptive stimulation which beneficial to stimulate orthodontic tooth movement. additionally, literatures have shown that the expression and production of some inflammatory mediators (pge2, il-1b are promoted by mechanical stimulation of the pdl. cyclooxygenase2 (cox-2) is induced in pdl cells by cyclic mechanical stimulation and is responsible for the augmentation of pge2 production. it was also demonstrated that compressive force up-regulated rankl expression and induction of cox-2 in human pdl cells.15 gum chewing is considered as cyclic mechanical stimulation. it is suggested that pdl cells under mechanical stimulation created by gum chewing may induce osteoclastogenesis through up-regulation of rankl expression via pge2 synthesis during orthodontic tooth movement, and resulted in faster tooth movement. it is in accordance with garat et al.,8 that light continuous bite force accelerates bone remodeling and prevents from excessive bone resorption. therefore, it is logical that gum chewing is able both accelerates orthodontic tooth movement, and prevents from excessive bone resorption. despite the benefits of gum chewing, there is a possibility that it may cause tmj problem.20 even though it is still a controversy, precautions should be conducted. it must be considered that mastication or chewing activities related to many physiological factors, like size, composition and mechanical advantage of jaw-closing muscles, sensitivity of the teeth, muscle and tmj can influence the generation of maximum bite force during mastication. it had been revealed that gender difference in bite force magnitude that boys being stronger than girls. moreover, boys were able to withstand pain more than girls. these differences are particularly noteworthy for numerous forms of musculoskeletal pain, including fibromyalgia, temporomandibular dysfunction (tmd) and myofascial pain.21 high bite force magnitude in gum chewing is undesirable, especially for the tmd sufferers, since it could impair the tmd. therefore, diagnosing tmd prior to continuous gum chewing activities during orthodontic treatment, especially in male patient, is mandatory. regarding to relationship between chewing activities and root resorption, a study related to hypofunctional periodontium by sringkarnboriboon et al.9 also supported this concept. in this study, an experimental hypofunctional periodontal condition of hypofunctional teeth and control were applied with heavy and continuous orthodontic forces which naturally induce root resorption in teeth with normal periodontium. the result revealed that root resorption was greater in the hypofunctional periodontium group. this implies that factors other than the applied force were responsible for root resorption. it was supposed that hypofunctional periodontium exhibited progressive atrophic changes in all functional structures, and accelerated the root destruction resulting from the mechanical stress of orthodontic force. due to the narrow periodontal space in the hypofunctional periodontium, the applied force might concentrate in the compression area. in addition, the narrow periodontal space and derangement of functional fibers would eliminate the normal cushioning effect of the periodontal ligament, and resulting in a high concentration of force.9,22 as the result, continuous chewing may also prevent from root resorption and extensive bone resorption during orthodontic treatment concerning the variability (i.e. stickiness, consistency, taste, and price) of commercial xylitol chewing gum products, there should be researches investigating the most efficient characteristics for ideal orthodontic xylitol chewing gum. stickiness is unfavorable characteristics for fixed orthodontic patients. in addition, durability of consistency is beneficial for maintaining adequate bite force to stimulate the pdl. however, it must be considered that gum chewing habit may lead to tmj problem in certain individuals, especially who chews with one side only.20 the duration of chewing and hardness of chewing gums is also essential to be evaluated to prevent from pain, muscle fatigue and tmj problem.22-24 in addition, besides gum chewing, it is plausible that other chewing actions could be done to stimulate the pdl. several actions such as increasing chewing frequency during eating; intermittently occlude teeth or jaw clenching, which conducted with continuous light pressure between meals is considered able to give the same results as gum chewing. for the concluding remarks, it should be noted that besides regularly consuming xylitol chewing gum; additional chewing frequency during eating food, and intermittent jaw clenching between meals are favorable for stimulating the pdl. stimulated pdl resulted in an increase of periodontal health and a wider pdl, thus enhance the cushioning effect that able to reduce bone and root resorptions. nevertheless, abusing gum chewing other than an adjunct of orthodontic treatment is prohibited. moreover, instructing gum chewing to tmd sufferers is not recommended. hence, it concluded that regularly consuming xylitol is beneficial for achieving safer and healthier orthodontic tooth movement. in order to refine this new concept, researches should be done to develop an effective orthodontic gum chewing method, 180 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 176-180 to discover deleterious effect of continuous gum chewing which lasted for months or years, and inventing an ideal orthodontic chewing gum. references 1. dfi-xylitol. xylitol history. available online at url http:// dficorp. net /xylitol/ index.htm. accessed november 2, 2007. 2. sengun a, sari z, ramoglu si, malkoc s. evaluation of the dental plaque ph recovery effect of a xylitol lozenge on patients with fixed orthodontic appliances. angle orthod 2004; 74:240–4. 3. steckesen-blicks c, lif holgerson p, olsson m, bylund b, sjo¨stro¨m i, sko¨ld-larsson k, kalfas s, twetman s. effect of xylitol on mutans streptococci and lactic acid formation in saliva and plaque from adolescents and young adults with fixed orthodontic appliances. eur j oral sci 2004; 112: 244–8. 4. graber tm, vanardsdall rl, vig kwl. orthodontics: current principles and techniques. 4th ed. st. louis: elsevier-mosby; 2005. p. 266–80. 5. covell d. can biology help us move teeth faster? presented by dr. david covell, on sept 20, 2004, annual session, palm springs. available online at http: www.pcsortho.org/bulletin/bulletin05/ spring05pdfs/p%2039-40%20covell.pdf. 6. lynn s. aoo wilckodontics 1 speed orthodontic surgery. arched wire. 26 mar. 2006. available online at url http://www.archwired. com/aoo1.htm. accessed nov 12, 2007. 7. deguchi t, yabuuchi t, ando r, ichikawa h, sugimoto t, takanoyamamoto t. increase of galanin in trigeminal ganglion during tooth movement. j dent res 85(7): 658–663. 2006. 8. garat ja, gordillo me, ubios am: bone response to different strength orthodontic forces in animals with periodontitis. j periodont res. 2005; 40: 441–5. 9. sringkarnboriboon s, matsumoto y, soma k. root resorption related to hypofunctional periodontium in experimental tooth movement. j dent res. 2003; 82(6):486–90. 10. lam m, riedy ca, coldwell se, milgrom p, craig r: children’s acceptance of xylitol-based foods. commun dent oral epidemiol. 2000; 28: 97–101. 11. ship ja, mccutcheon ja, spivakovsky s, kerr ar. safety and effectiveness of topical dry mouth products containing olive oil, betaine, and xylitol in reducing xerostomia for polypharmacy-induced dry mouth .j oral rehab. 2007; 34: 724–32. 12. xclearinc. how to use xylitol.. available online at url http:// www. xlear .com / xylitol/. accessed nov 19, 2007. 13. buschang ph. masticatory ability and performance: the effects of mutilatede and maloccluded dentitions. seminars in orthodontics 2006; 12(2): 92–5. 14. english jd, buschang ph; throckmorton g. does malocclusion affect masticatory performance? angle orthod 2002; 72(1): 21–7. 15. yamaguchi m, kasai k. inflammation in periodontal tissues in response to mechanical forces arch immunol ther exp, 2005; 53:388–98. 16. deguchi t, takeshita n, balam ta, fujiyoshi y, takano-yamamoto t. galanin-immunoreactive nerve fibers in the periodontal ligament during experimental tooth movement. j dent res. 2003; 82(9): 677–81. 17. polland ke, higgins f, orchardson r. salivary flow rate and ph during prolonged gum chewing in humans. j oral rehab 2003; 30; 861–65. 18. dawes c, kubieniec k the effects of prolonged gum chewing on salivary flow rate and composition. arch oral biol. 2004; 49(8): 665–9. 19. american dental associaton. chewing gum. available online at url http://www.ada.org/ada/seal/index.asp. accessed nov 20, 2007. 20. miyake r, ohkubo r, takehara j, morita m. oral parafunctions and association with symptoms of temporomandibular disorders in japanese university students. j oral rehab 2004; 31:518–23. 21. berkley kj. zalcman ss. simon vr. sex and gender differences in pain and inflammation: a rapidly maturing field am j physiol regul integr comp physiol. 2006; 291:r241–4. 22. verna c, dalstra m, melsen b. bone turnover rate in rats does not influence root resorption induced by orthodontic treatment. eur j orthod. 2003; 25(3):359–63. 23. bonjardim lr, gavia mbd, pereira olj, castelo pm. bite force determination in adolescents with and without temporomandibular dysfunction. j oral rehab. 2005; 32:577–83. 24. farella m, bakke m, michelotti a, martina r. effects of prolonged gum chewing on pain and fatigue in human jaw muscles. eur j oral sci 2001; 109:81–5. 187 vol. 44. no. 4 december 2011 research report the effectiveness of mimba oil (azadirachta indica a. juss) spray disinfectant on alginate impression hanoem eh1, wahjuni w1, and dinda dewi artini2 1 department of prosthodontics 2 dental student faculty of dentistry, airlangga university surabaya indonesia abstract background: alginate impression contaminated by saliva and blood could potentially cause cross contamination. to prevent this, the impression has to be disinfected by disinfectant liquid, such as mimba oil. mimba oil (azadirachta indica a.juss) has some chemical content, such as azadirachtin, which is a phenol group used as antibacterial and antimalaria, nimbolide used as antibacterial and antimalarial, and nimbidin used as antibacterial and antifungal. purpose: the purpose of this study was to find out the most effective concentration of mimba oil as disinfectant to decrease microorganism colony on alginate impression. methods: thirty six samples were taken from 9 respondents. this alginate impression was divided into 4 groups: group 1 sprayed with sterile aquadest (as control group), group 2 sprayed with mimba oil 50% for 30 seconds, group 3 sprayed with mimba oil 75% for 30 seconds, group 4 sprayed with mimba oil 100% for 30 seconds. the microorganism colony was counted by colony counter. the sample data then were analyzed with kolmogorov-smirnov test, and was tested with kruskal wallis test and mann whitney test for further analysis. results: there was significant difference among each group, p = 0.01 (p < 0.05). conclusion: in conclusion, usage of 50% concentration of mimba oil as disinfectant is effective to decrease microorganism colony on alginate impression. key words: alginate impressions, mimba oil, oral microorganism abstrak latar belakang: cetakan alginat yang terkontaminasi saliva dan darah dapat berpotensi terjadinya infeksi silang. untuk mencegah hal tersebut, cetakan didisinfeksi dengan bahan disinfektan cair seperti minyak mimba. minyak mimba (azadirachta indica a.juss) memiliki beberapa kandungan kimia, antara lain azadirachtin yang merupakan kelompok fenol yang memmiliki efek antibakteri dan antimalaria, nimbolide memiliki efek antibakteri dan antimalaria sedangkan nimbidin memiliki efek antibakteri dan antijamur. tujuan: tujuan dari penelitian ini adalah untuk menentukan konsentrasi yang paling efektif pada minyak mimba sebagai disinfektan untuk mengurangi jumlah koloni mikroorganisme pada cetakan alginat. metode: tiga puluh enam sampel diambil dari sembilan subyek. cetakan alginat dibagi menjadi empat kelompok: kelompok 1 disemprot dengan aquades steril (kontrol), kelompok 2 disemprot dengan minyak mimba 50% selama 30 detik, kelompok 3 disemprot dengan minyak mimba 75% selama 30 detik, kelompok 4 disemprot dengan minyak mimba 100% selama 30 detik, jumlah koloni mikroorganisme dihitung menggunakan colony counter. �ata dianalisa dengan uji kolmogorov-smirnov, dilanjutkan dengan uji kruskal wallis dan mann whitney. hasil: terdapat perbedaan bermakna pada masing-masing kelompok, p = 0,01 (p < 0,05). kesimpulan: �apat disimpulkan dengan konsentrasi 50% sebagai desinfektan cetakan alginate telah efektif menurunkan pertumbuhan microorganisme rongga mulut. kata kunci: cetakan alginat, minyak mimba, mikroorganisme rongga mulut correspondence: hanoem eh, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: subprostho@yahoo.com 188 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 187–191 introduction in dental care or denture making, oral tissue model is necessary to be made by doing impression with impression material and then fill it with plaster. the mostly used impression material in dentistry is alginate.1 however, in the impression process, the alginate impression can be contaminated with alginate pathogenic microorganisms causing cross infections, such as pneumonia, tuberculosis, herpes, hepatitis, and aids.2 but, the risk of those cross infections can be reduced by using disinfectant materials.1 the disinfection process of the alginate impression can be done by spraying and immersion in disinfectant material since the alginate material can not be sterilized.3 according to research results, disinfectant spraying technique can have the same antimicrobial activity as immersing one, but it does not affect the dimension stability of the alginate impression. 4 unfortunately, disinfectant materials sold in markets sometimes are not affordable for public because relatively high price. according to the who, traditional herbal medicine can become alternative disinfectant materials because of their easily known ingredients, safer, more efficient, and more easily obtained.5 mimba (azadirachta indica a. juss) can be used to solve tooth and gum problems, ulcers, malaria, typhoid, and gastroenteritis.6,7 mimba seeds contain oil that can be used to inhibit the growth of salmonella thyposa and staphylococcus aureus bacteria at concentrations of 50%, 75% and 100%.7 it is because mimba oil (azadirachta indica a.juss) has useful chemical ingredients, such as azadirachtin, nimbolide and nimbidin, which have antibacterial, antifungal and antimalarial effects.7 this research is aimed to determine the effectiveness of mimba oil (azadirachta indica a.juss) spraying with concentrations of 50%, 75% and 100% used as disinfectant to reduce the number of microorganism colonies on alginate impression. if the results of this research show that mimba oil is effective, it can become an alternative disinfectant material for alginate impression and may be developed for other health purposes because it has useful pharmacological characteristics. materials and methods this research was conducted from april to october 2011. the location of the research for the impression process took place in prosthodontic clinics of dentistry faculty, airlangga university. meanwhile, sample treatment process and measuring process of growing microorganism colonies were conducted ini microbiology laboratory of dentistry faculty, airlangga university. qualitative test and mimba oil concentration were conducted in phytochemistry pharmakognosis laboratory of pharmacy faculty, airlangga university. mimba oil was qualitatively examined through organoleptis examination, ph determination, specific gravity, and refractive index. the mimba oil was made into certain concentrations, 50%, 75%, and 100%, by using sterile aquadest diluent. nine subjects with certain criteria, 20–25 year old men with complete and normal dental structure, good oral hygiene, no dental caries and filling, good general health, no antibiotic treatment, no periodontal disorders, and no smoking habit were used in this research. alginate impression was made with a 7 grams of alginate powder and 15 ml of water mixed with a rubber spatula in bowl for 30 seconds, and then put into impression tray for 1.5 minutes.8 those nine subjects were asked to rinse by using water before alginate was impressed on their upper jaw for 1.5 minutes, and then the impressions were removed from their oral cavity. the alginate impressions were washed with running water for 15 seconds.8 four alginate pieces of their posterior palate were taken by using sterile aluminum ring with thickness of 2 mm and diameter of 10 mm.9 figure 1. ring with thickness of 2 mm and diameter of 10 mm. figure 2. the impression results of the alginate impressions cut by using ring. 189hanoem, et al.,: the effectiveness of mimba oil (azadirachta indica a. juss) the alginate in group i was sprayed with 2 ml of sterile aquadest for 30 seconds as control group, that in group ii was sprayed with 2 ml of 50% mimba oil for 30 seconds, that in group iii was sprayed with 2 ml of 75% mimba oil for 30 seconds, and that in group iv was sprayed with 2 ml of 100% mimba oil for 30 seconds.7,8 once given the treatment, all those groups were then washed with 2 ml of sterile aquadest for 15 seconds. each of alginate mold piece was inserted into test tubes containing bhi liquid media, and then was thinned, about 10–2. next, 0.1 ml of bhi liquid media were planted on petridish containing blood agar. it was then incubated for 24 hours at 37° c, and microorganism colonies were counted by using colony counter with cfu/ml units.8 results mimba oil obtained was qualitatively tested before being used in this research. the qualitative test results of neem oil can be seen in table 1. table 1. qualitative test result of mimba oil type of examination examination results references10 color yellow-brown color yellow-brown color aroma strong strong like onions flavor bitter bitter ph 5 5.3 density 0.918 0.922 g/ml refractive index 1.4650 1.4615–1.4705 based on the data obtained, mimba oil used in this research was in accordance with the description in the reference.10 based on the observation and counting results, the number of microorganism colonies on the alginate impressions sprayed in those 4 groups, namely a control group and 3 treatment groups sprayed with mimba oil for 30 seconds with concentrations of 50%, 75 % and 100% can be seen in table 2. the average number of microorganism colonies in the control group and those three treatment groups sprayed with 50%, 75% and 100% mimba oil was decreasing (table 2). in the table above, it is also known that the lowest number of the colonies was in the treatment group sprayed with 100% mimba oil. before analysis comparison test among those groups was conducted, normality test had been conducted first on each group by using kolmogrov-smirnov test. the result then showed that all those groups had greater p values than 0.05 (p > 0.05) indicating that the data on those groups were normally distributed. next, one-way anova test was conducted to see the homogeneity and significance of those groups, and in the levene test, it is known that p value obtained was 0.05 indicating that the data on all those groups were not homogeneous. since the data were not homogeneous, then non parametric test was conducted by using kruskal wallis test. the result then showed that p was 0.05 indicating that there was a significant difference among the four groups. to find out the differences among those treatment groups, mann whitney test was conducted (table 3). mann whitney test results between the control group and those groups sprayed with 50%, 75% and 100% mimba oil. table 3. the result of mann-whitney test between the control group and those three treatment groups with 50%, 75% and 100% mimba oil control 5% 75% 100% control 0.001* 0.001* 0.001* 50% 0.001* 0.001* 75% 0.002* 100% note: * = significant difference in each of those comparisons, there was significant difference in the number of microorganism colonies by using mann whitney test (table 3). discussion samples used were pieces of the alginate impressions of the posterior palate because there was no significant difference between the colonies of microbes growing on tooth region i, p and m so that it can be stated that the number of microbial colonies on the palate was generally more evenly.11 the alginate impressions of the posterior table 2. the mean and standard deviation results of the number of microorganism colonies in oral cavities after the alginate impressions sprayed with sterile aquadest and 50%, 75%, and 100% mimba oil sample mean standard deviation i 77.33 17.45 ii 28.44 7.63 iii 1655 4.58 iv 7.22 2.63 note: i: the alginate impression was sprayed with sterile aquadest (control); ii: the alginate impression was sprayed with 50% mimba oil; iii: the alginate impression was sprayed with 75% mimba oil; iv: the alginate impression was sprayed with 100% mimba oil 190 dent. j. (maj. ked. gigi), vol. 44. no. 4 december 2011: 187–191 palate were then cut by using sterile aluminum ring with thickness of 2 mm and diameter of 10 mm divided into 4 sections.9 after being taken out from the mouth, the alginate impressions should be washed with water to remove saliva, debris, and blood.12 alginate impression materials had reacted with water to form alginate calcium salts precipitating in the forms of woven fiber-like tissue with water molecules in its capillary space.3 capillary space and irregular alginate shape facilitated the attachment of microorganisms of oral cavity to the alginate impressions causing the microorganisms in the alginate impressions were not easily lost by washing with water only. in this research, spraying technique, moreover, was used to disinfect since the technique of disinfectant spraying showed the same antimicrobial activity as that of immersing, but did not affect the dimensional stability of the alginate impressions, such as dimensional accuracy, stability and wettability.4 and, concentrations of mimba oil used as disinfectant to reduce the number of microorganism colonies on the alginate impressions were about 50%, 75% and 100%. this is because based on a research it is known that neem oil can inhibit the growth of salmonella typhosa at concentration of 50% with resistance area diameter of 3 mm, at concentration of 75% with resistance area diameter of 4 mm, and at concentration of 100% with resistance area diameter of 5 mm. meanwhile, mimba oil can inhibit the growth of staphylococcus aureus at concentration of 50% with resistance area diameter of 4 mm, at concentration of 75% with resistance area diameter of 5 mm, and at concentration of 100% with resistance area diameter of 6 mm.7 based on the results, furthermore, there was significant difference of the number of microorganism colonies between on the alginate impression sprayed with sterile aquadest and on those sprayed with neem oil used as disinfectant. the average number of microorganism colonies on the alginate impression sprayed with 100% mimba oil was about 7.22. while, the average number of microorganism colonies on the alginate impression sprayed with 75% mimba oil was about 16.55. and, the average number of microorganism colonies on the alginate impression sprayed with mimba oil 50% was about 28.44. in short, it means that the greater the concentration of disinfectant that contact with the alginate impression, the lower the number of microorganisms grows. this is because their power depends on the concentration of antimicroorganism materials, time and temperature. thus, the greater the concentration of disinfectant is dissolved in water, the more effective the power to inhibit the growth of microorganisms.13 in the spraying of 100% mimba oil on the alginate impression, there was still microorganism colony grown since the microorganisms is considered as a kind of microorganisms that cannot be inhibited by mimba oil. that mimba oil can not effectively inhibit the microorganisms is because aaccording to some researches mimba oil is effective to inhibit the growth of several specific types of microorganisms, such as streptococcus mutants, staphylococcus aureus, salmonella thyphosa, staphylococcus coagulase, plasmodium falciparum, mycobacterium tuberculosa, tinea rubrum, and candida albicans.6,7,14,15 the ingredients contained in mimba oil, furthermore, are azadirachtin, nimbolide, and nimbidin which have antibacterial, antifungal and antimalarial characters. azadirachtin is a derivative phenol compound which has antibacterial and antimalarial characters that can inhibit the growth of streptococcus mutants and plasmodium falciparum.6 phenol can be used as disinfectant because it can damage the cell walls of microorganisms. the cell walls of microorganisms serve to maintain the integrity of the cell with osmotic pressure if the cells are in hypotonic condition in which the concentration of the fluid outside the cell is less than that inside the cell.16,17 besides, another active ingredients of mimba oil is a group of tetranotriterpenes nimbolide which has antibacterial character in the growth of plasmodium falciparum malaria, staphylococcus aureus and staphylococcus coagulase. meanwhile, a group of tetranotriterpenes nimbidin has antifungal and antibacterial characters in the growth of tinea rubrum and mycobacterium tuberculosis.14 in conclusion, 50% mimba oil as desinfectan is already effective decreases microorganism colonies in the alginate impression. references 1. powers jm, wataha jc. dental material properties and manipulation. 9th ed. usa: mosby; 2008. p. 172–3, 180. 2. van noort r. introduction to dental materials. 3rd ed. china: mosby; 2007. p. 204–5. 3. parnia f, hafezeqoran a, moslehifard e, mahboub f, nahael m, dibavar ma. effect of different disinfectants on staphylococcus aureus and candida albicans transfered to alginate and polyvinylsiloxane impression materials. j dent research dental clinic dental prospect 2009; 3(4): 122–5. 4. atabek d, alacam a, tuzuner e, polat s. in-vivo evaluation of impression material disinfection with different disinfectant agents. arastirma 2009; 33(2): 52–9. 5. masibo m, he q. in vitro antimicrobial activity and the major polyphenol in leaf extract of mangifera indica l. malaysian j microbiol 2009; 5(2): 73-80. 6. subramaniam sk, siswomihardjo, w, sunarintyas, s. the effect of different concentrations of neem leaves extract on the inhibition of streptococcus mutans (in vitro). maj ked gigi (dent j) 2005; 38(4): 176–9. 7. ambarwati. efektivitas zat antibakteri biji mimba (azadirachta indica) untuk menghambat pertumbuhan salmonella thyposa dan staphylococcus aureus. biodiversitas 2007; 8(3): 320–5. 8. ghahramanlod a, sadeghian a, sohrabi k, bidi a. a microbiologic investigation following the disinfection of irreversible hydrocolloid materials using spray methods. cda journal 2009; 37(7): 471–7. 9. sofou al, larsen t, fiehn ne, owall b. contamination level of alginate impressions arriving at a dental laboratory. clin oral invest 2002; 6(1): 161–5. 191hanoem, et al.,: the effectiveness of mimba oil (azadirachta indica a. juss) 10. benmhend. cold pressed neem oil. biopesticides registration action document; 2009. p. 17. 11. subianto a. efektivitas klorheksidin dan air ozon dalam air pencampur sebagai bahan disinfeksi cetakan alginat. karya tulis ilmiah, program spesialis. surabaya: fakultas kedokteran gigi universitas airlangga; 2001. 12. craig rg, o’brien wj, powers jm. dental materials properties and manipulation. 5th ed. usa: mosby year book; 1992. p. 163. 13. nolte wa. oral microbiology with basic microbiology and immunology. 4th ed. usa: mosby; 1982. p. 70. 14. biswas k, chattopadhyay i, banerjee rk, bandyopadhyay u. biological activities and medicinal properties of neem (azadirachta indica). current science 2002; 82(11): 1336–43. 15. kumar ps, mishra d, ghosh g, panda cs. biological action and medicinal properties of various constituent of azadirachtin indica (meliaceae) an overview. annals of biology research 2010; 1(3): 24–34. 16. bauman rw. microbiology. san fransisco: pearson benjamin cummings; 2004. p. 265. 17. jawetz e, melnick j, adelberg e. 1995. mikrobiologi kedokteran. nugroho e, editor. jakarta: egc; 1995. p. 54. isi vol 39 no 3 juli-september 2006.pmd 120 management of oral focal infection in patients with asthmatic symptoms haryono utomo dental clinic faculty of dentistry airlangga university surabaya indonesia abstract asthma is commonly related to allergic diseases, nevertheless only 40% of asthma patients are related to allergy or atopy. there are some unknown etiologies of asthma that are still in researches. one of the possible causes of asthma is the multiple chemical sensitivity syndrome (mcs) which related to the “neurogenic switching hypothesis”. since rhinitis, sinusitis and asthma are closely related, treatments which are successfully reduce or eliminate the rhinitis and sinusitis symptoms should also be advantageous to asthma management. there were a lot of sinusitis treatments which reduced asthma symptoms such as nasal corticosteroid, diathermy and surgery. it was also been reported that oral focal infection might cause sinusitis. however, the involvement of oral focal infection in the etiopathogenesis of asthma was seldom discussed. the objective of this study is to propose a mechanism of the relationship between oral focal infection and asthma which is explained by the “neurogenic switching hypothesis”. two asthmatic patients who also had periodontal disease, pulpal and periapical infection were treated with conventional and/or surgical dental treatments. after the dental and periodontal treatments were completed, the usual triggers of severe asthma attacks such as cold and house dust did not elicit the asthma symptoms. it concluded that regarding to the disappearing of asthma symptoms, the elimination of oral focal infection had a beneficial effect in reducing asthma symptoms. key words: asthmatic symptom, oral focal infection management correspondence: haryono utomo, c/o: fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id telp. 031-5053195. the neurogenic switching hypothesis was introduced by meggs7 which meant to explain the etiopathogenesis of allergic disease (i.e. urticaria, food allergy, and asthma), migraine, arthritis and other inflammatory diseases. it proposed that a local inflammation was able to initiate another inflammation in other parts of the body. in addition, the neurogenic switching mechanism may also occur in the oral tissues.8 according to li et al.,9 oral focal infections were able to elicit systemic symptoms by means of various mechanisms. evidence-based case reports had been revealed that oral focal infection might induce sinusitis. however, the relationship between oral focal infection and asthma was rarely discussed. the objective of this case report is to achieve a better knowledge about the relationship of oral focal infection and asthma based on the concept of neurogenic switching mechanism. case case 1: a male, 43 years, who came to the dental clinic faculty of dentistry, airlangga university. at that time he had a problem with the recurrent periapical abscess which introduction asthma is a chronic inflammatory disorder of the airways characterized by an obstruction of airflow, which may be completely or partially reversed with or without specific therapy. airway inflammation is the result of interactions between various cells, cellular elements, and cytokines. in susceptible individuals, airway inflammation may cause recurrent or persistent bronchospasm, which causes symptoms including wheezing, breathlessness, chest tightness, and cough, particularly at night or after exercise.1 it is important to understand the role of atopic or allergy in asthma. asthma and atopy may coexist, but not all asthmatics are atopic and only some atopic patients have asthma. forty percent of asthmatics have atopy, the rest has an unknown etiology of asthma. nevertheless, all asthmatics patients–regardless of the presence or absence of atopy–have the cardinal features that define asthma.2 epidemiological studies have clearly shown that rhinitis and asthma are frequently concurrent. therefore, the concept of “one airway–one disease” had been implicated in rhinitis and asthma management. allergen avoidance, specific immunotherapy and intra nasal corticosteroid were able to reduce symptoms in allergic rhinitis and asthma.4–6 121utomo: management of oral focal infection in patients elicited pain. an oral surgeon advised to have apical resection of the 16. he suffered from asthma and rhinitis symptoms for about 35 years. a lot of medications had been consumed. asthma attack could be triggered by chemical odor (i.e. alcohol and perfume), fermented food or drink, humid and cold weather. in order to prevent serious asthma symptoms, he always bring metered-aerosol bronchodilator. extra orally, there was a slight asymmetry on the right cheek and a dull pain was felt upon palpation. intra orally, the buccal fold of the 16 was somewhat raised, and palpation caused tenderness. there were a lot of restorations, including inlay in 46 and crowns in 16, 25, 35, 36. panoramic radiograph showed horizontal resorption on 16 15, 25 26. at the apex of 16 there was periapical abscess (figure 1). not solve the problem, an oral surgeon decided to conduct an apical resection. the apical resection was done in the operation room of oral surgery department, faculty of dentistry, airlangga university. the patient was scheduled for the next visit in 5 days for removing the sutures. a few days subsequent to the surgical procedures, the abscess disappeared and also the rhinitis symptoms were reduced, followed by the asthma symptoms. since then, the usual triggers of asthma such as chemical odors, fermented food, humid and cold weather did not elicit asthma symptoms. at second visit, the surgical wound was healed and the sutures were removed. at that time the rhinitis symptoms (i.e. nasal congestion and rhinorrhea) that always started in the morning till the afternoon were diminished. coincidentally, since the apical resection, the usual asthma symptoms also disappeared. the most recent evaluation was in june 2006, 3 years after the apical resection, the asthma symptoms did not reoccur. case 2: a male, 21 years who came to a private dental clinic. the chief complaint was pain on the upper left tooth, and also gingival bleeding. after the patient was told that oral infection might cause migraine, rhinitis and asthma symptoms, he told the dental practitioner about his suffering from rhinitis and asthma for about 10 years. a lot of medical treatment and alternative medicine had been tried to prevent asthma attack. the most recent prescription was aminophyllin tablets (oral bronchodilator) that had to be taken once daily. extra orally, the patient looked normal. intra oral inspection showed that the patient suffered from generalized gingivitis and a deep caries in 24. the upper and lower gingival tissues were easily bled. thermal testing on 24 revealed that the tooth remained vital, nevertheless it had increased sensitivity. panoramic radiograph (figure 2) showed that alveolar resorption was absent. apical dental radiograph indicated that perforation of the pulp chamber in 24 and periodontitis apicalis already occurred. case management case 1: since the chief complaint was persistent pain and periapical abscess in 16 and endodontic treatment could case 2: at the first visit, first attempt was the relief of pulpal pain followed by temporary restoration. scaling had been done in every region which resulted in a lot of oozed dark red blood. second visit was scheduled 4 days later. at the next visit the patient told that the asthma symptoms were disappearing, he only had a mild rhinitis in the morning; before that rhinitis could be happened all day long. the pulpal condition was diagnosed as irreversible pulpitis. root canal treatment was done by an endodontists which completed after 3 visits. evaluations were done monthly for about one year after the last visit, the latest evaluation was in june 2006; the asthma symptoms did not reappear. discussion in the first case, there were two possible causes of oral focal infection which was the source of bacterial endotoxin lipopolysaccharides (lps). the most possible cause was the periapical abscess in 16, although it had been treated endodontically, the periapical infection still in progress. the other cause was the chronic periodontitis which caused figure 2. panoramic radiograph pre-treatment. figure 1. panoramic radiograph pre apical resection of 16. 122 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:120–125 alveolar resorptions in 16 15 and 25 26. the indication was that after apical resection, the rhinitis and asthmatic symptoms gradually disappeared. among all animals, humans are the most sensitive to the effects of endotoxins, which makes the knowledge of their biological effects on tissues fundamentally important. endotoxins from vital or non vital, whole or fragmented bacteria act on macrophages, neutrophils and fibroblasts,10 and mast cells11 leading to the release of a large number of bioactive or cytokine chemical inflammatory mediators, such as tumor necrosis factor (tnf-α), interleukin-1 (il-1), il-5, il-8, alpha-interferon and prostaglandins. furthermore, lps is cytotoxic and acts as a potent stimulator of nitric oxide (no) production.9,10,12 according to leonardo et al.,10 the bacterial endotoxin (lps) adheres irreversibly to mineralized tissues, endodontic treatment and dressing only killed the bacteria. the inactivation of lps by eliminating its biologically toxic potential that was the lipid a should be done with certain materials such as calcium hydroxide. lipopolysaccharides and the products of degranulated mast cells were able to induced the neurogenic switching mechanism which then might elicit asthmatic symptoms.7,8,13 it seems plausible that the incomplete inactivation of lps after endodontic treatment in the first case caused the ongoing periapical infection; the apical resection treatment then eradicate the remaining lps. in the second case, since it was diagnosed as pulpitis irreversible and the periapical inflammation was not severe; the major contribution of the lps was suspected caused by the periodontal disease that was gingivitis. it was confirmed regarding to the immediate relief of asthma symptoms after scaling procedures whereas the endodontic treatment was not done yet. in the periodontal disease, lps originated from pathogenic gram negative bacteria such as porphyromonas gingivalis. periodontal pockets act as cytokines and biofilms reservoir which may continuously release proinflammatory mediators that may cause systemic effects. the propagation of oral focal infection to systemic could be conducted by several mechanisms, via the blood stream9 or the neurogenic switching mechanism.7,8,13 the interplay between immunogenic and neurogenic inflammation was called the neurogenic switching mechanism. the neurogenic switching hypothesis that was proposed by meggs, suggested that the etiopathogenesis of asthma was not merely related to atopy and immunogenic mechanism. the role of afferent nerve fibers stimulation which elicit neurogenic inflammation should also be considered.7 meggs7 postulates that “neurogenic switching”, or a crossover interaction of neurogenic and immunogenic inflammation, explains several puzzling aspects of inflammatory responses, including how an inflammatory stimulus applied to one tissue (e.g., skin) can result in inflammation in a different tissue (e.g., the lungs) and how neurogenic conditions such as migraine can be influenced by immunogenic agents.13 the interplay or cross-over of immunogenic and neurogenic inflammation which also termed the neurogenic switching mechanism was hypothesized to be involved in triggering asthmatic or other allergic symptoms.7,13 neurogenic switching is proposed to result when a sensory impulse from a site of activation is rerouted via the central nervous system to a distant location to produce neurogenic inflammation at the second location.13 it also provides a mechanism to explain how allergens, infectious agents, irritants, and possibly emotional stress can exacerbate conditions such as migraine, asthma, and arthritis. the difference mechanism between atopic asthma that mostly related to immunogenic reaction and neurogenic mechanism of asthma are described in table 1.7 according to lundy and linden, the neurogenic switching mechanism also occurred in the oral tissues.8 there were several factors which involved in neurogenic switching mechanism such as bacteria, toxins (lipopolysaccharides, lps and proteoglycans, pgn), neuropeptides (substance p, sp and calcitonin gene-related peptide, cgrp) which able to degranulate mast cells.8,15 afferent nerve fibers may in turn were stimulated by products of degranulated mast cells (i.e. histamine, tryptase) which then release neuropeptides. other stimulators are the pro-inflammatory cytokines produced by lps-induced macrophages, and bradykinin from damaged tissue.8 case reports related to oral focal infection by li et al. showed that sinusitis may be affected by oral infection. since asthma and sinusitis were closely related, it seems plausible that oral focal infection may also involved in the etiopathogenesis of asthma, especially through the neurogenic switching mechanism.7-9,13,14 in these two cases, table 1. immunogenic versus neurogenic asthma7 immunogenic neurogenic triggered by interacting with located on releasing stimulating producing manifesting as protein aeroallergens ige antibody mast cells histamine, leukotrines, prostoglanolins, chemotactic factors sensory nerve c-fibers bronchial inflammation asthma volatile organic chemicals chemical irritant receptors sensory nerve c-fibers neuropeptides: substance p, neurokinen a, calcitonin gene-related peptide mast cell degranulation bronchial inflammation asthma 123utomo: management of oral focal infection in patients the successful elimination of oral focal infection including the lps was indicated by the reducing of asthma symptoms. the possible explanation of the diminished asthma symptoms was that after dental and periodontal treatments, there was an immediate cut off of the neurogenic switching mechanism which initiated by oral tissue inflammation. in this case report, the patients also suffered from rhinitis which had a co-morbidity with asthma and may worsen asthma symptoms. allergic rhinitis which suffered by 80% asthmatics has several sequelae and co-morbidities which impaired the quality of life that related to asthma, such as sinusitis, mouth breathing etc. (figure 3).5 if the nose is obstructed, these individuals breathe with their mouth open, a practice that precipitates an asthma attack. mouth breathers may resulting in dry mouth (xerostomia) which reduces the immune function of saliva as a part of mucosal immunity. 16 regarding to the concept “one airway–one disease”, which involved rhinitis, sinusitis and asthma. there are some explanation about the pathophysiologic relationship between sinusitis and asthma (table 2), such as the aspiration of the mediators present in secretions. these mediators then reach the lower airway in inspired air, particularly at night, leading to deterioration in lung function, increased bronchial hyperreactivity, and symptoms on waking.4,6, 14,17 the main mechanism whereby rhinitis and asthma are interrelated had been proposed as a systemic dissemination of mediators which are eosinophils, intercellular adhesion molecule-1 (icam-1) and vascular cell adhesion molecule-1 (vcam-1) that were showed in nasal and bronchial mucosa after nasal provocation with allergen. nasal provocation induced eosinophilia, leukocyte activation in peripheral blood, systemic propagation of inflammation from nasal to bronchial mucosa.6 since the reducing of rhinitis and asthma symptoms in these patients happened in a short time without asthma medications, the possibility of neurogenic inflammation involvement was suspected. regarding to the neurogenic inflammation in the orofacial region, the relationship between rhinosinusitis, asthma and the trigeminal nerve (cn v) had been studied. experiments related to the nasobronchial reflex revealed that unilateral resection of the trigeminal nerve eliminated the bronchial resistance. the function of the trigeminal nerve is for the sense of irritation, also referred to chemestesis or the common chemical sense.6,18 further analysis of the interrelationship of the cn v and asthma was the existence of receptors in the nose and pharynx and, presumably, in the paranasal sinuses produce afferent fibers that form part of cn v. they passed to the brain stem and connects with the reticular formation of the figure 3. the sequelae and co-mordibidities of allergic rihinitis.5 table 2. proposed pathophysiologic mechanisms of asthma exacerbated by sinusitis14 spread of inflammatory mediators and chemotactic factors to lower airways triggers sinobronchial reflex mechanism. stimulation of autonomic nervous system causes acute bronchial hyperresponsiveness. bronchoconstrictive reflexes originating in extrathoracic airway receptors are stimulated. reversible partial beta-adrenergic blockade is enhanced. nasal congestion causes mouth-breathing, which leads to increased loss of water and heat in lower airways. depressed nitric oxide concentration promotes acute bronchial hyperresponsiveness. gastroesophageal reflux disease induces nasal mucosa edema, obstruction of sinus ostia, and stimulation of autonomic nervous system. allergic rhinitis impairment of quality of life sleep impairment food allergy atopic dermatitis sinusitis asthma allergic rhinitis sequelae and co-morbidities conjunctivities otitis media mouth breathing 124 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:120–125 dorsal vagal nucleus. from the vagal nucleus, parasympathetic efferent fibers travel in the vagus nerve to the bronchi.14 according to doty, some free nerve endings of the cn v might terminate 1 μm within the epithelial surface of the nose, such as in nasal vestibule and nasal chambers. they produce such sensations as irritation, tickling, burning, warming, cooling and stinging. in addition to serving chemosensory functions, cn v fibers within the nasal vestibule mediate the tactile sensations of temperature and pressure.19 trigeminal nerve stimulation can reflexively influence nasal engorgement, respiration rate, nasal secretion, and sneezing. since most trigeminal stimulants were lipid soluble, such as volatile chemicals; the stimulations were likely. stimulated cn v and the sphenopalatine ganglion (spg) may referred to multiple chemical sensitivity syndrome (mcs) which initiated in the nasal cavity.7,8,13,20,21 the periapical and periodontal infection in both cases might be able to conduct the neurogenic switching mechanism, which may antidromically stimulate the sphenopalatine ganglion (spg). the stimulation of spg, a parasympathetic ganglion also proposed as an etiology of mcs, which related to rhinitis, allergy, asthma, migraine etc. several medications which applied to the spg were able to relief migraine, rhinitis and asthma symptoms.20 since maxillary periodontal tissues are innervated by the sensory nerve fibers through the cn v2 and the parasympathetic nerve fibers through the spg, there might be a correlation between periodontal disease and the spg sensitization.8,22 activation of the spg by cn v2 which releases neuropeptides may cause the inflammation of the neighboring artery and mucosa. inflamed nasal mucosa resulting in nasal congestion, sinusitis; and migraine.21 regarding to the relief of the asthmatic symptoms, there was a case report regarding the possible relationship between periodontal inflammation, the spg and sinusitis. as sinusitis is closely related to asthma, dental treatment which successfully treated sinusitis patient such as the “assisted drainage” method, was expected to give the same result to asthma patient.22,23 periodontal treatment which conducted to the second case was similar to the “assisted drainage” method that was done due to assist drainage of pro-inflammatory mediators which may involve in the neurogenic switching mechanism in the periodontium. immediate cut off of the mechanism was suggested to reduce the sinusitis and asthma symptoms.22 the stimulation of afferent nerve or parasympathetic fibers by pro-inflammatory mediators in periodontal diseases and pulpal inflammation may propagate to distant site through the neurogenic switching mechanism. it may indirectly affect bronchial hyperresponsiveness via the nasal inflammation which probably caused by the sensitized spg.21,22 sensitization of the spg that might be caused by oral inflammation was able to induced nasal hypersensitivity. this condition increased the nasal susceptibility to provocations which stimulate the nasal sensory nerves that leads to sensations of pain and stuffiness. this reaction was created by type c nociceptive nerve fibers which releases neuropeptides that increase plasma extravasation and glandular secretion.19,24 this axonal response acts as an immediate protective mucosal defense mechanism. recruited parasympathetic reflexes cause submucosal gland secretion via acetylcholine and muscarinic m3 receptors. itching, sneezing, and other avoidance behaviors rapidly clear the offending agents from the upper airways and protect the lower airways.24 dysfunction of these nerves may contribute to allergic rhinitis, infectious rhinitis, nasal hyperresponsiveness, and possibly sinusitis. sympathetic arterial vasoconstriction reduces mucosal blood flow, sinusoidal filling, and mucosal thickness, and so restores nasal patency. loss of sympathetic tone may contribute to some chronic, nonallergic rhinopathies.22,24 regarding to the relief of asthma symptoms after the completion of dental and periodontal treatments; it concluded that the elimination of oral focal infection is mostly probable in reducing asthma symptoms. references 1. sharma g. asthma. available online at http://www.emedicine.com/ ped/pulmonology. accessed may 15, 2006. 2. terr ai. asthma. in: parlow tg, stites dp, terr ai, imboden jp, editors. medical immunology. 10th ed. boston: mcgraw-hill; 2003. p. 359. 3. ponsonby al, gatenby p, glasgow n, mullins r, mcdonald t, hurwitz m. which clinical subgroups within the spectrum of child asthma are attributable to atopy. chest 2002; 121:135–42. 4. lipworth bj, white ps. allergic inflammation in the unified airway: start with the nose. thorax 2000; 55:878–91. 5. fokkens w, bachert c. comorbidity of rhinitis. the ucb insitute of allergy, 2004. p. 5–13. 6. serrano c, valero a, picado c. rhinitis and asthma: one airway, one disease. arch bronconeomol 2005; 41:569–78. 7. meggs wj. neurogenic switching: a hypothesis for a mechanism for shifting the site of inflammation in allergy and chemical sensitivity. env health perspect 1997; 105(s2):1–10. 8. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 9. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microb rev. 2000; 13(4):547–58. 10. leonardo mr, da silva rab, assed s, nelson-fiho p. importance of bacterial endotoxin (lps) in endodontics. j appl oral sci 2004; 12(2):93–8. 11. supajatura v, ushio h, nakao a, akira s, okumura k, ra c, ogawa h. differential responses of mast cell toll-like receptors 2 and 4 in allergy and innate immunity. j clin invest. 2002; 109:1351–9. 12. madianos pn, bobetsis ya, kinane df. generation of inflammatory stimuli: how bacteria set up inflammatory responses in the gingiva. j clin periodontol 2005; 32(s6): 57–71. 13. cady rk, schreiber cp. sinus headache or migraine. neurology. 2002; 58:s10–s14. 14. muller ba. sinusitis and its relationship to asthma: can treating one airway disease ameliorate another? postgrad medicine 2000; 108(5):55–61. 15. walsh lj. mast cells and oral inflammation. crit rev oral biol med 2003; 14(3):188–98. 125utomo: management of oral focal infection in patients 16. laurikainen k. asthma and oral health: a clinical and epidemiological study. academic dissertation. tampere: tampere university press; 2002. p. 1–82. 17. volcheck gw. does rhinitis lead to asthma. evidence for the oneairway hypothesis. postgrad med 2004; 115(5):65–8. 18. shusterman d. review of the upper airway, including olfaction, as mediator of symptoms. env health perspect 2002; 100(s4): 649–53. 19. doty rl. intranasal trigeminal chemoreception: anatomy, physiology and psychophysics. in: doty rl, editor. handbook of olfaction and gustation. 1st ed. new york: marcell dekker. 1995. p. 821–33. 20. klinghardt dk. the sphenopalatine ganglion (spg) and environmental sensitivity. lecture on 23rd annual international symposium on man and his environment. june 9–12, 2005. dallas texas. available online at url http://www.naturaltherapy.com. accessed march 20, 2006. 21. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http://www.migraineprevention.com/index/html. accessed february 15, 2006. 22. utomo h. sensitization of the sphenopalatine ganglion by periodontal inflammation: a possible etiology for sinusitis and headache in children. majalah kedokteran gigi fkg universitas airlangga. 2006; 39(2):63–7. 23. grossan m asthma and sinusitis. available online at url http://www. emedicine.com/ent/contents.htm. accessed in june 6, 2006. 24. chih-feng t, baraniuk jn. upper airway neurogenic mechanisms. cur al clin immunol 2002; 2(1):11–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 85 volume 46 number 2 june 2013 research report new concept in allergy: non-allergic rats becomes allergic after induced by porphyromonas gingivalis lipopolysaccharide haryono utomo dental clinic faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: as a theory, seemingly it is impossible that allergic diseases, including asthma, are the result of exposure to a transmissible agent. the fact that nearly all children with asthma are allergic, but only a small proportion of allergic children have asthma, at least raises the possibility that other factors are involved. interestingly, non-allergic children become allergic after their parents came from working in allergic people for several months. recent research revealed that periodontal pathogens are also transmissible from mother and caregivers to infants.therefore, it is logical that non-allergic children could become allergic after exposed to periodontopathic bacteria. however, the mechanism is still unclear. purpose: the objective of this study is to verify a new concept that non-allergic rat may become allergic after exposed to porphyromonas gingivalis lipopolysaccharide. methods: randomized control series design experimental study was conducted to 24 male wistar rats, two experimental groups and one control group. one group was subjected to intrasulcular injection of pglps1435/1450. tissue examination were done for allergy biomarkers with peroxidase immunohistochemistry for leukotriene c4 (ltc4) and eosinophilic cationic protein (ecp) in bronchus tissue. serum level examination of interleukin 4 (il-4), and immunoglobulin e (ige) was done with elisa. data were analyzes using anova. results: after four days, ltc4 and ecp expression increased significantly (p=0.001); even insignificant, il-4 and ige serum level also increased. conclusion: pglps is able to stimulate immunocompetent cells which changed the host immune response of non-allergic rats. therefore, it is possible that they become allergic. key words: transmission, allergic, periodontopathic bacteria, lipopolysaccharide abstrak latar belakang: menurut teori, penularan penyakit alergi termasuk asma merupakan hal yang mustahil. fakta menunjukkn bahwa hampir semua anak penderita asma mempunyai alergi, tetapi tidak semua anak alergi menderita asma, sehingga mungkin ada faktor lain ya ng terlibat. hal yang menarik adalah timbulnya gejala alergi pada anak non-alergi setelah orang tua mereka bekerja beberapa bulan pada orang yang alergi. penelitian mutakhir juga menemukan bahwa bakteri periodontopatogen juga dapat ditularkan ke bayi dari ibu dan pengasuhnya. sebagai akibatnya, sangat nasuk akal bila anak non-alergi menjadi alergi setelah terpajan bakteri periodontopatogen. tujuan: untuk verifikasi konsep baru, yaitu bahwa tikus non-alergi dapat menjadi alergi setelah terpajan lipopolisakarida. metode: pada 24 tikus wistar jantan; dua kelompok perlakuan dan satu kontrol. satu kelompok diberikan injeksi intrasulcular dengan pglps1435/1450. pemeriksaan jaringan dilakukan pada biomarker alergi menggunakan imunohistokimia peroxidase untuk leukotriene c4 (ltc4) dan eosinophilic cationic protein (ecp) dari jaringan bronkus. pemeriksaan kadar serum pada interleukin 4 (il-4), dan immunoglobulin e (ige) menggunakan metode elisa. data dianalisis dengan anova. hasil: setelah empat hari, ltc4 ekspresi ecp meningkat secara bermakna (p=0.001); walau tidak bermakna, kadar il-4 and ige serum juga meningkat. simpulan: pglps dapat merangsang sel imunokompeten sehingga dapat merubah respons imun tikus non-alergi menjadi alergi. kata kunci: penularan, alergi, bakteri periodontopatogen, lipopolisakarida correspondence: haryono utomo, c/o: rumah sakit gigi dan mulut pendidikan, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id 86 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 85–91 introduction atopy or allergy is a personal and/or familial tendency to, usually in childhood or adolescence, become sensitized and produce immunoglobulin e (ige) instead of igm or igg antibodies in response to ordinary exposure to low doses of allergens, usually proteins. however, ige normally existed in non-allergic individuals eventhough it was the least amount among other immunoglobulins.1 atopy can be detected by specific serum ige or skin-test reactivity to environmental allergens and it is often associated with asthma. in some populations, the prevalence of asthma associated with allergies has increased more than that of non-atopic asthma, whereas in others the prevalence of the two types of asthma has increased to a similar degree. therefore, until today it is still not exactly known what factors cause asthma in a person with atopy or what factors cause atopy in a person with asthma.1,2 clinical studies of children and interventional studies of animals indeed suggest that the exposure to microbes through the gastrointestinal tract was able to develop immune function. in addition, the initial microbial exposure for children born by caesarean section is delayed compared with those born by vaginal delivery; thus have more asthma risk.2,3 this phenomenon was called as the “hygiene hypothesis” introduced by david strachan in 1989, it proposed that the increase in allergic diseases was caused by decrease o exposed infections during childhood.1 nevertheless, current cohort studies suggest that the risks of asthma are increased in children who suffer severe illness from a viral respiratory infection in infancy. moreover, researches revealed that asthma appeared first in adults returning to villages after working in a european influenced city. only thereafter did it appear in children; and another has found the rates of asthma to be nearly as high in adopted children of mothers with asthma as in natural children.4 as the result, yoo et al.4 in 2007 proposed a new concept that asthma is a transmissible disease. the possibility that oral bacteria infection may induce allergic asthma was confirmed by wiyarni et al.5 as well as utomo and harsono6 studies. it revealed that dental plaque control therapy improves respiratory quality and that the asymptomatic asthmatic children had significant lower gram-negative positive culture than uncontrolled asthma (wheezing and coughing)5 as well as significant decrease of histamine serum level6 in one week study. evaluation of randomly selected subjects after two months reported that their asthmatic symptoms and food allergy also diminished.7 coincidentally, a study by lee et al.8 showed that the periodontopathic bacteria in dental plaque are also transmissible. it was revealed that if at least one of the parents harbored a periodontal pathogen, then the child will exhibit the same genotype of bacteria. therefore, there is a possibility that allergic asthma is not merely inherited, but also transmissible to non-allergic individuals via periodontopatic bacteria. tanner et al.9 found that various anaerobic species colonize the edentulous mouths of infants, and that maternal or caregivers saliva may act as a source of some gramnegative anaerobes.7 several literatures had been suspected the involvement of periodontopathic bacteria in the pathogenesis of allergy such as netea et a.l,10 kato et al.,11 and card et al.,12 reported that allergic immune response could be modulated by gram-negative periodontopathic bacteria, could be worsen or attenuate the symptoms, depended upon the time of exposure. however, they still used ovalbumin (ova) injection for allergy sensitization while our study only used ova inhalation it was interesting that periodontopathic bacteria lipopolysaccharides (lps) have a unique characteristics; in some instances the enhanced type 1 immune response which release interferon-γ, i.e. lps from aggretibacter actinomycetemcomitans and porphyromonas gingivalis (high dose, ≥ 1.0 μg/ml); nevertheless low dose of p. gingivalis lps (pglps) (< 1.0 μg/ml) enhance the th2 immune response which release interleukin-4 (il-4) and il13.10 nevertheless, according to kumada et al.,14 synthetic lps only stimulated tlr4, thus was a tlr4-ligand. nevertheless, darveau et al,15 reported that pglps1435/1450 a particular synthetic pglps with major lipid a mass ion 1435 and 1450 m/z (pglps1435/1450) could act as an immunomodulator either to tlr2 or tlr4. therefore, in this study, it was used for mimicking naïve pglps characteristics which was both tlr2 and tlr4 ligands. the allergic reaction was measured based on the modulation of allergic reaction biomarkers that was leukotriene c4 (ltc4)16 which produced by mast cells and basophils; and eosinophilic cationic protein (ecp)17 which produced by eosinophils in gingival and bronchus tissues. other examination was il-418 and ige19 serum level. the objective of this study was to verify a new concept that non-allergic children may become allergic after exposed to p. gingivalis lipopolysaccharide using wistar rats as animal model. additionally, it also verify a new theory of asthma’s pathogenesis that allergy is not always inherited but also induced by periodontopathic bacteria such as p. gingivalis. materials and methods twenty four male wistar rats (120-150 grams) were randomly selected and divided into two experimental groups (a) and one control group (b). treatment group was divided into 3 subgroups (a1-3) which each consisted of 6 rats. subgroups 1, 2 and 3 were non allergic rats which subjected to intrasulcular (i.s.) injection of pglps1435/1450 according dumitrescu’s20 method to create chronic gingivitis in rats in first day and second day, there were injected one of three doses 0.3, 1.0 and 3.0 μg/ml consecutively (figure 1). these 3 doses represented low (0.3 μg/ml), cut off point (1,0 μg/ml) and high (3.0 μg/ml).10 control group 87utomo: new concept in allergy: non-allergic rats becomes allergics was injected with phosphate buffered saline (pbs). after 4 days, non-allergic rats were induced by ovalbumin (sigmaaldrich, germany) inhalation that was 1 mg/ml in sterile saline 1 ml dose using nebulizer (omron™, usa) for 30 minutes.21 each step (control, fourth day, day 4 after ova inhalation), tissue samples were taken by sectioning the gingiva and extrapulmonary bronchus (figure 2), and every time sacrifice was done according to the euthanasia protocol. blood serum was taken via tail vein during experimental procedures and from the heart after eutahanasia. the allergic reaction was measured based on the modulation of allergic reaction biomarkers that was leukotriene c4 (ltc4)16 which produced by mast cells and basophils; and eosinophilic cationic protein (ecp)17 which produced by eosinophils in gingival and bronchus tissues. allergic reaction biomarkers expressions of gingiva and bronchus immunohistochemistry samples were counted per view with light microscope (olympus™ cx-31). other examination was il-418 and ige19 serum level. tissue samples were examined with peroxidase immunohistochemistry using diamino benzidine (dab), and blood serum with serum enzyme-linked immunosorbent assay elisa. this research and its laboratory examinations were conducted in the biology department universitas brawijaya malang, october 2008 until february 2009. the research protocol had been approved by the animal care and use ethical committee, faculty of veterinary medicine airlangga university, surabaya. statistical analysis was done with anova to reveal the interaction between variable doses pglps1435/1450 and ovalbumin inhalation. results after intrasulcular injection of pglps 1435/1450 0.3, 1.0 and 3.0 μg/ml, in day 1 and 2, there were several modulation of allergic reaction biomarkers of gingival tissue and bronchus tissue in day 4. tissue examination results and statistical significance were shown in table 1 and 2. the expressions of the allergic reaction biomarkers (ltc4, ecp) after pglps 1435/1450 injection wistar rats in day 1 and 2. all variables increased significanly in bronchus tissue and insignificant in gingival tissue. the expressions of the allergic reaction biomarkers (ltc4, ecp) after pglps 1435/1450 injection wistar rats in day 4 as well as after ova inhalation. in gingival and bronchus tissues all variables increased significanly, except in bronchus tissue pglps1435/1450 1.0 μg/ml. table 3 showed the serum level after pglps 1435/1450 injections wistar rats on day 4 all variables were increased significantly except in il-4 pglps1435/1450 0.3 μg/ml. trachea extra pulmonary bronchus figure 2. location of extra-pulmonary bronchus sample section. figure 1. location of injection and assisted drainage therapy in wistar rats. table 1. allergic reaction in non-allergic rats with pglps 1435/1450 injection (gingival and bronchus tissues, day 4) dependent variable group b mean ± sd group a mean ± sd 0.3 μg/ml n = 6 p 1.0 μg/ml n = 6 p 3.0 μg/ml n = 6 p gingiva ltc4 ecp bronchus ltc4 ecp 2.333 ± 1.211 3.667 ± 1.632 2.167 ± 0.753 2.667 ± 1.211 3.50 ± 0.548 4.167 ± 1.472 12.833 ± 3.971 14.167 ± 3.061 0.520* 0.590* 0.001 0.001 3.167 ± 0.983 4.333 ± 1.366 10.667 ± 1.506 16.50 ± 2.881 0.757* 0.864* 0.001 0.001 2.333 ± 2.066 5.167 ± 0.753 12.333 ± 1.751 13.333 ± 2.658 1.000* 0.322* 0.001 0.001 *insignificant difference (p≥.05) 88 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 85–91 table 2. allergic reaction in non-allergic rats with pglps 1435/1450 injection (gingival and bronchus tissues, day 4) pre-inhalation and inhalation depend variable pglps1435/1450 0.3 μg/ml inj. μg/ml inj. pglps1435/1450 1.0 μg/ml inj. μg/ml inj. pglps1435/1450 3.0 μg/ml inj.μg/ml inj. pre inhalatione inhalation inhalationtion inhalation p pre inhalatione inhalation inhalationtion inhalation p pre inhalatione inhalation inhalationtionionon inhalation p mean ± sd ± sd mean ± sd ± sd mean ± sd ± sd mean ± sd ± sd mean ± sd ± sd mean ± sd± sd gingiva ltc4 ecp bronch ltc4 ecp 3.50 ± 0.548 4.167 ± 1.472 12.833 ± 3.971 14.167 ± 3.061 20.50 ± 0.548 22.50 ± 1.643 30.833 ± 1.835 25.00 ± 3.225 .001 .001 .001 .001 3.167 ± 0.983 4.333 ± 1.366 10.667 ± 1.506 16.50 ± 2.881 24.167 ± 3.764 22.00 ± 1.549 26.833 ± 4.021 19.50 ± 2.074 .001 .001 .001 .065* 2.333 ± 2.066 5.167 ± 0.753 12.333 ± 1.751 13.333 ± 2.658 24.67 ± 3.14 24.167 ± 1.17 28.833 ± 3.06 29.50 ± 1.049 .001 .001 .001 .001 *insignificant difference (p≥.05) a b figure 3. a) perfect match ab-ag; b) cross-reactivity ab-ag.22 figure 4. augmentation of allergic airway inflammation through the tlr4-mediated modification of mast cell.25 89utomo: new concept in allergy: non-allergic rats becomes allergics discussion the classic concept by strachan in 1989 “the hygiene hypothesis” which simply interpreted as “cleanliness makes allergy” was the prime barrier for our concept since it was believed by laymen or by allergic specialist. our concept so called as ”dental plaque-induced asthma hypothesis” was considered controversial before conducting a clinical study. oral infection such as periodontitis was considered protective to allergic asthma. despite these obstacles, however, the result of our study was very successful.5,6 dental plaque therapy reduced wheezing, improve respiratory quality based on forced expiratory quality in one second (fev1) in asthmatic children in one week study.5 within the same samples, utomo and harsono revealed decreased histamine serum level. moreover, based on the same study utomo reported that in randomly selected samples they were still symptomatic after two months later. nevertheless, since it was only a clinical study, in order to understand the mechanism a verification study with animal model should be conducted. utomo revealed that allergic asthma symptoms which caused by immunogenic as well as neurogenic inflammation also increased after injected with pglps1435/1450. interaction of these inflammations increased allergic symptoms even more. therefore,it is possible that pglps is a trigger of allergic reaction in non-allergic rats.27 a concept which proposed by yoo et al. in 2007 attracted us to reveal why non-allergic asthma children could become allergic eventhough not inherited by their parents, therefore, an animal study of non-allergic rats which induced for chronic gingivitis with pglps1435/1450 as well as evaluating the allergic reaction was conducted. after pglps1435/1450 injection in non-allergic rats, in day 4 examination revealed that in gingival tissue ecp expression increase significantly in all doses (p=0.003; p=0.006 and p=0.001). nevertheless, ltc4 expressions also increased eventhough insignificant (table 1). in extrapulmonary bronchus, ltc4 and ecp expressions increased significantly in all doses (p=0.001 in all variables). the increased expressions could be via the activation of toll-like receptor-2 (tlr2) and/or tlr4 in immunocompetent cells after pglps1435/1450 injection, which then released mediators. ovalbumin inhalation for 30 mins, as predicted did not change systemic immun response, since no significant change of il-4 and ige serum level (table not included). nevertheless, interestingly, ovalbumin inhalation resulted in significant increase of ltc4 and ecp expressions in gingival and bronchus tissues (p=0.001), except ecp expression in bronchus 1,0 μg/ml pglps1435/1450 injection (p=0.065) (table 2). it was logical since according to literatures ova inhalation increased ltc416 and ecp17 in ova-induced allergic rats. neverheless, this result was “out of the box” since without any previous ova-induced allergy injection, allergic reaction also occured. this phenomenon was considered the hardest thing to explain since it was an unusual finding. in a simple explanation, ova inhalation acts as specific allergen which cross-linked with the ova-specific iges that attached to mast cells and basophils in ova-induced allergy subjects and stimulated degranulation. nevertheless, even without previous ova-induced allergic sensitization, antigen-antibody reaction also happened, this mechanism is termed as cross reactivity.22 according to melton and landry,23 cross reactivity may happens if a protein which actually has a 3d shape, in this case the ovalbumin inhalation in our study, could attach to the non-specific iges even not perfect match as “lock and key”. (figure 3), in our study non-ova spesific ige recognized ova as its ligand and activate mast cells. the result of our study is supported by saluja et al.24 study in 2012, which reported that prolonged exposure (96 h) with tlr-ligands promoted mast cell reactivity following ige-receptor activation. tlr4 activation with lps generated the most pronounced effect, with an enhanced degranulation and secretion of leukotrienes, cytokines and chemokines. the effect of lps was mediated through a myd88-dependent pathway and the increased effect involved jnk-dependent pathway. in our study, this mechanism was suggested via fcer1-mediated mast cell reactivity amplification after stimulation of tlr2 and -4 with pglps1435/1450 (figure 4). this mechanism was also confirmed in this study, after pglps1435/1450 injection tlr2 and -4 expressions in rats’ bronchus were higher significantly (p=0.001) compared to control (table not included). there is an important question:”which cells are the first target of pglps injection as in our study?” it is not table 3. serum allergic biomarkers in non-allergic rats with pglps 1435/1450 injection (day 4) dependent variable group b mean ± sd group a mean ± sd adaptive immune reposnse 0.3 μg/ml n = 6 p 1.0 μg/ml n = 6 p 3.0 μg/ml n = 6 p il-4 allergy ige 0.833 ± 0.432 2.482 ± 0.073 3.983 ± 2.271 8.744 ± 1.874 0.790* 0.001 16.830 ± 8.256 9.337 ± 1.799 0.001 0.001 20.194 ± 6.324 7.784 ± 0.902 0.001 0.001 *insignificant difference (p≥.05) 90 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 85–91 easy to answer, but it will answer our research question. mast cells has a strategic location in the body; they are common at sites in the body that are exposed to the external environment, such as mucosa and the skin. in these locations, they are found in close proximity to blood vessels, where they can regulate vascular permeability and effector-cell recruitment.25 although they do not have direct cell to cell contact with local populations of antigenpresenting cells, mast cells can modulate the behaviour of these and other neighbouring effector cells through the release of mediators.26 according to kulka et al.,27 mast cells products such as histamine and tryptase stimulates nerve endings, thus neurogenic inflammation, which then secretes neuropeptides such as substance p or calcitonin gene-related peptide. these neuropeptides activates either basophils or mast cells via neurokinin recepters which then conducting a “vicious circle” that aggravated allergic reaction. berry et al.28 reported that these cells also secretes tnf-α which able to complicate asthma pathogenesis which termed as refractory or “difficult asthma”. moreover, these cells also produced il-4 that needed for isotype switching mechanism from igg or igm to ige, thus also increased ige in serum level. this mechanism was verified by the increase of il-4 serum level which was significant (p=0.001) in 1.0 and 3,0 μg/ml as well as an increase but insignificant (p=0.790) in 0.3 μg/ml (table 3). the presence of other specific iges which not induced by ova sensitization in this study also supported by hahn et al.29 study in 2012 which reported that chlamydia pneumonia-specific ige is prevalent in asthma and associated with asthma severity. therefore, there must be “still undiscovered” specific-ige which produced after pglps injection. the presence of this kind of iges may resulted to cross-reativity, thus increased mast cells or basophils activation which lead to increase allergic reaction. our study supported the successful dental plaque control therapy in reducing allergic asthma symptoms i.e. wheezing in wiyarni et al.6 study. in this study, asthmatic children who had been conducted dental plaque control therapy and dental health education had lower positive bacterial culture of gram negative bacteria significantly compared to control in o week study. moreover, a longer evaluation, that was until two month evalution, of randomly selected samples of this clinical study revealed that the allergic asthma symptoms of those children were still disappeared.7 it was not surprising since free specific ige (free/ unbounded) actually present for 1-2 days only, compared to 21 days if attached to mast cells or basophils, after that it would be degraded.30 consequently, decreasing allergic reaction by reducing the activity of mast cells and basophils that were stimulated by lps and neuropeptides is a better way than considering the ige level only. it was supported by gamble et al.31 in 2010, who reported that specific ige was not the best biologic predictor for allergic asthma severity, since it was racial dependent. the diminished activation of mast cells and basophils resulted in lowering interleukin-3 (il-3) excretion that functions as stimulator of mast cell proliferation and basophils apoptosis prevention.30 based on this study, it can be concluded that even in non-allergic individuals, a prolonged exposure of pglps is able to elicit allergic reaction, enhancing crossreactivity and increase fcεri expressions, thus mast cells and basophils reactivity which lead to increase allergic symptoms. therefore, eliminating periodontopathic bacteria is mandatory to prevent from allergy and allergic transmision. acknowledgement promotor and co-promotors of the doctorate program in universitas airlangga: prof. subijanto marto sudarmo, md., ph.d, pediatric gastroenterology consultant; prof. fedik abdul rantam, dvm., ph.d. and prof. m. rubianto, dds., ph.d, periodontist consultant. references 1. umetsu dt. early exposure to germs and the hygiene hypothesis. cell res 2012; 22(8): 1210–11. 2. roduit c, scholtens s, de jongste jc, wijga ah, gerritsen j, postma ds, brunekreef b, hoekstra mo, aalberse r, smit ha. asthma at 8 years of age in children born caesarean section. thorax 2009; 64(2): 107–13. 3. thavagnanam s, fleming j, bromley a, shields md, cardwell cr. a meta-analysis of the association between caesarean section and childhood asthma. clin exp allergy 2008; 38(4): 629–33. 4. yoo j, tcheurekdjian h, lynch sv, cabana m, boushey ah. microbial manipulation of immune function for asthma prevention inferences from clinical trials. proc am thorac soc 2007; 4(3): 277–82. 5. wiyarni p, imelda f, retno i, utomo h, anang e, harsono a. changes in bacterial profiles after periodontal treatment associated with respiratory quality of athmatic children. paediatr indones 2008; 48: 327–37. 6. utomo h, harsono a. rapid improvement of respiratory quality in asthmatic children after the assisted drainage therapy. pediatrica indonesiana 2010; 50(4): 199–206. 7. utomo h. reducing asthmatic symptoms through improving oral health: from imaginary to reality. j indon dent assoc special edition for 23th pdgi congress, march 2008. p. 28–33. 8. lee y, straffon lh, welch kb, loesche wj. the transmission of anaerobic periodontopathic organisms. j dent res 2006; 85(2): 182–6. 9. tanner acr, milgrom pm, kent r jr, mokeem sa, page rc, riedy ca, weinstein p, bruss j. the microbiota of young children from tooth and tongue samples. j dent res 2002; 81(1): 53–7. 10. netea mg, van der meer jwm, sutmuller rp, adema gj, kullberg bj. from the th1/th2 paradigm towards a toll-like receptor/ t– helper bias. antimicrob ag chemoth 2005; 49(10): 3991–6. 11. kato t, kimizuka r, okuda k. changes of immunoresponse in balb/c mice neonatally treated with periodontopathic bacterial endotoxin. fems immunol med microbiol 2006; 47(3): 420–4. 12. card jw, carey ma, voltz jw, bradbury ja, ferguson cd, cohen ea, schwartz s, flake gp, morgan dl, arbes sj jr, barrow da, barros sp, offenbacher s, zeldin dc. modulation of allergic airway inflammation by the oral pathogen porphyromonas gingivalis. inf immun 2010; 78(6): 2488–96. 91utomo: new concept in allergy: non-allergic rats becomes allergics 13. jaakkola jj, ahmed p, ieromnimon a, goepfert p, laiou e, quansah r, jaakkola ms. preterm delivery and asthma: a systematic review and meta-analysis. j allergy clin immunol 2006; 118(4): 823–30. 14. kumada h, haishima y, watanabe k, hasegawa c, tsuchiya t, tanamoto k, umemoto t. biological properties of the native and synthetic lipid a of porphyromonas gingivalis lipopolysaccharide. oral microbiol immunol 2008; 23(1): 60–9. 15. darveau rp, pham tt, lemley k, reife ra, bainbridge bw, coats sr, howald wn, way ss, hajjar am.. porphyromonas gingivalis lipopolysaccharide contains multiple lipid a species that functionally interact with both toll-like receptors 2 and 4. inf immun 2004; 72(9): 5041–51. 16. ilarraza r, wu y, adamko dj. montelukast inhibits leukotriene stimulation of human dendritic cells in vitro. int arch allergy immunol 2012; 159(4): 422–7. 17. koh gc, shek lp, kee j, wee a, ng v, koh d. saliva and serum eosinophil cationic protein in asthmatic children and adolescents with and without allergic sensitization. j asthma 2010; 47(1): 61–51. 18. maes t, joos gf, brusselle gg. targeting interleukin-4 in asthma. am j respir cell mol biol. 2012; 47(3): 261–70. 19. korn s, haasler i, fliedner f, becher g, strohner p, staatz a et al. monitoring free serum ige in severe asthma patients treated with omalizumab. resp med 2012; 106(11): 1494–500. 20. d u m it r e scu a l . h ist olog ica l c ompa r ison of p e r io dont a l inflammatory changes in two models of experimental periodontitis in rat: a pilot study. tmj 2006; 56(2): 211–7. 21. toward tj, broadley kj. early and late bronchoconstrictions, airway hyperreactivity, leucocyte influx and lung histamine and nitric oxide after inhaled antigen: effects of dexamethasone and rolipram. clin exp allergy 2004; 34(1): 91–102. 22. aalberse rc. assessment of allergen cross-reactivity. clin mol allergy 2007; 5(2): 1–9. 23. melton sj, landry sj. three dimensional structure directs t-cell epitope dominance associated with allergy. clin mol allergy 2008; 6(9): 1–12. 24. saluja r, delin i, nilsson gp, adner m. fcer1-mediated mast cell reactivity is amplified through prolonged toll-like receptor-ligand treatment. plos one 2012; 7(8): 43–7. 25. balzar s, fajt ml, comhair sa, erzurum sc, bleecker e, busse ww, castro m, gaston b, israel e, schwartz lb, curran-everett d, moore cg, wenzel se. mast cell phenotype, location, and activation in severe asthma. am j respir crit care med 2011; 183(3): 299–309. 26. amin k. the role of mast cells in allergic inflammation. respir med 2012; 106(1): 9–14. 27. kulka m, sheen ch, brian p, tancowny bp, grammer lc, schleimer rp. neuropeptides activate human mast cell degranulation and chemokine production. immunology 2010; 123(3):: 398–410. 28. berry ma, hargadon b, shelley m, parker d, shaw de, green rh, bradding p, brightling ce, wardlaw aj, pavord id. evidence of a role of tumor necrosis factor-α in refractory asthma. n engl j med. 2006; 354(7): 697–708. 29. hahn dl, schure a, patel k, childs t, drizik e, webley w. chlamydia pneumoniae-specific ige is prevalent in asthma and is associated with disease severity. plosone 2012; 7(4): 35–45. 30. lowe pj, tannenbaum s, gautier g, jimenez p. relationship between omalizumab pharmacokinetics, ige pharmacodynamics and symptoms in patients with severe persistent allergic (ige-mediated) asthma. br j clin pharmacol 2009; 68(1): 61–76. 31. gamble c, talbott e, youk a, holguin f, pitt b, silveira l, bleecker e, busse w, calhoun w, castro m, chung kf, erzurum s, israel e, wenzel s. racial differences in biologic predictors of severe asthma:data from the severe asthma research program. j allergy clin immunol 2010; 126(6): 1149–56.e1. 223223 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 223–228 effectiveness of line communication application as a social media on changes in tooth brushing behavior of junior high school students in banjarmasin w. widodo,1 r. darmawan setijanto,2 and agung sosiawan2 1department of dental public health, faculty of dentistry, universitas lambung mangkurat, banjarmasin indonesia 2department of dental public health, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: there were only 10.7% of junior school students in banjarmasin brushing their teeth before bedtime. using line (as one of the social media) can be assumed as an effective strategy to spread information. purpose: this study aimed to reveal changes in tooth brushing behavior before bedtime in students of class vii in all state junior high schools in banjarmasin after receiving information disseminated through line. method: pre and post test technique with control group design was used in this research. result: one week before the treatment, the mean frequency of tooth brushing behavior before bedtime in the line group was 1.90, while in the poster group was 1.93. during the treatment, the mean frequency of tooth brushing behavior before bedtime in the line group was 4.78 in the first 7 days, 5.07 in the second week, and 5.67 in the third week. on the other hand, the mean frequency of tooth brushing behavior before bedtime in the poster group was 4.66 in the first 7 days, 4.61 in the second week, and 5.18 in the third week. conclusion: messages/ information disseminated through both of line and poster can give a significant change in tooth brushing behavior before bedtime. nevertheless, line can trigger better effectiveness than poster in stimulating a change in tooth brushing behavior before bedtime. keywords: social media; line; poster; tooth brushing behavior before bedtime correspondence: widodo, department of dental public health, faculty of dentistry, universitas lambung mangkurat, jl. veteran no. 128b banjarmasin indonesia. e-mail: dodowident@yahoo.co.id introduction tooth decay will affect the overall health status of the body, resulting in disruption of daily activities. the impact of tooth decay can also be considered as one of the obstacles in improving both the quality of human resources and the standard of human life, especially in finding certain professions. oral health condition in indonesia is still poor. the prevalence of oral and dental problems in indonesia was 25.9% with a national dmf-t index value of 4.85.1 the prevalence of oral and dental problems in south kalimantan province even was 36.1%, the second largest after south sulawesi (36.2%). the prevalence of oral and dental problems in banjarmasin is 38.2% with a dmf-t index value of 5.54, the second highest in the province of south kalimantan after barito kuala based on who criteria.2 dental health promotion in banjarmasin is usually conducted through direct outreach and dissemination of information using conventional media, such as posters. unfortunately, these methods cannot be conducted evenly and continuously since the area of banjarmasin city is mostly surrounded by rivers and swamps. as a result, inadequate health workforce and geography become major obstacles for health personnel in running the health center service outside the building, resulting in unoptimal health education.3 the number of people in banjarmasin city brushing teeth before bedtime was only 36.7%. meanwhile, the number of junior high school students brushing teeth before bedtime was only 10.7%. 2 such conditions require dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 224 widodo, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 223–228 appropriate strategies to improve the behavior of the junior high school students in the city of banjarmasin, south kalimantan province, especially in brushing teeth properly before bedtime at night. a theory most commonly used in health education and health promotion is health belief model (hbm) theory. the basic concept underlying hbm theory is health behavior determined by personal beliefs or perceptions about the disease, as well as strategies to reduce the occurrence of disease. hbm depicts the considerations of a person before they adopt healthy behaviors. it means that hbm serves as a prevention model or preventative one. hbm can also be considered as a cognitive model demonstrating an individual’s behavior influenced by cognitive processes. these cognitive processes are influenced by several factors, such as demographic variables, sosiopsychological characteristics, and structural variables. demographic variables include class, age, and gender. meanwhile, sosiopsychological characteristics involve personality, peers, and group pressure.4 social media has an important role in changing human behavior. the existence of social media can make communication between users increasingly closer. a research on sma negeri 4 manado found that the use of social media can change learning behavior, resulting in improvement of school achievement scores. the learning behavior change is due to changes in motivation of the future (in order motive) that can be achieved.5 the interaction and communication among users in social media can be used to facilitate dissemination of information, motivation, and promotion in various business sectors, including health sector. line as one of social media is an instant messaging application using internet medium that is free of charge. line application can be used in various electronic devices, such as smartphones, tablets, and computers. various features are contained in line application, none of which is found on other applications, thus, it appeals to users and makes line developed very rapidly.6 features in line include free call, line cards, stickers in the form of emoticons, and online games without any cost, as a result, line is very popular among the youth, including schoolage children.7 in addition, line also allows students as its users to make a group of peers (peers group). therefore, related to the theory of hbm on the sosisopsychological characteristics of peer age, line can be assumed as a social media that can influence the health behavior change in students. by forming a group in line, the students can interact with each other as well as invite each other to change their health behavior.8 line is the most widely used type of social media at the young age, including school age with the age range of 12-24 years, reaching to 41.4%. motivation to use social media at the young age is mostly to make the social media as means of disseminating information.9 thus, those various facilities and excellent features offered in line as social media trigger this research to focus on the effectiveness of line as a social media in changing the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin compared to poster as the conventional media. consequently, all information or messages disseminated via line as the social media as well as poster as the conventional media in this research were focused on the benefits of tooth brushing before bedtime at night, the effects of the absence of tooth brushing before bedtime at night, and the benefits of brushing teeth diligently before bedtime at night. materials and methods this study used pre and posttest technique with control group design. samples in this research divided into two treatment groups with two different media. the first treatment group was exposed to information/ messages disseminated via a social media, line. meanwhile, the second treatment group was exposed to information/ messages disseminated via a conventional media, poster. the second group was used as a comparison group to the first group. moreover, this research was performed in four treatment periods, namely the first 7 days, the second 7 days, the third 7 days, and the fourth 7 days after the treatment was discontinued. independent variables in this research were the provision of information about the benefits of tooth brushing before bedtime at night, the effects of the absence of tooth brushing before bedtime at night, and the benefits of brushing teeth diligently before bedtime at night, transmitted/ disseminated via line and posters. meanwhile, dependent variables were changes in the tooth brushing behavior before bedtime at night. sampling was carried out in all state junior high schools in banjarmasin with several criteria. first, the schools had to have internet connection. students had to have a smartphone with line application. their score of health belief model questionnaire regarding dental health students had to be less than four. sampling of the population who met the criteria was performed using multistage random sampling technique10 on students of class vii in all of the state junior high schools in banjarmasin that met the criteria. the total of samples obtained were 360 students, divided into two groups, 180 of which were in the line group, and 180 of which were in the poster group. those samples were from four different state junior high schools in the city of banjarmasin, namely 100 students from smp negeri 1, 120 students from smp negeri 6, 70 students from smp negeri 7, and 70 students from smp negeri 26. this research then was conducted for 28 days, from june 6th to july 3rd, 2016. furthermore, this research also used a sheet of tooth brushing activity to measure changes in their tooth brushing behavior related to frequency of tooth brushing activity before bedtime at night. the sheet of tooth brushing activity was filled by parents of those samples every night, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 225225widodo, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 223–228 and then collected at the end of each period. the scale of measurement was in the form of ratio with a range of 0 (zero) up to 7 (seven). measurements then were made during those four periods, namely the first 7 days, the second 7 days, the third 7 days, and the fourth 7 days. the measurement on the first 7-days (from june 6th to june 12th, 2016) was performed by disseminating information about the benefits of tooth brushing before bedtime at night via posters and line. the posters were distributed to the poster group every day during school hours. on the other hand, for the line group, the information was disseminated through messages via line every evening at 21.00 pm. the measurement on the second 7 days (from april 13th to june 19th, 2016) was carried out by disseminating information about the effects of the absence of tooth brushing before bedtime at night via posters and line. similarly, the posters were distributed to the poster group every day during school hours, while for the line group, the information was disseminated through messages via line every evening at 21.00 pm. the measurement on the third 7 days (from june 20th to june 26th, 2016) was conducted by disseminating information about the benefits of brushing teeth diligently before bedtime at night via posters and line. like in the previous measurements, the posters were distributed to the poster group every day during school hours. for the line group, the information was disseminated through messages via line every evening at 21.00 pm. the measurement on the fourth 7 days (from june 27th to july 3rd, 2016) was focused on the frequency of tooth brushing activity before bedtime at night in both the line and poster groups after the provision of information via both posters and line was discontinued. the data then were collected at the end of this period. results data obtained in this research were about the frequency of tooth brushing activity before bedtime at night, measured before the treatment, during the treatment, and after the treatment. data about the frequency of tooth brushing activity before the treatment were collected from questionnaires distributed during the selection of samples that met the criteria with a certain range of scores, from 0 (zero/ never brushed their teeth) to 3 (rarely brushed their teeth). the data collected then were compared between the frequency of tooth brushing activity before the treatment, the frequency of tooth brushing activity during the treatment, and the frequency of tooth brushing activity after the treatment was discontinued. kolmogorov-smirnov test and shapiro-wilk test were performed to analyze the normality of the data with a significance value of 0.05. results of the test showed that the significance value obtained was 0.001, less than 0.05. thus, it indicates that all the data about the frequency of tooth brushing activity before the treatment, the frequency of tooth brushing activity during the treatment, and the frequency of tooth brushing activity after the treatment was discontinued in both the line and poster groups were not normally distributed. the results of this research also demonstrated that the mean frequency of tooth brushing activity before the treatment in the line group was 1.90, while in the poster group was 1.93. those mean frequencies of tooth brushing activity then increased during the treatment in both the line and poster groups. the mean frequency of tooth brushing activity on the first 7 days in the line group was 4.78, greater than in the poster group, about 4.66. results of the mann whitney test conducted with a significance value of 0.05 indicated the significance value obtained was 0.340, more than 0.05. it means that there was no significant table 1. change in the frequency of tooth brushing behavior before the treatment, during the treatment, and after the treatment in the line group and the poster group mann whitney test analysis media before the treatment during the treatment after the treatment the first seven days the second seven days the third seven days the fourth seven days line 1.90 4.78 5.07 5.67 5.52 poster 1.93 4.66 4.61 5.18 4.15 sig. 0.934 0.340 0.002 0.001 0.001 table 2. change in the mean frequency of tooth brushing behavior before the treatment, during the treatment, and after the treatment in the line group and the poster group change in the mean frequency of tooth brushing behavior before bedtime at night media of the treatment during the treatment after the treatment was discontinued on the fourth seven daysthe first seven days the second seven days the third seven days line 2.88 3.17 3.77 3.62 poster 2.73 2.68 3.25 2.22 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 226 widodo, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 223–228 difference in the increased frequency of tooth brushing activity before bedtime at night between in the line group and in the poster group (table 1). furthermore, the results of this research also indicated that the mean frequency of tooth brushing activity on the second 7 days of the treatment in the line group was 5.07, greater than in the poster group, about 4.61. results of the mann whitney test conducted with a significance value of 0.05 showed the significance value obtained was 0.002, less than 0.05. it means that there was significant difference in the increased frequency of tooth brushing activity before bedtime at night between the line group and the poster group. the results of this research also showed that the mean frequency of tooth brushing activity on the third 7 days of the treatment in the line group was 5.67, greater than in the poster group, about 5.18. results of the mann whitney test conducted with a significance value of 0.05 showed the significance value obtained was 0.001, less than 0.05. like on the second 7 days, it indicates that there was significant difference in the increased frequency of tooth brushing activity before bedtime at night between the line group and the poster group. meanwhile, the mean frequency of tooth brushing activity on the fourth 7 days after the treatment was discontinued in the line group was 5.52, greater than in the poster group, about 4.15. results of the mann whitney test conducted with a significance value of 0.05 showed the significance value obtained was 0.001, less than 0.05. similarly, it means that there was significant difference in the increased frequency of tooth brushing activity before bedtime at night between in the line group and in the poster group. in other words, changes in the mean frequency of tooth brushing activity before bedtime at night in the line group were greater than in the poster group after the treatment wad discontinued. the greatest change in the mean frequency of tooth brushing activity before bedtime at night was in the line group on the third seven days when the messages or information disseminated were about the benefits of brushing teeth diligently before bedtime at night. meanwhile, the smallest change in the mean frequency of tooth brushing activity before bedtime at night was in the poster group on the second seven days (2.68) when the messages or information disseminated were about the effects of the absence of tooth brushing before bedtime at night (table 2). discussion on the first 7 days of the treatment, the messages/ information disseminated in line and posters were about the benefits of tooth brushing before bedtime at night. consequently, the frequency of the tooth brushing activity increased after the provision of the messages/ information about the benefits of tooth brushing. this finding is in accordance with the hbm theory stating that a person’s behavior will change if given an understanding of the perceived benefits. 11 the provision of information about the perceived benefits of brushing teeth diligently could be pushed them to take an action (cues to action). the expected results to be obtained if they always brushed their teeth before going to bed at night, then it would persuade themselves (selfefficacy). as a result, they would soon make a change in behavior. the dissemination of the messages/ information was also expected to increase knowledge and awareness of those school children to adopt healthy behaviors. in other words, knowledge or cognition is the most essential domain in shaping a person’s behavior.4 on the second 7 days of the treatment, the messages/ information disseminated in line and posters were about the effects of the absence of tooth brushing before bedtime at night. similarly, the frequency of tooth brushing activity also increased during the second 7 days. this finding is in accordance with the belief component of the hbm theory stating that a person’s behavior will change if the individual is given an understanding of the seriousness of the disease as well as the severity of the disease, so preventive action must be taken (perceived severity). perceived severity component is in conjunction with the behavior change of theory transtheoritical model theory12 stating that most people have no desire to change their behavior because they do not realize that they have behavior problems and find no problem with unhealthy behaviors that they do. those school children in this research refused to brush their teeth diligently since they did not have problems with dental health so that they did not have a thought or consideration for brushing their teeth diligently. the provision of information containing the effects of the absence of tooth brushing before bedtime at night would give a dramatic relief impact, related to negative feelings, such as fear or anxiety about the risks if not brushing their teeth diligently. thus, those school children would discover and learn new facts supporting changes in their behavior into healthy one. furthermore, on the third 7 days of the treatment, the messages/ information disseminated in line and posters were about the benefits of brushing teeth diligently before bedtime at night. like the results on the first and second 7 days, the frequency of tooth brushing activity also increased since they have an expectation of rewards in their future. in other words, the rewards had a positive influence on their independence.13 the biggest change in the mean frequency of tooth brushing behavior was found on the third 7 days of the treatment on which the messages/ information disseminated in both line and posters were about the benefits of brushing teeth diligently before bedtime at night. a theory from skinner14 states that most of child’s behavior is operant response, response to an expectation. it means that expectation to gain rewards or prizes will trigger children to change their health behavior after receiving the advice. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 227227widodo, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 223–228 the increased frequency of tooth brushing activity before bedtime at night in this research results is also due to a right precipitating factor, which is the messages/ information disseminated through the media directly by their pe teachers. the right precipitating factor actually can strengthen individual to carry out the recommended action. this finding is also in conjunction with the hbm theory stating that one of the fundamental aspects of health behavior is a right precipitating aspect used as a reminder over and over again. nevertheless, the results of this research revealed that there was a significant difference in the frequency of tooth brushing activity between on the third 7 days of the treatment and on the fourth 7 days after the treatment was discontinued (sig=0.001 < α: 0.05). the frequency of tooth brushing activity began to decline again after the dissemination of the messages/ information through poster as a conventional media as well as line as a social media discontinued. thus, it can be said that unhealthy behavior is a chronic disease with remission and relapse. it means that although there are minimal and intensive interventions for healthy behavior, the targets generally will relapse and require repeated intervention before they can finally really manage themselves to behave healthy.12 therefore, maintenance stage is necessary to maintain changes in their behavior that have been made for the next 6 months by adding a reminder to make the targets stick to healthy behavior. in other words, the message/ information must continuously and repeatedly be delivered or disseminated without a break to change a person’s behavior, as a result, the behavior change will really become permanent. 15 the results of this research also showed that there was a significant difference in the effectiveness of line and posters as a means of disseminating information (sig=0.002 < α: 0.05). the effectiveness of line was higher than posters. it indicates that the provision of information on school children will be more effective when delivered in an interesting and attractive way, such as using a visual means of pictures that appeal to them.16 besides, the larger changes in the frequency of the tooth brushing activity in the line group is also because of the timely dissemination of the messages/ information, which is before bedtime. consequently, they could remember more easily. timely delivery of messages/ information is more effective to change a person’s behavior.17 on the other hand, the dissemination of the messages/ information via the posters is one-way delivery. as a result, the recipients of the posters cannot respond or interact with fellow receivers and the senders of the poster. meanwhile, the dissemination of the messages/ information via line as a social media is easier and more interesting for students. health education through line uses an extension method or a two-way method, consequently, fellow recipients of the messages can exchange comments and remind each other via line group already formed.18 another advantage of disseminating messages/information via line as a social media is that students can be involved actively and directly in response to messages/ information delivered and explained visually, thus, making them easy to understand. besides, line is more popular among the youth, including school-age children, than posters. images delivered via social media can be used as emoticons in line as well as status that can be disseminated to other users and be looked everywhere and every time.7 in general, the findings of this research are in accordance with nowak dan warneryd model theory stating that one of the elements considered in delivery of messages/ information is channel. therefore, channel used must be selected based on the characteristics of recipients and the type of messages delivered. to influence a person’s behavior, it will be more effective to use media with interpersonal channels, e.g. social networks like line. the effectiveness levels of line as a social media in the delivery of information based on calculation of aisas model are 91% for attention, 83% for interest, 83% for searching, and 80% for sharing. and, the effectiveness in delivering the most fundamental information before influencing a person’s behavior actually starts from the addition of knowledge first.19 in conclusion, information about the benefits of tooth brushing disseminated via line is effective to change the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin; information about the effects of the absence of tooth brushing disseminated via line is also effective to change the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin; information about the effects of brushing teeth diligently disseminated via line is also effective to change the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin; information disseminated in line as a social media is more effective than in poster as a conventional media to change the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin; information about the benefits of brushing teeth diligently disseminated via line has the highest effectiveness in changing the tooth brushing behavior before bedtime at night in those students of class vii in all state junior high schools in banjarmasin. references 1. soendoro t. riset kesehatan dasar 2013. jakarta: kementerian kesehatan republik indonesia; 2013. p. 110-9. 2. soendoro t. riset kesehatan dasar provinsi kalimantan selatan 2007. jakarta: departemen kesehatan republik indonesia; 2008. p. 130-40. 3. rudiansyah. profil kesehatan provinsi kalimantan selatan 2012. banjarmasin: banjarmasin pers; 2013. 4. simanulang b, masdiana. pengetahuan, sikap, kepercayaan dan perilaku budaya tradisional pada generasi muda di kota batam. jakarta: cv eka darma; 1997. p. 10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 228 widodo, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 223–228 5. nevi p, meity h. peranan blackberry massanger dalam meningkatkan prestasi belajar siswa sma negeri 4 manado. journal acta diurna 2014; iii (2): 11. 6. kaplan a. users of the world, the challenges and opportunities of social media. business horizons 2010; 53(1): 59–68. 7. anggra. kelebihan dan kelemahan line. silfi-anggra.blogspot.com /2013/05/ unduh 30 agustus 2016 pukul 14.00. 8. didno k. kelebihan dan kekurangan bbm, whatsapp, wechat, line, dan kakao talk. http//www.didno76.com/2013/09. accessed february 23, 2016 9. yasar m. hubungan antara frekuensi penggunaan fasilitas jejaring sosial dengan kejadian insomnia pada mahasiswa s.1 keperawatan semester iv di stikes muhammadiyah banjarmasin kalimatan selatan. banjarmasin: banjarmasin pers; 2012. p. 8. 10. watik ap. dasar-dasar metodologi penelitian kedokteran dan kesehatan. jakarta: pt raja grafindo persada; 2000. p. 64 -71. 11. houwink b, backer do, cramwinckle a. ilmu kedokteran gigi pencegahan. suryo s, editor. yogyakarta: gadjah mada university press; 1993. p. 58-77, 94-101. 12. saputra am. counseling with the transtheoritical model in changing smoking behavioral among adolescents. jurnal kesehatan masyarakat nasional 2013; 8(4): 154. 13. puspitasari r. pengaruh pemberian hadiah (reward) terhadap kemandirian belajar anak di tk tunas muda karas kabupaten magetan ta 2015/2016. proseding seminar nasional pendidikan, surakarta; 2013. p. 55. 14. notoatmojo s. promosi kesehatan dan ilmu perilaku. jakarta: rineka cipta; 1997. p. 121. 15. notoatmojo s. ilmu kesehatan masyarakat (prinsip-prinsip dasar). ed-2. jakarta: pt rineka cipta; 2003. p. 163-5. 16. kartono k. psikologi anak psikologi perkembangan. bandung: mandar maju; 1990. p. 133-46. 17. revyareza. kekurangan dan kelebihan media pembelajaran https:// revyareza wordpress.com/ /kekurangan-dan-kelebihan-mediapembelajaran 2013. accessed february 23, 2016 18. azwar. a. pengantar pendidikan kesehatan. jakart: sastra hudaya; 1983. p. 43-6. 19. a nast asia. efek t ivit as jeja r i ng sosia l li ne sebaga i me d ia penyampaian pesan kampanye w w f “tiggy tiger”. jur nal universitas atma jaya yogyakarta 2014; 18. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p223-228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p223-228 175175 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 175–180 effects of sarang semut (myrmecodia pendens merr. & perry) extracts on enterococcus faecalis sensitivity cut soraya,1 hendra dian adhyta dharsono,2 dudi aripin,2 mieke h. satari,2 dikdik kurnia,3 and danny hilmanto4 1department of conservative dentistry, faculty of dentistry, universitas syiah kuala, banda acehindonesia 2department of conservative dentistry, faculty of dentistry, universitas padjadjaran, bandung-indonesia 3department of chemistry, faculty of mathematics and natural science, universitas padjadjaran, bandungindonesia 4department of pediatric, faculty of medicine, universitas padjadjaran, bandung-indonesia abstract background: enterococcus faecalis (e. faecalis) is a gram positive oral pathogen that reported at the main agent infection of endodontic treatment. its activities are influenced by the virulence factors facilitating the interaction process between agents with host cells. like aggregation substance, cytolysin, extracellular superoxide, gelatinase, hyaluronidase, sex pheromones, and surface adhesions molecules. plant extracts are reported as the material antibacterial as well as e. faecalis in pathogenesis of endodontic infections. purpose: purpose of this study was to analyse of sarang semut extracts (myrmecodia pendens merr. & perry) towards sensitivity of e. faecalis. method: this research used the methanol extract of sarang semut, e. faecalis atcc 29212, and fosfomycin also chlorhexidine as the positive controls. whereas, bradford protein method was measured the concentration of the surface protein of e. faecalis and active component of the sarang semut extract. result: generally, the sarang semut extract possessed low sensitivity toward e. faecalis (≤ 13 mm), but on the concentrations of 100 µg/ml and 75 µg/ml better than inhibition of other concentrations, round 10.6-11.6 (mm). specifically, on 100 µg/ml has indicator the minimal bactericidal concentration (mbc) on e. faecalis. whereas minimal inhibition concentration (mic) on the concentration of 3,125 µg/ml. conclusion: based on mbc and mic assay, the extract of sarang semut has potential effects to adherence growth of e. faecalis, mainly on the highest concentration 100 µg/ml also mic on 3,125 µg/ ml. keywords: enterococcus faecalis; extracts of sarang semut; sensitivity; minimum bactericidal concentration; minimum inhibitory concentration correspondence: cut soraya, department of conservative dentistry, faculty of dentistry universitas syiah kuala. jln. teuku nyak arief darussalam, banda aceh, aceh 23111, indonesia. e-mail: cutsoraya1965@gmail.com introduction enterococcus faecalis (e. faecalis) bacteria are the most common pathogens isolated in root canal, especially after endodontic treatment. e. faecalis bacteria have properties to withstand a variety of conditions in the root canal. thus, despite eliminating them with various medication materials, their existence still can threaten tooth root tissue repair since they are able to survive in an acidic environment, even under conditions of nutrient deficiency and drug influence. the prevalence of e. faecalis bacteria in the case of endodontic infections reached 24 to 77%, in which the presence of these bacteria in the root canal is often associated with chronic apical periodontitis.2 the pathogenesis of e. faecalis bacterial infection begins with the formation of biofilms on the root canal tissue. this capability is facilitated by a number of other oral pathogens, then colonizing together in the root canal. e. faecalis bacteria decay a number of proteins to form acidic conditions, in which those pathogens facilitating colonization will die because of the increasing intensity of acidity in the root canal.3 one of virulence factors expressed by e. faecalis bacteria in the pathogenesis of their infection is liphoteichoic acid (lta), serving to contaminate the root canal and form colonies on the dentine surface, while surface proteins like collagen binding protein will interact with dentin collagen that can support the colonization of e. faecalis bacteria in the root canals.4 other virulent e. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 176 soraya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 175–180 faecalis proteins that inhibit other pathogens are cytolysin, aggregation substance (as-48) and bacteriosin sex pheromones, extraceluller superoxide production (esp), gelatinase lytic enzyme, hyalurodinase, as well as cytolysin toxin. in addition, carbohydrates or glycoconjugates are also categorized as virulence factors in pathogenesis of enterococcal infections.5,6 the prevention concept of e. faecalis infection is based on the sensitivity level of the anti-bacterial materials considered as irrigation materials.7 chlorhexidine (chx) is reported as very good irrigation material since it has higher sensitivity than calcium hydroxide.8 nevertheless, chx has limitations due to its insensitiveness against cell damage although it is able to prevent the formation of e. faecalis bacterial biofilms for five minutes.9 similarly, fosfomysin is reported to have anti e. faecalis bacteria by inhibiting phosphoenolpyruvate synthetase, but it can also trigger some negative effects, such as metabolic disorders in urinary system and kidney disfunction.10 therefore, using herbs is considered as an alternative effort to prevent bacterial infections, including e. faecalis infection.11 each plant actually has an excellent system of sensitivity perception, especially against bacteria, one of important virulence factors in the pathogenesis of bacterial infection.12 one of the important effects of herb extract as an anti-bacterial material is an ability to damage cells of pathogens (citotoxicity) by disrupting the membranes of the surface proteins, such as polysaccharide layer, fatty acids, and phospholipids, which eventually can degrade the structure of the cell membrane, thereby reducing the potential ca++.13 based on previous research, sarang semut has chemical compounds of flavonoid and terpenoids, playing a role as an anti-bacterial compound, unfortunately, whether flavonoids and terpenoids, active compounds, in the sarang semut can potentially inhibit e. faecalis has not clearly been known yet. 14 thus, this research aimed to analyze effects of sarang semut extract on the sensitivity of e. faecalis. materials and method anti-bacterial potency of sarang semut methanol extract was tested on e. faecalis (atcc 29212, tech atcc, manassas, usa). the sensitivity of the bacteria was analyzed using disc method, minimum inhibitory test, and minimum bactericidal test. the sensitivity of e. faecalis to fosfomycin (meiji inc, japan) and chx was also used as positive controls, while the medium without e. faecalis was used as a negative control. to determine the effectiveness of the methanol extract of the sarang semut to the development of e. faecalis, several steps were conducted. the sarang semut extract was obtained from the research laboratory of chemistry department, faculty of mathematics and natural sciences, universitas padjadjaran, bandung, indonesia. in addition to phytochemical test, a predictive analysis had been conducted for determining the bioactive compounds in the sarang semut extract based on prediction of activity spectra for substances (pass) approach with an indicator pass (pharmaexpert, moscow, rusia) value of ≥0.70. results of this analysis showed that the sarang semut had a value of more than 0.70. it means that flavonoids contained in the sarang semut have a complectivity value required based on the standard value of phytochemical analysis.15 e. faecalis atcc 29 212 bacteria were inoculated in 20 ml of mueller-hinton broth (mhb) (thermo fisher scientific inc, oxoid, uk) at 37°c for 24 hours, and then synchronized with the 0.5 mcfarland standard (1 x 108 cfu/ ml). multilevel dilution from 10-1 to 10-8 mha was performed, and then they were cultured in mha medium at 37° c for 24 hours. colonies growing as much as 30-300 cfu/ ml were used as references for inoculum candidate against the sensitivity of e. faecalis using the minimum inhibitory test and minimum bactericidal test.16 this research, 10-4 was used as a reference of dilution, then used as a reference of evaluation with an average colony of 53 cfu/ ml. 17 prior to the minimum inhibitory concentration and the minimum bactericidal concentration tests of the sarang semut extract against e. faeccalis, the concentrations of both the proteins of e. faecalis and the active compounds of the sarang semut extract were measured using bradford method (bio-rad). the proteins of e. faecalis were extracted with lysozyme extract (bioseutica b.v, zeewolde, netherlands).18 meanwhile, the active compounds of the sarang semut extract were extracted using hcl principle approach.19 moreover, to conduct this bradford test, bovine serum albumin (bsa) (polysciences inc, warrington, pausa) was used to obtain a standard protein concentration, ranging from 62.5 to 500 (pm/ ml). the e. faecalis proteins and the ant-nets extract were respectively put into elisa plate well as much as 160 µl (10 µl sample + 150 µl phosphate buffer saline (pbs)). another elisa plate well was given bsa as a standard protein as much as 160 µl (10 µl bsa + 150 µl pbs). afterwards, both the samples and bsa were added with 40 µl of protein assay (bradford), and then by using a multi-channel pipette they were resuspended and incubated at a room temperature for 1 hour. the concentration of the proteins then was measured using elisa reader based on optical density at a wavelength of 595 nm (bio-rad laboratories inc, ca, usa). furthermore, a sensitivity or susceptibility test was conducted on e. faecalis bacteria using diffusion method based on the clinical and laboratory standards institute (clsi), the standard for fosfomysin and chlorhexidine applications against e. faecalis bacteria with three categories, namely resistant if ≤13mm, intermediate if 14-16 mm, and susceptible if ≥17.20 experiments were performed in duplicate with repetition as much as 2 times. 1 ml of e. faecalis inoculum was spread in the mha medium. control and treatment discs were inserted. paper discs were dipped in fosfomysin and chx with a concentration of 25 mg/ 6.25 ml of 0.9% nacl, and then settled for 15 minutes. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 177177soraya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 175–180 the positive controls were put in the muller hilton agar (mha) (thermo fisher scientific inc, oxoid, uk) medium with tweezers. the sample discs were dipped into the 0.1 ml of the inoculum in 1 ml of the extract, and then settled for 15 minutes. the extract at the concentration of 100% (500 ug/ ml) was diluted for further concentrations, from 75, 50, 25, 12.5, 6.25, to 3.125%, and then incubated in an incubator at a temperature of 37°c. after the 24 hour incubation, the light zone (zone of inhibition) was measured by using a calipers (automation and metrology inc, oh, usa). the value (mm) of the diameter zone then was used as an indicator of the sensitivity of the sarang semut extract against e. faecalis. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) tests were conducted to determine the effects of the sarang semut extract on the development of e. faecalis. the e. faecalis atcc 29 212 bacteria in nutrient broth mc farland 0.5 (equivalent to 1.5 x 108 cfu/ ml) was diluted with serial techniques, and then the number of e. faecalis, 10-4, was diluted in order to be used as a reference to the mic and mbc tests0.1 ml of e. faecalis inoculum was respectively put into 1 ml of the sarang semut extract, the positive controls (fosfomycin and chlorhexidine), and the negative control (physiological saline). each sample was settled for 10 minutes, and then cultured on mha medium under an-aerobic atmosphere for 48 hours at a temperature of 37°c. e. faecalis colonies that grew were used as a reference to the ability of the sarang semut extract in inhibiting and killing e. faecalis with the positive controls and the negative control as references for assessment.21,22 results the concentrations of both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extract bradford protein test using bovine serum albumin as the standard was performed to determine the quantity of both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extracts, so the reactivity or interaction value of both would meet the analysis standard.23,24 both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extracts have a threshold concentration, approaching the bovine serum albumin’s concentration of 500%. discussion the results of the phytochemical test showed that the sarang semut extracts positively contains flavonoids, tannins, saponins, and alkaloids. flavonoids and saponins are known to act as anti-bacteria.15 the other active compounds can also play a role as anti-bacteria and antioxidant, and even saponins in particular have antibacterial properties with a wide spectrum.26 in addition, as shown in figure 1, the sarang semut extracts had active compounds sufficient to be used as anti-bacteria after calibrated with bovine albumin serum (bsa), as done by yesilada,27 using bsa to predict the active compounds of sambucus ebulus l. extract as an anti-bacterial material. in other words, the two samples equally possessed good sensitivity when interacted on the anti-bacterial test. moreover, pessione28 argues that in each test on an interaction between pathogens and anti-pathogens, the concentration of proteins should be measured before, especially proteins on cell wall of pathogens or bacteria using a recommended bradford assay method. for instance, bohle29 used bradford method to measure protein concentration of e. faecalis in order to examine the expression of proteins associated with response of stress. this is in line with schneewind30 explaining that the peptidoglycan of gram-positive bacteria serves as surface organelles to interact with the environment, particularly on the tissue of the host infected, and the concentration of the proteins contained of the cell walls determines the level of interaction with the host cell infected. based on the analysis of pass, furthermore, the sarang semut extract had a value of pa. if a compound 6 results the concentrations of both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extract figure 1. profiles of both the cell wall proteins of e. faecalis (left) and the active compounds of the sarang semut extracts (right). bradford protein test using bovine serum albumin as the standard was performed to determine the quantity of both the cell wall proteins of e. faecalis and the active compounds of the sarang semute extracts, so the reactivity or interaction value of both would meet the analysis standard.23,24 both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extracts have a threshold concentration, approaching the bovine serum albumin’s concentration of 500%. 6 results the concentrations of both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extract figure 1. profiles of both the cell wall proteins of e. faecalis (left) and the active compounds of the sarang semut extracts (right). bradford protein test using bovine serum albumin as the standard was performed to determine the quantity of both the cell wall proteins of e. faecalis and the active compounds of the sarang semute extracts, so the reactivity or interaction value of both would meet the analysis standard.23,24 both the cell wall proteins of e. faecalis and the active compounds of the sarang semut extracts have a threshold concentration, approaching the bovine serum albumin’s concentration of 500%. figure 1. profiles of both the cell wall proteins of e. faecalis (left) and the active compounds of the sarang semut extracts (right). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 178 soraya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 175–180 has a value of pa, greater than 0.7, it means that the compound has a specific biological activity.15 according to lagunin,31 pass and pharma expert methods can be used to evaluate biological activity of a natural product, including marine sponge alkaloids as well as triterpenoids and their derivatives, wherein the methods computationally demonstrate the ability to evaluate multiple targets from the effects of natural products, both additive/ synergetic and results of the potency test of the sarang semut extracts on the sensitivity of e. faecalis 7 results of the potency test of the sarang semut extracts on the sensitivity of e. faecalis figure 2. sensitivity of sarang semut extracts to e. faecalis. it shows that based on the clinical and laboratory standards institute (clsi), the sarang semut extracts at all concentrations had low sensitivity against e. faecalis (≤13mm). it can be said that the extracts can be categorized into resistance to e. faecalis. unlike the sarang semut extract, fosfomysin had a susceptible sensitivity at the concentration of 100% up to 12.5% (≥17), while at the concentrations of 6.25% and 3.125% (14-16 mm), it can be categorized into an intermediate level. at the concentrations of 100% and 75%, on the other hand, chlorhexidine had a potential anti-bacteria of e. faecalis, while at the concentrations of 50% to 3.125%, it can be categorized as an intermediate level.20, 25 figure 2. sensitivity of sarang semut extracts to e. faecalis. it shows that based on the clinical and laboratory standards institute (clsi), the sarang semut extracts at all concentrations had low sensitivity against e. faecalis (≤13mm). it can be said that the extracts can be categorized into resistance to e. faecalis. unlike the sarang semut extract, fosfomysin had a susceptible sensitivity at the concentration of 100% up to 12.5% (≥17), while at the concentrations of 6.25% and 3.125% (14-16 mm), it can be categorized into an intermediate level. at the concentrations of 100% and 75%, on the other hand, chlorhexidine had a potential anti-bacteria of e. faecalis, while at the concentrations of 50% to 3.125%, it can be categorized as an intermediate level.20, 25 results of mic and mbc tests of the sarang semut extracts on the development of e. faecalis 1 figure 3. minimal inhibition concentration value of sarang semut extract to e. faecalis. it shows that the sarang semut extract had a mic value at the concentration of 3.125%, while a mbc value at the concentration of 100%. unlike the sarang semut extract, fosfomysin had a mbc value at all concentrations. meanwhile, chx had a mbc value at the concentration of 100%. similar to the sarang semut extract. however, chx as the positive control had higher mbc value than the sarang semut extract and the negative control. figure 3. minimal inhibition concentration value of sarang semut extracts to e. faecalis. it shows that the sarang semut extracts had a mic value at the concentration of 3.125%, while a mbc value at the concentration of 100%. unlike the sarang semut extracts, fosfomysin had a mbc value at all concentrations. meanwhile, chx had a mbc value at the concentration of 100%. similar to the sarang semut extracts. however, chx as the positive control had higher mbc value than the sarang semut extracts and the negative control. antagonistic. another assumption of this information is that the sarang semut extract tested for its effectiveness against e. faecalis can be classified into synergetic and antagonistic groups, and have properties of adhesion, bio-tolerance, and bio-resistance against bacteria.32 figure 1 shows that e. faecalis, after the levels of their protein was measured using bradford method, had protein expression profiles proportional to bovine serum albumin dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 179179soraya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 175–180 at a concentration of 500%. dominica33 reported that the quantity and quality of the anti-bacterial activities of enterococcus spp. bacteria, such as arabian pea protein (glycate pea protein), are always determined by the protein concentration of the pathogens determined using bradford method with bovine serum albumin as the standard. figure 2 shows that the sarang semut extract had a low sensitivity (≤ 13mm). this value, based on the clinical and laboratory standard institute (clsi), is classified as resistance.22 bacteria can possibly become resistant to drugs since some bacteria are not capable of destroying expressions of β-lactamases produced by pathogens when interacting with drugs or medicinal plant extracts, as a result, the bacterial cell membrane is able to avoid the damage of the bioactive compounds contained in the drugs or medicinal plant extracts.34 besides, the resistance value is also related to the genitive property of gram-positive bacteria, expressing a gene behavior in the form of n-acyl homoserine lactone (ahl) signal when interacting with the environment. the principle of the ahl signal is to prevent interaction with bioactive components of plants as well as pathogenic, symbiotic, and saprophytic bacteria. a number of plant extracts, such as exudates pea (pisum sativum) have properties imitating bacterial ahl signal, consequently, it can affect bacterial adaptation to anti-bacteria.35 in addition, figure 3 shows that the mic value of the sarang semut extract was obtained at the concentration of 3.125%, while the mbc value was at the concentration of 100%. the abilities are related to the role of active flavonoid and tannin compounds to inhibit the growth of e. faecalis.15 similarly, garlic extract can inhibit trypsin-like enzyme and total protease activities of p. gingivalis at concentrations of 92.7% and 94.88%. it indicates that both the sarang semut extract and the garlic extract can inhibit the growth of oral pathogens, both in endodontic and periodontal therapies.36 the mic value of a. nilotica extract ranges from 4.9 to 313 ug/ ml. the mic value of the a. nilotica extract against e. faecalis is 9.75 ug/ ml, while the mbc value is 78 ug/ ml.37 this indicates that e. faecalis atcc 29 212 used as the subjects in this research had good sensitivity to anti-bacteria. another assumption is that the cellular and molecular role of flavonoids and tannins contained in the sarang semut extract is very important to inhibit dna synthesis, cytoplasmic membrane function, and energy metabolism.38 in conclusion, based on mbc and mic assay, the extract of sarang semut has potential effects to adherence growth of e. faecalis, mainly on the highest concentration 100 µg/ml also mic on 3,125 µg/ ml. acknowledgement we would like to express our gratitude to the research laboratory of chemistry department, faculty of mathematics and natural sciences, universitas padjadjaran, bandung for providing sarang semut extract from papua (myrmecodia pendens merr. & perry). we also would like to express our gratitude to basri a. gani, a lecturer of dentistry faculty, universitas syiah kuala, who has improved this article. references 1. stuart ch, schwartz sa, beeson tj, owatz cb. enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. j endod 2006; 32(2): 93-8. 2. wang z, shen y, haapasalo m. effectiveness of endodontic disinfecting solutions against young and old enterococcus faecalis biofilms in dentin canals. j endod 2012; 38(10): 1376-79. 3. saber s, el-hady sa. development of an intracanal mature enterococcus faecalis biofilm and its susceptibility to some antimicrobial intracanal medications; an in vitro study. eur j dent 2012; 6(1): 43-50. 4. narayanan ll, vaishnavi c. endodontic microbiology. j conserv dent 2010; 13(4): 233. 5. sava ig, heikens e, huebner j. pathogenesis and immunity in enterococcal infections. clin microbiol infect 2010; 16(6): 53340. 6. christo j, zilm p, sullivan t, cathro p. efficacy of low concentrations of sodium hypochlorite and low‐powered er, cr: ysgg laser activated irrigation against an enterococcus faecalis biofilm. int endod j 2016; 49(3): 279-86. 7. miller wr, munita jm, arias ca. mechanisms of antibiotic resistance in enterococci. expert rev anti infect ther 2014; 12(10): 1221-36. 8. evans m, davies j, sundqvist g, figdor d. mechanisms involved in the resistance of enterococcus faecalis to calcium hydroxide. int endod j 2002; 35(3): 221-8. 9. arias-moliz mt, ferrer-luque cm, espigares-garcía m, baca p. enterococcus faecalis biofilms eradication by root canal irrigants. j endod 2009; 35(5): 711-4. 10. naber kg, thomas p, fünfstück r. fosfomycin trometamol in patients with renal insufficiency and in the elderly. the international arabic journal of antimicrobial agents 2012; 2(1): 1-10. 11. du t, ma j, yang p, x iong z , lu x, cao y. evaluation of antibacterial effects by atmospheric pressure nonequilibrium plasmas against enterococcus faecalis biofilms in vitro. j endod 2012; 38(4): 545-9. 12. klančnik a, piskernik s, jeršek b, možina ss. evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. j microbiol methods 2010; 81(2): 121-6. 13. bakkali f, averbeck s, averbeck d, idaomar m. biological effects of essential oils–a review. food and chem toxicol 2008; 46(2): 446-75. 14. crisnaningtyas f. pemanfaatan sarang semut (myrmecodia pendens) asal kalimantan selatan sebagai antibakteri. jurnal riset industri hasil hutan 2010;2(2):31-35. 15. apriyanti e, kurnia dsd, dharsono hda, satari mh. flavonoid dari myrmecodia pendans dan aktivitas antibakteri terhadap enterococcus faecalis. unpad library 2015; 1(1): 1-9. 16. ross je, sader hs, schoenfeld zi, paukner s, jones rn. disk diffusion and mic quality control ranges for bc-3205 and bc-3781, two novel pleuromutilin antibiotics. journal of clinical microbiology 2012; 50(10): 3361-4. 17. m a r a k i s, sa mon i s g, r a fa i l id i s pi , vou lou m a nou e k , mavromanolakis e, falagas me. susceptibility of urinary tract bacteria to fosfomycin. antimicrob agents chemother 2009; 53(10): 4508-10. 18. gani ba, chismirina s, hayati z, bachtiar bm, wibawan iwt. the ability of igy to recognize surface proteins of streptococcus mutans. dental journal (majalah kedokteran gigi) 2009; 42(4): 189-93. 19. sasidharan s, chen y, saravanan d, sundram k, latha ly. extraction, isolation and characterization of bioactive compounds from plants’ extracts. afr j tradit complement altern med 2011; 8(1): 1-10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 180 soraya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 175–180 20. wolfensberger a, sax h, weber r, zbinden r, kuster sp, hombach m. change of antibiotic susceptibility testing guidelines from clsi to eucast: influence on cumulative hospital antibiograms. plos one 2013; 8(11): e79130. 21. jorgensen jh, turnidge jd. susceptibility test methods: dilution and disk diffusion methods. in: manual of clinical microbiology. eleventh edition. washington dc: asm press; 2015. p. 1253-73. 22. mulyani y, bachtiar e, kurnia mu. peranan senyawa metabolik sekunder tumbuhan mangrove terhadap infeksi bakteri aeromonas hydrophila pada ikan mas (cyprinus carpio l). jurnal akuatika 2013; iv(1): 1-9. 23. cath ro p, mcca r thy p, hoffma nn p, zilm p. isolation a nd identification of enterococcus faecalis membrane proteins using membrane shaving, 1d sds/page, and mass spectrometry. febs open bio 2016; 6(6): 586-93. 24. riadh h, imen f, abdelmajid z, sinda f. detection and extraction of anti-listeral compounds from calligonum comosum, a medical plant from arid regions of tunisia. afr j tradit complement altern med 2011; 8(3): 322–7. 25. gibson gw, kreuser sc, riley jm, rosebury-smith ws, courtney cl, juneau pl, hollembaek jm, zhu t, huband md, brammer dw, brieland jk, sulavik mc. development of a mouse model of induced staphylococcus aureus infective endocarditis. comp med 2007; 57(6):563-9. 26. dougnon tv, klotoé jr, sègbo j, atègbo jm, edorh ap, gbaguidi f, hounkpatin as, dandjesso c, fah l, fanou b, dramane k, loko f. evaluation of the phytochemical and hemostatic potential of jatropha multifida sap. afr j pharmacy and pharmacol 2012; 6(26): 1943-48. 27. yesilada e, gurbuz i. evaluation of the antiulcerogenic activity profile of a flavonol diglucoside from equisetum palustre l. j ethnopharmacol 2010; 131(1): 17-21. 28. pessione a, lamberti c, cocolin l, campolongo s, grunau a, giubergia s, eberl l, riedel k, pessione e. different protein expression profiles in cheese and clinical isolates of enterococcus faecalis revealed by proteomic analysis. proteomics 2012; 12(3): 431-47. 29. bøhle la, færgestad em, kent ev, steinmoen h, nes if, eijsink vgh, mathiesen g. identification of proteins related to the stress response in enterococcus faecalis v583 caused by bovine bile. proteome sci 2010; 8(1): 37. 30. schneewind o, missiakas dm. protein secretion and surface display in gram-positive bacteria. phil trans r soc b 2012; 367(1592): 1123-39. 31. lagunin a, filimonov d, poroikov v. multi-targeted natural products evaluation based on biological activity prediction with pass. curr pharm des 2010; 16(15):1703-17. 32. abouda z, zerdani i, kalalou i, faid m, ahami m. the antibacterial activity of moroccan bee bread and bee-pollen (fresh and dried) against pathogenic bacteria. research journal of microbiology 2011; 6(4): 376-84. 33. świątecka d, narbad a, ridgway kp, kostyra h. the study on the impact of glycated pea proteins on human intestinal bacteria. int j food microbiol 2011; 145(1): 267-72. 34. aqil f, khan msa, owais m, ahmad i. effect of certain bioactive plant extracts on clinical isolates of β‐lactamase producing methicillin resistant staphylococcus aureus. j basic microbiol 2005; 45(2): 106-14. 35. teplitski m, robinson jb, bauer wd. plants secrete substances that mimic bacterial n-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. mol plant microbe interact 2000; 13(6): 637-48. 36. bakri im, douglas cw. inhibitory effect of garlic extract on oral bacteria. arch oral biol 2005; 50(7): 645-51. 37. khan r, islam b, akram m, shakil s, ahmad a, ali sm, siddiqui m, khan au. antimicrobial activity of five herbal extracts against multi drug resistant (mdr) strains of bacteria and fungus of clinical origin. molecules 2009; 14(2): 586-97. 38. cushnie tt, lamb aj. antimicrobial activity of flavonoids. int j antimicrob agents 2005; 26(5): 343-56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p175-180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p175-180 55 volume 46 number 2 june 2013 research report enamel defect of primary dentition in sga children in relation to onset time of intrauterine growth disturbance willyanti soewondo sjarif department of pediatrics dentistry faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: prenatal disturbances disturb the development of organs resulting in small for gestational age (sga) babies and also causes enamel defects in primary teeth. there are disturbances occur in the beginning of pregnancy causing symmetrical sga, and asymmetrical type of sga, where the disturbances occur late in pregnancy. purpose: this research was to determined differences in severity of enamel defect of primary dentition in small for gestational age children based on the time of intrauterine growth restriction. methods: this was a clinical epidemiological cohort study. the ponderal index was used to determine sga type. the subjects were 129 sga children aged 9-42 months, 82 with asymmetrical sga and 47 with symmetrical sga. two hundred normal birth weight children were the control group. intra-oral examinations to determine enamel defect used the fdi modification of the developmental defect of enamel score at 3 months intervals. statistical t-tests were used to test the difference in severity of enamel defect, and chisquare to find out the difference of relative risk ratio (rrr). results: the results showed that the enamel defect scores of symmetrical sga were significantly higher than those with asymmetrical sga. rrr for severe defect was also significantly higher in symmetrical type for anterior and canines. conclusion: the study suggested that the severity of enamel defect for infants with symmetrical sga was higher than those with asymmetrical sga, indicating that the severity of the defect occurs in the beginning of pregnancy is more severe than in the late pregnancy. key words: enamel defect, small for gestational age, symmetrical, asymmetrical, intra-uterine growth restriction abstrak latar belakang: adanya gangguan prenatal mengganggu perkembangan organ, mengakibatkan terjadinya bayi lahir dengan kecil masa kehamilan (kmk) dan defek email pada gigi sulung. terdapat 2 tipe kmk yaitu tipe simetri; gangguan terjadi pada awal kehamilan; dimana lingkar kepala, berat dan panjang lahir lebih rendah dari normal. tipe asimetri dimana gangguan terjadi saat kehamilan lanjut: panjang dan berat badan lahir lebih rendah dari normal. tujuan: penelitian ini bertujuan meneliti perbedaan keparahan defek email gigi sulung pada anak kmk berdasarkan saat terjadinya gangguan hambatan pertumbuhan intrauterin. metode: jenis penelitian adalah epidemiologi dengan studi kohort. ponderal indeks digunakan untuk menentukan tipe kmk subjek terdiri dari anak kmk usia 9-42 bulan, 82 tipe asimetri dan 47 tipe simetri, 200 anak dengan berat lahir normal sebagai kontrol. pemeriksaan intra oral dilakukan untuk menentukan skor defek email yaitu dengan menggunakan skoring modifikasi dde indek dari fdi. subjek di teliti dengan interval 3 bulan, t-test digunakan untuk menentukan perbedaan defek email pada kmk simetri dan asimetri sedangkan chi square menentukan perbedaan rrr (rasio resiko relatif). hasil: hasil menunjukan bahwa skor defek enamel pada kmk simeteri lebih tinggi secara signifikan dibanding pada kmk asimetri dan rrr pada defek berat lebih tinggi pada kmk simetri 56 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 55–60 pada gigi anterior dan kaninus. simpulan: penelitian ini menunjukkan bahwa defek email lebih parah bila terjadi pada awal masa kehamilan (tipe simetri) dibanding bila terjadi pada saat kehamilan lanjut (tipe asimetri). kata kunci: defek email, kecil masa kehamilan, simetri, asimetri, hambatan pertumbuhan intrauterin correspondence: willyanti soewondo sjarif, c/o: kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan 1 bandung 40132, indonesia e-mail: willyantir@yahoo.com introduction small for gestational age (sga) describes a newborn infant with birth weight less than normal for its gestational age, to the extent of being under the 10th percentile of the intrauterine growth curve. these kinds of deliveries might. these kinds of deliveries might cause many problems in the future including morbidity and mortality. in general, sga neonates are in poor conditionare in poor condition and are at high risk for both their short term and long term health. intra uterine growth restriction (iugr) occurring in intra uterine growth restriction (iugr) occurring in) occurring inoccurring in the prenatal period can have an effect on foetal developmentcan have an effect on foetal development causing anomaly to several organs..1-8 the incidence of sga in the usa is about 3-10% of all deliveries, in cipto mangunkusumo hospital jakarta (1999) 4.42% and in hasan sadikin general hospital bandung it was 7.6-10% of all deliveries in 2005.9-10 the prenatal period is a criticalthe prenatal period is a critical time especially in forming primary dentition..11-18 overall, the incidence of enamel defect in primary dentition isdefect in primary dentition isin primary dentition is 22-33%. there are two types of sga, based on the onset of the restriction of inter-uterine growth i.e. symmetrical sga: which occur in the first trimester of pregnancy (embryonal phase) and a symmetrical sga: which occur in the second and third trimester of pregnancy (foetal period).1-7 symmetrical sga infants have disturbances in both brain and physical growth as shown by delayed growth of head circumference, body length and body weight, but the whole body is in good proportion. asymmetrical sga infants also have smaller body weight and length but relatively normal head circumference.1-6 determination of sga type therefore requires an accurate record of gestational age, birth length and head circumference. in general the clinical manifestation is worse in symmetrical sga infants than in asymmetrical ones. avery and chicago reported that anomalies of dentition which occurred during the embryonal period (symmetrical sga infants) is worse than in foetal period (asymmetrical sga infants).1-8 overall, 71% of enamel defect in primary dentition are caused by prenatal systemic factors, the effect of developmental disturbances occurring at the beginning of dental forming and calcification during the first, second or third trimester of pregnancy, and manifesting as hypoplasia or hypocalcification. the enamel defect of primary dentition becomes a problem because it is unregenerate, so the effect is permanent.11-18 enamel defect might also worsen enamel quality and cause easier accumulation of plaque that trigger caries occurence. untreated caries can then cause abscesses, resulting in premature loss of primary dentition. one of the predispositions to caries is the anomaly of enamel structure that is involved with prenatal developmental growth disturbances. the birth condition known as sga is caused by intra uterine growth restriction (iugr).1-8 in indonesia and other developing countries iugr is still a main health problem,with the highest mortality rate the cause of iugr might be placental, foetal and maternal characteristics such as the age of mother older than 35 years old or young mother, short and thin stature, or low increase of body weight during the third trimester of pregnancy. it might also caused by vascular disease during pregnancy such as hypertension and preeclampsia, or caused by severe infectious disease, lupus erythematosus, antiphospholipid syndrome, anemia, malignancy, nuliparity, smoking, alcohol, cocaine, and low socioeconomic status.1-5 family with low socio-economic conditions can result in bad nutrition in pregnant mothers that affects inter-uterine health. klaus and fanaroff, found that many poor pregnant women had bad nutritional status and gave birth to babies with low birth weight (less than 2500 g), with more than 60% being sga.1-5 the other factors are foetal characteristics, such as type of pregnancy (single/ multiple pregnancy), congenital anomalies (genetic and chromosomal), and placental factors (placental anomaly, infark, tumor and placentitis).1-8 several studies on enamel defect suggest that genetic and environmental factors might be implicated.11-17 even though a genetic factor could be the cause in some cases of the enamel defect, prevention is not an option, but early detection and intervention in all cases might facilitate managing the anomaly and minimizing its severity and subsequent effects.18-19 the goal of the study was to determine the differences in severity of enamel defect in the primary dentition of defect in the primary dentition ofin the primary dentition of sga infants, based on the onset of intrauterine growth disturbance. it is important to predict the severity of the it is important to predict the severity of the defect, to determine prognosis and treatment planning. materials and methods the subjects was of this study were 129 sga infants, thes was of this study were 129 sga infants, thewas of this study were 129 sga infants, the129 sga infants, the sga infants, the age range 9 to 42 months, born in hasan sadikin general hospital, universitas padjadjaran bandung indonesia. as bandung indonesia. as group consisted of 200 infants with normal birth weight consisted of 200 infants with normal birth weight (appropriate for gestational ageaga) in range of age 4 to) in range of age 4 to 42 months and caries free was used as control group. free was used as control group. the different of the sga and aga youngest subjectsyoungest subjects was in accordance with a study conducted by willyanti that 57sjarif: enamel defect of primary dentition in sga children sga children had delayed eruption of teeth compared toto aga children. children.20 inclusion criteria also required completealso required complete data of birth; for mothers and children, and an exclusion of birth; for mothers and children, and an exclusionfor mothers and children, and an exclusion criteria was infants with general anomalies (such as genetic (such as genetic(such as genetic anomaly). this was a clinical epidemiological ambispectiveical ambispective cohort study, with given sample sizes. after obtaining given sample sizes. after obtainingobtaining informed parental consent, and completing physical examination of sga (and aga/control) patients, the (and aga/control) patients, the(and aga/control) patients, thethe enamel anomaly or enamel defect and presence of dental caries were determined. scoring for hypoplasia and hypocalcification using for hypoplasia and hypocalcification usingand hypocalcification using modified developmental defect of enamel (dde) of federation dental internationale (fdi)..21 the subjects were examined three times, at one month intervals, to determineat one month intervals, to determine whether there were enamel defects on new teeth. the development of dentition was monitored in case there wasof dentition was monitored in case there waswas monitored in case there was any defect on the next erupted tooth. subjects with only subjects with only 1 or 2 teeth erupted were monitored, and examination on, and examination on subyects with primary dentition fully erupted.fully erupted.. small for gestational age (sga) is defined if the baby a is defined if the baby a defined if the baby a baby was born with birth weight under the 10th percentile of lubchenco curve of intrauterine growth and development of weight for gestational age. type of sga was determined was determined using the ponderal index. length birth 100 xweight birth = index ponderal type of sga was defined as symmetric if the ponderal index scores was 20 to 25 and asymmetric if the ponderal index scores was either less than 20 or more than 25. severity of enamel defect of primary dentition was identified as extent of hypoplasia/hypocalcification. hypoplasia was defined when there was pit, fissures, or cavity in the surface of the enamel while hypocalcification was defined when the teeth were unglistening and not transparent.11-19 dental examination was done using a mouth mirror, explorer, and probe with paper lighting. the teeth surface were cleaned, dried using a cotton role, then examined to record any defects on primary dentition. enamel hypoplasia/hypocalsification (ehp) score 1 (normal) was determined when the enamel transparent; score 2 (opacity) when the enamel opaque/ white, not transparent, or yellowish/ brownish; score 3 when there were pits and fissures on some of teeth surface; score 4 when there was un-neat vertical fissures; score 5 when there were exact horizontal fissures; and score 6 when most of the enamel missing or teeth were smaller. scoring used the fdi modification of the developmental defect of enamel (dde) for enamel hypoplasia/ hypocalcification (ehp) and an index of enamel defect severity (eds) was determined as follows;21 index enamel defect severity (eds) was determined using the fdi modification of developmental defect of enamel (dde) as follows: enamel defect score (eds) = ehp x total dentition with defect x 10 total teeth at risk the degree of severity of enamel defects of primary dentition was then classified relative to a statistical cut-off point of a median score of 12 determined from a kruskal wallis test (normal 0; mild/light 1-12; and severe >12). difference of enamel defect severity of primary dentition based on onset of intrauterine developmental growth disturbance was compared using a t-test. chi-square was used to determined differences in incidence of enamel defect based by type of sga and to differentiate enamel defect risk rates based on stage of intrauterine developmental growth disturbance. risk ratios of symmetric against asymmetric sga based on severity of defect, on anterior, canine and posterior teeth were separately tested by t-test. results small for gestational age (sga) infants had more severe enamel defect of primary dentition (eds mean: 12.27) than aga control infants (eds mean: 0.39) (table (table 1). type of sga related to incidence of enamel defect. enamel defect affects 100% of infants with symmetric sga and in asymmetric sga still high but less at 75.6% (table 2a), while the mean score on symmetric sga infants was significantly higher (15.29) than for asymmetric sga infants (10.38). it indicated that the enamel defect of primary dentition is more severe in the symmetric sga compared with the asymmetric sga condition (table 2a). symmetric sga infants were at significantly higher risk of both light and severe enamel defects on their anterior teeth than asymmetric sga infants -3.74 times at risk of light enamel defect and 7.11 times at risk of having severe defect (table 3). symmetric sga infants were atat significantly higher risk of both light and severe enamel defects on the caninus teeth than asymmetric sga infantscaninus teeth than asymmetric sga infantsthan asymmetric sga infants -2.19 times at risk to have light defects and 2.96 times at2.19 times at risk to have light defects and 2.96 times ats and 2.96 times at2.96 times at risk to have severe defects (table 4). symmetric sga. symmetric sga symmetric sga infants were at higher risk of both light and severe enamel defects on the posterior teeth than asymmetric sga infantsposterior teeth than asymmetric sga infants table 1. eds score means of sga and aga infantseds score means of sga and aga infantsscore means of sga and aga infantse means of sga and aga infants subject eds mean score 95% conf. interval sga 12.27 10.89 13.72 aga/control 0.39 0.295 0.475 t= 26.10, p < 0.001 table 2a. incidence of enamel defect based on type of sga type of subject enamel defect total defect no defect sga symmetric 47 (100%) 47 (100%) sga asymmetric 62 (75.6%) 20 (24.4%) 82 (100%) total 109 (84.49%) 20 (15.51%) 129 58 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 55–60 (although this was less significantly so for light defects) -1.42 times at risk to have light defects and 1.93 times at42 times at risk to have light defects and 1.93 times atand 1.93 times at risk to have severe enamel defects (table 5). discussion the study showed that enamel defect score (eds),enamel defect score (eds), based on the fdi modification of the developmental defect of enamel (dde) score for enamel hypoplasia/ hypocalcification (ehp), was higher in sga compared to aga infants, indicating that the enamel defect inprimary dentition is more severe for sga than for normal aga infants. this is because the iugr causing sga in infantsthe iugr causing sga in infants occursin the early foetal prenatal period, a critical period of primary dentition development..8 iugr at that stage causes disturbances/anomalies of the organs, including primary dentition..22,23 the results showed that enamelthe results showed that enamel defect of primary dentition in sga might affect several or even all types of teeth, bilaterally an interaction oftypes of teeth, bilaterally an interaction of genetic and environmental factors might effect the growth and development of dentition, and local environmental factors may have effect the growth of teeth environmental. may have effect the growth of teeth environmental.may have effect the growth of teeth environmental.e growth of teeth environmental.. stewart and mcdonald state that systemic factors might be the whole cause of enamel defect.16,24 our study has also shown that the enamel defect anomalies were more severe in sga infants. also thewere more severe in sga infants. also the. also the results indicate variation in severity of enamel defect according to the type of sga, in eds/dde score waswas higher (more severe defect) in the primary dentition of(more severe defect) in the primary dentition of defect) in the primary dentition ofdefect) in the primary dentition of infants with symmetrical type of sga. it seems that this is because for them the anomaly occurs earlier, i.e. during the first trimester or embryonic phase critical for dentition, or embryonic phase critical for dentition,ic phase critical for dentition, critical for dentition,critical for dentition, and for the asymmetric type it occurs later at the end of the second or third trimester or foetal phase. or foetal phase.. table 2b. eds mean score based on type of sga type of subject n eds mean std. err. conf. interval 95% asymmetric sga 82 10.38 0.79 8.86 11.98 symmetric sga 47 15.29 1.29 12.75 17.92 total 129 12.32 0.72 10.90 13.73 difference -4.91 1.42 -7.73 -2.09 note: difference = mean (asymmetric) – mean (symmetric); t=3.4457; p<0.001 table 3. anterior teeth relative risk ratio of symmetric against asymmetric sga based on light and severe defectanterior teeth relative risk ratio of symmetric against asymmetric sga based on light and severe defect relative risk ratio of symmetric against asymmetric sga based on light and severe defectrelative risk ratio of symmetric against asymmetric sga based on light and severe defectand severe defect type of sga total rrr 95% ci x2 p exposed unexposed light symmetrical 30 59 89 3.74 0.98-14.35 5.53 0.019 asymmetrical 2 17 19 total 32 76 108 severe symmetrical 21 10 31 7.11 1.86-27.18 17.20 0.001 asymmetrical 2 19 21 total 23 29 52 table 4. caninus teeth relative risk ratio of symmetric against asymmetric sga based on lightand severe defectcaninus teeth relative risk ratio of symmetric against asymmetric sga based on lightand severe defectand severe defect type of sga total rrr 95% ci x2 p exposed unexposed light symmetrical 29 18 47 2.19 1.21-3.99 8.60 0.003 asymmetrical 9 23 32 total 38 41 79 severe symmetrical 5 1 6 2.96 1.53-5.73 6.62 0.010 asymmetrical 9 23 32 total 14 24 38 59sjarif: enamel defect of primary dentition in sga children it was also seen in this study there was no hypoplasiainthis study there was no hypoplasiainwas no hypoplasiain the asymmetrical type, while in the symmetrical type there were both hypoplasia and hypocalcification.were both hypoplasia and hypocalcification. both hypoplasia and hypocalcification. hypocalcification in the asymmetrical type is a milder anomaly of the structure compared with hypoplasia. it is it isit is a disturbance sof enamel matrix calcification that occurssof enamel matrix calcification that occursf enamel matrix calcification that occursrix calcification that occurss after the 16th week of pregnancy. hypoplasia is an anomaly caused by disturbance of matrix forming by ameloblast-f matrix forming by ameloblast disturbance of matrix synthesis or the resorbtion that causes disturbance on the next mineralization. along lasting disturbance might result in incomplete enamel or no enamel forming at all..25-26 accordingly the asymmetrical typeaccordingly the asymmetrical type shows better clinical signs than symmetrical type..1-4 as a result of iugr during embryonal phase or foetal phase that might disturb dentition development, sga infants may have hypoplasia and hypocalcification. the manifestation of the anomaly is based on the time of disturbance and the organs’ development activities..11,14-17,24-26 in our study, the symmetrical type sga had more severe enamel defect than asymmetrical type because the defect occured during the embryonicthe embryonic phasecritical phase for organogenesis.critical phase for organogenesis.29,30 some of the sga (24.4%) had no anomalies because the calcificationecause the calcification process had completed when the disturbance/anomaly occured (table 2a). (table 2a). the results showed that the incidence of enamel defectresults showed that the incidence of enamel defectdefect of primary dentition in symmetrical sga was higher than in asymmetrical sga and the defect was more severe too.and the defect was more severe too.was more severe too. this is in accordance with statements in previous studies that anomalies which occur during the embryonic period (1st trimester) will be more severe than those occurring later during the foetal period (middle of 2 the foetal period (middle of 2the foetal period (middle of 2nd and 3rd trimester) because the foetus is in highly sensitive condition-cell proliferation and highly active with the cell number increaseand highly active with the cell number increase with the cell number increasethe cell number increase more than the cell size. disturbance during the embryonicdisturbance during the embryonic period may therefore decrease the amount of cells. during the foetal period (2nd and 3rd trimester) there is a decrease of the sensitivity from iugr disturbance – this is in accordance with taeusch who reports that symmetrical sga infants had more severe anomalies than infants with asymmetrical sga..27-29 table 5. posterior teeth relative risk ratio of symmetric against asymmetric sga based on the light and severe defectposterior teeth relative risk ratio of symmetric against asymmetric sga based on the light and severe defectand severe defect type of sga total rrr 95% ci x2 p exposed unexposed light symmetric 16 14 30 1.42 0.81 – 1,91 0.87 0.351 asymmetric 28 37 65 total 44 51 95 severe symmetric 5 1 6 1.93 1.23-3.05 3.58 0.058 asymmetric 28 37 65 total 33 38 71 figure 1. intrauterine deivelopment curve.1,5,6 small for gestational age appropriate for gestational age large for gestational age 60 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 55–60 enamel is the hardest tissue on which calcificationhich calcification might occur, consisting of crystals with 96% inorganic material, and only 4% water and organic material. enamel is formed by an extracellular matrix from the synthetic and protein secretion by ameloblasts. enamel, which is only formed once, is different to cartilage or bone and will not regenerate and resorb..8,25,30 enamel defect might occur in the amelogenesis period, i.e. matrixin the amelogenesis period, i.e. matrix aposition process and mineralization since the beginning of the 4th month of pregnancy. aposition is the end stage of morphodifferentiation. matrix forming consists of secretion and maturation, enamel matrix starts as an occlusal part of dentition. the first calcification begins on the 4th month prenatal up to the antenatal period. during up to the antenatal period. during amelogenesis the ameloblast is highly sensitive to whatto what might disturb its activities and any disturbance might result in enamel defect in the form of hypoplasia and alsodefect in the form of hypoplasia and also hypocalcification-hypoplasia being shortage of enamel-hypoplasia being shortage of enamel matrix, and hypocalcification being when the enamel matrix is sufficient but there is shortage of calcification. as mentioned, these disturbances might be caused by genetic or environmental factors during the perinatal or postnatal period..11-17,24 prenatal environmental factorss that might be the cause of enamel defect of the teeth aredefect of the teeth are maternal factors such as severe infectious disease at the beginning of pregnancy, chronic infection, long lasting malnutrition, premature and low birth weight. mother’s diseases during pregnancy such as maternal diabetes, hypertension, maternal alcoholism, torch, high fever at the beginning of pregnancy, might also cause hypoplasia,16 and might also result in sga. the possibility (relative risk ratio: rrr) of(relative risk ratio: rrr) of severe defect (>12) of the anterior and caninus teeth was significantly higher in symmetrical compared with asymmetrical sga (tables 3, 4, 5). the severe defect on. the severe defect on posterior teeth was less significant in symmetrical sgawas less significant in symmetrical sgaless significant in symmetrical sga because the posterior teeth, especially second primary molar second primary molar are the last teeth formed.h formed. previous studies had not looked at disturbances in prenatal growth.12-3,18-9,21 it was examined in this study and concluded that the severity of enamel structure anomaly of primary dentition was higher in infants with sga, and also, for those with symmetrical sga it is more severe than for those with asymmetrical sga. it means that the enamel defect/anomalies that occur in the beginning of pregnancy (embryonic phase) are more severe than those that occur later (during the foetal phase). this information will be important for assisting predictive prognosis and treatment planning. it was concluded that the severity of enamel defect which occurs in the beginning of pregnancy was more severe than in late pregnancy. references 1. klaus mh, fanaroff aa. care of the high risk neonate. 4th ed. philadelphia: wb saunders co; 2003. p. 69-83. 2. stoll bj, kliegman rm. the newborn infant. in: nelson, behrman re, kliegman rm, jenson hb, eds. textbook of pediatrics. 17th ed. philadelphia: wb saunders co; 2004. p. 524-31 3. gomella tl. assessment of gestational age. in: gomella tl, cunningham d, eyal fg, zenk ke, eds. neonatology, management, procedures, on-call problems, diseases, and drugs. 5th ed. appleton & lange; 2004. p. 21-8, 469. 4. anderson ms, hay ww. intrauterine growth restriction and the small for gestational age infant. in: avery gb, fletcher ma, mac donald mg, eds. neonatology, pathophysiology and management of the newborn. 5th ed. philadelphia. 1999. p. 411-44. 5. hay ww, thureen p, anderson ms. intrauterine growth restriction. neo reviews 2001; 2: 119–28. 6. battaglia fc, lubchenco lo. a practical classification of newborn infants by weight and gestational age. j pediatr 1967; 71(2): 15963. 7. shalitin s, lebenthal y, phillip m. children bor n small for gestational age: growth patterns, growth hormone treatment, and long-term sequelae. isr med assoc j 2003; 5(12): 877-82. 8. avery jk, chiego dj. essentials of oral histology and embryology: a clinical approach. 3rd ed. st louis: mosby co inc; 2006. p. 6375. 9. hamilton be, martin ja, ventura sj, sutton pd, menacker f. births preliminary data for 2004. natl vital stat rep 2005; 54(8): 1-17. 10. department of perinatology, hasan sadikin general hospital: annual report perinatology, 2005. 11. laskaris g. color atlas of oral diseases in children and adolescents. stuttgart: thieme; 2000. p. 20-3. 12. seow wk. effect of preterm birth on oral growth and development. aust dent j 1997; 42(2): 85-91. 13. seow wk, humphrys c, tudehope di. increased prevalence of developmental dental defects in low birth weight, prematurely born children: a controlled study. pediat dent 1987; 9(3): 221-5. 14. pinkham jr. pediatric dentistry: infancy through adolescence. 3rd ed. philadelphia: wb saunders co; 2005. p. 61-4. 15. mathewson rj, promosch re. fundamentals of pediatric dentistry. 3rd ed. chicago: quintessence publishing co. inc; 1995. p. 78-80. 16. stewart re, witkop cj, bixler d. the dentition. in: stewart re, barber tk, troutman kc, wei shy, eds. pediatric dentistry, scientific foundation and clinical practice. st. louis: the cv mosby co; 1982. p. 87-94. 17. welburry rr. pediatric dentistry. 2nd ed. oxford university; 2001. p. 294, 394-5. 18. aine l, backström mc, mäki r, kuusela al, koivisto am, ikonen rs, mäki m. enamel defects in primary and permanent teeth of children born prematurely. j oral pathol med 2000; 29(8): 403-9. 19. eastman dl. dental outcomes of preterm infants. wb saunders: nbin 2003; 3(3):93-8. 20. sjarif willyanti, oewen r. erupsi gigi sulung pada anak dengan kecil masa kehamilan. maj. ked. gigi (dent j) 2005; edisi khusus pertemuan ilmiah nasional kedokteran gigi anak i: 14-17. 21. agarwal kn, narula s, faridi mm, kalra n. deciduous dentition and enamel defects. indian pediatr 2003; 40(2): 124-9. 22. magnusson bo. pedodontic: a systemic approach. munksgaard: pj schmidt vojens; 1981. p. 78-95. 23. demirjian a. dentition in human growth. 15th ed. london: falknerr & tanner; 1978. p. 324-9. 24. mcdonald re, avery dr. dentistry for the child and adolescent. 9th ed. st. louis: cv mosby year book inc; 2011. p. 98-9. 25. simmer jp, hu jc. dental enamel formation and its impact on clinical dentistry. j dent educ 2001; 65(9): 896-905. 26. brauer. dentistry for children. 4th ed. new york: mcgrow hill book co inc; 1959. p. 43-55. 27. fisk nm, smith rp. fetal growth restriction; small for gestational age. in: chamberlain g, steer p, eds. turnbull’s obstetrics. 3rd ed. london: churchill livingstone; 2001. p. 197-209. 28. taeusch hw, ballard ra, gleason ca. avery’s diseases of the newborn. 8th ed. philadelphia: elsevier saunders; 2005. 29. cunningham fg, leveno kj, bloom sl, hauth jc, gilstrap l, wenstrom kd. williams obstetrics. 22nd ed. new york: mcgraw hill; 2005. p. 744-64. 30. balogh mb, fehrenbach mj. dental embryology, histology, and anatomy. 2nd ed. st. louis: elsevier saunders; 2006. p. 63079. 77 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 7–12 original article the effect of an 8% cocoa bean extract gel on the healing of alveolar osteitis following tooth extraction in wistar rats isnandar1, olivia avriyanti hanafiah1, muhammad fauzan lubis2, lokot donna lubis3, adzimatinur pratiwi4, yeheskiel satria yoga erlangga4 1department of oral and maxillofacial surgery, faculty of dentistry, universitas sumatera utara, medan, indonesia 2department of pharmaceutical biology, faculty of pharmacy, universitas sumatera utara, medan, indonesia 3department of histology, faculty of medicine, universitas sumatera utara, medan, indonesia 4student of the faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: alveolar osteitis is a well-known complication that occurs following a tooth extraction when the clot within the socket breaks down too early, causing increased localised inflammation and extreme pain. alveolar osteitis delays the wound healing process of the socket. the polyphenols in the cocoa bean (theobroma cacao l.) can stimulate the wound healing process. purpose: the aim of this study was to analyse the effect of an 8% cocoa bean extract gel on the healing of alveolar osteitis following a tooth extraction. methods: this study is an in vivo experiment with a posttest-only control group design. thirty-six male wistar rats were divided into three groups: a negative control, positive control and an 8% cocoa bean extract gel. a tooth extraction was performed on the mandibular incisor, and alveolar osteitis was induced by the application of adrenaline using a paper point on the socket. on the 3rd, 7th and 14th days, the clinical wound size of the extraction socket was measured, and the rats were sacrificed to observe the number of macrophages, fibroblasts and osteoblasts microscopically. a two-way analysis of variance test and post hoc least significant difference test were used to analyse the data (p < 0.05). results: the data analysis showed a significant difference in the clinical wound size of the extraction socket and the number of macrophages, fibroblasts and osteoblasts between the 8% cocoa bean extract gel and the control groups (p = 0.000). conclusion: an 8% cocoa bean extract gel stimulates the healing of alveolar osteitis following tooth extraction in wistar rats. keywords: alveolar osteitis; cocoa bean; socket healing; tooth extraction correspondence: isnandar, department of oral and maxillofacial surgery, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: isnandar@usu.ac.id introduction one of the most common procedures in oral and maxillofacial surgery is tooth extraction. a tooth extraction will cause an injury (or hole) called a socket. the procedure is considered successful if it is accompanied by an optimal wound healing process of the socket without any complications.1 alveolar osteitis is one of the most common complications following a tooth extraction. the prevalence of alveolar osteitis ranges from 1 to 5% for a routine extraction, but it can be as high as 40% following an extraction of the mandibular third molar.2,3 alveolar osteitis is a postoperative complication characterised by delayed wound healing of the extraction socket. alveolar osteitis causes significant pain that develops on the third or fourth day following a tooth extraction. on clinical examination, the socket appears empty with a partial or complete loss of a blood clot and exposed alveolar bone.2,3 the aetiology of alveolar osteitis is an early fibrinolysis in the extraction socket. the interaction between the excessive trauma during a tooth extraction and a bacterial invasion produce plasmin, which leads to a lysis of the blood clot. this condition delays the healing process due to the proliferation of the cells occurs from the circular gingival mucosa, which takes longer than a normal blood clot to form.1–3 the treatment of alveolar osteitis is performed by the placement of a local dressing, but the dressing can act dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 mailto:isnandar@usu.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 8 as a foreign material that can prolong the wound healing process.2,4 an alternative local dressing is required that is low in cost and has minimal side effects. presently, the use of an herbal dressing as an alternative is highly recommended.4,5 cocoa (theobroma cacao l.) is a type of plant that is known to have many health benefits. in indonesia, the cocoa bean is widely used as a food ingredient and in traditional medicine. cocoa beans contain polyphenols that have antioxidant, anti-inflammatory and antibacterial activities that have the ability to stimulate the wound healing process. the polyphenols in cocoa beans promote the activity and proliferation of cells through affecting growth factor receptors, which accelerates the wound healing process.6 according to a previous study by kurniawati et al.,7 an 8% cocoa bean extract gel can reduce the number of neutrophil cells in the healing process of tooth extraction wounds in wistar rats. based on this understanding, cocoa beans have much potential for use as a medicinal plant, but the effect of a cocoa bean extract on the healing process of alveolar osteitis is unknown. thus, the purpose of this study is to evaluate the effect of an 8% cocoa bean extract gel on the healing process of alveolar osteitis through a clinical and histological examination. materials and methods this study received ethical approval from the animal research ethics committees of the faculty of mathematics and natural sciences, universitas sumatera utara (0390/ keph-fmipa/2021). this study is an in vivo experiment with a posttest-only control group design. fresh cocoa beans were obtained from the pamah area, deli tua district, north sumatera, indonesia. the cocoa beans were dried and ground into a powder and then macerated by soaking them in 70% ethanol for 24 hours. the cocoa bean extract was filtered and evaporated to obtain a concentrated cocoa bean extract. the gel base was made by dissolving 3 g of carboxyl methyl cellulose sodium (cmc-na) powder into 100 ml of warm distilled water. to make the 8% cocoa bean extract gel, 8 g of cocoa bean extract were added homogeneously to the prepared gel base. the preparation of the experimental animals was carried out by acclimatising the rats for one week. thirtysix healthy wistar rats aged 2–3 months with weights of 200–250 g were divided into 3 groups. the negative control group was given 3% cmc-na, the positive control group was given aloclair® (alliance pharma, uk) gel and the experimental group was given the 8% cocoa bean extract gel. the rats were anesthetised intraperitoneally with 0.1 ml/100gbw ketamine (pantex holland, netherlands). then, the mandibular incisor was extracted using a needle holder (onemed health care, indonesia). following complete extraction, alveolar osteitis was induced by applying a 1:1000 adrenaline (ethica pharmaceutical, indonesia) solution using a paper point (gapadent, vietnam) for one minute in the extraction socket, then the socket was left for three days. following the induction of alveolar osteitis in the rats, 0.1 ml of the treatment was injected into the extraction socket using a 1 ml syringe (onemed health care, indonesia) twice a day (morning and evening) for 14 days. on the 3rd, 7th and 14th days post treatment, the mesiodistal and buccolingual widths were measured using a digital caliper (vernier, usa). the size of the clinical wound extraction socket was calculated by the formula used in the study by mokhtari et al.,8: mesiodistal width x buccolingual width. then, the rats were sacrificed via cervical dislocation and the mandibular socket tissue was excised using a blade and scalpel (onemed health care, indonesia). the fresh socket tissue was then put into a container and fixed in a 10% buffer formalin solution for tissue processing and hematoxylin-eosin staining (he). the observation of the number of macrophages, fibroblasts and osteoblasts was carried out using a light microscope (zeiss primo star, germany) in five fields of view at 400× magnification. the data were analysed using the two-way analysis of variance (anova) test and the post hoc least significant difference (lsd) test to compare the differences between all groups. results based on the histological observations, there was a decrease in the number of macrophages accompanied by an increase in the number of fibroblasts and osteoblasts over time in all groups, as shown in figures 1 and 2. there was an inflammatory response in the negative control group, which was characterised by the highest number of macrophages followed by the lowest number of fibroblasts (see figure 1) and osteoblasts (see figure 2). the positive control group and the 8% cocoa bean group showed the healing of alveolar osteitis, which was characterised by a lower number of macrophages with a higher number of fibroblasts and osteoblasts than the negative control group. the 8% cocoa bean group showed the best healing effect of alveolar osteitis, with a significant decrease in the number of macrophages and a significant increase in number of fibroblasts and osteoblasts. the two-way anova test results (table 1) showed a significant difference in the mean number of macrophages, fibroblasts and osteoblasts between all groups (p = 0.000). the mean difference in the number of macrophages, fibroblasts and osteoblasts using the post-hoc lsd test are shown in figure 3. for the number of macrophages, there was a significant difference between the 8% cocoa bean extract gel group and the negative control group on the 3rd and 7th days (p = 0.000 and p = 0.001, respectively), but there was no difference between the 8% cocoa bean extract gel group and the positive control group (p = 0.845 and p = 0.859, respectively). for the number of fibroblasts, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 isnandar et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 7–12 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 9 table 1. histological observation of the mean number of macrophages, fibroblasts and osteoblasts histological observation observation day negative control (mean ± sd) positive control (mean ± sd) cocoa bean 8% (mean ± sd) p-value macrophage 3 25.36 ± 3.86 5.50 ± 1.36 5.22 ± 1.00 0.000* 7 7.50 ± 3.85 2.07 ± 0.95 1.82 ± 0.33 fibroblast 3 80.85 ± 2.94 124.65 ± 3.14 128.72 ± 7.51 0.000* 7 97.85 ± 3.85 163.27 ± 0.66 199.95 ± 10.46 osteoblast 7 4.52 ± 1.11 18.75 ± 3.53 20.75 ± 1.26 0.000* 14 12.30 ± 0.77 33.82 ± 1.80 43.87 ± 8.91 *two-way anova test; statistically significant with p < 0.05 a b c d e f figure 1. histological description of the macrophages (black arrow) and fibroblasts (red arrow) in the negative control group on day 3 and day 7 (a, b), the positive control group on day 3 and day 7 (c, d) and the 8% cocoa bean extract gel group on day 3 and day 7 (e, f) using he staining at 400× magnification. a b c d e f figure 2. histological description of the osteoblasts (black arrow) in the negative control group on day 7 and day 14 (a, b), the positive control group on day 7 and day 14 (c, d) and the 8% cocoa bean extract gel group on day 7 and day 14 (e, f) using he staining at 400× magnification. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 isnandar et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 7–12 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 10 there was a significant difference between the 8% cocoa bean extract gel and the negative control group on the 3rd and 7th days (p = 0.000). on the 3rd day, there was no difference between the 8% cocoa bean extract gel and the positive control group (p = 0.155), but on the 7th day, there was a significant difference (p = 0.000). for the number of osteoblasts, there was a significant difference between the 8% cocoa bean extract gel group and the negative control group on the 7th and 14th days (p = 0.037 and p = 0.000, respectively). on the 7th day, there was no difference between the 8% cocoa bean extract gel group and the positive control group (p = 0.495), but on the 14th day there was a significant difference (p = 0.003). the results of the clinical wound size of the extraction socket decreased over time in all groups, as shown in table 2. the two-way anova test showed a significant difference in the wound size of the extraction socket on the 3rd, 7th and 14th days between all groups (p = 0.000). the post hoc lsd test results (figure 4) shows a significant difference between the 8% cocoa bean extract gel and the negative control group on the 3rd, 7th and 14th days (p = 0.000). on the 3rd and 7th days, there was no difference between the 8% cocoa bean extract gel group and the positive control group (p = 0.059 and p = 0.117, respectively), but on the 14th day there was a significant difference (p = 0.028). table 2. clinical observation of the wound size of the extraction socket clinical observation observation day negative control (mean ± sd) positive control (mean ± sd) cocoa bean 8% (mean ± sd) p-value wound size of extraction socket (mm2) 3 3.87 ± 0.03 2.20 ± 0.09 2.13 ± 0.03 0.000*7 3.52 ± 0.08 1.79 ± 0.03 1.73 ± 0.08 14 0.85 ± 0.02 0.44 ± 0.09 0.36 ± 0.01 *two-way anova test, statistically significant with p < 0.05 25 .3 6 7. 50 80 .8 5 97 .8 5 4. 52 12 .3 0 5. 50 2. 07 12 4. 65 16 3. 27 18 .7 5 33 .8 2 5. 22 1. 82 12 8. 72 19 9. 95 20 .7 5 4 3. 87 3 days 7 days 3 days 7 days 7 days 14 days macrophage fibroblast osteoblast negative control positive control cocoa bean 8% * * * * * * * * 250 200 150 100 50 0 figure 3. the mean number of macrophages, fibroblasts and osteoblasts in all experimental groups. *post hoc lsd test; statistically significant at p < 0.05. 3. 87 3. 52 0. 85 2. 20 1. 79 0. 44 2. 13 1. 73 0. 36 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 3 days 7 days 14 days clinical wound size of extraction socket negative control positive control cocoa bean 8% * * * * figure 4. the mean clinical wound size of the extraction socket in all experimental groups. *post hoc lsd test; statistically significant at p < 0.05. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 isnandar et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 7–12 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 11 discussion the wound healing process of the extraction socket is an organised sequence of biological activities that restores the integrity of the mucosa and alveolar bone. immediately following a tooth extraction, the socket will be filled by a blood clot through platelet aggregation and fibrin activation. the blood clot serves as a provisional matrix for healing cells to migrate to the wound area and becomes a reservoir for growth factors.1,9 leukocytes, fibroblasts and new blood vessels will migrate to the blood clot to perform their function in the healing process. in the inflammatory phase, macrophages, phagocytose microbes and cellular debris produce high levels of free radicals, cytokines and growth factors that are important in stimulating cell migration and proliferation.1,2,10 in the proliferative phase, fibroblasts begin to migrate and degrade the fibrin clots. fibroblasts play an important role in producing collagen to build an extracellular matrix to form connective tissue and support the growth of new alveolar bone in the socket.1,2,9 osteoblasts are cells that play an important role in the process of new alveolar bone growth. osteoblasts produce a bone matrix called an osteoid and organic components, such as osteocalcin, osteopontin and alkaline phosphatase. first, an amount of osteoid produced by the osteoblasts will form a woven bone. then, osteoblastic and osteoclastic activity remodels the woven bone, leading to the growth of mature lamellar bone.1,2,9 in the healing of alveolar osteitis, an early destruction of the blood clot before the clot is replaced by granulation tissue inhibits the migration and proliferation of healing cells, subsequently inhibiting the healing process of the extraction socket.1,3 in this study, the 8% cocoa bean extract gel group had a greater reduction in the number of macrophages compared with the control groups from day 3 to day 7. this is because cocoa beans contain secondary metabolites, one of which is proanthocyanin.6 proanthocyanin acts as an anti-inflammatory agent by inhibiting cyclooxygenase and lipoxygenase, which reduces the number of prostaglandin, thromboxane, prostacyclin, endoperoxide and leukotrienes produced, resulting in a reduction in the number of inflammatory cells that migrate to the injured area. thus, the inflammatory reaction is shortened in duration and the wound healing process can move to the proliferative phase.6,11 the results of this study are supported by a study conducted by dugo et al.,11 which found that cocoa polyphenol extracts reduced the reactions of proinflammatory macrophages (m1 macrophages) and induced changes in the phenotype of macrophages to antiinflammatory macrophages (m2 macrophages). human macrophages can be divided into two groups based on their activation stages: m1 macrophages and m2 macrophages. m1 macrophages play a role in initiating the inflammatory process, while m2 macrophages are involved in the resolution of inflammation. at the end of the inflammatory phase, m2 macrophages will produce growth factors, such as transforming growth factor-beta (tgf-β), vascular endothelial growth factor (vegf) and platelet-derived growth factor, which act as the initiators of the proliferative phase.11,12 macrophages are the most dominant cells in the inflammatory phase, with the highest number on day 3 and a gradual decrease on day 7. the reduction in the number of macrophages indicates the end of the inflammatory phase and the beginning of the proliferative phase.10,12 the proliferative phase is characterised by fibroplasia.9,10 in this study, the 8% cocoa bean extract gel group had the highest number of fibroblast cells on the 3rd and 7th days compared with the control groups. cocoa beans contain quercetin.6 based on a study by kant et al.,13 quercetin stimulates fibroblast proliferation by increasing tgf-β1 expression. transforming growth factor beta 1 is a growth factor that stimulates the migration and proliferation of fibroblasts and supports the synthesis and deposition of collagen in the wound healing process.10,13 one of the factors that inhibit the healing process in alveolar osteitis is bacterial invasion. the bacteria involved in the pathogenesis of alveolar osteitis are generally anaerobic pathogenic bacteria.2,4 the theobromine found in cocoa beans is a potent antibacterial agent. theobromine attaches to the bacterial cell wall, penetrates the biofilm generated by the bacteria and stimulates proteolytic enzymes, which damage the bacterial cell membranes. this causes the phagocytic activity of the bacteria that cause alveolar osteitis to be more easily carried out by inflammatory cells, speeding up the inflammatory phase so that the proliferative phase can begin, which is characterised by the increase in the number of fibroblasts.6 fibroblasts begin to migrate on the 3rd day after the injury and continue to proliferate until their number peaks on the 7th day.2,10 osteoblasts begin to migrate on the 7th day, and their number will continue to increase until the 14th day, after which the osteoblasts will differentiate into osteocytes. osteocytes are mature osteoblasts trapped within the osteoid.9,14 in this study, the 8% cocoa bean extract group had the highest number of osteoblasts compared with the control groups on the 7th and 14th days. this is because cocoa beans contain anthocyanidins.6 in the study conducted by xu et al.,15 anthocyanidins were found to increase the expression of vegf, which is needed in the process of angiogenesis. one of the crucial factors in the process of bone healing is vascularisation. the granulation tissue that forms on the surface of the alveolar bone during the wound healing process following a tooth extraction requires good vascularity. vascularisation is needed to provide a supply of nutrients and oxygen in the process of bone ossification.2,16 cocoa beans also contain catechins.6 according to a study by fajriani et al.,17 catechins can stimulate osteoblast proliferation by increasing the expression of tgf-β1 and bone morphogenetic-2 (bmp-2). transforming growth factor beta 1 is the most abundant growth factor in bone cells and stimulates osteogenic bone cell proliferation and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 isnandar et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 7–12 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 12 differentiation. in addition, tgf-β1 enhances osteoclast absorption and promotes new bone growth. meanwhile, bmp-2 increases mesenchymal stem cell migration, proliferation and osteogenic differentiation.11,16,17 the positive control group in this study used aloclair® gel, which contains 2% sodium hyaluronate. during the wound healing process, sodium hyaluronate binds well to the fibrin clot, promoting cell migration and proliferation to the wound area. in addition, sodium hyaluronate can also strengthen the bond between fibrin and collagen so that blood clots become more stable and less easily damaged.18 the wound healing process of the extraction socket can also be assessed clinically by the size of the socket wound. the faster the size of the wound decreases, the faster the wound healing process takes place.19 based on this study, the cocoa bean extract gel group had the lowest mean clinical socket wound size compared with the control groups on the 3rd, 7th and 14th days. this was because the cocoa bean extract gel group had the highest number of fibroblasts. an increase in the number of fibroblast cells increases the wound closure process because fibroblasts differentiate into myofibroblasts, which play a role in wound contraction, thereby causing the wound to shrink in size.2,10 there were significant differences in the mean socket wound size and the number of macrophages, fibroblasts and osteoblasts between the cocoa bean group and the control groups. the cocoa bean extract gel produced a better effect on the healing process of alveolar osteitis compared with the control groups. this study concludes that the application of an 8% cocoa bean extract gel stimulates the healing of alveolar osteitis following a tooth extraction in wistar rats, as evident from the decrease in the clinical wound size of the extraction socket, a decrease in the number of macrophages and an increase in the number of fibroblasts and osteoblasts. acknowledgements this study was funded by the universitas sumatera utara research institute based on the talenta usu 2021 research contract (126/un5.2.3.1/ppm/spp-talenta usu/2021). references 1. henry cj, stassen lfa. the non-healing extraction socket: a diagnostic dilemma case report and discussion. j ir dent assoc. 2016; 62(4): 215–20. 2. hupp jr, ellis e, tucker mr. contemporary oral and maxillofacial surgery. 7th ed. philadelphia: mosby elsevier; 2019. p. 44–52, 203. 3. ezhil i, kumar m. recent advances in the management of dry socket-a review. drug invent today. 2018; 10(4): 450–4. 4. supe nb, choudhary sh, yamyar sm, patil ks, choudhary ak, kadam vd. efficacy of alvogyl (combination of iodoform + butylparaminobenzoate) and zinc oxide eugenol for dry socket. ann maxillofac surg. 2018; 8(2): 193–9. 5. kumar g, jalaluddin m, rout p, mohanty r, dileep cl. emerging trends of herbal care in dentistry. j clin diagn res. 2013; 7(8): 1827–9. 6. k i m j, k i m j, sh i m j, l e e cy, l e e k w, l e e h j. cocoa phytochemicals: recent advances in molecular mechanisms on health. crit rev food sci nutr. 2014; 54(11): 1458–72. 7. kurniawati a, saputra dr, cholid z, putra hk. cacao seed (theobroma cacao l.) extract gel effect on the neutrofil number after tooth extraction. odonto dent j. 2020; 7(1): 60–7. 8. mokhtari s, sanati i, abdolahy s, hosseini z. evaluation of the effect of honey on the healing of tooth extraction wounds in 4to 9-year-old children. niger j clin pract. 2019; 22(10): 1328–34. 9. de sousa gomes p, daugela p, poskevicius l, mariano l, fernandes mh. molecular and cellular aspects of socket healing in the absence and presence of graft materials and autologous platelet concentrates: a focused review. j oral maxillofac res. 2019; 10(3): e2. 10. la r java h. o ra l wound hea l i ng: cel l biolog y a nd cl i n ica l management. singapore: wiley-blackwell; 2012. p. 43. 11. d ugo l , bel luomo mg, fa na l i c , russo m, c a c ciola f, maccarrone m, sardanelli am. effect of cocoa polyphenolic extract on macrophage polarization from proinflammatory m1 to anti-inflammatory m2 state. oxid med cell longev. 2017; 2017: 6293740. 12. koh tj, dipietro la. inflammation and wound healing: the role of the macrophage. expert rev mol med. 2011; 13: e23. 13. kant v, jangir bl, kumar v, nigam a, sharma v. quercetin accelerated cutaneous wound healing in rats by modulation of different cytokines and growth factors. growth factors. 2020; 38(2): 105–19. 14. vieira ae, repeke ce, de barros ferreira s, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to toot h ext ract ion i n m ice: m icro computed tomog raphy, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. 15. xu l, choi th, kim s, kim s-h, chang hw, choe m, kwon sy, hur ja, shin sc, chung j il, kang d, zhang d. anthocyanins from black soybean seed coat enhance wound healing. ann plast surg. 2013; 71(4): 415–20. 16. zhang z, zhang x, zhao d, liu b, wang b, yu w, li j, yu x, cao f, zheng g, zhang y, liu y. tgf-β1 promotes the osteoinduction of human osteoblasts via the pi3k/akt/mtor/s6k1 signalling pathway. mol med rep. 2019; 19(5): 3505–18. 17. fajriani, sartini, horax s, malik a, asmawati, balqis a, wulansari dp. role of green tea catechins toothpaste on transforming growth factor-β1 (tgf-β1) and bone morphogenetic-2 (bmp-2) on early childhood caries. syst rev pharm. 2020; 11(12): 42–7. 18. yang h, kim j, kim j, kim d, kim hj. non-inferiority study of the efficacy of two hyaluronic acid products in post-extraction sockets of impacted third molars. maxillofac plast reconstr surg. 2020; 42(1): 40. 19. putra ass, rahardjo, hasan cy, pangestiningsih tw. effect of concentration differences of snail mucus gel (achatina fulica) on collagen density and wound closure rate in wistar rat skin punch biopsy wounds. j int dent med res. 2021; 14(2): 574–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p7–12 isnandar et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 7–12 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p7-12 mkg vol 42 no 2 april 2009.indd 70 vol. 42. no. 2 april–june 2009 research report transformation analysis of oral epithelial dysplasia to carcinoma in-situ and squamous cell carcinoma by p53 expression and gene mutations mei syafriadi division of pathology, department of biomedical sciences faculty of dentistry, jember university jember indonesia abstract background: it is known that oral squamous epithelial dysplasia (sed) and carcinoma in-situ (cis) are precancerous lesion and it could transform to squamous cell carcinoma (scc). we had reported p53-protein over-expression and gene mutational of oral cis, such as basaloid, verrucous, and acanthothic/atrophic types, but demarcated between sed to cis and cis to scc and how their transformation is still unclear. it is considered that their molecular behavior related one another. purpose: to understand the molecular behavior of them we examined p53 exon 5-8 gene mutation and their protein expression in the sequential cases. methods: using 10 cases formalin–fixed paraffin sections that composed sed appearance, cis and scc in the same case were subjected to p53 immunohistochemistry. then all cases were subjected to p53 gene mutations analysis. by laser capturing microdissection dysplasia part, cis part and scc part were cutted, and followed by direct sequencing of pcr product for exon 5-8. result: sed p53-protein over-expression in some cells, and the expression was increased to cis and scc. mutational analysis for p53 gene showed that 60% of p53 gene mutation in cis also found in scc, therefore scc had additional mutation in other exon of p53 gene. while no particular mutations were found in sed part of all cases. conclusion: carcinoma in-situ is a squamous cell carcinoma eventhough not invasive yet, but squamous epithelial dysplasia is an early step to malignancy. it needs other genes examination to know any genes are involved in the precancerous to cancer transformation process. key words: dysplasia, carcinoma in-situ, squamous cell carcinoma, mutation, p53 correspondence: mei syafriadi, c/o: laboratorium patologi, bagian biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. email: mei_syafriadi@hotmail.com introduction cancer of oral cavity is one of several cancers in the body and oral squamous cell carcinoma (oscc) accounts for more than 95% of all cancer in the oral cavity1 and most of oral cancers arise in the tongue which majority of oral cancer present at an advanced stage iii or iv.2 epidemiological study and experimental evidence indicate a causal relationship between some carcinogenic with oral cancer such as chewing tobacco, betel quid chewing, smoking and drinking,2-6 but the exact cause of cancer is unknown. squamous epithelial dysplasia (sed) and carcinoma in-situ (cis) known as precancerous lesion that composed of dysplastic change in intra epithelial of squamous epithelium.7 according to carcinoma which arise only limited in intra epithelial, many authors using different term such as severe dysplasia,8 or squamous intraneoplasia (sin) high grade.9 whereas who classified oral cis as one classification that separate to squamous epithelial dysplasia (sed) classification.10 carcinoma in-situ (cis) has been defined as a true and non invasive neoplasm lying within an epithelial layer. the neoplastic cells are different from sed, because they have already been proliferated and subsequently differentiated. in epithelial dysplasia, the constituent cells are only proliferated but not differentiated yet.11 71syafriadi: transformation analysis of oral epithelial dysplasia the p53 gene structure consists of eleven exon which are exon 2-3 as transactivation domain, exon 5-8 as dna binding domain and exon 10 as oligomerization domain. they are multifunctional such as transcription factor, which regulates cell cycle progression and interacts with several key proteins which involved in dna replication, transcription and reparation.12 many studies reported that the p53 protein overexpression is frequently found in both malignant and dysplastic lesions which increased due to grades of dysplasia, and may be an early event in multistage carcinogenesis of head and neck cancer.13–17,18 the abnormal p53 protein expression reported in 10 to 80% of oral sed19–22,24 that some cases related to p53 gene mutation,13,14,24,25 and nearly 60% of human cancers are accompanied by mutation in p53 gene,13 however, in some cases it is possible to observe protein over expression but they did not show any mutation gene or p53 gene mutation.25 as reported before, oral cis have shown p53 gene mutation almost distributed in exon 7 and exon 8, eventhough this lesion is not invasive yet.26 based on this appearance, it is speculated that cis is carcinomatous lesion the same as squamous cell carsinoma (scc), but wether sed is carcinomatous lesion. it is still unclear, considering their molecular behaviors are the same. based on this p53 gene exon 5–8 and its protein expression were evaluated in sequential cases which each case composed of epithelial dysplasia part, carcinoma in-situ and squamous cell carcinoma. the purpose was understand their molecular behavior transformation from dysplasia to cis and scc. in this concept, it is considered that all of scc started from sed and cis. materials and methods formalin paraffin-fixed block selected from the surgical pathology files in the division of oral pathology, niigata university graduate school of medical and dental sciences, japan, during sixth year period 2002 to 2004, after critical reviewing of hematoxylin and eosin (he) stained sections. these researches consisted of ten cases which is each case compose oral epithelial dysplasia/sed (mild and moderate cases), cis case (basaloid types, verrucous or acanthothic type). two oral pathologists screened all the specimens, when the diagnoses of sed, cis and scc part were not identical those cases would be reevaluated together. all of the specimens were routinely fixed in 10% formalin and embedded in paraffin. serial 4 μm sections were cut from paraffin blocks. one set of the sections was stained with hematoxylin and eosin and used for reevaluation of histological diagnosis, and the other sets were toluidine blue staining used for microdissection as well as immunohistochemistry for p53 protein. the antibodies against p53-protein used in this study were mouse monoclonal antibody clone bp53-11, (igg2a) (progen, progen biotechnik gmbh, heidelberg, germany), that reacted to wild-type and mutant forms of human p53 antigen within n-terminal region epitope aa2031. for immunohistochemistry staining, tissue sections 4 μm in thickness were taken from tissue blocks. after deparaffinization and dehydration, sections were washed in 0.01 m phosphate buffered saline (pbs). to restore the antigenic sites, sections were autoclaved in 0.01 m citrate buffer (ph 6.0) for 15 min at 121° c and then kept standing for 20 min at room temperature. to block endogenous peroxidase activities, all the sections were quenched with 0.001% h2o2 in 100% methanol for 30 min at room temperature and rinsed with pbs containing 0.5% skim milk and 0.05% triton x-100 (pbst). after rinsing in pbst, the sections were incubated in 5% skim milk in pbs containing 0.05% tritonx-100 for 1 hr at 37° c to block non-specific protein bindings. the sections were then incubated with monoclonal primary antibodies against p53-protein were p53-protein/clone p53 ab-bp53-11 (1: 100, progen biotechnik gmbh, heidelberg, germany), for overnight at 4° c. after incubations with the primary antibodies, the sections were rinsed in pbst and then treated with polymer-immune complexes (envision+ peroxides, rabbit/mouse, dako, 1:1) for 1 hr at room temperature. the peroxidase reaction products were visualized by incubation with 0.02% 3, 3diaminobenzidine (dab, dohjin laboratories, kumamoto, japan) in 0.05 m tris-hcl solution (ph 7.6) containing 0.005% h2o2. the sections were counterstained with hematoxylin. cells were regarded as positive for p53 protein if nuclear staining was intense and could be readily visualized at 10 times magnification. positive staining for p53 protein staining were calculated quantitatively using a micrometer scale 1mm/square at 10 times magnification. calculation of positive cells had been done three times and the average was taken. for control experiments, the primary antibodies were replaced with pre-immune mouse igg2a (dako). after evaluation of immunostaining pattern for p53, each sample was stained by toluidine blue for laser microdissection (lmd). appropriate ten location of sed, cis and scc part were cutted due to appropriate area and collected in collection tube for extracted dna using proteinase k 10% for one night at 37° c. the dna solution was purified using phenol/chloroform/isoamyl alcohol mixture (25:24:1). pcr amplification was carried out for p53 exons 5–8. all primer sets were designed on intron sequences adjacent to each exon as follow: [exon 5] sense, 5'-gtt tct ttg ctg ccg tgt tc-3', antisense, 5'-agg cct ggg gac cct ggg ca-3' spanning 323 bps; [exon 6] sense, 5'-tgg ttg ccc agg gtc ccc ag-3', antisense 5'-gga ggg cca ctg aca acc a3' spanning 223 bps; [exon 7] sense, 5'-ctt gcc aca ggt ctc ccc aa-3', antisense 5'-tgt gca ggg tgg caa gtg gc-3' spanning 196 bps; [exon 8] sense, 5'-ttc ctt act gcc tct tgc tt-3', antisense 5' cgc ttc ttg tcc tgc ttg ct-3' spanning 201 bps. pcr were performed on a thermal cycler (pc-800, astec 72 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 70−75 co., ltd., fukuoka japan), after a pre-denaturation at 94° c for 5 minutes. the amplification step was carried out for 35 cycles in 100 μl of a pcr reaction mixture containing 5 units of takara ex taq polymerase (takara biotechnology, co., ltd., otsu, japan), 10x ex taq buffer, 2.5 mm each of dntp mixtures, and 20 pmol of each sense and antisense primers. the thermal cycling condition was as follow: denaturation at 94° c for 1 min, annealing at 63° c for exon 5, at 60° c for exon 6 and 8, and 62° c for exon 7 for 0.30 sec each, and extension at 72° c for 1 min. the last extension was prolonged by additional 7 min. amplification products were analyzed by electrophoresis on 3% agarose gel (nusieve 3:1 agarose, cambrex bioscience rockland inc., rockland, me usa) and the band were visualized by ethidium bromide upon exposure to an ultraviolet transilluminator. all pcr products were subjected to cycle sequencing by using thermo sequenase primer cycle sequencing kit with 7-deaza -dgtp (amersham biosciences corp., piscataway, usa). the sequence primers were synthesized based on the published data (lehman ta et al.) and labeled with texas red 5'-end. the labeled primers were as follow: 5'-gtt tct ttg ctg ccg tgt tc-3' for exon 5; 5'-gcc tct gat tcc tca ctg at-3' for exon 6; 5'-ctt gcc aca ggt ctc ccc aa-3' for exon 7; 5'-ttc ctt act gcc tct tgc tt-3' for exon 8. one tube sequencing reaction contained 3 μl of master mixes (appropriate nucleotides/ reaction buffer/thermo sequenase dna polymerase), 2 μl of the template pcr products, which were purified with gfx pcr dna and gel band purification kits (amersham biosciences corp., piscataway, usa), 8 μl of distillated water and 2 μl (2pm) of texas red-labeled primers. each sequencing reaction added 3 μl of a, c, g, t reagent. after denaturation at 95° c for 30 sec and annealing at 55° c for 30 sec. the reaction products were dissolved in 3 μl loading dye by vortexing and concentrated with vacuum desiccators. then 3 μl of samples for each lane were loaded on a gel (7% long ranger/6.1 m urea/1.2× tbe buffer (10 mm tris, 10 mm boric acid, and 2 mm edta). the electrophoresis was performed in a fluorescent dna sequencer (sq-5500-s, hitachi ltd., tokyo, japan), and the sequencing data were analyzed by using the sq5500 analysis software ver.3.03 (hitachi). statistical analysis was perform with calculating the numbers of p53 and ki-67 positive cells in a square unit 1 mm2 on a microscope equipped with a micrometer. ten fields were randomly counted per section at x100 magnification. one-way anova was used for statistical comparison of cell numbers between each group by using the spss software program (spss inc., chicago, il, usa). result immunohistochemical staining for p53 protein, when observed, it was found exclusively in the nuclei of epithelial cells of sed, cis and scc. their expression was increased figure 1. sequential he staining (sample 01-803) showed sed, cis and scc (a, c, e). immunohistochemistry of p53 protein found p53 protein over expressed in basal and parabasal layer of sed (b) and cis (d) and tumor nest of scc (f) (100×). figure 2. sequential he staining (sample 03-969) showed sed, cis and scc (a, c, e). p53 protein over expressed in basal and parabasal layer sed (b) but cis and scc had not showed p53 protein expression (d, f) (100×). 73syafriadi: transformation analysis of oral epithelial dysplasia figure 3. p53-protein expression showed positive cell were icreased significantly from dysplasia to cis and scc. table 2. p53 gene mutational analysis in sed, cis and scc no. no. of case sed cis scc 1 03-769 none e7:248, cgg-cag e7:248 cgg-cag 2 02-021 none e7:242, tgc-ttc e7:242, tgc-ttc e5:140, acc-atc 3 03-969 none e6:196, cga-tga e6:196, cga-tga 4 03-952 none none e5:150-154 deletion 13 base pair 5 02-1409 none e8:306, cga-tga e8:306, cga-tga 6 01-803 none e8:282, cgg-tgg e8:282, cgg-tgg 7 02-205 none e8:272, gtt-gtg e8:272, gtt-gtg e8:291, aag-tag e8:291, aag-tag 8 04-369 none e7:231, acc-gcc e5:139, aag-aac e5:183, tca-cca e8;303, agc-agt 9 03-573 none e8:282, cgg-tgg none 10 02-1683 none none e8:301, cca-gca e8:302, ggg-gag e8:303, agc-gac figure 4. point mutations of p53 gene found in cis and scc which cga (arginine) to tga (stop codon) (sample no. 03-969). from sed to cis and scc (figure 1, 2). the increasing of p53-protein expression in number was statistically significant (p < 0.05). in some cases sed part showed p53-protein expression had not showed p53 protein expression both in cis and scc (figure 2 and table 1). table 1. the p53-protein over expression positive cells in sed, cis and scc no. no. case sed cis scc 1 03-769 125 453 1210 2 02-021 34 100 289 3 03-969 42 0 0 4 03-952 72 0 0 5 02-1409 34 0 0 6 01-803 40 126 200 7 02-205 31 0 0 8 04-369 32 63 505 9 03-573 22 53 38 10 02-1683 23 402 911 all sequential cases of sed, cis and scc had shown p53 protein over-expression positive cells collected by laser microdissection. next step, those cells were sequencing for p53 gene analyses of exon 5-8. the result show 80% of cis and 90% of scc cases had p53 gene mutation which mutation type were 89% point mutation and 11% were frame shift/deletion of 13 base pair (table 2 and figure 4). meanwhile, 60% of p53 gene mutation in cis also found in scc cases, therefore scc had additional mutation in other exon of p53 gene (table 2). meanwhile no particular mutations were found in sed part of all cases (table 2 and figure 5). discussion oral sed is considered as an early step to malignancy because dysplastic cells could proliferate continuesly to form basaloid cells which replaced normal cell in whole layer that called cis and this cis is a step to scc that has behavior to invade the adjacent tissue or metastases to distant organs.14,26 74 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 70−75 figure 5. p53 gene analysis result in sed, no mutation were found in exon 5–8 (sample no. 03-769). alterations of p53 either at the gene or at the protein expression are a common feature in many human cancers. some reporting p53 mutation as an early event in the development of oral cancer and others suggesting that it is a late finding.14, 27 the transformation timing from oral sed to cis and scc has not clear yet. many study of p53 gene mutation and protein overexpression in oral sed and scc,14,17,22 but most of their study used sample from different case/patient. in this present study we use sequential cases included sed, cis and scc that were taken from the same case/sample, therefore with this method we can see the molecular behavior concomitantly. immunohistochemical study of this present study demonstrated that p53 protein was over-expression from sed to cis and scc which was increased according to severity of lesion. it could understand that p53 over expression could be able as a marker of malignant transformation; nevertheless, some case did not show any protein expression. it caused the mutation was stop codon or frame shifts resulting no protein can be detected by antibody. cruz, et al.22 reported that p53 protein expression in basal cell layer is an early even of malignant transformation.24 all of sed and cis cases showed preservation of basement membrane and without stoma induction,11 but cis should get more intention because the basaloid formation intra epithelial more dominant than sed, it is a sign of malignant transformation from precancerous lesion (sed), as syafriadi and saku reported basaloid cell is dysplastic cell that can replaced the normal intra epithelium, even though they have been toward keratinization but they are cancerous cells,27 and potentially transform to scc which ability to destruct basement membrane and carcinomatous foci perform, that invade to adjacent tissue followed by stromal formation. in this study showed p53 gene mutation of cis found in 80% cases and interestingly their mutation particularly same location with scc cases. it means cis is cancerous lesion but had not invasive yet as scc that showed tumor nest formation in the adjacent tissue or metastasis to other organs. saku,1 in his report called cis as superficial carcinoma and tend to recur. recently, in japan the superficial carcinomas are increasing in number. however in this study, mutation of p53 gene which involved exon 5 or 6 in scc cases was also found. it could be considered as progressive process of this lesion. this is supported by many reports demonstrated p53 gene mutation of head and neck scc showed similarity to oral cis p53 gene mutation in ths present study,1,5,24 it suggested scc was started from cis. the sed cases, several cases of cis and scc showed p53-protein over-expression but not any mutation of p53 gene exon 5-8, it was suggested that it involved other onco-supressor gene as possible factor, because it had been known that p53 gene produce protein to interact with other gene such as p21, mdm2, p63 or p73.6,12,13 the p53 gene mutation in cis and scc occurred in various exon but exon 8 more frequently (65% of total mutation cases) and was followed by exon 7 (29% of total mutation cases) and codon 248, 282 in exon 7 and 8 known as “hot spot”.13, 14 millward et al.,28 reported the p53 mutation in codon 157, 248, 249 and 273 is linked to the exogenous and endogenous carcinogenic factors such 75syafriadi: transformation analysis of oral epithelial dysplasia as found in head and neck, colorectal and breast cancer. in addition, other point mutation in exon 6 and exon 8 codon 196 (cga→tga) resulted from arginine to stop codon; codon 291 (aag→tag) resulted from lysine to stop codon and codon 306 (cga→tga) resulted from arginine to stop codon also found. these mutations were denoted as hot spots of p53 mutations. several study showed that arginine residue function is involving in dna repair,28, 29, 30 therefore, mutation in this amino acid could make dna fail to repair their dna damage. in conclusion, we have studied a series of oral precancerous lesion (sed) that have shown progression of p53 protein expression to cancerous lesion (cis and scc) and the p53 gene mutation location of cis and scc are similar and often involved in exon 7-8 moreover, some cases scc showed additional mutation in exon 56 that resulted frame shift or stop codon. the p53 gene mutation that occurred in hotspot codon of cis and scc may suggesting is related to extrinsic carcinogenic agent. the progressiveness of sed to cis and cis to scc may be detected by p53 protein expression and gene mutational. we suggested the oral surgeon should give attention to the dysplastic cells nearby the surgical margin that shows p53 protein over-expression. to understand clearly the progression of scc, more study need to be addressed to other gene which may involve in progression and metastasize of scc. references 1. saku t. differential diagnosis of oral mucosal lesions. proceeding. the 3rd national dental scintific meeting, 2008. faculty of dentistry, jember university. p. 1–5. 2. syafriadi m. patologi mulut. tumor neoplastik & non neoplastik rongga mulut. yogyakarta: cv andi offset; 2008. p. 31–7. 3. regezi ja, sciubba jj, jordan rck. oral pathology. clinical pathologic correlations. st louis: w.b saunders company; 2003. p. 68–110. 4. rubin e, gorstein f, rubin r, schwarting r, strayer d. pathology. clinicopathologic foundations of medicine. 4th ed. philadelphia: lippincott williams and wilkins; 2005. p. 1275. 5. reksoprawiro s. surgery in locally advanced oral cancer. proceeding. indonesian oral and maxillofacial association meeting. denpasar, 2008. p. 2–10. 6. harms kl, chen x. the c terminus of p53 family proteins is a cell fate determinant. journal of molecular and cellular biology 2005; 25:2014–30. 7. syafriadi m, ida-yanemochi h, ikarashi t, maruyama s, jen ky, cheng j, hoshina h, takagi r, saku t. carcinoma in-situ of the oral mucosa has a definite tendency towards keratinization. oral med pathol 2003; 8:43–4. 8. yanamoto s, kawasaki g, yoshitomi i, mizuno a. expression of p53r2, newly p53 target in oral normal epithelium, epithelial dysplasia and squamous cell carcinoma. cancer letters 2003; 190:233–43. 9. sakr wa, crissman jd. squamous intraepithelial neoplasia the upper aerodigestive tract in diagnostic surgical pathology of the head and neck. london: wb. saunders company; 2001. p. 1–9. 10. pindborg jj, reichart pa, smith cj, van der wall i. histological typing of cancer and precancer of the oral mucosa. 2nd ed. springerverlag, berlin: world health organization international histological classification of the tumors; 2005. p. 1–20. 11. the japanese society for oral pathology. guidelines for histopathological diagnosis of borderline malignancies of the oral mucosa. japan foundation 2005. p. 7–11. 12. national center for biotechnology information. the p53 tumor suppressor protein; genes and disease. united national institutes of health. 2008. p. 10. 13. glazko gv, koonin ev, rogozin ib. mutation hotspots in the p53 gene in tumors of different origin: correlation with evolutionary conservation and signs of positive selevtion. biochimica et biophysica acta 2004; 1679:95–106. 14. shahnavaz sa, regezi ja, bradley g, dube id, jordan rck. p53 gene mutation in sequential oral epithelial dysplasias and squamous cell carcinomas. j pathol 2000; 190:417–22. 15. klieb hbe, raphael sj, pindzola. comparative study of the expression of p53, ki67, e-cadherin and mmp-1 in verrucous hyperplasia and verrucous carcinoma of the oral cavity. j head and neck pathology 2007; 2:118–22. 16. panjwani s, sadiq s. p53 expression in benign, dysplastic and malignant oral squamous epithelial lesions. pak. j med sci 2008; 24:130–5. 17. piyathilake cj, frost ar, manne u, weiss h, heimburger dc, grizzle we. nuclear accumulation of p53 is a potential marker for the development of squamous cell lung cancer in smokers. chest 2003; 123:181–6. 18. piattelli a, rubini c, fioroni m, lezzi g, santinelli a. prevalence of p53, bcl2, and ki-67 immunoreactivity and of apoptosis in normal oral epithelium and in premalignant and malignat lesions of the oral cavity. j oral maxillofac surg 2002; 60:532–40. 19. alves fa, pires fr, de almeida op, lopes ma, kowalski lp. pcna, ki-67 and p53 expressions in submandibular salivary gland tumours. int j oral maxillofac surg 2004; 33:593–7. 20. kovesi g, szende b. changes in apoptosis and mitotic index, p53 and ki-67 expression in various types of oral leukoplakia. oncology 2003; 65:331–6. 21. farrar m, sandison a, peston d, gailani m. immunocytochemical analysis of ae1/ae3, ck 14, ki-67 and p53 expression in benign, premalignant and malignant oral tissue to establish putative markers of oral carcinoma. br j biomed sci 2004; 61:117–24. 22. cruz ib, meijer cjlm, snijders pjf, snow gb, walboomers jmm, van der waal i. p53 immunoexpression in non-malignat oral mucosa adjacent to oral squamous cell carcinoma: potential consequences for clinical management. j pathol 2000; 191:132–7. 23. mallofre c, castillo m, morente v, sole m. immunohistochemical expression of ck20, p53, and ki-67 as a objective markers of uroepithelial dysplasia. mod pathol 2003; 16:187–91. 24. scheneider-stock r, mawrin c, motsh c, boltze c, peters b, hartig r, buhtz p, giers a, rohrberck a, freigang b, roessner a. retention of the arginine allele in codon 72 of the p53 gene correlates with poor apoptosis in head and neck cancer. am j pathol 2004; 164:1233–41. 25. cruz i, snijders pj, van houten v, vosjan m, van der waal i, meijer cj. specific p53 immunostaining patterns are associated with smoking habits in patients with oral squamous cell carcinomas. j clin pathol 2002; 55:834–40. 26. syafriadi m, saku t. p53-protein over-expression and gene mutational of oral carcinoma in-situ. dental journal 2007; 40(2):55–60. 27. hussein sp, amstad p, raja k, sawyer m, hofseth l, shields pg, hewer a, phillips dh, ryberg d, haugen a, harris cc. mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (bpde) and the frequency of p53 mutations in nontumorous human lung. cancer research 2001; 61:6350–5. 28. millward h, samowitz w, wittwer ct, bernard ps. homogenous amplification and mutation scanning of the p53 gene using fluorescent melting curves. j clinical chemistry 2002; 48:1321–8. 29. blencowe bj. exonic splicing enhancer: mechanism of action, diversity and role in human genetic diseases. tibs 2000; 25:106–10. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. all manuscripts submitted to the journal must be written in english. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak1, matsuo hamada2, ninuk hartati3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur, malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima, japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne, australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in original articles should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in review articles should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (original articles, case reports, or review articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission  the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair. ac.id/mkg/about/submissions#onlinesubmissions  the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id 131131 research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 131–137 deoxypyridinoline and mineral levels in gingival crevicular fluid as disorder indicators of menopausal women with periodontal disease agustin wulan suci dharmayanti1 and banun kusumawardani2 1department of biomedical 2department of pathology anatomy faculty of dentistry, universitas jember jember-indonesia abstract background: menopause is a phase of a woman’s life marked by menstruation cycle cessation and an increased risk of periodontal disease. it can be caused by estrogen deficiency which alters the microenvironment in the sulcular gingival area and influences the composition and flow of gingival crevicular fluid (gcf). gcf has been widely studied as a non-invasive diagnostic and predictive tool for periodontal diseases. however, insufficient reports exist that explore its role as a predictive or diagnostic tool for bone loss detection in menopausal women. purpose: this study aimed was to investigate deoxypyridinoline (dpd) and mineral levels that could be utilized as disorder indicators in menopausal women with periodontal disease. methods: this study represents a form of analytical observation. eighty-four patients of the dental hospital, university of jember who fulfilled certain criteria were recruited. the subjects were divided into two main groups based on the presence of periodontal disease, (gingivitis=26; periodontitis=58) which were subsequently divided into three sub-groups based on their menopausal phase (pre-menopausal=26; perimenopausal=40; post-menopausal=18). gcf was collected using paper points from the buccal site of a posterior maxillary tooth with each subject having their gcf taken on only one occasion. dpd analysis was conducted by means of an elisa test. the analysis of calcium, magnesium and sodium incorporated the use of an atomic absorption spectroscope (aas), while that of phosphor was by means of a spectrophotometer. statistical analyses were performed using a comparison and correlation test (p<0.05). results: there were significant differences in dpd and the mineral level of gcf in menopausal women with periodontal diseases (p<0.05). dpd and mineral levels showed significant correlation to those of menopausal women with periodontal diseases and a ph of gcf. conclusion: dpd and mineral level in gcf could be used as disorder indicators in menopausal women with periodontal diseases key words: menopause; periodontal disease; deoxypyridinoline; mineral; gingival crevicular fliud correspondence: agustin wulan suci dharmayanti, department of biomedical, faculty of dentistry, universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: agutinwulan.fkg@unej.ac.id introduction the menopause is a phase in a woman’s life marked by menstruation cycle cessation and changes in sex hormone production, the latter of which increases the risk of periodontal disease in menopausal females. periodontal disease constitutes tooth support tissue inflammation caused by periopathogens ultimately resulting in tooth loss.1 a previous study showed that the incidence of periodontal disease in menopausal women was higher than in sexually productive women. this might be caused by a sex hormone deficiency, especially estrogen, which plays a pivotal role in periodontal tissue remodeling and repair. a number of researchers inferred that there was a relationship between periodontitis and the menopause. however, further study is required to explain that relationship more fully.1–6 estrogen deficiency is regarded as affecting the microenvironment in the sulcular gingival area, thereby dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 mailto:agustinwulan.fkg@unej.ac.id 132 dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 influencing the composition and flow of gingival crevicular fluid (gcf). gcf is a physiological fluid, as well as an inflammatory exudate, originating in the gingival plexus of blood vessels in the gingival corium, subjacent to the epithelium lining of the dentogingival space. gcf is composed of serum and locally generated components including: tissue breakdown products, inflammatory mediators, leukocytes, bacteria, and mineral electrolytes. recently, gcf has been explored and extensively investigated because of its many biochemical components offering several advantages, particularly that of detecting exchanges which occur both locally or systemically in the periodontal tissue.7,8 gcf has been widely studied as a non-invasive diagnostic and predictive tool in combating periodontal diseases. however, reports investigating the protein and mineral component of gcf as a predictive or diagnostic tool for bone loss detection in menopausal women remain insufficient in number.1,7,8 in this recent study, certain parameters potentially applicable as disorder indicators in menopausal women with periodontal disease were investigated. from the authors’ perspective, they offer encouraging potential as an early menopause diagnostic tool in the drive to improve women’s health. the parameters included deoxypyridinoline (dpd) representing a specific bone tissue breakdown product and mineral components such as: calcium, phosphor, magnesium, and sodium, constituting minerals or electrolytes active in inflammation and bone metabolism. materials and methods this study constitutes an analytical observation approved by the ethical commission of the faculty of dentistry, universitas gadjah mada. having been recruited by the dental hospital, universitas jember, all 84 patients signed an informed concern agreement giving them the legal status of research subjects. the participants were interviewed before completing a questionnaire regarding their menstruation cycles and menopausal symptoms. the criteria applied for participants to be accepted as study subjects included the following: female, 45 to 70 years old and systemic disease-free. subjects were excluded from the study if they were pregnant or nursing, were smokers, regularly consumed alcohol, and had received periodontal treatment during the previous 6 months, had taken antibiotics during the previous year, regularly used mouthwash, received hormone therapy or received calcium and/or mineral therapy. the study subjects (n=84) were divided into two main periodontal diseasebased groups determined according to a periodontal index (russel’s modification). the criteria of the index included the following factors: where gingivitis showed no loss attachment and pocket, but there was bleeding on probing (n=26); periodontitis showed loss attachment more than equal to 3 mm, a periodontal pocket more than 33 mm existed and bleeding on probing occurred (n=58).1thereafter, the main groups were divided into three sub-groups based on their menopausal phase: premenopausal (regular menstrual cycles in the past 3 months) or <50 years old (n=26), perimenopausal (experienced a menstrual period within the past 12 months but a missed period or irregular cycles during the past 3 months) or 50-55 years old (n=40), postmenopausal (no menstrual period during the previous 12 months) or >55 years old (n=18).9,10 gcf was collected between 08.00 and 13.00 because levels of dpd peak during the morning hours. gcf was taken from the buccal site of the posterior tooth infected with gingivitis or periodontitis. gcf was only taken once from each subject. prior to gcf sampling, the tooth element had to be cleaned of saliva, blood, plaque, debris, and calculus supragingiva. gcf was absorbed using three absorbent paper points number 20. each paper point was inserted in the buccal sulcus in a parallel position for approximately 60 seconds. it was then inserted into a 0.5 ml eppendorf tube and sealed with paraffin tape before being placed in a deep-freezer at -300c for dpd and mineral analysis.1 the paper point was centrifuged at 2200 rpms for 20 minutes. the tube lip of the eppendorf (1.5 ml) was subsequently perforated with a 30g needle and closed. after the paper point had been inserted into the tube, 50 ml 0.02 m 30g needle pbs ph 7.0-7.2 was added and incubated for five minutes. it was then centrifuged at 2200 rpms for 20 minutes, the resulting solution being collected in a 1.5 ml eppendorf tube. thereafter, 10 ml 0.02 m pbs ph 7.0-7.2 was added and centrifuged at 2200 rpms for 20 minutes. dpd analysis was conducted by means of an enzyme linked immunosorbent assay (elisa) kit (elabioscience, china). the procedures of elisa test followed those contained in the manual booklet.1 for the purposes of mineral analysis, this study used two paper points, one of which was subjected to calcium, magnesium and sodium analysis by an atomic absorbance spectrophotometer (aas), the other being subjected to phosphor analysis by a spectrophotometer. the sample preparation of gcf was the same as that for a dpd assay. however, the solution collected was added to100µl distilled water and 100 µl 0.02 m pbs ph 7.0-7.2, before being centrifuged at 2200 rpms for 20 minutes.1,11 all variables compared the dpd and mineral level of gcf in menopausal women with periodontal diseases using a one-way analysis of variance (anova) (p<0.05) test. then, the variables were analyzed by means of least significant differences (lsd) (p<0.05) multiple comparison. all variables were subsequently analyzed by multiple regressions and pearson’s correlation (p<0.05). this analysis was conducted to establish the association or correlation between variables. results the characteristics of 84 middle-aged women who participated as subjects in this study are shown in table 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 133133dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 the majority of participants who experienced periodontitis were aged approximately 49 years old. based on intra-oral status, they had approximately 26 remaining teeth and a periodontal index of around 2.8. moreover, the ph level of gcf participants suffering from periodontitis was just above 7 (slightly alkaline). table 1 show that there were significant differences between the dpd and mineral levels in the gcf of menopausal women with periodontal diseases (p<0.05). the pre-menopausal women with gingivitis registered the lowest dpd and mineral level in their gcf, with the exception of magnesium (2.67±2.21, mean±sd). the lowest magnesium level was recorded in postmenopausal women with periodontitis (0.96±0.21, mean±sd). the highest level could be seen among those women then experiencing the menopause. premenopausal women presented the highest magnesium level (gingivitis subjects=2.67±2.21; periodontitis subjects=1.64±0.23). in contrast, perimenopausal women presented the highest level of dpd (gingivitis subjects=77.28±9.26; periodontitis subjects=144±25.91) and phosphorous ( g i n g i v i t i s s u b j e c t s = 6 7 . 4 3 ± 6 . 4 7 ; p e r i o d o n t i t i s subjects=71.81±6.26), while postmenopausal individuals presented the highest level of calcium (gingivitis subjects=5.51±0.89; periodontitis subjects=5.28±0.11) and sodium (gingivitis subjects=621.33±6.65; periodontitis subjects=644.83±24.55). figure 1 illustrates multiple comparisons based on the mean difference in dpd and the mineral level of gcf between groups. with regard to the mean of the dpd level, there were significant differences between groups (p<0.05), except for menopausal women with gingivitis (p>0.05). within the mean of the mineral level, there were varying significance values. with regard to the calcium level, there were significant differences between the groups (p<0.05), except between the postmenopausal with gingivitis to periand postmenopausal with periodontitis, and the perimenopausal to postmenopausal with periodontitis (p>0.05). phosphor levels between groups showed almost no significant differences (p>0.05), except preand postmenopausal with periodontitis to pre and postmenopausal with gingivitis, and gingivitis to periodontitis in perimenopausal (p<0.05). turning to magnesium levels, almost all groups presented significant differences (p<0.05), except perimenopausal with gingivitis to premenopausal with periodontitis, and perimenopausal to postmenopausal with periodontitis (p>0.05). for sodium levels, there were significant differences between the groups (p<0.05), except perimenopausal to postmenopausal with gingivitis, premenopausal with periodontitis to peri and postmenopausal with gingivitis, and perimenopousal to postmenopausal with peridontitis (p>0.05). table 1. descriptions of characteristic of subjects (n=84) variable gingivitis periodontitis p value*premenopause (n=6) perimenopause (n=14) postmenopause (n=6) premenopause (n=20) perimenopause (n=26) postmenopause (n=12) age (years) 46.33 (2.73) 52.13 (2.28) 62.50 (3.73) 45.95 (2.26) 53.03 (2.58) 60 (2.27) teeth 26.83 (2.40) 26.79 (3.56) 23.33 (2.66) 26.60 (3.35) 24.38 (2.33) 21.75 (1.36) pi 0.43 (0.20) 0.52 (0.16) 0.45 (0.19) 2.81 (1.29) 2.86 (0.51) 3.04 (1.67) ph 6.92 (0.08) 6.82 (0.13) 6.92 (0.08) 7.21 (0.17) 7.18 (0.14) 7.24 (0.14) 0.000* dpd (nmol/l) 61.91 (1.74) 77,28 (9,26) 72.41 (2.14) 92,17 (9,21) 144,25 (25,91) 107.31 (10,00) 0.000* ca (ppm) 2.42 (0.39) 4.50 (1.07) 5.51 (0.89) 3.25 (0.68) 5.32 (0.84) 5.28 (0.99) 0.000* p (ppm) 63.83 (6.79) 67.43 (6.97) 64.17 (1.17) 69.55 (4.68) 71.81 (6.26) 67.00 (2.22) 0.003* mg (ppm) 2.67 (2.21) 1.68 (0.16) 1.22 (0.08) 1.64 (0.23) 0.99 (0.25) 0.96 (0.21) 0.000* na (ppm) 572.17 (12.35) 619.71 (14.41) 621.33 (6.65) 609.40 (16.30) 642.73 (20.42) 644.83 (24.55) 0.000* data was expressed as a mean (sd, standard deviation) for all variables. p value was derived from one-way analysis of variance * significantly different among the groups (p<0.05) n, number of study subjects; pi, periodontal index based on russel’s modification; ph, acid base degree of gcf; dpd, deoxypyridinoline; ca, calcium; p, phosphor; mg, magnesium; na, sodium. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 134 dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 according to a correlation analysis (table 2), a correlation existed in most of the variables between the periodontal disease of menopausal women and ph. although the correlation power varied for each variable, the levels of dpd, calcium, magnesium, and sodium demonstrated a significant and strong correlation with menopausal women suffering from periodontal diseases (p<0.05, r2>0.5), except for phosphor which had a moderate correlation with menopausal women presenting periodontal diseases (p<0.05, r2=0.3). moreover, dpd, phosphor, magnesium, and sodium levels demonstrated a significant correlation (p<0.05) and varying correlation power with the ph of gcf, a weak to strong correlation. however, there was correlation between the calcium level and ph of gcf (p>0.05, r2<0.1) (table 2). discussion this study confirmed gingival sulcus microenvironment changes indicated by significant ph shifts in the gcf. the ph of gcf in menopausal women with periodontitis was more alkaline than that of counterparts suffering from gingivitis (table 1). this could be due to initial inflammation in gingival triggered by acid-sensitive proteolytic bacteria colonization, such as porphyromonas gingivalis, in the gingival sulcus area where the bacteria induced protein metabolism and increased the ph of the gcf. saccharolytic bacteria, fusobacterium nucleatum and prevotella intermedia, are initially colonized on the surface of periodontal pocket and decrease the ph in the gingival sulcus area. the acidity is caused by the bacteria utilizing glucose in that location and producing lactic acid. there is subsequent bacteria change to asaccharolytic bacteria colonization, porphyromonas gingivalis. these bacteria result in the fermentation of amino acids into organic acids generating a high ph.8 in the present study, the highest level of dpd was found in perimenopausal subjects with periodontitis. moreover, the dpd level in such subjects was higher than that in subjects suffering from gingivitis. statistically, menopausal women with gingivitis demonstrated a similar dpd level. however, menopausal women with periodontitis presented a significant difference in dpd levels. this might provoke alveolar bone loss during the menopause with the periodontitis related to inflammation in periodontal tissue exacerbating estrogen deficiency in menopausal women. moreover, differences existed in the collagen composition between the gingiva and alveolar bone, where the gingiva is largely composed of type i, iii and v, with collagen and alveolar bone and periodontal ligament being formed from type i collagen. in contrast, dpd is a collagen cross-link of bone-specific type i collagen, resulting in high levels of dpd level in menopausal women with periodontitis.1 with regard to gcf mineral level observation, the study showed that the levels of minerals (calcium, phosphor and sodium) in the gcf of menopausal women with gingivitis were significantly lower than in those of menopausal women with periodontitis (p<0.05). magnesium level in the gcf of menopausal women with gingivitis was significantly higher than in their counterparts with periodontitis (p<0.05). these variations might be related to the severity of inflammation and hormonal changes in menopausal women. the highest calcium level occurred in post-menopausal women with gingivitis, the calcium level of periodontitis was higher than gingivitis. furthermore, the calcium level was statistically similar in perimenopausal and postmenopausal women with periodontitis (p>0.05). the calcium level in gcf in perimenopausal and postmenopausal females was higher than in their premenopausal counterparts with periodontitis and gingivitis. this suggests that the levels of steroid sex hormones and estrogen, in perimenopausal and postmenopausal individuals, are influenced to a greater extent by the calcium level in gcf than by the inflammation table 2. association between periodontal disease in menopausal women and ph to dpd level and minerals in gcf variable periodontal diseases in menopausal womena phb β coefficient r square p value* β coefficient r square p value* dpd (nmol/l) 24.399 0.569‡ 0.000* -481.999 0.521‡ 0.000* ca (ppm) 1.479 0.698‡ 0.039* 1.042 0.077 0.242 p (ppm) 71.543 0.332† 0.035* 21.591 0.236# 0.015* mg (ppm) 3.174 0.634‡ 0.000* 7.300 0.337† 0.001* na (ppm) 541.929 0.512‡ 0.000* 381.217 0.267# 0.007* a multiple regression test b pearson’s correlation test β coefficient was adjusted for periodontal disease, menopause phase, ph of gcf * significant correlation between variables (p<0.05) #significant but weak correlation †significant and moderate correlation ‡significant and strong correlation ph, acid base degree of gcf; dpd, deoxypyridinoline; ca, calcium; p, phosphor; mg, magnesium; na, sodium. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 135135dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 process of periodontal disease. moreover, estrogen levels tend to fluctuate during the perimenopausal phase before stabilizing fully in the postmenopausal phase. calcium enhancement was associated with calcium ion mobilization from bone surface to extracellular fluid, thereby inducing bone loss. several studies have suggested that estrogen deficiency in perimenopausal and postmenopausal women inhibited calcium absorption on bone surfaces and induced alveolar bone destruction. inhibition of calcium absorption may cause calcium on the bone surface to be released. this calcium may subsequently be discharged into the immediate blood stream and then be excreted through the epithelium of gingival into the sulcus gingiva that is dissolved in gcf.12,13 the amount of calcium release in gcf depends on the gcf flow during periodontitis which is higher than during gingivitis. the higher the gcf flow, the greater the calcium level in gcf.14 although the study revealed no significant differences between the phosphor level of gcf in the groups (p>0.05), the phosphor level of perimenopausal women with periodontal diseases was higher than that of their pre and postmenopausal counterparts. this could be related to calcium levels in gcf whose phosphor, in phosphate form, iss often bound with calcium. if the calcium level in gcf increases, the phosphor level in gcf also rises. most phosphor is located in bones and teeth, about 85% of the total within the human body, with small amounts bound with calcium, magnesium, and sodium in extracellular fluid.15 changes in phosphor levels can often be associated with bone and teeth disorders. however, phosphor levels in the osteoporosis serum of postmenopausal women was lower than that of their counterparts without osteoporosis.16 calcium and phosphate homeostasis is related to the biological effects of calctonin in bone metabolism.17 in this study, the lowest magnesium level in gcf was during the post-menopause with periodontitis phase. the level of periodontitis was significantly lower than that of gingivitis in menopausal women. however, perimenopausal women with gingivitis had a statistically similar magnesium level to that of preand postmenopausal women with periodontitis (p>0.05). it is suggested that inflammation and estrogen deficiency could decrease the magnesium level in gcf and cause bone loss. magnesium is one of the minerals involved in bone turnover that plays a role in bone formation.17 magnesium deficiency inhibits osteocalcin synthesis and secretion, subsequently inducing disruption of figure 1. bar diagrams showing deoxypyridinoline and mineral levels in gcf and the significant differences in inter-group comparisons of: a) deoxypyridinoline levels b) calcium levels c) phosphor levels d) magnesium levels and e) sodium levels. data presented includes: mean and standard errors and significant differences of the lsd multiple comparison test. * significant differences in the gingivitis and periodontitis of menopausal women (p<0.05); † significant differences in periodontitis (p<0.05) in menopausal women; § significant differences in gingivitis (p<0.05) in menopause women; * significant differences in gingivitis and periodontitis in menopausal women (p<0.05) except for gingivitis in perimenopausal individuals and gingivitis and periodontitis in one menopausal phase; † significant difference in periodontitis of menopausal women (p<0.05) except in one menopausal phase; § significant difference in the gingivitis of menopausal women (p<0.05) except in one menopausal phase. 13     † † 0 20 40 60 80 100 120 140 160 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis d p d l ev el in g c f (n m ol / l ) § § §    † 0 1 2 3 4 5 6 7 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis c a l ev el in g c f (p pm )        0 10 20 30 40 50 60 70 80 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis p l ev el in g c f (p pm ) § § §      0 0,5 1 1,5 2 2,5 3 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis m g l ev el in g c f (p pm ) § § §    † 0 100 200 300 400 500 600 700 800 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis n a l ve l o f g c f (p pm ) figure 1. bar diagrams showing deoxypyridinoline and mineral levels in gcf and the significant differences in inter-group comparisons of: a) deoxypyridinoline levels b) calcium levels c) phosphor levels d) magnesium levels and e) sodium levels. data presented includes: mean and standard errors and significant differences of the lsd multiple comparison test. * significant differences in the gingivitis and periodontitis of menopausal women (p<0.05); † significant differences in periodontitis (p<0.05) in menopausal women; § significant differences in gingivitis (p<0.05) in menopause women; *ʹ significant differences in gingivitis and periodontitis in menopausal women (p<0.05) except for gingivitis in perimenopausal individuals and gingivitis and periodontitis in one menopausal phase; †ʹ significant difference in periodontitis of menopausal women a b c d e 13     † † 0 20 40 60 80 100 120 140 160 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis d p d l ev el in g c f (n m ol / l ) § § §    † 0 1 2 3 4 5 6 7 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis c a l ev el in g c f (p pm )        0 10 20 30 40 50 60 70 80 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis p l ev el in g c f (p pm ) § § §      0 0,5 1 1,5 2 2,5 3 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis m g l ev el in g c f (p pm ) § § §    † 0 100 200 300 400 500 600 700 800 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis n a l ve l o f g c f (p pm ) figure 1. bar diagrams showing deoxypyridinoline and mineral levels in gcf and the significant differences in inter-group comparisons of: a) deoxypyridinoline levels b) calcium levels c) phosphor levels d) magnesium levels and e) sodium levels. data presented includes: mean and standard errors and significant differences of the lsd multiple comparison test. * significant differences in the gingivitis and periodontitis of menopausal women (p<0.05); † significant differences in periodontitis (p<0.05) in menopausal women; § significant differences in gingivitis (p<0.05) in menopause women; *ʹ significant differences in gingivitis and periodontitis in menopausal women (p<0.05) except for gingivitis in perimenopausal individuals and gingivitis and periodontitis in one menopausal phase; †ʹ significant difference in periodontitis of menopausal women a b c d e 13     † † 0 20 40 60 80 100 120 140 160 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis d p d l ev el in g c f (n m ol / l ) § § §    † 0 1 2 3 4 5 6 7 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis c a l ev el in g c f (p pm )        0 10 20 30 40 50 60 70 80 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis p l ev el in g c f (p pm ) § § §      0 0,5 1 1,5 2 2,5 3 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis m g l ev el in g c f (p pm ) § § §    † 0 100 200 300 400 500 600 700 800 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis n a l ve l o f g c f (p pm ) figure 1. bar diagrams showing deoxypyridinoline and mineral levels in gcf and the significant differences in inter-group comparisons of: a) deoxypyridinoline levels b) calcium levels c) phosphor levels d) magnesium levels and e) sodium levels. data presented includes: mean and standard errors and significant differences of the lsd multiple comparison test. * significant differences in the gingivitis and periodontitis of menopausal women (p<0.05); † significant differences in periodontitis (p<0.05) in menopausal women; § significant differences in gingivitis (p<0.05) in menopause women; *ʹ significant differences in gingivitis and periodontitis in menopausal women (p<0.05) except for gingivitis in perimenopausal individuals and gingivitis and periodontitis in one menopausal phase; †ʹ significant difference in periodontitis of menopausal women a b c d e 13     † † 0 20 40 60 80 100 120 140 160 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis d p d l ev el in g c f (n m ol / l ) § § §    † 0 1 2 3 4 5 6 7 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis c a l ev el in g c f (p pm )        0 10 20 30 40 50 60 70 80 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis p l ev el in g c f (p pm ) § § §      0 0,5 1 1,5 2 2,5 3 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis m g l ev el in g c f (p pm ) § § §    † 0 100 200 300 400 500 600 700 800 pr em en op au se pe ri m en op au se po st m en op au se pr em en op au se pe ri m en op au se po st m en op au se gingivitis periodontitis n a l ve l o f g c f (p pm ) figure 1. bar diagrams showing deoxypyridinoline and mineral levels in gcf and the significant differences in inter-group comparisons of: a) deoxypyridinoline levels b) calcium levels c) phosphor levels d) magnesium levels and e) sodium levels. data presented includes: mean and standard errors and significant differences of the lsd multiple comparison test. * significant differences in the gingivitis and periodontitis of menopausal women (p<0.05); † significant differences in periodontitis (p<0.05) in menopausal women; § significant differences in gingivitis (p<0.05) in menopause women; *ʹ significant differences in gingivitis and periodontitis in menopausal women (p<0.05) except for gingivitis in perimenopausal individuals and gingivitis and periodontitis in one menopausal phase; †ʹ significant difference in periodontitis of menopausal women a b c d e dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 136 dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 calcium and electrolyte homeostasis. estrogen deficiencyrelated menopause could induce inflammation.16magnesium levels decreased following the inflammation and severity of periodontal disease. 18inflammation causes magnesium deficiency which increases the levels of p substances. p substance levels increase pro-inflammatory cytokines that induce osteoclast activation and bone loss.19 sodium is contained in bone, extracellular fluid, serum and tissue. the sodium in bone constitutes a reserve source for the body and is utilized when the sodium level in serum is low in order to meet muscle tissue, heart and neural needs. moreover, in extracellular fluid, sodium is the major cation which plays a role in cellular osmotic gradient regulation. in females, its level is affected by age-related hormone production.20 the sodium levels in this study increased in postmenopausal women with periodontal diseases. statistically, there were significant differences in sodium levels between the groups (p<0.05), except for perimenopausal women with gingivitis compared to postmenopausal women with gingivitis, premenopausal women with periodontitis compared to postmenopausal women with gingivitis and perimenopausal women with periodontitis compared to postmenopausal women with gingivitis (p>0.05). it was suggested that menopause-related estrogen deficiency affected sodium levels in gcf. sodium level in the gcf of periodontitis patients was higher than in that of individuals with gingivitis. it was suggested that the change was related to gcf flow rate which is higher in cases of periodontitis compared to gingivitis. consequently, the amount of sodium extracted from intracellular and extracellular sources due to gcf in periodontitis-afflicted patients was also higher than in their counterparts suffering from gingivitis.14 borras described how the sodium level in gcf correlated with loss attachment in periodontal disease with sodium in gcf passing through damaged connective and alveolar bone tissue.21 moreover, sodium served as a transporter of other ions, such as calcium, magnesium, and phosphor, enabling them to pass through cell membranes and the epithelium cell layer.8 based on the results of the correlation test, significant correlation existed between dpd and minerals in the gcf of menopausal women with periodontal disease and the ph of gcf. however, there was no significant correlation between the calcium levels and ph of gcf. this might be explained by certain roles of calcium in gcf. two major points of view exist regarding calcium levels in gcf. in the first, calcium enhancement is associated with calcium ion mobilization from bone surface to extracellular fluid inducing bone loss. bone loss also occurs during the menopause because of periodontal disease. menopauserelated estrogen deficiency causes oral microenvironment changes triggering bacteria colonization on dental surface and gingival tissue. it also results in the loss of estrogen receptors on periodontal tissue that play a role in fibroblast proliferation and differentiation. bacteria colonization and virulence-induced inflammation and host response both activate cytokine pro-inflammation and induce bone matrix degradation and bone destruction. furthermore, the cytokines cause altered permeability in the junctional and sulcular epithelium with spaces between cells becoming wider, so that intercellular and extracellular components, such as leukocytes, electrolyte, bone degradation products and inorganic bone matrix components pass through the epithelium to the sulcus gingival area.1,5,8 it was also revealed that the calcium level increased in gcf owing to plaque genesis.14 initially, the colonization of saccharolytic bacteria on dental surfaces altered the acidity of gcf and induced inflammation. this condition promoted bacterial colonization, by saccharolytic and asaccharolytic strains, which resulted in the alkalinity of gcf. changes in ph to alkaline induced the proliferation of anaerobic bacteria which were acid sensitive and whose products negatively affected calcium absorption resulting in calcium being excreted into gcf. nevertheless, calcium in gcf promoted protein precipitation on enamel surfaces whose accumulation causes dental plaque and calculus.8,14 furthermore, changes in the mineral component in gcf might be related with the symptoms of menopause that middle-aged women often report. in this study, perimenopausal and postmenopausal women with periodontal disease had higher calcium and phosphate levels than their pre-menopausal counterparts, although the levels found in the serum might be equal. hypercalcemia and hyperphosphatemia in menopausal women result in cardiovascular and renal diseases.22 these conditions might be related to estrogen deficiency inducing oxidative stress in some organs. estrogen therapy in post-menopausal women can, therefore, reduce cardiovascular disease.23 while in the study reported here the magnesium level in periand post-menopausal women with periodontal diseases was lower than that of pre-menopausal women, it might occur in the serum of periand post-menopausal women. hypomagnesia associated with estrogen deficiency and periodontal inflammation could cause reactive oxidative stress (ros) accumulation that manifests itself in depressive syndrome.22 hirose et al.9 argue that the depressive symptoms of middle-aged women is related to mood disorders and oxidative stress. in conclusion, the dpd and mineral level in gcf could be used as disorder indicators in menopausal women with periodontal diseases. however, this study needs further research in order to investigate the mechanism of dpd and mineral component of gcf as specific and sensitive parameters for menopausal women. acknowledgement this research was supported by an universitas jember research grant. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 137137dharmayanti and kusumawardani/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 131–137 references 1. dharmayanti aws. deoxypyridinoline level in gingival crevicular fluid as alveolar bone loss biomarker in periodontal disease. dent j (maj ked gigi). 2012; 45(2): 102–6. 2. mariotti a. sex steroid hormones and cell dynamics in the periodontium. crit rev oral biol med. 1994; 5(1): 27–53. 3. markou e, eleana b, lazaros t, antonios k. the influence of sex steroid hormones on gingiva of women. open dent j. 2009; 3: 114–9. 4. yoshihara a, hayashi y, miyazaki h. relationships among bone turnover, renal function and periodontal disease in elderly japanese. j periodontal res. 2011; 46: 491–6. 5. bhardwaj a, bhardwaj s. effect of menopause on women's periodontium. j midlife health. 2012; 3(1): 5–9. 6. guiglia r, di-fede o, lo-russo l, sprini d, rini g, campisi g. osteoporosis, jawbones and periodontal disease. med oral patol oral cir bucal. 2013; 18(1): e93–9. 7. khurshid z, mali m, naseem m, najeeb s, zafar m. human gingival crevicular fluids (gcf) proteomics: an overview. dent j. 2017; 5: 1–8. 8. barros sp, williams r, offenbacher s, morelli t. gingival crevicular as a source of biomarkers for periodontitis. periodontol 2000. 2016; 70(1): 53–64. 9. hirose a, terauchi m, akiyoshi m, owa y, kato k, kubota t. depressive symptoms are associated with oxidative stress in middleaged women: a cross-sectional study. biopsychosoc med. 2016; 10: 1–6. 10. phipps ai, ichikawa l, bowles eja, carney pa, kerlikowske k, miglioretti dl, buist dsm. defining menopausal status in epidemiologic studies: a comparison of multiple approaches and their effects on breast cancer rates. maturitas. 2010; 67(1): 60–6. 11. ardiani dk, dharmayanti aws, pujiastuti p. kadar fosfor (p) dalam cairan sulkus gingiva pada penderita penyakit periodontal. idj. 2014; 3(1): 1–9. 12. sharma r, sharma p, kumar p, gupta g. role of magnesium in post-menopausal women with osteoporosis and osteopenia. asian j pharm clin res. 2016; 9:198–9. 13. hosthor ss, mahesh p, priya sa, sharada p, jyotsna m, chitra s. quantitative analysis of serum levels of trace elements in patients with oral submucous fibrosis and oral squamous cell carcinoma : a randomized cross-sectional study. j oral maxillofac pathol. 2014; 18(1): 46–51. 14. koregol ac, more sp, nainegali s, kalburgi n, verma s. analysis of inorganic ions in gingival crevicular fluid as indicators of periodontal disease activity: a clinico-biochemical study. contemp clin dent. 2011; 2(4): 278–82. 15. penido mgmg, alon us. phosphate homeostasis and its role in bone health. pediatr nephrol. 2012; 27: 2039–48. 16. razmandeh r, nasli-esfahani e, heydarpour r, faridbod f, ganjali mr, norouzi p, larijani b, khoda-amorzideh d. association of zinc, copper and magnesium with bone mineral density in iranian postmenopausal women – a case control study. j diabetes metab disord. 2014; 13: 1–6. 17. gur a, colpan l, nas k, cevik r, sarac j, erdogan f, duz mz. the role of trace minerals in the pathogenesis of postmenopausal osteoporosis and a new effect of calcitonin. j bone min metab. 2002; 20: 39–43. 18. k asu ma n. cor relat ion bet we en mag nesiu m a nd a l ka l i ne phosphatase from gingival crevicular fluid periodontal diseases. dent j (maj ked gigi). 2015; 48(3): 130–4. 19. he ly, zhang xm, liu b, tian y, ma wh. effect of magnesium ion on human osteoblast activity. braz j med biol res. 2016; 49(7): 1–6. 20. fijorek k, püsküllüoğlu m, tomaszewska d, tomaszewski r, glinka a, polak s. serum potassium, sodium and calcium levels in healthy individuals literature review and data analysis. folia med cracov. 2014; 54(1): 53–70. 21. boras vv, brailo v, rogić d, puhar i, bosnjak a, badovinac a, rogulj aa. salivary electrolytes in patients with periodontal disease. rjpbcs. 2016; 7(2): 8–14. 22. moe sm. disorders involving calcium, phosphorus, and magnesium. prim care. 2008; 35(2): 1–19. 23. terauchi m, honjo h, mizunuma h, aso t. effects of oral estradiol and levonorgestrel on cardiovascular risk markers in postmenopausal women. arch gynecol obstet. 2012; 285: 1647–56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p131-137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p131-137 194194 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 194–199 original article introduction warthin’s tumor is one of the tumors whose incidence rate ranks second among benign salivary gland tumors after pleomorphic adenoma tumors, which is 5%-22% of all neoplasm in the parotid gland.1,2 warthin’s tumor has a characteristic histopathological, a papillary pattern consisting of a two-layered epithelial structure and a cystic lumen as well as a tumor stroma containing lymphoid tissue with a germinal center.2,3 although this tumor is not a malignant tumor, several cases have reported malignant tumors such as lymphoma can develop from the stroma of warthin’s tumor.3,4 cytotoxic t-lymphocyte antigen 4 (ctla-4) is a surface receptor associated with immune blockade mechanisms in tumor cells.5 ctla-4 is one of the immune checkpoints. tumor cells can evade immune defense by producing excess ligand protein molecules that function as immune checkpoints, so that the excess ligand protein produced by tumor cells can be used by tumor cells to avoid recognition and attack by the immune system.6,7 ctla-4, which is normally produced by cells, functions as an inhibitory receptor to downregulate early-stage t-cell activation. it is well known that t cell activation requires antigen recognition by the t cell receptor to produce cd 28 protein which is stimulated by b7 protein. cd28 and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 role of cytotoxic t-lymphocyte antigen 4 (ctla-4) expression in the pathogenesis of warthin’s tumor growth alvionika nadyah qotrunnada1, tecky indriana1, jane kosasih2, meiske margaretha2, mei syafriadi1 1department of biomedical sciences, faculty of dentistry, university of jember, jember, indonesia 2department of patological anatomy, dr. soebandi hospital, jember, indonesia abstract background: one of the benign salivary gland tumors is warthin’s tumor, which is a benign tumor consisting of a papillary cystic structure covered by a double epithelial layer cells and lymphoid stroma with germinal center. several cases have reported the warthin’s tumor transformation into a malignant tumor such as lymphoma that develops from their stromal. expression of cytotoxic t-lymphocyte antigen 4 (ctla-4) as part of the immune checkpoint when highly expressed leads to a more rapid development or progression of tumors. purpose: to analyze ctla-4 expression in warthin’s tumors associated with the pathogenesis of its growth through an escape mechanism from immune checkpoints and analyze based on ctla expression whether this marker has the potential to be used as immunotherapy by administering anti ctla-4. methods: the tissue sections slides of warthin’s tumor (n=8) were stained with hematoxylin eosin and immunostained with recombinant anti-ctla4 antibody [cal49] (ab237712). the slide with positive ctla-4 is shown as staining on the cell membrane and/or cytoplasm. observations were carried out using optilab. the result is presented as figures. results: tumor cells expressed of ctla-4 show in cytoplasm and/or cell membranes of the epithelial and stromal components of warthin’s lymphoid. ctla-4 is expressed lymphoid stroma, which is associated with inhibition of t cell activity against tumor cells, while the exact mechanism of ctla-4 expression in epithelial components is not known but is thought to induce tumorigenesis and inhibit apoptosis. conclusion: ctla-4 is expressed in epithelial and stromal cells of warthin’s tumor and this expression indicates that warthin’s tumor cell growth is through the escape mechanism of the ctla-4 check point immune. further research is necessary to investigate whether ctla-4 expression in lymphoid stroma has relate to their transformation toward a malignant tumor of lymphoma. keywords: cytotoxic t-lymphocyte antigen-4 (ctla-4); immune escape; warthin’s tumor correspondence: mei syafriadi, department of biomedical sciences, faculty of dentistry, university of jember, jl. kalimantan no. 37, jember, 68121 indonesia. email: didiriadihsb@gmail.com mailto:didiriadihsb@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 195 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 194–199 ctla-4 have the same ligand, namely b7 but ctla-4 has a stronger affinity for b7. the stronger the stimulatory signal resulting from the binding of t cell receptor (tcr) cd28 to b7 on the surface of the antigen presenting cell will induce the production (upregulated) of ctla-4. if ctla-4 binds to b7, it will inhibit the immune system from fighting cancer cells (figure 1).8 ctla-4 which is expressed by tumor cells is triggered by infiltration of conventional t cells or treg cells and can also be expressed by tumor cells themselves.9,10 in addition, it was stated that ctla-4 is considered a “leader” of the inhibition of early activation of t cell, thus, it plays a very important role in the defense mechanism of the immune system.11 expression of ctla-4 as part of the immune checkpoint when highly expressed leads to a more rapid development or progression of tumors.12 based on the above description, this study aims to understand whether warthin tumors express ctla-4 and use the ctla-4 pathway to inhibit tumor infiltrating cells in order to escape from immune checkpoints for their growth and progression and also to understand the possible use of immunotherapy based on the location of ctla-4 expression in these tumor-forming cells. several studies reported that anti-ctla-4 therapy can be used as immune therapy in certain malignant tumors because ctla-4 immunotherapy inhibitors of monoclonal antibodies with immune checkpoint blockade mechanisms are being studied and developed.8–10 materials and methods this research uses a retrospective method and has received approval from the ethics committee at the dentistry faculty, university of jember no. 1272/un25.8/kepk/ dl/2021. the samples were paraffin embedded block cases warthin’s tumor patients who diagnosed at the anatomical pathology laboratory, dr. soebandi hospital, jember and have been treated. the sample selection was carried out using a purposive total sampling technique and obtained eight cases of warthin’s tumor and two cases breast cancer grade iii as the positive control of primary antibody. all paraffin embedded-tissue block were cut 4 µm in thick and stained with hematoxylin eosin (he) to confirm the diagnosis of sample and immunohistochemistry (ihc) anti-ctla-4.13 immunohistochemical staining was done according to datasheet protocol. all tissue samples after being dried for 3x24 hours on a slide warmer then followed by a series of processes such as removal of paraffin wax, rehydration, and washing with phosphate buffered saline (pbs) ph 7.4. the antigen retrieval site was carried out using a citrate buffer solution (ph 6) which was heated in an autoclave at 121°c for 15 min, following by cooling for 30 min at room temperature. all samples then washed in pbs. then added drops of h2o2 block at room temperature for 10 minutes to inhibit endogenous peroxidase, following by intense washing with pbs. all tissue samples were covered by protein block at room temperature for 10 min and rinsed with pbs. sections were applied with primary antibody recombinant anti-ctla-4 antibody [cal49] (ab237712) (abcam, cambridge, england) in a ratio of 1:100, then incubated at 4°c overnight and rinsed with pbs. added biotinylated goat anti-rabbit igg (abcam, cambridge, england) for the sections, incubation for 10 min at room temperature and rinsed with pbs. followed by adding streptavidin peroxidase for 10 min at temperature room and adding 3,3’-diaminobenzidine (dab) chromogen solution (abcam, cambridge, england) on the surface of tissue samples and incubating at temperature room for 10 min. tissue samples were counterstained with mayer’s hematoxylin as long as 1 min. the tissue samples were dehydrated in a graded series of alcohol (96% ethanol, 100% ethanol), cleared with xylene, covered with mounting medium and deck glass. this observation was carried out by four observers consisting of two anatomical pathology specialists, one consultant oral and maxillofacial pathology specialist and one researcher. the positive expression of pd-l1 was determined as the cytoplasm and/or membrane of immune cells and tumor cells stained by dab, while the cell nuclei stained purple figure 1. mechanism of ctla-4 functions in tumors. ctla-4 shares the same b7 ligands as cd28, including b7-1 (cd80) and b7-2 (cd86) with a negative effect on t cell activation. after activation of the tcr, ctla-4 induces idoleamine-2, 3-diocygenase (ido), promotes expression of the casitas-b-lineage lymphoma (cbl)-b protein, inhibits the formation of zeta-associated protein 70kda (zap-70) and also induces inhibitory pi3k/akt, cyclin d3-cdk4 /6 and nf-кb for negative regulation of t cell proliferation and activation by producing inhibitory signals to attenuate immune responses (source: zhao et al).8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 196qotrunnada et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 194–199 100x 400x 1000x he ihc (basophilic) by hematoxylin. immunohistochemical and he staining were evaluated by using binocular microscope (olympus cx43, tokyo, japan) and digitally scanned using optilab (miconos®, yogyakarta, indonesia). the result is presented as figures. results this study assessed the expression of ctla-4 protein in warthin’s tumors. based on histopathological analysis with he staining, it was found that all samples (100%) were warthin’s tumors showing the structure of bilayer oncotic epithelium and lymphoid stroma. all case samples of warthin’s tumor showed immunopositive of ctla-4 (100%) both in the epithelial cells and in the lymphoid stroma (figure 2), when viewed in the lymphoid stroma, even though they were immunopositive in the germinal center and dense lymphoid components of ctla-4, they were expressed with weak to strong expression (figure 3). in addition, figure 2 also shows that the expression of dense lymphoid components is less than that of the germinal center. expression of ctla-4 was seen in the cytoplasm and/or cell membranes of both epithelial and stromal lymphoid cells. of the eight case samples of warthin’s tumor, all of the epithelial cells (100%) expressed ctla-4 in the cytoplasm while six samples (75%) of the epithelial cells expressed ctla-4 in the cell membrane and there was anomalous expression of ctla-4 in the epithelial cell nuclei (figure 4). discussion based on the results of ctla-4 immunohistochemical staining, all samples showed a immunopositive expression of ctla-4 in tumor epithelial cells (oncocytes like cells) and lymphoid stroma. in t-cells, ctla-4 was a downregulate of t-cell immune function that could inhibit the early stages of t-cell activation. high ctla-4 expression leads to more rapid development or progression of tumors.12 in the early phase of tumorigenesis, ctla-4 could decrease t cell activity by producing inhibitory signals to weaken the immune system against tumors through binding of ctla-4 to cd80/cd86.8 the presence of high expression of ctla-4 in lymphoid stroma might be related to the pathogenesis of warthin’s tumor, which could transform to malignancy, such as malignant lymphoma. there have been several hypotheses regarding warthin’s stromal tumor to date. thus, further research was needed on the function of the lymphoid stroma of warthin’s tumor and the relationship between ctla-4 and malignancy.8 tumor cells have the ability to escape immune cells associated with pd-l1/pd-1 and ctla-4 overexpression.5 the presence of ctla-4 expression in the stroma of warthin tumors (lymphoid and/or germinal centre area) is thought to have a role in the immune escape mechanism. that is, it is likely that the germinal center containing lymphocyte cells in warthin’s stromal tumor is part of the tumor whose function is already abnormal and is not able to block the growth of tumor cells.8 figure 2. ctla-4 overexpression in epithelial and stromal cells of warthin’s tumor. figure (a-c) shows he staining and image (a’-c’) shows ihc staining. on ihc staining, the expression of ctla-4 protein was stained as brown (a’). ctla-4 expression is seen in epithelial (yellow arrow) and stromal cells (black arrow) (b’). warthin’s tumor. ctla-4 expression appears both in the program (white arrow) and cell membrane (red arrow) (c’). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 197 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 194–199 100x 400x 1000x he ihc he ihc figure 3. ctla-4 expression in the lymphoid stroma of warthin’s tumor. figure (a-b) shows he staining and image (a’-b’) shows ihc staining at 400x magnification. figure (a’) shows ctla-4 overexpression in the dense lymphoid component (black arrow) and germinal center (yellow arrow). figure (b’) shows less ctla-4 expression in the dense lymphoid component (black arrow) than in the germinal center (yellow arrow). figure 4. expression of ctla-4 on warthin’s tumor epithelial cells. figure (a-c) shows he staining and image (a’-c’) shows staining at 1000x magnification. the figure (a’) shows the expression of ctla-in the epithelium in the cytoplasm (yellow arrow) and cell membrane (black arrow). the figure (b’) shows the expression of ctla-4 on the epithelium in the cytoplasm (yellow arrow) and not on the cell membrane (black arrow). the figure (c’) shows the expression of ctla-4 on the epithelium in the cytoplasm (yellow arrow), membrane (black arrow), and cell nucleus (red arrow). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 198qotrunnada et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 194–199 in the lymphoid stroma of warthin’s tumor, expression of ctla-4 could be found in dense lymphoid components and germinal centers. ctla-4 is usually distributed in the germinal center and mantle zone of the lymphatic follicles.14 there were also samples that showed ctla-4 expression in the dense lymphoid component which was less than the germinal center. although many literatures suggest that ctla-4 is expressed on t cells, it is possible that the cells that express ctla-4 in the germinal center are b cells. ctla-4 expression on b cells is thought to be involved in the process of inhibiting the production of igm, igg, and ige. however, the specific mechanisms driving the expression and function of ctla-4 b cells have not been fully accepted beyond their t cell boundaries.15 moreover, until now there have not been many references that discuss the relationship between ctla-4 and the germinal center in a tumor, so further research is needed regarding it and its relation to the lymphoid stroma in warthin’s tumor. in some samples, ctla-4 was not expressed homogeneously in the lymphoid stroma. there were several reasons why ctla-4 is not expressed in t cells. first, the expression level of ctla-4 could be so low that it is not expressed in t-rest cells. ctla-4 predominantly appears to be due to t cell activation.16 second, the nonexpression of ctla-4 might also be related to mutations in the gene encoding the ctla-4 protein in the q33 band of chromosome 2. in addition to the lymphoid stroma, there was a positive expression of ctla-4 in oncocytic epithelial cells. until now, the mechanism regarding this is still unknown.17 zhang et al.18 reported that ctla-4 expression in melanoma cells induces tumorigenesis and inhibits apoptosis. ctla-4 expression on epithelial cells also showed strong expression. cases of malignant transformation in warthin’s tumor are more common from the lymphoid component than from the epithelial component.19 thus, further research is needed to determine whether the high expression of ctla-4 in epithelial oncocytic cells is indeed related to the transformation of warthin’s tumor to malignancy. t cells or other tumor cells that express intrinsic ctla-4 tumor cells are considered cells that have had different functions than t cells or other cells; therefore, studies with immuno-4 immuno-4 can bind to the intrinsic ctla-4 tumor cells and activate the epidermal growth factor receptor (efgr) pathway to induce programmed death-ligand 1 (pd-l1) expression so as to trigger apoptosis of tumor cells.20 the expression of ctla-4, programmed death-1 (pd-1), pd-l1, and egfr in tumor cells can predict the response of therapeutic immunosupnomic treatments by administering anti-pd-1 or anti-pd-l1 as predictive biomarkers of immunotherapy success. thus, the expression of ctla-4 in warthin’s tumor cells, both in tumor epithelial cells and in lymphoid stroma, may have the potential to be a better treatment response. in this research, it showed ctla-4 was expressed predominantly in the cytoplasm in both epithelial and stromal lymphoid cells. in t cells themselves, ctla-4 expressed in the cytoplasm or cell membrane can be seen by assuming that the brown color that approaches or touches the t cell nucleus; this matter indicates the presence of positive ctla-4.21 ctla-4 staining was predominantly cytoplasmic although rarely expressed on the membrane.16 structurally, ctla-4 was the same as cd28, but ctla-4 was an intracellular protein that can rotate between the cell surface and cytoplasm, in contrast to cd28 which is expressed on the cell surface.11 there was also ctla-4 in the study showing strong expression in the nucleus of warthin’s tumor epithelial cells. this possibility is related to the formation of ctla-4 proteins due to gene mutations which mean proteins cannot be transported out of the nucleus and accumulate in the nucleus. in the protein synthesis process, the transcription step was carried out in the cell nucleus and produced mrna, which will be released through the pores of the cell nuclear membrane to the ribosomes by trna.22 this unbalanced or dysfunctional trna problem will be related to the pathogenesis of tumor growth where tumor cells will be uncontrolled proliferation, and this is what is characteristic of a neoplasm. it is also known that trna abundance can affect mrna abundance, so that rna overexpression in cancer can increase protein synthesis.23 when the amount of trna is reduced, there is nothing to carry the mrna out to the ribosomes, and accumulation occurs in the cell nucleus. thus, it is possible that there is a relationship between the expression of ctla-4 in the epithelial cell nucleus of warthin’s tumor with the presence of gene mutations and further research is needed on this matter. from the results of this study, it can be concluded that ctla-4 is expressed in epithelial and stromal cells of warthin’s tumor and this expression indicates that warthin’s tumor cell growth is through the escape mechanism of the ctla-4 check point immune, and it is possible that ctla-4 expression in these tumor cells can be used as a marker for immunotherapy by administering anti ctla-4. however, the potential for ctla-4 to be used as an immunotherapy needs further research. references 1. diaz-segarra n, young lk, levin k, rafferty w, brody j, koshkareva y. warthin tumor of the oropharyngeal minor salivary gland. sage open med case reports. 2018; 6: 2050313x1881871. 2. neville b, damm d, allen c, chi a. oral and maxillofacial pathology. 4th ed. missouri: wb saunders, elsevier; 2016. p. 449–450. 3. kuzenko y v, romanuk am, dyachenko oo, hudymenko o. pathogenesis of warthin’s tumors. interv med appl sci. 2016; 8(2): 41–8. 4. ozkök g, taşlı f, ozsa n n, oztürk r, postacı h. diffuse large b-cell lymphoma arising in warthin’s tumor: case study and review of the literature. korean j pathol. 2013; 47(6): 579–82. 5. kumar v, abbas a, aster j. robbins basic pathology. 10th ed. elsevier; 2017. p. 225. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 199 qotrunnada et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 194–199 6. peng z, su p, yang y, yao x, zhang y, jin f, yang b. identification of ctla-4 associated with tumor microenvironment and competing interactions in triple negative breast cancer by co-expression network analysis. j cancer. 2020; 11(21): 6365–75. 7. jiang x, wang j, deng x, xiong f, ge j, xiang b, wu x, ma j, zhou m, li x, li y, li g, xiong w, guo c, zeng z. role of the tumor microenvironment in pd-l1/pd-1-mediated tumor immune escape. mol cancer. 2019; 18(1): 10. 8. zhao y, yang w, huang y, cui r, li x, li b. evolving roles for targeting ctla-4 in cancer immunotherapy. cell physiol biochem. 2018; 47(2): 721–34. 9. lawrenti h. perkembangan imunoterapi untuk kanker. cermin dunia kedokt. 2018; 45(8): 616–22. 10. seidel ja, otsuka a, kabashima k. anti-pd-1 and anti-ctla-4 therapies in cancer: mechanisms of action, efficacy, and limitations. front oncol. 2018; 8: 86. 11. buchbinder ei, desai a. ctla-4 and pd-1 pathways: similarities, differences, and implications of their inhibition. am j clin oncol. 2016; 39(1): 98–106. 12. szostak b, machaj f, rosik j, pawlik a. ctla4 antagonists in phase i and phase ii clinical trials, current status and future perspectives for cancer therapy. expert opin investig drugs. 2019; 28(2): 149–59. 13. syafriadi m, ummah dz, muna ai, surya mek. progressivity analysis of pleomorphic adenoma toward carcinoma ex pleomorphic adenoma. dent j (majalah kedokt gigi). 2022; 55(1): 1–6. 14. tiemann m, atiakshin d, samoilova v, buchwalow i. identification of ctla-4-positive cells in the human tonsil. cells. 2021; 10(5). 15. oyewole-said d, konduri v, vazquez-perez j, weldon sa, levitt jm, decker wk. beyond t-cells: functional characterization of ctla-4 expression in immune and non-immune cell types. front immunol. 2020; 11: 608024. 16. paulsen e-e, kilvaer tk, rakaee m, richardsen e, hald sm, andersen s, busund l-t, bremnes rm, donnem t. ctla-4 expression in the non-small cell lung cancer patient tumor microenvironment: diverging prognostic impact in primary tumors and lymph node metastases. cancer immunol immunother. 2017; 66(11): 1449–61. 17. navarrete-bernal mgc, cervantes-badillo mg, martínez-herrera jf, lara-torres co, gerson-cwilich r, zentella-dehesa a, ibarrasánchez m de j, esparza-lópez j, montesinos jj, cortés-morales va, osorio-pérez d, villegas-osorno da, reyes-sánchez e, salazar-sojo p, tallabs-utrilla lf, romero-córdoba s, rochazavaleta l. biological landscape of triple negative breast cancers expressing ctla-4. front oncol. 2020; 10: 1206. 18. zhang b, dang j, ba d, wang c, han j, zheng f. potential function of ctla-4 in the tumourigenic capacity of melanoma stem cells. oncol lett. 2018; 16(5): 6163–70. 19. nguyen ka, thai ta, giang ct. malignant transformation in a parotid warthin’s tumor: clinical peatures and histopathological examination. j cancer biol res. 2018; 6(1): 1115. 20. zhang h, dutta p, liu j, sabri n, song y, li wx, li j. tumour cell-intrinsic ctla4 regulates pd-l1 expression in non-small cell lung cancer. j cell mol med. 2019; 23(1): 535–42. 21. brown c, sekhavati f, cardenes r, windmueller c, dacosta k, rodriguez-canales j, steele ke. ctla-4 immunohistochemistry and quantitative image analysis for profiling of human cancers. j histochem cytochem. 2019; 67(12): 901–18. 22. nurhayati b, darmawati s. biologi sel & molekuler. jakarta: pusat pendidikan sumber daya manusia kesehatan, kementerian kesehatan republik indonesia; 2017. p. 71. 23. chen m, long q, borrie ms, sun h, zhang c, yang h, shi d, gartenberg mr, deng w. nucleoporin tpr promotes trna nuclear export and protein synthesis in lung cancer cells. plos genet. 2021; 17(11): e1009899. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p194–199 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p194-199 174174 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 174–178 case report bilateral ramus mandibulectomy with plate reconstruction in ameloblastic carcinoma patient eunike lay1,3, widodo ario kentjono2,3 1resident in department of otorhinolaryngology head and neck surgery, faculty of medicine, universitas airlangga, surabaya, indonesia 2department of otorhinolaryngology head and neck surgery, faculty of medicine, universitas airlangga, surabaya, indonesia 3dr. soetomo general hospital, surabaya, indonesia abstract background: ameloblastic carcinoma is a rare and malignant odontogenic tumour possibly arising de-novo from pre-existing ameloblastoma. it is aggressive and locally destructive. ameloblastoma is the most common benign odontogenic tumour of the mandible. it originates from the tooth-forming epithelium, where its aetiology remains unknown. ameloblastoma usually grows slowly, is asymptomatic, and destroys the surrounding bone tissue. malignant transformation of ameloblastomas may occur spontaneously. resection is the primary therapy for ameloblastic carcinoma with extensive bone destruction. mandibular resection causes instability due to the missing parts of bone, so reconstruction is needed. purpose: this study will report on an individual case of ameloblastic carcinoma that underwent a bilateral ramus mandibulectomy with reconstruction using the plate technique. case: bilateral ramus mandibulectomy with plate and reconstruction in an ameloblastic carcinoma patient. case management: two months after surgery, the patient could open her mouth functionally and aesthetically. conclusion: plate reconstruction is an option for reconstructing bilateral ramus mandibulectomy of a large ameloblastic carcinoma of the mandible. keywords: ameloblastic carcinoma; ameloblastoma; bilateral ramus mandibulectomy; plate reconstruction correspondence: widodo ario kentjono, department of otorhinolaryngology head and neck surgery, faculty of medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no.47, surabaya, 60132, indonesia. email: prof.ariounair@gmail.com introduction mandibular ameloblastoma is a benign odontogenic tumour that is slow-growing and invasive of surrounding structures.1 this tumour occurs between the ages of 30–60 and has a matching rate of occurrence in males and females.2,3 ameloblastoma is divided into unicystic, multicystic, peripheral, and desmoplastic ameloblastoma.3 the ameloblastoma keeps developing into a malignancy divided into metastatic ameloblastoma and ameloblastic carcinoma.4 ameloblastic carcinoma is a rare and malignant odontogenic tumour that contributes to 1% of all jaw tumours and possibly arises de-novo from pre-existing ameloblastoma.5 the diagnosis is made by panoramic radiography, computed tomography (ct) scan, and histopathology examination.6 management of mandibular ameloblastic carcinoma is by surgery, either conservative or radical. conservative surgeries include enucleation, curettage, excision, and marsupialization. radical surgeries include resections such as marginal, segmental, and total resection of the jaw (maxilla/mandible) with wide margins.7 there is relatively high recurrence with conservative surgery at 60%, whereas radical surgery is at 10%.1,4 the best chance of healing requires a wide resection with a margin of about 1–1.5 cm.4 post-resection reconstruction aims to restore shape for muscle attachment, chewing, swallowing, speaking functions, and cosmetics. several techniques commonly used to reconstruct the mandible are osteocutaneous vascularised bone graft, nonvascularised bone grafts, and plate reconstruction with/without soft tissue pedicle flaps.1 the purpose of this paper is to report on a case of ameloblastic carcinoma that underwent resection and reconstruction using a plate and screw. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p174–178 mailto:prof.ariounair@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p174-178 175 lay et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 174–178 case a 58-year-old woman came into the outpatient clinic with a five-year history of a gradually expanding painless mass in the lower left jaw. the patient could only eat soft food. physical examination revealed a solitary mass in the left mandibular region measuring 15 cm x 12 cm x 10 cm, causing a marked facial asymmetry. the mass was cystic to touch, fixed, hard consistency, and had normal mucosal colour resembling surrounding tissue. intraoral examination revealed a solitary mass in the left mandibular region, an almost entirely missing tooth, and no trismus. there were no palpable lymph nodes in the neck (figure 1). case management the panoramic photo showed a primary bone tumour suspected to be chondroblastoma. the ct scan of the head and neck showed a thin septate expansive cystic lesion and a geographic type of destruction. there was a narrow transitional zone, popcorn calcification, and soap bubble appearance of 19 hounsfield units (hu). also shown was its size ± 10 cm x 8.6 cm x 10.1 cm in the right and left mandibular up to the left mandibular angle with no significant contrast enhancement (23 hu) in the solid lesion, septa, and floating teeth. the ct scan suggested ameloblastoma or an odontogenic cyst (figure 2). the fine-needle aspiration biopsy (fnab) results revealed a benign cystic lesion. an open biopsy was not performed because the patient refused. the patient was diagnosed with mandibular ameloblastoma, and surgery was scheduled for a bilateral ramus mandibulectomy and mandibular plate reconstruction. the mandibular resection was carried out on march 14, 2019, and a pre-operative tracheotomy was performed. an incision was made to divide the lower lip from the midline along the mental region backward to the submandibular region. then a flap was made upwards past the lower border of the mandible to preserve the mandibular ramus and facial nerve. blunt dissection separated the masseter muscle from the bone and tumour mass. the right and left mandibles were cut with a gigli saw about 1.5 cm beyond the tumour margin, then the left and right mandibular ramus were left (± 2.5 cm). after removing the tumour, cauterization was performed in the centre of the residual bone segment to prevent a recurrence. mandibular reconstruction, along with plate and screw, was made of titanium. the plate was shaped according to the mandibular arch and later placed in order occlusion. it was installed with screws on the left mandibular with a two-piece segment and a three-piece segment on the right a b c figure 1. clinical appearance; a) front view of the face; b) side view; c) sublingual mass and the remaining teeth are shown. c b a figure 2. ct scan of multicystic head and neck. a) coronal section; shows the popcorn calcification. b) sagittal cut shows the soap bubble appearance. c) axial cut. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p174–178 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p174-178 176lay et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 174–178 b a d c g f e h figure 3. surgery stages: (a). disinfection of the surgical area after tracheostomy and intubation; (b). separation of tumour mass from nearby organs; (c). the mandible was cut using a gigli saw; (d). the left mandible resected; (e). the type and size of the plate used (arrow); (f). inserted plates and screws on the mandible, the left side with three screws and the right side with two screws; (g). gingivobuccal mucosa suturing; (h). multicystic tumour weighing 800 grams. a b 2 1 2 1 figure 4. a. 5th postoperative day: (1). shut mouth; (2). mouth open about 2 cm wide. b. two months postoperatively: (1). shut mouth; (2). open mouth, max-width. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p174–178 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p174-178 177 lay et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 174–178 mandibular. the mylohyoid muscle and soft tissue were sutured around the plate. the gingivobuccal mucosa was sutured to the floor of the mouth using the watertight and airtight principles (figure 3). vacuum drains were placed on the right and the left of the submandibular. the operation results showed the mandibular tumour was multicystic, measuring 17 cm x 12 cm x 8 cm, and weighed about 800 grams. postoperative histopathology was ameloblastic carcinoma with tumour-free resection margins. furthermore, the recovery treatment took ten days with nutrition provided through a nasogastric tube. by the fifth day, the surgical wound was dry, and the patient could open her mouth about 2 cm. at two months postoperative, her mouth could be opened normally, and she had oral nutrition intake (figure 4). we consulted with the patient at the dental and oral clinic, but a denture (prosthetic) could not be installed because there were no teeth for fixation. the patient did not receive chemoradiation because the plate and screw were attached. fortunately, no lymph node enlargement and tumour-free resection edges were found in the histopathological results. the patient was scheduled for an x-ray evaluation three months postoperatively, but the patient did not appear for control, and contact was lost. discussion these tumours could occur in anyone between 30–60 years old. the ratio of occurrence is the same for males and females.2 the signs and symptoms of mandibular ameloblastoma are swelling of the mandible, painlessness, loose teeth, and even loss of chewing and swallowing disorders.2,4 the patient in this study was a 58-year-old woman. chief complaints were a large lump in the lower left jaw that was painless and incomplete teeth. based on fnab results, benign cystic lesions were shown. furthermore, the ct scan showed ameloblastoma and odontogenic cysts. the ct scan of a multilocular type of ameloblastoma showed a classic soap bubble appearance.8 the patient was diagnosed with mandibular ameloblastoma. in case reports of the bilateral ramus, mandibulectomy is performed from the left mandibular ramus to the right. a bilateral bounded mandibulectomy is a resection of the anterior aspect of the mandible crossing the midline with an intact posterior mandibular segment bilaterally. it is further divided into five classes descriptively, one of which was bilateral ramus mandibulectomy.9 in this case, resection was performed about 1.5 cm outside the tumour with the remaining bone segments, with the right and left mandibular ramus (± 2.5 cm) as the plate placement. after the tumour was removed, the centre of the residual bone segment was cauterized. the best treatment option for ameloblastoma was a radical excision of the tumour mass, reaching the normal bone with a tumour-free margin of 1-2 cm.10,11 to prevent a recurrence, procedures were added by adjuvant therapeutic. this therapy was performed on the intraoperative bone margin through tissue fixation, drilling, or cauterizing bone tissue.8 there was also mandibular reconstruction with a plate and screw in this case. a fibula flap is considered the gold standard of choice owing to its length, bone stock, reliable pedicle, tolerance of dental implants, and low donor-site morbidity. although the free fibula flap provides rigid support, a soft tissue flap with a bridging plate, on the other hand, is often easier to perform as it allows for sample soft tissue and easily achieves defect closure.12 mandibular reconstruction was done using a plate and screw made of titanium and shaped to match the patient’s mandibular arch. the screws inserted were two pieces on the left and three on the right of the mandibular segment. mandibular reconstruction plates and screws are the most widely used alloplastic devices for mandibular reconstruction. the most common metals fabricating these plates are stainless steel, vitallium, and titanium.13 in the reconstruction stage, the operator formed a plate with a hand-forming technique that matched the contour of the patient’s bone. the ideal reconstruction would provide a solid arch to articulate the upper jaw that would restore swallowing, speech, mastication, and aesthetics.11 the mylohyoid muscle and soft tissue were sutured around the plate. the gingivobuccal mucosa was sutured to the mouth floor with watertight and airtight principles. later, the technique of suturing muscle and soft tissue, fixation of internal soft tissue, mucosa, and suturing the skin surface properly and correctly will affect the success of the function of the masticatory muscles in postoperative patients..14 in this case, there were differences in the pre and postoperative histopathological results. preoperatively, the ameloblastoma was determined. consequently, treatment was planned for a benign tumour. at the same time, the postoperative histology results showed ameloblastic carcinoma with tumour-free resection edges. ameloblastic carcinoma represents a malignant transformation of preexisting well-differentiated ameloblastoma or odontogenic cyst.15 because of the location of the plate and screw reconstruction, chemoradiation was not recommended. radiotherapy can be suggested to decrease tumour size before surgery and to improve local control when surgical margins are close or microscopically positive. experience with chemotherapy is minimal in treating ameloblastoma and is primarily limited to isolated cases.11 later, no enlarged lymph nodes were shown, with postoperative histopathological results of tumour-free resection ends. for patients with local recurrence or inadequate margins after surgery, adjuvant radiotherapy provides the potential for disease control.16 the patient was recommended to have regular control to detect recurrence and distant metastases. if recurrence and metastases occur, resection will be carried out.13 this technique was simple and uncomplicated and had a satisfactory success rate. this technique’s success rate is high without requiring microscopes, special techniques, or microsurgery. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p174–178 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p174-178 178lay et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 174–178 7. figueiredo nr, dinkar ad, meena m, satoskar s, khorate m. ameloblastoma: a clinicoradiographic and histopathologic correlation of 11 cases seen in goa during 2008-2012. contemp clin dent. 2014; 5(2): 160–5. 8. al sineedi f, aruveetil ya, kavarodi am, harbi so. bilocular unicystic ameloblastoma of the mandible in a 9 yr old child a diagnostic and management dilemma. saudi dent j. 2018; 30(3): 250–5. 9. adelusi ea. classification of mandibulectomy/mandibular defects. world j oral maxillofac surg. 2019; 2(3): 1032. 10. fahradyan a, odono l, hammoudeh ja, howell lk. ameloblastic carcinoma in situ: review of literature and a case presentation in a pediatric patient. cleft palate-craniofacial j. 2019; 56(1): 94–100. 11. abir b, abouchadi a, tourabi k, lakouichmi m. ameloblastic carcinoma of the mandible: a case report and review of the literature. médecine buccale chir buccale. 2017; 23(2): 95–8. 12. lin ja-j, loh cyy, tsai c-h, chang k-p, wu jc-h, kao h-k. free flap outcomes of microvascular reconstruction after repeated segmental mandibulectomy in head and neck cancer patients. sci rep. 2019; 9(1): 7951. 13. kumar bp, venkatesh v, kumar kaj, yadav by, mohan sr. mandibular reconstruction: overview. j maxillofac oral surg. 2016; 15(4): 425–41. 14. fonseca oliveira mt, soares f, batista jd, de moraes slc, zanettabarbos d. reconstruction of mandibular defects. in: a textbook of advanced oral and maxillofacial surgery. intech; 2013. p. 481–500. 15. gunaratne da, coleman hg, lim l, morgan gj. ameloblastic carcinoma. am j case rep. 2015; 16: 415–9. 16. kennedy wr, werning jw, kaye fj, mendenhall wm. treatment of ameloblastoma and ameloblastic carcinoma with radiotherapy. eur arch otorhinolaryngol. 2016; 273(10): 3293–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p174–178 acknowledgements the author thanks the department of otorhinolaryngology head and neck surgery, faculty of medicine, universitas airlangga, and dr. soetomo general hospital surabaya. references 1.a dham m, musa z, atmodiwirjo p, bangun k. ameloblastoma: hemimandibulectomy and reconstruction with free fibular graft — a case report and review of the literature. int j head neck sci. 2017; 1(4): 251–7. 2.o omens maem, van der waal i. epidemiology of ameloblastomas of the jaws; a report from the netherlands. med oral patol oral cir bucal. 2014; 19(6): e581-3. 3.v ishwani a, goyal k, suman d, arora v, arya s, yadav m. radical resection of giant ameloblastoma mandible with free fibula reconstruction: a case report and review of literature. int surg j. 2020; 7(2): 602–5. 4.m cclary ac, west rb, mcclary ac, pollack jr, fischbein nj, holsinger cf, sunwoo j, colevas ad, sirjani d. ameloblastoma: a clinical review and trends in management. eur arch otorhinolaryngol. 2016; 273(7): 1649–61. 5.p rashad k v, ramesh v, balamurali pd, premlatha b. ameloblastic carcinoma – a case report highlighting its variations in histology. j int oral heal. 2011; 3(6): 37–42. 6.e lmrini s, raiteb m, bahaa razem, hassani fza, slimani f. ameloblastoma giant: diagnosis, treatment and reconstruction: a case report. ann med surg. 2021; 68: 102589. https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p174-178 � vol. 45. no. 1 march 2012 case report immediate overdenture for improving aesthetic of anterior teeth with periodontal problem fx. ady soesetijo department of prosthodontics faculty of dentistry, jember university jember indonesia abstract background: the construction of overdenture is often applied because endodontic treatment usualy give very promising results and patient has high motivation to maintain their natural teeth. overdenture is a removable partial or complete denture that covers and rests on one or more remaining natural teeth, roots and/or dental implants. the presence of retained teeth can maximize retention, stabilization and prevent trauma to the oral mucosa. meanwhile, the presence of root in the bone can delay resorption of the alveolar process. the role of proprioceptor in the periodontal ligament abutment teeth remains effective. thus, it can be said the overdenture treatment is a preventive prosthodontic treatment. purpose: the purpose of this case report was to present a case of maxillary and mandibullary anterior teeth with periodontal disease, through endodontic and prosthodontic treatments for recovering its function of phonetic and aesthetic. case: the 25 years old female with periodontal problems (protrusive, wiggly °1–°2 and along with gingival retraction) on 12, 11, 21, 22 and 32, 31, 41, 42. the patient felt bad about his performance and affect his self confidence. the patient visited tthe dental hospital to restore her teeth and recovering aesthetic and phonetic functions. case management: the overdenture inserted immediately after one visit endodontic treatment and cutting off the clinical crown of the teeth. the adaptation of the denture is needed by relining using self cured acrylic resin. the patient was quite satisfied with the treatment. conclusion: in conclusion, the maxillary and mandibullary anterior teeth with periodontal problem could be managed through conservative and prosthotontic approach of treatment to recover of its performance and function. key words: immediate overdenture, endodontic, relining abstrak latar belakang: konstruksi overdenture sering diaplikasikan pada pasien, karena perawatan endodontik memberikan hasil perawatan yang sangat menjanjikan dan pasien memiliki motivasi tinggi untuk mempertahankan gigi asli mereka. overdenture adalah gigi tiruan lepasan sebagian atau lengkap yang bertumpu pada satu atau lebih gigi asli yang tersisa, akar dan/atau implan gigi. gigi asli yang tersisa di dalam mulut dapat memaksimalkan retensi, stabilisasi dan mencegah trauma pada mukosa oral. selain itu, dipertahankannya sisa akar gigi dapat menghambat resorpsi tulang alveolar serta peran proprioseptor ligamen periodontal tetap efektif. dengan demikian, dapat dikatakan bahwa perawatan overdenture adalah perawatan prostodontik pencegahan. tujuan: tujuan dari laporan kasus ini adalah untuk mempresentasikan kasus gigi-gigi anterior rahang atas dan rahang bawah dengan penyakit periodontal melalui perawatan endodontik dan prostodontik untuk memulihkan fungsi fonetik dan estetik. kasus: wanita usia 22 tahun dengan masalah periodontal (protrusi, goyang °1–°2 dan disertai dengan retraksi gingival) pada 12, 11, 21, 22 dan 32, 31, 41, 42. pasien datang ke rumah sakit gigi dan mulut ingin memperbaiki giginya untuk mengembalikan fungsi estetik dan fonetik. pasien merasa kurang percaya diri terhadap penampilannya. tatalaksana kasus: overdenture dipasang segera setelah perawatan one visit endodontik yang diikuti dengan pemotongan mahkota klinis. relining dengan resin akrilik diperlukan untuk menyesuaikan gigi tiruan terhadap jaringan pendukungnya. pasien cukup puas dengan perawatan ini. kesimpulan: disimpulkan bahwa, gigi-gigi anterior rahang atas dan rahang bawah dengan masalah periodontal dapat diperbaiki melalui pendekatan perawatan konservasi dan prostodonsi untuk memperbaiki penampilan dan fungsi. kata kunci: imidiat overdenture, endodontik, relining � dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 1–5 introduction a patient who still has complete natural teeth, but having problems with anterior teeth such as wiggling; protrusion; extrusion are cases frequently found in dental practice. these problems are usually resolved with extraction or replacing them with conventional denture. however, in a certain time, the user of this denture may perceive either reduction of its retention and stability, or looseness which generally relates to resorption in the alveolar bone occurred physiologically or pathologically. tooth extraction, subsequently replaced by removable denture, has some impacts. the patient needs to adjust himself to the new situation such as speaking, masticating, and swallowing. besides, the patient may get psychological and social problems. physically, there may be an occlusion change, vertical dimension, and alveolar bone reduction.1,2 it is common when a patient who has anterior teeth with poor condition, for instance, uneven structure affecting its aesthetic; or having periodontal problems, needs help from a dentist to get restoration or treatment. in this globalization era, many people ask for immediate, accurate, and qualified treatment, consequently the improvement of the dental health service related to prosthodontic treatment is necessarily conducted. one of services that can be provided in relation to the globalization demands is a treatment using overdenture (od). od may be a partial denture or full removable denture, which covers up and rests on one or more remaining natural teeth, toot root, or dental implant. in general, retained teeth have an endodontic treatment. they enable to inhibit the resorption process, which desirably can obtain a maximum retention and stability. thus, overdenture treatment is a preventive prosthodontic treatment. retained teeth, which lay under the overdenture basis, may preserve the height of alveolar ridge that make overdenture remain stable. this stability is possibly preserved any longer, because the masticating weight is more easily controlled and accepted evenly by the retained teeth and their adjacent tissue.3,4 periodontal retained teeth (the retained teeth under overdenture) are essential to consider since only the potential teeth that may be employed as props of overdenture. those teeth should have minimum mobility and depth of sulcus that can be treated on which the gingival attaches well.4,5 crown/root ratio if tooth crown cutting reaches the border of gingival margin, it may increase the crown/root ratio and tooth looseness may be reduced up to 40% allowing the functional stimulation accepted by those teeth possibly tolerate.6,7 likewise the conventional denture construction, od can be constructed immediately, therefore it is named immediate overdenture (iod), on which has previously been conducted endodontic and periodontal treatments. further, iod needs to be well-adjusted to its supporting/ adjacent tissue by grinding and relining before insertion, that is why it is also called temporary denture. it is also said that in the adjustment stage, it is advisably conducted relining by using tissue conditioner material. after the patient has adapted to the denture, furthermore, definitive denture can be made or replace the relining material on his denture with acrylic resin material.3,8 the purpose of this case report was to present a case of maxillary and mandibullary anterior teeth with periodontal disease, through endodontic and prosthodontic treatments for recovering its function of phonetic and aesthetic. case a 25 years old female student felt some distance among her front teeth since 7 months ago. there was much plaque on the upper and lower anterior teeth. the patient went to a dentist in order to have the plaque cleaned. the therapy conducted was a flap operation, foremost, on 12, 11, 21, 22, 32, 31, 41, 42. the result of the treatment showed there was no progress, precisely, it seemed to be severer. examination indicated that those teeth were wiggly between °1–°2. there were also gingival retraction (the severest were 11, 12, 21), and defect on the regio between 11 and 21. four upper incisive were found with multiple diastema, of which position was protrusion that made the patient feel less confident toward its aesthetic appearance because of having problem to close her upper lip (figure 1). therefore, the patient really needed restoration using denture to recover its aesthetic and phonetic functions. patient with fully devolved to the operator about the kinds of dentures. at the same time, x-ray photos were also conducted at 12, 11, 21, 22, 32, 31, 41, 42 for making the diagnosis and treatment plan. radiographic interpretation shows the ratio of crown/root 1:1, and no periapical abnormalities found. the treatment plan will do is make the iod for 12, 11, 21, 22, 32, 31, 41, 42. correspondence: fx ady soesetijo, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: fx.adys_drg@yahoo.com figure 1. early condition of the patient’s teeth. �soesetijo: immediate overdenture for improving aesthetic of anterior teeth case management when the patient came at the first time, the intra oral and extra-oral examinations were directly conducted. the anatomical impression of the maxillary and mandibulary teeth were made in order not only to get study models and dental records, to know the occlusion and relation, but also to determine the denture design and to prepare individual design tray. the next visit, the physiological impression of the maxillary and mandibulary teeth were made with elastomeric impression materials, and then reproduces with dental stone into the master model, where on the master model is constructed iod. the first stage, 11, 21, 22, 31, 32, 41, 42 on the master model were cut up to a 1 mm above the marginal gingiva. the selection of artificial teeth was adapted to the size and color of the retained teeth using shade guide. arrangement of artificial teeth with respect to the teeth next to them, overjet (3 mm) and overbite (2 mm), antero-posterior and lateral movement. the denture was made from acrylic resin with half jackson on 16, 26, 35, 45 as retention clasps (figure 2). on the last visit, endodontic treatment was done step by step, where it required local anaesthetic. endodontic methods used the traitement spad (laboratoire spad-b.p. n°7-21801 quetigny france). the first step was done on the 32, 31, 41, 42. this step was followed by cutting off their clinical crowns (figure 3). after denture insertion, path of insertion was checked (figure 4). the last step was done on the 12, 11, 21, 22. the procedure was similar with the first step. inter roots distance was checked in order to make the arrangement of the artificial the artificial teeth, and at the same time the root surface of the teeth were protected with fluoride (figure 5). the occlusion and articulation were grinding checked with articulating paper (occlusal grinding was done) and relining (using a tissue conditioner) was done on the denture base. removal of the denture to be cleaned of soft liner scraps, and then polishing and finishing. on the final insertion (figure 6), the occlusion, articulation and appearance of the patient were carefully observed (figure 7). in addition, patient education was important in order that she understand to maintain the denture. besides that, periodically control is needed, i.e. on 1 day, 3 days, 1 week and 1 month after the treatment. figure 2. removable partial denture upper and lower jaws element and basis from acrylic. mandibular and maxillary removable partial denture with half jackson on 16, 26, 35, 45 as retention clasps. figure 3. the cutting of clinical crowns after endodontic treatment on 32, 31, 41, 42. figure 4. trial of lower overdenture. figure 5. clinical crown cutting after endodontic treatment on 12, 11, 21, 22, 32, 31, 41, 42 and continued with fluoride protector application. � dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 1–5 in this case, if the financial standing becomes the main problem in relation to the patient’s inadequacy, the other alternative treatment may be provided with immediate denture (id). however, the use of iod obviously seems to be much more prominent compared to id. id is a temporary denture, because in the restoration process, alveolar ridge reduction leading to the denture instability or looseness. id requires continuous relining.12 it is different from iod, the presence of teeth or remained roots from the original teeth may provide proprioseptive stimulation from receptor appropriately, it makes the occlusal excess capable to send a signal to muscles in order to reduce their contractions.4,6,13 other alternative treatment possibly conducted is by making pin crown on the lower jaw, but the crown may be only constructed after endodontic treatment and teeth cutting, and waiting the reduction of teeth looseness that, of course, make the patient suffer from edentulous phase. however, this method is impossibly applied on the upper jaw because of poor aesthetic condition, foremost on the 11 and 21. in the case of denture fitting is done immediately, adjustment between denture base and adjacent tissue is achieved by relining. the first stage of relining is advisable to use tissue conditioner intended to adjust the adjacent tissue (especially tooth roots) in order to accept masticating force. after the adjacent tissue has been adjusted, the definitive relining material may be applied using self-cured acrylic. besides, to preserve the health of tooth roots, it needs to protect from either abrasion or caries, which may be conducted by applying protector fluoride, coping (extended casting metal), or filling (ionomer glass and composite).8,11,14 in this case, insertion is conducted through relining by using tissue conditioner (soft tissue treatment), on the last control (1 month), soft liner is disposed and replaced by self-cured acrylic. all steps of relining work are undergone directly (direct technique). in the first day of control, seemingly, there was a pressure spot presence and no significant complaint. iod basis was still contacted well to its adjacent tissue. further, in the following two weeks, it seemed a little distance between iod basis and its adjacent tissue, the patient also complained about the food remain attached in it. to resolve this problem, the first procedure conducted was relining by using soft liner. after 1 month it indicated that the patient felt satisfied with her denture, there was no complain and it seemed there was no distance between the denture base and its adjacent tissue. in the same time, the soft liner was subsequently replaced by self cured acrylic (figure 8). instruction and promotion about the importance of preserving oral hygiene are expected to grow the patient’s awareness of the importance of oral hygiene that leads her to change her habits. periodical examination within an interval of 6 months will serve appropriate maintenance to the patients’ prosthetic, restoration and periodontal condition. iod is a treatment using conservative and preventive approaches. if the teeth under the iod are retained, they can figure 6. post insertion of upper and lower od. figure 7. the patient’s performance with upper and lower iod. discussion some factors affecting the success of treatment using iod are the accuracy of diagnose, conscientious treatment plan, good co-operation between the dentist and the patient. in this case, some advantages obtained that there is no edentulous phase allowing to fit the denture immediately after conducting endodontic treatment and crown teeth cutting; improving the patient’s aesthetic, of which early condition is poor and disturbs the lips closing.4,6 a patient suffering from edentulous, mainly on the anterior, is risky to the resorption of alveolar bone. therefore, it is very beneficial to preserve the anterior teeth as the props of overdenture. besides, the anterior teeth have significant position to support and stabilize od.9 if the crown teeth is cut above the gingival margin, the wiggling teeth will 40% reduced due to the absence of pressure from the antagonistic teeth. besides, the presence of the retained roots may preserve the alveolar bone and neuromuscular function. the patient with od has masticating ability 1/3 bigger compared to those with conventional denture. however, the patient with od needs longer time to accomplish number of masticating movement.6,10,11 �soesetijo: immediate overdenture for improving aesthetic of anterior teeth protect and assist to preserve the height of alveolar bone. the patient does not necessarily get edentulous phase, and he or she may like this denture type more easily. in this case, the preparation time in needed relatively short due to one visit endodontic treatment. meanwhile, protecting the opened teetn roots surface are carried out by applying fluoride protector. the success of iod treatment highly depends on the patient’s ability to maintain his or her oral hygiene and denture as well as his or her discipline to visit a dentist. in conclusion, the maxillary and mandibullary anterior teeth with periodontal problem could be managed through conservative and prosthotontic approach of treatment to recover of its performance and aesthetic function. the patient was quite satisfied with the treatment. references 1. battistuzzi pgfcm, kayser af, keltjens hmam, plasmans pjjm. gigi tiruan sebagian: titik tolak pada diagnosa dan perawatan dari gigi-geligi yang rusak. kosasih ai, kosasih ar, editors. jakarta: widya medika; 1996 p. 243–51. 2. dupare ap. overdenture: an approach to preventive prosthodontics. jida 2011; 5(3): 366-8. 3. basker rm, harrison a, ralph jp. overdenture in general dental practice. br dent j 1993; 154: 285. 4. damayanti l. overdenture untuk menunjang perawatan prostetik. buku ajar. bandung: fakultas kedokteran gigi universitas padjadjaran; 2009. p. 17–22. 5. lord jl, teel s. overdenture: patients selection, using coping, and evaluation. j prosthet dent 1995; 32: 41–51. 6. hamada t. overdenture. ceramah ilmiah fkg unair; 1995. p. 3–5. 7. zmutzki j, chladek w, krukowska j. loading of overdenture attachments under stimulated bitting forces. int science j 2008; 32(1): 33–6. 8. marinus aj. treatment results with immediate overdenture: an evaluation of 4.5 years. j prosthet dent 1996; 32: 41–51. 9. roldan al, abad ds, bertomen ig, castillo eg. bone resorption process in patients wearing overdenture. med oral pathol 2009; 14(4): 203–9. 10. jonkman reg, van waas maj, van hoff ma, kalk w. an analysis of satisfaction with immediate (over) denture. j prosthet dent 1997; 25: 107–10. 11. borges tf, mendez fa, oliviera tr. overdenture with immediate load: mastication and nutrition. british j nutrition 2010; 105 (07): 990–4. 12. loo wd. ridge preservation with immediate treatment denture. j prosthet dent 1998; 19: 7–11. 13. rutkinas v, mitzutani h, takahashi h. evaluation of stable retentive properties of overdenture attachments. stomatol baltic dent maxillofacial j 2005; 7: 115–20. 14. keltjens hmam, schaeken mjm, hoeven js, hendriks jcm. caries control in overdenture patients: 18 months evaluation on fluoride and chlorhexidine therapies. caries res 1990; 24: 371–5. figure 8. the patient’s condition after wearing od for one month. 181 the application of methacrylate resin and the derivation as restorative material of damaged tooth tissue adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract the application of methacrylate resin and the derivation (composite resin and dentin bonding) in clinical conservative dentistry has been widely developed. this material could be used to restore class i-v cavity with good aesthetic due to the compatible color with tooth. composite resin adhesion hydrophobically in enamel that is due to mechanic retention in the form of resin tags which penetrates into enamel porosity. meanwhile hydrophilic dentin bonding adhesion due to the chemical reaction between functional groups of amino collagen with carbonyl in dentin bonding forming amide binding. in addition mechanical retention in which dentin bonding penetrating into nano inter fibrilar cavity then polymerized. the success of methacrylate resin adhesion restoration is decided by enamel porosity, wetting character of resin, wetting contact angle, good etching acid, optimal humidity of tooth surface, the accuracy of dentist during filling is done etc. key words: methacrylate resin, dentin bonding, hema, water chasing effect, nano filler resin, humidity correspondence: adioro soetojo, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction methacrylate resin and the derivation or in term of conservative dentistry is called composite resin as restoration material which is frequently used due to the strength property such as the color is compatible with the tooth so it has good aesthetic, adequate hardness and strength, good adhesion on enamel and dentin. this resin material was introduced by bowen in 1960’s under the name bis-gma which was mixed of bisphenol a with glycydil methacrylate1–4 this resin is hydrophobic meaning it is only possible well adhered on relatively dry region such as enamel. meanwhile for dentin tissue is generally wet due to the fluid from dentinal tubule, so the resin material would be difficult to perfectly adhere. bowen resin was classified as conventional or traditional resin.5 by the development of science, some kinds of composite resin are produced such as micro filled composite resin, hybrid composite resin and recently it has been introduced nano filler resin. the latest resin has very small/soft filler therefore it has advantage such as hardness and strength is high because the filler particle is more compact, resin surface is smooth high precision and low contraction after polymerization process.6,7 at the clinic, the surface of resin restoration would stay longer in cavity is determined by several factors such as : the operator in cases of preparation design, manipulation and method of resin application, the method of acid etching as well as proper indication of restoration. on the other side, influential factor is from the patient, himself whether the patient pays attention to restoration of his tooth, proper eating habit and hygiene factor on tooth and oral cavity. the purpose of this study is to describe clinical use of methacrylate resin and the derivation as restoration material of tooth decay, adhesive mechanism on tooth surface and the failure which occurs in adhesive process. adhesive resin on tooth structure adhesion process of restoration material on tooth structure is complex including methacrylate resin.1,3 many factors could contribute the failure of resin adhesion on tooth surface, such as whether fluor solution has never been applied in tooth preparation in which it could reduce resin wetting character, the presence of smear layer on tooth surface in which smear layer is debris leftover during cavity preparation or tooth cutting for sample preparation, non homogeneous tooth composition , very different component of organic and inorganic in enamel or dentin. a restoration which could adhere on organic part is not sure to be able to adhere on inorganic part. in contrast, saliva or blood contamination on tooth surface. dentin is a tissue which is always wet due to dentinal tubule. bis-gma resin could not adhere on dentin due to hydrophobic nature. meanwhile hema dentin bonding because of hydrophobic nature, could adhere in dentin tissue, even though the amount of water in dentin is another consideration, too much water content would distribute hema adhesion on dentin.3 physical adhesion of resin is correlated with tags i.e. adhesive resin penetrates into porous enamel after acid etching.1,3 researchers observed the effect crystal 182 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 181-185 orientation in enamel toward acid reaction, it is said that enamel dissolvent in acid is not always the same, depending on which direction acid would destroy the enamel and orientation of enamel prism3,8 and it is further stated that if enamel prism position is parallel with acid entrance so the structure of bee nest could be seen. it means that the most soluble part is the center of prism. in contrast, if the direction is perpendicular, so it would be seen like fibrous structure and the center of the prism would show more resistant to acid. resin adhesive process in dentin tissue is considered more complicated if it is compared with tooth enamel. unlike in enamel, dentin is alive tissue which consists of about 60% inorganic in the form of hydroxy-apatite: ca10(po4)6(oh)2: 30% organic and 10% water. 90% of organic material is collagen and the rest is non collagenous component. most of collagen is type i and only a few of type v. based on component of side chain (r group in amino acid molecule], so collagen is classified into several types i.e. type i until type vii. non collagenous matrix consist of phosphor-protein, proteoglicans, g-carboxy–glutamate which comprises of protein (such as: glaprotein), glycoprotein acid, growth factor and lipid. collagen is protein bio polymer and arranged in triple helix (that fibers plaited each other) the binding between fibers is formed by the presence of hydrogen bond, so, the fiber becomes stiff and strong. collagen fiber is generally in perpendicular direction toward dentinal tubule. collagen in tissue usually functions as formation structure or strengthening tissue such as tendon, dermis, bone, dentinal tooth. type i dentin collagen using no research material is usually a chain of amino proline, proline, glycin [hn-(pro–pro–gly)5–cooh]. 3,8 dentin elasticity is flexible bearing for the above enamel layer every location in dentin has different characteristic such as: the permeability in occlusal region on pulp horn is higher comparing with the center of tooth occlusal surface. dentin in proximal part is more permeable than in occlusal region while dentin corona is more permeable than root region. adhesion of resin material in dentin is determined by the presence of smear layer formed during cavity preparation. the thickness of smear layer is between 0.5–0.6 µm and should be previously removed in order that resin bonding material could bind fibril collagen to form hybrid dentin layer.8 hybrid dentin as a mixed molecule between collagen with polymer resin and it is prepared in dentin sub surface. which has been etched and also it is useful for monomer resin impregnacy in matrix. open fibril collagen is not pure collagen material, however it is covered by some proteins such as non collagen protein and proteoglycan. energy of dentin surface is quite low, so, an effort should be made in order that tensile surface of primer material could be compatible with dentin surface. in this case if the fluid could flow freely on solid surface, so, tensile fluid surface should be lower than energy of free surface of solid material, if the fluid could wet the whole solid surface, therefore this condition is called wet the surface. wetting capacity depends on contact angle formed by the fluid with substrate surface. at present, the exhaustion of some hybrid composite products is less 20 mm every year5. in this case it is almost similar with the mean exhaustion of amalgam is 10 mm per year. even though it is necessary to consider the advantage of new composite restoration which would be observed in short time that is about five years. nano filler composite it is not different from other composites, nano filler composite is generally based on bis–gma, urethane dimethacrylate, triethyleneglycol dimethacrylate and etc. due to very small sized filler, this composite has good physical–mechanical nature i.e. hardness, strength, smooth resin surface, accurate presition, and more easily manipulated.6,7 the size of filler particle is 1–25 nm and caco3, sio2, glass particle, boron, colloidal silicon are usually used. some researchers have done a study on sio2 was heated with 5% sodium peroxodisulfate solution, that followed by acetone cleansing. pretreatment process of coupling silane was then observed using x-ray, photoelectron spectrometry. the result is better composite in wear character and fatigue resistant.7 dentin bonding material in caries case which has involved dentin or cementum such as class v cervical erosion therefore filling with dentin bonding material is necessary. this case frequently occurs in elderly patient correlated with increasing of gingival retraction. dentin bonding adhesion in dentinal tissue could be chemical or physical mechanical bond.1,8 chemical bond occurs due to the presence of reaction between functional group of carbonyl dentin bonding with type i amino collagen in dentin. meanwhile physical-mechanical bond due to the presence of dentin bonding liquid penetrate into nano space inter fibriler, mechanical retention is tags that entering dentinal tubule and vander walls bonding between both material.1,2,3 dentin bonding material is usually based on three essential components i.e. primer, coupling agent and sealer, in this study, primer material is called dentin conditioner which consisting of acid. on reference or in daily term, coupling agent is also called primer while sealer functioning as dentin sealer, in which the liquid would flow into dentinal tubule.2 several years ago researcher believed that dentin bond was capable to form chemical bond with organic or inorganic group in dentin. this molecule could be described as m–r–x molecule in which m is methacrylate, r is spacer such as hydrocarbon chain and x is functional component which is useful to attach on dental tissue. during polymerization process, methacrylate group would react with resin forming chemical bond between resin and dentin.1,2 183soetojo: the application of methacrylate resin and the derivation dentin bonding material was firstly introduced in 1950’s and it was called first generation of dentin bonding the composed active material is glycerol–phosphoric acid. however, the weakness of this material is shrinking during polymerization and high expansion of thermal coefficient. in second generation which was produced in 1970’s, this material consisted of npg–gma (mix of n–phenylglysin and glycydil methacrylate). in clinical use it was seemingly lack of chemical binding between dentin bonding with dental tissue. in early 1980 the third generation was introduced this resin was based on halophosphorous ester of unfilled resin such as: bis –gma and hema (2 hydroxyethyl methacrylate). chemically, resin adhesion with dentin calcium, in which interaction ion process occurs between phosphate group and dentin calcium. the adhesion is too weak to balance the strength therefore phosphonate ester binding with dentin would be hydrolyzed in oral liquid. up to now dentin bonding which usually produced is from seventh generation which is called self etching adhesive consists of acid material, primer, catalyst and adhesive resin. therefore, in clinical application, the etching is not necessary.9,10 long term resin adhesion in dentin is still doubtful, because cleansing is not done, therefore, the salt could not be removed.11 in which the leftover of residual smear layer is not only consisted of inorganic but also organic material, denatured collagen fibril and bacteria. the adhesive strength of this material in dentin is lower compared with dentin bonding resin with total etched technique.12,13,15 self etching adhesive has been proved incapable to close perfectly, in dentin surface and interface resin, dentin is shown porous.14 some dentin bonding composed of multifunction monomer (primer or adhesive) with hydrophilic group to obtain good penetration and wetting.4,6 hydrophobic nature would polymerize and bind with composite resin on the upper layer. primer and adhesive is usually made in acetone dissolvent, alcohol or water, however, now days it is introduced in the form of solvent free, so it is not necessary drying before curing. hema as dentin bonding based material numerous hema based dentin bonding material has been found on the market such as scoth bond multi purpose (sbmp), all bond, single bond, clearfil se bond etc. hema is widely used because of some advantages such as: the making process is relatively easy, and having strong endurance capacity due to additional preservative substance is generally used such as: hydroquinone, butylated hydroxy toluene (bht) and relatively low viscosity.17,18 the formula of hema is c6h10o3 (figure 1), molecular weight is 130 and the use as dentin bonding material usually mixed with water, ethanol, or acetone. however, bonding with acetone dissolving for prolonged storage is less stable because acetone is easily evaporated therefore it would disturb dentin bonding material’s concentration.19,20 discussion the application of methacrylate based resin (composite resin, dentin bonding) is widely used at the clinic. this material could be applied as restoration for class i, ii, iii, iv, and v caries. composite resin is the main choice especially for restoration with aesthetic factor to be the main consideration, and composite resin is proper indication, even for posterior tooth cavity which is not too big. composite resin has many advantages, however, a dentist should be really careful and accurate during the treatment using this material. during acid etching period, operational part should be really dry, and should not be contaminated by water, saliva or blood, therefore rubber dam or saliva ejector is necessarily used. moreover, the treatment using composite resin initiated by dentin bonding material needs more application steps. at clinic, many patients were found with profound or cervical caries had involved many dentins, therefore treatments with dentin bonding material were needed. some recent years, a lot of 6 and 7 generation of dentin bonding have been produced which are usually called self dentin bonding. self etch means dentin bonding which is mixed with acid etching material. clinically, self etch is very beneficial because acid etching step, cleansing and drying are not necessarily done. in fact, cleansing is useful to remove the residual salt which is formed as a result of chemical reaction between acid and hydroxy-apatite dentin. the left over salt on tooth surface could disturb resin adhesion to the tooth, therefore, restorative resin is easily released. in addition, other materials which are also left on dentin surface are smear layer, protein/collagen which are denatured and some bacteria.11,12 it was reported that material which is left on dentin surface in certain period of time could penetrate into pulp space as a result pulpitis would occur, therefore some researchers still hesitate to apply self etch for prolonged use. researchers reported that the strength of total etching resin adhesion is higher comparing to self etch.12 the result of this study has proved that total etch dentin bonding adhesion (voco, excite) is significantly higher comparing to self etch (xeno, clearfil, lb) in p  0,05.21 figure 1. hema molecular structure (c6h10o3). 8 ch3 c = o och2 ch2oh h2c = c 184 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 181-185 ideal adhesion between dentin bonding with collagen dentin could de reached if the humidity condition on dentin surface in optimal condition.21 optimal humidity could contribute fibrilar collagen to be active and very permeable so the binding between both materials could easily occur. if collagen fibrilar is permeable, chemical reaction between amino collagen and carbonyl dentin bonding would take place and followed by amide bonding. besides, hydrogen binding also happens between both of them and continued by the formation of functional group (such as: carbonyl group, hydroxy, carboxilate, amine and amide).22 in this condition, complex reaction also happens between collagen and dentin calcium. in general, the success of dentin bonding adhesion in collagenous dentin depends on several factors i.e. low viscosity, type, concentration monomer, acid application as conditioner, temperature and humidity of adjacent collagen fibrilar.8,24 in vitro study was also performed on the effect of humidity on dentin bonding adhesion on dentin surface in which the humidity was 30%, 30%,75%, and 100%.25 it was reported that oral cavity humidity depends on whether rubber dam was used or not. if it was, the humidity was affected by humidity of dentist’s practicing room i.e. 50% ± at 23° c, but if rubber dam was not used, the humidity was about 80%–94%. the result in general shows increasing adhesion strength between dentin bonding with dentinal tooth simultaneously with increasing humidity, even though statistical analysis shows there is no significant difference. further more, it is also proved that bonding with hema content has the highest adhesion strength comparing to other adhesive materials. another study used two kinds of material i.e. scoth bond multi purpose (consisting of 47% hema, 40% water and 13% polyalkenoat) and clearfill se bond (consisting of hema, 10-methacryloyloxydecil–dihydrogen phosphate, alcohol), the humidity was: 30%, 50%, 65%, 80%, and 95%. the result has shown the increasing adhesion strength of both kinds of bonding in dentin correlated with decreasing humidity in adjacent dentin.26 the present study is also compatible with study done by chiba et al.27 reported that adhesion strength dentin bonding would decrease correlated with increasing humidity used in the study in which the humidity was about 50%, 80%, and 90%. the meaning of humidity is a condition correlated with water content in the adjacent material. humidity according to american encyclopedia is the amount of the water vapor containing in atmosphere. it is known as relative humidity that is comparison between water vapor in the atmosphere and the amount needed to make it saturated (in percentage) at a certain temperature. in this case, it means that humidity in the adjacent collagen would affect chemical characteristic and physical collagen bonding. the change would affect collagen binding on hema which is applied in the upper part either physical-mechanically or chemically. as it has been previously mentioned that water molecule in the adjacent would be capable to contribute physical change in collagen it self. up to the present there is not any study on the effect of humidity on chemical binding between hema and collagen which is observed simultaneously with adhesion strength mechanically between both materials. initiated by the previous study suggested that minimal humidity in oral cavity using rubber dam was 50%, so the same study is done. the result shows that minimal humidity of oral cavity using rubber dam is 60% (similar with humidity of practicing room at 23° c). this humidity of tropical countries in which it is higher than countries with cold climate.21 attaching or adhering two surface of solid materials is very difficult,1,2,3 even though with bare eyes both surface are smooth, but in microscopic level the surface is very rough, therefore if both surface are adhered, only the remarkable part is able to have adhesive contact, therefore, as a whole the contact field is not adequate and adhesion strength is low. strain force of two molecules would occur if the distance between them is less than 0.7 nm, higher than 0.7 nm would be very difficult. one of the ways to manage this problem is adding fluid in both surface. in order to obtain good adhesion, the fluid should be to flow and to wet the surface perfectly. in dentistry field, the capacity of adhesion to wet the surface is affected by several factors such as: the cleanness of the surface. thin layer of oxidation on metal surface could hinder adherence of adhesive material including inorganic fluid of acid etching on dentin surface could increase wetting, surface roughness and contribute dentinal tubule opening.28 in order to know the capacity of adhesive material wetting the surface which would be adhered, so it could be calculated the contact angle between adhesive fluid and solid surface of the material interface area1,4 if adhesive molecule which could perfectly attract material molecule which would be adhered, so, adhesive fluid would wet the whole surface. in this way, wetting contact angle is 0°, however, if contact angle is big meaning the wetting capacity of adhesive material is bad (figure 2). the kind of dissolvent material in dentin bonding fluid would affect adhesive process.22,29 as we know, acetone fluid is easily evaporated, and depleting resin figure 2. wetting of, material surface (solid).1,4 left, small contact angle (ø), good surface wetting. right, big contact angle, bad wetting. 185soetojo: the application of methacrylate resin and the derivation solution layer so it would decrease viscosity. when the solution is polished on dentin surface, it would penetrate into nano space between collagen fibrilar then drive water molecule and next, it would evaporate, so, it would leave dentin bonding resin to bind collagen fibrilar.29 acetone concentration would affect the thickness of bonding resin layer and the strain force. but the thickness of bonding resin will not correlate with strain force.29 the presence of cracking resin due to acetone evaporate, bad polymerization and low capacity/strength of strain of bonding resin (due to excessive amount of acetone). the other property of acetone is capable to drive water (water chasing effect), to increase vapor pressure of water especially water in adjacent collagen. it is proved that acetone ideal concentration is 37% of the weight, in this concentration, µm resin layer thickness and 63.5 mpa strain force is obtained. the study has been done and the result shows that the strain force in 60% humidity compared to 70% humidity, the number of water molecule is higher compared to 60% humidity, so the capacity of acetone to drive water molecule is higher and in this condition bonding resin would soon penetrate into collagen.21 the next chemical binding between resin and collagen is obtained and resin would penetrate into nano space interfibrilar forming mechanical retention. finally the value of strain force in 60% (control), 70%, 80%, and 90% humidity. chemical interaction between bonding resin which is believed to be the mean strength of resin adhesion and dentin.22,27 it is stated that by wrapping of n–methacryloxy–amino acid (n-maa) resin, the collapsed collagen would re-expand, develop collagen layer after acid etching and drying, so finally it would easily diffuse n-maa solution into collagen. after polishing of n-maa, significant increase of hybrid layer thickness until ten times higher and strain force would also increase. in fact the phenomena above is not supported by other experts. the thickness of hybrid layer has no correlation with strain force between resin and dentin.30 the conclusion is clinical use of methacrylate resin and the derivation (composite resin and denting bonding) could be used for restoration of class i-v caries. adhesion resin in enamel and dentin depends on humidity, etching acid technique, wetting contact angle, resin dissolvent, and the accuracy of dentist during resin application. references 1. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia: wb saunders co; 2003. p. 21–395. 2. noort rv. introduction to dental materials. 2nd ed. edinburgh london, new york, oxford: cv mosby co; 2002. p. 11–78. 3. summitt jb, robbins jw, hilton tj, schwartz r. fundamentals of operative dentistry. 3rd ed. chicago: quintess publ. co, inc; 2006. p. 183–242. 4. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 5. baum l, phillips rw, lund m. buku ajar operative dentistry. tarigan, editor. 1st ed. jakarta: penerbit buku kedokteran (egc); 1997. p. 251–65. 6. yu hj, wang l, shi q, jiang gh. study on nano caco3 modified epoxy powder coating. progress in organic coating 2006; 5:296–300. 7. shirai k, yoshida y, nakayama y. assessment of decontamination methods as pretreatment of silanization of composites glass filler. j biomed res 2000; 53:204–10. 8. nakabayashi np, pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 9. koibuchi h, yasuda n, nakabayashi n. bonding to dentin with a self-etching primer. dent mat 2001; 17:122–26. 10. zohairy aa, de gee aj, mohsen m. effect of conditioning time ofeffect of conditioning time of self-etching primers on dentin bond strength of three adhesive resin cements. dent mat 2005; 21:83–93. 11. harada n, nakajima m, pereira nr, yamaguchi s. tensile bond strength of a newly developed one-bottle self etching resin bonding systems to various dental substrates, dent in japan 2000; 36:47–53. 12. moll k, park hj, haller b. bond strength of adhesive/composite combinations to dentin involving total and self etch adhesive. j adhesive dent 2002; 3:171–80. 13. yoshiyama m, tay fr, doi j, nishitani y, yamada t, nakajima m. bonding of self etch and total-etch adhesives to carious dentin. j dent res 2002; 81:556–60. 14. tay fr, king nm, chan k. how can nanoleakage occur in self-etching adhesive systems that demineralize and infiltrate simultaneously? j adhes dent 2002; 4:255–69. 15. adioro s. the fractographic analysis of three dentin bonding agents on tooth surface. dental journal (majalah kedokteran gigi) 2006; 39(4):151–55. 16. swift ej, wilder ad, may kn, waddell sl. shear bond strength of one-bottle dentin adhesives using multiple applications. operative dent 1997; 22:194–99. 17. tay fr, pashley dh. aggressiveness of contemporary self-etching systems. dent mat 2001; 17:296–308. 18. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32:19–26. 19. maciel kt, carvalho rm, ringle rd. the effect of acetone, ethanol, hema and air on the stiffness of human decalcified dentin matrix. j dent res 1996; 75:1851–58. 20. leal jir, osorio r, terriza jah. dentin wetting by four adhesiveleal jir, osorio r, terriza jah. dentin wetting by four adhesivedentin wetting by four adhesive system. dent mat 2001; 17:526–32.17:526–32. 21. adioro s. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada berbagai kelembaban. dissertation. surabaya:dissertation. surabaya: airlangga university; 2006. p. 66–9. 22. xu j, stangel i, butler is, gilson dfr. an ft raman spectroscopy investigation of dentin and collagen surfaces modified by hema. j dent res 1997; 76:596–601. 23. renzo md, ellis th. chemical reactions between dentin and bonding agents. j adhesion 1994; 47:115–21. 24. brackett mg, brackett ww, haish ld. micro leakage of class v resin composites placed using self-etching resins. quintess int 2006; 37:109–13. 25. finger wj, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4:277–82. 26. besnault c, attal jp. influence of a simulated oral environmental on dentin bond strength of two adhesive systems. am j dent 2001; 14:367–72. 27. chiba y, miyasaki m, rikuta a. moore bk. influence of environmental conditions on dentin bond strengths of one application adhesive systems. oper dent 2004; 29:554–9. 28. rosales ji, marshall gw, watanabe lg. acid etching and hydration influence on dentin roughness and wet ability. j dent res 1999; 78:1554–9. 29. cho bh, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the micro tensile bond strength. dent mat 2004; 20:107–15. 30. wang y, spencer p. hybridization efficiency of the adhesive/dentin interface with wet bonding. j dent res 2003; 82:141–45. 186 vol. 43. no. 4 december 2010 pulp nerve fibers distribution of human carious teeth: an immunohistochemical study tetiana haniastuti department of oral biology faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: human dental pulp is richly innervated by trigeminal afferent axons that subserve nociceptive function. accordingly, they respond to stimuli that induce injury to the pulp tissue. an injury to the nerve terminals and other tissue components in the pulp stimulate metabolic activation of the neurons in the trigeminal ganglion which result in morphological changes in the peripheral nerve terminals. purpose: the aim of the study was to observe caries-related changes in the distribution of human pulpal nerve. methods: under informed consents, 15 third molars with caries at various stages of decay and 5 intact third molars were extracted because of orthodontic or therapeutic reasons. all samples were observed by micro-computed tomography to confirm the lesion condition 3-dimensionally, before decalcifying with 10% edta solution (ph 7.4). the specimens were then processed for immunohistochemistry using anti-protein gene products (pgp) 9.5, a specific marker for the nerve fiber. results: in normal intact teeth, pgp 9.5 immunoreactive nerve fibers were seen concentrated beneath the odontoblast cell layer. nerve fibers exhibited an increased density along the pulp-dentin border corresponding to the carious lesions. conclusion: neural density increases throughout the pulp chamber with the progression of caries. the activity and pathogenicity of the lesion as well as caries depth, might influence the degree of neural sprouting. key words: caries, dental pulp, nerve fibers, protein gene product 9.5 abstrak latar belakang: pulpa gigi manusia diinervasi oleh serabut saraf trigeminal yang berespon terhadap stimuli penyebab perlukaan dengan menimbulkan rasa sakit. perlukaan pada akhiran saraf dan komponen lain dari pulpa akan menstimulasi aktivasi metabolik dari neuron pada ganglion trigeminal sehingga mengakibatkan perubahan morfologi pada akhiran saraf perifer. tujuan: penelitian ini bertujuan untuk mengamati perubahan distribusi saraf pada pulpa gigi manusia yang disebabkan oleh proses karies. metode: penelitian ini menggunakan 15 buah gigi molar tiga yang mengalami karies dengan berbagai tingkat kedalaman karies dan 5 buah gigi molar tiga normal (tidak mengalami karies). gigi-geligi tersebut dicabut untuk keperluan perawatan ortodontik atau alasan perawatan lainnya. sebelum didekalsifikasi dengan menggunakan edta 10% (ph 7,4), seluruh sampel diamati dengan micro-computed tomography untuk mengetahui kondisi lesi secara tiga dimensi. spesimen kemudian diproses secara immunohistokimia menggunakan anti-protein gene products (pgp) 9,5 yang merupakan penanda spesifik untuk serabut saraf. hasil: pada pulpa gigi normal, serabut saraf yang menunjukkan ekspresi pgp 9,5 positif tampak terkonsentrasi di bawah lapisan odontoblast. distribusi serabut saraf tampak meningkat pada perbatasan dentin-pulpa di bawah lesi karies. kesimpulan: densitas serabut saraf pada kamar pulpa meningkat dengan bertambahnya kedalaman karies. aktivitas dan patogenisitas dari lesi serta kedalaman karies dapat berpengaruh terhadap penyebaran serabut saraf. kata kunci: karies, pulpa gigi, serabut saraf, protein gene product 9,5 correspondence: tetiana haniastuti, c/o: bagian biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i yogyakarta 55281, indonesia. e-mail: haniastuti@yahoo.com research report 187haniastuti: pulp nerve fibers distribution of human carious teeth introduction caries is an infectious and transmittable disease resulting from certain bacteria present within the oral cavity such as streptococcus mutans, streptococcus sobrinus, and lactobacilli.1,2 those bacteria produce acids following an individual’s sugar consumption which have ability to diffuse through the dental calcified tissues and drop the local ph to below 5.0, which in turn leads to dissolution of the mineral crystals.3,4 a variety of stimuli, including caries, have been demonstrated to have an effect on the pulp. carious lesion contains bacterial and antigenic substances which may affect the pulp through the dentin. caries can exert its effects on the dental pulp even before the infection breaches the dentin enamel junction. thereafter, the progression of infection exerts an increasing effect on the underlying pulp by eliciting defense and repair mechanisms mainly aimed at decreasing dentin permeability and eradicating pathogens.5,6 human dental pulp is richly innervated by trigeminal afferent axons that subserve nociceptive function. accordingly, they respond to stimuli that induce injury to the pulp tissue.7 an injury to the nerve terminals and other tissue components in the pulp stimulate metabolic activation of the neurons in the trigeminal ganglion which result in morphological changes in the peripheral nerve terminals. the results of previous studies have demonstrated a sprouting of pulpal nerve fibers following dental injury.8-10 protein gene product (pgp) 9.5 is a novel neuronspecific protein, widely distributed in both central and peripheral neurons. previous research on dental innervation clearly revealed that pgp 9.5 is a useful marker for identifying delicate nerve fibres such as a-delta and c-fibres. in addition, pgp 9.5 antigenicity is well preserved during demineralization process.11 although structural neural changes have been investigated following experimental pulp injury,8-10 there has been little attempt to study caries-induced neural changes in human. the purpose of the study was to observe caries-related changes in the distribution of human pulpal nerve using pgp 9.5. materials and methods twenty volunteers ranging in age from 20–40 years, who had been scheduled to undergo extraction for orthodontic or therapeutic reasons were enrolled in the study. informed consents were obtained from subjects after the proposed study was fully explained. fifteen third molars with caries at the occlusal site at various stages of decay and 5 intact third molars were extracted. the depth of the cavity judged by clinical examination was recorded. before decalcifying with 10% ethylene-diaminetetraacetic acid (edta) solution (ph 7.4) for 6 months at 4° c, all the samples were observed by micro-computed tomography (micro-ct) to confirm the lesion condition 3-dimensionally. the specimens were then processed for cryosection. the tissues were equilibrated in a 30% sucrose solution for cryoprotection. the specimens were cut at thickness of 50 µm on a freezing microtome, collected into cold phosphate-buffered saline, and treated as free-floating sections. frozen sections were processed for the avidin-biotin peroxidase complex (abc) method by using rabbit anti-human pgp 9.5 polyclonal antibody (chemikon international, temecula, usa). endogenous peroxidase was inhibited by treatment with 0.3% h2o2 in absolute methanol for 30 minutes. any non-specific immunoreaction was inhibited by preincubation in 2.5% normal goat serum (vector laboratories inc, ca, usa). following incubation with the primary antibodies, the sections were then reacted consecutively with biotinylated anti-rabbit igg and abc (vector laboratories inc, ca, usa). the sites of antigen-antibody reactions were visualized using 3–3’-diaminobenzidine tetrachloride in tris buffer and 0.002% h2o2 and counterstained with 0.05% methylene blue. the immunostained sections were thaw-mounted onto silane-coated glass slides and stained with 0.03% methylene blue. immunohistochemical controls omitting the primary antibody, the biotinylated anti-rabbit igg, or the abc complex resulted in no staining. results micro-ct observation showed intact teeth; no lesion was observed. the nerve density is greatest near the tip of the pulp horn. arrangement of positive fibers below the odontoblastic layer (rasckow’s plexus) was observed (figure 1). the nerves directed radially toward figure �. a specimen of intact tooth. rasckow’s plexus (arrows) was observed below the odontoblastic layer. the nerves directed radially toward the odontoblasts. most of pgp 9.5-positive nerve fibres penetrate into the predentine and dentine beyond the pulpodentinal border. 188 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 1867–189 the odontoblasts. the pulpal nerves, thin and frequently beaded in appearance, entered the odontoblast cell layer. some of pgp 9.5-positive nerve fibres terminated in the odontoblast layer, but the majority penetrated into the predentine and dentine beyond the pulpodentinal border. pgp 9.5-positive immunoreaction was also recognized in odontoblast cells. although micro-ct observation showed radiolucent area involving enamel, no dentinal injury was observed in histological specimens. pgp 9.5-positive nerve fibers demonstrated similar distribution to the intact teeth. radiolucent area involving dentin was showed. pgp 9.5 immunoreactivity exhibited an increase in density in the para odontoblastic region correspond to the area of inflammatory cells infiltration. there were sprouting nerve fibers under the lesions. reparative dentin was found beneath the lesion. pgp 9.5-positive nerve fibres were fewer subjacent the reparative dentin and terminated in the odontoblast layer; while in other areas pgp 9.5positive nerve fibers penetrated into the predentine and dentine beyond the pulpodentinal border. the rest of the pulp demonstrated a normal appearance. micro-ct observation showed lesion involving pulp. numerous pgp 9.5-positive nerve fibers were concentrated heavily in areas where pulp was inflamed (figure 2). in this region, nerve fibers displayed both extensive arborization and thickening of small nerve bundles to form bands of neural tissue. it appeared that increased neural density in the pulp horn was due to sprouting of nerve terminals rather than to increasing in the number of parent axons entering the tooth, since there were no apparent changes within the main nerve trunks passing up through the coronal pulp. discussion the present study has demonstrated the distribution of nerve fibers within the pulp with caries progression. neural density appeared to increase markedly throughout the pulp chamber with the progression of caries. it is likely that, in addition to caries depth, the activity and pathogenicity of the lesion may also influence the degree of neural sprouting. these findings concur with the previous studies of pulpal inflammation.8,9 this study used micro-ct to observe the depth and condition of the carious lesion three-dimensionally. micro-ct is an emerging technology that has been used as a research tool in various applications including morphometry of bone, connective tissue, teeth or root canals. the micro-ct technique is rapid and noninvasive. in addition, the results are reproducible and comparable with histology.12 pgp 9.5 is a novel neurone-specific protein. this protein is a useful marker for identifying delicate nerve fibers such as a-delta and c-fibers.11 a study by yoshiba et al.13 demonstrated that pgp 9.5 is a reliable marker for the demonstration of fine nerve terminals in human tooth pulp. caries-induced changes in neural distribution might be functionally important in the regulation of pulpal inflammation and healing. in this study, the nerve sprouting was most remarkable at the site where inflammatory cells were densely accumulated. these findings suggest a functional communication between neuropeptides and pulpal immunocompetent cells such as neutrophils, macrophages and t-lymphocytes. nerve fibers have demonstrated an extensive sprouting reaction in response to dentinal injury which probably results in metabolic activation of the neurons in the trigeminal ganglion to provide an increased local source of neuropeptides to the inflammatory region.14,15 previous studies revealed that the neuropeptides induce vasodilation and an increase in the permeability of the vessel walls; therefore, they regulate inflammatory cells invasion to the injury sites. such vascular reactions are an essential part of the inflammatory reaction and are also necessary to satisfy the nutritional needs related to the increased metabolic activity in connection with tissue repair and healing.16,17 pulp has ability to produce reparative dentin beneath a carious lesion as a mechanism for limiting the diffusion of toxic substances to the pulp.18 this study showed that the areas beneath the reparative dentin showed fewer number of pgp 9.5 immunoreactivity than normal. these findings indicating that sprouting of the nerve and neuropeptides upregulation continue as long as there is active inflammation that has not been walled off by scar formation. once an effective scar and reparative dentin were formed, the nerve sprouting decreases and neuropeptide levels return to normal range or are subnormal.15 in this study, the post mitotic mature odontoblasts also exhibited intense pgp 9.5 immunoreactivity as secretion of predentinal matrix was visible. pgp 9.5 immunohistochemical studies have shown that this protein is widely distributed in neuroendocrine cells in addition to figure �. a specimen with pulp lesion. numerous pgp 9.5positive nerve fibers were concentrated heavily in areas subjacent to the lesions (arrows). 189haniastuti: pulp nerve fibers distribution of human carious teeth central and peripheral neurons. a possible explanation of the immunoreactivity for pgp 9.5 in the human odontoblast may be due to their derivation from the neural crest, in common with neurons and neuroendocrine cells.19 in conclusion, neural density increases throughout the pulp chamber with the progression of caries. it is likely that the activity and pathogenicity of the lesion as well as the depth of the caries, might influence the degree of neural sprouting. references 1. love rm. invasion of dentinal tubules by root canal bacteria. endod top 2004; 9: 52–65. 2. marsh pd, nyvad b. the oral microflora and biofilms on teeth. in: fejerskov o, kidd e, editors. dental caries: the disease and its clinical management. oxford: blackwell munksgaard; 2008. p. 163–84. 3. featherstone jdb. the continuum of dental caries–evidence for a dynamic disease process. j dent res 2004; 83: c39–c42. 4. shen s, samaranayake lp, yip h. in vitro growth, acidogenicity and cariogenicity of predominant human root caries flora. j dent 2004; 32: 667–78. 5. bjørndal l. the caries process and its effect on the pulp: the science is changing and so is our understanding. j endod 2008; 34: s2–s5. 6. goldberg m, farges j, lacerda-pinheiro s, six n, jegat n, decup f, septier d, carrouel f, durand s, chaussain-miller c, denbesten p, veis a, poliard a. inflammatory and immunological aspects of dental pulp repair. pharmacol res 2008; 58: 137–47. 7. byers mr, narhi mvo. nerve supply of the pulpodentin complex and responses to injury. in: hargreaves km, goodis he, editors. seltzer and bender’s dental pulp. chichago: quintessence publishing co; 2002. p. 154–5. 8. haug sr, heyeraas kj. modulation of dental inflammation by the sympathetic nervous system. j dent res 2006; 85(6): 488–95. 9. rodd hd, boissonade fm. comparative immunohistochemical analysis of the peptidergic innervation of human primary and permanent tooth pulp. arch oral biol 2002; 47: 375–85. 10. yu c, abbott pv. an overview of the dental pulp: its functions and responses to injury. aust dent j 2007; 52 (1 suppl): s4–s16. 11. rood h, boissonade fm. immunocytochemical investigation of neurovascular relationships in human tooth pulp. j anat 2003; 202: 195–203. 12. jung m, lommel d, klimek j. the imaging of root canal obturation using micro-ct. int endod j 2005; 38: 617–26. 13. yoshiba k, yoshiba n, iwaku m. class ii antigen-presenting dendritic cell and nerve fiber responses to cavities, caries, or caries treatment in human teeth. j dent res 2003; 82(6): 422–7. 14. byers mr, suzuki h, maeda t. dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration. microsc res and tech 2003; 60: 503-15. 15. olgart l, bergenholtz g. the dentine-pulp complex: responses to adverse influences. bergenholtz g, hørsted-bindslev p, reit c, editors. textbook of endodontology. oxford: blackwell munksgaard; 2003. p. 36. 16. caviedes-bucheli j, camargo-beltran c, gomez-la-rotta a, moreno sc, abello gcm, gonzalez-escobar jm. expression of calcitonin gene-related peptide in irreversible acute pulpitis. j endod 2004; 30(4): 201–4. 17. caviedes-bucheli j, arenas n, guiza o, moncada na, moreno gc, diaz e, munoz hr. calcitonin gene-related peptide receptor expression in healthy and inflamed human pulp tissue. int endod j 2005; 38: 712–7. 18. kim s, trowbridge h, suda h. pulpal reaction to caries and dental procedures. in cohen s, burns rc, editors. pathways of the pulp. 8th ed. st. louis: mosby; 2002. p. 574. 19. arana-chavez ve, massa lf. odontoblasts: the cells forming and maintaining dentine. int j biochem cell biol 2004; 36: 1367–73. subject index volume 49 adherence, 214 aggregatibacter actinomycetemcomitans, 104, 196 alkaline phosphatase, 76 alveolar bone remodeling, 143 anadara granosa; 27 antibacterial, 93, 99, 158 effect, 104 antibiofilm; 148, 158 arabica extract coffee, 99 artificial mouth system, 67 benzylisothio-cyanate, 158 ß-1,3-glucanase, 81 beta defensins-2 expression, 49 biofilm, 67, 214 bone graft, 27 healing, 27 regeneration, 22 remodeling, 63 substitute, 181 bovine hydroxyapatite, 153 c. albicans biofilm, 71 cacao beans, 104 calcium hydroxide, 17 candida albicans, 148 cementum; 5 ceramic bracket, 190 channa striata extract, 125 chicken shank collagen, 22 children with down syndrome, 207 chitosan, 153 scaffold, 22 chlorophyll, 196 cinnamomum burmannii, 71 coagregation, 214 color stability, 185 compressive strength, 153 demineralized freeze-dried bovine bone xenograft, 37 dental caries prevention behavior, 218 health maintenance behavior, 207 denture base, 185 plaque, 81 dfdbbx, 43 diabetes, 133 dmf-t, 164 e. faecalis, 175 early detection, 54 elementary school-children, 164 enamel structure, 190 enterococcus faecalis, 93, 158 epithelial tongue, 10 epstein barr virus, 1 estrogen, 76 extracts of sarang semut, 175 family support, 164 fgf-2, 125 fibroblast,; 125 flavonoids of cassava leaves, 137 fusobacterium nucleatum, 93 gelatin, 153 gingival epithelium, 49 guided bone regeneration, 181 guinea pig, 169 herpes labialis, 230 high molecular nano chitosan, 185 hyperbaric oxygen, 63 immunohistochemical, 120 immunomodulator, 1 implant, 181 infectious mononucleosis, 1 inferior alveolar nerve, 59 inflammatory cells, 87 isoflavone genistein of soybean, 169 jengkol leaf extract, 148 l. reuteri, 49 lactic acid, 202 lactobacillus acidophilus, 99 laser diode, 196 line, 224 lower third molar, 59 macrophage, 133 magnetic beads, 32 mangosteen peel extract, 43 mda serum level, 110 methisoprinol, 1 minimum bactericidal concentration, 175 inhibitory concentration, 175 moringa oleifera, 196 oleifera leaf, 37 musculoskeletal complaints, 202 nordic body map, 202 odontectomy, 59 odontoblast-like cells, 17 ohis, 164 oral candidiasis, 10 health behavior, 164 orthodontic tooth movement, 143, 169 osteoblast, 27, 37, 43, 63 osteoclast, 37, 43 osteoporosis, 76 ovariectomy, 76 p-53, 120 papain, 71, 81 paraesthesia, 59 parental socio-economic conditions, 115 peer support education, 218 perceived parental demandingness, 207 responsiveness, 207 periodontal ligament, 63 periodontitis, 137 photodynamic inactivation, 196 pluchea indica less leaves extract, 93 porosity, 153 poster, 224 potentially malignant disorder, 54 prediction to get a new dental caries, 115 probiotic, 49 propolis, 17 radiographers, 110 rankl, 143 rat, 10 receptor, 214 residence area, 115 robusta coffee,143 extract coffee, 99 rood and shehab’s (1990), 59 root demineralization, 5 reba, 202 s. mutans, 49, 67 saline, 5 salivary neutrophils, 32 salvadorine, 158 sardinella longiceps oil, 27 scaffolds, 153 school age children, 218 s-ecc, 32 sensitivity, 175 shear bond strength, 190 silane, 190 siwak, 158 social media, 224 socket healing, 87 speckled leukoplakia, 54 squamous cell carcinoma, 120 sterile water, 5 stichopus hermanii extract, 10 taste buds, 110 tetracycline hcl, 5 tgf-ß1, 87, 143 the lower lip, 230 tnf-α, 137 tooth brushing behavior before bedtime, 224 extraction, 87 movement, 63 trimethylamine, 158 vigna unguiculata, 71 working positions, 202 wound healing, 125 x-ray irradiation, 87 znso4 1% gel, 133 authors index volume 49 adiana, ika devi, 185 apriasari, maharani laillyza, 1 atikah, ayu rafania, 104 aulia, agniz nur, 110 brahmanta, arya, 63 dwiandhono, irfan, 17 ferronika, shinta, 5 herniyati, 143 indrawati, retno, 81 kamadjaja, david b., 59 kartikasari, nadia, 153 kintawati, silvi, 120 kresnoadi, utari, 43 kusumaningsih, tuti, 49 luthfi, muhammad , 32, 71, 148 meilawaty, zahara, 137 morita, aryan, 67 mujayanto, rochman, 133 oentaryo, gunawan, 125 pargaputri, agni febrina, 93 prehananto, herlambang, 230 priyono, bambang, 115 pudyani, pinandi sri, 190 putra, ramadhan hardani, 87 putri, deby kania tri, 214 putriwijaya, fiory dioptis, 202 rahmitasari, fitria, 22 revianti, syamsulina, 10 romadlon, debby syahru, 218 rostiny, 37 salim, sherman, 76 sari, ika rhisty cendana, 158 sari, rima parwati, 27 soraya, cut, 175 suparwitri, sri, 169 suyono, benso sulijaya, 181 tampoma, selviana, 54 ulfah, siti fitria, 207 wibawa, i gde bagus yatna, 196 widiati, sri, 164 widodo, 224 wijaya, willy, 99 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 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country issue* 6 month 1 year surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " �� vol. 42. no. 1 january–march 2009 simple replantation protocol to avoid ankylosis in teeth intended for orthodontic treatment yuli nugraeni1, david buntoro kamadjaja2, and haryono utomo3 1department of conservative dentistry, dentistry study program, brawidjaja university, malang 2department of oral surgery, faculty of dentistry, airlangga university, surabaya 3dental clinic, faculty of dentistry, airlangga university, surabaya abstract background: dento-alveolar trauma resulted from accidents involving the oral regions mostly affect the upper central incisors. overjet that is beyond 5 mm and incompetent lip also contribute to increase the risk. several literatures had already discussed different methods of replantation of avulsed teeth. however, it was not meant for further orthodontic treatment. purpose: the objective of this review is to propose a simple replantation protocol of avulsed teeth which also prevent from ankylosis. reviews: protruded teeth usually need orthodontic treatment; therefore, an appropriate management should be done to avoid the development of ankylosis. ankylosis of the periodontal ligament (pdl) becomes a problem in orthodontic tooth movement in repositioned or replanted teeth. in addition, ankylosed teeth also more susceptible to root resorption. actually, it was caused by the endodontic treatment. in particular, severely protruded or unoccluded teeth are hypofunctional, therefore have narrow pdl, thus it may facilitate to ankylosis development. ideal management protocol such as the use of root canal sealer i.e. mineral trioxide aggregate (mta); the using of emdogain, and resilient wiring or semi-rigid fixation with brackets has become a solution in avulsed teeth arranged for orthodontic treatment. nevertheless, the presence of oral surgeon, endodontist and orthodontist in the same time, and also ideal preparations after an accident was difficult to achieve. conclusion: considering that reducing the ongoing pdl inflammation with intracanal medicaments and maintaining the functional force during mastication is possible; it is concluded that this simple replantation protocol is likely. key words: replantation protocol, ankylosis prevention, orthodontic treatment correspondence: yuli nugraeni, dentistry study program, brawijaya university, veteran 1, malang. e-mail: yulinugraeni@yahoo.com introduction dental trauma is one of the most serious oral health problems in active children and adolescents. it requires immediate initial emergency treatment followed by integrated procedures to restore damaged oral structures along with subsequent trauma prevention strategy. dentoalveolar injuries in the anterior region of the mouth are often characterized by tooth avulsion or intrusion, with or without fractures of the crown or root. the predisposing factors in dental trauma are maxillary teeth protruding more than 5 mm, which are two to three times more likely to suffer dental trauma, incompetent lip and obesity.1 tooth avulsion accounts for 0.5–16% of traumatic injuries in the permanent dentition and for 7–21% of injuries in the primary dentition. avulsions of permanent teeth occur most often in children at the age of 7–10 year, during which time the relatively resilient alveolar bone provides only minimal resistance to extrusive forces.2 avulsions cause severe pulpal and periodontal injuries. the status of the pulp is one decisive object for periodontal healing. pulp necrosis and consecutive infections cause ankylosis (or replacement resorption, rr),3 and infection-related resorption (irr, formerly called inflammatory resorption).4,5 unfortunately, tooth ankylosis leads to difficulties in orthodontic treatment i.e. retracting the avulsed protruded teeth. ankylosis is a known complication of replanted or severely intruded permanent incisors. despite considerable knowledge about the pathogenesis of ankylosis from animal studies and observation of human replanted teeth, there is no known treatment to arrest this condition. management techniques for ankylosis and its consequences are supported by little evidence, do not appear to be widely adopted and do not offer any proven long-term benefit. nevertheless, other literature review �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 25-30 literatures revealed that the antiresorptive-regenerative therapy (art) with the local application of glucocorticoids and enamel matrix derivative (emd, emdogain®) and the systemic administration of doxycycline combined with semi-rigid fixation was considered to have successful results.4,6 however, in private dental practice or even in small hospitals, the presence of dentists who are competent to avulsion management (i.e. special-trained dentists, oral surgeon, endodontist and orthodontist), and the provision of ideal preparations (i.e. mineral trioxide aggregate, mta and emd) at the same time of the accident are rarely achieved. therefore, a simple replantation protocol of avulsed tooth should be developed to achieve a successful treatment that also prevent from ankylosis. the objective of this review is to propose a new simple protocol of avulsed tooth management which also minimizes the risk of ankylosis; especially in tooth intended for further orthodontic treatment. this new concept is based on literatures regarding to the simplicity of methods and using the commonly available dental medicaments in most dental office. avulsion by definition, avulsion is a complete displacement of tooth out of its socket. the periodontal ligament is severed and fracture of the alveolus may occur. the diagnosis is made based on clinical and radiographic findings. radiographic assessment will verify that the tooth is not intruded when it is clinically missing. even though avulsion of permanent incisors is a relatively rare injury; in an emergency situation, a prompt decision should be made to treat it with replantation. therefore, the clinician must be prepared to learn about the management protocol for avulsed teeth and identify its negative consequences such as ankylosis and root resorption.6 there are contraindications to tooth replantation which are immunocompromise, severe congenital cardiac anomaly; severe mental disability, severe uncontrolled diabetes and lack of alveolar integrity.6 for better understanding of the avulsion treatment and to minimize its negative effects, the physiological and pathological aspects of the tooth and its surrounding tissue should be reviewed. the periodontal ligament recently it has been revealed that pdl tissue possesses multipotential mesenchymal stem cells that can differentiate into mineralized tissue-forming cells such as osteoblasts and cementoblasts. however, pdl tissue is never ossified in vivo under normal circumstances. in fact, in vitro maintained pdl cells have various osteoblast-like properties, including the capacity to form mineralized nodules, expression of bone-associated markers, and response to bone-inductive factors such as bone morphogenetic protein 2 (bmp-2) which lead to ankylosis.7 conversely, plap1/asporin which expressed predominantly in the pdl negatively regulates the mineralization of pdl cells. it also plays as a negative regulator of cytodifferentiation and mineralization probably by regulating bmp-2 activity to prevent the pdl from developing non-physiological mineralization such as ankylosis.8 ankylosis (replacement resorption, rr) ankylosis or rr is a pathologic fusion of the cementum or dentin of a tooth root to the alveolar bone. it is most likely to affect a replanted avulsed tooth or a severely intruded tooth within weeks following trauma. detection of ankylosis depends on clinical signs and radiographic interpretation. clinical diagnosis of ankylosis is based on qualitative assessment of the sound produced on percussion and of mobility.3ankylosis of teeth in the pre-adolescent can lead to infraocclusion and distortion of the gingiva and the underlying bone producing both functional and esthetic deficits with jaw growth. therefore, the benefit of early detection is that it gives earlier warning of growthassociated infraocclusion.2, 3, 7, 8 pathogenesis of ankylosis in healthy patients, pdl fibroblasts block osteogenesis within the periodontium by releasing locally acting regulators, such as cytokines and growth factors, thereby maintaining separation of tooth root from alveolar bone.8 necrosis of the periodontal ligament’s cellular elements by desiccation, crushing or mechanical damage, as in severe luxation injury, disrupts this normal homeostatic mechanism. ankylosis is established not only via inflammatory-mediated and mechanical alterations in the periodontal ligament but also because insufficient functional cellular elements survive to suppress osteogenic activity. this disruption allows growth of bone across the periodontal ligament and ankylosis (rr).3–5 inflammatory resorption (irr) sustained by bacterial infection of necrotic pulp tissue in the replanted tooth can be effectively arrested by pulpectomy followed by ca(oh)2 root canal filling. however, despite the ability to treat irr predictably, unfortunately, its arrest promotes rr.3,4 treatment of avulsion despite evidence that immediate replantation (i.e. within 5 min) is required for regeneration of the periodontal ligament (pdl) and its return to normal function. most school teachers and coaches would be reluctant to replant teeth if the circumstance arose. the reasons i.e. inadequate training, reluctance to induce pain or fear in the child, fear of replacing the tooth incorrectly and fear of possible legal consequences.2 general prognosis in avulsion treatment is vary, in the permanent teeth is primarily dependent upon formation of root development and extra oral dry time. in permanent teeth, there is risk for pulp necrosis, root resorption, ankylosis, and infraocclusion during adolescent growth. the tooth has the best prognosis if replanted immediately, if not be replanted within 5 min, it should be stored in a medium to maintain vitality of the periodontal ligament.9–12 moreover, the risk of ankylosis increases significantly with ��nugraeni, et al.: simple replantation protocol to avoid ankylosis an extra oral dry time of 15 min. an extra oral dry time of 60 min is considered the point where survival of the pdl cells is unlikely.6 transportation media for avulsed teeth include (in order of preference) viaspan, hank’s balanced salt solution, cold milk, saliva (buccal vestibule) or physiologic saline. water is an inappropriate media because it increases osmotic pressure and cells explode, not much better than dry. if avulsed tooth is stored in a physiologic media, storage time has not been found to be a significant factor.11, 12 antiresorptive-regenerative therapy (art) since 1998 the topical and systemic application of different medicaments was used to depress resorption activity and support regeneration in the pdl. antiresorptiveregenerative therapies (art) with application of topical corticosteroids and enamel matrix derivative (emd), combined with systemic doxycycline may have potential for enhancing the prognosis of avulsed teeth. treatment strategies are directed at avoiding or minimizing inflammation, increasing revascularization, and producing hard barriers in teeth with open apices.6 if the extra-oral dry time > 60 min, soak tooth for 5 min in 2.4% naf acidulated to a ph of 5.5 before replantation. immediately before replantation-after removal of the coagulum from the alveolus by rinsing with sterile isotonic saline–emd (emdogain®) was applied onto the root surface and into the alveolus. subsequently, topical corticosteroid (i.e. ledermix®) is applied intracanally; followed by systemic doxycycline therapy,4 and chlorhexidine mouth rinse for prevention of infection.12 splinting for tooth avulsion stabilizing the tooth with a functional, semi-rigid splint for one week for avulsion without alveolar fracture13 or two to three weeks with alveolar fracture will assist in re-establishing the pdl support of the tooth.1 semi-rigid splint wire is adapted to the facial or lingual surfaces of the teeth to be splinted and one sound tooth on either side of the traumatized tooth should be included.13 recent publications revealed that semi-rigid splint accommodate occlusal forces which are advantageous for pdl healing. successful replantation of permanent teeth without ankylosis is obtained when light occlusal forces were applied via archwire during the early stages of recovery.14,15 chen et al.,14 study showed that occlusal force increase nitric oxide (no) synthesis by inducible nitric oxide synthase (inos) which necessary for repair of injured pdl fibers, angiogenesis and nerve regeneration. moreover no induces osteoblast apoptosis and depresses bone formation. consequently, with the existence of inos, pdl width could be maintained, thus avoid from ankylosis. additionally, with the same stimulus hu et al.15 suggested that the increase of fibroblast growth factor (bfgf/fgf-2) is considered as a key factor in pdl healing. emdogain® future studies should help illuminate the reported benefits of emdogain®, an enamel matrix derivative (biora ab, malmo, sweden) extracted from developing embryonal enamel of porcine origin. it has been used to coat the entire root surface of the avulsed tooth prior to replantation. emdogain contains proteins of the amelogenin family and is presently thought to aid in the migration, attachment, proliferative capacity, and biosynthetic activity of pdl cells.4,16 emdogain has also been shown to enhance pdl cell proliferation and protein production, and it may act as a matrix for cells responsible for regenerating pdl at a wound site such as a replanted avulsed tooth. several case reports and other articles have shown promising results in terms of decreased replacement root resorption when it was used topically prior to replantation in several avulsion management protocols.4 endodontic treatment of avulsed teeth endodontic treatment is essential for the progress of the healing process of the replanted tooth. nevertheless, there are still controversies, especially in closed apex tooth (< 1 mm), should it be done extra or intra-orally. however, best results are obtained when the tooth is immediately, without being endodontically treated, inserted into the alveolar socket and pulp extirpation is directly done to prevent the initiation of irr. the canal should be debrided, dressed with a corticosteroid/ antibiotic or ca(oh)2 paste for one to three months, after which time the canal can be obturated.17 intra oral endodontic is provided intra-orally only after splinting.1, 10-14 endodontic therapy involving obturation with guttapercha or the placement of ca(oh)2 dressings at the time of replantation delays periodontal healing and accelerates ankylosis (rr) in mature teeth. thus, it is recommended that in mature teeth, endodontic therapy should be commenced 7–10 days following replantation. radiographs should be taken at regular intervals of one, three, six and 12 months.1,6 in immature teeth, when the time out of the mouth is short and the apex is open, revascularization of the pulp may occur. endodontic therapy can be delayed to establish whether revascularization will occur but only if patient compliance for follow-up treatment is assured. regular follow-up is essential and apexification procedures should be carried out at the first sign of resorption, discoloration, the presence of a draining sinus or periapical bone loss. the root canal should be accessed so the infected tissue and debris can be removed and the canal then be filled with ca(oh)2. root filling involving gutta-percha and/or mta can be carried out later.12 apexification apexification is the process by which the open apex of a tooth with pulp necrosis and an incompletely formed �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 25-30 root can be closed by deposition of hard tissue (probably cementum). to accomplish apexification, the necrotic pulp is removed, the canal is cleaned and the canal filled with ca(oh)2. after the apex is closed by hard tissue deposition, the root canal is filled with sealer and gutta percha (usually after 6 months) (figure 1).18,19 intracanal medicaments the pdl management in cases of extended dry times (i.e. > 60 minutes), recent animal studies have reported that roots treated with the intracanal medicament ledermix® (triamcinolone acetonide and demeclocycline calcium; lederle, uk) have significantly more favorable healing and less resorption than in those treated with ca(oh)2. in this case, time is not a big factor thus can be done extra or intra orally.20 calcium hydroxide has several advantages as it has antimicrobial activity, disintegrates endotoxin (lps); denatures il-1a, tnf-a, and neuropeptides; and stimulates apex development. combination of ca(oh)2 with other medicaments such as iodine potassium iodide 5% and camphorated chlor-phenol chamfer (chkm) increases its effectivity.18 nevertheless, it also interrupts healing process of pdl, thus the application should be delayed minimal until 7 days after replantation.18,19 pharmacological treatment tetanus prophylaxis and antibiotic coverage should be considered. treatment strategies are directed to avoid inflammation that may occur as a result of the tooth’s attachment damage and/or pulpal infection.6 pharmacological treatments manipulate the inflammatory response to minimize destruction and facilitate repair of the damaged root surface by new cementum and periodontal ligament. after completion of the art (or dental emergency treatment), doxycycline is prescribed for systemic use according to the patients’ weight (2 mg/kg body weight daily, maximum dose 100 mg/day) for 5 days. tetracycline derivates has been widely used in periodontal treatment because of its sustained antimicrobial effects and anti-resorptive properties. specifically, it has a direct inhibitory effect on resorption activity of osteoclast cells and collagenase.5 while tetracycline affects osteoclasts at the site of resorption, drugs are able to affect the recruitment of osteoclasts to the site of injury. thus the combination of the two types of drugs might have a synergistic effect on the inhibition of root resorption. glucocorticoids have been widely used to reduce the deleterious effects of inflammatory responses. therefore, they could also potentially be useful in manipulating the initial inflammatory response after attachment damage. thus, repair by cementoblasts rather than bone-derived cells would be encouraged. topical dexamethasone was found to be useful, while systemic usage was not.1 discussion it is clear that according to literatures ankylosis and root resorption in tooth avulsion treatment are mostly caused by: 1) improper management directly after injury (i.e. direct replantation of contaminated t o o t h , w a s h i n g w i t h c o n t a m i n a t e d w a t e r ) ; 2) dry time exceeding 60 minutes, 3) persistent inflammation of the pdl; 4) minimal or absence of occlusal force (i.e. severe protruded teeth, rigid wiring).1–5,14,15 however, until now, there were still inconsistency whether endodontic treatment should be conducted extra-orally or intra-orally in avulsed tooth exceeding dry time limit. it is interesting that notwithstanding with this controversy, studies revealed that with respect to pulpal healing, the revascularization success rate of replanted teeth has been reported to range from 8% in mature teeth to 25% to 34% in immature teeth relative to periodontal ligament healing, success has been reported to range from 24% to 57%.10 however, this article did not compare extra-oral and intra-oral endodontic treatment. thus, there are still other aspects that should be considered. according to becktor et al.,20 several aspects which may be related to ankylosis and root resorption are the existence figure 1. a) open apex filled with ca(oh)2; b) apex closing process. 21 ��nugraeni, et al.: simple replantation protocol to avoid ankylosis of epithelial rest of mallasez (erm) and hertwig’s epithelial root sheath (hers). fujiyama et al.,21 study showed that denervation in rats resulted in dentoalveolar ankylosis was also associated with decreased numbers of erm cells, therefore erm cells might protect the root against resorption. additionally, talic et al.,22 study have shown that erm might play a role for maintenance and remodelling of the pdm. it has been shown that erm synthesize and secrete latent collagenase, and that they proliferate and increase in size in connection with experimental tooth movement. thus, it is in coincident with mine et al.,23 research which revealed that low forces such as in orthodontic tooth movement is beneficial for dentoalveolar ankylosis prevention. moreover, according to macintosh et al.,24 hers and erm might play an important role in preventing calcification and ankylosis of the pdm has been illustrated in their comparing vertebrate ankylosistype attachment and mammalian ‘true’ periodontium. this study demonstrated how the root of a gecko was free of hers and erm and how it was connected to bone via ankylosis. nevertheless, since most avulsed teeth were not directly replanted, the reason of which being: 1) the patient and people surrounding the injury site are reluctant or do not know that avulsed tooth should be replanted directly; especially if the patient is unconscious; 2) the emergency treatment priority was for other injuries which may be more severe (i.e. skull fractures); 3) delayed intra-oral inspection. therefore, this article stresses to develop a simple management of the unreplanted avulsed teeth with dry time > 60 min, especially those with closed apex which are prone to ankylosis. after 60 min extra oral dry time, the survival rate of the pdl cells on the root surface is almost zero, and osseous replacement is predicted. therefore, direct extirpation the pulps and application of intraradicular medicaments is beneficial. it has been suggested that ledermix® inhibits the proliferation of dentinoclasts and it may prove effective when mixed with ca(oh)2. 25,26 moreover, according to studies, corticosteroids have anti-resorptive effect, and they are biocompatible. as a result, intracanal placement of corticosteroids can be used as standard treatment protocol at emergency visit for traumatic injuries, in which the root resorption is predicted. corticosteroids can also be placed in the canal space to intercept external irr caused by infected pulp as long as the source of infection has been cleared out.25 however, trope26 did not suggest overuse of this material because of the possibility of tooth discoloration, additionally, it is not easily available in indonesia. the promising results of emdogain® in replantation were excellent; however, future studies should provide much needed data in terms of the true value of its use especially when considering that it is considerably expensive compared to other less expensive or more affordable alternatives. it is shown that recent studies have found promising results using more novel, emerging alternatives, such as: 1) doxycycline; 2) minocycline; 3) alendronate; or 4) intracanal medicaments such as ledermix®.27 based on these literatures, the factors that may prevent replantation from ankylosis and root resorption are as follows: 1) dry time < 60 min; 2) decontamination of root surface; 3) application of preserving pdl agent (i.e. emdogain®); 4) direct extirpation without endodontic treatment; 5) art that are includes intracanal medicaments limited to topical corticosteroids and antibiotics, ca(oh)2 is applied after 7–10 days; chlorhexidine mouthwash and systemic doxycycline; 6) semi-rigid splinting for 2–3 weeks, and 7) endodontic treatment. these factors are beneficial for reducing the ongoing inflammatory process and promoting pdl healing. nevertheless, it is interesting that successful results were significantly higher in open apex teeth, even though they also have necrosed pulp. therefore, our new concept is to create an open apex figure 2. apex opening direction should be wider apically to facilitate maximal intracanal medicaments contact with the survived pdl on the tooth or the remaining pdl on the alveolar bone, and removes the “delta area”.18 apex opening direction “delta area” �0 dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 25-30 condition by widening the apical orifice which are also able to remove, although not perfectly, the “delta area” which contains accessory canals (figure 2). it is not the same procedure as apex resection which reduced root length and may be disadvantageous for avulsed teeth that are already prone to root resorption, thus may have shorter root predictably. this new concept that is considered as simple procedure had several benefits: 1) open apex facilitates the intracanal medicaments to enter the periapical area which reduce pdl inflammation; 2) it removes most of the accessory canals in “delta area” that are disadvantageous for pulp sterilization; and 3) it facilitates drainage of the pro-inflammatory mediators in the periapical area, thus also reduce inflammation. this “open apex” will gradually close with the apexification procedures, or with final root canal filling (i.e. with gutta percha). however, careful instrumentation should be done to prevent from direct stimulation of the healing pdl. additionally, other part of our simple procedures are the use of medicaments which usually available in dental office such as ca(oh)2, chkm and corticosteroid preparations i.e. endomethazon® (dexamethazone, hydrocortisone acetate, thymol iodide etc.). if minocycline gel is available, it is beneficial for topical application in the sulcular area. in case of the absence of ideal wire for splinting with brackets, twisted ligature wire can be used instead and attached to the tooth with glass ionomer cements or composites. patient must be also instructed to avoid biting at the avulsed tooth area for 2 weeks (periodontal healing time), to give periodontal stimulation, but the patient must avoid hard or tough food. for the concluding remarks, it is concluded that main principle to prevent from ankylosis is by reducing the ongoing inflammation of the pdl. inflammation leads to the altered function of the pdl transforming it into mineralized tissue-forming cells, which resulted to ankylosis. therefore, this simple replantation protocol that stresses on facilitating the application of anti-inflammatory medicaments is logical. however, since it is still a new concept, further laboratory and clinical researches are required. references 1. endodontic consideration in the management of traumatic dental injuries. endodontic: colleagues for excellence. published for the dental professional community by the american association of endodontists 2006. available online from url: http//www.aae. org/trauma_dental. accessed oct 11, 2008. 2. lin h. causes and prevalence of traumatic injuries to the permanent incisors of school children aged 10–14 years in maseru, lesotho. a thesis. 2006. 3. tekin u, filippi a, pohl y, kirschner h. expression of proliferating cell nuclear antigen in pulp cells of extracted immature teeth preserved in two different storage media. dental traumatol 2008; 24:38–42. 4. campbell km, casas mj, kenny dj. ankylosis of traumatized permanent incisors: pathogenesis and current approaches to diagnosis and management. j can dent assoc 2005; 71(10):763–8. 5. pohl y, filippi a, kirschner h. results after replantation of avulsed permanent teeth. ii. periodontal healing and the role of physiologic storage and antiresorptive-regenerative therapy. dent traumatol 2005; 21:93–101. 6. management of acute dental trauma. reference manual v29/no.7 07/08. available online from url: www.aapd.org/media/policies_ guidelines/gtrauma.pdf. accessed october 15, 2008. 7. yoshizawa t, takizawa f, iizawa f, ishibashi o, kawashima h, matsuda a, et al. homeobox protein msx2 acts as a molecular defense mechanism for preventing ossification in ligament fibroblasts. mol cell biol 2004; 24(8):3460–72. 8. yamada s, tomoeda m, ozawa y, yoneda s terashima y, kazuhiko i, et al. iplap-1/asporin, a novel negative regulator of periodontal ligament mineralization. j biol chem 2007; 282(32):23070–80. 9. saif ls, tordik pa. avulsed permanent teeth new treatment guidelines. clinical update 2004: 26(2):1–4. 10. mcintyre jd, lee jy, trope m, vann jr wf. management of avulsed permanent incisors: a comprehensive update. pediatr dent 2007; 29:56–63. 11. kenny dj, barret ej, casas mj. avulsions and intrusions: the controversial displacement injuries. j can dent assoc 2003; 69(5):308–13. 12. flores mt, andersson l, andreasen jo, bakland lk, malmgren b, barnett f, et al. guidelines for the management of traumatic dental injuries. ii. avulsion of permanent teeth. dental traumatol 2007; 23(3):130–6. 13. diangelis aj. bruised, broken and bedeviled teeth: contemporary management of traumatic dental injuries. available from: url www1.umn.edu/dental/courses/dent_5501/diangelis_handout. pdf. accessed october 7, 2008. 14. chen cc, kanno z, soma k. occlusal forces promote periodontal healing of transplanted teeth with enhanced nitric oxide synthesis. j med dent sci 2005; 52(1):59–64. 15. hu fw, hosomichi j, kanno z, soma k. the influence of occlusal stimuli on basic fibroblast growth factor expression in the periodontal healing of replanted teeth. j med dent sci 2008; 55:129–35. 16. kenny dj, barrett ej, johnston dh, sigal mj, tennenbaum hc. clinical management of avulsed permanent incisors using emdogain: initial report of an investigation. j can dent assoc 2000; 66(1):21. 17. moule aj, moule ca. the endodontic management of traumatized permanent anterior teeth: a review. aus d j supplement 2007; 52(suppl1):s122–s37. 18. lumley p, adams n, tomson p. practical clinical endodontics. 1st ed. burke fjt, editor. edinburgh: churchill livingstone; 2006. p. 82. 19. tsukiboshi m. treatment planning for traumatized teeth. 1st ed. chicago: quintessence pub; 2000. p. 66. 20. becktor kb, nolting d, becktor jp, kjær i. immunohistochemical localization of epithelial rests of malassez in human periodontal membrane. eur j orthod 2007; 29:350–3. 21. fujiyama k, yamashiro t, fukunaga t, balam ta, zheng l, takanoyamamoto t. denervation resulting in dento-alveolar ankylosis associated with decreased malassez epithelium. j dent res 2004; 83(8):625–9. 22. talic nf, evans ca, daniel jc, zaki ae. proliferation of epithelial rests of malassez during experimental tooth movement . am j orthod dentofac orthop 2003; 123:527–33. 23. mine k, kanno z, muramoto t, soma k. occlusal forces promote periodontal healing of transplanted teeth and prevent dentoalveolar ankylosis: an experimental study in rats. angle orthod 2005; 75(4):637–44. 24. mcintosh je, anderton x, flores-de-jacoby l, carlson ds, shuler cf, diekwisch tgh. caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian ‘true’ periodontium. micr res tech 2002; 59(5):449–59. 25. chen h, teixeras fb, ritter al, levin l, trope m. the effect of intracanal anti-inflammatory medicaments on external root resorption of replanted dog teeth after extended extra-oral dry time. dental traumatol 2008; 24:74–8. 26. trope m. clinical management of the avulsed tooth. mexico: oots summit v–monterrey; 2005. p. 1–5. 27. filippi a, pohl y, von arx t. treatment of replacement resorption by intentional replantation, resection of the ankylosed sites, and emdogain–results of a 6-year survey. dental traumatol 2006; 22(6):307–11. 71 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 71–75 original article introduction non-automated agitation of the irrigant can be produced using a file or a guttapercha cone that is inserted in the shaped and cleaned canal and subsequently moved periodically and rapidly by the operator.1,2 electrical ultrasonic and sonic devices are examples of automated agitation.3 these devices use a fine non-cutting polymer or metal tip that is vibrated within the canal space, with different frequencies according to manufacturer’s instruction. sonically activated instruments are driven with a frequency range of 1,000–6,000 hz and will generate a single node near the point at which the file is attached, as well as an antinode at the tip of the file.4–6 in ultrasonic solutions agitation, a tip oscillates at frequencies of 25 to 30 khz in a pattern of motion consisting of nodes and antinodes along its length.7 endoactivator, a sonically-driven canal irrigation system (dentsply tulsa dental specialties, tulsa, ok, usa), has been developed for activating root canal irrigants, and has recently been released onto the market. it comprises a portable hand-piece with three modes (low, medium, and high) and 3 types of disposable dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p71–75 visualization of bubbles generation of electrical-driven endoactivator tips during solutions activation in a root canal model and a modified extracted tooth: a pilot study harry huiz peeters1, elvira theola judith2, ketut suardita3, latief mooduto4 1laser research center in dentistry, bandung, indonesia 2faculty of dentistry, universitas maranatha, bandung, indonesia 3faculty of dentistry, iik bhakti wiyata, kediri, indonesia 4department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: endoactivator, a sonically-driven canal irrigation system (dentsply tulsa dental specialties, tulsa, ok), has been developed for activating root canal irrigants, and has recently been released onto the market. purpose: to obtain an initial understanding of bubbles generation of electrical endoactivator tips during activation of the irrigant in a transparent root canal model and a modified extracted tooth. methods: a modified extracted tooth and a straight glass model were filled with a solution containing 17% edta or 3% naocl. a medium activator tip 22-mm polymer noncutting #25, 0.04 file driven by an electrical sonic hand-piece at 190 hz (highest level) induced pressure waves that produced macroand micro-bubbles. the physical mechanisms involved were visualized using a miro 320s high-speed imaging system (phantom, wayne, nj, usa) with high temporal and spatial resolutions. the imaging system acquired images at 25,000 frames per second with 320×x240 pixels per image, and attached a 60-mm f/2.8 macro lens (nikon, tokyo, japan). results: the end of the tip did not generate bubbles formation. disruption of surface tension at the air–solution system in the glass canal model by an electrical sonic driven endoactivator tip generated bubbles in the solution. however, it did not occur at the system of solution–air interfaces in the glass canal and modified extracted tooth. conclusion: the physical mechanism of the solution activated by an electrical sonic driven endoactivator tip in generting bubbles formation is because the surface tension at the air–solution system disruption. no bubbles formation occurred in the solution in the restricted space either in the solution-air system or modified extracted tooth. better understanding of the physical mechanisms that relate specifically to the activation behaviour of endoactivator tips in solutions is key to improving the cleaning mechanism that applies during root canal treatment. keywords: bubbles formation; endoactivator; endodontics; sonic correspondence: harry huiz peeters, laser research center in dentistry, cihampelas 41 bandung, west java, 40174, indonesia. email: h2huiz@cbn.net.id mailto:h2huiz@cbn.net.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p71-75 72peeters et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 71–75 flexible non-cutting polymer tips of different sizes. its design allows for the safe activation of various irrigating solutions and could produce vigorous fluid dynamic in the canal space.8 electrical devices driven sonically has been found to be effective for cleaning root canals in many studies.3,9,10 however, in the last decade, some conflicting results have been reported in the literature on the efficacy of the system and its superiority over other systems. some researchers have found that sonically activated irrigation is less effective than ultrasonically activated irrigation, while others have suggested that the two methods produce comparable results.11,12 to our best knowledge, there have been no previous studies of the physical mechanism through which the electrical sonic driven endoactivator tip generates bubbles during activation of irrigation solution. in the present study, the mechanism was investigated by acquiring real-time data using a modified extracted tooth and a transparent glass model of root canal in order to visualize the oscillation amplitude of an endoactivator tip in the solution. hence, more knowledge on the mechanism underlying the activation behaviour of the endoactivator tips in the solutions is crucial to improve the outcomes of the root canal treatment. materials and methods a single-rooted human mandibular first premolar with straight canal and mature apex, and a tooth length of 25 mm was selected. the tooth was not decoronated, and the intact crown (10 mm) acted as a reservoir for the irrigation solution. the preparation of the root canal was completed to the working length (wl) using a pro taper hand file (dentsply maillefer) up to a size of 25/0.06 as the master apical file (maf) with the balanced force technique. the distal aspect of the root of the extracted tooth was carefully cut (from cervical to apex) using a fissure diamond bur (dentsply, tulsa, ok, usa) until the canal space was exposed (figure 1a and b), then the surface was ground successively with 240-, p400, and 600-grit sandpaper to provide a smooth surface. a glass cover was then carefully glued to the surface without entering the canal, and this was covered with composite (figure 1c and d). the apex was then sealed with composite resin and the outer surface of the tooth was covered with nail varnish, ensuring that there were no visible cracks, to simulate conditions within the root canal in vivo. the tooth was filled with the solution until it reached 5 mm below of the cervical level. the tip of the sonic instrument was inserted 2 mm shorter than the wl. a single modified extracted tooth was used for uniformity in the width and size of the root canal. the conditions within a straight root canal were simulated and visualized using a glass model with artificial canal and pulp cavity (which acted as a reservoir). the model was a glass root canal model (kimia farma, bandung, west java, indonesia), with canal inner diameter was 0.4 mm at the apex, crown height was 8 mm, crown diameter was 6 mm, a taper of 0.06 and an overall length of 25 mm. the canal was filled with a solution containing 17% edta or 3% naocl. the apex of the model was sealed with composite to allow the conditions within a root canal to be simulated. two approaches were used, one used the air–solution interface (in the coronal portion) and the other used the solution–air interface (in the apical portion). the solution–air interface system represents air entrapment in the apical region (figure 2). the models were filled with the solution until the level reached 5 mm below the coronal end (air-solution interface system), while in the solutionair interface system, the model was filled with solution starting 5 mm from the apex (air entrapment model). the tip of the sonic instrument was inserted into the solution at the air– solution or solution–air interface (10 mm and 4 mm from the interface, respectively). a single transparent glass model was used for uniformity in the width and size of the root canal. a 22-mm polymer noncutting # 25, 0.04 file (dentsply) was mounted on an endoactivator hand-piece (dentsply tulsa dental specialties, tulsa, ok) set at high mode (190 hz) and used to activate the irrigant. the models were subjected to active sonic irrigation. the tip of the sonic instrument was inserted into the solution at the air–solution (10 mm) or solution–air interface (4 mm), while the tip was inserted 2 mm shorter than the working length in the modified extracted tooth and activated passively without any filing motion. the hand-piece was fixed in a holder to ensure that the desired position was maintained. all the experiments with the different samples were performed by the same operator. figure 1. (a) a root sample with a longitudinal cut in the distal aspect of the root canal. (b) exposed root canal space. (c) exposed tooth with a glass cover (0.15 mm thick). (d) the glass cover was glued to the tooth and shaped to fit the shape of the root. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p71–75 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p71-75 73 peeters et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 71–75 figure 2. illustrations of the solution-air and air-solution systems. figure 3. representative images showing the disruption of surface tension at the air solution (a) and solution-air systems (b) in glass models, (c) modified extracted human root canal, (d) oscillation amplitude of the ea tip in the air. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p71–75 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p71-75 74peeters et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 71–75 condition. comprehensive visualizations were acquired using a high-speed camera and the glass root canal models together with modified teeth, to allow the basic physical mechanism involved in bubbles formation in the solution during sonic activation to be studied. ruddle8 claimed that cavitation occur when a solution is activated using an electrical-driven endoactivator tip. according to the formula for the cavitation number ca and the corresponding velocity threshold is 15 m/sec, the velocity of the file which can be calculated using the equation u = 2πfa (with f being the file oscillation frequency an a is the amplitude of oscillation). the ea tip oscillating at a frequency of 190 hz and oscillation amplitude of 5 mm oscillates with a velocity of 5.9 m/sec, it is not possible for cavitation to occur during an air or electrical sonic activation.13,14 it is not clear why bubbles occurred during activation using an electrical-driven endoactivator tip in solutions. cavitation is important to the efficient cleaning of the root canal system.15,16 there is a clear need to improve our understanding of the mechanism involved in bubble formation in a solution activated using an electricaldriven endoactivator tip during root canal treatment. two approaches were used, one using the air–solution interface system (in the coronal portion) and the other using the solution–air interface system (in the apical portion). the solution–air interface system represents air entrapment in the apical region. the process in the air–solution or solution–air systems can be observed closely using the modified extracted tooth and the glass canal model. the high-speed imaging method used enables the capture of images within intervals of mili-microseconds. surprisingly, we clearly observed that the end of the vibrating tip did not generate the formation of bubbles. furthermore, the tip vibrations disturbed the sonically activated solution interface. the vibrating activator tip transferred its energy to the solution and created waves, and these caused surface waves to form at the interfaces in both systems. in the air–solution interface system, the energy from the vibrating tip (at a certain power setting) was able to change the specific internal energy of the air-solution system to another form of energy.17 the energy was then transferred to the air-solution interface as pressure waves, disrupting the interface and creating individual surface tension in the form of bubbles. in other words, accumulation of input energy (from the vibrating tip) was used as kinetic energy for the vibrating tip to generate bubbles. this process created a series of waves or oscillations at the air-solution interface. finally, the formation of bubbles then occurred limited to the vicinity of the tip at around 75 µs post-ea tip activation. the life cycle of a bubble observed ranges from 250,000–700,000 µs with predominantly 700 µm in diameter. the authors have suggested that the bubbles are not cavitation because it occurred below the threshold needed for cavitation.13,14 the experiment showed that the vibration pattern of the tip at 190 hz generated the first mode shape, which has single node and an antinode. the node at the attachment and the antinode at the end of the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p71–75 the process was recorded using a phantom miro 320s high-speed digital imaging system (wayne, nj, usa) at 25,000 frames per second with 320x240 pixels per image using a macro lens (60 mm, f/2.8; nikon, tokyo, japan). the root canal model was illuminated using a fibre-lite lmi-6000 led continuous light source (dolan jenner industries, boxborough, ma, usa). measurements were repeated three times for each sample. results regardless of whether 17% edta or 3% naocl was used, the high-speed images and video clearly showed fluid streaming. the electrical sonic activated endoactivator tip generated hydrodynamic and finally bubbles formation occurred during the sonic activation process in the solution air system instead of in the restricted areas (the solution-air system and modified extracted tooth). oscillation amplitude of the ea tip in the air was approximately 5 mm. during the first 69 µs, the sonic energy was transferred to the tip and the tip vibrated at a frequency of approximately 190 hz, causing the solution surface (the air-solution interface) to move as a wave. after 75 µs, macro and micro bubbles formed in the solution. subsequently, accumulation of the energy during sonic activation at interface caused the micro and macro bubbles in the solution. finally, the bubbles formation occurred only the vicinity of the tip (figure 3a). (see supplemental video s1 at https://youtu. be/ut6vkwdgmj8). the life cycle of a bubble observed ranges from 250,000–700,000 µs with predominantly 700 µm in diameter. the end of the tip did not generate bubbles formation. a model with air trapped in the apical region was used; thus, there was a solution column and an air column. the energy of the tip vibration was not sufficiently enough to break the surface tension at the solution–air interface (figure 3b). (see supplemental video s2 at https://youtu.be/ rqzm0wmjuoy). no bubbles formation occurred during the activation process in the modified extracted tooth (air solution interface system) (figure 3c). (see supplement video s3 https://youtu.be/-bhdvlo3dka). discussion to be clinically relevant, in vitro studies should reproduce the clinical situation as much as possible. human dentin structure is not possible for direct visualization. all the in vitro models were made of transparent glass, and it is noted that glass may behave differently from root canal-wall material. the results of the in vitro studies may therefore not reflect what actually occurs in clinical settings. avoiding the bias results of in vitro study that it does not mimic the real condition of clinical settings, the authors, in this study, solve this dilemma using modified extracted teeth as models, which more relevant to clinical https://youtu.be/ut6vkwdgmj8 https://youtu.be/rqzm0wmjuoy https://youtu.be/-bhdvlo3dka https://e-journal.unair.ac.id/mkg/index https://youtu.be/ut6vkwdgmj8 https://youtu.be/rqzm0wmjuoy https://doi.org/10.20473/j.djmkg.v55.i2.p71-75 75 peeters et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 71–75 tip, which vibrates freely as a cantilever beam model (back and forth linear movement). a mode shape is defined as a specific pattern of vibration executed by a mechanical system at a specific frequency.18 the back and forth linear motion/pendulum of an electrical-driven endoactivator tip generated disruption of surface tension at the air–solution in the form of bubbles in the solution. the process involved the use of air (compressible fluid) and solution (incompressible fluid), which differ in density.19 the processes in the solution–air interface system showed that the accumulating kinetic energy reaching the surface of the solution (i.e., the solution–air interface) disturbed the surface and produced surface waves. however, the surface tension was just adequate to generate waves and streaming but not cavitation. we conclude that the energy transferred from the activator tip to the solution was not sufficient to disrupt the surface tension at the interface. surface tension is defined as the attractive force between molecules (internal energy) acting to decrease the surface area of a liquid. this produces a layer of surface molecules on the liquid that acts like a stretched membrane.20 in particular, this internal energy limits the flow of the liquid into narrow canals. it suggests that the surface tension internal energy of the solution, acting at the solution-air interface, has an important role in preventing bubbles generation.20 furthermore, in restricted areas, due to the oscillation amplitude of the ea tip to be 5 mm (figure 3d), the sonic driven tip will make more wall contact, which inhibits the tip vibration and may reduce the efficient streaming of the irrigant. this phenomenon occurred in the restricted areas such as modified extracted tooth.11 although each image was captured in a different root canal model, the dynamics of pressure waves, bubble formation, and wave formation proved to be reproducible in this in vitro study. to the best of our knowledge, this method of visualizing fluid dynamics and bubbles formation in real-time using real human tooth structures has not been used in any previous study. we believe that the results of this study improve our initial understanding of the basic mechanisms involved in the fluid dynamics in a solution used to treat root canals by means of an electrical-driven endoactivator tip. further studies are required to investigate the natural frequencies of the tip to improve its efficacy. regardless of the solution type, our results show that fluid streaming and bubbles formation occurred in a solution when activated by an electrical-driven endoactivator tip at the air-solution interface, instead of at the solution-air interface and modified extracted tooth. acknowledgments the authors deny any conflict of interest related to this study. the authors would like to thank professor zainal abidin phd at the institut teknologi bandung (bandung, indonesia), and mr. wowo watumas at the phantom company for their helpful contributions to discussions. references 1. andreani y, gad bt, cocks tc, harrison j, keresztes me, pomfret jk, rees eb, ma d, baloun bl, rahimi m. comparison of irrigant activation devices and conventional needle irrigation on smear layer and debris removal in curved canals. (smear layer removal from irrigant activation using sem). aust endod j. 2021; 47(2): 143–9. 2. andrabi sm-u-n, kumar a, mishra sk, tewari rk, alam s, siddiqui s. effect of manual dynamic activation on smear layer removal efficacy of ethylenediaminetetraacetic acid and smearclear: an in vitro scanning electron microscopic study. aust endod j. 2013; 39(3): 131–6. 3. mancini m, cerroni l, iorio l, armellin e, conte g, cianconi l. smear layer removal and canal cleanliness using different irrigation systems (endoactivator, endovac, and passive ultrasonic irrigation): field emission scanning electron microscopic evaluation in an in vitro study. j endod. 2013; 39(11): 1456–60. 4. tronstad l, barnett f, schwartzben l, frasca p. effectiveness and safety of a sonic vibratory endodontic instrument. endod dent traumatol. 1985; 1(2): 69–76. 5. plotino g, pameijer ch, grande nm, somma f. ultrasonics in endodontics: a review of the literature. j endod. 2007; 33(2): 81–95. 6. gulabivala k, ng y-l, gilbertson m, eames i. the fluid mechanics of root canal irrigation. physiol meas. 2010; 31(12): r49-84. 7. weller rn, brady jm, bernier we. efficacy of ultrasonic cleaning. j endod. 1980; 6(9): 740–3. 8. ruddle cj. hydrodynamic disinfection “tsunami” endodontics. int dent sa. 2009; 11(4): 6–18. 9. akman m, akbulut mb, aydınbelge ha, belli s. comparison of different irrigation activation regimens and conventional irrigation techniques for the removal of modified triple antibiotic paste from root canals. j endod. 2015; 41(5): 720–4. 10. haupt f, meinel m, gunawardana a, hülsmann m. effectiveness of different activated irrigation techniques on debris and smear layer removal from curved root canals: a sem evaluation. aust endod j. 2020; 46(1): 40–6. 11. swimberghe rcd, de clercq a, de moor rjg, meire ma. efficacy of sonically, ultrasonically and laser-activated irrigation in removing a biofilm-mimicking hydrogel from an isthmus model. int endod j. 2019; 52(4): 515–23. 12. rodrigues ct, ezeldeen m, jacobs r, lambrechts p, alcalde mp, hungaro duarte ma. cleaning efficacy and uncontrolled removal of dentin of two methods of irrigant activation in curved canals connected by an isthmus. aust endod j. 2021; 47(3): 631–8. 13. van der sluis lwm, versluis m, wu mk, wesselink pr. passive ultrasonic irrigation of the root canal: a review of the literature. int endod j. 2007; 40(6): 415–26. 14. macedo r, verhaagen b, rivas df, versluis m, wesselink p, van der sluis l. cavitation measurement during sonic and ultrasonic activated irrigation. j endod. 2014; 40(4): 580–3. 15. matsumoto h, yoshimine y, akamine a. visualization of irrigant flow and cavitation induced by er:yag laser within a root canal model. j endod. 2011; 37(6): 839–43. 16. verhaagen b. root ca na l clea n ing: th rough cavitation a nd microstreaming. [enschede, the netherlands]: enschede: university of twente; 2012. p. 359. 17. çengel ya, boles ma. thermodynamics: an engineering approach. 7th ed. new york: mcgraw-hill; 2011. p. 978. (cengel series in engineering thermal-fluid sciences). 18. scott as, fong e. body structures &amp; functions. thomson/ delmar learning; 2004. p. 495. 19. peeters hh, iskandar b, suardita k, suharto d. visualization of removal of trapped air from the apical region of the straight root canal models generating 2-phase intermittent counter flow during ultrasonically activated irrigation. j endod. 2014; 40(6): 857–61. 20. elger df, williams bc, crowe ct. engineering fluid mechanics. 10th ed. new jersey: john wiley & sons, inc.; 2012. p. 688. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p71–75 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p71-75 11 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 1–5 research report propolis extract as pulp capping material enhances odontoblastlike cell thickness and type 1 collagen expression (in vivo) ira widjiastuti, ari subiyanto, evri kusumah ningtyas, rendy popyandra, michael golden kurniawan and fauziah diajeng retnaningsih department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: propolis is a natural biocompatible material that has been widely studied in dentistry because of its inflammatory, anti-microbial and immunomodulatory properties. one of the active components is caffeic acid phenethyl ester (cape). cape is effective in stimulating collagen as well as inhibiting the inflammation and degeneration of dental pulp. purpose: to investigate the post-administration of propolis extract as pulp capping material enhances odontoblast-like cell thickness and type 1 collagen expression in wistar rats (rattus norvegicus) methods: this research was a true experimental design with a posttest-only control group design. sixty-three wistar rats were randomly divided into three groups, with each group consisting of 21 rats: group i: positive control; no capping material was administered; group ii: cape was administered; group iii: 11% of the propolis extract was administered. all samples were filled with glass ionomer cement. seven rats from each group were sacrificed after days 7, 14 and 28 of post-pulp capping administration, and their afflicted teeth were subsequently extracted for histologic analysis. results: no significant difference was seen in odontoblast-like cell thickness after the application of cape and propolis on days 7 and 14 (p > 0.05). however, a significant difference was noticed on day 28 (p < 0.05), with the thickness of odontoblast-like cell in cape being thinner than that in propolis. a significant difference in the expression of type 1 collagen was observed on days 7, 14 and 28 after the application of the propolis extract compared with cape (p < 0.05). conclusion: the post-administration of propolis extract as a pulp capping material could enhance odontoblast-like cell thickness and type 1 collagen expression in wistar rats. keywords: cape; odontoblast-like cell; propolis extract; pulp capping; type 1 collagen correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr moestopo 47, surabaya – indonesia. email: ira-w@fkg.unair.ac.id introduction dental caries can lead to degenerative changes in teeth structures. the products of bacterial metabolism destroy enamel and dentine, which can subsequently lead to pulp disease. basic measures to protect the pulp against caries include decreasing dentine permeability, reducing immune and inflammatory reactions and inducing tertiary dentine formation.1 pulp tissues consist of blood vessels, innervation, connective tissue fibres and cells, such as fibroblasts and odontoblasts. odontoblasts are unique cells that produce collagen and non-collagen proteins for the formation of dentine extracellular matrix; they are also the first response to exogenous stimuli or dental materials.2 dental pulp is similar to other connective tissues with the capability of restoration. the characteristics of pulp healing include the restoration of damaged soft tissues, differentiation of subodontoblasts into odontoblast-like cells and the construction of dentine bridges in perforated pulp tissues.3 cavity preparation often causes pulp perforation when removing the infected tissue in deep cavity lesion. this mechanical traumatic perforation results from the use of dental burs or other dental instruments. when the pulp is exposed, direct pulp capping can be performed to reduce the need for complicated treatment, such as root canal treatment or extraction.4,5 the regenerative pulp tissue treatment aims to regenerate normal tissues between pulp and dentine. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p1–5 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5 2 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 1–5 this tissue is responsible for regulating the formation of tertiary dentine. the pulp tissue consists of progenitor cells that proliferate and differentiate into odontoblasts. damaged odontoblasts can be replaced by odontoblastlike cells, originating from pulp fibroblast cells.6 the differentiation of odontoblasts involves several growth factors and extracellular matrices. at this stage, types 1 and 3 collagens play an important role. type 1 collagen can induce the differentiation and formation of the dentine component. type 1 collagen is a specific extracellular dense matrix produced at the beginning of the reparative dentine formation. type 1 collagen in odontoblast cells is considered the initial marker of the reparative dentine formation process.7 the most recognised and popular pulp capping material is calcium hydroxide. nevertheless, direct pulp capping with calcium hydroxide has some disadvantages. it can cause pulp inflammation for three months, have an unpredictable tissue response and have irregular reparative dentine formed below calcium hydroxide, probably forming the tunnel defect. these conditions can increase the dentinal bridge’s permeability, which can cause a bacterial invasion.8 nowadays, in dentistry, propolis is studied as a natural pulp capping material. propolis is a popular medicine in the world with therapeutic effects, such as anti-carcinogenic, anti-oxidant, anti-inflammatory, antibiotic, antifungal and antihepatotoxic.9–11 caffeic acid phenethyl ester (cape) is one of the active components of propolis, which has been investigated in vitro and in vivo. the function of cape has been shown to be effective in preventing cancer, inflammation and immunomodulation.12,13 cape can induce the formation of collagen in the dental pulp and reduce inflammation and degeneration of the pulp.9 thus, this study aims to investigate the effect of propolis extract as a pulp capping material on odontoblast-like cell thickness and type 1 collagen expression in wistar rats (rattus norvegicus). materials and methods this research was a true experimental design and approved by the ethics committee (no. 10/kkepk.fkg/ii/2015) of the faculty of dental medicine, universitas airlangga. sixty-three healthy, 8–16-month-old male wistar rats (rattus norvegicus), weighing 200–250 g and being fully erupted molars, were used in this study. the sample was fed a standard feed of 20 g/day per rat plus ad libitum drinking water. the wistar rats were anaesthetised using 100 mg of ketamine (ketalar®, warner–lambert, irlandia) and 10 mg/kg of xylazine hcl (rompun®, bayer, leverkusen, jerman) in sterile phosphate-buffered saline (pbs) placed on fixation board. the occlusal surface of the right maxillary first molar was perforated using a low-speed handpiece with a round diamond bur (diameter 0.46) until it reached the pulp chamber. the sample was divided into three groups, with each consisting of 21 rats (n = 7): on day 7, group i: positive control; no pulp capping material was administered, with glass ionomer cement (gic) restoration only; group ii: cape was administered; group iii: 11% of the propolis extract was administered. on day 14, group i: positive control; group ii: cape was administered; group iii: 11% of the propolis extract was administered. on day 28, group i: positive control; group ii: cape was administered; group iii: 11% of the propolis extract was administered. propolis was obtained from extract apis mellifera of bees and dissolved with 96% of ethanol. cape was obtained from the propolis extract and then analysed using visible spectrophotometer uv. for all groups, a micro applicator was used to put the pulp capping materials on dentine surface. then, all the samples were filled with restoration material (gic, fuji ix®, gc tokyo, japan). in addition, the samples were sacrificed after days 7, 14 and 28 post-pulp capping administration. the afflicted tooth and mandible were extracted and fixed in 10% of buffered formalin; they were then decalcified with 10% of ethylenediaminetetraacetic acid (edta) (onemed dental, pt. jaya mas mandiri, medika industri, sidoarjo, indonesia) for 28–30 days, embedded in paraffin and cut with microtome in a buccolingual plane parallel to the tooth vertical axis into sections of 6-micro thickness. the samples were then stained with hematoxylin–eosin (he) to analyse odontoblast-like cell thickness; they were viewed using a light microscope (nikon e100, tokyo, japan) in 400x magnification and counted in 1000x magnification on five different fields of view by two observers. to analyse type 1 collagen, the samples were stained with immunohistochemical (ihc) and monoclonal antibody (col1a1 antibody #sc-293182, santa cruz biotechnology, usa) and viewed using a light microscope (nikon e100, tokyo, japan) in 1000x magnification. spss software was used to analyse the data. one-way analysis of variance (anova) (p < 0.05) was used to compare the groups. subsequent pairwise comparisons were made among means by means of tukey’s honestly significant difference (hsd) (p < 0.05). results the thickness of odontoblast-like cells and type i collagen expression was higher in the propolis group than the other groups. the highest expression of type i collagen and thickness of odontoblast-like cells was found in group propolis (tables 1 and 2). the data on odontoblast-like cell thickness and type 1 collagen were analysed. anova test revealed significant differences in the thickness of odontoblast-like cells and type 1 collagen after days 7, 14 and 28 between the groups (p-value < 0.05). tukey’s hsd test was subsequently used to find out the differences between the groups. table 3 shows the significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p1–5 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5 3widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 1–5 figure 1. odontoblast-like cell in (a) positive control group, (b) cape group and (c) propolis group at 400x magnification. the black arrow shows the morphological of the odontoblast-like cell; the red line shows the thickness of the odontoblast-like cell; the yellow arrow shows the continuity of the odontoblast-like cell. figure 2. type i collagen expression in (a) positive control group, (b) cape group and (c) propolis group at 1000x magnification. the black arrow shows type i collagen expression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p1–5 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5 4 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 1–5 differences in the thickness of odontoblast-like cells between the cape and positive control groups on days 7 and 14, whereas no significant differences were seen on day 28. the odontoblast-like cells in the propolis group were significantly thicker than those in the positive control group on days 7, 14 and 28. meanwhile, between the propolis and cape groups, significant differences in the thickness of odontoblast-like cells were noticed on day 28 (figure 1). according to table 4, the propolis group showed more significant differences in the expression of type i collagen than the cape and positive control groups on days 7, 14 and 28 (figure 2). significant differences in type i collagen between the cape and positive control groups were recorded after days 7, 14 and 28 (p-value<0.05). discussion damage to the pulp tissue will set off an inflammatory reaction. inflammatory reactions are the initial stages of a series of healing processes. when the tissue is affected, fibroblasts migrate immediately to the wound, proliferate and produce collagen matrices; these later become hard tissue barriers against the remaining pulp tissues from irritants and set to repair the damaged tissues.14 the healing characteristics of the perforated pulp tissues include the restoration of the destroyed soft tissues, the differentiation of sub-odontoblasts into odontoblast-like cells and formation of reparative dentine bridge.3 in this research, the thickness of odontoblast-like cells on days 7, 14 and 28 showed significant differences across all groups. the odontoblast-like cells in the positive control group were thinner than those in the cape and propolis extract groups, perhaps because the positive control group was not treated with any pulp capping materials and that the cavity was only filled with restoration materials. karube et al.’s research suggests that sodium fluoride (naf) can induces apoptosis in odontoblast14 and that gic contains fluoride that is probably cytotoxic to odontoblast. the odontoblast-like cells on days 7, 14 and 28 in the propolis group were significantly thicker than those in the control group. these results may have been caused by the properties of propolis, which are known to have antibacterial, anti-inflammatory, antioxidant and immunomodulatory characteristics. these properties can cause the healing process in the dental pulp, which begins with the formation of collagen fibres, to occur more easily because propolis can prevent infection and accelerate cell regeneration.15 the active components of flavonoids and caffeic acid in the propolis extract play a crucial role in inhibiting table 1. mean and standard deviation of odontoblast-like cell thickness on days 7, 14 and 28 groups n mean ± sd day 7 day 14 day 28 positive control 7 0.01 ± 0.00 0.02 ± 0.00 0.04 ± 0.01 cape 7 0.07 ± 0.02 0.05 ± 0.01 0.06 ± 0.03 propolis 7 0.07 ± 0.01 0.07 ± 0.03 0.12 ± 0.01 table 2. mean and standard deviation of type i collagen expression on days 7, 14 and 28 groups n mean ± sd day 7 day 14 day 28 positive control 7 2.57 ± 1.13 2.71 ± 1.38 2.43 ± 0.78 cape 7 8.42 ± 2.07 7.43 ± 1.51 8.43 ± 1.51 propolis 7 13.14 ± 3.97 13.71 ± 3.72 17.57 ± 2.57 table 3. tukey hsd test results of odontoblast-like cell thickness on days 7, 14 and 28 days groups positive control cape propolis 7 positive control 0.000* 0.000* cape 0.657 propolis 14 positive control 0.008* 0.005* cape 0.107 propolis 28 positive control 0.032 0.000* cape 0.002* propolis *information: significant at p < 0.05 table 4. tukey hsd test results of type i collagen expression on days 7, 14 and 28 days groups positive control cape propolis 7 positive control 0.000* 0.000* cape 0.010* propolis 14 positive control 0.006* 0.000* cape 0.010* propolis 28 positive control 0.000* 0.000* cape 0.000* propolis *information: significant at p < 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p1–5 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5 5widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 1–5 the arachidonic acid lipoxygenase pathway, which can slow down the inflammatory response and increase the phagocytic activity and the stimulating cellular immunity. the propolis extract also functions as an antibacterial by destroying bacterial cell walls and preventing bacterial cell reproduction. the propolis extract offers better results arising from the dentine formation.16 the results of this study suggest that the propolis extract can accelerate the formation of odontoblast-like cells. the propolis extract is known to be able to stimulate tgfβ1 production.13 tgfβ1 stimulates the proliferation of fibroblasts and accelerates the formation of collagen content, which may participate in the healing process of the dental pulp.17 the potential of propolis to stimulate tgfβ1 production, differentiation of fibroblasts and inhibition of inflammation, was significantly thicker than that of the cape group. the use of cape as the pulp capping material did not inhibit the inflammatory response of the pulp tissue in the experimental animals, nor did it accelerate the healing and reparative processes of the dentin–pulp complex.18 the main compositions of propolis extracts comprise phenolic acids and esters, flavonoids (flavones, flavonones, flavonols, dihydroflavonols, chalcones), sesquiterpenes, terpenes, β steroids, naphthalene, aromatic aldehydes, alcohols, stilbene derivatives of benzopyran, caffeic acid, benzophenone, cinnamic acid derivatives and benzoic acid.19 cape is an active component in propolis, which inhibits the production of cytokines and chemokines, with t-cell proliferation and lymphokine production slowing down the inflammatory process. the anti-inflammatory activity of propolis concerns flavonoids, especially galangin and quercetin. the mechanism of cape involves inhibiting nf-kb; flavonoids, in turn, inhibit nf-kb and the activities of cyclooxygenase and lipoxygenase.20 flavonoids can also stimulate tgfβ1 induction and collagen synthesis by fibroblasts.18 in this study, the expression of type 1 collagen on days 7, 14 and 28 is higher in the propolis extract group than that in the control and cape groups. this explains why the odontoblast-like cells and the expression of type 1 collagen in the propolis extract group in this study were thicker than those in the cape group, which could be attributed to the propolis containing both cape and flavonoids, with some other substances containing quercetin, naringenin, salicylic acid, apigenin, ferulic acid, vestitol and galangin. these substances work together to accelerate the healing process by stimulating the expression of collagen type 1 during the initial stage of wound healing.20 in conclusion, the propolis extract produced thicker odontoblast-like cell layers and showed more expression of type 1 collagen than that in cape on days 7, 14 and 28. further study is required for the pulp capping material containing propolis to qualify as a medicine for human teeth. references 1. berman lh, hargreaves km, cohen s. cohen’s pathways of the pulp expert consult. 10th ed. st. louis: mosby elsevier; 2010. p. 8–9. 2. baldión pa, velandia-romero ml, castellanos je. odontoblast-like cells differentiated from dental pulp stem cells retain their phenotype after subcultivation. int j cell biol. 2018; 2018: 1–12. 3. parolia a, kundabala m, rao nn, acharya sr, agrawal p, mohan m, thomas m. a comparative histological analysis of human pulp following direct pulp capping with propolis, mineral trioxide aggregate and dycal. aust dent j. 2010; 55(1): 59–64. 4. kunarti s. pulp tissue inflammation and angiogenesis after pulp capping with transforming growth factor β1. dent j (majalah kedokt gigi). 2008; 41(2): 88–90. 5. zakaria mn. save the pulp is the essential issues on pulp capping treatment. j dentomaxillofacial sci. 2016; 1(2): 73–6. 6. be rgen holt z g, hør st e d-bi nd slev p, reit c. textb o ok of endodontology. 2nd ed. uk: wiley-blackwell; 2010. p. 3–7. 7. he g, george a. dentin matrix protein 1 immobilized on type i collagen fibrils facilitates apatite deposition in vitro. j biol chem. 2004; 279(12): 11649–56. 8. dwiandhono i, effendy r, kunarti s. the thickness of odontoblastlike cell layer after induced by propolis extract and calcium hydroxide. dent j (majalah kedokt gigi). 2016; 49(1): 17–21. 9. djurica g, vesna d, elena k. the effect of caffeic acid phenethyl ester on healing capacity and repair of the dentin-pulp complex: in vivo study. acta vet brno. 2008; 58: 99–108. 10. abbasi aj, mohammadi f, bayat m, gema sm, ghadirian h, seifi h, bayat h, bahrami n. applications of propolis in dentistry: a review. ethiop j health sci. 2018; 28(4): 505–12. 11. sabir a, tabbu cr, agustiono p, sosroseno w. histological analysis of rat dental pulp tissue capped with propolis. j oral sci. 2005; 47(3): 135–8. 12. elkhenany h, el-badri n, dhar m. green propolis extract promotes in vitro proliferation, differentiation, and migration of bone marrow stromal cells. biomed pharmacother. 2019; 115: 108861. 13. al-shaher a, wallace j, agarwal s, bretz w, baugh d. effect of propolis on human fibroblasts from the pulp and periodontal ligament. j endod. 2004; 30(5): 359–61. 14. karube h, nishitai g, inageda k, kurosu h, matsuoka m. naf activates mapks and induces apoptosis in odontoblast-like cells. j dent res. 2009; 88(5): 461–5. 15. yu c, abbott p v. an overview of the dental pulp: its functions and responses to injury. aust dent j. 2007; 52: s4–6. 16. sabir a. respons inflamasi pada pulpa gigi tikus setelah aplikasi ekstrak etanol propolis (eep) (the inflammatory response on rat dental pulp following ethanolic extract of propolis (eep) application). dent j (majalah kedokt gigi). 2005; 38(2): 77–83. 17. lin ps, chang hh, yeh cy, chang mc, chan cp, kuo hy, liu hc, liao wc, jeng py, yeung sy, jeng jh. transforming growth factor beta 1 increases collagen content, and stimulates procollagen i and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: role of mek/erk and activin receptor-like kinase-5/ smad signaling. j formos med assoc. 2017; 116(5): 351–8. 18. wang lc, lin yl, liang yc, yang yh, lee jh, yu hh, wu wm, chiang bl. the effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells. bmc immunol. 2009; 10: 39. 19. widjiastuti i, suardita k, saraswati w. the expressions of nf-kb and tgfb-1 on odontoblast-like cells of human dental pulp injected with propolis extracts. dent j (majalah kedokt gigi). 2014; 47: 13–8. 20. oryan a, alemzadeh e, moshiri a. potential role of propolis in wound healing: biological properties and therapeutic activities. biomed pharmacother. 2018; 98: 469–83. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p1–5 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5 �� understanding about the classification of pulp inflammation trijoedani widodo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract since most authors use the reversible pulpitis and irreversible pulpitis classification, however, many dentists still do not implement these new classifications. research was made using a descriptive method by proposing questionnaire to dentists from various dental clinics. the numbers of the dentists participating in this research are 22 dentists. all respondents use the diagnosis sheet during their examinations on patients. nonetheless, it can't be known what diagnosis card used and most of the dentists are still using the old classification. concerning responses given towards the new classification: a) the new classification had been heard, however, it was not clear (36.3%); b) the new classification has never been heard at all (63.6%). then, responses concerning whether a new development is important to be followed-up or not: a) there are those who think that information concerning new development is very important (27.2%); b) those who feel that it is important to have new information (68.3%); c) those who think that new information is not important (8%). it concluded that information concerning the development of classification of pulp inflammation did not reach the dentists. key words: classification, reversible pulpitis, irreversible pulpitis correspondence: trojoedani widodo, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the classification of a type of diseases is really needed to decide the treatment indication easily. the classification is also important for pulp disease, nonetheless, the exactly classification of pulp inflammation or pulpitis has never been found.1 it is mentioned that a challenge faced by dentists in classifying right on pulp diseases is due to the other symptoms often accompanying especially during the transitional period of the pulp diseases. in dentists’ office or in the dental clinics, the dentist use various classification of pulp inflammation and indication of treatment. previously some classifications have already been made, those were classifications made by bence 2 and knap3 at 1976 , grossman4 at 1981, and shafer et al.5 at 1983. smulson6 at 1984 stated that it is very important to assume the inflammation level happened in order to classify the pulp inflammation. we should distinguish the pulp tissues condition that could be kept under pulp capping treatment and the pulp tissues that need pulpectomy because it could not be maintained anymore. he classified the pulpitis into reversible pulpitis and irreversible pulpitis. reversible pulpitis is mild to medium pulp inflammation caused by stimulation and the defense system of the pulp tissue is still able to recover. while irreversible pulpitis is severe pulp inflammation caused by a type of stimulation and the defense system of the pulp tissue cannot overcome it longer, and this cannot be recovered or healed. other authors followed up this classification later on, for examples: grossman et al. 7 at 1988; wiene8 at 1989; simon et al.9 at 1994; walton and torabinejad10 at 1996; mount and hume11 at 1998, beer et al.12 at 2000, stock et al.13 and pitt ford14 at 2004. widodo15 at 1997 in her research classified the pulpitis into reversible pulpitis and irreversible pulpitis to express the immunopathological change happened in the pulp inflammation using immunopatological concept and morfofunctional on inflammation of pulp tissues. most authors using the reversible pulpitis and irreversible pulpitis classification, however, many dentists still do not implement these new classifications. based on this phenomena, there is a problem about is there a difference to determine the diagnosis of pulp inflammation and why the new classification is not been adopted by dentists in clinics. therefore, a research was made using a descriptive research method,16 by proposing questionnaires to dentists from various dental clinics covering: dental clinics in government hospitals, dental clinics in private hospitals, dental clinics in government hospitals in public health centers, and laboratory clinics in the area of faculty of dentistry airlangga university. then an analysis was made on the collected questionnaires, to know whether there was any change in the diagnostic techniques implemented to find the pulpitis classification, and some responses over the new classification. it is through this practice that the cause of not using the new classification method is found and how the effort should be made to get the same understanding about the pulpitis classification. �7widodo: understanding about the classification of pulp material and method the purpose of this research is to find an explanation concerning an objective situation, how the dentists-inclinics diagnosed in determining the classification of a disease and its treatment plan implemented. the data is collected from the dentists, and the method used in this research is descriptive research method.16 the population of this research were the dentists. some of the samples were the dentists at surabaya, working at the dental clinics in private hospitals, government hospitals, dental clinics in government hospitals in public health centers, and laboratory clinics in the area of faculty of dentistry airlangga university. while the analysis unit is the answer given through the questionaires. the questionnaires sheet is used as a supporting instrument for data collection. the questionnaires were distributed among the 22 dentists, consist of two dentists from two dental laboratories in the area of faculty of dentistry, airlangga university, two dentists from the public health center at surabaya, some dentists of the private hospital at surabaya, and some dentists who are participating a training in the hospital of dr. soetomo, surabaya. the questionnaires sheets were presented by giving some explanations that it will not go far beyond the expectation of the researchers. a descriptive analysis was made upon the collected data. the diagnosis performed to understanding concerning classification of pulp inflammation, their knowledge about the development of new classification; reversible pulpitis and irreversible pulpitis. moreover, it was also observed their responses towards the current development of dental sciences especially dental conservation. result the number of the dentists participating in this research is 22. they consisted of two dentists from dental laboratory in the area of faculty of dentistry, airlangga university, two dentists from the public health center at surabaya, five dentists of the private hospital at surabaya, two dentists from navy department i.e., from the dental laboratory of navy (ladokgi yos sudarso) in ujung pandang, dentist of the dental clinic of the hospital of tk.iii kasdam v brawijaya surabaya, and ten dentists coming from various hospitals participating a training in dr. sutomo hospital. the result of the questionnaire can be seen as shown in table 1. the questionnaire proposed to the dentists is accompanied by giving some explanations about how to fill the forms. all respondents use the diagnosis sheet during their examinations on patients as seen in table 1. nonetheless, it cannot be known the diagnosis card that the dentists used and most of the dentists were used old classification. concerning responses given towards the new classification: a) the new classification has ever been heard, but not clearly, responded by eight (8) dentists (36.3%); b) the new classification has never been heard at all, responded by fourteen (14) dentists (63.6%). the response of the dentists about whether a new development is important to be followed-up or not: a) the information about new development is very important, was responded by six dentists (27.2%); b) those who feel that it is important to have new information was responsed by fifteen dentists (68.3%); c) those who think that new information is not important was responded only by one person (8%). discussion the purpose of this research is to examine the understanding of classification of pulp inflammation among the dentists. the data obtained through the questionnaires and this research was the descriptive research. the dentists involved in the research were the dentists who live in surabaya, both as permanent residents or as graduate students who are studying in surabaya. it is expected that the information collected was more widespread and varies. each institution has their own standards, demands, or regulations to determine the diagnosis of a disease related to the classification of the disease. the classification is table 1. the result of questionnaire from 22 dentists about diagnostic techniques, classification reversible pulpitis and irreversible pulpitis diagnostic techniques using classification given toward new classification need new information using the diagnostic card 22 (100%) not using the diagnostic card 0 using old classification 22 (100%) using new classificatin 0 has already known 0 has ever heard but not very clear 8 (36.3% ) has never heard at all 14 (63.3% ) very important 6 (27.2%) important 15 (68.3%) not important 1 (8%) �� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 46–48 important as the basic steps to determine any indication of treatment. the dentists from two laboratories (oral surgery and pedodontic) of faculty of dentistry, airlangga university are chosen. in oral surgery laboratory, diagnosis of all the teeth are performed although extraction is the first priority, moreover, for the pedodontic laboratory, they perform diagnosis all of the teeth in detail. some data were also collected from the dentists from the private hospital at surabaya and the district hospital from various places, who are training in the hospital of dr. soetomo. by doing this, a better understanding can be obtained. the purposes of the research are to observe whether classification of pulpitis is well understood, to know whether there is any difference in the diagnostic techniques, and determine the factor causing the new classification of pulpitis is not implemented yet. concerning the diagnostic techniques implemented in the pulpitis, the dentists have already used the diagnostic card. however, this action does not guarantee that a good understanding concerning the pulpitis is alike. therefore, it is important to socialize the diagnostic techniques and the use of right diagnostic sheet. the basics of right choise of diagnosis implemented in local clinics given during studying seriously affect the understanding of the dentists concerning the classification of the pulpitis. it can be concluded that, it is very important for the dentists continuously follow the new development or techniques in the science of dental clinics. the classification of pulpitis has already developed year to year, moreover in the understanding of hyperemia. according to the old concept hyperemia was not a disease but a coincidence before inflammation.2,3,4 hyperemia is signed by the vasodilatation of the blood cells, it is a preliminary phase of an inflammation process that shafer et al.5 at 1983 started to change hyperemia term into focal reversible pulpitis, then it was emphasized by smulson6 at 1984 using new term the reversible pulpitis. reversible pulpitis as a mild to medium inflammation caused by a stimulation and the defense system of the pulp tissue is still able to overcome it that it can also be recovered. while the irreversible pulpitis is a severe inflammation which can't overcome it, so, it can't be recovered.6,7 the classification of the pulpitis turned to the reversible pulpitis and the irreversible pulpitis finally followed up by the other authors like: grossman et al.7 at 1988, weine8 at 1989; simon et al.9 at 1994; walton and torabinejad 10 at 1996; mount and hume 11 at 1998, beer et al.12 at 2000, stock et al.13 and pitt ford at 2004. although this new classification started to be used in conservative dentistry textbooks since 1984, most of the dentists still use the old classification, especially the use of hyperemia pulpae. actually, the good understanding concerning the hyperemia pulpae was not applicable and it is important to correct it by using the right term, the reversible pulpitis as it means that it can be cured for the defense system of the pulp tissue is still able to overcome it. it was said that focal reversible pulpitis is a science term for hyperemia pulpae.17 it concluded that information concerning the development of classification of pulp inflammation did not reach the dentists. however, some of them, especially the dentists who work in town or suburb, try to get a new information about conservative dentistry through seminars. there is only one dentist who expressed that knowing new development of pulpitis diagnosis is not important, the reason is that he has a lot of patients in his clinic and most of the patient prefer to pull the tooth out. references 1. widodo t. pulp inflammation classification and their treatment indication. journal of dentofasial, dental faculty hasaanudin university, makasar 2003; 256. 2. bence r. endodontik klinik. soendoro eh, editor. jakarta: penerbit universitas indonesia; 1976. p. 1–14. 3. knapp m. klasifikasi penyakit pulpa. in: gardjito k. perawatan gigi dengan diagnosis pulpitis partialis. majalah kedokteran gigi surabaya 1976. iv;6. 4. grossman li. endodontic practice. 8th ed. philadelphia: lea and febriger; 1981. p. 26–61. 5. shafer wg, hien mk, levy bm. a text book of oral pathology. 4th ed. philadelphia: wb saunders co; 1983. p. 479–510. 6. smulson mh. classification and diagnosis of pulp pathosis. the dent clin of north am 1984; 28:699. 7. grossman li, oliet s, del rio ce. endodontic practice. 11th ed. philadelphia: lea and febriger; 1988. p. 36–49. 8. weine fs. endodontic therapy. 4th ed. london: the cv mosby co; 1989. p. 74–153. 9. simon jhs, walton re, pashley dh, dowden we, bakland lk. pulpal pathology, in: ingle ji, bakland lk, editors. endodontics. 4th ed. philadelphia. 1994. p. 422–26. 10. walton re, torabinejad m. prinsip dan praktik ilmu endodonsi. narlan sumawinata, winiati sidharta, bambang nursasongko, editors. jakarata: penerbit buku kedokteran egc; 1996. p. 40–68. 11. mount gj, hume wr. preservation and restoration of tooth structure. london: the cv mosby; 1998. p. 37–44. 12. beer r, baumann ma, kim s. atlas of dental medicine, endodontology. new york: thiemu stuttgard; 2000. p. 1–16. 13. stock c, walker r, gulabivala k. endodontics. 3rd ed. london: eidenburg; 2004. p. 74. 14. pitt ford tr. harty’s, endodontics in clinical practice. 5th ed. london: eidenburg; 2004. p. 37–38. 15. widodo t. analisis perubahan imunopatologik pada pulpitis reversibel dan ireversibel untuk memperbaiki diagnosis atas dasar imunopatogenesis pulpitis. dissertation surabaya: universitas airlangga; 1997. 16. notoatmodjo s. metodologi penelitian kesehatan. jakarta: penerbit pt rineka cipta; 1993. p. 135–40. 17. crowford wh. oral pathology, 2003. available at: http://www.usc.edu/ hst/dental/opath/chapters08_main.html. accessed february 9, 2007. 161 volume 45 number 3 september 2012 penetration effect of prostaglandin e2 gel on oral mucosa of rats rafinus arifin1, retno widayati2, erni h purwaningsih3 and dewi fatma s4 1 orthodontic resident, faculty of dentistry, universitas indonesia 2 department of orthodontics, faculty of dentistry, universitas indonesia 3 department of medical pharmacy, faculty of medicine, universitas indonesia 4 department of oral biology, faculty of dentistry, universitas indonesia jakarta indonesia abstract background: several researches reported that prostaglandin e2 (pge2) injection on buccal mucosa combined with orthodontic pressure can faster tooth movement but has disadvantages such as high alveolar bone and root resorption furthermore pain from injection needle. pge2 gel was made to better replace the lacks of injectable pge2. purpose: this research was aimed to prove that pge2 gel can penetrate rat’s oral mucosa effecting the appearance of pmn cells. methods: this research was an in vivo laboratory experiment using 36 sprague dawley rats which were divided into 3 groups: normal group, topical pge2 gel group after 1, 2, 4, 8 hours (4 subgroups), and topical gel without pge2 group after 1, 2, 4, 8 hours (4 subgroups). each group consists of 4 rats, therefore the total sample for all research groups were 36 rats. gel with 25 µg/ml of pge2 and gel without pge2 were applied on oral mucosa for 2 minutes. then, the rats were sacrificed after 1 hour, 2 hours, 4 hours, and 8 hours application. after that, the samples were prepared for histological examination with hematoxyllin and eosin. the picture were taken with optilab view and pmn cells amount were counted with light microscope, set 400 times of magnification. results: penetration effect of pge2 gel on rat’s oral mucosa result in pmn inflammation cells distribution. one-way anova showed no significant difference on pmn cells count in rats’ lower jaws between groups of normal and gel without pge2. there was significant difference between groups of pge2 gel and gel without pge2 (p=0,001). pge2 gel application showed pge2 as inflammatory media, even though administered topically. conclusion: pge2 gel can penetrate rat’s oral mucosa, effecting pmn cells 1, 2, 4 and 8 hours after application of pge2 gel. key words: pge2, pmn, tooth movement abstrak latar belakang: beberapa penelitian melaporkan bahwa injeksi (prostaglandin e2) pge2 pada mukosa bukal yang dikombinasikan dengan tekanan ortodonti dapat mempercepat pergerakan gigi, tapi mempunyai kekurangan berupa resorpsi yang besar pada tulang alveolar dan akar gigi, serta adanya rasa sakit akibat penggunaan jarum suntik. gel pge2 dibuat untuk mengatasi kekurangan pemberian pge2 secara injeksi. tujuan: untuk membuktikan bahwa gel pge2 dapat berpenetrasi pada mukosa mulut tikus dengan efek munculnya sel pmn. metode: jenis penelitian adalah eksperimental laboratorik in vivo, menggunakan 36 tikus sprague dawley yang dibagi menjadi 3 kelompok, yaitu kelompok normal; kelompok pengolesan gel pge2 setelah 1 jam, 2 jam, 4 jam, 8 jam (4 sub kelompok); kelompok pengolesan gel tanpa pge2 setelah 1 jam, 2 jam, 4 jam, 8 jam (4 sub kelompok). masing-masing kelompok terdiri 4 sampel, sehingga total sampel seluruh kelompok penelitian 36 tikus. gel pge2 dosis 25 µg/ml dan gel tanpa pge2 dioleskan pada mukosa mulut rahang bawah selama 2 menit. tikus di sacrifice setelah 1 jam, 2 jam, 4 jam dan 8 jam pengolesan. kemudian dibuat sediaan histologi dengan pewarnaan hematoxylin dan eosin. foto preparat diambil menggunakan optilab view. hitung jumlah sel-sel pmn menggunakan mikroskop cahaya dengan pembesaran 400x. hasil: efek penetrasi gel pge2 pada mukosa mulut terlihat distribusi sel-sel inflamasi pmn. uji one-way anova menunjukkan tidak ada perbedaan jumlah sel pmn yang bermakna pada mukosa rahang bawah tikus antara kelompok gel tanpa pge2 dan normal. ada perbedaan bermakna antara jumlah sel pmn kelompok pengolesan gel pge2 dengan gel tanpa pge2. (p = 0,001). hasil aplikasi gel pge2 menunjukkan gel pge2 sebagai media inflamasi, meskipun research report 162 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 161–166 introduction the orthodontic treatment has the goal to achieve good occlusion. it needs relative longer treatment time than other kinds of dental treatment with mean 28.5–29 months.1,2 the longer of orthodontic treatment, may increase the adverse effect, such as caries,3 gingivitis, and root resorption.4 there are several ways to shorten the treatment time, e.g. self ligating system of brackets,5 electromagnetic usage,6 surgical corticotomy,7 and prostaglandin e2 (pge2) injection on buccal mucosa. the studies have shown that pge2 injection could accelerate the tooth movement 1.6–2 times faster than control.8,9 that is why, pge2 injection becomes an alternative to enhance the tooth movement in order to shorten the orthodontic treatment time. the study of pge2 was done on experimental animal with pge2 injection dose, in range of 0.1–10 µg/ml, in cycle of 2–3 weeks (21 days).8 although pge2 injection could enhance the tooth movement, there are adverse effects of over resorption on alveolar bone and tooth root, also pain during needle infiltration.10 this pain may be caused by the needle usage, the infiltration depth, needle penetration,11 and pge2 acted as inflammation trigger which could be painful. to overcome those effects, it is needed to develop a new kind of pge2 in a form of gel. gel has an advantage in simple usage. it could be applied on oral mucosa without pain,12 and in sequence, so that the effect are expected to be better.13 carboxylmethylcellulose (cmc) are the chosen gel.13–14 cmc is one of cellulose derivative, a natural structural polymer found in plants.11 physical properties of cmc are ph 2,5–3, white, fluffy, acidic,hygroscopic powder with a slight characteristic odour. characteristics of cmc as a bioadhesive polimer are common component in bioadhesive dosage forms, unaffected by temperature variations, hydrolysis, oxidation and resistant to bacterial growth. cmc is known as one of mucoadhesive polymers which are capable of attaching to oral mucosa surfaces.14 the dosage of pge2 gel is bigger than injection due to the thickness of mucosa, in order to have pge2 effect on the bones. in this study, the dosage of the pge2 is 25 µg/ml or 3 times dosage in sequence of 0 hour, 2nd hour, and 4th hour. orthodontic tooth movement means that a sustained force is directly delivered into tooth or teeth using orthodontic appliance. orthodontic force along with increased vascular zat aktif diberikan secara topikal. kesimpulan: pge2 gel dapat berpenetrasi ke mukosa mulut tikus, dengan efek adanya sel-sel pmn pada 1 jam, 2 jam, 4 jam dan 8 jam setelah pengolesan gel pge2. kata kunci: pge2, pmn, pergerakan gigi correspondence: retno widayati, c/o: departemen ortodonti, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4. jakarta 10430, indonesia. email: widayati22@yahoo.com permeability and cellular infiltration, trigger inflammatory processes in the involved dental and paradental tissues. neutrophil, lymphocyte and monocytes called as pmn cells were invade on the tissues, enhancing pge2 release which indirectly cause the elevation in pge2. 12 pge2 is an inflammation stimulator to trigger the capillary vasodilatation that brings the acute inflammation where the amount of pmn cells increased.15 pge2 in a form of topical application that could trigger tissue inflammation.16 microscopic observation shows that oral mucosa inflammation could be seen from capillary vasodilatation due to inter or extra cellular dilatation.15,16 higher amount of pmn cells could be found on acute inflammation tissue than mono nucleus cells, especially neutrophil cells that could be seen after 30-minute of application.16 on chronic inflammation, mono nucleus cells, especially limphocyte, are higher than pmn leucocyte cells.17 the purpose of this study was to prove that pge2 gel could penetrate into oral mucosa based on the observation of pmn cells –count in vivo in oral mucosa of rats. materials and methods thirty six rats of sprague dawley, under supervision of litbangkes ri vetenerinarian with criteria of male, 3 months old, 200–230 g, were in good condition to be studied. thirty six rats were divided into 3 groups: 16 rats with pge2 gel application (experiment), 16 rats with cmc gel only (control), and 4 rats without any application (normal). rats in normal group were used as a validity to rats in control group. this study had been approved by ethical commission of faculty of dentistry, university of indonesia no. 117/ethical clearance/fkg ui/iv/2012. pge2 gel was made recently before based on the preliminary study which consisted of 25 µg active pge2, 0.03 g cmc powder, and 0.97 ml aquabidest. cmc powder was crushed using mortar and pestle, mixed with aquabidest and pge2. cmc gel was made resenter paratus. gel without pge2 was cmc gel without active pge2. pge2 gel and cmc gel, each consisted of 100 mg, were applied on mesial area of 46 buccal mucosa. twenty five µg/ml cmc gel application was applied using cotton bud for 2 minutes with circular movement. sixteen rats of experiment and 16 rats of control were applied in sequence of 0 hour, 2nd hour, and 4th hour. rats were sacrificed after 1 hour, 2 163arifin, et al.: penetration effect of pprostaglandin e2 gel hour, 4 hour, and 8 hour of gel application of each group consisted of 4 rats. on the 1st day, pge2 gel was applied on 4 rats and cmc gel was applied on the other 4 rats. all of the rats were sacrificed after 1 hour of gel application. on the 2nd day, pge2 gel was applied on 4 rats and cmc gel was applied on the other 4 rats. all of the rats were sacrificed after 2 hour of gel application. on the 3rd day, pge2 gel was applied on 4 rats and cmc gel was applied on the other 4 rats at 0 hour and 2 hour. all of the rats were sacrificed after 4 hour of gel application. on 4th day, pge2 gel was applied on 4 rats and cmc gel was applied on the other 4 rats at 0 hour, 2 hour and 4 hour. the rats were sacrificed after 8 hour of gel application. four rats in normal group were sacrificed on the 4th day. furthermore, the mucosa and the bone of oral tissue were cut transversal on the mesial area of mandible first right molar. histological preparation was done on histology laboratory of faculty of medicine, university of indonesia. the fixation used 4% paraformaldehid for 12 hours, demineralized using 10% edta in 7.5% polyvinylpyrrolidone solution on 4°c until soft. samples were dehydrated using alcohol in sequence on 4°c, xylol alcohol, pure xylol, and paraffin xylol in room temperature, and then the tissue was cut with the thickness of ± 6 µm, and dyed with he.17 the pictures were taken with optilab view on the areas with the most of inflammation cells. pmn cells were counted using light microscope with enlargement of 400. callibration test was done on 10% of samples (4 samples between interobserver histological expert of faculty of medicine, university of indonesia and researcher). results interobserver reliability test was performed between histological expert of faculty of medicine, university of indonesia and researcher on 10% of the total sample to count the amount of pmn cells. unpaired t-test showed that p = 0.423, p > 0.05 and there was no significant difference which meant the reliability test was good. the group of cmc gel application as a control compared to normal group was needed to confirm the validity. oneway anova test was performed on normal to control group and the statistic result showed p = 0.099, which meant that there was no significant difference on pmn cells-count observed from the area of mandible mucosa of rats between control and normal group (table 1). one-way anova test was performed in order to know the difference of the amount of pmn cells-count of control and experiment. the result showed that there was significant difference between experiment and control group with p = 0.001, p < 0.05 (table 2). histology examination result (he) from each group after application of 1 hour, 2 hour, 4 hour, and 8 hour is presented on figure 1. all picture in figure 1 showed sprague dawley's oral mucosa layers and the arrow focus on inflammation cells. the picture in experiment groups were a, c, e, g showed increase inflammation cells or pmn cells-count compare to their control in picture b, d, f and h. picture i was normal sprague dawley's oral mucosa layer and also showed some inflammation cells. table 1. the different of amount pmn cells-count after 1 hour, 2 hours, 4 hours and 8 hours of topical application gel between control and normal group, using one-way anova normal group n control group n x ± sd p > 1 hour application 4 5.25 ± 0.96 0.099 (6.25 ± 2.06) 4 > 2 hour application 4 7.75 ± 0.96 > 4 hour application 4 7.75 ± 0.96 > 8 hour application 4 6.25 ± 1.71 table 2. the different of amount pmn cells-count after 1 hour, 2 hours, 4 hours and 8 hours of topical application gel between experiment and control group, using one-way anova n control experiment p x ± sd x ± sd > 1 hour experiment 4 5.25 ± 0.08 11.25 ± 1.500 p = 0.001* > 2 hour experiment 4 7.75 ± 0.957 24.25 ± 2.875 > 4 hour experiment 4 7.75 ± 0.957 27.00 ± 2.944 > 8 hour experiment 4 6.52 ± 1.708 30.75 ± 3.948 * p < 0.05 significant 164 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 161–166 a b c d c d e f g hg h i figure 1. oral mucosa on mesial area of 46 buccal's sprague dawley using light microscop with opti lab view, 400× magnification. arrow showed inflammation cells. (a) 1 hour after topical application pge2 gel; (b) 1 hour after topical application cmc gel; (c) 2 hour after topical application pge2 gel; (d) 2 hour after topical application cmc gel; (e) 4 hour after topical application pge2 gel; (f) 4 hour after topical application cmc gel; (g) 8 hour after topical application pge2 gel; (h)4 hour after topical application cmc gel, (i)normal oral mucosa layer. discussion several studies showed that pge2 injection on buccal mucosa combined with orthodontic force could enhance tooth movement, although it has a disadvantage of over root resorption, over resorption of alveolar bone, also a pain due to needle infiltration.8 to overcome those effect, it is needed to develop a new kind of pge2 in form of gel. gel has an advantage in simple usage in oral mucosa without pain.13 cmc gel are the chosen gel, because of it's stability on storage, good tolerance of water miscible solvents and good adhesive strength.14 in dentistry, until recently there is no pge2 gel. cmc is known as one of mucoadhesive polymers which are capable of attaching to oral mucosa surfaces. nowadays it has been accepted as a strategy of specific localization of drug delivery system on mucosa buccal area. advantages associated with buccal drug delivery have rendered this route of administration useful for a variety of drugs.14 pge2 gel was made by mixing cmc gel with pge2 as the active agent. cmc gel is a media for pge2 to penetrate into rats oral mucosa layer. the purpose of this study was to examine the penetration effect of pge2 gel on experimental rats mucosa, as an inflammation mediator. active agent pge2 is an inflammatory stimulator to trigger the capillary vasodilation that brings the acute inflammation where pmn cells increase.16 pmn number are an indicator of the degree of acute inflammation. to assested the quantifying their number in tissue section usually used standardized system, called pmn cells-count.17 if pge2 is given topically on human body, non-specific immunity response will appear, such as neutrophil, basophil, and macrofag as pmn and mn cells.15 the application of active pge2 gel showed that pmn cells were increased to submucosa layer (figure 1). rats oral mucosa structure is not different from epithel layer of human oral mucosa, but the thickness of rat's oral epithel is less than human, about 40–140 µm.18 to be able to make the small dosage of pge2, which is 25 µg and could penetrate into mucosa layer, the application of pge2 gel could be done in sequence, 3 times of 0, 2, and 4 hours. the active accumulation could continue to penetrate into deeper mucosa tissue until the alveolar bones. 165arifin, et al.: penetration effect of pprostaglandin e2 gel in this research, histological preparation was using hematoxyllin and eosin (he) because it could show the inflammation tissue and the morphology of pmn cells clearly.19,20 futhermore, pmn cells-count could be done through light microscope pictures and this slide were photographed by using opti lab view with 400 magnification. there are two main tissues component of the oral mucosa that consist of a stratified squamous epithelium, called the oral epithelium, and an underlying connective tissue layer, called lamina propice. lamina propria is composed a connective tissue with several different cells: fibroblasts, macrophages, and inflammatory cells. between lamina propria and alveolar bone there are submucosa layer.15 he staining showed that distribution of pmn cells were in submucosa layer, and the nucleus of the pmn cells appeared more red with violet colour. in figure 1, especially on experiment group, shown the it amount of pmn cells higher than control group. it means that pge2 gel as stimulatory mediator could penetrate into deeper oral mucosa layer. the control group was analyzed with one-way anova test compared to the normal group, and showed that there was no significant difference between them (table 1). it showed that the pressure during application could increase the pmn cells on control group; but it did not affect on endogenous formation of pmn cells on rats mucosa. so control group had a good validity as compared to the experiment group. pge2 is derived from 20-carbon essential fatty acids that contain three, four or five double bounds.20 pge2 is an inflammation stimulator that derived from arachidonat acid. pge2 is not stored on tissue but will be synthesized after the stimulation.20,21 topical application of pge2 could cause inflammation. inflammation is controlled by the presence of a group of substances called chemical mediator such as vasoactive amine histamine, serotonin, kinin, fibrinolytic system, complement system and arachidonic acid (prostaglandin and leukotrienes). vasoactive amine histamine is important in the initiation of early phase of acute inflammation as it mediates to increased vascular permeability. some chemical mediator are interrelated inducing arteriole dilatation, fibrinolytic system produce plasmin. plasmin does important things in inflammation. it can produced vasodilatation by generating fibrinopeptides.16 this condition will trigger on acute inflammation cells.15 that's why on group with active pge2 the amount of pmn cells were increase, compared to the control (table 2 and figure 1). this result showed that after 1 hour of pge2 gel application the mediator of inflammation increased even though the active agent was given topically. based on the inflammation theory, inflammation process on the tissue had started on 30 minutes after stimulation.16 in acute inflammation there is a reactionary response by immune system. the important factors in acute inflammation acted by granulocyte cells included netrofil, eosinofil, basofil which called as pmn cells, some antibody and others complement. histological examination result in this research showed that pmn cells were increased. increased pmn cells-count was positive, because it proved the effect of pge2 as an inflammatory agent could penetrated into rats oral mucosa using gel as a media. this study reports that pge2 gel could penetrate into rats oral mucosa based on pmn cells-count through of infammation process. for the next study we suggest to examine penetration effect of pge2 gel on rats oral alveolar bone. it is concluded that pge2 gel could penetrate into rats oral mucosa based on the observation of pmn cell-count. after 1 hour, 2 hours, 4 hours, and 8 hours of pge2 gel application, there was a significant difference increasing of the pmn cells-count compared to control. acknowledgement 1. this research was expensed by dana riset desentralisasi dikti dipa ui 2012, drpm ui. 2. dr. h. ahmad aulia jusuf, ahk., ph.d for his participation as histological expert from faculty of medicine, university of indonesia. references 1. vu cq, roberts we, hartsfield jk, jr ofner s. treatment complexity index for assessing the relationship of treatment duration and outcomes in a graduate orthodontic clinic. am j orthod dentofacial orthop 2008; 133(1): 9.e1–9.e13. 2. mavreas d, athanasiou ea. factors affecting the duration of orthodontic treatment: a systematic review. eur j orthod 2008; 30(4): 386–95. 3. chaussain c. interest in a new test for caries risk in adolescents undergoing orthodontic treatment. j clin oral invest 2010; 14(2): 177–85. 4. segal gr, schiffman ph, tuncay oc. longer orthodontic treatment may result in greater external apical root resorption. j orthod craniofac res 2004; 7(2): 71–8. 5. fleming ps, dibiase at, lee rt. randomized clinical trial of orthodontic treatment efficiency with self-ligating and conventional fixed orthodontic appliances. am j orthod dentofac orthop 2010; 137(6): 738–42. 6. ravindran kv. role of magnets in orthodontics-a review. indian j of dentistry 2011; 2(4): 147–55. 7. germec d, giray b, kocadereli i, enacar a. lower incisor retraction with a modified corticotomy. angle orthod 2006; 76(5): 882–90. 8. seifi m, eslami b, saffar s. the effect of prostaglandin e2 and calcium gluconate on orthodontic tooth movement and root resorption in rats. eur j orthod 2003; 25(2): 199–204. 9. valiathan a, dhar s. prostaglandin and enhanced orthodontic tooth movement: in search of the silver bullet. current science 2006; 90: 311–3. 10. hirsch l, gibney m, berube j, manocchio j. impact of modified needle tip geometry on penetration force as well as acceptability, preference and perceived pain in subject with diabetes. j diabetes science and technology 2012; 6(2): 328–32. 11. patel j, patel b, banwait hs, parmar k, patel m. formulation and evaluation of topical aceclofenac gel using different gelling agent. int j drug dev and res 2011; 3(1): 156–64. 166 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 161–166 12. bär kj, natura g, telleria-diaz a, teschner p, vogel r, vazquez r, schaible hg, ebersberger a. changes in the effect of spinal pge2 during inflammation: prostaglandin e (ep1-ep4) receptor in spinal nociceptive processing of input from the normal or inflammed knee joint. j neurosci 2004; 24(3): 642–51. 13. knuth k, amiji a, robinson jr. hydrogel delivery systems for vaginal and oral applications: formulation and biological considerations. advanced drug delivery reviews 1993; 11(1–2): 137–67. 14. khairnar ga, sayyad fj. development of buccal delivery system based on mucoadhesive polymer. int j pharm tech 2010; 2: 710–35. 15. ten cate ar, bartold pm, squer ca, nanci a. repair and regeneration of oral tissue. in: nanci a, editor. ten cat's oral histology: development, structure and function. 6th ed. st louis: cv mosby; 2008. p. 380–2. 16. trowbridge ho, emling rc. inflammation in: part 1. chemical mediators of the vascular response illinois. usa: quintessencce publishing co, inc; 1997. p. 19–38. 17. bystrom j, evans i, newson j, stables m, toor i, van rooijen n, crawford m, colville-nash p, farrow s, gilroy dw. resolutionphase macrophages possess a unique inflammatory phenotype that is controlled by camp. blood 2008; 112(10): 4117–27. 18. jansen rg, van kuppevelt th, daamen wf, kuijpers-jagtman am, von den hoff, jw. tissue reactions to collagen scaffolds in the oral mucosa and skin of rats: environmental and mechanical factors. arch oral biol 2008; 53(4): 376–87. 19. berkovitz bkb, holland gr, moxham bj. oral anatomy, histology and embriology. 3rd ed. edinburg, new york: mosby; 2002. p. 134–44, 168–78, 187–201, 213–7, 220–30. 20. morrow jd, robert j. lipid-derived autacoid. in: goodman, gillman. pharmacological basis of therapeutics. 3rd ed. 2001. p. 669–80. 21. liebermen m, marks a. mark's basic medical biochemistry: a clinical approach 3rd ed. philadelphia: lippincot williams and wilkins; 2009. p. 671–84. 210 vol. 42. no. 4 october–december 2009 research report dental health economics and diagnosis related groups/casemix in indonesian dentistry ronnie rivany department of health administration and policy faculty of public health, university of indonesia jakarta indonesia abstract background: dental health economics is a branch of transdiciplinary science that refers to the economic and public health science. on the other hand, in other developed countries, diagnosis related groups (drg’s) /casemix has been used as a basic in creating the same perception between providers, patients and insurance companies in many aspects such as health planning, healthcare financing and quality assurance. purpose: the objective of this review is to propose a new paradigm of economics to be applied in indonesian dentistry. reviews: the dental health economics should be considered as an important aspect in indonesian dentistry, which is used to determine the dental treatment fee based on unit cost, cost containment, and cost recovery rate analysis. referring to australian refined diagnosis related group, health care industry in indonesia has starting to try a more structured way in grouping disease pattern in order to come up with more precise health care services to their patients. the on going development of indonesian drg’s is meant to confirm the disease pattern and partition. conclusion: the development of indonesian drg’s concept, especially the dental & oral disorders, needs a new paradigm, so the practitioners and academics could group and calculate the unit cost from each dental treatment according to the indonesian drg version (ina-drg’s). key words: dental cost analysis, diagnosis related groups (drg’s) and casemix, australian refined-drg, inadrg’s correspondence: ronnie rivany, c/o: bagian administrasi kebijakan kesehatan, fakultas kesehatan masyarakat universitas indonesia. gedung f, lt. 1 kampus fkm ui depok. e-mail: ronnie_rivany@yahoo.com. introduction pricing in indonesian dentistry, either on private clinics or dental hospital, is generally done without calculating the cost analysis first. benchmark against competitor’s tariff is used as the main reference without noticing how much the actual cost of production in dental and oral healthcare. cost analysis to produce unit cost, cost containment, and cost recovery rate analysis to estimate profit and loss is still an academic theory in economics that is not yet applied in indonesian dentistry. on the other hand, there are a few undeveloped concepts such as: the making of clinical pathway that is more comprehensive than the dentistry medical standard which will be used for quality assurance. the other concept is the grouping of the dental & oral disorders which becomes part of major diagnostic categories (mdc) 03 diseases and disorders of the ear, nose, oral and throat from the australian refined diagnostic related groups (ar drg’s), where there is no specific mdc. there is still no grouping pattern and cost of treatment per indonesian drg, specifically on dental & oral disorders. in hospital economics, micro economy is applied on the tools used to calculate the costs that will calculate the unit cost, pricing of a health care service that the hospital would provide, or the selection of several alternatives presented in the investment of medical equipment based on the results of a cost benefit analysis for benefits that will be reaped.1 pharmaconomics is aimed more on which drugs would be more effective and efficient (cost effectiveness analysis) in an intervention procedure in the pharmaceutical field, plus the output which is expected to improve the quality of life or cure the patient. however, the characteristics of the dental & oral health care commodity differ from other healthcare commodities (table 1). the table showed the very principal characteristic 211rivany: dental health economics differences between normal product and dental product, which are commodity, consumer knowledge, supply induced demand and time to buy. on the other hand, the economic sciences applied in the dental & oral health is almost similar to the hospital, where it is used to: calculate the effective and efficient cost in selecting the dental healthcare equipment for private and institutionalized practices; analyzing the dental cost per cost unit that will be used for pricing; and also calculate the cost recovery rate to see whether the price for the products have been favorable or not. the objective of this review is to propose a new paradigm of economics to be applied in indonesian dentistry, where the indonesian drg’s development tries to have its own characteristic according to tropical disease pattern and its treatment. on the other side, health economics is the application of economic science in the public health,3,4 which pioneered the application on other health care sectors, such as hospital economics for its application in the hospital,5 pharmaconomics in the pharmaceutical field,6 and also in dental and oral health care, which is called dental health economics. dental cost analysis pricing a service or health care should take these variables into consideration:1,3 the unit cost, ability to pay and willingness to pay, benchmarking, policies, elasticity and the desired margin. theoretically, the cost calculation, usually called cost analysis, to get the unit cost of a product or procedure is based on two approaches, distribution and activity. the cost analysis based on distribution is simple distribution method, step-down distribution method, double distribution method and multiple distribution method. as the indonesian medical care standard for dentistry has been issued by ministry of health republic of indonesia in year 2002, the cost analysis for dentistry should be based on the activities or activity based costing (abc). technically, abc method calculates the direct and indirect costs of a service/health care or a resource used by a patient/consumer because of an activity.7 what should be recorded in this method is the identification of the activities from the admission, the medical procedure provided, until the patient returns home or is released. technically, the activities in the medical care aspect that called the clinical pathway are included in the medical care standard, only when the activities include administration aspects and others supporting medical aspects.8,9 the clinical pathway is an “integrated health care planning concept that includes all the steps the patient goes through based on the medical care standards, nursing care standards, and other health staff standards that are evidence based with measurable results and within a certain amount of time during the hospital visit.” technically, the clinical pathway includes the admission stage, which involves administrative staff; diagnostic stage, involving other health personnel; pretherapy-therapy procedures; follow up; and discharge of the patient when patient is cured, (figure 1). the problem is that the details in the dentistry medical standards for examination, ancillary table 1. characteristics of dental & oral healthcare commodities2 characteristic normal product dental commodity tangible in-tangible consumer knowledge good poor decision to buy/utilize consumer consumer + provider supply induced demand no yes decision quantity & quality consumer provider payer consumer consumer + 3rd party evaluator consumer provider time to buy predict un-predict non product/medic component no yes figure 1. the relations between clinical pathway and costing.9 212 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 210-215 examinations, consultation and therapy/medical care procedures are not clear, so it may vary at the private and institutional practices. according to the basic concepts of the clinical pathway that systematically combines the standard operating procedure, medical care standard, its administration and nursing aspects from the moment the patient enters the clinic until he/she is cured; the category of procedures must be approved according to its clinical pathway. for example: the activities of cavity cleaning is included in diagnostics or pre therapy. an agreement must be reached based on the current procedures/activities. the type of procedures in the conservation dentistry department of faculty of dentistry must be determined, so that there would only be five stages of clinical pathway, i.e.: admission, diagnostic, pre therapy, therapy, follow up, and discharge. similar analogy should be implemented for oral surgery department and others. this is what the academics, professional organizations or the indonesian dental association and other related organizations must be aware of. all activities or procedures in the clinical pathway must be approved first by all the stakeholders, then a cost analysis using the abc method can be performed. based on the activity stages that demonstrate the cost analysis calculation using the abc method, table 2 illustrates the output of a cost analysis for the conservative department of “x” specialist dental clinic.10 the output, which is very specific according to the process flow in the clinic, is showing the difference of the unit cost’s calculation result from the different of cost recovery rate for each services. cost analysis per unit is very powerful, because cost containment can be achieved and also taking into consideration the existing cost structures. the cost recovery rate also can be calculated, and therefore enabling us to see which service is more profitable and what is the differences in the revenue from the production amount multiplied by the price and the unit cost. the basic concept is crr = 100%, so if the crr < 100% it is a loss, and if the crr > 100% it is called a profit, so it can recover its own costs.11 in table 2, it was only a coincidence that there are activities with a crr exceeding 100%, because the pricing was not based on cost analysis. diagnosis related groups (drg’s) and casemix in modern countries, the cost of a procedure/treatment of a related disease group/diagnosis related group (drg’s) and its case mix with its complications and co morbidities usually already have a national cost range with certain standard deviations.8,12 based on the standard cost for the disease group, it helps the patient to estimate the cost size they need to prepare if they become ill, and also helps the insurance company for claiming both: their clients and the providers/hospitals. the disease grouping is based on the international classification of disease (icd) from the who that consists of 21 chapters of common diseases, which now has been revised for the tenth time as well as the international classification of disease volume 10 (icd x), that is used as the guidelines in the medical procedures. australian drg the who’s international classification of disease x (icd-x) was issued several years ago. the diseases were classified based on general anatomy and function of body organs.12 australia has endeavored to re-classify the diseases into a 23 category classification, which called major diagnostic categories (mdc)13 as shown in table 3. in australian version (australian drg), out of their 23 major diagnostic categories, australia then classify it into 661 diagnostic related group and coding it into the following format: a dd s, with the alphabet a stands for pre major diagnostic categories diagnosis related groups (mdc number nul), and so on until it displays lines of alphabet starting with mdc number one (mdc 01) with the alphabet b for disease and disorders of the table 2. cost analysis calculation using the activity based costing method in conservation department of “x” specialist clinic10 no type of activity to price (idr) uc (idr) tc (to × uc) (idr) tr (idr) crr (tr/tc × 100%) 1 plastic restoration class 150.000 resin dentures 405 201,717.40 81,695,545.29 60,750,000 74% amalgam ii class mod 28 180,943.97 5,066,431.18 4,200,000 83% 2 endo care class 200.000 visit i 369 250,129.55 92,214,427.27 73,733,333 80% visit ii 369 227,896.69 84,017,913.83 73,733,333 88% visit iii 369 204,575.67 75,420,231.86 73,733,333 98% 3 non plastic restoration class 600.000 on lay 94 379,960.66 35,716,301.58 56,400,000 158% inlay 17 379,960.66 6,459,331.14 10,200,000 158% 4 consultation 26 60.000 66,677.48 1,733,614.59 1,560,000 90% 5 polishing 71 50.000 44,807.70 3,181,346.35 3,550,000 112% to = total output: uc = unit cost; tc = total cost; tr = total revenue; crr = cost recovery rate 213rivany: dental health economics nervous system. the alphabet dd for drg’s partition which range 01–39 for surgical partition, range 40–59 for other partition, range 60–99 for medical partition. the alphabet s indicated for split indicator which a for highest resources drg, and b for second highest resources which caused by the presence of complications and co-morbidity (casemix). here, the australian refined drg, the dental and oral disorders are included in mdc 03, the classification for diseases and disorders of the ear, nose, oral and throat that may have surgical, medical, and other procedures. the list is alphabetical and therefore the dental and oral disorders are under the letter d with numbers 01–39 for surgical procedures, 40–59 for other procedures, and 60–99 for medical procedures. in this group, extraction and restoration procedures (others partition) are catalogued in mdc 03. d 40 z, while more medical procedures (nonextractions and restorations) are in group d 67 z. based on the ar-drg, the cost weight for those two groups is illustrated in table 4. the average cost for dental and oral disorders group (mdc 03. drg d 40 z) is 1,392 a$. 1,018 a$ in direct cost and 374 a$ in indirect costs/overhead. therefore, the cost for any extraction and restoration procedures of any dentist in australia is based on the amount set nationally. while the costs for mdc 03. drg d 67 z with medical nonextraction and restoration procedures is 1,359 a$. 1,005 a$ in direct costs and 355 a$ in indirect costs/overhead. the questions are: is it possible to classify the diseases into separate groups based on the functional organ, such as mdc 02 that only includes the diseases and disorders of the eyes, and what is the cost analysis for each drg for the indonesian version of dental and oral disorders. discussion indonesia, as a developing nation, should also begin to establish a planning, payment/funding, and quality assurance pattern such as the drgs and case mix in other developed countries. the ministry of health, as the organization that is responsible for this country’s health has collaborated with hospital universiti kebangsaan malaysia to adopt the malaysian case mix system, and is now in the process of socializing the pricing list for various government hospitals. the question is how did they apply the clinical pathway as a quality assurance measure before the pricing. table 3. major diagnostic categories–australian version13 no. category 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 diseases and disorders of the nervous system diseases and disorders of the eye diseases and disorders of the ear, nose, and throat diseases and disorders of the respiratory system diseases and disorders of the circulatory system diseases and disorders of the digestive system diseases and disorders of hepatobiliary system and pancreas diseases and disorders of the musculoskeletal system and connective tissue diseases and disorders of the skin, subcutaneous tissue, and breast endocrine, nutritional, and metabolic diseases and disorders diseases and disorders of the kidney and the urinary tract diseases and disorders of the male reproductive diseases and disorders of the female reproductive system pregnancy, child birth, and the puerperium newborn and other neonates with conditions originating in the perinatal period diseases and disorders of blood and blood forming organs and immunological disorders myeloproliferative disease and disorder, and poorly differentiated neoplasm infectious and parasitic disease (systemic or unspecified sites) mental diseases and disorders alcohol/drug use and alcohol/drug-induced organic mental disorders injuries, poisoning, and toxic effects of drugs burns factors influencing health status and other contact with health service table 4. mdc 03 cost weight diseases and disorders of the ear, nose, oral and throat13 drg direct cost indirect cost/overhead total cost d 40 z 1,018 374 1,392 d 67 z 1,004 355 1,359 214 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 210-215 another conceptual framework is the development of an indonesian drg (ina drg) by rivany.14 the objective of this act is to develop an indonesian classification for the in-patient diseases, whatever the clinical pathway template, the calculation of the cost of treatment and socialize it to all the stakeholders in the nation, without disregarding the basic idea. in specific, there are several goals of the academic terminology and concept of ina-drg. the first goal is to confirm whether the in-patient healthcare patterns with surgical/others/medical procedures and its co morbidities and complications in indonesian hospitals can be adjusted to or follow the in-patient disease patterns of other countries. the second is to identify all the activities and utilization (evidence based) related to in-patient healthcare with surgical/others/ medical procedures with its co morbidities and complications from the admission, diagnostics, pretherapy/therapy, and follow up as the basis for the clinical pathway. the third is to establish an inadrg based clinical pathway with the related professional organizations for evidence based in-patient healthcare patterns with surgical/others/medical procedures and its co morbidities and complications in indonesian hospitals. the fourth is to identify all the direct and indirect costs in the ina-drg based on clinical pathway of evidence based in-patient healthcare patterns with surgical/others/medical procedures and its co morbidities and complications in indonesian hospital. the fifth is to perform a cost analysis of all healthcare packages for in-patient healthcare patterns with surgical/others/medical procedures and its co morbidities and complications based on the established clinical pathway where the cost analysis using activity based costing method for the direct costs and simple distribution method for the indirect costs. the last goal is to perform a calculation sensitivity test on the costs for healthcare packages by creating a simulation of the cost of treatment without including the salary and drugs, to avoid double counting if it is completely funded by the government. technically, the outline of the concept is found on the thinking pattern of the indonesian diagnostic related group (ina-drg), with confirmation and cost analysis as the basic. the two conceptual framework are illustrated in figure 2 and 3. figure 2. ina-drg conceptual framework (1).9 icd mdc drg drg drg cost tariff casemix cost tariff figure 3. ina-drg conceptual framework (2).9 icd: international classification of disease; m d c : m a j o r d i a g n o s t i c c a t e g o r i e s ; drg: diagnosis related groups. 215rivany: dental health economics in the first conceptual framework (figure 2), the inadrg terminology and academic concept has to confirm and perform a cost analysis of the actual unit cost to obtain the current cost based on the clinical pathway issued for the cost of treatment at the hospital. the unit cost is calculated using the activity based costing method and simple distribution method, where the cost for in-patient care is a function of utilization and unit cost as shown in figure 3. in the second conceptual framework (figure 3), the idea is to obtain the actual costs of various procedures, drugs, and medical equipment based on the clinical pathway received by the patient, whereas the factors that influence the utilization are the main diagnosis based on icd x, the characteristics of the patient, and the case mix. pricing can be determined after the cost is analyzed, and may or may not have a margin, depending on the vision and mission of each hospital. assuming that the clinical pathway and unit cost is an independent variable of cost of treatment per diagnosis based on the degree of the disease (with or without co morbidities and/or complications), the main diagnostic aspect, case mix (complicating and ancillary diseases), characteristics of the patient (age and gender), length of stay, utilization of medical/non-medical procedures, drugs and medical equipment play a large role in the cost analysis for cost unit per procedure. keep in mind that creating the ina-drg requires money, time, manpower, and facilities. australia spent 5-10 years and more than 40 million a$ to develop their drg in 1996. for any current academics, especially with a medicine or dentistry backgrounds, the first problem that must be solved is preparing the clinical pathway of the various medical care standards. it can be established by their own professional organizations. with this clinical pathway, the cost analysis per disease can be calculated, and at least be used nationwide as preparation for healthcare pricing later on. referring to the scarceness of dental health economics based researches compared to those based on health economics alone, it is showed that clinical pathway and cost of treatment in indonesian dentistry is actually possible to be made and apply, and so is the grouping of disease pattern of the dental and oral disorders, which is not necessarily the same as drg d 40 z & d 67 z from mdc 03 australian drg. the main idea that comes from the lesson learned above is that on australian drg the grouping of the disease pattern of the dental and oral disorders is part of mdc 03 on the category of diseases and disorders of the ear, nose and throat, which probably needs to be taken into consideration first by the practitioners and academics in indonesian dentistry. it is concluded that the development of indonesian drg’s concept, especially the dental & oral disorders, needs a new paradigm, so the practitioners and academics could group and calculate the unit cost from each dental treatment according to the indonesian drg version (inadrg’s). references 1. feldstein pj. health care economics. 2nd ed canada: a willey medical publication, john willey & sons inc; 1983. p. 109. 2. sorkin al. health economics: an introduction. canada: lexington books, d.c heath & company; 1975. p. 114. 3. world health organization (geneva). hospital economics. geneva: journal of health economics 1992; 1: 12. 4. b o o t m a n j l , t o w n s e n d r j , m c g h a n w f . p r i n c i p l e s o f pharmacoeconomics. 2nd ed. cincinati: harvey whitney books company; 1996. p. 146. 5. tuominen r. health economics in dentistry. california: meded inc; 1994. p. 29. 6. drummond mf, stoddart gl, torrance gw. methods for the economics evaluation of health care programs. new york: oxford university press; 1987. p. 98. 7. brimson ja, antos j. activity based management for service industries: government entities and non profit organizations. canada: john wiley & sons inc; 1994. p. 163. 8. ministry of health republic of indonesia. clinical pathway. jakarta: health care services directorate; 2005. p. 4. 9. rivany r. relation of clinical pathway with diagnosis related groups: ina version [paper]. depok: faculty of public health, university of indonesia; 2006. p. 12. 10. soenardi ta. pricing analysis of dental conservation in “x” clinic. thesis. depok: faculty of public health, university of indonesia; 2002. p. 58. 11. hindle. casemix and financial management. new york: mcgrawindle. casemix and financial management. new york: mcgrawcasemix and financial management. new york: mcgraw and hill; 1997. p. 65. 12. rivany r. casemix: micro economic reformation on indonesia healthcare industry. dissertation. depok: faculty of public health, university of indonesia; 1998. p. 108. 13. australian casemix clinical committee. australian refined diagnosis related groups. canberra: australia; 2006. p. 8. 14. intellectual property rights. indonesian diagnosis related groups (ina drg’s) concept. jakarta: directorate general of intellectual property rights, department of law and human rights of the republic of indonesia 2008. p. 1. guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are 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background: the professional activity of dentists involves a relatively small treatment area, namely; the oral cavity. dental treatment requires a high level of precision with the result that dentists frequently perform their duties in a physically uncomfortable position over a relatively extended period of time. tooth extraction is the most common form of treatment performed in a standing position, with extraction of the posterior maxillary tooth being the most challenging. 80 per cent of students and dentists working in the faculty of dentistry at the university of indonesia present musculoskeletal disorders (msd). purpose: to evaluate the level of msd risk of oral and maxillofacial surgery clinic students at the universitas airlangga dental hospital following posterior maxillary tooth extraction. methods: the evaluation of msd risk level was performed over a period of three months on 73 subjects who had experienced posterior maxillary tooth extraction, categorized as extraction under anaesthesia, extraction involving the use of an elevator and extraction using forceps. evaluation was conducted by two observers by means of cctv video footage using a rapid entire body assessment (reba) worksheet. results: under anaesthetic sedation, 67.12% experienced medium risk, 31.51% high risk, and 1.37% low risk. during extraction using an elevator, 58.90% experienced high risk, 35.62% medium risk and 5.48% extremely high risk. during extraction using forceps, 57.53% ran medium risk, 39.73% high risk, and 2.74% extremely high risk. conclusion: students who performed posterior maxillary tooth extraction could be categorized as running a high risk of msd during extraction using an elevator, but medium risk when administering anaesthesia and performing extraction with forceps. keywords: ergonomics; musculoskeletal disorders; rapid entire body assessment; tooth extraction correspondence: ni putu mira sumarta, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: niputu.mira@fkg.unair.ac.id introduction the daily professional activity of dentists involves a relatively small treatment area, namely; the oral cavity, access to which requires a high degree of precision. as a result, dentists frequently execute their duties in physically uncomfortable positions over comparatively lengthy periods of time. moreover, it is not uncommon for dentists to maintain static hand and shoulder posture during treatment,1 prioritizing the comfort of their patients while devoting far less attention to their own. dentists consider it more appropriate to approach the patient, rather than adjust seated position of the latter in the dental chair.2 this, compounded by insufficient breaks, can increase their risk of musculoskeletal disorders (msd).3 according to brown et al. (2010), msd was the most common cause of retirement on the grounds of ill health amongst dentists (55%), followed by mental and behavioural disorders (28%).4 tooth extraction is one frequently performed procedure that is sufficiently difficult to necessitate dentists unconsciously adopting an uncomfortable posture that leads to difficulties requiring the application of specific appropriate techniques and strategies.5 msd constitutes a disorder of the muscles, tendons, joints, vertebrae, peripheral nerves and vascular system that can occur suddenly and acutely or, alternatively, slowly and chronically. these disorders can be induced or exacerbated dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 19prayudha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 18–23 by various factors including work-related ones such as poor posture, maintaining a static position for excessive periods and repetitive movements.6 msd can be assessed by a variety of methods, one of which is rapid entire body assessment (reba). reba was devised by hignett and mcatamney at the university of nottingham to analyze body postures associated with the risk of work-related msd. the reba method can be used in a range of contexts where the entire body is being employed, for example: static, dynamic or rapidly-changing (where posture is unstable, weights are lifted and modifications are made to the workplace, equipment, training or working practices). reba evaluation is carried out in several stages. first, work attitude and behaviour is observed. second, the posture to be studied is selected, including repetitive postures, prolonged unchanging postures, postures requiring high levels of energy expenditure or muscle activity, uncomfortable postures, awkward, extreme and unstable postures, or ones that can be corrected by intervention, control or other changes. lastly, posture is assessed and posture scores calculated using the reba worksheet.7 according to a study by hasibuan (2011), 80% of students and dentists who studied or practiced at the faculty of dentistry at universitas indonesia had experienced msd, especially that affecting the neck, shoulders, lower arms, hands and back. it was concluded that the most common procedure undertaken in a standing position was that of tooth extraction, with the posterior maxillary tooth being the most challenging to manipulate.8 this study aims to determine and evaluate the ergonomic aspects and risk level of msd affecting students of the universitas airlangga dental hospital during the extraction of posterior maxillary teeth using the reba method. hopefully, the results may raise awareness among students and dentists alike concerning the risks of developing msd during dental practice. materials and methods a descriptive observational study with a cross-sectional approach was conducted at the oral and maxillofacial surgery clinic of universitas airlangga dental hospital between september and november 2017. this study received ethical clearance by the universitas airlangga faculty of dental medicine health research ethical clearance commission with certificate number: 099/ hrecc.fodm/vii/2017. the research samples were students of the faculty of dental medicine, universitas airlangga. simple random sampling was employed to determine the minimum sample size for this study. given the 302 cases of posterior maxillary tooth extraction, a minimum of 73 samples were employed to ensure that sampling error remained within 10%. following a study by domingo et al. (2015), observation of the most difficult and most frequently performed tasks was conducted.9 after determining the most common procedure and the most difficult tooth to manipulate, it was decided that subjects included in this study should comprise faculty of dental medicine, universitas airlangga students experienced in extracting maxillary premolars and molars. samples excluded from this study were ones that provided incomplete information when observed by means of cctv video recordings and/or performed tooth extraction procedures without resort to anaesthesia, extraction using an elevator, and/or extraction employing forceps. a total of four cctv cameras were installed at strategic locations in the oral and maxillofacial surgery clinic of universitas airlangga dental hospital to record students performing tooth extraction procedures consisting of anaesthesia, extraction using an elevator and extraction utilising forceps. video recordings were monitored by two observers with the msd risk level being evaluated according to the reba method7 within which they assigned a score for each of the following body regions: neck, back, upper and lower arms, wrists, and legs. the scores were subsequently entered on an reba worksheet divided into two main sections, labelled a and b. section a contained analysis of the neck, trunk and legs, as presented in figure 1. section b contained analysis of the arm and wrist, as shown in figure 2. when using reba, only one side of the body (left or right) is assessed at a time. section a postures (trunk, neck and legs) are scored first, with whichever section b postures are dominant for the operator (either left or right upper arms, lower arms and wrists) being subsequently scored. for each region, as shown in figure 3, a posture scoring scale and additional adjustments needed to be considered and accounted for in the score. the postures to be assessed should be based on the following criteria: the most challenging posture and work task, the posture sustained over a protracted period, or the posture involving the highest force loads. after the data for each region had been collated and scored, the tables contained in the worksheet (figure 4) were then used to compile the risk factor variables, generating a single score that represents the level of msd risk as presented in table 1. where: n = minimum sample size n = population size d = tolerated margin of error used in this study z(1-α/2) = 1,96 p = proportion of incidence or prevalence rate, if unknown then 50% (0.5) was used dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 20 prayudha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 18–23 figure 3. additional adjustment in the reba worksheet.7 figure 4. compiling table in the reba worksheet.7 figure 1. section a of reba worksheet.7 figure 2. section b of the reba worksheet. 7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 21prayudha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 18–23 figure 5. distribution of students’ risk level of msd during extraction procedure of the posterior maxillary tooth. 0 1 2 3 4 5 6 7 8 9 extraction usinganesthesia elevator extraction using forceps sc or e procedures mean modus median figure 6. descriptive data of students’ reba score during extraction procedure relating to the posterior maxillary teeth. table 1. interpretation of risk levels according to reba7 risk levelscore negligible risk, no action required1 low risk, change may be needed2-3 4-7 medium risk, further investigation, change is required 7-10 high risk, investigate and implement change as soon as possible ≥ very high risk, implement change immediately11 table 2. test result of wilcoxon signed ranks test anaesthesia extraction using elevator extraction using forceps asymp. sig. value 0.3170.1571.000 results the study was conducted on 73 samples, consisting of 14 males and 59 females. as a result, 67.12% students were categorized as of medium msd risk level for undertaking anaesthetic procedures and 57.53% for performing extraction using forceps. while high risk students were most often found performing extractions involving the use of elevator procedures (81.13%), their low-risk counterparts were found to only implement anaesthetic procedures with the lowest amount (1.37%) compared to other levels of risk, as presented in figure 5. students who performed anaesthesia and extraction using forceps can be categorized as being at medium risk of msd as can be seen from the mean, mode and median values. in cases of extraction involving the use of elevators, the mean, mode, and median values are all categorized as involving a high risk of msd, as shown in figure 6. data from two observers was examined using the wilcoxon signed ranks test to eliminate the possibility of bias. the test showed there to be no significant difference between the two observers in all procedures (p ≥ 0.05), as indicated by the contents of table 2. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 22 prayudha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 18–23 discussion the results of the study showed that those students who performed anaesthesia and extraction using forceps could be categorized as having a medium risk of msd, whereas in cases of extraction involving elevators the risk run by students was categorized as high. one salient factor influencing the results was the static position maintained by the students for more than one minute when performing tooth extraction procedures. the higher the effort expended in maintaining a static position, the lower the blood supply to the muscles. blood supply obstruction can cause the by-products of muscle contraction, for example lactic acid accumulating in the muscles and causing acute pain in the area of the contracting muscle. when this static position is frequently maintained for a protracted period, the pain can extend to the joints, tendons and other tissues. this condition is also referred to as msd.10,11 another factor that influenced the high level of risk of msd was the relatively inclined posture widely adopted by the students. according to dul and weerdmeester (2008), the need to lean in this manner results from the height of work site being too low in relation to the elbow,11 with students compensating for this problem by bending further in order to change their posture. an inclined posture can cause compression in the area of the cervix, spine and surrounding muscles which, in turn, may inhibit the blood supply leading to intervertebral disc malnutrition and potential compromising of the integrity of the musculoskeletal system.12 another factor that needs to be considered is the condition of the facilities and infrastructure used while the students carry out extraction procedures. possible defects in these can affect the posture of the dentist, especially in the case of bending.11 malfunctioning dental chairs which cannot be set at the optimal height and angle can affect his/her posture, obliging him/her to bend or lift the arms to a position higher than that recommended. it is this factor that increases the level of risk of msd in the students and which can be exacerbated by the possibility of complicating factors in the extraction cases carried out. according to riawan (2009), this is because such factors necessitate greater effort, more complicated techniques and more protracted treatment5 this scenario produces an awkward or unstable posture, with the pressure to which the muscles is subjected also being greater and the risk of msd in the operator increased.13 the evaluation results suggest that the reba scores produced by the students during anaesthetic procedures constitute the lowest among the three tooth extraction procedures. according to gupta et al. (2014), this is due to extraction, either by means of elevators or forceps, being carried out by operators with repetitive movements.14 the musculoskeletal system can be damaged by a series of repetitive movements, a condition termed microtrauma. such movements can take the form of gripping, twisting, pushing and pulling, among others. when carried out continuously for a protracted period, they can lead to excessive energy expenditure resulting in fatigue and injury to the muscles in question. it is this process that increases the incidence of msd.13 a lower reba score during anaesthesia was also influenced by a relatively short process of extraction compared to that using elevators and forceps because, in general, a longer procedure is more likely to cause fatigue than one involving a shorter working time.3,11 moreover, rest periods play an important role in muscle fatigue which represents a reversible physiological process if balanced by an optimal rest period which, in the opinion of chakrabarty et al.(2016), should be approximately 15% of the total working time.3 the relaxation period referred to here is the time required to stretch the muscles or simply change the posture previously adopted. however, when forced to continue working pathological changes will gradually occur in the muscles which also affect the surrounding tissue. this situation can cause pain or msd that does not dissipate immediately, even after rest.3,11,12 therefore, a balanced and adjusted working time setting should be able to prevent excessive exposure to the source of injury.10,11 extraction using elevators was the primary procedure that placed students at high risk of msd. elevators are employed in tooth extraction procedures to separate the tooth from the surrounding soft tissue.15 this statement is supported by mamoun (2017), who argued that this procedure serves to expand the alveolar bone around the teeth and facilitate removal of the tooth from its socket by the use of forceps.16 therefore, it can be concluded that, in general, extraction using elevators requires greater effort and energy than that using forceps. according to research conducted by sholihah et al. (2016), providing knowledge of ergonomic concepts can reduce the number of complaints regarding msd.17 in the field of dentistry, understanding the concept of ergonomics and its application is very important in preventing msd and the occurrence of early retirement,4 thereby improving overall quality of life.18 the reba evaluation results support the conclusion that, from an ergonomic perspective, students who extracted posterior maxillary teeth at the oral and maxillofacial surgery clinic of universitas airlangga dental hospital ran a high risk of msd, especially when using elevators. in contrast, with regard to anaesthesia and extraction involving the use of forceps, the students are considered to have experienced a medium level of risk. limitations on the study include the fact that observation was conducted indirectly via a cctv camera feed. this meant that several variables such as differences in the right and left regions of the extracted teeth, complicating factors in tooth extraction, anthropometry, age, and gender were inadequately considered. it is anticipated that further research could conduct analytic studies which take these variables into account. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 23prayudha, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 18–23 references 1. wijaya at, darwita rr, bahar a. the relation between risk factors and musculoskeletal impairment in dental students: a preliminary study. j dent indonesia. 2011; 18(2): 33–7. 2. windi, samad r. penerapan postur tubuh yang ergonomis oleh mahasiswa tahap profesi fakultas kedokteran gigi universitas hasanuddin selama prosedur perawatan (application of ergonomic posture by clinical dental students of faculty of dentistry hasanuddin university during. dentofasial. 2015; 14: 32–7. 3. cha k raba r ty s, sa rka r k, dev s, das t, mitra k, sahu s, gangopadhyay s. impact of rest breaks on musculoskeletal discomfort of chikan embroiderers of west bengal, india: a follow up field study. j occup health. 2016; 58(4): 365–72. 4. brown j, burke fjt, macdonald eb, gilmour h, hill kb, morris aj, white da, muirhead ek, murray k. dental practitioners and ill health retirement: causes, outcomes and re-employment. br dent j. 2010; 209(5): 1–8. 5. riawan l. teknik dan trik pencabutan gigi dengan penyulit. in: prosiding temu ilmiah bandung dentistry 6. bandung: universitas padjadjaran; 2009. p. 2. 6. graveling ra. ergonomics and musculoskeletal disorders (msds) in the workplace: a forensic and epidemiological analysis. boca raton: crc press; 2018. p. 1–7. 7. hignett s, mcatamney l. rapid entire body assessment (reba). appl ergon. 2000; 31(2): 201–5. 8. hasibuan lb. evaluasi postur kerja praktik dan rancangan usulan standar prosedur operasional ekstraksi gigi posterior atas tingkat mahasiswa fakultas kedokteran gigi uninersitas indonesia dengan pendekatan virtual environment. thesis. depok: universitas indonesia; 2011. p. 3, 104–10. 9. domingo jrt, pano mtsd, ecat dag, sanchez nadg, custodio bp. risk assessment on filipino construction workers. procedia manuf. 2015; 3: 1854–60. 10. wilson jr, sharples s. evaluation of human work. 4th ed. boca raton: crc press; 2015. p. 419–444. 11. dul j, weerdmeester ba. ergonomics for beginners: a quick reference guide. boca raton: crc press; 2008. p. 5–39. 12. noll m, silveira ea, avelar is de. evaluation of factors associated with severe and frequent back pain in high school athletes. plos one. 2017; 12(2): 1–18. 13. putz-anderson v. cumulative trauma disorders. london: crc press; 2017. p. 5–7. 14. gupta a, bhat m, mohammed t, bansal n, gupta g. ergonomics in dentistry. int j clin pediatr dent. 2014; 7: 30–4. 15. fragiskos fd. medical history. in: oral surgery. berlin, heidelberg: springer-verlag berlin heidelberg; 2007. p. 1–20. 16. mamoun j. use of elevator instruments when luxating and extracting teeth in dentistry: clinical techniques. j korean assoc oral maxillofac surg. 2017; 43(3): 204–11. 17. sholihah q, hanafi as, bachri aa, fauzia r. ergonomics awareness as efforts to increase knowledge and prevention of musculoskeletal disorders on fishermen. aquat procedia. 2016; 7: 187–94. 18. mathew aj, chopra a, thekkemuriyil dv, george e, goyal v, nair jb, trivandrum copcord study group. impact of musculoskeletal pain on physical function and health-related quality of life in a rural community in south india: a who-ilar-copcord-bjd india study. clin rheumatol. 2011; 30(11): 1491–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p18–23 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p18-23 mkgs vol 44 no 1 jan-mar 2011.indd 54 vol. 44. no. 1 march 2011 expression of matrix metalloproteinase-8 gene in fixed orthodontic patients susilowati1, mansjur nasir1, imam mudjari2, and thalca hamid3 1department of orthodontics, faculty of dentistry, hasanuddin university, makassar-indonesia 2department of dental public health, faculty of dentistry, hasanuddin university, makassar-indonesia 3department of orthodontics, airlangga university, surabaya-indonesia abstract background: orthodontic treatment with fixed appliance produces structural and biochemical changes and breaking the balance between the synthesis and the breakdown of the collagen in the periodontium. matrix metalloproteinase-8 (mmp-8) plays an important role in the remodeling of periodontal ligament during orthodontic movement. purpose: the purpose of this study was to observe the expression of mmp-8 gene in the gingival crevicular fluid (gcf) of fixed orthodontic patients. it is expexted that the result can be used as a reference to decide the proper time for elastomeric chain to be reactivated. methods: orthodontic fixed appliances were placed on 8 patients and elastomeric chains exerting 75 grams were attached to produce canine distalization. gcf samples were collected from the distal side of upper canines before force application, 1-, 2-, 3-, and 4 weeks after application consecutively. the samples were analyzed by using rt-pcr. statistical analyses used were univariate analysis and mann-whitneyu test. results: the expression of mmp-8 in the gcf at t0 was 31.3% but the force application elevated its expression to 65.6% at t1, and then decreased continously at t2, t3, and t4. there was no statistically significant difference of mmp-8 gene expression between t0 and t3. conclusion: the highest level of mmp-8 gene expression due to orthodontic forces was occured in the first week, but it declined continously in the following weeks. the proper time to reactivate an elastomeric chain was 3 weeks after application. key words: mmp-8, fixed orthodontic appliance, reactivation time abstrak latar belakang: perawatan ortodontik dengan peranti cekat menghasilkan perubahan-perubahan stuktural dan biokimiawi pada jaringan periodontal dan mengganggu keseimbangan antara sintesis dan pemecahan kolagen pada periodonsium. matrix metalloproteinase-8mmp-8 memainkan peran yang penting dalam remodeling ligamentum periodontal selama pergerakan gigi ortodontik. tujuan: tujuan dari penelitian ini ialah untuk mengamati ekspresi gen mmp-8 dalam cairan krevikuler gingiva (gcf) dari pasien ortodontik cekat. diharapkan bahwa hasil penelitian ini dapat digunakan sebagai acuan untuk menentukan waktu yang paling tepat untuk mengaktivasi kembali rantai elastomer. metode: peranti ortodontik cekat dipasang pada 8 pasien dan rantai elastomer dengan kekuatan 75 gram dipasang untuk menarik gigi kaninus ke distal. sampel gcf dikumpulkan dari bagian distal gigi kaninus atas berturut-turut sebelum aplikasi gaya, 1-, 2-, 3-, dan 4 minggu setelah aplikasi. sampel dianalisis dengan menggunakan rt-pcr. analisis statistik yang digunakan adalah analisis univariat dan uji mann-whitney u. hasil: ekspresi gen mmp-8 di dalam gcf pada t0 adalah 31.3%, tetapi pemberian tekanan menaikkan ekspresinya menjadi 65,6% pada t1 dan kemudian menurun secara kontinyu pada t2, t3, dan t4. tidak ada perbedaan yang bermakna secara statistik antara ekspresi gen mmp-8 pada to dan t3. kesimpulan: tingkat ekspresi tertinggi dari gen mmp-8 akibat tekanan ortodontik terjadi pada minggu pertama, tetapi kemudian menurun pada minggu-minggu berikutnya. waktu yang paling tepat untuk mengaktivasi kembali rantai elastomer adalah 3 minggu setelah aplikasi. kata kunci: mmp-8, peranti ortodontik cekat, waktu reaktivasi correspondence: susilowati, c/o: kompleks universitas hasanuddin. jl. sunu cx-6 makassar, indonesia. e-mail: susmudjari@yahoo.co.id research report 55susilowati, et al.: expression of matrix metalloproteinase-8 gene introduction the main goal of orthodontic treatment is to obtain an optimal function of occlusion and facial aesthetics. research and clinical observation showed that the treatment will be stable if there is a balance between the teeth and surrounding soft tissue along with the development of orthodontics, people who seek help to improve their irregular position of their teeth are increased. the need for orthodontic treatment increases not only in indonesia but also in many other countries.1–3 the teeth will move if subjected to pressure, followed by changes in the connective tissue. in the past, orthodontic resorption process is related to the pressure side and the apposition process on the strain side. along with the development of science and technology, new discoveries have shown that bone tissue remodeling are seen from various sciences and clinical disciplines, which are all useful to human beings. recent research showed that a perspective on orthodontic tooth movement based on molecular biology and immunology focused on the metabolic function of the extracellular matrix of periodontal tissues and bone, can be used to identify biological and diagnostic tools to monitor orthodontic tooth movement.4 the initial phase of orthodontic tooth movement usually involves many reactions resembling inflammation characterized by vascular changes and migration of leukocytes out of periodontal ligament capillaries. these changes lead to cellular activation and release of biologically active substances, such as enzymes and cytokines in the periodontal tissue.5 after the application of pressure, there will be structural and biochemical changes that disrupt the molecular balance between synthesis and degradation of collagen in the periodontal tissue. it showed that orthodontic tooth movement in humans, causing an increase in collagenase activity in gingival crevicular fluid (gcf). matrix metalloproteinase (mmp) plays a very important role in periodontal tissue remodeling.6 mmp-8 hydrolyzes most effectively to collagen type i and iii which are the major interstitial collagenases in human gingival inflammation.7 it has been demonstrated that the expression of mmp-8 and mmp-13 mrna in periodontal ligament of rats was increased during active movement of teeth. morphological and histo-chemichal changes of periodontal ligament cells have been studied, but only few studies on mmp expression on the periodontal tissue due to mechanical pressure.8 is it possible to observe the expressions of mmp-8 gene gingival crevicular fluid and used them as a reference to determine the proper time for elastomeric chain to be reactivated. therefore of this study, the aim was to investigate the expression of mmp-8 gene in gingival crevicular fluid during orthodontic tooth movement on fixed appliance wearers. it is expected that the result can be used as a reference to decide the proper time to re-activate fixed appliance, in this case, elastomeric chain. materials and methods informed consent from the subjects was obtained after an explanation of the study protocol, which was reviewed by ethical committee of medical faculty at hasanuddin university, makassar. eight adult orthodontic patients (two males, six females, aged 19-25 years ± 2, 3 years) were enrolled in the study. all patients were treated at an academic orthodontic clinic, rsgmp-fkg unhas, makassar. they all had fixed appliances [mini roth bracket, 0.018 inch slot with elastomeric chain (ormco, usa)] that gave light forces of approximately 75 grams. inclusion criteria for sample selection are as follows: patients were suffering from maxillary protrusion and/ or anterior crowding, based on kesling’s space analyses, they required premolar extraction, good oral hygiene, no periapical/periodontal diseases, no root anomaly in terms of shape and length, never undergone orthodontic treatment, and use no medication. patients with the following conditions were excluded: suffering from systemic diseases (diabetes mellitus), extreme position of the canines, and gingival crevicular fluid mixed with blood. the experimental design of this study is clinical followup study. in each subject, the upper first premolars were extracted before placing the brackets and wire. the canine undergoing distal movement was used as the experimental tooth. orthodontic appliances were placed using an edgewise technique, in which 0.018 x 0.025-inch slot bands and brackets (ormco, usa) were used. the canines were retracted with elastomeric chain (ormco, usa) on a 0.018inch round wire (ormco, usa). the canines were moved distally using an elastomeric chain that exerted an initial force of 75 g. at the distal aspect of the canines, gcf samples were collected before, and 1, 2, 3, and 4 weeks after initiation of tooth movement. gcf samples were collected from distal sides (resorption sides) of gingival crevices of upper canines that orthodontically moved. the teeth at the sampling sites were isolated with cotton rolls and gently dried with air. two paper points were carefully inserted into the gingival crevice and allowed to remain there for one minute but then discarded. care was taken to avoid mechanical injury. the same method was repeated, but the paper points were placed into tubes with the buffered solution (l-6) insides. the samples were then frozen and kept at -20o until analysis. the gcf samples were extracted to get a total rna. the rt-pcr analysis was performed by putting the following reagents into a microfuge tube: 6 μl reverse transcription buffer (primecript, takara, japan), 1.5 μl specific primer for mmp-8 i.e. sense primer: tggacccaatggaatccttgc and antisense primer: 56 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 54-58 atagccactcagagcccagta which generate 544 bp fragment, 1.5 μl enzyme mixt, 19.5 μl h2o and 1.5 μl mrna sample (it comes from 50 pg mrna template). then, the tube was incubated at 37ο c for 15 minutes, it allows the reverse transcription to work. raise the temperature to 94° c for 2 minutes, 60° c for 2 minutes, and 72° c for 3 minutes. dna bands were observed after 37 cycles of pcr. glyceraldehyde 3-phosphate dehydrogenase (gapdh) was added to each sample served as an internal control/ house-keeping gene for the entire process. the pcr product was loaded onto 2% agarose gel for electrophoresis and visualized with uv light after gel incubation in ethidium bromide solution. the images were obtained by a digital camera. the expression levels of mmp-8 relative to gapdh can be scored through the photograph by three people which were previously calibrated. it was assumed that the higher concentration of rt-pcr product, the brighter the light of dna band would be. they were expressed in a semiquantitative score of 1–4 as follows: score 1 if the light of the dna band was less bright than the control, score 2 if the light of the dna band was the same bright with the control, score 3 if the light of the dna band was slightly brighter than the control, and score 4 if the light of the dna band was much brighter than the control. the data obtained from the study were processed electronically using spss software version 15.0, then analyzed by using mann-whitney u statistical method and univariate analysis. results results of this research consist of percentages of mmp8 gene expression according to rt-pcr test of 40 samples from 8 subjects treated with fixed orthodontic appliances and determination of time in which the level of mmp-8 expression is same with the baseline’s level using mannwhitney u test. from the univariate statistical analysis, it showed that the gene expression of mmp-8 before the attachment of elastomeric chain was 31.3%. after the attachment, it was up-regulated to 65.6 % in the first week, but then downregulated to 56.3% in the second week, decreased again to 34.4% in the third week, and the lowest was 31.3% in the fourth week (figure 1). the difference of mmp-8 gene expression between t0 and t2 was significant (p = 0.003). there was no significant difference between t0 and t3 (p = 0.602) (table 1). discussion mmp is a member of group of enzyme that can break down protein, such as collagen, that are normally found in the spaces between cells of tissue i.e. extracellular matrix proteins. they are known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands (such as fas ligand), and chemokine in/activation. mmps are also thought to play a major role on cell behaviors such as cell proliferation, migration (adhesion/ dispersion), differentiation, angiogenesis, apoptosis and host defense.10–12 mmp-8 (collagenase-2), is a collagen cleaving enzyme which present in the connective tissue of most mammals. in human, the mmp-8 is encoded by mmp-8 gene. it is produced primarily by pmns (polymorphonuclear cells) and released from the specific granules at sites of inflammation.13 examination of the cells found in gingival crevicular fluid (gcf) has consistently shown that neutrophils constitute the largest number (about 92%) of cells.14 this was one of the reasons why gcf was used as a sample fluid to investigate the mmp-8 gene in the present study. during orthodontic treatment with fixed appliances, a clinically healthy periodontium with no plaque or food debris accumulation is important. in the present study, all the patients had a clinically healthy periodontium. some believe that the flow of gcf is induced by microbial accumulation at the dento-gingival junction. this flow increases greatly with inflammatory changes of gingivitis and periodontitis. the expression and activity of mmps in adult tissues is normally quite low, but increases significantly in various pathological conditions that may lead into unwanted tissue destruction, such as periodontitis.15 orthodontic treatment is mainly aimed at tooth movement by remodeling and adaptive changes in paradental tissue. to affect this outcome, only small amounts of force (20 to 150 g) per tooth might be required. it is assumed that an optimal force moves teeth effi ciently into their desired position without causing discomfort or tissue damage to the patient.16 in this present study, an elastomeric chain exerting 75 g of force was used to move the upper canine distally. orthodontic tooth movement due to mechanical force of appliance can alter the pdl’s vascularity and blood fl ow, resulting in local synthesis and release of various key molecules, such as neurotransmitters, cytokines, growth factors, colony-stimulating factors, and arachidonic table 1. mann-whitney u test result of the difference between t0 – t2 and t0 – t3 to the expression of mmp-8 gene in fixed orthodontic patients duration of force (week) n mmp-8 gene expression significance (p) percentage (%) 0 8 31.3 0.003 2 8 65.6 0 8 31.3 0.602 3 8 34.4 57susilowati, et al.: expression of matrix metalloproteinase-8 gene acid metabolites. these molecules can evoke many cellular responses by various cell types in and around teeth, providing a favorable microenvironment for tissue deposition or resorption.17 the levels of those mediators in gcf have been well demonstrated to be responsive to orthodontic force in humans. it was also discovered that mechanical stresses alter the structural properties of tissues at the cellular, molecular, and genetic levels. however, only a few studies have been focused on the remodeling caused by mmp in gcf during orthodontic tooth movement.18 mmp-8 (collagenase 2) plays an important role in periodontal tissue remodeling. mmp-8 has ability to maintain the structure, integrity, and cellular activities and functions of the extracellular matrix of periodontal tissues. the main component of the extracellular matrix is tissue proteins, i.e. collagen, fi bronectin, and glikosamino glikan. orthodontic tooth movement causes a widespread degradation of collagen in the extracellular matrix of periodontal tissues and alveolar bone. it allows a release of cells from extracellular matrix environment, such as osteoblasts are moving into the apposition site and osteoclasts to the resorption site, causing a tooth movement. the present in vivo study demonstrated (figure 1) that the expression of mmp-8 gene at the baseline (t0) was 31.3%, then the orthodontic force up-regulated sharply the mmp-8 gene expression to 65.6 % in the fi rst week. it supported the result of previous study conducted by apajalahti et al.,19 that mmp-8 level in the gcf signifi cantly increased in the initial stage (at 4-8 hours from the application of fi xed orthodontic appliance). unfortunately, it was not reported when the mmp-8 level went down to the same level with that before force application. ingman et al.,20 in their study of mmp-1 and -8 in gcf during 1 month of follow-up after fi xed appliance activation using ifma method showed that the mmp-8 level was 12-fold higher than in control. in contrast with our present study, the level of mmp-8 on the fourth week was 2-fold higher than in the fi rst week. in this present study, mmp-8 gene expression was also analyzed after long term (four weeks) tooth movement by using rt-pcr technique. the mmp-8 gene expression down regulated on the second week, more decreased on the third week and the least was on the fourth week. it can be assumed that the force induced by the elastomeric chain decreases with the time. as a consequence, the mmp-8 gene expression will decrease too. in this study result, there was no difference significantly between the expression of mmp-8 gene before application (to) and that in the third week (t3) (table 1). it means that they had been more or less in the same level. at the time when the mmp-8 gene expression goes down to the same level with its expression before application, is assumed to be the proper time to reactivate the elastomeric chain. it can be concluded that expression of mmp-8 gene in the gcf was up-regulated by the orthodontic pressure. the highest level of mmp-8 gene expression was happened in the first week, and then decreased gradually in the second, the third and the fourth week. the proper time to reactivate elastomeric chain was three weeks after application. references 1. erbay ef, caniklioglu cm, erbay sk. soft tissue profile in anatolian turkish adults. part i: evaluation of horizontal lip position using different soft tissue analysis. am j orthod dentofac orthop 2002; 121(1): 57–64. 2. alkhatib mn, bedi r, foster c. ethnic variation in orthodontic treatment need in london schoolchildren. available from: http:// www.biomedcentral.com/1472-6831/5/8. accessed january 5, 2009. 3. artun j, kerosuo h, behbehani f, al-jame b. early orthodontic treatment need and experience among adolescents in kuwait. available from: http//iadr.confec.com/afrmde05 /preliminary program /indext.html. accessed october 21, 2009. 4. bildt mm, bloemen m, kuijpers-jagtman am, von den hoff jw. matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement. eur j of orthod 2009; 39: 529–35. 5. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofac orthop 2006; 129: 432–54. 6. surlin p, rauten am, manolea h. the involvement of metalloproteinase and their tissular inhibitors in the processes of periodontal orthodontic remodeling. am j of morphology and embryology 2009; 50(2): 181–4. 7. sasano y, jing-xu zhi, tsubota m. gene expression of mmp-8 and mmp-13 during embryonic development of bone and cartilage in the rat mandible and hind limb. j histochem & cytochem 2002; 50(3): 325–32. 8. takahashi i, nishimura m, mitani h. expression of mmp-8 and mmp-13 genes in the periodontal ligament during tooth movement in rats. j of dent res 2003; 82(8): 646–51. 9. casaccia gr, gomes jc, alviano ds. microbiological evaluation of elatomeric chains. j angle orthod 2007; 77(5): 890–3. 10. henneman s, maltha jc, kujper-jagtman am, hoff jw. cmt-3 inhibits orthodontic tooth displacement in the rat. available at: http://hdl.handle.net/2066/52939. accessed december 2009. 11. nagase h, visse r, murphy g. structure and function of matrix metalloproteinase and timp. cardiovascular research 2006; 69(3): 562–7. 0 1 2 3 4 70 60 50 40 30 20 10 0 m m p -8 (% ) week fgure 1. the percentages of mmp-8 gene expression according to rt-pcr test result, based on the duration of force. 58 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 54-58 12. dozier s, escobar gp, lindsey ml. matrix metalloproteinase-7 activates mmp-8 but not mmp-13. medical chemistry 2006; 2(5): 523–6. 13. t o f t h a n s e n h , n u t a l l k r , e d w a r d d r , o w e n s t . k e y metalloproteinases are expressed by specific cell type in experimental autoimmune encephalomyelitis. j of immun 2004; 173: 5200–18. 14. lehner t. immunology of oral disease. 3rd ed. massachusetts-victoria: blackwell sci publ; 1999. p. 18–27. 15. sorsa t, tjaderhane l, salo t. matrix metalloproteinase (mmps) in oral diseases. oral diseases 2004; 10(6): 311–8. 16. proffit wr. contemporary orthodontics. st. louis: cv mosby co; 2000. p. 150–2. 17. kohno t, matsumoto y, kanno z, warita h, soma k. experimental tooth movement under light orthodontic forces-rates of tooth movement and changes of the periodontium. j orthod 2002; 29: 129–35. 18. tae-yeon lee, kee-yeon lee, hyoung-seon baik. expression of il-1β, mmp-9 and timp-1 on the pressure side of gingival under orthodontic loading. the angle orthodontist 2008; 79(4): 733–9. 19. apajalahti s, sorsa t, railavo s, ingman t. the in vivo levels of matrix metalloproteinase-1 and -8 in gingival crevicular fluid during initial orthodontic tooth movement. j of dent res 2003; 82: 1018–22. 20. ingman t, apajalahti s, mantyla p, sorsa t. matrix metalloproteinase1 and -8 in gingival crevicular fluid during orthodontic tooth movement: a pilot study during 1 month of follow-up after fixed appliance activation. european j of orthod 2006; 27: 202–7. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 138 volume 45 number 3 september 2012 medication intake and its influence on salivary profile of geriatric outpatients in cipto mangunkusumo hospital yuniardini septorini wimardhani, winanda annisa and febrina rahmayanti department of oral medicine faculty of dentistry, universitas indonesia jakarta indonesia abstract background: it has been well documented that several diseases or conditions and their related medications could be the risk factors for several ailments found in the oral cavity. increased usage of medication in elderly could have impact on quality of saliva that affects oral health, eventually cause deterioration in quality of life. purpose: examine the salivary ph, buffering capacity, stimulatedand unstimulated salivary flow rate profile in elderly using medications. methods: seventy-six elderly were consented and agreed to participate in this study. interview and medical record analysis were performed to get data about their health status, chronic use of medications and complaints related to xerostomia. collection of unstimulated and stimulated saliva samples were completed in parallel with measurement of salivary ph and buffering capacity. results: the mean salivary ph was moderately acidic while having low salivary buffering capacity. the mean unstimulated salivary flow rate (ussfr) was 0.24 ± 1.8ml/min and 41of subjects (53%) were classified hyposalivation, while the stimulated salivary flow rate (ssfr) was 0.86 ± 0.49ml/min and 31 (40%) classified hyposalivation. number of drugs-induced xerostomia intake significantly correlated with the reduction in the ussfr of subjects (p<0.0001), however it was not the case with salivary ph and buffering capacity (p>0.05). it also showed correlation with complaints related to xerostomia. the mean ussfr did not correlate with xerostomia complaints. conclusion: medications intake influenced salivary profile and had more effect in changes in xerostomia complaints and salivary quantity than to salivary ph and buffering capacity in indonesian elderly population. key words: medication, xerostomia, hyposalivation, elderly abstrak latar belakang: telah lama diketahui bahwa beberapa penyakit atau kondisi sistemik dan medikasinya dapat menjadi faktor resiko terjadinya beberapa kelainan dalam rongga mulut. meningkatnya penggunaan medikasi sistemik pada lansia dapat mempengaruhi kualitas saliva sehingga berpengaruh pada kesehatan mulut yang akhirnya menyebabkan menurunnya kualitas hidup. tujuan: mengetahui profil ph saliva, kapasitas dapar, laju aliran saliva terstimulasi (last) dan tanpa stimulasi (lasts) pada lansia yang mendapatkan medikasi sistemik. metode: tujuh puluh enam lansia telah menandatangani inform consent dan setuju untuk berpartisipasi. wawancara dan analsis rekam medis dilakukan untuk mendapatkan data tentang status kesehatan, penggunaan medikasi sistemik jangka panjang dan keluhan xerostomia. pengumpulan saliva tanpa stimulasi dan terstimulasi dilakukan bersama-sama dengan pengukuran ph dan kapasitas dapar. hasil: ph saliva subyek adalah berada dalam kelompok asam sedang dengan kapasitas dapar yang rendah. rerata lasts adalah 0,24 ± 1,8ml/menit dan 41 subyek (53%) mengalami hiposalivasi, sementara last adalah 0,86 ± 0,49ml/menit dan 31 subyek (40%) mengalami hiposalivasi. jumlah medikasi yang dapat menginduksi xerostomia secara bermakna berhubungan dengan penurunan lasts (p < 0,0001), namun tidak demikian dengan ph dan kapasitas dapar (p>0,05). medikasi sistemik juga berhubungan dengan keluhan yang terkait xerostomia. rerata lasts tidak berhubungan dengan keluhan xerostomia. kesimpulan: medikasi sistemik pada populasi lansia indonesia mempengaruhi profil saliva dan mempunyai pengaruh yang lebih besar pada keluhan xerostomia dan kuantitas saliva dibandingkan ph dan kapasitas dapar. kata kunci: medikasi sistemik, xerostomia, hiposalivasi, lansia correspondence: yuniardini septorini wimardhani, departemen penyakit mulut, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta 10430, indonesia. e-mail: yuniardini@ui.ac.i.id, tel: +62-21-2303257. research report 139wimardhani, et al.: medication intake and its influence introduction it is well documented that growing population of the elderly is very fast over other age groups of the population worldwide, especially in developing countries.1 in less than 15 years, the number of elderly population worldwide would reach 600 million and the number would be doubled in 2050.1 as a developing country, indonesia is posing tremendous challenges to maintain the health status of its elderly population, since there would be shift of disease pattern. increase number of ageing population and burden of the related chronic diseases require definite health promotion and prevention. beside many physiological changes in the oral mucosa that increase risk of elderly to problems in the oral cavity,2-4 degenerative diseases such as cardiovascular disease, hypertension, cancer, and diabetes mellitus, that are prevalent in old age, would also have impact to the oral health status.1 saliva is one component in the oral cavity that plays an important role in maintaining oral health. saliva functions mainly to lubricate oral mucosa, as part of its role in the stomatognatic system. it is also important for protection against pathogen microorganisms and oral mucosal repair. reduction in the salivary bicarbonate composition would influence its buffering capacity which further interfering salivary ph and saliva remineralization function. 5 therefore, its alteration in terms of quantity, quality and composition would interfere the oral homeostasis.5 several studies have included salivary flow rate, ph and buffering capacity to assess salivary function and determine the salivary profile.6,7 although salivary function was thought to be physiologically decline in parallel with age, saliva composition is not significantly different among different age of healthy individuals.8 there is growing evidence of that quality and quantity of saliva significantly influence oral health of the elderly, thus affecting quality of life.8–10 studies have been shown that majority of salivary dysfunction cases in elderly are related to systemic disease medication, or the presence of radiotherapy in the head and neck region.8 complaints of xerostomia which is usually related to subjective information, could be objectively assessed by correlating it with the salivary flow rate. to date, data on the condition of salivary profile and its relation to medication intake in elderly population in indonesia is still not well documented. therefore, this study aimed to examine the salivary ph, buffer capacity, unstimulated and stimulated salivary flow rate profile in elderly using medications in elderly attending the outpatient geriatric clinic in cipto mangunkusumo general hospital, jakarta indonesia. this study would provide additional information regarding elderly in indonesia. materials and methods seventy-six elderly, who were attending the geriatric outpatient clinic in cipto mangunkusumo hospital, jakarta, were consented and agreed to participate in this study. interview about their health status, chronic use of medications and complaints related to xerostomia using modified xerostomia questionnaire was performed.11 this study collected the xerostomia complaints using three questions modified from xerostomia inventory,11 which were "do your mouth feel dry?", "do you get up at night to drink?" and "do you need to sip liquid to aid in swallowing food?". after that, the unstimulated salivary flow rate (ussfr) was collected in the morning of the next visit to the clinic approximately at 10 am. the subjects were ask to avoid eating or drinking about 1 hour prior saliva collection. the ussfr was collected in 5 minutes time, where patient was asked to expectorate the saliva every 60 seconds. the analysis of salivary ph and buffer capacity was performed using the ussfr sample. collection of the stimulated salivary flow rate (ssfr) sample was performed by asking the patient to chew paraffin wax for 5 minutes, then expectorating the saliva every 60 seconds. all the procedure was carried out using saliva-check buffer kit from gc (900200 gc america, inc.). one-way anova test was used to compare mean values of all measurement. correlation of salivary profile with xerostomia complaints was analyzed using pearson correlation test. spearman correlation was used to analyze any correlation between medication intake and xerostomia complaints. results the study showed that the mean of ussfr and ssfr for the sample group were 0.24 ± 1.8 ml/min and 0.86 ± 0.49 ml/min respectively. although the mean value of the ussfr and ssfr were not categorized as hyposalivation, this study showed 41 subjects (53%) were classified having hyposalivation based on ussfr measurement, while according to ssfr measurement, 31 subjects (40%) were classified having hyposalivation, and the difference was not significant (p > 0.05). analysis of salivary ph showed that the majority of this elderly population having moderately acidic ph, whilst having low buffering capacity according the gc saliva-check buffer kit (table 1). although, we found that 9 of subjects (11%) had highly acidic saliva with very low buffering capacity, quite high number of subjects still had normal salivary ph 27 (36%) and normal buffering capacity 25 (33%) (figure 1). 140 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 138–143 the majority of subjects were on medications to treat the related systemic diseases. this study noted 14 different types of systemic disease/conditions that required medications. data on patients medication was then classified into five types of drug-induced xerostomia according to previously published review (table 2).12 this study found that many subjects had taken more than one type of medications to treat multiple systemic diseases. we therefore analyzed the difference between salivary ph, buffering capacity and ussfr with the number of drugs-induced xerostomia intake in the subjects (table 3). there was no difference in salivary ph and buffering capacity between the three groups of patients (p > 0.05). however, number of drugs-induced xerostomia intake strongly influenced the ussfr of the subjects in this study (p < 0.0001). according to the modified xerostomia questionnaire that was used in this study, the number of subjects having complaints of xerostomia were 58 (76%), however 27 subjects (37%) with xerostomia complaints were found to have normal salivary flow rate (table 4). therefore there was no correlation table 1. salivary profile of the subjects saliva salivary flow rate (ml/min) mean ± sd number of subjects with hyposalivation n (%) ph mean ± sd buffering capacity mean ± sd ussfr 0.24 ± 1.8 41(53) 6.6 ± 0.5 7.6 ± 2.8 ssfr 0.86 ± 049 31(40) hyposalivation ussfr < 0.2 ml/min, ssfr < 0.7ml/min; ph score: highly acidic = 5.0–5.8, moderate acidic = 6.0–6.6, normal = 6.8–7.8; buffer capacity score: very low = 0–5, low = 6–9, normal = 10–12. ha m a n vl l n n um be r of s ub je ct s ph buffering capacity 50 40 30 20 10 0 40 (53%) 27 (36%) 9 (11%) 42 (56%) 25 (33%) 9 (11%) figure 1. salivary ph and buffering capacity of the ussfr sample of the subjects showed that mostly were having moderate acidic ph dan low buffer capacity. ha = highly acidic, ma = moderate acidic, vl = very low, l = low, n = normal. table 3. salivary ph and buffering capacity of subjects with different number of drug-induced xerostomia intake number of druginduced xerostomia intake n salivary ph mean ± sd buffering capacity mean ± sd mean ± sd ussfr 0 18 6.8 ± 0.58 7.80 ± 2.74 0.39 ± 0.17 1 29 6.75 ± 0.51 7.86 ± 2.2 0.22 ± 0.18 > 2 29 6.57 ± 0.62 8.06 ± 2.5 0.16 ± 0.09 p value* 0.32 0.93 < 0.0001 * one-way anova table 2. intake of drugs-induced xerostomia type of drugs number of subjects n (%) antihypertensive 58 (76) bronchodilator 14 (18) diuretics 3 (3) antidepressants 2 (2) cytokines 2 (2) 141wimardhani, et al.: medication intake and its influence between xerostomia and condition of hyposalivation in this study (r square = 0.02, p value = 0.8, pearson correlation). it was also confirmed that there was no significant difference between mean value of ussfr and xerostomia complaints (p > 0.05, one-way anova) (table 4) and there was also no correlation between number of drugs-induced xerostomia intake and xerostomia complaints (r = 0.86, p value > 0.05) (table 5). discussion saliva plays a very important role in maintaining an individual's oral mucosa health. aging was considered to be the cause of reduction of salivary function, although it is now accepted that the production of saliva and its composition are actually not influenced by age in healthy people.8,12 reduction in salivary function in the elderly is usually correlated with the effects of systemic diseases and the related medications. it is very common to find complaints of dry mouth or so called xerostomia, a consequence of reduction of salivary flow rate or hyposalivation, in the older people. many studies and reviews have shown information about the correlation between quantity and quality of saliva in the elderly in relation to the systemic diseases and medication intake.13-15 similar reports or reviews in relation to indonesia are not well documented, therefore this study provided additional information regarding elderly in indonesia.16 this study focused on the elderly population attending geriatric outpatient clinic in the cipto mangunkusumo hospital in jakarta, indonesia. this was a small survey study only sampled elderly population who came to cipto mangunkusumo hospital, since basic information about salivary profile in the elderly in terms of salivary ph, buffering capacity and flow rate in relation to systemic diseases and medication intake has not been available. since cipto mangunkusumo hospital is a type a general hospital with many patients referred from different part of indonesia, data generated from this study might be useful as baseline. further multicenter study using bigger sample size should be done to generalize the condition of indonesian elderly. medication intake was analyzed based on the type of medication and the number of medications used. several reviews have listed names of drugs that induced xerostomia.12 the salivary profile was also analyzed to see the possible correlation with xerostomia complaints. it showed that the types of medication having influences to xerostomia were antihypertensive, bronchodilators, diuretics, antidepressants and cytokines, as previously published.12,17 many subjects in this study took more than one type of drugs that could induce xerostomia, which is in line with other study and the subjects were divided into three groups (table 3). mechanism behind this phenomenon might be resulted from synergistic effect of different drugs consumed by the patients. differences at the number and type of drugs consumed by the subjects of the study showing table 4. salivary flow rate of patients with xerostomia complaints number of xerostomia complaints n mean ± sd ussfr number of subjects with hyposalivation 0 18 0.24 ± 0.15 1 1 29 0.20 ± 0.16 18 2 23 0.20 ± 0.09 13 3 6 0.35 ± 0.3 6 p value = 0.17* r = 0.17** 95% ci = –0.94 to 0.97 p value = 0.8 r square = 0.02 * : one-way anova, ** : pearson correlation table 5. number of drugs-induced xerostomia in subjects having xerostomia complaints number of drug-induced xerostomia intake number of xerostomia complaints correlation analysis* 0 1 2 3 0 8 7 2 1 r = 0.86 p value = 0.861 8 7 9 5 >2 12 4 9 4 n 28 18 20 10 * spearman correlation 142 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 138–143 importance of thorough assessment of patients' medical history. this information could be useful for the dentist to find out the results of presence oral complaints. the mean salivary ph for the whole subjects in this study was moderately acidic while having low salivary buffering capacity. however, there was no difference in salivary ph and buffering capacity between the three groups of subjects in this study (p>0.05). although the difference was not statistically significant, changes in ph and buffering capacity would have role in the presence of diseases or conditions found in the oral cavity of this group of population. possible explanation might be based on the fact that remineralization of teeth structure in the oral cavity is sustained by the presence of calcium and phosphate ions in the saliva that would happen in neutral salivary ph.8 several oral conditions have been found in the subjects of this study as previously published.2 further analyzes of salivary profile might be needed to elucidate other possible factors influencing those oral findings.2 the status of important enzymes, immunoglobulin a, lactoferrin, histatins and defensins that would provide antimicrobial activity of the saliva that protects oral mucosa and the teeth from dangerous agents as well as providing lubrication to helps chewing, swallowing, speech and preventing trauma to the oral tissue.8 xerostomia complaints found in this study were not correlated with the mean ussfr. it is a common oral discomfort symptom related to elderly population and could potentially be problematic.16,18 although it is a clinical complaint, many cases found to have been correlated with dysfunction of salivary gland. this study has found that 76% of subjects having xerostomia complaints, although only 37% of those were having hyposalivation. subjects who were not found to have hyposalivation should be encouraged to do several preventive measures of hyposalivation. recently, the use of 1% malic acid was proven to increase salivary flow rate in patients consuming antihypertensive drugs.19 the ones who have true hyposalivation should be considered to be treated aiming to reduce the symptoms, increase salivary flow rate or to use saliva substitute.10,20 drug-induced xerostomia would have impact on to the cholinergic and/or sympathetic systems of the autonomic nervous system.12,21 muscarinic receptors located on the cell surface are the major responsible aid for fluid secretion, while the protein secretion is controlled by adrenergic and other receptors. the stimulation of these receptors results in a complex cascade that is mediated by intracellular calcium, eventually causing saliva secretion.21 significant difference between number of medication intake and reduced salivary flow rate was found (p < 0.001) in this study. this finding is in agreement with several studies showing that medication is responsible for reduction of ussfr.14,22 the results of this study also showed possible synergistic effect of multiple drug intake to ussfr. possible mechanism of hyposalivation caused by medications in this study population may include dysfunction in neurotransmitter receptors, destruction or disorder of the parenchymal salivary gland, dysregulation of immune system, dna damage and alterations in fluid and electrolytes or combination of them.10 it is important to consider applying modification when choosing type of drugs in the elderly, since the systemic treatment is usually for a long term one. this might have influenced the findings of oral health problems related to xerostomia and/or hyposalivation in the elderly. management of patients having complaints of xerostomia and/or hyposalivation should be designed based on the underlying causes, which might not be applicable for every problem. however, it is important consider the quality of life of the elderly who is already a frail individual and is having many systemic diseases and medication usage. in conclusion, medications intake influenced salivary profile and had more effect in changes in salivary flow rate than to salivary ph and buffering capacity in this elderly population. the types of medication having influences to xerostomia were antihypertensive, bronchodilators, diuretics, antidepressants and cytokines. acknowledgement we would like to thank all the staff of department of internal medicine sub division of geriatric medicine for allowing us to do research on their patients. we thank the generous help of drs. ahmad ronal and indriasti indah wardhany for their kind assistance during patient examination. references 1. petersen pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health programme. community dent oral epidemiol 2005; 33: 81–92. 2. kurniawan a, wimardhani ys, rahmayanti r. oral health and salivary profile of geriatric outpatients in cipto mangunkusumo general hospital. j dent indonesia 2010; 17: 53–7. 3. lehl g, lehl ss. oral health care in elderly. j indian acad geriatr 2005; 1: 25–30. 4. imran mohammed khan kf, ravishankar tl, nikhath. dental health status and treatment need of institutionalized geriatric population: an indian scenario. j indian acad geriatr 2011; 7: 154–8. 5. bardow a, moe d, nyvad b, nauntofte b. the buffer capacity and buffer systems of human whole saliva measured without loss of co2. arch oral biol 2000; 45: 1–12. 6. fenoll-palomares c, muñoz montagud jv, sanchiz v, herreros b, hernández v, mínguez m, benages a. unstimulated salivary flow rate, ph and buffer capacity of saliva in healthy volunteers. rev esp enferm dig 2004; 96: 773–83. 7. moritsuka m, kitasako y, burrow mf, ikeda m, tagami j, nomura s. quantitative assessment of stimulated saliva f low rate and buffering capacity in relation to different ages. j dent 2006; 34(9): 716–20. 8. gupta a, epstein jb, sroussi h. hyposalivation in elderly patients. j can dent assoc 2006; 72: 841–6. 9. satoh-kuriwada s, shoji n, kawai m, uneyama k, kaneta n, sasano t. hyposalivation strongly influences hypogeusia in the elderly. j health sci 2009; 55: 689–98. 143wimardhani, et al.: medication intake and its influence 10. von bültzingslöwen i, sollecito tp, fox pc, daniels t, jonsson r, lockhart pb, wray d, brennan mt, carrozzo m, bandera b, fujibayshi t, navazesh m, rhodus nl, schiqdt m. salivary dysfunction associated with systemic diseases: systematic review and clinical management recommendations. oral surg oral med oral pathol oral radiol endod 2007; 103(suppl 1): s57, e1–15. 11. thomson wm, chalmers jm, spencer aj, williams sm. the xerostomia inventory: a mult-item approach to measuring dry mouth. comm dent health 1999; 16: 12–7. 12. scully c. drug effect on salivary glands: dry mouth. oral dis 2003; 9: 165–76. 13. ship ja, nolan ne, puckett sa. longitudinal analysis of parotid and sub-mandibular salivary flow rates in healthy, different-aged adults. j gerontol a biol sci med sci 1995; 50(5): m285–9. 14. shetty sr, bhowick s, castelino r, babu s. dr ug induced xerostomia in elderly individuals: an institutional study. contemp clin dent 2012; 3: 173–5. 15. scelza mf, sliva dde f, ahiadzro nk, da silva le, scelza p. the influence of medication on salivary flow of the elderly: preliminary study. gerodontology 2010; 27(4): 278–82. 16. hopcraft ms, tan c. xerostomia: an update for clinicians. aus dent j 2010; 55: 238–44. 17. ichikawa k, sakuma s, yoshihara a, miyazaki h, funayama s, ito k, igarashi a. relationships between the amount of saliva and medications in elderly individuals. gerodontology 2011; 28: 116–20. 18. kusdhany ls, sundjaja y, fardaniah s, ismail ri. oral health related quality of life in indonesian middle-aged and elderly women. med j indonesia 2011; 20: 62–5. 19. gerardo gm, javier g, antonio as, maribel ca, josé-eduardo msv, josé-luis cg. effectiveness of malic acid 1% in patients with xerostomia induced by antihypertensive drugs. med oral patol oral cir bucal 2012; doi:10.4317/medoral.18206. 20. chandu gs, hombesh mn. management of xerostomia and hyposalivation in complete denture patient. indian j stomatol 2011; 2: 263–6. 21. bergdahl m, bergdahl j. low unstimulated salivary flow and subjective oral dryness: association with medication, anxiety, depression and stress. j dent res 2000; 79(9): 1652–8. 22. sreebny lm, schwartz ss. a reference guide to drugs and dry mouth. gerodontology 1997; 14: 33–47. 115 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 115–121 original article the expression of bmp4 and fgf2 in wistar rats (rattus norvegicus) post application of gourami fish (osphronemus goramy) collagen chiquita prahasanti1, niken luthfiyya arini2, kurnia dwi wulan2, onge victoria hendro3, i komang evan wijaksana1, noer ulfah1, banun kusumawardani4, padmini hari5, shahabe saquib abullais6 1department of periodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3department of periodontology, faculty of dentistry, universitas hang tuah, surabaya, indonesia 4department of biomedical sciences, faculty of dentistry, universitas jember, jember, indonesia 5faculty of dentistry, mahsa university, kuala lumpur, malaysia 6department of periodontics and community dental sciences, college of dentistry, king khalid university, abha, saudi arabia abstract background: periodontitis is a chronic inflammatory disease of the periodontal tissue that is characterized by alveolar bone resorption. this occurs due to an imbalance of osteoblast and osteoclast during the bone formation and resorption processes. in order to obtain complete regeneration of periodontal tissue, bone grafting is frequently used in periodontal surgical therapy. although each material has disadvantages, safe graft materials derived from animal sources can be employed as an alternative to bone graft materials. osteoblast, osteoclast, calcified bone matrix, type i collagen, osteonectin, and hydroxyapatite can all be found in gourami scales, a form of food waste. bmp4 has osteoinduction functions, which are important in bone metabolism. through angiogenic activity, fgf2 also contributes to periodontal regeneration. purpose: the aim of the study was to assess the expression of bmp4 and fgf2 after the treatment group had been given gourami fish scale extract. methods: thirty-two experimental three-month-old male wistar rats (150-200g) were randomly divided into four groups: a seven-day control group, a seven-day treatment group, a 14-day control group, and a 14-day treatment group. one mandibular incisor was extracted from each wistar rat. the post-extraction socket was filled with blood for the control group and collagen extract for the treatment. results: the one-way anova test showed a significance level of 0.000 (p = <0.05). conclusion: the expression of bmp4 and fgf2 increased after the application of collagen extract from gourami scales. keywords: bmp4; collagen; fgf2; gourami fish scales; medicine article history: received 26 july 2022; revised 6 september 2022; accepted 22 september 2022 correspondence: chiquita prahasanti, department of periodontics, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo 47, surabaya 60132, indonesia. email: chiquita-p-s@fkg.unair.ac.id introduction periodontitis is defined as a chronic inflammatory disease indicated by the infiltration of immune cells in the gingiva, which leads to connective tissue damage, clinical attachment loss, and resorption of the alveolar bone.1,2 poor oral hygiene and lifestyle choices lead to the onset and progression of periodontitis, one of the most prevalent chronic inflammatory diseases in humans.3 periodontitis is a risk factor for a number of diseases, such as infectious endocarditis, cardiovascular disease, fatal or nonfatal stroke, premature birth, diabetes mellitus, pulmonary disease, and sinus disease.4 the most common periodontal diseases are gingivitis and periodontitis; in gingivitis, inflammation is limited to the gingival area, whereas in periodontitis, there is a destruction of connective tissue and alveolar bone.5 when there is an increase in attachment loss caused by periodontitis, both in terms of the number of teeth and the severity, this will affect a person’s quality of life. this is due to the possibility that it will enhance masticatory dysfunction by causing tooth migration, tooth extrusion, tooth hypermobility, and tooth loss.6 the use of collagen-based biomaterials in tissue engineering applications has increased over the last few copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 mailto:chiquita-p-s@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 116prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 decades. collagen is a versatile material that is frequently used in the fields of medicine, dentistry, and pharmacology because it has several advantages, including biocompatibility, biodegradability, adequate mechanical strength, flexibility, and the ability to absorb body fluids for nutrient transfer.7 collagen has great vascularity and a superior ability to repair wounds, which makes it biocompatible.8 an alternative source of collagen production is fish scales. type i collagen can be found in gourami fish scale extract (osphronemus goramy). the ideal porosity size for bone regeneration is between 100 and 500 µm; gourami scale collagen extract has pores ranging in size from 191.6 to 385.3 µm. the collagen scaffold’s porosity size contributes to the availability of spaces for cells to penetrate and grow.9 bone morphogenetic protein 4 (bmp4) is a member of the bone morphogenetic protein family which is part of the transforming growth factor-beta superfamily that plays an essential role in bone formation.10 in the early stages of bone formation, bmp4 acts as a stimulatory factor in cartilage ossification. bmp4 can be found in bone marrow cavities, periosteum, mesenchymal cells, cartilage cells, and muscle cells near fractured bones. bmp4 stimulates bone formation indirectly by inhibiting osteoclastogenesis, which is important in fracture repair.11 in periodontitis, the addition of fibroblast growth factor 2 (fgf2) is able to promote the establishment of cementum, periodontal ligament, and alveolar bone.12 fgf2 has been researched for its role in periodontal regeneration due to its angiogenic and mitogenic activities during the wound healing process. this protein elevates the expression of bone morphogenetic protein 2 (bmp2), which leads to an increase in bone deposits. it also stimulates fibroblast cell proliferation and increases angiogenesis. numerous studies have found that scaffolds derived from gourami fish scale extract have good biological properties that are safe for the viability of human gingival fibroblast,13 baby hamster kidney fibroblasts-21 fibroblast cell culture,9 and osteoblast cell culture.14 scaffolds derived from gourami fish scale extract can enhance the expression of osteoprotegerin (opg), receptor activator of nuclear factor kappa-β ligand (rankl),15 alkaline phosphatase (alp), transforming growth factor beta (tgf-β),16 bmp2, and vascular endothelial growth factor (vegf)17 in osteoblast cell cultures, which can accelerate angiogenesis and osteogenesis. however, no study has determined the effect of type i collagen scaffold derived from gourami fish scale extract (o. goramy) on the expression of bmp4 and fgf2 during the bone regeneration process. this study aims to assess the expression of bmp4 and fgf2 after the treatment group is given type i collagen scaffold derived from gourami fish scale extract (o. goramy) during the in vitro bone regeneration process. materials and methods the faculty of dental medicine, universitas airlangga, issued a certificate of ethical eligibility for this study with the number 423/hrecc.fodm/ix/2020, and the study was conducted in compliance with the standards of animal ethics and care. this study employed 32 experimental white male rats (rattus norvegicus) that were three months old and weighed 150–200 grams in vivo in an experimental lab setting with a post-test-only design. samples were chosen at random, and the lemeshow sample size formula was used to calculate the sample size, which led to eight samples for each sample group. for pain relief while extracting the mandibular incisor from each animal to create an alveolar bone defect, 0.2 ml of ketamine was intramuscularly injected into the gluteus muscle. each mandibular incisor socket was left with blood in the control group. extracted gourami scale collagen was used to fill each mandibular incisor socket in the treatment group. bmp4 and fgf2 markers were examined on seventh and 14 days after the rats in the treatment group were given 30 mg of gourami scales collagen extract. the gourami scales were obtained by washing fish scales and freezing them; then, 100 grams were taken and soaked in a 6% acetic acid solution for seven days. during the soaking process, the acetic acid solution was changed daily. after seven days, the process continued by running water over the fish scales until a ph of neutral was reached. collagen strands emerged during rinsing, and collagen clots formed. the collagen was then freeze-dried for 12 hours at a condensing temperature of -76°c and an ambient temperature of 23.6°c to completely eliminate all water content. the sterilizing of the collagen products was accomplished utilizing ethylene oxide gas. after being cultured for seven and 14 days, the levels of bmp4 and fgf2 expression in the osteoblast cells were analyzed using an immunohistochemical test. the data collection was based on the number of osteoblasts expressing bmp4 and fgf2. immunohistochemical representation of incisor socket preparations stained with immunohistochemical techniques, using monoclonal antibodies bmp4 (3c11c7) nbp2-52424 and fgf2 (c-2): sc-74412 (st cruz, us). table 1. mean and standard deviations of bmp4 and fgf2 expression marker group day mean ± sd bmp4 control 7 5.09 ± 1.49414 10.22 ± 1.863 treatment 7 9.50 ± 2.61514 15.75 ± 3.251 fgf2 control 7 5.56 ± 1.87014 8.69 ± 1.782 treatment 7 13.41 ± 2.07414 17.63 ± 1.350 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 117 prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 results the data collection was based on the number of osteoblasts expressing bmp4 and fgf2. table 1 shows that on days seven and 14, the average expression level of bmp4 in the treatment group was higher than that in the control group. the highest average was obtained in the treatment group on day 14. table 1 also suggests that when compared with the control group, the expression of fgf2 was significantly higher in the treatment group. the treatment group’s greatest average was achieved on day 14. images of the immunohistochemical representations of incisor socket preparations using monoclonal antibody bmp4 (3c11c7) nbp2-52424 stained using immunohistochemical techniques can be seen in figures 1 and 2. using various magnifications, images a, b, and c represent the expression of bmp4 in the control group. image a is magnified 40x, image b is magnified 200x, and image c is magnified 1000x until bmp4 is clearly visible. in contrast, images d, e, and f show the results of the treatment group with magnifications of 40x, 200x, and 1000x. in table 1, it can be seen that the treatment group had more bmp4 than the control group. it can be concluded that the expression of bmp4 on treatment group day 14 showed highest results, with an average number of 15.75. data retrieval research results were based on the number of osteoblasts expressing fgf2. images of the a b c d e f figure 1. expression of bmp4 in incisor socket preparations with monoclonal antibody bmp4 stained using immunohistochemical techniques. a–c show bmp4 expression in the control group after seven days at 40x, 200x, and 1000x magnification, respectively. d–f show bmp4 expression in the treatment group after seven days at 40x, 200x, and 1000x magnification, respectively. a b c d e f figure 2. expression of bmp4 in incisor socket preparations with monoclonal antibody bmp4 stained using immunohistochemical techniques. a–c show bmp4 expression in the control group after 14 days at 40x, 200x, and 1000x magnification, respectively. d–f show bmp4 expression in the treatment group after 14 days at 40x, 200x, and 1000x magnification, respectively. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 118prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 a b c d e f figure 3. expression of fgf2 in incisor socket preparations with monoclonal antibody fgf2 stained using immunohistochemical techniques. a–c show fgf2 expression in the control group after seven days at 40x, 200x, and 1000x magnification, respectively. d–f show fgf2 expression in the treatment group after seven days at 40x, 200x, and 1000x magnification, respectively. a b c d e f table 2. bmp4 and fgf2 normality tests marker shapiro–wilk statistic df sig. fgf2 0.945 32 0.101 bmp4 0.956 32 0.209 immunohistochemical representation of incisor socket preparations using monoclonal antibody fgf2 (c-2): sc-74412 stained using immunohistochemical techniques can be seen in figures 3 and 4. similar to figures 1 and 2, figures 3 and 4 show the expression of fgf2 in the control group (figures a, b, and c) and the treatment group (figures d, e, and f). in the treatment group, the amount of fgf2 also increased compared with the control group, with highest results on day 14, as shown in figure 4, with an average number of 17.63. the results of the study were analyzed using spss 16 software, and the output of the results of the analysis can be seen in the attachment sheet, while the explanation of the test results will be discussed below. the data needed to be normally distributed in order to be used in the parametric analysis. the testing of the data distribution for each group was carried out using the shapiro–wilk test. significant value of normality test with shapiro–wilk test for marker bmp4 was p: 0.101 (p > 0.05) and fgf2 was p:0.209 (p > 0.05), which meant that all the data were normally distributed (table 2). levene’s test of homogeneity showed p > 0.05, indicating that the bmp4 and fgf2 markers figure 4. expression of fgf2 in incisor socket preparations with monoclonal antibody fgf2 stained using immunohistochemical techniques. a–c show fgf2 expression in the control group after 14 days at 40x, 200x, and 1000x magnification, respectively. d–f show fgf2 expression in the treatment group after 14 days at 40x, 200x, and 1000x magnification, respectively. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 119 prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 showed homogeneous variations between the different groups. then, the data analysis was continued using the oneway analysis of variance (one-way anova) parametric test. this test was used to compare the levels of bmp4 and fgf2 expression in the control and treatment groups to see if there were any significant differences. the results showed that the treatment group’s levels differed significantly, with significance levels of bmp4 sig. 0.000 and fgf2 sig. 0.000 as shown in figure 5 and 6. a post hoc test was conducted using tukey’s honestly significant difference test (tukey’s hsd) after the results from the one-way anova test had been obtained, and it showed significant differences. this test was completed to determine the differences between the research groups, and they differed significantly. the significance levels using tukey’s hsd for markers bmp4, sig. 1.000 (p > 0.05), and fgf2, sig. 1.000 (p > 0.05), indicated that there was a substantial difference between the research groups based on the post hoc test (table 3). discussion the bone regeneration process necessitates three basic components, namely osteoprogenitor cells (stem cells, osteoblasts, cementoblasts, and fibroblasts), signaling 0 2 4 6 8 10 12 14 16 18 7th day 14th day control group treatment group (*) significantly different (p < 0.05) * * figure 5. expression of bmp4 in all groups. 0 2 4 6 8 10 12 14 16 18 20 7th day 14th day control group treatment group * * (*) significantly different (p < 0.05) figure 6. expression of fgf2 in all groups. table 3. result of bmp4 and fgf2 hsd testing groups n subset for alpha = 0.05 bmp4 fgf2 1 2 3 1 2 3 4 control 7 8 5.09 5.56 treatment 7 8 9.50 8.69 control 14 8 10.22 13.41 treatment 14 8 15.75 17.63 significance level 1.000 .932 1.000 1.000 1.000 1.000 1.000 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 120prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 molecules (bmp, fgf, and prp), and scaffolds (collagen, fibrin, polyglycolide, polylactide polymer, and copolymer), to regulate the differentiation and function of osteoprogenitor cells in damaged periodontal tissues to form new bone, cementum, and periodontal ligament.18 there are two cells that regulate bone homeostasis: osteoblasts for bone formation and osteoclasts for bone resorption. osteoblasts develop from mesenchymal stem cells (mscs) and play a major part in bone mass maintenance and regeneration, bone quality determination, and skeletal system function.19 collagen can be extracted from various sources; some of the most commonly used collagen sources for tissue engineering are bovine (skin, bone, and tendon), porcine (skin and bone), and marine animals (fish, prawn, octopus, squid, cuttlefish, starfish, jellyfish, sponges, sea urchin, and sea anemone). fish collagen can be extracted from bones, skin, fins, and scales.20 fish collagen has several advantages, including a high level of security (no hand, foot, and mouth disease from pigs or spongiform encephalopathy from cows), a high absorption rate, a low cost, biocompatibility, and no religious restrictions.21 this study used fish scale collagen, which was applied to the tooth sockets of rats and then analyzed on days seven and 14, which revealed that the treatment group’s mean expression of bmp4 and fgf2 was higher than that of the control group. the inflammatory process had stopped by day seven, and proinflammatory mediators had started to play a major part in the regeneration process. when specific mscs are activated, they can proliferate and differentiate into osteogenic cells, allowing for bone regeneration. bone formation can occur through intramembranous processes (direct) and endochondral processes (indirect). in both mechanisms, bone and cartilage induction occurs through epithelial–mesenchymal interactions initiated by specific cell differentiation. when mesenchymal progenitor cells are exposed to bmp, they can differentiate into osteoblasts and chondroblasts.22 this study shows that bmp can affect bone formation either directly or indirectly. in this study, day seven was selected not only because it was the end of the inflammatory process but also because the height of soft callus formation occurs in experimental rats on days seven to nine post-trauma. day 14 marks the height of the formation of hard callus and the process of mineralization of bone tissue. during hard callus formation, woven bone replaces the calcified cartilage, making the callus denser and mechanically harder. bmp4 is a key inducer of osteoblast differentiation and bone production.19 it also stimulates the ossification of soft bones during the period of bone development. prior to the production of soft bone and new bone, bmp4 is expressed in considerable numbers within six hours of a bone fracture, reaching 10 times the baseline value and gradually decreasing to the baseline level within 72 hours. collagen extract was added in this study, and day 14 saw a greater rise in bmp4 expression than day seven. on the seventh and 14th day of the experiment, there was less expression of bmp4 in the intervention group compared to the control group. this suggests a beneficial impact on the tooth socket’s bone-formation process. bmp4 was present in the soft bone cells, mesenchymal cells, bone marrow cavities, muscle cells in the vicinity of broken bones, and periosteum.11 this demonstrates how crucial a function bmp4 plays in fracture repair. by suppressing osteoclastogenesis, bmp4 indirectly promotes bone growth. the most popular fgf ligand for use in regenerative medicine, including bone regeneration, is fgf2.23 fgf2 is a fundamental fgf that, through a variety of mechanisms, including vasoformative processes, is thought to encourage cell proliferation and differentiation. angiogenesis, wound healing, and bone regeneration are all facilitated by fgf2. fgf2 acts as a trigger for mesenchymal stem cell differentiation and proliferation during bone repair. additionally, fgf2 has potent angiogenetic effects.24 rats lacking fgf2 saw a marked decline in bone mass and a reduction in bone formation.23 this assertion backs up research findings showing that administering collagen extract boosts fgf2 expression. another study discovered that the treatment of fgf2 can promote fibroblast cell proliferation, angiogenesis, and bone production, which can boost periodontal regeneration.12 moreover, in a study using individuals with a diagnosis of aggressive periodontitis, in comparison with the control group, it was observed that vertical bone defects had more alveolar bone.25 collagen extract can speed up bone development and be employed as an alternative regenerative material for bone production, according to this study’s findings of increased expression of bmp4 and fgf2. whereas fgf2 participates in angiogenesis, which is crucial for periodontal regeneration, bmp4 works to induce osteoblast differentiation. according to the findings of the immunohistochemical experiment, the osteoblast cell culture clearly expressed bmp4 and fgf2 after receiving collagen obtained from gourami (osphronemus goramy) fish scales. in conclusion, the use of gourami (osphronemus goramy) fish scale collagen in osteoblast culture increases the expression of bmp4 and fgf2. references 1. ramadhani nf, nugraha ap, gofur nrp, permatasari ri, ridwan rd. increased levels of malondialdehyde and cathepsin c by aggregatibacter actinomycetemcomitans in saliva as aggressive periodontitis biomarkers: a review. biochem cell arch. 2020; 20: 2895–901. 2. andriani i, medawati a, humanindito mi, nurhasanah m. the effect of antimicrobial peptide gel rise-ap12 on decreasing neutrophil and enhancing macrophage in nicotine-periodontitis wistar rat model. dent j. 2022; 55(2): 93–8. 3. wulandari p, widkaja d, nasution ah, syahputra a, gabrina g. association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients. dent j. 2022; 55(1): 16–20. 4. ramadhani nf, nugraha ap, gofur nrp, ridwan rd. elevation of c-reactive protein in chronic periodontitis patient as cardiovascular disease risk factor. biochem cell arch. 2020; 20: 2875–8. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 121 prahasanti et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 115–121 5. aprilianti na, rahmadhani d, rizqianti y, ridwan rd, ramadhani nf, nugraha ap. periodontal ligament stem cells, solcoseryl pasta incoporated nano-hydroxyapatite silica gel scaffold for bone defect regeneration in chronic periodontitis: a review. biochem cell arch. 2020; 20: 3101–6. 6. saskianti t, nugraha ap, prahasanti c, ernawati ds, suardita k, riawan w. immunohistochemical analysis of stem cells from human exfoliated deciduous teeth seeded in carbonate apatite scaffold for the alveolar bone defect in wistar rats (rattus novergicus). f1000research. 2020; 9: 1164. 7. khan r, khan m. use of collagen as a biomaterial: an update. j indian soc periodontol. 2013; 17(4): 539–42. 8. kakarla p, sai j, avula s, anche s, mjs p. collagen as a biomaterial in dentistry. int j innov med heal sci. 2016; 7: 1–4. 9. prahasanti c, wulandari dt, ulfa n. viability test of fish scale collagen (oshpronemus goura my) on baby ha mster k idney fibroblasts-21 fibroblast cell culture. vet world. 2018; 11(4): 506–10. 10. kajioka d, suzuki k, nakada s, matsushita s, miyagawa s, takeo t, nakagata n, yamada g. bmp-4 is an essential growth factor for the initiation of genital tubercle (gt) outgrowth. congenit anom (kyoto). 2020; 60(1): 15–21. 11. yang j, shi p, tu m, wang y, liu m, fan f, du m. bone morphogenetic proteins: relationship between molecular structure and their osteogenic activity. food sci hum wellness. 2014; 3(3–4): 127–35. 12. nagayasu-tanaka t, anzai j, takaki s, shiraishi n, terashima a, asano t, nozaki t, kitamura m, murakami s. action mechanism of fibroblast growth factor-2 (fgf-2) in the promotion of periodontal regeneration in beagle dogs. matsumoto t, editor. plos one. 2015; 10(6): e0131870. 13. ulfah n, rehuel santoso s, bargowo l, kurnia s, prahasanti c. the viability of collagen peptide from osphronemus goramy fish scale extract on human gingival fibroblast. res j pharm technol. 2022; 15(8): 3497–501. 14. krismariono a, wiyono n, prahasanti c. viability test of fish scales collagen from oshphronemus gouramy on osteoblast cell culture. j int dent med res. 2020; 13(2): 412. 15. wijaksana ik e, prahasanti c, bargowo l, sukarsono r m, krismariono a. opg and rankl expression in osteoblast culture after application of osphronemus gourami fish scale collagen peptide. j int dent med res. 2021; 14(2): 618–22. 16. tionardus m, dwija putra igna, ulfah n, k rismariono a, setiawatie e, prahasanti c. expression of alp and tgf-β in osteoblast cell cultures after administering collagen peptide derived from gouramy (osphronemus goramy) fish scales. dent hypotheses. 2021; 12(2): 73. 17. bargowo l, wijaksana ike, hadyan fz, riawan w, supandi sk, prahasanti c. vascular endothelial growth factor and bone morphogenetic protein expression after induced by gurami fish scale collagen in bone regeneration. j int dent med res. 2021; 14(1): 141–4. 18. prahasanti c, nugraha ap, saskianti t, suardita k, riawan w, ernawati ds. exfoliated human deciduous tooth stem cells incorporating carbonate apatite scaffold enhance bmp-2, bmp-7 and attenuate mmp-8 expression during initial alveolar bone remodeling in wistar rats (rattus norvegicus). clin cosmet investig dent. 2020; 12: 79–85. 19. chang y, cho b, kim s, kim j. direct conversion of fibroblasts to osteoblasts as a novel strategy for bone regeneration in elderly individuals. exp mol med. 2019; 51(5): 1–8. 20. silvipriya k, kumar k, bhat a, kumar b, john a, lakshmanan p. collagen: animal sources and biomedical application. j appl pharm sci. 2015; 5(3): 123–7. 21. chinh nt, manh vq, trung vq, lam td, huynh md, tung nq, trinh nd, hoang t. characterization of collagen derived from tropical freshwater carp fish scale wastes and its amino acid sequence. nat prod commun. 2019; 14(7): 1934578x1986628. 22. rao s, ugale g, warad s. bone morphogenetic proteins: periodontal regeneration. n am j med sci. 2013; 5(3): 161–8. 23. charoenlarp p, rajendran ak, iseki s. role of fibroblast growth factors in bone regeneration. i nf la m m regen. 2017; 37(1): 10. 24. kuroda y, kawai t, goto k, matsuda s. clinical application of injectable growth factor for bone regeneration: a systematic review. inflamm regen. 2019; 39(1): 20. 25. yoshinuma n, koshi r, kawamoto k, idesawa m, sugano n, sato s. periodontal regeneration with 0.3% basic fibroblast growth factor (fgf-2) for a patient with aggressive periodontitis: a case report. j oral sci. 2016; 58(1): 137–40. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p115–121 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p115-121 vol 51 no 3 jul sep 2018_pus.indd 114114 the effect of various concentrations of ha-tcp derived from cockle shell synthesis on scaffold porosity reyhan alvaryan ferdynanto, priska evita setia dharmayanti, putu tahlia krisna dewi, and widyasri prananingrum department of dental materials, faculty of dentistry, universitas hang tuah, surabaya indonesia abstract background: porosity is an important property that must be possessed by scaffold due to its role in new bone growth. hydroxyapatite is a scaffold material with a composition resembling that of bone that can be synthesized from cockle shell (anadara granosa). purpose: this research aimed to determine the effects of various ha-tcp concentrations (wt%) derived from cockle shell synthesis on scaffold porosity. methods: ha-tcp was synthesized from cockle shells using a hydrothermal method at 200o c with a 12-hour sintering process period. an xrd test was subsequently carried out to determine the composition of hydroxyapatite (ha) and tricalcium phosphate (tcp) compounds. eighteen scaffold samples (n=6) were then produced using a freeze dry method and divided into three groups, namely; group 1 (k1) treated with 5% ha-tcp, group 2 (k2) treated with 25% ha-tcp and group 3 (k3) treated with 50% ha-tcp. thereafter, a scaffold porosity test was conducted using liquid displacement method. scaffold porosity was observed by means of an sem image. a one-way anova test was subsequently performed, followed by an lsd post-hoc test (p <0.05). results: the results of the xrd test showed that the percentage of ha was 51.5%, while tcp was 16.8%. the porosity of the scaffolds was within the range of 67.24% 80.17%. the highest porosity was found in group 1, while the lowest occurred in group 3. there were significant differences in all groups. conclusion: the concentration of ha-tcp derived from the synthesis of cockle shells affects the porosity of scaffold. the lower the concentration of ha-tcp, the higher the scaffold porosity. keywords: ha-tcp concentration; gelatin; porosity; scaffold; cockle shells correspondence: widyasri prananingrum, department of dental materials, faculty of dentistry, universitas hang tuah, jl. arif rahman hakim no. 150, surabaya 60111, indonesia. e-mail: widyasri.prananingrum@hangtuah.ac.id dental journal (majalah kedokteran gigi) 2018 september; 51(3): 114–118 research report introduction repairing bone damage remains a problem for medical personnel since the bone healing process often proves ineffective, culminating in the need for a material to promote it. one method commonly used to restore the function of lost or damaged bone tissue is the application of bone graft1, a biomaterial used to fill damaged bone cavities which disappears when new bone cell growth has occurred.2 in general, there are three properties that a bone graft requires in order to form new bones, namely: osteoconductivity, osteoinductivity and osteogenetic.3 however, bone graft has been widely used in dentistry to overcome the problem of bone resorption by regenerating lost or severely damaged bones. bone grafts can be grouped into four types: autographs, allographs, xenographs and alloplasts. the most commonly used are xenografts, bone grafts from different species that possess osteoconductive properties and demonstrate high levels of biocompatibility. 4 hydroxyapatite (ca10(po4)6(oh)2) is the main inorganic component in bones and teeth generally used as a bone graft due to its excellent biocompatibility, osteoconductivity in relation to the chemical and biological affinity to bone tissue.5,6 synthetic hydroxyapatite currently constitutes an expensive imported product, costing approximately one million rupiah per gram. although hydroxyapatite can be synthesized from readily available natural ingredients, these have yet to be utilized.7 moreover, hydroxyapatite unfortunately demonstrates certain weaknesses, including fragility and poor absorbency.8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p114–118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p114-118 115 ferdynanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 114–118 tricalcium phosphate is a compound possessing physical and chemical properties similar to the mineral structure of human bones and teeth.9 tricalcium phosphate has bioactive, biocompatible and osteoconductive properties, while being readily absorbed. the combination of hydroxyapatite and tricalcium phosphate produced is assumed to possess higher quality and more stable compounds.10 the manufacture of ha-tcp combination compounds with a 12-hour sintering period produces a high tricalcium phosphate compound in which the longer the sintering time, the higher the production of tricalcium phosphate compounds.11 one of the natural ingredients possessing a high calcium content is cockle shell with a calcium carbonate level of 98%.12 cockles constitute one of the marine food commodities with a high commercial value that are favored by consumers in indonesia and throughout asia. the high consumption of cockles will, consequently, produce large quantities of shells as waste. the use of accumulated cockle shell waste can enhance waste management in reducing pollution and improving environmental aesthetics.13 this research, therefore, focused on converting cockle shell waste into ha-tcp bone graft. ha-tcp bone graft derived from cockle shells is formed into a scaffold enabling it to accelerate the process of biomineralization in the bone. an important property that must be possessed by scaffold is porosity. the porous characteristics of scaffold are very important to the process of new bone growth. scaffold represents a site to which growth cells can attach and develop to form new bone tissue. scaffold with high porosity, optimal pore size and pore interconnectivity plays a very important role in the growth of bone cells.14 hence, the hydroxyapatite present in porous scaffold is assumed to demonstrate more effective osteoconductivity and greater absorbability than dense scaffold. effective pore size for bone cell growth is approximately 40-100μm.15 the porosity of scaffold effectively employed as a bone graft is between 30% and 90%.16 consequently, there is a great demand for the development of porous scaffold synthesis. research conducted by narbat (2006) using ha scaffold at concentrations of 30%, 40% and 50% showed that the highest porosity was found in scaffolds with an ha concentration of 30%. however, ha has difficulty in absorbing.17 as a result, this research was conducted to synthesize ha-tcp from cockle shells (anadara granosa). theoretically, tcp will be more easily degraded and absorbed by the body, leading to the expectation of more rapid new bone formation.18 however, it has yet to be confirmed whether variations in the concentrations of ha-tcp synthesis derived from cockle shells can affect scaffold porosity. in this research, porous ha-tcp scaffold was produced with ha-tcp concentrations of 5%, 25% and 50% combined with gelatin, a biodegradable, biocompatible and soluble protein-based material..19 gelatin contains many protein bonds in aginine-glycine-apartic acid (rgd) which can increase cell attachment and cell growth.20 the addition of gelatin to scaffold with hydroxyapatite combination was intended to increase bone-forming cell differentiation and improve the mechanical properties of the scaffold.21 thus, this research aimed to determine the effects of ha-tcp concentration variations (wt%) derived from cockle shells synthesis on scaffold porosity. materials and methods this research constituted an experimental laboratory study with a posttest-only group design and was conducted in two stages, namely, a sampling process stage and a sample testing stage. the raw material used consisted of cockle shells (anadara granosa) extracted from waste present on the coast at probolinggo. the cockle shells were pulverized and converted to ha-tcp by a hydrothermal method at 200°c with a 12-hour sintering period, including calcination, sintering, pa methanol rinsing and drying processes. ha-tcp compounds were obtained from cockle shells which had been cleaned and ground to form a smooth texture. the filtered powder was then calcined at 100° for three hours in an oven furnace (naberterm, germany). as part of the hydrothermal process, the hydroxyapatite powder was mixed with ammonium dihydrogen phosphate (daishin, japan) and heated at 200°c in an oven furnace (naberterm, germany) with a sintering time of 12 hours. after completion of this process, the powder was rinsed with distilled water until the ph reached ±7, and was also rinsed with methanol pa (emsure, germany). the powder was then heated to a temperature of 50° for four hours and at 900°c for a further three hours. before manufacture of the scaffold, ha-tcp powder had been filtered with a 200mesh filter to produce a powder size of <74 μm. scaffold was subsequently produced by mixing ha-tcp with 10% gelatin (sigma, germany) (wt%) (1:1). in this research, three varieties of ha-tcp concentration (wt%) were used, namely 5% ha-tcp (k1), 25% ha-tcp (k2) and 50% ha-tcp (k3). the results of the mixing process were then deposited in a 6mm-diameter mold 10mm in height, frozen at a temperature of -80° c for five hours and freeze dried for 30 hours. an xrd test was conducted using an xpert-pro panalytical at an angle of 2θ= 5°60° to identify the crystallization phase and content of the material using x-ray electromagnetic radiation. the xrd test results were then presented in the form of spectrum charts and tables. the spectrum diffraction pattern of the xrd test results provides information about the angle of diffraction in the atomic material (2θ) on the horizontal axis and the intensity result on the vertical axis. an identification phase was then performed by comparing the hydroxyapatite diffraction pattern with data from international center for diffraction data (icdd). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p114–118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p114-118 116ferdynanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 114–118 porosity is the volume of empty space contained in a sample tested using a liquid displacement method determined by the percentage of liquid absorbed by scaffold after being immersed in ethanol. materials used in this test comprised ha-tcp scaffold derived from cockle shell synthesis and 96% absolute ethanol (merck, germany). the tools employed consisted of analytical scales, tweezers and vernier calipers (krisbow, indonesia). this research also used 18 cylindrical 6x10mm-sized (n=6) samples. a porosity test was subsequently carried out to determine the volume of empty space in each sample. a porosity test measured the volume of the samples, weighing their dry weight before soaking them in 96% absolute ethanol for 48 hours. the samples were weighed to determine the wet mass of the specimens. the porosity of the samples was then calculated using the following equation:22 x 100 porosity (%) = mb mk ρliquid x vb note: mb = wet mass of specimen (gram) mk = dry mass of specimen (gram) vb = volume of specimen (cm 3)ρliquid = density of water (1 gr/cm3) sem was conducted with an electron microscope at 1000x magnification in order to identify pores in the samples. pore size observed through an electron microscope lens was displayed on a computer screen using the sem imaging device. a photo was taken of the selected part of each sample at the desired magnification and the pore size was measured. the scaffold pore was represented by black and its size measured using a scale line found in the sem image. statistical analysis was performed on the porosity data using spss one-way anova with a significance level of 95% (0.05), followed by an lsd post-hoc test. results the xrd results of ha-tcp powder in figure 1 illustrated a diffractogram with high peaks indicating changes in the crystallization phase of the samples at each calcination temperature. the diffraction pattern of hydroxyapatite formation in the xrd results was shown figure 1 xrd spectrum graph. of ha-tcp derived from cockle shell synthesis. *p<0.05 figure 2. percentages of porosity in 5% ha-tcp scaffold (k1), 25% ha-tcp scaffold (k2), and 50% ha-tcp scaffold (k3). table 1. chemical compounds contained in ha-tcp powder derived from cockle shell synthesis. percentagename of compounds (%) chemical formulas ca16.8tri-calcium phosphate (tcp) 3(po4)2 ca51.5hydroxyapatite (ha) 5(po4)3(oh) caco20.8aragonite (caco3) 3 ca(oh)3.0calcium hydroxide 2 cao7.9calcium oxide caco-calcite 3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p114–118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p114-118 117 ferdynanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 114–118 at 2θ : 32°, and the formation of tricalcium phosphate at 2θ : 28°. the xrd results in table 1 showed that the levels of hydroxyapatite (ha) and tricalcium phosphate (tcp) produced were relatively high. other compounds produced from blood clams (anadara granosa) included: aragonite, calcium oxide and calcium hydroxide. as shown in figure 2, the percentages of porosity were high in all groups. the statistical test results showed significant differences between group 1 and group 2 (p = 0.037), group 2 and group 3 (p = 0.000), and group 3 and group 1 (p = 0.000). the percentage of porosity in the 5% ha-tcp group was 80.17%, 77.51% in the 25% ha-tcp group, and 67.24% in the 50% ha-tcp group. based on the sem image in figure 3, there were interconnected scaffold pores. in figure 4, the pore size was small, while in figure 5, the pore size varied. in general, the pore size in all groups varied and was unequally distributed. the average diameter of the 5% ha-tcp scaffold pore size was 75.99 μm, 25% ha-tcp scaffold was 45.08 μm and 50% ha-tcp scaffold was 90.31 μm. discussion an x-ray diffractometer (xrd) machine is essential not only to evaluate crystalline structures, crystallization phase and crystallinity degree, but also to determine types of elements or compounds contained in a material. the output of an xrd machine is a diffractogram with a high peak indicating the crystallization phase of a sample.23 in this research, the xrd machine results relating to cockle shell synthesis confirmed the presence of ha compounds (51.5%), tcp (16.8%), and others such as calcium carbonate in the form of aragonite. this is due to the decomposition of hydroxyapatite during combustion as a result of imperfections in the reactants. since natural material was used in this study, it proved difficult to achieve a high level of purity in the material.24 the results of this research indicated that the porosity of the samples varied. the highest porosity was demonstrated by group 1 that had been treated with 5% ha-tcp scaffold, while the lowest occurred in group 3 which had been treated with 50% ha-tcp scaffold. this signified that the porosity of ha-tcp scaffold samples decreased as the concentration of ha-tcp scaffold powder increased. the statistical test results also revealed significant differences between groups. in other words, the concentration of hatcp powder in the manufacture of scaffold affects the level of scaffold porosity. the higher the concentration of hatcp powder administered, the lower the level of porosity.5 this occurs because the formation of a salt bridge and cross-linking due to the hydroxyapatite reaction absorbed on the material matrix fills the gap between the particles in the material.25 the porosity of the scaffold is also known to be affected by porogen (porous-forming material/gelatin) concentration and the sintering process. the porogen concentration is directly proportional to the porosity of a scaffold. the higher the concentration of porogen used in making scaffold, the higher the porosity produced.26 in this research, the same concentration of porogen, 10% gelatin, was used in all groups. as a result, the effect of pa 1 = 69.90 μm pa 2 = 82.08 μm pa r1 pa r2 pa 1 pa 2 figure 3. sem image indicating porosity of 5% ha-tcp scaffold at 1000x magnification. pa 1 = 49.14 μm pa 2 = 41.02 μm pa 1 pa 2 pa r2 pa r2 figure 4. sem image indicating porosity of 25% ha-tcp scaffold at 1000x magnification. pa 2 = 88.68 μm pa 1 = 91.93 μm pa 1 pa 2 pa r2 pa r1 figure 5. sem image indicating porosity of 50% ha-tcp scaffold at 1000x magnification. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p114–118 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p114-118 118ferdynanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 114–118 the porogen factor on scaffold porosity was not detected. however, the porosity produced in this research was quite high. an effective level of porosity for bone cell growth is one of approximately 70%.27 thus, the manufacture of scaffold with 5% and 25% ha-tcp can produce porosity supportive of bone growth. in addition, sem images (figures 3, 4 and 5) showed that pores of varying sizes in ha-tcp scaffolds were connected to one another and spread unevenly in all groups. the research findings reported here indicate that pore size is not directly proportional to porosity. rather, a large number of small pores can increase porosity. the pore size of scaffold pores can be influenced by temperature and the freezing time required during the freeze-drying method. the lower the freezing temperature, the faster the ice crystal dendrite formed resulting in pores on the scaffold. the faster the freezing time, the larger the size of the pores formed.28 since variations in the freeze-drying method were not employed during this research, they did not affect scaffold porosity which has greater influence on bone formation compared to pore size. the number of endothelial cells, osteoblasts and bone mass is proportional to an increase in scaffold porosity. in conclusion, this research indicated that the smaller the concentration of ha-tcp powder (wt%), the higher the porosity of the scaffold. the highest porosity occurs in scaffold with 5% ha-tcp (80.17%), proving that the concentration of ha-tcp powder derived from cockle shell synthesis affects the porosity of the ha-tcp scaffold. acknowledgement this research was supported by the 2018 funding student creativity program (pkm) of the directorate of student affairs, directorate general of learning and student affairs, ministry of research, technology and higher education of the republic of indonesia. references ardhiyanto hb. peran hidroksiapatit sebagai bone graft dalam proses1. penyembuhan tulang. stomatognatic. 2011; 8(2): 118–21. nurmanta da, djoni ir, ady j. optimasi parameter waktu sintering2. pada pembuatan hidroksiapatit berpori untuk aplikasi bone filler pada kasus kanker tulang (osteosarcoma). thesis. surabaya: universitas airlangga; 2013. p. 1-19. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm3. bioallied sci. 2013; 5(suppl 1): s125-7. kurniawan h. efek pemberian kombinasi prf dengan xenograft dan4. alloplast terhadap jumlah osteoblas. denta j kedokt gigi. 2015; 9: 1–8. ismawati h, fadli a, akbar f. pengaruh penambahan dispersant5. dan waktu pengadukan pada pembuatan scaffold hidroksiapatit menggunakan sabut gambas sebagai template. jom fteknik. 2016; 3(2): 1–6. hoppe a, güldal ns, boccaccini ar. a review of the biological6. response to ionic dissolution products from bioactive glasses and glass-ceramics. biomaterials. 2011; 32(11): 2757–74. arrafiqie mf, azis y, zultiniar z. sintesis hidroksiapatit dari limbah7. kulit kerang lokan (geloina expansa) dengan metode hidrothermal. jom fteknik. 2016; 3: 1–8. warastuti y, abbas b. sintesis dan karakterisasi pasta injectable bone8. substitute iradiasi berbasis hidroksiapatit. j ilmiah aplikasi isotop dan radiasi. 2011; 7(2): 73–82. rasyid a, fadli a, akbar f. pembuatan trikalsium fosfat berpori9. menggunakan metode protein foaming-consolidation. jom fteknik. 2016; 3: 1–7. 10. naini a. potensi graft alloplast sebagai material augmentasi resorbsi ridge alveolar. in: proccedings book forkinas vi fkg unej. jember: universitas jember; 2016. p. 236–46. 11. pratama af. karakteristik hidroksiapatit hasil sintesis cangkang kerang darah (anadara granosa) menggunakan metode hydrothermal dengan variasi waktu sinterin. thesis. surabaya: universitas hang tuah; 2017. 12. ahmad i. pemanfaatan limbah cangkang kerang darah (anadara granosa) sebagai bahan abrasif dalam pasta gigi. j galung tropika. 2017; 6: 49–59. 13. kartono gs, widyastuti w, setiawan hw. biokompatibilitas hidroksiapatit graft dari cangkang kerang darah (anadara granosa) terhadap kultur sel fibroblas. denta j kedokt gigi. 2014; 8: 1–8. 14. nanda hs. preparation of porous scaffolds with controlled drug release for tissue engineering. thesis. tsukuba: university of tsukuba; 2014. 15. dehghani f, annabi n. engineering porous scaffolds using gas-based techniques. curr opin biotechnol. 2011; 22(5): 661–6. 16. vijayavenkataraman s, shuo z, fuh j, lu w. design of threedimensional scaffolds with tunable matrix stiffness for directing stem cell lineage specification: an in silico study. bioengineering. 2017; 4(3): 1–11. 17. narbat mk, orang f, hashtjin ms, goudarzi a. fabrication of porous hydroxyapatite-gelatin composite scaffolds for bone tissue engineering. iran biomed j. 2006; 10(4): 215–23. 18. nazir nm, mohamad d, seeni mohamed ma, omar ns, othman r. biocompatibility of in house �-tricalcium phosphate ceramics with normal human osteoblast cell. j eng sci technol. 2012; 7(2): 169–76. 19. rose jb, pacelli s, el haj aj, dua hs, hopkinson a, white lj, rose fraj. gelatin-based materials in ocular tissue engineering. materials (basel). 2014; 7(4): 3106–35. 20. kartikasari n, yuliati a, kriswandini il. compressive strength and porosity tests on bovine hydroxyapatite-gelatin-chitosan scaffolds. dent j (maj ked gigi). 2016; 49(3): 153–7. 21. hoque me, nuge t, yeow tk, nordin n, prasad rgs v. gelatin based scaffolds for tissue engineering – a review. polym res j. 2014; 9: 15–32. 22. kurniawan sb, ady j, djoni ir. sintesis dan karakterisasi mekanik mortar berbasis material komposit silika amorf dengan variasi penambahan sekam tebu. j fiska dan terapannya. 2013; 1(3): 28–36. 23. prabaningtyas rajms. karakterisasi hidroksiapatit kalsit (pt. dwi selogiri mas sidoarjo) sebagai bone graft sintesis menggunakan x-ray diffractometer (xrd) dan fourier transform infra red (ftir). thesis. jember: universitas jember; 2015. 24. kartikasari nd. sintesis dan karakterisasi hidroksiapatit dari cangkang keong sawah (pila ampullaceal) dengan porogen lilin sarang lebah sebagai aplikasi scaffold. thesis.surabaya: universitas airlangga; 2014. 25. mozartha m, praziandithe m, sulistiawati s. pengaruh penambahan hidroksiapatit dari cangkang telur terhadap kekuatan tekan glass ionomer cement. b-dent. 2015; 1(2): 75–81. 26. ardhiyanto hb, yustisia y. potensi limbah dental gypsum sebagai bahan baku material pengganti tulang. report. jember: universitas jember; 2017. 27. kutz m. standard handbook of biomedical engineering and design. new york: mcgraw-hill; 2003. p. 156-8. 28. ichsan mz, siswanto s, hikmawati d. sintesis komposit kolagenhidroksiapatit sebagai kandidat bone graft. j fisika dan terapannya. 2013; 1(1): 89–103. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p114–118 http://dx.doi.org/10.20473/j.djmkg.v51.i3.p114-118 http://e-journal.unair.ac.id/index.php/mkg mkgs vol 44 no 2 april-juni 2011.indd 82 vol. 44. no. 2 june 2011 relieving idiopathic dental pain without drugs haryono utomo1 and m. rulianto2 1dental clinic 2department of conservative dentistry, airlangga university faculty of dentistry, airlangga university surabaya indonesia abstract background: teeth are commonly obvious source of orofacial pain. sometimes the pain source is undetectable, thus called as idiopathic dental pain. since dentist wants to alleviate or eliminate the pains with every effort in their mind, a lot of drugs could be prescribed. moreover, it is make sense that endodontic treatment or even tooth extraction will be done. unfortunately, endodontic treatment may also initiate neuropathic tooth pain that is caused by nerve extirpation, thus worsen the pain. therefore, another cause of dental pain such as referred pain, periodontal disease, or stress which related to psychoneuroimmunology should be considered. in order to prevent from unnecessary drugs or invasive treatment such as root canal treatment and extraction, correct diagnosis and preliminary non-invasive therapies should be done. purpose: this review elucidates several therapies that could be done by dentists for relieving idiopathic dental pain which includes massage, the “assisted drainage” therapy, modulation of psychoneuroimmunologic status and dietary omega-3. reviews: understanding the basic pathogenesis of pain may help in elucidating the effects of non-drug pain therapy such as muscle massage, the “assisted drainage” therapy, omega-3 and psychological stress relieving. these measures are accounted for eliminating referred pain, reducing proinflammatory mediators and relieving unwanted stress reactions consecutively. psychological stress increases proinflammatory cytokines and thus lowered pain threshold. conclusion: as an individual treatment, this non-drug therapy is useful in relieving idiopathic dental pain; nevertheless, if they work together the result could be more superior. key words: idiopathic dental pain, without drugs therapy, pain relief abstrak latar belakang: gigi adalah suatu penyebab umum dari nyeri orofasial. kadang kala penyebab nyeri tidak dapat ditemukan, sehingga disebut sebagai nyeri gigi idiopatik. karena dokter gigi berupaya untuk mengurangi atau menghilangkan nyeri dengan segala cara maka banyak obat akan diresepkan ke pasien. bila gagal maka sangat mungkin dilakukan perawatan saraf gigi bahkan pencabutan gigi. akan tetapi, perawatan endodontik juga dapat menimbulkan nyeri neuropatik yang disebabkan oleh ekstirpasi saraf gigi, sehingga nyeri makin parah. sebab itu, penyebab lain nyeri gigi seperti nyeri yang dialihkan (referred pain), penyakit periodontal atau stres yang berhubungan dengan psikoneuroimunologi perlu dipertimbangkan.untuk mencegah kejadian konsumsi obat yang tidak perlu ataupun perawatan endodontik dan pencabutan gigi maka diagnosis yang tepat dan terapi non-invasif harus dilakukan terlebih dahulu. tujuan: studi pustaka ini menerangkan beberapa terapi yang dapat dilakukan dokter gigi untuk mengurangi nyeri gigi idiopatik yaitu masase, terapi assisted drainage,modulasi status psikoneuroimunologi dan diet omega-3. tinjauan pustaka: pengetahuan mengenai patogenesa nyeri dapat menerangkan efek terapi nyeri non-medikamentosa seperti masase otot, terapi assisted drainage, diet omega-3 dan mengurangi stres psikologis. berbagai terapi ini dapat mengurangi nyeri alihan, mediator proinflamasi dan mengurangi stres. stres psikologis akan mengingkatkan sitokin proinflamasi yang menurunkan ambang nyeri. kesimpulan: sebagai terapi individual, terapi non-medikamentosa ini berguna untuk mengurangi nyeri gigi idioaptik, akan tetapi bila bekerja sama dapat lebih baik lagi. kata kunci: nyeri gigi idiopatik, terapi non-medikamentosa, pereda nyeri correspondence: haryono utomo, c/o: klinik kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id literature review 83utomo and rulianto: relieving idiopathic dental pain without drugs introduction pain is a common presentation in general dental practice and usually its diagnosis and treatment is straightforward to the lesion. however, patients with non-dental causes of orofacial pain also seeking a dental solution to symptoms which may closely mimic toothache.1 according to linn et al.,1 22% population experience orofacial pain, 12.2% dental pain in 6 months period. ram et al.,2 revealed that approximately 45% orofacial pain patients went to dentist and wirz et al.,3 reported in their study that 17% of orofacial pain was atypical odontalgia or atypical tooth pain, other terms for this pain were neuropathic tooth pain or phantom pain.4 usually, the patient has self-diagnosed the problem as toothache and expects quick and efficient resolution of their problem.1,4 in practice, patient’s symptoms and complaints do not present so neatly and obviously as in textbooks, thus dentist may misleading the diagnosis and lead to irreversible treatments such as endodontic treatment or tooth extractions. these therapies are common attempts to decrease pain, but also cause more complications for patients and legal affairs for dentists.4 nixdorf et al.,5 study showed that 27.27% patients suffered from persistent tooth pain after root canal therapy which may be exaggerated by pulpal extirpation and became neuropathic tooth pain (ntp). interestingly, the prevalence of persistent pain occurred in the maxillary teeth were 87.5% compared to 12.5% in the mandibular teeth, and 68.8% relieved by tricyclic depressants.6 conventionally, relieving pain with drugs targeting biological factors by lowering the “pain triggers” mediators such as prostaglandins (pgs)7-9 or tumor necrosis α (tnf-α).10 nevertheless, prolonged or abuse of these drugs have adverse effects. therefore, a new non-invasive periodontal therapy that proposed termed as the “assisted drainage therapy” which had been verified significantly reduced either local of systemic tnf-α in an animal study is worthy to be tried.11 the effect of psychological stress towards inflammation and pain had been investigated in researches which refer to a “new” science so called psychoneuroimmunology that was founded and developed by ader in 1975. cortisol produced in chronic stress could potentiate proinflammatory cytokines rather than acts as antiinflammatory.12 moreover, stress may deteriorate body immune sytem13 and general health.14,15 some researchers revealed that dietary omega-3 fatty acid regulating and attenuating the stress response,16 as well as lowers proinflammatory cytokines.17,18 the aim of this article was to review the diagnostic procedures and treatment for orofacial pain, especially idiopathic dental pain without drugs. they should be done by dentists to avoid unnecessary drugs and dental treatments which may harmful to the patient, and may lead to medicolegal lawsuit in the future. what is orofacial pain? orofacial pain is every pain that happens in the orofacial region. nevertheless, according to okeson7 who wrote many books about orofacial pain, the definition of orofacial pain is evolving. at the present time the orofacial pain encompasses: masticatory musculoskeletal pain; cervical musculoskeletal pain; neurovascular pain; neuropathic pain; sleep disorders related to orofacial pain; orofacial dystonias; intraoral, intracranial, extra cranial, and systemic disorders that cause orofacial pain.7 the complexity of pain source detection in the trigeminal system pain sensation from the intra and extra oral structures of the head and face are carried to the central nervous system (cns) by the trigeminal system. pain transmission in the orofacial region is complicated because rather than a single nerve pathway. “trigeminal system” refers to a complex arrangement of nerve transmission fibers, interneurons, and synaptic connections which process incoming information from three divisions of the trigeminal nerve.2 the involvement of trigeminocervical complex makes the pain source detection more difficult.19–21 another factor complicating diagnosis and management is the very complexity and multidimensionality of pain, with its basis in biological processes but its impact on the emotional, psychological and social wellbeing of the patient (figure 1).22 the orofacial region has biological, emotional figure 1. the complex interrelationships among variables that affect pain.22 84 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 82–87 and psychological importance in eating, drinking, speech and the expression of our feelings, and facial appearance is also very important for most humans.4 modulators of pain perception psychological factors (i.e. stress, anxiety)22 and biological factors (i.e. female sexual hormones)23 may also modulate pain perception.19,20,22 the intensity of pain from physical injury relates to the attention given, if it is fully absorbed to other subject during the injury (distraction), no pain at all may be felt. moreover, recent concept of pain is changing; pain management should include body, mind and person. consequently, good therapy to pain begins with an attitude of caring and concern for the person more than for his or her body.7,21 diagnosing source of pain and site of pain in the orofacial region if someone accidently cut his finger, the source of pain is the same with the site of pain, because brain gives the same perception of pain for this area. in another type of pain, so called heterotopic pain or referred pain, the source of pain is different with the site of pain. in an inflammatory or pain process, a signal is sent only when a certain critical level of insult is reached, e.g. deep caries or an early pulpitis. brain may appreciate that there is a toothache somewhere but cannot localize it, and due to convergence factors, the brain experiences more difficulty in localizing the pain.7 the importance of referred pain in dentistry could be noted from a study in 400 patients with posterior tooth pain. referred pain was reported to be 89.9% and most common site for referred pain was neighboring teeth (80%), and the frequency of pain radiating to opposite dental arch was 24%.8 some criteria may helpful in determining the cause and source of non-odontogenic tooth pain (table 1).1 spesific pains in dentistry there are several pains that mimicking tooth pain even though it may not related to pulpalgia i.e.: atypical odontalgia/atypical tooth pain/idiopathic tooth pain;3 flareups (pain during endodontic treatment and post endodontic pain;24,25 periodontal pain paradox.11 it was interesting that endodontic treatment itself do not guarantee as all-round pain reliever since oshima6 study, ntps among post endodontic patients, 87.6% occurred in the maxilla (figure 3), since pulpal extirpation may also caused ntp which lead to persistent tooth pain.2 table 1. diagnostic categories-orofacial painnon odontogenic (adapted from linn1) condition characteristics investigations muscular (mpd, muscle tension headaches, neck paint, whiplash, fibromyalgia) arthralgia (internal derangement, osteoarthritis) psychogenic atypical facial pain atypical odontalgia pathology chronic infection – sinusitis – osteomyelitis malignancy – oral scc – brain tumour neuromuscular dystonia dystinesia chronic dull ache following muscular distribution muscular dysfunction pain, clicking, locking related to the tm joint abnormal often exaggerate description of symptoms abnornal response to treatment deep constant pains signs of inflammation usually painless unless advanced neuralgic pain abnormal involuntary movements with muscle pain tender muscles imaging normal diagnostic block no effect radiograph or ct may show bone morphology changes mri show disc abnormality objective tests normal subjective test atypical known previous psychiatric history and treatment abnormal imaging abnormal blood test – white cell shift – c-reactive protein abnormal ct – biopsy abnormal emg figure 3. distribution of pain locations (indicated by closed circles). patient number and % number are shown.6 atypical odontalgia that presents approximately 17% of orofacial pain.3 it mimics the symptoms of an acute toothache–severe throbbing, continuous pain starting in one quadrant and even crossing the midline. this condition is also called ntp,“phantom pain” or “dental migraine” and is often associated with patients suffering from unipolar depression. over 80% of the patients are female and over 90% of the time the pain is in the teeth, jaws or gingiva. almost a third of the pain is precipitated by dental procedure. tricyclic or monoamine oxidase inhibitor antidepression therapy relieves the pain in many cases.2,6 85utomo and rulianto: relieving idiopathic dental pain without drugs flare up means development of pain and swelling during or after endodontic treatment. various reasons have been attributed to these acute exacerbations of chronic conditions like: alteration of local adaptation syndrome; microbial factors; changes in periapical tissue pressure; effects of chemical mediators; immunological phenomena; and numerous psychological factors.24 according to utomo et al.,11 there was a particular tooth pain symptoms which was “out of the box”, and caused by non-specific pain causes. it was proposed termed as the “periodontal pain paradox” (paradox means unusual inverse effect). sharp pain could be triggered by rinsing with tap water in room temperature; moreover, hot or cold foods or drinks increased the pain even more. interestingly, scaling did not elicit pain, it was suggested caused by an increase sensitivity of the periodontal afferent sensory fibers since chronic gingivitis had higher pge2 in the gingival crevicular fluid if compared to normal gingival.26,27 general pain control techniques based on the “gate control theory”, painful stimuli can be altered at the spinal level, which means that pain is altered before it is felt. this theory is important in nondrug pain control which based on a mechanism that painful stimuli pass through a “gate” on the way to the central nervous system.7,28 to inhibit pain sensation, the gate can be closed by three main methods: non-painful sensory input can close the gate (i.e. stimulation by warmth and massage on trigger points, tps) which can help control pain (figure 4); the brainstem can project inhibitory impulses that close the gate to transmission of painful impulses (i.e. guided imagery or distraction is able to close the gate for other incoming stimuli); decreasing anxiety and increasing feelings of control over the situation.7 it is important to be remembered that tp-induced toothache is usually intermittent.7,8 pain therapy without drugs there are several pain relieving therapies without drugs that are: physical therapy, therapies which related to psychoneuroimmunology, and control of biological factors. the goal of physical therapy is the inactivation of myofascial trigger points, muscles relaxation, muscle rehabilitation, and postural education.15 physical therapy techniques are useful in treating muscle dysfunction and pain i.e. massage, superficial (warm) and deep heats (ultrasound). these modalities reduce muscle tension, decrease inflammation and inactivate myofascial tps.7,8 other therapies which related to psychoneuroimmunology could be stress relieving, empathy, caring, behavioural considerations and improving the immune system12–15, a new periodontal treatment so called the ”assisted drainage” therapy (adt) which is scaling and root planning that combined with subgingival massage had been successfully relieved periodontal pain paradox symptoms.11 in an animal study it was verified that this therapy was able to reduce either subgingival tissue or systemic (serum) tnf-α and sp in minutes. in addition, in this study, subgingival massage in chronic gingivitis animal model this therapy also released stress proteins such as heat shock protein 70 (hsp70) which suggested caused by increased of local temperature. heat shock protein 70 was beneficial for reducing periodontal inflammation, such as gingivitis since it act as antiinflammatory agent.29 down-regulate the inflammatory response with stress relieving and omega-3 ader12 who founded psychoneuroimmunology in 1975 said that human stress response has a number of checks and balances built in to ensure that various components do not become overactive. unfortunately, in the case of severe or overwhelming stress, the normal checks and balances fail, causing inflammation levels to be abnormally high. cortisol, which is normally anti-inflammatory, can change function under severe stress and potentiate the actions of il-1 and il-6.15,19 kiecolt-glaser et al.,16 noted that prior trauma “primes” the inflammatory response system so that there is heightened and more rapid rise in inflammation in response to stress. one key to improving the health of trauma survivors is downregulating this stress response and increasing resilience to stress. researches suggests a body-related adjunct to traditional trauma treatment that specifically downregulate the inflammatory response system by stress– relieving (i.e. music listening), consuming omega-3.16,17,19 omega-3 (from fish) in reduced inflammation by competing with aa which is a long chain omega-6 (from meat, egg and dairy products). excess aa has been associated with increased inflammation because it is converted into pgs and reduced the anti-inflammatory effects of omega-3.16–18 other than directly reduced inflammatory reaction, researchers have examined the impact of eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), the longchain omega-3, on stress. they may have an adaptogenic role in stress by regulating and attenuating the stress figure 4. referred pain patterns from trigger points, tp (x) in the temporalis muscle. a) anterior fibers; b and c: middle fibers; d= posterior fibers. 7 86 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 82–87 response. in higher levels of epa/dha population, they had a lower inflammatory response to stress.16 discussion pulpalgia is the most painful complaint found in dental patients. nevertheless, regarding the complexity of orofacial pain and referred pain, the diagnosis of the source of pain should be carefully done by dental practitioners. literatures showed that diagnosing pulpal pain is uneasy because if the infl ammation is limited to the pulpal tissues, it may be diffi cult for the patient to localize the offending tooth. it was due to the limited distribution of the discriminative touch receptors (proprioceptors) in the pulp.7,19,25 therefore, even though endodontic treatment is considered as a potent dental pain treatment and treatment of choice for irreversible pulpal pathosis, pain may still persist and become idiopathic dental pain or ntp in several cases.6 endodontic treatment itself may cause idiopathic dental pain (that may misinterpreted as “fl are ups”) at least by several reasons: nerve extirpation cause peripheral axonal injury;6 patients has to open their mouth for a long time that may cause muscle spasm. other persistent pains may cause from the referred pain (i.e. tmj pain, sinusitis) or habits (i.e bruxism).7 misdiagnosis of dental pain may be caused by referred pain in accordance to oshima et al.6 study, 87.6% of ntps were in maxillary teeth, which may be caused by sinusitis or temporal muscle (tps) (figure 4). consequently, diagnosing the dental pain should not limited to the dental and periodontal tissues examination; palpation of masticatory muscles to fi nd muscle spasms and tps is mandatory. additionally, orofacial pain is not just a simple matter because pain or related dysfunction that occurs in the face, mouth or jaws, especially when it becomes persistent or chronic, it can be associated with emotional, psychological and social disturbances that compromise the patient’s well-being and quality of life.7,22,23 thus, the management of a patient with chronic orofacial pain requires that the clinician appreciate this biopsychosocial basis, biological factors as well as understanding the basic knowledge of psychoneuroimmunology.12–15 eventhough treatments that based on this concept is diffi cult to be done, treatment with empathy, caring and patience may help in reducing emotional stress.15 biological factors of pain mostly affect women regarding the sexual hormonal fl uctuation.28 the transmission and modulation of pain signals may differ in men and women.23 normal hormonal variations and changes related to women’s reproductive functions can be sex-specifi c sources of pain. it was interesting that meana et al.,30 revealed that in women’s chronic pain management, physical therapy and cognitive-behavioral approaches i.e. ensuring patients that they can involve in controlling their own pain such as massaging tps, coping skills training such as relaxation and divert attention from pain (distraction) have been shown to be important components in treatment other than drugs alone. the importance of understanding the referred pain symptoms and treatment had been reported by okeson7 and mardani et al.8 they revealed that most of posterior tooth pain also showed referred pain in the head and neck area. it was interesting that okeson7 and lavelle et al.28 suggested massage therapy in myofascial tps which related to particular tooth to reduce referred pain (figure 4). massage therapy to tps is able to relief pain via the “gate control” theory, increasing blood circulation thus increase tissue oxygenation, stimulates endorphin (a morphine-like substance) release, as well as relief of mental stress and anxiety therefore, in idiopathic dental pain, especially if there is no obvious pathological fi nding, the non-drug tooth pain-reliever as in our concept i.e. massaging tps is worthy to be tried. treating tps for many pain symptoms had been known for centuries, nevertheless a “new” concept had been developed by travell and simmons in 1983.28 massaging tp is able to relief idiopathic tooth pain by relieving masticatory muscle spasm, this kind of tooth pain is usually termed as “muscular toothache”.7,25 this confusing symptom can be caused by several tps, chiefl y in the temporalis, digastric and masseter muscles. each tp has its own particular toothache pattern. therefore, during a long dental procedure, which often activates these tps, the patient should take periodic rests for exercise and relieve the jaw muscles. it should also be kept in mind that the level of infl ammatory mediators inside the circulation of our body is different between individuals.15,19 our body produces antiinfl ammatory such as cortisol, thus to keep in homeostasis, our body has the propensity to compensate noxious stimuli, proinflammatory mediators or neutralize antigens by itself.5,8,9 based on psychoneuroimmunology concept, in stressful individuals, cortisol level increased and plays a synergistic role with proinfl ammatory cytokines instead of suppressing them.12,15,16 this idea was supported with the facts that antidepression therapy relieves the pains.2,6 therefore, excess of circulating proinfl ammatory cytokines and omega-6,16,18,19 must be solved first or treated concomitantly with other non-drug pain therapies before conducting invasive dental procedures. the use of omega 3 diet for reducing infl ammation is also agreed by sharav and benoliel19 since it has the possibility as an alternative to nsaids for long term use. moreover, because high levels of epa and dha were related to lower levels of proinflammatory cytokines (il-1α, il-1β, il-6, and tnf-α) and higher levels of antiinfl ammatory cytokines, such as il-10.14,19 it decreases the pgs which is involved in lowering the pain threshold.16 the conversion of aa into pgs is done by the cyclooxygenases that is activated by tnf-α.31 since the assisted drainage therapy is able to decrease local and systemic tnf-α in minutes,29 application of this method is mandatory before conducting invasive treatment. 87utomo and rulianto: relieving idiopathic dental pain without drugs the most diffi cult management of orofacial pain was the psychogenic origin pain since it was related to anxiety, depression and other emotional disturbances which needed a lot of stress-relieving drugs. nevertheless, continuous consumption of these drugs may also cause adverse reactions, therefore alternative stress management should be discovered. other than stress relaxation therapy, dietary omega 3 had been reported as stress-reliever supplement by kiecolt-glaser.16 nevertheless the exact dose of dha and epa for anxiety and depression was still in controversy. recently, in a meta-analysis study, sublette et al., 32 in 2011 revealed that epa was more effective than dha for stressrelievers, that was supplement containing epa ≥ 60% of total epa + dha, in a dose range of 200 to 2,200 mg/day of epa in excess of dha. since daily dose for regular use is 150 mg dha and 1000 mg epa, twice of daily dosis was considered enough as stress-relievers. to summarize, the non-drug therapy for idiopathic dental pain consisted of several methods: the “assisted drainage” therapy that was able to diminish tooth pain by reducing local as well as systemic proinfl ammatory mediators, pgs, and acts as stimulator of hsp70 excretion; physical therapy including massage of the masticatory muscles that was able to reduce pain via “gate control” mechanism; omega-3s which down regulate the stress response as well as lowering levels of proinfl ammatory cytokines, and pge2; and other stress relieving procedures. therefore, it is concluded that the non-drug therapies for idiopathic dental pain is logical and should be conducted before and during treating pains in the orofacial region that manifest as dental pain. as an individual treatment, non-drug therapy is useful in relieving idiopathic dental pain; nevertheless, if they work together the result could be more superior. references 1. linn j, trantor i, teo n, thanigaivel r, goss an. the differential diagnosis of toothache from other orofacial pains in clinical practice. austr dent j 2007; 52: s100–4. 2. ram s, teruel a, satish k, kumar s, clark g. clinical characteristics and diagnosis of atypical odontalgia implications for dentists. jada 2009; 140: 223–8. 3. wirz s, wartenberg hs, nadstawek j. pain management procedures used by dental and maxillofacial surgeons: an investigation with special regard to odontalgia. head & face medicine 2005; 1: 14. 4. sessle bj. why are the diagnosis and management of orofacial pain so challenging?. jcda 2009; 75(4): 275–7. 5. nixdorf d, moana-filho ej, law as, mcguire la, hodges js, john mt. frequency of nonodontogenic pain after endodontic therapy: a systematic review and meta-analysis. j endod 2010; 36: 1494–8. 6. oshima k, ishii t, ogura y, aoyama y, katsuumi i. clinical investigation of patients who develop neuropathic tooth pain after endodontic procedures. joe 2009; 35(7): 958–61. 7. okeson jp. bell’s orofacial pain. 6th ed. carol stream: quintessence pub; 2005. p. 83, 277, 299, 313. 8. mardani s, eghbal mj, baharvand m. prevalence of referred pain with pulpal origin in the head, face and neck region. iranian endo j 2008; 3: 8–10. 9. bruno st-jacques b, ma w. role of prostaglandin e2 in the synthesis of the pro-inflammatory cytokine interleukin-6 in primary sensory neurons: an in vivo and in vitro study. j neurochemist 2011; 118(5): 841–54. 10. hess a,, axmann r, rech j, finzelb s, heindl c, kreitz s, et al. blockade of tnf-α rapidly inhibits pain responses in the central nervous system. pnas 2011; 108(9): 3731–6. 11. utomo h, kriswandini il, ernawati ds. the periodontal pain paradox: difficulty on pain assesment in dental patients. dent j 2006; 39(4): 143–6. 12. ader r. psychoneuroimmunology. 4th ed. san diego, usa: elsevier academic press; 2007. p. 15, 734–5. 13. ziemssen t, kern s. psychoneuroimmunology–cross-talk between the immune and nervous systems. j neurol 2007; 254(suppl 2): sii8–11. 14. kendall-tacket. psychological trauma and physical health: a psychoneuroimmunology approach to etiology of negative health effects and possible interventions. psychological trauma: theory, research, practice, and policy 2009; 1(1): 35–48. 15. alford l. psychoneuroimmunology for physiotherapists physiotherapy 2006; 92: 187–91. 16. kiecolt-glaser jk, belury ma, porter k, beversdoft d, lemeshow s, glaser r. depressive symptoms, omega-6: omega-3 fatty acids, and inflammation in older adults. psychosom med 2007; 69: 217–24. 17. cleland lg, james mj. marine oils for anti-inflammatory effect-time to take stock. j rheumatol 2006; 33(2): 207–9. 18. goldberg rj, katz j. a meta-analysis of the analgesic effects of omega-3 polyunsaturated fatty acid supplementation for inflammatory joint pain. pain 2007; 129: 210–23. 19. sharav y, benoliel r. orofacial pain and headache. edinburghlondon. mosby-elsevier; 2008. p. 205, 365. 20. conti pcr, pertes ra, heir gm, nasri c, cohen hv, de araujo crp. orofacial pain: basic mechanisms and implication for successful management. j app oral sci 2003; 11(1): 1–7. 21. mellick lb. treatment of acute orofacial pain with lower cervical intramuscular bupivacaine injections: a 1-year retrospective review of 114 patients. j orofac pain 2008; 57: 57–64. 22. caris hf. psychological techniques in the management of pain. anaesth intensive care med 2002; 13–5. 23. fillingim rb, king cd, ribeiro-dasilva mc, rahim-williams b. sex, gender, and pain: a review of recent clinical and experimental findings. j pain 2009; 10(5): 447–85. 24. mittal r. endodontic flare–ups: an overview. j oral health comm dent 2010; 4(3): 67–71. 25. nisha garg n, amit garg a. textbook of endodontics. 1st ed. new delhi: jaypee brothers medical publisher; 2010. p. 95. 26. burt b. epidemiology of periodontal disease. j periodontol 2005; 76: 1406–19. 27. vardeh d, wang d, costigan m, lazarus m, saper cb, woolf cj, et al. cox2 in cns neural cells mediates mechanical inflammatory pain hypersensitivity in mice. j clin invest 2009; 119(2): 287–94. 28. lavelle ed, lavelle w, smith hs. myofascial trigger points. anesthesiol clin 2007; 25(4): 841–51. 29. utomo h. immunoneuromodulatory mechanism of the “assisted drainage” therapy towards allergic reaction of rat induced by lipopolysaccharide from porphyromonas gingivalis. experimental study in rat subjects. dissertation. surabaya: postgraduate program airlangga university; p. 110, 121 30. meana m, cho r, desmeules m. chronic pain the extra burden on canadian women. whsr 2011; 1–19. available online at url. http://secure.cihi.ca/ cihiweb/products/ whsr_ chap_16 _e. pdf. accessed sept 24, 2011. 31. medeiros r, figueiredo cp, pandolfo p, duarte fs, prediger rd, passos gf, calixto jb. the role of tnf-alpha signaling pathway on cox-2 upregulation and cognitive decline induced by beta-amyloid peptide. behav brain res 2010; 209(1): 165–73. 32. sublette me, ellis sp, geant al, man jj. meta-analysis of the effects of eicosapentaenoic acid (epa) in clinical trials in depression. j pschyciatry 2011; 72 (9): 6–11. 61 volume 46 number 2 june 2013 gambaran densitas kamar pulpa gigi sulung menggunakan cone beam ct-3d (description of pulp chamber density in deciduous teeth using cone beam ct-3d) herdiyati y,1 epsilawati l,2 oscandar f2 dan nurianingsih r2 1 bagian kedokteran gigi anak 2 bagian radiologi kedokteran gigi fakultas kedokteran gigi universitas padjadjaran bandung indonesia abstract background: dental caries is the most common chronic diseases. detection of caries is needed, especially on the deciduous teeth. an examination such as radiological examination is essential. the radiographic figures distinguish radiolucent of the crown. digital radiography cone beam computed tomography (cbct) is able to show a more detailed picture. purpose: this study was aimed to get value of the density of pulp chamber of caries and non caries deciduous teeth using cbct radiographs. methods: the study was conducted by using simple descriptive. the samples were all the data cbct of pediatric patients aged 7-10 years who visited the dental hospital of the faculty of dentistry, university of padjadjaran. the samples were teeth with single and double root. results: the results showed that the value of the normal pulp density is 422.56 hu, while the condition of caries decreased becomes -77.89 hu. conclusion: the tooth with caries showed a lower density than the non caries/tooth. key words: density of pulp chamber, dental caries, deciduous tooth, cbct abstrak latar belakang: karies gigi merupakan penyakit kronis yang sering terjadi. deteksi terhadap karies sangat diperlukan terutama pada gigi decidius. pemeriksaan penunjang berupa pemeriksaan radiologis sangat diperlukan. secara umum gambaran radiografi dapat membedakan karies berupa gambaran radiolusent pada mahkota. radiografi digital cone beam computed tomografi (cbct), merupakan jenis radiografi yang mampu memperlihatkan gambaran yang lebih detail. tujuan: penelitian ini bertujuan mendapatkan nilai densitas kamar pulpa gigi sulung yang karies dan non karies menggunakan radiografi cbct. metode: penelitian dilakukan dengan metode simple deskriptif. sampel penelitian adalah semua data cbct dari pasien anak berusia 7 10 tahun yang berkunjung ke rsgm fakultas kedokteran gigi universitas padjadjaran. gigi yang dianalisa meliputi gigi berakar tunggal dan berakar ganda. hasil: hasil penelitian menunjukkan bahwa nilai densitas pulpa normal adalah 422,56 hu, sedangkan pada kondisi karies terjadi penurunan menjadi -77,89 hu. simpulan: pada gigi dengan karies menunjukkan densitas yang lebih rendah dibanding gigi yang tidak karies. kata kunci: densitas kamar pulpa, karies gigi, gigi sulung, cbct korespondensi (correspondence): yetty herdiyati, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan 1 bandung 40236, indonesia. e-mail: yettynonong@yahoo.com research report 62 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 61–64 pendahuluan kesehatan gigi dan mulut merupakan bagian dari kesehatan tubuh yang tidak dapat dipisahkan satu dengan yang lainnya, sebab kesehatan gigi dan mulut akan mempengaruhi kesehatan tubuh keseluruhan.1 kebersihan gigi dan mulut yang tidak diperhatikan, akan menimbulkan masalah salah satu kerusakan pada gigi adalah karies atau gigi berlubang. karies gigi bersifat kronis di mana dalam perkembangannya membutuhkan waktu yang lama, sehingga sebagian besar penderita mempunyai potensi mengalami gangguan seumur hidup. penyakit ini sering tidak mendapat perhatian dari masyarakat dan perencana program kesehatan, karena jarang membahayakan jiwa.2-4 kerusakan gigi terdapat di seluruh dunia tanpa memandang umur, bangsa ataupun keadaan ekonomi. penelitian di beberapa negara seperti eropa, amerika, asia, termasuk indonesia, ternyata 80-95% dari penduduk mengalami kerusakan gigi. prevalensi kerusakan gigi tertinggi terdapat di asia dan amerika latin dan terendah terdapat di afrika. kerusakan gigi didominasi oleh karies yang merupakan penyakit kronis yang sering terjadi. di amerika dilaporkan bahwa karies menempati peringkat kelima bahkan lebih tinggi dari kasus asma. berdasarkan survey kesehatan rumah tangga (skrt) pada tahun 2004, prevalensi karies mencapai 90,05%. berdasarkan laporan pada tahun 2007 dari centers for disease control and prevention, kerusakan gigi pada anak berusia 2-5 tahun berkisar 24-28% dan 70% disebabkan oleh karies.5-7 ada beberapa cara untuk mengetahui terjadinya kerusakan gigi.8,9 secara klinis gambarannya terkadang berbeda tetapi pada umumnya kerusakan gigi mempunyai penyebab yang sama. pada tahap awal karies gigi akan tampak berupa daerah berkapur namun berkembang menjadi lubang berwana kecokelatan. gigi sulung memiliki anatomi yang berbeda di mana email dan dentin lebih tipis, kamar pulpa yang cenderung lebih besar sehingga kondisi karies sering terdeteksi dalam kondisi lanjut di mana karies sudah terlanjur dalam. walaupun karies mungkin dapat saja dilihat dengan mata telanjang, pemeriksaan penunjang berupa pemeriksaan radiologis sangat diperlukan. hampir semua jenis radiografi baik ekstra maupun intraoral dapat dipergunakan untuk keperluan ini.10 secara umum gambaran radiografi dapat membedakan karies berupa gambaran radiolusent pada mahkota. radiografi digital cone beam computed tomografi (cbct), merupakan jenis radiografi yang mampu memperlihatkan detail dari gambaran yang diambilnya. dalam cbct, kita mampu menampilkan densitas atau kepadatan suatu jaringan. cbct mampu menampilkan detail dari kondisi densitas dari kamar pulpa. densitas suatu jaringan lebih umum diukur menurut skala hounsfield, yang merupakan suatu prinsip untuk sinar-x pada cbct. pada skala hounsfield, air suling memiliki nilai 0 hounsfield unit (hu), sementara udara ditentukan sebagai -1000 hu.11 perbedaan densitas pada jaringan termasuk kamar pulpa dapat menjadi penanda terjadinya kelainan. perubahan densitas atau kepadatan suatu jaringan sangat tergantung pada isi dari jaringan yang dinilai. pada kondisi karies, pulpa mengalami inflamasi sehingga gambaran densitas kamar pulpa dapat saja berubah. bagaimana perubahan densitas dan gambarannya masih belum banyak diteliti, salah satu peralatan yang digunakan untuk penilaian adalah cbct. penelitian ini bertujuan mendapatkan nilai densitas kamar pulpa gigi sulung yang karies dan non karies menggunakan radiografi cbct. bahan dan metode penelitian ini adalah penelitian deskriptif di mana hasil yang diperoleh berupa data kuantitatif.12 populasi adalah semua radiografi cbct rahang bawah dari januari 2009 sampai desember 2012. sampel yang dipergunakan adalah seluruh data cbct yang memenuhi kriteria yang ditetapkan. adapun kriteria populasi yang memenuhi syarat dalam penelitian ini bahwa data-data radiografi cbct-3d yang digunakan berasal dari pasien berusia antar 7–10 tahun di mana gender tidak dipisahkan, kondisi pulpa terlihat jelas sehingga memungkinkan untuk dilakukan pemeriksaan, kondisi pulpa dari gigi terpilih dalam keadaan normal dan kondisi pulpa dengan karies di mana kedalaman karies tidak dibedakan. dari data yang dikumpulkan, diperoleh sampel berjumlah 25 data pulpa normal dan 28 data untuk karies. gambar 1. memperlihatkan potongan sagital. daerah yang di dalam lingkaran menunjukkan dimana nilai densitas diukur. pengukuran ditarik dari mesial ke distal. gambar 2. nilai densitas yang diperoleh setelah penarikan garis profil pada potongan sagital, terlihat nilai atas 431 hu, nilai bawah 263 hu dengan demikian nilai densitas menjadi 347 hu. 63herdiyati: gambaran densitas kamar pulpa gigi sulung menggunakan cone beam ct-3d skala ataupun teknik pengukuran densitas yang dilakukan pertama dengan cara menarik garis profil dari sagital view dimulai dari permukaan bukal kamar pulpa ke arah lingual pada layar (gambar 1), kemudian pada layar akan tampak profil nilai densitas, kemudian kita mulai dapat menentukan nilai atas dan nilai bawah dari gambaran profil yang terlihat (gambar 2). setelah mendapatkan nilai atas dan nilai bawah, ditentukan nilai densitas yang diperoleh dari penjumlahan nilai atas dan nilai bawah kemudian dibagi dua. pengumpulan data nilai densitas dilakukan baik pada gigi normal maupun gigi dengan karies. penelitian ini menggunakan data radiografi yang dilakukan dan diolah dengan alat sinar-x jenis picasso trio; merek epx-impla, type b applied part impla, no seri 0165906; produksi vatech & e-woo korea. processor yang digunakan untuk mengolah data adalah satu unit komputer axio dengan spesifikasi pentium 4, memory 4g. soft-ware yang digunakan adalah program easydent 4 viewer dari vatech & e-woo korea.13 hasil hasil pengukuran terhadap densitas kamar pulpa dengan sampel 25 gigi normal dan 28 gigi karies diperoleh hasil bahwa pada densitas kamar pulpa normal rata-rata adalah 422,56 hu (grafik 1, bar warna biru), sedangkan batas atas 534,28 hu (grafik 1, bar warna hijau) dan batas bawah dari densitas 310,84 hu (grafik 1, bar warna orange). untuk gigi pada kondisi karies rata-rata densitas kamar pulpa 64, 52 hu (grafik 1, bar warna biru), batas atas dari densitas 200,93 (grafik 1, bar warna hijau), batas bawah -77,89 (grafik 1, bar berwarna orange) (gambar 3). pembahasan pulpitis atau peradangan pulpa dapat disebabkan oleh karies yang menembus enamel dan dentin dan mencapai pulpa. peradangan umumnya terkait dengan infeksi bakteri, tetapi juga dapat disebabkan oleh hal lain seperti trauma berulang atau penyakit periodontal.14 ketika pulpa meradang, tekanan di dalam kamar pulpa meningkat sehingga menekan saraf gigi dan jaringan sekitarnya. tekanan dari peradangan dapat menyebabkan rasa sakit mulai dari ringan sampai hebat, tergantung pada tingkat keparahan peradangan dan respons tubuh. tidak seperti bagian tubuh lain di mana tekanan dapat menghilang melalui jaringan lunak sekitarnya, kamar pulpa sangat berbeda. kamar pulpa dikelilingi oleh dentin, sebuah jaringan keras sehingga tidak memungkinkan dilakukan pembagian tekanan akibatnya rasa sakit yang timbul tidak dapat dibagi. peradangan pulpa diperhitungkan bukan saja pada saat proses pengrusakan jaringan pulpa terjadi, akan tetapi perhitungan telah dimulai pada saat produk bakteri atau toksin menyentuh jaringan pulpa.15 peradangan dapat bersifat akut atau kronis karena seperti jaringan lain dalam tubuh, pulpa akan bereaksi terhadap iritasi dengan respons imun bawaan dan/ atau adaptif.16,17 komponen dari respons inflamai setidaknya terjadi enam proses yaitu: 1) keluarnya cairan dentin; 2) aktifnya odontoblasts; 3) timbulnya reaksi neuropeptida dan neurogenik; 4) aktifnya sel imun seperti sel dendritik (dc), sel pembunuh (nk ), dan sel t seperti sitokin dan 5) kemokin, di mana dua hadirnya minimal dua item komponen merupakan tanda awal dalam respons inflamasi awal untuk karies.16,17 densitas atau kepadatan jaringan dalam hal ini adalah kamar pulpa gigi pada kondisi normal baik pada gigi dewasa memilki maksimal rata-rata bernilai 493, 04 hu.18 densitas atau kepadatan jaringan kamar pulpa gigi sulung diteliti pada penelitian ini dan nilai yang diperoleh adalah 422,56 hu. hal ini menunjukkan bahwa kepadatan jaringan pada gigi sulung lebih rendah dibandingkan gigi dewasa. hal ini dapat dipahami dikarenakan anatomi dari gigi sulung jauh lebih kecil dari gigi dewasa.19 dalam penelitian ini, juga diperoleh data bahwa pada gigi sulung pada kondisi karies nilai densitas berkurang dari kondisi normal yaitu dengan nilai rata-rata 64,52 hu. hal ini memenuhi asumsi bahwa pada kondisi karies diduga pulpa mengalami peradangan sehingga densitas menjadi menurun atau menjadi lebih hitam (radiolusentcy) meningkat. hal ini diduga karena pulpa gigi dewasa ataupun gigi sulung pada kondisi radang terjadi peningkatan kadar cairan. peningkatan kadar cairan di dalam kamar pulpa merupakan respons dari suatu organisme terhadap patogen pada jaringan. sebenarnya proses inflamasi adalah satu dari respons utama sistem kekebalan terhadap infeksi dan iritasi. inflamasi distimulasi oleh faktor kimia yang dilepaskan oleh sel yang berperan sebagai mediator radang di dalam sistem kekebalan untuk melindungi jaringan sekitar dari penyebaran infeksi.17 pada saat peradangan diproduksi cairan yang kaya protein dan sel darah putih, tertimbun dalam ruang ekstravaskular sebagai akibat reaksi radang disebut juga sebagai eksudat. eksudat adalah cairan radang ekstravaskular dengan berat jenis tinggi diatas 1.020 dan seringkali mengandung protein 2–4 mg % serta sel-sel darah putih yang melakukan emigrasi. cairan ini tertimbun sebagai akibat permeabilitas vascular yang memungkinkan protein plasma dengan molekul besar dapat terlepas, bertambahnya tekanan hidrostatik intravascular. protein plasma yang keluar dapat diasumsikan gambar 3. rata-rata densitas ruang pulpa pada kondisi normal dan karies. 64 dent. j. (maj. ked. gigi), volume 46 number 2 june 2013: 61–64 sebagai kadar darah yang keluar dengan konsentrasi tertentu.14, 20,21 hal inilah yang menyebabkan densitas kamar pulpa berubah menjadi lebih hitam bila dibandingkan dengan pulpa normal. penelitian mengenai densitas ruang pulpa dengan menggunakan cone bean ct masih kesulitan dilakukan sebelumnya, dan hasilnya belum lengkap. umumnya penelitian sebelumnya tentang densitas ruang pulpa menggunakan alat ct yang dipercaya keakuratannya. namun dengan alat ct kondisi ruang pulpa terlihat sangat kecil sehingga menyulitkan pengukuran.19 gambaran densitas menunjukkan bahwa dalam kondisi normal nilai densitas kamar pulpa berada pada nilai 422,56 hu, sedangkan adanya inflamasi menyebabkan densitas kamar pulpa berubah menjadi lebih cair bahkan mencapai 77, 89, lebih kental dari nilai densitas aquades yang nilainya 0 hu. ini menunjukkan bahwa cairan inflamasi sangat kental dan beragam isinya, bila dibandingkan dengan aquades yang lebih encer. oleh karena itulah maka dapat dipastikan bahwa kondisi karies atau pulpitis terjadi perubahan pada kondisi normal 97%,20,21 sehingga dapat dipastikan bahwa nilai densitas kamar pulpa pada gigi sulung akan menurun sejalan dengan lajunya inflamasi pada kamar pulpa. dengan melihat nilai rata-rata dari densitas kamar pulpa maka kita dapat mendeteksi terjadinya karies khususnya pada gigi sulung. penilaian terhadap kamar pulpa dilakukan dengan menilai gambaran kehitaman yang terlihat. pada penelitian ini kondisi pulpitis tidak dipisahkan reversible maupun irreversible sehingga hasil penelitian ini belum lengkap dan diperlukan penelitian lanjutan dengan variabel penelitian yang lebih lengkap dan sampel lebih banyak. berdasarkan pembahasan di atas dapat disimpulkan bahwa densitas kamar pulpa pada kondisi normal dan kondisi karies berbeda, dimana pada kondisi karies densitas akan menurun. penurunan densitas ini membuktikan bahwa dengan melihat perbedaan densitas kamar pulpa karies dapat ditemukan. kondisi inflamasi atau karies dapat dilihat dari perubahan nilai densitas. perubahan nilai densitas merupakan salah satu alat untuk mendeteksi kondisi karies terutama pada gigi sulung. daftar pustaka 1. soebroto. apa yang tidak dikatakan dokter tentang kesehatan gigi anda. jogja: bookmarks; 2013. h. 1. 2. taringan r. perawatan pulpa gigi. edisi 2. jakarta: hipokrates; 2004. h. 1. 3. jhon b. mengenal gigi anda. edisi 1. jakarta: arcan; 1996. h. 2. 4. herijulianty e, artini s, indriani t. pendidikan kesehatan gigi. jakarta: eg; 2002. h. 4 5. dye ba, tan s, smith v, lewis bg, barker lk, thorton-evans g. trends in oral health status in united states, 1988-1994 and 19992004. national center for health statistics. vital health stat 2007; (11): 248. 6. macek md, heller ke, selwitz rh, manz mc. percented of dental caries. public health dentistry j 2004; (64): 20-5. 7. vos t. years lived with disability (ylds) for 1160 sequelae of 289 diseases and injuries 1990-2010, an systematic analysis for the global burden of disease. geneva: lancet; 2012. h. 96. 8. rosenstiel sf. clinical diagnosis of dental caries: a north american perspective. maintained by the university of michigan dentistry library, along with the national institutes of health. national institute of dental and craniofacial research 2006. diakses pada 19 april 2013. 9. summit jb, robbins jw, schwartz rs. fundamentals of operative dentistry: a contemporary approach. 2nd ed. illinois: carol stream; 2001. p. 31. 10. grossman. ilmu endodntics dalam praktek. edisi 11. jakarta: egc; 1995. h. 149. 11. muhtadan hd. pengembangan aplikasi untuk perbaikan citra digital film radiografi. yogyakarta: sekolah tinggi teknologi nuklir; 2008. 12. sugiyono. statistika untuk penelitian. edisi 1. bandung: alphabetha; 2003. h. 62-3, 115. 13. vatech. current product picaso trio. 2008. diakses dari www. vatech.com. diakses 19 april 2013. 14. tarigan r. perawatan pulpa gigi. edisi 2. jakarta: egc; 2002. h. 183. 15. kakehashi s, stanley hr, fitzgerald rj. the effects of surgical exposures of dental pulps ingerm-free and conventional laboratory rats. oral surg oral med oral pathol 1990; 20: 340–9. 16. james ka. oral development and histology. 3th ed. new york: themes; 2001. p. 190-9. 17. guyton a. fisiologi kedokteran. setiawan i, editor. edisi 9. jakarta: penerbit buku kedokteran egc; 1996. h. 354. 18. epsilawati l, sitam s, oscandar f. deskripsi lebar, tinggi, ketebalan dan densitas ruang pulpa dengan menggunakan cbct (cone beam computed tomografi). departemen of dentomaxillofacial radiology, faculty of dentistry padjadjaran university, international associated dentomaxillo facial radiology conference proceeding, bergen, norwegia. 2013. 19. molteni r. from ct number to hounsfield units in cone beam volumetric imaging the effectt of artifacts. international association dentomaxillofacial j 2011; (62): 628. 20. byers mr, suzuki h, maeda t. dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration. microsc res tech 2003; (60): 503–15. 21. hahn cl, liewehr fr. innate immune responses of the dental pulp to caries. endod int j 2007; (33): 643. 6262 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 62–66 original article the antibacterial efficacy of calcium hydroxide–iodophors and calcium hydroxide–barium sulfate root canal dressings on enterococcus faecalis and porphyromonas gingivalis in vitro eric priyo prasetyo1, devi eka juniarti1, galih sampoerno1, dian agustin wahjuningrum1, ananta tantri budi1, dyanita hasri2, evelyn tjendronegoro3 1department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2dentistry programme, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3healthcare and research, irvine medical center, university of california, california, united states abstract background: a successful endodontic treatment is inseparable from the right choice of root canal dressing. the right choice of medicaments would result in patient satisfaction. enterococcus faecalis (e. faecalis) and porphyromonas gingivalis (p. gingivalis) are usually found in failed root canal treatments. calcium hydroxide is a gold standard dressing that creates an alkaline environment in the root canal and has a bactericidal effect. commercially, there are calcium hydroxide dressings with supporting additions, including calcium hydroxide–iodophors (ch–iodophors) and calcium hydroxide–barium sulfate (ch–barium sulfate). purpose: this study aimed to compare the antibacterial efficacy between ch–iodophors and ch–barium sulfate root canal dressings on e. faecalis and p. gingivalis. methods: ch–iodophors and ch–barium sulfate were obtained commercially. e. faecalis and p. gingivalis were obtained from stock culture taken from the root canal of failed endodontic treatment. e. faecalis and p. gingivalis were cultured in petri dishes, and for each bacterium, 12 wells were made in the media. six wells were used for the ch–iodophors group, and six wells were used for the ch–barium sulfate group. ch–iodophors and ch–barium sulfate were deployed in the wells in e. faecalis and p. gingivalis cultured media in the petri dishes. after incubation, the inhibition zone diameters were measured. an independent t-test was used for analysis, and the significance level was set at 5%. results: there is a significant difference in the antibacterial efficacy of ch–iodophors and that of ch–barium sulfate on e. faecalis and p. gingivalis (p = 0.00001). conclusion: ch–iodophors have a higher antibacterial efficacy than ch–barium sulfate on both e. faecalis and p. gingivalis. keywords: enterococcus faecalis; porphyromonas gingivalis; calcium hydroxide; iodophors; ch–barium sulfate; patient satisfaction correspondence: eric priyo prasetyo, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47, surabaya, 60132, indonesia. email: eric-p-p@fkg.unair.ac.id introduction the right choice of root canal dressing to eliminate bacteria in the root canal is important for a successful endodontic treatment. a successful endodontic treatment will result in the patient’s satisfaction. many attempts have been made to increase the success of root canal treatment, including finding efficient instrumentation, employing effective cleaning,1,2 using antibacterial dressings and improving irrigation materials.3–5 root canal treatment has a high success rate, but in some cases, there are failures. isolated bacteria from root canal treatment failures and the prevalence of these bacteria in the root canal system are caused by enterococcus faecalis (e. faecalis) in about 45.8% to 77% of cases and by porphyromonas gingivalis (p. gingivalis) in 28.17% of cases.6 both microorganisms are among the ones that survive disinfecting protocols.7 e. faecalis can invade dentine tubules and spread into the peri-radicular area, which causes the formation of periradicular lesions after root canal treatment.8 p. gingivalis can survive in the extra-radicular region, mostly in the area approximate with the root surface and responsible for periodontitis and peri-radicular lesions.6 p. gingivalis dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p62–66 mailto:eric-p-p@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p62-66 63 prasetyo et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 62–66 also contributes to root canal treatment failures through its by-product, lipopolysaccharides, which affect biological processes, inflammation and tissue destruction.9 different substances have been added to calcium hydroxide to increase its efficacy. the added substances are distilled water, saline, propylene glycol, chlorhexidine, glycerine, iodophors, ch–barium sulfate, corticosteroids, antibiotics, anesthetic solution, methyl cellulose and detergents. calcium hydroxide with added ch–barium sulfate and iodophors is commonly used in clinical practice. iodophors have been added to calcium hydroxide to work with different bacterial characteristics.10 ch– barium sulfate is added to calcium hydroxide; aside from its antibacterial effect, this substance functions to increase radiopacity.11 root canal treatment failure may happen even after applying dressing with calcium hydroxide if the dressing is done incorrectly, depending on the right instrumentation and irrigation to remove inorganic and organic smear layers. in this research, the efficacy of adding calcium hydroxide with iodophors (ch–iodophors) and calcium hydroxide with barium sulfate (ch–barium sulfate) was analysed on e. faecalis and p. gingivalis. based on this background, the authors would like to purposely compare the antibacterial efficacy of ch–iodophor and ch–barium sulfate root canal dressings on e. faecalis and p. gingivalis. materials and methods this is an experimental laboratory study conducted in the conservative dentistry section, universitas airlangga dental hospital and microbiology laboratory, research center, faculty of dental medicine. this study was ethically approved by the universitas airlangga faculty of dental medicine health research ethical clearance commission (166/kkepk.fkg). materials used in this research were commercially available calcium hydroxide dressings with iodophors (meta biomed, korea) and ch–barium sulfate (meta biomed, korea). enterococcus faecalis and porphyromonas gingivalis bacteria used in this study were stock bacteria previously cultured from patients who failed endodontic treatment. the method employed in this study was the agar diffusion method using mueller–hinton (mh) agar and brain heart infusion (bhi) broth. the method is followed according to alharthi et al.12 and balouiri et al.13, with modifications on the sum and position of the wells in the plates. the media were allocated for four groups of experiments. in the first group, 12 wells were prepared for ch–iodophors (six wells with e. faecalis and six wells with p. gingivalis), and in the second group, 12 wells were prepared for the ch–barium sulfate dressing (six wells with e. faecalis and six wells with p. gingivalis). both e. faecalis and p. gingivalis bacterial cultures from stock were moved into separate reaction tubes, each containing bhi broth, and stirred. incubation was done for both cultures at 37˚c for 24 hours in anaerobic condition. after 24 hours, 0.5 ml of the e. faecalis and p. gingivalis bacterial cultures in bhi broth were taken using a micropipette and poured into another reaction tube containing bhi broth until they were equal to 0.5 mcfarland standard of turbidity. the bacterial cultures were taken from the bhi broth using a sterile cotton swab and swabbed on the surface of each mh agar allocated for e. faecalis and p gingivalis. an antibacterial test was conducted by making wells for the tested dressing materials (ch–iodophors and ch–barium sulfate). the samples were incubated at 37˚c for 48 hours in anaerobic condition. after 48 hours, measurements were conducted on antibacterial efficacy through inhibition zone measurement using a vernier digital caliper (mitutoyo, japan). the clear zones of inhibition around the wells were measured, revealing no bacterial growth. data of measurements on inhibition zone diameters (in millimetres) were collected for each sample well. inhibition zone data were analysed statistically, and the significance level was set at 5%. spss 20.0 for windows (spss inc., chicago, illinois, usa) was used in this study. data normality was tested using the shapiro–wilk test. the significance was tested using an independent t-test. results the number of replications (n) for each treatment group was six. mean and standard deviations of the inhibition zone diameter of ch–iodophors and ch–barium sulfate on e. faecalis and p. gingivalis are shown in table 1. the mean of the e. faecalis inhibition zone from ch–iodophors was 11.8125 mm, and the mean from ch–barium sulfate was 6.3750 mm. the mean of the p. gingivalis inhibition zone from ch–iodophors was 12.7875 mm, and the mean from ch–barium sulfate was 6.6750 mm. an independent t-test was used in this study to check the significance between the ch–iodophors group and the table 1. mean, standard deviation, and significance from the inhibition zone diameter of ch–iodophors and ch–barium sulfate on e. faecalis and p. gingivalis groups n e. faecalis mean + sd p value p. gingivalis mean + sd p value ch–iodophors 6 11.8125 + 1.32001 0.00001* 12.7875 + 1.34961 0.00001* ch–barium sulfate 6 6.3750 + 0.19494 6.6750 + 0.51865 notes: n = replication; sd = standard deviation; * = statistically significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p62–66 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p62-66 64prasetyo et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 62–66 ch–barium sulfate group on e. faecalis and p. gingivalis. the significance between the inhibition zone diameter of ch–iodophors and that of ch–barium sulfate on e. faecalis and p. gingivalis growth is shown in table 1. we found a significant difference between the two treatment groups on both bacteria. the ch–iodophors group has higher antibacterial efficacy, with a wider antibacterial clear zone in both bacteria (p = 0.00001). the ch–barium sulfate group has lower antibacterial efficacy, with a narrower antibacterial clear zone in both bacteria. the inhibition zone diameter measurement is shown in figure 1. discussion enterococcus faecalis and porphyromonas gingivalis were used in this study because these bacteria are opportunistic, living in facultative anaerobic condition, found in the pathogenic state and associated with failed root canal treatments.6 these bacteria also have a major role in persistent root canal infections, can survive in the root canals and are resistant to commonly used intracanal dressings.14 the antibacterial efficacy of ch–iodophors and ch–barium sulfate dressing materials on e. faecalis and p. gingivalis growths were experimentally checked with the agar diffusion method, and inhibition clear zones were measured on the media. inhibition zone is a laboratory calculation method to check whether a material has the ability to inhibit bacterial growth.13 the diameter of clear zones would describe the strength of such material to inhibit bacterial growth; the more this material inhibits bacterial growth, the larger the clear zones would appear. in this study, mueller–hinton agar was used because this media can grow e. faecalis and p. gingivalis actively and sensitive to drug effects. inhibition zones appeared on both groups of ch–iodophors and ch–barium sulfate. this showed that each of these dressing materials has the ability to inhibit both e. faecalis and p. gingivalis. the antibacterial properties of both root canal dressing materials mainly come from calcium hydroxide, a mechanism that involves the hydroxyl ion to kill bacteria by protein denaturation; the cytoplasmic membrane and dna destruction will physically inhibit this bacterial growth in the root canal systems.15 antibacterial activity is connected with alkali formation, which can destroy lipid, the protein structure of bacteria and nucleic acid.16 direct contact of the hydroxyl ion in alkaline ph with a cytoplasmic membrane will destroy the hydrogen chain of the protein polypeptide. contact of the hydroxyl ion with dna will result in replication inhibition, causing a lethal mutation. dna is sensitive to temperature and ph change. in alkali condition, the dna structure and function will break and lead to bacterial cell death. the alkali condition from calcium hydroxide would impact the surrounding tissues, including healing, anti-inflammation17 and cytotoxic, leading to apoptosis.18,19 the result of this study showed a significant difference between ch–iodophors and ch–barium sulfate. the ability of ch–iodophors to inhibit e. faecalis and p. gingivalis growths is significantly greater than that of the ch–barium sulfate root canal dressing. in the ch–iodophor dressing, the iodophor substance will release iodine with high reactivity to promote protein oxidation. iodophors function as a disinfectant and infection control. thus, in the combination of ch and iodophors, the substances will synergistically strengthen each other. iodophors and calcium hydroxide can diffuse into dentinal tubules for added disinfection and eliminate the bacteria. ch–barium sulfate can diffuse into dentinal tubules and also perform antibacterial activity. in this study, the antimicrobial strength of ch–barium sulfate is not as good as that of ch–iodophors. this may be caused by the form and structure of the ch–barium sulfate used in the mixture. previous studies on the antimicrobial properties of ch–barium sulfate showed that ch–barium sulfate in micron particulate form would have potent antimicrobial properties.20 ch–barium sulfate is generally used for figure 1. agar diffusion assay containing e. faecalis (a) and p. gingivalis (b). the green dots indicate the inhibition area of each well. ch–iodophors are indicated by yellow arrows, and ch–barium sulfate is indicated by blue arrows. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p62–66 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p62-66 65 prasetyo et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 62–66 its radiopacity effect on radiographic examination.11 the addition of this material must take into account its properties, as these will affect the dressing’s consistency and application. e. faecalis and p. gingivalis can be killed with high ph levels. high ph or an extreme alkaline environment would disturb the survival of most bacteria.21,22 however, there are some studies stating that a ph higher than 11.5 is required for potent disinfection.6 this study showed that the inhibition zone of ch–iodophors is two times higher than that of ch–barium sulfate. this may be caused by the combination of these two materials, since calcium hydroxide and iodophors would create a synergistic effect on the antimicrobial activity to inhibit e. faecalis growth. even though this dressing material will not eliminate e. faecalis or p. gingivalis, the material is expected to weaken the bacteria, and eventually, the body’s defence mechanism will be able to eliminate these bacteria and their by-products. m u c h r e s e a r c h h a s b e e n d o n e t o f i n d n o v e l , biocompatible, antimicrobial and regenerating root canal dressing materials in order to support successful root canal treatment and enhance patient satisfaction. the success of root canal treatment, aside from the right choice of dressing, depends on other factors, such as the root canal system complexity, the diffusion ability of dressing materials within the dentinal tubules, the effect on mixed cultured bacteria or biofilm formation, and bacterial resistance, all of which need to be evaluated in further studies. as there are many microorganisms involved in failed root canal treatments, 23–25 even after biomechanical and chemical instrumentations during treatment, individual and personalised assessments of these bacteria or biofilms are needed for ideal treatment. there are limitations, as this is only an in vitro study, and there are many factors to consider, both in situ and in vivo. in conclusion, a calcium hydroxide–iodophors root canal dressing has a different (higher) antimicrobial efficacy on both e. faecalis and p. gingivalis. however, further studies need to be done regarding the effect on periodontal and periapical tissues with more complexities. acknowledgements the authors would like to thank the publication center, dental hospital, and the research center, faculty of dental medicine, universitas airlangga for the given facility. references 1. santoso cma, samadi k, prasetyo ep, wahjuningrum da. the differences between mangosteen peel extract irrigant and naocl 2.5% on root canal cleanliness. conserv dent j. 2020; 10(1): 40–3. 2. juniarti de, kusumaningsih t, soetojo a, sunur yk. antibacterial activity and phytochemical analysis of ethanolic purple leaf extract (graptophyllum pictum l.griff) on lactobacillus acidophilus. malaysian j med heal sci. 2021; 17(supp 2): 71–3. 3. astuti rhn, samadi k, prasetyo ep. antibacterial activity of averrhoa bilimbi linn leaf extract against enterococcus faecalis. conserv dent j. 2016; 6(2): 93–8. 4. prasetyo ep, saraswati w, wahjuningrum da, mooduto l, rosidin rf, tjendronegoro e. white pomegranate (punica granatum) peels extract bactericidal potency on enterococcus faecalis. conserv dent j. 2021; 11(2): 84–8. 5. harseno s, mooduto l, prasetyo ep. antibacterial potency of kedondong bangkok leaves extract (spondias dulcis forst.) against enterococcus faecalis bacteria. conserv dent j. 2016; 6(2): 110–6. 6. prada i, micó-muñoz p, giner-lluesma t, micó-martínez p, collado-castellano n, manzano-saiz a. influence of microbiology on endodontic failure. literature review. med oral patol oral cir bucal. 2019; 24(3): e364–72. 7. del fabbro m, samaranayake lp, lolato a, weinstein t, taschieri s. analysis of the secondary endodontic lesions focusing on the extraradicular microorganisms: an overview. j investig clin dent. 2014; 5(4): 245–54. 8. kaiwar a, nadig g, hegde j, lekha s. assessment of antimicrobial activity of endodontic sealers on enterococcus faecalis: an in vitro study. world j dent. 2012; 3(1): 26–31. 9. kuntjoro m, prasetyo ep, cahyani f, kamadjaja mjk, hendrijantini n, laksono h, rahmania pn, ariestania v, nugraha ap, ihsan is, dinaryanti a, rantam fa. lipopolysaccharide’s cytotoxicity on human umbilical cord mesenchymal stem cells. pesqui bras odontopediatria clin integr. 2020; 20: e0048. 10. najjar rs, alamoudi nm, el-housseiny aa, al tuwirqi aa, sabbagh hj. a comparison of calcium hydroxide/iodoform paste and zinc oxide eugenol as root filling materials for pulpectomy in primary teeth: a systematic review and meta-analysis. clin exp dent res. 2019; 5(3): 294–310. 11. ba-hattab r, al-jamie m, aldreib h, alessa l, alonazi m. calcium hydroxide in endodontics: an overview. open j stomatol. 2016; 06(12): 274–89. 12. alharthi ss, binshabaib m, saad almasoud n, shawky ha, aabed kf, alomar ts, albrekan ab, alfaifi aj, melaibari aa. myrrh mixed with silver nanoparticles demonstrates superior antimicrobial activity against porphyromonas gingivalis compared to myrrh and silver nanoparticles alone. saudi dent j. 2021; 33(8): 890–6. 13. balouiri m, sadiki m, ibnsouda sk. methods for in vitro evaluating antimicrobial activity: a review. j pharm anal. 2016; 6(2): 71–9. 14. jhamb s, singla r, kaur a, sharma j, bhushan j. an in vitro determination of antibacterial effect of silver nanoparticles gel as an intracanal medicament in combination with other medicaments against enterococcus fecalis. j conser v dent. 2019; 22(5): 479–82. 15. mohammadi z, dummer pmh. properties and applications of calcium hydroxide in endodontics and dental traumatology. int endod j. 2011; 44(8): 697–730. 16. sharma g, ahmed hma, zilm ps, rossi-fedele g. antimicrobial properties of calcium hydroxide dressing when used for longterm application: a systematic review. aust endod j. 2018; 44(1): 60–5. 17. prasetyo ep, kuntjoro m, cahyani f, goenharto s, saraswati w, juniarti de, hendrijantini n, hariyani n, nugraha ap, rantam fa. calcium hydroxide upregulates interleukin-10 expression in time dependent exposure and induces osteogenic differentiation of human umbilical cord mesenchymal stem cells. int j pharm res. 2021; 13(1): 140–5. 18. prasetyo ep, widjiastuti i, cahyani f, kuntjoro m, hendrijantini n, hariyani n, winoto er, nugraha ap, goenharto s, susilowati h, hendrianto e, rantam fa. cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells. pesqui bras odontopediatria clin integr. 2020; 20: e0044. 19. prasetyo ep, kuntjoro m, goenharto s, juniarti de, cahyani f, hendrijantini n, nugraha ap, hariyani n, rantam fa. calcium hydroxide increases human umbilical cord mesenchymal stem cells dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p62–66 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p62-66 66prasetyo et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 62–66 expressions of apoptotic protease-activating factor-1, caspase-3 and caspase-9. clin cosmet investig dent. 2021; 13: 59–65. 20. aninwene ii ge, stout d, yang z, webster tj. nano-baso4: a novel antimicrobial additive to pellethane. int j nanomedicine. 2013; 8(1): 1197–205. 21. jhajharia k, mehta l, parolia a, shetty kv. biofilm in endodontics: a review. j int soc prev community dent. 2015; 5(1): 1–12. 22. zancan rf, vivan rr, milanda lopes mr, weckwerth ph, de andrade fb, ponce jb, duarte mah. antimicrobial activity and physicochemical properties of calcium hydroxide pastes used as intracanal medication. j endod. 2016; 42(12): 1822–8. 23. dudeja p, dudeja k, srivastava d, grover s. microorganisms in periradicular tissues: do they exist? a perennial controversy. j oral maxillofac pathol. 2015; 19(3): 356–63. 24. pereira rs, rodrigues vaa, furtado wt, gueiros s, pereira gs, avila-campos mj. microbial analysis of root canal and periradicular lesion associated to teeth with endodontic failure. anaerobe. 2017; 48: 12–8. 25. siqueira jf, antunes hs, rôças in, rachid ctcc, alves frf. microbiome in the apical root canal system of teeth with posttreatment apical periodontitis. rittling sr, editor. plos one. 2016; 11(9): e0162887. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p62–66 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p62-66 7171 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 71–75 research report effectiveness of light-emitting diode exposure on photodynamic therapy against enterococcus faecalis: in vitro study nanik zubaidah, agus subiwahjudi, dinda dewi artini and karina erda saninggar department of conservative dentistry, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: a successful root canal treatment eliminates pathogenic bacteria from infected root canals. the most common bacteria in root canal infections is enterococcus faecalis (e. faecalis), due to its resistance to medicament and root canal irrigation. a photodynamic therapy (pdt) is a method of root canal disinfection that uses a combination of photosensitisers and light activation to eliminate bacteria in the root canal. the duration of the pdt irradiation results in the production of singlet oxygen and reactive oxygen species (ros) to eliminate the e. faecalis bacteria. purpose: to analyse the differences in the duration exposure of photodynamic therapy against the e. faecalis bacteria. methods: the e. faecalis bacteria culture was divided into seven eppendorf tubes. group i was a control group, and group ii, iii, iv, v, vi and vii were treated using pdt consisting of toluidine blue o (tbo) photosensitiser and light source irradiation for ten, 20, 30, 40, 50 and 60 seconds, respectively. after incubation, the number of bacteria was calculated by the quebec colony counter and analysed using the kruskal–wallis test and the mann–whitney test (p <0.05). results: there was a significant difference between the number of e. faecalis bacteria colonies in each treatment group (p <0.05). group vi and vii, which had a longer exposure to pdt, showed a smaller amount of e. faecalis bacteria. conclusion: the longer exposure of pdt results in a smaller amount of e. faecalis bacteria. the light irradiation of 50 seconds is the most effective to eliminate e. faecalis bacteria. keywords: enterococcus faecalis; irradiation time; light-emitting diode; photodynamic therapy; root canal treatment correspondence: nanik zubaidah, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo 47, surabaya – indonesia. email: nanik-z@fkg.unair.ac.id introduction endodontic infections occur due to the invasion of bacteria in the root canal. enterococcus faecalis (e. faecalis) is the most common pathogenic bacteria that is found in the root canal (4–40 per cent) and causes a 20–70 per cent failure of endodontic treatment.1,2 in several research studies, e. faecalis was found in the treated root canals, and this bacteria is reported to be resistant to some medicaments and antimicrobial irrigation during the root canal treatment.3–5 in dentinal tubules, e. faecalis can survive the intracanal medicament of calcium hydroxide (ca(oh)2) for more than ten days.6 the elimination of the pathogenic bacteria that is present in the root canal affects the success of root canal treatment. the complex structure and shape of the root canal is a major problem when cleansing the root canal to eliminate the pathogenic bacteria. the bacteria that is left in the root canal can penetrate the root dentinal tubules to a depth of 1000 µm, while irrigation disinfection materials can only reach a depth of 100 µm.4,5,7 over the last few decades, photodynamic therapy (pdt) was developed. pdt is a disinfection method that uses a light source (light-activated disinfection) of a specific wavelength, which consists of two components: a light source in the form of a light-emitting diode (led) or laser diode as photoactivation, and a photosensitising agent (photosensitiser), which causes photoinactivation against the bacteria. there is an energy transfer from the photosensitiser, which is activated by a light source, to the available oxygen. this results in the formation of a singlet oxygen reactive, which has a cytotoxic effect dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p71–75 mailto:nanik-z@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p71-75 72 zubaidah et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 71–75 against bacteria and can damage the structure of bacterial cells. the use of pdt after the root canal preparation and mechanical irrigation could effectively eliminate the pathogenic bacteria in the root canal. the light source of pdt can reach the root canal area, which is difficult to reach using conventional irrigation because the light can reach a depth of 0.5–1.5 centimetres of root dentinal tubules. in addition, the light source is reported to be non-toxic and has a high degree of selectivity to eliminate the bacteria through the reaction of photosensitisers and oxygen without damaging the host cell. in vivo studies also reported that pdt could effectively eliminate the bacteria that is resistant to several types of medicaments. fotosan, which is a pdt method, has been reported to eliminate gram-positive and negative bacteria, such as streptococcus mutans and e. faecalis.8,9 fotosan is a photodynamic therapy that utilises a red led with a 630-nanometre wavelength and a toluidine blue o (tbo) photosensitiser agent. when using fotosan in endodontic treatment, the photosensitiser agents are inserted into the root canal for 60 seconds so that the liquid comes into contact with the root canal wall. the endodontic tip from the device is then inserted into the root canal and irradiated for 30 seconds. this is consistent with schlafer’s in vitro and ex vivo research study, which shows that the use of fotosan for 30 seconds reduces the number of pathogenic microorganisms that cause endodontic infections (escherichia coli, e. faecalis, fusobacterium nucleatum, streptococcus intermedius), compared to ten seconds and 20 seconds. however, poggio’s study found a decrease in the number of e. faecalis, s. mutans and streptococcus sanguinis bacteria with a longer exposure time of 90 seconds. xhevdet et al.4 also reported that the irradiation time of five minutes against e. faecalis bacteria has a greater effect than irradiation of one minute and three minutes. however, there is no significant difference between the time exposure and the reduced number of bacteria.4,10,11 the effectiveness of pdt depends on the strength, duration, absorption of light in the tissue, geometry of the root canal and the distance of the tip to the target cell. the light absorption phenomenon by the photosensitiser is a photophysical process when a photosensitiser molecule that has an electron configuration in stable state (ground state) absorbs photon light. after absorbing the light, the molecular electron configuration changes to an unstable (excited state). from the excited state, the electron photosensitiser molecule can either return to a ground state if it loses energy or become a triplet state if it continues to get enough energy. this triplet state is a reactive state. a chemical interaction occurs between the electron molecules and oxygen, which have a stable state electron configuration. this results in the oxygen molecule becoming excited (unstable). the excited oxygen tends to flow towards the stable electron conditions, which means that it will interact with the surrounding biological systems. the interactions that occur between the excited oxygen and biological systems, such as the bacterial cells, will damage the cell’s system and structure. the major concept of irradiation time is to produce reactive oxygen to reduce the number of bacteria.12–14 the aim of this study was to analyse the differences in the duration exposure of pdt using red led lights and tbo photosensitisers against the number of e. faecalis bacteria. materials and methods this study was approved by the ethics committee of the faculty of dentistry at airlangga university with the reference number 160/hrecc.fodm/viii/2017. this research was a laboratory experimental study with a posttest only control group design. the sample that was used in this study was e. faecalis atcc 29212 bacteria. the determination of the number of samples using lemeshow et al.’s (1990) formula obtained 42 total samples. the samples were divided into seven groups; group i (i-c) was a control group without light exposure; group ii (ii-10) had pdt irradiation for ten seconds; group iii (iii-20) had 20 seconds; group iv (iv-30) had 30 seconds; group v (v-40) had 40 seconds; group vi (vi-50) had 50 seconds; and group vii (vii-60) had 60 seconds. the preparation of the e. faecalis bacterial culture was carried out by taking the e. faecalis bacterial culture preparations with the osse wire and placing it in a test tube, which contained brain heart infusion (bhi) broth i. it was then stirred and incubated at 37 degrees celsius (oc) for 48 hours in an anaerobic atmosphere.15 a 0.5 millilitre culture from the bhi broth i tube then was taken with a micropipette and inserted into a test tube that contained bhi broth ii and equalised with the mc farland scale to obtain a 1.5 x 108 cfu/ml bacterial suspension. the final samples were obtained from 0.5 ml bacterial suspension test tubes, which were taken with a micropipette to be put into 42 eppendorf tubes each. the eppendorf tube was coated with black tape15 to ensure that during the irradiation, the pdt light was not transmitted outside the tube wall. these 42 samples in the eppendorf tubes were divided into seven groups, with each group consisting of six eppendorf tubes. group i was the control group without light exposure and photosensitisers and only contained the e. faecalis bacteria sample. group ii was added with photosensitisers in the form of tbo liquid 0.5 ml, and after 60 seconds, it was irradiated with the led light for ten seconds. groups iii to group vii also were treated like group ii with the irradiation time of the led light 20 seconds, 30 seconds, 40 seconds, 50 seconds and 60 seconds, respectively.10,11 after the irradiation was carried out to all groups, a 0.1 millilitre sample was taken with a micropipette from each eppendorf tube (groups i–vii), cultured in a petri dish containing agar nutrient and incubated for 48 hours at 37oc in an anaerobic atmosphere. the number of bacteria dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p71-75 73zubaidah et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 71–75 colonies in the petri dish was calculated using the iuebec colony counter with colony-forming unit (cfu) method and used for the data analysis.9 results a statistical calculation was conducted to get the average results and standard deviation of the number of e. faecalis bacteria colonies after irradiation, as shown in table 1. from the average results, a normality test was performed using the shapiro–wilk test and a significance value or p value > 0.05 was obtained. this shows that all the groups have a normal data distribution. subsequently, a homogeneity test was conducted on the data using the levene test and obtained a significance value of homogeneity 0.007 (p <0.05) with levene statistics of 3.640. this shows that all the groups’ data did not have a homogeneous variance. after the normality and homogeneity tests were conducted, a kruskal–wallis test then was applied to assess the differences of the whole groups. it was obtained a significance value of 0.000 (p <0.05) for chi-square 40.038. this shows that there is a significant difference between the number of e. faecalis colonies in all treatment groups. the mann–whitney test has a requirement of p <0.05 to show that there are significant differences in each group. there are significant differences between group i (control) and other treatment groups (groups ii, iii, iv, v, vi and vii). the majority of p is less than 0.05; however, there are some groups that show p > 0.05: group vi (50 seconds) compared to vii (60 seconds), which is 1.000, and group vii (60 seconds) compared to vi (50 seconds), which is 1.000. this shows that there were no significant differences between groups vi and vii. it has been suggested that both groups could eliminate all the e. faecalis bacteria. discussion from the results, it was obtained that the mean number of e. faecalis bacteria after irradiation is significantly different in all treatment groups (control, ten seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds and 60 seconds). it has been suggested that the pdt method can significantly eliminate e. faecalis bacteria. in accordance with rios’s study, which states that pdt in combination with led light and tbo fluid has an antibacterial effect against e. faecalis bacteria, there is potential for it to be used as microbial disinfection for conventional endodontic treatment.16 for the irradiation times of ten seconds, 20 seconds, 30 seconds and 40 seconds, there was still a small amount of e. faecalis bacteria that was calculated by cfu (the mean was 33.67; 23.33; 16 and 12.50, respectively). this is not in accordance with schlafer’s study, which found that the use of fotosan for 30 seconds, according to the protocol for endodontic treatment, could effectively decrease the number of e. faecalis bacteria by 99.7 per cent compared to ten seconds and 20 seconds. however, the difference in the results of this study is due to the different research methods that were used, as well as the use of different fibre tip sizes for irradiation on the eppendorf tubes that contained bacterial suspension. a study reported that the use of the optical fibre tip size gives better results than when the light is used directly on the cavity of a tooth or root canal because the longer and smaller fibre tip size can help to emit the light of pdt that reaches the apical end root canal, which is difficult to access.9,10,17 in the groups with 50 seconds and 60 seconds irradiation, we found zero e. faecalis colonies, which suggests that 50 seconds of irradiation is effective enough to eliminate all e. faecalis bacteria. this is different to poggio’s study, which stated that the number of e. faecalis bacteria was reduced after 90 seconds of irradiation by 91.49 per cent table 1. statistical analysis data relating to the quantity of e. faecalis colonies after irradiation in each treatment group group mean ± sd normality test homogeneity test kruskalwallis test mann-whitney test i-c ii-10 iii-20 iv-30 v-40 vi-50 vii60 i-c 116.67 ± 4.67 0.896 0.007 0.000 0.004* 0.004* 0.004* 0.004* 0.002* 0.002* ii-10 33.67 ± 4.32 0.06 0.004* 0.004* 0.004* 0.002* 0.002* iii-20 23.33 ± 3.72 0.096 0.004* 0.004* 0.002* 0.002* iv-30 16.00 ± 2.19 0.783 0.044* 0.002* 0.002* v-40 12.50 ± 2.73 0.357 0.002* 0.002* vi-50 .00 ± .000 1.000 vii-60 .00 ± .000 *) there is a significant difference (p <0.05) note: a normality test score of p>0.05 means the data follows normal distribution; a homogeneity test score of p<0.05 means the data did not have a homogeneous variance; kruskal-wallis test score of p<0.05 means that significant difference exists; mannwhitney test score of p>0.05 in group vi (50 seconds) compared to vii (60 seconds) and group vii (60 seconds) compared to vi (50 seconds) means that there were no significant differences between groups vi and vii. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p71-75 74 zubaidah et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 71–75 compared to 30 seconds by as much as 87.72 per cent. prolonged exposure to fotosan could significantly reduce the percentage of bacteria compared to a short exposure. however, poggio’s study only compared 30 seconds and 90 seconds irradiation time and did not investigate the effect of 50 seconds irradiation, whereas this study conducted ten to 60 seconds of irradiation at ten second intervals and found that 50 seconds is adequate time to eliminate the bacteria.11 this study used fotosan as the pdt method for the root canal disinfection, as fotosan utilises a red led light with a wavelength of 628 nanometres and tbo photosensitisers. this is in accordance with hopp’s research, which states that a red light with a wavelength of 628 nanometres can activate tbo fluid to produce a singlet oxygen reactive that causes oxidative damage to bacterial cells. these light rays can reach up to 0.5–1.5 centimetres into the depths of the root dentinal tubules, which are difficult to reach by irrigation disinfecting materials and have a high degree of selectivity to eliminate the e. faecalis bacteria without damaging the host cell.13,18 the photosensitiser that was used in this study was tbo. tbo photosensitisers contain phenothiazine, which is a cation that will bind to the cell wall of the e. faecalis bacteria and is anionic. the bonding results in an electrostatic interaction, which further increases the bacterial cell wall’s permeability and causes the photosensitising cation to enter the cytoplasmic membrane of the bacteria and further disorganise the barrier’s permeability. from kikuchi’s research, tbo was reported to have antibacterial power because it can interact with the bacterial cell membrane lipopolysaccharides without irradiation. irradiation with a wavelength of 630 nanometres leads to the maximum absorption of photosensitiser fluid, which results in pdt photoinactivation. this kills the bacteria more effectively than photosensitiser fluid without irradiation. this is consistent with arneiro’s findings that the use of tbo without irradiating kills a smaller number of bacteria than tbo with irradiation.19,20 the led light in fotosan is a red light with a wavelength of 628 nanometres, an output power of 1000 mw and 30 j energy. the rays will cause the light absorption phenomenon by the photosensitiser, which is called the photophysical process. the first phase in this process is the ground state. in this phase, each electron is in a stable and paired state in its orbitals. after being exposed to the irradiation, there is an energy transfer, which causes the photosensitiser electron molecule in the ground state phase to change into an excited state. the paired electrons begin to become unstable and then increase to the triplet state phase where the electrons have separated from their pairs. therefore, they become reactive and look for pairs with other molecules. the triplet state is a reactive state, which occurs when there are chemical interactions between the electron molecules and oxygen that have electron configurations in a stable state, which results in the oxygen molecule becoming excited (unstable). the excited oxygen flows towards stable electron condition and interacts with the surrounding biological systems. the interactions that occur between the excited oxygen and biological systems, such as the bacterial cells, will damage the cell system and structure of bacteria cell.12,14 these interactions result in two types of mechanisms. in type i, there is an electron transfer between the photosensitiser and the substrate, which produces radical ions called ros. these consist of superoxide anion (o2 ●-), hydroxyl radical (●oh) and hydrogen peroxide (h2o2). these ions are oxidative to the cells. in type ii, there is an electron transfer between the photosensitiser and the oxygen receptor (o2), which will produce a singlet oxygen (1o2). this singlet oxygen is a reactive form of oxygen and a powerful oxidative agent. ros and singlet oxygen will cause damage to lysosomes, mitochondria and bacterial plasma membranes.14 that damage occurs because ros and singlet oxygen cause oxidative stress, which results in lipid peroxidation of the plasma membrane and organelles. the fatty acids that bond with the unstable free radicals can cause severe membrane damage, as well as the oxidation of amino acid chains, the formation of covalent protein bonds and protein oxidation. consequently, this will damage the structure of the protein by increasing the proteasomal protein degradation. in addition, it can cause prolonged dna chain crosslinking, inactivate the nadh succinate enzymes and lactate dehydrogenase, damage the balance of k+ ions and other ions and damage the bacterial cell dna, which will cause the death of the bacteria.4,14 from the test results in each group, group ii (irradiation of ten seconds) is the group that has the weakest ability to kill the e. faecalis bacteria. this is because the lack of irradiation time will result in a smaller concentration of radical ion formations and singlet oxygen. group vi (irradiation of 50 seconds) and group vii (irradiation of 60 seconds) had the best ability to kill the e. faecalis bacteria. in group vi, no bacteria colonies were found. therefore, it could be concluded that 50 seconds was the most effective time of irradiating the pdt to kill all the e. faecalis bacteria. this suggests that the importance of the irradiation time will result in the numerous concentration of photosensitiser molecules of the excited state and triplet state, so that produce reactive oxygen to kill the bacteria. the radical ions and singlet oxygen will damage the lysosomes, mitochondria and plasma membranes of the bacterial cells and kill more bacteria.12,14 in conclusion, the longer irradiation exposure during photodynamic therapy results in a smaller number of e. faecalis bacteria. the irradiation time of 50 seconds is the most effective time to eliminate all the e. faecalis bacteria. references 1. alagl as, bedi s, almas k. phytosolutions for enterococcus faecalis in endodontics: an update. oral health dent manag. 2016; 15(5): 332–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p71-75 75zubaidah et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 71–75 2. pourhajibagher m, kazemian h, chiniforush n, hosseini n, pourakbari b, azizollahi a, rezaei f, bahador a. exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of enterococcus faecalis from infected root canal during antimicrobial photodynamic therapy. photodiagnosis photodyn ther. 2018; 24: 206–11. 3. filipov i, markova k, boyadzhieva e. efficency of photoactivated disinfection on experimental biofilm scaning electron microscopy results. j imab annu proceeding (scientific pap). 2013; 19(4): 383–7. 4. xhevdet a, stubljar d, kriznar i, jukic t, skvarc m, veranic p, ihan a. the disinfecting efficacy of root canals with laser photodynamic therapy. j lasers med sci. 2014; 5: 19–26. 5. lins ccsa, melo ars, silva cc, oliveira jb, lima ga, castro cmmb, diniz fa. photodynamic therapy application in endodontic aerobic microorganisms and facultative anaerobic. formatex. 2015; : 559–63. 6. de la maza lm, pezzlo mt, shigei jt, tan gl, peterson em. color atlas of medical bacteriology. 2nd ed. washington: asm press; 2013. p. 67–90. 7. yildirim c, karaarslan e, ozsevik s, zer y, sari t, usumez a. antimicrobial efficiency of photodynamic therapy with different irradiation durations. eur j dent. 2013; 7(4): 469–73. 8. lópez-jiménez l, fusté e, martínez-garriga b, arnabat-domínguez j, vinuesa t, viñas m. effects of photodynamic therapy on enterococcus faecalis biofilms. lasers med sci. 2015; 30(5): 1519–26. 9. bago i, plečko v, gabrić pandurić d, schauperl z, baraba a, anić i. antimicrobial efficacy of a high-power diode laser, photo-activated disinfection, conventional and sonic activated irrigation during root canal treatment. int endod j. 2013; 46(4): 339–47. 10. schlafer s, vaeth m, hørsted-bindslev p, frandsen evg. endodontic photoactivated disinfection using a conventional light source: an in vitro and ex vivo study. oral surgery, oral med oral pathol oral radiol endodontology. 2010; 109(4): 634–41. 11. poggio c, arciola cr, dagna a, florindi f, chiesa m, saino e, imbriani m, visai l. photoactivated disinfection (pad) in endodontics: an in vitro microbiological evaluation. int j artif organs. 2011; 34(9): 889–97. 12. l est a r i w p, ast uti sd, setiawatie em. potensi ina k tivasi streptococcus mutans dengan penambahan fotosensitiser ekstrak daun kelor pada aplikasi fotodinamik light emitting diode (led). thesis. surabaya: universitas airlangga; 2016. 13. kishen a, shrestha a. photodynamic therapy for root canal disinfection. in: endodontic irrigation. springer international publishing; 2015. p. 237–51. 14. dai t, fuchs bb, coleman jj, prates ra, astrakas c, st. denis tg, ribeiro ms, mylonakis e, hamblin mr, tegos gp. concepts and principles of photodynamic therapy as an alternative antifungal discovery platform. front microbiol. 2012; 3: 1–16. 15. alison mr, poulsom r, forbes s, wright na. an introduction to stem cells. j pathol. 2002; 197(4): 419–23. 16. rios a, he j, glickman gn, spears r, schneiderman ed, honeyman al. evaluation of photodynamic therapy using a light-emitting diode lamp against enterococcus faecalis in extracted human teeth. j endod. 2011; 37(6): 856–9. 17. garcez as, fregnani er, rodriguez hm, nunez sc, sabino cp, suzuki h, ribeiro ms. the use of optical fiber in endodontic photodynamic therapy. is it really relevant? lasers med sci. 2013; 28(1): 79–85. 18. hopp m, biffar r. photodynamic therapies – blue versus green. laser. 2013; 1: 10–25. 19. kikuchi t, mogi m, okabe i, okada k, goto h, sasaki y, fujimura t, fukuda m, mitani a. adjunctive application of antimicrobial photodynamic therapy in nonsurgical periodontal treatment: a review of literature. int j mol sci. 2015; 16(10): 24111–26. 20. usacheva mn, teichert mc, biel ma. comparison of the methylene blue and toluidine blue photobactericidal efficacy against grampositive and gram-negative microorganisms. lasers surg med. 2001; 29(2): 165–73. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p71-75 117117 effectiveness of applied behavior analysis (aba) with regard to tooth brushing in autistic children felicia melati, ratna indriyanti and arlette suzy setiawan department of paediatric dentistry, faculty of dentistry, universitas padjadjaran, bandung – indonesia abstract background: children demonstrating autistic spectrum disorders tend to be uncooperative when receiving dental treatment. actions as simple as brushing the teeth with a prophylactic brush can constitute complex processes for children with such conditions. applied behavior analysis (aba) can train children in new positive behavior and it is, therefore, anticipated that an aba-based approach is capable of influencing the behavior of individuals with autistic spectrum disorder. purpose: this study aimed to assess the effectiveness of the aba-based approach for autistic children during dental brushing procedures. methods: the research constituted a quasiexperimental single subject investigation of children presenting autistic spectrum disorders who attended the lembaga pendidikan autisma prananda, bandung. potential changes in the behavior of subjects were monitored four times during treatment with a one-week interval between consultations. those subjects satisfying the inclusion criteria consisted of 11 boys and 4 girls. the data analysis used in this study consisted of an anova test and a non-parametric kruskal-wallis test with a p–value < 0.005. results: changes in scores between the initial and final consultations were statistically significant with a p-value (0.269) <0.05. statistically significant differences existed between changes in the behavior of level 1 and level 2 autistic subjects. conclusion: an aba-based approach effectively changes the behavior of autistic children with regard to prophylactic brushing. children with level 1 autistic spectrum disorder demonstrate greater capacity to follow instructions and consistently implement a prophylactic brushing technique. keywords: applied behavior analysis; autistic spectrum disorders; prophylaxis brush; picture cards correspondence: arlette suzy setiawan, department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, jl. sekeloa selatan no. 1, bandung 40132, indonesia. email: arlettesuzy@yahoo.com dental journal (majalah kedokteran gigi) 2019 september; 52(3): 117–121 research report introduction children with autism spectrum disorder (asd) tend to demonstrate limited ability to maintain their oral hygiene. activities such as brushing and flossing teeth can prove challenging for such individuals, while caregivers also usually experience a certain degree of difficulty in brushing their teeth.1–3 research conducted on 196 families by chan, et al. indicated that 42.3% of the population reported problems brushing their children’s teeth and that, consequently, they only attempted to do so once a day.4 individuals with asd enjoy eating sweets and soft foods, while also tending to pouch food inside their mouths (particularly in the vestibule, interdental and occlusal regions).1,5 oral hygiene levels in children with asd compared to those who do not present the condition contrast sharply. indeed, 59% of non-asd children demonstrated a high level of oral hygiene, whereas only 3% of their asd counterparts recorded a similar level.3 poor oral hygiene is responsible for caries and periodontal diseases among children afflicted with asd. the prevalence of dental caries among such individuals is as high as 77%, but only 46% in non-asd children. 97% of asd children suffer from gingivitis, whilst only 41% of their non-asd counterparts do so. given their dental condition, asd children require preventive action but, generally, prove uncooperative when receiving dental treatment. 3,6–8 one of the routine treatments administered during a dental consultation is that of prophylactic brushing. this procedure involves the use of a prophylactic brush dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p117–121 mailto:arlettesuzy@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p117-121 118 melati, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 117–121 and toothpaste to remove plaque from dental surfaces. such cleaning can be performed manually, but dentists prefer motor-driven prophylactic brushes for reasons of efficiency. a prophylactic brush is attached to a straight or angled hand-piece or a prophy angle. brushing should be performed at a recommended speed of 1500-3500 rpm, although it is difficult to measure the exact speed during actual brushing. a buzzing or high-pitched squeaking sound will be heard when the prophylactic brush is rotating at an excessive speed. the dental surface should brushed for 2-5 seconds at a moderate but consistent speed.9,10 this tooth cleaning procedure not only facilitates clinical observation but also introduces children to dental procedures. asd children who have learned dental cleaning techniques involving the use of prophylactic brushes are likely to become competent in other procedures. dentists can teach asd children such procedures by adopting a method-based behavioral approach known as applied behavior analysis (aba).10 aba is an instructional method commonly used with asd children which has also been employed within other disciplines to manage asd child behavior problems.1–3 an aba-based teaching approach involving the use of picture cards is one that can be adopted to amend the behaviour of asd children. the basic aba procedure consists of prompting, fading, shaping, and chaining which are employed repeatedly and consistently in combination with positive reinforcement. the effectiveness of the aba-based behavioral approach is evident from the change in the ability of children suffering from the condition to follow instructional steps. during each consultation, the subjects were rated in terms of their compliance with dental cleaning instructions. their scores, in addition to any indications of behavioral change, were observed and recorded. any increase in scores recorded between the initial and final meetings would indicate the effectiveness of the aba-based approach. the use of a prophylactic brush and dental micromotor constitutes a cleaning procedure for labial and buccal surfaces. the prophylactic brush employed during the research was of a pink-colored, soft-textured, nylon, latch flat type. this research aimed to measure the effectiveness of the aba approach in dental cleaning with a prophylactic brush with asd children. materials and methods approval for this research was granted by the research ethics commission of universitas padjadjaran in a letter numbered 374/un6.c1.3.2/kepk/pn/2016. the research subjects consisted of 15 asd children attending lembaga pendidikan autisma prananda who satisfied the inclusion criteria of the research project. subjects were selected on the basis of the following criteria: males and females diagnosed with level 1 and level 2 asd based on dsm-5 diagnostic criteria. children with specific limitations other than asd such as systemic diseases or physical disabilities were excluded. the research adopted a quasi-experimental single-subject design to assess the effectiveness of an aba-based approach in shaping dental cleaning behavior with a prophylactic brush among children with level 1 and level 2 asd. treatment was administered four times at intervals of one week. one day prior to treatment, the teacher prepared the subjects by introducing them to a set of picture cards that would be employed the following day (figure 1). the activities were completed in a room with only the teacher, children and researcher present. treatment was administered to six areas of the subjects’ oral cavities, commenced with region 2, continued with regions 5, 1, 3, and 4 and terminated with region 6. posterior region brushing was initiated with the most anterior tooth and culminated with the most posterior tooth. at each instruction step, the subjects were shown a picture depicting the respective treatment. subjects who were able to follow the instructions received verbal praise and a reward based on their preference indicated by a previously-administered questionnaire. any change in behavior was measured on the basis of the ability of subjects to follow instructions. scores of 1 and 0 were respectively awarded to those subjects who were able or unable to follow instructions as shown in table 1. no score would be awarded if the subject proved unable to follow an instruction. under such circumstances, the instruction would be repeated until the subject was able to perform the action as required. failure on the part of the subject to obey the instruction meant that the process would not continue to the subsequent step. the maximum a b c d figure 1. materials in picture cards: (a) researcher without face mask; (b) researcher wearing face mask; (c) subject seated during examination; (d) subject receiving dental cleaning treatment. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p117–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p117-121 119melati, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 117–121 treatment time was one of 15 minutes per consultation. in cases where the attention of the subject was distracted during treatment, his/her focus would be reoriented by eliminating the source of distraction and repeating the instruction. any step completed during one consultation would be repeated during the subsequent one before the subject was allowed to proceed to the next step. during each consultation, a record was made of the extent to which each subject was able to follow instructions and whether he/she demonstrated progress, retrogression, or no change. the data selected was then analysed through the administering of a kruskal-wallis test with a p-value <0.005 in order to address the research problem and test the hypothesis. results changes in the behavior of subjects between the initial and fourth meetings are presented in table 2. during the research, all subjects demonstrated varying degrees of behavioral change. subjects with level 1 asd proved capable of following all instructions, while of those with level 2 asd only two completed all the required steps. during the initial meeting, the subjects undertook different levels of tasks. one individual required more time before he/she was finally able to enter the observation room in a composed manner, while another sat calmly in the chair provided. three subjects were willing to wear a bib and one opened his/her mouth to allow the tool to be inserted, although with the power off. as for the remaining nine subjects, all were able to proceed to the prophylactic brushing stage. in general, positive behavioral change was identified during the second consultation with the exception of five subjects who demonstrated no change of any description. two subjects failed to present any behavioural change after transitioning from the second to the third consultation. the number of subjects capable of fully implementing the instructions increased from two to five during the third consultation. during the final consultation, eight subjects were able to complete their task effectively. overall, 12 subjects underwent dental cleaning with a prophylactic brush, although four underwent partial cleaning of specific areas of the oral cavity. as shown in table 2, only one subject failed to show any behavioral change after the third consultation. table 2. subjects’ behavioural change scores between 1st and 4th meeting asd level subject code 1st meeting score 2nd meeting score 3rd meeting score 4th meeting score level 1 (easy) a 8 11 11 11 b 7 7 9 11 c 7 11 11 11 d 7 9 11 11 e 7 9 11 11 f 8 9 11 11 level 2 (moderate) g 4 5 6 8 h 6 6 7 9 i 3 3 4 4 j 2 2 3 4 k 7 9 10 11 l 8 8 10 11 m 3 3 3 6 n 3 5 6 7 o 1 2 2 3 table 1. subject responses to instructions as assessment of behaviour no. instruction score 1 subject unwilling to enter treatment room 0 2 subject willing to enter room 1 3 subject willing to remain seated 1 4 subject willing to wear a bib 1 5 deactivated tool inserted in mouth 1 6 activated tool inserted in mouth, but brushing process cannot be performed 1 7 operator able to clean region 2 1 8 operator able to clean region 5 1 9 operator able to clean region 1 1 10 operator able to clean region 3 1 11 operator able to clean region 4 1 12 operator able to clean region 6 1 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p117–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p117-121 120 melati, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 117–121 based on the progression between the initial and final consultations, all subjects demonstrated varying degrees of positive behavioral change, while none showed behavioral retrogression. however, one subject was able to progress only one step further after the initial consultation. table 2 also contains the contrasting behavioral change of subjects with level 1 asd and those with level 2 asd. during the preliminary meeting, the subjects with level 2 asd proved capable of executing fewer instructions than their level 2 asd peers. all level 1 asd subjects were willing to open their mouths, thereby enabling the operator to perform dental cleaning, while only a trio of level 2 subjects accomplished this task. during the second meeting, two of the six level 1 asd subjects proved capable of performing the required steps. in the final meeting, however, all level 1 asd subjects accomplished these. unlike level 1 asd subjects, only two of their nine level 2 asd counterparts successfully followed the instructions. the statistical test results indicated a change in scores between the initial and the fourth consultations, with a p-value of <0.05 (0.026). as indicated by the scores achieved (p-value < 0.05), there was a statistically significant difference in behavioral change between level 1 and level 2 asd subjects of <0.05 (7.67e-06). discussion the initial aba procedure consisted of prompting which involved the use of picture cards to facilitate communication. this was followed by fading the objective of which was to reduce subject dependence on pictorial prompts. during the subsequent stage, referred to as shaping, instructions were repeated from the outset and the subjects were rewarded with complements or gifts. prophylactic brushing is a simple procedure appropriate for non-asd children. in contrast, for those with asd, the process can prove extremely complex. for this reason, the researcher adopted a chaining procedure. the prophylactic brushing procedure was broken down into several simpler instructional steps to facilitate subject comprehension and compliance. the researcher did not proceed to the next step until the subject were able to accomplish the current one. research has revealed that aba represents a potential approach to changing the behavior of children suffering from asd with regard to the use of prophylactic brushes for dental cleaning. all of the research respondents underwent contrasting degrees of behavioral change as indicated by the scores they achieved. the research findings were consistent with those of studies conducted by mochamant et al., and hidayatullah et al.11,12 foxx et al., (2016) even argued that aba technique represented the optimum approach whose effectiveness in changing the behavior of children with asd has been proven scientifically.13 the chaining process of breaking down dental procedures into several instructional steps was intended to facilitate the learning of new information and skills by asd children. chaining was employed because the human brain can more readily encode repeated short inputs in the synapses than process longer pieces of information that are provided only once. in addition to chaining, repetition is another important aspect of aba, while also constituting a basic learning principle.14,15 an aba approach is suitable for brain rewiring because it basically promotes continuous and consistent repetition of positive behaviour. behavioral change occurs because of the formation of new links between neurons during rewiring in the brain. which undergoes change by adapting to a novel activity or a repeated experience. behavioral change among children with asd varies because such individuals possess different levels of ability, intelligence, and performance.16–18 during the research, behavioral change occurred between the initial and fourth meetings, although not every subject presented this during every meeting. the number of subjects not experiencing behavioral change decreased with each consultation, but the number appeared to increase between the third and fourth consultation. this was because there were those who were able to complete the instructions in the course of that third meeting. in this case, non-occurrence of behavioral change was viewed in a positive light. generally, children with asd value stability and routine. altering the latter, therefore, requires careful preparation. individuals with level 2 asd tend to experience discomfort regarding any change to their daily routine. the situation differs in the case of children with level 1 asd who demonstrate greater flexibility in the face of change.19 one day prior to the consultation, the teacher was instructed to inform the subjects of a change in the following day’s schedule. he/she showed several picture cards and provided a brief explanation regarding the planned events of the subsequent day. the effective preparations of the teacher at lpa prananda rendered the subjects more tolerant of changes to their schedules. within the context of this research, the aba method applied involved the use of picture cards to facilitate communication. children with asd are generally good at processing visual information, but experience problems processing and interpreting verbal input. effective visual aids can, therefore, help to focus their attention on the task or instruction issued. pictures can both attract and retain the attention of such individuals. visual aids can also provide asd children with visually concrete tasks and instructions which support their processing of information. studies have proven the effectiveness of picture cards in introducing children to a new activity that is useful for their daily lives.10,19,20 picture cards can prove an effective tool in changing the behavior of children with asd . individuals with asd are capable of performing a task more effectively when provided with a visual cue rather than a verbal instruction. pictures support an improvement in their understanding by reducing dependence on abstract concepts and words. the researcher postulates that dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p117–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p117-121 121melati, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 117–121 individuals with asd tend to conceptualise visually owing to the intense level of activities in the parietal and occipital regions of their brain. the detailed and well-structured visual memory that they frequently demonstrate facilitates their adaptation to the environment.21 through the use of picture cards, the researcher was able to communicate instructions more effectively to the subjects. the majority failed to follow the verbal instructions issued during the initial meeting. however, when shown a set of cards with accompanying instructions, the subjects became more focused and were capable of following instructions. the length of time required to process information contained in a picture card varied from one subject to another. those with level 1 asd required less time to respond to and understand the content of a picture card. during the final meeting, only one of the subjects suffering from level 1 asd still required the use of visual aids. the others were able to follow verbal instructions effectively. in contrast to the subjects with level 1 asd, most of those suffering from level 2 asd needed to use visual aids up to and including the final meeting, the exception being respondent l. according to the teacher, this individual demonstrated a high level of compliance and extreme passivity in his/her daily life. the researcher used one picture card for each instruction step because, based on the findings of a study conducted by hidyatullah, subjects with asd would become confused when presented with two different picture cards for the same instruction. another difference from the picture cards used by hidayatullah was that, in this study, the picture operator donned a mask and gloves.12 this approach was adopted as a means of rendering the subjects more familiar with the researcher and understanding fully that he/she would clean their teeth with a prophylactic brush. one factor influencing the success of care is the level of recognition of the operator demonstrated by the subject.11 consequently, it is recommended that each subject is handled by a single operator. exposure to a photograph of the researcher proved effective because the subjects, particularly those with level 2 asd, became more cooperative when shown the photograph again and informed that this individual would perform the dental cleaning. the use of a photograph of the researcher as a form of introduction is an approach also adopted in other studies.1,22,23 it was concluded that aba has been proven to be an effective approach to changing its sufferers’ behaviour with regard to prophylactic brushing. the research found that subjects with level 1 asd experienced different degrees of change compared to those with level 2 asd. references 1. udhya j, varadharaja mm, parthiban j, srinivasan i. autism disorder (ad): an updated review for paediatric dentists. j clin diagnostic res. 2014; 8(2): 275–9. 2. delli k, reichart pa, bornstein mm, livas c. management of children with autism spectrum disorder in the dental setting: concerns, behavioural approaches and recommendations. med oral patol oral cir bucal. 2013; 18(6): e862–8. 3. nagendra j, jayachandra s. autism spectrum disorders: dental treatment considerations. j int dent med res. 2012; 5(2): 118–21. 4. chan dfy, chan shy, so hk, li am, ng rcm, tsang n. dental health of preschool children with autism spectrum disorder in hong kong. hong kong j paediatr. 2014; 19(3): 161–8. 5. sewell j. treating patients with autism in a dental setting. dentalcare. com; 2017. p. 1–37. 6. jaber ma. dental caries experience, oral health status and treatment needs of dental patients with autism. j appl oral sci. 2011; 19(3): 212–7. 7. carter ae, carter g, george r. autism spectrum disorders and the role of general dental practitioners: a review. j dent appl. 2015; 2(7): 254–60. 8. stein li, lane cj, williams me, dawson me, polido jc, cermak sa. physiological and behavioral stress and anxiety in children with autism spectrum disorders during routine oral care. biomed res int. 2014; 2014: 1–10. 9. sawai ma, bhardwaj a, jafri z, sultan n, daing a. tooth polishing: the current status. j indian soc periodontol. 2015; 19(4): 375–80. 10. hernandez p, ikkanda z. applied behavior analysis: behavior management of children with autism spectrum disorders in dental environments. j am dent assoc. 2011; 142(3): 281–7. 11. al mochamant i-g, fotopoulos i, zouloumis l. dental management of patients with autism spectrum disorders. balk j dent med. 2015; 19: 124–7. 12. hidayatullah t, agustiani h, setiawan as. behavior managementbased applied behavior analysis within dental examination of children with autism spectrum disorder. dent j (majalah kedokt gigi). 2018; 51(2): 71–5. 13. foxx r, mulick ja. controversial therapies for uutism and intellectual disabilities. 2nd ed. new york: taylor & francis; 2016. p. 256. 14. fisher ww, piazza cc, roane hs. handbook of applied behavior analysis. philadelphia: the guilford press; 2011. p. 221. 15. wirebring lk, wiklund-hörnqvist c, eriksson j, andersson m, jonsson b, nyberg l. lesser neural pattern similarity across repeated tests is associated with better long-term memory retention. j neurosci. 2015; 35(26): 9595–602. 16. granpeesheh d, tarbox j, dixon dr. applied behavior analytic interventions for children with autism: a description and review of treatment research. ann clin psychiatry. 21(3): 162–73. 17. andrzejewski me, mckee bl, baldwin ae, burns l, hernandez p. the clinical relevance of neuroplasticity in corticostriatal networks during operant learning. neurosci biobehav rev. 2013; 37(9): 2071–80. 18. sullivan k, stone wl, dawson g. potential neural mechanisms underlying the effectiveness of early intervention for children with autism spectrum disorder. res dev disabil. 2014; 35(11): 2921–32. 19. hume k, sreckovic m, snyder k, carnahan cr. smooth transitions: helping students with autism spectrum disorder navigate the school day. teach except child. 2014; 47(1): 35–45. 20. nirahma cp, yuniar ic. metode dukungan visual pada pembelajaran anak dengan autisme. j psikol klin dan kesehat ment. 2012; 1(2): 1–8. 21. jiang y v., palm be, debolt mc, goh ys. high-precision visual long-term memory in children with high-functioning autism. j abnorm psychol. 2015; 124(2): 447–56. 22. breslin cm, rudisill me. the effect of visual supports on performance of the tgmd-2 for children with autism spectrum disorder. adapt phys act q. 2011; 28(4): 342–53. 23. chandrashekhar s, bommangoudar js. management of autistic patients in dental office: a clinical update. int j clin pediatr dent. 2018; 11(3): 219–27. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p117–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p117-121 isi vol 39 no 3 juli-september 2006.pmd 89 is it possible to distinguish the understanding of denture adhesive between japanese dental students and indonesian peers by a questionnaire? shinsuke sadamori,* taizo hamada,* guang hong,* makoto kawamura,** nobuyuki nakai,* and arifzan razak*** * department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, hiroshima, japan ** preventive dentistry, hiroshima university hospital *** department of prosthodontics, faculty of dentistry, airlangga university, surabaya, indonesia abstract the purpose of this study was to compare cross-national differences in the recognition of denture adhesive between dental students. the design of the research was cross-cultural differences. the research was done in japan and indonesia. seventy-seven dental students from japan and indonesia were surveyed using a questionnaire regarding knowledge/comprehension of denture adhesive (in japanese and indonesian versions respectively). logistic regression using the wald method showed that it was possible to distinguish japanese dental students from indonesian peers with a probability of 96.1 per cent by using 3 items out of 12. for the question of “how many domestic products denture adhesive do you know?” 85 per cent of the japanese dentists answered “less than 3”, whereas 10 percent of indonesian subjects did so. it was concluded that there were big differences between japanese and indonesian dental students’ understanding and experience of denture adhesive. key words: denture adhesive, cross-national differences, dental students, questionnaire, japan, indonesia correspondence: dr shinsuke sadamori, department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, 1-2-3 kasumi, minami-ku, hiroshima, 734-8553, japan, tel: +81-82-257-5681, fax: +81-82-257-5684, e-mail: tsada@hiroshima-u.ac.jp according to forss and widström’s survey,12 the patients’ opinions had a strong influence on selection of restorative materials. the differences in the use of da between the two countries might be due to a difference in need of the patient, and/or in understanding the disadvantage of da. ozcan et al.13 stated that denture adhesive should be taught more intensively at dental school. as dental students have not enough knowledge about dental services, tv commercials about da may have an effect on awareness and interest of dental students. hence, it is interesting to know whether or not the similar results would be obtained between japanese and indonesian dental students. the purpose of this study was to clarify dental students’ recognition of denture adhesive between the two countries and to examine if there were differences in their understanding of denture adhesive (factors determined by their nationality). materials and methods in this study, 77 subjects were asked to complete and return a structured questionnaire. the subjects in this pilot study were selected from undergraduate dental students in japan (hiroshima university, hiroshima) and indonesia (airlangga university, surabaya). they included 47 students of fourth-year at hiroshima university and 29 introduction nowadays, there seems to be many differences among countries in the use of denture adhesive. grasso1 reported that 75% of dentists have recommended the use of denture adhesives. in general, denture wearers’ attitude toward denture adhesives is likely to be favorable: better retention of their dentures; more comfortable when chewing and speaking with denture adhesives than without. 2,3 meanwhile, opinions about denture adhesives have not been consent among dental professionals yet: 1) negative attitudes; prolongation of a wearing period of ill-fitting dentures;4 allergens and irritants to denture-bearing tissues;5,6 2) positive attitudes; prevention of food particles impaction under the denture, reduction of unfavorable mechanical irritation, and improvement in denture stability and retention.7–11 a recently published study reported that there were many differences in understanding and experience of denture adhesive in the clinic between japanese and indonesian dentists. japanese dentists had more information about denture adhesives. however, in the clinic, indonesian dentists tended to apply denture adhesive (da) to patients more often than japanese dentists did. the japanese dentists did not tend to apply denture adhesive to their patients in spite of having the opportunity to see tv commercials about such application. 90 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006: 89–92 students of fifth-year at airlangga university. mean age of the japanese students was not statistically significant compared to that of indonesian peers (23.6 and 23.1 yrs respectively). in the survey, the distribution and collection of the questionnaire was instituted by the staff of this survey in 2002. this questionnaire was administered after an explanation was given to all subjects about the aim of this survey, and understanding and consent from all subjects was gained. the questionnaire had been in japanese and then was translated into english. then, it was discussed among the staff at airlangga university, and this survey was instituted using the same estimation criterion between the two countries (in japanese and indonesian version respectively). the answers were evaluated in three steps of “no” or “nothing” (score 0), “yes, but a little” or “occasionally” (score 1) and “yes, very much” or “often” (score 2). logistic regression analysis was carried out on the dependent variable (country). the wald statistic was used to test the null hypothesis that the regression coefficients were zero. the nagelkerke r2 was used to discriminate how well the model is able to distinguish between the interest and knowledge of dental students in the two countries. all analyses were computed using spss for windows operating system (spss 10, spss japan inc., tokyo, japan). results the recovery rate was 98.7 per cent: hiroshima university 100 per cent, airlangga university 96.7 per cent. questionnaire items and percentage distribution of responses were shown in table 1. compared with indonesian students, more japanese students not only knew da but also had a clear understanding of its purpose and directions for its use (q1 and 2). the proportion of students who knew imported da products was higher in indonesia, while the proportion of students who knew domesticallyproduced da was higher in japan (q4 and 5). students in both countries were similarly educated about da in dental school or elsewhere (q6 and 7). japanese students also recognized da in tv commercial messages more often than indonesian students (q8). the proportion of students who were familiar with alternative products of da was slightly higher in japan, while more indonesian students were familiar with da in the clinic (q9, 10). there was a significant difference in the knowledge of the therapeutic effects of da between students in both countries (q11). as to the price of da, more indonesian students believed that it was reasonable than did japanese students (q12). table 2 shows the estimated coefficient and related statistics from the logistic regression model that predicts group membership. the model contained three variables. table 1. questionnaire items and percentage distribution of the answers by country category score item descriptions 2 1 0 p q1. q2. q3. q4. q5. q6. q7. q8. q9. q10. q11. q12. do you know the denture adhesive? do you know any purposes of the denture adhesive? do you know any disadvantages of the denture adhesive? how many imported products of denture adhesive do you know? how many domestic products of denture adhesive do you know? have you ever been taught about the denture adhesive? have you ever seen the denture adhesive in books or lecture meetings? have you ever seen any tv commercials about the denture adhesive? do you know any goods instead of the denture adhesive? have you ever seen the denture adhesive in the clinic? do you think the use of denture adhesive is more effective than medical intervention such as relining? do you think the price of denture adhesives is reasonable? a a a b b c c c a c a a jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina jpn ina 30 3 43 7 15 24 0 41 15 90 19 3 26 38 62 79 4 90 4 17 9 24 2 86 68 79 57 72 77 72 13 59 74 10 79 90 66 62 34 3 28 10 17 72 60 66 66 14 2 17 0 21 9 3 87 0 11 0 2 7 9 0 4 17 68 0 79 10 32 10 32 0 ** *** ns *** *** ns ns ** *** *** * *** a 2: yes, very much, 1: yes, but a little, 0: no b 2: three and more, 1: less than three, 0: nothing c 2: yes, often, 1: yes, occasionally, 0: no * : p < 0.05, **: p < 0.01, ***: p < 0.001, ns: not significant 91sadamori et al: is it possible to distinguish the understanding of denture adhesive by forward stepwise method (p<0.01):q2 (understanding of purposes of the use of da), q9 (knowledge of goods instead of da), q4 (number of imported products of da). table 3 showed that 45 japanese students (95.7%) and 28 indonesian students (96.6%) were correctly predicted by the model. the nagelkerke r2 statistic was 0.907; that is, 90.7 per cent of the variation in the outcome variable was explained by the model. also, the difference in the economic power between the countries would have affected their answers. in addition, the answers to the questions regarding imported and domestic da products may have reflected the difference in power of da production between the two countries. the results obtained in response to q5 and q8 could be due to the fact that mass media is more developed in japan than in indonesia. mass media about da in indonesia is unfortunately less developed than in japan, but, to take the converse point of view, it is anticipated that less harm would be exerted from mass media influence. in a study of diffusion of innovation it was reported that information flowed more directly from mass media while influences indirectly via personal communication (by health/care personnel et al.).14 the diffusion rate varies depending on the degree of “relative advantage”, such as economic profitability, alleviation of discomfort, retrenchment of time and labor, and immediacy of benefit. in actual clinics, patients often complain of instability of dentures, insufficient maintenance power, and so on. if we take these circumstances into consideration, an explosive diffusion of da in the general public, without the expert’s remarks, would be anticipated. in japan, mass media is more developed in comparison with indonesia, and it offers the appropriate information service from the specialist in mass media. we are in an age when medical care goods (such as denture adhesive) are diffused via tv and the internet. in days ahead it is necessary to send out accurate information via tv and the internet. students should be learned da based on evidence more properly in dental school, and the information of da might be provided for the people from specialists. using only 3 items out of 12 (q2: understanding of purposes of the use of denture adhesive, q9: knowledge of goods other than denture adhesive, and q4: number of imported products of denture adhesive) out of 12, nationality of the dental students in japan and indonesia was almost correctly predicted (96.1 per cent) by the model. acknowledgements acknowledgements this study was supported in part by grant-in-aid for scientific research (grant no.16390617) from japan society for the promotion of science. discussion using only 3 items regarding da out of 12, nationality of the dental students in japan and indonesia was almost correctly predicted (96.1 per cent) by the model. it was inferred that not only education of da in dental school but also information of da in tv commercial messages was more prevalent in japan. it is assumed to be important to teach particularly the advantages and disadvantages of denture adhesive in professional education. it is a problem that students learn da from tv instead of lectures in dental school. a lower proportion of japanese students thought that the price of da was reasonable, while almost all of the indonesian peers appeared to feel it reasonable. in our previous study, there was no difference in the answers between dentists in both countries. since students in either country equally seemed to have no clinical experiences of application of da and to have not realized its effects, this difference could be ascribed to the differences in information obtained mainly from mass media such as tv. table 2. results of binary logistic regression analysis using wald method (forward stepwise) item no. b s.e. wald chisquare freedom p exp (b) forward stepwise (wald) q2: understanding of purposes of the use of da* q9: knowledge about goods instead of da* q4: number of imported da* -4.90 1.98 3.31 1.36 0.84 1.28 12.90 5.58 6.74 1 1 1 0.000 0.018 0.009 0.01 7.24 27.44 * denture adhesive variables were entered in steps 1 to 3 q2, q9, q4 in that order. table 3. observed and predicted group membership using wald method predicted country country japan indonesia percentage correct forward stepwise (wald) japan indonesia total 45 1 2 28 95.7 96.6 96.1 the cut value is 0.50 nagelkerke r2 = 0.907 92 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006: 89–92 references 1. grasso je. denture adhesives: changing attitudes. j am dent assoc 1996; 127:90–6. 2. kelsey cc, lang br, wang rf. examining patients’ responses about the effectiveness of five denture adhesive pastes. j am dent assoc 1997; 128:1532–8. 3. benson d, rothman rs, sims tn. the effect of a denture adhesive on the oral mucosa and vertical dimension of complete denture patients. j south calif dent assoc 1972; 40:468–73. 4. instruments and equipment (us) council on dental materials. dentist’s desk reference. chicago: american dental association; 1981. p. 422–33. 5. heatwell cm, rahn ao. syllabus of complete dentures. 3rd ed. philadelphia: lea & febiger, 1980; p. 98. 6. hogan w. allergic reaction to adhesive denture powders. ny state dent j 1954; 20:65–6. 7. adisman ki. the use of denture adhesive as an aid to denture treatment. j prosthet dent 1989; 62:711–5. 8. boone m. analysis of soluble and insoluble denture adhesive and their relationship to tissue irritation and bone resorption. compend cont educ dent 1984; 4(supplement):22–5. 9. karlsson s, swartz b. effect of denture adhesive on mandibular denture dislodgment. quintessence int 1990; 21:625–7. 10. grasso je, rendell j, gay t. effect of denture adhesive on the retention and stability of maxillary dentures. j prosthet dent 1994; 72:399–405. 11. sadamori s, hamada t, hong g, nakai n, kawamura m, razak a. comparison of the recognition of denture adhesive between japanese and indonesia dentists: a pilot study. maj. ked. gigi (dent j) 2005; 38(4):189–93. 12. fors n, widstrom e. factors influencing the selection of restorative materials in dental care in finland. journal of dentistry 1996; 24:257–62. 13. ozcan m, kulak y, arikan a, silahtar e. the attitude of complete denture wearers towards denture adhesive in istanbul. j oral rehabil 2004; 31:131–4. 14. rogers em. diffusion of innovations. 3rd ed. new york: the free press; 1983. << 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� vol. 43. no. 1 march 2010 human-leukocyte antigen typing in javanese patients with recurrent aphthous stomatitis diah savitri ernawati, bagus soebadi, and desiana radithia departement of oral medicine faculty of dentistry, airlangga university surabaya indonesia abstract background: rec�rrent a��t���s st��atitis (ras) is a c����n �ral dis�rder t�at des�ite e�tensive researc�es, t�e eti�l�gy �� t�is ��en��en�n is still �nkn���n. beca�se t�is ��en��en�n �as �een ��served ��re ��ten in �a�ilies t�an in individ�al cases, genetic in�l�ence �as �een investigated in ��st researc�es. purpose: ��e ai� �� st�dy ��as t� eval�ate t�e ass�ciati�n �et��een ���an le�k�cyte antigen (hla) and ras in javanese ��re �recisely. method: ��e analysis �� hla-a, and hla-b in 85 javanese ras �atients and 71 �ealt�y c�ntr�l s��jects, ��ere �er��r�ed �y �sing t�e standard n�h �icr�ly���cyt�t��icity tec�niq�e. ����n��ist�c�e�istry ��as �er��r�ed ��r identi�icati�n �� hla-dr and hla dq antigen �sing ��n�cl�nal anti��dies anti hla-dr and dq. result: o�r res�lt revealed a cl�se ass�ciati�n �et��een hla-a9 and hla-b35 ras s��ject. a signi�icant increase in t�e �req�ency �� s��e antigens s�c� as hla-a9 (72,94%, � < 0,05;rr = 2,21), hla-a24 (65,82%; rr = 1,24) and hla-b35 in s��jects ��it� ras ��as ��served. analysis ��it� ����n��ist�c�e�istry hla-dr, hla-dq is e��ressed �n t�e s�r�ace �� e�it�elial cells �e��rane �� �ral ��c�sa and �acr���ages in ��t� �aj�r and �in�r ras �atients. conclusion: hla antigens are inv�lved in s�sce�ti�ility t� ras and t�e ��en�ty�es ��ere di��erence ��it� �t�er �revi��s st�dies. hlalinked genetic �act�rs �ay �lay a r�le in t�e devel���ent �� ras. key words: h��an le�k�cyte antigen, rec�rrent a��t���s st��atitis, �ral ��c�sal e�it�eli�� abstrak latar belakang: st��atitis a�t�sa rek�ren (sar) �er��akan sala� sat� gangg�an di r�ngga ��l�t yang �aling sering terjadi. fen��ena �enyakit ini �asi� �el�� jelas dan �asi� �e���t��kan �enelitian yang le�i� lanj�t. fakt�r ket�r�nan le�i� sering dari�ada kas�s individ�al. pengar�� �akt�r genetik tela� diteliti �le� �e�era�a �eneliti. tujuan: ��j�an �enelitian ini �nt�k �engeta��i adanya kaitan hla dengan sar �ada s�k� ja��a secara le�i� te�at. metode: analisis hla-a, hla-b �ada 85 �enderita ras dan 71 �enderita k�ntr�l yang �erasal dari s�k� ja��a di�it�ng dengan �engg�nakan teknik n�h �icr� ly����cyt�t��icity. �eknik ���n��ist�ki�ia dilak�kan �nt�k �engidenti�ikasi antigen hla–dr, hla dq dengan �engg�nakan anti��di ��n�kl�nal hla-dr & dq. hasil: �en�n�nj�kkan ����ngan yang k�at antara hla–a9 dan hla-b-35 �ada �asien sar. �erda�at �eningkatan yang signi�ikan dari �e�era�a antigen se�erti hla-a9 (72,94%, � < 0,05, rr = 2,21), hla–a24 (65,82%, rr = 1,24) dan hla–b35 �ada �asien sar yang di ��servasi. analisis dengan ���n��ist�ki�ia ta��ak hla–dr, dq dieks�resikan �ada �er��kaan �e��ran sel dan �akr��ag �ada �asien sar �ay�r �a���n �in�r. kesimpulan: antigen hla terli�at dengan ke�ekaan terjadinya ras, dan �en�ti�nya �er�eda dengan �asil �enelitian se�el��nya hla dan �akt�r genetik �er�eran �enting �ada terjadinya sar. kata kunci: h��an le�k�cyte antigen, st��atitis a�t�sa rek�ren, �ral ��c�sal e�it�eli�� c�rres��ndence: diah savitri ernawati, c/o: departemen oral medicine, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: savitri_glx@yahoo.com research report ��ernawati, et al.: human-leukocyte antigen introduction recurrent aphthous stomatitis (ras) is the most common inflammatory ulcerative condition of the oral mucosa. the lesions are localized, painful, shallow ulcers typically on nonkeratinized or poorly keratinized mucosa, often covered by a gray fibro membranous slough and surrounded by an erythematous halo. a recurrence rate of 1 outbreak every 1 to 3 months is considered typical. sites of predilection include the ventral surface of the tongue, the floor of the mouth, the buccal, labial, soft palatal, and oropharyngeal mucosa. the three main clinical types of ras are minor (80% of all ras), major and herpetiform ulcers. however, the significance of these distinctions is unclear, as they could be three distinct disorders. the etiopathogenesis of ras is not entirely clear, with many possible predisposing factors, including trauma, emotional stress, hormonal state, food hypersensitivity, viruses, bacteria, and immune dysregulation. evidence suggests a cytotoxic effect of peripheral-blood lymphocytes toward oral epithelial cells.1–3 genetic influences may play a role in the etiology of ras, because hla-b12 and hla-b51 has been shown to have an increased prevalence in ras, and hla-b5 is also increased in the closely related behçet disease.4 in addition, gallina reported that hla-dr7 was significantly decreased, however, another study has reported that there was no association between ras and hla class i antigen.5 these discordant result might be attributable to different ethnic background and/or disease heterogenity.with the aim of investigating whether or not the gene coding for hla antigens gene may affect the development of ras, we studied the hla class i antigen (hla-a and hla-b) and hla-dr, and dq in sample of healthy indonesian affected by minor forms of ras and compared it with a normal indonesian population, which have no history of the disease. material and method eighty-five subjects (23 men and 62 women), ranging in age from 10 to 59 years affected by ras, as clinically determined by the methodes of lehner,6 were typed for hla antigens. we included in the group under study only patients who have periodic ulcers, with no less than three recurrences appearing during 1-year period, and because of the high frequency of ras in the normal population (more than 20% of all persons are periodically affected by ulceration) we used a panel of 71 control subjects,who gave no history of ras (30 men and 41 women ranging in age from 19 to 59 years). affected subject and controls were javanese. peripheral blood lymphocyte were separated on a ficol-hypaque density gradient. hla-a, hla-b antigens were determined for 88 sera (one lambda) performed by the standard two stage national institutes of health (nih) microlymphocytotoxicity technique.7 peripheral blood was collected from each patients and lymphocytes were separated by ficoll hypaque gradient centrifugation for typing of class i antigens. for immunohistochemistry single immunoenzyme staining was performed by the biotin-streptavidinperoxidase method with the antibodies (from biosciencees) and the specificity of the antibodies was confirmed by replacing each with the respective isotype control. (to quantitate the infiltration of tissue by hla-dr,dq positive cells, light microscopy images were acquired with a nikon eclipse e600 microscope equipped with a color high resolution charge-coupled device ccd camera.8 scrapped specimen oral epithelial biopsy in oral mucosal and fixed on to object glass with alcohol 90% (15 minutes), and incubated in refrigator or directly blocked with bovine serum albumin 1% (bsa 1%) for 15 minute then incubated in co2 at the temperature 37° c for 45 minutes. after being washed by pbs, sample is reacted with monoclonal antibodi hla, anti hla-dr and hla-dq, reincubated in table 1. the profile of hla-a antigen in patients with recurrent aphthous stomatitis and control subject antigen hla patients (n = 85) control (n = 71) rr no % no % a1 a2 a3 a9 a10 a11 a19 a24 a28 a32 a33 a34 16 22 0 62 17 14 8 56 10 0 7 0 10.76 18.82 0 72.94 20 16.47 9.41 65.82 11.76 0 8.23 0 0 21 6 39 18 36 3 45 0 1 9 2 0 20.92 8.45 54.92 25.35 50.70 4.22 63.38 0 1.40 12.67 2.81 0 0.83 0 2.21 1.96 0.19 2.35 1.12 0.6 �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 26-30 co2 incubator at 37° c for one hour. after being washed by pbs, the sample was analyzed using immunofluorescent microscope with 40x magnified. to evaluate the association of class i hla antigens with ras, fisher’s exact test was employed (case in the small group < 5). relative risk, (rr) was evaluated by the formula (p+x c-): (p– x c+). where p+ or p– denotes the number of affected subjects positive or negative for specific antigen and c+ or c– denotes the number of controls positive or negative.9 result distribution of the frequences of representative class i antigens in patients with ras and the controls are shown in table 1 and 2 which consits of 12 alleles of hla-a and 20 alleles of hla-b. as can be seen from table 1, the frequency of hla-a9 increased in ras (72.94%) compared with the controls (54.92%), p value was 0.02 and relative risk was 2.21. conversely, hla a-11 deccreased (16.47%) compared to 50.70% in control subject, with p 0.02 and rr was 0.19. table 1 also shows that hla-a24 was significantly increased with rr 1.12. in the locus b the frequency of hla-b35 (34.1%) in ras patients significantly greater than the healthy control subject with p = 0.2 and relative risk (rr) was 1.525. however the frequency of hla-b15 decreased (41.2%) compared to 50.70% in control subject, with p = 0.261 and rr = 0.681. the phenotype frequencies of hla-a in 85 patients and 71 healthy control subjects are showed in table 1. we found that the phenotype frequency of hla-a9 (72.94%, p = 0.02, rr: 2.21) and hla-a24 (65.82%, p: 0.86; rr: 1.12) in ras patients was significantly greater than the phenotype frequency in healthy control subjects. however, the phenotype frequency of hla-a11 (16.47%, p = 0.0, rr = 0.19) in ras patients was significantly lower than the phenotype frequency in healthy control subjects. the hla phenotype frequencies of hla-b antigen in 85 ras patients and 71 healthy control subject are showed in table 2. we found that the phenotype frequency of hlab35 (34.1%) in ras patients significantly greater than the healthy control subject with p value was 0.2 and relative risk (rr) was 1.25. however the frequency of hla-b15 decreased (41.2%) compared to 50.70% in control subject, with p = 0.261 and rr = 0.681. a study has been conducted to 34 and 51 patients with major and minor ras, respectively, and to 30 non-ras patients as control in order to identify the presence of hla-dr, dq antigen in epithelial cells and macrophage of patients with (ras). this study revealed that hla-dr and hla-dq were expressed at the surface of epithelial cell membrane of oral mucosa and macrophage in both major and minor ras patients (figure1 & figure 2). hla-dr and dq is not expressed specifically in non-ras patients. table 2. the profile of hla-b antigen in patients with recurrent aphthous stomatitis and control subject antigen hla patients (n = 85) control (n = 71) rr no % no % b5 b7 b12 b13 b14 b15 b16 b17 b18 b21 b24 b27 b35 b40 b41 b44 b51 b60 b61 b63 6 3 16 9 3 35 7 6 3 0 0 16 29 3 1 3 4 3 0 11 7.05 3.52 18.82 10.58 3.52 41.18 8.23 7.05 3.52 0.52 0.52 18.82 34.11 3.52 1.17 3.52 4.70 3.52 0.52 12.94 9 12 0 6 0 36 6 9 3 3 1 0 18 3 0 0 0 6 1 3 12.60 16.90 0.52 8.45 0.52 50.70 8.45 12.60 4.22 4.22 1.40 0.52 25.35 4.22 0.52 0.52 0.52 8.45 1.40 4.22 0.52 0.17 1.28 0.68 0.97 0.52 0.82 1.13 0.82 0.39 7.36 ��ernawati, et al.: human-leukocyte antigen figure 1. hla-dq expression in this ras patients case is not well distributed in all cells, either at the cells were expressed at the surface of epithelial cell membranes of oral mucosa and macrophage. figure 2. hla-dr expression at surface epithelial cells membran in the oral mucosal mayor and minor ras patients reacted with hla dr monoclonal antibody. ras mayor and minor visualized with dab chromogen. discussion the ras lesions are usually noted in childhood or adolescence and recur with decreasing frequency and severity with age. the prevalence of ras varies from 5 to 66% in the general population. women are affected more commonly than men. lesions are classified into 3 groups: minor, major, and herpetiform ulcers. minor aphthous ulcers are most common, less than 1.0 cm, and resolve without scarring in 1 to 2 weeks. major aphthous ulcers are less common, usually greater than 1.0 cm, and deeper, and they heal slowly in 10 to 30 days with scarring. herpetiform ulcers are the least common variant, with numerous 1to 2 mm grouped ulcers that coalesce and heal in 7 to 30 days.1–3 the cause of ras is still unknown with many possible predisposing factors, including trauma, emotional stress, hormonal state, food hypersensitivity, bacteria, viruses and immune dysregulation. the ras may be the manifestation of a group of disorders of quite different etiology, rather than a single entity. immune mechanisms appear at play in persons with a genetic predisposition to oral ulceration. possible predisposing factors seen in a minority include trauma, hematinic deficiency, emotional stress, hormonal state, food allergies, and human immunodeficiency virus infection.1–3 in this study, the hla phenotype frequencies in ras patients were determined and compared with those in healthy control subjects. we found a significant increase in the phenotype frequency of hla-a9, hla-a24, hlab35 and hla-b15 in ras patients compared with the corresponding phenotype frequencies in healthy control subject. similar finding of a positive hla association with ras have also been reported by others.10 the prevalence hla-b51 in patients with ras was higher than control subjects, that in other studies was not increased,11 in our study, the prevalence was similarly low to that of healthy controls. analysis of hla antigens and associated disease is to examine the increase or decrease frequencies of the various hla markers in affected population. previous studies indicated there were not consistent differences in the frequency of hla antigens in patients with ras and controls. high frequency and relative risk of hlaa9 in ras subject were observed in this study. the high frequency of hla-a24 seems to be ralated with the increasing of hla-a9 since the hla-a24 allele is the subsets of hla-a9. our study demonstrated a significant association between ras and hla-a9 that might be involved in immunopathogenesis of ras. the hla-a9 antigen is not only the important contributor to development of ras in area in which the disease is prevalent, but also related to the severity of ras. furthermore the existence of hla-a11 in control subject might be contributes to the protective effect but this result need to be investigated. further, since both hla-a9 and hla-a11 alleles were detected in some individuals who do not have ras history. if hla-a9 and hla-b35 would be the most important gene for the development of ras, our result may support the role of environmental factor in persons having specific genetic background. expression of hla-dr and hla-dq determined by immunohistochemistry in oral mucosal epithelial cells of ras major patients. our result showed that most oral mucosal epithelial cells specimens expressed hla-dr and hla-dq weakly. this indicates that hla-dr and hladq might induce the occurance of ras which could be detected by expression hla-specific ras whether locally and sistemically. it has been proved immunohistochemically that hla-dr and dq can be detected at the surface of oral mucosal epithelium and cytoplasm of ras patients. hla localization has been widely related with immune cells and inflammation. epithelial cell in oral mucosa may related with many potential pathogenes, and hla expression will be relevant with immunity of oral mucosa. epithelium is the primary target of infectious agents. therefore, these epithelial cells play a pivotal role in inflammation (production of various cytokines and pro inflammatory cytokines).12 it is concluded that hla-dr and hla-dq has been expressed at the surface of cell membrane and macrophage in minor and major ras. hla specific ras was more �0 dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 26-30 predominantly expressed in major ras compared to minor ras. functional hla expression by oral mucosal epithelial cells had higher implications towards natural immune response and disease pathogenesis. it is suggested to undertake molecular characterization to determine specific hla against specific disease agents, so that it will be easy to identify the causing agent, with the result that ras disease management can be established comprehensively. acknowledgments this research are supported by hibah kompetensi dp2m (direktorat penelitian dan pengabdian masyarakat) ditjen dikti depdiknas 2009. references 1. robinson nd, gultrat j. recalcitrant. recurrent aphthous stomatitis with etanercept. arch dermatol 2003; 139: 1259–63. 2. jurge s, kuffer r, scully c, porter sr. recurrent aphthous stomatitis. oral dis 2006; 12: 1–21. 3. scully c, porter s. oral mucosal disease: recurrent aphthous stomatitis. british journal of oral and maxillofacial surgery 2008; 46: 198–206. 4. riggio mp, lennon a, wray d. detection of helicobacter pylori dna in recurrent aphthous stomatitis tissue by pcr. j oral pathol med 2000; 29: 507–13. 5. platz p, ryeder lp, donatsky o. no deviation of hla-a and b antigens in patients with recurrent aphthous stomatitis. tissue antigens 1996; 8: 279–80. 6. lehner t. immunology and autoimmun disorder of the oral mucosa immunological and autoimmun disorders of the oral mucosa 3rd ed. london: blackwell scientific publication; 1992. p. 150–7. 7. terasaki pi. micro droplet testing for hla-a, b and c and d antigen. am j of clin 1978; 69: 103–10. 8. ernawati ds. expression of tlr-2 and tlr-4 protein in the epithelial cells of the oral mucosal patients with recurrent apthous stomatitis (ras). proceeding of the international seminar on pharmacy, 2007. p. 45. 9. wilbelmsen nsw, weber r, monteiro f, kalil j, miziara id. correlation between histocompatibility antigens and recurrent aphthous stomatitis in the brazilian population. braz j otorhinolaryngol 2009; 75(3): 426–31. 10. shohat-zabarski r, kalderon s, klein t, weinberger a. close association of hla-b51 in person with ras. oral surgery oral med oral pathol 1992; 74: 455–8. 11. chang hk, kim ju, chung hr, lee kw, lee ih.hla-b51 and its allelic types in association with behcet’s disease and recurrent aphthous stomatitis in korea. clinical and experimental rheumatology 2001; 19(suppl 24): s31–s35. 12. lewkowicz n, lewkowicz p, kumatowska a. innate immune system is implicated in recurrent aphthous ulcer pathogenesis. j oral pathol med 2003; 32: 475–81. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none 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0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base isi vol 39 no 2 april 2006 file pertama.pmd 43 the density of collagen fiber in alveolus mandibular bone of rabbit after augmentation with powder demineralized bone matrix post incisivus extraction regina tc. tandelilin*, abdul salam m sofro**, al supartinah santoso*, marsetyawan hne soesatyo***, and widya asmara**** **** faculty of dentistry, gadjah mada university, yogyakarta **** faculty of medicine, yarsi university, jakarta **** faculty of medicine, gadjah mada university, yogyakarta **** faculty of veterinary medicine, gadjah mada university, yogyakarta abstract the bone defect due to tooth extraction contributes the most cases reported in the aspects of oral surgery. the defect can be preventively managed by adding powder bone matrix intended for augmentation which eventually induces the formation of new bones. this hard tissue wound healing is preceded by the presence of collagen fibers. the aim of this study was to determine the density of collagen fiber in the alveolus mandibular bone of rabbit which was augmented using powder demineralized bone matrix (dbm) post incisivus extraction. twenty four male rabbits aged 2.5–3 months weighed 900–1,100 grams were randomly divided into two groups. the treated rabbits were augmented with dbm after the incisivus extraction on mandible. the mucosa was then sutured. on the other hand, the controlled rabbits received similar treatments with those of the treated rabbits except there was no augmentation of dbm. decapitation of treated and controlled rabbits was made on day 5, 7, 10, and 14 days post surgery, each with three rabbits. mandibles were cut, decalcified, and imbedded in paraffin block. the staining was done using mallory. significant differences in the density of collagen were noted on day 10 and 14 post surgery, indicating that powder demineralized bone matrix successfully induced the stimulation of collagen. key words: demineralized bone matrix, augmentation, collagen correspondence: regina tc. tandelilin, c/o: fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii, sekip utara yogyakarta 55281. introduction the advancement of science in developing tissue transplantation has triggered an increase in the need for tissue replacement, so-called graft. the graft is required for either reconstruction or preventing further tissue deterioration on damaged body tissues. it has been reported that the abnormality of bone in the aspect of oral surgery is bone defect, in most cases engendered by tooth extraction.1 bone grafting is often essential in providing the best possible set of conditions for the anchorage of implants or augmentation in areas lacking in quality or quantity of bone to induce bone formation.2 there are heaps of different materials available for the augmentation of the alveolar bone. freeze dried powder demineralized bone graft or demineralized bone matrix (dbm) is a bone that has been treated with acid, then washed and followed by sterilization using γ (gamma) radiation to help extend shelf life. the utilisation of that type of allograft was reported effective with less risk of complication.3 previous studies show that dbm is able to induce ectopic bone formation in subcutaneous and intramuscular pockets in rodents.4,5 this bone induction process has been studied extensively.6,7 histological and biochemical analyses show that cartilage appears 5–10 days after implantation of active dbm.8 study of wang6 reports that this cartilage is mineralized by day 7–14 and subsequently replaced by bone. as dbm-related bone formation can be observed afterwards to occur at ectopic sites, it is assumed that pluripotent mesenchymal are attracted to the site of implantation. the use of allograft powder dbm leads bone tissue to be fully incorporated into patient’s tissue by a well established biological mechanism, and in treating bone defects has been proven beneficial for bone regeneration of both animals and humans.9,10 demineralized bone matrix (dbm) powder form comprises collagenous and non-collagenous. urist determined that the osteoinductive fraction could be extracted from the organic component of bone, which he termed bone morphogenic protein (bmp). the bmps are embedded in bone matrix.11 in addition to bmps, other growth factor such as platelet derived growth factor (pdgf) and transforming growth factor (tgf-β) were found.12 wound healing is basically a complex process in which a variety of cellular and matrix components act in concert to re-establish the integrity of injured tissues. the complexity of this process may be simplified into temporal sequence of normal healing: haemostasis, inflammation, cell proliferation (repair), and tissue remodelling. during 44 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 43–47 the evolution process, a group of structural protein develops, variously modified in terms of rigidity, elasticity, and strength levels, depending on the environmental influence and functional needs known as collagen. for instance, collagen on skin, bone, cartilage, and basal membrane. collagen is protein mostly found in humans and reaches 30% of the dry weight. the collagen consists of glysine amino acid (33.5%), prolin (12%), hidroxy prolin (10%), and the remaining substances such as other amino acids. type i collagen is the most spread one, found in the tissue as a classical structure renowned as collagen fiber. these fibers support bones, dentin, tendon dermis, etc. type ii collagen is mostly found in hyaline and elastic cartilage, whose shape is fibrous and very smooth.13 on day 7 in the process of wound healing on bone, the synthesis of type ii collagen appears whilst type i collagen generally prevails on day 10–12. 7 when observed by light microscope, the collagen fiber is acidophilic, appears to be pink by van geyson and blue by mallory trycom. collagen fibers comprise thick fibers solidly suppress each other with diameters approximately 75nm in mammals. in many parts of body, collagen fibers are conceived in parallel series forming collagen bundles.13 the objective of this study was to determine the density of collagen fibers on alveolus mandibular bone of rabbit which was augmented using powder dbm post-incisivus extraction. materials and methods this study was done pure experimentally using post– test only group control design. twenty-four male rabbits aged 2.5–3 months with weights of 900–1,100 grams were randomly divided into two groups. the first treated group i was augmented by powder dbm (370–710 μm) post-incisivus extraction, whilst the other group was not augmented, hence used as a controlled group. demineralized bone matrix (dbm) in the form of allograft made from cortical bone of rabbit was prepared by the bank of tissue dr soetomo rsud dr. soetomo, surabaya. the rabbits were injected with intra muscular with penobarbital sodium 100 mg/kg body weight; then after they were calm and sleepy, they were injected with 0,2ml/kg body weight of 2% lidocaine hcl (phapros, semarang) on incivus tooth labial. after the extraction ended, the wounds were cleaned by sterilized cotton, and gingival were pressed to control bleeding problem. subsequently, they were immediately augmented by dbm that has been mixed with the solution of lactate ringer. dbm was pressed to get in and fill the tooth sockets on mandible. mucosa crease was then sutured by non-absorbable suture no. 5 (ethycon silk suture). analgesic (250 mg) was also included into the liquid of ad libitum 250 ml only for two days after surgery. the food given was pellet (japfa comfeed, indonesia) and aquadest. on days 5, 7, 10 and 14 post-surgery, three rabbits from each group randomly took to be decapitated. mandible as wide as incisivus tooth was cut and submerged into the solution of lylis as the fixative solution and decalcification for 3–5 days. then the process was followed by 70% of alcohol as bone submersion solution for three days. subsequently, the bone was vertically cut in line with tooth axis using microtome and was imbedded in paraffin block. furthermore, the tissue was again cut as wide as 6 mm and put on object glass and processed for mallory staining. the observation on collagen fiber density was conducted on ten view fields under light microscope with the 4x 100 objective. criteria for valuation are: score 1 shows that the density of collagen fiber is low (figure 1-a); score 2 depicts that collagen fiber is medium (figure 1-b); and score 3 shows very high density (figure 1-c). in order to analyse the density data of the collagen fibers, mann-whitney test was utilized. results table 1 below shows the mean and standard deviation of the collagen fiber density of mandibular bone for each group. the pattern of collagen fiber density was summarized in figure 2. general description shown in figure 2 indicates that on day 7, collagen fiber density of dbm group was higher (denser) than that control group (without augmentation). meanwhile, histological description depicts that the formation of collagen fiber in dbm-augmented group is getting higher on day 7 after being wounded although histologically there has yet to show the prevalence of collagen bundle. this phenomenon is of difference from figure 1. (a) score of the collagen fiber density showing the range of low collagen density, (b) medium collagen density, and (c) high collagen density. a b c 45tandelilin et al: the density of collagen fiber in alveolus mandibular bone of rabbit that on day 5, showing that collagen fibers of the two groups are relatively few with similar mean values and without twist of collagen fiber. on day 10 after being wounded, dbm group shows the existence of collagen fiber bundle which appears to be denser after 14 days of treatment. on the other hand, the controlled group has also shown collagen bundle but the distance is not as dense as that of dbm group. subsequently, the mean rank of collagen fiber density in each group predicated upon treatment days is described in table 2. the difference significance of collagen fiber density of mandibular bone is graphed in table 3. as seen, there was no significant difference of collagen fiber density on day 5 and 7 after the extraction wounds. the difference was significant on day 10 and 14 in the wake of dbm augmentation. the results indicate that powder demineralized bone matrix induces the collagen fiber stimulation after the augmentation on rabbit alveolus mandibular postincisivus extraction. discussion results of this study tend to indicate that dbm powder of allograft can induce bone collagen fiber stimulation on mandible in post-extraction period. this fact substantiates previous studies that also show the capability of dbm of inducing collagen stimulation.7, 14 since the initial studies conducted by urist4 the osteoinductive capacity of dbm has been well established.15 dbm is produced by the extraction of human cortical bone and the components of the bone that remain behind including the non-collagenous proteins; bone osteoinductive growth factors, the most significant of which is bmp, and the type i collagen.14 dbm in this study is allograft in the form of freeze dried powder which could be stored at room temperature and is sterilized using γ (gamma) ray. these processes yield a dry granular material that consists of collagen matrix in which bmps and other insoluble growth factors are embedded. this condition is also in line with the use reported as effective grafting materials.3 the major phases of the osteoinduction are chemotaxis, mitosis, and differentiation.16 chemotaxis may be defined as the directed migration of cells in response to a chemical gradient. implantation of allograft dbm powder promotes chemotaxis of cells to vicinity. plasma fibronectin binds avidly to the implanted bone matrix.17,18 fibronectin is a protein that has affinity for collagen, fibrin, and heparin, the major components in the site of any skeletal trauma. it is well known that peptides of fibronectin are chemotactic and perhaps mitogenic.7 the major constituents of the demineralized bone matrix (powder) are collagen and other matrix proteins. the mineralization process that occurs in hard tissues of the body relies upon this extracellular matrix. it has generally been agreed that fibrillar collagen, the primary component of this matrix, has the capacity to serve as the structural framework in tissue that is undergo mineralization.19 one of the bone healing parameters is the growing collagen fibers. this study’s results indicate a significant difference of collagen fiber density on day 10 and 14 after the tooth extraction. it is apparent that collagen fiber table 1. mean and standard deviation of the density of mandibular fiber collagen on control and dbm groups time after augmentation n control x ± sd dbm x ± sd day 05 day 07 day 10 day 14 30 30 30 30 1.53 ± 0.73 1.80 ± 0.76 1.90 ± 0.84 1.93 ± 0.63 1.56 ± 0.62 2.11 ± 0.79 2.43 ± 0.67 2.50 ± 0.57 table 2. mean rank and sum of ranks of the density of the mandibular collagen fiber on control and dbm groups group time after augmentation n mean rank sum of ranks control dbm control dbm control dbm control dbm day 5 day 7 day 10 day 14 30 30 30 30 30 30 30 30 29.60 31. 40 26.70 34.30 25.25 35.75 23.78 37.22 888.00 942.00 801.00 102. 90 757.50 1072.50 713.50 1116.50 note: dbm = powder demineralized bone matrix. table 3. mann-whitney test of the density of the mandibular collagen fiber on control and dbm groups time after augmentation assym. sig (2 –tailed) day 05 day 07 day 10 day 14 0.653 0.73 0.013** 0.010** figure 2. the increase in density of collagen fiber on the mandibular bone post surgery based on observation day of control and dbm groups. 0 0,5 1 1,5 2 2,5 3 3,5 5 7 10 14 day s co re o f co ll ag en fi be r d en si ty control dbm 46 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 43–47 density, supposed to be type i and ii on dbm-augmented alveolus, is denser. collagen fibers on mandibular alveolus are mostly type i and ii. this condition is in line with the study of reddi et al.7 reporting that the synthesis of type i collagen prevails between day 10 and 12 in the process of bone induction of dbm implantation. bone matrix consists mainly of collagen fibers (approximately 90%) and noncollagenous proteins. type i collagen, which plays structural as well as morphogenic roles and provides scaffolding for mineral crystals, is the major species roles and within lamellar bone, the fibers are forming arches for optimal bone strength.20 accordingly, allograft powder of dbm as the augmentation materials is capable of hastening bone reconstruction by inducing biosynthesis of collagen fibers stimulation for bone strength. the effect of bone healing augmented by powder allograft of dbm is possibly caused by several growth factors that it contains, such as bmp, pdgf, gdf and tgfβ1, that synergically cooperating with local recipient cells that influence the proliferation of cells involved in bone healing. the main action of bmps is to commit undifferentiated pluripotential cells to differentiate into cartilage and bone-forming cells.21,22 bmps are basically abundant in bone and belong to the transforming growth factor-β (tgf-β) superfamily, which consists of a group of related peptide growth factors.23 the mechanisms of polypeptide growth and differentiation factors (gdfs) on the repair and regeneration of tissues have been shown to have pleitropic effects on wound repair in nearly all tissues.22,24 the expression of various gdfs following bone and soft tissue injury may regulate the repair and or regenerative process. previous studies have shown that acidic and basic fgf are found in bone matrix25 and in vitro; both forms stimulate dna synthesis and cell replication.26 of significance, the bfgf potently stimulates angiogenesis that is critical for the vascular invasions of bone.27 tgf-β1 has itself shown to be a strong promoter of extracellular matrix production in many cell types. the effects of tgf-β1 appear to be highly dependent upon bone cell source and local environment.28 this growth factor stimulates type i collagen, fibronectin, and osteonectin biosynthesis as well as bone matrix deposition and chemotaxis.29 this study’s results substantiate that of contran et al.,30 finding that the collagen synthesis in the healing process is influenced by some factors such as pdgf, fgf, tgf-β, and cytokins il-1 and il-4 secreted by leukocyte and fibroblast. the conclusion of the stimulation of collagen synthesis on alveolus of rabbit mandibular bone post-incicivus extraction has proven significantly induced by demineralized bone matrix augmentation. the density of collagen fiber per se is of significant difference on day 10 and 14 after the augmentation of demineralized bone matrix post incisivus extraction. acknowledgements this study was supported by bpps of directorate of higher education, department of national education. we confer sincere appreciation to dr. totok utoro, ph.d for our invaluable discussion on collagen fiber criteria. eventually, we sincerely thank to dr. didik setyo h., dr. rini maya puspita, and andreas ak. tandelilin, for their helps as blind reviewers on collagen scoring in this study. references 1. mercier p. resorption patterns of the residual ridge. in: block ms, kent jn, editors. endosseus implants for maxillofacial reconstruction. philadelphia, london, toronto, montreal, sidney, tokyo: wb saunders co; 1997. p. 10–16. 2. marx re. mandibular reconstruction. j oral maxillofac surg 1993; l 51:466. 3. wilkins rm, stringer ea. demineralized cortical bone powder: use in grafting space occupaying lesions of bone. int orthop 1994; 2: 71–8. 4. urist mr. bone: formation by autoinduction. science 1965; 150:893–9. 5. van de putte ka, urist mr. osteogenesis in the anterior of intramuscular implants of decalcified bone matrix. clinical orthopaedic and related research 1965; 257–70. 6. wang ea. bone morphogenetic proteins (bmps): therapeutic potential in healing bony defects. trends in biotechnology 1993; 11:379–83. 7. reddi ah, wientroub, muthukumaran n. biologic principles of bone induction. orthopaedic of north america 1987; 18:207–12. 8. sampath tk, reddi ah, homology of bone-induction proteins from human, monkey, bovine and rat extracellular matrix. proceeding of the national academy of science of the usa 1983; 80:6591–95. 9. gepstein r, weiss re, hallel t. bridging larged defects in bone by demineralization bone matrix in the form of a powder. a radiographic, histological, and radioisotope-uptake study in rat. j bone and joint surgery, american 1987; 69:984–92. 10. einhorn ta, lane jm, burstein ah, kopman cr, vigrota vj. the healing of segmental bone defects. j bone and joint surg, american 1984; 66:274–79. 11. urist m, strates b. bone morphogenetic protein. j dent res 1971; 50(suppl 6):1392. 12. wozney jm. overview of bone morphogenetic proteins. spine 2002; 27(165):s2–s8. 13. jungueira lc, carneiro j, kelley r. basic histology. 8th ed. jakarta: egc publisher; 1995. p. 91–120. 14. tuli sm, singh ad. the osteoinductive property of decalcified bone matrix: an experimental study. j bone joint surg br 1978; 60: 116–23. 15. ludwig sc, kowalski jm, boden sd. osteoinductive bone graft substitutes. j eur spine 2000; 9(supple 1):s119–s125. 16. reddi ah. extracellular matrix and development. in: piez ka, reddi ah, editors. extracellular matrix biochemistry. new york: elsevier; 1984. p. 375–412. 17. weiss re, reddi ah. synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentation of cartilage and bone in vivo. proc nat acad sci usa 1980; 77: 2074–78. 18. weiss re, reddi ah. role of fibronectin in collagenous matrixinduced mesencymal cell proliferation and differentation in vivo. exp cell res 1981; 133:247–54. 19. miller ej, martin gr. the collagen of bone. clin orthop1968; 59:195. 47tandelilin et al: the density of collagen fiber in alveolus mandibular bone of rabbit 20. groeneveld hj, van den bergh jpa, holzman p, ten bruggenkate cm, tuinzing db, burger eh. mineralization processes in demineralization bone grafts in tissue floor elevations. j biomed mat and res 1999; 48:393–402. 21. zhang m, powers rm, wolfinbarger l. effect(s) of the demineralization process on the osteoinductivity of demineralized bone matrix. j periodontol 1997; 68:1085–92. 22. ripamonti u, reddi ah. periodontal regeneration. potential role of bone morphogenetic proteins. j periodont res 1994; 29:225–35. 23. groeneveld ehj, burger eh. bone morphogenic proteins in human bone regeneration. eur j endocrin 2000; 142:9–21. 24. graves dt, concran dl. mesencymal cell growth factors. crit rev oral biol med 1990; 1:17–36. 25. globus rk, plouet j, gospodarowicz d. cultured bovine osteoblasts synthesize basic fibroblast growth factor and store it in their extracellular matrix. j endocrin 1989; 124: 1529–47. 26. canalis e, mc carty t, centrella m. effects of basic fibroblast growth factor on bone formation in vitro. j clin invest 1988; 81: 1572–77. 27. folkamn j, klasburn m. angiogenic factors. science 1987; 235: 442–47. 28. centrella m, mc carhty tl, canalis e. transforming growth beta is a bifunctional regulator of replication and collagen synthesis in osteoblast–enriched cell cultures from fetal rat bone. j biol chem 1987; 262:2869–74. 29. bonewald lf, mundy gr. role of transforming growth factor-beta in bone remodelling. j clin orthop 1990; 250:261–76. 30. contran r, kumar v, collins t, tissue repair: cellular growth, fibrosis, and wound healing. in: robbins pathologic basic of disease. 8th ed. philadelphia: wb sounders co; 1999. p. 89-112. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 49 no 3 juli-sept 2016.indd 125125 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 125–132 acceleration of fibroblast number and fgf-2 expression using channa striata extract induction during wound healing process: in vivo studies in wistar rats gunawan oentaryo,1 istiati,2 and pratiwi soesilawati 3 1department of oral biology, faculty of dentistry, universitas lambung mangkurat, banjarmasin-indonesia 2department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, surabaya-indonesia 3department of oral biology, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: wound healing is a biological process associated with tissue growth and regeneration. wound healing process, is important to repair damaged tissue. wound healing process consists of coagulation and hemostasis, inflammation, proliferation, as well as remodeling phases. the process can be accelerated by taking synthetic or non synthetic drugs. one of them is channa striata extract. the extract contains albumin, copper, and zinc, which can be assumed to increase inflammatory cell infiltration, fibroblast proliferation, and collagen secretion. purpose: this study aimed to reveal the effects of channa striata extracts on fibroblast number and fgf-2 expression in mucosal wound healing process of the wistar rats’ lower lip. method: this research was a true laboratory experimental research with randomized post test only control group design. samples of experiment were devided to experiment and control group that consist five samples each. experimental group was treted with channa striata extract and ethanol at concentration of 25%, 50%, and 100%. the fibroblast number and fgf-2 expresion were examined. result: the number of fibroblasts in the treatment groups receiving channa striata extract at concentrations of 25%, 50%, and 100% was higher than in the control group. the highest number of fibroblasts was found on day 3 at the concentration of 100% (p<0.05). similarly, fgf-2 expression in the treatment groups receiving channa striata at concentrations of 25%, 50%, and 100% was higher than in the control group. the highest expression of fgf-2 was found on day 3 at the concentration of 50% (p<0.05). conclusion: channa striata extract increased fibroblast number and fgf-2 expression in mucosa wound healing process. keywords: channa striata extract; fgf-2; fibroblast; wound healing correspondence: istiati, department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: prof.istiati@gmail.com introduction oral cavity is an integral part of the body often traumatized when performing its functions. trauma can occur intentionally or unintentionally causing wounds to the oral mucosa omitted.1,2 wounds constitute a change of continuity in anatomical and cellular tissues that can occur on skin or mucosa. damage to the continuity, can be improved through wound healing process.3 wound healing process is a complex cellular and vascular process. the process aims to restore the integrity of the structure and function of damaged tissue. wound healing process generally consists of four phases, namely coagulation and homeostasis, inflammation, proliferation, and remodeling. histologically, the results of wound healing process can be indicated by density of collagen fibers produced by fibroblasts in new connective tissue in the phase of proliferation.4 fibroblasts first appears on day 2-3 after the injury in conjunction with the formation of new capillaries which will provide sufficient supply of nutrients for cell proliferation. the proliferation of fibroblasts and the formation of new capillaries are triggered by growth factors, such as vascular endothelial growth factor (vegf) and fibroblast growth factor-2 (fgf-2). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 126 oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 fgf-2 is one of the growth factors secreted by macrophages. fgf-2 expression increases immediately after the injury, and reaches its peak on days 5-8. fibroblasts regulate angiogenesis via secretion of these growth factors. in addition, fibroblasts also produce collagen type i, iii, v and other extracellular matrix components.5,8,9 in the final phase of wound healing process or remodeling process, the expression of collagen type iii drops and is replaced with increased expression of collagen type i.10 fibroblasts then migrate into the wound tissue and reach the maximum number on days 7-14 after the injury. 11 in experimental researches on traumatic ulcers on the buccal mucosa of wistar rats show that on day 7 after injuries, the ulcers will experience cicatrix, the color of mucosa will be normal, and the edge of ulcers will disappear.12 wound healing process actually can be accelerated by using certain agents, one of which is channa striata extract. channa striata extract plays an important role in wound healing process.13,14 this is because channa striata extract contains high albumin. albumin is indispensable in human body every day, especially in the process of wound healing. other researches also have reported that channa striata extract also contains compounds essential for the human body, including zinc (zn), copper (cu), and iron (fe), which play a role in enhancing immunity. in addition, cu is reported to increase vegf, so angiogenesis increases.13-15 in other researches, copper is reported to increase fgf-2 that plays a role in the proliferation of fibroblasts and the expression of collagen type 1, so tissue structure in the wound healing process can be repaired faster.16,17 channa striata extract is one of the traditional medicines with huge potential. in the last decade, traditional medicine is increasingly popular. traditional medicinal products can be made from animals or plants, widely used as an alternative treatment to meet the basic needs of people in the health field. the advantages of using traditional medicine are cheap, easy to obtain, and minimal side effects compared to chemical drugs.14 indonesia, especially kalimantan, is a natural habitat for channa striata. in addition to its distinctive taste, eating channa striata for south kalimantan people can accelerate wound healing process. for those reasons, this research aimed to determine the effects of channa striata extract on the mucosal wound healing process of wistar rats’ lower lip histopathologically and immunohistochemically on days 3, 5, and 7. in other words, this research focused on analyzing acceleration of fibroblasts count and fgf-2 expression in the mucosal wound healing process of wistar rats’ lower lip. materials and method this research was a true laboratory experimental research (true experimental design). this research used randomized design post test only control group design. in this research, the number of samples to be examined was calculated using lemmeshow’s formula. the total samples of each group was five samples. the research materials and instruments used in this research were channa striata samples, aquadest, 50% ethanol, buffer formalin, cotton buds, ether, xylol, paraffin, reagents fgf-2, hematoxylin eosin staining, becker glass, stirer, pipette, oven, scales, mixer glass, vibrator, test tube racks, thermometers, autoclave, measuring cup, tweezers dentistry, glass mouth, burnisher, burner, disposible syringe 2.5 ml, excavators, sample bottle, label, slide and cover glass, petri disks, lights, gloves, and cover mouth. independent variables of this research were channa striata extract and ethanol at a concentrations of 25%, 50%, and 100% as well as aquadest as a control group. before conducting an examination on experimental animals, this research design was submitted to the health research ethics commission in faculty of dental medicine, universitas airlangga in order to be approved. animals in the control and treatment groups were anesthetized in order to make them feel painless at the beginning of treatment. those animals were put into glass tubes essentially given a cloth that had been soaked in a solution of 10% ether, and then sealed. they were waited to fall asleep. the mucosa of their lower lip then got asepsis with 0.12% clorhexidine digluconate. the mucosa of their lower lip was injured with a scalpel heated for 1 minute and touched for 1 second. the size of the wound was 10 mm with a depth of 1 mm. after the rats free of anesthetic, they were kept in a cage and fed in moderation. treatment given can be seen in table 1. the fibroblast number and fgf-2 expression were examined, results based on the results of observation and measurement with fibroblast parameters, there were four groups, namely control group, treatment group with a concentration of 25%, treatment group with a concentration of 50%, and treatment group with a concentration of 100%. the mean number of fibroblasts at each concentration of the extract on days 3, 5, and 7 can be seen in table 2. table 2 shows that the mean number of fibroblasts in the treatment groups was higher than in the control groups. the highest number was found in the treatment group receiving channa striata extract at the concentration of 100% on day 3, but decreased from day 5 to 7. however, the mean number of fibroblasts in the treatment group receiving channa striata extract at the concentration of 100% was generally higher on days 3, 5 and 7 than the other treatment groups at the other concentrations. the results seen in table 2 were also confirmed by microscopic histopathological examination on fibroblasts in each research group on days 3, 5, and 7 as depicted in the following figure 1. figure 1 shows that based on the results of the histopathological examination, the number of fibroblasts on day 3 in the treatment groups receiving channa striata dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 127127oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 table 1. classifying and treatment of the experimental animals group types of treatment doses decapitation 1 aquadest 2.5 ml/ day day-3 2 aquadest 2.5 ml/ day day-5 3 aquadest 2.5 ml/ day day-7 4 25% channa striata extract 10 ml/kgbm/ day day-3 5 25% channa striata extract 10 ml/kgbm/ day day-5 6 25% channa striata extract 10 ml/kgbm/ day day-7 7 50% channa striata extract 10 ml/kgbm/ day day-3 8 50% channa striata extract 10 ml/kgbm/ day day-5 9 50% channa striata extract 10 ml/kgbm/ day day-7 10 100% channa striata extract 10 ml/kgbm/ day day-3 11 100% channa striata extract 10 ml/kgbm/ day day-5 12 100% channa striata extract 10 ml/kgbm/day day-7 table 2. the mean and standard deviation of fibroblasts at each concentration of the extract on days 3, 5, and 7 day concentration of the extract mean standard deviation 3 control 3.4000 1.14018 25% 4.2000 1.30384 50% 4.8000 .83666 100% 5.6000 .89443 5 control 2.6000 .89443 25% 3.4000 .54772 50% 4.0000 .00000 100% 5.4000 .54772 7 control 2.0000 1.00000 25% 3.0000 1.00000 50% 3.8000 .83666 100% 4.8000 .83666 a b c d figure 1. the results of histopathological examination on fibroblasts on day 3 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). a b c d figure 2. the results of histopathological examination on fibroblasts on day 5 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). a b c d figure 3. the results of histopathological examination on fibroblasts on day 7 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 128 oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 extract at the concentrations of 25% (b), 50% (c), and 100% (d) was higher than in the control group (a). the highest number of fibroblasts was found in the treatment group receiving channa striata extract at the concentration of 100% (d). furthermore, the number of fibroblasts on day 5 and 7 can be seen in figures 2 and 3. figure 2 shows that based on the results of the histopathological examination, the number of fibroblasts on day 5 in the treatment groups receiving channa striata extract at the concentrations of 25% (b), 50% (c), and 100% (d) was higher than in the control group (a). the highest number of fibroblasts was found in the treatment group receiving channa striata extract at the concentration of 100% (d). based on figure 1, the number of fibroblasts on day 5 reduced compared to the number of fibroblasts on day 3. figure 3 shows that based on the results of the histopathological examination, the number of fibroblasts on day 7 in the treatment groups receiving channa striata extract at the concentrations of 25% (b), 50% (c), and 100% (d) was higher than in the control group (a). the highest number of fibroblasts was found in the treatment group receiving channa striata extract at the concentration of 100% (d). based on figures 1 and 2, the number of fibroblasts on day 7 reduced compared to on day 3 and day 5 (in case of measurement). observation and measurement of on fgf-2 expression w e r e c o n d u c t e d t h r o u g h i m m u n o h i s t o c h e m i s t r y examination. the samples were divided into four groups, namely the control group, the treatment group with the concentration of 25%, the treatment group with the concentration of 50%, and the treatment group with the concentration of 100%. the mean expression of fgf-2 at each concentration on days 3, 5, and 7 can be seen in table 2. table 3 shows than the mean expression of fgf-2 in the treatment groups was higher than in the control table 3. the mean and standard deviation of fgf-2 expressions at each concentration of the extract on days 3, 5, and 7 day concentration of the extract mean standard deviation 3 control 8.6000 1.67332 25% 14.6000 1.67332 50% 18.0000 3.39116 100% 17.8000 1.30384 5 control 6.6000 2.07364 25% 10.0000 3.08221 50% 12.0000 1.58114 100% 12.8000 1.92354 7 control 3.6000 1.14018 25% 7.8000 2.04939 50% 10.0000 1.87083 100% 13.4000 3.13050 a b c d figure 4. the results of histopathological examination on fgf-2 expressions on day 3 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). a b c d figure 5. the results of histopathological examination on fgf-2 expressions on day 5 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). a b c d figure 6. the results of histopathological examination on fgf-2 expressions on day 7 in the control group (a); the treatment groups receiving channa striata extract the concentration of 25% (b); 50% (c); and 100% (d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 129129oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 group. the highest expression of fgf-2 was found in the treatment groups on day 3. the mean expression of fgf-2 in the treatment groups decreased from day 5 to day 7, and the highest ones at the concentration of 100%, except in the treatment groups on day 3, the highest one at the concentration of 50%. the results as shown in table 3 were supported by the results of microscopic histopathological examination on fgf-2 expressions in each group on days 3, 5, and 7 as seen in the following figure 4. figure 4 shows that the results of histopathological examination on fgf-2 expression on day 3 in the treatment groups receiving channa striata extract at the concentrations of 25% (b), 50% (c), and 100% (d) were higher than in the control group (a). the highest expression of fgf-2 was found in the treatment group receiving channa striata extract at the concentration of 50% (d). the number of fibroblasts on days 5 and 7 can be seen in the following figures 5 and 6. figure 5 shows that the results of histopathological examination on fgf-2 expression on day 5 in the treatment groups receiving channa striata extract at the concentrations of 25% (b), 50% (c), and 100% (d) were higher than in the control group (a). the highest number of fibroblast was found in the treatment group receiving channa striata extract at the concentration of 100% (d). based on figure 4, fgf-2 expression on day 5 reduced compared to on day 3. figure 6 shows that the results of histopathological examination on fgf-2 expression on day 7 in the treatment groups receiving channa striata extract at the concentrations of 25% (b), 50% (c), and 100% (d) were higher than in the control group (a). the highest expression of fgf-2 was found in the treatment group receiving channa striata extract at the concentration of 100% (d). based on figures 4, 5 and 6, fgf-2 expression on day 7 reduced from day 3 to day 5. tables 4 show that the mean values of the three parameters in the treatment groups were higher than in the control group. the mean values in the treatment groups were higher as the concentration of the extract increased. the differences in the mean values between the treatment groups and the control group can be seen in the diagram below. figure 7 shows that the mean values of the three parameters in the three treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100% fibroblas fgf2 group va lu es co nt ro l co nt ro l figure 7. the mean values of the three parameters in the control group and the treatment groups. table 4. the significance values of difference in fibroblasts between the control group and the treatment groups on day 3 day parameter group control 25% 50% 100% day 3 fibroblasts control 0.250 0.053* 0.005* 25% 0.384 0.053* 50% 0.886 100% * there were significant differences fgf-2 day 3 day 5 day 7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 130 oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 were higher than in the control group. the highest mean value in almost all parameters was found in the treatment group receiving channa striata extract at the concentration of 100%. data analysis was continued with statistical tests to analyze the significance difference in the parameters of collagen, fibroblasts, and fgf-2 among each group research. the results of the tests showed significance values among the research groups. there were significant differences in fibroblasts on day 3 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%, between the treatment group receiving channa striata extract at the concentration of 25% and the treatment groups with the concentrations of 50% and 100%, as well as between the treatment group receiving channa striata extract at the concentration of 50% and the treatment group with the concentration of 100%. there were significant differences in fgf-2 expressions on day 3 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 50%, and 100%, between the treatment group receiving channa striata extract at the concentration of 25% and the treatment group with the concentration of 100%, as well as between the treatment group receiving channa striata extract at the concentration of 50% and the treatment group with the concentration of 100%. nevertheless, there was no significant difference between the control group and the treatment group receiving channa striata extract at the concentration of 25% as well as between the treatment group receiving channa striata extract at the concentration of 25% and the treatment group with the concentration of 50% (table 5). there were significant differences in fibroblasts on day 5 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%, between the treatment group receiving channa striata extract at the concentration of 25% and the treatment groups with the concentrations of 50% and 100%, as well as between the treatment group receiving channa striata extract at the concentration of 50% and the treatment group with the concentration of 100% (table 6). there were significant differences in fgf-2 expressions on day 5 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%, between the treatment group receiving channa striata extract at the concentration of 25% and the treatment group with the concentration of 50%, as well as between the treatment group receiving channa striata extract at the concentration table 5. the significance values of difference in fgf2 expressions between the control group and the treatment groups on day 3 day parameter group control 25% 50% 100% day 3 fgf-2 control 0.133 0.012* 0.000* 25% 0.224 0.002* 50% 0.022* 100% * there were significant differences table 6. the significance values of difference in fibroblasts between the control group and the treatment groups on day 5 day parameter group control 25% 50% 100% day 5 fibroblasts control 0.126* 0.025* 0.000* 25% 0.070* 0.000* 50% 0.005* 100% * there were significant differences table 7. the significance values of difference in fgf-2 expressions between the control group and the treatment groups on day 5 day parameter group control 25% 50% 100% day 5 fgf-2 control 0.029* 0.002* 0.000* 25% 0.176 0.650 50% 0.579 100% * there were significant differences dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 131131oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 of 50% and the treatment group with the concentration of 100% (table 7). there were significant differences in fibroblasts on day 7 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%, as well as between the treatment group receiving channa striata extract at the concentration of 25% and the treatment groups with the concentrations of 50% and 100%. however, there was no significant difference between the treatment group receiving channa striata extract at the concentration of 50% and the treatment group with the concentration of 100% (table 8). there were significant differences in fgf-2 expressions on day 7 (p<0.05) between the control group and the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%, between the treatment group receiving channa striata extract at the concentration of 25% and the treatment groups with the concentrations of 50% and 100%, as well as between the treatment group receiving channa striata extract at the concentration of 50% and the treatment group with the concentration of 100% (table 9). discussion wound healing process is a complex cellular and vascular process consisted of four phases, coagulation and hemostasis, inflammation, proliferation, and remodeling. the process can be accelerated using synthetic or nonsynthetic ingredients, one of which is channa striata extract. in this study, observation on fgf-2 expression and fibroblast number was focused in wound healing process of wistar rats’ oral mucosa given and not given channa striata extract at the concentrations of 25%, 50%, and 100%. the number of fibroblasts in the treatment groups receiving channa striata extract at the concentrations of 25%, 50% and 100% was higher than in the control group. the highest number of fibroblasts was found in the treatment group receiving channa striata extract at the concentration of 100% about 5.6 on day 3, but declined into 5.4 on day 5 and 4.8 on day 7 as seen in table 1 and figure 1. based on table 3, 5 and 7, the number of fibroblasts decreased on days 5 and 7. this finding is consistent with a theory stating that fibroblasts will first appear on day 3. the decline in the number of fibroblasts can also be associated with reepithelization process of mucosal ulcer healing in wistar rats on day 7.12 the number of fibroblasts increased in the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100%. this is because channa striata extract contains copper (cu). izzati et al.18 reported that channa striata extract contains 0.447 mg/ l of cu. copper plays a role in the growth and replication of cells that can trigger the proliferation of fibroblasts in areas that experience healing process. besides copper, fgf-2 expression can also increase, triggering the proliferation of fibroblasts and the expression of collagen type 1. as a result, the proliferation of fibroblasts increase, so they can repair tissue structures faster during the wound healing process. the expression of fgf-2 in the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100% declined from day 3 to day 7. however, the expression of fgf-2 generally increased in the treatment groups receiving channa striata extract at the concentrations of 25%, 50%, and 100% as shown in table 2 and illustrated in figure 4 as well as proved by statistical significance values in table 4, table 6, and table 8. similarly, the researches conducted by soepribadi6 and nagayasu-tanaka7 show that the expression of fgf-2 will increase immediately after the injury, and reach its peak table 8. the significance values of difference in fibroblasts between the control group and the treatment groups on day 7 day parameter group control 25% 50% 100% day 7 fibroblasts control 0.106* 0.007* 0.000* 25% 0.189* 0.007* 50% 0.106 100% * there were significant differences table 9. the significance values of difference in fgf-2 expressions between the control group and the treatment groups on day 7 day parameter group control 25% 50% 100% day 7 fgf-2 control 0.007 0.000* 0.000* 25% 0.128* 0.001* 50% 0.025* 100% * there were significant differences dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 132 oentaryo, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 125–132 on day 3. this is because fgf-2 which stimulates the proliferation of fibroblasts has been reduced, but the amount of collagen will continue to increase until the fourteenth day. it also affects the amount of collagen expression since fgf accumulates collagen. based on the data obtained, channa striata extract has effects on the number of fibroblasts and the expression of fgf-2. similarly, the researches conducted by restiana et al. and soemaridini et al. show that channa striata plays a role in wound healing process due to the high albumin level contained in the fish.13,14 thus, channa striata extract at a concentration of 100% indicates that there are 7.568 mg/ l of albumin, 6.7 mg/ l of zn, 0.72 mg/ l of fe, and 0.447 mg/ l of cu.18 albumin is one type of protein. this protein plays a role in increasing the proliferation of fibroblasts, thus increasing the synthesis, accumulation, and remodeling of collagen. albumin also plays a role in oxygen transport. zinc, moreover, plays a role in the growth and replication of cells. zn also plays a role in immune response. cu serves to improve angiogenesis process through increased expression of vegf and fgf-2. fe plays a role in dna replication. fe also plays a role in the formation of collagen. in addition, cu is reported to increase vegf triggering the increasing of angiogenesis.13-15 in the other researches, copper is also reported to increase fgf-2 that plays a role in the proliferation of fibroblasts and the expression of collagen type 1, thus, the repairing process of tissue structure and the process of wound healing can be accelerated.16,17 however, this study still has several limitations. this research still cannot be able to determine the total number of active substances contained in channa striata extract at a concentrations of 25%, 50%, and 100% affecting the number of fibroblasts and the expression of fgf-2. this research also still cannot determine the standardization of channa striata quality and its extract quality affecting the number of fibroblasts and the expression of fgf-2. in coclusion, channa striata extract increased the fibroblasts number and fgf-2 expression in mucosa wound healing process. references 1. chandra s, chandra s. textbook of community dentistry. delhi, india: jaypee brothers; 2007. p. 105, 185. 2. girish ms, anandakrishna l, chandra p, nandlal b, srilatha kt. iatrogenic injury of oral mucosa due to chemicals: a case report of formocresol injury and reviews. iosr journal of dental and medical sciences 2015; 14(4): 01-05. 3. miloro m, cohali ge, larsen pe, waite pd. peterson’s principles of oral and maxillofacial surgery. third edtion. london, england: bc decker; 2011. p. 3-6. 4. yuhernita, juniarti, aryenti. pengaruh pemberian gel dari ekstrak metanol daun jarak tintir (jatropha multifidi l) terhadap kepadatan serabut kolagen dan jumlah angiogenesis dalam proses penyembuhan luka. prosiding seminar nasional dan workshop perkembangan terkini sains farmasi dan klinik iv. jakarta, 2014; p. 47-55. 5. la r java h. o ra l wou nd hea l i ng: cel l biolog y a nd cl i n ica l management. uk: john wileys sons; 2012. p. 138, 141. 6. soepribadi i . regenerasi dan penyembuhan. jakarta: sagung seto; 2013. p. 23-30. 7. nagayasu-tanaka t, anzai j, takaki s, shiraishi n, terashima a, asano t. action mechanism of fibroblast growth factor-2 (fgf-2) in the promotion of periodontal regeneration in beagle dogs. plos one 2015; 10(6): e0131870. 8. nguyen vt. mechanism of delayed wound healing in various models in human disease. human health and pathology. thesis. paris: universito pierre at marie curie; 2015. p. 23-5. 9. pan x, chen z, huang r, yao y, wa g. transforming growth factor beta1 induces the expression of collagen type i by dna methylation in cardiac fibroblast. plosone, 2013; 8(4): e60335. 10. olczyk p, mencner l, komosinska-vassev k. the role of the extracellular matrix components in cutaneous wound healing. biomed reasearch international 2014; 2014: 1-8. 11. bazzaz aa, bukhari fo, bazzaz za, chelebi na. in vivo assesment of growth promoting activity of a synthetic β-fgf in wound healing of rat’s skin. european scientific journal 2013; 3: 222-39. 12. cavalcante gm, de paula rjs, de souza lp, sousa fb, mota mrl, alves apnn. experimental model of traumatic ulcers in the cheek mucosa of rats. acta cir bras 2011; 26(3). 13. restiana, taslim n, bukhari a. pengaruh pemberian ekstrak ikan gabus terhadap kadar albumin dan status gizi penderita hiv/ aids yang mendapatkan terapi arv. makassar: fk universitas hasanuddin; 2013. p. 2. 14. soemardini, permaningtyas k, chandra d. pengaruh pemberian ekstrak ikan gabus (channa striata) terhadap kadar nitric oxide pada tikus rattus norvegicus jantan strain wistar model diabetes melitus. malang: fk universitas brawijaya; 2011. p. 1-3. 15. suprayitno e. profile albumin fish cork (opicephalus striatus) of different ecosystems. international journal of current research and academic review 2014; 2(12): 201-8. 16. ahn hj, lee wj, kwon yd. fgf-2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of jnk signaling. science direct, 2009; 583(17): 29226. 17. parenteau-barell r, gauvin r, berthod ff. collagen-based biomaterials for tissue engineering application. materials 2010; 3: 1863-77. 18. izzaty a, dewi n, pratiwi din. ekstrak haruan (channa striata) secara efektif menurunkan jumlah limfosit fase inflamasi dalam penyembuhan luka. dentofasial 2014; 13(3): 176-81. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p125-132 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p125-132 202 volume 45 number 4 december 2012 the effect of nickel as a nickel chromium restoration corrosion product on gingival fibroblast through analysis of bcl-2 fx ady soesetijo1 and mandojo rukmo2 1department of prosthodontics, faculty of dentistry, universitas jember, jember – indonesia 2department of conservative dentistry, faculty of dentistry, universitas airlangga, surabaya – indonesia abstract background: restoration of nicr may undergo corrosion process in artificial saliva. corrosion product is soluble ni substances in salivary electrolytes. ni2+ may freely enter the cells through passive transport dmt-1. ni2+ in the cell causes initiation of the ros formation,which subsequently can conduct the redoxs reactions leading to dna damage. the damage dna affects the genetic expression, especially bcl-2, and even triggers apoptosis. purpose: the aim of this study was to reveal the mechanism of ni toxicity as a corrosion product of nicr restoration on gingival fibroblasts through expression analysis of bcl-2. methods: cells with a density of 105 planted on each coverslip in 72 wells to the treatment group and 24 wells to the control group (24 hours incubation). in the treatment groups, each well exposed with 20 μl artificial saliva containing ni concentration results immerse each restoration, whereas the control group was exposed to 20 μl artificial saliva (incubation 1, 3, and 7 days). the data collected were subsequently analyzed using two-ways anova, followed by one-way anova. comparing between experimental groups after one-way anova was conducted using fisher’s lsd. whereas, the calculation and documentation of bcl-2 expression was performed camera of olympus microscope bx-50 japan. results: statistical analysis of two-ways anova showed the presence of interaction between the increasing ni concentration and exposure duration on the expression of bcl-2 gingival fibroblasts (p=0.021<a=0.05). conclusion: it can be concluded that the higher concentration of ni exposed to gingival fibroblasts, and the longer incubation time will decreased bcl-2 expression. key words: ni2+, dmt-1, dna damage, bcl-2 abstrak latar belakang: restorasi nicr dapat mengalami proses korosi di dalam saliva artificial. produk korosi yang dihasilkan adalah substansi ni yang terlarut di dalam elektrolit saliva. ni2+ bebas dapat memasuki sel (fibroblas gingiva) melalui transport pasif dmt-1. ni2+ di dalam sel menginisiasi pembentukan ros, yang selanjutnya dapat menjalankan reaksi redoks dan dapat menimbulkan kerusakan dna. dna yang rusak mempengaruhi ekspresi genetik, terutama bcl-2 dan bahkan dapat memicu apoptosis. tujuan: tujuan penelitian ini adalah untuk mengungkap mekanisme toksisitas ni sebagai suatu produk korosi restorasi nicr pada fibroblas gingiva melalui analisis ekspresi bcl-2. metode: sel dengan kepadatan 105 ditanam pada tiap-tiap coverslip di dalam 72 well untuk kelompok perlakuan dan ditanam pada tiap-tiap coverslip di dalam 24 well untuk kelompok kontrol (inkubasi selama 24 jam). pada kelompok perlakuan, masing-masing well dipapar dengan 20 μl saliva artificial yang mengandung konsentrasi ni hasil perendaman tiap-tiap restorasi, sedangkan pada kelompok kontrol dipapar 20 μl saliva artificial (inkubasi 1,3 dan 7 hari). data yang terkumpul selanjutnya dianalisis menggunakan anova dua arah dan anova satu arah. perbandingan antar kelompok eksperimental setelah analisis anova satu arah menggunakan uji fisher’s lsd. penghitungan jumlah sel yang mengekspresikan bcl-2, kemudian dilanjutkan dengan dokumentasi dengan menggunakan kamera olympus microscope bx-50 japan. hasil: analisis statistik anova dua arah menunjukkan adanya interaksi antara peningkatan konsentrasi ni dan lama paparan terhadap ekspresi bcl-2 fibroblas gingiva research report 203soesetijo and rukmo: the effect of nickel as a nickel chromium restoration corrosion product (p = 0,021 < á = 0,05). kesimpulan: dapat disimpulkan bahwa semakin tinggi konsentrasi paparan ni pada fibroblas gingiva dan semakin lama masa inkubasi, maka akan menurunkan ekspresi bcl-2. kata kunci: ni2+, dmt-1, dna damage, bcl-2 correspondence: fx ady soesetijo, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: adysoesetijo@ymail.com introduction nickel-chromium (nicr) alloy was widely used in dentistry for restoration construction purposes requiring strength, e.g. crowns, bridges and metal frame dentures. the consideration of the use of these alloys is mainly due to their relative low price compared to noble alloy, possess adequate hardness, and fine physical and mechanical properties. nicr alloy consists of main components of ni 65%, cr 22.5%, as well as additional components of mo, fe, si, ce and nb.1-3 biocompatibility nicr restoration is a critical issue because of its existence in the long term of intimate contact with oral tissue. the presence of water, ions in saliva, changes in temperature and ph are factors that play a role in the occurrence of tarnish and corrosion. this was correlated with the release of alloy elements that impacts on unfavorable the biological effects.4 corrosion may cause chemical changes of nicr restoration. chemical change occurring was an increase in restoration oxidation i.e. the formation of metal oxides easily detached from the surface of the restoration becoming cations. nicr restoration recasting results in the increased toxicity in gingival fibroblasts due to the increase of ni cations release in saliva.5,6 ni is an unstable transition class and easy-released into saliva.7 nicr alloy potentially causes toxic effects, for the reason that it may cause cellular damage. ni elements released may affect the ability of cellular metabolism, particularly proliferation.8 ni exposure on cultured cells may decrease the activity of several fe-s enzymes associated with energy metabolism and induces oxidative stress by increasing the reactive oxygen species (ros). generation of ros in the cells affects the expression of b-cell lymphoma-2 (bcl-2).9-11 bcl-2 was a negative regulator of cell death, extends cell apoptosis and inhibits cell cycle. bcl-2 family and the products were identified as a key regulator in the process of apoptosis in many cell types. excessive expression of bcl-2 increases cell survival. apoptosis induced by ni2+ is marked by the decrease of the expression of anti-apoptotic protein bcl-2 and bcl-xl, and induces the increase of the expression of bad, bclxs, bax, and caspase sit c (9,3 and 6).12 ni2+ accumulation in cells can increase dna damage response genes, decreased the expression of bcl-2 gene and triggers apoptosis.13 therefore, this study aims to determine the effect of toxicity of ni as a product of nicr restoration corrosion results in gingival fibroblasts through analysis of bcl-2 expression. materials and methods this study began with a laboratory test to obtain a sample i.e the concentrations of ni were dissolved in 5 ml of artificial saliva after immersion of restoration nicr casting, recasting 1× and 2× for 7 days.14 restoration nicr (diameter of 10 mm, thickness of 1 mm) obtained from casting alloys nicr (4all nicr alloy white ceramic, ivoclar vivadent-usa). ni concentrations were obtained with the atomic absorption spectrophotometer (aas) assay (model ag analytikjena zeenit 700), subsequently exposed on gingival fibroblast culture. negative control group (describes artificial saliva containing no ni on gingival fibroblasts). gingival obtained from patients after conducting some procedures and filling out informed-consent, and subsequently undergoing gingivectomy. this study was approved by the ethical appropriateness of the research ethics committee of the faculty of medicine, jember university number: 125/h.25.i.ii/ke/2011. the next stage was to conduct the process of tissue culture. cell cultures were grown in dulbeco, s eagle.s modified medium (dmem) fetal bovine serum containing (fbs) 10% (v/v), penicillin-streptomycin 1% (v/v). the cells were then harvested from the culture dish/flask with 0.025% trypsin-edta (gibco). cells with a density of 105 planted on coverslip in 72 wells as treatment group and 24 wells as control group (24-hour incubation), then the media were removed and washed with pbs. in the treatment group, each well was exposed to 20 ml artificial saliva containing ni concentration resulted from the immersion of each restoration, whereas the control group was exposed to 20 ml artificial saliva (incubation 1, 3, and 7 days). coverslip containing the cells were taken and placed on an object glass, then performed immunocytochemistry assay with bcl-2 staining. expression of bcl-2 gingival fibroblasts was performed by immunocytochemistry assay with monoclonal antibody anti bcl-2 (bcl-2 kit: bioworld, cat. no. bs1511, usa). in this study, observations were carried out using light microscope (olympus bx-50 japan) with magnification of 400 x, where the cells that expressed bcl-2 in the cytoplasm was yellow-brown. percentage of bcl-2 = the number of positive cells bcl-2 divided by the total number of cells x 100%.15 the data were analyzed by two way anova, followed by one way anova and multiple comparison least different significance test with 95% significance level (a = 0.05). 204 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 202–207 results nickel solubility in artificial saliva performed by aas with a wavelength of 323 nm, the parameters per parts million (ppm) and the units used are mg/l. the result is [ni] casting = 0.780; recasting 1 x = 3.002 and recasting 2 x = 6.320 (tabel 1). the mean and standard deviation of bcl-2 expression progressively decreased with increasing ni concentration and increasing duration of exposure. based on this interpretation, it was necessary to analyze using two way anova toward the possibility of interaction between increasing ni concentration and duration of exposure (table 2 and figure 1). the result of two way anova showed that there were significant differences in the mean of bcl-2 expression among the different concentrations of ni (p < 0.0001), the mean expression of bcl-2 also varied significantly among the durations of exposure (p < 0.0001), as well as the interaction between ni concentration and duration of exposure (p = 0.021) (table 3). the result of one way anova showed that the expression of bcl-2 gingival fibroblasts exposed to 0 mg/l (control), duration of exposure among the treatment groups are 1, 3 and 7 days, there was no difference between the mean of 1 day and 3 dayexposures, while there were significant differences in the mean of 1 day and 7 days, and the mean of 3 days and 7 days. expression of bcl-2 of gingival fibroblasts exposed to 0.780 mg/l shows that the mean of exposure duration of 3 days and 7 days was not significant difference, whereas the mean and standard deviation of exposure duration of 1 day and 3 days, as well as 1 day and 7 days demonstrate significant differences. in the mean time, expression of bcl-2 gingival fibroblasts exposed to 3.002 mg/l shows that the mean exposure duration between 1 day and 3 days was not significant difference, whereas the mean of exposure duration between 1 day and 7 days, as well as 3 days and 7 days shows significant differences. expression of bcl-2 of gingival fibroblasts exposed to 6.320 mg/l shows that the mean of exposure duration between 1 day and 3 days was not varied, whereas the mean of exposure duration between 1 day and 7 days, as well as between 3 days and 7 days demonstrates significant differences (table 4 and figure 2). discussion rust was the result of corrosion, a form of metal oxides. corrosion or rusting process was an electrochemical process. restoration of nicr acts as a reductant and o2 dissolved in the saliva acts as an oxidant. rust formed on restoration accelerates the subsequent corrosion process, therefore, rust was also known as autocatalysis. oral environment was very conducive to the formation of tarnish and corrosion, as always wet and ph changes. corrosion occurs when elements in the restoration of the lost electrons and ionized, so that the ions are formed is released into the saliva. corrosion reactions in the oral cavity was aquaeus corrosion. genetic expression was an essential process activity in living organisms, as this process was the disclosure by the genes in the form of proteins, both structural proteins and protein enzymes. structural support protein cellular components to perform its function, while protein enzymes catalyze change various compounds, including the genetic expression itself. thus the process was the process of figure 1. the mean and standard deviation of bcl-2 expression in the various [ni] and duration of exposure. table 1. [ni] in the various restoration after immersion into artificial saliva for 7 days restoration [ni] (mg/l) casting 0.780 recasting 1x 3.002 recasting 2 x 6.320 table 2. the mean and standard deviation of bcl-2 expression in the various [ni] and duration of exposure [ni] (µg/l) x ± sd duration of exposure (days) 1 3 7 0 18.75±1.49 17.63±1.41 15.38±1.69 0.780 10.25±1.04 8.00±1.07 7.50±0.93 3.002 4.88±1.13 4.25±0.46 3.25±0.71 6.320 2.88±0.83 2.50±0.76 1.75±0.89 table 3. the result of two way anova of bcl-2 expression in the various [ni] and duration of exposure source df mean square f p valued corrected model 11 298.030 252.874 <0.0001 intercept 1 6272.667 5322.263 <0.0001 [ni] 3 1060.250 899.606 <0.0001 days 2 39.385 33.418 <0.0001 interaction [ni]*days 6 3.135 2.660 0.021 r2 = 0.967 days 205soesetijo and rukmo: the effect of nickel as a nickel chromium restoration corrosion product isocitrate leading to decreased atp production. disruption of mitochondrial function is followed by a decrease or loss of expression of bcl-2. transport ni + + into cells may disrupt fe + + homeostasis by inhibiting the extracellular iron into the cell, thereby inhibiting iron-dependent enzymes in the cytoplasm and mitochondria. ni++ may also replace co-factor of fe2+ in the prolyl hydroxylases, an enzyme playing a role in the activation of hif-1a.16 ni increases the activity of malate dehydrogenase (mdh) and isocitrate dehydrogenase (idh). increasing mdh and idh reflect that there is an attempt to restore the function of the citric acid cell cycle. it was also explained that ni exposure may increase the activity of glycolysis and the ratio of nadh/nad. cell oxidative phosphorylation depends on the iron to transfer electrons from nadh to oxygen, oxidative phosphorylation disruption result in the accumulation of nadh. disruption of iron homeostasis results in atp depletion and decreased expression of bcl-2.11 bcl-2 plays an important role in the regulation of programmed cell death apoptosis that was genetically regulated, where it was a balance between the expression of anti-apoptotic bcl-2 and pro-apoptotic bax. it showed a high degree of homology to bcl-2. over-expression of bax leads to heterodimer bond with bcl-2, as a result, bcl-2 functions become inhibited and apoptosis induced. whereas, if bcl-2 was over-expressed, the protein will be bond with each other in homodimer, causing the cell to survive.17 the process of apoptosis is regulated through two pathways, extrinsic pathway (cytoplasm) through the activity of fas death receptor by activating the fasfas ligand interactions (fasl), and intrinsic pathway (mitochondria), which triggers the release of sit-c which depends on the settings of bcl-2 protein as an anti-apoptotic protein and bax as pro-apoptosis protein.18 there are two key components that cause the release of sit-c, i.e. mitochondrial permeability transition pore (mptp) and apoptotic protein bax. mptp was located at the meeting point between the outer membrane and the inner mitochondrial membrane, which in an open state allowing compounds with a molecular weight less than 1.5 kd pass freely between the matrix and the cytosol. the opening of mptp is affected by ca accumulation, oxidant table 4. the result of one way anova of bcl-2 expression in the various [ni] and duration of exposure [ni] (µg/l) duration of exposure (days) p valued 1 3 7 0 18.75±1.49a 17.63±1.41a 15.38±1.69b 0.001 0.780 10.25±1.04c 8.00±1.07d 7.50±0.93d <0.0001 3.002 4.88±1.13e 4.25±0.46e 3.25±0.71f 0.002 6.320 2.88±0.83g 2.50±0.76g 1.75±0.89h 0.038 p valued <0.0001 <0.0001 <0.0001 note: vary of letter vary of significance (p valued<0.05) based on the result of multicomparison test figure 3. bcl-2 expression (magnitude 400x) notes : a. exposure of 0,780 µg/l, incubated for 7 days, showed cells strong positive expression ( ) and negative expression ( ) b. exposure of 3,002 µg/l, incubated for 7 days, showed cells weak positive expression ( ) and negative expression ( ) c. exposure of 6,320 µg/l, incubated for 7 days, showed all of the cells negative expression d. control, incubated for 7 days, showed all of the cells strong positive expression figure 2. bcl-2 expression (magnitude 400x). notes: a. exposure of 0.780 mg/l, incubated for 7 days, shower cells strong positive expression ( ) and negative expression ( ); b. exposure of 3.002 mg/l, incubated for 7 days, showed cells week positive expression ( ) and negative expression ( ); c. exposure of 6.320 mg/l, incubated for 7 days, showed all of the cell negative expression; d. control, incubated for 7 days, showed all of the cell strong positive expression. translating genetic expression of genes in the sequence of nitrogen bases form a protein amino acid sequence. bcl2 gene is one of the genes that contribute to carcinogenesis. the role of the expression of bcl-2 inhibits apoptosis by ptotecting mitochondrial transition pore, so that the sit-c was not released. the role of bcl-2 expression was in contrast to p 53, if p 53 is active, the bcl-2 was inactive, whereas if p 53 is inactive bcl-2 was active, so that apoptosis does not occur, and proliferation continues. this study suggests that there was the interaction between the ni concentrations with the exposure duration on the expression of bcl-2. the exposure to artificial saliva within 7 days may decrease the expression of bcl2. it was caused by the anion present in the electrolyte, especially clmay affect fe2+ transport into cells by forming fecl2 compounds. it may cause disruption in the activities of fe (ii)-dependent enzyme that was important in aerobic catabolism in the mitochondria that was aconitase converting citrate into 206 dent. j. (maj. ked. gigi), volume 45 number 4 december 2012: 202–207 and low mitochondrial transmembrane potential. mptp is actually too small to be by passed by sit-c (13 kd), but bonding with bax forms a channel specifically for sit-c. the bonding between bax and mptp in pore formation activity is inhibited by anti apoptotic protein bcl-2. so the balance among pro-apoptotic protein, anti-apoptosis and their interaction with mptp largely determine the cells viability.19 phagocytosis ni by cells in the region of membrane ruffling, when there was a contact between ni particles and the surface of cell membranes. the time required from the first contact up to the ni endocytosis into cells is varied, it takes about 7–10 minutes. that process shows the effort salvatory motion of cells. ni accumulation in and around the cell nucleus takes about 24–48 hours. ni inside the cell may cause dna fragmentation directly or indirectly through the formation of ros. damaged dna stimulates the p53, which will further suppress the expression of bcl-2 via induction bax.20 ni compounds dissolved may affect intracellular ca transport through a calcium ionophore ionomicyn channel, thus ni was known as calcium channel blockers. decrease in intracellular ca concentration would lead to an increase in the compensation process from the free ca supplies inside the cell (endoplasmic reticulum and mitochondria). changes in the intracellular concentration signals the changes in the expression of bcl-2.21,22 ca possesses very important role, especially, its responsibility to regulate various physiological processes as well as involvement in maintaining homeostasis in pathological conditions and immune system. therefore, it was also mentioned that the mitochondria serve as a buffer against ca ions. ca exits from endoplasmic reticulum along with sit-c release from mitochondria which was the phenomenon of apoptosis. ca is identified as a message carrier to coordinate mitochondrial and endoplasmic reticulum that interact on apoptosis through suppression of bcl-2, caspase activity, sit-c and nuclease enzyme.23, 24 statistical analysis shows the determination coefficient of expression bcl2 (r2 = 0.967). thus, in this case the ability of the independent variables (ni concentration and duration of exposure) in explaining the variance of the dependent variable (bcl-2 expression gingival fibroblasts) amounted to 97%, while the remaining 3% of variance of the dependent variable was caused by other factors. viewed from the size of the particles of each metal constructing nicr alloys, then all of them may diffuse into the cell, because the size was smaller than the ion channels or pores of the membrane (300–400 ao), which has a negative charge. when viewed from the electrode potential, the ability of the metal to retain or release electrons, the ni2 + enters first because it possesses the highest electrode potential. the electrode potential possessed by ni2+, ni2 + causes a higher affinity for electrons or negative charge than the other cathions. based on this, it may be presumed that the ni2 + enters into cells via ion channel divalent metal transporter-1 (dmt-1). the presence of ni2 + in the cell may replace co-factor in the cytosolic enzyme, especially mg 2 + co-factor required by g6pd. eviction co-factor of mg 2 + by ni2 + causes the enzyme catalytic function was lost, so the anaerobic catabolism in the cytosol may not undergo, so that the next process in the mitochondria for aerobic catabolism may not undergo either, and make the atp as a source of cellular energy may not be generated. the situation mentioned above may reduce the expression of bcl-2. another possibility was ni2 + within cells act as free radicals that can cause oxidative stress in dna. injured/damaged dna activates p 53 in the nucleus. p 53 stimulates and activates dna repair genes. if the process of dna repair does not succeed, then the p53 in the nucleus may be out into the cytosol. in the cytosol, p 53 will suppress bcl-2 via activation of bax, and eventually the cells will execute themselves through apoptosis mechanism. ni++ diffuse into the gingival fibroblasts presumably through dmt-1. in the cells, ni++ directly or indirectly, through stimulation of ros formation, cause injury/damage dna. the injured/damaged dna leads to the suppression of bcl-2 with bax activation, and eventually the cell executes themselves through the mechanism of apoptosis. it can conclude that the higher concentration of ni exposed to gingival fibroblast, and the longer incubation time will decreased bcl-2 expression. references 1. wataha jc. biocompatibility of dental casting alloy: a review. j prosthet dent 2000; 83: 223–34. 2. anusavice jk. phillips science of dental materials. 11th ed, philadelphia: wb saunders co; 2003: 653. 3. can g, alkpinar g, can a. effects of base–metal casting alloys cytoskeletal filaments in cultured human fibroblasts. int j prosthodont 2004; 17: 45–51. 4. strietzel r. recasting dental alloysa contradiction of the medical product act? 2000; available from: http:/www.bego.com/pdf/public/ e/recasting.pdf, accessed november 25, 2005. 5. al-hiyasta, darmani h. the effects of recasting on the cytotoxicity of base metal alloy. j prosthet dent 2005; 93(2): 158–63. 6. imirzalioglu p, alaaddinoglu e, �ilmaz z, oduncuoglu b. influence of recasting different types of dental alloys on gingival fibroblast cytotoxicity. j prosthet dent 2010; 107(1): 24–33. 7. nejatidanesh f, savabi o, �asdanparast a. an investigation on metallic ion release from four dental casting alloys. j prosthet dent 2005; 2(4): 168–73. 8. gulsen c, akpinar g, aydin a. the release of elements of dental casting alloy into cell culture medium and artifisial saliva. euro j dent 2007; 1: 86–9. 9. bumgardner jd, doeller j, lucas lc. effect of nickel based dental casting alloys on fibroblast metabolism and ultrastructural organization. j biomed mater 2004; 29(5): 611–617. 10. chen h, costa m. effect of soluble nickel on cellular energy metabolism in a549 cells. experiment biol med 2006; 231: 1474–80. 11. carlisle dl, pritchard de, singh j, owen bm, blankenship, orenstein jm, patierno sr. apoptosis and p53 induction in human lung fibroblast exposed to ni (ii). j toxicol 2000; 55: 60–8. 12. wang �f, shyu hw, chang �c, tseng wc. ni (ii)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ros and mitochondria mediated pathway. j toxicol pharmacol 2012; 259(2): 177–86. 207soesetijo and rukmo: the effect of nickel as a nickel chromium restoration corrosion product 13. danial nn. bcl-2 family proteins: critical checkpoints of apoptotic cell death. clin cancer research j 2007; 13: 7254. 14. mareci d, nemtoi gh, aelenei n, bocanu c. the electrochemical behaviour of various non-precious ni and co based alloys in artificial saliva. euro cells and mater 2005; 10: 1–7. 15. kokawa k, shikone t, otani t, nishiyama r, ishii �, �agi s, apoptosis and the expression of bax and bcl-2 in hyperplasia and adenocarcinoma of the uterine endometrium. am j hum gene 2001; 16: 2211–18. 16. garrick md, singleton s, vargas f, kuo hc, zao l, knopfel m,davidson t, costa m, paradkar p, roth ja and garrick lm. dmt: which metals does it transport. j biol research 2006; 39: 79–85. 17. tilly cmlj, kooy ajws, ramaekers fcs, neumann ham. bax exspression and growth behaviour of cell. j pathol 2002; 29: 79–87 18. ghobrial im, witzig te, adjei aa. targeting apoptosis pathways in cancer therapy. cancer j clinicians 2005; 55: 178–94. 19. eposti md, hatzinisirou i, mc lennan h, ralph s. bcl-2 and mitochondrial oxygen radicals: new approaches with reactive oxygen species sensitive probes. j biol chem 1999; 98: 134–43. 20. chen h, davidson t, singleton s, garick m, costa m. nickel decrease celluler iron level and convert cytosolic aconitase to iron regulatory protein 1 in a549 cells. j toxicol pharmacol 2005; 206(3): 275–87. 21. willis s, catherine ld, hinds mg, huang dcs. the bcl-2-regulated apoptotic pathway. j cells science 2003; 116: 4053–6. 22. valko m, morris h, cronin mtd. current medicinal chemistry. j med chem 2005; 12: 1161–208. 23. boehning d, patterson rl, sedagat l, glebova no, snyder sh. cytochrome binds inositol (1,4,5) triphosphate receptors, amplifying calcium-dependent apoptosis. nat cell biol 2003; 5: 1051–61. 24. mattson mp, chan sl. calcium orchestrates apoptosis. nat cell biol 2003; 5: 1041–3. 62 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 62–66 research report a comparative study of the e. faecalis antibiofilm efficacy of photoactivated curcumin, chlorophyll and riboflavin riski setyo avianti, sri kunarti and ari subiyanto department of conservative dentistry, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: photosensitisers play a vital role for reactive oxygen species (ros) production in diode laser 405 nm therapy. curcumin, chlorophyll and riboflavin have been used and viewed in several studies as effective photosensitisers for the elimination of enterococcus faecalis (e. faecalis), a persistent microorganism that may cause endodontic failure. while each has given valuable and promising results as an alternative endodontic irrigant, no study has compared the efficacy of these three natural dyes. purpose: to prove that the photosensitiser curcumin in diode laser 405 nm therapy is more effective for e. faecalis biofilm degradation than chlorophyll and riboflavin, and that a duration of irradiation of 90 seconds is more effective than 60 seconds. methods: the biofilm monospecies e. faecalis was divided into two microplates with 96-well according to the irradiation periods: 60 seconds (group 1) and 90 seconds (group 2). each group contained one control (without a photosensitiser) and three treatments were carried out by adding the photosensitisers curcumin, chlorophyll and riboflavin, where each treatment contained eleven samples. after irradiation for 60 seconds and 90 seconds, a crystal violet assay was carried out for each group. results: the one-way anova test showed a significant difference between groups based on the irradiation period. tukey’s test showed each treatment in each group also showed a significant difference (p<0.05) with curcumin as the best substance to cause biofilm degradation in both groups. the duration of the irradiation showed that the longer the biofilm was illuminated, the lower the absorbance value or optical density (od). conclusion: curcumin irradiated for 90 seconds gives better biofilm degradation on e. faecalis due to its natural properties and molecular structure, highlighting its efficacy in photodynamic therapy. keywords: chlorophyll; curcumin; enterococcus faecalis; photosensitiser; riboflavin correspondence: sri kunarti, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47 surabaya, indonesia 60132. email: sri-k@fkg.unair.ac.id introduction the success of a root canal treatment is mainly related to the process of cleaning and shaping. the use of chemicals during root canal treatment is necessary to eliminate the bacteria that colonise the dentine tubules.1 of the several chemical agents used as root canal irrigation, sodium hypochlorite (naocl) has a non-specific ability as a strong proteolytic agent in disrupting and damaging biofilms. it can also dissolve necrotic tissue. nonetheless, naocl has disadvantages. it has a bad odour, it can cause tooth discoloration when it reacts with certain antibiotics such as tetracycline or when combined with other irrigation solutions such as chlorhexidine (chx), it is corrosive to metal instruments and it can cause irritation when pushed into periapical.2 an alternative that is being developed is photodynamic therapy using laser light. the process of photodynamic therapy requires three essential components, namely a photosensitiser (ps), a light source and oxygen. a ps is a molecule that is local to the target cell or tissue and can only be activated by light.3 behind its contribution in the treatment of pathogenic microorganisms in the field of medicine and dentistry, some synthetic chemical pss such as toluidine blue (tb) have weaknesses. research says that although it can inhibit bacterial adhesion and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p62–66 mailto:sri-k@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p62-66 63avianti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 62–66 biofilm formation, irradiated ortho-toluidine (tb-o) has toxic effects on fibroblast cells.4 therefore, natural pss are now widely used in photodynamic therapy. a ps made from natural materials is claimed to have almost the same potential and function as chemically synthesised photodynamic agents, even exceeding them. in addition, safety must be considered when choosing natural pss, because these ingredients are not toxic and are safe for consumption. the product of the solution is therefore safe and environmentally friendly.5 some natural ingredients used as pss in photodynamic therapy include curcumin (cur), chlorophyll (chl) and riboflavin (rfv).6 these three ingredients, according to the literature, are non-toxic and safe materials because they do not cause adverse effects in normal cells.7–9 in this study, monospecies biofilms containing enterococcus faecalis (e. faecalis) bacteria were used. e. faecalis was chosen because a number of studies have been conducted showing that this bacterium is associated with secondary infections in the failed treatment of root canals. this species is also found in primary intraradicular infections, that is when the pulp is open because of caries, trauma or other factors that cause the integrity of the hard tooth tissue to be disrupted.10 the superior effect of curcumin as a ps in photodynamic therapy on the e. faecalis biofilm has been shown in several previous studies.11,12 however studies comparing curcumin with ps from other natural ingredients have not been conducted. the natural ps ingredients used as a comparison in this study were chlorophyll and riboflavin. chlorophyll can eliminate e. faecalis better than irrigation material chx, and ethylenediaminetetraacetic acid (edta).13 riboflavin also provides good antibacterial power against grampositive and gram-negative bacteria and fungi, including e. faecalis.14 curcumin, chlorophyll and riboflavin were chosen as ps in this study because all three of these materials have been used in many photodynamic therapy studies mainly to overcome chronic and recurrent infections caused by pathogenic microorganisms. curcumin, chlorophyll and riboflavin are natural compounds that when irradiated with light will produce reactive oxygen species (ros), which are effective in causing damage to cell structures and bacterial biofilms. the purpose of this study is to prove that ps curcumin with 405 nm diode laser irradiation has a better biofilm degradation ability against e. faecalis than ps chlorophyll and riboflavin. materials and methods this study was approved by the ethics commission of the faculty of dental medicine, universitas airlangga, with number 022/hrecc.fodm/i/2020. this type of research is a purely experimental laboratory research post test control group design. the sample in this study was the e. faecalis biofilm which was cultured from the atcc 29212 bacterial stock by making an e. faecalis colony suspension in the brain heart infusion broth (bhib) medium. the sample size was obtained by the federer formula, and each treatment group had a sample size of 11. the formation of the in vitro biofilm e. faecalis was carried out by culturing bacterial cells in 4 ml of bhib media for 24 hours at 37°c. these cells were then harvested by centrifugation at 16,000 rpm for 5 minutes at 4°c and washed twice with sterile bhib. a suspension of bacterial cells was obtained with a density of 1.5 × 108 cells/ml (0.5 mcfarland standard), then as much as 200 ml of sediment was taken with a pipette and put on 96-well polystyrene microtiter plate. the plate was then covered with parafilm and incubated for 48 hours at 37°c without agitation. curcumin ps material was prepared by dissolving standard curcumin with a molecular weight of 368.38 g/ mol (c1386, sigma-aldrich, merck kgaa, darmstadt, germany) in a 5% polyethylene glycol solution (sigmaaldrich, merck kgaa, darmstadt, germany), until it reached a concentration of 2.5 mg/ml. it was maintained at ph 6.12 meanwhile, the chlorophyll ps comes from alfalfa, medicago sativa l. (k-link liquid chlorophyll, south jakarta, indonesia), with a concentration of 1.6mg/ ml in a normal saline solution.14 the riboflavin ps was made by dissolving 25 grams of standard riboflavin, with a molecular weight of 376.36 g/mol (47861, supelco, merck kgaa, darmstadt, germany), in a 25 ml (1g/ml) sterile phosphate buffer saline (pbs) solution. it was then stored in a dark room.15 after incubating each 1.5 × 108 cfu/ml suspension on a sterile microplate at 37ºc for 48 hours, the well microplate was filled with each of the following materials: a) the control group (+): 20μl e. faecalis biofilm, 90μl bhib; b) treatment group i: 20μl e. faecalis biofilm, 90μl bhib, 90μl ps curcumin; c) treatment group ii: 20μl e. faecalis biofilm, 90μl bhib, 90μl ps chlorophyll; and d) treatment group iii: 20μl e. faecalis biofilm, 90μl bhib, 90μl riboflavin ps. the microplate was labelled for 60 seconds (group i) and 90 seconds (group ii). each treatment group was numbered to 11 so that each microplate had 44 wells filled. before irradiation, the two microplates were incubated in a dark room for 5 minutes at room temperature (25 ± 2ºc). the microplate containing the control group (+) and treatments i, ii, and iii was then inserted into a laser diode computer numerical control (cnc biophysics laboratory, faculty of science and technology, airlangga university, surabaya) for irradiation with a wavelength of 405 nm. the instrument was set at a 10 mm irradiation distance, a 60 second irradiation duration for microplate 1 and a 90 second duration for microplate 2. after the irradiation, the microplate was incubated in an anaerobic jar for 48 hours. the crystal violet (cv) test began with media removal and the gentle rinsing of the plate with 200μl pbs. then, staining with 0.1% cv 50μl took place in order to dye the bacterial biofilm at room temperature for 15 minutes. after that, the cv was discarded and rinsed with pbs dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p62–66 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p62-66 64 avianti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 62–66 200μl. the plates were added with 96% 50μl ethanol and left for 10 minutes at room temperature to dry. then, each microplate well was filled with 33% 150μl acetic acid. the plate was then inserted in the microplate reader to determine the degradation of the biofilm. this was measured by calculating the absorbance of the solution on the microplate reader with a wavelength of 570nm. the absorbance value produced by the microplate reader (epoch, biotek instruments, inc., usa) showed the absorption of light received by the biofilm formed on the well wall of the microplate with optical density (od) units. the results of the study were then analysed using the statistical package for the social sciences software (spss statistics 17.0, ibm, usa) by conducting normality, homogeneity and one-way anova tests. results the results of this study is absorbance values or optical density (od) and biofilms formation (figure 1). the data that shows the description and the analysis of the results in this study can be seen in table 1. the one-way anova test was carried out to compare between the irradiation duration groups and all treatments in each group. there were significant differences between the irradiation length groups, which were shown through the one-way anova test with p = 0.000. in the treatment group with a 60-second irradiation period, because the variance with the levene test showed an inhomogeneous result, p = 0.008 (p < 0.05), a non-parametric (kruskal– wallis) test was carried out and obtained a significant 60 seconds 90 seconds laser diode+riboflavin laser diode+curcumin laser diode+chlorophyll laser diode average of optical density (od) 1,355.82 1,039.18 1,798.00 2,835.73 1,564.27 1,153.73 1,935.73 2,849.18 figure 1. a diagram of the mean optical density (od) of each ps with a long irradiation of 60 seconds and 90 seconds. table 1. description and data analysis of the results of a comparative study of ps type and exposure time type of treatment mean ± std. deviation comparison between groups comparison of treatments between groups comparison between treatments in each group group 1 (irradiation of 60 sec) laser diode 2,849.18 ± 0.020663 * ** *** laser diode + curcumin 1,153.73 ± 0.035429 laser diode + chlorophyll 1,935.73 ± 0.067088 laser diode + riboflavin 1,564.27 ± 0.027821 group 2 (irradiation of 90 sec) laser diode 2,835.73 ± 0.037990 * * *** laser diode + curcumin 1,039.18 ± 0.034289 laser diode + chlorophyll 1,798.00 ± 0.063786 laser diode + riboflavin 1,355.82 ± 0.048410 note: significance values for comparing between groups were obtained through the one-way anova test with p < 0.05*; intergroup 1 treatment was obtained through the kruskal–wallis test with p < 0.05**, and intergroup 2 treatment was obtained through the one-way anova test with p < 0.05. while the comparison between treatments in each group was obtained through the tukey/hsd test with p < 0.05***. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p62–66 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p62-66 65avianti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 62–66 result p = 0.000. in the treatment group with a 90 second irradiation time, the anova test results showed significance, which was p = 0.000. furthermore, the tukey test in each time group showed a significant difference in each treatment, p = 0.000. ps curcumin had the best antibiofilm effect, followed by riboflavin and chlorophyll (p < 0.05). discussion the results of this study indicate that curcumin has the best antibiofilm effect, which is indicated by the lowest average optical dentisy (od), followed by riboflavin and chlorophyll. this is because curcumin has the lowest molecular weight, which is 368.38 g/mol so it has a better diffusion ability. consequently, in the same incubation period, curcumin can be present in biofilms in greater amounts than riboflavin and chlorophyll with molecular weights of 376.36 g/mol and 893.51 g/mol16 respectively. to carry out its function as an antibiofilm, ps in photodynamic therapy can work in two ways: destroying the biofilm matrix, or entering further past extracellular polymeric substance (eps) and damaging the microbial cells in it. penetration of ps material further into the area will produce massive damage.17 therefore, diffusion of ps, its localisation and the duration of irradiation are all important in photodynamic therapy. diffusion is the movement of a substance from a high concentration to a low concentration. ps diffusion is related to the molecular weight of the material. the higher the molecular weight of a ps, the slower it diffuses into cells/tissues.18,19 this may be the reason why curcumin is more effective in causing damage to the e. faecalis biofilm structure than chlorophyll and riboflavin. the duration of irradiation and the characteristics of the ps become important factors that influence the outcome of photodynamic therapy. based on previous studies, the irradiation duration that was effective in causing damage to the e. faecalis cell wall was 60 seconds and 90 seconds.13 in this study, there were significant differences between the time groups of 60 seconds and 90 seconds. each ps showed a decrease in e. faecalis biofilm density with an increasing duration of irradiation. the penetration of ps material in order to induce intracellular damage depends on the length of the incubation before the irradiation, which gives the ps time to interact with the cells. therefore, ps material must be left at least 5 minutes to allow time for ps material to penetrate the cell/tissue so that the photodynamic process can work optimally. however, in this study, each ps has not reached the maximum inhibitory power against e. faecalis biofilm. this is because the biofilm structure is more complex than the planktonic cells and thus requires a longer incubation time for the ps material penetration. diogo et al.13 recommends allowing an incubation period of 15 minutes before irradiation. the superior ability of curcumin to damage biofilms is in line with several previous studies. neelakantan et al.11 concluded in their study that light-activated curcumin provides an excellent antibiofilm effect, even more effective in killing bacteria in the root canal, including e. faecalis, than light-activated naocl. other studies suggest that light-activated curcumin has a very good antibiofilm effect on e. faecalis when compared with some commonly used intracanal medicaments such as antibiotic paste (trimix), 2% chlorhexidine, and calcium hydroxide.12 masoule et al.20 investigated the antibacterial and antibiofilm effects of curcumin on two endopathogenic species e. faecalis and pseudomonas aeruginosa, both in the form of planktonic bacteria and biofilms. the results when doing antibacterial photodynamic therapy (apdt) with ps curcumin showed a significant decrease in cell viability and od biofilm when compared with the administration of curcumin or irradiation with a light-emitting diode (led) alone. the advantage of curcumin as a ps is the presence of three reactive functional groups, a ketone and two phenol groups that play a role in the phototherapeutic activity. the phenol group is an essential factor in the interaction of the curcumin and the biological membrane where the attachment of the hydrogen group plays an important role.21 biological chemical reactions that occur are as follows: hydrogen donations that cause oxidation, the addition of reversible and irreversible nucleophilic (michael reaction), hydrolysis, degradation, and enzymatic reactions.22 research using curcumin as a ps for root canal disinfection was first reported to show that the canal curcumin base in the presence of two conjugated electron systems made curcumin an effective ps.11 riboflavin, vitamin b2 and flavin (as part and product of degradation) are pss that are efficient in inducing oxidative damage to tissue exposed to light. phenolic, n-heterolytic amino acids and their peptides and proteins deactivate triplet-excited riboflavin states in controlled diffusion processes, which efficiently compete with deactivation by oxygen, resulting in the degradation of direct proteins (referred to as type i) through electron transfer or protonelectron pair transfer.23 chlorophyll is an important pigment that plays a role in the process of photosynthesis. chlorophyll is very efficient at absorbing light and sending excitation energy to the photosystem reaction centre before a photosynthetic electron transport occurs. excited chlorophyll will produce singlet oxygen, which can cause severe cellular damage. the chlorine portion obtained in chl-a is a conjugated π-tetrapyrrole group that acts as a potential phototoxic chromophore.24 moreover, pss with tetrapyrrole groups (except bacteriochlorin) tend to produce oxygen singlet (type ii), which is more dominant than ros (type i), which is usually produced by pss with other structures, in photodynamic therapy.6 the three ps materials used in this study can produce an effective ros in causing biofilm damage, which is characterised by a lower optical density (od) value than dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p62–66 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p62-66 66 avianti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 62–66 the control group. the penetration of ps material into the structure of biofilms, matrices and the microbial cells in it, is not a major factor. wherever the ps is located, ros will be generated during the irradiation to initiate the second step, namely multitarget oxidative damage. the abundant production of ros will weaken the antioxidant defences of microbial cells. as soon as ros is produced, it will attack many related cell molecules, including targets in the biofilm matrix (polysaccharides), on the cell surface (lipids) and in the cells (proteins and dna), which results in the collapse of the biofilm matrix and the disintegration of microbial cells.17 it can be concluded that curcumin as a ps in photodynamic therapy gives a better antibiofilm effect on e. faecalis compared to chlorophyll and riboflavin. in addition, the 90-second irradiation duration of all pss was shown to be more effective in the degradation of e. faecalis biofilms. this relates to the nature of curcumin and its molecular structure, which enhance its function in photodynamic therapy. however, further research needs to be carried out in order to identify and compare the production of ros from each ps. references 1. prażmo ej, godlewska ar, sałkiewicz m, mielczarek ab. effects of 980 nm diode laser application protocols on the reduction of enterococcus faecalis intracanal biofilm: an in vitro study. dent med probl. 2017; 54(4): 333–8. 2. kumavath rn. antibacterial agents. kumavath rn, editor. croatia: intech; 2017. p. 89–90. 3. wan mt, lin jy. current evidence and applications of photodynamic therapy in dermatology. clin cosmet investig dermatol. 2014; 7: 145–63. 4. li x, liu z, liu h, chen x, liu y, tan h. photodynamic inactivation of fibroblasts and inhibition of staphylococcus epidermidis adhesion and biofilm formation by toluidine blue o. mol med rep. 2017; 15(4): 1816–22. 5. sperandio ff, huang y-y, hamblin mr. antimicrobial photodynamic therapy to kill gram-negative bacteria. recent pat antiinfect drug discov. 2013; 8(2): 108–20. 6. abrahamse h, hamblin mr. new photosensitizers for photodynamic therapy. vol. 473, biochemical journal. portland press ltd; 2016. p. 347–64. 7. mandrol ps, bhat k, prabhakar ar. an in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts. j indian soc pedod prev dent. 2016; 34(3): 269–72. 8. fiedor l, zbyradowski m, pilch m. tetrapyrrole pigments of photosynthetic antennae and reaction centers of higher plants: structures, biophysics, functions, biochemistry, mechanisms of regulation, applications. in: advances in botanical research. academic press inc.; 2019. p. 1–33. 9. pinto jt. riboflavin. adv nutr. 2016; 7(5): 973–5. 10. singh h. microbiology of endodontic infections. j dent oral heal. 2016; 2(5): 1–4. 11. neelakantan p, cheng cq, ravichandran v, mao t, sriraman p, sridharan s, subbarao c, sharma s, kishen a. photoactivation of curcumin and sodium hypochlorite to enhance antibiofilm efficacy in root canal dentin. photodiagnosis photodyn ther. 2015; 12(1): 108–14. 12. devaraj s, jagannathan n, neelakantan p. antibiofilm efficacy of photoactivated curcumin, triple and double antibiotic paste, 2% chlorhexidine and calcium hydroxide against enterococcus fecalis in vitro. sci rep. 2016; 6: 1–6. 13. diogo p, fernandes c, caramelo f, mota m, miranda im, faustino maf, neves mgpms, uliana mp, de oliveira kt, santos jm, gonçalves t. a ntim icrobial photodynam ic therapy aga inst endodontic enterococcus faecalis and candida albicans mono and mixed biofilms in the presence of photosensitizers: a comparative study with classical endodontic irrigants. front microbiol. 2017; 8: 1–11. 14. ahgilan a, sabaratnam v, periasamy v. antimicrobial properties of vitamin b2. int j food prop. 2016; 19(5): 1173–81. 15. astuti sd. an in-vitro antimicrobial effect of 405 nm laser diode combined with chlorophylls of alfalfa (medicago sativa l.) on enterococcus faecalis. dent j (majalah kedokt gigi). 2018; 51(1): 47–51. 16. american chemical society. chlorophyll american chemical society. 2019. available from: https://www.acs.org/content/acs/en/ molecule-of-the-week/archive/c/chlorophyll.html. accessed 2020 mar 5. 17. hu x, huang yy, wang y, wang x, hamblin mr. antimicrobial photodynamic therapy to control clinically relevant biofilm infections. front microbiol. 2018; 9: 1–24. 18. clark ma, choi j, douglas m. passive transport. in: biology 2e. houston: openstax biology 2nd edition; 2018. p. 355–81. 19. yecyec jda. effect of molecular weight in the rate of diffusion. ukessays. 2018. available from: https://www.ukessays.com/essays/ sciences/effect-molecular-weight-rate-diffusion-1198.php?vref=1. accessed 2020 mar 5. 20. masoule sf, pourhajibagher m, khoobi m, safari j. photodynamic inactivation of endopathogenic microbiota using curcuminmediated antimicrobial photodynamic therapy. j sci islam repub iran. 2018; 29(3): 205–9. 21. haukvik t, bruzell e, kristensen s, tønnesen hh. a screening of curcumin derivatives for antibacterial phototoxic effects studies on curcumin and curcuminoids. xliii. pharmazie. 2011; 66(1): 69–74. 22. jankun j, wyganowska-swiatkowska m, dettlaff k, jelinska a, surdacka a, watróbska-swietlikowska d, skrzypczak-jankun e. determining whether curcumin degradation/condensation is actually bioactivation (review). int j mol med. 2016; 37(5): 1151–8. 23. cardoso dr, libardi sh, skibsted lh. riboflavin as a photosensitizer. effects on human health and food quality. food funct. 2012; 3(5): 487–502. 24. kashiyama y, yokoyama a, kinoshita y, shoji s, miyashiya h, shiratori t, suga h, ishikawa k, ishikawa a, inouye i, ishida ki, fujinuma d, aoki k, kobayashi m, nomoto s, mizoguchi t, tamiakia h. ubiquity and quantitative significance of detoxification catabolism of chlorophyll associated with protistan herbivory. proc natl acad sci. 2012; 109(43): 17328–35. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p62–66 https://www.acs.org/content/acs/en/ https://www.ukessays.com/essays/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p62-66 vol 38 no 3 2005 124 peran komunikasi interpersonal dalam pelayanan kesehatan gigi (the role of interpersonal communication integrated with medical dental care) hanindio soelarso, roesanto heru soebekti, dan achmad mufid bagian ilmu kesehatan gigi masyarakat fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract this article is a literature study, concerning the interpersonal communication role in conjunction with technical dental and oral health care conducted by the dentists towards the patient individually. in addition, interpersonal communication means to be synergic communication among dentist and the patient. in relation with the verbal or non verbal dental care process, the effectiveness of interpersonal communication is identified through the perception of the messages and it’s translated by recipient perception, and it will be the same meaning as the messager’s perception. in this case, the dentist and the patient will be capable to send or accept mutual messages as messanger and message recipient. in conclusion, in the dental and oral medical care on the procedure point of view, the similar perception determines very much the successfulness of the wholedental and oral health care process toward diagnosis, determination therapy plan, treatment and post treatment process. key words: interpersonal communication, dentist and patient communication koresponsdensi (corresponsdence): hanindio soelarso, bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan telah diketahui bahwa masalah kesehatan gigi dan mulut di indonesia sampai saat ini merupakan masalah klasik, ini ditandai dengan angka prevalensi karies gigi dan penyakit periodontal masih tetap tinggi. artinya bila kita amati pergeseran pola penyakit gigi secara epidemiologis walaupun ditunjang oleh teknologi perawatan canggih dan sistem pelayanan kesehatan gigi yang terpadu, angka kesakitan gigi mulut sampai saat ini tetap sama. dari aspek penyelenggara layanan kesehatan gigi, salah satu faktor pendukung klasik masalah kesehatan gigi dan mulut adalah layanan medis teknis kesehatan gigi dan mulut yang tidak optimal, sebagai contoh: kegagalan hasil perawatan, timbulnya masalah kesehatan gigi baru, rasa sakit yang tidak reda, penderita tidak puas terhadap hasil pelayanan medik gigi yang diterimanya. hasil penelitian yang dilakukan pada pekerja industri pertanian di northwest, inggris tentang hubungan antara pola kunjungan mereka ke dokter gigi dengan status kesehatan gigi, disimpulkan bahwa meningkatnya frekuensi kunjungan ke dokter gigi untuk mendapatkan pelayanan medik gigi memang benar dapat menghilangkan risiko kehilangan gigi, tetapi tidak dapat mencegah timbulnya masalah kesehatan gigi baru, seperti karies sekunder dan karies gigi yang lain.1 evaluasi terhadap 48 molar sulung yang ditumpat glass ionomer kelas ii di klinik pedodontia fakultas kedokteran gigi universitas airlangga didapatkan bahwa, 33% diantaranya mengalami kegagalan perawatan setelah 6 bulan ditumpat.2 evaluasi klinis satu tahun setelah tumpatan resin komposit kelas vi yang dilakukan rahardjo dkk.3 di klinik konservasi gigi fakultas kedokteran gigi universitas airlangga mendapatkan 34% dari 30 subyek yang ditumpat mengalami kegagalan perawatan. dari jumlah tersebut 50% disebabkan adanya karies sekunder, sisanya karena tumpatan yang patah. jika diamati menurut prosesnya, pelayanan medik gigi dan mulut dapat dipandang sebagai transaksi terapeutik antara dokter gigi dengan penderita. transaksi terapeutik didefinisikan sebagai transaksi untuk menentukan terapi yang paling tepat terhadap penderita dan sebagai transaksi berarti mengandung kegiatan komunikasi antara dokter gigi dengan penderita.4 dari pandangan ini salah satu penyebab pelayanan medik gigi yang tidak optimal adalah proses komunikasi yang tidak optimal. menurut teori komunikasi, komunikasi yang terjadi selama transaksi terapeutik adalah komunikasi interpersonal. naude cit. santosa5 menyebutkan bahwa pada proses pelayanan medik gigi terjalin suatu hubungan kerja sama antara dokter gigi dengan penderitanya yang dikenal dengan komunikasi interpersonal. komunikasi interpersonal adalah komunikasi yang terjadi antara dua orang atau lebih, bisa secara verbal dan nonverbal proses ini dapat terjadi secara langsung tanpa melibatkan media komunikasi sebagai penghantar pesan. dalam proses komunikasi interpersonal sumber komunikasi 125soelarso, dkk.: peran komunikasi interpersonal (komunikator) dan penerima pesan (komunikate) dapat berganti peran, pergantian peran seperti ini disebut komunikan, dan proses pergantian peran tersebut dapat timbul saat itu juga (saat yang sama) misalkan bila ada interupsi.6 pendekatan layanan medik kesehatan gigi lebih ditekankan pada layanan medik gigi secara perorangan yang meliputi layanan medik dan layanan administratif. karena merupakan layanan medik gigi perorangan, maka titik awalnya adalah pertemuan antara dokter gigi dengan penderitanya dan titik akhirnya adalah tindakan medik gigi yang sudah dinyatakan selesai oleh dokter gigi. dengan demikian sebenarnya secara episodik layanan medik gigi meliputi: episode layanan administrasi (pencatatan identitas penderita), episode rencana perawatan, perawatan, dan pasca perawatan. namun demikian mengingat kompleksitas layanan medik gigi dalam setiap episode, maka dalam kajian ini lebih diarahkan pada layanan medik dasar atau layanan medik gigi tingkat pertama yang diharapkan dapat menggambarkan layanan medik gigi secara keseluruhan, termasuk layanan spesialistik. tujuan yang ingin diwujudkan dalam kajian ini adalah selain penanganan medik gigi dengan teknologi modern dan perawatan yang canggih, diperlukan suatu instrumen lain dengan mengoptimalkan komunikasi interpersonal antara dokter gigi sebagai provider dan penderita sebagai konsumer, sehingga diharapkan dapat menumbuhkan kesadaran providers akan pentingnya komunikasi interpersonal pada pelayanan medik teknis gigi dan mulut dengan harapan angka kesakitan gigi secara epidemiologis mengalami pergeseran (menurun secara signifikan). konunikasi interpersonal komunikasi interpersonal diterjemahkan sebagai komunikasi yang terjadi antara dua orang atau lebih, di mana komunikator sebagai sumber komunikasi yang mengirim pesan dan komunikate sebagai penerima pesan. pesan tersebut diterima oleh komunikate sebagai stimulus dan komunikate memberikan jawaban berupa respons, demikian juga bisa terjadi sebaliknya. menurut rakhmat, 6 karakteristik komunikasi interpersonal adalah proses komunikasi terjadi tanpa melalui media komunikasi, sehingga dalam proses komunikasi interpersonal mempunyai ciri sebagai berikut: 1) pesan dari komunikator tidak terbatas pada pesan verbal tetapi juga pesan nonverbal seperti ekspresi wajah, gerakan anggota tubuh, sehingga pesan tersebut mempunyai makna yang beragam, selanjutnya dapat menimbulkan respons yang beragam pula. sangat mungkin stimulus yang paling menonjol yang diterima komunikate adalah ekspresi wajah (nonverbal) tanpa disadari oleh komunikator, sehingga respons yang diterima komunikator tidak sesuai dengan isi yang diinginkannya; 2) komunikate dapat berganti peran sebagai komunikator pada saat yang bersamaan (selama proses komunikasi berlangsung), contoh munculnya interupsi. pergantian peran secara bersamaan merupakan suatu mekanisme umpan balik (feed back mecanism), sehingga kemungkinan hambatan komunikasi tidak dapat segera diketahui oleh kedua belah pihak. secara psikologis selama proses komunikasi interpersonal berlangsung maka dalam diri komunikan akan terjadi proses sensasi, memori, persepsi, dan berpikir. keempat proses ini merupakan tahapan ketika seorang menerima pesan hingga menghasilkan respons.6 sensasi adalah saat stimulus ditangkap oleh indera manusia (senses) selanjutnya dirubah menjadi impuls melalui syaraf dan dipahami oleh otak manusia.6 oleh sebab itu ketepatan dan kecepatan pemahaman terhadap stimulus tergantung kepekaan indera manusia, sehingga ini berpengaruh pada proses selanjutnya. proses persepsi akan melibatkan memori dan proses berpikir, karena persepsi merupakan proses ketika otak manusia memberi makna atau menafsirkan stimulus. sedangkan memori adalah proses menyimpan informasi yang dapat dipakai sebagai kerangka rujukan (frame of references), dan akan dikeluarkan kembali bila informasi tersebut dibutuhkan. selanjutnya berpikir adalah suatu proses untuk menetapkan keputusan, memecahkan masalah dan memproduksi respons.6 ketika proses komunikasi interpersonal berlangsung keempat tahap ini berjalan tanpa dapat dirasakan atau diraba, walaupun terjadi dalam satu episode, sebagai contoh: ketika seorang dokter gigi bertanya kepada pasiennya tentang berapa lama merasakan sakit gigi, atau seberapa parah sakit gigi yang dideritanya, maka persepsi penderita dalam mengelola stimulus tersebut sangat tergantung dari kerangka rujukan yang ada di memorinya, apa yang dimaksud dengan berapa lama sakitnya atau seberapa parah sakitnya. hal ini sangat menentukan dalam menciptakan makna pesan pada proses persepsinya. dari contoh tersebut dapat dicermati pentingnya proses persepsi dalam optimalisai komunikasi interpersonal. sehingga bila keliru dalam mempersepsi stimulus maka respons yang diproduksi tidak sesuai dengan makna pesan yang diinginkan dokter giginya. desiderato cit. rakhmat6 menyatakan bahwa proses persepsi juga melibatkan atensi (perhatian), ekspektasi (harapan), motivasi, dan memori, yang semua ini sangat berpengaruh dalam memproduksi respons. rakhmat 6 membagi faktor penentu ketepatan persepsi menjadi faktor struktural dan faktor personal. faktor struktural sebagai faktor yang berada di luar diri komunikate, oleh karena itu sangat berperan terhadap kualitas pesan (stimulus), sebagai contoh: kualitas pertanyaan dokter gigi merupakan faktor struktural yang sangat menentukan persepsi penderitanya. sedangkan faktor personal adalah berbagai faktor yang ada dalam diri komunikate, seperti: emosi, mood, sugesti serta kerangka rujukan komunikate.6 sebagai contoh: bila penderita tidak mempunyai kebutuhan tinggi terhadap kesembuhan sakit giginya, atau jika penderita takut terhadap perawatan gigi maupun figur dokter giginya, atau jika penderita miskin 126 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 124–129 akan kerangka rujukan, maka besar kemungkinan pertanyaan dokter gigi terhadap kondisi sakit gigi dan rencana perawatan gigi akan dipersepsi secara tidak tepat oleh penderitanya. oleh karena itu efektifitas komunikasi interpersonal sangat bergantung pada persepsi interpersonalnya.6 naude cit. santosa5 menyatakan bahwa instruksi dan informasi dapat diterima dengan baik oleh penderita, jika ia memberikan perhatian penuh terhadap informasi, pengertian atas pesan serta penerimaan pesan secara psikologis. pelayanan medik kesehatan gigi dan mulut pelayanan medik kesehatan gigi dan mulut adalah bagian dari pelayanan kesehatan (health care services) dan didefinisikan sebagai setiap upaya kesehatan secara mandiri atau bersama dalam suatu organisasi untuk memelihara derajat kesehatan yang ditujukan kepada individu, keluarga, kelompok dalam masyarakat.7 menurut jenisnya pelayanan kesehatan dibedakan menjadi dua yaitu, pelayanan kedokteran (medical services) dan pelayanan kesehatan masyarakat (public health services), yang membedakan antara kedua jenis pelayanan kesehatan tersebut adalah cara pengorganisasiannya.8 pelayanan kedokteran bersifat individu (solo practice) atau bersama-sama dalam suatu organisasi (institusi) seperti rumah sakit dan klinik swasta, sebagai sasaran adalah individu atau keluarga. tujuannya untuk menyembuhkan penyakit atau memulihkan kesehatan. sedangkan pelayanan kesehatan masyarakat pengorganisasiannya secara bersama dan tujuannya untuk memelihara dan meningkatkan derajat kesehatan masyarakat serta pencegahan terhadap penyakit. pelayanan kedokteran dikaji dari kemampuan memberikan pelayanan, dibedakan menurut tiga tingkatan, yaitu: pelayanan tingkat pertama (primary health services), pelayanan tingkat kedua (secondary health services), dan layanan tingkat ketiga (tertiary health services). layanan tingkat pertama adalah layanan kesehatan pokok (basic health services) sebagai contoh layanan rawat jalan (ambulatory atau out patient services). sedangkan tenaga medis yang dibutuhkan pada layanan tingkat pertama adalah dokter umum (general practitioner). layanan kesehatan tingkat kedua adalah layanan kesehatan lebih lanjut (layanan spesialis), dan ini dibutuhkan tenaga dokter spesialis. sedangkan layanan kesehatan tingkat ketiga adalah layanan kesehatan lebih kompleks, dan untuk penyelenggaraannya dibutuhkan dokter superspesialis.8–10 seperti telah diketahui bahwa layanan kesehatan gigi dan mulut merupakan subsistem layanan kesehatan umum, artinya uraian dan pembidangan pelayanannya berlaku pula untuk layanan kesehatan gigi. sebagai bentuk layanan medik karakteristik layanan kesehatan gigi dan mulut dapat dikaji menurut cara pendekatannya (characteristic approach) terhadap perorangan maupun kepada kelompok masyarakat. pola pendekatan perorangan akan terjadi tatap muka secara langsung antara tenaga medis (dokter gigi) dengan individu (penderita) tanpa menggunakan media komunikasi, hal ini merupakan ciri atau karakteristik penting dalam layanan medik gigi. pandangan ini tercermin dalam definisi transaksi terapeutik yang dinyatakan sebagai suatu perjanjian layanan medik untuk upaya penyembuhan penderita.4 ini sesuai dengan pernyataan naude cit. santosa5 bahwa layanan medik kesehatan gigi dan mulut akan terjadi komunikasi interpersonal. pelayanan kesehatan gigi dan mulut merupakan suatu proses yang berlangsung dalam suatu kurun waktu tertentu, dimana titik awal dari proses tersebut adalah pertemuan penderita dengan dokter gigi hingga titik akhir proses yaitu tindakan layanan medik sudah dianggap selesai oleh dokter gigi. sehingga tatap muka antara dokter gigi dengan penderita berlangsung selama kurun waktu proses perawatan medik kesehatan gigi. secara teknis proses layanan medik kesehatan gigi dan mulut dapat dipilah dalam beberapa episode, antara lain: episode layanan administrasi, episode diagnosis, episode rencana perawatan, episode pelaksanaan perawatan dan episode pasca perawatan. nurdjanah cit. kristina11 dalam tulisannya tentang panduan kegiatan komunikasi terapeutik menyatakan bahwa komunikasi terapeutik dilaksanakan sejak pertemuan pertama (fase prainteraksi) hingga akhir layanan medik (fase terminasi). pembahasan telah diketahui bahwa karakteristik pendekatan (approach) layanan medik gigi dan mulut secara perorangan (individual) terjadi proses komunikasi interpersonal, melalui beberapa episode yaitu: episode adminisitrasi medik, diagnosis, rencana perawatan, perawatan, dan pasca perawatan. kualitas proses komunikasi interpersonal antara dokter gigi dengan penderitanya merupakan salah satu instumen penting agar proses maupun hasil layanan medik gigi dan mulut menjadi optimal. pendapat ini didasari oleh beberapa fakta sebagai berikut: 1) dinyatakan bahwa optimalisasi proses dan hasil layanan medik gigi dan mulut sebagian besar tergantung pada respons penderitanya, beberapa bentuk respons penderita yang dimaksud adalah: a) jawaban penderita terhadap pertanyaan dokter giginya dalam rangka episode administrasi medik, diagnosis, penetapan rencana perawatan, proses perawatan maupun dalam kerangka membangun peran serta penderita; b) informasi dari penderita tentang status dan riwayat penyakit gigi dan mulut, pertanyaan tentang rencana dan proses perawatan, proses kesembuhan hasil perawatan; c) peran serta dan sifat kooperatif penderita terhadap seluruh proses perawatan gigi dan mulut. 127soelarso, dkk.: peran komunikasi interpersonal ketergantungan kepada respons penderita terlihat pada seluruh episode layanan medik gigi dan mulut,misalkan pada episode diagnosis kelainan medik gigi dan mulut, diperkirakan bahwa sebagian besar validitas metode untuk mendiagnosa kasus kesehatan gigi terutama karies gigi dan kelainan periodontal sangat bergantung pada respons penderita terhadap stimulus yang diterimanya. artinya bahwa walaupun semua metoda pemeriksaan untuk menunjang diagnosis seperti pemeriksaan intra oral, rontgenologis dan histopatologis sebesar 100%, maka diasumsikan 80% diantara metoda tersebut validitasnya bergantung pada respons penderita. ini sejalan dengan pendapat bender,12 pollock et al.,13 maupun cailleleau et al.14 yang menggunakan electric pulp vitalitester menunjukkan besarnya kepekaan jaringan pulpa subyek penelitian pada skala vitaliteser pada dasarnya bergantung pada respons (rasa linu) dari subyek penelitian ketika gigi mereka diberi stimulus listrik. wong and baker cit. haroen15 menggunakan metode pengukuran kualitas rasa nyeri gigi dengan menggunakan alat ukur visual analog scale (vas) untuk mengukur, lalu diterjemahkan dengan menggunakan skala garis kontinum (uni dementional check list) dengan ukuran skala 1 hingga 3. rasa nyeri ringan berada pada skala 1, rasa nyeri sedang berada pada skala 2 dan rasa nyeri berat berada pada skala 3. pengukuran dilakukan dengan cara meminta penderita memilih skala pada vas sesuai dengan penafsiran (perception) nyeri gigi akut yang sedang dirasakan. tentu saja validitas hasil ukur metode ini bergantung pada kecermatan dan kecepatan penafsiran penderita terhadap rasa nyeri akutnya, dan atas dasar penafsiran inilah penderita menghasilkan respons berupa pilihan skala vas. penelitian mc. daniel16 tentang kesediaan penderita untuk menceritakan kelainan sistemik yang diidapnya sampai pada kesimpulan, bahwa penderita usia dewasa enggan memberikan informasi secara benar tentang penyakitnya kepada dokter, karena mereka berpendapat bahwa layanan medik gigi dan mulut tidak memerlukan informasi penyakit sistemik. padahal diketahui bahwa sangat mungkin terdapat kontra indikasi pemberian obat pada perawatan gigi mereka yang berkaitan dengan riwayat penyakitnya. dari penelitian ini dapat dilihat bahwa respons penderita dalam bentuk riwayat penyakit ikut berperan dalam menetapkan rencana perawatan gigi dan mulut. runkat17 maupun delcanho18 menyatakan bahwa untuk mendapatkan informasi dari penderita tentang riwayat penyakit termasuk kebiasaan yang berkaitan dengan fungsi oklusi dan artikulasi merupakan salah satu metode untuk membantu menegakkan diagnosa kelainan sendi temporo mandibular. soenartyo 19 bahkan menyatakan pentingnya mendapatkan informasi penderita tentang kondisi sosial, situasi rumah (housing) dan pekerjaan untuk keperluan menegakkan diagnosa kelainan sendi mandibula tersebut. pemahaman penderita terhadap tentang penyebab kelainan mandibula dan peran sertanya untuk mengikuti petunjuk dokter giginya merupakan salah satu faktor penentu keberhasilan perawatan kelainan sendi temporo mandibular. widodo20 dalam tulisannya tentang paradigma baru perawatan endodontik, menyatakan bahwa diagnosis keradangan jaringan pulpa yang dilakukan sampai saat ini lebih berdasarkan atas keluhan rasa sakit atau respons penderita saja, ini disebabkan jaringan pulpa merupakan jaringan ikat yang berada di dalam jaringan keras gigi. dengan demikian secara klinis sangat sulit untuk melihat kondisi yang sebenarnya, terutama untuk mendeteksi proses keradangan secara tepat. sehingga dalam proses perawatan dan pascaperawatan ketergantungan pada respons penderita masih juga dapat dilihat melalui bentuk peran serta dan dukungan penderita terhadap proses perawatan dan pascaperawatan gigi dan mulut. savitri21 mengkaji kasus exfoliative cheilitis penderita di klinik fakultas kedokteran gigi universitas airlangga menyimpulkan bahwa untuk mendapatkan hasil maksimal dari perawatan kasus ini diperlukan kerja sama antara dokter gigi dengan penderitanya. dan karakteristik perawatannya diasumsikan bahwa 80% perawatan dengan upaya rehabilitasi pengunyahan, seperti pemasangan gigi tiruan atau mahkota jembatan. usaha rehabilitasi ini jelas memerlukan peran serta penderita hingga tahap pascaperawatan, karena diperlukan proses penyesuaian terhadap hasil pemasangan gigi tiruan tersebut. proses penyesuaian penderita tersebut disebut sebagai periode habituasi.22 basket et al. cit. soeprapto.22 menyatakan bahwa proses habituasi pada dasarnya adalah proses belajar, dalam proses belajar berarti terjadi proses komunikasi. dari kajian dari berbagai respons tersebut dapat dipastikan bahwa dalam melakukan layanan medik gigi dan mulut, dokter gigi akan menuntut respons penderitanya yang sesuai dengan pesan dan instruksinya. karena respons penderita diproduksi berdasarkan persepsi terhadap pesan yang diterimanya, maka apabila persepsi terhadap pesan tidak tepat, maka respons yang diproduksi tidak sesuai dengan makna pesan yang diinginkan dokter giginya. ada dua hal pokok sebagai penyebab persepsi yang tidak tepat, antara lain pertama adalah kapasitas kerangka rujukan penderita dan kedua adalah kualitas pesan dokter giginya. dari hasil penelitian januar23 tentang interaksi dokter gigi dengan penderitanya di empat puskesmas di jakarta selatan, disimpulkan adanya perbedaan tingkat intelektual antara dokter gigi dengan penderitanya sehingga menyebabkan penderita ragu-ragu dan kurang percaya diri untuk mengajukan pertanyaan kepada dokter giginya. namun demikian rendahnya kapasitas kerangka rujukan penderita ini seharusnya dapat diimbangi dengan kualitas pesan, baik materi pesan maupun cara penyampaian pesan kepada penderita. sehingga materi dan cara penyampaian yang disesuaikan dengan kerangka 128 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 124–129 rujukan penderita akan lebih memudahkan dalam proses persepsi penderita, dan selanjutnya produksi respons akan sesuai dengan harapan yang diinginkan dokter gigi tersebut. jika dikaji dari persepsi masyarakat tentang masalah kesehatan gigi dan mulut pada umumnya masyarakat memiliki pola pikir (mind set) bahwa masalah kesehatan gigi dan mulut bukan merupakan masalah yang dapat mengancam jiwa atau tidak menimbulkan hal-hal yang fatal seperti kematian dan sebagainya. pandangan ini didukung pendapat reisiene dan locker24 meyatakan anggapan masyarakat bahwa masalah kesehatan gigi dan mulut bukan masalah yang dapat mengancam kehidupannya, sehingga pola pikir demikianlah yang membentuk perilaku mereka dalam mengatasi masalah kesehatan gigi dan mulut. salah satu bentuk perilaku dalam mengatasi masalah kesehatan gigi dan mulut di masyarakat adalah menolak rencana perawatan yang sudah diindikasikan terhadap dirinya, misalkan takut sakit lagi, tidak memiliki waktu untuk perawatan yang berulang kali dan alasan lain. data yang dikumpulkan oleh kent cit. januar25 terhadap dokter gigi di inggris menemukan bahwa permasalahan yang paling banyak dialami dokter gigi adalah keputusan menetapkan rencana perawatan, karena ikut sertanya penderita menetapkan perawatan tersebut. persoalan yang timbul adalah ketika jenis perawatan yang diinginkan atau yang dipilih penderita justru merupakan perawatan kontra indikasi baginya. dalam kondisi seperti inilah justru peran komunikasi interpersonal untuk merubah perilaku khususnya kerangka rujukan penderita menjadi sangat bermakna. di bidang kesehatan gigi masyarakat timbul paradigma baru tentang the person behind the theeth, di mana penerapan paradigma ini dalam berbagai layanan kesehatan gigi dan mulut pada masyarakat tidak bisa terlepas dari dari persoalan manusia seutuhnya, artinya perawatan kesehatan gigi dan mulut kepada individu atau masyarakat bukan terhadap kasus penyakitnya saja tetapi justru terhadap orang atau manusia secara utuh (as human being).23 dengan demikian unsur manusia dengan berbagai karakteristiknya menjadi bagian penting dalam upaya penanganan masalah kesehatan gigi dan mulut di masyarakat, sehingga dengan penekanan aspek karakteristik manusia dalam layanan medik kesehatan gigi dan mulut menyebabkan peran komunikasi antara dokter gigi dengan penderitanya akan lebih menonjol. dari ketiga fakta yang telah diuraikan baik secara teori maupun berbagai kegagalan komunikasi maka dapat digambarkan peran komunikasi interpersonal antara dokter gigi dengan penderita menjadi lebih optimal apabila kualitas komunikasi interpersonal mampu membangun hubungan interpersonal antara dokter gigi dengan penderitanya. ini sesuai dengan analisis milgrom et al.26 tentang pengelolaan rasa sakit pada penderita anak-anak menyimpulkan bahwa, komunikasi interpersonal yang dibangun dokter gigi terhadap penderita anak-anak ternyata sangat efektif untuk menekan rasa sakit gigi yang dirasakan anak-anak tersebut. sedangkan dari hasil penelitian januar23 yang mempelajari konsep kepuasan penderita terhadap perawatan gigi yang sudah dijalaninya terutama tentang kualitas interaksi atau komunikasi interpersonalnya. penelitian yang dilakukan untuk mengamati tingkat kepuasan penderita pengunjung poli gigi di empat puskesmas di jakarta selatan ini didapatkan bahwa kurangnya interaksi dokter gigi dengan penderitanya terutama dalam pemberian informasi layanan medik gigi dan mulut, menyebabkan kesulitan untuk mengatasi pengetahuan penderita terhadap layanan medik gigi dan mulut yang sangat minim, temuan ini menjadi sangat menarik sebab pengetahuan penderita tentang hal tersebut sebenarnya merupakan gambaran kapasitas kerangka rujukan penderita terhadap layanan medik gigi dan mulut. apabila kita mengacu pada proses persepsi maka kerangka rujukan ini faktor penyebab utama terhadap timbulnya persepsi yang tidak cermat, dan dalam skala populasi dapat diasumsikan bahwa persepsi yang tidak cermat terhadap layanan medik gigi dan mulut terjadi pada masyarakat secara umum, akibatnya akan menimbulkan respons yang tidak tepat pula. hal inilah yang menghambat setiap program peningkatan kesehatan gigi masyarakat, sehingga program tersebut sering mengalami kegagalan. dari kajian di atas dapat disimpulkan bahwa peran komunikasi interpersonal antara dokter gigi dengan penderitanya sangat penting, agar proses layanan medik gigi dan mulut akan lebih optimal, dan pemahaman tentang peran komunikasi interpersonal diharapkan mampu mendorong peningkatan kemampuan dan penguasaan berkomunikasi dengan penderita dan akhirnya timbul hubungan interpersonal yang optimal antara dokter gigi dengan penderitanya. daftar pustaka 1. peterson pe, holst d. utilization of dental health services. in: cohen lk, gift hc, editors. desease prevention and oral health promotion. copenhagen: federation dentaire intenational; 1995. p. 352–3. 2. kuntari sa six month evaluation of glass ionomer cement restoration in class ii caries of primary molar. maj. ked. gigi (dent j) 1999; 32(2):73. 3. rahardjo p, rukmo m, rulianto m. evaluasi klinis satu tahun pada tumpatan resin komposit kelas vi. maj. ked. gigi (dent j) 2002; 35(3):120–1. 4. ariadi bss, moerdiati s, poerwoleksono de, shomad a, kabat s. tinjauan yuridis di dalam perjanjian penyembuhan. jurnal penelitian universitas airlangga 1989; 5(2):20–22. 5. santosa lm. komunikasi interpersonal antara dokter gigi dan pasien di ruang praktek. majalah kedokteran gigi 1998; 31(4):137. 6. rakhmat j. psikologi komunikasi. edisi ii. bandung: cv remaja karya; 1986. h. 16, 61–86, 100–14. 7. azwar a. sebagai guru besar tetap pada fakultas kedokteran universitas indonesia. jakarta. 1977. h. 2. 8. azwar a. pengantar administrasi kesehatan. edisi i. jakarta: binarupa aksara; 1996. h. 35–40. 9. mc mahon r, barton e, piot m. manajemen pelayanan kesehatan primer. poppy kumala. jakarta: penerbit buku kedokteran egc; 1995. h. 44. 129soelarso, dkk.: peran komunikasi interpersonal 10. trihono, analisis rumusan rencana intervensi monitoring evaluasi (aprime). pedoman manajemen puskesmas. proyek kesehatan keluarga dan gizi. depkes ri. 2002. h. 54–57. 11. kristina d. peran komunikasi terapeutik dalam perilaku kepatuhan berobat pasien gigi di puskesmas. maj. ked. gigi (dent. j) 2003; 36(4):147–150. 12. bender ib. the optimun placement site of the electrode in electric pulp testing of the 12 anterior teeth. in dental review 1990; 2(1):14. 13. pollock rm, levenson md. the endodontic emergency. practical method of management in dental review 1990; 2(1):17. 14. cailleleau jg, ludington jr. using the electric pulp tester with gloves a simplified approach in dental review 1990; 2(1):18. 15. haroen er. pengaruh kualitas nyeri gigi akut terhadap derajad kecemasan pasien. majalah kedokteran gigi 2002; 35(3):129. 16. mc daniel tf. are patients willing to reveal health history information?. in dental review 1995; 7(5):19–20. 17. runkat j. a new paradigm of the solving on surgically tmj dysfunction. majalah kedokteran gigi 2002; 35(3):120–1. 18. delcanho r. temporo mandibullar disorder screening assesment. in dental review 1996; 8(1):10. 19. soenartyo h. tatalaksana nyeri wajah karena kelainan fungsi temporo mandibullar joint. majalah kedokteran gigi 2004; 37(1):33–37. 20. widodo t. paradigma baru perawatan endodontik. majalah kedokteran gigi 2002; 35(3):134–5. 21. savitri d. exfoliative cheilitis. suatu laporan kasus. maj. ked. gigi (dent j) 1996; 29(4):120. 22. soeprapto. periode habituasi pemakai geligi tiruan lengkap. majalah kedokteran gigi (dent. j) 1999; 32(2):57–59. 23. januar p. konsep pasien tentang kepuasan terhadap perawatan gigi. maj. ked. gigi (dent j) 2001; 34(3a):341–6. 24. reisine s, locker d. social, psichological and economic impact of oral onditions and treatments. in: cohen lk, gift hc, editors. desease prevention and oral health promotion. copenhagen: federation dentaire international; 1995. p. 34-35. 25. januar p. pengaruh ciri perseorangan pasien terhadap interaksi dengan tenaga kesehatan gigi, kepuasan dan ketaatan berobat pada perawatan gigi di puskesmas. disertasi. surabaya: pascasarjana universitas airlangga; 1996. h. 5. 26. milgrom p, weinstein p, golletz d, leroux b, domoto p. pain management in children. in dental review 1995; 7(5):26. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 172 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 172–177 original article the characteristics of swelling and biodegradation tests of bovine amniotic membrane-hydroxyapatite biocomposite titien hary agustantina1, elly munadziroh1, anita yuliati1, muhammad riza hafidz bahtiar2, octarina3,4, rizki fauziah salma2, ajeng putri meyranti2, fathilah abdul razak5 1department of dental materials, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3doctoral program, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4department of dental materials, faculty of dentistry, universitas trisakti, jakarta, indonesia 5department of oral and craniofacial sciences, faculty of dentistry, universiti malaya, kuala lumpur, malaysia abstract background: a good biocomposite is a structure that can provide opportunities for cells to adhere, proliferate, and differentiate. it is affected by the characteristics of a material. as bone tissue regeneration occurs, biomaterials must have a high swelling ability and low biodegradability. the high swelling capability will have a larger surface area that can support maximal cell attachment and proliferation on the biocomposite surface, which accelerates the regeneration process of bone defects. purpose: the study aimed to analyze the characteristics of swelling and biodegradation of bovine amniotic membrane-hydroxyapatite (bam-ha) biocomposite with various ratios. methods: the bam-ha biocomposite with a ratio of 30:70, 35:65, and 40:60 (w/w) was synthesized using a freeze-dry method. the swelling test was done by measuring the initial weight and final weight after being soaked in phosphate-buffered saline for 24 hours and the biodegradation test was done by measuring the initial weight and final weight after being soaked in simulated body fluid for seven days. results: the swelling percentage of bam-ha biocomposite at each ratio of 30:70, 35:65, and 40:60 (w/w) was 303.90%, 477.94%, and 574.19%. the biodegradation percentage of bam-ha biocomposite at each ratio of 30:70, 35:65, and 40:60 was 9.43%, 11.05%, and 12.02%. conclusion: the bam-ha biocomposite with a ratio of 40:60 (w/w) has the highest swelling percentage while the 30:70 (w/w) ratio has the lowest percentage of biodegradation. keywords: biocomposite; biodegradation; bovine amniotic membrane; hydroxyapatite; socket preservation; swelling article history: received 10 october 2022; revised 23 november 2022; accepted 12 january 2023; published 1 september 2023 correspondence: elly munadziroh, department of dental materials, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya, 60132, indonesia. email: elly-m@fkg.unair.ac.id introduction the desired outcomes of biomaterials related to tissue engineering for clinical applications should include the attraction of natural resident stem cells to the site of injury and the suppression of inflammation, reduction of scar formation, enhancement of vascularization, and prevention of infection. the amniotic membrane is recommended for usage as an ideal biomaterial for wound healing due to its protein, cytokine, and growth factor concentration. the amniotic membrane can be used as a single or even double sheet to cover wounds on the body’s exposed outer surfaces, such as the skin and cornea.1 the amniotic membrane has many different applications in medicine, but it is particularly useful for treating skin burns and preventing tissue adhesion during head, neck, abdomen, larynx, and genitourinary tract surgeries. the amniotic membrane can also serve as a barrier that protects internal organs.2 for instance, wrapping the peritoneal cavity, tendon, spinal cord, and peripheral nerves in the amniotic membrane could prevent adhesion and minimize the formation of scars.1 amniotic membrane is a biomaterial used widely in tissue regeneration because it has anti-bacterial and anti-inflammatory properties.3 amniotic membrane has growth factors, such as vascular endothelial growth factor (vegf), fibroblast growth factor (fgf), epidermal growth factor (egf), tissue inhibitor metalloproteinase (timp), transforming growth factor-β (tgf-β), and plateletderived growth factor (pdgf).4 one source of the amniotic copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 mailto:elly-m@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 173agustantina et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 172–177 membrane is bovine. the bovine amniotic membrane has some benefits over the human amniotic membrane, such as being easier to legalize, being considered more ethical, and not being impacted by religious rituals.5 the limitation of the amniotic membrane is its low mechanical properties and rapid degradation, which makes it easy to decompose and difficult to maintain the structural integrity required for bone regeneration.6 the amniotic membrane is also easily torn by this feature when it is applied. the integrity of the structure, which is vital when mending extraction wounds with long-term treatment, will be impacted by how easily the amniotic membrane degrades. modifying the amniotic membrane is needed to find a solution to this issue.7 the amniotic membrane in combination with a bone graft will produce biocomposites that have the potential to support bone formation and provide better outcomes.8 one of the bone graft materials that is often used to support the bone healing process is hydroxyapatite.9 hydroxyapatite is a bioceramic material with a mineral composition similar to bones and teeth.10,11 hydroxyapatite has been used widely as a biomaterial for bone tissue replacement and repair because of its high osteoconductivity, nontoxicity, and good biocompatibility.12 hydroxyapatite can be obtained from bovine bone (bovine hydroxyapatite).13 bovine bone material consists of 93% hydroxyapatite and 7% β-tricalcium phosphate (ca3(po4)2, β-tcp). 14 bovine hydroxyapatite (bha) has the same chemical composition as human bone, which affects bone metabolism.15 a combination of bovine amniotic membrane and hydroxyapatite will produce a new material in the form of a sponge-shaped biocomposite. the bovine amnionhydroxyapatite membrane was expected to preserve the socket after tooth extraction. the characteristic properties of a biomaterial are important for successful tissue regeneration and must be able to adapt to the tissue that will be replaced.16 a good biocomposite is a structure that can provide opportunities for cells to adhere, proliferate, and differentiate. it is affected by the characteristics of a material.17 the swelling test is a method used to determine the capacity of the material to absorb liquid. the swelling properties of the biomaterial have an impact on good cell proliferation. higher swelling abilities increase the surface area of the biomaterial, thus facilitating cell attachment to the biomaterial.18 with more cells attached, it is hoped that cell growth will also be faster. swelling properties also play an important role in increasing the absorption of fluids from the body or media, as well as the transfer of nutrients and metabolic wastes.19 the increase in the swelling ratio is caused by the presence of hydrophilic properties. hydrophilic conditions are suitable for cell attachment and proliferation, therefore it accelerates the regeneration process.20 biomaterials implanted in the body and in contact with biological systems can trigger a series of reactions between the biomaterials and host tissues. the ability of biodegradation is an important role in biomaterials’ formation of new tissues because of its properties that can affect cell viability and proliferation. the biodegradation test is a parameter needed to see the time required for biomaterial to be degraded according to the formation of new tissue. the biodegradation test can indicate the biodegradability of a material.21 biomaterials that have been implemented in the body must be able to maintain sufficient mechanical properties and structural integrity so that cell adaptation goes well and can store their extracellular matrix. biomaterials that have biodegradable properties are expected to be able to create space for new bone tissue to grow.22 based on this, the study aimed to analyze the characteristics of bovine amniotic membrane-hydroxyapatite (bam-ha) biocomposite through a swelling and biodegradation test. materials and methods ethical approval, which is managed as a condition for conducting research, has been obtained from the research ethics commission of the faculty of medicine, universitas airlangga surabaya (no. 400/hrecc.fodm/vii/2021). this research is an experimental laboratory with a posttest-only control group design. the data used primary data, which was directly collected by the researcher. the manufacture of bam-ha biocomposite was done at the biomaterial center installation of the tissue bank hospital by dr. soetomo surabaya. the dry amniotic membrane was prepared at 3 grams (30:70 ratio), 3.5 grams (ratio 35:65), and 4 grams (ratio 40:60). the amniotic membrane was cut into pieces of about 2 cm and added with 40 ml of 0.9% nacl. then, the amniotic membrane with nacl was soaked for 5 minutes until the liquid was absorbed. the amniotic membrane was blended for 10 minutes to produce a smooth amniotic slurry. the amniotic slurry was added with 7 grams of hydroxyapatite powder (30:70 ratio), 6.5 grams of hydroxyapatite powder (ratio 35:65), and 6 grams of hydroxyapatite powder (ratio 40:60), then stirred until homogeneous and put into a petri dish with a diameter of 10 cm. the petri dish was put into the freezer (thermo, usa) at -80oc for 24 hours, then freeze-dried (virtis benchtoptm “k” series) for 24 hours at -100oc (figure 1). sponge-shaped biocomposite with a diameter of 10 cm was cut into pieces of 1.5x1.5 cm using a scalpel with a no. 15 blade and handle scalpel no. 3 (figure 2). the biocomposite is in the form of a sponge with a sizing of 1.5x1.5 cm and various ratios weighed as the initial weight (wi). the biocomposite was immersed in 10 ml of phosphate buffer saline (pbs) solution at 37oc for 24 hours. after that, the biocomposite was taken and then drained using whatman filter paper for 3 seconds, and the final weight (wf) was determined. the swelling ratio was calculated using the formula: copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 174 agustantina et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 172–177 table 1. the average value of the swelling test biocomposite ratio swelling ratio (%) mean sd bovine amniotic membranehydroxyapatite 30:70 (control) 303.90 10.75 35:65 477.94 10.90 40:60 574.19 35.44 table 2. the results of one-way welch anova statistical analysis on the swelling test robust tests of equality of means swelling test statistic df1 df2 sig. welch 745.760 2 16.455 .000 table 3. post-hoc tukey hsd statistical test results of swelling percentage ratio 30:70 35:65 40:60 30:70 0.000* 0.000* 35:65 0.000* 40:60 *significant (p < 0.05) a b c figure 1. the results of making bam-ha ratios of 30:70 (a), 35:65 (b), and 40:60 (c). a b c figure 2. the results of the preparation of bam-ha biocomposite ratios of 30:70 (a), 35:65 (b), and 40:60 (c). the biodegradation test was performed using a biocomposite is in the form of a sponge with a sizing of 1.5x1.5 cm and various ratios weighed as the wi. the biocomposite was immersed in simulated body fluid (sbf) solution at 37°c for seven days. after seven days, the biocomposite was taken and then dried using a freezedrier for 48 hours at a temperature of -100oc. after that, the biocomposite was weighed again to determine the wf. the biodegradation of biocomposites was calculated using the formula: results the average value of the swelling level of the bam-ha biocomposite (table 1) increased after immersion for 24 hours. the bam-ha biocomposite with a ratio of 40:60 (w/w) obtained an average swelling rate of 574.19%, which was the highest rate compared to the ratios of 35:65 and 30:70 (w/w), which were 477.94% and 303.90%, respectively. statistical analysis of variance (one-way welch anova) is one of the parametric statistical tests to copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 175agustantina et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 172–177 determine differences between groups of data as a whole. the results of the one-way welch anova test showed a significance value of p = 0.000, which means that there was a significant difference between groups with a p-value of less than (p < 0.05) (table 2). therefore, further testing was carried out using the post-hoc tukey honestly significant difference (hsd) to find out which groups had significant differences. the post-hoc tukey hsd test has been summarized and presented in table 3. there was a significant difference in the average level of swelling between the bam-ha biocomposite ratio of 30:70 with a ratio of 35:65 (w/w) (p = 0.000), bam-ha biocomposite ratio of 30:70 with a ratio of 40:60 (w/w) (p = 0.000), and the bam-ha biocomposite ratio of 35:65 with a ratio of 40:60 (w/w) (p = 0.000) (p < 0.05). the average value of the biodegradation rate of bamha biocomposite in various ratios is shown in table 4. the bam-ha biocomposite 40:60 (w/w) ratio obtained the highest average value of the biodegradation rate at 12.02%, followed by 11.05% at 35:65 (w/w), and 9.54% at 30:70 (w/w). the results of the one-way welch anova test showed a significance value of p = 0.000, which means that there was a significant difference between groups with a p-value of less than 0.05 (table 5). therefore, the post-hoc tukey hsd analysis test was continued to find out which groups had significant differences. the post-hoc tukey hsd test has been summarized and presented in table 6. data analysis was performed using the post-hoc tukey hsd test. it showed that there was a significant difference in the mean level of biodegradation between the bam-ha biocomposite group with a ratio of 30:70 and a comparison ratio of 35:65 (w/w) (p = 0.000) and bam-ha biocomposite with a ratio of 30:70 and a comparison ratio of 40:60 (w/w) (p = 0.000). discussion the selection of biomaterials for making bone tissue regeneration material is a significant matter in the socket preservation procedure. the requirements for biomaterials in bone tissue regeneration, include having a porous structure that promotes cell proliferation and distribution so that it can support the formation of new bone tissue. additionally, it can provide a temporary structure that will degrade over time with the formation of new tissue.23,24 socket preservation biomaterial candidates used in this study were obtained by synthesizing bovine amniotic membrane biomaterials and bha with a combined ratio of 30:70, 35:65, and 40:60 (w/w) using the freeze-drying method. the freeze-drying process can form a porous structure, which can affect the outer surface of the biocomposite to become hydrophilic.25,26 the characteristic properties of a biomaterial are important for the success of tissue regeneration and must be able to adapt to the tissue to be replaced.16 swelling is one of the important properties in the application of biomaterials in bone tissue regeneration. swelling in biomaterials can play a role in cell infiltration into biomaterials.27 the results of the swelling test showed that the average swelling ratio was increasing from a ratio of 30:70 (w/w) of 303.90±10.75%, 35:65 (w/w) of 477.94±10.90%, 40:60 (w/w) of 574.19±35.44%, and after the one-way anova test, showed that there was a significant difference between groups with a p-value of less than 0.05. this study showed that the bam-ha biocomposite with the larger bovine amniotic membrane ratio of 40:60 (w/w) obtained the highest percentage of swelling results compared to the 35:65 and 30:70 (w/w) ratios. that is because the bovine amniotic membrane biomaterial in this study contains a polymer in the form of collagen.28 table 4. the average value of the swelling test biocomposite ratio biodegradation ratio (%) mean sd bovine amniotic membranehydroxyapatite 30:70 (control) 9.54 1.43 35:65 11.05 1.42 40:60 12.02 0.48 table 5. the results of one-way welch anova statistical analysis on the biodegradation test robust tests of equality of means biodegradation test statistic df1 df2 sig. welch 14.048 2 14.307 .000 table 6. post-hoc tukey hsd statistical test results of biodegradation percentage ratio 30:70 35:65 40:60 30:70 0.024* 0.000* 35:65 0.180 40:60 *significant (p < 0.05) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 176 agustantina et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 172–177 these findings are appropriate with several findings from previous research that demonstrates collagen is one of the polymers with a high-water absorption capacity. a high amount of collagen will increase the water absorption properties of the biomaterial. collagen is a polymer with many hydrophilic groups, which can form hydrogen bonds with the surrounding solution. the low contact angle of biomaterials with hydrophilic characteristics can result in low surface tension. if the surface tension of the liquid is low, the surface of the biocomposite material will contact it more easily. as a result, the biocomposite can absorb liquids more readily.29 several other studies have shown that the increase in the percentage of swelling is directly proportional to the increase in the amount of gelatin, which is widely known as a biomaterial that has hydrophilic properties, while the increase in the amount of hydroxyapatite is inversely proportional to the percentage of swelling. the higher hydroxyapatite ratio in the biocomposite caused a decrease in the percentage of swelling and its swelling ability.19,30 by attaching calcium and phosphate to the hydrophilic groups of cooh or nh2, hydroxyapatite forms a crosslink between polymer chains and reduces the biomaterial’s hydrophilicity.31 another property that plays a role in the success of bone tissue regeneration is biodegradability. biodegradable biomaterials were expected to provide a space for the growth of new bone tissue.22 according to the data, the bam-ha biocomposite ratio of 40:60 (w/w) had the highest average value of biodegradation at 12.020±0.483%, followed by 11.045±1.415% at a ratio of 35:65 (w/w). meanwhile, the bam-ha biocomposite with a ratio of 30:70 (w/w) had the lowest percentage of biodegradation, which was 9.541±1.428%. the results of the one-way welch anova test showed a value of significance of p = 0.000, which means that there is a significant difference between groups with a p-value of less than 0.05. the lowest percentage of a biodegradation rate was obtained at a 30:70 (w/w) ratio, with the highest composition hydroxyapatite ratio. that is because hydroxyapatite can reduce the pore size of the biocomposite.30 as a result, the lowest percentage level was attained at the 30:70 (w/w) ratio, which had a higher hydroxyapatite content than the 40:60 and 35:65 (w/w) ratios. meanwhile, the average percentage of degradation rate was high for the 40:60 (w/w) ratio because it contained less hydroxyapatite and more bovine amniotic membrane than the ratios of 35:65 and 30:70 (w/w). the inclusion of the hydroxyapatite ratio can result in a reduction in porosity size. therefore, it may have an impact on a biocomposite’s ability to access liquids. when the biomaterial immerses in the sbf solution, the solution forms a link with the biomaterial’s surface. by capillary action, the fluid will progressively seep into the biomaterial. during this phase, the pore wall will serve as a new connection between the liquid and the biomaterial. a decrease in porosity size will result in a smaller surface area. as a result, liquid accessibility reduces, which results in a slower rate of biodegradation.32 the level of biomaterial degradation is influenced by chemical properties, polymer composition, and environmental conditions.18 this study seeks to identify the appropriate combination ratio between the bovine amniotic membrane and hydroxyapatite and develop a bone biocomposite that produces swelling and biodegradation properties suitable for bone tissue regeneration. as bone tissue regeneration occurs, biomaterials must have a high swelling ability and low biodegradability. the high swelling capability will have a larger surface area that can support maximal cell attachment and proliferation on the biocomposite surface, which accelerates the regeneration process of bone defects.27 biocomposite can be used as bone graft material, preferably having a low level of biodegradation for bone regeneration to occur.26 the results show that the percentage of swelling increased with the concentrations of bovine amniotic membrane increasing and the concentrations of hydroxyapatite decreasing. additionally, the percentage of biodegradation decreased with the concentrations of hydroxyapatite increasing and the concentrations of bovine amniotic membrane decreasing. the bam-ha biocomposite with a ratio of 40:60 (w/w) has high swelling and biodegradation ability, while the ratio of 30:70 (w/w) has low swelling and biodegradation ability. references 1. elkhenany h, el-derby a, abd elkodous m, salah ra, lotfy a, el-badri n. applications of the amniotic membrane in tissue engineering and regeneration: the hundred-year challenge. stem cell res ther. 2022; 13(1): 8. 2. schmiedova i, dembickaja a, kiselakova l, nowakova b, slama p. using of amniotic membrane derivatives for the treatment of chronic wounds. membranes (basel). 2021; 11(12): 941. 3. kumar a, chandra rv, reddy aa, reddy bh, reddy c, naveen a. evaluation of clinical, antiinf lammatory and antiinfective properties of amniotic membrane used for guided tissue regeneration: a randomized controlled trial. dent res j (isfahan). 2015; 12(2): 127–35. 4. faadhila t, valentina m, munadziroh e, nirwana i, soekartono h, su rboyo mc. bovine sponge a m n ion stimulates socket healing: a histological analysis. j adv pharm technol res. 2021; 12(1): 99. 5. putra nhd, matulatan f, wibowo md, danardono e. effects of dried bovine amniotic membrane as prosthetics of abdominal fascial defect closure observed by the expression of platelet-derived growth factor in rattus norvegicus wistar strain. syst rev pharm. 2020; 11(6): 987–91. 6. leal‐marin s, kern t, hofmann n, pogozhykh o, framme c, börgel m, figueiredo c, glasmacher b, gryshkov o. human amniotic membrane: a review on tissue engineering, application, and storage. j biomed mater res part b appl biomater. 2021; 109(8): 1198–215. 7. dadkhah tehrani f, firouzeh a, shabani i, shabani a. a review on modifications of amniotic membrane for biomedical applications. front bioeng biotechnol. 2021; 8: 606982. 8. nurhaeini csw, komara i. socket preservation. padjadjaran j dent. 2015; 27(3): 133–8. 9. arifin a, mahyudin f, edward m. the clinical and radiological outcome of bovine hydroxyapatite (bio hydrox) as bonegraft. j orthop traumatol surabaya. 2020; 9(1): 9–16. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 177agustantina et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 172–177 10. kattimani vs, kondaka s, lingamaneni kp. hydroxyapatite–-past, present, and future in bone regeneration. bone tissue regen insights. 2016; 7: btri.s36138. 11. rujit a napa n ich s, ku mpapa n p, wa nja noi p. synt hesis of hydroxyapatite from oyster shell via precipitation. energy procedia. 2014; 56: 112–7. 12. ardhiyanto hb. peran hidroksiapatit sebagai material bone graft dalam menstimulasi kepadatan kolagen tipe l pada proses penyembuhan tulang. stomatognaic j kedokt gigi. 2012; 9(1): 16–8. 13. yuliati a, kartikasari n, munadziroh e, rianti d. the profile of crosslinked bovine hydroxyapatite gelatin chitosan scaffolds with 0.25% glutaraldehyde. j int dent med res. 2017; 10(1): 151–5. 14. saputra aah, triyono j, triyono t. bovine bone hidroksiapatite materials mechanics properties at 900°c and 1200°c of calcination temperature. mek maj ilm mek. 2017; 16(1): 26–30. 15. ratnayake jtb, mucalo m, dias gj. substituted hydroxyapatites for bone regeneration: a review of current trends. j biomed mater res part b appl biomater. 2017; 105(5): 1285–99. 16. shoichet ms. polymer scaffolds for biomaterials applications. macromolecules. 2010; 43(2): 581–91. 17. bružauskaitė i, bironaitė d, bagdonas e, bernotienė e. scaffolds and cells for tissue regeneration: different scaffold pore sizes—different cell effects. cytotechnology. 2016; 68(3): 355–69. 18. kartikasari n, yuliati a, listiana i, setijanto d, suardita k, ariani md, sosiawan a. characteristic of bovine hydroxyapatitegelatin-chitosan scaffolds as biomaterial candidate for bone tissue engineering. in: 2016 ieee embs conference on biomedical engineering and sciences (iecbes). kuala lumpur, malaysia: ieee; 2016. p. 623–6. 19. azhar ff, olad a, salehi r. fabrication and characterization of chitosan-gelatin/nanohydroxyapatitepolyaniline composite with potential application in tissue engineering scaffolds. des monomers polym. 2014; 17(7): 654–67. 20. mohamed kr, beherei hh, el-rashidy zm. in vitro study of nanohydroxyapatite/chitosan–gelatin composites for bio-applications. j adv res. 2014; 5(2): 201–8. 21. maji k, dasg upt a s, p ra ma n i k k, bissoyi a. p repa ration and evaluation of gelatin-chitosan-nanobioglass 3d porous scaffold for bone tissue engineering. int j biomater. 2016; 2016: 9825659. 22. bose s, roy m, bandyopadhyay a. recent advances in bone tissue engineering scaffolds. trends biotechnol. 2012; 30(10): 546–54. 23. melek l n. tissue eng i ne e r i ng i n or a l a nd m a x i l lofa cia l reconstruction. tanta dent j. 2015; 12(3): 211–23. 24. edgar l, mcnamara k, wong t, tamburrini r, katari r, orlando g. heterogeneity of scaffold biomaterials in tissue engineering. materials (basel). 2016; 9(5): 332. 25. tam tt, todo m, cheong ky, hamid zaa. evaluation of cell viability of porous scaffold fabricated via freeze-drying technique for vascular tissue engineering. in: aip conference proceedings. 2020. p. 020007. 26. wattanutchariya w, changkowchai w. characterization of porous scaffold from chitosan-gelatin/hydroxyapatite for bone grafting. in: the international multiconference of engineers and computer scientists vol ii. hongkong: chiang mai university; 2014. p. 1–5. 27. asaeli ap, yuliati a, budhy ti, wilda s, silalahi i, santoso d. the profile of cross-linked chitosan and collagen derived-chicken shank scaffold as biomaterials in tissue engineering. j int dent med res. 2019; 12(1): 6–11. 28. gunasekaran d, thada r, jeyakumar gfs, manimegalai np, shanmugam g, sivagnanam ut. physicochemical characterization and self-assembly of human amniotic membrane and umbilical cord collagen: a comparative study. int j biol macromol. 2020; 165: 2920–33. 29. anusavice kj, shen c, rawls hr. phillips’ science of dental materials. 12th ed. philadelpia: saunders; 2013. p. 592. 30. ari mda, yuliati a, rahayu rp, saraswati d. the differences scaffold composition in pore size and hydrophobicity properties as bone regeneration biomaterial. j int dent med res. 2018; 11(1): 318–22. 31. l ee j, yun h. hyd roxyapatitecont a in ing gelatin /ch itosa n microspheres for controlled release of lysozyme and enhanced cytocompatibility. j mater chem b. 2014; 2(9): 1255–63. 32. wang q, wang q, wan c. the effect of porosity on the structure and properties of calcium polyphosphate bioceramics. ceram silikaty. 2011; 55(1): 43–8. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p172–177 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p172-177 vol 51 no 1 jan-mrt 2018.indd 55 nickel ion release from stainless steel brackets in chlorhexidine and piper betle linn mouthwash tanti deriaty,1 indra nasution,2 and muslim yusuf3 1 orthodontist, medan-indonesia 2 department of mechanical engineering, faculty of engineering ,universitas sumatera utara 3 department of orthodontics, faculty of dentistry, universitas sumatera utara medan-indonesia abstract background: orthodontist prescribe mouthwash for their patients especially since most of patients do not have a satisfactory oral hygiene and have high risk of dental caries. stainless steel brackets that exposed by mouthwash may have nickel ion release. corrosion and nickel ion release can induced allergic reaction and make more friction during orthodontic treatment. purpose: this study aimed to measure nickel ion release of stainless steel bracket that immersed in chlorhexidine and piper betle linn mouthwash. methods: thirty-six stainless steel bracket immersed in artificial saliva, chlorhexidine, and piper betle linn mouthwash. all brackets stored in incubator for 1, 3, 5, and 7 weeks. nickel ion release was measured by atomic absorption spectrophotometry (aas). results: the results showed a significant differences of nickel ion release in all groups (p<0.05). conclusion: in conclusion, among the mouthwash, chlorohexidine has the highest nickel ion release from stainless steel brackets, followed with piper betle linn mouthwash. keywords: nickel ion release; stainless steel bracket; chlorhexidine mouthwash; piper betle linn. mouthwash correspondence: tanti deriaty, orthodontist. jl. sei ular baru no.4 medan indonesia. e-mail: tantisitepudds@gmail.com. research report introduction orthodontic brackets are an important component in orthodontic appliances. brackets should have the correct hardness and strength to deliver the exact force from the wire to the teeth. they also should have a smooth archwire slot to reduce frictional resistance and plaque deposition. orthodontic brackets should be accurately manufactured to reflect the prescription type of each bracket. they should also have a good biocompatibility and high corrosion resistance.1–3 brackets are usually placed in oral cavity between two and three years. during this time, brackets are contaminated by substances from the inside and outside mouth. this situation can destroy brackets physically and chemically then leading to corrosion and ion release. thus, high corrosion resistance metals or alloys are the best choice to prevent orthodontic brackets corrosiveness.1,2 stainless steel bracket is one of metal orthodontic bracket. this brackets have some primary advantages such as greater strength, lower cost, good modulus of elasticity and formability than any other brackets, and have high corrosion resistance in the oral cavity.1–4 there are some types of stainless steel brackets, such as aisi type 304 l ss, 316 l ss, and 17-4 ph ss. stainless steeel 304 l consist of 18–20% chromium, 8–10% nickel, and less than 0.03% manganese, silicon, and carbon. stainless steel 316 l has a higher nickel content than 304 l, 2–3% molybdenum, and consist of lower carbon to improved intergranular corrosion resistance and for better welding results. stainless steel 17-4 ph has similar corrosion resistance and higher mechanical property than 304 type.1–4 orthodontic corrosion and ion release in oral environment have two important concerns. first, when corrosion products absorb by body and caused local and systemic toxic effect. nickel ion release was known as the most common allergic substance that caused contact dermatitis in women and others hypersensitive reaction in 10% of general population. nickel is a strong medium immune reaction that can cause dental journal (majalah kedokteran gigi) 2018 march; 51(1): 5–9 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p5–9 mailto:tantisitepudds@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p5-9 6 deriaty, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 5–9 hypersensitivity reaction, contact dermatitis, gingival enlargement, asthma, hypercytotoxicity, and mutagenic. cultured human cells study reported that nickel ion release was moderately cytotoxic in cells. nowadays, there are alternative low-nickel and nickel-free alloys to replace stainless steel. however, the biocompatibility of lownickel alloys have not been accurately evaluated. second, metal corrosion could affect stainless steel physical and clinical properties. metal surface corrosion could increase friction of two different type of metal that cause prolonged treatment time and uncomfortable tooth movement.5–12 good oral hygiene is an essential part of a successful orthodontic treatment. orthodontist prescribes mouthwash due to most of patients have lack oral hygiene and high risk of dental caries. mouthwashes are effective to prevent formability of microbial plaques. among mouthwashes, chlorhexidine has known to be highly effective in prevention of dental plaques and reduction streptococcus mutans. however, some studies report that chlorhexidine mouthwash could make high ion release due to irrigation effect it self.13,14 recently, the use of herbal mouthwash is increasing. natural products that extract from herbal plants are found to be highly efficient to prevent the dental caries/plaque found in fixed orthodontic appliances patients undergoing orthodontic treatment. one of most common herbal plant in indonesia is piper betle linn. this plant has known as traditional medication including to prevent bad breath and dental caries. piper betle leaves contains several active compounds such as eugenol and its isomers, chavibetol, hydroxychavicol, pentatriacontanol, piperol, piperbetol, carotenes, and ascorbic acid. hydroxychavicol has been examined as an antimicrobial ingredient, and it shows promising results for several applications as an oral care agent. antimicrobial profiles of hydroxychavicol are well suited for an active ingredient for oral care products. corrosion behaviour of this plant is still unknown.14–16 this study report measured the levels of nickel ion release from stainless steel brackets immersed in chlorhexidine and piper betle linn mouthwash. these results should help orthodontist to prescribe the best choice of mouthwash for their patient needs. materials and methods thirty six brackets (first premolar bracket, stainless steel, 0.018-in, roth prescription, mini-gamma sd orthodontic, usa) were used for this study. the brackets divided into 12 groups. each group immersed for 1, 3, 5, and 7 weeks in different solutions. group 1-4 is a control group that immersed in artificial saliva. group 5-8 immersed in chlorhexidine mouthwash 0.2%. group 9-12 immersed in piper betle linn mouthwash 3%. direction of use mouthwash is usually rinse for about one minute twice a week, and after having mouthwash, patient should not be eating food, drinking, and rinsing their mouth to ensure that its components remain present for period of time. to calculate the presence of mouthwash active ingredient on mouth especially on brackets, we estimated that, if an individual followed this regime, the mouthwash components would be present in a patient’s mouth for 6 hours (twice a week for 24 months is equal 69.000 minutes). therefore, on this study the immersion and incubation time of brackets was 49 days (49 days being almost equivalen to 69.700 minutes).16 each bracket was placed in individual glass tube containing 10 ml of immersion solution and incubated at 37° c for 1, 3, 5, and 7 weeks. after incubation, the immersion solution was measured with atomic absorption spectrophotometer (aas) (shimadzu aa-7000). aas is spectroanalytical procedure to measuring the ion concentration in immersion solution using energy absorption from certain wavelength of light (commonly 190-900 nm). aas typically include a flame burner to atomize the sample (in this research we used a hollow cathode lamp as a flame burner), a monochromator and a photon detector. wavelength to measured nickel ion is 232.10 nm. first, we have to make standard solutions of three different concentrations, determine the absorbance then make a calibration curve from the values. fitting nickel light source lamp to the lamp housing to measure the absorbance then switch on the instrument. switch on the source lamp and set at nickel’s wavelength (232.10 nm). ignite the mixture of these gases. adjust the gas flow rate and pressure, and make the zero adjustment after nebulizing the solvent into the flame. the absorbance for the sample was measured then determined concentration from the previous curve of calibration.17 results results on table 1 showed nickel ion release mean levels in the groups. therefore, to look the presence of different nickel ion release we need to tested data with kruskal-wallis test in table 2. a non-parametric test (kruskal-wallis) in table 1 showed nickel ion release statistically significant differences in artificial saliva among 1, 3, 5 and 7 weeks groups p= 0.030 (p< 0.05); in chlorhexidine was significantly different p= 0.015 (p<0.05); in piper betle linn was significantly different p=0.015 (p<0.05). this research not only report based on type solution but also based on immersion time (table 3). kruskal-wallis test reported nickel ion release statistically significant differences in artificial saliva, chlorhexidine, and piper betle linn for 1 week p=0.023 (p<0.05), 3 weeks p=0.020 (p<0.05), 5 weeks p=0.017 (p<0.05); and 7 weeks p= 0.015 (p<0.05). discussion metal corrosion could be happen in the mouth environment and released metal ion into saliva. orthodontist dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p5–9 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p5-9 7deriaty, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 5–9 commonly recommended mouthwash for their patients to decrease the risk of plaque and caries formability. however, lack of study reported the effects of various mouthwashes on ion release of orthodontic brackets.18 this study reported nickel ion release from stainless steel brackets that immersed in artificial saliva, chlorhexidine, and piper betle linn. mouthwash increased over time. this result relevant with amini’s study that measured chromium and nickel concentration in gingival crevicular fluid before treatment, 1 month, and 6 months after using orthodontic appliance from 24 patients using aas. the nickel levels were increased over time same with this study.19 among various mouthwash solutions, chlorhexidine showed the maximum level of nickel ion released, the next highest being in piper betle linn mouthwash, and the lowest in artificial saliva. this result relevant with danaei et al. study that immersed 160 stainless steel brackets divided randomly in four solution groups (chlorhexidine, oral-b fluoride, persica mouthwash, and distillated deionized water) and incubation time for 45 days at 37° c. nickel, table 1. mean levels of the ions released in all groups groups (solution) immersion time (weeks) pn 1 3 0.030*33artificial saliva 35 37 31 0.015*33chlorhexidine 35 37 31 piper betle linn 0.015*33 35 37 * p<0.05 table 2. kruskal-wallis test of nickel ion released in the artificial saliva, chlorhexidine, and piper betle linn. mouthwash in 1, 3, 5, 7 weeks groups immersion time (weeks) artificial saliva (control) (n=3) χ ± sd (ppm) chlorhexidine (n=3) χ ± sd (ppm) piper betle linn. (n=3) χ ± sd (ppm) 0.033 ± 0.006670.185 ± 0.050.0073 ± 0.005771 0.099 ± 0.010000.5463 ± 0.006670.0103 ± 0.005773 0.154 ± 0.020000.9313 ± 0.006670.0113 ± 0.005775 0.203 ± 0.006671.333 ± 0.006670.0133 ± 0.005777 * p<0.05 table 3. kruskal-wallis test in immersion time groups groups immersion time (weeks) pnsolution 3artificial saliva 0.023*31 chlorhexidine piper betle linn 3 3artificial saliva 0.02033 chlorhexidine * piper betle linn 3 3artificial saliva 0.0173chlorhexidine5 * piper betle linn 3 3artificial saliva 0.01537 chlorhexidine * piper betle linn 3 * p<0.05 chromium, iron, copper, and manganese ion release were measured by inductively coupled plasma spectrometer. the highest nickel ion release showed in chlorhexidine solution compared with the other mouthwash.20 it reported on a fixed appliance simulator that described full upper arch. the samples were measured at 1, 7, 14, 21, and 28 days by flame atomic absorption spectrophotometer. the highest levels of metal ion release at day 7, and all releasing ion is finished within 28 days. this results is not relevant with our study.21 corrosion occurs from either dissolve of metal ions into solution or progressive release of a protective layer, usually an oxide or a sulphide. the corrosion and metal ion release mechanism from stainless steel alloy started from dissolved the protective film that consist of chromium oxide and chromium hydroxide, and then forms on contact with oxygen on the stainless steel’s surface.20 corrosion made from two simultaneous reactions: oxidation and reduction (redox). for example we were using iron in a weak acid. oxidation (anodic) reaction results dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p5–9 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p5-9 8 deriaty, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 5–9 in dissolution of the iron as ferrous ions are produced (fe fe2++ 2e-). reduction occurs at the cathode, with hydrogen ions reduced to hydrogen gas (2h++ 2e h2). this reaction will continue until all of the metal consumed, unless the metal can form a protective surface layer (passivation), or until the cathodic reactant is consumed (exhaustion of dissolved oxygen in solution).22,23 corrosion levels of any metal depends on chemical reaction of the immersion solution (figure 1). the formation of passive oxide film can delay the corrosion process on the orthodontic appliances. however, this protective layer can dissolve mechanically and chemically. this passive oxide films can slowly dissolve (passivation) without chemical or mechanical abrasion only to reform (repassivation) as the metal surface is exposed to oxygen from the air or the surrounding medium. the passivation reaction could accelerated by chloride ions and acidic environment that happen because of sodium chloride, acidic carbonated drinks intake, and fluoride-containing products, such as toothpaste and mouthwash. some studies reported, the corrosion resistance of some metals, especially titanium, are decreased in a fluoridated, acidic environment,.22–25 the orthodontic metal alloy risk of corrosion depends on the oral environment, which is determined by mechanical and chemical factors. mechanical factors including foods, liquids, and tooth brushes abrasion. chemical factors including quantity and quality of saliva, and ph of food and beverages. corrosion will occur continuously in the mouth over time.27 some factor that effect different results betwen studies are study design, measuring system, immersion time, and immersion solution. moreover, same products on different manufacturers have been shown different results. surface area of the bracket is one of an important factor to determine the amount of corrosion and ion release, but calculating orthodontic bracket’s comprehensive surface area was on our exclusion criteria in this study because of their complex shape and geometry. the corrosion of brackets could influence the process of orthodontic treatment. nickel ion release can result allergic reactions and cytotoxicity. since the nickel allergic reaction is one of our concern, orthodontist should be aware that nickel ion release might cause some allergic reaction including contact allergy and gingivitis. contact allergy clinically seems at oral soft tissue that contact with brackets. in orthodontic patient, severe gingivitis not only related to lack of oral hygiene but also to nickel allergic reaction from stainless steel brackets. we also need to determine patient history of hypersensitivity by anamneses the patient.20 parameters that affect the corrosion and ion release of metals in saliva including immersion time, presence of oxygen, ph, and temperature. metal ion released into the oral environment with saliva as the medium. high level of chloride contain in saliva, various intake of foods and drinks with a low ph could lead to acidic condition that increase the amount of corrosion and ion release. furthermore, the characteristics of saliva change according to the patient’s health and the time of day can affect the ion release.22–24 in this study, mouthwash was used in a static environment. however, in real life, mouthwash were used in dynamic environment. increasing metal ion release could happen in dynamic condition not only because of the saliva fluidity and ph but also abrasion by tooth brushing and mastication mechanism. a large number of metal ion release after using an oral functioning simulator apparatus to describe the dynamic condition of oral environment.28 in this study, we did not use adhesive resin to coverage base of brackets so the involved bracket surface was larger than clinical conditions.29,30 average nickel intake a day from foods are 5–100 mg and 300–500 mg, respectively. nickel ion intake in beverages is commonly under 20 mg per liter. the amount of nickel ion released from in this study is lower than daily food and water nickel intake. however, patient with nickel intolerant reacted even with a small amount of nickel ion release.20 this study clearly identifies the risk of corrosion and nickel ion release when mouthwashes are used. corrosion could make some clinical problems when orthodontic treatment occurs including allergic reaction and uncomfortable sliding movements. it also could negatively impact the aesthetic result of orthodontic treatment. patients who have metal ion hipersensitivity especially nickel is recommended not having mouthwash for a long period of time. it seems to be a need for a new type of mouthwash containing both anti-caries and corrosion inhibitors which could be used without restriction by patients undergoing orthodontic therapy. in conclusion, among the mouthwash, chlorohexidine has the highest nickel ion release from stainless steel brackets, followed with piper betle linn mouthwash. figure 1. corrosion on orthodontic bracket.26 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p5–9 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p5-9 9deriaty, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 5–9 references 1. amini f, jafari a, amini p, sepasi s. metal ion release from fixed orthodontic appliances—an in vivo study. eur j orthod. 2012; 34: 126–30. 2. tanabe m, takahashi t, shimoyama k, toyoshima y, ueno t. effects of rehydration and food consumption on salivary flow, ph and buffering capacity in young adult volunteers during ergometer exercise. j int soc sports nutr. 2013; 10: 49. 3. kolokitha oeg, chatzistavrou e. allergic reactions to nickelcontaining orthodontic appliances: clinical signs and treatment alternatives. world j orthod. 2008; 9(4): 399–406. 4. english jd, peltomaki t, pham-litschel k. mosby’s orthodontic review. st. louis: elsevier health sciences; 2008. p. 239-43. 5. gautam p, valiathan a. ceramic brackets: in search of an ideal! trends biomater artif organs. 2007; 20(2): 117–22. 6. matos de souza r, macedo de menezes l. nickel, chromium and iron levels in the saliva of patients with simulated fixed orthodontic appliances. angle orthod. 2008; 78(2): 345–50. 7. bakhtari a, bradley tg, lobb wk, berzins dw. galvanic corrosion between various combinations of orthodontic brackets and archwires. am j orthod dentofac orthop. 2011; 140: 25–31. 8. arezou j, shahabi m, mokhber n, ardakani t. comparison of nickel ion release and corrosion sites among commonly used stainless steel brackets in iran. j mashhad dent sch. 2009; 33: 17–24. 9. amini f, khazali m, mahmodei f, hematpour s. salivary content of nickel, chromium and cobalt ions in subjects with fixed orthodontic appliances. iran j orthod. 2010; 5: 63–6. 10. costa mt, lenza ma, gosch cs, costa i, ribeiro-dias f. in vitro evaluation of corrosion and cytotoxicity of orthodontic brackets. j dent res. 2007; 86(5): 441–5. 11. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 11th ed. philadelphia: saunders elsevier; 2011. p. 235–7. 12. hafez hs, selim emn, kamel eid fh, tawfik wa, al-ashkar ea, mostafa ya. cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study. am j orthod dentofac orthop. 2011; 140(3): 298–308. 13. omidkhoda m, sahebnasagh z, poosti m, yaghoobi m, izadpanahi a. evaluation of mechanical properties of niti orthodontic wire after immersion in three different mouthwashes. iran j orthod. 2011; 6: 32–8. 14. farah cs, mccullough mj, mcintosh l. mouthwashes. aust prescr. 2009; 32(6): 162–4. 15. singgih m, damayanti s, pandjaitan n. antimicrobial activity of standardized piper betel extract and its mouthwash preparation. int j pharm pharm sci. 2014; 6(7): 243–6. 16. sandhya r. herbal product as mouthwash a review. int j sci res. 2017; 6(7): 1334–7. 17. dubey rc. a textbook of biotechnology. new delhi: s. chand publishing; 2007. p. 190–4. 18. rafeeq ra, saleem ai, nissan lmk. ions release from fixed orthodontic appliance in two different mouthwashes. j baghdad coll dent. 2014; 26(4): 152–5. 19. amini f, shariati m, sobouti f, rakhshan v. effects of fixed orthodontic treatment on nickel and chromium levels in gingival crevicular fluid as a novel systemic biomarker of trace elements: a longitudinal study. am j orthod dentofac orthop. 2016; 149(5): 666–72. 20. danaei sm, safavi a, roeinpeikar smm, oshagh m, iranpour s, omidekhoda m, omidekhoda m. ion release from orthodontic brackets in 3 mouthwashes: an in-vitro study. am j orthod dentofac orthop. 2011; 139(6): 730–4. 21. mikulewicz m, chojnacka k, wołowiec p. release of metal ions from fixed orthodontic appliance: an in vitro study in continuous flow system. angle orthod. 2014; 84(1): 140–8. 22. patel r, bhanat s, patel d, shah b. corrosion inhibitory ability of ocimum sanctum linn (tulsi) rinse on ion release from orthodontic brackets in some mouthwashes: an invitro study. natl j community med. 2014; 5: 135–9. 23. kuhta m, pavlin d, slaj m, varga s, lapter-varga m, slaj m. type of archwire and level of acidity: effects on the release of metal ions from orthodontic appliances. angle orthod. 2009; 79: 102–10. 24. house k, sernetz f, dymock d, sandy jr, ireland aj. corrosion of orthodontic appliances—should we care? am j orthod dentofac orthop. 2008; 133(4): 584–92. 25. gajapurada j, ashtekar s, shetty p, biradar a, chougule a, bhalkeshwar, bansal a, zubair w. ion release from orthodontic brackets in three different mouthwashes and artificial saliva: an in-vitro study. iosr j dent med sci. 2016; 15(4): 76–85. 26. luft s, keilig l, jäger a, bourauel c. in-vitro evaluation of the corrosion behavior of orthodontic brackets. orthod craniofac res. 2009; 12: 43–51. 27. kao ct, huang th. variations in surface characteristics and corrosion behaviour of metal brackets and wires in different electrolyte solutions. eur j orthod. 2010; 32(5): 555–60. 28. chaturvedi tp, upadhayay sn. an overview of solutions. dent mater j. 2010; 29: 53–8. 29. fábián tk, fejérdy p, csermely p. saliva in health and disease, chemical biology of. in: wiley encyclopedia of chemical biology. hoboken, usa: john wiley & sons, inc.; 2008. p. 1–9. 30. iijima m, yuasa t, endo k, muguruma t, ohno h, mizoguchi i. corrosion behavior of ion implanted nickel-titanium orthodontic wire in fluoride mouth rinse solutions. dent mater j. 2010; 29: 53–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p5–9 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p5-9 vol 51 no 1 jan-mrt 2018.indd 20 antibacterial activity of mixed pineapple peel (ananas comosus) extract and calcium hydroxide paste against enterococcus faecalis intan fajrin arsyada, devi rianti, and elly munadziroh department of dental materials faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: enterococcus faecalis (e. faecalis) is the bacteria most commonly resulting from failed root canal treatment. intracanal medicament is used to enhance the success of root canal treatment. a material widely used for this purpose is calcium hydroxide,. however, its ineffectiveness in eliminating e. faecalis requires the addition of other antibacterial substances, such as iodoform which has the disadvantage of having toxic effects on tissues. pineapple peel has antibacterial properties because it contains chemical compounds, such as flavonoid, saponin, tannin, as well as the enzyme bromelain. purpose: the aim of the study was to determine the antibacterial activity of a mixture of pineapple peel extract at 6.25% and 12.5% concentrations and calcium hydroxide paste at a ratio of 1:1 compared to100% calcium hydroxide and a mixture of calcium hydroxide and iodoform paste against e. faecalis. methods: the research was laboratory-based experiment in nature. sample groups were divided into two control groups (one featuring100% calcium hydroxide paste and a second featuring a mixture of calcium hydroxide and iodoform paste) and two treatment groups (mixture of pineapple peel extract and calcium hydroxide paste in 6.25% and 12.5% concentrations with ratio 1:1). the method was using agar diffusion. the result data were analyzed by one way anova test. results: the highest average of the inhibitory zone occurred in group with a mixture of pineapple peel extract 12,5% and calcium hydroxide paste while the smallest average was that of group with a mixture of calcium hydroxide and iodoform paste conclusion: mixture of pineapple peel extract in 6.25% and 12.5% concentrations and calcium hydroxide paste ratio 1:1 has higher antibacterial activity than paste of 100% calcium hydroxide and mixture of calcium hydroxide and iodoform paste againts e. faecalis. keywords: calcium hydroxide; pineapple peel; zone inhibit; enterococcus faecalis; intracanal medicament correspondence: devi rianti, department of dental materials, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: devi-r@fkg.unair.ac.id research report introduction dental caries constitute the most prevalent infectious disease worldwide. if dental caries are ignored, bacteria may reach the pulp chamber through open dentinal tubules resulting in the colonization of microorganisms. this may, in turn, lead to infection of the root canal.1 root canal treatment is the treatment of choice for such infections. failure of root canal treatment due to the persistence of bacterial infection within the root canal is referred to as a secondary infection.2 microorganism is a factor associated with root canal treatment failure.3 enterococcus faecalis (e. faecalis) is the most commonly bacteria found in cases of failed root canal treatment.2 studies reported that 77% of failed root canal treatment cases suffered reinfection due to e. faecalis resistance. the high resistance of e. faecalis because of various virulence factors, including its ability to compete with other microorganisms in invading dentinal tubules and to survive in high temperatures and broad ph range.4 the principle of root canal treatment is removal of the organic and inorganic debris, microorganisms and toxic products from the root canal.5 preparation procedures cannot eliminate bacteria effectively. therefore, it is dental journal (majalah kedokteran gigi) 2018 march; 51(1): 20–24 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p20–24 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p20-24 21arsyada, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 20–24 necessary to administer intracanal medicaments prior to obturation of the root canal.6 one material widely used as an intracanal medicament is calcium hydroxide which is a bactericide, but it is not effective in eliminating microbes from dentin tubules because e. faecalis is resistant to it.7 the optimization of the antibacterial activity of calcium hydroxide should be supplemented with another antibacterial substance such as iodoform. iodoform can increase antibacterial activity, but has some shortcomings such as it can be dissolved rapidly by root canal microorganisms and have toxic effects on tissues.8 it is necessary to develop a herbal material as an alternative to substitute these chemicals. one of the herbal materials possessing antibacterial properties is pineapple peel (ananas comosus). pineapple peel has antibacterial properties because it contains chemical compounds. a phytochemical test showed that ethanol extracted from pineapple skin contains alkaloids, phenols, flavonoids, phytosterols, steroid, saponins, tannins and terpenoids.9 in addition, there is another component of pineapple peel, namely; bromelain enzyme.10 previous research indicated that the inhibitory zone of pineapple peel extract against staphylococcus aureus measured 15.06 mm.11 other research indicated that the minimum inhibitory concentration (mic) of pineapple peel extract against streptococcus viridans was 1.56% and minimum bactericidal concentration (mbc) was 3.125%.12 according to previous studies, the minimum bactericidal concentration of pineapple peel extract against the growth of e. faecalis was 6.25%. therefore, it was necessary to investigate the antibacterial activity of the pineapple peel extract (concentration of 6.25% and 12.5%) and calcium hydroxide paste mixture as an alternative intracanal medicament against e. faecalis. the aim of the study was to determine the antibacterial activity of a mixture of pineapple peel extract in 6.25% and 12.5% concentrations and calcium hydroxide paste at a ratio of 1:1 compared to100% calcium hydroxide and a mixture of calcium hydroxide and iodoform paste against e. faecalis. materials and methods this research was approved by the ethics review committee of the faculty of dental medicine, universitas airlangga, surabaya, no. 147/kkepk.fkg/vii/2016. pineapple peel extract was obtained from phytochemicals laboratory, materia medica, batu, indonesia. e. faecalis atcc 29912 bacteria were drawn from the stock of the microbiology laboratory, faculty of dental medicine, universitas airlangga, surabaya. this research constituted a laboratory experiment with post test only control group design. sample groups divided into four groups, including two positive control groups and two treatment groups. the positive control groups (group a and b) contained 100% calcium hydroxide paste (hydroxidocalcio pa), and a mixture of calcium hydroxide and iodoform paste (calplus). meanwhile, the treatment groups (group a and b) received a mixture of 12.5% pineapple peel extract and calcium hydroxide paste, and mixture of 6.25% pineapple peel extract and calcium hydroxide paste. pineapple peel extract was extracted by macerating with 96% ethanol. the results of extraction produced 45 ml of 100% pineapple peel extract. the dilution processes of pineapple peel extract using distilled water to obtain 12.5% and 6.25% concentrations. pineapple peel extract was combined with calcium hydroxide powder at a ratio of 1:1 and mixed until homogeneous. preparation of e. faecalis bacteria begins with culturing e. faecalis in bhib and incubating it in an anaerobic atmosphere at 37° c for 24 hours. subsequently, a suspension of e. faecalis bacteria comparable to a mcfarland standard 0.5 of 1.5 x 108 cfu/μ was produced (1.5 x 108 cfu/ ml). the determination of the antibacterial activities of sample groups against e. faecalis employed an agar diffusion method. e. faecalis was cultured in nutrient agar media. a petridish containing nutrient agar media was divided into four zones, separated by a line marker on its underside. each well zone contained 0.5 gram sample groups. the petridish was incubated anaerobically in an incubator at 37° c for 24 hours. the diameter of the inhibitory zone of the resulting culture was measured. the inhibitory zone could be seen as a clear area around the wells that showed no bacterial growth. measurement of the diameter of the inhibitory zone to the nearest 0.1 mm was effected using calipers. a kolmogorov-smirnov normality statistic test was performed, while the difference between the groups was established by the conducting of a one-way anova test, followed by a multifactorial comparison test using a tukey hsd test and a preceding levene homogenity statistic test. e 1. diameter of inhibitory zone a b c d figure 1. diameter of inhibitory zone in each sample groups againts enterococcus faecalis. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p20–24 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p20-24 22 arsyada, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 20–24 results the subjects of the research into the antibacterial activity of a mixture of pineapple peel extract and calcium hydroxide paste against e. faecalis were divided into four groups, two treatment groups and two positive control groups with seven replications. the calculation results of the averages and standard deviations in each sample group can be seen in table 1. the results showed that the highest average of the inhibitory zone occurred in group c with a mixture of pineapple peel extract 12.5% and calcium hydroxide paste, while the smallest average was that of group b with a mixture of calcium hydroxide and iodoform paste. the terms of parametric statistical tests were normality and homogenity. a kolmogorof smirnov normality test showed that each sample group had a value of p>0.05. consequently, it can be seen that the data from all the research groups had normal distribution.the results of a subsequent homogenity test using levene test showed a value of p 0.125, indicating that all the research groups had a homogen variance (p>0.05). with the prerequisite of normal and homogeneous distribution, a one-way anova test was then conducted. based on its results, the significance value obtained was 0.001, smaller than α=0.05, indicating that there was a significant difference in the inhibitory zone of all sample groups against e. faecalis. a tukey hsd test was performed to identify the significant differences between the sample groups, the results of which showed a significance value less than 0.05. this means that there were significant differences in the inhibitory zone between each group against e.faecalis. the probability value of the tukey hsd test results can be seen in table 2. discussion the results showed the inhibition zone of the mixture of pineapple peel extract and calcium hydroxide paste and 100% calcium hydroxide paste, except for the mixture of calcium hydroxide and iodoform paste. an anova test showed there to be significant differences in the inhibitory zone in all sample groups againts e. faecalis. the highest average inhibitory zone was found in the group containing a mixture of pineapple peel extract 12.5% and calcium hydroxide paste, while the smallest inhibitory zone occurred in the group featuring a mixture of calcium hydroxide and iodoform paste. based on the research findings, the mixture of calcium hydroxide paste and pineapple peel extract with respective concentrations of 6.25% and 12.5% yielded 19.8 mm and 21.58 mm of inhibitory zone diameters. these inhibitory zone diameters are greater than that of 100% calcium hydroxide paste, which yielded 17.58 mm of inhibitory zone. the inhibitory zone of the group containing 100% calcium hydroxide paste indicated that calcium hydroxide possesses antibacterial properties. these results were due to the mechanical action of calcium hydroxide against bacterial growth through the release of hydroxyl ions that can damage the cytoplasmic membrane of bacteria.13 hydroxyl ions with an alkaline ph when coming into contact with the bacterial cytoplasmic membrane protein, will break the hydrogen bonds in the protein structure. the damage caused by hydrogen bonding will lead to changes in the polypeptide chain binding protein, and protein denaturation or loss of natural functions of the protein will occur. when hydroxyl ions come into contact with bacterial dna, this will inhibit bacterial replication and mutations. this resulted in the breakdown of the structure and function of dna, lead to bacterial cell death. based on its antibacterial activities, calcium hydroxide was able to kill bacteria.14 iodoform added to calcium hydroxide paste was expected to increase the antibacterial activities of calcium hydroxide. the antibacterial mechanism of iodoform through the release of iodine which increases protein precipitation and oxidation of essential enzymes. iodoform plays an important role in controlling infection and has an intermediate antibacterial level.15 in this research the opposite occurred since the group b, which was a mixture of calcium hydroxide and iodoform paste, showed no inhibition zone against e. faecalis. it could potentially be influenced by the inadequacy of the iodoform and could be dissolved rapidly by root canal microorganisms,8 including e. faecalis. tabel 1. averages (mm) and standard deviations of inhibitory zone in each sample groups againts e. faecalis nsample groups average (mm) standard deviation 0.3368217.58867a 007b 0.1973921.57577c 0.4335519.80437d note: group a : 100% calcium hydroxide paste group b : mixture of calcium hydroxide and iodoform paste group c : mixture of pineapple peel extract 12.5% and calcium hydroxide paste (1:1) group d : mixture of pineapple peel extract 6.25% and calcium hydroxide paste (1:1) n : sample size table 2. the result of tukey hsd test dcbasample groups 0.000--a * 0.000* 0.000--b * 0.000* 0.000---c * ----d note: * = there was a significant difference (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p20–24 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p20-24 23arsyada, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 20–24 the data relating to the results of a mixture of pineapple peel extract and calcium hydroxide paste showed that the inhibitory zone was higher than 100% calcium hydroxide paste. according to the results of phytochemical test referred to in previous research, the highest concentration of active compound in pineapple peel extract was that of flavonoids.16 ether group in flavonoids tends to be unstable due to the easy release of electrons and have an opposite charge equal to that of the hydrogen atom of calcium hydroxide. this makes binding possible to form an alcohol group or hydroxyl. the release of hydroxyl ions can damage the bacterial cytoplasmic membrane. this reaction explains the ability of pineapple peel extract in synergism with calcium hydroxide to increase the inhibitory zone against e. faecalis. previous research showed that the inhibition zone increases linierly with the increasing concentration of the pineapple peel extract mixed with calcium hydroxide paste. one of the factors influencing the values of inhibitory zone is the concentration of antibacterial compounds.17 a mixture of pineapple peel extract 12.5% and calcium hydroxide paste produces a higher inhibitory zone than the group containing a mixture of pineapple peel extract 6.25% and calcium hydroxide paste. the higher inhibitory zone due to antibacterial compounds in pineapple peel extract is more elevated at higher concentrations. the higher antibacterial compound, the greater ability to inhibit bacteria within the extract. pineapple peel extract demonstrates antibacterial properties because it contains chemical compounds. in addition to flavonoids as the highest concentration of chemical compounds in pineapple peel extract, there are other active compounds, namely: saponins, tannins, and enzymes bromelain. each of these chemical compounds has a specific role in inhibiting the growth of e. faecalis. flavonoid compounds have the ability to form complex bonds with extracellular protein through hydrogen bonding. hydrogen bonds with extracellular proteins cause bacterial cell membrane structures containing proteins to become unstable. consequently, the permeability of bacterial cell wall will be disrupted, resulting in bacterial cell death.18 tannin compounds can damage bacterial cell membranes and result in reduced permeability, so the growth and activities at bacterial cells is inhibited.19 in other chemical compounds, saponin, as an antibacterial agent, can reduce the surface tension leading to the increasing permeability of cell membranes. changes in cell membrane permeability cause release of intracellular compounds from the cell that leads to bacterial lysis.20 the action mechanism of the bromelain enzyme as an antimicrobial lies in altering or damaging the wall structure of bacteria-containing protein. the bromelain enzyme will break down and denature the protein constituent of bacterial cell walls. as the cell weakens it can allow small moleculer to leak out21 antibacterial activity based on the value of the inhibitory zone was categorized into three groups, a weak level inhibitory zone of 0.1 mm-11.5 mm, a middle level inhibitory zone of 11.5 mm-19.7 mm and a high level inhibitory zone of more than 19.7 mm.22 the results of inhibitory zone in group featuring a mixture of pineapple peel extract and calcium hydroxide were 19.8 mm and 21.58 mm respectively. this indicates that antibacterial activities of a mixture of pineapple peel extract and calcium hydroxide paste are included in the strong level. based on these analysis, the mixture of pineapple peel extract and calcium hydroxide paste can be considered to be an alternative intracanal medicament in root canal treatment. from the forgoing analysis, it can be concluded that a mixture of pineapple peel extract at 6.25% and 12.5% concentrations and calcium hydroxide paste with a ratio of 1:1 demonstrates a higher antibacterial activity than 100% calcium hydroxide paste and mixture of calcium hydroxide and iodoform paste against e. faecalis. references 1. narayanan ll, vaishnavi c. endodontic microbiology. j conserv dent. 2010; 13(4): 233–9. 2. shailaja s, suresh bs. endodontic microfloraa review. j oral heal community dent. 2014; 8(3): 160–5. 3. punathil s, bhat ss, bhat sv, hegde sk. microbiolgical analysis of root canal flora of failed pulpectomy in primary teeth. internstional j curr microbiol appl sci. 2014; 3(9): 241–6. 4. fraser sl. enterobacter infections. 2015. p. 346. available from: https://emedicine.medscape.com/. accessed 2016 jun 3. 5. gjorgievska e, apostolska s, dimkov a, nicholson jw, kaftandzieva a. incorporation of antimicrobial agents can be used to enhance the antibacterial effect of endodontic sealers. dent mater. 2013; 29(3): e29–34. 6. mattigatti s, ratnakar p, moturi s, varma s, rairam s. antimicrobial effect of conventional root canal medicaments vs propolis against enterococcus faecalis, staphylococcus aureus and candida albicans. j contemp dent pract. 2012; 13(3): 305–9. 7. kandaswamy d, venkateshbabu n, gogulnath d, kindo aj. dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide. int endod j. 2010; 43(5): 419–23. 8. da silva lab, leonardo mr, de oliveira dsb, da silva rab, de queiroz am, hernández pg, nelson-filho p. histopathological evaluation of root canal filling materials for primary teeth. braz dent j. 2010; 21: 38–45. 9. kalaiselvi m, gomathi d, uma c. occur rence of bioactive compounds in ananus comosus (l.): a quality standardization by hptlc. asian pac j trop biomed. 2012; 2(3): s1341–6. 10. shweta ap. bromelain a cysteine protease: helps to reduce infection caused by acinetobacter spp., a nosocomial pathogen. int j adv biotechnol res. 2014; 5(3): 415–22. 11. manaroinsong a, abidjulu j, siagian krista v. uji daya hambat ekst ra k k ul it na nas (a na nas comosus l) terhadap ba k ter i staphylococcus aureus secara in vitro. pharmacon. 2015; 4(4): 27–33. 12. yustisia bca. daya antibakteri ekstrak kulit nanas (ananas comosus) terhadap pertumbuhan streptococcus viridans. thesis. surabaya: universitas airlangga; 2015. p. 45. 13. gautam s, rajkumar b, landge sp, dubey s, nehete p, boruah lc. antimicrobial efficacy of metapex (calcium hydroxide with iodoform formulation) at different concentrations against selected microorganisms--an in vitro study. nepal med coll j. 2011; 13(4): 297–300. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p20–24 https://emedicine.medscape.com/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p20-24 24 arsyada, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 20–24 14. bauman rw. microbiology: with diseases by taxonomy. 2nd ed. london: pearson education; 2007. p. 287-310. 15. estrela c, estrela cr de a, hollanda acb, decurcio d de a, pécora jd. influence of iodoform on antimicrobial potential of calcium hydroxide. j appl oral sci. 2006; 14: 33–7. 16. putri rma. daya antibakteri kulit nanas (ananas comosus) terhadap pertumbuhan bakteri enteococcus faecalis. thesis. surabaya: universitas airlangga; 2015. p. 38. 17. markey bk, leonard fc, archambault m, cullinane a, maguire d. clinical veterinary microbiology. 2nd ed. st. louis: mosby elsevier; 2013. p. 80. 18. manimozhi dm, sankaranarayanan s, sampathkumar g. evaluating the antibacterial activity of f lavonoids extracted from ficus benghalensis. int j pharm biol res. 2012; 3: 7–18. 19. damayanti a. efektivitas antibakteri ekstrak etanol biji alpukat (persea americana) sebagai bahan irigasi saluran akar terhadap pertumbuhan bakteri enterococcus faecalis. thesis. surakarta: universitas muhammadiyah surakarta; 2014. p. 10. 20. ngajow m, abidjulu j, kamu vs. pengaruh antibakteri ekstrak kulit batang matoa (pometia pinnata) terhadap bakteri staphylococcus aureus secara in vitro. j mipa unsrat online. 2013; 2(2): 128–32. 21. eshamah h, han i, naas h, rieck j, dawson p. bactericidal effects of natural tenderizing enzymes on escherichia coli and listeria monocytogenes. j food res. 2013; 2: 8–18. 22. kriplani r, thosar n, baliga ms, kulkarni p, shah n, yeluri r. comparative evaluation of antimicrobial efficacy of various root canal filling materials along with aloevera used in primary teeth: a microbiological study. j clin pediatr dent. 2013; 37(3): 257–62. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p20–24 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p20-24 119 the pattern of p53 gene mutations on oral squamous cell carcinoma peter agus department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract oral squamous cell carcinoma (oscc) is the result of accumulation genetic lesion and caused by specific mutation in specific key regulation genes. p53 gene is key target specific regulatory genes which function as negative regulators in cell cycle control. the highest mutation rates found in human cancers and the etiology in high risk populations and the pattern of molecular pathogenesis mechanism involved in the oscc remain unclear. the purpose of this study was to determine the presence of alteration or mutation of p53 gene and to associate these mutations histopathological status of the patients such as well differentiated and poorly differentiated in oscc in order to elucidate the molecular pathogenesis mechanism of oscc based on the pattern of p53 gene. using 40 untreated well and poorly differentiated oscc biopsy samples and 16 normal patients were analyzed for the presence of mutation in the conserved region of the p53 gene exons 5 and or 7 by pcr-sscp mutational analysis for p53 gene showed 70% of total samples : exon 5: 27.5% with heterozygous mutation 81.8%, exon 7: 55% with heterozygous mutation 100%. the incidence of p53 mutation was not significantly associated with well and poorly differentiated oscc with the exception in exon 5 of p53 gene (p = 0.013) using contingency coefficient. this study concludes that mutation of p53 gene especially in exon 7 may not indirectly play in the progressivity of oscc with the exception of mutation in exon 5 of p53 which indicates the essential role in the progressivity of oscc. key words: p53, exon5, exon7, mutation, oscc, well and poorly differentiated correspondence: peter agus, c/o: bagian bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: peter_a@indo.net.id introduction oral squamous cell carcinoma (oscc) is the most common malignant tumor of oral cavity, it is account for 80-90% of all malignancies in oral cavity and the mortality rate remains high about 5% in worldwide and 2,4%-3,57% in indonesia.2 up to now, the molecular pathogenesis of oscc is still unknown and the diagnosis is only based on clinicopathological examination. consequently, the most frequent oscc cases are found in advanced state approximately 76,3%, therefore, the management of oscc has not shown any satisfactory outcome.1,2 disruption of the cell cycle as well as component regulatory genes which involved in controlling cell cycle is the main factor in the development process of all malignancies, p53 tumor suppressor gene has an important role in malignancy development process especially in early phase of cell cycle g1-s and loss of function p53 gene most common cause of oscc.3 futhermore, p53 gene has shown a direct in activation and has an essential role to control and to regulate cell cycle as well as to induce apoptosis.4,5 subsequently, inactivation of p53 gene would cause the activity of p53 to be disrupted resulting in uncontrolled cell proliferation and if it continuously occurs as a result it could contribute malignancy in all cancers. the aim of this study was to determine the molecular pathogenesis of oscc and by detecting the presence of p53 mutation in oscc and to deserve the association of mutation with histopathological grade. the advantage of this study is expected to be able to support clinical diagnosis and histopathological of oscc consequently the patient could be treated better. material and methods in this study, analytical observational comparative study by cross sectional design was used. the subjects of observation were patients who clinically diagnosed as suspected having oscc, patients who underwent histhopatological diagnosis suffering form oscc and normal patients who fulfilled the observation’s requirement. furthermore, patients were classified into two groups consisted of well differentiated oscc and poorly differentiated oscc and controlled group with normal tissue taken from impacted third molar was taken with surgery indication. detection of p53 gene mutation on 40 samples freshed tissue with hpa diagnostic as well and poorly differentiated oscc and controlled group with normal tissue using pcr-sscp with primer design p53 of exon 5 and 7 (gene bank accession number u 94788) as follows : exon 51 : 5’ tgt tca ctt gtg gtg ccc tga ct-3’, 5’-agc aat cag tga gga atc ag-3’ and exon 52-cag ccc tgt cgt ctc tcc ttc ct-3’, 5’-cag ccc tgt cgt 120 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 119-122 ctc tcc ag-3’ while exon 71: 5’-ctt gcc aca ggt ctc ccc aa-3’, 5’-agg ggt cag cgg caa ga-3’ and exon 72: 5’-agg cgc act ggc ctc atc tt-3’, 5’-tgt gca ggg tgg caa gtg gc-3’. next, dna isolation was performed using procedure of modification dna isolation by taking biopsy tissue oscc and normal tissue (1 × 1 × 1 cm) with scalple and washed with pbs solution in eppendorf tubes, added with 700 µl buffer consisted of 50 mm tris hcl ph 8, 100 mm edta, 100 mm nacl, 1% sds, 50 ml of 10-20 mg/ml proteinase – k and incubated at 55 °c overnight until cell suspension was achieved. the dna was extracted from oscc tissue and controlled tissue using 0,5 ml phenol/ciaa and put into eppendorf shaker (eppendorf mixer 5432) for 2 hours. it was centrifuged for 5 minutes and cell suspension was put into fresh eppendorf tube and transferred into another tube added with 500 ml isopropanol/ethanol until precipitated from was obtained then, the supernatant was disposed. the next step, 500 ml te buffer was added into cell suspension in eppendorf tube and incubated overnight at room temperature. afterward, eppendorf tube was shaken manually until it was well mixed. dna identification was done using 2% agarose gel electrophoresis and visualized by uv light. dna concentration test was done by spectrophotometry.6 sscp analysis for dna resulted from p53 gene pcr product (exon 5.7). 40 samples were divided in which every group consisting of 10 samples then, 5 ml dna of pcr resulted from optimal product of p53 gene pcr, every group was marked with number of samples and k for control group. next step, 10 ml formamid was added as loading buffer, 15 ml denaturation solution was achieved in eppendorf, shaken eppendorf with vertical rotater was done followed by centrifuged. denaturation was continued in boiling water at 100 °c for 10 minutes, and chilled using nitrogen liquid at –80 °c for 5–10 minutes. each pcr tube contained 15 µl of dna p53 gene was added with buffer loading, followed by analysis using 12% non denaturation acrylamide gel electrophoresis at room temperature for 5 hours with constant voltage 150 °c. furthermore, silver nitrate staining was done with following procedure; fixer solution (7.5% acetic acid) for 30 min and washed (3×) using dionized water (dh2o) for 10 minutes. then added with 100ml silver nitrate solution (1.5 g/l agno3 and 0.056% formaldehyde) for 45 minutes and rinsed with dionized water for 15 seconds. furthermore, image development was done with developer solution 30 g/l na2co3, 0,056% formaldehyde 2 mg/l sodium thiosulfate for 2–5 min. stop solution with fixer solution 7,5% acid at (–4 °c) for 30 seconds until 5 min. the result of sscp analysis the product of p53 gene pcr exon 5 and 7 showed the migration pattern or single stranded conformation polymorphism (sscp) could exhibit 2 bands in unsimilar position with 2 band of dna resulted from controlled sscp showed the presence of homezygote mutation. indicating positive (+) or positive mutation (score 1), if the result was 3 bands or 4 bands of dna p53 gene exon 5.7 unsimilar position with 2 bands of normal dna sscp (controlled pcr-sscp) showed the presence of heterozygote mutation indicating positive (+) or score 1, if the result was 2 bands in p53 exon 5.7 in similar position with 2 bands in controlled pcr-sscp indicating the absence of mutation negative (–) or score 0.6 cross sectional data analysis was done using spss program version 11. the data resulting from observation of gene mutation by pcr-sscp method in all samples was useful to know the absence or the presence of gst p53 gene mutation (exon 5.7) and the correlation with hpa data: well and poorly differentiated oscc. the contingency coefficient test was used for statically analysis to know the strength and the weakness of hpa correlation: well or poorly differentiated oscc with tumor suppressor p53 gene mutation resulted from p53 gene mutation exon 5.7 in oscc. result the study outcome of 40 samples of tumor gene suppressor p53 of oscc compared with controlled group mutation of p53 detected in exon 5: 11/40 (27.5%) with homozygous mutation 2/11 (18.2%), heterozygous 9/11 (81.8%) while the absence of mutation 29/40 (72.5%). in p53 exon 7 showed 22/40 (55%) mutation with heterozygous mutation 22/22 (100%) while the absence of mutation 18/40 (45%) shown on table 1. the analysis outcome of 40 patient with well differentiated hpa of oscc, 20 mutation cases of p53 exon 5: 2/20/ (10%) and exon 7: 10/20/ (50%). the analysis outcome of 40 patient with poorly differentiated hpa of oscc, 20 mutation cases of p53 exon: 9/20(45%) and exon 7: 12/20 (60%) the data shown below on table 2. significant associated with value 0,365 and p = 0.013 between histopathological feature of well and poorly differentiated of oscc and mutation of p53 exon 5 of oscc, shown on table 3. table 1. pcr-sscp analysis of p53 of oscc oscc incidence of mutations p53 (%) mutation (+) no mutation (–) total homozygote heterozygote total p53 e5 11 (27.5%) 29 (72.5%) 40 2 (18.2%) 9 (81.8%) 11 e7 22 (55%) 18 (45%) 40 0 (0%) 22 (100%) 22 121agus: the pattern of p53 gene mutations the result of statistical test of the association of histophatological feature between well poorly differentiated of p53 exon 7 of oscc, no significant association with value 0.100 and p = 0.525 between well and poorly differentiated p53 exon 7, shown on table 4. discussion the incidence of p53 mutation over 50% was frequently found in exon 5-8 in all cancers especially exon 5 and 7 for colorectal and laryngeal carcinoma.7,8 in this study the incidence of p53 mutation in oscc has shown 28/40 (70%), exon 7; 22/40 (55%) and exon 5; 11/40 (27.5%). the result indicated that mutation of gene p53 more frequently involved in molecular control of oscc cell cycle and it is not influenced by alteration of other tumor suppressor gene inactivation in oscc cell cycle. seventy percent of p53 mutation showed that p53 gene has controlling function of cell cycle and encountered 70% inactivation or loss stability of p53 function as factor of transcription gene p21 walf1/cip1, mdm2, gadd45, 14-3-3a bax and killer/dr5 which has an important role in controlling and regulating cell cycle, improvement of damaged dna as well as apoptosis. in this case, p53 showed more direct role in g1/s phase, comparing with gene 21 which also has direct activation in g1/s phase, however, further study is necessarily done. the dysregulation of the molecular events governing cell cycle control is emerging as a central theme of oral carcinogenesis. regulatory pathways responding to extracellular signaling or intra cellular stress and dna damage converge on the cell cycle apparatus. abrogation of mitogenic and anti-mitogenic response regulatory proteins, such as the retinoblastoma tumor suppressor protein (prb), cyclin d1, cyclin-dependent kinase (cdk) 6, and cdk inhibitors (p21(waf1/cip1), p27 (kip1), and p16 (ink4a), occur frequently in human oral cancers. in this study, 30% mutation of p53 gene was not found and probably caused by other gene mechanism such as : gene p21, prb, p27 and oncogen cyclin d1, cdk6 involving directly in mechanism of cancer cell cycle control especially in g1-s phase of cell.9 in this study exon 7 (55%) with heterozygous mutation 100% was found higher than exon 5 (27.5%) with homozygous mutation 18,2%. the result of this study also suggested that in oscc mutation of p53 exon 7 more frequently occurred than exon 5 and mostly involved in molecular pathogenesis of oscc. meanwhile, p53 gene exon 5 with 81.8% heterozygous mutation and 18.2% homozygous mutation could induce uncontrolled cell proliferation, therefore p53 exon had an important role in controlling cancer cell cycle. tumor suppressor gene p53 has the highest heterozygous mutation in exon 7 comparing with the result of study done in foreign country: 22/22 (100%), homozygous mutation 0/22 (0%) and exon 5 with heterozygous mutation 9/11 (81.8%) homozygous mutation 2/11 (18.2%). the result of the study is not equivalent to the outcome of some studies done in foreign countries which suggested p53 with heterozygous mutation 65% in china,9 heterozygous mutation p53 (exon 2-9) 55% in england,10 table 2. hpa feature of p53 gene mutation of oscc hpa mutation of p53 exon 5 mutation of p53 exon 7 mutation (+) no mutation (–) mutation (+) no mutation (–) w diff 10 90 50 50 p diff 45 55 60 40 note: w diff = well differentiated, p diff = poorly differentiated table 3. the result of statistical test of the association of histopathological feature between well and poorly differentiated p53 exon 5 of oscc variable test df value p (sig) hpa oscc w diff mutation of p53 ekson5 contingency coefficient 1 0,365 0,023 p diff table 4. the result of statistical test of association of well and poorly differentiated of p53 exon 7 of oscc variable test df value p (sig) hpa oscc w.diff mutation of p53 exon7 contingency coefficient 1 0.100 0.525 p.diff 122 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 119-122 and less frequent heterozygous mutation p53 in india.11 p53 gene especially exon 7 (55%) with homozygous mutation 100% would induce loss of p53 function in which dominant mutation in one of both alelle of chromosome resulting more uncontrolled cell cycle proliferation as the cause of malignancy in oscc, therefore, p53 exon 7 is ideal to be molecular indicator to detect the level of malignancy especially oscc. meanwhile, although in p53 exon 5 less possibilities (27.5%) of other genes mechanism was found involved in controlling cell cycle of oscc, consequently, mutation could occur in the other region of exon 5 or in other hot spot region. interestingly, in p53 exon 5 with high heterozygous mutation (81.8%) was found and it shows that p53 exon 5 also has essential role in controlling cell cycle of cancer as the cause of oscc and could be targeted as molecular indicator to detect the level of oscc malignancy. in this study, no significant association (p = 0.168) was found between well and poorly differentiation oscc with p53 mutation of exon 7 (55%, p = 0.525), the possibility of other gene mechanism which directly plays an essential role in the mechanism of molecular pathogenesis such as p21 gene which could directly inhibit the activation of excessive cell proliferation in cell cycle. significant association (p = 0.0013) was found in p53 gene exon 5 (27.5%) with heterozygous mutation 81.8% and homozygous mutation 18.2% in well and poorly differentiated oscc. this study is not equivalent to the outcome of study done in germany for head and neck cancer and in china for breast cancer.12 so the result of this study shows that in tumor suppressor gene p53 exon 5 although the incidence of mutation is relatively small, however, it has an essential role in the progressivity of cancer, it could be shown by the presence of p53 mutation exon 5 in well differentiated oscc (20%) and the increase of mutation (80%) in poorly differentiated oscc, therefore, p53 exon 5 is ideal to be an indicator to detect the level of malignancy especially when well differentiated oscc is found. therefore, it could be used either as diagnosis indicator or as a molecular prognosis of oscc. it could be concluded that detection of p53 mutation of oscc in oral cancer 70% of total samples was found p53 exon 7 (55%) with heterozygous mutation 100%, therefore, p53 exon 7 is ideal to be targeted indicator of molecular diagnosis and prognosis of oscc but it is not specific for histopathological feature of well and poorly differentiated oscc. meanwhile, p53 exon 5 (27.5%) has significant association in histopathological feature between well and poorly differentiated oscc (p = 0.013) therefore, p53 exon 5 is ideal to be targeted indicator of molecular diagnosis and prognosis of oscc especially well differentiated oscc. in addition, it is necessary to perform a study on p53 detection using clinical feature parameter such as: age, gender, tumor location, tumor staging, tnm system and sequencing procedure is done to detect the type and location af dna mutation so, it would be useful to increase and to confirm the outcome of this study such as: performing a study on other tumor suppressor gene which frequently involving in the mechanism of molecular pathogenesis of cell cycle such as : p21, prb, p27, and other oncogene: cyclin d1, cdk4, e2f, mdm2 and c-myc so it would be useful to elucidate molecular pathogenesis of cancer especially oscc. references 1. neville bw, day ta. oral cancer and precancerous lessions. ca cancer j clin 2002, july–aug 2002; 52(4):195–216. 2. agus p. analisis molekuler patogenesis karsinoma sel skuamosa rongga mulut berdasarkan pola mutasi gen p53 dan p16. disertasi. surabaya: pascasarjana universitas airlangga; 2004. p. 1–7. 3. vielba r, bilbao j, ispizua a, zabalza i, alfaro j, rezola r, moreno e, elorriaga j, alonso i, baroja a, de la hoz c. p53 and cyclin d1 as prognostic factors in squamous cell carcinoma of the larynx. laryngoscope 2003 january; 113(1):167–72. 4. kawamata h, omotehara f, nakashiro k, uchida d, shinagawa y, tachibana m, imai y, fujimori t. oncogenic mutation of the p53 gene derived from head and neck cancer prevents cells from undergoing apoptosis after dna damage. int j oncol 2007 may; 30(5):1089–97. 5. chang ns. the non-ankyrin c terminus of ikappa b alpha physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, dna damage, and transforming growth factorbeta 1-mediated growth suppression. j biol chem 2002 march 22; 277(12):10323–31. 6. sambrook j, fritsch ef, maniatis t. molecular cloning: a laboratory manual. 2nd ed. new york: cold spring laboratory press; 1989. p. 6.4–6.60, 14.2–14.35. 7. hsieh js, lin sr, chang my, chen fm, lu cy, huang tj, huang ys, huang cj, wang jy. apc, k-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance. am surg 2005 apr; 71(4):336–43. 8. russo a, corsale s, agnese v, macaluso m, cascio s, bruno l, surmacz e, dardanoni g, valerio mr, vieni s, restivo s, fulfaro f, tomasino rm, gebbia n, bazan v. tp53 mutations and s-phase fraction but not dna-ploidy are independent prognostic indicators in laryngeal squamous cell carcinoma. j cell physiol 2006 january; 206(1):181–8. 9 todd r, hinds pw, munger k, rustgi ak, opitz og, suliman y, wong dt. cell cycle dysregulation in oral cancer. crit rev oral biol med 2002; 13(1):51–61. 10. bai wl, gao h, ren z, pan zm. detection of mutation in exon 5 and exon 8 of pten in laryngeal squamous cell carcinoma. zhonghua er bi yan hou ke za zhi 2004 january; 39(1):13–6. 11. kannan s, yokozaki h, jayasree k, sebastian p, mathews a, abraham ek, nair mk, tahara e. infrequent loss of heterozygosity of the major tumour suppressor genes in indian oral cancers. int j oral maxillofac surg 2002 august; 31(4):414–8. 12. hong y, miao x, zhang x, ding f, luo a, guo y, tan w, liu z, lin d. the role of p53 and mdm2 polymorphisms in the risk of esophageal squamous cell carcinoma. cancer res 2005 october 15; 65(20):9582–7. 13. kumar s, walia v, ray m, elble rc. p53 in breast cancer: mutation and countermeasures. front biosci. 2007 may 1; 12:4168–78. 139139 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 139–143 original article dental traumatic injuries during the covid-19 pandemic: a retrospective study mehmet veysel kotanli1, mehmet emin doğan2, sedef kotanli2, mehmet sinan doğan1 1department of pediatric dentistry, faculty of dentistry, harran university, sanliurfa, turkey 2department of dentomaxillofacial radiology, faculty of dentistry, harran university, sanliurfa, turkey abstract background: although dental trauma has been reported at various frequencies worldwide, as far as we know there are few data regarding the frequency of dental trauma during the pandemic period. purpose: this study aims to retrospectively evaluate the data of pediatric patients who were admitted to the department of pedodontics in a university hospital due to dental trauma during the covid19 pandemic. methods: between april 2020 and december 2020, the clinical records regarding admissions to the harran university faculty of dentistry pedodontics clinic due to dental trauma were examined. the age and gender of the patient, type of trauma, teeth affected by the trauma, and treatment methods applied after the trauma were recorded. the data obtained were analyzed using the independent sample t-test for intergroup comparisons and pearson chi-square test for categorical variables with the help of statistical package for the social sciences version 23.0. results: the data of 43 patients (27 males and 16 females) aged between 1–13 years (mean age: 9.13±3.20) who were admitted to the clinic with trauma complaints during an eight-month period were evaluated. it was determined that the most common trauma types in the pandemic period were simple crown fracture (25.6%) and subluxation/lateral luxation (23.3%). during the pandemic, 34.9% of all cases were “falling at home”, while “falling at school” was 14.0% (using the pearson chi-square test). conclusion: the covid-19 pandemic period has affected many aspects of social life as well as the number of patients who were admitted for dental trauma and the causes of trauma. multicenter studies are needed for more accurate results. keywords: covid-19; dental traumatic injuries; medicine; pediatric dentistry article history: received 8 november 2022; revised 27 january 2023; accepted 8 february 2023; published 1 september 2023 correspondence: sedef kotanli, department of dentomaxillofacial radiology, faculty of dentistry, harran university. 63300 haliliye, sanliurfa, turkey. email: sedefakyol@harran.edu.tr introduction traumatic dental injury (tdi) is a public health problem with high prevalence in childhood.1,2 with the widespread use of preventive medicine, the incidence of caries, tooth loss, and periodontal problems in childhood and adolescence has been reduced, but the frequency of tdi has not decreased.3 tdi can cause temporary (bleeding, pain, discoloration, and tooth loss) or permanent (anterior teeth malformation, alveolar bone loss, and insufficient jaws growth) effects in the teeth. such injuries negatively affect not only the patients but also the parents physically, emotionally, and psychologically.4 while tdi in the preschool period usually occurs because children are unable to balance, school-age children are more likely to suffer from falls while playing sports or playing games.5 in addition, tdi in children is associated with reasons such as collisions, falls, sports activities, car and bicycle accidents, and predisposing anatomical factors such as overjet and inadequate lip coverage.6 however, susceptibility to injury is often related to the physical, social, and personal environment.1,7 it is suggested, therefore, that investigation of the determinants of tdi should not be limited to individual factors. social and living conditions, income distribution, and health policies are important aspects of the distribution of such diseases and should be studied with the same intensity and attention.8 according to the world literature, rates of adolescents presenting evidence of tdi varies from 7.9% to 26.6%.9,10 in studies conducted, the incidence of tdi in deciduous teeth was reported to vary from 7% to 42%,11 while this rate in permanent teeth was found to be 20% in the age range 8–12 years.12,13 although tdi has been copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p139–143 mailto:sedefakyol@harran.edu.tr https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p139-143 140 kotanli et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 139–143 reported at various frequencies around the world and has been found to be associated with many different factors, there is no data on the frequency of tdi during the covid19 pandemic period. the significant impact of covid-19 includes the decrease in the number of patients, where cases have been reported to visit the dental clinic mainly due to dental trauma and oral infection.14 the covid-19 pandemic process has affected many areas of our lives. it is known that in this process, applications to dental hospitals have decreased, and dentists only intervene in emergencies. the aim of this study is to investigate the effects of the pandemic process, which significantly changed social life, on tdi and to compare the results of our study with the studies conducted before the pandemic. materials and methods in this study, the records of patients aged 0–13 years who were admitted to harran university faculty of dentistry, department of pediatric dentistry with a complaint of dental trauma in 2020 were examined. ethics committee approval (22/03/24) was obtained from the ethics committee of harran university faculty of dentistry for the study. all data of the patients (age, gender, affected tooth, cause of trauma/type, and treatment procedure) were obtained from standard trauma-registration forms. causes of trauma were classified as falling at home, falling at school, sports/cycling, fighting/assault, and collision/hitting any place or object. trauma types have been classified as simple crown fracture, complicated crown fracture, root fracture, complicated crown-root fracture, alveolar fracture, intrusion, avulsion, rotation, subluxation/lateral luxation, and concussion. the treatments applied to the patients were recorded as restorative treatment, pulp capping treatment, amputation, fixation, reimplantation, root canal treatment, reposition plus root canal treatment, and follow-up. also, the patients who had a splint applied were identified, and the relationships between splint application and trauma types and causes were evaluated. the data were analyzed using statistical package for the social sciences version 23.0 (spss inc., chicago, il, usa). the median, minimum–maximum, and percentage values were calculated for descriptive statistics. the normality of the data distribution was tested using histograms and the kolmogorov-smirnov test (p>0.05). as the data were normally distributed, the independent sample t-test was used for intergroup comparisons. the relationships between categorical variables were calculated using the pearson chi-square test (p<0.05). results data of 43 patients (27 males and 16 females) aged 1–13 years (mean age: 9.13±3.20) who were admitted to the clinic with trauma complaints during an eight-month period were evaluated. table 1 shows the distribution of trauma types by gender in the pandemic period. it was determined that the most common trauma types in the pandemic period were simple crown fracture (25.6%) and subluxation/lateral luxation (23.3%). no statistically significant relationship was found between trauma types and gender. the distribution of trauma causes by gender in the pandemic period is shown in table 2. in the pandemic table 1. trauma type distribution by gender in the pandemic period type of trauma men n (%) women n (%) total n (%) p value simple crown fracture 7 (16.3%) 4 (9.3%) 11 (25.6%) 0.506 complicated crown fracture 4 (9.3%) 4 (9.3%) 8 (16.3%) root fracture 1 (2.3%) 1 (2.3%) 2 (4.7%) complicated crown-root fracture 1 (2.3%) 0 (0.0%) 1 (2.3%) alveolar fracture 3 (7.0%) 0 (0.0%) 3 (7.0%) i̇ntrusion 1 (2.3%) 2 (4.7%) 3 (7.0%) avulsion 3 (7.0%) 0 (0.0%) 3 (7.0%) rotation 1 (2.3%) 0 (0.0%) 1 (2.3%) subluxation/lateral luxation 6 (14.0%) 4 (9.3%) 10 (23.3%) concussion 0 (0.0%) 1 (2.3%) 1 (2.3%) total 27 (62.8%) 16 (37.2%) 43 (100.0%) table 2. trauma cause distribution by gender in the pandemic period cause of trauma men n (%) women n (%) total n (%) p value falling at home 12 (27.9%) 3 (7.0%) 15 (34.9%) 0.509 falling at school 3 (7.0%) 3 (7.0%) 6 (14.0%) sports/cycling 5 (11.6%) 3 (7.0%) 8 (18.6%) fighting/assault 3 (7.0%) 3 (7.0%) 6 (14.0%) collision/hitting any place or object 4 (9.3%) 4 (9.3%) 8 (18.6%) total 27 (62.8%) 16 (37.2%) 43 (100.0%) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p139–143 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p139-143 141kotanli et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 139–143 period, 34.9% of all cases were “falling at home,” while those for “falling at school” were 14.0%. no statistically significant relationship was found between the causes of trauma and gender. the distribution of treatment methods applied to trauma patients in the pandemic period by gender is shown in table 3. it was observed that the most common treatment during the pandemic period was restorative treatment (27.9%). a comparison of the patients with and without splint treatment, according to the causes of trauma, is given in table 4. splint treatment was unnecessary in 65.1% of trauma cases in the pandemic period. no statistically significant relationship was found between the causes of trauma and splint treatment (p>0.05). the comparison of the patients with and without splint treatment, according to trauma types, is given in table 5. a statistically significant relationship was found between trauma types and splint treatment (p<0.05). while splints were not used in 90.9% of simple crown fractures, splints were applied in 100% of avulsions, alveolar fractures, and rotations. discussion tdis constitute a serious oral-dental problem, as their treatment is costly, has a significant impact on the oral health-related quality of life of the population, and is often required due to preventable injuries.15 traumas related to deciduous and permanent teeth can negatively affect the ongoing development of teeth and jaws of the individual. with the correct diagnosis and effective treatment, the effects of trauma are minimized, and in this way the development of teeth and jaws can continue in a healthy manner. in studies conducted before the pandemic, it was reported that men were more exposed to trauma.16–18 in our study, however, no statistically significant difference was found between gender and trauma. when the studies in the literature have been examined, it has been determined that the incidence of crown fractures varies from 10% to 50%.3,17–19 it has been reported that luxation injuries are more common than crown fractures in primary dentition.19,20 rocha and cardoso stated that table 4. relationship between trauma cause and splint application cause of trauma splint used n (%) splint not used n (%) total n (%) p value falling at home 7 (46.7%) 8 (53.3%) 15 (100.0%) 0.381 fighting/assault 3 (50.0%) 3 (50.0%) 5 (100.0%) falling at school 1 (16.7%) 5 (83.3%) 6 (100.0%) collision/hitting any place or object 1 (12.5%) 6 (87.5%) 7 (100.0%) sports/cycling 3 (37.5%) 5 (62.5%) 8 (100.0%) total 15 (34.9%) 28 (65.1%) 43 (100.0%) table 3. distribution of treatment method applied to trauma patients by gender treatment men n (%) women n (%) total n (%) p value amputation 1 (2.3%) 1 (2.3%) 2 (4.7%) 0.093 fixation 3 (7.0%) 0 (0.0%) 3 (7.0%) root canal treatment 6 (14.0%) 4 (9.3%) 10 (23.3%) pulp capping treatment 1 (2.3%) 0 (0.0%) 1 (2.3%) reimplantation 3 (7.0%) 0 (0.0%) 3 (7.0%) reposition + root canal treatment 1 (2.3%) 0 (0.0%) 1 (2.3%) restorative treatment 7 (16.3%) 5 (11.6%) 12 (27.9%) follow-up 5 (11.6%) 6 (14.0%) 11 (25.6%) total 27 (62.8%) 16 (37.2%) 43 (100.0%) table 5. relationship between trauma type and splint application type of trauma splint used n (%) splint not used n (%) total n (%) p value avulsion 3 (100.0%) 0 (0.0%) 3 (100.0%) 0.005* intrusion 1 (33.3%) 2 (66.7%) 3 (100.0%) complicated crown fracture 3 (37.5%) 5 (62.5%) 8 (100.0%) complicated crown-root fracture 0 (0.0%) 1 (100.0%) 1 (100.0%) simple crown fracture 1 (9.1%) 10 (90.9%) 11 (100.0%) root fracture 2 (100.0%) 0 (0.0%) 2 (100.0%) subluxation/lateral luxation 1 (10.0%) 9 (90.0%) 10 (100.0%) alveolar fracture 3 (100.0%) 0 (0.0%) 3 (100.0%) rotation 1 (100.0%) 0 (0.0%) 1 (100.0%) concussion 0 (0.0%) 1 (100.0%) 1 (100.0%) total 15 (34.7%) 28 (65.3%) 43 (100.0%) copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p139–143 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p139-143 142 kotanli et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 139–143 the incidence of both types of injuries (51.5%, 48.5%) was similar, and there was no statistically significant difference.21 ludwig et al., in a study examining the effect of covid-19 on trauma, reported that dental trauma was seen least in the pandemic period, and this rate was 18%.22 yang et al., in their study during the pandemic period in 2020, determined that the rate of complicated crown fracture was 62.5% and that of subluxation was 31.0%.23 in our study, simple crown fracture was the most common type, with a rate of 25.6%, followed by luxation injury, with a rate of 23.3%. it appears that this proportional difference is due to the difference in the prevention measures and bans taken in the countries during the covid-19 period and the sample size. when the literature has been reviewed, it has been determined that traumatic injuries mostly occur as a result of “fall” and, second, as a result of “impact.”24–26 gabris et al., ramaiah et al., and gassner et al. reported that traumatic injuries are more common as a result of sports injuries.27–29 yang et al., in their study investigating the causes of tdi, found most occurrences were as a result of falls and traffic accidents (35.0%, 36.7%) in 2019, while tdi due to falls occurred at a rate of 89.5% in 2020.23 in the study by elbay et al., they determined that 27.4% of tdis occurred in the park, 26.9% at school, and 21.1% at home.30 in our study, “falling at home” ranks first with a rate of 34.9%. the authors think that this difference is due to the rules and bans applied within the scope of covid-19 measures. when the treatment procedure after trauma is examined in our study, the first order is “treatment with restoration” and the second is “follow-up.” these results are consistent with similar studies in the past.24,31–33 in addition, unlike the studies in the literature, a statistically significant relationship was found between trauma type and splint treatment in our study. while splints were not used in 90.9% of simple crown fractures, splints were applied in 100% of avulsions, alveolar fractures, and rotations. since there are very few studies examining the effect of the covid-19 period on dental traumas, some parameters could not be discussed in conclusion, the covid-19 pandemic period has affected many aspects of social life as well as the number of patients who have been admitted for dental trauma and the causes of trauma. the increase in the rates of dental trauma at home during the pandemic period is an indication regarding the awareness of parents about dental trauma and the need to increase social awareness in terms of early intervention in trauma cases. references 1. baxevanos k, topitsoglou v, menexes g, kalfas s. psychosocial factors and traumatic dental injuries among adolescents. community dent oral epidemiol. 2017; 45(5): 449–57. 2. bomfim ra, herrera dr, de-carli ad. oral health-related quality of life and risk factors associated with traumatic dental injuries in brazilian children: a multilevel approach. dent traumatol. 2017; 33(5): 358–68. 3. guedes oa, alencar ahg de, lopes lg, pécora jd, estrela c. a retrospective study of traumatic dental injuries in a brazilian dental urgency service. braz dent j. 2010; 21(2): 153–7. 4. walker a, brenchley j. it’s a knockout: survey of the management of avulsed teeth. accid emerg nurs. 2000; 8(2): 66–70. 5. gassner r, garcia jv, leja w, stainer m. traumatic dental injuries and alpine skiing. dent traumatol. 2000; 16(3): 122–7. 6. eslamipour f, iranmanesh p, borzabadi-farahani a. cross-sectional study of dental trauma and associated factors among 9to 14-year-old schoolchildren in isfahan, iran. oral health prev dent. 2016; 14(5): 451–7. 7. ramchandani d, marcenes w, stansfeld sa, bernabé e. problem behaviour and traumatic dental injuries in adolescents. dent traumatol. 2016; 32(1): 65–70. 8. fonseca rcl da, antunes jlf, cascaes am, bomfim ra. individual and contextual factors associated with traumatic dental injuries in a population of brazilian adolescents. dent traumatol. 2019; 35(3): 171–80. 9. oyedele ta, jegede at, folayan mo. prevalence and family structures related factors associated with crown trauma in school children resident in suburban nigeria. bmc oral health. 2016; 16(1): 116. 10. oliveira filho pm, jorge ko, ferreira ef, ramos-jorge ml, tataounoff j, zarzar pm. association between dental trauma and alcohol use among adolescents. dent traumatol. 2013; 29(5): 372–7. 11. hasan aa, qudeimat ma, andersson l. prevalence of traumatic dental injuries in preschool children in kuwait a screening study. dent traumatol. 2010; 26(4): 346–50. 12. andreasen jo, andreasen fm. essentials of traumatic injuries to the teeth: a step-by-step treatment guide. 2nd ed. wiley-blackwell; 2010. p. 192. 13. díaz ja, bustos l, brandt ac, fernández be. dental injuries among children and adolescents aged 1-15 years attending to public hospital in temuco, chile. dent traumatol. 2010; 26(3): 254–61. 14. hudyono r, bramantoro t, benyamin b, dwiandhono i, soesilowati p, hudyono ap, irmalia wr, nor nam. during and post covid-19 pandemic: prevention of cross infection at dental practices in country with tropical climate. dent j. 2020; 53(2): 81–7. 15. borum mk, andreasen jo. therapeutic and economic implications of traumatic dental injuries in denmark: an estimate based on 7549 patients treated at a major trauma centre. int j paediatr dent. 2008; 11(4): 249–58. 16. kovacs m, pacurar m, petcu b, bukhari c. prevalence of traumatic dental injuries in children who attended two dental clinics in targu mures between 2003 and 2011. oral health dent manag. 2012; 11(3): 116–24. 17. aren g, sepet e, pinar erdem a, tolgay cg, kuru s, ertekin c, guloglu r, aren a. predominant causes and types of orofacial injury in children attending emergency department. turkish j trauma emerg surg. 2013; 19(3): 246–50. 18. dua r, sharma s. prevalence, causes, and correlates of traumatic dental injuries among seven-to-twelve-year-old school children in dera bassi. contemp clin dent. 2012; 3(1): 38–41. 19. unal m, oznurhan f, kapdan a, aksoy s, dürer a. traumatic dental injuries in children. experience of a hospital in the central anatolia region of turkey. eur j paediatr dent. 2014; 15(1): 17–22. 20. lam r. epidemiology and outcomes of traumatic dental injuries: a review of the literature. aust dent j. 2016; 61: 4–20. 21. de carvalho rocha mj, cardoso m. traumatized permanent teeth in brazilian children assisted at the federal university of santa catarina, brazil. dent traumatol. 2001; 17(6): 245–9. 22. ludwig dc, nelson jl, burke ab, lang ms, dillon jk. what is the effect of covid-19-related social sistancing on oral and maxillofacial trauma? j oral maxillofac surg. 2021; 79(5): 1091–7. 23. yang y, zhang w, xie l, li z, li z. characteristic changes of traumatic dental injuries in a teaching hospital of wuhan under transmission control measures during the covid‐19 epidemic. dent traumatol. 2020; 36(6): 584–9. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p139–143 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p139-143 143kotanli et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 139–143 24. gümüş h, öztürk g, kürem b. profiles of traumatic dental injuries among children aged 0–15 years in cappadocia, turkey: a retrospective cohort study. dent traumatol. 2021; 37(3): 419–29. 25. aktas s. multigravidas’ perceptions of traumatic childbirth: its relation to some factors, the effect of previous type of birth and experience. med sci | int med j. 2018; 7(1): 203–9. 26. rajab ld, baqain zh, ghazaleh sb, sonbol hn, hamdan ma. traumatic dental injuries among 12-year-old schoolchildren in jordan: prevalence, risk factors and treatment need. oral health prev dent. 2013; 11(2): 105–12. 27. gábris k, tarján i, rózsa n. dental trauma in children presenting for treatment at the department of dentistry for children and orthodontics, budapest, 1985-1999. dent traumatol. 2001; 17(3): 103–8. 28. ramaiah sd, raghuramaiah s, h v s. evaluation of prevalence and etiological factors of traumatic dental injury among school children. j evol med dent sci. 2015; 4(89): 15455–8. 29. gassner r, tuli t, hächl o, rudisch a, ulmer h. craniomaxillofacial trauma: a 10 year review of 9543 cases with 21067 injuries. j cranio-maxillofacial surg. 2003; 31(1): 51–61. 30. elbay m, şermet elbay ü, uğurluel c, kaya c. bir üniversite hastanesindeki pedodonti kliniğine başvuran 156 dental travma olgusunun değerlendirilmesi: retrospektif araştırma. selcuk dent j. 2016; 3(2): 48–55. 31. zuhal k, semra oem, huseyin k. traumatic injuries of the permanent incisors in children in southern turkey: a retrospective study. dent traumatol. 2005; 21(1): 20–5. 32. özgür b, ünverdi ge, güngör hc, mctigue dj, casamassimo ps. a 3‐year retrospective study of traumatic dental injuries to the primary dentition. dent traumatol. 2021; 37(3): 488–96. 33. güngör hc. management of crown-related fractures in children: an update review. dent traumatol. 2014; 30(2): 88–99. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p139–143 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p139-143 221 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 221–225 original article cleft lip and palate based on birth order and family history at mitra sejati general hospital, indonesia hendry rusdy, isnandar, indra basar siregar, veronica department of oral surgery, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: cleft lip and palate is one of the most common congenital abnormalities in infants and is caused by more than one factors, which can be genetic and environmental. defects in a family gene can result in cleft lip and palate. the study found a high family history relationship with the incidence of cleft lip and palate. birth order studied by several researchers also has different results, which were influenced by folic acid consumption, maternal knowledge, lack of antenatal care visits and maternal age. to determine the cases of cleft lip and palate based on the birth order of the children and family history of the patients at mitra sejati general hospital. purpose: the study aimed to investigate cleft lip and cleft palate patients by birth order and family history. methods: this research was a retrospective descriptive study using survey method. researchers gave 13 questions through a questionnaire and data were collected and counted manually. results: the results showed that based on the birth, the cleft case in the first birth order amounted to 25 people, the second 30 people, the third 19 people, and the fourth or more as many as 20 people. based on family history, 27 patients had a family history of cleft lip and palate, while 67 patients did not have family history. conclusion: the cases of clefts at mitra sejati general hospital happened more frequently in the second child and most incidence did not have family history. keywords: cleft lip; cleft palate; genetic; risk factors correspondence: hendry rusdy, department of oral surgery, faculty of dentistry, universitas sumatera utara. jl. alumni no.2, medan 20155, indonesia. email: hendry_rusdy@yahoo.co.id introduction clefts of the lip and palate are congenital anomalies that take place between the fifth and tenth weeks of fetal life. the lip, alveolar ridge, and hard or soft palates are the most common site affected by oral clefts. individuals with congenital anomalies may experience problems with teeth, malocclusion, as well as feeding, speaking, hearing, and social integration problems.1,2 globally, the prevalence of cleft lip was 0.3 in every 1000 live births.3 meanwhile, in indonesia, the 20132018 basic health research or riset kesehatan dasar (riskesdas) report showed an increasing the incidence of cleft lips (from 0.08% to 0.12%) among children 24 to 59 months of age.4,5 in most cases, the causes of cleft lip and palate are multifactorial, including genetic and environmental factors. medication during pregnancy (amoxicillin, phenytoin, and oxprenolol), nutrient deficiency, radiation, hypoxia, virus, teratogen, smoking, also excess or deficiency of vitamins can be identified as environmental factors. genetic factor, in addition, is known from the family history.1,6 several studies showed that family history was strongly associated with an increased risk of cleft lip or palate.7,8 in line with that, an epidemiology study conducted by acuñagonzález et al.9 showed that the presence of cleft lip or palate background for father, mother, or siblings increased the incidence of the anomalies. based on the birth order, a study by noorollahian et al.10 showed that most of the patients with malformations were first-born children (40.8%), followed by second-born children (28.19%), third-born children (14.97%), and fourth-born children or higher (16.74%). this statement was supported by jac okereke who reported that the firstborn children accounted for most patients, but didn’t have any significance relation to cleft lip and palate.11 however, kesande et al.12 reported that, regarding birth order, most children with orofacial clefts were in the fourth and fifth dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p221–225 mailto:hendry_rusdy@yahoo.co.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p221-225 222rusdy et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 221–225 rank (45.0%) compared to one to third rank (25.0%) and those in the sixth to seventh rank (30.0%). one of the hospitals in medan that treats patients with clefts is mitra sejati general hospital. it is partner with smile train, which is an international children’s charity that provides cleft repair surgery and comprehensive cleft care to children in more than 85 countries. according to data from mitra sejati general hospital, in 2019 there were 96 patients with cleft lip or palate and in 2020 there were 107 patients with cleft lip or cleft palate.13 in a previous study,14 there was a study on the prevalence of cleft lip and palate regarding gender in other cities and hospitals. however, in this study, the research examined cases of cleft lip and palate regarding birth order, which never had been researched and with no recent information at mitra sejati general hospital, and also regarding family history which was found to have various results. therefore, this study aimed to provide data on the distribution of cleft lip and palate based on the birth order of the children and family history at mitra sejati general hospital. materials and methods this retrospective descriptive study using survey method was carried out in may 2021 at mitra sejati general hospital as partner of smile train, the world’s largest cleft-focused organization that provides 100% free cleft surgery. the research ethics commission of universitas sumatera utara approved this study (no.510/kep/ usu/2021). the sample selection was made using the purposive sampling technique and involved 94 samples after we added 10%. it applied inclusion and exclusion criteria. the patients included in the current study had a cleft lip and/ or palate diagnosis registered in the hospital from january 2015december 2020 for surgical treatment or other management. inclusion criteria were (a) patients who had received a treatment at mitra sejati general hospital; (b) patients with cleft lip and/or palate without systemic conditions; and (c) parents or guardians of the children with cleft lip and/or palate who were willing to be interviewed. the following were excluded: (a) parents or guardians, who do not cooperate or were unwilling to be interviewed; (b) unreachable parents or guardians (i.e., by phone); (c) infants with chromosomal anomalies or other birth defects of known etiology and syndrome such as trisomy 13, fryns syndrome, meckel syndrome, and van der woude syndrome; and (d) patients who were treated at mitra sejati general hospital but with missing or incomplete medical records. the participants were informed of their right to participate or drop out of the study without affecting their relationship with the investigators. informed consent was given through digital platform such as google form in the beginning of the interview because this study was conducted during of the covid-19 pandemic. the primary data were conducted by interviewing parents or guardians via phone. the interview was voice recorded to help countercheck any information missing. there were 13 questions in each questionnaire, which related to things experienced by the mother during pregnancy. information was obtained from questions number one to four regarding the birth order of the children and questions number five to seven regarding the family history of cleft lip and palate. the remaining questions were administered to figure out the risk factors of the cleft incidence. the medical records were collected at mitra sejati general hospital with the visit year of 2015-2020 as the secondary data, which included information on name, age, gender, ethnic, address, telephone and birth order. there were two observers involved in this study to minimize the bias and, before the interview, the investigators were provided brief and training for the calibration. data processing was manually put in microsoft excel, the information was tabulated, by which frequencies and percentages were calculated. results the socio-demographic data is shown in table 1. of 94 patients with cleft incidence, 25 patients were first-born children (26.60%), 30 patients were second-born children (31.91%), 19 patients were third-born children (20.21%), and 20 patients were fourth-born children or higher (21.28%). based on the family history, 27 patients (28.72%) had parents or siblings with cleft lip and/or palate, while 67 patients (71.28%) showed negative family history of oral clefts. the distribution of cleft incidence regarding birth order is shown in table 2. cleft lip and palate were the most common cases found among all groups. the majority of cleft cases from first-born until last were cleft lip and palate 80%, 86.67%, 84.21%, and 80%, respectively. the prevalence of cleft lip and palate was studied in the first-degree relatives and found to be 25% in patients with affected fathers and 12.50% in affected mothers, then followed by siblings as shown in table 3. the distribution of cleft incidence regarding the family history of second-degree relatives is shown in table 4. the result showed that cleft lip and palate were mostly patients with affected grandparents. in addition, the incidence of oral clefts was found patients with affected uncles/aunties, where one patient had a cleft palate, while the other six patients had both cleft lip and palate. in the third-degree relatives, a total of nine patients (100%) had cousins with clefts. four patients suffered from cleft lip only, while five patients suffered from both cleft lip and palate as shown in table 5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p221–225 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p221-225 223 rusdy et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 221–225 table 1. the characteristics of study subjects (n=94) respondent characteristic n (people) % birth order of the children 1 25 26.60 2 30 31.91 3 19 20.21 ≥4 20 21.28 family history yes 27 28.72 no 67 71.28 table 2. the distribution of clefts incidence based on the birth order of the children (n=94) cleft cases birth order 1 2 3 ≥4 n % n % n % n % cleft lip only 5 20 3 10 2 10.53 2 10 cleft palate only 0 0 1 3.33 1 5.26 2 10 cleft lip and palate 20 80 26 86.67 16 84.21 16 80 total 25 100 30 100 19 100 20 100 table 3. the distribution of clefts incidence based on the family history of first-degree relatives cleft cases first-degree relatives father mother sibling child n % n % n % n % cleft lip only 0 0 0 0 1 20 0 0 cleft palate only 0 0 0 0 2 40 0 0 cleft lip and palate 2 100 1 100 2 40 0 0 total 2 25 1 12.5 5 62.5 0 0 table 4. the distribution of clefts incidence based on the family history of second-degree relatives cleft cases second-degree relatives grandparents uncle/ aunt nephew/niece step sibling grandchildren n % n % n % n % n % cleft lip only 0 0 0 0 0 0 0 0 0 0 cleft palate only 0 0 1 14.29 0 0 0 0 0 0 cleft lip and palate 6 100 6 85.71 0 0 0 0 0 0 total 6 46.15 7 53.85 0 0 0 0 0 0 table 5. the distribution of clefts incidence based on the family history of third-degree relatives cleft cases third-degree relatives great grandparents great uncle/aunt great grandchildren cousin n % n % n % n % cleft lip only 0 0 0 0 0 0 4 44.44 cleft palate only 0 0 0 0 0 0 0 0.00 cleft lip and palate 0 0 0 0 0 0 5 55.56 total 0 0 0 0 0 0 9 100 discussion the present study showed that clefts incidence was found to be more prevalent in second-born children compared to other groups. similarly, a study by acuña-gonzález et al.9 also stated that the prevalence of cleft lip and/or palate in first or second-born children was higher compared to third or fourth-born children. however, a study conducted by noorllahian et al.10 showed a different result, where the incidence of cleft lip and/or palate was the highest in first-born children. this may be due to consanguineous marriages between two individuals who are related as first cousins or closer from both paternal and maternal sides. consanguineous marriages can lead to various disorders, such as congenital abnormalities, stunted growth, epilepsy, mental retardation or learning disabilities, blood disorders, unexplained neonatal death, and an increase in autosomal recessive disorders.15 we also reported a different result from a study conducted by kesande et al,12 where the fourth and fifth-born children had the highest prevalence of oral clefts. these results may be influenced by several factors, such as approximately 20% of mothers have a history of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p221–225 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p221-225 224rusdy et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 221–225 abortion and most of them live in hillside areas.12,16 a higher maternal age is also associated with the risk of cleft incidence, where the energy in embryonic development decreases. as a result, deformity during the cell division stage to the organogenesis stage may occur.17 the deficiency of folic acid during embryonic development and the lack of maternal knowledge can affect the incidence of cleft lip or palate. folic acid is well-known for its role as a one-carbon donor, which is essential for dna synthesis and proliferation. it also plays an important role in synthetizing enzyme methylenetetrahydrofolate reductase (mthfr) for folate-dependent metabolism of homocysteine. as a result of low mthfr activity due to polymorphisms, higher homocysteine or lower plasma folate cells may occur, of which both are associated with neural tube defect.18 the deficiency of folic acid may also lead to the inability of methionine production; hence, the production of antioxidants (glutathione) and sulfurcontaining amino acid that eliminate toxins in the body, strengthens tissues, and maintains cardiovascular health also decreases.19,20 in addition, lack of maternal knowledge about prenatal nutrition, such as folic acid and zinc, can affect fetal growth and development.8,9,21 deficiency of zinc, for instance, causes impaired reabsorption of folate which affects the production of methionine. other possible factors are the impact of unwanted pregnancy among married women, which affects the mother’s mental and psychological condition in the process of child care and nutrition.22 during pregnancy, psychological stress causes disruption in endocrine, nervous and immune systems. it can strongly activate the hypothalamo-pituitaryadrenal (hpa) stress response, increases corticotropinreleasing hormone (crh), and stimulates the production of inflammatory cytokines. as a result of altered immune system due to stress, mothers are more susceptible to infection and illness during pregnancy.23 the majority of the patients with oral clefts do not have positive family history of the anomalies. the result was similar to a study conducted by figueiredo et al.8 where clefts incidence mostly occurred in patients without positive family history and it was statistically significant. it was assumed that environmental factors, such as exposure to cigar smoke or mothers who did not take folic acid, played a bigger part than the genetic factors.8 according to kummet et al.,24 passive smoking women have a greater risk of giving birth to children with clefts. smoking is one of the risk factors for cleft lip and palate. cigarettes contain nicotine, polycyclic aromatic hydrocarbons, tar, carbon particles, and carbon monoxide. exposure to cigarette smoke to embryonic tissue depends on the number of cigarettes smoked, the frequency of inhalation, and the depth of inhalation. the mechanism by which cigarette smoke affects pregnancy is still not well-understood.25 several studies also reported significant relationship between tobacco exposure and development of orofacial clefts. however, the mechanism by which cigarette smoke affects pregnancy is not well-understood.25–27 most of the cleft cases this study were cleft lip and palate, where 78 people were affected. in line with that, lin et al.28 also reported a similar study result. among all possible factors, it may be related to maternal level of knowledge and age during pregnancy.28 according to bui, et al.,26 cleft lip and palate were the most cases if compared to cleft palate, which were caused by father or mother’s smoking. based on the result in table 3, 4 and 5, cousins had the most frequencies of cleft lip and palate. similarly, martelli et al.2 reported that cousins were the most affected by clefts incidence. the plausible factor is autosomal recessive inheritance, where both parents of cousins are a carrier that inherits clefts incidence. autosomal recessive inheritance occurs when two individuals with a recessive allele meet, so that the newborn individuals have a phenotype. when both parents have recessive type, there is a chance whereby 25% of the babies will inherit the anomalies, 50% will be a carrier, and 25% will be normally born. in the present study, we may assume that both parents have carrier gene since most of the patients do not have positive family history of clefts incidence.29 this study, however, had several limitations because we found it hard to obtain more detailed information from medical records at mitra sejati general hospital due to inefficient medical record storage system and also respondents who were less fluent in sharing information, so that it can affect the interview process with researchers. we suggested this research can be a theoretical base for further research and a guideline for regional health services, especially mitra sejati general hospital, to provide guidance and first aid to babies with cleft lip and palate, and hope this study can be used as additional education material to add new information and knowledge to public for increase awareness about the risk factors for the occurrence of cleft lip and palate. in conclusion, the highest distribution of cleft lip and palate regarding birth order at mitra sejati general hospital in 2015-2020 was among second-born children with a total of 30 people (31.91%), individuals with negative family history with a total of 67 people (71.28%), and cousins who were affected by clefts (100%). acknowledgment we would like to thank all the staff of mitra sejati general hospital, who granted access to the medical records and facilitated the process of this research activity. references 1. ellis e. management of patients with orofacial clefts. in: hupp j, tucker m, ellis e, editors. contemporary oral and maxillofacial surgery. 7th ed. philadelphia: mosby elsevier; 2018. p. 608, 610, 615. 2. martelli drb, coletta rd, oliveira ea, swerts mso, rodrigues lam, oliveira mc, martelli júnior h. association between maternal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p221–225 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p221-225 225 rusdy et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 221–225 smoking, gender, and cleft lip and palate. braz j otorhinolaryngol. 2015; 81(5): 514–9. 3. salari n, darvishi n, heydari m, bokaee s, darvishi f, mohammadi m. global prevalence of cleft palate, cleft lip and cleft palate and lip: a comprehensive systematic review and meta-analysis. j stomatol oral maxillofac surg. 2022; 123(2): 110–20. 4. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2013. jakarta: kemetrian kesehatan republik indonesia; 2013. p. 188–9. 5. badan penelitian dan pengembangan kesehatan. laporan nasional r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 435. 6. agbenorku p. orofacial clefts: a worldwide review of the problem. isrn plast surg. 2013; 2013: 1–7. 7. jamilian a, sarkarat f, jafari m, neshandar m, amini e, khosravi s, ghassemi a. family history and risk factors for cleft lip and palate patients and their associated anomalies. stomatologija. 2017; 19(3): 78–83. 8. figueiredo rf, figueiredo n, feguri a, bieski i, mello r, espinosa m, damazo as. the role of the folic acid to the prevention of orofacial cleft: an epidemiological study. oral dis. 2015; 21(2): 240–7. 9. acuña-gonzález g, medina-solís ce, maupomé g, escoffieramírez m, hernández-romano j, márquez-corona m de l, islas-márquez aj, villalobos-rodelo jj. family history and socioeconomic risk factors for non-syndromic cleft lip and palate: a matched case-control study in a less developed country. biomedica. 2011; 31(3): 381–91. 10. noorollahian m, nematy m, dolatian a, ghesmati h, akhlaghi s, khademi gr. cleft lip and palate and related factors: a 10 years study in university hospitalised patients at mashhad--iran. afr j paediatr surg. 2015; 12(4): 286–90. 11. jac-okereke c, onah i. epidemiologic indices of cleft lip and palate as seen among igbos in enugu, southeastern nigeria. j cleft lip palate craniofacial anomalies. 2017; 4(3): 126. 12. kesande t, muwazi lm, bataringaya a, rwenyonyi cm. prevalence, pattern and perceptions of cleft lip and cleft palate among children born in two hospitals in kisoro district, uganda. bmc oral health. 2014; 14: 104. 13. smile train. rsu mitra sejati | smile train indonesia. 2020. available from: https://www.smiletrainindonesia.org/id/node/1618. accessed 2021 aug 3. 14. triwardhani a, permatasari g, sjamsudin j. variation of nonsyndromic cleft lip/palate in yayasan surabaya cleft lip/palate center surabaya, indonesia. j int oral heal. 2019; 11(4): 187–90. 15. hamamy h. consanguineous marriages. j community genet. 2012; 3(3): 185–92. 16. küchler ec, silva la da, nelson-filho p, sabóia tm, rentschler am, granjeiro jm, oliveira d, tannure pn, silva ra da, antunes ls, tsang m, vieira ar. assessing the association between hypoxia during craniofacial development and oral clefts. j appl oral sci. 2018; 26: e20170234. 17. suryandari ae. hubungan antara umur ibu dengan klasifikasi labioschisis di rsud prof. dr. margono soekarjo purwokerto. indones j kebidanan. 2017; 1(1): 49–56. 18. pan x, wang p, yin x, liu x, li d, li x, wang y, li h, yu z. association between maternal mthfr polymorphisms and nonsyndromic cleft lip with or without cleft palate in offspring, a meta-analysis based on 15 case-control studies. int j fertil steril. 2015; 8(4): 463–80. 19. wahl se, kennedy ae, wyatt bh, moore ad, pridgen de, cherry am, mavila cb, dickinson ajg. the role of folate metabolism in orofacial development and clefting. dev biol. 2015; 405(1): 108–22. 20. hoshi r, alves lm, sá j, peixoto medrado a, de castro veiga p, de almeida reis sr. nonsyndromic cleft lip and/or palate. the role of folic acid 30. brazilian j med hum heal. 2014; 2(1): 30–4. 21. phyu mn, lin z, tun k, myint wei t, maung k. maternal stressful events and socioeconomic status among orofacial cleft families: a hospital-based study. j cleft lip palate craniofacial anomalies. 2020; 7(1): 24. 22. suryani l, rosyada a. the effect of unintended pregnancy among married women on the length of breastfeeding in indonesia. j ilmu kesehat masy. 2020; 11(2): 136–49. 23. coussons-read me. effects of prenatal stress on pregnancy and human development: mechanisms and pathways. obstet med. 2013; 6(2): 52–7. 24. kummet cm, moreno lm, wilcox aj, romitti pa, deroo la, munger rg, lie rt, wehby gl. passive smoke exposure as a risk factor for oral clefts-a large international population-based study. am j epidemiol. 2016; 183(9): 834–41. 25. ozturk f, sheldon e, sharma j, canturk km, otu hh, nawshad a. nicotine exposure during pregnancy results in persistent midline epithelial seam with improper palatal fusion. nicotine tob res. 2016; 18(5): 604–12. 26. bui ah, ayub a, ahmed mk, taioli e, taub pj. maternal tobacco exposure and development of orofacial clefts in the child. ann plast surg. 2018; 81(6): 708–14. 27. campos neves a de s, volpato lr, espinosa m, aranha af, borges a. environmental factors related to the occurrence of oral clefts in a brazilian subpopulation. niger med j. 2016; 57(3): 167. 28. lin y, shu s, tang s. a case-control study of environmental exposures for nonsyndromic cleft of the lip and/or palate in eastern guangdong, china. int j pediatr otorhinolaryngol. 2014; 78(3): 545–51. 29. moura e, cirio sm, pimpão ct. nonsyndromic cleft lip and palate in boxer dogs: evidence of monogenic autosomal recessive inheritance. cleft palate craniofac j. 2012; 49(6): 759–60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p221–225 https://www.smiletrainindonesia.org/id/node/1618 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p221-225 vol 51 no 3 jul sep 2018_pus.indd 143 the effects of breadfruit leaf (artocarpus altilis) extract on fibroblast proliferation in the tooth extraction sockets of wistar rat darin hulwani rinaldi, david b. kamadjaja and ni putu mira sumarta department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: a prolonged tooth extraction socket healing process can affect the well-being of the patient and increase the risk of infection. fibroblast proliferation in the proliferation phase is an important stage in the healing process. fibroblast formed from extracellular matrix and collagen fibers support bone formation in the socket. breadfruit leaves, extremely common in indonesia, contain polyphenol, flavonoid, tannin and alkaloid substances which accelerate the wound healing process because of their anti-inflammatory, anti-bacterial and anti-oxidant properties. a previous study showed that 16% breadfruit leaf gel extract administered to wistar rats produced an encouraging anti-inflammatory effect, but its capacity for increasing fibroblast proliferation remains to be fully understood. purpose: the aim of this study was to observe the effect of applying breadfruit leaf extract on fibroblast proliferation on the healing process in tooth extraction sockets. a preliminary phytochemical study was undertaken. methods: 24 wistar rats were divided into four groups: two control groups and two experimental groups. 16% breadfruit leaf gel extract was applied to the experimental groups, while none was applied to the control groups. the number of fibroblasts was counted on both the third and fifth days post-extraction. data was analyzed statistically using an independent t-test. results: there were significant differences in the number of post-extraction fibroblasts in wistar rat tooth sockets on day 3 (p=0.000; p < α=0.05) and day 5 (p=0.000; p < α=0.05). conclusion: breadfruit leaf gel extract application increases fibroblast proliferation during the healing process in the tooth extraction sockets of wistar rats. keywords: tooth extraction; wound healing; fibroblasts; breadfruit leaves correspondence: david b. kamadjaja, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: davidbk65@gmail.com; dental journal (majalah kedokteran gigi) 2018 september; 51(3): 143–146 research report introduction tooth extraction is the removal of a tooth from its socket as the final treatment option when it has been severely damaged and can no longer be preserved. tooth extraction causes tissue injury leading to inflammation, followed by a healing process in order to restore normal tissue function. the healing process can be divided into four sequential phases, namely: haemostasis and coagulation, inflammation, proliferation and remodeling.1 the healing process is complex and protracted. a prolonged healing process can affect patient well-being and cause postextraction infection. it will also increase the number of visits to the dentist, requiring the patient to invest more time and money in treatment.2 in 2013, world health organization (who) data revealed that more than 80% of the world population lives in developing countries and depends primarily on plant-based medicines to meet their basic healthcare needs. plant-based medicine generally involves a wide range of biological and medical activities which are safer, more readily available and cheaper.3 artocarpus altilis or breadfruit is extremely common in indonesia and offers numerous benefits since it contains polyphenol, phenolic, flavonoids, jacalin, lectin, stilbenoids, alkaloids and tannin.4,5 previous studies have shown that breadfruit has anti-cancer, anti-austeric, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p143–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p143-146 144rinaldi, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 143–146 anti-oxidant, anti-inflammatory, anti-bacterial and antiatherosclerotic properties.6,7 another study indicated that 16% breadfruit leaf gel extract administered to wistar rats induced effective anti-inflammatory activities.8 an important factor in the inflammatory process is cyclooxygenase (cox), mainly cox-2, which is formed by macrophages. the presence of this enzyme is extremely strong during the inflammatory process and plays a role in the formation of prostaglandin from arachidonic acid. another study found that after breadfruit leaf extract application cox-2 expression and activity decreased. this showed that breadfruit leaf extract possesses antiinflammatory properties with cox-2 expression and activities as the target.6,9 it was expected that, with the suppression of cox-2 expression, fibroblast proliferation and differentiation, together with angiogenesis, will ensue. fibroblasts constitute extremely important cells in the early stages of wound healing and start to form during the proliferative stage from the third day after injury. between day 3 and day 5, fibroblasts start to migrate to the wound site and proliferate, resulting in their numbers on the wound site being dominant.10 fibroblasts act to break up blood clots, forming the extracellular matrix (ecm) and collagen fibers to support effective new bone formation in the socket.11 the ability of breadfruit leaf extract to induce fibroblast proliferation is not yet fully understood. the aim of this study is to observe the effect of its application on this process in tooth socket healing. the effectiveness of breadfruit leaf extract was observed histopathologically, with fibroblast numbers being counted manually. materials and methods this research constituted an in vivo experimental laboratory study with post-test only control group design on wistar strain rattus novergicus obtained from the biochemistry laboratory of the faculty of medicine at universitas airlangga. breadfruit leaf samples were cleaned before being dehydrated at room temperature for seven days. the dehydration process was undertaken in order to obtain suitable breadfruit leaf weight for deriving extract. dehydrated samples were cut into small pieces and subsequently ground in an electric blender. ten grams of breadfruit leaf powder were macerated through continual mixing with ethanol (mrc, ts-400 orbotal shaker) for three days at room temperature. a phytochemical study was conducted using uv-visible spectrophotometry to evaluate the chemical composition of the extract which was subsequently centrifuged at 3000 rpm for ten minutes and filtered using whatman filter grade 1 paper (ika® rv 065 basic) in a high-pressure vacuum pump. the extract was scanned with a wavelength ranging from 300-1100nm using a perkin elmer spectrophotometer, resulting in the characteristic peaks being detected. the relative percentage of each component was calculated by comparing its average peak area to the total area. the extract was made into gel form at a concentration of 16%. twenty-four male wistar rats aged 2-4 months and weighing 200-250 grams were quarantined for a period of one week. male specimens were selected in order to avoid the potential hormonal impact affecting female wistar rats. they were divided into four groups of six subjects comprising two control groups (k1 and k2) and two experimental groups (p1 and p2). k1 represented a control group with no gel extract applied and its fibroblasts being counted on the third day after tooth extraction. k2 constituted a control group with no gel extract applied and the number of fibroblasts calculated on the fifth day after tooth extraction. p1 was an experimental group with gel extract applied and its fibroblasts counted on the third day after tooth extraction and gel extract application. p2 represented an experimental group to which gel extract was applied and whose fibroblasts were computed on the fifth day after extraction and gel extract application. the gel extract used with the experimental groups was applied once, immediately after extraction. before the tooth was extracted, the wistar rats were anesthetized intramuscularly using a mixture of ketamine and xylazin. the left mandibular incisors were extracted with a needle holder and the socket subsequently irrigated with 0.2 ml aquadest in order to eliminate any residual debris in the socket before being dried with sterile gauze. thereafter, 0.1ml of 16% breadfruit leaf gel extract was applied to the experimental group using a syringe followed by the application of a suture. the subjects were sacrificed on the third and fifth day post-extraction. incision of the mandibular body was performed before it was placed in a sterile tube containing 10% formalin solution to inhibit changes in post-mortem tissues. therefore, the samples did not rot and autolysis was inhibited. the samples were then processed into histopathological slides for further examination using harris hematoxylin and eosin (he) staining. these slides were observed under a light microscope at 400x magnification and the fibroblast numbers in five different areas were counted with the average for each group being calculated. the fibroblast numbers in the control and experimental groups were compared with data being analyzed statistically by means of a kolmogorov-smirnov test followed by an independent t-test. results preliminary phytochemical analysis was performed before the extract was made into a gel in order to evaluate the content of the breadfruit leaves. the phytochemical analysis results are shown in table 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p143–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p143-146 145 rinaldi, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 143–146 the fibroblast density in h&e staining during examination on day 3 and day 5 is shown in figure 1. the experimental groups had a denser fibroblast appearance and a higher number of fibroblasts than the control groups on both day 3 and day 5 post-extraction. the fibroblast appearance in each group is shown in figure 1. a comparison of the average numbers of fibroblasts on day 3 and day 5 is shown in figure 2. the data obtained was tested statistically using spss. a normality test was conducted by means of a kolmogorovsmirnov test. data from every group showed normal distribution, p > α (α = 0.05). an independent t-test was subsequently conducted to compare the difference in the average amount between the study groups. the results showed that there were significant differences between all groups p = 0.000; p < α (α = 0.05) on both day 3 and day 5. discussion breadfruit leaf extract was made into gel form in order to ease its application to the tooth socket. a concentration of 16% was selected based on research conducted by abdassah et al.8 that found this concentration of breadfruit leaf extract administered to wistar rats displayed satisfactory antiinflammatory properties. these research results showed that control groups had lower fibroblast numbers compared to the experimental group following the administering of 16% breadfruit leaf gel extract. the significant difference in fibroblast numbers between the control and experimental groups was probably caused by polyphenol compounds at a concentration of 4.92%. polyphenol is the most common compound present in the breadfruit leaf extract used in this experiment indicating that it is more likely to play a key role in fibroblast proliferation. polyphenol can inhibit pro-inflammatory enzyme activity and increase anti-inflammatory enzyme activity. this statement corresponds to the fact that polyphenol can inhibit pro-inflammatory gene expression such as interleukin (il) receptors, toll-like receptors (tlr-4), nuclear factor kappa b (nf-kb), activator protein (ap-1) and c-jun-n-terminal kinases (jnk), while also increasing the production of anti-inflammatory molecules, for example il-4, il-10, il-13 and adiponectin.12 together with polyphenol, flavonoid at a concentration of 2.11% in the breadfruit leaves used in this experiment also acts as an anti-inflammatory. flavonoid inhibits proinflammatory enzymes such as cox-2, lipoxygenase, and inducible nitric oxide synthase (inos, tnf α, il-1, nf-kb, ap-1 and mapk).13 pro-inflammatory enzymes inhibited by polyphenol and flavonoids causing stimulation of the phospholipid cell membrane decrease with the result that arachidonic acid cannot be released from the phospholipid cell membrane by phospholipase activation. the inhibited cyclooxygenase and lypoxygenase cycle will suppress prostaglandin, endoperoxidase, thromboxane, hydroperoxidase acid and leukotriene with the result that the inflammatory phase can be reduced and promote more rapid fibroblast proliferation.14 polyphenol and tannin at high concentrations (2.56%) in the extract used, as well as flavonoid compounds, contribute to anti-oxidant effects on the breadfruit leaves a c d b figure 1 histopathological appearance on post extraction. socket, group a (k1), b (p1), c (k2), d (p2). the arrows point on fibroblast formed in h&e staining, with 400x magnification. figure 2. average amount of fibroblast for each group. the fibroblast number were counted on five different areas using microscope with 400x then averaged on each groups. fibroblast numbers then compared between control (blue bar) and experimental (red bar) groups on both day 3 and day 5. experimental group on day 3 and day 5 show a higher number of fibroblast proliferation compared to control group. table 1 phytochemical analysis result of breadfruit leaves. extract. concentration (%)compoundsno. polyphenol 4.921 alkaloid 3.82 tannin 2.563 2.11flavonoid4 1.74saponin5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p143–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p143-146 146rinaldi, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 143–146 used in this experiment. the anti-oxidant effect of breadfruit leaves works by donating one of its electrons to an oxidant substance impeding the activity of that substance.15 unstable free radicals that bind to the anti-oxidant can reduce cell membrane breakdown and suppress inflammation leading to acceleration of the proliferative phase.10 another study posited that breadfruit leaves (artocarpone) inhibit nitric oxide (no) and inos production. no is a free radical substance promoting macrophage cell activation in inflammatory sites. an antibacterial effect is produced by the alkaloid, tannin and flavonoid in the leaves used. it is believed that the antibacterial effect of breadfruit leaves is caused by their elevated alkaloid content. alkaloid can disrupt bacteria cell membranes as well as inhibit dna and rna synthase, while also being toxic to microorganisms.16 the antibacterial effect of tannin can be achieved in several ways, including creating complex bacterial and fungal compounds which disrupt the metabolism of microorganisms by inhibiting oxidative phosphorylation.17 in contrast, flavonoids exert an antibacterial effect by rendering the microbe adhesion, enzymes and protein cell transport non-active.7 these antibacterial activities can minimize the pathogenic bacteria and their potential interference with the healing process. the properties mentioned above are supported by the fact that breadfruit leaf extract possesses anti-inflammatory, antibacterial and antioxidant properties.4 these properties are derived from chemical substances contained in the extract such as polyphenol, alkaloid, tannin and flavonoid which promote more rapid fibroblast proliferation. based on the findings of the experiment conducted, there are significant differences in fibroblast proliferation between the groups with the application of gel extract and those to which it is applied on the third and fifth days. these findings show that 16% breadfruit leaf gel extract can increase fibroblasts leading to faster than normal wound healing. it can be concluded that 16% breadfruit leaf gel extract increases fibroblast proliferation during the healing process in the tooth extraction sockets of wistar rats. references velnar t, bailey t, smrkolj v. the wound healing process: an1. overview of the cellular and molecular mechanisms. j int med res. 2009; 37(5): 1528–42. vowden p. hard-to-heal wounds made easy. wounds int. 2011; 2(4):2. 1–6. beyene b, beyene b, deribe h. review on application and manage-3. ment of medicinal plants for the livelihood of the local community. j resour dev manag. 2016; 22: 33–9. jagtap ub, bapat va. artocarpus: a review of its traditional uses,4. phytochemistry and pharmacology. j ethnopharmacol. 2010; 129(2): 142–66. utami r, yuliawati k, syafnir l. pengaruh metode ekstraksi terhadap5. aktivitas antioksidan daun sukun (artocarpus altilis (parkinson) fosberg). thesis. bandung: universitas islam bandung; 2015. p. 283. fakhrudin n, hastuti s, andriani a, widyarini s, nurrochmad a.6. study on the antiinflammatory activity of artocarpus altilis leaves extract in mice. int j pharmacogn phytochem res. 2015; 7(6): 1080–5. kumar s, pandey ak. chemistry and biological activities of fla-7. vonoids: an overview. sci world j. 2013; 2013: 1–16. abdassah m, sumiwi sa, hendrayana j. formulasi ekstrak daun8. sukun (artocarpus altilis (parkins.) fosberg) dengan basis gel sebagai antiinflamasi. j farm indones. 2009; 4(4): 199. steer sa, moran jm, maggi lb, buller rml, perlman h, corbett9. ja. regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded rna and viral infection. j immunol. 2003; 170(2): 1070–6. 10. ardiana t, kusuma apk, firdausy md. efektivitas pemberian gel binahong (adredera cordifolia) 5% terhadap jumlah sel fibroblast pada soket pasca pencabutan gigi marmut (cavia cobaya). odonto dent j. 2015; 2: 64–70. 11. bainbridge p. wound healing and the role of fibroblasts. j wound care. 2013; 22(8): 407–12. 12. tsai y-s, maeda n. ppargamma: a critical determinant of body fat distribution in humans and mice. trends cardiovasc med. 2005; 15(3): 81–5. 13. serafini m, peluso i, raguzzini a. flavonoids as anti-inflammatory agents. proc nutr soc. 2010; 69(3): 273–8. 14. sabir a. pemanfaatan flavonoid di bidang kedokteran gigi. maj ked gigi (dent j). 2003; 36(timnas iii): 81–7. 15. winarsi h. antioksidan alami & radikal bebas. yogyakarta: kanisius; 2007. p. 77. 16. aniszewski t. alkaloids : chemistry, biology, ecology and applications. 2nd ed. amsterdam: elsevier; 2015. p. 359. 17. dhanasekaran d, thajuddin n, panneerselvam a. fungicides for plant and animal diseases. mexico: intech; 2012. p. 308. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p143–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p143-146 4141 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 41–47 original article chitosan’s effects on the acidity, copper ion release, deflection, and surface roughness of copper-nickel-titanium archwire ika devi1, erliera sufarnap2, finna3, eric rionaldi p. pane3 1department of pediatric dentistry, faculty of dentistry, universitas sumatera utara, medan, indonesia 2department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia 3professional student, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: chitosan has an antimicrobial effect in oral hygiene control. orthodontists sometimes prescribe mouthwash to adolescent patients. copper-nickel-titanium (cuniti) orthodontic archwire is widely used in orthodontic treatment. chitosan’s effects on the cuniti properties of orthodontic archwire are not generally known. purpose: this study aimed to measure the acidity, copper ion release, deflection, and surface roughness of cuniti orthodontic archwire immersed in artificial saliva and 2% chitosan. methods: this study comprised experimental laboratory research. forty-two cuniti orthodontic archwires were divided into three groups. group a consisted of 18 archwires immersed in artificial saliva, group b consisted of 18 archwires immersed in 2% chitosan, and group c was six archwires for the baseline sample. the two intervention groups (a and b) were divided into three subgroups of six samples and were subjected to different immersion times—i.e., two, four, and six weeks. acidity, copper ion release, deflection, and surface roughness were measured using ph meters, atomic absorption spectrophotometry (aas), a universal testing machine (utm), and a scanning electron microscope (sem). results: the results showed that group a was more alkaline than group b, and it was significantly different only in week 2. group b’s copper ion release was significantly lower than group a for all the time observations (p<0.05), and the deflection analysis showed no significant difference in any of the groups (p>0.05). furthermore, the sem images showed cuniti in group a at week-6 had the most porosities and defects. conclusion: the chitosan produces buffer effects on the ph; it also exhibits lower copper ion release, no differences in unloading forces, and subjectively has better surface roughness. keywords: cuniti; chitosan; copper ion release; deflection; surface roughness article history: received 7 april 2022, revised 16 may 2022, accepted 19 july 2022 correspondence: erliera sufanap, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan, indonesia, 20155. email: erliera@usu.ac.id introduction adolescent patients undergoing orthodontic treatment are usually unable to maintain adequate oral hygiene because compliance is a significant issue at this age.1 therefore, practitioners recommend mouthwash.2 one well-known mouthwash contains chlorhexidine (chx) and is commonly used due to its clinical efficacy in chemical plaque control.3 however, using chlorhexidine mouthwash daily may lead to undesirable side effects, such as tooth and tongue staining, an unpleasant taste, and a sense of burning or dryness in the mouth.3,4 on the other hand, recent studies have found that chitosan has an antimicrobial effect on many organisms.3,5 chitosan is a linear and primary polysaccharide produced from the deacetylation of chitin and can be used as a chemical agent for mouthwashes.3,6 it can be extracted from fungi, the exoskeletons of insects, or the shells of crustaceans.5 chitosan is widely used in biomedicals, cosmetics, and foods in agriculture because of its biodegradability, biocompatibility, renewability, absorptivity, non-immunogenicity, non-toxicity, and noncarcinogenicity.5,6 brackets and archwires are essential in orthodontic appliances for moving teeth to targeted positions.7 nowadays, archwire made of cuniti is frequently used in orthodontic treatment due to its better tooth movement. the addition of copper to niti wire creates temperature copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 mailto:erliera@usu.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 42 devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 transition range (ttr) control and gives better spring back properties. cuniti orthodontic archwire also has greater material strength, produces more constant but gentle force, and is more resistant to permanent deformation.8–10 the acid contained in different food and beverages can release nickel ions from the wire.11 allergic reactions to these nickel ions are also associated with chrome and cobalt.12 moreover, excess copper in the body can cause liver damage and gastrointestinal symptoms. nevertheless, copper toxicity rarely occurs in individuals who do not have hereditary copper homeostasis defects because copper is an essential mineral in the formation of hemoglobin and thus helps to prevent anemia. it also has an effect as an antimicrobial agent.13–15 copper is naturally present in the human body and some foods, such as offal, shellfish, seeds, and nuts.16 however, copper toxicity has been reported in populations that consume water containing high levels of the element, when the corrosive action of water causes it to leach from copper pipes in buildings. who revealed that domestic plumbing can be subject to corrosion. high levels of dissolved oxygen have also been shown to accelerate corrosion in some cases. water that flows to residents contains copper oxide, increasing daily copper exposure.14 the copper ions released from dental alloys ranged from 0.045–0.098 ppm/day, and copper ions reached a cytotoxic level at a concentration of 10 ppm.17 deflection is the response of a wire (bending or twisting) when a force is exerted to move the teeth and needs to be activated periodically.2 the amount of deflection in each wire may differ depending on its brand, size, and composition.18–20 moreover, deflection may alter during application in the oral cavity, after contact with saliva, mouthwash, soft drinks and wire sterilization fluid.21–24 there is a great deal of interest in investigating and analyzing the properties of orthodontic appliances, such as ion release and surface roughness.4 material composition, manufacturing geometry, and surface roughness are considered essential parameters in determining the corrosion behavior of metals. surface roughness also plays a significant role in the effectiveness of archwires, such as in guided tooth movement, biocompatibility, surface contact and friction, esthetics, hygiene, and color stability.7,25 the effects of chitosan in the corrosion process of cuniti orthodontic archwire have not been studied extensively. thus, this study aimed to measure the changes in the ph levels, copper ion release, deflection, and surface roughness of cuniti orthodontic archwire after being immersed in artificial saliva and 2% chitosan. materials and methods this study was experimental with group control and sought to observe the ph changes for three types of property analysis: copper ion release, the unloading force for deflection, and cuniti orthodontic archwire surface roughness. the research was carried out in a number of locations: the immersion process and acidity analysis of the solution were conducted in the faculties of dentistry and pharmacy at universitas sumatera utara; the ion release procedure was observed at balai standardisasi dan pelayanan jasa industri (baristand) medan; the deflection was analyzed in the impact fracture research center (ifrc) laboratory, faculty of engineering, universitas sumatera utara; and the surface roughness was investigated in the integrated research laboratory at universitas sumatera utara. ethical clearance had been received in advance from the health research ethics committee of universitas sumatera utara, medan, north sumatra, indonesia, letter no. 557/kep/usu/2021. forty-two samples of archwire, 4 cm in length, composed of cuniti tanzo (american orthodontics) were analyzed. according to sastroasmoro and ismael,26 the calculated minimum number of samples required were six for each group. the size of the cuniti orthodontic archwire was 0.016 x 0.025 in. one liter of artificial saliva with a ph value of 7.5, which consisted of nahco3 9.8%, na2hpo4, 12h2o 9.3%, nacl 0.47%, kcl 0.57%, cacl2 orp cacl2.2h2o 0.04% (0.045%), mgcl or mgcl2.2h2o 0.06% (0.065%) was formulated at oral dental hospital (faculty of dentistry, universitas sumatera utara). the chitosan powder concentration was 2% (2/100 ml of distilled water) from prawn shells was formulated at the laboratory of research centre (faculty of mathematics and science, universitas sumatera utara). the 42 cuniti orthodontic archwires were divided into two groups (n=18), and each group was further divided into three subgroups based on observation time—i.e., two, four, and six weeks (n=6). group a was the control group and used artificial saliva, while group b used artificial saliva with 2% chitosan. the last six samples formed the baseline and did not undergo intervention. group b simulated daily use in the mouth (two times a day for one minute). all samples were incubated at 37°c. the 2% chitosan was dissolved in artificial saliva and immersed for 28 minutes for the two-week subgroup, 54 minutes for the four-week subgroup, and 84 minutes for the six-week subgroup. the acidity of the solution was analyzed by the digital ph meter (hanna, hi98107) at the end of the observation, atomic absorption spectrophotometry (aas, shimadzu aa7000) was used to study the immersed solution of copper ion release after two, four, and six weeks with a wavelength of 324.8 nm and a slit width of 0.5 nm. first, the standard solutions of different concentrations determined the absorbance and continued to make a calibration curve from the value. then, the measurement of the absorbance and concentration of the sample was determined from the last calibration curve. after this, observation continued with a three-point bending test pressuring a 5-mm load to the wire with a universal testing machine (tensilon, rtf-1350, japan) to examine the unloading value of cuniti orthodontic archwire deflection in newtons. the final stage examined the copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 43devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 surface roughness of cuniti orthodontic archwire in three areas with a scanning electron microscope (sem, hitachi tm3000) at 2000x magnification. statistical package for social sciences software version 26.0 was used for the statistical analysis. a shapiro-wilk analysis concluded that the ph of copper ion release and deflection data were distributed normally with a value of p>0.05. subsequently, these three variables were statistically analyzed using a one-way anova between group a (the control) and group b (2% chitosan). this was followed by a least significant different (lsd) post hoc analysis. results the artificial saliva ph baseline was 7.5, but after the cuniti was immersed, the ph of both groups rose. the ph for group a (the control) was significantly more alkaline than that of group b at week 2 (table 1 and figure 1a). the lsd post hoc test analysis was based on the time intervals and revealed that the alkaline effects were significantly different only in weeks 2–4 for group a, and group b did not show any difference in acidity (table 1). the copper ion release mean levels for both groups were significantly different amongst all the subgroups (at weeks 2, 4, and 6), while group b demonstrated considerably lower copper ion release than group a (the control). the copper ion release showed a gradual but significant reduction over time during observation, according to the lsd post hoc analysis, except for group b from week 2 to week 4 (table 2 and figure 1b). the unloading force of the baseline sample was 45.049±2.99n. the deflection test for unloading forces showed that group b had higher unloading forces than group a; however, there were no significant differences between both groups in any of the observations over time (table 3 and figure 1c). based on the time interval, the post hoc lsd anova test showed the unloading value of the cuniti orthodontic archwire deflection had only altered significantly in group b, with a p-value of 0.001. the sem micrographs of the cuniti archwire surface roughness were subjectively analyzed because the exact quantitative result could not be provided. however, the micrograph obtained in this study showed that the immersed cuniti archwires in group a at week 2 had the fewest porosities and defects. in contrast, the cuniti archwires in the control group (figure 2) at week 6 showed the most porosities and defects. table 1. acidity (ph) solution between groups and subgroups (n=6). the baseline ph was 7.5 solution mean ± sd week 2 p-value** week 4 p-value** week 6 artificial saliva 8.29±0.032 0.034 8.13±0.073 0.095 7.99±0.204 2% chitosan 7.82±0.186 0.055 7.99±0.152 0.724 8.02±0.068 p-value* 0.001 0.086 0.754 significance level of p<0.05; * p-value from one-way anova; ** p=value from the lsd post hoc analysis between time observations. table 2. copper ion release between the groups and subgroups (n=6) solution mean±sd (µg/l) week 2 p-value** week 4 p-value** week 6 artificial saliva 16.215±0.343 0.001 14.178±0.290 0.001 12.818±0.162 2% chitosan 10.707±0.435 0.952 10.720±0.401 0.001 9.756±0.265 p-value* 0.001 0.001 0.001 significance level of p<0.05; * p-value from the one-way anova; ** p=value from the lsd post hoc analysis between time observations. table 3. mean levels and independent t-test of t h e unloading value of cuniti orthodontic archwire deflection (n=6). the baseline unloading force was 45.049n solution mean±sd (µg/l) week 2 p-value** week 4 p-value** week 6 artificial saliva 47.639±0.866 0.172 45.936±0.699 0.301 47.207±0.933 2% chitosan 49.349±0.299 0.001 45.428±0.698 0.001 48.621±0.352 p-value* 0.092 0.619 0.187 significance level of p<0.05; * p-value from the one-way anova; ** p-value from the lsd post hoc analysis between time observations. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 44 devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 47.6 49.3 45.9 45.4 47.2 48.6 43 44 45 46 47 48 49 artificial saliva chitosan week-2 week-4 week-6 ** * * ** ** ** 8.29 7.82 8.13 7.997.99 8.02 7,5 7,6 7,7 7,8 7,9 8 8,1 8,2 8,3 8,4 artificial saliva chitosan (a) acidity (ph) * 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 ** (b) copper ion release 18 week 2 week 4 week 6 week 2 week 4 chitosan week 6 artificial saliva 16 14 12 10 8 6 4 2 0 16.22 14.18 12.82 10.71 10.72 9.76 (c) cuniti deflection 50 figure 1. (a) acidity (ph), (b) copper ion release, and (c) cuniti deflection for all groups. a significance level of p<0.05; * p-value from the one-way anova; ** p-value from the lsd post hoc analysis between time observations. b1 b2 b3 a c1 c2 c3 figure 2. the surface roughness of the cuniti archwire. a. baseline, b. group a (artificial saliva), c. group b (2% chitosan), where c1, c2, and c3 are at weeks 2, 4, and 6, respectively. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 45devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 discussion corrosion can be interpreted as an electrochemical process in metal orthodontic wire exposed to saliva in the oral cavity. this process causes the material’s surface to become rough, weakening the appliance and resulting in dissolution or ion release in the oral cavity, either partially or entirely.10,27 orthodontic appliances themselves can alter salivary components and thus characteristics, such as flow rate, ph, buffer capacity, and calcium. the ph and the activity of maintaining the buffer capacity in orthodontic patients were significantly more alkaline than in non-orthodontic patients.28 this study was an in vitro experiment and simulated the conditions in the mouth. the results were analogous to the previous research in that the ph baseline of the artificial saliva was 7.5, and after the cuniti archwire was immersed in the artificial saliva (group a) and artificial saliva with 2% chitosan (group b), the ph became more alkaline in all groups at all time observations. moreover, group a was more alkaline than group b. chitosan is a derivative of chitin with amino groups on its surface and functions by generating positive zeta potential.29 this means that chitosan’s amino groups tend to remain protonated in acidic or neutral ph conditions, so in this experiment, it stabilized the materials. furthermore, chitosan as a nanolayer particle is used as an efficient ph buffer to reduce metal corrosion caused by the acidity of chemical compounds. here, the chitosan served as a proton sponge to protect the wire from surface-mediated ph changes.29,30 biocompatibility in orthodontic archwire must be the main requirement of orthodontic appliances. it can be determined by evaluating the amount of metal ion release from the archwire under many different conditions.31 the release of ion in the oral cavity may cause allergic or even toxic effects both locally and systemically.12,32,33 orthodontists usually prescribe a mouthwash to adolescent patients who cannot maintain adequate oral hygiene.1,32 however, some mouthwashes have been reported to cause the release of ions and decrease corrosion resistance.34,35 this study observed that copper ion release from the cuniti orthodontic archwire in group b (2% chitosan, which simulated a mouthwash) was lower than that of the control. the research on copper ion release of different brands of cuniti archwire immersed in neutral and acid solutions showed higher release in the neutral solution than in the acid solution.9 group a in this study additionally proved more alkaline than group b; therefore, the copper ion release in the former was higher than the latter. copper ion release from orthodontic appliances is unavoidable. what should be noted, however, is the safe threshold for ion release in the body.36 the recommended daily intake of copper ions is 2 mg/day.13 this study revealed that the highest copper ion release of all the samples was in the artificial saliva at week 2, with a concentration of 0.016 mg/l or 16 µg/l, which was still lower than the recommended daily intake. thus, it can be concluded that the copper ion release was within the safe threshold. the results showed that copper ions released from cuniti archwire decreased over time. this study was contrary to another study, which showed that copper ions released from polypropylene composite with copper metal had increased with time and temperature.37 the differences in the results of this study were due to the research sample and different solutions used. in this research, the copper ion released by the polypropylene composite was predicted to be significant due to antimicrobial activity: higher copper ion release means higher microbial activity. chitosan can be used as a chemical agent for gel and mouthwashes that provide clinical benefits for plaque control because it has an antimicrobial effect against a wide range of organisms.3,38 the 2% chitosan mouthwash is not significantly different from chlorhexidine mouthwash on the plaque and gingivitis index.3 the chitosan gel was also studied for its antimicrobial effects on mini orthodontic implants, and it was concluded that chitosan could reduce the bacterial count at the mini implant site in the patient, even though it was less effective than chlorhexidine. chitosan could be used as an alternative antimicrobial agent in gel or mouthwash products as it may not cause side effects.3,38 cuniti archwire is one of the superelastic archwires and tends to offer a constant, light force, which is needed to move teeth into alignment over a longer activation time.39 orthodontic appliances change the salivary conditions in the mouth; however, food consumption, temperature fluctuations, acidity from drinks, and additionally mouthwash used as a biological or chemical agent may also potentially damage the condition of orthodontic archwires.27,39,40 these factors may alter the stiffness and condition of the cuniti archwire, which can be assessed using a three-point bending method to determine its load-deflection properties. these analyses were the most important parameters in identifying the biological environment needed to move the tooth.25,36 the deflection for the unloading forces in this study showed that group b had higher unloading forces than group a, but there were no significant differences between the groups in any of the time observations. a similar study found an increase in the unloading force after immersion in a naf solution compared to a saliva group at 2.5-mm deflection.22 the mechanical behavior of chitosan coated in polyethylene, which is used in biomedical/food packaging, improved the surface hardness of the material as well as its frictional properties, owing to the rough surface of the coating. the study was carried out using nano-indentation, a scratch test to the surface, and measured by an atomic force microscope (afm).41 the unloading force is also affected by friction, which occurs due to the resistance between the wire and bracket; in this in vitro experiment, the effects of friction were not analyzed.42 even though the previous study found that the polyethylene coated with the chitosan nanoparticle copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 46 devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 improved friction due to its rough surface, according to the afm analysis, the archwire with the chitosan nanoparticle coated in stainless-steel (ss) instead reduced friction, as confirmed by a sliding test carried out with a universal testing machine, and exhibited a smoother surface, according to a subjective analysis of the sem images.41,43 the unloading force of cuniti archwire decreased from weeks 2 to 4 and increased again from weeks 4 to 6, with both groups exhibiting almost the same force at week-2. a study was discussed which examined hysteresis or force loss between nickel-titanium (niti) and cuniti when stored in water, subjected to temperature changes, and in an acidic condition for 60 days, which simulated an orthodontic treatment in the mouth. the data revealed that the control group (not immersed) had the highest loading force, while the immersed condition had a reduced loading force. as chitosan has a buffer effect on cuniti in an immersed condition, this study showed that the chitosan groups had higher loading forces than the control group. this parallels the control group of the previous study, which also found that cuniti archwire had a lower hysteresis or force loss than niti archwire, so that the force remained stabilized throughout the 60 days.39 the longest observation time in our study was six weeks (42 days), which corresponds to the aforementioned study that noted a reduced force due to intervention, but the cuniti archwire had low hysteresis of force, and the force stayed the same throughout the periods of observation. unfortunately, the chitosan effects of deflection analysis on the mechanical behavior of cuniti archwire for observations over several time intervals were still not found. the last observation of corrosion in the cuniti archwire analyzed the surface roughness with an sem at 2000x magnification. from the beginning of the immersion, group b showed more porosity and defects, though observations over time showed both of these had increased; however, these features remained the same until week-6. in group a, meanwhile, porosity and defects emerged slowly but continued to increase over time. the acidity comparison of groups a and b only found a significant difference from weeks 2 to 4, but the copper ion release in group a was significantly higher than in group b for all subgroups. the sem analysis revealed that group a at week 6 had the greatest number of defects and highest porosity. the limitation of this study was that the images for the sem analysis were analyzed subjectively, and a further study would be required to quantify the surface roughness with an afm. another study observed the surface roughness of orthodontic brackets and ss archwire coated in chitosan. they were analyzed with an sem and a sliding effect test of friction, which concluded that the ss coated with the chitosan nanoparticle had less friction and a smoother surface.43 on the contrary, it was found that the polyethylene coated with the chitosan nanoparticle exhibited improved frictional properties in the rough surface, which were analyzed with an afm.41 from the results obtained in this study using two solutions, group b (2% chitosan) demonstrated buffer effects in the ph with lower copper ion release, but there was no significant difference in the unloading force. further investigation is needed to evaluate the effects of 2% chitosan on another chemical element in terms of ion release, deflection simulating orthodontic treatment, and surface roughness using afm analysis or an sem mathematical analysis. references 1. dean ja. mcdonald and avery’s dentistry for the child and adolescent. 10th ed. st. louis: elsevier; 2016. p. 700. 2. proffit w, fields h, larson b, sarver d. contemporary orthodontics. 6th ed. philadelphia: elsevier; 2019. p. 744. 3. uraz a, boynueǧri d, özcan g, karaduman b, uç d, şenel s, pehlivan s, öǧüs e, sultan n. two percent chitosan mouthwash: a microbiological and clinical comparative study. j dent sci. 2012; 7(4): 342–9. 4. razavi e-se, nik th, hooshmand t, farazdaghi h, arefi ah. surface characterization and frictional force between stainless steel brackets and archwires in orthodontic patients using chlorhexidine and persica-containing mouthrinses: a randomized controlled trial. dent res j (isfahan). 2021; 18: 21. 5. atay hy. antibacterial activity of chitosan-based systems. in: functional chitosan. singapore: springer singapore; 2020. p. 457–89. 6. thakur vk, thakur mk, kessler mr. handbook of composites from renewable materials. hoboken: wiley; 2017. p. 116, 118. 7. a m i n i f, r a k hsha n v, poust i m, r a h i m i h, sha r iat i m, aghamohamadi b. variations in surface roughness of seven orthodontic archwires: an sem-profilometry study. korean j orthod. 2012; 42(3): 129–37. 8. sunny s, reghunathan n, dileep a, alex a. newer nickel titanium archwires in orthodontic treatment: an overview. iosr j dent med sci. 2020; 19(9): 40–3. 9. furlan tpr, barbosa ja, basting rt. nickel, copper, and chromium release by cuni-titanium orthodontic archwires is dependent on the ph media. j int oral heal. 2018; 10(5): 224–8. 10. mahalaxmi s. materials used in dentistry. 2nd ed. new delhi: wolters kluwer; 2018. p. 79,143,633. 11. lubis hf, purba ya. release of nickel ion and surface microstructure of niti archwire after immersion in tomato and orange juice. iop conf ser earth environ sci. 2021; 912(1): 012017. 12. pazzini ca, marques ls, pereira lj, corrêa-faria p, paiva sm. allergic reactions and nickel-free braces: a systematic review. braz oral res. 2011; 25(1): 85–90. 13. farrukh ma. atomic absorption spectroscopy. croatia: intech; 2012. p. 145–6. 14. world health organization. guidelines for drinking-water quality; incorporating the 1st addendum. 4th ed. who; 2017. p. 224, 340. 15. vincent m, hartemann p, engels-deutsch m. antimicrobial applications of copper. int j hyg environ health. 2016; 219(7): 585–91. 16. gritsch l, lovell c, goldmann wh, boccaccini ar. fabrication and characterization of copper(ii)-chitosan complexes as antibiotic-free antibacterial biomaterial. carbohydr polym. 2018; 179: 370–8. 17. milheiro a, nozaki k, kleverlaan cj, muris j, miura h, feilzer aj. in vitro cytotoxicity of metallic ions released from dental alloys. odontology. 2016; 104(2): 136–42. 18. pratomo hg, mardiati e, soemantri es, evangelina ia. deflection test on different orthodontic wire materials sized 0.016 x 0.022 inches. maj kedokt gigi indones. 2019; 4(3): 142–8. 19. lombardo l, marafioti m, stefanoni f, mollica f, siciliani g. load deflection characteristics and force level of nickel titanium initial archwires. angle orthod. 2012; 82(3): 507–21. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 47devi et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 41–47 20. doshi uh, mahindra rk. comparison of the load def lection characteristics of esthetic and metal orthodontic wires on ceramic brackets using three point bending test. j indian orthod soc. 2013; 47(4 suppl 3): 400–4. 21. hasyim hs, devi ls, sumono a. pengaruh perendaman kawat nikeltitanium termal ortodonti dalam minuman teh kemasan terhadap gaya defeksi kawat (the effect of immersion thermal nickel-titanium achwire in the bottled te drinks to the achwireforce deflection). e-jurnal pustaka kesehat. 2016; 4(2): 375–80. 22. ahrari f, ramazanzadeh b-a, sabzevari b, ahrari a. the effect of fluoride exposure on the load-deflection properties of superelastic nickel-titanium-based orthodontic archwires. aust orthod j. 2012; 28(1): 72–9. 23. hosseinzadeh nik t, ghadirian h, ahmadabadi mn, shahhoseini t, haj-fathalian m. effect of saliva on load-deflection characteristics of superelastic nickel-titanium orthodontic wires. j dent (tehran). 2012; 9(4): 171–9. 24. sreekanth c, durga pg, santhanakrishnan k, vijaya prasad ke. a comparative evaluation of effects of different kinds of sterilizations on modulus of elasticity and surface topography of copper niti wires-an invitro study. ann essences dent. 2012; 4(3): 1–8. 25. toloei a, stoilov v, northwood d. the relationship between surface roughness and corrosion. in: asme international mechanical engineering congress and exposition volume 2b: advanced manufacturing. california: american society of mechanical engineers; 2013. p. 1–10. 26. sastroasmoro s, ismael s. dasar-dasar metodologi penelitian klinis. 4th ed. jakarta: sagung seto; 2011. p. 359. 27. eliades t, brantley wa. orthodontic applications of biomaterials. cambridge: elsevier; 2017. p. 32, 110, 141. 28. lindawati y, sufarnap e, munawarah w. the effect of fixed orthodontic treatment on salivary component. dentika dent j. 2019; 22(2): 30–3. 29. nurunnabi m, revuri v, huh km, lee y. polysaccharide based nano/microformulation: an effective and versatile oral drug delivery system. in: nanostructures for oral medicine. elsevier; 2017. p. 409–33. 30. andreeva d v, kollath a, brezhneva n, sviridov d v, cafferty bj, möhwald h, skorb e v. using a chitosan nanolayer as an efficient ph buffer to protect ph-sensitive supramolecular assemblies. phys chem chem phys. 2017; 19(35): 23843–8. 31. eliaz n. corrosion of metallic biomaterials: a review. mater. 2019; 12(3): 407. 32. deriaty t, nasution i, yusuf m. nickel ion release from stainless steel brackets in chlorhexidine and piper betle linn mouthwash. dent j (majalah kedokt gigi). 2018; 51(1): 5–9. 33. mikulewicz m, chojnacka k, wołowiec p. release of metal ions from fixed orthodontic appliance: an in vitro study in continuous flow system. angle orthod. 2014; 84(1): 140–8. 34. fatene n, mansouri s, elkhalfi b, berrada m, mounaji k, soukri a. assessment of the electrochemical behaviour of nickel-titaniumbased orthodontic wires: effect of some natural corrosion inhibitors in comparison with fluoride. j clin exp dent. 2019; 11(5): e414– 20. 35. brar as, singla a, mahajan v, jaj hs, seth v, negi p. reliability of organic mouthwashes over inorganic mouthwashes in the assessment of corrosion resistance of niti arch wires. j indian orthod soc. 2015; 49(3): 129–33. 36. rasyid ni, pudyani ps, heryumani jcp. pelepasan ion nikel dan kromium kawat australia dan stainless steel dalam saliva buatan. dent j (majalah kedokt gigi). 2014; 47(3): 168–72. 37. palza h, quijada r, delgado k. antimicrobial polymer composites with copper microand nanoparticles: effect of particle size a nd polymer mat r ix. j bioact compat polym. 2015; 30(4): 366–80. 38. anggani hs, rusli v, bachtiar ew. chitosan gel prevents the growth of porphyromonas gingivalis, tannerella forsythia, and treponema denticola in mini-implant during orthodontic treatment. saudi dent j. 2021; 33(8): 1024–8. 39. sofar mk, rafeeq ra. evaluation of mechanical properties of niti and cuniti archwires in as received and after artificial aging. j res med dent sci. 2021; 9(2): 73–9. 40. aghili h, yasssaei s, ahmadabadi mn, joshan n. load deflection characteristics of nickel titanium initial archwires. j dent (tehran). 2015; 12(9): 695–704. 41. stoleru paslaru e, tsekov y, kotsilkova r, ivanov e, vasile c. mechanical behavior at nanoscale of chitosan-coated pe surface. j appl polym sci. 2015; 132: 42344. 42. mu raya ma m, na mu ra y, ta mu ra t, iwa i h, sh i m i z u n. relationship between friction force and orthodontic force at the leveling stage using a coated wire. j appl oral sci. 2013; 21(6): 554–9. 43. elhelbawy n, ellaithy m. comparative evaluation of stainlesssteel wires and brackets coated with nanoparticles of chitosan or zinc oxide upon friction: an in vitro study. int orthod. 2021; 19(2): 274–80. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p41–47 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p41-47 vol 44 no 3 sept 2011.indd 159 vol. 44. no. 3 september 2011 sensitivity difference of streptococcus viridans on 35% piper betle linn extract and 10% povidone iodine towards recurrent apthous stomatitis maharani laillyza apriasari1, bagus soebadi2, and hening tuti hendarti2 1department of oral medicine, study program of dentistry, faculty of medicine, lambung mangkurat university, kalimantan selatan-indonesia 2department of oral medicine, faculty of dentistry, airlangga university, surabaya-indonesia abstract background: oral ulceration often becomes the main reason for the patients to see a dentist. therapy of the oral ulceration is by giving the palliative therapy with topical antiseptic. nowadays, there are many researches concerning with the traditional medicines as alternative therapy. one of them is piper betle linn which contains the antiseptic agent. purpose: this research is aimed to observe the sensitivity difference of streptococcus viridans on 35% piper betle linn extract and 10%povidone iodine. methods: this laboratory research was conducted by the post test only design with random complete design. the research sampel is streptococcus viridans culture that was scrapped from the ulcer of the recurrent aphthous stomatitis patient, then it was replicated by using the federer theory. results: inhibitory zone of 35% piper betle linn extract is bigger than 10% povidone iodine. conclusion: streptococcus viridans are more sensitive to 35% piper bittle linn extract than 10% povidone iodine. 35% piper betle linn extract has more antibacterial effect than 10% povidone iodine. key words: bacteriocid test, 35% piper betle linn extract, 10% povidone iodine, streptococcus viridans, recurrent aphthous stomatitis abstrak latar belakang: ulserasi rongga mulut seringkali menjadi alasan utama bagi pasien untuk memeriksakan diri ke dokter gigi. terapi ulserasi rongga mulut adalah pemberian terapi paliatif kepada penderita, seperti: pemberian obat topikal yang mengandung antiseptik. saat ini banyak penelitian dalam pengembangan obat tradisional yang dapat dijadikan sebagai obat alternatif. salah satu diantaranya adalah daun sirih yang mengandung zat antiseptik. tujuan: penelitian ini bertujuan mengetahui perbedaan sensitivitas streptococcus viridans terhadap ekstrak daun sirih 35% jika dibandingkan dengan povidone iodine 10%. metode: penelitian laboratoris yang dilakukan dengan post test only design dengan rancangan acak lengkap. sampel penelitian adalah kultur streptococcus viridans yang diambil melalui swab dari hapusan ulser pada pasien yang menderita stomatitis aftosa rekuren, kemudian dilakukan replikasi dengan rumus federer. hasil: zona hambat ekstrak daun sirih 35% lebih besar daripada zona hambat povidone iodine 10%. kesimpulan: streptococcus viridans lebih sensitif terhadap ekstrak daun sirih 35%. ekstrak daun sirih 35% memiliki efek daya antibakteri yang lebih tinggi jika dibandingkan dengan povidone iodine 10%. kata kunci: uji bakteriosid, ekstrak daun sirih 35%, povidone iodine 10%, streptococcus viridans, stomatitis aftosa rekuren correspondence: maharani laillyza apriasari, program studi kedokteran gigi fakultas kedokteran gigi universitas lambung mangkurat. jl. a. yani km 36 banjarbaru kalsel, e-mail: rany.rakey@gmail.com research report 160 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 159–163 introduction ulceration in oral cavity often becomes a major reason for patients to see a dentist. complaints of oral ulceration can even involve recurrent ulceration, once occurred ulceration or ulceration persistently occured. the etiology of ulcers on the oral mucosa actually involve trauma, recurrent aphthous stomatitis, microbial infections, mucocutaneous disease, drug therapy, and squamous cell carcinoma.1,2 recurrent aphthous stomatitis (ras) is ulcerative oral lesions often found. ras occurred about 20% of the population, 2% of whom got severe pain.1,2 the number of women who suffer from ras is more than men, and more common at age of 20–30 years.1 oral streptococci are actually gram-positive bacteria that can be obtained on all sides of the human oral cavity. most of oral streptococci are classified into viridans group that are opportunistic pathogens. these bacteria can reach the bloodstream because of trauma through oral lesion. streptococcus sanguis and streptococcus mitis are classified into streptococcus viridans group that can cause secondary infections in ras, so it can inhibit healing process.6–8 recurrent aphthous stomatitis is actually considered as urcerative lesions often occured on oral cavity. the cases of ras in patients who arrived at the department of oral medicine, faculty of dentistry airlangga university in 2009 was about 38.99% which was quite high compared to other cases. the diagnosis of oral mucosal ulceration is established based on the history of the disease, and the clinical feature of the ulcer. the biopsy will then be conducted if malignancy or persistent lesions is suspected more than 2 weeks after the causative factors are ommited.1,4,5 therapy for oral ulceration is conducted by giving palliative therapy to patients, such as antiseptic mouthwash (eg. 0.2% chlorhexidine, 1% hydrogen peroxide, 1% povidone iodine) or by giving simple covering agent, such as 10% povidone iodine ointment and topical corticosteroids which can eliminate the symptoms and help healing process.1,4,5 nowadays, a lot of researches concern with the development of traditional medicine that can be used as alternative medicine since the material is easily available with affordable price.10,11 piper betle linn is a medicinal plant that has many benefits and contains antiseptic substances in all its parts. piper betle linn is widely used to treat bleeding nose, itchy eyes, sore throath, bad breath, bleeding gums, and sore mouth (ulcers).11,12 mouthwash containing 25% piper betle linn can kill dental plaque bacteria, streptococcus sanguis for 30 seconds. research conducted by hendratini and rasyaad shows that mouthwash containing with 25% piper betle linn extract could inhibit plaque better than that containing with 1% povidone iodine, 1.5% hydrogen peroxide, and 0.1% chlorhexidine gluconate.13 a research conducted by siswanto, moreover, shows that piper betle linn extract ointment 35% is the fastest solution of wound healing process in the cheek mucosa of white rats when compared with 15% and 25% ones.13,14 in vivo research conducted in female rats induced with everfescent tablet containing with piper betle linn with the highest toxic dose even did not show any organ damage or death in those examined animals.15 clinical research was also ever conducted in humans through patch test showed that 35% piper betle linn extract was a non-allergenic material.16 considering to antibacterial effect possessed by piper betle linn, it has already been able to be used in oral cavity since there are many toothpaste and mouthwash products containing with piper betle linn extract sold commercially to protect gingival and dental health. based on the description above, it is necessary to study more about the benefits of piper betle linn extract as a natural antimicrobial in oral cavity therapeutics. thus, piper betle linn extract later can be used as a therapeutic oral ulceration made in the form of an ointment. the antibacterial effect of piper betle linn extract also can prevent secondary infections. it is also known that piper betle linn extract in the form of ointment can be used as covering agent to accelerate the ulcer healing process. before clinical test was conducted, the sensitivity test towards streptococcus viridans was conducted on 35% piper betle linn extract and then compared with the test on 10% povidone iodine commercially available in the form of ointment. clinical test showed that 10% povidone iodine in the form of ointment is able to cure recurrent aphthous stomatitis ulcers on the fifth day.9 antimicrobial sensitivity test is a test conducted to determine the sensitivity of microbial pathogens towards antimicrobials. diffusion method of antibacterial test, moreover, is the most frequently used method, agar diffusion method. the greater the inhibitory zone around the medicine is, the more sensitive bacteria to the medicine is.17 therefore, the aim of this research is to observe the differences of the sensitivity of streptococcus viridans on 35% piper betle linn extract and 10% povidone iodine. materials and methods javanese piper betle linn leaves are obtained from balai materia medica batu. they were then extracted in the phytochemistry laboratory of upt of balai materia medica batu by the following steps. first, fresh piper betle linn leaves were separated from their stems, and then washed and dried by being aerated for ±3 days. then, they were weighed and finely ground to make it become dry powder. after that, they which wanted to be extracted were weighed. the material of piper betle linn powder used was about as much as 75.524 g (put into six reaction tubes). the piper betle linn powder put into those six tubes was given 161apriasari, et al.: sensitivity difference of streptococcus viridans ethanol solvent 95% about 1020 ml. the extraction unit was programmed into five extraction cycles with drying programs for 60 minutes. the result obtained was 100% piper betle linn extract about 155 ml. the result of the 100% pure extract was finally given sterile aquades, and then diluted up to 35%. this research was an experimental clinical research conducted by post test only design with complete randomized block design and has been declared by the ethical clearance commission of health research, faculty of dentistry, airlangga university. samples of this research is streptococcus viridans bacterial culture derived from ulcer swabs on recurrent aphthous stomatitis based on the certain following criteria: namely untreated ulcer diagnosed as major type of recurrent aphthous stomatitis with diameter > 3 mm. retrieval specimens then were obtained from lesions of recurrent aphthous stomatitis in adult patients aged between 22–44 years old (figure 1). patients were given oral and written explanation about the purpose and methods of how the research will be conducted, and the patients were then asked to fill out and sign informed consent voluntarily. afterwards, the patients were asked to rinse with water. their ulcers were then dried with sterile cotton. then, swab was conducted by using cotton bud that had been sterilized by autoclave. the results were then sent to the microbiology laboratory to be cultured and also to have bacterial identification. figure 1. patient with recurrent aphthous stomatitis on lower labial mucosa (arrow = ulcer). the bacterial identification of streptococcus viridans was conducted. first, the swab results were inserted into brain heart infusion (bhi) media. they were incubated for 1 day at 37 centigrade degree. then, they were planted in blood agar plate (bap) for 1 day at 37 centigrade degrees. the colonies identified as streptococcus were then planted in chocolate agar slant (cas) for 1 day at 37 centigrade degrees. in other word, the results which were seemed as green strains around the colonies in cas could be identified as streptococcus viridans, and then were planted in bap for antimicrobial sensitivity test. bap containing with streptococcus viridans was then divided into 3 parts, namely the filter paper with 35% piper betle linn extract (as the treatment group), the filter paper, 10% povidone iodine (as the positive control group), and the untreated group (negative control). the inhibitory zone diameters of those both groups, the treatment group and the positive control group were then compared after one day incubation. next, those groups were replicated as much as 9 times by using federer formula. the date was analyzed using independent t test. results based on the observation and calculation results of the inhibitory zone diameter of oral streptococci viridans on the groups using 35% piper betle linn extract and 10% povidone iodine with nine times of replication for each the mean of the inhibitory zone diameter of streptococcus viridans on the group using 10% povidone iodine is about 10.22 mm lower than that on the group using 35% piper betle linn extract about 13.77 mm (table 1). table 1. the mean and standard deviation of the inhibitory zone diameter of streptococcus viridans on the two research groups group n mean (mm) standard deviation 10% povidone iodine 9 10.2222 0.97183 35% piper betle linn extract 9 13.7778 1.56347 before conducting the test and analysis on those research groups, normality test was conducted on each of those groups by using kolmogorov smirnov test. the result of the test showed that all of those groups had greater values than 0.05 (p > 0.05). it indicates that the data of those groups has normal distribution. next, a different parametric test, independent t-test, was also conducted to see the significance differences among those research groups. after conducting independent t-tests to see the comparison of the inhibitory zone diameters of streptococcus viridans between the group using 35% piper betle linn extract and the group using 10% povidone iodine, it is finally known that there were significant differences in inhibitory zone diameter of streptococcus viridans among those groups since the significance value was smaller than 0.05 (p = 0.001 or p < 0.05). discussion based on the results of bacteriocid test on piper betle linn extract against streptococcus viridans in recurrent aphthous stomatitis patients, it is known that there was significant difference in inhibitory zone diameter on the sample group using 35% piper betle linn extract (p < 0.05). 162 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 2011: 159–163 compared with 10% povidone iodine group, 35% piper betle linn extract even had greater inhibitory zone diameter. in this research, there were actually three given treatments, 35% piper betle linn extract, 10% povidone iodine, and control. those three treatments then were replicated by using the federer formula since this research is considered as a purely experimental research using homogeneous and randomized samples derived from the cultured colonies of streptococcus viridans. streptococcus viridans was taken from a sample through a swab on the patients with recurrent aphthous stomatitis ulcer to maintain the homogeneity of the research sample. then the bacteria are cultured and incubated at 37 degrees for 1 day. sensitivity test of streptococcus viridans was then conducted by diffusion method. it aims to know the diameter size of inhibitory zone of 35% piper betle linn extract compared with 10% povidone iodine. the larger the diameter of the zone is the higher the inhibitory properties of its bacteriocid streptococcus viridans is actually more sensitive to 35% piper betle linn extract than 10% povidone iodine because of high enough antibacterial properties of phenolic component in the essential oil of piper betle linn extract. toothpaste with essential oil of piper betle linn extract has high antiseptic power against streptococcus colonies α.18 essential oil actually consists of phenol component (propenyl phenol) as much as 60% and non-phenol component. phenol is antiseptic component consisting of eugenol, estragol, chavibetol (betle phenol),9 and chavikol which can kill some bacteria, such as grampositive and gram-negative bacteria.12 propenyl phenol is a toxic compound that can disturb and open three-dimensional structure of streptococcus viridans, which then becomes a random structure without causing damage to the structure of the covalent skeleton, but causing denatured proteins of streptococcus viridans. after the denaturation process, amino acid sequence of protein remains intact, but the biological activities of protein are broken, so it cannot implement its function.12 the content of propenyl phenol on piper betle linn extract is very strong and able to kill bacteria since it has bacteriocid, five times greater than phenol. there is great phenolic components in 35% piper betle linn extract, such as eugenol, estragol, chavibetol, and chavikol. this condition makes the diameter of inhibitory zone of 35% piper betle linn extract greater than that of 10% povidone iodine. as standard antiseptic, povidone iodine has high antiseptic power by interacting in the cell walls of bacteria causing the formation of permanent pores, so it then causes the loss of cytoplasmic material and the reducing of enzyme activity, and later the bacteria become lysis.19 to obtain high antibacterial power, the selection of piper betle linn must be considered. the use of piper betle linn that is still young is better than the old one since the content of volatile oil in the young is higher than the old one.18 another thing that must be considered to get the essential oil of piper betle linn is selecting the fresh one with bright color, perfect shape, free of disease (fungus or pests), and without color changing.10 in this research, 95% ethanol solvent was used. in the extraction process of piper betle linn, organic solvents, such as ether, alcohol, and chloroform, should be used. it is because the essential oil is not soluble in the water solvent.20 twenty five percent piper betle linn extract and methanol solvent has better antibacterial power that that with bacitracin 10 u, chloramphenicol 30 μg, streptomycin 10 μg, sulfonamides 300 g, and vancomycin 30 μg.21 it can be concluded that streptococcus viridans is more sensitive to 35% piper betle linn extract since it has higher antibacterial inhibitory effect than 10% povidone iodine. thus, clinical test research is needed on 35% piper betle linn extract as the active ulcer therapeutic agent to be applied in patients with oral ulcers. furthermore, bio molecular research is then also needed to know the content mechanism of 35% piper betle linn extract during the healing process of oral cavity ulcer. references 1. field a, longman l. tyldesley’s oral medicine. 5th ed. new york, united states: oxford university press inc; 2003. p. 52–9. 2. silverman s. mucosal lesions in older adults. j am dent assoc 2007; 138: 41–6. 3. wray d, lowe gd, dagg jh, felix dh, scully c. textbook of general and oral medicine. london: churchill livingstone; 2001. p. 238–9. 4. langlais rp, miller cs. color atlas of common oral disease. 3rd ed. usa: lippincott williams & wilkins; 2003. p. 156–7. 5. laskaris g. treatment of oral disease. thieme, stuttgart, new york: consice textbook; 2005. p. 169. 6. xiaojing l, kristin m, trunstad l, olsen i. systemic diseases caused by oral infection. j of clinical microbiology reviews 2000; 10: 547–88. 7. jurge s, kuffer r, scully c, porter sr. mucosal diseases: recurrent aphthous stomatitis. oral diseases. 2006, p. 1–21. available at: http:// www.blackwellmuntsgaard.com. accessed november 8, 2009. 8. glen h. diseases of the oral mucosa: traumatic ulcer. emedicine article, department of oral and maxillofacial pathology, university of oklahoma health science center, 2006; 1–5. 9. nafi’ah. perbedaan waktu sembuh klinis pengobatan ekstrak daun jambu biji 0,1% dengan povidone iodine 10% pada stomatitis aftosa rekuren. karya tulis akhir. surabaya: universitas airlangga; 2007. p. 38. 10. mahendra b. panduan meracik herbal. jakarta: penebar swadaya; 2006. p. 21. 11. muhlisah f. tanaman obat keluarga (toga). jakarta: seri agrisehat; 2007. p. 67–8. 12. astuti dh, praba fw, ayu iy, roeslan bo, sjahruddin l. efek aplikasi topikal laktoferin dan piper betle linn pada mukosa mulut terhadap perkembangan karies gigi. majalah ilmiah kedokteran gigi 2007; 22(1): 1–4. 13. siswanto y. konsentrasi optimal salep ekstrak daun sirih dalam proses penyembuhan luka pada mukosa pipi tikus putih. skripsi. surabaya: universitas airlangga; 2007. 14. sari r, isadiartuti d. studi efektivitas sediaan gel antiseptik tangan ekstrak daun sirih. majalah farmasi indonesia 2006; 17(40): 163–9. 15. utaminingrum w. ketoksikan akut tablet effervescent dari ekstrak daun sirih pada tikus betina putih galur wistar. jurnal rac center, fmipa uii, 2006; 11(22): 1. 16. apriasari ml, soebadi b, hendarti ht. patch test dengan ekstrak daun sirih 35% sebagai bahan aktif terapi ulser rongga mulut. denta jurnal kedokteran gigi 2010; 5(1): 44–9. 163apriasari, et al.: sensitivity difference of streptococcus viridans 17. winkelhoff j, newman g. antibiotic and antimicrobial use in dental practice. 2nd ed. usa: quintessence publishing co, inc; 2001. p. 25–7. 18. praptiwi r, priyono h. identifikasi senyawa kimia dan aktivitas antibakteri ekstrak piper sp. asal papua. jawa barat: bidang botani, puslit. biologi lipi csc cibinong; 2002. p. 1–6. 19. freedrick p. the comprehensive resource for phycians, drug, and illness information. 2004. available at: http://www.pordue.ca.pdf. betadine. accessed june, 2008. 20. sugianti b. pemanfaatan tumbuhan tradisional dalam pengendalian penyakit ikan. bogor: makalah falsafah sains, pascasarjana institut pertanian bogor; 2005. p. 1–5. 21. poeloengan m, komala i, noor m, andriani r. aktivitas air perasan, minyak atsiri, etanol daun sirih terhadap bakteri yang diisolasi dari sapi mastitis subklinis. seminar nasional teknologi peternakan dan veteriner, 2006; p. 250–5. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 190 vol. 43. no. 4 december 2010 the management of dental fracture on tooth 61 in a child with attention deficit hyperactivity disorders veranica� and mochamad fahlevi rizal� 1resident in department of pediatric dentistry 2department of pediatric dentistry faculty of dentistry, university of indonesia jakarta indonesia abstract background: attention deficit hyperactivity disorder (adhd) is often characterized as a neurobehavioral developmental disorder, impaired concentration, impaired motor skills, impulsivity, and hyperactivity, and also diagnosed as psychiatric disorders. children with adhd would have a tendency of the traumatized anterior teeth because of their hyperactive behavior. dental trauma is actually one of factors causing the damages of the deciduous teeth and the permanent teeth. dental and mouth care for children with special needs, such as children with adhd, requires special treatment. purpose: this study is aimed to report the case management of the dental fracture of the tooth 61 in a child with adhd. case: a four-year old girl suffered from both adhd and dental fracture involving the dentin of the tooth 61. case management: the examination of the patient with dental fracture consists of emergency examination and further investigation. the emergency examination covers general condition and clinical situation. based on the dental radiographic assessment, it is known that the dental fracture of the tooth 61 had involved the dentine, the resorption had reached 1/3 of the apical teeth and the permanent teeth had been formed. the application of calcium hydroxide on the opened dentin is aimed to improve the formation of the secondary dentin served as pulp protector. next, the restoration of the traumatized teeth used compomer since it does not only meet all the aesthetic requirements, but it also releases fluoride. management of the patient’s behavior with adhd was conducted by non-pharmacological method; tell show do (tsd) method combined with restrain method. conclusion: it can be concluded that the application of calcium hydroxide and the restoration of the teeth with compomer could provide maximum results through the combination of tsd and restrain methods that can effectively increase the positive value to replace the negative behaviors that have been formed. key words: attention deficit hyperactivity disorder, traumatized anterior teeth, compomer, tell show do, restrain abstrak latar belakang: gangguan pemusatan perhatian-hiperaktivitas (gpph), sering dikarakteristikan sebagai gangguan perilaku, gangguan konsentrasi, motorik, impulsif, dan hiperaktivitas dan didiagnosa sebagai gangguan psikiatrik. dan didiagnosa sebagai gangguan psikiatrik. anak penderita gpph mempunyai kecenderungan mengalami trauma gigi anterior karena perilaku hiperaktivitasnya. trauma gigi anak merupakan salah satu penyebab kerusakan pada gigi sulung maupun pada gigi tetap. perawatan gigi dan mulut pada anak berkebutuhan khusus seperti anak penderita gpph memerlukan pendekatan khusus. tujuan: makalah ini bertujuan melaporkan kasus penatalaksanaan fraktur gigi 61 pada anak dengan gpph. kasus: seorang anak perempuan berusia 4 tahun menderita gpph dan mengalami fraktur yang melibatkan dentin pada gigi 61. tatalaksana kasus: pemeriksaan pasien yang mengalami fraktur terdiri dari pemeriksaan darurat dan pemeriksaan lanjutan. pemeriksaan darurat meliputi keadaan umum dan keadaan klinis. penilaian radiografis memperlihatkan fraktur gigi 61 melibatkan dentin, resopsi mencapai 1/3 apikal dengan benih gigi tetap telah terbentuk. aplikasi kalsium hidroksida pada dentin terbuka bertujuan untuk meningkatkan pembentukkan dentin sekunder dan berfungsi sebagai pelindung pulpa. restorasi gigi yang mengalami trauma menggunakan kompomer karena selain memenuhi persyaratan estetik juga melepaskan fluor. penanganan manajemen perilaku pada anak gpph dilakukan dengan metode non farmakologi, yaitu melalui pendekatan tell show do (tsd) yang case report 191veranica and rizal: the management of dental fracture dikombinasikan dengan metode restrain. kesimpulan: aplikasi kalsium hidroksida dan restorasi gigi dengan kompomer memberikan hasil maksimal, melalui kombinasi tsd dan restrain, efektif meningkatkan nilai positif untuk menggantikan perilaku negatif yang telah terbentuk. kata kunci: gangguan pemusatan perhatian-hiperaktivitas, trauma gigi anterior, kompomer, tell show do, restrain correspondence: veranica, c/o: ppdgs ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salembalmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat 10430, indonesia. e-mail: veranica_drg@yahoo.com introduction attention deficit hyperactivity disorder (adhd), is often associated with cerebral dysfunction characterized as neurobehavioral developmental disorder, impaired concentration, impaired motor skills, impulsivity, and hyperactivity. thus, adhd can be diagnosed as psychiatric disorders. 3–5% of the total children even have these early symptoms before the age of seven.1 children with adhd actually have a tendency of the traumatized anterior teeth because of their hyperactive behavior.2 this dental trauma is actually one of factors causing the damages of the deciduous teeth and the permanent teeth with the prevalence ranging between 22–30%. the fracture of the anterior teeth can be caused by the direct or indirect trauma and the high-risk games, such as playing ball and trampoline.3,4 the treatment of the traumatized anterior teeth is generally divided into two, namely emergency care and advanced treatment. if the treatment is conducted on the traumatized dental pulp as early as possible, the irritation can be possibly prevented further. dental and mouth care for children with special needs, such as children with adhd, nevertheless, requires a special approach. generally, the approach of the child’s abnormal behavior consists of non-pharmacology, involving tell-show-do (tsd) method, modeling method, retraining method, desensitization method, and so on. on the other side, pharmacologic approach becomes an alternative option in the management of the child’s abnormal behavior.5 therefore, this study is aimed to report on the management of dental fracture of the tooth 61 in children with attention deficit-hyperactivity disorder. case a four year-old girl (weight: 16 kg, height: 100 cm) came to the clinic of pediatric dental clinic, university indonesia on july 1, 2009, escorted by her mother with a chief complaint of broken upper front teeth because of being knocked on a chair in dining room a week ago while she was playing. at the time of the incident, the patient’s parents, however, did not take their child to the dentist and not give any medical treatment. her teeth were not either bleeding or rocking. the child even did not have any complaints of pain. based on the information derived from her mother, it was also known that according to diagnose of a psychologist, the patient had a behavioral disorder adhd. the results of psychological test suggested that the patient had impaired concentration of attention, fine motor disorder, and delays in speaking. during the examination of the status of teeth and supporting tissues, it was known that the crown fracture of the teeth had involved the dentine of the tooth 61. similarly, based on the radiographic assessment, it was also known that the crown fracture of the tooth 61 had involved the dentin, the resorption had reached 1/3 of the apical teeth, and the permanent teeth had been formed. case management the treatment was started with dental health education and oral prophylaxis. during the comprehensive examination, the operator conducted the special approach by using tell-show-do technique since the patient could not sit calmly, had a lot of moves (hyperactivity) and attention defisit, and also made less eye contact. for the reason, the behavior of the patient could not be controlled during the dental restoration procedures of the tooth 61. as a consequence, the operator then conducted the modeling approach by showing other patients who were next to them and behave cooperatively towards dental care. afterwards, the operator conducted sensitized technique, for example, if the patient is afraid of dental light units, the operator will hold the lamp in a reasonably safe distance and then explain to the patient that the dental light unit is not painful, but useful to see the condition of the teeth and mouth. since the patient remains uncooperative and getting out of control behavior, for efficiency of time and examining the behavior of the patient who were not restrained at the time of restoration, the operator then decided to use the restrain method to control the restrain movement of the patient. next, the operator explained the methods to the patient’s mother who fully supported. the patient then lay in the lap of the patient’s mother who sat on the dental chair and held the patient’s hands in order to hold her if she moved excessively and abruptly. afterwards, the operator used a low-speed round burr to make the retention of dental tissue which would be restored, and then applied calcium hydroxide in the opened dentin which was continued with the compomer restoration of the tooth 61 (figure 1). based on the evaluation of the second visit, one week later, it is known that the patient had no complaints, that 192 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 190–194 the dental restorations of the tooth 61 were still in good condition, and that there were no abnormalities in the dental supporting tissue. at that time, the patient was more cooperative and willing to receive oral prophylactic. similarly, on the third visit, one month later, the condition of the dental restoration was also in good condition. however, before starting the dental care in each visit, the operator always began with the tell-show-do approach to the patient. discussion general guide of adhd patients defines the attention defisit hyperactivity disorder with three major symptoms: inattention, impulsivity, and hyperactivity. there are actually two proposed types of adhd: attention deficit disorder accompanied with hyperactivity indicated by those three symptoms found; and attention deficit disorder without being accompanied by hyperactivity indicated by attention defisit and impulsivity symptomps.1 etiology factors of adhd are not specifically known yet, however, structural disease and brain trauma are playing important roles in which the suspected adhd child will have attention defisit, hyperactivity, and impulsivity because of the reflection of the frontal lobe dysfunction.1 one of clinical descriptions of adhd is that the child is unable to concentrate for long periods of time or the child’s attention can easily be distracted. besides that, the patients of adhd also suffer from hyperactivity and impulsivity. the impulsivity may manifest as impatience, while the effect of the hyperactivity can make the patients tend to get trauma easily.1 trauma is actually a state of emergency condition that must require a treatment immediately to relieve the pain in order to reduce the risk of the increasing dental damage.5 dental trauma in children can occur directly or indirectly. the direct trauma can occur if the teeth are directly hit by objects, such as a hard ball, stick, or fist. meanwhile, the indirect one can be caused by the hard collision on the chin because of being fallen, fighting, getting traffic accidents, and so on.3,4 the dental fractures can be classified into an enamel fracture, dentine fracture, periodontal tissue fracture, and root fracture.6 in addition, the examination conducted on the patients suffering from those dental fractures consists of the emergency examination and further examination. the emergency examination involves general and clinical conditions. examining the general condition, moreover, involves identity, case histories, and medical history. the identity includes name, age, address, and gender. case histories must not only concern with the complaints or symptoms of the spontaneous pain in the teeth during chewing, and the sensitive taste towards the occurrence of injury; but must also concern with the location of the trauma occurred. for medical history, the history of general health and systemic conditions of the patients must be recorded, such as allergies to certain medications, and immunization status of anti-toxoid serum. clinical examination is then conducted after trauma-affected areas are cleaned with warm water, and the types of fracture, extension, tooth disposition, wounds, bleeding, and swelling are recorded. palpation is conducted around the soft tissue in order to see the degree of tooth unsteadiness. vitality test is not recommended at the time of imminent trauma because it will increase the burden of the pulp newly exposed to trauma so that the vitality test becomes inaccurate.7 further investigations involving complete clinical examinations consist of extra oral and intra-oral examinations, and radiological examination. these investigations are aimed to determine not only the growth and development of the teeth, the shape of the pulp, and the expansion of the fracture, but also the existence of the root fractures, the alveolar bone fractures, and the presence of both foreign bodies in the tissues and abnormalities in the areas experiencing trauma. with the careful and complete examinations, diagnosis according to the classification of dental damages due to trauma then will be obtained, and the treatments can be properly planned.7 in this case report, the patient is a 4-year-old girl with crown fracture involving the dentine of tooth 61 since it was hit by chair in the dining room a week ago when she was playing. the condition of the patient is actually the same as what is found in the research conducted by beltrao et al.8 in brazil stating that the teeth often traumatized are upper central incisor which has the highest prevalence involving one tooth only. based on information gathered from the patient’s mother, it is known that the patient suffering from adhd is under therapy and counseling with a psychologist. usually, the patients with attention defisit-hyperactivity disorder are associated with cerebral dysfunction, which is characterized with impaired concentration, motoric disorders, impulsivity, and hyperactivity. hyperactivity is figure �. the intra-oral photographs (a,b) and dental restorations of the tooth 61 (c). c a b 193veranica and rizal: the management of dental fracture a word used to describe excessive motor behavior. children suffering from adhd generally have a tendency to suffer from traumatized anterior teeth due to their hyperactive behavior.1,3 on the first visit, the attitude of the patient was considered to be brave to conduct a complete examination. however, the dental and mouth cares conducted in our faculty clinic were started with the education of dental health and oral prophylaxis. the parents were involved in the education of dental health in order to be able to train the patient. as a result, the patient could be more independent, especially in maintaining oral hygiene. the treatment was conducted together with the application of calcium hydroxide on tooth 61, followed by the restoration of compomer. crown fracture involving enamel and dentin needs aesthetical and functional restoration. besides that, maintaining the pulp vitality is also necessary. if the fracture of the dentine and the enamel occurs, a large number of the dentin tubules will open, as a result, it gives access to bacteria and environmental toxins from the mouth into the pulp resulting inflammation. the application of calcium hydroxide on tooth 61 must be conducted because the fracture had caused the opening of the dentin. calcium hydroxide is very effective not only in increasing the formation of the secondary dentin, but also in producing a thick layer of dentin that can be used to protect the pulp from irritants causing the inflammation of pulp.4 aesthetic restorative materials should be able to resemble the natural teeth in color, translucency, texture, strength, edge adaptation, and adhesion. they must also be both soluble uneasily and biocompatible. restorative material often used for anterior deciduous teeth is polyacidmodified composite resin, known well as compomer. this material can be used for restoring damaged teeth caused by caries. this compomer, contains calcium aluminum fluorosilicate glass filler and polyacid component. both these materials have the basic components of glass ionomer cement without water contain, so the acid-base reactions can occur. the acid-base reactions occured in the compomer accompanied with moist circumstances in the mouth then make the fluoride released. the success of adhesion depends on the use of dentine-bonding primer before this material is applied.4,9,10 the behavioral approach generally consists of nonpharmacologic approaches, involving tell-show do technique, positive reinforcement technique, distraction technique, modeling technique, and desensitization technique. the tell-show do technique is widely used for children with special needs, such as adhd. in this technique, eye contact must be done when instructions are given. in short, technical procedures of the tell-show do are as follows: a) the dentist explains to those children what will be done by using language that is easily understood by those children; b) the dentist then shows them how the procedure is performed; c) the dentist finally acts as what he said and showed to them. other non-pharmacologic approaches are modeling technique, a technique using the children’s ability to imitate their parents, brothers/sisters, friends, or other children who have similar experiences and have succeeded. desensitization technique is a way to reduce fear or anxiety of children by providing stimulation of fear or anxiety continuously until the children are not afraid or worried again.5 tsd technique can actually be conducted by an operator to a patient since this tell-show-do technique is considered as a method that can be used for all ages and all children’s behavior, including for children with special needs. in a complete examination, those children with special needs are brave, but when they get restorations on their tooth, their behavior become unmanageable. the other techniques, such as modeling and desensitization techniques, have been conducted, but the patients sometimes still cannot cooperate. because of this inconducive situation, restrain method must be conducted as treatment. restrain method is a non-pharmacologic approach used not only for very young children under 3 years of age, for children with health and mental disorders, for children with disabilities who cannot control sudden movements, but also for children who can be potentially uncooperative. in other words, this method is a technique designed to promote positive values used for replacing the negative behaviors that have been formed. before the restrain method is conducted, the cause of bad behavior in those children must be known in order to make restrain process more effective. therefore, before the restrain method is conducted, those children and their parents must be notified in advance about the aims of this method.5,11 in this case, moreover, the impulsive behaviors of the patient could be obviously visualized. impulsivity may actually manifest as impatience, which is one of the typical symptoms in patients with adhd. unlike normal children who have no psychiatric disorder and psychological trauma, the patient must be generally explained by the daily language about the dental treatment that will be conducted by using tsd approach. unlike three year old normal children who are be able to sit quietly, the patient suffered from adhd could not control her behavior. it can be concluded that dental trauma in children is one of the causes of the damages of the deciduous teeth and the permanent teeth. this situation often occurs in young patients of adhd because of their hyperactive behavior. the application of calcium hydroxide on the opened dentin and the use of compomer restoration must be conducted because of possesing a good aesthetics and a character of releasing fluoride. for those reasons, it can be finally said that through such non-pharmacologic approaches like tellshow-do technique combined together with the method of restrain, co-operative attitudes of children towards dental care can be increased. counseling with a psychologist is needed, so that not only positive behaviors of patients can be improved, but impulsive and hyperactive behaviors can also be suppressed. 194 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 190–194 references 1. lumbantobing sm. anak dengan mental terbelakang. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 2006. p. 68–81. 2. oredugba fa, akindayomi y. oral health status and treatment needs of children and young adults attending a day centre for individuals with special with special health care needs. bmc oral health 2008; 8: 30. available from: url: http://www.biomedcentral.com/14726831/8/30. accessed november 1, 2009. 3. millett d, welbury r. clinical problem solving in orthodontics and pediatric dentistry. philadelphia: elsevier; 2005. p. 99–101. 4. koch. g, thessleff i, kreiborg i. tooth development and disturbances in number and shape of teeth. in: koch g, poulsen s, editors. pediatric dentistry. a clinical approach. 2nd ed. denmark: blackwell; 2009. p. 186–7. 5. chadwick bl, hosey mt. child taming: how to manage children in dental practice. london: quintessence; 2003. p. 37–47. 6. holand g, mctigue dj. managing traumatic injuries in the young permanent dentition. in: pinkham jr, mctigue dj, casamassimo ps, fields, hw, nowak aj. pediatric dentistry: infancy through adolescence. 4th ed. philadelphia: wb saunders; 2005. p. 237–54. 7. camp jh, stewart c. dental trauma. available from: url: http:// www.thrombosisconsult.comarticles/textbook/139dental.htm. accessed january 5, 2010. 8. beltrao em, cavalcanti al, albuquerque ssl, duarte rc. prevalence of dental trauma in children aged 1–3 years in joao (brazil). european archives of pediatric dentistry. 2007. available from: url: http://www.thefreelibrary.com/ prevalenceofdentaltraumainchildrenage3yearsinjoaopesoa. accessed january 5, 2010. 9. drummond b, kilpatrick n. dental caries and restorative pediatric dentistry. in: cameron ac, editor. handbook of pediatric dentistry. 2nd ed. london: mosby; 2003. p. 52. 10. powers jm, sakaguchi rl. craig’s restorative dental materials. 12th ed. london: mosby; 2006. p. 204–7. 11. wilson s. no pharmacologic issues in pain perception and control. in: pinkham jr, mctigue dj, casamassimo ps, fields, hw, nowak aj, editors. pediatric dentistry: infancy through adolescence. 4th ed. philadelphia: wb saunders; 2005. p. 105. isi vol 39 no 2 april 2006 file pertama.pmd 85 surface hardness of hybrid ionomer cement after immersion in antiseptic solution anita yuliati and ajeng kartika sri wardani department of dental material faculty of dentistry airlangga university surabaya indonesia abstract hybrid ionomer cement or resin modified glass ionomer cement is a developed form of conventional glass ionomer cement. this hybrid ionomer cement can be eroded if in direct contact with acid solution which will affect surface hardness. the aim of this study is to learn surface hardness of hybrid ionomer cement after immersion in methyl salicylate 0.06% (ph 3.6) and povidon iodine 1% (ph 2.9) solution. sample of hybrid ionomer cement with 5 mm diameter and 3 mm thickness was immersed in sterile aquadest solution (control), methyl salicylate ph 3.6, povidon iodine ph 2.9 for 1 minute, 7 and 14 minutes. surface hardness was measured with micro vickers hardness tester. the obtained data was analyzed statistically with anova followed by lsd test. the result of hybrid ionomer cement after immersion in sterile aquadest, methyl salicylate 0.06% ph 3.6 and povidon iodine 1% ph 2.9 for one minute, showed no significant difference; while immersion for 7 and 14 minutes showed a significant difference. the conclusion states that hybrid ionomer cement after 14 minutes immersion in povidon iodine 1% ph 2.9 has the lowest surface hardness. key words: hybrid ionomer cement, antiseptic solution, surface hardness correspondence: anita yuliati, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. maintain oral health, although they have brushed their teeth regularly. antiseptic is a chemical agent utilized on mucosa surface or skin to kill microorganism by destroying or inhibiting microorganism growth.7 preliminary survey by researcher had found brand of antiseptic solution commonly used and circulated in the market was antiseptic liquid containing methyl salicylate 0.06% and povidon iodine 1%. ph of methyl salicylate 0.06% was 3.6 and povidon iodine 1% had ph 2.9. manufacturer’s formula has indicated that the antiseptic solution can reduce foul breath, eliminate plaque and decrease oral ulceration. it is recommended to gargle 30 seconds twice/day, maximal in 14 days. hybrid ionomer cement will contact directly with antiseptic solution in oral cavity, where the antiseptic solution is acid in nature. hybrid ionomer cement can undergo erotion if in contact with acid solution and it affects surface hardness.8 this study used a conversion of antiseptic solution against hybrid ionomer cement, as follows: hybrid ionomer in contact with antiseptic solution in 1 minute (duration of antiseptic solution for 1 day), 7 minutes (7 days duration of antiseptic solution usage) and 14 minutes (2 weeks duration).therefore, problems rise in pertaining to surface hardness of hybrid ionomer cement after immersion in acidic antiseptic solution. the purpose of this study is to learn surface hardness of hybrid ionomer cement after immersion in methyl salicylate 0.06% (ph 3.6) and povidon iodine 1% (ph 2.9) solution for 1, 7 and 14 minutes. the benefit of this study introduction conventional glass ionomer cement has been used for esthetic filling in cavity with low stress bearing, for example in class iii and iv restoration, in patient with high caries risk, or on pit and fissure. the most common problem found in conventional glass ionomer cement is its sensitivity to moisture and its low initial strength. in the early 1990, modified resin glass ionomer cement was released into market to produce good physical nature similar to those of composite resin, and still it could maintain the basic features of the conventional glass ionomer cement. these new material was called as resin modified glass ionomer cements or hybrid ionomers.1, 2 the composition of hybrid ionomer cement is similar with conventional glass ionomer cement. hybrid ionomer cement contains polyacrilyc acid or modified polyacrilyc acid with monomer hydroxyethyl methacrylate (hema) or bis-gma in the liquid.3 hybrid ionomer cement has better qualities than conventional ionomer cement in: controllable working time, less sensitivity to moisture, wear resistance solubility, low, good esthetics, fluoride release, biocompatibility, adhesion to a moist tooth structure, more elastic, good dimentional stability, and higher surface hardness.1,4,5 based on in vitro analysis, hybrid ionomer cement releases fluoride in equal amount with what is released by conventional glass ionomer.6 at present, people tend to keep the hygiene of their mouth cavity using mouthwash as an antiseptic liquid to 86 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 85–88 is to understand the effect of acidic antiseptic solution usage inside mouthwash liquids towards hybrid ionomer surface, so that erotion process can be prevented. materials and methods this is an experimental laboratory study. carried out in material and technology department, school of dentistry, airlangga university and laboratory of technical engineering, institute of sepuluh nopember, surabaya. the materials used are: type ii hybrid ionomer cement (fuji ii lc, gc corporation, tokyo, japan, reorder no. 000225); methyl salicylate 0.06% (listerine, pt. bayer indonesia, reg.pom cd 1302101501); and povidon iodine 1% (isodine, pt. mahakam beta farma, depkes ri.bpd.05100021). the tools are: cylindric teflon 5 x 3 mm,9 plastic spatula, glass slab, 1 kg weight scale, magnifier glass, celluloid strip, striped visible light unit (litex) and micro vickers hardness tester (shimadzu). the sample of hybrid ionomer cement was made as follows: the lower side of sample cast was given celluloid strips and put on top of glass slab. according to manufacturer’s formula, hybrid ionomer cement and powder was mixed and put into plastic ring until the sample cast was full. further procedure was performed following previous researchers.9 the samples were clustered based on treatments and sample number in every treatment cluster was 6. the samples were treated like this, group i were immersed in aquadest sterile, group ii were immersed in methyl salicylate 0.06% ph 3.6 and group iii were soaked in povidon iodine 1% ph 2.9. every treatment group were immersed for 1 minute, 7 and 14 minutes. to measure sample surface hardness, the surface of sample which was already immersed was dried with tissue paper and marked with black marker pen. then, the sample was placed in the middle of micro vicker hardness tester platform with 250 gram weight load. the picture in the lens was focused with 200 times enlargement, pressed red button, the diamond penetrator rised. the lens was changed with 400 times enlargement, then the result of sample surface penetration was seen in rhombus shaped cut. the result of diagonal length measurement was placed in a formula in micro vicker hardness tester manual i.e. 2 d p)(1.854 v × = v = surface hardness (kg/mm2) p = load (kg) d = average diagonal length (1/1000 mm) measurement was done in three different sites, averaged and the result of the average (mean) was taken as sample surface hardness. results mean (average) and standard deviation of hybrid ionomer cement surface hardness after immersion in sterile aquadest, methyl salicylate 0.06% ph 3.6, povidon iodine 1% ph 2.9 in 1 minute, 7 and 14 minutes (kg/mm2) were presented in table 1. table 1 showed that hybrid ionomer cement after immersion in povidon iodine antiseptic solution 1% ph 2.9 had the lowest surface hardness compared with the two other groups. data inside table 1 was homogen, normal distribution, tested with one sample kolmogorovsmirnov test. furthermore, it was analyzed with one-way anova test, and p < 0.05. this result showed that there was a significant difference of surface hardness after immersion in sterile aquadest and antiseptic solution with different ph. the different treatment was tested with lsd as seen in table 2. hybrid ionomer cement after immersion in methyl salicylate 0.06% ph 3.6 and povidon iodine 1% ph 2.9 for 1 minute showed no significant difference, it meant there was no surface hardness decrease. while after immersion for e•7 minutes, there was a significant difference, meaning there was a decrease of surface hardness. discussion another vehicle for delivering active agents with desirable effects to the surfaces of the teeth and gingival is mouthwash. this liquid is a commonly used antiseptic solution to enhance oral hygiene, esthetic and breath sterile aquadest (control) methyl salicylate 0.06% ph 3.6 povidon iodine 1% ph 2.9 duration of immersion n x ± sd x ± sd x ± sd 1 minute 7 minutes 14 minutes 6 6 6 41.29 ± 0.77 38.19 ± 0.56 39.13 ± 1.03 41.06 ± 1.04 38.15 ± 0.92 35.17 ± 0.43 40.95 ± 1.17 37.11 ± 1.09 32.85 ± 1.61 table 1. mean and standard deviation of hybrid ionomer cement surface hardness after immersion in sterile aquadest, methyl salicylate 0.06% ph 3.6, povidon iodine 1% ph 2.9 in 1, 7 and 14 minutes (kg/mm2) 87yuliati and wardani: surface hardness of hybrid ionomer cement freshnness. two natures of antiseptic solution which has to be watched is the acid and ethanol content. these two natures are very damaging to composite resin, compomer and sealant restoration materials.1 thus, the usage of mouthwash can affect physical and mechanical natures of dental restoration materials. the result of this study showed that the longer hybrid ionomer cement immersed in sterile aquadest, methyl salicylate 0.06% ph 3.6 and povidon iodine 1% ph 2.9 solutions, the more surface hardness decreased. hybrid ionomer cement surface hardness is easily affected by storage media.5 other researchers stated that the longer hybrid ionomer cement immersed in pure water, the more water can be absorbed, so that surface hardness becomes softer. physical nature of hybrid ionomer after being soaked in water will be softer. water will be absorbed by hema inside hybrid ionomer cement because of hema’s hydrophilic character. water absorption will inhibit metal ion cross ties because metal ion will be dissolved in water. metal ion which was detached from hydroxyl compound inside hybrid ionomer cement will cause the material to be fragile.10 hybrid ionomer cement will absorb the liquid which will dissolve matrix forming cation and anion to surrounding areas. this happening will produce fragile cement dissolved easily, causing transparent significant alteration.6 antiseptic solution used as mouthwash has composed of three main ingredients, namely active agents, surfactants, and flavoring agents. methyl salicylate 0.06% inside mouthwash is a flavoring agent functioning as breath freshnness. active agents inside mouthwash can be liquefied in water and/or alcohol.1 active agents inside antiseptic solution used in this study is dissolved in alcohol. it is written outside the packaging of these two antiseptic solutions that it has high alcohol level (ethanol 21.6%) for methyl salicylate, whereas povidon iodine has not shown any alcohol percentage. alcohol inside gargle liquid antiseptic solution can influence surface hardness. this study has proven that the longer hybrid ionomer cement immersion in methyl salicylate 0.06% ph 3.6 and povidon iodine 1%, the weaker hybrid ionomer cement surface will be. thus, a significant decrease of surface hardness is persistent. ionomer cement is sensitive towards acid. acid media causing erosion is saliva, plaque and alcoholic beverages. hardened glass ionomer cement consisted of glass particle clumps which did not react and covered by silica gel. unreacted particle clumps are covered by silica gel in amorphous matrix of calcium hydrate and aluminum salt mixture.6 alcohol inside antiseptic solution can dissolve part of hybrid ionomer cement matrix, causing damage at hybrid ionomer cement structure and decreased surface hardness. hybrid ionomer cement immersed in povidon iodine 1% ph 2.9 has the lowest surface hardness compared to other groups. the decreasing surface hardness is caused by erosion due to direct contact with acid, in particular strong acid. in acidic milieu, ionomer cement matrix will be dissolved, physical and mechanical nature will also decrease. this can cause detachment of metal ion inside hybrid ionomer.8 the result of this study is in accordance with hypothesis that glass ionomer cement surface hardness (fuji lc ii) can decrease along with the decrease of ph and time. observation with electron microscope shows that after 5 hours of immersion in citric acid added with artificial saliva ph 3, the microstructure of hybrid ionomer cement does change. the surface splits, more porous and after 72 hours immersion, surface texture lessen.5 erosion mechanism can be illustrated as follows: h+ ion solution enters inside cement and change place with metal cation in polycarboxilate molecule cross ties inside cement matrix. 9 hybrid ionomer cement solution containing hema in acidic milieu will cause excess hydrogen ion (h+). this will replace metal ion which has cross tied with polyalkenoat chain, causing breakage of cement matrix ties. this is the beginning of degradation process for dissolving material. erosion and hybrid ionomer cement dissolvement has been declared by other researcher, also that hybrid ionomer cement can dissolve, releasing substances during immersion in distilled water, but when tested with lactic acid, only small portion dissolve in erosion test. it is further said that basically resin cement is not easily dissolved, only releasing small amount of substances i.e. unreacted monomer. unreacted monomer will definitely weaken ionomer resin cement surface hardness.12 conclusion of this study reveals that hybrid ionomer cement immersion for 14 minutes in povidon iodine 1% ph 2.9 has the lowest surface hardness. references 1. craig rg, powers jm. restorative dental material. 11th ed. st louis, london, philadelphia, sydney, toronto: mosby a harcourt health sciences company; 2002. p. 203–5, 614–8. table 2. lsd test of hybrid ionomer cement surface hardness after immersion in aquadest and antiseptic solution with different ph sterile aquadest ph 7 (control) methyl salicylate 0.06% ph 3.6 povidon iodine 1% ph 2.9 immersion duration 1 mnt 7 mnt 14 mnt 1 mnt 7 mnt 14 mnt 1 mnt 7 mnt 14 mnt 1 minute 7 minutes 14 minutes s s s ns s s s s s s s s ns s s s s s s s s note: s = significant; ns = non-significant 88 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 85–88 2. nagaraja up, kishore g. glass ionomer cement–the different generations. trends biomater artif organs 2005; 18(2): 158–65. 3. hatrick cd, eakle ws, bird wf. dental materials. clinical applications for dental assistants and dental hygienists. philadelphia, london, new york, st louis, sydney, toronto: saunders; 2003. p. 183. 4. hara at, serra mc, rodrigues al. radiopacity of glass-ionomer/ composite resin hybrid materials. braz dent j 2001; 12(2): 85–9. 5. koppari s, persson m. the effect of storage in different ph on the surface microhadness of a resin-modified glass ionomer cement and resin composite. department of dental biomaterials science. karolinska institutet huddinge, sweden. 2005. 427–38. available at www.ki.se/odont/caridogi_endodonti/kov/tio stina_koppari_maria_persson.pdf. accessed april 30, 2005. 6. anusavice kj. phillips science of dental material. 11th ed. usa: elsevier science; 2003. p. 358–61, 471–8. 7. samaranayake lp. essential microbiology for dentistry. edinburgh, london, new york, philadelphia, st louis, sydney, toronto: churchill livingstone; 2002. p. 262. 8. fukazawa m, matsuya s, yamane m. the mechanism for erosion of glass-ionomer cement in organic-acid buffer solutions. j dent res 1990; 69(5): 1175–9. 9. farihah sh, meizarini a, yuliati a. variasi ketebalan celluloid strip terhadap kekerasan permukaan resin komposit sinar tampak. majalah kedokteran gigi (dent j) 2001; 34(4): 753–5. 10. nicholas jw, antice hn, mclean jw. a preliminary report on the effect of storage in water on properties of commercial light cured glass ionomer cements. br dent j 1992; 151: 98–101. 11. mc cabe jf, walls awg. applied dental materials. 8th ed. blackwell science; 2000. p. 212–7. 12. jones dw. dental cements: a further update. j can dent assoc 1998; 64: 788–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� placement of fin type dental implant in three different surgical situations of alveolar bone coen pramono d department of oral and maxillofacial surgery faculty of dentistry, airlangga university/dr. soetomo, general public hospital surabaya indonesia abstract three different dental implant placements according to surgical implant bed situations were observed in its bone integration 3 months after dental implant insertion. this observation was done on implant system which has plateau or fin system. elf implants were placed in the upper jaw in two patients. in case one, two implants were inserted immediately after tooth extraction, and the other six implants were placed in the alveolar crest regions in delayed implantation or in which the teeth had been extracted over 6 months of period. in case two, three implants were inserted in the post trauma region in the anterior maxilla, which the labial plate had been lost and reconstructed with bone grafting procedure using a mixture of alloplastic and autogenous bones. the alveolar reconstruction was needed to be performed due to only thin alveolar crest width was left intact. all of those implants observed showed in good integration. key words: fin implant system, immediately dental implant placement, alveolar crest width reconstruction, mixture of autogenous and allogenic bones correspondence: coen pramono d, c/o: bagian bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction a modern technique in oral rehabilitation using dental implant brought dentistry into a new chance in giving a good service to the patients who need for dental rehabilitation after missing their teeth. the use of dental implant may lead a comfortable result and may preserve the neighboring teeth while the dental implant needed only a single retainer i.e. bone, differs when the tooth rehabilitation is done using fixed denture using a bridge technique which at least two teeth are necessary to be half cut in creating new attachment for the lost tooth. many implant systems have been produced with its advantages and disadvantages. implant system using fin or plateau form surface colored the dental implant world with its unique system which has a different system as usually found in other implant system, while this system is applied not in screw type but by inserting the implant into precise prepared bone holes exactly according to the implant diameter needed to be inserted. the unique implant systems using fin type found in its implant body, which has a fin design and offers at least 30 % more surface area than a screw implant type of the same size and affords direct resistance to vertical forces of occlusion on the surrounding haversian bone. additional implant retention is achieved through bone growth between each fin and integrated into implant surface as seen in the histologic level. retention of fin type implant is achieved through three mechanisms: first, precession placement of the dental implant body into the alveolar crest and the initial fitting is achieved, secondly bone growth between each fins this finding supported by the histological study showed that mature haversian bone surrounding the fins,1 and tertiary is integration between bone and implant surface.2-4 according to the advantage of this dental implant system as reported by some authors,5-13 this type of implant was selected for observation. the observation on immediate and delayed implant placement was done based on experiences in using screw-shaped type implant system which sometimes found with difficulty in its utility during the drilling process on thin and narrow alveolar crest as it with the risk of alveolar wall perforation. this problem might be happened when a high speed drilling is used. as shown in the previous report, placement of screwshaped type implant using both immediate and delayed technique had been proved with a good result.14 problem lead on the drilling speed during screw type implant placement which need about 800 rpm for bone preparation therefore using this high speed rotation in thin alveolar width might give a risk to alveolar perforation. different with what given in the utility of fin type implant that need only low speed drilling of 50 rpm or less and in such cases when slowest drilling rotation is needed a hand drilling instrument also available to prevent alveolar bone from perforation or rupture. therefore dental implant with fin system was chosen as an implant system used for observation. �0 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 49–53 cases c a s e 1 : a 5 6 y e a r o l d m a n a s k e d f o r t e e t h replacement with dental implant. he was complaining of uncomfortable removable partial denture and felt badly disturbed as the partial denture influenced his phonetic quality, unpleasant by eating and feeling of boring in the maintainance of the denture. the lower jaw partial denture was than thrown away, because of the vomiting senses every time he wore the denture. twelve teeth were observed and needed to be replaced, and planned with dental implant placement for the dental rehabilitation, eight dental implants in the upper jaw and four implants in the lower jaw. the teeth were 11, 12, 14, 15, 16, 24, 25, 26, 34, 36, 44, 46 (figure 1). the upper jaw was the first priority to be restored as it is more related with aesthetic and those in the lower jaw were ceased temporarily waiting for the next setting. the problems in this case were the alveolar crest in the regions of teeth 11 and 12 which were found atrophic in the labial site and atrophic maxilla in the region of tooth 26. three point three millimeter (3.3 mm) of implant diameter figure 1. (a) panoramic x-ray shows edentulous region in the upper and lower jaws. twelve teeth implants were planned in the upper and lower jaws in teeth: 11, 12, 14, 15, 16, 24, 25, 26, 34, 36, 44, 46. in teeth 15 and 24 planned with immediate implant placement. severe radiolucent areas shows around tooth 15 and 24; (b) tooth extraction before implant placement; (c) bone drilling and; (d) five implants were inserted in the right upper jaw: 11, 12, 14, 15, and 16. immediate implant placement after extraction of teeth 15 and 24; (e) panoramic x-ray four months after placement of eight implants in the region of teeth: 11, 12, 16, 15, 17, 24, 25, and 26. immediately placement in teeth 15 and 24; (f) osseointegration evaluation method: guide pin inserted into each implants and mobilization test is done; (g) eight temporary abutments were placed for sulci forming; (h) temporary removable denture was made for cosmetic reason seated above the temporary abutments; (i) example of three sulci and implants wells successfully formed. situation in five days after temporary abutment removal; (j) eight integrated crown-abutment ready for insertion; (k) mouth mirror reflection: eight integrated crown-abutment inserted in teeth 11, 12, 14, 15, 16, 24, 25 and 26. a b c d e f g h i j k ��pramono: placement of fine dental implant was used in the anterior regions and in tooth 26, a short implant of 5.7 mm long and 6 mm diameter were inserted to cope the problem with success. interesting problems were found in teeth 15 and 24 where in these regions the teeth were surrounded with granulation tissues. two implants were immediately inserted into fresh extraction sites of teeth 15 and 24 presented with well integrated as no were found mobile implants. the other six implants which had been placed in delayed placement and also presented with a good result. the sequence of those implants placement can be seen as shown in figure 1b to k. case 2: a 19 year-old female asked for teeth replacement after having accident 3 months previously. in the post trauma region showed with width defect presented with a thin paper alveolar crest exhibited had lost its labial bone plate which thought to be impossible for an implant placement (figure 2a). alveolar reconstruction was done for rebuilding the alveolar width using a mixture of autogenous and allograft bones. the autogenous bones were taken from the chin and retromolar regions and prepared in a mixture with allogaft bone material liquefy with her own blood and harvested into the region of alveolar crest of teeth 21, 22 and 23 and covered with an allograft membrane using double layer technique (figure 2b-e). one year after bone implantation the reconstructed alveolar crest formed and healed perfectly and three dental implants were inserted in this region. one year after the alveolar crest reconstruction the alveolar width had increased positively and showed with matured bone (figure 2f). although it has a different quality in its density comparing to the original bone beside the grafted region but still presented as an acceptable bone for implant placement. this bone quality was qualify during the initial drilling and marked softer comparing to the healthy bone, therefore hand reamer was obligatory to be used to prevent the labial bone from fracture. periapical xray taken one month after implant insertion presented bone had been grown between the implant fins showed with no bone gab between the surrounding bone and all implants sites (figure 2g). one month after implants insertion showed bone had been integrated between the implant fins as seen in figure 1g. definitive integrated crown-abutment will be inserted three months after the implants placement. cases management two patients asked for dental restoration in their partial edentulous jaws. both patients were wearing removable dentures and complaining of discomfort. therefore they asked for another alternative restorative treatment and dental implant was offered for those teeth rehabilitation and bicon implant system with hydroxyapatite (ha)-coated surface was used. in case 1, two problems had been mentioned as the atrophic site in the region of upper left first molar therefore a short type implant dimension was needed. replacement of teeth 15 and 24 were planned with immediate implant placement into fresh alveolar socket after the teeth being extracted. a short dimension with only 5,7 mm length and diameter of 6 mm was used. therefore problems of short figure 2. (a) defect of the alveolar crest in the region of 11, 12 and 21 presented with no the labial bone and prepared for bone grafting; (b-c) autogenous bone taken from the lateral retromolar and chin regions, bone puncher was used; (d) a mixture of autogenous-allograft bone materials and blood; (e) harvested into the alveolar crest region of teeth 11, 12 and 21; (f) one year situation after alveolar crest width reconstruction; (g) periapical x-ray one month after insertion of three dental implants for replacement of teeth 11, 12 and 21 presented with no dental implant-bone gap. a b c d e f g �2 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 49–53 available bone in molar region of left upper jaw may be solved with this system. in case 2, the necessary of alveolar crest reconstruction after alveolar crest trauma made this case included in this observation. coen’s measurement technique was used to qualify the available bone height in all sites needed to be replaced with dental implant.15 discussion denture restoration using dental implant is now widely accepted through out the world. all of the dental implant system offered a success in its utilization and those had been reported by many authors that all of implants market can be well integrated with the surrounding bone as all implants usually designed with treated surface to ensure bone to be well integrated with the implant surface. easy and safety in its utilization during implant placement is the most important part in dental implant placement. one of important factor for success in implant placement is depend on a step during bone preparation procedure and it should be importantly considered as complication during bone preparation for example implant wall perforation will complicate the implant placement while it may lead of a failure. there are three types of implant systems known, screwshaped, screw-hollow cylinder and hollow-cylinder. the screw-hollow and hollow-cylinder system had never been market in ours before, only the screw-shaped type implant is available. the implant system with fin or plateau surface seemed had been developed as a combination from hollowcylinder and screw-shaped implant systems. the fin or plateau system which developed by bicon has a unique body design and available in three surface types. these three treated types surfaces are: tio2-blasted, ha coated and titanium plasma sprayed coated (tps). those implant surfaces treatment has been made to allow a better contact between bone and implants.1–4 three types of retentions can be expected in fin system, as 1) the precision bone hole according to the implant diameter, 2) the implant plateau or fins, affords direct resistance to vertical forces on surrounding haversion bone, and 3) the ha coated permitted a good osseointegration between implant and the surroundings bone. studies on anchorage of implant surfaces to the surrounding bone had been studied by some authors. gotfredsen et al.2 in 26 rabbits with totally 156 implants placed in the tibia. three types of implant surface had been observed, as tio2-blasted surface, tio2-blasted coated with hydroxyapatite and the control was machine type implant. the result showed that tio2 blasting on implant surface improves the implant anchorage and in tio2blasted surface coated with ha also presented improvement of bone contact but not yet practicable. histological and histomorphometric comparasion of bone to implant contact of immediately placed ha coated and tps implants studied by karabuda et al.3 showed that the ha coated surface can achieve better bone contact than given in tps when placed into fresh extraction socket. the investigation from novous et al.4 also presented with the same result, where the ha coated implant may improved better bone contact comparing to machined and tps sprayed implants but less superior comparing to soluble particle (sbm) surface implant. this observation made base on experiences using screwshaped type implant placed both in immediate placement into fresh extraction socket or in delayed placement.11 although in that previous report placement of immediate and delayed implant placement using screw type implant presented with a good result,11 but in such cases, the use of bone drill driven into a speed of 800 rpm as usually used in the screw type dental implants placement might be risky comparing to the drilling speed needed for placement of dental implant used in the dental implant has a fin system. besides the alveolar height, preservation of the alveolar crest width during bone drilling should be considered important as perforation of the labial or palatal walls would lead a failure. as it might facilitate the fibrous tissues grown in between the dental implant and the alveolar bone wall sites and promoted of those surrounding implant socket walls absorption. in cases of thin alveolar crest width, in the regions which had been received bone grafting or in the soft bone region, bone drilling using a low speed drilling with 50 rpm or less or using hand reamer technique proved to be a safe procedure because alveolar wall perforation or even rupture can be easily ovoid and may also providing safely distal bone margin. using hand reamer or a low-speed drilling with less than 50 rpm might also provide bone debris that can be used for autogenous bone transplant material which usually necessary to be harvested immediately in the region around the dental implant neck after fin implant type insertion. retention of the dental implant into the surrounding bone bed plays as an important role to achieve success in all types of dental implant placement. in this fin type mplant, initial fitting can be easily provided therefore bone integration into implant surface can be expected. achieving of initial retention in any implant type placement had been ruled out very important step as it related with the integration process would be proceed between the dental implant surface and the surrounding bone. failure in achieving an initial retention will allow the dental implant placed unstable and might promoted an implant placement from failure through the present of gap between bone and implant. the gap will fulfill with fibrous tissue therefore the bone would be failed to integrate with the implant surface and the inflammatory process would be than taken part. in this paper presented three different types of implant placement according to the period after tooth extraction. �3pramono: placement of fine dental implant as shown in case 1, which has short and thin alveolar crest width in the anterior region as well as a narrow alveolar crest in the region of tooth 26, a short implants dimension and the use of hand bone reamer were helpful as it avoid to vital structures and implant wall perforation. observation of 4 months after immediately insertion of two implants into fresh alveolar socket in the region of teeth 15 and 24 resulted with success showed with no implants mobility and presented with healthy surrounding mucosa. those two implants were found stable the same as given by the other implants which had been inserted in the alveolar crest of 6 months to 1 year after tooth extraction as shown in teeth 11, 12, 14, 16, 25, 26, 34, 36. as well as in case 2, in the alveolar bed which had been received bone graft with a mixture of autogenous and allograft bones and placed by three dental implants showed with a good result. all those dental implants had been inserted in those three different alveolar crest situations showed had been integrated with success. in my opinion three primary important consideration should be taken to achieve success in implant placement are: 1) placement of the implants into a good host with no systemic factors problem, 2) the surgical procedure according to the dental implant type should be carefully followed, and 3) the initial fitting of the implant body to the bone must be achieved, lost of that initial fitting may lead a failure as the bone may failed to integrate with the dental implant surfaces. references 1. bicon dental implants bulletin. available at: http://www.bicon. com/news/n_publications.html. accessed february 27, 2007. 2. gotfredsen k, wennerberg a, johansson c, skovgaard lt, hjortighansen e. anchorage of tio2-blasted, ha-coated, and machined implants; an experimental study with rabbits. j biomed mater res 1995 october; 29(10):1223–31. 3. karabuda c, sandalli p, yalcens s, steflik de, parr gr. histologic and histomorphometric comparasion of immediately place hydroxyapatite-coated and titanium plasma-sprayed implants: a pilot study in dogs. int j oral maxillofac implants 1999 jul-aug; 14(4):510–5. 4. n o v o u s a b j r , s o u z a s l , d e o l i v e i r a p t , s o u z a a m . histomorphometric analysis of the bone-implant contact obtained with 4 different implant surface treatments place side by side in the dog mandible. int j oral maxilollifac implants 2000 may-june; 17(3):377–83. 5. yong chan l. the unique quality of bicon implant system clinical application. dental success 2004; 24(6):732. 6. she hi h. use of bicon implant, placement without the need of sinus lift (i); procedure, clinical implant,11/12. 2004. 7. she hi h. use of bicon implant, placement without the need of sinus lift (ii). process of prosthetic restoration. clinical implant, 01/01, 2005. 8. urdaneta r, chuang sk, marincola aw. two-year retrospective evaluation of a unique restoration for single implants. iadr/aadr/ cadr 80th general session, honololu, hi, march 2004. available at: http://www.bicon.com/news/n_publications.html. 9. woo vv, chuang sk, daher s, muftu a, dodson tb. dentoalveolar reconstructive procedures as a risk factor for implant failure. j oral max fac surg 2004 july; 62(7):773–80. accessed february 27, 2007. 10. leary j, hiryama m. use of integrated abutment crowns to enhance the aesthetics of maxillary anterior restoration. ao general session, san francisco, ca, 2004 (march). available at: http://www.bicon. com/news/n_publications.html. accessed february 27, 2007. 11. yoo rh, chuang sk, erakat m, weed m, dodson tb. changes in crestal bone levels in the setting of immediately loaded implants. int j oral and maxillofacial implants 2005 march-april; 21(2):253. 12. gentile m, chuang sk, dodson t. survival estimates and risk factors for failure with 6 x 5.7 mm implant. int j oral and maxillofacial implants 2005 november-december; 20(6): 930–37. 13. abadallah j, assaf a. immediate abutment placement for one-stage implant surgery. arab dental j 1999; 37:55–61. 14. coen p. placement of replace select ti-unite-coated type implants using combination of immediate and submerge technique after tooth extraction. dent j (maj ked gigi) 2006 april-june; 39(2):48–53. 15. coen p. surgical technique for achieving implant parallelism and measurement of the discrepancy in panoramic radiograph. j. oral max fac surg 2006 may; 64(5):799–803. 95 research report dental journal (majalah kedokteran gigi) 2018 june; 51(2): 95–98 differences in photodynamic therapy exposure time and staphylococcus aureus counts adeline jovita tambayong, ira widjiastuti, and cecilia g.j. lunardhi department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: the success of endodontic treatment can be achieved when pathogenic bacteria are eliminated from the root canal and periapical tissue resulting in healing of such tissue. one of the bacteria located in root canals is staphylococcus aureus (s. aureus) reportedly found to be in severe periapical abscesses. photodynamic therapy is one current technology that can help eliminate microorganisms without causing damage to human body cells. average of research has been conducted using different tools and bacteria to evaluate the effects of exposure time used in photodynamic therapy on the number of bacteria. purpose: the research reported here aimed to determine the correlation between the exposure time of photodynamic therapy and the number of s. aureus bacteria. methods: the s. aureus bacteria used in this research were divided into seven treatment groups: a control group and six treatment groups with respective exposure times of 10, 20, 30, 40, 50 and 60 seconds. all of the bacteria were administered a photosensitiser and radiated according to the treatment intended for each group. they were then planted in nutrient agar and incubated for 48 hours. the colonies of bacteria formed were calculated using the quebec colony counter and subsequently analyzed by means of both kruskal wallis and mann whitney u tests. results: after calculating the number of bacterial colonies, the average number of staphylococcus aureus bacteria in the non-irradiated group was 119 cfu/ml, 29 cfu/ml in the group with a 10-second exposure time, 20 cfu/ml in the group with a 20-second exposure time, 13 cfu/ml in the group with a 30-second exposure time, 7 cfu/ml in the group with a 40-second exposure time, but none in the groups with exposure times of 50 or 60 seconds. conclusion: the longer the photodynamic therapy exposure time, the greater the number of s. aureus bacteria eliminated. an exposure time of 50 seconds was found to be sufficient to exterminate all s. aureus bacteria present. keywords: photodynamic therapy; staphylococcus aureus; exposure time correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicine universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: ira-w@fkg.unair.ac.id introduction staphylococcus aureus (s. aureus) is a gram-positive bacterium considered to be a facultative anaerobe. concentrations of s. aureus bacteria varying between 0.7% to 15% are present in acute dental abscesses.1 thus, successful endodontic treatment may occur when these pathogenic bacteria have been eliminated from the root canal and periapical tissue, resulting in healing of the latter.2 various procedures have actually been undertaken to achieve successful endodontic treatment, such as root canal preparation, irrigation material application and intrachannel medication. nevertheless, they fail to ensure the eradication of bacteria present in the root canal system which, if allowed to remain there, will trigger re-infection and render root canal treatment ineffective.3 consequently, root canal preparation and irrigation are performed to remove dead and infected vital tissue as well as forming the root canal in order that it can be cleared easily and obturated effectively. the processes of preparation and microbiological irrigation are intended to exterminate and eliminate microorganisms in root canals.4 the most widely-used irrigation materials include sodium dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p95–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p95-98 96tambayong, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 95–98 chloride (naocl) and chlorhexidine. however, they are unable to destroy all pathogenic microorganisms. the smear layer formed after root canal preparation can actually reduce the effectiveness of the disinfectant agent, thereby allowing potential re-infection and causing the failure of root canal treatment. moreover, the formation of narrow root canals makes it difficult for all dental surfaces to be irrigated.2 therefore, new methods of endodontic treatment are required to facilitate and improve treatment success. photodynamic therapy (pdt) is the latest technology employing photoactivated disinfection to destroy pathogenic microorganisms during endodontic treatment. pdt was first introduced for cancer treatment. however, given the increasing cases of bacterial resistant to antibiotics, pdt technology has subsequently been developed to eliminate bacteria. various microorganisms can be destroyed by pdt without proving toxic to the surrounding tissue2,5 and this form of therapy has also recently been used as a root canal disinfectant.6 pdt consists of three components, namely: light source (photoactivation), photosensitiser material and oxygen.7 this therapy is used after mechanical preparation and chemical irrigation and prior to obturation, while it may or may not be accompanied by intracanal medication.8 pdt is also known to have the advantages of greater selectivity in the destruction of bacteria, with the result that it does not induce bacterial resistance, and ease of use. several cases of research have even shown a dose of photoactivation administered to destroy bacteria to be lower than one causing damage to keratinocyte cells and fibroblasts.5 extensive research has been conducted to determine the ability of pdt to destroy various bacteria. fotosan, a photosensitiser tool with a wavetime of 380–450 nm, is known to be capable of killing gram-positive and negative bacteria, such as s. mutans and e. faecalis.3 moreover, the research conducted by arneiro suggests that pdt is effective in reducing e. faecalis bacteria in the root canal. consequently, pdt is considered an appropriate disinfectant material in endodontic treatment.9 for these reasons, the research reported here aimed to investigate the correlation between pdt exposure time and s. aureus counts in order to reveal the effects of pdt exposure time on the number of s. aureus bacteria. materials and methods s. aureus bacteria were employed as samples for the purposes of this research, being divided into seven groups: a control group and six treatment groups with respective exposure times of 10, 20, 30, 40, 50 and 60 seconds. each group consisted of six samples. the s. aureus bacteria culture provided by the laboratory of microbiology, faculty of dentistry, universitas airlangga was standardized using mc farland 1.5  108 cfu/ml. then, 0.5 ml of the culture was drawn by means of micropipette and inserted into each of the 42 eppendorf tubes whose walls had been coated with black insulation in order to approximate the conditions inside the tubes to those within opaque root canals.10 this research utilised a pdt tool manufactured by fotosan 630 (cms dental aps, copenhagen denmark) consisting of activation rays and a photosensitiser liquid. in group i (control), the test tubes were not subjected to the photosensitiser and radiation, while for the other groups the test tubes were exposed to the photosensitiser for one minute, before being radiated for a precise exposure time based on the specific treatment designed for each group. subsequently, each group was inoculated and grown in petridish nutrients while incubated for 48 hours at 37°c in an anaerobic atmosphere. the number of bacterial colonies in each group was then calculated using the cfu method with a quebec colony counter. the results obtained were tested for their normality (distribution of abnormal data) and the differences between groups evaluated by means of kruskal wallis and mann-whitney u tests. results the effects of pdt exposure time on the number of s. aureus bacteria studied during this research were illustrated by the average number of s. aureus colonies as shown in table 1 and the bacterial colony growth as presented in figure 1. in order to evaluate the difference between groups, a kruskal wallis test was performed the results of which indicated there to be a significant difference in the number of s. aureus bacteria colonies among all treatment groups with a p value of 0.000 (p<0.05) furthermore, a mann whitney u test was conducted to determine the differences between two groups within the entire research population. the results of the mann whitney u test are contained in table 2. discussion a variety of measures have been taken to ensure the success of endodontic treatments, such as root canal preparation, irrigation material usage and intrachannel medication. however, such measures still do not guarantee the elimination of bacteria in the root canal table 1. the average number of s. aureus bacterial colonies after exposed to pdt groups n x sd control 6 119.17 8.976 10 seconds 6 28.67 1.633 20 seconds 6 19.83 1.472 30 seconds 6 12.67 1.862 40 seconds 6 6.50 1.871 50 seconds 6 0.00 0.000 60 seconds 6 0.00 0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p95–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p95-98 97 tambayong, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 95–98 system.11 furthermore, the use of chemicals such as naocl or chlorhexidine cannot eradicate all pathogenic microorganisms. a smear layer formed after root canal preparation can reduce the effectiveness of the disinfectant to the extent that re-infection by the bacteria and consequent failure in root canal treatment remain as possibilities. small root canals can also produce toughness in all tooth surfaces to be irrigated.2 technological developments in endodontic treatment have involved the use of pdt as a disinfectant. pdt is applied after mechanical and chemical irrigation preparation which serves to eliminate pathogenic bacteria and consists of three components: a light source (photoactivation), photosensitiser material and oxygen.5,7 if the photosensitiser or photoactivation are used separately, there will be no antimicrobial effect.8 the mean value obtained indicated the number of bacteria capable of surviving pdt application. the results of the difference test subsequently revealed there to be a significant difference between the treatment groups (p<0.05). this result is consistent with research conducted by de oliveira which posited that pdt can help to eliminate microorganisms in root canals.5 when photosensitiser is administered to the root canal, the photosensitiser containing phenothiazines will bind to the bacterial cell wall. this occurs because phenothiazines are positively charged (cation), while the bacterial cell wall is negatively charged (anion). both will bind to produce an electrostatic interaction causing the release of ca2+ and mg2+ ions located on the bacterial wall which, in turn, results in increased permeability of the bacterial cell wall. such increased permeability will cause the photosensitiser to diffuse into the plasma membrane and the cytoplasm into bacterial dna. consequently, on completion of this process, photoactivation is conducted which provokes formation reactions of ros and singlet oxygen. the ros and singlet oxygen generated from the process can then produce cytotoxic effects in the bacteria, while also causing various problems, including: elongation of crosslink plasma membrane proteins, inactivation of succinate nadh enzymes and lactate dehydrogenase, reduced balance between k+ ions and other ions, as well as that destruction of bacterial cell dna that leads to death table 2. results of the mann whitney u between the groups groups 10 seconds 20 seconds 30 seconds 40 seconds 50 seconds 60 seconds control 0.004* 0.004* 0.004* 0.004* 0.002* 0.002* 10 seconds 0.004* 0.004* 0.004* 0.002* 0.002* 20 seconds 0.004* 0.004* 0.002* 0.002* 30 seconds 0.004* 0.002* 0.002* 40 seconds 0.002* 0.002* 50 seconds 1.000 note: *) significant difference 8 figure 1. s. aureus bacterial colonies in nutrient agar media after exposed to pdt. note: a. control group; b. group ii (10 seconds); c. group iii (20 seconds); d. group iv (30 seconds); e. group v (40 seconds); f. group vi (50 seconds); g. group vii (60 seconds). table 2. results of the mann whitney u between the groups groups 10 seconds 20 seconds 30 seconds 40 seconds 50 seconds 60 seconds 0.004* 0.002* 0.002*control 0.004* 0.004* 0.004* 0.004* 0.002* 0.002*10 seconds 0.004* 0.004* 0.004* 0.002* 0.002*20 seconds 0.004* 0.004* 0.002* 0.002*30 seconds 0.002* 0.002*40 seconds 1.00050 seconds note: *) significant difference a b c d e gf figure 1. s. aureus bacterial colonies in nutrient agar media after exposed to pdt. note: a. control group; b. group ii (10 seconds); c. group iii (20 seconds); d. group iv (30 seconds); e. group v (40 seconds); f. group vi (50 seconds); g. group vii (60 seconds). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p95–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p95-98 98tambayong, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 95–98 of the bacterial cells.12 as a result of this research, 10 seconds exposure to pdt radiation was known to produce an antibacterial effect, yet numerous bacteria survived. similarly, after 20, 30, and 40 seconds of irradiation, although the number of bacteria decreased, not all were eradicated. in contrast to these results, fotosan’s protocol states that 30 seconds of irradiation is required during endodontic treatment. indeed, the research reported here demonstrated that this period of irradiation still proved insufficient to destroy all the bacteria. these results also differ from those of a 2014 study conducted by xhevdet which stated that exposure to radiation for 60 seconds did not provide significantly contrasting results compared to those of the control group. however, during this research 50 seconds of irradiation proved sufficient to destroy all bacteria. many factors can influence the differences between these research results and those of previous investigations. variations in the tools used may have caused differences in the power and wavelength produced. the use of large-capacity photoactivations may also have produced side-effects such as thermal injuries on periodontal tissue, and they should, therefore, be used carefully.9 moreover, the photosensitivity agents used in this research varied, consisting of photosensitisers containing toluidine blue which were derived from fotosan, a class of phenothiazines that can kill bacteria at low concentrations without causing toxicity in the surrounding tissue.13 another potential factor is the use of optical fiber. fiber in photoactivation can produce a superior effect because it can help to reach difficult-to-access areas.5 thus, this research used fiber optics since they are able to irradiate the bottom of the eppendorf tube. at exposure times of 50 and 60 seconds, no live bacteria survived. this suggests that the concentrations of ros and singlet oxygen formed as a result of photosensitiser activation reactions had proved capable of eradicating all bacteria present. in other words, when photoactivation of a longer duration is performed, the reaction between the formation of ros and singlet oxygen that occurs will increase in intensity resulting in the destruction of a greater number of bacteria. this means that a longer pdt exposure time triggers the decrease in bacteria. similarly, research conducted by xhevdet confirmed that the longer the pdt exposure time, the greater the decrease in the number of bacteria.2 finally, it can be concluded that longer photodynamic therapy exposure will decrease the number of s. aureus bacteria and an exposure time of 50 seconds can destroy all s. aureus bacteria. references 1. robertson d, smith aj. the microbiology of the acute dental abscess. j med microbiol. 2009; 58(2): 155–62. 2. xhevdet a, stubljar d, kriznar i, jukic t, skvarc m, veranic p, ihan a. the disinfecting efficacy of root canals with laser photodynamic therapy. j lasers med sci. 2014; 5: 19–26. 3. maisch t, wagner j, papastamou v, nerl hj, hiller ka, szeimies rm, schmalz g. combination of 10% edta, photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro. j appl microbiol. 2009; 107(5): 1569–78. 4. haapasalo m, endal u, zandi h, coil jm. eradication of endodontic infection by instrumentation and irrigation solutions. endod top. 2005; 10: 77–102. 5. de oliveira b, aguiar c, camara a. photodynamic therapy in combating the causative microorganisms from endodontic infections. eur j dent. 2014; 8(3): 424–30. 6. diogo p, gonçalves t, palma p, santos j m. photodynam ic antimicrobial chemotherapy for root canal system asepsis: a narrative literature review. int j dent. 2015; 2015: 269205. 7. konopka k, goslinski t. photodynamic therapy in dentistry. j dent res. 2007; 86(8): 694–707. 8. sivieri-araujo g, santos lms, queiroz íoa, wayama mt, martins cm, dezan-junior e, cintra lta, gomes-filho je. photodynamic therapy in endodontics: use of a supporting strategy to deal with endodontic infection. dent press endod. 2013; 3(2): 52–8. 9. arneiro ras, nakano rd, antunes laa, ferreira gb, fontes kbfc, antunes ls. efficacy of antimicrobial photodynamic therapy for root canals infected with enterococcus faecalis. j oral sci. 2014; 56(4): 277–85. 10. mattiello fdl, coelho aak, martins op, mattiello rdl, ferrão júnior jp. in vitro effect of photodynamic therapy on aggregatibacter actinomycetemcomitans and streptococcus sanguinis. braz dent j. 2011; 22(5): 398–403. 11. bhatia s, kohli s. lasers in root canal sterilization a review. int j sci study. 2013; 1(3): 107–11. 12. basrani b. endodontic irrigation. london: springer international publishing; 2015. p. 237–51. 13. yildirim c, karaarslan e, ozsevik s, zer y, sari t, usumez a. antimicrobial efficiency of photodynamic therapy with different irradiation durations. eur j dent. 2013; 7(4): 469–73. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p95–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p95-98 65 thinking pattern of first grade students towards edentulous replacement eri h. jubhari department of prosthodontics and dental materials faculty of dentistry hasanuddin university makassar indonesia abstract many people ignore their oral hygiene and do not use denture after extraction. this may caused by their thinking pattern. thinking pattern indicates the degree of comprehension to solve a problem. this research aims to find out the thinking pattern of first year students towards edentulous replacement. the questionnaire was filled in by students. the oral cavity state of students who have edentulous was inspected. the study finds that only 3 men and 27 women had edentulous, all of them did not use denture. however, more than 96% of the edentulous and not-edentulous groups said that edentulous need denture, due to aesthetic factor. the reasons for not using denture are, for example, not enough time, not disturbed by the absence of denture, and its cost. it can be concluded that the 2005 batch students of faculty of dentistry (fkg) unhas had shown a good understanding about edentulous replacement. their views on the profits and detriments using denture are the main reasons to determine whether they will use denture. key words: thinking pattern, first grade students, edentulous replacement correspondence: eri h. jubhari, c/o: bagian prostodonsia dan ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas hasanuddin. jl. kandea no. 5 makassar, indonesia. e-mail:erihjubhari@lycos.com introduction for many people, dental health is unimportant and sometimes is ignored. this statement, however, is not right at all. stoll,1 said that teeth are the source of many physical and mental problems. from the early of one’s life, teeth can cause pain and inconvenient. first, a baby feels glad when sucked, but the happiness disappears when his teeth erupt. then, the child looks very fearful when the deciduous is extracted unless his parents explain that it is natural in growing. basically everyone will feel fear to lose part of their body including teeth. if people lose one or more of their teeth, they will be unable to chew and speak, and it will influence their aesthetic. mcguire2 said that loss of teeth can cause specific problems in brushing teeth especially at the upper or the lower parts of the edentulous and at the empty space due to the extraction. this is caused by the inadequately processed self-cleansing. the loss of teeth which is the main cause of nutrient alteration causes difficulty in chewing some food, meat, fruit and vegetable. if edentulous is not replaced immediately, general health will be disturbed. in general the loss of teeth could affect the way of speaking, because the absence of teeth will make it difficult to pronounce words.3 besides that acharya4 and franks (as cited in zarb5) said that damage of temporomandibular joint (tmj) is related with loss of teeth which further accelerates resorbtion of alveolar ridge.6 as the effects of extraction without replacement can be risky, the edentulous must be replaced with denture. however, clinically, the edentulous space is neglected without treatment. there are some cases that make people do not replace their teeth, one of them is people’s thinking pattern. some say that the loss of only one tooth especially with no inconvenienced, does not need denture. many things can affect people’s thinking pattern, attitude and behavior like income, experience, ideals, and sociocultural factors including norms and standards in family, community and especially education. education is the changing process of attitude and behavior of a man or people to establish their maturity via study, training, and ways of educating.7 the higher the education, the higher the awareness and knowledge about dental treatment. thus, prevalence of dental diseases like periodontal diseases, which are the main cause of teeth loss, can be minimized.8 based on the above explanation, it is necessary to find out the thinking pattern of students of faculty of dentistry hasanuddin university, especially the 2005 batch, as a part of community, as these freshmen are still early in their comprehension about dental hygiene. this article aims to give description about thinking pattern of first grade student of faculty of dentistry hasanuddin university towards edentulous replacement. materials and methods this descriptive study was conducted to all first year students of faculty of dentistry hasanuddin university. data was collected at the lecture room on the december 2005. 66 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 65–69 sampling was based on purposive sampling technique.9 criterion of the sample is to have at least one edentulous tooth. based on that criterion from 102 participants, 30 students were determined to be the sample of this study. the answers given are expected to inform the thinking pattern of the students towards edentulous replacement. because some respondents gave contradictory answers, interview was carried out to students. examination and interview were carried out to 30 students who stated edentulous in their questionnaire. in this study, edentulous is defined as the loss of teeth, at least one tooth, caused by periodontal diseases, caries, orthodontic treatment, and trauma. in other words, edentulous is a state without natural teeth in mouth.10 definition of thinking pattern in this study is student’s comprehension towards edentulous replacement. thinking pattern is comprehension of people to solve a problem.11 data which was descriptively analyzed by systematically explain the results of the study and then distributed data in tables. results this study shows the non-edentulous 72 students consisted of 18 males and 54 females and the 30 edentulous students having consisted of 3 males and 27 females, as can be seen from table 1 which shows respondents’ identity. table 2 shows the answers of some questions. all the answers to the question “have you replaced the edentulous with denture,” are “not use denture.” from the question “is there any family using denture,” 26 sample of edentulous and 50 of non-edentulous answer “having family using denture with various types of family relationship.” regarding the question “if your teeth are extracted, will you use dentures?” 23 edentulous and non-edentulous 69 people respectively “yes.” for the question “in your opinion, caries is better treated or extracted,” 57 students answered “caries need to be treated.” table 3 shows that 96.67% edentulous students said it is necessary to replace edentulous with denture and 3.33% considered unnecessary. while 98.61% of the nonedentulous students answered it is necessary. table 4 showed that for the edentulous group aesthetic reasons (56.67%) are the main factor to consider in using denture while both aesthetic and chewing (20%) seem to be the second important factor. while the non-edentulous students need to use denture due to aesthetic and chewing reasons (43.06%) and aesthetic reason only (22.22%). table 2. distribution of answers of the edentulous people no question answer answer answer answer y n ev ed wed ed wed t ex dcy n y n y n y n 1 have you replaced your edentulous with denture 30 2 has any your family used denture?: father mother grandfather grandmother uncle aunt cousin sibling 26 6 5 4 14 3 2 1 1 4 50 9 7 5 25 6 13 2 22 3 if your teeth are extracted, will you use dentures 23 7 69 3 4 in your opinion, caries is better treated or extracted 57 4 41 note: y: yes; n: no; ev: ever; t: treated; ex: extracted; dc: depending on the case; ed: edentulous; wed: without edentulous. on question no. 2: there were some students answered more than one family relation. table 1. respondents' identity no identity students total (students) e we 1 2 3 gender male (%) female (&) age (year) 17 18 19 20 parents occupation civil servants entrepreneur official retired miscellany 3 27 4 13 11 2 20 10 18 54 7 46 16 3 49 14 4 3 2 21 81 11 59 27 5 69 24 4 3 2 note: e: edentulous; we: without edentulous 67jubhari: thinking pattern of first grade students table 5. distribution of reasons not to use denture no reasons total 1 2 3 4 5 6 7 do not have sufficient time do not feel disturb expensive do not care about it afraid do not yet think about it orthodontic 12 7 5 3 1 2 6 total 36 note: some students answered more than one reason. table 5 showed factors that make edentulous students not use denture; first, do not have sufficient time to see dentist (12 students), do not feel disturb (7 students), and high cost (5 students). there was 1 student who feels fear using denture because of hearing complaint and inconvenience from his family. two students answered they do not care about using denture. discussion this study of thinking pattern of fresh students at faculty of dentistry hasanuddin university towards edentulous replacement shows that females more often visiting dentists than males do (table 1). however, this finding indicates that their visits are generally for extraction. this is line with stoll,1 who said that females more often visit to dentist than males do. this may be caused by the fact that men are not patient enough to observe their teeth. if caries or periodontal diseases exist, which are the main causes of tooth loss,12 they tend to be neglected. moreover, from an observation, some roots are left at their position, so they can not be identified as edentulous. besides gender, age is another factor that affects someone to treat their teeth. based on a finding cited in stoll,1 50% people who treated their teeth were younger than 45 years old, while only 26% people who are older than 65 years. in this study the effect of age to dental treatment has not been seen because the age of samples did not vary and the number of sample is very view (table 1). condition of family also determines dental health of children and adolescent especially in making decision whether to use denture. some families are of highereducation parents and others are not.13 socio-economy condition of a family can be determined by their education, occupation, and income. those factors affect each others. occupation, for instance, can be affected by education grade, or family income that can be affected by occupation.9 generally, there are some occupations which are covered by insurance subsidy; this affects someone in determining to look for best dental treatment appropriate with his finance. payment planning made someone does not make a problem out of treatment payment. all of above, it is education grade, parents income that can be known from the occupation, or the type of health insurance subsidy from the occupation, affect the family in looking for health service.14 origin town can affect someone to look for dental health service. there are areas that can be found dental health service easily (community health centre, hospital, and dentist), until the community can choose easily. on the other side, there are areas that have only a little even nothing dental health service, until complicate them to choose dental health service. in this study, majority of the respondents came from makassar which has many dental health services. table 3. distribution of answers to the question “is it necessary to replace edentulous with denture?” question edentulous without edentulous number of students number of students necessary not necessary necessary not necessary is it necessary to replace edentulous with denture? 29 96.67% 1 3.33% 71 98.61% 1 1.39% table 4. distribution of reasons to use denture no reasons to use denture edentulous without edentulous total % total % 1 2 3 4 5 6 7 aesthetic chewing phonetic aesthetic and chewing aesthetic and phonetic phonetic and chewing chewing, aesthetic and phonetic 17 1 – 6 1 1 4 56.67 3.33 – 20.00 3.33 3.33 13.34 16 9 – 31 – 1 15 22.22 12.50 – 43.06 – 1.39 20.83 total 30 100 72 100 68 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 65–69 it can be hoped that this factor is not constitute a problem for someone gaining dental health service. table 2 showed answers of some questions. the majority answers of question “will you use denture if you have edentulous?” is “yes.” this pointed that they have had comprehension towards the important using denture. but from question “have you replaced the edentulous with denture,” all samples answered “no.” if being observed from education that they are having, actually surprised, but this thing may be caused by they still have great effects from their family and environment, because they are still in the early grade. this point contradiction enough with data on table 3 that showed majority of respondents that have edentulous said “it’s necessary replace edentulous because there are many negative effect of edentulous,” for instance disturb chewing,3-5,15-17 aesthetic,4-6,15,18 and phonetic.3,15,18 behavior has an important role in affecting dental health condition, because besides of affect dental health condition directly, behavior can affect environment factor and health service.19 bloom that cited by astoeti,19 said that behavior could be divided into 3 types, it is cognitive, affective, and psychomotor. then sears et al,20 said that cognitive consists of understanding of someone about something objects, it is fact, knowledge, and conviction about object. affective consists of all feeling or emotion of someone to the object, mainly evaluation. while psychomotor consists of practice or action did by someone. from the conditions above, can be seen that majority of respondents have known the important of replacing edentulous with denture; the cognitive component plays. now, they think that replace edentulous is very useful and can avoid from negative effect of tooth loss; affective component plays. furthermore, “will they use denture?” from this study, there was no student used denture. with other words, like said by sears et al,20 that psychomotor component does not always similar with cognitive and affective. the unconformity between behavior components may be caused by some matters which considered why they do not replace their edentulous. sears et al.,20 said that behavior of someone affected by their knowledge or comprehension, but generally behavior mainly affected by all evaluation of those people, positively or negatively. a man always acts that based on the profit and the loss of behavior. in this case, the respondents understood the important using denture and feel very useful, but the other side they think that for gaining that benefit, they must sacrifice many things. they feel the profit and the loss does not comparable, so the actions be done were the more profit and nothing loss. still from table 2, the question “is there any family using denture,” majority respondents have family that using denture. people around respondents were very affecting them, to use denture or not. since born, a child had contacted with people around him. first, with his family mainly father and mother, then brother and sister, uncle, aunt, etc. in children development, family role, mainly father and mother, is very important and determine the formation of their personality in the future.13 table 3, which shows the necessity of edentulous replacement, shows that majority of edentulous and nonedentulous respondents, answered ”necessary” with vary reasons that can be seen at table 4. the main reasons are aesthetic and chewing factors. this matter similar basker’s statement that cited by kusmawati,21 it is that main motivation of a patient using denture in the first time usually aesthetic and chewing factors.5 from this point, can said that aesthetic still be the main purpose of treatment, whereas generally tooth loss causes complaint in phonetic and chewing, too. although respondents on average answer “it is necessary to replace edentulous,” but from students that having edentulous, in fact, shows there is no students using denture. the considerations of the students who did not use denture as can be seen at table 5 are as follows. first, there was no sufficient time to visit dental health service because they were busy, the health service centres were far from home, or they thought edentulous as unimportant.14 second, they felt their daily activities were not disturbed by tooth loss even they had pain and inconvenience. the pain often neglected because they think it will leave immediately. besides of that, there are people do self-treatment just to relief the pain.14 third, the fee was very expensive. this factor is still form a constraint that can not be overcome by the community. whereas many people can surpass that problem, but fee factor can not be neglected. suppose to get health insurance subsidy, many people do not exploit it because its arrangement is difficult and its administration service is not satisfied.14, 21 fourth, they did not care, and they were lazy and indifferent. this matter is a personality problem that affected by environment or follows the family. fifth, they felt fearful using denture after hearing complaint and inconvenience from denture user, then the feeling closes to children mind and develops greater if they does not gain a right and clear explanation. sixth, the effect of edentulous was not felt yet. and the seventh, the space will close in the future after orthodontic treatment. from this study, it can be concluded that majority respondents have known the important replacing edentulous. as dental students, 2005 batch have shown right comprehension towards edentulous replacement. aesthetic still be the main factor in using denture. however, behavior in using denture has not been formed because it is affected by external and internal factors. external factors are family, time, economy, education, official attitude, and environment. internal factors consist of knowledge, thinking pattern, and behavior. from those factors, time is the main external factor in using denture. while thinking pattern, that is internal factor, seen plays important role. their evaluation about profit and loss in using denture will be the main reason using denture or not. 69jubhari: thinking pattern of first grade students acknowledgement the author is indebted to miss. atri, who had participated in this study by helping collected data in questionnaire, interview, and oral examination. references 1. stoll fa. dental health education. 5th ed. philadelphia: lea & febiger; 1977. p. 17–24. 2. mcguire. tooth fitness your guide to healthy teeth. nevada: st. michaels’s press; 1994. p. 139. 3. university of texas health science center at san antonio. effects of tooth loss. 2001. available from: url: http://www.teachhealthk12.uthsca.edu/pa/pa09/pa09pdf/0906lsn.pdf. accessed november 19, 2005. 4. acharya v. save your teeth. 2004 jun. available from: http://www. hindu.com/thehindu/mag/2004/06/20/stories/2004062000410600. htm. accessed november 22, 2005. 5. zarb ga, bergman bo, clayton ja, mackay hf. prosthodontic treatment for partially edentulous patients. saint louis: the cv mosby company; 1978. p. 26. 6. merck & co. inc. mouth and dental disorders. 2005. available from: http://www.merck.com/pubs/mmanual_ha/sec3/ch39/ch39e.html. accessed november 19, 2005. 7. budiharto. perilaku kelompok masyarakat rendah di perkotaan terhadap kesehatan gigi tahun 1999. jurnal kedokteran gigi universitas indonesia 2000; 7(2):40–1. 8. carranza fa. glickman’s clinical peridontology. 9th ed. london: wb saunders company; 2002. p. 90. 9. notoadmodjo s. metodologi penelitian kesehatan. jakarta: pt rineka cipta; 2005. p. 68, 70, 88–9. 10. harty fj, ogston r. kamus kedokteran gigi. sumawinata n, editor. jakarta: egc; 1995. p. 102. 11. bono e. lateral thinking. 2006. available from: http://en.wikipedia. org/wiki/lateral_thinking. accessed february 5, 2006. 12. odusanya sa. tooth loss among nigerians: causes and pattern of mortality. int j oral maxillofac surg 1987; 16 (2): 184-9. available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retri eve&db=pubmed%list-uids=10946463&dopt=citation. accessed december 11, 2005. 13. purwanto mn. psikologi pendidikan. bandung: pt remaja rosdakarya; 2002. p. 104, 158, 161. 14. notoadmodjo s. pendidikan dan perilaku kesehatan. jakarta: pt rineka cipta; 2003. p. 10, 13, 121, 124–5, 179, 195–6, 202. 15. gordon j. missing teeth? weigh your options. available from:http:// www.dentalcomfortzone.com/archive/missingteeth.html. accessed november 22, 2005. 16. ferreira l. teeth in a day. 2005. available from http://www. dentalimplants-usa.com/misc?newsrelease/naturalhealthweb/ naturalhealthweb.html. accessed november 22, 2005. 17. hutton b, feine j, morais j. is there an association between edentulism and nutritional state?. j can dent assoc 2002; 68(3):182-3. 18. prajitno hr. ilmu gigitiruan jembatan. jakarta: egc; 1991. p. 2. 19. astoeti te, boesro s. pengaruh tingkat pengetahuan terhadap kebersihan gigi dan mulut murid-murid sdn dki jakarta. jurnal kedokteran gigi universitas sumatera utara 2003; 2(8):149. 20. sears do, freedman jl, peplau la. psikologi sosial. adryanto m, soekrisno s, editors. jakarta: erlangga; 1994. p. 14, 138–41. 21. azwar a. pengantar administrasi kesehatan. 3rd ed. jakarta: binarupa aksara; 1996. p. 157–8. guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews 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author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 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signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month 1 year 2 years surabaya q rp80.000,00 q rp160.000,00 q rp320.000,00 java island (pulau jawa) q rp90.000,00 q rp180.000,00 q rp360.000,00 outside java island (luar pulau jawa) q rp100.000,00 q rp200.000,00 q rp400.000,00 other countries (negara lain) q us $ 27 q us $ 54 q us $ 108 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 033-01-11343-16-0 name of bank : bank niaga cabang dharmahusada name of beneficiary : drg. sianiwati goenharto " � vol. 45. no. 1 march 2012 orthodontic treatment considerations in down syndrome patients sianiwati goenharto department of orthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: down syndrome is an easily recognized congenital disease anomaly, a common autosomal chromosomal anomaly with high prevalence of malocclusion. orthodontic treatment demand should be high but it seems difficult to be done because of specific condition of disability. purpose: the purpose of this literature review was to discribe the orthodontic problems found in down syndrome patients and several consideration that shoud be done to treat them. reviews: many studies report the high prevalence of malocclusion among people with down syndrome. there is a greater frequency of clas iii relationship, crossbite, crowding and also open bite. several problems might appear in the treatment because of dental, medical, mental, and behavioural factor. conclusion: it is concluded that orthodonic treatment can be performed in down syndrome patient, although several difficulties may appear. good consideration in mental, behavior, medical and also dental condition will influence whether the treatment will success or not. special care and facilities will support the orthodontic treatment. key words: down syndrome, orthodontic, malocclusion abstrak latar belakang: sindroma down adalah suatu kelainan congenital yang mudah dikenali, merupakan kelaian kromosom autosomal yang cukup banyak terjadi, dengan prevalensi maloklusi cukup tinggi. seharusnya permintaan akan perawatan ortodonti juga tinggi meskipun tampaknya sulit dilakukan karena adanya kondisi ketidakmampuan/cacat yang spesifik. tujuan: tujuan studi pustaka ini adalah untuk menggambarkan problem perawatan ortodonti pada penderita sindroma down dan pertimbangan apa yang sebaiknya diambil untuk mengatasi masalah tersebut. tinjauan pustaka: banyak penelitian melaporkan tentang prevalensi maloklusi yang tinggi pada penderita sindroma down. maloklusi yang sering dijumpai adalah relasi klas iii, gigitan silang, berdesakan dan juga gigitan terbuka. problem dapat terjadi saat perawatan ortodonti karena adanya faktor dental, medis, mental dan tingkah laku penderita. kesimpulan: disimpulkan bahwa perawatan ortodonti masih dapat dilakukan pada penderita sindroma down meskipun dengan beberapa kesulitan. pertimbangan mental, tingkah laku, kondisi medis sistemik dan kondisi dental, akan mempengaruhi hasil perawatan. tindakan dan fasilitas khusus diperlukan untuk menunjang suksesnya perawatan ortodonti pada penderita sindroma down. kata kunci: down syndrome, ortodonti, maloklusi correspondence: sianiwati goenharto, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: sianiwati.goenharto@yahoo.id literature review introduction down syndrome is the most common clinically recognizable syndrome characterized by generalized physical and mental deficiencies. it was firstly reported in 1866 by john l. down and the prevalence is variative, in between 1 in 7501 and 1 in 1250 live births.2 statistic in united states showed an increasing prevalence, currently observed at 11.8 per 10,000 births.3 the prevalence increases with the age of the mother.4 in indonesia, �goenharto.: orthodontic treatment considerations in down syndrome patients nowadays there are approximately 300,000 people with down syndrome, whereas in jawa timur every year there are 689 down syndrome patients. in 1959 lejeune, gautier dan turpin found the association between down syndrome and chromosome 21.5 normal person has 46 chromosomes, but 95% down syndrome patients have extra chromosome #21. nondisjunction problem makes total chromosome become 47; therefore it is also known as trisomy #21.6-8 there is a typical mongoloid appearance and virtually all patients have learning disabilities.7 forty percents of down syndrome patients can be accompanied with congenital heart problem such as mitral valve prolapse (mvp). other medical conditions are upper respiratory tract infections, hepatitis, leukemia and even that 100% of person with down syndrome over 35 years old develop neurological signs of alzheimer disorder. besides, there are still problem of speech and hearing, eye problem, and epilepsy.4 the prevalence and the variety of malocclusion on down syndrome patients have already been studied. study on 112 subjects with down syndrome was found 92% of the subjects had malocclusion. class iii malocclusion was most frequently observed (43.8%). crowding and unilateral cross bite were found in 15% of the subjects respectively. bilateral cross bite was present in 5.4% of the subjects.9 study on 57 children with down syndrome in mexico also found that the prevalence of anterior open bite was 31.6%.10 down syndrome patients are like other normal children, who need orthodontic treatment. parents of down syndrome children in france often have difficulties to access oral health care and the patients are less likely to receive dental services.11 on the other hand, down syndrome is categorized to a person with disabilities, so they need special management. down syndrome often accompanied with systemic problem besides the malocclusion itself which require special attention. the systemic disease can influence the orthodontic treatment, but the orthodontic treatment can worsen the general condition. another problem is the mental retard condition and behavior disorder that can disturb the communication between the patient and dentist. motivation to treatment usually not come from the patient but from the parents, so support from the surrounding people should be always provided. because of that, several considerations must be taken during the treatment so the goal can be achieved. the purpose of this literature study was to describe the problems of orthodontic treatment for down syndrome patients and several considerations that should be taken to overcome the problem, so treatment result can be obtained and the patient can get a better life. dental problem on down syndrome patients generally, down syndrome patients have several dental problems. it is important how they can get good oral hygiene. study about the oral health condition of individuals with down syndrome in nigeria showed that the subjects had poorer oral hygiene, with no significant sex difference. this condition need frequent oral health assessment and routine treatment is expected to be done.12 study of dental treatment needs of 300 children (aged 9–13 years) with disabilities in melbourne found that 41 % of them required simple treatment; including caries treatment, periodontal therapy and oral health promotion.13 dental anomaly is often found in down syndrome patient. the incidence is about 73%. congenital missing teeth is about 10x times more common in individuals with down syndrome than in general population.14,15 with a higher frequency in males than in females. agenesis occurred more frequently in mandible than in maxilla and most oftenly on the left side. the main components in the pattern of agenesis observed in down syndrome are supposed to be related to the peripheral nervous system and abnormal cartilagenous tissue.15 research through longitudinal panoramic radiograph in canada showed that hypodontia was found in 92% of samples when third molars were considered and in 56% when third molars were not considered. the most frequently agenetic teeth were maxillary and mandibular third molars, maxillary lateral incisors, mandibular second premolars, mandibular incisors, maxillary second premolars, and maxillary second molars. hypodontia was more prevalent and severe in females.3 another study in netherland found 59.6% of 114 down syndrome patients had missing teeth. absence of both mandibular central incisors was a high predictor for oligodontia. congenital heart disease and hypothyroidism are parameters involved in tooth agenesis.16 the incidence of canine impaction is ten times higher than normal individual.17 delayed eruption of teeth is often occured, with an abnormal sequence. primary teeth may not appear until the age of two, with complete dentition delayed until age 4 or 5. some primary teeth are then retained in some children until they are 14 or 15.6 abnormality in tooth formation such as microdontia (35-55%) is also seen in people with down syndrome. crowns tend to be smaller, and roots are often small and conical, which can lead to tooth loss from periodontal disease. severe illness or prolonged fevers can lead to hypoplasia and hypocalcification.6,18 taurodontism is also found in down syndrome patients with the prevalence of 0,54–5,6%.19 it is characterized by a large pulp chamber and it is most commonly seen in molars.20 malocclusion on down syndrome patients compare with general population, malocclusion in down syndrome patients is more frequent, more severe and more skeletally based which can adversely affect functions.21 malocclusion is often found in down syndrome patients particularly because of delayed eruption of permanent teeth and underdevelopment of maxillary arch, leading to poor positioning of teeth.6 study on 136 chilean children with down syndrome, showed a higher frequency � dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 6–11 of malalignments in both the deciduous and permanent dentitions. the frequency was higher in the permanent teeth than in deciduous dentition. higher frequency of malalignment was found in the upper central incisor, lateral incisor, and canine regions.22 the maxilla of down syndrome patients showed hypoplasia in the vertical plane and the sagittal plane. sagittal maxillary growth is relatively constant from the age of 8 to 18 years, with an average increase of 0.12 mm/year measured at the level of point a. in the vertical plane it grows at an average rate of 0.62 mm/year and 0.70 mm/year, measured at the level of the anterior nasal spine and posterior nasal spine respectively.2 down syndrome may affect the size and shape of the palate. palatal length, width, and height were significantly influenced.23 the palate of down syndrome patients may appear highly vaulted and narrow, due to the unusual thickness of the sides of the hard palate. this thickness restricts the amount of space the tongue can occupy in the mouth and affects the ability to speak and chew.18 this v shaped palate is caused by deficient development of the midface, and it affects the length, height, and depth of the palate, but rarely affects the width.6 cephalometric measurement held in canada showed the reduced dimension height of maxilla and mandible, short teeth, maxilla and mandible, forward rotation of maxillary and mandibular plane that lead to deep bite and mandibular prognatism.3 the characteristic of muscle hypotonia is found on perioral muscles, lip and chewing muscles and a protruding tounge, followed by active tounge protrusion or tounge thrush.24 a small oral cavity with a relatively large and fissured tongue leads to mouth breathing, which is a common cause of chronic periodontitis, xerostomia,18 drolling, angular cheilitis and halitosis.6 the prevalence of malocclusion on down syndrome patients is quite high. meštrović et al.,9 found 92% children with down syndrome had malocclusion, whereas study on 112 down syndrome patients in rio de janeiro, brazil showed a prevalence of malocclusion was 74%; cause by vertical and transversal occlusal alteration. age, nail or finger biting habit, mouth posture, and cold or sore throat were the variables associated with the peavalence of malocclusion in these subjects.25 maxillary anteroposterior hypoplasia makes 54% of down syndrome patients have angle class iii tendencies.17 craniofacial dysplasia that has already occured at birth, become more severe with increasing age. an anterior open bite and class iii malocclusion may be due to proclination of the incisor, under-development of the maxilla and a more anterior position of the hypoplastic mandible.26 posterior crossbite occurred in 65% of patients due to maxillary transverse hypoplasia. a smaller maxilla also contributes to an open bite, poor positioning of the teeth and increases periodontal disease and dental caries. this finding is accordingly with study of meštrović et al.,9 who found class iii malocclusion, crowding and anterior or posterior cross bite. systemic and mental problems in down syndrome patients about 40-50 % of the children with down syndrome are born with a congenital heart disease, but most of them had received surgical correction within the first few years of life. the decrease in number of t cells can affects the immune system and contributes to a higher rate of infections and makes greater incidence of periodontal diseases, stomatitis, oral candida infection and gingivitis.19 history of the patients should be examined thoroughly. patients can be asked to fill the questionnaire about their general health, because down syndrome is often related to other health problems such as epilepsy, diabetes, leukemia, hypotiroidism,19 and also seven times higher as hepatitis virus carrier.6 down syndrome patients have variation in intelligence condition. there are some patients with an iq above 69, but the most typical is moderate or severe retardation (iq 20-50).4 in early infancy they are in the range of low typical development, iq decreases in the first decade of life and in adolescent years cognitive function reaches a plateau that continues into adulthood. children with down syndrome have more behavioral and psychiatric problems than other children. there are 17.6% of individuals with down syndrome aged less than 20 years have a psychiatric disorder, most frequently a disruptive behavior such as hyperactivity disorder, oppositional disorder or aggressive behavior. firstly, the parents are disappointed when they found their newborn is diagnosed with down syndrome. however, within a few months, they become attached, because child with down syndrome is happier and more loveable.5 other problems are speech and language difficulties that affect communication, although research showed that there in no association existed between speech disorders and anterior open bite in down syndrome mexican children.10 there is individual variation in the delay of language acquisition in children with down syndrome. patients have great difficulty in communicating with people who do not know them well and their ability to develop the relationship with society is limited. parents, patient’s family or caregiver will be able to help reaching better communication with the patients. it is important that the dentist communicate directly with the patient in order to build a level of trust to improve their confidence. maybe it will need more time for explaining the orthodontic procedure, but if down syndrome patients has already willing to be treated and trust the operator, they can be cooperative.19 discussion a child with down syndrome is a handicapped child with disabilities and special needs. actually there should be a high orthodontic treatment need for down syndrome patients because of an increased prevalence and severity of malocclusion.21 nevertheless, orthodontic services for �goenharto.: orthodontic treatment considerations in down syndrome patients the handicapped have generally been neglected,27 although handicapped children including down syndrome patients are able and willing to undergo orthodontic treatment.28 orthodontic treatment held in special need child is different with normal child; especially if the child has systemic disorder and mental problems. the treatment procedures need to be simplified, so it will be possible to be done. treatment objective for down syndrome patients should be the same as that of normal patients, although the treatment plan may need to be adapted to each individual’s condition.29 it is often found that the parents have highly motivated to seek the orthodontic treatment for their children, because they want the best for their children i.e. better appearance, better oral hyhiene to gain better quality of life. motivation from the patiens themselves is often not clear. it must be realized that there is still possibility of treatment failure due to patient’s limitation. in this condition, maybe the objective must be changed from ideal condition to compromise, only to achieve esthetic improvement in order the child can be easily accepted in social community.30 another treatment objective is to overcome the narrow palate which can influence the general health. it is reported that there was positive improvement on oral motoric and articulation function31 and also there was significant orofacial function increase after treatment with palatal plate.32 to several persons, orthodontic treatment will be beneficial, but to another persons may be not. nevertheless, down syndrome is not always a contra indication to orthodontic treatment. orthodontic treatment still can be done in selected patients. if it has been decided to do orthodontic treatment, attention must not directed towards the malocclusion itself, but several considerations and good care should be taken particularly if accociated with systemic, mental and behavior condition of the patients. during orthodontic treatment there may be systemic condition that affects the oral environment. child’s health history and clinical descripsion should be well recorded and remembered to acknowledge the sensitivity of the patients. about half of the children with down syndrome are born with a congenital cardiac anomaly.14 orthodontist should be aware about the sign and symptom manifestation in oral cavity that probably associated with specific disease. sometimes multidiciplinary approach is necessary to prevent bacterial endocarditis. there are always some possibilities of infections such as upper respiratory tract infections, hepatitis, epilepsy, leukemia, and other conditions, so medical consultation with the internist and pediatrist should always be done. premedication like antibiotic prophylaxis, vitamin and improved immune response are needed to be done. mental condition of the patients may be the most important factor that influences orthodontic treatment procedures. parent’s agreement may be advantageous to support their psychological condition. parent’s ability to collaborate with the orthodontist is usually helpful for successful completion of treatment.28 it is important to always remember the wise advice that: “a person with a disability is still just a person who deserves to be treated with dignity and respect. we should never assume someone doesn’t understand what we are saying because they don’t speak as well as we do”.17 based on that advice, better communication with the patients should be achieved. the hearing loss experienced by many of these individuals should be taken into consideration when communicating with the patient. good doctor-patient relationship will increase the confidence level of the patients to overthrough the difficult time during orthodontic treatment. behaviour disorder is often a problematic in treating down syndrome patients because of lack of understanding, increased apprehension, short attention, and limited tolerance.21 communication to the patients with down syndrome who usually has intelegency below average should be always developed, so good doctor-patient relation can be achieved. to overcome the communication problem, every phase shoud be done slowly, in order not to shock the patients. every step that will be done should be explained clearly with tell-show-do technique, so the children brave enough to experience the treatment. in every step, operator should always think about the comfort of the patients. maybe they require more chairside time and there will be an increased number of appointments. orthodontic treatment can be done if the patient is cooperative. this behavior is needed because orthodontic treatment is multivisit and need a long periods. the patients should keep their appointment to control, keep their oral hygiene well, and avoid hard and sticky child.28 patient’s ability to keep their oral hygiene is required to gain good treatment result. it is advised to take frequent drink or oral rinse to minimize the dry mouth sensation. caries frequency in down syndrome patients can be minimized with preventive measures such as fluoride topical application, fissure sealant, fluoride tooth paste suggestion and non cariogenic food and beverage consumption. parents and caregivers play an important role in keeping good oral hygiene, maintaining the diet and consumption pattern, and going to the dental office to get routine treatment.6 failure in keeping oral hygiene can increase the possibility of gingival hyperplasia, periodontal disease, and bacterial endocarditis risk. orthodontic treatment is started with impression and radiographic taking. the first problem is how to get the study models. down syndrome patients often have strong gag reflex due to tounge condition (macroglossia) and anciety or phobia. this condition usually can be overcome with simple explanation and good communication.21 impression must be taken as quick as possible, with fast set type or low viscosity impression material. this kind of material needs skillful and fast working operator. failure in the first impression may increase the difficulty to get the second impression and so on. if it is not possible, impression can be taken under conscious sedation, intravenous sedation or even with general anesthesia. �0 dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 6–11 panoramic radiograph is considered to be useful for orthodontic assessment. however, it requires patient’s cooperation, the patients stay still minimally during the rotation of the tube of the panoramic radiographic machine. down syndrome patients often have difficulty to stay still because of uncontrolled head and limb movements. problems will also arise if frightened child shoud be restricted in a cephalostat since it will increase their fear and even generate panic. periapical photo maybe more difficult to obtain since the film must be held intra orally during the x-ray exposure and this procedure may be uncomfortable and increase the anciety of the patients. in certain cases, computed tomography (ct) scan can be performed under sedation.21 generally, orthodontic appliance is bulky, uncomfortable and painful. on the other hand, the appliance needs day-today maintenance.28 these conditions will make the patients reluctant wearing the appliance. the treatment should be planned in a simple way, not in a hurry to reach the goal, slowly but sure to get the improvement. it was reported that orthodontic treatment problems with fixed appliances were more difficult than with removable appliances. treatment period with fixed appliance was harder on 47% patients, compared with 11.8% removable appliance users.28 the problems of wearing removable orthodontic appliances are mastication and speech difficulties. it is beneficial that adjustment of removable orthodontic appliances can be done extra orally. furthermore, oral health maintaining is better with removable orthodontic appliances. successful orthodontic treatment with removable orthodontic appliances has already reported by becker and shapira.33 in severe malocclusion cases, combination treatment with fixed appliance may be needed. from the doctor’s point of view, orthodontic treatment with fixed appliance is hard to be done. the patients should sit in dental chair for a long enough periods of time and the teeth must be dry during bracket placement. sedation or general anesthesia will make the procedures easier to be done, as started by jackson in 1967.30 the problem is that the equipments for sedation are rarely provided in dental private practice. it should be done in a clinic or more sophisticated hospital. it will be easier if bracket placement is done indirectly, since it can be accurate and the process will be less time consuming. thus, the most time consuming step shoud be done out of the mouth including steps in dental laboratory. light forces should be given to move the teeth. it’s not advisable to give heavier forces, since it will make the patient more uncomfortable. straight wire technique can be chosen because it has minimal wire changes. treatment with self ligating bracket is also beneficial since it can reduce the visiting time and no ligature wire or elastomeric modules make teeth brushing easier to be done. maintaining oral hygiene is more difficult with fixed than with removable orthodontic appliances. because of that reason, it is recommended for down syndrome patients to wear removable orthodontic appliance rather than fixed appliances. fixed appliances should be worn only in limited period. besides of that, bonding procedure must be done correctly so it is not easily debond. replacement of bracket will difficult and need more time.21 activation, wire changing, and other procedures can give unpleasant sensation because of many instruments used inside the mouth. fixed appliance is the only choice of treatment for non cooperative down syndrome patients, since the patients are unable to remove the appliance by their own efforts. nevertheless, the immature condition can make the patients do something that damage the appliance, so it can not work properly and even hurt the oral mucosa. after active treatment has finished, retention period is needed in order to maintain the treatment result. in children with skeletal discrepancies, or with large tongue, non elimited bad habits, treatment result can not be maintained and tends to relapse. to achieve long period of stability, removable retainer can be used, but it depends on the willingness of the patient to wear it. retainer such as hawley retainer, wraparound, or clear retainer can be used.34,35 if the cooperation is doubtful, it is better to use permanent retainer such as bonded lingual retainer. in cases with hypodontia, if there are still residual spaces after the treatment is completed, prosthodontic approach is needed for planning the suitable denture. succsessful orthodontic treatment is not only increase the estethic factor but also improve the other function including swallowing, speech and mastication. failures can occur because of unpredicted condition such as: the patients become ill, uncontrolled behavior and inadequate oral hygiene.21 it is recommended to remove the appliance in these conditions, although the treatment result has not achieved yet in order to prevent further negative effects. it is concluded that orthodontic treatment still can be done on selective down syndrome patients, although some difficulties may occur. medical, mental and behavior condition besides the malocclusion itself will affect the treatment result. several considerations, skillful orthodontist and chairside, specific effort and facilities such as sedation equipment and well trained operator are needed to gain the treatment objectives. references 1. kliegman rm, behrman re, jenson hb, stanton bf. nelson textbook of pediatrics. 18th ed. philadelphia: saunders; 2007. p. 507–9. 2. alió j, lorenzo j, iglesias mc, manso fj, ramírez em. longitudinal maxillary growth in down syndrome patients. angle orthod 2011; 81(2): 253–9. 3. suri s, tompson bd, atenafu e. prevalence and patterns of permanent tooth agenesis in down syndrome and their association with craniofacial morphology. angle orthod 2011; 81(2): 260–9. 4. southern association of institutional dentist (said). down syndrome: a review for dental professionals. self-study course. module 3. available from: http://www.saiddent.org/modules/ 11module3.pdf. accessed july 12, 2011. ��goenharto.: orthodontic treatment considerations in down syndrome patients 5. roizen nj, patterson d. down’s syndrome. the lancet 2003; 361: 1281–9. 6. national institute of dental and craniofacial research (nidcr). practical oral care for people with down syndrome. available from: http://www.nichd.nih.gov nichdinformationresourcecenter@ mail.nih.gov. accessed august 15, 2011. 7. scully c, welbury r, flaitz c, de almeida op. a color atlas of orofacial health and disease in children and adolescents. 2nd ed. st louis: martin dunitz; 2002. p. 214. 8. cawson ra, odell ew. cawson’s essentials of oral pathology and oral medicine. 7th ed. edinburg: churchill livingstone; 2008. p. 419–21. 9. me≥trovi s, mik≥i m, ≠tefana -papi j, stipetic j. prevalence of malocclusion in patients with down’s syndrome. acta stomatologica croatica 2002; 36(2): 239–41. 10. lopez-perez r, borges-yanez a, lopez-morales m, anterior open bite and speech disorders in children with down syndrome. angle orthod 2008; 78(2): 221–7. 11. allison pj, hennequin m, faulks d. dental care access among individuals with down syndrome in france. special care in dentistry 2000; 20(1): 28–34. 12. oredugba fa. oral health condition and treatment needs of a group of nigerian individuals with down syndrome. down syndrome research and practice 2007; 12(1): 72–6. 13. desai m, messer lb, calache h. a study of the dental treatment needs of children with the abilities in melbourne, australia. aust dent j 2001; 46(1): 41–50. 14. norderyd j, kling-karlberg m. orthodontic treatment strategies in five patients with down syndrome, congenital heart defect, and oligodontia. available from: www.lj.se/index.jsf?childid=4748&no deid=25819&nodetype. 15. russell bg, kjær i. tooth agenesis in down syndrome. arch oral biol 1993; 38(1): 85–9. 16. reuland-bosma w, reuland mc, bronkhorst e, phoa kh. patterns of tooth agenesis in patients with down syndrome in relation to hypothyroidism and congenital heart disease: an aid for treatment planning. am j orthod dentofacial orthop. 2010; 137(5): 584–9. 17. musich dr. orthodontic intervention and patients with down syndrome: the role of inclusion, technology, and leadership. angle orthod 2006; 76: 734–5. 18. desai ss, flanagan tj. orthodontic considerations in individuals with down syndrome: a case report. angle orthod 1999; 69(1): 85–8. 19. pilcher es. dental care for the patient with down syndrome. down syndrome research and practice 1998; 5(3): 111–6. 20. nawa h, oberoi s, vargervik k. taurodontism and van der woude syndrome. is there an association?. angle orthod 2008; 78(5): 832–7. 21. becker a, chaushu s, shapira j. orthodontic treatment for the special needs child. semin orthod 2004; 10: 281–92. 22. ondarza a, jara l, bertonati mi, blanco r. tooth malalignments in chilean children with down syndrome. j periodontol 1993; 55(4): 466–71. 23. dellavia c, sforza c, orlando f, ottolina p, pregliasco f, ferrario vf. three-dimensional hard tissue palatal size and shape in down syndrome subjects. eur j orthod 2007; 29(4): 417–22. 24. limbrock gj, fisher-brandies h, avalle c. castillo-morales orofacial therapy: treatment of 67 children with down syndrome. developmental medicine & child neurology 1991; 33(4): 296– 303. 25. oliveira acb, paiva sm, campos mr, czeresnia d. factors associated with malocclusions in children and adolescents with down syndrome. am j orthop dentofac orthop 2008; 133(4): 489. 26. fisher-brandies h, cephalometric comparison between children with and without down’s syndrome. eur j orthod 1988; 10 (1): 255–63. 27. rao db, hegde am, munshi ak. malocclusion and orthodontic treatment need of handicapped individual in south canara, india. international dental journal 2003; 53: 13–8. 28. becker a, shapira j, chaushu s. orthodontic treatment for disabled children-a survey of patient and appliance management. j orthod 2001; 28: 39–44. 29. khan r, abdallah ai, antony vv. down syndrome: a case report. the orthodontic cyber journal 2009 january. 30. chadwick sm, asher-mcdade c. the orthodontic management of patients with profound learning disability. br j orthod 1997; 24: 117–25. 31. backman b, grever-sjolander ac, holm ak, johansson i. children with down syndrome: oral development and morphology after use of palatal plates between 6 and 18 months. int j paediatr dent 2003; 13(5): 327–35. 32. carlstedt k, henningsson g, dahllof g. a four-year longitudinal study of palatal plate therapy in children with down syndrome: effects on oral motor function, articulation and communication preferences acta odontol scand 2003; 61(1): 39–46. 33. becker a, shapira j. orthodontics for handicapped child. eur j orthod 1996; 18: 55–67. 34. yanez eer, white l, araujo rc, galuffo amg, yanez ser. 1001 tips for orthodontics and its secrets. miami: amolca; 2007. p. 312–51. 35. staley rn, reske nt. essentials of orthodontics diagnosis and treatment. oxford: wiley-blackwell; 2011. p. 239–52. p-issn: 1978-3728 e-issn: 2442-9740 volume 53, number 4, december 2020 editorial team of dental journal (majalah kedokteran gigi) sk: 07/un3.1.2/2020 january 2nd – december 31st, 2020 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: saka winias, drg., m.kes., sp.pm (department of oral medicine, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); aulia ramadhani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); beta novia rizky (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); irna sufiawati (deparment of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); sianiwati goenharto (vocational faculty, universitas airlangga, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); desiana radithia (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); titiek berniyanti (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); dini setyowati (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); eha renwi astuti (department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia); alida (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 53, number 4, december 2020 p-issn: 1978-3728 e-issn: 2442-9740 1. the relationship between dental fear, anxiety and sociodemography in jakarta, indonesia lisa prihastari, rima ardhani iswara, ghina al afiani, fajar ramadhan, mega octaviani, willy anugerah hidayat, muhammad al faqih and ahmad ronal .......................................... 175–180 2. effect of different final irrigation solutions on push-out bond strength of root canal filling material rahmatillah, isyana erlita and buyung maglenda ...................................................................... 181–186 3. burning mouth syndrome caused by xerostomia secondary to amlodipine tengku natasha eleena binti tengku ahmad noor .................................................................... 187–190 4. orthodontic camouflage treatment using a passive self-ligating system in skeletal class iii malocclusion fransiska monika and retno widayati ......................................................................................... 191–195 5. acceleration of post-tooth extraction socket healing after continuous aerobic and anaerobic physical exercise in wistar rats (rattus norvegicus) aqsa sjuhada oki, moch febi alviansyah, christian khoswanto, retno pudji rahayu and muhammad luthfi ................................................................................................................... 196–200 6. maxillary anterior root resorption in class ii/i malocclusion patients post fixed orthodontic treatment fransiska rima tallo, ida bagus narmada and i. g. a. wahju ardani ................................... 201–205 7. electronic application for oral health school programme enhances the quality of the information in dental health data records nurul fatikhah, gilang yubiliana and fedri ruluwedrata rinawan ........................................ 206–211 8. combining 10% propolis with carbonated hydroxyapatite to observe the rankl expression in a rabbit’s alveolar bone nungky devitaningtyas, ahmad syaify and dahlia herawati .................................................... 212–216 9. oral health profile of the elderly people in the pandalungan community amandia dewi permana shita, zahreni hamzah, zahara meilawaty, tecky indriana, ari tri wanodyo handayani and dyah indartin setyowati ....................................................... 217–222 10. the difference between begg and straightwire appliances on molar position, occlusal plane angle, and anterior and posterior facial height changes dewi sartika santoso, christnawati and cendrawasih andusyana farmasyanti .................... 223–228 11. lemuru fish oil gel as host modulation therapy in periodontal ligaments induced with porphyromonas gingivalis widyastuti, dian widya damaiyanti, dian mulawarmanti, cindy aprilia sari and diah ayu siwi ........................................................................................................................... 229–234 contents page mkgs vol 44 no 2 april-juni 2011.indd 88 vol. 44. no. 2 june 2011 research report effectivity of 0.15% benzydamine on radiation-induced oral mucositis in nasopharynx carcinoma remita adya prasetyo division of oral medicine installation/department of dental & oral health, dr. soetomo hospital surabaya indonesia abstract background: nasopharynx carcinoma is the most common malignant tumour in head and neck region. radiotherapy is the first choice of treatment for nasopharynx carcinoma that had not been metastases. the most common oral complications in radiotherapy is mucositis (± 80%). 0.15% benzydamine hydrochloride (hcl) oral rinse can be used to prevent radiation-induced oral mucositis. purpose: the aim of this research was to study the effectivity of 0.15% benzydamine hcl oral rinse for prevention of radiation-induced oral mucositis in nasopharynx carcinoma. methods: samples were divided into 2 groups. group a was using 0.15% benzydamine hcl oral rinse for 10 days. group b was using placebo oral rinse for 10 days. evaluation was conducted 3 times: first day, fifth day and tenth day of radiotherapy. the scoring used spijkervet’s mucositis α score. results: independent t test analysis for initial occurrence of oral mucositis showed no significant difference between 2 groups. paired t test analysis showed significant difference between initial mucositis α score and mucositis α score in tenth day in each group. independent t test analysis showed no significant difference in mucositis α score in tenth day between 2 groups. conclusion: in conclusion 0.15% benzydamine hcl oral rinse was not effective to prevent radiation-induced oral mucositis in nasopharynx carcinoma. key words: 0.15% benzydamine hydrochloride, prevention, radiation-induced oral mucositis, nasopharynx carcinoma abstrak latar belakang: karsinoma nasofaring (knf) merupakan tumor ganas terbanyak di daerah kepala-leher. radioterapi merupakan terapi pilihan utama knf yang belum mempunyai metastasis jauh. komplikasi akibat radioterapi dalam rongga mulut yang terbanyak adalah mukositis (± 80%). salah satu obat untuk pencegahan mukositis akibat radioterapi adalah benzydamine hydrochloride (hcl) 0,15%. tujuan: tujuan penelitian ini adalah untuk mempelajari efektivitas penggunaan obat kumur benzydamine hcl 0,15% sebagai pencegah mukositis akibat radioterapi pada karsinoma nasofaring. metode: sampel dibagi ke dalam 2 kelompok. kelompok a yang menggunakan obat kumur benzydamine hcl selama 10 hari. kelompok b menggunakan obat kumur plasebo selama 10 hari. evaluasi dilakukan pada tahap awal, hari ke-5 radioterapi dan hari ke-10 radioterapi. alat ukur adalah skor mukositis α spijkervet. hasil: analisis independent t-test menunjukkan awal terjadinya mukositis antara kedua kelompok tersebut tidak berbeda bermakna. hasil uji t berpasangan antara skor mukositis α awal dengan skor mukositis α evaluasi ii pada masing-masing kelompok tersebut menunjukkan perbedaan yang bermakna. berdasarkan uji t, skor mukositis α evaluasi ii antara kelompok a dengan b tersebut tidak berbeda bermakna. kesimpulan: disimpulkan bahwa obat kumur benzydamine hcl 0,15% tidak efektif sebagai pencegah mukositis akibat radioterapi pada penderita knf. kata kunci: obat kumur benzydamine hydrochloride 0,15%, pencegahan, oral mukositis akibat radiasi, karsinoma nasofaring correspondence: remita adya prasetyo, c/o divisi penyakit mulut, instalasi/smf kesehatan gigi dan mulut, rsud dr. soetomo. jl. mayjend. prof. dr. moestopo no. 6-8 surabaya, indonesia. e-mail: mitaprasetyo@gmail.com 89prasetyo: effectivity of 0.15% benzydamine on radiation introduction nasopharynx carcinoma (npc) is the most common of malignant tumor in head and neck region and also in the department of ear, nose and threat. npc also showed increasing number from year to year.1,2 npc is a malignant tumor located in the nasopharynx, which manifestation including initial symptoms in the nose and ear, and late symptoms because of the expansion of primary tumor to surrounding organ in nasopharynx, or regional metastases to lymph nodes in the neck.2 radiotherapy is the first choice of treatment for npc that had not been distant metastases.1,3 this therapy is aimed to eradicate cancer cells with ionizing radiation. radiotherapy is also occasionally associated with dysfunction and disintegration of healthy tissue during and after therapy, including oral mucosa, through delayed of cell maturation and development.4 during the treatment of the head and neck radiotherapy, oral cavity is always in the risk of exposure of radiation. therefore, oral complications are expected, such as radiation-induced oral mucositis, which is the most common oral complications (± 80%). oral mucositis generally begin about ± 1–2 weeks after the start of radiotherapy (± 1000 cgy–2000 cgy). oral mucositis is associated with significant pain, inability to tolerate food and fluids, affect speech, and further compromising patients’ response to complete planned radiotherapy, thus it can prolong the duration of radiotherapy.4–10 planning of the precise therapy before radiotherapy is aimed to prevent radiation-induced oral mucositis.9,11 in several literatures, to prevent radiation-induced oral mucositis, 0.15% benzydamine hydrochloride (hcl) oral rinse was used. benzydamine hcl is a nonsteroidal rinse with anti-inflammatory, local anesthetic, antipyretic and antimicrobial activities.9,12 this oral rinse can be effective in preventing oral mucositis.5,7,13 according to the medical records in the ent oncology outpatient clinic of dr. soetomo hospital, there had not been any effort to prevent radiation-induced oral mucositis in npc’s patients. no attention to radiation-induced oral complications is given yet. the purpose of this research was to study the effectivity of 0.15% benzydamine hcl oral rinse for prevention of radiation-induced oral mucositis in npc. materials and methods the design of this research used randomized controlled trial. population of this research was npc patients in the department of radiotherapy dr. soetomo hospital who received radiotherapy. inclusion criteria of the sample was cooperative sample, stage iii & iv npc without distant metastases (loco-regional advanced), histopathology results showed undifferentiated carcinoma (who type 3), which planned to receive fractional dose radiation 200 cgy per day, five times a week, man & woman, 30–60 years old, not undergoing chemotherapy, no symptoms about oral mucositis and xerostomia, no infection disease, no allergy, no systemic disease (liver and nephrotic disease, hypertension and diabetes mellitus), no consumption of drugs that could cause xerostomia (antidepressant, antihistamin, antihypertension, opiate, sedative and diuretic drugs), and no consumption of systemic analgesic drugs. exclusion criteria of the sample was absent of visit and not using oral rinse as it was planned, could not undergo radiotherapy as it was planned, allergy to benzydamine hcl or other signs of side effect, and refused to continue this research. samples were divided into 2 groups. group a was using 0.15% benzydamine hcl oral rinse 120 ml for 10 days, rinse or gargle 15 ml for 60 seconds, three times daily. each time before the radiation was conducted, samples were using oral rinse under supervision from researcher. group b was using placebo oral rinse 120 ml for 10 days, with the same protocol as group a. there was no intervention before, including dental treatment. evaluation was conducted 3 times: first day, fifth day and tenth day of radiotherapy. the results of evaluations were recorded in the dental records. the assessment of oral mucositis used spijkervet’s mucositis α score. this scoring technique was specifically developed to measure tissue changes relative to doseresponse relationships and the effects of preventative mucositis strategies. spijkervet states that the mucositis scores developed by this technique are basically useful for research and are of limited value clinically because the total score α does not always reflect the clinical condition of the patient. this scoring distinguishes the most common and significant local clinical signs of radiation mucositis that represent the order or progression of mucosal radiation damage (k) that includes no mucositis, white discoloration, erythema, pseudomembranes, and ulceration. eight anatomical areas (n) of the mouth are scored (right and left buccal mucosa, hard palate, soft palate, dorsum of tongue, right and left border of tongue, and floor of mouth), although any one area might include several subareas with different local signs of mucositis observed in that area. the length (k) of each identical local sign for each subarea is measured (in centimetres) and then summed and corresponds with a value e (1 ≤1 cm, 2 = 1.0–2.0 cm, 3 = 2.1–4.0 cm, 4 ≥ 4 cm). the degree of mucositis for each subarea was defined as the product of the values k and e; the score for mucositis in an area was defined as the sum of these products. finally, the overall spijkervet’s mucositis α score is calculated as the mean of the scores assigned to the number of irradiated areas (n).6 data analysis used descriptive and inferential (independent t-test, paired t-test, mann-whitney test and fisher’s exact test), with level of significance (α) was 0.05 (5%). 90 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 88–92 table 1. homogenity test between group a and group b data type group a goup b statistic test p sex: man woman 7 (70%) 3 (30%) 7 (77.88%) 2 (22.22%) fisher’s exact test 1.000** age: rate (years) sd 45.10 7.95 45.11 6.95 independent t-test 0.997** level of education: elementary junior school high school 7 (70%) 2 (20%) 1 (10%) 5 (55.56%) 2 (22.22%) 2 (22.22%) mann-whitney test 0.476** npc stage: iii iv 4 (40%) 6 (60%) 4 (44.44%) 5 (55.56%) fisher’s exact test 1.000** ohi-s (first day) rate sd 2.60 0.88 2.26 0.96 independent t-test 0.426** notes: **: no significant different (p > 0.05) table 2. independent t-test analysis for initial occurence of oral mucositis between group a and group b group n rate sd p initial occurence of oral mucositis a b 10 9 7.20 7.56 1.14 1.13 0.504** notes: ** : no significant difference (p > 0.05) table 3. paired t-test analaysis for mucositis α score in group a (the 1st, 5th and 10th day of radiotherapy) group a n rate sd p mucositis α score (1st day) mucositis α score (5th day) mucositis α score (1st day) mucositis α score (10th day) 10 10 10 10 0.00 0.00 0.00 1.37 0.00 0.00 0.00 0.79 *** 0.00* notes: *: significant difference (p < 0.05); ***: could not be analyzed table 4. paired t-test analaysis for mucositis α score in group b (the 1st, 5th and 10th day of radiotherapy) group b n rate sd p mucositis α score (1st day) mucositis α score (5th day) mucositis α score (1st day) mucositis α score (10th day) 9 9 9 9 0.00 0.00 0.00 1.86 0.00 0.00 0.00 0.96 *** 0.00* notes: *: significant difference (p < 0,05); ***: could not be analyzed results this research was completed in 3 months and included 19 samples. group a was 10 samples and group b was 9 samples. statistic analysis (table 1) showed the homogenity of sex, age, level of education, npc stage and oral hygiene index simplified (ohi-s) in the first day of radiotherapy, between group a and b (p > 0.05). independent t-test analysis for initial occurence of oral mucositis between group a and group b (table 2) showed p = 0.504 (p > 0.05), it meaning there was no significant difference between 2 groups. paired t test analysis showed significant difference between initial mucositis α score and mucositis α score in tenth day in each group (table 3 & 4). independent t test analysis showed no significant difference in mucositis α score in tenth day between 2 groups (table 5). discussion the design of this research was randomized controlled trial. this clinical trial was an experimental trial with human being as the sample. this research was phase iii clinical trial because it aimed to evaluate new treatment, compared with placebo.14 the homogenity between 2 groups (table 1) must be tested to know about the factor that could affect mucositis α score. if there was any difference between 2 groups, the reason of difference was only because of the experiment that were given in both groups.14 table 1 showed that group a and b were homogen. initial occurence of oral mucositis between 2 groups showed no significant difference among them (table 2). it meant that 0.15% benzydamine hcl was not effective to delay the initial occurence of oral mucositis, as in placebo. this was similar with putwatana et al.,15 that comparing benzydamine with natural agents, glycerine payayor (herbal product) was found to be superior in preventing and relieving radiation-induced oral mucositis than benzydamine hydrochloride. although 0.15% benzydamine hcl also had antimicrobial effect, it could 91prasetyo: effectivity of 0.15% benzydamine on radiation table 5. independent t-test analysis for mucositis α score between group a and group b in 1st, 5th and 10th day of radiotherapy group n rate sd p mucositis α score (1st day) mucositis α score (5th day) mucositis α score (10th day) a b a b a b 10 9 10 9 10 9 0.00 0.00 0.00 0.00 1.37 1.86 0.00 0.00 0.00 0.00 0.79 0.96 *** *** 0.245** notes: **: no significant difference (p > 0.05); ***: could not be analyzed not delay this disorders. it might because rinsing was not guaranteed to have enough contact between antimicrobial agent with microorganism, so 0.15% benzydamine hcl as antimicrobial could not help the antiinflammation effect. but the research of epstein et al.13 and worthington et al.,16 stated the opposite things. these differences because the method to assess oral mucositis was different. epstein et al.,13 used mucositis score based on subjective and clinical manifestation of oral mucositis (erythema, ulceration and pain). worthington et al.16 recommended in additional large trials to determine benefit, dosage, and administration method. while spijkervet’s mucositis α score used in this research was a special method for research so the assessment is accurate. this method assessed clinical changes of radiation-induced oral mucosa in qualitative and qualitative ways (white discoloration, erythema, pseudomembrane and ulceration), not subjective complaint or dysfunction of oral cavity. it is important to note that, whereas the score developed by the spijkervet technique will not always reflect the clinical state of the patient, it does quantify the degree of tissue change or damage.6 besides that, the differences might be caused by initial occurence of oral mucositis was not due to the microba, but because of the radiotherapy’s side effect. thus the use of antimicrobial agent did not have effect, and the antiinflammation was playing the role. the antiinflammation effect depends on oral hygiene, tissue resistance to radiotherapy, total dose of radiotherapy and how long the patient received radiotherapy. in this research, oral hygiene in the first day of radiotherapy between 2 groups was homogenous (table 1), but it meant both groups had bad oral hygiene, so it might stimulate the initial occurence of oral mucositis. this was similar with köstler et al.,12 berger & kilroy,17 cheng et al.,18 which reinforced oral hygiene as a important direct factor that could affect the degree of severity and duration of mucositis. besides oral hygiene, there were also radiation source, daily doses, cumulative doses and irradiated mucosa volume. the side effect of radiotherapy, especially sensitive to cell with faster proliferation such as tumor cell, but this effect also affect healthy tissue in the radiation field, so tissue resistance was decreased because of radiotherapy.4 there was significant differences between the rates of mucositis α score in the first and the tenth day of radiotherapy in each group (table 3 and 4). this fact could be caused by the initiation of oral mucositis in both groups, so mucositis α score could be assessed already. there was no significant different of mucositis α score’s rates between group a and group b in the tenth days. (table 5). it meant that 0.15% benzydamine hcl was not effective, as in the placebo. this was similar with rosenthal and trotti8, hancock et al.11 which stated that the risk for developing radiation-induced oral mucositis depends on different factors, such as anti cancer treatment protocol, age and diagnosis of the patient, level of oral hygiene during therapy, genetic factors. kartabrata et al.4 and beck,19 said that disintegrity of lining mucosa was port d’entry of microorganism and caused local infection which potentially disseminated through blood stream. according to stokman et al.,7 epstein et al.13 and kazemian et al.,20 there was significant different of mucositis score between group using benzydamine with placebo as a prevention because it proved could prevent or reduce the severity and the risk of secondary infection and bleeding because of benzydamine’s antiinflamation effect. besides, those research used different definition of prevention, that was to prevent or reduce clinical manifestation of oral mucositis. while in this research, the definition was to prevent the occurence of oral mucositis. therefore, it can be concluded that 0.15% benzydamine hcl oral rinse was not effective to prevent radiation-induced oral mucositis in nasopharynx carcinoma. it will need further research and better cooperation between specialists of oncology radiation and oral medicine. references 1. kentjono wa. penatalaksanaan karsinoma nasofaring masa kini. simposium kanker nasofaring dan demo biopsi dengan tehnik aspirasi jarum halus, 2003; p. 24–6. 2. mulyarjo. epidemiologi dan gambaran klinik karsinoma nasofaring. simposium kanker nasofaring dan demo biopsi dengan tehnik aspirasi jarum halus, 2003; p. 1–3. 3. mulyarjo. diagnosis dan penatalaksanaan karsinoma nasofaring. pendidikan kedokteran berkelanjutan iii ilmu penyakit tht-kl. perkembangan terkini diagnosis dan penatalaksanaan tumor ganas tht-kl, 2002; p. 38–48. 4. kartabrata m, hendarti ht, ayu s. prevalensi kandidiasis mulut pada penderita yang mendapat terapi radioterapi kanker kepala dan leher. maj ked gigi (dent j) 2001; 34(3a): 376–9. 92 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 88–92 5. roopashri g, jayanthi k. radiotherapy and chemotherapy induced oral mucositis-prevention and current therapeutic modalities. ijda 2010; 2(2): 174–9. 6. schubert mm. measurement of oral tissue damage and mucositis pain. 2004. available at http://painresearch.utah.edu/cancerpain/ch.15. html. accessed april 26, 2004. 7. stokman ma, spijkervet fkl, boezen hm, schouten jp, roodenburg jln, de vries ege. preventive intervention possibilities in radiotherapy and chemotherapy-induced oral mucositis: results of meta-analyses. j dent res 2006; 85(8): 690–700. 8. rosenthal di, trotti a. strategies for managing radiation-induced mucositis in head and neck cancer. semin radiat oncol 2009; 19: 29–34. 9. wardhany ii, subita gs. meningkatkan kualitas hidup pasien kanker kepala dan leher yang menjalani radioterapi melalui pengendalian mukositis. maj ked gigi (dent j) 2001; 34(3a): 582–5. 10. harrison js, dale ra, haveman cw, redding sw. oral complications in radiation therapy. oral medicine, oral diagnosis 2003 novemberdecember; 552-60. available at http://www.acd.org. accessed july 30, 2004. 11. hancock pj, epstein jb, sadler gr. oral and dental management related to radiation therapy for head and neck cancer. j can dent assoc 2003; 69(9): 585–90. 12. köstler wj, hejna m, wenzel c, zielinski cc. oral mucositis complicating chemotherapy and/or radiotherapy: options for prevention and treatment. ca cancer j clin 2001; 51: 290–315. 13. epstein jb, silverman s jr, paggiarino da, crockett s, schubert mm, senzer nn, lockhart pb, gallagher mj, peterson de, leveque fg. benzydamine hcl for prophylaxis of radiation-induced oral mucositis: results from a multicenter, randomized, double-blind, placebo-controlled clinical trial. cancer 2001; 92: 875–85. 14. harun sr, putra st, wiharta as, chair i. uji klinis. in: sastroasmoro s, ismael s, eds. in: dasar-dasar metodologi penelitian klinis. jakarta: sagung seto; 2002. p. 145–65. 15. putwatana p, sanmanowong p, oonprasertpong l, junda t, pitiporn s, narkwong l. relief of radiation-induced oral mucositis in head and neck cancer. cancer nurs 2009; 32(1): 82–7. 16. worthington hv, clarkson je, eden ob. interventions for preventing oral mucositis for patients with cancer receiving treatment. cochrane database syst rev 2006 .apr; 19(2): cd000978. available at: http://www.ncbi.nlm.nih.gov/pubmed/16625538. accessed august 16, 2011. 17. berger am, kilroy tj. oral complications. in: devita jr. vt, hellman s, rosenberg sa, eds. in: cancer principles and practice of oncology. 6th ed. philadelphia: lippincott williams & wilkins; 2001. p. 2881–93. 18. cheng kk, chang am, yuen mp. prevention of oral mucositis in paediatric patients treated with chemotherapy; a randomised crossover trial comparing two protocols of oral care. eur j cancer 2004; 40: 1208–16. 19. beck s. mucositis in cancer. 2000. available at http://www. cancersource.com/ nursing/ce/cecourse.cfm?courseid=56&cont entid=19481. accessed june 21, 2004. 20. kazemian a, kamian s, aghili m, hashemi fa, haddad p. benzydamine for prophylaxis of radiation-induced oral mucositis in head and neck cancers: a double-blind placebo-controlled randomized clinical trial. eur j cancer care (engl) 2009; 18(2): 174–8. isi vol 39 no 3 juli-september 2006.pmd 93 mandible vertical height correction using lingual bone-split pedicle onlay graft technique coen pramono d department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract as edentulous mandible become atrophic, a denture bearing area will also be reduced. difficulty in the removable prosthesis rehabilitation will be present as well. the purpose of this paper reports an innovative surgical technique to cope a problem of unstable complete lower denture due to bone atrophy and resulted of vertical height reduction of the anterior region of the mandible necessary for denture retention. vertical advancement of the lower jaw using lingual bone split pedicle onlay graft technique in the anterior region of the mandible and followed by secondary epithelization vestibuloplasty in achieving the vertical height dimension. the surgery was achieved satisfactorily as the vertical dimension of the mandible anterior region had increased and the denture seated more stable comparing with the previous denture worn by the patient. it concluded that the surgery was achieved with a great result as the vertical height of the anterior region of the mandible had increased positively therefore lead the denture seated more stable. key words: mandible atrophy, alveolar vertical height correction, lingual bone split, pedicle lingual bone, bone onlay graft, vestibuloplasty correspondence: coen pramono d, c/o: bagian ilmu bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. surgical technique in oral vestibular sulcus extension in an atrophied alveolar process has a long evolutionary history of success and gives the credit for pioneering the clinical practice of pre-prosthetic surgery. some articles have documented the clinical versatility and outcome of sulcus extension surgery1-20 and those techniques till now days still uses as a basic surgical knowledge for any sulcus extension surgery. review study from hillerupp et al.20 concluded that lowering of the floor of the mouth provide a substantial benefit to patients with denture problem due to alveolar ridge atrophy. several pre-prosthetic surgical procedures that have been used successfully are little known or applied in ours. among these are sub-mucosal vesitybuloplasty, secondary epithelization vestibuloplasty, alveolar ridge-skin grafting vestibuloplasty, buccal sulcus-skin grafting vestibuloplasty, tuberoplasty, and upper jaw sandwich osteotomy with immediate vestibuloplasty. all of those techniques are applicable in both maxilla and mandible correction and conjunction with the oral soft manipulations. although some recent surgical techniques and appliances of denture rehabilitation have been developed, such as the used of dental implant and distraction osteogenesis,21–27 the previous techniques of ridge correction for dental bearing enlargement which had been developed still gave a great benefit in many settings, especially in patients who can not afford of those advanced appliances. introduction the height of the alveolar process in edentulous patients gives an important contribution in prosthetic dentistry. although the present of dental implant give a significant contribution in dental rehabilitation, correction of the vertical height in atrophis jaw to achieve a retentive removable dental prosthesis by surgery are sometimes needed to be performed in many settings. pre-prosthetic surgery were improved since the end of world war ii, through the developments of better materials, the improved accuracy of diagnostic technique and better understanding of oral physiology, dental prosthetics has made great strides in increasing the successful use of prosthetic appliances in edentulous patients. nonetheless, there remain a number of patients who can never be made to use denture effectively because the process of bone atrophy, soft tissue hypertrophy, or both have developed beyond the point of prosthetic accommodation. in these patients, surgery offers a significant contribution. in case of atrophy of the alveolar process in the posterior region found with sufficient depth of the lingual sulcus and sufficient high of the vertical height of the anterior alveolar process, stable denture retention can be expected. a difficult situation would be occur when the lost of the denture bearing suffered in the anterior region of the mandible, this situation might contribute a difficulty in achieving complete denture prosthesis stabilization. 94 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006: 93–97 the surgical technique for denture bearing extension basically can be done by two way basic methods, soft tissues extension, and secondly is combination of soft tissuebone surgery. the soft tissue surgery sometimes can be done in simple way in comparing to soft tissue-bone surgery. surgical combination of bone-soft tissue surgery usually done in two stages of surgeries, as bone advancement for the first step and followed by vestibuloplasty to built a new sulcus. the lack of bone mass associated with relatively high muscle attachments and insufficient vestibular depth complicates the prosthetic restoration of the atrophic mandible. bell et al.28 in his experimental studies of interpositional bone grafts to the maxilla and mandible indicate that the palatal and labio-buccal mucoperiosteum in the maxilla and lingual mucoperiosteum in the mandible provide an adequate vascular pedicle for single stage repositioning of athropic edentulous maxilla by le fort i osteotomy or superior repositioning of mandibular basal bone. various surgical reports of ridge augmentation in the mandible by davis et al.,8 sander and cox9 ewers and haerle,12 tideman et al,15 schettler,18 had shown its superiority in achieving an augmented ridge and denture stability. the complexity of the surgical methods as had been presented by some authors, therefore applying another simple modification of surgical technique in improving the vertical height of the anterior part of atrophic mandible was thought to be useful. case a 65 year old female was visited department of oral and maxillofacial surgery, airlangga university refered by department of prosthodontic with mandible atrophic especially in the anterior region. the patient appeared in our clinic was wearing her upper and lower complete dentures, but she was complaining of unstable lower jaw denture, therefore she expected for a new dental prosthesis. the prostodontist also reported the difficulty to arrange a stable denture due to the insufficient vertical height of the mandible anterior region. the patient was observed and planned with new full denture replacement both upper and lower jaws. the denture bearing in the upper jaw was found with sufficient ridge height, differed from what given in the lower jaw. the lingual sulcus in posterior region and buccal sulcus were found in between acceptable depth for placing a removable denture, but she had lost the alveolar process, labial sulcus depth in the region of canine to canine. flabby and movable soft tissue also presented, increasing the denture seated unstable. a labial depth correction was presumed to be necessary to perform by increasing the vertical height and sulcus depth. panoramic radiography showed a mark atrophied of the anterior region therefore a vestibuloplasty alone would not be enough to ensure the sulcus depth as the vertical bone height had been lost (figure1). combination of bone augmentation and vestibuloplasty were planned in two stages. a vertical bone osteotomy of the lingual part of the mandible was done and the bone fragment was onlayed above the mandible bone and fixed with t-miniplate in combination with wire osteosynthesis (figure 2-a, b, c and d). three months after the surgery, the t plate was removed and followed by immediate secondary epithelization vestibuloplasty in the region of canine to canine in the labial side (figure 3-a). two weeks after the vestibuloplasty, the vertical height of the anterior part of the mandible shown increased and the lower jaw impression was taken for a definitive complete denture prosthetic (figure 3-b, c). the stability of the denture was reported by the prosthodontist with satisfactory (figure 3-d). case management in atrophic edentulous mandible, advancement of the vertical height is necessary as the mouth had failed its denture bearing for placement of the denture. a new method of mouth rehabilitation using dental implant is now widely used, otherwise that method is still difficult to be proceeded in many setting as that technique of dental implant need of a highly cost, therefore correction of the alveolar process height using bone split technique can be use as an alternative method to cope problem of unstable denture due to alveolar atrophy. the basic idea of this surgical technique is done by creating a new vertical height of the anterior region of the mandible. the lingual part of the mandible in the region from left to right first premolar was osteotomized vertically and followed by bone separation from the mandible with the lingual mucoperiosteum and muscles preserved on its attachment. the osteotomized bone is than extended figure 1. preoperative situation: the anterior region of the lower jaw presented with flat ridge and narrowed labial sulcus. 95pramono: mandible vertical height correction figure 3. (a) immediate secondary epithelization vestibuloplasty simultaneously done after the t plate removal; (b) six months after surgery, anterior region of the mandible shows with vertical height and labial sulcus depth improvement, facilitated to a good denture retention; (c) the result of lower jaw impression shows a positive ridge improvement in the anterior region; (d) the lower jaw denture fitted nicely as well as the upper denture. figure 2. (a) vertical and horizontal osteotomy the anterior part of the mandible in the lingual region from right first premolar to left first premolar; (b) the lingual bone separated from mandible; (c & d) the osteotomized lingual bone raised and placed above labial bone and fixed with bended t miniplate and stainless steel wires. a b c d (figures c and d published under the courtesy of drg. roestiny and drg. mefina from the department of prosthodontic, faculty of dentistry, airlangga university). a b c d 96 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006: 93–97 superiorly and onlayed above the mandible bone as a pedicle bone graft to create a new alveolar process. t form miniplate osteosynthesis can be used as an alternate method for bone graft fixation. the plate can be removed 3 months after the first surgery and an immediate vestibuloplasty using secondary epithelization technique is done following the plate removal. discussion extensive changes may occur in the morphology of the jaw after tooth loss. the jaws are consisted of alveolar and basal bone. the alveolar bone and periodontal tissues supported the teeth, but neither have any physiological function once the teeth lost, and are therefore resorbed.29 three disadvantage problems simultaneously existed in this case, as: a) lost of vertical height of the anterior region, b) a flabby tissue arose following the atrophied alveolar bone and c) unstable denture due to muscles activites. the activity of the orbicularis oris and mentalis muscles interfered strongly in the anterior region of the mandible. many of fibers that are contained entirely within orbicularis oris pass obliquely through the thickness of the lips from the dermis of the skin on outer labial surface to the mucous membrane on inner aspect. contraction of the orbicularis oris compresses the lips against the teeth as well as closing the oral cavity. another small slip of muscle, the mentalis, passes from front of the mandible near to midline to be inserted into the skin of the chin. it lies just to the side of the frenulum of the lower lip.30 in patient with atrophied mandible who wearing a lower complete denture prosthesis, the activity of 2 muscles, the lower part of the orbicularis and mentalis muscles might be easily dislodge the denture. ridge augmentation, sulcus extention, and muscle release are three major surgical objectives should be done to achieve denture stability. augmentation of the atrophic edentulous mandibular ridge has long been a problem for the oral and maxillofacial surgeon. various types of transoral onlay bone grafting techniques in the mandible have been tried.8-9,12,15 davis et al.31 have reported the use of autogenous transoral onlay rib grafting. in long term follow-up, these authors reported of excessive resorption of 30% to 50% of the bone had been grafted.8 ewers and haerle in year 1980 reported an absolute elevation of the ridge in which the mandible had osteotomized vertically and displaced like a visor. the fiveyear results of 10 patients observed were reported. the average of bone grafting resorption shown of 18 % of the elevation of the alveolar ridge in the first year, 10 % in second year, 8 % (i.e 0.6 mm), in the third year and 3% (i.e 0.2 mm) in the fourth and fifth years.12 the sources of bone had grafted as reported by davis8 and ewers and haerle12 were different, which davis used an autogenous rib as a bone sources and ewers and haerle used an autogenous bone which taken from the part of the mandible and placed like a visor, therefore it might be the reason why the rate of bone resorption as reported by davis31 found higher then given by ewers and haerle.8 mandible bone resorption in the anterior region in this case leads the difficulty in the complete denture prosthetic rehabilitation. an augmentation of the anterior region was proofed to be possible using lingual bone split pedicle onlay graft technique. this surgical method was ensured based on two considerations, the bone used for this grafting procedure was an autogenous bone type as it was taken from the same mandible and applied by maintaining its vascularity through muscle attachment and lingual mucoperiosteum, means that the bone had been grafted to the recepient site in a pedicle form of bone graft. application of this pedicle bone graft method expecting of minimum of bone resorption and would be adequately accepted by the recipient as it has a same type of intramembranous bone. in short observation of eight months period after surgery concluded that this technique of alveolar process augmentation in the anterior region of lower jaw had augmented nicely, the anterior sulcus depth shown had been maintained and the lower jaw prosthesis stability is achieved successfully. this surgical technique lingual bone split pedicle onlay graft technique might be used as an alternative surgical technique for correction of atrophy in the anterior region of the mandible. furthers observations are needed to determine the long-term results of this surgical technique. references 1. schuchardt von k. die epidermistransplantation bei der mundvorhofplastik. dtsch zahnaerzl z 1952; 32:132–35. 2. trauner r. die alveoloplastik im unterkiefer auf der lingualen seite zur loesung des problems der unteren prothese. dtsch zahnaerzl z 1952 march; 7(1):256–369. 3. obwegeser hl. die submukoese vestibulumplastik. dtsch zahnaerzl z 1959; 14:629–35. 4. obwegeser hl. die totale mundbodenplastik. schweiz mschwr zahnheilk 1963; 73(7): 565–72. 5. obwegesser hl. surgical preparation of maxilla for prosthesis. j oral surg 1964; 22:127–34. 6. macintosch rb, obwegeser hl. preprosthetic surgery: a scheme for its effective employement. j oral surg 1967; 25:397–413. 7. steinhauser ew. vestibuloplasty-skin grafts. j oral surg 1971 november; 29:777–85. 8. davis wh, delo ri, waard wb, terry b, patakas b. long term ridge augmentation with rib graft. j max fac surg 1975; 3:103–6. 9. sanders b, cox r. inferior-border rib grafting for augmentation of the atrophic edentulous mandible. j oral surg 1976; 34:897-900. 10. obwegeser hl. der atrophische kiefer aus der sicht des kieferchirurgen. schweiz mschwr zahnheilk 1977; 879(9):946–58. 11. bell wh, buckles rl. correction of the alveolar ridge by interpositional bone grafting: a progress report. j oral surg 1978; 36:693–700. 12. ewers von r, haerle f. langezeitresultate nach visierosteotomi. dtsch zahnaerzl z 1980; 35: 1046–47. 13. haerle f. indikation, methoden und ergebnisse zur absoluten alvelarkammerhoehung des unterkiefers. dtsch zahnaerzl z 1982; 37:121–26. 14. schwenzer von n. prinzipien und standardverfahren zur operativen verbesserung des prothesenlagers. dtsch zahnaerzl z 1982; 37:127–31. 97pramono: mandible vertical height correction 15. tideman von h, stoelinga pjw, de koomen de. die erhoehung des atrophierten unterkiefers mit beckenknochentransplantat. erweitertes autoreferat. dtsch zahnaerzl z 1980; 35:1011–13. 16. de koomen von h, stoelinga pjw, tideman, hendricks fhj. resultate bei der erhoehung des atropischen unterkiefers mit beckenknoochentransplantat. dtsch zahnaerzl z 1980; 35:1014-16. 17. tesch von p, jacobi-hermanns e. die indikation zur relative alveolarkammerhoehung des unterkiefers. dtsch. zahnaerzl z 1980; 35:1046–47. 18. schettler von d. spaeterergibnisse der absoluten kieferkammerhoehung im atropischennterkiefer durch die ”sandwichplastik”. dtsch zahnaerzl z 1982; 37:32–135. 19. schmelzle von r, schwenzer n. 10jaehrige klnische erfahrung mit cialitr konserviertem stuetzgewebe in der praeprotischen chirurgie. dtsch zahnaerzl z 1982; 37:136–38. 20. hillerupp s. preprosthetic mandibular vestibuloplasty with split-skin graft. a 2-year follow-up study. int j oral maxillofac surg 1987; 16:270–78. 21. lew d, hinkler, unhold g. reconstruction of the severely atrophic edentulous mandible by means of atugenous bone grafts and simultaneous placement of osseointegrated implants. j oral maxillofac surg 1991; 49: 228. 22. jensen j, sindet-pendersen s, oliver aj. varying treatment strategies for reconstruction of maxillary atrophy with implants: result in 98 patients. j oral maxillofac surg 1994; 52:210–16. 23. keller ee, eckert se, tolman e. maxillary antral and nasal onestage inlay composite bone graft: preliminary report on 30 recipient sites. j oral maxillofac surg 1994; 52:438–47. 24. betts nj, miloro m. modification of sinus lift procedure for septa in the maxillary antrum. j oral maxillofac surg 1994; 52:332–33. 25. donovan mg, dickerson nc, hanson lj, gustafson rb. maxillary and mandibular reconstruction using calvarial bone grafts and branemark implants: a preliminary report. j oral maxillofac surg 1994; 52:558–94. 26. sawaki y, ohkubu h, hibi h, ueda m. mandibular lengthening by distraction osteogenesis using osseointegrated implants and an intraoral device: a preliminary report. j oral maxillofac surg 1996; 54:594–600. 27. carlson er, marx re. mandibular reconstruction using cancellous cellular bone graft. j oral maxillofac surg 1996; 54:889–97. 28. bell wh. bone healing and vascularization after maxillary osteotomy. j oral surg 1975 april; 33–7. 29. matthew ir, frame jw. surgical aids to prosthodontic, including osseointegrated implants. in: pedlar j, frame jw, editors. oral and maxillfofacial surgery: an objective-based textbook. 3rd ed. churchill livingstone: printed in spain; 2004. p. 143–62. 30. johnson dr, moore wj. anatomy for dental student. hongkong: oxford 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 51 no 4 okt-des 2018.indd 158158 variations of gelatin percentages in ha-tcp scaffolds as the result of 6and 12-hour sintering processes of blood cockle(anadara granosa) shells against porosity desak putu sudarmi ari, firda dean yonatasya, gita saftiarini, and widyasri prananingrum department of dentistry material and technology sciences faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: porous scaffold is one type of biomaterial primarily employed as a bone substitute material which demonstrates superior osteoconductive and osteointegrative properties than solid scaffold since it can stimulate and accelerate the growth of new tissue. for the purposes of this study, porous scaffold was produced using hydroxyapatite-tricalcium phosphate (ha-tcp) powder derived from a synthesis of blood cockle (anadara granosa) shells and gelatin. purpose: the aim of this study was to reveal the effects of the percentage of gelatin in ha-tcp scaffolds derived from 6and 12-hours sintering processes involving blood cockle shells on porosity. methods: ha-tcp powder was derived from a synthesis of anadara granosa shells using a hydrothermal method at 200oc with sintering periods of 6 and 12 hours. a xrd test was subsequently conducted to reveal the compositions of ha-tcp powder. the 24 scaffold samples (n=6) employed were manufactured using a freeze dry method before being divided into four groups, namely; group 1 using 25% ha-tcp powder (a six-hour sintering process) combined with 20% gelatin, group 2 using 25% ha-tcp powder (a six-hour sintering process) combined with 10% gelatin, group 3 using 25% ha-tcp powder (a twelve-hour sintering process) combined with 20% gelatin; and group 4 using 25% ha-tcp powder (a twelve-hour sintering process) combined with 10% gelatin. a scaffold porosity test was subsequently carried out using a liquid displacement method. a one-way anova test was performed using spss, followed by a post-hoc lsd (p<0.05). results: the statistical results for scaffold porosity were within the range of 67.21 -77.51%. the highest porosity was found in group 3, while the lowest was in group 4. significant differences were also present in all groups. conclusion: variations in the percentage of gelatin can affect the porosity of ha-tcp scaffolds derived from 6-and 12 hours sintrering processes blood cockle shells. the smaller the percentage of gelatin, the higher the porosity. keywords: anadara granosa shell; ha-tcp; percentage of gelatin; porosity; scaffold correspondence: widyasri prananingrum, department of dentistry material and technology sciences, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya, indonesia. e-mail: widyasri.prananingrum@hangtuah.ac.id introduction bone substitute material serves to assist reconstruction, stabilize the structure and bonding of bone and stimulate osteogenesis and healing processes within bone defects.1 bone substitute material must, therefore, be biocompatible, non-toxic, non-cariogenic and non-allergenic, while possessing a biological mechanism that is osteoconductive, osteoinductive and osteogenic.2 in general, there are four types of bone substitute material (bone graft), namely; autograft, allograft, xenograft and alloplast. autograft is a substitute for bone material taken from the patient’s body, while allograft is derived from that of another human being. contrastingly, xenograft is extracted from the body of an animal or a different species, while alloplast is composed of synthetic material.3 hydroxyapatite (ha), with the chemical formula ca10 (po4)6 (oh)2, is an inorganic compound capable of binding to bone4 which can be produced synthetically from blood cockle (anadara granosa) shells. indonesia is one of the countries producting significant amounts of seafood, one example being blood cockle. dental journal (majalah kedokteran gigi) 2018 december; 51(4): 158–163 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 159 ari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 158–163 according to data released by the directorate general of capture fisheries in 2011, shellfish production in indonesia during the previous year reached 34,929 tons of which the volume of blood cockle production amounted to 34,482 tons.5 until recently, blood cockles had been harvested only for their meat, the consumption of which results in high levels of unutilized shell waste. as a result, hydroxyapatite derived from the synthesis of blood cockle shell waste is used to produce scaffold. scaffold applied to tissue engineering requires open porosity construction and adequate pore size to support cell nutrient transport, cell proliferation and migration resulting from tissue vascularization.6 a porous surface also serves to facilitate mechanical interlocking between the scaffolds and surrounding tissue to improve the mechanical stability of the implant. in addition, the network structure of the pores assists in guiding and promoting new tissue formation.7 to promote ideal scaffold production the addition of supporting materials is required, one of which is gelatin. gelatin is a collagen-rich polypeptide bond derived from the hydrolysis of bone and animal skin. it is non-toxic, biocompatible, biodegradable and nonimmunogenetic in character, rendering it useful as a coating for implants, wound dressings and scaffold in cell culture. therefore, as a natural polymer, gelatin can be used as a scaffold for tissue engineering.8,9 a mixture of 10% gelatin and 80% nanoparticles cockle shells bone graft is effective in increasing the number of osteoblast cells in the bone healing process. 10% gelatin produces a high viscosity liquid with significant potential for tissue engineering.10,11 on the other hand, scaffold containing 20% gelatin shows a strong expansive character and induces biological responses such as cell attachment, cell proliferation and effective cell spread.12 gelatin plays an important role in making scaffold because of its ability to cross-link and modify other materials that can significantly change their mechanical properties to become stronger and porous.13 the addition of gelatin in the manufacture of scaffold is also intended to increase adhesion, migration and mineralization of osteoblast cells that play an important role in bone formation process.14 the pore structures in each sample of macroporus scaffold will differ. furthermore, variations in sintering period will also affect the composition and structure of the ha powder produced, while also causing differences in crystallinity. the longer the sintering period, the greater the crystallinity produced which affects the regularity of ha atom arrangement.15 in this research, porous ha scaffold was derived from the synthesis of blood cockle shells and 25% ha and 10% or 20% gelatin after 6and 12-hour periods of hydrothermal circulation. hence, this study aimed to determine the effect of adding variations of gelatin percentage on the porosity of hydroxyapatite-tricalcium phosphate (ha-tcp) scaffold as a result of blood cockle shells synthesis with hydrothermal sintering of 6and 12hour duration. materials and methods the sample manufacture stage includes producing scaffold from the synthesis of blood cockle shells. ha was obtained by processing blood cockle shells which involved boiling them for 30 minutes before cleaning and drying. the shells were subsequently pounded to form a powder which was filtered through a 100-mesh sieve. the powder was calcined at 100°c for three hours to produce calcium carbonate (caco3) powder, 10 grams of which were then dissolved in distilled water to produce 1m caco3. thereafter, 1m of caco3 was mixed with 0.6m of nh4h2po4 obtained from 6.9 grams of nh4h2po4 which was dissolved in 100 ml of distilled water. by means of a hydrothermal method at 200oc for 6 hours for p1 and 12 hours for p2, caco3 powder was further processed and rinsed using distilled water to obtain ph ± 7. thereafter, final rinsing was performed using methanol pa after which it was warmed at 50oc for 4 hours before being subjected to the final sintering process at 900oc for 3 hours until ha powder was produced. scaffold was then produced by mixing up to 25% (wt%) ha powder with 10% or 20% (wt%) gelatin and putting it into 360 μl (6–10 mm) well plates. the scaffold material was subsequently frozen at -80o c for 5 hours before being freeze dried for 30 hours. a x-ray diffraction (xrd) test was carried out to determine the crystal system, lattice parameter, crystallinity degree and substance type contained in each sample. x-rays were directed at the sample inducing the xrd detector to rotate according to the range of diffraction angles employed. a diffractogram graph depicting the relationship between the intensity and diffraction angle was created, before being presented on the computer screen. the diffractogram graph was subsequently interpreted using software match which provided information about the crystal structure contained in the sample in the form of percentage results. a porosity test was carried out to determine the percentage volume of void space contained in the samples. in this research, the samples were divided into 4 groups (n = 6) according to differences in the ratio between ha and gelatin. 96% absolute ethanol was subsequently used as a liquid to determine the wet mass value. moreover, a digital balance was also used to quantify the mass of objects. a porosity test was carried out using the liquid displacement method which involved soaking the samples in 96% absolute ethanol for 48 hours before wet mass measurement was carried out.16 the percentage porosity of each sample was then quanitified after the object mass measurement results of the wet mass of the samples had been obtained by means of the following equation (figure 1). a scanning electron microscope (sem) test was performed to identify the microstructure of the samples with the results being observed through a photo. a sample preparation had previously been produced for observation using the sem imaging device. coated scaffold samples were then cut on the side to be studied and observed through dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 160ari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 158–163 an electron microscope and displayed on a computer screen. an interpretation was subsequently conducted by searching for a field of view, selecting pores randomly and measuring them with the sem imaging device at 1000x magnification. a statistical test analysis was carried out using one-way anova, followed by lsd post-hoc test (p<0.05). results xrd was performed to determine the lattice parameters, crystallinity degree and substances contained in a sample. the results of an xrd test on ha-tcp powder derived from the synthesis of blood cockle shells with a 6-hour sintering period produced a diffractogram with sharp peaks, the highest being in the range of 30°-40°, as well as high intensity. this signifies the sample having undergone perfect crystallinity. moreover, this diffraction pattern also indicated that the predominant content of the sample was ha. the results of an xrd test on ha-tcp powder derived from the synthesis of blood cockle shells during a 12-hour sintering period produced a diffractogram similar to that of a 6-hour sintering period with sharp peaks, the highest being in the range of 30°-40° as well as elevated intensity. the similarity between the xrd results of the two samples indicated that ha was the main content of ha-tcp powder derived from the synthesis of blood cockle shells. table 1 shows that ha-tcp powder derived from the synthesis of blood cockle shells with a 6-hour sintering period had the most dominant ha level of 54.5% and a tcp level of 9.1%. the table above also indicates that ha level in the group with a 12-hour sintering period was less than in the group with a 6-hour sintering period. however, the tcp level in the group subject to a 12-hour sintering period was double that of the group subject to one of 6 hours. table 2 shows that k4-generated scaffold had the highest percentage of porosity compared to the other three sample groups. furthermore, the porosity test results revealed that the group using 25% ha-tcp combined with 10% gelatin and a 6-hour sintering period demonstrated a higher average percentage porosity than the group using 25% ha-tcp combined with 20% gelatin and a 6-hour sintering period. similarly, the group using 25% ha-tcp combined with 10% gelatin and a 12-hour sintering period had a higher average percentage of porosity than the group using 25% ha-tcp combined with 20% gelatin and a 12hour sintering period. x 100 porosity (%) = x mb mk ρliquid x vb figure 1. porosity test equation.16 note: mb = wet mass of sample (gram) mk = dry mass of sample (gram) vb = volume of test object s(cm 3) ρliquid = density of liquid table 1. list of chemical compounds contained in scaffold based on xrd test on ha-tcp powder derived from the synthesis of blood cockle shells sintering times lasting 6 and 12 hours. sintering period (hours) percentage (%)chemical formulacompounds caha 5(po4)3 6 54.5(oh) catcp 3(po4)2 6 9.1 caha 5(po4)3 12 51.5(oh) catcp 3(po4)2 12 16.8 table 2. the porosity of the ha-tcp scaffold with variations in the percentage of gelatin and the results of the comparative test between groups pmean ± sdscaffold sample codesgroups 25% ha-tcp combined with 20% gelatin (with a 6-hoursk1 sintering period) 67.65 ± 0.872 0.000* 25% ha-tcp with 10% gelatin (with ak2 6-hours sintering period) 72.98 ± 2.250 25% ha-tcp with 20% gelatin (with ak3 12-hours sintering period) 67.21 ± 1.977 k4 25% ha-tcp with 10% gelatin (with a 12-hours sintering periiod) 77.51 ± 2.858 note: * p<0.05 (significantly different) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 161 ari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 158–163 from table 3 it can be seen that the results of the post hoc lsd test showed significant differences between each study group, except between k1 and k3 both of which showed a significance level of more than 0.05. a sem test at 1000x magnification was then conducted on pores derived from the randomly selected sample to be measured digitally through the computer. in all the samples tested, the scaffold pore size varied within the range of 41.02 μm to 73.63 μm. figure 3. xrd graph of ha-tcp powder derived from the synthesis of blood cockle shells with a sintering period lasting 12 hours. table 3. results of the post hoc lsd test k4k3k2groups k1 0.000*0.7220.000*k1 k2 0.001*0.000* k3 0.000* *p < 0.05 (significantly different) a b c figure 4. sem results in scaffold porosity at 1000x magnification. (a) 25% ha combined with 10% gelatin after a 12-hours sintering period; (b) 25% ha combined with 20% gelatin after a 12-hours sintering period; (c) 25% ha combined with 10% gelatin after a 6-hour sintering period. figure 2. xrd graph of ha-tcp powder derived from the synthesis of blood cockle shells with a sintering period lasting 6 hours. subsequently, a one-way anova test with a significance level of 95% (0.05) was performed, followed by an lsd post-hoc test. the one-way anova test showed significant differences between groups, where the value was one of p<0.05. in table 2, the results for significance between groups were 0 with a p value of less than 0.05 (0 <0.05). it indicates that there was a significant difference in the mean percentage of porosity between all groups. therefore, an lsd post hoc test was then performed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 162ari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 158–163 figure 4 shows that pores od various sizes were interconnected in all groups. the porosity of ha-tcp scaffold combined with 10% gelatin and a 12-hour sintering period formed pores with an average diameter of between 41.02-49.14 μm. meanwhile, the porosity of ha-tcp scaffold combined with 20% gelatin and a 12-hour sintering time formed pores with an average diameter of between 72.14 μm -73.63 μm. in addition, the porosity of ha-tcp scaffold combined with 10% gelatin and a 6-hour sintering period formed pores with an average diameter of between 41.76 μm 64.87 μm. discussion in xrd characterization, the main property of a material observed using x-ray waves is crystallinity. the longer the processing time, the greater the semicrystalline nature of the calcium phosphate phase. this is because the more protracted the duration of calcium phosphate formation, the more the particles will combine with others to form agglomerates. this situation causes changes in the crystalline properties of calcium phosphate and also widens the xrd diffractogram. in other words, the longer the duration of calcium phosphate formation, the greater the crystallite size because particles will combine with others (agglomeration).17 the ha-tcp samples with a 6-hours sintering period had the highest peak at 2θ = in the range 31˚-32˚ (figure 2). meanwhile, the ha-tcp samples with a 12-hours sintering period had the highest peak at 2θ = in the range 31˚-32˚ (figure 3). the majority of peaks identified from ha-tcp samples subject to varying sintering duration (6 and 12 hours) were the same, thereby signifying the presence of ha. this proves that, during the synthesis process, the ha composition is dominant. although the ha level at the end of the 6-hours sintering period was higher than the ha level at the end of the 12-hours sintering period, the tcp level at the end of the 6-hours sintering period was less than the tcp level at the end of the 12-hours sintering period. this is due to the hydrothermal method. the highest peak of ha level was at the end of the 6-hour sintering period. at the end of the 12-hours sintering period, the ha level decreased while the tcp level increased. the ha level at the end of the 12-hours sintering period decreased since ha has a peak point after which ha will decrease and be converted to tcp with the result that the tcp percentage increases, while the ha percentage decreases.18 porosity, the ratio of void space volume to the mass volume of a solid material, can be measured using the ratio between dry mass, wet mass and sample volume. there was a significant difference between porosity in ha-tcp scaffold combined with 25% gelatin (6-hours sintering) and that in ha-tcp scaffold combined with 10% gelatin (6-hours sintering). moreover, there was also a significant difference between the porosity of ha-tcp scaffold combined with 25% gelatin (12-hours sintering) and that in ha-tcp scaffold combined with 10% gelatin (12-hours sintering). the results of this research indicate that the composition of ha-tcp scaffold compounds is influenced by variations in the percentage of gelatin which is a natural polymer employed as a scaffold in tissue engineering. the combination of ha-tcp and gelatin will form covalent bonds between ca2+ ions and r-cooions derived from gelatin molecules. this crosslinking will then cause a reduction in the distance between ha-tcp-gelatin fibers.19 the greater the percentage of gelatin formed the more numerous the bonds resulting in a shorter distance between the fibers and reduced porosity. the freeze dry method applied during the manufacture of scaffolds removed liquid from the scaffolds20 with the result that rough structure patterns formed. this is because the ha-tcp particle powder used in this research was less than 74 μm in size, while the largest pore diameter was derived from ha-tcp scaffold combined with 20% gelatin. the largest pore diameter of ± 70 μm was found in the group using ha-tcp scaffold combined with 20% gelatin. meanwhile, the smallest pore diameter of ± 40 μm was in the group using ha-tcp scaffold combined with 10% gelatin. therefore, it is assumed that the addition of 20% gelatin can generate ± 70 μm pore diameter that is open and interconnected. finally, it can be concluded that the addition of various percentages of gelatin can affect the porosity of ha-tcp scaffolds as a result of the synthesis of blood cockle shells after a 6or 12-hours sintering period. the lower the percentage of gelatin, the higher the porosity of the scaffold. the highest porosity is found in ha-tcp scaffold with the addition of 10% gelatin. references 1. ardhiyanto hb. peran hidroksiapatit sebagai bone graft dalam proses penyembuhan tulang. stomatognatic. 2011; 8(2): 118–21. 2. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125–7. 3. zhang s. hydroxyapatite coatings for biomedical applications. boca raton: crc press; 2013. p. 202-5. 4. darwis d, warastuti y. sintesis dan karakterisasi komposit hidroksiapatit (ha) sebagai graft tulang sintetik. j ilmiah aplikasi isotop dan radiasi. 2008; 4(2): 143–53. 5. ha ra hap aw, helwa ni z , zultinia r z , yelm ida y. sintesis hidroksiapatit melalui precipitated calcium carbonate (pcc) kulit kerang darah dengan metode hidrotermal pada variasi ph dan waktu reaksi. jom fteknik. 2015; 2(2): 1–8. 6. salerno a, di maio e, iannace s, netti pa. tailoring the pore structure of pcl scaffolds for tissue engineering prepared via gas foaming of multi-phase blends. j porous mater. 2012; 19(2): 181–8. 7. loh ql, choong c. three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. tissue eng part b rev. 2013; 19(6): 485–502. 8. schönwälder sms, bally f, heinke l, azucena c, bulut öd, heißler s, kirschhöfer f, gebauer tp, neffe at, lendlein a, brenner-weiß g, lahann j, welle a, overhage j, wöll c. interaction of human plasma proteins with thin gelatin-based hydrogel films: a qcm-d and tof-sims study. biomacromolecules. 2014; 15(7): 2398–406. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 163 ari, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 158–163 9. agustin at, sompie m. kajian gelatin kulit ikan tuna (thunnus albacares) yang diproses menggunakan asam asetat. pros sem nas masy biodiv indon. 2015; 1(5): 1186–9. 10. mahmood sk, zakaria mzab, razak isba, yusof lm, jaji az, tijani i, hammadi ni. preparation and characterization of cockle shell aragonite nanocomposite porous 3d scaffolds for bone repair. biochem biophys reports. 2017; 10: 237–51. 11. schuurman w, levett pa, pot mw, van weeren pr, dher t wja, hut macher dw, melchels f pw, k lei n tj, ma lda j. gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. macromol biosci. 2013; 13(5): 551–61. 12. nojehdehyan h, torshabi m, tabatabaei fs. preparation and in vitro evaluation of 2 composite scaffolds containing gelatin for hard tissue engineering applications. j dent med. 2014; 27(3): 152–60. 13. imaniar acd. efektivitas bone graft kombinasi cangkang kerang darah (anadara granosa) dan gel minyak ikan lemuru (sardinella longiceps) terhadap peningkatan jumlah sel osteoblas pada proses penyembuhan tulang. thesis. surabaya: universitas hang tuah; 2017. p. 6, 17-8. 14. azami m, samadikuchaksaraei a, poursamar sa. synthesis a nd cha racter ization of a la m inated hyd roxyapatite/gelatin nanocomposite scaffold with controlled pore structure for bone tissue engineering. int j artif organs. 2010; 33(2): 86–95. 15. kusrini e, sontang m. characterization of x-ray diffraction and electron spin resonance: effects of sintering time and temperature on bovine hydroxyapatite. radiat phys chem. 2012; 81(2): 118–25. 16. han f, dong y, su z, yin r, song a, li s. preparation, characteristics and assessment of a novel gelatin-chitosan sponge scaffold as skin tissue engineering material. int j pharm. 2014; 476: 124–33. 17. sinambela f, windarti t, parsaoran p. pengaruh waktu pada pembentukan kalsium fosfat dengan sistem membran selulosa bakterial. j kim sains dan apl. 2012; 15(3): 105–10. 18. pratama af. karakteristik hidroksiapatit hasil sintesis cangkang kerang darah (anadara granosa) menggunakan metode hydrothermal dengan variasi waktu sintering. thesis. surabaya: universitas hang tuah; 2018. p. 1-58. 19. khan mn, islam jmm, khan ma. fabrication and characterization of gelatin-based biocompatible porous composite scaffold for bone tissue engineering. j biomed mater res part a. 2012; 100a(11): 3020–8. 20. porrelli d, travan a, turco g, marsich e, borgogna m, paoletti s, donati i. alginate-hydroxyapatite bone scaffolds with isotropic or anisotropic pore structure: material properties and biological behavior. macromol mater eng. 2015; 300(10): 989–1000. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p158–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p158-163 88 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 88–92 research report efficacy of topical hydrogel epigallocatechin-3-gallate against neutrophil cells in perforated dental pulp kun ismiyatin, ari subiyanto, michelle suhartono, paramita tanjung sari, olivia vivian widjaja and ria puspita sari department of conservative dentistry, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: one cause of pulpitis is mechanical trauma such as pulp perforation. the emergency treatment of pulpitis in a clinic uses eugenol. eugenol in a high concentration causes cytotoxicity, which causes local necrosis and inhibits the recovery process, while in lower doses it can cause oral mucosal hypersensitivity. due to these side effects, it is worth considering other biocompatible materials with minimal side effects, such as epigallocatechin-3-gallate (egcg), which is found in green tea. as a polyphenol, egcg has a radical scavenging ability, which has an effect on reducing the number of neutrophils. the application of egcg is expected to reduce neutrophils on the second day after injury so the rehabilitation process is completed more quickly and ongoing inflammation and pulp necrosis is prevented. purpose: to analyse the efficacy of topical hydrogel egcg in reducing the number of neutrophils after 48 hours in the perforated dental pulp of wistar rats. methods: 20 wistar rats were divided equally into four groups, which were designated control (c) and treatment groups (t1, t2, t3). the upper first molar teeth of each rat were perforated and then t1, t2, and t3 were given 60 ppm, 90 ppm and 120 ppm hydrogel egcg respectively. on the second day, the rats were sacrificed. hpa preparations were made to calculate the number of neutrophils in each group. data was analysed using kolmogorov–smirnov, levene’s, one-way anova and tukey hsd test (p<0.05). results: there were significant differences between t2 and t3 compared with c and t1 (p<0.05), but no significant differences in the comparison of t1 with c and of t2 with t3 (p>0.05). conclusion: 90 ppm hydrogel egcg is effective in reducing the number of neutrophils in the perforated dental pulp of wistar rats. keywords: epigallocatechin-3-gallate (egcg); inflammation; neutrophil; pulp perforation correspondence: kun ismiyatin, department of conservative dentistry, faculty of dental medicine, universitas airlangga, ji. mayjen. prof. dr. moestopo 47, surabaya 60132, indonesia. email: kun-is@fkg.unair.ac.id introduction according to the indonesian health profile of 2010, pulpitis was seventh out of the top ten causes of outpatient care in hospitals in indonesia and dental pulp treatment had the highest rate compared to other dental treatments.1 according to the american association of endodontics (aae) 2013, reversible pulpitis is a dental pulp inflammation that should be resolved and the pulp return to normal following appropriate management of the etiology. one of the treatments is excavation of the infected tissue, which can cause mechanical trauma such as iatrogenic errors. pulpal perforation due to iatrogenic errors occurs in approximately 2–12% of teeth receiving endodontic treatment.2,3 inflammation that continues to be chronic can cause failure in the tissue and this can lead to pulp necrosis.4 neutrophils are the first immune cells that arrive to lesions5 and will undergo apoptosis after 1–2 days.6 if neutrophils remain in injured tissue then the transition of proinflammatory m1 phenotype macrophages (classically activated macrophages) to reparative m2 phenotype macrophages (alternatively activated macrophages) will be inhibited, resulting in the slowing of the tissue-repair process by the reparative m2. at the site of infection or injury, neutrophil cells recognise and phagocyte microbes, then kill pathogens dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p88–92 mailto:kun-is@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p88-92 89ismiyatin et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 88–92 through cytotoxic damage. this method instigates the production of reactive oxygen species (ros) as well as the release of antimicrobial peptides.7 emergency treatment of pulpitis currently uses eugenol with concentrations of up to 74.3%.8 eugenol can have cytotoxic effects and also adversely affects fibroblast cells and osteoblasts, which causes local necrosis and inhibits the healing process. while at lower doses, eugenol can cause hypersensitivity reactions in the oral mucosa, inducing contact stomatitis and contact dermatitis.9 in view of these side effects, it is important to consider the use of other biocompatible materials with minimal side effects, such as the epigallocatechin-3-gallate (egcg) in green tea. egcg is the most concentrated polyphenol in green tea, which makes up around 50–80% of the total catechins.10 as a polyphenol, the structure of egcg has eight -oh groups.11 with more -oh groups, egcg is expected to be more effective in reacting with and binding to ros compared to eugenol, causing a more effective radical scavenging. the more effective radical scavenging is expected to lead to a faster decrease in the number of neutrophils that move toward the affected tissue, meaning the inflammatory and healing processes can be completed more quickly. egcg has been shown to affect several cellular mechanisms, including inflammation. egcg in cells inhibits neutrophil migration through endothelial cells and decreases the number of oxidative stress markers.12 in the pulpal inflammation of a rat tooth, topical 0.01% and 0.1% egcg were shown to inhibit pain distribution.13 as a polyphenol, egcg has a radical scavenging ability to clear ros, either directly by reacting with ros or indirectly by regulating the pathways that control the clearance of ros and enzymes.14 the study was carried out with the consideration that pure egcg is toxic to gingival fibroblast cells at a concentration of 150 μm, which is equal to 68.7 ppm.15 in general, hydrogels are used as a drug delivery system because of their ability to regulate drug release, protect drug contents from the outside environment16 and effectively disperse.17 in dental pulp regeneration therapy, hydrogel preparations can induce the release of fibroblast growth factor-2 (fgf-2) gradually and continuously.18 the most commonly used gel base in hydrogel preparations is polyethylene glycol (peg).16 peg is a hydrophilic polymer with low toxicity, immunogenicity and antigenicity but with excellent biocompatibility. the basic property of peg is hydrophilic, which makes peg the best choice of polymer for the hydrogel base.19 the good biocompatibility of peg leads to a better maintenance of cell viability.20 until now, there has been no research conducted to examine the concentration of topical hydrogel egcg that is effective in reducing the number of neutrophil cells as acute inflammatory cells in the dental pulps which are given lesions until perforated. this study was conducted to examine the effect of 60 ppm, 90 ppm, and 120 ppm hydrogel egcg application on the number of neutrophil cells in a tooth cavity that has been perforated. materials and methods ethical clearance was approved by the ethical eligibility committee of the dentistry faculty, universitas airlangga (number: 412/hrecc.fodm/vi/2019). this study was laboratory in vivo experimental research with posttestonly control group design that used 20 healthy male wistar rats (rattus norvegicus), approximately 3 months old and weighing 200–300 grams, as animal subjects. the subjects were divided equally into four groups (n=5): a control group (c) that received cavity preparation but no egcg application and three treatment groups that received cavity preparation and topical application of 60 ppm (t1), 90 ppm (t2) and 120 ppm (t3) hydrogel egcg respectively. peg hydrogel was produced by mixing 80% peg 400 (schuchardt ohg, germany) with 20% peg 4000 (sigmaaldrich, st. louis, usa).15 egcg hydrogel was produced by mixing egcg (xi’an rongsheng biotechnology co., ltd., shaanxi, china. batch number: 190702) with 80% peg 400 and 20% peg 4000. before cavity preparation, wistar rats were anaesthetised using a 0.2 cc intra-muscular injection of a mixture of ketamine (kepro b.v., deventer, holland) and xyla® xylazine base (pt tekad mandiri, bandung, indonesia) with a 1:1 ratio. preparation was performed on the occlusal surface of the upper right first molar tooth using a 0.8 mm diameter round bur (edenta®, edenta corp., switzerland) at low speed until it reached the pulp.21 the depth of preparation was as large as the bur head. perforation of the pulp chamber was performed using a 0.8 mm diameter round bur (edenta®, edenta corp., switzerland). to dry the cavity and confirm the presence of bleeding, which is a sign of pulp perforation, a fine paper point (inline®, b.m. dentale s.a.s., torno, italy) was used. wateronetm saline (pt jayamas medica industri, indonesia) was used to clean up the bleeding. egcg hydrogel was measured using a micropipette (acura® manual 825, socorex isba sa, switzerland) then applied using a microbrush (tpc®, tpc advances tech. inc., usa) to the base of the cavity of the upper right first molar tooth in the treatment groups that had been prepared. 60 ppm, 90 ppm and 120 ppm of egcg hydrogel were applied respectively to the t1, t2 and t3 groups. after the application, the cavities were filled with glass ionomer cement (gic) (fuji 9, gc corp, tokyo, japan). the wistar rats were sacrificed 2x24 hours after completion of the treatment in order to obtain analysis specimens by surgically removing the upper right first molar tooth along with the jaw. the maxillae were then fixed with 10% buffered formalin (polysciences, polysciences inc., usa) and decalcified using 10% ethylen diamin tetraacetyc acid (edta) (rpi, rpi corp., usa) at ph 7.4 with the solution being replaced every three days during 30 days of immersion at room temperature. the samples were taken from the dental pulp of the teeth. the specimens were dehydrated by soaking in stratified alcohol followed by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p88–92 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p88-92 90 ismiyatin et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 88–92 the purifying material. next, the specimens were placed into xylol-paraffin (1:1), followed by pure paraffin i, pure paraffin ii and pure paraffin iii for 60 minutes each. pure paraffin was poured into the box, up to the brim, without any air bubbles inside the paraffin block. the specimen was then inserted into the paraffin block using pointed tweezers. the tissue position was arranged so that when the block was cut it would provide longitudinal pieces. after the paraffin block hardened, a 6 μm thick paraffin slice was cut to be painted with hematoxylin eosin (he) staining. observation of the number of neutrophils in each specimen was carried out using a light microscope (olympic, usa) under 400x magnification and then a photo preparation was made. calculation of the number of neutrophil cells was carried out in the area under the cavity preparation with eight different fields of view and counting was performed manually through photos with the help of ocular micrometer (graticule). the results were divided into eight, according to the number of fields of view, to obtain the average number of neutrophil counts for each sample in each group. total calculation of the average for each sample in each group was then divided by the number of samples in each group to obtain the average number of neutrophils in each group. the averages and the standard deviation of the study were calculated. all the data obtained was analysed with the statistical package for the social sciences (spss) version 20 (ibm, new york, usa), using the kolmogorov-smirnov test to find out whether the data was normally distributed. after confirming that the data was normally distributed, levene’s test was used to evaluate homogeneity. a one-way anova was then carried out, followed by a tukey hsd test to determine significant differences between groups. significant differences were considered to be present in p<0.05. results the data in table 1 shows the mean and the standard deviation (sd) of neutrophil cells in the control and treatment groups. the expression of neutrophil cells can be seen in figure 1. the results of normality and homogeneity table 1. the mean number (𝐱 ̅) and standard deviation (sd) of neutrophil cells in each control and treatment group. groups n mean and sd (𝐱̅ ± sd) c 5 11 ± 1 t1 5 11 ± 1.225 t2 5 7.2 ± 0.837 t3 5 6.2 ± 0.837 notes: n= number of samples; c= given injury but not given any treatment; t1= treated with 60 ppm hydrogel egcg; t2= treated with 90 ppm hydrogel egcg; t3= treated with 120 ppm hydrogel egcg. table 2. tukey hsd test results of neutrophil cells between groups. c t1 t2 t3 c 1.000 0.000* 0.000* t1 0.000* 0.000* t2 0.406 t3 notes: *= significantly different; c= given injury but not given any treatment; t1= treated with 60 ppm hydrogel egcg; t2= treated with 90 ppm hydrogel egcg; t3= treated with 120 ppm hydrogel egcg. t2 t3 t1 c figure 1. expression of neutrophil cells in hpa preparation with 400x magnification. the black arrows indicate positive expressions. c group was given injury but not given any treatment, t1 group was treated with 60 ppm hydrogel egcg, t2 group was treated with 90 ppm hydrogel egcg and t3 group was treated with 120 ppm hydrogel egcg. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p88–92 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p88-92 91ismiyatin et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 88–92 tests showed that the data was normally distributed and homogeneous so that it fulfilled the requirements for parametric tests using one-way anova. the one-way anova test results showed that there were significant differences in the number of neutrophils between groups (p<0.05). the tukey hsd test (table 2) was then carried out to investigate significant differences between groups and it showed that there was no significant difference (p>0.05) in the decrease of neutrophil cells between c group and t1 group. the neutrophil cells were significantly lower (p<0.05) in groups t2 and t3 than in the groups c and t1, but there was no significant difference (p>0.05) in the decrease of neutrophil cells between t2 group and t3 group. discussion in the inflammatory process, nitric oxide (no) triggers the formation of reactive radical species through chemical reactions with oxygen. in the first 48 hours, no mainly has a role in vasodilation, antimicrobial activity, antiplatelet aggregation activity and in the induction of vascular permeability.22 no can cause vasodilation so that inflammatory cells, including neutrophils as acute inflammatory cells, migrate towards injury.23,24 egcg extract, which was used in this study, is a polyphenol that acts as a radical scavenger. no, which is a free radical, also binds to the -oh groups of egcg. furthermore, egcg as an anti-inflammatory inhibits the activation of nuclear factor-κb (nf-κb) and activator protein-1 (ap-1), thereby reducing inducible nitric oxide synthesis (inos) expression. the application of egcg in lesions also has the effect of inhibiting the production of inos, which is induced by interleukin-1β (il-1β) and interferon-γ (ifn-γ), and decreasing mrna inos and protein inos, causing a decrease in no expression in blood vessels.25 a previous study showed that various cells could express il-6 and il-8, both of which are believed to participate in tissue injury related to inflammation or neutrophils.26 this results in vasoconstriction of blood vessels, which in turn results in capillary permeability decrease, so that the migration of neutrophil cells into the affected tissue is inhibited and the number of neutrophil cells that reach the affected tissue is reduced, causing the acute phase of inflammation to end more quickly. modulation of the decrease in neutrophil cells facilitates the safe clearance of neutrophils from lesions, which is through macrophage cells, which happens within a few days, with minimal damage to tissue. this is beneficial because the mobilisation and ongoing activity of neutrophils can trigger chronic pathological responses.12 it was found that administration of egcg extract can reduce the number of neutrophil cells in the dental pulp of wistar rats that have perforated tooth cavities. when compared to the control group, significant reduction in neutrophils was found in the group that was given 90 ppm hydrogel egcg and the group that was given 120 ppm hydrogel egcg. significant differences between the number of neutrophils in the control group and the groups that were given 90 ppm and 120 ppm hydrogel egcg showed that there was a decrease in the number of neutrophil cells toward the lesion in the groups that were given 90 ppm and 120 ppm hydrogel egcg. the results of this study are in line with the previous study that stated that egcg with higher concentrations has a greater antioxidant activity than egcg with lower concentrations. in that study, the comparison was between 100 ppm egcg and 50 ppm egcg in the form of emulsion.27 a decrease in the number of neutrophil cells present at an injury can lead to an increase in neutrophil apoptosis, which can shorten the inflammatory process and minimise tissue damage.28 however, the group that was given 60 ppm hydrogel egcg did not experience a significant decrease in neutrophils compared to the control group, which suggests that 60 ppm hydrogel egcg was not concentrated enough to effectively perform radical scavenging activities. if the number of hydroxyl groups increases, the radical scavenging will become stronger.11 therefore, there was a significant decrease in the number of neutrophil cells in the group given 90 ppm and the group given 120 ppm hydrogel egcg compared to the control group. there was no significant decrease in neutrophils in the group that was given 120 ppm hydrogel egcg compared to the group that was given 90 ppm hydrogel egcg. but an insignificant difference between the groups that were given 90 ppm and 120 ppm hydrogel egcg was shown by the number of neutrophils in the same amount. this shows that hydrogel egcg at a concentration of 90 ppm is effective in reducing the number of neutrophil cells in the perforated dental pulp of wistar rats and increasing the number of neutrophils is almost the same as 120 ppm hydrogel egcg. this is possibly related to the egcg saturated concentration binding to peg29 and the self-oxidation properties of egcg. in the mixture of egcg with peg, sediment is produced due to the bond between egcg and the polymer that produces colloids and aggregates. colloids increase the difficulty in formulation and reduce the efficacy of polyphenols.30 several studies have shown that high concentrations of egcg can cause self-oxidation and function as pro-oxidants by producing hydroxyl radicals, hydrogen peroxide and quinonoid intermediates, which cause cytotoxicity. the study by chen et al.31 found that catechol-quinone produced by self-oxidation of egcg and egc can crosslink with erythrocyte membrane proteins as a crosslinking link, thus leading to membrane protein aggregates; the galloyl group is an important group of catechins that have a pro-oxidative effect. furthermore, in physiological concentrations (1–2 μm to 10 μm), egcg can produce a small number of reactive oxygen species to activate several signalling pathways and generate appropriate cellular protection mechanisms, thus dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p88–92 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p88-92 92 ismiyatin et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 88–92 presenting an antioxidant effect.32 the biological effects of egcg are likely to be related to its metabolic product.33 the pro-oxidant effect of egcg can result in neutrophil infiltration.34 this study proves that the application of egcg on the dental pulp in the perforated tooth cavity of wistar rats can cause a decrease in the number of neutrophil cells on the second day after the administration to a mechanical trauma lesion. however, the limitation of this study is that it was conducted on wistar rats. further studies need to be done before clinical application can be conducted. references 1. kementerian kesehatan republik indonesia. profil kesehatan indonesia 2010. ja ka r ta: kementer ia n kesehata n republik indonesia; 2011. p. 1–220. 2. tsesis i, fuss z. diagnosis and treatment of accidental root perforations. endod top. 2006; 13(1): 95–107. 3. widjiastuti i, subiyanto a, ningtyas ek, popyandra r, kurniawan mg, retnaningsih fd. propolis extract as pulp capping material enhances odontoblast-like cell thickness and type 1 collagen expression (in vivo). dent j (majalah kedokt gigi). 2020; 53(1): 1–5. 4. garg n, garg a. textbook of endodontics. new delhi: jaypee brothers medical publishers; 2010. p. 59–60. 5. greenlee-wacker mc. clearance of apoptotic neutrophils and resolution of inflammation. immunol rev. 2016; 273(1): 357–70. 6. kristanti s, hendrianingtyas m. hubungan neutrophils/lymphocytes ratio dan c-reactive protein pada infeksi neonatal. j nutr heal. 2017; 5(3): 187–94. 7. mayadas tn, cullere x, lowell ca. the multifaceted functions of neutrophils. annu rev pathol mech dis. 2014; 9(1): 181–218. 8. bendre rs, rajput jd, bagul sd, karandikar ps. outlooks on medicinal properties of eugenol and its synthetic derivatives. nat prod chem res. 2016; 4(3): 1–6. 9. deshpande a, verma s, macwan c. allergic reaction associated with the use of eugenol containing dental cement in a young child. austin j dent. 2014; 1(2): 1–3. 10. dewi k. pengaruh ekstrak teh hijau (camellia sinensis var. assamica) terhadap penurunan berat badan, kadar trigliserida dan kolesterol total pada tikus jantan galur wistar. j kedokt maranatha. 2008; 7(2): 1–11. 11. singh bn, sha n ka r s, sr ivastava r k. g reen tea catech in, epigallocatechin-3-gallate (egcg): mechanisms, perspectives and clinical applications. biochem pharmacol. 2011; 82(12): 1807–21. 12. donà m, dell’aica i, calabrese f, benelli r, morini m, albini a, garbisa s. neutrophil restraint by green tea: inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis. j immunol. 2003; 170(8): 4335–41. 13. ismiyatin k, soetoyo a, wahluyo s, safitri i. therapeutic efficacy of topical epigallocatechin-gallate as a new therapeutic strategy for inhibition of pain conduction on rat models with acute pulpal inflammation. in: international medical device and technology conference. johor bahru: universiti teknologi malaysia; 2017. p. 107–10. 14. shay j, elbaz ha, lee i, zielske sp, malek mh, hüttemann m. molecular mechanisms and therapeutic effects of (-) -epicatechin and other polyphenols in cancer, inf lammation, diabetes, and neurodegeneration. oxid med cell longev. 2015; 2015: 1–13. 15. weisburg jh, weissman db, sedaghat t, babich h. in vitro cytotoxicity of epigallocatechin gallate and tea extracts to cancerous and normal cells from the human oral cavity. pharmacol toxicol. 2004; 95(4): 191–200. 16. ismiyatin k, wahluyo s, purwanto da, rahayu rp, soetojo a, mukono is. effect of topical epigallocatechin-gallate on lipopolysaccharide-induced pulpal inflammation in rat models. iran endod j. 2018; 13(4): 528–33. 17. setiawan i, lindawati ny, amalia b. formulasi dan uji antiinflamasi sediaan hidrogel ekstrak jahe merah. media farm indones. 2018; 13(1): 1330–4. 18. silna ea, krishnakumar k, nair sk, narayanan a v, dineshkumar b. hydrogels in topical drug delivery a review. int j innov drug discov. 2016; 6(2): 87–93. 19. srivastava a, yadav t, sharma s, nayak a, kumari a, mishra n. polymers in drug delivery. j biosci med. 2016; 4: 69–84. 20. zavisova v, koneracka m, kovac j, kubovcikova m, antal i, kopcansky p, bednarikova m, muckova m. the cytotoxicity of iron oxide nanoparticles with different modifications evaluated in vitro. j magn magn mater. 2015; 380: 85–9. 21. sabir a, tabbu cr, agustiono p, sosroseno w. histological analysis of rat dental pulp tissue capped with propolis. j oral sci. 2005; 47(3): 135–8. 22. broughton g, janis je, attinger ce. the basic science of wound healing. plast reconstr surg. 2006; 117(7 suppl): 12s-34s. 23. enggardipta ra, haniastuti t, handajani j. efek eugenol terhadap jumlah sel inflamasi pada pulpa gigi molar tikus sprague dawley. maj kedokt gigi indones. 2016; 2(2): 66–73. 24. dwiandhono i, effendy r, kunarti s. the thickness of odontoblastlike cell layer after induced by propolis extract and calcium hydroxide. dent j (majalah kedokt gigi). 2016; 49(1): 17–21. 25. han mk. epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic β-cell damage. exp mol med. 2003; 35(2): 136–9. 26. erdemir eo, hendek mk, keceli hg, apan tz. crevicular fluid levels of interleukin-8, interleukin-17 and soluble intercellular adhesion molecule-1 after regenerative periodontal therapy. eur j dent. 2015; 9(1): 60–5. 27. liu tt, yang ts. effects of water-soluble natural antioxidants on photosensitized oxidation of conjugated linoleic acid in an oil-inwater emulsion system. j food sci. 2008; 73(4): c256–61. 28. mccracken jm, allen lah. regulation of human neutrophil apoptosis and lifespan in health and disease. j cell death. 2014; 7(1): 15–23. 29. peter b, farkas e, forgacs e, saftics a, kovacs b, kurunczi s, szekacs i, csampai a, bosze s, horvath r. green tea polyphenol tailors cell adhesivity of rgd displaying surfaces: multicomponent models monitored optically. sci rep. 2017; 7: 1–16. 30. cao y, teng j, selbo j. amorphous solid dispersion of epigallocatechin gallate for enhanced physical stability and controlled release. pharmaceuticals. 2017; 10(4): 1–17. 31. chen r, wang jb, zhang xq, ren j, zeng cm. green tea polyphenol epigallocatechin-3-gallate (egcg) induced intermolecular crosslinking of membrane proteins. arch biochem biophys. 2011; 507(2): 343–9. 32. yang h, guo j, deng d, chen z, huang c. effect of adjunctive application of epigallocatechin-3-gallate and ethanol-wet bonding on adhesive-dentin bonds. j dent. 2016; 44: 44–9. 33. chu c, deng j, man y, qu y. green tea extracts epigallocatechin-3gallate for different treatments. biomed res int. 2017; 2017: 1–9. 34. goodin mg, bray bj, rosengren rj. sexand strain-dependent effects of epigallocatechin gallate (egcg) and epicatechin gallate (ecg) in the mouse. food chem toxicol. 2006; 44(9): 1496–504. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p88–92 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p88-92 163 vol. 43. no. 4 december 2010 literature reviews clinical consideration of thrombocytopenia in children s. ratna laksmiastuti department of pediatric dentistry faculty of dentistry, trisakti university jakarta indonesia abstract background: pediatric patient with the history of bleeding disorder as thrombocytopenia is considered as a clinical case in dentistry. the patient with platelet count below normal has potential risk of bleeding disorders. the situation would be more dangerous if the dentist could not identify the problem. purpose: the aim of this review is to describe how a dentist must understand the step and management that should be performed in pediatric patient with history of bleeding disorder due to thrombocytopenia. reviews: bleeding disorders might be the result of thrombocytopenia, a condition that alter the ability of blood vessels, platelet and coagulation factors in normal hemostatic system. thrombocytopenia is defined as a platelet count of less than normal (150.000/mm3–400.000/mm3). etiology, risk factor and preventive method of thrombocytophenia are still unknown. conclusion: it is concluded that special attention is needed for pediatric patient with thrombocytopenia. a dentist should understand well about this disorder on how to take the history, to do clinical examination, to establish the diagnosis and to decide treatment plan as well as to consult to related collongues. key words: bleeding disorder, thrombocytopenia, dentist abstrak latar belakang: penderita anak-anak dengan riwayat gangguan perdarahan yang ditandai dengan adanya trombositopenia merupakan masalah klinis yang ditemukan dalam bidang kedokteran gigi. beberapa perawatan di bidang kedokteran gigi beresiko menimbulkan terjadinya perdarahan. keadaan akan berbahaya bila dokter gigi tidak dapat mengidentifikasi masalah. tujuan: artikel ini menunjukkan pentingnya seorang dokter gigi memahami dan mengetahui langkah dan tindakan apa yang harus dilakukan, bila suatu saat menghadapi penderita anak-anak dengan riwayat gangguan perdarahan akibat trombositopenia. review: gangguan perdarahan adalah suatu kondisi terjadinya penurunan kemampuan dari pembuluh darah, platelet, dan faktor pembekuan pada fungsi normal hemostasis. trombositopenia adalah jumlah trombosit/platelet dalam darah berada pada jumlah di bawah normal (150.000/mm3–400.000/mm3), yang dapat mengakibatkan terjadinya gangguan perdarahan. penyebab, faktor risiko dan cara pencegahan kasus trombositopenia belum diketahui dengan pasti. kesimpulan: diperlukan perhatian khusus pada penderita anak yang mengalami gangguan perdarahan. seorang dokter gigi harus mengetahui dengan baik bagaimana menggali informasi tentang riwayat penyakit, melakukan pemeriksaan klinis yang tepat, menegakkan diagnosis, menentukan rencana perawatan serta melakukan rujukan bila diperlukan. kata kunci: gangguan perdarahan, trombositopenia, dokter gigi correspondence: s. ratna laksmiastuti, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas trisakti. kampus b, jl. kyai tapa (grogol) jakarta barat, indonesia. e-mail: ratna_oct@hotmail.com introduction the sign and symptom of thrombocytopenia that connected to hematologic disease, commonly caused by the decreasing or increasing of erythrocyte (anemia or erythrocytosis); leukocyte (leucopenia or leucocytosis); thrombocyte (thrombocytopenia or thrombocytosis), hemostatic disturbance (bleeding or coagulation) or 164 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 163–167 neoplasia in lymphoreticular system (lymphoma or dyscrasia of plasma cell).1,2 thrombocytopenia defined as platelet count of less than normal, is the most common cause of defective primary hemostasis that can lead to significant bleeding in children.3 this condition is sometimes associated with abnormal bleeding. bleeding disorders are conditions that alters the ability of blood vessel, platelet and coagulation factor in normal hemostatic function. genetic factor also plays an important role in bleeding disorders but without genetic factor, it should be due to disease related to the integrity of blood vessel wall, platelet and coagulation factor and can also be contributed by the use of medicine, radiation or chemotherapy in cancer case. most of bleeding disorders are iatrogenic.4 pediatric patient with the history of bleeding disorder indicated to have thrombocytopenia is considered clinical case in dentistry. bleeding is also a potential risk in dental treatment. in normal patient, the risk of bleeding can be minimized. in patient with the decrease of bleeding control ability due to medication or a certain disorder, this condition would endanger the patient if the dentist could not identify the problem. correct diagnosis and management are really necessary. a dentist is highly requested to understand the step and the management that should be performed in pediatric patient with history of bleeding disorder due to thrombocytopenia. the management include medical history, review all medications, proper clinical examination, laboratory screening, establishing diagnosis and correct treatment plan. in addition it is also important to know family history of the patient and with consult to related colleagues. definition and etiology of thrombocytopenia thrombocytopenia is one of bleeding disorders indicated by decreasing number of thrombocyte/platelet in blood circulating (<150.000/mm3). platelet/thrombocyte is an important cell in the process of blood coagulation. the lower number of thrombocytes the higher the risk of bleeding will occur. the normal number of platelet is 150.000/mm3– 400.000/mm3.1,2 etiology, risk factor and method of prevention are still unknown. many conditions can cause thrombocytopenia.1 platelet disorders may devided into two catagories by etiology, congenital and acquired and into two additional categories by type, thrombocytopenias (quantity platelet disorders) and thrombocytopathies (qualitative platelets disorders).5 the decrease number of platelet/thrombocyte is caused by the failure or decreasing of platelet production, disturbance of platelet distribution and increasing of platelet destruction. decreasing or failure of thrombocyte production is due to cytotoxic drug (eg: chemotherapy, radiation in malignancy). radiation therapy or chemotherapy destroys megakaryocytic, the precursor cells that produce platelets in the bone marrow. aplastic anemia can impair platelet production, spinal cord transplantation, nutrition deficiency (eg: b12, folat, cobalamin) and short-term low platelets is also associated with some viral infection (eg: rubela, varicela, mumps, hiv, epstein barr virus). disturbance of thrombocyte distribution is caused by abnormal circulating thrombocyte in the spleen or splenomegaly. meanwhile, increasing of thrombocyte destruction is contributed by immune system disturbance that is: shortening survival time of platelet from 10 days into at the longest 1 day.2,6–8 genetic abnormalities, may impair production of normal platelets. a cancer, such as lymphoma, in the bone marrow can inhibit production of platelets. certain drugs, especially thiazide diuretics or alcohol, depresses production of precursor cells that produce platelets in the bone marrow.2 idiophatic thrombocytopenic purpura (itp) is the most common cause of acute onset thrombocytopenia in otherwise healthy children. it characterized thrombocytopenia due to autoantobody binding platelet antigen, and causing premature destruction of platelet. this condition is frequently associated with history of in viral illness in 50–60% of cases.9 other autoimmune diseases, such as hiv infection, systemic lupus erythematosus (sle), lymphoproliferative disorders, myelodysplasia, hypogammaglobulinemia, drug induced, can cause thrombocytopenia.10 classification of bleeding disorders b l e e d i n g d i s o r d e r s a r e d e v i d e d i n t o n o n thrombocytopenic purpuras, thrombocytophenic purpuras, and disorders of coagulant. the first, non-thrombocytopenic purpuras consist of vascular wall alteration (eg: scurvy, infection, chemicals, allergy) and disorders of platelet function (eg: genetic defects/bernard-soulier disease; drugs like aspirin, nsaid, alcohol, beta-lactam antibiotics, penicillin, cephalotins; allergy; autoimmune disease, von willebrand’s disease/secondary factor viii deficiency, uremia). the second, thrombocytopenic purpuras consist of primary-idiophatic and secondary (eg: chemicals, physical agents like radiation, systemic disease like leukemia, metatastic cancer to bone, splenomegaly, drugs (like alcohol, thiazide diuretics, estrogens, and gold salts), vasculitis, mechanical prosthetic heart valves, and viral or bacterial infections). the third, disorders of coagulant is catagorized into inherited and acquired. the inherited group consist of hemophillia a (deficiency of factor viii), hemophillia b (deficiency of factor ix) and others. the acquired group consist of liver disease, vitamin deficiency (eg: biliary tract obstruction, mal-absorption, excesscive use of broad-spectrum antibiotics), anticoagulation drugs (eg: heparin, coumarin, aspirin and nsaid), disseminated intravascular coagulation and primary fibrinogenolysis.4 pathophysiology of thrombocytopenia pathophysiology of thrombocytopenia frequently cannot be understood clearly (idiophatic).1 sixty percent of thrombocytopenic patients caused by autoimmune disorder, as a result the antibody would be against membrane of glycoprotein platelet as igg. platelet which has been covered by igg susceptible to phagocytic macrophage of the spleen. igg autoantibodies might also contribute destruction of megakaryocyte as precursor platelet. consequently in bone marrow the number of platelet would decrease.11–13 165laksmiastuti: clinical consideration of thrombocytopenia clinical symptoms and oral manifestation of thrombocytopenia thrombocytopenia is bleeding disorders with the sign of bleeding in epidermal or mucosa, resulting in petechiae (small red patches) or ecchymosis (small hemorraghic spot) in oral mucosa, membrane mucosa and gingiva. another sign of thrombocytopenia is bruises in epidermal layer or membrane mucosa with unknown cause and if the size of bruises is wider, it is called hematoma.2,7 when the platelet drastically decreases until below 40.000/mm3, in general, the patient will have petechiae, ecchymosis, spontaneous bleeding in gingival, urinary and gastrointestinal tract. the most common character and symptom of thrombocytopenia is manifested in oral cavity, spontaneous bleeding after tooth brushing or small trauma. gingival bleeding occur easily and repeatedly, followed by blood clot in gingival margin and the color will change gradually into dark color and finally cover the teeth.2,4 the classical features in children from high income countries such as sudden on set of excessive bruising, petechiae, and or mucous membrane bleeding 1–4 weeks following viral infection.10 laboratory examination a dentist can recommend the patients suspected with bleeding disorder due to thrombocytopenia to undergo laboratory examination. items which should be observed are complete blood count which show the size, number and maturity of blood cells. platelet count is also important to understand the possibility of bleeding disorder due to thrombocytopenia through the number of thrombocyte/ platelet: normally between 150.000/mm3–400.000/mm3 or 140.00/mm3–400.000/mm3. the patient with platelet number 500.000/mm3–100.000/mm3 has potential risk of bleeding more serious than normal if he has major trauma, while the patient with platelet number less than 50.000/mm3 he will have symptom of pupura on the skin and mucosa if he has minor trauma. the patient will have spontaneous bleeding if the number of platelet is less than 20.000/mm3. bleeding time must also be examined to understand the bleeding duration and this examination is performed to find congenital disorder of platelet function. the normal bleeding duration is 1–6 minutes.7 platelet function analyzer 100 should be observed, to detect platelet dysfunction. the normal value is < 175 seconds. finally the urine test is used to detect the possibility of infection.2,4 dental management of thrombocytopenia patient’s identification is very essential to be done by a dentist before performing the treatment. four methods applied to indentify a patient: to understand the history of illness, to perform physical examination, laboratory examination and to observe postoperative bleeding.2 to understand the history of illness, some questions are necessary to be given to the patient such as: whether there is bleeding problem in the family (spontaneous bleeding), serious bleeding after trauma, surgery or tooth extraction; what kind of past and present disease suffered by the patient. patient with thrombocytopenia is not recommended to have regular dental treatment until good general condition is obtained. emergency treatment should be done palliatively and the patient should be referred and consulted to related colleagues to establish the diagnosis and to decide proper treatment plan. advice to increase oral hygiene, regular and periodical check up and treatment are really necessary. a dentist can recommend laboratory examinations to detect suspected patient with bleeding disorder by screening the complete blood count, platelet count, bleeding time, platelet function analyzer 100, urine test.2,4 the essential treatment in dentistry for thrombocytopenic patient is to reduce the inflammatory by elemination of local irritating factor. periodontal treatment are scaling and root planning, usually safe for platelet count < 60.000/mm3. operation will be done if the platelet count is > 80.000/mm3, platelet transfusion is possible to be done before surgery if it is necessary. tooth extraction is the last choice of treatment but it should be performed at the hospital equipped with platelet transfusion.7 spontaneous gingival bleeding can be managed with oxidizing mouthwashes. good oral hygiene and conservative periodontal therapy help to remove the plaque and the calculus that trigger the bleeding. platelets level of 50.000/mm3 are desirable before dental treatment and further transfusion should be given as needed to maintain hemostasis.14 mandibular block anesthetic should be avoided to prevent complication such as: hematoma and obstruction of air way. anti fibrinolytic administration such as: tranexamic acid 15–20 mg/kg body weight is recommended.15 the history of illness, the history of medication and the type of anticoagulant are essential to know, therefore, consulting to releated colleagues is needed. trauma of the operation should be as minimal as possible and local hemostatic agent should be applied. in general, patient with thrombocytopenia is treated using corticosteroid and tends to stress during dental treatment, consequently stress control is necessary to be done. interaction between barbiturate and anticoagulant must be observed. in thrombocytopenia patient with adequate hemoglobin, nitrous oxide-oxygen is well accepted anxiolytic. analgesic selection should be well, acetaminophen and/or codein, aspirin and nsaid, should be avoided due to antiprostaglandin effect which will increase the possibility of bleeding. there are some medicines that are potential to contribute thrombocytopenia such: quinine and quinidine.2,4,7 penicillin per oral is the main choice to prevent and to treat infection with the main choice to prevent and to treat infection with condition that the patient has no hypersensitivity. tetracycline is effective to increase coumarin anticoagulant. prolonged antibiotic administration might disturb the balance of intestinal flora which has important in vitamin k absorption, therefore it might increase the risk of bleeding.16 166 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 163–167 discussion some dental treatments are risky to the occurrance of bleeding. in normal patient, the risk of bleeding can be minimized but in patient with decreasing ability to control bleeding due to medication or a certain disease, this condition would be dangerous if the dentist could not identify the problem.4 prolonged bleeding can occur when haemostasis is disturbed. primary haemostasis is initiated after injury to a blood vessel with the formation of a primary platelet plug. the process is mediated by interactions between platelets, plasma coagulation factors and vessel wall. defect of primary haemostasis generally result in bleeding from the skin or mucosal surfaces. in patient with thrombocytopenia, the number of platelet is less than normal due to decreasing ability to control bleeding. decreasing thrombocyte or thrombocyte function will cause initial clot formation failure. children with thrombocytopenia will have instant bleeding after trauma or surgery. this case is very different from children with hemophilia who suffer from bleeding 4 hours after the onset of trauma.15 acquired thrombocytopenia is classified into immune and non-immune. the example of immune classification is itp currently, it is known as idiopathic thrombocytopenic purpura is acquired bleeding disorder which most commonly happens in children.17 the incidence of itp is 50–100 in 1.000.000 individual/year, and the percentage of children is 50%. this condition is not malignancy and the prognosis is good. according to a research, 15–30% children suffer from acute itp and alter into chronic itp.18 as noted, acute itp is the most common bleeding disorder of childhood. it occurs most frequently in children aged 2–5 years and often follows infection with viruses such as rubella, varicella, measles, or epstein-barr. most patients recover spontaneously within a few months.19 childhood itp is acute and generally seasonal in nature, suggesting that infectious or environmental agents may trigger the immune response to produce platelet-reactive autoantibodies 4 to 8 weeks following an infection. in general, the patient is well apart from the diffuse bruising and petechiae indicative of a profound thrombocytopenia. the peak age of acute itp is 2 to 5 years of age, a period when children experience the greatest frequency of viral infections.20 in infection condition, inbalance of thrombocyte number frequently happens consequently abnormal bleeding can occur such as spontaneous epistaxis in children when having an infection.21 the cause, risk factor and preventive method of thrombocytopenic are still unknown. mostly due to igg auto body, which bind the platelet by itself.13 misdiagnosis of thrombocytopenia sometimes happen. in a research by bader-meunier et al., was found that children the suspected diagnosis on referral was autoimmune thrombocytophenic purpura and the final diagnosis was inherited thrombocytopenia. the suggestive of inherited thrombocytopenia included a history of familial thrombocytopenia, failure of steroid and or intravenous ig to raise the platelet count to normal level.22 thrombocytopenia and splenomegaly was also found as unusual presentation in children with congenital hepatic fibrosis. a 10-year-old girl of caucasian was reported that the bood count showed thrombocytopenia, platelet count (68×103/mm3). congenital hepatic fibrosis is a rare autosomal recessive desease that affects hepatobiliarry and renal systems.23 children with severe iron deficiency has been reported have less platelet count. but the validity of the association and the mechanism of thrombocytopenia are not well established.24 thrombocytopenia also had a link with vaccination in childhood. the immunization monitoring program active data on vaccine-associated thrombocytopenia, conducted by the canadian paediatric society, reports 103 cases vaccineassociated thrombocytopenia since 1992. the median age was 13 months, and 61% of those affected were boys. petechiae and bruising were the typical presenting sign. thrombocytopenia is a rare, but important adeverse event following vaccination. the clinical sign and management are similiar to itp.25 itp in children is usually self-limiting disorder presenting most commonly with short history of bruising and purpura. it may follow a firal infection or immunization and caused by an appropriate response of the immune system.26 ryugo sato and massimo franchini found the correlation between heliobacter pylori infection and itp. the patient is given antibiotic to eliminate bacterial infection causing drastically decrease of platelet number.16,27 oral care providers like dentist and dental hygienist, must be aware of the impact of bleeding disorders on the management of dental patients. initial recognition of a bleeding disorder, which may indicate the presence of a systemic pathologic process, may occur in dental practice.28 thrombocytopenia is a common hematologic finding in patients infected with the human immunodeficiency virus. multiple mechanisms may contribute to the development of chronic thrombocytopenia as immune-mediated platelet destruction, enhanced platelet splenic sequestration and impaired platelet production.29 congenital abnormalities, of platelet function or production for example glanzmann’s thromboasthenia and wiskottaldrich syndrome are rare. clinical symptoms are bruising, epistaxis, gingival hemorrhage or bleeding, palatal petechiae and menorrhagia.5 acute malaria is often associated with mild or moderate thrombocytopenia in non immune adult and children from malaria-endemic areas. it is not specific indicator of infection with malaria parasites.30 promoting oral hygiene motivation and plaque control are crucial problem to prevent gingival bleeding, inflammation and severe periodontal disease in patient with thrombocytopenia. understanding clinical finding in oral cavity, good cooperation among dentist, patients and related colleagues are essentially needed in patient’s management. good dental management can support to increase general 167laksmiastuti: clinical consideration of thrombocytopenia health.18 medicines causing thrombocytopenia can be classified into 3 group: medicine related with decreasing thrombocyte production (chemoteraphy, diuretic, thiazide, alcohol, estrogen, cloramphenicol, ionization radiation), medicine related with trombocyte destruction (sulfonamide, quinidine, kinine, carbamazepine, valproic acid, heparin, digoxin), and medicine related with the alteration of thrombocyte function (aspirin, dipiridamol).32 it is concluded that special attention is needed for pediatric patient with thrombocytopenia, therefore a dentist should understand well about this disorder on how to take the history, to do clinical examination, to establish the diagnosis and to decide treatment plan as well as to consult to related colleagues. references 1. rose lf, kaye d. buku ajar penyakit dalam untuk kedokteran gigi. edisi 2 jilid 1. jakarta: penerbit binarupa aksara; 1997. p. 465–69. 2. bricker sl, langlais rp, miller cs. oral diagnosis, oral medicine, and treatment planning. 2nd ed. philadelphia: lea and febiger; 1994. p. 399–407. 3. consolini dm. thrombocytopenia in infants and children. available from http://pedsinreview.aappublications.org/. accessed august 11, 2010. 4. little jw, falace da, miller cs , rhodus nl . dental management of the medically compromised patient. 7th ed. missouri: mosby elsevier company; 2008. p. 396–421. 5. patton ll. bleeding and clotting disorders. in: greenberg ms, glick m, editor. burket’s oral medicine diagnosis & treatment. 10th ed. ontario: bc decker; 2003. p. 460–1. 6. reed e, drews md. critical issues in hematology: anemia, thrombocytopenia, coagulopathy, and blood product tranfussions in critically ill patient. clin chest med 2003; 4: 607–22. 7. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 10th ed. philadelphia: saunders elsevier co; 2006. p. 668. 8. kömür m, bayram i, erbey f, kücükosmanoğlu. a rare cause of thrombocytopenia in infants: vitamin b12 deficiency. eur j gen med 2010; 7(1): 107–10. 9. gupta v, tilak v, bhatia bd. immune thrombocytopenic purpura. indian j of pediatrics 2008; 75: 723–8. 10. rehman a. immune thrombocytopenia in children with reference to low-income countries. eastern mediterranean health journal 2009; 15(3): 729–36. 11. silverman ma, dyne pl. idiophatic thrombocytopenic purpura. medscape reference. available from http://www.emedicine. medscape.com. accessed august 11, 2010. 12. s e m p l e j w . i m m u n e p a t h o p h y s i o l o g y o f a u t o i m m u n e thrombocytopenia purpura. elsevier blood reveiews 2002; 16(1): 9–12. 13. ahn ys, horstman ll. idiophatic thrombocytopenic purpura: pathophysiology and management. int j hematology 2002; 76: 123–31. 14. koch g, poulsen s. pediatric dentistry a clinical approach. 2nd ed. united kingdom: blackwell publishing; 2009. p. 327. 15. cameron ac, widmer rp. handbook of pediatric dentistry. 2nd ed . sydney: mosby elsevier company; 2003. p. 237–9. 16. stasi r, sarpatwari a, b jodi, osborn j, evangelista ml, cooper n, provan d, newland a, amadari s, bussel jb. effects of eradication of helicobacter pylori infection in patients with immune thrombocytopenic purpura: a systematic review. blood j of hematology 2009; 113: 1231–40. 17. bennet cm, tarantino m. chronic immune thrombocytopenia in children: edpidemiology and clinical presentation. clinical review article hematology/oncology clinics of north america 2009; 23(6): 1223–38. 18. guzeldemir e. the role of oral hygiene in a patient with idiopathic thrombocytopenic purpura. int j dent hygiene 2009; 7: 289–93. 19. hay ww, hayward ar, levin mj, sondheimer jm. current pediatric diagnosis & treatment. 15th ed. new york: mcgraw-hill; 2001. p. 770–3. 20. nugetntf dj. children immune thrombocytopenia purpura. blood reviews elsevier 2002; 16: 27–9. 21. hasan r, alatas h, latief a, napitupulu pm, pudjiadi a, ghazali mv, putra st. buku kuliah ilmu kesehatan anak. jilid 2. jakarta: infomedika; 2007. p. 929. 22. brigitte bm, valerie p, catherine t, dominique d, martine g, yvart j, marie d. misdiagnosis of chronic thrombocytopenia in childhood. j of ped hematology 2003; 25: 548–52. 23. poala sb, bisogno g, colombatti r. thrombocytopenia and splenoegaly: an unuaual presentation of congenilat hepatic fibrosis. orphanet j of rare disease 2010; 5: 4. 24. perlman mk, schwab j, nachman j. thrombocytopenia in children with severe iron deficiency. j of ped hematology 2002; 24(5): 380–4. 25. sauve lj, scheifele d. do childhood vaccines cause thrombocytopenia?. available from http://www.ncbi.nlm.nih.gov/pmc/articles/. accessed april 10, 2010. 26. tanir g, aydemir c, tuygun n, kaya ö, yarali n. immune thrombocytopenia as sole manifestation in case of acute hepatitis a. turk j gastroenterol 2005; 16(4): 217–9. 27. israels s, schwetz n, boyar r, mc nicol a. bleeding disorders: characterization, dental considerations and management. j can dent assoc 2006; 72(9): 827a–827k. 28. gupta a, epstein jb, cabay rj. bleeding disorders of importance in dental care and related patient management. j can dent assoc 2007; 73(1): 73–83. 29. barboni g, candi m, bayon m, balbaryski j, gaddi e. prevalence of thrombocytopenia in hiv infected children. j of medicina b aires 2010; 70(5): 421–6. 30. pascual cc, kai o, newton crjc, peshu n, robert dj. thrombocytopenia in falciparum malaria is associated with high concentrations of il-10. am j trop med hyg 2006; 75(3): 434–6. 31. markum ah, ismael s, alatas h, akib a, firmansyah a, sastroasmoro s. buku ajar ilmu kesehatan anak. jilid 1. jakarta: percetakan gaya baru; 1991. p. 324–5. 32. permono b, sutaryo, ugrasena idg, windiastuti e, abdulsalam m. buku ajar hematologi-onkologi anak. cetakan ke-2. jakarta: badan penerbit idai; 2006. p. 138–9. vol 49 no 1 jan-mrt 2016.indd 3737 research report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 38–43 the effect of combined moringa oleifera and demineralized freeze-dried bovine bone xenograft on the amount of osteoblast and osteoclast in the healing of tooth extraction socket of cavia cobaya rostiny, eha djulaeha, nike hendrijantini, and agus pudijanto department of prosthodontic faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: alveolar bone has an important role in providing support to teeth and dentures. loss of support caused by alveolar resorption will cause functional and aesthetic problems. preservation socket using bone graft is one way to maintain the dimensions of the alveolar bone. moringa oleifera leaf can increase the activity of bone graft in the formation of new bone. purpose: this study was aimed to evaluate the effect of combined moringa oleivera leaf extract and demineralized freeze-dried bovine bone xenograft (dfdbbx) towards the formation of osteoblasts and osteoclasts in the tooth extraction sockets of cavia cobaya. method: this study used 28 cavia cobayas divided into four groups. the combination of moringa oleifera leaf extract and dfdbbx was inducted into the sockets of lower incisor tooth with certain dose in each group, ointment 1 containing peg (a mixture of peg 400 and peg 4000) for control group, ointment 2 containing moringa oleifera leaf extract and dfdbbx and peg (at active subtance consentration of 0.5%) for group 1, ointment 3 containing moringa oleifera leaf extract and dfdbbx and peg (at active substance concentration of 1%) for group 2, and ointment 4 containing moringa oleifera leaf extract and dfdbbx and peg (at active substance consentration of 2%) for group 3. paraffin block preparations were made for histopathology examination using hematoxylin eosin staining. result: the results showed that there were significant differences of the number of osteoblasts and osteoclasts in each treatment group (p < 0.05). conclusion: it can be concluded that the combination of moringa oleifera leaf extract and dfdbbx at 2% cocentration can increase the number of osteoblasts and decrease osteoclasts in the healing of tooth extraction sockets of cavia cobaya. keywords: demineralized freeze-dried bovine bone xenograft; moringa oleifera leaf; osteoblast; osteoclast correspondence: rostiny, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: rostiny54@gmail.com introduction alveolar bone has an important role in providing support to teeth and dentures.1 making dentures require sufficient structure and volume of alveolar bone as a buffer for removable dentures, fixed dentures and implant placement. loss of the structure and volume of alveolar bone will affect stability, retention and comfort in the use of dentures in patients. this condition is usually caused by resorption process of alveolar bone due to mechanical factors, pathological processes, trauma on face and resorption process triggered by tooth extraction.2 therefore, it is essential to maintain alveolar ridge after tooth extraction. one of techniques aimed to maintain alveolar ridge after tooth extractions by using graft materials either with or without membrane.3,4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42 38 rostiny, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 37–42 graft materials are natural or synthetic subtances used to correct defect or deficiency in tissue by providing extra cellular matrix in the form of scaffold during bone regeneration process.5,6 however, the use of graft materials in post-extraction sockets is still questionable because it can interfere with healing process in the socket when the socket will be used for the placement of implant.3,4 the formation of new bone derived from graft materials moreover also depends on time, therefore a substance that can simulate the activity of the graft materials is needed to accelerate new bone formation process.7,8 moringa oleifera, is a plant that has many benefits because it contains many nutritions, including vitamin c, α and β tochoferol, α and β-carotene, and 12 kinds of flavonoids including kaempferol and quercetin with high concentration.9 flavanoid compound serves as an anti-inflammatory, anti oxidants and osteoprotection.10-12 several other researchers even claim that moringa oleifera plant can inhibit cyclooxygenase enzyme, prostaglandin synthesis and nitrous oxide production in macrophages cells induced by lipopolysacaride (lps).12,13 tooth extraction can cause trauma triggering inflammation. inflammatory reaction is a sign of first activated defense cells.14 this process occurs as a result of prostaglandin synthesis. this prostaglandin synthesis then will increase prostaglandin e2 (pge2). pge2 will directly lead to the increasing of osteoclast activity. in addition, macrophages infiltration as protection against infection will also be increased in the area of trauma and will induce nuclear factor kappa β (nf-ĸβ). consequently, nf-ĸβ will trigger the secretion of pro-inflammatory mediators, namely interleukin-1 (il-1), interleukin 6 (il-6) and tumor necrosis factor α (tnfα) to strength immune response and also accelerate metabolic process. those pro-inflammatory mediators then can regulate the receptor activators of nuclear kappa β ligand (rankl) to bind to the receptor activators of nuclear kappa (rank) causing the increasing of the differentiation of pre-osteoclasts into osteoclasts, later accelerating bone resorption process.15 in the healing process of the sockets after tooth extraction, osteoblast cells have been formed, derived from pluri-potential cells of periodontal ligament. this process indicates osteogenic function occured in the third week. in this week, cortical bone in the sockets will also remodel, and the crest of the alveolar bone will be rounded by the activity of osteoclastic resorption.16,17 based on the description above, therefore, this research is aimed to evaluate the effect of combine bonegraft and moringa leaf extract on the formation of osteoblasts and osteoclasts in the tooth extraction. materials and method this was an experimental laboratory research using experimental animals with true experimental design (post only control group design). subjects in this research were healthy male cavia cobayas weighed 300-350 grams, aged 3-3.5 months obtained from biochemistry laboratory unit of experimental animal, faculty of medicine, universitas airlangga. peg is a compound serves as carrier for extract of moringa oleifera leaf and dfdbbx, making them readily absorbed by human’s body. twenty-eight cavia cobaya were divided into four groups, each consisting of seven samples. mixture of moringa oleifera leaf extract, dfdbbx and peg were made. ointment 1 containing 25 grams of peg (a mixture of peg 400 and peg 4000), ointment 2 containing 0.5 grams of moringa oleifera leaf extract, 0.5 grams of dfdbbx and 99 grams of peg (at active substance concentration of 0.5%), ointment 3 containing 0.5 grams of moringa oleifera leaf extract 0.5 grams of dfdbbx and 49 grams of peg (at active substance concentration of 1%), and ointment 4 containing with 0.5 grams of moringa oleifera leaf extract,0.5 grams dfdbbx and 24 grams of peg (at active substance concentration of 2%).18 those ointments were applied on the tooth extraction sockets of lower left incisors of cavia cobayas previously got anesthesia injection with ketamine. ointment 1 was applied on the sockets in the control group, ointment 2, 3 and 4 were applied to the sockets in group 1, 2 and 3. on day 28th, all of those animals were killed and mandibular were taken and decalcified with edta solution for 30 days. then, paraffin blocks were made and cut with rotary microtomes at 4 microns of thickness. deparafinitation was conducted by dissolving them in xylol for 2 x 3 minutes. residual xylol was washed with absolute alcohol 99%, 95%, 90%, 80%, and 70%, respectively for 2 x 1 minutes. the residual alcohol was washed with running water. he staining was conducted for 30 seconds and then rinsed with water. eosin staining was conducted for 1-2 minutes, and then washed with absolute alcohol 70%, 80%, 90%, 95%, and 99% for 2 x 3 minutes and with xylol for 2 x 2 minutes. each preparation was closed with a cover glass previously dropped with canadian balsam. histology examination of osteoblasts and osteoclasts were done by using a light microscope at 400x magnification.18,19 the data collected from the observation was analysed statistically. kolmogorov smirnov test, levene test and one way anova were done, followed with multifactorial comparison test using tukey-hsd. results it can be seen that the highest mean number of osteoblast cells was in the group treated with the active substance concentration of 2%, while the lowest in the control group. furthermore, the highest mean number of osteoclasts was in the control group, while the lowest was in group 3 treated with the active substance concentration of 2% (figure 1). the increased number of osteoblasts and the decreased of osteoclasts in the control group and the treatment groups can be seen in figure 2 and 3. in addition, the results of normality test showed that the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42 3939rostiny, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 37–42 distribution of data in all groups of osteoblasts was normal (p = 0.160). based on the results of homogenity test, it is also known that the variance of data obtained from those groups was homogeneous (p = 0.975). it was shown that all data in the groups of osteoclasts was normally distributed (p = 0.395) with homogeneous variance (p = 0.083). the results of anova test showed that there were significant differences among the treatment groups (p < 0.05). the results of multiple comparison using, tukey hsd test (table 1). showed that there was no significant difference of the 0 5 10 15 20 25 control group 1 group 2 group 3 osteoblasts osteoclasts figure 1. graph of the mean and standard deviation of the number of osteoblasts and osteoclasts. ab a b c c d d figure 2. description of osteoblast cells as a result of he examination with a light microscope at 400x magnification. a) control group; b) group 1; c) group 2; and d) group 3. the number of osteoblasts was different among the groups, getting increased from control group to group 3. (arrow heads indicate osteoblast). a a c c b b d d figure 3. description of osteoclast cells as a result of he examination with a light microscope at 400x magnification. a) control group; b) group 1; c) group 2; and d) group 3. the number of osteoclasts was different among the groups, getting decreased from control group to group 3. (arrow heads indicate osteoclast). tabel 1. results of tukey hsd test on the number of osteoblasts and osteoclasts in each treatment group control group 1 group 2 group 3 osteoblasts control * * group 1 * * group 2 * * * group 3 * * * osteoclasts control * * * group 1 * * * group 2 * * group 3 * * *= no significant difference (p<0.05) note: control: control group; group 1: at active substance concentration of 0.5%; group 2: at active substance concentration of 1%; group 3: at active substance concentration of 2%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42 40 rostiny, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 37–42 number of osteoblasts between control group and group 1. while there were significant differences in the number of osteoblasts between control group and both group 2 and group 3. there were also significant differences of the number of osteoblasts between group 1 and both group 2 and group 3. similarly, there were significant differences in the number of osteoblasts between group 2 and control group, and also between group 1 and group 3. there were significant differences of the number of osteoblasts between group 3 and the other treatment groups. the result of tukey hsd showed that there was a significant difference in the number of osteoclasts between the control group and all of those treatment groups. there were significant differences of the number of osteoclasts between group 1 and the control group, and also between group 2 and group 3. it is also known, furthermore, that there were significant differences between group 2 and both the control group and group 1. nevertheless, there was no significant difference between group 2 and group 3. it also shows that there were significant differences between group 3 and both the control group and group 1. there was no significant difference between group 3 and group 2. discussion based on data analysis, the mean number of osteoblast cells was getting increased from the control group to group 3. it is also known that based on the results of one way anova test, there was a significant difference of the number of osteoblast cells among the treatment groups. it indicates that the combination of moringa oleifera leaf extract and both peg and dfdbbx can influence the formation of osteoblasts in the tooth extraction sockets. similarly, a previous research also claims that moringa oleifera leaf extract has osteogenic effects.20,21 a compound playing a role in this condition is flavonoid, especially kaempferol and quercetin. osteogenic effects derived from flavonoid can be detected from the increasing of alkaline phosphatase (alp) enzym in cultured osteoblast cells. in addition to the increasing of alp, ethanol derived from moringa oleifera leaf extract can also improve other remodelling bone markers, namely calcium serum and phosporus serum.22 alp playing a role in mineralization process is aimed to create alkaline atmosphere in osteoid tissue formed, as a result, calcium can be easily deposited in the tissue.23 meanwhile, specific alp plays a role to make bone synthesized into osteoblasts and reflects osteoblast cell activities during bone formation.24 in addition to osteogenic effects, kaempferol also has estrogenic effects. kaempferol can bind to estrogen receptors in osteoblasts. kaempferol through the estrogen receptors then can increase biomarkers of osteoblast differentiation, such as alp and osteoblastic genes transcription, namely collagen type 1, osteocalsin and osteonectin.20 another cause of the condition is by the participation of a graft material combined with moringa oleifera leaf extract. a graft material used in this study is dfdbbx, a kind of xenograft produced by batan. xenograft is useful to stimulate the proliferation of osteoblasts, fibroblasts and endothelial cells.25 xenograft also has an ability to regenerate tissue osteoconductively because it has inner surface properties, porosity, calcium ratio, and mineral factors, such as hydroxyapatite owned by bovine similar to human bone minerals.26 dfdbbx plays a role as osteo conductor in osteoinduction.27 but, inorganic materials derived from this graft can make the attachment and proliferation of osteoblast cells as the first step for the formation of osteoblasts to form bone.15 unlike the number of osteoblast cells, the highest number of osteoclasts in this research was in the control group treated with only the administration of peg on the tooth extraction sockets. meanwhile, the lowest number of osteoclasts was in group 3 treated with the administration of the active substance concentration of 2%. the decreasing of the number of osteoclasts is caused by kaempferol and quercetin contained in moringa oleifera leaf extracts playing an important role as anti-inflamation. in inflammatory reaction triggered by tooth extraction, pro inflammatory mediators are released from macrophages in the form of cytokines, including il-1, il-6, tnfα and prostaglandin e2 (pge2). these components then will stimulate the formation of osteoclasts either directly or indirectly. pge2 can directly induce vascular changes in inflammation process, and also induce bone resorption in the absence of inflammatory cells with a few of multinucleated osteoclasts when attached directly to the surface of bone.28 cytokine then is produced by macrophages as a process of bone destruction, namely il-1 that can improve the regulation of rankl in osteoblasts. rankl binds to rank as the initial process of bone destruction. the increasing of osteoclast differentiation process is influenced by the increasing of rank fusion on the precursor of the surface of osteoclasts, resulting in bone resorption.29 moringa oleifera leaf, contains the large amount of flavonoids, namely kaempferol and quercetin.30 flavonoids can be used in all inflammatory conditions because they can inhibit cyclooxygenase2 enzyme playing a role in the formation of inflammatory mediators derived from arachidonic acid. it can also inhibit the release of histamine by mast cells in basophils that have anti-inflammatory effects.31,32 the effects of this enzyme then will reduce a number of pro-inflammatory mediators, such as il-1, il-6 and tnfα. kempferol and quercetin activities can also directly inhibit tnfα activity and rankl expression.33 both of these flavonoids have a tendency to bind atoms or act as scavenging for free radical atoms, so reactive oxygen species (ros) can not be formed redundantly. ros can stimulate phosphorylation process of inhibitor kappa β (ikβ), which serves to bind nfĸβ, so it can be inactive in cytoplasm. if ikβ is phosphorylated, the bond of the bond ikβ and nfĸβ will be separated, so nfĸβ becomes active and moves to cell nucleus. this process is called activation process of nfĸβ. therefore, with the barriers of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42doi: 10.20473/j.djmkg.v48.i4.p173-176 4141rostiny, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 37–42 flavanoids in forming ros, the activation of nfĸβ will also be inhibited.34 nfĸβ is known as gene transcription that will induce the formation of pro-inflammatory cytokines, such as tnfα. as mentioned before, tnfα and rankl are precursors of osteoclast cell maturation playing a role in bone resorption. in addition, quercetin is also able to inhibit differentiation and activation as well as to induce apoptosis of osteoclast cells.35,36 thus, in this research, it is known that moringa oleifera leaf extract can reduce the number of osteoclasts, but on the other hand it can increase the number of osteoblasts. this is caused by the suppression phase of differentiation terminal of osteoclasts, so the formation of pre-osteoblast cells become more accelerated. the combination extract of moringa and dfdbbx accelerates bone formation due to its good compatibility and osteoconductivity. accelaretion of bone regeneration mechanically might be caused by combination substanced which acts as scaffold, stabilizing blood clot and preventing epithelia from growing inward the socket, while biologically the subtances provide extra minerals.37 combination of subtances could be caused resorption and gradation in formation of new bone so that enabling osteocompetent penetration and endothelial cells and vascularization in order to accelerate new bone formation.38 it can be concluded that the combination of moringa oleifera leaf extract and dfdbbx at 2% cocentration can increase the number of osteoblasts and decrease osteoclasts in the healing of tooth extraction sockets of cavia cobaya references 1. allegrini s jr, koening b jr, allegrini mr, yoshimoto m, gedrange t, fanghaenel j, lipski m. alveolar ridge sockets preservation with bone grafting–review. ann acad med stetin 2008; 54(1): 70–81. 2. irinakis t. rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. j can dent assoc 2007; 72(10): 917–22. 3. nevins m, camelo m, de paoli s, friedland b, schenk rk, parmabenfenati s, simion m, tinti c, wagenberg b. a study of the buccal wall of extraction socket of teeth with prominent roots. int j periodonti cs restorati ve dent 2006; 26(1): 19-20. 4. oghli aa, steveling. ridge preservation following tooth extraction: a comparison between a traumatic extraction and socket seal surgery. quint int 2010; 4(7): 605-9. 5. gark k, lynch se, robert g, robert em. tissue engineering applications in maxillofacial surgery and periodontics. illinois: quintessence public inc; 1999. p. 83-9. 6. weiss cm, weis a. principle and practice of implant dentistry. 1st ed. united state of america: mosby inc; 2001. p. 659. 7. baharuddin na, kamin s, samsuddin ar. the use of demineralized freeze-dried bovine bone xenograft in reducing post surgical periodontal pocket depth. annals of dentistry. university of malaya 2003; 10: 33-6. 8. nevins m, camelo m, de angelis n, hanratty jj, khang wg, kwon jj, rasperini g, rocchietta i, schupbach p, kim dm. the clinical and histological efficacy of xenograft granules for maxillary sinus floor augmentation. int j periodontics restorative dent 2011; 31(3): 227-35. 9. copin ja. study of the nutritional and medicinal values of moringa oleifera leaves from sub-saharan africa: ghana, rwanda senegal and zambia.thesis. the state university of new jersey: graduate school-new brunswick rutgers; 2008. p. 5-99. 10. kasolo jn, bimenya gs, ojok l, ochieng j, ognel jw. phytochemical and uses of moringa oleifera leaves in ugandan rural communities. journal of medicinal plants research 2010; 4(9): 753-7. 11. patel c, rangrez a, parikh p. the anti-osteoporotic effect of moringa oleifera on osteoblastic cells: saos2. journal of pharmacy and biological sciences 2013; 5(2): 10-7. 12. coppin jw, xu y, chen h, pan mh, ho ct, juliani r, simon je, wu. determination of flavonoid by lc/ms anti inflammatory activity in moringa oleifera. journal of functional foods 2013; 5: 1892-9. 13. araújo lc, aguiar js, napoleão th, mota fv, barros al, moura mc, coriolano mc, coelho lc, silva tg, paiva pm. evaluation of cytotoxic and anti-inflammatory activities of extracts and lectins of moringa oleifera seeds. plos one 2013; 8(12): e81973. 14. weijen fv, actqua d, slot de. alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. j clin periodontol 2009; 36(12): 1048-58. 15. yustina ar, suardita k, agustin d. peningkatan jumlah osteoklas pada keradangan periapikal akibat induksi lipopolisakarida p or phy r omona s g i ng iva l is (su at u p enel it ia n lab or at or ie s menggunakan tikus). journal biosains 2012; 14(3): 140-4. 16. ghom a, mhaske s. textbook of oral phatology.1st ed. new delhi, india: jaypee brothers medical publisher ltd; 2009. p. 86-7. 17. rajendran s. textbook of oral phatology. 6th ed. india: elseiver; 2010. p. 599-60. 18. kresnoadi u, rahayu rp. the inf luence of aloe vera bnp 2 expression and amount of osteoblast of alveolar bone induced into tooth extraction socket (cavia cobaya). journal of biological researches 2014; 19(2): 2-10. 19. vindani d. efektivitas kombinasi ekstrak jinten hitam (nigella sativa) dan graft terhadap peningkatan osteoblas tulang alveol pada cavia cobaya. thesis. surabaya: pendidikan dokter gigi spesialis universitas airlangga; 2013. p. 24-35. 20. jiang yang guo, chiyan choi, yuzhong zheng, ping chen, tingxia dong, zheng-tao wang, günter vollmer, taiwai lau, wah-keung tsim. kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling. chinese medicine 2012; 7(10): 1-7. 21. yamaguchi m. the osteogenic effect of bioactive f lavonoid p-hydroxycinnamic acid: development in osteoporosis treatment. oa biotechnology 2013; 01: 2(2): 15. 22. jiaying g. mechanistic studies on the osteogenic effects of dietary flavonoids in cultured osteoblasts: potential drug development against osteoporosis. thesis. hong kong: hong kong university of science and technology; 2011. p. 146-50. 23. thomas sdc. bone turnover markers. aust prescr 2012; 35: 156-8. 24. yudaniayanti is. aktifitas alkaline phosphatase pada proses kesembuhan patah tulang femur dengan terapi caco3 dosis tinggi pada tikus jantan. media kedokteran hewan 2008; 21(1): 15-8. 25. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc 2007; 73(6): 513. 26. baharuddin na, kamin s, samsuddin ar. the original article use of demineralized freeze-dried bovine bone xenograft in reducing post-surgical periodontal recession. annals of dentistry. university of malaya 2005; 12: 37–40. 27. purnomo a, adji d. ekspresi bone morphogenetic protein-2 untuk mengukur efektivitas biomaterial freeze dried bovine bone xenograft (fdbbx) sebagai bahan penyambung tulang. jurnal sain veteriner 2012; 30(1): 1-6. 28. khan sn, cammisa fp jr, sandhu hs, diwan ad, girardi fp, lane jm. the biology of bone grafting. j am acad orthop surg 2005; 13(1): 77-86. 29. takanyangi. ostoimunology: share mechanism and crosstalk between the immunes and bone system. 7th ed. tokyo: nature publishing group; 2007. p. 292-302. 30. sreelatha s, jeyachitra a, padma pr. antiproliferation and induction of apoptosis by moringa oleifera leaf extract on human cancer cells. food chem toxicol 2011; 49(6): 1270-5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42 42 rostiny, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 37–42 31. thorne research. 2000. alternative medecine review. vol.3(2). available from: www.thorne.com/altmedrev/.fulltex/3/2/140.pdf. accessed on 10 may 2015. 32. della l. anti inflammatory activity of benzopyrones that are inhibitors of cyclo and lipo-oxygenase. pharmacological ressearch communications 2001; 20: 91-4. 33. amijaya app, murwani s, wardhana aw. 2012. efek ekstrak air daun kelor (moringa oleifera) terhadap kadar tumor necrosis factor alpha (tnfα) dan gambaran histopatologi sel endotel arteri coronaria pada tikus putih (rattus norvegicus) yang diberi diet aterogenik. program studi pendidikan dokter hewan. program kedokteran hewan. universitas brawijaya. 1-12. available from: http://pkh.ub.ac.id/wp-content/uploads/2012/10/0911310033-ari purnamasari p.pdf. accessed on 9 may 2015. 34. wihastuti ta, sargowo d, rohman ms. efek ekstrak daun kelor (moringaoleifera) dalam menghambat aktifasi nfkb, ekspresi tnfα dan icam-1 pada huvecs yang dipapar ldl teroksidasi. jurnal kardiologi indonesia 2007; 28: 181-8. 35. wattel a, kamel s, mentaverri r, lorget f, prouillet c, petit jp, fardelone p, brazier m. potent inhibitory effect of naturally occuring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. biochem pharmacol 2003; 65: 35-42. 36. woo jt, nakagawa h, notoya m, yonezawa t, udagawa n, lee is, ohnishi m, hagiwara h, nagai k. quercetin suppressses bone resorption by inhibiting the differentiation andivation of osteoclast. biol pharm bull 2004; 27(4): 504-9. 37. de risi v, clementini m, vittorini g, mannocci a, de sanctis m. alveolar ridge preservation techniques, a systematic review and metaanalysis of histological and histomorphometrical data. clin oral implants res 2015; 26(1): 50-68. 38. logea r t-avramoglou d, anagnostou f, bizios r, petite h. engineering bone chalanges and obstacles. j cell mol med 2005; 9(1): 72-84. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p37-42 1111 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 11–15 original article the comparative micro-ct analysis on trabecular bone density between hydroxyapatite gypsum puger scaffold application and bovine hydroxyapatite scaffold application amiyatun naini department of prosthodontics, faculty of dentistry, universitas jember, jember – indonesia abstract background: generally, after tooth extraction, trauma is caused by bone damage, which leads to a decreased bone density. bone damage repair should be conducted using a bone graft containing hydroxyapatite (ha). ha can be synthesised from gypsum puger powder, which is abundant and easy to obtain. hydroxyapatite gypsum puger (hagp) was successful with 100% hydroxyapatite purity level. purpose: to compare the ratio of trabecular bone density in wistar rats between hagp scaffold application and bovine hydroxyapatite (bha) scaffold application. methods: this study is a laboratory experiment using 6 treatment groups, namely k (-) polyethylene glycol (peg) 7, k (-) peg 28, hagp + peg 7, hagp + peg 28, bha + peg 7, and bha + peg 28. hagp scaffold freeze-drying. the rats were anaesthetised intramuscularly, and their left mandibular incisor was removed. the scaffold was applied to the mouse socket, followed by tissue decapitation after 7 and 28 days. the examination was carried out with micro-computed tomography (micro-ct). next, statistical analysis using a one-way analysis of variance (anova) test was conducted (p <0.05). results: the anova test result showed a difference in bone density between the treatment and control groups on days 7 and 28. the least significant difference (lsd) test result revealed that there was no significant difference between k (-) peg 28 and hagp + peg 7 (p=0.133). nevertheless, there were significant differences between the other groups. conclusion: based on the micro-ct analysis, the trabecular bone density in wistar rats following hagp scaffold application is higher than that of bha scaffold application. keywords: bone graft; bovine hydroxyapatite; hydroxyapatite gypsum puger; micro-computed tomography correspondence: amiyatun naini, department of prosthodontics, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. email: amiyatunnini.fkg@unej.ac.id introduction trabecular bone is a part of the bone that appears spongy and is found near the ends of all bones, as well as in the middle region of the vertebral bones. an example is the jawbone, where the bone is not solid but full of holes connected by thin rods. the shape and structure of the trabecular bones are optimally regulated to withstand the loads imposed by functional activities. in dentistry, trabecular bone density is crucial in the dental implant system. usually, the percentage density of trabecular bone ranges from 0.2–0.8 g/cm3, while its porosity is 75–95%.1 extraction of numerous teeth will lead to bone damage trauma, causing the bone density to decrease. bone density has a high correlation with fracture risk.2 cases of trabecular bone damage are generally caused by trauma, post-tooth extraction and bone resorption. repair of severe bone damage cases can be performed with the help of bone fillers, whereas the most preferred bone filling material is hydroxyapatite (ha) since it has a chemical composition similar to human bones and teeth.3 in this study, the gypsum used as a base material was taken from the gamping mountain in the puger subdistrict as it is abundant, easy to obtain, and inexpensive; its calcium content is higher than commercial gypsum.4 the gypsum puger was then successfully synthesised into dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p11–15 mailto:amiyatunnini.fkg@unej.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p11-15 12 naini/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 11–15 hydroxyapatite. the hydroxyapatite gypsum puger (hagp) powder was subsequently developed into an hagp scaffold with a 100% purity level of hydroxyapatite. based on x-ray diffraction (xrd) and scanning electron microscope (sem) scaffold examinations, the hydroxyapatite obtained had significant quantities of scaffold pores similar to the bovine hydroxyapatite (bha) scaffold, which was the gold standard.5 according to naini et al.,6 at 10% concentration, an ha scaffold from puger gypsum material can increase the number of osteoblasts and the area of bone trabeculae detected by histopathology anatomy (hpa) examination. osteoblasts as cells bone constituents are formed from osteoprogenitor mesenchymal stem cells (msc). 7 osteoblast cells as bone matrix constituents can even improve bone physical properties.8 physical properties of bones in the form of mechanical strength are influenced by trabecular bone density.9 moreover, the volume of trabecular bone density can be measured using a 3-dimensional micro-ct tool. this can also display good trabecular structures with extremely high resolution using x-rays. it then adequately correlates with the histology of bone morphology so that it can be used as a standard.10 this study hypothesised that the trabecular bone density in wistar rats after hagp scaffold application would be higher than that of the bha scaffold application. the aim of this research is therefore to analyse trabecular bone density in wistar rats following hagp scaffold application and compare the findings to that of a bha scaffold application. materials and methods this study is a laboratory experiment with a post-test only control group design. it is approved with a research ethic number: 247/kkepk.fkg/x/2016. there were six treatment groups in this study: (a) control group (k (-)) using polyethylene glycol (peg) for 7 days; (b) control group (k (-)) using peg for 28 days; (c) treatment group 1 using hagp + peg for 7 days; (d) treatment group 2 using hagp + peg for 28 days; (e) treatment group 3 using bha + peg for 7 days; and (f) treatment group 4 using bha + peg for 28 days. the replication of each group consisted of 4 samples. since there were 6 groups, 24 rats were needed. the sample size was determined with the lemeshow formula.6 furthermore, to make the hagp scaffold, 250 mg of hagp powder was mixed into 300 mg of gelatin and 10 ml of distilled water until homogeneous. the homogeneous mixture was then placed into a mould and a freeze-drying process was carried out.5 the bha scaffold was obtained from the tissue bank of dr soetomo general hospital surabaya. there were several parameters for determining the wistar rats used in this study: 12–14 weeks of age, male, 200–250g weight, had one-week adaptation before treatment and looked after for 35 days. certain treatments were given to these rats. first, they were anaesthetised intramuscularly at a dose of 20–40 mg/kg body weight.11 second, asepsis of the mandibular left incisor was created with povidoneiodine and then the mandibular left incisor was extracted. third, a scaffold was applied to the socket of each rat and sutured with a specific sewing thread, dr. sella silk braided asp 3/0 75 cm. fourth, the sewing thread was removed on the seventh day after the extraction. after 7 and 28 days, the rats were sacrificed using 5 ml of ether in cotton and placed in a closed glass box for 5 minutes. afterwards, each rat’s left lower jaw tissue was carefully cut from the anterior to the posterior and washed with phospate buffer saline (pbs). the tissue was eventually placed in a 10% neutral buffer formalin fixation solution for 24 hours before scanned with the micro-ct device. all the treatments were conducted in the biochemistry laboratory of the medical faculty, universitas airlangga, surabaya. subsequently, tissue scanning was conducted using a micro-ct device (bruker skyscan 1173 high energy micro-ct, kontich, belgium) with ctvox software version 3.3.1 (64-bit) and ctan version 1.20.8.0 (64-bit). the scanning stage generates a grey-scale projection image in 16-bit tagged image file format (tiff) format, having an index interval between 0–65,535. the separation of tissue structure will ultimately occur, producing a visual image in 3d space. the analysis of each sample was carried out by calculating the mean of grayscale index and the average thickness of the trabecular (trabecular thickness).12 the micro-ct examination was conducted in the micro-ct laboratory of the faculty of mathematics and natural sciences, institute of technology bandung. afterwards, statistical analysis was performed using a data normality test, the shapiro wilk test and a homogeneity test, the levene test. the results of both tests showed that the data were normally distributed and homogeneous. hence, a one-way anova parametric test was performed using the ibm statistical package for social science (spss) version 23 for windows (armonk, new york, usa). when the test results indicated a significant one, the lsd multiple comparison test was then conducted to determine which pair of groups were different. the results of the lsd multiple comparison test revealed that there was no significant difference between k (-) peg 28 and hagp + peg 7 with a p-value of 0.133. however, there were significant differences between the other groups with an anova significant level of < 0.05. results the results of the hagp scaffold and bha scaffold application analysis using micro-ct are illustrated in figure 1. the results of bone density analysis using the micro-ct tool are shown in figure 2 and table 1. subsequently, the results of bone density data analysed with the shapiro wilk normality test showed a p-value of > 0.05, indicating that the data were normally distributed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p11–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p11-15 13 a b figure 1. the morphology of the hagp scaffold with an approximate size of 500 µm (a) and the morphology of the bha scaffold with an approximate size of 250 µm (b). table 1. description of bone density volume groups n bone density volume (mm3) anova (p)mean sd minimum maximum k (-) peg 7 4 0.2200 0.09557 0.0679 0.3721 0.000 k (-) peg 28 4 3.1300* 0.14583 2.8980 3.3620 hagp+peg 7 4 3.4400* 0.13589 3.2238 3.6562 hagp+peg 28 4 14.9200 0.22891 14.5558 15.2842 bha+peg 7 4 6.3900 0.55731 5.5032 7.2768 bha+peg 28 4 9.0100 0.23209 8.6407 9.3793 significant at α = 0.05; the same superscript (*) showed no differences between groups using lsd multiple comparisons. figure 2. the results of bone density volume analysis are shown in red: (a) k (-) peg 7, (b) k (-) peg 28, (c) hagp + peg 7, (d) hagp + peg 28, (e) bha + peg 7, and (f) bha + peg 28. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p11–15 naini/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 11–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p11-15 14 naini/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 11–15 next, levene homogeneity test was carried out and revealed a p-value of 0.180, denoting that the data variance between groups was homogeneous. thus, the anova test analysis was then conducted, which resulted in a p-value of 0.000. this indicates that there was a difference in bone density between the treatment and control groups on days 7 and 28. to find out which group pairs were different; the lsd multiple comparison test was then employed. it showed a p-value of 0.133 between k (-) peg 28 and hagp + peg 7, implying that there was no significant difference. comparatively, the lsd multiple comparison test result for the other groups were significantly different. the bone density volume observed with micro-ct can be seen in figure 2. the images showing mostly red sections were obtained from the hagp + peg 28 group relative to the other groups. correspondingly, the highest average volume of bone density up to 14.9200 mm3 was found in the hagp + peg 28 group, followed by the bha + peg 28, bha + peg 7, hagp + peg 7, k (-) peg 28 and k (-) peg 7 groups. discussion severe cases of bone damage can be rehabilitated with the aid of bone grafts to restore bone density and thickness. in this study, bone substitution material from hagp and bha scaffold was used as a comparison. the morphology of both hagp and bha scaffold sizes along with alveolar bone density was analysed using micro-ct.13 the micro-ct analysis results showed that the morphology of hagp scaffold size was unequal with that of the bha scaffold size. the hagp scaffold was approximately 500 µm, while the bha scaffold was approximately 250 µm. this may be due to particle diffusion during the scaffold manufacturing process. particle diffusion causes the movement of particles in the crystal toward the surface, forming a dendrite. the particle diffusion would bring the material above the dendrite at a rate greater than the surrounding surface. this process results in different shapes, once freeze-drying is done. next, different pores will form on the hagp and bha scaffolds. the larger pore shape will facilitate larger and faster tissue formation and scaffold bioresorption.14 moreover, alveolar bone density was analysed using micro-ct to obtain both quantitative data as presented in table 1 and qualitative data as illustrated in red in figure 2. the data were then statistically analysed. the data analysis findings revealed variations in alveolar bone density between the hagp, bha and control groups. on days 7 and 28, the hagp group had a higher alveolar bone density than the bha group. this may be due to the different particle composition and morphology of the two scaffolds. the composition of calcium in hagp (82.2%) was greater than bha (81.86%). according to crosman, the level of calcium can affect the absorption of x-rays in the bones.15 if there is an increase in the volume of mineralised bone density, it can increase bone calcium as well as the absorption of x-rays. calcium ions can stimulate the expression of bone markers on osteoblast cells for bone regeneration.16 thus, the accumulation of calcium in the hagp scaffold can provide a favourable environment for alveolar bone density to form new bone tissue growth.6 furthermore, the two research materials compared in this study contain ha. ha is an alloplastic material used as a bone graft substitution that is bioactive and osteoconductive. ha in the form of particles can even have a regenerating effect on alveolar bone defects. ha is also known to have high crystallisation due to the sintering process and larger particle size than bone apatite.17 a porous ha scaffold can trigger good bone regeneration in vivo; the formation has a porosity size and three-dimensional shape that has the potential to be osteoconductive and osteogenic in the tooth socket.18 in naini et al.’s5 previous research, based on a degradation test, the release of calcium and phosphate ions in the hagp scaffold at the beginning of immersion was higher than in the bha scaffold, resulting in a molecular chain-breaking process.19 the initial stage of biodegradation can raise the ion concentration in the bone defect, increasing bone growth/bone regeneration.20 if there is rapid degradation, osteoblasts can be easily absorbed to accelerate the healing of the defect.21,22 naini et al.6 stated that hagp scaffold can increase osteoblasts and decrease osteoclasts during tooth socket healing with hematoxillin eosine (he) examination. osteoblast activity is higher in bone-forming parts than in bone-absorbing osteoclasts; connectivity is established and bone density increases, leading to a new bone growth process.23 micro-ct analysis is typically used to determine bone mass and microstructure that can be used in the study of bone metabolism in mice. bone formation and mineralisation would then affect bone strength and mechanical stability, depending on the number and density of bones. the novelty of this study is to compare trabecular bone density in wistar rats using the hagp scaffold application with that of the bha scaffold application. it can be concluded that the trabecular bone density in wistar rats is higher after the application of the hagp scaffold than after the application of the bha scaffold. bone density analysis was conducted with micro-ct tools. to develop better research on analysing trabecular bone density, it is suggested that further study should be performed using the same scaffold particle size with other parameters. references 1. lita ya, azhari a, firman rn, epsilawati l, pramanik f. aspek radiografis dan biologis tulang dalam penilaian kualitas tulang pada osteoporosis. j radiol dentomaksilofasial indones. 2019; 3(2): 47–9. 2. meyers ma, chen py, lopez mi, seki y, lin aym. biological materials: a materials science approach. j mech behav biomed mater. 2011; 4(5): 626–57. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p11–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p11-15 15 3. sadat-shojai m, khorasani mt, dinpanah-khoshdargi e, jamshidi a. synthesis methods for nanosized hydroxyapatite with diverse structures. acta biomater. 2013; 9(8): 7591–621. 4. naini a, rachmawati d. composition analysis of calcium and sulfur on gypsum at the puger district jember regency as an alternative gypsum dental material. dentika dent j. 2010; 15(2): 179–83. 5. na in i a, sudia na ik, rubia nto m, ferdia nsya h, mufti n. characterization and degradation of hydroxyapatite gypsum puger (hagp) freeze dried scaffold as a graft material for preservation of the alveolar bone socket. j int dent med res. 2018; 11(2): 532–6. 6. naini a, sudiana ik, rubianto m, kresnoadi u, latief fde. effects of hydroxyapatite gypsum puger scaffold applied to rat alveolar bone sockets on osteoclasts, osteoblasts and the trabecular bone area. dent j (majalah kedokt gigi). 2019; 52(1): 13–7. 7. veni mac, rajathi p. interaction between bone cells in bone remodelling. j acad dent educ. 2: 1–6. 8. sims na, martin tj. coupling signals between the osteoclast and osteoblast: how are messages transmitted between these temporary visitors to the bone surface? front endocrinol (lausanne). 2015; 6: 1–5. 9. feng x, mcdonald jm. disorders of bone remodeling. annu rev pathol mech dis. 2011; 6: 121–45. 10. azhari a, suprijanto s, prafiadi h, juliastuti e. analisis kemampuan citra radiografi panoramik dalam mendeteksi kerapatan trabekula tulang dengan micro ct sebagai baku standard -image analysis capability of detectinc panoramic radiographic trabecular bone density as standard with standard micro ct. indones j appl sci. 2014; 4(1): 1–5. 11. kusumawati d. bersahabat dengan hewan coba. yogyakarta: gadjah mada university press; 2004. p. 5–22. 12. latief fde, sari ds, fitri la. applications of micro-ct scanning in medicine and dentistry: microstructural analyses of a wistar rat mandible and a urinary tract stone. j phys conf ser. 2017; 884: 012042. 13. rochmatulloh ak, fawziah uz, sumaryono rf, feranie s, latief fde. analisis citra digital untuk sampel batuan menggunakan micro-ct scanner skyscan 1173. in: prosiding seminar nasional fisika (e-journal) snf2017 unj. pendidikan fisika dan fisika fmipa unj; 2017. p. 81–8. 14. moisenovich mm, arkhipova ay, orlova aa, drutskaya ms, volkova s v, zacharov se, agapov ii, kirpichnikov mp. composite scaffolds containing silk fibroin, gelatin, and hydroxyapatite for bone tissue regeneration and 3d cell culturing. acta naturae. 2014; 6(1): 96–101. 15. cosman f, de beur sj, leboff ms, lewiecki em, tanner b, randall s, lindsay r, national osteoporosis foundation. clinician’s guide to prevention and treatment of osteoporosis. osteoporos int. 2014; 25(10): 2359–81. 16. lü l-x, zhang x-f, wang y-y, ortiz l, mao x, jiang z-l, xiao z-d, huang n-p. effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo. acs appl mater interfaces. 2013; 5(2): 319–30. 17. jang sj, kim se, han ts, son js, kang ss, choi sh. bone regeneration of hydroxyapatite with granular form or porous scaffold in canine alveolar sockets. in vivo. 2017; 31(3): 335–41. 18. kim jm, son js, kang ss, kim g, choi sh. bone regeneration of hydroxyapatite/alumina bilayered scaffold with 3 mm passage-like medullary canal in canine tibia model. biomed res int. 2015; 2015: 1–6. 19. da silva hm, de lima ir, bezerra pgp, peregrino g, de almeida soares gd. in vitro dynamic degradation of strontium-hydroxyapatite granules. key eng mater. 2011; 493–494: 205–8. 20. sumathi s, gopal b. in vitro degradation of multisubstituted hydroxyapatite and fluorapatite in the physiological condition. j cryst growth. 2015; 422: 36–43. 21. wang y, yang x, gu z, qin h, li l, liu j, yu x. in vitro study on the degradation of lithium-doped hydroxyapatite for bone tissue engineering scaffold. mater sci eng c. 2016; 66: 185–92. 22. lin wc, chuang cc, yao c, tang cm. effect of cobalt precursors on cobalt-hydroxyapatite used in bone regeneration and mri. j dent res. 2020; 99(3): 277–84. 23. tanaka h, mine t, ogasa h, taguchi t, liang ct. expression of rankl/opg during bone remodeling in vivo. biochem biophys res commun. 2011; 411(4): 690–4. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p11–15 naini/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 11–15 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p11-15 197197 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 197–203 research report analysis of the relationship between human cytomegalovirus dna and gb-1 genotype in the saliva of hiv/aids patients with xerostomia and salivary flow rate irna sufiawati,1 s. suniti,1 revi nelonda,1 rudi wisaksana,2 agnes rengga indrati,3 riezki amalia4 and isabellina dwades tampubolon5 1department of oral medicine, faculty of dentistry, universitas padjadjaran 2department of internal medicine, faculty of medicine, universitas padjadjaran 3department of clinical pathology, faculty of medicine, universitas padjadjaran 4department of pharmacology and clinical pharmacy, faculty of pharmacy, universitas padjadjaran 5molecular biology laboratory, rajawali hospital bandung – indonesia abstract background: human immunodeficiency virus (hiv) infection increases vulnerability to opportunistic viral infection, including human cytomegalovirus (hcmv) infection, that has been detected in saliva. the hcmv envelope glycoprotein b (gb) is highly immunogenic and has been associated with hcmv-related diseases. purpose: the purpose of this study is to assess the prevalence of hcmv and gb-1 genotype in the saliva of hiv/aids patients and to analyse their relationship with xerostomia and salivary flow rate (sfr). methods: this cross-sectional study involved 34 hiv/aids patients. saliva was tested for the presence of hcmv dna using pcr microarrays, and nested pcr for gb-1 genotype detection. xerostomia was measured using a fox’s questionnaire. unstimulated whole saliva flow rate was measured by means of the spitting method. results: the composition of the research population consisting of 73.5% males and 26.5% females with hiv/aids. hcmv was found in 64.7% of hiv/aids patients, while gb-1 genotype was detected in 59.1%. xerostomia was closely associated with the presence of hcmv in saliva (p<0.05), but not with gb-1. there was no significant relationship between xerostomia and sfr rates in the research subjects with hcmv positive saliva (p> 0.05). conclusion: the presence of xerostomia-associated hcmv in saliva was elevated among hiv/aids patients. further investigation is required to identify other gb genotypes that may be responsible for xerostomia and sfr changes in hiv/aids patients. keywords: glycoproteins b-1; human cytomegalovirus; human immunodeficiency virus; xerostomia correspondence: irna sufiawati, department of oral medicine, faculty of dentistry, universitas padjadjaran. jl. sekeloa selatan no. 1, bandung 40132, indonesia. e-mail: irna.sufiawati@fkg.unpad.ac.id introduction human cytomegalovirus (hcmv) or human herpesvirus 5 is a beta-herpesvirus classified as an opportunistic virus pathogen in patients infected with human immunodeficiency virus (hiv). hcmv may also play a role in hiv disease progression.1 epidemiological studies have confirmed a high seroprevalence of hcmv infection worldwide estimated at between 50% and more than 90% in hivinfected individuals.2–4 previous studies conducted in west java, indonesia also indicated a high seroprevalence of hcmv in excess of 90%.5 in addition to sera, several studies have investigated hcmv dna in saliva by means of pcr microarrays as a useful method for the diagnosis of cmv infection.6,7 the prevalence of hcmv in the saliva of hiv/aids patents has been reported as ranging from 5% to 50%.8–11 of the various herpes virus (hhvs) families, hcmv is the largest hhvs member virus 100-nm in diameter, an icosahedral nucleocapsid containing a linear 230 kb double-stranded dna surrounded by a protein layer called tegument. all particles are wrapped in a lipid bilayer envelope containing 6 gp, namely; gp ul55 (gb), gp ul73 (gn), gp ul74 (gh), gp ul100 (gm), and gp ul115 (gl). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 198 sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 the gp ul55 or gb is a highly immunogenic virus envelope and plays an important role in the process of inserting the virus into the host cell, the spread from cell to cell, cell fusion, and ripening of the virion. hcmv gb genotype is classified into four main variants of genotypic gb (gb 1-4) based on the sequence of gb.12–15 the gb genotype distribution among hiv/aids patients has been extensively studied with varying results.16–18 the responsibility of hcmv for a variety of diseases, including salivary gland dysfunction, with the most common symptom being xerostomia in hiv/aids patients, has been widely investigated.19–22 xerostomia is the subjective sensation of a dry mouth usually associated with low salivary flow rate (hyposalivation). however, xerostomia can occur with or without a decrease in saliva production and, thus, may not always be associated with salivary gland dsyfunction.23 a previous study reported a strong relationship between the presence of hcmv dna in saliva with xerostomia and salivary flow rate which suggests that hcmv may be a cause of salivary gland dysfunction in aids patients with low cd4 counts.24 a compromised immune system in hiv-infected patients causes reactivation of hcmv.25 the hcmv gb genotypes have also been studied to determine their role in the pathogenesis of hcmv-associated diseases.26 the role of hcmv and its gb-1 gene as the risk factors causing xerostomia and salivary flow rate in hiv/aids patients remains unclear and has not been widely investigated. the present study was conducted to investigate the prevalence of hcmv and gb-1 genotype in the saliva of hiv/aids patients and to analyze its relationship with xerostomia and salivary flow rate (sfr). materials and methods this cross-sectional study enrolled 34 hiv/aids patients, selected by consecutive sampling, who were not undergoing anti-retroviral therapy (art) at dr. hasan sadikin general hospital, bandung, west java, indonesia. the study included hiv/aids patients aged 18 or over, excluding those who were taking xerogenic drugs (except art). xerostomia was assessed by means of a fox’s questionnaire whose validity and reliability when written in indonesian had previously been assessed.27 the questionnaire consisted of the following four questions: “(1) does the amount of saliva in your mouth seem to be a). too little, b). too much, or c). noticeable” (2) do you have difficulties swallowing any particular foods? (3) “does your mouth feel dry when eating a meal? (4) do you sip liquids to help you swallow dry foods?”. positive responses to any of the preceding questions was considered to be evidence of xerostomia.27 unstimulated whole saliva flow rate was collected using the spitting method under standardized conditions with the rate being measured as previously reported.28 subjects expectorated the saliva into a test tube once a minute for a period of five minutes and the flow rate was recorded in ml/min. the research subjects were allocated to one of three groups (very low < 0. 1/min, low 0.1-0.2 ml/min, and normal > 0.2 ml/min).29 ethical approval was granted by the ethics committee of the faculty of medicine, universitas padjadjaran no. 1433/un6. kep/ec/2018. pcr microarrays were used to investigate positive hcvm dna in saliva. the pcr used a primary sequence of 5’-tcatctacggggacacggac-3’ (forward primer) and 5’cgcaccagatccacg ccctt-3’ (reverse primer) and a positive control probe sequence of 5’-acgaaagcggacaaacacg-3’. to detect gb-1 hcmv positive saliva, a nested pcr was carried out at the biomolecular laboratory of rajawali hospital, bandung. nested pcr of primary pcr used primary primer with a sequence of 5’ggc atc aag caa aaa tct-3’ (foward primer) and 5’cag ttg acg gta ctg cac-3’ (reverse primer) hcmv to obtain an amplicon of gb-1 hcmv.6. the primers in the second stage of the inner pcr were 5’tgg aac tgg aac gg 3 gtt’ (foward primer) and 5 ‘-gaa acg cgc ggc aat cgg-3’ (reverse primer). the pcr-nested reaction was conducted with a final volume of 25 ul for each stage. for stage 1 pcr, 12.5 ul gotaq green master mix 2x (promega) was added to each 2.5 ul outer primer (macrogen) fhcmv1 and rhcmv2, 6.5 ul dh2o free rnase (promega) and 1 ul sample dna. the homogeneous mixture was then placed in an analytik jena biometra thermal cycler and followed a stage 1 pcr program, which consists of an initial cycle at 95o c for two minutes, followed by 30 cycles of denaturation at 95o c for one minute, primary attachment at 60oc for 30 seconds, installation of nucleotide base (extension) at 72oc for one minute, and one final cycle extension at 72oc for five minutes. during stage 1, a negative control was included, where the dna sample was replaced with dh2o free rnase. for pcr stage 2, 12.5 ul gotaq green master mix 2x (promega) was added to each 2.5 ul of primary inner (inner primer) (macrogen): fhcmv3 and rhcmv4, 5.5 ul dh2o free rnase (promega) and 2 ul dna from the results of stage 1 pcr. the homogeneous mixture was then placed into an analytik jena biometra thermal cycler and followed a first stage pcr program consisting of one cycle at 95oc for two minutes, followed by 35 denaturation cycles at 95oc for one minute, primary attachment at 68oc for 30 seconds, installation of nucleotide (extension) at 72o c for 1.5 minutes, and a final cycle extension at 72oc for seven minutes. in stage 2, the negative control dna template was taken from the results of the first pcr negative control. the results of pcr electrophoresis were carried out using agarose gel (promega) with 2% concentrated gel to which 2 ul of etidium bromide (sigma) dye were added. the agarose gel was placed into the electrophoresis tank and tae 1xbuffer was added until it was flooded. 5 ul negative control, 5 ul of 100 bp dna marker (thermo fischer) and 5 ul of pcr sample were subsequently added to the gel wells. electrophoresis was carried out at a voltage of 75v dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 199sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 for 25 minutes. electrophoretic gel was placed on top of the uv laminator to visualize the dna bands obtained which were recorded with digital cameras. the data was analyzed using frequency variables with the results being presented in percentages. the relationship between the presence of hcmv in saliva and xerostomia and salivary flow rate were analyzed using chi-square and mann whitney tests. results the present study was conducted on 34 hiv/aids patients consisting of 25 males (73.5%) and 9 females (26.5%). the highest percentage (61.8%) occurred in the 30-39 years age group and the lowest (2.9%) in the ≥50 years age group. the cd4 counts varied from 17 to 790 cells/mm3, with up to 38.3% with cd4 counts <200 cells/mm3, while up to 50% had received art. the characteristics of the research subjects can be seen in table 1. in order to detect hcmv in the saliva of hiv/aids patients in this study, a microarray pcr technique was performed. the results showed that 22 (64.7%) of all subjects had hcmv dna in their saliva (figure 1). furthermore, nested pcr was examined to detect the presence of gb-1 hcmv. the results of the analysis under an illumination beam in 2% algarose gel were detected in 13 positive patients (59.1%) and 9 negative patients (40.9%) (figure 2). the positive results of nested pcr gb-1 hcmv amplification can be seen from the presence of a band which is indicated in the dna marker location of 100 bp (m) with a location at 500bp (figure 3). the fox’s questionnaire results indicated that of the 22 hcmv positive subjects, 15 (68.2%) had complained of xerostomia and 12 (54.5%) had low sfr (figure 4). a chi-square test was performed and confirmed a significant relationship between xerostomia and the presence of hcmv in saliva (p<0.05). the median salivary flow rate in hcmv positive subjects was 0.2 ml per minute lower than that of hcmv negative subjects of 0.4 ml/min, but no significant difference between the two groups on a statistical test (p>0.05) was detected. low sfr (0.1–0.2 ml/min) in 64.70% 35.30% hcmv dna positive hcmv dna negative figure 1. prevalence of hcmv dna in the saliva of hiv/aids patients. 59.1 40.9 0 10 20 30 40 50 60 70 gb-1 positive gb-1 negative th e nu m be r o f h iv /a id s pa tie ns w ith h cm v po si tiv e (% ) figure 2. prevalence of gb-1 genotype in saliva among hcmvpositive hiv/aids patients. 68.2 31.8 54.5 45.5 0 10 20 30 40 50 60 70 80 present absent low normal xerostomia salivary flow rate th e nu m be r o f h iv /a id s pa tie ns w ith h cm v po si tiv e (% ) figure 4. the prevalence of xerostomia and salivary flow rate among hcmv positive hiv/aids patients. figure 3. the band of gb-1 gene detected in the saliva of hcmv-positive hiv patients at 500bp with 100bp marker (m) dna using nested pcr. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 200 sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 hcmv positive subjects was 54.5%, whereas only 25.0% in hcmv negative subjects, but no statistically significant different was found between the two groups (p>0.05) (table 2). when identifying the relationship between xerostomia and salivary flow rates in hcmv positive saliva, statistical analysis revealed that there was no significant relationship between the two conditions (p> 0.05) (table 3). furthermore, eight (66.7%) of subjects with gb-1 genotype positive were found to have experienced xerostomia. however, as seen from the contents of table 4, there was no statistically significant relationship between xerostomia and gb-1 genotype (p<0.05). discussion in the present study, hcmv in saliva was detected in 64.7% of hiv/aids patients. this finding showed that the prevalence of hcmv in saliva was higher than in the published studies that had reported it as ranging from 5% to 50%.8–11 it is well known that hcmv is transmitted through direct contact with the saliva or other bodily fluids of a hcmv-infected person. hcmv can be transmitted vertically (from mother to child in utero or through breastfeeding) and horizontally (person-to-person either through sexual contact and or contact with infected body fluids). in comparison with serologic studies that indicated the higher prevalence of hcmv positive in the sera of hiv patients (ranging from 50% to 90%)2–4 than in saliva specimens, indicating that risk factors for horizontal hcmv transmission is more common than vertical. in children, hcmv is most frequently found in their urine, but it is also often present in their saliva. among adults, sera and genital secretions are both common fluids for hcmv shedding indicating that sexual transmission is considered a major route of hcmv transmission. however, hcmv can also be detected in saliva and, therefore, spread through kissing or oral sex between adults.30,31 in hiv positive individuals, one suggested mechanism of viral opportunistic transmission (including hcmv) may be the sub-epithelial and intra-epithelial immune cells in the oral cavity becoming infected with hiv. hiv gp120 and tat protein may induce tight junction disruption and lead the opportunistic virus to penetrate the oral mucosal epithelium.32 the distribution of hcmv gb genotypes in aids patients has also been widely investigated through analysis table 1. characteristics of the research subjects basic characteristics number gender male female n = 34 25 (73.5%) 9 (26.5%) age 18-29 years 30-39 years 40-49 years > 50 years 32 ± 0.88 12 (35.3%) 19 (55.9%) 2 (5.9%) 1 (2.9%) cd4 (cells/mm3) median (min-max) > 500 350-499 201-349 <200 262 (17-790) 6 (17.6%) 7 (20.6%) 8 (23.5%) 13 (38.3%) received anti-retroviral therapy yes no 17 (50%) 17 (50%) table 2. relationship between xerostomia and the flow rate of saliva containing hcmv in hiv/aids patients human cytomegalovirus dna in saliva p-value (+) n=22 (-) n=12 xerostomia, n (%) present absent 15 (68.2) 7 (31.8) 4 (33.3) 8 (66.7) 0.051a saliva flow rate (ml/min) median (min–max) 0.2 (0.2 – 0.7) 0.4 (0.2 – 0.6) 0.230b saliva flow rate, n (%) 0.1 – 0.2 (ml/min) > 0.2 (ml/min) 12 (54.5) 10 (45.5) 3 (25.0) 9 (75.0) 0.097a aanalysis using chi-square test, bmann whitney test table 3. relationship between xerostomia and the flow rate of saliva in hcmv-positive hiv/patients saliva flow rate p-value0.1 – 0.2ml/ min n=12 >0.2 ml/ min n=10 xerostomia, n (%) present absent 10 (83.3) 2 (16.7) 5 (50) 5 (50) 0.172* *analysis using the fisher’s exact test table 4. relationship between xerostomia and gb-1 genotype in hiv/aids patients xerostomia hcmv gb-1 genotype p-value present n=12 absent n=18 present absent 8 (66.7) 4 (33.3) 6 (33.3) 12 (66.7) 0.135* *analysis using fisher’s exact test dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 201sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 of blood, urine, semen, vitreous, and saliva specimens. the gb-1 hcmv present in saliva was detected in 57.1% of hiv/aids patients. this finding is consistent with those of previous studies showed that gb1 was found to be the predominant glycoprotein genotype (86.96%) among hcmv-infected aids patients.16 similar results have been reported that the most frequent hcmv genotype was gb1 followed by other genotypes among organ transplant patients in turkey.33 however, other studies confirmed that gb3 and gb2 were the most prevalent genotypes in the sera of aids patients and hcmv-infected neonates and a high incidence of mixed infection with the gb1 and gb3 genotypes.34,35 hcmv is known to be responsible for a variety of diseases, for example, in the oral cavity; persistent and atypical mucosal ulcers and xerostomia potentially accompanied by salivary gland dysfunction.36 the prevalence of xerostomia among hiv-infected patients, has been estimated to range from 1.2 to 40%22 and reduced salivary flow rate occurs in 2-30% of subjects.37 the findings of the research reported here indicated that 68.2% of the subjects whose saliva contained hcmv positive had xerostomia, while 54.5% of the subjects experienced a low unstimulated salivary flow rate (0.1-0.2 ml/min). statistical analysis confirmed a significant relationship between the presence of hcmv in saliva and xerostomia. this finding is consistent with that of a prior study demonstrating a link between hcmv in saliva and salivary gland dysfunction in hiv-infected patients.24,38 meanwhile, this investigation revealed no significant relationship between the presence of hcmv in saliva and the salivary flow rate. however, the median of salivary flow rate in hcmv positive saliva was lower at 0.2 ml/min than that of hcmv negative saliva at 0.4 ml/min. in contrast, a previous study observed that significant xerostomia, reduction in salivary flow rate and flavor alteration were all evident in hiv-positive patients receiving highly active antiretroviral therapy (haart).39 as seen in the present study, xerostomia, a subjective complaint of dry mouth, is not always correlated to hyposalivation as objective reduction of salivary flow rates40,41. xerostomia can also be experienced by patients with a normal salivary flow rate. there are multiple causes of salivary gland dysfunction related to hcmv and hiv with various mechanisms. several researchers have suggested that hcmv is often detected in the salivary gland during primary infection and reproduces in the oral epithelium. the local hcmv reactivation affecting the major salivary glands is responsible for xerostomia.21,42 salivary gland disfunction associated with hiv/aids has also been suggested as the result of diffuse infiltration of cd8+ lymphocyte in salivary glands causing suppression of salivary gland functions.43,44 a number of investigators have also reported that oral manifestation of hcmv correlates with the severity of immunosuppression in aids patients with cd4 counts below 100 cells/mm3 in the disseminated form of the disease.37 this indicates that hcmv reactivation in hiv-infected patients may occur under advanced immunosuppressive conditions. hiv infection induces the loss of and dysfunction in cd4+ t cells, a failure to support cd8+ t cells which leads to an increase in their expansion and causes greater hcmv replication. signals from hcmv infection may also promote hiv persistence in cd4+ t cells. cd8+ t cell expansion, coupled with a loss of cd4+ t cells, is linked to morbid outcomes of hcmv and hiv infections.44 furthermore, antiretroviral drugs (including nucleoside transcriptase inhibitors and protease inhibitors) may also cause xerostomia or hyposalivation.37 however, the exact mechanism by which this art can lead to salivary disfunction remains unclear. the suggested mechanism may be due to alteration of the structure and composition of saliva due to the chemical structure of antiretroviral drugs leading to a reduced salivary flow rate. in addition, antiretroviral drugs can alter adipose tissue deposition within the salivary gland itself.44 differences in the gb hcmv genotype may play an important role in the pathogenesis of the disease.26,34 it has been also reported that gb hcmv in immunocompromised individuals contributes to the molecular epidemiology and genetic variability of viruses in clinical manifestations and prognoses.45 the research findings reported here indicated that there was no statistically significant relationship between the gb-1 genotype and the occurrence of xerostomia, although the majority of subjects (66.7%) with positive gb-1 genotype experienced xerostomia. other gb genotypes or mixed infection with more than one gb in hiv/aids patients might be responsible for the occurrence of xerostomia and changes in salivary flow rate, as reported in previous studies. therefore, further research is required to confirm the situation. in conclusion, the high prevalence of hcmv and gb-1 gene in the saliva of hiv/aids patients supports the hypothesis that saliva constitutes an important reservoir for hcmv. there was a statistically significant relationship between xerostomia and the presence of hcmv in the saliva of hiv/aids patients. however, there was no statistically significant relationship between hcmv gb-1 and salivary flow rate. studies featuring larger sample sizes are required to identify other gb genotypes as the specific risk factors associated with hcmv-related xerostomia and hyposalivation in hiv/aids patients. acknowledgements the authors express their gratitude to all study participants who have made a significant contribution to this study. they would also like to thank the members of the teratai hiv clinic and the clinical pathology laboratory at dr. hasan sadikin general hospital bandung, west java, indonesia for their assistance. this project was supported by an internal research grant provided by universitas padjadjaran. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 202 sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 references 1. patel eu, gianella s, newell k, tobian aar, kirkpatrick ar, nalugoda f, grabowski mk, gray rh, serwadda d, quinn tc, redd ad, reynolds sj. elevated cytomegalovirus igg antibody levels are associated with hiv-1 disease progression and immune activation. aids. 2017; 31(6): 807–13. 2. micol r, buchy p, guerrier g, duong v, ferradini l, dousset jp, guerin pj, balkan s, galimand j, chanroeun h, lortholary o, rouzioux c, fontanet a, leruez-ville m. prevalence, risk factors, and impact on outcome of cytomegalovirus replication in serum of cambodian hiv-infected patients (2004-2007). j acquir immune defic syndr. 2009; 51(4): 486–91. 3. fielding k, koba a, grant ad, charalambous s, day j, spak c, wald a, huang ml, corey l, churchyard gj. cytomegalovirus viremia as a risk factor for mortality prior to antiretroviral therapy among hiv-infected gold miners in south africa. plos one. 2011; 6(10): 1–6. 4. meh rk ha n i f, ja m s, sabzva r i d, fatta h i f, kouror ia n z , seyedalinaghi s, jabbari h, mohraz m. cytomegalovirus coinfection in patients with human immunodeficiency virus in iran. acta med iran. 2011; 49(8): 551–5. 5. suf iawat i i, widyaput ra s, djaja k usuma h ts. a st udy of cytomegalovirus serology among hiv-infected patients in the highly active antiretroviral therapy era. maj kedokt bandung. 2013; 45(2): 112–7. 6. mwaanza n, chilukutu l, tembo j, kabwe m, musonda k, kapasa m, chabala c, sinyangwe s, mwaba p, zumla a, bates m. high rates of congenital cytomegalovirus infection linked with maternal hiv infection among neonatal admissions at a large referral center in sub-saharan africa. clin infect dis. 2014; 58(5): 728–35. 7. ross sa, novak z, pati s, boppana sb. overview of the diagnosis of cytomegalovirus infection. infect disord drug targets. 2012; 11(5): 466–74. 8. basso m, andreis s, scaggiante r, franchin e, zago d, biasolo ma, del vecchio c, mengoli c, sarmati l, andreoni m, palù g, parisi sg. cytomegalovirus, epstein-barr virus and human herpesvirus 8 salivary shedding in hiv positive men who have sex with men with controlled and uncontrolled plasma hiv viremia: a 24-month longitudinal study. bmc infect dis. 2018; 18: 1–10. 9. carvalho kss, silvestre e de a, maciel s da s, lira hig, galvão ra de s, soares mj dos s, costa chn, malaquias lcc, coelho lfl. pcr detection of multiple human herpesvirus dna in saliva from hiv-infected individuals in teresina, state of piauí, brazil. rev soc bras med trop. 2010; 43(6): 620–3. 10. munawwar a, singh s. human herpesviruses as copathogens of hiv infection, their role in hiv transmission, and disease progression. j lab physicians. 2016; 8(1): 5–18. 11. sahin s, saygun i, kubar a, slots j. periodontitis lesions are the main source of salivary cytomegalovirus. oral microbiol immunol. 2009; 24(4): 340–2. 12. schottstedt v, blümel j, burger r, drosten c, gröner a, gürtler l, heiden m, hildebrandt m, jansen b, montag-lessing t, offergeld r, pauli g, seitz r, schlenkrich u, strobel j, willkommen h, von könig ch. human cytomegalovirus (hcmv)-revised. transfus med hemotherapy. 2010; 37(6): 365–75. 13. isaacson mk, compton t. human cytomegalovirus glycoprotein b is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress. j virol. 2009; 83(8): 3891–903. 14. shepp dh, match me, lipson sm, pergolizzi rg. a fifth human cytomegalovirus glycoprotein b genotype. res virol. 149(2): 109–14. 15. feire al, roy rm, manley k, compton t. the glycoprotein b disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry. j virol. 2010; 84(19): 10026–37. 16. jiang xj, zhang j, xiong y, jahn g, xiong hr, yang zq, liu yy. human cytomegalovirus glycoprotein polymorphisms and increasing viral load in aids patients. plos one. 2017; 12(5): 1–14. 17. baloyi ss, selabe s, kyaw t. human cytomegalovirus gb genotype distribution among hiv infected patients from pretoria. int j infect dis. 2014; 21: 450–1. 18. goossens vj, wolffs pf, van loo ih, bruggeman ca, verbon a. cmv dna levels and cmv gb subtypes in art-naive haarttreated patients: a 2-year follow-up study in the netherlands. aids. 2009; 23(11): 1425–9. 19. verma n, patil r, khanna v, singh v, tripathi a. evaluation of salivary flow rate and gustatory function in hiv-positive patients with or without highly active antiretroviral therapy. eur j dent. 2017; 11(2): 226–31. 20. sharma g. salivary gland disease in human immunodeficiency virus/acquired immunodeficiency syndrome: a review. world j dermatologyy. 2015; 4(1): 57–62. 21. ebrahim s, singh b, ramklass ss. hiv-associated salivary gland enlargement: a clinical review. sadj. 2014; 69(9): 400–3. 22. mortazavi h, baharvand m, mohammadi m, movahhedian a, khodadoustan a. xerostomia due to systemic disease: a review of 20 conditions and mechanisms. ann med health sci res. 2014; 4(4): 503–10. 23. niklander s, veas l, barrera c, fuentes f, chiappini g, marshall m. risk factors, hyposalivation and impact of xerostomia on oral health-related quality of life. braz oral res. 2017; 31: e14. 24. greenberg ms, dubin g, stewart jc, cumming cg, macgregor rr, friedman hm. relationship of oral disease to the presence of cytomegalovirus dna in the saliva of aids patients. oral surg oral med oral pathol oral radiol endod. 1995; 79(2): 175–9. 25. anuradha b, pratibha mm, vijayadurga s. the reactivation of the cytomegalovirus (cmv) infection in hiv infected patients. j clin diagnostic res. 2011; 5(4): 749–51. 26. fidouh‐houhou n, duval x, bissuel f, bourbonneux v, flandre p, ecobichon jl, jordan mc, vildé jl, brun‐vézinet f, leport c. salivary cytomegalovirus (cmv) shedding, glycoprotein b genotype distribution, and cmv disease in human immunodeficiency virus–seropositive patients. clin infect dis. 2001; 33(8): 1406–11. 27. fox pc, busch ka, baum bj. subjective reports of xerostomia and objective measures of salivary gland performance. j am dent assoc. 1987; 115(4): 581–4. 28. mavazesh m, kumar sks. measuring salivary flow: challenges and opportunities. j am dent assoc. 2008; 139(suppl. 2): 35s-40s. 29. sreebny lm, valdini a. xerostomia. part i: relationship to other oral symptoms and salivary gland hypofunction. oral surg oral med oral pathol. 1988; 66(4): 451–8. 30. hyde tb, schmid ds, cannon mj. cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv. rev med virol. 2010; 20(5): 311–26. 31. cannon mj, schmid ds, hyde tb. review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. rev med virol. 2010; 20(4): 202–13. 32. sufiawati i, tugizov sm. hiv-associated disruption of tight and adherens junctions of oral epithelial cells facilitates hsv-1 infection and spread. plos one. 2014; 9(2): 1–14. 33. eran daǧlar d, öngüt g, çolak d, özkul a, mutlu d, zeytenoǧlu a, midilli k, gökahmetoǧlu s, günseren f, ögünç d, gültekin m. determination of cytomegalovirus glycoprotein b genotypes in different geographical regions and different patient groups in turkey. mikrobiyol bul. 2016; 50(1): 53–62. 34. tarragó d, quereda c, tenorio a. different cytomegalovirus glycoprotein b genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested pcr. j clin microbiol. 2003; 41(7): 2872–7. 35. alwan sn, shamran ha, ghaib ah, kadhim hs, al-mayah qs, al-saffar aj, bayati ah, arif hs, fu j, wickes bl. genotyping of cytomegalovirus from symptomatic infected neonates in iraq. am j trop med hyg. 2019; 100(4): 957–63. 36. doumas s, vladikas a, papagianni m, kolokotronis a. human cytomegalovirus-associated oral and maxillo-facial disease. clin microbiol infect. 2007; 13(6): 557–9. 37. hirata chw. oral manifestations in aids. brazilian journal of otorhinolaryngology. 2015; 81(2): 120–3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 203sufiawati, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 197–203 38. greenberg ms, glick m, nghiem l, stewart jc, hodinka r, dubin g. relationship of cytomegalovirus to salivary gland dysfunction in hiv-infected patients. oral surg oral med oral pathol oral radiol endod. 1997; 83(3): 334–9. 39. jeffers l, webster-cyriaque jy. viruses and salivary gland disease (sgd): lessons from hiv sgd. adv dent res. 2011; 23(1): 79–83. 40. hijjaw o, alawneh m, ojjoh k, abuasbeh h, alkilany a, qasem n, al-essa m, alryalat sa. correlation between xerostomia index, clinical oral dryness scale, and espri with different hyposalivation tests. open access rheumatol res rev. 2019; 11: 11–8. 41. löfgren cd, wickström c, sonesson m, lagunas pt, christersson c. a systematic review of methods to diagnose oral dryness and salivary gland function. bmc oral health. 2012; 12: 1–16. 42. islam nm, bhattacharyya i, cohen dm. salivary gland pathology in hiv patients. diagnostic histopathol. 2012; 18(9): 366–72. 43. freeman ml, lederman mm, gianella s. partners in crime: the role of cmv in immune dysregulation and clinical outcome during hiv infection. curr hiv/aids rep. 2016; 13(1): 10–9. 44. lópez-verdín s, andrade-villanueva j, zamora-perez al, bolognamolina r, cervantes-cabrera jj, molina-frechero n. differences in salivary flow level, xerostomia, and flavor alteration in mexican hiv patients who did or did not receive antiretroviral therapy. aids res treat. 2013; 2013: 1–6. 45. cunha aa, aquino vh, mariguela v, nogueira ml, figueiredo ltm. evaluation of glycoprotein b genotypes and load of cmv infecting blood leukocytes on prognosis of aids patients. rev inst med trop sao paulo. 2011; 53(2): 82–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p197–203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p197-203 130130 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 130–136 original article cytotoxic test of different solvents of soursop (annona muricata) leaf extract against hsc-3 cell line areta vania bhanuwati,1 alfred pakpahan2 1dental student, faculty of dentistry, universitas trisakti, jakarta, indonesia 2department of oral biology, faculty of dentistry, universitas trisakti, jakarta, indonesia abstract background: soursop (annona muricata) leaves have been researched extensively and found to have anticancer properties. the use of soursop as an anticancer treatment is increasingly popular due to its selective cytotoxic activity by acetogenins. the polarity of the extract solvent contributes to the biological activity of the plant, namely cytotoxicity. purpose: to determine the cytotoxicity of a. muricata leaf extract with ethanol, ethyl acetate and hexane fractions against human oral squamous carcinoma (hsc-3) cell lines. methods: this experimental laboratory study consisted of twenty four treatment groups tested against the hsc-3 cell line. the ethanol, ethyl acetate and hexane fractions of a. muricata leaves were administered to seven different concentrations, namely 0.3 μg/ml, 3 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 150 μg/ml and 300 μg/ml. the control group consisted of three groups: negative control, solvent control and positive control. the percentage of cell viability was calculated by absorbent enzyme-linked immunosorbent assay (elisa) reader. the cytotoxicity of a. muricata leaf extract against hsc-3 cells was determined by cell counting kit-8 (cck-8) assay and expressed by ic50 value. the results were analysed using one-way analysis of variance (anova) followed by tukey’s honestly significant difference (hsd). results: the results show that the leaf extracts of a. muricata are moderately cytotoxic to hsc-3 cells. the highest cytotoxic activity was found in the ethyl acetate extract with an ic50 value of 76.66 μg/ml – making it the best solvent – then hexane (ic50: 84.14 μg), then ethanol (ic50: 101.32 μg/ml). statistical analysis using one-way anova and tukey’s hsd is considered significant p < 0.001. conclusion: ethanol, ethyl acetate and hexane fractions of a. muricata leaf extract are moderately cytotoxic, with ic50 values in the range of 21–200 μg/ml. keywords: annona; cytotoxicity; antineoplastic agent; squamous cell carcinoma of head and neck correspondence: alfred pakpahan, department of oral biology, faculty of dentistry, universitas trisakti, jl. kyai tapa no. 260, jakarta,11440, indonesia, email: alfred@trisakti.ac.id introduction cancer is a group of diseases characterised by the uncontrolled growth and spread of abnormal cells.1 according to the world health organization, in 2018, cancer became the second leading cause of death in the world, causing 9.6 million people or one in six people to die. oral cancer is one of the top ten forms of cancercausing deaths in the world, with an estimated 657,000 new cases and more than 330,000 deaths annually.2 until now, the exact aetiology of oral cancer has been unknown because of its complex and multifactorial nature. some of the influencing factors can be local, such as poor oral hygiene, irritation or trauma to restorations or dental caries. in addition, there are also external factors that can influence its occurrence, namely bad habits such as smoking, alcohol consumption, chewing betel nut, viral infections, or it can be influenced by the host’s genetics, age, gender and immunity.3 the most common oral cancers are oral squamous cell carcinoma (oscc), with a prevalence of 80%–90% of all malignancies of oral neoplasms.4 oscc can occur in the lip, oral cavity, nasopharynx or pharynx. the treatment depends on several factors, such as the cancer’s stage of progression, its location and the general health of the patient.5 some of the therapies that are often used today include surgery, radiotherapy, chemotherapy, gene therapy and/or hormonal therapy, alone or in combination. commonly dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 mailto:alfred@trisakti.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 131 bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 used chemotherapy drugs include antimetabolites or those that inhibit enzymes, dna-interactive compounds, antitubulin compounds, hormones and molecular targeting compounds. however, the side effects of chemotherapy treatment are caused by the drugs’ attack on normal cells, causing hair loss; bone marrow suppression; reduced levels of haemoglobin, platelets and white blood cells; drug resistance; weak body; etc.6,7 the number of side effects from common chemotherapy drugs has led to numerous studies on herbal ingredients and their potential for being alternative treatments to cancer. herbal ingredients are believed to have minimal side effects and are also more economical. the anticancer properties of herbal ingredients can be in the form of plant extracts or single active compounds that have been isolated.7 indonesia has biodiversity with the potential to be used as medicinal plants or herbal ingredients. one of which is the soursop plant (annona muricata), which is generally very easy to find throughout the island of indonesia because they can grow any place, especially in areas that are watery. in addition to being consumed, this plant can also be used as a medicinal plant because it has anticancer, anti-tumour, antiviral, anti-inflammatory, antidepressant, antidiabetic, antihypertensive and antibacterial properties.8 chemical compounds that can be found in a. muricata include acetogenins, alkaloids, flavonoids, phenolic compounds and other compounds. however, the secondary metabolite acetogenins have the most dominant anticancer effect and have been reported ethnobotanically regarding their possible selective cytotoxic activity against cancer cells through adenosine triphosphate (atp) intake.9 flavonoids were found to induce apoptosis more strongly than the clinically proven anti-tumour agent camptothecin.10,11 a previous study showed the cytotoxic activity of a. muricata leaf extract with ethanol, ethyl acetate and hexane fractions against cervical cancer (hela) cell lines at concentrations of 1.5 µg/ml, 3.125 µg/ml, 6.25 µg/ml, 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml.12 the leaves of the a. muricata have been investigated extensively for their diverse pharmacological applications and were found to be superior for their anti-inflammatory and anticancer properties. soursop as an anticancer treatment is becoming increasingly popular, so it has been reported ethnobotanically regarding its possible selective cytotoxic activity. bioactivity is considered selective because there are several extracts that have been shown to be more toxic to cancer cells than to normal cells. this selective activity is reported to induce healing with minimal side effects.13 however, most of the studies that have been previously carried out did not use the bioactive isolates responsible for the activity but were based on crude plant extracts.14 meanwhile, the polarity of the extract solvent used contributes to the biological activity of the plant, namely its cytotoxicity. the secondary metabolites contained tend to be more soluble in solvents of the same polarity.15 therefore, we hypothesise that the secondary metabolites contained in the ethanol, ethyl acetate and hexane fraction of a. muricata leaf extract may have cytotoxic effects on the human oral squamous carcinoma (hsc-3) cell line. this study will explore the cytotoxic activity of a. muricata in three solvents with different polarities, namely ethanol (polar), ethyl acetate (semi-polar) and hexane (nonpolar), against oral cancer cells in vitro. materials and methods the materials used in this study were soursop (a. muricata) leaves obtained from manoko solvents, such as ethanol, ethyl acetate and hexane; supporting materials such as phosphate buffer saline (pbs); cell counting kit-8 (cck-8) reagent; dulbecco’s modified eagle medium-fetal bovine serum (dmem-fbs) 10% as media for the cells and as a negative control; dimethyl sulfoxide (dmso) 1% as solvent control, to dissolve each fraction of extract and to see whether the solvent contributes to the cytotoxic activity; dmso 10% as a positive control to obtain the desired effect of the treatment, which is the cytotoxic effect, and as an antibiotic (penicillin-streptomycin) and antimycotic (amphotericin b) 1%. the tools used for the analysis included a blender, macerator, beaker glass, rotary evaporator, separatory funnel, erlenmeyer, 96-well plate, tissue culture flask, co2 incubator, 450 nm microplate reader, micropipette, biosafety cabinet and microscope. soursop leaves originated in bandung, west java, indonesia and were then extracted using maceration with 96% ethanol and continued to be fractionated using three solvents of different polarity: ethanol (polar), ethyl acetate (semi-polar) and hexane (nonpolar) using the liquid– liquid extraction method. maceration and fractionation were performed at natural material organic chemistry laboratory, bandung institute of technology, west java, indonesia. afterwards, the three extracts of a. muricata were diluted into seven different concentrations: 0.3 µg/ ml, 3 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 150 µg/ ml and 300 µg/ml. hsc-3 cells stored in liquid nitrogen were placed in a water bath and heated to 37°c until dissolved. they were then diluted with culture medium with ten times the amount of cells. then, they were centrifuged to obtain a cell palette, resuspended in medium, and the number of cells present were counted. the growing cells were then seeded using a t-flask, incubated for 24 hours, and the cells were ready approximately one week after seeding. phytochemical screening of a. muricata leaf extract with ethanol, ethyl acetate and hexane was performed at the natural materials organic chemistry laboratory, bandung institute of technology to obtain the active compound using the method ascribed to j.b. harborne.16 identification of alkaloid compounds was carried out by mixing 0.1 mg of extract with 10 ml of chloroform and a few drops of ammonia. to separate and acidify the chloroform fraction, a dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 132bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 few drops of concentrated h2so4 were added, then divided into three tubes, and dragendorff’s, mayer’s and wagner’s reagents were added. if there was a red (dragendorff), yellowish white (mayer) or brown (wagner) precipitate, it indicated the presence of alkaloids. to identify flavonoid compounds, samples were given 0.1 mg magnesium powder, 0.4 ml amyl alcohol (a mixture of 37% hcl and 95% ethanol in a ratio of 1:1) and 4 ml of alcohol, which was then shaken. the presence of flavonoids was indicated by the formation of a red, yellow or orange precipitate on the amyl alcohol layer. for phenolic compounds, 1 g of sample was extracted using 20 ml of 70% ethanol; 1 ml of the extract was then given two drops of fecl3 5% solution. if it produced a green or blue–green colour, it contained phenolic compounds. saponin compounds were identified using the foam test in hot water. if the foam did not disappear when one drop of 2 n hcl was added, and it was stable for 10 minutes, then there were saponins. to identify tannin compounds, 1 g of extract was mixed with 10 ml of distilled water and brought to a boil. then, after the filtrate had cooled, 5 ml of fecl3 was added. if a deep blue colour formed, this indicated the presence of tannins. the hsc-3 cell line obtained from the japan health science research resources bank was isolated from a 64-year-old man in japan. the main tumour from the hsc-3 cell culture was on the tongue with lymph node metastases, belonging to the category of moderately differentiated oscc.17 the method used to test cytotoxicity was cck-8 assay using cck-8 reagent. in this method, water-soluble tetrazolium salts (wst-8) dye is reduced using dehydrogenase in cells to produce formazan or orange colour, which is soluble in water through nicotinamide adenine dinucleotide (nadh) and nicotinamide adenine dinucleotide phosphate (nadph) produced by cellular activity.18 the amount of formazan formed depends on dehydrogenase activity.19 the amount of formazan produced indicated the viability of the cells. this could be seen from the optical density (od) of formazan by using a 450 nm microplate reader. first, the estimated number of cancer cells were calculated at 20.000 cells/well and the number inserted into the 96-well plate. then, the concentration of the diluted extract with the culture medium was adjusted so that it reached concentrations of 0.3 µg/ml, 3 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 150 µg/ml and 300 µg/ml. after that, it was incubated at 37oc in a 5% co2 incubator for 24 hours. then, the treatment medium was rinsed with 90 µl of pbs, and 10 µl of cck-8 was added to each well and incubated for 1 hour. this was performed three times for each group at each concentration. od results were determined using a microplate enzyme-linked immunosorbent assay (elisa) reader with a wavelength of 450 nm. cytotoxicity was measured by examining the reduction in cell viability compared with the control group using ic50 value. the ic50 value was determined by plotting a graph of cell viability versus concentration. the higher the ic50 value, the less cytotoxic the extract was because high concentrations were needed to inhibit cells by 50%. absorbance data could be obtained from each well using a microplate reader or spectrophotometer (biochrom anthos zenyth, usa). this data was converted to cell inhibition rate using the formula below. the percentage of inhibition was calculated using the formula:12 % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 1 – � 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑔𝑟𝑜𝑢𝑝 𝑤𝑖𝑡ℎ 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝 � 𝑥 100% the results were analysed using the shapiro–wilk data normality test. statistical significance of the data was calculated using one-way analysis of variance (anova) in addition to tukey’s honestly significant difference (hsd) post hoc test to determine the level of significance. the results were declared significant if p < 0.05. this analysis was carried out using spss 28.0 (spss inc., chicago, il, usa) software. results the results of the phytochemical screening can be seen in table 1. from the results obtained, od was converted into the number of cells through the equation obtained through the normal curve. in figure 1, it can be seen that as the table 1. phytochemical screening results of ethanol, ethyl acetate and hexane fractions of a. muricata leaf extract using j.b. harborne’s method fractions of a. muricata leaf extract chemical compound result ethanol saponin + tannin + ethyl acetate alkaloid + flavonoid + hexane steroid + triterpenoid + figure 1. colour change that occurred after the human oral squamous carcinoma (hsc-3) cells were treated with various concentrations of a. muricata leaf extract and were given cell-counting kit (cck-8) reagent. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 133 bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 7.87% 2.30% 0.92% 8.03% 7.85% 4.91% 3.14% 3.42% 5.36% 10.17% 8.94% 7.20% 6.80% 2.72% 3.14% 0.98% 1.95% 4.92% 4.16% 4.08% 4.08% 2.08% 0.26% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% solvent control positive control 0.3 3 25 50 100 150 300 0.3 3 25 50 100 150 300 0.3 3 25 50 100 150 300 ethanol ethyl acetate hexane percentage of inhibition standard deviation %inhibition figure 2. ethanol, ethyl acetate and hexane fractions of a. muricata extract and the average inhibition percentage of human oral squamous carcinoma (hsc-3) cells after being treated with the test materials. figure 3. linear regression formula to calculate the ic50 values of each fraction. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 134bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 lowest ic50 value. however, the ic50 values of the hexane and ethanol fractions show results that do not differ much from the ethyl acetate fraction. therefore, the three fractions can be categorised as moderately cytotoxic according to the u.s. national cancer institute because they are in the range of 21–200 µg/ml.20 discussion the chemical compounds in the a. muricata are known to show bioactivity, such as antioxidant, antimicrobial, anti-inflammatory and cytotoxic properties, against cancer cells.8 a previous study by qorina et al. shows that ethanol, ethyl acetate and hexane fractions of a. muricata extract have high cytotoxic activity against hela cells and have an ic50 value of under 20 µg/ml. 12 the results of the phytochemical tests that have been carried out in the study show that saponin and tannin compounds are present in the ethanol fraction, alkaloid and flavonoid compounds are in the ethyl acetate fraction and triterpenoid and steroid compounds are in the hexane fraction. this may have been caused by the different polarities of the solvents used.21 the results of the cytotoxicity evaluation using the cck-8 assay show that the ethyl acetate and hexane fraction extracts have a cytotoxic effect on hsc-3 cells that increases with the concentration of the extracts. however, the ethanol fraction extract had the greatest cytotoxic effect at a concentration of 100 g/ml, which started decreasing at a concentration of 150 µg/ml. at the concentration of 300 µg/ml, the number of viable cells in the ethanol fraction approached the negative control. from these results, it can be seen that the hexane fraction extract contains steroid and triterpenoid compounds, which can produce almost the same cytotoxic activity as the ethyl acetate fraction. in the ethanol fraction extract, there are saponin compounds that are known to be involved in dna replication, prevent cancer cell proliferation pathways and inhibit cancer cell proliferation by stopping the cell cycle in the g1/s and g2/m phases.13 meanwhile, the ethyl acetate fraction extract contains alkaloids and flavonoids. in previous studies on the cytotoxicity of a. muricata, ethanol, ethyl acetate and hexane fractions against hela cell lines proved that the three fractions were strongly cytotoxic, with ic50 in the ethanol fraction of 5.91 µg/ml, in the ethyl acetate fraction of 7.56 µg/ml and in the hexane fraction of 8.39 µg/ml.12 the three values of ic50 in this study are close to the ic50 value of the positive control, namely cisplatin (ic50 value: 1.78 µg/ml). cisplatin is one of the well-known chemotherapy drugs and has been used in the treatment of various cancers, one of which is head and neck cancer. the mode of action of this drug is related to its ability to cross-link with purine bases in dna, interfere with dna repair mechanisms, cause dna damage and induce cancer cell apoptosis. however, for the treatment of head and neck cancer, cisplatin is not an table 2. viable human oral squamous carcinoma (hsc-3) cells after being treated with various concentrations of each fraction as well as the negative control, solvent control and positive control for 24 hours test material: a. muricata leaf extracts and controls mean ± standard deviation (cells) negative control 36.751 ± 4.317 solvent control (dmso 1%) 26.754 ± 2.893 positive control (dmso 10%) 2.365 ± 846 ethanol 0.3 µg/ml 35.114 ± 3.353 ethanol 3 µg/ml 32.310 ± 2.951 ethanol 25 µg/ml 19.066 ± 2.886 ethanol 50 µg/ml 15.900 ± 1.802 ethanol 100 µg/ml 15.566 ± 1.154 ethanol 150 µg/ml 18.233 ± 1.258 ethanol 300 µg/ml 34.013 ± 1.968 ethyl acetate 0.3 µg/ml 34.299 ± 3.737 ethyl acetate 3 µg/ml 33.389 ± 3.286 ethyl acetate 25 µg/ml 16.400 ± 2.645 ethyl acetate 50 µg/ml 14.900 ± 2.499 ethyl acetate 100 µg/ml 11.400 ± 1.000 ethyl acetate 150 µg/ml 5.233 ± 1.154 ethyl acetate 300 µg/ml 1.181 ± 360 hexane 0.3 µg/ml 35.749 ± 2.421 hexane 3 µg /ml 34.183 ± 1.809 hexane 25 µg /ml 14.066 ± 1.527 hexane 50 µg /ml 13.900 ± 1.500 hexane 100 µg /ml 12.900 ± 1.500 hexane 150 µg /ml 12.233 ± 763 hexane 300 µg /ml 2.087 ± 95 table 3. ic50 value of a. muricata leaf extracts a. muricata leaf extract ic50 value (µg/ml) ethanol 101.32 ethyl acetate 76.66 hexane 84.14 concentrations increase, the resulting colour is lighter in the well. the darker the resulting colour, the greater the number of surviving cells. the results of the mean ± standard deviation of the number of surviving cells can be seen in table 2. after obtaining the number of surviving cells from each group, the percentage of inhibition was calculated using the formula. the results of the calculation of the percentage of inhibition can be seen in figure 2. the ethyl acetate and hexane fractions’ percentages of inhibition escalate as the concentration increases. however, in the ethanol fraction, the highest percentage of inhibition was at a concentration of 100 µg/ml and decreased at a concentration of 150 µg/ml. the ic50 value can be found to determine at what concentration the extract causes 50% cell death. the higher the ic50 value, the less toxic the extract. from the calculation of the number of surviving cells, ic50 values were also calculated for the ethanol, ethyl acetate and hexane fraction extracts using the regression formula (figure 3), and as can be seen in table 3, the ethyl acetate fraction extract had the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 135 bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 effective drug, so it must be combined with various other drugs, such as doxorubicin, methotrexate, vinblastine and others. in addition, cisplatin can cause various side effects, such as kidney problems, decreased immunity to infection, bleeding and others.22 in addition, previous studies also showed an increase in the percentage of inhibition in the three fractions along with an increase in the concentration of a. muricata.12 however, the results of the ethanol fraction in this study were different. the percentage of inhibition of the ethanol fraction of a. muricata at a concentration of 100 µg/ml was 57.64% and then decreased with increasing concentration. the difference in results may have been caused by the different cell lines or the extraction method used; the research conducted in 2020 used the vacuum dry method. in addition, according to several other previous studies, a. muricata was cytotoxic to the hepg2 liver cancer cell line, the ethyl acetate fraction was cytotoxic against leukaemia cancer cell lines u937 and lung cancer a549 and the hexane fraction was cytotoxic against hela cell lines and pancreatic cancer capan-1.14 in conclusion, there was cytotoxic activity of a. muricata leaf extract in the ethanol, ethyl acetate and hexane fractions against hsc-3 cell lines. extracts with the ethyl acetate solvent had the highest cytotoxic activity, making it the best solvent. however, this study required the use of 1% dmso due to the limitations associated with the dissolution of the extract. the study was also performed twice and in stages, adding three sets of extract concentrations to obtain the ic50 values. in addition to the secondary metabolite content in the extracts, there are other factors that can affect the efficacy of a. muricata ethanol, ethyl acetate and hexane fractions, such as biological variations of the plants, the plants’ habitats and the leaves used. environmental factors, such as humidity, temperature, types of nutrients from the soil and solar radiation, also play an important role in physiological functions, anatomical forms and plant life cycles.23 further research is needed on the cytotoxic ability of a. muricata leaf extract against hsc-3 cells in order to learn the optimal dose, long-term safety and side effects on normal cells. quantitative research is needed to phytochemically test and determine the levels of active compounds contained in the extract. this research shows that three extracts of a. muricata have a moderate cytotoxicity against hsc-3 cancer cells, with the greatest cytotoxicity in the ethyl acetate extract (ic50 = 76.66 µg/ml). thus, extracts of a. muricata need to be researched further to find the most effective concentration with the potential to be developed as a novel alternative therapy for oral cancer due to its antineoplastic agents. acknowledgements the authors acknowledge the support of the faculty of dentistry, universitas trisakti, jakarta. they also would like to express gratitude to dr indra kusuma, m. biomed from universitas yarsi, jakarta, who provided substantial help during this research. references 1. mathur g, nain s, sharma p. cancer: an overview. acad j cancer res. 2015; 8(1): 1–9. 2. rivera c. essentials of oral cancer. int j clin exp pathol. 2015; 8(9): 11884–94. 3. sirait am. faktor risiko tumor/kanker rongga mulut dan tenggorokan di indonesia (analisis riskesdas 2007). media litbangkes. 2013; 23(3): 122–9. 4. pires fr, ramos ab, de oliveira jbc, tavares as, da luz psr, dos santos tcrb. oral squamous cell carcinoma: clinicopathological features from 346 cases from a single oral pathology service during an 8-year period. j appl oral sci. 2013; 21(5): 460–7. 5. gharat sa, momin m, bhavsar c. oral squamous cell carcinoma: current treatment strategies and nanotechnology-based approaches for prevention and therapy. crit rev ther drug carrier syst. 2016; 33(4): 363–400. 6. vinken m, rogiers v. protocols in in vitro hepatocyte research. vinken m, rogiers v, editors. new york: springer; 2015. p. 1–390. (methods in molecular biology; vol. 1250). 7. zafrial rm, amalia r. anti kanker dari tanaman herbal. farmaka. 2018; 16(1): 15–6. 8. lestari r, solihah sm, aprilianti p, wawangningrum shh, agustin ek, sahromi, wibowo aru, munawaroh s, permatasari pa. koleksi tumbuhan buah kebun raya katingan. witono jr, yuzammi, editors. jakarta: lipi press; 2017. p. 28–9. 9. widyastuti da, rahayu p. antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate. biol med nat prod chem. 2017; 6(1): 1–4. 10. fertilita s, sandhika w, suprabawati dga. the cytotoxic activity of annona muricata linn leaves ethanolic extract (amee) on t47d breast cancer cell line. med lab technol j. 2020; 1(1): 32–9. 11. indrawati l, pramono s, ascobat p, bela b, abdullah m, surono is. cytotoxic activity of soursop “annona muricata” leaves extracts and their phytochemical contents. j glob pharma technol. 2017; 9(2): 35–40. 12. qorina f, arsianti a, fithrotunnisa q, tejaputri na, azizah nn, putrianingsih r. cytotoxicity of soursop leaves (annona muricata) against cervical hela cancer cells. pharmacogn j. 2020; 12(1): 20–4. 13. moghadamtousi sz, fadaeinasab m, nikzad s, mohan g, ali hm, kadir ha. annona muricata (annonaceae): a review of its traditional uses, isolated acetogenins and biological activities. int j mol sci. 2015; 16(7): 15625–58. 14. abdul wahab sm, jantan i, haque ma, arshad l. exploring the leaves of annona muricata l. as a source of potential antiinflammatory and anticancer agents. front pharmacol. 2018; 9: 661. 15. thouri a, chahdoura h, el arem a, omri hichri a, ben hassin r, achour l. effect of solvents extraction on phytochemical components and biological activities of tunisian date seeds (var. korkobbi and arechti). bmc complement altern med. 2017; 17(1): 248. 16. syafitri ne, bintang m, falah s. kandungan fitokimia, total fenol, dan total flavonoid ekstrak buah harendong (melastoma affine d. don). curr biochem. 2014; 1(3): 105–15. 17. ribeiro ip, rodrigues jm, mascarenhas a, kosyakova n, caramelo f, liehr t, melo jb, carreira im. cytogenetic, genomic, and epigenetic characterization of the hsc-3 tongue cell line with lymph node metastasis. j oral sci. 2018; 60(1): 70–81. 18. dojindo laboratories. measuring cell viability/cytotoxicity: cell counting kit-8. cell viability and cytotoxicity assay: protocol. dojindo molecular technologies, inc.; 2020. p. 4–15. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 136bhanuwati and pakpahan/dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 130–136 19. cai l, qin x, xu z, song y, jiang h, wu y, ruan h, chen j. comparison of cytotoxicity evaluation of anticancer drugs between real-time cell analysis and cck-8 method. acs omega. 2019; 4(7): 12036–42. 20. damasuri ar, sholikhah en, mustofa. cytotoxicity of ((e)-1-(4aminophenyl)-3-phenylprop-2-en-1-one)) on hela cell line. indones j pharmacol ther. 2020; 1(2): 54–9. 21. gavamukulya y, wamunyokoli f, el-shemy ha. annona muricata: is the natural therapy to most disease conditions including cancer growing in our backyard? a systematic review of its research history and future prospects. asian pac j trop med. 2017; 10(9): 835–48. 22. dasari s, tchounwou pb. cisplatin in cancer therapy: molecular mechanisms of action. eur j pharmacol. 2014; 740: 364–78. 23. pramadya pp, hendryana ma. efek ekstrak metanol daun sirsak (annona muricata) dalam menghambat pertumbuhan bakteri salmonella typhi secara in vitro. j med udaya. 2020; 9(9): 65–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p130–136 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p130-136 159159 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 159–163 case report leukoplakia in hiv patients and risk of malignancy: a case report yuli fatzia ossa,1 anandina irmagita soegyanto,1 diah rini handjari2 and endah ayu tri wulandari3 1department of oral medicine, faculty of dentistry, universitas indonesia, jakarta, indonesia 2department of anatomical pathology, faculty of medicine, universitas indonesia, jakarta, indonesia 3division of oral medicine, department of dentistry, dr. cipto mangunkusumo general hospital / faculty of medicine, universitas indonesia, jakarta, indonesia abstract background: oral potentially malignant disorder (opmd) was defined by the world health organization (who) as an oral lesion which shows abnormalities with potential to become malignant. leukoplakia is one such opmd that may present in patients with human immunodeficiency virus (hiv), with the condition of hiv presenting a heightened risk of malignancy. purpose: this case report aims to describe the initial finding and case management of an oral lesion that was suspected as leukoplakia in an hiv-positive patient on antiretroviral therapy (art). case: a 34-year-old male patient was referred to the oral medicine division from an hiv clinic at dr. cipto mangunkusumo general hospital, jakarta, with a chief complaint of dental caries. the patient was hiv and hepatitis c positive and already undergoing art. case management: during the intraoral examination, we found multiple caries, poor oral hygiene and a single thin white plaque lesion on the right buccal mucosa around the second and third mandibular molar region. the white plaque was painless, irregular, clearly demarcated, could not be rubbed off and did not disappear when stretched. history of trauma in this area was non-contributory and the lesion had gone unnoticed prior to oral examination. the patient had an existing smoking habit of 25 years. as clinical examination suggested leukoplakia, a biopsy was arranged. conclusion: leukoplakia is defined as a potentially malignant lesion. the risk of shifting into malignancy can be higher for patients who are hiv positive. especially in hiv-positive patients, special measures are needed to prevent shifting into malignancy, such as early detection, elimination of risk factors, performing excisional biopsy and regular intraoral examination. keywords: hiv; leukoplakia; oral potentially malignant disorder correspondence: yuli fatzia ossa, department of oral medicine, faculty of dentistry, universitas indonesia, jl. salemba raya 4 jakarta 10431, indonesia. email: yuliossa92@gmail.com. introduction in conjunction with a workshop with the centre for oral cancer/precancer, the world health organization (who), in 2007, outlined the characteristics of patients who have the potential to develop malignant disorders, or what is called a potentially malignant disorder, classifying potentially malignancy disorders into subgroups as follows. the first subgroup includes precancerous lesions which are benign with morphologically altered tissue and have a greater risk becoming malignant than normal lesions.1,2 the second group describes a precancerous condition in which a disease or patient’s oral hygiene habits do not necessarily alter the clinical appearance of local tissues, but are associated with greater than normal risk of a precancerous lesion or the development of cancer in the affected tissue.1,3 leukoplakia is defined as a white plaque lesion that cannot be clinically or histologically characterised by other conditions or diseases.4 the prevalence of leukoplakia is estimated at 2% of the global population, with this condition known to occur in individuals who have a habit of smoking and drinking alcohol.1,4 leukoplakia can occur in all parts of the oral mucosa and is clinically divided into two types, namely homogenous lesions, which are flat, thin and a uniform white in colour, and non-homogenous lesions, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p159–163 mailto:yuliossa92@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p159-163 160 ossa et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 159–163 which have a white-red appearance and can have irregular surface texture (speckled) or be nodular.4,5 human immunodeficiency virus (hiv) is a viral infection that attacks the immune system, with its main target being cd4+ t cells, causing the body to lose its ability to fight infection.6 based on the latest report from the indonesian ministry of health regarding the spread of human immunodeficiency virus/acquired immune deficiency syndrome (hiv/aids) from 1987 to june 2018, infection rates continue to rise.7 to date, the reported number of people living with hiv/aids in indonesia is 301,959.7 leukoplakia falls into the category of precancerous lesions. as hiv is considered a precancerous condition, both leukoplakia and hiv have the potential to become malignant.1 in this case report, the authors describe the management of leukoplakia in hiv-positive patients undergoing antiretroviral therapy (art), with case management carried out with the aim of implementing preventive measures that will reduce the risk of transformation into malignancy. case on 25 february 2019, a 34-year-old male patient was referred from the hiv clinic to the oral medicine devision of dr. cipto mangunkusumo general hospital, with a working diagnosis of dental caries and a request for evaluation and management of the condition. case management the patient was hiv positive and had been on art since 2005. he was also known to have had chronic hepatitis c infection since 2003. during the first visit, the patient reported that the hepatitis c virus was at such a level that it was no longer detectable. the art taken was a combination of nevirapine and duviral with a prescribed dosage frequency of two times per day. during the patient history, it was confirmed that the patient had actively smoked for the past 25 years, with an amount given of one pack of cigarettes per day. upon undertaking oral examination using a light-emitting diode light, white plaque lesions were detected on the right buccal mucosa of the second and third mandibular molars. the white plaque lesion was painless, irregular, clearly demarcated, could not be rubbed off, did not disappear when stretched and was around 5mm in diameter. the white plaque lesion had gone unnoticed prior to oral examination and the patient denied history of trauma due to tooth friction in the area. based on the patient history and clinical examination, it was determined that the white lesion was suspected leukoplakia. thus, an excisional biopsy (figure 1) was scheduled. as shown in the panoramic radiograph (figure 2), figure 1. clinical finding of white lesion on right buccal mucosa (see yellow arrow). figure 2. panoramic radiograph. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p159–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p159-163 161ossa et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 159–163 poor oral hygiene was evident in the form of suband supragingival calculus, tooth radices and pulp necrosis. an improvement of oral hygiene, multiple dental extraction and scaling were subsequently planned. prior to the excisional biopsy, the white plaque lesion was treated with topical application of 100,000 iu vitamin a three times a day, with the additional suggestion that the patient take a once-daily dose of antioxidant supplement. we informed the patient of the oral findings, the aetiological factor of leukoplakia and the risk of malignant transformation of leukoplakia in hiv-positive patients, moreover recommending that the patient make changes toward a healthier lifestyle, including stopping smoking. the complete blood count on 26 february 2019 showed erythrocyte levels, activated partial thromboplastin time (aptt), blood creatinine and blood urea below normal range, while mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch) and mean corpuscular haemoglobin concentration (mchc) levels were above normal range. an hepatitis c virus ribonucleic acid test had shown undetectable levels of the virus on 25 january 2019, while the february 2019 blood test indicated absolute lymphocyte count (cd45+), percentage of t cells (cd3+), t cells absolute (cd3+) and t cell absolute (cd4+) all above normal range (6629/µl, 74%, 4876 cell/µl and 1627 cell/µl respectively). only the percentage of cd4 t cells was below normal range (25%). the patient underwent an excisional biopsy for the white plaque lesion and multiple tooth extraction on 6 march 2019. the specimen from the excisional biopsy was then sent to the anatomical pathology laboratory for histopathological examination (figure 3). on 15 march 2019, the patient came for a follow-up visit post-biopsy, in which they were advised of good hygiene practices and informed of the histopathological examination result. they were instructed to cease application of the topical ointment but continue to take the antioxidant supplement at a dose of one tablet per day, with a routine follow-up visit scheduled for every three months going forward. they were also informed of the need for a repeat biopsy at the same area one year after the first, especially should the white plaque lesion reappear. discussion in the above case, the patient was initially unaware of the white lesion, which was only found during intraoral examination by an oral medicine specialist. the white lesion was only present on the right buccal mucosa and persisted even when blanched. furthermore, it could not be rubbed off. the patient denied history of trauma due to tooth friction in the area. based on the clinical appearance, the history of heavy smoking for the past 25 years to date and the level of patient alcohol consumption, the white lesion was identified as suspected leukoplakia. according to lima et al.8 individuals with a smoking habit have a six times higher risk of leukoplakia compared to those who do not smoke, with the most common location in which leukoplakia occurs being the buccal mucosa. this supports the conclusion that the white lesion identified on the buccal mucosa of the patient, who was a heavy smoker of one pack of cigarettes per day over the preceding 25 years, was highly suggestive of leukoplakia. the causes of leukoplakia are attributed to several factors, with smoking being the main one. leukoplakia can present as single or multiple lesions, with any oral site having the potential to be affected. clinically, leukoplakia is divided into two types: homogenous and non-homogenous.5,9 homogenous lesions can be flat, thin and a uniform white in colour, while non-homogenous legions can be white or red and may be either of irregular surface texture (speckled), nodular or verrucous, the latter variety of which has an elevated, proliferative or corrugated surface appearance.10 leukoplakia is categorised as a lesion with the potential to become malignant.10 the estimated rate of change toward malignancy is 1–2%.10,11 the causes of leukoplakia are divided into two categories: idiopathic leukoplakia and leukoplakia associated with habitual use of tobacco.10,11 while leukoplakia can occur in all parts of the oral cavity, occurrences located at the base of the mouth, soft palate and tongue have a high risk of becoming malignant, whereas leukoplakia in other areas such as the buccal mucosa have a low risk of such transformation.11 in this case report, the patient was hiv positive. based on the ec clearinghouse on oral problems related to hiv infection study of 1992,9 manifestations of hiv/aids figure 3. histopathological result with haematoxylin eosin (h&e) staining. squamous epithelium hyperkeratosis, spongiotic gingivitis, acanthosis and elongated rete ridges (see black arrow) are present, while the basal membrane is still intact and there is no sign of dysplasia (see blue arrow). furthermore, there is an infiltration of lymphocyte cell in the subepithelial area. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p159–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p159-163 162 ossa et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 159–163 in the oral cavity were divided into three groups: first, lesions that were strongly associated with hiv infection; second, lesions with a strong association with hiv; and third, lesions that could found in hiv-positive patients.9,10 leukoplakia is not included in the ec clearinghouse categorisation. in terms of the white lesion examined in this study, an excisional biopsy was planned to confirm its nature. the choice to perform an excisional biopsy was made due to the relatively small size of the lesion (less than 6mm). once the procedure was complete, the biopsy specimen was taken to the anatomy pathology laboratories for histopathological examination. histopathological features of leukoplakia have various appearances, including epithelial hyperkeratosis and hyperplasia, and can be accompanied by the presence or absence of dysplasia as well as carcinoma in situ.12 dysplasia is a histopathological change that is characterised by a loss of architectural form of epithelial cells and can be associated with impaired cell proliferation and impaired cell maturation.10 in this case report, the histopathological features present in the specimen were hyperkeratosis, prolonged rete ridges, presence of acanthosis, hyperplasia in the basal cell area and an infiltration of subepithelial lymphocyte cells. however, there was no dysplasia found in the histopathology examination. although leukoplakia located on the buccal area has a low risk of becoming malignant when compared that on other locations, the presence of an hiv infection leads to the persistent assumption of a possibility that lesion may still be malignant, as hiv patients have a higher risk of malignancy when compared to general population.13 referring to farah et al.’s14 examination of several previous studies, it can be seen that, due to immune system deficiency issues, patients who are hiv positive have a high risk of developing cancer, including lip cancer, oral cancer, pharyngeal cancer and oesophageal cancer, compared to the general population. immunodeficiency contributes to malignancy because an impaired immune system can reduced immune surveillance for malignant cells and also impair the ability to suppress oncogenic factors.13,15 in hiv patients, the risk of developing oral cancer is increased by immune deficiency and can also be related to tobacco use.14 we therefore informed the patient that risk of oral cancer may not be eliminated by excision of the lesion, recommending regular follow-up visits every three months. we also informed the patient that the lesion may recur within a matter of time, ranging from weeks, months to several years. management of leukoplakia can be accomplished through surgical excision, pharmacological therapy with drugs or a combination of both. for the patient described in this study, both pharmacological therapy and surgical excision were used. prior to the excisional biopsy, we prescribed a topical application of 100,000 iu vitamin a cream to aid cellular growth and differentiation of epithelial cells after modulating cellular gene expression.16 retinoids are also a class of antioxidant compound that are responsible for the balance of cellular growth, differentiation and apoptosis, and have the capacity to prevent recurrence of leukoplakia.16,17 topical rather than systemic vitamin a was selected due to the lower side effects of topical versus systemic consumption. in addition to this topical application of vitamin a, an antioxidant supplement containing lycopene was also prescribed. lycopene not only has antioxidant properties, but is also able to modify the intercellular exchange junction, meaning it functions as an anti-cancer mechanism.17 the purpose of the treatment was to prevent the lesion from becoming malignant. as leukoplakia has the potential to become malignant, it is important for clinicians to facilitate early diagnosis and remove possible contributing factors such as smoking, thus reducing the rate of malignant transformation. in conclusion, leukoplakia is categorised as a potentially malignant disorder, while in patients with hiv, the risk of becoming malignant can be even higher. thus, in order to reduce the rate of malignant transformation, early detection and treatment, as well as patient education to avoid all risk factors, are of great importance. furthermore, due to the potential recurrence of leukoplakia, it is highly recommended that patients have regular follow-up visits. acknowledgment we would like to express our appreciation to the patient who featured in this case report. references 1. mortazavi h, baharvand m, mehdipour m. oral potentially malignant disorders: an overview of more than 20 entities. j dent res dent clin dent prospects. 2014; 8(1): 6–14. 2. speight pm, khurram sa, kujan o. oral potentially malignant disorders: risk of progression to malignancy. oral surg oral med oral pathol oral radiol. 2018; 125(6): 612–27. 3. warnakulasuriya s, ariyawardana a. malignant transformation of oral leukoplakia: a systematic review of observational studies. j oral pathol med. 2016; 45(3): 155–66. 4. van der waal i. historical perspective and nomenclature of potentially malignant or potentially premalignant oral epithelial lesions with emphasis on leukoplakia—some suggestions for modifications. oral surg oral med oral pathol oral radiol. 2018; 125(6): 577–81. 5. van der waal i. oral leukoplakia, the ongoing discussion on definition and terminology. med oral patol oral cir bucal. 2015; 20(6): e685–92. 6. bobat r, archary m. hiv infection. in: green rj, editor. viral infections in children, volume i. spinger; 2017. p. 69–100. 7. ditjen p2p kementrian kesehatan republik indonesia. laporan perkembangan situasi hiv-aids & pims di indonesia januaridesember 2017. jakarta; 2018. p. 560. 8. lima js, pinto dds, de sousa som, corrêa l. oral leukoplakia manifests differently in smokers and non-smokers. braz oral res. 2012; 26(6): 543–9. 9. villa a, woo s bin. leukoplakia—a diagnostic and management algorithm. j oral maxillofac surg. 2017; 75(4): 723–34. 10. kayalvizhi eb, lakshman vl, sitra g, yoga s, kanmani r, megalai n. oral leukoplakia: a review and its update. j med radiol pathol surg. 2016; 2: 18–22. 11. ribeiro as, salles pr, da silva ta, mesquita ra. a review of the nonsurgical treatment of oral leukoplakia. int j dent. 2010; 2010(2): 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p159–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p159-163 163ossa et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 159–163 12. deliverska eg, petkova m. management of oral leukoplakia analysis of the literature. j imab annu proceeding (scientific pap. 2017; 23(1): 1495–504. 13. borges a, dubrow r, silverberg m. factors contributing to risk for cancer among hiv-infected individuals and evidence that earlier cart will alter risk. curr opion hiv aids. 2014; 9(1): 34–40. 14. farah cs, jessri m, currie s, alnuaimi a, yap t, mccullough mj. aetiology of oral cavity cancer. in: kuriakose ma, editor. contemporary oral oncology. springer; 2017. p. 31–76. 15. silverberg mj, chao c, leyden wa, tang b, horberg ma, klein d, charles p, jr q, towner wj, abrams di. hiv infection and the risk of cancer with and without a known infectious cause. aids. 2009; 23(17): 2337–45. 16. seo j, utumi er, zambon ce, pedron ig, ceccheti mm. use of retinoids in the treatment of oral leukoplakia: review. rev clín pesq odontol curitiba. 2010; 6(2): 149–54. 17. salati na. clinico-pathologic evaluation & medical treatment of oral leukoplakia. int j pharm sci invent. 2014; 3(2): 7–12. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p159–163 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p159-163 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak1, matsuo hamada2, ninuk hartati3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur, malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima, japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne, australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in original articles should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in review articles should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (original articles, case reports, or review articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, 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editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair.ac.id/ mkg/about/submissions#onlinesubmissions the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id isi vol 39 no 2 april 2006 file pertama.pmd 48 placement of replace select ti-unite-coated type implants using a combination of immediate and submerge techniques after tooth extraction coen pramono d department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract the high success rate of immediate implant placement both in the anterior and posterior regions were reported by many authors, therefore applying this techniques can be considered as a safe surgical procedure and minimizing the dental office visit for patient satisfaction. this paper reports the outcome of immediate placement of implants following extraction of anterior maxillary teeth. combination technique of immediate and submerge implant placements including bone grafting procedure were used. four implants with tiunite surface type were placed immediately in two patients with the short-term result indicated that this technique may serve as a simple and safe procedure for immediate implant placement. it was concluded that immediate implant placement technique combined with tiunite implant surface was successful in treating region directly after tooth extraction therefore this technique can be use as an alternative surgical method for dental implant rehabilitation. key words: immediate implant placement, ti-unite-coated implant surface, osseointegration correspondence: coen pramono d, c/o: bagian bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. intoduction in may 1982 conference held in toronto, the north american dental profession was introduced to a body of scientific literature on swedish research into bone-to implant interface, a concept called osseointegration. this concept is based on a-traumatic implant placement and delayed implant loading. the swedish research team led by pi branemark reported the high success of implant placement in the mandible over15 years.1 the recent success of dental implants relates directly to the discovery of method to maximize the amount of bone and implant contact. extraction and immediate use of immediate denture is common solution to aesthetic problem. however, the subsequent healing of the extraction socket may result in collapse of the local bone and soft tissue, leading in turn to poor esthetic outcome. acceptable esthetic outcomes after augmentation is uncertain, another treatment option as suggested by schwartz-arad and chaushu in 19972 and kirketerp et al.3 placement of implant combined with tooth extraction has been demonstrated as a viable option. pedlar and frame4 disagreed with immediate implant placement as the prognosis for successful osseointegration is poor, especially in the presence of apical pathology prior implant insertion. other problems likely to be encountered including the differences between the width of the natural tooth and the endosseous implant and insufficient bone at the coronal aspect, therefore a bone graft may be required to close the defect. a further disadvantage is that nonkeratinized mucosa may have to be advanced to cover the implant. they suggested that 5 to 6 weeks after tooth extraction, the soft tissue will have healed but the residual ridge should not have undergone excessive resorption. the recent success of dental implants relates directly to the discovery of method to maximize the amount of bone and implant contact. osseointegration is a histologic definition meaning as: a direct contact between living bone and loaded-bearing endoseous implant at the microscopic level.1 this concept of osseointegration has brought a revolutionary progress of dental implantology in dentistry. placement of 36 implants replace select ha immediately after teeth extraction reported by kirketerp et al.3 showed that 35 of 36 immediately replaced maxillary front teeth were clinically stable and asymptomatic after one year loading. whorle in 1998 reported immediate replacement of 14 extracted teeth in the anterior maxilla. all implants remained stable during the 9 to 36 months follow-up.4 glauser et al.5 and rocci et al.6 reported the excellent clinical outcome of immediately loaded partial fixed bridge. glauser et al.5 reported success rate of 97% following immediate load of tiunite implant placed predominantly in soft bone and implant placed in bone quality 4 showed a 100% success rate.6 study by rocci et al.6 comparing between immediate loading of tiunite and machined implants in the posterior mandible, a 10% higher success rate was found for tiunite implants (95.5%). the present 49pramono: placement of replace select ti-unite-coated type implants histological evaluation confirmed that successful osseointegration of tiunite implant placed in soft bone is possible when the implant is immediately loaded. the 93.3% bone-implant contact achieved might even imply the immediate loading had stimulated the bone healing process. recently an implant surface modification has been developed for enhancing early bone response and this surface has been shown to enhance primary implant stability,7,8 and to achieve secondary stability earlier than a machined surface.9,10 cases two cases of patients treated with immediate implant placement in the anterior regions of the upper jaw are reported. those two patients are 41 year old man presented in case 1 and 25 year old women presented in case 2. both patients asked for dental implant treatment as they refused for a fixed crown and bridge denture rehabilitation after their teeth being extracted. both patients also have limited time for having their dental treatment, therefore they asked for the possibility of an alternative treatment used a direct implant placement after teeth extractions for time saving. the treatment planning included measurement of implant depth using coen’s measurement technique,11 a-traumatic tooth extraction using periotome, careful cleaning of the socket and placement of the implant with raising a flap. due to the bone available after teeth extraction an implant loading were delayed for a minimum period of 2.5 months after implant placement. torch of 35 ncm were un-reached in both cases as the alveolar bone condition had mostly resorbed by an inflammatory processes, but relative primary implant stability was achieved as the implants were inserted more palatal and an additional bone stability was taken by passing the implants slightly through the alveolar apical bone area to reach a healthy and compact bone. replace select tiunite from nobel biocare dental implants were used in these cases, one dental implant was inserted in case 1 and three in case 2. in both cases removable partial dentures were prepared pre-operatively for cosmetic reason, designed with freed in the cervical area to avoid soft tissues pressure in the implant sites during its use after the implants had been placed. case management case 1: a 41 year old male complained of recurrent abscesses in the labial site of tooth 12 (figure 1-a). panoramic radiograph presented with a radiolucent area surrounded the apical region of tooth 12 almost closed to the alveolar crest (figure 1-b). preservation of that tooth with root resection was presumed with un-successful result, therefore tooth extraction followed by immediate implant placement was chosen as a choice of treatment as the patient refused for a fixed crown and bridge denture. intraoperative steps were done with extraction of tooth 12 (figure 1-c) and a wide base mucoperiosteal flap was made and deflected (figure 1-d). the cyst and the remnants soft tissue were curettage and a thin labial bone was carefully preserved to be intact (figure 1-e). step by step bone drilling was made with initial direction more to the palatal bone and afterward the drilling angle direction changed parallel to the axis of adjacent anterior teeth (figure 1-f). the drilling process continued into the required implant depth and diameter. a 4.3 mm tiunite implant from nobel biocare was placed, some gaps around the implant sites were showed therefore a bone graft which had taken from the region of right zygoma was grafted, the mucosa then sutured back covered the whole implant body. the implant abutment was connected to the implant body 5 months after implant placement and followed by single ceramic crown rehabilitation (figure 1-h and i). no clinical symptom was reported by the patient. clinical observation 5 months after implant placement showed the implant had been reached its stability in satisfactory (figure 1b-i). case 2: a 25 year old female with chief complaint of pain sensation in the apical region of teeth 11, 21 and 22 asked for dental treatment. the patient had undergone for apical teeth resection on those teeth 5 years before. radiographic examination showed with apical radiolucent on those three teeth, and had an indication of teeth extraction (figure 2-a). immediate implant placement was planned for teeth replacement. tiunite implant from nobel biocare with 5.0 mm diameter for teeth 11 and 21 and 4.3 mm diameter for tooth 22 were used. pre-operatively a removable denture was prepared for cosmetic consideration. the teeth 11, 21 and 22 were extracted carefully and a wide based mucoperiosteal flap was made with posterior incision sides at the distal ends of the extracted teeth. a deep curettage was done to clean the socket from cyst and remnants tissues. the labial bone was carefully maintained to avoid from bone lost. step by step drilling for 4.3 mm and 5.0 mm diameter dental implants were made and left the labial bone intact, and followed by implant body placement. up to one week showed all implants were free from clinical symptoms and inflammatory process. panoramic radiograph ten days after implant placement showed all implants were well placed (figure 2-b). clinical and panoramic radiography observation 2.5 months after three implant placement showed with good results (figure 2-c). clinical symptoms as pain, implants mobility and sign of infection were not presented. a-traumatic gingival opening using punch technique was done for exposing those three dental implants (figure 2-d). fifteen degree type of abutment was connected to implant bodies in teeth 11 and 21, and a normal abutment in tooth 22 and screwed until the 15 ncm torch had reached and three single ceramic crowns were inserted by her private dentist (figure 2-e–g). 50 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 48–53 figure 1. (a) tooth 12 with recurrent of gingival abscess. (b) periapical cystic lesion grown almost involving the entire distal alveolar crest of tooth 12. (c) tooth extracted left good dental socket. (d) flap deflected and the socket deeply curettage. (e) socket curettage and left the thin labial alveolar bone intact. (f) step by step direct bone drilling on the fresh socket for implant immediate placement and left the labial-bone un-disturb. (g) panoramic x-ray:5 months after implant placement. a new bone grown seen fulfill the bone defect. (h) the abutment fixed on the implant. (i) final single ceramic crown situation 6 months after implant placement. a b c d e f g h i 51pramono: placement of replace select ti-unite-coated type implants discussion immediate implant placement was performed based on considerations that placement of implant and waiting for the bone has to be matured which usually taken about 3 to 6 months would be time consuming. the high affinity and degree of bone formation promoted into the implant surface due to the available modified implant surface with anodic oxidation technique had proved by some authors with significant osseointegration result,3,5-12 therefore the success of those cases reported had been taken as a basic knowledge on predicting the success these implant placement. implant placement need to be inserted in a good bone quality, and dental implant usually being performed after the bone had consolidated which can be found in the range of time about 3 to 12 months after tooth extraction. some authors had reported a good result of direct implant placement into a good bone quality and directly loaded with direct loading. in such situation implant placement needed to be placed immediately as patient has a limited visiting time to visit the dental office. direct implant placement immediately after tooth extraction can be used as an alternative method then placed waiting for the bone had reached its maturity. figure 2. (a) pre-operative panoramic radiography view: teeth 11, 21 and 22 with apical radiolucent post roots resection. (b) enlarged panoramic radiography 10 days after implant placement. (c) two and a half months after days implant placement. (d) a soft tissue punch method gives a-traumatic gingival opening. (e) three stable implants 2,5 months after insertion and exposed a-traumatically. (f) situation 4 days after implants exposure and connection of fifteen degrees abutment in teeth 11, 21 and straight form abutment in tooth 22. (g) final single ceramic crowns on teeth 11, 12 and 22. a b c d e f g 52 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 48–53 two cases of immediate dental implant placement are reported. implant placement in the extraction socket often leads no primary bone contact surrounding the implant body. if the implant stability is achieved, it is usually due to bone contact at the palatal bone side and in the apical end of the socket. the coronal end and some part of the labial site usually can be found with slightly open or even the implant threads might be found exposed due to the implant diameter is smaller than the tooth size which needed to be replaced. therefore bone graft is usually necessary to be placed around the implant body necessary to cover some of the exposed implant with bone graft. bone integration of such an implant depends on bone being formed from adjacent bone surfaces toward the implant surface. histological evidences were found by carlsson et al. and cited by salata et al.12 and akimoto et al.13 that large gaps between a turned implant and bone will not be filled with bone. clinical study by schwartzarad and chaushu2 presented a good long-term result of placing implants into fresh socket. in this surgical report, in both cases bone graft procedure were done, as some gaps were presented between bone-implant. in these cases the bone–implant gaps were harvested with an autogenous bone which had taken from the bone residues during bone drilling process and added with some bones taken from the zygoma area. this bone graft procedures were performed to enhance the process of osseointegration. a mucosa tissue split technique for bone covering was done to stabilize the harvested bone graft material remain stable in the grafted areas. the periosteum was separated from the mucosa, adapted and sutured around the neck implants, followed by mucosa adaptation and suturing. a recent study found that the junctional epithelium effectively attached via hemidesmosome to uniquely porous tiunite surface much like natural teeth. the connective tissue interposed between junctional epithelium and the alveolar crest is dominated by both circumferentially and longitudinally running collagen fibers. as a result, the connective tissue around tiunite implants is stable and healthy.14 a number of studies in implant type which its surface modified by anodic oxidation have shown a higher affinity and degree of bone formation.9,10 a study by salata et al.12 using resonance frequency analysis on (rfa) np mkiii tiunite, nobel biocare ab supported the previous studies.9,10 they concluded that oxidized implant surface has a significant influence on bone formation when placed in bone defects and the implant stability increase more rapidly. the excellent clinical outcome of this direct implant placement reported in this case is nearly closed with the data reported by glauser et al.5 a different situation given in these case reported, an immediately implant loaded was considered to be too risky to performed as only a thin part of the labial bone left intact and the maximum implant primary stability of 30 to 35 n/cm2 was not achieved, therefore implant placement was done in combination, direct implant placement immediately after tooth extraction and submerged technique. two and a half months after the implant placement, good stability is shown in all 4 implants observed. this situation can be assumed that the 4 walls bone of the dental sockets had grown and the implantsbones osseointegration had achieved properly. some critical steps during the surgical procedure were noted as: 1) the bone gaps between implants and bones, therefore bone grafting was considered necessary to be applied to close those gaps, 2) during the drilling procedures highly consideration should be mentioned: a) the initial drilling direction must be directed to the palatal wall direction of the post extraction socket to achieve more compact bone for implant placement in achieving primary stability and b) change of the drill’s trajectory from palatal into parallel direction with the adjacent teeth to achieve good implant axis. this step can be paid a consequence while it related with the result of implant angulations. as shown in case 2, two implants were placed in the region of teeth 21 and 22 with the implants axis position more palatal, a 15 degree angled abutments was necessary to be used to compensate the implant position. according to my early experience can be concluded that combination of immediate and submerge implant placement technique and additionally with bone grafting procedure to cover the uncovered implant surface presented constitutes a simple and safe procedure for replacement of hopeless anterior teeth in the maxilla. application of this technique gave a favorable experience as the implant can be placed with clear surgical axis angulations following the alveolar socket of the extracted teeth. additionally bone stability can be achieved by placement the dental implant slightly to the palatal bone and passing through the implants end to the alveolar apical bone area to reach a healthy and compact bone. references 1. peterson lj, ellis e, hupp jr, tucker mr. contemporary oral and maxillofacial surgery. 4th ed. st louis: mosby; 2003. p. 305–42. 2. schwartz-arad d, chaushu g. the way and where forces of immediate placement of implants into fresh extraction sites: a literature review. j periodontol 1977; 68(9):15–23. 3. kirketerp p, andersen jb, goeran u. replacement of extracted anterior teeth by immediately loaded replace select ha-coated implants. a one-year follow-up of 35 patients. appl osseointegration res 2002; 3(1):40–3. 4. pedlar j, frame jw. oral and maxillofacial surgery. an objectivebased text book, churchill livingstone. printed in spain. 2001. p. 157. 5. glaucer r, gottlow j, lundgren ak, senneerby l, portmann m, rusthaller p, haemmerle chf. immediate occlusal loading of branemark mkiv tiunite implants placed in bone quality 4. appl osseointegration res 2002; 3:22–4. 6. rocci a, martiggoni m, sannerby l, gottlow j. immediate loading of a branemark system implant with the tiunite surface. histological evaluation after 9 months. appl osseointegration res 2002; 3(1): 25–8. 53pramono: placement of replace select ti-unite-coated type implants 7. rompen e, dasilva d, lundgren ak, gottlow j, sennerby l. stability measurements of double-threaded titanium implant design with turned or oxidized surfaces. an experimental resonance frequency analysis study in dog mandible. appl osseointegration res 2000; 1:18–20. 8. glauser r, portmann m, ruhstaller p, lundgren ak, haemmerle c, gottlow j. stability measurements of immediately loaded machined and oxidized implants in the posterior maxilla. a comparative clinical study using resonance frequency analysis. appl osseointegration res 2001; 2:27–9. 9. albrektsson t, johansson c, lundgren ak, sul y, gottlow j. experimental studies on oxidized implants. a histomorphometrical and biomechanical analysis. appl osseointegration res 2000; 1: 21–4. 10. henry p, tan a, allan b, hall j, johansson c. removal torque comparison of tiunite and turned implants in greyhound dog mandible. appl osseointegration res 2000; 1:15–7. 11. pramono c. introducing a simple innovative technique in achieving implant parallelism and an equation concept in measurement the discrepancy in panoramic radiograph. accepted for publication in the j oral maxillofac surg for 2006. 12. salata l, rasmusson l, novaes jr a, papalexiou v, sennerby l. the influence of anodic oxidation on implant integration and stability in bone defects. an rfa study in dog mandible. appl osseointegration res 2002; 3(1):32–4. 13. akimoto k, becker w, persson r, baker da, rohrer md, o’neal rb. evaluation of titanium implants placed into stimulated extraction sockets: a study in dogs. int j oral maxillofac implants 1999; 14: 351–60. 14. glauser r, schuepbach p, gottlow j, lundgren ak, ruhstaller p, haemmerle chf. soft tissue barrier at non-submerged one-piece micro-implants with different surface topography retrieved from human, poster presentation: 12th annual meeting european academy of osseointegration, october 9–11, viena 2003. nobel biocare, nobel direct biologic one-piece implant. clinical procedure and product catalog, 2004. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 9393 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 93–98 research report correlation between age and dental arch dimension of javanese children atiek driana rahmawati,1, 2 iwa sutardjo rus sudarso,3 dibyo pramono4 and eggi arguni5 1doctoral program, faculty of dentistry, universitas gadjah mada 2department of pediatric dentistry, dental school, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta 3department of pediatric dentistry, faculty of dentistry, universitas gadjah mada 4department of preventive dentistry and dental public health, faculty of dentistry, universitas gadjah mada 5department of child health, faculty of medicine, public health and nursing, universitas gadjah mada yogyakarta – indonesia abstract background: dental arch form and dimension are fundamental factors in orthodontic diagnosis and treatment planning. its dimension will increase, due to the eruption of teeth, and is also affected by ethnicity, nutrition, systemic disease, hormonal factors, and gender. many teeth are erupting in 8–10-year-old children. purpose: this study aimed to assess the correlation between age and dental arch dimension of javanese children in good nutritional status for consideration of orthodontic treatment. methods: this was a crosssectional study with 66 children aged 8–10 years in a normal dentoskeletal relationship, grouped based on age as the subject. each group consisted of 22 pairs of dental study models, male and female. anterior and posterior size of dental arch length were measured by digital sliding calipers from the midpoint between the right and left permanent central incisors perpendicular to the inter-canines and inter-molars. the width was measured at the inter-canines and inter-molars. results: pearson’s correlation test showed that there were significant correlations between age and maxillary dental arch lengths (p = 0.01, r = 0.31 for anterior, and p = 0.043, r = 0.249 for posterior). conclusion: based on this study, it can be concluded that there was a positive correlation between age and dental arch length of 8–10-year-old javanese children in good nutritional status, especially in maxillary dental arch length. keywords: arch length; arch width; children; dental arch; dimension correspondence: atiek driana rahmawati, doctoral program, faculty of dentistry, universitas gadjah mada, jl. denta no. 1, sekip utara, yogyakarta 55281, indonesia. e-mail: atiek.driana@umy.ac.id introduction in patients who are growing, tooth development and bone maturation are widely used to determine the time of orthodontic treatment and the selection of treatment modalities.1 the dental arch size will increase due to permanent tooth eruptions. it is influenced by the environment, nutrition, genetics, race, sex, and age.2 children aged 8–10 years are in the mixed dentition period. there are some permanent tooth eruptions that replace some primary teeth. the erupting teeth that occur in an 8-year-old child are permanent maxillary lateral incisors; in a 9-year-old, they are permanent mandible canines, and in a 10-year-old, they are permanent maxillary and mandible first premolars, and also permanent maxillary second premolars.3 changes in the growth of the dentocraniofacial complex caused by poor nutrition can be reflected in the reduced space for tooth eruption4. mack1 states a significant relationship between weight status, determined by the bmi percentile, and dental age and cervical bone maturity. the bmi percentile increases with the increasing development of the teeth and bones. bmi is an easy measurement and calculation method, which is the most widely used diagnostic tool to identify the nutritional status of a population, and usually determines whether a person is underweight, healthy, or overweight.5 lombardo et al.6 find that dental arches can be affected by ethnicity. its size, both in width and length, can be influenced by genetic factors, and the effect is very visible in size difference between maxilla and mandible.7 the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 mailto:atiek.driana@umy.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 94 rahmawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 93–98 dental arch dimensions were not similar for each different ethnic population.8 different ethnic populations will also have significant differences in the size of their teeth.9 the javanese are the most populous ethnicity in indonesia. according to jacob, javanese are included in the deuteromalay ethnic group, which has characteristics such as wide nostrils and alae nasi and medium-thickness lips.10 comprehensive diagnosis and treatment planning are factors that greatly determine the success of orthodontic treatment. dental arch form and its dimensions are one of the fundamental factors in the diagnosis.11 the dental arch dimension is explained by arch width, arch length, and arch perimeter.12 the growth and development of dental arches are a continuous process with several changes during the period of child development. changes occur in all dimensions continuously in adolescence to adulthood.13 this condition is important for determining the diagnosis and orthodontic treatment planning, as well as post-treatment stability.14 many studies about the dimension of the dental arch, especially in indonesia and the javanese, are focused on adults.15–18 this study aimed to know the correlation between age, dental arch length, and width in 8–10-year-old javanese indonesian children for consideration of orthodontic treatment. materials and methods all procedures performed in this study involving human participants were approved by the ethical committee of the faculty of medicine and health sciences, universitas muhamadiyah yogyakarta, no: 455/ep-fkik-umy/ x/2018. this research was observational and analytic with a cross-sectional design, carried out in grogol state elementary school, bantul regency, yogyakarta special region. the parents of the subjects had been informed of all the procedures of this study. only children who were permitted by their parents could be the subjects of this study, and informed consent had been given by the parents of each child. the subjects of this study were taken by simple random sampling, and the sample size was calculated using this formula: � ��� (�)� n = ; n = number of samples needed; n = number of population (154) 1; d = validity level selected (d = 0.1). the minimum sample size was 61, and in this study, the number of subjects was 66 children, male and female. inclusion criteria were javanese children (until filial 2, descendants from java), aged 8– 10 years old, good nutritional status, normal occlusion, overjet and overbite of 2–4 cm, and normal tooth position at contact points and contact surfaces. meanwhile, the other criteria for the subjects were 8-year-old children whose maxillary permanent central incisors and mandible lateral incisors had erupted, 9-year-olds whose maxillary permanent lateral incisors had erupted, and 10-year-olds whose mandible permanent canines had erupted. the teeth which were measured were to be free of restorations, fractures, or proximal caries. exclusion criteria were that the children have ever had or are in orthodontic treatment and facial trauma with clinical symptoms. the other exclusion criteria were that they had a systemic disease and had radices or caries/fractures/attrition involving the proximal wall and anomalous form of the teeth that were measured. nutritional statuses were determined based on body mass index (bmi) for age, as stated in the indonesian minister of health decree number 1995/menkes/ sk/xii/2010 concerning anthropometric standards for assessing children’s nutritional status,19 and good (normal) nutritional criteria were used in this study. the child’s weight was measured using an electronic digital scale (qc pass p: es-bg00 do01193281, the capacity of up to 180 kg) placed on a flat surface for recording weight. the height was recorded by using ordinary measuring tape fixed at the wall. the child was straight, the frankfurt plane was horizontal, and the head-pressing piece was gently lowered until it was touching the top of the child’s head. the subjects aged 8–10 years in good nutritional status had their dental impressions taken using alginate impressions (aroma fine plus normal set, gc corporation, tokyo, japan) to get dental study models grouped by age. reference points were determined for each dental study model for measuring its dimension, both for dental arch length and width. the reference points were the midpoint between the right and left permanent central incisors, the cusp tip of the right and left canines, and the tip of the mesiobuccal cusp of the right and left permanent first molars. using a digital sliding caliper (mitutoyo digimatic caliper, code no. 573-721-20, model no. ntd12-p6”m, serial no. 0000644, japan), the dental arch length was measured from the midpoint between the right and left permanent central incisors perpendicular to the midpoint of the line which was connecting the right-left cusp tip of the canine teeth. this measure resulted in an anterior arch length. then the posterior arch length was measured from the vertical line which was the distance from the middle of the central incisors perpendicular to the line formed between the tips of the mesiobuccal cusps of the right and left first molars. anterior dental arch width was measured from the inter-canine width, and posterior dental arch width was measured from the inter-molar width. inter-canine width was measured from the cusp tip of one side to the cusp tip of the other side for each canine, and inter-molar width was taken from the mesiobuccal cusp tip of the right side to the left side. these measurements were done in both arches. all measurements and assessments of all required parameters were carried out by one operator to reduce the error measurement. the study models were assessed twice, and individual measurements that differed by more than 0.1 mm were measured a third time to resolve the discrepancy. the data were analysed by pearson’s correlation to find the correlation between age and dental arch dimension (figure 1) of javanese indonesian children in good nutritional status. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 95rahmawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 93–98 results the study of the correlation between age and dental arch dimension of javanese indonesian children aged 8–10 years old was done by measuring both maxillary and mandibular anterior and posterior length and width of the dental arch, and the data were analysed by pearson’s correlation. the descriptive statistics (mean, standard deviation) of anterior and posterior dental arch length and width in maxilla and mandible are shown in table 1. the mean of the maxillary anterior and posterior dental arch length increases in all of the age groups (table 1), and correlation analysis shows that there were significant weak correlations between age and both maxillary anterior dental arch length (p = 0.011; r = 0.31) and posterior dental arch length (p = 0.043; r = 0.249) (table 2). the mean of the maxillary anterior dental figure 1. dental arch dimension. anterior dental arch length (a); posterior dental arch length (b); anterior dental arch width (c); posterior dental arch width (d). table 1. mean ± sd maxillary and mandibular dental arch length and width in 8–10-year-old javanese children age (years) maxillary dental arch mandibular dental arch length (cm) width (cm) length (cm) width (cm) anterior posterior anterior posterior anterior posterior anterior posterior 8 7.17 ± 1.10 29.88 ± 1.94 33.34 ± 2.34 52.23 ± 2.08 4.03 ± 1.03 24.95 ± 2.14 27.32 ± 2.09 46.46 ± 1.96 9 7.37 ± 1.26 30.22 ± 2.27 32.74 ± 1.76 52.81 ± 2.31 4.51 ± 1.04 24.77 ± 2.75 26.28 ± 2.09 45.69 ± 1.97 10 8.08 ± 1.20 31.67 ±3.47 34.23 ± 1.65 53.40 ± 2.37 4.49 ± 1.21 25.12 ± 1.76 26.95 ± 1.75 46.03 ± 2.47 table 2. correlation between age and maxillary dental arch length maxillary anterior dental arch length maxillary posterior dental arch length age maxillary anterior dental arch length coefficient correlation 1 0.578 0.310 sig. (2-tailed) 0.000 0.011 n 66 66 66 maxillary posterior dental arch length coefficient correlation 0.578 1 0.249 sig. (2-tailed) 0.000 0.043 n 66 66 66 age coefficient correlation 0.310 0.249 1 sig. (2-tailed) 0.011 0.043 n 66 66 66 table 3. correlation between age and maxillary dental arch width maxillary anterior dental arch width maxillary posterior dental arch width age maxillary anterior dental arch width pearson correlation 1 0.369 0.187 sig. (2-tailed) 0.002 0.133 n 66 66 66 maxillary posterior dental arch width pearson correlation 0.369 1 0.211 sig. (2-tailed) 0.002 0.089 n 66 66 66 age pearson correlation 0.187 0.211 1 sig. (2-tailed) 0.133 0.089 n 66 66 66 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 96 rahmawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 93–98 arch width increased from the age of 8 years old to 10 years old, and it happened in the posterior too (table 1). there was a significant correlation between maxillary anterior and maxillary posterior dental arch width (p = 0.002; r = 0.37), but neither maxillary anterior nor maxillary posterior dental arch width had a significant correlation with age (table 3). the mean of the mandibular anterior dental arch length increases from the age of 8 to 10 years old, and this happens in the posterior from 8 to 10 years old too, but there were decreases in both mandibular anterior and posterior dental arch width. a significant correlation between mandibular anterior and posterior dental arch length (p = 0.024; r = 0.277) could be seen in table 4, and significant correlation between mandibular anterior and posterior dental arch width (p = 0.014; r = 0.301) could be seen in table 5. table 5 also showed that there was a negative value for pearson’s correlation coefficient between the mandibular anterior (r = −0.075) and posterior (r = −0.082) dental arch width and age, but p > 0.05, which indicated that the correlations were not significant. discussion the development of the dental arch is a continuous process with some changes during the mixed developmental period. the results of this study showed that there were differences in the length and width of the dental arch between children aged 8, 9, and 10 years. table 1 showed that there was an increase of anterior and posterior dental arch length, both in maxilla and mandible. these results are in accordance with bisara et al.’s study, which stated that in children aged from 3–13 years old, maxillary arch length increased significantly, and on the other hand the increase in mandibular arch length was complete by 8 years.20 table 2 showed a correlation between maxillary anterior and posterior dental arch length (p < 0.05; r = 0.578). this table also showed that there were correlations between age and both maxillary anterior and posterior dental arch length. the correlation coefficient has a positive value; which indicates a relationship between two variables in which both variables move in the same direction. when the ages increase, the maxillary dental arch length will increase too. this increase in the size of the dental arch is due to the change at this age of deciduous teeth into permanent teeth, which take a larger dental arch; this is caused by the mesiodistal size of the permanent teeth being larger than the mesiodistal size of deciduous teeth. it was supported by foster21, who states that the mesiodistal size of permanent teeth is larger than the mesiodistal size of primary teeth. the length of the maxillary anterior dental arch increases due to the eruption of permanent anterior teeth. the age of 8–10 years is the age of maxillary lateral incisor eruption, which affects the increase in the arch size of the anterior teeth. table 4. correlation between age and mandibular dental arch length mandibular anterior dental arch length mandibular posterior dental arch length age mandibular anterior dental arch length pearson correlation 1 0.277 0.170 sig. (2-tailed) 0.024 0.172 n 66 66 66 mandibular posterior dental arch length pearson correlation 0.277 1 0.030 sig. (2-tailed) 0.024 0.813 n 66 66 66 age pearson correlation 0.170 0.030 1 sig. (2-tailed) 0.072 0.813 n 66 66 66 table 5. correlation between age and mandibular dental arch width mandibular anterior dental arch width mandibular posterior dental arch width age mandibular anterior dental arch width pearson correlation 1 0.301 −0.075 sig. (2-tailed) 0.014 0.551 n 66 66 66 mandibular posterior dental arch width pearson correlation 0.301 1 −0.082 sig. (2-tailed) 0.014 0.512 n 66 66 66 age pearson correlation −0.075 −0.082 1 sig. (2-tailed) 0.551 0.512 n 66 66 66 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 97rahmawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 93–98 this is supported by previous research conducted by ogodescu et al., which states that the eruption of permanent central incisors, permanent lateral incisors, and permanent canines can cause the increase of anterior dental arch length.22 these results are in accordance with the study of thilander, which states that there were some increases in the length of the anterior and posterior mandibular dental arch and that these could be caused by the change of primary canines into permanent canines since the mesiodistal permanent canine is larger than the primary canine, and due to the eruption of incisor teeth in a proclined position.13 in children aged 8 years old, there were deciduous canine teeth that were smaller in size than permanent canine teeth. at the age of 9 years old the mandibular permanent canines have erupted and moved rapidly, so the average inter-canine distance increases at this age due to the size of the permanent teeth being larger than the primary canines.8 the mean of the mandibular anterior dental arch length increases with age between 8 and 10 years old, and it happens in the posterior from 8 to 10 years old too. a significant correlation between mandibular anterior and posterior dental arch length (p = 0.024; r = 0.277) could be seen in table 4. the maxillary anterior and posterior arch width were increased at these ages (table 1). these conditions were in accordance with the results of the study by thilander, which states that in the maxilla there was an increase of arch width recorded up to 16 years of age, especially between 5 and 10 years.13 these results are also consistent with heikinheimo et al.’s research, which stated that the maxillary canine width increased from 7 to 12 years, and the increase continued up to the age of 15.23 these might have occurred due to the size differences of deciduous and permanent canines.24 this study showed that there was a correlation between maxillary anterior and posterior dental arch width (p = 0.002; r = 0.37), but neither maxillary anterior nor maxillary posterior dental arch width had a significant correlation with age (table 3). this result is in accordance with the study of skripsa el al. which stated that there was a significant relationship between inter-canine and inter-molar width.25 the mandibular anterior and posterior arch width decreased with age between 8 and 10 years (table 1). these were in line with the result of sinclair et al.’s research, as cited by loulyi et al., that found a decrease in mandibular inter-canine width between mixed and early permanent dentitions.26 this result accords with the study of thilander, which states that the permanent first mandibular molars will drift mesially, resulting in a decrease in the depth and width of the dental arch.13 a significant correlation between mandibular anterior and posterior dental arch width (p = 0.014; r = 0.301) could be seen in table 5. this relationship was supported by the result of research from skripsa et al. that said that inter-canine and inter-molar widths exhibited a significant relationship.25 table 5 showed that the pearson’s correlation coefficient between mandibular anterior and posterior dental arch width and age had negative values. it means that there was an inverse correlation between those variables, whereby they moved in opposite directions: when the ages increase, then the mandibular dental arch width decreases. but the value of p > 0.05; this indicated that the correlations were not significant. neither mandibular anterior nor posterior dental arch width had significant correlation with age. in louly’s study, there was a nonsignificant slight increase for the maxillary inter-canine width and a decrease for the mandibular inter-canine width. these differences could be related to genetic or ethnic variations.26 based on the result of this study, it can be concluded that there was a positive correlation between age and dental arch dimension of javanese children in good nutritional status. this relation is especially in maxillary dental arch length, and it was in a weak correlation. references 1. mack kb, phillips c, jain n, koroluk ld. relationship between body mass index percentile and skeletal maturation and dental development in orthodontic patients. am j orthod dentofac orthop. 2013; 143(2): 228–34. 2. alam mk hurshee., shahid f, purmal k, ahmad b, khamis mf adhl. bolton tooth size ratio and its relation with arch widths, arch length and arch perimeter: a cone beam computed tomography (cbct) study. acta odontol scand. 2014; 72(8): 1047–53. 3. dean ja, jones je, walker vinson lqa. mcdonald and avery’s dentistry for the child and adolescent. 10th ed. st. louis: mosby elsevier; 2015. p. 349–74. 4. khan sh, hasan mn, anjum s, rafique t. is there is any relationship between malocclusion and nutritional pattern of children. updat dent coll j. 2015; 4(2): 9–13. 5. al-refeidi e, gammash m, hameed ms, sadatullah s, togoo ra. crowding , spacing and closed dentition in primary dentition and its relationship with bmi among saudi children. sch j dent sci. 2016; 3(12): 312–6. 6. lombardo l, coppola p, siciliani g. comparison of dental and alveolar arch forms between different ethnic groups. int orthod. 2015; 13(4): 462–88. 7. švalkauskienė v, šmigelskas k, šalomskienė l, andriuškevičiūtė i, šalomskienė a, vasiliauskas a, šidlauskas a. heritability estimates of dental arch parameters in lithuanian twins. stomatol balt dent maxillofac j. 2015; 17: 3–8. 8. lee kj, trang vtt, bayome m, park jh, kim y, kook ya. comparison of mandibular arch forms of korean and vietnamese patients by using facial axis points on three-dimensional models. korean j orthod. 2013; 43(6): 288–93. 9. proffit wr, fields hw, sarver dm. contemporary orthodontics. 4th ed. st louis-missouri: mosby elsevier; 2007. p. 24–94. 10. jacob t. beberapa pokok persoalan tentang hubungan antara ras dan penyakit di indonesia. berkala ilmu kedokteran. 1978; 10(2): 105–14. 11. alam mk, noor mim, tajuddin mf, basri r, purmal k, rahman sa. comparison of variation in tooth size and arch dimension in malaysian malay and malaysian chinese subject with torus palatinus. int med j. 2014; 21(2): 184–7. 12. howe rp, mcnamara ja, o’connor ka. an examination of dental crowding and its relationship to tooth size and arch dimension. am j orthod. 1983; 83(5): 363–73. 13. thilander b. dentoalveolar development in subjects with normal occlusion. a longitudinal study between the ages of 5 and 31 years. eur j orthod. 2009; 31: 109–20. 14. al-k hatib a, rajion za, masudi sm, hassan r, anderson pj, townsend gc. tooth size and dental arch dimensions: a stereophotogrammetric study in southeast asian malays. orthod craniofacial res. 2011; 14(4): 243–53. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 98 rahmawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 93–98 15. erliera, alamsyah rm, harahap nz. hubungan status gizi dengan kasus gigi berjejal pada murid smp kecamatan medan baru. dentika dent j. 2015; 18(3): 242–6. 16. ardani igaw, kannayyah d, triwardhani a. correlation of maxillary and mandibular arch form and tooth size ratio in ethnic javanese malocclusion patient. j int oral heal. 2019; 11(2): 75. 17. park sj, leesungbok r, song jw, chang sh, lee sw, ahn sj. analysis of dimensions and shapes of maxillary and mandibular dental arch in korean young adults. j adv prosthodont. 2017; 9(5): 321–7. 18. rieuwpassa ie, toppo s, haerawati sd. perbedaan ukuran dan bentuk lengkung gigi antara laki-laki dan perempuan suku bugis, makassar, dan toraja. j dentomaxillofacial sci. 2012; 11(3): 156–60. 19. kementerian kesehatan republik indonesia. keputusan menteri kesehatan republik indonesia nomor: 1995/ menk es/sk / xii/2010 tentang standar penilaian antropometri penilaian status gizi anak. jakarta: kementerian kesehatan republik indonesia; 2011. p. 5, 19–20, 37–8. 20. bishara se, jakobsen jr, treder t, nowak a. arch length changes from 6 weeks to 45 years. angle orthod. 1998; 68(1): 69–74. 21. foster td. buku ajar ortodonsi. 3rd ed. jakarta: egc; 2016. p. 48–67. 22. ogodescu ae, tudor a, szabo k, daescu c, bratu e, ogodescu a. longitudinal changes of dental arches in growing children. jurnalul pediatrului. 2011; xiv(55–56): 12–7. 23. heikinheimo k, nystrom m, heikinheimo t, pirttiniemi p, pirinen s. dental arch width, overbite, and overjet in a finnish population with normal occlusion between the ages of 7 and 32 years. eur j orthod. 2012; 34(4): 418–26. 24. salzmann ja. practice of orthodontics. philadelphia: j.b. lippincott company; 1966. p. 1074. 25. skripsa th, rizal mf, sutadi h, budiardjo sb, soetopo ms, indriati is, fauziah e, wahono na. relationship between inter-molar, intercanine, and inter-gonion widths in children aged 6-9 years. j phys conf ser. 2018; 1073(2): 022010. 26. louly f, nouer pra, janson g, pinzan a. dental arch dimensions in the mixed dentition: a study of brazilian children from 9 to 12 years of age. j appl oral sci. 2011; 19(2): 169–74. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p93–98 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p93-98 �� vol. 43. no. 1 march 2010 mengkudu (morinda citrifolia linn.) gel affect on post-extraction fibroblast acceleration christian khoswanto department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: ���t� e�tracti�n is �ne �� treat�ent �req�ently d�ne �y dentists in clinics, ��s�ital, and even �rivate �ractices. one t�ing t�at is needed t� �e ��served a�ter t�e treat�ent is t�e s�eed �� ����nd rec�very �r�cess. �engk�d� is c����nly �sed as �edicinal treat�ents, s��e �� t�e� t� �eal ����nds, ��t t�ere �ad never �een researc� �� t�e �se �� �engk�d� �r�it �n ����nd rec�very a�ter t��t� e�tracti�n. purpose: ��e ai� �� t�is st�dy ��as t� investigate t�e e��ect �� �engk�d� gel in accelerating t�e escalati�n �� �i�r��last ��st t��t� e�tracti�n �n da��ley rats. method: ��is st�dy ��as �sed ��st test �nly c�ntr�l gr��� design. ��irty �ale da��ley rats ��eig� �et��een 250–300 gra�s, 3 ��nt�s �� age are �eing �sed. ���t� e�tracti�n is �eing d�ne �n l���er le�t incis�r. ��e 30 rats are divided int� t�ree gr���s, t�ere are �engk�d� (��rinda citri��lia linn.) gel, ��vicl�ne i�dine, and c�ntr�l gr���. ��e data ��ere analyzed statistically �sing one-way anova and lsd. result: ��e res�lt �� every tested gr��� ��it� k�l��g�r��-s�irn�v test s����ed � > 0.05. e�a�inati�n s����ed t�ere ��as signi�icant di��erence in �i�r��last a���nt �et��een t�e gr��� ��it� �engk�d� gel and t��� �t�er gr���s (� < 0.05). conclusion: ��e a��licati�n �� �engk�d� gel can accelerate t�e escalati�n �� �i�r��last a�ter t�e t��t� e�tracti�n �n da��ley rats. key words: fi�r��last, �engk�d� (��rinella citric�lia linn.) gel, e�tracti�n ����nd abstrak latar belakang: ekstraksi gigi �er��akan �era��atan yang sering dilak�kan �le� d�kter gigi �aik di klinik, r��a� sakit, dan �raktek �ri�adi. sat� �al yang �erl� di�er�atikan setela� �enca��tan gigi adala� kece�atan �enye����an l�ka �ekas ca��t. �engk�d� �er��akan �a�an yang sering dig�nakan �nt�k �eng��atan, sala� sat� diantaranya adala� �nt�k �enye����an l�ka, na��n �enelitian �nt�k kese����an l�ka �asca ca��t gigi yang �engg�nakan �engk�d� �el�� �erna� dilak�kan se�el��nya. tujuan: ��j�an �enelitian ini �nt�k �engeta��i e�ek gel �engk�d� dala� �e��erce�at �eningkatan j��la� �i�r��las setela� �enca��tan gigi tik�s da��ley. metode: penelitian ini �engg�nakan rancangan �enelitian ��st test �nly c�ntr�l gr��� design. �iga ��l�� ek�r tik�s da��ley jantan, �erat 250–300 gra�, �sia 3 ��lan. gigi yang dica��t dilak�kan �ada insisi� ra�ang �a��a�. �iga ��l�� ek�r tik�s da��ley di�agi dala� tiga kel����k �enelitian, yait� gel �engk�d� (��rinda citri��lia linn.), ��vicl�ne i�dine, dan kel����k k�ntr�l. data sa��el yang dida�at dianalisa �engg�nakan one-way anova dan lsd. hasil: hasil dari tes �ada �asing-�asing gr��� dengan �engg�nakan k�l��g�r��-s�irn�v �en�nj�kkan � > 0,05. hasil �er�it�ngan �en�nj�kkan ada �er�edaan �er�akna �ada j��la� �i�r��las antara gel �engk�d� dengan 2 kel����k lain (� < 0.05). kesimpulan: a�likasi gel �engk�d� da�at �e��erce�at �eningkatan j��la� �i�r��last setela� �enca��tan gigi �ada tik�s da��ley. kata kunci: fi�r��las, gel �engk�d�, l�ka �enca��tan gigi c�rres��ndence: christian khoswanto, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya. e-mail: christiankhoswanto@hotmail.com research report �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 31-34 introduction indonesia is tropical country rich in many different kinds of plants that useful for health. one kinds of plants which is known for its many usefulness for health is mengkudu (��rinda citri��lia linn.).1,2 mengkudu fruit has complex chemical contents, some of them are: ant�raq�in�ne, xer�nine, pr��er�nine, sorbic acid, terpenoid compound, anti bacterial substance, sc���letin, and da�nacant�al. this substance acts as antimicrobial, especially bacteria, fungus, and it also has an important role in taking care of inflammation and allergy, as well as one of the important nutrition which is needed for wound recovery.3 mengkudu assists in the availability of xeronine in body. this enzyme is crucially needed in the every metabolism activity of the human body. that is why this fruit can help in strengthening body immune system and useful as adaptogen, to balance the work of cell in human body. the enzyme exists in mengkudu in the �r��er�nine compound.2 wound recovery process from tooth extracted sometimes have an experiences disturbance so that complication may happen. some researchers claim that the use of medicine post tooth extraction can reduce the possibility of complication and it’s often expected to be able to accelerate blood coagulation process, so that it will also accelerate the process of wound recovery.4 healing or recovery of damaged tissue basically is a replacement of the damaged tissue with new normal tissue. the process of tissue recovery is the first stage of dynamic processes.5 the healing process is important for normal structure maintenance, function, and life perpetuity of an individual, one of which has important role is fibroblast. fibroblast synthesize collagen, elastin, glycoaminoglycans, proteoglycans and multiadhesive glycoproteins. fibroblast are the most common cells in connective tissue and are responsible for the synthesis of extracellular component such as collagen fiber. collagens constitute the most abundant proteins found in the body. all collagens are composed of three polypeptide alpha chains coiled around each other to form the typical collagen triple helix configuration. common features include the presence of the amino acid glycine in every third position, a high proportion of proline residues. hydroxylation of proline and lysine occurs after these amino acids are incorporated into polypeptide chains, hydroxylation begins after the peptide chain has reached a certain minimum length and is still bound to ribosomes. the two enzymes involved are peptidyl proline hydroxylase and peptidyl lysine hydroxylase.4 cells with intense synthetic activity are morphologically distinc from the quiescent fibroblasts that are scattered within the matrix they have already synthesized. the quiescent fibroblast is smaller and tends to be spindle shaped, it has fewer processes, a smaller, darker, elongated nucleus.6,7 fibroblast, particularly those activated and responding to some type of stimulation, such as inflammation or mechanical forces, secrete a number of growth factor, cytokines, and inflammatory mediators. the repertoire of factors varies depending on the location and type of fibroblast but may include interleukin-1, interleukin-6, interleukin-8, tumor necrosis factor a, prostaglandin e2, platelet-derived growth factor, insulin-like growth factor, transforming growth factor b, vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and keratinocyte growth factor. in response of damage tissue, fibroblast proliferates and actively synthesizes matrix components and upon more specific observation on cellular level, the active fibroblast has an abundant and irregularly branched cytoplasm and also appears bigger and more basophilic. its nucleus is ovoid, large, and pale staining, with fine chromatin and a prominent nucleolus. the cytoplasm is rich in rough endoplasmic reticulum, and the golgi complex is well developed. fibroblast starts to appear on the wounded area three days after the laceration happens.6 wound is a damage on body tissue which is caused by several kinds of factors. wound recovery is an attempt to fix the damage. the main component in wound recovery process is fibroblast. fibroblast is the cell which responsible for collagen synthesis. fibroblast is a cell which comes from a mesenchymal tissue which is also embryonic tissue for connective tissue, bone tissue, cartilage, etc. fibroblast produce extracellular component from growing connective tissue. fibroblast exist in all fibrous connective tissue in the body and responsible to synthesize precursors from collagen, reticular and elastic fibres.8 mengkudu is commonly used as medicinal treatments, some of them to heal wounds, but there had never been research of the use of mengkudu fruit in wound recovery after tooth extraction. the purpose of this research is to know the effect of mengkudu gel in accelerating the escalation of fibroblast amount post tooth extraction on dawley rats. material and method this study is an experimental laboratory research using the post-test only control group design. thirty male dawley rats weigh between 250–300 grams, 3 months of age are being used. have well condition, food and drink water given ad libitum. this animal is used because tooth extraction on dawley rats is easier with sufficiently wide socket extraction wound for applying mengkudu gel. tooth extraction is being done on lower left incisor. the choosing of lower incisor is based on the structure and anatomical form of rat’s teeth which enable extraction to be done. the 30 rats are divided into three groups. on the first group, after the extraction is done, mengkudu gel is applied on the extraction wound. on the second group, povidone iodine is applied on the extraction wound, and on the third group, sterile aquades is applied on extraction wound. ��khoswanto: mengkudu (morinda citrifolia linn.) gel effect mengkudu fruit which is made into gel will be easier to be put into the extraction wound socket because of its solid, soft and elastic characteristics. this gel forms makes the substance durable in extraction wound socket, so that it helps the body in wound recovery process. the making of mengkudu fruit gel is uses the mixture of caeboxyl methyle celluloses (cmc) na and distillation of mengkudu fruit. cmc na is derivative of cellulose and often being uses in food industry.9 the characteristics of cmc na are for thickening, stabilizer, gel maker and in some things as emulsifiers. in hydrocolloid emulsion system it doesn’t function as emulsifiers, but more as substance which gives stabilization. this cmc na is easily soluble in hot or cold water, so it is easy to use. it is used as stabilizer because it’s easily obtainable and reasonably priced.10 after three days, all animal was decapitation and the socket post extraction preserved on the slide. buffered isotonic solution of 4% formaldehyde was used for fixatives. 70% to 100% ethanol was used to extracted the water from the fragment. the ethanol then replaced with a solvent miscible with the embedding medium. in paraffin embedding, the solvent used is xylene. once the tissue is impregnated with the solvent, it is placed in melted paraffin in the oven at 58–60° c. the heat causes the solvent to evaporate, and the space within the tissue become filled with paraffin. the tissue together within its impregnating paraffin hardens taken out of the oven. tissues embedded with plastic resin dehydrated in ethanol. the hard blocks containing the tissues are then taken to a microtome, and sliced into thin sections 1–10 µm. the sections are floated on water and transferred to glass slide to be stained. staining tissue with masson trichrome was done to make the various tissue component conspicuous. under the light microscope, tissue are examined via a light beam that is transmitted through the tissue using image magnified 400 times.4 every tested group date were analyzed by one-way anova test with 5% significant rate and continued by lsd test if there was significant difference. result the mean and standard deviation of the amount of fibroblast post extraction on dawley rats is shown in the table 1. the kolmogorof-smirnov test was carried out on the data to determine the normality of distribution. the result of every tested group showed p > 0.05, therefore all the data had a normal distribution. therefore, a one-way anova test with 5% significant rate was done and continued by lsd test if there was a significant difference. the result on the third day examined via a light beam that is transmitted through the tissue using image magnified 400 times shows that the amount of fibroblast on the group which is given mengkudu gel is much more than the control group and the povidone iodine given group. table 2 shows there is significant difference in each group treatment p < 0.05 (p = 0.001). after that, the data was continued with lsd test. table 1. mean amount of fibroblast and standard deviation in treatment group group mean sd mengkudu gel 49.80 3.19 povidone iodine 39.70 1.33 control 41.10 1.85 table 2. the anova result from fibroblast cell in treatment group variation source free level mean square f. account probability between group 2 299.433 58.331 0.001 in group 27 5.133 total 29 post-hoc test showed there is no significant difference in fibroblast amount between control group and the povidone iodine given group but the comparison between mengkudu gel given group with two other groups shows the significant difference in fibroblast growth (table 3). table 3. the result of lsd test of fibroblast amount between mengkudu gel with two other groups mengkudu povidone iodine control mengkudu povidone iodine * control * – note: * = significant – = not significant discussion after an injury to either oral mucosa or socket post extraction, blood clot is formed in the area and the inflammatory response is triggered. if the source of injury is removed, tissue repair can begin within the next few days. the epithelial cells at the periphery of the injury will lose their desmosomal intercellular junction and migrate to form a new epithelial surface layer beneath the clot. it is very important in repairing the connective tissue and must be retained in the first day of repairing because it acts as a guide to form a new surface. after the epithelial surface is repaired, the clot is broken down by enzymes because it is no longer needed. repair of the epithelium is tied to the repair taking place in the deeper connective tissue.11 fibroblast synthesize proteins, such as collagen and elastin, that form collagen, reticular and elastic fibers. they also involved in the production of growth facrtors that influence cell growth and differentiation.4 �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 31-34 the significant escalation of fibroblast amount on the use of mengkudu is caused by the existence of substance in mengkudu, one of them is anthraquinone. anthraquinone is the main component in mengkudu, the antibacterial characteristic from anthraquinone helps body to avoid infection, fever and all bacterial-related disease. anthraquinone increases the biosynthesis regulation from type i collagen, polypeptide a chains are assembled on polyribosomes bound to rough endoplasmic reticulum membranes and injected into the cisternae as procollagen molecules. collagens constitute the most abundant proteins found in the body, that plays important role in wound healing process. this study showed that fibroblast is more active, so it has an abundant and irregularly branched cytoplasm and also appears bigger and more basophilic. its nucleus become ovoid, large, and pale staining, with fine chromatin and a prominent nucleolus. it is also participate in the remodeling of connective tissue through the degradation of collagen and other matrix molecules and their replacement by newly synthesized molecules. two mechanisms for the degradation of collagen which is the secretion by cells of enzymes that sequentially degrade collagen and other matrix molecules extracellularly and the selective ingestion of collagen fibrils by fibroblast and their intracellular degradation.12 the collagen triple helix is highly resistant to proteolytic attack. the matrix metalloproteinase (mmp) family is a large family of proteolytic enzymes that includes collagenases (mmp-1, mmp-8 and mmp-13), gelatinases (mmp-2 and mmp-9), metalloelastase (mmp-12), stromelysin (mmp-7). these enzyme are capable of degrading collagen and other matrix macromolecules into small peptides extracellularly. mmps are synthesized and secreted by fibroblast, inflammatory cells and some epithelial cells. extracellular degradation often occurs in inflammatory lesions or when large amount of collagen must be degraded rapidly. the mmps are secreted as in active precursorand must be cleaved proteolytically themselves to become active. fibroblast secrete the activators and the inhibitors of mmps, which allow these cells to participate in regulating extracellular degradation. the most important mechanism for the physiologic turnover and remodeling of collagenous was intracellular degradation. this process involves recognition of the fibrils to be degraded, possibly through binding to fibroblast integrin receptors, partial digestion of the fibrils into smaller fragments, probably by gelatinase a, formation of a phagolysosome, and intracellular digestion of the collagen fragments within the acidic environment of the phagolysosome by lysosome enzymes, particularly the cathepsins.11,13 the essence of mengkudu also helps the availability of xeronine in body. this enzyme is really needed in every metabolism activity in human body, that’s why this fruit can strengthen body immunity system and also useful as adaptogen, to balance the work of cells in human body. the enzyme exist in mengkudu fruit in the form of proxeronine compound.2 sorbic acid in mengkudu fruit is the abundant source of vitamin c, sorbic acid is essentially needed by fibroblast to produce collagen.14 vitamin c has a crucial role in wound recovery process as first antioxidants defence in plasma against reactive oxygen species (ros) and free radicals, which can heavily damage cell and interfere with wound recovery process. the escalation of ros will cause damage in dna which will cause cell death.11 another contents such as terpenoids and flavanoids have been known in their role as astringent to help wound recovery process, which is actively seen in wound contraction and increasing of epithelization process.15,16 in conclusion, the application of mengkudu gel can accelerate the escalation of fibroblast amount after the tooth extraction on dawley rats. it is showed that mengkudu gel was a better modulator to recovery from damaged tissue. refferences 1. maria gw. sehat dengan mengkudu. jakarta: pt. kesaint blanc;jakarta: pt. kesaint blanc; 2000. p. 1–10. 2. mengkudu. anugrah alam sebagai obat tradisional. 2009. availableanugrah alam sebagai obat tradisional. 2009. available2009. available at: http://www.sarimengkudu.com/mengkudu anugrah alam.html. accessed january 3, 2009. 3. ruli, iwan. toga tahun 1 seri 2. surabaya: pt jawa pos printed; 2000. p. 11. 4. junqueira lj, carneiro j. basic histology. 11th ed. california: lange medical publications; 2005. p. 89–97, 113–4. 5. saptoyono b. pengaruh aplikasi lokal getah pisang pada penyembuhan luka pasca pencabutan gigi cavis cobaya. maj kedok gigi (dent. j)gigi (dent. j) 1996; 29: 17–20. 6. young b, heath jw. functional histology. 4th ed. churchill livingstone; 2005. p. 69–70. 7. sharon c a. wound healing. the journal of care management. 2005. available at: http://www.dermascience.com/clinical/pressure-ulcers. asp. accessed january 3, 2009. 8. burns er, cave md. histology and cell biology. 2nd ed. st. louis: mosby co; 2007. p. 53–5. 9. fennema or, karen m, lund db. principle of food science.principle of food science. connecticut: the avi publishing; 1996. p. 15–20.1996. p. 15–20. 10. jenral. pengaruh penambahan na-cmc (natrium carboxyl methyl cellulose) dan gula terhadap kualitas sirup nanas. malang: universitas muhammadiyah; 2004. p. 1–15. 11. bath mb, fehrenbach mj. dental embriology, histology, and anatomy. 2nd ed. st louis: elsevier saunders; 2006. p. 110–3. 12. bachraini r. pengaruh vitamin c terhadap jumlah sel fibroblas pada proses penyembuhan luka di mukosa rongga mulut. skripsi. surabaya: fkg unair; 2006. p. 10–27. 13. nanci a. oral hystology development, structure, and function. 6th ed. st louis: mosby co; 2003. p. 60–77. 14. mackay d, miller al. nutritional support for wound healing. 2003. available at: http://highwire.stanford.edu/cgi/medline/. accessed june 2, 2009. 15. scortichini m, pia rm. preliminary in vitro evaluation of the antimicrobial activity of triterpenes and terpenoids towards erwinia amylovora. j bacteriol 1991; 71: 109–12. 16. tsuchiya h, sato m, miyazaki t, fujiwara s, tanigaki s, ohyama m. comparative study on the antibacterial activity of phytochemical flavonones against methicilin-resistant staphylococcus aureus. j ethnopharmacol 1996; 50: 27–34. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base vol 49 no 3 juli-sept 2016.indd 163163 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 163–167 oral health status of elementary-school children varied according to school they attended sri widiati,1 al. supartinah santosa,2 yayi suryo prabandari,3 and johana endang prawitasari4 1department of preventive and public health dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta-indonesia 2department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta-indonesia 3department of health behavior, environment health and social medicine, faculty of medicine, universitas gadjah mada, yogyakarta-indonesia 4faculty of psychology, faculty of humaniora and social sciences, universitas kristen krida wacana, jakarta-indonesia abstract background: oral health has been promoted in elementary school. oral health status is worsening among children aged 12 in indonesia. schools are an ideal social environment where dental health promotion strategies could be implemented to improve children’s oral health and to develop lasting good oral health behavior. purpose: this study aimed to determine the association of sex, age, oral health behavior (tooth brushing practice, eating sweets and snacks, and routine dental health care visit) and family support, with oral health status among elementary school-children. method: a school-based survey was carried out in 45 public elementary schools served by15 community health centers in sleman, yogyakarta. all fifth grade students (a total of 1191 students) in the schools were recruited as study participants after informed consent being given to parents. questionnaires on health behavior and family support were administered to students, and examinations for ohis and dmf-t were conducted by trained research assistants. regression analyses (with r) were performed to identify whether sex, age, oral health behavior, family support and schools were significant determinants of oral health. result: females had higher dmf-t compared to males (1.93 vs 1.56), older children showed higher dmf-t. effects oral health behavior and family support on ohis and dmf-t were not significant after adjusting for school. school was significantly associated with ohis and dmf-t. conclusion: sex and age were determinants of dmf-t. oral health behavior and family support were not associated with ohis and dmf-t. school was a consistent predictor of ohis and dmf-t. school-based programs, especially targeted to certain schools with worse oral health, should be strengthened. keywords: oral health behavior; family support; ohis, dmf-t; elementary school-children correspondence: sri widiati, department of preventive and public health dentistry, faculty of dentistry, universitas gadjah mada. jl. denta no. 1 sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: widiati_kusnanto@yahoo.co.id introduction oral health is an essential component of general health and wellbeing. school-based oral health program was started in 1951 in indonesia. children attended health promoting schools enjoyed better oral health quality of life.1 poorer oral health status was associated with dental pain, school absenteeism, and poor school performance.2 if untreated, poor oral health may compromise the children’s quality of life.3 oral health behavior, including toothbrushing practices, habitual eating of sweets and snacks, using tooth-paste and routine dental health care visit, is known to influence oral hygiene and the incidence of dental caries among school children.4,5 according to the indonesian basic health surveys,6,7 25.9% of respondents experienced oral health problems in 2013, an increase from 23.4% in 2007. a high percentage (93.8%) of the respondents brushed their teeth everyday in 2013, an increase from 91.1% in 2007. decayed, missing and filled teeth (dmf-t) index in children 12 years of age became worse from 0.9 in 2007 to 1.4 in 2013. schools are an ideal social environment where dental health promotion strategies could be implemented to improve children’s oral health and to develop lasting good oral health behavior. healthy life style has been promoted in schools as an essential determinant of oral and dental health.8 school age children are subject to behavioral change through intervention at schools9 and improvement dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p163-167 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p163-167 164 widiati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 163–167 in family environment.10 school-based oral health preventive programs have shown significant effects on oral and dental health status after program implementation.11 poorly performing school-based dental programs are associated with worse quality of life of participating school-children.12 variations in oral and dental health outcomes among children attending different schools may possibly arise due to school related programs or other social determinants associated to the students and their families. certain social characteristics of students may lead to their attendance in particular schools. school-based oral health education in west java, indonesia, does not significantly improve selfreported oral health behavior and levels of dental caries.13 social factors are more strongly associated with dental caries, while direct association between school-based health programs and dmf-t is not found.14 the purpose of this study was to determine the association of sex, age, oral health behavior (tooth brushing practice, eating sweets and snacks, and routine dental health care visit) and family support, with oral health status among elementary school-children. material and methods a survey (cross-sectional study) was carried out among children attending elementary schools in sleman district yogyakarta. participants of this study were children, at fifth grade of public elementary schools. three schools were randomly selected from each of service areas of 15 community health centers (puskesmas), giving a total of 45 schools. all fifth grade students attending the selected schools became the study subjects after informed consent was obtained from their parents or guardians. questionnaires asking for frequency of tooth brushing, habitual eating of sweet and snacks, visits to dentists or dental nurses (oral health behavior) were completed by the students in the classrooms (oral health behavior measures). questions about family support to own a toothbrush, regular tooth-brushing, to refrain from consumption of sweet and snacks, and to regularly visits dentists or dental nurses were also being asked to the school-children (family support measures). sex and age of study participants were recorded and taken into account as potential confounding factors. anonymity of the answers to the questionnaires was assured, so the students were expected to express their real situations. examinations of oral hygiene were based on the scoring of debris and calculus (oral hygiene index–simplified or ohis) according to greene and vermillion.16 dental caries was measured as dmf-t according to methods developed by world health organization (who).17 regression analyses on ohis and dmf-t (dependent variables) of sex, age, oral health behavior, family support and school attended by the children were carried out. this study was approved by the ethical committee (internal review board) at the school of dentistry, universitas gadjah mada. data analyses used multiple regression (general linear model) available in r version 3.3.1 (open source statistical software). results there were 45 elementary schools with 1191 schoolchildren participated in this study. demographic characteristics of the study participants indicated that most children were 11 years of age (fifth grade), and the number of boys was higher than girls (table 1). the mean and standard deviation of ohis and dmf-t, both are dependent variables in this study, showed that girls had a slightly better ohis, but worse dmf-t (table 2). the proportion of caries-free children was 34.8%. the average score of oral health behavior was 41.1 standard deviation (sd) = 5 with a range of 25 to 56, and family support was 63.6 (sd = 7) with a range of 33 to 80. the average ohis in each school varied from 0.67 sekolah dasar (sd) mlesen to 2.38 (sd banyurejo), and the average of dmf-t varied from 0.609 (sd semarangan) to 3.1 (sd banyurejo). a regression analysis was conducted to identify factors (sex, age, oral health behavior and family support) associated with each of oral health indicators ohis and dmf-t (table 3). oral health behavior and family support were significantly associated with ohis, not with dmf-t. no association between oral health behavior and dmf-t was found (p = 0.083). sex was not associated with ohis, but female showed higher level of caries experience table 1. sex and age of the study participants sex frequency (%) female 572 (48%) male 619 (52%) age 10 years 146 (12%) 11 years 759 (64%) 12 years 238 (20%) 13 years 38 (3%) 14 years 10 (1%) table 2. mean and standard deviation of ohis and dmf-t oral health status mean (standard deviation) median (minimum maximum) ohis (total) female male 1.24 (0.9) 1.19 (0.9) 1.27 (0.9) 1.17 (0 – 5.67) 1 (0 – 4.67) 1 (0 – 5.67) dmf-t (total) female male 1.74 (1.9) 1.93 (2) 1.56 (1.8) 1 (0 – 13) 1 (0 – 6) 1 (0 – 13) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p163-167 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p163-167 165165widiati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 163–167 table 3. multiple regression analyses on ohis and dmft determinants ohis * (regression coefficient and p-value) dmf-t ** (regression coefficient and p-value) regression model 1 regression model 2 regression model 1 regression model 2 intercept 2.20 (0.001) 2.21 (0.001) 0.17 (0.890) 0.69 (0.546) sex -0.01 (0.838) 0.44 (0.001) 0.46 (0.001) age 0.070 (0.06) 0.071 (0.053) 0.27 (0.001) 0.27 (0.001) behavior -0.012 (0.039) -0.012 (0.038) -0.021 (0.083) -0.015 (0.175) family support -0.010 (0.016) -0.011 (0.012) 0.01 (0.247) note: *regression model 1: ohis = 2.20 0.01 sex (female = 1, male = 0) + 0.070 age – 0.012 behavior 0.01 family support regression model 2: ohis = 2.21 + 0.071 age – 0.012 behavior – 0.011 family support **regression model 1: dmft = 0.17 + 0.44 sex (female = 1, male = 0) + 0.27 age – 0.021 behavior + 0.01 family support regression model 2: 0.69 + 0.46 sex (female = 1, male = 0) + 0.27 age – 0.015 behavior table 4. determinants of ohis (oral hygiene status) including schools determinants *ohis (regression coefficient and p-value) regression model 1 regression model 2 regression model 3 intercept 2.08 (0.001) 2.21 (0.001) 2.36 (0.001) age 0.071 (0.053) 0.027 (0.438) behavior -0.012 (0.038) -0.001 (0.821) family support -0.011 (0.012) -0.004 (0.346) school** -0.86 (<0.001) -0.85 (<0.001) note: *regression model 1: ohis = 2.08 0.86 school (sd semarangan = 0) regression model 2: ohis = 2.21 + 0.071 age – 0.012 behavior – 0.011 family support regression model 3: ohis = 2.36 + 0.027 age – 0.001 behavior – 0.004 family support – 0.85 school **sd semarangan (with the lowest average dmf-t) as the reference school table 5. determinants of dmf-t (caries status) including schools determinants *dmf-t (regression coefficient and p-value) regression model 1 regression model 2 regression model 3 intercept 2.38 (0.001) 0.69 (0.546)) 0.82 (0.413) sex 0.46 (0.001) 0.46 (0.001) age 0.27 (0.001) 0.30 (0.001) behavior -0.015 (0.175) -0.007 (0.545) school** 1.40 (0.006) 1.41 (0.005) note: *regression model 1: dmf-t = 2.38 + 1.40 school regression model 2: dmf-t = 0.69 + 0.46 sex + 0.27 age – 0.015 behavior regression model 3: dmf-t = 0.82 + 0.46 sex + 0.30 age – 0.007 behavior + 1.41 school **sd semarangan (with the lowest average dmf-t) as the reference school dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p163-167 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p163-167 166 widiati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 163–167 or dmf-t. age was associated with dmf-t, and associated with ohis (p = 0.053). schools attended by the study participants were consistently associated with ohis (table 4) and dmf-t (table 5). school attended by the children was a confounding factor for the association between all other variables (demographic, oral health behavior and family support) with ohis. sex and age of the study participants remained significant determinants of dmf-t, even if the school was entered in the regression model. girls had higher caries experience than boys. higher caries prevalence was also found as the children got older. the findings in this study revealed that the average of dmf-t among the study participants (1.74) was higher than national average, especially in girls. this is consistent with the national data (riskesdas 2013) indicating that caries prevalence is higher among women than men. higher prevalence of caries in women could be due to earlier eruption of teeth in girls and frequent snacking during food preparation.18 discussion the proportion of caries free children (34.8%) in this study was similar to that of the low prevalence group of children in campinas, brazil (32.4%),19 and in vadodara, india (30.9%).20 children of the same age in italy have better caries free prevalence (64.2%),21 while the figure is worse, only 15% in qatar.22 caries experiences among children in developed countries have decreased significantly in the past few decades, due to improved oral hygiene practices, dietary habit with less sugar intake, and regular visits to dental clinics. however, caries levels increase with age, and becomes health related burdens in adults.23 in this study, significant variation of dmf-t among children attending different schools was found. children attending public schools in bharatpur, india, show higher prevalence of caries compared to children from private schools in the same city.24 all students who participated in this study attended public schools. ohis was different among children attending different schools, while no other variables were significantly associated with ohis (table 4). dmf-t was only associated with sex and age. school remained to be the significant determinant of dmf-t. place of residence25 or schools where children attended26 may account for access to dental care and risks for dental health problems. children with similar social background may attend the same school. better schools may offer better dental health programs. in this study, all schools are owned by the government, and there is not much variation in the activities of school-based programs. significant effects of schools on ohis and dmf-t could be related to average social status of the neighborhood where the schools are located.27 the lack of effectiveness of oral health behavior intervention may be responsible for the high level of ohis among children attending certain schools,28 and in the longterm, caries level of these children may increase. household support for oral health behavior of children plays significant role in caries prevention. dmf-t in children 12 years of age is predicted by caries level at the age of 6 years.29 for ohis and dmf-t, schools give contextual-level effects,30 which confounded individual-level effects of oral health behavior and family support to oral health behavior. type of school is also a significant determinant of caries experience in brazil.31 school-based intervention focusing on tooth brushing and other preventive measures, however, may not be effective in preventing caries32 except in certain controlled trials, such as dental sealant programs.33 this study also found that girls and students at older ages were at higher risk for developing caries, which might indicate dietary habits and direct biological effects as factors contributing to caries experiences. differences in caries levels are attributed to social factors affecting schoolchildren, such as household food insecurity,34 nonworking parents,35 and parental education status.36 it can be concluded that sex and age were associated with dmf-t, suggesting the importance of biological factors as proximate determinants of caries. oral health behavior was associated with oral hygiene (ohis) but not with caries status (dmf-t). family support to oral health behavior, similarly, was associated with ohis and was not associated with dmf-t. school was a consistent determinant of ohis (no other variables were associated with ohis) and dmf-t (together with age and sex). adjustment to school should be made when estimating predictors of oral health status. references 1. yusof zym, jaafar n. health promoting schools and children’s oral health quality of life. health qual life outcome 2013; 11: 25. 2. jackson sl, vann wf, kotch jb, pahel bt, lee jy. impact of poor oral health on children’s school attendance and performance. am j pub health 2011; 101(10): 1900-6. 3. jurgensen n, petersen pe. promoting oral health of children through schools–results from a who global survey 2012. comm dent health 2013; 30: 204-18. 4. cheng yc, huang hk, wu ch, chen cc, yeh ji. correlation betwee dental caries and diet, oral hygiene habits, and other indicators among elementary school students in xiulin township, hualien county, taiwan. tzu chi med j 2014; 26(4): 175-81. 5. colagoklu n, has e. the evaluation of the effects of the sociodemographic factors on oral and dental health: a study on the ages 6-12. proc soc behav sci 2015; 195: 1278-87. 6. kementrian kesehatan ri. riset kesehatan dasar 2007. jakarta: kementrian kesehatan ri; 2008 p. 130-47. 7. kementrian kesehatan ri. riset kesehatan dasar 2013. jakarta: kementrian kesehatan ri; 2013 p. 110-19. 8. kementrian kesehatan ri. pedoman usaha kegiatan gigi sekolah. jakarta: kementrian kesehatan ri; 2012 p. 1-2. 9. rosario r, araujo a, padrao p, lopes o, moreira a, abreu s, vale s, pereira b, moreira p. impact of a school-based intervention to promote food intake. pub health 2016 public health 2016; 136: 94 100. 10. de castilho arf, mialhe fl, barbosa td, puppin-rontani rm. influence of family environment on children’s oral health: a systematic review. j pediatria 2013; 89: 116-23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p163-167 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p163-167 167167widiati, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 163–167 11. antonia ag, de carvalho vianna rb, quintanilha lel. oral health conditions in children with and without school-based oral preventive program. ped dent j 2006; 16: 163-9. 12. amalia r, schaub rm, stewart re, widyanti n, groothoff jw. impact of school-based dental program performance on the oral health-related quality of life in children. j investig clin dent 2015; doi: 10.1111/jicd.12179. 13. ha r tono sw, la mbr i se , va n pa lenstei n helder ma n w h. effectiveness of elementary school-based oral health education in west java, indonesia. int dent j 2002; 52: 137–43. 14. amalia r, schaub rm, widyanti n, stewart r, groothoff jw. the role of school-based dental programme on dental caries experience in yogyakarta province, indonesia. int j paediatr dent 2012; 22(3): 203–10. 15. poutanen r, lahti s, tolvanen m, hausen h. parental influence on children’s oral health behavior. acta odontol scand 2006; 64: 288–92. 16. greene jc, vermillion jr. the simplified oral hygiene index. j am dent assoc 1964; 68: 7–13. 17. world health organization (who). oral health surveys. basic methods. 5th ed. geneva: who; 2013. p. 85. 18. martinez-mier fa, zandona af. the impact of gender on caries prevalence and risk assessment, dent clin n am 2013; 57: 301-15. 19. cypriano s, hoffmann rhs, de sousa mlr, wada rs. dental caries experience in 12-year-old schoolchildren in southeastern brazil. j appl oral sci 2008; 16(4): 286-92. 20. joshi n, sujan sg, joshi k, parekh h, dave b. prevalence, severity and related factors of dental caries in school going children of vadorada city–an epidemiological study. j int oral health 2013; 5(4): 35-9. 21. ferrazzano gf, sangiantoni g, cantile t, ingenito a. relationship between social and behavioural factors and caries experience in schoolchildren in italy. oral health prev dent 2016; 14: 55-61. 22. al-darwish m, ansari we, bener a. prevalence of dental caries among 12-14 years old children in qatar. saudi dent j 2014; 26(3): 115-25. 23. bernabe e, sheiham a. age, period and cohort trends in caries of permanent teeth in four developed countries. am j pub health 2014; 104(7): e115-21. 24. inge na, dubey hv, kaur n, gupta r. prevalence of dental caries among school children of bharatpur city, india. j int soc prev community dent 2014; 4(1): 52-5. 25. fisher-owens sa, soobader mj, gansky sa, isong ia, weintraub ja, platt lj, newacheck pw. geography matters: state-level variation in children’s oral health care access and oral health status. pub health 2016; 134: 54–63. 26. engelma n n j l, tomazon i f, machado m d, a rdengh i t m. association between dental caries and socioeconomic factors in schoolchildren – a multilevel analysis. braz dent j 2016; 27(1): 72-8. 27. tellez m, sohn w, burt ba, ismail ai. assessment of the relationship between neighborhood characteristics and dental caries severity among low-income african-americans: a multilevel approach. j pub health dent 2006; 66: 30-6. 28. widiati s. k nowledge, attitude, practice and motivation as potential determinants of oral and dental health among elementary schoolchildren in sleman, yogyakarta province. proceeding of the indonesian journal of dental research 2013; p. 156–60. 29. masood m, yusof n, hassan mia, jaafar n. assessment of dental caries predictors in 6-year old school children – results from 5-year retrospective cohort study. bmc pub health 2012; 12: 989–96. 30. ha dh, lalloo r, jamieson ln, giang l. trends in caries experience and associated contextual factors among indigenous children. j public health dent 2015; doi: 10.1111/jphd.12134. 31. paula js, ambrosano gmb, mialhe fl. the impact of social determinants on schoolchildren’s oral health in brazil. braz oral res 2015; 29(1): 1-9. 32. hilgert la, leal sc, mulder j, creugers nh, frencken je. caries preventive effect of supervised toothbrushing and sealants. j dent res 2015; 95(9): 1218–24. 33. muller-bolla m, pierre a, lupi-pequrier l, velly am. effectiveness of school-based dental sealant programs among children from lowincome backgrounds: a pragmatic randomized clinical trial with a follow-up of 3 years. community dent oral epidemiol 2016; doi: 10.1111/cdos.12241. 34. santin gc, pintarelli tb, fraiz fc, de oliveira acb, paiva sm, ferreira fm. association between untreated dental caries and household food insecurity in schoolchildren. 2016; 21: 573–84. 35. al-meedani la, al-diaigan yh. prevalence of dental caries and associated social risk factors among preschool children in riyadh, saudi arabia. pak j med sci 2016; 32: 452-6. 36. bhayade ss, mittal r, chandak s, bhondey a. assessment of social, demographic determinants and oral hygiene practices in relation to dental caries among the children attending anganwadis of hingna, ngapur. j indian soc pedod prev dent 2016; 34: 124-7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p163-167 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p163-167 201201 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 201–205 research report maxillary anterior root resorption in class ii/i malocclusion patients post fixed orthodontic treatment fransiska rima tallo, ida bagus narmada and i. g. a. wahju ardani department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: previous studies on root resorption were reviewed by panoramic radiographs. cone-beam computed tomography (cbct) showed that 41.5% of teeth experienced resorption when panoramically examinated, while 68% of teeth experienced resorption when the examination method used was cbct. root resorption occurs in the maxillary central incisor (as much as 74%) and in the maxillary lateral incisor (as much as 82%). the maxillary canines have the most resorption, followed by the lateral maxillary incisors. purpose: the aim of this study was to determine the differences of apical resorption in anterior maxillary teeth before and after orthodontic treatment in skeletal class i/ii cases of extraction. methods: samples from this study were the results of panoramic photographs of 50 patients treated by fixed orthodontic appliances at the dental and oral hospital airlangga university. these were selected according to the sample criteria. the evaluation method consists of measuring root and crown lengths with a digital application (radiant dicom viewer). subsequently, the measurements were evaluated using cbct images. results: the data were statistically analysed using normality tests with shapiro–wilk and kolmogorov–smirnov tests. based on the results of paired sample tests, it was found that every treatment group had significant differences in the average length of the crowns and roots, with a result of p=0.000 (p<0.05). conclusion: the use of cbct is considered quite effective and accurate in evaluating root resorption compared to panoramic photographs. keywords: cbct; class ii division i malocclusion; maxillary anterior; panoramic photographs; root resorption correspondence: fransiska rima tallo, department of orthodontics, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no.47, surabaya 60132, indonesia. email: rimatallo@yahoo.com introduction malocclusion is a type of connection between the maxilla and mandible that deviates from the standard form, but is accepted as a normal form. malocclusion can be caused by dentofacial balance. this dentofacial balance is not caused by one factor alone, but several factors that influence each other. these factors influence heredity, environment, growth and development, ethnicity, function and pathology.1,2 malocclusion can be treated using orthodontic appliances to obtain normal occlusion and a proportional facial profile.2 the prevalence of malocclusion in indonesia is still high (seen in around 80% of the population) and may increased dental and oral health problem3 orthodontic treatment always uses mechanical force to move teeth. the mechanical force on the tooth that will be moved orthodontically will be transmitted to the entire tooth’s supporting tissue, which starts a remodelling process to help the tooth move through the bone.4 orthodontic treatment has a positive effect but can have undesirable secondary effects. during orthodontic treatment, the application of various procedures, tools and materials can cause side effects, both local and systemic. one of the side effects is root resorption, which is clinically difficult to identify when radiographs are made, especially in cases of orthodontic treatment.5 external apical root resorption (earr) is a state of permanent loss of the apex structure of the tooth. crosssectional studies show that earr is a common iatrogenic consequence and minor problem for the average orthodontic patient, with the mean radiographic resorption being less than 2.5 mm.6,7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p201–205 mailto:rimatallo@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p201-205 202 tallo et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 201–205 one study found that the risk factors involved in root resorption were dental trauma, bone density, root morphology, age and length of treatment. most of the research on root resorption has focused more on maxillary incisors because they are thought to be more prone to root resorption than other teeth. more specifically, root resorption often occurs in maxillary incisors and other teeth with an abnormal root shape; for example, those that are pipette-like, blunt or macerated. the maxillary lateral incisors were most frequently subjected to root resorption, followed by the maxillary central incisors.4 root resorption occurs in the maxillary central incisor (up to 74%) and in the maxillary lateral incisor (as much as 82%). the maxillary canines have the most resorption, followed by the maxillary lateral incisors, which are measured using cone-beam computed tomography (cbct).8 research shows that root resorption in maxillary first molars has a mean resorption of 53.3%-63.3%. this absorption occurs because the force applied to the molars is greater than that on the premolars. in addition, the resorption in the extraction case was 3.72 times greater than that in the non-extraction case.7,8 several studies of root resorption and its relationship with orthodontic treatment have found that numerous factors influence root resorption: age, sex, nutrition, genetics, type of appliance, the amount of force used during treatment, extraction or non-extraction, length of treatment and distance of tooth movement. there is positive correlation between the strength of orthodontic style, length of treatment and increased resorption.9,10 individuals with skeletal anterior open bite have a greater risk of resorption during orthodontic treatment compared to those with other types of malocclusion. dental intrusions are four times more likely to cause earr than extrusion movements.4,11 previous studies on root resorption were reviewed by panoramic radiographs and cbct. it was found that 41.5% of teeth experienced resorption when panoramically examined, while 68% of teeth experienced resorption when the examination method used was cbct.9 an advantage of cbct is its accuracy in measuring root resorption, but its disadvantage is that radiation levels are 1.5 to 33 times higher than levels in panoramic photographs; thus, careful consideration is needed when using cbct.9 in this study, panoramic radiographs show some degree of distortion, which was caused by the lack of a three-dimensional image. cbct imaging allows the threedimensional evaluation of teeth and adjacent anatomical structures, which provides a more detailed visualisation of the tooth and surrounding structures and can diagnose earr with accuracy. based on the above, the authors were interested in researching the evaluation of maxillary anterior tooth root resorption that occurred before and after orthodontic treatment in class i/ii malocclusion by using panoramic radiographs and cbct. the aim of this study was to determine the differences of apical resorption in anterior maxillary teeth before and after orthodontic treatment in skeletal class i/ii cases of extraction and to help the operator prevent the occurrence of more severe root resorption when finding it on radiographs. materials and methods this study is an observational analytic research. the sample of this study was made up of patients who had been treated by fixed orthodontic appliances at dental and oral hospital airlangga university from 2014 to 2018. they were selected according to the sample criteria: patients needed to have class ii division 1 skeletal patterns, have had both first maxillary premolars extracted. ethical clearance was obtained from the health research ethics commission of the faculty of dental medicine, universitas airlangga (number: 614/hrecc.fodm/iv/2019). the minimum sample size needed was 35 to avoid drop out. it is necessary to over-sample, so this study also used 50 samples from the data obtained at the dental and oral hospital airlangga university. these samples qualified according to the lameshow formula.7 panoramic measurements: methodology developed by fontana et al.11 demonstrates periapical radiographic measurements of central incisors with root lengths (reference teeth) taken before treatment and after treatment. the evaluation method involves measuring the length of the roots and crowns using a digital application (radiant dicom viewer) (figure 1). the root apex, incisal edge and cemento enamel junction (cej) of each tooth were a b figure 1. a) before orthodontic treatment. b) after orthodontic treatment. an example of calculating tooth length before and after treatment using radiant dicom software. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p201–205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p201-205 203tallo et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 201–205 determined by making a point. the longitudinal axis of each tooth is projected from the tip point of the root to the edge of the incisal following the root canal. the perpendicular axis is then directed down the longitudinal axis from the mesial to the distal side of the cej. the value of the crown length is automatically calculated from the incisal edge to the cej projection and the root length from the cej projection to the apex of the root (figure 2). the difference between the two measurements shows earr.11 measurements on cbct: then, the measurements were evaluated using the cbct image. first, the cbct image of the maxillary central incisor obtained after orthodontic treatment was aligned using the cej angle. next, the amount of apical root resorption was calculated as the distance between the root apex before and after orthodontic treatment on the axis of the maxillary central incisor. the root resorption area was measured using a digital application (radiant dicom viewer) and classified as labial and palatal after identifying tooth axes (figure 3). the ratio of labial root resorption is defined as the ratio of the area of labial root resorption to the resorption area for all roots, and the ratio of palatal root resorption is defined as the ratio of the palatal area to the area of resorption for all roots. the measurements of the lengths of the crowns and roots that were obtained were then tested using ibm spss 26 for mac to determine the distribution of the data. the first data analysis that was performed focused on data normality. analysing data is needed to determine the use of the next statistical test or whether parametric or non-parametric tests should be used next. if the data is normally distributed, parametric statistics can be used.13 the normality tests used in the data analysis of this study were the kolmogorov–smirnov test and the shapiro–wilk test. these tests can be used on both research with small samples and research with large samples.8 results based on the measurements of the lengths of the crowns and roots using a digital application (radiant dicom viewer), several results were obtained. the results of paired sample tests found that almost every treatment group had significant differences in their average lengths of crowns and roots, as p=0.000 (p<0.05) (table 1). this significant difference indicates the presence of root resorption after orthodontic treatment. as shown in this diagram (figure 4), tooth 11 had an average root resorption of 0.98 mm, tooth 12 had an average of 1.17 mm, tooth 13 had an average of 0.86 mm, tooth 21 had an average of 0.93 mm, tooth 22 had an average of 1.13 mm and tooth 23 had an average of 0.87 mm. based on the diagram above, the highest root resorption was in tooth 12 and the lowest was in tooth 13. in this study, the authors included 10 participants who had completed orthodontic treatment and were then recalled figure 2. a) anatomic landmarks for measuring earr: cementoenamel junction (cej). b) a reference for measuring x-rays.12 table 1. mean and standard deviation of the calculations of crown and root lengths with panoramic measurements teeth mean ± sd delta (%) p valuebefore treatment after treatment 11 29.34±3.48 28.46±3.25 -2.93 <0.0001 12 27.44±3.05 26.27±3.11 -3.11 13 31.58±3.89 30.72±3.75 -2.83 21 29.51±3.26 28.58±3.25 -2.98 22 27.81±3.23 26.68±3.23 -3.08 23 31.76±3.84 30.89±3.76 -2.89 figure 3. measurement of the degree of absorption in cbct with the use of radiant dicom software. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p201–205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p201-205 204 tallo et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 201–205 for cbct photos. the authors compared root resorption on panoramic radiographs after treatment with cbct photographs (table 2). the results obtained indicated that seven samples showed mild resorption by panoramic (an average of 1.2 mm) and three samples showed moderate resorption (around 2.5 mm). however, using cbct revealed that eight samples showed moderate resorption and two samples showed mild resorption. this is consistent with dudic’s study in which 275 teeth were evaluated with panoramic radiographs and cbct to measure apical root resorption. discussion in general, class ii malocclusion often occurs with tooth root resorption of mild to moderate severity.14 patients with class ii malocclusion have an increased frequency of tooth root resorption. patients with class i malocclusion had tooth root resorption with an average value of 1 mm, while patients with class ii malocclusion had an average of 2 mm. maxillary central incisors had greater resorption values in class ii malocclusion patients.15 the classification of class ii malocclusion in this study was based on skeletal anteroposterior discrepancy. the antero-posterior relationship between the maxilla and mandible was evaluated through the a point, nasion, b point (anb) angle, where the anb size was significantly greater in skeletal class ii than in skeletal class i. in patients treated using bracketed maclaughlin, bennet and trevisi (mbt), root resorption was 18.26%, while patients treated using edgewise brackets had a root resorption of 14.82%. tooth root resorption in mbt patients was greater than in edgewise patients. this was due to the tooth root in mbt. torque with an increasing angle will affect the severity of tooth root resorption. the increase in torque angle and the duration of torque usage causes the apical tooth to have a hollow hyaline zone. the accumulation of this hollow hyaline zone will result in a short tooth root and will reduce the dimensional ratio between the root and crown of the tooth.6 root absorption often occurs in the apical part of softer teeth and contains less of sharpey’s fibre. another cause of this is the use of torque, as it presses the periodontal tissue at the apical part so that the tooth is more susceptible to root resorption.6 significant differences were observed between the two methods and for all levels of resorption. one hundred and forty-five teeth evaluated panoramically showed no resorption, whereas, out of those evaluated by cbct, only 80 teeth showed no resorption. ninety-two teeth showed mild apical root resorption with panoramic evaluation, and this increased to 128 teeth with cbct. only 21 teeth had moderate panoramic resorption, but this increased to 48 teeth with cbct. in addition, two teeth had severe resorption when assessed by cbct. cbct imaging allows for a three-dimensional evaluation of teeth and their adjacent anatomical structures, resulting in a detailed visualisation of the tooth as well as its neighboring structures. subsequently, the area of the tooth resorption can be detected easily. in contrast, the panoramic radiographic image is a two-dimensional radiographic image that experiences distortion. this results in difficult interpretation with minimal accuracy, which makes it less helpful in measuring root resorption. in conclusion, this study has proven the existence of root resorption after orthodontic treatment in class ii/i malocclusion cases. the highest resorption (a value of more than 1 mm) was found in tooth 12 (1.13 mm) and the lowest was found in tooth 13 (0.86 mm). the use of cbct is considered quite effective and accurate in evaluating root resorption compared to panoramic photos. references 1. fleming ps. timing orthodontic treatment: early or late? aust dent j. 2017; 62: 11–9. 2. sharaf rm, jaha hs. etiology and treatment of malocclusion: overview. int j sci eng res. 2017; 8(12): 101–14. 3. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 110. 4. mauès cpr, do nascimento rr, vilella o de v. severe root resorption resulting from orthodontic treatment: prevalence and risk factors. dental press j orthod. 2015; 20(1): 52–8. 0.87 1.13 0.930.86 1.17 0.98 1.4 1.2 1 0.8 0.6 0.4 0.2 0 mean of tooth resorption (mm) 11 12 13 21 22 23 figure 4. diagram showing the mean of crown and root length calculations with panoramic measurements. table 2. mean and standard deviation of the crown and root length calculations with cbct teeth mean ± sd 11 29.82 ± 3.21 12 26.83 ± 3.13 13 34.02 ± 3.45 21 25.99 ± 3.11 22 25.01 ± 3.07 23 29.13 ± 3.14 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p201–205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p201-205 205tallo et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 201–205 5. marinescu ir, bănică ac, mercuţ v, gheorghe ag, drăghici ec, cojocaru mo, scrieciu m, popescu sm. root resorption diagnostic: role of digital panoramic radiography. curr heal sci j. 2019; 45(2): 156–66. 6. weltman b, vig kwl, fields hw, shanker s, kaizar ee. root resor ption associated with or thodontic tooth movement: a systematic review. am j orthod dentofac orthop. 2010; 137(4): 462–76. 7. de castilhos bb, de souza cm, simas netta fontana mls, pereira fa, tanaka om, trevilatto pc. association of clinical variables and polymorphisms in rankl, rank, and opg genes with external apical root resorption. am j orthod dentofac orthop. 2019; 155(4): 529–42. 8. oktaviani ma, notobroto hb. perbandingan tingkat konsistensi normalitas distribusi metode kolmogorov-smirnov, lilliefors, shapiro-wilk, dan skewness-kurtosis. j biometrika dan kependud. 2014; 3(2): 127–35. 9. alhammadi ms, halboub e, fayed ms, labib a, el-saaidi c. global distribution of malocclusion traits: a systematic review. dental press j orthod. 2018; 23(6): e1–10. 10. gay g, ravera s, castroflorio t, garino f, rossini g, parrini s, cugliari g, deregibus a. root resorption during orthodontic treatment with invisalign®: a radiometric study. prog orthod. 2017; 18: 12. 11. fontana mlssn, de souza cmh, bernardino jf, hoette f, hoette ml, thum l, ozawa to, capelozza filho l, olandoski m, trevilatto pc. association analysis of clinical aspects and vitamin d receptor gene polymorphism with external apical root resorption in orthodontic patients. am j orthod dentofac orthop. 2012; 142(3): 339–47. 12. li y, deng s, mei l, li z, zhang x, yang c, li y. prevalence and severity of apical root resorption during orthodontic treatment with clear aligners and fixed appliances: a cone beam computed tomography study. prog orthod. 2020; 21: 1–8. 13. dos santos jb, mateo-castillo jf, nishiyama ck, esper la, de castro pinto l, pinheiro cr. external root resorption: diagnosis and treatment. clinical case report. j dent heal oral disord ther. 2018; 9(2): 160–4. 14. mclaughlin rp, bennett jc. evolution of treatment mechanics and contemporary appliance design in orthodontics: a 40-year perspective. am j orthod dentofac orthop. 2015; 147(6): 654–62. 15. roscoe mg, meira jbc, cattaneo pm. association of orthodontic force system and root resorption: a systematic review. am j orthod dentofac orthop. 2015; 147(5): 610–26. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p201–205 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p201-205 vol 49 no 3 juli-sept 2016.indd 137137 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 137–142 effect of cassave leaf flavonoid extract on tnf-α expressions in rat models suffering from periodontitis zahara meilawaty and banun kusumawardani department of biomedical science faculty of dentistry, universitas jember jember-indonesia abstract background: bacteria playing a role in periodontitis are gram-negative anaerobic bacteria that can release endotoxin or lipopolysaccharide (lps). lps acts as a stimulus to a variety of host cells that can stimulate expression of pro-inflammatory cytokines in periodontal disease, such as il-1α, il-1β, and tnf-α. increased tnf-α then can lead to periodontal tissue destruction. furthermore, cassava leaves have many health benefits due to flavonoid organic compound contained, known to possess anti-inflammatory activity are used as a medicine. purpose: this study aimed to determine the effect of cassava leaf flavonoid extract as a basic ingredient of anti-inflammatory gel on tnf-α expression in wistar rats suffering from periodontitis induced with escherichia coli (e. coli) lps. method: this study used 24 male wistar rats. those rats were divided into six groups. group 1 consisted of four rats induced with e. coli lps for 2 weeks, and then decapitation was performed on day 3. group 2 consisted of four rats induced with e. coli lps for 2 weeks, and then decapitation was conducted on day 7. group 3 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel (manihot esculenta) at a concentration of 25%, and then decapitation was performed on day 3. group 4 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel (manihot esculenta) at a concentration of 25%, and then decapitation was conducted on day 7. group 5 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel (manihot esculenta) at a concentration of 50%, and then decapitation was performed on day 3. and, group 6 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel (manihot esculenta) at a concentration of 50%, and then decapitation was conducted on day 7. the topical cassava leaf flavonoid extract gel was inserted into gingival sulcus on the first right molar of their lower jaw by using a blunted syringe needle. the gel was given two times a day for 7 days. result: the expression of tnf-α in the control group was more than that in the treatment groups given the cassava leaf flavonoid extract gel at the concentrations of 25% and 50%. the expression of tnf-α in the treatment groups given the cassava leaf flavonoid extract gel at the concentration of 50% was lower than that in the treatment groups given the cassava leaf flavonoid extract gel at the concentration of 25%. conclusion: cassava leaf flavonoid extract gel could be used as an anti-inflammatory gel characterized by a decrease in tnf-α expression in rat models suffering from periodontitis. keywords: periodontitis; flavonoids of cassava leaves; tnf-α correspondence: zahara meilawaty, department of biomedical science, faculty of dentistry, universitas jember. jl. kalimantan no. 37 jember 68121, indonesia. e-mail: zhr_mel@yahoo.com introduction periodontal disease is an oral health problem with a fairly high prevalence in all age groups in indonesia, approximately about 96.58%.1 periodontal disease is a disease attacking tissues supporting teeth in children, adults, and parents. periodontal disease sometimes can only attack gingival tissue, or attack periodontal tissue entirely, namely gingiva, periodontal ligament, cementum, and alveolar bone, called as periodontitis. periodontitis is mainly caused by plaque bacteria.2,3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 138 meilawaty and kusumawardani/dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 137–142 bacteria playing a role in periodontitis are gram-negative anaerobic bacteria, secreting a variety of products including biologically active endotoxin or lipopolysaccharide (lps).4 lps is one of factors triggering periodontal disorders. lps can stimulate biological activity causing inflammation. the inflammatory response caused by lps is the first part of the immune system against pathogens. lps acts as a stimulus to a variety of host cells which will ultimately result in expression of pro-inflammatory cytokines in periodontal disease, such as interleukin (il)-1α, il-1β, and tumor necrosis factor-α (tnf-α).5,6 in the last few years, many researchers in indonesia have developed many studies on medicinal plants that are useful as alternative drugs subtituting to chemical drugs on markets, including cassava leaves (manihot esculata). cassava leaves have many health benefits since they have a high level of vitamin c and some organic compounds, such as flavonoids, triterpenoids, tannins, and saponins. flavonoids are known to have anti-inflammatory activity. flavonoids are polyphenolic compounds that occur ubiquitously in plants having avariety of biological effects both in vitro an in vivo. they have been found to have antimicrobial, antiviral, anti-ulcerogenic, cytotoxic, antineoplastic, mutagenic, antioxidant, antihepatotoxic, antihypertensive, hypolipidemic, antiplatelet ant antiinflammatory activities. flavonoids also have biochemical effects, which inhibit a number of enzymes such as aldose reductase, xanthine oxidase, phosphodiesterase, ca+2atpase, lypoxygenase, cycloxygenase.7 flavonoids are assumed to suppress tnf-α expression released during inflammation. similarly, a research conducted by peluso shows that flavonoids can reduce tnf-α.8 some previous in vitro studies, futhermore, have shown that cassava leaf extract at concentrations of 12.5% and 25% can reduce cox-2 expression, an enzyme that plays a role during inflammation, and also can improve the viability of monocytes exposed by escherichia coli (e. coli) lps. 9 for those reasons, this research aimed to determine the effectiveness of the cassava leaf flavonoid extract as a basic ingredient of anti-inflammatory gel on tnf-α expression in wistar rats suffering from periodontitis induced with e. coli lps. materials and method all procedures in this research were approved by the ethics and advocacy commetee of faculty of dentistry, university of gajah mada (no. 00 366/ kkep/ fkg ugm/ ec/ 2015). cassava leaves were identified at the indonesian institute of sciences in plant conservation center in purwodadi. cassava leaves as much as 450 grams were washed, cut into small pieces, and dried using aerated technique for 2 days in a room at a room temperature without being exposed to direct sunlight. they were dried in an oven for 24 hours at a temperature of 400 c. after being dried in the oven, the weight of the dried cassava leaves became into 238.54 grams. the dried cassava leaves were smashed in a blender, and then sieved with a sieve of 80 maze to obtain as much as 207.25 grams of fine powder. the powder was macerated with 96% ethanol for 3 days, and stirred every 24 hours. the solution was concentrated by rotary evaporator at a temperature of 500 c and a speed of 90 rpm/ rotation in order to become crude cassava leaf extract at a concentration of 100%, as much as 20 grams. the crude extract of cassava leaves as much as 20 grams was added with 100 ml of absolute ethanol, and then was processed ultrasonically for 10 minutes. it was added with 10 ml of 5% h3po4, heated at a temperature of 800 c for 30 minutes, and then settled for 8 hours. the top layer formed was taken, and then vaccum filtration was conducted. the filtrate was extracted with 10 ml of petroleum ether (repeated 3 times). the extract result was roasted at a temperature of 600 c. to reduce the amount of ethanol, it was added with water to a volume of 5 ml. 20 ml of acetonitrile was added, and sonication process was conducted for 5 minutes. it was centrifuged at 4000 rpm for 5 minutes. the upper layer formed was taken and dried in order to obtain cassava leaf flavonoid extract. the cassava leaf flavonoid extract then was tested using liquid chromatography-tandem mass spectrometry (lc-ms/ ms) to determine the level of flavonoid. the procedure was based on a modification of two different protocols proposed by docheva et al. and muhammad et al.10,11 the extract was altered into gel at the laboratory of pharmaceutical faculty of pharmacy, universitas jember. the manufacturing process of gel base was started with carbopol developed in hot water in a mortar, and then stirred until homogeneous before added with triethanolamine (tea) in small increments until gel mass was formed. the cassava leaf flavonoid extract was mixed with propylene glycol until homogeneous. the mixtures of the extract and propylene glycol were then mixed into the gel base, and stirred until homogeneous. the distilled water remained was added to the gel in small increments until homogeneous. the procedure of making the gel was based on a modification of protocol proposed by ahmed et al.12 propylene glycol is a solvent that can dissolve a variety of materials, such as corticosteroids, phenol, sulfa drugs, barbiturates, vitamins a and d, alkaloids, and many local anesthesia.13 this study used 24 male wistar rats divided into 6 groups. group 1 consisted of four rats induced with e. coli lps for 2 weeks, and then decapitation was performed on day 3. group 2 consisted of four rats induced with e. coli lps for 2 weeks, and then decapitation was conducted on day 7. group 3 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel at a concentration of 25%, and then decapitation was performed on day 3. group 4 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel at a concentration of 25%, and then decapitation was conducted on day 7. group 5 consisted of four rats induced with e. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 139139meilawaty and kusumawardani/dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 137–142 coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel at a concentration of 50%, and then decapitation was performed on day 3. group 6 consisted of four rats induced with e. coli lps for 2 weeks, treated with the topical cassava leaf flavonoid extract gel at a concentration of 50%, and then decapitation was conducted on day 7. afterwards, in the early stage, those wistar rats were anaesthetized using ketamine (ktm 100) at a dose of 0.5 ml/ kg bm,14 injected into their quadriceps muscle/ triceps muscle of their right rear-foot. e. coli lps (sigma) at a concentration of 1 mg/ ml in pbs was injected at the gingival sulcus of their first right mandibular molar, as much as 5 ml into lingual part and 5 ml into buccal part. it was injected every three days for two weeks using a tuberculin syringe with a 30 gauge syringe to trigger periodontitis. this method was based on the modification of a method proposed by buduneli et al.15 after the rats suffering from periodontitis, their gingiva enlarged and became reddish. there were also pockets. radiographically, their alveolar bone declined (figure 1 and 2). the provision of the cassava leaf flavonoid extract gel with the concentrations of 25% and 50%9 was applied to the gingival sulcus of their first right mandibular first molar using the blunted syringe needle. the gel was applied twice a day for 7 days. this procedure was based on a procedure proposed by sato et al.16 the exceess gel on the gingival sulcus then was cleaned with a cotton pellet. decapitation was performed on those rats on days 3 and 7 after the administration of cassava leaf flavonoid extract gel. decapitation on day 3 was considered as inflammatory phase. it was then followed with proliferative phase considered as wound healing process on day 7.17 this treatment was performed in biomedical laboratory of faculty of dentistry, universitas jember. after the decapitation, their lower jaw was taken, and then fixed in a buffered formalin solution. the process of decalcification was conducted using edta for ±4 weeks. after their tissues softened, the tissues were excised. the tissues were then cleaned and washed with running water for 60 minutes. the softened and cleaned tissues were soaked in alcohol at concentrations of 70%, 80%, and 90%, as well as absolute alcohol (100%) i, absolute alcohol (100%) ii, and absolute alcohol (100%) iii to remove the water in the tissues. the tissues were successively soaked at each concentration for 60 minutes. the tissues then were soaked in solutions of xylol, xylol ii, and xylol iii respectively for 60 minutes. paraffin infiltration process was gradually carried out in an oven at a temperature of 600 c. the preparations then were put into pure paraffin i, pure paraffin ii, and pure paraffin iii respectively for 60 minutes. after that, embedding and labeling processes were performed. cutting then was conducted using a microtome. heating was performed at a temperature of 400 c to dry on a hot plate. all of these treatments were conducted in biomedical laboratory of faculty of dentistry, universityas jember. the procedure was a modification of two different procedures conducted by leitao18 and jimson.19 immunohistochemical staining was performed in accordance with a staining procedure was a modification by schiessl20 and olsen.21 deparaffinization of the tissues was carried out using xylol iii for 2 minutes, xylol ii for 2 minutes, xylol i for 2 minutes, absolute alcohol iii for 2 minutes, absolute alcohol ii for 2 minutes, absolute alcohol i for 2 minutes, 90% alcohol for 2 minutes, 80% alcohol for 2 minutes, and 70% alcohol for 2 minutes. those tissues then were washed three times with pbs each for 5 minutes. the tissues were put in a solution of 0.3% h2o2 in methanol for 20 minutes. those tissues were washed again with running water for 10 minutes, washed with distilled water for 3-5 minutes, and then washed three times with pbs, each for 5 minutes. they were incubated in antigen retrieval (citrate buffer) using a microwave for 10 minutes, and then cooled at a room temperature for 30 minutes. they were washed three times with pbs, each for 5 minutes. ultra v block was applied, and then incubation was conducted for 5 minutes at a room temperature. the tissues were washed three times with pbs, each for 5 minutes. primary antibody (polyclonal antibody tnf-α) in a ratio 1: 100 (10μ antibody : 1cc pbsa) was given, and then figure 1. a swollen and reddish rat gingiva. figure 2. a rat dental radiography picture (arrows indicating radiolucent area where alveolar bone decreased). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 140 meilawaty and kusumawardani/dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 137–142 settled for 24 hours at 40 c. they were washed again three times with pbs, each for 5 minutes. biotinylated goat anti-polyvalent (secondary antibody) was applied, and then incubation was conducted for 5 minutes at a room temperature. they were washed three times with pbs, each for 5 minutes. streptavidin peroxidase was applied, and then incubation was performed for 5 minutes at a room temperature. they were washed three times with pbs, each for 5 minutes. incubation then was carried out in dab chromogen dye (1,3-diamino benzidine), and settled for 10-20 minutes. they were washed again three times with pbs, each for 5 minutes. they then were washed with running water for 10-15 minutes. counterstain with mayer’s hematoxylin was performed for 1-5 seconds. dehydration (as opposed to deparaffinization) was carried out. mounting then was performed using canada balsam and then covered with a coverslip. tnf-α expressions on mesial gingival fibroblasts which cell cytoplasm was brown were observed. observation was performed under a microscope with a magnification of 400 times. the research data obtained were the mean number of tnf-α expressions calculated per three visual fields. the data then were tested using a normality test, shapiro-wilk test. the results of the shapiro-wilk test showed that the data had normal distribution. as a result, a parametric statistical test was performed using one way anova test to determine differences in tnf-α expression in all groups. lsd test was carried out to compare tnf-α expressions in between the treatment groups. results microscopically, the color of cells expressing tnf-α was brownish, whereas the color of cells not expressing tnf-α was purplish blue. more details can be seen in figure 3. the mean and standard deviation of tnf-α expressions in each treatment group can be seen in the following table 1. in the control group, the highest mean number of tnf-α expressions on the 7th day was 13.83, whereas in the treatment groups given the cassava leaf flavonoid extract gel at the concentration of 50% on the 7th day, the lowest mean number was 8.22. results of the one way anova test showed that there were significant differences in tnf-α expressions in between the treatment groups. discussion the results of this research showed that the mean number of tnf-α expressions in the control group was higher than that in the groups given the cassava leaf flavonoid extract gel at the concentrations of 25% and 50% as anti-inflammatory. tnf-α expressions in the control group increased on the 7th day, while tnf-α expressions figure 3. the brown cell nuclei indicated tnf-α expressions (black arrows). meanwhile, the blue cell nuclei blue did not show any tnf-α expressions (blue arrows). table 1. mean, standard deviation, and one way anova test results of tnf-α expressions in the treatment groups based on decapitation time group tnf-α expressions sig.x ± sd on day -3 control 11.33 ± 0.44 0.02 25% gefds 9.67 ± 0.28 50% gefds 8.56 ± 0.68 on day -7 control 12.23 ± 1.05 0.01 25% gefds 8.56 ± 0.41 50% gefds 8.17 ± 0.31 note: 25% gefds: cassava leaf flavonoid extract gel at a concentration of 25%; 50% gefds: cassava leaf flavonoid extract gel at a concentration of 50%; x : mean; sd: standard deviation; sig: significance of one way anova test results in the groups given the cassava leaf flavonoid extract gel at the concentrations of 25% and 50% decreased on the 7th day compared to on the 3rd day. thus, it can be said that the provision of the cassava leaf flavonoid extract gel at the concentrations of 25% and 50% could decrease tnf-α expressions. tnf-α is produced primarily by activated monocytes and macrophages. however, it can also be produced by b cells, t cells, and fibroblasts, playing a role in inflammation.21 tnf-α is also considered as a powerful immune response modulator mediating induction of adhesion molecules and other cytokines, as well as activation of neutrophils. nevertheless, excessive tnf-α can damage endothelial cells, causing vascular occlusion and improving endothelium permeability.22 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 141141meilawaty and kusumawardani/dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 137–142 cassava leaves, known as a medicinal plant for mild diseases, can be used as analgesics and anti-inflammatory. 23,24 cassava leaves contain some organic compounds, such as flavonoids, saponins, tannins, and triterpenoids. cassava leaves play a role in suppressing inflammatory process. flavonoids are potential to suppress the inflammation by blocking the cycle path of cyclooxygenase (cox) and lipoxygenase. as a result, inflammatory cells that migrate are limited, and clinical signs of inflammation are reduced. inhibition of cox and lipoxygenase pathways also directly causes inhibition of the biosynthesis of eicosanoid. 25 the results of this research, moreover, also showed that the highest number of tnf-α expressions was found in the control group (lps). lps is a potent stimulus to secrete tnf since lps can induce inflammation. lipopolysaccharide, a product of microorganisms, can involve tlr4 system that can activate nf-κb and generate genes encoding the protein components of the nonspecific immune responses, including inflammatory cytokines (tnfα, il-1 and il-12).26 in other words, the inflammatory response is triggered by an immune reaction in the cellular level, so other pro-inflammatory cytokines, such as tnf-α, can be improved.27 furthermore, the number of tnf-α expressions in the groups given the cassava leaf flavonoid extract gel at the concentrations of 25% and 50% as anti-inflammatory was less than that in the control group. this is presumably due to anti-inflammatory effects of flavonoids. the antiinflammatory effects of flavonoids may be due to their action in inhibiting the accumulation of leukocytes at inflammation sites. during inflammation, many endothelial derived mediators and complement factors may lead to adhesion of leukocytes to endothelial wall, as a result, the leukocytes become immobilized and stimulate neutrophil degranulation.28 therefore, it can be said that the provision of flavonoids can reduce both the number of immobilized leukocytes and the activation of complements resulting in lower adhesion of leukocytes to the endothelium and can decrease body inflammatory response.25 in addition, flavonoids play a role as an anti-inflammatory by blocking iκb kinase. consequently, degradation of iκb preventing activation of nf-kβ will not occur. thus, tnf-α levels will not increase. this condition may be due to quercetin contained. quercetin flavonoids then can trigger a decrease in tnf-α levels through inhibition of nuclear factor kappa b (nf-κb). nf-κb plays a role in controlling expressions of genes encoding proinflammatory cytokines and chemokines, such as tnf-α, il-1β, il-6, and other proteins.29 it can be concluded that the administration of cassava leaf flavonoid extract gel as anti-inflammatory can reduce tnf-α expressions in rat models suffering periodontitis. acknowledgement this research was funded by universitas jember dipa (daftar isian pelaksanaan anggaran) year 2015 based on a decree no. dipa-042.04.2.400073/ 2015. references 1. tampubolon ns. dampak karies gigi dan penyakit periodontal terhadap kualitas hidup. http://library.usu.ac.id. 2005. accessed december 15, 2016. 2. kurniawati a. hubungan kehamilan dan kesehatan periodontal. j biomed 2005; 2(2): 43-51. 3. fitria e. kadar il-1b dan il-8 sebagai penanda periodontitis, faktor resiko kelahiran prematur. j pdgi 2006; 56(2): 60-4. 4. djais ai. periodontitis sebagai faktor resiko jantung koroner aterosklorosis. j pdgi 2006; 56(2): 53-9. 5. ren l, leung k, darveau rp, jin l. the expression profile of lipopolysaccharide-binding protein, membrane-bound cd 14, and toll-like receptors 2 and 4 in chronic periodontitis. j periodontol 2005; 76(11): 1950-9. 6. indahyani d, santoso as, utoro t, marsetyawan hne. pengaruh induksi lipopolisakarida (lps) terhadap osteopontin tulang alveolaris tikus pada masa erupsi gigi. ind j dent 2007; 14(1): 2-7. 7. rathee p, chaudhary h, rathee s, rathee d, kumar v, khli. mechanism of action of flavonoids as anti-inflammatory agents: a review. j inflammation & allergy 2009; 8(3): 229-35. 8. peluso i, raquzzini a, serafini m. effect of flavonoids on circulating levels of tnf-α and il-6 in humans: a systematic review and metaanalysis. mol nutr food res 2013; 57(5): 784-801. 9. meilawaty z. potensi ekstrak daun singkong (manihot utilissima) dalam memodulasi cox-2 pada monosit yang dipapar lps. dental journal (majalah kedokteran gigi) 2013; 46(4): 212-7. 10. docheva m, dagnon s, statkova-abeghe s. flavonoid content and radical scavenging potential of extracts prepared from tobacco culvitars and waste. natural product research 2014; 28: 1328-34. 11. muhammad aa, pauzi nas, arulselvan p, abas f, fakurazi s. in vitro wound healing potential and identification of bioactive compounds f rom mor inga oleifera ra m. biomed resea rch international 2013, 2013: 1-10. 12. ahmed mg, choudhari r, acharya a. formulation and evaluation of in situ gel of atorvastatin for the treatment of periodontitis. rguhs j.pharm sci 2015; 5(2): 53-60. 13. rowe rc, sheskey pj, owen sc. handbook of pharmaceutical excipients. 5th edition. london: pharmaceutical; 2006. p. 111-3. 14. duarte pm, assis dr, casati mz, sallum aw, sallum ea, nociti jr, fh. alendronate may protect againts increased periodontitis-related bone loss in estrogen-deficient rats. j periodontol 2004; 75(9): 1196202. 15. buduneli e, vardar s, buduneli n, berdeli ah, turkoglu o, baskesen a, atilla g. effect of combined systemic administration of low-dose doxycycline and alendronate on endotoxin-induced periodontitis in rats. j. periodontol 2004; 75(11): 1516-23. 16. sato s, fonseca mjv, ciampo jod, jabor jr, pedrazzi v. metronidazole-containing gel for the treatment of periodontitis: an in vivo evaluation. braz oral res 2008; 22(2): 145-50. 17. velnar t, bailey t, smrkolj v. the wound healingprocess: an overviewof the cellular and molecular mechanisms. j int med res 2009; 37(5): 1528-42. 18. leitao rfc, rocha fac, chaves hv, lima v. locally applied isosorbide decreases bone resorption in experimental periodontitis in rats. j periodontol 2004; 75(9): 1227-32. 19. jimson s, balachander n, masthan kmk, elumalai r. a comparative study in bone decalcification using different decalcifying agents. int j of sci and res 2014; 3(8): 1226-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 142 meilawaty and kusumawardani/dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 137–142 20. schiessl b, mylonas i, hantschmann p, kuhn c, schulze s, kunze s, friese k, jeschke u, expression of endothelial no synthase, induchible no synthase, and estrogen receptors alpha and beta in placental tissue of normal, preeclmptic, and intrauterine growthrestricted pregnancies. j of histochemistry & cytochemistry 2005; 53(12): 1441-9. 21. olesn t, goll r, cui g, christiansen i, florholmen j, tnf-alpha gene expression in clorectal mucosa as a predictor of remission after induction therapy with infliximab in ulcerative colitis. j cytokine 2009; 46(2009): 222-7. 22. brennan fm, mcinnes ib. evidence that cytokines play a role in rheumatoid arthritis. j clin invest. 2008; 118(11): 3537-45. 23. abbas ak, lichtman ah. cytokines. in: cellular and molecular immunology. 5th ed. philadelpia: wb. saunders company; 2005. p. 243-54. 24. fasuyi ao, aletor va. protein replacement value of cassava (manihot esculenta, crantz) leaf protein concentrate (clpc) in broiler starter: effect on performance, muscle growth, haematology and serum metabolites. international journal of poultry science 2005; 4(5): 339-49. 25. garcia d, domingues mv, rodrigues e. ethnopharmacological survey among migrants living in the southeast atlantic forest of diadema, sao paulo, brazil. j ethnobiol ethnomed 2010; 6: 29. 26. nijveldt rj, van nood dec, van hoorn pg, boelens k, van norren pam, van leeuwen. flavonoids: a review of probable mechanisms of action and potential applications. am j clin nutr 2001; 74(4): 418-25. 27. sargowo d, sumarno ik, muliartha d, kamaruddin m. peran lipopolisakarida helicobacter pylori terhadap aktivitas neutrofil pada penderita infark miokard akut melalui degradasi kolagen tipe iv. j kardiol ind 2007; 28: 327-37. 28. ahmed e. immune mechanisms in atherosclerosis. dissertation. konferensrummet, centrum för molekylär medicin, karolinska sjukhuset; 2001. 29. hidayati na, listyawati s, setyawan ad. kandungan kimia dan uji antiinflamasi ekstrak etanol lantana camara l. pada tikus putih (rattus norvegicus l.) jantan. j bioteknologi 2008; 5(1): 10-7. 30. nieman dc, henson da, davis jm, angela murphy e, jenkins dp, gross sj, carmichael md, quindry jc, dumke cl, utter ac, mcanulty sr, mcanulty ls, triplett nt, mayer ep. quercetin’s influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mrna. j appl physiol 2007; 103(5): 1728-35. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p137-142 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p137-142 123 the difference of tensile bond strength between total etch and self etch dentin bonding on dentin surface adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract total etch dentin bonding agents had been used extensively in operative dentistry. these materials were used on dentin surfaces before application of the resin adhesive restorative. the purpose of this research was to prove the difference of tensile bond strength between total etch and self etch dentin bonding agent on dentin surface. the manner of preparing total etch dentin bonding agent was : bovine dentin as sample was grounded to give flat surface which was then etched with 37% phosphoric acid for 15 seconds, washed with 20 ml water and dried with blot dry technique. sample was placed in a desiccator for one hour 60% humidity covered with bonding agent and put into tensile tool plunger and stored at room temperature (± 28 °c) for 24 hours. sample was tested using autograph instrument. the manner of preparing self etch dentin bonding was equal with total etch manner but without acid etching, washing and drying. the data analyzed using one-way anova test at a = 0.05 and followed tukey hsd test. the result indicated that the tensile bond strength of total etch was higher than self etch dentin bonding at 60% humidity (p  0.05). in conclusion, the total etch dentin bonding agent with acetone solvents have a higher tensile bond strength compared with self etch dentin bonding agent also with dentin bonding in alcohol solvents. key words: total etch technique, self etch primer, tensile bond strength, dentin collagen, blot dry technique correspondence: adioro soetojo, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. in recent years primer self etch dentin bonding has been extensively used. resin has been added by acid etching, therefore in clinical application resin is really practical, because etching, washing and drying stage are not necessarily done. since washing stage is not done consequently residual material would be left on dentin surface such as : salt which is resulting from the reaction between acid and hydroxy-apatite dentin, smear layer, denatured protein/collagen and bacteria. in certain period of time, those materials would infiltrate into the pulp which can cause pulp inflammation.9-12 the other character of self etch dentin bonding is to absorb more water comparing with total etch dentin bonding. the tensile bond strength is lower than total etch dentin bonding.13 low viscosity of dentin bonding agent which has good wetting capacity would increase the energy of dentin surface.1,2 hema bonding agent (hydroxyethyl methacrylate) functions as hydrophilic moisturizer (hydrophilic humectants agent).14 the first time hema bonding agent was used as an agent increasing adhesive restoration in dentin to improve adaptation between both surfaces.15 on this study, 60% humidity was used due to the most optimal tensile bond strength of dentin bonding agent located on dentin surface.16 the aim of the study was to know the difference of tensile bond strength between total etch and self etch dentin bonding agent on dentin surfaces. introduction in recent years dentin bonding agent is extensively used in conservative dentistry, especially for class v erosive lesion restoration involving dentin tissue. dentin bonding adhesive agent on dentin part (mainly collagen fibril tissue) can be in the form of chemical or physical-mechanical binding.1,2,3 the chemical binding is due to the interaction between amino collagen and carbonyl dentin bonding which would form amide (peptide) binding. the occurrence of physical-mechanical binding is due to penetration of bonding agent into nano space interfibriler and eventually bonding resin would polymerize to form mechanical retention, in addition physical bonding occures because of vander walls tensile bond between both agents.4,5,6 to reach maximal bending between dentin bonding material and dentin collagen, it is important that collagen fibril should be in permeable condition/active.1,7 permeable collagen fibril is strongly influenced by the moist surrounding dentin surface. some studies reported that the optimal humidity that enables collagen to be permeable is moist condition not wet or dry condition. if the condition surrounding the dentin is wet, bonding resin will be difficult to penetrate into collagen tissue because it is obstructed by water molecule. if the condition is too dry collagen fibril will collapse, as a result dentin bonding cannot bond the collagen.1,8 124 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 123-126 material and methods bovine’s incisivus teeth was taken from slaughtery house of pegirian, surabaya. total etch dentin bonding agents were: voco (germany), excite (ivoclar-vivadent, schaan/leichtenstein). self etch dentin bonding agents were: xeno (dentsply de trey gmbh, germany), clearfil liner bond 2 (kuraray, japan). hard gypsum (glass tone 2000, dentsply, germany). adhesive tape (indonesia). liquid of acid etching (ivoclar-vivadent), self curing acrylic: vertex (dentimex, holland). the composition of the four dentin bonding agents is shown on table 1. table 1. dentin bonding agent composition material composition voco (total etch i) bis-gma, hema, butylated hydroxyl toluene (bht), acetone, organic acid excite (total etch ii) hema, dimethacrylate, phosphoric acid, acrylate, silicon dioxide, alcohol xeno (self etch i) tetra-methacrylate-ethylpyro-phosphate clearfil liner bond 2 (self etch ii) h2o, phenyl-p, 5-nmsa, etoh/hema the support tools: diamond disk, diamond drill, grease paper no. 400 and no 100 (fuji star, japan), desiccator with vacuum valve (china), hygrometer (haar, synth. hygro, germany) compressor/air suction (schuco, usa), spuit injection, autograph ag-10 te (shimadzu, japan), plunger made of metal. the treated tooth was carefully and accurately washed using brush and sharp scalpel for soft an hard tissue. during the washing process, the tooth was in wet condition. further, the tooth was cut using diamond disk and planted in dental stone. the dentin part was placed upward and prepared using fissure form diamond drill. dentin surface was fined using silicon grease paper no. 400 and continued no. 1000. then, dentin was covered by adhesive tape with 3 mm holes and attached in the middle of dentin surface. dentin was polished once using cotton pellets for 15 minutes, next, it was washed using 20 cc aquades from injection syringe and dried by cotton pellets. this drying procedure is called blot dry technique. the next step, the tooth was put into desiccator for one hour with 60% humidity by filling 150 cc water into desiccator and put into hygrometer which has been calibrated. in this condition hygrometer showed the humidity was between 94–95%. the air was pumped out using air suction from the valve on desiccator and the humidity reached 60%. to save the time, active silica gel was filled into desiccator and the samples were ready to be treated. the procedure was done for total etch dentin bonding sample was to remove sample from desiccator, polished by primer solvent and bonding mixture. dentin surface was polished by disposable brush and left it dry for 30 seconds and lighting was done using light curing unit for 20 seconds (according to manufacture reference). every control sample was put into cylinder and placed into the plunger. the opposite plunger was filled with self cured acrylic as restoration on dentin bonding with plastic filling instrument acrylic mixtured was put into plunger hole connected with opposite plunger and fixed by lock peg. then, sample was kept at room temperature (± 28 °c) for 24 hours. (according to manufacture reference). the procedure of self etch dentin bonding sample was almost the same with total etch dentin bonding without etching, washing or drying after the sample was removed from desiccator, dentin surface was polished with dentin bonding, lighting with light curing for 20 seconds (according to manufacture reference). the next step was: to perform tensile bond strength test using autograph with cross head speed =10 mm/minute, range = 5, load cell capacity = 5 kn/500 kgf. surface width of control dentin = 7,1 mm2. the collected data was analyzed using one-way anova test. result the mean and standard deviation of tensile bond strength between total etch and self etch could be shown on table 2. table 2. tensile bond strength of total etch and self etch bonding agent on dentin surface (mpa) material n x and sd total etch i 8 16.67 ± 1.99 total etch ii 8 13.10 ± 2.05 self etch i 8 10.97 ± 1.67 self etch ii 8 10.77 ± 2.12 note: x = mean of tensile bond strength, sd = standard deviation, n = number of samples on table 2 data analysis was done by kolmogorovsmirnov test to achieve data normality. the result was: p = 0.580 (p > 0.05) for total etch i, p = 0.677 (p > 0.05), total etch ii p = 0.795 (p > 0.05) self etch i and p = 0.756 (p > 0.05) self etch ii. the table shows that all control groups have normal distribution. meanwhile, levene test was done to prove that the data was homogenous. the result showed that all control groups have p = 0.905 (p > 0.05). it is means that four bonding agents of control groups are homogeneous. statistical analysis using anova test was performed to know whether there is difference of tensile bond strength among the four control groups. the result showed significant value of the four boding agents was 0.001 (p < 0.05) and it showed that significant difference was 125soetojo: the difference of tensile bond strength found among the four groups. test tukey-hsd was done to know the difference in every group and the result was shown on table 3. table 3. the result of lsd test on total etch and self etch dentin bonding material significant total etch i total etch ii self etch i self etch ii 0.006* 0.001* 0.001* total etch ii total etch i self etch i self etch ii 0.006* 0.160 0.109 total etch i total etch i self etch ii self etch ii 0.000* 0.160 0.997 total etch ii total etch i self etch ii self etch i 0.000* 0.109 0.997 note: there is significant difference a = 0.05 table 3 shows significant difference of tensile bond strength comparing total etch i with total etch ii. self etch ii (p < 0.05). there is no significant difference (p > 0.05) of tensile bond strength comparing total etch ii with self etch i and self etch ii, even though tensile bond strength of total etch ii is higher than self etch i and self etch ii (table 2). discussion maximal adherence of dentin bonding agent in collagen occurs because collagen fibril is in permeable condition/ active has been proved.1,17 permeable condition is possible if the environment surrounding dentin is moist but not dry or wet. if the environment is dry, collagen would collapse as a result amino collagen group is covered by the remain of fibril and further result carbonyl group in dentin bonding cannot chemically bind to amino collagen. physically, nano space inter fibriler disappears because every collagen fibril would closely contact each other, so, dentin bonding is unable to enter into nano space forming mechanical retention. if the environment surrounding dentin is wet dentin bonding is not capable to bind either chemically or mechanically collagen fibril due to the excessive water molecule surrounding dentin.7,10 the result of this study shows tensile bond strength of total etch i has the highest significant value (16.67 mpa) comparing to total etch ii, self etch i and self etch ii because total etch i is dentin bonding with acetone solvent. acetone is volatile agent and capable of depleting therefore the viscosity will decrease.18 when total etch i is polished on dentin surface, it will penetrate into inter fibrile nano space, then, chase the water molecule and finally it will evaporate. in this way it will let resin bonding bind fibril collagen. the capability of acetone to chase water molecule is called water chasing effect. chemically, acetone will not chase but bind water molecule then acetone as well as water will simultaneously evaporate. total etch ii dentin bonding with alcohol solvents which does not evaporate as fast as acetone. therefore during the evaporation the remain of water surrounding collagen will obstruct dentin bonding to interact with collagen. acetone concentration will influence the thickness of resin bonding layer and tensile bond strength. resin thickness does not correlate with tensile bond strength. the occurrence of resin crack is due to bad resin bonding polymerization and low resin strain (it is caused by excessive acetone amount). the other character of acetone is capable to increase vapour pressure of water especially water surrounding collagen. the ideal acetone concentration is 37% which can produce 30.2 mm thickness of resin bonding layer. the comparison between total etch and self etch resin bonding shows that tensile bond strength of total etch i dentin bonding is significantly higher than self etch i and self etch ii (p < 0.05). the result of this study is similar to the result of previous study reported that the remain of self etch dentin bounding procedure were not done and the remain would disturb adhesion mechanism of resin and dentin.13 significant difference of total etch ii tensile bond strength (p > 0.05) was not found comparing with self etch i and self etch ii resin bonding, because total etch ii with alcohol solvent which has slow vaporizing capacity, therefore, penetration into collagen fibril was also slow, so, it made adhesive strength low. in the process of dentin bonding adhesion in collagen, the competition of water molecule and resin bonding agent with collagen fibril occurred, therefore, water concentration surrounding dentin would determine the tensile strength. mechanical bonding is a process of strong adhesion of one substance and the other which it could be reached through mechanical bounding or refention.1 in general, the adhesion is better than tensile bond strength among molecules. for example: in dentistry: penetration of adhesive resin agent into macroscopic irregularities of a surface (porous, micro space, crack). low concentration or semiviscus of adhesive resin is good material for this procedure because of great penetration capability. cementation of gold crown, inlay, onlay, post endodontic and metal core also is mechanical bonding. to achieve adhesion, both of interfaces should have tensile bond activity.1,2 it is also reported that this condition could occur without paying attention to the phase of the substance, whether it is solid, liquid or gas agent, with exception that adhesion between two kinds of gas is difficult to occur because lack of characters between the interfaces. the energy on the outer surface of solid material generally is bigger than the energy of the inner part due to molecular geometric lattice pattern. inner molecular lattice of the entire atom has equal tensile bond and equal atom’s distance therefore, the energy is minimal. on the surface of molecular lattice, the energy increases because the most distant atom has no equal tensile bond. the increasing 126 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 123-126 energy of every unit area on the surface correlates with surface energy or surface tensile. adhering or attaching surface of two solid materials is very difficult1,4,5 even though without microscopic sight the surface is smooth, however, it is very rough in microscopically level. therefore, if the two solid materials are adhered the adhesive contact would only occur on the rough part of the surface. as a whole, surface contact area is only small part, the adhesive strength is low. the tensile bond between two molecules would occur if the distance of both molecules is less than 0.7 nm consequently if the distance is higher than 0.7 nm tensile bond would be difficult to occur. one of the methods to manage the problem is by adding liquid material to the surface to achieve good adhesion in condition that the liquid is able perfectly to flow and to wet the surface. in operative dentistry, wetting capability of adhesive material on the surface would be deter mined by the cleanness of material surface, thin oxidation layer on metal surface could obstruct adhesive process including organic liquid. acid etching on dentin surface could increase wetting and surface roughness resulting the opening of dentin tubules.19 the ability of adhesive surface material could be considered by the contact angle between adhesive liquid on the solid surface in interface area.1,2 if the adhesive molecule could perfectly bind the molecule of material which would be adhered consequently adhesive liquid would wet the whole surface, therefore wetting contact angle is 0°, but if the contact angle is big, meaning that the wetting capability of adhesive material is bad. the conclusion of this study is the tensile bond strength of total etch dentin bonding is higher compared with self etch dentin bonding. references 1. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia: wb saunders co; 2003. p. 21–395. 2. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 3. noort rv. introduction to dental materials. 2nd ed. edinburgh, london, new york, oxford: cv mosby co; 2002. p. 11–78 4. brackett mg, brackett ww, haish ld. microleakage of class v resin composites placed using self-etching resins. quintess int 2006; 37:109–13. 5. jacques p, hebling j. effect of dentin conditioners on the micro tensile bond strength of a conventional and a self-etching primer adhesive systems. dent mat 2005; 21:103–9. 6. carrilho mr, tay fr., pashley dh. mechanical stability of resindentin bonding components. dent mat 2005; 232–41. 7. nakabayashi np. pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 8. chiba y, miyasaki m, rikuta a, moore bk. influence of environmental conditions on dentin bond strength of one application adhesive systems. oper dent 2004; 29:554–9. 9. accorinte mlr, loquercio ad, reis a, muench a. adverse effect of human pulps after direct pulp-capping with the different components from a total etch, three step adhesive system. dent mat 2005; 21:599–607. 10. frankenberger r, tay fr. self etch vs etch-and-rinse adhesive: effect of thermo-mechanical fatigue loading on marginal quality of bonded resin composite restorations. dent mat 2005; 21:397–412. 11. zohairy aa, de gee aj, mohsen m. effect of conditioning time of self-etching primers on dentin bond strength of three adhesive resin cements. dent mat 2005; 21:83–93. 12. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32:19–26. 13. moll k, park hj, haller b. bond strength of adhesive/composite combinations to dentin involving total and etch adhesive. j adhesive dent 2002; 3:171–80. 14. leal jir, osorio r, terriza jah. dentin wetting by four adhesive system. dent mat 2001; 17:526–32. 15. xu j, stangel i, butler is, gilson dfr. an ft raman spectroscopy investigation of dentin and collagen surface modified by hema. j dent res 1997; 76:596–601. 16. soetojo a. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada bebagai kelembaban. dissertation. surabaya: airlangga university; 2006. p. 66–9. 17. summitt jb, robbins jw, hilton tj, schwartz r. fundamentals of operative dentistry. 3rd ed. chicago: quintess publ co, inc; 2006. p. 183–242. 18. cho bh, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the microtensile bond strength. dent mat 2004; 20:107–15. 19. rosales ji, marshall gw, watanabe lg. acid etching and hydration influence on dentin roughness and wettability. j dent res 1999; 78:1554–9. vol 51 no 1 jan-mrt 2018.indd 42 dental journal (majalah kedokteran gigi) 2018 march; 51(1): 42–46 building team agreement on large population surveys through inter-rater reliability among oral health survey examiners sri susilawati,1 grace monica,2 r. putri n. fadilah,3 taufan bramantoro,4 darmawan setijanto,4 gilang rasuna sadho,4 and retno palupi4 1 department of dental public health, universitas padjadjaran, bandung – indonesia 2 department of dental public health, universitas maranatha christian, bandung – indonesia 3 department of dental public health, universitas jenderal achmad yani, bandung – indonesia 4 department of dental public health, universitas airlangga, surabaya indonesia abstract background: oral health surveys conducted on a very large population involve many examiners who must be consistent in scoring different levels of an oral disease. prior to the oral health survey implementation, a measurement of inter-rater reliability (irr) is needed to know the level of agreement among examiners or raters. purpose: this study aimed to assess the irr using consensus and consistency estimates in large population oral health surveys. methods: a total of 58 dentists participated as raters. the benchmarker showed the clinical sample for dental caries and community periodontal index (cpi) score, with the raters being trained to carry out a calibration exercise in dental phantom. the consensus estimate was measured by means of a percent agreement and cohen’s kappa statistic. the consistency estimate of irr was measured by cronbach’s alpha coefficient and intraclass correlation. results: the percent agreement is 65.50% for photographic slides of dental caries, 73.13% for photographic slides of cpi and 78.78% for calibration of dental caries using phantom. there were statistically significant differences between dental caries calibration using photographic slides and phantom (p<0.000), while the consistency of irr between multiple raters is strong (cronbrach’s alpha: >0.9). conclusion: a percent agreement across multiple raters is acceptable for the diagnosis of dental caries. consistency between multiple raters is reliable when diagnosing dental caries and cpi. keywords: inter-rater reliability; calibration; training; oral health survey correspondence: taufan bramantoro, department of dental public health, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: taufan-b@fkg.unair.ac.id research report introduction when an oral health survey is conducted on a large population, it might involve many team members as examiners. at times, these individuals score different levels of oral disease inconsistently. the question of consistency or agreement between examiners will arise due to variations in the diagnosis of oral disease between two or more such individuals or for the same examiner on more than one occasion. the other factor influencing consistency is the variability between examiners due to factors such as fatigue or differences in visual acuity and tactile sensation. in order to diagnose oral disease consistently in oral health surveys, all examiners must have been trained in standardization and calibration. it is important to train examiners who will be involved in oral health surveys, especially for epidemiological studies based on world health organization (who) basic oral health survey methods (2013).1 oral health surveys are needed to plan and evaluate oral health programs and services, with control of the methodological biases in such surveys being exercised. according to who methodology, prior training and calibration of examiners represents the essential initial steps of oral health surveys. the calibration allows standardized interpretation of diagnostic criteria between examiners or raters. the general percentage agreement (gpa) and kappa statistics have been proposed for this task.2 the extent of agreement between examiners or raters is referred to as “inter-rater reliability (irr)”. irr is, to a greater or lesser degree, a concern in most large-scale studies due to the fact that multiple individuals collecting dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p42–46 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p42-46 43susilawati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 42–46 data may experience and interpret the phenomena of interest differently.3,4 irr refers to the level of agreement between a specific set of judges on a particular instrument at a particular time.5,6 calibration is needed to ensure that all raters examine to the same standard. it is recommended that the training and calibration processes adhere to the methods propounded by the who basic oral health survey. the purpose of the training and calibration process is to minimize variation between examiners, to synchronize interpretation and to understand and apply the criteria for oral conditions such as dental caries that will be observed and recorded.1 the training stages consist of theoretical discussions, calibration exercises on dental phantom models and practical activities involving patient simulation. a benchmarker examiner or gold standard conducted the training with theoretical and practical activities. in theoretical activities, a benchmarker examiner explains who basic oral health survey methods (2013), principles, code and criteria of dental caries and periodontal examination, the data collection procedure and data management. the study objective is to assess the irr using consensus and consistency estimates in a large population oral health survey. materials and methods a total of 58 dentists from faculties of dental medicine throughout indonesia participated in the training and calibration of an oral health survey. the training was held at the faculty of dental medicine, universitas airlangga, in may 2017. a benchmarked examiner (gold standard) conducted the training program consisting of theoretical and practical activities. the examiner (gold standard) should meet the following requirements: he/she has followed who guideline-based training drawing on the oral health survey and passed with a kappa score of at least 0.8, holds a calibration and simulation trainer qualification, has calibration training instructor experience, and has participated in research on the who oral health survey. the training and calibration procedure were delivered at the faculty of dental medicine, universitas airlangga, in conjunction with the dental public health association meeting. the six trainers as benchmarker examiners were drawn from the faculties of dentistry of universitas padjadjaran, universitas jenderal achmad yani, universitas kristen maranatha and universitas indonesia. the kappa scores among the benchmaker examiners from all the faculties varied between 0.6 and 0.7. first, after the theoretical session, the benchmarker presented the clinical sample of 25 photographic slides for each criterion of healthy and decaying teeth. the benchmarker also displayed 13 photographic slides showing the periodontal condition for each community periodontal index (cpi) score using cpi-modified scoring. the second stage in the training consisted of a calibration exercise on a dental phantom head. a total of 36 healthy and decaying teeth were mounted in 36 plaster blocks for examination with a ball-ended probe in accordance with who criteria. all raters examined the clinical diagnosis of both healthy and decaying teeth and the assessment criteria. the purpose of the first and seconds steps was to determine the inter-rater reliability based on the percent agreement across multiple raters, cronbach’s alpha and the intraclass correlation. to obtain the measure of percent agreement, a matrix in which the columns represented the different raters and the rows represented variables for which the raters had collected data was created. the cells in the matrix contained the rater scores entered for each variable. this technique allowed the researcher to identify variables that may be problematic.3,7 percent agreement is useful but, because it does not account for chance agreement, it should not be relied upon as the only measure of inter-rater consensus. in this study, intraclass correlation, as one of the most popular and consistent inter-rater reliability methods for numerous raters has been adopted. the last step of the training, after parents/teachers had signed an informed consent form, was calibration by examining school children subjects with healthy and decayed teeth, which was granted ethical clearance by the faculty of dental medicine, universitas airlangga. the simulation of examination activity involved six school children as standard patients. the participants examined the condition of dental caries. otherwise, the cpi score examined through slides simulation. before the raters examined the school children, a gold standard examination of the children’s dental condition based on who basic oral health survey methods was conducted. each rater was helped during the study by a recorder, but did not discuss their findings with the gold standard. the examination was carried out indoors, the school children lying on a chair or table with the examiner seated at their heads and the recorder sitting in front of the chair. the examination of dental caries was conducted using a dental mirror and a 0.5 mm diameter ball-ended probe. table 1. calculation of the kappa score for dental caries examination examiner 1 examiner 2 total decayhealthy a + ccahealthy b + ddbdecay a + b + c + dc + da + btotal a = proportion of teeth both examiners consider to be healthy, b = proportion of teeth examiner 1 considers to be decayed and examiner 2 consider to be healthy, c = proportion of teeth examiner 1 considers to be healthy and examiner 2 considers to be decayed, d = proportion of teeth both examiners consider to be decayed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p42–46 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p42-46 44 susilawati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 42–46 the results of such examinations carried out by raters were compared with those conducted by the gold standard. a more reliable means of assessing the overall agreement between examiners is the kappa statistic which relates the actual measure of agreement with the degree of agreement which would have occurred by chance.3,8,9 the kappa score in examining for dental caries can be calculated using a 2 x 2 table3,8 as seen in table 1. kappa formula: k = pa–pc 1–pc pa = the percentage of assessments that are consistent across raters, pc = the percentage of assessments that vary between raters. this figure can be calculated using the following formula: pa = (a+d) (a+b+c+d) pc = (a+c)×(a+b)+(b+d)×(c+d) (a+b+c+d)2 the kappa score is interpreted as follows: <0.20 poor agreement, 0.21-0.40 fair agreement, 0.41-0.60 moderate agreement, 0.61-0.80 substantial agreement, 0.81-1.00 almost perfect agreement. results the irr, based on a calculation of the percent agreement for each rater regarding the code of dental caries, cpi use of photographic slides and phantom, can be seen in table 2 which shows the percent agreement between multiple raters. table 2 exhibits a percent agreemeent of 65.50% for photographic slide of dental caries, 73.13% for photographic slides of cpi and 78.78% for calibration of dental caries using a phantom head. table 3 shows the variables of photographic slides and phantom heads. rater agreement for each variable can be seen which shows that the raters achieved 65.79% agreement for all variables of photographic slide of dental caries, 73.76% for cpi and 79.27% for dental caries variables using phantom heads. according to the contents of table 3, the raters achieved between 25% and 98.21 % agreement for photographic slide of dental caries, between 10.71% and 100.00% in cpi and between 20.70% and 98.30% for dental caries using phantom heads. table 4 shows that statistically significant differences existed between dental caries calibration using photographic slides and phantom head (p<0.000). with reference to the kolmogorov-smirnov test, neither percent agreement was normally distributed, so that a differences test of non parametric tests was employed. based on the results of a wilcoxon test, the difference between the mean of percent agreement between photographic slides and phantom heads is very significant (p=0.000). the method of calculating the percent agreement does not account for chance agreement. in this study, the interrater reliability was based on cronbach’s alpha and an intraclass correlation method to analyze the consistency and agreement among raters based on the contents of table 5, the cronbrach’s alpha score for all calibration methods was >0.9, indicating that the consistency of irr between multiple rater was strong. this means that all raters were reliable in diagnosing dental caries using photographic slides and phantom heads. all raters were also reliable in determining the cpi code using photographic slides. table 2. the percent agreement between multiple raters ncalibration method mean (%) sd min (%) max (%) dental caries (slide) cpi (slide) dental caries (phantom) 56 56 58 65.50 73.13 78.78 73.29 10.16 12.61 12.00 46.00 16.67 84.00 92.00 94.44 table 3. the percent agreement among multiple raters for each variables item total variables mean (%) sd min (%) max (%) dental caries (slide) cpi (slide) dental caries (phantom) 25 13 36 65.79 73.76 79.27 22.25 26.09 19.21 25.00 10.71 20.70 98.21 100.00 98.30 table 4. differences in percent agreement between photographic slide and phantom mean (%) pzsd photographic slide phantom n=53 65.81 80.66 12.37 8.5 0.000*-5.635 * significant table 5. inter-rater reliability based on cronbach’s alpha cronbrach alphancalibration method dental caries (slide) cpi (slide) dental caries (phantom) 56 56 58 0.942* 0.973* 0.946* *) p=0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p42–46 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p42-46 45susilawati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 42–46 table 6 shows the average score of the 56 raters using photographic slides to diagnose dental caries to be reliable (an interval of 0.903 to 0.937 with 95% confidence). the average score of the 56 raters using photographic slides to determine the cpi code was reliable (an interval of 0.946 to 0.9990 with 95% confidence). the average score of the 56 raters using phantom heads to diagnose dental caries was also reliable (an interval of 0.917 to 0.968 with 95% confidence). this suggests that, despite their apparent differences in diagnosis dental caries and determining cpi using various methods, the process was successful in training the examiner to determine the code of dental caries and cpi based on who oral health survey methods. in the final calibration session, the simulation of dental caries examination was carried out on the students. table 7 shows the results of the examination simulation performed by the examiners on students. the kappa score is 0.23 which, being below 0.4, represents fair agreement. discussion the limitation of information about the irr among examiners in training and the calibration of oral health surveys based on who methods in indonesia underlies this study. the data of irr in this study was collected by several methods using the percent agreement, cronbach’s alpha, consistency using intraclass correlation and kappa statistics.10,11 irr constitutes the degree of agreement between raters. if raters agree, irr is 1 (100%), whereas if they disagree, the irr is 0 (0%). this produces a score of how much homogeneity or consensus exists between the ratings awarded by raters. based on the percent agreement, the irr in this study fell within the range of 60-90%. in general, above 75 % is considered acceptable for diagnosing dental caries using photographic slides and phantom heads and determining cpi using photographic slides. there are some factors potentially influencing the low percent agreement between rater/examiners in this study. first, some of the examiners might not yet have been familiar with the code and criteria of dental caries and cpi based on who basic oral health survey methods (2013). second, the quality of photographic slides or phantom heads remains in question due to their unclear appearance. the low percent agreement in diagnosing dental caries is found in photographic slides, but increases when the raters follow the calibration using phantom heads. based on a wilcoxon test, the difference in the mean of the percent agreement between photographic slides and phantom heads is very significant (p=0.000). it means that the perception and understanding of all raters about the code of dental caries based on who methods increased after they had followed the second training stage. the most popular method for computing a consensus estimate of inter-rater reliability is through the use of the percent agreement between multiple raters. percent agreement is easy to calculate and explain.12 the calculation of percent agreement does not take chance agreement into account. that is one of the disadvantages of the percent agreement method. in this study, the kappa statistic was used as the other method of irr to determine the consensus or agreement between two raters. cohen’s kappa was designed to estimate the degree of consensus between two raters after correcting the percent agreement figure for the amount of agreement that could be expected due to chance alone based upon the values of the marginal distributions.13 kappa statistics are used for the assessment of agreement between two or more raters when the measurement scale is categorical. kappa agreement is simply an adjusted form of percent agreement that takes chance agreement into account. a kappa score is usually expressed as a proportion, rather than a percentage, and is not multiplied by 100 as with percent agreement. in this study, the kappa score for one sample falls within the fair agreement category.4,5 the factors that affect the fair agreement category in this sample of the study might be that the raters lack familiarity with the dental caries code based on the who method. the other factors might be related to the condition of mixed dentition which is confusing for raters to determine the coding for deciduous or primary teeth. in this study, the consistency of raters is assessed by cronbach’s alpha and intraclass correlation method. cronbach’s alpha coefficient is a measure of internal consistency reliability and is useful for understanding the extent to which the ratings from a group of raters can be taken together to measure a common dimension.13,14 the consistency between raters using various calibration methods in this study is rigorous. table 6. consistency among raters based on intraclass correlation 95% ci ncalibration method average measures lower bound upper bound dental caries (slide) cpi (slide) dental caries (phantom) 56 56 58 0.942* 0.973* 0.946* 0.903 0.946 0.917 0.937 0.990 0.968 *) p = 0.000 table 7. calculation of the kappa score for dental caries examination examiner 1 examiner 2 total decayhealthy 241311healthy 1192decay 13 22 35total using the kappa formula, a pc score of 0.23 (fair agreement) is obtained dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p42–46 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p42-46 46 susilawati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 42–46 intraclass correlation is one of the most popular irr methods to measure two or more raters.14,15 the results of an intraclass correlation method of both dental caries and cpi are reliable, as can be seen from the average score of raters for all the calibration methods. the consistency of multiple raters using various calibration methods in this study was strong, when computed by both cronbach’s alpha and intraclass correlation. in conclusion, the percent agreement across multiple raters in this study are considered acceptable for diagnosing dental caries, but the agreement based on kappa statistics must increase if raters, particularly those with low kappa scores, follow the same training. the consistency between multiple raters using cronbach’s alpha and intraclass correlation in this study was fair and reliable in diagnosing dental caries and cpi score in large population oral health surveys based on who oral health survey methods. references 1. world health organization. oral health surveys : basic methods. 5th ed. france: world health organization; 2013. p. 25-7. 2. tonello as, silva rp da, assaf av, ambrosano gmb, peres sh de cs, pereira ac, meneghim m de c. interexaminer agreement dental caries epidemiological surveys: the importance of disease prevalence in the sample. rev bras epidemiol. 2016; 19(2): 272–9. 3. mchugh ml. interrater reliability: the kappa statistic. biochem medica. 2012; 22(3): 276–82. 4. mandrekar jn. measures of interrater agreement. j thorac oncol. 2011; 6: 6–7. 5. stem ler se. a compa r ison of consensus, consistency, a nd measurement approaches to estimating interrater reliability. pract assess res eval. 2004; 9(4): 1–11. 6. gisev n, bell js, chen tf. interrater agreement and interrater reliability: key concepts, approaches, and applications. res soc adm pharm. 2013; 9(3): 330–8. 7. lebreton jm, burgess jrd, kaiser rb, atchley ek, james lr. the restriction of variance hypothesis and interrater reliability and agreement: are ratings from multiple sources really dissimilar? organ res methods. 2003; 6: 80–2. 8. sim j, wright cc. the kappa statistic in reliability studies: use, interpretation, and sample size requirements. phys ther. 2005; 85(3): 257–68. 9. watson pf, petrie a. method agreement analysis: a review of correct methodology. theriogenology. 2010; 73(9): 1167–79. 10. ma r uster i m, baca rea v. compa r ing groups for statistica l differences: how to choose the right statistical test? biochem medica. 2010; 20: 15–32. 11. pieper d, jacobs a, weikert b, fishta a, wegewitz u. inter-rater reliability of amstar is dependent on the pair of reviewers. bmc med res methodol. 2017; 17: 98. 12. stolarova m, wolf c, rinker t, brielmann a. how to assess and compare inter-rater reliability, agreement and correlation of ratings: an exemplary analysis of mother-father and parent-teacher expressive vocabulary rating pairs. front psychol. 2014; 5: 509. 13. mccrae rr, kurtz je, yamagata s, terracciano a. internal consistency, retest reliability, and their implications for personality scale validity. personal soc psychol rev. 2011; 15: 28–50. 14. vilella kd, assunção lr da s, junkes mc, de menezes jvnb, fraiz fc, ferreira f de m. training and calibration of interviewers for oral health literacy using the breald-30 in epidemiological studies. braz oral res. 2016; 30: e90. 15. hallgren ka. computing inter-rater reliability for observational data: an overview and tutorial. tutor quant methods psychol. 2012; 8: 23–34. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p42–46 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p42-46 vol 44 no 3 sept 2011.indd 164 vol. 44. no. 3 september 2011 odontoblast layer structure alteration as a response to carious lesions tetiana haniastuti department of oral biology faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: dental caries is a bacterial disease affecting the hard tissue of the teeth as well as the pulp. the human dental pulp consists of odontoblast which are organized as a densely packed cell layer. odontoblasts is located at the periphery of the pulp; therefore, they are the first cells encountered by cariogenic bacteria and their products that are represented in the carious lesion. purpose: this study aimed to elucidate the effect of cariogenic bacteria to odontoblasts of human teeth. methods: five intact third molars and 15 third molars with occlusal caries at various stages of decay were extracted because of orthodontic or therapeutic reasons. the tooth specimens were fixed, decalcified with 10% edta solution (ph 7.4), and embedded in paraffin. serial sections of 5 μm thickness were cut and stained with haematoxylin eosin and gram’s, in addition to nestin immunohistochemistry. the specimens were then examined under light microscopy. results: in normal teeth, odontoblast layer were aligned along the pulp chamber showing normal morphology of the cells. slight disorganization of odontoblast layer was seen in the cases of carious lesions confined to enamel. in the cases of carious lesions confined to dentin, odontoblast layer was not observed in the areas subjacent to the lesions, only single cells showing flattened cell morphology were found. odontoblasts beneath the lesion suffered severe damage and diminished nestin immunoreaction were observed in all cases of carious lesions with pulp exposure. conclusion: cariogenic bacteria invasion may damage the odontoblasts by affecting the morphology and vitality of the cells. the severity of the damage of the odontoblasts may increase as the bacterial invasion progresses toward the pulp. key words: dental pulp, odontoblast, carious lesion abstrak latar belakang: karies merupakan penyakit yang disebabkan oleh bakteri, yang dapat memengaruhi jaringan keras gigi maupun pulpa. pada pulpa gigi manusia terdapat sel odontoblas yang tersusun atas lapisan sel. odontoblas terletak pada tepi kamar pulpa, sehingga sel ini merupakan sel yang pertama kali bertemu dengan bakteri kariogenik dan produk-produknya yang terdapat dalam lesi karies. tujuan: penelitian ini bertujuan untuk mengetahui pengaruh invasi bakteri kariogenik terhadap sel odontoblas gigi manusia. metode: lima buah gigi molar ketiga utuh dan 15 gigi molar ketiga yang mengalami karies pada permukaan oklusal dengan berbagai tingkat keparahan diekstraksi karena akan menjalani perawatan ortodontik atau perawatan lainnya. gigi-geligi tersebut kemudian difiksasi, didekalsifikasi dengan larutan edta 10% (ph 7,4), dan ditanam dalam parafin. spesimen gigi tersebut kemudian dipotong dengan ketebalan 5 μm dan diwarnai dengan hematoksilin eosin dan gram, serta immunohistokimia dengan nestin. spesimen kemudian diamati di bawah mikroskop cahaya. hasil: pada gigi normal, lapisan odontoblas terdapat di sepanjang tepi kamar pulpa dengan morfologi sel normal. disorganisasi ringan pada lapisan odontoblas tampak pada kasus-kasus karies dengan kedalaman enamel. pada kasus-kasus lesi karies dengan kedalaman dentin, lapisan odontoblas tidak tampak pada daerah di bawah lesi, hanya ditemukan sel odontoblas tunggal dengan dengan morfologi sel yang pipih. odontoblas di bawah lesi mengalami kerusakan yang parah dan tidak menunjukkan nestin immunopositif merupakan gambaran dari kasus-kasus karies dengan pulpa terbuka. kesimpulan: invasi bakteri kariogenik dapat menyebabkan kerusakan sel odontoblas dengan menyebabkan perubahan morfologi dan memengaruhi vitalitas selnya. kerusakan sel akan semakin parah dengan semakin dalam invasi bakteri ke arah pulpa. kata kunci: pulpa gigi, odontoblas, lesi karies correspondence: tetiana haniastuti, c/o: departemen biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: haniastuti@yahoo.com research report 165haniastuti: odontoblast layer structure alteration introduction dental caries, the most common chronic infection in humans, is a classic biofilm disease that develops when changes in the oral environment enhance the growth ofcariogenic bacteria. microbiological assessment of caries-active sites and studies with experimental animals implicated that carious lesions may be caused by a range of bacteria, but the principal among the cariogenic flora are gram-positive bacteria such as streptococcus, lactobacillus, and actinomyces spp.1,2 previous study showed that gram-positive bacteria are frequently found in dentinal tubules of teeth with carious lesions and in teeth with irreversible pulpitis.3 the microflora changes its composition as the carious infection progresses to the pulp-dentin interface. it characterized by a decrease of the proportion of gram-positive aerobic bacteria and an increase of gram-negative anaerobic ones mainly fusobacterium, prevotella and tannerella spp.2 cariogenic bacteria are highly efficient at converting carbohydrates to the organic acids that able to demineralize tooth enamel.4 this process may result in access for bacteria to the pulp tissue through the dentinal tubules and subsequently induce pulp inflammation.5,6 odontoblasts are cells which is located at the periphery of the pulp chamber and organized as a densely packed cell layer. due to their peripheral situation, odontoblasts are the first cells encountered by cariogenic bacteria that are represented in the carious dentin. odontoblasts become exposed to the bacteria as the bacteria and their products progressively demineralize enamel and dentin and enter the disrupted tissues to gain access to the pulp.7 odontoblasts are responsible for both development and reparative formation of dentin. odontoblasts localized beneath the damaged region can up-regulate their dentin secretory activity during mild tissue injury. injury of greater intensity causes localized odontoblasts necrosis which are subsequently replaced by an odontoblast-like cell population.8 whilst the bacterial aetiology of caries is well established, there is limited understanding of the dynamic nature of the tissue changes within the dentin-pulp complex in response to lesions varying in their rate of progression. the purpose of the present study was to elucidate the effect of cariogenic bacteria to odontoblasts of human teeth. materials and methods the protocol for this study was reviewed and approved by the ethical committee of medical faculty of universitas gadjah mada. twenty volunteers ranging in age from 20 to 40 years who had been scheduled to undergo extraction for various therapeutic reasons, were enrolled in the study. informed consent was obtained from all subjects after the proposed study was fully explained. lesion depth assessments were performed clinically using explorer and confirmed using micro-computed tomography. five intact third molars and 15 third molars with carious lesion involving enamel (n = 5), dentin (n = 5), and pulp (n = 5) were extracted. the teeth were fixed in 10% neutral buffered formalin solution, and demineralized using 10% ethylene diaminetetraacetic acid disodium salt (edta) solution (ph 7.4). the specimens were then embedded in paraffin andserially sectioned at 5 μm thickness. all sections coming through the cavity floor or pulp exposure site were stained with hematoxylin-eosin and gram’s for identification of the bacteria, in addition to nestin immunohistochemistry, a specific marker for the odontoblast.9 nestin immunohistochemistry procedure were done by processed the sections with avidin-biotin-peroxidase complex (abc) method, using polyclonal antibody to nestin (chemikon international, temecula, usa). inhibition of endogenous peroxidase was done by treated the sections with 0.3% h2o2 in absolute methanol for 30 minutes. any non-specific immunoreaction was inhibited by preincubation in 2.5% normal goat serum (vector laboratories inc, ca, usa). after incubation with the primary antibodies, the sections were reacted consecutively with biotinylated anti-rabbit igg and abc (vector laboratories inc, ca, usa). the sites of antigen-antibody reactions were visualized using 3-3’diaminobenzidine tetrachloride in tris buffer and 0.002% h2o2 and counterstained with 0.05% methylene blue. immunohistochemical controls were performed by omitting the primary antibody, the biotinylated anti-rabbit igg, or the abc complex. these immunostained sections showed no specific immunoreaction. sections of all 15 teeth were examined under light microscopy and evaluated for the quality of the odontoblast layer below the lesion. results in normal teeth, no bacteria was observed in all specimens. odontoblast layer were seen aligned along the pulp chamber, showing normal cell morphology. their cells appeared palisade tall columnar cells and a nucleus located in a basal position, adjacent to the predentin. cell bodies and processes of the odontoblasts revealed an intense nestin positive-immunoreaction indicating that odontoblasts were vital. cell-free zone located immediately subjacent to the odontoblast layer was observed (figure 1). in the cases of carious lesions confined to enamel, based on the clinical asessment and micro-computed tomography observation, the lesion already affected the enamel; however, no bacteria was stained in all samples. although slightly disorganized, odontoblast layer was maintained its continuity and observed aligned along the pulp chamber in all specimens (figure 2a). some vacuolizations were observed in the odontoblastic layer subjacent to the lesion. the odontoblasts showed an intense 166 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 20111: 164–168 nestin-positive immunoreaction indicating that odontoblasts were vital (figure 2b). cell-free zone could not be identified in the area localized to the affected dentinal tubules in all specimens. a slight increased number of cells in the location of cellfree zone was observed. these cells had a morphology corresponding to that of fibroblasts and undifferentiated cells in the rest of the pulpal tissue. a few inflammatory cells were also found in this area. in the cases of carious lesions confined to dentin, stained bacteria along the dentinal tubules was observed in all specimens. all specimens showed that odontoblast layer lost their continuity and could not be identified in the areas subjacent to the lesions, only some individual odontoblast cells were sparsely observed (figure 3a). the cells had flattened cell morphology and showing nestinpositive immunoreaction. however, odontoblast layer was recognized in other areas of the roof of the pulp chamber showing an intense nestin-positive immunoreaction (figure 3b). the odontoblast layer was aligned along approximately 30% (3 cases) and 50% (2 cases) of the roof of the pulp chamber. no predentin was observed in the area corresponding to the lesions. inflammatory cells infiltration was found in the area adjacent to the lesions. polymorphonuclear (pmn) cells were infiltrated the odontoblast layer, occasionally they were observed close to the predentin. nestin-positive filamentous structures were also shown surrounding the inflammatory cells infiltration. in the cases of carious lesions with pulp exposure, all samples showed stained bacteria penetrated the pulp chamber. odontoblasts beneath the lesion suffered severe damage. odontoblast layer could not be identified in their pseudostratified appearance at roof of the pulp chamber in all samples. some individual nestin-immunopositive odontoblast cells showing flattened cells morphology were sparsely observed. nestin-immunopositive odontoblast layer was found at the area at a distance from the lesions sites. figure 1. specimen of a normal tooth. a) odontoblast layer is aligned along the pulp chamber. the tall columnar cells appear palisade with nucleus located in a basal position adjacent to the predentin. cell-free zone is located immediately subjacent to the odontoblast layer. b) cell bodies and processes of the odontoblasts are showing an intense nestin positive-immunoreaction (red arrows). a b figure 2. specimen of the tooth with carious lesion confined to enamel. a) slight disorganization of odontoblast layer. no cell-free zone can be identified in the the subodontoblastic region. b) the odontoblasts are showing an intense nestin-positive immunoreaction (red arrows). a b 167haniastuti: odontoblast layer structure alteration severe inflammation characterized by intense infiltrations of pmn and mononuclear cells was observed in the pulp chamber particularly in the area corresponding to the lessions. nestin expression was found up-regulated in the dental pulp cells surrounding the inflammatory cells infiltration. discussion dental caries is an infectious and transmittable disease which is caused by bacteria.1 this study showed that invasion of cariogenic bacteria in dentinal tubules may damage the odontoblasts by affecting the morphology and vitality of the cells. odontoblasts, the most highly specialized post-mitotic cells of the pulp, is located at the periphery of the pulp. due to their peripheral situation, they are the first cells encountered by the cariogenic bacteria. odontoblasts become exposed to cariogenic oral bacteria as the bacteria progressively demineralize enamel and dentin and enter the disrupted tissues to gain access to the pulp.10 in the cases of carious lesions confined to enamel, no bacteria was observed in all specimens. enamel was dissolved due to the decalcification process. therefore, it was proven that the bacteria had not invade the dentinal tubule yet. all specimens showed odontoblast layer aligned along the pulp showing nestin-positive immunoreaction, indicating that odontoblast cells were vital thus, they were capable of elaborating reactionary dentin. however, there were some extracellular vacuolization in the odontoblast layer subjacent to the lesion indicating early damage of the odontoblast cells due to the penetration of soluble substance of bacterial origin to the dentinal tubule. during bacterial multiplication, various product such as bacterial enzymes, metabolic products, and other extracelluar substances are released. components of the bacterial cell structure may also be liberated after lysis and disintegration of the bacterial cells. previous study has been demonstrated that a bacterial endotoxin was able to diffuse through dentinal tubules to the pulp chamber.11 in the cases of carious lesions confined to dentin, all specimens showed that bacteria already penetrate the dentinal tubules. all samples showed disorganization of the odontoblast layer at the roof of the pulp chamber.the odontoblast layer lost its continuity particularly at the area subjacent to the lesions, however, some individual odontoblast cells were identified showing nestin-positive immunoreaction. nestin was expressed in the processes of odontoblasts surrounding the carious lesion suggesting a role for nestin in the elaboration of the reactionary dentin.12 cariogenic bacteria and their products have ability to demineralize enamel and dentin, and penetrate the dentinal tubule. as bacteria invade enamel and enter the dentin, changes commence in the pulp. carious lesion provide constraints on the free diffusion of ions and small molecules, therefore, the rate of ingress of microbial metabolic products might bear a linear relationship to disease progression. the diffusion kinetics of both these microbial products and the degradation products arising from their action will influence disease progression.11 no odontoblast layer was observed in the cases of carious lesion with pulp exposure, only few single cells were observed revealed nestin positive-immunoreaction. previous studies13,14showed that nestin could be used for monitoring the degeneration and regeneration processes of damaged odontoblasts under pathological conditions in animal experimental models using mice and rats. nestin contributes to the signaling cascade, resulting in odontoblast-like cell differentiation. continuous bacterial invasion of dentinal tubules overcomes the pulp-dentin complex resulting in the infection of the pulp and may cause the death of odontoblasts. in addition, nestin expression was found up-regulated in the dental pulp cells surrounding the inflammatory cells infiltration in this case. expression of figure 3. specimen of the tooth with carious lesion confined to dentin. a) odontoblast layer lost their continuity and can not be identified in the areas subjacent to the lesion (black arrows). no predentin in the area corresponding to the lesions. b) odontoblast layer is recognized in other areas showing nestin-positive immunoreaction (red arrows). a b 168 dent. j. (maj. ked. gigi), vol. 44. no. 3 september 20111: 164–168 this molecule at the injured pulp may help to coordinate cell fate decisions as well as proliferative, migratory, and differentiation activities.15 various degree of inflammation were observed in all caries cases. it seemed that the severity of the inflammation was advanced as the caries progressed toward the pulp. previous study showed that odontoblasts exposed to bacteria and their by-products expressed interleukin 8 mrna and protein. interleukin 8 is a potent chemotactic factor for neutrophil, which is the predominant inflammatory effector cell observed in those cases. neutrophilic degranulation liberates lysosomal enzymes that digest host as well as bacterial cells.16 no predentin was observed beneath the lesions in the cases of carious lesion confined to the dentin and with pulp exposure. odontoblasts are responsible for both the developmental and reparative formation of dentin. they produce the components of the organic matrix of predentin and dentin, including proteoglycans, collagens, and noncollagenous proteins as well.17 therefore, disorganization or death of the odontoblasts may affect their function in producing predentin. in conclusion, cariogenic bacteria invasion may damage the odontoblasts by affecting the morphology and vitality of the cells. the severity of the damage of the odontoblasts may increase as the bacterial invasion progresses toward the pulp. references 1. garcía-godoy f, hicks j. maintaining the integrity of the enamel surface: the role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization. j am dent assoc 2008; 139: 25s–34s. 2. marsh pd, nyvad b. the oral microflora and biofilms on teeth.in fejerskov o, edwina k, editors. dental caries the disease and its clinical management. 2nd ed. oxford: blackwell publishing ltd; 2008. p. 163–85. 3. love rm, jenkinson hf. invasion of dentinal tubules by oral bacteria. crit rev oral biol med 2002; 13: 171–83. 4. shen s, samaranayake lp, yip h. in vitro growth, acidogenicity and cariogenicity of predominant human root caries flora. j dent 2004; 32: 667–78. 5. raslan n, wetzel we. exposed human pulp caused by trauma and/or caries in primary dentition: a histological evaluation. dent traumatol 2006; 22: 145–53. 6. bjørndal l. the caries process and its effect on the pulp: the science is changing and so is our understanding. j endod 2008; 34: s2–5. 7. horst ov, horst ja, samudrala r, dale ba. caries induced cytokine network in the odontoblast layer of human teeth. bmc immunol 2011; 12: 1–13. 8. murray pe, hafez aa, smith aj, windsor lj, fox, cf. histomorphometric analysis of odontoblast-like cell numbers and dentin bridge secretory activity following pulp exposure. int endod j 2003; 36: 106–16. 9. struys t, krage t, vandenabeele f, raab whm, lambrichts i. immunohistochemical evidence for proteolipid protein and nestin expression in the late bell stage of developing rodent teeth. arch oral biol 2005; 50: 171–4. 10. arana-chavez ve, massa lf. odontoblasts: the cells forming and maintaining dentine. int j biochem cell bio 2004; 36: 1367-73. 11. love rm. invasion of dentinal tubules by root canal bacteria. endod topics 2004; 9: 52–65. 12. about i, laurent-maquin d, lendahl u, mitsiadis ta. nestin expression in embryonic and adult human teeth under normal and pathological conditions. am j pathol 2000; 157: 287–95. 13. hasegawa t, suzuki h, yoshie h, ohshima h. influence of extended operation time and occlusal force on determination of pulpal healing pattern in replanted mouse molars. cell tissue res 2007; 329: 259–72. 14. kuratate m, yoshiba k, shigetani y, yoshiba n, ohshima h, okiji t. immunohistochemical analysis of nestin, osteopontin, and proliferating cells in the reparative process of exposed dental pulp capped with mineral trioxide aggregate. j endod 2008; 34: 970–4. 15. mitsiadis ta, rahiotis c. parallels between tooth development and repair: conserved molecular mechanisms following carious and dental injury. j dent res 2004; 83: 896–902. 16. okiji t. pulp as a connective tissue. in: hargreaves km, goodis he, editors. seltzer and bender’s dental pulp. chicago: quintessence publishing co; 2002. p. 105–6. 17. levin lg. pulpal irritants. endod topics 2003; 5: 2–11. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 9999 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 99–103 antibacterial ability of arabica (coffea arabica) and robusta (coffea canephora) coffee extract on lactobacillus acidophilus willy wijaya, rini devijanti ridwan, and hendrik setia budi department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: dental caries is the most commonly dental health problem found in indonesia. lactobacillus acidophilus (l. acidophilus) is bacteria playing a role in the development and continuation of caries. some researches in dentistry faculty show that many plants are efficacious for oral health. one of them is coffee bean. coffee bean containing caffeine, phenolic, trigonelline, and chlorogenic acid is reported to have antimicrobial activity. purpose: this research aimed to determine the differences in the inhibition of arabica and robusta coffee extract to l. acidophilus. method: this research was an laboratory experimental research. the method used was well diffusion method using seven samples for each treatment group. bhi-a and inoculated l.acidophilus bacteria was poured into each petri dish, and then 8 pitted holes were made with a diameter of 5mm and a depth of 3mm using a ring. next, arabica or robusta coffee extracts at a concentration of 100%, 75%, 50%, 12.5%, 6.25%, and 3.125% were put into each of the pitted hole until it was full, and a negative control was also prepared. they then were put in an incubator at a temperature of 37 °c for 24 hours. afterwards, measurements and observations were conducted on inhibition zone area. result: robusta coffee extract at the concentrations of 100% and 75% had greater inhibitory than arabica coffee extract (p<0.05). meanwhile, arabica and robusta coffee extracts at the concentrations of 50% and 25% had no significant inhibitory difference (p>0.05). conclusion: in conclusion, robusta and arabica coffee extracts have inhibitory effects on l.acidophilus. robusta coffee bean extract, nevertheless, has better inhibitory effects than arabica coffee bean extract. keywords: arabica extract coffee; robusta extract coffee; lactobacillus acidophilus; antibacterial correspondence: rini devijanti ridwan, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: devi.rini@yahoo.co.id introduction the teeth are part of masticatory apparatus of the digestive system in the human body. a dental health problem mostly found in indonesia is dental caries. based on the results of basic health research in 2013 conducted by the agency for health research and development, the national prevalence of oral health problems was amounted to 25.9%, with the highest proportion of 30.5% in the productive age of 35-44 years and 31.9 % in the age of 44-45 years, thus indicating an increase in the national prevalence of oral health problems compared to in 2007, about 23.5%.1 caries is a demineralization process of dental hard tissue due to metabolic activity of bacteria. this involve of vulnerable hosts, bacteria causing caries, and substrate for bacteria. the bacteria causing caries include streptococci, lactobacilli, and actinomycetes.2 streptococcus mutants play a role in the initiation of dental caries, while lactobacillus play a role in the development and continuation process of caries. the most dominant lactobacillus species causing dental caries are lactobacillus acidophilus (l. acidophilus).3 the amount of lactobacillus in dental plaque ranges 104-105 cells/ mg in patients with active caries. the amount of l. acidophilus identified in the saliva of the subjects exposed to caries is as dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p99-103 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p99-103 100 wijaya, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 99–103 much as 3-24%. l. acidophilus can ferment carbohydrates and produce acid so that ph of plaque will decrease. decrease in ph repeated within the specified time results in demineralization of vulnerable tooth surface and even caries process is started.4 many researches on plants are useful as an effective herbal treatment with minimal side effects have been conducted. coffee plant is an export commodity that has relatively high economic value in the world market, in addition to the one commodity developed in indonesia. coffee is favored because it has a special taste and aroma. 5 coffee beans naturally contain various types of volatile compounds, such as aldehydes, furfural, ketones, alcohols, esters, formic acid, and acetate acid. 6 in addition to volatile compounds responsible for the aroma of coffee, the coffee also contains caffeine, phenolic compounds, trigonelline, and chlorogenic acid which reportedly have antimicrobial activity.7 chemical composition of the beans may vary depending on types of coffee and geographic conditions in which the coffee is planted.8 coffee, moreover, is a beverage that has been consumed since the days of our ancestors, and now coffee is one of the world’s favorite beverage consumption level of 6.7 million tons per year.9 according to statistics from the international coffee organization in 2000-2010, world coffee consumption continues to rise by 3-4% annually. generally, there are two types of coffee most often consumed namely arabica and robusta coffee. arabica coffee contains caffeine from 0.4 to 2.4% of the total dry weight, while robusta coffee contains 1-2% of caffeine and 10.4% of organic acid.8 furthermore, a research conducted by daglia et al.,10 shows that coffee can help to prevent caries. other researches also show that coffee made from roasted coffee beans has antibacterial ability against certain microorganisms, both gram positive and gram negative bacteria, including s. mutans as the main cause of dental caries. a research conducted by aroma states that the smallest concentration of robusta coffee bean extract even still has inhibitory effects on s. mutans growth of 12.5%. thus, the researchers want to conduct this research on the effects of the antibacterial ability of robusta and arabica coffee extracts on l. acidophilus bacteria with the objective of verifying and comparing inhibition of arabica and robusta coffee extract to l. acidophilus. materials and method this research was a laboratory experimental research using post-test only control group design. materials used were arabica and robusta coffee beans, l. acidophilus bacterial culture, sterile distilled water, 96% ethanol, bhi-b media, bhi-a media, standard comparator of caffeine, trigonelline, caffeic acid, polyphenols, chlorogenic acid, diterpene ester, caffeic acid, and coffee oil compounds. manufacture of arabica and robusta coffee extract was performed in upt materia medica, malang. this research was conducted in the laboratory of microbiology, faculty of dental medicine, universitas airlangga, surabaya. in addition, arabica and robusta coffee extracts were made in several stages. arabica and robusta coffee powder that had been weighed was macerated with 1000 ml of 96% ethanol solution, and then shaken with digital shaker at a speed of 50 rpm for 24 hours. the coffee extract was filtered and accommodated in erlemeyer. the liquid extract obtained by the evaporation process was carried out using a rotary evaporator for 1 hour. the results obtained were re-evaporated above water bath for 2 hours to obtain concentrated extracts of coffee arabica or robusta with 100% concentration. moreover, to make the extracts with a concentration of 75%, 0.75 ml of 100% extracts was mixed with 0.25 ml of sterile distilled water. to make the extracts with a concentration of 50%, 1 ml of 100% extract was mixed with 1 ml of sterile distilled water. to make the extracts with a concentration of 25%, 1 ml of 50% extract was mixed with 1 ml of sterile distilled water. to make the extracts with a concentration of 12.5%, 1 ml of 25% extract was mixed with 1 ml of sterile distilled water. to make the extracts with a concentration of 6.25%, 1 ml of 12.5% extract was mixed with 1 ml of sterile distilled water. and, to make the extracts with a concentration of 3.125%, 1 ml of 6.25% extract was mixed with 1 ml of sterile distilled water. after arabica and robusta coffee extracts with various concentrations were completely made, the extracts then were added with sodium carboxyl to change the extracts into the form of a gel. sodium carboxyl was put little by little as 1-3 grams to achieve the desired consistency. furthermore, antibacterial inhibition test was performed using two methods, namely dilution tube method and well diffusion method. dilution tube method was used to determine the minimum inhibitory concentration and the minimum bactericidal concentration (mic and mbc) required by arabica and robusta coffee extracts in inhibiting the growth of l. acidophilus. meanwhile, well diffusion method was used to determine the inhibitory power generated large extracts of arabica and robusta coffee to l. acidophilus. furthermore, dilution tube method was conducted by serial dilution. arabica or robusta coffee extract with the concentration of 100% on the first tube as much as 5ml was poured into the second tube containing 5ml of bhi-b media. the solution in the second tube was taken about 5 ml, then added to the third tube, and so on to obtain the extracts with the concentrations of 100%, 50%, 25%, 12.5%, 6.25% and 3.125%. for arabica and robusta coffee extracts with a concentration of 75% was prepared by mixing the arabica coffee extract or robuta coffee extract with the concentration of 100% as much as 7.5 ml into tubes that had contained 2.5 ml of bhi-b media. after the serial dilution, 0.1 ml of l. acidophilus bacteria equivalent to 0.5 mc farland standard was put into each tube with various concentrations. next, the bhi-b media dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p99-103 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p99-103 101101wijaya, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 99–103 were incubated for 1x24 hours with a temperature of 37° c. after the incubation, each tube was planted in the bhi-a media, and then the media were incubated for 1 x 24 hours with a temperature of 37° c. observation then was performed on the results to know whether there was bacterial growth or not. mic value was obtained from the lowest concentration, indicating there was no bacterial growth.11 meanwhile, to determine mbc, the number of l. acidophilus bacterial colony growth on the bhi-a media emerged was observed.12 on the other hand, the initial steps in well diffusion method was to make a well hole on each petri dish using a ring as much as 8 wells with a diameter of 5 mm and a depth of 3 mm in the bhi-a media which had been inoculated by l. acidophilus bacteria. next, robusta or arabica coffee bean extract with the concentration of 100%, 75%, 50%, 25%, 12.5%, 6.25%, and 3.125% was put into each well that had been tagged previously until the pitting holes were filled. one hole then was used for a negative control. after that, they were incubated for 24 hours at 37° c. the amount of inhibition zone was measured using calipers, and then data analysis was conducted. finally, normality test was performed using kolmogorvsmirnov test, and then homogeneity test was carried out using levene’s test. if the results of both tests show that the data were normal and homogeneous (p>0.05), then t test was conducted to determine whether there were significant differences between the effects of arabica coffee extract and the effects of robusta coffee extract on l. acidophilus. results the results of dilution method in this research showed that mic value obtained in arabica and robusta coffee extract to inhibit the growth of l. acidophilus was 25%, while mbc was 50% (figure 1). the mean diameter of the inhibition zone in arabica and robusta coffee extracts can be seen in table 1. figure 1 shows the new l. acidophilus bacterial growth was emerged at the concentration of 12.5%, while at the concentrations of 100% -25% there was no l. acidophilus bacterial growth. based on these results, it can be concluded that a concentration of arabica and robusta coffee extracts, which still can inhibit the growth of l. acidophilus is 25%. and, for determining mbc required, the number of l. acidophilus colonies was measured. table 1 shows the number of bacterial colonies growing on the media bhi-a at each concentration of the arabica and robusta coffee extracts. it also shows that there was no growth of l. acidophilus bacterial colonies at the concentrations of 100%, 75%, and 50%. the growth of new l. acidophilus bacterial colonies was also emerged at the concentration of 25%. the number of l. acidophilus bacterial colonies increased at the concentration of 12.5%. based on these results, it can be concluded the mbc value of arabica and robusta coffee extracts is equal at the concentration of 50%. table 2 shows that the largest diameter mean of the inhibition zones found on robusta coffee extract at the concentrations of 100% and 75% were 13.83 mm and 12.62 mm. meanwhile, the largest diameter mean of the inhibition zones found on arabica coffee extract at the concentrations of 50% and 25% were 9.31 mm and 8.14 mm. the normality of data obtained was tested using kolmogorv-smirnov test. the results of the normality test showed that the data obtained was normally distributed (p> 0.05). after that, homogeneity test was conducted using levene’s test. the results of levene’s test showed the value of p was less than 0.05 in the inhibition zone of arabica and robusta coffee extracts at the concentration of 100%, indicating the data were not homogeneous. meanwhile, the value of p was more than 0.05 in the 9 figure 1. planting coffee extract dilution culture: (a) arabica and (b) robusta on bhi-a media at concentrations of 100-3.125%. table 1. the number of l. acidophilus bacterial colonies given with arabica and robusta coffee extracts 100% 75% 50% 25% 12.5% arabica 0 0 0 38 62 robusta 0 0 0 51 77 c+ 109 c0 note: g+: positive control group; g-: negative control group table 2. the calculation results of the mean inhibition zone diameter of arabica and robusta coffee extract note: mean: average diameter of inhibition zone; sd: standard deviation; n: number of samples; ab: indicating the presence of significant value difference; aa: indicating the absence of significant value difference. concentration mean(mm) ± sd arabica robusta 100% 12.53 ± 0.307a 13.83 ± 0.71b 75% 10.66 ± 0.373a 12.62 ± 0.42b 50% 9.31 ±1.003a 9.23 ± 1.07a 25% 8.14 ± 0.821a 8.03 ± 0.89a a b figure 1. planting coffee extract dilution culture: (a) arabica and (b) robusta on bhi-a media at concentrations of 100-3.125%. table 1. the number of l. acidophilus bacterial colonies given with arabica and robusta coffee extracts 100% 75% 50% 25% 12.5% arabica 0 0 0 38 62 robusta 0 0 0 51 77 c+ 109 c0 note: g+: positive control group; g-: negative control group table 2. the calculation results of the mean inhibition zone diameter of arabica and robusta coffee extract concentration mean(mm) ± sd arabica robusta 100% 12.53 ± 0.307a 13.83 ± 0.71b 75% 10.66 ± 0.373a 12.62 ± 0.42b 50% 9.31 ±1.003a 9.23 ± 1.07a 25% 8.14 ± 0.821a 8.03 ± 0.89a note: mean: average diameter of inhibition zone; sd: standard deviation; n: number of samples; ab: indicating the presence of significant value difference; aa: indicating the absence of significant value difference. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p99-103 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p99-103 102 wijaya, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 99–103 inhibition zone of arabica and robusta coffee extracts at the concentrations of 75%, 50%, and 25%, indicating the data were homogeneous. after the homogeneity test was conducted, t test then was performed then to know the differences in inhibiting the growth of l. acidophilus bacteria between arabica coffee extract and robusta coffee extract. the results of t test showed that the value of p was less than 0.05 at the concentrations of 100% and 75%. it means that there was a significant difference in inhibiting the growth of l. acidophilus bacteria between arabica coffee extract and robusta coffee extract at the concentrations of 100% and 75%. on the other hand, the value of p was more than 0.05 at the concentrations of 50% and 25%. it indicates that there was no significant difference in inhibiting the growth of l. acidophilus bacteria between arabica coffee extract and robusta coffee extract at the concentrations of 50% and 25%. discussion in this research, the antibacterial inhibitory effects of arabica and robusta coffee extracts at various concentrations on the growth l. acidophilus were observed using well diffusion method in the bhi-a media. the antibacterial inhibitory effects of arabica and robusta coffee extracts were indicated with the existence of a clear zone around the well hole. in other words, the larger the diameter of the clear zone is formed, the greater the inhibitory effects are.13 before doing the research on the amount of inhibition zone on arabica and robusta coffee extracts, however, the values of mic and mbc of arabica and robusta coffee extracts to inhibit and kill the growth of l. acidophilus were measured using dilution tube method on the bhi-b media. the results showed that the mic values of arabica and robusta coffee extracts to inhibit the growth of l. acidophilus were obtained at the concentration of 25%, while the mbc value was obtained at the concentration of 50%. on the other hand, the minimum concentration of robusta coffee extract in inhibiting s. mutans bacteria was at a concentration of 12.5%. 7 the results of this research, furthermore, showed that the inhibitory zone of robusta coffee extract was greater than the inhibitory zone of arabica coffee extract at the concentrations of 100% and 75%. on the other hand, there was no difference in the inhibitory zone of arabica and robusta coffee extracts at the concentrations of 50% and 25%. the differences in the diameter of the inhibition zone at each concentration may be due to a large difference in the active substances contained in arabica and robusta coffee extracts that are antibacterial, such as caffeine, trigonelline, caffeic acid, and chlorogenic acid. it means that the greater the concentration is, the greater the components of the active substances contained are, as a result, the inhibition zone formed is also different in each concentration.14 in addition, according to butler et al., an increase and a decrease in inhibition zone are caused by the component substances contained in medicinal plants that can mutually weaken, strengthen, improve, or change completely the effects of the medicinal plants. the quality and quantity of the substances contained in the medicinal plants are determined by environmental factors, such as growing climate, soil, sunlight, and growing conditions until harvesting date.15 based on examination results conducted by in the laboratory of research, and industrial consultation agency (balai penelitian dan konsultasi industri) in surabaya, east java, the greatest components contained in arabica coffee bean extract were caffeine (0.18%), trigonelline (0.17%), diterpene ester (0.05%), caffeic acid (0.15%), chlorogenic acid (0.07%), polyphenols (0.54%), and coffee oil (0.09%). on the other hand, the greatest components contained in robusta coffee bean extract were caffeine (0.21%), trigonelline (0.12%), diterpene ester (0.08%), caffeic acid (0.11%), chlorogenic acid (0.09%), polyphenols (0.72%), and coffee oil (0.08%). caffeine and trigonelline are ones of the largest components of the alkaloid compounds found in coffee beans serving as antibacterial.10 this statement is supported by a research conducted by nuhu et al.,16 that trigonelline contained in robusta coffee bean extract is positively correlated to a decrease in s. mutans biofilm formation through its bacteriostatic action. according to a research conducted by almeida et al.,17 trigonelline, caffeine, and chlorogenic acid contained do not differ in their antimicrobial activity. caffeic acid and trigonelline are known to have the same inhibitory effect on the growth of microorganisms. caffeine and chlorogenic acid are also known to have a very strong antibacterial effect in inhibiting the growth of serratia marcescens and enterobacter cloacae. it can be concluded that robusta and arabica coffee bean extracts have inhibitory effects on l. acidophilus. nevertheless, robusta coffee bean extract has a greater inhibition ability than arabica coffee extract. 10 figure 2. the results of inhibition zone diameter of arabica coffee extract (a) and robusta coffee extract; (b) at concentrations of 100-25%. 10 figure 2. the results of inhibition zone diameter of arabica coffee extract (a) and robusta coffee extract; (b) at concentrations of 100-25%. a b figure 2. the results of inhibition zone diameter of arabica coffee extract (a) and robusta coffee extract; (b) at concentrations of 100-25%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p99-103 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p99-103 103103wijaya, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 99–103 references 1. worotitjan i. pengalaman karies gigi serta pola makan dan minum pada anak sekolah dasar di desa kiawa kecamatan kawangkoan utara. jurnal e-gigi 2013; 1(1): 59-68. 2. quivey rg, koo h, seils j, abranches j, robert a, william h. influence of apigenin on gtf gene expression in streptococcus muta ns ua159. a ntim icrobia l agents a nd chemotherapy. j microbiology 2006; 50(2): 542-6. 3. sanders me, klaenhammert tr. the scientific basis of lactobacillus acidophilus ncfm functionality as a probiotic. 2012. p. 319-31. 4. fitrianti d, noorhamdani, setyawati s. efektifitas ekstrak daun ceplukan sebagai antimikroba terhadap methicillin-resistant staphylococcus aureus in vitro. jurnal kedokteran brawijaya, 2011; 26(4): 3. 5. wang y, ho ct. polyphenolic chemistry of tea and coffee: a century of progress. journal of agricultural and food chemistry 2009; 57: 8109-14. 6. widyotomo s, sri m. ekstraksi kafein dari dalam biji kopi. warta pusat penelitian kopi dan kakao indonesia 2007; 22(3): 13-41. 7. suhad ams, ekbal ak, ali as. anti microbial investigation of suaeda baccata (chenopodiaceae). j plant science 2004; 2(1): 49-51. 8. farida a, ristanti e, kumoro ac. penurunan kadar kafein dan asam total pada biji kopi robusta menggunakan teknologi fermentasi anaerob fakultatif dengan mikroba nopkor mz-15. jurnal teknologi kimia dan industri 2013; 2(3): 70-5. 9. baylin a, hernandez-diaz s, kabagambe ek, siles x, campos h. transient exposure to coffee as a trigger of a first nonfatal myocardial infarction. j epidemiology 2006; 17(5): 506-11. 10. namboodiripad a, kori s. can coffee prevent caries?. j conserv dent 2009; 12(1): 17-21. 11. mardiyaningsih a, aini r. pengembangan potensi ekstrak daun pandan (pandanus amaryllifolius roxb) sebagai agen antibakteri. j pharmaciana 2014; 4(2): 188. 12. ayini u, hanina s, dewi tc. efek antibakteri ekstrak daun mimba (azadirachta indica a. juss) terhadap bakteri vibrio alganoliticus secara in vitro. journal of biology & biology education 2014; 6(1): 4-6. 13. taguri t, kouno ta. antimicrobial activity of 10 different plant polyphenols against bacteria causing food borne disease. biol pharm bull 2004; 27: 1965-9. 14. quivey rg. influence of apigenin on gtf gene expression in streptococcus mutans ua159. antimicrobial agents and chemotherapy 2006; 50(2): 542-6. 15. mendez cr, badet c, yanez a, domingue ml, giono s, richard b, nancy j, dorignac g. identification of oral strains of lactobacillus species isolated from mexican and french children. journal of dentistry and oral hygiene 2009; 1(1): 9-16. 16. nuhu a. bioactive micronutrient in coffee: recent analytical ap-nuhu a. bioactive micronutrient in coffee: recent analytical approaches for characterization and quantification. nigeria: hindawi publishing corporation isrn nutrition; 2013. p. 1-13. 17. almeida aa, farah a, daniela am, elziria, beatriz m. anti-almeida aa, farah a, daniela am, elziria, beatriz m. antibacterial activity of coffee extracts and selected cofee chemichal compounds against enterobacteria. j agric food chem 2006; 54 (23): 8738-43. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p99-103 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p99-103 195 vol. 43. no. 4 december 2010 multidisciplinary management of a mandibular buccal plate perforation yuli nugraeni� and chiquita prahasanti� 1department of conservative dentistry, dentistry study program, brawijaya university, malang indonesia 2 department periodontic, faculty of dentistry, airlangga university, surabaya indonesia abstract background: endodontists often have difficulty in the management of endo-perio cases, because they cannot visually detect the condition of outer root surfaces, especially in bucco-lateral roots. the bone defect is rare and its treatment needs collaboration of endodontists and periodontists. an endodontist treats cases based on dental history, radiograph of root canals to measure root canals, to uncover abnormalities and to diagnose, but as the endodontists cannot directly see the affected parts, the unseen portion of the tooth could only be seen after flap surgery. purpose: this case presents the importance of multidisciplinary approach by the endodontist and periodontitis to treat bucal plate perforations in endo-perio cases. case: the first patient, a 47-year-old female had endodontic treatment and a porcelain crown restoration; however, after several months she felt pain. the second patient, a 45 year-old female had endodontic treatment and after six months she feel painful. case management: the first patient, was referred to a periodontist. the opening flap surgery has been done, a bone defect was found in tooth 45. subsequently, the exposed crater was filled with a bone graft and the pain disappeared. the second patient, with improper endodontic treatment. the flap surgery was conducted, there was found a bone defect in tooth 36. the last treatment, a bone graft has been covered and then the pain was not present. conclusion: proper diagnosis and treatment of perforations on the buccal aspect of a root was able to eliminate pain and avoid tooth extraction. key words: bucco-lateral defect, endo-perio lesion, management abstrak latar belakang: endodontis sering mengalami kesulitan dalam menangani kasus endo-perio karena secara visual kondisi ini tidak tampak diluar permukaan akar, khususnya pada akar bukolateral. defek tulang sangatlah jarang dan perawatannya membutuhkan kolaborasi endodotis dan periodontis. seorang endodontis merawat kasus-kasus berdasarkan riwayat gigi, radiografis saluran akar untuk melihat saluran akar, melihat abnormalitas dan diagnosis, tetapi ketika endodontis tidak dapat secara langsung melihat bagian yang terlibat, bagian gigi yang tidak terlihat ini hanya dapat dilihat setelah dilakukan bedah flap. tujuan: kasus ini menunjukkan pentingnya pendekatan multidisipliner oleh endodontis dan periodontis dalam menangani perforasi aspek bukal akar pada kasus endoperio. kasus: pasien pertama, seorang wanita 47 tahun telah mendapatkan perawatan endodontik dan restorasi mahkota porselen, akan tetapi selama beberapa bulan pasien tersebut mengeluh nyeri. pasien kedua, seorang wanita 45 tahun telah mendapatkan perawatan endodontik dan setelah 6 bulan juga mengeluh nyeri pada gigi tersebut. tatalaksana kasus: pasien pertama dirujuk ke periodontis. bedah pembukaan flap dilakukan dan ditemukan defek tulang pada gigi 45, kemudian crater yang terbuka diisi graft tulang dan nyeri berangsur hilang. pada pasien kedua ditemukan perawatan endodontik yang kurang baik. pada pasien ini ilakukan bedah flap dan ditemukan defek tulang pada gigi 36, pada akhir perawatan graft tulang dipasang dan nyeri berangsur hilang. kesimpulan: diagnosa dan perawatan perforasi yang tepat pada aspek bukal akar dapat menghilangkan nyeri dan menghindari ekstraksi gigi. kata kunci: defek bukolateral, lesi endo-perio, manajemen correspondence: yuli nugraeni, c/o: bagian konservasi gigi, program studi kedokteran gigi, universitas brawijaya. jl. veteran 1 malang. e-mail: yulinugraeni@yahoo.com. case report 196 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 195–200 introduction endodontics represents a real challenge in daily practice and establishing an accurate diagnosis is the first step in successful endodontic therapy. it is well known that clinical endodontic diagnosis is based on the patient’s dental history, clinical evaluation and the radiographic appearance of the inflamed tissue. the diagnosis of necrotic pulps may be difficult to establish due to signs and symptoms being inconclusive and to abnormal root positions.1 endodontists may not directly see a problem and need to take great care in patient management such as using a series of radiography to decide on optimal treatment. in many cases the diagnosis is easy to established, but there are certain cases where it becomes more complex, especially when problems coexists with periodontal diseases. sometimes root perforations may occur during endodontic treatment and bring difficulties in its successful completion. if the perforation occurs below the level of the periodontal attachment, infection with loss of attachment and pocketing can spread.2 in this case, the patient had been treated endodontically and a porcelain crown was fitted. the radiography showed that the apical obturation looked hermetic as well. unfortunately, after nearly two years, the periodontal symptoms appeared and a radiography showed a lesion which had expanded along the lateral of the root. consequently, the periodontal surgery was conducted and it was determined that there was a bone defect, a perforation on a bucco-lateral aspect. the diagnosis of tooth with a defect which also accompanied by a periodontal lesion after the endodontic treatment is rare and may be difficult to establish for reasons of inconclusive signs and difficult location.3 in this case, the perforation was localized in bucco-lateral aspect of the root, involving the alveolar bone, so called an endo-perio lesion. therefore the poor prognosis of these infections requires an interdisciplinary approach between endodontics and periodontics specialists. the relationship between periodontal and pulpal disease was first described by simring and goldberg in 1964. since then the term of endo-perio lesion has been used to describe lesions due to inflammatory products found in varying degrees in both the periodontium and the pulpal tissues. the simultaneous existence of pulped problems and inflammatory periodontal disease can complicate diagnosis and treatment planning. an endo-perio lesion can have a varied pathogenesis which ranges from quite simple to relatively complex.4 figure �. tooth 45 within crown and bridge before endodontic treatment (arrow). this study reports a rare case of root perforation on the bucco-lateral aspect, involving the alveolar bone. the treatment of root perforation with associated periodontal defects can be challenging to the most clinician. in these cases, a multidisciplinary approach between endodontist and periodontist was performed to treat the problems. case case �: a 47 year-old-female patient came with referred pain and bad condition on tooth 45 within crown and bridge. in examination, there was a sensitivity on palpation. the radiography showed a periapical lesion around apical of tooth 45, and the tooth was diagnosed as necrotic pulps and treated endodontcally (figure 1). two years later, patient reported with a chief complaint about spontaneous pain, swelling, and pus from 45. the figure �. tooth 45 within a new crown and bridge after endodontic treatment and showed that there was a widening lesion a year forward (arrow). 197nugraeni and prahasanti: multidisciplinary management of a mandibular buccal plate perforation radiography showed a widening of the periodontal ligament in the lateral of root. the periodontal probing of the remaining teeth was within normal range. an endo-perio lesion associated with 45 was diagnosed (figure 2). inspite of this, patient had asked to take some medicine for reducing the pain while preparing for periodontal surgery procedure. the treatment plan involved exploratory periapical surgery. the patient was made aware of a high possibility of a root perforation. case �: a 45 year-old-female patient came with persistent pain after root canal treatment and porcelain crown restoration. there was a sensitive response on palpation. however, six months later the patient felt pain and taken analgesic for relief of pain. sometimes, this patient has a spontaneous pain, swelling and pus came through and on its buccal aspect there was a deep pocket on probing. the radiographic showed that there was a widening lesion area around furcation of tooth 36 and the apical obturation was improper (figure 4). the tooth was diagnosed as an endo-perio lesion. consequently the periodontist had to check with the probe, the tooth was in uncommon condition. subsequently, the flap surgery was planned soon and then followed by retreatment. while preparing for periodontal surgery procedure, some medicine should be prescribed for relief of pain. case management case �: endodontic treatment was done on tooth 45. an access cavity was prepared and the root canal system was cleaned in the first session with 5.25% sodium hypochlorite irrigation and shaped by using protaper™. the canal was given temporary dressing with rockless and the access cavity was sealed with cavit cement. on the fourth visit there was an absence of pain or signs of inflammation therefore obturation was done with gutta-percha and a root canal sealer. finally, a new crown and bridge was placed. a one-month recall revealed a stable mastication and there were no pain reported. however, two years later, the patient reported with chief complaint of pain, swelling, and pus discharge from the right mandibular second premolar. the tooth was previously sensitive, but there was no any pockets persisted around the figure ��. at three month later, the radiographic showed that there was alveolar bone gain (arrow). figure �. the tooth 36 was diagnosed as an endo-perio lesion. the endodontic treatment showed improper obturation. 198 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 195–200 tooth. based on the case and radiography, a periodontist decided to correct the problem by opening flap surgery, and the crater was filled by using bone graft substitute. one week after the surgery, the patient returned and there was asymptomatic. at three month later as showed at the figure 3, patient reported, there was no pain and the radiographic showed that there was alveolar bone gain. case �: on the other patient, the tooth 36 was diagnosed as an endo-perio lesion that showed at figure 4. based on the situation, patient was referred to the periodontist immediately. periodontal surgery was done first, which included scaling and root planning and apical curettage on the tooth. while the operation has been exploring, there was a defect as a crater on the buccal plate. consequently the inflammation area was cleaned-up and the crater was packed by bone graft substitution and some antibiotics was be taken. the defect was packed with an autobone graft. a week later, the patient was recalled and reported that there was not any symptom. then the retreatment was followed by an endodontist. after the cavity access was done, the root canal was cleaned with 5.25% sodium hypochlorite irrigation and shaped properly by using protaper™ for several visits. as the final point, the canal was completed by apical obturation. finally, both the endodontist and the periodontist advised the patients to take evaluation in several visits to make sure that the bone was healed properly. discussion the dental pulp is closely connected with the periodontal ligament through the apical foramen, accessory canals and dentinal tubules. due to this relationship, pulp diseases may influence periodontal health, and the periodontal infection may affect the pulp integrity. it is estimated that pulp and periodontal problems are responsible for more than 50% of tooth mortality.5 the apical dental foramen could serve as an open way of entrance for periodontal bacteria in the dental pulp, promoting situations of degenerative and necrotic nature. some studies had suggested that the communication between pulp and periodontal tissue can occur not only by apical dental foramen. rubach and mitchell6 in 1965 suggested that the periodontal disease may affect pulp health when it has exposition of the accessory canals to the oral cavity, what it would allow bacteria proceeding from this region to invaded the pulp provoking a chronic inflammatory reaction followed by necrosis to the pulp. the relationship between pulp and periodontal disease can be traced from embryological development, since the pulp and the periodontium are derived from a common mesodermal source. the pulp can be invaded from the periodontium through dentin tubules, lateral and accessory canals and the apical foramen; although evidence exists for such canals of communication, a mechanism for the direct transmission of periodontal infection into the pulpal tissues remains unclear.6 in both of cases, defect on alveolar bone area of teeth 45 and 36 could be a sign of a lateral canal accessory existence that had not been detected during root canal treatment. the origin of the endo-perio lesion was rarely unknown and establishing the original cause is not usually straightforward. serial radiographs and the state of the pulp on first entering the root canal system can provide useful clues. the patient was diagnosed as an endo-perio lesion and treated endodontically and followed by regenerative therapy to treat the defect. in most cases of endo-perio lesions, clinical symptoms disappear following successful endodontic therapy. however, it becomes essential to correct the periodontal defect simultaneously in these cases to prevent recurrence, and to improve the functional status of the tooth.3 the majority of accessory canals are found in the apical part of the root and lateral canals in the molar furcation regions. tubular communication between the pulp and periodontium may occur when dentinal tubules become exposed to the periodontium due to the absence of overlying cementum. these are the pathways that may provide the means by which pathological agents pass between the pulp and periodontium, thereby creating the perio-endo lesion.3 figure 5. the opening of flap operation on a first management showed that there was a bone defect as a crater (arrow). 199nugraeni and prahasanti: multidisciplinary management of a mandibular buccal plate perforation both endodontic and periodontal diseases are caused by mixed anaerobic infections. bacteria and their inflammatory by-products can penetrate through the pathways connecting the periodontium and the pulp. lateral and accessory canals appear mainly in the apical area and in the furcation area of molars.4 both of these cases, bone defects were found that assumed from lateral canal accessory. it has been suggested to be direct pathways between the pulp and the periodontium which contain connective tissue and blood vessels that connect the circulatory systems of the two tissues. the presence of patent accessory canals is a potential pathway for the spread of bacteria and toxic substances from the pulp, resulting in an inflammatory process within the periodontal ligament.7 therefore, a great success of the obturation on the radicular space is necessary to eliminate leakage. the obturation prevents coronal leakage and bacterial contamination, seals the apex from the periapical tissue fluids, and seals the remaining irritants in the canal.15 the root perforations due to the existence of canal accessory are undesired complications of endodontic treatment which result in loss of integrity of the root and further destruction of the adjacent periodontal tissues.9 root perforations adversely affect the prognosis of teeth. inadequacy of the repair materials has been a contributing factor to the poor outcome of repair procedures. mineral trioxide aggregate (mta) is a relatively new material that is being successfully used to repair perforations.13 several in vitro and in vivo studies have shown that mta has good sealing ability, biocompatible, and can promotes tissue regeneration when placed in contact with the dental pulp or periapical tissue.14 the closing of defects can be packed by bone graft substitute. the ideal bone graft material would be biocompatible, completely biodegradable, osteo-conductive, inexpensive, easy to handle, and able to support the defect area until bone growth is complete.8 the destruction in both of the cases involve the alveolar bone which create a crater. therefore the autogenous bone graft has been regarded as an ideal material for bone defect. on the second case, the root perforation especially in furcal area was restored with mta, the root surface was chemically conditioned with tetracycline and the defect was filled with bone graft and then prescribed post-op medications: clindamycin 300 mg qid for 10 days, mouth rinse and mefenamic acid as needed for pain. three months post-op clinical evaluation showed significant periapical bone fill with periodontal probing depth of 2 mm. the prognosis and treatment of each endodontic periodontal disease type varies. primary endodontic disease should only be treated by endodontic therapy and has a good prognosis. primary periodontal disease should only be treated by periodontal therapy. both of these cases, the prognosis depends on the severity of periodontal disease and patient response. both of the patient were in good condition. nevertheless, on the second case, open flap surgery should be done first due to the pus discharge and deep pocket. at typical situation, the lower first molar showed a sign of periodontal disease with loss of attachment on the buccal aspect root. commonly, in this case, the symptom was misinterpreted and the diagnosis was of localized periodontitis; consequently, the applied treatment consists of scaling and root-planning, associated with local antiseptics and irrigations with clorhexidine was recommended. actually, as a primary endodontic disease with secondary periodontal involvement should first be treated with endodontic therapy.10 in the present cases, as the radiography examination the healing of bone was getting healthy. however, because of the high resorption rate of the autogenous bone graft, a longer observation interval may be needed to confirm the stability of the clinical outcome, as observed after six months.11 healing was unevent and the three months post-op clinical evaluation showed significant periapical bone fill with no evidence of a sinus tract and labial periodontal probing depth of 2 mm. guided tissue regeneration and guided bone regeneration can prove invaluable in endodontic-periodontal cases that may otherwise have a poor prognosis which may ultimately lead to tooth loss. augmentation materials can assist in the regeneration of new attachment cells and bony support for previously compromised cases.12 the tooth anatomy and the etiology of endodontic-periodontal lesions offer a strong base for establishing a correct diagnosis. endodontists often misdiagnosed just because of the limitation in management, they can treat based on the history taking, the symptoms and the radiography proved, also even especially of a common sense. on the other hand, the periodontist can treat clearly by opening flap surgery. therefore, multidisciplinary approach of endodontist and periodontist is mandatory in especially endo-perio lession cases. moreover, due to the complexity of these affections, an interdisciplinary approach with a good collaboration between endodontists, periodontologists and microbiologists, is recommended.6 furthermore, the endodontists is responsible for the improvement of their knowledge through the management. however, essential collaboration between endodontists and periodontists to correct the periodontal defect simultaneously in these cases was aimed to prevent the recurrence, and to improve the functional status of the tooth. in conclusion, an endo-perio lesion consequent, retreatment or collaboration with periodontist should be done to prevent further tooth extraction. the proper diagnosis and treatment of root perforation involving bone defect was able to eliminate pain symptoms, thus preventing from tooth extraction. references 1. koyees e, fares m. referred pain: a confusing case of differential diagnosis between two teeth presenting with endo-perio problems. case report. int endodon j 2006; 39: 1–2. 200 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 195–200 2. shuman ie. repair of a root perforation with a resin-ionomer using an intentional replantation technique. general dentistry july–agustus 1999; 47(4): 21–5. 3. singh s. management of an endo-perio lesion in a maxillary canine using platelet-rich plasma concentrate and an alloplastic bone substitute case report. 2009; 13(2): 97–100. 4. nadir b. the pathogenesis and treatment of endo-perio lesions. the preliminary program for iadr saudi arabian section meeting january, 2006; p. 28–31. 5. didilescu a, iliescu r, rusu d, iliescu aa, ogodescu a, ogodescu e, stratul s. current concepts on the relationship between pulpal and periodontal disease. tmj 2008; 58(1): 99–103. 6. silva mf, piva, reinheimer, dejean. spread of odondogenicsilva mf, piva, reinheimer, dejean. spread of odondogenicspread of odondogenic infection originating from endo-periodontal-case report: available at: saqmartins@hotmail.com/www.actiradentes.com. br/.../19revistaato-spread_of_odontogenic_infection-2009.pdf. accessed january 16, 2009. 7. trombelli l, annunziata m, belardo s, farina r, scabbia a, guida l. autogenousbone graft in conjunction with enamel matrix derivative in the treatment of deep periodontal intra-osseous defects: a report of 13 consecutively treated patients. j clin periodontol 2006; 33: 69–75. 8. white jr c. combined therapy of mineral trioxide aggregate and guided tissue regeneration in the treatment of external root resorption and an associated osseous defect. j of periodontol 2002; 73(12): 1517–21. 9. agrawal pk. combined periodantal endodontic lesion. sr. prof and head dept. of periodontia govt. dental college, jaipur. available at: www.gdcaaj.comwww.gdcaaj.com/articles%20pdf/pkagrawal. pdf/2006. accessed december 17, 2006. 10. yildirim g, dalci kk. treatment of lateral root perforation with mineral trioxide aggregate: a case report. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology, 2006. 102(5): e55–e58. 11. mamidwar ss, ricci jl, alexander h. bone regeneration with calcium-sulfate–based bone grafts. inside dentistry 2006; 2: 1–8. 12. gold a. guided tissue regeneration (gtr) can promote or guide the proliferation of periodontal ligament cells and attachments onto denuded root surfaces. j indian soc periodontol 2009 oct; 13: 97–100. 13. main c, mirzayan n, shabahang s, torabinejad m. repair of root perforations using mineral trioxide aggregate: a long-term study. j of endod 2001; 30(2): 80–3. 14. chong. harty’s endodontics in clinical practice. 6th ed. elsevier; 2010. p. 146. 177177 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 177–182 research report effectiveness of anadara granosa shell-stichopus hermanni granules at accelerating woven bone formation fourteen days after tooth extraction rima parwati sari1 and hansen kurniawan2 1department of oral biology, 2department of periodontics, faculty of dentistry, universitas hang tuah, surabaya – indonesia abstract background: post-extraction complications can cause alveolar bone resorption. hydroxyapatite-tricalcium phosphate (ha-tcp) is one potential bone graft material that can be synthesized from anadara granosa shell. another biomarine, stichopus hermanni, contains hyaluronic acid which can accelerate bone formation on the fourteenth day. purpose: this study aims to prove the effectiveness of anadara granosa shell-stichopus hermanni granules in weaving bone formation fourteen days after tooth extraction. methods: twenty-five male wistar rats were divided into five groups. their lower left incisor was extracted with gelatin being administered to the control group (c) and granule scaffold derived from anadara granosa (ag) shell and anadara granosa shell-stichopus hermanni at concentrations of 0.4%-0.8%-1.6% (agsh1-agsh2-agsh3) to the treatment group. this study developed a ha-tcp synthesized from anadara granosa combined with whole stichopus hermanni to create granule scaffolds by means of a freeze-dried method. the jaw was removed on the fourteenth day post-tooth extraction. observation of hpa involved the use of an image raster®. the resulting data was subjected to analysis by anova and tukey-hsd tests (p<0.05). results: data showed the mean of c=0.157±0.078; ag=1.139±0.371; agsh1=1.595±0.291; agsh2=1.740±0.308; and agsh3=1.638±0.286. statistical analyses showed significant differences in the woven bone area (mm2) between c and the treatment groups ag;agsh1;agsh2; agsh3; and between ag and the agsh2 groups. conclusions: scaffold granules from anadara granosa shells and stichopus hermanni effectively accelerate the bone formation process with the most effective being stichopus hermanni at a concentration of 0.8%. keywords: anadara granosa; bone formation; granule scaffold; stichopus hermanni; tooth extraction correspondence: rima parwati sari, department of oral biology, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: rima.parwatisari@hangtuah.ac.id introduction in 2013, indonesia’s national basic health research body produced a missing teeth (mt) index of 2.9, indicating that of the permanent teeth extracted, 290 tooth roots per 100 people remained in the jaw.1 following tooth extraction, the alveolar bone is gradually resorbed and offset by a remodeling process which still results from vertical to palatal resorption.2 schropp et al. (2003),3 estimated that two-thirds of hard and soft tissue changes occur within the initial three months. in another study, alveolar bone resorption occurred in the vertical and horizontal directions ranging between 40-60% during a 2-3 year period.4 this post-extraction process will continue in an integrated manner ultimately resulting in disruption to the stomatognathic system.5 one week after extraction, the blood clots with which the socket had initially been filled were almost completely replaced by granulated tissue. this process was followed by an inflammatory response capable of triggering proliferation which was characterized by the presence of growth factors. these played a role in new blood vessel formation which, in turn, increased the number of fibroblasts and pre-osteoblasts and promoted their differentiation into mature osteoblasts. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 178 sari, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4):177–182 these osteoblasts will appear in the bone cortex surrounding the defect site. mature osteoblasts synthesize bone matrix, especially type i collagen, and regulate newly-formed bone mineralization.6,7 in this phase, granulation tissue and provisional matrix (pm) are filled with mesenchymal cells, collagen fibers, and blood vessels. within two weeks, woven bone had begun to form in the apical region of the tooth socket.8,9 therapy to stimulate bone formation and regeneration of bone damage due to tooth extraction generally utilizes bone graft10 which must have a physical structure and properties similar to bone in order for it to accelerate the healing process. bone grafts must satisfy certain conditions, firstly, that they are biocompatible or acceptable to the body; secondly, that they facilitate bone formation through a mechanism that contains bone-forming cells (osteogenesis); thirdly, that they serve as a scaffold for bone formation (osteoconduction); and, lastly, that they contain boneinducing ingredients (osteoinduction).11 the basic ingredient of bone graft is calcium phosphate (ha) which plays a role in bone mineralization. research has been carried out on ceramic materials with ha/tcp combination at different ratios. a study conducted by kim et al. (2017),12 indicated a good ha/tcp ratio of 70:30, while other analyses have posited the ha/tcp ratio to be one of 60:40 13. this does not constitute a significant difference with the ha/tcp ratio of 80:20 identified by the biomaterial test. one natural ingredient with the potential for use as a candidate bone substitute material is that of blood clam shells (anadara granosa). the compound found in blood vessels was extracted from cleaned restaurant waste containing 98.8% caco3. 14 in the research conducted by sari (2018),15 after implementing a series of synthesizing processes using the hydrothermal method, it was found that the ha and tcp content were 72% and 21% respectively, with the remaining 7% being accounted for by calcium carbonate. in certain cases, the use of calcium phosphate compounds as bone graft requires the addition of polymeric material to the bone graft structure. ah polymer, a naturally-occurring, hydrophilic, nonimmunogenic material, is found in the cytoplasm of osteoprogenitor cells16 and plays a complex role in cell adhesion, cell proliferation and cell movement. in the extracellular matrix, ah represents a framework that triggers post-injury tissue recovery, suppresses antiinflammatory activity, modulates tissue hydration, osmotic balance and collagen recognition and contractions during the repair process.17 ah in the form of hydrogel can reduce inflammatory cells on day 7 and increase bone density and blood vessel length on day 14. this study identified a microscopic change in the inflammatory process and angiogenesis on the seventh day of the wound healing process.18 research using ah at a concentration of 0.8% suggests that it accelerates bone regeneration through chemotaxis, proliferation and differentiation of mesenchymal cells.19 stichopus hermanni, a natural resource found on the island of raas, in sumenep regency, is high in hyaluronic acid (ah) component (75.7%), and collagen (29.47%).15 granule is one of the scaffolds, small in size and porous in nature, that provides room for cells to attach to bone and grow into new bone tissue.20 granule constitutes gelatin derived from collagen in the skin, bones and connective tissue of animals that has been hydrolyzed by an acid or base. hyaluronic acid-rich gelatin with stichopus hermanni acts as a binding material enabling the graft to turn into a hydrogel which will subsequently undergo a freeze-drying process to produce granular formations.21 this study seeks to explore the potential role of natural wealth in the form of marine life present in indonesian waters to produce innovations in bone substitute materials. these would constitute a combination of ha-tcp synthesized anadara granosa shell and stichopus hermanni polymer at various concentrations in granular form within bone formation after tooth extraction on the fourteenth day. materials and methods this research constitutes a complete randomized design involving the use of wistar strain rattus novergicus which was issued ethical approval number 002/hrecc.fodm/ i/2018 by the faculty of dental medicine, universitas airlangga, surabaya. preparation for the study began with the production of a graft from an anadara granosa (ag) shell synthesized in the form of ha-tcp. blood clam shells (anadara granosa) were boiled, cleaned, crushed and sifted through a 100 mesh to produce smaller particles. ag shell powder containing up to 1m and 0.6m of ammonium dihydrogen phosphate (nh4h2po4) solution mixed with a magnetic stirrer was transferred to the reactor which had been heated in an electric oven at a temperature of 200oc for 12 hours. the results obtained were cooled at room temperature, repeatedly washed with distilled water, separated and showed normal ph (7). the latter was washed with methanol to limit the agglomeration of ha particles during the process of drying. the sample was dried in an electric oven at 50oc for four hours before being sintered at 900°c for three hours to remove impurities and increase its crystallinity.15 preparation of other stichopus hermanni materials entailed washing the material with distilled aquadest before finely chopping it at a mixer ratio of 500 grams to 1 liter of distilled water until a smooth consistency was obtained. the next stage involved a drying process utilizing the freeze-drying method, the results of which were finely milled and sieved through a 50 micron mesh (297 µm). producing powder of an even smaller micro size required high energy milling performed with an ellipse 3d motion (hem-e3d) by nanotech® indonesia.22 scaffold was produced by mixing 5% ha-tcp with 50ml of distilled water and 10 grams of gelatin before agitating the solution for four hours with a magnetic stirrer, placing it in a 96-well plate mold and conducting freezedental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 179sari, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4):177–182 0.0000 0.5000 1.0000 1.5000 2.0000 2.5000 c ag agsh1 agsh2 agsh3 area of woven bone (mm2) figure 1. extensive graph of woven bone showing bone graft application involving a combination of anadara granosa and stichopus hermanni shells on 2/3 apical post-extraction sockets. figure 2. wide histological picture of woven bone with bone graft application involving a combination of anadara granosa shells and stichopus hermanni on the apical 2/3 post-extraction socket on day 14. observations were made by means of he painting employing a light microscope at 100x magnification. table 1. post-hoc test results (tukey hsd) average mean difference (ij) p-value group (i) group (j) c ag agsh1 agsh2 agsh3 -0.9817 -1.4381 -1.5823 -1.4804 0.000* 0.000* 0.000* 0.000* ag agsh1 agsh2 agsh3 -0.4564 -0.6006 -0.4988 0.122 0.024* 0.078 agsh1 agsh2 agsh3 -0.1442 -0.0424 0.927 0.999 agsh2 agsh3 0.1018 0.979 note: * indicates a significant difference information: this table shows a comparison of woven bone area between groups. the area of woven bone in the treatment group is greater than that of the control group. the negative difference means that the area of woven bone is smaller. the agsh2 group has the largest woven bone area compared to the other three groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 180 sari, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4):177–182 drying.15 the final stage of producing this scaffold consisted of sterilization by gamma ray irradiation of 25 kgy supplied by the national nuclear energy agency (batan).23 research on the experiment subjects commenced with a seven-day period of acclimatization. subjects were weighed and marked before being divided into groups and denied food until midnight when the anesthetic, namely; a dose of 10% ketamine, 0.1 cc/kgbb and 0.01 cc/100 g bw xylazine was injected intramuscularly into the right upper thigh24. once cleaning of the aseptic extraction areas of the socket with water spray and antiseptic had been completed, extraction of the left mandibular incisor was effected using a needle holder and scaffold application. application of the treatment involved dividing the 30 male rats into five groups. gelatin alone was applied to the control group, scaffold from anadara granosa shell was applied to the ag group, while the agsh1-3 group received scaffold from the combination of anadara granosa shell and stichopus hermanni at concentrations of 0.4%; 0.8% and 1.6%. suturing of the socket ensued with silk braid (usp. 3/0) supplied by dr. sella®. novalgin® 0.09 cc/200gr bb analgesics and interflox® 0.1cc/100gr bb antibiotics were applied to control any resulting swelling and pain. fourteen days after application to the socket, the experiment subjects were sacrificed and their mandibular preparations subsequently removed and placed in a 10% buffered formalin solution. after tissue fixation, decalcification was carried out by application of ethylenediaminetetraacetic acid (edta) for one month. mandibular specimens were made in the form of sagittal fragments with hematoxillin eosin staining. the area of woven bone was measured in the socket area with an image raster® at 100x magnification with resulting data from each group being tabulated. the statistical analysis employed was a one-way anova parametric test followed by a tukey-hsd test. results the effects of the granule scaffold combination anadara granosa shell and stichopus hermanni administered were evaluated on day 14 after application to the socket. formation of woven bone in the control group (c) occurred on day 14. bone graft application from ag shells indicated an increase in woven bone formation. the combined administering of ag sh shell bone graft (0.4; 0.8; 1.6) increased the formation of wider woven bone (figure 1). the application of bone graft from ag shells did not sufficiently expand the area of woven bone (ag). broad woven bone was increased through the application of bone graft shell in groups agsh1; agsh2; agsh3, while the largest expansion in woven bone area was found in the agsh3 (combination anadara granosa shell and stichopus hermanni 0.8%) group (figure 2). statistical analysis carried out using spss version 23 ibm® 2015 indicated normal and homogeneous distribution of data (0.05), confirmed by the results of a shapiro-wilk test and a levene’s test. in the one-way anova test results (p>0.05) significant differences existed in the woven bone variables across all treatment groups. however, significant differences between one group with another did not occur. this can be seen in the multiple comparison test using tukey-hsd (table 1). discussion the process of socket healing commenced with the occurrence of vascular damage in the socket resulting in platelet aggregation and blood clot formation.25 the inflammatory response, characterized by infiltration of inflammatory cells and macrophages, released proinflammatory cytokines and growth factor. this growth factor played a role in the process of angiogenesis and stimulated the formation of fat. one growth factor actively stimulating an increase in the number of fibroblasts and preosteoblasts and greater differentiation into mature osteoblasts is fgf.26 mature osteoblasts secrete osteoid, type i collagen, growth factors, alkaline phosphatase. calcified irregular collagen tissue will form woven bone.27 formation of new woven bone was initiated on the seventh day and became visible along the lateral alveolar wall and the base of the socket.28,29 woven bone formation increased during the second week of the socket healing process. the newly formed woven bone was surrounded by numerous newly differentiated osteoblasts. these produced a bone matrix with a high proportion of osteocytes in the lateral wall of the socket and extending to its center, thereby reaching the old trabecular bones.30 the application of blood shell granules (anadara granosa) containing ha-tcp serves as a framework for the growth and development of mesenchymal cells into osteoprogenitor cells.31 the hydroxyapatite structure has a stoichiometry similar to that of bone mineral, while tcp is a biodegradable compound that can release calcium ions.32,33 in the study conducted by zhang et al. (2015),34 providing scaffold of gelatin/β-tcp composites induced osteogenic differentiation of bone marrow stem cells (bmsc) in vitro through activation of ca 2+ -sensing receptor signaling (casr) which is proven to increase the expression of runx2, bmp2, col-1 and ocn. hyaluronic acid contained in the stichopus hermanni interacts with cd44 to initiate signal transduction through mapk activation which induces erk1/2 phosphorylation and ap-1 activation resulting in cell migration due to the release of various growth factors.35,36 this activation, in turn, triggers the proliferation and differentiation of osteoprogenitor cells into osteoblast cells which play an important role in the formation of bone matrix. this process was demonstrated by this study in which groups which had been administered with a combination of anadara granosa and stichopus hermanni shells experienced a greater dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 181sari, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4):177–182 increase in their area of woven bone on the fourteenth day compared to that of the c and ag groups. the largest area of woven bone was found in the group to which bone graft from a combination of anadara granosa shell and stichopus hermanni 0.8% had been administered. the different concentrations of whole stichopus hermanni also affected the formation of woven bone due to the initial process of interaction with cd44. this was indicated by the insignificant difference between c and ag with the combination group following the addition of whole stichopus hermanni 0.4% and 1.6%. however, a significant difference was shown between the c and ag groups following the addition of whole stichopus hermanni 0.8 %. this is because stichopus hermanni contains other glycosaminoglycans, such as chondroitin sulfate and keratin sulfate, which can affect the ability of cd44 to bind ah. modification of bonds due to n and o chains in chondroitin sulfate produces a negative effect on the ability of cd44 to bind ah.37 it can be concluded, therefore, that the administration of scaffold granules from a combination of anadara granosa shells and stichopus hermanni can accelerate the formation of woven bone on the fourteenth day after tooth extraction in order to prevent alveolar bone resorption (socket preservation). the concentration of stichopus hermanni 0.8% is the most effective in terms of accelerating the formation of woven bone on the fourteenth day after tooth extraction references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar. jakarta: kementerian kesehatan republik indonesia; 2013. p. 118–9. 2. pagni g, pellegrini g, giannobile w v., rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 1–13. 3. schropp l, kostopoulos l, wenzel a. bone healing following immediate versus delayed placement of titanium implants into extraction sockets: a prospective clinical study. int j oral maxillofac implants. 2003; 18(2): 189–99. 4. ashman a. postextraction ridge preservation using a synthetic alloplast. implant dent. 2000; 9(2): 168–76. 5. hamzah z, kar tikasari n. pencabutan gigi yang ir rasional mempercepat penurunan struktur anatomis dan fungsi tulang alveolar. stomatognatic. 2015; 12(2): 61–6. 6. huldani h. biomarker remodeling tulang. thesis. banjarmasin: universitas lambung mangkurat; 2012. p. 3–17. 7. rustam a, syahbani an, fahruddin am. kolagen sisik ikan nila (oreochromis niloticus) sebagai barrier membrane alternatif untuk reregenerasi tulang alveolar pada kasus periodontitis. bimkgi. 2016; 4: 37–43. 8. la r java h. o ra l wound hea l i ng: cel l biolog y a nd cl i n ica l management. singapore: wiley-blackwell; 2013. p. 350–1. 9. van der weijden f, dell’acqua f, slot de. alveola r bone dimensional changes of post-extraction sockets in humans: a systematic review. j clin periodontol. 2009; 36(12): 1048–58. 10. jamjoom a, cohen r. grafts for ridge preservation. j funct biomater. 2015; 6(3): 833–48. 11. mao t, kamakshi v. bone grafts and bone substitutes. int j pharm pharm sci. 2014; 6(2): 88–91. 12. kim kh, park jy, park hs, kim ks, chin dk, cho ye, kuh su. the influences of different ratios of biphasic calcium phosphate and collagen augmentation on posterior lumbar spinal fusion in rat model. yonsei med j. 2017; 58(2): 407–14. 13. zhao n, wang y, qin l, guo z, li d. effect of composition and macropore percentage on mechanical and in vitro cell proliferation and differentiation properties of 3d printed ha/β-tcp scaffolds. rsc adv. 2017; 7(68): 43186–96. 14. hazmi aja, zuki abz, noordin mm, jalila a, norimah y. mineral composition of the cockle (anadara granosa) shells of west coast of peninsular malaysia and it’s potential as biomaterial for use in bone repair. j anim vet adv. 2007; 6(5): 591–4. 15. sari rp. mekanisme kerja bone graft dari cangkang kerang darah (anadara granosa) dan teripang emas (stichopus hermanii) pada penyembuhan tulang alveolar pasca pencabutan. dissertation. surabaya: universitas airlangga; 2018. p. 124–44. 16. polo-corrales l, latorre-esteves m, ramirez-vick je. scaffold design for bone regeneration. j nanosci nanotechnol. 2014; 14(1): 15–56. 17. mariggiò ma, cassano a, vinella a, vincenti a, fumarulo r, lo muzio l, maiorano e, ribatti d, favia g. enhancement of fibroblast proliferation, collagen biosynthesis and production of growth factors as a result of combining sodium hyaluronate and aminoacids. int j immunopathol pharmacol. 2009; 22(2): 485–92. 18. browne s, monaghan mg, brauchle e, berrio dc, chantepie s, papy-garcia d, schenke-layland k, pandit a. modulation of inflammation and angiogenesis and changes in ecm gag-activity via dual delivery of nucleic acids. biomaterials. 2015; 69: 133–47. 19. dahiya p, kamal r. hyaluronic acid: a boon in periodontal therapy. n am j med sci. 2013; 5(5): 309–15. 20. alexander rw. adipose tissue complex (atc): cellular and biocellular uses of stem/stromal cells and matrix in cosmetic plastic, reconstructive surgery and regenerative medicine. in: regenerative medicine and plastic surgery. cham: springer international publishing; 2019. p. 45–69. 21. faruq o, kim b, padalhin ar, lee gh, lee bt. a hybrid composite system of biphasic calcium phosphate granules loaded with hyaluronic acid-gelatin hydrogel for bone regeneration. j biomater appl. 2017; 32(4): 433–45. 22. baláž p. high-energy milling. in: mechanochemistry in nanoscience and minerals engineering. berlin, heidelberg: springer; 2008. p. 103–32. 23. warastuti y, abbas b. sintesis dan karakterisasi pasta injectable bone substitute iradiasi berbasis hidroksiapatit. j ilm apl isot dan radiasi. 2011; 7(2): 73–82. 24. tanideh n, dehghani nazhvani s, mojtahed jaberi f, mehrabani d, rezazadeh s, pakbaz s, tamadon a, nikahval b. the healing effect of bioglue in articular cartilage defect of femoral condyle in experimental rabbit model. iran red crescent med j. 2011; 13(9): 629–33. 25. nanci a, ten cate ar. ten cate’s oral histology: development, structure, and function. 7th ed. st. louis: mosby elsevier; 2009. p. 380–3. 26. marie pj. fibroblast growth factor signaling controlling osteoblast differentiation. gene. 2003; 316: 23–32. 27. shapiro f. bone development and its relation to fracture repair. the role of mesenchymal osteoblasts and surface osteoblasts. eur cells mater. 2008; 15: 53–76. 28. kim bs, kim sj, kim hj, lee sj, park yj, lee j, you hk. effects of nicotine on proliferation and osteoblast differentiation in human alveolar bone marrow-derived mesenchymal stem cells. life sci. 2012; 90(3–4): 109–15. 29. al-obaidi mmj, al-bayaty fh, al batran r, hassandarvish p, rouhollahi e. protective effect of ellagic acid on healing alveolar bone after tooth extraction in rat a histological and immunohistochemical study. arch oral biol. 2014; 59(9): 987– 99. 30. vieira ae, repeke ce, de barros ferreira s, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to toot h ext ract ion i n m ice: m icro computed tomog raphy, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 182 sari, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4):177–182 31. harsas na. efek pemberian graft tulang berbentuk pasta dengan berbagai komposisi dan konsentrasi terhadap viabilitas sel osteoblas (in vitro). thesis. jakarta: universitas indonesia; 2008. p. 5–14. 32. mao k, zhou f, cui f, li j, hou x, li p, du m, liang m, wang y. preparation and properties of α-calcium sulphate hemihydrate and β-tricalcium phosphate bone substitute. biomed mater eng. 2013; 23(3): 197–210. 33. nakamura s, matsumoto t, sasaki j-i, egusa h, lee ky, nakano t, sohmura t, nakahira a. effect of calcium ion concentrations on osteogenic differentiation and hematopoietic stem cell niche-related protein expression in osteoblasts. tissue eng part a. 2010; 16(8): 2467–73. 34. zhang x, meng s, huang y, xu m, he y, lin h, han j, chai y, wei y, deng x. electrospun gelatin/β-tcp composite nanofibers enhance osteogenic differentiation of bmscs and in vivo bone formation by activating ca2+ -sensing receptor signaling. stem cells int. 2015; 2015: 1–13. 35. schwertfeger kl, cowman mk, telmer pg, turley ea, mccarthy jb. hyaluronan, inflammation, and breast cancer progression. front immunol. 2015; 6: 1–12. 36. litwiniuk m, krejner a, grzela t. hyaluronic acid in inflammation and tissue regeneration. wounds. 2016; 28(3): 78–88. 37. ruffell b, johnson p. the regulation and function of hyaluronan binding by cd44 in the immune system. glycoforum. 2009; 13(a1): 1–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p177–182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p177-182 186186 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 186–193 review article oral microbiota in oral cancer patients and healthy individuals: a scoping review irna sufiawati1, alamsyah piliang2, vatchala rani ramamoorthy3 1department of oral medicine, faculty of dentistry, universitas padjadjaran, bandung, indonesia 2oral medicine residency program, faculty of dentistry, universitas padjadjaran, bandung, indonesia 3department of oral and maxillofacial pathology, faculty of dentistry, jamia millia islamia, jamia nagar, new delhi, india abstract background: oral microbiota has been suggested to have a role in the etiopathogenesis of oral cancer; however, the oral microbiota diversity in patients with oral cancer compared to healthy individuals remains unclear. purpose: this scoping review aimed to provide an overview of the current evidence regarding the oral microbiota composition colonized in oral cancer patients and its comparison with healthy individuals. reviews: this study was conducted according to the preferred reporting items for systematic reviews and metaanalyses (prisma) guidelines. eligible studies were searched in pubmed, scopus, web of science, and sciencedirect databases from january 2015 to march 2022. a total of 20 relevant studies were included according to the inclusion and exclusion criteria, including 14 cross-sectional studies and 6 cohort studies. all studies have identified various oral bacteria, but only one study has detected viruses and parasites diversity. a variety of oral microbiota found were 6 phyla of bacteria, 6 phyla of viruses, 7 phyla of fungi, and 7 phyla of parasitic. seventeen studies proved that oral microbiota compositions were statistically significant differences compared to healthy controls, but not in 3 studies. conclusion: the majority of studies showed various oral bacteria in oral cancer patients which were statistically significant difference compared to healthy controls. this study indicates the need for more research to evaluate viruses and parasites composition and diversity in oral cancer patients. moreover, future research should focus to clarify whether the changes of oral microbial composition as a community may play a critical role in the etiopathogenesis of oral cancer. keywords: microbiota; cancer; oral cavity correspondence: irna sufiawati, department of oral medicine, faculty of dentistry, universitas padjadjaran, jl. sekeloa selatan i no. 1, bandung, 40132, indonesia. email: irna.sufiawati@fkg.unpad.ac.id introduction global cancer statistics (globocan) data in 2020 showed that there were 19.3 million new cases of cancer and 10 million deaths from 36 types of cancer in 185 countries in the world. among them, cancer of the lip and oral cavity (oral cancer) is the 11th most common malignancy in various regions of asia. the number of new cases of oral cancer (icd-10 codes c00-c06) was 377,713 (2%) and the number of deaths was 177,757 (1.8%). the incidence and mortality rate of oral cancer was higher among men compared to women.1–3 oral cancer is a persistent health issue. detecting and diagnosis in early stage of oral cancer are essential for patient survival. however, due to diagnostic delay (including the patient, professional and system delay), diagnosis of this type of cancer at late stage causes poor survival of patients with oral cancer.4 numerous risk factors or possible causative agents have been associated with oral cancer. the most common risk factors include tobacco smoking, consumption of alcohol, betel quid chewing, human papillomavirus (hpv) infection, and nutritional factors.5–7 to date, recent studies have shown that the oral microbial community (oral microbiota) may play an important role in the initiation and progression of head and neck cancer, including oral cancer, suggesting that oral microbiota is a new risk factor for cancer development.8–10 the oral microbiota plays an essential role within the maintenance of a normal oral physiological environment. they are living in a symbiotic relationship with one another and the host immune system.11,12 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 mailto:irna.sufiawati@fkg.unpad.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 187 sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 however, direct effect of metabolites reaction of chemical carcinogens and inflammatory process caused by oral microbiota may play an important role in cancer patients. the oral microbiota and their metabolites have both direct and indirect effects on the dna damage causing cell mutations which lead to carcinogenesis. the oral microbial composition changes also result in chronic inflammation that can increase risk of oral cancer. in a chronic inflammatory process, the immune system’s host defense activates host cells to produce proinflammatory cytokines, growth factors, chemokines. generating free radicals that can damage dna, causing cell mutations that lead to the initiation of oral cancer. the generation of reactive oxygen and nitrogen species (rons) by proinflammatory cytokines during the inflammation process causes cell dna damage causing cell mutations that lead to the initiation of cancer, and may also contribute in angiogenesis and metastasis. cell proliferation stimulation and apoptosis resistance can be influenced by growth factors. chemokines can promote cell migration and invasion by inducing the proliferation of cancer cells and preventing their apoptosis.13–16 a l t h o u g h c o m p e l l i n g e v i d e n c e s h o w e d t h a t carcinogenesis can be modulated by microbiota, specifically an association between microbiota in the oral cavity with oral cancer, its composition and diversity in oral cancer patients remain unclear. a big question remains: what are the oral microbiota composition and diversity found in oral cancer patients that may influence an individual’s oral cancer risk? are there any differences with healthy individuals? the aim of this present review is to identify and summarize the existing evidence regarding the oral microbiota composition and diversity colonized in patients with oral cancer and its comparison with healthy individuals, which may help in the understanding the role of a community microorganism in the oral cavity in the etiopathogenesis of oral cancer. methods the method of this review complies with the preferred reporting items guidelines for methods of systematic review and meta-analysis (prisma).17 according to the picos schema to construct the literature search, the following criteria were used: (p=patients) patients diagnosed with oral cancer, (i=intervention) examination of oral microbiota composition without restriction of the type of microbiological technique used, (c=comparison) healthy control, (o=outcome) to summarize the oral microbiota composition. four electronic databases were searched through pubmed, scopus, web of science, and sciencedirect from january 2015 to march 2022 for the studies containing data related to “oral microbiota,” “phyla“ and “oral cancer.” the inclusion criteria in the present review were no age and gender restrictions, clinical trials and studies in humans. exclusion criteria are articles are not written in english and not indexed with scopus. the relevant articles were as many as 15484 articles. the amount of data after eliminating duplication in the database was 1412 articles, with screened records that fulltext articles assed for eligibility were 102 articles. articles were excluded because of non-oral cancer (n = 23), nonphyla of microorganism classification (n = 45), articles not published in english (n = 9) and animal studies (n=5). the final number of full text articles that were checked for eligibility was 20 articles (figure 1). records identified through pubmed (n = 1078) scopus (n = 366), web of science (n = 426) science direct (n = 13614) total database searching (n = 15484) records after duplicates and abstract only removed (n = 1412) records excluded: non-oral microbiota phyla (n = 45) non-oral cancer (n = 23) non-english language (n = 9) animal studies (n = 5) records screened full-text articles assessed for eligibility (n = 102) records inclusion: clinical trial human studies id en tif ic at io n sc re en in g e lig ib ili ty studies included in qualitative synthesis (n = 20) in cl ud ed figure 1. prisma flow diagram showing the process of study search, selection, inclusion, and exclusion. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 188sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 results this systematic review obtained 19 studies examining the oral microbiota in oral cancer. the studies were conducted in 10 asian countries,8,18–25 two studies in australia,14,26 and six studies in united states of america (usa),9,27–29 and two studies in europe.30,31 the types of studies used were cohort (n=6) and cross-sectional (n=14). from all the studies, a total of 947 oral cancer and 761 healthy controls samples were obtained. the results of this review showed that oral cancer patients have a diversity of microbiota in the oral cavity. various species of oral microbiota in oral cancer patients that have been identified consist of bacteria, fungi, viruses, and parasites. six bacterial phyla were identified in oral cancer patients, mostly firmicutes, bacteroidetes, proteobacteria and the rest were actinobacteria, fusobacteria, spirochaetes and tenericutes. six phyla of viruses found in a study, they were reoviridae, herpesviridae, poxviridae, orthomyxoviridae, retroviridae and polyomaviridae. seven phyla of fungi that were identified include fonsecaea, malassezia, pleistophora, rhodotorula, cladophialophora, cladosporium, and glomeromycotan. a study found seven phyla of parasitic hymenolepis, centrocestus, dipylidium, prosthodendrium, trichinella, contracaecum and toxocara. the majority of studies (17 studies) proved that oral microbiota composition were statistically significant differences compare to healthy control. however, three studies found no statistically significant differences. a summary of the oral microbiota composition and diversity identified in oral cancer patients compared to healthy controls can be seen in table 1. table 1. summary of oral microbiota identified in oral cancer patients. authors country sample type of study methods oral microbiota in oral cancer patients oral microbiota in healthy controls results zixuan li et al. (2021) china 10 oral squamous cell carcinoma (oscc), 10 healthy controls cross sectional 16s rrna gene sequencing bacteria: bacteroidetes 24.02% firmicutes 19.47% actinobacteria 2.60% proteobacteria 2.06% bacteria: bacteroidetes 9.69% firmicutes 39.82% actinobacteria 6.98% proteobacteria 4.56% statistically significant difference (p < 0.05) hezi li et al. (2021) china 33 oscc, 35 healthy controls cross sectional 16s rrna gene sequencing bacteria: firmicutes 34.0% bacteroidetes 25.3% proteobacteria 17.0% fusobacteria 10.9% bacteria: firmicutes 31.1% bacteroidetes 24.9% proteobacteria 20.1% fusobacteria 10.3% statistically significant difference (p < 0:05) ling zhang et al. (2020) china 50 oscc, 50 healthy controls cross sectional 16s rrna gene sequencing bacteria: firmicutes 12.13% bacteroidetes 17.85% proteobacteria, 12.57% fusobacteria 11.03% actinobacteria 1.32% bacteria: firmicutes 25.54% bacteroidetes 11.99% proteobacteria 12.55% fusobacteria 3.29% actinobacteria 3.73% statistically significant difference (p < 0.05) madhusmita panda et al. (2020) india 8 oropharyngeal (op) and hypopharyngeal squamous cell carcinoma patients (hp), 10 healthy controls cross sectional 16s rrna gene sequencing bacteria: actinobacteria bacteroidetes proteobacteria firmicutes spirochaetes bacteria: actinobacteria bacteroidetes proteobacteria firmicutes spirochaetes statistically significant difference (p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 189 sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 yasuharu takahashi et al. (2019) japan 60 oscc, 80 healthy controls cross sectional 16s rrna gene sequencing bacteria: bacteroidetes 0.87% firmicutes 1.31% proteobacteria 1.54% bacteria: bacteroidetes 0.81% firmicutes 0.98% proteobacteria 1.40% statistically significant difference (p < 0.05) jenn-ren hsiao et al. (2018) taiwan 138 oscc, 151 healthy controls cross sectional 16s rrna gene sequencing bacteria: bacteroides 1.20% fusobacterium 1.05% bacteria: bacteroides 0.49% fusobacterium 0.57% statistically significant difference (p < 0.05) chia-yu yang et al. (2018) taiwan 197 oscc, 51 healthy controls cross sectional 16s rrna gene sequencing bacteria: firmicutes 59.76% proteobacteria 35.57% actinobacteria 11.56% bacteroidetes 8.39% fusobacteria 7.92% bacteria: firmicutes 58.40% proteobacteria 23,22% actinobacteria 8.36% bacteroidetes 5.65% fusobacteria 2.98% statistically significant difference (p < 0.0001) susan yost et al. (2018) usa 4 oscc, 4 healthy controls cohort 16s rrna gene sequencing bacteria: fusobacteria firmicutes bacteroidetes bacteria: firmicutes proteobacteria statistically significant difference (p < 0.05) yenkai lim et al. (2018) australia 11 occ and opc, 10 healthy controls cohort 16s rrna gene sequencing bacteria: firmicutes proteobacteria actinobacteria bacteroidetes fusobacteria bacteria: firmicutes proteobacteria actinobacteria bacteroidetes fusobacteria statistically significant difference (p < 0.01) m. perera et al. (2018) australia 25 oscc, 27 healthy controls cohort 16s rrna gene sequencing bacteria: firmicutes 37% proteobacteria 20% bacteroidetes 15% fusobacteria 17% actinobacteria 11% bacteria: firmicutes 41% proteobacteria 19% bacteroidetes 9% fusobacteria 13% actinobacteria 11% not statistically significant difference (p > 0.05) zhao h et al. (2017) china 80 oscc, 80 healthy controls cross sectional 16s rrna gene sequencing bacteria: bacteroidetes 37.6% proteobacteria firmicutes fusobacteria actinobacteria bacteria: bacteroidetes proteobacteria firmicutes fusobacteria actinobacteria statistically significant difference (p < 0.05) nezar noor al-hebshi et al, (2017) saudi arabia 20 oscc, 20 healthy controls cross sectional 16s rrna gene sequencing bacteria: fusobacteria 30% proteobacteria 28% bacteroidetes 19% firmicutes 17% actinobacteria 6% bacteria: fusobacteria 20% proteobacteria 19% bacteroidetes 21% firmicutes 24% actinobacteria 16% statistically significant difference (p < 0.01) wei-hsiang lee et al. (2017) taiwan 125 oscc, 127 healthy controls cross sectional 16s ribosomal dna (rdna) sequencing bacteria: firmicutes bacteroidetes, proteobacteria, actinobacteria, fusobacteria. bacteria: firmicutes, bacteroidetes, proteobacteria, actinobacteria, fusobacteria. statistically significant difference (p < 0.01) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 190sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 axel wolf et al. (2017) austria 11 oscc, 11 healthy controls cross sectional 16s rrna gene sequencing bacteria: firmicutes 48% bacteroidetes 17% actinobacteria 15% proteobacteria 12% fusobacteria 5% bacteria: firmicutes bacteroidetes actinobacteria proteobacteria fusobacteria statistically significant difference (p < 0.05) abdrazak amer et al. (2017) ireland 36 oscc, 36 healthy controls cross sectional 16s rrna gene sequencing bacteria: bacteroidetes proteobacteria fusobacteria firmicutes bacteria: bacteroidetes proteobacteria fusobacteria firmicutes statistically significant difference (p < 0.007). sagarika banerjee et al. (2017) usa 100 oscc, 20 healthy controls cross sectional 16s rrna gene sequencing bacteria: proteobacteria 41% firmicutes 35% actinobacteria 31% bacteroides 27% virus: reoviridae, herpesviridae, poxviridae, orthomyxoviridae, retroviridae and polyomaviridae. fungi: fonsecaea, malassezia, pleistophora, rhodotorula, cladophialophora and cladosporium. parasites: hymenolepis, centrocestus, dipylidium, prosthodendrium, trichinella, contracaecum and toxocara bacteria: proteobacteria 25% firmicutes 24% actinobacteria 36% bacteroides 5% virus: reoviridae, herpesviridae, poxviridae, orthomyxoviridae, retroviridae and polyomaviridae. fungi: fonsecaea, malassezia, pleistophora, rhodotorula, cladophialophora and cladosporium. parasites: hymenolepis, centrocestus, dipylidium, prosthodendrium, trichinella, contracaecum and toxocara statistically significant difference (p < 0.05) pranab k. mukherjee et al. (2017) usa 39 oscc, 39 healthy controls cross sectional 16s rrna gene sequencing bacteria: firmicutes 48% actinobacteria 20% fungi: glomeromycota 2.2% bacteria: firmicutes 40% actinobacteria 11%, fungi: glomeromycota 2.7%, not statistically significant difference (p > 0.05) daniela börnigen et al. (2017) usa 121 oral cancer, 242 healthy controls cohort 16s rrna gene sequencing bacteria: firmicutes actinobacteria bacteroidetes bacteria: firmicutes actinobacteria bacteroidetes statistically significant difference (p < 0.01) rafael guerreropreston et al. (2017) usa 17 hnscc, 25 healthy controls cohort 16s rrna gene sequencing bacteria: firmicutes, proteobacteria, bacteroidetes, fusobacteria, actinobacteria bacteria: firmicutes, proteobacteria, bacteroidetes, fusobacteria, actinobacteria statistically significant difference (p < 0.05) rafael guerreropreston et al. (2016) usa 25 oscc, 25 healthy controls cohort 16s rrna gene sequencing bacteria: firmicutes 67% bacteroidetes 13.4% proteobacteria 10.24% bacteria: firmicutes 47.1% bacteroidetes 21.2% proteobacteria 22.7% statistically significant difference (p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 191 sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 discussion the oral cavity host complex communities of microbial, called oral microbiota, are comprised of a wide variety of bacteria, fungi, viruses, archaea, and protozoa.11,32 the oral microbiota has a symbiotic relationship with the host. the oral microbiota contributes to critical metabolic, physiological, and immunological functions.33 disruption in the symbiotic microbiota compositions (dysbiosis) can have significant consequences on the development of oral health and disease.34–36 oral dysbiosis, a shift in the oral microbiota composition, can be predisposed by many factors. poor oral hygiene is a major factor cause of dysbiosis of oral microbiota. poor oral hygiene is frequently seen in oral cancer patients and potentially oral dysbiosis leads to the emergence of potential pathogens that can promote the progression of oral cancers. other factors such as inflammation of gingival/periodontal, genetic variation, salivary dysfunction, dietary habits, and smoking can also lead to oral dysbiosis.35–37 this review identified the various oral microbiota among oral cancer patients. the three major phyla (firmicutes, bacteroidetes, and proteobacteria) were the most prevalent bacterial found in oral cancer patients. the high levels of bacteria have been known to be related to infection and inflammation in the oral cavity. accordingly, the mechanisms of how bacteria may contribute in carcinogenesis of oral cancer are by chronic inflammation induction and carcinogenic bacterial metabolites production.38 a prior study reported that the microecological composition of bacteria in saliva was different from the tumor site. firmicutes were predominant in saliva of oral cancer patients followed by bacteroidetes, while proteobacteria were found more in tumor site followed by bacteroidetes, firmicutes, and others such as fusobacteria and actinobacteria. based on the location of oscc, both phyla bacteroidetes and fusobacteria were more found at the site of the tongue, while firmicutes was detected more in gingiva and proteobacteria in oropharynges. the bacteria composition was also different in the various stages of oscc. bacteroidetes and fusobacteria were more found in the early stage, while firmicutes and proteobacteria were more identified in the late stage.39 in addition to bacteria, fungi were also detected in two studies with different results. it is well known that fungi are a small part of the oral microbiota; however, these opportunistic pathogens have been linked to carcinogenesis.40,41 candida albicans (c. albicans) is well-known as the predominant species in the oral cavity and involved in oral cancer development. the mechanisms by which c. albicans may initiate or promote oral carcinogenesis are still not well-established. however, it has been suggested that it can occur through several mechanisms. first, it was proposed that c. albicans produce carcinogenic substances such as acetaldehyde that promote dna damage-induced apoptosis and contribute to carcinogenesis. the production of endogenous nitrosamines through hyphal invasion may also induce c. albicans in the development of oral cancer. second, candidalysin secreted during the infection by candida is able to cause epithelial damage and immune activation via mitogen-activated protein kinase (mapk) signaling pathways. third, c. albicans infection could enhance the production of several inflammatory cytokines. these immune responses may affect metabolic pathways and promote oral cancer development. fourth, reducing the antimicrobial peptide β-defensin caused by chronic cigarette smoking and heavy alcohol consumption may also be associated with the development of oral cancer. human β-defensin is a broad spectrum of antimicrobial activity that also has an important component of innate host defense against microbial colonization. fifth, the influence of candida infection on the tumor suppressor gene p53, cell proliferation nuclear antigen ki-67, and (cox-2) expression that is related to malignant transformation through the upregulation of inflammatory and epithelial proliferatition.40,42–44 these possible mechanisms may provide our understanding of the oral cancer development associated with fungal dysbiosis. the viruses and parasites in the oral cavity among oral cancer patients were found only in a study conducted by banerjee et al. (2017). several viruses have been strongly associated with oral cancer, particularly papilloma virus (hpv), and other viruses include herpes simplex virus (hsv), epstein-barr virus (ebv), and hepatitis c virus (hcv). the possible mechanisms by which viruses are responsible for the development of oral cancer are through both direct mechanism (genomic instability, increased cell proliferation, altered cell and tissue differentiation, apoptosis resistance, accumulation of dna damage and defect), and indirect mechanism (through immunosuppression, chronic inflammation, or chronic antigenic stimulation).45–47 unlike bacteria and viruses, there are few studies showing an association between parasites and oral cancer. similar mechanisms has been suggested to underlie parasite infections in modulating carcinogenesis, including the modulation of immune system, genomic instability and mutation, insufficient proliferation, chronic inflammation, stimulation of angiogenesis, deregulation of apoptosis, and activation of cancer invasion and metastasis.48 however, future studies are needed to confirm the association between parasites and oral cancer. the different results obtained on the oral microbiota composition and diversity may be related to different types of samples used and different sample collection methods. however, the present review showed that the majority of studies proved that the oral microbiota in cancer patients were statistically significant difference compared to healthy controls. the findings suggest that the oral microbiota may undergo distortions that lead to imbalance and promote the development and progression of cancer. the effect of oral dysbiosis in the etiopathogenesis of oral cancers has not been completely elucidated.49 recently, four possible dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 192sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 mechanisms of oral microbiota dysbiosis have been found to contribute to the etiopathogenesis of oral cancer as proposed by la rosa et al. (2020), including (1) stimulate chronic inflammatory responses; (2) genetic damage in oral mucosa epithelial cells induced by oxygen and nitrogen reactive species, also oncogenic metabolites produced by bacteria; (3) changes in the integrity of epithelial barrier that can lead to irreversible damage of genomic dna; and (4) epigenetic alterations.50 it has also been elucidated another mechanism in the process of carcinogenesis called a tightly interdependent triangle; they are dysbiosis of microbiota, dysfunction of epithelial barrier, and dysregulation of immune responses/inflammation.51 this study has a limitation. assessment of the quality of studies is beyond scoping review parameters; therefore, we couldn’t assess the quality of each study included in this review. however, the present review provides a comprehensive map of the studies on the oral microbiota composition and diversity. the significant differences in the composition of the oral microbiota between oral cancer patients and healthy individuals may emerge the potential of oral microbiota as potential biomarkers associated with the progression of the cancer and therapeutic target in the management of the disease. identification of the oral microbiota allows the diagnosis of oral cancer to be possible and even allows early treatment intervention to occur before the onset of oral cancer. in conclusion, the majority of studies in this review showed various oral bacteria in oral cancer patients (dominated by firmicutes, bacteroidetes, proteobacteria, and fusobacteria), and statistical analysis revealed significant differences compared to healthy controls. this study indicates the need for more research to evaluate viruses and parasites composition and diversity in oral cancer patients. moreover, future research should focus to determine whether the changes of oral microbial composition as a community may contribute to oral cancer development and progression. references 1. sung h, ferlay j, siegel rl, laversanne m, soerjomataram i, jemal a, bray f. global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2021; 71(3): 209–49. 2. international agency for research on cancer. world health organization. global cancer observatory. 2021. available from: https://gco.iarc.fr/. accessed 2021 jun 6. 3. ferlay j, colombet m, soerjomataram i, parkin dm, piñeros m, znaor a, bray f. cancer statistics for the year 2020: an overview. int j cancer. 2021; 149(4): 778–89. 4. rakhmania h, sufiawati i. impact of delay on diagnosis and treatment of oral squamous cell carcinoma: three cases report. j int dent med res. 2017; 10(3): 1017–20. 5. khan z, tönnies j, müller s. smokeless tobacco and oral cancer in south asia: a systematic review with meta-analysis. liotta la, editor. j cancer epidemiol. 2014; 2014: 394696. 6. smith em, rubenstein lm, haugen th, pawlita m, turek lp. complex etiology underlies risk and survival in head and neck cancer human papillomavirus, tobacco, and alcohol: a case for multifactor disease. longatto-filho a, editor. j oncol. 2012; 2012: 1–9. 7. sufiawati i, putra ingj, herawati dmd, indrati ar. low serum 25-hydroxyvitamin d levels in oral cancer patients. j int dent med res. 2021; 14(1): 216–20. 8. zhao h, chu m, huang z, yang x, ran s, hu b, zhang c, liang j. variations in oral microbiota associated with oral cancer. sci rep. 2017; 7(1): 11773. 9. banerjee s, tian t, wei z, peck kn, shih n, chalian aa, o’malley bw, weinstein gs, feldman md, alwine j, robertson es. microbial signatures associated with oropharyngeal and oral squamous cell carcinomas. sci rep. 2017; 7(1): 4036. 10. mok sf, karuthan c, cheah yk, ngeow wc, rosnah z, yap sf, ong hka. the oral microbiome community variations associated with normal, potentially malignant disorders and malignant lesions of the oral cavity. malays j pathol. 2017; 39(1): 1–15. 11. radaic a, kapila yl. the oralome and its dysbiosis: new insights into oral microbiome-host interactions. comput struct biotechnol j. 2021; 19: 1335–60. 12. sampaio-maia b, caldas im, pereira ml, pérez-mongiovi d, araujo r. the oral microbiome in health and its implication in oral and systemic diseases. in: sariaslani s, michael gadd gbt-a in am, editors. advances in applied microbiology. academic press; 2016. p. 171–210. 13. ma s. research progress on the relationship between oral microbial community and tumor. infect int. 2016; 5(1): 16–22. 14. lim y, fukuma n, totsika m, kenny l, morrison m, punyadeera c. the performance of an oral microbiome biomarker panel in predicting oral cavity and oropharyngeal cancers. front cell infect microbiol. 2018; 8(aug): 267. 15. landskron g, de la fuente m, thuwajit p, thuwajit c, hermoso ma. chronic inflammation and cytokines in the tumor microenvironment. j immunol res. 2014/05/13. 2014; 2014: 1–19. 16. yang g, lei c, xue-dong z. research progress on the relationship between oral microbial community and tumor. hua xi kou qiang yi xue za zhi. 2014; 32(5): 527–31. 17. page mj, mckenzie je, bossuyt pm, boutron i, hoffmann tc, mulrow cd, shamseer l, tetzlaff jm, akl ea, brennan se, chou r, glanville j, grimshaw jm, hróbjartsson a, lalu mm, li t, loder ew, mayo-wilson e, mcdonald s, mcguinness la, stewart la, thomas j, tricco ac, welch va, whiting p, moher d. the prisma 2020 statement: an updated guideline for reporting systematic reviews. bmj. 2021; 372: n71. 18. li h, luo z, zhang h, huang n, li d, luo c, wang t. characteristics of oral microbiota in patients with esophageal cancer in china. biomed res int. 2021; 2021: 2259093. 19. zhang l, liu y, zheng hj, zhang cp. the oral microbiota may have influence on oral cancer. front cell infect microbiol. 2019; 9(january): 476. 20. panda m, rai ak, rahman t, das a, das r, sarma a, kataki ac, chattopadhyay i. alterations of salivary microbial community associated with oropharyngeal and hypopharyngeal squamous cell carcinoma patients. arch microbiol. 2020; 202(4): 785–805. 21. yang s-f, huang h-d, fan w-l, jong y-j, chen m-k, huang c-n, chuang c-y, kuo y-l, chung w-h, su s-c. compositional and functional variations of oral microbiota associated with the mutational changes in oral cancer. oral oncol. 2018; 77(200): 1–8. 22. hsiao j-r, chang c-c, lee w-t, huang c-c, ou c-y, tsai s-t, chen k-c, huang j-s, wong t-y, lai y-h, wu y-h, hsueh w-t, wu s-y, yen c-j, chang j-y, lin c-l, weng y-l, yang h-c, chen y-s, chang js. the interplay between oral microbiome, lifestyle factors and genetic polymorphisms in the risk of oral squamous cell carcinoma. carcinogenesis. 2018; 39(6): 778–87. 23. yang c-y, yeh y-m, yu h-y, chin c-y, hsu c-w, liu h, huang p-j, hu s-n, liao c-t, chang k-p, chang y-l. oral microbiota community dynamics associated with oral squamous cell carcinoma staging. front microbiol. 2018; 9: 862. 24. al-hebshi nn, borgnakke ws, johnson nw. the microbiome of oral squamous cell carcinomas: a functional perspective. curr oral heal reports. 2019; 6(2): 145–60. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://gco.iarc.fr/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 193 sufiawati et al./dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 186–193 25. lee w-h, chen h-m, yang s-f, liang c, peng c-y, lin f-m, tsai l-l, wu b-c, hsin c-h, chuang c-y, yang t, yang t-l, ho s-y, chen w-l, ueng k-c, huang h-d, huang c-n, jong y-j. bacterial alterations in salivary microbiota and their association in oral cancer. sci rep. 2017; 7(1): 16540. 26. perera m, al-hebshi nn, perera i, ipe d, ulett gc, speicher dj, chen t, johnson nw. inflammatory bacteriome and oral squamous cell carcinoma. j dent res. 2018; 97(6): 725–32. 27. mukherjee pk, wang h, retuerto m, zhang h, burkey b, ghannoum ma, eng c. bacteriome and mycobiome associations in oral tongue cancer. oncotarget. 2017; 8(57): 97273–89. 28. yost s, stashenko p, choi y, kukuruzinska m, genco ca, salama a, weinberg eo, kramer cd, frias-lopez j. increased virulence of the oral microbiome in oral squamous cell carcinoma revealed by metatranscriptome analyses. int j oral sci. 2018; 10(4): 32. 29. börnigen d, ren b, pickard r, li j, ozer e, hartmann em, xiao w, tickle t, rider j, gevers d, franzosa ea, davey me, gillison ml, huttenhower c. alterations in oral bacterial communities are associated with risk factors for oral and oropharyngeal cancer. sci rep. 2017; 7(1): 17686. 30. guerrero-preston r, white jr, godoy-vitorino f, rodríguez-hilario a, navarro k, gonzález h, michailidi c, jedlicka a, canapp s, bondy j, dziedzic a, mora-lagos b, rivera-alvarez g, ili-gangas c, brebi-mieville p, westra w, koch w, kang h, marchionni l, kim y, sidransky d. high-resolution microbiome profiling uncovers fusobacterium nucleatum , lactobacillus gasseri/johnsonii , and lactobacillus vaginalis associated to oral and oropharyngeal cancer in saliva from hpv positive and hpv negative patients treated with surgery an. oncotarget. 2017; 8(67): 110931–48. 31. guerrero-preston r, godoy-vitorino f, jedlicka a, rodríguezhilario a, gonzález h, bondy j, lawson f, folawiyo o, michailidi c, dziedzic a, thangavel r, hadar t, noordhuis mg, westra w, koch w, sidransky d. 16s rrna amplicon sequencing identifies microbiota associated with oral cancer, human papilloma virus in fection a nd su rgica l t reatment. oncot a rget. 2016; 7(32): 51320–34. 32. baker jl, bor b, agnello m, shi w, he x. ecology of the oral microbiome: beyond bacteria. trends microbiol. 2017; 25(5): 362–74. 33. kilian m, chapple ilc, hannig m, marsh pd, meuric v, pedersen aml, tonetti ms, wade wg, zaura e. the oral microbiome – an update for oral healthcare professionals. br dent j. 2016; 221(10): 657–66. 34. cho i, blaser mj. the human microbiome: at the interface of health and disease. nat rev genet. 2012; 13(4): 260–70. 35. sudhakara p, gupta a, bhardwaj a, wilson a. oral dysbiotic communities and their implications in systemic diseases. dent j. 2018; 6(2): 10. 36. gupta p. microbial communities in healthy oral mucosa. int j adv res. 2019; 7(8): 613–22. 37. mathur r, singhavi hr, malik a, nair s, chaturvedi p. role of poor oral hygiene in causation of oral cancer—a review of literature. indian j surg oncol. 2018/12/07. 2019; 10(1): 184–95. 38. parsonnet j. bacterial infection as a cause of cancer. environ health perspect. 1995; 103(suppl 8): 263–8. 39. zhang z, yang j, feng q, chen b, li m, liang c, li m, li z, xu q, zhang l, chen w. compositional and functional analysis of the microbiome in tissue and saliva of oral squamous cell carcinoma. front microbiol. 2019; 10: 1439. 40. sultan as, theofilou vi, alfaifi a, montelongo-jauregui d, jabra-rizk m-a. is candida albicans an opportunistic oncogenic pathogen? chowdhary a, editor. plos pathog. 2022; 18(4): e1010413. 41. huët mal, lee cz, rahman s. a review on association of fungi with the development and progression of carcinogenesis in the human body. curr res microb sci. 2022; 3: 100090. 42. di cosola m, cazzolla ap, charitos ia, ballini a, inchingolo f, santacroce l. candida albicans and oral carcinogenesis. a brief review. j fungi (basel, switzerland). 2021; 7(6): 476. 43. ghosh sk, mccormick ts, weinberg a. human beta defensins and cancer: contradictions and common ground. front oncol. 2019; 9: 341. 44. shukla k, vun i, lov i, laparidis g, mccamley c, ariyawardana a. role of candida infection in the malignant transformation of oral leukoplakia: a systematic review of observational studies. transl res oral oncol. 2019; 4: 2057178x1982822. 45. gupta k, metgud r. evidences suggesting involvement of viruses in oral squamous cell carcinoma. tosi p, editor. patholog res int. 2013; 2013: 1–17. 46. metgud r, astekar m, verma m, sharma a. role of viruses in oral squamous cell carcinoma. oncol rev. 2012; 6(2): 21. 47. sand l, jalouli j. viruses and oral cancer. is there a link? microbes infect. 2014; 16(5): 371–8. 48. machicado c, marcos la. carcinogenesis associated with parasites other than schistosoma, opisthorchis and clonorchis: a systematic review. int j cancer. 2016; 138(12): 2915–21. 49. lim y, totsika m, morrison m, punyadeera c. oral microbiome: a new biomarker reservoir for oral and oropharyngeal cancers. theranostics. 2017; 7(17): 4313–21. 50. la rosa g, gattuso g, pedullà e, rapisarda e, nicolosi d, salmeri m. association of oral dysbiosis with oral cancer development. oncol lett. 2020/03/03. 2020; 19(4): 3045–58. 51. pang x, tang y, ren x-h, chen q-m, tang y-l, liang x-h. microbiota, epithelium, inflammation, and tgf-β signaling: an intricate interaction in oncogenesis. front microbiol. 2018; 9: 1353. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p186–193 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p186-193 57 research report effects of silkworm fiber position on flexural and compressive properties of silk fiber-reinforced composites ariyani faizah, dendy murdiyanto, yulita nur widyawati and narawidya laksmi dewi department of biomaterials faculty of dentistry, universitas muhammadiyah surakarta surakarta – indonesia abstract background: fiber-reinforced composites represent a combination of fiber-reinforced composite materials. the availability of fiber within dentistry in indonesia is limited and, therefore, requires lengthy advance ordering. the increasing use of fiber derived from natural materials, such as silk, is of greater concern due to its considerable mechanical strength, biocompatibility and wider availability. the application of fiber will increase the mechanical strength of fiber-reinforced composites, including both flexural and compression strength. one factor affecting the mechanical strength of fiber is the laying of fiber or fiber position. purpose: the purpose of this research is to establish the influence of silkworm fiber position on both the flexural and compression strength of silk fiber-reinforced composites. methods: flexural strength and compression strength tests using a universal testing machine involved the division of the research population into three treatment groups: compression side, neutral side and tension side. results: the results of data analysis indicated that the tension side group possessed the highest flexural strength (121.42 mpa), while the compression side group demonstrated the highest compression strength (337.65 mpa). a one-way anova analysis test produced a significant result of p = 0.000 (<0.05) both for silkworm fiber position effect and compression strength of silk fiber reinforced composites. conclusion: the position of silkworm fiber will affect its flexural strength as well as that of the compression of silk fiber-reinforced composites. keywords: silk worm fiber; flexural strength; compression strength; fiber position; silk fiber reinforced composite correspondence: ariyani faizah, department of biomaterials, faculty of dentistry, universitas muhammadiyah surakarta, jl. kebangkitan nasional no. 101 penumping, laweyan, kota surakarta, jawa tengah 57141, indonesia. e-mail: ariyani.faizah@ums. ac.id introduction tooth loss can inhibit chewing ability as well as reduce an individual’s aesthetic appeal. in cases where large or old teeth are lost, it even reduces performance of the temporomandibular joints (tmj). other problems arising from tooth loss are impaired speech function and the psychological aspect related to aesthetics, especially for certain professions that require an attractive dental profile.1 treatment to replace a missing tooth usually necessitates use of a fixed denture (gtc).2 many materials are used in making gtcs, one of which is fiber-reinforced composite (frc)3 which has several advantages including: corrosionfree metal, non-toxic material, minimal preparation and limited maintenance time.4,5 frc is a combination of fiber-reinforced polymer matrix consisting of a polymer monomer which serves to hold the fibers, maintain the pressure between the fibers and, finally, protect the fibers from the external environment. the use of fiber in frc, thus, has the function of increasing its strength, stiffness and resistance to fracture.6 fibers commonly used in dentistry include: glass fiber, aramid fiber, carbon fiber and polyethylene fiber or ultra-high molecular weight polyethylene fiber (uhmwpe). e glass fiber is most commonly used in dentistry7, having several advantages one of which is attractive aesthetic properties. nevertheless, the availability of e glass fiber in indonesia remains extremely limited with lengthy supply time frames. dental journal (majalah kedokteran gigi) 2018 june; 51(2): 57–61 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p57-61 58faizah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 57–61 in order to overcome limited e glass fiber availability, certain natural fibers have been developed as alternatives to replace dental fiber. one such natural material widely available in indonesia is silk fiber, with the village of regaloh in the pati regency8 being one of the centers of the silk fiber processing industry. in fact, silk fibers have long been developed by the textile industry, their use being due to strong mechanical properties. other advantages they offer comprise: strong environmental stability, biocompatibility and flexible shape.9 therefore, with such mechanical properties, fibers derived from silkworms are expected to be employed as biomaterials for non-dental fibers.10 the use of gtc in the oral cavity will, furthermore, generate a variety of pressures during the mastication process, namely: compressive strength, tensile strength and shear strength. such clinical conditions should be taken into account when making gtc, especially that produced from frc. moreover, frc used in the manufacture of gtc should possess strong mechanical properties with the aim of avoiding marginal degradation and restoration cracking.11 the mechanical properties of the fibers are generally influenced by composite filler resin particles, fiber volume and fiber position.12 many factors influence the mechanical strength of fibers including: their composition, volume, orientation and position. fiber position refers to the laying position of fiber12, differences in which can affect the amount of pressure distributed from the matrix to the fiber. for example, fiber placed on the compression side, neutral side and tension side will distribute pressure across all frc layers.13 therefore, research into fiber position placed in the frc component, needs to be conducted since fiber position is one of the factors that will support the mechanical strength of frc following its application within the mouth. hence, this research aimed to reveal the effects of certain silkworm fiber positions on the flexural and compressive strength of silk fiber-reinforced composites. materials and methods the materials used in this research comprised packable composite resin (filtek z250 xt, 3m espe, usa), silkworm fiber (pati, central java) and silane coupling agent (monobond n, vivadent ivoclar, liechtenstein). preparation was conducted by first storing silkworm fibers in a desiccator for 24 hours in order to minimize their internal water concentration. these fibers were cut with scissors to a length of 25 mm suitable for a flexural strength test and also to a length of 3 mm for a compressive strength test. having been weighed, 3 mg of the cut fibers were subjected to a flexural strength test, while 0.7 mg, equivalent to a layer of dental fiber, underwent a compressive strength test. finally, the fibers used as samples for the flexural strength test were molded into 2 mm  2 mm  25 mm beams, while those used for the compressive strength test were formed into a cylinder 3 mm in height and 6 mm in diameter.14 the molds used for this purpose were constructed from metal. in the next stage, the samples were divided into three groups, namely: group i (compression side), group ii (neutral side) and group iii (tension side), each consisting of nine samples. thus, the research required a total of 27 specimens for each test. in group i with the compression side position, up to three quarters of each mold was filled with the composite, i.e. about 1.5 mm from the top of the mold for the flexural strength samples and 4 mm for the compressive strength samples. the height of the composite filling used was marked using a small ruler, specifically designed to indicate the one-third, half-way and threequarter points of the total height of the mold. each of the silkworm fibers was subsequently placed on a glass plate, with one drop of silane coupling agent being applied via micropipette. the fibers were allowed to stand for one minute and then dried for another minute with an electric fan. the silanized fiber was then placed with tweezers on the top of the composite. the composite resin was injected again over the fibers until they were fully immersed and flattened with a plastic implement. polymerization was then carried out using a light curing unit. all samples were divided into four parts to enable each to undergo perfect polymerization due to its ability to adapt to the size of the light cure unit tip. each part of the frc was irradiated for 40 seconds, while the unlit part was covered with aluminum foil until all parts were irradiated. the sample was removed from the mold with any excess polymer being removed by disk polishing before the position of the fiber was indicated by means of a marker. the same procedure was conducted with both the tension side and neutral side groups, but the fibers were placed in one-third of each mold with the laying position on the tension side group. meanwhile, the fibers were placed in the middle of each mold on the neutral side. prior to testing, each of the prepared samples was soaked in 20 ml of distilled water contained in a conical tube which was placed in an incubator at 37° c for 24 hours after which time the sample was removed and dried with absorbent paper. then, flexural strength and compression strength tests were conducted by means of a universal testing machine at the materials laboratory, mechanical engineering faculty, universitas gajah mada (ugm). both the flexure strength test and three-point bending test were performed using a universal testing machine. the samples were placed on a support board at 20 mm intervals (1). each sample was then subjected to 1 mm/min loading (p) at its center until a fracture occurred. thereafter, a generated value (p) emerged on screen as the maximum load acceptable by the sample. the magnitude of flexural strength was then expressed in mpa with the data being dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p57-61 59 faizah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 57–61 analyzed using the following formula: 4 again over the fibers until they were fully immersed and flattened with a plastic implement. polymerization was then carried out using a light curing unit. all samples were divided into four parts to enable each to undergo perfect polymerization due to its ability to adapt to the size of the light cure unit tip. each part of the frc was irradiated for 40 seconds, while the unlit part was covered with aluminum foil until all parts were irradiated. the sample was removed from the mold with any excess polymer being removed by disk polishing before the position of the fiber was indicated by means of a marker. the same procedure was conducted with both the tension side and neutral side groups, but the fibers were placed in one-third of each mold with the laying position on the tension side group. meanwhile, the fibers were placed in the middle of each mold on the neutral side. prior to testing, each of the prepared samples was soaked in 20ml of distilled water contained in a conical tube which was placed in an incubator at 37° c for 24 hours after which time the sample was removed and dried with absorbent paper. then, flexural strength and compression strength tests were conducted by means of a universal testing machine at the materials laboratory, mechanical engineering faculty, universitas gajah mada (ugm). both the flexure strength test and three-point bending test were performed using a universal testing machine. the samples were placed on a support board at 20mm intervals (1). each sample was then subjected to 1mm/min loading (p) at its center until a fracture occurred. thereafter, a generated value (p) emerged on screen as the maximum load acceptable by the sample. the magnitude of flexural strength was then expressed in mpa with the data being analyzed using the following formula: = 22 σ (3p.1) = bd note: : flexural strength (mpa.) p: maximum load that can be supported by the sample before the object fractures (newton). l: distance between support board and pedestal (mm). b: sample width (mm). d: thickness of the tested sample (mm). meanwhile, a universal testing machine was used to measure compressive strength by placing each sample on the metal plate in the center of the machine and activating an engine moving at a speed of 1 mm/min which suppressed the sample until a fracture occured. the value of compression strength (mpa) could then be obtained using the following formula:15 note: s : flexural strength (mpa.) p : maximum load that can be supported by the sample before the object fractures (newton) l : distance between support board and pedestal (mm). b : sample width (mm). d : thickness of the tested sample (mm). meanwhile, a universal testing machine was used to measure compressive strength by placing each sample on the metal plate in the center of the machine and activating an engine moving at a speed of 1 mm/min which suppressed the sample until a fracture occured. the value of compression strength (mpa) could then be obtained using the following formula:15 cs = f / a note: cs : compressive strength (mpa) f : compression load (n) a : sample base area l (mm2) data on the flexural and compressive strengths were analyzed with a one-way anova statistic test. results the results of the flexural strength test on the silk fiber reinforced composites of several silkworm fiber positions can be seen in table 1. its contents indicate that certain increases in the mean flexural strength value of silkworm fiber reinforced composites occurred, namely: 121.42 mpa in the tension side group, 95.25 mpa in the neutral side group and 65.84 mpa in the compression group. the highest flexural strength was found in the tension side group (121.42 ± 1.07 mpa), while the lowest was in group i, the compression side group (65.84 ± 0.86). the data was then subjected to a saphiro-wilk normality test in order to determine whether the data on the flexural strength was normally distributed. the results for each group indicated that all data relating to flexural strength was normally distributed (p>0.05). in the next stage, the data on the flexural strength was subjected to homogeneity analysis using a levene’s test whose results showed that all data on the flexural strength was homogeneous (p>0.05). thereafter, a oneway anova test was conducted to analyze the effects of silkworm fiber positions on the flexural strength of silk fiber reinforced composites. the results of the one-way anova test indicated that the silkworm fiber positions had significant effects on the flexural strength of silkworm fiber reinforced composites (p<0.05). a 95% confidence level lsd test was then carried out to determine the significance of flexural strength differences between the fiber silkworm position groups. based on the contents of table 2 above, there was a significant difference (p<0.05) between the compression side group and both the neutral side and tension side groups. a significant difference also existed between the neutral side group in relation to the compression side and tension side groups. similarly, there was a significant difference between the tension side group and the compression side and neutral side groups (p<0.05). the results of the compression strength test on the fiber silkworm positions can be seen in table 3. table 3 showed the highest mean compressive strength value of silkworm fiber reinforced composites to be found in group i, namely; the compression side group (337.65 ± 1.05 mpa). the data on the compressive strength was then tested further to analyze their normality using a saphiro wilk test. the results of the normality test indicated that the data of all three groups relating to compressive strength were normally distributed (p>0.05). the data on the compressive strength was assessed for homogeneity using a levene’s test the results of which showed that the data on compressive strength was homogeneous with a significance value of 0.455 (p>0.05). a one-way anova test was then conducted to analyze the effects of silkworm fiber positions on the compressive strength of silk fiber reinforced composites. its results indicated that the silkworm fiber positions had significant effects on the compressive strength of silkworm fiber table 1. mean and standard deviation of the flexural strength of the silk fiber reinforced composites (mpa) groups n x̄ sd i 9 65.84 ±0.86 ii 9 95.25 ±1.03 iii 9 121.42 ±1.07 table 2. results of the lsd test on the flexural strength positions compression side neutral side tension side compression side 0.000 0.000 neutral side 0.000 tension side table 3. mean and standard deviation values of the fiber positions on the compressive strength of silkworm fiber reinforced composites (mpa) groups n x̄ sd i 9 337.65 ±1.05 ii 9 275.78 ±0.86 iii 9 259.02 ±0.77 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p57-61 60faizah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 57–61 reinforced composites (p<0.05). the least significant difference test (lsd) with a 95% confidence level was then performed to determine the significance of compressive strength differences between the fiber silkworm position groups, the results of which are illustrated in table 4. based on the lsd test results as depicted in table 4, there were significant differences in the compressive strength of silkworm fiber reinforced composites between the compression side group, the neutral side group and the tension side group. discussion differences in flexural strength are influenced by several factors, namely; fiber volume, position, type and orientation, as well as the impregnation of resin composite. these factors can determine the mechanical strength of fiber-reinforced composites since fiber is a component that acts as a pressure distributor.16 the fiber type used in this study was that produced by silkworms due to the mechanical strength resulting from its fibroin content. since fiber is a potential component in the distribution of pressure, high mechanical strength is also influenced by fiber position. flexural force is a mechanical force constituting a combination of compressive strength, tensile strength and shear strength. increased flexural strength will occur if the fiber is placed on a weak denture area located on the tension side. the tension side position will then induce maximum tensile pressure in the fiber affecting the elongation of denture dimensions.13 thus, the maximum tensile pressure was detected in the tension side group within which the pressure will be distributed evenly within the frc.12 on the other hand, the neutral and compression side groups did not demonstrate high flexural strength value. the placement of fibers on the middle or neutral side alone generated sufficient flexural strength and minimum tensile strength, but maximum shear strength. these observations suggest that the neutral side position exerts limited influence on flexural strength.16 meanwhile, the placement of fibers on the compression side is ineffective since pressure present there is not directly distributed to the frc but, initially, to the composite.17 consequently, the position of the fibers in three-quarters of the mold base received only limited tensile strength with the result that flexural strength decreased. conversely, fibers on the compression side will gain in compressive strength.18 based on the results of the compressive strength test contained in table 3, the compression side group generated the highest compressive strength compared to that of the tension side and neutral side groups. the results of the anova test analysis then indicated that the different fiber positions significantly affected the compressive strength of silk with a significance value of 0.000 (p<0.05). the results of the post hoc test analysis combined with those of an lsd test showed that there were significant differences in the compressive strength of silk fiber reinforced composites between the groups (p<0.05) (table 4). therefore, it can be said that the placement of fibers in a position corresponding to the pressure received by frc can distribute the pressure efficiently and effectively resulting in increased mechanical strength, including compressive strength. it means that the position of the fiber on the compression side will distribute the pressure more appropriately in a manner producing higher compressive strength. in other words, the position of fiber on the compression side is more dominant in increasing the compressive strength of composite resin. the compression side position approaching the surface of the composite will experience a compression load. therefore, the laying of fiber on the compression side will distribute the pressure and increase its resistance to the applied pressure. meanwhile, the neutral side position will experience maximum shear strength and maximum tensile strength which prevents the effective distribution of compression pressure.17 fiber-reinforced composite is now selected as a fixed denture material. fixed dentures or gtc must possess sufficient mechanical strength to withstand mastication loads.13 the mastication loads exerted on gtc include: compressive strength, shear strength and tensile strength. compressive strength is received by the gtc at its top, shear strength at the center and tensile pressure at the bottom. this situation needs to be taken into consideration when frc is employed as a material in the manufacture of gtc in order to produce the necessary mechanical strength to withstand the load experienced during the process of mastication. as a result, fiber serving as a component that can distribute the pressure must be placed in an appropriate position to facilitate the effective and timely distribution of the pressure exerted on the gtc. the research findings showed that in order to achieve high flexural strength, fiber should be positioned on the tension side, while achieving good compressive strength required the placing of fiber on the compression side. finally, it can be concluded that silkworm fiber positioned on the tension side will possess the greatest flexural strength of all silk fiber reinforced composites, while silkworm fiber placed on the compression side will demonstrate the highest compressive strength. this indicates that in order to obtain the desired mechanical strength results in the manufacture of gtc by using frc, fiber placement should be conducted through a combination of tension side and compression side positions. the combination of fiber placement will then allow table 4. results of the lsd analysis of the compressive strength compression side neutral side tension side compression side 0.000 0.000 neutral side 0.000 tension side dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p57-61 61 faizah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 57–61 appropriate pressure distribution according to the pressure placed on the gtc during the mastication process. acknowledgement this research was undertaken with lppm perekom f i n a n c i a l s u p p o r t p r o v i d e d t h r o u g h u n i v e r s i t a s muhammadiyah surakarta. references 1. agtini md. persentase pengguna protesa di indonesia. media penelitian dan pengembangan kesehatan. 2010; 20(2): 50–8. 2. gunadi ha, margo a, burhan lk, suryatenggara f, setiabudi i. ilmu geligi tiruan sebagian lepasan. 2nd ed. jakarta: egc; 2012. p. 22–3. 3. rosyida nf, sunarintyas s, pudyani ps. the effect of silanated and impregnated fiber on the tensile strength of e-glass fiber reinforced composite retainer. dent j (maj ked gigi). 2015; 48(1): 22–5. 4. madhok s, madhok s. evolutionary changes in bridge designs. iosr j dent med sci. 2014; 13(6): 50–6. 5. khetarpal a, talwar s, verma m. single visit rehabilitation with anterior fiberreinforced resin composite bridges : a review. indian j appl res. 2013; 3(2): 287–9. 6. maghrabi aa. reinforcement of fiber-reinforced composites crowns with variant margin designs. pakistan oral dent j. 2010; 30: 264–8. 7. zhang m, matinlinna jp. e-glass fiber reinforced composites in dental applications. silicon. 2012; 4: 73–8. 8. nurjayanti ed. budidaya ulat sutera dan produksi benang sutera melalui sistem kemitraan pada pengusaha sutera alam (psa) regaloh kabupaten pati. mediagro. 2011; 7(2): 1–10. 9. chen f, porter d, vollrath f. structure and physical properties of silkworm cocoons. j r soc interface. 2012; 9(74): 2299–308. 10. chandramohan d, marimuthu k. a review on natural fibers. int j res rev appl sci. 2011; 8(2): 194–206. 11. della bona a, benetti p, borba m, cecchetti d. flexural and diametral tensile strength of composite resins. braz oral res. 2008; 22: 84–9. 12. widyapramana w, widjijono w, sunarintyas s. pengaruh kombinasi posisi fiber terhadap kekuatan fleksural dan ketangguhan retak fiber reinforced composite polyethylene. insisiva dent j. 2013; 2(2): 1–8. 13. septommy c, widjijono w, dharmastiti r. pengaruh posisi dan fraksi volumetrik fiber polyethylene terhadap kekuatan fleksural fiber reinforced composite (the effect of position and volumetric fraction polyethylene fiber on the flexural strength of fiber reinforced composite). dent j (maj ked gigi). 2014; 47(1): 52–6. 14. klymus me, shinkai rsa, mota eg, oshima hms, spohr am, burnett lh. influence of the mechanical properties of composites for indirect dental restorations on pattern failure. stomatol balt dent maxillofac j. 2007; 9(2): 56–60. 15. mosharraf r, givechian p. effect of fiber position and orientation on flexural strength of fiber-reinforced composite. j islam dent assoc iran. 2012; 24(2): 21–7. 16. fonseca rb, favarão in, kasuya avb, abrão m, luz nfm, naves lz. influence of glass fiber wt% and silanization on mechanical flexural strength of reinforced acrylics. j mater sci chem eng. 2014; 2014(2): 11–5. 17. spyrides smm, do prado md, simão ra, bastian fl. effect of plasma and fiber position on flexural properties of a polyethylene fiber-reinforced composite. braz dent j. 2015; 26(5): 490–6. 18. iso 10477:2004. dentistry -polymer-based crown and bridge materials. 2nd ed. switzerland: international organization for standardization; 2004. p. 1–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p57-61 mkgs vol 44 no 1 jan-mar 2011.indd 17 vol. 44. no. 1 march 2011 titanium ceramic restoration: how to improve the binding between titanium and ceramic harry laksono department of prosthodontics faculty of dentistry, airlangga university surabaya-indonesia abstract background: titanium alloys has been used as an alternative to nickel-chromium alloys for metal-ceramic restorations because of its good biocompatibility and mechanical properties. this indicated that it was possible to design coping according to standards established for metal-ceramics. however, titanium is chemically reacting strongly with gaseous elements which causes problems when ceramics are fused to titanium. purpose: to provide information about improving the bonding between titanium and ceramic. review: titanium has two crystal modifications, the close-packed hexagonal (α) structure, up to 880° c, and above this temperature the bodycentered cubic (β) structure. the principal problems is the extensive dissolution of oxygen resulting in thick, oxygen-rich titanium layers called α-case that harms the bonding of ceramic to titanium and the great mismatch in the coefficient of thermal expansion of conventional ultra-low fusing ceramic. methods have been developed for fusing ceramic to titanium like processing methods, the used of ultra-low fusing titanium ceramic, bonding agent, and protocol for ceramic bonding to titanium. conclusion: titanium and titanium alloys, based on their physical and chemical properties suitable for titanium-ceramic restorations, but careful selection of processing methods, ceramic materials, laboratory skill and strict protocol for ceramic bonding to titanium are necessary to improve the bonding between titanium and ceramic. key words: dental ceramic, titanium, bond strength abstrak latar belakang: logam campur titanium telah dipakai sebagai salah satu bahan alternatif untuk logam nikel-krom pada pembuatan restorasi keramik taut logam karena mempunyai biokompatibilitas dan sifat mekanik yang baik. hal ini menunjukkan bahwa logam titanium dapat dipakai untuk pembuatan koping logam berdasarkan standar yang dipakai untuk pembuatan restorasi keramik taut logam. meskipun, secara kimiawi logam titanium bereaksi dengan elemen-elemen gas yang menyebabkan masalah pada perlekatan keramik pada titanium. tujuan: memberikan informasi tentang cara meningkatkan kekuatan perlekatan antara keramik dengan titanium. tinjauan: titanium mempunyai 2 struktur kristal, struktur close-packed hexagonal (α) diatas 880°c dan struktur bodycentered cubic (β) dibawah 883°c. masalah utama adalah pelepasan gas oksigen yang menghasilkan lapisan titanium kaya oksigen yang tebal disebut α-case yang menghalangi perlekatan keramik pada titanium dan koefisien ekspansi panas dari bahan ultra-low fusing ceramic yang berbeda dengan titanium. berbagai cara telah dikembangkan untuk mendapatkan perlekatan keramik pada titanium seperti teknik pembuatan, pemakaian bahan ultra-low fusing titanium ceramic, bahan bonding dan protokol untuk perlekatan bahan keramik pada titanium. kesimpulan: titanium dan logam campur titanium, berdasarkan sifat-sifat mekanik dan kimiawinya dapat dipakai untuk pembuatan restorasi keramik taut logam, tetapi pemilihan teknik pembuatan, bahan keramik, ketrampilan peteknik gigi dan mengikuti protokol untuk mendapatkan perlekatan keramik pada titanium dengan benar diperlukan untuk meningkatkan kekuatan perlekatan antara keramik dan titanium. kata kunci: keramik kedokteran gigi, titanium, kekuatan perlekatan correspondence: harry laksono, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. literature review 18 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 17–24 introduction metal-ceramic restorations have been increasingly used in esthetic crown and fixed prosthesis restorations since the 1960.1 rational selection of an alloy for metalceramic restorations should be based on physical properties, chemical properties, biocompatibility, laboratorium workability, and ceramic compatibility.2 selection of high quality materials and their appropriate manipulation are the most effective means to protect the patient from material-induced injury.3 the fabrication of metal ceramic restoration is a technical sensitive procedure, including fabrication of metal coping, metal surface treatment for increasing bond strength which is considered the most important step for the success of ceramic fused-to-metal in the whole procedure, and application and firing of ceramic onto the metal to complete restoration.4 in the early 1970, titanium has been used in cast dental prostheses because of excellent biocompatibility, light weight, high strength to weight ratio, low modulus of elasticity and excellent corrosion resistance.5–7 titanium is currently considered a key metal for successful implantology, published by branemark et al. recently, there have been increasing efforts to develop titanium technologies for other applications in prosthetic dentistry, including fusing to dental ceramics to commercially pure titanium (cp ti).8 according to the american society for testing and materials (astm), cp ti is available in four different grades (grade i to iv) and three titanium alloys. it is based on the incorporation of small amounts of oxygen, nitrogen, hydrogen, iron, and carbon during purification procedures where by each grade has different physical and mechanical properties.9 titanium-ceramics has become a topic of interest for prosthetic applications as an alternative for patients sensitized to nickel-chrome or chrome-cobalt alloys.10 a number of concepts have been presented, including metal frameworks (coping) produced by casting or milling and a number of materials and methods for veneering.11 based on research, the highly oxidative nature of titanium surface in high temperature has been thought as the potential problem of the weak bond between titanium and ceramic.5 thus, although titanium-ceramic systems are available today, there still seem to be unsolved problem related to the fusing of ceramics to titanium. the purpose of this article is to provide information about improving the bonding between titanium and ceramic, because there are reasons to believe that titanium-ceramic restorations will become an important clinical concept. commercially pure titanium (cp ti) and titanium alloys the american society for testing and materials recognizes four grades of cp ti and three titanium alloys: ti-6 al-4 v, ti-6 al-4 v extra low interstitial (low components) and ti-al-nb.9 clinically two forms of titanium have received the most interest, the cp ti and an alloy of titanium is titanium-6% aluminium-4% vanadium (ti-6al-4v).5 the titanium is used as an alternative to chrom-type alloys because of its high strength and rigidity, low density and light weight, ability to withstand high temperatures and resistance to corrosion, low cost and mechanical properties that resemble those of hard and extra-hard casting golds.2,9,10 for fixed dental prostheses, titanium alloys can consider as viable option to more traditional noble and base metal alloys, but careful selection of processing methods and laboratory skill are necessary to ensure success.9 “commercially pure” disclaimed 100% purity and acknowledges that small amounts of oxygen (0.18 to 0.40% by weight) and iron (0.20 to 0.50% by weight) are combined with titanium.2 the oxygen is in solution so that the metal is single phase. elements such as oxygen, nitrogen and carbon have a greater solubility in the hexagonal closed-packed structure of the α-phase than in the cubic from of the β-phase. this elements form interstitial solid solution with titanium and help to stabilize the α-phase. transition elements such as molybdenum, niobium and vanadium act as β stabilizers.5 titanium can be alloyed with element such as aluminium, vanadium or niobium resulting in improved mechanical properties. only a few titanium alloys are being used for the commercial production of fixed and removable dental prosthesis. a titanium alloy, ti-6al-4v, has been used for metal-ceramic restorations with special low thermal expansion ceramic (coefficient of expansion of 9×10–6/°c) similar to that of titanium and baking technique, the bond strengths between titanium and ceramic have some what improved.6 aluminium and vanadium are added in only small quantities. the strength of the alloy is much increased over that of cp ti, aluminium is an α stabilizer with vanadium acting as a β-stabiliser. when these materials are added to titanium, the temperature at which the α-β transition occurs is depressed such that both the α and β forms can exist at room temperature. thus, ti-6al-4v has a two-phase structure of α and β grains.5 the effects of interactions with atmospheric gases during melting, casting and other high-temperatures laboratory procedures are as destructive to titanium alloys as they are unalloyed titanium. the problems encountered in producing cast restorations from titanium alloys are not unlike those experienced with titanium of high or commercial purity.2 physical and chemical properties pure titanium is a white, lustrous metal, which has the attraction of low density, good strength and an excellent corrosion resistance.5 and present two major problems for metal ceramic restoration, a high melting point (16800 c) and high reactivity.12 titanium, in its metallic form at ambient temperature, with hexagonal, close-packed crystal lattice (α phase) at low temperature, which transform into a body-centered cubic form (β phase) above 880° c.5,9,12 the tensile strength and elongation of pure titanium are about 250 mpa and 50%, respectively. however, seemingly slight variations in oxygen and iron content substantial and lasting effects on titanium’s properties. generally, as oxygen or iron content increases, strength increases and ductility 19laksono: titanium ceramic restoration decreases. furthermore, the consequences of titaniumoxygen interaction seem vary according to melting and casting practices.2,5 titanium’s elastic modulus of about 17 million psi (117 gpa) is higher than those of type iii and type iv casting gold (≈100 gpa), but lower than those of most chromium-type alloys (171 to 218 gpa). the vickers hardness number (vhn) of cast cp ti is 210. the strength and rigidity of titanium are comparable to those of other noble or high noble alloys commonly used in dentistry, and titanium ductility, when chemically pure, is similar to that of many dental alloys.2 titanium alloy are a mixture of the α and β phase, where the α phase is relatively soft and ductile while the β phase is harder and stronger but also less ductile. the ti-6 al-4 v considerably higher tensile properties than pure titanium which makes it attractive for partial dentures. an important feature of these materials is the fatique resistance of titanium alloys, and the fatique limit is approximately 450 mpa.5 except for elastic modulus, the tensile properties of titanium alloys are similar to those of chromium type alloys.2 good mechanical performance is corrosive-resistant in addition to having a neutral taste and a vhn of 180 to 250, and low thermal conductivity, less to experience sensitivity to hot and cold, and titanium will not react to surrounding materials.13 the potential for corrosion of titanium in the biological environment has been studied and has confirmed its excellent corrosion resistance.5 the metal can also be highly polished to help reduce tartar accumulation and the radiolucency of the titanium helps enable the dental team to view the underlying tooth structure for caries detection without removing the crown, the patient highly appreciates this, especially for more extensive restoration.12 titanium as a “biometal” because of its good biocompatibility and mechanical performance. this material is hypoallergenic (impervious to body fluids and is not recognized by the body as a foreign materials), making it very diserable for use in these with metal allergies.1,8 titanium as a coping material for metal-ceramic restorations has received attention in dentistry, with the idea that it could be as an affordale alternative for expensive precious metal alloys because it has a thermal expansion coefficient of 9 × 10–6 k–1, low thermal conductivity, low density and biocomp atibility.6,11,13,14 titanium-oxide (ti-o) system or α-ti (o) useful biological properties of titanium, especially biocompatibility, are based on the existence of ti-o layers, which are naturally formed in oxygen-containing environments. in the hexagonal structure of titanium, each atom has one octahedral site which can be occupied by oxygen atoms. for example, one mole of oxygen atoms dissolves in titanium, forming a dilute solid solution, a large amount of energy is released. thus, the ti-o solid solution is thermodynamically very stable.1,5 even a low percentage of oxygen in the solid-solution phase makes it brittle, but not until the oxygen content reaches about 30% in the solution, the formation of the first oxide layer begin at elevated temperatures (> 700° c). titanium has an exceptional ability to form several stoichiometric and non-stoichiometric oxides e.g., ti-o, ti2o3, ti3o5 and ti o2. the non-stoichiometric titanium monoxide is one of the most interesting phases, because of its wide range of homogenity and several crystal modifications. ti ox is in “equilibrium” with the oxygen-rich bulk metal. despite these formation conditions, the outer most oxide layer is always (the most oxygen-rich) titanium dioxide (ti o2). 1 titanium-ceramic system (ti-si-o system) “titanium ceramic” is used in the literature for a feldspathic ceramic used for veneering at titanium.9 according to fusing temperature, ceramic can be classified into high fusing (1300° c) for denture tooth fabrication, medium fusing (1100–1300° c) for denture tooth fabrication, low fusing (850–1100° c) for crown and bridge construction and ultra low fusing (< 850° c) for crown and bridge construction. the high and medium fusing ceramics are similar in both composition and microstructure. in terms of actual use, denture teeth are made from the fusing materials, whereas the medium fusing most often are used to produce prefabricated pontics. the low fusing ceramic containing leucite (feldspar-based) became thermally compatible with metal-ceramic alloys. ultra low fusing ceramic were designed to veneer a metal coping at even lower firing temperature.4 as for metallic coping materials they are also classified into a) high noble (au-pt-pd, au-pdag, au-pd); b) noble (pd-au, pd-au-ag, pd-ag, pd-cu, pd-co, pd-ga-ag); and c) base metal (cp ti, ti-al-v, ni-cr-mo-be, ni-cr-mo, co-cr-mo, co-cr-w ).15 when si o2-based dental ceramics are fused to titanium, the most important reaction occur among ti, o and si. when sio2-based dental ceramics and cp ti are in contact at firing temperatures (720–750° c) for a given time, the dissociation of oxide layers by ti (after dissolving its own native oxides) and the subsequent dissolution of the elements in ti will occur (ti-sio2 bond). 1 the ti-ceramics system can be considered as a double-layer structure, comprising of at least titanium substrate and ceramic body, including bonding agent.15 in contrast with most dental alloys for metal-ceramic restorations, titanium cannot be veneered with conventional feldspathic dental ceramic for several reasons. the use of adapted ceramics for veneering is therefore required. titanium is known for its increasing oxidation at elevated temperatures and an increase of grain size and coarsening of microstructure nearby and passing through the β-transus at 880° c. this can cause ill-fitting copings. during the β titanium phase, the ceramic will form a white dioxide layer that is unsuitable for bonding, and its leads to impurity and brittleness.13 therefore, the firing temperature for fusing ceramic to titanium is restricted to a maximum of 800° c.11 in addition to compatible thermal expansion coefficient of ceramics and metals, knowledge of the microstructures and compositions of the reaction zones is of great importance for optimizing the ceramic-fused-to20 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 17–24 metal system and their processing. several bonding agent have been used as interlayers in conventional metal-ceramic systems to improve the mechanical compatibility of the joint. gold containing interlayers and ceramic interlayers have been applied to the titanium-ceramic system to increase bond strength.1 processing methods of titanium coping several methods was incorporated to make the titanium coping fabricationare: casting, spark erosion and cad/cam. during the casting process, the high melting temperature of titanium has made the casting process difficult, rapid oxidation and the high affinity of molten titanium to investment materials has created reactive α-case layers (oxygen-rich surface layer) on the surface of the casting and results in inferior titanium-ceramic bonding.14,16 titanium melting is best done in specially designed furnaces with an argon atmosphere.2,5 titanium must be melted in a vacuum or under inert gas to prevent oxidation and the incorporation of oxygen that can lead to embrittlement of the cast metal. contamination with even low concentration of atmospheric oxygen can lead to significant loss of ductility. the molten alloy also can react readily with refracting investment materials, requiring careful selection of compatible materials, removal of the surface-reacted layer of metal or both. casting of titanium commonly are used to fabricate crowns, bridge copings and full partial denture frameworks. several commercial machines for casting titanium are available, but their cost is considerably higher that for standard dental casting equipment. materials with low reacting are used to prevent surface reaction with the molten metal, and materials with high setting expansion are used to compensate for the high casting shrinkage of titanium.9 to avoid formation of the α-case layer and to make coping fabrication process simpler and faster, computer aided design/computer aided machine (cad/cam) system have been used to mill coping from a prefabricated titanium block enables to control the uniformity of the alloy composition.13,14,17 a wide variety of materials are available for use by cad/cam restorative systems, including ceramic, composite and titanium. the milling process using cad/cam technology enables the laboratory technician to fabricate an extremely well fitting crown,13 excellent marginal accuracy to less 10 μm18 and can create full anatomic crowns with automated computer-generated cut-backs, to ensure a uniform ideal thickness of the coping by reducing the full-anatomic virtual wax-up. optical scanning is followed by cad/cam fabrication of crown coping. once the coping has been verified to fit on die, it is ready for ceramic firing. dental cad/cam system have advantages that cannot be matched with respect to the strength of materials and precision of the restorations. these materials are generally better in quality, strength and durability that those used to make dental restorations in the traditional casting process.13,14 protocol for ceramic bonding to titanium the more recent ceramic bonding success has been attributed to the strict protocol for ceramic bonding to titanium. the steps are as follows: unidirectional grinding of the coping with a specialized bur (titanium cutters, cross cut burs) is use for roughening the surface with slow speed and low pressure (do not overheat the framework), diamonds and stones should not be used, and then decontamination of the coping by placing it in an ultrasonic bath of either alcohol for 30 minutes, this is a precautionary step because titanium copings and bridges are milled under a mixture of water and oil to keep the milling unit’s burs cool. because they do not undergo sintering, it is logical to conclude that a light coating of oil may remain on the coping, which could affect the reaction with ceramic under the firing sequences. after that, sandblaster machine is used for sandblasting the coping surface with aluminum oxide 120 μm to 150 μm grit, which is preformed to removed α oxidation layer prior to ceramic veneering. then, steamcleaning and passivation able done. the coping must be allowed to interact with the open air for 5 to 10 minutes, but no more than 30 minutes, because at room temperature, titanium has a strong tendency to spontaneously form a very fine oxide layer. this layer prevents corrosion and therefore is responsible for the biocompatibility of the material. the coping is ready to be layered and the bonding agent is applied for surface wetting. do not use of acid or etching agents. spraying the bonding agent completely cover the marginal area in order to obtain excellent adhesion of the ceramic. after that, the opaque dentin is applied, then a second opaque dentin layer and the main build up occurs, followed by optional stain and glazing. discussion bonding ceramic to dental alloys is accomplished during firing, a sintering process. dental ceramic-metal bonding is frequently conducted at temperatures at which chemical reaction between dissimilar materials are to be expected. ceramic-metal bonding is frequently conducted at temperatures at which chemical reactions between dissimilar materials are to be expected. therefore, in addition to compatible thermal expansion coefficients of ceramics and metals, knowledge of the microstructure and compositions of the reaction zones is of great importance for optimizing the ceramics-fused-to-metal systems and their processing.1 however, titanium is chemically an exceptional metal, reacting strongly with gaseous elements like oxygen, hydrogen, and nitrogen and also dissolving them extensively. this high reactivity causes problems related to the fusing of dental ceramics to titanium.1,8 it should also be mentioned that cp ti has a low thermal expansion coefficient that makes it a difficult substrat for ceramics to bond onto.19 21laksono: titanium ceramic restoration among the various titanium alloys, the ti-6al-4v system, or grade v, is the mostly used, because of its better physical and mechanical properties in comparison to cp ti. ti grade v shows greater bending strength (890 mpa against 340 mpa) and greater hardness (350 vhn against 210 vhn) than ti grade ii.20 at temperature above 800° c, which are required for firing conventional ceramic (about 950° c), titanium oxides rapidly, producing a thick oxide layer (up to 1 mm) with a rather weak bond to the underlying titanium, resulting in inadequate metal-ceramic bond strength. although low fusing ceramic are available for veneering cp ti, the use of these ceramics is limited owing to the great mismatch in the coefficient of thermal expansion, since cp ti has a much smaller thermal expansion coefficient (in units of 106 m/m° c, or ppm) than does conventional ceramics.15,20 from the technical of ceramic over titanium requires a special protocol.20 several studies have been reported that the bond strength of grade ii and v titanium substrate combined to low-fusing ceramics were significantly weaker than conventional noble-metal ceramic.20,21 failure of the titanium-ceramic predominantly occurred at the titanium-oxide interface because only o, ti and al elements were found at the titanium surface and ti-ceramic interface.2 although titanium has many stable oxides, it is distinct from other strong oxide-formers such as aluminum and magnesium by its having exceptionally great potential for dissolving large amounts of oxygen or nitrogen. this unique property is utilized, for example, in diffusion bonding or in the joining of metals to oxide or nitride ceramics. however, it is to be noted that too extensive dissolution of oxygen into β-titanium at high temperature (> 880° c) stabilizes the α-titanium. this later phase, having the hexagonal crystal structure, is formed on the top of β-titanium. therefore, there is a relatively large difference in oxygen contents across the α/β-interface. during the cooling of the oxygen-contaminated surface of titanium, the β-titanium will transform into α-titanium, forming the so-called “α-case” (oxygen rich titanium layers), which detaches from the underlying α-titanium of low oxygen content. the solubility of oxygen in titanium is very great as compared with that of aluminum and magnesium. this ti-o system produces the α-case, which impairs the mechanical compatibility of the titanium-ceramic joint. an additional problem is the formation of chemical reaction produces, especially oxides and silicides, generated during the firing, which can fracture under the influence of thermal stress.1 an interfacial oxide layer, some 100–1000 nm (10–100 ä) thick, forms during firing and the thicker this layer becomes, the weaker the bonding strength between the ceramics and the titanium. fabricated titanium structures consisting mainly of β phase are stronger but less ductile than comparable structure with a dominant α phase. thus, to obtain consistenly reproducible mechanical properties, the solidified metal’s cooling rate and the time and temperature of subsequent heat treatment must be controlled.9 methods have been developed for fusing dental ceramic to titanium for fixed partial prosthesis, but the choice of ceramic is limited by two critical factors: the ceramic fusion temperature must be below 800° c to avoid the α to β phase transition and the coefficient of thermal expansion of the ceramic must match that of the metal. the principal problem in the fusing of dental ceramic to titanium is the extensive dissolution of oxygen into the titanium lattice, resulting in thick, oxygen-rich titanium layers. a recent study showed that ceramic fired under inert atmosphere resulted in improved bonding. furthermore, it is difficult to maintain consistency in titanium dental casting because of their inherently poor castability, and few laboratories are able to provide this service. though titanium is economical, biocompatible and readily available, the technologies necessary for casting, machining and veneering this metal are relatively new and more expensive than those used for conventional dental metals. for these reasons, the use of titanium for dental casting has not become a prevalent laboratory and clinical practice.9 the low density of titanium (4.5 gcm–3) lower than those of gold and palladium-silver alloys (18.3 and 10.7 gcm–3 and of the systems ni-cr and co-cr 8.0 and 9.0 gcm–3) provides feather-light restorations.20 the low density has also technical advantages during firing ceramic into a titanium coping.11,13 firing of appropriate ceramic masses is performed at nearly 800° c. due to its specific combination of low density and high melting point, titanium exhibits a high sag resistance. this enables firing of even large span restoration without any thermally induced dimensional changes and with no need for joining processes after firing.11 one study has been reported that the titanium alloys is a good alternative to gold alloys to metal-ceramic restorations emphasizing that it is biocompatible and is has a low density.22 corrosion can be a serious problem, both in terms of degradation of the prosthesis and the release of potentially toxic or allergenic compounds.5 the corrosion resistance and biocompatibility of titanium at room, oral, and body temperatures are attributed to the formation of a stable oxide film with a thickness of less then 1 mm (10–9 m). if the film is scratch or abraded, the involved area repassivates instaneously. at high temperatures, the oxide film is not protective because it thickness and becomes nonadherent. numerous reports document the superior biocompatibility of titanium. the reaction of tissue that contacts titanium or its alloy, ti-6al-4v is extremely mild and direct bone ingrowth or osseointegration does occur.2 one study has been reported that ti-6al-4v did not cause elevated interleukin-1 beta release from cells at non toxic levels. interleukin-1 beta seems to play a central role in the inflammatory reaction.23 the casting of titanium alloy can cause many challenges for the dental technician because the material extremely light and does not flow well after reaching its high melting temperature. in addition, many concerns have been raised 22 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 17–24 about the casting and firing procedure leading to the marginal accuracy and creation of a “reactive layer” that inhibited the bond between titanium-ceramic systems. this problem led to the fracture of ceramic from the titanium coping.13 micro and macroporosity remain consistent features of dental restorations cast from titanium. one study reports that the use of a double-sprue technique for titanium copings produces smoother cast surface and less internal porosity than a single-sprue design. also remaining to be resolved definitively is the inordinate amount of difficulty experienced in making relatively thin casting. to make a suitable titanium coping for a ceramicfused-to-metal restoration, the pattern is usually waxed to a thickness of about 0.6 mm. the resultant casting must be machined to desired thickness. when heated in air at temperatures in the vicinity at 750° c, titanium becomes embrittled through the absorption of oxygen, hydrogen and nitrogen. such embrittlement may cause thin margins of restorations to fracture during burnishing. when heated at temperatures below 800° c for short periods, titanium of high purity forms a compact, adherent oxide scale. moreover, at higher temperatures and extended periods of heat treatment, titanium form a porous and poorly adherent scale. during a few successive firing of a low fusing (750° c), low expansion (7.1 × 10–6/° c on cooling) ceramic to titanium, the smooth adherent oxide produced on degassing at 750° c thickness and becomes flaky. an oxide of this type precludes attainment of reliable ceramic-to-metal bond. when melted and cast with the use of an argon arccentrifugal casting machine, yield strength (0.2% offset), ultimate tensile strength, and elongation for grade 1 cp ti are about 579 mpa, 701 mpa and 18%, respectively. comparatively, castings made in an argon-tungsten arc vacuum pressure machine exhibit greater ductility (elongation = 31%), but yield strength and tensile strength drop to 285 mpa and 365 mpa, respectively.2 cad/cam titanium-ceramic restorations were developed with the potential for replacing expensive, high noble ceramic restorations, and the materials are not subjected to the high casting temperatures that can lead to problems, such as α-case layer formed as a result of the surface reaction with the molten metal are eliminated.13,24 in terms of a restoration precision, because of the accuracy of the scanner, software and milling machine in a cad/cam system, the fit of the dental restorations is quite predictable.14,25 several studies have been reported that marginal accuracy of cad/cam systemfabricated titanium copings significantly better than casting technique.18,25 the clinical performance of cad/cam titanium-ceramic restoration, a study has been reported that the cad/cam titanium-ceramic crowns were acceptable with no biologic complications and high cumulative survival rate for 3 years.14 for fixed partial dentures (fpds), a study has been reported that the cad/cam titaniumceramic fpds survived in the mouths of patients without major complication for 3 years, although the risk of ceramic fracture appeared to be relatively high.25 the quality of bonding between low-fusing ceramic to titanium has been extensively evaluated. a study by using a low fusing ceramic system, showed that the threepoint flexure bond strength to degassed titanium was comparable to the strength of the ceramic to gold alloy.26 several researches have presented that such bonding was acceptable but variations could be occurred if different titanium-ceramic systems were used. it was found that there are several factors that enhance the titanium-ceramic bonding. these include alteration of the titanium surface using pre-oxidation treatment, airbone-particle abrasion, acid etching and application of a bonding agent prior to ceramic application.27 in the effect of surface texture on the titanium-ceramic system, a study has been reported that the morphology of the titanium surface influences the mechanical integrity of the joints. the joints of ceramic-fused-to sandblasted with pure al2o3 particles were structurally better than those fired on the electropolished.1 this study has been supported by another study that the bond strength of ceramic-titanium can be extremely improved by the application of sandblasting with silica-coated aluminum as well as the additional treatment of steam cleaning following sandblast regardless of the sand media.28 in the effects of interlayers on mechanical performance of the titanium-ceramic system, several studies have been carried out to minimize the formation of the non-adherent oxide layer involving an intermediate layer deposited on ti prior to the application of ceramic.26 the use of au (gold),6,29 si3n4, chrome, sio2 and tio2 as intermediate layers has been investigated.30 several study showed that the titanium ceramic adhesion could be improved by coating cast titanium surface with au.29 this can be explained by the formation a ti-au intermetallic compound and surpressed the formation of a ti-deficient intermediate layer, resulting in improved adherence between ceramic and titanium. the ceramic fused to-titanium without gold coating produced a ti-deficient intermediate layer exposed of armorphous titania and highly oxygen-dissolved titanium on the titanium side, this layer was considered to be a cause of cohesive failure at the interface. the gold coated titanium did not have a ti-deficient layer. besides, there was fairly close contact between ceramic and titanium via the ti2au and ti2al phases under ti-au intermetallic compound layer. these result suggested that gold coating suppressed the formation of a ti-deficient intermediate layer and contributed to the adhesion between ceramic and titanium.6 a gold-containing bonding agent lowered the mechanical compatibility of the joint compared with that of the titanium-ceramic system without the agent. according to thermodynamic calculations, this can be explained by the formation of brittle au-ti intermetallic compounds during the firing procedure at given temperatures. when ceramic based bonding agents are used, there are always chemical reactions between titanium and oxygen as well as with metal elements of the ceramics. since the brittleness of the titanium-oxygen solid solution is known, not to mention that 23laksono: titanium ceramic restoration of intermetallic compounds, it is difficult to understand how these agents can improve the bond strength. as to proper soft metallic interlayers, they can reduce thermal stress of the titanium-ceramic joint due to plastic deformation and have an important influence on the formation of reaction layers generated during fusing. therefore, the interlayers should be selected so that the structures generated during the firing are not destroyed in intermetallic reactions or in oxygen reactions. an ideal interlayer should maintain its original properties as perfectly as possible. however, its reactions should still be minimal regarding titanium and the ceramics, so that the driving force allows for controlled fusing reactions. reactivity is necessary for chemical bonding, whereas in titanium-ceramic systems, brittle reactions products may impair the mechanical compatibility of the joint.1 coating the titanium surface with sio2 has also been used because it was considered that sio2 is one of the main composition of the conventional dental ceramic powder (k2o-al2o3-6sio2 or na2o-al2o3-6sio2). silica on titanium surface would serve as an oxygen diffusion barrier while forming an oxide layer to which the ceramic would be more bondable.2 silica (sio2) coating was an effective intermediate layer to improve titanium-ceramic adhesion,30 it could be suggested that the oxidation of the ti-ceramic sysem; during the ceramic fusion, minute amounts of oxygen were able to penetrate the cracks and caused localized oxidation of the ti-substrate. the sio2 coating prevented the diffusion of oxygen to the titanium surface and improved the mechanical and chemical bonding between titanium and ceramic and another study reported that when ceramic was fired in vacuum in the presence of the gold layer, the titanium-ceramic bonding was weakened in as-cast titanium and was not affected in machined titanium.20 oxidation is one of the principal steps in the preparation of the coping for ceramic bonding and there is no single standard oxidizing technique for all alloys on the market. on the contrary, the type of atmosphere (vacuum or air) and the high temperature setting or duration differ among the numerous base metal-ceramic alloys.4,31 according to one hypothesis, the oxide layer is permanently bonded to the coping on one side with the ceramic on the other side. the oxide layer itself is sandwiched between the coping and the opaque ceramic under this so-called sandwich theory and the possible presence of a thick oxide layer would weaken the bonding of metal to ceramic.4 in the effect of oxidation on the bonding strength of ti-ceramic, the pre-oxidation treatment of tio2 which was participate in the interfacial reaction was increased, and resulted in the thickening of reaction layer. failure of the ti-ceramic with pre-oxidation treatment predominantly occurred at the ti-o interface, this suggested that the temperature of pre-oxidation had a great effect on the bond strength of ti-ceramic. pre-oxidation treatment did not increase the bonding strength of ticeramic, it could be suggested that the titanium surface after oxidation treatment revealed the α-ti(o) as the major phase and the rutile (mineral consisting of tio2) as a secondary phase. it revealed that a rutile layer was formed on the titanium surface after oxidation treatment. the rutile layer was more strongly bonded to the ceramic than titanium. the poor adhesion of the rutile with substrate was due to the thermal stress arising from large lattice mismatch and the large difference in coefficient of thermal expansion between titanium and rutile during cooling. therefore, it is favourable to select slow cooling to improving the bond strength. the effect of cooling rate is due to the change of heat stress of ti-ceramic interface.30 as a conclusion, the bond of ceramic over titanium is a sensitive technique influenced by the effects provoked mainly by the layer of surface oxide. titanium and titanium alloys, based on their physical and chemical properties suitable for titanium-ceramic restorations, but careful selection of processing methods, ceramic materials, laboratory skill and strict protocol for ceramic bonding to titanium are necessary to improve the bonding between titanium and ceramic. references 1. kononen m, kivilahti j. fusing of dental ceramics to titanium. j dent res 2001; 80(3): 848–54. available at: http://jdr,sagepub. com/contact/80/5/848. accessed december 29, 2010. 2. o’brien wj. dental materials and their selection. 4th ed. chicago, berlin, tokyo, london, paris, milan, barcelona, instanbul, sao paolo, mumbai, moscow, praque, and warsaw: quintessence publ co; 2008. p. 202–11, 238–42. 3. wirz j, hoffmann al. electroforming in restorative dentistry. new dimension in biologically based prostheses. chicago, berlin, london, tokyo, paris, barcelona, sao paolo, moscow, praque and warsaw: quintessence publ co., inc; 2000. p. 16–27. 4. naylor wp. introduction to metal-ceramic technology. 2nd ed. ouintessence publ co, inc; 2009. p. 112–3, 146–61. 5. van noort r. introduction to dental materials. 3rd ed. edinburg, london, new york, oxford, philadelphia, st. louis, sydney, toronto: mosby elsevier; 2007. p. 227, 234–6, 253–4. 6. tanaka y, watanabe i, okabe t. cross-sectional tem analysis of porcelain fused-to-gold coated titanium. j dent mat 2007; 26(1): 84–8. 7. klaasen cd. casarett, doulls. toxicology the basic science of poisons. 7th ed. new york, chicago, san fransisco: quintessence pub; 2008. p. 970. 8. haag p, nilner k. bonding between titanium and dental porcelain: a systemic review. acta odont scandinavia 2010; 68: 154-64. available at:http://informahealthcare.com/doi/pdf/10.3109/801635 0903575260. accessed january 6, 2011. 9. ada council on scientific affairs. titanium applications in dentistry. jada 2003; 134: 347–49. 10. scmalz g, arenhold-bindslev d. biocompatible of dental materials. berlin: springe-verlag berlin herdelberg; 2009. p. 117, 234–35, 344. 11. leyens c, peters m. titanium and titanium alloys: fundamentals and applications. weinheim: wiley-vch gmbh & co; 2003. p. 458–66. 12. curtis rv, watson tf. dental biomaterials. imaging, testing and modelling. cambridge england: woohead publ limited; 2008. p. 482–85. 13. maclean s. treatment using a porcelain-fused-to-titanium crown 2009. available at: http://nobelbiocare.dentalaegis.com/pdfs/nobelmaclean.pdf. accessed december 29, 2010. 14. boeckler af, lee h, stadler a, setz jm. prospective observation of cad/cam titanium ceramic single crowns. a three-year follow-up. j prosthet dent 2009; 102: 290–7. 24 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 17–24 15. oshida y. bioscience and bioengineering of titanium materials. the boulevard, kidlingtone, oxford: elsevier; 2007. p. 259–65. 16. guilin y, nan l, yousheng l, yining w. the effects of different types of investment on the alpha-case layer of titanium castings. j prosthet dent 2007; 97: 157–64. 17. loevgren r, andersson b, carlsson ge, odman p. prospective clinical 5-year study of ceramic-veneered titanium restorations with the procera system. j prosthet dent 2000; 84: 514–21. 18. shokry te, attia m, mosleh i, elhosary m, hamza t, shen c. effect of metal selection and porcelain firing on the marginal accuracy of titanium-based metal ceramic restorations. j prosthet dent 2010; 103(10): 45–52. 19. vasquez v, ozcan m, nishioka r, souza r, mesquita a, pavanelli c. mechanical and thermal cycling effects on the flexural strength of glass ceramics fused to titanium. j dent mater 2008; 27(1): 7-15. available at: http://www.soc.nii.ac.jp/jsdmd/2008/27-1cc-2.pdf. accessed january 7, 2011. 20. garbelini wj, elias g, henriques p, junior mt, masquita mf, dezan cc. evaluation of low fusing ceramic systems combined with titanium grades ii and v by bending test and scanning electron microscopy. j appl oral sci 2003; 11(4): 354-60. available at: http://www.scielo. br/pdf/jaos/v11n4/ a14v11n4.pdf. accessed january 6, 2011. 21. sadeq a, cai z, woody rd, miller aw. effects of interfacial variables on ceramic adherence to cast and machined commercially pure titanium. j prosthet dent 2003; 90(1): 10–7. 22. olivieri kan, neisser mp, bottino ma, miranda me. bond characteristics of porcelain fused to cast and milled titanium. brazil j oral sci 2005; 4(15): 923-28. available at: http://www.bioline.org. br/request?os05041. accessed february 2, 2011. 23. ozen j, ural au, dalkiz m, beydemir b. influence of dental alloys and an all-ceramic material on cell viability and interleukin-1 beta release in a three-dimentional cell culture model. turk j med sci 2005; 35: 203–8. 24. witkowski s, komine f, gerds t. marginal accuracy of titanium copings fabricated by casting and cad/cam techniques. j prosthet dent 2006; 96(1): 47–52. 25. boeckler af, lee h, psoch a, setz jm. prospective observation of cad/cam titanium-ceramic fixed partial denture: 3 year follow-up. j prosthodont 2010; 19(8): 592–7. available at: http://onlinelibrary. willey.com/doi/10.1111/j.1532-849x.2010.00638.x/full. accessed february 14, 2011. 26. liu j, atsuta m, watanabe i. bond strength of porcelain to degassed cast titanium. int chin j dent2002; 2: 67–74. available at: http:// www.kssfp.jp/pdf/icjd2-2 liu 67-74 pdf. accessed february 2, 2011. 27. loonta p, sukjai h, amornkitbamrung v. the adhesion between dental porcelain and titanium coated with silica using the solgel dip coating technique. kdj 2010; 13(2): 71–9. available at: http://thailand.digitaljournals.org/index.php/kkudj/article/ viewfile/4657/4180. accessed february 2, 2011. 28. wang cs, chen kk, tajima k, nagamatsu y, kakigawa h, kozono y. effects of sandblasting media and steam cleaning on bond strength of titanium-porcelain. j dent mat 2010; 29(4): 381–91. 29. choi th, park s w, vang ms, yang hs, park ho, lim hp, oh gj, kim hs, lee km, lee kk. the bond characteristic of porcelain fused by titanium surface modification. j korean acad prosthodont 2007; 45(2): 169–81. available at: http://www.koreamed.org /search basic. php? rid=0084 jkp/2007.45.20169&dt=1. accessed february 2, 2011. 30. guo l, liu x, zhu y, xu c, gao j, guo t. effect of oxidation and sio2 coating on the bonding strength of ti-porcelain. j mater engineering and performance 2010; 19(8): 1189–92. available at: http://www.bioline.org.br?request?os. accessed february 2, 2011. 31. rosenstiel sf, land mf, fujimoto j. contemporary fixed prosthodontics. 4th ed. mosby elsevier; 2006. p. 744–5. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 168 vol. 43. no. 4 december 2010 the increasing of odontoblast-like cell number on direct pulp capping of rattus norvegicus using chitosan widyasri prananingrum department of material science and technology faculty of dentistry, hang tuah university surabaya indonesia abstract background: pulpal perforation care with direct pulp capping in the case of reversible pulpitis due to mechanical trauma was performed with chitosan which has the ability to facilitate migration, proliferation, and progenitor cell differentiation. purpose: the purpose of this study was to determine the increasing number of odontoblast-like cells in direct pulp capping dental care of rattus norvegicus using chitosan for seven and fourteen days. methods: samples were molars of male rattus norvegicus strain wistar, aged between 8–16 weeks, divided into two treatment groups, namely group i given chitosan and group ii as a control group given ca(oh)2. those rattus norvegicus’ occlusal molar teeth were prepared with class i cavity, and then chitosan and ca(oh)2 were applied as the pulp capping materials. afterwards, glasss ionomer cement type ix was used as a restoration material. their teeth and jaw were then cut on the seventh day and the fourteenth day. next, histopathological examination was carried out to observe the odontoblast like cells. all data were then analyzed by t test. degree of confidence obtained, finally, was 95%. results: the results obtained showed that the significant differences of odontoblast like cells on the seventh day observation was 0.001 (p = 0.001), and on the fourteenth day observation was 0.002 (p = 0.002). conclusion: the number of odontoblast-like cells in direct pulp capping dental care of rattus norvegicus using chitosan is higher than the one using ca(oh)2 for seven and fourteen days. key words: chitosan, calcium hydroxide, direct pulp capping, odontoblast-like cells abstrak latar belakang: perawatan perforasi pulpa pada kasus pulpitis reversible karena trauma mekanis bur dilakukan direct pulp capping dengan cara pemberian bahan secara topikal pada daerah perforasi. kitosan memiliki kemampuan untuk memfasilitasi migrasi, proliferasi dan diferensiasi sel progenitor pulpa. tujuan: tujuan penelitian ini adalah untuk menentukan jumlah peningkatan odontoblas-like cell pada perawatan direct pulp capping gigi rattus norvegicus menggunakan kitosan selama 7 dan 14 hari. metode: sampel adalah gigi molar rattus norvegicus jantan strain wistar, berusia antara 8–16 minggu, dibagi menjadi 2 kelompok perlakuan yaitu kelompok i yang diberi kitosan dan kelompok ii sebagai kontrol yang diberi ca(oh)2. oklusal gigi molar rattus norvegicus dipreparasi kelas i kemudian kitosan dan ca(oh)2 diaplikasikan sebagai bahan pulp capping. glass ionomer cement tipe ix digunakan sebagai bahan restorasi. gigi beserta rahang tikus dipotong pada 7 dan 14 hari. pemeriksaan histopatologi dilakukan untuk mengamati odontoblas-like cell. semua data dianalisis dengan uji t. tingkat kepercayaan = 95%. hasil: hasil penelitian menunjukkan perbedaan yang signifikan dalam odontoblas like cell pada pengamatan hari ke-7 (p = 0,001) dan pengamatan hari ke 14 (p = 0,002). kesimpulan: jumlah odontoblas like cell pada perawatan direct pulp capping gigi rattus norvegicus menggunakan kitosan lebih tinggi dibandingkan dengan ca(oh)2 selama 7 dan 14 hari. kata kunci: kitosan, kalsium hidroksid, direct pulp capping, odontoblast-like cells correspondence: widyasri prananingrum, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas hang tuah. jl. arif rahman hakim 150 surabaya. e-mail: widyaerlangga@yahoo.com telp. 031-5945894. research report 169prananingrum: the increasing of odontoblast-like cell number introduction pulpal perforation care in the case of reversible pulpitis due to mechanical trauma or deep caries cleaning can be conducted by direct pulp capping using topical materials applied in the perforation area. the application of wound covering materials on the opened pulp is for bacterial prevention and to iniate soft tissue healing as well as to repair the dentin tissue of the opened area, therefore the wound will not progress to pulpitis irreversible which can eventually cause pulpal death. calcium hydroxide (ca(oh)2) is considered as the standard material used for direct pulp capping care until now. nevertheless, this material has some disadvantages, for example, it can cause necrosis in the superficial layers of pulp since ca(oh)2 can be ionized into ca+ + and ohwhich form strong alkali. this alkaline character can trigger the risk of pulp and apical lesion abnormalities.2 the lack of ca(oh)2, can be solved by developing chitosan biomaterial that is safe (not toxic), biocompatible, and biodegradable, since it can accelerate wound healing activity, adsorption, and anti-infection.3 chitosan used in this study was derived from white shrimp shell with a 88.96% degree of deacetylation. chitosan is actually polycationic complex carbohydrate that is able to facilitate the migration and proliferation of progenitor cells.4 biocompatibility test of chitosan derived from shrimp shell was conducted in this study by using a skin patch test which cannot cause allergic reaction in individuals with a history of allergies or seafood allergies.5 chitosan test with deacetylation degree of 88% towards antimicrobial also showed lower growth of streptococcus mutans and candida albicans. 6 thus, this study was finally aimed to determine the ability of chitosan in increasing the number of odontoblast-like cells on direct pulp capping of rattus norvegicus. materials and methods the type of this study was an experimental research with completely randomized design using samples of molars of male rattus norvegicus strain wistar weighting 200–250 g and aged between 8–16 weeks. the samples of this study were divided into two treatment groups, namely group i given chitosan and group ii as a control group given the ca(oh)2. each treatment group, were divided in two samples of observations, seven day observation and fourteen day observation, with 8 samples in each group. next, the occlusal part of the rattus norvegicus’ molar was prepared with class i cavity by using low-speed tapered round diamond bur, and then was perforated with the tip of explorer. the pulp capping materials, chitosan and ca (oh)2, were applied as much as 0.01 grams into the cavity, and then the cavity was covered with type ix glass ionomer cement as a restoration material. the teeth and jaw of rats were cut on the seventh and fourteenth day. all samples were stored in fixation solution for 48 hours, and then were decalsificated until soft. after that, it was embedded in paraffin blocks to be prepared for 4–5 μ cutting, and then was continued with he staining in order to observe the odontoblast-like cells. the odontoblast-like cells on both group on the seventh day can be seen on figure 1. the calculation was finally conducted by using light microscope with magnification view of 400×/, to obviously the number of odontoblast-like cell in the left and right edges of the perforation. results analysis result of statistical calculations showed that the mean and standard deviation of odontoblast-like cells number in the chitosan group was higher than that in the ca(oh)2 group (table 1). the result of t test showed that in comparison between the chitosan group and the ca(oh)2 group in the seventh and fourteenth day observations, there was a significant difference in the seventh day observation at p = 0.001 (p < 0.05). meanwhile, the result of t test in the comparison between the chitosan group and the ca(oh)2 group in the fourteenth day observation showed that there was significant difference of odontoblast-like cell number at p = 0.002 (p < 0.05). discussion based on table 1, the results of this study could indicate that the mean number of odontoblast-like cells in the chitosan group was higher than in the ca(oh)2 group. the comparison of chitosan and ca(oh)2 towards odontoblast-like cells on the seventh day and the fourteenth day after the treatment could also indicate that there were significant differences among them (table 2). it is because chitosan as direct pulp capping material has a 88.957% degree of deacetylation which has a high percentage of acetyl groups that are more active so that chitosan could stimulate the differentiation of odontoblast-like cells. the degree of deacetylation of chitosan, could also affect the biological character of chitosan, including biodegradation of chitosan caused by lysozyme enzyme produced by inflammatory cells and macrophages neutrophil.7 actually, chitosan is a source of the active n-acetyl-d-glucosamine dimer, therefore if it is applied to the wound area, it will make inflammatory cells release lysozyme.3 neutrophils produced by inflammatory cells, furthermore, will migrate into the wound area several hours after the injury and will reach a maximum concentration in about 24 hours. similarly, macrophages, the dominant cells, will migrate into the wound area after 24 hours and for about five days,8 and the inflammatory cells will decline after seven days.9 therefore, if chitosan containing the active n-acetyld-glucosamine dimer experiences biodegradation, it will form cross-linked with glycosaminoglycan and 170 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 168–171 glycoprotein which plays a role in biological processes, including both the cell and matrix interactions and the activation of growth factors.7 growth factors, such as bone morphogenetic protein-2 (bmp-2) which is a superfamily of transforming growth factor b (tgf-b) then will stimulate the differentiation of osteoblastic-cell.10 thus, chitosan is able to accelerate wound healing process through fibrinogenic mediators, such as growth factors. the increasing of the expression of growth factors will also be able to increase the activity of fibroblasts. this is because of the ability of chitosan in forming polyelectrolyte complex by using polyanion heparin to improve and extend the half-life of growth factors in stimulating cell differentiation. through in vitro studies, it is also known that mesenchymal cells exposed with chitosan showed a higher differentiation than control, indicating that chitosan could stimulate the differentiation of osteoprogenitor cell and bone formation.11 table �. the mean and standard deviation of the number of odontoblast-like cell in the chitosan group and the ca(oh)2 group for seven and fourteen days materials seven days fourteen days mean sd mean sd chitosan 17.75 1.28 19.00 1.30 ca(oh)2 13.63 2.32 14.88 2.85 table �. the significance rate of odontoblast like cell in the comparison of the chitosan group and the ca(oh)2 group on the seventh day and the fourteenth day variable p score chitosan– ca(oh)2 the seventh day the fourteenth day odontoblast like cell 0.001* 0.002* note: * = significant difference chitosan is a natural cation that plays a role in electrostatic interactions with anionic glycosaminoglycan and proteoglycan that will improve the effectiveness of growth factors.10 osteoblast cell cultures stimulated by chitosan, as a result, can cause the increasing of the expression of both alkaline phosphatase (alp) mrna after 3 days and bmp-2 mrna after seven days.9 it is also because chitosan is able to directly stimulate the differentiation of multipotent mesenchymal progenitor cells into osteogenic cells. another evidence even stated that chitosan implantation of absorbable collgen sponge on rat calvarials can increase the formation of new bone, greater than just giving absorbable collagen sponge only, after eight weeks of treatment. it means that chitosan is a potential material used to accelerate the regeneration of bone since chitosan can trigger the differentiation into osteogenic cells.4 finally, it may be concluded that the number of odontoblast-like cells on direct pulp capping of rattus norvegicus using chitosan is higher than using ca(oh)2 for seventh and fourteenth days. references 1. mitsiadis ta, rahiotis c. parallels between tooth development and repair: conserved molecular mechanism following carious and dental injury. j dent res 2004; 83(12): 896–902. 2. bergenholtz g. textbook of endodontology. oxford: blackwell; 2003. p. 56–7. 3. alsarra ia. chitosan topical gel formulation in the management of burn wounds. int j biol macromol 2009; 45(1): 16–21. 4. pang ek, paik jw, kim sk, jung uw, kim cs, cho ks, kim ck, choi sh. effects of chitosan on human periodontal ligament fibroblast in vitro and on bone formation in rat calvarial defects. j periodontol 2005; 76(9): 1526–33. 5. maretaningtias da, yuliati, tokok a. toxicity testing of chitosan from tiger prawn shell waste on cell culture. dent j 2009; 42(1): 15–20. 6. tania a. efek derajat deasetilasi dan konsentrasi kitosan dalam menghambat pertumbuhan streptococcus mutans dan candida albicans. tesis. surabaya: pascasarjana universitas airlangga; 2009. p. 21–30. a b figure �. a) odontoblast-like cell in the chitosan group on the seventh day, b) odontoblast-like cell in the ca(oh)2 group on the seventh day. 171prananingrum: the increasing of odontoblast-like cell number 7. ikeda t, yanagiguchi k, matsunaga t, yamada s, ohara n, ganno t, hayashi y. immunohistochemical and electron microscopic study of the biodegradation processes of chitin and chitosan implanted in rat alveolar bone. j oral med pathol 2005; 10: 1–138. 8. nanci a. oral histology development, structure and function. 7th ed. united states of america: mosby elsevier; 2008. p. 391. 9. matsunaga t, yanagiguchi k, yamada s, ohara n, ikeda t, hayashi y. chitosan monomer promotes tissue regeneration on dental pulp wounds. j biomed mater res a. 2006; 76(4): 711–20. 10. muzzarelli, belmonte m, pugnaloni a, biagini g. biochemistry, histology and clinical uses of chitin and chitosans in wound healing. 1999. available at: http://www.mavicosmetics.it/pdf/nanofibrille/ 2%20biochemistryhistology_%20clinical%20uses chitins&chitosans. pdf. accessed september 1, 2010. 11. lahiji a, sohrabi a, hungerford ds, frondoza cg. chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. j biomed mater res 2000; 51(4): 586–95. �� vol. 45. no. 1 march 2012 the effective concentration of red betel leaf (piper crocatum) infusion as root canal irrigant solution fani pangabdian1, slamet soetanto2, and ketut suardita2 1resident of conservative dentistry 2department of conservative dentistry faculty of dentistry, airlangga university surabaya-indonesia abstract background: smear layer is a debris consisting of organic and inorganic particles of calcified tissue, necrotic tissue, pulp tissue, and dentinoblast and microorganism processes that can close the entrance to the dentin tubuli. smear layer, will not only inhibit the penetration of disinfection materials and sealers to the dentin tubuli, but will also reduce the attachment of root canal filling material so that root canal irrigation solution is needed to dissolve the smear layer. red betel leaf (piper crocatum) infusion, on the other hand, contains saponin characterized as “surfactants” which can dissolve smear layer. nevertheless, the effective concentration of the red betel leaf infusion has still not been known clearly. purpose: this study is aimed to determine the effective concentration of the red betel leaf infusion for cleaning root canal walls from smear layer. methods: fiveteen extracted human teeth with straight single roots were randomized into 5 groups (n=3). the specimens were then shaped by using rotary instruments up to a size of 25/.07. during instrumentation, each canal was irrigated with 10, 20, 30 and, 40% red betel leaf infusion for treatment groups, while another was irrigated with aquadest for the control group. root canal cleanliness was observed by using scanning electron microscope (sem). results: there were significant differences among treatment groups (p<0.05), except in the treatment groups irrigated with red betel leaf infusion with concentrations of 30% and 40% (p>0.05). conclusion: it can be concluded that red betel leaf infusion with a concentration of 30% is effective for cleaning the root canal walls from the smear layer. key words: red betel leaf infusion (piper crocatum), smear layer, saponin, surfactant abstrak latar belakang: smear layer adalah suatu debris yang mengandung partikel organik dan anorganik dari jaringan terkalsifikasi, jaringan nekrotik, proses dentinoblas, jaringan pulpa dan mikroorganisme yang dapat menutup jalan masuk ke tubuli dentin. smear layer akan menghalangi penetrasi dari bahan disinfeksi dan sealer terhadap tubuli dentin dan mengurangi perlekatan bahan pengisi saluran akar, sehingga dibutuhkan larutan irigasi yang dapat membuang smear layer tersebut. infusa daun sirih merah (piper crocatum) mengandung saponin yang dikarakteristikkan sebagai surfaktan yang dapat melarutkan smear layer, tetapi sampai sekarang belum ada penelitian tentang hal tersebut. tujuan: penelitian ini dilakukan untuk mengetahui konsentrasi efektif daya pembersih infusa daun sirih merah (piper crocatum) dapat membersihkan dinding saluran akar dari smear layer. metode: 15 gigi premolar bawah manusia yang mempunyai akar lurus dibagi menjadi 5 kelompok secara acak (n=3). gigi dipreparasi menggunakan rotary instrumen sampai protaper f2 (30/0.02). selama instrumentasi, dilakukan irigasi dengan infusa daun sirih merah (piper crocatum) dan konsentrasi 10, 20, 30, 40% dan diirigasi aquadest untuk grup kontrol. setelah itu kebersihan dinding saluran akar diperiksa dengan menggunakan scanning electron microscope (sem). hasil: terdapat perbedaan yang signifikanantara masing-masing kelompok (p<0,05), kecuali kelompok yang diirigasi infusa daun sirih merah (piper crocatum) konsentrasi 30% dan 40% (p>0,05). kesimpulan: dapat disimpulkan infusa daun sirih merah (piper crocatum) dengan konsentrasi 30% efektif untuk membersihkan dinding saluran akar dari smear layer. kata kunci: infusum daun sirih merah (piper crocatum), smear layer, saponin, surfaktan research report ��pangabdian, et al.: the effective concentration of red betel leaf (piper crocartum) introduction endodontic treatment is actually considered as an effort to maintain teeth as long as possible in oral cavity by taking the entire pulp tissue, either from pulp chamber or root canal.1-3 the principles of endodontic treatment, moreover, involves preparation, sterilization and filling root canals. the preparation of root canal can be considered as one of the important stages in the endodontic treatment.1 this stage involves smear layer cleaning and root canal shaping, so it can facilitate sterilization and filling the root canal.1,4 smear layer can be defined as a debris composition consisting of organic and inorganic particles of calcified tissue, necrotic tissue, and odontoblast and microorganism processes. smear layer, therefore, can contain bacteria, and this layer even can close the entrance to the dentine tubuli so that it will reduce the attachment of root canal filling material.4-6 during and after the preparation stage, or every changing of preparation number, the irrigation of root canal should always be conducted, in general, the term of irrigation means watering, washing, or cleaning by using irigation liquid.3,5 this root canal irrigation procees is aimed to remove necrotic tissue, to water root canals, and to dispose smear layer, so it can simplify the implementation of the preparation as well as can reduce the number of microorganisms in root canals.7-11 red betel leaf (piper crocatum) is one of the natural materials that could potentially be used as an ingredient of root canal irrigation.5,9,11 red betel leaf contains saponin compound characterized as “surfactants” which can lower the surface tension so that it has an ability to dissolve organic materials, inorganic materials, and microorganisms in root canal.12,13 as root canal irrigation material, red betel leaf infusion must have a certain concentration, but until now there has been no research on it.15-17 thus, it is necessary to find the effective concentration of red betel leaf infusion used as a biocompatible root canal irrigation solution.18-20 the purpose of this reseach is to determine the effective concentration of red betel leaf infusion for cleaning the root canal walls of smear layer. materials and methods fifteen lower premolars that had been extracted for orthodontic purposes were immersed in normal saline solution, and then classified into five groups, each group consisted of three teeth. dental radiography concerned with buccal and mesial aspects was then conducted on those correspondence: ketut suardita, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: suardita@yahoo.com teeth in order to ensure closed apical. both of the thickness of the root canal, especially in one third of the apical, and the length were confirmed by radiographic images using a file with a size of 20/.07. the preparation of root canal was then conducted with rotary protaper until protaper f2 (25/.07) with the preparation duration of 10 seconds for every protaper. during the changing of the instruments, the irrigation of the root canal was conducted based on the irrigation solution used. group 1 using 10% red betel leaf; group 2 using 20% red betel leaf; group 3 using 30% red betel leaf; group 4 using 40% red betel leaf; and group 5 using aquadest as control. these irrigation processes were conducted by using a tool that had been prepared so that the air pressure became the same, about 50 mpa/kg. the irrigation solution used for each irrigation process was about 3 ml for 5 seconds, so the total amount of red betel leaf infusion used for each tooth sample was about 15 ml. after the irrigation processes, the root canal was dried with sterile paper for three times. afterwards, those samples were cut horizontally at one third of the apical, about 4 mm from the edge of stale, by using disc bur (save side disk). then, they were cut longitudinally with a chisel in order to become two parts with the same size. those samples cut already were planted or placed on sample holder with root canal surface facing up. then, it was inserted into air barrier tool from coating unit. coating process with gold was conducted for 40 seconds, and then was ready to be examined by scanning electron microscopy (sem). at first, the entire the apical region was observed with 150x magnification in the middle. later, this area was enlarged to the size of 1500× which contrast and brightness were set. by pressing a certain button, one third of the area apical could also be observed. the amount smear layer of each specimen were scored by four blinded evaluator. the scores were determined as follows (5): 1 = there was no smear layer, and all dentin tubuli were open and clean; 2 = there were few smear layers, and some dentin tubuli were open and clean; 3 = there was homogeneous smear layer covering some root canal walls; 4 = there was homogeneous smear layer covering the entire root canal walls; 5 = there was nonhomogeneous smear layer covering the entire root canal walls (heavy). cohen’s kappa statistical analysis was used to analyze agreement among the evaluator. the differences within each group were then analyzed by using non-parametric analysis of kruskal-wallis test. and, mann-whitney test was finally conducted followed with control median test. �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 12–16 results the absence and presence of smear layer are presented on figure 1. based on cohen kappa’s test, values obtained was above 0.07 indicating a good grade in which there was the same perception of the valuation methods among the observers. the assessment results of the root canal cleaning power of red betel leaf infusion at concentrations of 10%, 20%, 30%, and 40%, and that of aquadest as in the control group can be seen in table 1. table 1. the mean and standard deviation of the smear layer in each group groups mean standard deviation 10% 3.44 0.52 20% 1.88 0.60 30% 1.22 0.44 40% 1.22 0.44 aquadest 4.66 0.50 in this research, several variables were controled in order to obtain homogeneous samples. non-parametric test was conducted with the kruskal-wallis test to determine whether there was any difference among all of those treatment groups. based on the results of the test, it is then known that the significance level was about 0.001, smaller than 0.05 (p <0.05). this indicates that there were significant differences in all treatment groups. afterwards, mann-whitney test was conducted to determine the differences in each of those treatment groups. based on the results of the test, it is then known that the significance level of in each of those treatment groups was about 1.000, (p>0.05). this indicates that there was no significant difference in those treatment groups. control median test was conducted to find out the median of each group. based on the results of the test, it is then known that median value of red betel leaf infusion with a concentration of 30% was the same as that of red betel leaf infusion with a concentration of 40%, about 1.000, considered as the smallest value compared with the other groups. this suggests that red betel leaf infusion with a concentration of 30% was the most effective value. discussion at the preparation stage, during and after the stage, or every changing of file number, the irrigation of root canal should always be conducted, therefore, the use of irrigation solution during the preparation is important.2-4 smear layer is used as an indicator of the level of cleanliness because every act of root canal preparation will occur friction endodontic tool with root canal wall resulting in the formation of a layer of debris attached to the walls of the root canal known as smear layer.5,26,27 the effective concentration of red betel leaf infusion can be known by conducting laboratory research and then by using sem showing the absence of smear layer and all the open and clean dentine tubuli. thus, the more open dentin tubuli is, the more clean root canal walls of the smear layer.21-23 sem was chosen because this tool can be a b c d e figure 1. sem photograph of the surface rootcanals in group: a) control: the entire root canal walls were covered with non-homogeneous smear layer (heavy), (b) 10% red betel leaf infusion: the entire root canal walls were covered with homogeneous smear layers, c) 20% red betel leaf infusion: the entire root canal walls were covered with homogeneous smear layers, d) 30% red betel leaf infusion: there was no smear layer, and all dentin tubuli were open and clean, e) 40% red betel leaf infusion: there was no smear layer, and all dentin tubuli were open and clean. ��pangabdian, et al.: the effective concentration of red betel leaf (piper crocartum) used to see the cleanliness of the canal wall, and can also be considered as one of the parameters of the successful results of the root canal preparation.23-27 based on the assessment results conducted by using photomicrographs, it can be seen that in the control group irrigated with aquadest there was nonhomogeneous smear layer that covered the entire root canal wall. this is because aquadest has no active substances, such as saponins, and only serves to water the root canal walls but without any abilities to dissolve the smear layer, so it can not clean the root canal walls of the smear layer.28,29 meanwhile, in the two treatment groups irrigated with red betel leaf infusion with concentrations of 10% and 20%, there were homogeneous smear layers covering the roots, but there were few open dentin tubuli. this suggests that red betel leaf infusion with those concentrations contains little saponins, so it still has not worked effectively to clean the root canal walls of the smear layer.30 while the other two treatment groups irrigated with red betel leaf infusion with concentrations of 30% and 40% did not have smear layer so that all dentine tubuli could be open and clean. both those groups even showed the same quality of cleaning power that was not statistically and significantly different. this is because at these concentrations the amount of active substance, saponin, contained in red betel leaf infusion at concentrations of 30% and 40% could work well and effectively to dissolve smear layer.21,31,32 red betel leaf infusion actually contains active substance, saponin, a type of glycoside found in many plants with characteristics as “surfactants”.4,7,12 saponins (phytonutrients) has properties resembling soap, so often called as “natural detergent”, that is a foaming solution classified by the structure of the complex aglykon into triterpenoid and steroid saponins.4,26,33 this compound has a long hydrocarbon chain with ion tip which consists of clusters of non polar (hydrophobic) and polar (hydrophilic) groups. non-polar (hydrophobic) cluster will divide smear layer molecules into smaller particles so that water is not only easy to form an emulsion with smear layer, but also easy to be separated. meanwhile, polar (hydrophilic) cluster will be dissolved in water to form bubbles and binding particles of smear layer causing the formation of emultion.32-34 however, because of the long hydrocarbon chains, not all smear layer molecules are soluble in water so that saponins will emulsify or suspense smear layer in water. in this process, anions form colloidal micelle particles, the collections of (50-150) molecules, which hydrocarbon chain cluster with the ion tips facing the water so that the ions have higher solubility than the single insoluble ones.4,11,35 the longer the hydrocarbon chain is, the higher value of solubility is. thus, the ability of saponin to dissolve smear layer is getting better. therefore, saponins can be characterized based on its ability as a “surfactant” that is capable of lowering the surface tension of root canal walls, so the smear layer containing dentine debris, organic materials, inorganic materials, and microorganisms in root canals can be dissolved later.36-39 it can be concluded that red betel leaf infusion with a concentration of 30% is effective for cleaning the root canal walls of the smear layer. references 1. walton, torabinejad m. principles and practice of endodontics. 3rd ed. philadelphia: wb saunders; 2002. p. 360–78. 2. shahravan a, haghdoost aa. effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. j endod 2007; 33(2): 96–105. 3. moon ym, shoon wj, baek sh, bae ks, kum ky, lee wc. effect of final irrigation regimen on sealer penetration in curved root canals. j. endod 2010; 36(4): 732–6. 4. agustin dw. perbedaan khasiat anti bakteri bahan irigasi saluran akar antara hidrogen peroksida dan infusum daun sirih merah 20% terhadap bakteri mix. maj ked gigi (dent j) 2005; 38(1): 45–7. 5. de moor rjg, meire m, goharkhay k, moritz a, vanobbergen j. efficacy of ultrasonic versus laser-activated irrigation to remove artificially placed dentin debris plugs. j endod 2010; 36(9): 1580–3. 6. george r, rutley eb, walsh lj. evaluation of smear layer: a comparison of automated image analysis versus expert observer. j endod 2008; 34 (8): 999–1002. 7. de menezes acsc, zanet cg, valera mc. smear layer removal capacity of desinfectant solution used with and without edta for the irrigation of canals: a sem study. pasgui odontol braz 2003; 17(4): 1. 8. sen bh, wesselink pr, turkun m. the smear layer aphenomenon in root canal therapy. int endodont j 1995; 28(3): 141–8. 9. ajizah a. sensitivitas salmonella typhimurium terhadap ekstrak daun psidium guajava l. bioscientiae. j pertanian indonesia 2004; 1(1): 31–8. 10. akiyama h, fujii k, yamasaki o, oono t, iwatsuki k. antibacterial action of several tannins agains staphylococcus aureus. j of antimicrobial chemotherapy 2001; 48(4): 487–91. 11. ariesdyanata c. perbedaan daya hambat ekstrak daun sirih hijau (piper bettle lynn) dengan daun sirih merah (piper crocatum) terhadap staphylococcus aureus. adln journal. 2008. available at: http://www.adln.lib.unair.ac.id/go.php?id=gdlhub-gdl-s1-2010ari esdyana11276&phpsessid=bfbf537fe39d6a589d1d2fi7c232a00b. accessed march 20, 2010. 12. mahvi ah. removal of anionic surfactants in detergen wastewater by chemical coagulation. iran pak j biol sci 2004; 7(12): 2222–6. 13. depkes ri. farmakope indonesia. 4th ed. 1995. p. 12–3. 14. cohen s, hargreaves km. pathway of the pulp. 9th ed. st. louis: mosby; 2006. p. 339. 15. ebadi m. pharmacodynamic basis of herbal medicine. london: crc press; p. 393–400. 16. farida c. manfaat sirih merah sebagai agen anti bakterial terhadap bakteri gram positif dan negatif. j ked dan kesehatan indonesia 2010; 37(5): 1–9. 17. grossman li, oliet s, rico ce. endodontic practice. eleventh edition. philadelphia, pennsy ivania, u.s.a: lea & febiger; 1998. p. 196–247. 18. gunawan ja. reevaluasi terapi endodontik: berbagai inovasi baru dalam pengisian saluran akar. majalah ilmiah kedokteran gigi fkg usakti 2001; 16: 13–4. 19. hostettmann k, marston a. chemistry and pharmacology of natural products. cambridge: cambridge university press; 1990. p. 239. 20. neviyanti. biokompatibilitas larutan irigasi saluran akar. e-journal usu 2004; 33: 1–4. 21. gutma n n j l. p roblem solving in endodontics: prevention, identification, and management. 5th ed. st louis: mosby; 2011. p. 209. 22. hülsmann m, heckendorff m, schäfers f. comparative in vitro evaluation of three chelator pastes. international endod j 2002; 35(8): 668–80. �� dent. j. (maj. ked. gigi), vol. 45. no. 1 march 2012: 12–16 23. isik ag, tarim b, hafez aa, yalcin fs, onan u, cox cf. a comparative scanning electron microscope study on the charac teristics of demineralized dentin root surface using different tetracycline hcl concentrations and application times. j periodontal 2000; 71(2): 29. 24. katzung bg. basic and clinical pharmacology. 9th ed. san francisco; 2009. p. 449–65. 25. manoi f. sirih merah sebagai tanaman multi fungsi. warta puslitbangbun 2007; 13(2): 2–7. 26. daut el-mor a z i n a, vu lca i n j m, bon nau re -ma l let m. a n ultrastructural study of smear layer: comparative aspects using secondary electron image and backscattered electron image. j endod 1994; 20(11): 531. 27. canderasari nm. perbedaan sitotoksisitas larutan tetrasiklin hidroklorida 1% dengan natrium hipoklorit 2,5%. karya tulis akhir. surabaya: airlangga; 2004. p. 1. 28. orstavik d. intracanal mediation. endodontic. 4th ed. norwegian: wright; 1996. p. 312–25. 29. parwata imoa, dewi pfs. isolasi dan uji antibakteri minyak atsiri dari rimpang lengkuas (alpina galanga l). j kimia 2008; 2(2): 100–4. 30. phillipson jd. chemistry and pharmacology of natural product. london: cambridge university press; 1990. p. 239–40. 31. ford ptr, rhodes js. endodontics: problem solving in clinical practice. uk: martin dunitz comp; 2002. p. 45, 111. 32. sadono jm. pengembangan dan pemberdayaan bahan alam di bidang kedokteran gigi. jakarta: penerbit buku kedokteran egc; 2009. p. 1–20. 33. sanjaya h. khasiat anti bakteri bahan irigasi infusum daun sirih 20%, sodium hipoklorit 2,5%, dan hidrogen peroksida terhadap streptococcus viridans. thesis. surabaya: fkg unair; 2007. 34. sassone lm, fidel ras, fidel sr, dias m, junior rh. antimicrobial activity of different concentrations of naocl and chlorhexidine using a contact test. braz int j 2003; 14(2): 157–169. 35. shahravan a, haghdoost aa, adl a. effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. j endod 2007; 33: 96–105. 36. sudewo b. basmi penyakit dengan sirih merah. jakarta: agromedia pustaka; 2008. p. 2, 4, 9, 25, 29, 44. 37. torabinejad m, khademi aa, babagoli j, cho y, johnson wb, bozhilov k. a new solution for the removal of the smear layer. j endod 2003; 29(3): 170. 38. zehnder m. root canal irrigants. j endod 2006; 32(5): 389-98. 39. weine fs. endodontic therapy. 6th ed. st louis: mosby co; 2004. p. 221–4, 498–503. subject index volume 53 10% propolis, 212 17% edta, 181 2.5% naocl, 181 20% citric acid, 181 3d imaging, 50 abalone gel, 99 acid etching, 16 adherence, 20 aesthetic, 126 alkalisation, 57 aloe vera, 115 alveolar bone mineral density, 164 amlodipine, 187 antithrombotic, 111 arch length, 93 width, 93 arginine, 99 aspirin, 111 bimaxillary and bidental protrusion, 223 bmi, 144 body mass index, 140 bovine bone graft, 10, 40 burning, 187 caffeine in chocolate, 164 cape, 1 carbonate hydroxyapatite, 76, 212 caries severity, 36 cbct, 201 cephalometry, 133 child health, 122 children, 93 chitosan, 115 chlorophyll, 62 class ii division i malocclusion, 201 malocclusion, 133 class iii malocclusion, 191 collagen, 76 density, 229 composite resin, 16, 57 continuous aerobic physical exercise, 196 anaerobic physical exercise, 196 correlation value, 30 covid-19, 81 cross infection, 81 curcumin, 62 daily performance, 122 dental arch, 93 caries, 36, 122, 153 fear and anxiety, 175 practice, 81 dentist visit, 175 dimension, 93 dmft, 140 double crown, 126 dry mouth, 187 edgewise technique, 170 effective dose, 40 egg yolk, 20 elderly, 144, 217 electronic application, 206 enterococcus faecalis, 62, 71 epigallocatechin-3-gallate (egcg), 88 eruption of central incisors and molars, 140 ethanolic extract of moringa oleifera, 107 extraction, 133 facial landmark distance, 30 vertical height, 223 fibroblasts, 196 fixed orthodontic treatment, 170, 223 flavonoid, 107 flexural strength, 57 forensic dentistry, 50 ftu, 144 glycosaminoglycans, 111 healing index, 45 heat treatment, 6 hiv, 159 holothuria scabra, 111 howe’s analysis, 149 index, 149 hsf1, 107 human β-defensin 3, 76 hypersensitivity dentin, 99 igy, 20 inclination, 133 indonesia, 175 inflammation, 88 inner and outer coping, 126 iron-deficiency, 153 irradiation time, 71 isothiocyanate, 107 javanese, 217 lemuru fish oil, 229 leukoplakia, 159 light-emitting diode, 71 madurese, 217 mastication, 144 maxillary anterior, 201 microhardness, 6 military officer, 187 molar position, 223 nanohybrid resin composite, 6 neovascularisation, 196 neutrophil, 88 nickel ion, 67 occlusal plane, 133, 223 odontoblast-like cell, 1 ohi-s, 36 open wound, 45 oral cancer, 107 oral health, 217 impact on daily performance, 122 potentially malignant disorder, 159 school programme, 206 orthodontics, 149 archwires, 67 camouflage treatment, 191 tooth movement, 164 osteoblast, 10, 40 osteoclast, 10, 40 overdenture, 126 pandalungan 217 panoramic photographs, 201 paradoxical sleep deprivation, 24 passive self-ligating system, 191 periodontal dressing, 45 surgery, 45 periodontitis, 76, 212 width of the periodontal ligament, 229 photodynamic therapy, 71 photosensitiser, 62 pophyromonas gingivalis, 20 post-tooth extraction wound healing, 196 preheating, 6 prevention, 81 proinflamatory cytokines, 24 propolis extract, 1, 10, 40 proto-malay, 149 pulp capping, 1 perforation, 88 push-out bond strength, 181 quality information, 206 rankl expression, 212 recording and reporting, 206 riboflavin, 62 room disinfection, 81 root canal treatment, 71 resorption, 201 s. mutans bacteria, 170 scaffold pore size, 115 silk fibre, 57 sleep deprivation, 24 recovery, 24 smartphone, 50 sociodemography, 175 socket preservation, 10 sodium lauryl sulphate, 67 stainless steel, 67 straightwire technique, 170 streptococcus mutans, 36 sobrinus, 36 students, 153 superimposition, 50 tamarind extract, 16 telescopic denture, 126 tensile strength, 16 tooth paste, 67 total sleep deprivation, 24 tubule occlusion, 99 type 1 collagen, 1 vegf, 115 vertical dimension of occlusion, 30 volume fraction, 57 white spot lesions, 170 wound healing index, 45 woven alveolar bone, 115 xerostomia, 187 authors index volume 53 ahliawan, jevina sicilia, 144 andriani, ika, 76 arnanda, bramita beta, 164 avianti, riski setyo, 62 barunawati, sri budi, 99 budisidharta, yoeliani, 45 devitaningtyas, nungky, 212 dianawati, nur, 36 fatikhah, nurul, 206 ginting, rehulina, 30 hepitaria, nova andriani, 20 hudyono, rikko, 81 ismiyatin, kun, 88 lubis, hilda fitria, 67, 149 maharani, elfira, 170 monika, fransiska, 191 mulawarmanti, dian, 111 nizar, much, 10 noor, tengku natasha eleena binti tengku ahmad, 187 oki, aqsa sjuhada, 196 ossa, yuli fatzia, 159 prabowo, teguh setio yuli, 40 prihastari, lisa, 175 rahmatillah, 181 rahmawati, atiek driana, 93 santoso, dewi sartika, 223 septyarini, brelian elok, 6 setijanto, darmawan, 122 shita, amandia dewi permana, 217 subiakto, birgitta dwitya swastyayana, 126 sularsih, 115 syahputri, vania, 107 tallo, fransiska rima, 201 utomo, haryono, 50 widjiastuti, ira, 1 widyaningsih, pratiwi nur, 24 widyasrini, dyah anindya, 57 widyastuti, 229 wulandari, erawati, 16 yani, ristya widi endah, 153 yohana, nelvi, 133 younus, mohamed salim, 140 zubaidah, nanik, 71 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and 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author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be selfexplanatory, containing data that is not duplicated within the text and figures. online submission the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair.ac.id/ mkg/about/submissions#onlinesubmissions the author can also submit the manuscript by sending email via the 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... ... ... ... ... ... ... ... ... . d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal nbkbmbi!lfeplufsbo!hjhj faculty of dental medicine, universitas airlangga editorial address c/o: jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone/fax: +6231 5039478 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/mkg/index i want to subscribe the dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) ................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... e-mail: ..................................................................................... date/tanggal: 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combined with hyaluronic acid metronidazole gel increased the quantity of osteoblasts in the alveolar bone wistar rat ernie maduratna setiawatie1,2 1department of periodontics, faculty of dental medicine, universitas airlangga 2institute of tropical disease, universitas airlangga surabaya indonesia abstract background: bone graft material have been used extensively in bone healing and periodontal treatment. alloplast such as hydroxyapatite are frequently used to repair and reconstruct bone defects. by merely applying hydroxyapatite for the treatment of bone is not fully effective yet to produce new bone regeneration. locally applied high molecular hyaluronic acid (ha) has been shown to stimulate differentiation and migration of mesenchymal cells. recent studies on regenerative surgical procedures indicate that reduction of bacterial burden at the wound site may improve the clinical outcome of regenerative therapy. metronidazole has the greatest bacteriostatic effect. a clinical application of ha metronidazole gels during the surgical therapy may reduce the bacterial contamination of surgical wound site. purpose: the purpose of this study was to examine the effect of combination hydroxyapatite and ha metronidazole gel 1% on osteoblast cell number after wound healing process in the wistar rats incisor tooth extraction socket. method: twenty seven wistar rats were divided randomly into 3 groups. the first group consisted of wistar rats given hydroxyapatite were subjected to the mandibular incisor extraction socket. the second group were given hydroxyapatite combined with ha metronidazole gel 1%. the control group were filled with blood. wistar rats were euthanized on day 14 and then preparation for histological examination was stained using hematoxylin-eosin and then the numbers of the osteoblasts were calculated. result: the differences in each group were tested by one way anova test (α=0.05). the numbers of osteoblasts in each group had a significant difference (p<0.05): the highest numbers of osteoblasts were found in the group that was given hydroxyapatite combined with ha metronidazole, followed by the lower numbers of osteoblasts in the hydroxyapatite group and the lowest numbers of osteoblasts were found in the control group. conclusion: adjunctive application of ha metronidazole to hydroxyapatite after tooth extraction increase the number of osteoblast in the treatment of bone defects on a wistar rat model. keywords: hydroxyapatite; hyaluronic acid metronidazole; osteoblast correspondence: ernie maduratna setiawatie, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: erniemaduratna@gmail.com introduction bone augmentation techniques are commonly employed in dentistry. the role of bone graft materials in regenerative procedures is based on that osteogenic, osteoinductive or osteoconductive potential. alloplast include hydroxyapatite, collagen-based matrices, calcium sulfate, and tri-calcium phosphate is a synthetic body material that has ostoeconductive capacity. however, hydroxyapatite is not fully effective yet to produce new bone regeneration. treatment of bone defects by using hydroxyapatite alone cannot be maximized in new bone forming. indeed, hydroxyapatite were found enhance osteoblast differentiation, but combined hydroxyapatite collagen or growth factor together were shown to accelerate osteogenesis.1,2 one of many approaches of tissue engineering is to create a device of similar mechanical and biological properties to the one of the substituted tissue. the use of regenerative materials and antibacteria which is used in conjunction with hydroxyapatite can increase dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p204-208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p204-208 205205setiawatie/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 204–208 the accomplishment of bone augmentation. it is expected that the use of regenerative materials such as hyaluronic acid, and antibacteria such as metronidazole which is used in conjunction with hydroxyapatite can increase the accomplishment of the bone healing. bone healing is a complex process that involves local and systemic organic activity and osteoblast are some of the cells directly involved in this mechanism. there are three phases in bone healing including reactive phase (phase inflammation and granulation tissue formation), reparative phase (cartilage callus formation and deposition of lamellar bone) and remodeling phase (formation of bone contour). osteoblasts plays an important role in the mineralization process. osteoblast proliferating from the pre-existing bone beneath the calcified cartilage, and in conjunction with angiogenic activity, form bone on the calcified cartilage stratum. this bone formation process is endochondral ossification. small bone defects, such as socket after tooth extraction, heal by direct synthesis of new bone to fill the defect. after extraction no bone formation occurred for the first week. at 8 days, new bone formation was noted throughout the alveolar bone, particularly under the wall but not on the surface of the bone lining the extraction socket. at 10 days, bone formation was noted on the surface of the socket wall. at 12 days, new bone formation continued along the socket wall and in the trabecular spaces surrounding the extraction site. osteoblasts regulates calcium and phosphate concentrations. in addition osteoblasts also express alkaline phosphatase in high quantities to the plasma membrane. osteoblasts as secretory metabolically active, producing a number of bmp-2, bmp-7 and growth factor. osteoblasts also express rankl and opg expression products of osteoblasts occurs during bone remodeling.3,4 in the early stages of inflammation, the tissues will contain a large amount of hyaluronic acid which is much needed for the process of bone regeneration. hyaluronic acid (ha) is hyaluronan/hyaluronate which is a non-sulfated polysaccharide component from the glycosaminoglycan group with high molecular weight (10,000–10,000,000 da). ha serves to accelerate the process of regeneration by way of chemotaxis, proliferation and differentiation of mesenchymal cells and share the role as bone induction with osteogenic substance such as bone morphogenetic protein-2 (bmp-2) and osteopontin.4,5 ha is one of the essential components of extracellular matrix, which plays a predominant role in tissue morphogenesis, cell migration, differentiation, and adhesion which will contribute bone healing properties. in the medical world apart from being used for therapeutic in dentistry, hyaluronic acid is also used for the treatment of the skin, joints, hair and eyes. 6 recent studies on regenerative surgical procedures indicate that reduction of bacterial burden at the wound site may improve the clinical outcome of regenerative therapy. metronidazole has the greatest bacteriostatic effect, particularly on porphyromonas gingivalis, aggregatibacter actinomycetemcomitans, prevotella oris and staphylococcus aureus strains, which are commonly found in oral lesions. a clinical application of ha metronidazole gels during the surgical therapy may reduce the bacterial contamination of surgical wound site, thereby, lessening the risk of postsurgical infection and promoting more predictable regeneration.7 considering that, no previous study exists on the use of hydroxyapatite combined with ha metronidazole as adjunctive to grafting process. the purpose of this study was to examine the effect of combination hydroxyapatite and ha metronidazole gel on osteoblast cell number after wound healing process in alveolar bone wistar rat. materials and methods the study is a laboratory experimental research with randomized post test only control group design. the sample used 27 wistar rats aged 8-16 weeks were divided randomly into 3 groups which had 9 wistar rat each. the study protocol had been approved by ethical committee of faculty of dental medicine universitas airlangga, indonesia (no. 197/kkepk.fkg/xi/2015). the first group consisted of wistar rats given hydroxyapatite were subjected to the mandibular incisor extraction socket. the second group consisted of wistar rats that were given hydroxyapatite combined with ha metronidazole on the mandibular incisor extraction socket. the third group as control which the mandibular incisor extraction socket was filled with blood. the materials used in the present study were ha metronidazole with a concentration 1% in a gel form and hydroxyapatite. ha metronidazole gel 0.5 mg was mixed with hydroxyapatite 0.5 mg in the same ratio 1: 1 were applied into the socket of dental extraction. the extraction cavity was then covered with coronally repositioned flap, sutured and compressed with the moist gauze. animals were placed on a soft diet for two days post surgically. prior to the treatment, the wistar male rats were acclimatized for one week. the rats were maintained in a 40 cm x 30 cm x 15 cm size plastic cage. they were given the same food and drink during the adjustment period. the plastic cage was covered with woven wire, and the cage floor was covered with husk. the cage was placed in an isolated room with good ventilation and natural lighting. the rats was decaputated 14 days after application by injection of ketamine 50 mg/kg. immediately after sacrifice, animals’ jaws were carefully dissected, and the experimental teeth with their surrounding bone tissue were block-sectioned using electric surgical saw. blocks were immediately fixed in a 70% buffered formalin solution to prevent tissue changes so they do not decay, or harden, increase the refractive index of various tissue components and increase tissue affinity against paint materials. after complete decalcification, blocks were routinely processed and embedded in paraffin. six mµ sections were cut in a buccolingual direction, mounted on glass slides, deparaffinized, hydrated and stained. after the 48 hours, the fixative was replaced with a new one and the tissues dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p204-208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p204-208 206 setiawatie/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 204–208 were cut much smaller so that the fixation could penetrate evenly into the tissues. in the second stage, the fixation was left in the solution for 48 hours. after fixation the tissues were rinsed with running water for 6-9 hours, and then put in a hno3 5% decalcification solution for 1 hour. on the next stage, hematoxylin and eosin (h & e) stain was performed on the preparations for histology. histomorphometric analysis was carried out on the healing sites. in each he stained slide, three digital images were captured at (×400) magnification. each group was observed with an olympus bx41 series microscope which is equipped with a d70 digital camera using the olysia software. the intensity of the color in the osteoblast areas could be seen as a quantitative value. this research data was analysed by one way anova statistical test and continued with tukey hsd. results osteoblast obseved were found in the apical third of the tooth socket. histological examination of all treated cavities sites demonstrated newly osteoblast of implemented materials. figure 1 shows histological imaged that observed under the microscope. normality and homogenity tests were carried out first to obtain data of the research. the kolmogorovsmirnov test was applied for normality test while the levene test was used for homogeneity test. results of the kolmogorov-smirnov test denoted the probability of each treatment group which were bigger than 0.05 (p>0.05). the values mean that all the data were taken from the symmetrical or normal distributions. in the levene test result on osteoblasts sig = 0.977 means p>0.05 data was homogeneous. the data acquired was known normally and distributed homogeneously, next step one way anova test was to carry out to determine the differences in the number of osteoblasts in all the three treatment groups. one way anova test continued tukey hsd test showed that the probability figure obtained was 0,000 (p<0.05). therefore the average of osteoblast cell on each group was a significant difference (table 1 and 2). discussion hydroxyapatite has been used for a variety of biomedical applications, including matrices for drug release control and bone tissue engineering materials. hydroxyapatite is chemically similar to the inorganic component of bone matrix–a very complex tissue with general formula ca10(oh)2(po4)6. hydroxyapatite exhibits strong affinity to host hard tissues. chemical bonding with the host tissue offers hydroxyapatite a greater advantage in clinical applications compared to most other bone substitutes such as allografts or metallic implants. the main advantages of hydroxyapatite are its biocompatibility, slow biodegradability in situ, and good osteoconductive capabilities. hydroxyapatite exhibits excellent biocompatibility with soft tissues such as skin, muscle and gums and also has been widely used to repair hard tissues. common uses include bone repair, bone augmentation, as well as coating of implants or acting as fillers in bone or teeth.8,9 bone healing includes a series of highly reproducible and rigidly controlled biologic events (inflammation, granulation tissue formation, epithelium formation and tissue remodelling) which begin with chemo attraction of cells that accumulate and debride the injured tissue, foreign material, and microbial cells. ha possesses various 11 figure 1. alveolar bone tissue preparations showing the existence of osteoblast quantity on (a) hydroxyapatite group; (b) hydroxyapatite combined with ha metronidazole group; and (c) control group. table 1. the mean and standard deviation of the number of osteoblasts cells no. group n x +sd sig 1. hydroxyapatite 9 14.00 + 2.739 0.00 2. hydroxyapatite + ha metronidazole 9 19.00 + 1.500 0.00 3. controlled 9 3.56 +1.236 0.00 table 2. significancy of tukey hsd test tukey hsd multiple comparisons dependent variable (i) group (j) group mean difference (ij) std. error sig. osteoblast ha hap + ha -8,222* ,651 ,000 control 7,222* ,651 ,000 hap + ha ha 8,222* ,651 ,000 11 figure 1. alveolar bone tissue preparations showing the existence of osteoblast quantity on (a) hydroxyapatite group; (b) hydroxyapatite combined with ha metronidazole group; and (c) control group. table 1. the mean and standard deviation of the number of osteoblasts cells no. group n x +sd sig 1. hydroxyapatite 9 14.00 + 2.739 0.00 2. hydroxyapatite + ha metronidazole 9 19.00 + 1.500 0.00 3. controlled 9 3.56 +1.236 0.00 table 2. significancy of tukey hsd test tukey hsd multiple comparisons dependent variable (i) group (j) group mean difference (ij) std. error sig. osteoblast ha hap + ha -8,222* ,651 ,000 control 7,222* ,651 ,000 hap + ha ha 8,222* ,651 ,000 11 figure 1. alveolar bone tissue preparations showing the existence of osteoblast quantity on (a) hydroxyapatite group; (b) hydroxyapatite combined with ha metronidazole group; and (c) control group. table 1. the mean and standard deviation of the number of osteoblasts cells no. group n x +sd sig 1. hydroxyapatite 9 14.00 + 2.739 0.00 2. hydroxyapatite + ha metronidazole 9 19.00 + 1.500 0.00 3. controlled 9 3.56 +1.236 0.00 table 2. significancy of tukey hsd test tukey hsd multiple comparisons dependent variable (i) group (j) group mean difference (ij) std. error sig. osteoblast ha hap + ha -8,222* ,651 ,000 control 7,222* ,651 ,000 hap + ha ha 8,222* ,651 ,000 figure 1. alveolar bone tissue preparations showing the existence of osteoblast quantity on (a) hydroxyapatite group; (b) hydroxyapatite combined with ha metronidazole group; and (c) control group. table 1. the mean and standard deviation of the number of osteoblasts cells no. group n x ±sd sig 1. hydroxyapatite 9 14.00 ± 2.739 0.00 2. hydroxyapatite + ha metronidazole 9 19.00 ± 1.500 0.00 3. controlled 9 3.56 ±1.236 0.00 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p204-208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p204-208 207207setiawatie/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 204–208 physiological and structural functions, which include cellular and extracellular interactions with growth factors and regulation of the osmotic pressure and tissue lubrication. all these functions help in maintaining the structural and homeostatic integrity of the tissue. these biopolymers are completely biodegradable and support the growth of fibroblasts, osteoblast, chondrocytes and mesenchymal stem cells. many proteins play a role in binding ha, namely, fibrinogen, fibrin, fibronectin, and collagen. all these molecules play an important role in wound healing.10 the mechanisms by which ha promote bone formation were attributed to some actions. ha accelerated bone regeneration by means of chemotaxis, proliferation and successive differentiation of mesenchymal cells. ha increased alkaline phosphatase and hence stimulate cell mineralization. ha allowed the early deposition of osteoid tissue by providing a scaffold on which osteoprogenitor cell attached and so stimulated osteoblastic differentiation. furthermore, the anti-inflammatory ability of exogenous ha may reduce the postsurgical inflammation and bacterial contamination when added to the surgical site and finally improve the surgical outcomes. through recognition of its viscoelastic nature, ha can influence the cell functions that modify the surrounding cellular and the extracellular micro and macro environments. the viscoelastic properties of the material may slow the penetration of viruses, and bacteria, a feature of particular interest in the treatment of periodontal diseases.11 in this study a good response is seen in the use of a combination of hydroxyapatite and ha metronidazole compared with merely using hydroxyapatite. it is seen from the increasing number of osteoblasts in the healing process of the male wistar rats’ sockets, because ha is distributed by polysaccharide components of the extracellular matrix of connective tissue and bone marrow which has a function, among others, to facilitate cell migration, cell movement, cell interaction, cell matrix adhesion, differentiation during the formation and tissue repair and regeneration. ha acting on mineralized tissue and non-mineralized is found in the extracellular space of the entire tissues and synthesized in the cellular plasma membrane. ha serves to accelerate the process of regeneration by way of chemotaxis, proliferation and differentiation of mesenchymal cells and share the role as a bone induction with osteogenic substance such as bone morphogenetic protein-2 and osteopontin.12 in vitro studies ha is known to have a role in improving the formation of osteoblasts of the bone by increasing differentiation and migration of mesenchymal cells.13 ha shares a role in inducing bone with the osteogenic substrate such as calcitonin and bone morphogenic protein (bmp). bmp are the growth factors generally known to stimulate new bone formation.12-14 ha is recognized by specific cell receptor cd44 acting as selective and protective quote around cell membrane. cd 44 can exist in numerous isoforms. ha binding properties of cd44 are determined by the isoform and the cell type on which it is expressed. ha participates in tissue repair and wound healing and is used topically as anti inflammatory and anti-oedematous agent. the antiinflammatory effect may be due to action of exogenous hyaluronan as a scavenger by draining prostaglandins, metalloproteinases and other bioactive molecules increases proliferation, metabolism and cell migration, thereby accelerating bone healing.15,16 recent studies on regenerative surgical procedures indicate that reduction of bacterial burden at the wound site may improve the clinical outcome of regenerative therapy. the most common chemotherapeutic agents are antimicrobials and anti-inflammatory drugs. they are administered either systemically or topically. topical antimicrobial agents for the treatment of bacterial anaerob include metronidazole. metronidazole is a derivative of nitroimidazole, it has antiprotozoal and antibacterial action. the mechanism of action of metronidazole is in biochemical reconstruction of its 5-nitrogroup by intracellular transport proteins of anaerobic bacteria and protozoa. recovered 5-nitrogroup of metronidazole is reacted with the dna of microorganism cell by inhibiting the synthesis of nucleic acids, which leads to the death of the bacteria. 7 tissue damage or impaired bone healing causes bacterial infection is a secondary result. acute alveolar osteitis is a relatively common complication of the routine extraction of teeth. antibacterial strategies have shown more promise. preoperative good results have been obtained with topical antibiotics including clindamycin, metronidazole and tetracycline.7 in the research, metronidazole was choosed as tropical antibiotic because its good safety profile, low risk of allergy and effectiveness against pathogens which cause oral table 2. significancy of tukey hsd test tukey hsd multiple comparisons dependent variable (i) group (j) group mean difference (i-j) std. error sig. osteoblast ha hap + ha -8,222* ,651 ,000 control 7,222* ,651 ,000 hap + ha ha 8,222* ,651 ,000 control 15,444* ,651 ,000 control hap -7,222* ,651 ,000 hap + ha -15,444* ,651 ,000 *. the mean difference is significant at the 0.05 level. hap : hydroxypatite dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p204-208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p204-208 208 setiawatie/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 204–208 infections. metronidazole is particularly suitable due to its restricted spectrum of activity against obligate anaerobes and its limited side-effects. metronidazole is classified in the who essential medicines list as antiamoebic, antigiardiasis, and antibacterial. since infection control is one modifiable factor, which can be controlled by the clinician, to so attempts have been made to incorporate metronidazole which is an antimicrobial drug along with hyaluronic acid gel to prevent bacterial contamination to be utilized as a graft material. gel with metronidazole improved wound healing during the 1st week after surgery owing to maximum growth factor concentration and antibiotic action for the first 5-7 days. it was also observed that significant improvement in bone regeneration (reduction in defect size) occurred over the entire course of healing as evident by the rapid and more complete bone growth.17 probably, the control of microbial population has resulted in successful healing and a good regeneration response. for the research, ha was used as carrier of metronidazole because ha are promising in drug delivery applications. ha affects the permeability of tissues and the transfer of other medical substances. ha plays invaluable role not only as an independent medicine, but also as an instrument of slow transfer of other therapeutic agents to the tissues, providing also their controlled release. biologically active components may be covalently or non-covalently bound to ha by varying of the concentration. it is possible to control the rate of its degradation or diffusion and, therefore, the speed of delivery of a medicine to the tissues. ha creates a depot of a medicine in the place of application and gradually collapsing frees a medicine, improving its pharmacological profile and preventing the development of possible adverse reactions. viscoelastic properties and shear thinning character was strongly dependent on ph. structured systems were obtained at ph 3, with an increase of several orders of magnitude in zero-shear viscosity values.18 the most recent research shows that ha assists the process of repair in both the hard and soft tissues. ha has a high capacity of water holding-one molecule of hyaluronic acid binds 200-300 water molecules. together with other proteoglycans hyaluronic acid is a member of the extracellular matrix. due to its physical and chemical properties such as high viscosity contributes to the manifestation of numerous functions of connective tissue. ha affects the permeability of tissues and the transfer of other medical substances. a clinical application of ha membranes, gels, and sponges during the surgical therapy may reduce the bacterial contamination of surgical wound site, thereby, lessening the risk of postsurgical infection and promoting more predictable regeneration.19,20 in conclusion adjunctive application of ha metronidazole to hydroxyapatite after tooth extraction increase the number of osteoblast in the treatment of bone defects on a wistar rat model. references 1. elkaragy a. alveolar sockets preservation using hydroxyapatite/ beta tricalcium phosphate with hyaluronic acid (histomorphometric study). j am sci 2013; 9(1): 556-63. 2. joung yk, heo jh, park km, park kd. controlled release of growth factors from core-shell structured plgs microfibers for tissue engineering. biomat res 2011; 15: 78. 3. yun yr, kim hw, jang jh. application of growth factors in tissue regeneration. biomaterial research 2013; 17: 133. 4. radhi hi, ghaban nm. evaluation the effect of hyaluronic acid on bone healing process in rabbits (immunohistochemical study for tgf-β. j bagh college dentistry 2015; 27: 111-6. 5. necas j, bartosikova l, brauner p, kolar j. hyaluronic acid (hyaluronan): a review. veterinarni medicina 2008; 53(8): 397411. 6. dahiya p, kamal p. hyaluronic acid: a boon in periodontal therapy. n am j med sci 2013; 5(5): 309–15. 7. zamani m, morshed m, varshosaz j, jannesari m. controlled release of metronidazole benzoate from poly(ε-caprolactone) electrospun nanofibers for periodontal diseases. eur j pharm biopharm 2010; 75(2): 179-85. 8. haider a, gupta kc, kang ik. morphological effects of ha on the cell compatibility of electrospun ha/plga composite nanofiber scaffolds. biomed res int 2014; 7: 306-9. 9. krishnamurty g. a review on hydroxyapatite-based scaffolds as a potential bone graft substitute for bone tissue engineering applications. jummec 2013; 16: 1-6. 10. bezzerra b, brazao ma, casati mz, sallum ea, sallum aw. association of hyaluronic acid with a collagen scaffold may improve bone healing in critical-size bone defects. clin oral implants res 2012; 23(8): 938-42. 11. gontiya g, galgali sr. effect of hyaluronan on periodontitis: a clinical and histological study. j indian soc periodontol 2012; 16(2): 184-92. 12. nie h, wang ch. fabrication and characterization of plga/hap composite scaffolds for delivery of bmp-2 plasmid dna. j control release 2007; 120(1-2) :111-21. 13. patterson j, siewa r, herringb sw, linc asp, guldbergc r, staytona ps. hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration. biomaterials 2010; 31(26): 6772–81. 14. joung yk, heo jh, park km, park kd. controlled release of growth factors from core-shell structured plgs microfibers for tissue engineering. biomaterial research 2011; 15: 78. 15. park hk, lee sj, jong-suk , lee sg, jeong y, lee hc. smart nanoparticles based on hyaluronic acid for redox-responsive and cd44 receptor-mediated targeting of tumor. nanoscale research letters 2015; 10: 288. 16. jordan a, racine rr, hennig jp, lokeshwar vb. the role of cd44 in disease pathophysiology and targeted treatment. front immunol 2015; 6: 182. 17. tripthi ps, girieh r, reethi b. prf gel as an medium for local delivery of metronidazole. ijhs 2014; 1: 113-22. 18. takeda k, sakai n, shiba h, kurihara, mizuno h, hiroaki. characteristics of high-molecularweight hyaluronic acid as a brain derived neurotrophic factor scaffold in periodontal tissue regeneration. tissue eng part a 2011; 17: 955–67. 19. baldini a dds, zaffe d md, nicolini g. bone-defects healing by high-molecular hyaluronic acid: preliminary results. ann stomatol (roma) 2010; 1(1): 2-7. 20. francesco b, enrico b, roberto b, carlo c. treatment of infrabony periodontal defects using a resorbable biopolymer of hyaluronic acid. a randomized clinical trial. quintessence int 2013; 44: 231–40. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p204-208 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p204-208 144 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 144–148 research report the influence of functional tooth units on body mass index in the elderly of the jember regency jevina sicilia ahliawan,1 zahreni hamzah1 and mei syafriadi2 1department of biomedical science, 2department of oral pathology, faculty of dentistry, universitas jember, jember – indonesia abstract background: tooth loss can disrupt the mastication process. mastication disorders limit the amount of food a person consumes, affecting nutrition. the number of functional tooth units (ftus) can affect nutritional status, measured using the body mass index (bmi). purpose: the purpose of this study is to analyse the effect of the number of ftus on the bmi in the elderly. methods: this research was conducted in four villages in the jember regency. the number of subjects was 189, aged 45 years and over. the number of ftus was calculated based on the anterior and posterior teeth that have contact with the antagonist and the bmi using the bmi formula. the data was analysed using a linear regression test; the level of significance is 0.005. results: a simple linear regression test showed that there was significant correlation between ftu and bmi (p=0.366). conclusion: the number of ftus affects bmi; the higher the number of ftus, the greater the bmi. keywords: bmi; elderly; ftu; mastication correspondence: zahreni hamzah, department of biomedical science, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. e-mail: zahreni.fkg@unej.ac.id introduction an elder is someone who has reached the age of 60 years.1 in 2017, the elderly population of the world amounted to 962 million people, double that of 1980. this number is expected to double by 2050 to around 2.1 billion.2 most elders aged ≥ 65 years have a health problem that is an effect of the aging process. this situation shows that the elderly deserve special attention in terms of health, including oral health.3 the aging process is partly caused by the failure of body cells to function normally or produce new cells to replace dead or non-functioning cells.4 in biological terms, the aging process is related to the gradual accumulation of various molecular and cellular damage. over time, this damage causes a gradual decrease in physiological reserves, an increased risk of disease and a general decline in the capacity of individuals, which ultimately results in death.5 in general, the aging process is associated with physical changes that make individuals more susceptible to chronic diseases. these changes can also be observed in the oral cavities of the elderly. in the oral cavity of an elder, there are changes in the structure of the teeth, oral mucosa, periodontal tissue, salivary glands and salivary secretions.3 there are degenerative changes in both major and minor salivary glands and also a decrease in salivary secretions.6 various changes that occur in the oral cavity of an elder can lead to tooth loss. the most significant factor that can cause tooth loss is caries and periodontal disease.7 according to the world health organization (who), elders aged 65 years and over in indonesia have tooth loss problems, with an average of 17 teeth per individual.8 a large amount of tooth loss in the elderly can disrupt the mastication process, which can affect the absorption of nutrients. this condition shows that tooth loss can cause a decrease in the quality of life of an elder.9 this includes decreased chewing ability, which makes people reluctant to enjoy food with family or friends (e.g. psychological domains, social relationships and the environment) and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p144–148 mailto:zahreni.fkg@unej.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p144-148 145ahliawan et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 144–148 may interfere with one’s relationships. chewing ability is also likely to affect general health – when the ability to masticate is reduced, the selection of food that is consumed is affected.10 the ability to masticate is influenced by the number of teeth that come in contact with the antagonist. the pair of teeth that come into contact with the antagonist are called the functional tooth units (ftus). these ftus are used to assess the ability of a person’s mastication because teeth that do not have contact with their antagonist do not achieve good masticatory functions.11 research on ftus was conducted by indrasari et al.11 and naka et al.12 regarding the effect of the number of ftus, especially posterior teeth, on the ability to masticate. the results of the studies showed that the number of ftus positively affected the ability to masticate.10,11 similar research conducted by shakina et al.13 and adhiatman et al.14 states that there is no a significant relationship between tooth loss and nutritional status. the difference between the previous study and this study is that the previous study only used posterior teeth, while this study used both anterior and posterior teeth. in addition, there is still little research on ftus, especially in indonesia. the purpose of this study was to analyse the effect of the number of ftus on the body mass index (bmi) in the elders of the jember regency. materials and methods this study is an observational analysis with a crosssectional study approach. it was conducted between january and february 2020 in four villages representing jember regency: 1) coastal area: kecamatan puger and desa puger wetan; 2) mountainous area: kecamatan jelbuk and desa sucopangepok; 3) urban area: kecamatan sumbersari and kelurahan kebonsari and 4) agricultural area: kecamatan sumberbaru and desa sumberagung. data was collected in the village hall of each village using total sampling, which is the examination the entire population. the study used the cluster-sampling technique. jember regency consists of four regions: urban, agricultural, coastal and mountainous areas. each of the four regions consists of several districts and one district was chosen as a research location. each district consists of several villages where one village was chosen as the research location, so the total number of villages used as research subjects to represent jember regency was four. jember was chosen as the research location because the researcher is located in the regency. the subjects of the study were divided into three age groups of the who: 1) middle-aged: 45–59 years; 2) elderly: 60–74 years; 3) old: ≥75 years.15 the variables in this study consisted of the independent variable that is ftu and the dependent variable that is bmi. age, gender, eating frequency, physical activity, tooth brushing frequency and plaque index were obtained with a questionnaire. the turesky-gilmore-glickman plaque index was used. the elders were instructed to brush their teeth and were then given a disclosing agent in order to see the plaque remaining in the oral cavity. the plaque that was assessed was plaque found on the facial and lingual parts of the teeth. the plaque index is calculated by dividing the total plaque score by the number of tooth surfaces examined.16 inclusion criteria were an age of 45 years and over, willingness to be responsive and cooperative and to be at the research site (village hall) at the time of the study. the exclusion criterium was diabetes mellitus. the ftus in the study used anterior and posterior teeth. each pair of anterior teeth in the upper and lower jaw were assessed as one ftu, so six ftus were complete anterior teeth. each premolar pair in the upper and lower jaw was assessed as one ftu, so the four ftus were complete premolar teeth. each pair of molar teeth in the upper jaw and lower jaw was assessed as two ftus, so eight ftus were complete molar teeth. a maximum of 18 ftus in the oral cavity were complete teeth.17 the teeth that were counted as ftus were as follows: natural teeth or dentures with implants and crowns restoration, permanent crown restoration, bridge dentures and removable dentures (i.e. teeth that can still function for chewing, restored teeth and carious teeth, which includes enamel caries, dentin caries and caries that have reached the pulp but have not caused severe crown damage). criteria for teeth that are not counted as ftu are: natural crowns lost due to caries, teeth with third-degree mobility and pain during percussion tests, teeth with severe natural crown damage (to be extracted), root-residual and m3 teeth, as not all individuals have m3 teeth.18–21 bmi is calculated using the following formula: weight (in kilograms) divided by the square of the height (in meters). then, the bmi score is classified into several who categories (table 1). body weight was measured using a weight scale, while height was measured using a microtoise – from the highest point of the head, called the vertex (crest of the head), to the lowest point of the calcaneus bone (calcanei tuberosity), called the heel.22 the results of the examination of the number of ftus and the bmi were then recorded on the examination form. this form is complemented by the identity of the subjects table 1. distribution of the elderly according to bmi classification bmi (kg/m2) age group (n) total 45-59 60-74 >75 underweight < 18.5 6 7 7 20 normal 18.5 – 24.9 26 57 15 98 pre-obesity 25.0 – 29.9 19 27 4 50 class i obesity 30.0 – 34.9 4 7 0 11 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p144–148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p144-148 146 ahliawan et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 144–148 and the questions were used as supporting data, which are eating frequency, physical activity, tooth brushing frequency, and plaque index score. the results were then tabulated and analysed using a simple linear regression test. this research has received ethical clearance from the research ethics commission of the dentistry faculty of gadjah mada university, with reference number 00353/ kkep/fkg-ugm/ec/2020. results this cross-sectional study approached 189 subjects aged 45 years and over. of these, 10 people were excluded because of the exclusion criteria, so the total number of research subjects included was 179. the participants were grouped by gender and age group. the percentage of women is 83.8%, while the percentage of men is 16.2%. the percentage of elderly people between 45 and 59 years of age is 31%, 55% are between 60 and 74 while those 75 years and over make up only 14% (table 2). tooth loss can affect the number of ftus. the average number of lost teeth shows an increase with age. the results indicate that average tooth loss is lowest between 45 to 59 years and highest at 75 years and over (table 3). table 4 shows that, on average, the elderly brush their teeth twice a day. however, only 12% have a plaque index score of 0 (no residual plaque), while most received scores of 2 and 3 (table 4). table 5 shows that the average elder eats three times a day, while most eat two or three times. the number of those that eat once a day or more than three times a day is very small (table 5). the bmi results were divided into four categories. the percentage of those with a normal bmi is 54.74%, 11.17% are in the underweight category, 27.93% are pre-obese and 6.14% are grade i obese. elders with class ii or class iii obesity were not found in this study (table 1). the average number of ftus decreases with age, as does the average bmi. this is shown in the graph in figure 1. although average bmi decreases with age, the bmi of the elders in all of the age categories is normal. the average number of ftus increases with increasing bmi (figure 1). figure 1. the average number of ftus and bmi according to age group. table 2. elderly distribution according to age and gender age group ∑ females ∑ males ∑ total middle-aged (45-59 th) 52 3 55 elderly (60-74 th) 84 14 98 old (>75) 14 12 26 total 150 29 179 table 3. average and standard deviation of tooth loss age group tooth loss average ± sd 45-59 10 ± 7.6 60-74 14 ± 9.7 >75 20 ± 8 table 4. tooth brushing frequency and plaque index score age group tooth brushing frequency/day plaque index score no tooth brushing <2x ≥2x 0 1 2 3 4 5 45-59 1 54 12 6 23 12 2 0 0 60-74 4 74 6 4 33 16 5 0 20 >75 0 17 0 6 3 6 2 0 9 total 5 145 18 16 59 34 9 0 29 table 5. eating frequency age group eating frequency/day 1x 2x 3x >3x 45-59 0% 29.09% 69.09% 1.81% 60-74 0% 34.69% 63.26% 2.04% >75 0.5% 30.76% 65.38% 0% total 0.5% 32.4% 65.36% 1.68% table 6. the average and standard deviation of ftus, bmi and tooth loss age group average ± sd bmi ftu tooth loss 45-59 24.25 ± 4.2 8 ± 5.2 10 ± 7.7 60-74 23.65 ± 4.2 6 ± 5.4 14 ± 9.8 >75 20.93 ± 3.9 3 ± 2.8 20 ± 8 table 7. linear regression test result model r r2 adjusted r2 error estimation std. sig. 1 0.366 0.134 0.129 0.40754 0.00 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p144–148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p144-148 147ahliawan et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 144–148 also, average bmi is inversely related to average tooth loss; therefore, tooth loss decreases with increasing bmi (table 6). tooth loss affects the number of ftus. the relationship between tooth loss and ftu was tested using a linear regression test for each age group; test results from each age group showed a significant correlation. the correlation coefficient of tooth loss and ftu shows a negative value, which means that the relationship is inverse. this means that the less the tooth loss, the greater the number of ftus (table 6). the relationship between age and ftus was tested using the pearson correlation test and a weak correlation was the result. likewise, the relationship between age and bmi has weak correlation. this study uses a simple linear regression test. the data was first tested using assumption tests to find out whether the data could be tested using linear regression. the simple linear regression test provided a significance value of 0.000. this value indicates that the number of ftus influences the bmi. value r in the linear regression test is 0.366, which is moderate. value r2 is 0.134, which means that the effect of the number of ftus on the bmi is 13.4% (table 7). discussion as people age, their number of ftus decreases (figure 1). this statement is in accordance with the average tooth loss in table 3, which shows that the older the person, the higher the number of lost teeth. research conducted by bashiru and oyenashia7 also explains that the number of lost teeth increases with age. this increasing tooth loss causes the number of ftus to decrease (table 1). the inverse relationship between ftu and the amount of tooth loss can be caused by the more number of tooth loss, the less likely the teeth to come in contact so the fewer the number of ftus. based on the results of the correlation test, age category shows that the number of lost teeth is related to the number of ftus; the number of ftus is also influenced by other factors. the position of the teeth that are in contact also affects ftus. when a tooth does not come into contact with its antagonist, it does not count as an ftu. the difference between anterior and posterior teeth also influences the number of ftus. posterior teeth, especially molars, were considered to have a larger role than anterior and premolar teeth in the masticatory function; therefore, molar teeth have a value of 2 ftus, while anterior and premolar teeth have a value of 1 ftu.7,17 this study shows that 96.67% of the elderly brush their teeth ≥2 times a day, but many still have a plaque index score of ≥2 after brushing their teeth (table 4). this shows that the average elder still does not brush their teeth properly. a lack of knowledge about how to brush teeth properly can result in remaining residual plaque or debris after brushing teeth. the accumulation of plaque attached to the tooth surface is likely to persist if it is not cleaned properly, dissolving enamel or dentin. if caries occur over time, a tendency for tooth loss can occur. tooth loss in the elderly is also related to the aging process, where changes occur within the soft and hard tissues. the aging process has an impact on dental and oral hygiene, including the increase in dental caries and periodontal disease. these, if not treated, can result in a decrease in alveolar bone density over time, which causes tooth loss.23,24 in this study, 54.74% of the elderly had a normal bmi, 11.17% were classified as underweight and the remaining 34.07% were pre-obese or obese (table 1). this shows that most had a normal bmi and only a few were underweight. therefore, the average elder has fairly good nutritional status, and few have poor nutritional status. the average normal bmi can be related to the number of meals eaten a day. most eat three a day, which is 65.36%, and the rest eat less than three times a day (table 5). obesity and pre obesity can be associated with physical activity. as people age, their physical activity decreases, which leads to weight gain or increased bmi.25 the average elder has a normal bmi. the bmi results shows that with age, bmi decreases (table 6); this is related to the aging process. body weight and muscle mass tends to decrease above 60 years of age.26 the relationship between the number of ftus and bmi can be seen in the graph in figure 1. in the graph, declining ftu is directly proportional to decreasing bmi. table 6 also shows that mean bmi increases with increasing ftu. in addition, the results of the statistical tests indicate that there is an influence of the independent variable, which is the number of ftus on the dependent variable, which is bmi. the effect of ftus on bmi was 13.4% and the rest was influenced by other factors. other factors that can influence bmi are age, physical activity, gender, variety of diet, genetic factors and the presence of systemic diseases. the influence of ftus on bmi is supported by research conducted by indrasari et al.10 that explains that ftus are related to the ability to masticate – the higher the ftu value, the better the ability to masticate and vice versa. good mastication is obtained when the minimum number of ftus in the oral cavity is 7.5.11 the increasing number of ftus in the elderly affects their ability to masticate. if an elder has good mastication ability, they are not too worried about their choice of food. conversely, elderly people with mastication disorders consume fewer fruits, vegetables and fibrous foods than those with normal mastication.27 this can ultimately affect nutritional status. based on research conducted by samnieng,17 a low number of ftus is associated with a poor ability to masticate and low fibre intake, so it can ultimately lead to poor nutritional status. this study has some limitations. it used a cross-sectional approach, so the distribution of research subjects in each group was uneven. also, it did not analyse the variables that have the potential to become a confounding factor. these variables, such as eating frequency, plaque index score dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p144–148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p144-148 148 ahliawan et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 144–148 and tooth brushing frequency, are only used as supporting data. this study also did not examine whether dentures or natural teeth influenced bmi, so the number of ftus was only calculated in total. future studies are suggested to include the analysis of other factors that can affect bmi. the conclusion of this study is that the number of ftus influences bmi moderately. the greater the ftu value, the better the mastication, resulting in a better bmi. acknowledgements this research was funded by the ‘environment, aging, and health’ elderly research group at the faculty of dentistry, university of jember. the author thanks the parties who collaborated and participated in this research. references 1. pusat data dan informasi kementerian kesehatan republik indonesia. situasi lanjut usia (lansia) di indonesia. 2016. available from: https://pusdatin.kemkes.go.id/article/view/16092300002/ infodatin-situasi-lanjut-usia-lansia-di-indonesia.html. accessed 2020 may 6. 2. department of economic and social affairs population division united nations. world population ageing. world population ageing 2017 highlights. new york: united nations; 2017. p. 1–46. 3. razak pa, richard kmj, thankachan rp, hafiz kaa, kumar kn, sameer km. geriatric oral health: a review article. j int oral heal jioh. 2014; 6(6): 110–6. 4. pathath aw. theories of aging. int j indian psychol. 2013; 4(3): 15–22. 5. world health organization. world report on ageing and health 2015. geneva: who; 2015. 6. ha mza h z , i nda r tin d, meilawaty z. t he progressive low chronic inflammation on oral tissue in elderly. in: proceeding of 1st international conference on medicine and health sciences (icmhs). jember: jember university; 2016. p. 177–81. 7. bashiru bo, oyenashia a grace. prevalence, causes and pattern of tooth loss among elderly people in port harcourt, nigeria. cent african j public heal. 2019; 5(2): 98–101. 8. yuniendra gg, rahardjo a, adiatman m, maharani da, thuy pav. relationship between oral health status and masticatory performance with nutritional status in the elderly. j phys conf ser. 2018; 1073(4): 042007. 9. rosli ti, chan ym, kadir ra, hamid taa. association between tooth loss and body mass index among older adults in kuala pilah, negeri sembilan. j sains kesihat malaysia. 2018; 16: 81–6. 10. samnieng p, lekatana h. oral health and quality of life among elderly in thai. j dent indones. 2016; 23(2): 40–7. 11. indrasari m, dewi rs, rizqi aa. the influence of the number of functional tooth units (ftus) on masticatory performance. j int dent med res. 2018; 11(3): 982–7. 12. naka o, anastassiadou v, pissiotis a. association between functional tooth units and chewing ability in older adults: a systematic review. gerodontology. 2014; 31(3): 166–77. 13. shakina t, masulili c, indrasari m. hubungan antara kemampuan mastikasi dan indeks massa tubuh (imt). j dent indones. 2014; : 1–20. 14. adhiatman a. gw, kusumadewi s, griadhi pa. hubungan kehilangan gigi dengan status gizi dan kualitas hidup pada perkumpulan lansia di desa penatahan kecamatan penebel tabanan. odonto dent j. 2018; 5(2): 145–51. 15. cakır o. accessibility of information technologies in the resting home. int j comput sci inf technol. 2012; 4(6): 1–10. 16. alse ass, anandkrishna l, chandra p, ramya m, kamath ps, shetty ak. educational intervention on the plaque score among hearing impaired children. j adv clin res insights. 2015; 2: 26–30. 17. samnieng p. relationship of nutritional status with oral health status in visual impairment. makara j heal res. 2015; 19: 34–8. 18. ueno m, yanagisawa t, shinada k, ohara s, kawaguchi y. masticatory ability and functional tooth units in japanese adults. j oral rehabil. 2008; 35(5): 337–44. 19. hsu kj, yen yy, lan sj, wu ym, chen cm, lee he. relationship between remaining teeth and self-rated chewing ability among population aged 45 years or older in kaohsiung city, taiwan. kaohsiung j med sci. 2011; 27(10): 457–65. 20. zelig r, byham-gray l, singer sr, hoskin er, marcus af, verdino g, radler dr, touger-decker r. dentition and malnutrition risk in community-dwelling older adults. j aging res clin pract. 2018; 7: 107–14. 21. adiatman m, ueno m, ohnuki m, hakuta c, shinada k, kawaguchi y. functional tooth units and nutritional status of older people in care homes in indonesia. gerodontology. 2013; 30(4): 262–9. 22. snell rs. anatomi klinik untuk mahasiswa kedokteran. 6th ed. jakarta: egc; 2006. 23. tiwari t, scarbro s, bryant ll, puma j. factors associated with tooth loss in older adults in rural colorado. j community health. 2016; 41(3): 476–81. 24. newman m, takei h, klokkevold p, carranza f. newman and carranza’s clinical periodontology. 13th ed. california: elsevier saunders; 2018. p. 944. 25. al-zeidaneen sa, hdaib mt, najjar yw, al-zidaneen ma. gender and physical activity: are they associated with body mass index in elderly jordanian people? rom j diabetes nutr metab dis. 2018; 25(3): 237–42. 26. estrella-castillo df, gómez-de-regil l. comparison of body mass index range criteria and their association with cognition, functioning and depression: a cross-sectional study in mexican older adults. bmc geriatr. 2019; 19(1): 339. 27. kwon sh, park hr, lee ym, kwon sy, kim os, kim hy, lim ys. difference in food and nutrient intakes in korean elderly people according to chewing difficulty: using data from the korea national health and nutrition examination survey 2013 (6th). nutr res pract. 2017; 11(2): 139–46. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p144–148 https://pusdatin.kemkes.go.id/article/view/16092300002/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p144-148 mkg vol 39 no 1 jan 2006 isi.pmd 1 lip and allar base dimensions among adolescence in healthy and post unilateral cleft lips correction in deutro-malaid population coen pramono d department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract the purpose of this study is to observe the differences between some anthropometric values of the lip and allar base area in normal and those who had undergone incomplete unilateral cleft lip surgical correction using millard’s i technique. the investigation was done on male und female adolescence of 12–15 years of age. thirteen anthropometric dimension variables were observed, as x1 = width of the allar base, x2 = the distance between two corners of the mouth, x3 = the distance between the center of the lip and the right mouth corner, x4 = the distance between the center of the lip and the left mouth corner, x5 = the distance between the center of the allar base and the center of the of upper lip, x6 = the distance between upper part of two philtrum pillars, x7 = the distance between two philtrum pillars base, x8 = the height of right philtrum pillar, x9 = the height of left philtrum pillar, x10 = the distance between the right allar base to the right mouth corner, x11 = the distance between the left allar base to the left mouth corner, x12 = the width of the right nostril, and x13 = the width of the left nostril. the t-test analysis showed the average of the anthropometric lip and allar base dimensions among male adolescence groups in variables of x1, x2, x4, x5, x6, x7, x10, x11, x12, and x13 shown with significant differences from those of some variables which had undergone incomplete unilateral cleft lip surgical correction (p < 0.05), and no significant differences shown in the variables of x3, x6, x8, and x9 (p > 0.05). the result of the test among the female group presented a significant difference in variables of x4, x5, x6, x9, x11, x12 and x13 (p < 0.05), differed what given in variables of x1, x2, x3, x7 and x8 showed with no significant difference (p > 0.05). it concluded that there are differences in dimensions of the lips and allar bases among normal and post-surgical groups both in adolescence males and females. key words: lip and allar base anthropometric dimensions, incomplete unilateral cleft lip post correction anthropometric dimensions, bird face millard's i surgical technique correspondence: coen pramono d, c/o: bagian ilmu bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction cleft lip is congenital abnormal space or gap in the upper lip, alveolus, or palate, the more appropriate terms are cleft lip, cleft palate or cleft lip and palate. this abnormality of growth occurs with unknown etiology. some presumable factors are genetic, diseases, malnutrition, environment, and lack of certain substances in the intrauterine during the growing process. to understand the causes of oral clefts, a review of nose, lip, and palate embryology is necessary. the entire process takes place between the fifth and tenth weeks of fetal life.1 cleft lip result from a failure of merging of the middle portion of the upper lip which taken place between the fourth and eight weeks fetal life.2 the occurrence of oral clefts in united states has been estimated as 1 in 700 births3 and in indonesia no accurate data of cleft incidence is found. cleft of the lip may range from a minute notch on the edge of the vermilion border to a wide cleft that extends in to nasal cavity and thus divides the nasal floor. cleft lip is the most common serious congenital anomaly to affect the oral region as this anomaly appearance may be grotesque because the deformity can be clearly seen therefore it might constitute a serious affliction to those who have it. clinical appearance of individuals who have this defect sometimes can not be covered as they usually have a specific face and lip pattern which can only be seen in cleft lip patients. there are many cleft surgical techniques developed to reconstruct the cleft lip with their advantage and disadvantages. some famous cleft lip surgical techniques were presented by rose, mirault, hagedron-lemeurier, tennyson, tennyson-cronin, wynn, millard, lemesuire, and skoog.4 all those surgical methods are focusing on skin and muscle closure, and used the opposite side as a guide in taking the measurement. often seen un-proportional surgical result as the surgery is done can not exactly base on the opposite side of its healthy lip. two surgical report given by o’neal et al.5 and puccket et al.,6 reported a surgical technique of lip reconstruction focusing on a necessary step to do a muscle reconstruction to achieve a good anatomical lip form. 2 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 1–7 amaratunga7 reported his work in observing the surgical result of cases with complete unilateral cleft lips had undergone surgery using millard i and lemesuire surgical techniques, done by comparing some lips components using symmetrical index. his study showed that the millard i technique has an advantage in achieving of a good nostril and cupid’s bow reformed, and the lemesuire presented a good result for vermellion and philtrum correction. in many publications of surgical techniques for cleft lips surgery are mentioned to the healthy site as the surgical guidance, the result of the surgery mostly is not based on the dimensions as usually used in orthodontic field. the surgical technique used is usually chosen by the surgeon and applied based on the surgeon experience and the surgical result expectation is the symmetrical result of the lip form in both sites. study of anthropometric of soft and hard facial tissues are given by many authors. winoto8 in her study of indonesian facial profile in surabaya was done by analyzing the cephalometric radiography x-ray and reported that in the population she had observed has the facial profile characteristic of a deutro malaid population. study of facial profile by camper in 1768,8 he reported his observation on the facial angle, which is created from perpendicular of two lines, line from points of glabella (g) to maxilla (a) points and a line from porion (po) to anterior spina nasalis (ns). this facial angle is important in determining the exact position of maxilla to cranial base. hamilah9 reported her study of facial soft tissue development related to the facial skeletal bone growth in adolescence. schwarz in19669 reported his study of the prerequisite of harmonious facial soft tissue. he suggested a harmonious profile region named by kiefer profil felt (kpf). this region is located behind the vertical line which taken in perpendicular from the soft tissue nasal tip to the horizontal line from nasion (n) to orbita (o) points defined as the kfp region. a harmonious face is categorized when lips and chin are located inside this vertical line. lips print pattern in yogyakarta population was studied by munakhir10 and he found that lips have its specific character according to its genes and the result of his study presented that lips have a print pattern which can be used for identification both in forensic and non forensic needs. in general, human body has its own specific body pattern that might be grouped or differentiated from races and ethnic to other ethnic groups. therefore lips and allar bases might have also a specific dimension in such ethnic groups indonesia has some varieties of ethnic groups believed to be the result of the migration of people from north mongolia, china, arab and europe.8 glinka11 reported his investigation on indonesian population presented that indonesian population gave a characteristic of at least 2 characters from 3 racial characters. some other racial characters also found existed in other races and fischer in year 1964 categorized the javanese population in a group of deutro-malaid.8 javanese population was recorded by citizen census in 1980 as the largest group population in indonesia,12 therefore an anthropological observation on normal lip dimensions in deutro-malaid ethnic group was done for a control group and might be necessary to be used as a reference point in any anthropological setting. observation of the surgical result in incomplete unilateral cleft lip patients from deutro-malaid group used a millard i surgical technique was done and comparing to those groups of the normal lips and allar bases region group from the same population. materials and methods the study was carried out on 70 adolescence deutromalaid population varying from 12-15 years old males and females, consisted of 40 with healthy lips (hl) and 30 selected patients with incomplete unilateral cleft lip (iucl) who had undergone surgical treatment using millard i technique in the department of oral and maxillofacial surgery, faculty of dentistry, gadjah mada university during the year 1994–1995. the hl group presented by 40 healthy lips students taken randomly from 440 students of the state junior high school in kotamadia yogyakarta. all of 440 students taken for sample groups have a familial back ground of javanese ancestor in 3 steps above. some anthropometric lip and allar base reference landmarks according to martin and knussmann13 were used and measured included: the width of the allar base (ac – ac) = x1, the distance between two mouth corners (ch – ch) = x2, the distance between the middle of upper lip to right mouth corner (ls – chr) = x3, the distance between the middle of upper lip to le ft mouth corner (ls – chl) = x4, the distance between the middle of allar base to the middle of the upper lip (sn – ls) = x5, the distance between two point of the upper philtrum pillar (x – y) = x6 the distance between two point in the philtrum pillar base (x1 – y1) = 7, the height of right philtrum (x – x1) = x8, the height of left philtrum (y – y1) = x9, the distance between right allar base to right mouth corner (acr – chr) = x10, the distance between left allar base to left mouth corner (acr – chr) = x11, the width of right nostril (np – np’r) = x12, the width of left nostril (np – np’r) = x13 and measured with sliding ruler caliper in millimeter (mm). two way analysis of variance was used and followed by t-test to analysis the statistical differences between the data. fi 1 s th t i l d k d 3pramono: lip and allar base dimensions among adolescence anthropometric landmarks notes: np-np’ = inner nasal diameter x – x1 = philtrum pillar height right side y – y1 = philtrum pillar height left side sn = nasal base point ls = upper lip center point results the result of measurement on lips and allar bases landmarks measured in hl group and ucl group can be seen as follows: tabel 1. the result of measurements in average (cm) of lips and allar bases in hl and iucl adolescence males groups hl-a1b1 iucl-a2b1 variable average sd n min max average sd n min max x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 3,200 4,330 2,780 2,660 1,285 0,732 1,255 1,250 1,250 2,092 2,037 0,780 0,745 0,283 0,334 0,399 0,307 0,195 0,144 0,172 0,158 0,158 0,187 0,280 0,189 0,146 20 20 20 20 20 20 20 20 20 20 20 20 20 2,500 3,600 2,200 2,100 1,100 0,550 0,950 1,050 1,050 1,800 1,800 0,550 0,550 3,600 4,800 3,700 3,200 1,700 0,900 1,500 1,650 1,650 2,500 2,500 1,050 0,950 3,447 4,607 2,860 2,907 1,127 0,800 1,427 1,293 1,327 2,367 2,400 1,053 1,120 0,220 0,335 0,203 0,252 0,183 0,165 0,294 0,167 0,308 0,226 0,151 0,223 0,345 15 15 15 15 15 15 15 15 15 15 15 15 15 3,00 4,100 2,700 2,700 0,600 0,500 0,900 1,100 0,900 2,00 2,200 0,700 0,700 3,700 5,100 3,200 3,200 1,300 1,100 2,100 1,600 1,900 2,700 2,700 1,500 1,400 hl and iucl in male groups adolescence female group table 2. the result of measurements in average (cm) of lips and allar bases in hl and iucl adolescence females groups hl-a1b2 iucl-a2b2 variable average sd n min max average sd n min max x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 3,220 4,325 2,760 2,595 1,135 0,865 1,232 1,160 1,160 2,092 2,070 0,877 0,842 0,283 0,334 0,399 0,307 0,195 0,144 0,172 0,158 0,158 0,187 0,280 0,189 0,146 20 20 20 20 20 20 20 20 20 20 20 20 20 2,500 3,600 2,200 2,100 1,100 0,550 0,950 1,050 1,050 1,800 1,800 0,550 0,550 3,600 4,800 3,700 3,200 1,700 0,900 1,500 1,650 1,650 2,500 2,500 1,050 0,950 3,347 4,173 2,787 3,033 0,927 0,640 1,280 1,200 1,347 2,213 2,240 1,107 1,087 0,220 0,335 0,203 0,252 0,193 0,165 0,294 0,167 0,308 0,224 0,151 0,223 0,345 15 15 15 15 15 15 15 15 15 15 15 15 15 3,000 2,900 2,600 2,700 0,600 0,500 1,000 0,800 0,800 2,100 2,100 0,800 0,700 3,700 4,600 2,900 3,200 1,200 0,800 1,400 1,600 1,600 2,400 2,400 1,500 1,300 figure 1. some antrhopometric landmarks and some points were created for his purpose of investigation.13 4 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 1–7 table 3. summary measurement result of 2 way analysis of variance on lips and allar bases dimensions between adolescence hl and iucl males and females adolescence group sources variable ssq df msq f r2 p between a (hl-iucl) x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 0,597 0,067 0,049 2,011 0,575 0,106 0,206 0,030 0,297 0,669 1,215 1,082 1,643 1 1 1 1 1 1 1 1 1 1 1 1 1 0,597 0,067 0,049 2,011 0,575 0,106 0,206 0,030 0,297 0,669 1,125 1,082 1,643 8,448 0,589 0,707 27,309 14,494 6,571 4,294 1,033 6,539 16,985 29,035 22,995 30,061 0,112 0,007 0,010 0,286 0,155 0,069 0,058 0,014 0,088 0,194 0,291 0,251 0,307 0,005* 0,548 0,592 0,000* 0,001* 0,012* 0,040* 0,314 0,012* 0,000 0,000* 0,000* 0,000* between b (gender) x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 0,017 0,622 0,032 0,005 0,516 0,001 0,100 0,146 0,032 0,076 0,044 0,108 0,030 1 1 1 1 1 1 1 1 1 1 1 1 1 0,017 0,622 0,032 0,005 0,516 0,001 0,100 0,146 0,032 0,076 0,044 0,108 0,030 0,244 5,463 0,466 0,070 13,015 0,005 2,093 5,073 0,707 1,920 1,045 2,296 0,550 0,003 0,069 0,007 0,001 0,139 0,001 0,028 0,070 0,010 0,022 0,010 0,025 0,006 0,629 0,021* 0,504 0,768 0,001* 0,810 0,149 0,026* 0,592 0,147 0,311 0,131 0,532 between ab x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 0,062 0,786 0,012 0,157 0,011 0,367 0,066 0,000 0,052 0,101 0,159 0,000 0,073 1 1 1 1 1 1 1 1 1 1 1 1 1 0,062 0,786 0,012 0,157 0,011 0,367 0,066 0,000 0,052 0,101 0,159 0,000 0,073 0,873 6,903 9,177 2,138 0,265 22,662 1,379 0,002 1,141 2,559 3,794 0,178 1,342 0,012 0,087 0,003 0,022 0,003 0,238 0,019 0,000 0,015 0,029 0,038 0,002 0,014 0,644 0,010* 0,679 0,145 0,165 0,000* 0,243 0,967 0,289 0,111 0,053 0,678 0,249 inner x1 x2 x3 x4 x5 4,667 7,518 4,553 4,860 2,617 66 66 66 66 66 0,071 0,114 0,069 0,074 0,040 5pramono: lip and allar base dimensions among adolescence the average difference among variables measured to hl and iucl males and females adolescence groups. the result of the t test statistical analysis can be shown in table 4 as follow. tabel 4. t test measurement result average difference of lip and allar base size of hl and iucl male and female group to inter ab source variation sources a1b1–a2b1 (male) a1b2–a2b2 (famele) variables t-test p t-test p x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 2,176 2,400 0,892 2,661 2,328 1,554 2,295 0,747 1,053 4,045 5,188 3,689 4,696 0,008* 0,018* 0,621 0,010* 0,022* 0,121 0,023* 0,536 0,297 0,000* 0,000* 0,001* 0,000* 1,395 1,316 0,297 4,729 3,506 5,179 0,635 0,690 2,563 1,783 2,433 3,093 3,058 0,164 0,190 0,765 0,000* 0,004* 0,000* 0,535 0,500 0,012* 0,076 0,017* 0,003* 0,004* notes: a1b1 = healthy lip adolescence male group a2b1 = post incomplete cleft lip surgery to adolescence male group a1b2 = healthy lip adolescence female group a2b2 = post incomplete cleft lip surgery to adolescence female group hl1 = healthy lips group male iucl1 = post correction of unilateral cleft lip male hl2 = healthy lips group female iucl2 = post correction of unilateral cleft lip female x1 = allar base width (ac-ac) x2 = mouth width (ch-ch) x3 = distance between the middle of lip to right mouth corner (ls-chr) x4 = distance between the middle of lip to left mouth corner (ls-chl) x5 = distance between allar base and the middle of the lip (sn-ls) x6 = distance between two highest points of philtrum pillars (x-y) x7 = distance between two lowest points of philtrum pillars (x1-y1) x8 = right philtrum height (x-x1) x9 = left philtrum height (y-y1) x10 = distance between right lateral allar base to right mouth corner (acr-chr) x11 = distance between left lateral allar base to left mouth corner (acl-chl) x12 = right nostril width (np-np’r) x13 = left nostril width (np-np’l) sources variable ssq df msq f r2 p inner x6 x7 x8 x9 x10 x11 x12 x13 1,068 3,164 1,902 3,000 2,598 2,762 3,106 3,607 66 66 66 66 66 66 66 66 0,016 0,048 0,029 0,045 0,039 0,042 0,047 0,055 total x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 5,343 8,994 4,646 7,034 3,718 1,542 3,536 2,078 3,381 3,443 4,180 4,305 5,354 69 69 69 69 69 69 69 69 69 69 69 69 69 table 3 (lanjutan) 6 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 1–7 discussion this research was done to observe the lips and allar bases dimensions on 70 adolescence of 12–15 years of age, males and females with healthy lips group (hl) and post surgical correction of incomplete unilateral cleft lips group (iucl) used a millard i surgical technique. the differences of lips and allars bases situation between 2 groups of hl and iucl can be explained as follows. in males group the result of the analysis in male group presented that the surgical treatment can not reestablish the anthropometry dimensions, shown in variables of x1 (ac–ac), x2 (ch–ch), x4 (lschl), x5 (sn–ls), x7 (x1–y1), x10 (acr–chr), x11 (acl–chl), x12 (np–np’r) and x13 (np–np’l) not as given in control group of (hl). this situation might be due to those variables of x1 (ac–ac), x5 (sn–ls), x10 (ac–chr), x11 (ac–chl), x12 (n–np’r) and x13 (n–np’l) are variables which included in the cleft side. in variable x2 (ch–ch) presumed to be a difficult landmark to measure as the landmark points are located in both this mouth corners known very unstable due to movements of muscles of the mouth. the orbicularis oris muscle consists anatomically and functionally of two parts, the superficial and the deep layers. the fibers of the superficial portion of the orbicularis oris muscle connect with the maxilla and septum above and with the mandible below. in the upper lip these fibers consist of two bands: the lateral band arises from alveolar process of the maxilla, opposite the lateral incisor tooth, the medial band connects the muscle with the septum. it is joined by the muscle of expression, which intermingle with it and participate in its function by their dilating or stabilizing effect or both. the superficial portion of the orbicularis oris muscle also brings the lips together and its fibers contract independently to provide fine shades of expression. the deep layers of muscles encircle the orifice of the mouth and function solely as mouth constrictor.14 in variables x3 (ls–chr) and x4 (ls–chl) shown with different result, this might be due the x3(ls–chr) not located in the cleft region but located in the normal side and the x4 (ls–chl) is included in the cleft side. in variables x3 (ls–chr), x6 (x–y), x8 (x–x1, x9 (y–y1) shown with no significant differences between hl group and ucl group (p > 0.05). in case of iucl the millard i used in the correction of group iucl, resulted a good result in the correction of the philtrum, presented by variables, the distance between 2 points in the upper philtrum pillars abbreviated in x6 (x–y), and 2 philtrum pillars abbreviated in x8(x–x1) and x9 (y–y1) statistically shown with no significance differences in these variables between hl and iucl groups. in fameles group the result of analysis in females group presented that the surgical treatment can not reestablishing the anthropometry dimensions in some variables shown by no significance differences (p > 0.05) in, x1 (ac–ac), x2 (ch–ch), x3 (ls–chr), x7 (x1–y1), x8 (x–x1) and x10(acr–chr) and 7 variables shown with significantly difference (p < 0.05) in x4 (ls–-chl), x5 (sn–ls), x6 (x–y), x9 (y–y1), x11 (acl–chl), x12 (np–np’r) and x13 (np–np’l). the same result as presented in the iucl males group, surgical correction in iucl can not perform the lips and allar bases dimensions perfectly when compared to the hl group. in 6 variables observed found with no significant difference are not related closely to the incomplete unilateral cleft side which had undergone surgical correction, differed what shown in variables which closely related to the cleft side in male and famele groups from those 2 groups of males and females observed, shown that these groups have the same differences in variables of x4, x5, x11, x12 and x13 to control group of hl (p < 0.05). this result represented that the lips form in males and females groups have the same post surgical result, that the lips form in iucl and hl are different significantly (p < 0.05). in both groups of iucl in males and females, a variable of x4 represented an important result for symmetrical situation of the upper lips. in iulc, the lateral defect of the orbicularis oris muscle related to the muscle discontinuity and lost part of the muscle tissue. closure of one site muscle discontinuity would be brought the lip become shorter in its horizontal dimension therefore it is difficult to find a lip in symmetrical form or has a dimension as given by hl after a cleft lip surgical reconstruction. the result of the observation in males on 13 variables taken from adolescence of 12–15 years old males and females from deutro-malaid groups shown that 9 variables were found different in x1, x2, x4, x5, x7, x10 x11, x12 and x13. in 4 other variables of x3, x6, x8, x9 found with no significantly differences. between males group, 7 variables were found difference in x4, x5, x6, x9, x11, x12 and x13. in other 6 variables of x1, x2, x3, x7, x8 and x10 found with no significant difference. the result of this observation concluded that the lips and allar bases regions between iucl group and hl group have different in dimensions. in complete unilateral cleft lip, the fibers of the orbicularis oris muscle, proceeding horizontally from corner of the mouth towards the midline, turn upwards along the margins of the cleft. they terminate laterally beneath the base of the alla of the nose and medially beneath 7pramono: lip and allar base dimensions among adolescence the base of columnella, in which most of them attach themselves to the periosteum of the maxilla, a few disappearing in the subcutis.14 in cleft lip patients the maxillo-premaxilla structures usually disturbed during the growing process, therefore an anomaly in this region usually clinically presented. changes in the maxilla and premaxilla regions might lead to develop a nose in asymmetry. the cartilage bone raised and bended to the direction of uncleft side, and might cause the collumella shorten and slanting to the cleft side. the amount of soft tissue lost in patients with unilateral cleft lips might present with different clinical appearance from patient to another patient. the lost of the orbicularis oris muscle continuity related to the degree of anatomical lip defect severity and in this observation can be presented by 7 variables observed, in which this muscle should be proceed horizontally in nature. the lips defect which given by birth caused by the orbicularis oris discontinuity usually also followed by skeletal defect. in patients who had suffered from cleft lip in any grades of severity and had undergone surgical correction therefore should not be compared to a healthy lip, as in the cleft lips, the total length of lips in born given could be found shorter then normal. the symmetrical lip analysis as reported by amaratunga7 might be the best method can be used to evaluate the surgical result in any degrees of cleft lips. the post surgical lip correction found in symmetry would be clinically more acceptable and found with beauty then corrected into an anthropology millimeter norm but found with unrealistic lip form. the control groups of healthy lips in adolescence deutro-malaid population at kotamadia yogyakarta had taken as a preliminary group for having the lips and allar bases anthropometric dimensions data should be further observed by increasing the number of the sample groups to achieve more representative and accurate data. those dimension values found in the control group might be important in contributing the anthropology literatures in the future. acknowledgement i would like to express my gratitude to the the department of higher education, students, teachers, head misters of the state secondary high school at kotamadia jogjakarta for their great contribution made this research possible to be done. references 1. langman j. medical embryology. 3rd ed. baltimore: william and wilkin; 1975. p. 1–6. 2. stevenson re, hall jg, goodman rm. human related anomalies. new york: oxford university press; 1993. p. 39–79. 3. jones c. the genetic of cleft lip and palate: information for families, chapel hill, nc. booklet cleft palate foundation, 2000. 4. krueger go. textbook of oral surgery. 4th ed. st louis: the cv mosby co; 1974. p. 192–293. 5. o’neal rm, greer dm, nobel gl. secondary correction of bilateral cleft lip deformities with millard’s midline muscular closure. plastic and reconstructive surgery j 1974; 54(1):45–51. 6. puccket cl, reinisch jf, warner rs. late correction of orbicularis discontinuity in bilateral cleft. cleft palate j 1980 january; 17(1):34–9. 7. amaratunga na. a comparasion of millard’s and lemesuirer’s methods of of repair of the complate unilateral cleft lip using a new symetry index. j oral maxillofac surg 1988; 46:353–6. 8. winoto ns. studi profil fasial indonesia di surabaya dengan pendekatan sefalometrik. desertasi. 1981. 9. hamilah d. pola pertumbuhan jaringan lunak kraniofasial serta kaitannya dengan pola pertumbuhan jaringan keras kraniofasial dan pertumbuhan umum. kajian sefalometrikrontgenografik dan fotometrik pada anak usia 6–18 tahun. desertasi. jakarta: universitas indonesia; 1991. 10. munakhir ms. pola sidik bibir penduduk propinsi daerah istimewa yogyakarta. kajian pada kelompok populasi jawa dan cina di sekolah menengah atas kotamadya yogyakarta. thesis. yogyakarta: universitas gadjah mada; 1995. 11. glinka j. gestelt und herkunft: betrag zur antrophologischer kleiderung indonesiens. studia instituti antropos. angestin. bein bonn verlag des antropos institute 1978; 35:155–6. 12. rasyid a. penduduk indonesia: hasil sensus penuduk 1980. biro pusat statistic jakarta; 1980. seri no. 2. 13. martin r, knussman g. handbuch der vergleichenden biologi des menschen. band i. stuttgart: gustav fischer verlag; 1988. p. 192–293. 14. fara m. the musculature of cleft lip and palate. in: reconstructive plastic surgery. principles and procedures in 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isi vol 39 no 3 juli-september 2006.pmd 98 effects of orthodontic forces on pulp tissue pinandi sri pudyani department of orthodontic faculty of dentistry gadjah mada university yogyakarta indonesia abstract numerous researches on pulp tissue changes caused by orthodontic forces have been performed, among others are: pulp angiogenesis, pulp tissue respiration rate, alkaline phosphatase and aspartate aminotransferase enzyme activities; micro vascular response inside the pulp and the effect of dental movement i.e. extrusion, intrusion, and torque. the result is still controversial, as some researchers claim that orthodontic force has a negative effect, others deny by saying there is no such effect on pulp tissue. key words: pulp tissue, orthodontic force correspondence: pinandi sri pudyani, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii sekip utara yogyakarta 55281, indonesia. of both groups. after 40 days dental extrusion, the first group showed two cases of light pulp inflammation reaction and big pulp stone forming; while in the second group there was no inflammation cell infiltration, no soft tissue changes and no deposition of reparative dentin on all cases.5 pulp tissue response caused by dental intrusive force several researchers believed that intrusive orthodontic force can harm microcirculation of the pulp.2,8,9 intrusion or other dental movement can obstruct pulp inside circulation causing pulp damage. one of the researchers said that the use of light force interrupted by several breaks will definitely decrease pulpal iatrogenic process, therefore, it was advisable to give an orthodontic force not more than a capillary pressure of 20 mm/hg, because blood constriction can be followed by necrosis. excessive intrusion and extrusion will lead to necrotic pulp tissue without any odontoblast layer regeneration. it was noticed that teeth with open apical foramen was prone to receive the impact of dental intrusion.8 statement in pertaining to the effect of apical foramen was still controversial, some researchers considered that the impact of intrusion depended on root forming stage. teeth with opened apex had better prognosis. the majority of researchers stated that orthodontic force will cause hyperemi, diapedesis, white blood cell marginalization, and vacuoles forming on odontoblast layer.8 although the intrusive pressure was light, it can cause hyperemic pulp and decreased blood circulation into the pulp.8,10 excessive force will end in an irreversible pulp damage.11,12,13 the decreases in pulpal blood flow was of brief duration (averaging approximately 32 minutes) and was followed by a prolonged period (48 hours). of the possible force vectors that can be applied to teeth during orthodontic introduction for years, pulp tissue response to orthodontic forces has been studied by researchers, so far, the results are controversial.1-3 histological test on previous studies show severe pulp degeneration in every observed case. it is said that the orthodontic forces has blocked collateral circulation, while pulp is the most sensitive tissue in human body. it is concluded that pulp tissue damage can not be avoided during orthodontic treatment.1 previous researchers stated that orthodontic forces had a negative effect on pulp tissue. the research result through a histological test, showed that dental pulp was influenced by orthodontic dental movement, it was proven by pulp reaction i.e. disrupted blood circulation, and necrosis.4 on the contrary, other researchers testified that orthodontic forces had no prolonged significant negative effect on pulp.3 numerous researches on pulp tissue changes caused by orthodontic forces have been performed, among others are: pulp tissue respiration rate,1 pulp angiogenesis,2 pulp tissue response due to dental extrusion,4 aspartate aminotransferase3,5 and alkaline phosphatase6,7 enzyme activities and pulp micro vascular response.8 the writing aims at studying the effects of dental pulp tissue after subjected to various orthodontic forces. pulp tissue response caused by dental extrusive force to learn the pulp tissue response after dental extrusion, the study was conducted on 2 research groups i.e. dental extrusion group with sectional system of 75 g force, and dental extrusion group with ¼ inch elastic -4.5 oz. force. after 10 days extrusion observation, both groups showed no inflammation response either in the pulp or in the hard tissue, but there was a pulp stone forming on several cases 99pudyani: effects of orthodontic forces on pulp tissue treatment, intrusion is thought to have the greatest impact on the apical region, it can continue to constrict the pulp, inhibiting pulpal blood supply. intrusive forces might move the apex of the tooth into close proximity with the base of the bony socket and thus compress the periapical blood vessels.10,13 a light intrusive force of 50 gm in 60 days on premolars caused a compression on monkey’s periodontal ligament.14 light force of 5–25 gm per maxillary incisor was recommended for clinical intrusion, although higher force can be utilized with anterior high pull head gear.15,16 short duration dental intrusion impact on pulpal blood circulation had been analyzed using a laser doppler flowmetry probe. the results were: no changes of blood circulation in the pulp after a brief intrusive orthodontic force and there was only 19% blood circulation decrease compared to control group, meaning a non-significant difference.10 the orthodontic force had an impact on pulp tissue by decreasing 27% of pulp tissue respiration rate. there was a positive correlation between age and the pulpal tissue respiration decrease. it was concluded that a short duration orthodontic force can produce biochemical and biologic changes on pulp tissue, and there was a bigger risk for older orthodontic patients.1 pulp angiogenesis during orthodontic dental movement angiogenic changes in pulp tissue during orthodontic teeth movements remain unknown. angiogenesis is the growth of new capillary structures before the organization into larger structures through the neovascularization process. angiogenesis may be found in embryo formation, tissue growth, healing of wounds, in an inflammation process and pathology related to growth of blood vessels.2 the research was done on 30 premolars which were extracted due to orthodontic treatment and contra-lateral teeth were used as control. the instrument used was a straight wire system on 0.5–1 n power for 2 weeks. brackets were placed from the second right to the second left premolars except on the teeth being used as control. the technique utilized was a 3-dimensional proliferative assay based on a previous research. observation of tissue culture was done on the 5th and 10th day. it was reported that micro-blood vessels were discovered on the 5th day and their growth was to reach its apex on the 8th day to the 10th. the amount of micro-blood vessels was more easily found significantly on pulp tissue that was moved than the control group on the 5th and 10th day.2 alkaline phosphatase activity on pulp tissue after subjected to orthodontic force alkaline phosphatase (alp) is an enzyme which is related to mineralization of tissue6,7 and found on the matrix vesicle of mineralized tissue. alkaline phosphatase plays a role in the early process of mineral deposition and calcification; and matrix vesicle plays an important role in the growth of extra-cellular matrix and tissue mineralization. a high alp activity can be seen on the pulp and can be detected in cells under the odontoblast cellular layer.6 several cells in the dental pulp such as fibroblast and odontoblast can synthesize alp. alkaline phospatase activity in human pulp is 8 times higher in reversible pulp inflammations and in irreversible inflammations is almost equal to normal pulp.6 this shows how alp plays a role in the metabolic shift of dental pulp and other organs. perinetti et al.7 conducted a research on alp activity in the pulp during orthodontic treatment on 16 patients. the measurement of alp activity was done on premolars which were extracted due to orthodontic treatment. dental extraction was done after 7 days of treatment. measurement of alp activity was done using visible uv spectrophotometer on 405 mm for 4 minutes. there was a significant decrease in alp activity on orthodontic patients. aspartate aminotransferase (ast) activity aspartate aminotransferase is an intra-cellular cytoplasmic enzyme and released to the extra-cellular in dead cells, and its activity in the extra-cellular environment can be considered as a sign of necrosis.3,5 even so the base ast level in the extra-cellular environment can also be detected as a turnover of the physiological tissue which results in the possibility of detecting ast in healthy and inflammed pulp tissue.3 the results showed an increase of ast in reversible pulp inflammations (7.98 ± 2,1 unit/mg of pulp tissue), a decrease in irreversible inflammations (2.28 ± 1,7 unit/mg) while in normal pulp tissue is 4.8 ± 0,7 unit/mg. perinetti et al.3 conducted a research on 17 patients where the first premolar tooth was extracted due to treatment with the straight wire system. the force used was 30-90 g. measurement was done after 7 days of treatment using visible uv spectrophotometer. the results showed a significant increase of ast activity in dental pulp being subjected to orthodontic force (6.7 ± 1.9 unit/mg of pulp tissue) and in the control group the result was 3.6 ± 1.4 unit/mg. the increase of ast activity was also found in the gingival crevicular liquid due to inflammation process17 and orthodontic force on teeth.18 changes in dental pulp tissue histology due to orthodontic force the research was conducted by applying torque movements on mandible incisors and bodily movements on mandible incisors in another group. treatment used the edgewise system by placing bands on the teeth and the size of the bracket slots were 0.18 inches with anchors on the canines. length of treatment was 21 days. the results of the treated group were as follows: teeth which were moved bodily showed an increase in the quantity of collagen fibers in the whole pulp and a decrease in cellular concentration. a decrease in fibroblast cells and 100 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006: 98–101 an increase in inflammed cells especially in the pulp crown area were also happened. blood flow was disrupted, number of blood vessels went down and were narrower compared to the control group. there was a extravasation of red blood cells and odontoblast damage which was varied according to the force applied to teeth. on torque movement to the lingual, there was odontoblast damage on the middle third of the root, and cellular damage on the labial and lingual side of apical section of dental pulp.9 discussion the effect of orthodontic force on varied dental orthodontic movements on pulp tissue so far is a controversial problem faced by researchers. most of researchers1,3,7,9 stated that orthodontic force presents a negative impact on pulp tissue, whereas other researchers said that orthodontic force creates no specific impact on pulp tissue.4,10,19 the result research on pulp tissue reaction towards orthodontic force, found histological tissue changes on almost all observed cases. the most frequent changes discovered were damages on odontoblast layer. the damage level depended upon the given force. on torque movements, the damage was in the middle third of the root, which was in accordance with the fulcrum of the teeth. on bodily movements, odontoblast damage was found on the pressuredas well as on the stretched-areas. the time-span of cellular damage fitted to the length of cellular activity on alveolar bone next to the bodily-moved tooth. odontoblast damage on bodily movements spread out to the dental root. this histological change was due to dental internal structure which was unable to stand an orthodontic force causing an impact on dental internal histomorphology,9 but further studies on pulp tissue after subjected to orthodontic force, found no significant difference. pulp hyperemi occurred only after the activation. it was concluded that orthodontic force yielded no significant difference on pulp vascular system.19 in relation to the rate of pulp tissue respiration caused by orthodontic force, it was reported that 72 hours of orthodontic force can decrease 27% of the respiration rate of human premolars. it was assumed that the decrease of tissue respiration will give a negative impact to cellular activities, particularly to the intracellular metabolism (in a biochemical manner).the decrease of pulp tissue respiration was not caused by cellular changes alone, but also by the changes of pulp tissue stroma around dental supporting tissues. the influencing factors to the changes were blood constriction and halted blood flow to the pulp.1,9 orthodontic force caused lots of micro-blood vessels forming found on the 5thday observation, and the peak forming was on the 10th day. this phenomenon was believed as a response of an increased angiogenic development factor in pertaining to orthodontic force.2 orthodontic force generated changes alkaline phosphatase (alp)7 and aspartate aminotransferase (ast) activities.3 alkaline phosphatase played an important role in the physiologic and pathologic pulp tissue mineralization. studies of alp activity on pulp tissue after subjected to orthodontic force, showed a decrease. it was concluded that orthodontic force did not broaden the predentin layer and did not form pulp stone either. the decrease of alp activity -as a consequence of the presence of orthodontic strengthwas probably caused by damaged cells inside the pulp which were responsive to alp enzyme. due to the fact that pulp cells i.e. fibroblast and odontoblast also synthesized alp, the decrease of alp could also be caused by disturbances of the cells.7 studies on orthodontic impact towards pulp tissue were also conducted on aspartate aminotransferase (ast) activity. the ast activity was used to predict pulp necrosis.3,9 a necrotic process was related to blood constriction and the halted pulp blood flow after the teeth were subjected to orthodontic force, causing the decrease of pulp oxygen.1,20 upon receiving a 30–90 g orthodontic force for 7 days, the teeth showed a significant increase of ast activity in dental pulp, and it assumed to be the initiation of an inflammation process. the decrease of tissue respiration rate and the increase of pulp tissue apoptosis were factors to influence ast increased activity.8,21 these changes had constricted and stopped pulp blood flow. the decrease of blood flow to the pulp caused the decrease of oxygen content and alkalinity of pulp tissue.3,19,21 orthodontic force impact on apoptosis i.e. cell death program was already examined.22 measurement was done by tunel (terminal deoxynucleotidyl by transferasemediated dutp nick and labeling) reaction, by counting the tunel-positive cells. the study showed that maximal apoptosis occurred 3 days after application of the appliance. most tunel-positive cells were found in the periapical area and inside the pulp tissue of the first and second molars. possibly, it was caused by high proliverative activity and cell turnover in the pulp cavity. tunel-positive cells might be osteocytes, osteoblast and macrophages. generally the apoptotic body will be deleted by macrophages and there was no inflammation reaction during apoptosis process. various orthodontic force to move teeth will produce pulp tissue changes, among other is a dental extrusion movement.4 many studies show no inflammation reaction on treated teeth, but it shows an odontoblast aspiration into dental tubules (22.5%) on several teeth. histological data shows that there is no pathological changes on pulp tissue on 10–40 days dental extrusion with 75 g initial force. the same thing happens to 10–40 days dental intrusion with 4.5 oz elastic. it is said that 25–30 g dental extrusion force on adults can prevent the damage of pulp tissue. it is concluded that the extrusive force used in this research, has not caused significant degenerated odontoblast.4 intrusive force must be carefully counted so that no impending damage occur to pulp or to dental supporting101pudyani: effects of orthodontic forces on pulp tissue tissues as well.10,23 a 4500 gm intrusive force is the force to be used to incisor teeth.10 the force utilization is based on an estimation that a tooth will move 0.028 mm as a response to 1000 gm force in 15 seconds. this is the force commonly used in orthodontic clinics, where no disturbances of pulpal blood flow take place, as a result of the incapability of the intrusive force to move the apical part. short duration force is presumably not strong enough to press periodontal ligament. dental movement of 0.028 mm, the average of periodontal ligament width at the apex is between 0.18–0.21 mm, then the resulting reduction of less than 17% periodontal space may be insufficient to compress the apical vasculature. it is assumed that compression of the periodontal ligament of one third or less will not alter its blood circulation. moreover, even quite heavy force may not easily cause excessive apical tooth movement because of the mechanical properties of the periodontal ligament that resist displacement. collagen fibers inside the periodontal ligament are oriented to resist intrusive forces and remain rigid when force application is brief.10 the conclusion reveals that it is compulsory to measure thoroughly the force to move teeth in orthodontic manner. several factors to be taken into account are: movement type, the size of given force, and patient’s age in relation to dental periapical condition. this consideration is based on studies which stated the negative impact of orthodontic force on pulp tissue. it is advisable to give force not more than a capillary pressure of 20 mm/hg in order to prevent the disruption of blood circulation into the pulp which can be followed by a pulp tissue necrosis. references 1. hamersky pa, weimer ad, taintor jf. the effect of orthodontic force application on the pulpal tissue respiration rate in the human premolar. am j orthod 1980; 77(4):368–78. 2. derringer ka, jaggers dc, linden rwa. angiogenesis in human dental pulp following orthodontic tooth movement. j dent res 1996; 75(10):1761–66. 3. perinetti g, varvara g, festa f, esposito p. aspartate aminotransferase activity in pulp of orthodontically treated teeth. am j orthod dentofac orthop 2004; 125(1):88–92. 4. subay rk, kaya h, tarim b, subay a, cox cf. response of human pulpal tissue to orthodontic extrusive applications. j endod 2001; 27(8):508–11. 5. spoto gs, fioroni m, rubini c, tripodi d, perinetti g, piatelli a. aspartate aminotransferase activity in human healthy and inflammed dental pulps. j endod 2001; 27(6):394–95. 6. spoto g, fioroni m, rubini c, tripodi d, stilio md, piattelli a. alkaline phosphatase activity in normal and inflammed dental pulps. j endod 2001; 27(3):180–2. 7. perinetti g, varvare g, salini l, tete s. alkaline phosphatase activity in dental pulp of orthodontically treated teeth. am j orthod dentofac orthop 2005; 128(4):492–6. 8. guevara m, mcclugage sg. effects of intrusive forces upon the microvasculature of the dental pulp. angle orthod 1980; 50(2):129–34. 9. anstendig hs, kronman jh. a histologic study of pulpal reaction to orthodontic tooth movement in dogs. am j orthod 1972; 42(1):50–5. 10. barwick pj, ramsay ds. effect of brief intrusive force on human pulpal blood flow. am j orthod dentofac orthop 1996; 110(3):273–9. 11. graber tm. orthodontic principles and practice. 3rd ed. philadelphia, pennsylvania: wb saunders; 1972. 12. graber tm, swain bf. current orthodontics concepts and techniques. 2nd ed. philadelphia, pennsylvania: wb saunders; 1975. 13. dermaut lr, de munk a. apical root resorption of upper incisors caused by intrusive tooth movement: a radiographic study. am j orthod 1986; 90(4):321–6. 14. dellinger el. a histologic and cephalometric investigation of premolar intrusion in the macaca speciosa monkey. am j orthod 1969; 53(3):325–55. 15. burstone cr. deep over bite correction by intrusion. am j orthod 1977; 72(1):1–22. 16. melsen b, agerbaek n, markenstam g. intrusion of incisors in adult patients with marginal bone loss. am j orhod dentofac orthop 1989; 96(3):232–41. 17. chambers da, crawford jm, mukerjee s, cohen rl. aspartate aminotransferase in crevicular fluid during experimental gingiva in beagle dogs. j periodontol 1984; 55(4):525–30. 18. perinetti g, paolantonio m, d attilio m, d archivio d, dolci m, femminella b. et al. aspartate aminotransferase activity in gingival crevicular fluid during human orthodontic treatment. a controlled shrt-term longitudinal study. j periodontol 2003; (1):145–52. 19. nixon ce, saviano ja, king gj, keeling sd. histomorphometric study of dental pulp during orthodontic tooth movement. j endod 1993; 19(1):13–6. 20. unterscher re, nieberg lg, weimer ad, dyer jk. the response of human pulpal tissue after orthodontic force application. am j orthod dentofac orthop 1987; 92(3):220–4. 21. stenvik a, mjor ia. pulp and dentine reactions to experimental tooth intrusion: a histologic study of the initial changes. am j orthod 1970; 57(3):370–85. 22. rana mw, pothisiri v, killiany dm, xu xm. detection of apoptosis during orthodontic tooth movement in rats. am j orthod dentofac orthop 2001; 119(5):516–21. 23. mc fadden wm, engstrom c, engstrom h, anhoim jm. a study of the relationship between incisor intrusion and root shortening. am j orthod dentofac orthop 1989; 96(5):390–6. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 157 vol. 43. no. 3 september 2010 indirect veneer treatment of anterior maxillary teeth with enamel hypoplasia devi eka juniarti department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: nowadays, aesthetic rehabilitation becomes a necessity. it is affected by patient’s background, especially career, social and economic status. the aesthetic abnormality of anterior teeth i.e discoloration, malposition and malformation can affect patient’s appearance, especially during smile. these dental abnormalities, as a result, can decrease patient’s performance. dental malformation, for instance, can be caused by developmental tooth defect, such as enamel hypoplasia. enamel hypoplasia is a developmental defect caused by the lack of matrix amount which leads to thin and porous enamel. enamel hypoplasia can also be caused by matrix calcification disturbance starting from the formation and development of enamel matrix causing defect and permanent changes which can occur on one or more tooth. purpose: the aim of the study is to improve dental discoloration and tooth surface texture on anterior maxillary teeth with enamel hypoplasia by using indirect veneer with porcelain material. case: a 20 years-old woman with enamel hypoplasia came to the dental hospital, faculty of dentistry airlangga university. the patient wanted to improve her anterior maxillary teeth. it is clinically known that there were some opaque white spots (chalky spotted) and porous on anterior teeth’s surface. case management: indirect veneer with porcelain material had been chosen as a restoration treatment which has excellent aesthetics and strength, and did not cause gingival irritation. as a result, the treatment could improve the confidence of the patient, and could also make their function normal. conclusion: indirect veneer is an effective treatment, which can improve patient’s appearance and self confidence. key words: indirect veneer, porcelain, enamel hypoplasia, aesthetics abstrak latar belakang: saat ini perbaikan estetik menjadi suatu kebutuhan. kebutuhan akan estetik dipengaruhi latar belakang penderita, terutama karir, status sosial dan ekonomi. hal ini disebabkan, kelainan estetik seperti diskolorasi, malposisi, malformasi, dapat mempengaruhi penampilan penderita terutama saat tersenyum. kelainan gigi tersebut pada akhirnya dapat memperburuk penampilan penderita. malformasi gigi dapat disebabkan oleh kelainan pada masa perkembangan gigi, misalnya hipoplasia enamel. hipoplasia enamel adalah kelainan perkembangan yang disebabkan sedikitnya matriks enamel sehingga terjadi ketipisan dan porusnya enamel. hipoplasia enamel dapat disebabkan gangguan kalsifikasi matriks saat pembentukan dan perkembangan matriks enamel, kerusakan dan perubahan permanen ini dapat melibatkan satu atau beberapa gigi. tujuan: tujuan perawatan ini memperbaiki diskolorasi dan tekstur permukaan gigi depan rahang atas akibat hipoplasia enamel menggunakan veneer indirek berbahan porselen. kasus: seorang penderita wanita 20 tahun dengan hipoplasia enamel datang ke rumah sakit gigi dan mulut pendidikan fakultas kedokteran gigi universitas airlangga. penderita ingin memperbaiki gigi depan rahang atas yang secara klinis tampak bercak putih opak seperti kapur dan porus pada permukan labial gigi. tatalaksana kasus: veneer indirek dengan bahan porselen dipilih sebagai restorasi oleh karena segi estetik, kekuatan dan tidak mengiritasi ginggiva. restorasi ini dapat meningkatkan kepercayaan diri penderita dan memperbaiki fungsi gigi. kesimpulan: veneer indirek merupakan perawatan yang efektif, dapat meningkatkan penampilan dan kepercayan diri penderita. kata kunci: veneer indirek, porselen, hipoplasia enamel, estetik correspondence: devi eka juniarti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl.mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: yuniartidevieka@yahoo.com case report 158 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 159–161 introduction aesthetic appearance is a major concern for most people, especially women. abnormality on the anterior teeth, especially upper jaw, will make everyone less comfortable and less confident.1 dental malformation, dental malposition, dental crowded, diastema, fractures, dental discoloration with or without caries, and developmental abnormalities caused by disturbances occured during tooth growth, such as enamel hypoplasia and tetracycline; can reduce the aesthetic appearance. one of the developmental abnormalities causing dental discoloration is enamel hypoplasia. enamel hypoplasia is caused by disturbances occurred during enamel matrix calcification process, initiated by the process of the formation and the subsequent growth of enamel matrix in the form of defect, i.e. permanent changes which can occur on one or more teeth.2 enamel hypoplasia can be considered as a developmental disorder caused by the reduced number of enamel matrix, causing hollow and thin enamel. degrees of hypoplasia that are caused by systemic disorders reflected in the severity, timing, and duration of occurrence of the disorder; as a result, the systemic disorders usually attack many teeth, and the defect positions of their enamel are related to enamel formed during the disturbances occured.3 the treatment of enamel hypoplasia can be postponed until patients began to care about her appearance. the purpose of the treatment is to improve the discoloration aesthetically. several techniques even have already been developed to treat teeth with enamel hypoplasia, such as composite resin restorations, composite direct veneer, porcelain indirect veneers and porcelain crowns. the selection of restoration depends on the severity of enamel hypoplasia.1,3,4 veneer technique is not a new way in improving the shape and color of teeth. nevertheless, precision factor is needed in the procedure of this technique which is still less understood by practitioners in indonesia. in the dictionary, veenering means to cover anything with a coating in order to have a better appearance. current development in dentistry and the various technologies supporting both manufacturing techniques and materials, especially with the discovery of the 7th generation of bonding technology, the dental veneers gives more satisfactory results. it can stimulate the development of cosmetic dentistry as the bonding technology and the veneer can mutually support each other.5 based on the treatment technique, it is known that there are two kinds of veneer, direct veneer and indirect veneer. the main material used for direct veneer is resin composite (partial veneer and entire veneer), especially nanofill type, since this material can be polished, so the result will look like the actual enamel and the polishing results can survive for long periods of time. materials used for indirect veneer are composite resin, acrylic resin, porcelain and ceromer. among those materials, porcelain can give the best results in durability and aesthetic color. porcelain is also considered as the material that can give the best results, especially in durability, color, translucency, dental adaptation, the edge density, biocompatibility, insolubility in oral fluids and a good surface texture. however, this material tends to become brittle.3,5 dental veneer is indicated for tooth discoloration with severe degrees such as tetracycline, restoration which has color changes, light diastema either central or multiple, enamel defect, and deformity of dental anatomy. nevertheless, it can be contraindicated if the patient wants cosmetic instant with a class iii malocclusion, edge to edge relationships and bad habits (such as bruxism, nails and pencils biting), short crowns, and enamel which can not be enough to be etched.5 the aim of this case report is to show how teeth with enamel hypoplasia can be treated by indirect veneer with porcelain material. case a twenty year old female patient came to dental hospital of dental faculty of airlangga university surabaya, with her main complaint about the color of her anterior teeth suffering from patches of white and porous. moreover, she had no pains, and the palatal of 11 was filled 2 years ago. based on the objective examination, teeth 13, 12, 11, 21, 22, 23 got color changes and had calculus (figure 1). there was no complaint on pressure and percussion tests. the teeth was vital and clinical diagnosis were enamel hypoplasia. the treatment plan was indirect veneer restorations with porcelain materials. finally, the prognosis of the treatment known was also good. case management the diagnosis and the treatment plans as well as digital photographs were conducted before the treatment. after obtaining patients approval, the suitable tooth color was selected through vitalumin shade guide, as a result, incisal section was a2, body was a2, and cervical was a3. the impression was then taken in order to obtain the working model of the temporary veneer. in the second visit, preparation on the labial surface of study model was done in order to make temporary veneer and it can be use as the guide of the preparation that would be conducted on the patient teeth later. tooth preparation was done on the third visit after gingival management was conducted by using retraction thread, so the margin of the preparation could reach sub gingiva, not exceed than 0.5 mm from the gingival margin. after infiltration anesthesia was given, preparation of the teeth was done according the preparation on the working model. the preparation on the tooth labial surface, was started by using depth cut bur to create the depth of preparation, 2 mm, so on the dental labial niche was then 159juniarti: indirect veneer treatment formed and continued by flattening the entire labial surface by using a two-grit diamond bur. the preparation in the cervical area, was precisely conducted on gingival crest with a depth of 0.5 mm and chamfer shaped. in the proximal area, the chamfer marginal preparation was done without eliminating the proximal contact area. the incisal area was cut about 1 mm parallel to the incisal area (figure 2). after all of those procedures were accomplished, the result of the preparation was smoothed with a finishing bur. after the preparation was completed, the entire tooth surface was then cleaned with pumice powder and water by using a rotary brush. nevertheless, before the first impression was conducted, the gingival management was conducted again by using retraction thread, so the result of the preparation, especially in the cervical area, could clearly be visible. then, the impression was taken by using double impression material. after that, bite registration was conducted, and temporary veneer was inserted with freegenol material. after obtaining a working model, the cervical area was ditched in order to obtain a clear preparation border. in the fourth visit, the porcelain veneer trial was conducted. the relationship between the teeth and the veneers were observed, whether there was a gap; whether the position of the veneer was stable; and whether the incisal area of the veneer was in conformity with the incisal area of the teeth. finally, the occlusion was examined. however the preparation of the teeth and the veneer restoration was started, the color selection of flowable composite resin was also conducted to maximize the aesthetic appearance. the teeth were cleaned with a mixture of pumice powder and water in order to remove the contamination of plaque and temporary crown-cement residue. in order not to impede the process of enamel etching, the pumice powder must not contain oil and fluorine. the working area was isolated with cotton roll and celluloid ribbon set on the interproximal teeth. etching process was conducted on the teeth with 37% phosphoric acid for about 20 seconds. the teeth were then washed with water spray and dried. after that the bonding material was applied, thinned by using air spray, and then exposed with visible light for 20 seconds. the preparation of the veneer restoration was conducted by etching procedure on the surface of the porcelain veneer using hydrofluoric acid, 9.5% for 30 seconds. next, it was washed with distilled water, and dried with air spray. silanes was applied as coupling agent, let stand for 1 minute, thinned with air spray, and then exposed with visible light for 20 seconds. a thin layer of flowable resin composite was then applied and smoothened on the entire surface of the veneer. afterwards, it was gently placed and pressed on the tooth surface that had been given the celluloid on the mesial and distal areas. the strong pressure, however, could cause cracks in the veneer porcelain. the excess composite around the veneer was then cleaned, and the veneer was exposed with visible light for 40 seconds on each side. during the exposure, the veneer must always be pressed with fingers. after the insertion was completed, the occlusion was then re-examined, and should be free from traumatic occlusion (figure 3). finally, the patient was advised to control further. the first controls were then conducted in one week, two weeks, and one year after the insertion process, not only based on the anamnesa results whether there were not any complaints from patients, but also based on the extraoral examination whether there were no abnormality and swelling. during intra oral examination, it is also known figure �. the condition of the anterior teeth before the treatment. figure �. tooth preparation. figure ��. the insertion of the porcelain veneer. 160 dent. j. (maj. ked. gigi), vol. 43. no. 3 september 2010: 159–161 that the veneer was not cracked or broken. based on the percussion and pressure test, it was known that there were no complaints and gingiva around the teeth was normal, and t teeth 13, 12, 11, 21, 22, 23 are known vital. finally, instructions must be informed to the patient in order to improve her oral hygiene. discussion the main objective of the restorative dentistry is not only to repair tooth defects through the restoration process of dental anatomy and function, but also to protect dental supporting tissues.6 veneer is a thin layer of tooth-colored materials applied to the labial surface of teeth in order to restore one or more teeth suffering from defects on their enamel, discoloration, malposition, and malformation.7 indirect veneer even has become an alternative treatment to restore anterior teeth because of the conservative principles of its preparation and its high aesthetic value. in recent years, porcelain veneer has been widely accepted as a restoration treatment in the aesthetic dentistry. porcelain veneer can also be used to correct the dental abnormalities related with its color, shape, and size, diastema, and dental fracture.8 diagnosis is the first important stage in the treatment and it is important to design the future treatment planning.7 thus, information about patient’s motivation, patient’s history of dental and general health, and patient’s problems related with his/her daily habits should be explored as complete as possible. future treatment plan is determined based on the clinic examination. in indirect veneer treatment, the selection of material used will be based on both patients’ expectation of care and their economic status.8 in this case, it is diagnosed that the patient got an intrinsic discoloration due to enamel hypoplasia. some of her anterior maxillary teeth even were clinically known suffering not only from white spots, but also from rough surface texture and porous. this discoloration of the tooth surface could be caused by enamel defects occured during periods of tooth growth. demineralization and failures in the process of calcification of the enamel, can produce white spot of hypoclasification.1 therefore, some enamel hypoplasia treatments, such as: tooth whitening, abrasion techniques, direct or indirect veneer, and jacket crown, can be choosed depend on its severity level. in enamel hypoplasia, in which lesions are clinically chalky white, tooth whitening can be conducted as the treatment. abrasion technique, on the other side, can be used for mild cases of enamel hypoplasia in which there are clinically small superficial ditches. in enamel hypoplasia, in which the majority of tooth enamel, ± 2 to 2.5 mm is attacked or only a thin layer of enamel is left, the direct and indirect veneers can become the appropriate treatments.9-11 veneer restoration is considered to be more profitable than jacket crown since the preparation only occurs on the enamel, so it will not disturb the vitality of the pulp and the health of the periodontal tissue.12 the selection of the indirect veneer with porcelain material in this case is actually because it can give aesthetic and strength as same as those in the original teeth, such as the color quite stable and natural; the periodontal health can be controlled since the surface of the porcelain can reduce the accumulation of plaque more compared to other kinds of veneer; the resistance to abrasion and the usage are high; it can also resist to fluid absorption; the rate of fractures is low; and the level of recurrent caries is low. porcelain veneer needs hydro fluoric acid 9.5% etching, which has high strength of adhesion on the etched enamel through the medium of resin-bonding, so the strength of this veneer is higher than that of other types.13,14 in addition, the success of the aesthetic restoration treatment on the anterior teeth related with the satisfaction of patients towards the results obtained is also determined by the communication not only between dentists and patients, but also between dentists with the laboratory technician.14 instruction after the insertion process must be informed to the patient. besides that, the veneer care related with its daily use, such as avoiding eating hard foods and chewing excessive burden, also needs to be informed to the patient. the patient is then asked to come to control a week later, and to have regular control every 6 months. with these recommended procedures, a good prognosis for long-term can be expected to be obtained. it can be concluded that the indirect porcelain veneer is an alternative treatment that can conservatively improve the aesthetic appearance by taking the enamel tissue as little as possible and leaving the healthy tooth tissue as much as possible. it can give better color reproduction and more natural form of teeth, get the optimal tooth function, including the optimal protection of tooth supporting tissues. in other words, the indirect veneer is an effective treatment that can improve the appearance of the patient. references 1. theodore m r, harald oh, edward js. art and science of operative dentistry.5th ed. carolina: mosby co; 2002. p. 623, 648–62. 2. spear fm, kokich vg, mathews dp. interdisciplinary management of anterior dental esthetics. j am dent assoc 2006; 137(2): 160-9. 3. powers jm, sakaguchi rl. craig’s restorative dental materials. 12th ed. philadelphia: elsevier inc; 2006. p. 454–6. 4. roulet jf. indirect aesthetic restoration. j adv aes rest dent 2003; 5: 15–9. 5. finke pm. the all ceramic system of the future. j adv aes rest dentj adv aes rest dent 2003; 7: 25–6. 6. ferrari m, vichi a, feilzer aj. materials and luting cements formaterials and luting cements for indirect restorations. in: roulet jf, wilson nhf, fuzzi m, eds. advances in operative dentistry. contemporary clinical practice. chicago: quintessence publishing co inc; 2001. p. 95–6. 7. heymann ho. additional conservative esthetic procedures. in: roberson tm, heymann ho, swift ej, eds. sturdevant’s art and science of operative dentistry. 4th ed. st. louis: mosby co; 2002. p. 12–24, 30, 606–7. 8. summit jb, robbins jm, hilton tj, schwartz rs. fundamentals of operative dentistry: a contemporary approach. 3rd ed. chicago: quintessence publishing; 2006. p. 571–84. 161juniarti: indirect veneer treatment 9. goracci g. direct posterior restorations-techniques of effective placement. in: roulet jf. wilson nhf, fuzzi m, eds. advances in operative dentistry. contemporary clinical practice. chicago: quintessence publishing co inc; 2001. p. 73. 10. hilton tj. direct posterior esthetic restorations. in: summit jb, robbin jw, schwartz rs. fundamentals of operative dentistry-a contemporary approach. chicago: quintessence publishing co inc; 2001. p. 265–70. 11. tracy r. enamel hypoplasia causes and treatment options. available from: www.docstoc.com. accessed december, 2008. 12. fuzzi m. clinical application of ceramic bonded retorations. in: roulet jf, wilson nhf, degrange m, eds. adhesion. the silent revolution in dentistry. chicago: quintessence publishing co inc; 2000. p. 8, 10, 303. 13. robbins jw. porcelain veneers. in: summit jb, robbins jw, schwartz re, eds. fundamentals of operative dentistry-a contemporary approach. chicago: quintessence publishing co inc; 2001. p. 432, 36–8. 14. robeson tm, heyman ho, swift ej. sturdevant’s art and science of operative dentistry. 5th ed. philadelphia: mosby inc; 2006. p. 610–1. vol 49 no 1 jan-mrt 2016.indd guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative 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i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be 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of beneficiary : ketut suardita 70 the response of periodontal ligament collagen fibres and the thickness of inserting periodontal ligament fibre bundles at cementum pressure sites of fixed orthodontic appliances noengki prameswari laboratory of orthodontics faculty of dentistry hang tuah university surabaya indonesia abstract previous research has indicated that there were several reactions in cellular activity and periodontal ligament collagen fibre as a response after orthodontic force application. cementum has function to give attachment to collagen fibres of the periodontal ligament, maintaining the integrity of the root, helping to maintain the tooth in its functional position in the mouth, and being involved in tooth repair and regeneration so in the orthodontic tooth movement can induce changes in the cementum. the aim of this research is to investigate that fixed orthodontic appliance can change the amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles at pressure site of cementum. this experimental study was held in laboratory with post test only control group design. twenty two (22) premolar sample from 11 patient were divided into 2 groups. k group as control group (without treatment) and p group as treatment group (with using fixed orthodontic appliance). the amount of periodontal ligament collagen fibre and thickness of inserting periodontal ligament fibre bundles was examined by light microscopy and measured by image tool program. in the summary, there are increasing amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles at cementum pressure sites as a normal response to remodeling and regenerating to orthodontic appliance and have function for strengthen adhering tooth cementum to the periodontal ligament. key words: fixed orthodontic appliance, collagen, cementum correspondence: noengki prameswari, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas hang tuah. jln. arif rahman hakim 150 surabaya 60111, indonesia. email: noengki_prameswari@yahoo.com introduction the use of orthodontics appliances has a principal to give continually pressure, until the tooth movement happens. it involves bone remodeling process, whereas resorption happens on the moved side, and apposition with a new bone at the opposite side. the pressure which is given to the teeth cause a pressure area and tension area at the teeth. on the fixed appliance, there is a light continuously forces and produced bodily movement. there are some changes on tooth movement in using fixed appliance, such as pain, cellular changes on the periodontal ligament, undermining resorption at the alveolar bone, and a changes on a root of teeth, including the cementum.1 after the treatment of orthodontics appliance is completed, relapse of teeth that move back to the beginning position can happen. the possibility of relapse, 40% came from the previous tooth movement.2 the teeth relapse process can occurs over 4–6 months period, and also can occurs until 1 year after the orthodontics appliance is removed.1 mechanism of relapsing itself is not clearly yet. factors that involve to unstable orthodontic treatment result are: the gingival and periodontal tissues are affected by orthodontic tooth movement and require time for reorganization when the appliance are removed, changes produced by growth may alter the orthodontic treatment result, the teeth may be in an inherently unstable position after the treatment. experiences held on the mouse molar relapse teeth show that fastly remodeling occur on the periodontal ligament and around alveolar bone, and hyalinization process and mineralization occurs on the pressure sites that can be resorpt by osteoclas, macrophagelike cells and fibroblast-like cells.3 so, it is important to know about root structure in functional position, ligament periodontal, bone, cellular response, extra cellular matrix that bind with cementum and changes in response to the orthodontics movement. there are many researches that has been done to human related with cellular changing, because of the force orthodontics appliance, but there are still much things that not clearly yet, especially about the changes of cell quantity on the root during the treatment using orthodontics apparatus. so it needs an advanced research. in experiment using proliferating cell nuclear antigen (pcna), during the use of orthodontics appliances, obtained on the tension areas, cell proliferation on the periodontal ligament reduce at the 3rd day until the 28th day, whereas on the pressure area at the bone surface area, cell proliferation increase at the 3rd day until the 10th day, and then reduce until 28th day, and cells that found at bone surface is the multinucleated 71prameswari: the response of periodontal ligament osteoclast, at the periodontal ligament is fibroblast, osteoblast, and cementoblast precursor cell. beside that, the increasing of cell apoptosis either at pressure area and tension area are founded.4 at the other research results there are some picnotic nuclei which is found at the periodontal ligament with the shrinkage signs and chromatin margination along the nucleus of membrane that it is a signs of necrosis.5 the research did to the maxillary first incisive, resulted that mechanics stresses that give to apical root during the movement of teeth cause the increase of the apical cementum thickness.6 at the other research found any collagen synthesis that increase at the periodontal ligament pressure sites compared at the tension area that showing a resorption area and remodeling that works very active to the pressure area followed by loosing of the hyalinization.7,8 one of the tooth structure that responsive to the force orthodontics appliance is cementum. primary function of cementum is giving adhering to the collagen fibre from periodontal ligament. beside it, cementum keeps the integrity of the tooth root, helps keeping the position of functional tooth in mouth, and help either to repair and regeneration of tooth. as the cellular process implication to the orthodontics treatment is the amount change of periodontal ligament collagen fibre and thickness of inserting periodontal ligament fibre bundles. this research using fixed orthodontics appliances that is used to the premolar subject that have extraction indication on the orthodontic treatment and after that examined on the pressure site of cementum, so that can knowing amount change of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles. biology of cementum cementum is the thin layer of calcified tissue covering the dentine of the root. cementum contains on a wet-weight basis 65% inorganic material, 23% organic material and 12% water. cementum varies in thickness at different level of the root. it is the thickest at the root apex and in the interradicular areas of multirooted teeth, and the thinnest cervically. the thickness cervically is 10–15 µm, and apically 50–200 µm. cementum is a vascular and has no innervation.9 the various types of cementum encountered may be classified in three different ways : the presence or absence of cells, the nature and origin of the organic matrix and combination of both. cellular cementum contains cells (cementocytes); acellular cementum does not. acellular cementum covers the root adjacent to the dentine, whereas cellular cementum is found mainly in the apical area and overlying the acellular cementum.9 acellular cementum consists of the first layer of cementum deposited at dentinocementojunction and also called primary cementum. it is formed at a slow rate and contains no embedded cementocytes. acellular cementum appears relatively structure less. in the outer region of the radicular dentine, the granular layer of tomes can be seen and outside this hyaline layer of hopewell-smith. a dark line may be discerned between the hyaline layer and the acellular cementum; this may be related to the afibrilar cementum that is patchily present at this position. cellular cementum or secondary cementum consists of the last layers of cementum, mainly in the apical one-third of the root. cellular cementum is formed at a faster rate than acellular, and thus many embedded cementocytes are found within it. at its periphery are the cementoblasts in the periodontal ligament, which allow for the future production of more cellular cementum if needed.9,10 cementum derives its organic matrix from two sources: from the inserting sharpey’s fibres of the periodontal ligament, and from the cementoblasts. classification cementum is decided according to the nature and origin of the fibrous matrix. when derived from the periodontal ligament, the fibres are referred to as the extrinsic fibres. these sharpey’s fibres continue into the cementum in the same direction as principal fibres of the ligament (perpendicular or oblique to the root surface). when derived from cementoblast, the fibres are referred to as intrinsic fibres. these run parallel to the root surface and approximately at right angles to the extrinsic fibres. where both extrinsic and intrinsic fibres are present, the tissue may termed mixed fibre cementum.9 the organic matrix of cementum is composed primarily of collagen. the principle collagen in cementum is type i with lesser amounts of types iii,v, and vi. sharpey’s fibres, which represent a major volume feature of cementum comprise most mostly type i, with type iii apparently coating type i collagen in these fibres. cementum contains two major non-collagenous proteins, bone sialoprotein (bsp) and osteopontin (opn).11,12 organic matrix and inserting periodontal ligament fibre bundle three cementum types differing in the presence of cells and collagen fibres are distinguished in humans. acellular extrinsic fibre cementum/periodontal ligament collagen fibre/periodontal ligament collagen fibre which serves the primary attachment function, covers cervical and middle portions of the roots, and it is usually confined to the coronal half of the root. it consists of a dense fringe of collagenous fibres implanted into dentinal matrix and perpendicular to the root surface. for this type of cementum all the collagen is derived as sharpey’s fibres from the periodontal ligament (the ground substance itself may be produced by the cementoblasts. the fibres are generally well mineralized.9,11,12 cellular intrinsic fibre cementum is composed only of intrinsic fibres running parallel to the root surface. the absence of sharpey’s fibres mean intrinsic fibre cementum has no role in tooth attachment. it may be found in patches in the apical region. it may be a temporary phase, with extrinsic fibres subsequently gaining a reattachment, or may represent a permanent region without attaching fibres.9 72 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 70–75 in mixed-fibre cementum, the collagen fibres of the organic matrix are derived from both extrinsic fibres (from the periodontal ligament) and intrinsic fibres (from cementoblast). the extrinsic and intrinsic fibres can be readily distinguished.9 the extrinsic fibres seen in ground sections may have unmineralised cores and called as inserting periodontal ligament fibre bundles. with total internal reflection of transmitted light, giving the appearance of thin black lines. that inserting periodontal ligament fibres bundle have an ovoid or round bundles about 5–7 µm in diameter.9 fixed orthodontic appliance and the response of cementum and matrix cementum to orthodontic movement orthodontic treatment is based on the principle that force application to a tooth causes remodeling of the periodontal ligament and the bone surrounding the root, which will finally result in displacement of that tooth. sandstedt reported already in 1904 on bone resorption on the ‘pressure’ side and bone deposition on the ‘tension’ side after force application to a tooth. it is necessary to consider the biologic control mechanisms that lead from the stimulus of sustained forced application to the response of orthodontic tooth movement. two possible control elements, biologic electricity and pressure-tension in the periodontal ligament (pdl) that affect blood flow, are contrasted in the two major theories of orthodontic tooth movement. the bioelectric theory relates tooth movement at least in part to changes in bone metabolism controlled by the electric signals that are produced when alveolar bone flexes and bends. the pressure-tension theory relates tooth movement to cellular change produced by chemical messengers, traditionally thought to be generated by alterations in blood flow through the pdl. pressure and tension within the pdl, by reducing (pressure) or increasing (tension) the diameter of blood vessels in the ligament space, could certainly after blood flow. the two theories are neither incompatible nor mutually exclusive. from a contemporary perspective, it appears that both mechanisms may play a part in the biologic control of tooth movement.1 alterations in the periodontal tissue during orthodontic tooth movement specifically affect the alveolar bone, periodontal ligament, and root surface of cementum orthodontic. orthodontic force initiation stimulates the remodeling of alveolar bone, which result in tooth movement. before this remodeling, initial changes in response to a local compression of the periodontal ligament and affect to the cementum, which have attachment to collagen fibres of the periodontal ligament.2,7 the response of cementum to the orthodontic movement is less readily resorbed, a feature is unknown but it may be related to differences in physicochemical or biological properties between bone and cementum, the properties of the precementum, the increased density of sharpey’s fibres particularly in acellular cementum, and the proximity of epithelial cell rests to the root surface.9 although cementum is less susceptible to resorption than bone under the same pressure (with orthodontics forces), most roots of permanent teeth still show small localized areas of resorption. the resorption is carried out by multinucleated odontoclasts. resorption deficiencies may be filled by deposition of mineralized tissue. a line known as reversal line may be seen separating the repair tissue from the normal underlying dental tissues. in this section, odontoclasts have resorbed through the thin layer of acellular cementum.7,9 changes also occur in the matrix of cementum. in the pressure sites at 14 days, the course of the collagenous fibres was completely disturbed. the process of removal of hyalinized tissue and revascularization had begun, new collagenous fibres had been formed. in the tension areas at 14 days sharpey’s fibres, consisting of type i collagen were embedded in the osteoid and in cementum. collagenous fibres appeared to be stretched although the width of the periodontal ligament was hardly larger than normal.2,7 repair is occurring and a thin layer of formative cells (cementoblasts) have deposited a thin layer of matrix (precementum) in the deficiency. the repair tissue resembles cellular cementum. the formative cells have a similar ultra structure to cementoblasts; its crystals are smaller; and calcify globules are present, suggesting that mineralization is not proceeding evenly.2,9 material and methods patients and orthodontic treatment of study group researches was done at department of orthodontics, dr ramelan hospital surabaya for patient’s research, laboratory of pathology anatomy of dr sutomo hospital to make a histology section, and laboratory of histology and anatomy of medical school airlangga university surabaya to make photomicroscopics and measurement calculation using image tool program. twenty two (22) maxillary first premolar with suitable indication to extract because an orthodontics reason from 11 patients (age 16–20, females), attending the department of orthodontic dr ramelan hospital surabaya, east java, indonesia were used as subjects. patients with carries and periodontitics will not use as a subject. all patients were given an enough explanation about the purpose and benefit of research, and asked for their willingness with signing an informed consent as a agreement.13 subjects were divided into 2 groups. right maxillary first premolar for each patient under treatment using fixed orthodontic appliance with 0.012 inch nitinol arch wire is placed in an 0.018 bracket with adjusting first ordered bends out step in the horizontal plane of space using angled utility arch pliers so that first premolar were retracted to bucal served as the test teeth (treatment group) and contra lateral first premolar as a control group (without treatment). after 10 days, the tooth were extracted. the extract procedure by using local anesthetics technique on bucal fold of first 73prameswari: the response of periodontal ligament premolar teeth and removal the teeth by luksation at bucalpalatal direction. teeth that extracted must be intact a whole of root structure, so that cementum can be seen fully. teeth that extracted yet kept in 10% buffer formalin solution. histological procedure after 7 days the teeth were kept in 10% buffer formalin solution, then do the decalcification so that the hard tissue loss their calcium. first, decalcify sections in large quantities of 5% aqueous solution of nitric acid for 1 to 4 days with changing the solution daily. then, washing in running water for 24 hours. after that, neutralizing in 10% formalin to which an excess of calcium or magnesium carbonate has been added, dehydrate, and clear.14,15 after that, cut sagitally specimen on bucal-palatal direction with thin section in the middle and prepare the preparation of section (figure 1). the specimens were embedded in paraffin with a conventional technique. section, 4 to 5 µm thick, were stained with malory acid.14,15 histology slides were examined by light microscoph nicon skt. s-123355 that is completed with digital camera viewing 400× enlargement. then, needed to decide part of section to measure. measured part is a half from apical to cervical cementum pressure area and cementum tension area at the other side per unit viewing area microscoph and then take a photograph. the measurement do with two observer dependent variable group control treatment x sd x sd amount of periodontal ligament collagen fibre and (amount fibre per unit viewing area) 20.8182 3.7635 24.3636 2.3779 thickness of inserting periodontal ligament fibre bundles (micron) 6.0418 1.0746 8.9773 1.4380 table 1. mean and standard deviation data of amount of periodontal ligament collagen fibre and (amount fibre per unit viewing area), and thickness of inserting periodontal ligament fibre bundles (micron) inserting periodontal ligament fibre bundles periodontal ligament collagen fibre figure 2. light micrograph of periodontal ligament collagen fibre and inserting periodontal ligament fibre bundles (×400). figure 1. sagital section of first premolar at middle bucal– palatal direction. for counting amount of periodontal ligament collagen fibre, and thickness of inserting periodontal ligament fibre bundle per unit viewing area (figure 2). ¾ crown ¾ neek ¾ root bucal first premolar (pressure sites) histology section measures part 74 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 70–75 result this experiment is investigating amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles as a effect of orthodontic appliance. data is observed from light micrograph photo of periodontal ligament collagen fibre and inserting periodontal ligament fibre bundles. descriptive analysis test shows that mean data of amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles is increased. mean of treatment group have amount periodontal ligament collagen fibre 24.3636 compare with control group 20.8182 and thickness of inserting periodontal ligament fibre bundles 8.9773 compare with control group 6.0418. manova test shows that amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles is increased significantly difference compare with control group (sig 0.01; p < 0.05) as a effect orthodontic appliance. discussion cementum prime function is to give attachment to collagen fibres of the periodontal ligament, is a highly responsive mineralized tissue, maintaining the integrity of the root, helping to maintain the tooth in its functional position in the mouth, and being involved in the tooth repair and regeneration.9 cementum is the site where soft tissue attachment has to be re-established and cementum matrix is a rich source of many growth factors which influence the activities of periodontal cell.12 cementum is unique tissue to respond orthodontic movement. cementum is less readily resorbed, a feature that is important for permitting orthodontic tooth movement and may be related to density of sharpey’s fibres, properties of precementum, and physicochemical/ biological bone.9,16 sample are taken in 10 days after tooth retracted because tooth movement happened after 7 days tooth retracted.1 in mabuchi,4 experiment, the difference cellular proliferation during the use of orthodontics appliances happened at 3rd day until 10th day, obtained on the tension areas, cell proliferation on the periodontal ligament reduce at the 3rd day until the 28th day, whereas on the pressure area at the bone surface area, cell proliferation increase at the 3rd until the 10th day, and then reduce until 28th day. cells changing as a normal response to remodeling and regenerating to orthodontic movement that found is the multinucleated osteoclast, fibroblast, osteoblast, and cementoblast precursor cell.6,7 during tooth retracted, angiogenesis, collagen synthesis and periodontal ligament collagen fibre component become active. in the pressure site there are cell changes involves migration, adhesion, proliferation, and differentiation of several cell type. all these activities are triggered when polypeptide mediators bind to their cell-surface receptor and when integrins bind to periodontal ligament collagen fibre component.12 in this experiment, amount of periodontal ligament collagen fibre, and thickness of inserting periodontal ligament fibre bundle were examined as a respond indicator of periodontal ligament collagen fibre activity at the cementum pressure sites to the orthodontic appliances. in this experiment, after orthodontic appliance, the amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles is increased. periodontal ligament collagen fibre and inserting periodontal ligament fibre bundle are produced by fibroblast, one of cells can change during orthodontic appliances. measurement can not be done direct to the fibroblast because of difficulty of seeing fibroblast at the malory acid staning. in the orthodontic appliance, in the cementum pressure site, there are up regulation activity of cell like osteoclasts, fibroblasts, osteoblasts and osteocytes precursor.2,6,17 one important function of fibroblasts is to increase periodontal ligament collagen synthesis as a cellular respond of homeostasis mechanism to tooth retracted. collagen that is produced giving adhering between cementum and periodontal ligament and keep the integrity of the teeth root, help keep the position of functional teeth in mouth to the mechanical stress such as orthodontic movement.2,17 remodeling collagen in the periodontal ligament during orthodontic movement have half lives 2 days.11 increasing amount of periodontal ligament collagen fibre, and thickness of inserting periodontal ligament fibre bundle in the pressure site related to increasing periodontal ligament collagen fibre synthesis. periodontal ligament collagen fibre determines the three-dimensional cell architecture and transmits and translates external mechanical and tensional forces to appropriate response signals. adhesion to periodontal ligament collagen fibre is essential for cell cycle. periodontal ligament collagen fibre also regulates gene expression of growth factors, growth factor receptors, and other protein, and determines the outcome of a cell’s response to growth factors. growth factors that regulate periodontal ligament collagen fibre function is platelet-derived growth factor (pdgf), which mitogenic to fibroblasts. other growth factors: transforming growth factor-b (tgf-b) and connective tissue growth factor (ctgf) activate collagen synthesis.12,18 increasing the amount change of periodontal ligament collagen fibre and thickness of inserting periodontal ligament fibre bundles related to mechanosensitive process in regulating cell’s volume because of mechanical stress such as orthodontic appliance.1,2,7,19 as a response to the mechanical force, cells bind to periodontal ligament collagen fibre through integrin, and the binding initiates a cascade of signaling reaction. signaling reactions activated include tyrosine phosphorylation of focal adhesion kinase (fak) and other signaling proteins, activation of mitogen-activated protein kinase (map kinase) cascade, expression of c-fos, and elevation of certain cyclin levels. 75prameswari: the response of periodontal ligament signaling pathways induced by extra cellular matrix components cooperate with those activated by growth factors in mediating their biological function, and both integrin-and growth-factor-induced-signals are necessary for expression og g1 cyclins and cell cycle progression from g0/g1 to s-phase. cyclin regulation is important for cell division.12,20,21,22 inserting periodontal ligament fibre bundles is a inserting bundle fibre from periodontal ligament to the cementum. the thickness of inserting periodontal ligament fibre bundles is important for strengthen tooth cementum in responding mechanical force with periodontal ligament.9 theory of inserting periodontal ligament fibre bundles is not knowing clearly yet. the thickness of inserting periodontal ligament fibre bundles is increase related to respond of mechanical stress orthodontic appliance. this function require active participation of integrins.12,20,21,22 one theory about inserting periodontal ligament collagen fibre is derived from periodontal ligament collagen fibre which change because of mechanical pressure from orthodontic appliance. mechanical pressure from orthodontic appliance induce cell migration and adhesion that need integrins function. in integrin expression, proteolysis is necessary for cell migration during tissue remodeling. proteolysis need matrix metalloproteinase (mmps) activity. activated inflammatory cells produce matrix metalloproteinase (mmps) than can degrade collagen and leads new transformation and composition adapted depending on the functional requirements of the tissue such as inserting periodontal ligament collagen fibre.11,12,23 in the summary, there are many changes to response mechanical force of orthodontic appliance. increasing amount of periodontal ligament collagen fibre and the thickness of inserting periodontal ligament fibre bundles at cementum pressure sites is a normal response to remodeling and regenerating to orthodontic appliance and have function for strengthen adhering tooth cementum to the periodontal ligament, keeping integrity of the tooth, and helping to maintain the tooth in its functional position in the mouth. acknowledgement this research was supported by a penelitian dosen muda grant from dikti, depdiknas, indonesia. references 1. proffit w. contemporary orthodontics. 2nd ed. st louis: mosby year book, inc; 1993. p. 266–339. 2. pillon j. orthodontic forces and tooth movement: orthodontic forces and relapse, an experimental study in beagle dogs. thesis. medical sc iences of the university of nijmegen. available from www.orthodontics.nl/research/phd_thesis/jjgm-pilon.htm. accessed january 13, 2005. 3. yoshida y, sasaki t. cellular roles in relapse processes of experimentally-moved rat molars. journal electron microscopy 1999; 48(2): 147–57. 4. mabuchi r, matsuzaka k, shimono m. cell proliferation and cell death in periodontal ligaments during orthodontic tooth movement. journal periodontal res 2002; 37:118–24. 5. hatai t, yokozeki m. apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement. oral disease 2001; l7:287–90. 6. shaw a, sameshima g, vu h. mechanical stress generated by orthodontic forces on apical root cementum: a finite element model. orthodontics & craniofacial research 2004; 7(2):98. 7. bishara s. textbook of orthodontics. philadelphia: wb saunders company; 2001. p. 468–72. 8. buman a, carvalho r, schwarzer c, yen e. collagen synthesis from human periodontal ligament cells following orthodontic tooth movement. the european journal of orthodontics 1997; 19(1):29–37. 9. berkovitz b, holland g, moxham b. oral anatomy histology and embryology. 3rd ed. st louis: mosby; 2002. p. 168–77. 10. nanci a. ten cate’s oral histology development, structure and function. 6th ed. st louis: mosby; 2003. p. 191–209. 11. waddington r, embery g. proteoglycans and orthodontic tooth movement. journal of orthodontics. british orthodontic society 2001; 28:281–90. 12. grzesik w, narayanan a. cementum and periodontal wound healing and regeneration. critical review oral biology and medicine 2002; 13(6):474–84. 13. pudjirahardjo w, poernomo h, machfoed h. metode penelitian dan statistik terapan. surabaya: airlangga university press; 1993. p. 57–8. 14. suntoro h. metode pewarnaan (histologi dan histokimia). penerbit bharatara karya aksara; 1983. p. 30–303 15. sudiana i. teknik praktis untuk jaringan sel. negara: cv dharma sandi; 1993. p. 2–35. 16. diekwisch t. developmental biology of cementum. international journal developmental biology 2001; 45:695–706. 17. bartold m, narayanan s. periodontal connective tissues. chicago: quintessence publishing co, inc; 1988. p. 173–92. 18. smith a. vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. journal of dental education 2003; 67(6):678–87. 19. foster t. a textbook of orthodontics. 3rd ed. oxford: blackwell scientific publications limited; 1997. p. 240–1. 20. schwartz m, assoia r. integrin and cell proliferation. journal of cell science 2001; 114:2553–60. 21. setyawan s. mechanotransduction-integrins and cellular responses. makalah seminar ikatan ahli ilmu faal indonesia cabang surabaya pada 22 agustus 2005. p. 1–5. 22. shyy j, chien s. role of integrin in endothelial mechanosensing of shear stress. circulation research 2002; 7(2):769–75. 23. kerrigan j, mansell j, sandy j. matrix turnover. journal of orthodontic 2000; 27(3):227–33. guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and 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endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case 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................................................................................................. ................................................................................................. 131 antibacterial force of the luting-type of glass ionomer cement toward lactobacillus species and streptococcus mutans dwi warna aju fatmawati and ekiyantini widyowati the conservation division of dental hospital the faculty of dentistry, the university of jember jember indonesia abstract rigid restoration is attached within oral cavity using adhesive cement layer. the hardening adhesive cement fills and tights the rough tooth surface with reciprocal opposing restorations. the luting type of glass ionomer cement was mostly used in the clinic for crown cementation as well as poured restoration. we can be taken a problem how strong is antibacterial effect of the luting type of glass ionomer cement to lactobacillus species and streptococcus mutans. the purpose of the research was to know the antibacterial force of the luting-type of glass ionomer cement toward lactobacillus species and streptococcus mutans. this research was a laboratory experiment. the samples of the research were divided into two groups, treated-group (fuji and shofu) and controlled-group. the numbers of samples in each group consisted of 7 pieces. taking 0,5 ml of artificial saliva in which the sample of the luting-type of glass ionomer cement (5 mm in diameter and 2 mm thick) had been immersed and storing it into petri dish containing warm mrsa and 0,1 ml lactobacillus sp. using poured technique. the mixture was subsequently incubated, and the colony was counted on the observation of 1st day, 7th day, and 14th day. the data were analyzed using anova and lsd. the result of the research showed that the greatest mean value of the bacterial colony presented in the controlled-group and the smallest was in the group of shofu. statistical analysis showed a significant difference (p < 0.05). the released fluoride from glass ionomer cement occurred in the damaging phase caused by polyacrylate that released h+ ion from carboxyl group (cooh). the fluoride influenced the growth of bacteria by decelerating the activity of gycolytic enolase enzyme. the luting-type of glass ionomer cement had antibacterial force toward streptococcus mutans and lactobacillus sp. the luting-type of glass ionomer cement of shofu possessed greater antibacterial force than fuji and controlledgroup. key words: antibacterial force, luting-type of glass ionomer cement, lactobacillus sp, streptococcus mutans correspondence: dwi warna aju fatmawati, the conservation division of dental hospital the faculty of dentistry, the university of jember. jln. kalimantan 37 kampus tegal boto jember, indonesia. e-mail: warna_ayu@yahoo.co.id. telp. 0331-333536. introduction rigid restoration is attached within oral cavity using thin-layered adhesive cement. the hardening adhesive cement fills and tights the rough tooth surface with reciprocal opposing restorations. adhesive cement is used to prevent edge leaking, and the cement layer should be made as thin as possible (approximately 80 um) so that it is not soluble within saliva.1 this hardening material not only covers the space but also retains the restoration. the material used in this requirement is classified as the type 1 cement.2 in the last two decades, adhesive cement has been developed into adhesive attachment and possessed therapeutic effects such as glass ionomer cement.3 glass ionomer cement was initially used by wilson and kent in 1971. the luting type of glass ionomer cement was mostly used in the clinic for crown cementation as well as poured restoration.4 the main advantage of this cement is its adhesive force upon dentin and email. according to subiyanto,5 this material performed chemical binding between atoms or molecules from the material and enamel or dentin resulting in stronger ion bindings or covalence bindings compared to physical binding this cement is also adhesive on the surface oxidized from alloy used for metalceramic restoration and upon the surface of tin-layered gold restoration1. this material functions to fill the roughness on the preparation and crown surface or pouring to provide mechanical attachment on the rough surface and result in an excellent restoration.6 subiyanto5 stated that fluoride ion consisted in ionomer cement was the effort to obtain anticariogenic and antibacterial traits. craig7 and summit8 said that there was no improvement in the number of bacteria during 56 days after crown cementation using glass ionomer cement. it was because the height of fluoride ion degree released from the glass ionomer cement. the most essential antibacterial effect of fluoride was disturbing the metabolism of bacteria.5 budi9 said that the main reason of restoration failure was caused by secondary caries. streptococcus mutans had important roles on the carries occurrence, or the restoration failure caused by very acidogenic streptococcus mutans which rapidly produced acid leading to the demineralization of apatite hydroxide. lactobacillus species is often called as predominant micro-flora within the lesion of deep dentine caries. it has 132 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 131-135 made lactobacillus species stated as the influential factor in the caries.10 ford1 said that lactobacillus species was the bacteria mostly found along dentine tube. it indicated that the ability of lactobacillus species to survive in the acid circumstance was as well as their ability to form acid itself. meanwhile, the number of bacteria within plaque on the tooth surface was very small and caused no email caries, however, it involved in damaging dentin. based on above elaboration, can be taken a problem how strong is antibacterial effect of the luting type of glass ionomer cement to lactobacillus species and streptococcus mutans. this research was aimed at knowing the antibacterial force of the luting-type of glass ionomer cement toward lactobacillus species and streptococcus mutans. the result of the research was expected to be advantageous not only to the development of dental material science of restoration but also to used for everyone who has competence in dental conservation. material and method this research was a laboratory experiment. the method used in this research was post-test only control group design. the objects of the research were divided into two groups, treated-group (fuji and shofu) and controlled-group. all of the instruments and materials used in the research were firstly sterilized in an autoclave within temperature of 121 °c for 30 minutes. the procedure of making suspension of streptococcus mutans was conducted by planting the culture in a reacting tube containing 5 ml sterile tripticase soy broth (tsb) unaerobically, and storing it in an incubator with temperature of 37 °c for 24 hours and subsequently followed by taking 1 cc streptococcus mutans from tsb using a disposable syringe, diluting it as many as 10–7. similar procedure was also carried out to make suspension of lactobacillus sp, however, it used manitol rogosa sharpe broth (mrsb) as the medium. the procedure of making sample (fuji and shofu) was through measuring the powder and the luting-type of glass ionomer cement according to the instruction of the manufacturer and mixing them to be a homogenous mixture and its consistence could be lifted and not cut off as high as ± ½–¾ inch, and storing in a brass plate cast with 5 mm in diameter and 2 mm thick and let it to setting (for 5 minutes). after setting, the sample was immersed in artificial saliva. only the controlled-group that used artificial saliva. the sample was then incubated in an incubator with temperature of 37 °c for 1 day, 7 days, and 14 days. the treatment procedure was carried out by taking 0,5 ml of sterile artificial saliva (containing immersed sample of the luting-type of glass ionomer cement (fuji and shofu brands)) and putting it in a sterile petridish. afterward, the saliva was added by 0.1 ml of streptococcus mutans and warm 25 ml tripticase soy agar (tsa). on the other petridish (poured plate technique), 0.5 ml of artificial saliva was mixed with 0.1 ml of lactobacillus sp. and 25 ml manitol rogosa sharpe agar (mrsa). the mixture was then stirred by spinning the petridish (poured plate techniqu) to be homogenous mixture. after tsa and mrsa media were hard, they were stored in the decisator with reversed position of petri dish and incubated in an incubator with temperature of 37 °c for 24 hours. observation was conducted on the 1st day, 7th day, and 14th day. the colony number was counted using a colony counter. the counting result of the streptococcus mutans colony was in the entity of colony forming unit (cfu) per milliliter.11 streptococcus mutans and lactobacillus sp were counted three times using a colony counter to obtain the mean. the counting results of streptococcus mutans and lactobacillus sp were in the entity of cfu per milliliter.11 the data were analyzed using one-way anova statistic test, and further differences of both materials used were analyzed using multiple comparison test by least significant difference (lsd). result table 1 showed that on the observation of the 1st day, 7th day, and 14th day, the mean of the number of lactobacillus sp in the controlled-group was greater compared to shofu and fuji. meanwhile, the mean of least number of lactobacillus sp was found in shofu. table 1. mean and deviation standard of the number of lactobacillus sp colony in the observation on the 1st day, 7th day, and 14th day samples the number of lactobacillus sp. (cfu/ml) 1 7 14 mean ds mean ds mean ds controlled 380.71 6.87 380.86 6.01 381.57 6.85 shofu 183.57 4.93 286.57 4.72 324.86 4.30 fuji 268.29 2.56 313.29 5.28 349.29 4.31 133fatmawati and widyowati: antibacterial force of the luting-type table 2. the result of one-way anova test toward the number of lactobacillus sp colony df sig. 1st day between groups within groups total 2 18 20 .000 7th day between groups within groups total 2 18 20 .000 14th day between groups within groups total 2 18 20 .000 the result of the normality and homogeneity test found that the significant value was greater than p > 0.05, it meant that the data of analyses obtained in each variable were normal homogeneity. the subsequent test was one-way anova test and lsd with significant level p < 0.05. this is showed in the table 2 and 3. in the table 2, the result of anova test showed that the significant value of antibacterial test of the luting-type of glass ionomer cement toward lactobacillus sp was p < 0.05. the result of lsd test showed that there was a significant difference between the controlled-group and the treated-group. table 4 showed that in the observation of 1st day, 7th day and 14th day, the mean of colony number of the streptococcus in the controlled-group was greater than treated-group. meanwhile, the least number was found in shofu group. the result normality and homogeneity test showed that significant value was greater than p > 0.05, it meant that the data of analyses obtained in each variable were normal homogeny. the one-way anova test and lsd showed the significant level p < 0,05 as showed in table 5 and 6. in the table 5, the result of anova test showed that the significant value of antibacterial force of the lutingtype of glass ionomer cement toward lactobacillus sp was p < 0.05. the lsd test (table 6) showed that there was a significant difference between the controlled-group and the treated-group. discussion the result of the research showed that mean value of antibacterial force of the luting-type of glass ionomer cement toward lactobacillus sp and streptococcus mutans was higher than treated-group, while glass ionomer cement of shofu had greater antibacterial force than glass ionomer cement of fuji. analytical test showed that there was significant difference. it was because the powder table 3. the result of lsd test toward lactobacillus sp colony (i) (j) controlled shofu fuji 1st day controlled shofu fuji 197.1429* 112.4286* 84.7143* 7th day controlled shofu fuji 94.2857* 67.5714* 26.7143* 14th day controlled shofu fuji 56.7143* 32.2857* 24.4286* note: * significance table 4. the result of mean and deviation standard of the number of streptococcus mutans colony on the observation of 1st day, 7th day, and 14th day samples the number of streptococcus mutans colony (cfu/ml) 1 7 14 mean ds mean ds mean ds controlled 451.71 10.70 475.28 9.69 491.28 12.29 fuji 273.14 5.08 356.00 7.07 385.42 5.56 shofu 128.71 7.38 243.00 5.91 351.57 6.29 134 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 131-135 composition of the luting-type of glass ionomer cement shofu had additional tannin-fluoride (hy-agent), while the luting-type of glass ionomer cement fuji had no such material. according to ikeda et al.,12 tannin-fluoride (hyagent) was able to decelerate the growth of bacteria in saliva and tooth plaque. the presence of tannin-fluoride (hy-agent) in large number would be very advantageous to eradicate the bacteria in the oral cavity and to assist controlling and preventing secondary caries. this was also confirmed by yamaga et al.13 that tannin-fluoride (hyagent) would result in penetrating fluoride into dentine more deeply so that it allowed to form binding with more stable apatite that reduced of caries resistance. according to subiyanto5 and summit et al.,8 the fluoride releasing from glass ionomer cement happened in the damaging phase caused by polyacrilate acid, it released ion h+ from carboxyl cluster (cooh) resulted in the separation of f– from the glass composition into diluted cement phase. anusavice2 revealed that high concentration fluoride directly resulted in reducing the population of bacteria. the higher separation of fluoride caused by the lutingtype of glass ionomer cement made the ph of saliva reduced, while the concentration of saliva increased. on the other hand, the ph of bacterial intracellular liquid was higher than saliva, while the concentration of bacterial intracellular liquid was less than saliva. the difference of ph and concentration between saliva and bacterial intracellular liquid allowed fluoride to enter within the bacteria, affecting the carbohydrate metabolism leading to the change of cell structure. the fluoride released by glass ionomer cement influenced the growth of streptococcus mutans by decelerating the activity of glicolotic enolase enzyme. the decrease of glicolotic enolase enzyme would reduce the number of phosphoenolpyruvate (pep) that was necessary to transport glucose into the cells so that made glycolysis and synthesis of the intracellular glucose decelerated. streptococcus mutans entered into the bacteria in the oral cavity that were sensitive toward the lower degree of fluoride in the lower ph environment.14 normally, streptococcus mutans live well in the ph of 7,4-7,6 and the optimum temperature for their growth is 37 °c, their growth reduces rapidly in the temperature of 40 °c.15 summit et al.8 stated that ph influenced the fluoride separation. it was assumed that the ph of the artificial saliva reduced because of the ion h+ that was released by polyacrylate acid caused erosion the surface of glass ionomer cement. kuhn and wilson in the seppä et al.16 revealed that the separation of glass ionomer cement fluoride was based on three reasons; surface wash-off, dissolution from cracks and fissures, and dissolution by solid-state diffusion from the bulk. the result of forsten's table 5. the result of one-way anova test toward the number of streptococcus mutans collony df sig. 1st day between groups within groups total 2 18 20 .000 7th day between groups within groups total 2 18 20 .000 14th day between groups within groups total 2 18 20 0.00 table 6. the result of lsd test toward sreptococcus mutans colony (i) (j) controlled fuji shofu 1st day controlled fuji shofu 178.5714* 323.0000* 144.4286* 7th day controlled fuji shofu 119.2857* 232.2857* 113.0000* 14th day controlled fuji shofu 105.8571* 139.7143* 33.8571* note: * significance 135fatmawati and widyowati: antibacterial force of the luting-type research in subiyanto5 showed that lower ph released more ion fluoride. in the ph of 6,1 ion fluoride released in the 7th day was 2,3 ppm, while in the ph of 5.0 the ion fluoride separation was 4,1 ppm. in the observation of 1st day, it showed the least number of bacterial colony compared to the 7th day and 14th day, it could be assumed that fluoride is mostly released in the 1st day, and then decreased regularly. perin et al. cit. summit et al.,8 carey et al.17 and weidlich et al.18 reported that in the measurement of separation of glass ionomer cement fluoride, the greatest separation occurred on the first day, decreased rapidly on the second day, and reduced regularly more than 3 weeks to the lower level on the long-term separation. summit et al.8 said that no glass ionomer cement was able to retain its acidity more than 48 hours. the material that released ion would also release the other fluoride and ion with decreased degree in line with time so that the direct bactericidal effect from the fluoride separation was limited. craig7 research stated that there was no improvement on the number of bacteria during 56 days after crown cementation using glass ionomer cement. after 1 year, however, the glass ionomer cement still released fluoride with the concentration of 0.05 ppm each day. the separation of fluoride from a restoration material was influenced by intrinsic and extrinsic factors. the intrinsic factor was influenced by the comparison of powder and fluid, mixing time, temperature, specimen shape, surface coating and polishing. the extrinsic factor was related to the mediator (ph), temperature, composition, immersion fluid volume and the frequency of immersion replacement.19 granfield et al. cit. summit et al.8 reported that fluoride separation was influenced by the shape of specimen. the specimen with more extended surface would release more fluoride. they also reported that ph influenced the fluoride separation. smith and ruse in davidson and mjör3 also reported that there was ph change during setting (initial acidity). the low ph in the initial mixing of powder and fluid was ph 2 in 1–5 minutes mixing, ph 3 after 10 minutes mixing. this low ph of glass ionomer cement was unable to retain over 48 hours after application. this was influenced by the separation of fluoride from glass ionomer cement which was the process of solvent selective decomposition from the artificial saliva so that the number of fluoride separation was greater and the ph of artificial saliva tended to more acidity. based on the result of the research, it was concluded that the luting-type of glass ionomer cement possessed antibacterial force toward streptococcus mutans and lactobacillus sp. the luting-type of glass ionomer cement of shofu had greater antibacterial force than fuji and the controlled-group. as consideration to choose cementation material in clinic, it is necessary to conduct further research on the various brands of luting-type of glass ionomer cement and other adhesive materials which are not classified into glass ionomer cement. acknowledgement thank you very much to trisia fajarini and didit hidayat for your helping. references 1. ford ptr. restorasi gigi. 2nd ed. sumawinata n, editor. the restoration of teeth. jakarta: egc; 1993. p. 10, 117, 118. 2. anusavice kj, phillips. buku ajar ilmu bahan kedokteran gigi. 10th ed. budiman ja, purwoko s. philip's science of dental materials. jakarta: egc; 2003. p. 447, 471. 3. davidson cl, mjör ia. advances in glass ionomer cements. germany: quintinessence publishing co inc; 1999. p. 149, 155. 4. van noort r. introduction to dental materials. 2nd ed. usa: mosbyyear book, inc; 2002. p.124. 5. subiyanto a. daya antibakteri semen gelas ionomer tipe perekat dan tumpatan terhadap streptococcus mutans. majalah kedokteran gigi (dental journal) 2002; 35(93):111–3. 6. baum, lloyd, phillips, ralph w, lund, melvin r. buku ajar ilmu konservasi gigi. 3rd ed. tarigan r, editor. textbook of operative dentistry. jakarta: egc; 1994. p. 669. 7. craig rg. restorative dental materials. 10th ed. usa: mosby-year book, inc; 1996. p. 194. 8. summit jb, et al. fundamentals of operative dentistry. a contemporary approach. 2nd ed. quintessence publishing co, inc; 2001. p. 379, 380, 382. 9. budi at. pengaruh antibakteri dari bahan restorasi semen gelas ionomer dengan teknik art terhadap streptokokus mutans. majalah kedokteran gigi (dental journal) 2001; 34(3a):154–56. 10. kusumaningsih t. pengujian antibodi saliva terhadap streptococcus mutans 1 dan streptococcus mutans 3 pada karies aktif dan karies terawat anak umur 4, 6 tahun di tk. ki hajar dewantoro. majalah kedokteran gigi (dental journal) 1992; 25(1):172. 11. alcamo ej. laboratory fundamentals of microbiology. new york: addison-wesley publishing; 1983. p.73. 12. ikeda s, igarashi t, goto n. miyazaki t. antibacterial effect of glass ionomer cement combined with tannin-fluoride (hy-agent) on oral bacterial. j dent mate 2000; 19(5):456-63. abstract. [on line] available at: http://www.soc.nii.ac.jp/jsdmd/2000/19-5je.html. accessed june 21, 2006. 13. yamaga m, koide t, hieda t. fluorine uptake and cristallinity of dentin treated with glass ionomer cement containing tannin-fluoride preparation. dent mater j 1994 jun; 13(1):89-102. abstract. [on line] available at: http://www.ncbi.nlm.nih.gov. accessed july 3, 2006. 14. marsh, martin. oral biology. cornwell great britain. mpg books ltd; 1999. p. 96-97. 15. syahrurachman a, et al. buku ajar mikrobiologi kedokteran. revised edition. jakarta: binarupa aksara; 1993. p. 112. 16. seppä l, forss h, øgaard b. the effect of fluoride application on fluoride release and the antibacterial action of glass ionomers. j dent res 1993 september; 72(9):1310-4. [serial on line] available at: http://www.jdr.iadrjournals.org. accessed august 30, 2006. 17. carey cm, spencer m, gove rj, eichmiller fc. fluoride release from a resin-modified glass-ionomer cement in a continuous-flow system: effect of ph. j dent res 2003; 82(10):829-32. international and american associations for dental research. [serial on line] available at: http://jdr.iadrjournals.org/cgi/content/full. accessed june 22, 2006. 18. weidlich p, miranda la, maltz m, samuel smw. fluoride release and uptake from glass ionomer cements and composite resins. braz dent j 2000; 11(2):89-96. [serial on line] available at: http://forp. usp.br/bdj.htm. accessed may 3, 2006. 19. nuraini p. pelepasan fluorida maksimal semen gelas ionomer. majalah kedokteran gigi (dental journal) 2001 august; 34 (3a):545–7. 55 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 5–10 original article effectiveness of reminder sticker books at increasing dental health knowledge and oral hygiene muhammad chair effendi1, edina hartami1, merlya balbeid2 and ghea dewi hapsari3 1department of pediatric dentistry, 2department of community and preventive dentistry, 3undergraduate program in dentistry, faculty of dentistry, universitas brawijaya malang – indonesia abstract background: health education games can be an effective way for elementary school children to increase their dental health knowledge and oral health. purpose: this study aims to test the effectiveness of using reminder sticker books by showing the increase in dental health knowledge and improvement in the simplified oral hygiene index (ohi-s) in sevento eight-year-olds. methods: the study was quasi-experimental with a pretest-posttest group design and descriptive analysis method. it involved 54 elementary school students. a reminder sticker book was used to measure the levels of knowledge and oral hygiene. intraoral examinations were conducted, and the children’s frequency of attaching stickers in the morning and afternoon was measured. frequency was divided into three categories. a questionnaire measured the children’s levels of knowledge, which were divided into five categories. results: the reminder sticker game increased dental health knowledge, with the mean of the control group at 64.4, and the treatment group at 92.5. ohi-s means were 1.68 for the control group and 0.78 for the treatment group. an independent t-test measured the levels of knowledge before and after the study. ohi-s and anova tests measured the increase of dental health knowledge, which was divided into five categories, and decrease in the ohi-s index (divided into three categories). conclusion: reminder sticker books can increase oral health knowledge and reduce ohi-s scores in sevento eight-year-old children. keywords: educational game; oral hygiene index simplified; reminder sticker correspondence: muhammad chair effendi, department of pediatric dentistry, faculty of dentistry, universitas brawijaya. jl. veteran, malang, 65145 indonesia. email: chaireffendi@gmail.com introduction the global dental caries rate is 60–90% for school-aged children and almost 100% for adults. this decay causes pain and discomfort.1 dental and oral problems were found in 57.6%, and 10.2% of the general population received services from dental professionals. proper toothbrushing behaviour was found in 2.8% of the general population, starting at 3 years of age.2 game features such as tasks, rewards and achievements challenge the players, which motivates them to achieve their goals.3 games are also a tool for learning and forming healthy habits, owing to an increase in awareness, resulting in improved oral health and tooth and gum quality.4 educational games are an effective way to provide health education for elementary school children.5 oral hygiene must be taught and practiced at early ages since it is one of the determinants of health conditions in later life.6 causes of dental and oral health problems in the community include behavioural factors and a lack of knowledge of the importance of maintaining oral health.7 habits form as people pursue goals in daily life. when repeatedly performing a behaviour in a particular context, people develop implicit associations in their minds between contexts and responses.8 learning is a type of communication, and the message senders, the messages themselves and the recipients are all factors. according to the concept of edutainment, learning will not succeed if it dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 mailto:chaireffendi@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 6 effendi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 5–10 occurs in tense or frightening conditions. for children, this means that learning will only be effective if the children are relaxed.9 the simplified oral hygiene index (ohi-s) is an index that measures the surface area of teeth covered by oral debris and calculus. oral hygiene is assessed by the presence of food scraps and calculus (tartar) on the surface of a tooth using ohi-s measurements as described by greene and vermillion.10 the degree of plaque reduction when brushing teeth can be examined in 30-second to three-minute time spans. plaque removal when brushing teeth can be performed quickly, but optimal results come from longer brushing.11 the reminder sticker books used as morning and afternoon play tools in this study were designed to measure the effectiveness of games and to increase dental and oral knowledge while reducing the ohi-s scores of sevento eight-year-old children. there is no previous research into the use of reminder sticker books as a helpful and fun way to teach children about oral hygiene. materials and methods the study is quasi-experimental, with a pretest-posttest group design. inclusion criteria are that the children are seven to eight years old at the time of the study, willing to participate by asking their parents for informed consent, and cooperative. exclusion criteria are children with behavioural disorders, such as oppositional defiant disorder (odd), conduct disorder (cd), attention deficit hyperactivity disorder (adhd), autism, cerebral palsy (cp) and epilepsy; children without parental permission; and children who use orthodontic brackets. before the study was conducted, parents were asked to provide informed consent. the study started with 63 students but, due to qualification factors, only 54 participated in the research. the study was conducted at the ketawanggede elementary school in malang. the research focused on sevento eight-year-olds because this age range is the most susceptible to dental caries and, therefore, needs to understand the importance of dental health, treatment and prevention.12 the study meets the ethical feasibility requirements set by the ethics commission of the faculty of medicine, universitas brawijaya through ethical eligibility statement no.225/ec/kepk-s1-fkg/08/2019. a reminder sticker book is a tool for developing children’s interest in brushing their teeth more effectively and efficiently while playing. the use of the reminder stickers in the morning and in the afternoon was expected to help children remember to brush their teeth regularly. based on preliminary studies, the frequency indicators of morning and afternoon toothbrushing were divided into three categories by counting the number of stickers posted in the sticker book: very routine (40–60 stickers total, with 20–30 in the morning and 20–30 in the afternoon), routine (20–39 stickers total, with 10–19 in the morning and 10–19 in the afternoon) and not routine (0–19 stickers total, with 0–9 stickers in the morning and 0–9 in the afternoon). the goal was to associate toothbrushing with the fun activity of putting their stickers in the books. the reminder sticker books were given to the treatment group after counselling on how to maintain dental and oral hygiene, including toothbrushing. the books, which were 21.6 by 26.5 centimetres large and printed on wood-free paper (hvs) weighing 70 grams per square meter (gsm) with 180 gsm art paper covers, were given to 27 students. the two-centimetre by three-centimetre stickers were made with chromo paper (figure 1). the books were designed as games, which have long been used as innovative and challenging teaching tools in both child and adult education to promote autonomous learning and participation. through repetition and reiteration, games appear to increase retention and application.13 the books (figure 1) include the following pages: the front cover (a); a book ownership page (b); a sticker corner page for three games –teeth sudoku, count it up and find the missing me (c); a sticker corner page for the game copy and stick (d); a children’s story about how dental caries are formed (e); the game teeth sudoku, which consists of boxes of different sizes and requires players to put stickers into the boxes (f); the game copy and stick, played by placing stickers in accordance with the images in the box (g); the game count it up, played by adding the values of the pictures (h); the game find the missing me, featuring empty boxes that players fill by attaching matching pictures (i); a story about a diligent child who goes to the dentist (j); a page for attaching sun-shaped stickers in the morning – there are 30 boxes, meaning it must be used for 30 days (k); a page for attaching moonshaped stickers in the afternoon, also with 30 boxes and a 30-day commitment (l); a sticker page (m); and the back cover of the book (n). the students were randomly divided into two groups, each with 27 students, by drawing numbers. students who drew odd numbers became the control group (k), while students who drew even numbers became the treatment group (p). a 10-minute pretest was conducted with each group to ascertain their dental and oral hygiene knowledge (table 1). after the pretest, the children received counselling about maintaining their oral and dental hygiene, with a similar focus to the pretest. the counselling used a phantom teeth, toothbrush and toothpaste. counselling topics included dental and oral functions and dental anatomy (five minutes), ways to maintain oral and dental hygiene (10 minutes) and the importance of maintaining oral and dental hygiene (five minutes), with a question and answer session included after each topic. a 10-minute posttest followed the counselling. pretest and posttest results were recorded and assessed. each answer was worth 10 points, with possible scores ranging from 0 to 100. based on the preliminary studies, pretest and posttest results were divided into five categories: very good (80–100), good (60–79), acceptable (40–59), poor (20–39), and very poor (0–19). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 7effendi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 5–10 table 1. pretest and posttest knowledge questions question answer a b how many times per day should you brush your teeth? once twice* when should you brush your teeth? in the morning after breakfast and at night before sleeping* anytime when should you go to the dentist? every six months* when i have a toothache which of the following is a tool for cleaning teeth? toothbrush* spoon which of the following foods can cause caries? spinach candies* which of the following techniques should you use to brush your teeth? rubbing technique rolling technique* how much toothpaste should you use? the size of a bean * as much as possible how long does it take to brush your teeth? 1-3 minutes* 10-15 minutes what is the name of a tooth’s outer layer? gum enamel* which of the following foods can cause toothaches? apple ice cream* *: correct answer figure 1. reminder sticker book. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 8 effendi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 5–10 before giving the reminder sticker books to the treatment group, both groups’ ohi-s scores were measured. the treatment group then received the books, which were meant to be used routinely as a reminder to maintain oral hygiene. students in the treatment group attached stickers every morning and afternoon after brushing their teeth. after 30 days, both groups’ ohi-s scores were measured again to compare the differences between the two groups. ohi-s scores include debris and calculus. debris is leftover food that remains in the form of soft deposits on the surface of the teeth after eating,14 and calculus is a mineralised bacterial plaque that is formed on natural teeth surfaces where there is a constant supply of saliva. there are two types of calculus: supragingival and subgingival.15 each component of the debris index (di) and calculus index (ci) is scored on a scale of 0 to 3. the examination only uses a mouth mirror and a dental explorer, with no disclosing agent. these criteria are scored based on the state of soft deposits, or debris, and calculus tartar. index calculation for each individual includes two components (debris index and calculus index). the debris index (di) is the total number of debris scores divided by the total number of examined teeth, and the calculus index (ci) is the total number of calculus scores divided by the total number of examined teeth. the di and ci scores are then added to determine an ohi-s score. the categories of dental and oral hygiene based on ohi-s scores are as follows: good (0.0–1.2), fair (1.3–3.0) and poor (3.1–6.0).16 ohi-s examinations are conducted on six teeth: 16, 11, 26, 36, 31 and 46. in teeth 11 and 31, the labial section is examined; in teeth 16 and 26, the buccal section is examined; and in teeth 36 and 46, the lingual section is examined.17 an independent t-test measured the differences in knowledge test scores and ohi-s scores between the control and treatment groups, while an anova test assessed the differences between the treatment group’s frequency of attaching the stickers and brushing their teeth (with three categories: very routine, routine and not routine). the anova test result was significant, so a pearson’s correlation was calculated to determine the relationship between the frequency of attaching the stickers and brushing teeth and ohi-s scores. the frequency of attaching the stickers was identical to the frequency of brushing teeth (figure 1: k, l, m) results figure 2 shows the increase in the means of dental health knowledge test scores for the control group (64.4) and the treatment group (92.5). the results of the independent t-test show that the means of the knowledge test scores between the control and treatment groups are significant (p=.000). figure 3 indicates that there was a decrease in the mean of ohi-s scores for the control group (1.68) and figure 2. mean of knowledge post-test; **) p<0.01. figure 3. mean of post ohi-s examination after 30 days; **) p<0.01. figure 4. mean of ohi-s based on the frequency of using reminder stickers for 30 days; *) p<0.05. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 9effendi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 5–10 the treatment group (0.78). the results of the independent t-test reveal that the means of ohi-s between the control and treatment groups are significant (p=.000). the results of the anova test (figure 4) that measured the means of ohi-s scores and the frequency of attaching the stickers show that there is no significant difference between the not routine and routine categories (p=.089) or between the routine and very routine (p=.070). however, the differences between the not routine and very routine categories are significant (p=.047). because these anova test results were significant, the study continued with a pearson’s correlation between the frequency of attaching the stickers and ohi-s scores. this calculation produced a significant relationship and negative correlation coefficient in the moderate range (table 2). statistical results reveal that games can significantly increase children’s knowledge about dental health. the game used in this study was a reminder sticker book with educational stories (figure 1: e, j). discussion reminders in the form of stickers can be widely used in the health field. one of the reasons for the success the reminder stickers is that they can increase mindfulness (figure 2), which is the process of bringing one’s attention and awareness to the present time.16 games, which are the best strategy for improving children’s oral health behaviours,18 are valuable learning tools that make learning more interesting and have been widely used in all fields of education by both students and teachers. game-based oral health interventions reinforce messages about oral health knowledge through crosswords and quizzes.19 educational media are necessary to increase the success of achievement goals in learning communication. 20 this communication and dental health education (dhe) in schools can significantly increase knowledge of oral health and oral hygiene.21 games can also reduce ohi-s scores. statistical test results indicated that ohi-s scores significantly decreased after students were given the reminder stickers (figure 3) and had a significant relationship with the negative correlation coefficient between the frequency of attaching the stickers in the morning and in the afternoon (table 2). the negative correlation indicates that the mere routine of attaching the stickers can reduce ohi-s scores. these scores decreased significantly in the very routine group compared to the not routine group (figure 4). the frequency of attaching the stickers in the morning and in the afternoon is an indicator of how routinely to brush the teeth (figure 1: k, m, l). a toothbrushing habit that is not routine can cause food debris and calculus. several studies show a decrease in ohi-s scores can correlate with the knowledge gained from health education methods such as games, that include health and hygiene messages. these games are an effective way to teach basic health concepts and significantly reduce index plaque scores.19,22 the increased knowledge and decreased ohi-s scores in this study were also influenced by the sample groups: children who still enjoy interactive activities like games and simple quizzes and who have basic dental health knowledge.23 although there are many levels of toothbrushing frequency, we suggest that brushing twice per day, once in the morning and once at night, is sufficient.24 the reminder sticker book in this study included sun and moon stickers that mirror toothbrushing times. though twice-daily toothbrushing is effective at reducing bacterial load in oral cavities, supplementing brushing with routine tongue cleaning would result in dramatic and significant improvements in dental health and hygiene.25 reminder sticker books are a tool for developing children’s interest in brushing their teeth more effectively and efficiently by playing games. the means of the knowledge posttest increased after they were given the reminder stickers, which were easy and fun to play with. this was significantly different from the control group; the ohi-s index differed significantly between the treatment group and the control group. the reminder sticker books can increase dental and oral health in sevento eight-yearold children. acknowledgments our thanks to the principal and the teachers of the state elementary school of ketawanggede in malang, indonesia, the staff of the creative and visual art (canva) in bekasi, indonesia and everyone who assisted in this study. references 1. peterson pe, ogawa h. prevention of dental caries through the use of fl uoride – the who approach. community dent health. 2016; 33: 66–8. 2. badan penelitian dan pengembangan kesehatan. hasil utama riset kesehatan dasar. jakarta: kementerian kesehatan republik indonesia; 2018. p. 103–4. 3. shi y-r, shih j-l. game factors and game-based learning design model. int j comput games technol. 2015; 2015: 1–11. 4. ávila-curiel bx, solórzano-mata cj, avendaño-martínez ja, luna-vásquez b, torres-rosas r. playful educational intervention for improvement of oral health in children with hearing impairment. int j clin pediatr dent. 2019; 12(6): 491–3. 5. hutami ar, dewi nm, setiawan nr, putri nap, kaswindarti s. penerapan permainan molegi (monopoli puzzle kesehatan gigi) sebagai media edukasi kesehatan gigi dan mulut siswa sd negeri 1 bumi. j pemberdaya masy univ al azhar indones. 2019; 1(2): 72–7. 6. osadolor oo, iwuoha ce. oral hygiene status of primary school children. int j dent res. 2019; 4(3): 104–7. 7. anwar ai, abdat m, ayub aa, yusrianti m. status kebersihan mulut berdasarkan indeks oral hygiene index simplified (ohi-s) table 2. correlation between the frequency of sticking the stickers and ohi-s variable correlation r p note frequency of attaching the stickers – ohi-s -0.387 0.046 significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 10 effendi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 5–10 pada siswa sekolah usia 9, 10 dan 11 tahun. cakradonya dent j. 2019; 11(2): 86–90. 8. carden l, wood w. habit formation and change. curr opin behav sci. 2018; 20: 117–22. 9. fadlillah m. buku ajar: bermain & permainan anak usia dini. jakarta: prenada media group; 2017. p. 7–12. 10. singh k, ayurved p, haridwar h, singh p, oberoi g. effect of yoga on dental care: pranayama techniques or rhythmic breathing exercises on the oral hygiene and gingival bleeding. int j appl dent sci. 2017; 3(3): 91–5. 11. gallagher a, sowinski j, bowman j, barrett k, lowe s, patel k, bosma ml, creeth je. the effect of brushing time and dentifrice on dental plaque removal in vivo. j dent hyg. 2009; 83(3): 111–6. 12. li nt a ng jc, pa la ndeng h, l ema n m a. hubu nga n t i ngkat pengetahuan pemeliharaan kesehatan gigi dan tingkat keparahan karies gigi siswa sdn tumaluntung minahasa utara. e-gigi. 2015; 3(2): 567–72. 13. kumar y, asokan s, john b, gopalan t. effect of conventional and game-based teaching on oral health status of children: a randomized controlled trial. int j clin pediatr dent. 2015; 8(2): 123–6. 14. eastabrooks d. oral hygiene assessment: soft and hard deposits. in: darby ml, walsh m, editors. dental hygiene: theory and practice. 4th ed. st. louis, missouri: saunders elsevier; 2015. p. 290. 15. balaji vr, niazi tm, dhanasekaran m. an unusual presentation of dental calculus. j indian soc periodontol. 2019; 23(5): 484–6. 16. rohl a, eriksson s, metcalf d. evaluating the effectiveness of a front windshield sticker reminder in reducing texting while driving in young adults. cureus. 2016; 8(7): e691. 17. pawlaczyk-kamieńska t, torlińska-walkowiak n, borysewiczlewicka m. the relationship between oral hygiene level and gingivitis in children. adv clin exp med. 2018; 27(10): 1397– 401. 18. melo p, fine c, malone s, frencken je, horn v. the effectiveness of the brush day and night programme in improving children’s toothbrushing knowledge and behaviour. int dent j. 2018; 68(suppl 1): 7–16. 19. malik a, sabharwal s, kumar a, samant ps, singh a, pandey vk. implementation of game-based oral health education vs conventional oral health education on children’s oral health-related knowledge and oral hygiene status. int j clin pediatr dent. 2017; 10(3): 257–60. 20. widodo sa, wahyudin. selection of learning media mathematics for junior school students. turkish online j educ technol. 2018; 17(1): 154–60. 21. sadana g, gupta t, aggarwal n, rai hk, bhargava a, walia s. evaluation of the impact of oral health education on oral hygiene knowledge and plaque control of school-going children in the city of amritsar. j int soc prev community dent. 2017; 7(5): 259–63. 22. hashmi s, mohanty vr, balappanavar ay, yadav v, kapoor s, rijhwani k. effectiveness of dental health education on oral hygiene among hearing impaired adolescents in india: a randomized control trial. spec care dent. 2019; 39(3): 274–80. 23. pratt jm. oral health education and promotion. in: noble sl, editor. clinical textbook of dental hygiene and therapy. 2nd ed. birmingham: wiley-blackwell; 2012. p. 136–7. 24. zeng x-t, leng w-d, zhang c, liu j, cao s-y, huang w. metaanalysis on the association between toothbrushing and head and neck cancer. oral oncol. 2015; 51(5): 446–51. 25. winnier jj, rupesh s. tongue cleaning methods: a review. int j dent heal sci. 2016; 3(1): 141–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p5–10 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p5-10 vol 38-no4-2005-isi.pmd 185 the recurrent aphthous stomatitis' healing duration differences in female students between the sufferer and non sufferer of chicken pox isidora karsini soewondo department of oral medicine faculty of dentistry airlangga university surabaya indonesia abstract chicken pox (cp) is a generalized primary infection that occurs the first time an individual contacts the virus. the etiology of cp is a vzv virus, and the replication of virus allowing recovery in two to three weeks. during the process, the vzv may progress along sensory nerves to the sensory ganglia, where it can reside in a latent, undetectable form; and can be reactivated at any time. recurrent aphthous stomatitis (ras) is a recurrent ulcer in the mouth, painful, disturbing the mouth's function and esthetic when occurs in the lips. one of the etiology of ras is the reactivation of the latent virus in the mouth. the aim of this study was to know the contribution of the latent virus in the ganglia, intervered with the differences of the healing duration of ras in female students, between the cp sufferer and non cp sufferer. by cross-sectionally, clinical examination, after filling the questioner that included informed consent, 307 students of the st. yusup senior high school, karangpilang surabaya, were examined. in the 3rd class, there were only 6 female students that suffered ras had cp history, while 11, student did not. levene's test for equality of variances was done, and p: 0.698, while 2-tail sign: 0.512. according to this statistical analysis, there was no significant difference between the two groups examined. it was suggested that the female students of ras' sufferer should maintain their balanced food intake, so the ulcer of ras would heal quickly. key words: recurrent aphthous stomatitis, healing duration, female students correspondence: isidora ks, department of oral medicine, faculty of dentistry, airlangga university, 47th prof. dr. mustopo st. surabaya 60132, indonesia. introduction chickenpox (cp) is a generalized primary infection that occurs the first time an individual contacts the virus. the etiology of cp is varicella zoster virus, that responsible for two major clinical infections of man: chickenpox (varicella) and singles (herpes zoster ).1 viral infection may result in cytolysis, chronic metabolic dysfunction, or transformation of the cell, or there may be no pathologic effect.2 the change occurs in the middle and deeper layers of the prickle cells of the epidermis, and consists of the ballooning degeneration of the cells and the outpouring of a great deal of intra cellular oedema. in the walls and floors of the chickenpox vesicles multinucleated giant cells are found and intra nuclear inclusion bodies.3 these cause blistering skin condition. they include the common cold sore around the mouth, which is due to herpes simplex virus (hsv) type 1, and the sexually transmitted genital herpes caused by hsv type 2. infection with varicella zoster virus (vzv) causes the lesions of chickenpox (varicella) and singles (herpes zoster). chickenpox arises as an epidemic form after direct contact within individuals who have either chickenpox or singles, and causes an acute vesicular rash associated with fever, malaise or lymphadenopathy. thereafter the virus remains dormant within the nerve tissue until reactivation in the form of singles.4 changes in the mucous membranes of the mouth and pharynx are similar to those in the skin, but the thin roof of the lesions breaks down quickly, so that shallow ulcer rather than vesicles are formed.3 an oral ulcer is any breakdown of the lining of the mouth, which includes the cheeks, tongue, gums, lips, and roof of the mouth. the raw area of an ulcer is often very sensitive and painful.3,5,6 in childhood, chickenpox leads to wide spread blisters. after recovery, the virus may survive in the posterior root ganglia and become reactivated within the dermatome involved as singles. the lesion of hsv and vzv are similar histologically, with oedema of keratinocytes leading to intraepidermal vesicle formation, within which damaged epidermal cells with prominent intranuclear viral inclusions may be seen.3,4,5 the fluid in the chickenpox vesicles rapidly becomes turbid as polymorphonuclear leukocytes migrate up from the corium, inflammatory in the corium are slight.3 this is an acute specific fever, and analogous to the acute herpetic gingivo stomatitis of herpes simplex virus. after the primary diseased is healed, vzv becomes latent in the dorsal root of ganglia of spinal nerve or extra medullary ganglia of cranial nerve.1,7 186 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 185–188 dentists should bear in mind that one of every 38 dental patient is potentially a latently infections patients.8 after the initial infection, the virus will remain dormant until reactivated. the frequency of reactivation with clinical recurrence has been reported as occurring in 40%, and 10– 15% of those with the latent virus.9 reactivation can occur as a result of several factors that suppress the immune system. these include but are not limited, to emotional stress, trauma, cold, sunlight, extreme fatigue, fever and menstrual cycle.9,10 the oral cavity consists of the lips, teeth, gums, oral mucous membranes, palate, tongue and oral lymphoid system. the oral cavity plays essential roles in many bodily functions, including nutrition (mastication and swallowing), respiration and communication. just as examining any are of the body, it is important to inspect directly and systematically all areas of the oral cavity. many disease processes, benign and malignant, localized and systemic, may present as an ulcerative lesion in the oral cavity.10 recurrent aphthous stomatitis (ras) is an inflammatory ulcerative disease of unknown etiology. recurrent discrete areas of ulcerations, which are almost always painful, characterize this condition. ras frequently referred to as canker sore, are among the most common lesions of the mouth. their recurrent pattern and associated discomfort make them extremely bothersome and at times, debilitating patients. recurrent aphthous stomatitis (ras) may occur as occasional single ulceration or may be manifested as a never-ending continuum of severe ulcerative lesions.1,2,1113 recurrent aphthous stomatitis (ras) are usually small, but can, in rare cases, become fairly large. they generally start as erythematous papules, which soon undergo necrosis and acquires a fibrin coating. the small crateriform ulcers have a white to yellow membrane, surrounded by an erythematous halo. most ras ulcers heal in one to two weeks. patients usually complain of these lesions being very painful and intervering with eating. nearly all aphthae healing without scarring. recurrent aphthous stomatitis (ras) can be distinguished from other diseases with similar appearing oral lesions, such as certain viral exanthems, by their tendency to recur, their multiplicity and chronicity. in ras there is a history of recurrence, and lack of systemic symptoms such as fever, malaise and cervical lymphadenopathy seen in primary herpes.1,2 in some instances, the mucosal surface may become erythematous or ulcerated. oral mucosal macules and nodules have also been described in 14% of individuals receiving hemodialysis. other lesions that can occur intra-orally in allograft recipient are: uremic stomatitis, geographic tongue, erythematous patch, and macules/nodules. uremic stomatitis may manifest as white, red or grey area of the oral mucosa. the erythematous form consists of grey pseudomembrane overlying painful erythema patches, while an ulcerative form is red with a pultaceous covering. uremic stomatitis suggested may be due to chemically based trauma from elevated levels of nitrogenous compounds. 14 the patient history, the physical examination, and the results of any indicated tests are important to the diagnosis process. if the patient history is accurate and the physical examination allows the clinician to see the lesion(s), other tests may not be necessary. the history, location and appearance of the lesions should allow the knowledgeable clinician to establish a presumptive diagnosis.9 the etiologies of ras are unknown and are multiple factors. many suggestions have been made. an autoimmune or hypersensitivity mechanism is widely considered possible, but ras is not typical of either disease type.12 since the etiology of ras is indeterminate, research has focused upon a variety of potentiating factors. studies of these are not conclusive, but precipitating factors that have been identified include: stress, nutritional deficiencies, trauma, hormonal changes, diet and immunologic disorders. other contributors that have received attention are: food, allergens, progesterone levels, psychologic (anxiety and depress) factors, local and oral factors, viral, bacterial and a family history.9,15,16,17 the aim of this study was want to know, whether the latent virus vzv in the ganglia after reactivated by some trigger to become an ulcer in the mucous membrane, different in healing duration with the one without the latent virus. by knowing that, it can be bare in mind, that the one with the sign of cp ( that can be appeared/ seen as one or some craterlike cicatrixes in the face), ha to be adviced, always maintain their good health by intake balance nutrition, in hoping the ulcer of ras that may be occur will heals quickly. materials and method this study was done cross-sectionals; clinical examination after filling the questioner that included informed consent. the population studied were all of the students in the st.yusup senior high school, karangpilang surabaya in the year 1998. the students were between 15–19 years old. the equipment for the study was the routine dentist's examination needed (mouth mirrors, sondes, pinsets, alcohol, cotton, towels, waste-baskets etc). after preparing the administration letters for permission of the study, the students were given the questioner that included the informed consent. the informed consent had to be signed by them. after returning the questioner, they were examined clinically by three dentists who were already trained by the author. the examination using the sun light (outdoor) since the study was in august, 1998, the dry season in surabaya. the students had to wash their mouth or gargle first, before seated on a chair. the teeth and the mucosa were examined in a secure way with a systematically procedures, using the equipment needed. the cp's collecting data were by looking at their face carefully to search for one or more craterlike cicatrixes, as the result of the herald spot in the skin. the questioner 187soewondo: the recurrent aphthous stomatitis filled with data, were collected and tabulated, and then continued by statistical analysis. results from the study that had already done, there were only 6 female students from the third class of this school, which suffered ras with the history of cp, while 11 female students with ras without history of cp. from the whole 307 students examined, only these 17 female students that fulfilled the study's criteria, the data was seen in the table 1. the differences of healing duration between the two groups was statistically analysis by using levene's test for equality of variances: p = 0.698, while 2 – tail sig. = 0.512. discussion according to the students, as a public understanding, in indonesian people, ras has some synonyms such as: sariawan, jampien, lumpangen or panas dalam. this is a condition of the occurring ulcer(s) in the oral cavity, whether it's because of a trauma, recurrent intraorally herpes or actually ras.the anamnesis or tracking history about suffering cp, usually easy to answer or remember. the itching of the disease or secondary infection that triggering to become crusting and scarring, leave a mark that difficult to be erased. may be it made a permanently some small craterlike defect on the skin, that can't be forgotten. the population of the respondents that filled the study's criterions was the female students from the third class of the st.yusup senior high school at the month of august 1999. the student's old were between 15–19 years old. this period was the productive period; the growth hormone is in an optimal one. when there is an ulcer or an aphthae happened in the oral mucosa, the healing process will take place in a short time. this is when there is no underlying illness in the body.1,9 in indonesia, the begin of the school's lecture is in july, and up till 20th august, most of the time filled with activities of the celebration of national's independence day. the stressor for the lecture's activities is begin after that, so the third class' students on the time examined, still enjoy the school's environment. the study was permitted to be done, according to this condition too. the excitement of the future final examination didn't affect them greatly, so the filling of the questioner's items go fluently. from the table 1 above, it can be seen that the female student who suffered ras and had been with cp, the duration of healing ulcer of ras was 10 days, so was the group without cp history. it can be meant that the ulcers from both groups were healed just as the literature said, between 10–14 days.1,10,16 this was showed that the respondent's body defence still in a good condition. the minimum healed duration was three days in group i, while one day in group ii. because the students from this school had already settled with the school's regulation schedule, so the body defence was good too. in group i, the body defence was able to block the latency of the virus, so it couldn't be reactivated in a broad or deep location of the mucosa, or the part of the body, that innervated by the sensory nerve.1,9 in group ii, the eruption of the ulcer can be covered in a fast or short duration of time, because the body defence had no latency virus. the occurrence of the ulcer may be just as a result of another etiology, such as a trauma. the destruction of the epithelium didn't become large ulcer, because the body defence had rolled it out, so the epithelium will be healed. this is just as the other authors said.1,9 in group i, the ras sufferer with history of cp, the longest duration of healing ulcer was 10 days, and it happened before the menstrual cycle. according to terri,9 the reactivation of the latent virus can be manifest in the oral cavity as a ras lesion. during this period, the estrogen hormone was in the lowest of the curve, so the maintenance of the mucosal barrier was very low. oral mucosa is a very fragile epithel, and received so many injuries, that's why in this period of menstrual cycle, it can be occur an ulcer, which is assumption as a ras ulcer. in group ii, although there was no history about cp, the ras longest duration of healing was 10 days too. the ulcer happened before or after the menstrual cycle. it can be assumption that the oral mucosa still fragile during this period, because the estrogen hormone is still not in a standard level. the barrier of the oral mucosa was still not in optimal level, so the injury on them can be make a result as an ulcer, which is called a ras lesion.9 by statistical analysis the levene's test for equality of variances, p = 0.698, while 2-tail sign. = 0.512, this was mean that there was no significant difference between the ras' healing duration, between the two groups examined. the conclusion of this study was, that the body defence of the female students in the third class of the senior high school of st.yusup in karangpilang surabaya, still in good level. the ulceration caused by ras could be healed in a short duration time, no longer than 14 days, just as the former authors said.1,2,11,18,19 there were no differences between the duration of healing processed, in the groups of ras sufferer, with and without the history of chickenpox infection. it can be suggested for these groups to maintain their good whole body's condition, especially for the oral mucosa, by intake balance nutrition, for avoiding the recurrency of the ras, and so the ulcer of ras will heals quickly. 188 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 185–188 table 1. the duration of healing ras' ulcer in the female students with and without history of cp conditions mean ± sd signficance chicken pox (–) chicken pox (+) 5.167 ± 2.787 4. 773 ± 2.533 p = (> 0.05) acknowledgement with deepest great full to all of the students in st.yusup senior high school, karangpilang surabaya, 1998, for their good contribution during the study, such as fulfilled the questioner, signed the informed consent and being examined. especially for mrs. dina arie, the head mistress, and the whole staff of this school for the permission in doing this study. references 1. greenberg ms, glick m. ulcerative, vesicular and bullous lesions. chapter 4. in: burket's oral medicine diagnosis & treatment. 10th ed. bc decker inc; 2003. p. 17, 50–7. 2. sonis st, fasio rc, fang l. principles and practice of oral medicine. 2nd ed. philadelphia, london, toronto, montreal, sydney, tokyo: wb saunders co; 1995. p. 14–18, 27–31. 3. christie ab. infectious disease: epidemiology and clinical practice. 4th ed. vol. i. edinburgh, london, melbourne, new york: churchill livingstone; 1987. p. 355. 4. wray d, lowe gdo, dagg jh, felix dh, scully c: vesiculobullous disorders–text book of general and oral medicine. edinburgh, london, new york, oxford, philadelphia, st louis, sydney, toronto: churchill livingstone; 2003. p. 235–42. 5. mcmahon rft, sloan p. essentials of pathology for dentistry. chapter 15. in: diseases of the skin. 1st ed. edinburgh, london, new york, philadelphia, st louis, sydney, toronto: churchill livingstone. 2000. p. 220–22. 6. mouth ulcers. available at http://www.kindandental.com/is/mouthulcers.html. accessed may 20, 2005. 7. cawson ra, binnie wh, barnett aw, wright jm. mucocutaneous diseases and other forms of stomatitis in oral disease clinical and pathological correlations. 6th ed. edinburgh, london, new york, philadelphia, st. louis, sydney, toronto: bailliere tindall, churchill livingstone. mosby, wb saunders; 2001. p. 1326–35. 8. matsusaka t, ikawa k, iwakura m. latently infected patients comprise 2.6% of dental out patients. j dent res 82 (spec iss c) 2003; (125):c–413. 9. terri ss, tillis rdh, mcdowell j. differential diagnosis: "is it herpes or aphthous?". the journal of contemporary dental practice 2002 february 15; 3(1):2–7. 10. yang gy. the effect of psychological intervention on anxiety and depress in-patient with recurrent oral ulcer. j dent res 82 (spec iss c) 2003; (075):c–339. 11. quin fb, ryan mw. ulcerative lesions of the oral cavity. available at http://www.utmb.edu/otoref/grnds? ulcer-oral-021016/ulceroral-021016.htm. accessed may 20, 2005. 12. aphthous dot net. presented by aphthous. p. 2. net: in lieu of the dead site www.umds.ac.uk.june.2003. available in http:// www.users.quest.net/-dallas7/ daphtrt. txt. assecced juny 14, 2005. 13. verdu s, nalli g, harada l, scola m, gonzales rl, verges v, maturana s, lence a, lanfianchi h. oral recurrent aphthous preliminary paper, clinical-therapeutic study. j dent res 82 (spec iss c) 2003; (195):c–32. 14. proctor r, kumar n, stein a, moles d, porter s. oral and dental aspects of chronic renal failure. critical reviews in oral biology & medicine. journal of dental research 2005 march; 84(3):202–3. 15. hoy dm. stomatitis. 2005. available on: http://www.5mcc.com/ assets/summary/tp0877. html. accessed may 20, 2005. 16. field a, longman l. tyldesley's oral medicine. chapter 4. infectious of the gingivae and oral mucosa. 5th ed. oxford university press; 2004. p. 42–3. 17. ngan v. aphthous ulcers dermnet nz. available at: http:// www.dermnetnz.org/site-age-specific/aphtjae.html. accessed april 28, 2005. 18. dayan s, stashenko p, niederman r, kupper ts. oral epithelial overexpression of il-1 alpha causes periodontal disease. journal of dental research 2005 october; 83(10):786. 19. barron rw.treatment strategies for recurrent oral aphthous ulcers. am j health-syst pharm 2001;58(1):41–53, @ 01. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 8181 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 81–86 the differences of effectiveness of β -1,3-glukanase vigna unguiculata and papain carica papaya enzymes in hydrolysis of denture plaque retno indrawati, muhammad lutfi, and erina fatmala yuli andari departement of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: accumulation of denture plaque can lead to pathological changes in oral mucosa, such as denture stomatitis, halitosis, and caries. plaque matrix is mostly formed by protein (30%) and polysaccharide complexes. thus, an alternative enzyme solution as denture cleanser is required for hydrolysis of denture plaque. papain is a proteolytic enzyme hydrolyzing proteins, while β-1,3-glucanase is a hydrolase enzyme hydrolyzing polysaccharides. purpose: this study aimed to analyze the differences of effectiveness of ß-1,3glucanase vigna unguiculata enzyme and papain carica papaya enzyme in hydrolysis of denture plaque. method: this research was a laboratory experimental research with post test only control group design. after using denture for 24 hours, the denture was soaked in a solution of 100 ml pbs, papain enzyme, and β 1-3 glucanase enzyme at a concentration of 0.5 mg/ml, 1 mg/ml, and 2 mg/ml for 10 minutes. the solution from plaque hydrolysis was soaked in pbs and vortex enzyme for 2 minutes, then soaked in ice water for 15 minutes, and centrifuged at 3000 rpm 5-10º for 10 minutes. the supernatant was separated and analyzed. turbidity readings then were performed in spectrofotometer with a wavelength of 480 nm. result: 2 mg/ml of ß-1,3 glucanase enzyme generated the highest values of hydrolysis with a mean percentage of 68.77% compared to papain enzyme (44.86 %). the lowest values of hydrolysis were generated by pbs with a mean percentage of 3.24%. conclusion: ß-1,3-glucanase enzyme is more effective in hydrolysis of denture plaque than papain enzyme. keywords: papain; ß-1,3-glucanase; denture plaque correspondence: retno indrawati, department of oral biology, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: retno_in2007@yahoo.co.id introduction economic condition and population growth have rapidly developed, triggering an increase in the use of complete dentures. dentures are artificial teeth together with their surrounding tissue, replacing some or all of the lost natural teeth and their surrounding tissue, so the function, appearance, and health of teeth can be restored. according to the basic health survey (riskesdas) in 2013, the percentage of denture prosthesis users in indonesia reached 4.5% of the population, 14.5% of which was from older people aged above 65 years old using dentures.1,2 according to oral health data from who, the prevalence of patients who lost all the teeth at the age of 65-75 years was 16.9% in france, 24.8% in germany, and 26-36% in the united states.1 the number of denture or removable denture users in indonesia currently reaches approximately 20 million people. however, the majority of them still do not consider the importance of cleaning dentures because material used for denture base are acrylic resin, poly metyl methacrylate, which easily polymerizes, thus forming microporosity. consequently, the material can facilitate attachment of microorganisms, leading to plaque formation.3-5 using dentures for 30 can make the oral cavity surface covered by sediment with a thickness of 0.5-1.5 derived dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 82 indrawati, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 81–86 from salivary glycoprotein and immunoglobulin, called as acquired denture pellicle (adp). the pellicle then provides substrates, such as mucin, food particles, and squamous epithelial cells so that microorganisms (bacteria and fungi) are easily to attach. the adhesion of microorganisms, consequently, will change the materials, such as sucrose and glucose in the oral cavity so that the microorganisms can multiply and proliferate. as a results, the colonies of microorganisms will increase gradually, creating denture plaque.6-8 thus, dentures that are not cleaned in a long period of time will cause pathological changes in the oral mucosa, such as denture stomatitis, halitosis and caries.9-11 various ways to keep dentures have been developed, such as mechanically by rubbing the denture base or chemically by soaking dentures in a cleaner. however, some cleaning materials available are less satisfying in cleaning dentures. for instance, alkaline perborate is less effective in cleaning thick calculus, and also has a negative effect on a soft liner. besides that, sodium hypochlorite and hydrochloric acid can cause discoloration on denture base, causing an unpleasant smell, corrosive, and abrasive.12 based on data of organic components, plaque matrix is mostly formed by protein complexes (30%) and polysaccharide. pellicle composed of proteins and polysaccharides first attaches to the denture. alternative denture cleanser is needed to break down the organic components of protein and polysaccharide matrix so that the arrangement of regular plaque becomes damaged, and plaque even can be removed from the dentures.13,14 in indonesia, papaya (carica papaya) is easy to grow and easy to obtain. carica papaya is considered as a medicinal plant that has been used for traditional medicine.14 a research conducted by sunarintyas even reports that hydrolysis activity of papain enzyme derived from carica papaya as much as 1 mg/ ml used as effective denture cleanser against plaque is 15.66 tu/ mg within 10 minutes. papain enzyme does not generate both cytotoxic effects triggered by either exposure below the ic50 value of 75.688 tu/mg, or hypersensitivity reactions in healthy people.15 β-1,3-glucanase enzyme derived from vigna unguiculata easily found in indonesia, on the other hand, can be considered as natural material used for hydrolyzing polysaccharides. β-1,3-glucanase enzyme plays a role in cutting glucose residue from the edge of a polymer or oligomers.15 a research conducted by afrilliana even shows that hydrolysis activity of β-1,3-glucanase enzyme derived from crude vigna unguiculata extract against candida biofilms is 3.1528 u/ mg. 16,17 based on the above reasons, therefore, this laboratory experimental research aimed to reveal the differences of effectiveness of ß-1,3-glucanase vigna unguiculata enzyme and papain carica papaya enzyme used as an alternative denture cleanser in hydrolysis of denture plaque. materials and method this research was conducted after approved by the health research ethics committee of faculty of dentistry, universitas airlangga (no. 68/kkepk. fkg/viii/2015). in this research, samples were divided into three groups. first, as control group i, dentures were soaked in pbs. second, as group ii (treatment), dentures were soaked in papain enzyme at concentrations of 0.5mg/ ml, 1mg/ ml, and 2mg / ml. third, as group iii (treatment), dentures were soaked in ß-1,3 glukanase enzyme at concentrations of 0.5mg / ml, 1mg / ml, 2mg / ml). materials for making those enzymes were obtained from research and industry consultation center (balai penelitian dan konsultasi industri) in surabaya. papain enzyme was obtained from crude carica papaya latex extract purified. meanwhile, ß-1,3glukanase enzyme was obtained from crude vigna unguiculata germination extract purified. to obtain the certain concentrations, 50mg, 100mg, and 200mg of the enzymes were dissolved in 100 ml of pbs at ph 7.2. in other words, the preparation of the enzymes was performed using dilution method to obtain papain upper removable dentures were used as samples with the consideration that they have a larger surface area. the dentures were cleaned using a toothbrush without paste cleaners in pbs solution. denture hygiene control then was carried out by smearing the dentures with disclosing solution. if there was a red color of the disclosing solution on the surface of the dentures, it would mean that the dentures were not clean and needed to be scrubbed. thus, the dentures had to be rinsed with distilled water before being returned to the oral cavity of the respondents. the respondents were asked to wear their removable dentures for 16 hours (from 22:00 pm to 6:00 am). all of the respondents then had to follow diet instructions (menu of food controlled). after 16 hours of usage, their removable dentures were rinsed with distilled water to clean food debris stuck. afterwards, the dentures were put in a clear tubular jar in accordance with groups i, ii, and iii with varying concentrations for 10 minutes. after 10 minutes, the dentures were taken, and solution resulted from plaquehydrolysis process using pbs and enzymes was stored in a cooler box to stop the enzymatic reaction in order to be analyzed in the laboratory. the solution resulted from plaque-hydrolysis process using pbs and enzymes then was vibrated for 2 minutes. it was soaked in ice water for 15 minutes. after centrifuged at 3000 rpm at 5° c for 10 minutes, supernatant was separated and analyzed. turbidity reading then was performed with a wavelength of 480 nm using a spectrophotometer. plaque remained on the dentures not hydrolyzed by the enzyme was brushed in 100 ml of pbs to be used as a reference amount of total plaque. total plaque is the sum of plaques hydrolyzed with residual plaque. plaque levels in solution were determined dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 8383indrawati, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 81–86 by reading optical density (od) of the sample solution. the percentage of plaque concentration dissolved was calculated by using lamber’s–beer’s formula. concentration of a substance in solution is proportional to the absorbance resulted from optical density reading with a absorbance range of 0.2 to 0.8. several statistical tests were conducted, such as normality test by using kolmogorov-smirnov test, homogeneity test by using levene’s test of variance, nonparametric test by using kruskal wallis, and significance of difference test by using tukey hsd (honestly significant different) to determine the differences between the control group and the treatment groups as well as between one treatment group and another treatment group. results this research aimed to compare the effectiveness of ß-1,3-glucanase vigna unguiculata enzyme and papain carica papaya enzyme in hydrolysis of denture plaque. examination of plaque hydrolysis was performed on the surface of the upper removable dentures in those of 12 samples of each group by soaking the dentures that had been worn for 16 hours in solutions of papain enzyme and ß-1,3 glucanase enzyme at the concentrations of 0.5mg/ ml, 1mg/ ml, and 2mg/ ml within 10 minutes (table 1). the content of ß-1,3 glucanase enzymes was lower than papain enzyme. the results of their extraction were difference in terms of particle size and color. based on the reading results of plaque hydrolysis by pbs in papain and ß-1,3 glucanase enzymes using a spectrophotometer, the mean hydrolysis obtained is as follows: the smallest mean value of hydrolyzed plaque absorbance was found in the negative control group with table 1. the mean and standard deviation of hydrolyzed plaque absorbance by pbs, papain enzyme, and ß-1,3 glucanase enzyme group number of samples mean standard deviation group 1 pbs 12 0.0161 0.0299 group 2 with 0.5mg/ ml of papain enzyme 12 0.0668 0.0275 group 2 with 1mg/ ml of papain enzyme 12 0.0897 0.0268 group 2 with 2mg/ ml of papain enzyme 12 0.1056 0.0147 group 3 with 0.5mg/ ml of ß-1,3 glukanase enzyme 12 0.1556 0.0473 group 3 with 1mg/ ml of ß-1,3 glukanase enzyme 12 0.2706 0.0581 group 3 with 2mg/ ml of ß-1,3 glukanase enzyme 12 0.4232 0.0792 table 2. the mean and standard deviation of hydrolyzed plaque absorbance remained on the dentures group number of samples mean standard deviation group 1 pbs 12 0.4891 0.0256 group 2 with 0.5 mg/ml of papain enzyme 12 0.1188 0.0412 group 2 with 1 mg/ml of papain enzyme 12 0.0880 0.0802 group 2 with 2 mg/ml of papain enzyme 12 0.1320 0.0567 group 3 with 0.5 mg/ ml of ß-1,3 glukanase enzyme 12 0.1372 0.0582 group 3 with 1 mg/ ml of ß-1,3 glukanase enzyme 12 0.1923 0.0570 group 3 with 2 mg/ ml of ß-1,3 glukanase enzyme 12 0.2256 0.0972 table 3. the mean percentage of hydrolyzed plaque absorbance (%) by pbs, papain enzyme, and ß-1,3glukanase enzyme group number of samples mean group 1 pbs 12 3.24% group 2 with 0.5 mg/ ml of papain enzyme 12 34.23% group 2 with 1 mg/ ml of papain enzyme 12 43.84% group 2 with 2 mg/ ml of papain enzyme 12 44.86% group 3 with 0.5 mg/ ml of ß-1.3 glukanase enzyme 12 53.68% group 3 with 1 mg/ ml of ß-1.3 glukanase enzyme 12 59.36% group 3 with 2 mg/ ml of ß-1.3 glukanase enzyme 12 68.77% table 4. differences between groups concentration n subset for alpha = 0.05 1 2 3 4 5 pbs 12 .01608 p0.5 12 .06675 .06675 p1 12 .08967 p2 12 .10558 .10558 g0.5 12 .15558 g1 12 .27058 g2 12 .42317 sig. .085 .329 .092 1.000 1.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 84 indrawati, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 81–86 pbs, about 0.0161. the highest mean value of hydrolyzed plaque absorbance was found in the negative control group with pbs, about 0.4891 (table 2). the lowest mean percentage was found in the treatment group with pbs, about 3.24%. the highest mean percentage was found in group 3 treated with ß-2mg/ ml of 1,3 glucanase enzyme, about 68.77% (table 3). plaque hydrolysis power generated by pbs was lower than the ones generated by ß-1,3 glucanase and papain enzymes with significant differences. the low concentration of the enzymes produced low absorbance values so that the concentration of plaques of hydrolyzed was also low. in other words, the greater the concentration of enzyme is, the greater the percentage of plaque hydrolyzed is. based on kolmogorov-smirnov test, all the data obtained had a normal distribution (p>0.05). based on levenne test, the significance of p obtained was 0.000 (p<0.05). it indicates that the variant data were not homogeneous. as a result, the data did not meet the requirements of anova test. the data were analyzed statistically using the nonparametric kruskal-wallis test. based on the results of kruskal-wallis test there was a difference between the two groups (p<0.05). to determine the differences between the control and the treatment groups as well as between in treatment group, tukey hsd test then was performed. from table 4 the best hydrolysis results are shown with the highest rates of the enzymes ß-1,3 glucanase 2 mg / ml, at 0.42317 (table 4). discussion in oral cavity, dentures made from acrylic resin will be in contact with saliva, then will absorb glycoproteins from saliva, called as acquired denture pellicle evolved into plaques within 24 hours, and will continue to form the plaque that is more mature for up to 7 days.13,18 dental plaque is composed of 70-80% of microbes and 20-30% of intercellular matrix. the intercellular matrix contains organic and inorganic materials derived from the saliva, crevicular fluid and bacterial products.19 organic materials include polysaccharides, proteins, glycoproteins, and fat. the main organic component of intermicrobial matrix is a protein-polysaccharide complex produced by microorganisms in plaque.13 there are two ways to remove plaque, stain, and calculus, namely re-polishing dentures and soaking dentures routinely. these ways aim to keep dentures moisture and to avoid them from being dry. soaking dentures in enzyme materials (mutanase and protease) has been reported to reduce plaque significantly.12 considering the main component of plaques, protein-polysaccharide complex in intercellular matrix, two enzymes then were selected, namely papain enzyme and ß-1,3-glucanase enzyme hydrolyzing plaque. examination of plaque hydrolysis was conducted on the surface of the upper dentures. this is because with the consideration that there would be a extensive contact between the surface of the denture with palatal mucosa tissue. as a result, the plaque was formed more easily, and then measured by the researcher. in this research, the examination was conducted by soaking the dentures that had been worn for 16 hours in a solution of ß-1,3 glucanase and papain enzymes at the concentrations of 0.5mg/ ml, 1mg/ ml, and 2mg/ ml within 10 minutes. plaque in general was formed after 4 hours. the use of the dentures for 16 hours is in reference to the previous research.15 the selected concentrations of the enzymes were 0.5mg/ ml, 1mg/ ml, and 2mg/ ml with 10 minutes immersion time. the concentration and length of immersion were determined based on the results of the previous research stating that the dose required by papain enzyme to hydrolyze protein plaques attached to dentures used for 24 hours is 1mg/ ml with the enzyme activity of 15.66 tu/ mg. it means that the selection of three concentrations was done as variations in the concentration range of enzymes.15 the results of sodium dodecyil sulfate (sds) examination showed that within 10 minutes papain could hydrolyze all types of protein plaques found in denture acrylic resin. soaking time more than 10 minutes will make papain protein residue left on the dentures, about 0.009 ± 0.005 g, causing toxicity and allergic symptoms in people with papain allergies.15 solution resulted from plaque hydrolysis on the dentures by pbs and the enzymes then was soaked with ice water in order to inhibit the enzymatic reaction of the release of plaque on the dentures, so the hydrolysis time to be expected was exactly 10 minutes. based on the results of the research, there were differences in the effectiveness of the ß-1,3-glucanase vigna unguiculata and papain carica papaya enzymes at the certain concentrations of 0.5mg/ ml, 1mg/ ml, and 2mg / ml in hydrolyzing the release of the denture plaque. this finding can be seen from the results of optical density readings of hydrolyzed plaque. the mechanism of enzyme reaction to plaque on both of the enzymes was equal to hydrolyze macromolecules into smaller molecules, papain enzyme is a protease hydrolyzing peptide chains of proteins, while ß-1,3-glucanase enzyme is carbohydrates hydrolyzing 1,3-ß chain on polysaccharides. the substrate differences lead to different results of hydrolysis.16,20 the reaction mechanism of ß-1,3-glucanase enzyme in hydrolyzing polysaccharides is by cutting ß-1,3 chain at random site along the polysaccharide chain found in plaque by releasing smaller oligosaccharides. the active site responsible for cutting polysaccharide chain found in two catalytic glutamic acid residues, namely e231 as a cutter of catalytic nucleophile and e288 as a proton donor to continue the enzyme reaction in some sites of polysaccharide chains.21 hydrolyzing of the bonds between molecules of ß-1,3-glucan as the main constituent components of biofilm/ plaque make plaques on the dentures can be removed easily. reaction mechanism of papain enzyme in hydrolyzing protein, on the other hand, is by cutting peptide chain in almost all amino acid residues contained in intracellular dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 8585indrawati, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 81–86 proteins and pellicle (the layer was first attached to the dentures). one of active sites responsible is cys-25 damaging carbon atoms of carbonyl groups in the peptide chain, so sh amino acids at the terminal becomes free. another active site is ast-158 helping to direct the imidazole ring of histidine 159 to deprotonation. meanwhile, his-159 donates protons in sulfur atom of cys-25, which removes acid from the substrate. after removing acid, the enzyme catalyze the next reactions.22 stabilization of papain enzyme’s structure, moreover, is composed of carbon element as hydrophobic core and hydrophilic part on the surface to interact with the medium. interaction of hydrophobic and hydrophilic parts then will encourage the folding of the enzyme so that the enzyme structure will be more stable and work more optimal.23,24 the differences in the percentage of plaque hydrolysis, furthermore, may be because of the difference in the substrate of the enzyme materials and environmental influences, such as temperature, ph, activators, inhibitors, and hydrolysis reaction of each enzyme. most plaque intracellular matrix component is 6.5 mg of polysaccharide and 2.3 mg of protein. in other words, it is similar to the results of this research showing that the percentage of plaque hydrolyzed by ß-1,3-glucanase enzyme was higher.13,25 papain enzyme works slower without the addition of activator, such as cysteine and edta. cysteine is added to reduce s-s bond in the active site of papain to form -sh bonds and also to activate the enzyme. edta, on the other hand, is added to eliminate metal ions binding to the essential thiol group of -25 cysteine on the active side of papain. therefore, it can be concluded that without activator, the activity of papain will not be optimal and the time required to hydrolyze plaque will also be longer.15 similarly, the results of this research show that the mean percentage of plaque hydrolysis generated by ß-1,3glucanase enzyme was higher than the one generated by papain enzyme. generally, enzymes require other compounds which are not proteins in their activities. one substance that can function as compounds activating or inhibiting the activity of the enzyme is a metal ion. at certain concentration, metal ions can activate the function of the enzyme (as activator) and can also inhibit the action of the enzyme (as inhibitor). in the research of micro elements, fe2+ and other ions are found in plaque. this enables the inhibition function of papain enzyme stimulated by fe 2+ ions, as a result, its activity decreases.26 temperature also can affect the action of the enzyme. temperature is very influential in thermodynamic motion of protein or enzyme molecule. a low temperature leads to a lack of collisions between molecules of the enzyme and the substrate, whereas at higher temperatures, the thermodynamic motion of enzyme molecules is large enough so that collisions between molecules of the enzyme and substrate will happen quickly. at the higher temperature, protein will also denaturize resulting in a change in the structure of the enzyme protein so that the active site of the enzyme will change.18 temperature range for papain enzyme is 30-60º c. thus, the treatment temperature was equated approximately to the room temperature of 3537º c. however, the optimal temperature for papain enzyme is 50-60º c. therefore, this condition was thought to be one of the factors leading to less optimal activity of papain enzyme generated in hydrolyzing plaque on the dentures. consequently, the hydrolysis activity of papain enzyme was lower than the one of ß-1,3-glukanase enzyme (table 2). it may be influenced by several factors, such as the optimum dose of papain enzyme, about 1mg/ ml. thus, increasing the concentration will not affect the work of the enzyme significantly and the non-optimal work of the enzyme without activators.15 ß-1,3-glucanase enzyme, on the other hand, generated more optimal plaque hydrolysis activity with all of those concentrations than papain enzyme and pbs. in other words, the higher the concentration is, the greater plaque is hydrolyzed. the properties of the enzyme including a large temperature range, inhibitor, temperature, ph, and stabilization can be considered as factors triggering the work of the enzyme more optimum and the plaque hydrolysis power of the enzyme larger. it is possible that there are maximum concentration above 2mg/ ml of ß-1,3glucanase enzyme to hydrolyze plaque on dentures. it can be concluded that ß-1,3-glucanase enzyme is more effective than papain enzyme in releasing denture plaque. the results of this research are useful as a basis for the development of alternative denture cleanser. references 1. balitbang kemenkes ri. riset kesehatan dasar; riskesdas. jakarta: balitbang kemenkes ri; 2013. p. 19. 2. agtini md. percentage of the artificial denture usage in indonesia. media litbang kesehatan 2010; xx (2): 50–8. 3. vargas cm, kramarow ea, yellowitz ja. the oral health of older americans, aging trends. 3rd ed. national center for health statistics; 2001. p. 1. 4. craig rg, powers jm, wataha jc. dental materials: properties and manipulation. usa: elsavier; 2004. p. 264. 5. wahyuningtyas e. pengaruh ekstrak graptophyllum pictum terhadap pertumbuhan candida abicans pada plat gigi tiruan resin akrilik. skripsi. yogyakarta: ugm; 2008. p. 47. 6. gharechahi m, moosavi h, forghani m. effect of surface roughness and materials composition on biofilm formation. journal of biomaterials and nanobiotechnology 2012; 3(4a): 541-6. 7. rebouillat s, fernand.p. recent strategies for the development of biosourced-monomers, oligomers and polymers-based materials: “a review with an innovation and a bigger data focus. journal of biomaterials and nanobiotechnology 2016; 07(04): 167-213. 8. shay k. denture hygiene. the journal of contemporery dental practice 2000; 1(2): 11-7. 9. mandali g, sener id, turker sb, ulgen h. factors affecting the distribution and prevalence of oral mucosal lesions in complete denture wearers. gerodontology 2011; 28(2): 97-103. 10. coulthwaite l, verran j. review article: potential pathogenic aspects of denture plaque. british journal of medical science 2007; 64(4): 181. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 86 indrawati, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 81–86 11. naeem a, amrit r, sumit m, nisha s, pankaj k, taseer b. denture hygiene: a short note on denture cleansers. journal of science 2015; 5(3): 131-3. 12. car ranza fa, newman mg, takei h.h. car ranza’s clinical periodontology. 11th ed. st. louis, missouri: saunders el sevier; 2012. p. 239-48. 13. jagadeesh js, shalini n. an overview of carica papaya and its medicinal uses. research journal of pharmaceutical, biological and chemical sciences 2014; 5(2): 641. 14. sunarintyas s. peran papain pada pelepasan plak gigi tiruan serta sifat biokompatibilitas. disertation. surabaya: program pascasarjana universitas airlangga; 2002. p. 25, 95, 97, 129-30. 15. katatny mhei, somitsch w, robra kh, katatny msei, gubitz gm. production of chitinase and 1,3-glucanase by trichoderma harzianum for control of the phytopathogenic fungus sclerotium rolfsii. j food technol biotechnol 2000; 38(3): 170-80. 16. afrilliana f, baktir a, harsini m. karakterisasi enzim β-1,3glukanase dari beberapa sereallia serta amobilisasi ekstrak kasar enzim terbaik sebagai kandidat biosensor kandidiasis. skripsi. surabaya: fakultas sains dan teknologi universitas airlangga; 2014. 17. putri mh, herijulianti e, nurjannah n. ilmu pencegahan penyakit jaringan keras dan jaringan pendukung gigi. preventive dentistry. jakarta: penerbit buku kedokteran egc; 2010. p. 257. 18. eley bm, manson jd. periodontics. 5th edition. london: elsevier ltd; 2004. p. 21-8, 133-40. 19. am ri e, mamboya f. papain, a plant enzyme of biological importance: a review. american journal of biochemistry and biotechnology 2012; 8(2): 99-104. 20. varghese nj, garret tpl, colman pm, hou pb, fincher gb. three dimensional structures of two plant β-glucan endohydrolases with distinct substrate specifities. proc natl acad science usa 1994; 91(7): 2785-9. 21. xian m, chen x, liu z, wang k, wang pg. inhibition of papain by s-nitrosothiols. formation of mixed disulfides. j biol chem 2000; 275(27): 20467-73. 22. wang l, sun n, terzyan s, zhang x, benson dr. histidine/ tryptophan π-stacking interaction stabilizes the heme-independent folding core of microsomal apocytochrome b5 relative to that of mitochondrial apocytochrome b5. biochemistry 2006; 45(46): 13750-9. 23. chamani j, heshmati m, rajabi o, parivar k. thermodynamic study of intermediate state of papain induced by n-alkyl sulfates at two different ph values: a spectroscopic approach. open sur sci j 2009; (1): 20-9. 24. cury ja, rebelo ma, del bel cury aa, derbyshire mt, tabchoury cp. biochemical compositin and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. caries res 2000; 34(6): 491-7. 25. gafar m, grigorescu g, andrescu c, dumitriu h, petrescu l, farcaşiu m, popescu r, maliţa c. a study of the microelements in the dental plaque. rev chir oncol radiol orl oftalmol stomatol ser stomatol 1989; 36(3): 161-6. 26. harris elv. concentrition of extract. in: harris elv, angal s, eds. protein purification methods: a practical approach. new york: oxford university irl. press; 1998. p. 123–61. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p81-86 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p81-86 181181 case report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 181–184 contemporary guided bone regeneration therapy for unaesthetic anterior peri-implantitis case benso sulijaya, sandra olivia kuswandani, and yuniarti soeroso department of periodontology faculty of dentistry, universitas indonesia jakarta – indonesia abstract background: dental implant is one of an alternative solutions reconstruction therapy for missing teeth. complication of dental implant could occurs and leading to implant failure. in order to restore the complication, surgical treatment with guided bone regeneration (gbr) is indicated. the potential use of bone substitutes is widely known to be able to regenerate the bone surrounding the implant and maintain bone volume. purpose: the study aimed to demonstrate the effectiveness of implant-bone fully coverage by using sandwich technique of biphasic calcium phosphate (bcp) and demineralized freeze-dried bone allografts (dfdba) bone substitutes combined with collagen resorbable membrane. case: a 24-year-old male came with diagnosis of peri-implantitis on implant #11. clinical finding indicated that implant thread was exposed on the labial aspect. case management: after initial therapy including oral hygiene improvement performed, an operator did a contemporary gbr to correct the defect. bone graft materials used were 40% β-tri calcium phosphate (β-tcp)-60% hydroxyapatite (ha) on the outer layer and dfdba on the inner layer of the defect. resorbable collagen membrane was used to cover the graft. conclusion: gbr with sandwich technique could serve as one of the treatment choices for correcting an exposed anterior implant that would enhance the successful aesthetic outcome. keywords: guided bone regeneration; implant; bone substitute correspondence: benso sulijaya suyono, department of periodontology, building b, 2nd floor. faculty of dentistry, universitas indonesia. jl. salemba raya no. 4. jakarta pusat 10430, indonesia. e-mail: bensosulijaya@gmail.com; benso.sulijaya87@ui.ac.id; tel. (+6221) 3911502/ fax. (+6221) 3911502. introduction p e r i i m p l a n t i t i s i s o n e o f t h e d e n t a l i m p l a n t complications. it defines as an inflammatory condition on tissues surrounding implant characterized with loss of supporting bone and inflammation.1 koldsland et al.2 did an observed in university of oslo on 109 canadian subjects and showed the prevalence of peri-implantitis was 11.3% to 47.1%.2 moreover, tarnow3 stated that the average amount of bone loss in peri-implantitis was about 30% of implant length. contrary a tarnow, nicolo et al.4 revealed that the incidence of peri-implantitis was lesser than most of researches. several studies have been conducted to cure periimplantitis. heitz-mayfield et al.5 explained that generally there are two interventions, non-surgical (e.g. local and or systemic delivery antibiotic, mechanical debridement, antiseptics, air-powder abrasive, er:yag laser) and surgical (regenerative treatment with or without additional regiment). schwarz et al.6 reported that treatment with graft material and collagen membrane for peri-implantitis cases showed pocket depth reduction from 7.1 mm to 4.4 mm. sandwich technique of guided bone regeneration is defined as different bone allografts were used to encourage the composition of natural bone. different layers are consisted of autograft and allograft materials.7 fu et al.8 described the effectiveness of sandwich technique for augmenting bone in implant placement simultaneously. the use of gbr has been accepted as therapy modality because it reported has a predictable bone gain. this case report aimed to demonstrate the effectiveness of implant-bone fully coverage by using sandwich technique dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p181-184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p181-184 182 sulijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 181–184 of biphasic calcium phosphate (bcp) (containing 40% β-tri calcium phosphate (β-tcp)-60% hydroxyapatite (ha)) and demineralized freeze-dried bone allografts (dfdba) bone substitutes combined with collagen resorbable membrane. case a 24 year-old-male came with diagnosis of periimplantitis on implant #11 (figure 1). dental history reported implant placement in last 6 months. patient was an active smoker (6 cigarettes per day). patient used removable partial denture made from flexible material at that moment. from clinical examination, the operator found that patient had an average smile line and thick periodontal biotype. periodontal parameter was evaluated and the oral hygiene index-score (ohi-s) was 1.7 (based on silness and loe).9 pocket depth on labial was 5 mm. bleeding on probing score was negative. implant thread was exposed on the labial aspect. no mobility and suppuration were found. lang et al.,10 classified the protocol concept of cumulative interceptive supportive therapy (cist)modified akut as: stage a (pocket depth (pd) <3 mm) is indicated to mechanically cleaning and polishing; stage b (pd 4-5 mm) is indicated to mechanically cleaning with anti-infective regiments; stage c (pd>5 mm) is indicated to mechanically cleaning, microbiological test and antiinfective regiments; stage d (pd >5 mm) is indicated to respective or regenerative therapy.10 based on that protocol, this case was categorized as stage d (pocket depth (pd) >5 mm; bone loss >2 mm) that needed a regenerative surgery. case management two weeks before surgery, patient fulfilled the initial therapy including scaling, root planing, polishing and correcting the denture. patient also informed to use minocycline hydrochloride (minocycline hcl) 2% gargle, twice a day. patient was also educated to reduce his smoking habit. no systemic antibiotic was given since patient had no systemic disease. a written informed consent was signed before the surgery. periodontal reconstructive surgery with sandwich technique of gbr was planned. local anesthesia of 2% lidocaine (1 : 50,000 epinephrine) (indofarma, indonesia) was administrated in labial and palatal sides. papilla preservation flap was indicated in this case. vertical incisions were made on the 2/3 mesial angles of adjacent teeth. full thickness flap was elevated. it noted that the second and forth threads of implant were exposed and not covered by labial bone. the width of bone defect was estimated 3 x 5 mm width. granulation tissue and necrotic bone were excavated. the implant surface was cleaned with plastic-made instrument. bone decortication was made using slow-rotating small diameter bur on the cortical bone surround the implant (figure 2). 8 figure 1. the implant on #11 was detected on labial aspect (seen by arrow). figure 2. a) the implant tread on #11 was seen when the flap is raised. there was bone loss at the labial area. b. implant was cleaned and de-cortication procedure was done on the bone surrounding the implant (seen by arrow); c) bone graft materials bcp was placed on the outer layer and dfdba on the inner layer of the defect; d) collagen membrane was used to cover the graft; e) flap was repositioned back and sutured using nylon 5.0; f) two weeks after surgery. a b c d f e figure 1. the implant on #11 was detected on labial aspect (seen by arrow). 8 figure 1. the implant on #11 was detected on labial aspect (seen by arrow). figure 2. a) the implant tread on #11 was seen when the flap is raised. there was bone loss at the labial area. b. implant was cleaned and de-cortication procedure was done on the bone surrounding the implant (seen by arrow); c) bone graft materials bcp was placed on the outer layer and dfdba on the inner layer of the defect; d) collagen membrane was used to cover the graft; e) flap was repositioned back and sutured using nylon 5.0; f) two weeks after surgery. a b c d f e 8 figure 1. the implant on #11 was detected on labial aspect (seen by arrow). figure 2. a) the implant tread on #11 was seen when the flap is raised. there was bone loss at the labial area. b. implant was cleaned and de-cortication procedure was done on the bone surrounding the implant (seen by arrow); c) bone graft materials bcp was placed on the outer layer and dfdba on the inner layer of the defect; d) collagen membrane was used to cover the graft; e) flap was repositioned back and sutured using nylon 5.0; f) two weeks after surgery. a b c d f e figure 2. a) the implant thread on #11 was seen when the flap is raised. there was bone loss at the labial area. b. implant was cleaned and de-cortication procedure was done on the bone surrounding the implant (seen by arrow); c) bone graft materials bcp was placed on the outer layer and dfdba on the inner layer of the defect; d) collagen membrane was used to cover the graft; e) flap was repositioned back and sutured using nylon 5.0; f) two weeks after surgery. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p181-184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p181-184 183183sulijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 184–184 ossifi (equinox medical technology b.v., holland) bone graft materials used were biphasic calcium phosphate (bcp) (containing 40% β-tri calcium phosphate (β-tcp)60% hydroxyapatite (ha)) on the outer layer and dfdba (batan, jakarta, west java, indonesia) on the inner layer of the defect. osseoguard (collagen matrix, inc., franklin lakes, nj, usa) resorbable collagen membrane was used to cover the graft. tension-free flap was made in order to promote the healing process. five hundred grams of paracetamol (indofarma, indonesia) was prescribed 3 times a day for 3 days as an analgesic. two weeks after surgery, the clinical outcome of implant-bone dehiscence was corrected and the gingiva was healthy and firm. patient used temporary abutment screw retained crown made by using 3m espe filtek z250xt nano hybrid universal (3m, espe, st paul, usa) composite material. twelve months follow-up, implant was completely healed with no sign of inflammation and no shadow of implant treads exposure (figure 3). discussion peri-implant mucositis and peri-implantitis are frequently occurred as a complication caused by an inflammatory host response.11,12 mir-mari et al.13 found 9.1% peri-implantitis cases from total 946 implants and almost 40% peri-mucositis cases in spain. in italy, marrone et al.14 revealed 37% subjects from 103 patients from 2002 to 2012 had suffered peri-implantitis. in this particular patient, an exposed dental implant is categorized as periimplantitis since there was a bone loss on labial side of implant #11. this complication might have happened as a result of inappropriate treatment planning and or surgical procedure. labial bone dehiscence has risen within 6 months period after implant placement. early detection of this condition will brighten our prediction about outcome’s possibilities. treatment option to manage peri-implanitis is depend on the situation. the goal is to discontinue further bone loss, rebuild a healthy peri-implant mucosal seal that could regenerate both hard and soft tissue-implant integration.8,15 oral hygiene improvement was done to eliminate bacterial biofilm on peri-implant surfaces. plastic, teflon, carbon and titanium instruments are recommended to be used in managing peri-implant diseases.16 this is because metallic instrument could roughening implant surface that directly could increase plaque retention.15 lisa et al.5 explained that to treat peri-implantitis, it is essential to improve oral hygiene, remove prosthesis, debride non-surgically and to use of bone substitute. surgical approach is applied after initial therapy was performed. aims of this phase are to decontaminate implant surface, to fill the osseous defect surround the implant and also to improve soft tissue condition.1 rafl et al.16 explained that the principle of treating peri-implantitis is mainly similar with periodontitis. potential use of bone substitutes are widely known could regenerate the bone surrounding the implant and maintain bone volume. in some of severe bone atrophy cases that are not sufficient for implant, bone grafting procedure might be required.17 analyzing on this case, author followed the protocol of cumulative interceptive supportive therapy (cist) and modified into akut-concept produced by lang et al.10 it stated that stage a (pocket depth (pd) <3 mm) was indicated to mechanically cleaning and polishing; stage b (pd 4-5 mm) was indicated to mechanically cleaning with anti-infective regiments; stage c (pd>5 mm) was indicated to mechanically cleaning, microbiological test and anti-infective regiments; stage d (pd >5 mm) was indicated to respective or regenerative therapy. based on the stage mentioned, this case described as stage d (pocket depth (pd) >5 mm; radiology bone loss >2 mm) that need a regenerative surgery. bone substitutes used in this case was biphasic calcium phosphate (bcp) and dfdba. biphasic calcium phosphate was a mixture of an alloplastic or synthetic graft material with the composition of 40% β-tri calcium phosphate (β-tcp)-60% hydroxyapatite (ha), whereas dfdba was an allograft that collected from human. even though autograft is a gold standard for gbr, but both of bcp and dfdba has a quite high osseo-inductive and osseoconductive ability. in some studies the use of bone graft and membrane are still debate-able, but rafl et al.10 have concluded that the result studies of gbr using bone graft and membrane were higher than gbr using membrane or bone graft alone. schwarz at al.18 treated 22 peri-implantitis patients randomly with open flap debridement combined with the application of nano-crystalline hydroxyapatite and collagen membrane. he stated that there was no significant differences were found. the combination of alloplastic, allograft and or xenograft material could substitute and mimic the characteristic of autologous graft.16 wang et al.7 explained that sandwich technique could be used to improve the composition of native bone. autograft layer is placed over the exposed implant could serves as cancellous bone. then, the outer layer is covered by cortical bone allograft or alloplast material. collagen membrane was applied as a barrier membrane and it serves to protect bone regeneration from soft tissue proliferation. based on the previous study described by wang et al.,7 we applied bcp bone graft 9 figure 3. twelve months follow-up post-surgery. figure 3. twelve months follow-up post-surgery. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p181-184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p181-184 184 sulijaya, et al./dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 181–184 material (40% β-tcp 60% ha) on the outer layer and dfdba on the inner layer of the defect. beside the osseoinductive and osseo-conductive characters, the combination of bcp and dfdba give a better osseogenic character. the treatment goals in this case are the absent of inflammation, pocket depth reduction, no further bone loss, soft and hard peri-implant tissues establishment. important things stated in american academy of periodontology consensus,19 that principles of regular implant’s evaluation and structure beyond should be maintained. in conclusion, guided bone regeneration (gbr) with sandwich technique could serves as one of the treatment choices for correcting an exposed anterior implant that will enhance the success of aesthetic outcome and maintain long-term implant stability. references 1. mombelli a, moene r, decaillet f. surgical treatments of periimplantitis. eur j oral implantol 2012; 5(suppl): s61–s70. 2. koldsland oc, scheie aa. prevalence of peri-implantitis related to severity of the disease with different degrees of bone loss. j periodontol 2010; 81(2): 231-8. 3. tarnow dp. increasing prevalence of peri-implantitis: how will we manage?. j dent res 2016; 95(1): 7-8. 4. cavalli n, corbella s, taschieri s, francetti l. prevalence of periimplant mucositis and peri-implantitis in patients treated with a combination of axial and tilted implants supporting a complete fixed denture. scientific world journal 2015; doi: 10.1155/2015/874842. 5. heitz-mayfield lj, mombelli a. the therapy of peri-implantitis: a systematic review. int j oral maxillofac implants 2014; 29(suppl): 325-45. 6. schwarz f, sahm n, bieling k. surgical regeneration treatment of peri-implantitis lesions using nanocrystalline hydroxy-apatite or a natural bone mineral in combination with collagen membrane: a four-year clinical follow-up report. j clin periodontol 2009; 36: 807-14. 7. wang hl, misch c, neiva rf. ‘‘sandwich’’ bone augmentation technique: rationale and report of pilot cases. int j periodontics restorative dent 2004; 24(3): 232-45. 8. fu j, wang h-l. the sandwich bone augmentation technique. clin adv peridontics 2012; 2: 172-7. 9. loe h. the gingival index, the plaque index and the retention index systems. j periodontol 1967; 38(6 suppl): 610-6. 10. lang np, berglundh t, heitz-mayfield lj, pjetursson be, salvi ge, sanz m. consensus statements and recommended clinical procedures regarding implant survival and complications. int j oral maxillofac implants 2004; 19(suppl): 150-4. 11. berglundh. t, persson l. a systematic review of the incidence of biological and technical complications in implant dentistry reported in prospective longitudinal studies of at least 5 years. j clin periodontol 2002; 29(suppl 3): 197-212. 12. lindhe j, meyle j. peri-implant diseases: concensus report of the sixth european workshop on periodontology. j clin periodontol 2008; 35 (suppl. 8): 282–5. 13. mir-mari j, mir-orfila p, figueiredo r, valmaseda-castellón e, gay-escoda c. prevalence of peri-implant diseases. a crosssectional study based on a private practice environment. j clin periodontol 2012; 39(5): 490-4. 14. marrone a, lasserre j, bercy p. prevalence and risk factors for periimplant disease in belgian adults. clin oral implants res 2012; 2: 247-50. 15. popat rp, bhavsar nv, popat pr. peri-implantitis: management of ailing, failing & failed dental implants. iosr journal of dental and medical sciences 2014; 13(1): 43-6. 16. smeets r, henningsen a, jung o, heiland m, hammächer c, stein jm. definition, etiology, prevention and treatment of periimplantitis: a review. head face med 2014; 10: 34. 17. chiapasco m, zaniboni m. augmentation procedures for the rehabilitation of deficient edentulous ridges with oral implants. clin oral implants res 2006; 17(2): 136-59. 18. schwarz f, bieling k, latz t, nuesry e, becker j. healing of intrabony peri-implantitis defects following application of nanocrystalline hydroxyapatite (ostim) or a bovine-derived xenograft (bio-oss) in combination with a collagen membrane (bio-gide). a case series. j clin periodontol 2006; 33(7): 491-9. 19. cohen re. research, science and therapy committee, american academy of periodontology. position paper: periodontal maintenance. journal of periodontology 2003; 74(9): 1395-401. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p181-184 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p181-184 127 the microbiological detection of mycobacterium tuberculosis from oral lesion atik kurniawati department of oral biology faculty of dentistry jember university jember indonesia abstract tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis. the oral manifestations of tuberculosis have been reported. the objectives of this study was to detect mycobacterium tuberculosis from oral lesions by swabbing. samples were received from collected primary data on annually clinical report status and then clinical intra oral examination was performed. oral lesions were swabbed and cultured using lowenstein jensen media, middlebrook 7h9 and middlebrook 7h10. positive culture were recorded and observed everyday until 8 weeks. from 66 samples, there were 2 patients with ulcer as an oral clinical manifestation with location on the tongue. culture examination revealed 2 positive samples. it was inferred that mycobacterium tuberculosis could be detected in oral lesion by swabbing. key words: microbiological detection, m. tuberculosis, oral lesion correspondence: atik kurniawati, c/o: bagian biologi oral, fakultas kedokteran gigi jember. jln. kalimantan no. 37 jember 68121, indonesia. e-mail: atikku_fkgunej@yahoo.com introduction tuberculosis is a chronic infectious granulomatous disease caused by mycobacterium tuberculosis, less frequently by mycobacterium bovis or by other atypical mycobacteria. depending on the portal of entry of infection and on the degree of haematogenous many organs and system of the body could be affected. the common site of infection is the lungs.1 tuberculosis is a disease of global importance. one third of the world population is estimated to have infection with mycobacterium tuberculosis and eight million new cases tuberculosis arise each year. the tuberculosis crisis is likely to escalate since the human immunodeficiency virus (hiv) epidemic, as a result greater increased in the number of tuberculosis cases. the majority of tuberculosis patients are 15 to 45 years of age, mostly in productive years of live. tuberculosis kills over two million people world wide each year, more than any single infectious disease, including aids and malaria.2,3 in indonesia, new cases of this disease exceed 150 000 per year or 8 per 10 000 persons and those with latent infection are estimated 24 per 10 000 persons, based on survei of kesehatan rumah tangga (skrt 1996). tuberculosis in indonesia was the third current incidence in the world after the republic of china and india.4 primary infection of tuberculosis in human ussually occurs in the lungs and consist of a localized lesion at the site of impaction of the bacilli and, at a later stage, regional adenitis. initially, litlle or no tissues reaction take place but after a period of weeks or months delayed hipersensitivity to the tubercle bacillus develope and the host tissues react with an intense inflammatory response with caseations. the lesion may heal by fibrosis and calcification, or an equilibrium between the host and tubercle bacilli may develope, which, if disrupted, produce symptomatic disease many years later. the lesion may progress, spreading locally in the lungs and eroding in to bronchi, producing open tuberculosis, or in the bloodstream, to produce a disseminated form of the disease, including in oral cavity.3,5,6,7 the oral manifestations of tuberculosis have been reported.7,8,9,10 oral lesions were usually secondary to primary tuberculosis infection elsewhere in the body. primary infections of the oral mucosa by mycobacterium tuberculosis have been described even they are rare. the source of infection is infected sputum or blood-borne bacilli in the case of secondary infection, while in primary infection the source is unknown. lesions are found more commonly in the posterior parts of the mouth and it has been suggested that there is related to the relative distribution of lymphoid tissue. there is wide variation in the clinical presentation of tuberculosis lesions of the oral mucosa, with ulceration and pain being commonly reported. however, diffuse inflammatory lesions, granulomas and fissures have also been described and pain may be mild or absent. the tongue is probably most commonly affected, but lesions have been noted on the buccal mucosa, gingival, floor of mouth, lips and the hard and soft palate as well.8,9,10,11 pulmonary tuberculosis is suggested by persistent productive cough for three weeks or longer, weight loss, night sweats and chest pain. pulmonary tuberculosis usually occurs in the apex of the lungs. these developed cavities which contain large population of tubercle bacilli can be 128 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 127-130 detected in a sputum specimen. the diagnosis can only be made reliable on demonstrating the presence of tubercle bacilli or acid fast bacilli (afb) the sputum by means of microscopy and/or culture in the laboratory.1,2,13,14 but not all patients that have been diagnosed suspect tuberculosis pulmonary, the sputum could not enough be turned out with coughing when examined, so the afb test was negative and diagnosed as non tuberculosis, whereas they potentially for spreading tuberculosis disease. therefore, in this research, the method of taking samples by swabbing within oral lesion, used one of alternative sampling method to diagnosed tuberculosis disease. the purpose of the research was to detect mycobacterium tuberculosis from oral lesion by culture. material and method the research was a laboratory observation, conducted on the population of lung tuberculosis with criteria: the patients were diagnosed as tuberculosis based on the lung tuberculosis guidance stated by health department of republic of indonesia,4 productive-aged (15–60 years old), carrying no other systemic diseases, male or female, new tuberculosis patients (initially diagnosed) with positive afb, or they who were still in curing process. the material, purchased from the specimens were lesions taken from the oral cavity of lung tuberculosis patients. oral lesion is a scar resulting in damage and inflammation on oral mucosa. conducting swab with particular swab instrument. swab was a process of wiping on the mucosa of the lesion after the exudates was previously cleaned.5 the result of swabbing subsequently stored directly into liquid media middlebrook 7h9 as the carrier. the growth shape of bacteria within the liquid media was marked by the presence of muddiness. the specimen should be delivered to laboratory soon for further procedure of decontamination. the decontamination was conducted in respect with the recommendation of who using sodium hydroxide method. this method provide very effective result, foremost, if fulfilling some factors such as limitation of decontamination time (15 minutes), centrifugation upon the speed of 3500 rpm because it was possible a lot of bacteria contained in the swab result, rinsing using pz sterile to reduce or remove further effect of bactericidal sodium hydroxide that only optimally eradicated other bacteria except m. tuberculosis. after the swabbing result had been centrifuged, the supernatant was disposed into lisol. the sediment and the media remnant were respectfully suspended by vortaking. the result of this suspension was wholly cultured and planted in the solid media in order to be askew lowenstein jensen (l.j media) and middlebrook 7h10. the culture was stated positive if it had been mycobacterium tuberculosis, exhibiting rough colony, white cream-coloured, broccoli likeflowershaped on the lowenstein-jensen media and rough colony formation, dry, scaling, yellowish cream-coloured on the surface of culture media on the middlebrook 7h10.13,14,15 result the research observed 66 tuberculosis patients, and oral examination was conducted. it was found that 2 patients had oral lesions, or as much as 3%. the lesions were ulcers on the tongue. both lesions were subsequently swabbed, and the result showed that there was positive culture growth. discussion the accuracy of detection m. tuberculosis from swabbing oral lesions of tuberculosis patients was influenced by the factor of swabbing, delivering, maintaining or the process of decontamination concentration as well as the examining method of microbiological laboratory.16 swabbing from oral lesion was conducted using sterile swab by pressing and spinning swab on the lesion surface and putting it in the liquid media of middlebrook 7h9. it was liquid media as the carrier of specimen so it was not dry and dead regarding the result which was frequently very little so it needed maintaining the life circumstance as in the lesion environment. besides, it helped the growth of m. tuberculosis since this media was abundant in nutrition so that improved the detecting sensitivity toward m. tuberculosis in the specimen. in this research, growing media was conducted through duplo using lowestein-jensen media and middlebrook 7h10 that was aimed at increasing the detecting sensitivity toward m. tuberculosis, increasing the detecting performance of m. tuberculosis in small number from 1 up to 100 micro-bacteria such as in the swab or sputum of the patients that were still in the curing treatment. thus, this research improved the detecting sensitivity toward m. tuberculosis in the lesion swabbing specimens with a method using liquid media for the culture and transportation, and was subsequently followed by culture in the two solid media (duplo) of lowenstein-jesen (l-j) and middlebrook 7h10. after the swabbing result had been centrifuged, the supernatant was disposed into lisol. the sediment and the media remnant were respectfully suspended by vortaking. the result of this suspension was wholly cultured and planted in the solid media in order to be askew lowenstein jensen (l.j media) and middlebrook 7h10. culture method was still gold standard method since from the infection source, the m. tuberculosis bacteria, as the cause of infection according to postulate koch, could be isolated. the advantage of this culture method that it has sensitivity more than 95% and enabled to detect m. tuberculosis 1-100 per ml. sputum, and its specification nearly 100% especially if the standard procedure is conducted appropriately.16,17 solid media was used because it could detect the morphology of m. tuberculosis colonies. the solid media used in this research was a lowenstein jensen (conventional standard media), that included into egg base media group. this media was used to isolate m. tuberculosis primer. the characteristic shape of colony m. tuberculosis in the lowenstein jensen (figure 1) was rough characteristic 129kurniawati: the microbiological detection of micobacterium tuberculosis colonies, yellowish-coloured, non-pigmented, resembling to broccoli flower-shaped.13,14,15 culture examination was carried out once a week and if there was no growth after 8 weeks, the culture would be stated negative. after observing the first week, there had not been any growth yet, in the third week (the 22nd day) there was growth of characteristic colony. this result was not far different with pfyffer’s research17 where examination time for all types of mycobacterium with lowenstein-jensen was averagely 23,1 days, while the result of susilo’s research18 of which samples were taken from the sputum of lung tuberculosis patients, the mean of their growth were 16.48 days. in the middlebrook media 7h10, that is one of the groups of commercial agar base media, the culture result was the same with the lowenstein jensen media. the culture examination was conducted once a week, and if there was no growth after 8 weeks, it was stated that the culture was negative. in this research, after observing the first week (the 6th day), the growth was present, the growth of colony was faster and more obvious because of the transparent media. the shape of characteristic colony of m. tuberculosis bacteria in this media (figure 2) was rough colonies in dry shape, scaling, cream-colored.13,14,15 this result was not far different from pfyffer researchr17 where the examination time toward all types of mycobacterium using middlebrook media 7h10, the mean was 6.4 days, while the result of susilo’s research18 in which samples were taken from the sputum of lung tuberculosis patients, the growth mean was 6,41 days. thus, the use of duplo media, lowenstein-jensen (l-j media) and middlebrook 7h10 not only improved the detecting performance or sensitivity but also accelerated the positive growth. in both media, the growth of bacteria was showed by the presence of characteristic colony figures (figure 1 and 2). in observing the growth of m tuberculosis bacteria, generally, the growth decreased after the fourth week, the growth was absent through the sixth week and the death was started in the eighth week. the observation toward the growth of m. tuberculosis bacteria from swabbing the oral lesion that was cultured according to the growth pattern of bacteria in general where the cell division of m. tuberculosis bacteria is 18–24 hours. the growth of a bacteria colony in the solid media was expressed after the age of colony had been more than a week, however, if the number of bacteria coming from the specimen planted in the media was bulk, the characteristic colony would be detected faster15 it supported the research conducted by pfyffer17 and susilo18 where the mean time of the growth of bacteria colony was faster compared to the result of this research due to the specimens such as the sputum of tuberculosis sufferers, while in this research the samples were taken from the swabbing applied in the oral lesion in which the bacteria number was less compared to the sputum. conclusion: it was inferred that mycobacterium tuberculosis could be detected from the oral lesion by swabbing using culture method, and the growth pattern of mycobacterium tuberculosis in general as well. references 1. davis bd, dulbecco reirsen hn, ginsberg hs. growth and death of bacteria, protein synthesis & location, mycobacteria. in microbiology. 4th ed. philadelphia: jb lippincot & company; 1990. p. 51–63, 105-21, 647–64. 2. gloucester. essentials of tuberculosis control for the practising physician. canadian thoracic society canadian medical association journal 1994; 150: 561–71. 3. martin g, lazzarus a. epidemiology and diagnosis of tuberculosis: recognition at risk patient is key to prompt detection. journal simposium 2000 august; 108(2). 2000, post graduate medicine. 4. depkes ri. pedoman nasional penanggulangan tuberkulosis oleh pusat kesehatan kerja. 2005. available at: http:/www.depkes.go.id/ index.php?option=articles?&task=viewarticles&article=154&itemd =3. accessed july 5, 2005. figure 1. culture of m. tuberculosis on the lowenstein-jensen media: organism exhibiting typical rough colonies, white cream-coloured (no pigmented), broccoli like flower-shaped (right tube). figure 2. culture of m. tuberculosis on the midllebrook 7h10 media, organism exhibiting typical rough colonies formation, dry, scaling, yellowish cream-coloured on the surface (right tube). 130 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 127-130 5. jones jh, mason dk. oral manifestation of systemic disease. london, philadelphia, toronto: wb saunders company ltd; 1990. p. 349–52. 6. sonis st, fazio rc, fang lf. principal and practice of oral medicine. philadelphia, london, toronto, mexico, city, rio de janeiro, sydney, tokyo: wb saunders company; 1995. p. 192–3, 197. 7. lynch ma. burket’s oral medicine diagnosis and treatment. 10th ed. philadelphia, toronto: jb lippincott company; 2003. p. 441–4. 8. fujibayashi t, takahashi y, yoneda t. tuberculosis of the tongue: a case report with immunologic study. oral surg 1979; 47:427. 9. hashimoto y, tanioka h. primary tuberculosis of the tongue : report of case. j oral maxillofac surg 1989; 47:746-7. 10. mignogna md, muzio llo, favia g, ruoppo e, sammartino g, zarelli c, bucci c. oral tuberculosis : a clinical evaluation of 42 cases. mac millan published ltd, oral disease 6. 2000. p. 25–30. 11. bricker sl, langlais rp, miller cs. oral diagnosis, oral medicine and treatment planning. pensyluania: lea and febiger; 1994. p. 180–91. 12. crofton sj, horne n, miller f. general background for clinical tuberculosis; tuberculosis in children; pulmonary tuberculosis in adult; treatment tuberculosis. in: clinical tuberculosis. london and basingstoke: mac milan press ltd; 1992. p. 1–27, 29-82, 89–115, 139–71. 13. richard j, wallace jr, barbara a, ellison b, hall l, roberts g, linda b. clinical and laboratory feature of mycobacterium tuberculosis. j clin microbiol 2002; 40:2930–5. 14. baron ej, peterson lr, finegold sm. mycobacteria. in: bailey, scott’s, editors. diagnostic microbiology. st louis, baltimore, boston, chicago, london, madrid, philadelphia, sydney, toronto: mosby year book, inc; 2002. p. 550–633. 15. boyd rf. mycobacterial genetics, mycobacterium tuberculosis. in: basic medical microbiology. 5th ed. boston, new york, toronto, london: little brown and company; 1995. p. 61–81, 331–4. 16. de kantor in, kim sj, frieden t. laboratory services in tuberculosis control. culture part iii. world health organization. 1998. 17. pfyffer, patel am, abrahams ew. micobacteria growth indicator tube. j clin micr 1997; 35:767–70. 18. susilo b. studi komparasi pemeriksaan kultur m.tuberculosis dengan media lowenstein jensen, middlebrook 7h10 komersial dan middlebrook 7h10 modifikasi. thesis. surabaya: fakultas pascasarjana universitas airlangga; 2003. p. 60–90. isi vol 39 no 2 april 2006 file pertama.pmd 59 tensile bond strength of hydroxyethyl methacrylate (hema) bonding agent to bovine dentine surface at various humidity adioro soetojo department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract one factor that make bonding agent adhere to dentine surface maximally is the humidity condition around the dentine surface. the best bond strength between bonding agent with dentine surface is depending on the moist surface. it mean that the dentine surface should neither too dry or wet. the objective of this research is to know the tensile bond strength of hydroxyethyl methacrylate (hema) bonding agent to bovine dentine surface at various humidity. the bovine dentine was grounded to give flat surface, which was then etched with 37% phosphoric acid for 15 seconds. dentine was washed with 20 ml water and dried with blot dry technique. the dentine, except the control group, was placed in a desiccator for one hour at difference humidity. dentin was removed from desiccator, then covered with bonding agent and put into tensile tool plunger. self-cured acrylic resin was applied on this bonding agent layer, which was placed on opposite-plunger. after 24 hours, tensile bond strength was measured with autograph instrument. data was statistically analyzed with one-way anova at 95% confidence level, continued with lsd test. results of this study showed that 60%–90% humidity gave produce the lower of tensile bond strength of bonding agent to dentine surface (p ≤ 0.05). in conclusion, the treatment in 60% humidity gave the greatest tensile bond strength. key words: humidity, dentine bonding agent, bovine dentine, tensile bond strength correspondence: adioro soetojo, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. generally, the bond efficacy of resin bonding materials to dentin collagen depend on some factors that are: the lowness of monomer viscosity, concentration and type of monomer, acid application as conditioner, humidity and temperature around fibril kolagen.4–7 research concerning the influence of humidity to tensile bond strength of dentin bonding materials at dentin surface was done by in vitro experiment. in this research, they used 33%, 50%, 75%, and 100% humidity. humidity of oral cavity depends on the usage of rubber dam. if we use rubber dam, the humidity is 50% at 23 °c that influenced by the humidity of the dental practice room. but, if we do not use rubber dam, the humidity is about 80–94%.8 in this research, we used humidity that ranging from 60–90% with temperature 25 °c. this 60% humidity is minimum humidity according to the research previously. at 50 % and 65 humidity, the tensile bond strength of bonding materials at dentine is higher than 80% and 90% humidity.9 the purpose of this research is to know the tensile bond strength between hema dentine bonding agent to bovine dentine surface at various humidity. this research will be explain the mechanism of tensile bond strength of hema dentine bonding agent to bovine dentine surface at various humidity, so clinically will be introduction in operative dentistry field, drying is one step of the application of dentine bonding based on hydroxyethyl methacrylate (hema) to dentine surface. this step is done based on total etched technique. the purpose of the procedure is to blot up excess water that were used for washing dentine surface after etching process.1–3 the purpose of washing is to removed the salt that formed as reaction of acid etched materials and dentine mineral. some researchers said that the dentine surface should neither too dry nor wet.4 if the surface is too dry, the dentine collagen will collapse so that the bonding between resin and dentine collagen will be difficult. on the contrary, if the dentine surface is too wet, there are too many water molecules around collagen, so hydrogen bond between water and amino collagen will inhibit bonding agent to bond with collagen. therefore an optimal condition of dentin surface is needed to obtain a maximal bonding between dentine bonding agent and dentine collagen. a good moisture characteristic of dentin bonding materials is referring to the low level of viscosity so that can improve surface energy. hema is a humectants hydrophilic agent that has ability to wet dentin surface.5 60 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 59–62 achieve maximum tensile bond strength of hema dentine bonding agent to dentine surface. materials and methods materials that were used: bovines incisive, acid etch (ivoclar vivadent, schaan/liechtenstein), hema dentine bonding (voco, germany). appliances that were used: diamond disk, diamond drill, emery number 400 and 1000 (fuji star, japan), desiccator with vacuum faucet, hygrometer and air thermometer (haar. synth. hygro, germany); compressor/air suction tool (schuco, usa), autograph ag-10 te, (shimadzu, japan). tooth sample was cleaned carefully by removed dirt at tooth surface using brush, while for hard or soft tissue using sharp scalpel. during cleaning, tooth was always in wet condition. tooth was cut with diamond disk and planted in hard gypsum cylinder log. the dentin part was facing upward. preparation was done until dentin surface using fissure diamonds bur. the surface of dentin was attenuated with silicon emery number 400 and continued with number 1000. after that, dentin was covered with adhesive tape which 3 mm diameter and attached precisely in the middle of the dentin surface.10 the preparation above as according to procedure that was done previously.11 next, dentin specimen was coated with 37% phosphoric acid etch with cotton pellets for 15 second, then washed with 20 ml aquades using injection spuit and dried smoothly using cotton pellets. this drying techniques is call blot dry technique.12 samples were placed into desiccator for 1 hour at 60% humidity for group i; 70% for group ii; 80% for group iii; 90 % for group iv, and 65% for group of v (control group) with 8 samples each.10 immediately, after released from desiccator, primary condensation and bonding were mixed and then applied to dentine surface using disposable brush for 30 second, after that were exposed with light curing unit for 20 second (as according to manufacturer’s guide). next, the cylinder log was inserted into plunger. the opponent plunger was filled with self cured acrylic as filling materials over dentine bonding and then united with the other plunger and fixed by installing lock dowel. samples were kept at room temperature (± 28 °c) for 24 hours. after 24 hours, the tensile bond strength of the samples was measured with autograph (cross head speed = 10 mm/minute, range: 5, capacities of load cell: 5 kn/500 kgf). the result which shown at screen have set of kgf (1 kgf = 9.81 n. 1mpa = n/mm2). wide of dentine surface sample = àr2 = 7.1 mm2. data was statistically analyzed with one-way anova at 95% confidence level and was continued with lsd test. results the tensile bond strength, mean, and standard deviation of hema dentine bonding agent to bovine dentine surface can be seen in table 1. the differences of all samples in this experiment were analyzed with one-way anova at 95% confidence level. the result showed that tensile bond strength of hema dentine bonding agent to bovine dentine table 2. lsd of the tensile bond strength between dentine bonding agent based on hema with bovine dentine surface humidity 60% 70% 80% 90% control ( 65% ) 60% 70% 80% 90% control (65%) xxx + + + - + xxx - + + + - xxx - + + + - xxx + - + + + xxx description: +: significance difference, —: not significance table 1. mean and standard deviation of tensile bond strength between dentine bonding agent based on hema with bovine dentine surface (mpa) humidity n x sd 60% 70% 80% 90% control 65% 8 8 8 8 8 16.74 12.74 11.59 10.02 15.50 1.8 2.5 2.4 2.1 1.8 description: n = sample amount, x = mean of tensile bond strength, sd = standard deviation surface at 60%–90% humidity level significantly different (p < 0.05). lsd test was used to determine the difference of each sample (table 2). the data distribution of tensile bond strength was normal that analyzed with kolmogorov-smirnov test. sample group at 60–90% humidity have p value more than 0.05, it mean the variable data has normal distribution. lavene test was used to prove that the tensile bond strength sample was homogenous. from the calculation, it obtained that sample group of tensile bond strength at 60–90% humidity was homogeneous (p > 0.05). 61soetojo: tensile bond strenght of hydroxyethyl methacrylate (hema) bonding agent the effect of 60–90% humidity to the tensile bond strength between resin bonding materials and dentin surface showed the significance in each sample group (p < 0.05). to know the significance of each sample group the data was analyzed using lsd test (table 2). the result showed that sample group at 60% humidity have p value smaller than 0.05 compared to sample group at 70%, 80%, and 90% humidity. it mean the tensile bond strength between resin bonding and dentin at 60%, 70%, 80%, and 90% humidity was significantly different. the tensile bond strength at 60% humidity compared to sample group at 65% humidity was not significantly different (p > 0.05). tensile bond strength of resin bonding to dentin was significantly different between at 70% and at 60%, 90% and 65% (control group) humidity (p < 0.05), but at 70% humidity and 80% have no significance (p > 0.05). if we compared, sample group at 80 % humidity and 90% humidity had p value more than 0.05. this matter means there was no significance of the tensile bond strength between sample group at 80% and 90% humidity. the tensile bond strength of sample group between at 80% humidity and to control sample group show the significance. if compared to control group, tensile bond strength of sample group at 90% humidity was significantly different (p < 0.05). discussion tensile bond strength between resin bonding and dentine surface is caused by chemical interaction and mechanical retention. chemical bond happened because of the interaction between carbonyl ester of hema with amino of collagen dentine. in the other hand, mechanical bonding obtained by penetration of hema into inter-fibril cavity (nano-space) which later polymerized. microscopically, there are no collagens at whole dentine surface, while mechanical bonding entangle all areas of dentin surface. therefore, mechanical bond always more dominant than chemical bond. to prove that chemical bond also influence in the process of mechanical bond between dentine bonding resin and dentine collagen, a research that caused collagen collapse was done.10 in this experiment the mean value of tensile bond strength between dentine bonding resin and dentine collagen decrease become 11.88 ± 2.91 mpa (equal to ± 29% degradation). the higher tensile bond strength happened because the efficacy of monomer infiltration is totally at demineralized dentine layer. if monomer infiltration just at the top, it will leave matrix of demineralized dentin unprotected. this matter caused collagen hydrolyzed so that the tensile strength is low.4 in this research, the influence of humidity to tensile bond strength of resin bonding agent to dentine can be seen in table 1. the highest value of tensile bond strength is obtained at 60% humidity, then decreased significantly at 70%, 80% and 90% humidity. high humidity will decrease tensile bond strength. this result is same as besnault and attal research’s.9 so 60% humidity is the best humidity where the amount of water molecules is ideal enough to re-expansion collagen fibril so that resin will easy to penetrating into cavity between fibril. chemically, interaction between resin and collagen is strong. dentine bonding agent based on hema contains acetone as a solvent. acetone is a solution that evaporate easily, can slight resin liquid so decrease the viscosity. when this liquid applied at dentine surface, it will penetrate into micro cavity between collagen fibril, and then chasing water molecule and then evaporated. thereby, it will leave dentine bonding resin bind with collagen fibril.13 acetone concentration influence the thickness of resin bonding layer and it’s tensile bond strength, but the thickness of bonding resin have no relation with it’s tensile bond strength. resin material leakage is caused by acetone evaporation, bad polymerization, and the low strain strength of dentine bonding resin (because the acetones amount is too much). another characteristic of acetone are: water chasing effect, able to increase vapor pressure of water, especially water around collagen. the optimum concentration of acetone is 37% weight. in this concentration the thick of resin bonding layer is 30.2 μ and the tensile bond strength is 63.5 mpa. in this research, the tensile bond strength at 60% humidity was higher than at 70% humidity, hence at 70% humidity the amount of water molecules is higher than at 60%, so the acetone ability to chase water also low at that humidity. at 60 % humidity the acetone ability to chase water is higher, and in this condition bonding resin will immediately penetrate to collagen. in conclusion, maximum tensile bond strength value between hema dentine bonding agent and bovine dentine surface obtained at 60% humidity. as suggestion, further research is needed to examine the tensile bond strength of hema dentine bonding agent with dentine collagen with various ph of hema, hema concentration, acetone concentration, and temperature in connection with humidity of dentine surface. references 1. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 2. noort rv. introduction to dental materials. 2nd ed. edinburgh, london, new york, oxford: cv mosby co; 2002. p. 11–78. 3. anusavice kj. phillip’s science of dental materials. 11th ed. philadelphia, london, toronto: wb saunders co; 2003. p. 21–395. 4. nakabayashi np, pashley dh. hybridization of dental hard tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 5. leal jir, osorio r, terriza jah. dentin wetting by four adhesive system. dent mat 2001; 17: 526–32. 6. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32: 19–26. 62 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 59–62 7. breschi l, gobbi p, marzotti g, falconi m. high resolution sem evaluation of dentin etched with maleic and citric acid. dent mat 2002; 18: 26–35. 8. finger wj, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4: 277–82. 9. besnault c, attal jp. influence of a simulated oral environmental on dentine bond strength of two adhesive systems. am j dent 2001; 14: 367–72. 10. soetojo a. kekuatan perlekatan antara bahan bonding hema dengan kolagen dentin pada berbagai kelembaban. disertation. surabaya: airlangga university; 2006. h. 66–9. 11. soetopo. adhesi komposit resin dengan tehnik etsa asam untuk restorasi kerusakan gigi. disertation. surabaya: airlangga university; 1980. h. 39–44. 12. meerbeek bv, yoshida y, lambrechts p, vanherle g. a tem study at two water based adhesive systems bonded to dry and wet dentin. j dent res 1998; 77: 50–9. 13. cho bh, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the micro tensile bond strength. dent mat 2004; 20: 107–15. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 49 no 1 jan-mrt 2016.indd 4949 research report beta-defensins-2 expressions in gingival epithelium cells after probiotic lactobacillus reuteri induction tuti kusumaningsih department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: beta-defensins (bd) are antimicrobial peptides that play a role in defense against pathogens. beta-defensins (bd) are expressed by a variety of epithelial cells, including gingival epithelium, salivary glands, saliva and salivary duct. bd-1 is expressed constitutively, while bd-2 and bd-3 expressions can be induced by commensal bacteria. probiotics are commensal bacteria, thus l. reuteri as probiotic bacteria may act as “inducer” for bd-2 in epithelial gingiva. s. mutans is the main bacteria causing dental caries and sensitive to bd-2. purpose: this study was aimed to prove that the administration of probiotic l. reuteri may improve bd-2 expressions in the gingiva epithelium. method: this study was conducted in vivo using twenty-four male rattus norvegicus wistar strains aged 10-12 weeks and weighed 120-150 g. those rats were randomly divided into four groups, namely negative control group (not induced with l. reuteri or s. mutans), positive control group (induced with s. mutans for 14 days), treatment group 1 (induced with l. reuteri for 14 days and s. mutans for 7 days), and treatment group 2 (induced with l. reuteri and s. mutans for 14 days concurrently). the concentration of l. reuteri used was 4x108cfu/ml, while the concentration of s. mutans was 1x 1010cfu/ml. 0.1 ml of each was dropped in the region of the mandibular incisors. bd-2 expression was calculated using immunohistochemical method. the difference of bd-2 expressions in gingival epithelial cells in the respective groups was analyzed by anova/spss. results: there were significant differences in bd-2 expressions in gingival epithelial cells in each group based on the results of anova test (p=0.001). conclusion: the administration of probiotic l. reuteri is able to increase bd-2 expressions in gingival epithelial cells. keywords: beta defensins-2 expression; gingival epithelium; probiotic; l. reuteri; s. mutans corespondence: tuti kusumaningsih, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tutikusumaningsih@yahoo.com introduction gingival epithelium is a defense against bacteria in oral cavity, not only physically but also chemically. defense function of the gingival epithelium is characterized by their unique structure and integrity of the anti-microbial peptide (amp), such as human beta defensins (hbds).1 defensins are antimicrobial peptides first discovered in mammals. defensins in humans consist of two sub-families, namely alpha and beta-defensins. alpha-defensins are produced by polymorphonuclear leukocytes and panet cells, while beta-defensins are produced by epithelial surface of skin, intestine, trahea, and oral cavity.2 defensins have broad activities against bacteria, fungi, and viruses. in the optimal conditions, antimicrobial activities of defensins work at low concentration of 1-10mg /ml.3 human beta-defensins (hbds) are widely expressed in tissues of the oral cavity, including gingival epithelium, salivary glands, saliva and salivary duct. these peptides are involved in defense against bacteria that colonizes in the oral cavity. defensins in the oral cavity have an important role to protect the structure of the teeth from bacteria causing caries. human beta-defensins have broad antimicrobial activity against oral microorganisms, such as streptococcus mutans, porphyromonas gingivalis, and actinobacillus actinomycetemcomitans.3 changes in lifestyle and diet can affect the composition and amount of the normal flora in someone’s oral cavity. several factors causing the dental journal (majalah kedokteran gigi) 2016 march; 49(1): 50–54 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p49-53 50 kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 49-53 occurrence of dental caries are physical factors, biological factors, environmental factors, habits, and life style.4 dental caries is an example of a disease that one of causes is too often consuming sucrose being offset by the improper process of oral cavity cleaning. dental caries is an infectious disease caused by an imbalance of homeostasis between host and microbe.5 streptococcus mutans (s. mutans) and streptococcus sobrinus (s. sobrinus) are the main causes of dental caries.5-7 excessive amount of s. mutans in the oral cavity can be considered as an early occurrence of dental caries. recently, there have been many outstanding supplement products containing probiotics. according to the who / fao, probiotics are life micro-organisms which, when administered in adequate amount, confer a health benefit on the host. meanwhile, international life science institute (ilsi) europe defines probiotics as a life of microbial food ingredient that, when ingested in sufficient quantities, exerts health benefits on the consumer.8 based on both definitions, it can be concluded that probiotics are live microorganisms causing an effect on health. one of the many probiotics often used in dentistry is lactobacillus reuteri (l. reuteri). probiotics including l. reuteri are commensal bacteria. commensal bacteria are excellent inducers to bd-2 in epithelial cells of the oral cavity, so it is possible for probiotic bacteria to act as an inducer for bd-2.9 there are two species of bacteria causing dental caries, namely s. mutans and s. sobrinus, sensitive to bd-2.10 thus, it indicates that bd-2 can be used as an alternative material for preventive and therapeutic potential against dental caries. therefore, this research was aimed to determine whether the administration of probiotic l. reuteri, induced into gingival mice could increase bd-2 expressions in the gingival epithelium. materials and method this research was approved by komisi kelaikan etik penelitian kesehatan-kkepk (the commission on health research ethics airworthiness), faculty of dental medicine, universitas airlangga. twenty-four male rats (rattus norvegicus wistar strain) aged 10-12 weeks with body weight of 120-150 grams were divided into four groups, namely negative control group, positive control group, treatment group 1, and treatment group 2. there were five rats in the negative control group, which were not induced by s. mutans or l. reuteri. in the positive control group, there were five rats induced with s. mutans for 14 days. in the treatment group 1, furthermore, there were seven rats induced with l. reuteri from day 1 to day 14 (14 days), and s. mutans from day 8 to day 14 (7 days). on the other hand, in the treatment group 2, there were seven rats induced with l. reuteri and s. mutans from day 1 to day 14 simultaneously. on day 15, those rats were sacrificed, and then their jaws and gingival tissue were cut for the preparation of paraffin blocks for immunohistochemical examination (ihc). the concentration of the bacterial suspension used was 4 x 108 cfu/ml of l. reuteri, and 1 x 1010 cfu / ml of s. mutans.9 dsm 17 938 + atcc pta 5289 was added to brain heart infussion (bhi) liquid, and then incubated in anaerobic condition for 1x24 hours using gas generating kit with oxoid brand. after removing from the incubator, turbidity and sediment will appear, indicating l. reuteri growth. planting to mrs agar (de man, rogosa and sharpe; merck gmbh, damrstadt, germany) was performed, and then incubated for 2x24 at a temperature of 370 c. after that, several colonies were taken and planted into a liquid bhi medium, and then incubated for1 x 24 hours at a temperature of 370 c. after removing from the incubator, bacterial density were observed to know whether it had reached 4 x 108cfu / ml, and then examined using spectrophotometry with a wavelength lambda of 625 nm and an optical density of 0.080.10 identical to the mc farland 0.5.9 s. mutans used in this research were s. mutans serotype c taken from the stock in the form of freeze dry. s. mutans were taken about 1 ose, added to liquid bhi medium, and then incubated for 1 x 24 hours at a temperature of 370c. after removing from the incubator, turbidity and sediment will appear, indicating s. mutans growth. planting to a blood agar medium was performed, and then incubated for 1x24 at a temperature of 370 c. after that, several colonies were taken and planted into a liquid bhi medium, and then incubated for1 x 24 hours. after removing from the incubator, bacterial density were observed to know whether it had reached 1 x 1010 cfu / ml, and then examined using spectrophotometry with a wavelength lambda of 625 nm and an optical density of 0.080.10 identical to the mc farland 0.5.9 examination of beta-defensin-2 (bd-2) expressions was performed. there are several preparation stages of histopathology conducted in this research as stated in humason’s method cited in sudiana, 2005. calculating method of the results of immunohistochemical staining, according to soini et al.11,12 consists of several stages. first, gingival tissue was fixated in paraformaldehid 4% for 4 hours at a temperature of 40c. after washed in pbs, the tissue was planted in oct compound, and then immediately frozen. then the tissue was cut into a thickness of 6 μm, and then incubated with primary antibody anti bd-2 (lab vision) for 24 hours at a temperature 40c. the tissue sections were incubated with secondary antibody rat monoclonal, anti opg (lab vision), and hrp kid for 24 hours at a temperature 40c. chromogenics used in this research were 3.3 diaminobenzidine tetrahydrochloride, and then counterstained with he.9 immunohistochemical staining with indirect method was carried out to show cells expressing bd-2 protein. the cells expressing bd-2 were shown with a brown cytoplasm, and then observed with a light microscope with a magnification of 400 x. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p49-53 5151kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 49-53 anova test, was performed to determine the differences in bd-2 expressions in the gingival epithelium of each group. hsd test was conducted to determine the significance of the differences between groups. results immunohistochemical examination was performed to determine the distribution of cells expressing bd-2 in the gingival epithelium after induced with probiotics, l. reuteri bacteria, in each group (table 1). based on table 1, the results show that there was a significant difference of bd-2 expression in the gingival epithelium of wistar rats among the treatment groups (p = 0.001). a decline in the mean of bd-2 expressions in the gingival epithelium was found in the positive control group (group induced by s. mutans), compared to the negative control group, namely from 15.80 into 4.80. an increase in the mean of bd-2 expressions in gingival epithelium was found in the treatment group 2 (induced by probiotic l. reuteri for 14 days and s. mutans for 14 days), compared to the treatment group 1 (induced with probiotic l. reuteri for 14 day and s. mutans for 7 days), namely from 24.00 into 27.00 the overall bd-2 expressions were significantly different among the groups. based on the results of hsd test, it is known that there was a significant difference in bd-2 expressions between the negative control group and the positive control group. there were also significant differences in bd-2 expressions between the negative control group and the treatment group 1 as well as between the negative control group and the treatment group 2. similarly, there were also significant differences in bd-2 expressions between the positive control group and the treatment group 1 as well as between the positive control group and the treatment group 2. however, there was no significant difference between the treatment group 1 and the treatment group 2. discussion the administration of l. reuteri as probiotics in this research could increase the expressions of bd-2 at gingival epithelial cells (table 1 and figure 1). gingival epithelium is stratified squamous (flat stratified epithelium) which serves as a defense against pathogenic bacteria. epithelial tissues in the oral cavity are continuously in contact with various kinds of bacteria, even so most individuals can maintain the balance of their health. epithelial tissues of the oral cavity play a role in protecting the host not only with the physical defense, but also through the innate immune responses in the form of antimicrobial peptides. bds are small antimicrobial peptides, which form cautions produced by epithelial cells playing an important role in mucosal defense and skin.13 based on figure 2, the results of immunohistochemical examination for bd-2 expressions in the gingival epithelium showed that bd-2 is expressed in stratum spinosum and stratum granulosum. the results is in line with the results of a research conducted by weinberg et al.,14 stating that within the gingival tissue, mrna from figure 1. immunohistochemical examination results of bd-2 expressions in the gingival epithelium in each group (400x magnification); black arrow (cells expressing bd-2); red arrows (cells not expressing bd-2). a) bd-2 expressions in the gingival epithelium in the negative control group; b) bd-2 expressions in the gingival epithelium in the positive control group; c) bd-2 expressions in the gingival epithelium in the treatment group 1; d) bd-2 expressions in the gingival epithelium in the treatment group 2. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p49-53 52 kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 49-53 sw-1 and sw-2 is located in the suprabasal stratified epithelium, while peptide bd-2 is detected in the upper epithelial layers.15 gingiva, hard palate, and the dorsal part of the tongue is the keratinized epithelium of the oral cavity, while the floor of the mouth and the buccal area are not keratinized epithelium. the results of immunohistochemical examination conducted to observe bd-2 expressions in the keratinized epithelial in oral cavity (including gingiva) showed a weak positive result, while in the non-keratinized epithelial showed a negative result. this situation suggests that keratinization in the epithelium of the oral cavity plays an important role for the retention of peptides. 14 the results found in the negative control group in this research can be associated to the results found in normal epithelium condition as stated by abiko y et al. since the group was not treated. bd-2 expressions in the gingival epithelium in the negative control group were 15.80, while bd-2 expressions in the treatment group 1were 24.00, and 127.00 in the treatment group 2 (table 1). it indicates that bd-2 expressions increased after the induction of probiotic bacteria, namely l. reuteri and s. mutans bacteria. the induction of probiotics in the treatment group 1 was assumed as a precaution. in this treatment group, l. reuteri bacteria were first induced from day 1 to day 14, whereas s. mutans bacteria causing caries were induced from day 8 to day 14. on the other hand, the induction of probiotics in the treatment group 2 was assumed as a therapeutic action. in the treatment group 2, l. reuteri and s. mutans were administered together from day 1 to day 14. in table 1, the number of gingival epithelial cells expressing bd-2 in the negative control group the group not induce l. reuteri and s. mutans bacteria was lower than in the treatment groups. this condition showed that bd-2 expressions were still expressed both at the gingival epithelium in normal circumstances and at the gingival epithelium in inflammation circumstances. in contrast, in most other epithelia, such as skin epithelium, tracheal epithelium, and intestinal epithelium, bd-2 is expressed only in inflammation circumstances. 16 this statement is supported by the results of a research conducted by whasun & dale, stating that in most tissues, bd-2 is induced and expressed only in inflammation circumstances, but in epithelial tissues of the oral cavity, bd-2 is expressed only in normal circumstances (without inflammation) since the oral mucosa is continuously exposed to a variety of bacteria.17 the lowest number of bd-2 expressions was found in the positive control group (table 1). the positive control group is a group only induced with s, mutans. thus, it can be said that s. mutans is not able to induce bd-2. a research conducted by wehkamp et al showed that e. coli nissle 1917 strains can induce bd-2, while the other 40 isolates of e. coli cannot induce bd-2. the results of the isolation and purification of flagellant protein derived from e. coli nissle 1917 are able to induce bd-2, while flagellin deficient mutans derived from the bacteria are not able to induce defensins. lps of e. coli nissle 1917 even cannot stimulate the production of bd-2. therefore, it indicates that flagellant protein is decisive mamp in e. coli nissle 1917 probiotics that can stimulate bd-2 excessively.17 based on these results, it can be said that the administration of probiotics, l. reuteri bacteria, for 14 days with a concentration 4x108cfu/ml could increase bd-2 expression significantly (p=0.001). this result can be seen in table 1 and figure 2 which show that the number of bd-2 expressions found in gingival epithelial cells in the treatment group 2 induced with probiotics, l. reuteri bacteria, for 14 days, was the highest one. the reason is that the active molecule on the cell wall of the probiotic bacteria, peptidoglycan, activates nod-2 receptors in the cytoplasm. activation of signaling through nod-2 can trigger a recruitment of protein adaptor (rick). rick then will be activated, resulting in phosphorylating complex ikk. nf-kb is in the inactive form, which binds to ikb, inhibitor protein in the cytoplasm. this stimulus will result in phosphorylation, ubiquitination, and degradation of ikb proteins that will cause the translocation of nf-kb into the nucleus and the activation of nf-kb.18,19 it will activate target genes for synthesis of bd-2 protein. in conclusion, probiotics, l. reuteri, can improve bd-2 expressions in the epithelial cells of the gingiva. table 1. the mean and standard deviation of bd-2 expressions in gingival epithelium after induced with probiotic l. reuteri in each group. group mean standard deviation significance *) negative control 15.80 2.28 positive control 4.80 1.30 treatment group 1 24.00 2.94 treatment group 2 27.00 2.58 p=0.001 table 2. the results of hsd test among the groups group negative control positive control treatment group 1 treatment group 2 negative control _ 0.000* 0.000* 0.000* positive control _ 0.000* 0.000* treatment group1 _ 0.334 treatment group 2 _ dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p49-53 5353kusumaningsih/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 49-53 references 1. ji s, shin je, kim ys, oh je, min bm, choi y. toll-like receptor 2 and nalp2 mediate induction of human beta-defensins by fusobacterium nucleatum in gingival epithelial cells. infect immun 2009; 77(3): 1044-52. 2. gomes ps, fernandes mh. defensin in the oral cavity: distribution and biological role. j oral pathol med 2010; 39(1): 1-9. 3. barrera gj, tortolero gs, rivas a, flores c, gonzales je. increased expression and levels of human β defensins (hbd2 and hbd3) in adults with dental caries. j health sci 2013; 3(2): 88-97. 4. karpiński tm, szkaradkiewicz ak. microbiology of dental caries. j biol earth sci 2013; 3(1): m21-4. 5. negrini tc, duque c, hofling jf, goncalves rb. fundamental mechanisms of immune response to oral bacteria and the main perspectives of a vaccine against dental caries: a brief review. rev. odonto cienc 2009; 24(2): 198-204. 6. forssten sd, bjorklund m, ouwehand ac. streptococcus mutans, caries and stimulation models. nutriens 2010; 2(3): 290-8. 7. li l, guo l, lux r, eckert r, yarbrough d, et al. targeted antimicrobial therapy against streptococcus mutans establishes protective non-cariogenic oral biofilm and reduces subsequent infection. int j oral sci 2010; 2(2): 66-73. 8. bhardwaj a, bhardwaj sv. role of probiotics in dental caries and periodontal disease. arch clin exp surg 2012; 1(1): 45-9. 9. kusumaningsih t. the increasing of beta-defensin-2 level in saliva after probiotic lactobacillus reuteri administration. dent j (majalah kedokteran gigi) 2015; 48(1): 31-4. 10. nishimura e, eto a, kato m, hashizume s, imai s, nisizawa t, hanada n. oral streptococci exhibit diverse susceptibility to human β-defensin-2: antimicrobial effects of hbd-2 on oral streptococci. curr microbiol 2004; 48(2): 85-7. 11. soini y, paakko p, lehto vp. histopathological evaluation of apoptosis in cancer. am j pathol 1998; 153(4): 1041-51. 12. sudiana k. tehnologi ilmu jaringan dan imunohistokimia. jakarta: sagung seto; 2005. p. 4-20. 13. chung wo, dale ba. innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species. infection and immunity 2004; 72(1) : 352-8 14. weinberg a, jin g, sieg s, mccormick ts. the yin and yang of human beta-defensins in health and disease. front immunol 2012; 3: 294. 15. abiko y, nishimura m, kaku t. defensin in saliva and the salivary glands. med electron microsc 2003; 36(4): 247-52. 16. reddy s, sumanthi j, anuradha ch, shekar ch, chandra k, reddy bvr. oral defensin the antimicrobial polypeptides. j orofac sci 2010; 2(2): 64-70. 17. schlee m, wehkamp j, altenhoefer a, oelschlaeger ta, stange ef, fellermann k. induction of human beta-defensin 2 by the probiotic escherichia coli nissle 1917 is mediated through flagellin. infect immun 2007; 75: 2399-407. 18. tripathi p, aggarwal a. nf-kb transcription factor : a key player in the generation of immune response. curr sci 2006; 90(4): 519-31. 19. carpenter s, o’neill la. how important are toll-like receptors for antimicrobial responses? cell microbiol. 2007; 9(8): 1891-901. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p49-53 isi vol 39 no 2 april 2006 file pertama.pmd 54 nursing habits and early childhood caries in children attending hospital university science malaysia (husm) widowati witjaksono*, mon mon tin oo**, and khamisah awang kechik*** * department of periodontic, university science malaysia school of dental sciences and airlangga university faculty of dentistry ** department of community dentistry, university science malaysia school of dental sciences *** year 5 student, university science malaysia school of dental sciences abstract the habit of nocturnal bottle or breast-feeding has been reported to be a potential cause for early childhood caries (ecc) in very young children. the aim of this study was to determine the prevalence of ecc in children 2-5 years of age attending out patient clinic husm, in relation to the nursing habits. in this cross-sectional study, 90 children were randomly selected to examine their caries status using torch and disposable mirror. data on mothers’ educational level, nursing habits and oral hygiene practices, were gather by using structured questionnaire. it has been found that 16.7% of subjects were caries free while 83.3% of them had caries with mean dmf score 6 (sd 5.3). with regard to nursing habits, 29% of subjects had breast-feeding alone, 16% had bottle-feeding alone and 55% had both breast and bottle-feeding. ninety-three percent of children had been nursed beyond 14 months and 47% had been fed with liquids other than breast milk, infant formula or water. twenty-seven percent of children were allowed to sleep with nursing bottle in mouth and 52% were allowed to sleep with breast nipple in the mouth which shows significantly associated with ecc (p = 0.03). tooth brushing habit was reported for 91% of children using toothpaste. mean age of the children (in months) when the mothers started brushing the teeth was 19.1 (sd 10.8) and has significant association with ecc (p < 0.05). this study demonstrates that the habit of allowing infants to sleep with breast nipple in their mouth and the late start of tooth brushing are associated with prevalence of ecc. educational programs for pregnant women and mothers of young children should be emphasized to enhance the knowledge and awareness of mothers in preventing ecc. key words: early childhood caries, nursing habits, nursing bottle correspondence: widowati, periodontic department university science malaysia, school of dental sciences, health campus, k. kerian 16150, kelantan, malaysia. e-mail: widowati@kb.usm.my introduction children’s oral health is dependent on the interest and ability of parents. hence, oral health education is given to expectant mothers, so that home care can be started in early infancy. despite this, in the national oral health survey of school children 1997, the researchers found that the proportion of 6 years old children with 1 or more carious teeth in deciduous dentition was 80.6%.1 rampant caries in infants and young children has long been recognized as a clinical syndrome referred to by various names, including nursing caries, nursing bottle syndrome, night bottle mouth, and baby bottle tooth decay. the common theme among these terms is the perceived central role of inappropriate use of the baby bottle in the etiology and progression of carious lesions. early childhood caries (ecc) is a unique pattern of dental caries in very young children due to prolonged and improper feeding habits. even as the first primary tooth starts erupting, the oral environment can be conducive to the initiation of demineralization. in spite of the fact that dental caries is diminishing in our youth, there are still a large number of children with advanced multiple carious lesions, victims of parental ignorance of nursing caries which is a distinct clinical entity. the primary factor is a pattern of sugar consumption that has been established early in life of the child. other factors include feeding practices such as putting the child to bed at naptime, or bedtime with a bottle containing sweetened milk or beverages and inappropriate practice using a bottle containing milk, juice or other cariogenic substance as a pacifier for non-nutritive reasons. prevalence of caries and maxillary anterior decay varies greatly in developed and underveloped countries and among socioeconomic groups in developed countries. however, the onset and progression of the disease or the antecedents of ecc, such as high-risk behaviors, cultural norms, health beliefs and attitudes, or health care delivery factors is relatively unknown. furthermore, studies that do exist are limited by small and possibly biased samples and a lack of clear case definition. evidence exists suggesting that the experience of ecc puts children at a significantly higher lifetime risk for dental caries and its sequelae, both in their primary and permanent 55witjaksono et al: nursing habits and early childhood caries in children dentitions.2 children with ecc had more severe fissure caries and were more likely to develop posterior proximal caries than children without ecc.3 early childhood caries (ecc) is a significant risk factor for later caries, and find that ecc children have more than twice the number of carious, extracted or restored teeth over the time spanned by their fourth to sixth years.4 extraction and restoration are expensive procedures, and tooth loss may have a significant impact on the child’s future dental development. the pain and discomfort associated with ecc may be responsible for the phenomena of low body weight and adversely affected growth observed in ecc children.5 the main purposes of this study were: to identify the prevalence of early childhood caries and the nursing habits amongst children age 2–5 years old attending husm and to determine the association between the nursing habits and caries status. materials and methods a cross sectional study was conducted during may to june 2004 among randomly selected 90 children aged 2-5 years old who attended out patient clinic husm. children with chronic or severe illness were excluded. a written informed consent form was obtained from the mother following an explanation of procedures. mothers were interviewed using structured questionnaire to obtain information regarding mother’s educational level, feeding history and dietary habits of their child and oral hygiene practices. the oral examination of children was performed with the help of a mouth mirror and a pen light source. no attempt was made to probe the teeth and dental radiographs were not used in any of the examinations. caries experience was recorded using dmf index: (d = the number of decayed teeth, m = the number of missing teeth which have been extracted due to caries, and f = the number of filled teeth). the criteria used for caries diagnosis were that of united surveilances summaries.6 data were then compiled and analyzed using spss version 11.0. the analyses to be used were descriptive statistics that is mean and standard deviation. as for the hypotheses, chi2 test, fisher’s exact test, and independent t-test were used. results table 1 showed the description of demographic variables among respondents. the children’s ages ranged from 2 to 5 with median age of 4. mean age of mothers is 35.6 years and majority of them completed secondary school. among them 66.7% are housewives and the rest are working mother. for caries experience and prevalence of caries, this study showed 16.7% of children had dmf score 0 (caries free) while 83.3% of them had caries experience (figure 1). caries experience (dmf score) among children is shown in table 2 by frequency distribution, the mean dmf score was 6 (sd 5.3). from statistical analysis, it was 95% sure that the prevalence of caries in the population will be between 75% and 91. in this study population prevalence of caries in male subjects was higher than female, which was 91.7% in male and 77.8% in female (figure 2). as for nursing habits of subjects 29% percent of subjects had breast-feeding alone, 16% had bottle-feeding alone while other 56% had both breast and bottle-feeding. ninety-three percent of them had been nursed beyond 14 months. fifty-three percent of them had been fed with liquids other than breast milk, formula or water during their bottle-feeding. 27.8% were allowed to sleep with infant formula bottle in their mouth while 52% were allowed to sleep with breast nipple in the mouth. only 10% of them had been allowed to take bottle without restriction, 40% of children were given water prior to sleep. most of the mothers (91%) reported that they brush their child’s teeth using toothpaste. mean age (in months) when mother start encouraging subjects to drink in cup was 20.5 (sd ± 10.5). mean age when mother started to brush their table 1. description of study population variable mean (sd) median (iqr) freq.(%) mother's age mother's educational level primary school secondary school higher education mother's working status working housewife 35.6 (6.6) 4.0(2.0)a 6 ( 6.7) 73 (81.1) 11 (12.2) 30 (33.3) 60 (66.7) sd = standard deviation iqr = interquartile range freq = frequency ª the distribution is skewed to the left subject's caries experience 16,70% 83,30% 0,0% 20,0% 40,0% 60,0% 80,0% 100,0% no caries caries figure 1. caries experience of the study population. 56 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 54–58 teeth (in months) was 19.1 (sd ± 10.8). summary of nursing habits of the children is shown in table 3. caries experience and association factors are shown in table 4. among all categorical variables, the habit of allowing the infant to sleep with breast nipple in mouth is associated with caries experience, which is statistically significant (p = 0.03). for numerical variables, age of subjects and age when started brushing teeth were significantly difference between caries and non-caries subjects (p < 0.05) table 5. table 2. frequency distribution of dmf score among the study population dmf score frequency percent cum. % 0 1 – 5 6 – 10 11 – 15 16 – 20 15 31 25 12 7 16.7 34.4 27.8 13.3 7.8 16.7 51.1 78.9 92.2 100 total 90 100.0 100.0 mean dmf score = 6 (sd 5.3) severe ecc = 48.9% (dmf >6) caries experience by gender 77,8% 91,7% 70,0% 75,0% 80,0% 85,0% 90,0% 95,0% male female gender % c ar ie s ex pe ri en ce figure 2. caries experience of children showing by gender. table 3. nursing habit of children variable yes freq (%) no freq (%) breast feeding alone bottle feeding alone breast feeding and bottle feeding nursed beyond 14 months fed with liquid other than breast milk, formula or water sleep with infant formula bottle in mouth sleep with breast nipple in mouth had bottle without restriction given plain water before sleep tooth-brushing practice 26 (28.9) 14 (15.6) 50 (55.6) 84 (93.3) 48 (53.3) 25 (27.8) 47 (52.2) 9 (10.0) 36 (40.0) 82 (91.1) 64 (71.1) 76 (84.4) 40 (44.4) 6 (6.7) 42 (46.7) 65 (72.2) 43 (47.8) 81 (90.0) 54 (60.0) 8 (8.9) table 4. factors (categorical variables) associated with early childhood caries variable n caries freq (%) no caries freq (%) x2 statisticª ( df ) p value gender male female mother's educational level primary and secundary school higher educational level mother's working status working housewife mother had received information about oral health yes no breast feeding alone yes no bottle feeding alone yes no breast feeding and bottle feeding yes no mother nursed child beyond 14 months yes no 36 54 79 11 30 60 27 63 26 64 14 76 50 40 84 6 33(91.7) 12(22.2) 66(83.5) 9(81.8) 23(76.7) 52(86.7) 21(77.8) 54(85.7) 24(92.3) 51(79.7) 4(28.6) 65(85.5) 41(82.0) 34(85.0) 69(82.1) 6(100.0) 3(8.3) 42(77.8) 13(16.5) 2(18.2) 7(23.3) 8(13.3) 6(22.2) 9(14.3) 2(7.7) 13(20.3) 10(71.4) 11(14.5) 9(18.0) 6(15.0) 15(17.9) 0(0.0) 3.00(1) 1.44(1) 0.14(1) 0.083 1.000* 0.230 0.368* 0.215* 0.240* 0.704 0.584* 57witjaksono et al: nursing habits and early childhood caries in children discussion early childhood caries is one frequently encountered problem for preschool age children in world wide. it is also well known that good oral hygiene and proper feeding habits should be established as early as possible in order to prevent ecc, which is certainly depending on mother’s knowledge and practice. there is an association of low socio-economic status with parenting stress and the adoption of poor techniques for feeding and pacifying the child.7 mother’s education is one of the associated factors in streptococcus mutan’s count in children who were undergone treatment for ecc.8 from family factors mother’s basic education comes to be significant during early childhood and poor dental health at 10 years of age.9 although there were relationship between the children’s caries experience and the number of missing teeth, dental attendance pattern and the level of education of the mother,10 in the present study the mother’s education level is not related to ecc. this reality can be due to the fact that most of the mothers had received the information about children’s oral health but they were not practicing the proper way to nurse their child and taking care of their child’s teeth. the study of syahril et al.11 also revealed that these attitude were not reflected in the dental health status of their children, eventhough parents had adequate knowledge and positive attitude to their children’s dental care. the prevalence of ecc in this study population is high (48.9% with dmf > 6) compared to western population in greater manchester,12 whereby 32% children affected by caries and dmfs was 2.8 which has to be considered as severe ecc.12,13 in south east asia countries including malaysia, the prevalence of caries is also still high, such as caries lanjutan tabel 4. ª population variance were significantly different (levene’s test p value=0.010), therefore t’ test without assuming equal variance was used. ª chi square test for independence * fisher’s exact test table 5. factors (numerical variable) associated with early childhood caries variable n caries freq (%) no caries freq (%) x2 statisticª ( df ) p value mother use liquid other than breast milk,formula or water to bottle feed yes no child fall asleep with infant formula bottle yes no child fall asleep with breast nipple inside mouth yes no child have bottle without restriction yes no mother give plain water prior to sleep yes no mother brush child's teeth yes no mother use toothpaste when brushing child;s teeth yes no 42 48 25 65 47 43 9 81 36 54 82 8 77 6 34(81.0) 41(85.4) 21(84.0) 54(83.1) 43(91.5) 32(74.4) 6(66.7) 69(85.2) 29(80.0) 46(85.2) 68(82.9) 7(87.5) 65(84.4) 4(66.7) 8(19.0) 7(14.6) 4(16.0) 11(16.9) 4(8.5) 11(25.6) 3(33.3) 12(14.8) 7(19.4) 8(14.8) 14(17.1) 1(12.5) 12(15.6) 2(33.3) 0.32(1) 4.71(1) 0.33(1) 0.571 1.000* 0.030 0.169* 0.564 1.000* 0.266 variable caries (n=75) mean (sd) no caries (n=15) mean (sd) mean differ (95% ci) t statistic (df) p value child's age (years) age when started to have drink from cup (months) age when started to brush teeth (months) 3.8(0.944) 21.2(10.948) 20.3(4.413) 3.0(1.000) 16.9(6.503) 13.4(11.346) -0.9(-1.4,-0.3) -4.37(-10.4,1.7) -6.90(-10.5,-3.3) -3.26(88) -1.44(87) -3.82(53)ª 0.002 0.153 0.000 58 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 54–58 prevalence of children aged 2–6 years old in northern philippines that were 59–92%.14 the high prevalence of caries shows their low level of caries awareness and its consequences in those countries. regarding nursing habits of respondents this study indicated the significant association between ecc and the habit of allowing the infants to sleep at night with breast nipple in their mouth (ad libitum nocturnal breast feeding). similarly in the dutch study, weerheim et al.15 found that nursing caries group was significantly morelikely to have been given the breast frequently at night through the age of 18 months. these significant association may be due to given breast-feeding together with bottle-feeding until they were over 14 months and the age encouraging their infants to have drink from a cup was about 20.5 months. these were far different from the american academy of paediatric dentistry (aapd) suggestions that infants should be weaned from the bottle at 12 to 14 months of age and should be encouraged to have drinks from a cup as they approach their first birthday.16 despite the changing of feeding habit offend a young child, it is parent’s responsibility to adopt the appropriate feeding practices for prevention of ecc. in this study 48 mothers used nursing bottle to feed liquids other than breast milk or formula of water. sweetened liquids, which have a minimal nutrient content, mainly contain carbohydrate, increase the risk of caries due to prolong contact between sugar and cariogenic bacteria on susceptible teeth. in addition, allowing the infant to fall asleep at naps or bedtime with the bottle inside mouth is also inappropriate use of nursing bottles. although these nursing habits were not significantly associated, it was obvious that mothers who participated in this study did not practice a proper way of feeding. american academy of paediatric dentistry (aapd) encourages that oral hygiene measures should be implemented by the time of eruption of the first primary tooth.16 although 91.1% of respondents from this study reported cleaned their infant teeth by using toothbrush and toothpaste however, mean age when started to brush (in months) was 19.1 (sd + 10.8) which is rather late. there was statistically significant relationship among the age of start brushing teeth and ecc, whereas mean age (in months) in caries group is 20.3 (sd + 4.4) while in non-caries group 13.4 (sd + 11.3). this result showed the importance of early practice of tooth brushing that can prevent from developing ecc. this study also showed an association between age of the child and caries prevalence where mean age of caries group (in years) was 3.8 (sd + 0.9) while for non caries group was 3 (sd + 1.0). this result also supported by the study of parents and nursing-bottle caries,17 that reported children with caries and non caries were differ in two respects; caries children on the whole are older and they have been sick more often than non caries children. this might be due to increase exposure of sugary food and containing fermentable carbohydrate when the child’s age increases. as the mother plays an important role in maintaining their children’s oral health, the authors suggest that early educational, intervention programs should be developed for pregnant mothers and mothers of young children. comprehensive anticipatory guidance in the areas of nursing habits, diet and nutrition, oral hygiene practices, fluoride use, causes and sequelae of ecc and early dental visits should be emphasized to enhance the knowledge and awareness of mother in prevention of ecc. references 1. ministry of health malaysia. the national oral health survey of school children. 1997. 2. nih consensus statement. diagnosis and management of dental caries throughout life. on line march 26–28, 2001; 18(1):1–24. 3. douglass jm, dm o’sullivan, n tinanoff. temporal changes in dental caries levels in a native american pre-school population. j public health dent 1996; 56(4):171–5. 4. thakib, et al. primary incisor decay before age 4 as a risk factor for future dental caries. paediatric dentistry 1997; 19:37–41. 5. ahyan h, suskan e, yildirim s. the effect of nursing or rampant caries on height, body weight and head circumference. journal of clinical paediatric dentistry 1996; 20(3):209–12. 6. united states surveilance summaries. surveilance for dental caries, dental sealants, tooth retention, edentulism, and enamel fluorosis. august 26, 2005; 54(03):1–44. 7. reisine s, litt m. social and psychological theories and their use of dental practice. international dental journal 2004; 43 (3 supplement 1):279–87. 8. peretz b, sarit f, eidelman e, steinberg d. mutans streptococcus counts following treatment for ecc. journal of dentistry for children 2003 may–august; 70(2):111–4. 9. mattila ml, rautava p, aromaa m, ojanlatva a, paunio p, hyssala l, helenius h, sillanpaa m. behavioural and demographic factors during early childhood and poor dental health at 10 years of age. j of caries res 2005 march-april; 39(2):85–91. 10. grytten j, rossow i, holst d, steele l. longitudinal study of dental health behaviours and other caries predictors in early childhood. community dent oral epidemiology 1988 december; 16(6):356–9. 11. syahril d, abdul-kadir r, yassin z, jali nm. knowledge and attitudes of parents of children with nursing bottle syndrome in serdang, malaysia. j nihon univ sch dent 1995 sept; 37(3): 146–51. 12. ismail al, sohn w. a systematic review of clinical diagnosing criteria of ecc. j public health dent 1999; 59:171–91. 13. davies gm, blinkhorn fa, duxbury jt. caries among 3-year-olds in greater manchester. br dent j 2001; 190:381–4. 14. carino km, shinada k, kawaguchi y. early childhood caries in northern philippines. community dent oral epidemiol 2003 apr; 31(2):81–9. 15. weerheim kl, uyttendaele-speybrouck bf, euwe hc, groen hj. prolong demand breast-feeding and nursing caries. caries res 1998; 32(1):46–50. 16. the american academy of paediatric dentistry. policy on early childhood caries (ecc): classifications, consequences and preventive strategies. 2003. 17. van everdingen t, eijkman ma, hoogstraten j. parents and nursing bottle caries. asdc journal of dentistry for children 1996; 63(6):426–33. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkgs vol 44 no 2 april-juni 2011.indd 93 vol. 44. no. 2 june 2011 research report constraints on the performance of school-based dental program in yogyakarta, indonesia: a qualitative study rosa amalia1, niken widyanti1, johan w. groothoff2, and rob m.h. schaub3 1 department of community and preventive dentistry, faculty of dentistry, gadjah mada university, yogyakarta indonesia 2 department of health sciences, university medical center groningen, groningen, the netherlands 3 department of clinical epidemiology and oral health care, center for dentistry and oral hygiene, university medical center groningen, groningen, the netherlands abstract background: a high prevalence of caries at ages ≥ 12 in yogyakarta province (dmft = 6.5), raises the question of the effectiveness of the school-based dental program (sbdp) which, as a national oral health program in schools, is organized by community health centers (chcs). purpose: the aim of this study is to explore the possible constraints on work processes which might affect the performance of sbdps in controlling caries. methods: in-depth interviews was conducted in twelve chcs, covering all five districts both in urban and rural areas. subjects were 41 dentists and dental nurses working in these chcs. the interviews were structured according to the following themes: resources and logistics; program planning; target achievement; monitoring and evaluation; and suggestions for possible improvements. the data were analyzed using content analysis. results: the main constraints identified were limited resources and inflexible regulations for resource allocation in the chc, and inadequate program planning and program evaluation. inadequate participation of parents was also identified. another constraint is thatpolicy at the district level orientates oral health towards curative intervention rather than prevention. suggestions from interviewees include encouraging a policy for oral health, task delegation, a funding program using school health insurance, and a reorientation towards prevention. conclusion: the weakness of management processes and the unsupported policy of the sbdp at the local level result in a lack of effectiveness. the constraints identified and suggestions for improvements could constitute a basis for improving program quality. key words: school-based dental program, program constraints, yogyakarta abstrak latar belakang: tingginya prevalensi karies pada usia ≥ 12 tahun (dmft = 6.5) di provinsi daerah istimewa yogyakarta (diy) menimbulkan pertanyaan akan efektifitas usaha kesehatan gigi sekolah (ukgs). ukgs adalah salah satu program nasional di bidang kesehatan gigi dan mulut yang dilaksanakan oleh puskesmas. tujuan: tujuan dari penelitian ini adalah untuk mengeksplorasi hambatan pada pelaksanaan ukgs yang dapat mempengaruhi kinerja dari ukgs untuk mengontrol karies. metode: penelitian kualitatif dengan menggunakan in-depth interview dilakukan di 12 puskesmas, meliputi lima kabupaten di diy baik di daerah pedesaan maupun perkotaan. subyek terdiri dari 41 dokter gigi dan perawat gigi di puskesmas. struktur tema dari wawancara adalah sumber daya dan logistik; perencanaan program; pencapaian target; monitoring dan evaluasi; saran untuk perbaikan program. data dianalisis menggunakan analisis konten. hasil: hambatan pada program ukgs yang teridentifikasi adalah sumber daya yang terbatas dan regulasi yang tidak fleksibel untuk alokasi sember daya di puskesmas, perencanaan dan evaluasi program yang tidak memadai dan rendahnya peran serta orang tua. hambatan lain adalah kebijakan pada tingkat kabupaten yang berorientasi pada pelayanan kuratif daripada preventif. saran yang dikemukakan adalah penguatan kebijakan untuk kesehatan gigi, delegasi tugas, asuransi kesehatan sekolah dan reorientasi pelayanan preventif. kesimpulan: proses manajemen yang lemah dan kurangnya dukungan kebijakan untuk ukgs pada tingkat daerah menjadi salah satu sebab kurang efektifnya program. hambatan dan saran yang teridentifikasi pada studi ini dapat menjadi dasar untuk meningkatkan mutu program. kata kunci: usaha kesehatan gigi sekolah, hambatan program, yogyakarta 94 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 93–100 introduction over the years, caries experience index (decay missing filling teeth (dmft)) of 12-year-old indonesian children increased from dmft = 0.7 in the 1970s, to 2.3 in the 1980s, and 2.7 in the 1990s.1 from a national epidemiological survey in 2007, it appears that yogyakarta province, situated on the island of java, had a very high caries experience on aged ≥12 years (dmft = 6.5), compared with an average experience for all java (dmft = 4.8) and for all indonesia (dmft = 4.9).2 the caries experience was higher than that of thailand (dmft = 3.7) and india (dmft = 3.9) which have comparable characteristics of developing countries.3 these data show that in indonesia, and in yogyakarta in particular, it is very difficult to control the progression of caries. the high rate of caries prevalence was unsatisfactory finding related to the national effort of school health. in indonesia, children are expected to benefit from the school-based dental program (sbdp), a national oral health program aimed at prevention and promotion which has been a part of national school health policies. this program is organized by community health centers (chcs) which play an important role in the national health program by delivering comprehensive and integrated health services to the community under the authorization of the district health office (dho). the organization and operation of the school-based dental program is regulated by the ministry of health of indonesia, which has published a manual on it.4 dental staff (dentists and dental nurses) working in chcs are responsible for the implementation of the sbdp, together with other tasks in the dental clinic or serving in the community. in the sbdp, a dentist is in charge of program management, while dental nurses help with the implementation of the program, for instance, by recording, reporting, and carrying out the program in schools. in practice, dentists and dental nurses work closely together in implementing the program. according to the manual, the activities in sbdp including training teachers in matters of oral health, oral health screening followed by oral health education in the classroom, practice tooth brushing in school and complete treatment upon referral to the chc. within the sbdp, screening and dental health education are free of charge, but treatment in a chc dental clinic upon referral is charged for. the charges vary depending on district policy. the increasing caries rate of children raises the question of whether the sbdp is adequate in controlling caries. this assumption has been strengthened by unpublished reports of chcs in yogyakarta showing that the sbdps often fail to achieve their operational targets. it is well known that in developing countries, the insufficient infrastructure and lack of financial means are major problems in the provision of health programs for the community.5 this sort of information is rather broad, while to improve the performance of programs like the sbdp, there is a need to look deeper within the program. therefore, the research question of this study is; what are possible constraints on work processes which might affect the performance of sbdps in controlling caries. this study aimed at exploring the major factors of constraints in four cathegories: resources and logistics; action plans; achievement of targets; monitoring and evaluation. materials and methods the study was carried out as a part of evaluation study on school-based dental program in 2009–2010 in yogyakarta province, located on the island of java. it is the second smallest province in indonesia and has a high population density: 3.5 million people in an area of 3185.8 km2.6 yogyakarta province consists of five districts: sleman, gunung kidul, bantul, kulonprogo, and yogyakarta city, which together have 117 chcs, each of which must run a sbdp.7 a qualitative study was conducted using in-depth interviews such as those used previously to gain a more in-depth insight of the subjects under study.8 sampling technique was non probabilistic–which is called purposive sampling. this technique was chosen on the assumption that the investigator wants to discover, understand and gain insight and therefore must select a sample from which the most can be learned.9 first step, the chcs were grouped on the basis of rural and urban area. the distinction between urban and rural areas was determined at first because of the socioeconomic differences between the areas, which may affect the work processes variation and differentiation of the objects under study.10 second step, the chcs were selected on the basis of target sample criteria. dental professionals (dentists and dental nurses) were chosen as target sample because of their experience and competence in the work processes of sbdp. the criteria of selection was dentists or dental nurse who runs sbdp in the same chc at least five-years to ensure that each participant understood the workprocess of the programme. other consideration on the selection of chcs was the availability of yearly reports and activity records from the last two years. size of the sample was considered finalized at the point of saturation.11 this is the point at which no new correspondence: rosa amalia, c/o: bagian ilmu kedokteran gigi pencegahan & ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas gadjah mada. jl. denta sekip utara yogyakarta 55581, indonesia. e-mail: rosadewanto@yahoo.com. telp: +62 81578804082 95amalia, et al.: constraints on the performance of school-based dental program concepts emerge from reviewing of successive data from a theoretically sensitive sample of participants. finally, twelve chcs which responded and matched with the criteria were then included in this study (2 chcs from kulon progo, 2 chcs from yogyakarta city, 2 chcs from sleman and 3 chcs from gunung kidul and 3 chcs from bantul). yearly reports and activity records from these twelve chcs were reviewed in terms of work processes and possible constraints in program implementation. the review of the chcs’ reports was based on the four categories of procedures from the sbdp manual: resources and logistics, including human resources, budget, materials, and facilities; action plans, including a situational analysis of the area based on epidemiological data, problem formulation, problem solutions, and resources distribution; achievement of targets; and monitoring and evaluation. issues emerging from these four themes became the basis for the in-depth interviews aimed at eliciting explanations for problems in the execution of the sbdp in each chc. specifically, potential solutions suggested by respondents were also discussed in the interview. in developing guidelines for conducting the in-depth interview, kvale’s principles were employed to keep the original vision of the study. the purpose of the interviews, the questioning technique, and the strategy for data analysis were determined in advance.12 the interviews took place in each chc. the data from each interviewee were handled separately. the interviews were semi-structured, but open discussion was encouraged. in each interview the interviewer (ra) began with an introduction, encouraged discussion, avoided being placed in the role of expert, and made a summary. the interview lasted between 90 and 150 minutes. an audio recorder was used in each session to preserve the actual words spoken. an assistant was present to help with recording and data handling during the interview. data were kept confidential after analysis. a verbatim transcript was produced of each interview, reflecting the session and using the participants’ own words which were then analyzed by manual thematic analysis. thematic analysis is a research method which pays attention to the qualitative aspects of the material analysed through the systematic classification process of coding and identifying themes and patterns emerging from the transcribed text.13 the analysis followed the theory of fereday et al.14 the first step in the data analysis was reading all data repeatedly to achieve immersion and obtain a sense of the whole and identify themes. after this, data were read word-by-word, adding codes to note patterns in the data and relate them to themes. labelling these patterns was designed to allow distinctions to be drawn and research question to be answered. each relevant expression was classified and coded for every aspect of the limitations in sbdp performance presented. emphasis was put on elucidating areas of thematic importance and exploring major constraints in the work processes of the sbdp as perceived by dental staff. codes then were sorted into categories based on relationships between the different codes. comparable explanations were combined under a single code. to improve reliability, these coded findings were then discussed with the research team in order to arrive at consensus on interpretation.15 written permission from all participants was obtained prior to the start of the interview. ethical approval was obtained from the ethical committee of the faculty of dentistry,gadjah mada university, yogyakarta, indonesia. permission for the study was granted by various government offices both on the district and the provincial level. results among 41 respondents, 16 were dentists and 25 were dental nurses. subjects were 17 percent male and 83 percent female. average work experience was 10 years for dentists and 13 years for dental nurses. number of dentist and its working experience in each district is in table 1. four categories, each related to sub categories were table 1. respondents characteristics districts average work experience (in year) dental nurse dentist bantul 10.9 7.2 kulon progo 18 19.5 sleman 10.5 11 yogyakarta city 16.7 6 gunung kidul 7.8 11 table 2. perceptions of dental staff regarding constraints in the school-based dental programme themes major issues resources and logistics shortage of dental staff comparing with burden task insufficient programme budget scarce material for prevention strict regulation in resource allocation programme planning unfamiliar procedure deficient training in programme management low priority for dental health in the chc level limited use of epidemiological survey target achievement limited time to go to schools poor links to parents unclear financing scheme on referral policy curatively oriented unclear target on dental status or behaviour changes from dho poor socioeconomic circumstances monitoring and evaluation ineffective means for evaluation at chc level poor feedback from dho lack of integrated information system 96 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 93–100 generated in the analytical process, which is presented in diagram in table 2. in general, dentists and dental nurses in yogyakarta province face similar problems, which imply on the consequence that there is no specific condition that could be determined as a basis for differentiation per district. barriers of program implementation in rural area was complicated by the socioeconomic condition including poverty, distance and lack of transportation which affect on access to health care. generally speaking, the respondents noted that the number of dentists and dental nurses in yogyakarta province both in urban and rural area did meet the national standard. the tasks of providing treatment in the chc dental clinic, together with community activities like the sbdp, were hard to manage. the respondents indicated shortages in the budget to run the program. one dentist explained that the limitations of the budget made some activities hard to carry out, for instance, training teachers about dental health, and conducting meetings to foster cooperation with the education office or the parents. the budget available for the program was only sufficient for administrative purposes such as paper or book procurement for recording these activities. they also indicated their limited ability to manage the resources at the chc level, because decisions over resource allocation per chc program came from the district health office (dho).the regulation of resources was strict, which made it impossible to use excess funds from other chc health programs for the sbdp. the interviews showed a sense that dentists and dental nurses were dissatisfied with the resources available and desired procedures to make flexible allocation between programs possible. one dentist phrased it as follows: the respondents indicated the material support provided in the chc’s dental clinic for dental care was mostly for curative treatment while the materials for prevention were scarce. this led to curative-oriented treatment, while preventive treatment for caries such as fissure sealant or topical application was neglected. one dental nurse related the problems providing prevention treatment to that of resources as follows: “i understand that efforts in controlling caries will be effective if we provide preventive care like fissure sealant or topical application for those who are not affected or just have an initial lesion. but for years… even since i began work here for the first time… eight years ago… it was absent. if the materials are missing, we cannot offer this treatment to children. our focus now is on clinical treatment or acute problems.” dentists and dental nurses agreed that unfamiliarity with the sbdp manual made them produce a plan of action (poa) in their own format. their poa usually only consisted of the number of schools to be served and a time schedule instead of a complete planning program based on a situational analysis of the area that was, in turn, based on epidemiological data, problem formulation, problem solutions, and resources distribution. they also mentioned a lack of skills and knowledge regarding program management as being a constraint. there had been no training in public health, especially dental public health, before they started work in the community health centers, which was a constraint on developing a successful planning for the program. one dentist phrased it as follows: “…after starting to work in the chc, we have had to manage the program without any training first. it was hard because we knew little about sbdp management or organization. the last sbdp manual was published more than 10 years ago and the book was nowhere to be found…” a dental nurse mentioned that, although they had the task of undertaking an epidemiological survey, they seldom did so, since existing data were only barely taken into consideration in the planning of the program. a schedule for screening and referral was considered sufficient for running the program. one dental nurse mentioned that other health programs had a higher priority than dental health. this was reflected in meetings held to discuss the program planning of every unit in the chc. time allocated for discussing the dental program was limited, since other health programs were considered more urgent. therefore, this situation led to difficulties in communicating effective plans. “there are many health programs that must be prioritized, such as dengue, tuberculosis, nutrition, and maternity. discussing program planning in chcs sometimes consumes a lot of time and we do not feel comfortable in speaking up for the schoolbased dental program. dental disease never causes death, does it?” participants from disadvantaged locations mentioned poverty and difficulties with transportation as constraints for compliance with referral after screening. another constraint in the achievement of targets was the role of parents. they play an important role in the sbdp, because the results of the screening are collected by the teacher and handed over to the parents. one dental nurse from a rural area explained that parent committees rarely discussed the school health program, since they considered this a task for the chc. “only a few parents who live near the chc or have enough money will bring their children to the chc. disadvantage geographic area and lack of transportation still become a problem. yes, we do the screening, but without parents’ participation; that is a problem. i was once involved in the parents’ committee discussion, but they care more about computers or books than oral health. they considered it a chc task.” another important constraint expressed by a dentist was the limited time for visiting schools, since they have 97amalia, et al.: constraints on the performance of school-based dental program a large number of patients in the dental clinic to treat and also have tasks in other programs such as the mother and toddler program. time constraints also have an impact on dental personnel doing administrative work such as baseline surveys, reporting, and recording. one dental nurse said that the standard form for dental screening provided by the ministry of health was complex and took time to complete. therefore, they recorded only children with positive screening, generally for caries disease and prolonged primary teeth retention, who were then given a referral letter. “i cannot leave the clinic easily because many patients come everyday. therefore, i have a dedicated time just for screening at least once a year. this chc has to serve more than twenty schools. we also have to run community programs in more than 30 villages. the clinic would be closed then and people would complain.” –dentist “the form is complex. it includes many oral malfunctions and malformations. when i visit schools, i have to serve about 200 children in each school. i also have to deliver dental health education after screening –when can i do the recording? i cannot do everything at once.” – dental nurse respondents mentioned that the data required for the annual reports mostly concerned curative interventions such as filling and extracting on referral, which discouraged preventive efforts in the sbdp. one dentist mentioned that the format of the annual reports also did not require comparison of program outcomes with those of the previous year, explaining why the chcs lacked progress reports: “the school-based dental program was always interpreted as a screening, filling, and extracting program. that is because the report to the dho only asked for that. if we do screening at least once a year, the program is considered done. we don’t have the burden of considering changes to status or behavior.” respondents claimed that the lack of a specific department with responsibility for oral health after the decentralization of health services in 2001 was a constraint on sbdp monitoring and evaluation. no attention was paid to oral health, resulting in ineffective monitoring and evaluation. one dentist stressed that developing a clear blueprint for the roles and responsibilities of the oral health sector might help improve services. a lack of any formal approval by the dho discouraged improvement of the programs. another dentist highlighted the absence of an integrated information system in the district health office as a crucial factor in the evaluation system, because referred children could also be treated in private practice. “the district health office never requests anything from this program. it has never been evaluated and never monitored. even if the coverage of the program were zero, they would not bother. we want the district health office to reorganize the program.”–dentist “the lack of an integrated information system in the district health office makes us difficult to track the status of children after getting referral letter. it is because children could also get care from private practice. if the system exist then we will know how many children get referral treatment outside the chc.” –dentist then, some respondents shared ideas for improving the target achievement of the program. a dentist who was not able to carry out program activities routinely in all schools mentioned the possibility of delegating some tasks to school teachers, for instance, demonstrating toothbrushing, administering simple medication, and identifying and referring children with oral problems to the chc. another respondent stated that the chc had previously circumvented the referral barrier by administering treatment – such as extraction of persistent deciduous teeth, art fillings, and medication for dental pain – in schools directly after screening. this strategy was no longer possible, because the government had launched new regulations on medical practice in 2004 which forbade all medical services outside formal health facilities. a dental nurse in an urban area mentioned a school health insurance plan which improved referral compliance. it increased the willingness of parents to send their children for dental treatment to a chc: “there are many parents who don’t really care about dental health. most parents of low socioeconomic status objected to paying for the treatment. however, since all schools in our catchment area manage school health insurance, referral compliance here is actually quite high. this system really works in overcoming that problem.” in the interview, a majority of the respondents expressed positive opinions about prevention. they did not consider filling and extracting to be caries-preventing activities. respondents also agreed that prevention yields a sustained improvement in the oral health status of children and minimizes the risk of dental problems in the future. the respondents also expressed the fact that delivering preventive services to children was preferable. one dentist expressed this as follows: “children will get more lasting benefit if the target to be achieved applies the principle of real prevention rather than just filling and extracting. it’s like waiting for the disease to come and then fixing it. the classic problem is resources and facilities. we have no choice because no preventive treatment is available in the chc, so we cannot refer children for prevention. reorientation towards prevention should be started, and we really support it.” 98 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 93–100 the respondents agree that an emphasis on prevention would be a challenge for the sbdp, since the existing policy is curative oriented. most respondents highlighted the role of the district health office in influencing their activities in terms of delivering health services. they agreed that the dho should begin a reorientation of services towards prevention, and provide adequate resources and facilities. discussion this study has provided some information about the constraints on the work process of the sbdp as experienced by dental staff. dental staff in the five districts faced similar problems as sbdp is a national program which routinely carried out in all over chc in a standard format. significant constraints were identified in each of the four categories: resources and logistics, program planning, target achievement, and monitoring and evaluation. the constraints found were limited resources and inflexible regulation of resource allocation in the chc. inadequate program planning and program evaluation is a weakness in the management process. this situation is worsen because of the limited time for visiting schools and low parental cooperation. despite the national guidelines and the sbdp manual, the policy at the district level orientates dental health towards more curative intervention rather than prevention. suggestions were made regarding task delegation, improving procedures for small curative interventions in schools, funding programs through school health insurance, and reorientation towards preventive treatment in the schools. concerning resources, dentists and dental nurses both in urban and rural area face pressures in terms of time allocation in balancing their obligations to treat patients in the clinic and their task of serving out in the community. therefore, it appears that in practice, human resources in the oral health sector are insufficient despite the fact that the number of dentists and dental nurses are theoretically in compliance with the standard. moreover, there is an insufficient budget to finance the program completely. an inadequate workforce together with a lack of resources is also recognized in other public health programs as a classic problem in developing countries.16 however, this finding indicates the need to reassess the resource allocation for the oral health sector, adjusted to the actual need to develop a successful program. poor program planning is identified as a constraint on the work process. the majority of respondents agreed that the skills for analyzing and planning sbdp activities were limited. this could be what leads dentists to continue to implement the program without considering what is actually needed to deal with current children’s dental health problems. this is in line with a study in developing countries suggesting that effective public health programs need skills in analysis and planning.17 setting a practical, feasible, and evidence-based plan has to be based on objective information about the situation rather than on subjective impressions. it is suggested that dental personnel should be encouraged and trained to produce realistic and qualified planning reports as tools for program improvement. the majority of respondents mentioned that a low response on referral was mostly generated by poor relationships with parents. it was explained further that the poor cooperation of parents was enhanced by the feebased mechanism of referral payment.this is in line with a study which mentioned that access to care is determined largely by individual’s ability to pay.18 therefore, a mechanism for financing should be considered carefully. a financing mechanism for referral, making use of school health insurance as suggested by one of the dentists interviewed, could intensify this problem if it is only aimed at curative treatment for dental disease. this could generate dependency on professionals and move the program away from prevention.19 the important thing that emerged from this study concerning achievement of target was the socioeconomic inequality between chcs in urban and rural area, which affected on the awareness to visit dental care. it seemed here that rural children at a greater risk of poor oral health outcomes. the optimization of available resources was experienced by a study the philippines, could become an important discourse. 20 reactivation of good oral health behaviour in school by promoting oral health supported by healthy environment in school and good connections with parents may also become an initial step for tackling the boundaries. 21 the curative orientation which was blamed as one of the causes of increasing caries is common in developing countries and indicated as the most significant barriers in reducing caries in children.22 achieving oral health, which should be the goal, should not be meant as oral treatment. in a situation like yogyakarta, where the caries level remains quite high, the combination of curative and preventive care for those who suffer from caries, and prevention care for those who are free of caries, is the best choice for providing oral health care. in accordance with the program of monitoring and evaluation, it appears that dental staff experienced deficient monitoring and poor reporting compliance even at the chc level itself. most of the respondents agreed that the major constraints were the scant attention paid to dental health by the dho. it is common that limited health-system resources leads governments to concentrate on a few high-priority items.23 however, it should be noted that globally, dental caries shows a trend towards being on the increase in the years to come and therefore the dho as regulator needs to make some improvements in various important technical aspects.24 furthermore, neglecting the oral health program will negatively affect the motivation and performance of dental staff in the chc. supportive supervision from the dho is critical in strengthening and enhancing performance in primary health facilities.25 cooperation with private practice in developing an integrated oral health information system in 99amalia, et al.: constraints on the performance of school-based dental program the district could be considered. a feasible computerized system could prove effective in supporting the monitoring capacity.26 however, it should be noted that the end result of the system should not just be providing data but using that data to provide information for decision-making aimed at improving the oral health program. despite the weaknesses and constraints found, there is a positive side regarding school-based dental programs in yogyakarta. this system is an established program supported by a national policy for screening the oral health of the children on a regular basis, which ensures that children have contact with a dental service. in this study, dentists and dental nurses also showed that they were in favor of dental prevention. the absence of preventive treatment is triggered more by conditions such as material and equipment shortages than by their attitudes. the importance of this finding is that reorientation toward prevention could be successful, if it is supported at all levels by the chc staff, the district health office, and the ministry of health. the results of this study provide the decision-maker in dental health policy with information for developing strategies to improve sbdp. in order to implement an effective and efficient program policy for oral health, assessing the constraints on the program through the experience of dental staff is a necessary step. this qualitative study provides a better understanding of the context and personal experiences in the day-to-day processes of the sbdp. however, some limitations in this study have to be taken into account. the study was based on qualitative data that might be a limitation as the findings only provide hypotheses of deficiencies, but this information can be a useful information in disease specific program like sbdp. bias reporting may have confounded some of the responses, but previous studies report similar issues suggesting that the results are externally valid.27 the findings in this study may be generalizable to other chcs in yogyakarta due to similar health system infrastructures, socio-cultural environments, and topography. optimizing all available resources and implementing efficient work might be the most recommendable, since obtaining more resources from the district health office cannot realistically be expected. for instance, one study has shown that the delegation of basic community oral health services such as dental education or emergency care to other health professionals in the chcs can help dentists and dental nurses improve the program .28 however, this delegation should be supervised by a dental professional in order to maintain quality. regulation that forbids all clinical services outside formal health facilities is also unfavorable in terms of optimizing resources. it is recommended that an exception should be made for certain treatment with low risk for complications, such as extraction of primary prolonged teeth and filling with atraumatic restoration treatment (art), with concern on preventing cross-infection. the participation of parents is particularly important to ensure that the oral health education for children are addressed adequately and effectively. beside clinical prevention, effort on community prevention level could be implemented with consideration on practicable effort like water quality improvement or fluoride mouth rinsing and the use of fluoridated toothpaste in school. these methods have been proven to be more cost-effective than professional application and could be equally distributed to all children.29 clear guidelines of dental health strategy to enhance preventive effort should be developed in terms of a broad strategy on oral health prevention and promotion. however, further research is also required to assess the oral health outcome of the sbdp including oral health status and behaviour of the children, in order to evaluate the existing strategy. in conclusion, the school-based dental program in yogyakarta is carried out with weak management processes, low support from parents, and a curative orientation with little priority for oral health at district level. these shortcomings are one explanation for the failure to be succesful in controlling caries, despite the continuing efforts being made. this study could serve as a basis for developing recommendations that would be valuable for future sbdp work. references 1. who oral health country/area profile programme: indonesia. available at http://www.whocollab.od.mah.se/searo/indonesia/data/ indonesiacar.html. accessed on july 7, 2010. 2. departemen kesehatan ri. riset kesehatan dasar (riskesdas) 2007. jakarta: badan penelitian dan pengembangan kesehatan depkes ri; 2008. p. 133–47. 3. who oral health country/area profile program. available at http:// www.collab.od.mah.se/index.html. accessed on july 7, 2010. 4. departemen kesehatan ri. pedoman pelaksanaan usaha kesehatan gigi sekolah. jakarta: direktorat jendral pelayanan medik, depkes ri; 1996. p. 7–8. 5. pappaioanou m, malison m, wilkins k, otto b, goodman ra, churchil re, white m, thacker sb. strengthening capacity in developing countries for evidence-based public health. soc sci med 2003; 57(10): 1925–37. 6. resosudarmo bp, sugiyanto c, kuncoro a. livelihood recovery after natural disasters and role of aid: the case of the 2006 yogyakarta earthquake. available at http://rspas.anu.edu.au/economics/ publications.php. accessed on november 25, 2010. 7. bps-statistics of d.i. yogyakarta province. daerah istimewa yogyakarta in figures 2007/2008. yogyakarta: bps-statistics of d.i. yogyakarta province; 2008; p. 158 8. hansen ec. successful qualitative health research: a practical introduction. alien and unwin crows nest, p. 95–8., nsw; 2006. 9. merriam sb. qualitative research: a guide to design and implementation. san fransisco, ca: john willey & sons; 2009. p. 55–84. 10. patton mq. qualitative evaluation and research methods. 3rd ed. thousand oaks, ca: sage publications; 2002. p. 234–5. 11. flick u. an introduction to qualitative research. 4th ed. london, uk: sage publication ltd; 2009. p. 114–24. 12. steinar k. doing interviews (the sage qualitative research kit). london: sage publication; 2007. p. 35–6. 13. joffe h, yardley l. content and thematic analysis. in: research methods for clinical and health psychology. marks df, yardley l, editors. london, uk: sage pub; 2004. p. 56–68. 100 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 93–100 14. fereday j, muir-cochrane e. demonstrating rigor using thematic analysis: a hybrid approach of inductive and deductive coding and theme development. international j qualitative methods 2006; 5(1): 80–92. 15. bradley eh, curry la, devers kj. qualitative data analysis for health services research: developing taxonomy, themes and theory. health serv res 2007; 42(4): 1758–72. 16. travis p, bennett s, haines a, pang t, bhutta z, hyder aa, pielemeier nr, mills a, evans t. overcoming health-systems constraints to achieve the millennium development goals. lancet 2004 sep; 364(9437): 900–6. 17. world health organization. integrated management of child health: guide to planning for implementation of imci at district level. cairo: who regional office for the eastern mediteranean; 2008. available at: http//www.emro.who.int/cah/pdf/cah_184.pdf. accessed september 20, 2010. 18. watson mr, manski rj, macek md. the impact of income on children’s and adolescents’ preventive dental visits. j am dent assoc 2001; 132: 1580–7. 19. gift hc, andersen rm. the principles of organisation and models of delivery of oral health care. in: pine c, harris r. community oral health. 2nd ed. uk: quintessence publishing co. ltd; 2007. p. 429. 20. monse b, naliponguit e, belizario v, benzian h, van helderman wp. essential health care package for children--the ‘fit for school’ program in the philippines. int dent j 2010; 60(2): 85–93. 21. cornwell l, hawley sr, st romain t. implementation of a coordinated school health program in a rural, low-income community. j sch health 2007; 77(9): 601–6. 22. edelstein bl. the dental caries pandemic and disparities problem. bmc oral health 2006; 6(suppl 1): s2. 23. beaglehole r, epping-jordan ja, patel v, chopra v, ebrahim s, kidd m, haines a. improving the prevention and management of chronic disease in low-income and middle-income countries: a priority for primary health care. the lancet 2008 september; 372(9642): 940–9. 24. bagramian ra, garcia-godoy f, volpe ar. the global increase in dental caries. a pending public health crisis. am j dent 2009 february; 21(1): 1–8. 25. enkhtuya b, badamusuren t, dondog n, khandsuren l, elbegtuya n, jargal g, surenchimeg v, grundy j. reaching every districtdevelopment and testing of a health micro-planning strategy for reaching difficult to reach populations in mongolia. rural remote health 2009; 9(2): 1045. 26. ludwick da, doucette j. adopting electronic medical recors in primary care: lessons learned from health information systems implementation experience in seven countries. int j med inform 2009; 78(1): 22–31. 27. heywood p, choi y. health system performence at the district level in indonesia after decentralization. bmc int health and hum rights 2010; 10: 3. 28. cane rj, butler dr. developing primary health clinical teams for public oral health services in tasmania. aust dent j 2004 dec; 49(4): 162–70. 29. centers for diseases control and prevention. recommendations for using fluoride to prevent and control dental caries in the united states. mmwr 2001; 50(n.rr-14): 19–24. 201 vol. 43. no. 4 december 2010 candida albicans adherence on acrylic resin plates immersed in black tea steeping soebagio department of dental materials faculty of dentistry, airlangga university surabaya indonesia abstract background: black tea or cournelia sinensis is one of known tea varieties in indonesia. actually, black tea is consumed daily as beverage that can function as antiseptics and fungicides. black tea containing antibacterial and fungicide properties can reduce the number of candida albicans (c. albicans) colony attachment on the surface of acrylic resin plates. purpose: this study was done to determine the effective concentration of black tea steeping used as the immersion material of acrylic resin plates towards the number of candida albicans colonies. methods: in this study, acrylic resin plates with the size of 10 × 20 × 1 mm were immersed in black tea steeping with the following concentrations, 3.33%, 6.66%, and 13.33%, for one hour. the growth of c. albicans colonies were then detected by counting the number of colonies growing on sabouraud’s dextrose medium. data was analyzed by using both one-way anova with the significance level of 5%, and tuckey’s multiple comparison tests (tuckey’s hsd test). results: there were significant differences in the number of c. albicans colonies in acrylic resin plates immersed in black tea with the concentration of 3.33%, 6.66%, and 13.33%. conclusion: it can be concluded that the higher concentration of black tea (13.33%) used to immerse acrylic resin plates, the greater the decreased number of c. albicans colonies. key words: black tea, candida albicans, acrylic resin plate abstrak latar belakang: teh hitam atau cournelia sinensis adalah salah satu macam teh yang dikenal di indonesia. teh hitam seringkaliteh hitam seringkali dipakai sebagai minuman seharihari dan berkhasiat sebagai antiseptik maupun fungisid. teh hitam yang mengandung sifat antibakteri dan fungisid dapat menurunkan jumlah koloni candida albicans (c. albicans) yang melekat pada permukaan lempeng resin akrilik. tujuan: untuk mengetahui konsentrasi efektif seduhan teh hitam sebagai bahan perendam lempeng resin akrilik terhadap jumlah koloni c. albicans. metode: pada penelitian ini lempeng resin akrilik ukuran 10 × 20 × 1 mm direndam dalam seduhan teh hitam konsentrasi 3,33%, 6,66%, 13,33% selama 4 jam. pertumbuhan jumlah koloni c. albicans dilakukan dengan cara menghitung jumlah koloni yang tumbuh pada media sabouraud’s dextrose. analisis data menggunakan uji anova satu arah dengan taraf kemaknaan 5%,analisis data menggunakan uji anova satu arah dengan taraf kemaknaan 5%, uji pembanding ganda tuckey hsd. hasil: terdapat perbedaan yang bermakna jumlah koloni c. albicans yang melekat pada lempeng resin akrilik yang direndam dalam seduhan teh hitam dengan konsentrasi 3,33%, 6,66% dan 13,33%. kesimpulan: konsentrasi 13,33% atau makin pekat seduhan teh hitam yang digunakan untuk merendam lempeng resin akrilik menyebabkan penurunan jumlah koloni c. albicans. kata kunci: teh hitam, candida albicans, lempeng resin akrilik correspondence: soebagio, c/o: departemen material kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend.jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. research report 202 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 201–204 introduction denture base material commonly used is polymethyl methacrylate resin. the properties of the heat cured acrylic resin are non-toxic, easy manipulated, non-irritating,easy manipulated, non-irritating,, undissolved in oral liquid though soluble in water, fulloral liquid though soluble in water, full, full of aesthetics, look-alike gingiva color, stable, repairable, has small dimensional changes, relatively cheaper, and commonly used.1 wearing of dentures will make the mucosa covered with removable denture for a long time so that it can block either the mucosal surface or tooth surface from being cleaned by the tongue and saliva. as a result, it can cause the formation of denture plaque.2,3 microbial plaque formed on the denture surface facing the mucosal surface is considered as a factor stimulating the pathogenesis of the denture stomatitis. it even has been reported that 65% of users suffer from denture stomatitis.4 food accumulation and dental plaque will increase the frequency and density of c. albicans. in other words, denture stomatitis caused by the dentures is related to the proliferation of c. albicans on the plaque attaching to the dentures. the infection of c. albicans is significantly reported as the cause of denture stomatitis.2 the prevention of denture stomatitis is essential, including the elimination of c. albicans. nevertheless, the infection of c. albicans can be prevented by cleaning and removing dentures at night.5 the cleaning of the dentures can be conducted in two ways, mechanical and chemical. the mechanical cleaning can be conducted with a toothbrush or ultrasonic equipment, while the chemical cleaning can be conducted by immersing the dentures in the cleaning solution.4 actually, tea can be used as the immersion material of acrylic resin plate since tea is considered as antiseptics. one kind of tea known in indonesia is camellia sinensis or black tea.6 black tea is often used as a beverage since it is cheap, easily obtained, and considered as disinfectants. these disinfectant properties are generated by the content of fluoride, polyphenols, catechins and other oxidated derivatives.7 however, the optimal level of black tea steeping is determined by the optimal proportion between the amount of tea and water used for steeping the tea. generally, the standard proportion of black tea steeping is 20 grams of black tea and 300 ml of water equivalent to the concentration of 6.66%.8 for those reasons, it becomes essential to know the concentration of the steeping black tea that can effectively be used to reduce the number of c. albicans colonies. the study is then aimed to determine the effective concentration of black tea steeping used as the immersion material of acrylic resin plates towards the number of c. albicans colonies. materials and methods this study is considered as a laboratory experiment with factorial design. the materials used are heat cured acrylic resin, black tea as immersion solution, and the suspension of c. albicans. the sample used, moreover, is a box-shaped acrylic resin with the size of 10 × 20 × 1 mm. and, the location of the study is in microbiology laboratory and materials department of dentistry, faculty of dentistry. to make the suspension, c. albicans was put in 5 ml of sabouraud’s broth medium, and was incubated for 48 hours at 37° c. one oose of the suspension was taken and put into 5 ml of sabouraud’s broth, and it was then incubated for 48 hours at 37° c in order to be used to contaminate with acrylic resin plates (10 × 10 × 1 mm). sterile saliva was obtained from a person without any stimulation, and it was then disentrifuged for 20 minutes at 2000 rpm.9 finally, the saliva supernatan was put into 5 cc syringe of injection, and it was then filtered with 0.2 μm millipore unit of filter mounted on the syringe needle.10 the attachment of c. albicans on acrylic resin plates was measured through the following procedures. acrylic resin plates were immersed in water for 48 hours in order to reduce the residual monomer.11 after that, the acrylic resin plates were sterilled by using autoclave at 121° c for 18 minutes.12 then, the acrylic resin plates were immersed in sterile saliva for 1 hour at the room temperature to form pellicle. the acrylic resin plates were rinsed twice with phosphate buffered saline solution.9 they were inserted into the test tube containing the suspension of c. albicans, and then were incubated again for 24 hours at 37° c. after that, the acrylic resin plates were inserted into the test tube, each of which contained with black tea solution with the concentration of 10 gr/300 ml (3.33%), 20 gr/300 ml (6.66%) and 40 gram/300 ml (13.33%) for 1 hour, and with sterile distilled water as control. furthermore, the acrylic resin plates immersed in black tea were rinsed twice with pbs and then were inserted into 10 ml of sabouraud's broth medium, and were vortexed for 30 seconds to release c. albicans attaching to the plate. the suspension of c. albicans in sabouraud's dextrose was taken for about 1 ml for spreading, and then was incubated for 48 hours at 37° c.10 finally, the number of c. albicans colonies was measured in cfu/ml. the result of the measurement of the number of c. albicans colonies then was tabulated based on the groups. afterwards, it would be tested by using one-way anova at 5% significance level. if there were significant differences, it would continually be tested by using tuckey’s multiple comparison test (tuckey’s hsd test). 203soebagioi: candida albicans adherence on acrylic resin results the results of the mean value and the standard deviation of the number of c. albicans colonies attaching to the acrylic resin plates immersed in the black tea steeping can be seen in table 1. table �. the mean value and the standard deviation of the number of c. albicans colonies attaching to the acrylic resin plates immersed in the black tea steeping (cfu/ ml) concentration n x sd control 6 623.66 2.50 3.33% 6 204.16 9.17 6.66% 6 142.50 2.25 13.33% 6 106.66 6.50 note: x : mean, sd: standard deviation table �. the results of the analysis of tuckey’s multiple comparison test (tuckey’s hsd test) on the number of c. albicans attaching to the acrylic resin plates 3.33% 6.66% 13.33% 3.33% s s 6.66% s s 13.33% s s note: s = significances = significance before conducting the parametric tests to determine the significance of the differences, this study needed to conduct normality test using kolmogorov-smirnov test in order to with the result p = 0.6274 (p > 0.05). it means that those three treatment groups and the control group have normal distribution. the comparative test of those three treatment groups and the control group then was also conducted by one-way anova test. from one-way anova test, it is known that there was significant difference among those treatment groups with p = 0.000 (p < 0.05). to analyze the differences among those treatment groups further, the data then was tested by using the tuckey’s hsd test. the result of tuckey’s hsd test, finally, indicated that there were significant differences between the treatment groups and the control group. in other words, the increasing of the concentration of the black tea will reduce the number of c. albicans colonies (table 2). discussion based on the result, it is known that the average number of c. albicans colonies adherence on the acrylic resin plates immersed in the black tea indicates that the higher concentration of the black tea stepping, 13.33%, makes the number of c. albicans colonies decreased. meanwhile, the lower the concentration is, the higher the number of c. albicans colonies significantly is. it is because the black tea steeping used as the immersion material of acrylic resin plates is considered as disinfectant by the content of fluoride, polyphenols, catechins and other oxidated derivates.7 polyphenols is actually soluable in water,13 thus, polyphenols contained in tea leaves can also be dissolved in water. nevertheless, the tea containing polyphenols is still influenced by the concentration of the solution. the more concentrated the solution is, the higher the number of polyphenols in the black tea steeping is. therefore, it becomes more effective to be used to decrease the number of c. albicans colonies adherence to the acrylic resin plates. besides that, polyphenols classified into phenol group widely used as a disinfectant that has a good antimicrobial activity. it is considered as the fast bacteriacide and fungucide. however, this microbial activity can be decreased due to the dilution since the effectiveness of disinfectant materials is influenced by concentration, duration and temperature.15 based on the mechanism of antimicrobial activity, phenols can kill vegetative cells of fungi and bacteria by conducting protein denaturation and by decreasing the surface tension, as a result, the permeability of those fungi and bacteria is increased.16 this mechanism actually involves the reaction to protein cells through restricting or killing process by damaging the colloidal system, as a consequence, the coagulation and precipitation of protein occur. this protein coagulation of microbial cell then causes both the disruption of metabolism and the changing of the permeability of membrane cells, and also decreases the surface tension which later causes the increasing of the permeability of membrane cells, the absorption of fluid, and the death of microba.17,18 finally, it can be concluded that the increasing of the concentration (13.33%) or the higher the concentration of black tea used to immerse the acrylic resin plates, the lower the number of c. albicans colonies attached to the plates. however, further researches about antimicrobial activity of black tea against other bacteria in the oral cavity are needed. references 1. combe ec. notes on dental material. 6th ed. edinburg: churchill livingstone; 1992. p. 189–97. 2. rianti d. efektivitas lama perendaman resin akrilik dalam ekstrak daun coleus amboinicus terhadap keberadaan candida albicans. maj ked gigi (dent j) 2003; 36(4): 129–33. 3. rianti d. daya antimikroba ekstrak coleus amboinicus. lour terhadap candida albicans pada resin akrilik. j kedokteran gigi indonesia 2003; edisi khusus: 845–51. 4. buddtz je. materials and methods for cleaning denture. j prosthet dent 1979; 42: 619–22. 5. devenport jc. the oral distribution of candida in denture stomatitis. brit dent j 1970; 129: 151–6. 6. nazarudin. perbandingan dan pengelolaan teh. jakarta: penebar swadaya; 1993. p. 7, 26, 34, 205–14. 7. krisnowati, rahardjo mb, sunariani j. laporan wisata kerja teh hitam produksi pt. perkebunan nusantara xii (perkebunan teh wonosari pabrik teh santoon lawang) fakultas kedotkeran gigi universitas airlangga, 1997. p. 7–10. 204 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 201–204 8. soekarno. upaya mengenal lebih dekat jenis teh hitam (black tea) dan menentukan khasiatnya. surabaya: pt perkebunan nusantara xii; 1998. p. 5–7. 9. evans rt, baker pj, coburn ra, genco rj. comparison of antiplaque agents using an in vintro assay refleching oral condition. j dent res 1977; 56: 559–66. 10. darwazeh amg, mac farlane tw, mccuish a, larney pj. mixed salivary glucose levels and candidal carriage in patient with diabetes mellitus. j oral pathol med 1991; 20: 280–3. 11. tamamoto m, hamada t, miyake t, suginka h. ability of enzyme to remove candida. j prosthet dent 1985; 53: 214–16. 12. rostiny. pengaruh proses kuring basis gigi tiruan terhadap kekasaran permukaan dan perlekatan streptococcus mutans dan candida albicans. tesis. surabaya: pascasarjana universitas airlangga; 1995. p. 113–5. 13. ganguly dk. pharmacoterapeutics of black tea. shanghai china: proc. int. tea quality human health; 1995. p. 59–61. 14. boedi s. aspek klinis dan penetapan diagnosis kandidiasis mulut. majalah ilmiah kedokteran gigi fakultas kedokteran gigi usakti 2001; 16(44): 86–95. 15. rianti d, yogyarti s. antimicrobial effects of coleus amboinicus. lour folium infusum towards candida albicans and streptococcus mutans. maj ked gigi (dent j). 2006; 39(1): 12–5. 16. rahardjo mb. perbedaan daya antibakteri allium sativum linn dan kaempferia galanga terhadap streptococcus mutans dan bermacammacam bakteri yang berasal dari saluran akar gigi gangraena pulpae. tesis. surabaya: universitas airlangga; 1993. p. 13. 17. melville ph, russel c. microbiology for dental student. 3rd ed. london: williem heinewmann medical book ltd; 1981. p. 155–76. 18. minagi s, miyage y, inagaki k, tsurn h, suginaka h. hydrophobic interaction in candida albicans and candida tropical adherence to various denture base resin materials. infect immune 1985; 47: 11–3. mkgs vol 44 no 2 april-juni 2011.indd 101 vol. 44. no. 2 june 2011 orthodontic treatment with skeletal anchorage system arya brahmanta1 and jusuf sjamsudin2 1department of orthodontics, faculty of dentistry university of hang tuah 2department of orthodontics, faculty of dentistry university of airlangga surabaya indonesia abstract background: correction of class i malocclusion with bimaxillary dental protrusion and unilateral free end right upper ridge in adult patient is one of difficult biomechanical case in orthodontics. due to this case that needs proper anchorage for upper incisor retraction with missing teeth in the right posterior segment. purpose: the aim of this study to find an effective therapy for correction of bimaxillary protrusion with unilateral free and ridge. case: a female patient, 36 year old complaining for the difficulty of lip closure due to severe bimaxillary protrusion with incompetence lip. case management: firstly correction of the maxillary and mandibular incisor proclination were done by extraction of the mandibular first premolar, the maxillary second premolar on left side and finally placement of miniplates implant in the zygomatic process on right side as an absolut anchorage. conclusion: skeletal anchorage system (sas) can be considered as an effective therapy for corection of bimaxillary protrusion with unilateral free end ridge. key words: skeletal anchorage system, bimaxillary protrusion, unilateral free end ridge abstrak latar belakang: koreksi dari maloklusi klas i dari penderita dewasa yang disertai protrusi bimaksiler dengan kehilangan gigi posterior pada regio kanan atas merupakan salah satu kasus sulit untuk dikerjakan terutama berhubungan dengan biomekanik pergerakan giginya dalam perawatan ortodonti. tujuan: tujuan dari penulisan artikel ini adalah untuk menemukan terapi yang efektif untuk perbaikan protrusi bimaksiler dan kehilangan gigi posterior pada satu sisi. kasus: seorang penderita wanita usia 35 tahun datang dengan keluhan utama kesulitan untuk menutup mulut oleh karena gigi rahang atas dan rahang bawahnya maju dan bibirnya tidak kompeten. tatalaksana kasus: koreksi pada gigi insisivus rahang atas dan insisivus rahang bawah yang protrusi dilakukan dengan melakukan pencabutan terlebih dahulu pada gigi premolar pertama dirahang bawah sisi kanan dan sisi kiri serta pencabutan pada gigi premolar kedua di rahang atas sisi kiri dan pemasangan miniplate implant di regio prosesus zigomatikus di sisi kanan sebagai penjangkar absolut. kesimpulan: sistem penjangkar absolut pada perawatan ortodonti merupakan pilihan terapi perawatan yang efektif pada kasus penderita dewasa dengan protrusi bimaksiler dan kehilangan gigi posterior pada regio kanan atas. kata kunci: sistem penjangkar absolute, protrusi bimaksiler, kehilangan gigi posterior satu sisi correspondence: arya brahmanta, c/o: bagian ortodonti, fakultas kedokteran gigi universitas hang tuah. jl. arif rahman hakim 150 surabaya, indonesia. email: arya.brahmanta@yahoo.com. telp. (031) 5912191. case report introduction orthodontics anchorage can also be explained by the third law of newton which stated, every action creates reaction and reaction is equal in size and opposite in direction. the anatomic unit, antagonizes the active force and is being called as anchorage in orthodontics.1 anchorage preparation is a very important in orthodontic treatment. the success of orthodontics treatment generally depend on the anchorage protocol, especially for particular case. to prepare good anchorage, the clinician must be realistic to predict the possibility of anchorage loss. anchorage loss 102 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 101–105 is a consequences from unstable construction and lack of patient cooperation as well. type of anchorage based on the type of tooth movements.1,2 anchorage is the greatest problems in orthodontics. orthodontists always faced with difficulties in trying to achieve maximal anchorage due to the orthodontic movements in response to orthodontic forces. therefore, to maximize the anchorage, patients need to use headgear as additional anchorage. reinforced anchorage with extraoral appliances has severe limitations because it requires excellent patient compliance.2,3 the use of implants in orthodontic involves tooth replacement or intraoral rigid anchorage assistance in the movement of teeth. recent developments in osseointegration made it possible to use implants for orthodontic anchorage. since the implant is known like an ankylosed tooth, it can be used as a reliable anchorage unit for orthodontic tooth movements. experimental biomechanic studies on animal models and clinical investigations showed that dental implants placed in the alveolar bone is resistant to orthodontic force.3 it is important to achieve maximal anchorage in correction of severe maxillary protrusion especially in cases with excesive molar anchorage loss on free end ridge. skeletal anchorage system (sas) was developed for correcting class ii malocclusions with maxillary protrusion. using this system, the anterior retraction can be done without unfavorable side effects.4 the purpose of this article is to deliver a case of an adult patient with severe bimaxillary protrusion, treated with sas. case a patient 36 year old woman, presented a bimaxillary dental protrusion with class i malocclusion and unilateral free end ridge on upper arch came to the orthodontic specialist clinic at airlangga university dental hospital. she complained complaining about the difficulty of lip closure due to severe bimaxillary dental protrusion. her facial profile was convex with a protrusive upper lip without facial asymmetry. over jet and over bite 2 mm (figure 1b). occlusal contact recognized only at the premolar and molar on the left side (figure 1-c). there is no occlusal contact at premolar and right molar. the upper left first molar, second molar and lower first molar were missing (figure 1-a, e). cephalometric analysis showed a skeletal class i jaw base relationship sna 88º; snb 84º; anb 4º. the facial profile was convex fh-np 88º; nap 10º; y-axis 62.5º; the upper and lower incisor were labially inclined i -na line 11.5 mm; i-na angle 35º; i-nb line 16 mm; i-nb angle 45º; interinsisal angle 96º. the mandibular plane angle was steep 30º and the gonial angle was large 110º (figure 1-d). case management the case was diagnosed as class i malocclusion with bimaxillary dental protrusion and unilateral free end right upper ridge, skeletal class i jaw base relationship. the treatment objectives were extraction of the bilateral figure 1. intra oral photographs: a) right side; b) front side; c) left side; d) cephalometric; and e) panoramic photographs before treatment. a b c d e 103brahmanta: orthodontic treatment with skeletal anchorage mandibular lower first premolar and the maxillary second premolar because it is in poor condition. placement of edgewise standart braket with 0.018 inch slot on upper and lower arch, followed by placement of miniplates implant in the zygomatic process as an absolut anchorage. correction of the maxillary and mandibular incisor by retraction using elastik chain. retention using hawley retainer in both jaws. before starting orthodontic treatment, the patient received periodontal treatment. periodontal treatment involved oral hygiene instruction and scaling. the upper left second molar was extracted because of poor condition. bilateral mandibular lower first premolar were extracted to gain space for retraction. initial phase was levelling with 0.012 inch round niti archwire. after leveling with a 0.016 inch niti arch wire, the miniplates implanted onto the zygomatic process of the maxilla through buccal mucosa (figure 2-a,b). the miniplates contoured to fit the bone surface. the head portion of miniplates intraorally exposed and positioned outside the dentition (figure 2-c). figure 2. the miniplates implant placement on to zygomatic processa) miniplates implant placement; b) miniplates implant; and c) suturing a b c figure 3. treatment progress a) right side; b) front side; and c) left side. a b c e figure 4. intra oral photographs: a) right side; b) front side; c) left side; d) cephalometric; and e) panoramic photographs after treatment. d a b c 104 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 101–105 after a month of healing, integration and adaptation retraction of the anterior teeth was started with elastic chain. an elastic chains was applied from the upper right premolar region to the miniplate as absolute anchorage for retraction with sliding mechanic (figure 3-a, b, c). this treatment made spacing in the upper dentition were closed. the upper incisor inclined palatally and the lower incisor lingually inclined. acceptable occlusion achieved and the overjet and overbite come to normal. the caninus relation were class i on the both sides (figure 4-a, b, c). facial photographs showed overall facial balance was improved. the lips becomes less tension on closure (figure 5-a, b, c, d). cephalometric analysis by comparing the superimposing pretreatment and posttreatment cephalometric tracings is indicated the type of retraction movement of the maxillary incisor by relative movement of the incisor crown and the incisor root (figure 4-d). cephalometric superimposing analysis showed a normal sna 86º; snb 84º; anb 2º. the facial profile was becoming straight fh-np 85º; nap10º; y-ax 62,5º; the upper and lower incisor have been corected: i -na line 7 mm; 21º; inb line 12 mm; 40º; inter incisal 114º. comparison the pretreatment and posttreatment cephalometric tracings showed that maxillary and mandibular incisor crown had moved posteriorly (figure 6). retention phase with hawley retainer provided acceptable occlusion. facial profile was also maintained, indicating a stable occlusion (figure 7). discusion facial esthetics is a major concern of many orthodontic patients. the negative impacts on the facial profile with upper lip protrusion often lead patients to seek orthodontic treament. increased upper lip procumbency is commonly associated with protrusive maxillary dentition in angle figure 5. facial photographs: a) right side; b) front side before treatment; c) right side; and d) front side after treatment. a b dc figure 6. superimposing cephalometric tracing. ----: pre treatment ----: progress ----: post treatment figure 7. retention phase: a) right side; b) front side; and c) left side. a b c 105brahmanta: orthodontic treatment with skeletal anchorage class ii division 1 malocclusions and class i malocclusions with bimaxillary protrusion.2 in such circumtances, the major orthodontic treatment goal is to reduce the proclination of the maxillary incisors. consequently, the treatment plan often includes extraction of the bilateral maxillary premolars, followed by retraction of the anterior teeth with maximum anchorage. maximum anchorage was added to prevent forward movement of the maxillary posterior teeth during anterior teeth retraction and can be provided with different approaches.5,6 extraoral headgear are commonly used to reinforce posterior anchorage during anterior tooth retraction or are directly applied to retract anterior teeth. full time headgear wearing is demanding for most patients, extraoral appliances are often rejected by adults for social reasons. patients cooperation is an important factor for the effectiveness of extraoral appliances.6,7 application of bony anchorage for tooth movements is effficient, because it is not depending on patient cooperation in wearing extraoral appliance. several methods of bony anchorage have been reported, such are: dental implants, titanium screws and miniplates. the use of miniplate implant for absolute anchorage has proved to have many advantages. absolute anchorage makes the treatment plan more reliable and enables treatment time reduction. this anchorage system obviates the dependency on patient compliance.5,6,8 skeletal anchorage developed from mini plate implant are placed by screwing to engage the cortical bone. the most common areas for mini implant placement are in the zygomatic area and the buccal aspect of the body of the mandible.8 in this case, orthodontic treatment was performed in adult patient diagnosed as angle class i malocclusion with bimaxillary dental protrusion and unilateral free end upper right posterior segment. the only option to correct the proclination of anterior teeth was to move the anterior teeth distally using absolute anchorage. therefore, sas offered the best benefit therapy choice. miniplates were placed in the zygomatic process in the maxilla. the titanium l–shaped (hook of miniplates) facilitated adjustment of the direction of force to retract the upper incisors.9,10 patients with bimaxillary dental protrusion have specific characteristics, including incisor proclination and convec facial profile. to correct dentoalveolar protrusion, extraction of the premolar is indicated. the treatment mechanic for space closure of the extraction sites was closed by sliding mechanics. the use of miniplates as skeletal anchorage system for patient with insufficient teeth for anchorage is almost 100% succesfull, if the right type of implant is used and the clinical situation is properly evaluated.10–12 retraction of the maxillary incisor can be assessed by comparing pre treatment and post treatment on cephalometric tracings. the tipping control assigned by moving maxillary incisor crown posteriorly with the center of rotation at the root of the tooth. uncontrolled tipping makes the maxillary central incisor crown move posteriorly although the roots move anteriorly. the mechanotherapy control was important for satisfactory correction of dentoalveolar protrusion, leading to a positive soft-tissue response, with lip protrusion reduction.11,12 the patient’s main complaints, in which difficulty of lip closure due to severe bimaxillary dental protrusion was improved by the treatment. since the proclination was corected, the upper lip became more relaxed and the lips showed less tension. it is concluded that sas can be considered as an effective therapy choice for correction bimaxillary dental protrusion with unilateral free end ridge. references 1. keles a, erverdi n. bodily molar distalization with absolute anchorage. angle orthod 2003; 73: 471–82. 2. nanda r. biomechanics and esthetic strategies in clinical orthodontics. st. louis: saunders elsevier; 2005. p. 295–309. 3. sugawara j. jco interviews, dr. junji sugawara on the skeletal anchorage system. j clin orthod 2000; 33: 689–96. 4. tanaka e, sasaki-nishi a, hasegawa t, nishio c, nobuhiko k, tanne k. skeletal anchorage for orthodontic correction of severe maxillary protrusion after previous orthodontic treatment. angle orthod 2008; 78: 181–8. 5. sugawara j, nagasaka h, umemori m, mitani h, kawamura h. skeletal anchorage system (sas). dental outlook 2002; 99: 397–406. 6. lee js. application of orthodontic mini–implants. hanover park, quintessence 2007; 7(179): 202–6. 7. fukunaga t, kuroda s, kurosaka h, yamamoto t. skeletal anchorage for orthodontic correction of maxillary protrusion with adult periodontitis. angle orthod 2006; 76: 148–55. 8. wahl n. orthodontics in 3 millennia. chapter 15: skeletal anchorage. american j orthod and dentofac orthop 2008; 134: 707–10. 9. bong kc, dong sc, jang i, brinkmann j, peter ngan. maxillary protraction using miniplates as skeletal anchorage. hong kong dent j 2010; 7: 87–93. 10. yao cj, hualai eh, chang c, chen i. comparison of treatment outcomes between skeletal anchorage and extraoral anchorage in adults with maxillary dentoalveolar protrusion. am j orthod dentofac orthop 2008; 134: 615–24. 11. labanauskaite b, jankauskas g, vasiliauskas a, haffar n. implants for orthodontic anchorage. meta–analysis. stomatologija baltic and maxillofacial j 2005; 7: 128–32. 12. lee k, leung c, wong k, rabie. versatility of skeletal anchorage in orthodontics. world j orthod 2007; 9: 221–32. vol 51 no 1 jan-mrt 2018.indd 4747 an in-vitro antimicrobial effect of 405 nm laser diode combined with chlorophylls of alfalfa (medicago sativa l.) on enterococcus faecalis suryani dyah astuti 1,2 1 biomedical engineering magister program, post graduate school, universitas airlangga 2 department of physics, faculty of science and technology, universitas airlangga surabaya indonesia abstract background: enterococcus faecalis (e. faecalis) is a bacterium commonly detected in the root canals of teeth with post-treatment apical periodontitis or advanced marginal periodontitis. it has the ability to live in an extreme environment and survive as an organism with its virulence factor possibly contributing to the pathogenesis of post-treatment apical and marginal periodontitis. photodynamic therapy (pdt) is an urgently required alternative method of improving therapy effectiveness. photodynamic therapy combined with conventional endodontic treatment decreases the number of antibioticresistant bacteria and biofilms. chlorophyll is one of the photosensitizers added to enhance the absorption of light in photodynamic therapy. purpose: the purpose of this study was to determine the antimicrobial effect of the combination of photodynamic laser therapy and alfalfa chlorophyll in e. faecalis. methods: in vitro study using e. faecalis distributed between negative control (c-) and positive control (c+), treatment groups using various energy doses of a 405 nm diode laser (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 j/cm2) with (g1) and without alfalfa chlorophyll as organic photosensitizer (g2). the suspension was inoculated on tryptocase soy agar (tsa) and incubated at 37° c for 24 hours. the number of colonyforming units per milliliter (cfu/ml) was determined. the results were analyzed by anova with p value ≤0.05. results: a 405 nm irradiating laser with or without a photosensitizer can decrease e. faecalis viability percentage through the administering of various energy doses. the highest decrease (42%) was obtained in the group without a photosensitizer using 20 j/cm2, while 10 j/cm2 in the group with a photosensitizer proved the most effective dose (25%). conclusion: the results of this study showed a decrease in the viability of e. faecalis exposed to a 405 nm (40 mw) laser. an irradiating process using a 405 nm laser without a photosensitizer (alfalfa chlorophyll) resulted in the highest percentage decrease (42%) in e. faecalis bacterial viability. keywords: antimicrobial photodynamic therapy; enterococcus faecalis; diode laser 405 nm; alfafa chlorophyll correspondence: suryani dyah astuti, department of physics, faculty of science and technology, universitas airlangga. jl. mulyorejo, mulyorejo, surabaya 60115, indonesia. e-mail: suryanidyah@fst.unair.ac.id research report introduction enterococcus faecalis (e. faecalis), a gram-positive bacterium commonly found in the root canal, is ovoid in shape with a diameter of between 0.5 and 1 μm. this bacterium is a facultative anaerobe and possesses the ability to survive in an extreme environment such as in a highly alkaline ph and high salt concentrated condition.1 e. faecalis is resistant to calcium hydroxide and antibiotics.2 tetracycline produce a poor antimicrobial effect on periodontal e. faecalis, with more than 50% of the e. faecalis periodontal isolates showing resistance. furthermore, e. faecalis isolates from root canals demonstrate a high prevalence of the genetic determinant of tetracycline resistance (tetm). recent studies have indicated that tetm genes were detected in approximately 50% of isolates from the root canal.1 dental journal (majalah kedokteran gigi) 2018 march; 51(1): 47–51 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p47–51 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51 48 astuti/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 47–51 e. faecalis demonstrate high frequency in cases of poor response to either endodontic or periodontal treatment.3 therefore, alternative methods are urgently needed to improve the therapy effectiveness, one such method being photodynamic therapy which is a medical treatment that utilizes light to activate photosensitizer agents. exposure of the photosensitizer to light produces a wide range of oxygen species and free radicals that cause localized damage and the death of bacteria.3 photodynamic therapy is also known as an antimicrobial photodynamic therapy (apdt) and photodynamic inactivation (pdi).4 photodynamic therapy uses light sources and lightsensitive photosensitizer agents. the suitability of the light spectrum and photosensitizer agent to photodynamic therapy will produce ros. light sources such as light emitting diodes (led) and a variety of lasers can be used to activate the photosensitizer agent.3 these lasers generally have complicated systems resulting in a high cost. currently, diode lasers are widely used because they offer the advantages of simple systems, portability and low cost.5 research results showed that photodynamic therapy combined with conventional endodontic treatment decreases the prevalence of bacteria and biofilms.6,7 certain photosensitizer agents have been tested for their ability to efficiently produce reactive oxygen species (ros) formed through photochemical type i or ii that inactivate microbial cells.8 antimicrobial photodynamic therapy success relies heavily on the type of photosensitizer, the light source wavelength and output power and the irradiation time used.9 the use of low power laser photodynamic therapy with different exogenous photosensitizers, toluidine blue (tbo),10,11 methylene blue (mb),12–14 and organic photosensitizer curcumin15 has been investigated. for these reasons, chlorophyll is more appropriate for development as a photodynamic therapy in cases of tumors and cancer.16 chlorophyll is a substantial bioorganic molecule with the core function of absorbing light and transferring excitation energy to the reaction center in photosynthetic devices. high absorption energy during the photosynthesis process is caused by a relatively long excitation process of chlorophyll (≤ 10-8 seconds). the longer the process of singlet excitation of chlorophyll, the greater the electronic energy converted. the conversion process may occur from a basic level to ones of triplet excitation. the excess energy produced by chlorophyll at the level of triplet excitation provides an opportunity to transfer energy to oxygen molecules, a process producing reactive singlet oxygen.17 in this study, chlorophyll was extracted from the leaves of alfalfa (medicago sativa l), a type of perennial leguminous plant with high chlorophyll content.18 other research showed that chlorophyll a and b, in addition to pheophytin are all powerful antioxidants that can reduce ros and prevent lipid peroxidation and dna damage.19 the purpose of this study was to determine the antimicrobial photodynamic effect of the photodynamic therapy laser diode in combination with alfalfa chlorophyll in e. faecalis bacteria. materials and methods the sample strains used in this research consisted of purely cultured bacteria from e. faecalis atcc 29212. the bacteria strains were grown in tryptic soy agar (oxoid, england, uk), subsequently inoculated in triptic soy broth (tsb) solution (merk, darmstadt, germany), incubated for 24 hours and, finally, diluted to a value of optical density of od600nm = 1.6/0.142, which is equal to ~ 10 3 cfu/ml. alfalfa chlorophyll (k-link liquid chlorophyll, jakarta selatan, indonesia) at a concentration of 1.6 mg/ml was diluted in normal saline. the absorption spectrum of chlorophyll was measured using a shimadzu uv-vis 1800 spectrometer (shimadzu, tokyo, japan). laser irradiation was carried out using sony diode lasers with output wavelengths of 405 nm. the power outputs were 40 mw with a focal spot ®xed: 0.3 cm2 this research was conducted using post-test only control group design. the samples were distributed into four groups: (1) e. faecalis exposed to neither a 405 nm diode laser nor alfalfa chlorophyll as the negative control (c-); (2) e. faecalis exposed only to 1.6 mg/ml alfalfa chlorophyll as the positive control (c+); (3) e. faecalis exposed to various energy doses of 405 nm diode laser (2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 17.5; 20.0) and 1.6 mg/ml alfalfa chlorophyll as group 1 (g1); (4) e. faecalis exposed to various energy doses of diode laser 405 nm (2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 17.5; 20.0 j/cm2) without alfalfa chlorophyll as group 2 (g2). after treatment, the suspension was inoculated on tsa and incubated at 37o c for 24 hours. the number of colony-forming units per milliliter was counted manually to determine the antimicrobial effect on e. faecalis. the percentage of decrease bacterial viability was defined as: (ʃ control colony – ʃ treatment colony) ×100% ʃ control colony a broth microdilution method was applied to determine the minimum inhibitory concentration (mic) of alfafa chlorophyll against e. faecalis. testing was conducted using a 96-well flat-bottomed microplate (nunc, sjælland, denmark). each well contained 90 μl tsb, 90 μl alfafa chlorophyll at various concentrations (0.8, 1.0, 1.4, 1.6 mg/ml), and 20 μl of e. faecalis culture at a concentration of 10 cfu/ml1. the microplates were incubated for two hours at 37° c under microaerophilic conditions. the mic was defined after two hours of incubation. all tests were repeated at least four times. the number of colony-forming units per milliliter (cfu/ml) was calculated manually to determine the mic test results which were log-transformed and analyzed with spss version 10.05 (spss inc., chicago, illinois, usa) using anova (p=0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p47–51 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51 49astuti/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 47–51 results the photosensitizer used in this study was alfalfa chlorophyll, the absorption spectrum of which20 is shown in figure 1.the mic test for alfalfa chlorophyll confirmed that the dye had no toxic effects on e. faecalis. statistical results showed that the chlorophyll concentrations (0.8, 1.0, 1.4, 1.6 mg/ml) did not significantly differ from each other at p=0.12 (p>0.05). figure 2 shows the viability of e. faecalis exposed to 405 nm diode laser at the same concentration of photosensitizer (chlorophylls). the 405 nm diode laser treatment group resulted in statistically significant increases and decreases of cfu p=0.00 (p<0.05) compared to the control group. the statistical test results showed the largest percentage of reduction in bacterial viability to be in the treatment of 405 nm 20.0 j/cm2 laser exposure to be 41.75%. figure 1. the absorption spectrum of chlorophyll alfalfa (medicago sativa l.) figure 2. the viability of e. faecalis exposed to diode laser 405 nm. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p47–51 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51 50 astuti/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 47–51 figure 3 shows the percentage (%) of decreasing bacterial viability at various irradiating laser energy doses with chlorophyll as a photosensitizer. the results in figure 3 indicate that the irradiating process using a 405 nm laser with and without chlorophyll can reduce the viability of e. faecalis bacteria. a 405 nm laser without a photosensitizer (alfalfa chlorophyll) produced the highest decrease in the e. faecalis bacterial viability percentage (42%) and that of the photosensitizer (25%). discussion antimicrobial photodynamic therapy requires a light source at a specific wavelength that activates the ps. this study used a laser diode at an output power wavelength of 405 nm with a range of 40 mw. for medical applications, the mechanism of photochemical interaction plays a significant role in photodynamic therapy. similarly with biostimulation, photochemical interactions occur at a very low power density (1 w/cm2) and an exposure time of one second.21 the results of temperature measurements in figure 3 showed that during irradiation the temperature stablized below 45o c, which remained in the optimum growth range of the e. faecalis bacterium. this suggested that bacterial death was not caused by an increase in temperature but, rather, was due to irradiation. the photosensitizer used in this study was alfalfa chlorophyll at a concentration of 1.6 mg/ml. the absorption spectrum of exogenous photosensitizer in figure 4 indicated effective absorption at a blue and red wavelength. based on the data, the quantum yield of laser diode with a wavelength of 405 nm could be calculated. the chlorophylls absorption stood at 92.97%.22 the absorption percentage of photosensitizer affects the production of ros. the concentration of photosensitizer and conformity of the light wavelength with the absorption spectrum of the photosensitizer leads to successful antimicrobial photodynamic therapy.23 this study used a diode laser with a 405 nm wavelength and the same focal spot. table 1 shows the energy doses and time duration of laser irradiation applied in this study. irradiation at different wavelengths produces a variety of quantum yields. the similarity of the wavelength of the light source with the photosensitizer absorption spectrum will produce a photophysical mechanism, i.e. the absorption of light energy. absorption of light energy will excite photosensitizer molecules that trigger the occurrence of photochemical reactions resulting in radical oxygen species. another deciding factor was the radiation energy dosage. the appropriate dose of energy activates a chemical reaction producing a wide range of reactive oxygen species that causes photoinactivation in bacteria. the energy dose of laser irradiation per total area of irradiation (power density, unit j/cm2) is the magnitude of radiation energy (power multiplied by the longer exposure time) divided by the total area of irradiation. this determines the time duration of the laser irradiation adjusted to the energy dose and quantum yield. the results of this study showed a decrease in the viability of e. faecalis that were exposed to a 405 nm laser with a power output of 40 mw. the irradiating process using a 405 nm laser at an energy dose of 20 j/cm2 without a photosensitizer (alfalfa chlorophyll) resulted in the highest decrease in the e. faecalis bacterial viability percentage (42%). an irradiating 405 nm 10 j/cm2 laser with a photosensitizer resulted in the highest percentage decrease (25%) in the bacterial viability of e. faecalis. singlet dioxygen is capable of causing permanent damage to various parts of the cells, including: the plasma, mitochondria, lysosomes, nuclear membranes, in addition to the modification of proteins. photodynamic therapy within various photosensitizer molecules is more effective in immobilizing gram-positive than gram-negative figure 3. the percentage of the e. faecalis decrease exposed to laser 405 nm. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p47–51 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51 51astuti/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 47–51 bacteria.24,25 differential susceptibility to photodynamic therapy arises because of contrasting cell wall structure of the respective groups. the outer wall (15–80 nm thick) of gram-positive bacteria consists of more than 100 layers of peptidoglycan-related lipotechoic and teichuronic acids. this layer is relatively porous allowing macromolecules with a molecular weight of 30,000–60,000 da to diffuse across the plasma membrane. therefore, a photosensitizer with a molecular weight of 1500-1800 da can be diffused. gram-negative bacterial cells consist of the cytoplasmic membrane and outer membrane separated by peptidoglycan-containing periplasm. moreover, the latter is of heterogeneous composition, including: proteins that function as porin, lipopolysaccharide and lipoprotein. the outer membrane forms an effective barrier to permeability. only hydrophilic compounds with a low molecular weight of 600-700 da can diffuse and limit the penetration of photosensitizer. type 1 photochemical reactions occur in gram (+), while type ii occur in gram (-). photoinactivation in bacteria consists of several processes: (a) photosensitizer translocation to the plasma inner membrane, (b) photoinactivation of the photosensitizer, (c) the generating of ros, (d) oxidative modification of the target, (e) a decrease in cell function and metabolism and (f) inhibition of cell growth and cell death.25 the results of this study showed a decrease in the viability of e. faecalis that had been exposed to a 405 nm (40 mw) laser. an irradiating process using a 405 nm laser without a photosensitizer (alfalfa chlorophyll) resulted in the highest percentage decrease (42%) in e. faecalis bacterial viability. the use of photosensitizer (alfalfa chlorophyll) decreases bacterial viability to 25%. acknowledgement the research reported here was funded by a 2016 grant from the ministry of research, technology, and higher education. references 1. cath ro p, mcca r thy p, hoffma n n p, zilm p. isolation a nd identification of enterococcus faecalis membrane proteins using membrane shaving, 1d sds/page, and mass spectrometry. febs open bio. 2016; 6(6): 586–93. 2. de andrade ferreira fb, silva e souza p de ar, do vale ms, de moraes ig, granjeiro jm. evaluation of ph levels and calcium ion release in various calcium hydroxide endodontic dressings. oral surg oral med oral pathol oral radiol endod. 2004; 97(3): 388–92. 3. konopka k, goslinski t. photodynamic therapy in dentistry. j dent res. 2007; 86(8): 694–707. 4. grossweiner li, jones lr, grossweiner jb, rogers bhg. the science of phototherapy: an introduction. dordrecht: springer; 2005. p. 374. 5. kübler a, niziol c, sidhu m, dünne a, werner j. eine kosteneffektivitäts-analyse der photodynamischen therapie mit foscan® (foscan®-pdt) im vergleich zu einer palliativen chemotherapie bei patienten mit fortgeschrittenen kopf-halstumoren in deutschland. laryngo-rhino-otologie. 2005; 84(10): 725–32. 6. garcez as, ribeiro ms, tegos gp, núñez sc, jorge aoc, hamblin mr. antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. lasers surg med. 2007; 39: 59–66. 7. soukos ns, goodson jm. photodynamic therapy in the control of oral biofilms. periodontol 2000. 2011; 55: 143–66. 8. sperandio ff, huang y-y, hamblin mr. antimicrobial photodynamic therapy to kill gram-negative bacteria. recent pat antiinfect drug discov. 2013; 8(2): 108–20. 9. chiniforush n, pourhajibagher m, shahabi s, kosarieh e, bahador a. can antimicrobial photodynamic therapy (apdt) enhance the endodontic treatment? j lasers med sci. 2016; 7(2): 76–85. 10. gergova rt, gueorgieva t, dencheva-garova ms, krasteva-panova az, kalchinov v, mitov i, kamenoff j. antimicrobial activity of different disinfection methods against biofilms in root canals. j investig clin dent. 2016; 7(3): 254–62. 11. pinheiro sl, silva jn da, gonçalves ro, villalpando kt. manual and rotary instrumentation ability to reduce enterococcus faecalis associated with photodynamic therapy in deciduous molars. braz dent j. 2014; 25(6): 502–7. 12. de oliveira bp, aguiar cm, câmara ac, de albuquerque mm, correia acr de b, soares mf de lr. the efficacy of photodynamic therapy and sodium hypochlorite in root canal disinfection by a single-file instrumentation technique. photodiagnosis photodyn ther. 2015; 12(3): 436–43. 13. silva ej, coutinho-filho wp, andrade ao, herrera dr, coutinhofilho ts, krebs rl. evaluation of photodynamic therapy using a diode laser and different photosensitizers against enterococcus faecalis. acta odontol latinoam. 2014; 27(2): 63–5. 14. miranda rg, santos eb, souto rm, gusman h, colombo ap v. ex vivo antimicrobial efficacy of the endovac system plus photodynamic therapy associated with calcium hydroxide against intracanal enterococcus faecalis. int endod j. 2013; 46(6): 499– 505. 15. da frota mf, guerreiro-tanomaru jm, tanomaru-filho m, bagnato vs, espir cg, berbert flcv. photodynamic therapy in root canals contaminated with enterococcus faecalis using curcumin as photosensitizer. lasers med sci. 2015; 30(7): 1867–72. 16. derosa mc, crutchley rj. photosensitized singlet oxygen and its applications. coord chem rev. 2002; 233–234: 351–71. 17. limantara l, heriyanto h. photostability of bacteriochlorophyll a and its derivatives as potential sensitizers for photodynamic cancer therapy: the study on acetone-water and methanol-water solvents. indones j chem. 2011; 11(2): 154–62. 18. radovic j, sokolovic d, markovic j. alfalfa-most important perennial forage legume in animal husbandry. biotechnol anim husb. 2009; 25(5–6): 465–75. 19. hsu c-y, chao p-y, hu s-p, yang c-m. the antioxidant and free radical scavenging activities of chlorophylls and pheophytins. food nutr sci. 2013; 4(8a): 1–8. 20. hamblin mr, hasan t. photodynamic therapy: a new antimicrobial approach to infectious disease? photochem photobiol sci. 2004; 3(5): 436–50. 21. hamblin mr, huang y-y. handbook of photomedicine. boca raton: crc press; 2013. p. 854. 22. astuti sd, zaidan a, setiawati em, suhariningsih. chlorophyll mediated photodynamic inactivation of blue laser on streptococcus mutans. in: aip conference proceedings. surabaya: aip publishing llc; 2016. p. 120001. 23. nagata jy, hioka n, kimura e, batistela vr, terada rss, graciano ax, baesso ml, hayacibara mf. antibacterial photodynamic therapy for dental caries: evaluation of the photosensitizers used and light source properties. photodiagnosis photodyn ther. 2012; 9(2): 122–31. 24. kharkwal gb, sharma sk, huang y-y, dai t, hamblin mr. photodynamic therapy for infections: clinical applications. lasers surg med. 2011; 43(7): 755–67. 25. jori g, fabris c, soncin m, ferro s, coppellotti o, dei d, fantetti l, chiti g, roncucci g. photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. lasers surg med. 2006; 38(5): 468–81. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p47–51 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p47-51 mkgs vol 44 no 2 april-juni 2011.indd 59 vol. 44. no. 2 june 2011 research report the effectivity of toothpick tooth brushing method on plaque control chiquita prahasanti1, iwan ruhadi1, and agus sobar mulyana2 1 department of periodontics 2 dental practicion faculty of dentistry, airlangga university surabaya indonesia abstract background: periodontal diseases are associated with bacteria species which present in biofilms that colonize on dental surfaces. several tooth brushing methods had been known and proved to be effective in maintaining oral hygiene. among them, tooth pick technique was a relatively new method and its superiority in removing interproximal plaque was better than other methods. purpose: the purpose of this study was to examine the effectivity of toothpick tooth brushing method to conventional method on periodontal health. methods: this research was designed as an analytical observational study. thirty samples selected from five hundred and twelve males indonesian air-force members in malang, aged 18–40 yrs, with periodontal pockets (≤ 5 mm) in upper or lower teeth, without crowding, gingival index minimal > 1 (moderate gingivitis), ohi-s score minimal ≥ 1.3 (moderate), without systemic diseases, do not undergone medical therapy/drug prescriptions, without using mouth rinse during study, and without prosthesis. there were thirty samples in this research and devided to two groups, fifteen samples easch. the groups were toothpick tooth brusing method and conventional method (control group). in this study oral hygiene index simplified (ohi-s), gingival index (gi), bleeding on probing (bop) and pocket depth were examined. results: there were significant differences (p = .001) in ohi-s, gi, bop, and pd before and after conducting each toothbrushing method, as well as differences between means (quarrel means), that were p = .003; p = .001; p = .001 and p = .001 consecutively. conclusion: toothpick brushing method was more effective in plaque control compared to conventional method. key words: effectiveness, toothpick tooth brushing method, periodontal health abstrak latar belakang: penyakit periodontal berhubungan dengan bakteri yang berkoloni dalam biofilm yang terdapat di permukaan gigi. saat ini telah dikenal berbagai macam metode menyikat gigi tetapi masih belum ada penelitian tentang efek metode tersebut terhadap ohi-s. penelitian in ingin menunjukkan efek menyikat gigi dengan metode toothpick terhadap kesehatan jaringan periodontal. tujuan: tujuan dan penelitian ini adalah untuk mengetahui efektivitas metode toothpick dibandingkan metode konvensional dalam kontrol plak. metode: jenis penelitian ini adalah analitik observasional. sampel sejumlah tiga puluh orang diseleksi dari 512 anggota tni angkatan udara skadron pasukan khas 464 wing ii lanud abdulrahman saleh malang, usia 18–40 tahun, dengan kedalaman poket periodontal (≤ 5 mm) pada gigi rahang atas/bawah dengan, susunan gigi yang tidak berdesakan, memiliki skor gingival indeks minimal > 1 (gingivitis sedang), memiliki skor ohi-s minimal ≥ 1,3 (sedang), tanpa ada kelainan sistemik, tidak dalam perawatan dokter/mengkonsumsi obat-obatan, tidak menggunakan obat kumur selama penelitian, tidak menggunakan protesa. sampel dibagi menjadi dua kelompok, lima belas orang menggunakan metode toothpick sedangkan lima belas orang menggunakan metode konvensional sebagai kelompok kontrol. hasil: terdapat perbedaan yang bermakna (p = .001) pada ohi-s, gi, bop dan kedalaman poket sebelum dan sesudah menyikat gigi dengan masing-masing metode menyikat gigi yang ditetapkan. selain itu, terdapat perbedaan bermakna 60 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 59–62 introduction since the beginning of modern dentistry, emphasis has been placed on the importance of oral hygiene. toothbrushing is the most common form of oral hygiene habit practiced by people in developed countries. indeed, mechanical oral hygiene procedures are thought to be essential for proper plaque control and maintenance of periodontal heath. in fact, the single most continuous, cornerstone of preventive and public health dentistry has always been the cleaning of teeth. the presence of food remnants, dental plaque and calculus in the oral cavity showed the degree of oral hygiene. effective oral hygiene performed regularly by the patient disrupts the plaque biofilm on the tooth surface and is considered an important factor in helping to reduce the incidence of periodontal disease. poor oral hygiene has been shown to increase the occurence and progression of periodontal disease. the simultaneous presence of plaque and poor oral hygiene practices can cause the initiation of periodontal inflammation/gingivitis. these factors alone were able to elicit gingival inflammation which will progress to periodontal diseases. relationship between oral hygiene and periodontal disease severity was existed, therefore, optimal oral hygiene is needed to prevent and cure periodontal disease as well as maintaining tissue health.1,2 inflammation is now known to play a critical role in diseases that are not usually classified as inflammatory diseases, such as cardiovascular and alzheimer’s diseases. periodontal diseases are associated with bacteria species which present in biofilms that colonize on dental surfaces. several tooth brushing methods had been renowned and proved to be effective in maintaining oral hygiene.3,4 among them, tooth pick technique was a relatively new method and its superiority in removing interproximal plaque is better than other methods. interproximal area cleaning is important since it is the location where periodontal pathogenesis initializes. it also stimulates the secretion of immunoglobulin a which functions as mucosal defense mechanism. tooth pick tooth brushing method was done by placing the tip of toothbrush bristles on the gingival edge facing tooth crown and forms 30° angle with tooth long axis. these bristles are pushed into the interdental space and pulled out with, the same action of tooth pick movement; applied in the buccal and lingual side. this forward and backward movement was repeated 8-9 times every region.5,6 this removal method of interdental plaque could be done without dental floss or interdental brush. a small amount of tooth brush bristles were able to enter the narrow interdental space, and one or two tufts could enter the wide interdental space.7 since this method was less known by indonesian people, therefore we were obliged to conduct a research regarding the use and ability of toothpick toothbrushing method in reducing pocket depth, gingival index and increasing oral hygiene index. materials and methods the population was members of indonesian air force special squadron troop 464 wing ii abdulrachman saleh airport. sample criteria were as follows: male 18–40 years; periodontal pocket (< 5 mm) in upper and lower jaw; uncrowded teeth; gingival score index minimal >1 (moderate gingivitis); ohi-s score minimal >1.3 (moderate); without the presence of systemic disease; did not undergone medical therapy; did not use oral rinse during research; and did not use prosthesis. examination was done to all population in 10 days, and there were 30 members selected. the selected subjects were divided into 2 groups, toothpick tootbrusing methods and convensional methods (control group), 15 samples each. in toothpick method group, they were instructed how to conduct toothpick method one week before, and were evaluated in the third day in the same week. one week after pre-experimental period, in the toothpick method group pocket depth, gingival index, bop and ohi-s were measured and the location was recorded. in ohi-s examination, scoring was recorded in a form. afterward, subjects were reeducated about toothpock toothbrushing method until they understand and were able to demonstrate this technique. operator also show directly in each subject how to toothbrush with this method. subsequently subjects were instructed to brush their teeth using toothpick method at home for 2 weeks with the same toothbrush and toothpaste brands and toothbrush frequency was twice daily (in the morning and at night); tooth paste length was ±1 cm and leaflet about toothpick toothbrushing method instruction. evaluations were done in day 3, 6, 9 and 12 in order to was given see the ability of subjects to apply this method. two weeks later pocket depth (in the same side of the tooth), gingival index (gi), bop and ohi-s were measured. in day 12, measurement of pocket depth was conducted towards teeth in the same side, gingival index, bop and ohi-s. in order to achieve good examination reliability and antara selisih rerata sebelum dan sesudah menyikat gigi (p = .003; p = .001; p = .001 and p = .001) antara kedua kelompok tersebut. kesimpulan: metode toothpick brushing lebih efektif uuntuk kontrol plak dibanding dengan kelompok kontrol. kata kunci: efektifitas, metode menyikat gigi toothpick, kesehatan periodontal correspondence: chiquita prahasanti drg, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: chiquita_prahasanti@yahoo.com 61prahasanti, et al.: the effectivity of toothpick tooth brushing validity, examination was done by a periodontist and each group were divided into 3 subgroups which consisted of 5 samples. this group division method was done for treatment groups as well as control group, and examination of each subgroups was done separatedly by one day. in control group as done another day, measurements were done directly, then subjects were undergone scaling and root planing. they were also instructed to brush their teeth twice/day using the same tootbrush and toothpaste (tooth paste length ±1 cm) with the toothpick group. after 2 weeks, subjects were scheduled for evaluation. both groups evaluations were recorded. statistical data analysis was done with wilcoxon signed rank test; paired t-test and mcnemar test. results table 1. means and difference significancy of ohi-s, gi, bop and pocket depth (pd) before and after tooth pick method (treatment group) toothpick mean ± sd significancy before after ohi-s gi pd (mm) bop 3.14 ± .44 2.13 ± .44 4.50 ± 2.88 .91 ± .29 1.17 ± .52 .88 ± .25 2.76 ± .58 .11 ± .31 p = .001 p = .001 p = .001 p = .001 table 2. means and difference significancy of ohi-s, gi, bop and pocket depth (pd) before and after common method (control group) control mean ± sd significancy before after ohi-s gi pd(mm) bop 3.15 ± .47 2.40 ± .26 4.38 ± .23 .95 ± .23 2.10 ± .75 1.55 ± .40 3.93 ± .33 .73 ± .44 p = .001 p = .001 p = .001 p = .001 table 3. difference between means and significancy of pre and post treatment in treatment and control groups quarrel mean before and after from 2 weeks significancy (2-tailed) toothpick control ohi-s gi pd (mm) bop 1.97 1.25 1.74 .80 1.05 .85 .45 0,22 .03 .01 .01 .01 the result of this study revealed that greene-vermillion ohi-s status and gi in toothpick group before and 2 weeks after toothpick toothbrushing method (treatment) had the p = 0.001 (p < 0.05), thus had significant difference (table 1). the control group had the same result (table 2). bop and pd before and 2 weeks after in toothpick and control groups showed significant difference p = 0.001 (p < 0.05). statistical tests results regarding the difference between means before and after using toothpick (treatment group) compared to control group revealed significant difference in all parameters measured (ohi-s, bop, pd and gi). discussion periodontal status may influence the performance of oral hygiene and the response of tissues to brushing. indeed the importance of plaque control and its effectiveness at reducing inflammation. it is appreciated that the toothbrush alone is capable of removing up to 1 mm of subgingival plaque, but is ineffective in the interproximal region so toothpick method is effective in the interproximal.8 mechanical stimulation by toothbrushing promotes healing of gingivitis through accelerating cel proliferation, junctional epithelium proliferates at periodontal pocket formation. proliferation of basal cell and fibroblast play a major role in maintaining healthy periodontal tissue. fibroblast as the most predominant cell in connective tissue are engaged in production of collagen fibers.9 before and after study in toothpick method group revealed significant difference which meant that toothpick toothbrushing method was able to increase 0hi-s, decrease gi and bop; the same result occured with control group (p = 0.001; p < 0.05). increase of ohi-s index accompanied with increase of gingival index, bop and pocket depth from toothpick method group was caused by the improvement of dental plaque control pattern with toothpick toothbrushing method. with toothpick toothbrushing method, stimulation proliferation basal cells would reduce gingival bleeding in gingivitis and periodontitis.9 other possibility was the healing effect from scaling therapy. previous studies revealed the decrease of pd after initial treatment the change of pd condition was influenced by several factors which interrelated to each other. result analysis showed relations among variables which account for the desease of pd, such as periodontal condition, periodontal disease development and the width of attached gingival.10,11 generally, the result of initial treatment was not only affect pd decrease, but also improves other indexes such as gi and bop. the result of both groups showed significant difference between two weeks before and after treatment. it meant that both groups had the ability to improve ohi-s, gi, bop and pd. nevertheless, if considering the difference of means in ohi-s, gi, bop and pd before and after treatment, in toothpick group had greater value compared to control group (p < 0.05) (table 3). toothbrushing promotes the proliferation activity of gingival cells adjacent to the cementum, gingival and the alveolar bone9. proliferation of fibroblasts were consistent with stress distribution at toothbrushing, but no effects on proliferation of periodontal ligament. 62 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 59–62 therefore, it could be confirmed that toothpick toothbrushing method allows dental plaque control superior to common method. a study in japan compared two toothbrushing method, the toothpick and bass methods in supragingival plaque removal showed that toothpick method superiorly remove more plaque than bass method.5,6 a 6 months research that was conducted in a japanese community with toothpick technique revealed that in the 1st and 2nd months observation showed decrease of gi, bop, and cpitn.12,13 tooth brushing is simple and effective ways to reduce plaque and gingivitis and thus maintain periodontal health. ohrn and jonsson11 reported a relationship between periodontal disease and oral health. toothbrushing contributes to the repair of gingival inflammation and pocket formation.the effects of mechanical stimulation by toothbrushing can reduce gingival bleeding.9 duration and strength of mechanical stimulation were resulted in the activation and proliferation of junctional epithelium basal cell, collagen and gingival cells synthesis, increase of gingival oxygen saturation and stimulates iga secretion in mucous membrane as humoral immunity towards antigens.14,15 immunoglobulin a is an immunoglobulin class that secreted by gingival crevicular fluid (gcf) and acts as oral mucosal immunity. gingivitis and periodontitis severity should increase the volume and composition of gcf. stimulation by tooth brushing enhances the secretion of gcf which carries important components such as secretory iga.16 neutrophils (pmn) are within the frst line of host defense, and by their ability to phagocytize microbes, they can protect the host from infection. they can also give rise to pmn dependent vascular injury and contribute to increased vascular permeability, edema and further release of chemoattractants.9 in the periodontal tissue, immune activity works by preventing the penetration of bacteria and its products into the gingival tissue. tooth brushing has various effect towards periodontal tissue, for example triggering oral epithelium keratinization, enhance gingival capillary circulation, fibroblast proliferation and decreasing inflammatory cells.9,15 host immune factors also plays an important role in tissue healing response; therefore, periodontal tissue healing is affected by various factors which interrelates to each other. the inconsistent method and time caused the accumulation of plaque which leads to periodontal disease such as gingivitis and periodontitis. poor oral hygiene is one prime local factor of periodontal disease other than involved factors such as systemic disorders.1,3 good oral hygiene has long been associated with better periodontal heath than poor oral hygiene. in order to maintain good oral health to prevent periodontal disease, twice a day toothbrushing frequency is recommended internationally.17 gingivitis diminution could be gained by mechanical plaque removal by constant toothbrushing with the right method, in other words, good gingival condition influence to oral health status. the changes of ohi satus, gi, bop and pd were regarded better in toothpick method group after treatment compared to control. it was concluded that toothpick method is more effective in plaque control compared to common method. further comprehensive research should be done to evaluate the effectivity of this method towards oral health, especially periodontal disease. references 1. s g a n c o h e n h d . o r a l h y g i e n e : p a s t h i s t o r y a n d f u t u r e recommendations. int j dent hygiene 2005; 3: 54–8. 2. macdonald e, north a, maggio b, sufi f, mason s, moore c, addy m, west nx. clinical study investigating abrasive effects of three toothpastes and water in an situ model. journal of dentistry 2010; 38: 509–16. 3. van dyke te. inflammation and periodontal diseases: a reappraisal. j periodontol 2008; 79(80): 1501–2. 4. uzel ng, teles fr, teles rp, song xq, torresyap g, socransky ss, haffajee ad. microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease. j clin periodontol 2011; 38: 612–20. 5. watanabe t, morita m, nishi k. comparison of 2 tooth brushing methods for efficacy in supragingival plaque removal the toothpick method and the bass method. j clin periodontol 1998; 25: 829–31. 6. watanabe t. the nippon dental review. 64(1): 735. available from: url http://www.hyoron.co.jp. accessed november 20, 2009. 7. horiuchi m, yamamoto t, ishikawa a. tooth brushing promotes gingival fibroblast proliferative more effectively than removal plaque. j. clin periodontol 2004; 29: 791–5. 8. claydon nc. curent concepts in toothbrushing and interdental cleaning. periodontology 2000, 2008; 48: 10–22. 9. tomofuji t, sakamoto t, ekumi d, yamamoto t, watanabe t. location of proliferating gingival cell following toothbrush stimulation. oral diseases 2007; 13: 77–81. 10. van dyke te, serhan cn. resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases. j dent res 2003; 82(2): 82–90. 11. ohrn k, jonsson b. a comparison of two questionnaires measuring oral health-related quality of life before and after dental hygiene treatment in patients with periodontal disease. int j dent hygiene 2011; 1: 1–6. 12. morita m, sakamoto. preventive medicine in japan. toothpick tooth brushing. available from: http://www.pmjp7.co.jp. accessed january 7, 2006. 13. ishikawa a, kimura t, tomozane t. effect of repeated tooth brushing instructions on periodontal health in a community. available at: uids=8534. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=re trieve&db=pubmed&list. accessed december 21, 2009. 14. herrera d, roldan s, gonzalea i. the periodontal disease. clinical and microbiological findings. j clin periodontol 2000; 27: 387-94. 15. jansson. h, bratthall g, soderhalm g. clinical outcome observed in subjects with recurrent periodontal disease. j periodontol 2003; 74: 372–7. 16. yamamoto t, tomofuji t, ekuni d. effects of tooth brushing frequency on proliferation of gingival cells and collagen synthesis. j clin periodontol. 2004; 31: 40–4. 17. maes l, vereecken c, vanobbergen j, honkala s. tooth brushing and social characteristics of families in 32 countries. int dent j 2006; 56(3): 159–67. 8686 effects of moringa oleifera leaf extract combined with dfbbx on type-1 collagen expressed by osteoblasts in the tooth extraction sockets of cavia cobaya soekobagiono, sherman salim, hanoem eka hidayati, and karina mundiratri department of prosthodontics faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: tooth extraction is a common procedure in dentistry after which the residual ridge will no longer receive stimulus leading to volume, height and width loss. these anatomical changes can then result in difficulties with future denture fabrication and implant placement. preservation of the alveolar ridge, therefore, assumes considerable importance after tooth extraction. moringa oleifera, on the other hand, can enhance bone formation. type-1 collagen is a marker of osteoblast formation. purpose: this research aimed to analyze the effects of moringa oleifera leaf extract combined with dfbbx on type-1 collagen expressions in tooth extraction sockets. methods: 56 cavia cobaya subjects were divided into eight groups. their lower left incisors were then extracted prior to the sockets of the first and fifth groups being filled with peg, those of the second and sixth groups with dfbbx, those of the third and seventh groups with moringa oleifera leaf extract and a combination of dfbbx and moringa oleifera leaf extract in those of the fourth and eighth groups. the sockets were then examined on days 7 and 30 by means of an immunohistochemical technique. the data collected was subsequently subjected to analysis by one way anova and tukey hsd tests. results: there were significant differences between the control group and the treatment group administrated with moringa oleifera leaf extract combined with dfbbx. on days 7 and 30, the groups treated with the combination of dfbbx and moringa oleifera leaf extract had the highest number of type-1 collagen expressions. conclusion: a combination of dfbbx and moringa oleifera leaf extract is effective in increasing type-1 collagen expressions in tooth extraction sockets. keywords: alveolar bone; dfbbx; moringa oleifera leaf extract; type-1 collagen; socket preservation. correspondence: soekobagiono, department of prosthodontics, faculty of dental medicine, university airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: soekobagiono@fkg.unair.ac.id dental journal (majalah kedokteran gigi) 2018 june; 51(2): 86–90 research report introduction tooth extraction, one of the most common procedures performed in dentistry, can unfortunately cause alveolar ridge resorption. the largest bone loss usually occurs in the horizontal dimension, especially on the facial side of the ridge and the buccal side of the vertical dimension. postextraction alveolar bone resorption is, thus, an unavoidable physiological process.1 in the manufacture of dentures and dental implants, bone resorption is one of the complications potentially impeding the success of the therapy.2 as a result, it is important to maintain the height of the alveolar ridge after the tooth extraction process has been completed. in order to maintain the alveolar ridge post-extraction, graft material with or without a membrane is usually applied.3 graft material is a natural or synthetic material that can be used to repair defects.4 xenograft is a graft material transferred from one species to another. one of the most commonly used xenograft materials is demineralized freeze bovine bone xenograft (dfbbx). derived from cow bone whose particles are of a specific size. the removal of minerals contained in the bone was effected by soaking it in an acidic solution.5 more recently, however, treatments have been developed and improved by the use of drugs derived from plants or herbs whose medicinal properties have been doi: 10.20473/j.djmkg.v51.i2.p86–90 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p86-90 87 soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 86–90 known for many years. one such medicinal herb is moringa oliefera which can be used to accelerate the process of bone formation and prevent that of bone resorption.6 a study conducted by chirag patel argues that flavonoid compounds contained in moringa oliefera leaf extract can generate alkaline phosphatase (alp) and hydroxyproline when applied to saos-2 cell line culture.7 another piece of research indicated that moringa oliefera leaf extract is capable of increasing cell proliferation and survival, as well as cell migration of human dermal fibroblasts (hdf).8 moreover, research conducted by pudianto reveals that a combination of moringa oliefera leaf and demineralized freeze bovine bone xenograft (dfbbx) at an effective dose of 2% can generate osteoblasts, leading to the acceleration of alveolar bone formation after tooth extraction in cavia cobaya subjects.9 similarly, research conducted by wirawan also found that a combination of moringa oliefera leaf extract and dfbbx can significantly increase the number of osteoblasts in their tooth extraction sockets.10 during the bone formation process, many markers can be observed, one of which is type 1 collagen expression. type 1 collagen is the most dominant extracellular matrix protein present in bone and plays an important role in the bone formation process.11 nevertheless, the effects of moringa oliefera leaf extract combined with dfbbx on type-1 collagen expressions are still unclear. as a result, this research aimed to analyze the effects of moringa oleifera leaf extract combined with dfbbx on type-1 collagen expressions in post-extraction sockets by observing osteoblast cells. materials and methods this research was a laboratory experiment involving a sample size of 56 cavia cobaya subjects that filled certain inclusion criteria, namely: male, body weight of 300-350 grams, aged 3-3.5 months, healthy and active, having a normal appetite, free of limb injuries or skin complaints and able to run freely. ethical approval was granted by the ethics commission, faculty of dental medicine, universitas airlangga with no. 0009/hreccfodm/i/2017. the 56 cavia cobaya subjects were subsequently divided into eight groups, each of which contained seven members. their left mandibular incisor was then extracted. following extraction of their incisors, the resulting sockets of groups i and v as the control groups were filled with polyethylene glycol (peg). the animals in group i were then terminated on day 7, while those in group v were terminated on day 30. meanwhile, the empty sockets in groups ii and vi were filled with moringa oliefera leaf extract and peg. the subjects in group ii were executed on day 7, while those in group vi were executed on day 30. furthermore, the tooth extraction sockets in groups iii and vii were filled with dfbbx and peg. the animals in group iii were terminated on day 7, with those in group vii being terminated on day 30. meanwhile, the tooth extraction sockets of groups iv and viii were filled with a combination of moringa oliefera leaf extract, dfbbx and peg. the members of group iv were executed on day 7, while those in group viii were executed on day 30. all of these animals were executed using a cervical dislocation method. their tooth extraction sockets were then subjected to immunohistochemical analysis. immunohistochemical staining was conducted using polyclonal anti-collagen type i antibody (d-bio system usa®) with the number of type 1 collagen expressions being calculated by observing osteoblasts with a light microscope at a magnification of 1000x. the data collected was subsequently subjected to a normality test (a saphirowilk statistical test). a one-way anova was carried out to calculate the difference in the number of type 1 collagen expressions between the groups. results figures 1 and 2 show type-1 collagen expressed by osteoblasts in the tooth extraction sockets on days 7 and 30, subjected to immunohistochemical examination by light 2 figure 2. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 30. a: group i; b: group ii; c: group iii; d: group iv figure 3. the diagram of type-1 collagen expressions on days 7 and 30. a b c d figures 2. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 30. a: group i; b: group ii; c: group iii; d: group iv 1 table 1. the mean and and standard deviation values of type-1 collagen group n mean± standard deviation group i 7 3.00 ± 0.816a group ii 7 11.42 ± 1.511b group iii 7 15.14 ± 1.672c group iv 7 15.85 ± 1.772c group v 7 10.57 ± 1.902de group vi 7 13.28 ± 1.380d group vii 7 15.71 ± 2.690e group viii 7 19.57 ± 1.902f note: different superscripts showed a statistically significant difference (p<0.05) figure 1. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 7. a: group i; b: group ii; c: group iii; d: group iv. a b c d figures 1. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 7. a: group i; b: group ii; c: group iii; d: group iv. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p86-90 88soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 86–90 table 1. the mean and and standard deviation values of type-1 collagen group n mean ± standard deviation group i 7 3.00 ± 0.816a group ii 7 11.42 ± 1.511b group iii 7 15.14 ± 1.672c group iv 7 15.85 ± 1.772c group v 7 10.57 ± 1.902dew group vi 7 13.28 ± 1.380d group vii 7 15.71 ± 2.690e group viii 7 19.57 ± 1.902f note: different superscripts showed a statistically significant difference (p<0.05) microscope at a magnification of 1000x. table 1 contains the mean number of osteoblast cells expressing type 1 collagen in the tooth extraction sockets in each treatment group. on day 7, the highest number of type 1 collagen expressions was found in group iv, while the lowest was in group i. meanwhile, on day 30, the highest number of type 1 collagen expressions was found in group viii, with the lowest in group v. the results of the normality test in the form of a saphiro-wilk test showed that the data on days 7 and 30 was normally distributed with respective p values of 0.383 and 0.340 (p>0.05). the results of the levene's test showed that the data on the 7th and 30th days were homogeneous with p values of 0.056 and 0.089 (p>0.05). the results of the oneway anova test conducted on days 7 and 30 indicated that there were significant differences in the treatment groups with p values of 0.000 and 0.000 (p<0.05). the results of the tukey hsd test carried out also indicated that there were significant differences between the treatment groups as illustrated in table 1. as figure 3 shows, the highest level of type 1 collagen expression was found in group viii, while the lowest occurred in group i. the number of type 1 collagen expressions in the tooth extraction sockets on day 7 also showed statistically significant differences between the treatment groups, except between group iii and group iv. similarly, the number of type 1 collagen expressions in the tooth extraction sockets on day 30 indicated statistically significant differences between the treatment groups, except between group v and group vi and between group v and group vii. the number of type 1 collagen expressions on day 30 was higher than that on day 7. the number of type 1 collagen expressions in the groups with the combination of moringa oleifera leaf extract and dfbbx on both days 7 and 30 was significantly higher than in the other groups. discussion in this research, the lower left mandibular incisors of the cavia cobaya subjects were extracted. the resulting extraction sockets were then filled with peg, dfbbx, moringa oleifera leaf extract or a combination of moringa oleifera leaf extract and dfbbx. xenograft is known to have osteoconductive properties with porous internal surfaces allowing for revascularization and osteoblast migration from the socket base which will support osteogenesis. the inorganic bone matrix structure and content of xenograft also renders it more osteoconductive which facilitates bone formation.12 consequently, there were no significant differences in the amount of type-1 collagen between several groups treated with dfbbx on days 7 and 30. this may be because the xenograft inserted into the tooth extraction sockets serves as a scaffold for new bone growth, derived from the osteoblasts at the base of the sockets.13 on the other hand, moringa oleifera leaf extract contains flavonoid compounds, especially kaempferol and quercetin, which inhibit prostaglandin synthesis, especially pge-2 which decreases macrophage infiltration.14,15 the decrease in macrophage cells will be subsequently followed by a decrease in inflammatory mediators, such as histamine, serotonin, and all three proinflammatory cytokines (tnf a, il-1, il-6).14 the decrease in proinflammatory cytokines then induces a decrease in bone resorption.16 prostaglandins (pge-2) is known to play a role in stimulating osteoclast formation directly or indirectly through rankl, resulting in differentiation and fusion of osteoclast precursors into osteoclasts. hence, the presence of barriers to pge-2 and cytokine synthesis can serve as an inhibitor of osteoclast formation so that the number of those osteoclast cells and proinflammatory cytokines are also capable of inhibiting osteoprotegerin (opg).17 in other words, the decrease in pge-2 synthesis indirectly induces new bone formation through biological cascade activation of osteoblastogenesis by deactivating rankl.16 certain researchers have already indicated that moringa oleifera leaf has many phytochemical variants, especially phytoestrogen that produces a positive effect on bone formation.18 phytoestrogen is a flavonoid compound belonging to the isoflavonoid group. phytoestrogen demonstrates a similar bioactivity to estrogen because of structural similarities between phytooestrogens and estradiol (17-beta-estradiol), which is a naturally-produced estrogen in the body.19 the presence of estrogen activity can then lead to increased osteoblast activity.20,21 osteoblast is one of the cells that can synthesize type-1 collagen.22 thus, in the research reported here, osteoblasts were also expected to express type 1 collagen. moringa oleifera leaf, on the other hand, is assumed to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p86-90 89 soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 86–90 increase the number and activity of osteoblasts indicated by an increase in the concentration of anti-collagen type i antibody observed using immunohistochemical techniques. therefore, the combination of moringa oleifera leaf extract and dfbbx (in groups iv and viii) generated type-1 collagen expressed by osteoblasts on days 7 and 30. in this research, the mean number of type-1 collagen expressions in those treatment groups on day 30 tended to increase compared to those on day 7. this indicates that the number of osteoblasts on day 30 was higher than on day 7. the results above are consistent with the findings of research conducted by guskuma et al.23 showing that bone defect on day 7 is still in the inflammation stage and starts to experience the early stage of resorption. on day 30, the bone defect then starts to experience the early stage of bone formation process. the bone deposition process in the tooth extraction sockets is known to occur on day 28. at that time, osteoblasts and other osteogenic tissues begin to form significantly.24 similarly, research conducted by kresnoadi et al. also revealed that the number of osteoblasts on day 30 increase significantly compared to the previous day.25 in conclusion, the post-extraction preservation of sockets using a combination of moringa oleifera leaf extract and dfbbx may increase the activity of alveolar bone formation as indicated by an increase in type 1 collagen expressions. the selection of materials used in the preservation of tooth extraction sockets, nevertheless, plays an important role in the process of bone formation. dfbbx, according to research,26 has osteoconductive properties that function as scaffolds for new bone growth, derived from the osteoblasts at the base of the sockets. on the other hand, moringa oleifera leaf extract possesses osteoinductive properties since it can increase the proliferation and differentiation of osteoblasts.27 therefore, the combination of moringa oleifera leaf extract and dfbbx in this research can significantly increase type-1 collagen expressions. the increased type 1 collagen expression indicates the occurrence of osteoconduction and osteoinduction activities in the tooth extraction sockets. this is likely to further enhance the success of socket preservation, enabling bone dimensions and volume after the tooth extraction to be maintained. however, further research is needed to improve 2 figure 2. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 30. a: group i; b: group ii; c: group iii; d: group iv figure 3. the diagram of type-1 collagen expressions on days 7 and 30. a b c d 2 figure 2. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 30. a: group i; b: group ii; c: group iii; d: group iv figure 3. the diagram of type-1 collagen expressions on days 7 and 30. a b c d 2 figure 2. the arrows indicate type-1 collagen expressions by osteoblasts in the tooth extraction sockets on day 30. a: group i; b: group ii; c: group iii; d: group iv figure 3. the diagram of type-1 collagen expressions on days 7 and 30. a b c d figures 3. the diagram of type-1 collagen expressions on days 7 and 30. the efficacy of the combination of moringa oleifera leaf extract and dfbbx. references 1. mezzomo la, shinkai rs, mardas n, donos n. alveolar ridge preservation after dental extraction and before implant placement: a literature review. rev odonto ciência. 2011; 26(1): 77–83. 2. van der weijden f, dell’acqua f, slot de. alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. j clin periodontol. 2009; 36(12): 1048–58. 3. salmen fs, oliveira mr, gabrielli ma, piveta acg, filho vap, gabrielli mfr. bone grafting for alveolar ridge reconstruction. review of 166 cases. rev col bras cir. 2017; 44(1): 33–40. 4. murphy cm, o’brien fj, little dg, schindeler a. cell-scaffold interactions in the bone tissue engineering triad. eur cells mater. 2013; 26: 120–32. 5. kamadjaja db, harijadi a, soesilawati p, wahyuni e, maulidah n, fauzi a, rah ayu f, simanjuntak r, soesanto r, asmara d, rizqiawan a, agus p, pramono c. demineralized freeze-dried bovine cortical bone: its potential for guided bone regeneration membrane. int j dent. 2017; 2017: 1–10. 6. coppin j. a study of the nutritional and medicinal values of moringa oleifera leaves from sub-saharan africa: ghana, rwanda, senegal and zambia. thesis. new jersey: the state university of new jersey; 2008. p. 5-99. 7. patel c, rangrez a, parikh p. the anti-osteoporotic effect of moringa oliefera on osteoblastic cells : saos 2. iosr j pharm biol sci. 2013; 5(2): 10–7. 8. bakar a. kedokteran gigi klinis. 2nd ed. yogyakarta: quantum sinergis media; 2012. p. 117. 9. pudianto a. dosis efektif kombinasi ekstrak daun kelor (moringa oliefera) dengan bone graft pada soket pencabutan gigi terhadap osteoblas dan osteoklas tulang alveolar cavia cobaya. thesis. surabaya: universitas airlangga; 2015. p. 50-72. 10. wirawan s. pemberian ekstrak daun kelor (moringa oliefera) dan bone graft serta kombinasi keduanya pada soket pencabutan gigi terhadap pembentukan sel osteoblas dan sel osteoklas (cavia cobaya). thesis. surabaya: universitas airlangga; 2015. p. 30-50. 11. lu y, kamel-el sayed sa, wang k, tiede-lewis lm, grillo ma, veno pa, dusevich v, phillips cl, bonewald lf, dallas sl. live imaging of type i collagen assembly dynamics in osteoblasts stably expressing gfp and mcherry-tagged collagen constructs. j bone miner res. 2018; 33(6): 1166–82. 12. al-ghamdi h, mokeem sa, anil s. current concepts in alveolar bone augmentation : a critical appraisal. saudi dent j. 2007; 19(2): 74–90. 13. gupta r, pandit n, malik r, sood s. clinical and radiological evaluation of an osseous xenograft for the treatment of infrabony defects. j can dent assoc. 2007; 73(6): 513–513f. 14. tan ws, arulselvan p, karthivashan g, fakurazi s. moringa oleifera flower extract suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated raw 264.7 macrophages via nfκb pathway. mediators inflamm. 2015; 2015: 1–11. 15. karthivashan g, tangestani fard m, arulselvan p, abas f, fakurazi s. identification of bioactive candidate compounds responsible for oxidative challenge from hydro-ethanolic extract of moringa oleifera leaves. j food sci. 2013; 78(9): c1368–75. 16. schett g. effects of inflammatory and anti-inflammatory cytokines on the bone. eur j clin invest. 2011; 41(12): 1361–6. 17. park hj, baek k, baek jh, kim hr. tnfα increases rankl expression via pge2-induced activation of nfatc1. int j mol sci. 2017; 18(3): 1–15. 18. burali sc, kangralkar v, sravani os, patil sl. the beneficial effect of ethanolic extract of moringa oleifera on osteoporosis. int j phamaccutical appl. 2010; 1: 50–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p86-90 90soekobagiono, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 86–90 19. patisaul hb, jefferson w. the pros and cons of phytoestrogens. front neuroendocr. 2010; 31(4): 400–19. 20. schilling t, ebert r, raaijmakers n, schütze n, jakob f. effects of phytoestrogens and other plant-derived compounds on mesenchymal stem cells, bone maintenance and regeneration. j steroid biochem mol biol. 2014; 139: 252–61. 21. barrett k, brooks h, boitano s, barman s. ganong’s review of medical physiology. 23rd ed. new york: mcgraw hill medical; 2010. p. 261-72. 22. henriksen k, karsdal ma. type i collagen. in: karsdal ma, editor. biochemistry of collagens, laminins and elastin. amsterdam: academic press; 2016. p. 1–11. 23. guskuma mh, hochuli-vieira e, pereira fp, rangel-garcia i, okamoto r, okamoto t, filho om. evaluation of the presence of vegf, bmp2 and cbfa1 proteins in autogenous bone graft: h istomet r ic a nd i m munoh istochem ica l a na lysis. j cra n io maxillofacial surg. 2014; 42(4): 333–9. 24. tomlin em, nelson sj, rossmann ja. ridge preservation for implant therapy: a review of the literature. open dent j. 2014; 8: 66–76. 25. kresnoadi u, rahayu rp, rubianto m, sudarmo sm, budi hs. tlr2 signaling pathway in alveolar bone osteogenesis induced by aloe vera and xenograft (xcb). braz dent j. 2017; 28(3): 281–6. 26. kresnoadi u, ariani md, djulaeha e, hendrijantini n. the potential of mangosteen (garcinia mangostana) peel extract, combined with demineralized freeze-dried bovine bone xenograft, to reduce ridge resorption and alveolar bone regeneration in preserving the tooth extraction socket. j indian prosthodont soc. 2017; 17(3): 282–8. 27. rostiny r, djulaeha e, hendrijantini n, pudijanto a. the effect of combined moringa oleifera and demineralized freeze-dried bovine bone xenograft on the amount of osteoblast and osteoclast in the healing of tooth extraction socket of cavia cobaya. dent j (maj ked gigi). 2016; 49(1): 37–42. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p86-90 volume 53, number 1, march 2020 dental journal accredited no. 32a/e/kpt/2017 published quarterly per year 33724207719789 5 effect of various inductions of sleep deprivation stress on proinflammatory cytokine levels in gingival crevicular fluids of white male wistar strain rats (rattus novergicus) • the distribution of streptococcus mutans and streptococcus sobrinus in children with dental caries severity level • simple smartphone applications for superimposing 3d imagery in forensic dentistry p-issn: 1978-3728 e-issn: 2442-9740 volume 53, number 1, march 2020 editorial team of dental journal (majalah kedokteran gigi) sk: 07/un3.1.2/2020 january 2nd – december 31st, 2020 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: saka winias, drg., m.kes., sp.pm (department of oral medicine, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); nastiti faradilla (department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia) . peer-reviewers boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); sri oktawati (department of periodontics, faculty of dentistry, universitas hasanuddin, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); retno palupi (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); priyawan rachmadi (department of dental materials, faculty of dental medicine, universitas airlangga, indonesia); maretaningtyas dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); y. yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); andra rizqiawan (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); ratri maya sitalaksmi (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 53, number 1, march 2020 p-issn: 1978-3728 e-issn: 2442-9740 1. propolis extract as pulp capping material enhances odontoblast-like cell thickness and type 1 collagen expression (in vivo) ira widjiastuti, ari subiyanto, evri kusumah ningtyas, rendy popyandra, michael golden kurniawan and fauziah diajeng retnaningsih ................................................ 1–5 2. the different effects of preheating and heat treatment on the surface microhardness of nanohybrid resin composite brelian elok septyarini, irfan dwiandhono and dian n. agus imam ....................................... 6–9 3. the effect of propolis extract and bovine bone graft combination on the number of osteoclast and osteoblast as an effort to preserve post-extraction socket (on cavia cobaya) much nizar, utari kresnoadi and soekobagiono ......................................................................... 10–15 4. potential of 5% tamarind extract gel as an etching agent: tensile strength and scanning electron microscope (sem) evaluation erawati wulandari, faiqatin cahya ramadhani and nadie fatimatuzzahro .......................... 16–19 5. the potency of immunoglobulin y anti porphyromonas gingivalis to inhibit the adherence ability of porphyromonas gingivalis on enterocytes nova andriani hepitaria, indeswati diyatri, markus budi rahardjo and rini devijanti ridwan .............................................................................................................. 20–23 6. effect of various inductions of sleep deprivation stress on proinflammatory cytokine levels in gingival crevicular fluids of white male wistar strain rats (rattus novergicus) pratiwi nur widyaningsih, fitranto arjadi and erlina sih mahanani ..................................... 24–29 7. correlation of the vertical dimension of occlusion with five distances between facial landmarks among those of batak toba ethnicity rehulina ginting and debora lovelisa hinson simbolon ............................................................ 30–35 8. the distribution of streptococcus mutans and streptococcus sobrinus in children with dental caries severity level nur dianawati, wahyu setyarini, ira widjiastuti, rini devijanti ridwan and k. kuntaman ............................................................................................................................. 36–39 9. effective dose of propolis extract combined with bovine bone graft on the number of osteoblasts and osteoclasts in tooth extraction socket preservation teguh setio yuli prabowo, utari kresnoadi and hanoem eka hidayati .................................. 40–44 10. the effects of zinc oxide non-eugenol and cellulose as periodontal dressings on open wounds after periodontal surgery yoeliani budisidharta, ahmad syaify and sri pramestri lastianny ......................................... 45–49 11. simple smartphone applications for superimposing 3d imagery in forensic dentistry haryono utomo, mieke sylvia margaretha amiatun ruth, levina gita wangsa, rodrigo ernesto salazar-gamarra and luciano lauria dib ..................................................... 50–56 contents page mkgs vol 44 no 1 jan-mar 2011.indd 25 vol. 44. no. 1 march 2011 acupuncture analgesia: the complementary pain management in dentistry abdurachman department of anatomy-histology medical faculty, airlangga university surabaya-indonesia abstract background: pain is the most common reason for medical consultation in the united states. pain is a major symptom in many medical conditions, and can significantly interfere with a person’s quality of life and general functioning. one of the very unpleasant pain is toothache. conventional treatments for toothache are improving oral hygiene, prescribing analgesics, anti-inflammatory, and also antibiotics if there are infection even extractions are performed if necessary. another way to conventional approaches, patients may consider acupuncture method. acupuncture involves the insertion of needles with the width of a human hair along the precise points throughout the body. this process triggers body’s energy normal flow through extra anatomy pathway called meridian. purpose: this case report is aimed to emphasize the existence of teeth-organ relationships through communication channels outside the lines of communication that has been known in anatomy. case: two patients with toothache complaints in the lower right molars came to an acupuncturist who was a medical practitioner. in these cases pain were relieved by acupuncture analgesia. case management: two patients were subjected to acupuncture analgesia with different acupuncture points that were customized to the affected tooth, case 1 with the large intestine-4 (li-4) which located in the hand and case 2 with bladder-25 (bl-25) which located in the back of the body. ninety percent of pain was relieved in 40 seconds. conclusion: pain in toothache can be relieved using acupuncture analgesia technique, using meridian as an extra anatomy pathway. nevertheless, treating the source of pain by dental practitioner is mandatory. key words: acupuncture analgesia, pain, dental abstrak latar belakang: nyeri adalah alasan paling umum yang menyebabkan orang datang berkonsultasi kepada profesional medis di amerika serikat. nyeri merupakan gejala utama dalam kasus medis, dan dapat mengganggu kualitas hidup dan kegiatan umum seseorang secara signifikan. salah satu nyeri yang sangat tidak menyenangkan adalah nyeri gigi. pengobatan selalu diarahkan kepada menjaga kebersihan mulut, memberikan analgesik, mengurangi inflamasi dan menambahkan antibiotik jika ada infeksi bahkan jika perlu dilakukan ekstraksi gigi. cara lain dari pendekatan konvensional di atas adalah pasien dapat mempertimbangkan metode akupunktur. akupunktur dilakukan dengan menusukkan jarum seukuran rambut manusia di sepanjang titik yang tepat di seluruh tubuh. proses ini memicu aliran normal energi tubuh melalui jalur ekstra anatomi disebut meridian. tujuan: laporan kasus ini untuk menekankan adanya hubungan gigi-organ melalui jalur komunikasi di luar jalur komunikasi yang telah dikenal dalam anatomi. kasus: dua penderita dengan keluhan nyeri gigi geraham kanan bawah dilakukan terapi analgesia akupunktur oleh akupunkturis yang adalah dokter umum. dalam kasus ini ditunjukkan bahwa rasa nyeri pada gigi dapat dihilangkan menggunakan teknik akupunktur. tatalaksana kasus: pasien dengan keluhan nyeri gigi, kasus #1 pada rahang bawah dilakukan terapi akupunktur pada titik li-4 pada tangan dan kasus #2 pada titik bl-25 pada pinggang. pemilihan titik disesuaikan dengan keluhan pasien, menurut teori meridian dalam akupunktur. nyeri berkurang sampai 90% dalam 40 detik. kesimpulan: keluhan nyeri pada gigi dapat dihilangkan menggunakan teknik akupunktur analgesia, menggunakan meridian sebagai jalur komunikasi di luar jalur komunikasi anatomi. walaupun demikian perawatan kedokteran gigi pada penyebab nyeri yang utama adalah yang paling penting. case report 26 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 25–29 kata kunci: akupunktur analgesia, nyeri, kedokteran gigi correspondence: abdurachman, c/o: departemen anatomi-histologi, fakultas kedokteran universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60131, indonesia. email: rachman1166@yahoo.com. telp: +62 31 5020251 fax: +62 31 5022472 introduction pain is the most common reason for medical consultation in the united states.1 pain is a major symptom in many medical conditions, and can significantly interfere with a person’s quality of life and general functioning.2 one of the very unpleasant pains is toothache. conventional treatment are improving oral hygiene, prescribing analgesics and anti-inflammatory, and add antibiotics if infection involved, or even extractions are performed if necessary.3 another way from conventional approaches, patients may consider acupuncture method. acupuncture involves the insertion of needles with the width of a human hair along the precise points throughout the body. this trigger maintains the normal body’s energy (chi) flow through extra anatomy pathway called meridian, a communication path that is existed aside from nerve, blood and lymph vessels. in acupuncture theory, it is mentioned that chi flows through the body’s meridians. if this chi flows is disrupted, complains (included pain) or symptoms according to the degree of disruption and the affected meridian where disrupted will appear. figure 1. large intestine meridian.4 as for severe pain in toothache, acupuncture can stir up the chi of meridian, regulate chi, clear channels which then acts as natural pain relief for toothache. the effect of acupuncture as pain reliever is commonly termed as acupuncture analgesia. acupuncture is able to ease muscle spasm, promote blood circulation and remove blood stasis, diminish infl ammation and swelling and promote tissue repair function. in a word, acupuncture is able to cure for pain in toothache by addressing both the symptoms and the primary cause. the map of energy communication path (part of large intestine meridian) in a human body’s is shown in the following picture (figure 1).4 according to gellman,5 the body’s vital energy flows through specific channels so called meridian and regulates the whole body function of the body’s organ. meridian is channels which connect all the body’s components. aside from connecting all of the body’s energy internally, meridian also connects the body’s internal energy with external energy (natural energy) through “doors” called acupuncture points. stimulation on acupuncture points will be transmitted via meridian communication path. stimulation will affect circulation of the existing energy system, then creating a healing effect, especially to meridian that is connected directly to the stimulated acupuncture point. diameters of the acupuncture points are approximately between one to three millimeters.5 the depths from the surface of the skin are according to the place and are different individually. it has long been known that acupuncture points have some specific characteristics. superficial acupuncture points have high electric potential (can reach as high as 300 mv), high electric capacitance (0.1–l mf), low electric resistance, ability to increase skin respiration, high local temperature, radiating light which spontaneously visible from jing and yuan points, and sound signals (frequency 2–15 hz, amplitude: 0.5–l mv). deep acupuncture points have low perception threshold to electric stimulation, high capacity, electric resonance with the other acupuncture points, high conductivity to isotopic tracers.6 in the following case report it will be shown that pain in toothache can be relieved using acupuncture technique, another way from conventional treatment in dentistry. case case 1: female 19 years old came with toothache complaint to an acupuncturist who is a medical practitioner. extraoral examination showed swollen cheek in the lower right cheek area. intra-oral examination showed periodontal abscess approximately in the location of #47 (figure 2). the toothache had been felt since ten days before and 27abdurachman: acupuncture analgesia resulting in chewing difficulty. the patient suddenly felt pain in that area and continued for about 3 days. patient treated her pain by consuming antibiotics and analgesics. nevertheless, the periodontal abscess were still increasing and the pain was not disappear (figure 3). body temperature was not elevated. figure 2. a) the location of complaint, first case; b) gum swell. figure 3. point punctured at li-4. case 2: female 36 years old came with toothache complaint. extra-oral examination showed normal appearance. intra-oral examination showed redness in gingival color, and more pronounced in the location of #46 and #47 (figure 4). the pain was suspected caused by periodontal disease. toothache had been felt since two days before which caused chewing difficulty. the gingiva was swollen and the pain was mimicking canker sores. pain was increased suddenly and the teeth were felt like going off. body temperature was not elevated. (figure 5). figure 4. location of complaint. figure 5. there were no swelling and no difference color between right and left gum. case management case 1: the medical practitioner performed acupuncture therapy using acupuncture needle (stainless steel) sterile size 0.25 × 25 mm. the needle was punctured perpendicularly into the large intestine-4 (li-4) acupuncture point, in the middle of the 2nd metacarpal bone on the radial side (figure 4). point selection was customized to the patient’s complaint, according to the theory of meridians in acupuncture.7 acupuncture needle was rotated in clockwise then counterclockwise direction and vice versa. alternating rotation was conducted 40 seconds. once the needle is removed, ninety percent of the pain was disappeared and patient is not complain anymore. afterwards, patient referred to a dental practitioner for further treatment. a b 28 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 25–29 case 2: in some patients, for reasons of fear of needles, stabbing can be done in other places far from the complaints but still related to the large intestine meridian. another point that could be used for this purpose is an equal at the low back, below the spinous process of the 4th lumbar vertebra, 1.5 cun lateral to the posterior midline (figure 6). figure 6. bladder meridian and back-shu (bl-25) acupuncture point.4 the medical practitioner performed acupuncture therapy by acupuncture needle (stainless steel) sterile size 0.25 × 40 mm. acupuncture needle was punctured perpendicularly at the bladder-25 (bl-25) (figure 7). acupuncture needle was rotated clockwise direction then counter-clockwise direction and vice versa. alternating rotation was conducted 40 seconds for each point. once the needle is removed, the pain disappeared. afterwards, patient referred to a dental practitioner for further treatment. discussion acupuncture has a different system of communication lines with the lines of communication as it is commonly known in the science of anatomy. these communication channels are called meridians, the extra anatomy pathway. in western medicine, channels of communication is known through the nerves, lymph vessels and blood vessels. as a communication pathway, meridian is not mentioned above, but it is a separate path, a signal transduction pathway. along the meridians there are acupuncture points.8 each meridian correlated with each organ. each meridian has its own main indication. large intestine meridian for example (figure 1), the pathway start up at the posterior aspect of the arm, enters the lung in the chest. a branch goes down and connects with the large intestine. another branch goes up around the mouth to the other side of the body.8,9 pain is classified into the bi syndrome in acupuncture. bi syndrome caused by the blockade of the body’s energy flow. therapy for pain relief which termed as acupuncture analgesia is done by re-launching the body’s energy flow.5 point li-4 was chosen as an effective point for the body’s energy flow in large intestine energy lines, especially for unblocked of body energy flow in #47 (figure 9). bl-25 acupuncture points are the sited of the back-shu points of large intestine. the back-shu points are the points on the back where chi of the respective solid-hollow organs is infused (solid organ like lung, heart, liver and hollow organ i.e. large intestine, small intestine, gall bladder). they are located on either side of the vertebral column. each of the solid-hollow organs has a back-shu point, as does the large intestine (bl-25), a total of twelve.9 stimulation at point bl-25 for example, will cause the effect of improvements in organ colon and other organs related. in accordance with chinese medicine, colon organ associated with the fi rst and second molar (figure 8, 9). in the fi rst case, li-4 acupuncture point is used because this point is consistent with the complaint in patient, according to the selection point according to traditional chinese medicine. in the second case, the point bl-25 was chosen because patient felt fear do to acupuncture therapy in the visible region. point bl-25 is the back-shu point of the large intestine meridian, which also can be used to perform therapeutic measures in accordance with patient complaints.7,9–11 the relationship between an organ and its acupuncture point had been proven through laboratory experimental research by sagiran,12 and abdurachman13 had scientifi c based that the effect of laser at bladder-20 (bl-20) point, the back-shu point for pancreatic gland, can make β cell increase the number as well as the function. figure 7. the site location of acupuncture needle (bl-25). 29abdurachman: acupuncture analgesia stimulation in gall bladder-34 (gb-34) point can relieve pain in dextral hypochondrial area that caused from an old wound in infra malleolus lateralis dextra.14 the wound is appropriate with gb-40 point in acupuncture. this finding impressed the existence of extra anatomy pathway called meridian in acupuncture. based on the literatures which support the acupuncture analgesia concept, it can be concluded that: pain in toothache can be relieved using acupuncture technique. this finding impressed the existence of extra anatomy pathway called meridian in acupuncture, furthermore this finding support that acupuncture’ technique can relief pain in toothache quickly, the site of acupuncture point due to toothache can be located at a distant site from the main complaint. this finding also impressed the relation between special tooth and special organ in the body. nevertheless, treating the source of pain by dental practitioner is mandatory. references 1. turk dc, dworkin rh. what should be the core outcomes in chronic pain clinical trials?. arthritis res ther 2004; 6(4): 151–4. 2. breivik h, borchgrevink pc, allen sm, rosseland la, romundstad l, hals ek, kvarstein g, stubhaug a. assessment of pain. br j anaesth 2008; 101(1): 17–24. 3. bautista d, shiel jr. wc. toothache. available at: http://www. medicinenet.com/toothache/article.htm. accessed april 2, 2011. 4. dupuis c. ying yang house. available at: http://www.yinyanghouse. com/acupuncturepoints/li1. accessed april 2, 2011. 5. gellman h. acupuncture treatment for musculoskeletal pain. a text book for orthopedics, anesthesia and rehabilitation. new york: taylor and francis; 2002. p. 3–7. 6. starwynn d. electrophysiology and the acupuncture systems. medical acupuncture 2001; 13(1): 41–5. 7. yanfu z. chinese acupuncture and moxibution. shanghai, china: publishing house of shanghai university of traditional chinese medicine; 2002. p. 41–143. 8. shang c. the mechanism of acupuncture beyond neurohumoral theory. available at: http://acupuncture.com/education/theory/ mechanismacu.htm. accessed april 2, 2011. 9. yin g, liu z. advanced modern chinese acupuncture therapy. china: new world press; 2000. p. 91–115. 10. lerner dl. the centre for holistic dentistry. 2008. available at: http://www.holisticdentist.com/image-pages/dental-acupunturepoints.html. accessed april 14, 2011. 11. revins t, weisz s. tooth/ organ relationship. available at: http://www. ravecoholistic.com/toothchart.html. accessed at april 2, 2011. 12. sagiran. the effect of pancreatic ß cell damage to the electrical voltage profile of difference acupuncture points. thesis. yogyakarta: post graduate program university of gajah mada; 2001. 13. abdurachman. effect of laser at pishu point on amount and function of pancreatic β cell (rattus norvegicus) injected by streptozotocin. an experimental study. folia medica indonesiana 2005; 42(1): 8–16. 14. abdurachman. acupuncture therapy to relieve pain in dextral hypochondrial area. case report. folia medica indonesiana 2009; 45(3): 232–6. figure 8. dental-acupunture-points.10 figure 9. correlation chart of each tooth with body organs.11 << /ascii85encodepages 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 209209 the amount of macrophages and activated plasma cells on wound healing process affected by spirulina regina purnama dewi iskandar,1 retno indrawati,1 ira arundina,1 and retno pudji rahayu2 1department of oral biology 2department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: spirulina which grows abundantly in tropical seas have been investigated to enhance immune system. the administration of spirulina in tooth extraction sockets was expected to optimise the function of immunocompetent cells. therefore, wound healing process would be improved. purpose: the aim of this study was to prove that administration of spirulina could influence immune system in tooth extraction sockets. method: there were 28 cavia cobayas used in this study and were put in group of four. mandibular left incisive were extracted from each of them. the basis made from mixture of polyethylene glycol (peg) 400 and peg 4000 was administrated into each socket in control group (tg0). in addition, spirulina 12% was administrated into group tg1, spirulina 24% was administrated into group tg2, and spirulina 48% was administrated into group tg3. all of the cavia cobaya were decapitated and the jaws were removed in day 5 after tooth extraction. the jaws were decalcified in edta solution, formed into paraffin block, processed for hematoxylin and eosin (h & e) and immunohistochemistry staining afterwards. datas were analysed statistically using anova method. result: there was an augmentation in the number of macrophages and activated plasma cells after spirulina application. the administration of higher concentrations of spirulina leads to greater amount of macrophages and activated plasma cells in each groups. conclusion: in conclusion, spirulina is able to increase the amount of macrophages and activated plasma cells which play important role in healing process. keywords: spirulina; macrophages; plasma cells correspondence: retno pudji rahayu, departemen patologi mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: retnorahayu@yahoo.com research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 209–212 introduction tooth extraction is traumatic to oral mucosa, especially gingiva. the wound would heal after days. there are possible complications interfering healing process, such as excessive bleeding, pain, infection, swelling, and dry socket in certain individuals might occur due to impaired immune response which leads to unproper formation of various components that involved in healing process.1 anti-inflammatory agents are commonly given following tooth extraction ito support eliminating inflammation. however, synthetic drugs that are widely used are relatively unreachable and unaffordable by community so exploring traditional herbs are worth considering. the use of herbal drug has reached greater development in the past twenty years.2 algaes are abundantly grown in tropical sea, including indonesia. one of the benefits of spirulina is to substistute synthetic anti-inflammatory drug.3 spirulinas contain protein, iron, gamma-linoleic acid, carotenoid, vitamins, and have been widely used as health supplement.4,5 according to its structure, spirulina is digestible and absorbable due to minerals, fibers, and vitamins, nucleic acid, fatty acid, and carbohydrate contained in spirulina.6 moreover, spirulina is allegedly as an anti-allergic agents due to its ability to decrease the level of specific immunoglobulin e (ige),7 and histamine.8 spirulina is classifed as class 1 food, which is safe for consumption dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p209-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p209-212 210 iskandar, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 209–212 in accordance with the united states of pharmacopeial convention (usp).9 macrophage is one of chronic inflammatory cells which is responsible for phagocytosis infectious microbes following tooth extraction. spirulina works by targeting macrophages, but the molecular mechanism about the role of spirulina in wound healing process has not been clearly noted.10 another component involved in healing process is plasma cells which produces antibodies as response to antigen exposure. secretory immunoglubulin a (siga) is typically antibodies found in oral mucosa. previous studies reported that spirulina increases the amount of siga.11 the variables of this study are the amount of macrophages and activated plasma cells due to their vital roles in healing process. the purpose of this study is to demonstrate the ability of spirulina gel to affect the amount of macrophages and activated plasma cells in wound following tooth extraction. materials and methods this research was an experimental laboratory with samples of 28 males cavia cobaya weighing 300-350 grams, aged 2-3 months which have been adapted to the environment for 1 week. the mandibular left incisives were extracted and spirulina was administrated into each sockets. the spirulina have been dried previously and formed into gel using mixture of polyethylene glycol (peg) 400 and peg 4000 (3:1) as basis. the concentrations of spirulina gels used in this study were 12%, 24%, and 48%. there were 4 groups in this study: control group (tg0) which was only administrated with basis, spirulina 12% was administrated into treatment group 2 (tg2), spirulina 24% was administrated into treatment group 3 (tg3), spirulina 48% was administrated into treatment group 4 (tg4). spirulina was administrated to the animals right after the tooth extraction. the experimental animals were eliminated in order to remove the lower jaws in day 5. the samples were processed for hematoxylene and eosin (h & e) to observe the number of macrophages and immunohistochemistry staining using monoclonal antibody anti-igg to observe the amount of activated plasma cells which contain immunoglobulin (ig) in the cytoplasms. quantities of macrophages and activated plasma cells were counted using light microscope (400x). data obtained were analysed using anova and hsd test afterwards. results the results obtained from control group and treatment groups are described in table 1 and 2. the results demonstrate that there were more macrophages and activated plasma cells observed in treatment groups than in control group. figure 1 indicate that higher concentration of spirulina leads to greater amount of macrophages and activated plasma cells. table 1. mean and standard deviation of macrophages groups n mean standard deviaton control 7 19 3.19 spirulina 12% 7 37 6.80 spirulina 24% 7 52 4.58 spirulina 48% 7 58 7.42 table 2. mean and standard deviation of activated plasma cells groups n mean standard deviaton control 7 5 1.58 spirulina 12% 7 7 2.56 spirulina 24% 7 13 4.58 spirulina 48% 7 26 7.42 8 table 1. mean and standard deviation of macrophages groups n mean standard deviaton control 7 19 3.19 spirulina 12% 7 37 6.80 spirulina 24% 7 52 4.58 spirulina 48% 7 58 7.42 table 2. mean and standard deviation of activated plasma cells groups n mean standard deviaton control 7 5 1.58 spirulina 12% 7 7 2.56 spirulina 24% 7 13 4.58 spirulina 48% 7 26 7.42 figure 1. the amount of macrophages and activated plasma cells. 19 37 52 58 5 7 13 26 0 10 20 30 40 50 60 70 control spirulina 12 % spirulina 24 % spirulina 48 % macrophage activated plasma cells figure 1. the amount of macrophages and activated plasma cells. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p209-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p209-212 211211iskandar, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 209–212 tg4 (spirulina 48%) has the greatest number of macrophages. the anova test showed significant differences of the amount of macrophages and activated plasma cells with sig. value 0.000 (p<0.05). thus, the administration of spirulina could enhance immune response through increasing the number of macrophages and activated plasma cells. figure 2 illustrates macrophages after the administration of spirulina in post-extracted tooth sockets of cavia cobaya using h & e staining, and were observed under light microscope (400x). the black arrows indicate macrophages, whereas the yellow and green ones indicate plasma cells. however plasma cells appeared in he staining were not counted. figure 3 illustrates activated plasma cells after the administration of spirulina in post-extracted tooth sockets of cavia cobaya using immunohistochemistry staining, and were observed under light microscope (400x). the black arrows indicate activated plasma cells which contain antibodies in the cytoplasms. the blue shadows formations which are numerous in figure 3a are incativated plasma cells due to absence of antibodies in their cytoplasms. discussion the increasing numbers of macrophages and activated plasma cells verify that bioactive components contained in spirulina could enhance immune system through affecting macrophages and activated plasma cells,13 and stimulates vegf to form collagen which is beneficial for healing process.14 the ability of spirulina in increasing the function of innate immunity is due to polysaccharide called immulina which is 100 times more effective in maximising the activity of macrophages mediated by tlr-2 and cd14, as well as increasing the production of tnf-α and il-1β. the elevation of the cytokines activates fibroblasts to generate fgf2.13 the modulation of immune response by spirulina is initiated by increased proliferation of macrophages which is the target cell of spirulina.16 macrophages act as antigen presenting cell (apc) mainly in epithelial tissue, responsible for phagocytosis of immunogenes and presents them in order to be identified by t effector cells. extracellular immunogenes are identified by macrophages 9 figure 2. macrophages in h & e staining. black arrows indicates macrophages (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. figure 3. activated plasma cells in immunohistochemistry staining with monoclonal antibody anti-igg. the black arrows indicate activated plasma cells (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. figure 2. macrophages in h & e staining. black arrows indicates macrophages (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. 9 figure 2. macrophages in h & e staining. black arrows indicates macrophages (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. figure 3. activated plasma cells in immunohistochemistry staining with monoclonal antibody anti-igg. the black arrows indicate activated plasma cells (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. figure 3. activated plasma cells in immunohistochemistry staining with monoclonal antibody anti-igg. the black arrows indicate activated plasma cells (a) control group; (b) spirulina 12%; (c) spirulina 24%; (c) spirulina 48%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p209-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p209-212 212 iskandar, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 209–212 and b lymphocytes, and presented by mhc class ii afterwards. cd4+ which functions as t helper would then recognise the peptides and help b lymphocytes to produce antibodies. the interaction between apc and t cell is called immunological synapse.17 there are two possible ways in activating t effector cells by macrophages. the macrophages express costimulator to bind t naive cells, activate t cells, secrete il-12 to stimulate the differentiation of t naive cells into effector cells and initiate cell mediated immunity. the responses of t cells occur around 12-18 hours after antigen exposure.12 another variable observed in this study was the amount of activated plasma cells which produce antibodies and are generated from differentiated and matured b cells. the life span of plasma cells ranges up to several years.18 the increasing number of plasma cells in this study are proportional to the production of antibodies involved in humoral immunity needed in healing process. antibodies work as effector in humoral immune response that bind to antifens and outgrow antigens through neutralising process that enhances phagocytic cells. the result of this study corresponds to previous study reported that spirulina could enhance cellular and humoral immune response.4,11 the enhancement of spirulina-induced proliferation and activity of macrophage leads to secretion of il-1β and il-6. the cytokine il-1β stimulates proliferation of t cell, enhance the function of t cytotoxic and nk cells, and to induce b cells to differentiate into plasma cells. whereas il-6 stimulates the production of antibodies by plasma cells.19 the mechanism of increased antibodies production following administration of spirulina is due to increasing number of cd11b+ or through augmenting level of il-6 which is vital in the development of b cells.11 complements are essential in humoral immune response. extracellular immunogens activate complement system through alternative pathway. one of the generated protein is c3d which is able to bind immunogenes. when b lymphocyte recognises antigens through its receptors, b cells also recognises the binding of c3d with immunogenes through such specific receptors for c3d. the binding of c3d and antigens provide signal for b cells to differentiate into plasma cells and produce antibodies subsequently. it shows that complement play a role as signals in humoral immune response.20 the stimulation by antigens alters b lymphocytes to have more interaction with t helper cells. the increasing expression of b7 costimulator induced by b cell activation provides signal to activate t cell and its receptors. the antibodies response to antigen requires assistance by t helper cells.21 t helper cells recognise antigens presented in b cells by expressing cd40 ligand (cd40l) and produce cytokines. cd40l subsequently bind with cd40 expressed by b lymphocytes. that binding delivers signal to b cell to stimulate proliferation, synthesis, and secretion of antibodies. the cytokines produced by t helper cells bind with receptors in the surface of b lymphocyte to induce proliferation and differentiation of b cells, and to induce antibody production.12 hence, there is a synergistic relationship between macrophage and activated plasma cells through the mechanism of immunological synapse.17 in conclusion, spirulina is able to increase the amount of macrophages and activated plasma cells which play important role in healing process. references 1. choi wy1, kang do h, lee hy. enhancement of immune activation activities of spirulina maxima grown in deep-sea water. int j mol sci 2013; 14(6): 12205-21. 2. ghaeni mj. the effect of spirulina (fresh and dry) on some biological factors in and peneaus semiculcatus larvae. j marine sci res dev 2013; 3(3): 222. 3. moreira lm, ribeiro ac, duarte fa, de morais m, de souza soares. spirulina plantesiss biomass cultivated in southern brazil as a source of essential minerals and other nutrients. afr j food sci 2013; 7(12): 451-5. 4. oh sh, han jg, ha jh, kim y, jeong mh, kim ss, jeong hs, choi gp, park uy, kang dh. enhancement of immune activity of spirulina maxima by low temperature ultrasonification extraction. korean j food sci technol 2009; 41: 313–9. 5. ravi m, de sl, azharuddin s, paul sfd. the beneficial effects of spirulina focusing on immunomodulatory and antioxidant propoerties. nutrition and dietary supplements 2010; 2: 73-83. 6. marles rj, barrett ml, barnes j, chavez ml, gardiner p, ko r, mahady gb, low dt, sarma nd, giancaspro gi, sharaf m, griffiths j. united states pharmacopeia safety evaluation of spirulina. crit rev food sci nutr 2011; 51(7): 593-604. 7. casanova jl, abel l. revisting crohn’s disease as a primary immunodeficiency of macrophages. j exp med 2009; 206(9): 1839-43. 8. balachandran p, pugh nd, ma g, pasco ds. toll-like receptor 2-dependent activation of monocytes by spirulina polysaccharide and its immune enhancing action in mice. int immunopharmacol 2006; 6(12): 1808-14. 9. abbas ak, lichtman ah. basic immunology: functions and disorders of the immune system. philadelphia: saunders elsevier; 2007. p. 46-132. 10. capelli b, cysewski gr. potential health benefits of spirulina microalgae. nutrafoods 2010; 9(2): 19-26. 11. løbner m, walsted a, larsen r, bendtzen k, nielsen ch. enhancement of human adaptive immune responses by administration of a high-molecular-weight polysaccharide extract from the cyanobacterium arthospira platensis. j med food 2008; 11(2): 31322. 12. nuhu a. spirulina (arthospira): an important source of nutritional and medical compounds. journal of marine biology 2013; article id 325636: 8. 13. gordon s. the macrophage: past, present, and future. eur j immuno 2007; 37(suppl 1): s9-17. 14. krummel mf, cahalan md. the immunological synapse: a dynamic platform for local signalling. j clin immunol 2010; 30(3): 364-72. 15. mesin l, di niro r, thompson km, lundin kea, sollid lm. long-lived plasma cells from human small intestine biopsies secrete immunoglobulins for many weeks in vitro. j immunol 2011; 187: 2867-74. 16. chen hw, yang ts, chen mj, chang yc, wang eic, ho cl, lai yj, yu cc, chou jc, chao lkp, liao pc. purification and immunomodulating activity of c-phycocyanin from spirulina platensis cultured using power plant flue gas. process biochemistry 2014; 49: 1337-44. 17. del nagro cj, kolla rv, ricker rc. a critical role for complement c3d and the b cell coreceptor (cd19/cd21) complex in the initiation of inflammatory arthritis. j immunol 2005; 175: 5379-89. 18. maglione pj, chan j. how b cells shape the immune response against mycobacterium tuberculosis. eur j immunol 2009; 39: 676-86. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p209-212 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p209-212 �� vol. 43. no. 1 march 2010 special considerations for orthodontic treatment in patients with root resorption haru s. anggani department of orthodontic faculty of dentistry, university of indonesia jakarta indonesia abstract background: ort��d�ntic treat�ent needs g��d c�nsiderati�n es�ecially ���en t�ere are �n�av�ra�le c�nditi�ns ��r �rt��d�ntic treat�ent, s�c� as �eri�d�ntal diseases �r t��t� ��it� r��t res�r�ti�n. r��t res�r�ti�n s���ld n�t �ec��e ���rse d�e t� �rt��d�ntic treat�ent., all risk �act�rs s���ld �e eli�inated �e��re �rt��d�ntic treat�ent is started. ot�er��ise, t�e g�al �� �rt��d�ntic treat�ent c��ld �e di��ic�lt t� ac�ieve �eca�se �� ���r dental and �r �ral �ealt�. purpose: ��e ��r��se �� t�is st�dy ��as t� learn ��re a���t �ec�anical �act�rs t�at c��ld ���rsen t�e r��t res�r�ti�n t�at �as already �een t�ere �r even �r�v�ke r��t res�r�ti�n t� devel�� d�ring �rt��d�ntic treat�ent. reviews: res�r�ti�n �� dental r��t s�r�ace is t�e c�nditi�n in ���ic� ce�ent�� is de�raved and t�e da�age c��ld als� incl�de dentin �� dental r��t. �t can �cc�r eit�er ��ysi�l�gically �r �at��l�gically d�e t� s��e ca�ses. ��e �cc�rrence �� t�e r��t res�r�ti�n is s�s�ected �eca�se �� t�e �i�l�gical �act�r, t�e t��t� c�nditi�n, t�e s����rtive tiss�e and t�e �ec�anical �act�rs. pan�ra�ic �-ray ���ic� r��tinely �sed t� s����rt diagn�se in �rt��d�ntic cases, can detect r��t res�r�ti�n in general, alt���g� s��eti�es �eria�ical �-ray ��it� �arallel tec�niq�e is needed t� en�ance t�e diagn�sis. be��re starting a treat�ent, t�e risk �act�rs t�at s�s�ected as t�e ca�ses �� r��t res�r�ti�n s���ld �e eli�inated, t��s t�e �ec�anical treat�ent can �e calc�lated. conclusion: ort��d�ntic treat�ent in �atient ��it� r��t res�r�ti�n s���ld n�t escalate t�e r��t res�r�ti�n ���ic� already �cc�rs. ��e treat�ent s���ld �e d�ne e��ectively �y �sing ��ti�al ��rces. giving disc�ntin�ed ��rces and av�iding intr�si�n and t�rq�e ��ve�ents c��ld red�ce t�e risk �act�rs �� r��t res�r�ti�n. key words: r��t res�r�ti�n, �rt��d�ntic treat�ent abstrak latar belakang: keadaan gigi dan jaringan �end�k�ng yang k�rang �eng�nt�ngkan �agi �era��atan �rt�d�ntik �endaknya �e���t��kan �er�atian ekstra �ara klinisi. k�ndisi terse��t �isalnya adanya �enyakit �eri�d�ntal ata���n adanya gigi dengan res�r�si akar. pera��atan �rt�d�ntik yang dilak�kan �endaknya tidak �ena��a� �ara� res�r�si akar yang tela� ada se�el��nya. se�el�� �e��lai �era��atan, sel�r�� �akt�r yang did�ga se�agai �akt�r resik� di�ilangkan terle�i� da��l�. se�aliknya, t�j�an �era��atan �rt�d�ntik akan s�lit dica�ai aki�at ��r�knya keadaan gigi dan jaringan �end�k�ngnya. tujuan: �e��elajari le�i� ja�� �engenai �akt�r �ekanik yang da�at �enye�a�kan res�r�si akar ata� �a�kan �e��er�ara� terjadinya res�r�si akar yang tela� ada aki�at �era��atan �rt�d�ntik. tinjauan pustaka: res�r�si �er��kaan akar gigi adala� k�ndisi r�saknya jaringan se�ent�� akar gigi yang da�at �erlanj�t �ingga ke jaringan dentin akar gigi. res�r�si akar da�at terjadi secara �isi�l�gis ata� �at�l�gis. �erjadinya res�r�si akar ini did�ga karena adanya �akt�r �i�l�gis, k�ndisi gigi dan jaringan �end�k�ng serta adanya �akt�r �ekanik. f�t� r�nsen �an�ra�ik yang r�tin dig�nakan se�agai �en�njang diagn�sa �ada �era��atan �rt�d�ntik da�at �endeteksi secara ���� adanya res�r�si akar, �eski��n terkadang di��t��kan ��t� r�nsen �eria�ikal teknik �aralel �nt�k �e��erjelasnya. se�el�� �e��lai �era��atan, �akt�r resik� yang did�ga se�agai �enye�a� terjadinya res�r�si akar �endaknya di�ilangkan le�i� da��l�, �ar� ke��dian �e��erti��angkan �i��ekanika �era��atan �rt�d�ntik. kesimpulan: pera��atan �rt�d�ntik �ada �asien dengan res�r�si akar �endaknya tidak �e��er�ara� res�r�si akar yang tela� ada. pera��atan yang dilak�kan �ar�sla� see�ekti� dan see�isien ��ngkin review article �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 35-39 dengan �engg�nakan gaya yang ��ti�al. selain it� �e��erian gaya secara disk�ntiny� dan �eng�indari �e��erian gaya intr�si dan t�rq�ing da�at �eng�rangi terjadinya res�r�si akar gigi. kata kunci: res�r�si akar gigi, �era��atan �rt�d�ntik c�rres��ndence: haru s. anggani, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta, indonesia. e-mail: haruanggani@yahoo.com occurs as the result of biological, dental, periodontal, and mechanical factors.9,10 age is one of biological factors that are considered playing role in the occurrence of dental root resorption. this issue has been studied a lot by many researchers, but the results differs from each other.10–14 besides age, sex is also considered as risk factor in occurrence of dental root resorption, but it is still a controversial issue.9,11,14 the other biological risk factor suspected having relationship with dental root resoprtion is genetic. in 1997, harris et al.9 found that genetic has a susceptibility relation with root resorption in 103 pair of siblings whom were treated with the same technique with one orthodontist. although the pattern of hereditary remains unclear. al qawasmi et al.15 found that low production of il-1b cytokines inflammatory protein is considered to play role with dental root resoprtion. systemic condition is also presumed having a relationship with dental root resoption.16 systemic condition is also presumed having a relationship with dental root resorption.16 whereas, the local factor considered having relationship with dental root resorption is the unfavorable periodontium, such as the condition of traumatic occlusion, parafunctions, and bad habit including tongue thrusting and nail biting.1,17,18 in those condition, periodontium receives continuous excessive loading, which lead to the damage of cementum and the exposure of underlying dentin.1 another local factor that is the adjacent impacted tooth. rimes et al.19 studied about resorption of incisive caused by impacted canine and the result showed that the resorption has tendency to occur extensively, reaches the two third of apical and causes symptoms to patients.20 the form of apical portion could also play role in the occurrence of dental root resorption. lavender and malmgren cit. nigul and jomagi14 divided the form of dental root into 5 categories which are: normal, short, dilacerated, pipette like, and blunt. similarly, mirabella and artun22 classified the form of apical portion into 6 types that are: normal, blunt, eroded, pointed, curved, and cork like. tooth with more root length is more susceptible to resorption because it needs more force to be moved and the movement of the root is also greater during tipping and torque movements. beside, traumatic tooth is suspected having greater risk to root resorption compared to non-traumatic tooth.8,23 meanwhile, a vital tooth has greater risk to root resorption compared to a non-vital tooth that has undergone endodontic treatment.22,24. reitan cit. graber1 stated that an endodontically treated tooth has harder dentin compared to a vital tooth. it may explain why an endodontically introduction the goals of orthodontic treatment are to achieve harmonic relations among teeth and jaws, and to attain good esthetic without neglecting healthy condition of periodontium.1 it could be achieved only if the patient has healthy general condition, dental, and periodontium. however, some patients come with unfavorable conditions for orthodontic treatment. for example, there is a periodontal disease or a tooth with root resorption.2,3 dental root resorption can occur either physiologically or pathologically.4 it should be considered by practitioners in order not to escalate the root resorption that already occurs before an orthodontic treatment begins.5 if a patient with root resorption look for orthodontic treament, the treatment should be done without increasing the root resorption. ideally, practitioner could detect it before starting the orthodontic treament and eliminate the risk factors that can be suspected to promote root resorption. beside that, the mechanotherapy should be considered to prevent the worsening of root resorption that already occurs. since a practitioner must take all known measures to prevent the root resorption before orthodontic treatment starts, the purpose of this study was to learn more about mechanic factors that could worsen the root resorption that has already been there or even provocating root resorption to develop during orthodontic treatment. the types of root resorption resorption of dental root surface is the condition in which cementum is depraved and the damage could also include dentin of dental root. it can occur due to osteoclast and cementoclast activities.6 based on the degree of severity, dental root resorption could be classified into 3 types which are surface resorption, inflammatory resorption, and replacement resorption.7 graber divided dental root resorption into superficial resorption of root surface and apical root resorption.1 malmgren et al.8 divided root resorption into 4 types: irregular contour of dental root, apical resorption less than 2 mm, apical resorption 2 mm to a third of root length, and root resorption more than a third of previous root length. risk factors of dental root resorption superficial resorption of root surface is caused presumptively by some factors. this type of resorption is developed by an imbalance between resistance capacity and repairing ability of periodontal tissues toward forces received by dental root and presumptively ��anggani: special considerations for orthodontic treatment treated tooth has less resorption. it also occurs in teeth with incomplete root formation because they have thick predentin layer.24,25 another factor suspected to play role in surface root resorption is mechanical factor of orthodontic treatment. the consideration of the magnitude of forces, the methods of force applying, the duration and the direction of forces influence the occurrence of dental root resorption.1,6,10,26,27 besides the magnitude of forces, the methods of force applying is one of the factors suspected to trigger dental root resorption. there are 3 known methods of force applying: continuing, interrupted or intermittent.26,28 a factor that is not less important is duration of orthodontic force. it is equivalent with the longevity of treatment.29,30,31 another mechanical factor that can cause root resorption is the types of dental movement. it is stated that intrusion and torque are the most frequent movements causing dental root resorption.1,32,33 pathophysiology of dental root resorption dental cementum is an independent tissue; unlike bone, cementum does not involve in metabolic process such as calcium homeostatic. the process of resorption of dental root surface is developed by imbalance between resistance capacities and repairing ability of periodontal tissues toward forces applied to dental root surface.9 the process of root resorption is an elimination of hyalinization zone.1,7 hyalinization is a process marked with the presence of cellular and vascular changes that cause degeneration to cells and vascular structures. if this condition persists, necrotic tissues and hyalinization zone will be formed. hyalinization zone would be selfeliminated by body and at that time the resorption would occur.1,6,7,27,33 the detection of dental root resorption the detection of dental root resorption can be performed by some methods, some of them are through radiography, histology, scanning electron microscopy (sem) or micro computed tomography (micro-ct). histological detection, sem, or micro ct could only be done in extracted tooth.33,35 lateral cephalogram and panoramic x-rays are routinely used prior to orthodontic treatment. however, sometimes additional radiographs are required, such as dental, periapical, occlusal photos, and other projections.10,11,26 periapical radiograph gives more accurate details, less distortion and less error in superimposition compared to panoramic and lateral cephalogram. by periapical projection, pathologic condition in periapical tissue could be seen in more detail compared to panoramic projection.36,37 mcnicol cit. brezniak and wasserstein12 stated that the most favorable radiograph technique in detecting root resorption is periapical with parallel technique. geometrically, the result is accurate and it is the most preferable technique for observing root resorption, as shown in the picture below.36 discussion some mechanical factors of orthodontic treatment are related to the increase of the risk of surface root resorption. so it is easy to understand that to perform orthodontic treatment at a patient with dental root resorption as special considerations are needed. orthodontic treatment should be done without enhancing the severity of resorption. before discussing about the management of root resorption, early detection is necessary to be completed first. routine panoramic x-rays used as supporting diagnostic tool for orthodontic cases could show the appearance of existing root resorption, although additional periapical radiograph is necessary to be indicated.11,36,37 orthodontic treatment could be performed without deteriorating the existing root resorption. it was stated that local risk factor should be eliminated first and afterward the magnitude, the direction, and the duration of force applied and mechanics used should also be considered.1,3,6,7 the magnitude of forces applied should be light. the light force can be achieved by reducing friction, space closing with non-sliding mechanic, or by adding anchorage teeth.23,26 sliding mechanism produces kinetic friction which will produce higher orthodontic forces.38,39 a number of studies have observed the influencing factors toward frictional force. some of them are the material of the bracket, the design of bracket slot, the material of wire, the sectional form of wire, the diameter of wire, and the ligation used.38,41 it was stated that stainless steel bracket has lower friction compared to ceramic bracket because the surface of the ceramic is coarser than of stainless steel. moreover, the design with larger intra-bracket distance and self ligating bracket has lower friction.27,42,43 besides bracket selection, another factor influences frictional force is orthodontic wire. it is stated that nickel titanium wire has coarser surface than stainless steel. however, the greatest friction is obtained from beta titanium wire.27 round wire has lower friction than square form. smaller wire has smaller fiction too. the friction produced could be reduced by ligating wire to bracket technique. ligation using elastomer rubber gives higher friction compared to ligation using wire.38,39 another effort to reduce friction can be seen from the method of space closing. the post extraction space closing process could be performed by sliding or non-sliding mechanics. according to profitt,26 the non-sliding method gives lower friction compared to the sliding one. however, the development of brackets nowadays based on sliding mechanic movement. to minimize the friction, modification to bracket, wire or ligation methods are performed. the principle to produce light force can be achieved by adding anchorage to anchoring side so that the force applied to the tooth which is going to be moved is relatively low toward the anchoring teeth.27 �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 35-39 another consideration in orthodontic treatment of tooth with root resorption is the method of force application. continuous force application is more susceptible for producing root resorption compared to intermittent method. in intermittent method, cementum tissue has an opportunity to do self repairing and to prevent the occurrence of more severe resorption. the cementum repair occurs after force application is stopped and starts from periphery area of resorption because of cementoblast migration.1 acar et al.44 observed the effect between continue and discontinue techniques in force application toward root resorption and they stated that the discontinue force application has less resorption area compared to the continue method. the duration of force application is proportional with the longevity of orthodontic treatment performed. the presence of root resorption occurring in the beginning of the treatment indicates that the tooth has root resorption risk towards orthodontic treatment. the regular radiograph x-rays is necessary to monitor root resorption occurred.1 orthodontic treatment in patient with existing dental root resorption should be done as efficiently as possible.29,30 harry et al.29 stated that the duration of force application factor is more crucial than the magnitude of force applied. in their study, it was found that with applied force as big as 40–60 gram in the day of 14, it was started to show small area of root resorption; in the day of 35, bigger zone of resorption was seen; and so did in the day of 200. the result showed that the extent of root resorption goes bigger as duration of force increasing. another thing that should be noticed in orthodontic treatment with dental root resorption is the types of tooth movement. it is reported that the movement conducts with avoiding intrusion in teeth with existing root resorption.2,3 sometimes intrusion and torque movement cannot be avoided in treating certain cases. graber et al.1 asserted that intrusion and torque can be performed with minimal force along with resting phases in the interval. intrusion with light force would be more effective. one of the methods of intrusion is using intrusion arch. it gives intrusion effect to anterior teeth as well as extrusion effect to molar tube. the important mechanical factors in intrusion are the magnitude of forces, the constant force, the point of force applying and the molar tip back moment.27 besides the consideration of the magnitude of force applied, the point of force application is another factor that is needed to be considered. if the force is applied to the center of resistance then pure intrusion force will be produced; but if the force is applied though bracket attaching to tooth, the moment of force will emerge. the usage of intrusion arch with chinch back in molar will avoid flaring in anterior teeth that is caused by the moment.27 according to the third law of newton, the usage of intrusion arch has is a contributory effect to the third molar, that is tip back moment of molar. it could be advantageous in class ii molar relation, but in class i molar relation, anticipation should be performed by adding anchorage in molar teeth.27 although theoretically the force should be applied as light as possible, but practically it is difficult for clinician to measure the magnitude of force applied to teeth. yet, in the market, the tool that is able to measure the magnitude of force is already available; it is known as stress and tension gauge. the tool has two ends with hook or fork like forms. on the body of the tool, there is a line scale which shows the magnitude of force. stress and tension gauge is used by hooking one of its end to the orthodontic wire, or to other orthodontic appliances, followed by counting the number of lines seen on the tool. on the 16 oz gauze, there are 16 lines, each line represents 1 oz. the importance of prevention is avoiding deterioration of the existing root resorption. the point is that the magnitude of force applied should be as light as possible. it can be achieved by reducing friction which could be attempted by selecting the types of bracket, wire and closing technique, applying ligating method, and adding anchorage. it is important to remember that the intermittent method of force application is the safest one because it gives a chance for cementum tissue to do self repairing so that the duration of treatment can be controlled by scheduling dental visits in order to allow roots to rest. in addition, it is necessary to measure the magnitude of force and if it is possible to posses the tool used for measuring the magnitude of force that is already available in the market. it is concluded that panoramic x-ray which is routinely used to support diagnosis in orthodontic cases, can detect root resorption in general, although sometimes periapical x-ray with parallel technique is needed to enhance the diagnose. besides the importance of early detection, for patient who already has root resorption as the result of local factor, the orthodontic treatment should eliminate the factor so it should not escalate the root resorption that already occurs. in this case, the force should be applied as light as possible and discontinuous force is recommended to give opportunity for cementum tissue to do self repairing and to prevent the occurrence of more severe resorption. the orthodontic treatment also should be performed as effective and as efficient as possible. intrusion and torque forces give risk in root resorption caused by the great stress in apical part of a tooth. references 1. graber tm, vanarsdall rl, vig kwl. orthodontics. currentorthodontics. current principles and technique. 4th ed. st. louis: elsevier inc; 2005. p. 4, 209–11, 263–6. 2. hisano m, ohtsubo k, chung cj, nasution f, soma k. vertical control by combining a monoblock appliance in adult class iii over closure treatment. angle orthod 2006; 76(2): 226–35. 3. kokich vg. orthodontic and non-orthodontic root resorpion: their impact in clinical dental practice. j dent educ 2008; 72(8): 895–902. 4. harris ef, robinson qc, woods ma. an analysis of causes apical root resorption in patients not treated orthodontically. quintessence int 1993; 24: 417–28. ��anggani: special considerations for orthodontic treatment 5. lupi je, handelman cs, sadowsky c. prevalence and severity of apical root resorption and alveolar bone loss in orthodontically treated adults. am j orthod dentofac orthop 1996; 109(1): 28–37. 6. bishara se. textbook of orthodontics. philadelphia: saunders company; 2001. p. 463–75. 7. brezniak n, wasserstein a. root resorption after orthodontic treatment: part 2. literature review. am j orthod dentofac orthop 1993; 103(2): 138–46. 8. malmgrem o, goldson l, hill c, orwin a, lundberg m. root resorption after orthodontic treatment of traumatized teeth. am j orthod 1982; 82(6): 487–91. 9. harris ef, kineret se, tolley ea. a heritable component for externala heritable component for external apical root resorption in patients treated orthodontically. am j orthod dentofac orthop1997; 111: 301–9. 10. brezniak n, wasserstein a. root resorption after orthodontic treatment: part ii. literature review. am j orthod dentofac orthop 1993; 103(1): 62–6. 11. sameshima gt, sinclair pm. predicting and preventing root resorption: part i. diagnostic factors. am j orthod dentofac orthop 2001; 119(5): 505–10. 12. mcfadden wm, engstrom c, engstrom h, anholm jm. a study of the relationship between incisor intrusion and root shorthening. am j orthod dentofac orthop 1989; 96(5): 390–6. 13. beck bw, harris ef. apical root resorption in orthodontically treated subjects: analysis of edgewise and light wire mechanics. am j orthod dentofac orthop 1994; 105(4): 305–61. 14. nigul k, jagomagi t. factors related to apicalroot resorption of maxillary incisor in orthodontic patient. baltic dental and maxillofacial journal 2006; 8(3): 76–9. 15. al-qawasmi ra, hartsfield jk jr, everett et, flury l, liu l, foroud tm, macri jv, roberts we.genetic predisposition to external apical root resorption. am j orthod dentofac orthop 2003; 123(3): 242–52. 16. verna c, dalstra m, melsen b. bone turnover rate in rats does not influence root resorption induced by orthodontic treatment. eur j orthod 2003; 25: 359. 17. harris ef, butler ml. patterns of incisor root resorption before and after orthodontic correction in cases with anterior openbite. am j orthod dentofac orthop 1992; 101(2): 112–9. 18. lee ry, artun j, alonzao ta. are dental anomalies risk factors forare dental anomalies risk factors for apical root resorption in orthodontic patients? am j orthod dentofac orthop 1999; 116: 197–95. 19. rimes jr, mitchell cnt, willmot dr. maxillary incisor root resorption in relation to the ectopic canine: a review of 26 patients. eur j orthod 1997; 19(1): 79–84. 20. saldarriaga jr, patino mc. ectopic eruption and severe root resorption. am j orthod dentofac orthop 2003; 123(3): 259–65. 21. heimisdottir k, bosshardt d, ruf s. can the severity of root resorption be accurately judged by means of radiograph? a case report with histology. am j orthod dentofac orthop 2005; 128(1): 106–9. 22. mirabella ad, artun j. risk factors for apical root resorption of maxillary anterior teeth in adult orthodontic patients. am j orthod dentofac orthop 1995; 108(3): 48–55. 23. linge l, linge bo. patient characteristics and treatment variables associated with apical root resorption during orthodontic treatment. am j orthod dentofac orthop 1991; 99(1): 35–43. 24. spurrier sw, hall sh, joondeph dr, shapiro pa, riedel ra. a comparison of apical root resorption during orthodontic treatment in endodontically treated and vital teeth. am j orthod dentofac orthop 1990; 97(2): 130–4. 25. hendrix i. carels c, kuijpers-jagtman a, van’t hof m. a radiographics study of posterior apical root resorption in orthodontic patients. am j orthod dentofac orthop1994; 105: 345–9. 26. proffitt. wr. contemporary orthodontic. 3rd ed. philadelphia: cv mosby company; 1999. p. 296–315, 345–7. 27. nanda r. biomechanics and esthetic strategies in clinical orthodontics. missouri: elsevier inc; 2005. p. 135, 28–9, 140–3, 199–208. 28. singh. text book of orthodontics. new delhi: jbm publisher; 2004. p. 208–9. 29. harry mr, sims mr. root resorption in bicuspid intrusion. a scanning electron microscope study. angle orthod 1982; 52: 235–58. 30. kurol j,owman-moll p, lundgren d. time related root resorption after application of a controlled continuous orthodontic force. am j orthod dentofac orthop 1996; 110: 303–10. 31. smale i, artun j, behbehani f, doppel d, van hof m, kuijpersjagtman a. apical root resorption 6 months after initiation of fixed orthodontic appliance therapy. am j orthod dentofac orthop 2005; 128: 57–67. 32. costopoulos g, nanda r. an evaluation of root resorption incidet to orthodontic intrusion. am j orthod dentofac orthop 1996; 109: 543–8. 33. chan e, darendiler ma. physical properties of root cementum: part 7. extent of root resorption under areas of compresion and tension. am j orthod dentofac orthop 2006; 129: 504–10. 34. jimenez-pellegrin c, arana-chavez ve. root resorption in human mandibular first premolars after rotation as detected by scanning electron microscopy. am j orthod dentofac orthop 2004; 126: 178–85 35. wierzbicki t, el-bialy t, aldaghreer s, li g, doschak m. analysis of orthodontically induced root resorption using micro-computed tomography (micro-ct). angle orthod 2008; 79: 91–6. 36. leach ha, ireland aj, whaites e j. radiology: radiographic diagnosis of root resorption in relation to orthodontics. br dent j 2001; 190: 16–22. 37. sameshima gt, asgarifar ko. assessment of root resorption and root shape: periapical vs panoramic films. angle orthod 2000; 71: 185–9. 38. cacciafesta v, sfondrini mf, ricciardi a, scribante a, klersy c, auricchio f. evaluation of friction of stainless steel and esthetic self-ligating brackets in various bracket-archwire combination. am j orthod dentofac orthop 2003; 124: 395–406. 39. hain m, dhopatkar a, rock p. the effect of ligation method on friction in sliding mechanics. am j orthod dentofac orthop 2003; 123: 416–22. 40. southard te, marshall sd, grosland nm. friction does not increase anchorage loading. am j orthod dentofac orthop 2007; 131: 412–4. 41. braun s, bluestein m, moore k, benson g. friction in perspective. am j orthod dentofac orthop 1999; 115: 19–27. 42. mcdonald f, toms ap. a scanning electron microscopic investigation of ceramic orthodontic bracket. saudi dental journal 1990; 2(3): 91–5. 43. jeena ak, duggal r, mehrota ak. physical properties and clinical characteristic of ceramic bracket: a comprehensive review. trends biomater artif organs 2007; 20(2): 101–15. 44. acar a, canyurek u, kocaaga m, erverdi n. continous vs. discontinous force application and root resorption. angle orthod 1999; 69: 159–63. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base isi vol 39 no 3 juli-september 2006.pmd 102 clinical evaluation in periodontitis patient after curettage widowati witjaksono,* roselinda abusamah,** and tp. kannan*** * department of periodontic, school of dental sciences university science malaysia and faculty of dentistry airlangga university ** dds student, school of dental sciences university science malaysia *** department of oral biology, school of dental sciences university science malaysia abstract curettage is used in periodontics to scrap off the gingival wall of a periodontal pocket, and is needed to reduce loss of attachment (loa) by developing new connective tissue attachment in patients with periodontitis. the purpose of this study was to evaluate the success of curettage by the formation of tissue attachment. this clinical experiment was done by comparing loa before curettage, 2 weeks and 3 weeks after curettage on 30 teeth with the indication of curettage. study population were periodontitis patient who attending dental clinic at hospital university science malaysia (husm) with inclusion criteria good general health condition, 18 to 55 years old male or female and presented with pocket depth > 3mm. the teeth were thoroughly scaling before intervention and evaluated by measuring the periodontal attachment before curettage, two weeks and three weeks after curettage. repeated measure anova and paired t test were used to analyze the data. the result of the study showed that there was reduction in the periodontal attachment loss in periodontitis patient after curettage either in the anterior or posterior teeth which were supported by statistical analysis. this study concluded that curettage could make reattachment of the tissue key words: loss of attachment, periodontitis, periodontal pocket correspondence: widowati, department of periodontic, school of dental sciences university science malaysia, health campus 16150 k. kerian, k. bharu, kelantan, malaysia. curettage is legally sanctioned duty in many states.10,11 based on the controversions, the aim of the study was to evaluate the success of curettage by the formation of tissue attachment. materials and methods this clinical experiment compared loa before curettage, 2 weeks and 3 weeks after curettage. the samples were patients who visited husm dental clinic, in range of age 18 to 55 years old, general health in good condition, and suffered chronic periodontitis with periodontal pocket >3mm. the examinations were done on 30 teeth from 15 patients who match the criteria. informed consent was obtained from all volunteers, and all procedures were in accordance to ethical guidelines established for human subjects which approved by the elective committee of university science malaysia, school of dental sciences. the instruments were prepared and sterilized by dental surgery assistant including mouth mirror, tweezer, william probe, gracey curettes (hu-friedy), explorer, examination tray, gauze and cotton pellets. a week before curettage (0 day), whole mouth scaling and prophylaxis were done (figure 1). then, loa evaluation was done at the same day and repeated at 2 weeks and 3 weeks after curettage. loss of attachment was measured from the cemento enamel junction to the base of the pocket on the deepest site 3 (figure 2). after rinsing with introduction curettage is used in periodontics by scraping off the gingival wall of a periodontal pocket to separate the diseased soft tissue and remove the chronically inflammed granulation tissue formed in the lateral wall of the periodontal pocket.1,2 curettage is needed to reduce loss of attachment (loa) by developing new connective tissue attachment.1,2,3 there are so many opinions on curettage. some investigators report that the removal of the pocket lining and junctional epithelium by curettage is not complete.4,5,6 however, other investigators report that both epithelial lining of the pocket and junctional epithelium, sometimes including underlying inflamed connective tissue, are removed by curettage.1,3,7 the reason why curettage no longer being frequently used, are because the procedure technically difficult to master and time consuming.3 short and long-term clinical trials have confirmed that gingival curettage provides no additional benefit in terms of probing depth reduction, attachment gain, or inflammation reduction,8,9 when compared to scaling and root planning alone. thus, some dental schools do not apply curettage in their daily practice activity. the american dental association has deleted curettage as a method of treatment on their 1989 world workshop in clinical periodontics.4 however, 80% of dental hygiene programs in the united states still apply the gingival curettage procedure with the reason that 103witjaksono et al: clinical evaluation in periodontitis patient after curettage chlorhexidine 0.2 % solutions, local infiltrative anaesthesia was applied to the region of 13,14 and 15 (figure 3). then, removal of any soft and hard deposits from the root surface and also smoothening of the root surface (root planing) were done (figure 4). a universal curette was inserted inversely into the pocket. the inner surface of the pocket was carefully peeled (figure 5a, 5b and 6a, 6b). finally, the area was flushed with physiologic saline 0.9 % to remove debris (figure 7a, 7b), and the tissue was partly adapted to the tooth by gentle finger pressure. the clinical evaluations for periodontal attachment loss were repeated at 2 weeks and 3 weeks after curettage results figure 8-a showed before curettage, the gingiva appeared hemorrhagic and bright red in the region 13, 14, 15. the normal conditions especially in color, figure 5a–b. curettage on buccal site of 14. a b figure 1. scaling and prophylaxis a week before intervention (0 day). figure 2. loa measurement before curettage, 2 weeks and 3 weeks after curettage. figure 3. local infiltrative anaesthesia. figure 4. scaling and root planing. 104 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:102–106 figure 6a–b. curettage on palatal site of 14. figure 7a–b. irrigation of pocket by 0.9 % saline. figure 8. (a) before curettage, (b) after curettage. consistency, surface texture, and contour of the gingiva were attained at 3 weeks after curettage and the gingival margin was well adapted to the tooth (figure 8-b). this result also can be interpreted on the table 1. table 1 showed that all samples (100%) were 30 cases with loa > 3mm. not a single had a loa between 1 and 3mm. there were reduction from 30 cases to 26 cases within 2 weeks after curettage and to 10 cases within 3 weeks after curettage. table 2 was showing loa in the anterior and posterior teeth before curettage, 2 weeks and 3 weeks after curettage. in the anterior region, 2 weeks after curettage the loa > 3 mm reduced from 10 cases to 8 cases and at 3 weeks after curettage reduced again from 8 cases to 4 cases. in the posterior region, 2 weeks after curettage the loa > 3 mm reduced from 20 cases to 18 cases and reduced a b a b a b 105witjaksono et al: clinical evaluation in periodontitis patient after curettage again at 3 weeks after curettage to 6 cases. it showed that there was an increase of tissue attachment at 2 weeks and 3 weeks after curettage in the anterior and posterior teeth. figure 9 showed that there was a significant reduction of loa between before curettage and 2 weeks and 3 weeks after curettage. the reduction of loa between before and after 2 to 3 weeks was more significant than the reduction between 2 weeks and 3 weeks after curettage. discussion these clinical observations were done on the day of loa measurement, 2 weeks and 3 weeks after curettage. the healing process was observed and professional plaque control were done during clinical examination and 3 weeks after intervention. this procedure was supported by the report which stated that healing of the epithelial lining of the pocket after periodontal debridement and gingival curettage can be expected to take 5 to 12 days11 while another study said that restoration and epithelization of the sulcus generally require from 2 to 7 days.1 from the clinical and statistical analysis, it showed that curettage could re-attach the tissue with reduction in loa at 2 weeks and 3 weeks after curettage. according to the previous study, curettage could reduced pocket depth by developing new connective tissue attachment and tissue shrinkage.3 other clinical study which also evaluate the effect of curettage in patients with periodontitis also concluded that curettage could make tissue re-attachment.12 this clinical experiment revealed that there were reduction in loa after curettage in the anterior and posterior teeth. the statistical analysis showed that more reduction loa obtained at before and 2 weeks after curettage as well as before and 3 weeks after curettage. however, less reduction from 2 weeks to 3 weeks after curettage may caused by several factors, such as short duration of observation (only a week), systemic factor or the immune status of the patient, and patients were not taking a good care of oral hygiene at home. this condition was also supported by study that stated, if the area has not completely healed in 7 to 10 days, a disturbance in healing should be suspected.7 this is most commonly due to the presence of local irritants, either calculus that has not been removed or plaque that reaccumulated.2 if generalized delay in the healing of the entire curetted area occurs, a systemic interference should be suspected.13 the study revealed that periodontitis patient who undergone curettage procedure showing reduction of loa clinically, either in the anterior or posterior teeth. anyhow, the authors could support the american academy of periodontology statement4 to delete the curettage in the guidelines of periodontal therapy if the curettage was separated with scaling and prophylaxis procedure in periodontal treatment. in this study, curettage should always be preceded by scaling and prophylaxis which every body knows is the basic periodontal therapy,11,14 so there is no curettage can be done without scaling and prophylaxis. this study showed that curettage could make tissue attachment by reduction of periodontal attachment loss. it means leaving or deleting curettage from the basic periodontal therapy should be aimed mainly to the mastered table 1. loss of attachment before curettage, 2 weeks and 3 weeks after curettage (analyzed by repeated measure anova) treatment loa before curettage 2 weeks after curettage 3 weeks after curettage loa ≥ 3 mm 30 (100%) 26 (87%) 10 (33%) table 2. loss of attachment before curettage, 2 weeks and 3 weeks after curettage in the anterior and posterior teeth (analyzed by paired t test) treatment region of loa before curettage 2 weeks after curettage 3 weeks after curettage anterior loa ≥ 3 mm posterior loa ≥ 3 mm 10 (33%) 20 (67%) 8 (27%) 18 (60%) 4 (13%) 6 (20%) figure 9. loss of attachment (before, 2 weeks and 3 weeks after) curettage. l o a ( m m ) weeks 1 2 3 2.5 3 3.5 4 4.5 2 1 1.5 0 106 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:102–106 clinical periodontist since their hand skill in doing fully mechanical debridement. finally, the authors would like to emphasize that although scaling, prophylaxis, and curettage are difficult, time-consuming and often tedious procedures, but they are basic to periodontal therapy and should be mastered by all general dental practitioners. acknowledgements curettage has not applied anymore on a daily clinical practice curriculum in the school of dental sciences university science malaysia. this procedures was conducted mainly for the purpose of elective project 2005–2006 as prerequisite for professional examination in the doctor of dental surgery course. special thanks are extended to the dental surgery assistant’s students who help much in running this project. references 1. newman, takei, carranza. carranza’s clinical periodontology. 9th ed. philadelphia: wb saunders company; 2003. p. 744–47. 2. simon h. what are the procedures for treatment of periodontal disease? (cited 2002 december). available at: http:www.umm.edu/patiented/articles/ what_procedures_treatment_of_periodontal_disease_000024_8.htm. accessed august 27, 2005. 3. lindhe j, karring t, lang np. clinical periodontology and implant dentistry. 4th ed. oxford: blackwell publishing company 2003; p. 406–08. 4. american academy of periodontology statement regarding gingival curettage. j.periodontol, october 2002, 73 (10): 1229–30. available at: http://www.perio.org/resources_products/pdf/38_curettage.pdf. accessed september 18, 2005. 5. aukhil i. biology of wound healing. periodontology 2000; 2000. 22:44. 6. cobb cm. clinical significance of non-surgical periodontal therapy: an evidence-based perspective of scaling and root planing. j clin periodontol 2002; 29(supplement 2):6. 7. goldman hm, cohen dw. periodontal therapy. 6th ed. st louis, missouri: the cv mosby; 1980. p. 677–82, 760–61. 8. greenwell h, harris d, pickman k, burkart j, parkins f, myers t. clinical evaluation of nd: yag laser curettage on periodontitis and periodontal pathogens. j dent res 1999; 78(spec. issue): 138 (abstr. 2833). 9. neil me, melloning jt. clinical efficacy of the nd:yag laser for combination periodontal therapy. pract periodontics aesthet dent 1997; 9:1–5. 10. perry, beemsterboer, taggart. periodontology for the dental hygienist. 2nd ed. philadelphia: wb saunders company; 2001. p. 222–29. 11. esther m, wilkins. clinical practice of the dental hygienenist. 9th ed. philadelphia: lippincot williams & wikins; 2005. p. 646. 12. prahasanti c. kehilangan perlekatan jaringan pada penderita periodontitis setelah dirawat kuretase. maj. ked. gigi (dent j.) 2001; 34(3a):199–201. 13. american academy of periodontology. treatment of plaque–induced gingivitis, chronic periodontitis, and other clinical conditions. endorsed by the american academy of pediatric dentistry 2004; p.169–78. 14. cohen es. atlas of cosmetic and reconstructive periodontal surgery. 2nd ed. boston massachusets: lea and febiger; 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 175175 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 175–180 research report the relationship between dental fear, anxiety and sociodemography in jakarta, indonesia lisa prihastari,1 rima ardhani iswara,1 ghina al afiani,1 fajar ramadhan,1 mega octaviani,1 willy anugerah hidayat,1 muhammad al faqih1 and ahmad ronal2 1department of preventive and dental public health, 2department of oral medicine, faculty of dentistry, universitas yarsi, jakarta – indonesia abstract background: the anxiety associated with dental visits is one of the obstacles preventing dentists from improving oral health and is also a significant predictor of dental visit evasion, which is frequently observed in indonesia. purpose: to identify the level of dental fear and anxiety in the population of jakarta, indonesia and establish the relationship with sociodemographic factors. methods: a cross-sectional method was used with a sample size of 1811 respondents aged 17–65 years old who were asked to complete the validated indonesian versions of modified dental anxiety scale (mdas) and dental fear scale (dfs) questionnaires. the data obtained was then analysed using nonparametric and chi-square tests. results: the prevalence of subjects with moderate to high dental anxiety and fear was 16.3% (295 respondents) and 36.1% (654 respondents), respectively. the primary sources of dental fear and anxiety were dental drilling and anaesthesia before tooth extraction. the results of the nonparametric and chi-square tests show that both are significantly related to gender, age, educational status, income level, insurance and history of dental visits (p = < 0.05). conclusion: several sociodemographic factors are associated with dental fears and anxiety among the participants in jakarta, indonesia. keywords: dentist visit; dental fear and anxiety; sociodemography; indonesia correspondence: lisa prihastari, department of preventive and dental public health, faculty of dentistry, universitas yarsi, kav. 13, jl. letjend suprapto, jakarta, 10510, indonesia, lisa.prihastari@yarsi.ac.id introduction the fear of dental care is a major obstacle to preventing problems related to dental and oral health, which are known to interfere with daily activities. dental fear is a challenge for dentists because it complicates medical procedures and leads to irregular dental visits (and ultimately poor oral health).1–4 this type of fear, which can be defined as an emotional response to a threat or danger involving dental treatment, is a common phenomenon in dentistry.5,6 in indonesia, a survey on dental fear and anxiety measured the prevalence of anxiety towards certain dental treatments: tooth extraction, dental fillings and oral hygiene; the results showed that around 20–30% of subjects felt fear and anxiety towards the treatments. there is limited data on the common causes of dental fear and anxiety in indonesian society. however, research in other countries reports that the frequency of dental anxiety ranged from 5% to 20% and was higher in females.7,8 furthermore, the prevalence of dental anxiety among children ranged from 6% to 20%, and in adolescents, this increased to 11%.6 the prevalence of dental anxiety varies from 4% to 30%.9 the percentage of dental care utilisation in jakarta province is only 16.4%;10 therefore, it can be concluded that while jakarta is the province with the fastest rate of progress and development in indonesia, residents are less likely to participate in dental health care. the low number of dental visits or avoidance of dental treatment may both be a result of dental anxiety and fear. several instruments have been developed to measure anxiety and fear related to dental care. one of the most common instruments is the dental anxiety scale (das). the das is widely used and has been updated by dailey et al.11 to the modified dental anxiety scale (mdas), which is more dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 mailto:lisa.prihastari@yarsi.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 176 prihastari et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 175–180 concise and is both valid and reliable.6 it consists of five questions, with each question offering responses ranging from 1 to 5 (‘not anxious’ to ‘very anxious’, respectively). the minimum total score was 5 and the maximum was 25; 19 and above indicated high dental anxiety, which may require special attention from the dentist.12 the kleinknecht’s dental fear scale is the second most frequently used instrument, and it focuses on specific situations and procedures.13 the updated version contains 20 questions rated on a five-point scale where 1 is ‘no fear’ and five means ‘extreme fear’; hence, the total scores ranged from 20 to 100.14 the purpose of this study is to provide data on the prevalence of fear and anxiety levels associated with dental care in indonesia – especially in the province of jakarta – and identify the main causes of these and their relationship with sociodemography using the mdas and dfs. data on the prevalence of dental fear and anxiety is critical for indonesian dentists and governments in order to see how much this will affect dental care and how to address this phenomenon. materials and methods this study used an analytic cross-sectional design to determine the relationship between sociodemographic factors and the fear and anxiety levels towards dental care. the population included 17–65-year-old residents from the thousand islands and regions in the jakarta province (central, east, west, north and south jakarta). the study was conducted from september to november 2017 following the review and approval of the research ethics protocol by the yarsi university ethics commission (certificate number 316/kep-uy/bia/xi/2017). the participants signed informed consent, and multistage cluster sampling was used: the sample was taken randomly up to the district level and in accordance with the proportion of the population of jakarta with a total of 1811 respondents. the procedure was conducted by requesting permission from the relevant agencies and testing the validity and reliability of the mdas and dfs questionnaires (0.844 and 0.935, respectively) using cronbach alpha and a p value of < 0.05. the calculated r value is greater than r table for all question items from the two questionnaires using the pearson product moment correlation test. the dfs and mdas scoring, which initially consisted of a five-point scale, was converted into two-point scale for analysis in logistic regression. the scores in mdas are divided as follows: 0–5 for not anxious, 6–10 for somewhat anxious, 11–14 for moderately anxious, 15–18 for highly anxious and 19–25 for extremely anxious. for dfs, the scores are divided as ≥ 60 for high dental fear, 34–59 for moderate fear, 21–33 for low fear and scores < 20 for no fear. the surveys were first calibrated by six interviewers then administered by researchers and extended for approximately two months. furthermore, each respondent was asked to provide sociodemographic data consisting of age, region, sex, education level, income, questions related to health insurance and history of dental visits. the indonesian versions of the dfs and mdas were made available. the subjects’ ages were classified into 17–25, 26–35, 36–45, 46–55 and 56–65, and education level was divided into basic (elementary–junior high school), secondary (high school) and higher (higher education/university). income level was categorised into non-income and income below and above the jakarta umr (regional minimum wage).15 the data obtained was analysed using spss software with a nonparametric test to compare the medians because the data did not have a normal distribution or chi-square values for proportions and binary logistic regression. results based on sociodemographic status, the frequency distribution of research respondents is shown in table 1. the data in table 1 shows a gender variable characterised by 1012 male respondents (55.9%) and 799 female respondents (44.1%). in the age variable, the largest proportion was in the group of 17–25-year-olds (818 respondents, 45.2%) while the smallest was 37 respondents (2%) in the 56–65 age group. table 1. sociodemographic status of research subjects variable n % gender male female 1012 799 55.9 44.1 region north jakarta central jakarta east jakarta west jakarta south jakarta thousand islands 300 201 414 404 382 110 16.6 11.1 22.9 22.3 21.1 6.1 age of subject 17–25 years old 26–35 years old 36–45 years old 46–55 years old 56–65 years old 818 491 273 190 37 45.2 27.1 15.1 10.5 2.0 level of education higher education secondary education basic education 508 921 381 28.1 50.9 21.0 income no income < minimum wage ≥ minimum wage 479 433 899 26.4 23.9 49.6 insurance yes no 1251 560 69.1 30.9 ever been to dentist yes no 1370 441 75.6 24.4 total 1811 100.0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 177prihastari et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 175–180 table 2. frequency of sources of fear in dfs questionnaire items questions no fear some fear moderate fear high fear extreme fear total the source of fear during treatment promise to visit the dentist 1137 (62.8%) 519 (28.7%) 110 (6.1%) 24 (1.3%) 21 (1.2%) 1811 (100%) approach the dentist’s clinic 1310 (72.3%) 391 (21.6%) 82 (4.5%) 19 (1.0%) 9 (0.5%) 1811 (100%) sit in the dentist’s waiting room 1140 (62.9%) 526 (29.0) 112 (6.2%) 21 (1.2%) 12 (0.7%) 1811 (100%) sitting in the dental care chair 898 (55.7%) 671 (37.1%) 184 (10.2%) 42 (2.3%) 16 (0.9%) 1811 (100%) smell of the dental clinic 1307 (72.2%) 365 (20.2%) 96 (5.3%) 31 (1.7%) 12 (0.7%) 1811 (100%) seeing the dentist enter the room 1133 (62.6%) 509 (28.1%) 132 (7.3%) 23 (1.3%) 14 (0.8%) 1811 (100%) the sight of syringe for anaesthesia 579 (32.0%) 667 (36.8%) 372 (20.5%) 113 (6.2%) 80 (4.4%) 1811 (100%) the feeling of injected syringe 554 (30.6%) 744 (41.1%) 309 (17.1%) 127 (7.0%) 77 (4.3%) 1811 (100%) seeing the dental drill 623 (34.4%) 655 (36.2%) 347 (19.2%) 100 (5.5%) 86 (4.7%) 1811 (100%) the sound of the dental drill 636 (35.1%) 688 (38.0%) 321 (17.7%) 89 (4.9%) 77 (4.3%) 1811 (100%) the vibration of the dental drill 565 (31.2%) 714 (39.4%) 347 (19.2%) 100 (5.5%) 85 (4.7%) 1811 (100%) after the teeth cleaning process 1287 (71.1%) 381 (21%) 94 (5.2%) 32 (1.8%) 17 (0.9%) 1811 (100%) table 3. mdas and dfs nonparametric test results based on sociodemographic status variable mdas median (mean±sd) p value dfs median (mean±sd) p value gender male 9.00(9.85±4.08) 0.0001* 30.00(33.13±12.06) 0.0001*female 10.00(10.86±4.12) 33.00(35.42±12.24) region north jakarta 9.00(10.02±3.77) 0.0001* 30.00(32.28±10.74) 0.0001* central jakarta 9.00(9.55±4.03) 31.00(33.25±11.81) east jakarta 10.00(10.92±4.13) 33.00(35.65±12.44) west jakarta 10.00(10.49±4.54) 31.00(34.86±13.27) south jakarta 9.00(9.83±3.88) 31.00(33.09±11.38) thousand islands 11.00(11.13±4.06) 33.50(36.04±13.08) age of subject 17–25 years old 10.00(10.51±4.09) 0.0001* 32.00(34.93±12.18) 0.0001* 26–35 years old 10.00(10.34±4.18) 32.00(34.47±12.07) 36–45 years old 9.00(10.06±4.05) 30.00(32.98±11.83) 46–55 years old 9.00(9.86±4.335) 29.50(32.40±12.86) 56–65 years old 9.00(9.08±3.507) 25.00(30.00±11.19) education higher education 9.00(9.68±4.06) 0.0001* 30.00(32.92±12.31) 0.0001*secondary education 10.00(10.36±4.00) 31.00(34.51±12.13) basic education 10.00(10.98±4.41) 33.00(34.90±12.08) income no income 10.00(10.74±3.99) 0.0001* 32.00(34.96±12.17) 0.0001*< minimum wage 10.00(11.11±4.66) 33.50(36.64±13.59) ≥ minimum wage 9.00(9.67±3.88) 30.00(32.50±11.21) insurance yes 9.00(9.77±3.98) 0.0001* 31.00(33.48±11.77) 0.0001*no 10.00(10.98±4.37) 33.00(35.63±12.97) ever been to dentist yes 9.00(9.91±3.86) 0.0001* 30.00(33.07±11.17) 0.0001* no 11.00(11.50±4.68) 34.00(37.48±14.43) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 178 prihastari et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 175–180 the sociodemographic data for the education variable showed that the highest proportion had a high school education (921 respondents, 50.9%), while 381 (21%) had a basic education. furthermore, 899 respondents (49.6%) received income above the minimum wage (minimum wage in jakarta is rupiah 3,648,035), and a majority (1251, 69.1%) also had either government (bpjs) or private insurance. a total of 1370 respondents (75.6%) have been to the dentist, while the remaining 441 (24.4%) have not. in this study, the levels of dental anxiety in 1811 respondents were as follows: high-level anxiety in 216 respondents (11.9%), extreme anxiety in 79 respondents (4.4%), moderate anxiety in 461 respondents (25.5%), low anxiety in 842 respondents (46.5%) and no anxiety in 213 respondents (11.8%). moreover, the dfs questionnaire (table 2) attributed the main source of fear to the sight and sensation of dental drills and anaesthesia (questions 7–11). this was indicated by the likert scale, with scores of 4 (high fear) and 5 (extreme fear). the percentages of dental fear table 4. the mdas and dfs chi-square test results based on sociodemographic status variable dental anxiety (%) dental fear (%) not anxious somewhat anxious moderately anxious highly anxious extremely anxious p value no fear low fear extreme fear p value gender male 14.7 47.6 23.5 10.5 3.7 0.0001* 67.8 24.5 7.7 0.001 female 8.0 45.1 27.9 13.8 5.3 58.9 31.0 10.0 region north jakarta 10.7 49.0 27.7 10.35 2.3 0.0001* 69.0 24.0 7.0 0.088 central jakarta 16.9 48.8 20.4 10.4 3.5 67.7 25.9 6.5 east jakarta 6.8 44.9 30.0 13.0 5.3 58.7 31.6 9.7 west jakarta 15.1 43.8 21.0 13.6 6.4 62.9 26.0 11.1 south jakarta 13.9 49.2 22.8 11.3 2.9 64.9 28.3 6.8 thousand islands 4.5 41.8 37.3 10.9 5.5 62.7 25.5 11.8 age of subject 17–25 years old 8.8 46.7 28.2 11.5 4.8 0.0001* 60.3 30.6 9.2 0.024* 26–35 years old 11.6 46.4 25.3 11.6 5.1 64.0 28.3 7.7 36–45 years old 15.0 46.2 22.0 13.9 2.9 67.4 23.1 9.5 46–55 years old 18.4 45.3 20.5 12.1 3.7 71.6 19.5 8.9 56–65 years old 21.6 51.4 18.9 8.1 0.0 78.4 16.2 5.4 education higher 14.2 53.0 19.5 9.4 3.9 0.0001* 70.7 22.2 7.1 0.003*secondary 11.2 44.7 28.1 12.6 3.4 62.1 28.2 9.7 basic 10.0 42.3 27.0 13.4 7.3 59.3 32.0 8.7 income no income 7.5 45.1 29.4 13.4 4.6 0.0001* 60.3 29.4 10.2 0.0001*< minimum wage 11.5 38.6 27.0 15.7 7.2 55.0 33.0 12.0 ≥ minimum wage 14.1 51.1 22.6 9.3 2.9 70.1 23.6 6.3 insurance yes 10.5 41.3 24.3 18.0 5.9 0.0001* 57.3 31.6 11.1 0.0001* no 12.3 48.8 26.0 9.2 3.7 66.8 25.5 7.7 ever been to dentist yes 9.3 37.9 27.4 16.6 8.8 0.0001* 53. 3 33.1 13.6 0.0001* no 12.6 49.3 24.8 10.4 2.9 67.3 25.5 7.2 *p = < 0.05: significant table 5. binary logistic regression analysis with dfs and mdas variable mdas dfs p value odds ratio p value odds ratio gender 0.05 0.631 0.0001* 0.660 age 0.253 0.621 0.008* 0.646 education 0.017* 0.543 0.169 0.843 income 0.780 1.080 0.507 0.925 insurance 0.138 1.428 0.001* 1.411 ever been to dentist 0.0001* 2.854 0.0001* 1.756 *p = < 0.05: significant; nagelkerke r2 dfs = 0.05; nagelkerke r2 mdas = 0.066 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 179prihastari et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 175–180 in this study were as follows: high fear in 76 participants (4.2%), moderate fear in 693 participants (38.3%), low fear in 872 participants (48.2%) and no fear in 170 participants (9.4%). bivariate analysis was performed to establish a relationship between gender, region, age, education, income, insurance and history of dental visits (independent variables) and mdas and dfs (dependent variables). this analysis involved the use of nonparametric tests (table 2) and chi-square tests (tables 3 and 4). table 3 shows the nonparametric test results obtained using the mann-whitney/kruskal-wallis tests and indicates a significant difference in mdas and dfs scores in terms of all the variables. mdas and dfs scores were higher for females (10.00[10.86±4.12]; 33.00[35.42±12.24]) than males (9.00[9.85±4.08]; 30.00[33.13±12.06]), and the age group of 17–25-year-olds scored the highest (10.00[10.51±4.09]; 32.00[34.93±12.18]). the scores declined with the subsequent increase in age groups. furthermore, the education variables show higher mdas and dfs scores in participants with lower levels of education (10.00[10.98±4.41]; 33.00[34.90±12.08]) and in those with no or low income below the minimum wage (10.00[10.74±3.99]; 33.50[36.64±13.59]). individuals without insurance and those who had never visited a dentist indicated a higher level of fear and anxiety (11.00[11.50±4.68]; 34.00[37.48±14.43]). the chi-square test results in table 4 showed significant differences in the gender variables: females had a higher proportion of moderate to extreme anxiety and fear towards dental care compared to males. this phenomenon is also higher among the age groups of 17–45-year-olds compared to individuals between 46 and 65 years old. this study also identified differences based on education level, income, insurance and history of dental visits. discussion sociodemographic factors (gender, age, education, etc.) play a role in determining an individual’s fear and anxiety towards dental care.16 the gender variable in this study based on bivariate nonparametric and chi-square analysis of the mdas and dfs scores had a p value of < 0.05, indicating the presence of statistically significant differences, with a higher mean score for females than males (tables 4 and 5). this outcome was congruent with the studies conducted by saatchi et al.14 and fayad et al.7. furthermore, physiological conditions in the nature of phobias, panic, stress, depression and fear are also more common in females; hence, there is a possibility that dental anxiety is related to these. age is one of the factors commonly reported in various studies; although this study reported a significantly higher level of dental fear and anxiety in adolescents than in adults, this contradicts previous studies that found increased dental anxiety in adults 31–35 years of age and which decreased after 60+ years.8 do nascimento et al.17 found that the highest scores (found in the age group of 30–39year-olds) were a result of the relationship between age, an individual’s experiences and views and their maturity level.18 this assumption is supported by the research conducted by fayad et al.7 this study demonstrates that a person’s educational status (primary, secondary or higher) affects the level of fear and anxiety because of the significant differences based on the nonparametric and chi-square tests; the results of this study indicate that the higher a person’s education, the lower the level of dental fear and anxiety. these findings are consistent with other studies that associated a higher level of education to reduced anxiety related to dental care.7,17 an individual with a higher level of education is more likely to maintain better oral health and visit the dentist more frequently.8 this finding is contrary to other studies that reject the assumptions that stipulate the presence of a relationship.7,14 an assessment of the socioeconomic factors related to fear and anxiety towards dental care shows less fear among individuals with higher socioeconomic status. this is congruent with a study conducted by armfield et al.18 in australia, although other studies showed different results.17 the ability to pay for dental care or affiliated insurance premiums is directly related to an individuals’ job status, income and wealth.19 the results obtained from the bivariate analysis with nonparametric tests between mdas and dfs scores with the coverage value showed a p value of < 0.05, which indicates a statistically significant difference between anxiety and fear between people who have insurance and those who do not; this may be because insurance can lower cost-related stress related to dental treatment. however, there are several types of insurance that have limited coverage, especially in terms of some dental procedures, so this can cause concern. there is a need for a broader level of insurance for someone with a higher level of fear, followed by a demand for more care needs. this study provides the same results as previous studies that reported a relationship between dental fear and individuals who have private insurance.18 the regularity of dental visits is considered another important contributor to fear and anxiety for dental care. in this study, the results of the bivariate analysis using nonparametric tests between mdas and dfs scores showed a p value of < 0.05, which means that the difference is statistically significant between the mdas and dfs scores of respondents who visited the dentist (mdas: 9.00[9.91±3.86]; dfs: 30.00[33.07±11.17]) and those who never visited the dentist (mdas: 11.00[11.50±4.68]; dfs: 34.00[37.48±14.43]). this was in line with the research conducted by doganer et al.,20 which showed higher anxiety in participants evading dental treatment than those attending regular appointments. therefore, patients with fear tend to keep their dental appointments only when necessary (e.g. when they can no longer endure the pain); they also avoid routine dental visits.20 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 180 prihastari et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 175–180 the chi-square test results between mdas and dfs scores in relation to respondents’ visits to the dentist indicated the absence of statistically significant differences (p = > 0.05). furthermore, the results show an absence of any possible influence on fear and anxiety towards dental care due to the higher proportion of respondents that regularly visit the dentist. this outcome was in contrast with the results of svensson et al.,8 which demonstrated a significant difference between dental anxiety and the rate of visits. based on the results and discussion, the percentages of dental fear and anxiety in the province of jakarta were 4.2% (high fear), 38.3% (moderate fear), 4.4% (extremely anxious) and 25.5% (moderately anxious). in this study, several sociodemographic factors were confirmed to be related to dental fear and anxiety. in addition, nonparametric test results showed an association between gender, age and income level (p < 0.05), while the chi-square results confirmed a correlation between gender and dental anxiety (p < 0.05) as well as income level and dental fear (p < 0.05). the limitation of this study is that it cannot explain the relationship between sociodemographic factors and dental fear and anxiety, so further longitudinal research is required. however, there are several other factors that are expected to be analysed in future research: the relationship between dental fear and anxiety and respondent behaviours, dental treatment experiences, dentist attitudes and others. acknowledgements the author is grateful to the yarsi university research institute for providing the financial support for this research and to the lecturers for input and revisions to the article. there were no conflicts of interest while conducting the investigation and write up. references 1. yildirim tt. evaluating the relationship of dental fear with dental health status and awareness. j clin diagnostic res. 2016; 10(7): 105–9. 2. brahm co, lundgren j, carlsson sg, nilsson p, corbeil jl, hägglin c. dentists’views on fearful patients. problems and promises. swed dent j. 2012; 36(2): 79–89. 3. pohjola v, mattila ak, joukamaa m, lahti s. anxiety and depressive disorders and dental fear among adults in finland. eur j oral sci. 2011; 119(1): 55–60. 4. armfield jm, heaton lj. management of fear and anxiety in the dental clinic: a review. aust dent j. 2013; 58(4): 390–407. 5. appukuttan dp. strategies to manage patients with dental anxiety and dental phobia: literature review. clin cosmet investig dent. 2016; 8: 35–50. 6. diercke k, ollinger i, bermejo jl, stucke k, lux cj, brunner m. dental fear in children and adolescents: a comparison of forms of anxiety management practised by general and paediatric dentists. int j paediatr dent. 2012; 22(1): 60–7. 7. fayad mi, elbieh a, baig mn, alruwaili sa. prevalence of dental anxiety among dental patients in saudi arabia. j int soc prev community dent. 2017; 7(2): 100–4. 8. svensson l , ha keberg m, wideboma n u. dent a l a n x iet y, concomitant factors and change in prevalence over 50 years. community dent health. 2016; 33(2): 121–6. 9. armfield jm. how do we measure dental fear and what are we measuring anyway? oral health prev dent. 2010; 8(2): 107–15. 10. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 181–222. 11. daiiey ym, humphris gm, lennon ma. the use of dental anxiety questionnaires: a survey of a group of uk dental practitioners. br dent j. 2001; 190(8): 450–3. 12. arslan s, ertaş e, ulker m. the relationship between dental fear and sociodemographic variables. erciyes med j. 2011; 33(4): 295–300. 13. mărginean i, filimon l. dental fear survey: a validation study on the romanian population. j psychol educ res. 2011; 19(2): 124–38. 14. saatchi m, abtahi m, mohammadi g, mirdamadi m, binandeh e. the prevalence of dental anxiety and fear in patients referred to isfahan dental school, iran. dent res j (isfahan). 2015; 12(3): 248–53. 15. pemerintah dki jakarta. peraturan gubernur provinsi dki jakarta nomor 182 tahun 2017 tentang upah minimum provinsi tahun 2018. 2018 p. 1–3. 16. pohjola v. dental fear among adults in finland. oulu: oulu university press; 2009. p. 32–4, 81–2. 17. do nascimento dl, da silva araújo ac, gusmão es, cimões r. anxiety and fear of dental treatment among users of public health services. oral health prev dent. 2011; 9(4): 329–37. 18. armfield jm, spencer aj, stewart jf. dental fear in australia: who’s afraid of the dentist? aust dent j. 2006; 51(1): 78–85. 19. duncan l, bonner a. effects of income and dental insurance coverage on need for dental care in canada. j can dent assoc (tor). 2014; 80: e6. 20. doganer yc, aydogan u, yesil hu, rohrer je, williams md, agerter dc. does the trait anxiety affect the dental fear? braz oral res. 2017; 31: e36. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p175–180 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p175-180 53 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 53 research report dental journal (majalah kedokteran gigi) 2015 june; 48(2): 53–58 expression of bone morphogenetic protein-2 after using chitosan gel with different molecular weight on wound healing process of dental extraction sularsih1 and endah wahjuningsih2 1departement of dental material 2departement of oral biology faculty of dentistry, universitas hang tuah surabaya indonesia abstract background: bone morphogenetic protein-2 (bmp-2) is bone stimulator which capable of inducing differentiation of mesenchymal cells into osteoblast, stimulating bone formation in wound healing process of dental extraction. chitosan is polymer composed n-acetyld-glucosamine unit that has been used in various applications in wound healing process and bone tissue engineering. purpose: the objective of this research was to analyzed expressions of bmp-2 for 7,14 and 21 days after using chitosan gel with different molecular weight on wound healing process of dental extraction. method: the research was an experimental laboratory study. rattus nornegicus strain wistar male, aged 8-16 weeks, divided into 3 treatment groups namely group 1 and ii which given chitosan gel 1 % with high and low molecular weight and group iii as control which were not given chitosan gel. chitosan gel were applied into the socket of dental extraction. rat was decaputated 7,14 and 21 days after chitosan gel application and the jaw in the treated regions and control group were cut for immunohistochemical examination to observe bmp-2. data were analyzed using anova test. result: the result of this research showed significant differences on bmp-2 for 7,14 and 21 days observation (p<0,05). the increasing of bmp-2 were found in the group which given chitosan gel with high molecular weight. conclusion: it may be concluded that chitosan gel with high molecular weight can enhance the expresion of bmp-2 on wound healing process of dental extraction. keywords: chitosan; bone morphogenetic protein-2; wound healing correspondence: sularsih, c/o: departemen ilmu material kedokteran gigi, fakultas kedokteran gigi universitas hang tuah. jl. arif rachman hakim 150 surabaya 60111. e-mail: larsihdentist@gmail.com introduction bone morphogenetic protein (bmp)-2 has been widely used as an effective growth factor in bone tissue engineering. it is a member of the transforming growth factor-β (tgfβ) superfamily of multifunctional cytokines which has remarkable ability to induce bone formation and bone tissue reconstruction. it plays critical roles in osteogenesis and bone metabolism.1 the bone matrix is rich in growth factors, among which bone morphogenetic proteins (bmps), that are synthesized and secreted by osteoblast and incorporated into the matrix during bone formation. the bmps, released during osteoclastic bone resorption, are capable of inducing differentiation of mesenchymal cells into osteoblasts (osteinduction), stimulating bone formation in both remodeling and repairing process.1,2 chitosan is carbohydrate copolymer composed of glucosamine and n-acetyl-d-glucosamine units. chitosan has a number of properties including biocompatibility, biodegradability, mucoadhesiveness and wound healing capabilities that make it useful as a biomaterial.3 chitosan is very versatile and can be prepared as films, gels, sponges and other forms and has been used in various applications including wound healing and bone tissue engineering.2,3 biological activity of chitosan depend on their molecular weight and deacetylation degree. the deacetylation degree dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 54 sularsih and wahjuningsih/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 53–58 and molecular weight are important parameter, which could influences the performance of chitosan in many its application. the deacetylation degree is related to the ability of chitosan to form isoelectric interaction with other molecules. the deacetylation degree more than 75% will make more amide group formed, so chitosan will become have high chemical reactivity, and be able to interact with proteins and other organic matrices, such as anionic glycosaminoglycans and proteoglycans, and extracellular matrix macromolecule. the molecular weight is the sum of the atomic masses of molecule.4,5 it is one factor that determine the mucoadhesive properties. chitosan with high molecular weight powder become chitosan gel with good physical characteristic, higher viscosity, neutral ph and have good mucoadhesive.6 chitosan is able to increase transforming growth factor beta 1 (tgf β1), platelets release transforming growth factor (pdgf), fibroblasts growth factor-2 (fgf2), bmp expression of mrna on the seventh day.6,7 in previous study, injectable chitosan gel has been found significantly enhance the osteoblastic differentiation of mouse osteoblast precursor cells. chitosan gel formulation can release bmp-2 that enhance osteoblastic activity of bone cells by measuring alkaline phosphatase (alp) specific activity of preosteoblast mouse bone marrow stromal cells.8 bmp-2 has been widely investigated for bone healing due to its properties. bmp-2 recruits stem cells to the bone healing, promotes angiogenesis and causes differentiation of stem cells into osteoblast.3,6 in the wound healing process of dental extraction, it have different phases of alveolar healing were recognized by histological examination in socket. at the end of 1st week, the socket were filled granulation tissue and there was osteoid matrix deposition. at the end of 2nd week, there was progressive bone formation, and the end of 3rd week, most socket filled with thicker bone trabecula surrounding interconnecting space.2 the increasing expression of bmp-2 on wound healing process of dental extraction could be achieved by using chitosan gel. the aim of this study was to analyzed expressions of bmp-2 for 7, 14 and 21 days after using chitosan gel with different molecular weight on wound healing process of dental extraction. materials and methods the material in this experiment were chitosan powder purchased from sigma chemical, st. louis, usa. the degree of deacetylation was more than 75%. chitosan with high molecular weight (sigma, product number: 419419, lot number: mkbh5816v) and chitosan with low molecular weight (sigma, product number: 448869, lot number: mkbh7256v), acetic acid 2% p.a (merck, germany), buffer formalin 4% and 10%, ketamin (ketalar,pfzer), xylazine (merck, germany), alkohol 80%, alkohol 95%, alkohol 100 % (absolute), xylene (merck, germany), buffer parafin, edta 10 % (jt baker, usa), naso4 2% (merck, germany), pbs, tripsin 0,125%, h202 0,5%, methanol (merck, germany), naoh 1,25% (merck, germany) and monoclonal antibody bone morphogenetic protein-2 rattus norvegicus. chitosan gel 1% (w/v) was made with diluted one gram of chitosan powder in 100 ml acetic acid 2%. it added with naoh 1,25% solution to get neutral ph. the mixture was stirred until the gel was completely formed. after homogenization, the gels were stored in closed containers at ambient temperature until use. the characteristic of chitosan gel was evaluated includes solubility, ph, viscosity, physical characteristic, homogeneity, consistency, and duration of storage time. the homogeneity test of gel carried out using glass plates after the powder diluted in acetic acid 2%. it should be observed on optimized homogeneous. consistency test could be done by using a penetrometer or mechanically sentrifugator. gel without precipitation will produce a good consistency. physical characteristic test or organoleptic analysis during the storage time includes change of colour, form of formulation gel and odorless. measurement of viscosity using viscometer ostwald (capillary method).8,9 the research was an experimental laboratory study. rattus nornegicus strain wistar male, aged 8-16 weeks, divided into 3 treatment groups namely group 1 and ii which given chitosan gel 1% with high and low molecular weight and group iii as control which were not given chitosan gel. 0.1 ml chitosan gel were applied into the socket of dental extraction. rat was decaputated 7, 14 and 21 days after chitosan gel application and the jaw in the treated regions and control group were cut for immunohistochemical examination to analyze expression of bmp-2. fixation was performed using 10% buffer formalin and decalcification applying edta. further process was dehydration and continued by clearance. the tissue could be cut using microtome in 4-6 µm thickness. deparafin and rehydration were subsequently performed. bmp-2 monoclonal antibody was diluted by antibody diluents. it was washes by pbs. streptavidin-biotin was dropped and incubated for 30 minutes, washed by pbs. counterstained using haematoxyline and washed by flowing water and dried. it was given entelan and covered by cover glass. light microscope was applied and the evaluation was done. the measuring result were analyzed using anova test. it analyzed the comparison between chitosan treated with high molecular weight group, lower molecular weight group and the control groups (p<0.05). results table 1 shown the evaluation of chitosan gel formulation. chitosan gel considered as ideal formulation forms for wound healing dressing could have good characteristic and easy to applicate on dental socket. the expressions of bmp2 for 7, 14 and 21 days observation after using chitosan gel with different molecular weight on wound healing process 55 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 55sularsih and wahjuningsih/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 53–58 table 1. measurement solubility, ph, viscosity, homogeneity, consistency of chitosan gel   ph  viscosity  homogeneity consistency solubility formula of chitosan         high molecular weight 7,02 high excellent excellent good low molecular weight 6,78 low good  good good 11 table 1. measurement solubility, ph, viscosity, homogeneity, consistency of chitosan gel ph viscosity homogeneity consistency solubility formula of chitosan high molecular weight high excellent excellent good 7,02 low molecular weight low good good good 6,78 figure 1. the expressions of bone morphogenetic protein-2 (bmp-2) at 7 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. figure 2. the expressions of bone morphogenetic protein-2 (bmp-2) at 14 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. figure 1. the expressions of bmp-2 at 7 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. 11 table 1. measurement solubility, ph, viscosity, homogeneity, consistency of chitosan gel ph viscosity homogeneity consistency solubility formula of chitosan high molecular weight high excellent excellent good 7,02 low molecular weight low good good good 6,78 figure 1. the expressions of bone morphogenetic protein-2 (bmp-2) at 7 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. figure 2. the expressions of bone morphogenetic protein-2 (bmp-2) at 14 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. figure 2. the expressions of bmp-2 at 14 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. 12 figure 3. the expressions of bone morphogenetic protein-2 (bmp-2) at 21 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. table 2. the mean and standard deviation bmp-2 of each group at 7, 14 and 21 days after treatment 7 days 14 days 21 days variable treatment mean± sd mean± sd mean± sd control 11.17 ± 0.75 13.57 ± 1.81 11.50 ± 1.22 the expressions of bmp-2 chitosan, high bm 18.25 ± 4.42 21.33 ± 2.67 19.29 ± 4.19 chitosan, low bm 11.80 ± 1.92 14.88 ± 1.45 14.00 ± 2.53 figure 4. the graphic of expressions of bmp-2 on 7, 14 and 21 days using chitosan gel with high and low molecular weight group and control group. figure 3. the expressions of bmp-2 at 21 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. of dental extraction shown in figure 1, 2 and 3. the region which would be evaluated and observed in third apical socket. the expressions of bmp-2 in third apical socket was done 400 times magnification in all sub groups. in our study, the expressions of bmp-2 on wound healing process of dental extraction using chitosan was more increasing compared to the control group. the expressions of bmp-2 in 7, 14 and 21 days observation after using chitosan gel with high molecular weight was more increasing than the other groups. table 2 and figure 4 shown the mean and standard deviation of each group from immunohystochemistry study done in 7, 14 and 21 days after treatment. the expressions of bmp-2 for 7, 14 and 21 days observation after using chitosan gel with different molecular weight on wound healing process of dental extraction. the data a a b c b c a b c dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 56 sularsih and wahjuningsih/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 53–58 was analyzed using kolmogorov-smirnov statistical test. it showed normal distribution (p>0.05) in which fulfiiling the requirement of parametric test. the anova test showed, expressions of bmp-2 in treatment group using chitosan gel with high molecular weight were significant difference compared to the treatment group using chitosan gel with low molecular weight and control group (p<0.05). discussion the proliferation and differentiation of sufficient progenitor cells are critical for bone healing. these processes are regulated by growth factors such as tgf-β and bmps. bmp-2 can efficiency induce bone formation. many materials, especially the natural polymers, have been researched for controlled release of bmp-2.10,11 chitosan is naturally derived polysaccharide. it has gained much attention as biomaterial in tissue engineering application, due to its antimicrobial activity and biocompatibility.10 chitosan is reported to be biocompatible, biodegradable and non toxic, having some biomedical properties such as healing accelerator, antifungal, hemostatic, antimicrobial and analgesic. several study have shown the activity of chitosan in healing process, where it enhanced the infiltration of inflamamatory cells in the injured area and release growth factor.6,12 chitosan is the partially deacetylated form poly-(1,4)-2-amino-2-deoxy-d-glucose) of chitin, and is a potential biomaterial for bone tissue engineering application.1,10,12 in the present study, the chitosan powder was mixed with blood of each person and filled in dental socket, and it found that bone tissue regeneration will be faster in chitosan-filled socket than untreated dental socket.13 in our study it was used chitosan gel material with different molecular weight to analyzed expressions of bmp-2 rattus norvegicus on wound healing process of dental extraction. it showed the expressions of bmp-2 in 7 and 14 days observation after using chitosan gel was increasing and decreasing at 21 days observation. chitosan table 2. the mean and standard deviation bmp-2 of each group at 7, 14 and 21 days after treatment 7 days 14 days 21 days variable treatment mean± sd mean± sd mean± sd   control 11.17 ± 0.75 13.57 ± 1.81 11.50 ± 1.22 the expressions of bmp-2 chitosan, high bm 18.25 ± 4.42 21.33 ± 2.67 19.29 ± 4.19   chitosan, low bm 11.80 ± 1.92 14.88 ± 1.45 14.00 ± 2.53 12 figure 3. the expressions of bone morphogenetic protein-2 (bmp-2) at 21 days observation, magnification 400x. (a) control group, without using chitosan; (b) treatment group using chitosan gel with high molecular weight; (c) treatment group using chitosan gel with low molecular weight. table 2. the mean and standard deviation bmp-2 of each group at 7, 14 and 21 days after treatment 7 days 14 days 21 days variable treatment mean± sd mean± sd mean± sd control 11.17 ± 0.75 13.57 ± 1.81 11.50 ± 1.22 the expressions of bmp-2 chitosan, high bm 18.25 ± 4.42 21.33 ± 2.67 19.29 ± 4.19 chitosan, low bm 11.80 ± 1.92 14.88 ± 1.45 14.00 ± 2.53 figure 4. the graphic of expressions of bmp-2 on 7, 14 and 21 days using chitosan gel with high and low molecular weight group and control group. figure 4. the graphic of expressions of bmp-2 on 7, 14 and 21 days using chitosan gel with high and low molecular weight group and control group. b m p -2 control 7 days high bm 7 days low bm 7 days control 14 days high bm 14 days low bm 14 days control 21 days high bm 21 days low bm 21 days group 57 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 57sularsih and wahjuningsih/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 53–58 is a polycationic complex carbohydrate with a structure similar to those of glycosaminoglycans specifically hyaluronic acid. hyaluronic acid is thought to facilitate the migration and proliferation of progenitor cells. previous studies have demonstared that chitosan might function as a substrate that enhances the migration and differentiation of osteoblast cells and conversely, it might inhibit the function of fibroblast and so indirectly facilitate osteogenesis.14,15 its biological properties of chitosan, including chitosan biodegradation by lysozim that can change chitosan polymer form (n-acetyl-d-glucosamine) to dimer active form. n-acetyl-d-glucosamine dimer active form crosslinked with matrix macromolecules extracellular as well as stimulate increased tgf-ß and bmp expression of mrna.6,15 chitosan loading rhbmp-2 can facilitate bone regeneration of periodontal tissue. in the cavities filled with chitosan, not only the bone regeneration was faster, but also the density was similar to the density of the bone of the subject under study.13 according to the figure 4, the expressions of bmp-2 in 7 and 14 days observation after using chitosan gel with high molecular weight was more increasing than the other groups. chitosan was the material used to form gels due to its good gel forming property, biodegradability, biocompatibility and non toxic. gels could be considered as ideal pharmaceutical forms to treat the dental socket. it have the ability to absorb exudates, which moisture on the wound surface. besides, gels have high water vapor and oxygen permeability, as well as mechanical properties that resemble physiological soft tissue. chitosan gel has a strong tissue-adhesive property.12,17,18 the characteristic of chitosan gel is related with its molecular weight. the absorbtion mechanism also depend upon the particle size of chitosan powder. the molecular weight related to particle size of powder. molecular weight and particle size has been linked with viscosity, low molecular powder which small particle size will result formulation with a low viscosity. in addition the molecular weight is also directly related to length of molecular chain, more longer the molecular chain, the viscosity more higher.19-21 the viscosity of chitosan gel with high molecular weight more higher than formulation with low molecular weight. it have high viscosity and better mucoadhesive properties due to strong blood clot is formed in order to eliminate complications of dental extraction such as dry socket.19,20,22 chitosan with high molecular weight have more biodegradable induce n-acetyl-dglucosamine dimer active of chitosan cross-linked with glycosaminoglycan and glycoprotein that part of matrix macromolecules extracellular as well as stimulate increased bmp-2 that is growth factor of bone healing. however, it can promote the accleleration of wound healing process on dental socket.15,16,22 bmp-2 can efficiently induce bone formation. it has been widely use as an effective growth factor in bone tissue engineering. using c1c12 myoblast cells as in vitro models, the enhanced bioactivity of bmp-2 was attributed primarily to the stimulation of 6-o sulfated chitosan. a low dose of 6-o sulfated chitosan showed significant enhancement alkaline phosphatase activity and mineralization induced by bmp-2, as well as the expression of alp and osteocalcin mrna.23 alp is an enzyme regarded to be important in the process of mineralization, since high levels of alp would promote hydrolysis of phosphate, produce orthophosphate and increase the calcium deposition. alp activity and calcium deposition are widely used as marker for early and late differentiation of osteoblast cells, respectively.15 mature osteoblast produce mineralized bone matrix, and the component of this matrix, such as calcium deposition and osteogenic markers (osteocalcin and osteopontin), are usually used as markers for late stage of osteoblast differentiation. osteocalcin and osteopontin expression marks the late stage of osteoblast differentiation, and bmp-2 is an important factor for osteoblast maturation and osteoblast activity.2,3,15 in our study, the expressions of bmp-2 in 21 days observation after using chitosan gel with high molecular weight was decreasing. probably, the activity of alp has decreased. there was most socket filled with thicker bone trabecula surrounding interconnecting space in the end of the 3nd week. the major proportion of bone formation and the maximum mineral density has occurred in the end of the 2nd week. in the end of the 2nd week the activity of alp has decreased 2 the using of chitosan gel with high molecular weight could increasing the expression of bmp-2. bmp-2 is major growth factor in bone healing. it may be concluded that chitosan gel with high molecular weight could accelerate bone healing process of dental extraction. references 1. zhou h, qian j, wang j, yao w, liu c, chen j, cao x. enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-n6-o-sulfated chitosan in vitro and in vivo. biomaterials 2009; 30(9): 1715-24. 2. calixto r f, teófilo jm, brentegani lg, lamano-carvalho tl. alveolar wound healing after implantation with a pool of commercially available bovine bone mor phogenetic protein (bmps)–a histometric study in rats. braz dent j 2007; 18(1): 2933. 3. reves b, jennings j. osteoinductivity assessment of bmp-2 loaded composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pounch. materias j 2011; 4: 1360-74. 4. park jk, chung mj, choi hn, park yi. effect of molecular weight and deacetylation degree of chitosan oligosaccharides on antitumor activity. int j mol sci 2011; 12(1): 266-77. 5. khan t, peh k. mechanical, bioadhesive strength and biological evaluation of chitosan films for wound dressing. j pharm pharmaceut sci 2000; 3(3): 303-11. 6. sularsih, wahjuningsih e, yoshe y. the characterization chitosan gel on wound healing process of dental extraction. proceedings of 8th international dental scientific meeting dentistry faculty of hasanudin university, makasar, 2014 juni 20-22. 7. kim s. neuroprotective properties of chitosan and its derivatives. marine drug j 2010; 8(10): 2117-28. 8. kim s, kang y, kruegar c. sequential delivery of bmp-2 and igf-1 using chitosan gel with gelatin microspheres enhances early osteoblastic differentiation. acta biomater j 2012; 8(5): 17. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 58 sularsih and wahjuningsih/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 53–58 9. matsunaga t, yanagiguchi k, yamada s, ohara n, ikeda t, hayashi y. chitosan monomer promotes tissue regeneration on dental pulp wounds. j biomed mater res a 2006; 76(4): 711-20. 10. mappa t, edy hj, kojong n. formulation gel of extract sasaladahan leave (peperomia pellucida (l.) h.b.k.) and effectivity test to wound healing process of burn wound. pharmacon jurnal ilmiah farmasi–unsrat 2013; 2(02): 49-55. 11. anggraeni y, hendradi e, purwanti t. the characteristic of formulation natrium diklofenak in system niosom with base gel of carbomer 940. pharma scientia 2012; 1(1): 1-15. 12. yun y, kim s, kang e. the effect of bone morphogenetic protein-2 (bmp-2) immobilizing heparinized-chitosan scaffolds for enhanced osteoblast activity. j tissue engineering and regenerative medicine 2013; 10(3): 1308-777. 13. kim s, kang y, kruegar c.sequential delivery of bmp-2 and igf-1 using chitosan gel with gelatin microspheres enhances early osteoblastic differentiation. acta biomater j 2012; 8(5): 17. 14. nascimento eg. evaluation of chitosan gel 1% silver sulfadiazine as an alternative for burn wound treatment in rats. j acta cirurgica brasileira 2009; 24(6): 460-5. 15. ezoddini a, azam a, yassei s. effect chitosan on dental bone repair. j health 2011; 3(4): 200-5. 16. silberman m. use of chitosan for stimulating bone healing and bone formation. available from http://d:/chitosan/bmp/bmp/patente wo2006057011a2.2006. accessed may 13, 2015. 17. pang e, paik j, kim s, jung u. effect of chitosan on human periodontal ligament fibroblast in vitro and on bone formation in rate calvarial defect. j periodontal 2005; 75: 1526-33. 18. chin l, halim as. in vitro models in biocompability assessment for biomedical-grade chitosan [derivatives in wound management. j molecular science 2009; 10(3): 1300-13. 19. karthikeyan g, adsorbtion dynamics and equilibrium studies of zn onto chitosan. j chem sci march 2004; 116(2): 119-27. 20. zeng l. absrobtion and distribution of chitosan in mice after oral administration. j carbohydrat polymer 2008; 71: 435-40. 21. rochima e, suhartono mt, syah d, sugiono. the viscosity and molecular weight chitosan from chitin deasetilate enzymatic reaction isolat bacillus papandayan. national congress of agricultural assosiation (patpi), bandung 2007. p. 2-10 22. sularsih. type 1 collagen on wound healing process of dental extraction with different molecular weight of chitosan. proceeding of international congress dentisphere 3, november 2013. p. 1-6 23. kim s. chitin chitosan oligosaccarides and their derivatives. usa: crc press; 2011. p. 233-8. mkg vol 39 no 1 jan 2006 isi.pmd 8 study on the cytotoxicity and chromosome aberration following implantation of sea coral in rabbits kannan tp, freeda woon faiza woon tai keat, widowati witjaksono and abdul rani samsudin school of dental sciences, university sains malaysia kubang kerian, kelantan malaysia abstract coral has been used as a bone substitute in many experimental studies. it has been proven to be biocompatible, biodegradable and easy to handle: and it has not been found to cause any inflammatory responses. the present study was undertaken to determine the cytotoxicity in terms of mitotic index as well as the clastogenic effect (chromosome aberration) of sea coral implantation in rabbits. the animals comprised of five male adult healthy new zealand white (oryctolagus cuniculus) rabbits. the biomaterial, sea coral granules used in this study was obtained from porites species and processed by the tissue bank of universiti sains malaysia, health campus, kubang kerian, malaysia. the blood samples were collected twice from the rabbits, once before the implantation of the sea coral granules (which acted as the control) and the other, one week after the implantation (which acted as the treatment) and lymphocyte cultures were set up. the cultures were then harvested and the chromosomes were prepared for analysis. the diploid number of chromosomes in the rabbits (oryctolagus cuniculus) was found to be 44. mean mitotic indices of 3.84 ± 0.54 per cent and 3.76 ± 0.23 per cent were obtained before and after implantation of sea coral granules respectively. there were no structural or numerical chromosomal aberrations observed in both the cases. the mitotic index values and chromosomal analyses in this preliminary study carried out indicate that the biomaterial, sea coral granules is non-cytotoxic and non-clastogenic under the present test conditions. key words: sea coral, cytotoxicity, chromosome aberration correspondence: kannan tp, school of dental sciences, university sains malaysia, 16150 kubang kerian, kelantan, malaysia. telp: 60–9–7663684 e-mail: tpkannan@kb.usm.my introduction autogenous bone graft has been considered the gold standard for bone repairing procedures as it contains triggering ingredients necessary for bone formation in bone defect. however, the availability of the autograft bone is limited and the harvesting of autografts bone causes morbidity at the donor site. therefore, allografts are used as an alternative to autografts. eventually, alternative bone substitute materials or biomaterials have been developed.1 most corals are colonial organisms consisting of thousands of individual polyps.2 natural coral graft substitutes are derived from the exoskeleton of marine madreporic corals. coral skeletons are composed primarily of calcium carbonate (99%) in the form of aragonite and the remaining 1% is composed of simple amino acids.3 researchers first started evaluating corals as potential bone graft substitutes in the early 1970s in animals and in 1979 in humans. the structure of the commonly used coral, porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. the exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site and the species. coral grafts act as an adequate carrier for growth factors and allow cell attachment, growth, spreading and differentiation. when applied appropriately and when selected to match the resorption rate with the bone formation rate of the implantation site, natural coral exoskeletons have been found to be impressive bone graft substitutes.4 coral has been used as a bone substitute in many experimental studies.5,6 it has been proven to be biocompatible, biodegradable and easy to handle: and it has not been found to cause any inflammatory responses.7,8 an ideal bone substitute should be biocompatible, which means acceptance of the implant to the tissue surface.8 recognition of an implant material as biocompatible nowadays depends on a large number of factors such as absence of cytotoxicity, mutagenicity, carcinogenicity, exclusion of allergenic properties, physical-chemical and biological inertia and its stability in its biological environment.9 the chromosome aberration test using cultured mammalian cells is one of the sensitive methods to predict mutagens and/or carcinogens and is a complementary test to the salmonella/microsome assay.10 the aim of this study was to determine whether the implantation of sea-coral granules (obtained from the porites species and processed by tissue bank of universiti sains malaysia, health campus, kubang kerian, malaysia) produced any cytotoxicity in terms of mitotic index and chromosome aberration in the lymphocytes of rabbits. 9kannan et al: study on the cytotoxicity and chromosome materials and methods the animal ethics committee of health campus, universiti sains malaysia, approved this experimental study. five adult male rabbits of new zealand white breed were used in the present study. blood samples were collected prior to (which acted as the control) and one week after (which acted as the treatment) the implantation of the biomaterial. the coral used in this study is derived from a species of dead sea coral, porites species that was harvested from the malaysian coastal region. this coral was cleaned from debris and washed with distilled water, made into granular form and then chemically treated followed by freezedrying. after that this sample was triple packed and sterilized using gamma radiation. the animals were anaesthetized using xylazine and ketamine and a cavity of size 2.5 cm × 0.5 cm × 0.5 cm was made in the femur of the rabbits using a surgical bur. 500 mg of the biomaterial, sea coral granules was implanted in the cavity (figure 1). blood samples were collected from the ear vein of rabbits prior to and after the implantation of the biomaterial. rpmi 1640 medium, mitogen, phytohaemagglutinin m, fetal bovine serum, l-glutamine, antibiotic solutions, penicillin and streptomycin and 0.3 ml of whole blood were the constituents of the cultures set which were incubated at 37 °c co2 incubator for 72 hours (figure 2). the cultures were mixed twice daily by gentle shaking during the incubation period. colcemid was added to the cultures 1½ hours prior to harvesting to arrest the cell division at metaphase. hypotonic treatment was given using 0.56 percent potassium chloride solution for 30 minutes to swell the cells and then the cells were fixed using 3 : 1 methanol : acetic acid. the cultures were left in the refrigerator overnight at 4 °c for effective fixation of cells. next day, the cultures were repeated with fresh fixative washes twice and the chromosomes were prepared on clean grease free slides. the slides were stained with leishman’s stain for analysis. the mitotic index was calculated as follows. 100 ×= counted cells ofnumber total metaphase incell ofnumber indexmitotic the slides were screened for both the numerical and structural aberrations in all the samples collected prior to and after the implantation of the biomaterial. results good metaphase chromosomes were obtained with rpmi 1640 as medium and the addition of colcemid 1½ hours prior to harvesting for culturing the lymphocytes of rabbits. the protocols of the culture like hypotonic treatment using 0.075 m potassium chloride solution (0.56 per cent) at 37 °c for 30 minutes and fixation of cells overnight at 4 °c resulted in consistent results with regard to chromosome preparation. the diploid number of chromosomes in new zealand white rabbit (oryctolagus cuniculus) was found to be 44. a total of 1000 cells were counted per culture to determine the mitotic index values to assess the cytotoxicity of the sea coral granules. the mean mitotic index values of the lymphocyte cultures from the five rabbits, before and after implantation of sea coral granules were 3.84 ± 0.54 and 3.76 ± 0.23 percent respectively as given in table 1. figure 1. implantation of sea coral granules. figure 2. incubation of culture. table 1. mean mitotic indices of lymphocytes cultured from five rabbits mean ± se parameter n before implantation of sea coral granules after implantation of sea coral granules mitotic index (%) 1000 3.84 ± 0.54 3.76 ± 0.23 n = number of cells counted/sample 10 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 8–11 two hundred metaphase spreads per culture sample were analyzed to screen for any chromosomal aberrations that might have been caused due to the implantation of sea coral granules. however, in the present study, no chromosome aberrations, either numerical or structural were observed as shown in the metaphase photos of chromosomes taken before (figure 3) and after implantation of the biomaterial (figure 4). produced synthetically. the sea coral granules used in the present study went through material characterization studies, biological validation studies, in vitro and in vivo and finally followed by a controlled clinical trial. this biomaterial has similar characteristics to bone with a pore size ranging from 60 to 800 μm. chemical analysis using x-ray diffraction showed that the processed coral material is composed of 99 percent calcium carbonate in the form of aragonite and was also free of other elements and toxic compounds. the current status of a new bone graft substitute is made by conversion of calcium carbonate skeleton of the coral into hydroxyapatite. hydroxyapatite does not cause a chronic inflammatory response, toxic reactions and have excellent biocompatibility.13 in addition efforts have been made to enhance the mechanical properties of coralline hydroxyapatite to extend the field of application.14 if such materials are intended for use in medicine, the combination of two different mutagenesis studies (e.g. bacteria and mammalian cells) continues to be necessary. many of them are based on the principle that genotoxicity of mutagenicity serves as an indicator for the carcinogenic potential of the substance.15 under the organization for economic co-operation and development (oecd) high production volume (hpv) testing program, chromosomal aberrations tests of the hpv industrial chemicals have been conducted using chinese hamster lung cells to examine the induction of chromosomal aberration.16 similarly, genotoxicity of dental amalgams were assessed using chromosome aberration test in syrian hamster embryo cells.17 the culture technique for rabbits followed in the present study for whole blood yielded consistent results which are in agreement with the reports of earlier workers.18-21 the diploid chromosome number for rabbits was reported to be 2n = 44 first by painter22 in the year 1962. the modal chromosome number of rabbit (oryctolagus cuniculus) in the present study was also found to be 2n = 44. the mitotic index is a measure of cytotoxic/cytostatic effects and depends on the time after treatment. at the time of harvesting, a reduction in degree of mitotic index (greater than 50 per cent) is considered as an indication of cytotoxicity.23 a reduction greater than 50 per cent in the mitotic index value after treatment when compared to the control indicates that the biomaterial is cytotoxic in nature. however, the values of the mitotic indices obtained in the present study does not show greater than 50 per cent reduction 3.76 ± 0.23 per cent (after treatment) when compared to the control, 3.84 ± 0.54 per cent (before treatment) which indicates the non-cytotoxic nature of the biomaterial. in this preliminary study carried out, no chromosome aberrations, either numerical or structural were observed before or after implantation of sea coral granules. a normal complement of 2n = 44 xy devoid of any chromosomal aberrations were observed in the cells that were analyzed, both in the control and treatment groups. this shows that the result is negative and that the biomaterial, sea coral figure 3. metaphase spread of male rabbit ( o r y c t o l a g u s c u n i c u l u s ) b e f o r e implantation of sea coral granules. figure 4. metaphase spread of male rabbit (oryctolagus cuniculus) one week after implantation of sea coral granules. discussion when a bone substitute with some mechanical strength is needed, hydroxyapatite appears most appropriate, as it represents the natural mineral in human bone and approximates the natural structure of cancellous bone.11 the recent developments in the use of resorptive biomaterials as osseous substitutes show a growing interest by the potential users (orthopaedic and maxillo-facial surgeons, aestheticians, etc.12 some hydroxyapatite bone substitutes are made from bovine bone while others are 11kannan et al: study on the cytotoxicity and chromosome granules does not cause any chromosome aberrations in cultured mammalian cells.23 the mitotic index values indicate the non-cytotoxic nature of the sea coral granules. similarly, the chromosomal analyses show neither structural (clastogenicity) nor numerical aberrations (aneuploidy) in this study, which shows that the biomaterial is nonclastogenic. hence, it can be concluded that the noncytotoxic and non-clastogenic nature of the sea coral granules may be attributed to the reason that it is devoid of any toxic compounds as well as due to the biocompatible nature of the hydroxyapatite. acknowledgements the authors would like to thank dr. suzina sheikh abdul hamid and the staff of tissue bank for providing the biomaterial for conducting this research. thanks are also due to cik. rodiah mohd. radzi and en. shaharol anuar abd. latif for their help rendered in animal house during this study. references 1. tuominen t, jämsä t, tuukkanen j, nieminen p, lindholm tc, lindholm ts, jalovaara p. native bovine bone morphogenetic protein improves the potential of biocoral to heal segmental canine ulnar defects. international orthopaedics 2000; 24:289–94. 2. souter dw, linden o. the health and future of coral reef systems. ocean and coastal management 2000; 43:657–88. 3. ouhayoun jp, shabana ah, issahakian s, patat j-l, guillemin g, sawaf mh, forest n. histological evaluation of natural coral skeleton as a grafting material in miniature swine mandible. j material sciences 1992; 3:222–8. 4. demers c, hamdy cr, corsi k, chellat f, tabrizian m, yahia l. natural coral exoskeleton as a bone graft substitute: a review. biomedical materials and engineering 2002; 12:15–35. 5. begley ct, doherty mj, ram m, wilson dj. comparative study of the osteoinductive properties of the bioceramic, coral and processed bone graft substitutes. biomaterials 1995; 16:1181–95. 6. damien cj, ricci jl, christel p, alexander h, patat jl. formation of a calcium phosphate rich layer on absorbable calcium carbonate bone graft substitutes. calcif tissue int 1994; 55:151–8. 7. guillemin g, patat jl, fournie j, chetail m. the use of coral as a bone graft substitute. j biomed mater res 1987; 21:557–67. 8. suchanek w, yoshimura m. processing and properties of hydroxyapatite-based materials for use as hard tissue replacement implants. j mater res 1998; 13:94–103. 9. katzer a, marquardt h, westendorf j, wening jv, von foerster g. polyetheretherketone-cytotoxicity and mutagenicity in vitro. biomaterials 2002; 23:1749–59. 10. ishidate m, miura kf, sofuni t. chromosome aberration assays in genetic toxicology testing in vitro. mutation rresearch 1998; 404:167–72. 11. cooke fw. ceramics in orthopaedic surgery. clin orthop 1992; 276:135–46. 12. braye f, irigaray, jallot e, oudadesse h, weber g, deschamps n, deschamps c, frayssinet p, tourenne p, tixier h, terver s, lefaivre j, amirabadi a. resorption kinetics of osseous substitute, natural coral and synthetic hydroxyapatite. biomaterials 1996; 17:1345–50. 13. constantino pd, friedman cd, lane a. synthetic biomaterials in facial plastic and reconstructive surgery. facial plastic surgery 1993; 9:1–15. 14. sivakumar m, manjubala i. preparation of hydroxyapatite/ fluoroapatite-zirconia composites using indian corals for biomedical applications. materials letter 2001; 50:199–205. 15. wiliams g. batteries of short term tests for carcinogen screening. in: williams g, et al., editors. the predictive value of short term screening tests in carcinogenicity evaluation. north holland: elsevier biomedical press; 1980. p. 237. 16. kusakabe h, yamakage k, wakuri d, sasaki k, nakagawa y, watanabe m, hayashi m, sofuni t, ono h, tanaka n. relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals. mutation research 2002; 517:187–98. 17. akiyama m, oshima h, nakamura m. genotoxicity of mercury used in chromosome aberration tests. toxicology in vitro 2001; 15:463–7. 18. stranzinger gf, miller rc, fechheimer ns. an improved and simple leucoctye culture technique for chromosomal preparation in rabbits. cytologia 1974; 39:161–4. 19. chan fph, sergovich fr, shaver el. banding patterns in mitotic chromosomes of the rabbit (oryctolagus cuniculus). can j genet cytol 1977; 19:625–32. 20. schröder j, suomalainen h, van der loo w, schröder e. karyotypes in lymphocytes of two strains of rabbit and two species of hare. hereditas 1978; 88:183–8. 21. schröder j, van der loo w. comparison of karyotypes in three species of rabbit: oryctolagus cuniculus, sylvilagus nuttallii, and s. idahoensis. hereditas 1979; 91:27–30. 22. painter ts. studies in mammalian spermatogenesis. vi. the chromosomes of the rabbit. j morph physiol 1926; 43:1–43. 23. health effects test guidelines – oppts 870.5375. in vitro chromosome aberration test – united states environmental protection agency. 1998 august. 1–11. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1313 dental journal (majalah kedokteran gigi) 2023 march; 56(1):13–16 case report creeping attachment post-gingival recession treatment using a vestibular incision subperiosteal tunneling access technique combined with a connective tissue graft hapsari kartika prathivi,1 rezmelia sari2 1periodontics resident, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of periodontics, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: the exposed roots of teeth due to gingival recession can generate dentin hypersensitivity and esthetic problems because a patient feels that the teeth, especially the anterior teeth, look long. recession in thin anterior gingiva often brings unsatisfactory treatment results, so mucogingival surgery, such as vestibular incision subperiosteal tunneling access (vista) with the addition of a connective tissue graft (ctg), can be chosen as an appropriate treatment technique. purpose: this case report describes the creeping attachment phenomenon after treatment of anterior gingival recession with vista and ctg techniques. case: a 28-year-old female patient came with miller class i gingival recession in thin anterior gingiva and malposition teeth, complaining about pain and esthetic problems. case management: the patient was treated with a vista technique combined with ctg. the creeping attachment phenomenon seen at three months postoperatively obtained good root coverage so that the patient’s complaints were resolved even though periodic control was needed to evaluate oral hygiene. conclusion: vista and ctg techniques are appropriate for treating anterior mandibular gingival recession with minimal trauma and provide significant results. keywords: anterior gingiva; creeping attachment; ctg; medicine; vista article history: received 16 april 2022; revised 6 june 2022; accepted 26 july 2022 correspondence: rezmelia sari, department of periodontics, faculty of dentistry, universitas gadjah mada. jl denta sekip utara, sinduadi, mlati, sleman, special region of yogyakarta 55281, indonesia. email: rezmelia_sari@mail.ugm.ac.id introduction gingival recession is clinically characterized by an apical shift of the gingival margin away from the cementoenamel junction.1 pathologically, gingival recession can be caused by dental malposition, frenulum traction, orthodontic treatment, surgical procedures, and hard tooth brushing.2–4 this condition delivers dentin hypersensitivity, complicates the cleaning process, and interferes with esthetics.5,6 various treatment modalities can cover the root surface. still, in patients with thin gingiva, it is necessary to choose the proper technique to reduce the risk of treatment failure.7 thin gingiva is visible from its smooth and transparent surface. the bone beneath thin gingiva is likely to be fenestrated, dehydrated, and susceptible to resorption due to the lack of keratinized gingiva covering (<1.5 mm).8,9 in patients with thin gingiva, recession treatment can be performed using the vestibular incision subperiosteal tunneling access (vista) technique to avoid tissue trauma due to gingival margin rupture. this technique is the tunneling modification with a vestibular access incision to maintain the blood supply and integrity of the interdental papillae.10 gingival grafts with a connective tissue graft (ctg) were added to increase the thickness of keratinized gingiva with minimal trauma to the donor area while achieving better esthetics.11 mucogingival surgery has been carried out to cover the root surface. an interesting phenomenon that often occurs after mucogingival surgery is the coronal migration of the gingival margin, known as creeping attachment.12,13 this case report aimed to present the gingival creeping attachment after recession treatment with the vista technique combined with ctg. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p13–16 mailto:rezmelia_sari@mail.ugm.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p13-16 14 prathivi and sari. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 13–16 case a 28-year-old female patient came to the periodontics specialist clinic, dental and oral hospital, universitas gadjah mada, prof. soedomo, yogyakarta, indonesia. the chief complaints were long teeth and pain when drinking cold water and brushing her teeth starting two months ago (april 2021). clinical examination presented a labioversion #41 with a 4-mm recession height and a 3.5-mm recession width. the patient had a class ii division i angle’s malocclusion with a 6-mm overjet, a 3.5-mm overbite, and a shallow vestibule. thin gingiva with a keratinized gingival width of <1 mm was seen (figure 1a). radiographic examination showed a normal alveolar bone crest and furcation (figure 1b). based on the analysis, a miller class i gingival recession was diagnosed due to the accumulation of plaque and calculus, which was exacerbated by thin gingiva and malposition teeth. case management the treatment plan was to remove the causative factor, followed by surgery. the patient was informed about the treatment procedures, risks, complications, and alternative treatments and asked to sign an informed consent. dental health education and plaque control were performed at the first visit, followed by scaling, root planing, and polishing. on the next visit, the gingival recession was treated with vista and ctg. the procedure was initiated with asepsis and anesthesia infiltration in the recipient area (#41, #31). a vertical incision was made in distal #41 from the mucogingival junction towards the incisal as high as a cementoenamel junction (figure 2a), followed by forming a subperiosteal tunnel distal #31 to mobilize the gingival margin and facilitate repositioning toward the coronal (figure 2b).10 the donor area was cleaned of deposits, and ctg was taken from the palate area of #14–16 (figure 3a). a a b figure 1. gingival recession #41 with thin gingiva and malposition teeth: (a) clinical, (b) radiographic. b a figure 2. (a) vertical incision distal #41, (b) subperiosteal tunnel until distal #31. a b c figure 3. (a) harvesting ctg of #14–16 palatal area, (b) collagen membrane inserted, (c) suturing donor area with blue nylon #5.0. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p13–16 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p13-16 15prathivi and sari. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 13–16 collagen membrane was inserted to prevent bleeding and maintain palatal volume (figure 3b), then sutured with blue nylon #5.0 (figure 3c). a ctg was applied to the recipient area through the tunnel area of the vestibule (figure 4a) and fixed using a #5.0 monophyte (figure 4b). the flap was then pulled coronally, the incision margin was sutured, and the recipient area was covered with a periodontal dressing (figure 4c). post-surgical medication was amoxicillin 500 mg every eight hours for five days, mefenamic acid, and 0.2% chlorhexidine mouth rinse. the patient was advised to consume soft foods during recovery, avoid hot and spicy foods, and use the modified stillman brushing method.14 the periodontal dressing was removed on day 7, and the sutures were removed on day 14. one month after surgery, the patient’s pain complaints were reduced, the recipient and donor areas had healed, and the gingiva had a coral pink color (figure 5a). two months postoperatively, the pain disappeared, and the gingiva appeared thickened with a recession height of 3 mm (figure 5b). the following month, the gingiva appeared thickened, was a coral pink color, and the recession height had reached 1 mm, meaning the patient’s esthetics were achieved (figure 5c). discussion the choice of surgical technique in gingival recession treatment needs to consider several factors, including the recession size, the thickness of the keratinized tissue around the recession, the width and height of the interdental soft tissue, the vestibule depth, and the frenulum involvement. another factor to consider is esthetics and the patient’s expected outcome, with the minimum possible number of surgeries and intraoral surgical sites.15 treatment of localized miller class i gingival recession on the mandibular incisors was performed using vista and ctg techniques. a vestibular access incision in the vista technique aims to reduce the risk of trauma to the thin gingival margin that often occurs in intrasulcular tunneling. in this technique, careful subperiosteal dissection should be performed to reduce the tension of the gingival margin in coronal movement by avoiding papillary reflection to maintain the integrity of interdental papillae.10 using ctg by placing deepithelialized connective tissue into the recipient area will obtain perfect chromatic integration and optimal esthetic results. another advantage is the availability of ctg blood supply from the recipient area and the flap that covers it, affecting the graft tissue’s survival. it also minimizes a patient’s discomfort because the ctg donor area will heal faster.16 gingival wound healing after mucogingival surgery begins with a blood clotting process followed by a granulation phase characterized by a thickening of the gingiva and a maturation phase characterized by creeping attachment.17 creeping attachment phenomenon refers to the coronal migration of the gingival margin after mucogingival surgery. the achievement of creeping attachment can be influenced by the recession’s width, the graft’s position on the root surface, the position of the teeth in the arch, the height of the interproximal bone, and the patient’s oral hygiene. several clinicians have reported that the creeping attachment phenomenon is best in the localized recession of the mandibular anterior teeth after mucogingival surgery with an autologous graft. this phenomenon can occur from the second month and continue until 12 months after or even longer.18 figure 4. (a) application of ctg through tunnel vestibule, (b) suture with monophyte #5.0, (c) the recipient area is covered with a periodontal dressing. a b c figure 5. (a) one-month follow-up, (b) two-month follow-up, (c) three-month follow-up. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p13–16 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p13-16 16 prathivi and sari. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 13–16 in their study of two case reports, soldatos et al.19 described that gingival recession treatment with the same gender and same age and similar cases showed creeping attachment at the two-month post-operative visit. correspondingly, the 3-mm gingival growth in the patient with the localized recession of #41 that appeared three months postoperatively was also explained as a creeping attachment phenomenon. gul et al.20 described that the amount of creeping attachment gained in their study was proportional to the depth of recession defect. the physiological mechanism that influences this phenomenon comes from the proliferation of periosteal connective tissue cells as a tissue response to the surgical process and the characteristics of the graft material that can bridge the root surface, proliferate, and mature.20 ctg is a gingival graft procedure that has been widely used and is predictable in root coverage. however, ctg needs to be careful with the palate anatomy to minimize the risk of palatine artery trauma.21 the treatment of a single recession with ctg is presented in a study by bautista et al.22 they handled the wide and deep single recession with a one-stage surgical procedure with ctg and started showing the result three months postoperatively.22 the vista technique combined with ctg can provide good results in treating localized mandibular incisor recession. creeping attachment in the treatment of gingival recession is unpredictable, but this phenomenon can promote esthetics and improve gingival quality. research with more samples and a longer observation period is recommended to further study the creeping attachment phenomenon. references 1. jepsen s, caton jg, albandar jm, bissada nf, bouchard p, cortellini p, demirel k, de sanctis m, ercoli c, fan j, geurs nc, hughes fj, jin l, kantarci a, lalla e, madianos pn, matthews d, mcguire mk, mills mp, preshaw pm, reynolds ma, sculean a, susin c, west nx, yamazaki k. periodontal manifestations of systemic diseases and developmental and acquired conditions: consensus report of workgroup 3 of the 2017 world workshop on the classification of periodontal and peri-implant diseases and conditions. j periodontol. 2018; 89(suppl 1): s237–48. 2. dörfer ce, staehle hj, wolff d. three-year randomized study of manual and power toothbrush effects on pre-existing gingival recession. j clin periodontol. 2016; 43(6): 512–9. 3. heasman pa, holliday r, bryant a, preshaw pm. evidence for the occurrence of gingival recession and non-carious cervical lesions as a consequence of traumatic toothbrushing. j clin periodontol. 2015; 42(suppl 1): s237-55. 4. seong j, bartlett d, newcombe rg, claydon nca, hellin n, west nx. prevalence of gingival recession and study of associated related factors in young uk adults. j dent. 2018; 76: 58–67. 5. changi kk, greenstein g, tarnow d, royzman d, kang p. creeping clinical attachment after acellular dermal matrix augmentation to attain root coverage. clin adv periodontics. 2020; 10(2): 75–80. 6. cieślik-wegemund m, wierucka-młynarczyk b, tanasiewicz m, gilowski ł. tunnel technique with collagen matrix compared wit h con ne ct ive t issue g ra f t for t reat ment of p er io dont a l recession: a randomized clinical trial. j periodontol. 2016; 87(12): 1436–43. 7. cosgarea r, juncar r, arweiler n, lascu l, sculean a. clinical evaluation of a porcine acellular dermal matrix for the treatment of multiple adjacent class i, ii, and iii gingival recessions using the modified coronally advanced tunnel technique. quintessence int. 2016; 47(9): 739–47. 8. abraham s, deepak kt, ambili r, preeja c, archana v. gingival biotype and its clinical significance – a review. saudi j dent res. 2014; 5(1): 3–7. 9. manjunath rgs, rana a, sarkar a. gingival biotype assessment in a healthy periodontium: transgingival probing method. j clin diagnostic res. 2015; 9(5): zc66-9. 10. reddy s, prasad mgs, bhowmik n, singh s, pandit hr, vimal sk. vestibular incision subperiosteal tunnel access (vista) with platelet rich fibrin (prf) and connective tissue graft (ctg) in the management of multiple gingival recessiona case series. int j appl dent sci. 2016; 2(4): 34–7. 11. agudio g, chambrone l, pini prato g. biologic remodeling of periodontal dimensions of areas treated with gingival augmentation procedure: a 25-year follow-up observation. j periodontol. 2017; 88(7): 634–42. 12. meza-mauricio j, tavelli l, marx m, maximiano h, mafra ij, garcia jp. creeping attachment following treatment of multiple gingival recession defects with xenogeneic collagen matrix: two case reports. j int acad periodontol. 2021; 23(3): 253–8. 13. graziani f, gennai s, roldán s, discepoli n, buti j, madianos p, herrera d. efficacy of periodontal plastic procedures in the treatment of multiple gingival recessions. j clin periodontol. 2014; 41(suppl 1): s63-76. 14. suhasini j, valiathan m. brushing techniques. eur j mol clin med. 2020; 7(2): 6601–11. 15. imber j-c, kasaj a. treatment of gingival recession: when and how? int dent j. 2021; 71(3): 178–87. 16. mohamed a, marssafy l. comparative clinical study between tunnel and vista approaches for the treatment of multiple gingival recessions with acellular dermal matrix allograft. egypt dent j. 2020; 66(1): 247–59. 17. hämmerle chf, giannobile w v, working group 1 of the european workshop on periodontology. biology of soft tissue wound healing and regeneration--consensus report of group 1 of the 10th european workshop on periodontology. j clin periodontol. 2014; 41(suppl 1): s1-5. 18. wan w, zhong h, wang j. creeping attachment: a literature review. j esthet restor dent. 2020; 32(8): 776–82. 19. soldatos nk, font k, powell ca. creeping attachment after xenogenic collagen matrix placement for multiple recessions treatment: a discussion of two case reports. j implant adv clin dent. 2016; 8(6): 6–14. 20. gul ss, zardawi fm, sha am, rauf am. assessment of creeping attachment after free gingival graft in treatment of isolated gingival recession. j int acad periodontol. 2019; 21(3): 125–31. 21. zuhr o, bäumer d, hürzeler m. the addition of soft tissue replacement grafts in plastic periodontal and implant surgery: critical elements in design and execution. j clin periodontol. 2014; 41(suppl 1): s123-42. 22. bautista ca, cafferata ea, vernal r, cárdenas am. treatment of a single gingival recession with a subepithelial connective tissue graft with a double papilla flap: a case report. sage open med case reports. 2022; 10: 2050313x221078706. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p13–16 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p13-16 vol 51 no 3 jul sep 2018_pus.indd 133 cytoxicity test of naocl and mangosteen (garcinia mangostin l.) peel extract used as an irrigation solution in human periodontal ligament fibroblast cells (hpdlfc) tamara yuanita, dina rystiawati and karlina samadi department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: root canal irrigation is an important stage in root canal treatment as it is requires to eliminate necrotic and debris tissue as well as root canal wetting. unfortunately, root canal irrigation can cause the material utilised to pass into the apical foramen leading to periapical complications. consequently, the irrigation solution should have low toxicity. sodium hypochlorite (naocl) is a commonly used irrigation solution since it has antibacterial properties. moreover, naocl is also known to have the ability to dissolve necrotic tissue, vital pulp tissue and organic components of dentin and biofilms. nevertheless, it can still cause damage when coming into contact with periapical tissues. on the other hand, mangosteen peel extract (garcinia mangostana l.), also has antibacterial activities. hence, mangosteen peel extract is assumed to be employable as an alternative irrigation solution. purpose: this research aimed to reveal the toxicity levels of naocl and mangosteen peel extract (garcinia mangostin l.) used as irrigation solution in human periodontal ligament fibroblast cells (hpdlfc). methods: hpdlfc were obtained from periapical tissues taken from one third of the first premolar teeth cultured. these cells were subsequently divided into several groups exposed to naocl and mangosteen peel extract at certain concentrations. a toxicity test was then conducted using mtt assay. the results were analyzed with an elisa reader. cell deaths and lc50 were then calculated. results: naocl became toxic at a concentration of 0.254 μl/ml or 0.025%, while mangosteen peel extract became so at one of 2.099 ug/ml or 0.209%. conclusion: naocl can be toxic at a concentration of 0.254 μl/ml or 0.025% and mangosteen peel extract at one of 2.099 μg/ml or 0.209%. keywords: cytotoxicity; naocl; mangosteen peel extract; hpdlfc correspondence: tamara yuanita, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tamara-y@fkg.unair.ac.id dental journal (majalah kedokteran gigi) 2018 september; 51(3): 133–137 research report introduction root canal treatment is the most common form applied in the field of endodontics. its main goal within endodontic therapy is to improve and regenerate periapical tissue. in achieving this, root canals must be biomechanically cleaned of bacteria and debris without causing irreversible damage to the surrounding periapical tissues.1 root canal treatment can be divided into three stages: biomechanical preparation (cleaning and shaping), dressing and obturation. biomechanical preparation is a process intended to remove not only all infected pulp tissues, both in vital and non-vital conditions, but also microorganisms from inside the root canal. however, root canal irrigation is still required to eliminate necrotic and debris tissue, as well as root canal wetting. the number of microorganisms in the root canals can, as a result, be reduced since unclean root canal walls can become a breeding ground for bacteria, reducematerial attachment, increase apical cleft and cause root canal blockage.2 the most commonly used irrigation solution is sodium hypochlorite (naocl) which has antibacterial abilities. moreover, naocl also has the ability to dissolve necrotic tissue, vital pulp tissue and organic components of dentin and biofilms and is commonly used as an irrigation solution at concentrations of 0.5-5.25%.3 unfortunately, naocl has dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p133-137 134yuanita, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 133–137 several disadvantages, such as increasing toxicity as its concentration increases, producing unpleasant odors and tastes and causing damage when coming into contact with periradicular tissue.4 in addition, when naocl passes from the root canal into the periapical tissues it can also cause complications, one of which is chemical burns which can ultimately result in tissue necrosis. inflammatory reactions then will occur with the potential to cause both swelling and pain in the surrounding mucosa.5 in recent years, there have been a number of reports on the activities and possible applications of natural ingredients as a form of root canal disinfection. natural irrigation can be used as an alternative in order to avoid the toxic effects of chemical irrigation materials. for instance, mangosteen (garcinia mangostana l.) is a plant indigenous to indonesia and several parts of southeast asia. mangosteen peel contains active ingredients, including: xanthones, flavonoids, saponins, tannins, garcinon, gartanin, vitamins b1, b2, terpenes, anthocyanins, phenol and other bioactive substances.6 mangosteen (garcinia mangostana l.) peel extract also performs antioxidant, anti-inflammatory and antibacterial biological functions. xanthone, an active ingredient of α-mangostain derivates, even has the ability to protect against increased oxidative stress and antioxidant deficiency. meanwhile, γ-mangosten inhibits cox-1 and cox-2 activities at concentrations of 0.8 and 2 μm.7 matsuo8 argues that flavonoids can act as antioxidants, protect against oxidative stress, while also inhibiting endothelial human umbilical vein cells (huve) and pc12 cells from lipid peroxidase. nevertheless, at high concentrations, mangosteen peel extract can also cause mitochondrial cell disorders, an increase in reactive oxidation stress (ros) and loss of potential mitochondrial membranes, leading to cell death.9 irrigation solutions can come into contact with pulp and periapical tissues. blood and irrigation solutions that are expelled from the apical foramen can then lead to periapical complications. cytotoxic effects of materials used in endodontic treatment are, consequently, considered to be of particular concern since they can cause damage and irritation leading to periapical tissue degeneration and wound healing delay.1 since irrigation solutions must also be biocompatible, cytoxicity tests can be used as an initial biocompatibility assessment of the irrigation solutions used against certain organisms. one cytoxicity test method is the enzymatic test using (3(4.5-dimethylthiaziol-2yl) -2.5diphenyl-tetrazolium bromide (mtt) assay. the basis of the mtt test is to measure the ability of living cells based on the mitochondrial activities of cell cultures. by using the cytoxicity test, an irrigation solution can be confirmed as toxic-based according to the toxicity parameters of median lethal concentration (lc50), indicating the ability of the material to cause 50% death of cell cultures.10 in other words, an irrigation solution is considered to be toxic if the post-exposure percentage of living cells is below 50%.11 other previous research conducted by karkehabadi1 on the cytoxicity test of naocl irrigation solution in human periodontal ligament fibroblast cells (hpdlfc) found that naocl became toxic at a concentration of 0.025%, while at one of 0.4%, it caused the death of all cells. unfortunately, the cytoxicity test of mangosteen peel extract in human periodontal ligament fibroblast cells (hpdlfc) has yet to be conducted. according to freshney,12 the initial assessment of biocompatibility and toxic effects of a material are supposed to be directly performed on cell culture. the cytotoxicity of naocl and mangosteen peel extract (garcinia mangostana linn.) used as irrigation solutions for human periodontal ligament fibroblast cells (hpdlfc) needs to be established since they have an important role in the development, function, and regeneration of periodontal tissues. furthermore, hpdlfc are considered the first cells to come into contact with the irrigation solutions emerging from the root canal. consequently, these cells, commonly studied for periodontal regeneration, are also recognised as the main cells reacting to endodontic material in the periapic tissues.13,14 materials and methods this research was a laboratory experimental study with post-test only control group design. the materials used in this research were sodium hypochlorite (naocl), mangosteen peel extract, the first premolar teeth, dulbecco’s modified eagle medium culture media (dmem), 10% fetal bovine serum (fbs), mtt reagent [2(4,5-dimethylthiazol2-yl) -2, 5-diphenyl tetrazolium bromide], phosphate buffer saline (pbs), 70% ethanol and sterile distilled water. the research was conducted at the integrated research and testing laboratory, universitas gajah mada, yogyakarta. mangosteen peel extract was produced and identified by materia medika, batu, malang. mangosteen peels were extracted and dried by maceration method using 70% ethanol solvent. the internal and external surfaces of the mangosteen peels were washed with running water to remove any dirt, before being drained and dried in an oven at 50° for 24 hours. the peels were milled, sieved, weighed and divided into 200gm batches and then immersed in 70% ethanol solvent for 24 hours, agitated in a digital shaker at a speed of 50 rpm, filtered and placed in a rotary evaporator for three hours. the extract was dissolved in dmem medium in a series of doses at concentrations of 4μg/ml, 2μg/ml, 1μg/ml, 0.5μg/ml, 0.25μg/ml and 0.125μg/ml. human periodontal ligament fibroblasts cells (hpdlfc) were obtained by culturing the first premolar teeth extracted during orthodontic treatment. immediately after extraction, each tooth was placed in a 10ml tube containing dmem medium to which fungizone and penstrep had been added. the periodontal ligament of each tooth was then carefully removed from one third of the periapical with a scalpel. the resulting fragment was placed in dmem with a combination of 10% serum fetal bovine serum (fbs) and antibiotics before being cultured at 37°c with an dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p133-137 135 yuanita, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 133–137 atmospheric humidity of 5% co2 and 95% water. once every four days, the medium was discarded and replaced. the cells were observed until the level of confluence reached 90% . a cytotoxicity test was subsequently carried out using mtt assay. there were 12 groups, namely: a 1μl/ ml naocl treatment group, a 0.5μl/ml naocl treatment group, a 0.25μl/ml naocl treatment group, a 0.125μl/ml naocl treatment group, a 0.0625μl/ml naocl treatment group, a 0.03125μl/ml naocl treatment group, a 4μg/ ml mangosteen peel extract treatment group, a 2μg/ml mangosteen peel extract treatment group, a 1μg/ml mangosteen peel extract treatment group, a 0.5μg/ml mangosteen peel extract treatment group, a 0.25μg/ml mangosteen peel extract treatment group and a 0.125μg/ ml mangosteen peel extract treatment group. each group consisted of three samples. the fibroblast cells were then divided according to the criterion of a cell density of 2 x 104/ 20,000 cells/ well in 96 well plates. thereafter, 100ul naocl and 100ul mangosteen peel extract were added at various concentrations, with the plates being incubated in 5% co2 at 37º c for 24 hours. following the incubation period, the cell medium was discarded and a maximum of 10μl. 100ul mtt was added to each well. stop solution was also introduced into each of the wells and incubated overnight. the optical density values of the formazan crystalline formed were read by spectrophotometry using an elisa reader at a wavelength of 550 nm. the percentage of cell deaths was then calculated using the following formula: results the research findings were presented in the form of reading results produced by an elisa reader and revealed the absorbance levels or optical density values quantifying both the living cells and cell death percentages. at this point in the procedure, the observation and reading results of the absorbance values of the toxicity tests on the naocl and mangosteen peel extract in human periodontal ligament fibroblasts cells (hpdlfc) were divided into 12 groups (each undergoing the treatment on three occasions) accompanied by the controls depicted in figures 1 and 2. according to the content of figure 1, the higher the concentration, the higher the cell death rate. the highest percentage recorded, namely 87.195%, was that at a concentration of 1μl/ml, while the lowest occurred at one of 0.0625μl/ml. based on the statistics in figure 2, the higher the concentration of mangosteen peel extract, the greater the number of cells which died. the highest percentage of cell death, in this case 57.17%, was at a concentration of 4mg/ ml, while the lowest was recorded at one of 0.125μg/ml. the results of a shapiro-wilk test, conducted to analyze the normality of the data distrbution, indicated that it was normal. moreover, those of a levene test confirmed that the data was homogeneous. a one-way anova test was then carried out to identify any differences between the groups (table 1). probit analysis was subsequently performed to determine the lc50 value of each irrigation solution based on its concentration and cell death percentage. the lc50 value of naocl, based on the probit analysis, was obtained at a concentration of 0.25μl/ml (as illustrated in figure 3). on the other hand, the lc50 value of mangosteen peel extract, was obtained at one of 2.09μg/ml (as demonstrated in figure 4). figure 1. the mean percentage of the cell death of human periodontal ligament fibroblasts cells (hpdlfc) exposed to naocl. figure 2. the mean percentage of the cell death of human periodontal ligament fibroblasts cells (hpdlfc) exposed to mangosteen peel extract. table 1. the one-way anova test sum of squares meandf square sig.f 0.000288.321 22.14961729.925between groups 13.01714182.244within groups 201912.169total dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p133-137 136yuanita, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 133–137 discussion in this research, the cytoxicity tests of the naocl and mangosteen peel extract (garcinia mangostin l.) used as irrigation solutions were carried out by mtt assay method to measure the biocompatibility of these solutions before they could be applied clinically. the optical density value results were then read by a spectrophotometer with the data being calculated to reveal the percentage of cell death. probit analysis was then performed to determine the values of a lethal concentration of 50% (lc50). sodium hypochlorite (naocl) is one of the most widely known and used endodontic irrigation solutions due to its antimicrobial activity and ability to dissolve organic and necrotic tissue remnants. unfortunately, naocl can become toxic when exposed to cells and tissues around the root canal. the death of periodontal ligament fibroblast cells is likely to be blocked by the presence of ros in oxidative stress conditions. reactive oxygen compounds, including hydroxyl groups, are strong oxidants. negative effects on cells can arise due to reactivation of these compounds which can also damage cell components important to maintaining the integrity of cell life. reactive oxygen species (ros) are reactive oxygen compounds, well-known as free radicals which can be derived from the mitochondrial respiration chain and reactive chemical auto-oxidation.15 naocl, when in contact with the tissue, will produce hydroxyl ions and hypochlorous acid (hocl¯). the naocl-produced hydroxyl ions will then react with oxygen produced by mitochondria in human periodontal ligament fibrous cells to form hydroxyl radicals through a process of autoxidation. if ros production exceeds the capture capacity of antioxidants, it will lead to a condition called oxidative stress.14 the presence of oxidative stress will cause lipid peroxidation reactions in the plasma membrane and organelles. thereafter, the unstable bond of fatty acids with free radicals will form lipid radicals that react with oxygen to form peroxyl radical lipids. these then react with other lipid radicals to become lipid peroxide, resulting in more severe membrane damage. ros can also cause oxidation of amino acid chains, formation of covalent protein bonds and oxidation of proteins, leading to damage to the protein structure and an increase in proteasomal protein degradation. in addition, ros can cause dna damage and cross-linking dna chains. this mechanism is what causes cell death indicated by high levels of naocl cytotoxicity.14 in this research, a cytoxicity test was also conducted on mangosteen peel extract obtained by means of a maceration method at upt materia medica, batu, malang. based on probit analysis, the lc50 value of mangosteen peel extract was found at a concentration of 2.099μg/ml. in other words, the concentration of mangosteen peel extract killing 50% of periodontal ligament fibroblasts cells was 2.099μg/ml or 0.209%. this means that mangosteen peel extract at this concentration can become toxic. the results showed that mangosteen peel extract must be more concentrated to cause 50% of cell death. several active compounds contained in mangosteen peel were reported to be responsible for several pharmacological activities. the active compounds identified at the industrial research and consultation center in surabaya included: xanthones (4.01%), flavonoids (2.38%), tannin (12.05%) and saponin (4.38%). the death of human periodontal ligament firoblast cells due to the application of mangosteen peel extract in this research may also have been caused by an increase in ros which causes an imbalance in oxidant and antioxidant status as well as a proloferation of pro-oxidants, referred to as oxidative stress.13 xanthones are categorized into a polyphenolic class. the most important xanthone derivative in mangosteen peel extract is α-mangostin. when reacting with oxygen produced by mitochondria in human periodontal ligament fibroblast cells through an auto-oxidation process, the hydroxyl group of α-mangostin will result in the formation of free radicals/ ros/semiquinone radicals. as a result, oxidative stress can compromise mitochondrial integrity. the active ingredient of flavonoids which plays a role is quercetin. the hydroxyl groups of quercethane then interact with oxygen through the autoxidation process also producing ros/free radicals, figure 3. determination of lc50 value of naocl. naocl probit analysis concentration figure 4. determination of lc50 value of mangosteen skin extract. mangosteen peel extract probit analysis concentration dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p133-137 137 yuanita, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 133–137 namely o-quiones. the overload of ros will cause the release of ca2+ due to the opening of mitochondrial pores (mptp).9 excessive ca 2+ will, in turn, lead to distubances in membrane permeability as well as excessive reaction to the synthesis of the tricarboxylic acid chain. it can increase electron flow, resulting in oxidative phosphorillation failure and atp depletion, while stimulating a reaction to lipid peroxidation in plasma membranes and organelles. bonding fatty acids with unstable free radicals can then cause more damage to the membrane.16 like xanthones, tannins and saponins at inappropriate concentrations can also interfere with the permeability of fibroblast cell membranes. tannins have a high affinity for proteins that can form complex bonds with them, causing cytoplasmic disturbances (moosophin et al., 2010). meanwhile, saponins can break down the lipids in cell membranes, leading to disruptions to membrane permeability resulting in an imbalance of influx and exflux of ions (ca2+, na2+, k+). those reactions then cause cell death.16 mangosteen peel extract at high concentrations has antioxidant ability9 due to the xanthone, flavonoids and tannin it contains. its hydrogen chain can release and combine with o2• ¯, leading to the stable formation of phenoxil radicals. besides inhibiting the occurrence of lipid peroxidation, it can prevent changes in the globally harmonized system (ghs) and superoxide dismutase (sod) and also return to near normal levels. hence, mangosteen peel extract can prevent sod depletion, in this case, glutathione (gsh), the best antioxidants playing an important role as antioxidant enzymes.9 in other words, mangosteen peel extract is very effective at reducing the formation of reactive oxygen species (ros), resulting in reduced cell death. its antioxidant ability causes mangosteen peel extract to demonstrate lower cytotoxicity. previous research conducted by kumar17 also found that the anti-inflammatory effect of garcinia mangostana l extract occurs through the down regulation of nitric oxide (no) production. at concentrations of 0.976μg/ ml to 15.625μg/ml, it can cause a down regulation in no production. its cytotoxic potency measured during this research using mtt assay was at the same concentration, i.e. at concentrations of 0.906μg/ml to 15.625μg/ml. in conclusion, naocl irrigation solution can become toxic at a concentration of 0.254μl/ml or 0.025%, while mangosteen peel extract becomes toxic at one of 2.099μg/ ml or 0.209%. references karkehabadi h, yousefifakhr h, zadsirjan s. cytotoxicity of endo-1. dontic irrigants on human periodontal ligament cells. iran endod j. 2018; 13(3): 390–4. gutmann jl, lovdahl pe. problem solving in endodontics. 52. th ed. missouri: mosby elsevier; 2011. p. 209-12. hargreaves km, berman lh, rotstein i. cohen’s pathways of the3. pulp. 11th ed. st. louis: mosby elsevier; 2016. p. 251-2. harris j. comparison of steriplex4. tm hc and sodium hypochlorite cytotoxicity on primary human gingival fibroblasts. thesis. virginia: virginia commonwealth university; 2012. p. 1-25. faras f, abo-alhassan f, sadeq a, burezq h. complication of im-5. proper management of sodium hypochlorite accident during root canal treatment. j int soc prev community dent. 2016; 6(5): 493–6. kaomongkolgit r, jamdee k, pumklin j, pavasant p. laboratory6. evaluation of the antibacterial and cytotoxic effect of alpha-mangostin when used as a root canal irrigant. indian j dent. 2013; 4: 12–7. pedraza-chaverri j, cárdenas-rodríguez n, orozco-ibarra m,7. pérez-rojas jm. medicinal properties of mangosteen (garcinia mangostana). food chem toxicol. 2008; 46(10): 3227–39. matsuo m, sasaki n, saga k, kaneko t. cytotoxicity of flavonoids8. toward cultured normal human cells. biol pharm bull. 2005; 28(2): 253–9. martínez-abundis e, garcía n, correa f, hernández-reséndiz s,9. pedraza-chaverri j, zazueta c. effects of α-mangostin on mitochondrial energetic metabolism. mitochondrion. 2010; 10(2): 151–7. 10. zhang m, aguilera d, das c, vasquez h, zage p, gopalakrishnan v, wolff j. measuring cytotoxicity: a new perspective on lc50. anticancer res. 2007; 27: 35–8. 11. khoswanto c, arijani e, soesilawati p. cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using mtt assay on culture cell line. dent j (maj ked gigi). 2008; 41(3): 103–6. 12. freshney ri. culture of animal cells: a manual of basic technique and specialized applications. 6th ed. new jersey: john wiley & sons, inc.; 2011. p. 1-8, 111-4, 187-206, 365-77. 13. scanlon cs, marchesan jt, soehren s, matsuo m, kapila yl. capturing the regenerative potential of periodontal ligament fibroblasts. j stem cells regen med. 2011; 7: 54–6. 14. ok e, adanir n, hakki s. comparison of cytotoxicity of various concentrations origanum extract solution with 2% chlorhexidine gluconate and 5.25% sodium hypochlorite. eur j dent. 2015; 9: 6–10. 15. saraswati w. apoptosis sel odontoblas pulpa akibat resin bonding agent hema. thesis. surabaya: universitas airlangga; 2009. 16. arabski m, w�gierek-ciuk a, czerwonka g, lankoff a, kaca w. effects of saponins against clinical e. coli strains and eukaryotic cell line. j biomed biotechnol. 2012; 2012: 1–6. 17. kumar v, abbas ak, aster jc. robbins and cotran pathologic basis of disease. 9th ed. philadelphia: elsevier saunders; 2015. p. 44-52. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p133–137 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p133-137 136 prevalence of xerostomia on type 2 diabetes mellitus in hajj hospital surabaya kus harijanti,1 bagus soebadi,1 and irvanda mulyaningsih2 1 departement of oral medicine 2 student faculty of dentistry airlangga university surabaya indonesia abstract diabetes mellitus (dm) is a chronic disorder of carbohydrate, fat and protein metabolism. a defective or deficient of the insulin secretory, which is translated into impaired carbohydrate (glucose) use, is characteristic feature of dm, as the resultant is hyperglycemia. there is variability among patients, however, morphologic changes are likely found in arteries (atherosclerosis), basement membrane of the blood vessel wall tissues (micro angiopathy), kidneys (diabetic nephropathy), retina (retinopathy), nerves (neuropathy) and other tissues. if it involves salivary glands, the clinical sign is xerostomia. the type 2 of dm is caused by a combination of peripheral resistance to insulin action and an inadequate secretory response of the pancreatic b-cell. approximately 80% to 90% of patients have type 2 diabetes. the purpose of this study was to determine the prevalence of xerostomia and its relation with level of blood glucose in type 2 dm. the data was taken using cross sectional method on the diabetic patients of internal medicine clinic, hajj hospital surabaya from february to march 2006. the result that showed among 50 samples of the type 2 dm, the prevalence of xerostomia were 38 patients (76%). most of the patients (32 patients = 84%) on bad regulation of dm with level of fasting glucose  126 mg/dl and level of post prandial glucose  180 mg/dl. the study showed that bad regulation of type 2 dm could develop complication on salivary glands, with xerostomia as the clinical sign. key words: xerostomia, diabetes mellitus type 2 correspondence: kus harijanti, c/o: bagian oral medicine, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction diabetes mellitus (dm) is a metabolic disease with clinical feature such as hyperglycemia and glycosuria. it may also appear with polyuria, polyphagia or decrease of body weight. the symptoms are chronic or asymptomatic.1 the two metabolic defects that characterized type 2 dm are: 1) decrease ability of peripheral tissue to respond insulin (insulin resistance) and 2) b-cell pancreatic dysfunction that is manifested as inadequate insulin secretion, and the cardinal manifestation of diabetes mellitus is hyperglycemia. in most cases, the first process is insulin resistance and is followed by increasing degrees of b-cell pancreatic dysfunction. insulin resistance is defined as resistance to the effects of insulin on glucose uptake, metabolism or storage. these are the characteristic feature of most patients with type 2 dm. insulin resistance often detected 10 to 20 years before the onset of dm in predisposed individual (e.g., offspring of type 2 diabetics).2,3,4 the eighty percent to 90% of the dm patients are the type 2. the onset is slow and usually occurs on patients over 40 years old with obesity frequently obese. however, with the increase of obesity and sedentary lifestyle in our society, type 2 diabetes is now found in children and adolescents with increasing frequency.2,3 oral diabetic is a manifestation of dm in the mouth, present on patients with uncontrolled or undiagnosed dm. there are differences in reaction and ability between normal person and patients with dm toward injury, infection and local irritation in the mouth. the oral diabetic has various manifestations in the mouth, such as xerostomia diabetic. xerostomia is defined if unstimulated saliva secretion is less or equal to 0.15 ml/min. the term is used to encompass the spectrum of oral complaints voiced by patients with dry mouth. patients complain of difficulty chewing, swallowing and speaking. many patients reported that they to need fluids while eating to ease the swallowing process or enable to swallow dry foods.3,5 xerostomia diabetic is permanent condition, which can not be treated by drinking-water or administering sialogog.6 the aim of the study was to determine the prevalence of xerostomia and its relation with level of blood glucose in type 2 dm at hajj hospital surabaya. material and method the research had been done at interne clinic of hajj hospital on february until march 2006 by cross-sectional method and selective random sampling. criterias of the 137harijanti et al: the prevalence of xerostomia sample were 1) patient with type 2 dm, 2) the age over 40 years old, 3) non smoking, 4) non alcoholic, 5) non dental prosthetic user 6) patient did not undergo radiation treatment, and 7) for women, samples they were not menopause. the primary data was obtained by measuring unstimulated saliva secretion of the patients and answering questioner. the secondary data was taken from the registered medical status. the diagnosis of diabetes is established by noting elevation of blood glucose. a fasting blood glucose level of type 2 dm is more than 126 mg/dl, and 2 hours post prandial blood glucose level is more than 200 mg/dl. one hour before collecting the saliva, patients had no meal. while the patients were waiting, we checked the fasting and 2 hours post prandial blood glucose level from the medical status. after that, we examined the intra oral mucosa and anamnesed the oral mouth complaints. after one hour, the patient seat on the chair with the head down, the saliva collected in the floor of the mouth and spited into the measuring tube. then volume of the saliva was measured in milliliter per minute. result fifty patients with type 2 dm, that matched the criteria samples were found (36 male and 14 female). most of patients with type 2 dm were over than 60 years old (19 patients -38%), 51–60 years old were 17 patients (34%), 40–50 years old were 14 patients (28%). the suffering duration of dm less than 1 year consist of 10 patients (20%), 1–5 years was 19 patients (38%), 6–10 years was 13 patients (26%) and the suffering duration over 10 years was 8 patients (16%). good regulation of fasting blood glucose level was found on 5 patients (10%), moderate regulation was 14 patients (28%), and bad regulation was 31 patients (62%). while two hours post prandial blood glucose level with good regulation was found on 9 patients (18%), moderate regulation was 6 (12%) and bad regulation 35 patients (70%). thirty eight out of 50 patients (76%) of type 2 dm experienced xerostomia which saliva secretion less than 0.15ml/mint. among the patients with xerostomia, six patients had suffered less than 1 year, 17 patients had suffered 1–5 years, 9 patients had suffered 6–10 years and 6 patients had suffered more than 10 years. from 38 patients which suffering xerostomia, 32 patients had bad regulation on fasting and the 2 hours post prandial blood glucose level. five patients had moderate regulation and only one patient had good regulation on blood glucose level. table 2 showed, that suffering duration less than 1 year were 10 patients, and had experienced xerostomia were 6 patients (12%), and all of the patients felt various subjective symptoms. four patients had not experienced xerostomia, the two patients felt subjective symptoms and two others patients were not. in the group of suffering duration of 1–5 years were 19 patients and had experienced xerostomia were 17 patients (34%), and all of the patients felt various subjective symptoms. in the group of suffering duration of 6–10 years were found 13 patients, and felt subjective symptoms. the xerostomia is patients were 9 patients (18%) and saliva secretion of the four patients were normal. likewise, in the group of suffering duration over than 10 years were found 8 patients, six patients (12%) were saliva secretion of less than normal. either, the xerostomia patients or not, felt subjective symptom. table 1. distribution of suffering duration, age, gender, saliva secretion, regulation of fasting blood glucose, and regulation 2 hours post prandial suffering duration age (years) gender saliva secretion (ml/mint) fasting blood glucose level (mg/dl) 2 hours-post prandial blood glucose level (mg/dl) < 1 years 1–5 years 6–10 years > 10 years 40–50  5 51–60  3 > 60  2 40–50  2 51–60  7 > 60  10 40–50  5 51–60  3 > 60  5 40–50  2 51–60  4 > 60  2 10 px : 7 3 19 px : 14 5 13 px: 8 5 8 px : 7 1  0.15   4 < 0.15  6  0.15  2 < 0.15  17  0.15  4 < 0.15  9  0.15  2 < 0.15  6 good  2 moderate  2 bad  6 good  2 moderate  8 bad  9 good  0 moderate  2 bad  11 good  1 moderate  2 bad  5 good  1 moderate  0 bad  9 good  5 moderate  2 bad  12 good  2 moderate  4 bad  7 good  1 moderate  0 bad  7 n = 50 px 40–50  14 51–60  17 > 60  19 14 36 normal 12 xerostomia 38 good  5 moderate  14 bad  31 good  9 moderate  6 bad  35 138 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 136-139 discussion approximate 80% to 90% of dm patients are type 2,2 that was the reason why we chooce type 2 dm as sample, it can represent almost all patient with dm. the result of this study at hajj hospital surabaya showed the prevalence of male patient was 72% and female 38%. this was different from previous research that was done at dr. soetomo hospital and dr. saiful anwar hospital, which the comparison between male by female patients were equal.7,8 one of the criteria sample of this study, was the women had not been menopause (even over 40 years old). therefore, the women samples were less than men. in this study on patients with type 2 dm , increased along the age and the result was similar with the epidemiological finding previously.9 according to maitra & abbas,2 most of the patients with type 2 diabetes mellitus are (over 40 years old). the suffering duration of this disease was measured from the initial diagnosis and treatment given as dm. the result, was very from 1 month until more than ten years. the characteristic feature of the most type 2 dm is insulin resistance.3,4 insulin resistance is defined as resistance to the effects of insulin on glucose uptake, metabolism or storage, and is frequently detected 10 to 20 years before the onset of dm in predisposed individual.2 according to the perkeni,10 the criteria of good regulation fasting blood glucose level is 80-109mg/ dl, moderate regulation is 110–125 mg/dl and bad regulation is equal or higher 126 mg/dl. good regulation of two hours post prandial blood glucose level is 80–144 mg/dl, moderate regulation is 145–179 mg/dl and bad regulation  180mg/dl. insulin is known as the most potent anabolic hormone, and principally has a metabolic function to increase the rate of glucose transport into certain cells in the body. these are the striated muscles including myocardium cell, and to a less extend adiposities representing collectively about two thirds of the entire body weight. insulin resistance can decrease uptake of the glucose in muscle and adipose tissue and unable glucagons hormone to suppress hepatic gluconeogenesis, resulted the increase blood glucose level and in the long term would cause serious complication including macro and micro vascular disease.2 saliva secretion equal 0.15 ml/min is the borderline of xerostomia. the two patients that were not experience xerostomia, one of them felt subjective symptoms. in these groups, the process of micro vascular glands disturbance might occured, but clinical sign had not revealed. that was proved, that some patients had subjective symptoms although saliva secretion was normal. most of the patients complained dryness of the mouth, dryness while eating, difficulty of swallowing, needed fluid for swallowing, dryness of the mouth at night. some of them complained difficulty of tasting, soreness of the tongue and dryness while speaking. the longer suffering period of dm the more complication will appear. saliva is produced by salivary glands, the fluid formation in salivary glands occurs in the end pieces (acini) where serous cells produce a watery seromucous secretion and mucous cell produce a viscous mucin rich secretion. these secretions arise by the formation from blood in capillaries of interstitial fluid which is then modified by the end piece cells to produce the fluid which is secreted into the lumen. from the lumen it passes the ductal system where it is further modified. most of the modification occurs in the striated duct. the composition of saliva is further modified in the excretory duct before it is finally secreted into the mouth. the blood supply to the glands also influences secretion. there is a concentration of capillaries around the striated ducts. the process of salivation indirectly dilates the blood vessels thus providing increased nutrition as needed. salivary secretion is usually accompanied by a large increase in blood flow.11 an extensive blood supply is table 2. distribution of suffering duration, saliva secretion, and subjective symptom in the mouth suffering duration saliva secretion ml/mint dryness of the mouth dryness at eating difficulty of swallowing needed fluid for swallowing dryness of the mouth at night difficulty of tasting soreness of the tongue dryness at speaking < 1 year 10 px (20%)  0.15: 4 < 0.15: 6 4 4 1 2 1 2 1 2 1 2 2 1–5 years 19 px (38%)  0.15: 2 < 0.15:17 7 1 12 1 11 1 10 8 2 4 6–10 years 13 px (26%)  0.15: 4 < 0.15: 9 5 2 7 3 4 3 4 1 7 1 1 1 1 > 10 years 8 px (16%)  0.15: 2 < 0.15: 6 4 1 5 1 6 1 6 6 1 1 1 6 s  0.15: 12 (4%) < 0.15: 38 (76%) 20 px 4 28 6 23 6 22 2 23 3 1 6 1 13 139harijanti et al: the prevalence of xerostomia required for the rapid secretion of saliva, when in the blood supply is occurred disturbance as micro and macro vascular disease on dm therefore saliva secretion decreased. moore and vernillo said that blood glucose level concentration is significant correlation to salivary flow.12,13 the functions of saliva are not only protective, but also have other functions. such as to coat mucosa and to protect against irritation, to assist smooth air flow, to produce speech and to swallow.11,14 if the saliva secretion is less than normal, it will the decrease the functions, finally subjective symptoms would appear. according to this research, the prevalence of xerostomia on type 2 dm in hajj hospital surabaya was high (76%) and most of the patients with xerostomia (84,21%) were in bad regulation of dm. therefore, saliva can be used as the important indicator for serious systemic disease as diabetes mellitus. references 1. tjokroprawiro a. diabetes mellitus (kapita selekta 1997). pkbxii. lab.upf ilmu penyakit dalam fk unair-rsud dr. soetomo, surabaya. 1997. p. 3. 2. maitra a, abbas ak. the endocrine of the pancreas. in pathologic basic of disease. pennsylvania: elsevier inc; 2005. p. 1189–205. 3. ibsen oac, phelan ja. oral pathology for the dental hygienist. 2nd ed. philadelphia: wb saunders co; 1996. p. 391–4. 4. power, alvin c. harrison’s. principle of internal medicine. vol. ii. 16th ed. new york: mcgraw-hill; 2005. p. 2152–80. 5. wilkins em. clinical practice of the dental hygienist. 8th ed. philadelphia: lippincot william & wilkins; 1999. p. 880–9. 6. darby ml, walsh mm. dental hygiene theory and practice. philadelphia: wb saunders co; 1995. p. 568, 814, 898–907, 922–3. 7. harlina, hernawan. hubungan antara kadar glukosa darah dan kadar glukosa saliva pada penderita diabetes mellitus. majalah kedokteran gigi (dental journal) 2003; 36(2):64–7. 8. dewi ar. prevalensi xerostomia pada penderita diabetes mellitus tipe 2 poli diabetes instalasi rawat jalan rsud dr. saiful anwar malang. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2005. p. 18–30. 9. tjokroprawiro a. garis besar kuliah diabetes mellitus. surabaya: lab. upf ilmu penyakit dalam fk unair rsud dr. soetomo; 1993. p. 2. 10. perkeni. petunjuk praktis pengelolaan diabetes mellitus tipe 2. pusat diabetes dan lipid. universitas indonesia; 2002. p. 1–10, 43–47. 11. whelton h. introduction the anatomy and physiology. in saliva and oral health. 2nd ed. london: the british dental association; 1996. p. 1–7. 12. moore. diabetes and oral health promotion: a survey of disease prevention behavior. journal american dental association 2000; 13 (9):1333–41. 13. vernillo a. dental considerations for the treatment of patients with diabetes mellitus. journal american dental association 2003; 134 (1):24s–33s. 14. sreebny ml. xerostomia: diagnosis, management and clinical complication.in saliva and oral health. 2nd ed. london: the british dental association; 1996. p. 43–7. 178 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 178–183 original article toxicity test of stenochlaena palustris extract based on kidney histopathology examination maharani laillyza apriasari1, tiara intan permata sari2, i wayan arya krishnawan firdaus2 1department of oral medicine, faculty of dentistry, universitas lambung mangkurat, banjarmasin, indonesia 2department of oral biology, faculty of dentistry, universitas lambung mangkurat, banjarmasin, indonesia abstract background: kalimantan’s people consume stenochlaena palustris leaf extract as food and for traditional medicine. the bioactive components of stenochlaena palustris leaf extract are flavonoids, alkaloids, saponins, and tannins. an in vitro study shows that the leaf extract has no toxic effect, so it can be used as an alternative drug in oral health, such as in mouthwashes or topical ulcer drugs. purpose: this study aims to analyze the toxic effects of stenochlaena palustris leaf extract based on the bleeding and lesions resulting from necrosis in kidney by using histopathology examination. methods: the stenochlaena palustris leaves were extracted using 95% ethanol and then given to male wistar strain (rattus norvegicus) with a 2,000, 2,500, and 3,000 mg/kg/body weight two times a day for fourteen days. the kidneys were collected and subjected to histopathology examination. results: there are higher bleeding and necrosis lesion rates in the 2,500 and 3,000 mg/kg/body weight of stenochlaena palustris leaves extract group compared to the control and 2,000 mg/kg/body weight of stenochlaena palustris leaves extract group (p<0.05). conclusion: stenochlaena palustris leaf extract showed no toxic effect at doses of 2,000 mg/kg/body weight. keywords: antioxidant; bleeding; necrosis; stenochlaena palustris leaves; toxicity test; medicine article history: received 1 december 2022; revised 20 january 2023; accepted 9 february 2023; published 1 september 2023 correspondence: maharani laillyza apriasari, department of oral medicine, faculty of dentistry, universitas lambung mangkurat. jl. veteran 128b, banjarmasin, indonesia email: maharaniroxy@gmail.com introduction south kalimantan has various types of ferns that are potentially medicinal plants. based on empirical data, stenochlaena palustris is eaten as a vegetable and used as a breast milk enhancer and alternative medicine to cure anemia and allergies and relieve fever.1 the public demand for herbal ingredients is increasing because of science and technology’s rapid development and progress, which is encouraging the development of naturally derived alternative medicines. in addition, these natural ingredients are more affordable and easier to obtain compared to drugs made with chemicals and are considered to have minimal side effects.2 stenochlaena palustris leaves contain various secondary metabolites. based on the results of phytochemical screening by debnath et al.,3 the ethanolic extract of stenochlaena palustris leaves contains flavonoids, saponins, alkaloids, steroids, and tannins. flavonoids have a content of 503.56 mg qe/g. alkaloids, saponins, steroids, tannins, and total phenol were 11.56%, 8.06%, 3.43%, 23 mg gae/g, and 51.69 ± 1.28 mg gae/g, respectively. tannins are antioxidants that accelerate the epithelialization process. steroids and saponins are antiinflammatory and antiseptic.3–5 there have been several studies on the extract of stenochlaena palustris leaves. one tested the toxicity of bhk-21 fibroblast cells and concluded that 10%, 20%, and 30% concentrations of the stenochlaena palustris leaf extract were toxic to bhk-21 fibroblasts. in comparison, the concentrations of 40%, 50%, 60%, 70%, 80%, and 90% were proven to be non-toxic to bhk-21 fibroblast cells. this study concluded that the extract of stenochlaena palustris leaves could increase cell viability because it contains flavonoids with non-enzymatic antioxidant functions that inhibit cell damage caused by reactive oxygen species (ros).6 therefore, antioxidants in stenochlaena palustris leaves have the potential to heal wounds because of their copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 mailto:maharaniroxy@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 179apriasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 178–183 granular tissue formation ability, fibroblast proliferation, and collagen fiber production.7 research has not found the toxicity or side effects of stenochlaena palustris herbal products used as medicine to heal wounds. it has been widely used in the community. several factors, including the accuracy of the dose, timeliness, and method of use, must be considered when taking natural treatments, such as stenochlaena palustris leaves, as a drug to heal wounds. therefore, it is necessary to carry out further testing to protect the user community from potentially detrimental effects and to ensure the safety of its use.8 hazard data is obtained using a toxicity test that detects the poisonous effect of a substance on a biological system and receives specific dose-response data from the test preparation. the test detects the presence of toxins in a substance and determines the toxin’s target organ and its sensitivity after the acute administration of the compound for 14 days. this toxicity test’s parameters include clinical symptoms, subject death, and organ histopathology.9 the kidneys often receive the unwanted effects of drugs. suppose the content of the extract that enters the body exceeds normal conditions. in that case, it will decrease the kidneys’ ability to concentrate xenobiotic substances in the cells, resulting in the accumulation of toxic substances that cause damage to the kidneys.10 the relatively high blood flow to the kidneys exposes them to substances carried in the circulatory system, so toxic substances can quickly damage the kidney, causing changes in structure and function. damage to the nephrons can occur in the tubules, renal corpuscles, and capillaries. the conditions of toxicity commonly found in the kidneys include cell necrosis, cell degeneration, and bleeding.11 furthermore, the study analyzes the toxic effects of three different stenochlaena palustris leaf extracts (2,000, 2,500, and 3,000 mg/kg/body weight) based on the bleeding and necrosis found in kidney tissue using a histopathology examination. materials and methods the test used 16 male wistar rats (rattus novergicus) aged 8–12 weeks, with body weights between 200–250 g, and all in good health. the wistar rats usually ate 20% of their total body weight, so in a laboratory adaptation, they were fed 40 g once daily for one week. boiled water was given in 500 ml bottles. the subjects were grouped by simple random sampling and divided into four groups, each consisting of four rats. the protocol of this study was approved by the ethical committee of the faculty of dentistry, the universitas lambung mangkurat, with registration number 016/kepkg-fkgulm/ec/iii/2022. greenish mature leaves (12 kg) of stenochlaena palustris were taken in the anjir barito kuala region. the leaves were washed, cut into small pieces, and dried in an oven at 40°c for four hours. after, the leaves were crushed in a blender and macerated with 95% ethanol for 72 hours.12 upon finishing, the solution was filtered with wh40 filter paper until a clear brownish liquid was obtained. in the next stage, a vacum rotary evaporator evaporated the solvent for four to six hours. it was then heated with a water bath to get a brownish liquid residue. ethanol-free was tested with potassium dichromate (k2cr2o7) on 3 ml of ethanol. the extract was then stored in a 10°c refrigerator to prevent oxidation. the doses of the extract were prepared by diluting the extract with distilled water.1 the toxicity test was performed by giving the stenochlaena palustris per-oral to the subject with a gastric probe two times a day for 14 days. the subjects were divided into four groups listed in table 1. at the end of the day’s treatment, the subjects were fasted and given only water for 8–12 hours. on the 15th day, the subjects were sacrificed by giving intraperitoneal injection using 0.5 ml of ketamine.13 the kidneys were collected, cleaned, wrapped in a white cloth, and buried at a depth of ± 50 cm.14 the kidneys were then washed with nacl and soaked with 10% bnf solution for tissue fixation and process for further processing. the bleeding and necrosis lesions were analyzed using histopathology with hematoxylin–eosin (he) staining. the bleeding lesions were characterized by red blood cells migrating from the vasculature into the tissue between the proximal tubular spaces. in contrast, the necrosis lesions were characterized by changes in shape and cell nucleus (darker nucleus), indicating the presence of pyknosis. the bleeding and necrosis lesions on kidney cells were counted and scored as listed in table 2.15 table 1. the distribution of subjects in the toxicity test group dose control 1 ml of distilled water p1 2000 mg/kg/body weight stenochlaena palustris extract (1 ml) p2 2500 mg/kg/body weight stenochlaena palustris extract (1 ml) p3 3000 mg/kg/body weight stenochlaena palustris extract (1 ml) table 2. bleeding and necrosis lesion scoring bleeding appearance score no bleeding lesion 0 mild bleeding (<25% area) 1 moderate bleeding (25–50% area) 2 severe bleeding (>50% area) 3 necrosis lesion no necrosis lesion 0 mild necrosis (<25% area) 1 moderate necrosis (25–50% area) 2 severe necrosis (>50% area) 3 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 180 apriasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 178–183 results the histopathology analysis of the bleeding lesion was characterized by red blood cells migrating from the vasculature into the tissue between the proximal tubular spaces (black arrow) (figure 1). the kidney tissue showed mild intratubular bleeding (< 25% area) in groups that were given stenochlaena palustris extract with 2,500 and 3,000 mg/kg/body weight (figure 1c–d). the type of bleeding lesion was petechial, measuring 1–2 mm. there were no histopathological changes in bleeding in control and 2,000 mg/kg/body weight stenochlaena palustris extract groups (figure 1a–b). the 2,500 and 3,000 mg/kg/body weight stenochlaena palustris extract groups showed higher bleeding lesions than the control and 2,000 mg/kg/body weight stenochlaena palustris extract (p<0.05). there is no significant difference in bleeding lesions in the control and 2,000 mg/kg/body-weight stenochlaena palustris extract groups (p>0.05). figure 1. the histopathology of a bleeding lesion in the kidney tissue. (a) control; (b) 2,000 mg/kg/body weight stenochlaena palustris extract; (c) 2,500 mg/kg/body weight stenochlaena palustris extract; and (d) 3,000 mg/kg/body weight stenochlaena palustris extract. magnification 400x, hematoxylin–eosin staining. figure 2. the histopathology of a necrosis lesion in the kidney tissue. (a) control; (b) 2,000 mg/kg/body weight stenochlaena palustris extract; (c) 2,500 mg/kg/body weight stenochlaena palustris extract; and (d) 3,000 mg/kg/body weight stenochlaena palustris extract. magnification 400x, hematoxylin–eosin staining. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 181apriasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 178–183 the necrosis lesion in histopathology analysis is characterized by the changes in shape and cell nucleus (darker nucleus) indicating the presence of pyknosis (black arrow) (figure 2). the kidney showed mild necrosis (<25% area) in groups with 2,500 and 3,000 mg/kg/body weight stenochlaena palustris extract (p<0.05) (figure 2c–d). no necrosis lesion was observed in the control and 2,000 mg/kg/body-weight stenochlaena palustris extract groups (figure 2a–b). discussion the histopathological changes in the kidneys are usually caused by substances that enter the bloodstream and that are toxic to the body. inappropriate use of natural ingredients can potentially cause organ damage, such as the kidney. this is because the kidney is an organ that often accumulates unwanted effects from the use of drugs taken orally.10,11 based on the results of statistical tests on the histopathological appearance of bleeding, it was found that the 2,000 mg/kg/body weight group had no significant difference from the control. in the treatment group at a dose of 2,500 mg/kg/body weight and the treatment group at a dose of 3,000 mg/kg/body weight, there was a significant difference with the control group, suggesting the treatment was potentially toxic to the kidneys of wistar rats. in the control group that was given distilled water, there was no histopathological appearance of bleeding. this indicates that the administration of distilled water is safe for the kidney of rats. according to research by maliangkay et al.,16 distilled water does not have the potential to have a toxic effect because it is not an irritant. the administration of a dose of 2,000 mg/kg/body weight of stenochlaena palustris leaf extract did not cause any signs of bleeding. in the p1 group who were given the 2,000 mg/kg/body weight stenochlaena palustris extract, no bleeding was seen. this is in line with the research by alsawaf et al.17 who found that the use of flavonoids at the right dose will not cause toxic effects on the kidneys of wistar rats. sudira et al.18 stated that the presence of flavonoids strengthens blood vessels and inhibits lipid peroxidation through peroxidase activation of hemoglobin in anticipation of damage caused by free radicals. flavonoids can prevent hemolysis of red blood cells caused by free radicals.19 the treatment group at a dose of 2,500 mg/kg/body weight and the treatment group at a dose of 3,000 mg/kg/ body weight had a significant difference from the control group. in both groups, there was a histopathological appearance of mild bleeding <25%. according to research by rafe et al.,19 tubular bleeding that resulted from toxic effects occurred in the treatment group that consumed a high dose of the extract. saponin is one of the secondary metabolites that affect the occurrence of bleeding with the use of stenochlaena palustris leaf extract. saponins are membranolytic which causes disintegration of the capillary endothelial layer, causing bleeding in the rats’ kidneys. saponins injure the lipid bilayer of the protein membrane of red blood cells, resulting in an inflammatory response.18 the two stages of inflammation are the vascular stage and the late stage. the vascular stage is associated with vasodilation and increased capillary permeability where blood substances leave the plasma. the late stage occurs when leukocytes infiltrate the inflamed tissue. this causes the release of various mediators such as histamine, prostaglandins, and leukotrienes which are produced from plasma. prostaglandins have a vasodilating effect, relax smooth muscles, and increase capillary permeability. histamine plays a role in the earliest changes, causing vasodilation in arterioles which is preceded by initial vasoconstriction and increased capillary permeability. this causes changes in the distribution of red blood cells, such as in slow blood flow when red blood cells clump and are consequently pushed to the edge. white blood cells stick to the walls of blood vessels with slower blood flow. changes in permeability that occur cause blood to come out of the blood vessels and collect in the tissues.20,21 the results of statistical tests on the histopathological appearance of necrosis showed that the treatment group at a dose of 2,000 mg/kg/body weight had no significant difference from the control group. in the treatment group at a dose of 2,500 mg/kg/body weight and the treatment group at a dose of 3,000 mg/kg/body weight, there was a significant difference with the control group. according to research, makiyah et al.,15 if the treatment group had a significant difference from the control group, the group was potentially toxic to the kidneys based on necrosis. in the microscopic observation of the control group that was only given distilled water, there is no histopathological change in necrosis because water is not an irritant, so it is not potentially toxic to organs. the p1 group, which was given a dose of 2,000 mg/ kg/body weight of stenochlaena palustris leaf extract, did not show any histopathological appearance of necrosis. the right dose of flavonoids has antioxidant potential to inhibit oxidation reactions by neutralizing free radicals and preventing cell damage. this is due to the content of secondary metabolites possessed by the extracts of the stenochlaena palustris leaves, such as flavonoids and phenolics, which have the ability to inhibit free radicals and oxidative reactions.22 as antioxidants, the direct mechanism of action for flavonoids is to stabilize free radicals by complementing their lack of electrons and inhibiting the chain reactions that form the celldamaging free radicals. the indirect mechanism of action of antioxidants is the inhibition of the enzymes involved in the production of free radicals by neutralizing and suppressing the formation of ros so that their number decreases and the activity of superoxide dismutase (sod) enzymes increases. antioxidants can modulate free radical reactions by reducing lipid hyper peroxides and hydrogen peroxide (h2o2), thereby preventing the occurrence of copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 182 apriasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 178–183 lipid peroxidation. sod plays a role in catalyzing the dismutation reaction of superoxide anion free radicals (o-) into hydrogen peroxide and oxygen, thereby turning them into products that are more stable and harmless to cells.23 in the treatment group, a dose of 2,500 mg/kg/body weight and a dose of 3,000 mg/kg/body weight showed a histopathological appearance of necrosis. the results of statistical tests stated that the treatment group at a dose of 2,500 mg/kg/body weight and the treatment group at a dose of 3,000 mg/kg/body weight had significant differences from the control group so that both treatment groups were potentially toxic to the kidneys of rats. tubular epithelial cell necrosis is caused by excessive intake of secondary metabolites contained in the extract that are nephrotoxic. flavonoids is one of the potentially toxic contents in stenochlaena palustris leaf extract. flavonoids in high concentrations can be pro-oxidants. quercetin is one of the compounds that can be prooxidant among these flavonoid compounds.24 when there are too many free radicals, the endogenous antioxidants will deplete their rate of use compared to their rate of regeneration. an imbalance between prooxidants and antioxidants in the body will result in an increased rate of free radical formation, resulting in an increase in oxidative stress that causes lipid peroxidation, oxidized dna, and misfolded proteins. this results in the accumulation of covalent bonds in the tubular membrane with free radicals, resulting in damage to the renal tubules. pro-oxidants cause oxidative stress and mitochondrial dysfunction. mitochondrial dysfunction is a condition characterized by impaired mitochondrial biogenesis, altered membrane potential, reduced number of mitochondria, and altered protein oxidative activity due to the accumulation of ros in cells and tissues. mitochondrial dysfunction causes hydropic degeneration due to impaired active transport. the disturbed osmosis process causes water to enter the cell so that the cell swells with the vacuole, and the cell nucleus enlarges. cells undergoing hydropic degeneration and that are continuously exposed to toxic compounds will become necrotic.25–27 it can be concluded that there was no toxic effect on the administration of 2,000 mg/kg/body weight dose of stenochlaena palustris leaf extract to the kidney of wistar rats based on the histopathological appearance of bleeding and necrosis. there was a toxic effect on the administration of the stenochlaena palustris leaves extract at a dose of 2,500 mg/kg/body weight and a dose of 3,000 mg/kg/body weight on the kidney of wistar rats based on the histopathological appearance of bleeding and necrosis. acknowledgement the authors thank to faculty of dentistry, universitas lambung mangkurat that have supported the research. references 1. syamsul es, hakim yy, nurhasnawati h. penetapan kadar flavonoid ekstrak daun kelakai (stenochlaena palustris (burm. f.) bedd.) dengan metode spektrofotometri uv-vis. j ris kefarmasian indones. 2019; 1(1): 11–20. 2. firdaus iwak, dewi n, fuady ri, apriasari ml. antibacterial effect of kelakai leaf extract (stenochlaena palustris (burm) bedd.) for inhibiting enterococcus faecalis. odonto dent j. 2022; 9(1): 110–8. 3. debnath sl, kundu p, ahad mf, saha l, biswas nn, sadhu sk. investigation of phytochemical and pharmacological assessment of ethanol extract of stenochlaena palustrisan edible fern of sundarbans. j med plants stud. 2021; 9(3): 226–32. 4. chear nj-y, khaw k-y, murugaiyah v, lai c-s. cholinesterase inhibitory activity and chemical constituents of stenochlaena palustris fronds at two different stages of maturity. j food drug anal. 2016; 24(2): 358–66. 5. awang-kanak f, abu bakar mf. traditional vegetable salad (ulam) of borneo as source of functional food. food res. 2019; 4(1): 1–12. 6. panche an, diwan ad, chandra sr. flavonoids: an overview. j nutr sci. 2016; 5: e47. 7. ahmed om, mohamed t, moustafa h, hamdy h, ahmed rr, aboud e. quercetin and low level laser therapy promote wound healing process in diabetic rats via structural reorganization and modulatory effects on inflammation and oxidative stress. biomed pharmacother. 2018; 101: 58–73. 8. arundina i, tantiana, diyatri i, surboyo mdc, adityasari r. acute toxicity test of liquid smoke of rice hull (oryza sativa) on mice (mus musculus). j int dent med res. 2020; 13(1): 91–6. 9. badan pengawas obat dan makanan republik indonesia. peraturan kepala badan pengawas obat dan makanan republik indonesia nomor 7 tahun 2014 tentang pedoman uji toksisitas nonklinik secara in vivo. 2014. p. 3–4.available from: https://peraturanpedia. id /perat u ra n-bada n-pengawasobatda n-ma ka na n-nomor-7 tahun-2014/. 10. meles dk, wurlina w, mustofa i, zakaria s, basori a, hariadi m, safitri e, cempaka putri dks, suwasanti n. toxicity, stability and renal histopathology of alkaloid of jarong (achyranthes aspera linn.) (caryophyllales: amaranthaceae) leaf on mice. philipp j vet med. 2018; 55(special issue): 35–42. 11. sudjarwo, widiastuti h, primaharinastiti p, prihatiningtyas s. toxicity test from gloriosa superba l leaves extract in rats (rattus novegicus). int j pharm pharm sci. 2014; 6(5): 183–7. 12. setyorini d, firdaus iwak, oktiani bw. comparison of inhibitory activity of kelakai leaves extract with ciprof loxacin against aggregatibacter actinomycetemcomitans atcc® 6514tm. dentino j kedokt gigi. 2019; 4(2): 199–204. 13. silverman j, suckow ma, murthy s. the iacuc handbook. 3rd ed. st louis: crc press taylor & francis group; 2014. p. 377–416. 14. fajriani n, carabelly an, apriasari ml. the effect of toman fish extract (channa micropeltes) onneutrophilin diabetes mellitus wound healing. in vivo study in the back of male wistar mice (ratus novergicus). dentino j kedokt gigi. 2018; 3(1): 15–21. 15. makiyah a, tresnayanti s. uji toksisitas akut yang diukur dengan penentuan ld50 ekstrak etanol umbi iles-iles (amorphophallus variabilis bl.) pada tikus putih strain wistar. maj kedokt bandung. 2017; 49(3): 145–55. 16. maliangkay hp, rumondor r, walean m. uji efektivitas antidiabtes ekstrak etanol kulit buah manggis (garcinia mangostana l) pada tikus putih (rattus novergicus) yang diinduksi aloksan. chem prog. 2018; 11(1): 15–21. 17. a lsawa f s, a l nua i m i f, a fza l s, t homas r m, chela k kot al, ramadan ws, hodeify r, matar r, merheb m, siddiqui ss, va zhappilly cg. pla nt f lavonoids on oxidative st ressmediated kidney inflammation. biology (basel). 2022; 11(12): 1717. 18. sudira w, merdana m, winaya ibo, parnayasa ik. histopathological changes in white rat’s kidney given ant nest extract induced paracetamol toxic dose. bul vet udayana. 2019; 11(2): 136–46. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 https://peraturanpedia https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 183apriasari et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 178–183 19. rafe masr, gaina cd, ndaong na. gambaran histopatologi ginjal tikus putih (rattus norvegicus) jantan yang diberi infusa pare lokal pulau timor. j vet nusant. 2019; 3(1): 61–73. 20. rezkita f, wibawa kgp, nugraha ap. curcumin loaded chitosan nanoparticle for accelerating the post extraction wound healing in diabetes mellitus patient: a review. res j pharm technol. 2020; 13(2): 1039–42. 21. narmada ib, laksono v, nugraha ap, ernawati ds, winias s, prahasanti c, dinaryanti a, susilowati h, hendrianto e, ihsan is, rantam fa. regeneration of salivary gland defects of diabetic wistar rats post human dental pulp stem cells intraglandular transplantation on acinar cell vacuolization and interleukin-10 serum level. pesqui bras odontopediatria clin integr. 2019; 19(1): 1–10. 22. jeane m, asih iara, bogoriani nw. asupan glikosida flavonoid terong belanda (solanum betaceum cav.) terhadap aktivitas superoksida dismutase dan kadar malondialdehid tikus wistar yang diberi aktivitas fisik maksimal. j media sains. 2018; 2(1): 32–6. 23. shafira n, ayu pr, susianti. potensi bit merah (beta vulgaris l.) sebagai nefroprotektor dari kerusakan ginjal akibat radikal bebas. medula med prof j univ lampung. 2019; 9(2): 322–7. 24. lazuardi m, suharjono s, chien c-h, he j-l, lee c-w, peng c-k, sukmanadi m, sugihartuti r, maslachah l. toxicity test of flavonoid compounds from the leaves of dendrophthoe pentandra (l.) miq. using in vitro culture cell models. vet world. 2022; 15(12): 2896–902. 25. purnamasari p, purnawati rd, susilaningsih n. pengaruh ekstrak daun sukun dan madu terhadap gambaran mikroskopik ginjal tikus wistar yang diinduksi dietilnitrosamin. diponegoro med j. 2018; 7(2): 1391–405. 26. ardiaria m. disfungsi mitokondria dan stress oksidatif. jnh (journal nutr heal. 2019; 7(3): 50–5. 27. budi hs, kriswandini il, iswara ad. antioxidant activity test on ambonese banana stem sap (musa parasidiaca var. sapientum). dent j. 2015; 48(4): 188–92. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p178–183 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p178-183 3939 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 39–45 original article cultivation and expansion of mesenchymal stem cells from human gingival tissue and periodontal ligament in different culture media banun kusumawardani1,4, dwi merry christmarini robin1, endah puspitasari2, irma josefina savitri3 and dea ajeng pravita suendi4 1department of biomedical sciences, faculty of dentistry, universitas jember, jember, indonesia 2department of pharmaceutical biology, faculty of pharmacy, universitas jember, jember, indonesia 3department of periodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4postgraduate program, graduate school of biotechnology, universitas jember, jember, indonesia abstract background: gingival tissue and periodontal ligament act as sources of mesenchymal stem cells (mscs) that play a vital role in periodontal regeneration, but they both have limitations for cell availability. mscs cultivated and expanded in various media formulations could be used as a basis for the development of cell therapy protocols. purpose: this study aimed to determine the optimum culture media formulation for cultivation and expansion of human gingival-derived mesenchymal stem cells (hgmscs) and human periodontal ligament stem cells (hpdlscs). methods: the hgmscs and hpdlscs were obtained from gingival tissue and periodontal ligament specimens from an adult patient. the two different culture media formulations used were: 1) α-minimum essential media (α-mem) supplemented with 10% fbs, 100 u/ml penicillin, 100mg/ml streptomycin and 2.5 µg/ml amphotericin b; and 2) dulbecco’s minimum essential media-low glucose (dmem-lg) supplemented with 10% fbs, 2 mmol/l l-glutamine, 100 u/ml penicillin, 100mg/ml streptomycin and 2.5 µg/ml amphotericin b. the minced-gingival tissue and periodontal ligament samples were seeded in 3 cm tissue culture dishes with one of two experimental culture media, and incubated at 37oc in a humidified atmosphere of 5% co2. results: cell morphology was observed on days two and five of the third passage. the gingival tissue and periodontal ligament primary cells exhibited fibroblast-like morphology, long processes and were spindle-shaped. the hpdlscs grown in α-mem exhibited a significant increase in cell viability and proliferation rate compared to the hpdlscs grown in dmem-lg. however, hgmscs displayed similar cell viability and proliferation rate on both types of experimental media. both the hgmscs and hpdlscs expressed msc markers, including cd105, cd146, and cd90, but did not express cd45. conclusion: culture media formulations of α-mem and dmem-lg can be used for the cultivation and expansion of both hgmscs and hpdlscs. keywords: cultivation; expansion; gingival-derived mesenchymal stem cells; periodontal ligament stem cells; proliferation rate. correspondence: banun kusumawardani, department of biomedical sciences, faculty of dentistry, universitas jember, jl. kalimantan 37 jember, 68121 indonesia. email: banun_k.fkg@unej.ac.id introduction human dental tissue is a rich source of easily obtainable adult stem cells, and has a multipotent feature appropriate for use in tissue engineering and regenerative medicine. periodontal ligament is an effective cell source that encourages bone regeneration, while gingival tissue is an easily available source of mesenchymal stem cells (mscs) that have excellent proliferation abilities.1–4 both these cell types exhibit high differentiation capability and can be used to heal various types of periodontal disease. there are however, some major similarities and differences between gingival-derived mesenchymal stem cells (gmscs) and periodontal ligament stem cells (pdlscs). for example, cd146, cd105 and cd90 are markers frequently used to determine cells with multi-lineage differentiation potential, and are expressed in mscs from human dental tissue and bone marrow.2 mscs have to fulfil three criteria, namely: 1) cells are capable of attaching to the cell culture matrix, 2) cells can differentiate into osteocytes, chondrocytes, and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 mailto:banun_k.fkg@unej.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 40 kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 adipocytes,5,6 and 3) cells capable of expressing surface markers cd146, cd105 and cd90, but may not express surface markers cd34 and cd45 at the same time.7 culture media is one component that maintains the biological properties of mscs. dulbecco’s minimum essential media (dmem) and α-minimum essential media (α-mem) are widely used to grow mesenchymal stem cells, because they contain l-glutamine and l-ascorbicacid-2-phosphate, so they can preserve stem cell phenotypes, such as expressing cd146, stro-1, cd44, and cd105, until the eighth passage.8 however, there is no consensus on the most effective culture media to optimise the culture conditions of human gmscs (hgmscs) and human pdlscs (hpdlscs). optimising cell quality and standardising the manufacturing method is very important for the development and application of periodontal tissue regenerative therapy. therefore, the determination of the most suitable media in which to cultivate and expand hgmscs and hpdlscs is undoubtedly very necessary. this study aimed to analyse the stemness maintenance of hgmscs and hpdlscs originating from the same patient using α-mem media formulation compared to using dmem-low glucose (dmem-lg) media formulation. materials and methods tooth extraction was performed in the department of oral surgery at persaudaraan djama’ah haji indonesia (pdhi)-islamic hospital, yogyakarta, indonesia, and all the participants involved in this study had given their informed consent in writing prior to the study. the experimental protocol was approved by the ethics committee of faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia, with document number of 001607/kkep/fkgugm/ec/2018. the hgmscs and hpdlscs were isolated from extracted third molar teeth of a healthy patient (34year-old female) with no history of periodontal disease. the culture media consisted of two experimental media: 1) α-mem supplemented with 10% fbs, 100 u/ml penicillin, 100mg/ml streptomycin and 2.5 µg/ml amphotericin b; and 2) dmem-lg supplemented with 10% fbs, 2 mmol/l l-glutamine, 100 u/ml penicillin, 100mg/ml streptomycin and 2.5 µg/ml amphotericin b. gingival tissue samples attached to the cervical teeth were collected and minced, whereas pdl tissue samples were obtained from one-third to apical root of the third molar, and then minced. for the human gingival and periodontal ligament primary cells, the minced-tissue samples were plated in 3 cm tissue culture dishes with one of two experimental culture media, and incubated at 37oc in a humidified atmosphere of 5% co2. the culture media was freshened up twice a week throughout the 14-day incubation period. before harvesting, the human gingival and periodontal ligament primary cells were visually examined with an inverted microscope. next, the subculture of human gingival and periodontal ligament primary cells was carried out on day 14 of incubation. the cells in two experimental culture media attained up to 80% confluency, and they were then harvested with a 0.25% trypsin-edta solution. for the consecutive experiments, human gingival and periodontal ligament primary cells were cultured using one of two experimental culture media until passage two (p2). culture media was refreshed every three days. then, the following p3 was evaluated for cell morphology, cell proliferation and immunophenotype. both human gingival and periodontal ligament primary cells were regularly observed under the inverted microscope to check for any morphological changes. the cell morphology was identified by cell shape into round, branched and spindle cell. images were recorded at a magnification of ××100 and were taken of five fields in randomly selected dishes from each group. for morphological analysis of hgmscs and hpdlscs, they were categorised as round-shape (rs), spindle-shaped (ss), and flat-shape (fs). the proliferation rate and cell viability of hgmscs and hpdlscs were determined by mtt assay [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide].9 briefly, the cells were harvested from a 25 cm2 flask, reseeded at 5 × 103 cells/cm2 into a 96-well plate, and incubated at 37oc in a co2 incubator for periods of 24, 48, 72 and 96 h. the media were removed, and 20 µl of mtt dye (5 mg/ml of phosphate buffered saline (ph 7.2) was added into all wells. the plate was incubated at 37oc in a co2 incubator for 4 h, and 150 µl of dimethylsulfoxide (dmso) was added to dissolve the formazan crystals. the optical density (od) was measured at 570 nm of wavelength using a microplate reader. the experiments were conducted in triplicate wells. proliferation rate (pr) was calculated from the formula pr = od of cells at tn/od of cells at t0. surface markers for hgmscs and hplscs were analysed using flow cytometry incorporating four fluorochrome-conjugated antibodies (human mesenchymal stem cell: multi-color flow cytometry kit. catalog no. fmc020. r & d system, mn-usa). confluent cells were detached from their place by 0.25% trypsin-edta solution, adjusted to 1 × 106 cells in 100 µl, washed with 2 ml of staining buffer, and centrifuged at 300 xg for five minutes. fc receptor blocking reagents were added, then blocking and staining took place using 1 µg of pre-immune igg. cells were incubated along with mouse monoclonal antibodies (10 µl) specific for human cd105, cd146, cd90, and cd45, or isotype-matched control immunoglobulin gs for 30–45 minutes at room temperature in darkness. at the end of the incubation period, the cells were washed with 2 ml of staining buffer, and then the cell pellet was resuspended in 200–400 µl of staining buffer for flow cytometric analysis. data for proliferation rate and cell viability were stated as mean ± standard deviation (sd). statistical analysis was carried out using one-way analysis of variance, followed by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 41kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 tukey’s post hoc multiple-comparison tests to determine differences between the groups. the degree of significance was considered to be p < 0.05. results the hgmscs and hpdlscs were maintained in an adherent culture, and three morphologically distinct cell types were observed: rounded-shape (rs) cells, spindleshaped (ss) cells and flatten-shape (fs) cells (figure 1). cell morphology was observed on day two and day five of p3. the gingival and periodontal ligament primary cells showed fibroblast-like morphology, long processes and were spindle-shaped. cells exhibited differences in cell morphology using both experimental media. the hgmscs (g1 group) and hpdlscs (pl1 group) had predominantly ss cells on dmem-lg supplemented with 10% fbs, 2 mmol/l l-glutamine, 100 u/ml penicillin, 100mg/ml streptomycin and 2.5 µg/ml amphotericin b. however, rs cells were found abundantly from hgmscs (g2 group) and from hpdlscs (pl2 group) that had been grown on α-mem supplemented with 10% fbs, 100 u/ml penicillin, 100 mg/ml streptomycin and 2.5 µg/ml amphotericin b. besides, few cells were determined to be binucleated-cells and fs cells on both experimental media. the effects of culture media formulations were analysed on the hgmscs and hpdlscs viability (table 1 and figure 2), and proliferation rate (figure 3). the comparison of the cell viability demonstrated that the hpdlscs grown in α-mem presented a significant increase in cell viability and proliferation rate compared to the hpdlscs grown in dmem-lg. the growth curve of pdlscs showed that the media formulation of α-mem was more able to induce cell g1 g2 pl1 pl2 g1 pl1 g2 pl2 a b figure 1. cell morphology of gingival stem cells in dmem-lg (g1) and α-mem (g2), and periodontal ligament stem cells in dmem-lg (pl1) and α-mem (pl2), observed on day 2 (a) and day 5 (b) on the third passage, phase-contrast inverted microscopy, index 100 µm. all cells were tightly adherent, spread apart, and displayed spindle-shaped fibroblastic-like cells. the hgmscs and hpdlscs cultured in dmem-lg formulation grew faster with earlier confluence. shape indicators: spindle-shape (black arrow), rounded-shape (white arrow) and flat-shape (yellow arrow). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 42 kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 proliferation. besides, the cell viability and proliferation rate were similarly recorded at hgmscs on both types of experimental media. the hgmscs and hpdlscs expressed the positive msc surface markers cd105, cd146, and cd90, and expressed the negative hematopoietic cell marker cd45 (table 2, figure 4 and figure 5). the expression levels of cd146 in all groups were very low, whereas the surface marker expressions of cd105 and cd90 were high in all groups. the hgmscs and hpdlscs showed an absence of cd45 expression. these results indicated that hgmscs and hpdlscs had similar msc-like potential. table 1. cell viability of gingival stem cells and periodontal ligament stem cells on experimental media of dmem-lg and α-mem for 0, 24, 48, 72 and 96 hours incubation duration (h) cell viability gmscs plscs dmem-lg α-mem dmem-lg α-mem 0 84.67 ± 6.13 118.50 ± 8.31 68.12 ± 17.60 154.03 ± 42.39* 24 98.03 ± 10.56 102.77 ± 10.50 76.01 ± 7.66 132.42 ± 12.89† 48 88.60 ± 9.20 113.65 ± 11.34 79.09 ± 4.33 126.70 ± 6.95§ 72 93.90 ± 1.93 106.52 ± 2.20 78.47 ± 2.63 127.54 ± 4.25‡ 96 100.62 ± 15.74 100.98 ± 15.41 70.87 ± 10.67 143.13 ± 20.34# *†§‡# significant difference in relation between groups 0 50 100 150 200 250 c el l v ia bi lit y (o d a t 5 95 n m ) experimental group 0 hr 24 hrs 48 hrs 72 hrs 96 hrs figure 2. cell viability of gingival stem cells in dmem-lg (g1) and α-mem (g2), and periodontal ligament stem cells in dmemlg (pl1) and α-mem (pl2) for 0, 24, 48, 72 and 96 hours incubation. 0,00 0,50 1,00 1,50 2,00 0 hr 24 hrs 48 hrs 72 hrs 96 hrs p ro lif er at io n r at e (o d a t 5 95 n m ) incubation time (hours) g1 g2 pl1 pl2 table 2. flow-cytometric analysis of mesenchymal stem cell markers cells cd105 cd146 cd90 cd45 gingival primary cells + dmem (g1) 79.16 4.03 75.95 1.81 gingival primary cells + α-mem (g2) 67.35 3.21 75.00 2.61 periodontal ligament primary cells + dmem (pl1) 89.98 13.29 79.42 4.34 periodontal ligament primary cells + α-mem (pl2) 77.20 9.94 71.27 9.37 figure 3. proliferation rate of gingival stem cells in dmem-lg (g1) and α-mem (g2), and periodontal ligament stem cells in dmem-lg (pl1) and α-mem (pl2) for 0, 24, 48, 72 and 96 hours incubation. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 43kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 g1 pl1 pl2 g2 figure 4. the dot plot of gingival stem cells in dmem-lg (g1) and α-mem (g2), and periodontal ligament stem cells in dmemlg (pl1) and α-mem (pl2). g1 g2 pl1 pl2 figure 5. flow-cytometric analyses for gingival stem cells in dmem-lg (g1) and α-mem (g2), and periodontal ligament stem cells in dmem-lg (pl1) and α-mem (pl2) have similar surface molecule phenotype. there was a lack of cd146 and cd45, but a positive showing for cd105 and cd90. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 44 kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 discussion gmscs and pdlscs play a pivotal role in periodontal tissue regeneration.10 functionally, gmscs and pdlscs can maintain their biological properties in vitro, and can rejuvenate periodontal tissue in vivo.11 however, the number of gmscs and pdlscs that can be obtained from a single sample are very limited.2,12 consequently, gmscs and pdlscs require expansion before application. this study of the third passage of hgmscs and hpdlscs, examined the morphology, viability and proliferative ability of the cells in two culture media formulations of α-mem and dmem-lg. on the second an fifth day of expansion, the predominantly ss cells were found from hgmscs (g1 group) and hpdlscs (pl1 group) in the culture media formulated with dmem-lg. the hgmscs demonstrated no statistically significant difference in viability or proliferation rate when they were grown in dmem-lg and α-mem formulation, whereas hpdlscs exhibited a significant increase in viability and proliferation rate in α-mem formulation compared to dmem-lg formulation. this study suggested that culture media strongly influences the biological properties of hgmscs and hpdlscs to maintain the cell stemness. this study reported that dmem-lg served as a suitable culture medium for cultivating hgmsc. lower glucose concentrations may be most appropriate for large-scale hgmsc culture, but α-mem formulation does not serve as a proper media for hgmscs, although α-mem has the lower concentration of glucose. otherwise, hpdlscs cultivated in α-mem formulation had greater proliferation rates than hpdlscs cultivated in dmem-lg formulation. this maybe due to the fact that α-mem has a higher concentration of amino acids, vitamins, and nucleotides compared to dmem.8,13 in addition, glucose and glutamine used in culture media are essential for providing energy (atp), which is necessary for cell growth and cell maintenance.14,15 however, l-glutamine can be degraded to ammonium ions, which are harmful to cell viability under culture conditions. based on the immunophenotypic profile of the hgmscs and hpdlscs, the data exhibited that more than 70% of the cells expressed cd105 and cd90, low expression of cd146 and negative expression of cd45 (table 2, figure 4 and figure 5). generally, mscs have to express cd73, cd105 and/or cd90, and do not express cd14 or cd11b, cd34, cd45, hla class ii, cd79a or cd19.16,17 other study also reported that approximately 60% of the hdpscs expressed cd105, and that they did not express cd14 or cd45.18 furthermore, pdlscs express cd105, cd73, cd166, cd90, and they show a negative expression for cd14, cd34, cd31, cd45, cd40, cd79a, cd54, hladr, cd80 and cd86,11,18 whereas gmscs were positive for cd29, cd73, cd90, cd105, cd146, and stro-1, and they show a negative expression for cd45 and cd34.19,20 more specifically, this study also found that hpdlscsexpanded with dmem-lg formulation expressed cd146. this study demonstrated that hpdlscs have pericyte-like characteristics, which may originate from pericytes located in the perivascular wall of the periodontal ligament. in this case, pdlscs have similarities to pericytes in morphology, phenotype, differentiation potential, and that they have the ability to form capillary structures in vitro.21,22 this study concluded that α-mem and dmem-lg culture media maintained cell morphology, proliferation rate and immunophenotypes of hgmscs and hpdlscs. both culture media formulations were suitable media culture for the cultivation and expansion of hgmscs and hpdlscs. more importantly, these findings favour the pursuit of standardised protocols in cultivation and expansion of hgmscs and hpdlscs. nevertheless, more studies are required to define the effects of culture media formulation on the osteogenic, chondrogenic and adipogenic potential of hgmscs and hpdlscs. acknowledgement this research has been supported by research institute of universitas jember, indonesia (grant no. 3240/un25.3.1/ lt/2019). references 1. yang jw, shin yy, seo y, kim h-s. therapeutic functions of stem sells from oral cavity: an update. int j mol sci. 2020; 21(12): 4389. 2. zhu w, liang m. periodontal ligament stem cells: current status, concerns, and future prospects. stem cells int. 2015; 2015: 1–11. 3. assem m, kamal s, sabry d, soliman n, aly rm. preclinical assessment of the proliferation capacity of gingival and periodontal ligament stem cells from diabetic patients. open access maced j med sci. 2018; 6(2): 254–9. 4. lang np, bartold pm. periodontal health. j clin periodontol. 2018; 45(suppl 20): s9–16. 5. cheng k-h, kuo t-l, kuo k-k, hsiao c-c. human adipose-derived stem cells: isolation, characterization and current application in regeneration medicine. genomic med biomarkers, heal sci. 2011; 3(2): 53–62. 6. mccormack sw, witzel u, watson pj, fagan mj, gröning f. the biomechanical function of periodontal ligament fibres in orthodontic tooth movement. plos one. 2014; 9(7): e102387. 7. alipour f, parham a, kazemi mehrjerdi h, dehghani h. equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials. cell j. 2015; 16(4): 456–65. 8. jung i-h, kwon b-s, kim s-h, shim h-e, jun c-m, yun j-h. optimal medium formulation for the long-term expansion and maintenance of human periodontal ligament stem cells. j periodontol. 2013; 84(10): 1434–44. 9. nirmala mj, durai l, gopakumar v, nagarajan r. preparation of celery essential oil-based nanoemulsion by ultrasonication and evaluation of its potential anticancer and antibacterial activity. int j nanomedicine. 2020; 15: 7651–66. 10. el fattah ma, ding g, wei f, zhang c, ezz ea, wang s. identification and cementoblastic / osteoblastic differentiation of postnatal stem cells from human periodontal ligament. maced j med sci. 2011; 4(1): 37–43. 11. bartold pm, gronthos s. standardization of criteria defining periodontal ligament stem cells. j dent res. 2017; 96(5): 487–90. 12. iwata t, yamato m, zhang z, mukobata s, washio k, ando t, feijen j, okano t, ishikawa i. validation of human periodontal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 45kusumawardani et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 39–45 ligament-derived cells as a reliable source for cytotherapeutic use. j clin periodontol. 2010; 37(12): 1088–99. 13. salehinejad p, alitheen nb, nematollahi-mahani sn, ali am, omar ar, janzamin e, hajghani m. effect of culture media on expansion properties of human umbilical cord matrix-derived mesenchymal cells. cytotherapy. 2012; 14(8): 948–53. 14. yuan x, logan tm, ma t. metabolism in human mesenchymal stromal cells: a missing link between hmsc biomanufacturing and therapy? front immunol. 2019; 10: 977. 15. sánchez-kopper a, becker m, pfizenmaier j, kessler c, karau a, takors r. tracking dipeptides at work-uptake and intracellular fate in cho culture. amb express. 2016; 6: 48. 16. petrenko y, vackova i, kekulova k, chudickova m, koci z, turnovcova k, kupcova skalnikova h, vodicka p, kubinova s. a comparative analysis of multipotent mesenchymal stromal cells derived from different sources, with a focus on neuroregenerative potential. sci rep. 2020; 10: 4290. 17. viswanathan s, shi y, galipeau j, krampera m, leblanc k, martin i, nolta j, phinney dg, sensebe l. mesenchymal stem versus stromal cells: international society for cell & gene therapy (isct®) mesenchymal stromal cell committee position statement on nomenclature. cytotherapy. 2019; 21(10): 1019–24. 18. soares imv, fernandes gv de o, larissa cordeiro c, leite ykp de c, bezerra d de o, carvalho mam de, carvalho cmrs. the influence of aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats. j appl oral sci. 2019; 27: e20180103. 19. mrozik k, gronthos s, shi s, bartold pm. a method to isolate, purify, and characterize human periodontal ligament stem cells. methods mol biol. 2017; 1537: 413–27. 20. hung bp, hutton dl, kozielski kl, bishop cj, naved b, green jj, caplan ai, gimble jm, dorafshar ah, grayson wl. platelet-derived growth factor bb enhances osteogenesis of adipose-derived but not bone marrow-derived mesenchymal stromal/stem cells. stem cells. 2015; 33(9): 2773–84. 21. lei m, li k, li b, gao l-n, chen f-m, jin y. mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation. biomaterials. 2014; 35(24): 6332–43. 22. iwasaki k, komaki m, yokoyama n, tanaka y, taki a, kimura y, takeda m, oda s, izumi y, morita i. periodontal ligament stem cells possess the characteristics of pericytes. j periodontol. 2013; 84(10): 1425–33. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p39–45 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p39-45 216 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 216–220 original article introduction class ii skeletal malocclusion is often encountered in orthodontics and is one of the main reasons for patients to seek treatment. previous studies have shown that the occurrence of class ii malocclusion is quite high among populations around the world.1 a study in denmark found a 25% occurrence of class ii skeletal malocclusion in their population.2 in asia, a study with people of mongoloid ethnicity that showed 33.1% of this population had class ii malocclusion3 and another study in india showed 30.1% class ii malocclusion in the population.1 a study in singapore with people of chinese ethnicity showed a 23.1% occurrence of class ii malocclusion4 and 70% occurrence was found in iran.5 rosenblum6 found out that in 103 class ii skeletal malocclusion cases, 56.3% had maxillary prognathism and only 27% showed mandibular retrognathism. the differentiation of the osteoblast from its progenitor requires the activity of specific transcription factors which are expressed during development. runt-related transcription factor-2 (runx2) is a member runt family of transcription factors that is essential for skeletal development.7-9 runx2 is essential for osteoblast differentiation in endochondral and intramembranous ossification.10 the runx gene consists of runx1, runx2 and runx3. runx2 plays an important role in osteogenesis, cartilage formation, cell migration, vascular bone invasion and tooth formation.8,11,12 runx2 is essential dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p216–220 runx2 rs59983488 polymorphism in class ii malocclusion in the indonesian subpopulation fadli jazaldi1, benny m. soegiharto1, astrid dinda hutabarat2, noertami soedarsono3 and elza ibrahim auerkari3 1department of orthodontics, 2orthodontic postgraduate program, 3department of oral biology, faculty of dentistry, university of indonesia, jakarta, indonesia abstract background: class ii malocclusion is one of the main orthodontic issues for patients in seeking treatment. the prevalence of class ii malocclusion varies in different populations. variation in skeletal profile is mainly controlled internally by a regulatory gene. runt-related transcription factor-2 (runx2) plays a role in osteoblast differentiation and is highly expressed during development. purpose: this study aimed to evaluate the relation of regulatory gene variation in the runx2 promoter with class ii malocclusion. methods: dna samples were acquired from 95 orthodontic patients in jakarta, indonesia, who were divided into two groups: class i skeletal malocclusion (control group) and class ii malocclusion. a single nucleotide polymorphism was investigated using the polymerase chain reaction and restriction fragment length polymorphism techniques. the distribution of alleles was assessed using the hardy-weinberg test. the relationship between polymorphism and skeletal variation was assessed with the chi-square test and logistic regression. results: the frequency distributions of genotypes and alleles were tested for hardy-weinberg equilibrium and found to be slightly deviated. there was an equal distribution of g and t alleles throughout class ii and class i skeletal malocclusions and the chi-square test showed that this relationship was not significant (p=0.5). conclusion: runx2 rs59983488 polymorphism was found in the indonesian subpopulation; however, an association between runx2 rs59983488 polymorphism and class ii skeletal malocclusion was not found. keywords: class ii; g330t; indonesia; polymorphism; runx2; rs59983488 correspondence: fadli jazaldi, department of orthodontics, faculty of dentistry, university of indonesia. jl. salemba raya no.4, jakarta pusat, jakarta 10430, indonesia. email: fadli.jazaldi@ui.ac.id mailto:fadli.jazaldi@ui.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p216-220 217jazaldi et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 216–220 in bone development and chondrogenesis as an upstream controller for several effector genes. runx2 is required in the preosteoblast maturation process.10,11,13 runx2 regulates matrix protein genes and indian hedgehog transcription factor, an important regulator for chondrogenesis.11,14 runx2 is upregulated by bone morphogenetic proteins (bmps), fibroblast growth factors (fgfs) and retinoid acid. the runx2 gene is located at 6p21.1 with 250 kb spans. runx2 consists of two transcription factors, the distal p1 promoter and the proximal p2. runx2 in osteoblasts was found mainly as transcripts from the p1 promoter. both promoters are specifically expressed in osteoblast and chondrocytes. 8,11,15,16 genetic variation of runx2 has been investigated for its relation to skeletal variation and bonerelated disease.2,17 single nucleotide polymorphism (snp) is the most common variation that occurs in the human genome and happens in more than 1% of the population.18 the association of runx2 rs59983488 polymorphism with class ii malocclusion was inconsistent in previous studies,16,19 therefore it is important to do further research to determine this relationship in this population. this study aimed to assess the relationship of runx2 rs59983488 polymorphism to class ii skeletal malocclusion in the indonesian subpopulation. materials and methods the research participants were orthodontic patients aged between 20 and 45 years from the university of indonesia’s dental hospital. all study participants signed an informed consent document. ethical clearance of this study was granted by the ethical committee, faculty of dentistry, university of indonesia (no. 26/ethical approval/fkgui/ vii/2015, protocol number: 09370515). age, sex, ethnicity and socioeconomic status were recorded. malocclusion classification was based on sagittal relationship of steiner’s analysis measuring sella-nasion-a point (sna), sellanasion-b point (snb) and a point-nasion-b point (anb). study participants included 48 class ii and 52 class i malocclusion patients. class i subjects were recruited with sna 82°±2°, snb 78°±2°, anb 2°±2°. class ii subjects were recruited as: class ii with mandibular retrusion (sna 82°±2°, snb <76°, anb >4°, overjet >4 mm), class ii with maxillary protrusion (sna >84°, snb 78°±2°, anb >4°, overjet >4 mm), and class ii with maxillary protrusion and mandibular retrusion (sna >84°, snb <76°, anb >4°, overjet >4 mm). all lateral cephalometric images were traced the by same operator (f.j), who is an experienced orthodontist. to reduce the effect of operator measurement error, the tracing was analysed three separate times at two weeks intervals with single blinding. genomic dna was collected from the peripheral blood of all 95 subjects. dna extraction was performed by using 3 ml peripheral blood and 9 ml red blood cell lysis solution (1.45 m nh3cl, 5 mm anhydrous edta, and 0.1 khco3, [instagene matrix, bio-rad, california, united state of america]) incubated at room temperature for 10 min. the mixture was then centrifuged at 1500 rpm for 10 min and the supernatant was removed. 2 ml cell lysis solution was added and incubated at 37℃ for 60 min. 1.3 ml protein precipitation (pp) solution [promega, wisconsin, united state of america] was added and then vortexed and centrifuged at 40℃ at 3000 rpm for 5 min. the supernatant was transferred into a new tube containing 2.3 ml isopropanol, and then inverted to dry. this process was repeated until the dna turned white. after that, the dna was rehydrated with 200–300 µl tris-hcl edta, incubated in 37℃ for 2 h and stored at -20℃. runx2 rs59983488 g330t polymorphism was detected by using the polymerase chain reaction and restriction fragment length polymorphism (pcr-rflp) method. pcr amplification was done by using the forward primer 5’-aaa gca aag gag gtt gac cgg-3’ and reverse primer 5’-ccc tgc cct tct ttc tct ctc-3’ with perkin elmer gene amp pcr system 9700 [kumamoto, japan]. each reagent contained 10 µl buffer solution [promega, wisconsin, united state of america], 2 µl forward and reverse primers, 7.5 µl ddh2o, and 0.5 µl genomic dna. the conditions for this pcr were 94℃ for 6 min followed by 35 cycles of the amplification denaturation phase at 94℃ for 60 s, the annealing phase at 62℃ for 30 s, elongation at 72℃ for 30 s and final elongation at 72℃ for 5 min. the pcr product was digested with bsaj1 enzyme restriction [new england biolabs (neb), massachusetts, united states of america]. the mixture consisted of 1 µl restriction enzyme bsaj1 (10u/µl), 2 µl enzyme buffer and 18 µl ddh2o was added to 10 µl pcr product. the mixture was incubated at 60℃ for 4 h and inactivated by incubating at 80℃ for 20 min. the restriction products were subjected to electrophoresis on 2% agarose gel [promega, wisconsin, united state of america] in 1x tae buffer solution (0.04 m tris–acetate, 0.002 m edta, ph 8.0) at 80 v, 400 ma, for 60 min, resulting in three possible genotypes: gg, gt and tt. the restriction products were visualised with geldoc 2000 [bio-rad, california, united state of america] and showed qualitative data for the g allele of 205 bp and 20 bp and the t allele of 225 bp. this study used the hardy-weinberg equilibrium to evaluate genetic variation that occurred in the population. intra-observer reliability was evaluated using the intraclass correlation coefficient (icc). chi-square and logistic regression tests were used to assess the relation of runx2 rs59983488 polymorphism with class ii skeletal malocclusion. statistical analysis was performed using the program for social science (ibm spss) version 22 [chicago, united state of america]. results a total of 95 orthodontic patients, consisting of 48 class ii skeletal malocclusion cases and 52 class i skeletal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p216–220 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p216-220 218 jazaldi et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 216–220 malocclusion cases, were recruited at the dental hospital, faculty of dentistry, university of indonesia. the reliability of the cephalometric measurement was good (icc > 0.820). genotyping using the pcr-rflp methods showed g allele cuts in 205 bp and 20 bp and t allele cuts in 225 bp (figure 1). therefore, the gg genotype (wildtype) showed two bands, the gt genotype (heterozygous) showed three bands and the tt genotype (mutant) showed a single band. the distributions of genotypes and alleles are shown in table 1. this population was tested against hardy-weinberg equilibrium and showed a slight deviation (p=0.04959). the distributions of the genotype and allele moi (class i skeletal malocclusion) and moii (class ii skeletal malocclusion) were assessed with the chi-square test to identify their relationship within each group and were not found to be significant. the distributions of genotype and allele g and t were equal in class ii skeletal malocclusion and the control (class i skeletal malocclusion). a logistic regression test was used and found significant (p<0.05) with a slight reduction of the odds ratio 0.908 (95% confidence interval :0.51–1.617). discussion runx2 rs59983488 shows bases alteration from g to t at -330 in the p1 region. genotyping using pcr-rflp methods showed gg genotype cuts in 205 bp and 20 bp, gt genotype cuts in 225 bp, 205 bp and 20 bp, and tt genotype cuts in 225 bp (figure 1). the 20 bp fragment is hard to see in figure 1 because traditional agarose gels are most effective in separating dna fragments between 100 bp and 25 kb. agarose gel is popular for separating moderate to large-sized nucleic acids which have a wide range of separations. there are few alternate methods for separating small dna fragments. polyacrylamide gel can be used as an alternative since this gel has a higher concentration resulting in better resolution when run vertically.20 another method to separate smaller dna fragments would be to use agarose gel doped with graphene oxide.21 the genotype and allotype frequency distributions in this study are slightly deviated from the hardy-weinberg equilibrium (hwe) (p<0.05). this finding is consistent with previous runx2 studies in indonesian and chinese populations.22,23 mokhtar et al.24 found different results table 1. distributions of genotypes and alleles of runx2 t330g genotype moi moii p n % n % tt 7 13.5 6 13.9 0.831 gt 32 61.5 24 55.9 gg 13 25 13 30.2 t allele 46 44.2 36 41.8 0.5 g allele 58 55.8 50 58.2 figure 1. genotyping runx2 rs59983488; a and d. gt genotype (225 bp, 205 bp, 20 bp); b and f. tt genotype (225 bp); c. gg genotype (205 bp, 20 bp); lad. ladder 50 bp; g. positive control; h. negative control. moi: class i skeletal malocclusion; moii: class ii skeletal malocclusion. chi-square test, p<0.05 was considered significant. logistic regression test p<0.05, or 0.908, confidence interval 95% 0.51–1.617 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p216–220 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p216-220 219jazaldi et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 216–220 in the malaysian population that showed consistency of genotypes and alleles with the hwe. another study of runx2 polymorphism in indonesia has been done in relation to osteoporosis, which had similar findings to ours, but showing a higher deviation in hwe value.23 this study involved a different population from that study, although they shared the same location, and therefore the populations had similar characteristics. the reason for this deviation was not clear. we suspect it was due to population stratification and a limited number of study participants.25-27 the population in this study was relatively small – 95 subjects in total, with 13 having the tt genotype (13.7%) and only 6 (0.06%) in the class ii skeletal malocclusion group. in this study, an association between runx2 rs59983488 polymorphism and class ii skeletal malocclusion was not found. the t and g alleles were spread evenly in both groups of class i and class ii malocclusion. küchler et al.19 found a different result in the caucasian population. he studied two sites of runx2 promoter and found an association between rs59983488 polymorphism and maxillary protrusion, although he did not find any association between rs1200425 and either maxillary protrusion or class ii malocclusion. the study also found a strong interaction between runx2 rs59983488 snp and smad6 rs3934908 snp in the class ii malocclusion model.19 another study in malaysia investigated the relation of runx2 rs6930053 polymorphism with class ii malocclusion and found a significant difference in both allele and genotype.24 bigger sample size and additional maxillary measurement are recommended to evaluate any association of runx2 snp with class ii skeletal malocclusion and maxillary growth, and the possible mechanism for this. runx2 polymorphism at various sites has been widely investigated in its relation to different phenotypes of skeletal growth (table 2). chang et al.28 reported that there was an association between runx2 polymorphism and ossification of the posterior longitudinal ligament. auerkari et al.23 and bustamante et al.16 reported no association between runx2 rs59983488 polymorphism and osteoporosis. there were also studies that reported on the relationship between runx2 polymorphism and non-syndromic cleft lip and palate.22,29 there are still inconsistencies regarding the relationship of polymorphism in runx2 with skeletal phenotypes, and further research is needed to assess the role of runx2 in skeletal growth and development. the level of runx2 expression and its activity fluctuate during the developmental process. the activity of the runx2 protein is regulated by various post-translational modifications, such as phosphorylation, methylation and acetylation or ubiquitination. bmp-2 activity was reported to stimulate runx2 acetylation.11 post-translational regulation by phosphorylation and acetylation is essential for fgf-induced cranial development, and this has been investigated for premature fusion of cranial sutures and the midfacial hypoplastic changes that occur because of lack of growth at the fused sutures. enhanced stability of runx2 by post-translational regulation could offer a methodology for bone regeneration therapy.30 one study shows that epigenetic histone modifications on receptors for vitamin d (vdr) and bone cell differentiation affect the binding of runx2 to the genome, modify and restrict patterns of gene expression and alter cellular response to the vitamin d hormone.31 yan et al.32 found that mettl3 could directly induce m6a methylation of runx2 mrna to enhance its cellular stability and indirectly upregulate the cellular level of runx2 by m6a methylation of pre-mir-320. ling et al.33 reported that nicotinamide phosphoribosyltransferase (nampt) promotes osteogenesis through the epigenetic regulation of runx2 expression, thus upregulating runx2 expression. future studies involving runx2 posttranslational regulation modification should be carried out to investigate its relation with maxillary growth and class ii skeletal malocclusion. table 2. runx2 polymorphism studies rs p phenotype reference rs967588 rs16873379 rs3749863 rs6908650 rs1321075 rs1406846 rs2677108 c>t t>c a>c g>c a>c t>a c>t not significant significant not significant not significant not significant significant significant ossification posterior longitudinal ligament chang et al., 201728 rs59983488 g>t not significant osteoporosis auerkari et al., 201423 deletion 17a>11a significant bone fracture morrison et al., 201334 rs6930053 significant class ii malocclusion mokhtar et al., 201824 rs545239 rs1200425 rs16873396 significant non-syndromic cleft lip and palate wu al., 201222 rs1934328 significant non-syndromic cleft lip and palate jung et al.,201429 rs59983488 rs1200425 rs59983488 rs59983488 g>t g>a g>t g>t significant not significant not significant not significant maxillary protrusion osteoporosis class ii malocclusion küchler et al., 202019 bustamante et al., 200716 this study dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p216–220 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p216-220 220 jazaldi et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 216–220 in conclusion, runx2 rs59983488 polymorphism was detected in the indonesian subpopulation. however, a relationship between the runx2 polymorphism promoter and class ii skeletal malocclusion was not found. a larger sample of this population and an additional maxillary component of measurement are recommended to further assess the link between runx2 rs59983488 polymorphism and class ii skeletal malocclusion and maxillary growth. acknowledgement this study was supported by post graduate grant 2015, dprm, university of indonesia. references 1. joshi n, hamdan am, fakhouri wd. skeletal malocclusion: a developmental disorder with a life-long morbidity. j clin med res. 2014; 6(6): 399–408. 2. proffit wr, fields hw, larson b, sarver dm. contemporary orthodontics. 6th ed. st. louis: mosby; 2018. p. 685–715. 3. wahab rma, idris h, yacob h, ariffin shz. cephalometric and malocclusion analysis of kadazan dusun ethnic orthodontic patients. sains malaysiana. 2013; 42: 25–32. 4. chan gxl, tan ely, chew mt, wong hc, foong kwc, yow m. prevalence of class i, ii and iii skeletal relationships and its association with dental anomalies in an ethnic chinese orthodontic population. proc singapore healthc. 2021; : 201010582110007. 5. oshagh m, ghaderi f, pakshir hr, baghmollai am. prevalence of malocclusions in school-age children attending the orthodontics department of shiraz university of medical sciences. east mediterr heal j. 2010; 16(12): 1245–50. 6. rosenblum re. class ii malocclusion: mandibular retrusion or maxillary protrusion? angle orthod. 1995; 65(1): 49–62. 7. takarada t, hinoi e, nakazato r, ochi h, xu c, tsuchikane a, takeda s, karsenty g, abe t, kiyonari h, yoneda y. an analysis of skeletal development in osteoblast-specific and chondrocyte-specific runt-related transcription factor-2 (runx2) knockout mice. j bone miner res. 2013; 28(10): 2064–9. 8. komori t. roles of runx2 in skeletal development. in: groner y, ito y, liu p, neil j, speck n, van wijnen a, editors. runx proteins in development and cancer advances in experimental medicine and biology. singapore: springer; 2017. p. 83–93. 9. lam k, zhang d-e. runx/cbf transcription factors☆. in: reference module in biomedical sciences. elsevier; 2015. p. 1–5. 10. long f. building strong bones: molecular regulation of the osteoblast lineage. nat rev mol cell biol. 2012; 13(1): 27–38. 11. wysokinski d, pawlowska e, blasiak j. runx2: a master bone growth regulator that may be involved in the dna damage response. dna cell biol. 2015; 34(5): 305–15. 12. bruderer m, richards r, alini m, stoddart m. role and regulation of runx2 in osteogenesis. eur cells mater. 2014; 28: 269–86. 13. kawane t, qin x, jiang q, miyazaki t, komori h, yoshida ca, matsuura-kawata vk dos s, sakane c, matsuo y, nagai k, maeno t, date y, nishimura r, komori t. runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating fgfr2 and fgfr3. sci rep. 2018; 8(1): 13551. 14. ono k, hata k, nakamura e, ishihara s, kobayashi s, nakanishi m, yoshida m, takahata y, murakami t, takenoshita s, komori t, nishimura r, yoneda t. dmrt2 promotes transition of endochondral bone formation by linking sox9 and runx2. commun biol. 2021; 4(1): 326. 15. komori t. the functions of runx family transcription factors and cbfb in skeletal development. oral sci int. 2015; 12(1): 1–4. 16. bustamante m, nogués x, águeda l, jurado s, wesselius a, cáceres e, carreras r, ciria m, mellibovsky l, balcells s, díezpérez a, grinberg d. promoter 2 -1025 t/c polymorphism in the runx2 gene is associated with femoral neck bmd in spanish postmenopausal women. calcif tissue int. 2007; 81(4): 327–32. 17. carlson ds, buschang ph. craniofacial growth and development: developing a perspective. in: graber lw, vanarsdall rl, vig kwl, huang gj, editors. orthodontics: current principles and techniques. 6th ed. st. louis: mosby elsevier; 2017. p. 1–30. 18. alberts b, hopkin k, johnson a, morgan d, raff m, roberts k, walter p. essential cell biology. 5th ed. uk: w. w. norton; 2018. p. 864. 19. küchler ec, reis clb, carelli j, scariot r, nelson‐filho p, coletta rd, paza ao, matsumoto man, proff p, kirschneck c. potential interactions among single nucleotide polymorphisms in bone‐ and cartilage‐related genes in skeletal malocclusions. orthod craniofac res. 2021; 24(2): 277–87. 20. l e e py, cost u mbrado j, hsu c-y, k i m y h. aga rose gel electrophoresis for the separation of dna fragments. j vis exp. 2012; (62): 3923. 21. li j, yang y, mao z, huang w, qiu t, wu q. enhanced resolution of dna separation using agarose gel electrophoresis doped with graphene oxide. nanoscale res lett. 2016; 11(1): 404. 22. wu t, fallin md, shi m, ruczinski i, liang ky, hetmanski jb, wang h, ingersoll rg, huang s, ye x, wu-chou y-h, chen pk, jabs ew, shi b, redett r, scott af, murray jc, marazita ml, munger rg, beaty th. evidence of gene-environment interaction for the runx2 gene and environmental tobacco smoke in controlling the risk of cleft lip with/without cleft palate. birth defects res part a clin mol teratol. 2012; 94(2): 76–83. 23. auerkari ei, suryandari da, umami ss, kusdhany ls, siregar twa, rahardjo tbw, talbot c, hogervorst e. gene promoter polymorphism of runx2 and risk of osteoporosis in postmenopausal indonesian women. sage open med. 2014; 2: 205031211453157. 24. mokhtar ki, abu bakar n, kharuddin af. determination of runx2 single nucleotide polymorphism rs6930053 in class i, ii and iii malocclusions. iium med j malaysia. 2017; 16(2): 1060. 25. salanti g, amountza g, ntzani ee, ioannidis jpa. hardy–weinberg equilibrium in genetic association studies: an empirical evaluation of reporting, deviations, and power. eur j hum genet. 2005; 13(7): 840–8. 26. chen b, cole jw, grond-ginsbach c. departure from hardy weinberg equilibrium and genotyping error. front genet. 2017; 8: 167. 27. zintzaras e. impact of hardy–weinberg equilibrium deviation on allele-based risk effect of genetic association studies and metaanalysis. eur j epidemiol. 2010; 25(8): 553–60. 28. chang f, li l, gao g, ding s, yang j, zhang t, zuo g. role of runx2 polymorphisms in risk and prognosis of ossification of posterior longitudinal ligament. j clin lab anal. 2017; 31(4): e22068. 29. jung sh, lee a-y, park jw, baek s-h, kim yh. investigation of parental transmission of runx2 single nucleotide polymorphism and its association with nonsyndromic cleft lip with or without palate. cleft palate-craniofacial j. 2014; 51(2): 234–9. 30. kim w-j, shin h-l, kim b-s, kim h-j, ryoo h-m. runx2modifying enzymes: therapeutic targets for bone diseases. exp mol med. 2020; 52(8): 1178–84. 31. pike jw, meyer mb, st. john hc, benkusky na. epigenetic histone modifications and master regulators as determinants of context dependent nuclear receptor activity in bone cells. bone. 2015; 81: 757–64. 32. yan g, yuan y, he m, gong r, lei h, zhou h, wang w, du w, ma t, liu s, xu z, gao m, yu m, bian y, pang p, li x, yu s, yang f, cai b, yang l. m6a methylation of precursor-mir-320/runx2 controls osteogenic potential of bone marrow-derived mesenchymal stem cells. mol ther nucleic acids. 2020; 19: 421–36. 33. ling m, huang p, islam s, heruth dp, li x, zhang lq, li d-y, hu z, ye sq. epigenetic regulation of runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase. cell biosci. 2017; 7(1): 27. 34. morrison na, stephens as, osato m, pasco ja, fozzard n, stein gs, polly p, griffiths lr, nicholson gc. polyalanine repeat polymorphism in runx2 is associated with site-specific fracture in post-menopausal females. grant sfa, editor. plos one. 2013; 8(9): e72740. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p216–220 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p216-220 p-issn: 1978-3728 e-issn: 2442-9740 volume 52, number 4, december 2019 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2019 january 2nd – december 31st, 2019 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (department of dental health techniques, faculty of vocational studies, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); i. b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); ernie maduratna setiawati (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 52, number 4, december 2019 p-issn: 1978-3728 e-issn: 2442-9740 1. a gender-based comparison of intermolar width conducted at padjajaran university dental hospital, bandung, indonesia adriana azlan, endah mardiati and ida ayu evangelina ........................................................... 168–171 2. the expression of nuclear factor of activated t cell c1 and receptor activator of nuclear factor kappa β induced by enterococcus faecalis in osteoclastogenesis (laboratory experiment on wistar rats) nirawati pribadi, rosita rahmawati, mandojo rukmo, adelina kristanti tandadjaja, hendy jaya kurniawan and ratna puspita hadi ......................................................................... 172–176 3. effectiveness of anadara granosa shell-stichopus hermanni granules at accelerating woven bone formation fourteen days after tooth extraction rima parwati sari and hansen kurniawan .................................................................................. 177–182 4. proliferation of odontoblast-like cells following application of a combination of calcium hydroxide and propolis ira widjiastuti, sri kunarti, fauziah diajeng retnaningsih, evri kusumah ningtyas, debby fauziah suryani and andrie handy kusuma ................................................................... 183–186 5. the correlation between exposure to cigarette smoke and the degree of mucosal epithelium-based dysplasia in rattus norvegicus tongues dorisna prijaryanti, diah savitri ernawati, desiana radithia, hening tuti hendarti and rosnah binti zain ..................................................................................................................... 187–191 6. microleakage difference between total-etch and self-etch bonding in bulk fill packable composite restoration after carbonic acid immersion widya saraswati, dian pramita ayu kumalasari and adioro soetojo ....................................... 192–196 7. analysis of the relationship between human cytomegalovirus dna and gb-1 genotype in the saliva of hiv/aids patients with xerostomia and salivary flow rate irna sufiawati, s. suniti, revi nelonda, rudi wisaksana, agnes rengga indrati, riezki amalia and isabellina dwades ............................................................................................ 197–203 8. peripheral ossifying fibroma of the anterior maxillary gingiva ganendra anugraha and ni putu mira sumarta ......................................................................... 204–208 9. the effects of shark liver oil on fibroblasts and collagen density in the periodontal ligaments of wistar rats induced with porphyromonas gingivalis dian mulawarmanti, dwi andriani, dian widya damaiyanti, farizia putri khoirunnisa and alifati nita juliatin ................................................................................................................... 209–214 10. minimum inhibitory concentration of cocoa pod husk extract in enterococcus faecalis extracellular polymeric substance biofilm thickness tamara yuanita, latief mooduto, reinold christian lina, fajar agus muttaqin, ika tangdan, revina ester iriani marpaung and yulianti kartini sunur ................................ 215–218 11. the potency of andrographis paniculata nees extract to increase the viability of monocytes following exposure to porphyromonas gingivalis yani corvianindya rahayu, didin erma indahyani, sheila dian pradipta and anis irmawati ............................................................................................................................ 219–223 11 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 1–4 original article epidemiological survey for dental trauma among 12-year-old children in erbil city, iraq mohamed salim younus and karam ahmed faculty of dentistry, tishk international university, erbil, iraq abstract background: dental injury and trauma are some of the most important public health concerns among school children that may require immediate management, as they could result in consequences affecting the child aesthetically, psychologically and socially. purpose: the aim of this study was to determine the prevalence of dental trauma, the type of fracture, the most commonly affected tooth and the effect of gender on the occurrence of dental trauma. methods: the methodology of this study was a cross-sectional survey consisting of a questionnaire and an oral examination which was conducted on 12-year-old public primary school children. schools were selected from five different areas of erbil city in iraq. results: the results indicate that 16.3% of the sample were affected with dental trauma in both genders. maxillary central incisor was the most commonly affected tooth, and the majority of dental traumas were untreated. the most common type of dental trauma was simple enamel fracture. conclusions: child students are at great risk regarding dental trauma, which may affect their social activity especially within their school life. keywords: dental trauma; prevalence; school students correspondence: mohamed salim younus, faculty of dentistry, tishk international university, erbil, kurdistan region, iraq. email: mohamed.salim@tiu.edu.iq introduction trauma was defined as a physical damage or wound caused by an outside force (external) which may cause death or permanent disabilities,1 and the term dentoalveolar trauma represented alveolar injuries (mandible and maxilla), teeth injuries (fractures, avulsions and luxation) and all soft tissue wounds (lips, gingiva and tongue), ranging from bruising to extreme laceration.2 damage implies offended, hurt or harmed tissue that will stimulate a dystrophic and/or provocative response from the affected area.3 dental injury may be a harm to the mouth—including teeth, lips, gingiva and tongue—and commonly incorporates a broken or lost tooth.1 tooth wounds were portrayed as a break, luxation or separation, in spite of the fact that a combination of wounds may happen within the same tooth.4 dental injury is a significant and serious health problem, as it may lead to many problems including pain and loss of function, as well as aesthetic, social and psychological issues for the child and their guardians (the entire family). traumatic wounds primarily influence children, with an incidence peak of 2–4 years and 8–10 years of age, affecting boys more than girls.5,6 most dental damage was related to falls, mainly while using bicycles or skateboards. others were related fundamentally to playing football as well as other contact sports, particularly hockey, rugby, judo and karate.7–9 the predominance of dental trauma was high throughout the world and affected primary and permanent teeth, in spite of the fact that the oral cavity comprises as little as 1% of the entire body.10 it has been depicted in some studies that the predominance of dental and facial wounds within a population may reach as high as 35 % of registered cases.7 study on iraqi mothers showed a low level of knowledge regarding the methods of management and prevention of dental trauma.11 though the researchers found many interesting aspects in this field, the study was planned to evaluate the prevalence of dental injuries, the type of fracture, the most commonly affected tooth, and the effect of gender on the occurrence of dental trauma. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p1–4 mailto:mohamed.salim@tiu.edu.iq https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p1-4 2 younus and ahmed/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 1–4 materials and methods before starting the research, written approval was obtained from the ethical committee at tishk international university (tiu) (no. 27 on 20/11/2019). the sample chosen for this cross-sectional survey consisted of 416 child students (188 boys and 228 girls), and this sample was selected from 5 different geographic areas (to cover the entire topography of erbil city) including 10 primary schools randomly selected in erbil city, iraq. the subjects were all 12-yearold school students (according to their birth date). the purpose and importance of the research were explained and discussed with the authorities in the selected schools to ensure their full cooperation during the diagnosis of the students. written consent forms were sent to students’ parents, explaining the procedure and the reasons for the study through the school authority. the questionnaires included information about where, when, and how many times tooth trauma occurred, to supplement the visual examination of traumatic tooth or teeth recording. only the children who submitted duly signed forms were included in the study (all the students had the choice as to whether or not they wanted to participate in the research). the ethical statement of this research will include: approval from the directorate of education was taken before starting the diagnosis. the following instruments were used for diagnosis: disposable dental mirrors, disposable dental probes, disposable tweezers, gloves and masks. the examined students were seated on their chair in their classes, and the examiner was standing in front of the chair for diagnosis. the criteria of garcía-godoy was used for assessing the dental trauma.12 visual and tactile examinations were used for determining the type of dental injuries. no radiographs were taken so the fracture root was not recorded. anterior teeth with restorations were excluded if the cause of the restoration was not confirmed to be due to trauma. we chose this index because it is simple and has been widely used in most of the previous studies. furthermore, this classification was appropriate for retrospective studies. the inclusion criterion in this study was that the 12year-old students (their parents) agreed to participate in the survey. the exclusion criteria were students younger or older than 12 years old, parents or children that refused to participate in the research or cases with incomplete documentation, and students under orthodontic treatment. statistical package for the social sciences software (spss), version 20 (ibm, new york, usa), was used for data analysis and processing. the collected data was grouped and placed in tables and a chi-square test was used to compare the results (χ2). all the values were considered statistically significant at p<0.05. results the sample consisted of 416 primary school students from ten different public schools in erbil governorate. 188 of the sample subjects were male (45.2%) and 228 were female, (54.8%) all with an age of 12 years. the distribution of students according to gender is summarised in table 1. the prevalence of dental trauma was shown to be 16.3% in the total sample and girls showed a higher prevalence of dental trauma (18.4%) in contrast with the boys (13.9%). however, this difference in prevalence was statistically non-significant (p>0.05), as shown in table 2. table 3 shows that from 68 students who experienced trauma, the most prevalent kind was enamel fracture, while the least common was enamel dentine pulp fracture with high significant differences at p<0.001. the result showed that the teeth most commonly affected by trauma were upper centrals, while the least affected teeth were lower canines with high significant differences at p<0.001, as shown in table 4. table 5 shows that from the 68 students who had trauma, the dental trauma occurred mainly at home, followed by school. the least common places were in the street and park, table 1. distribution of sample according to gender gender no. percentage (%) boys 188 45.2 girls 228 54.8 total 416 100 table 2. prevalence of dental trauma among male and female students dental trauma boys girls total no. % no. % no. % no 162 86.1 186 81.6 348 83.7 yes 26 13.9 42 18.4 68 16.3 total 188 100 228 100 416 100 (χ2=0.67, p=0.238) table 3. the types of dental trauma types of trauma percentage (%) e. fracture 91.2 d. fracture 5.9 e.d.p fracture 2.9 (χ2=210.67, p<0.008) table 4. the teeth affected with trauma tooth percentage (%) upper central 67.6 upper lateral 8.8 upper canine 5.9 lower central 2.9 lower lateral 5.9 lower canine 2.9 (χ2=121.35, p<0.001) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p1–4 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p1-4 3younus and ahmed/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 1–4 with a statistically significant difference (p<0.05). from the total number of traumatised students, falling down and being struck by an object were the most prevalent reasons for the dental trauma in both genders, followed by the other causes with high significant differences at p<0.001, as shown in table 6. the number of students visiting the dentist for dental treatments was only 20% (23% female and 15% male), while the number of students not visiting the dentist was 79.4% (77% female and 85% male) with statistically non-significant differences (p>0.05), as seen in table 7. discussion the most common dental injury could be a broken or displaced tooth. the study showed that (16.3%) of the sample had a dental trauma, which was in agreement with other studies that found the prevalence to be 15.1%, 14.4%, 14.5%, 18.5% and 16.3% respectively; this study was higher than that founded by others which found dental trauma prevalence to be 10.2%, 5.68% and 6.3%.13–19 within the present study, the high prevalence of dental injury requires proficient planning and intervention to prevent and decrease its rate. girls were found to have more dental injuries in comparison with boys, similar to the result found by rajab et al.,20 while other studies showed different results. 21,22 this finding may be because of a higher number of girls than boys in our sample. enamel fracture (91.5%) was most prevalent in the school children followed by enamel-dentin fractures (5.9%), which was comparable to the result found by other studies.22–24 the tooth that was most highly affected with dental trauma was the maxillary central incisor (67.6%), followed by upper laterals, (8.8%) which confirms by bendo et al. and prasad et al.25,26 this finding could be attributed to the early eruption and the protrusive position of these teeth. home was the most common place for an injury to happen, followed by school. this finding was comparable to other studies. 27,28 this result may be due to the fact that the students spend most of their time at home instead of at school or other places. from the whole number of traumatised students, falling down and being struck by an object were the most common reasons for dental injury in both genders, which was comparable to results found by other studies.17,18,23,24 the rate of children that visit the dental practitioner for dental treatment was (20.6%), which was comparable to research by prasad et al.,26 which found that the majority of dental injuries were untreated. this result might be related to the fact that most of the traumas were enamel-dentin fractures which are not seen as situations that need urgent treatment. dental trauma is a critical public dental health issue among school children which needs prompt treatment. dental injury can result in aesthetic, psychological, social and therapeutic problems. it can be concluded from this study that dental trauma was highly prevalent among the sample of students. moreover, dental trauma was more common among girls than boys, simple enamel fractures were discovered to be the most widely recognised type of dental trauma, and the maxillary central incisors were the most influenced tooth by dental injury. the most widely recognised cause of dental trauma was falling, followed by being struck by an object, and dental trauma most frequently occurred at home followed by school. finally, high levels of untreated traumatised teeth were found in this study. references 1. powers mp, quereshy fa, ramsey ca. diagnosis and management of dentoalveolar injuries. in: fonseca rj, walker r v, betts nj, table 5. the students’ distribution according to the place of trauma dental trauma home school street park total male 14 10 2 0 26 female 30 10 0 2 42 total 44 20 2 2 68 (χ2=23.46, p<0.026) table 6. the distribution of students according to the cause of trauma dental trauma falling down struck by object bicycle accident violence during playing unknown cause rta total male 10 4 2 0 4 4 2 26 female 20 16 2 2 0 2 0 42 total 30 20 4 2 4 6 2 68 (χ2=42.32, p<0.001) table 7. the percentage of students visiting the dentist for treatment gender dental visit by percentage (%) yes no boy 15 85 girl 23 77 total 20.6 79.4 (χ2=4.817, p=0.108) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p1–4 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p1-4 4 younus and ahmed/dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 1–4 editors. oral and maxillofacial trauma. 3rd ed. st. louis: elsevier saunders; 2005. p. 427–78. 2. casey rp, bensadigh bm, lake mt, thaller sr. dentoalveolar trauma in the pediatric population. j craniofac surg. 2010; 21(4): 1305–9. 3. teusner dn, chrisopoulos s. dentist labour force projections 2005 to 2020: the impact of new regional dental schools. aust dent j. 2008; 53(3): 292–6. 4. hussien aa. traumatic dental injuries among 6 – 13 – year old school children in tikrit city. mustansiria dent j. 2016; 13(1): 46–51. 5. altun c, ozen b, esenlik e, guven g, gürbüz t, acikel c, basak f, akbulut e. traumatic injuries to permanent teeth in turkish children, ankara. dent traumatol. 2009; 25(3): 309–13. 6. ahlawat b, kaur a, thakur g, mohindroo a. anterior tooth trauma: a most neglected oral health aspect in adolescents. indian j oral sci. 2013; 4(1): 31. 7. priya m, sharmin d, amarlal d, thomas e, pooja y. knowledge and attitudes of coaches regarding sports related oro-facial injuries in chennai, india. j dent oral disord ther. 2016; 4(3): 01–5. 8. qudeimat ma, al-hasan aa, al-hasan ma, al‐k hayat k, andersson l. prevalence and severity of traumatic dental injuries among young amateur soccer players: a screening investigation. dent traumatol. 2019; 35(4–5): 268–75. 9. güngör hc. management of crown-related fractures in children: an update review. dent traumatol. 2014; 30(2): 88–99. 10. atak n, karaoğlu l, korkmaz y, usubütün s. a household survey: unintentional injury frequency and related factors among children under five years in malatya. turk j pediatr. 2010; 52(3): 285–93. 11. yassen gh, chin jr, younus ms, eckert gj. knowledge and attitude of dental trauma among mothers in iraq. eur arch paediatr dent. 2013; 14(4): 259–65. 12. garcía-godoy f. a classification for traumatic injuries to primary and permanent teeth. j pedod. 1981; 5(4): 295–7. 13. ravishan ka r t l, kuma r ma, naga rajappa r, chaitra tr. prevalence of traumatic dental injuries to permanent incisors among 12-year-old school children in davangere, south india. chinese j dent res. 2010; 13(1): 57–60. 14. kumar a, bansal v, veeresha k l, sogi gm. prevalence of traumatic dental injuries among 12to 15-year-old schoolchildren in ambala district, haryana, india. oral health prev dent. 2011; 9(3): 301–5. 15. sulieman ag, awooda em. prevalence of anterior dental trauma and its associated factors among preschool children aged 3-5 years in khartoum city, sudan. int j dent. 2018; 2018: 2135381. 16. dighe k, kakade a, takate v, makane s, padawe d, pathak r. prevalence of traumatic injuries to anterior teeth in 9-14 year school-going children in mumbai, india. j contemp dent pract. 2019; 20(5): 622–30. 17. juneja p, kulkarni s, raje s. prevalence of traumatic dental injuries and their relation with predisposing factors among 8-15 years old school children of indore city, india. clujul med. 2018; 91(3): 328–35. 18. al-ansari a, nazir m. prevalence of dental trauma and receipt of its treatment among male school children in the eastern province of saudi arabia. sci world j. 2020; 2020: 7321873. 19. eltair m, pitchika v, standl m, lang t, krämer n, hickel r, kühnisch j. prevalence of traumatic crown injuries in german adolescents. clin oral investig. 2020; 24(2): 867–74. 20. rajab ld, baqain zh, ghazaleh sb, sonbol hn, hamdan ma. traumatic dental injuries among 12-year-old schoolchildren in jordan: prevalence, risk factors and treatment need. oral health prev dent. 2013; 11(2): 105–12. 21. patel mc, sujan sg. the prevalence of traumatic dental injuries to permanent anterior teeth and its relation with predisposing risk factors among 8-13 years school children of vadodara city: an epidemiological study. j indian soc pedod prev dent. 2012; 30(2): 151–7. 22. al-obaidi w, al-mashhadani a. traumatic injury among 5-30 years in sheha village. iraqi dent j. 2002; 29: 299–304. 23. lin h, naidoo s. causes and prevalence of traumatic injuries to the permanent incisors of school children aged 10-14 years in maseru, lesotho. sadj j south african dent assoc. 2008; 63(3): 152, 154–6. 24. jabbar nsa, aldrigui jm, braga mm, wanderley mt. pulp polyp in traumatized primary teeth a case-control study. dent traumatol. 2013; 29(5): 360–4. 25. bendo cb, paiva sm, oliveira ac, goursand d, torres cs, pordeus ia, vale mp. prevalence and associated factors of traumatic dental injuries in brazilian schoolchildren. j public health dent. 2010; 70(4): 313–8. 26. prasad s, tandon s, pahuja m, wadhawan a. prevalence of traumatic dental injuries among school going children in farukhnagar, district gurgaon. int j sci study. 2014; 2(2): 44–9. 27. navabazam a, farahani ss. prevalence of traumatic injuries to maxillary permanent teeth in 9to 14-year-old school children in yazd, iran. dent traumatol. 2010; 26(2): 154–7. 28. gojanur s, yeluri r, munshi ak. prevalence and etiology of traumatic injuries to the anterior teeth among 5 to 8 years old school children in mathura city, india: an epidemiological study. int j clin pediatr dent. 2015; 8(3): 172–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p1–4 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p1-4 153153 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 153–158 research report the correlation between dental caries and serum iron (fe) levels in female students of mamba’ul khoiriyatil islamiyah (mhi) madrasa in jember, east java, indonesia ristya widi endah yani,1 taufan bramantoro,2 farida wahyuningtyas3 and talitha zulis islaamy4 1department of dental public health, faculty of dentistry, universitas jember, jember – indonesia 2department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 3department of nutrition, faculty of public health, universitas jember, jember – indonesia 4health sciences postgraduate program, universitas jember, jember – indonesia abstract background: dental caries may cause discomfort and pain when chewing food, which in turn leads to insufficient absorption of nutrients, including iron, by the body. lack of iron intake can cause iron-deficiency anaemia. iron deficiency may also decrease salivary secretion and buffer capacity due to reduced salivary gland function, which may lead to dental caries. purpose: this study aims to analyse the correlation between dental caries and serum iron (fe) levels in the students of mamba’ul khoiriyati islamiyah (mhi) madrasa aliyah in bangsalsari jember. methods: out of 71 students aged 15-18 years, 24 students were examined for caries with the dmf-t index and blood samples were also collected using the phlebotomy technique to assess the serum iron (fe) level in µg/dl. the data were analysed using the spearman’s correlation test (α = 0.05). results: the average dmf-t score was high (7 ± 3.4) and the average serum iron (fe) level was below normal (38 ± 11.5 µg/dl). the spearman’s correlation test for dental caries and serum iron (fe) levels showed significant results (p <0.05). conclusion: the occurrence of dental caries correlates with serum iron (fe) levels. the higher the level of serum iron (fe), the lower the level of dental caries. keywords: dental caries; iron-deficiency; students correspondence: ristya widi endah yani, department of dental public health, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. email: ristya_widi@unej.ac.id introduction caries is the most common dental disease, and it is considered to be a major global dental and oral health problem.1 caries occurs due to the demineralisation process caused by acid bacterial activity from fermentable carbohydrates. within a certain period of time, this process may damage tooth enamel, causing a cavity.2 the prevalence of dental caries in indonesia is relatively high, and has increased from 43.4% in 2007 to 53.2% in 2013.3 the basic health research (bhr) carried out in 20133 also showed that the prevalence of caries tends to increase with age, as it was 1.4% in the 12-14 year age group and 1.8% in those aged 15-24 years. the national health and nutrition examination survey states the prevalence of caries in the age groups of 12-15 years and 16-19 years was 14.5% and 22.6% respectively.4 anaemia or low haemoglobin levels in the blood denote a condition in which the body cannot meet physiological needs.5 anaemia is caused by iron deficiency in high-risk groups including children, women of childbearing age and pregnant women.5 younger women also commonly have increased iron needs as a result of rapid growth and mestruation.6 in 2005, the number of people worldwide with anaemia was 1.62 million. women of childbearing age were among those with anaemia, who in 2011 accounted for 29% of the world population (528.7 million people).5 the bhr in 2007 showed that the percentage of anaemia in indonesia in women of childbearing age who were not pregnant (≥ 15 years) in urban areas was 19.7%. furthermore, the percentage of anaemia in childbearing women aged 15-44 years was 35.3% in 2013. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 mailto:ristya_widi@unej.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 154 yani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 153–158 this study aims to analyse the correlation between dental caries and serum iron (fe) levels in the students of mamba’ul khoiriyatil islamiyah madrasa, bangsalsari, jember. according to the primary healthcare of bangsalsari, there is a high prevalence of anaemia in female students at this school. most of them live in dormitories and have separate classrooms from male students, making sampling easier. there are female students aged 15-18 years who need early intervention to avoid anaemia in future pregnancies. materials and methods an analytic observational study was performed on 71 female students aged 15-18 years in mamba’ul khoiriyatil islamiyah (mhi), a madrasa aliyah in jember. the respondents were chosen by using purposive sampling. blood serum was only taken from 24 students who met the inclusion criteria (willing to be respondents for dental check, physically healthy, not currently menstruating, having normal menstrual periods and not having a chronic disease). all respondents had signed a consent form stating that they were willing to participate in this study. the respondents were also asked to fill in a form giving personal details (name, home address, age, level of parents’ education, parents’ occupation, family income, amount of pocket money and residence). dental caries was measured by using the decayed, missing, filled (teeth) (dmf-t) index. the tooth was diagnosed as decayed (d) if there was primary or recurrent caries. the missing (m) component related to teeth that had been extracted due to caries, or were indicated for extraction due to caries. the last component, filling (f), related to teeth that had been filled due to caries. the level of dental caries in each respondent was calculated by the sum of the components d, m and f. the dmf-t index assessment was performed by a clinician using a disposable dental instrument kit (dochem). the dental caries categories based on the dmf-t index were very low (6.5).7 serum iron (fe) levels were measured in µg/dl. blood samples were taken intravenously up to 3 cc, then put into a vacutainer tube gp and put in a cooler box (marvel hro-b35l) that had been filled with ice packs. the samples were then sent to the laboratory to measure the serum iron (fe) levels using the phlebotomy technique. the procedure of taking blood serum was as follows. first, all the equipment needed for the procedure (sterile glass or plastic tubes with rubber caps, vacuum-extraction blood tubes, syringe, tourniquet, alcohol hand rub, 70% alcohol swabs for skin) was collected. next, the patient was prepared for the procedure. the site for drawing blood was selected by extending the patient’s arm and inspecting the antecubital fossa or forearm. the tourniquet was then applied about 4–5 finger widths above the venepuncture site and the vein re-examined. the operator then cleaned their hands, washing with soap and water if not visibly contaminated, otherwise cleaning with alcohol rub. after performing hand hygiene, the operator put on well-fitting, non-sterile gloves. the site was then cleaned with a 70% alcohol swab for 30 seconds and allowed to dry completely (30 seconds). the syringe was then injected and the blood drawn. following this, the blood was put into a test tube. finally, it was labelled and sent to the laboratory.8 the normality test showed that only the serum iron (fe) level data were not normally distributed. the data were then analysed using the spearman’s correlation test with α = 0.05 to find the correlation between dental caries and serum iron (fe) levels. all the procedures performed in this study have been reviewed and approved by the institutional ethical committee in the faculty of dentistry, university of jember (certificate number 824/un25.8/ kepk/dl/2019). results the data in table 1 shows that the age range of students in mhi madrasa was 15 to 18 years, with the majority (22 students) aged 15 years (31%). parental education (father and mother) was mostly at elementary school level. there were 51 respondents who had fathers with elementary school education (71.8%), and 59 respondents who had mothers with elementary school education (83.1%). there were 39 respondents with fathers who were working as entrepreneurs (54.9%), and 36 respondents who had mothers who were housewives (50.7%). the majority of the respondents (45) were from low income families (≤ idr 1,500,000/month) (63.4%). there were 57 students with an allowance of less than idr 5000 per day (80.3%). the data revealed that 66 respondents (92.9%) lived in the dormitory and only 5 students (7.1%) lived with their parents. there were 24 students who were asked about food consumption using food recall 2x24 hours. most respondents were exposed to deficits in energy, protein, iron (fe) and vitamin c (table 2). according to kusharto and supariasa,9 someone who has a daily intake of less than 89% of the recommended dietary allowance (rda) has an energy and nutrient deficit. adolescent girls who consume less food containing protein, iron (fe) and vitamin c are more at risk of anemia.10 from the 71 students selected using the inclusion criteria, there were 24 students who were examined for dental caries and serum iron (fe) levels. the dental caries examination was carried out using the dmf-t index. there were 23 respondents who had dental caries (95.8%). the results showed that the dmf-t score for the 24 students of mhi madrasa was in the range of 0 to 13 with an average of 7 ± 3.4, which can be grouped into the very high category (table 3). the results of the examination of serum iron (fe) levels showed an average of 38 ± 11.5 µg/dl and ranged from dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 155yani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 153–158 12 µg/dl to 50 µg/dl (table 2). normal serum iron (fe) levels in adolescent girls are 50 µg/dl.11 this indicated that the levels in these students were below normal, and some (10 respondents) might suffer from iron deficiency anaemia (83.3%). the correlation between dental caries and serum iron (fe) levels was then analysed using the spearman’s correlation test with a significance of p <0.05. the results showed a significant correlation between the dmf-t scores and serum iron (fe) levels with a p value of 0.007. this showed that there is a correlation between dental caries and serum iron (fe) levels (table 4). the correlation is inverse, meaning the higher the level of serum iron (fe), the lower the level of dental caries. discussion a high prevalence of dental caries is associated with low socioeconomic conditions.12 socioeconomic conditions can be measured using level of parental education, parental income and social status in the community.13,14 the majority of respondents had parents with a low level of education. this may lead to dental caries as parents with a lower level of education may make poor choices about the level of acid intake of their children.15 the socioeconomic level of these students was middle to low, due to the fact that the school is not close to any urban areas. this means that medical care, including dental and oral health care, is in shorter supply .16 table 1. characteristics of respondents characteristics total (n) % age 15 years old 22 31 16 years old 15 21.1 17 years old 18 25.4 18 years old 16 22.5 total 71 100 parents’ education level father elementary school 51 71.8 middle school 15 21.1 high school 5 7.1 total 71 100 mother elementary school 59 83.1 middle school 8 11.3 high school 4 5.6 total 71 100 parents’ occupation father entrepreneur 39 54.9 farmer 19 26.8 trader 8 11.3 construction worker 5 7 total 71 100 mother entrepreneur 9 12.7 farmer 15 21.1 trader 11 15.5 housewife 36 50.7 total 71 100 family income low (≤ idr1,500,000/month) 45 63.4 medium (> idr1,500,000 idr 2,500,000/month) 26 36.6 total 71 100 pocket money ≤ idr 5,000/day 57 80.3 > idr 5,000/day idr 10,000/day 14 19.7 total 71 100 residence dormitory 66 92.9 outside dormitory 5 7.1 total 71 100 table 2. total intake of energy, protein, iron (fe) and vitamin c per day students of mhi madrasa, bangsalsari, jember variable n category average/day/ personinadequate adequate energy (kcal) 24 23 1 1376 protein (g) 24 24 0 36.5 iron (fe) (mg) 24 24 0 5.9 vitamin c (mg) 24 24 0 13.4 table 3. data on the results of the level of dental caries and serum iron (fe) levels of students of mhi madrasa, bangsalsari, jember variable n minimum maximum average sd dental caries 24 0 13 7 3.4 serum iron (fe) level 24 12 50 38 µg/ dl 11.5 table 4. spearman’s correlation test of dental caries with serum iron (fe) levels variable p-value correlation coefficient notes dental caries serum iron (fe) 0.007 -0.539 there is correlation between dental caries and serum iron (fe) levels. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 156 yani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 153–158 parental education, parental work and family income are linked. a higher level of education will lead to jobs with higher salaries.17 julianto and utari18 state that the level of education significantly influences the level of individual income, where the higher the level of education, the higher the income level. for these students, the low level of education of their parents (mainly their fathers) means that they often work as entrepreneurs with a low income (≤ idr 1,500,000/month). this can lead to parents believing that dental care for their children is not urgent.14 children who come from low socioeconomic backgrounds do not buy food based on nutritional value, but good taste and low price.19 the majority of these students had an allowance of less than idr 5000 per day. this amount of money can be used to buy snacks outside the home (dormitory) that are high in sodium, sugar and fat as well as high-sugar drinks.20 the children who live with their parents eat food cooked by their mothers, who pay more attention to the nutritional content of the food being served to their families.21 the female students often consumed food containing less iron due to the problems associated with a low socioeconomic background.22 low haemoglobin levels can affect the inflammatory response in the pulp.23 haemoglobin in the blood carries oxygen to the tissues. if the level of haemoglobin in the blood decreases, hypoxia might occur in the tissues, which decreases the inflammatory response in the pulp and increases the level of dental caries.23 the dmf-t scores of the respondents averaged 7 ± 3.4. this score, according to the world health organization (who), is very high. this is probably caused by the consumption of snacks and beverages during the day, such as wafers and sweet tea. food containing high sugar levels and improper tooth brushing behaviour causes dental caries.24 sugary foods tend to increase the occurrence of dental caries compared to fibrous foods.25 the type of food consumed relates to the formation of dental caries. consumption of frequent and repeated cariogenic foods will cause the ph of the oral cavity to become more acidic, which facilitates the demineralisation of enamel and formation of dental caries.24 a good diet and regular dental care are very important to maintain the teeth and prevent vulnerability to cavities. widayati26 suggests that there is a need for information regarding the importance of 6 monthly dental and oral examinations for children. the results of the serum iron (fe) level examinations showed that the average serum iron (fe) levels of mhi bangsalsari jember female students were below normal. serum iron (fe) is a microelement iron that is essential for the body. this substance is required in haematopoiesis (blood formation), specifically for the synthesis of haemoglobin (hb). haemoglobin (hb) as oxygen that delivers erythrocytes is essential for the body. haemoglobin consists of fe (iron), protoporphyrin and globin (1/3 hb weight consists of fe).27 anaemia generally occurs throughout the world, especially in developing countries. overall, anaemia occurs in 45% of women in developing countries and in 13% of women in developed countries.28 anaemia is characterised by low concentrations of haemoglobin (hb) or haematocrit of the threshold value. it is caused by low production of red blood cells (erythrocytes) and hb, increased erythrocyte damage or excessive blood loss.29 there are several types of anaemia, but the most common is iron deficiency anemia.29 it occurs due to an increased need for iron in the body such as during menstruation, pregnancy or childbirth, while only a small amount of iron is entering the body.30 adolescent girls are at risk from iron deficiency anaemia because they menstruate every month and lack knowledge about the condition.31 the volume of blood lost during menstruation ranges from 30 to 50cc per month. this causes women to lose as much iron as 12-15 mg per month or 0.4-0.5 mg per day for 28 to 30 days. during menstruation, women also not only experience iron loss but also experience basal loss. in total, women can experience as much iron loss as 1.25 mg per day.29 iron deficiency anaemia is caused by a lack of iron in the body for erythropoiesis. this is a condition characterised by hypochromic-microscopic red blood cells, decreasing levels of serum iron (fe) and transferrin as well as a rise in the total iron binding capacity (tibc). also, reservation of the iron in the bone marrow and elsewhere is very poor or non-existent. many factors can cause iron deficiency anaemia, such as profuse bleeding during injury, surgery, menstruation and an increased need for iron in pregnant women and for growth. other causes include a lack of intake of iron and protein from food and impaired absorption of fluids caused by parasites in the body, such as hookworm or tapeworm. in these cases daily iron intake is needed to replace iron lost through faeces, urine and skin.27 the spearman’s correlation test for dental caries and serum iron (fe) levels showed significant results (p <0.05). the results indicated a correlation between dental caries and serum iron (fe) levels. patients with high serum iron (fe) levels have a low dmf-t score indicating a low prevalence of dental caries, while patients with low serum iron (fe) levels have a high dmf-t score indicating a high prevalence of dental caries. from this study, it can be concluded there is an inverse correlation between serum iron (fe) levels and dental caries. these results are in line with research conducted by daryani et al.32 untreated caries also causes acute and chronic inflammation such as pulpitis, periapical aphasia and fistulas that release various mediators, especially interleukin i and cytokines. pulp pain at night causes sleep disturbance and decreases glycosteroid production. this decrease causes suppression of haemoglobin production in the blood, leading to anaemia caused by chronic inflammation due to the restrained erythropoiesis.33 dental caries causes discomfort and pain when chewing. as a result, the food cannot be chewed properly, and thus the absorption of nutrients in the intestine is reduced. this can cause nutritional deficiencies, which also affect the iron levels in the body.34 the most abundant source of iron is dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 157yani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 153–158 found in meat, chicken and fish. if the food is not chewed properly, then iron cannot be optimally absorbed by the small intestine.33 research by nagarajan et al.35 in children with iron deficiency anaemia showed an improvement in nutritional status after overall oral cavity treatment. the treatment relieves pain and therefore increases iron intake. the theory of the correlation between serum iron (fe) levels and dental caries is a two-way effect theory.23 serum iron (fe) levels affect the occurrence of dental caries. conversely, dental caries can also influence the risk of low serum iron (fe) levels, which can lead to anaemia. the level of dental caries in those with anaemia can also increase due to the formation and decrease in calcification of dentin and enamel.23 thus, teeth are easily decalcified and caries might occur. in addition, people who experience severe anaemia usually consume drugs that contain sucrose and are taken frequently and continuously.23 sucrose is one of the components that can cause caries if consumed continuously. higher levels of dental caries can also occur in patients with anaemia who have complications and require inpatient care. inpatients have a habit of not maintaining oral hygiene properly due to sufferers having difficulty in brushing their teeth routinely, worsening their oral hygiene and increasing the frequency of caries occurrence.23 another theory states that iron deficiency affects salivary gland function. the function of the salivary glands of iron deficient patients tends to decrease. as a result, salivary secretion and buffer capacity are reduced, which increases the risk of dental caries.36 also, the function of the salivary glands can be disrupted due to iron deficiency, causing reduced salivary secretion and low buffer ability. salivary buffering is a mechanism to restore the ph of the oral cavity to normal. a low ph can cause tooth decay and damage mucosal surfaces. saliva has a buffer function to prevent enamel demineralisation based on the phosphate and carbonic/bicarbonate system. when the buffer capacity is low, there is an increased risk of dental caries.36 in addition, reduced salivary secretion also reduces simple mechanical rinsing, antimicrobial activity, calcium phosphate binding, immune surveillance and antimicrobial peptide secretion in the oral cavity. these are the main natural defence systems in the oral cavity.37 the current study supplements the evidence of correlation between dental caries and serum iron (fe) levels in female students aged 15-18 years. early intervention is needed to prevent health problems and achieve good health in future pregnancies. our paper is unique, due to the fact that determination of dental caries using blood serum achieves better results than using saliva samples in general studies. unfortunately, this study had limited samples due to the fact that many samples did not meet the inclusion criteria. expansion of location and additional samples in further research are needed to gain the best understanding of oral health and nutritional problems in young women. in conclusion, there is a correlation between dental caries and serum iron (fe) levels. the higher the level of serum iron (fe), the lower the level of dental caries. references 1. boy h, khairullah a. hubungan karies gigi dengan kualitas hidup remaja sma di kota jambi. j kesehat gigi. 2019; 6: 10–3. 2. rostinawati t, aryani h, iskandar y. identification of bacteria causing dental caries through genetic testing and activity assay of toothpastes. j pharm sci res. 2018; 10(3): 511–3. 3. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar. jakarta: kementerian kesehatan ri; 2013. p. 1–384. 4. fleming e. national health and nutrition examination survey. cdc: natioanal center for health statistics. 2014. available from: https:// www.cdc.gov/nchs/nhanes/. accessed 2019 dec 9. 5. gautam s, min h, kim h, jeong h-s. determining factors for the prevalence of anemia in women of reproductive age in nepal: evidence from recent national survey data. plos one. 2019; 14(6): e0218288. 6. abbaspour n, hurrell r, kelishadi r. review on iron and its importance for human health. j res med sci. 2014; 19(2): 164–74. 7. world health organization. oral health surveys: basic methods. geneva: world health organization; 2013. p. 1–125. 8. world health organization. who guidelines on drawing blood: best practices in phlebotomy. geneva: who document production services; 2010. p. 35–37. 9. kusharto cm, supariasa idn. survei konsumsi gizi. yogyakarta: graha ilmu; 2014. p. 1–62. 10. sholicha ca, muniroh l. hubungan asupan zat besi, protein, vitamin c dan pola menstruasi dengan kadar hemoglobin pada remaja putri di sman 1 manyar gresik. media gizi indones. 2019; 14(2): 147–53. 11. kumar chowdhury s, ahmed s, ara hossain i, yasmin r, faruquee mh, salim afm. status of iron deficiency anemia among bangladeshi children: urban and rural settings. acta sci paediatr. 2019; 2(8): 08–12. 12. moses j, rangeeth bn, gurunathan d. prevalence of dental caries, socio-economic old school going children of chidambaram status and treatment needs among 5 to 15 year old school going children of chidambaram. j clin diagnostic res. 2011; 5(1): 146–51. 13. costa sm, martins cc, bonfim m de lc, zina lg, paiva sm, pordeus ia, abreu mhng. a systematic review of socioeconomic indicators and dental caries in adults. int j environ res public health. 2012; 9(10): 3540–74. 14. jumriani. gambaran kejadian karies gigi dengan status sosial ekonomi siswa kelas vii di smp darul hikmah kota makassar. media kesehat gigi. 2017; 16(2): 72–80. 15. maharani da, zhang s, gao ss, chu c. dental caries and the erosive tooth wear status of 12-year-old children in jakarta, indonesia. int j environ res public health. 2019; 16: 2994. 16. wala hc, wicaksono da, tambunan e. gambaran status karies gigi anak usia 11-12 tahun pada keluarga pemegang jamkesmas di kelurahan tumatangtang i kecamatan tomohon selatan. e-gigi. 2014; 2(1): 1–8. 17. kurniasari ad, nurhayati f. hubungan antara tingkat pendidikan, pekerjaan dan pendapatan orang tua dengan status gizi pada siswa sd hangtuah 6 surabaya. j pendidik olahraga dan kesehat. 2017; 05(02): 163–70. 18. julianto d, utari pa. analisa pengaruh tingkat pendidikan terhadap pendapatan individu di sumatera barat. ikra-ith ekon. 2019; 2(2): 122–31. 19. amalia l, endro op, damanik mrm. preferensi dan frekuensi konsumsi makanan jajanan pada anak sekolah dasar di kecamatan cijeruk, kabupaten bogor. j gizi dan pangan. 2012; 7(2): 119–26. 20. aprillia ba, dieny ff. faktor yang berhubungan dengan pemilihan makanan jajanan pada anak sekolah dasar. thesis. semarang: universitas diponegoro; 2011. p. 1–63. 21. kurniawan mww, widyaningsih td. hubungan pola konsumsi pangan dan besaran uang saku mahasiswa manajemen bisnis dengan dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 https://www.cdc.gov/nchs/nhanes/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 158 yani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 153–158 mahasiswa jurusan teknologi hasil pertanian universitas brawijaya terhadap status gizi. j pangan dan agroindustri. 2017; 5(1): 1–12. 22. kim jy, shin s, han k, lee kc, kim jh, choi ys, kim dh, nam ge, yeo hd, lee hg, ko bj. relationship between socioeconomic status and anemia prevalence in adolescent girls based on the fourth and fifth korea national health and nutrition examination surveys. eur j clin nutr. 2014; 68(2): 253–8. 23. bansal k, goyal m, dhingra r. association of severe early childhood caries with iron deficiency anemia. j indian soc pedod prev dent. 2016; 34(1): 36–42. 24. salamah s, rahmawati i, danan. hubungan perilaku menyikat gigi dengan indeks dmf-t pada murid kelas iii dan iv sekolah dasar negeri gambut 5 pematang panjang kabupaten banjar. j sk keperawatan. 2016; 3(38): 1–6. 25. gayatri rw. hubungan tingkat pengetahuan dengan perilaku pemeliharaan kesehatan gigi anak sdn kauman 2 malang. j heal educ. 2017; 2(2): 201–10. 26. widayati n. faktor yang berhubungan dengan karies gigi anak pada usia 4-6 tahun. j berk epidemiol. 2014; 2(2): 196–205. 27. susiloningtyas i. pemberian zat besi (fe) dalam kehamilan. maj ilm sultan agung. 2012; 50(128): 1–27. 28. sjahriani t, faridah v. faktor-faktor yang berhubungan dengan kejadian anemia pada ibu hamil. j kebidanan. 2019; 5(2): 106–15. 29. kristianti s, wibowo ta, winarsih w. hubungan anemia dengan siklus menstruasi pada remaja putri di sma negeri 1 imogiri, bantul, yogyakarta tahun 2013. j stud pemuda. 2016; 3(1): 33–8. 30. ruqoiyah s. hubungan kepatuhan konsumsi tablet tambah darah dengan kejadian anemia pada remaja putri kelas xi di sma negeri 1 sentolo kulon progo. thesis. yogyakarta: universitas ’aisyiyah; 2019. p. 1–65. 31. suryani d, hafiani r, junita r. analisis pola makan dan anemia gizi besi pada remaja putri kota bengkulu. j kesehat masy andalas. 2015; 10(1): 11–8. 32. daryani kk, s pk, neeraj deshpande a, dinesh bargale s, khoja m, patel ks. comparative evaluation of serum iron level, serum ferritin level and salivary ph with dental caries in children with iron deficiency anaemiaan observational cross-sectional study. j adv med dent sci res. 2019; 7(4): 16–8. 33. hashemi a, bahrololoomi z, salarian s. relationship between early childhood caries and anemia: a systematic review. iran j pediatr hematol oncol. 2018; 8(2): 126–38. 34. venkatesh babu ns, bhanushali pv. evaluation and association of serum iron and ferritin levels in children with dental caries. j indian soc pedod prev dent. 2017; 35(2): 106–9. 35. nagarajan u, dhingra r, chaudhuri p, karunanand b, arora p. influence of full mouth rehabilitation on iron deficiency anemia status in children with severe early childhood caries. j appl dent med sci. 2017; 3(2): 25–32. 36. mahantesha t, parveen reddy km, ellore vpk, ramagoni nk, iitagi v, anitha ks. evaluation and association of iron deficiency anaemia with salivary ph and buffering capacity. natl j physiol pharm pharmacol. 2014; 4(3): 229–32. 37. hegde mn, attavar sh, shetty n, hegde nd, hegde nn. saliva as a biomarker for dental caries: a systematic review. j conserv dent. 2019; 22(1): 2–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p153–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p153-158 vol 49 no 1 jan-mrt 2016.indd 54 case report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 55–59 early detection and treatment of speckled leukoplakia selviana tampoma1 and iwan hernawan2 1 oral medicine resident, faculty of dental medicine, universitas airlangga 2 department of oral medicine, faculty of dental medicine, universitas airlangga abstract background: leukoplakia is one of potentially malignant disorders that can be found on oral mucosa. speckled leukoplakia is a rare type of leukoplakia with a very high risk of premalignant growth. approximately 3 % of worldwide population has suffered from leukoplakia, 5-25% of which tend to be malignant leukoplakia. purpose: this case report was aimed to discuss about early detection of speckled leukoplakia as one of potentially malignant disorders. case: a 62 year old male patient came with chief complaint of bald and painful tongue since one month ago. the patient has a history of allergic reaction, hypertension, uric acid, and hepatitis b. he had been a heavy smoker since young until 10 years ago. intra oral examination showed a firm, rough, non scrapable white plaque lesion with a size of 1 x 1.5 cm, surrounded by painful erosion with diffuse boundary. case management: based on cytology examination, the patient was reffered to oncologist to get an excisional biopsy. next, the patient succesfully underwent the excisional biopsy and came for control. the results showed the healing process of the lesion with a minimal complaint of bald tongue, especially when eating spicy or hot meal. to improve healing process, the patient then was given an antibacterial mouth rinse containing zinc and mulvitamin. conclusion: speckled leukoplakia could show high malignant transformation rate, therefore, early detection and treatment are necessary. keywords: potentially malignant disorder; speckled leukoplakia; early detection correspondence: selviana tampoma, oral medicine resident, faculty of dental medicine universitas airlangga. jl. mayjen. prof. dr. moestopo 42, surabaya 60132, indonesia. email: selvianatampomadrg@gmail.com. introduction public awareness of lesions that can potentially be a malignancy in oral cavity has been increasing. one lesion that can be found in the oral cavity is leukoplakia. leukoplakia is derived from the word “leuko” which means white, and “plakia” which refers to the word plaques or patches. thus, leukoplakia can be defined as a white plaque that cannot be scraped off. its etiology, however, is still questionable after eliminating all risk factors that do not have a tendency toward malignancy.1,2,3 approximately, 3% of the worldwide population has suffered from leukoplakia, 5-25% of which are pre-malignant lesions. after verified through histopathological examination, all lesions of leukoplakia can be considered as a potentially malignant lesion.3 one of leukoplakia types on oral mucosa is speckled leukoplakia or erythroleukoplakia, a non-homogeneous type. speckled leukoplakia (sl) according to who, is a leukoplakia with a mix of white and red plaque lesions.2,4 sl is a form of leukoplakia that is rarely found with very aggressive high risk for transformation into malignancy, and also considered as precursor lesion for squamous cell carcinoma.4 early diagnosis of sl can be based on clinical conditions in which there are white plaques with unknown etiology. if there is a local trauma, such as a tooth or sharp restoration, then the trauma factor should be eliminated. if within two weeks of healing it does not happen again, then the tissue biopsy should be done to eliminate the possibility dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p54-58 5555tampoma and hernawan/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 54-58 of malignancy.2 therefore, early detection and treatment of potentially malignant lesions in the oral cavity are important as a precautionary measure for the development of squamous cell carcinoma lesions. case a male patient aged 62 years came to the oral and dental hospital of faculty of dental medicine, universitas airlangga on october 27, 2014 on the referral of a private hospital in surabaya with complaints of pain and thickening of the bottom left of the tongue. his tongue had felt bald and stinging since one month ago if exposed to spicy foods. there was also a wound near the thickened area that could bleed easily when touched. the tooth on the area near the tongue thickened was patched with black dental filling material two years ago. before the tooth was patched, there was no complaint about the tongue. the patient also has a history of allergy to the cold, high blood pressure, gout and high cholesterol, as well as hepatitis b. therefore, he had regularly taken high blood pressure medicines, especially calcium antagonist class (amlodipine or nifedipine). in addition, he had also regularly consumed herbal drinks, such as turmeric and ginger solution. he had been smoking since the age of approximately 20 years, but he has stopped the habit since 10 years ago. there is a genetic predisposition in which his brother suffers from mammary carcinoma and bladder carcinoma. based on submandibular gland examination, his left submandibular gland was palpable, soft, supple, and pain. on the left ventral tongue (figure 1), there was a single, oval, white plaque lesion sized 1 x 1.5 cm that could not be scraped with rough surface, regular edges, clear boundaries, and pain. in addition, there were also two oval-shaped ulcers sized 2 x 3 mm and 7 x 3 mm with redness color, pseudomembranous coated surface, regular edges with diffuse boundaries, easy bleeding, and pain. on tooth 37, there was amalgam filling with sharp edges. case management based on the history and the extra-oral and intra-oral clinical examination, a temporary diagnosis of leukoplakia was set with a diagnosis of traumatic keratosis. the patient then was instructed to perform a complete blood count and liver function (sgot/sgpt) test. he was also referred to the conservative dentistry to improve his sharp tooth filling. as symptomatic and supportive therapy, the patient then was given topical steroid anti-inflammatory drugs (triamcinolone acetonide in ora base 0.1%) three times a day, applied at the lesion. the patient was also advised to avoid the consumption of spicy food, and come back after the filling was improved and the laboratory results were received. in the second visit, the patient denounced the recommended laboratory tests, and went to dr. ramelan navy hospital surabaya to seek another opinion on his own initiative. his dental filling still was not repaired. in dr. ramelan navy hospital, cytology biopsy examination was performed with scraping on the lesion. the results of the histopathological examination showed squamous epithelial cell dysplasia, from mild to moderate, and also atypical cells. thus, the patient was advised to undertake an excisional biopsy, but the patient refused. the patient was then educated to follow the advice to perform an excisional biopsy as the results of the histopathological examination, but the patient still was not willing. in the third visit, three months later, the patient came with complaints of pain in the tongue getting worse, difficulty of swallowing, and pain terraces up to the neck. on the examination of submandibular gland, moreover, the left submandibular gland was palpable, chewy, pain, and static. based on the results of the intra-oral examination, figure 1. there was a single white lesion and two ulcers on the ventral surface of the tongue. figure 2. there was an amalgam filling on tooth 37 with a sharp edge. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p54-58 56 tampoma and hernawan/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 54-58 there was a single white oval plaque lesion sized 1 x 1.5 cm with clear boundaries, regular edges, and rough surfaces on the left lateral tongue, that could not be scraped and caused pain. there was also a redness erosion emerged on the area around the plaque extending to the base of the tongue with diffuse boundaries and pain (figure 3). the dental filling was then replaced with composite materials. finally, the patient was willing to be referred to an oncologist to perform the excisional biopsy in dr. ramelan navy hospital. excisional biopsy surgery was conducted on february 11, 2015 by an oncologist. the results of histopathological examination showed leukoplakia with mild dysplasia. two months after the surgery, the patient began to feel comfortable, but his tongue still felt bald when exposed to hot or spicy food. during the intra-oral examination, a single erosion sized 5 x 3 cm with irregular edges, diffuse boundaries, and painless was found (figure 4). this condition can be considered as the healing process of the excisional biopsy surgery. the patient was prescribed with antibacterial mouthwash, made of sodium chlorite, aloe vera, and zinc three times a day, as well as vitamin supplements (b complex, vitamin e, vitamin c, and zinc) once a day. the patient was also advised to avoid spicy and hot food/beverage. six months after the surgery, the patient still felt pain in tongue when exposed to spicy or fried food. tongue and left cheek were often bitten, causing injuries. intraoral examination of the left lateral tongue showed macular erythematous with diffuse boundaries no pain, and no nodule or induration found on the palpation (figure 5). on the left buccal region of the right upper second molar (27), there was a single ulcer with irregular edges, clear boundaries, and pain. the occlusal surface of the premolars, (34 and 35) and the left mandibular second molar (37) and left upper jaw (27) was noticeably sharper. examination using velscope® was also conducted on the entire mucosal surface, and the results did not reveal any areas that absorbed light (dark). to reduce the risk of trauma to the buccal and lateral tongue, occlusal grinding was performed on the surface of the teeth 34, 35, and 27. for traumatic ulcer, a treatment was conducted by applying topical steroid anti-inflammatory drugs (triamcinolone acetonide in ora base 0.1%). the patient was also educated to increase the consumption of fruits and vegetables containing high beta-carotene, such as tomatoes and carrots, as well as to avoid spicy and fried food. based on the results of the clinical features and the final diagnosis of the histopathological examination, it was diagnosed as speckled leukoplakia. figure 3. the widespread of erosion on the tongue (on visit 3). figure 4. the condition of the patient’s tongue two months after the excisional biopsy surgery. figure 5. the wound healing process after the excisional biopsy with erythematous macular on the left lateral surface of the tongue. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p54-58 5757tampoma and hernawan/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 54-58 discussion during the first examination, the patient was diagnosed with leukoplakia with traumatic keratosis diagnosis based on clinical description in which there are white nodular lesions and dental filling using amalgam materials with sharp edges. but after the dental filling was repaired, in the next two month control, the lesion was not improved, but getting worse by erosion around the nodular lesion. the patient was then referred to the oncology department for further examination and treatment. this is in accordance that if local factors have been eliminated and there is no improvement in the lesion, the patient should be referred for biopsy examination.2 sl diagnosis can be made on clinical and histopathologic examinations. sl is a type of non-homogeneous leukoplakia with the clinical picture in the form of plaques, nodular, or white granular with reddish basis.2,5,6 sl is often accompanied by pain and discomfort4,7 as perceived by the patient. l e u k o p l a k i a i s p r i m a r i l y a c l i n i c a l t e r m . histopathological findings of the biopsy consists of surface epithelial hyperplasia and hyperkeratosis, atrophy with or without dysplasia cells. dysplasia cells can be mild, moderate, or severe.2,7 epithelial dysplasia is commonly found in homogeneous leukoplakia, but less in nonhomogeneous leukoplakia.2 the results of histopathological examination after the excisional biopsy in the patient showed hyperkeratosis epithelium, mild dysplasia, and intact basement membrane. predisposing factors of sl in this case were old smoking habit (± 30 years) and chronic trauma of sharp dental filling edge. tobacco in various forms is the primary etiology of leukoplakia, especially tar contained in tobacco considered as toxics and carcinogens. furthermore, mechanical trauma is also considered as a factor that plays a role in the pathogenesis of leukoplakia.7 based on animal studies, chronic trauma accompanied by the risk factors and carcinogenic materials (such as tar) will trigger epithelial cell transformation.8 therapy for sl is an aggressive surgery, excisional biopsy. in addition, the elimination of risk factors (smoking and alcohol consumption) and etiological factors (sharp teeth, metallic restorations, and dentures bridges that do not fit) can be a precaution that can be conducted.4 control regularly every three months in the first year is also recommended. if the lesions are not recurrent or there is no change in the mucosa, so the control time can be increased to six months. if there is a change in the mucosa, biopsy should be performed again. if after five years, there is no change in the mucosa, patients are advised to observe themselves.2,8 for the patient in this case, the excisional biopsy therapy was chosen based on the results of the initial cytologic examination showing mild to moderate dysplasia cells in the presence of atypical cells. similarly, surgical therapy, is chosen based on the discovery of the epithelial cells that undergo displasia.7 symptomatic therapy given to the patient, was an antibacterial mouthwash made of sodium chlorite, aloe vera, zinc, and vitamin b complex supplements plus zinc. the antibacterial mouthwash was given to improve the oral hygiene of the patient. sodium chlorite, aloe vera, and zinc contained are expected to help the healing process of the tissue. sodium chlorite will produce oxygen, which is essential for cell metabolism, specifically the production of energy via adenosine triphosphate (atp). oxygen can prevent infection in wounds, stimulate angiogenesis, increase differentiation, migration as well as keratinocytes re-epithelialization, increase fibroblast proliferation and collagen synthesis, and trigger wound contraction. in addition, level of superoxide production by polymorphonuclear leukocytes, necessary to kill bacteria is highly dependent on the level of oxygen. 10 the administration of zinc is also expected to enhance the regeneration of the epithelial cells since zinc can activate transforming growth factor beta (tgfβ), which plays a role in the early wound healing process. 11 zinc may also play a role in the activation of metalloproteinase enzymes that play a role in the process of collagenase. 12 the administration of multivitamin containing vitamin b complex, vitamin e and vitamin c, and zinc, moreover, aims to help the wound heal process. vitamins e and c have antioxidant and anti-inflammatory effects on the wound healing process.10 vitamin b complex plays a role as co-enzymes that catalyze biochemical reactions in body.13 there are several factors triggering malignant leukoplakia. 4,7,14 first, age factor can make the elderly people increasingly at risk. second, size factor of lesions can trigger the risk if it is larger than 2 cm. third, habits factor can lead to the risk of malignancies, especially more common in smokers than non-smokers. fourth, the location of the lesions on the tongue and floor of the mouth is more at risk than the buccal mucosa and commissure. fifth, sex factor can also trigger the risk, which means women have higher risk of malignancies than men do. sixth, clinical types can lead to the risk more than non-homogeneous type. seventh, epithelial dysplasia can be considered to have a higher risk for experiencing malignancies. eight, leukoplakia accompanied with candidiasis infection can trigger more the risk of malignancies. ninth, the presence of ulcers, erosions, or nodules can also trigger the risk. this patient had six of the nine factors that influence the malignant tendencies of sl suffered. therefore, early detection and excisional biopsy are essential to prevent potential malignancy of the lesion nowadays, a tool to detect epithelial dysplasia has been developed and set in dental chair. velscope® (visually enhanced lesion scope) is a tool that does not require dye and needs shorter examination time. this tool is based on dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p54-58 58 tampoma and hernawan/dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 54-58 the principle that normal cells will glow when exposed to fluorescent light, while abnormal cells will absorb the fluorescent light and be looked dark. 15 this tool, can assist dentists in detecting the early presence of an abnormality in oral mucosa so that prevention from the malignant transformation can be performed early. the results of the examination using velscope® during visits six months after the excisional biopsy did not reveal any areas that absorb light (dark). it means that there were no cells suffering from dysplasia. therefore, there was no sign of malignancy. in conclusion, leukoplakia, especially sl, is a lesion that can lead to high malignancy, especially if there is epithelial cell dysplasia. dentists play an important role in early detection of suspicious lesions leading to malignancy, which then could affect the prognosis for patients. in other words, early detection and treatment of lesions are important to prevent the possibility of lesion transformation into a malignant lesion. references 1. kardam p, rehani s, mehendiratta m, sahay k, mathias y, sharma r. journey of leukoplakia so far an insight on shortcomings of definitions and classifications. j dent oral disord ther. 2015; 3(2): 1-6. 2. jontell m, holmstrup p. red and white lesions of the oral mucosa. in: glick m, editor. burket’s oral medicine. 12th ed. connecticut: people’s medical publishing house; 2015. p. 100-3. 3. lingen mw. head and neck. in: kumar v, abbas ak, aster, jc. editors. robbins and cotran-pathologic basis of disease. 9th ed. philadelphia: elsevier; 2015. p. 731. 4. suresh kv, shenai p, chatra l, bilahari n, ashir kr. bilateral recurrent speckled leukoplakia: a case report. pac j med sci 2012; 10(1): 51-6. 5. radwan-oczko m, mendak m. differential diagnosis of oral leukoplakia and lichen planus on the basis of literature and own observations. j stoma 2011; 64(5): 355-70. 6. monu y, taseer b, shipli c, vijay k, naeem a, pankaj. review article-leukoplakia; a mysterious white patch. international journal of scientific research and education 2014; 2(9): 1824-30. 7. abidullah m, kiran g, gaddikeri k, raghoji s, ravishankar ts. leukoplakia review of a potentially malignant disorder. j clin and diagn res 2014; 8(8): ze1-4. 8. epstein j, elad s. oral and oropharyngeal cancer. in: glick m, editor. burket’s oral medicine. 12th ed. connecticut: people’s medical publishing house; 2015. p. 176. 9. scully c. oral and maxillofacial medicine the basis of diagnosis and treatment. 3rd ed. london: elsevier; 2013. p. 195. 10. guo s, dipietro la. factors affecting wound healing. j dent res 2010; 89(3): 219-29. 11. cario e, jung s, harder d’heureuse j, schulte c, sturm a, wiedenmann b, goebell h, dignass au. effects of exogenous zinc supplementation on intestinal epithelial repair in vitro. eur j clin invest 2000; 30(5): 419-28. 12. truong-tran aq, carter j, ruffin r, zalewski pd. new insights into the role of zinc in the respiratory epithelium. immunol cell biol 2001; 79(2): 170-7. 13. report of a joint fao/who expert consultation [internet]. bangkok (th): fao/who; c2002. human vitamin and mineral requirements available from: ftp://ftp.fao.org/docrep/fao/004/y2809e/y2809e00. pdf. accessed july 10, 2015. 14. rao pkj. potentially malignant lesion-oral leukoplakia. global advanced research journal of medicine and medical sciences. 2012; 1(11): 286-91. 15. balevi b. evidence-based decision making: should the general dentist adopt the use of the velscope for routine screening for oral cancer?. j can dent assoc. 2007; 73(7): 603-6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p54-58 vol 38-no4-2005-isi.pmd 189 comparison of recognition about denture adhesive between japanese and indonesian dentists: a pilot study shinsuke sadamori,* taizo hamada,* guang hong,* nakai nakai,* makoto kawamura,* and arifzan razak** *department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, hiroshima, japan **department of prosthodontics, the faculty of dentistry, airlangga university, surabaya, indonesia abstract the purpose of this study was to compare cross-national differences of the recognition of denture adhesive among dentists. the design of the research was cross-cultural differences. the research was done in japan and indonesia. one hundred and ten dentists from japan and indonesia were surveyed using a questionnaire regarding knowledge/comprehension of denture adhesive (in japanese and indonesian versions respectively). logistic regression model (forward stepwise method) showed that it was possible to distinguish japanese dentists from indonesian peers with a probability of 96.0 per cent by using 4 items out of 16. for the question of "how many domestic products of denture adhesive (da) do you know?" approximately a half of the japanese dentists answered "less than 3", whereas 93 per cent of indonesian subjects answered "nothing". it was concluded that there were much differences in dentists' understanding and experience of denture adhesive in the clinic, between japan and indonesia. key words: denture adhesive, cross-national differences, dentists, japan, indonesia correspondence: dr shinsuke sadamori, department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, 1-2-3 kasumi, minami-ku, hiroshima, 734-8553, japan, tel: +81-82-257-5681, fax: +81-82-257-5684, e-mail: tsada@hiroshima-u.ac.jp introduction denture adhesive has been used by denture wearers as a means to enhance denture retention, stability, and function.1–6 in the clinic, a negative opinion toward denture adhesive has existed,7–10 and dentists have been slow to accept it in their clinic. the first patent for denture adhesive was issued in 1913 in the us, with other patents following in the 1920s and 1930s.11 however, the american dental association first reported the use of denture adhesive in 1935.12 denture adhesive was imported into japan in 1951. after that, many japanese original denture adhesives were selling on the market in the 1970s and 1980s.13 several reports noted that the main reasons for the use of denture adhesive are to improve fit, comfort, chewing ability, and also to improve patient confidence in wearing dentures.5,14 fifteen per cent of denture wearers in the us used denture adhesives in 1980.15,16 wilson et al.17 reported that 30 per cent of denture wearers used, or had used, denture adhesive. however coates18 reported that a significant number of subjects in his study did not know that denture adhesives existed. denture adhesives at present would have a legitimate and indispensable place in prosthetic dental treatment. testing method, mechanical properties, and cytotoxicity of denture adhesives have been widely investigated.19–23 although the knowledge level of denture wearers about denture adhesive has been investigated,14,18,24 knowledge/ experience about denture adhesive among dentists has not been clarified. if japanese dentists could be distinguished from indonesian peers only by using the questionnaire, it could indicate a difference between the degree of knowledge and interest of dentists regarding denture adhesive use. the synthetic recognition about denture adhesive by dentists seems to, in future, motivate their patients and affect its use in the clinic. therefore, the purpose of this study was to clarify dentists' recognition about denture adhesive and to examine cross-national differences in dentists' recognition. materials and methods in this study, 110 subjects were surveyed to complete and return a structured questionnaire (see table 1). the subjects in this pilot study were selected from dentists in japan (hiroshima university, hiroshima) and indonesia (airlangga university, surabaya). they included 43 dentists (men: 27, women: 16) at hiroshima university and 65 dentists at airlangga university. (men: 30, women: 35) mean age of the japanese dentists was not statistically significant compared to that of indonesian peers (30.1 and 38.6 years respectively). in the survey, the distribution and collection of the questionnaire was instituted by the staff of this survey in hiroshima and airlangga university in 2002. this questionnaire was administered after explanation was given to all subjects about the aim of this survey, and understanding and consent from all subjects was gained. 190 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 189–193 the questionnaire was produced in japanese and then translated into english. then, it was discussed with the staff in indonesia, and this survey was instituted by the same estimation criterion. the answers were evaluated in three steps of "no" or "nothing" (score 0), "yes, but a little" or "occasionally" (score 1) and "yes, very much" or "often" (score 2). chi-square tests were used to examine the difference of responses for each item on the questionnaire between the two countries. then, two stepwise logistic regression analyses were carried out on the dependent variable (country). the wald statistic was used to test the null hypothesis that the regression coefficients were zero. the negelkerke r2 statistic was used to discriminate how well the model is able to distinguish between the interest and knowledge of dentists in the two countries. all analyses were computed using spss for windows operating system (spss 10, spss japan inc., tokyo, japan). results the recovery rate was 99.1 per cent. the recovery rate of the subjects at hiroshima university was 100 per cent, and that of airlangga university was 98.5 per cent. table 1 presents questionnaire items and percentage distribution of responses. significant differences between the two countries were found for 12 questions. for the question of "how many domestic products of denture adhesive (da) do you know?" about a half of the japanese dentists answered "less than 3", whereas 93 per cent of subjects in indonesia answered "nothing". category score item descriptions 2 1 0 χ2 test q 1. do you know the denture adhesive? jpn 42 56 2 a ns ina 31 66 3 q 2. do you know any purposes of the denture adhesive? jpn 42 58 0 a ns ina 32 65 3 q 3. do you know any disadvantages of the denture adhesive? jpn 33 53 14 a ** ina 16 44 40 q 4. how many imported products of denture adhesive do you know? jpn 14 26 60 b *** ina 10 62 28 q 5. how many domestic products of denture adhesive do you know? jpn 35 51 14 b *** ina 0 7 93 q 6. have you ever been taught about the denture adhesive? jpn 21 58 21 c *** ina 0 52 48 q 7. have you ever taught the denture adhesive to your students? jpn 5 14 81 c ns ina 3 25 72 q 8. have you ever seen the denture adhesive in books or lecture meetings? jpn 28 58 14 c * ina 8 70 22 q 9. have you ever seen any tv commercials about the denture adhesive? jpn 37 56 7 c *** ina 2 3 95 q10. do you know any goods instead of the denture adhesive? jpn 16 42 42 a * ina 2 44 54 q11. have you ever applied the denture adhesive to patients? jpn 2 26 72 c ** ina 5 55 40 q12. have you ever seen the denture adhesive in the clinic? jpn 14 49 37 c * ina 19 65 16 q13. have you ever let your patients use the denture adhesive? jpn 7 28 65 c ** ina 9 61 30 q14. have you ever applied the denture adhesive for stability of the base jpn 5 30 65 c *** plate in the clinic? ina 3 66 31 q15. do you think the use of denture adhesive is more jpn 5 67 28 a ns effective than medical intervention such as relining? ina 2 58 40 q16. do you think the price of denture adhesives is reasonable? jpn 5 65 30 a * ina 5 39 56 a 2: yes, very much, 1: yes, but a little, 0: no b 2: three and more, 1: less than three, 0: nothing c 2: yes, often, 1: yes, occasionally, 0: no table 1. questionnaire items and percentage distribution of the answers by country 191sadamori: comparison of recognition about denture adhesive table 2 shows the estimated coefficient and related statistics from the logistic regression model that predicts group membership. the model contained four variables by forward stepwise method (p < 0.01): q14 (application of da for stability), q9 (tv commercial about da), q5 (number of domestic da) and q2 (understanding of purposes of the use of da). the model contained six variables by backward stepwise method (p < 0.05): q1 (understanding of da), q3 (understanding of disadvantage of da), q5 (number of domestic da), q9 (tv commercial about da), q12 (utilization of da in the clinic) and q14 (application of da for stability). table 2. results of binary logistic regression analysis using 2 wald methods item no. b s.e. wald chi-square freedom p exp (b) forward stepwise (wald) q14: application of da* for stability 3,43 1,20 8,11 1 0,004 30,90 q 9: tv commercials about da* -2,64 0,80 10,86 1 0,001 0,07 q 5: number of domestic da * -4,02 1,35 8,90 1 0,003 0,02 q 2: understanding of purposes of the use of da* 2,35 0,78 9,12 1 0,003 10,51 backward stepwise (wald) q 5: number of domestic da * -4,60 1,57 8,59 1 0,003 0,01 q12: utilization of da* in the clinic 2,46 1,08 5,21 1 0,022 11,76 q14: application of da* for stability 2,81 1,24 5,16 1 0,023 16,57 q 9: tv commercials about da* -1,67 0,77 4,75 1 0,029 0,19 q 1: understanding of da* 2,32 1,08 4,65 1 0,031 10,22 q 3: understanding of disadvantage of da* -2,14 1,08 3,91 1 0,048 0,12 * denture adhesive • for forward stepwise, variables were entered in steps 1 to 4•q14, q9, q5, q2 in that order. •• for backward stepwise, variables were removed in steps 1 to 11•q11, q8, q13, q7, q16, q2, q4, q6, q10, q15 in that order. table 3 presents that 41 japanese dentists (95.3 per cent) were correctly predicted by the former model. similarly, 56 indonesian dentists (96.6 per cent) were correctly predicted. the nagelkerke r2 statistic was 0.853; that is, 85.3 per cent of the variation in the outcome variable was explained by the logistic regression model. table 3. observed and predicted group membership using 2 wald methods predicted country country japan indonesia percentage correct forward stepwise (wald) japan 41 2 95,3 indonesia* 2 56 96,6 total 96,0 backward stepwise (wald) japan 41 2 95,3 indonesia 3 55 94,8 total 95,0 the cut value is 0.50. negalkerke r2 = 0.853 (forward stepwise), 0.878 (backward stepwise). * seven dentists were delated because they did not give complete answers in the questionnaire. discussion there are various differences in the environment between the two countries. these differences could affect dental treatment, and affect the use of denture adhesive. from the answers of q1 (understanding of da) and q3 (understanding of disadvantage of da), there was a difference in understanding about denture adhesive between japanese and indonesian dentists. there was a difference in the answers for q9 (tv commercials about da) "have you ever seen any tv commercials about denture adhesive?". these results are likely caused by differences in the popularity of mass media, commercials on television, and so on. the number of televisions per 1000 people in japan was 613 and that in indonesia was 59 in 1999.25 there seems to be a difference between countries as to the quantity of information from television. no difference between countries was seen in response to q6 (receiving instruction about da) or q7 (instruction about da to students). there would be little, or no difference in school education of both countries concerning denture adhesive. the results support the same opinion reported by ozan et al,14 which should be taught more intensively at dental schools. the basic concept of the diffusion of innovation model proposed by rogers and shoemaker26 is how different 192 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 189–193 people pass through the five stages: awareness, interest, trial, decision, and adoption. they described five groups of adopters: innovators, early adopters, early majority, late majority, and laggards. those who adopt an innovation earliest, the innovators, tend to be middleclass people who are more adventurous and actively seek information about new ideas. in the first three groups, people adopt a change primarily on the basis of their reasoning about costs, and benefits of an innovation. tv commercials about denture adhesive may have an effect on awareness and interest of those with dentures. the adoption of an idea, like the adoption of any product, requires a deep understanding of people's needs and perceptions, preferences of the reference groups, and behavioral patterns of the target audience. the late majority is lower in social influence and social status, and learns of new ideas primarily from peers through social influence, rather than from media channels. all people are probably receivers of the message, but only the most influential and well informed have special qualifications about how the message should be understood and assessed. further knowledge needed about the tailoring of messages, the effect of media, 'costs', and facilities to maximize the case for adopting the idea. the percentage of answers for q5 (number of domestic da) is very different in japan than in indonesia. for the answer "nothing" is 14 per cent in japan, compared to 93 per cent in indonesia. the problem of whether dentists know the product in the country is not only the problem of whether information on that product is abundant. in japan, gross national product per capita (gnp) was 28,190 (current us $, 1992) and gni per capita was 33,660.27 in indonesia, gnp was 670, and gni was 720. these results might indicate, that there is a difference in economic conditions between the countries, such as the creation of adhesives in that country; the capacity to buy the product by dentists or patients. answers to q12 (utilization of da in the clinic) and q14 (application of da for stability) indicate that indonesian dentists tend to apply denture adhesive to patients more often than japanese dentists do. differences in use of denture adhesive between the two countries may be due to a difference in need of the patient, and/or in understanding the disadvantage of da (q3). the use of denture adhesive is influenced by various factors. however, the methods used in this study clearly distinguished japanese dentists from indonesian peers. forward selection starts without any variables in the model, whereas backward elimination starts with all of the variables. at each step in the forward selection, the variable with the smallest significance level for the score statistic, provided it is less than the chosen cutoff value, is entered into the model. all variables in the forward stepwise block that have been entered are then examined to see if they meet removal criteria. if the wald statistic is used for deleting variables, the wald statistics for all variables in the model are examined and the variable with the largest significance level for the wald statistic, providing it exceeds the chosen cutoff value, is removed from the model. if no variables meet removal criteria, the next eligible variable is entered into the model. if a variable is selected for removal and it results in a model that has already been considered, variable selection stops. otherwise, the model is estimated without the deleted variable and the variables are again examined for removal. this continues until no more variables are eligible for removal. then, variables are again examined for entry into the model. the process continues until either a previouslyconsidered model is encountered (which means the algorithm is cycling), or, no variables meet entry or removal criteria. in univariate analysis, there was no correlation between country and q1 or q2. however, statistical significance, was seen in the regression coefficient of q1 by backward method, and that of q2 by forward method. other questions, which also had significant associations with countries, were significant in univariate analysis. these contradictions may happen when the sample size is not large. in such a case, input variables chosen in the logistic analysis have much influence in the result, because their interrelations affect regression coefficients. it is necessary, therefore, to conduct future research in a larger sample. some limitations can be identified in this study. first, indirect translation from japanese to indonesian might influence the results. second, sample sizes were small. third, there were significant differences in age between the two countries. fourth, the school environments in this study may not be representative of other schools in the two countries due to factors such as educational setting, training program, and geographic origin. it is unknown whether the results will generalize to other samples, therefore, it is necessary to conduct future research in more diverse samples. although caution should be exercised generalizing based on the results of this study, the variation in dentists' understanding and experiences in the clinic toward denture adhesive appeared to reflect effects of mass media, such as tv, but not their clinical training experience. the results also suggested that it was possible to distinguish japanese dentists from indonesian peers with a probability of more than 95% by using the questionnaire. these methods may be useful for comparison of synthetic recognition of denture adhesive by country, and may contribute to the evaluation of denture adhesive. a difference in dentists' understanding and experience of denture adhesive in the clinic was exhibited between japan and indonesia. dentists in japan had more information about denture adhesives. however, in the clinic, dentists in indonesia tended to see and use denture adhesives more often than dentists in japan. the method used in this study clearly distinguished japanese dentists from indonesian peers, and is available to compare differences in recognition about denture adhesive by country. 193sadamori: comparison of recognition about denture adhesive acknowledgement this research was supported in part by a grant-in-aid (no. 14406027) for scientific research from the ministry of education, culture, sports, science, and technology, japan. references 1. tarbet w, boone m, schmidt nf. effect of a denture adhesive on complete denture dislodgement during mastication. j prosthet dent 1980; 44: 374–8. 2. chew cl, boone me, swartz ml, phillips rw. denture adhesives: their effects on denture retention and stability. j dent 1985; 13: 152–9. 3. adisman ik. the use of denture adhesives as an aid to denture treatment. j prosthet dent 1989; 62: 711–715. 4. chew cl. retention of denture adhesives an in vitro study. j oral rehabil 1990; 17: 425–434. 5. coates aj. denture adhesives: a review. aust prosthodont j 1995; 9: 27–31. 6. grasso je. denture adhesives: changing attitudes. j am dent assoc 1996; 127: 90–6. 7. woelfel jb, kreider ja, berg t. deformed lower ridge caused by the relining of a denture by a patient. j am dent assoc 1962; 64: 763–9. 8. means cr. the home reliner materials: the significance of the problem. j prosthet dent 1964; 14: 1086–90. 9. lamb dj. denture adhesives: a side effect. j dent 1980; 8: 35–42. 10. grasso je, rendell j, gay t. effect of denture adhesive on the retention and stability of maxillary dentures. j prosthet dent 1994; 72: 399–405. 11. yankell sl. overview of research and literature on denture adhesives. the compendium of continuing education in dentistry 1984; 4 (supplement): 518–21. 12. accepted dental remedies. chicago: am dent assoc, 1935, 172–3. 13. hamada t, murata h, yuuda s. et al. denture adhesive, 1st ed. dental diamond; 2003. p. 78–82. (in japanese). 14. ozcan m, kulak y, arikan a, silahtar e. the attitude of complete denture wearers towards denture adhesives in istanbul. j oral rehabil 2004; 31: 131–4. 15. tarbet w, grossman e. observations of denture-supporting tissue during six months of denture adhesive wearing. j am dent assoc 1980; 101: 789–91. 16. shay k. denture adhesives. choosing the right powders and pastes. j am dent assoc 1991; 122: 70–6. 17. wilson mj, mccord jf, watts dc. denture adhesives: an in vitro evaluation. j dent res 1990; 69: 970. 18. coates aj. usage of denture adhesives. j dent 2000; 28: 137–40. 19. ellis b, al-nakash s, lamb dj. the composition and rheology of denture adhesives. j dent 1980; 8: 109–18. 20. love wb, biswas s. denture adhesives – ph and buffering capacity. j prosthet dent 1991; 66: 356–60. 21. ekstrand k, hensten-pettersen a, kullmann a. denture adhesives: cytotoxicity, microbial contamination, and formaldehyde content. j prosthet dent 1993; 69: 314–17. 22. william dg, millicent g, donald k. microbial contamination in four commercially available denture adhesives. j prosthet dent 1994; 71: 154–8. 23. koppang r, berg e, dahm s, flöystrand f. a method for testing denture adhesives. j prosthet dent 1995; 73: 486–91. 24. kelsey cc, lang br, wang rf. examining patient's responses about the effectiveness of five denture adhesive pastes. j am dent assoc 1997; 128: 1532–8. 25. unicef. the state of the world's children 1995. oxford university press; 1994. p. 72–3. 26. rogers em, shoemaker ff. communication of innovation. a crosscultural approach (2nd ed.). the free press; 1971. 27. world bank group (http://devdata.worldbank.org/data-query/) << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice � vol. 43. no. 1 march 2010 research report relationship between trauma mechanism and etiology on mandibular fracture patterns fakhrurrazi dentistry study program faculty of medicine, syiah kuala university darussalam banda aceh abstract background: �andi��lar �ract�re �cc�rs ��re c����nly t�an �a�illary �ract�re �eca�se �� its �r��inent ��siti�n and its arr����andi��lar �ract�re �cc�rs ��re c����nly t�an �a�illary �ract�re �eca�se �� its �r��inent ��siti�n and its arr��� arc� like ��ne anat��y. �any �act�rs �ay ca�se �andi��lar �ract�re. ��t�rcycle accident is t�e �ain eti�l�gy �� �andi��lar �ract�re in t�e ���rld. based �n t�e literat�re, 43% �andi��lar �ract�res are ca�sed �y ��t�rcycle accident, 34% �y vi�lence, 7% �y accident at ���rk, 7% �y �all, 4% �y s��rts and t�e �t�ers ��ere ca�sed �y vari��s t�ings. purpose: ��e ��r��se �� t�is st�dy ��as t� kn��� t�e��e ��r��se �� t�is st�dy ��as t� kn��� t�e relati�n �et��een t�e eti�l�gy and �ec�anis�s �� tra��a and t�e �atterns �� �andi��lar �ract�re at hasan sadikin h�s�ital, band�ng, �r�� jan�ary 2006 t� oct��er 2007. method: ��e st�dy ��as taken �n �atients ��it� �andi��lar �ract�res ���� ca�e t� hasan sadikin��e st�dy ��as taken �n �atients ��it� �andi��lar �ract�res ���� ca�e t� hasan sadikin h�s�ital band�ng. ��e data ��ere taken retr�s�ectively �y d�c��enting t�e eti�l�gies �� �andi��lar �ract�re, t�e �ec�anis�s �� �ract�re, and t�e l�cati�n �� �andi��lar �ract�re. ��e data ��ere analyzed ��it� c�i sq�are statistic test. result: ��e res�lt s����ed��e res�lt s����ed t�at ��ere ��ere 83 �andi��lar �ract�res. ��e �andi��lar �ract�re ��re c����nly attacks �en a���t 77%, and ����en a���t 22.9%. �andi��lar �ract�re �cc�rs ��re ��ten �et��een t�e age gr��� �� 21–30 years �ld, a���t 31 �e��le (37.3%). �andi��lar �ract�re ��as ��stles ��ten ca�sed �y ��t�rcycle accident, a��ecting a���t 71 �e��le (85.5%). parasy���ysis �ract�re is t�e ��st c����n �ract�re l�cati�n a��ng �andi��lar �ract�re cases, a���t 47 �e��le (56.6%). conclusion: �t can �e c�ncl�ded t�at t�ere is n� signi�icant�t can �e c�ncl�ded t�at t�ere is n� signi�icant relati�ns�i� �et��een t�e eti�l�gy and �ec�anis�s �� tra��a and t�e �attern �� �andi��lar �ract�re. key words: �andi��lar �ract�re l�cati�n, eti�l�gy, tra��a �ec�anis�s abstrak latar belakang: frakt�r �andi��la le�i� sering terjadi di�andingkan dengan �rakt�r �aksilla karena ��sisinya yang le�i� �r��inen dan �ent�k anat��i t�lang se�erti ��s�r �ana�. banyak �akt�r yang da�at �enye�a�kan terjadinya �rakt�r �andi��la. kecelakaan kendaraan �er��t�r �er��akan eti�l�gi �ta�a �enye�a� �rakt�r �andi��la di d�nia. literat�r �enye��tkan �a���a 43% �rakt�r �andi��la dise�a�kan �le� kecelakaan kendaraan �er��t�r, 34% dise�a�kan �le� kekerasan, 7% kecelakaan kerja, 7% aki�at jat��, 4% �ada kecelakaan �la�raga dan sisanya �le� �er�aca�-�aca� se�a� lainnya. tujuan: �enelitian ini �nt�k �engeta��i a�aka� ada ����ngan antara eti�l�gi dan �ekanis�e tra��a dengan ��la �rakt�r �andi��la �ada �enderita �rakt�r �andi��la di r��a� sakit hasan sadikin band�ng dari ��lan jan�ari 2006 sa��ai okt��er 2007. metode: penelitian dilak�kan �ada �asien dengan �rakt�r �ani��lar yang datang ke r��a� sakit hasan sadikin band�ng. data dik����lkan secara retr�s�ekti� dengan cara �encatat eti�l�gi,�ekanis�e, dan l�kasi terjadinya �rakt�r �andi��la. data dik����lkan dan dikel����kkan ke��dian dianalisis dengan �ji statistik c�i-sq�are. hasil: dari �asil dida�atkan 83 kas�s �rakt�r �andi��la. frakt�r �andi��la le�i� sering terjadi �ada laki-laki yait� se�anyak 77% di�andingkan ��anita 22,9%. frakt�r �andi��la sering terjadi �ada �sia 21–30 ta��n, yait� se�anyak 31�rang (37,3%). frakt�r �andi��la le�i� �anyak dise�a�kan ta�rakan ��t�r yait� 71 �rang (85,5%). frakt�r �arasi��isis �er��akan yang ter�anyak yait� 47 �rang (56,6%). kesimpulan: penelitian ini da�at disi���lkan �a���a antara eti�l�gi dari �rakt�r �andi��la, �ekanis�e tra��a dengan ��la �rakt�r �andi��la tidak terda�at ����ngan yang �er�akna. kata kunci: l�kasi �rakt�r �andi��lar, eti�l�gi, �ekanis�e tra��a c�rres��ndence: fakhrurrazi, prodi kedokteran gigi, fakultas kedokteran universitas syiah kuala darussalam banda aceh 23111. e-mail: abunidafahiza@gmail.com � dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 1-5 introduction mandibular fracture actually can be classified into many terminologies that have not been standardized yet, which are simple, compound, greenstick, comminuted, pathologist, multiple, impaction, atrophic, indirect and complex fracture. nevertheless, mandibular fracture sometimes is also classified based on the involved anatomy, such as symphysis, corpus, angulus, ramus, processus condyloideus (condylus), coronoideus, and alveolaris.1,2 there are some factors causing mandibular fracture. motorcycle accident is the main etiology causing mandibular fracture in the world. some literatures even mention that 43% of mandibular fracture are caused by motorcycle accident, 34% are caused by violence, 7% are caused by working accident, 7% are caused by falling, 4% are caused by sport accident, and the rest is caused by other causes.2 in the united states, trauma is considered not only as the third factor causing death in all levels of age, but also as the first factor causing death in children, teenagers, and youth, who are in the age of 1-44 years old. motorcycle accident is considered as the most common factor causing both trauma on face and multiple injuries. violence and falling from the height also occur in developing countries. the type and direction of trauma power actually can help to conduct diagnosis. thus, the big power resulted during collision, and motorcycle and car accidents usually tends to make patients get multiple, compound, comminuted mandibular fracture, in which at the first time of hit the patients tend to get single, simple, and nondisplaced facture. the object of collision target also affects the type and the number of fracture.3,4,5 in addition, the direct hit on chin can cause bilateral fracture in condylus, and the hit on the angle of parasymphysis can cause condylus contralateral fracture or angulus fracture. if the patient sits on the car, moreover, it can help to diagnose mandibular injury or other injuries. for instance, injury on the chest is caused by being hit on the handlebar, and facial fracture is caused by being hit on the dashboard and by facial laceration.3 the reason of conducting the study, furthermore, is because there was no published study about the relation between the etiology and mechanisms of trauma and the pattern of mandibular fracture. the objective of the study, was to find out whether there is a relation between the etiology and mechanisms of trauma with the pattern of mandibular fracture on mandibular fracture patients at hasan sadikin hospital, bandung, from january 2006 to october 2007. material and method the study had retrospectively been conducted for a year and 10 months, from january 2006 to october 2007, with the number of patients about 83 people hospitalized at hasan sadikin hospital, bandung. moreover, the data of patient were taken based on the characteristic of demographic data, which were gender, age, the cause of trauma, the mechanisms of trauma, and the location of mandibular fracture. the inclusive criteria involved patients with mandibular fracture visiting at hasan sadikin hospital, bandung. the data then were collected, classified, and analyzed with spss program for windows version 13.0 using chi-square test. result the characteristics of the subject based on the gender and age can be explained in the following table 1. the cases of mandibular fracture, based on the etiology of trauma, the speed of trauma, the location of mandibular fracture, and the object of collision target, can be explained in table 2. table 1. the characteristics of mandibular fracture based on gender and age variable n % gender male female group of age (year) 0–10 11–20 21–30 31–40 41–50 51–60 64 19 5 27 31 11 6 3 77.1 22.9 6.0 32.5 37.3 13.3 7.2 3.6 total 83 100.0 table 2. the cases of mandibular fracture based on the mechanisms of trauma, the location of mandibular fracture, and the object of collision target variable n % mechanisms of trauma motorcycle accident car accident falling violence location of mandibular fracture parasymphysis fracture angulus fracture corpus fracture ramus fracture condylus fracture parasymphysis, angulus fracture parasymphysis, corpus fracture parasymphysis, condylus fracture object of collision target asphalt handlebar of motorcycle hand car 71 1 7 4 47 9 7 4 2 5 4 5 71 5 3 4 85.5 1.2 8.4 4.8 56.6 10.8 8.4 4.8 2.4 6.0 4.8 6.0 85.5 6.1 3.6 4.8 total 83 100.0 �fakhrurrazi: relationship between trauma mechanism the cases of mandibular fracture more commonly attacked motorcycle drivers wearing helmet, about 45 people (61.6%), meanwhile those not wearing helmet, about 28 people (38.4%). moreover, the relation of the mechanisms of trauma and the location of mandibular fracture can be explained in table 3. from 71 people whose face hit on asphalt, most of them had fracture location in parasymphysis, about 41 people (57.7%), and the least of them had fracture location in condylus and in both parasymphysis and condylus, each of which was 2 people (2.8%). meanwhile, for those whose face hit on handlebar of motorcycle, 4 (80%) of them had fracture location in parasymphysis. for those whose face was hit by hand, 66.7% of them had fracture location in both of parasymphysis and condylus. for those whose face hit on car, furthermore, they had fracture location in parasymphysis, in angulus, in corpus and in both of parasymphysis and condylus, each of which was about 22.5%. from 45 people wearing the safety equipment, there were 29 people (64.4%) who had fracture location in parasymphysis, and among those not wearing the safety equipment there were also 13 people (46.4%) who had fracture location in parasymphysis. the relation between the object of the collision target and the number mandibular fracture location, can be explained in table 4. discussion in this study, it found that mandibular fracture attacked more men, about 64 people (77.1%) than women, about 19 people (22.9%). this phenomenon was appropriate with some literatures discussing that the ratio of mandibular fracture patients, between male and female patients, is 3-6:1. the reason is because men drive or get involve in violence or fight more than women. moreover, it is also known that mandibular fracture commonly attacked the age group of 21-30 years old, about 37.3%; the age group of 11–20 years old, about 32.5%; the age group of 31–40 years old, about 13.3%; the age group of 41–50 years old, about 7.2%; and the age group of 0–10 years old, about 6% (table 1). this finding is appropriate with the result of another research stating that mandibular fracture commonly attacks the age group of 15–30 years old since that group is considered as the productive age group.1,4,6–8 actually, there are some factors causing mandibular fracture. the motorcycle accident is the main etiology causing facial trauma in the world.7–12 some literatures mentions that 43% of mandibular fracture are caused by motorcycle accident; 34% are caused by violence; 7% are caused by working accident; 7% are caused by falling; 4% are caused by sport accident; and the rest is caused by many others. the location of mandibular fracture is table 3. the relation between the mechanisms of trauma and the location of mandibular fracture location of mandibular fracture mechanisms of trauma total motorcycle accident car accident falling violence parasymphysis angulus corpus ramus condylus parasymphysis, angulus parasymphysis, corpus parasymphysis,condylus 42 (59.2%) 7 (9.9%) 6 (8.5%) 3 (4.2%) 2 (2.8%) 4 (5.6%) 4 (5.6%) 3 (4.2%) 0 (0.0%) 1 (100.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 3 (42.9%) 1 (14.3%) 1 (14.3%) 1 (14.3%) 0 (0.0%) 1 (14.3%) 0 (0.0%) 0 (0.0%) 2 (50.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (50.0%) 47 9 7 4 2 5 4 5 total 71 (100.0%) 1 (100.0%) 7 (100.0%) 4 (100.0%) 83 score of p=0.709 table 4. the relation between the object of collision target and the number of mandibular fracture location location number mandibular fracture object of collision target total face hit on asphalt face hit on handlebar of motorcycle face was hit by hand face hit on car 1 location 2 locations 3 locations 54 (76.1%) 15 (21.1%) 2 (2.8%) 4 (80.0%) 1(20.0%) 0 (0.0%) 1 (33.3%) 2 (66.7%) 0 (0.0%) 1 (25.0%) 3 (75.0%) 0 (0.0%) 60 21 2 total 71 (100.0%) 5 (100.0%) 3 (100.0%) 4 (100.0%) 83 score of p = 0.256 � dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 1-5 more commonly found in parasymphysis than in other areas.1,3,7,9,12–14 those phenomena are also appropriate with the result of this study finding that mandibular fracture was commonly caused by motorcycle accident, about 71 people (85.5%); the most common type of collision target was collision to asphalt, about 71 people (85.5%); and the location of mandibular fracture was at parasymphysis, about 47 people (56.6%), meanwhile, other fracture locations was about 2.4% to 10.8% (table 2). in this study, mandibular fracture more commonly attacked motorcycle drivers wearing helmet. in other words, the result of the study indicates that mandibular fracture more commonly attacked motorcycle drivers wearing helmet than those not wearing helmet. the reason is because most of motorcycle drivers in indonesia seldom wear standard helmet for protecting head and face. based on the result of study conducted by fridrich et al.,15 it is also known that mandibular fracture caused by car accident is more often located in condylus. mandibular fracture caused by motorcycle accident often occurs at symphysis or parasymphyisis, meanwhile when caused by violence often occurs at angulus. this fact is also found in this study that from 71 people with trauma mechanisms obtained from motorcycle accident, most of them had fracture location in parasymphysis, which were about 42 patients (59.2%) and among cases of falling from the height, most of them also had fracture location in parasymphysis, about 42.9%, while in violence cases, the fracture location often occurred in parasymphysis and in both parasymphysis and condylus, each about 50%. nevertheless, the statistic test result, chi square test, with the reliability degree about 95% showed that there were no significant relation between the mechanisms of trauma and the location of mandibular fracture, with the score of p=0.709 (p>0.05) (table 3). based on the result of some studies, it was found that most of mandibular fractures caused by motorcycle accident often occur in parasymphysis.4,7,12,14 the reason was because in the motorcycle accidents, most of patients face hit on asphalt. this finding was appropriate with the result of this study showing that from 71 people whose face hit on asphalt, most of them had fracture location in parasymphysis, and the fewest of them had fracture location in condylus. however, based on the chi square test, with the reliability degree 95% it was shown that there were no significant relation between the object of collision target and the location of mandibular fracture with the score of p = 0.550 (p>0.05). it was shown that from 45 people wearing the safety equipment there were 29 people who had fracture location in parasymphysis, and among those not wearing the safety equipment there were also 13 people who had fracture location in parasymphysis. nevertheless, based on the statistic test result, chi square test, with the reliability degree 95% it is shown that there were no significant relation between the using of safety equipment and the location of mandibular fracture with the score of p=0.369 (p>0.05). it means that based on the study result, the location of fracture in parasymphysis occurs mostly either in patients wearing safety equipment or those not wearing safety equipment. the reason was because most of motorcycle drivers in indonesia seldom wear standard helmet for protecting their head and face. the study also finds that mandibular fracture often occur in motorcycle accident, and most of them located in parasymphysis.4,7,12,15 specifically, based on the study result, from 71 people whose face hit on asphalt, there were 54 people (76.1%) who had 1 site fracture location; there were 21.1% who had 2 site fracture locations; and there were 2.8% who had 3 site fracture locations (table 4). those findings were appropriate with some literatures mentioning that mandibular fracture occurs in 1 site fracture location (unilateral), about 53%, in 2 site fracture locations, about 37%, and 3 site fracture locations, about 9%.1 this finding was appropriate with the result of ajmal et al.,9 study stating that single mandibular fracture was found in 54% while multiple mandibular fractures were seen in 46% of patients. however, based on the chi square test, with the reliability degree 95% it was shown that there was no significant relation between the object of collision target and the location number of mandibular fracture with the score of p = 0.256 (p>0.05). based on the result it can be concluded that there was no relation between the etiology the mechanisms of trauma and the pattern of mandibular fracture. there is some limitation in this study, the secondary data and the writer had no opportunity to do exploration on the cases. the exploration that was supposed to do involves things describing trauma specifically, such as the speed of the vehicles, the position of falling/target of collision, and the power of collision. it needs further studies with the larger sample number in order to analyze whether there is a cause-effect relation between the etiology and the mechanisms of trauma and the pattern of mandibular fracture. references 1. barber dh, bahram r, woodburry sc, silverstein ke, fonseca rj. mandibular fracture. in: fonseca rj, eds. oral and maxillofacial trauma. 3rd ed. st. louis: elsevier saunders; 2005. p. 485–90. 2. ochs mw, tucker mr. management of facial fractures. in: peterson lj, ellis e, hupp jr, tucker mr, eds. contemporary oral and maxillofacial surgery. 4th ed. st louis: mosby co; 2003. p. 527–36. 3. fonseca rj, vigliante ce, chen pm. the societal impact of maxillofacial trauma. in: fonseca rj, eds. oral and maxillofacial trauma. 3rd ed. st. louis: elsevier saunders; 2005. p. 1–9. 4. lee kh. epidemiology of mandibular fractur in a tertiary trauma centre. (abstract). emergency medicine journal 2008; 25: 565-68. available at: <http://emj.bmj.com/cgi/content/abstract/25/9/565>. accessed october 28, 2008. 5. thomas dw, hill cm. etiology and changing patterns maxillofacial trauma. in: booth pw, schendel sa, hausamen je, eds. maxillofacial surgery. 2nd ed. st. louis: churchill livingstone elsevier; 2007. p. 2-8. 6. bamjee y, lownie jf, cleaton-jones pe, lownie ma. maxillofacial injuries in a group of south africans under 18 years of age. br j oral maxillofac surg. 1996 aug; 34(4): 298–302. �fakhrurrazi: relationship between trauma mechanism 7. sirimaharaj w, pyungtanasup k. the epidemiology of mandibular fractures treated at chiang mai university hospital: a review of 198 cases. j med assoc thai 2008; 91(6): 868–74. 8. patrocínio lg, patrocínio ja, borba bhc, bonatti bs, pinto lf, juliana vieira v, et al. mandibular fracture: analysis of 293 patients treated in the hospital of clinics, federal university of uberlândia. brazilian journal of otorhinolaryngology 2005; 71(5): 560–5. 9. ajmal s, khan ma, jadoon h, malik sa. management protocolmanagement protocol of mandibular fractures at pakistan institute of medical sciences, islamabad, pakistan. men j ayub med coll abbottabad 2007; 19(3): 51–5. 10. shah a, mushtaq m, qureshi zur. frequency of mandibular fractures at the angle as a result of maxillofacial trauma. pakistan oral & dental journal; 28(1): 29–32. 11. dhaif g, r ramaraj r, magra a, yasser a, al-sammak n. mandibular fractures in bahrain a 10 year study. bahrain medical bulletin september 1996; 18(3). 12. elgehani ra, orafi ma. incidence of mandibular fractures in eastern part of libya. med oral patol oral cir bucal. 2009; 14 (10): 529–32. 13. king re, scianna jm, petruzzelli gj. mandibular fracture patterns:mandibular fracture patterns: a suburban trauma center experiance. am j otolaryngol 2004; 25(5): 301–7. 14. tuncali d, barutcu ay, aslan g. the relationship between the fracture site and etiology in mandibular fractures. kulak burun bogaz ihtis derg 2005; 14(1–2): 25–8. 15. fridrich kl, pena-velasco g, olson raj. changing trends with mandibular fractures: a review of 1067 cases. j oral maxillofac surg. 1992; 50: 586. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 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bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base mkgs vol 44 no 2 april-juni 2011.indd 63 vol. 44. no. 2 june 2011 research report effects of different saliva ph on hybrid composite resin surface roughness nirawati pribadi and adioro soetojo department of conservative dentistry faculty of dentistry, of airlangga university surabaya indonesia abstract background: currently, hybrid composite resin is the mostly used fi lling material to restore esthetic and function. during function, this material is in contact with various ph from food consumption, which is acidic and alkali which may effect the physical properties of composite resin, including surface roughness. purpose: the research was conducted to determine the effect of ph in saliva on surface roughness of hybrid composite resin. methods: this research used artifi cial saliva and composite resin samples divided into 3 groups based on different ph of immersion (ph 4, ph 7 and ph 10) for 30 days. results: there were signifi cant differences (p > 0.05) among those three treatment groups of hybrid composites soaked in artifi cial saliva with different ph for 30 days. and, with lsd test it is also known that there were signifi cant differences between the artifi cial saliva with ph 4 and ph 7, whereas there was no signifi cant difference between ph 4 and ph 10 and between ph 7 and ph 10. conclusion: it can concluded that the changes of salivary ph affect the surface roughness of the hybrid composite resin. acidic ph has increase the surface roughness of hybrid composite resin, whereas alkaline ph has no effects on the surface roughness of hybrid composite resin. key words: hybrid composite resin, acid, alkali, surface roughness abstrak latar belakang: saat ini tumpatan komposit merupakan bahan tumpatan yang paling sering digunakan untuk memperbaiki estetik dan fungsi. dalam rongga mulut, bahan ini kontak dengan berbagai macam ph dari konsumsi makanan, baik asam maupun basa yang dapat mempengaruhi perubahan sifat fisik resin komposit, diantaranya yaitu kekasaran permukaan. tujuan: penelitian ini dilakukan untuk mengetahui tentang efek ph saliva terhadap kekasaran permukaan tumpatan resin komposit hybrid. metode: penelitian ini menggunakan saliva buatan yang dibagi dalam 3 kelompok sampel yaitu masing-masing dengan perendaman ph yang berbeda (ph 4, ph 7 dan ph 10) selama 30 hari. hasil: terdapat perbedaan yang bermakna (p > 0,05) antara kelompok perlakuan komposit hybrid yang telah direndam saliva buatan dengan berbagai ph selama 30 hari. uji lsd menunjukkan adanya perbedaan yang signifikan antara perendaman pada saliva buatan antara ph4 dengan ph 7, sedangkan perbedaan yang tidak signifikan antara ph4 dengan ph 10 dan ph 7 dengan ph 10. kesimpulan: dapat disimpulkan bahwa perubahan ph saliva berpengaruh terhadap kekasaran permukaan resin komposit hybrid, ph asam meningkatkan kekasaran permukaan resin komposit hybrid, sedangkan ph basa tidak berpengaruh terhadap kekasaran permukaan resin komposit hybrid. kata kunci: resin komposit hybrid, asam, basa, kekasaran permukaan correspondence: nirawati pribadi, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. 64 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 63–66 introduction as the public demand for the services of aesthetic composite restoration increases, people are affected with their diet, especially various fast foods that are widely available. this condition, as a result, impact on rapid changes in acidic and alkaline condition in mouth which can affect the physical properties of composite resin filling materials including surface roughness changes. there are several factors that can cause the damage of composite resin filling materials. one of them is the effect of low saliva ph caused by diet and tooth brushing. the low ph in the saliva can increase the occurrence of polymer erosion.1 if the surface of the composite is rough due to the low ph in the saliva, it may cause discoloration, plaque, and finally secondary caries.2 at neutral ph, such as water, the degradation of the physical properties of composite resin can still occur.3 the degradation occurs when water enters polymer chains through porosity and intermolecular space so that there is an expansion accompanied by the loss of uncreated components, such as polymerization promoters and oligomers of filler particles causing both the decreasing of the hardness and other properties, such as diametric pulling power tensile, and the roughness of the cast surface roughness.1,3,4 surface roughness affects the attachment of debris and bacteria on the surface of the composite cast. as a result, it can easily trigger secondary caries. salivary fluid actually consists of 99.5% water with inconstant ph in the oral cavity. the changes of ph can be caused by the influence of bacteria, enzymes, hormones and other factors. when restorative materials are immersed in oral fluid, there may be degradation process, because there are separation of particles in the restoration between the matrix which will contribute to the mechanical and physical properties, one of which is the surface roughness.4 composite roughness can also be caused by the effects of acid produced by bacteria, as a result, it can damage composite base materials (bis-gma) and the significant reduction of the composite mechanical properties after 30 days of storage in artificial saliva with acidic ph.4,5 alkaline environment can increase the dissolution of barium filler particle, exfoliation of resin, and destroy resin structural surface.6 therefore, it is needed to know the influence of acidic and alkaline of artificial saliva towards the surface roughness of hybrid composite resin. this research is aimed to analyze whether the hybrid composite resin have greater roughness when immersed in the artificial saliva with low ph than that with high ph. the results of this research is to prove and provide information to dentists about the changes of surface roughness of the hybrid composite resin cast after immersed in the artificial saliva with low ph (acidic) and with high ph (alkaline). materials and methods this research is considered as a laboratory experimental research with post-test design conducted only for control groups. the total samples is 18 samples, and were devided into three groups 6 samples each. group i is hybrid composite immersed in artificial saliva with ph 4 for 30 days; group ii is hybrid composite immersed in the artificial saliva with ph 7 for 30 days; group iii is hybrid composite immersed in the artificial saliva with ph 10 for 30 days. the samples are hybrid composite resin tablet with 5 mm in diameter and 3 mm in height. the samples were made of plastic rings with 5 mm diameter and 3 mm in height. next, by using the tube hybrid composite was inserted into the plastic rings about 1.5 mm (half of the plastic rings) and light cured for 40 seconds. after polymerized, the rings were filled again, and acetate celluloid strip was put on the top of it in order to make the surface of the composites fl at and smooth. afterwards, the irradiation was conducted for 24 hours. the samples were stored in erlenmeyer tubes containing artifi cial saliva (ph 7) in a room at ± 25° c for 30 days. after 24 hours these samples were divided into 3 groups according to the ph of each, and then stored in a room at 25° c. then, the samples were removed with tweezers, and dried with tissue paper and dryers for one minute.4 the composites were fixed with paraffin, and their surface roughness measured areas were then marked. afterwards, unit surftest was applied on driving shaft and drive unit, and the tip of the needle was placed on the composites that would be measured. after the set meter indication was green, the tool was run with 2000x magnification on the graph. then, the graph result could be calculated. the data was analyzed using anova test. results the results of this research concerning on the effects of acid and alkali of artifi cial saliva on the surface roughness of the hybrid composite resin can be seen in table 1. the normality test was conducted on the results using one-sample kolmogorov-smirnov test. the test result showed normal distribution. next, variance homogeneity test was conducted in those three groups. the result then indicated the above data was homogeneous, (p>0.05). afterwards, one-way anova statistical test with α=0.05 was conducted in order to determine the differences among the groups. the result showed signifi cant difference, (p=0.01), between those treatment groups soaked in various ph of artifi cial saliva for 30 days. it indicates that the 30-day immersion of hybrid composites affects the composite 65pribadi: the effects of the different ph table 1. the average of the surface roughness of the hybrid composite resin cast that has been soaked in the artificial saliva with ph 4, 7, 10 (in μ mm) ph n x 4 6 3.5 7 6 1.25 10 6 2.15 note: ph: the acidic concentration of the artificial saliva; x: the average; n: the number of samples table 2. the result of lsd test to compare the differences of the surface roughness among the treatment groups with various ph of artificial saliva ph significant level 4 and 7 0.003* 4 and 10 0.058 7 and 10 0.175 note: *) there is significant difference, p < 0.05 surface roughness. after that, lsd test was conducted to compare the differences of the surface roughness among those sample groups. and, the result then shows that there was signifi cant difference between ph 4 and ph 7, meanwhile there was no signifi cant difference between ph 4 and ph 10 and between ph 7 and ph 10 (table 2). discussion currently, resin composites are more widely used than amalgam and glass ionomer cast materials since they can aesthetically restore the teeth. besides that, the composite cast materials have already been used for anterior and posterior tooth fi llings for years.5 though the superior composite is aesthetically stronger than glass ionomer, this composite is susceptible to low ph (acid). in the saliva with low ph (acid), the composite is susceptible to damage as the increasing of erosion on polimer.5 some factors, often causing both the damages of the composite resin materials and the changes of the ph of saliva in the oral cavity, are eating and tooth brushing patterns. it can be indicated by the fact that low ph used to soak the composite can increase erosion on polymers. it is also known that water can cause degradation of physical properties of composites by entering polymer chains through porosity and inter-molecular space, so the bond of the polymer chains gets hydrolysis. the separation of the polymer chains by molecules which do not form principal chemical bond chains can cause the reducing of physical properties of the composites, such as diametric pulling power.9 catalyst is a substance that can accelerate the reaction rate at a certain temperature without changing or being used by the reaction itself. in other words, catalyst can have a role in reaction, but not as a reactant or product. the catalyst can also either make the reaction faster or allow the reaction at lower temperatures due to changes triggered on the reagent by providing an optional pathway with lower activation energy, thus, it can reduce the energy required for the reaction progress.7 however, the effect of ph of the saliva on the composite surface roughness is only as catalyst, so ph does not react with the saliva or composite, but affects reaction rates. the separation process of bonding at low ph is caused by the heterogeneous erosion of poly or lactic acid that causes anti catalyst. the results of the formation of monomer is carboxylic acid which can accelerate polymer degradation by lowering the ph, so the acidity is influenced not only from outside, but also from inside. the degradation process occurs in the components of tegdma or bis-gma causing the breaking of polymer chains into monomer ones, so it can reduce the physical power of the materials. the effects of alkaline medium on the composite materials can be described through the interaction with oh-ions during the hydrolysis process. actually, alkaline ph can give more million times of hydroxyl ions than in the saliva with neutral ph. besides the possibility of breaking ties, there is also the possibility of the hydrolysis occurrence in filler.1 the use of composite resins cast with hybrid type is because this type is not only more oftenly used for both anterior and posterior casts, but it also has better physical, mechanical and aesthetic properties than those in conventional ones (macrofilled and microfilled).11 surface roughness was measured in this research since it is the important mechanical properties in many dental restorative materials. restorative materials degraded due to the influence of ph changes in the oral cavity can cause rougher surface. this roughness can not only cause the atmosphere become acidic, but can also make the degradation progress faster. the use of low ph (acidic) in this research is because the acidic ph is considered to be the lowest ph found mostly in plague.3 the composites resin immersed in the artificial saliva with low ph are rougher than that with high ph due to the influence of ph as a catalyst which character in high ph is better than that in low ph. low ph can accelerate the hydrolysis reaction of the composite to be faster than the high ph, while the surface of the cast becomes rougher than immersed in the high ph. it can be concluded that the change of salivary ph can affect the surface roughness of the hybrid composite resin. acidic ph increase on the surface roughness, whereas the alkaline ph has no effect on the surface roughness of hybrid composite resin. references 1. igor m. diametral tensile strength and vickers hardness of composite after storage in different solutions. j oral sci 2007; 49(1): 61–6. 66 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 63–66 2. gedik r, hurmuzlu f, akisi c, beletas oo, ozdemir ak. surface roughnes of new microhibrid resin-based composites. jada 2005; 136: 1106. 3. prakki, anuradha, north rv. influence of ph environment on polymer based dental material properties. elsevier journal of dentistry 2005; 33: 91–8. 4. svanberg m, mjor ia, orstavik d. mutans streptococci in plaque from margins of amalgam, composite, and glass-ionomer restorations. j dent 1990; 21: 861–4. 5. kidd e. leeds dental institute. available at: www.dentistry.leeds. ac.uk/. accessed may, 2008. 6. agha-hosseini f, mirzaii di, amirkhani s. the composition of unstimulated whole saliva of healthy dental students. journal of contemporary dental practice 2006; 7: 1–5. 7. soetojo a. kekuataan kompresi bahan semen glass ionomer modifikasi resin setelah perendaman didalam saliva buatan. maj ked gigi (dent j) 2000; 33(1): 9–13. 8. baum l, phillips rw, lund mr. 1995. buku ajar ilmu konservasi gigi. edisi 3. tarigan r, editor. jakarta: penerbit egc; 1997. p. 253–65. 9. gopferich aa. mechanisms of polymer degradation and erosion. biomaterials 1996; 4: 103–14. 10. somterre jp, shaji lbw, levng. relation of dental composite formulations to their degradation and the release of hydrolyzed polymeric-resin-derived products. toronto: university of toronto; 2001. p. 136–50. 11. craig rg, powers jm, watacha jc. dental material properties and manipulation. 8th ed. st. louis, toronto, london, phidelphia, sydney: cv mosby company; 2002. p. 60, 66–9. 12. hatrich ed, eakle ws, bird wf. dental material: clinical applications for dental assistants and dental hygienis. philadelphia: wb saunders; 2003. p. 62–73. 164 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 164–169 research report effect of caffeine in chocolate (theobroma cacao) on the alveolar bone mineral density in guinea pigs (cavia cobaya) with orthodontic tooth movement bramita beta arnanda, sri suparwitri and pinandi sri pudyani department of orthodontics, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: the benefits of chocolate have attracted significant attention from clinicians, especially the active compound of caffeine on bone metabolism. the bone density significantly affected the rate of tooth movement. purpose: this study aims to analyse the effect of the dose and the duration of caffeine consumption in chocolate on alveolar bone mineral density in orthodontic tooth movement. methods: forty-eight male guinea pigs (cavia cobaya) aged between 3-4 months and weighing 300-350 grams were divided into four groups (group a control, group b caffeine dose of 2.3 mg, group c caffeine dose of 3.45 mg, and group d caffeine dose of 4.6 mg). an open coil spring was applied to the mandibular inter-incisor with an orthodontic force of 35 grams. guinea pigs were sacrificed using lethal doses of anaesthetics on days 0, 1, 7, and 14 after an orthodontic appliance installation. mandibular alveolar bone mineral density in compression sites was analysed with an atomic absorption spectrophotometer (aas). experiment data results were analysed using two-way anova with a 95% degree of confidence. results: caffeine consumption with a dose of 4.6 mg on day 7 had the lowest alveolar bone mineral density and the highest was at a dose of 2.3 mg on day 14, but there were no differences between the dose groups, the duration groups and interactions between both of them (p>0.05). conclusion: the consumption of caffeine in chocolate did not decrease the bone mineral density in the compression site of orthodontic tooth movement. keywords: orthodontic tooth movement, caffeine in chocolate, alveolar bone mineral density correspondence: bramita beta arnanda, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta i, sekip utara, yogyakarta 55281, indonesia. email: bramita.beta.a@mail.ugm.ac.id introduction orthodontic treatment is one of the treatments in the field of dentistry that aims to obtain good dental structure and occlusal contact so that facial occlusion and aesthetic functions can be achieved as well as stable treatment results.1 however, orthodontic treatment periods are generally completed within a long time period since orthodontic devices are installed, which make it difficult to maintain oral hygiene, thus patients are more prone to caries and periodontal disease.2 recently, natural materials have been used, developed, and produced for treatment purposes, one of which is to accelerate the movement of teeth when consuming caffeine in chocolate.3 orthodontic tooth movement is influenced by many factors, one of them being bone density.4 bone density is produced by the deposition of calcium and phosphorus, which act as hydroxyapatite during the bone mineralization process.5 bone density can be analysed in the laboratory by measuring bone calcium levels.6 during orthodontic tooth movement, bone mineral density is regulated by osteoblasts and osteoclasts cells through interactions between the receptor activator of nuclear factor-ligand (rankl), receptor activator of nuclear factor-кb (rank), and osteoprotegerin (opg). osteoclasts play a role in alveolar bone surface resorption by forming an acidic environment that causes bone demineralization and collagen matrix degradation, while osteoblasts play dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 mailto:bramita.beta.a@mail.ugm.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 165arnanda et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 164–169 a role in bone formation through collagen secretion and glycoprotein to form osteoids.7 osteoclasts are regulated by two major cytokines, namely macrophage colonystimulating factor (m-csf) and rankl produced by osteoblasts that bind to osteoclasts receptors, namely c-fms and rank.8 chocolate (theobroma cacao) is a processed cocoa product that has a diverse mixture of chemical compounds. chocolate generally contains 55% fat, 17% carbohydrates, 11% protein, and the rest are tannins. the amount of caffeine in chocolate varies by the percentage of cocoa it contains, with 100% cocoa chocolate (unsweetened chocolate) containing around 240 mg caffeine/100g, 55% cocoa (bittersweet chocolate) containing 124 mg caffeine/100g, and 33% cocoa (milk chocolate) containing 45 mg caffeine/100g.9 however, the effect of caffeine on bone metabolism is still being debated. the potential impact of caffeine on bone is its ability to increase calcium excretion, although in several studies using experimental animals it has not demonstrated a definitive effect of caffeine on bone.10 the biochemical role of caffeine is known to influence the process of bone apposition and resorption which is the basis of orthodontic tooth movement.11 alhasyimi and rosyida,3 stated that giving chocolate during the active period of orthodontic treatment has been shown to increase rankl expression and decrease opg on the compressed side that causes the acceleration of orthodontic tooth movement. this is due to the biochemical mechanism of the methylxanthine content, which is caffeine in chocolate, that can induce the release of rankl by osteoblasts through the increased production of cyclic adenosine monophosphate (camp) so that there is an increase in the number of osteoclasts that will absorb bone.3 the effect of caffeine on bone metabolism depends on the dose consumed. an in vitro study demonstrated that by giving caffeine at a dose of 0.005-0.1 mm for 24 hours can increase osteoclast cell differentiation through increased rankl but does not affect the viability and differentiation of osteoblasts. an in vivo study of male guinea pigs with a caffeine administration of 22 mg/day (0.1%) and 44 mg/ day (0.2%) for 20 weeks showed a decrease in bone mineral density through increased osteoclastogenesis.12 research conducted by lacerda et al.10 with an administration of caffeine at a dose equivalent to 240 ml/day in humans for 42 days showed an increase in urine calcium levels and a decrease in bone mineral density. according to the u.s. food and drug administration (fda), the allowable dose of caffeine is 100-200 mg/day, whereas according to sni 01-7152-2006 the maximum limit of caffeine in food and drink is 150 mg/day or 50 mg/serving.13,14 decreased bone mineral density after caffeine consumption is known to be temporary. patients who have high bone density such as hypoparathyroidism patients, athletes, and women with early menarche and who use oral contraceptives containing estrogen can utilize caffeine as an intermittent therapy during orthodontic treatment to restore the balance of bone resorption and apposition processes.15–17 based on the data that has been described, we felt the need to conduct research on the effect of caffeine on bone mineral density in relation to the evidence that caffeine consumption in chocolate can increase the osteoclast activity that plays a role in bone matrix resorption and the dissolution of mineral content in the alveolar bone on the compressed side, so it is expected to be used to accelerate the rate of orthodontic tooth movement. this study aims to analyse the effects of the dose and the duration of caffeine consumption from chocolate on alveolar bone mineral density in orthodontic tooth movement. materials and methods all research procedures involving animals were performed according to in vivo experimental guidelines. an ethics permit was obtained from the research ethics commission of the faculty of dentistry, universitas gadjah mada with the number 00320/kkep/fkg-ugm/ec/2019. the experimental animal study was conducted at the pharmacology and clinical pharmacy laboratory, faculty of pharmacy, universitas gadjah mada with 48 male guinea pigs weighing between 300-350 grams. acclimatization was performed for one week to allow for their adaptation to shelter and food before being given treatment. pellet-shaped food (pollard) was given to minimize the likelihood of the bracket breaking. a drink for guinea pigs was given ad libitum. the temperature of the guinea pigs’ maintenance room was 25oc with 50% humidity. the guinea pigs’ lighting was 12 hours off (to simulate a night-like condition in the cage) and 12 hours on (to simulate a day-like condition in the cage).3 the guinea pig cages were made of pvc with a size of 60 x 50 x 40 cm. the caffeine dose used was based on a fda recommended safe dose of 100-200 mg/day, which converted to a guinea pig dose resulted in a caffeine level of 2.3 mg in 1.37 grams of chocolate; 3.45 mg in 2.05 grams of chocolate, and 4.6 mg in 2.74 grams of chocolate (hershey’s natural cocoa unsweetened, usa). experimental animals were randomly divided into 4 groups, each consisting of 12 animals which included a group a (control); a group b (caffeine dose of 2.3 mg); a group c (caffeine dose of 3.45 mg), and a group d (caffeine dose of 4.6 mg). these groups were then randomly divided into 4 subgroups according to the day of observation, i.e. days 0, 1, 7, and 14 after orthodontic mechanical induction. each subgroup consisted of three male guinea pigs. the number of samples was determined based on the federer formula. the installation of orthodontic devices was performed firstly by administering ketamine anaesthesia with a dose of 50 mg/kg bw (kepro, netherland) and xylazine with a dose of 5 mg/kg bw (xyla, netherland) intramuscularly on the lower thigh. the separator installation was carried out between the two mandibular incisors, followed by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 166 arnanda et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 164–169 etching on the labial surface of the tooth and a 0.022 inch single wing straight roth mini bracket (marquis™, ortho technology®, usa) and a 0.016 inch stainless steel round wire and nickel-titanium open coil spring (american orthodontics, usa) with a length of 1.5 times the interbracket distance paired between the brackets. the activated open coil spring provided a 35-gram strength measured using a tension gauge (medkraft orthodontics, usa) and was not reactivated after observing the 7th day (figure 1). the consumption of caffeine in chocolate was conducted from the beginning of the installation of the orthodontic devices for up to 14 days, when each cocoa powder was dissolved in 3 ml of distilled water and given orally using a gastric tube twice a day in divided doses.18 the guinea pigs were sacrificed with lethal doses of anaesthesia. dissection was performed on the right lower alveolar bone until the distal of the right lower incisor and the root tip of the tooth could be removed. the samples that had been obtained were then calcined at 700oc for 24 hours aimed at removing the bone organic matrix and dissolving minerals, then dissolved in concentrated hno3 at 80oc and diluted to obtain concentrations of 1-5 ppm. the samples obtained were analysed using the atomic absorption spectrophotometer (aas) (perkin elmer 3110, usa) with a wavelength of 422.7 nm and a lamp current of 10 ma which was calculated in percentage units (%).19 the data obtained were presented as mean ± standard deviation. the data were subjected to a test of normality and homogeneity and then were analysed using the two-way anova test with a significance value of 95%. statistical analysis was processed with the spss 22.0 software system (ibm, chicago, illinois, usa). results the results of the measurement of the alveolar mandibular bone mineral density of the compressed side in guinea pigs are shown in table 1, which showed that the lowest right compressed side of alveolar bone mineral density occurred in the caffeine 4.6 mg dose group with a duration of caffeine consumption for 7 days. the highest of the alveolar bone mineral density of the right compressed side was seen in the treatment group with a dose of 2.3 mg of caffeine and a duration of caffeine consumption in chocolate for 14 days. the alveolar mandibular bone mineral density of the right compressed side in the control group appeared to increase on day 1 and decreased on days 7 to 14, inversely proportional to the treatment group with a dose of caffeine consumption in chocolate 2.3 mg which experienced a decrease on the 1st day and gradually increase from the 7th to 14th days. the changes in the right compressed side of table 1. the mean values and standard deviations (sd) of the right compressed side of alveolar mandibular bone mineral from guinea pigs in orthodontic tooth movement in groups a, b, c and d group mean ± sd (%) day 0 day 1 day 7 day 14 a 25.36 ± 0.78 27.39 ± 3.44 26.01 ± 1.49 24.92 ± 0.79 b 26.15 ± 0.16 23.39 ± 193 26.68 ± 1.45 27.88 ± 1.15 c 26.27 ± 2.99 23.77 ± 3.45 26.80 ± 0.96 25.35 ± 0.52 d 25.01 ± 3.25 25.84 ± 1.43 22.95 ± 1.35 25.29 ± 2.48 3 2 1 figure 1. installation of an open coil spring with a length of 1.5 times the distance of the lower incisors interbracket. (1) archwire 0.016” ss, (2) open coil spring, (3) power o. 0 5 10 15 20 25 30 35 h0 h1 h7 h14 alveolar bone mineral density (%) kelompok a kelompok b kelompok c kelompok dgroup a group b group c group d day 0 day 1 day 7 day 14 figure 2. alveolar bone mineral density changes in the compressed side of the mandible during the guinea pigs’ orthodontic tooth movement. note: a : control group (distilled water); b: treatment group with a caffeine dose of 2.3 mg; c: treatment group with a caffeine dose of 3.45 mg; d: treatment group with a caffeine dose of 4.6 mg dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 167arnanda et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 164–169 alveolar mandibular bone mineral in the treatment group with a caffeine dose of 3.45 mg on days 0 to 7 days were the same as the caffeine dose group of 2.3 mg but decreased on the 14th day. the changes in the alveolar bone mineral density in the treatment group with a caffeine dose of 4.6 mg from day 0 to day 7 were the same as in the control group but increased on day 14 (figure 2). the research data were then analysed using the two-way anova test to determine differences between groups of doses, between groups of duration and interactions between groups of doses, and doses of caffeine consumption in chocolate. the two-way anova test results (table 2) showed that there was no significant difference in the right compressed side of alveolar mandibular bone mineral density between group a (without caffeine consumption in chocolate), group b (caffeine dose of 2.3 mg), group c (caffeine dose of 3.45 mg), and group d (caffeine dose of 4.6 mg). it also showed no significant difference in the mineral density of the right compressed side of alveolar mandibular bone between 0, 1, 7, and 14 days of observation and no significant difference in interaction between the dose group and the duration of caffeine consumption in chocolate (p> 0.05). discussion the results of this study on the effect of the dose and the duration of caffeine consumption in chocolate on the mineral density of the right compressed side of alveolar mandibular bone showed a decrease in calcium levels in the bones in the control group starting from day 1 to day 14. this indicates that there has been remodeling of the alveolar bone that was dominated by bone resorption. this is in line with research conducted by wang et al.7 in mice using a closed coil spring which shows a decrease in bone mineral density on days 3 to 14 and returns to normal on day 28. a decrease in bone mineral density in the control group is likely due to the mechanical induction of orthodontics to stimulate osteoblasts to release rankl and m-csf which then binds to c-fms and rank on osteoclast precursors so that osteoclast differentiation and activation occurs for bone resorption.8 alveolar bone mineral density in the caffeine consumption group in chocolate at a good dose of 2.3 mg, 3.45 mg, and 4.6 mg appears to vary or fluctuate. this is probably caused by differences in the systemic conditions of experimental animals used in the study. systemic conditions, especially in the liver, affect the process of caffeine metabolism in the body. caffeine metabolism in the liver is determined by the cytochrome p450 1a2 (cyp1a2) enzyme which varies by subject. subjects with high cyp1a2 enzyme levels have a fast caffeine metabolic rate so that it has a small effect on the body. conversely, low cyp1a2 enzyme levels will reduce the metabolic rate and maintain caffeine concentration in the body longer so that it will have a significant effect on body tissues.20 the ups and downs phenomenon of alveolar bone mineral density also correspond to the yerkes-dodson caffeine activity curve. rozenek et al.21 stated that in the yerkes-dodson curve, the effect of caffeine has an optimum limit to achieve efficient performance which means that at certain doses of caffeine the effect will increase at a certain point, then the effect will again decrease and these dynamics are influenced by the sensitivity of the subject to caffeine which is determined by genetic factors and the quality of health and the rate of metabolism of caffeine in the body. a decrease in the right-sided lower jaw alveolar bone mineral density showed no significant differences between the dose groups, between the duration and interaction of doses, and the duration of caffeine consumption in chocolate. this indicates that the consumption of caffeine in chocolate does not cause a decrease in the mineral density of the alveolar bone in the orthodontic movement of guinea pig teeth. the effect of caffeine on bone metabolism is still being debated. the potential impact of caffeine on the bone is its ability to increase calcium excretion in urine which causes a decrease in bone calcium levels, but several studies using experimental animals have not demonstrated the definitive influence of caffeine on bone. this is in line with research conducted by yi et al.22 which stated that alveolar bone mineral density in the caffeine group at a dose of 25 mg/kg bw and controls induced by orthodontic strength were not significant differences. this is due to the presence of a number of confounding factors, including differences in dosage, the duration of consumption, the method of administration and type, and the age range of experimental animals used.22 according to research conducted by hallstrom,23 caffeine has a biphasic dose-dependent effect. an increased excretion of calcium in the urine only occurs over a short duration (initial acute rise) in the consumption of caffeine with a low concentration of 0.005-0.1 mm (equivalent to 0.97-19.4 mg). then caffeine will quickly induce differentiation osteogenic from primary adipose-derived stem cells and bone marrow stromal cells, thereby returning calcium levels to baseline. the effect of caffeine on bones also depends on the concentration of the caffeine metabolite, namely paraxanthine. the concentration of caffeine consumed is relatively the same as the amount table 2. the anova test results of two pathways affecting the dose and the duration of caffeine consumption in chocolate on the mineral density of the depressed alveolar mandibular bone on the orthodontic tooth movement of guinea pigs variables f p value dose 0.892 0.456 duration 0.314 0.815 dose*duration 1.921 0.086 note: the difference is not significant if p> 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 168 arnanda et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 164–169 of paraxanthine concentration in the body, so with a low dose of caffeine the concentration of paraxanthine is also low.11,12,23 gavrieli et al.24 stated that the effect or potential of caffeine on the body is proportional to the subject’s body weight and height, which means that the greater the body weight, the dose of caffeine needs to be increased. the results of the study were insignificant, possibly due to not weighing the subject so that the caffeine dose in chocolate that was given did not follow the weight gain of experimental animals during the study causing a decrease in the potential for caffeine in alveolar bone tissue. in line with research by hallstrom23 and yi et al.22 a significant decrease in alveolar bone mineral density is likely caused by the source of caffeine used in this study of chocolate. the caffeine content in 5 grams of hershey’s chocolate powder is 8.4 mg which is lower than the caffeine content in coffee or tea, so that the effect of caffeine on decreasing bone calcium levels is not optimal. in addition, the route or method of administration can also affect the amount of active concentration of caffeine on the body’s physiological response.25 the oral administration of gavage tends to have a slow onset of therapeutic effect, causing the caffeine potential to decrease due to metabolic processes in the gastrointestinal tract and liver. the insignificant research results may also be caused by the difficulty of controlling the type of tooth movement during the administration of orthodontic force induction. according to kirschneck et al.26 experimental studies on experimental animals tend to occur primarily with tipping movements and to a lesser degree bodily tooth movement that causes uneven distribution of pressure in the periodontal tissue. this results in differences in biological responses to the strength of orthodontics given. research by ahn et al.27 stated that in the movement of tipping, there is a greater accumulation of pressure in the alveolar crest region so that more resorption occurs in the area. yu et al.28 added that the decrease in alveolar bone mineral density is greater in the cervical region (alveolar crest) than in the apical region. in line with the study of ahn et al.27 and yu et al.28 an insignificant decrease in bone mineral density is likely caused by alveolar bone used as samples are alveolar bone that covers the cervical to apical regions so that calcium levels dissolved by osteoclast activity are only modest because osteoclast activity is less dominant in the intermediates and apical regions. based on research conducted by alihasyimi and rosyida,3 consumption of caffeine in chocolate 2.7 mg (hershey’s, usa) in male wistar mice can accelerate tooth movement through increased rankl expression and decrease opg on days 0, 1, and 7 on side stress. this indicates that the dose and the duration of the study used is 2.3 mg to 4.6 mg for 14 days safe for consumption and provides a positive impact or correlation because it can accelerate the orthodontic movement of guinea pigs but does not cause decreased alveolar bone mineral density. based on the results of the study, it can be concluded that the consumption of caffeine in chocolate did not decrease in the bone mineral density of the lower jaw alveolar bone in guinea pigs (cavia cobaya) with orthodontic tooth movement. acknowledgement this research is fully supported by the hibah penelitian dana masyarakat grant, faculty of dentistry, universitas gadjah mada, republic of indonesia, for the fiscal year 2019 (contract no. 4319/un1/fkg1/set.kg1/pt/2019). references 1. ardhana w. identifikasi perawatan ortodontik spesialistik dan umum. maj kedokt gigi indones. 2013; 20: 1–8. 2. yi j, zhang l, yan b, yang l, li y, zhao z. drinking coffee may help accelerate orthodontic tooth movement. dent hypotheses. 2012; 3(2): 72–5. 3. alhasyimi aa, rosyida nf. cocoa administration may accelerate orthodontic tooth movement by inducing osteoclastogenesis in rats. iran j basic med sci. 2019; 22(2): 206–10. 4. graber l, vanarsdall r, vig k, huang g. orthodontics: current principles and techniques. 6th ed. philadelphia: mosby; 2017. p. 133. 5. kranioti ef, bonicelli a, garcía-donas jg. bone-mineral density: clinical significance, methods of quantification and forensic applications. res reports forensic med sci. 2019; 9: 9–21. 6. song l. calcium and bone metabolism indices. adv clin chem. 2017; 82: 1–46. 7. wang c, cao l, yang c, fan y. a novel method to quantify longitudinal orthodontic bone changes with in vivo micro-ct data. j healthc eng. 2018; 2018: 1–8. 8. kim jh, kim n. signaling pathways in osteoclast differentiation. chonnam med j. 2016; 52: 12–7. 9. temple jl, bernard c, lipshultz se, czachor jd, westphal ja, mestre ma. the safety of ingested caffeine: a comprehensive review. front psychiatry. 2017; 8: 80. 10. lacerda sa, matuoka ri, macedo rm, petenusci so, campos aa, brentegani lg. bone quality associated with daily intake of coffee: a biochemical, radiographic and histometric study. braz dent j. 2010; 21(3): 199–204. 11. shirazi m, vaziri h, salari b, motahhari p, etemad-moghadam s, dehpour ar. the effect of caffeine on orthodontic tooth movement in rats. iran j basic med sci. 2017; 20(3): 260–4. 12. liu sh, chen c, yang r sen, yen yp, yang yt, tsai c. caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats. j orthop res. 2011; 29(6): 954–60. 13. fahmi arwangga a, raka astiti asih ia, sudiarta iw. analisis kandungan kafein pada kopi di desa sesaot narmada menggunakan spektrofotometri uv-vis. j kim. 2016; 10: 110–4. 14. food and drug administration. highly concentrated caffeine in dietary supplements: guidance for industry. us dep heal hum serv. 2018; (april): 1–8. 15. rubin mr, bilezikian jp. hypoparathyroidism: clinical features, skeletal microstructure and parathyroid hormone replacement. arq bras endocrinol metabol. 2010; 54(2): 220–6. 16. national institutes of health. osteoporosis: peak bone mass in women. nih osteoporos relat bone dis natl resour cent. 2015; (juni): 1–2. 17. kacprzak a, strzecki a. methods of accelerating orthodontic tooth movement: a review of contemporary literature. dent med probl. 2018; 55(2): 197–206. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 169arnanda et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 164–169 18. herniyati, narmada ib, devi ls. caffeine increases pge2 levels at compression and tension areas during orthodontic tooth movement. int j chem. 2018; 11(6): 177–82. 19. figueiredo m, cunha s, martins g, freitas j, judas f, figueiredo h. influence of hydrochloric acid concentration on the demineralization of cortical bone. chem eng res des. 2011; 89: 116–24. 20. langer jw. genetics, metabolism and individual responses to caffeine. coffee heal inst sci inf coffee. 2018; : 1–13. 21. rozenek eb, górska m, wilczyńska k, waszkiewicz n. in search of optimal psychoactivation: stimulants as cognitive performance enhancers. arh hig rada toksikol. 2019; 70(3): 150–9. 22. yi j, yan b, li m, wang y, zheng w, li y, zhao z. caffeine may enhance orthodontic tooth movement through increasing osteoclastogenesis induced by periodontal ligament cells under compression. arch oral biol. 2016; 64: 51–60. 23. hallström h, wolk a, glynn a, michaëlsson k. coffee, tea and caffeine consumption in relation to osteoporotic fracture risk in a cohort of swedish women. osteoporos int. 2006; 17(7): 1055–64. 24. gavrieli a, karfopoulou e, kardatou e, spyreli e, fragopoulou e, mantzoros cs, yannakoulia m. effect of different amounts of coffee on dietary intake and appetite of normal-weight and overweight/ obese individuals. obesity. 2013; 21(6): 1127–32. 25. hines rm, khumnark m, macphail b, hines dj. administration of micronized caffeine using a novel oral delivery film results in rapid absorption and electroencephalogram suppression. front pharmacol. 2019; 10: 983. 26. kirschneck c, bauer m, gubernator j, proff p, schröder a. comparative assessment of mouse models for experimental orthodontic tooth movement. sci rep. 2020; 10: 12154. 27. ahn hw, moon sc, baek sh. morphometric evaluation of changes in the alveolar bone and roots of the maxillary anterior teeth before and after en masse retraction using cone-beam computed tomography. angle orthod. 2013; 83(2): 212–21. 28. yu jh, huang hl, liu cf, wu j, li yf, tsai mt, hsu jt. does orthodontic treatment affect the alveolar bone density? med (united states). 2016; 95(10): 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p164–169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p164-169 isi vol 39 no 2 april 2006 file pertama.pmd 63 sensitization of the sphenopalatine ganglion (spg) by periodontal inflammation: a possible etiology of sinusitis and headache in children haryono utomo dental clinic faculty of dentistry airlangga university surabaya indonesia abstract sinusitis is a frequent complication of allergic rhinitis. theoretically, sinusitis could be found in human since infancy. the prevalence of diagnosed sinusitis is 20% of ambulatory patients in daily practice. unfortunately, a lot of sinusitis cases must be treated by surgical operation. other cases are treated conservatively with decongestants, corticosteroids, antibiotics and diathermy. however, dental treatment approach for sinusitis management is rarely discussed. headache, especially migraine is also a common problem in children. sinusitis and migraine, are closely related; sinusitis sufferers often accompanied by migraine and vice versa. this phenomenon resulting in misdiagnosis of the main etiology of sinusitis and migraine; if this case happens in young children, the diagnosis should be more complicated. dental procedures which may directly reduce the periodontal inflammation were done to children diagnosed as sinusitis by otolaryngologist and pediatrician. in a short period of time, the sinusitis and headache symptoms subsided. the objective of this case reports is to propose the possible explanation of the neurogenic switching mechanism cut off, that resulting in the instant relief of sinusitis and headache symptoms. regarding the immediate relief of the symptoms, the role of autonomic nervous system should also be considered. since parasympathetic innervations of nasal, sinus mucosa and maxillary periodontal tissues originated from the sphenopalatine ganglion; the conclusion is that the periodontal inflammation may sensitize the sphenopalatine ganglion which may trigger sinusitis and headache in children. key words: sphenopalatine ganglion, periodontal inflammation, sinusitis, headache correspondence: haryono utomo, c/o: fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id. telp. +6231 5053195. introduction sinusitis is an inflammation involving the mucous membrane of one or more paranasal sinuses, accompanied by symptoms i.e. nasal congestion, facial pain; and thick and purulent nasal discharge. it is a relative common problem in daily practice; in the usa (1995), 13.4–25 million visits to medical practice were related to sinusitis and/or their subsequent effects.1 most sinusitis are caused by viral infection, and recovered without antibiotic therapy,1 which is coincident to reported studies that a proportion of sinusitis patients had a negative culture.2 conservative treatments of sinusitis include decongestants, corticosteroids, antibiotics and diathermy. if conservative treatments failed; surgical procedures had to be done to treat sinusitis.1 unfortunately, dental treatment procedure is not included in the conservative treatment of sinusitis. the most common headache in children is migraine without aura. it may not be hemicranial in children and is less intense compared with the migraine in adults.2,3 parasympathetic activation,4 as well as the hypothesized mechanism of neurogenic or immunogenic switching (i.e. crossover interactions of neurogenic and immunogenic inflammation), may account for both the frequent occurrence of nasal symptoms in migraine; and the possibility that sinus inflammation can sometimes act as migraine trigger.3 the sphenopalatine ganglion (spg) is related to the autonomic nervous system; the parasympathetic and sympathetic nerves. stimulation of the parasympathetic nerves lead to nasal congestion which also called “stuffy nose”. recently, the role of spg as a treatment target of rhinitis, sinusitis or migraine symptoms; such as blocking the spg with injections,5,6 or nasal spray forms, had been popularized.6 however, the role of dental and periodontal inflammation in spg sensitization is rarely discussed. previous case reports which related to headache7 and allergic rhinitis in children,8,9 revealed the possibility of oral inflammation as a trigger of headache and rhinitis. nevertheless, the proposed mechanism limited only by immunological mechanism; the possibility of neurogenic mechanism involvement was not apparently discussed. the objective of this case report is to propose a new concept in propagation of oral inflammation through the 64 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 63–67 neurogenic switching mechanism involving the periodontal tissues. sensitization of the spg by periodontal inflammation may induced rhinitis, sinusitis and headache. cases case 1: a 7 years old boy, suffering from sinusitis and diagnosed as maxillary sinusitis by an otolaryngologist, came to a private dental practice. at that time he had already suffered for more than 3 years. when he was 4 years old, he had already treated by another otolaryngologist. the diagnostic of maxillary sinusitis was confirmed with water’s projection radiograph. treatment which done by the first otolaryngologist, was medication and diathermy for more than 10 times. the result was insignificant, he still suffered from sinusitis symptoms including headache. he also became temperamental and had an impaired hearing which caused speaking difficulty of some consonants sound, especially “s”. approximately three years later, he consulted another otolaryngologist and was also diagnosed as sinusitis, which confirmed by water’s projection radiograph. at that time, he was also given medication and diathermy for 10 times. these treatments gave satisfying results, most of the symptoms disappeared. however, when the weather was humid, the severe symptoms reappeared. he was also told to study music to improve his impaired hearing, and he had done his best in music playing, especially the violin. extraorally, the patient appeared fatigue, and there was a small amount of thin nasal discharge in the nostrils. intraoral inspection showed moderate dental plaque in every region, abundant dental plaque was seen in the upper posterior regions. inflamed gingival was also seen in several regions, especially the 16 55 and 65 26, on which pseudopockets were detected and contained impacted food. the patient had no caries or filling. case 2: a 7 years old girl, came to a private dental practice as an orthodontic patient. at that time, she had already suffered from sinusitis for about 3 years as diagnosed by an otolaryngologist and a general practitioner. the diagnosed was confirmed with water’s projection radiograph. she had been treated with a lot of medications, and diathermy for more than 40 times without significant improvement. her mother said that the latest medications given by a general practitioner were helpful, but if it were stopped the symptoms recurred. extraorally, the patient looked normal, except a small amount of watery discharge came out from her nostrils. intraoral inspection showed moderate dental plaque in every region. inflamed gingival also seen in several regions, especially the 16 55, on which pseudopocket was detected. the patient had no caries or filling. case management oral plaque control therapy was done with rotating brush, pumice and contra-angled handpiece. the interdental spaces were irrigated with hexetidine 0.1%; after about 1 minute, the interdental spaces and pseudopockets were searched for food remnants or debris using a sickle shaped explorer. dark red blood oozed from the interdental spaces and pseudopockets, especially in the upper left and right posterior teeth. approximately one minute later, the patients could breathe easily through his/her nostrils. the parents said that before the dental procedures, despite many medications and diathermy, the patients could not breathe easily through the nose. the patients and their parents were taught how to conduct oral plaque control procedures and were prescribed hexetidine 0.1% mouthwash. the patients were scheduled for another visit in a week time. on the second visit; extraorally, the patients looked more cheerful. the parents said that all the symptoms related to sinusitis (i.e. nasal congestion, headache) had already disappeared, they only had watery (thin) discharge. especially in the first case, watery discharge was found came out from the right nostril; the intraoral inspection coincidentally found that the gingiva in the buccal area of 16 55 was still inflamed and bleed easily. the interdental and pseudopockets exploration procedures were done until dark red blood did not oozed anymore. in the second case, intraorally, the gingiva had regained its normal color and consistency; and the sinusitis symptoms were disappeared. the latest evaluations were in april 2006, 6 months after the first case and 2 years after the second case management. severe sinusitis symptoms did not exist, only occasional sneezing and rhinorrhea. discussion sinusitis may affect everyone since infancy, since the maxillary sinuses have already developed in the third month of fetal life, followed by the ethmoid sinuses.1 as sinusitis mostly accompanied by migraine or vice versa,3 diagnosis of the main etiology could be difficult . consequently, it will become more difficult if happen to infants or young children. there are several symptoms which resulting in misdiagnosis of sinusitis and migraine. the migraine symptoms are facial pain, facial flushing, lacrimation, rhinorrhea, nasal congestion and vertigo,10 whereas sinusitis symptoms are nasal congestion, facial pain, malaise2 and thick and purulent nasal discharge.3,4 in addition, several mechanisms were also proposed related to the interrelationship between sinusitis and migraine, that are: autonomic symptoms caused by parasympathetic cranial 65utomo: sensitization of the sphenopalatine ganglion (spg) activation,10 and neurogenic and immunogenic switching mechanism.3 while acute sinusitis is widely accepted and recognized as a cause of headache, chronic sinusitis or sinonasal abnormalities as a cause of headache has been more controversial. chronic sinusitis is not validated as a cause of headache unless relapsing into an acute phase.4 migraine and tension-type headache are often confused with true sinus headache because of “similarity in location”.4 on the other hand, migraine may cause autonomic symptoms mimicking sinus problem (i.e. nasal congestion and rhinorrhea) is proposed through the cranial parasympathetic activation by sensitized trigeminal nucleus caudalis (figure 1).10 hypothesis consists of the crossover mechanism between immunogenic and neurogenic inflammation.3,13 neurogenic and immunogenic switching can be explained as follows: afferent sensory nerve fibers can be stimulated by histamine and tryptase from degranulated mast cell, proinflammatory cytokines secreted from lps stimulated macrophages, bradykinin, and nitric oxide (no). subsequently, stimulated afferent nerve fibers release neuropeptides that are substance p (sp) and calcitonin generelated peptide (cgrp) which in turn stimulate mast cells.13 mast cells degranulation induced by antigens, bacteria, proteoglycans, lps, chemokines, and physical factors (i.e. cold temperatures, ultraviolet b radiation, exercise).14 these neurogenic switching mechanism is a vicious circle until something “cut off” the process. considering the connection of sinusitis and migraine, much attention has been given to the sinuses and nose as a source of acute and chronic head and face pain. it is therefore worth a brief review of sinonasal innervation. the general sensory innervation of the mucosa of the nose and paranasal sinuses is from the ophthalmic (v1) and maxillary branches (v2) of the trigeminal nerve, with minor contributions from the greater superficial petrosal branch of the n vii (facial nerve) (figure 2).4,15 some researches found that sensitization of the spg had already proposed to trigger rhinitis, sinusitis, asthma, headache and other ailments. it maybe related to the anatomical characteristics of spg which contains sensory, parasympathetic and sympathetic nerves.5,15 sagittal dissection illustration of the nose and its adjacent tissues shows the parasympathetic innervations of nasal mucosa and sinus passages by the spg (figure 2).15 the spg has sensory innervation by v2 which connected by fine fibers; and the sphenopalatine nerve. the collaterals of v2 are able to store sp in the ganglion.5 the parasympathetic nerves originated from the vagus nerve and the superior cervicalis ganglion. it has either a secreto-motor function to the nasal and sinuses, or soft and hard palate.5 activation of the spg by v2 which releases neuropeptides (i.e.sp, cgrp) may cause the inflammation of the neighboring artery and mucosa. inflamed nasal mucosa resulting in nasal congestion, sinusitis; and migraine.16 maxillary periodontal tissues are innervated by the sensory nerve fibers through the v2; the parasympathetic nerve fibers through the spg, and sympathetic nerve fibers through the superior cervical ganglion.13 from this point of view, it seems possible that oral inflammation which involving the periodontal tissues, in some instances could elicit sinusitis and migraine. the correlation between the spg, sinusitis and migraine also proved by blocking the spg with xylocaine and cortisone-type injections to relieve allergic rhinitis and sinusitis,5,6 injection could be done through the greater palatine foramen. the others are nasal applicator/ transnasal pain in migraine is believed elicited by the activation of trigeminovascular structures which manifests as headache.4 nevertheless, the pathogenesis of pain in migraine was not completely understood, but three factors merit consideration: 1) the cranial blood vessels, 2) the trigeminal innervation of vessel, and 3) the reflex connection of the trigeminal system with the cranial parasympathetic outflow.11 nasal congestion is the most common symptom in sinusitis, which associated with turbinate dysfunction. the etiology of turbinate dysfunction is multifactorial. because the turbinate have a very rich blood supply and are governed by the parasympathetic nervous system, anything that affects either of these 2 systems affects the turbinates and hence, the nose.12 the autonomic nervous system provides the general innervation to the nose, with the parasympathetic nerves supplying the resting tone and controlling secretions. the nerve supply originates from the n vii (facial nerve) at the inferior salivatory nucleus and follows along the distribution of the n vii through the spg.12 according to cady and schreiber, the connection between sinusitis and migraine should be related to the neurogenic and immunogenic switching hypothesis.3 this figure 1. autonomic symptoms by cranial parasympathetic activation in migraine.10 66 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 63–67 approach of medicaments (i.e. essential oil),5 or the application of nasal spray forms of naratriptan (antimigraine).4 the possible correlation of oral inflammation and allergic rhinitis which caused by dental plaque had been discussed in several literatures.8,9 since rhinitis, sinusitis and migraine related mostly to the neurogenic and immunogenic switching mechanism; the oral plaque control therapy which mostly related to the immunogenic inflammation should take several hours to give a significant effect. a plausible explanation to this mechanism was that after the sudden fall of bacteria population; the available immune response could by itself reduces the inflammation by gradually eliminate the infectious agents, neutralizes the toxins and secretes anti-inflammatory mediators. subsequently, the resolution of immunogenic inflammation also reduced the neurogenic inflammation. in this case report, the patients were selected as caries free and had no filling. it was meant to exclude the interference of pulpal inflammation which may also triggers the v2 afferent sensory nerves. this study was limited only on the possibility of mucosal and periodontal sensory or parasympathetical nerves induction to the spg, which acted through the neurogenic switching mechanism. instant relief of the symptoms (i.e. reduced nasal congestion and headache) that only took several minutes to give a significant effect is proposed to be the “assisted drainage” procedure to the chronic inflamed tissues. blood which oozed after the exploration of the pseudopockets using sickle-shaped explorer should contained bacteria, toxins (proteoglycans, pgn and lipopolysaccharides, lps), pro-inflammatory mediators and enzymes,17 which may induced neurogenic inflammation of the maxillary nerve (v2) or the parasympathetic nerves of maxillary periodontal tissues. rapid decrease of toxins and pro-inflammatory mediators level may cause instant resolution of inflammation,18 and the neurogenic switching mechanism “cut off”. it may indirectly diminish the sensitization the parasympathetic nerves of the spg by v2, or directly from the sensitized maxillary periodontal parasympathetic nerve fibers, thus decreasing the nasal congestion and migraine. the remarkable result of treatment procedures in this case report was similar with the previous article about headache in children.7 this “assisted drainage” is a simple and effective procedure in sinusitis and headache management in children, which are suspected to be induced by periodontal inflammation. in order to minimize unnecessary or prolonged treatments and medications for the sinusitis and headache in children; the collaboration of general practitioners, otolaryngologist, paediatrician and dental practitioners is important. continuing dental health maintenance by proper brushing, using mouthrinses and flossing should be taught to the patients and their parents for obtaining maximal results. regular dental check-up should be done to maintain optimum oral health. the sphenopalatine ganglion is the target of recent sinusitis and migraine treatments. concerning to the direct effect of “assisted drainage” of the chronic inflamed pseudopockets, which resulted in the immediate disappearing of sinusitis and headache symptoms; it is concluded that periodontal inflammation is able to sensitize the spg which eventually may elicit sinusitis and headache. references 1. widodo ak. rinosinusitis: etiologi dan patofisiologi. pendidikan berkelanjutan iv ilmu kesehatan telinga hidung tenggorok. 1–3. 2. behrman re, kliegman rm, jenson hb. nelson textbook of pediatrics. 17th ed. philadelphia: saunders; 2004. p. 760, 1832. 3. cady rk, schreiber cp. sinus headache or migraine. neurology 2002; 58:s10–4. 4. stroud rh, bailey bj, quinn fb. headache and facial pain. dr. quinn’s online textbook of otolaryngology grand rounds archive. 2001. available online at url. http://www.utmb.edu/otoref/grnds/ ha-facial-pain-2001-0131/ha-facial-pain-2001. doc. accessed march 20, 2006. 5. klinghardt dk. the sphenopalatine ganglion (spg) and environmental sensitivity. lecture on 23rd annual international symposium on man and his environment. june 9–12, 2005. dallas texas. 6. mirkin g. chronic stuffy nose, nasal polyps and fungus. available inline at url http://www. drmirkin.com. accessed april 15, 2006. 7. pradopo s, utomo h. nyeri kepala pada anak.. majalah kedokteran gigi (dent j) 2005; edisi khusus pertemuan ilmiah nasional ilmu kedokteran gigi anak i: 60–66. 8. utomo h, soehardjo i. hubungan respons imun dan kesehatan rongga mulut dengan rinitis alergika pada anak. majalah kedokteran gigi (dent j) 2005; edisi khusus timnas iv:69–76. 9. utomo h, setijanto d. apakah terapi pengendalian plak gigi dapat menurunkan keparahan rinitis alergika pada anak. majalah kedokteran gigi (dent j) 2005; 38(2):96–102. 10. green mw. diagnosing and treating migraine: low tech diagnosis, high tech treatment. available online at url http://www.amaassn.org/ama1/pub/ upload/ mm/31/24pres-green.pdf. accessed february 20, 2006. 11. goadsby pj, lipton rb, ferrari md. migraine-current understanding and treatment. n eng j med 2002; 346(4):257–70. 12. archer sm. turbinate dysfunction. emedicine. cm/ent/ reconstructive–sv. accessed april 10, 2006. figure 2. sagittal dissection of the nose.15 sphenopalatine ganglion 67utomo: sensitization of the sphenopalatine ganglion (spg) 13. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 14. walsh lj. mast cells and oral inflammation. crit rev oral biol med 2003; 14(3):188–98. 15. gray’s anatomy of the human body. the trigeminal nerve. available online at url. http://education.yahoo.com/reference/gray. accessed march 15, 2006. 16. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http:// www.migraineprevention.com/index/html. accessed february 15, 2006. 17. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microb rev 2000; 13(4):547–58. 18. rabson a, roitt im, delves pj. really essential medical immunology. 2nd ed. oxford: blackwell pub; 2005. p. 91, 116–7. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 51 no 2 apr-jun 2018.indd 71 behavior management-based applied behaviour analysis within dental examination of children with autism spectrum disorder taufiqi hidayatullah,1 hendriati agustiani,2 and arlette suzy setiawan3 1faculty of dentistry, universitas syiah kuala, banda aceh indonesia 2faculty of psychology, universitas padjadjaran, bandung indonesia 3department of paediatric dentistry, faculty of dentistry, universitas padjadjaran, bandung indonesia abstract background: autism spectrum disorder (asd) is a developmental disorder with three main characteristics: communication disorders, social interaction disorders and repetitive behavior. the main problem faced when treating child patients with this disorder is the difficulty of establishing communication with the result that they are unable to understand instructions. one form of therapy frequently applied in cases of autism is that of applied behavior analysis (aba). it is easier for children with asd to absorb information visually. purpose: the purpose of this study was to evaluate the effectiveness of aba-based behavior management using visual media in the form of picture cards for oral examination of children with asd. methods: the study design was observational descriptive in nature and the sample selection was based on purposive sampling. the study was conducted by observing changes in childrens’ behavior during treatment administered four times a month. the subjects were 13 children with asd who met the study criteria and were receiving treatment for autism at prananda special school in bandung. the collated data related to changes in subjects’ behavior observed during four meetings assessed on the basis of score 1 confirming compliance with instructions and 0 indicating non-compliance. a kruskal-wallis statistical analisysis test was used to analyze the data. results: the results showed a general increase in the former over the latter initial behavior during treatment. statistical analysis showed that the coefficient of kruskal-wallis was meaningful in terms of statistical significance with a p-value of 26.947 (<0.05). the multiple comparison value for average ranks was 15.68 (sd 18.69). conclusion: the conclusion of this study is that the application of a behavior management-based analytical methodology is effective in supporting the oral examination of children with asd. keywords: autism spectrum disorder, applied behavior analysis, behavior management correspondence: arlette suzy setiawan, department of paediatric dentistry, faculty of dentistry, universitas padjadjaran, jl. raya bandung sumedang km. 21, jatinangor 45363, indonesia. e-mail: arlettesuzy@yahoo.com dental journal (majalah kedokteran gigi) 2018 june; 51(2): 71–75 research report introduction autism is defined as a developmental disorder featuring the three main characteristics of impaired communication, social interaction disorder and repetitive behavior. studies on autism reveal it to be a disorder with a broad spectrum of symptoms. autistic children possess unique and diverse characteristics and exhibit individual symptoms with varying degrees of severity in terms of both quality and quantity. autism is now often referred to as asd.1,2 autistic spectrum disorder is one of the fastest growing forms of developmental disability. the number of cases continues to increase every year. in 2012, the data center for disease control and prevention (cdc) has reported the increasing incidence of autism. the average prevalence of individuals identified with asd in asia, europe and north america is between 1% and 2%.3,4 a 2013 literature review by putri highlighted that there are approximately 6900 children suffering from asd in indonesia with the number expected to increase annually. putri also cited several researchers who conclude the number of asd people in the country to have increased over the last 20 to 30 years. nevertheless, in indonesia there has been no further detailed study, with the result that the number of people with asd cannot be definitively ascertained.4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p71–75 mailto:arlettesuzy@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p71-75 72hidayatullah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 71–75 a key area of challenge when dealing with sufferers of asd is to identify a means of communicating effectively in order that information can be successfully conveyed. communication problems with individuals affected by asd are often accompanied by restricted intellectual capacity, uncontrolled body movements, hyperactivity, limited attention span and a propensity to become easily frustrated or angry and to self-harm when something takes them out of their comfort zone.5,6 children with asd need treatment for their overall well-being as well as their oral and dental health. caries and inadequate oral hygiene represent common problems. a sufferer of asd, like any child, should enjoy access to dental care. communication barriers constitute a major problem to be resolved by dentists. a child afflicted by asd is generally difficult and unable to understand direct instructions or accept strangers present in the vicinity. modified communication is necessary if such children are to prove cooperative. children with asd experience significant difficulties in comprehending oral information effectively. their understanding of the environment is based more on body language cues or alternative forms of non-verbal communication. many studies contained in the literature on the subject reveal that children with asd process visual information more effectively. at present, numerous educational strategies have been developed for children with asd through the use of visual media as a means of interaction.1,3,7,8 one common method of the educational processes or therapies employed with children suffering from asd is applied behavior analysis (aba). aba is a branch of psychology whose application focuses on the analysis and modification of human behavior.6 aba-based procedures have been accepted by the american academy of paediatrics dentistry (aapd) as a form of behavioral management of children with limited communication. such procedures have the potential to improve the results of conventional behavior management practices. the approach is expected to support asd children in behaving more appropriately so that they can receive enhanced dental and oral care treatment. dentists can minimize the need for interventional behavioral management such as restraint, sedation and care delivered under general anesthesia.7 the application of aba can employ various tools. for example, visual media is suitable for promoting the learning of children with asd. one simple form of visual media is a picture card created as required which can help dentists to communicate with children suffering from asd. appropriate methods will support the successful use of these media, while moulding the child's behavior to better receive dental and oral care procedures.5–7 the purpose of this study was to analyze the effectiveness of aba-based management methods using image cards within behavior-based management of children with asd during dental and oral examinations. materials and methods the research population of this study comprised children diagnosed with autism spectrum disorder who attended sekolah prananda, a special school in bandung. diagnoses of the condition were based on dsm v and ranged in severity from mild to moderate (level 1 and level 2) encompassing individuals who still require assistance with certain activities, but who have started to follow instructions and do not display extreme behaviour such as uncontrolled tantrums. the study was conducted between early july 2015 and the end of august 2015. in this research, determining the sample was performed by a combination of non-probability sampling and purposive sampling. the design of this study constituted an observational descriptive case study type, being an intensive review of an individual/group of individuals deemed to fall within a particular case. the research employed a single subject approach design. such methodology is used to conduct in-depth exploration of specific events, the focus being on a small number of events investigated in depth with a set time span. in addition, it also focuses on individual data as samples.9 single subject research is used to highlight or change the behavior of individuals with an important issue as the result of an intervention.10,11 treatment was conducted four times at one-week intervals for a month. subjects were treated using an ababased method with the aid of picture cards and assessed after each treatment session. parents and teachers were asked to fill in a questionnaire about the child's daily behavior prior to treatment, including: the parent and child profile, the child's medical/systemic condition, the child's ability to execute daily activities (e.g. going to the toilet, using cutlery and brushing his/her teeth), the child's reaction to being given assistance such as having his/her hair and nails cut and questions about his/her general everyday behavior. questions to the teacher covered the child’s asd level, severity of intellectual disability, daily behavior at school, conditions potentially triggering aggressive behavior, conditions conducive to maintaining concentration during therapy, objects or conditions that make him/her content and suitable learning media for the child to internalise information or follow instructions. the teacher was also asked about characteristics of the child’s behavior such as the ability to maintain eye contact, self-mutilation tendencies, pica and the frequency of tantrums. a day prior to treatment, the teacher prepared the child by showing a picture card related to the following day’s treatment. four people were present in the treatment room, namely: operators, assistant operators, teachers and children. before each treatment session, the teacher and the operator showed the child image cards depicting its successive steps. every time the child succeeded in following instructions, a verbal praise was given in the form of his/her favorite complements interspersed with personalized rewards identified from the feedback provided through the parents dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p71-75 73 hidayatullah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 71–75 and teacher questionnaires. if a child refused to follow instructions the reward was withheld. the instructions were repeated until the child proved successful in executing the task. failure to follow instructions resulted in his/her not proceeding to the next stage. the duration of treatment was limited to 15 minutes per meeting. if the child's attention was suddenly distracted during treatment, his/her focus was restored by removing the cause of the distraction, repeating instructions and giving direction as required. during a particular session, successfully completed stages were repeated before the child proceeded to the next stage. observation was conducted of those stages determined by the indicator. in each session, activities which according to the indicators were considered appropriate to the child’s developmental stage were graded as follows: increase in level of engagement = 1; no change or the child is unwilling to enter the room = 0. the stages of instruction amounted to 10 sequential criteria. all stages were worth 1 except the first which was rated at 0. the various stages were as follows; first, the child did not want to enter the room/was not yet calm (0); second, the child was calm but distracted when in the room (1); third, the child was able to sit calmly in the room (1); fourth, the child was willing to shake hands with operator and assistant (1); fifth, the child sat in a chair and opened his/her mouth (1); sixth, the child sat in a chair, opened his mouth and was willing to be examined with a dental mirror (1); seventh, the child sat in a chair, opened his mouth and was willing to be examined with an explorer (1); eighth, the child was willing to have his/her teeth smeared with disclosing agent (1); ninth, the child accepted the cleaning of his/her teeth using cotton/cotton roll (10; and tenth, the child was able to brush his/her teeth and gargle (1). the lowest value for each treatment was 0 and the highest was 9. any increase, decrease or lack of persistence of behavioral stages was observed during the intervals between each treatment session. results the research population consisted of children being treated for asd at prananda special school in bandung. based on the number of available samples, research subjects were selected, according to the following predefined criteria: subjects were still actively receiving therapy at prananda special school for autism in bandung, subjects had never undergone a routine dental check up, and subjects suffered from category 1 and 2 (mild and moderate) autism spectrum disorder as defined by the dsm v diagnostic criteria. the number of children receiving therapy at the school was 27. nine were absent due to temporary leave, while five did not meet the inclusion and exclusion criteria. consequently, the study sample consisted of 13 children divided into 11 boys and two girls ranging in age from 5 to 18 and with a median of 2-3 years. generally, they received autistic therapy shortly after a diagnosis of asd. table 1 contains the score/value of the children recorded at each meeting. the results suggest that there was an increased value in their behavior. two children were able to complete all stages of behavior before the final stage of treatment. at that stage, the child was prepared to sit in a chair, open his/her mouth and be examined with an oral mirror. in general, the stage of behavior that can be completed by children in this study is the willingness to open their mouths without the use of tools. table 1. scores relating to respondents' behavior stages during four consultations fourth consultationthird consultationsecond consultationfirst consultationno. respondent 53311 54312 86543 44314 43315 33316 96317 65538 54439 995410 543111 543312 543113 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p71-75 74hidayatullah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 71–75 during the first meeting, all the children succeeded in entering the room. eight children recorded an unreliable response because their attention was distracted. three children were able to sit quietly in the room, shake hands with the operators and assistants, while the other two, in addition to being able to sit quietly and shake hands also proved capable of sitting in a dental chair and calmly open their mouths without the use of a tool. during the second meeting, nine children were able to sit quietly in the room and shake hands with the operators and assistants. one child was able to sit quietly and was willing to open his/her mouth without tools, while another three children voluntarily opened their mouths to enable examination with tools. the result of the second meeting was an improvement in behavior in 12 children with only one child not improving or demonstrating worsening behavior. the third meeting confirmed six children as being capable of following the instruction to open their mouths without tools. one child opened his/her mouth and was examined with an oral mirror, while another two children opened their mouths and were checked using an explorer. one child proved able to complete this behavioral stage, but three children merely wanted to shake hands with the operators and assistants. the result of the third meeting was an improvement in behavior in eight children, five other children proved to be at the same stage of behavior as during the second meeting. the fourth meeting revealed that two children were able to complete all stages including one who was able to undergo all stages during the third meeting. one child was already willing to accept a disclosing agent and clean his teeth with cotton, although not to brush them. two children wanted to merely sit with their mouths open. one child would open his/her mouth and was examined using the explorer, while another simply wanted to play with it. six children proved able to complete the five stages of opening their mouths and being examined with an oral mirror. the conclusion of the last meeting was an improvement in behavioral stages in 11 children including one who was able to complete all stages during the third meeting. however, there were two children who still demonstrated no improvement in the behavioral stages. statistical analysis in the form of a kruskal-wallis test (table 2) was used to test the effectiveness of aba-based behavioral management using picture cards of dental and oral examination of asd children. a kruskal-wallis test is a rank-based nonparametric test whose purpose is to determine whether there are statistically significant differences between two or more independent variable groups on numerical and ordinal scaled dependent variables. a statistical test conformed a statistically significant kruskal-wallis coefficient with chi-square value = 26.947 and p-value (6.04e-06) <0.01. the conclusion reached was that the application of the aba method using effective or statistically significant static cards proved successful in managing behavior during oral and dental examination in children with asd. discussion the main focus of this study was to apply aba methods that are often utilised during behavioral therapy of children with asd and to combine them with visual media in the form of picture cards. the study aimed to establish the effectiveness of the method applied in relation to the child's cooperative level during oral and dental examinations. the results showed its effectiveness in boosting the child's level of cooperation as evident from the increase in behavioral stages that can be completed by his/her following instructions at each consultation. the application of an aba-based method in this study used visual media in the form of picture cards to enhance the effectiveness of asd child behavior during dental and oral examination. according to the literature on the subject, visual media represent one of the most suitable forms of media for the treatment of children with asd who respond positively when shown picture cards. the cards shown to the child each time with instructions are useful when he/she becomes distracted or anxious. at times during a study, researchers also demonstrated how to receive such treatment. for example, when instructing a subject to open his/her mouth, the researchers gave an example of how to do so. choirunissa emphasized that table 2. kruskal-wallis test analysis for establishing the effectiveness of aba-based behavior management using a picture card in comparison to dental and mouth examination of children with asd avg. rankmedian n w111.19131.00 w223.08133.00 w331.92134.00 w439.81135.00 total524.00 h (corrected for ties)26.947 d.f.3 6.04e-06 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p71-75 75 hidayatullah, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 71–75 body language is also a form of visual support.3 children who have begun to communicate verbally are generally not so affected by the cards because they already understand instructions. a child will better understand when given a direct example of body language and the use of tools is explained directly. nevertheless, the average asd child is incapable of effective verbal communication. during the study, the behavior of children at all levels can vary with each meeting, being cooperative on one occasion yet uncooperative on others. the conclusions of this study show that visual media support in the form of an effective picture card supports the application of aba principles when performing dental and mouth examination of child with asd. this is because generally such individuals readily receive visual instruction. it is suggested that further research of longer duration needs to be undertaken involving a larger number of research subjects with more diverse characteristics. in addition, the type of visual media in electronic media can be tested on children with asd, given that technology has developed sufficiently rapidly that dentists can apply it in one form of media. references 1. riandini s. pengaruh pola pengasuhan dengan perkembangan komunikasi anak autis kepada orang tua. med j lampung univ. 2015; 4(8): 99–106. 2. udhya j, varadharaja mm, parthiban j, srinivasan i. autism disorder (ad): an updated review for paediatric dentists. j clin diagn res. 2014; 8(2): 275–9. 3. nirahma cp, yuniar ic. metode dukungan visual pada pembelajaran anak dengan autisme. j psikol klin dan kesehat ment. 2012; 1(2): 1–8. 4. putri spnmdaa, astrini a. persepsi orang tua tentang pola attachment anak dengan autism disorders di klinik “x” jakarta barat. thesis. jakarta: binus university; 2013. p. 1-11. 5. dewi r. peran orangtua terhadap terapi biomedis untuk anak autis. thesis. depok: gunadarma university; 2009. p. 1-20. 6. astutik ip. penerapan metode aba (applied behaviour analysis) dengan media kartu bergambar dan benda tiruan secara simultan untuk meningkatkan pengenalan angka pada siswa kelas ii di sdlb autis harmony surakarta tahun pelajaran 2009/2010. thesis. surakarta: universitas sebelas maret; 2010. p. 1-56. 7. hernandez p, ikkanda z. applied behavior analysis: behavior management of children with autism spectrum disorders in dental environments. j am dent assoc. 2011; 142(3): 281–7. 8. morisaki i, ochiai tt, akiyama s, murakami j, friedman cs. behaviour guidance in dentistry for patients with autism spectrum disorder using a structured visual guide. j disabil oral heal. 2008; 9(3): 136–40. 9. delli k, reichart pa, bornstein mm, livas c. management of children with autism spectrum disorder in the dental setting: concerns, behavioural approaches and recommendations. med oral patol oral cir bucal. 2013; 18(6): e862–8. 10. marienzi r. meningkatkan kemampuan mengenal konsep angka melalui metode multisensori bagi anak autis. j ilm pendidik khusus. 2012; 1(3): 320–31. 11. margaretha sepm. efektifitas video self modelling terhadap kemampuan menggosok gigi pada anak dengan autisme spectrum disorders di karesidenan banyumas. thesis. depok: universitas indonesia; 2012. p. 1-100. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p71–75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p71-75 8080 dental journal (majalah kedokteran gigi) 2023 june; 56(2):80–86 original article the relationship between tooth loss and pre-elderly nutritional status and quality of life widiya ulfa1, rosa amalia2, al supartinah santoso3 1master of dentistry study program, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of preventive and community dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 3department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: tooth loss leads to decreased masticatory function, which affects nutritional intake. tooth loss accompanied by poor nutritional status affects quality of life both physically and psychosocially. purpose: this study was conducted to identify the relationship between number of teeth lost due to nutritional status and quality of life. methods: a study conducted at gulai bancah primary health care in bukittinggi. participants were recruited using a non-probability purposive sampling technique. 178 pre-elderlies aged between 45 and 59 years took part in this study. tooth loss was reported in the dental examination format while nutritional status and quality of life pertaining to dental and oral health were determined using the mini nutritional assessment (mna) and the geriatric oral health assessment index (gohai). pearson correlation and path analysis were utilized to analyze the data. results: the pearson correlation test indicated a moderate correlation between tooth loss and nutritional status (r = -0.549) and a strong correlation between the number tooth lost and quality of life (r = -0.742). pathway analysis confirmed that the direct effect value (beta coefficient = 0.552) was greater than the indirect effect value (beta coefficient = 0.189). the findings indicated that, indirectly, the amount of tooth loss mediated with nutritional status had no significant relationship to quality of life. conclusion: an increase in tooth loss leads to pre-elderly poor nutritional status and quality of life. however, nutritional status is not an intermediate factor between the number of teeth lost and pre-elderly quality of life. keywords: nutritional status; oral health related quality of life; pre-elderly; tooth loss article history: received 27 july 2022; revised 2 oktober 2022; accepted 8 november 2022 correspondence: widiya ulfa, faculty of dentistry, universitas gadjah mada. jl denta sekip utara, sinduadi, mlati, sleman, special region of yogyakarta 55281, indonesia. email: widiyaulfa@mail.ugm.ac.id introduction increases in life expectancy are the proof of successful health developments; however, this poses separate challenges. one challenge that occurs is that as people age, they will face declining physical condition. in the pre-elderly population, degenerative processes have already begun, both physiologically and biologically. the aging process is a cumulative change in the body that decreases functional capacity. the regenerative ability of the preelderly is limited, meaning that they are more susceptible to various diseases. if no prevention efforts to provide health services are carried out properly then there is a decline in body functions.1,2 tooth loss is one of the oral health problems that occurs in the pre-elderly period. tooth loss experienced by the pre-elderly due to the aging processes is associated with the changes of oral cavity tissue structure, which are stipulated by complex factors such as caries, periodontitis, and trauma.3 in 2018, the prevalence of tooth loss in west sumatra in adults between the ages of 45–54 years was 23.98%. specifically in bukittinggi town, the statistic reported was 19.68% and the prevalence of low nutritional status was 9.56%.4 the function of the oral cavity will be optimal if there is a sufficient amount of occlusion of the teeth or there are dentures that function as replacements for missing teeth.5 pre-elderly who experience significant tooth loss not supported by a sufficient number of dental occlusions will have difficulty chewing food, which can affect the quality of life. they will also experience difficulty in the mastication process, which will cause a negative impact copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 mailto:widiyaulfa@mail.ugm.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 81 ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 on the functional limitation scale on related quality of life dental and oral health. the impact on nutritional risk related to weight conditions will also interfere with psychosocial quality of life and cause discomfort.6 an adequate number of teeth can be expected to support the functions of the oral cavity, with the minimum number being 20 teeth. one previous study indicated that missing 16 or more teeth as a risk factor for malnutrition.7,8 tooth loss leads to decreased masticatory function, thereby reducing an individual’s ability to digest solid and fibrous foods including meat, nuts and, fruits, which interferes with the mechanism of the nutritional absorption process. this alters the body’s metabolism, which affects nutritional intake.9,10 the mini nutritional assessment (mna) is a clinical assessment tool for grading nutrient status and assessing the malnutrition threat in senior cases without necessitating a dietician or nutritionist. the mna is a well-validated technique with high perceptivity, particularity, and trustability.11 it has been cross-validated in a multicenter study and is grounded on anthropometric measures, a global assessment of general health status, a salutary questionnaire, and a subjective assessment of health and nutrition confines. it can also give veritably useful information for patients by indicating the factors that contribute to their altered nutrient status. advancements in mna scores have been observed after interventions have been performed.12,13 the geriatric oral health assessment index (gohai) is a quality-of-life instrument related to dental and oral health that measures quality of life from the individual’s perceptions, including including physical, psychosocial, and comfort dimensions. affected physical quality of life includes chewing difficulties while social dimensions include communication disturbance. the gohai is a 12point assessment primarily developed by atchinson and dolan in 1990 in the usa.14 it covers a range of fields including functional (eating, speaking, and swallowing), psychological (concerns about oral health, dissatisfaction with appearance, tone-conscious about oral health, and avoidance of social connection because of oral problems), and pain or discomfort (medicines, gingival perceptivity, teeth perceptiveness, and discomfort when biting certain foods). the gohai has been restated and validated in many languages including persian, chinese, arabic, german, and indonesian.15,16 tooth loss, especially anterior, affects psychosocial dimensions and comfort due to aesthetic and personal appearance deviations, whereas posterior tooth loss affects functional aspects that interfere with the masticatory process associated with nutritional status. preventive and promotive activities could be started from the pre-elderly age to ensure that the health, nutrition, and psychosocial condition of the pre-elderly is maintained so that it remains productive.17,18 a preliminary study of the pre-elderly between the ages of 45 and 59 years (accounting for 58% of the population) discovered a tooth loss proportion among 8 out of 10 preelderly patients, which indicated frequent tooth loss in the gulai bancah primary health care working area. this study was conducted to identify the relationship between tooth loss and nutritional status and quality of life, and to illustrate the indirect relationship between tooth loss on the oral health related quality of life (ohrqol) by using nutritional status. materials and methods this study has been approved by the research ethics commission of the faculty of dentistry, universitas gadjah mada with certificate number 0019/kkep/fkgugm/ec/2022. this is a quantitative study with a cross sectional design. the population in this research were pre-elderly patients served in the working area of the gulai bancah primary health care. purposive sampling with a non-probability technique was employed to recruit study participants based on inclusion and exclusion criteria. participants were pre-elderly and aged between 4 and 59 years, according to the world health organization (who),19 willing to participate, able to communicate verbally, and had experienced tooth loss. the exclusion criteria were uncooperative patients and those using dentures or diagnosed with systemic diseases. the variables studied were tooth loss, nutritional status, and ohrqol. the number of teeth lost was reported in the dental examination format. nutritional status was measured using the scores obtained from the mna questionnaire. the scoring is as follows: good nutritional status if the score is 24–30, a risk of malnutrition if the score is 17–23.5, and malnutrition if the score < 17. the mna consists of four assessment components: global health evaluation, food intake assessment, anthropometric assessment, and respondent perception. quality of life related to dental and oral health was appraised using the gohai. this questionnaire was completed for each subject by one investigator through the methodology of surveying and clinical examination. the responses from the participants were recorded on a 5-point likert scale. responses to statements points 3, 5, and 7 were rear-scored. the methodology used in this study was the accretive strategy, which corresponds to casting up the scores attained for each of the 12 gohai questions. the ohrqol of each participant was determined as good if the score was 57–60, average when 51–56, and poor when < 50. bivariate statistical analysis in a form of pearson correlation illustrates the relationship between variables, namely the relationship between the number of teeth lost with nutritional status and quality of life. a multivariate test using the path analysis test was employed in this study. the path analysis illustrated an indirect relationship between tooth loss on the ohrqol by using nutritional status as the mediating variable. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 82ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 results there were 178 participants in this study who were all preelderly patients within the age range of 45–59 years. their characteristics are presented in table 1. the average age of the study participants was 51.6 years and 72% of them were female. the incidence of posterior tooth loss was higher than anterior teeth. a secondary level of education occupied the largest proportion among the participants’ level of education. the data in table 2 presents the number of missing teeth, the average mna score, and gohai index. it is inferred from the table that the highest number of teeth lost was four teeth with an average mna score of 24.5, which indicates a good nutritional status, and a gohai score of 54.2, which indicates a moderate quality of life. participants who had lost six or more teeth were at risk of malnutrition with an average mna score of between 17 and 23.5. pre-elderly patients who had lost seven or more teeth associated with poor quality of life, which was indicated by a gohai score of less than or equal to 50. based on the mna indicators shown in table 3, 24.7% of the study participants experienced weight loss, 22.4% reported a lower food intake, 55% consumed two servings of protein per day, 79.7% did not consume fruit and vegetable daily, 10.6% had a bmi score of < 19, and 58.9% did not know their health status. the data in table 4 presents the gohai score based on functional, psychosocial, and comfort dimensions. the data showed that participants in this study limit their type of food (5.6%), are concerned about their oral cavity (23.5%), and are unable eat comfortably (13.5%). table 1. characteristics of the participants characteristics frequency average±sd n % age 51.6 ± 4.37 gender: male 50 28 female 128 72 tooth loss: anterior and posterior 12 6.7 posterior 166 93.3 education: no education 10 5.6 elementary school 51 28.7 high school 105 59 higher education 12 6.7 table 2. tooth loss distribution number of teeth lost frequency average mna score average gohai score 1 22 27.1±1.5 58.3±1.4 2 28 26.2±2.1 56.9±2.7 3 19 25.5±1.6 55.7±3.6 4 29 24.5±2.4 54.2±4.0 5 23 24.6±2.2 52.8±3.3 6 26 23.5±2.2 50.1±4.0 7 14 22.7±2.3 48.3±3.6 8 9 22.1±1.9 43.4±4.9 9 7 23.8±3.4 47.4±4.8 10 1 19±0 43±0 table 3. respondents’ answers based on the mna questionnaire20 mna questionnaire answer n % global health evaluation: weight loss during in the last three months does not know between 1–3 kg no weight loss 15 44 119 8.4 24.7 66.8 mobility normal 178 100 other disease problems no 178 100 how to eat eat alone 178 100 food intake assessment: has food intake declined over the last three months due to loss of appetite, digestive problems, biting, or swallowing difficulties? severe moderate no decrease 0 40 138 0 22.4 77.5 how many full meals does the patient eat daily? 1 time 2 times 3 times 10 105 63 5.6 58.9 35.3 selected consumption markers for protein intake 1 protein intake 2 protein intakes 3 protein intakes 10 98 70 5.6 55.05 39.3 consumes two or more servings of fruit or vegetables per day? yes no 35 142 19.6 79.7 how much fluid (water, juice, coffee, tea, milk) is consumed per day? < 3 cups 3–5 cups > 5 cups 15 129 34 8.4 72.4 19.1 anthropometric assessment: body mass index < 19 19–21 21–22 23 or more 19 33 50 76 10.6 18.5 28.1 42.6 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 83 ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 mid-arm circumference < 21 21–22 23 5 19 154 2.8 10.6 86.5 calf circumference < 31 31 7 171 3.9 96.1 respondent perception: self-view of nutritional status views self as being malnourished is uncertain of nutritional state views self as having no nutritional problem 9 84 85 5.1 47.1 47.7 in comparison with other people of the same age, how does the patient consider his/her health status? not as good does not know as good better 5 105 63 5 2.8 58.9 35.3 2.8 table 4. participants’ response based on the gohai dimensions not question functional often sometimes seldom never n % n % n % n % 1. limit the kinds or amount of food 10 5.6 24 13.4 34 19.2 110 61.8 2. have trouble chewing food 9 5 30 16.9 36 20.2 103 57.9 3. able to swallow comfortably 0 0 1 0.6 24 13.4 153 86 4. prevention of speaking the way participants wanted 1 0.6 2 1.1 31 17.4 144 80.9 psychosocial 1. limiting self due to oral conditions 0 0 13 7.3 89 50 76 42.7 2. unpleasant with the oral cavity condition 0 0 35 19.7 43 24.2 100 56.1 3. worry 0 0 42 23.5 89 50.1 47 26.4 4. panic due to teeth problems 1 0.6 2 1.1 31 17.4 144 80.9 5. uncomfortable eating in front of people 0 0 13 7.3 105 59 60 33.7 pain/discomfort 1. unable to eat comfortably 0 0 24 13.5 57 32.0 97 54.5 2. use medication to relieve pain or discomfort 0 0 6 3.3 31 17.5 141 79.2 3. teeth or gums sensitive to hot, cold, or sweets 1 0.5 15 8.4 26 14.7 136 76.4 table 5. relationship between tooth loss and dependent variables variable correlation coefficient p value nutritional status (mna) -0.549 0.00 quality of life (gohai) -0.742 0.00 number of teeth lost (x) nutritional status (y) quality of life (z) β=-0.549 β=0.345 β=-0.552 e2= 0.45 e1= 0.83 figure 1. formulation of the path analysis model. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 84ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 bivariate analysis was carried out to determine the pattern and relationship between dependent and independent variables, which were the association between tooth loss and nutritional status, and tooth loss and quality of life. the results of the pearson correlation analysis are provided in table 5. the bivariate analysis confirmed a significant relationship between the number of teeth lost and dependent variables, which includes nutritional status and quality of life, with a significance value of 0.00 (0.00 < 0.05). the mna nutritional status indicated a moderate relationship with r = -0.549. in addition, the quality-of-life variable revealed a strong relationship with r = -0.742. the negative correlation represented higher tooth loss being associated with a worse nutritional status and lower quality of life related to dental and oral health. multivariate analysis was conducted using path analysis. the analysis illustrated an indirect relationship between the independent variables on the dependent variable by using the mediating variable. as the data retrieved in this study has a ratio and interval scale with normal data distributed based on a normality test, the multicollinearity test yielded no strong relationship between independent variables, which was recorded by the tolerance value and variance inflation factor (vif) value. in this test, there was no indication of multicollinearity, which was declared by tolerance > 0.01 and vif < 10. it is inferred that all the assumption tests have met the requirements. this study successfully designed and created a structural equation that represents the problem formulation. the test was carried out to identify the indirect relationship between variables through the mediating variable. figure 1 illustrates a structural model that has been validated. based on the correlation between variables, there was a significant relationship between x and y, x and z, and y and z with p < 0.05. the indirect effect showed that the relationship analysis between the number of teeth lost mediated by nutritional status on quality of life, had a direct effect of x on y with 0.552, while the indirect effect of x mediated by y on z is the beta coefficient of 0.549 x 0.345 = 0.189. the calculation imposed that the direct effect was greater than the indirect effect and interpreted indirectly, tooth loss mediated by nutritional status did not have a significant relationship with patients’ quality of life. discussion this study involved 178 participants who were in the working area of the gulai bancah health center, bukitinggi city, west sumatra. the results of this study indicate that there is a relationship between variables, but there is no indirect relationship between the number of teeth lost and quality of life through the mediating variable of nutritional status. the study showed that there was a relationship between the number of teeth that had been lost and nutritional status as a greater number of lost teeth led to the individual’s nutritional status being lower. the pre-elderly experienced chewing difficulties and were expected to choose types of food and determine the timing of eating. other factors that influenced patient comfort was the food positioning in the oral cavity to the remaining teeth, which also affects nutritional status.21 there are other factors that affect the decreased nutritional status of the pre-elderly. based on the results of the study, it was found that posterior tooth loss (93.3%) had a higher percentage than anterior teeth. posterior tooth loss was more common and results in low nutritional intake. this was evidenced by participants with greater posterior tooth loss associating with having to choose foods types. the first loss of posterior teeth, especially mandibular molars was frequent.22,23 the analysis used to see the relationship between the amount of tooth loss and nutritional status was the pearson correlation, which shows a relationship between the number of lost teeth and nutritional status with a moderate correlation. tapsell et al.24 state that many other factors affect nutritional status including the amount of food intake, diet, variety of food, nutritional content, and how to process food. based on this statement, this moderate correlation indicates that other factors can affect nutritional status. having six or more lost teeth was reported to indicate a risk of malnutrition. however, this study, in contrast with iyota et al.7 which stated the minimum number of teeth for proper function as 20, did not discover any cases of malnutrition. the risk of malnutrition was demonstrated by the mna results. the mna not only presented an objective point of view, but also measured indicators based on the respondent’s perception. this study revealed that the level of tooth loss strongly correlated with quality of life. rocha’s study presented similar findings in that a greater number of lost teeth was linked to lower quality of life, indicated by a poor ohrqol. the loss of posterior teeth, especially mandibular first molars, is very common and causes concern about dental and oral diseases.25 individuals over 40 years of age are also associated with an inability to chew so can be considered to have a fairly poor oral quality of life. this statement is supported by the research that revealed that oral health status influences ohrqol, which can affect the gohai dimensions including functional, psychosocial, and discomfort dimensions.26,27 poor quality of life was revealed to be associated with seven or more lost teeth. this study is incongruent with the findings of khan et al.18, which reported that patients aged between 30 and 45 years with three lost teeth experienced poor quality of life. the findings were associated with psychosocial aspects due to changes in aesthetics. quality of life was evaluated using the gohai, which assessed the participants’ perceived dental and oral health problems. based on the responses, it can be inferred that participants were concerned about their oral cavity. psychosocial is an important aspect in addition to functional dimensions related to the masticatory process. the study copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 85 ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 confirmed that participants have different responses based on their experiences and perceived health conditions.21 it is suggested the number of teeth lost had no significant effect on the quality of life if mediated by nutritional status. nutritional status is not a particular contributor that links the number of teeth lost with quality of life. the findings presented table 3 demonstrated that 6.7% of the participants experienced anterior tooth loss. according to khan et al.18 and haag et al.28, the loss of anterior teeth is correlated with aesthetics and associated with psychosocial aspects. maxillary anterior teeth contributed to a lower ohrqol compared to missing teeth from another quadrant. individuals who had one anterior tooth loss had a lower gohai score compared to respondents who had lost two or three teeth in the posterior region with no further association to nutritional status.18,28 this research acknowledged that the mediating variable of nutritional status had no significant effect. research conducted by yin et al.29 aimed to examine whether eating and communication mediated the loss of dental and functional difficulties among the elderly. the study concluded that eating and communication difficulties served as a mediating role, which infers that communication difficulties can be an indirect factor of tooth loss. the scope of quality of life related to dental and oral health does not only measure functional dimensions related to the masticatory process but also discuss psychosocial aspects and pain/discomfort, therefore perceptions of aesthetic function and phonetic function will also affect quality of life. respondents who have lost several teeth and do not use dentures will have affected perceptions. the research explains that the perception of the elderly who use removable dentures has a good category.30 based on the study, it was found that the psychosocial dimensions and a sense of discomfort also affect respondents’ quality of life. acknowledgments we kindly thank universitas gadjah mada for making this study possible. we also kindly thank all co-workers for their support in the data collection. references 1. washio m, kiyohara c. health issues and care system for the elderly. singapore: springer singapore; 2019. p. 196. (current topics in environmental health and preventive medicine). 2. kholifah sn. keperawatan gerontik: modul bahan ajar cetak keperawatan. jakarta: kementerian kesehatan republik indonesia; 2016. p. 1–105. 3. saskianti t, nugraha ap, prahasanti c, ernawati ds, tanimoto k, riawan w, kanawa m, kawamoto t, fujimoto k. study of alveolar bone remodeling using deciduous tooth stem cells and hydroxyapatite by vascular endothelial growth factor enhancement and inhibition of matrix metalloproteinase-8 expression in vivo. clin cosmet investig dent. 2022; 14: 71–8. 4. badan penelitian dan pengembangan kesehatan. laporan nasional riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 207. 5. tôrres lh do n, da silva dd, neri al, hilgert jb, hugo fn, sousa m da lr de. association between underweight and overweight/ obesity with oral health among independently living brazilian elderly. nutrition. 2013; 29(1): 152–7. 6. de medeiros mmd, pinheiro ma, de figueredo omc, de oliveira lfs, wanderley rl, cavalcanti yw, rodrigues garcia rcm. masticatory function in nursing home residents: correlation with the nutritional status and oral health-related quality of life. j oral rehabil. 2020; 47(12): 1511–20. 7. iyota k, mizutani s, oku s, asao m, futatsuki t, inoue r, imai y, kashiwazaki h. a cross-sectional study of age-related changes in oral function in healthy japanese individuals. int j environ res public health. 2020; 17(4): 1376. 8. angraini di, supartinah a, wachid dn. status kesehatan mulut dan asupan makan sebagai faktor risiko underweight pada lansia. j gizi klin indones. 2013; 9(4): 188–96. 9. kosaka t, kida m. tooth loss leads to reduced nutrient intake in middle-aged and older japanese individuals. environ health prev med. 2019; 24(1): 15. 10. patel p, shivakumar k, patil s, suresh k, kadashetti v. association of oral health-related quality of life and nutritional status among elderly population of satara district, western maharashtra, india. j indian assoc public heal dent. 2015; 13(3): 269–73. 11. khusumawerdanie ek, maulina m. status gizi lansia berdasarkan m in i nut r itiona l assessment ( m na) di pa nti sosia l tresna werdha lhokseumawe aceh utara. j samudera. 2015; 9(2): 29–40. 12. kostecka m, bojanowska m. an evaluation of the nutritional status of elderly with the use of the mna questionnaire and determination of factors contributing to malnutrition. a pilot study. rocz panstw zakl hig. 2021; 72(2): 175–83. 13. machado rsp, coelho masc, veras rp. validity of the portuguese version of the mini nutritional assessment in brazilian elderly. bmc geriatr. 2015; 15: 132. 14. atchison ka, dolan ta. development of the geriatric oral health assessment index. j dent educ. 1990; 54(11): 680–7. 15. gkavela g, kossioni a, lyrakos g, karkazis h, volikas k. oral health related quality of life in older people: preliminary validation of the greek version of the geriatric oral health assessment index (gohai). eur geriatr med. 2015; 6(3): 245–50. 16. gita f, wir yasmoro t, sagala dm, hendr y, hoger vorst e, kusdhany ls. oral health status and oral health-related quality of life in indonesian elderly (analysis using the indonesian version of the oral health assessment tool and the geriatric oral health assessment index). j int dent med res. 2017; 10(special issue): 533–9. 17. batista mj, lawrence hp, de sousa m da lr. impact of tooth loss related to number and position on oral health quality of life among adults. health qual life outcomes. 2014; 12: 165. 18. khan su, ghani f, nazir z. the effect of some missing teeth on a subjects’ oral health related quality of life. pakistan j med sci. 2018; 34(6): 1457–62. 19. pusat data dan informasi kementerian kesehatan republik indonesia. situasi lanjut usia (lansia) di indonesia. 2016. available from: https://pusdatin.kemkes.go.id/article/view/16092300002/ infodatin-situasi-lanjut-usia-lansia-di-indonesia.html. accessed 2020 may 6. 20. nursilmi n, kusharto cm, dwiriani cm. hubungan status gizi dan kesehatan dengan kualitas hidup lansia di dua lokasi berbeda. media kesehat masy indones. 2017; 13(4): 369–79. 21. dable ra, nazirkar gs, singh sb, wasnik pb. assessment of oral health related quality of life among completely edentulous patients in western india by using gohai. j clin diagn res. 2013; 7(9): 2063–7. 22. kikutani t, yoshida m, enoki h, yamashita y, akifusa s, shimazaki y, hirano h, tamura f. relationship between nutrition status and dental occlusion in community-dwelling frail elderly people. geriatr gerontol int. 2013; 13(1): 50–4. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://pusdatin.kemkes.go.id/article/view/16092300002/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 86ulfa et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 80–86 23. perera r, ekanayake l. relationship between nutritional status and tooth loss in an older population from sri lanka. gerodontology. 2012; 29(2): e566-70. 24. tapsell lc, neale ep, satija a, hu fb. foods, nutrients, and dietary patterns: interconnections and implications for dietary guidelines. adv nutr. 2016; 7(3): 445–54. 25. guimarães rocha ekt, vanderlei ad, beder ribeiro cm, de oliveira lima al, dos santos af, trindade filho em. impact of tooth loss on quality of life. pesqui bras odontopediatria clin integr. 2016; 16(1): 69–78. 26. bortoluzzi mc, traebert j, lasta r, da rosa tn, capella dl, presta aa. tooth loss, chewing ability and quality of life. contemp clin dent. 2012; 3(4): 393–7. 27. echeverria ms, wünsch is, langlois co, cascaes am, ribeiro silva ae. oral health-related quality of life in older adults-longitudinal study. gerodontology. 2019; 36(2): 118–24. 28. haag dg, peres kg, brennan ds. tooth loss and general quality of life in dentate adults from southern brazil. qual life res. 2017; 26(10): 2647–57. 29. yin z, yang j, huang c, sun h, wu y. eating and communication difficulties as mediators of the relationship between tooth loss and functional disability in middle-aged and older adults. j dent. 2020; 96: 103331. 30. tulandi jdg, tendean l, siagian kv. persepsi pengguna gigi tiruan lepasan terhadap fungsi estetik dan fonetik di komunitas lansia gereja international full gospel fellowship manado. e-gigi. 2017; 5(2): 1–9. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p80–86 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p80-86 205 vol. 43. no. 4 december 2010 case report a combination of endodontic therapy and root resection in furcation involvement case ernie maduratna setiawati department of periodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: management of furcation involvement is one of major problems in clinical periodontology. the prognosis and treatment choices depend on the degree and severity of the lesion. thus the decision for a specific treatment of furcation-involved tooth certainly depends on several factors. tooth anatomy and the degree of furcation involvement are considered as the most important factors affecting the decision for one or more treatment plans mode. purpose: the aim of this case report is to determine the management of class iv furcation involvement with the combination of endodontic therapy and root resection. case: this study reported a case of 47 years old male patient who came to periodontics clinic with tooth mobility, hypersensitivity and furcation involvement. case management: one of the treatment is a combination of endodontic therapy and distal root resection. the results of these case could help the patient to have a better treatment for his furcation defect. conclusion: combining endodontic treatment and root resection were considered as an appropriate choice for retaining clinically-important trifurcation-involved tooth. root resection had a better prognosis to treat periodontal problems than for non-periodontal problems. however, complex interdisciplinary treatment is important to be performed in the overall treatment plan. key words: furcation involvement, endodontic therapy, root resection abstrak latar belakang: penatalaksanaan furkasi merupakan salah satu masalah di bidang periodontik. rencana perawatan dan prognosis tergantung pada derajat keparahan furkasi. untuk menentukan perawatan yang spesifik tergantung pada beberapa faktor. anatomi gigi dan derajat keparahan furkasi merupakan faktor yang paling berpengaruh terhadap keputusan rencana perawatan. tujuan: tujuan laporan kasus ini adalah untuk menjelaskan penatalaksanaan furkasi kelas iv dengan perawatan kombinasi endodontik dan reseksi akar. kasus: penderita laki-laki usia 47 tahun datang ke klinik periodonsia dengan keluhan gigi goyang, hipersensitif dan tampak adannya furkasi. tatalaksana kasus: dilakukan kombinasi perawatan endodontik dan reseksi akar distal. hasil perawatan menunjukkan hasil yang lebih baik pada defek furkasi. kesimpulan: kombinasi perawatan endodontik periodontik dapat menjadi pilihan untuk mempertahankan gigi dengan furkasi. reseksi akar dapat meningkatkan prognosis yang lebih baik. perawatan interdisipliner yang kompleks dibutuhkan dalam membuat semua rencana perawatan. kata kunci: furkasi, perawatan endodontik, reseksi akar correspondence: ernie maduratna setiawati, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: setiowati_ernie@yahoo.co.id introduction furcation involvement is actually considered as a periodontal disorder involving open bifurcation or trifurcation because of the loss of alveolar bone located among the roots. furcation involvement is actually the most common case found in the mandibular and maxillary first molar. the problem often raised is the difficulty to 206 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 205–209 reach furcation areas during plaque control, especially in the area of furcation with mesial-distal direction or on the roots adjacent to each other. it is because furcation is an area that has a complex anatomy which even makes it is difficult to be cleaned with care at home; as a result, the periodontal disease is aggravated.1 the etiology of furcation involvement lesions may originate from plaque, pulp, abnormal occlusion, or the combination of those three causes. however, the main etiology of the aggravated furcation defect is caused by the presence of bacterial plaque in situ for a long time. it is also affected by the anatomical variations of root morphology and the anomalies of dental form, such as cervical enamel projections. pulp inflammations may cause furcation involvement because of the additional root canals are connected with the area of furcation. products of these necrosis and traumatic occlusion pulps then can cause inflammation in the area of furcation.2 the classification of the furcation involvement can actually be divided into four classes based on the degree of alveolar bone loss in the horizontal area of furcation.1 class i: in the early stage of furcation defect, there are suprabony pockets where furcation involvement still cannot be observed. class ii: the furcation defect may involve one or more furcation located on the same side, but alveolar bone is still attached to the teeth. class iii: the furcation defect has already been covered by soft tissue, so it cannot be observed anymore. probe even cannot pass the area of furcation. the radiolucent area in the bifurcation or trifurcation areas can be observed through radiographic examination. class iv: the soft tissue is lowering to the apical area. the furcation involvement can clinically be observed, and probe can pass through the area of furcation. the treatment of furcation involvement is selected based on the classification of furcation involvement, the expansion and position of bone resorption, and the tooth anatomy. the goals of the treatment are aimed to facilitate the self-maintenance of oral hygiene, to prevent the more severe attachment loss, and, whenever possible, to close the furcation defect. types of treatment of furcation involvement, furthermore, are various depending on the degree of damage in the inter-radicular area. in this case report, furcation involvement was treated with a combination of endodontic therapy and root resection. case a 47-year-old man came to periodontics clinic in faculty of dentistry, airlangga university, with main complaint in the upper left posterior area. the patient felt sensitive in the maxillary left area, especially when exposed to cold, and also felt that the tooth was shaky and the gums receded. medicine that has been taken was paracetamol. intra oral examination showed calculus, inflammation, bleeding, and gingival recession on the upper left posterior area. case management based on the anamnesis of clinical and radiographic examinations on the first visit, it was known that there were chronic periodontitis cases, on teeth 24, 25, 26, and 27, with furcation involvement on 26 (figure 1). on the first visit, full mouth scaling and root planning were performed. then on the second visit, the shaky tooth was splinted with wire-composite splinting along the 24, 25, 26, and 27. on the third visit, the patient still felt sensitive on tooth 26 when exposed to cold; as a consequence, the patient was consulted to the endodontic division for root canal treatment on 26. during the post-endodontic therapy, the patient had not felt any pain on the tooth 26, but the tooth was still loose; thus, it still could not used optimally for chewing. during intraoral examination, it was known that there were bleeding and inflammation on teeth 26 and 27, and gingival recession on the distobuccal root of tooth 26. therefore, the resection of the distal root of tooth 26 was planned to be performed on the next visit (figure 2). on the fourth visit, the resection of the distal root of the tooth 26 was conducted (4 months after the first visit). after reviewing the patient's medical history and treatment plan, the patient was prepared for periodontal surgery and anesthetized. access to the root surface was gained by elevating buccal and lingual full-thickness mucoperiosteal flaps. the extent of the flap must be sufficient to provide access and visibility for instrumentation and to facilitate proper wound closure. all chronic inflammatory tissue was removed with curettage, exposing the bone and root surfaces. in the case of a single root resection, a long fissure or diamond bur is used to section the root by positioning it at the most coronal portion of the root (the roof of the furcation) and gently penetrating through the furcation. complete root separation was verified by inserting a probe through the furcation and removing it through-and-through or by testing the mobility of each root individually. once the root was clearly separated from the remaining roots and the crown of the tooth, it can be elevated carefully from its socket with elevators to avoid luxation or damaging the remaining roots. once the resected root has been removed, odontoplasty should be performed to ensure that no "lip" of tooth structure, which would act as a plaqueretentive ledge, was left in the dome of the furcation. after the removal of the root, the remaining root surfaces were planed to remove deposits, all soft tissue in the furcation area is curetted, and an ostectomy or an osteoplasty was performed to eliminate the remaining bony deformities and provide a biologic width for the dentogingival complex after healing.3 the flaps were then re-approximated and sutured. postoperative instructions were similar to other periodontal surgical procedures, and the patient should be reinstructed on oral hygiene procedures, which were specific for the new dento-radicular morphology. on the fifth visit, the patient did not feel any pain on the tooth 26 after the resection was done, and even the patient could clean the area by himself easier than before. during intraoral examination, 207setiawati: a combination of endodontic therapy the gingiva had partially covered the area of the distal root of the tooth 26 which had been resected (figure 3). thus, the gradual treatment was needed in the division of periodontics. to obtain the best result of the treatment, periodontal tissue regeneration, the regenerative therapy by using bone graft, growth factor and coronally positioned flap then was planned to be conducted on the next treatment. the patient was satisfied were the result of endodontic and root resection and agreed to do maintenance and supportive periondontal therapy every 3 months. discussion periodontal attachment loss associated with marginal periodontitis around multi-rooted teeth can progress to the furcation area. because of the physical inaccessibility for hygiene procedures, infections in this area present a considerable therapeutic challenge with the potential for continued periodontal breakdown. consequently, the objective of periodontal furcation treatment is to eliminate the plaque-retentive areas in the exposed furcation and make the area more accessible for maintenance. success depends on the magnitude to which the periodontal infection has invaded the furcation area. early furcation involvement may be treated by therapeutically debriding the area, addressing the etiologic factors (eg, overhanging restorations, enamel projections) and improving hygiene access to the furcation entrance through odontoplasty. moderate to advanced furcation involvement, or through-and-through furcation involvement, often requires surgical intervention.2 the presence of furcation involvement is one clinical finding that can lead to a diagnosis of advanced periodontitis and potentially to a less favorable prognosis for the affected tooth. furcation involvement therefore presents both diagnostic and therapeutic dilemmas. the etiologies of furcation involvement include extension of inflammatory periodontal disease, local anatomic factors, trauma from occlusion, pulpo-periodontal disease and root fractures involving furcation. pulp and periodontal inflammation problems are tightly interconnected, both of which affect the diagnosis, the treatment planning, and the treatment procedure. the reason is because bacteria and inflammatory products of periodontitis can reach the pulp through the additional canal, apical foramen, or dentin tubule. inflammatory processes in the periodontium associated with necrotic dental pulp and periodontal disease have an infectious etiology. the essential difference between the two diseases entities is their respective source of infection. a potential pathway for infectious elements in the root canal in such instances may be lateral canals. acute manifestations of root canal infections can result in rapid and extensive destruction on the attachment apparatus. the effect of periodontal inflammation on the pulp is controversial. it has been suggested that periodontal disease has no effect on the pulp, at least until it involves the apex. on the other hand, the effect of periodontal disease on the pulp may include an increase in calcifications, fibrosis and collagen resorption. it seems that the pulp is not directly affected by periodontal disease, until recession has opened up an accessory canal to the oral environment.4 combined periodontic-endodontic lesions are localized, circumscribed areas of bacterial infection originating from either dental pulp, periodontal tissues surrounding the involved tooth. combined periodontic-endodontic lesions can originate from each or both of two distinct figure �. the radiographical examination, showed the resorption on the horizontal bone in the area of alveolar crest, 24, 25, 26, 27, and there was also radiolucency in the area of 26 furcation. the treatment of furcation involvement class iv by using the combination of endodontic figure �. the treatment of furcation involvement class iv by using the combination of endodontic therapy and root resection. figure ��. the result of endodontic therapy and root resection treatment combination. 208 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 205–209 locations and may be informally subclassified as follows: endodontic–periodontic, infection from the pulp tissue within a tooth may spread into the bone immediately surrounding the tip, or apex, or the root, forming a periapical abscess. this infection may then proliferate coronally to communicate with the margin of the alveolar bone and the oral cavity by spreading through the periodontal ligament; periodontic–endodontic, infection from a periodontal pocket may proliferate via accessory canals into the root canal of the affected tooth, leading to pulpal inflammation. treatment includes conventional endodontic therapy followed by periodontal therapy. endodontic therapy involves the removal of these structures, the subsequent cleaning, shaping, and decontamination of the hollows with tiny files and irrigating solutions, and the obturation (filling) of the decontaminated canals with an inert filling such as gutta percha and typically a eugenol-based cement. methods for periodontal therapy with the treatment of furcation involved molars have shown varying degrees of success. grade i early furcation involvements are generally treated well with effective plaque control and scaling and root planning. early grade ii and iii furcations require surgical management. surgery permits access or root debridement, odontoplasty, osseous recontouring and periodontal regeneration. advanced defects require endodontic therapy and resection of the root or part of tooth with advanced bone loss.3 the first step in performing a root resection is to determine which root will be removed. radiographs and clinical examinations are used to assess the extent of the furcation involvement, the amount of attachment loss, the morphology and proximity of the roots, the ability to perform endodontic therapy, the proximity of anatomical structures, and the existence of caries or root resorption. it is typical that the root with the least amount of remaining bony support or the most difficult for the endodontist or restorative dentist to treat should be removed.5 because of the high incidence of furcation involvement in maxillary teeth and the anatomy and relation of the first and second molars, the root most commonly resected is the disto-buccal root of the maxillary first molar.6 it is generally agreed that whenever possible, endodontic treatment should be performed before a root resection. this facilitates the performance of the endodontic obturation and allows the endodontist to determine whether the canals can be adequately instrumented. in addition, performing endodontic treatment before a root resection may minimize the potential for postoperative pain.4,6 root resection can be a valuable procedure when the tooth in question has a very high strategic value or when specific problems exist that cannot be solved by any other therapeutic approach. root resections may be the treatment of choice when the proximity to anatomical landmarks (eg, maxillary sinus, mandibular canal) limits the amount of bone available for dental implants.6 in this case report, furthermore, the patient had furcation involvement degree iv, in which gingival recession. the type of the treatment of furcation involvement degree iv can be the combination of restorative endodontic therapy and surgical therapy. the reason is because of the severe periodontitis indicated by the opening of the root surface and the loss of attachment induced inflammatory products and bacterial toxins spread to the pulp through additional canal, apical foramen, or penetration dentin tubule, which can lead to pulpitis or pulp necrosis. in this case, endodontic treatment can reduce acute symptoms suffered by the patient although the patient will still have sensitive pain to percussion. endodontic therapy and the root resection then can be indicated to be good for the treatment of furcation involvement class iv. actually, the root resection can be conducted on vital tooth that has been treated with endodontic therapy, but it better to conduct the endodontic therapy prior to the root resection.7 root resection is the process by which one or more of the roots are removed at the level of the furcation while leaving the crown and remaining roots in function. root resections have been performed in dentistry since the late 1800s. with proper long-term monitoring and maintenance, a root resection is accepted as a valid treatment with reasonable long-term effectiveness. in a recent report on periodontal outcomes in a private practice setting in teeth followed a minimum of 10 years after active treatment, 90% of root-resected teeth were maintained in a stable state long term. complications and failures were mainly of an endodontic nature and tended to occur 10 years or more after the resection procedure.7 the result of root resection in this treatment showed that the gingiva had been covered the surgical site. the patient even did not complain of pain anymore and could easily clean the area by himself. through root resection therapy, furcation-involved molars can be converted to non-furcated single–root teeth and provide a favorable environment for oral hygiene for patient and clinicians. root resection therapy had poor long term results unless a high level of expertise was available in all applicable disciplines. root resection therapy for molars with periodontal problems was based on the periodontal pathology and could obtain a good prognosis.8 root resection therapy can remove the deposited periodontal bacteria as well as unfavorable anatomic features, which can act as an a future bacteria reservoir. in addition, bone defects can be resolved by healing after removing the involved roots and a positive architecture can be achieved.9,10 according to the standardized reports on root resection, 89% of root resected teeth survived over a 7-year period.7 after the resection, regular maintenance treatment, consisting of subgingival instrumentation might be needed for the resected molars to prevent progressivity of periodontal disease. in this case, the teeth were still shaky since the splinting was often loose. it was suggested to have further treatment with regenerative therapy or with fixed splint. surgical debridement of the furcation alone may not be sufficient to improve the long-term prognosis. the regeneration therapy is indicated to be a good treatment for the furcation defect vertically similar with 2 walls or 3 wall-defects. surgical therapy involving regenerative 209setiawati: a combination of endodontic therapy procedures is indicated in grade iii and iv furcation involvements. the regenerative procedures used in these cases include bone grafts and guided tissue regeneration. results obtained from studies have revealed the positive effects of bone grafts for the treatment of furcation defects especially vertical defect fill. in addition to this, platelet rich factor (prf) which is an autologous source of platelets was used to enhance the results of the regenerative procedure.5 extraction is used to indicate severe furcation involvement with extensive bone resorption surrounding one or two roots, especially in patients who cannot maintain oral hygiene by themselves, has the high risk of dental caries, or cannot comply with program maintenance because of socioeconomic factors.11 as a conclusion, that understanding the periodonticendodontic continuum is a vital part of successful endodontic and periodontal treatment. treatment and prognosis of primarily endodontic and primarily periodontal disease are very straightforward. endodontic therapy is more predictable and completion of this therapy before periodontal procedures has a positive effect on periodontal healing. the most guarded prognosis is given for true combined lesions. in cases of combined disease, the success of endodontic therapy is dependent on the completion of periodontal therapy. the complete treatment of both aspects of perio-endo lesions is essential for long term results. therefore it is absolutely essential that the periodontal problem also be treated to obtain optimal therapeutic outcomes. references 1. newman mg, takei h, carranza fa. clinical periodontology. 9th ed. philadelphia, london, new york: wb saunders company; 2006. p. 825–37. 2. caffesse rg, quinones cr. surgical, non surgical, occlusal and furcation therapies. periodontol 2000, 2000; 9: 69–90. 3. manson jd, eley bm. outline of periodontics. 4th ed. oxford, auckland, boston: bath press, somerset; 2000. p. 227–70. 4. sunitha r, emmadi p, rajaraman v. the periodontal–endodontic continuum: a review. j conserv dent 2008; 11: 54–63. 5. sangeta s. management of an endo-perio lesion in a maxillary canine using platelet rich plasma concentrate and an alloplastic bone substitute. j indian soc periodontol 2009; 13: 97–100. 6. romito ga, pustioglioni fe. biometric study of furcation area of first maxillary molars. braz dent j 2004; 15(2): 155–8. 7. shin sy, yang sm. factor influencing the outcome of root resection therapy in molars. j periodontol 2009; 80: 32–40. 8. sallum e, casati mz, bittencourt s, nociti fh, vale hf, ribeiro ed. radiographic characteristic of furcation involvement in mandibular molars as prognostic indicators of healing after nonsurgical periodontal therapy. j am dent assoc 2009; 140: 434–40. 9. sánchez-pérez a, moya-villaescusa mj. periodontal disease affecting tooth furcations. a review of the treatments available. med oral patol oral cir bucal 2009; 14(10): 554–7. 10. jeremias f, santos-pinto l, porciúncula hf. root trifurcation of upper permanent second molars: anatomical aspects of clinical interest. int j morphol 2009; 27(3): 649–53. 11. polson am, blieden t. long-term outcomes after periodontal therapy. j periodontol 2002; 73: 1092. 9999 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 99–106 research report effects of manufacturing methods of abalone gel as a desensitisation material on the closing of dentinal tubules sri budi barunawati,1 wayan tunas artama,2 suparyono saleh,3 siti sunarintyas4 and yosi bayu murti5 1doctoral program, faculty of dentistry, universitas gadjah mada 2department of biochemistry, faculty of veterinary medicine, universitas gadjah mada 3department of prosthodontics, faculty of dentistry, universitas gadjah mada 4department of biomaterial, faculty of dentistry, universitas gadjah mada 5department of pharmaceutical biology, faculty of pharmacy, universitas gadjah mada yogyakarta – indonesia abstract background: abalone (haliotis varia linnaeus) shells possess a high arginine content and are expected to be an alternative desensitisation material that is both insoluble and able to properly close dentinal tubules. different methods of manufacturing abalone gel affect the molecular weight, hydrophilic or hydrophobic properties, and protein content of the lysis. purpose: this study aimed to determine the effects of different manufacturing methods on the dentinal tubule closure of abalone desensitisation gel. methods: this study involved the extraction of abalone shells followed by preparative and thin-layer chromatography. the drying of the samples was carried out by the precipitation, drying, and addition methods. the research was divided into eight treatment groups, each consisting of three samples (f1, f2, f3). each sample was applied to two study subjects’ post-extracted third molars, which were cut into disc shapes and subsequently etched with 6% citric acid. the percentage of dentinal tubule occlusion was calculated by image j (nih, usa) software. data were analysed using three-way anova. results: the results showed that there were significant differences (p < 0.05) both in terms of the effects of the samples with deposition and addition on the occlusion of the dentinal tubules and in terms of the interactions between the samples with drying and addition. there was no significant difference (p > 0.05) in terms of the interactions of the samples’ three manufacturing methods. conclusion: the manufacture of abalone gel as a desensitisation material requires a minimum of two interactions between the sample-making method and the addition, deposition, and drying methods. the best method was deposition. keywords: abalone gel; arginine; hypersensitivity dentin; tubule occlusion correspondence: sri budi barunawati, doctoral program, faculty of dentistry, universitas gadjah mada, jl. denta no. 1, sekip utara, yogyakarta 55281, indonesia. email: barunawati@ugm.ac.id introduction the tooth preparation procedures that are necessary to the manufacture of fixed denture restorations have the potential to trigger discomfort afterwards, particularly in the forms of dentin hypersensitivity and pulp irritation.1 full crown preparation results in the exposure of 1 cm2 of dentin, containing more than 3 million tubules/cm2. the dentinal pulp complex deposits a tertiary layer of dentin under the severed tubules during the cavity preparation procedure, which forms a prepared dentinal wall, preventing the entry of bacteria into the pulp.2 this response by the pulp is influenced by many factors, including: remaining dentin thickness (rdt), heat friction and bur vibration generated during crown preparation, dentin drying, local anaesthetic effects, gingival haemostatic material, and the moulding and cementing of the restoration. the increase in complications related to endodontic treatment after the fixed denture fabrication procedure is generally due to the preparation of teeth that are too deep. this results in the thickness of the dentin remaining above the surface of the dentin-pulp complex on the prepared teeth being less than 2 mm, which can endanger the vitality of the pulp.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 mailto:barunawati@ugm.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 100 barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 the current biological approach to dentin hypersensitivity therapy is to mimic the natural desensitisation process, which triggers the spontaneous closure of the dentinal tubules.4 the disadvantages of current hypersensitivity therapy, such as fluorine, hydroxyapatite, strontium, zinc chlorides, and potassium oxalate, are the short durability and poor effectiveness of their therapeutic effects, namely that the desensitisation material has only a short effect due to the daily brushing process, dissolution in acidic beverages, and closure of the tubules is incomplete. several studies have shown that, to date, there is no agreement on a gold standard for desensitisation materials.5 the latest technological development in desensitisation materials is based on the understanding of the natural processes of reducing dentin hypersensitivity such as that effected by the arginine contained in saliva. an important component of this latest desensitising agent is arginine, an alkaline amino acid with a physiological ph (i.e. 6.5–7.5). arginine is physically absorbed into the surface of calcium carbonate in vivo, forming positively charged agglomerates that bind negatively to the dentin on the exposed surfaces of the dentin layer and inside the dentinal tubules. the interaction of arginine and calcium carbonate in vivo triggers phosphate, calcium, and carbonate deposition on the surface of the dentin and in the dentinal tubules.6 when a desensitisation agent is applied to exposed dentin, the arginine and calcium carbonate in the saliva work together to accelerate the natural mechanism of closure, meaning that the desensitisation agent binds to the negatively charged surface of the dentin, forming a dentin-like mineral deposit as a covering over the dentinal tubules and a protective layer on the dentin’s surface.4 one instance of marine biota that contains high amounts of the amino acid arginine is abalone. abalone has an expensive shell that contains iodine, zinc, iron, potassium, vitamin a, vitamin e, vitamin b12, omega-3 fatty acids, and magnesium.7 the dominant part of the abalone is its muscles, which attach to the shell and legs. one species of abalone that is bred is haliotis varia linnaeus; arginine is the highest average of its amino acid contents.8 some studies also mention that abalone meat has a high mineral content along with its protein content, especially the amino acid arginine.9 abalone brings such health benefits as being anti-inflammatory, accelerating bone growth, containing vitamins and minerals, and containing omega-3 fatty acids, which are antioxidants.7 the method of manufacturing desensitisation gel from abalone shell extraction take place over several steps. some characteristics of a good desensitisation material are its ability to properly close dentinal tubules and not be easily dissolved by saliva or acid in the oral cavity.5 therefore, this study aims to investigate the effects of different sampling methods from abalone shell extraction on dentinal tubule closure. materials and methods the abalone clam used in this study was a male, the third offspring (f3) from a parent of natural origin (f0) cultivated by the center for marine fisheries research, gondol, buleleng, bali. species identification – as haliotis varia linnaeus, 1758 – was carried out at the animal systematics laboratory, faculty of biology, gadjah mada university. the extraction procedure was carried out using abalone clam meat that had been frozen at a temperature of −26°c. as much as 50 g was taken and washed, dried, cut into pieces, and blended with 150 ml of 0.1 n hcl. this solution was then centrifuged in 15 ml tubes for 20 minutes at a speed of 5,000 rpm. one tablet-shaped protease inhibitor was added to each 15 ml tube for samples undergoing the addition method. then, 60 ml of a solution consisting of 0.5 m tris (hydroxymethyl aminomethane), 30 mm 2-mercaptoethanol, and 2 mm edta disodium dihydrate was added to the resulting supernatant. the resulting mixture was then centrifuged in 15 ml tubes for 20 minutes at a speed of 5,000 rpm. this supernatant was taken and freeze-dried for 24 hours. the results of which were dissolved into 18 ml of a 50 mm acetate buffer eluent (ph 5.0) and then filtered with filter paper.10 preparative chromatography was then carried out after the extraction process with the stationary phase of a sephadex g-25 and the mobile phase of the acetate buffer 50 mm (ph 5.0).10 the instrument used was a glass column with a height of 60 cm x 2 cm. a 20-g sephadex g-25 matrix was packed in a column, and 150 ml of acetate buffer were added and allowed to condense for 24 hours. the acetate buffer eluent was added until the glass column was full and allowed to drip until within ± 2 cm from the deposited g-25 sephadex. then, 3 ml of abalone extract were dropped circularly along the column wall. the results of fractionation incorporated as much as 8 ml into a tube with a flow rate of 1 ml per minute. the grouping of fractionation active substances with qualitative thin layer chromatography (tlc) tests performed with the stationary phase were tlc silica gel 60 f254 and the mobile phase was n-butanol 3.5 ml, acetone 3.5 ml, glacial acetic acid 1 ml, and distilled water 1 ml. the tests of active substances in each fraction was carried out by taking every 10 μl of liquid per fraction and dropping it on tlc silica gel and placing it into a chamber containing a mobile phase. the grouping of active substances was made visible by painting ninhydrin so that three fractions (f1, f2, f3) were obtained for each group of protease inhibitors and without protease inhibitors. the results of the f1, f2, f3 fractions were dried using two methods: freeze-drying for 24 hours and oven-drying at 40°c for 24 hours.11 the centrifuge and non-centrifuge methods were performed using cold centrifuge procedures at 4°c. a 20 mg samples obtained by both ovenand freeze-drying procedures were dissolved into 500 μl of acetate buffer dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 101barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 solvent and cold centrifuged in five-minute intervals at 7,000 rcf, 9,000 rcf, 11,000 rcf, 13,000 rcf, 15,000 rcf, 17,000 rcf, and 19,000 rcf. the supernatant, which yielded as much as 200 μl, was put into a microtube and dried in an oven at 40°c for 24 hours, producing a precipitate. at this stage, 24 sample groups were obtained, namely the f1, f2, f3 samples, in the following groups: npi fd s (without protease inhibitor, freeze-drying, centrifuge), npi o s (without protease inhibitor, oven, centrifuge), pi fd s (protease inhibitor, freeze-drying, centrifuge), pi o s (protease inhibitor, freeze-drying, centrifuge), npi fd ns (without protease inhibitor, freeze-drying, without centrifuge), npi o ns (without protease inhibitor, oven, without centrifuge) pi fd ns (protease inhibitor, freezedrying, without centrifuge), and pi o ns (protease inhibitor, freeze-drying, without centrifuge). to obtain dry samples before they were dissolved into cmcna, all samples were dried in a 40°c oven for 24 hours. to make a sample into gel, 5 mg of dry sample were dissolved into 250 μl of a 1% cmcna solution. the samples were then stirred until all of them were dissolved. for this study, three methods of dry sampling were categorised for observing dentinal tubule occlusion: 1) with and without the addition of protease inhibitors, 2) with and without the use of a centrifuge, and 3) oven-drying at 40°c and freeze-drying for 24 hours. in vitro research was carried out on 48 third molars (two subjects for each sample), eight treatment groups, and 24 samples (two samples for each treatment group). the caries-free third molars were extracted from healthy adult patients according to a protocol that has been accepted by the research ethics commission of the faculty of dentistry, gadjah mada university, no. 001126/kkep/fkg-ugm/ec/2017. teeth were cleaned and stored in a pbs solution (ph 7.4) at 4°c with no more than one month to be used as research subjects. specimens of disc-shaped dentin with a thickness of 1 mm was prepared by cutting parallel to the dental axis on a cement enamel junction (cej) using a slow-speed bur with water spray (isomet, buehler ltd., lake bluff, il, usa). the formation of a standard smear layer on the surface of the dentin was made using 600-grit silicon carbide paper for 30 seconds under a constant flow of water, followed by 1200-grit silicon carbide paper.5 this study was divided into eight treatment groups with each group consisting of three samples (f1, f2, f3); each sample was applied to two research subjects, which were caries-free, extracted third molars. subjects had 6% citric acid applied to them for two minutes to ensure the opening of the pulp suffix from the dentinal tubules. the surfaces of the subjects were observed with an optical microscope (olympus, tokyo, japan) to examine the exposed dentinal tubules. twenty-four of the treatment samples were treated with an application of abalone desensitisation gel to the dentin surface with a microbrush, left for seven minutes and washed with distilled water. the subjects were stored in artificial saliva at 37°c, and the procedure was repeated for seven days.12 after seven days, the subject was dried and stored in a desiccator. the specimens were mounted on aluminium stubs and sputter-coated in platinum before examination using a scanning electron microscope (jeol, jsm6510la, japan). for each region of each subject, typical fields were photographed at a magnification of 500x. quantitative analysis of dentinal tubule closure on the surface of the dentin was performed by an assessment of the occlusion or non-occlusion of dentinal tubule closure, which was measured using image j (nih, usa) software.13 the percentage of dentinal tubule closures was determined by dividing the total number of closed dentinal tubules by the total area of the subject in the sem image area. data were analysed using the three-way anova method. the probability level for statistical significance was set at α = 0.05.14 results results showed that the percentage of dentinal tubule closure was different in each treatment group with different manufacturing methods, as shown in table 1 and figure 1. the data in table 1 shows the highest incidence of dentinal tubular closure, seen in the pi o ns treatment group (98.692 ± 0.306). the lowest incidence of dentinal tubular closure was seen in the npi fd ns treatment group (91.817 ± 1.386). figure 1 shows that the treatment group without the precipitation method (ns) had a higher rate of closure table 1. the mean and standard deviation of dentinal tubule occlusion (%) sample method deposition drying addition centrifuge (s) without centrifuge (ns) oven (o) protease inhibitor (pi) 94.825 ± 1.327 98.692 ± 0.306 without protease inhibitor (npi) 94.108 ± 1.054 97.492 ± 2.004 freeze-drying (fd) protease inhibitor (pi) 96.767 ± 1.247 97.183 ± 0.934 without protease inhibitor (npi) 91.817 ± 1.386 95.850 ± 1.236 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 102 barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 86.000 88.000 90.000 92.000 94.000 96.000 98.000 100.000 102.000 n pi f d n s n pi o n s pi f d n s pi o n s n pi f d s n pi o s pi f d s pi o sd en tin al t ub ul e o cc lu si on (% ) treatment groups figure 1. average closing of dentinal tubules from eight treatment groups. 84.000 86.000 88.000 90.000 92.000 94.000 96.000 98.000 100.000 102.000 d en tin al tu bu le o cc lu si on (% ) sample groups figure 2. percentage of dentinal tubule closure in each sample. notes: f1: first fraction; f2: second fraction; f3: third fraction; npi: without the addition of protease inhibitor; pi: with addition of protease inhibitor; fd: drying with freeze-drying; o: drying with oven; s: centrifuge; ns: without centrifuge. a b c d e f figure 3. closure of the dentinal tubules is seen in samples without the addition of protease inhibitors and without a centrifuge. (a), (b), (c): drying by freeze-drying. (d), (e), (f): drying with an oven (1000x magnification). figure c shows more open dentinal tubules than closed dentinal tubules. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 103barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 g h i j k l figure 4. closure of the dentinal tubules is seen in samples with the addition of protease inhibitors and without a centrifuge. (g), (h), (i): drying with freeze-drying. (j), (k), (l): drying with an oven (1000x magnification). s t u v w x m n o p q r figure 6. closure of the dentinal tubules is seen in samples by the addition of protease inhibitors and by centrifuge. (s), (t), (u): drying with freeze-drying. (v), (w), (x): drying with an oven (1000x magnification). figure 5. closure of the dentinal tubules is seen in samples without the addition of protease inhibitors and by centrifuge. (m), (n), (o): drying with freeze-drying. (p), (q), (r): drying with an oven (1000x magnification). figures m, n, and o show more open dentinal tubules than closed dentinal tubules. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 104 barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 than that in which the precipitation method was used (s). incidence of closure in the treatment group with the addition method (pi) was higher than in the group without (npi). the treatment group in which oven drying was used (o) was higher than that in which freeze-drying was used (fd). the percentage of dentinal tubule closure in 24 samples, which comprised three samples (f1, f2, f3) for each group of eight treatment groups, is shown in figure 2. the results showed that the percentage of dentinal tubule closure was different for each sample within the eight different treatment groups shown in figure 2. the highest dentinal tubular closure was seen in the f3 pi o ns sample. the lowest dentinal tubular closure was seen in the f3 npi fd s sample. closure of the dentinal tubules was observed with a scanning electron microscope (sem), as shown in figures 3, 4, 5, and 6 (at 1000x magnification). the sem figure displays the different percentages of dentinal tubule closure in each sample. the results of the three-way anova test (table 2) showed that there was a significant difference (p < 0.05) in dentinal tubule closure between the samples prepared with the centrifuge deposition method and those without (g1). there was no significant difference (p > 0.05) in dentinal tubule closure between the samples prepared using the freeze-drying method and those using oven-drying (g2). there was no significant difference (p > 0.05) in dentinal tubule closure between the samples prepared by adding protease inhibitors and those without protease inhibitors (g3). there was no significant difference (p > 0.05) in dentinal tubule closure between the interaction of the method of making the deposition sample (g1) and drying sample (g2). there was a significant difference (p < 0.05) in dentinal tubule closure between the interaction of sampling methods between deposition (g1) and addition (g3). there was a significant difference (p < 0.05) in dentinal tubule closure between the interaction of sampling methods with drying (g2) and addition (g3). no significant difference (p > 0.05) in dentinal tubule closure was seen in the interactions of the sampling methods with deposition (g1), drying (g2), or addition (g3). discussion one effective way to overcome dentin hypersensitivity is to develop a new material that can close dentinal tubules well; last for a long time; not be dissolved by saliva, acidic food, or drink; and not be lost to abrasion or erosion. arginine is just such an alternative ingredient. natural proteins play a role in the formation of teeth, such as dentin phosphophoryn, dentin matrix protein-1 (dmp-1), which is rich in glutamic acid, and phosphoprotein, which facilitates the nucleation of hydroxyapatite (hap) and modifies the formation of the mineralised collagen fibres in dentinal tubules.15 abalone is a natural, alternative desensitisation material due to its high arginine content, its anti-inflammatory properties, its rich in mineral content, and other factors. the centrifuge procedure for abalone extraction produces deposits with high molecular weights, resulting in small to medium molecular weights remaining in the supernatant. the primary concern with dentin regeneration is the realisation of mineralisation between the fibres in the collagen matrix. collagen requires a material with a molecular weight ranging from 6–40 kda. however, the molecular weight of l-arginine is 174.2 kda, which is much higher.16 this study used the supernatant of abalone extraction, which resulted in more material with low-tomoderate molecular weight in the sample. tubule closure with samples that were prepared using the centrifuge method was less than those that were prepared without the centrifuge. oven-drying and freeze-drying for 24 hours evaporated the acetate buffer solvent. the oven-drying procedure was carried out at 40°c for 24 hours, which eased the evaporation of the buffer without damaging the protein content. this is consistent with research that has found that the amino acid profile in the oven-drying process produces hydrophobic residues, whereas freeze-drying produces hydrophilic residues – both without damaging the protein content.17 neither the freeze-drying nor the oven-drying procedure affected the protein composition of the samples, and there was no significant difference in tubule closure. table 2. the results of three-way anova source of variance sum of squares free degrees average square f sig. g1 0.012 1 0.012 48.358 0.000 g2 7.465 1 7.465 0.028 0.869 g3 0.001 1 0.001 3.712 0.057 g1 * g2 0.001 1 0.001 2.667 0.106 g1 * g3 0.011 1 0.011 42.679 0.000 g2 * g3 0.007 1 0.007 26.836 0.000 g1 * g2 * g3 0.000 1 0.000 1.176 0.281 error 0.023 88 0.000 total 88.240 96 corrected total 0.055 95 notes: g1: with centrifuge (s) and without centrifuge (ns) depositional treatment groups; g2: freeze-drying (fd) and oven-drying (o) treatment groups; g3: with added protease inhibitors (pi) and without protease inhibitors (npi); (*): interaction. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 105barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 the current study showed that the mean closure of dentinal tubules with oven-drying was higher because samples that underwent freeze-drying had a hollower structure with hydrophilic properties, meaning that when they attached to the dentin surface, they became more soluble. the dentin matrix is principally composed by mineralised collagen fibres, which are hydrophilic; it therefore becomes difficult to wet or infiltrate the surface of dentin that has been etched with hydrophobic material, although some studies do mention having been done using a hydrophilic monomer 2-hydroxyethyl methacrylate or ethanol wetting technique; however, these desensitisation materials could not cover the dentinal tubules for long.15 this is reinforced by the fact that the patient’s oral cavity is filled with flowing saliva. a desensitisation material will dissolve if it is not able to sufficiently bind to dentin.18 amino acids, as the basic building blocks of proteins, play a biochemical role in the remineralisation process of etched dentin. arginine, as a desensitisation material, is an amino acid that is positively charged under physiological environmental conditions if it forms a complex with calcium carbonate as a source of calcium; thus, it can close exposed tubules well and remain resistant to acids. they are thus believed to be constitute a long-lasting prevention of dentin hypersensitivity.15 protease inhibitors catalyse the hydrolytic breakdown of proteins into peptides or amino acids, produce protein degradation, and deactivate or inhibit protease in the cell lysis buffer or cell extract.19 this research shows that samples that included protease inhibitors kept the protein content of the samples from going into lysis. the longer the peptide chain, the higher the ability to bind hydroxyapatite and collagen. peptides bind to the positively charged side of demineralised hydroxyapatite and collagen fibres via electrostatic interactions. then, nucleation templates occur to absorb calcium ions through negatively charged groups or secondary structures, which allows calcium ions to join with phosphate ions to form deposited minerals on the dentinal tubules.18 this study showed that the samples with added protease inhibitors had a greater effect on dentinal tubule closure than those without. this is consistent with the opinion of kleinberg (2002), who asserted that the combination of arginine and calcium carbonate would form a positively charged complex that would bind to the surface of the negatively charged dentin and inside the dentinal tubules. the alkali nature of the arginine compound with calcium carbonate would also facilitate the deposition of calcium and phosphate from saliva or dentinal fluid in permanently closing the dentinal tubules.20 table 2 shows that the deposition method (g1) had a significant effect (p < 0.05) on dentinal tubule closure. however, the drying (g2) and addition methods (g3) had no significant effect (p < 0.05) on dentinal tubule closure. it positively asserted that the manufacture of samples by the g1 method alone can affect dentinal tubule closure. the results of the three-way anova analysis showed the presence of interaction factors. interaction factors that significantly influenced (p < 0.05) dentinal tubule closure were the g1–g3 and g2–g3 methods. this shows that the most important factor was the g1 method, while the g2 method in this case did not have a significant effect on tubule closure. the g2 method needs to be carried out in conjunction with the g3 method in order to significantly influence tubule closure, whereas the g1 method did not require the presence of the other methods to influence tubule closure. the interaction of the g1 and g3 methods affected the ability of the abalone gel to close dentinal tubules. deposition using the centrifuge separated the results of abalone extraction with low-to-moderate molecular weight more widely than without the use of the centrifuge. adding protease inhibitors extended peptide bonds, thereby further increasing the ability of the abalone gel to bind hydroxyapatite in the dentinal tubules. the interaction of the g2 and g3 methods also showed a significant difference (p < 0.05). the most suitable drying process is that which does not damage the protein content, as it further enhances the ability of the abalone gel to adhere to the walls of the dentinal tubules due to its insoluble, hydrophobic nature. the interaction of the g1and g2 methods was not significant (p > 0.05) to tubule closure, meaning that the presence of the g2 method did not support the g1 method. the interaction of g1, g2, and g3 was not significant (p > 0.05) because the g2 method did not significantly influence dentinal tubule closure. the results of the current study showed that manufacturing abalone gel for desensitisation by causing dentinal tubule closure requires a minimum of two interactions of sample-making methods, such as the interactions of the methods of deposition (g1) and addition (g2), or the methods of drying (g2) and addition (g3). the best method for manufacturing abalone desensitisation gel samples is the deposition method (g1). references 1. gupta n, reddy un, leela vasundhar p, sita ramarao k, pratap varma kvv, vinod v. effectiveness of desensitizing agents in relieving the preand postcementation sensitivity for full coverage restorations: a clinical evaluation. j contemp dent pract. 2013; 14(5): 858–65. 2. abu-nawareg mm, zidan az, zhou j, chiba a, tagami j, pashley dh. adhesive sealing of dentin surfaces in vitro: a review. am j dent. 2015; 28(6): 321–32. 3. berani r, sveqla m. remaining dentine thickness following tooth preparation and its impact on dentine – pulp complex. int j bus technol. 2018; 6(2): 1–8. 4. singh s. p ro -a rgin: a brea k t h rough tech nology for dentin hypersensitivity treatment. int j sci study. 2013; 1(3): 133–7. 5. wang t, yang s, wang l, feng h. use of poly (amidoamine) dendrimer for dentinal tubule occlusion: a preliminary study. plos one. 2015; 10(4): e0124735. 6. uraz a, erol-şimşek ö, pehlivan s, suludere z, bal b. the efficacy of 8% arginine-caco3 applications on dentine hypersensitivity following periodontal therapy: a clinical and scanning electron microscopic study. med oral patol oral cir bucal. 2013; 18(2): e298–305. 7. je jy, park sy, hwang jy, ahn cb. amino acid composition and in vitro antioxidant and cytoprotective activity of abalone viscera hydrolysate. j funct foods. 2015; 16: 94–103. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 106 barunawati et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 99–106 8. widayati n, pratiwi r. kandungan protein dan komposisi asam amino pada abalon (haliotis volcanicus) dan (haliotis diversicolor varia linnaeus) di pantai selatan gunung kidul daerah istimewa yogyakarta. thesis. yogyakarta: universitas gadjah mada; 2011. 9. latuihamallo m, iriana d, apituley d. amino acid and fatty acid of abalone haliotis squamata cultured in different aquaculture systems. procedia food sci. 2015; 3: 174–81. 10. ishmayana s, kristina y, kamara ds, hindersah r, soemitro s. komposisi asam amino peptida γ-glutamilsistein yang diisolasi dari tajuk selada (lactuca sativa l.) dengan dan tanpa inokulasi azotobacter sp. lkm6. in: seminar nasional biokimia. jakarta: universitas indonesia; 2008. p. 1–7. 11. rubiyanto d. teknik dasar kromatografi. yogyakarta: deepublish; 2016. p. 7–23. 12. sales-peres sh de c, de carvalho fn, marsicano ja, mattos mc, pereira jc, forim mr, da silva mfgf. effect of propolis gel on the in vitro reduction of dentin permeability. j appl oral sci. 2011; 19(4): 318–23. 13. garofalo sa, sakae lo, machado ac, cunha sr, zezell dm, scaramucci t, corrêa aranha ac. in vitro effect of innovative desensitizing agents on dentin tubule occlusion and erosive wear. oper dent. 2019; 44(2): 168–77. 14. guo x, yu j, smales rj, chen h, si h, wu y. effect of different irradiation times on the occlusion of dentinal tubules when using a nd:yag laser: an in vitro sem study. open j stomatol. 2015; 5: 72–9. 15. wang r, wang q, wang x, tian l, liu h, zhao m, peng c, cai q, shi y. enhancement of nano-hydroxyapatite bonding to dentin through a collagen/calcium dual-affinitive peptide for dentinal tubule occlusion. j biomater appl. 2014; 29(2): 268–77. 16. xu x, chen x, li j. natural protein bioinspired materials for regeneration of hard tissues. j mater chem b. 2020; 8(11): 2199– 215. 17. elavarasan k, shamasundar ba, badii f, howell n. angiotensin i-converting enzyme (ace) inhibitory activity and structural properties of ovenand freeze-dried protein hydrolysate from fresh water fish (cirrhinus mrigala). food chem. 2016; 206: 210–6. 18. liang k, xiao s, liu h, shi w, li j, gao y, he l, zhou x, li j. 8dss peptide induced effective dentinal tubule occlusion in vitro. dent mater. 2018; 34(4): 629–40. 19. nurrohman h, carneiro kmm, hellgeth j, saeki k, marshall sj, marshall gw, habelitz s. the role of protease inhibitors on the remineralization of demineralized dentin using the pilp method. mishra yk, editor. plos one. 2017; 12(11): e0188277. 20. chen cl, parolia a, pau a, celerino de moraes porto ic. comparative evaluation of the effectiveness of desensitizing agents in dentine tubule occlusion using scanning electron microscopy. aust dent j. 2015; 60(1): 65–72. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p99–106 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p99-106 11 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 1–6 original article progressivity analysis of pleomorphic adenoma toward carcinoma ex pleomorphic adenoma mei syafriadi, dina zakiyatul ummah, aisyah izzatul muna, maria evata krismawati surya department of biomedical sciences, faculty of dentistry, university of jember, jember, indonesia abstract background: pleomorphic adenoma (pa) is a benign salivary gland tumour with high incidence and recurrence after treatment. it may recur with the same appearance or develop toward malignancy, namely as carcinoma ex pleomorphic adenoma (cxpa). how this tumour can transform into a cxpa remains unclear. purpose: the aim of this study was to analyse the possibility of pathogenesis and progressivity of pa to cxpa. methods: twenty-four samples of pa and three control samples of cxpa were stained with haematoxylin and eosin (he), mallory’s trichrome, and periodic acid–schiff (pas). all of the pa cases were identified through different kinds of stroma, tumour cells types, morphologic patterns, or else through atypical appearance of the pa similar to the cxpa. results: twenty-four samples of pa demonstrated that the most dominant stroma was myxofibrous, and the dominant tumour cell type was plasmacytoid cells with a trabecular pattern. additionally, in the pleomorphic histological picture of adenomas we found several patterns of malignant tumour behaviour, including pseudopodia, metaplasia and hyalinisation, and cholesterol crystals that are thought to come from fat cell necrosis derived from adipose metaplasia. conclusions: pa displays several atypical characteristics that have the potential to develop into malignancies such as cxpa, due to capsular infiltration, necrosis, hyalinization and high mitotic activity of cells, but all these atypical characteristics that we observed still cannot be clearly classified as cxpa because they require other specific examinations. keywords: pathogenesis; pleomorphic adenoma; progressivity correspondence: mei syafriadi, department of biomedical sciences, university of jember, jl. kalimantan no. 37, jember, 68121 indonesia. email: didiriadihsb@gmail.com introduction pleomorphic adenoma (pa) is a type of salivary gland neoplasm with an incidence of over 80%. it is also called mixed benign tumour due to its heterogeneous appearance.1,2 histopathologically, this tumour structure consists of duct cells, myoepithelial cells, and mesenchymal cells. it has the appearance of various extracellular matrices, called stroma, such as myxoid, fibrous, chondroid, myxofibrous and myxochondroid stroma. pleomorphic adenoma often occurs in major salivary glands, mainly the parotid gland.3,4 carcinoma ex pleomorphic adenoma (cxpa) is often referred to as the growth of de novo malignancy. the growth of de novo malignancy in cxpa is a malignancy that arises not from the benign tumour cells that are transformed into malignant tumour cells but the malignant cells derived from normal cells around the tumour cells. however, there are reports of cxpa which is a recurrence of pa.5 the pathogenesis of pa recurrence that develops towards malignancy cannot yet be explained with certainty because the recurrence rate of this tumour is low and if there is a recurrence of pa after treatment it may appear in the same pattern, or it can develop into malignancy. valstar et al.6 reported that of the 125 pa patients who had a recurrence, 20 patients (16%) had a recurrence for the second time, and 2 in 20 patients (10%) experienced a recurrence for the third time. however, only 4 patients (3.2%) of all patients who experienced recurrence showed a transformation towards malignancy. the type of malignant tumour which appears after removal is called carcinoma ex pleomorphic adenoma (cxpa).6 cxpa, although known as the result of pleomorphic adenoma recurrence, was also reported as de novo malignancy growth. this means that the malignancy dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 mailto:didiriadihsb@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 2 characteristic appeared without previous history. who classifies cxpa into three groups according to its carcinomatous stroma’s ability to invade the capsule; widely, minimally, and non-invasive.6,7 the purpose of this study is to analyse the pathogenesis and progressiveness of pleomorphic adenoma towards carcinoma ex pleomorphic adenoma based on the picture of changes in the atypia of its cells in order to explain whether the cases of cxpa are de novo malignant or a continuation or transformation of the pa. materials and methods this study is a retrospective study that was approved by the medical research ethics committee at the faculty of dentistry, university of jember no.791/un25.8/kepk/ dl/2019. in this study, cases of pa and cxpa were used which had been diagnosed in the anatomical pathology laboratory of dr soebandi hospital, jember, indonesia, between 2017 and 2019. the research samples were obtained through purposive sampling. samples were selected from all pa and cxpa cases based on the completeness of patient data in the histopathology report such as patient age, gender and tumour location and paraffin-embedded tissue blocks that were still in good condition. incomplete histopathology report samples (absent hpa report data or defective paraffin blocks) were excluded from the study. from the sample requirements above, twenty-four pa cases and four cxpa case controls were selected and used in this study. all tissue on the paraffin-embedding block was cut using a sliding microtome (tissue-tek, ivs-410, sakura finetek, tokyo, japan) for a thickness of 4 mµ on as many as three slides; each slide was then stained with haematoxylin and eosin staining (merck kgaa, darmstadt, germany), mallory’s trichrome (biognost d.o.o, croatia, europe) and periodic acid–schiff (scytek laboratories, logan, united states). the histopathological appearance was observed by two examiners under a light microscope (olympus cx 43, tokyo, japan) with 40x, 100x and 400x magnification. the heterogeneity of pa was examined through extracellular matrices, cell type, and morphology type. the progressivity from pa to cxpa was observed through the premalignancy atypical appearance (increase of nucleus and cytoplasm ratio, high level of mitotic activity, pleomorphism nucleus and cell, giant nucleoli, loss of cell attachment, capsule infiltration, hyalinisation, and necrosis/anaplastic) in the samples.8 we used optilab advance (miconos, yogyakarta, indonesia) to perform the observation. the data has been presented in tables and images. figure 1. variation of stroma in pa cases. a: myxoid stroma with cribriform type showing duct-like cell appearance (black arrow), (he, 40x). b: fibrous stroma showing collagen fibres (black arrow), (mallory trichrome, 40x). c: myxofibrous stroma, a blend of myxoid stroma and fibrous stroma (black arrow) that was stained with mallory trichrome (mallory trichrome, 40x). d: chondroid stroma (black arrow), (pas, 40x). e: myxochondroid stroma, a blend of myxoid stroma (m) and chondroid stroma (c), (he, 40x). f: cystic type showing pseudocyst and some microcysts (black arrow), (he, 40x). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 syafriadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 1–6 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 3 results all pa samples consisted of twelve men and twelve women, each with an average age of 44.88 years. pa most often occurs in the parotid gland (54%), followed by the submandibular gland (25%). it is not usual for pa to occur in the minor salivary glands; the latest study shows that 12.5% of pa cases occur in the palatal salivary glands. the stroma of pa was categorized into five types: myxoid, fibrous, chondroid, myxofibrous, and myxochondroid (table 1 and figure 1), and 11 of 24 samples (45.8%) were myxofibrous stroma. during observation of tumour cells, the plasmacytoid cell type was frequently observed; other common cell types were spindle, epithelioid, and clear cells. morphologic patterns of pa included trabecular, cribriform, cystic and solid cell types. we observed metaplasia in squamous, chondrocyte and adipocyte cells with atypical characteristics such as capsule infiltration or pseudopodia, necrosis hyalinisation marked by cholesterol crystals, and high level of mitotic activity (table 2 and figure 2, figure 3). some of the same characteristics were also found in cxpa such as having a myxofibrous stroma and also containing high mitotic tumour cells, hyalinization and specifically found carcinomatous foci among pa tumour cells which infiltrated forming tumour nest into the capsule like pseudopodia in case of pa (figure 4). the carcinomatous foci of cxpa displayed high mitotic levels, pleomorphism and stromal hyalinisation. table 1. prevalence of pleomorphic adenoma stroma stroma myxoid myxofibrous fibrous myxochondroid chondroid total 7 (29.2%) 11 (45.8%) 2 (8.3%) 3 (12.5%) 1 (4.2%) table 2. prevalence of cell type, morphology type, and other variants of histopathology in pa stroma ps ch h mi metaplasia cell type morphology type sq c a pl sp ep cc tr cr cs so myxoid 0 0 2 0 1 2 0 5 5 4 2 4 4 2 3 myxofibrous 2 1 0 0 4 1 0 8 8 6 0 5 7 3 2 fibrous 0 0 0 0 0 0 1 2 1 1 1 1 1 2 0 myxochondroid 0 0 0 0 0 1 0 3 1 1 0 2 0 1 0 chondroid 0 0 0 1 0 0 0 1 1 0 0 1 0 0 0 total 2 1 2 1 5 4 1 19 16 12 3 13 12 8 5 notes: ps (pseudopodia); ch (cholesterol crystal); h (hyalinization); mi (mitosis activity); metaplasia: sq (squamosal), c (cartilagenous), a (adipose); cell type: pl (plasmacytoid), sp (spindle), ep (epithelioid), cc (clear cell); morphology type: tr (trabecular), cr (cribriform), cs (cyst), so (solid) figure 2. cell type in pa cases. a: plasmacytoid type with mitotic activity (black arrow) in chondroid stroma (he, 100x). b: clear cell type (black arrow) in fibrous stroma (he, 100x). c: spindle type (black arrow), (he, 100x). d: epithelioid type (black arrow) in myxoid stroma with trabecular pattern type (he, 100x). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 syafriadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 1–6 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 4 figure 3. atypical appearance (pre-malignancy) in pa cases. a: squamous metaplasia produces keratin and showing keratin pearls appearance (black arrow), (he, 40x). b: cartilage metaplasia (black arrow), (he, 40x). c: adipose metaplasia (black arrow) is rarely metaplasia in pleomorphic adenoma, (he, 40x). d: pseudopodia (black arrow); there are two pseudopodia in one view that are surrounded by fibrous capsules (c), (he, 40x). e: cholesterol crystal (black arrow), result of adipose necrosis (he, 40x). f: hyalinisation appearance (black arrow) in solid pattern (he, 40x). figure 4. atypical appearance (malignancy) in cxpa cases. a: hypercellularity with high mitotic activity (black arrow), (he, 100x). b: capsule of tumour (c) with tumour nest infiltration (black arrow), (he, 100x). c: hyalinization with necrosis appearance (black arrow), (he, 100x). d: loss of cell attachment (he, 400x). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 syafriadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 1–6 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 5 discussion histopathologically, pleomorphic adenoma demonstrates pleomorphism of its stroma and various cells. the stroma types include myxoid, fibrous, chondroid, myxofibrous and myxochondroid stroma. the formation of pa is mainly attributed to the plag1 (pleomorphic adenoma gene 1) and p63/p40 genes. p63 is a known tumour suppressor gene located inside the myoepithelial cell.9 p63 and p40 are related to the p53 gene, which is the most commonly mutated oncogene in malignant head and neck tumours. p53 is the last defence when over-proliferation occurs; if this gene undergoes mutation, the apoptosis cycle will be disrupted. mutations in the p53 gene result in errors in protein formation that will trigger a failure to stimulate and attach other proteins in dna binding domains (dbd). their mutations, such as translocation, deletion, amplification or point mutation in several codons, can occur in exons 5 to 10. previous studies have shown that p53 activation was observed in more than 50% of neoplasm cases, especially in malignant cases.10 in a normal environment, myoepithelial cells could differentiate into ductal cells as cell regeneration occurs. it is one of the reasons why this cell is presumed to play an important role in pa formation when the p63 and p40 genes are mutated and plag1 is activated. in pa, this cell can differentiate into other cell types, such as plasmacytoid, spindle, epithelioid, and clear cells.11 plasmacytoid and spindle cells are the most common types of cell in pa composition. however, koutlas et al.12 showed that plasmacytoid cells are not the differentiation form of myoepithelial cells. the negative result of a myoepithelial cell marker test was the reason they concluded that plasmacytoid cells are the result of epithelial-mesenchymal transformation.12 pleomorphic adenomas have several morphological patterns: trabecular, cribriform, cystic, and solid. the cribriform pattern is strongly dominated by cells such as ducts, differentiated types of myoepithelial cells or the ductal cells themselves, which are also capable of producing mucus (mucoid) secretions. accumulation of mucoid material may occur between tumour cells, resulting in a myxomatous background.10 in this study, it was seen in eosinophilic images in duct-like cells, which also showed myxoid stroma images. dead cells observed with haematoxylin and eosin staining showed cell remnants and cytoplasmic components in the form of pseudocysts/microcysts. myoepithelial cells’ proliferation and differentiation ability to other cell types is quite recognisable. it may differentiate into epithelial cuboid cell of ductus, and become a hallmark of malignancy. the duct-like cell also could undergo non-cancerous change (metaplasia) into a squamous cell, which is known as squamous metaplasia. squamous metaplasia also produces keratin and causes the appearance of keratin pearls. furthermore, this cell mutation sometimes continues to occur and develops into dysplasia, marked by the increasing presence of mitotic figures and pleomorphism nuclear that signal the beginning of a malignant tumour. however, there are researchers who report that the pleomorphic adenoma transformation into cxpa is not affected significantly by the number of keratin pearls appearing. adipocyte and chondrocyte cell metaplasia was also observed in pa, which supports the possibility of metaplastic change in plasmacytoid.13 pseudopodia in pa are tumour nodules bulging from the tumour edges and separated by fibrous tissue from the main tumour mass, but still localised within the main tumour capsule because it appears inside the capsule of pa; this is known as capsule infiltration.14 it is possible that this infiltration into the capsule occurs due to mutations in the metastatic gene allowing tumour cells to destroy capsules through their proteolytic enzymes. the components of the plasminogen activation system (pas) and the metalloproteinase family [mainly matrix metalloproteinases (mmps)] are overexpressed in malignant tumours.15 it has been reported that pseudopodia are found in the myxoid matrix, and this increases the risk of recurrence, mostly in cases with enucleated treatment choice. our study of pa also found necrotic foci inside the tumour; we observed tissue necrosis caused by insufficient blood supply. the tumour cell has no supply of oxygen or nutrition which leads to its death.16 normal cells surrounding tumour cells can be necrotised due to tumour cells producing pro-inflammatory cytokines such as tnf-α, ifn. il-1β, il-3, il-5, il-6, il-8.17 in this study, we observed one case showing cholesterol crystal, the hallmark of adipocytes necrosis, metaplastic adipocytes also experienced no oxygen and nutrition supply which led to a necrotic state. this process is important to limit the tumour cells’ growth; however, it is also one of the main indicators of malignancy. the rapid growth of malignant cells is often impossible to stop through the necrosis process. the tumour only needs several months or even weeks to develop into malignancy.5,6 hyalinisation is also a signature of malignancy. pa with hyalinisation is usually the turning point condition into premalignancy.15,18 we also observed a high level of mitotic cells in one case of pa samples, even though it was not as high as the cxpa (figure 4). from all the pa samples, only three cases (12.5%) of atypical appearance were observed as similar to cxpa appearance, such as mitotic figure appearance, squamous metaplasia with keratin pearls, nuclear pleomorphism and capsule infiltration (pseudopodia). based on this study, we can conclude that the pathogenesis and progression of pleomorphic adenoma to cxpa can be seen in atypical features that serve as markers of premalignancy, such as metaplasia (squamous, adipose, and cartilage), pseudopodia, cholesterol crystals, hyalinisation, lymphoid tissue, and mitotic features. these factors can give pleomorphic adenomas the potential to transform into malignancy, although in those cases we have not been able to classify them as cxpa because specific examination (immunostaining) is required to confirm transformation from pa to cxpa. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 syafriadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 1–6 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 6 acknowledgments the authors would like to thank all those who participated in the study, particularly the director, head of the department of pathological anatomy, and laboratory members of dr soebandi hospital who permitted and facilitated our use of all the paraffin-embedding block cases in this study. we declare that no conflicts of interest took place before, during or after this study. references 1. soni a. duct pseudopodia: a new finding in parotid pleomorphic. ann clin otolaryngol. 2019; 4(1): 1036. 2. fonseca fp, carvalho m de v, de almeida op, rangel alca, takizawa mch, bueno ag, vargas pa. clinicopathologic analysis of 493 cases of salivary gland tumors in a southern brazilian population. oral surg oral med oral pathol oral radiol. 2012; 114(2): 230–9. 3. garcía jj. atlas of salivary gland pathology. cham: springer international publishing; 2019. p. 153–60. 4. fa rhat f, asn i r r a, yud h isti ra a, daulay er, saga la i p. a n uncom mon occur rence of pleomor phic adenoma in the submandibular salivary gland: a case report. open access maced j med sci. 2018; 6(6): 1101–3. 5. mariano fv, noronha alf, gondak ro, de a.m. altemani am, de almeida op, kowalski lp. carcinoma ex pleomorphic adenoma in a brazilian population: clinico-pathological analysis of 38 cases. int j oral maxillofac surg. 2013; 42(6): 685–92. 6. valstar mh, de ridder m, van den broek ec, stuiver mm, van dijk bac, van velthuysen mlf, balm ajm, smeele le. salivary gland pleomorphic adenoma in the netherlands: a nationwide observational study of primary tumor incidence, malignant transformation, recurrence, and risk factors for recurrence. oral oncol. 2017; 66: 93–9. 7. di palma s. carcinoma ex pleomorphic adenoma, with particular emphasis on early lesions. head neck pathol. 2013; 7(suppl 1): s6876. 8. auclair pl, ellis gl. atypical features in salivary gland mixed tumors: their relationship to malignant transformation. mod pathol. 1996; 9(6): 652–7. 9. chiosea si, thompson ldr, weinreb i, bauman je, mahaffey am, miller c, ferris rl, gooding we. subsets of salivary duct carcinoma defined by morphologic evidence of pleomorphic adenoma, plag1 or hmga2 rearrangements, and common genetic alterations. cancer. 2016; 122(20): 3136–44. 10. reisner e, reisner h. crowley’s an introduction to human disease: pathology and pathophysiology correlations. 10th ed. burlington: jones & bartlett learning; 2017. p. 808. 11. shah aak, mulla af, mayank m. pathophysiology of myoepithelial cells in salivary glands. j oral maxillofac pathol. 2016; 20(3): 480–90. 12. koutlas ig, dolan m, lingen mw, argyris pp. plasmacytoid cells in salivary pleomorphic adenoma: an alternative interpretation of their immunohistochemical characteristics highlights function and capability for epithelial-mesenchymal transition. oral surg oral med oral pathol oral radiol. 2019; 128(5): 515–29. 13. non it ha s, yogesh t l , na nd apr a sa d s, ma heshwa r i bu, ma ha la ksh m i i p, veerabasava ia h bt. h istomor phologica l comparison of pleomorphic adenoma in major and minor salivary glands of oral cavity: a comparative study. j oral maxillofac pathol. 2019; 23(3): 356–62. 14. dulguerov p, todic j, pusztaszeri m, alotaibi nh. why do parotid pleomorphic adenomas recur? a systematic review of pathological and surgical variables. front surg. 2017; 4: 26. 15. wyga nowsk a-świąt kowsk a m, ta r nowsk i m, mu r t ag h d, skrzypczak-jankun e, jankun j. proteolysis is the most fundamental property of malignancy and its inhibition may be used therapeutically (review). int j mol med. 2019; 43(1): 15–25. 16. lopes mld de s, barroso kma, henriques ácg, dos santos jn, martins md, de souza lb. pleomorphic adenomas of the salivary glands: retrospective multicentric study of 130 cases with emphasis on histopathological features. eur arch otorhinolaryngol. 2017; 274(1): 543–51. 17. mahyudin f, edward m, basuki mh, prawira ks. analysis of immunity in osteosarcoma a basis for development following therapy. j orthop traumatol surabaya. 2019; 7(2): 139–48. 18. bhat v, biniyam k, aziz a, yeshwanth s. carcinoma ex-pleomorphic adenoma of submandibular salivary gland: a case report and review of literature. j dr ntr univ heal sci. 2017; 6(3): 185–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p1–6 syafriadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 1–6 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p1-6 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format 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triglyceride serum level in periodontitis rima parwati sari and syamsulina revianti department of oral biology faculty of dentistry, university of hang tuah surabaya indonesia abstract background: the level of triglyceride can be used as a parameter of hypercholesterolemia. periodontitis can make the condition of hypercholesterolemia worse. stichopus hermanii extract is a source of saturated fatty acid containing omega-3 which can decrease triglyceride blood level. purpose: the aim of this research was to investigate the effect of stichopus hermanii extract in triglyceride blood level of wistar rats which got periodontitis. methods: the samples of this research were 30 rats divided into 5 groups, namely group k(–) as negative control group (without treatment), group k(+) as positive control group (induced with periodontopathogen mix), group p1 as treatment group1 (induced with periodontopathogen and stichopus hermanii extract mix, 0.09 ml/kgw), group p2 (induced with periodontopathogen and stichopus hermanii extract mix, 0,18 ml/kgw), and group p3 (induced with periodontopathogen and stichopus hermanii extract mix, 0,36 ml/kgw). then, all of those rats were sacrificed and all serum was measured for their level of triglyceride. results: all data was analyzed with anova test showing a significant result. lsd test showed a significant different between group k(–) and group k(+), and between group k(+) and group p2 and p3. conclusion: stichopus hermanii extract can decrease the triglyceride blood level in wistar rats with periodontitis. key words: hypercholesterolemia, triglyceride, stichopus hermanii extract, periodontitis, omega-3 abstrak latar belakang: kadar trigliserida dalam darah dapat digunakan sebagai parameter hiperkolesterolemia. periodontitis dapat memperburuk kondisi hiperkolesterolemia. stichopus hermanii ekstrak mengandung asam lemak jenuh terutama omega-3 yang dapat berfungsi menurunkan kadar trigliserida dalam darah. tujuan: tujuan dari penelitian ini adalah untuk mengetahui pengaruh ekstrak stichopus hermanii terhadap kadar trigliserida dalam darah tikus wistar yang mengalami periodontitis. metode: sampel penelitian ini adalah 30 ekor tikus wistar yang dibagi dalam 5 kelompok. kelompok k(–) sebagai kelompok kontrol negatif (tanpa perlakuan), kelompok k(+) sebagai kelompok kontrol positif (tikus diinduksi periodontopatogen campuran), kelompok p1 sebagai kelompok perlakuan 1 (tikus diinduksi periodontopatogen campuran dan stichopus hermanii ekstrak 0,09 ml/kgw), kelompok p2 (tikus diinduksi periodontopatogen campuran dan stichopus hermanii ekstrak 0,18 ml/kgw) dan p3 (tikus diinduksi periodontopatogen campuran dan stichopus hermanii ekstrak 0,36 ml/kgw). kemudian semua tikus dikorbankan dan diukur kadar trigliserida serumnya. hasil: semua data dianalisis dengan uji anova dan menunjukkan hasil yang signifikan, dan uji lsd menunjukkan terdapat perberbedaan yang bermakna antara kelompok k(–) dengan k(+) dan kelompok k(+) dengan p2 dan p3. kesimpulan: ekstrak stichopus hermanii dapat menurunkan kadar trigliserida darah pada tikus wistar yang mengalami periodontitis. kata kunci: hiperkolesterolemia, trigliserida, ekstrak stichopus hermanii, periodontitis, omega-3 correspondence: rima parwati sari, c/o: bagian biologi oral, fakultas kedokteran gigi universitas hang tuah. jl. arif rahman hakim no. 150 surabaya 60111, indonesia. e-mail: rima.sari@yahoo.com research report 107sari: the activity of stichopus hermanii introduction periodontal disease is recognized as an infection of periodontal tissues caused by bacteria related with the immune response of hosts which can lead to periodontal tissue destruction. meanwhile, periodontitis is an inflammation disease of tissues supporting teeth caused by a specific organism that later can cause progressive destruction of periodontal ligament and alveolar bone accompanied by pocket depth formation, gingival recession or a combination of both.1 periodontal disease, can also become a predisposing factor for cardiovascular disease due to several reasons, such as invasion of gram-negative bacteria, levels of pro inflammatory cytokines, immune system, fibrinogen, and the number of white blood cells.2 this is supported by a research conducted by kartika et al.,3 stating that the induction of bacterial periodontopathogen can increase triglyceride serum level. similarly, the high-cholesterol diet can also exacerbate inflammatory response of periodontitis caused by bacterial pathogens due to high cholesterol foods that can increase the amount of lipid deposition in various organs. as a result, the number of inflammatory molecules that circulate in the systemic tissues is increased.4,5 triglycerides are the major fats in the body that is closely associated with cholesterol, which both have a relation that cannot be separated in metabolism process.6 triglycerides are even a form of three basic human fats. unlike cholesterol stored in liver tissue or blood vessel walls, triglycerides are stored in fatty cells under the skin. high triglyceride levels will alter cholesterol metabolism of very low density lipoprotein (vldl) into a form of largevldl (l-vldl). this l-vldl form then will become the low-density lipoprotein (ldl), which finally will aggravate bad cholesterol content in blood vessels.7 the mechanism of triglycerides formation into bad fat can be clearly seen in figure 1. the consumption of foods containing fat (cholesterol and triglycerides) then can trigger the releasing of gallbladder bile acids (produced by liver) to intestine. bile acids are actually needed to form micelles or droplets of emulated fat. considering that the most of the human body contains water, cholesterol and other fatty nutrition then should be in the form of emulated ones (an emulsifier is need), so they are easily absorbed in intestine. in intestine, mixed micelles (cholesterol, triglycerides, bile acids, proteins) are absorbed, while some are discarded. mixed micelles absorbed (chylomicrons) are then secreted in liver into vldl. vldl actually contains most of fat, cholesterol, and small amounts of protein. vldl is known as bad cholesterol which then enters bloodstream and causes atherosclerosis, the narrowing or hardening of arteries. this condition causes heart disease and stroke. thus, when atherosclerosis occurs in arteries leading to brain (carotid artery), it will trigger to stroke. meanwhile, when it occurs in arteries leading to the heart (coronary arteries), it will trigger to heart attack.8 foods that are low in fat and rich in omega-3 are considered to be an effort to cope with atherosclerosis. in early observations based on empirical facts, it is known that eskimo community whose life is inseparable from fish consumption does not easily get heart diseases. the level of total cholesterol, triglycerides, and ldl cholesterol were lower in eskimo population who has fish as their daily food than the population that has left the daily consumption of fish.9 therefore, it needs to pursue marine resources for the treatment of atherosclerotic disease. indonesia is a country that has the largest marine resources, with a variety of marine resources and its utilization. one of the marine resources rich in omega 3 is a sea cucumber or gamat. gamat has many kinds, one of which is sea cucumber/gold gamat (stichopus variegates/stichopus hermanii). stichopus hermanii contains 86% protein, 80% collagen, minerals, mucopolysaccharide, glicosaminoglycans (gags), natural antiseptics, chondroitin sulphate, omega-3, 6, and 9, amino acid.10 since it is rich in omega-3 epa and dha, it can lower cholesterol levels in rats infected with porphyromonas gingivalis, and can also be useful as an additional treatment for periodontal diseases.11 based on the description above, it is necessary to do a research focused on the effects of gold sea cucumber extract (stichopus hermanii) on triglyceride serum levels in rats infected with periodontitis. figure 1. the changing mechanism of tg to vldl. 8 figure 2. the mean of triglyceride serum levels. 0 20 40 60 80 100 k(-) k(+) p1 p2 p3 108 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 106–110 materials and methods this research is a laboratory experimental research with completely randomized design.12 this research was conducted in laboratory of biochemistry, faculty of medicine, university of airlangga, in laboratory of microbiology, university of airlangga, and in laboratory of regional public health, surabaya. the samples were 30 white male rat (rattus novergicus), age 2–3 months with the weight of 200 grams. the samples were devided into 5 groups, 6 rats each before treated, the adaption process of the rats to the environtment was conducted for 1 week under monitoring. bacteria then were taken from the wobbly tooth of patients suffering from periodontitis by using sterilized paper points entered on its pocket. the revocation was actually conducted four times by using different sterilized paper points, and then incubated in incubator for 3 days. afterwards, by using a micropipette, it was taken little for making preparations followed by coloring process in order to see what kind of bacterial colonies dominated. then, it was known that bacteria dominating were gram (–) bacteria. colonies of bacteria were then diluted equivalent to mc farland 0.5 in order to be ready to be given to control group, treatment group 1, treatment group 2, and treatment group 3.13 the procedures of this research began with the acclimatization of the research animals for 7 days in a laboratory. before given treatment, white rats were fasted approximately 18 hours, but were still given water to drink. if there was rat which got pain, it would be excluded from this research. those white rats were divided into five groups: first, negative control group, k(–), which was not induced with the mix of periodontopathogen bacteria and stichopus hermanii extract; second, positive control group, k(+), which was induced with periodontopathogen bacteria, but not induced with stichopus hermanii extract; third, treatment group 1, (p1), which was induced with periodontopathogen bacteria and stichopus hermanii extract about 0.09 ml; treatment group 2, (p2), which was induced with periodontopathogen bacteria and stichopus hermanii extract about 0.18 ml; and treatment group 3, (p3), which was induced with periodontopathogen bacteria and stichopus hermanii extract about 0.36 ml. afterwards, the induction of periodontopathogen mix was conducted for 4 weeks, in which 5 days were for induction process, and 2 days were not for induction.11 during induction process, a syringe was used to put the extract into the mouth of wistar rats which was then washed manually by the researcher. the procedures of stichopus hermanii extract induction was conducted on the same day, namely in groups p1, p2 and p3 for in 14 days. furthermore, those rats in all the three groups were sacrificed, and their blood then was taken for the examination process of serum triglyceride levels. data obtained from the examining results of triglyceride serum levels in each group then were tabulated. next, the statistical test, anova test, was also conducted, and then the test was followed by least significant difference (lsd) for significance test in each group. results the mean of serum triglyceride levels in each group can be seen in figure 2. the results of anova test showed the differences of the mean of triglycerides serum levels. meanwhile, the results of lsd test shows that there were significant differences (sig < 0.05) between k(–) group and k(+) group (p = 0.006), k(+) group and p1 group (p = 0.026), p2 group (p = 0.001) and p3 group (p = 0.000), p1 group and p3 group. there was no significant differences (sig > 0.05) between k(–) group and p1 group (p = 0.541), p2 group (p = 0.559) and p3 group (p = 0.103), p1 group and p2 group (p = 0.237), and p2 group and p3 group (p = 0.282) as can be seen in table 1. discussion triglycerides are fats contained in blood which tends to rise due to alcohol consumption, weight gain, sugar and fat rich diet, and lack of exercise. the increasing of triglycerides triggers the risks of heart disease and stroke. from chemistry point of view, triglyceride is considered as a substance consisting of glycerol binding fatty acid chain. thus, consuming foods containing fat will increase triglycerides in blood and cholesterol levels.7 besides that, poor oral hygiene, such as periodontitis, can also increase triglycerides. this fact is also supported by some researches showing that the severity of periodontal disease is positively related to cholesterol plasma levels. it means that periodontitis is associated with the increasing of triglycerides.3 thus, this research also prove that the table 1. the results of lsd test dependent variable treatment group k(+) p1 p2 p3 levels of tg serum k (–) 0.006* 0.541 0.559 0.103 k (+) 0.026* 0.001* 0.000* p1 0.237 0.029* p2 0.282 note: *signifi cant difference 109sari: the activity of stichopus hermanii induction of periodontopathogen mix can significantly elevate the levels of triglycerides serum. pathogenesis of these conditions are actually due to lipopolysaccharide (lps) produced by periodontopathogen (on a negativegram bacteria) that can inhibit the metabolism of fats resulting in the increasing of triglycerides later. under the conditions of inflammation and periodontitis, body attempts to localize by producing cytokines, namely interleukin-1β (il-1β) and tumor necrotizing factor-α (tnf-α) which have effect on fat metabolism by influencing the production of other cytokines and the use of amino acid and other ingredients included in the metabolism of fat. thus, if il-1β and tnf-α are increasing, hyperlipidaemia will potentially occur. it is because pro-inflammatory cytokines can increase intracellular cholesterol levels in some cell types, for instance, il-1β can increase cholesterol accumulation in mesengial cells by regulating receptor of receiver via ldl receptor dysregulation. this leads to il-1β and tnf-α that can accelerate the increasing of triglycerides serum.14,15 in addition, lps derived from periodontopathogen can also trigger the decreasing of lipoprotein lipase activities during infection, while the function of lipoprotein lipase is breaking triglycerides. if triglycerides cannot be broken down, it then can be buried and can cause the increasing of cholesterol levels.14 this possibility can occur since triglycerides are metabolized in liver from fatty acid derived from carbohydrate lipolysis, proteins, fats, and alcohol consumed daily which with apo-b lipoproteins can form vldl excreted into blood circulation. vldl then will be separated from triglycerides by lipase lipoprotein enzyme altering into intermediate density lipoprotein (idl) and then into ldl that can very easily be oxidized and can also damage high-density lipoprotein (hdl) later, as a result, it finally will aggravate cholesterol content in blood vessel.7 patients with periodontal disease also have high levels of c-reactive protein serum (crp) and fibrinogen, called as acute-phase proteins (acute phase proteins), in which patients have more white blood cells than those in healthy individuals. crp can only be synthesized by hepatocytes, and its making is controlled by il-6, a cytokine that has proagglomeration and pro inflammatory activities often isolated from bacterimist patients. the levels of crp serum can be increased 100 times, while the levels of fibrinogen can be increased 10 times in response to antimicrobial diseases. fibrinogen is actually an essential component for agglomerating working in platelet function and considered as an ingredient for the formation of fibrin. fibrinogen is also considered as an acute phase protein which can be increased as a result of systemic infections. thus, the increasing level of fibrinogen in thrombosis can become a risk indicator of atherosclerosis.16 the induction of stichopus hermanii containing omega3 was proved to be effective in lowering triglyceride serum levels based on the results of lsd test which showed that there were no significant differences between k(+) group and all the other treatment groups induced with stichopus hermanii (p > 0.05). omega-3 actually consists of eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), but gold sea cucumber (stichopus hermanii) has high levels of epa and dha, 25.69% and 3.69%. the high level of epa in stichopus hermanii can indicate that the speed of repairing damaged tissue and the prevention of the prostaglandins formation causing inflammatory and cholesterol reduction in blood.17 meanwhile, dha is considered as the major fatty acid in sperm, brain, and eye’s retina. thus, the high level of dha intake can lower blood triglycerides causing heart disease.18 the mechanism of omega-3 fatty acids disease in lowering hyperlipidemia is by increasing the excretion of cholesterol in feces and suppressing the synthesis of triglycerides in liver so that it can alter the composition of fatty acids contained in lipoproteins. as a result, lipoprotein fluidity does not only become increasing, but also affects the activity of lipolytic enzymes as well as increases the speed of synthesis and catabolism of vldl in plasma. on the other words, it can also be said that omega-3 can affect lipolysis of fatty tissue, so triglycerides are not formed by the reaction of free fatty acids and glycerol.19 it can be concluded that golden sea cucumbers extract (stichopus hermanii extract) decreasing of triglyceride serum levels in rats which got periodontitis. references 1. newman mg, takei h, carranza fa. clinical periodontology. 9th ed. philadelpia: wb saunder co; 2002. p. 65–339. 2. li x, kolltveit km, tronstad l, olsen i. systemic disease caused by oral infection. clin microbiol rev 2000; 13: 547–58. 3. kartika lw, sari rp, revianti s. pemberian ikan lemuru terhadap kadar serum trigliserida tikus yang hiperkolesterol dan periodontitis. proceeding sem kel; 2011. p. 131–5. 4. saijo y, kiyota n, kawasaki y, miyasaki y, kashimura j, fukuda m. relationship between c-reactive; protein and visceral adipose tissue in healthy japanese subject. diabetes obes metab 2004; (6): 249–58. 5. warnberg j, moreno la, mesana mi, marcos a. inflammatory mediators in overweight and obese spanish adolescent. the avena study. int j obes relat metab disord 2004; 28: s59–s63. 6. noer s. ilmu penyakit dalam. edisi ketiga. jakarta: balai penerbit fku i; 2006. p. 718. 7. soeharto i. kolesterol dan lemak jahat, kolesterol dan lemak baik, dan proses terjadinya serangan jantung dan stroke. cetakan 2. jakarta: gramedia pustaka utama; 2002. p. 47–8, 182–3. 8. rogers a, vaughan p, prentice t, edejer t, evans d, lowe j. reducing risks, promoting health life. world health report 2002, who october 2002. p. 257–60. 9. simopoulos, artemis p. omega-3 fatty acids in inflammation and autoimmune diseases. j of the american college of nutrition 2002; 21(6): 495–505. 10. sendih g. keajaiban teripang penyembuh mujarab dari laut. jakarta: agromedia pustaka; 2006. p. 21–6. 11. kesavalu l. omega-3 fatty acid effect on alveolar bone loss in rats. j dent res 2006; 85(7): 648–52. 12. hanafiah ka. rancangan percobaan, teori dan aplikasi. fakultas pertanian universitas sriwijaya, jakarta: pt raja grafindo persada; 2003. p. 56–60. 13. sari rp. black seed (nigella sativa) effect to the number of lymphocytes and plasma cells in mice (mus musculus) gingival induced by mix bacteria. maj ked gigi 2009; edisi khusus timnas v: 97–100. 110 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 106–110 14. fentoglu o, bozkurt fy. the bi-directional relationship between periodontal disease and hiperlipidemia. eur j dent 2008; 2: 142–6. 15. persson j, nilsson j, lindholm mw. interleukin-1 beta and tumour necrosis factor-alpha impede neutral lipid turnover in macrophagederived foam cells. bmc immunology 2008; 9(70): 1–11. 16. korman s, kenneth, thomas wg. fundamentals of periodontitis. chicago: quintessence publishing co, inc; 2003. p. 573–577. 17. yulianto p. pengaruh ekstrak gamat (stichopus hermanii) terhadap poliferasi sel fibroblast pada proses penyembuhan luka tikus wistar. skripsi. surabaya: fakultas kedokteran gigi, universitas airlangga; 2008. 18. ryan as, keske ma, hoffman jp, nelson eb. clinical overview of algal-docosahexaenoic acid: effects on triglyceride levels and other cardiovascular risk factors. am j therapeutics 2008; 0(0): 1–9. 19. nurjanah. omega-3 dan kesehatan. disertation. program pascasarjana institut pertanian bogor; 2002. 6262 case report management of palatal perforation in systemic lupus erythematosus patient dwi setianingtyas,1,2 paulus budi teguh,3.4 widyastuti,5 neken prasetyaningtyas,1 ramadhan hardani putra6, and felicia eda haryanto2 1 oral medicine specialists of the dental department, dr. ramelan naval hospital 2 department of oral medicine, faculty of dentistry, universitas hang tuah 3 prosthodontics specialists, dental department, dr. ramelan naval hospital 4 department of prosthodontics, faculty of dentistry, universitas hang tuah 5 department of periodontics, faculty of dentistry, universitas hang tuah 6 department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: systemic lupus erythematosus (sle) is an autoimmune disease which damages tissues and causes chronic inflammation with an idiopathic etiology. it has been suggested that oral lesions in patients with sle can be grouped clinically as erythema, discoid lesions and oral ulcerations. the latter have been said to indicate the onset of a severe systemic disease flare and that oral ulcers represent cases of mucosal vasculitis. palatal lesions generally present in the form of ulcers or, in more severe forms, as perforation. acquired palatal perforations can be caused by several etiologies including: developmental disorders, malignancy and infections. purpose: to report the management of palatal perforation in an sle patient. case: a 14-year-old female patient attended the dr. ramelan naval hospital, with both a perforated palate that often caused her to choke when eating or drinking and maxillary anterior tooth mobility. case management: the treatment for the patient in this case consisted of debridement and dhe, pharmacological therapy including aloclair gel and minosep mouthwash to maintain oral hygiene and prevent re-infection. at the end of the first consultation, the patient was prescribed an obturator in order to avoid oro-anthral infection. during the second consultation, the patient’s orthodontic bracket was removed to facilitate scaling and splinting of the anterior maxillary teeth carried out to prevent their movement. during the third consultation, a swab was taken by an oral surgeon who also administered antifungal therapy. during the fourth and final consultation, the patient was examined a prosthodontic specialist due to an obturator which was causing discomfort. conclusion: the management of palatal perforation lesions in an sle patient requires a multidisiplinary approach. keywords: systemic lupus erythematosus; palatal perforation; oral manifestation; candida; obturator correspondence: dwi setianingtyas, dental department, dr. ramelan naval hospital, surabaya. jl. gadung no. 1, surabaya, indonesia. e-mail: dwi.setianingtyas.anik@gmail.com. introduction systemic lupus erythematosus (sle) constitutes a chronic systemic autoimmune disease1,2 whose precise etiology remains unexplained. however, there are certain pre-dispositional factors underlying it, including: genetics, infection, hormones, antibodies, the immune system, sunlight, diet, stress and physical fatigue.2 sle can present itself in a wide variety of ways. it may affect one or more organs, being characterized by widespread inflammation of the blood vessels and connective tissues, and tends to be episodic with intermittent periods of remission. according to the american college of rheumatology (acr), a key symptom of sle is the presence of a malar rash affecting both cheeks known as a facial butterfly rash3 which is usually accompanied by joint and muscle pain, fatigue, hair loss (alopecia), anemia, oral ulcers, high fever, decreased appetite and chest pain or chest tightness among other symptoms. while individual patients present contrasting symptoms, the presence of four of these support a diagnosis of sle3,4 which can attack parts of the body such as the skin, oral cavity, joints, blood and even the internal organs, dental journal (majalah kedokteran gigi) 2018 june; 51(2): 62–66 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p62–66 mailto:dwi.setianingtyas.anik@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p62-66 63 setianingtyas, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 62–66 for example the kidneys and lungs to name but a few. the type of illness and organs affected differ from one patient to another with the result that it is referred to as an individual disease.4,5 approximately 75% of people with sle present various symptoms in the oral cavity including an oral ulcer located in the buccal mucosa, palate, periodontitis with floating teeth and a dry mouth accompanied by a burning sensation, especially when eating spicy or hot foods.1–3,5 according to sete, et al.1 several studies exist confirming the association between chronic periodontitis and sle conditions, characterized by damaged periodontal tissue resulting in floating teeth and ulcers on the oral mucosa, and most of them on the hard palate. a diagnosis of sle can be established through anamnesis, characteristic clinical manifestations and supporting investigation, such as complete blood tests and an antinuclear antibody (ana) test. from this examination, the existence of the autoimmune process can be determined.4 the main treatment for sle is the administering of corticosteroids as an immunosuppressant because when the condition occurs the pre-dispositional factors result from abnormalities in the immune system. further therapy may consist of anti-malarial drugs or other symptomatic treatment. in immunocompromised states, long-term treatment with corticosteroids and antibiotics may cause the microorganisms to develop into pathogenes.6 corticosteroid therapy should be conducted under the supervision of competent medical personnel due to the side effects of long-term treatment with corticosteroids including: osteoporosis, elevated blood sugar levels, hypertension (resulting in a full moon face) in addition to the risk of infection, both viral and bacterial, and fungal infections.7 the most common type of fungus found in the oral cavity is candida two strains of which, candida albicans and non-candida albicans (ncac), cause fungal infection referred to as oral candidiasis. 84.8% of cases of oral candidiasis, including: candida crusei, candida tropicalis, candida parapsilosis, candida kefyr, and candida famata, are caused by candida albicans and ncac.8 in recent years, the prognosis for individuals suffering from sle has improved considerably when treated with appropriate therapy and the greater ease of access to both information and the health services equipped to treat the condition.2 the purpose of this paper was to report the management of palatal perforation in sle patients which can provide insight for dentists working in an integrated multidisciplinary manner in other fields in order to offer optimal care in sle cases requiring holistic treatment. case a 14-year-old female referred by the prosthodontics department attended the oral medicine department at dr. ramelan naval hospital in surabaya together with her father. the purpose of this prosthodontic referral was to ascertain whether a patient diagnosed with sle could undergo a safe palate perforation involving the creation of an obturator, since the prosthodontist himself had been referred by an oral surgeon. the patient’s chief complaint was that of frequent choking while eating and drinking due to palate perforation, although she experienced little pain. her father stated that between january and march 2017 the patient had already been hospitalized at rsud bojonegoro with dengue fever on four occasions. her blood test results confirmed that she had experienced a decrease in both platelets and hemoglobin. on march 17 2017, the doctor treating the patient referred her to dr. ramelan naval hospital in surabaya where she was admitted as an in-patient. the provisional diagnosis was one of suspected hypovolemic shock and febrile neutropenia with pancytopenia. the patient had undergone a complete blood examination and ana test at the clinical pathology laboratory of dr. ramelan naval hospital, surabaya, the results of which supported a diagnosis of sle. when the patient was hospitalized at dr. ramelan naval hospital, rashes suddenly developed on both cheeks which, while not itchy or tender themselves, were accompanied by painful cracked lips. in early april, following the patient’s discharge from hospital, a red ulcer appeared on her palate which, on april 25, turned white. despite an increase in the 10 figure 1. a) palate middle: ulcer, with painful bone exposure. b) patient with a fixed orthodontic brace. figure 2. the results of splinting using palatal wire. figure 3. a patient’s dental impression. a b figure 1. a) palate middle: ulcer, with painful bone exposure. b) patient with a fixed orthodontic brace. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p63–67 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p62-66 64setianingtyas, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 62–66 ulcer’s size, the fact that it was pain-free led to its being regarded as merely an ordinary in nature. by the end of june 2017, the ulcer had a needlepoint hole which gradually grew to its current size (figure 1a, 1b). on extra-oral examination, a blackish ulcer was found on the skin of the hand. the patient, who had been fitted with a removable orthodontic appliance, was also found to be suffering from lymphadenopathy. intra-oral examination of the palate region revealed an ulcer and necrotic exposure of the palate bone which caused considerable pain. moreover, the maxillary front teeth appeared to be mobile. although her general condition appeared to be relatively good, the patient appeared somewhat depressed. the patient had been placed on a range of medication by various doctors. the internist had prescribed methylprednisolone, chloroquine, folic acid, sandimmune, and cavit-d3; the oral surgeon mefenamic acid and cefadroxil; and the psychiatrist based at rsud bojonegoro a concoction of mood-enhancing medicines. case management visit i (july 6th, 2017): according to the medical records, on the first visit, notes existed on the results of the panoramic photo, blood test and ana test previously conducted. an ana test examination confirmed the patient to be ana ifm positive as determined by previous diagnoses of sle cases manifested in the oral cavity. management of the condition consisted of a combination of debridement and dental health education (dhe), while pharmacologic therapy combined the application of aloclair gel (aloe vera extract) to reduce pain and minosep mouthwash to maintain oral hygiene and prevent secondary infection. in order to address prosthodontists’ uncertainty regarding whether the use of obturators as a symptomatic and rehabilitative therapy was permitted, the authors, as oral medicine specialists, sanctioned their manufacture. instructions governing the use of obturators stated that they should not be used continuously but, rather, only during eating in order to avoid the possibility of an allergic reaction should the patient suffer from an autoimmune disease. another piece of advice stated that the obturator must be removed and cleaned at night to avoid fungal infections potentially exacerbating the patient’s existing condition. the treatment plan to be implemented in sle cases included several essential steps. first, implementation of an integrated approach involving cooperation with other specialists such as periodontists, orthodontists, prosthodontists and oral surgeons. second, removal of the orthodontic bracket. third, completion of scaling and splinting of the teeth in question. fourth, taking an impression for the manufacture of an obturator. fifth, swabbing the mouth in order to produce a tissue culture as a means of identifying the microorganisms present. sixth, referral to an anatomical pathology specialist for assessment of the condition of the surrounding tissue. lastly, regular attendance of the control by the patient. visit ii (july 7th 2017): the processes of anamnesis and clinical examination conducted during the second consultation were identical to those of the patient’s first visit. in accordance with the original plan, the management on the second day included: removal of the orthodontic bracket, scaling and splinting work in the resorbing region (figure 2) involving teeth 11, 12, 13, 21, 22, and 23 (loose teeth with 2-3 degrees of mobility), and taking an impression for the manufacture of an obturator (figure 3). visit iii (july 10th. 2017): on the third visit (second control), the patient’s pain had been reduced due to her having obeyed fully all the instructions issued during the previous consultation. on this occasion, management consisted of performing a swab examination in order to identify the type of fungus present which was conducted by an oral surgeon experienced in the investigation of fungal infections. in the case reported here, the presence of a deep fungal infection was suspected because of the existence of perforation. consequently, the patient was administered an antifungal therapy involving fluconazole 200 mg which is regarded as the gold standard for such treatment. in the final step, an obturator was inserted. visit iv (july 18th 2017): on the fourth visit, the anamnesis conducted confirmed that the patient felt more comfortable with the obturator, despite the continued 10 figure 1. a) palate middle: ulcer, with painful bone exposure. b) patient with a fixed orthodontic brace. figure 2. the results of splinting using palatal wire. figure 3. a patient’s dental impression. a b figure 2. the results of splinting using palatal wire. 10 figure 1. a) palate middle: ulcer, with painful bone exposure. b) patient with a fixed orthodontic brace. figure 2. the results of splinting using palatal wire. figure 3. a patient’s dental impression. a b figure 3. a patient’s dental impression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p62–66 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p62-66 65 setianingtyas, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 62–66 leakage she experienced when eating or drinking. her swab result confirmed the presence of both candida crusei and candida tropicalis. the subsequent patient management involved a prosthodontic check-up to correct the functional use of the obturator (figure 4). the final step consisted of referring the patient to the anatomy pathology department in order to ascertain the recent condition of the lesion on her palate. discussion infection constitutes the entry of microorganisms into the tissues or body fluids, accompanied by both local and systemic clinical symptoms, which can occur on the skin, in soft tissues, in ulcers and in surgical wounds. infectious diseases caused by a variety of viruses, bacteria and fungi are a major cause of morbidity and mortality.9 in the past, fungal infections have been continually underestimated. therefore, in the present day, they should attract serious attention not only because of a failure to diagnose them correctly, but also due to their potential to lead to higher mortality. the incidence of fungal infection is classified as an endemic condition, for example: histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis and opportunistic fungal infections. 10 fungal infections that are primarily opportunistic might demonstrate severe progression, particularly in patients with immunocompromised conditions including: diabetes mellitus, malignancy and sle among others.4 in the oral cavity, this species is found commensally in 40% of the intra oral healthy population and dominates 90% of isolates from patients with candida albicans.4 although the most frequently found species is candida albicans, recently the incidence of ncac infection has continued to increase.8 over 30% of sle patients often present problems with the salivary gland where an unstimulated saliva flow rate results in decreased salivary production and, in turn, severe sjögren’s syndrome, oral candidiasis and xerostomia.2 oral ulcers, painless lesions often found in the buccal mucosa and palate, have been identified as the major diagnostic manifestation of sle by the american rheumatism association committee on diagnostic therapeutic criteria. such ulcerative lesions are usually small (less than 1 cm), although sle-related oral lesions observed in the palate tend to be longer and larger, resembling red plaques surrounded by white areas.2 the patient’s major complaint in this case was that of an oral complication arising from palate perforation which caused her discomfort when eating or drinking because she often started to choke. from analysis of the swab taken, the presence of candida crusei and candida tropicalis was confirmed. similarly, an anatomical pathology examination revealed the role of fungi and bacteria. the possibility of etiopathogenesis from growth of the ulcer to perforation of the palate was investigated through several processes. as previously explained, the intact mucosa constitutes one of the body’s innate immunity system against fungal infections. when an ulcer is present in a patient’s mucosa, the mucosa is no longer intact a condition which facilitates invasion by microorganisms. long-term use of corticosteroid therapy results in the emergence of candida tropicalis and candida crusei which both belong to the candida albicans family. these generally cause diseases in tropical climates, especially in patients with leukopenia, neutropenia and immunocompromised conditions, as in the case of sle, and facilitate the development of a fungemia pathogen. candida tropicalis is one ncac species demonstrating the highest level of virulence due to its attachment ability, the strongest among epithelial cells, which can secrete protein at medium levels.8 the fungus entering the body induces a response from the host’s immune system. igm and igg are produced and circulated by the bloodstream in response to fungal infections. in fact, these two kinds of candida are commensal in nature, but they can develop into pathogens. in the presence of fungal exposure, this phenomenon can depend on the type and degree of immune response from the host. cellular immune responses are a major mediator of the body’s resistance to fungal infections. neutrophils and phagocytes play an important role in eliminating fungi.10 sete, et al.1 state that, with regard to sle, there are several possible causes such as: disruption of t cell function, genetic defects in the immune response control system, abnormal macrophage functioning, b cell damage, ineffective host response to an infectious agent or a combination of several of these elements. candida tropicalis and candida crusei are primarily commensal in healthy individuals, but can cause oral candidiasis accompanied by painful symptoms in immunocompromised individuals. in the case of sle, because of a compromised immune system highlighted by sete, et al.1, severe damage may result in defects in the bone palate which can develop into necrosis potentially culminating in perforation of the bone palate. kumar, et al.11 added that, in sle, the lesion initially 11 figure 4. application of an obturator by a prosthodontist. figure 4. application of an obturator by a prosthodontist. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p63–67 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p62-66 66setianingtyas, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 62–66 appears as an irregular whitish area that then extends towards the peripheral, after which its center reddens and develops into an ulcer with a hyperkeratotic edge. in this case, according to the anamnesis obtained from the patient’s closest blood relative (her father), she initially had a reddish ulcer which then turned white and grew in size until, finally, a small hole appeared in the palatal mucosa. this gradually expanding hole occurs due to deficient tissue healing and the failure of the patient’s immune system which enables the candida to develop into a pathogen. this statement is supported by the immunocompromised condition of the patient diagnosed with sle, an autoimmune disease, which renders the tissue more easily infected. the treatment for the patient in this case consisted of provision of the debridement and dhe, pharmacological therapy using aloclair gel and minosep mouthwash to maintain oral hygiene and prevent a second infection after the first visit. at the end of that visit, the patient used an obturator in to order to avoid an oro-anthral infection. during the second visit, the patient’s orthodontic bracket was removed and scaling and splinting of the anterior maxillary teeth performed to prevent movement of the anterior maxillary teeth. the third visit witnessed a swab being undertaken and antifungal therapy administered by the oral surgeon. although nystatin and amphotericin b are the drugs most commonly used locally, fluconazole oral suspension is proving to be very effective in treating oral candidiasis. fluconazole was found to be the drug of choice for the systemic treatment of oral candidiasis.12 during the final visit, the last patient consulted the prosthodontic specialist due to an obturator which was causing discomfort. in addition to the reduced host immune response, sle was suspected based on several symptoms, including xerostomia, characterized by a dry mouth which results in reduced saliva production. since saliva contains several antifungal elements such as lysozyme, histatin, lactoferrin, and calprotectin, its reduced production causes a decrease in cellular and humoral immunity.13 sle disease is an autoimmune disease that affects many women of reproductive age with clinical symptoms affecting many organs, one being the oral cavity. the chief complaints arising from palate perforation include: impaired intake processing of food, difficulty in talking or communicating and the production of a nasal sound during speech. moreover, the ongoing infection caused by the disease leads to halitosis. all of the above effects can undermine the self-confidence of children and, indeed, the teenage patient who was the subject of this case study was very concerned about her appearance.2 sle disease affecting the oral cavity can negatively impact the quality of life of the patient. therefore, the handling of oral manifestations of this disease should be undertaken carefully to ensure that patients receive proper care supportive of the success of the treatment. in conclusion, commensal candida may develop into pathogens in immunocompromised hosts such as sle. references 1. sete mrc, figueredo cm da s, sztajnbok f. periodontitis and systemic lupus erythematosus. rev bras reumatol. 2016; 56(2): 165–70. 2. prihantini nr, masulili slc. perawatan periodontal pada pasien lupus eritematosus sistemik. maj ked gi ind. 2012; 19: 72–6. 3. uva l, miguel d, pinheiro c, freitas jp, marques gomes m, filipe p. cutaneous manifestations of systemic lupus erythematosus. autoimmune dis. 2012; 2012: 1–15. 4. glick m, feagans wm. burket’s oral medicine. 12th ed. shelton: people’s medical publishing house; 2015. p. 495–500. 5. waluyo s, putra bm. 100 questions & answers: lupus, manis namanya, dahsyat gejalanya. jakarta: pt elex media komputindo; 2012. p. 164. 6. dangi ys, soni ml, namdeo kp. oral candidiasis: a review. int j pharm pharm sci. 2010; 2(4): 36–41. 7. mozayani a, raymon lp. buku ajar interaksi obat: pedoman klinis & forensik. jakarta: egc; 2008. p. 261–5. 8. lukisari c, setyaningtyas d, djamhari m. penatalaksanaan kandidiasis oral disebabkan candida tropicalis pada anak dengan gangguan sistemik. dentofasial. 2010; 9(2): 78–85. 9. baghela a, thungapathra m, shivaprakash mr, chakrabarti a. multilocus microsatellite typing for rhizopus oryzae. j med microbiol. 2010; 59(12): 1449–55. 10. nasronudin. penyakit infeksi di indonesia: solusi kini & mendatang. 2nd ed. surabaya: airlangga university press; 2011. p. 507–9. 11. kumar ps, leys ej, bryk jm, martinez fj, moeschberger ml, griffen al. changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16s cloning and sequencing. j clin microbiol. 2006; 44(10): 3665–73. 12. garcia-cuesta c, sarrion-pérez m-g, bagán j v. current treatment of oral candidiasis: a literature review. j clin exp dent. 2014; 6(5): e576–82. 13. setyaningtyas d, hardiyanti s, ivan n, revianti s, ramadhan hp. thrush pada pasien diabetes melitus disertai lesi premalignant ( t h r ush i n pat ient wit h d iab et es mel l it us susp e ct e d wit h premalignant lession). j pdgi. 2015; 64(3): 136–41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p62–66 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p62-66 213213 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 213–216 application of chitosan scaffolds on vascular endothelial growth factor and fibroblast growth factor 2 expressions in tissue engineering principles ariyati retno pratiwi,1 anita yuliati,2 istiati soepribadi,3 and maretaningtias dwi ariani4 1master program in dental health science 2department of dental material 3department of oral pathology and maxillofacial 4department of prosthodontics faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: tissue engineering has given satisfactory results as biological tissue substitutes to restore, replace, or regenerate tissues that have a defect. chitosan is an organic biomaterial often used in the biomedical field. chitosan has biocompatible, antifungal, and antibacterial properties. chitosan is osteoconductive, suitable for bone regeneration applications. bone defect healing begins with inflammatory phase as a response to the presence of vascular injury, so new vascularization is required. vascular endothelial growth factor (vegf) and basic fibroblast growth factor-2 (fgf2) are indicators of the beginning of bone regeneration process, playing an important role in angiogenesis. purpose: this research was aimed to determine the effects of chitosan scaffold application on the expressions of vegf and fgf2 in tissue engineering principles. method: chitosan was dissolved in ch3cooh and naoh to form a gel. chitosan gel was then printed in mould to freeze dry for 24 hours. those rats with defected bones were divided into two groups. group 1 was the control group which defected bones were not administrated with chitosan scaffolds. group 2 was the treatment group which defected bones were administrated with chitosan scaffolds. those rats were sacrificed on day 14. tissue preparations were made, and then immunohistochemical staining was conducted. finally, a statistical analysis was conducted using kruskal wallis test. result: there was no significant difference in the expressions of vegf and fgf2 between the control group and the treatment group (p>0.05). conclusion: chitosan scaffolds do not affect the expressions of vegf and fgf2 during bone regeneration process on day 14 in tissue engineering principles keywords: chitosan; vegf; fgf2; tissue engineering correspondence: ariyati retno pratiwi, c/o: peserta program s2 ilmu kesehatan gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ariyatiretnop@gmail.com introduction tissue engineering is an application of the principles and methods of engineering used to restore, maintain, or improve tissue function. the principles of tissue engineering are conducted by providing appropriate materials to trigger the cells in order to regenerate or combine the performance of the cells in the body by using scaffold to trigger the growth of new tissue. tissue engineering has given satisfactory results as biological tissue substitutes to restore, replace, or regenerate tissue suffering from defects.1 cells, scaffold, and growth stimulating signals are generally called as tissue engineering triad, a major component of tissue engineering. scaffold is an important material in tissue engineering. scaffold is a very porous artificial extracellular matrix used for cell accommodation, cell growth, and tissue regeneration.2 scaffold must meet some requirements, such as has interconnecting pores appropriate to support tissue integration and vascularity. it made from materials that have dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p213-216 214 pratiwi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 213–216 certain properties, such as biodegradation or bioresorption. as a result, the tissue will eventually be replaced with scaffolds, which have a surface suitable for supporting cell attachment, cell differentiation, and cell proliferation, as well as have good mechanical properties, easily made into a variety of shape and size.3 organic biomaterials are often used in the biomedical field, especially chitosan application. chitosan has biocompatibility, antifungi, and antibacteria properties.4 chitosan also is osteoconductive, so it is suitable for hard tissue regeneration application, but the mechanical properties and biological activities need to be improved.5 the biological properties of chitosan that very influential in the process of wound healing are biodegradability, biocompatibility, and antimicrobial activity. the high biodegradation rates can trigger chitosan to be increasingly inadequate in accelerating wound healing. biodegradability of chitosan also affects its biocompatibility because hight degradation will increase amino sugar accumulation and inflammatory response.6 bone healing process is a series of molecular and cellular process as well as tissue transformation, from resorption to hard and soft tissue formation. bone defect healing process begins with inflammatory phase, which is vascular response to injury, requiring new vascularization. vascularization plays an important role in osteogenesis during the bone healing process.7-9 increased formation of blood vessels in the area indicates that wound epithelialization process occurs faster.10 bone regeneration can be affected by vascular endothelial growth factor (vegf) directly or indirectly. vegf plays an important role in the formation of new blood vessels serving to mobilize and recruit endothelial progenitor cell (epc), a well as to differentiate and proliferate endothelial cells.8 vegf induces angiogenic process through endothelial cells. bone-forming precursor cells migrate through the bloodstream to the callus that will differentiate into osteoblasts.9 vegf affects osteogenesis from day 14 to day 21 after the defect occurred. in addition to vegf, basic fibroblast growth factor 2 (fgf2) plays an important role in the process of vascularization. vegf and fgf2 can also stimulate fibroblasts to migrate to the defect and trigger collagen synthesis. fgf2 expression occurs in the early phase of bone healing process to form osteoblasts.11 fgf2 also plays a role in mitogenesis of mesenchymal cells, proliferating and differentiating into progenitor cells. progenitor cells will differentiate into osteoblasts as bone formation cells.13 this research was aimed to determine the effects of chitosan scaffold application on vegf and fgf2 expressions in tissue engineering principles. materials and method this research was a laboratory experimental research with post control group design. animals used were male rats (rattus norvegicus) aged 3 months old and weighed 250 grams. materials used in this research were chitosan with deacetylation 81% (sigma 93646, usa), a solution of acetic acid (ch3cooh) and naoh (merck, germany), vegf polyclonal antibody (biossusa), and fgf2 polyclonal antibody (biossusa). tools used in this research were a glass beaker, connicle tube, freezer (royal chest freezer bd 195, china), spatula glass, scales (pioneer, usa), magnetic stirrer (hanna, usa), and freeze-dryer (heto fd3, en 87 164, japan). chitosan scaffold was synthesized by dissolving chitosan powder into a solution of acetic acid (ch3cooh 0.5m) and sodium hydroxide (naoh 0.1m), and then centrifuged at a speed of 9000 rpm for 10 minutes. supernatant in the form of chitosan gel was inserted into the mold. the mold already containing chitosan gel was frozen at a temperature of -20° c using deep freezer for 2 hours, and then freeze-drying was conducted for 24 hours to form a porous three-dimensional structure, known as scaffold. research procedure was performed into several phases. those six rats were divided into two groups. group 1 was the control group, while group 2 was the treatment group. those rats were acclimatized for one week. the manufacture of bone defects was performed in both groups by drilling the rats’ femoral and dextral areas. during the making of bone defects, irrigation was made using ringer solution. in the defect area of the control group, placebo scaffold was applied to each defect using tweezers and escavator, and then sutured with 3/0 non-absorbable blacksilk thread on muscles. in the defect area of the treatment group, chitosan scaffold was applied to each defect using tweezers and escavator, and then sutured with 3/0 nonabsorbable black-silk thread on muscles. on the 14th day after the closure of the defect, those rats were anesthetized using 10% ether as asphyxiation, and then sacrificed. the defected tissues of those rats were cut. the pieces of the tissues were fixed with 10% formalin for two days. they were decalcified using edta in order to soften the tissues to facilitate the process of cutting. the tissues were cut to a thickness of 0.3 mm. dehydration process was carried out using alcohol. those pieces of the tissues were put in xylol solution three times. paraffin infiltration was conducted by melting solid paraffin. embedding tissue was performed by pouring paraffin into molding devices. the tissues were taken using tweezers, and then implanted into the mold, which had been filled by paraffin. the mold, which had been filled by paraffin and tissue, was cooled to form paraffin blocks. those rats that had been sacrificed were then buried properly. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p213-216 215215pratiwi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 213–216 immunohistochemical preparations were performed by cutting the paraffin block using a rotary microtome. the pieces placed in the glass object was then deparaffinized using xylol solution. the preparations were dripped with normal serum evenly on a glass object, and then put in a preparation box (humidity chamber) with tissue paper, etched with pbs to keep the moisture. the preparations were put in an incubator at a temperature of 37º c for 45 minutes. the tissues were dripped with primary antibody against inducible nitric oxyde synthase (inos) enzyme, and incubated at 4° c for one night. the tissues then were dripped with secondary antibody (biotinylation), and incubated at 37º c for 35 minutes. the administration of chromogen was performed by dripping a solution of 3,3’diaminobenzidine (dab), then incubated at 37° c for 35 minutes. counter stain was conducted by dripping hematoxylin shed evenly, then left for 15 seconds and washed with running water. dehydration was carried out with alcohol solution 70%, 80%, 90%, and 100%. clearing with xylol (i, ii, and iii) and closing the preparat (mounting) then were carried out immediately using the cover glass. expressions of vegf and fgf2 in each sample were assessed semiquantitatively using the modification of remmele method. remmele scale index (immuno reactive score/irs) is the result of multiplying immunereactive cells percentage score to color intensity score on the immunoreactive cells. data then were obtained from the average value of the irs in each sample observed in five different fields of view at 400x magnification. the whole of this examination was performed using light microscope (nikon n600l) equipped with a 300 megapixel ds fi2 digital camera and image processing software (nikkon image system). results the vegf and fgf2 distribution were show in figure 1. the average of vegf and fgf2 expression in the control and treatment groups were shown in figure 2. based on the results of the statistical kruskal wallis test, p value obtained was less than 0.05, which means that there was no significant difference in vegf and fgf2 expressions between the control group and the treatment group. discussion tissue engineering involves cells as a building block, scaffold as a template, and growth factor as a biochemical signal that indicates there has been a growth of tissue. the primary function of scaffold is as cell support, an artificial extracellular matrix. it is not only providing sufficient mechanical environment of cells, but also causing cell attachment, proliferation, differentiation, and metabolism signals.4 selection of biomaterials for scaffold design is essential to cell growth and proliferation in threedimensional matrix. chitosan is a natural polysaccharide which is similar to glycosaminoglycans and has a good interaction with cell membrane.6 natural polysaccharides can stimulate the activity of growth factors, which can maintain cell phenotype in particular morphology and play an important role as scaffold component of soft tissue and hard tissue.7 chitosan-containing n-acetyl-d-glucosamine can bind to receptors that recognize macrophages. macrophages produce vegf directly to stimulate endothelial cell proliferation.9 chitosan may provide more amino groups 8 figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. figure 2. the average of vegf and fgf2 expression. a 8 figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. figure 2. the average of vegf and fgf2 expression. b 8 figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. figure 2. the average of vegf and fgf2 expression. c 8 figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. figure 2. the average of vegf and fgf2 expression. d figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p213-216 216 pratiwi, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 213–216 for the attachment and proliferation of endothelial cells because of the affinity between cations of the ammonium group of chitosan and the anion surface of the endothelial cell membrane.13 in this research, chitosan with a degree of deacetylation of 81% was used. deacetylation degree can increase the attachment and stimulate fibroblast proliferation. deacetylation degree of chitosan can also indicate free amine groups presented in the structure of chitosan. cationic amine chitosan group provides a suitable environment for cell attachment. cell attachment, is not only influenced by acetylation degree, but also by molecular weight and porosities. chitosan, furthermore, can increase the proliferation of fibroblasts indirectly through the formation of poly-electrolyte complex with serum as heparin.13 the results of this research showed that there was no significant difference in vegf expressions between the treatment group and the control group on day 14 during the process of bone regeneration. chitosan could stimulate inflammatory cells and growth factors in the early phase of wound healing process. this is supported by a research conducted by inan and saraydin indicating that the expression of vegf in post-administration of chitosan will decline on day 14.14 chitosan could also stimulate the formation of granulation tissue. at the same time, chitosan could stimulate both fibroblasts to proliferate and extracellular matrix formation. this study also showed that there was no significant difference in fgf2 expressions between the treatment group and the control group on day 14 during the process of bone regeneration. the number of fgf2 expressions on the 14th day is lower than on the 3rd day.14 this condition is possible because fibroblasts have produced collagen fiber on the 14th day. consequently, fibroblasts will grow into inactive fibroblasts, known as fibrocyte. in this study, however, chitosan scaffold on the 14th day could still interact with the endothelial cells and fibroblasts although not optimal. finally, it may be concluded that chitosan scaffold cannot affect vegf and fgf2 expressions on the 14th day during bone regeneration process in tissue engineering principles. references 1. karp jm, langer r. development and therapeutic applications of advanced biomaterials. curr opin biotechnol 2007; 18(5): 454-9. 2. chan bp, leong kw. scaffolding in tissue engineering: general approaches and tissue-specific considerations. eur spine j 2008; 17(suppl 4): 467–9. 3. sachlos e, czernuszka jt. making tissue engineering scafollds work. review: the application of solid free form fabrication technology to the production of tissue engineering scaffolds. euro cell mater 2003; 5: 29-39. 4. shin ja, choi jy, kim st, kim cs, lee yk, cho ks, chai jk, kim ck, choi sh. the effect of hydroxyapatite-chitosan membrane on bone regeneration in rat calvarial defects. j korean acad periodontal 2009; 39: 213-4. 5. isikli c, hasirci v, hasirci n. development of porous chitosangelatin/hydroxiapatite composite scaffold for hard tissue engineering application. j tissue eng reg med 2012; 6(2): 135-43. 6. aranaz i, mengibar m, harris r, panos i, miralles b, acosta n, galed g, heras a. functional characterization of chitin and chitosan. current chemical biology 2009; 3: 203-30. 7. hankenson kd, dishowitz m, gray c, schenker m. angiogenesis in bone regeneration. injury 2011; 42(6): 556-61. 8. saran u, piperni sg, chatterjee s. role of angiogenesis in bone repair. arch biochem and biophys 2014; 561: 109-17. 9. stegen s, gastel nv, carmeliet g. bringing new life to damaged bone: t he impor t a nce of a ngiogenesis i n bone repa i r a nd regeneration. bone 2014; 70: 19-25. 10. mackay d, miller a. nutritional support for wound healing. altern med rev 2003; 8(4): 359-77. 11. dimitriou r, tsiridis e, giannoudis pv. current concept of molecular aspects of bone healing. injury 2005; 36(12): 1392-404. 12. arvidson k, abdallah bm, applegate la, baldini n, cenni e, gomez-barrena e, granchi d, kassem m, konttinen yt, mustafa k, pioletti dp, sillat t, finne-wistrand a. bone regeneration and stem cells. j cell mol med 2011; 15(4): 718-46. 13. ma l, gao c, mao z, zhou j, shen j, hu x, han c. collagen/ chitosan porous scaffold with improved biostability for skin tissue engineering. biomaterials 2003; 24(26): 4833-41. 14. deniz i, serpil s. investigation of the wound healing effects of chitosan on fgfr3 and vegf ımmunlocalization in experimentally diabetic rats. int j biomed mat research 2013; 1: 1-8. 8 figure 1. vegf expressions on the control group (a) and the treatment group; (b) fgf2 expressions on the control group; (c) and the treatment group; (d) vegf and fgf2 expressions were evenly distributed in the control and treatment groups. figure 2. the average of vegf and fgf2 expression. figure 2. the average of vegf and fgf2 expression. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p213-216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p213-216 16 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 16–19 research report potential of 5% tamarind extract gel as an etching agent: tensile strength and scanning electron microscope (sem) evaluation erawati wulandari,1 faiqatin cahya ramadhani1 and nadie fatimatuzzahro2 1department of conservative dentistry, 2department of biomedical science, faculty of dentistry, universitas jember, jember – indonesia abstract background: acid etching is a stage in obtaining bonds between composites and enamel. the application of acid to the enamel surface, however, can cause dissolution of hydroxyapatite and demineralisation of the enamel surface. phosphoric acid, a strong acid, is an etching material that can reduce enamel hardness. excessively reducing hardness can interfere with attachment to the restorative material. one medicinal plant that can be used as an alternative material in acid etching is tamarind. purpose: this study aims to determine the effect of 5% tamarind extract gel on the tensile strength of composite resins. methods: this is an experimental research study with a post-test-only control-group design. the study used 14 mandibular incisors. the labial part of the incisor was prepared using a diamond fissure bur with a diameter of 4 mm and a depth of 2 mm. the control group was then etched with 37% phosphoric acid gel, while the experimental group was etched with 5% tamarind extract gel. bonding resins and micro-hybrid composite resins were applied, based on the manufacturers’ instructions. next, a tensile strength test and seeing formation resin tags by scanning electron microscope (sem) were performed. data were analysed using an independent t-test (p < 0.05). results: the average tensile strength of composite resins in the group etched with 5% tamarind extract gel was the same as in the 37% phosphoric acid group (p > 0.05). sem images also show that enamel etched with 5% tamarind extract gel produced a tag similar to that etched with 37% phosphoric acid gel. conclusion: 5% tamarind extract as an etching material can generate tensile strength of composite resin and trigger formation of resin tags in the same way as 37% phosphoric acid. keywords: acid etching; composite resin; tamarind extract; tensile strength correspondence: erawati wulandari, department of conservative dentistry, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. e-mail: era.fkg@unej.ac.id introduction composite resin is an anterior and posterior restoration material often chosen because it has good aesthetics, a similar colour to natural teeth and good mechanical strength so that it can withstand mastication.1–3 this material is micromechanically adhesive on the surface of enamel. one of the stages in obtaining bonding between composite and enamel is acid etching. the etching process starts with removing the enamel by as much as 10 µm, forming a microlayer as deep as 5–50 µm, removing surface contaminants and smear layers and then producing micro-irregularity/micro-porosity in prismatic enamel surfaces to provide micromechanical retention for resins.4,5 next, the application of acid to the enamel surface causes dissolution of hydroxyapatite and the enamel surface becomes demineralised. if resin bonding material is applied to the etched surface, it will flow to fill the irregular surface and form a resin tag; there will also be a mechanical bond between the composite resin and the enamel. the acid etching material most often used is 37% phosphoric acid.6,7 phosphoric acid is a strong acid that can reduce enamel hardness.8,9 excessive reduction of hardness can interfere dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p16–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p16-19 17wulandari, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 16–19 with the attachment of the restorative material.9 the length of the tag produced from etching using phosphoric acid does not contribute to the strength of adhesion.10 in addition, phosphoric acid causes pulp irritation.8,11 these side effects can be minimised by using alternative ingredients from medicinal plants. medicinal plants have advantages: as natural basic ingredients they are considered safer and have less serious side effects.12 one medicinal plant considered as an alternative is tamarind (tamarindus indica). ripe tamarind flesh contains organic acids, such as citric acid, lactic acid, malic acid and tartaric acid,12 which are classified as chelation agents, similar to ethylenediaminetetraacetic acid (edta) and phosphoric acid.2 tamarind extract at a concentration of 5% as an irrigation agent is able to dissolve calcium in root canal dentine.13 the toxicity of 5% tamarind extract in root canal irrigation is lower than 3% hydrogen peroxide.14 an ingredient that can remove and bind metal ions, such as calcium, can be used as an etching material,2 therefore 5% tamarind extract has the potential to be used as an etching material. to determine the ability of a material and to maintain its attachment under a received load, a tensile strength test can be performed. measurement of the bond between the dental tissue and the restoration material can be carried out to assess the ability of the restoration material to remain in place.15 hence, this study aims to determine the potential of 5% tamarind extract as an alternative agent for acid etching, measuring the tensile strength of composite resins and descriptively seeing the formation of resin tags using a scanning electron microscope (sem). materials and methods this study has been approved by the ethics commission of the medical faculty of jember university, number 1138 / h25.1.11 / ke / 2017. this is an experimental research study with a post-test-only control-group design. this study used 14 lower mandibular incisors, which were single root and caries free, with no fracture and no abrasion on the buccal surface. tamarind extract was made from 300 g of ripe tamarind meat that had been separated from the seeds; 1 l of distilled water was added and the mixture stirred until homogeneous. the mixture was then centrifuged (centrifuge hermle z-306, germany) for 15 minutes, filtered and dried to produce extract in the form of crystals. the tamarind crystal extract then was stored in a freezer (-3oc) to keep it stable. tamarind extract gel at a concentration of 5% was prepared by mixing 5 g of carboxymethyl cellulose sodium (cmc-na) powder (yanxing, china) and 100 ml of sterile distilled water. it was then stirred in a porcelain cup until it reached the phase of homogeneous gel. then, 5 g of tamarind crystal extract was added and stirred until completely dissolved. the gel was stored in a glass jar. the ph of this gel had to be 4. subsequently, dental samples were prepared. first, tooth samples were cut at the cemento-enamel junction using a carborundum disc (zhengzhou shengxin medical instrument co., china), so that the crown and root were separated. second, cylindrical moulds (made of pvc) were prepared to fix the sample. the moulds were filled with investment material (super gips dental plaster, indonesia). third, the samples were placed on the surface of the moulds (precisely in the middle), with the labial part facing upwards. fourth, the labial part was prepared using a diamond fissure bur (baistra, china) with a diameter of 4 mm and a depth of 2 mm. fifth, the enamel surface of each sample was washed with distilled water and dried with air spray. sixth, the samples in the control group were etched with as much as 0.1 ml of 37% phosphoric acid gel (magnumdental, usa), while those in the experimental group were etched with as much as 0.1 ml of 5% tamarind extract gel, for 25 seconds. the etching materials were applied until the cavities were fully filled. seventh, the cavities were washed with distilled water for 20 seconds and dried using air spray. eighth, as much as 0.1 ml of bonding resin (master bond biodinamica, brazil) was dripped on a micro-brush and applied to the etched surfaces.16 ninth, the cavities were dried with light air pressure and then irradiated for 20 seconds (according to factory instructions). tenth, the cavities were filled with a micro-hybrid composite resin (master fill a2 biodinamica, brazil) using layer-by-layer plastic filling instruments, where each layer was condensed with a cement stopper and then shined for 40 seconds (according to the manufacturer’s instructions). eleventh, an acrylic ring (4 mm in diameter and 2 mm in height) was attached to each cavity that had been restored, using double-sided tape. twelfth, the ring was filled with a micro-hybrid composite resin and shined. thirteenth, polymethyl-methacrylate (pmma) with a diameter of 10 mm and a height of 20 mm was glued on top of the acrylic ring with self-cured acrylic (figure 1).16 after completion, 1.5 mm pmma rod 2 mm bovine teeth (labial) composite resins molding tool (ring) composite resins (inside of ring) composite resins (inside of tooth cavity) bonding system figure 1. shape of sample specimen for tensile strength test (side view). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p16–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p16-19 18 wulandari, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 16–19 the sample was stored at room temperature (20–25oc) for 24 hours. fourteenth, the samples were tested for tensile strength using a universal testing machine (shimadzu, japan). the result that appears on the universal testing machine monitor indicates the tensile strength of the composite resin, i.e. the force required to break it away from the tooth surface, in newton units (n) and in megapascal units (mpa). the research data were analysed statistically by independent t-test using spss statistics for windows, version 20 (p < 0.05) (ibm, usa). after the tensile strength test was performed, the tooth sample was removed from the investment material and cleaned of the remnants of the investment material. the tooth then was cut using a carborundum disc to obtain a sample 5 mm in length, 3 mm in width and 2 mm in thickness. after that, photographs were taken by sem (hitachi, japan). results the data in table 1 show the tensile strength of composite resins in the group etched with 5% tamarind extract gel as well as in the 37% phosphoric acid group. the results of the independent t-test show a value of p = 0.175 (p > 0.05), which means there is no significant difference between two groups. sem images in the two groups can be seen in figure 2, descriptively showing the presence of resin bonding that fills the tag/micro-porosity formed from etching using 5% tamarind acid extract and 37% phosphoric acid. discussion restoration with a composite resin requires etching on tooth enamel, which aims to clean the smear layer as well as to produce micro-porosity on the surface of the tooth and the formation of tags. the application of the bonding material will fill the tags, forming a resin tag. this results in a mechanical interlocking bond between the composite resin and the dental tissue.6 the results of this study show that the samples in the group etched with 5% tamarind extract generated an average tensile strength value as large as samples in the group etched with 37% phosphoric acid. this is presumably because the tamarind extract contains several organic acids, such as citric acid, lactic acid, tartaric acid, acetic acid and malic acid, which, although they are weak acids, have almost the same effect as phosphoric acid (a strong acid).2,12 as a strong acid, phosphoric acid releases more hydrogen ions (full ionisation) than a weak acid (half ionisation).17,18 as more hydrogen ions are released, demineralisation of active teeth occurs, so that the microhardness of the enamel is reduced.19 the decrease in microhardness influences the attachment of the restorative material to the hard tissue of the tooth.20,21 sem photographs were taken to show the teeth after application of 5% tamarind extract and 37% phosphoric acid. in teeth etched with 5% tamarind extract, radiopaque lines as a result of the formation of resin tags are apparent (figure 2); these also appear in the teeth etched with 37% phosphoric acid. this proves that 5% tamarind extract has the same potential for etching as 37% phosphoric acid. the organic acid content in tamarind extract is a chelating agent similar to edta and phosphoric acid, which can change the ratio of calcium/phosphate (ca/p) to hydroxyapatite.13 the application of acids on the enamel surface causes demineralisation and hydroxyapatite dissolution, leading to micro-irregularity/micro-porosity on the surface of the prismatic enamel, providing micromechanical retention for the resin.4 table 1. mean tensile strength score of composite resins groups n mean (mpa) sd 5% tamarind extract 7 5.442 0.07 37% phosphoric acid 7 5.376 0.09 n: number of samples; sd: standard deviation. figure 2. formation of resin tags (arrows) after teeth were etched with (a) 5% tamarind extract and (b) 37% phosphoric acid (500x magnification sem). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p16–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p16-19 19wulandari, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 16–19 in addition, the statistical analysis shows that there is no significant difference between the average tensile strength of composite resins after etching with 5% tamarind extract and after etching with 37% phosphoric acid. this indicates that the demineralisation caused by the two materials on the surface of teeth is similar. although the organic acid content in tamarind is weak, this weak acid still can release hydrogen ions. these ions will accumulate and stimulate further demineralisation,21 with an insignificant difference in the tensile strength of composite resins between 37% phosphoric acid and 5% tamarind extract. in conclusion, 5% tamarind extract as an etching agent can generate tensile strength of composite resins and trigger the formation of resin tags to the same extent as 37% phosphoric acid. references 1. arhun n, celik c, yamanel k. clinical evaluation of resin-based composites in posterior restorations: two-year results. oper dent. 2010; 35(4): 397–404. 2. garg n, garg a. textbook of operative dentistry. 3rd ed. new delhi: jaypee brother medical publishers; 2015. p. 241, 502. 3. domingos pa dos s, garcia ppns, de oliveira albm, palma-dibb rg. composite resin color stability: influence of light sources and immersion media. j appl oral sci. 2011; 19(3): 204–11. 4. benarjee a, watson tf. pickard manual konservasi restoratif. 9th ed. irmaleni, puspitasari d, editors. jakarta: egc; 2014. p. 92. 5. low im. advances in ceramic matrix composites. philadelphia: woodhead publishing; 2014. p. 659. 6. soetojo a. penggunaan resin komposit dalam bidang konservasi gigi. surabaya: pt. revka petra media; 2013. p. 107, 135–6. 7. reddy sk. dental pulse volume 1. 9th ed. hyderabad: swapna medical publishers; 2015. p. 88, 92. 8. hatrick cd, eakle ws. dental materials: clinical applications for dental assistants and dental hygienists. 3rd ed. st. louise: elsevier; 2015. p. 50, 58, 60. 9. zafar ms, ahmed n. the effects of acid etching time on surface mechanical properties of dental hard tissues. dent mater j. 2015; 34(3): 315–20. 10. shinchi mj, soma k, nakabayashi n. the effect of phosphoric acid concentration on resin tag length and bond strength of a photo-cured resin to acid-etched enamel. dent mater. 2000; 16(5): 324–9. 11. walton re, torabinejad m. endodontics : principles and practice. saunders/elsevier; 2009. p. 474. 12. utami p, puspaningtyas de. the miracle of herbs. jakarta: agro medika pustaka; 2013. p. 3. 13. fatimatuzzahro n, wulandari e. dekalsifikasi dentin saluran akar gigi setelah diirigasi dengan ekstrak asam jawa 5% dan 2,5%. spirulina. 2012; 7(1): 19–24. 14. wulandari e. cytotoxicity of 5% tamarindus indica extract and 3% hydrogen peroxide as root canal irrigation. dent j (majalah kedokt gigi). 2008; 41(3): 107–9. 15. mandava d, ajitha p, narayanan ll. comparative evaluation of tensile bond strengths of total-etch adhesives and self-etch adhesives with single and multiple consecutive applications: an in vitro study . j conserv dent. 2009; 12(2): 55. 16. susra w, nur dl, puspita s. perbedaan kekuatan geser dan kekuatan tarik pada restorasi resin komposit microhybrid dengan bonding generasi v dan bonding generasi vii. insisiva dent j maj kedokt gigi insisiva. 2013; 2(2): 69–76. 17. bateman n, jefferson r, thomas s, thompson j, vale a. oxford desk reference: toxicology. london: oxford university press; 2014. p. 42. 18. malhotra s, dhawan p, arora sa. complete refresher science for class x. new delhi: tata mcgraw hill; 2010. p. 27. 19. belitz hd, grosch w, schieberle p. food chemistry. 4th ed. food chemistry. heidelberg: springer; 2009. p. 455. 20. cruz-filho am, sousa-neto md, savioli rn, silva rg, vansan lp, pécora jd. effect of chelating solutions on the microhardness of root canal lumen dentin. j endod. 2011; 37(3): 358–62. 21. adebayo oa, burrow mf, tyas mj, adams gg, collins ml. enamel microhardness and bond strengths of self-etching primer adhesives. eur j oral sci. 2010; 118(2): 191–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p16–19 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p16-19 144 volume 45 number 3 september 2012 maternal endotoxin-induced fetal growth restriction in rats: fetal responses in toll-like receptor banun kusumawardani1, marsetyawan hne. soesatyo2, djaswadi dasuki3 and widya asmara4 1 departement of biomedical, faculty of dentistry, jember university, jember-indonesia 2 departement of histology and cell biology, faculty of medicine, universitas gadjah mada, yogyakarta indonesia 3 departement of obstetric and gynecology, faculty of medicine, universitas gadjah mada, yogyakarta indonesia 4 departement of microbiology, faculty of veterinary medicine, universitas gadjah mada, yogyakarta indonesia abstract background: porphyromonas gingivalis as a major etiology of periodontal disease can produce virulence factor, lipopolysaccharide/lps, which is expected to play a role in the intrauterine fetal growth. trophoblast at the maternal-fetal interface actively participates in response to infection through the expression of a family of natural immune receptors, toll-like receptor (tlr). purpose: the aims of study were to identify endotoxin concentration in maternal blood serum of porphyromonas gingivalis-infected pregnant rats, to characterize the tlr-4 expression in trophoblast cells, and to determine its effect on fetal growth. methods: female rats were infected with live-porphyromonas gingivalis at concentration of 2 x 109 cells/ml into subgingival sulcus area of the maxillary first molar before and/or during pregnancy. they were sacrified on 14th and 20th gestational day. fetuses were evaluated for weight and length. endotoxin was detected by limulus amebocyte lysate assay in the maternal blood serum. the tlr-4 expression in trophoblast cells was detected by immunohistochemistry. results: the mean of lps concentrations in maternal blood serum was significantly different (p < 0.05) among the four maternal periodontal infection groups. the tlr4 expressions in syncytiotrophoblast, spongiotrophoblast and trophoblastic giant cells from porphyromonas gingivalis-infected periodontal maternal groups were significantly higher than the control group (p < 0.05). maternal endotoxemia affected (p < 0.05) the fetal weight and fetal length. conclusion: the increased lps concentration in maternal blood serum resulted in the decreased fetal weight and fetal length. syncytiotrophoblast, spongiotrophoblast and trophoblastic giant cell were able to recognize porphyromonas gingivalis lps through the tlr-4 expression. this findings strengthened the link between periodontal disease and fetal growth restriction. key words: porphyromonas gingivalis, periodontitis, endotoxin, pregnancy, fetal growth restriction abstrak latar belakang: porphyromonas gingivalis sebagai etiologi utama penyakit periodontal dapat menghasilkan faktor virulensi, lipopolisakarida/lps, yang positif berperan dalam pertumbuhan janin intrauterin. trofoblas pada antarmuka maternal-janin aktif berpartisipasi dalam respon terhadap infeksi melalui ekspresi suatu famili reseptor imun alamiah, toll-like receptor (tlr). tujuan: penelitian ini bertujuan untuk mengidentifikasi konsentrasi endotoksin dalam serum darah maternal dari tikus hamil yang terinfeksi porphyromonas gingivalis; mengkarakterisasi ekspresi tlr-4 pada sel trofoblas, dan mengetahui efeknya pada pertumbuhan janin. metode: tikus betina diinfeksi dengan porphyromonas gingivalis hidup pada konsentrasi 2 x 109 sel/ml ke area sulkus subgingiva molar pertama maksilaris sebelum dan/atau selama kehamilan. tikus tersebut dikorbankan pada hari kehamilan ke 14 dan 20. janin dievaluasi untuk berat dan panjang janin. endotoksin dideteksi dengan uji limulus amebocyte lysate dalam serum darah maternal. ekspresi tlr-4 pada sel trofoblas dideteksi secara imunohistokimiawi. hasil: rerata konsentrasi lps dalam serum darah maternal berbeda nyata (p < 0,05) di antara empat kelompok infeksi periodontal maternal. ekspresi tlr-4 pada sinsitiotrofoblas, spongiotrofoblas dan giant cell trofoblas dari kelompok periodontal maternal yang terinfeksi porphyromonas gingivalis secara signifikan lebih tinggi daripada kelompok kontrol (p < 0,05). endotoksemia maternal mempengaruhi (p < 0,05) berat janin dan panjang janin. kesimpulan: peningkatan konsentrasi lps meningkat dalam serum darah maternal mengakibatkan penurunan berat janin dan panjang janin. sinsitiotrofoblas, spongiotrofoblas dan giant cell trofoblas mampu mengenali lps porphyromonas gingivalis melalui ekspresi tlr-4. temuan ini memperkuat hubungan antara penyakit periodontal dan restriksi pertumbuhan janin. kata kunci: porphyromonas gingivalis, periodontitis, endotoksin, kehamilan, restriksi pertumbuhan janin correspondence: banun kusumawardani, c/o: departemen biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: kusumawardani_banun@yahoo.co.id research report 145kusumawardani, et al.: maternal endotoxin-induced fetal growth restriction introduction periodontal disease is a multifactorial chronic infection resulted that destruction of the periodontium. the primary microorganism which caused of periodontal disease is gramnegative rod-shaped facultative anaerobes. porphyromonas gingivalis is a periodontal pathogenic bacteria which has potential virulence factors such as proteolytic enzymes, leucotoxin, endotoxin (lipopolysaccharide/lps), evasion of host responses, invasion of host tissues, and induction of inflammatory mediators.1,2 porphyromonas gingivalis is not only exhibit the pathogenic properties on periodontal disease but also on systemic diseases such as cardiovascular disease and abnormal pregnancies.3,4 in humans, gram-negative bacteria infections have been recognized as a cause of stillbirth and perinatal death5 whereas lps has been associated with embryonic resorption, intra-uterine fetal death, intrauterine growth restriction and preterm birth in rodents.6,7 these findings indicate that periodontal pathogens may play a role in the development and progression of systemic disease. placental growth and development are very dependent on trophoblast cells. apoptosis and the factors involved in the regulation are associated with almost all stages of development and trophoblast differentiation.8 one of the factors that can lead to the increased apoptosis of trophoblast during pregnancy is intrauterine infection.9 the exact mechanism of infection resulted the progression of this disorder is not clearly understood, but recent studies suggested that bacterial products have a direct effect on the trophoblast. trophoblast can respond to infection through the expression of a family of natural immune receptors, toll-like receptor (tlr). tlr capable of recognizing conserved sequences on the surface of microorganisms.10 ligation of tlr-4 with lps led to the first trimester human trophoblast cells to produce cytokines, including tumor necrosis factor-a (tnf-a), which induces apoptosis of trophoblast cells.11 therefore, we hypothesized that porphyromonas gingivalis and its lipopolysaccharide from periodontal tissue can spread into the uterus through the circulatory system, then induces placental inflammatory response resulting in fetal growth restriction. the aims of the present study were to identify endotoxin level in maternal blood serum of porphyromonas gingivalis-infected pregnant rats, to characterize the tlr-4 expression in trophoblast cells, and to determine its effect on fetal growth. materials and methods all procedures were approved by the ethics committee, faculty of medicine, universitas gadjah mada, yogyakartaindonesia. this study had taken female sprague-dawley rats, adult, 2 months, 150–250 g and primiparous. the rats were maintained on the controlled and standardized conditions. the subjects of study were consisted of two blocks, they were sacrificed on gestational day (gd) 14 and gd 20. each block was subdivided into four groups, which consisted of the control group, no porphyromonas gingivalis infection; the pg-bd group, an infection of porphyromonas gingivalis before and during pregnancy; the pg-b group, an infection of porphyromonas gingivalis before pregnancy; and the pg-d group, an infection of porphyromonas gingivalis during pregnancy. each group consisted of five pregnant rats. induction of experimental periodontitis was performed by injection of 0.05 ml live-porphyromonas gingivalis atcc 33277 with a concentration of 2 x 109 cells/ml into the distopalatal and distobuccal gingival sulcus area of maxillary first molar. injection was repeated every 3 days for 30 days. for infection after pregnancy, it was also performed by a repeated injection every 3 days for 19 days. control group rats were injected saline 0.05 ml as the treatment schedule of the treatment groups. then, the female rats were mated with the same strain of male rat overnight ratio 2:1. the next morning, female rats were removed from the cages and examined the vaginal plug. if the vaginal plug was found, the day was recorded as gd 1. each fetus was taken post-mortem from the chorioamniotic sac on gd 14 and gd 20. placental weight, fetal weight and fetal length were recorded for each maternal. furthermore, the placental immunohistochemically was undertaken to determine the expression of tlr-4. samples were incubated overnight at 4°c with primary antibody, rabbit polyclonal anti-tlr4 antibody dilution 1:500 (abbiotec, san diego, ca, 1:100-1:500), while negative control was incubated with secondary antibody as a substitute for the primary antibody. tlr-4 is expressed on the cell wall and cytoplasm. data were presented as mean number of cells expressing tlr-4 in each type of cell. these specimens were evaluated in macrophages of labyrinth zone (lm), junctional zone (sm) and decidua zone (dm), as well as syncytiotrophoblast (ls), spongiotrophoblast (st) and trophoblastic giant cell (dg). maternal blood serum was taken on gd 13 and gd 19, and it was performed to endotoxemia test. endotoxin in maternal blood serum was tested by limulus amebocyte lysate (lal) pyrochrome method according to the manufacturer's instructions (cape cod, u.s.). this method is easy to do in a timely, specific, and highly sensitive. pyrochrome was added as soon as possible to all of the negative control samples, endotoxin standards and specimens with a ratio 1:1 and was incubated 37°c for 30 seconds in an incubator. furthermore, the reaction was stopped with 0.05 ml sodium nitrite in hcl, and it was added 0.05 ml ammonium sulfamate, and 0.05 ml n-(1-naphthyl)-ethylenediamine (neda) to each well. magenta color would be formed quickly. the test was read at 540–550 nm. standard curve was used to determine the concentration of endotoxin in the specimen. endotoxin concentrations of the positive control were determined by 146 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 144–149 standard endotoxin dilution consisting of 0.005, 0.05, 0.5, 5 and 50 endotoxin units (eu)/ml. sensitive detection limit was 0.005 eu/ml. numerical variables which consisted of maternal lps concentration, tlr-4 expression, placental weight, fetal weight and fetal length were performed by statistical analyzes to identify endotoxin level in maternal blood serum of porphyromonas gingivalis-infected pregnant rats, to characterize the tlr-4 expression in trophoblast cells, and to determine its effect on fetal growth. one-way anova with post hoc test was performed to compare the endotoxin levels of maternal periodontal infection. linear regression analysis was to analyze the linear relationship between numerical variables. value of significance was determined as p < 0.05. numerical data were presented in mean ± standard deviation. results endotoxemia test showed that the mean of lps concentration in maternal blood serum from control group was 1.11±0.62 eu/ml on gd 14 and 4.19 ± 2.45 eu/ml on gd 20. furthermore, the mean of lps concentration in maternal blood serum on gd 14 and gd 20 were significantly different (p < 0.05) in the four maternal periodontal infection groups. the control group was significantly different (p < 0.05) with the pg-bd, pg-b and pg-d groups. the mean of lps concentration in the maternal blood serum from pg-bd group was significantly different (p < 0.05) with pg-b group, but was not significantly different (p > 0.05) with pg-d group. similarly, the mean of lps concentration in maternal blood serum from pg-b group was significantly different (p < 0.05) with pg-d group (figure 1). this study also showed that tlr-4 expressions in the labyrinth zone, junctional zone and decidual zone (figure 2) were significantly different (p < 0.05) from the control, pg-bd, pg-b and pg-d groups on gd 14. however, tlr-4 expressions of the control, pg-bd, pg-b and pg-d groups on gd 20 were significantly different only in the macrophages, syncytiotrophoblasts and trophoblastic giant cells. both gd 14 and gd 20, the pg-bd, pg-b and pg-d groups had a higher tlr-4 expression than control group (table 1). the linear regression analysis showed that maternal endotoxemia on gd 14 and gd 20 affected (p < 0.05) the placental weight, fetal weight and fetal length. the increased lps concentration in maternal blood serum resulted in the decreased fetal weight and fetal length. the results can be seen in table 2. disscusion the lps concentration in porphyromonas gingivalisinfected periodontal maternal groups were higher than control group. the increased lps concentration was directly proportional to the severity of periodontal disease. the increased lps concentration was in accordance to long-term maternal chronic periodontal infection. lps concentration in the maternal blood serum was 2-fold higher than in amniotic fluid. the dynamic changes of lps were correlated with disease severity and suggested lps causing secondary hepatic injury.12 much evidence indicates that bacterial lps (endotoxin) is removed from the bloodstream mainly by the liver, yet the hepatic uptake mechanisms remain uncertain and controversial. in plasma, lps can be either 'free' (as aggregates, bacterial membrane fragments or loosely bound to albumin, cd14, or other proteins) or 'bound' (complexed with lipoproteins). whereas most free lps is taken up by kupffer cells, lipoprotein-bound lps has seemed to be cleared principally by hepatocytes.13 figure 1. maternal endotoxemia caused by porphyromonas gingivalis infection in maternal periodontal tissues. maternal blood serum samples were taken 40 minutes after the bacteria exposure. data were presented in mean±sem and were compared with anova (* p<0.05) by the maternal periodontal infection. 147kusumawardani, et al.: maternal endotoxin-induced fetal growth restriction table 1. tlr-4 expression in the rat placenta of porphyromonas gingivalis-infected periodontal maternal on gd 14 and gd 20 variable maternal periodontal infection p control (n = 23) pg-bd (n = 10) pg-b (n = 13) pg-d (n = 10) gd 14: tlr4-lm tlr4-ls tlr4-sm tlr4-st tlr4-dm tlr4-dg gd 20: tlr4-lm tlr4-ls tlr4-sm tlr4-st tlr4-dm tlr4-dg 1.13 ± 0.92*†§ 1.57 ± 0.79*†§ 1.48 ± 0.79*†§ 1.65 ± 1.03*†§ 1.35 ± 0.65*†§ 1.74 ± 0.92*†§ 2.00 ± 0.95*† 2.78 ± 1.09*†§ 2.22 ± 1.04* 2.96 ± 1.07 2.35 ± 1.03 2.26 ± 1.05*†§ 2.50 ± 1.84*† 3.30 ± 1.16*† 2.60 ± 0.97*† 6.70 ± 1.89*†§ 2.60 ± 1.65*† 3.90 ± 0.88*†§ 3.70 ± 1.70*†§ 7.50 ± 1.72*†§ 2.60 ± 1.17 3.80 ± 1.39 2.50 ± 1.18 3.40 ± 0.69*† 2.62 ± 1.19*§ 2.92 ± 1.04*§ 2.15 ± 0.38*§ 4.92 ± 1.85*†§ 2.46 ± 0.66*†§ 3.08 ± 0.64*†§ 2.31 ± 1.18†§ 3.85 ± 1.41*†§ 2.23 ± 0.83§ 3.38 ± 1.33 2.00 ± 0.71 2.85 ± 0.69*§ 2.50 ± 1.08* 3.30 ± 1.95* 2.20 ± 1.03* 4.80 ± 1.48*† 1.90 ± 1.10 3.60 ± 1.35*† 3.60 ± 2.07*§ 6.90 ± 1.73*§ 3.30 ± 0.95*§ 3.40 ± 0.52 2.90 ± 1.19 3.30 ± 0.48* 0.001 0.001 0.002 0.001 0.002 0.001 0.003 0.001 0.037 0.249 0.224 0.001 macrophages of labyrinth zone (lm), junctional zone (sm) and decidua zone (dm); syncytiotrophoblast (ls); spongitrophoblast (st); trophoblastic giant cell (dg) periodontal infection before and during pregnancy (pg-bd); periodontal infection before pregnancy (pg-b); periodontal infection during pregnancy (pg-d) *, †, §: mean difference was significant at 0.05 a b a d b c d c a b a d b c d c a b a d b c d c a b a d b c d c figure 2. tlr-4 expression on placenta of control a) pg-bd; b) pg-b; c) and pg-d; d) groups at gd 20. tlr-4 was expressed weakly a) and strong; b-d) on the cell wall and cytoplasm of macrophage (a), syncytiotrophoblast (b), spongiotrophoblast (c) and trophoblastic giant cell (d). 400√ magnification table 2. effect of maternal endotoxemia to placental weight, fetal weight and fetal length on gd 14 and gd 20 variable maternal endotoxemia gd 14 gd 20 n r2 b p n r2 b p placental weight, gram 141 0.562 -0.003 0.001 141 0.434 -0.006 0.001 fetal weight, gram 141 0.626 -0.003 0.001 141 0.548 -0.056 0.001 fetal length, mm 141 0.584 -0.099 0.001 141 0.526 -0.478 0.001 148 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 144–149 this study identified that the endotoxin was contained in the maternal blood serum from the control group. the mean of lps concentration in the maternal blood serum from the control group was 1.11 ± 0.62 eu/ml on gd 14 and 4.19 ± 2.45 eu/ml on gd 20. however, the maternal endotoxemia in the control group was not adversely affect fetal growth. in accordance to our study, previous study showed that low-dose lps pretreatment greatly attenuated lps-induced increases in tnf-a protein in fetal liver and fetal brain. taken together, these results indicate that perinatal exposure to low-dose lps induces a reduced sensitivity to subsequent lps challenge.14 porphyromonas gingivalis lps plays an important role in the induction of inate and acquired immune responses. differential cytokine response to live-porphyromonas gingivalis indicates that live-porphyromonas gingivalis and its components play different roles. live-porphyromonas gingivalis can lead to a relatively minor inflammatory infiltration and less intense in antigen-specific immune responses.15 porphyromonas gingivalis from maternal periodontal tissue can spread into the placenta,16 it is assumed that porphyromonas gingivalis can also achieve chorio-decidual space and then penetrate through the amnion into the amniotic fluid. finally, the fetus can be infected if the amniotic fluid enters the fetal lungs and gastrointestinal tract. on gd 14 and gd 20, tlr-4 were expressed by syncytiotrophoblasts and spongiotrophoblasts. this indicated that tlr-4 in syncytiotrophoblasts and spongiotrophoblasts were able to respond porphyromonas gingivalis that previously have been through the decidual compartment. thus, porphyromonas gingivalis will only pose a threat to fetus if the syncytiotrophoblast layer was breached, so that porphyromonas gingivalis can enter the fetal blood vessels. toll-like receptor-4 were strong expressed in placental on gd 14 and 20, especially the trophoblasts in labyrinth zone and junctional zone which are a frontal barrier between maternal and fetus. trophoblast was expected to have important functions in regulating the host immune response against bacterial infection. stimulation of tlr4 is required for the reliable signaling to synergize lps binding with tlr-4 in tnf release from macrophages and trophoblasts. thus, expression of tlr-4 in macrophages and trophoblast of the labyrinth and junctional zone could potentially be a security against destructive infection. it is assumed that expressions of tlr-4 are increased their regulation in placenta as a defense mechanism that can be easily mobilized to protect the fetus from infection during pregnancy. tlr-4 may be an important regulator of the immune system from placental infection, but also may be required for the maturation of fetal immune response. porphyromonas gingivalis exposure in trophoblastic giant cell can increase the expression of tlr-4. trophoblastic giant cell has been reported as a differentiated trophoblast precursor cells. it is possible that porphyromonas gingivalis can cause cells of maternal-fetal interface to degenerate and die, thus it stimulates trophoblasts to perform phagocytosis and eliminate damaged cells. phagocytosis act as a biological mechanism for the elimination of dead or degenerated cells. it was expected that the degeneration and deterioration of trophoblastic giant cells eventually lead to reduced trophoblasts. therefore, tlr-4 serve as an important sensor for macrophages and trophoblasts cells, which makes it possible to coordinate the local immune response and to enhance cell invasion and placental formation. tlr-4 also can provide a bridge for placental recognition to the danger signal, and the generated responses can have harmful consequences for the pregnancy. this study also analyzed that the increasing concentration of lps in the maternal blood serum resulted from porphyromonas gingivalis infection on maternal periodontal tissues can result in decreased placental weight, fetal weight and fetal length. the decreased fetal weight and fetal length were caused by a decrease placental weight. the placenta provides a better characterization about the intrauterine environment, in particular the specific changes in immune responses leading to environmental changes in pro-inflammatory and anti-inflammatory. it will affect the activity of nutrient transport to fetus from maternal, resulting in decreased fetal weight and fetal length. thus, changes in placental morphologic condition caused by exposure to toxic agents can play a role as markers of intrauterine environmental disturbance. in conclusion, the present study indicates that the increased lps concentration in maternal blood serum resulted in the decreased fetal weight and fetal length. syncytiotrophoblast, spongiotrophoblast and trophoblastic giant cell were able to recognize porphyromonas gingivalis lps through the tlr-4 expression. this findings strengthened the link between periodontal disease and fetal growth restriction. acknowledgement we would like to thank dr. retno pujirahayu; sitarina widyarini, ph.d; tri wibawa, ph.d; dr. totok utoro; dr. indwiani astuti and prof. al. supartinah, each of whom has made invaluable contributions to our work. their efforts, collaborations, insights, and discussions are greatly appreciated. this study was supported by grant no. 481/sp2h/pp/dp2m/vi/2010 from directorat general of higher education, ministry of national education – indonesia. references 1. socransky ss, haffajee ad. periodontal microbial ecology. periodontol 2000 2005; 38: 135–87. 2. gursoy uk, könönen e, uitto vj. stimulation of epithelial cell matrix metalloproteinase (mmp-2, -9, -13) and interleukin-8 secretion by fusobacteria. oral microbiol immunol 2008; 23: 432–4. 149kusumawardani, et al.: maternal endotoxin-induced fetal growth restriction 3. gibson fc, genco ca. porphyromonas gingivalis mediated periodontal disease and atherosclerosis: disparate diseases with commonalities in pathogenesis through tlrs. curr pharm des. 2007;13(36): 3665–75. 4. arce rm, barros sp, wacker b, peters b, moss k, offenbacher s. increased tlr4 expression in murine placentas after oral infection with periodontal pathogens. placenta 2009; 30: 156–62. 5. han yw, fardini y, chen c, iacampo kg, peraino va, shamonki jm, redline rw. term stillbirth caused by oral fusobacterium nucleatum. obstet gynecol 2010; 115: 442–5. 6. xu d, chen y, wang h, zhao l, wang j, wei w. tumor necrosis factor alpha partially contributes to lipopolysaccharide-induced intra-uterine fetal growth restriction and skeletal development retardation in mice. toxicology letters 2006; 163: 20–9. 7. xu d, chen y, wang h, zhao l, wang j, wei w. effect of nacetylcysteine on lipopolysaccharide-induced intra-uterine fetal death growth retardation in mice. toxicol sci 2005; 88: 525-33. 8. straszewski-chaves sl, abrahams vm, mor g. the role of apoptosis in the regulation of trophoblast survival and differentiation during pregnancy. endocrine rev 2005; 26: 877–97. 9. von dadelszen p, magee la. could an infectious trigger explain the differential maternal response to the shared placental pathology of preeclampsia and normotensive intrauterine growth restriction?. acta obstet gynecol scand 2002; 81: 642–8. 10. medzhitov r, janeway jr ca. decoding the patterns of self and nonself by the innate immune system. science 2002; 296: 298–300. 11. abrahams vm, straszewski-chavez sl, guller s, mor g. first trimester trophoblast cells secrete fas ligand which induces immune cell apoptosis. mol hum reprod 2004; 10: 55–63. 12. pan c, gu y, zhang w, zheng y, peng l, deng h, chen y, chen l, chen s, zhang m, gao z. dynamic changes of lipopolysaccharide levels in different phases of acute on chronic hepatitis b liver failure. plos one 2012; 7: 1–7. 13. shao b, munford rs, kitchens r, varley aw. hepatic uptake and deacylation of the lps in bloodborne lps-lipoprotein complexes. innate immun 2012; 18(6): 825-33. 14. ning h, wang h, zhao l, zhang c, li x, chen y, xu d. maternallyadministered lipopolysaccharide (lps) increases tumor necrosis factor alpha in fetal liver and fetal brain: its suppression by low-dose lps pretreatment. toxicol lett 2008; 176: 13–9. 15. vitiello pf, shainheit mg, allison em, adler ep, kurt ra. impact of tumor-derived ccl2 on the t cell effector function. immunol lett 2004; 91: 239–45. 16. kusumawardani b, soesatyo m, dasuki d, asmara w. fetal growth restriction in porphyromonas gingivalis-infected pregnant rats. dentika dent j 2011; 16: 26–30. vol 51 no 3 jul sep 2018_pus.indd 119 socioeconomic status and orthodontic treatment need based on the dental health component hilda fitria lubis and hilda paula laturiuw department of orthodontics faculty of dentistry, universitas sumatera utara medan indonesia abstract background: the dental health component (dhc) is used to assess different malocclusions by evaluating the number of teeth, overjet, overbite and contact points of each tooth. this index is subjectively influenced by socioeconomic factors. purpose: this study aimed to determine the overview of the socioeconomic status and orthodontic treatment need based on the dhc of state 15 medan junior high school students. methods: this study represented a descriptive research featuring cross-sectional design. the subjects of this study consisted of 100 high school students. this study was conducted by making dental impressions and distributing questionnaires. data analysis was conducted with spss 17.0 software using a descriptive statistical test and presented in a distribution and frequency table. results: 89% of subjects demonstrated dhc levels of 1-2. 66.3% of subjects with parents of secondary education level had dhc grades 1-2, followed by 31.5% with parents of higher education level and 2.2% with parents of basic education level. the majority of subjects with dhc grades 1-2 had parents occupying class 2 jobs and the lowest number of students had parents holding class 4 jobs. the majority of the subjects with dhc grades 1-2 (77.5%) were of low income parents. conclusion: the largest distribution of orthodontic treatment need based on dhc among students of state 15 junior high school occurred at levels 1-2 which either required or did not require minor orthodontic treatment. the majority of the students with dhc grades 1-2 have parents of secondary education level, a class 2 job and a low monthly income. keywords: malocclusion; orthodontic treatment need; dental health component; socioeconomic status; junior high school students correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, padang bulan, kampus usu, padang bulan, medan baru, medan, sumatera utara 20155, indonesia. e-mail: hildadrgusu@gmail. com dental journal (majalah kedokteran gigi) 2018 september; 51(3): 119–123 research report introduction malocclusion is defined as the deviation of tooth position beyond the normal jaw arch1 whose prevalence varies worldwide. based on a 2013 indonesian national health research report, 14 provinces record oral health problems (24.9%) and the prevalence of malocclusion in indonesia remains high, at approximately 80% of the population.2 based on research conducted by wijayanti et al.3 the prevalence of malocclusion among 12-14 year olds attending junior high schools in jakarta was 83.3%, making it the third most frequent oral and dental problem after tooth caries and periodontal disease. the results of the study showed that most young children experienced malocclusion due to a lack of awareness of dental treatment and bad habits such as thumb sucking and lip biting. therefore, efforts should be made to curb the incidence rate by means of, among other approaches, early detection and prevention of malocclusion.3,4 the severity of malocclusion will have an impact on oral function, occurrence of periodontal diseases and facial aesthetics.5 reviews of psychological studies suggest that malocclusions affecting physical appearance may have an unfavorable impact on the psychological development of adolescents.6 adolescence is an important stage in the life of any individual because it is the transitional period from childhood to adulthood, whereby physical, mental and psychosocial changes rapidly affect various aspects of life.7 the need for orthodontic treatment is not only influenced by objective research, but also by subjective dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p119–123 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p119-123 120lubis and laturiuw/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 119–123 judgment of aesthetics, sociocultural and socioeconomic factors.8 according to rahayu,9 socioeconomics constitute a combination of education, employment and income. the research conducted by oley et al.5 suggests that socioeconomic status affects the need for orthodontic treatment. parents of high socioeconomic status have a more elevated level of awareness and therefore prioritize regular visits to the dentist, whereas parents of lower socioeconomic standing lack the economic means to afford dental treatment.5 the index of orthodontic treatment need (iotn) had been internationally accepted and judged to be valid, reliable and easy to use. the index of orthodontic treatment consists of two components, the aesthetic component (ac) and the dental health component (dhc).10,11 the latter is used to objectively assess multiple malocclusions using hypodontia, overjet, crossbite, contact point and overbite measurements. dhc consists of a malocclusion severity scoring system, whereby a score of 1-2 indicates the need for mild orthodontic treatment or no treatment. a score of 3 indicates the need for borderline or moderate treatment and a score of 4-5 indicates the need for utmost care (table 1).8,11–13 the study by oley et al5 involving 390 tondano state 3 junior high school students showed that, based on the dhc, 51.56% did not require treatment, 35.94% needed mild care and 12.5% were in desperate need of treatment. this high level of awareness about the desirability of visiting a dentist stems from the higher socioeconomic status of the students.5 in addition, a study by badran et al.14 of 550 13-17 year old students in four public and private schools in amman using iotn confirmed 17.1% of children of low socioeconomic table 1. dental health components of the iotn.13 grade 1 (none) extremely minor malocclusions including contact point displacements less than 1 mm.1. grade 2 (mild treatment need) increased overjet greater than 3.5mm, but less than or equal to 6mm with competent lips.2.a reverse overjet greater than 0 mm but less than or equal to 1 mm.2.b anterior or posterior crossbites with less than or equal to 1mm discrepancy between the retruded contact and intercuspal2.c positions. contact point displacements greater than 1mm, but less than or equal to 2mm.2.d anterior or posterior open bite greater than 1mm, but less than or equal to 2mm.2.e increased overbite greater than or equal to 3.5mm without gingival contact.2.f pre-normal or post-normal occlusions with no other anomalies (includes up to half a unit discrepancy).2.g grade 3 (borderline/ moderate treatment need) increased overjet greater than 3.5mm but less than or equal to 6mm with incompetent lips.3.a reverse overjet greater than 1mm,but less than or equal to 3.5mm.3.b anterior or posterior crossbites with greater than 1mm but less than or equal to 2mm discrepancy between retruded contact3.c position and intercuspal position. contact point displacements greater than 2mm, but less than or equal to 4mm.3.d lateral or anterior open bite greater than 2 mm,but less than or equal to 4mm.3.e deep overbite complete on gingival or palatal tissues, but without trauma.3.f grade 4 (great need of treatment) increased overjet greater than 6mm, but less than or equal to 9mm.4.a reverse overjet greater than 3.5mm with no masticatory or speech difficulties.4.b anterior or posterior crossbites with greater than 2mm discrepancy4.c severe contact point displacements greater than 4mm.4.d extreme lateral or anterior open bite greater than 4mm.4.e increased and completed overbite with gingival or palatal trauma.4.f less extensive hypodontia requiring pre-restorative orthodontic or orthodontic space closure to obviate the need for4.h prosthesis. 4.l posterior lingual crossbite with no functional occlusal contact in one or both buccal segments. 4.m reverse overjet greater than 1mm but less than 3.5mm with reported masticatory or speech difficulties. 4.x presence of supernumerary teeth. grade 5 (great need of treatment) increased overjet greater than 9mm5.a extensive hypodontia with restorative implications (more than one tooth missing in any quadrant) requiring pre-restorative5.h orthodontics. impeded eruption of teeth (except for third molars) due to crowding, displacement, the presence of supernumerary teeth,5.i retained deciduous teeth and any pathological cause. reverse overjet greater than 3.5 mm with reported masticatory or speech difficulties.5.m defects of cleft lip and palate and other craniofacial anomalies.5.p submerged deciduous teeth.5.s dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p119–123 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p119-123 121 lubis and laturiuw/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 119–123 status as having no malocclusion or requiring only mild care, whereas 20.3% desperately needed care. in contrast, 8.3% of children of high socioeconomic status presented no malocclusions or required only mild treatment, while 5.9% were in great need of care.14 the lack of research into the level of orthodontic treatment need based on the dental health component (dhc) and socioeconomic status in medan interests researchers in determining an overview of the socioeconomic status and level of dhc-related orthodontic treatment need based among students of state 15 junior high school, medan. materials and methods this study constituted descriptive research featuring a cross-sectional design. a total of 100 students of state 15 junior high school in medan were selected as subjects of this research with a minimum sample size of 87, determined by using a categorical descriptive formula. the institution selected constituted a public school whose students were drawn from various socioeconomic backgrounds. this study was conducted from september 2016 to february 2017. a simple random sampling method was employed whereby subjects were indiscriminately selected to participate in this study with the estimated sample size being calculated using an approximated proportion of the population. prior to initiation of this research, ethical approval was obtained from the university of north sumatera health research ethics committee (76/270217/kepk fk usu-rsup ham/2017). the inclusion criteria were as follows: 15-17-year old state 15 junior high school male and female students, willing to participate as research subjects, with no history of requiring orthodontic treatment and possessing complete dentition (excluding the third molars). students suffering dental decay, with a previous history of orthodontic treatment or who were currently undergoing it or who presented observable asymmetry of the jaw were excluded from this research. the rejection criteria applied to subjects during this study included: ill-health, inability to attend school and interrupted study. all subjects were informed that the provision of written informed consent would ensure their inclusion in this study. data on the socioeconomic status of each consenting respondent was obtained by means of a questionnaire containing open-ended items. the intraoral condition of each subject was examined to verify the presence of decay and malocclusions before dental impressions were taken using normal-setting alginate (hygedent, inc., beijing, china). dental stone type iii (heraeus-kulzer gmbh, hanau, germany) was immediately poured into the dental impressions to obtain the study model (figure 1). assessment of the need for orthodontic treatment was conducted by measurement of the study model applying the examination criteria of overjet, overbite, crossbite, contact point shift and hypodontia. repeated measurements were taken by inter-operators (h.f.l. and h.p.l.) to confirm the measurement results and reduce bias. furthermore, the results were later classified according to the level of orthodontic treatment need based on dhc.12,14 all statistical analysis was performed with the statistical package for the social sciences (spss), version 17.0 (spss, inc., chicago, il, usa) using the descriptive statistical test and presented in a distribution and frequency table. results the results showed that majority of the subjects did not require orthodontic treatment (dhc grades 1-2), with only a handful of students in desperate need of it (dhc grades 4-5) (figure 2). the recording of the highest level of parental education and orthodontic treatment requirements of students based on dhc showed that a majority of those with dhc grades 1-2 had parents with a secondary education. for dhc grade 3, some subjects had ones with a secondary education, whereas some subjects had parents with a higher education. as for dhc grades 4-5, most of the students have ones who progressed to higher education. (figure 3). orthodontic treatment needs based on dhc figure 2. distribution of orthodontic treatment needs based on dhc. figure 1. clinical malocclusion as observed from the study model. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p119–123 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p119-123 122lubis and laturiuw/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 119–123 the results of parental employment level and orthodontic treatment need based on the dhc of students showed that a majority of the subjects with dhc grades 1-2 had parents with a class 2 job, while the lowest number of students had ones occupying a class 4 job. for dhc grade 3, most subjects have parents with class 2 jobs. as for dhc grades 4-5, a majority of the subjects have ones also holding a class 2 job (figure 4). the results for the monthly parental income of state 15 junior high school students and the orthodontic treatment need based on dhc showed that a majority of the subjects with dhc grades 1-2 had low income parents and the lowest percentage were those with high income parents . for dhc grade 3, a large number of subjects had low income parents. as for dhc grades 4-5, all subjects had low income parents (figure 5). discussion the dental health component (dhc) is an index that objectively assesses malocclusions using overjet, overbite, crossbite, contact point shift and hypodontia.9 dhc is used to record the worst features of malocclusion that affect longterm dental function and health.1,15 the results of this study are in line with those of the research conducted at theodorus kotamobagu catholic junior high school which showed that 112 students (85.3%) required minor orthodontic treatment, 19 students (13.3%) needed moderate care and two students (1.4%) were in urgent need of treatment.6 research conducted on 15-17 year old adolescents showed that 33 students (51.56%) required no or only minor treatment, 23 students (35.94%) needed moderate care and 8 students (12.5%) were in desperate need of attention.5 adolescents possess a higher level of awareness of their physical appearance and are, consequently, more self-conscious about dental malocclusions.5,16 contrasting results were produced by a study conducted on 61 students in state 1 tareran junior high school. among 13-14 yearold students, there were 10 students (16.39%) who required no or only minor treatment, 11 students (18.04%) were slightly in need of care, while 40 students (65.57%) were in dire need of it.12 this proves that the use of different indices or indicators may yield contrasting results. moreover, there were differences in the characteristics of each population and race.3 the level of education is determined by the duration of formal schooling which, according to indonesian law no.20 of 2003, consists of basic education, secondary education and higher education. basic education is delivered at preschool and middle schools, whereas secondary education is provided by junior and senior high schools. higher or tertiary education consists of diploma, bachelor, masters, specialist and doctoral-level qualifications obtainable through a university.17 the results of this study failed to definitively prove that the higher the level of formal education, the superior the knowledge and attitude towards a healthy lifestyle that might be gained.8,18,19 it is also possible that the occurrence of malocclusion is strongly influenced by genetic and environmental factors. 3 according to barker’s job classification, there are five job classes: class 1 consists of professions such as doctor, prosecutor, architect, notary amongst others requiring a higher education; class 2 are careers accessed by possession of a medium-level education including: civil servant, private employee and teacher ; class 3 included those occupations necessitating only a basic education, for example drivers, security guards, tailors and craftsmen, class 4 were those requiring no basic education such as labor figure 3. distribution of parental education and orthodontic treatment needs based on dhc. figure 4. distribution of parental job class and orthodontic treatment needs based on dhc. figure 5. distribution of parental income per month and orthodontic treatment needs based on dhc. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p119–123 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p119-123 123 lubis and laturiuw/dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 119–123 workers and domestic workers, while class 5 consisting of the unemployed.14 the highest percentage of students requiring either no or only minor orthodontic treatment (68.5%) had parents with class 2 jobs. these results were due to children from higher socioeconomic groups possibly expressing higher demands for orthodontic treatment and their parents possessing greater oral and dental hygiene awareness and concern for their health.14,18 however, the majority of subjects with a moderate or desperate need for orthodontic treatment had parents occupying class 2 jobs. this was probably due to some parents being unable to monitor the dietary habits of their children because of hectic work schedules culminating in nutritional deficiency.19 this would, in turn, result in underdeveloped dentofacial structures, leading to either dental or skeletal malocclusion.20 for the purposes of this study, income levels were determined according to the monthly family income. susi et al.21 define family income as the money earned by parents (either individually or combined) from various daily economic activities. in this study, based on feedback from subjects, the range of parental income was divided into three categories: less than 6.5 million rupiah, between 6.5 and 13 million rupiah and in excess of 13 million rupiah. although the majority of students with low income parents had a minor need for orthodontic treatment, all subjects with high income parents shared this level of need. as with the results of previous studies,22 no significant associations were found between parental wage levels and malocclusion as genetic and environmental factors can affect children regardless of social class. it is also important to note that all subjects in desperate need of orthodontic treatment had low income parents. this was probably due to the higher caries rate among students with such parents resulting in premature loss, molar tooth drifting and crowding. conversely, high income parents were able to afford dental treatment and prevent further tooth decay in their children by having them visit the dentist at an younger age.23 the prevalence of malocclusions requiring minor treatment or none was the largest group, but it did not include the presence of dental anomalies such as crowding, crossbite or supernumerary teeth.24 it was concluded that the largest distribution of orthodontic treatment need based on dhc among students of state 15 junior high school was at levels 1-2. these individuals either required minor or no orthodontic treatment. the majority of the students with dhc levels 1-2 have parents with a secondary education level, a class 2 occupation and a low monthly income. references rahardjo p. ortodonti dasar. 21. nd ed. surabaya: airlangga university press; 2012. p. 64-204. rorong gfj, pangemanan dhc, juliatri j. gambaran maloklulsi pada2. siswa kelas 10 di sma negeri 9 manado. j e-gigi. 2016; 4: 2–7. wijayanti p, krisnawati k, ismah n. gambaran maloklusi dan3. kebutuhan perawatan ortodonti pada anak usia 9-11 tahun (studi pendahuluan di sd at-taufiq, cempaka putih, jakarta). j pdgi. 2014; 63: 25–9. laguhi va, anindita p., gunawan pn. gambaran maloklusi dengan4. menggunakan hmar pada pasien di rumah sakit gigi dan mulut universitas sam ratulangi manado. j e-gigi. 2014; 2(2): 1–7. oley ab, anindita ps, leman ma. kebutuhan perawatan orthodonti5. berdasarkan index of orthodontic treatment need pada usia remaja 15-17 tahun. j e-gigi. 2015; 3(2): 292–7. hansu c, anindita ps, mariati ni wayan. kebutuhan perawatan6. ortodonsi berdasarkan index of orthodontic treatment need di smp katolik theodorus kotamobagu. j e-gigi. 2013; 1(2): 99–104. dewi o. analisis hubungan maloklusi dengan kualitas hidup pada7. remaja smu kota medan tahun 2007. thesis. medan: universitas sumatera utara; 2008. p. 17-36. indraswari r, agusni t, sylvia m. besarnya tingkat kebutuhan8. perawatan ortodonti pada populasi jawa (orthodontic treatment need in javanese). orthod dent j. 2010; 1(1): 26–9. rahayu wp. analisis intensitas pendidikan oleh orang tua dalam9. kegiatan belajar anak, status sosial ekonomi orang tua terhadap motivasi belajar dan prestasi belajar siswa. j pendidikan dan pembelajaran. 2011; 18: 72–80. 10. kaolinni w, hamid t, winoto er. dental student’s perception to aesthetic component of iotn and demand for orthodontic treatment. dent j (maj ked gigi). 2013; 46(2): 97–100. 11. hamid t. treatment results evaluation using the index of orthodontic treatment need. dent j (maj ked gigi). 2009; 42(4): 204–9. 12. wilar la, rattu ajm, mariati nw. kebutuhan perawatan orthodonsi berdasarkan index of orthodontic treatment need pada siswa smp negeri 1 tareran. j e-gigi. 2014; 2(2): 1–8. 13. cobourne mt, dibiase at. handbook of orthodontics. philadelphia: mosby; 2010. p. 22-6. 14. badran sa, sabrah ah, hadidi sa, al-khateeb s. effect of socioeconomic status on normative and perceived orthodontic treatment need. angle orthod. 2014; 84(4): 588–93. 15. gill ds. ortodonsia at a glance. suta t, editor. jakarta: egc; 2014. p. 28-30, 135-6. 16. rumampuk ma v., anindita ps, mintjelungan c. kebutuhan perawatan ortodonsia berdasarkan index of orthodontic treatment need pada siswa kelas ii di smp negeri 2 bitung. j e-gigi. 2014; 2(2): 1–6. 17. setyaningsih r, prakoso i. hubungan tingkat pendidikan, tingkat sosial ekonomi dan tingkat pengetahuan orangtua tentang perawatan gigi dengan kejadian karies gigi pada anak usia balita di desa mancasan baki sukoharjo. j ilmu kesehatan kosala. 2016; 4: 13–24. 18. ngantung ra, pangemanan dh., gunawan p. pengaruh tingkat sosial ekonomi orang tua terhadap karies anak di tk hang tuah bitung. j e-gigi. 2015; 3(2): 542–8. 19. abuaisha aa, huda bz. dental caries and its associated factors among children aged 8-12 years in libyan schools, klang valley, malaysia. asian j agric biol. 2018; : 55–61. 20. singh n, tripathi t, rai p, gupta p. nutrition and orthodonticsinterdependence and interrelationship. res rev j dent sci. 2017; 5(3): 18–22. 21. susi s, bachtiar h, azmi u. hubungan status sosial ekonomi orang tua gengan karies pada gigi sulung anak umur 4 dan 5 tahun. majalah kedokteran andalas. 2012; 36: 96–105. 22. de sousa rv, pinto-monteiro ak de a, martins cc, granville-garcia af, paiva sm. malocclusion and socioeconomic indicators in primary dentition. braz oral res. 2014; 28: 1–7. 23. prabu d, naseem b, manish j, mathur a, dhanni c, saify m, goutham b, kulkarni s. a relationship between socio-economic status and orthodontic treatment need. virtual j orthod. 2008; 8(2): 9–16. 24. kolonio fe, anindita ps, mintjelungan cn. kebutuhan perawatan ortodonsi berdasarkan index of orthodontic treatment need pada siswa usia 12-13 tahun di smp negeri 1 wori. j e-gigi. 2016; 4(2): 259–64. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p119–123 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p119-123 140 accuracy of a fourth generation apex locator-an in vitro evaluation dalia abdullah1 and mariam abdullah2 1 department of operative dentistry faculty of dentistry, ukm 2 department of conservative dentistry, faculty of dentistry, um kuala lumpur abstract the new fourth generation electronic apex locator (eal), elements (sybronendo, usa) has been introduced recently in the market. this study aims to investigate the accuracy of this eal and to compare the result with a well-known apex locator, root zx and the radiographic technique using an in vitro model. thirty anterior teeth with straight canals stored in 10% formalin were used. access cavities were prepared followed by coronal flaring of the canals. water was used as an irrigant. after the actual lengths (al) were measured, the teeth were then embedded in an alginate model. periapical radiograph of each tooth was taken using a digital sensor and the radiographic lengths (rl) were measured 0.5 mm short of the radiographic apex. electronic tooth length measurements (el) were carried out using both eal. canals were then irrigated with 2.5% naocl and el was taken again. results showed that both eals were highly accurate to within + 0.5 mm of the apical foramen, with mean differences between the al and el of elements 0.23mm (se = 0.04) and root zx was 0.31mm (se = 0.05). rl was significantly less accurate compared to the readings from both eal. no significant difference was found in the reading between both apex locators when measurements were taken in naocl solution. both elements and propex proved to be as reliable as root zx. presence of sodium hypochlorite solution did not affect the accuracy of the measurements. key words: electronic apex locator, elements, root zx, alginate model correspondence: dalia abdullah, department of operative dentistry, faculty of dentistry, ukm. jalan raja muda abdul aziz, 50300 kuala lumpur. email: daliaabd@medic.ukm.my/daliaabd@hotmail.com; tel: 03 4040 5769, fax: 03 4040 5794. introduction the objective of root canal treatment is to provide an environment that allows healing of periradicular tissues. one of the important steps to achieve this objective is to clean and shape the entire canal system from the orifice to the minor apical foramen. this entire length of canal is commonly called as the “working length”. before the development of electronic apex locators, working length was determined by measuring tooth length in pre-operative radiographs. kuttler1 in his study reported that the minor apical foramen is commonly located about 0.5–1 mm from the anatomical apex. based on this fact, the common practice of estimating working length was to measure the length of the root from the radiographic apex to the crown and then deducting 0.5–1 mm from the length. however, this method has been reported to be unreliable and inaccurate as the radiographic images, most of the time was found to be distorted.2 electronic apex locator (eal) is an important adjunct in root canal treatment. it helps to locate the minor apical foramen and therefore reduces the error during working length determination. sunada3 introduced the first apex locator after he discovered that he could use the constant value of impedance of both periodontal ligament and the oral mucosa to locate the position of the apical foramen. the earlier two generations of apex locators however, tend to give poor readings and its performance could be influenced by canal contents such as presence of pulp tissues and irrigants.4 since then, improvements were made to the device that led to the development of apex locators using multiple frequencies to increase the accuracy. one of the third generation apex locators, root zx (j. morita, tokyo, japan), had been extensively studied and was reported to be 95-100% accurate.5,6 it was proven to be reliable even in the presence of body fluid, pulp tissues and irrigants.7 in 2003, elements diagnostic unit (sybronendo, anaheim, usa), a fourth generation eal was newly introduced in the market. it is a 2-in-1 unit with the second function of vitality testing. to determine the length, it uses the principle of measuring the impedance value by calculating both the resistance and capacitance and comparing this value with the database built in the device. this technique of measurement was claimed by manufacturer to increase the accuracy and reliability of the device.8 the purpose of this study were, to investigate the accuracy of elements eal using an in vitro model, to compare its accuracy with root zx and to the length obtained from radiographs, and to investigate its accuracy of measurement in the presence of 2.5% sodium hypochlorite (naocl) solution. 141abdullah: accuracy of a fourth generation apex locator materials and methods thirty single-rooted anterior extracted teeth were collected from various dental clinics and kept wet in 10% formalin. these were mature teeth with completely formed apices and straight roots with the absence of apical resorption. presence of a patent apical canal was confirmed by allowing a #6 file to protrude through the apical foramen. the incisal edges were trimmed and leveled with a diamond bur to provide fixed coronal reference points to get accurate and reproducible measurements. standard endodontic access cavities were made at the palatal surfaces. canals were flared coronally using gates glidden drills (dentsply, switzeland) size 2 and 3. canals were irrigated with water using a 3 ml syringe and 25g hypodermic needle. determination of actual length (al): al was measured by introducing a size 15 k-file (dentsply, switzeland) into each canal until the tip of the file was just visible at the main foramen using the microscope ompi pico (carl zeiss, germany) under 2.5 × magnification (from 0, 45 and 90 degree angle). each measurement was repeated 3 times and an average was computed. alginate model specially developed by kaufmann et al.9 for testing apex locators was used in this study. the model consisted of teeth embedded in alginate in a plastic box (figure 1). the alginate model was kept moist in the refrigerator when not in use throughout the experiment. pink wax was used to block an area next to the embedded teeth to accommodate placement of the radiographic digital sensor for taking radiographs. figure 1. alginate model. d e t e r m i n a t i o n o f r a d i o g r a p h i c l e n g t h ( r l ) : radiovisiograph (rvg, trophy) was used to take the images of the teeth. the rvg sensor was placed in the allocated slot, which was parallel vertically and horizontally to the teeth. the sensor was placed as near as possible to the teeth, distance from the sensor and x-ray cone was fixed at 4 cm and with the exposure time fixed at 0.17s. software trophy windows ver 5.04 was used to visualize the images. the tooth length was calibrated, measured and recorded under 100% magnified images by measuring the coronal reference point to the radiographic apex. a 0.5 mm was deducted from this length to give the rl as what was usually practiced clinically. determination of electronic length (el): lip clip was attached to alginate and the file clip was attached to the file in each tooth. measurement was taken with k-files of appropriate sizes (first file that binds to the apex). for root zx, measurement of the length was taken when the device indicated ‘apex’. for elements (figure 2), measurement was taken when reading was at ‘0’ (although the instructional manual stated that from ‘0’ reading, the file should be withdrawn to 0.5 mm mark to achieve working length clinically). measurements were repeated 3 times for each tooth and the third working length measurement was taken for analysis. determination of electronic length (el) in the presence of 2.5% naocl: all canals were irrigated with 15 ml 2.5% naocl solution. excess solution in the canal was aspirated using the irrigating syringe and el was taken again using both eals. to prevent bias, measurement for all 30 teeth was done at a random order. figure 2. elements apex locator. distribution of differences in measurements between el/rl and al 0 2 4 6 8 10 12 14 16 x>-1 -0.5<x=<-1 0<x=<-0.5 x=0 0<x=<0.5 0.5<x=<1 x > 1 range (mm) n u m b e r o f t e e th root zx elements radiograph figure 3. distribution of the difference between rl and el with al (n = 30). data analysis: all the measurements of rl and el were deducted from the al for each tooth to give a value of the difference. the negative value indicated the length was short of the apex, zero indicated that length was at the apex and positive value indicated that the length went 142 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 141-143 beyond the apex. mean difference of rl and el (for all 30 teeth) were computed. results were subjected to statistical analysis (paired t-test and anova with repeated measurements). statistical significance was accepted at p value of less than 0.05. results water as irrigant-measurement obtained from elements (table 1) was found to be closer to al with the mean difference of 0.23 mm. however, the result was not statistically significant as the mean value of measurement taken from root zx eal was at 0.31mm (p = 0.108). the rl was significantly less accurate compared to the readings taken from both eals with mean difference of 0.60mm with the p = 0.00 for element and p = 0.002 for root zx. naocl as irrigant-mean difference of el taken with both elements and root zx in the presence of naocl solution were shown in table 1. statistical analysis of mean difference using anova (post hoc test/turkey’s method) showed no significant difference between the reading of both eals taken with and without the presence of naocl (p = 1). difference of el with al for all teeth were categorized into 7 groups according to distance from apical foramen (figure 3). distribution of differences in measurements between els and als in sodium hypochlorite solution 0 2 4 6 8 10 12 14 16 18 20 x>-1 -0.5<x=<-1 0<x=<-0.5 x=0 0<x=<0.5 0.5<x=<1 range (mm) n u m b e r o f t e e th root zx element figure 4. distribution of differences in measurement between el and al in 2.5% sodium hypochlorite. water as irrigant-ninety percent (n = 27) of the el readings taken from elements fell within the range of 0 or 0.5 mm shorter than al, for root zx it was 80% (n = 24), whilst only 46% (n = 4) of the readings taken from radiographs fell in within this range. in comparison, 43% of the rl readings were about 0.5 mm longer than the actual length (figure 3). naocl as irrigant-eighty percent (n = 24) of the el readings taken from both elements and root zx fell within the range of 0 or 0.5 mm shorter than al (figure 4). discussion the results obtained from this study indicate that elements apex locator was able to locate the minor apical diameter 0–0.5 mm short of the root length, and its performance was at par with root zx. the mean values of both devices were below 0.5 mm with no statistical significant difference and both values were shorter than actual length. this apical position of less than 0.5 mm from the root end could be considered clinically acceptable as the distance of the apical constriction to the major apical foramen (end of the root) is approximately 0.5–0.8 mm in all tooth types.1 apical constriction represents the boundary between the pulpal and periodontal tissue with the smallest diameter of the canal (minor apical foramen). this location was considered to be an ideal position to end the preparation and obturation of root canals, as irritation to the periodontal tissues (by irrigants, obturating materials) was kept minimal (creating a small wound site) and therefore would provide an optimal condition for healing of the periapical tissues.10 even epidemiological study had shown that the best prognosis is when the root filling lies within 2 mm of the apex.11 however, determining the location of this boundary could not be done accurately by using the radiographs. modern apex locators, as shown in this study have the ability to determine this position with greater accuracy. root zx uses two frequencies to determine distance from the end of the apex, which accounts for its accuracy.12 the results from our study showed that 80% of the 30 readings fell within 0.5 mm and the percentage increased to 90% if 1.0 mm distance from the apex was taken as a reference point. these readings were reproducible even when taken in the presence of sodium hypochlorite. elements apex locator showed similar result in which 90% of the 30 readings fell within 0.5 mm from the apex. the readings were reproducible when taken in the presence of sodium hypochlorite with no significant difference in the mean value. the manufacturer claimed that its consistent readings were attributed mainly to its mechanical measurement whereby a composite waveform of two signals, 0.5 and 4 khz were used. these signals go through a digital-to-analogue converter to be converted into table 1. mean differences between rl, el with al (n = 30) solution used for irrigation root zx (se) elements (se) radiograph (se) water 2.5% naoci 031 (0.50) 0.48 (0.14) 0.23 (0.03) 0.43 (0.13) 0.60 (0.60) 143abdullah: accuracy of a fourth generation apex locator an analogue signal, which then goes through amplification and then to the patient circuit model. the feedback signal waveforms are then fed into a noise reduction circuit to give the reading.8 one point to note is that a smaller percentage of readings (10–20%) from both eal were 0.5 mm longer than the actual length. verification of the electronic measurements by radiographs may indeed be necessary to avoid trauma to the periapical tissues. during the study, it was observed that the readings from both apex locators tend to fluctuate when the canals were flooded with the solution and both devices performed much better when the excess solution was aspirated from the canal. radiographic method has been known to be unreliable in accurately determining working length. the measurements obtained from the radiographs were constantly longer than the actual length (mean difference of 0.60 mm) and longer compared to the readings from both ealss. these differences were statistically significant. this result was similar with stein & corcoran,2 who found a difference of 0.7 mm between the real file positions to the measurements calculated on radiographs. the alginate model proved to be a good and easy-tomake method to test the accuracy of apex locators in vitro. the electronic length was taken only after coronal flaring was carried on all the teeth to simulate the clinical practice. early coronal flaring will ensure no change in length occurs after canal preparation. during the study, unstable readings were observed when a small file (eg file #10) was used in a relatively bigger canal. a bigger file (the first file that engaged the canal wall at the apical end) was therefore used to take the measurement. in conclusion, this study shows that elements eal is able to measure canal length within a clinically acceptable range and its accuracy is comparable to root zx. presence of sodium hypochlorite does not affect the accuracy of the measurement. radiographic measurements, at most time, results in overestimation of the actual length. references 1. kuttler y. microscopic investigation of root apexes. j am dent assoc 1955; 50:544–52. 2. stein tj, corcoran jf. radiographic working length revisited. oral surg oral med oral pathol 1992; 74:796–800. 3. sunada i. new method for measuring the length of the root canal. j dent res 1962; 41:375–87. 4. fouad af, krell kv, mckendry dj, koorbusch gf, olson ra. clinical evaluation of five electronic root canal length measuring instruments. j endod 1990; 16: 446–9. 5. mcdonald nj, pileggi r, glickman g, varella c. an in vivo evaluation of third generation apex locators [abstract]. j dent res 1999; 78:373. 6. vajrabhaya l, tepmongkol p. accuracy of apex locator. endod dent traumatol 1997; 13:180–2. 7. shabahang s, goon ww, gluskin ah. an in vivo evaluation of root zx electronic apex locator. j endod 1996; 22:616–8. 8. gordon mpj, chandler np. electronic apex locators. int endod j 2004; 37:425–37. 9. kaufman ay, keila s, yoshpe m. accuracy of a new apex locator: an in vitro study. int endod j 2002; 35:186–92. 10. wu mk, wesselink pr, walton re. apical terminus location of root canal treatment procedures. oral surg oral med oral path oral radiol endod 2000; 89:99–03. 11. sjogren u, hagglund b, sundqvist g, wing k. factors affecting the long-term results of endodontic treatment. j endod 1990; 16:498–504. 12. kobayashi c, okiji t, kaqwashima n, suda h, sunada i. a basic study on the electronic root canal length measurement: part 3. newly designed electronic root canal length measuring device using division method. japanese journal of conservative dentistry 1991; 34:1442–8 223223 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 223–228 research report the difference between begg and straightwire appliances on molar position, occlusal plane angle, and anterior and posterior facial height changes dewi sartika santoso, christnawati and cendrawasih andusyana farmasyanti department of orthodontics, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: bimaxillary and bidental protrusion class i angle malocclusions have a characteristic convex facial profile and protrusion lips due to the labial inclination of the anterior teeth. extraction of the first four premolars is the most common choice for orthodontic treatment of these cases when all the permanent teeth are complete and in good condition. orthodontic treatment can be performed using the begg or straightwire techniques. purpose: this study aims to investigate the difference in the effect of orthodontic treatment with begg and straightwire appliances on molar position, occlusal plane, and anterior and posterior facial height. methods: sixty pairs of lateral cephalograms before and after the treatment of patients with bimaxillary and bidental protrusive angle malocclusion class i, aged 18–35 years old, who underwent orthodontic treatment using the begg and straightwire techniques with the extraction of all first premolars that met the inclusion criteria. data analysis was performed using two-way repeated analysis of variance (p<0.05) and pearson correlation (p<0.05). results: molar position, occlusal plane angle, and anterior and posterior facial heights increased significantly after the begg technique treatment and decreased significantly after the straightwire technique treatment (p<0.05), but there were no significant differences between the four variables in the two techniques (p>0.05). medium correlation was found between variables in both the begg and straightwire techniques. conclusion: molars were extruded and mesialized and the occlusal plane angle and height of the anterior and posterior faces increased after the begg appliances treatment. the molars moved mesially and occlusally and there was a decrease in the occlusal plane angle, as well as the height of the anterior and posterior faces, after treatment with the straightwire appliances. however, there was no difference between the two techniques. keywords: bimaxillary and bidental protrusion; fixed orthodontic treatment; molar position; occlusal plane; facial vertical height correspondence: christnawati, department of orthodontics, faculty of dentistry, universitas gadjah mada, jl. sekip utara, bulaksumur, yogyakarta, 55281 indonesia. email: christnawati_fkg@ugm.ac.id introduction malocclusion is a condition deviating from the normal occlusion that occurs due to a discrepancy between the dental arch and the jaw arch.1 this situation can occur in the upper and lower jaw and results in disturbances in chewing, phonation and aesthetics.1–3 the prevalence of class i angle malocclusion in the indonesian deuteromalay population is 48.8%.4,5 class i bimaxillary protrusion malocclusion has a convex profile.2 the orthodontic treatment objective is the correction of the malrelation and malposition of teeth to achieve stable occlusion function and pleasant facial aesthetics.2,6 the vertical dimension of the face of the patient undergoing orthodontic treatment is an important aspect to consider because it determines facial aesthetics.7 the height of the vertical dimension of the face is influenced by the angle of the occlusal plane, the height of the anterior face, the height of the posterior face and the movement of the molar in the horizontal and vertical directions.8–10 the begg technique is a fixed orthodontic treatment technique that has long been used.11 round section dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 mailto:christnawati_fkg@ugm.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 224 santoso et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 223–228 archwires provide the free tipping motion of crowns.11,12 the function of the anchorage bend is to open the anterior bite and control anchorage, thereby preventing the tipping of the mesial anchoring molars.11,12 correction of a malocclusion angle class i bimaxillary protrusion uses z elastic (intramaxillary elastic combined with intermaxillary elastic class ii) from the first stage of treatment.11–13 the anchorage bend creates a force vector that acts on the anterior mandibular teeth down and front, whereas on the mandibular molars it is down and back.10,11,14 intermaxillary elasticity causes forward and upward force vectors on the mandibular molars as well as down and backward on the anterior mandibular teeth.10,11,14 an extrusion of the molars will result in a rotation of the mandible backwards and downwards so that the occlusal plane angle increases and the facial height increases.10,11,14–16 the straightwire technique is one of the orthodontic treatment techniques.2 orthodontic tooth movement uses sliding mechanics.1,2 stainless steel bow wire measuring 0.016 x 0.022 inches is used at the anterior retraction stage so that there is bodily tooth movement, and maximum anchorage is required.1–3 maximum anchorage is obtained by bonding the buccal tube to the first and second molars.2,3 a gable bend is used during retraction functions to increase anchorage control in the molars.2,3,17 the vector of forces acting on the mandibular molars is forward and downward, whereas in the anterior mandibular teeth it is backward and upward due to archwire deflection.17 a gable bend on the mesial buccal tube will create a forward and upward force vector of the mandibular molar, whereas in the anterior teeth the force vector is forward and down.17 anchorage loss causes the molars to move mesially and there is a forward and upward rotation of the mandible resulting in smaller mandibular plane angles and a shortening of facial height.17–19 alkumru et al. disclosed that the vertical dimension of the face is not affected by the movement of the molar to the mesial.20 the research results of tarvade et al. are contrary to the popular opinion that the begg technique causes greater vertical dimensional height increases than the preadjusted appliances technique (edgewise and mbt).15 based on the data that has been described, it is necessary to conduct a study to investigate the differences between the orthodontic treatments using the begg and straightwire techniques on molar position, angle of the occlusal plane, and anterior and posterior facial height. materials and methods an ethics permit was obtained from the research ethics commission of the faculty of dentistry, universitas gadjah mada with the number 00435 / kkep / fkg-ugm / ec / 2020. the research object was secondary data, in the form of an initial 120 lateral cephalograms followed by fixed orthodontic treatments using the begg or straightwire techniques, which are appropriate standard requirements by the faculty of dental surgery, the royal college of surgeons of england, which provide a clear contrast and sharpness of the image.21 lateral cephalograms were calibrated using corel draw x5 (corel corp., ottawa, canada). inclusion criteria: a. 18–35 years old; b. angle class i malocclusion is bimaxillary and bidental protrusion; c. anb angle 0°–4°; d. upper and lower lips in front of the s line; e. index of orthodontic treatment need/iotn (dental health component/dhc) scores 1–3; f. complete number of teeth except third molars; g. network periodontal healthy; h. do not have systemic diseases; i. treatment plan the first four premolars were removed. exclusion criteria: a. anodontia; b. there are edentulous; c. badly crowded teeth; d. impacted other than the third molars. determination of the position of the left mandibular first molar in the horizontal direction was calculated using the pancherz parameter, namely the linear distance from the mesiobuccal molar cusp to the vertical mandible (figure 1a). the position of the left mandibular first molar in the vertical direction was calculated using the pancherz parameter, namely the linear distance of the mesiobuccal molar cusp to the horizontal mandible (figure 1b). the position of the left maxillary first molar in the horizontal direction was calculated using the pancherz parameter, which is the linear distance between the mesiobuccal molar cusp to the maxillary vertical (figure 2a). the position of the first molar of the left maxilla in the vertical direction was calculated using the pancherz parameter, namely the linear distance of the mesiobuccal molar cusp to the horizontal maxillary (figure 2b). the occlusal plane angle was calculated using the steiner parameter (figure 3a), which is the angle formed from the occlusal plane (overlapping lines of the first molar and premolar) and sella-nasion. the anterior face height was calculated using the gebeck parameter (figure 3b), which is the distance between the palatal plane (ans-pns) perpendicular to the menton. the posterior facial height was calculated using the gebeck parameter (figure 3c), which is the distance between the articular and the mandibular plane (gonion-menton). the data obtained in this study were tabulated and tested for normality and homogeneity, then analysed using figure 1. measurement of the horizontal mandibular molar position (a) and vertical mandibular molar position (b) using corel draw x5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 225santoso et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 223–228 the parametric test. the change in molar position, angle of the occlusal plane, anterior facial height and posterior facial height before and after orthodontic treatment were analysed by means of a two-way repeated analysis of variance (anova). the relationship between changes in molar position, angle of the occlusal plane, and height of the anterior face as well posterior facial height after orthodontic treatment were analysed using pearson’s parametric correlation and regression. the level of trust that was used in the study was 95% (α = 0.05). analysis was carried out using the statistical package for social science (spss) (ibm, illinois, us) version 23. results the results showed an increase in vertical maxillary molars, vertical mandibular molars, occlusal plane angle, and anterior and posterior facial height after the begg technique orthodontic treatment. decreased horizontal maxillary molars, horizontal mandibular molars, occlusal plane angle, and anterior and posterior facial height were found after fixed orthodontic treatment with the straightwire technique (table 1). there were significant differences between molar positions, occlusal plane angles, and anterior and posterior facial heights after treatments using the begg and straightwire techniques (p = 0.000) (table 2). there was no significant difference (p> 0.05) in molar position, occlusal plane angle, and anterior and posterior facial height after the begg and straightwire orthodontic treatments (table 3). figure 2. measurement of the horizontal maxillary molar position (a) and vertical maxillary molar position (b) using corel draw x5. figure 3. measurement of the angle of the occlusal plane (a), and height of the anterior face (b) and posterior face (c) using corel draw x5. table 1. mean (x) and standard deviation (sd) values of molar positions, angle of the occlusal plane, and height of the anterior and posterior faces of subjects with orthodontic treatment using the begg and staightwire techniques variable mean ± standard deviation (sd) begg straightwire before after before after horizontal maxillary molar (mm) 66.02±6.60 64.94±5.72 67.30±9.34 66.43±7.07 vertical maxillary molar (mm) 22.25±2.10 22.92±2.41 22.52±2.93 22.88±2.79 horizontal mandibular molar (mm) 68.35±6.78 67.37±5.54 70.13±9.58 69.66±8.60 vertical mandibular molar (mm) 27.80±3.48 28.24±3.89 27.54±4.49 28.65±3.76 occlusal plane angle (o) 22.27± 6.62 23.75±5.29 23.74±5.63 22.61±4.44 anterior facial height (mm) 64.78±5.26 66.43±5.44 68.16±8.06 66.55±6.46 posterior facial height (mm) 41.94±5.50 43.17±7.29 41.62±4.96 40.81±4.94 table 2. two-way repeated anova test results, molar position, occlusal plane angle, and anterior and posterior facial height of subjects with orthodontic treatment using the begg and straightwire techniques group f sig. treatment stage 73.829 0.000* treatment stage * type of treatment 3.296 0.075 treatment effect 1327.033 0.000* effect of treatment * type of treatment 1.417 0.207 treatment stage * treatment effect 1275.541 0.000* * significant difference p < 0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 226 santoso et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 223–228 table 4. pearson correlation of fixed orthodontic treatment using the begg and straightwire techniques begg straightwire correlation coefficient sig. correlation coefficient sig. mmxh-mmxv 0.442 0.020 0.442 0.020 mmxh-mmdh 0.442 0.020 0.412 0.020 mmxh-mmdv 0.442 0.020 0.442 0.020 mmxh-sbo 0.415 0.018 0.401 0.020 mmxh-twa 0.456 0.010 0.455 0.010 mmxh-twp 0.373 0.023 0.363 0.023 mmxv-mmdh 0.443 0.020 0.423 0.020 mmxv-mmdv 0.443 0.020 0.443 0.020 mmxv-sbo 0.411 0.024 0.411 0.024 mmxv-twa 0.491 0.006 0.481 0.006 mmxv-twp 0.414 0.021 0.373 0.023 mmdh-mmdv 0.420 0.020 0.420 0.020 mmdh-sbo 0.391 0.022 0.391 0.022 mmdh-twa 0.605 0.000 0.565 0.000 mmdh-twp 0.365 0.022 0.363 0.023 mmdv-sbo 0.420 0.017 0.389 0.022 mmdv-twa 0.620 0.000 0.590 0.000 mmdv-twp 0.425 0.014 0.373 0.023 sbo-twa 0.420 0.017 0.412 0.019 sbo-twp 0.397 0.021 0.389 0.024 twa-twp 0.404 0.019 0.414 0.019 information: mmxh: horizontal maxillary molars mmxv: vertical maxillary molar sbo: occlusal plane angle mmdv: vertical mandibular molar mmdh: horizontal mandibular molar swa: straightwire twa: anterior face height twp: posterior face height table 5. regression of fixed orthodontic treatment using the begg and straightwire techniques model r r square adjusted r square std. error of the estimate begg 0.784 0.581 0.359 1.23654 straightwire 0.759 0.529 0.098 1.67787 table 6. the results of regression analysis of molar position, angle of the occlusal plane, and height of the anterior and posterior faces on the begg and straightwire appliances begg straightwire b t sig. b t sig. mmxh-mmxv 0.223 0.232 0.019 0.196 0.557 0.023 mmxh-mmdh 0.199 0.764 0.020 0.344 1.170 0.000 mmxh-mmdv 0.158 0.272 0.028 0.383 1.677 0.017 mmxh-sbo 0.106 0.350 0.010 0.211 0.800 0.032 mmxh-twa 0.194 0.805 0.019 0.239 1.031 0.013 mmxh-twp 0.356 0.556 0.013 0.281 0.233 0.011 mmxv-mmdh 0.258 0.138 0.008 0.383 0.557 0.023 mmxv-mmdv 0.116 0.189 0.010 0.212 1.170 0.000 mmxv-sbo 0.159 0.033 0.015 0.196 0.715 0.022 mmxv-twa 0.181 0.318 0.025 0.265 0.752 0.020 mmxv-twp 0.123 0.055 0.019 0.274 0.112 0.011 mmdh-mmdv 0.139 0.182 0.048 0.383 1.677 0.007 mmdh-sbo 0.139 0.329 0.045 0.101 0.800 0.032 mmdh-twa 0.450 0.044 0.043 0.281 1.031 0.013 mmdh-twp 0.379 0.605 0.021 0.239 0.455 0.033 mmdv-sbo 0.233 0.049 0.023 0.129 1.871 0.044 mmdv-twa 0.638 0.422 0.006 0.426. 1.871 0.044 mmdv-twp 0.379 0.264 0.011 0.426 0.126 0.039 sbo-twa 0.214 0.025 0.013 0.204 0.273 0.048 sbo-twp 0.146 0.030 0.019 0.124. 0.032 0.025 twa-twp 0.233 0.542 0.033 0.462 0.780 0.011 table 3. the results of the two-way repeated anova test for molar position, occlusal plane angle, and anterior and posterior facial height between the begg and straightwire techniques variable df f sig. type of treatment 1 0.742 0.392 * significant difference p <0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 227santoso et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 223–228 the fixed orthodontic treatment using the begg and straightwire techniques showed a correlation in direction and moderate closeness. the effect of vertical mandibular molars on the angle of the occlusal plane has the greatest value when compared to other molars in the begg technique. horizontal maxillary molars have the greatest value when compared to other molars in the straightwire technique (table 4). the contribution of molar position, anterior face height and posterior face height is 58.1% on the angle of the occlusal plane in the begg technique and 52.9% in the straightwire technique (table 5). there was significant influence between the four variables in both the begg and straightwire techniques (p> 0.05). each 1 mm increase in horizontal maxillary molars, vertical maxillary molars, horizontal mandibular molars and vertical mandibular molars caused the occlusal plane angles to increase by 0.106°, 0.159°, 0.139° and 0.233°. an increase in the angle of the occlusal plane by 1° cause the anterior and posterior facial heights to increase by 0.214 mm and 0.146 mm. the results of the regression analysis on the straightwire technique showed that every 1 mm increase in the horizontal maxillary molar caused the occlusal plane angle to increase by 0.211o (table 6). discussion this study found that there were changes in molar position, occlusal plane angle, and anterior and posterior facial height after orthodontic treatments with the begg and straightwire techniques. the after-treatment effect of the begg technique causes the maxillary molars to extrude and move mesially, and the angle of the occlusal plane and the anterior and posterior facial height to increase, while the treatment effect of the straightwire technique causes the maxillary and mandibular molars to move mesially and extrude, and the angle of the occlusal plane and the anterior and posterior facial height to decrease, which shows a statistically significant difference. molar extrusion and mesialization in the begg technique are probably due to the use of intramaxillary elastic. maxillary molars receive orthodontic force from the use of intramaxillary elastic for retraction and the use of an anchorage bend is intended to prevent anchorage loss as well as to avoid a deep bite due to anterior retraction.10,11 use of an anchorage bend to the mesial buccal tube causes the molar to tip distally. this force can be neutralised by using intramaxillary elastic as the molars will receive an anterior force from the intramaxillary elastic. the anchorage bend angle used in the treatment of the subject was 30–45o and dynamic because it was adjusted to the subject’s overbite each time the control is carried out. intramaxillary strength was ¼ light to ¼ medium or 2.5–4.5 oz. the maxillary molars remain in the initial position, but if the anchorage bend angle is too small or the use of the intramaxillary elastic is too strong, the maxillary molars can extrude and move mesially as in the results of this study. the retraction in the straightwire technique consists of two stages, namely canine retraction followed by anterior/ incisor retraction, which can cause the molars to move mesially. efforts to prevent molar mesialization include using a gable bend that serves as anchorage preparation. the decrease in the occlusal plane angle and facial height are due to molar mesialization. maxillary and mandibular molars receive orthodontic force through the use of a powerchain and gable bend that are fixed during orthodontic treatment. the molars tip distally due to the use of a gable bend.17 it is intended that the molars remain in their position when the retraction stage starts using the powerchain because the molars act as anchorage.2,11 not all orthodontists use a gable bend as this can make it easier for molars to move mesially. the use of force for retraction is 100–250 grams, however, the use of this force is less certain because a tension gauge is not used. a force that is too large is also one of the causes for a molar to move mesially.11 the shorter height of the anterior face despite molar extrusion could be due to the vertical movement of the molar being smaller than the horizontal movement, or the molar extrusion being smaller than the mesial to the molar shift. the movement of molar to mesial can also be caused by the principle of bodily motion in the straightwire technique so that the molar as anchorage moves mesial.2,11 during the finishing stage of this technique, both box elastic and intermaxillary class ii elastic were used.11,18 the use of elastics can cause the extrusion of molars. other factors that lead to molar extrusion include placing the buccal tube deeply into the gingival.2,11 the changes that occurred after orthodontic treatment using both the begg and the straightwire techniques were not statistically significant. this could be due to the difference in the values of the two techniques that are not very dissimilar. the results of this study are the same as those of tarvade et al., who stated that there was no significant difference in facial height increase between the begg and preadjusted (mbt) technique treatment groups.15 the results of the correlation test for fixed orthodontic treatment using the begg and straightwire techniques show that there was a moderate correlation between the angle of the occlusal plane, molar position, and anterior and posterior facial height. in the begg technique, vertical mandibular molars have the greatest correlation with the angle of the occlusal plane compared to other molar positions, indicating that the tooth has the greatest influence on the angle of the occlusal plane. for each increase in vertical mandibular molars by 1 mm, there is an increase in the angle of the occlusal plane by 0.233o. this could be due to the use of intramaxillary elastic, class ii intermaxillary elastic and the use of an anchorage bend to open the anterior bite. bratu et al. stated that when intermaxillary elastic is used throughout the day, the effect of the vertical component is much greater than that of the horizontal component.22 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 228 santoso et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 223–228 in the straightwire technique, horizontal maxillary molars have the greatest value compared to other molar positions. for each increase in horizontal maxillary molar by 1 mm, there will be an increase in the angle of the occlusal plane by 0.196o. a study by chandra et al. reported that mandibular molars moved mesially by 2.26 mm.23 this could be due to the application of excessive force and the eruption of the third molars.23,24 some of the study subjects had impacted third molars and some of the third molars had erupted. according to nanda, a maximum anchorage with two molars as anchors will still have a 25% chance of moving the posterior teeth mesially.17 this study used different cephalogram instruments. efforts to overcome this, namely by calibration of the lateral cephalogram and a validity test of the head length (glabella-occipital), were carried out between the patient and lateral cephalogram. suggestions for further research need to be carried out in prospective studies with the same cephalogram instrument. based on the research results, it can be concluded that the molars extruded and mesialized, and the occlusal plane angle and height of the anterior and posterior faces increased after the begg technique treatment. the molars moved mesially and occlusally and there was a decrease in the occlusal plane angle and the heights of the anterior and posterior faces after treatment with the straightwire technique. however, there was no difference between the two techniques. references 1. foster td. buku ajar ortodonsi. 3rd ed. jakarta: egc; 2012. p. 23–5. 2. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st. louis: mosby elsevier; 2013. p. 5–7, 196–207, 382– 400. 3. singh g. textbook of orthodontics. 3rd ed. new delhi: jaypee brothers medical publishers; 2015. p. 109–13, 172–80. 4. wahab rma, idris h, yacob h, ariffin shz. cephalometric and malocclusion analysis of kadazan dusun ethnic orthodontic patients. sains malaysiana. 2013; 42: 25–32. 5. mizoguchi i, toriya n, nakao y. growth of the mandible and biological characteristics of the mandibular condylar cartilage. jpn dent sci rev. 2013; 49(4): 139–50. 6. ardhana w. identifikasi perawatan ortodontik spesialistik dan umum. maj kedokt gigi indones. 2013; 20: 1–8. 7. burstone cj, marcotte mr. problem solving in orthodontics. journal of orthodontics. chicago: quintessence publishing co inc.; 2001. p. 31–50. 8. cobourne mt, dibiase at. handbook of orthodontics. philadelphia: mosby; 2010. p. 160–5. 9. chhibber a, upadhyay m, shetty vs, mogra s. cephalometric comparison of vertical changes between begg and preadjusted edgewise appliances. eur j orthod. 2011; 33(6): 712–20. 10. fletcher ggt. the begg appliance and technique. boston: john wright & sons; 1981. p. 273–8. 11. begg pr, kesling pc. begg orthodontic theory and technique. philadelphia: saunders; 1977. p. 411–6. 12. setyowati p, ardhana w. perawatan maloklusi kelas iii dengan hubungan skeletal kelas iii disertai makroglosia menggunakan alat ortodontik cekat teknik begg. maj kedokt gigi indones. 2013; 20(2): 184. 13. winarti hs, heryumani jcp, soehardono d. hubungan antara perubahan inklinasi gigi anterior rahang atas dan bawah dengan perubahan tinggi wajah anterior bawah pada maloklusi angle klas i protrusif bimaksiler. j kedokt gigi. 2014; 5(3): 263–70. 14. parkhouse r. tip-edge orthodontics. st. louis: mosby; 2003. p. 38–43. 15. tarvade s, chaudhari c, satish ha. vertical changes during begg’s and pea-a comparative study. iosr j dent med sci. 2013; 9(4): 48–53. 16. yuliastanti d, soehardono, heryumani. hubungan antara sudut bidang oklusal terhadap perubahan tinggi wajah anterior pada maloklusi angle klas ii divisi 1 setelah perawatan ortodonti dengan teknik begg. j kedokt gigi. 2014; 5(3): 247–52. 17. nanda r. esthetics and biomechanics in orthodontics. 2nd ed. philadelphia: saunders; 2014. p. 80–5. 18. hayasaki sm, henriques jfc, janson g, de freitas mr. influence of extraction and nonextraction orthodontic treatment in japanesebrazilians with class i and class ii division 1 malocclusions. am j orthod dentofac orthop. 2005; 127(1): 30–6. 19. danaryudho bp, sjafei a. treatment of angle class i malocclusion with crossbite anterior using preadjusted technique (case report). maj ortod. 2014; 12(2): 12–5. 20. alkumru p, erdem d, altug-atac at. evaluation of changes in the vertical facial dimension with different anchorage systems in extraction and non-extraction subjects treated by begg fixed appliances: a retrospective study. eur j orthod. 2007; 29(5): 508–16. 21. faculty of dental surgery, the royal college of surgeons of england. methodologies for clinical audit in dentistry. london: the royal college of surgeons of england; 2000. p. 32. 22. bratu cd, fleser c, glavan f. the effect of intermaxillary elastics in orthodontic therapy. tmj. 2004; 54(4): 406–9. 23. chandra p, kulshrestha rs, tandon r, singh a, kakadiya a, wajid m. horizontal and vertical changes in anchor molars after extractions in bimaxillary protrusion cases. apos trends orthod. 2016; 6(3): 154–9. 24. nahidh m, al azzawi am, al-badri sc. understanding anchorage in orthodontics-review article. j dent oral disord. 2019; 5(2): 1117. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p223–228 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p223-228 8181 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 81–87 review article during and post covid-19 pandemic: prevention of cross infection at dental practices in country with tropical climate rikko hudyono,1 taufan bramantoro,2 benni benyamin,3 irfan dwiandhono,1 pratiwi soesilawati,4 aloysius pantjanugraha hudyono,5 wahyuning ratih irmalia6 and nor azlida mohd nor7 1educational dental hospital, faculty of medicine, universitas jendral soedirman, purwokerto – indonesia 2department of public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 3sultan agung dental hospital, faculty of dentistry, universitas islam sultan agung, semarang – indonesia 4department of oral biology, faculty of dental medicine, universitas airlangga, surabaya – indonesia 5prima medistra dental specialist clinic, kudus – indonesia 6indonesian health innovation and collaboration institute, surabaya – indonesia 7department of community oral health and clinical prevention, faculty of dentistry, university of malaya, kuala lumpur – malaysia abstract background: covid-19 has been regarded as a new pandemic in the world. this disease is highly contagious and can be transmitted easily through droplets and air. this matter is considered as a red flag to all dentists all over the globe. until today, there is only a few specific guideline in regards to dental practice during and after the pandemic. the protocol only revolves around the limitation of patients’ appointments and using level 3 personal protection equipment. there is no specific mention on the preparation method of the practice room especially in indonesia. purpose: this study aims to review literature on infection control in dental settings during covid-19 pandemic and discuss possible recommendations based on available evidence. review: the review also discussed the background of covid-19, transmission, clinical findings, physicochemical properties, and cross infection in dental practice. despite the usage of personal protective equipment, the rooms need to be set to specific requirement to reduce contamination inside the room. until today, covid-19 transmission must be prevented with the best method available. conclusion: no single protocol may fully guarantee the safety of the patients and dental workers. we suggest to combine the protocol listed above to minimize to self and crosscontamination ’new normal’ practice. keywords: covid-19; cross infection; dental practice; prevention; room disinfection correspondence: taufan bramantoro, department of public health, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: taufan-b@fkg.unair.ac.id introduction coronavirus infection diseases (covid)-19 is a new strain of human coronavirus (hcov). who previously termed this virus as 2019 novel coronavirus (2019-ncov). on february 11th, 2020, who introduced a new term for this novel coronavirus known as coronavirus disease (covid 19).1 the coronavirus is not a new virus recorded in history. it was first described in 1931, by schalk and hawn as a new respiratory disease. when it first spread in north dakota, this virus infected newborn chicks, causing gasping and listlessness.2 the term human coronavirus (hcov) had been described in 1960s, published in bmj 1965 by a group of researchers led by virologist david tyrrell. the team found a strange virus, referred as b814 after studying nasal swab. this new virus is unrelated to any previously known human respiratory tract virus.3 in indonesia, the first two cases of covid-19 was first reported in march 2nd, 2020 from depok, west java. and until may 9th, 2020 data shows the number of confirmed cases as 13,112 cases and 943 death. mortality rate of covid-19 in indonesia was 7.27% with recovery rate of 2.381 cases (19.02%).4 in jakarta, the capital city of indonesia, it was estimated that more than 100 hospital healthcare workers were infected with covid-19.5 indonesian medical association released data of 25 doctors dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 mailto:taufan-b@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 82 hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 and 6 dentists who had passed away during this pandemic until may 8th 2020.6 sars-cov-19 virology coronaviruses are categorized as enveloped virus. according to international committee on taxonomy of viruses (ictv), this virus is belongs to the realm of ribovirus, nidovirales order, in the family of coronaviridae and subfamily of ortho coronaviridae. the subfamily of orthocoronavirinae consists of four genera alpha-, beta-, gammaand delta-coronavirus. sars-cov-2 belongs to genus betacoronavirus which usually infects mammals, and cause mild to severe respiratory syndrome.3 this virus possess a positive-sense single-stranded rna (+ssrna) and a nucleocapsid of helical symmetry.3 the virus size is around 50-150 nm in diameter. under the electron micrographs, the virus has 80 nm envelope in the middle and 20 nm club-shaped spike project from their surface. its image resembles solar corona, from which the name obtained. coronavirus genome size range is around 26-32 kilobases, and considered as one of the largest rna viruses.7 the spike, a glycoprotein membrane developed by the enveloped virus, plays an important role in facilitating entrance into the host cells. infection of host cells is begun by the binding of the spike and receptors on the cell’s surface. spike protein attaches to host cells and facilitate synthesis of the virus and the host membranes, thus permitting the inclusion of the genome into the host cell’s cytoplasm.5 transmission and mode of transmission the sars-cov-2 as its antecessor is a zoonotic virus. it is closely related to the chinese rufous horseshoe bat (rhinolophus sinicus). at the beginning, it was suggested that the virus originated from the huanan seafood market, wuhan, china. and pangolin (manis javanica) had been proposed as intermediary host. however, another research of phylo-epidemiologic analysis concluded that sarscov-2 could have been imported from other places. until today, the exact origin of this virus remains unknown.5 china’s cdc revealed the incubation time of covid19 around 3 7 days and may last to 2 weeks. this data also showed that covid-19 doubled about every 7 days. preliminary study shows that the basic reproduction number (ro) varies between 2.24-3.58. in other words, each patient, on average, may infects 2.2 more individuals. this reproduction number is much higher than seasonal influenza (ro=1.27-1.80) and indicates that this virus is highly contagious.8 most viruses will attach to the surface and recognize cell surface receptor of the host cell for invasion. study had identified that the angiotensin converting enzyme-2 (ace-2) act as receptor for sars-cov-2, similar to its antecessor. the invasion was begun when spike protein create bond with ace-2 receptor (figure 1a). then the complex was processed proteolytically by transmembrane protease type 2 (tmprss2). ace-2 receptor was cleaved and the spike protein was activated (figure 1b), and thus facilitating the entrance of virus into the target cell (figure 1c).9 this process is similar to the mechanism of human metapneumovirus and influenza. this new virus shares the same host’s receptor as the sars-cov-1 but in a higher affinity. any cells expressing surface receptor ace-2 are susceptible to sars-cov-2. ace2 was easily found expressed in lungs, arteries, esophagus, ileum, colon, and bladder.10 several studies have also revealed oral cavity have ace2 receptor. this receptor can be found on tongue and salivary gland, suggesting saliva may have a role in transmission of covid-19.9 according to current evidence, covid-19 virus is primarily transmitted between people through respiratory droplets and contact routes. who announced that touching contaminated surfaces without washing hands may transmit the disease. droplets, from coughing, sneezing or talking, generated by an infected people may also transmit sars-cov-2. even very small droplets less than 5μm in diameter, referred as droplet nuclei, may transmit the disease.10 airborne, the virus may be detected in the air for up to 3 hours and may be 4 meters away from the patient. another research also found that the virus has high survival rate in low temperature. after 60 mins post aerosolization, the presence of living particle is more than 63% in 25°c. the presence of living particle is reduced significantly to 4.7% in 38°c. this data suggests that sars cov may have potential occupational hazard to health and dental workers.11 figure 1. host cell recognition and invasion.9 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 83hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 clinical findings of covid-19 the symptoms of this disease vary from asymptomatic, presymptomatic to respiratory failure that requires ventilator.12 covid-19 may also lead to septic shock, sepsis, and multiple organ dysfunction syndromes (mods).13 clinical manifestations of this disease may be best described by its severity: mild disease: occurred in 81% cases of non-pneumonia and mild pneumonia. only mild to moderate respiratory disturbances and may recover without any special medical treatment; severe disease: occurred in 14% of cases. characterized with dyspnea, respiratory frequency less than 30x/min, oxygen saturation (spo2) less than 93%, horowitz index (p/f ratio) less than 300, or the presence of lung infiltrates more than 50% within 24 to 48 hours; critical disease: occurred in 5% of all cases, and characterized by respiratory failure, septic shock, or the presence of multiple organ dysfunction syndrome (mods). computerized tomography (ct) scans of confirmed covid-19 pneumonia demonstrated a various pattern with affect both the interstitium and lung parenchyma.12 most typical finding is airspace opacities, or commonly described as consolidation or ground glass opacity (ggo) or mixed ggo. lesions, the ground-glass or consolidative opacity, tend to have bilateral, peripheral, basal predominant distribution and multifocal.13 nosocomial infection in dental practice in the current time, when the covid-19 outbreak has become more serious, the most recommended guidelines are: dentists should limit the procedures in their practice, and reduce the number of dental appointments. during the covid-19 pandemic, some protocols i.e physical distancing, limit interpersonal contact and reduce patient’s queue on the waiting room, shall be applied to the dental care. dentist are recommended to use personal protective equipment level 3 while performing emergency treatment. it is also recommended to postpone all elective care for unspecified time.14 impact on dental practice significant impact of covid-19 that has been reported includes the decrease in the number of patients, where only 38% patients visited the dental clinic during the covid-19 pandemic. the cases were dominated by dental trauma and oral infection. the other report suggested that the regular visit reduced by five percent during the pandemic which in turn bring some financial impact on dental practices around the world.14 discussion in dental practice, the most common splatter will be on nose and inner eyes.15 the indonesian dental association had proposed a special issue for ppes which include proper donning and doffing, and also hand washing technique, corresponding to who.14 even though no protective measure is guaranteed, we believe that we have to do extra precautions to prevent self and crosscontamination. until today, guidelines for dental practice re-opening is limited. we would like to review all recommendation to provide specific guidelines to dental practice in indonesia. indonesia may have a little difference with the other country. it has tropical climate, average daily temperature is hot with relatively high humidity. we divided this section into: personal protective equipment (ppes) and hand hygiene, protect dentist, dental team, and patients as well; patient selection to reduce possibility of positive covid19 transmission during dental treatment; aerosol reduction, when the use of agps is inevitable, we have to be able to reduce the viral load and aerosol spread across the room; and room disinfection, after the treatment, we have to avoid cross-contamination to the next patient.16,17 there are some personal protective equipment (ppes) for dental practice, such as filtering face piece ppe : surgical mask or n95 respirator, face shield, protectives goggles and hand hygiene. first ppes is filtering facepiece ppe: surgical mask or n95 respirator. the most common filtering facepiece utilized by health care workers are n95 respirator or facemask (sometimes called surgical mask). these filtering facepiece is parts of protective personal equipment to protect the healthcare workers from inhaling contaminated airborne particle. these devices are disposable and worn on the face, covering the nose and mouth.17 numerous studies had been conducted in order to test the efficacy between surgical mask and n95 respirator in protecting the wearer especially from viral infection.18 even though n95 respirator offers high protection, this kind of respirator may inhibit air exchange and thus increase additional metabolic workload.17 dentists should be warned of negative effect in the protracted use of n95, which may cause discomfort, shortness of air due to the increase in breathing resistance, and co2 level. some wearers also complain of headache, lightheadedness, and difficulty communicating.19 research had confirmed that such changes minimally affect physical work performances.20 second ppes is face shield. the efficacy of face shield in reducing the aerosol exposure to the wearer’s had been proposed and tested in various condition. some studies had been conducted to test the efficacy of face shield in order to reduce the expelled aerosols and splatter of body fluids.21 splash and splatter from surgical procedures may result from the use of various high speed drills. the incidence is quite high, despite being not in a dental setting.22 the third ppes that is used by dental practice is protective goggles. the use of protective goggles is mandatory. previous study had indicated that sarscov-2 may be present in tears. despite being low in incidence, however, the virus may have a second route of transmission via conjunctiva. therefore, protective goggles, is mandatory.23–25 besides that, hand hygiene is important for dental practice. who always recommended to wash hand in dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 84 hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 healthcare facilities. the procedure of hand hygiene had been widely spread. use clean and running water for hand hygiene. use and apply the enough amount of soap to cover all surfaces. it is estimated that 40-60 seconds is enough to clean all surfaces. the use of disposable towel is preferable.26 in patient evaluation, the most important thing is patient triage. the indonesian dental association had proposed some guidelines for the treatment that shall be done during the pandemic. it is also suggested to postpone elective treatment and do telemedicine. telemedicine can be used for taking anamnesis, visual examination, make a diagnosis even for prescribe a medicine.27 once you have determined that the patient is in need of urgent emergency care, then we have to screen the patient. ask them via telephone or another telemedicine platform to take anamnesis. some questions had been developed to pre-screen the patients: 1) have you got any fever for the last 14 days?; 2) have you experienced any breathing difficulties such as cough in the last 14 days?; 3) have you, within 14 days, travelling in the red zone area of covid-19 as stated by indonesian government?; 4) have you, within 14 days, get in touch to person with fever, or cough or breathing difficulties or person shall be suspected with covid-19?; 5) have you participated in any social gathering or meeting and got in touch with many unacquainted participants recently? if a patient answer ‘yes’ for any question, advise them not to continue dental treatment, they should be selfquarantined, and referred to the nearest hospital or public health facility.28 dentist could only continue treatment if the patient answer ‘no’ for all question and the body temperature is below 37.3°c before entering clinic, otherwise, patient need to be referred to the nearest hospital or public health facility for further assessment. the use of non-contact forehead thermometer for thermal body screening is recommended before patient can enter the dental clinic.28 social distancing protocol, hand hygiene protocol for patient can be introduced to patient. eye protector, and disposable gloves can also be dispensed to each patient before entering the clinic and start the treatment. during the dental treatment it is imperative to reduce the aerosol exposure (figure 2). avoid the use of dental handpiece whenever possible. however, if agps cannot be avoided, it is required that we to reduce the infection risk from aerosol exposure. mouthwash, such as hydrogen peroxide 1.5% or povidone iodine 0.2% before treatment, had been proposed as effective protocol to reduce the viral and bacterial load on oral cavity, and in turn reduce the viral load on aerosol or splatter.29–31 aerosol containment box: development of a containment box from acrylic or pvc pipe and wrap had been seen as an effort to prevent the aerosol spread. however, this type of box may hinder the operator sight and restrict hand movement which eventually limits the type of dental procedures that can be performed. the other innovation seen on social media is dentist tries to place a round plastic sheet with a hole for handpiece or scaler on the middle. until today no study had been conducted to test its effectiveness in reducing aerosol spread. aerosol suction and high vacuum evacuator: very few studies had been conducted regarding the use of central vacuum system found in pubmed. liu mh et al., reported that central vacuum system with 28.5 liter per minute is effective in reducing aerosol by 36%.32 vacuum system was reported more effective in reducing the aerosol exposure on dentist than the use of filtering respirator alone. another study also emphasizes the use of high vacuum evacuator as it may reduce the ultrafine particle exposure on patient and dentists more than 80% compared to conventional dental suction. some dentists also modify the vacuum cleaner unit to be an aerosol suction. uvgi requires time to kill the virus. the question still remains as the coronavirus size is 0.125 micron and hepa filter is effective in capturing particle larger than 0.30 micron. literature stated that it is 70% of 0.3-μm particles and 95% of 1.0-μm particles.33 the result may be different, there are also other claims in which the efficiency of hepa filter in capturing virus is less than 0.1 μm by means of electrostaticity.33–35 however, due to the lack of data supporting the use of hepa in filtering coronavirus, we recommend to release figure 2. example of modification on the concept of vacuum cleaner to reduce the aerosol. (originally designed by dr. ap. hudyono). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 85hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 the output of the aerosol suction outside the room. the virus will be easily inactivated by the hot temperature and humidity outside.36 the routine use of rubber dam on root canal treatment and restoration can reduce the amount of saliva splashes that will become droplets. we can disinfect cavities and reduce the amount of virus in the distribution of droplets use rubber dam. rubber dam can lessen the spread of microorganisms significantly (90-98%).37 some chemicals are known to have the ability to remove caries tissue without using a bur. the main principle of chemo-mechanical caries removal (cmcr). instead of drilling, this technique works by chemically softening the caries tissue so that caries can be easily cleaned using a hand instrument. cmcr can now be classified into sodium hypochlorite-based agents (naocl) or enzyme-based agents.38,39 indonesia has tropical climate with hot temperature in daily average. a room conditioner is highly required in every dental practice. however, this appliance may complicate the room disinfection and may facilitate the virus spread across the room. for room disinfection we should prepare the condition, such as air ventilation, ozone and hydroxyl agents and cleaning on floor and surfaces of dental unit. the importance of air ventilated room in dental clinic is well described in some studies. it was known that dental clinic is associated with various pollutants, pathogenic microbes, chemical or ultrafine particles. a study found that a high level of co2, total vocs and particulate matter were found during the operational hours, and were reduced significantly in close hour.40 the ventilation rate is measured as air change per hour (ach, sometimes abbreviated as acph). it is a measure of how much air volume is changed, either added or removed, from a room in an hour41. generally said this is to measure of how many times the air in a room is replaced. in order to achieve equilibrium pressure, the amount of air leaving and entering the space must be the same. the ach is measured by dividing air flow changes in a room for an hour with the volume of the room itself. the ach is simply counted by equation below: ach = 60 q p x l x t x c where: ach = air change rate per hour q = air flow per minute in cubic feet per minute (cfm) or liter per minute or meter cubic/minute pxlxt = room dimension in meter c = conversion value (if q is in liter per minute, c is 1000; if q is in cubic feet (cfm), c is 35,29; if q is in meter cubic per minute, then c is 1) the air change rate is not the only factor. the design of ventilation system, flow pathways, and its distance from contaminant source shall be considered to be more important than ach number or flow rate itself.40,41 consideration of contaminant source suggests the importance of aerosol suction for the first line to remove the contaminant from the air. uv is an electromagnetic radiation with short wavelengths. it is shorter than visible light but longer than x-rays. corresponding to its wavelength, uv is divided into three categories, uva, uvb, and uvc, and the germicidal effect is only seen in uvc. natural uvc is present in very little amount as the ozone layer blocked it. however, the uv-c can be artificially generated by some method, i.e mercury-based lamps, light emitting diode, or pulse xenon. the germicidal effect of uv-c is achieved by damaging the nucleic acid of microbes. wavelengths between about 200 nm and 300 nm are strongly absorbed by nucleic acids. the principal mode of inactivation occurs when the absorption of a photon forms pyrimidine dimers between adjacent thymine bases and renders the microbe incapable of replicating. uvgi is mainly used today to disinfect water, air, and surfaces. the surfaces disinfection is limited due to shadows or protective layer. the uvgi had been widely used for water and air sterilization. the uvgi is also employed in many industrial lines to help disinfection processes. water disinfection is currently the most advanced and proven application. uvgi is also widely used for air disinfection. several methods are available, including a full-room irradiation (when it is not occupied) or only the upper room, or irradiating air as it passes through enclosed aircirculation and heating, ventilation, and air-conditioning (hvac) systems.42 the susceptibility of sars-cov-2 to uvgi has not been documented yet. however, uvgi effect had been studied to previously known coronavirus, included the sars-cov-1 and mers-cov. even though the use of uvgi is effective in laboratory, the case may be different in clinical setting. the uvgi effectivity is mainly affected by distance from the uv light source, and the presence of obstacles in the room may prevent the uv light to reach the target.42 we found that some data which state that the uvgi is partially effective in inactivating coronavirus for dental practice. ozone, a trioxygen, is a chemical compound with symbol o3. it is considered to be very reactive to organic compounds. ozone is regarded as a promising method to inactivate viruses. when ozone breaks down to dioxygen, it produces free radicals of oxygen which is very damaging to organic compounds. virus inactivation by ozone is influenced by ozone concentration, contact time, different capsid architecture of viruses, and relative humidity (rh). the authors observed that the survival fraction of viruses on surfaces decreased with the increasing ozone dose. the ozone dose is stated by min (mg/m3) which means contact time (in minutes) multiplied by ozone concentration (in mg/m3). viruses required ozone doses of 20-112 min dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 86 hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 (mg/m3) for 90% inactivation and 47-223 min (mg/m3) for 99% inactivation. the ozone dose for 99% inactivation is two times higher than for 90% inactivation. the ozone generator usually produces 500 mg/m3 in a minute (depends on the manufacturer). for a room 3 x 3 x 2.5 meters, it can be calculated that 10-15 minutes is required to inactivate coronavirus in a room. the low relative humidity may increase the ozone concentration required for virus inactivation. in summary, ozone should be an effective method for reducing the viral number on surfaces.43 another study also found that low ozone exposure may be beneficial in inactivation of enveloped virus. concentration of 1.13 ppm ± 0.26 ppm was enough to yield the same result.44 the occupational safety and health administration (osha) in usa, has set a public health air standards limit for ozone exposure as much as 0.1 ppm for 8 h or 0.3 ppm for 15 min. using appropriate generators at appropriate ozone concentrations, ozone will help to decontaminate rooms, hospital room, public transport, etc. ozone is toxic when inhaled, therefore, room decontamination must be free of people and animals.45 cleaning shall be done following the room disinfection, before new patient enters the room. the rationale is, the longer the period, the aerosol and splatter will come on the floor by gravitation, and the viral count on the ambient air will be reduced by means of uvgi, or ozone, and exhaust fan. this arrangement may prevent the cleaning staff from being exposed to aerosol, and the cleaning will be more effective as the aerosol and splatter has fallen to the floor and the dental unit. cdc recommends to use damp soft mop to clean floor surfaces and damp microfiber cloth for all surfaces of dental unit.44 we found that cleaning is a beneficial and may provide a clean environment. rapid change in guidelines for dental practice reopening may occur based on recent research on covid-19 and the situation in the community. for the concluding remarks, until today no protection method has been proven against sars-cov-2. no single protocol may guarantee the prevention of cross or self-contamination. we find that the use of ppes and patient triage is mandatory in reducing the risk of infection. air ventilation rate, control on the aerosol spread, and disinfection of floor and dental unit surface are beneficial in reducing the contaminant in room. however, the use of air conditioned has not been resolved. until today, there is no protocol that can be used to sterilize the air conditioner in dental practice facility. ozone may be promising, but it needs to be researched further. we recommended to limit the use of air conditioner unit as little as possible. or place an air conditioner unit in the waiting room, and let the cool air enter the room by means of negative pressure from exhaust fan. overall, can be concluded that no single protocol may fully guarantee the safety of the patients and dental workers. we strongly suggest to combine the protocol listed above to minimize to self and cross-contamination. references 1. world health organization. novel coronavirus (2019-ncov) situation report 1. who bull. 2020; (jan): 1–7. 2. banat gr, tkalcic s, dzielawa ja, jackwood mw, saggese md, yates l, kopulos r, briles we, collisson ew. association of the chicken mhc b haplotypes with resistance to avian coronavirus. dev comp immunol. 2013; 39(4): 430–7. 3. cui j, li f, shi zl. origin and evolution of pathogenic coronaviruses. nat rev microbiol. 2019; 17(3): 181–92. 4. kementerian kesehatan indonesia. dashboard kasus covid-19 di indonesia. 2020. available from: https://www.kemkes.go.id/ article/view/20031900002/dashboard-data-kasus-covid-19-diindonesia.html. accessed 2020 may 9. 5. singhal t. a review of coronavirus disease-2019 (covid-19). indian j pediatr. 2020; 87(4): 281–6. 6. dinas kesehatan kota jakarta. data pantauan covid-19 jakarta. 2020. available from: https://corona.jakarta.go.id/id. accessed 2020 may 9. 7. ul qamar mt, shahid f, ashfaq ua, aslam s, fatima i, fareed mm, zohaib a, chen l-l. structural modeling and conserved epitopes prediction against sars-cov-2 structural proteins for vaccine development. research square. 2020. p. 1–49. 8. zhao s, lin q, ran j, musa ss, yang g, wang w, lou y, gao d, yang l, he d, wang mh. preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak. int j infect dis. 2020; 92: 214–7. 9. rabi fa, al zoubi ms, al-nasser ad, kasasbeh ga, salameh dm. sars-cov-2 and coronavirus disease 2019: what we know so far. pathogens. 2020; 9(3): 1–14. 10. li y, zhou w, yang l, you r. physiological and pathological regulation of ace2, the sars-cov-2 receptor. pharmacol res. 2020; 157(april): 104833. 11. pyankov o v., bodnev sa, pyankova og, agranovski ie. survival of aerosolized coronavirus in the ambient air. j aerosol sci. 2018; 115: 158–63. 12. zhou s, wang y, zhu t, xia l. ct features of coronavirus disease 2019 (covid-19) pneumonia in 62 patients in wuhan, china. am j roentgenol. 2020; 214(6): 1287–94. 13. cheng z, lu y, cao q, qin l, pan z, yan f, yang w. clinical features and chest ct manifestations of coronavirus disease 2019 (covid-19) in a single-center study in shanghai, china. am j roentgenol. 2020; 215(1): 121–6. 14. indonesian dental association (pdgi). surat edaran nomor: 2776/ pb pdgi/iii-3/2020 tentang pedoman pelayan kedokteran gigi selama pandemi virus covid-19. jakarta; 2020. 15. nejatidanesh f, khosravi z, goroohi h, badrian h, savabi o. risk of contamination of different areas of dentist’s face during dental practices. int j prev med. 2013; 4(5): 611–5. 16. coulthard p. dentistry and coronavirus (covid-19) moral decision-making. br dent j. 2020; 228(7): 503–5. 17. radonovich lj, simberkoff ms, bessesen mt, brown ac, cummings dat, gaydos ca, los jg, krosche ae, gibert cl, gorse gj, nyquist ac, reich ng, rodriguez-barradas mc, price cs, perl tm. n95 respirators vs medical masks for preventing influenza among health care personnel: a randomized clinical trial. jama j am med assoc. 2019; 322(9): 824–33. 18. smith jd, macdougall cc, johnstone j, copes ra, schwartz b, garber ge. effectiveness of n95 respirators versus surgical masks in protecting health care workers from acute respiratory infection: a systematic review and meta-analysis. cmaj. 2016; 188(8): 567–74. 19. zhu j, lee j, wang d, lee hp. effects of long-duration wearing of n95 respirator and surgical facemask: a pilot study. j lung, pulm respir res. 2014; 1(4): 97–100. 20. rebmann t, carrico r, wang j. physiologic and other effects and compliance with long-term respirator use among medical intensive care unit nurses. am j infect control. 2013; 41(12): 1218–23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 https://www.kemkes.go.id/ https://corona.jakarta.go.id/id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 87hudyono et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 81–87 21. roberge rj. face shields for infection control: a review. j occup environ hyg. 2016; 13(4): 239–46. 22. leong xya, yee fzy, leong yy, tan sg, amin ibm, ling ml, tay sm. incidence and analysis of sharps injuries and splash exposures in a tertiary hospital in southeast asia: a ten-year review. singapore med j. 2019; 60(12): 631–6. 23. napoli pe, nioi m, d’aloja e, fossarello m. the ocular surface and the coronavirus disease 2019: does a dual ‘ocular route’ exist? j clin med. 2020; 9(5): 1269. 24. xie h-t, jiang s-y, xu k-k, liu x, xu b, wang l, zhang m-c. sars-cov-2 in the ocular surface of covid-19 patients. eye vis. 2020; 7: 23. 25. zhou y, duan c, zeng y, tong y, nie y, yang y, chen z, chen c. ocular findings and proportion with conjunctival sars-cov-2 in covid-19 patients. ophthalmology. 2020; 127(7): 982–3. 26. world health organization. who guidelines on hand hygiene in health care. first global patient safety challenge clean care is safer care. geneva: world health organization; 2009. p. 270. 27. kementerian kesehatan republik indonesia. surat edaran nomor hk.02.01/menkes/303/2020 tentang penyelenggaraan pelayanan kesehatan melalui pemanfaatan teknologi informasi dan komunikasi dalam rangka pencegahan penyebaran covid-19. jakarta; 2020. 28. peng x, xu x, li y, cheng l, zhou x, ren b. transmission routes of 2019-ncov and controls in dental practice. int j oral sci. 2020; 12: 9. 29. sharma k, acharya s, verma e, singhal d, singla n. efficacy of chlorhexidine, hydrogen peroxide and tulsi extract mouthwash in reducing halitosis using spectrophotometric analysis: a randomized controlled trial. j clin exp dent. 2019; 11(5): e457–63. 30. kariwa h, fujii n, takashima i. inactivation of sars coronavirus by means of povidone-iodine, physical conditions and chemical reagents. dermatology. 2006; 212(suppl. 1): 119–23. 31. kanagalingam j, feliciano r, hah jh, labib h, le ta, lin jc. practical use of povidone-iodine antiseptic in the maintenance of oral health and in the prevention and treatment of common oropharyngeal infections. int j clin pract. 2015; 69(11): 1247–56. 32. liu mh, chen ct, chuang lc, lin wm, wan gh. removal efficiency of central vacuum system and protective masks to suspended particles from dental treatment. plos one. 2019; 14(11): 1–9. 33. vijayan vk, paramesh h, salvi ss, dalal aak. enhancing indoor air quality -the air filter advantage. lung india. 2015; 32(5): 473–9. 34. rupf s, berger h, buchter a, harth v, ong mf, hannig m. exposure of patient and dental staff to fine and ultrafine particles from scanning spray. clin oral investig. 2015; 19(4): 823–30. 35. malaithao k, kalambaheti t, worakhunpiset s, pongrama r. evaluation of an electronic air filter for filtrating bacteria and viruses from indoor air. southeast asian j trop med public heal. 2009; 40(5): 1113–20. 36. dee sa, deen j, cano jp, batista l, pijoan c. further evaluation of alternative air-filtration systems for reducing the transmission of porcine reproductive and respiratory syndrome virus by aerosol. can j vet res. 2006; 70(3): 168–75. 37. casanova lm, jeon s, rutala wa, weber dj, sobsey md. effects of air temperature and relative humidity on coronavirus survival on surfaces. appl environ microbiol. 2010; 76(9): 2712–7. 38. hamama h, yiu c, burrow m. current update of chemomechanical caries removal methods. aust dent j. 2014; 59(4): 446–56. 39. ganesh m, parikh d. chemomechanical caries removal (cmcr) agents: review and clinical application in primary teeth. j dent oral hyg. 2011; 3(3): 34–45. 40. memarzadeh f, xu w. role of air changes per hour (ach) in possible transmission of airborne infections. build simul. 2012; 5(1): 15–28. 41. grosskopf k. air change rate vs airf low pathway: bioaerosol containment and removal in patient rooms. antimicrob resist infect control. 2015; 4(s1): p94. 42. reed ng. the history of ultraviolet germicidal irradiation for air disinfection. public health rep. 2010; 125(1): 15–27. 43. tseng c, li c. inactivation of surface viruses by gaseous ozone. j environ health. 2008; 70(10): 56–62. 44. dubuis me, dumont-leblond n, laliberté c, veillette m, turgeon n, jean j, duchaine c. ozone efficacy for the control of airborne viruses: bacteriophage and norovirus models. plos one. 2020; 15(4): 1–19. 45. hudson jb, sharma m, vimalanathan s. development of a practical method for using ozone gas as a virus decontaminating agent. ozone sci eng. 2009; 31(3): 216–23. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p81–87 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p81-87 5353 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 53–57 original article physical characterization and analysis of tissue inflammatory response of the combination of hydroxyapatite gypsum puger and tapioca starch as a scaffold material amiyatun naini,1 dessy rachmawati2,3 1department of prosthodontics, faculty of dentistry, universitas jember, jember, indonesia 2department of dental biomedical science, faculty of dentistry, universitas jember, jember, indonesia 3center of excellent of agromedicine (ceamed), universitas jember, indonesia abstract background: cases of bone damage in the oral cavity are high, up to 70% of which consist of cases of fracture, tooth extraction, tumor, and mandibular resection. the high number of cases of bone damage will cause the need for bone graft material to increase. the bone graft material that we have developed is a combination of hydroxyapatite gypsum puger (hagp) and tapioca starch (ts) scaffold. purpose: this study analyzes the physical characterization and tissue inflammatory response of the combination of hagp+ts as a scaffold for bone graft material. methods: eighteen wistar rats were used. hagp+ts were installed into the molar 1 socket for 7 and 14 days. first, hagp was evaluated using xrf and sem before setting up the in vivo experiment. a blood sample was drawn and then tested for tnf-α levels using elisa. results: the xrf revealed that the main constituents of hydroxyapatite were ca and p. next, sem characterization on the hagp+ts showed an average pore size of 112.42 µm2, which is beneficial for cell activity to grow as new bone tissue. in addition, tnf-α on days 7 and 14 on the hagp+ts scaffold did not elicit an inflammatory response. conclusion: the combination of hagp+ts contains a high amount of ca and also has excellent interconnectivity between pores. it also does not trigger an inflammatory response in the tissue; therefore, it is a good candidate as an alternative bone graft material. keywords: bone graft; characterization; hydroxyapatite gypsum puger scaffold; tapioca starch; inflammation article history: received 17 april 2022, revised 19 august 2022, accepted 15 september 2022 correspondence: amiyatun naini, department of prosthodontics, faculty of dentistry, universitas jember. jl. kalimantan 37 jember, indonesia. email: amiyatunnaini.fkg@unej.ac.id introduction the occurrence of bone destruction related to oral health is prevalent. there are several factors causing bone destruction, such as fracture, tooth extraction, periodontitis, tumor, mandibular resection, alveolar cleft, and cleft palate.1,2 the high number of bone destruction cases is causing a rise in the demand for bone replacement materials, bone implants, and bone grafts.3 in prosthodontics, the technological advancement of bone grafting techniques can help cases of bone destruction and implant placement. the ideal bone graft material should be biocompatible, osteoconductive (i.e., providing a framework or scaffold for the new bone to grow), and osteoinductive (i.e., growth-stimulating materials).4 materials that can be used as bone substitutes or bone grafts include autograft, allograft, xenograft, alloplastic, or synthetic bone substitute (bioceramic).5 a bioceramic material that a previous study has recently developed is hydroxyapatite gypsum puger (hagp). the gypsum puger has been synthesized into a hydroxyapatite scaffold, and thus it becomes an alternative graft material at a more affordable price and is easy to obtain.6,7 however, it still poses some weaknesses, such as low biomechanical properties, low porosity, and brittleness.8 therefore, to improve its material properties, it needs to be enhanced by combining it with biopolymer materials. one of the natural biopolymer materials is cassava. cassava is an abundant raw material at a relatively affordable price, and it is easy to obtain. cassava is a tuber plant with the latin name manihot utilissima from the euphorbiaceae family. cassava is a polysaccharide copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p53–57 mailto:amiyatunnaini.fkg@unej.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p53-57 54 naini and rachmawati. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 53–57 containing starch with amylopectin and amylose content. cassava can be processed to produce tapioca starch (ts) with many benefits. tapioca starch is beneficial for health since it is a source of carbohydrates, high in calories and proteins, and contains b complex vitamins, minerals, fibers, and vitamin k, which are beneficial for building bone mass.9 hagp is a new development in bone graft material that has improved mechanical properties and porosity when combined with cassava starch; therefore, the hagp+ts scaffold material is firm and has good mechanical characteristics. in the case of fractured bones, tissue engineering procedures are needed to accelerate the healing process and formation of new bone. one of the components of tissue engineering is a scaffold. a scaffold is a component for inducing cell growth.10 the scaffold material that will be used as a bone graft must have characterization in the form of an elemental composition test/x-ray fluorescence (xrf), a morphological test using scanning electron microscopy (sem), and an inflammation test to determine the tissue response to the incoming scaffold/foreign object. inflammation is a reaction to a foreign agent entering the body. the tissue damage is caused by the invasion of microorganisms, harmful chemicals, and physical factors. inflammatory signs are usually seen as redness, heat, swelling, pain, and impaired function. tnf-α is a cytokine that plays an important role in the inflammatory response.11 inflammation is an initial response from the body tissues if there is an injury. a bone graft applies foreign matter to the tissues of the human body. therefore, it may cause injury, such as inflammation. the purpose of this study is to analyze the elemental composition and morphological characterization of the combination of hagp+ts scaffold as a candidate new bone graft material. next, we also examine the tissue inflammatory response of the combination to get an overview of the biocompatibility of the new bone graft material. materials and methods the hagp sample was made using the following procedure. weigh 0.5 g of gypsum, 0.5 g of diammonium hydrogen phosphate (dhp), and 500 ml of distilled water. mix them in a beaker and then put the beaker on a magnetic hotplate for 15 minutes. then, put the beaker in an oven at 100°c for 30 minutes. wash the solution using distilled water, and at the same time, filter it using filter paper several times until the ph is neutral. then, dry the powder in a microwave at 50°c for 5 hours to create hydroxyapatite powder. the process of making tapioca starch generally comprises peeling, washing, grating, extracting, settling, and milling. first, weigh and wash 500 g of cassava, and then grate it. add 1 l of water and filter it. next, let the contents rest for 12 hours to settle the starch at the bottom. then dry, mash, and sieve the starch. the composite hagp+ts scaffolds were made through the following procedure. weigh 250 mg hydroxyapatite and 300 mg solid gelatin. add 10 ml distilled water and heat to 40°c. then add 250 mg of hagp and mix until homogeneous. then add 250 mg ts and 10 ml distilled water and mix using an ultrasonic homogenizer for 6 minutes. put the mixture into cylindrical molds with a diameter of 5 mm and a height of 5 mm. then freeze and dry with a sublimation/freeze-drying system. sterilize using gamma irradiation. next, characterize them to see the concentration of scaffold content using xrf and sem. this study was an in vivo laboratory experiment using a posttest-only control group design. the independent variable was the hagp+ts scaffold, and the dependent variable was tumor necrosis factor alpha (tnf-α) levels. eighteen wistar rats with the following inclusion criteria : 12-to-14-week-old male wistar rats with a body weight of around 200–250 g were used. the rats were kept with the same feed, the rats’ drinks (distilled water), and the rats’ caring method. in addition, also the evaluation time, the skin area for injection, the time and level of material given, and the application method were controlled. wistar rats were divided into 6 sampling groups, with 3 samples (n=3) in each group. inflammatory responses were evaluated by enzyme-linked immunosorbent assay (elisa) analysis method, the prepared hagp+ts scaffold was ready to be applied to the rats with the following steps. first, anesthetize the wistar rats intra-muscularly using 100 mg/ml ketamine and 20 mg/ml xylazine base and xylazine ratio with a dose of 0.08–0.2 ml/kg body weight. once the rats were anesthetized, the first molar on the left mandible was extracted using a needle holder. then, put hagp+ts scaffold material into the extraction socket. sew it using a 75 cm sewing thread (dr. stella silk braided usp 3/0). then, wait for 7 and 14 days. collect the rats’ blood for elisa analysis by using 5 ml of ether on a cotton swab. put the cotton swab in a closed glass box. put the rats into the glass box singly for 5 minutes. dissect the thoracic area of the rats and collect 2 ml of blood from the heart. determine tnf-α levels using elisa. the data obtained were subjected to statistical analysis with a statistical package for the social sciences (spss) software, version 22 (ibm, usa). all scale variables were analyzed for normality and homogeneity tests. the data were normally distributed; therefore, parametric tests using one-way analysis of variance (anova) were conducted, followed by least significant difference (lsd), with p < 0.05 indicated as statistical significance. results the results of the characterization analysis using xrf, the hagp scaffold, and the hagp+ts scaffold produced elemental percentages that can be seen in table 1. the copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p53–57 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p53-57 55naini and rachmawati. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 53–57 results of sem characterization used to determine the morphology (including the shape, pore diameter, and pore area) of the hagp+ts composite scaffold are shown in figure 1. the sem test results of the hagp scaffold and hagp+ts scaffold in figure 1 show an irregular pore edge shape and inhomogeneous interconnectivity with different sizes among samples. the results of the diameter and the pore area data for the hagp scaffold and the hagp+ts scaffold are presented in tables 2 and 3. the diameter and pore size of the hagp+ts and hagp groups were analyzed using the shapiro-wilk normality test. the analysis showed p > 0.05, which indicated that the data is normally distributed. a homogeneity test was also conducted using the levene test. the results showed that the significancy of pore diameter p = 0.13 and pore area p = 0.06. it indicated that the variance of the data is homogeneous between groups. moreover, an anova test was also conducted. the results of pore diameter showed p = 0.03, and pore area showed p = 0.02. it showed the difference in pore diameter and pore area between groups. the tissue inflammatory response analysis was conducted to analyze the tnf-α levels after the application of the hagp+ts group and hagp group. negative controls on days 7 and 14 were analyzed using the shapiro-wilk normality test. the results showed p > 0.05, which indicated that the data was normally distributed. a homogeneity test using the levene test was also conducted. the results showed p = 0.22, which indicated that the data variance was homogeneous between groups. furthermore, an anova test was conducted. the results showed p = 0.75, indicating no difference in tnf-α levels between groups. the results of tnf-α levels are presented in table 4. a b c d e f figure 1. sem analysis of hagp scaffold and hagp+ts scaffold. a. hagp scaffold 250x magnification, b. hagp scaffold 500x magnification, c. hagp scaffold 750x magnification, d. hagp+ts scaffold 250x magnification, e. hagp+ts scaffold 500x magnification, f. hagp+ts scaffold 750x magnification. table 1. the results of xrf test on hagp and hagp+ts scaffold compound p ca cr fe ni cu yb s hagp scaffold 34% 49.5% 5.8% 5.0% 3.4% 1.9% 0.9% hagp+ts scaffold 13.5% 82.9% 0.39% 0.081% 0.46% 2.70% table 2. pore diameter of hagp scaffold and hagp+ts scaffold group mean sd minimum maximum p hagp scaffold 143.97 µm 36.15 105.20 µm 217.90 µm 0.03 hagp+ts scaffold 112.42 µm 18.80 85.33 µm 142.90 µm table 3. pore area of hagp scaffold and hagp+ts scaffold group mean sd minimum maximum p hagp scaffold 16155.08 µm2 6615.77 8687.62 µm2 27272.10 µm2 0.02 hagp+ts scaffold 10168.69 µm2 3321.66 5715.74 µm2 16030.01 µm2 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p53–57 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p53-57 56 naini and rachmawati. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 53–57 discussion the results of the xrf test showed ca and p elements, which were the main elements of hydroxyapatite with the chemical formula ca10(po4)6(oh)2 contained in the hagp+ts scaffold. the addition of ts to the process of hagp scaffold-making resulted in a greater amount of ca compared to the hagp scaffold without ts. a greater amount of ca can improve the material properties of the scaffold. the presence of element p is related to the deposition of phosphate from hydroxyapatite.12 there was a hydrothermal reaction in the process of making the scaffold to obtain single crystals. hydroxyapatite crystals are the same size as bone hydroxyapatite crystals.13,14 the scaffolds used in this research were made of hagp powder mixed with ts, while the scaffolds for the control group were processed using a freeze-drying system. microstructural characterization of the hagp scaffold using sem obtained a three-dimensional interconnected pore structure with an average diameter of 143.97 µm, and the hagp+ts scaffold showed an average diameter of 112.42 µm. for the pore area (this pore is a hole formed on the surface of the hagp scaffold), the hagp scaffold showed 16155.08 µm2, and hagp+ts showed 10168.69 µm2. the hagp+ts scaffold showed better interconnectivity between each pore than the hagp scaffold. it can be assumed that the addition of ts to gelatin and hagp caused interactions with ts particles; thus, separating gelatin particles from one another, which caused the formation of interconnectivity and more even pore distribution.15 the hagp+ts scaffold had a smaller pore size than the hagp scaffold. this was due to the constant freezing temperature and the addition of ts that helped decrease the pore size.16 ts can lower pore size by forming hydrogen bonds that increase the strength of the scaffold through an interlocking mechanism.16 the small pore size exists because of the presence of hydroxyapatite (ha), causing the pore size and pore wall to decrease after freeze-drying because of the increasing number of ice crystals and the closing distance among ice crystals.17 the addition of cassava starch polymer allows bond formation between the cassava starch polymer and hagp. the concentration of cassava starch polymer influences the pore size. by triggering hydrogen bonds to form, the pores shrink.. it is conducive to cell activity so that it enters and grows in it, which is useful for bone remodeling and bone tissue engineering.17,18 the minimum scaffold pore size was between 75–100 µm. however, if the pore size diameter is > 300 µm, it will be better for bone formation since it is easier to facilitate and vascularize the tissue.19 scaffold pores have a retentive shape for cell attachment, being a place for cell proliferation and differentiation into osteoblast cells.20 in this study, all sample groups produced pore sizes with the potential to assist bone growth. this is ideal as it meets the requirements for bone graft material, i.e., being osteoconductive, that is, providing a framework or scaffold to grow.4 in this study, tnf-α levels were observed on days 7 and 14. the results of the tnf-α level of the negative control group showed that the hagp scaffold and hagp+ts scaffold were almost the same. in the hagp+ts group, the levels were higher but statistically within the range of the hagp+ts group. for the hagp and negative control groups, the results showed p = 0.75, which indicated no significant difference, and thus did not cause an inflammatory response. inflammation is an important mechanism needed by the body to defend itself from dangers such as tissue damage and invasion of microorganisms, antigens, and foreign materials that disrupt the balance of the tissue. as a foreign object that enters the rats’ body through the tissue, the scaffold can activate macrophages and other cells to produce and release various cytokines, including tnf-α.21 tnf-α levels on days 7 to 14 were associated with the recruitment of osteoclast precursors and differentiation into mature osteoclasts in the defect. it corresponds to other research findings that defects treated with ha in the first week are associated with tnf-α expression from the surface osteoblasts markers.22 on day 7, the inflammation peaks after the application of the material to the tissues, while day 14 has a decrease in the inflammatory response and begins to form tissue regeneration. tnf-α levels report levels of cytokines, which play an important role in the inflammatory response.21 in this study, there was no significant difference between the control and treatment groups concerning the inflammation test. thus, the hagp+ts scaffold material is safe for the body (biocompatible) and an ideal candidate for bone graft material.4 hagp+ts scaffold is a type of alloplastic bone graft material or synthetic bone replacement material (bioceramic), which can be referred to as a regenerative material. based on a systematic review study on graft materials related to alveolar regeneration, there were no significant differences between regenerative materials and iliac crest grafts in the meta-analysis. it indicates that this regenerative material still meets the requirements for treating bone destruction.3 the conclusions of this study are that the combination of hagp+ts scaffold contains high amounts of ca and p by xrf test, has excellent inter-pore interconnectivity by sem test, and does not trigger an inflammatory response in tissue by elisa test; therefore, this material is a good table 4. the results of tnf-α levels scaffold n tnf-α mean sd p k (-) 7 3 0.10 0.02702 0.75 k (-) 14 3 0.12 0.01493 hagp 7 3 0.11 0.00902 hagp 14 3 0.12 0.00987 hagp+ts 7 3 0.12 0.00513 hagp+ts 14 3 0.12 0.02178 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p53–57 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p53-57 57naini and rachmawati. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 53–57 candidate for alternative bone graft material. however, further research is needed using other biomarkers before it can be applied clinically. acknowledgements the authors would like to thank all those who have participated in this research and in particular the bioscience laboratory of dental and oral hospital, universitas jember which has allowed and facilitated this research. references 1. kheirallah m, almeshaly h. present strategies for critical bone defects regeneration. oral heal case reports. 2016; 2(3): 127. 2. mattiola a, bosshardt d, schmidlin p. the rigid-shield technique: a new contour and clot stabilizing method for ridge preservation. dent j. 2018; 6(2): 21. 3. alkaabi sa, alsabri ga, natsirkalla ds, alavi sa, mueller weg, forouzanfar t, helder mn. a systematic review on regenerative alveolar graft materials in clinical trials: risk of bias and metaanalysis. j plast reconstr aesthet surg. 2022; 75(1): 356–65. 4. kolk a, handschel j, drescher w, rothamel d, kloss f, blessmann m, heiland m, wolff k-d, smeets r. current trends and future perspectives of bone substitute materials from space holders to innovative biomaterials. j craniomaxillofac surg. 2012; 40(8): 706–18. 5. tal h, artzi z, kolerman r, beitlitum i, goshe g. augmentation and preservation of the alveolar process and alveolar ridge of bone. in: bone regeneration. intech; 2012. p. 139–84. 6. na in i a, sudia na ik, rubia nto m, ferdia nsya h, mufti n. characterization and degradation of hydroxyapatite gypsum puger (hagp) freeze dried scaffold as a graft material for preservation of the alveolar bone socket. j int dent med res. 2018; 11(2): 532–6. 7. naini a, sudiana ik, rubianto m, kresnoadi u, latief fde. effects of hydroxyapatite gypsum puger scaffold applied to rat alveolar bone sockets on osteoclasts, osteoblasts and the trabecular bone area. dent j (majalah kedokt gigi). 2019; 52(1): 13–7. 8. tripathi g, basu b. a porous hydroxyapatite scaffold for bone tissue engineering: physico-mechanical and biological evaluations. ceram int. 2012; 38(1): 341–9. 9. ndubuisi nd, chidiebere acu. cyanide in cassava: a review. int j genomics data min. 2018; 2: 118. 10. bucholz rw. rockwood and green’s fractures in adults. 7th ed. vol. 1. lippincott williams & wilkins; 2010. p. 113–4. 11. baratawidjaja kg, rengganis i. imunologi dasar. 11th ed. jakarta: fakultas kedokteran universitas indonesia; 2014. p. 860. 12. naini a, rachmawati d. composition analysis of calcium and sulfur on gypsum at the puger district jember regency as an alternative gypsum dental material. dentika dent j. 2010; 15(2): 179–83. 13. choi ah, ben-nissan b, matinlinna jp, conway rc. current perspectives: calcium phosphate nanocoatings and nanocomposite coatings in dentistry. j dent res. 2013; 92(10): 853–9. 14. remya ns, syama s, gayathri v, varma hk, mohanan p v. an in vitro study on the interaction of hydroxyapatite nanoparticles and bone marrow mesenchymal stem cells for assessing the toxicological behaviour. colloids surf b biointerfaces. 2014; 117: 389–97. 15. ramadoss p, subha v, kirubanandan s. gelatin-silk fibroin composite scaffold as a potential skin graft material. j mater sci surf eng. 2018; 6(2): 761–6. 16. qi y, wang h, wei k, yang y, zheng r-y, kim i, zhang k-q. a review of structure construction of silk fibroin biomaterials from single structures to multi-level structures. int j mol sci. 2017; 18(3): 237. 17. kaviani z, zamanian a. effect of nanohydroxyapatite addition on the pore morphology and mechanical properties of freeze cast hydroxyapatite scaffolds. procedia mater sci. 2015; 11: 190–5. 18. k ha n y, yaszemsk i m j, mikos ag, laurencin ct. tissue engineering of bone: material and matrix considerations. j bone joint surg am. 2008; 90(suppl 1): 36–42. 19. chocholata p, kulda v, babuska v. fabrication of scaffolds for bone-tissue regeneration. materials (basel). 2019; 12(4): 568. 20. ariani md, matsuura a, hirata i, kubo t, kato k, akagawa y. new development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. dent mater j. 2013; 32(2): 317–25. 21. abbas a, lichtman a, pillai s. cellular and molecular immunology. 9th ed. philadelphia: elsevier; 2016. p. 359–81. 22. cardemil c, elgali i, xia w, emanuelsson l, norlindh b, omar o, thomsen p. strontium-doped calcium phosphate and hydroxyapatite granules promote different inflammatory and bone remodelling responses in normal and ovariectomised rats. plos one. 2013; 8(12): e84932. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p53–57 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p53-57 99 research report dental journal (majalah kedokteran gigi) 2018 june; 51(2): 99–103 changes in the number of macrophage and lymphocyte cells in chronic periodontitis due to dental x-ray exposure alhidayati asymal,1 eha renwi astuti,1 and rini devijanti2 1department of dentomaxillofacial radiology 2department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: periodontitis is an inflammatory disease caused by specific microorganisms that attacks tooth-supporting tissues, p. gingivalis bacteria are mostly found in patients suffering from chronic periodontitis which is usually diagnosed by means of clinical and radiographic examination. the latter play important roles in the management of periodontitis, including: establishing diagnosis, determining treatment plans and evaluating the results of treatment. unfortunately, the use of x-rays to perform such radiographic examination has negative effects since the body’s various parts, especially the head, are not well protected from the effects of x-ray radiation. purpose: this research aimed to analyze the effects of dental x-ray exposure on the number of macrophages and lymphocytes in experimental subjects suffering from periodontitis. methods: 36 rats that had been diagnosed with chronic periodontitis were divided into three groups, namely: a control group, treatment group i (exposed to a 0.16 msv dose of radiation) and treatment group ii (exposed to a 0.32 msv dose of radiation). these subjects were subsequently sacrificed on the third and fifth days after treatment. thereafter, histopathological examination was performed to identify any changes in the number of macrophages and lymphocytes. results: the results of an hsd test confirmed that, on the third day, there were significant differences in the number of lymphocytes between the control group and treatment group i, as well as between the control group and treatment group ii. on the fifth day, there were also significant differences in the number of lymphocytes between the control group and treatment group i, as well as between treatment group i and treatment group ii. similarly, there was a significant difference in the number of macrophage cells on the third day between the control group and treatment group i. on the fifth day, there were also significant differences in the number of macrophage cells between the control group and treatment group i, as well as between treatment group i and treatment group ii. conclusion: dental x-ray exposure at a dose of 0.16 msv can elevate the number of macrophages and lymphocytes on the third and fifth days. on the other hand, dental x-ray radiation at a dose of 0.32 msv can reduce the number of macrophages on day 3 as well as the number of lymphocytes on the third and fifth days. keywords: dental x-ray radiation; macrophages; lymphocytes; chronic periodontitis correspondence: alhidayati asymal, department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: alhidayatidrg@gmail.com introduction periodontitis is an inflammatory condition that affects tooth-supporting tissue caused by specific microorganisms and characterized by damage to periodontal tissue and alveolar bone surrounding the tooth.1 periodontitis is caused by gram-negative bacteria such as: porphyromonas gingivalis (p. gingivalis), tannerella forsythia (tf) and actinobacillus actinomycetemcomitans (aa). however, the most common bacteria found in patients with chronic periodontitis are p. gingivalis at a percentage of 48%.2 p. gingivalis contains lipopolysaccharide (lps), toxic membrane vesicles and protease enzymes that play a role in inflammation.3 although bacteria and their bi-products doi: 10.20473/j.djmkg.v51.i2.p99–103 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg mailto:alhidayatidrg@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p99-103 100asymal, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 99–103 play a role in periodontitis, the main causal factor of soft and hard tissue damage associated with periodontitis is that of the host immune response against the bacteria.4 the immune system components that contribute to periodontitis include macrophages and lymphocytes, the latter being immune cells characteristic of the host immune response to injury during chronic inflammation, whereas macrophages are immune and inflammatory cells that play an important role in host defense against periodontal pathogen infection.5 macrophages are crucial to the occurrence of non-specific immunity through the action of microbial phagocytosis and the production of cytokines which will then activate inflammatory mediators.6 several pieces of research have even shown that about 5-30% of inflammatory cells infiltrated in periodontitis tissue are macrophages, whereas the number of macrophages in gingival tissue suffering from periodontitis is higher than that in healthy gingival tissue.7 the diagnosis of periodontitis is carried out by means of clinical examination, including: establishing the extent of bleeding on probing, quantifying the depth of infection and radiographic analysis. the last mentioned is important in the management of periodontitis since it involves evaluating surrounding hard tissues and alveolar peak conditions, monitoring the extent of bone loss and increasing periodontal space. in addition, radiographic examination may also assist dentists in the areas of diagnosis, defining treatment plans and evaluating treatment outcomes. unfortunately, the use of x-rays to produce radiography has a negative effect since certain parts of the body, especially the head, are not well protected from the effects of x-ray radiation.8 x-ray radiation is one form of radiation that can induce an ionization process in the media through which it passes. x-ray radiation used to produce intraoral radiographic images is administered at low doses ranging from 0.01 to 10 msv.9 although included in low-dose radiation, the principle of radiation protection remains very important to the manufacture of radiographic images. this is because radiation at the lowest doses can still cause biological effects in the body due to x-ray ionization that can damage deoxyribonucleic acid (dna).9 in the field of radiology, the radiation to which each patient is exposed is set at a dose limit value (nbd) required by the international commission on radiological protection (icrp). the limit value of radiation exposure doses received should not exceed 0.3 milisievert (msv) per year. meanwhile, the dose administered in a single periapical radiographic examination was 0.08 msv.10 nevertheless, the effects of dental x-ray radiation on patients with periodontitis have still not been fully explained. therefore, the results of the research reported here are expected to reveal the effects of such exposure on the number of macrophages and lymphocytes in experimental subjects suffering from periodontitis. materials and methods this research used 36 male wistar rats aged 1.5-2 months and 150-200 grams in weight. these subjects were divided into three groups consisting of a control group, treatment group i and treatment group ii, each consisting of 12 male wistar rats. in the control group, the subjects were not exposed to x-ray radiation, while those in treatment group i were exposed to x-ray radiation at a dose of 0.16 msv and those in treatment group ii were exposed to x-ray radiation at a dose of 0.32 msv. the research was conducted at the laboratory of biochemistry, faculty of medicine, universitas airlangga and at the research center of the faculty of dental medicine, universitas airlangga. chronic periodontitis was subsequently induced in all subjects by the administering of 0.03 ml of p. gingivalis atcc 33277 and 2 x 106 cfu/ml injected into the gingival sulcus of their lower right and left incisors once every three days for two weeks.11 clinical signs of chronic periodontitis, such as gingival hyperplasia, pocket formation figure 1. the results of hpa on the lymphocyte cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in treatment ii group on day 3; (f) in treatment ii group on day 5. figure 2. the results of hpa on the macrophage cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in the treatment ii group on day 3; (f) in treatment ii group on day 5. table 1. the mean value and standard deviation of macrophage and lymphocyte cell counts in the control group, treatment i group and treatment ii group control treatment i treatment ii macrophages day-3 7.16 ± 0.989 10.5 ± 2.73 7.5 ± 2.42 day-5 8.33 ± 1.86 12.83 ± 3.43 8.5 ± 2.25 lymphocytes day-3 2.16 ± 0.98 7.0 ± 1.78 6.16 ± 1.47 day-5 2.33 ± 0.98 9.0 ± 1.78 4.5 ± 1.04 a b c d e f a b c d e f figure 1. the results of hpa on the lymphocyte cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in treatment ii group on day 3; (f) in treatment ii group on day 5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p99–103 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p99-103 101 asymal, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 99–103 and periodontal attachment loss were then observed. hpa examination was also carried out in order to confirm the diagnosis of periodontitis, after which treatment group i was exposed to a 0.16 msv dose of x-ray radiation, while treatment group ii was exposed to one of 0.32 msv. the subjects were sacrificed by means of ether inhalation on days 3 and 5 after treatment since on those specific days they would experience the initial and final phases of inflammation respectively. their mandibular was then removed and fixed in 10% formalin solution. thereafter, tissue processing was performed, prior to he staining being conducted. the preparations were observed by using he staining technique with a light microscope at a magnification of 400x from five (5) fields of view. results the results of the observation of the number of macrophages and lymphocytes can be seen in table 1 and figures 1 and 2. the table above shows that the lowest number of macrophage cells was found in the control group sacrificed on day 3, but not exposed to x-ray radiation (7.16 ± 1.16). meanwhile, the highest number of macrophage cells was in treatment group i exposed to x-ray radiation at a dose of 0.16 msv and subsequently sacrificed on day 5 (12.83 ± 3.43). on the other hand, the lowest number of lymphocytes was found in the control group sacrificed on day 3, but not exposed to x-ray radiation (2.16 ± 0.98), whereas the highest number of lymphocytes was in treatment group i sacrificed on day 5 (9.0 ± 1.78). based on the results of the kolmogorov smirnov test illustrated in the table above, the number of macrophage and lymphocyte cells in all groups demonstrated normal distribution (p>0.05). the results of the one-way anova test indicated the existence of a significant difference in the study groups. the hsd test results showed that there were significant differences between the research groups. for example, there was a significant difference in the number of macrophage cells on day 3 between the control group and treatment group i (p= 0.049). there were also significant figure 1. the results of hpa on the lymphocyte cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in treatment ii group on day 3; (f) in treatment ii group on day 5. figure 2. the results of hpa on the macrophage cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in the treatment ii group on day 3; (f) in treatment ii group on day 5. table 1. the mean value and standard deviation of macrophage and lymphocyte cell counts in the control group, treatment i group and treatment ii group control treatment i treatment ii macrophages day-3 7.16 ± 0.989 10.5 ± 2.73 7.5 ± 2.42 day-5 8.33 ± 1.86 12.83 ± 3.43 8.5 ± 2.25 lymphocytes day-3 2.16 ± 0.98 7.0 ± 1.78 6.16 ± 1.47 day-5 2.33 ± 0.98 9.0 ± 1.78 4.5 ± 1.04 a b c d e f a b c d e f figure 2. the results of hpa on the macrophage cells (yellow arrows) of the subjects suffering from chronic periodontitis using he staining technique. (a) in the control group on day 3; (b) in the control group on day 5; (c) in treatment i group on day 3; (d) in treatment i group on day 5; (e) in the treatment ii group on day 3; (f) in treatment ii group on day 5. table 1. the mean value and standard deviation of macrophage and lymphocyte cell counts in the control group, treatment i group and treatment ii group control treatment i treatment ii macrophages day-3 7.16 ± 1.16 10.5 ± 2.73 7.5 ± 2.42 day-5 8.33 ± 1.86 12.83 ± 3.43 8.5 ± 2.25 lymphocytes day-3 2.16 ± 0.98 7.0 ± 1.78 6.16 ± 1.47 day-5 2.33 ± 0.98 9.0 ± 1.78 4.5 ± 1.04 table 2. the results of a kolmogorov smirnov test and one-way anova test on the number of macrophage cells on day 3 groups significance of the normality test significance of the one-way anova test control 0.4572 0.0378*treatment i 0.9321 treatment ii 0.2729 *p<0.05 there was a significant difference dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p99–103 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p99-103 102asymal, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 99–103 differences in the number of macrophage cells on day 3 between the control group and treatment group ii (p=0.963), as well as between treatment group i and treatment group ii (p=0.08). similarly, there were significant differences in the number of macrophage cells on day 5 between the control group and treatment group i (p=0.023) as well as between treatment group i and treatment group ii (p=0.028). meanwhile, there was no significant difference between the control group and treatment group ii (p=0.993). on the other hand, with regard to the number of lymphocyte cells, there were also significant differences on day 3 between the control group and treatment group i (p=0.0001) as well as between the control group and treatment group ii (p=0.0006). however, there was no significant difference between treatment group i and treatment group ii (p=0.592). there were significant differences in the number of lymphocyte cells on day 5 between the control group and treatment group i (p=0.001) as well as between treatment group i and treatment group ii (p=0.001). however, no significant differences existed between the control group and treatment group ii (p=0.108). discussion the results of the hsd test on day 3 showed that there was a significant difference in the number of macrophages between the control group and treatment group i. this indicates that when treatment group i was exposed to x-ray radiation at a dose of 0.16 msv, the macrophage cells could still neutralize free radical damage. at the time of exposure, immune cells responded to the radiation exposure in the form of a wound so that activated macrophages proliferated more strongly in the affected area as the body's defense response. there was no significant difference between the control group and treatment group ii or between treatment group i and treatment group ii. however, the number of macrophage cells in treatment group ii was higher than in the control group. this may be due to the higher dose of x-ray radiation (0.32 msv), which causes the number of free radicals to increase and damage the chains of dna, proteins, carbohydrates and macrophage cell lipids, leading to apoptosis of the macrophage cells. in addition, when the body responds to injury, it needs time to repair the damage resulting in inhibition of macrophage cell proliferation.12 the results of the hsd test on the number of macrophage and lymphocyte cells on day 5 revealed there to be significant differences between the control group and treatment group i as well as between treatment group i and treatment group ii. the number of macrophages and lymphocytes was higher in treatment group i than in treatment group ii and the control group. this could be caused by the radiation dose in treatment group i being lower than that in treatment group ii so that the macrophage and lymphocyte cells in treatment group i could still improve the free radical damage and immediately proliferate in response to the injury. meanwhile, in the second treatment group there was a decrease in the number of macrophages and lymphocyte cells. this may have occurred because the dose of x-ray radiation administered was higher so that macrophages and lymphocytes experienced a higher level table 3. the results of a kolmogorov smirnov test and one-way anova test on the number of macrophage cells on day 5 groups significance of the normality test significance of the one-way anova test control 0.3577 0.0139*treatment i 0.6085 treatment ii 0.3018 *p<0.05 there was a significant difference table 4. the results of a kolmogorov smirnov test and one-way anova test on the number of lymphocyte cells on day 3 groups significance of the normality test significance of the one-way anova test control 0.0935 0.0001*treatment i 0.5431 treatment ii 0.5245 *p<0.05 there was a significant difference table 5. the results of a kolmogorov smirnov test and one-way anova test on the number of lymphocyte cells on day 5 groups significance of the normality test significance of the one-way anova test control 0.0935 0.0001*treatment i 0.5431 treatment ii 0.7624 *p<0.05 there was a significant difference dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p99–103 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p99-103 103 asymal, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 99–103 of apoptosis than treatment group i. radiation-induced cell apoptosis will intensify in tandem with the increase of the ionization radiation dose.13 free radicals resulting from radiation can subsequently cause dna damage such as the impeding of hydrogen bonds between chains, cross linking and breaking of dna chains. disruption of dna will eventually lead to cell death or genetic mutations. x-rays can also interfere with cell mitochondrial function, resulting in the oxidation of carbohydrates, lipids and cell proteins. this, in turn, can induce disruption of the energy cycle in the cells. when free radicals cannot be neutralized by the body this can lead to inactivation of cell proliferation, retention of cell cycle check point, induction of cell apoptosis and inhibition of cell cycle.12 chronic periodontitis is a disease in which the interaction between bacteria and the host immune response greatly affects the severity of the periodontal condition. following the bacterial attack, an inflammatory reaction constitutes the body's defense response during which a number of immune cells such as macrophages and lymphocytes will congregate on the infected side. macrophages represent one of the immune and inflammatory cells that play an important role in the host’s defense against periodontal pathogen infection.5 meanwhile, lymphocytes are white blood cells that help the body's immune system fight infection. these cells produce antibodies against antigens in the inflammatory sites.14 the presence of exposure to low to mid-dose x-ray radiation can then lead to apoptosis, whereas high-dose radiation will lead to the death of cells resulting in necrosis.13 free radicals formed due to exposure to x-ray radiation can also cause intracellular stress which signals to the mitochondria causing them to undergo change. transformation in the mitochondria begins in the open outer membrane, followed by swelling of the matrix and the loss of transmembrane potential which causes the mitochondria to lose their electron transport function. this leads to mitochondrial proteins such as cytocrom-c being released. the detached cytocrom-c can then activate caspase 9 which may eventually lead to apoptosis.13 apoptosis that occurs in macrophages and lymphocytes can induce the immune system to work harder since, on the one hand, it must respond to the invasion of bacteria which, on the other hand, it must inhibit proliferation due to free radical effects caused by radiation. when immune cells undergo proliferative inhibition, the host's immune response to bacterial invasion will decrease which, in turn, will affect the body's defense mechanism to act against infectious diseases.15 finally, it can be said that exposure to dental x-ray radiation potentially leads to changes in the number of macrophages and lymphocytes in rats suffering from periodontitis. exposure to x-ray radiation at a dose of 0.16 msv can increase the number of macrophages and lymphocytes on days 3 and 5, whereas exposure to x-ray radiation at a dose of 0.32 msv can decrease the number of macrophages on day 3 and decrease the number of lymphocytes on days 3 and 5. therefore, the principle of radiation protection is very important to consider in the manufacture of periapical radiographic images. although the radiation used in producing such images is at relatively low doses, it can still cause cellular changes in the body since radiation at the lowest possible doses produces biological effects in the body.9 in conclusion, exposure to x-ray radiation at a dose of 0.16 msv can increase the number of macrophages and lymphocytes on days 3 and 5, whereas exposure to x-ray radiation at a dose of 0.32 msv can decrease the number of macrophages on day 3, while also reducing the number of lymphocytes on days 3 and 5. references 1. cochran dl. inflammation and bone loss in periodontal disease. j periodontol. 2008; 79(8 suppl): 1569–76. 2. mane ak, karmarkar ap, bharadwaj rs. anaerobic bacteria in subjects with chronic periodontitis and in periodontal health. j oral heal community dent. 2009; 3(3): 49–51. 3. k a t o h , ta g uch i y, tom i n a ga k , um e d a m , ta n a k a a . porphyromonas gingivalis lps inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells. arch oral biol. 2014; 59(2): 167–75. 4. savitri ij, ouhara k, fujita t, kajiya m, miyagawa t, kittaka m. irsogladine maleate inhibits porphyromonas gingivalis-mediated expression of toll-like receptor 2 and interleukin-8 in human gingival epithelial cells. j periodontal res. 2015; 50(40): 486–93. 5. yang j, zhang l, yu c, yang xf, wang h. monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. biomark res. 2014; 2: 1–9. 6. duque ga, descoteaux a. macrophage cytokines: involvement in immunity and infectious diseases. front immunol. 2014; 5: 1–12. 7. poole n m, mamidanna g, smith r a, coons lb, cole ja. prostaglandin e2 in tick saliva regulates macrophage cell migration and cytokine profile. parasit vectors. 2013; 6: 1–11. 8. white sc, pharoah mj. oral radiology: principles and interpretation. 7th ed. missouri: mosby; 2013. p. 91-130. 9. alatas z. efek kesehatan pajanan radiasi dosis rendah. in: aspek keselamatan radiasi dan lingkungan pada industri non-nuklir. jakarta; 2003. p. 27–39. 10. whaites e, drage n. essentials of dental radiography and radiology. 5th ed. philadelphia: churchill livingstone; 2013. p. 488. 11. krismariono a. the decreasing of nfκb level in gingival junctional epithelium of rat exposed to porphyromonas gingivalis with application of 1% curcumin on gingival sulcus. dent j (maj ked gigi). 2015; 48: 35–8. 12. azzam ei, jay-gerin jp, pain d. ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. cancer lett. 2012; 327(1–2): 48–60. 13. supriyadi s. evaluasi apoptosis sel odontoblas akibat paparan radiasi ionisasi. indones j dent. 2008; 15: 71–6. 14. kumar v, abbas ak, aster jc, perkins ja. robbins and cotran pathologic basis of disease. 9th ed. philadelphia: saunders; 2015. p. 11-31. 15. widyasari e, listyawati s, pangastuti a. pengaruh iradiasi sinar-x terhadap produksi antibodi mencit galur balb/c dengan pemberian vaksin toksoid tetanus. bioteknologi. 2007; 4: 13–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p99–103 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p99-103 mkgs vol 44 no 1 jan-mar 2011.indd 30 vol. 44. no. 1 march 2011 management of anterior teeth damage caused by complex caries through aesthetic endorestoration nanik zubaidah department of conservative dentistry faculty of dentistry, airlangga university surabaya-indonesia abstract background: dental caries is a microbiological disease that result in localized dissolution and destruction of the calcified tissue. it is multifactorial, therefore prevention must be based on a multifactorial approach. the damage of anterior teeth due to complex caries, for certain person may interfere their performance and decrease their self confidence aesthetically. restoration of tooth form and function, especially on anterior teeth is highly valuable. purpose: to present a case of maxillary anterior teeth with complex caries, through endorestoration treatment for recovering its original function and aesthetic. case: the 21 years old male patient with complex carries on maxillary anterior teeth number 12, 11, 21, 22 and 23. the patient felt bad about his performance and affect his self confidence. the patient visited the clinic to repair his teeth and to get its form and function aesthetically. case management: the endorestoration treatment was performed for carious teeth through pulpectomy followed by insertion of post retention and porcelain fused to metal crowns. conclusion: anterior teeth with severed complex caries can be managed through endorestoration treatment to recover its performance and function aesthetically. key words: complex caries, anterior teeth, aesthetic, endorestoration treatment abstrak latar belakang: dental karies adalah penyakit infeksi yang berakibat kerusakan jaringan kalsifikasi dan bersifat multifactorial. oleh karena itu pencegahan dilakukan dengan pendekatan multifactorial. kerusakan gigi anterior karena karies kompleks untuk orang-orang tertentu mungkin berdampak pada penampilan dan penurunan kepercayaan diri karena factor estetik. perbaikan gigi anterior dari berbagai kerusakan baik dalam hal bentuk maupun fungsinya sangat besar nilainya. tujuan: untuk menunjukkan kasus gigi anterior rahang atas karena karies kompleks melalui perawatan endorestorasi untuk mengembalikan fungsi gigi asli dan estetik. kasus: laki-laki usia 21 tahun dengan karies kompleks pada gigi anterior rahang atas 12, 11, 21, 22, dan 23. penderita dating ke klinik untuk perawatan tentang giginya dan mengembalikan bentuk maupun fungsi estetiknya. tatalaksana kasus: perawatan endorestorasi dilakukan untuk gigi karies melalui pulpektomi, insersi pasak dan mahkota porselen fuse to metal. kesimpulan: gigi dengan karies kompleks yang berat dapat diperbaiki melalui perawatan endorestorasi untuk mengembalikan penampilan dan fungsi estetiknya. kata kunci: karies kompleks, gigi anterior, estetik, perawatan endorestorasi correspondence: nanik zubaidah, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: nanikzubaidah@yahoo.com case report 31zubaidah: management of anterior teeth introduction in today’s era of globalization, the development of science and technology of instruments and materials used in dentistry have already been improved. as a result, the need of community for oral health services even is also increasing. for instance, the number of people who need dental care, especially the one with caries on anterior teeth is increasing. it means that people now become more aware with the importance of dental care, especially for preventing their anterior teeth from any dental caries, since the anterior teeth can support their physical appearance.1 however, almost all people have ever suffered from dental caries which can occur on one or more dental surface. dental caries is actually a multifactorial disease, but still can be prevented. preventive efforts must be based on a multifactorial approach. dentine caries, deriving from latin which means cavities, is caused by the progressive destruction of enamel and dentin due to metabolic activity of bacterial plaque. fortunately, the prevalence of dental caries in developing countries is very large and reaches more than 90%.1,2 dental caries can be classified based on the degree of severity or speed of attacking, involving the number and the location of teeth. according to pickard et al.,3 dental caries can actually be classified into following categories: mild caries, if it only attacks on teeth with most vulnerable surfaces, such as pit and fissure on the occlusal area; moderate caries, if it attacks on occlusal and proximal surfaces of posterior teeth; and severe caries, if it attacks on anterior teeth. and, the term of complex dental caries can be interpreted as dental caries attacking on more than one dental surface. dental caries can not only attack the smooth surface of enamel which normally occurs around the cervical of molars and premolars, but can also attack the surface of cervicolabial of incisive teeth. dental caries on the labial and cervical surfaces, moreover, often occurs in maxillary and mandibulary incisive teeth. but, if gingival recession occurs, dental caries will often occur on dentino enamel junction, which then can disturb the enamel. if this condition is not treated, the dental caries will be more severe, and then fractures can occur in the cervical area of anterior teeth.3,4 anterior teeth decay due to dental caries will usually show several symptoms in the later stages, such as pain; visible discoloration, changing into brown or black color; visible hole on teeth that can not only be felt and touched by tongue, but can also be seen when laughing. severe pain is caused by pulpitis and advanced propagation of caries. if this condition is not treated, it will cause the death of dental pulp, increase the fragility and the teeth will easily fractured.3,5 therefore, the prevention and treatment of dental caries are important to be conducted according patient’s need, especially in terms of restoration of dental function and aesthetics. dental esthetics is an important part of patient’s self-appearance. natural and appropriate appearance is an aesthetic form most widely expected. similarly, aesthetics in dentistry is considered as a philosophy closely related to self-appearance created by a restoration process that can be achieved through color and natural shapes.6 in dentistry, especially in dental aesthetic conservative dentistry, aesthetic treatments are usually needed by people who suffer tooth decay, including active dental caries, tooth discolorations due to the death of pulp (after endodontic treatment), fractures, anomaly of tooth, malposition, crowding, central diastema, abrasion, attrition, and dental erosion. moreover, if all of those abnormalities occur in anterior teeth, they may reduce patients’ confidence when smiling since anterior teeth is the most prominent element when laughing. besides that, since interesting and pleasant smile can make someone more acceptable in society, people do not only demand for their anterior tooth aesthetics, but also motivate to obtain good dental and facial cares.7,8 maxillary anterior teeth decay due to complex dental caries can reduce the patients’ confidence, this condition can still be solved by conducting endorestoration treatment to restore the form, function and aesthetic of those teeth in order to be seen like the original teeth.9 it means that endodontically treatment teeth can get another treatment which is restoring their roots and crowns with retentive and stable crown cast, so it can be used as long as possible. the success of the restoration is determined by some factors, such as retention, stability, aesthetics (especially the anterior teeth), and biological aspects.3 for those reasons, the following case of maxillary anterior teeth with complex dental caries which has gotten endorestoration aesthetic treatment would be reported. the purpose of this case report is to provide information that teeth with severe dental caries (complex dental caries) do not need to be removed, but they can still be treated and maintained through endorestoration treatment, which is endodontic treatment followed with the insertion of cast core and the porcelain fused to metal jacket crowns. case figure 1. the initial condition of the patient’s teeth, 12, 11, 21, 22, and 23 before treatment. the patient is a 21 year old man suffering from complex dental caries on his maxillary anterior teeth, 12, 11, 21, 22, and 23 (figure 1). as a result, he felt less confident because 32 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 30–34 of his dental appearance. thus, he desperately needed a dental treatment that could not only improve the aesthetic defects, but could also eliminate the psychological disorder that he had suffered for long time. in other words, the patient wanted that those of his teeth could not only regain their normal form and aesthetics, but could also have their original tooth function. in the first visit, intra-oral examination was conducted on the patient’s teeth, 12, 22, and 23 which suffered from severe dental caries causing some parts of his teeth, started from the entire surface of their tooth crown to the entire layer of their enamel, lost and black.11 based on the result of the intra-oral examination, it was then known that the tooth 11 suffered not only from class v of dental caries, especially in its cervical, but also from class iii of dental caries, especially in its proximal distal. it was also known that the tooth 21 has perforation caries. panoramic and periapical x-rays were also conducted for making the diagnosis and treatment plan. there was radiolucent on the periapical area of those teeth 12, 21, 22, and 23; and they were diagnosed as irreversible pulpitis. tooth 11 got reversible pulpitis. however, all of those teeth were in vital condition. therefore, the dental treatment plan for those teeth, 12, 21, 22, and 23, was endorestoration treatment involving pulpectomy by using cast post-core as retention, and also by making porcelain fused to metal jacket crowns, as restoration. case management when the patient came at the first time, the intra oral and extra-oral examinations were directly conducted. and then, the printed images of his teeth, 12, 11, 21, 22, and 23 which front, left and right sides suffering from dental caries, were taken both before and after the treatment. next, the anatomical impression of the maxillary and mandibulary teeth were made in order not only to get study models and dental records, to know the occlusion and relation, but also to prepare the temporary jacket crown that would be insert on those teeth, so that the dental aesthetics would not be reduced during the treatment. moreover, the tooth 11 with a diagnosis of reversible pulpitis would be restored with class v and class iii of composite resin which preparations were simultaneously conducted. the preparation of class v composite restorations was then conducted on enamel with an undercut additional retention. after that, kidney-shaped cavity was prepared with a depth up to the dentin and with color adjusted with shade guide of restorative materials. next, etching and bonding processes (generation 6) were conducted, and curing process then was conducted for 20 seconds with visible light (according to the factory instruction). afterwards, it was filled with microfilled composite resin by using aluminum cervical matrix, and then curing process was conducted for 20 seconds with visible light. similar procedures were also conducted in the class iii, but only the location and shape of dental preparation were different. it is because the location and the form of the class iii preparation must be adjusted to the size of caries occurred in the distal proximal. the endorestoration treatment was conducted on those teeth, 12, 21, 22, and 23, involving endodontic and restoration treatments, which are pulpectomy with crown down technique root canal preparation, by using pro taper until the file # f2 (the teeth no. 12, 21, and 22), and until file # f3 (the tooth no. 23), based on the working length. on the next stage, the impression of those teeth that had already been treated with endodontic treatment were made by using elastomers, in order not only to make both their cast post and core reinforcing, but also to make their temporary jacket crowns adjusted to their normal position in the good dental arch. the purpose of making these temporary jacket crowns was not only to protect the cast core that had been inserted during the treatment, but also to be used as a description of the normal anterior tooth position in a good dental arch with normal overbite and overjet. figure 2. the insertion of cast post into the core of teeth, 12, 21, 22, and 23. figure 3. the condition of teeth, 12, 11, 21, 22, and 23, after the treatment. the insertion of cast post and core was conducted one by one into the root canal of teeth 12, 21, 22, and 23 with zinc phosphate cement. after that, the repair of inserted cast post and core preparation were conducted in order to obtain the good position and alignment so the insertion of porcelain fused to metal jacket crowns can be easily done (figure 2). the impression of the teeth 12, 21, 22 and 23 with double impression materials as well as the description of the bite registratiion was conducted in order to make them as same as the original ones. next, those teeth were covered by 33zubaidah: management of anterior teeth the temporary jacket crown. the maxillary and mandibular models accompanied with its information then were sent to the dental laboratory in order to make the porcelain fused to metal jacket crowns. figure 4. photo of the patient’s teeth, 12, 11, 21, 22, and 23, one year after the treatment. the final stage of this treatment was to try the porcelain fused to metal jacket crowns. on the teeth 12, 21, 22, and 23. since during the trial the initial fit was looked good, the form and color were matched, and there was also no premature contacts, the permanent insertion with glass ionomer luting cement then could be conducted (figure 3). finally, the patient was asked to have controls 1 week, 1 month, 6 months and 1 year after the treatment in order to be evaluated (figure 4). discussion dental caries is a microbiology dental infectious disease causing the multifactorial destruction and breakage of tissue calcification. therefore, its prevention efforts must be based on a multifactorial approach. however, dental caries still becomes one of the most common disease largely infecting humans. the prevalence of dental caries in developing countries could reach more than 90%. the management of those complex anterior tooth damages caused by dental caries can be solved by endorestoration treatment.2 generally, patients who suffer from severe dental caries on teeth, 12, 11, 21, 22 and 23, require aesthetic treatments to repair anterior teeth which entire surface of their crown is looked almost black color. as a result, this condition makes the patients feel both ashamed with their physical appearance and less confident, especially when they laugh. then, patients will expect optimal results from the aesthetic treatments that they take, as a result, the form and function of their anterior teeth can become normal again. actually, according to antune et al.,10 maxillary anterior teeth can be functionally and aesthetically rehabilitated to restore a good smile and increase self-confidence. one visit endodontic treatment was conducted on the teeth 12, 21, 22, and 23 aimed to prevent the spread of the disease from the pulp to the periapical tissue; or if it occurred, the treatment would be aimed to change or return the periapical tissue into its normal condition. the treatment also provides benefits of not only reducing the risk of infection possibly occurred among the visits, saving time, but also reducing the risk of infrequent flare-up.7 the root canal preparation then was conducted on the teeth 12, 21, 22, and 23 by using crown down technique with pro taper instruments. this technique is very beneficial not only because most of the microorganisms located in 1/3 coronal and 1/3 center has been drawn before entering into the apical area, but also because the irrigation is more perfect in 1/3 apical.11 endodontic treatment teeth need to be restored both root and crown of the tooth by using retentive and stable cast and core crowns, so it is not easily removed and can be used in the oral cavity as long as possible as the original teeth. however, the internal moisture of teeth that have undergone the endodontic treatment is getting reduced so it becomes brittle, relatively easy to fracture (broken), and subject to color changing. it also makes the remaining tooth structure weak as a result of the reduction of dentin during the endodontic treatment. therefore, the comprehensive cast must be conducted not only by using both casts, post and core, but also by making porcelain fused to metal jacket crowns in order to make those teeth get no fractures.12 in this case, the teeth 12, 21, 22 and 23 would use cast post and core since those casts could make both cast post and core unify, and then could closely follow the form of root canal preparation. the application of cast post is aimed to repair and form the position, so it can be in accordance with the normal anterior tooth position in a good dental arch with normal overbite and overjet.13 the success of the use of cast post and core is about 90.6% in an average, and it needs six years for the foundation of a restoration. 14 the insertion of cast post and core on teeth 12, 21, 22, and 23 must be conducted one by one in the same time. it is aimed to get not only the shape and size that are in accordance with the normal ones, but also the anterior dental arch with normal overbite and overjet, as a result, the good and harmonic aesthetic form can hopefully be accepted. in conclusion the anterior teeth decay caused by complex dental caries can be treated by endorestoration aesthetic treatments followed with both the application of retention, such as cast post and core, and the application of the final restoration, such as porcelain fused to metal jacket crown. references 1. walmsley ad. restorative dentistry. 2nd ed. edinburg, london, new york, oxford, philadelphia, st louis, sydney, toronto: churchill livingstone; 2007. p. 73–115. 2. shivakumar km, vidya sk, chandu gn. dental caries vaccine. indiana j dent res 2009; 20(1): 154–6. available at: htpp://www. ijdr.in. accessed october 15, 2009. 3. pickard hm, kidd eam, smith bgn. manual konservasi restorasi. sumawinata n, editor. edisi ke-6. jakarta: penerbit widia medika; 2002. p. 6–15. 34 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 30–34 4. lunardhi cgj. resin komposit untuk restorasi gigi posterior. simposium sehari mempertahankan gigi selama mungkin, lustrum vii unair, 1998; 89: 51–8. 5. ford ptr. restorasi gigi. sumawinata n, editor. edisi ke-2. jakarta: ecg; 1993. p. 1–20. 6. jablonski s. illustrated dictionary of dentistry. 2nd ed. philadelphia: wb saunder co; 1982. p. 230–99. 7. grossman li, oliet s, del rio ce. ilmu endodontik dalam praktek. 1st ed. jakarta: penerbit buku kedokteran ecg; 1995. p. 196–380. p. 196–380. 8. tjan ahl, miller gd, josephin gp. some esthetics factors in smile. j of prost dent 1984; 51(1): 24–8. 9. baritcigil c, harorli ot, yildiz m. restoration of crown fracture with a fi ber post, polyethylene and composite resin. rev clin pesq odontol curitiba 2009; 5(1): 73–7. 10. antune rpa, magalhaes f, matsumoto w, orsi ia. anterior aesthetic rehabilitation of all ceramic crown. quint int 1998; 29: 38–40. 11. nesha g, amit g. textbook of endodontic. 1st ed. new delhi, india: jaype brothers. medical ltd; 2007 p. 196. 12. chan dcn, myers ml, chipped. fracture, or endodonticcally treated teeth. in: goldstein re, editor. esthetics in dentistry. 2nd ed. hamilton, london: bc decker inc; 2002. p. 537–9. 13. yuzugullu b, canay s. metal–ceramic dowel crown restoration for severely damaged teeth: a clinical report. indiana j dent res 2009; 20(1): 110–2. 14. bitter k, kielbassa am. post-endodontic restoration with adhesively luted fi ber reinforced composite post systems: a review. american j of dent 2007; 20(6): 354–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isi vol 39 no 3 juli-september 2006.pmd 107 periodontal tissue damage in smokers hutojo djajakusuma department of oral medicine faculty of dentistry airlangga university surabaya indonesia abstract dental plaque is the primary etiological factor in periodontal diseases. however, there are many factors that can modify how an individual periodontal tissue will respond to the accumulation of dental plaque. among such risk factors, there is increasing evidence that smoking tobacco products alters the expression and rate of progression of periodontal diseases. the aim of this study was to find out the loss of periodontal tissue adhesion in smokers by measuring pocket depth using probe, and by measuring alveolar bone damage using bone loss score (bls) radiographic methods on teeth 12, 11, 21, 22, 32, 31, 41, 42. based on t test statistical analysis, there were significant differences in pocket depth damage of alveolar bone in smokers and non smokers. in conclusion there were increasing pocket depth and alveolar bone damage in smokers. key words: smoking, periodontal adhesion loss correspondence: hutojo djajakusuma, c/o: bagian oral medicine, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. previous research of has been reported. based on the above background, the problem in this study i.e. is there any difference of periodontal tissue damage in smokers and in non-smokers by clinical and radiographic examination? materials and methods the tipe of this research was an observational analytical study, and comparative, according to its purpose of comparing periodontal tissue damage in smokers and nonsmokers. materials used were: periapical extra-speed film ep 21, fixing solution and developer. equipments used were: dental photo x-ray unit, x-ray film holder, caliper, oral mirror, periodontal standard probe, millimeter grid and viewer. samples were taken from patients of airlangga university faculty of dentistry consisted of 10 males who had smoked minimal one year ago with an average of 20 cigarettes per day. non-smoker samples were also taken from patients of airlangga university faculty of dentistry consisted of 10 males who had never smoked. sample quantity was determined according to sample formula of sugiarto et al.9 to ensure sample’s accuracy to research requirements, criteria were added as follows: good general condition (no systemic disorder), aged 20–35, whole anterior teeth were well positioned in dental arch. patients were examined to detect clinical periodontal adhesion loss in upper jaw anterior region and lower jaw 12, 11, 21 22, 32, 31, 41, 42 by measuring pocket depth introduction smoking has hazardous effect for health. many studies reveal correlation of smoking to varied diseases such as lung cancer, pharynx and larynx, coronary heart disease, peripheral arteries disorder,1 also the correlation of smoking to higher risk of infertility in women2 and increased risk of impotency in men.3 in dentistry, the effect of smoking to oral changes has been reported in many researches, such as: dry socket occured four times higher at smokers than non-smokers, keratoses, decreased saliva ph, changes of oral micro-floral organism, even malignancy. various researches of the impact of smoking to periodontal diseases have been reported, among others are smoking caused more severe periodontitis and higher incidence of acute ulcerative gingivitis compared to non-smokers.4 recently showed that passive smokers have 1.5 times higher risk of gum disease than non-smokers.5 the world cigarette consumption obviously has increased. in 1996, the developing countries amass 68% of world cigarette consumption and it rises to 72% in the year of 2001.6 this condition will lead to higher incidence of gingivitis and periodontitis which will ultimately cause fall-out teeth. furthermore, periodontitis is chronic and destructive. smokers in general have no knowledge of the damaging process, they come to the dentist long overdue making it more difficult to handle. the aim of, this research at smokers to discover the scale of periodontal tissue damage by measuring clinical periodontal loss using probe7 and radiography,8 so that diagnosis and treatment plan can be determined. as far as the knowledge of the author, no 108 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:107–111 using probe from gingival margin until the base of the pocket. measurement was done at mesial and distal side of each tooth, followed by a radiographic examination using parallel technique. standard probe technique was used against the proximal side of dental pocket to measure the pocket depth. the periodontal tissue damage was measured according to the percentage of alveolar bone resorption in vertical direction (bls) in proximal side, based on adams and nystrom criteria.10 results research result of 10 smokers and 10 non-smokers towards pocket depth using probe on teeth 12, 11, 21, 22, 32, 31, 41, 42 is seen in figure 1 and 2. figure 1 and 2 showed the average of pocket depth using probe in smokers is bigger than non-smokers on teeth 12, 11, 21, 22, 32, 31, 41, 42 from mesial and distal sides. figure 3 and 4 showed the average of alveolar bone damage in smokers and non-smokers, measured by bls radiographic method. figure 3 and 4 showed that in smokers, the damage of alveolar bone using bls based on radiographic examination was higher than non-smokers in region 12, 11, 21, 22, 32, 31, 41, 42, in mesial as well as in distal. prior to applying the t test 2 free sample, a normality test was performed with kolmogorov-smirnov. the test result showed all groups had significant value > 0.05 meaning data was a normal distribution. further, levene’s test was done to know the homogeneity of two groups variance. if the significant value was > 0.05, the two groups variance were homogenous. the result of hypotheses test using t 2 free sample produced significant value ≤ 0.05 meaning there was a significant difference of pocket depth in smokers and non-smokers using probe and using bls method based on radiographic examination, both on 12, 11, 21, 22, 31, 32, 42, 41 region in mesial and distal sides. figure 2. pocket depth average in smokers and non-smokers using probe on lower jaw (mm). smokers non smokers p oc ke t de pt h (m m ) 42 41 d m d m 31 32 m d m d 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.7 1.7 4.1 2.1 4.0 4.1 2.2 4.0 2.0 3.9 1.9 4.4 2.5 3.9 2.0 d = distal m = mesial figure 1. pocket depth average in smokers and non-smokers using probe on upper jaw (mm). smokers non smokers d = distal m = mesial p oc ke t de pt h (m m ) 2.1 4.1 2.1 4.0 1.7 3.9 2.1 4.4 2.4 4.2 4.3 2.4 4.4 1.9 3.9 22 21 11 12 d m d m m d m d 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 109djajakusuma: periodontal tissue damage in smokers discussion test result of clinical periodontal adhesion loss periodontal tissue damaged is most often tested by measuring the loss of clinical periodontal adhesion and by radiographic examination.11-12 both measuring methods are easy, fast, inexpensive and painless. periodontal adhesion loss is measuring by pocket depth i.e. the distance of gingival edge until the base of penetration of the probe tip. the study shows that the average of pocket depth in smokers is bigger than non-smokers in teeth 12, 11, 21, 22, 31, 32, 42, 41 in mesial and distal sides. this condition shows that the pocket depth in smokers is bigger than in non-smokers. statistical test of the t 2 free sample presents a significant value < 0.05 meaning there is a significant difference of smokers and non-smokers pocket depth using probe method in 12, 11, 21, 22, 31, 32, 42, 41 in mesial and distal. this is in accordance with several studies stating smokers are prone to gingivitis and periodontitis than non-smokers.13,14 in this condition, periodontal pocket forming and alveolar bone damage can be found.15 the finding is supported by akhmad et al.16 who stated that the prevalence of smoking habit is more dominant as the cause of the prevalence of periodontal diseases. presumably, it is triggered by smoker’s condition, where a potential variation change of reduction-oxidation at gingival area takes place indicating the presence of figure 3. the average of alveolar bone damage in smokers and non-smokers using bls method (%) based on upper jaw radiographic examination. a lv eo la r bo ne d am ag e (% ) smokers non smokers 31 32 m d m d 42 41 d m d m 9.17 6.61 10.62 8.31 9.84 9.55 7.48 10.35 8.40 9.39 7.66 10.29 7.43 9.84 6.44 12.00 10.00 8.00 6.00 4.00 2.00 0.00 d = distal m = mesial figure 4. the average of alveolar bone damage in smokers and non-smokers using bls method (%) based on lower jaw radiographic examination. d = distal m = mesial smokers non smokers a lv eo la r bo ne d am ag e (% ) 31 32 m d m d 42 41 d m d m 12.00 10.00 8.00 6.00 4.00 2.00 0.00 9.87 7.51 9.27 7.00 9.51 10.14 7.54 10.15 7.52 9.71 7.38 10.44 8.20 9.59 6.86 110 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:107–111 anaerobic microorganism. the decline of reductionoxidation will increase anaerobic plaque bacteria.4 in addition, tobacco influence oral cavity immune system and smoking decreases oral cellular immunity by way of decreasing chemotactical response, phagocytic of leukocyte and it causes vasoconstriction which in the end it will decrease tissue immune response.17 as the result of an infection, epithelia adhesion proliferate along dental root’s surface towards the apical side, producing the lessening of desmosome adhesion, causing the detachment of epithelium surface from dental root surface, thus creating a deeper sulcus base.18 yet, several studies state that this manner can not measure the size of adhesion loss accurately, because the measurement initial pole is the edge of gingival which are unsteady. the suggested method is transgingival probing (sounding depth probing).19 the clinical adhesion loss and the transgingival probing have a similar measuring method, except for pressure difference, more or less by 100 gr. the pressure difference will produce a different result of measurement i.e. the clinical adhesion loss measurement describes the pocket base, while the transgingival probing measurement describes the base of alveolar bone damage.7,11,20 the examination is rarely performed due to its unpleasantness. test result of alveolar bone damage the dentistry world has recognized the importance of dental radiography for diagnoses tool, dental therapy plan, and treatment result assessment. the quality of radiography depends on several factors, among others are radiography technique and film processing.8 the most sophisticated dental x-ray unit has not guarantee to produce a good radiograph, if it lacks suitable radiography technique. wei21 said, in order to make a radiography of dental anterior upper and lower jaws, for intra oral radiography, the method-ofchoice is periapical technique because the picture is more accurate and the distortion is smaller should the film placing is correct. test result of alveolar bone damage shows that the average of alveolar bone damage in smokers is greater than in non-smokers in 12, 11, 22, 32, 31, 41, and 42 in mesial and distal sides. the result is supported by the statistical t 2 free sample test, with a significant value ≤ 0.05 pointing that there is a significant difference of alveolar bone damage between smokers and non-smokers using bls method of radiographic examination in region 12, 11, 21, 22, 32, 31, 41, 42 in mesial and distal sides. the statement is in accordance with previous studies stating that a radiographic examination exposes an increase in alveolar bone resorption paralleled to an increase of tobacco use, and the prevalence of periodontal disease and alveolar bone resorption is higher in smokers compared to non-smokers. also from an observation and assessment to bone density, smokers show bigger resorption than in non-smokers, and it also reveals a correlation of smoking with more severe periodontitis, that is the incidence of periodontitis is faster in smokers than in non-smokers.17 the increase of alveolar bone damage can be the result of increased infection17 and infection will result in faster bone alveolaris resorption so that it causes the resorption and the height of alveolar bone will be shortened.22 a radiograph of an alveolar bone damage demonstrates a crest alveolar resorption picture. alveolar bone density declines more than 3 mm starting from the tip of the bone, email margin and cementum.8 a radiographic examination using parallel technique, will bring about the result of correct radiograph. a radiographic examination applying parallel technique will produce a better, more accurate measurement towards the height of alveolar bone tissue, in relation to cement enamel junction.23 in addition, using bls method will enable us to find out the exact percentage of alveolar bone damage, so that the treatment will be precise. based on this experiment exposing the pocket depth and alveolar bone damage in smokers compared to non-smokers, it is suggested to dentists as oral and dental hygiene personnel to play an important role promoting and stressing community for awareness of the danger of smoking in general and oral health in particular. by knowing the percentage of alveolar bone damage using bls method with radiographic examination, a definite treatment plan can be executed correctly. there is no preliminary study in this writing, therefore to clarify whether the periodontal damage happened because of smoking or prior to smoking, it is strongly suggested for other researchers to do supportive examinations preceding the study. references 1. who. tobacco and health impact. available at: http://www.who.int/ tobacco/health_impact/en/. accessed may 13, 2006. 2. hull, et al . delayed conception and active and passive smoking. the avon longitudinal study of pregnancy and childhood study team. fertil steril. 2000 oct; 74(4):725–33. available at: http://www.ncbi.nim.nih.gov/entrez/query.fogi?cmd = retrieve & db = pubmed&dopt-citation&list_uida = 11020514. accessed may 13, 2006. 3. tengs to, osgood nd. the link between smoking and impotance: two decades of evidence. prev med 2001 jun; 32(6):447–52. available at: http://www.ncbi.nim.nih.gov/entrez/query.fcgi? cmd = retrieve&db = pubmed&dopt = citation&list_uids = 11394947. accessed may 13, 2006. 4. ruslan g. efek merokok terhadap rongga mulut. available at: file://g:/cermin%20 dunia%20 kedokteran.htm. accessed march 28, 2006. 5. jaga-jaga. perokok pasif dapat menderita gangguan gusi. availabel at: http://www.jaga-jaga.com/an 1 hatiyook.php? ida = 236. accessed march 28, 2006. 6. yurekli, bayer. bank dunia 2002. available at: http:// www1.worldbank.org/tobacco/pdt/indonesian.pdf. accessed may 13, 2006. 7. theil em, heaney tg. the validity of periodontal probing as method of measuring loss pf attachment. j clin periodontol 1991; 18:648–53. 8. white sc, pharoah mj. oral radiology: principles and interpretation. 5th ed. new delhi: el sevier; 2004. p. 321. 9. sugiarto, siagian d, sunaryanto lt, oetomo ds. teknik sampling. cetakan kedua. jakarta: penerbit pt gramedia pustaka utama; 2003. h. 32. 111djajakusuma: periodontal tissue damage in smokers 10. adams ra, nystrom gp. periodontitis severity index. journal of periodontology 1986; 57: 177. 11. pihlstrom bl. measurement of attachment level in clinical trials: probing methods. j periodontol 1992; 63:1072–77. 12. kelly gp, cain rj, knowles jw, nissle rr, burgett fg, shick ra, ramfjord sp. radiographs in clinical periodontal trial. j periodontol 1975; 46:381–86. 13. bastian, waite im. effects of tobacco smoking on plaque development and gingivitis. j periodontol 1978; 49:480–82 14. hidalgo rf. smoking and periodontal disease. j periodontol 1986; 57:61724. 15. carranza fa. glickman’s clinical periodontology.7th ed. philadelphia, london, toronto, montreal, sydey, tokyo: wb saunders company; 1990. p. 51-61, 234–49, 513–7. 16. akhmad, hasan h, syamsuddin. available at: http://litbang.kaltim.go.id/ hasil%20penelitian bidang%20kemasyarakatan/ s e k t o r % 2 0 s o s i a l % 2 0 p o l i t i k / a b s t r a k % 2 0 sektor%sosial%20politik%203.10.12.4.html/. accessed may 15, 2006. 17. the colgate periodontal education program. smoking and periodontal disease. available at: http://www.adelaide.edu.au/spdent/ dperu/cpep/smoking.htm. accessed may 15, 2006. 18. stahl ss. speculations on periodontal attachment loss. j clin periodontol 1986; 8:98–106. 19. yehouda ab, machtei ee. sounding depth measurement: a method for evaluating various surgical technique. j periodontol 1991; 62:565–69. 20. velden vdu. probing force and the relationship of the probe tip to the periodontl tissue. j clin periodontol 1979; 16:106–14. 21. wei shy. pediatric dentistry: total patient care. philadelphia: lea and febiger; 1998. p. 115–38. 22. halazonetis td, haffajee ad, sockransky ss. relatioship of clinical parameters to attachment loss in subsets of subjects with destructive periodontal disease. j clin periodontol 1989; 16:563–8. 23. the colgate periodontal education program. radiography and periodontal diagnosis. http://www.adelaide.edu.au/spdent/dperu/ cpep/radio.htm. accessed may 15, 2006. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice subject index volume 52 adjustment disorder, 163 aggregatibacter actinomycetemcomitans, 81 alginate, 36 alveolar bone, 13 anadara granosa, 177 andrographis paniculata nees extract, 219 angular cheilitis, 76 anti-inflammatory, 90 antioxidant, 90 apoptosis, 138 applied behavior analysis, 117 arab population, 154 autistic spectrum disorders, 117 bone formation, 177 repair, 61 bovine bone graft (bbg), 126 bulk fill packable composite, 192 caffeine, 1 calcium hydroxide, 183 candida, 110 carbonic acid, 192 carcinogenesis, 138 caries, 86 caspase-1, 105 cell proliferation, 142 cell viability, 142 chronological age, 100 cigarette smoke, 133 closed reduction, 147 cocoa pod husk extract, 159, 215 collagen density, 209 coronally advanced flap, 8 cortisol level, 163 cox-2, 105 dental caries, 66 dental trauma, 57 diabetes mellitus, 76 diabetes type, 110 diabetes, 51 differential diagnosis, 32 dmf index, 66 dry, 41 edta, 122 eighth-generation bonding, 41 endodontic, 215 infection, 172 enterococcus faecalis, 172, 215 epithelial dysplasia, 187 er,cr:ysgg, 57 ergonomics, 18 ethanol, 41 exposure to cigarette smoke, 187 extracellular polymeric substance biofilm, 215 fibroblasts, 126, 209 fusobacterium nucleatum, 81 gelatine, 36 gestational, 110 gingival enlargement, 204 overgrowth, 204 recession, 8 glycerin, 95 glycoproteins b-1, 197 granule scaffold, 177 hand foot mouth disease, 32 hardness of nanofilled composite resin, 95 herpes labialis, 76 human cytomegalovirus, 197 immunodeficiency virus, 197 periodontal ligament fibroblast cells, 142 hyperplasia, 133 igy, 81 il-17, 105 inflammation, 105 irradiation, 24 isopropanol, 41 isothiocyanate, 71 laser diode, 142 lemuru, 51 lipotheicholic acid, 172 malocclusion, 168 management, 76 mandibular fracture, 147 mesenchymal stem cells, 36 micro-computed tomography, 1 microleakage, 192 mixed dentition analysis, 154 mmp-8 expression, 51 moist, 41 monocytes viability, 219 moringa oleifera extract, 71 moyers method, 154 mta, 57 musculoskeletal disorders, 18 neglected mandibular fracture, 147 nfatc1, 172 nutrient intake, 86 odontoblast like cell, 183 oral cancer, 71, 133, 187 health, 66 thrush, 76 orthodontic, 168 tooth movement, 1 osphronemus goramy, 45 osteoblasts, 13, 61, 126 osteoclasts, 13, 126 p53, 138 packable composite, 24 pericoronitis, 105 periodontitis, 51, 61 induced with porphyromonas gingivalis, 209 peripheral ossifying fibroma, 204 permanent incisors, 57 tooth eruption, 100 picture cards, 117 pineapple peel extract, 122 platelet-rich fibrin, 8 porcelain repair, 27 porphyromonas gingivalis, 81, 219 preschoolers, 66 primary varicella zoster infection, 32 prophylaxis brush, 117 propolis extract, 126 , 159, 183 protein biofilm, 86 density, 86 pulp therapy, 57 rank, 172 rapid entire body assessment, 18 rattus norvegicus, 187 reccurent aphthous stomatitis, 163 red pomegranate, 90 residual monomer, 24 root canal irrigation, 122, 159 scaffold hydroxyapatite gypsum puger, 13 scales, 45 self-etch bonding, 192 sex, 100, 168 shark liver oil, 209 shear bond strength, 27 sidestream cigarette smoke, 138 silane, 27 smear layer, 122 snail mucin (achatina fulica), 61 socioeconomic status, 66 sockets, 13 preservation, 126 stichopus hermanni, 177 streptococcus mutans, 45, 86 sanguinis, 81 tamarind soft drink, 95 tanaka-johnston method, 154 thorns, 45 timp-1 expression, 51 tongue mucosa, 138 tooth extraction, 18, 177 total-etch bonding, 192 traumatic ulcer, 90 ulser, 163 umbilical cord, 36 upeneus moluccensis, 45 vegf expression, 71 viability, 45 wistar rats, 172 xerostomia, 197 authors index volume 52 angriany, dian, 138 anugraha, ganendra, 204 apriasari, maharani laillyza, 32, 76 ayuningtyas, nurina febriyanti, 133 azlan, adriana, 168 balan, gülşah, 57 damaiyanti, dian widya, 51 dewanti, i dewa ayu ratna, 45 dewi, asti rosmala, 8 dwiandhono, irfan, 41 gunardi, olivia jennifer, 147 handayani, fani tuti, 154 titis mustikaningsih, 95 hartono, dwicha rahma nuriska, 71 hendrawati, h., 61 hendrijantini, nike, 36 hernawati, sri, 90 herniyati, h.,1 ismiyatin, kun, 142 kriswandini, indah listiana, 86 lestari, oktaviani suci, 81 lunardhi, louisa christy, 126 marjianto, agus, 100 melati, felicia, 117 mulawarmanti, dian, 209 nain, amiyatun, 13 noaman, bushra rashid, 66 pangestu, ayu ragil destrian, 110 prayitno, adi, 105 prayudha, anggy, 18 pribadi, nirawati, 122, 172 prijaryanti, dorisna, 187 rahayu, yani corvianindya, 219 rosha, jayanti, 24 saraswati, widya, 192 sari, rima parwati, 177 sufiawati, irna, 197 widjiastuti, ira, 27, 183 yuanita, tamara, 159, 215 yuliana, y., 163 guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those 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citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on 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....................... ........................................................................................... account no. : 988.01010.00000.135 bank : bank bni account holder : fkg dental journal ................................................................................................. ................................................................................................. 3131 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 31–34 original article correlation between salivary zinc levels and salivary volume on taste disorders in elderly patients dewi kania intan permatasari1, tenny setiani dewi1 and dewi marhaeni diah herawati2 1 departement of oral medicine, faculty of dentistry, padjadjaran university 2 departement of public health, faculty of medicine, padjajaran university bandung – indonesia abstract background: taste disorders often occur in the elderly, which can have serious consequences on their health status. zinc and saliva volume have a role in maintaining taste acuity, especially in the elderly. purpose: this study was to determine the correlation between salivary zinc levels, salivary volume, and taste disorders in elderly patients. methods: this was a cross-sectional research. elderly patients with and without taste disorders were included in this study. salivary zinc levels were measured using the atomic absorption spectrometry (aas) method. salivary volume was measured using the spitting method. the correlation between salivary zinc levels, salivary volume, and taste disorders was analysed using the chi-square test. multivariate analysis was performed to control for confounding variables with logistic regression. results: based on our findings, no significant correlation was determined between the levels of zinc in saliva and taste disorders. however, there was a significant correlation between salivary volume and taste disorders. salivary volume and smoking were determined to be associated with taste disorders. conclusion: this research showed that salivary zinc levels were not correlated with taste disorders; on the contrary, salivary volume was correlated with taste disorders. smoking was determined to be a confounding variable on taste disorders in elderly patients. keywords: elderly; salivary volume; salivary zinc; taste disorder correspondence: dewi kania intan permatasari, department of oral medicine, faculty of dentistry, padjadjaran university. jl. sekeloa selatan no. 1 bandung 40132, indonesia. email: dewi.kania.ip@gmail.com introduction worldwide, there has been a rapid increase in the number of older individuals, both in developed and developing countries. ‘elderly’ has been defined as someone who is 60 years old and above. in indonesia, the number of elderly people is estimated to increase from 23.66 million in 2017 to 33.69 million in 2025.1 the elderly often complain of reduced taste, sometimes having no taste for certain types of food. this is commonly referred to as ageusia. this complaint often occurs with ageing and is related to a decrease in the number of taste cells on the tongue, which affects the sense of taste and is marked by a decreased taste sensation. these changes will naturally have consequences, one of which is reduced appetite and enjoyment in eating.2–6 zinc as a micronutrient has been determined to play an essential role in taste acuity. as a person gets older, the zinc concentration in the body decreases, which in turn can cause taste problems in the elderly; therefore, zinc intake should be of concern as it affects appetite and ultimately affects the quality of life for them.5,6 the reference value of zinc content in saliva is 88–135 µg/l.7 in general, the sense of taste has been considered to be less important than the other senses because decreased function or taste disturbance is rarely fatal and does not require special medical attention. however, disturbance in the sense of taste can reduce the enjoyment of life, thereby making the patient uncomfortable as it affects their ability to enjoy food, drinks and pleasant aromas; furthermore, this disorder also affects the patient’s ability to recognise harmful chemicals, which can have serious consequences.8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p31–34 mailto:dewi.kania.ip@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p31-34 32 in addition to nutritional intake that is often deficient, there are other factors that can affect the sharpness of taste, namely systemic diseases that often occur in the elderly in the form of neurological diseases; cancer; kidney, lung, and endocrine disorders; rheumatoid diseases; gastrointestinal diseases; cardiovascular disorders such as hypertension; and drugs that elderly patients need to take for treating their illnesses. anti-hypertensive drugs have been identified as being widely and routinely consumed by elderly patients, and one of their side effects is that they can interfere with taste acuity. other factors that have to be considered are drinking alcohol, poor oral hygiene and tobacco consumption, which is a habit of elderly men.4,9 the purpose of this study was to analyse and determine the effects of salivary zinc levels, salivary volume and confounding variables such as zinc intake, hypertension and smoking on taste disorders in elderly patients. materials and methods the selection of study subjects was by means of consecutive sampling, consisting of 50 subjects each with and without taste disorders. the research subjects were elderly patients at the babatan health centre in bandung city with the following inclusion criteria: age ≥ 60 years, willing to be research subjects and able to provide informed consent; having complaints of taste disorders and not having complaints of taste disorders; being able to open their mouths as wide as two to three fingers; and without cognitive impairment, as tested in the mini-mental state examination (mmse) where the test score > 24. confounding factors such as zinc intake and hypertension were obtained from secondary data, while smoking habits were obtained from interviews. this study has received ethical clearance from the ethics committee of the faculty of medicine, padjadjaran university, bandung number: 1233/un6.kep/ec/2019 with the principles of respect for human dignity; respect for privacy and confidentiality; and respect for justice, inclusiveness, beneficence, and non-maleficence. the sample was collected from the subjects using the spitting test method, i.e. saliva collection by measuring the total saliva flow rate, without stimulation, for five minutes. the stopwatch was activated at one-minute intervals during saliva collection; the subject was then asked to spit into the measuring cup. this was continued for a period of five minutes.10 the collected saliva sample was then measured for zinc levels using the atomic absorption spectrometry (aas) method. the taste stimulation threshold was measured for sweetness (0.01 m and 0.1 m sucrose), saltiness (0.01 m and 0.1 m nacl), sourness (0.00032 m and 0.0032 m citric acid) and bitterness (quinine 0.000008 m and 0.00008 m) by placing a drop on the tongue according to the region of taste.11 bivariate analysis used the chi-square test, while multivariate analysis used logistic regression with p-value < 0.05. results this study consisted of 100 respondents with an age range of 60–99 years. the respondents comprised 36 men and 64 women. the distribution of research subjects, based on age, shows that the predominant age group is 60–69 years. the correlation between salivary zinc levels, salivary volume and taste disorders in the elderly can be seen in table 1. in this study, the average salivary zinc levels were determined to decrease in both respondents, with and without taste disorders. statistically, there is no significant correlation between salivary zinc levels and taste disorders. the average volume of saliva in the two groups of respondents was considered normal; however, respondents with taste disorders experienced a higher decrease in salivary volume. in this statistical test, there was a significant correlation between salivary volume and taste disorders. in this study, we measured several confounding factors that can influence taste disorders, namely zinc intake, hypertension and smoking. the results of statistical analysis show that there is a significant correlation between zinc intake, smoking and taste disorders, but no significant correlation was determined between hypertension and taste disorders. the effects of zinc intake, hypertension, and smoking on taste disorders have been summarised in table 2. the results of the multivariate logistic regression statistical test found that the volume of saliva, and smoking had a significant effect on taste disorders. individuals with a salivary volume < 1.45 ml/5 minutes are 3.23 times more at risk of developing taste disorders, after controlling for confounding variables – zinc intake, hypertension, and smoking. the correlation between salivary volume and taste disturbance has been determined to be controlled by confounding variables (zinc intake and smoking), as can be seen in table 3. table 1. the correlation between salivary zinc levels, salivary volume and taste disorders in the elderly variable taste disorders p-valueyes n=50 no n=50 salivary zinc (µg/l) mean 57.690 66.196 median 37.396 37.655 range 0.100– 304.405 0.100– 589.231 < 88 44 (88%) 39 (78%) 0.183 88–135 or more 6 (12%) 11 (22%) salivary volume (ml/5 minutes) mean 1.5 1.9 median 1.4 1.5 range 0.1–4.5 0.7–7.3 < 1.45 28 (56%) 18 (36%) 0.045* ≥ 1.45 22 (44%) 32 (64%) note: this analysis uses the chi-square test, * = significant (p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p31–34 permatasari et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 31–34 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p31-34 33permatasari et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 31–34 discussion in this study, a difference was determined in the average levels of zinc saliva in respondents with taste disorders and without taste disorders. watanabe, et al. observed that patients with taste disorders had lower levels of zinc saliva when compared to healthy patients.6 in this study, the respondents were divided into two groups: with and without taste disorders. like their study, this research was also conducted on elderly respondents; however, the number of respondents in this study was larger. in this study, other metallic elements were not measured. meanwhile, selow, et al. in their research showed that there was no significant difference in salivary zinc levels in subjects of various age ranges.12 their study measured zinc levels in respondents aged 18–59 years old, while in this study, we only measured zinc levels in respondents aged 60 years and above. some of the factors that can influence the low zinc levels in this study are advanced age, low zinc intake by all study respondents, and smoking, which inhibit zinc absorption in the digestive tract. there was a difference in the average volume of saliva among respondents with taste disorders compared to those without. the average volume of saliva among respondents who experienced taste disorders was found to be lower than those of the respondents without taste disorders. the volume of unstimulated normal saliva is 0.29–0.41 ml per minute. the volume of normal saliva obtained in five minutes is 1.45–2.05 ml.13,14 decreased salivary secretion usually occurs with age, but can be attributed to systemic diseases such as diabetes mellitus or hypertension, drugs, and radiotherapy in the neck and head area.15,16 salivary secretion in hypogeusia subjects is significantly reduced when compared to normal subjects.17 hershkovich and nagler’s research showed that the salivary flow rate in subjects with taste disorders was lower than in controlled group, but it was not statistically significant.18 they conducted a study on respondents aged 15–88 years by comparing a healthy group to a group with taste disorders, burning mouth syndrome, and xerostomia. in accordance with research conducted by hershkovich and nagler, healthy respondents have a greater volume of saliva than respondents who have complaints of taste disorders. hershkovich and nagler also took sialochemical measurements in the form of na, k, total protein, albumin, uric acid, lysozyme, amylase, ig m, ig g, ig a and sig a but in this study no sialochemical measurements were made.18 in this study, the results of the multivariate logistic regression statistical test showed that zinc levels in saliva did not have a significant correlation with taste disorders. this is in contrast to the shatzman and henkin study, where it was shown that there was a decrease in zinc levels in the saliva of patients with taste disorders.19 their study, by giving zinc therapy, showed an increase in zinc levels after therapy with improved taste function. in this study, no such method was used. the results of research conducted by watanabe, et al. showed that there was a decrease in zinc levels in saliva in subjects with taste disorders.6 their study was the same as this study, in that there was no zinc therapy or supplementation. salivary volume has a significant relationship with taste disturbances according to the literature, which indicates that the saliva function includes the transport of taste substances and the protection of taste receptors.16 this study differs from the study conducted by watanabe, et al., which showed no significant association between saliva volume and taste disturbances. the difference may occur because of the fairly large age range of the respondents in their study when compared to this study, which was conducted only with the elderly. the results of the research conducted by pushpass, et al. showed that salivary flow and elasticity were reduced in older persons, and this may play a role in impaired taste function.20 in this study, a significant correlation between salivary volume and smoking was determined as the smoking process has been proven to effect changes in salivary secretion, leading to decreased salivary secretion that in turn can cause taste disturbances. smoking has a significant correlation as a confounding variable to taste disturbances, as smoke and heat caused by smoking affect the action of papillae and taste buds found on the tongue. the smoke and heat of the cigarette will eventually make the papillae on the dorsal tongue dull, thereby killing the gustatory cells. these gustatory cells play a role in taste table 2. the correlation of confounding variables – zinc intake, hypertension, and smoking – on taste disorders variable taste disorders p-valueyes n = 50 no n = 50 zinc intake insufficiently 50 (100%) 44 (88%) 0.027b* sufficiently 0 (0%) 6 (12%) hypertension yes 27 (54%) 18 (36%) 0.070a no 23 (46%) 32 (64%) smoking yes 24 (48%) 8 (16%) 0.001a* no 26 (52%) 42 (84%) note: this analysis uses the achi-square test, bfisher’s exact test, * = significant (p < 0.05) table 3. the correlation between salivary volume and taste disorders with controlled confounding variables (zinc intake and smoking) variable multivariate logistic regression adjusted or (95% ci) p-value salivary volume < 1.45 ml/5 minutes 3.23 (1.25–8.37) 0.016* insufficient zinc intake 18.08 (0.88–369.64) 0.060 smoking 5.30 (1.86–15.11) 0.007* dependent variable: taste disturbance, * = significant (p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p31–34 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p31-34 34 sensitivity in humans because if their function is disrupted the result will be a decrease in taste sensitivity.21 in this study, smoking can be one of the causes of decreased zinc levels in saliva, because smoking can inhibit the process of zinc metabolism in the digestive tract. the results showed that 73% of study respondents had zinc salivary levels below 88 µg/l and as many as 32% of study respondents had a history of smoking. in addition, chéruel, et al. showed that there was a decrease in the threshold of taste stimulation between respondents who smoked and those who did not smoke.21 their study was conducted on smoking and non-smoking respondents, and the changes that occurred after respondents stopped smoking were observed, without measuring zinc levels. however, this study did not focus on respondents who smoked but rather on measuring their salivary zinc levels. based on the literature, continuous cigarette use can cause injury to the mucosal lining of the digestive tract as well as inflammation. histological observations of the digestive tract in experimental animals showed bleeding from the postcapillary veins that led to damaged tissue function. another form of damage caused by cigarette smoke occurred with ethanol-induced gastric damage that resulted in decreased prostaglandin e2, increased myeloperoxidase activity, and increased accumulation of neutrophils in the gastric mucosa.22 this study has several limitations, namely the effect of high respondent subjectivity. this study did not intervene with zinc supplementation so salivary zinc levels before and after supplementation could not be compared. this study did not analyse confounding factors for menopause in elderly women. thus, this study concludes that there is no correlation between salivary zinc levels and taste disorders in elderly patients. however, there is an association between salivary volume and taste disorders in elderly patients. this study also shows that among the three confounding variables (zinc intake, hypertension and smoking), smoking is highly correlated with taste disorders in elderly patients. it is necessary to do further research on taste disorders with zinc supplementation. acknowledgements the author would like to thank the babatan health centre, bandung city as a location for research; the integrated research laboratory of the faculty of dentistry, padjadjaran university, sekeloa; and the central laboratory of padjadjaran university, jatinangor for sample measurement. references 1. pusat data dan informasi kementerian kesehatan republik indonesia. analisis lansia di indonesia. buletin kemenkes. 2017; : 1–10. 2. razak pa, richard kmj, thankachan rp, hafiz kaa, kumar kn, sameer km. geriatric oral health: a review article. j int oral heal. 2014; 6(6): 110–6. 3. amarya s, singh k, sabharwal m. changes during aging and their association with malnutrition. j clin gerontol geriatr. 2015; 6(3): 78–84. 4. imoscopi a, inelmen em, sergi g, miotto f, manzato e. taste loss in the elderly: epidemiology, causes and consequences. aging clin exp res. 2012; 24(6): 570–9. 5. aliani m, udenigwe cc, girgih at, pownall tl, bugera jl, eskin mna. zinc deficiency and taste perception in the elderly. crit rev food sci nutr. 2013; 53(3): 245–50. 6. watanabe m, asatsuma m, ikui a, ikeda m, yamada y, nomura s, igarashi a. measurements of several metallic elements and matrix metalloproteinases (mmps) in saliva from patients with taste disorder. chem senses. 2005; 30(2): 121–5. 7. ismawati r, wirdjatmadi b, yoes priyatna d, mertaniasih nm. the effect of zinc, lysine, and vitamin a supplementation to increase cellular immune response of pulmonary tuberculosis patients. biochem physiol open access. 2015; s5: 5–7. 8. risso d, drayna d, morini g. alteration, reduction and taste loss: main causes and potential implications on dietary habits. nutrients. 2020; 12(11): 3284. 9. ambaldhage vk, puttabuddi jh, nunsavath pn, tummuru yr. taste disorders: a review. j indian acad oral med radiol. 2014; 26(1): 69–76. 10. kasuma n. buku fisiologi dan patologi saliva. padang: andalas university press; 2015. p. 1–26. 11. aeran h, seth j, saxena s, sharma g. taste perception a matter of sensation. int j oral heal dent. 2015; 1(2): 88–93. 12. selow mlc, lunelli f, vieira i, al e. analysis of zinc concentration in the saliva of individuals at different age ranges. j dent sci. 2016; 31(1): 12–5. 13. niklander s, veas l, barrera c, fuentes f, chiappini g, marshall m. risk factors, hyposalivation and impact of xerostomia on oral health-related quality of life. braz oral res. 2017; 31: e14. 14. villa a, connell cl, abati s. diagnosis and management of xerostomia and hyposalivation. ther clin risk manag. 2014; 11: 45–51. 15. takeuchi k, furuta m, takeshita t, shibata y, shimazaki y, akifusa s, ninomiya t, kiyohara y, yamashita y. risk factors for reduced salivary flow rate in a japanese population: the hisayama study. biomed res int. 2015; 2015: 1–7. 16. pedersen aml, sørensen ce, proctor gb, carpenter gh, ekström j. salivary secretion in health and disease. j oral rehabil. 2018; 45(9): 730–46. 17. sa sa no t, satoh-ku r iwa d a s, k a net a n, shoji n, k awa i m, uneyama h. incidence of taste disorder and umami taste disorder among the japanese elderly and youth. j nutr food sci. 2012; s10: 1–4. 18. hershkovich o, nagler rm. biochemical analysis of saliva and taste acuity evaluation in patients with burning mouth syndrome, xerostomia and/or gustatory disturbances. arch oral biol. 2004; 49(7): 515–22. 19. shatzman ar,henkin ri. gustin concentration changes relative to salivary zinc and taste in humans. proc natl acad sci usa. 1981; 78(6): 3867–71. 20. pushpass rg, daly b, kelly c, proctor g, carpenter gh. altered salivary flow, protein composition, and rheology following taste and trp stimulation in older adults. front physiol. 2019; 10(652): 1–11. 21. chéruel f, jarlier m, sancho-garnier h. effect of cigarette smoke on gustatory sensitivity, evaluation of the deficit and of the recovery time-course after smoking cessation. tob induc dis. 2017; 15(1): 1–8. 22. berkowitz l, schultz bm, salazar g, pardo-roa c, sebastian vp, alvarez-lobos mm, bueno sm. impact of cigarette smoking on the gastrointestinal tract inflammation: opposing effects in crohn’s disease and ulcerative colitis. front immunol. 2018; 9: 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p31–34 permatasari et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 31–34 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p31-34 174 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 174–180 case report management of a complete 180° rotation of bilateral maxillary canines putri intan sitasari1, niken merrystia2 and ida bagus narmada2 1department of orthodontics, faculty of dental medicine, universitas mahasaraswati, denpasar, bali, indonesia 2department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: dental anomalies usually lead to complicated decisions having to be made in terms of the orthodontic treatment of permanent dentition; tooth rotation is the most common of these irregularities. the prevalence rate of this phenomenon is 2.1–5.1% in patients who have not received orthodontic treatment. purpose: this case report aimed to manage the complete bilateral rotation of maxillary canines with couple force by using a nance appliance modification. case: a 17-year-old male patient came in wanting to straighten his teeth. he complained about his bilateral canines, which were not in a normal position. there was an impacted left maxillary second premolar and an ectopically erupted right maxillary first premolar. he also had protrusions in the upper and lower anterior teeth and crowding in the lower anterior teeth as well as upper and lower midline deviations. case management: a clinical examination showed a class i relationship between the dental and cephalometry measurements and highlighted a class i skeletal pattern. the upper right first premolar was extracted and the left second premolar had undergone an odontectomy to allay protrusion and correct crowding. bilaterally rotated upper canines were derotated using a modified nance appliance and an elastomeric chain with couple force. conclusion: the success of the orthodontic treatment was influenced by the specific nature of the patient’s dental and medical history, extraoral and intraoral examination, diagnosis and treatment planning, which was followed by a systematic approach to treatment. the nance appliance modification reduced the total treatment time by achieving controlled anchorage and derotation of the canines. keywords: canine rotation; couple force; derotation; nance appliance correspondence: ida bagus narmada, department of orthodontics, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo 47, surabaya 60132, indonesia. email: ida-b-n@fkg.unair.ac.id introduction dental anomalies usually mean complicated decisions with regard to the orthodontic treatment of permanent dentition have to be made.1 the aetiology of these dental anomalies is caused by genetic and environmental factors,2 and developmental versions of these irregularities are an essential part of dental morphologic variations. abnormalities in the tooth shape, tooth number and tooth structure result from disruption during the developmental stage of morphological differentiation, while abnormalities in the tooth position stem from developmental disruption in the eruption pattern of the permanent teeth. rotation, impaction and ectopic eruption are all related to irregularities in the tooth position.3 tooth rotation is intra-alveolar displacement in the mesiolingual or distobuccal direction of the tooth around a longitudinal axis of at least 20°, and the prevalence of tooth rotation is 2.1–5.1% in patients who have not received orthodontic treatment.3 rotation of permanent dentition can be caused by pre-eruptive and post-eruptive disturbances. furthermore, several factors relate to preeruptive disturbance, such as displaced and misaligned development of tooth buds due to pre-maxillary region injury in childhood, and pathological conditions that can interfere with tooth eruption like a cyst, odontogenic tumour or mesiodens (supernumerary tooth). elements that can cause post-eruptive disturbances are usually local/environmental, habitual or mechanical. although the aetiology of dental malpositioning is multifactorial, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 mailto:ida-b-n@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 175sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 environmental reasons that must be considered as a cause of tooth rotation include the presence of space, the tooth eruption pathway and functional effects that are influenced by the tongue and lips. rotations often occurs in relation to crowded teeth, but an excess number of rotations might also occur in instances that relate to the degree of space.4 gupta et al.3 categorised rotation into three groups: <45°, 45° to 90° and >90°. in their research, the majority of tooth rotation was between 45° and 90° (58%), followed by <45° rotations (31%) and then >90° rotation (11%). out of all of the instances of rotation, there were only two cases that presented with a complete 180° rotation (the buccal side is on the palatal side, and vice versa).4 it can therefore be interpreted that this case was rare. in this study, the patient had a complete 180° rotation of the bilateral maxillary canine and other anomalous conditions, such as an impacted maxillary second premolar on the left side and an ectopically erupted maxillary first premolar on the right side. the routine treatment of rotated teeth is to use fixed orthodontic and modified nance appliances; however, this case report aimed to manage the complete bilateral rotation of maxillary canines with couple force using a nance appliance modification. case a 17-year-old man came to the dental and oral hospital/ rumah sakit gigi dan mulut pendidikan (rsgm-p) at the orthodontics department, faculty of dental medicine, universitas airlangga, wanting to straighten his teeth because his canines were not in a normal position. the patient said that the posterior upper left tooth was never replaced after the primary tooth was extracted. the general condition of the patient was good and he had never had orthodontic treatment before. moreover, the patient wanted to be treated to improve the appearance of his smile, particularly with regard to the aesthetic of his teeth. an extraoral examination showed that he had a straight face profile, a medium face type, a mesocephalic head shape, competent lips, normal speech function and no bad habits (figure 1). meanwhile, an intraoral examination revealed normal oral mucosa, tongue and palate, with a mild caries frequency and good oral hygiene. the maxillary arch was a normal shape, but the mandibular arch was not typically formed. there was a protrusion in the anterior upper and lower teeth, crowding among the lower anterior teeth, an ectopically erupted #14, a large space between #24 and #26, bilateral rotations in #13 and #23 and a midline deviation of one mm to the right in the upper and one mm to the left in the lower. sagittally, the relation of the right canine was mesioclusional, the left canine was neutroclusional and the right and left molar were also neutroclusional. the overbite and overjet were found to measure one mm. at the same time, dental cast analysis indicated a discrepancy in the upper and lower arch of -8mm and the curve of spee was one mm (figure 2). figure 1. patient’s pre-treatment extraoral photographs. figure 2. patient’s pre-treatment intraoral photographs. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 176 sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 no pathological conditions were detected on the panoramic radiograph, yet there were several impacted teeth at #18, #25, #28, #38 and #48 (figure 3). cephalometric analysis indicated the patient had a straight profile with ∠ n a s i o n p o i n t a p o g o n i o n ( n a a p o g ) 3 º a n d ∠ frankfort horizontal-nasion-pogonion (fh-npog) 85º. the maxilla and mandible relation to the cranium base highlighted a skeletal class i relationship with ∠ sella-nasion-point a (sna) 84º, ∠ sella-nasion-point b (snb) 81º and ∠ point a-nasion-point b (anb) 3º; also, there was a wits appraisal of two mm (figure 4). the dental inclinations of the maxillary and mandibular incisors were protrusive, having the respective values of ∠ upper incisornasion-point a (i-na) 27º, ∠ lower incisor-nasion-point b (i-nb) 32º, ∠ inter incisal 125º, ∠ incisor mandibular plane angle (impa) 98º and ∠ frankfort mandibular incisive angle (fmia) 57º. meanwhile, use of rickett’s and steiner’s soft tissue and lip analyses showed that the lower lips were in front of the e-line and the s-line, which meant the patient had protrusive lower lips (table 1). case management the treatment objectives were to correct the maxillary bilateral rotation of the canines, deal with mandibular crowding, reduce upper and lower incisor protrusion and lower lip protrusion, correct midline deviation and maintain a class i relationship with regard to the canines and molars with an ideal arch form and attain a normal overjet and overbite. from the results of clinical examinations and diagnostic records, including a dental and orthodontic history, dental cast, intraoral and extraoral photographs and radiograph photos, the clinician planned to use fixed orthodontic appliances, which would be accompanied by the extraction of #14, #35 and #45 and an odontectomy on #25. there would also be a retention phase using removable retainers. the first step of the treatment was the utilisation of bonding brackets using 0.022 slots of mbt, a pre-adjusted edgewise appliance, after the extraction of #14, #35 and #45, and an odontectomy was completed on #25. the molar bands figure 3. patient’s pre-treatment panoramic and occlusal radiography. figure 4. patient’s pre-treatment cephalogram. table 1. pre and post-treatment cephalogram measurements measurement surabaya mean subject pre post ∠ na-apog 84.5 º 85º 85º ∠ fh-npog 6.1 º 3º 2º ∠ sna 84.3º 84º 83º ∠ snb 81.4º 81º 82º ∠ anb 3º 3º 2º ∠ op sn 15–32º 18º 17º ∠ mp sn 20–40º 32º 32º ∠ i-na 26º 27º 20º i-na (mm) 6.3mm 10mm 5mm ∠ i-nb 29º 32º 25º i-nb (mm) 7.9mm 8mm 5mm nasolabial angle 110–120º 92º 100º upper lips: e line -2–3mm +1mm -1mm lower lips: e line -1–2mm +2 mm 0mm upper lips: s line 0mm +2mm +1mm lower lips: s line 0mm +3mm 0mm dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 177sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 were cemented to the upper and lower first molar; those in the upper jaw have a slot for inserting the nance appliance. levelling and aligning begins with 0.012 nickel-titanium (niti), followed by 0.014, 0.016, 0.016 x 0.016 and 0.016 x 0.022 niti for the upper and lower arches. retraction of #24, #34 and #44 was also undertaken using a 0.016 x 0.022 stainless steel (ss) wire with an omega stopper in the mesial of #26, #36 and #46 along with an elastomeric chain. the next step was the derotation of #13 and #23. apart from being a conventional anchorage system, the nance appliance has been modified by adding a hook for the derotation of the canines. the hooks were soldered on the nance appliance around the palatal area of the canines (figure 5), and derotation took place using elastomeric chains with couple force as well as the joining together of the premolar and both molars using a ligature wire to reinforce the posterior anchorage. these anchor units would be used to hold the posterior teeth during derotation to prevent loss of anchorage. the derotation of t he canine was done via the employment of elastic chains from the palatal surface, which were then attached to the hook on the molar band and from the buccal surface to the hook of the nance appliance (using lingual buttons). the derotation had to be achieved on both the left and right sides (figure 6). the retraction of the anterior segment began with canine retraction, which first used 0.016 x 0.022 ss wire with an omega stopper in the mesial of #16, #26, #36 and #46; subsequently, a power chain was utilised. after the canine retraction was done, the upper and lower anterior retraction was started using a t-loop in 0.017 x 0.025 ss. then, the mesialization of #16, #26, #36 and #46 took place using a power chain. after the overbite and overjet were corrected, a class i canine and molar relationship had been attained. almost two years and two months later, all the fixed appliances were removed, and a wraparound retainer was chosen for both arches. discussion during the diagnosis of malocclusion, dental anomalies comprise 10% to 20% of cases in orthodontic patients and usually involve complicated decisions having to be made in terms of the orthodontic treatment of permanent dentition.1 the orthodontist is perhaps the first individual who will detect dental anomalies and diagnose malocclusion in the patient. furthermore, they will carry out an examination to detect any additional defects in these patients to provide the best treatment.2 dental anomalies can relate to shape, number, structure and position.3 irregularities in dental position occur when the tooth moves from its development area lead-up to the functional position,5 and such issues in relation to tooth position could be down to tooth rotation, impaction and ectopic eruption. in their study, gupta et al.3 concluded that anomalies in the dental position were the most common occurrence among this group. nevertheless, the aetiology of rotation in permanent dentition is multifactorial and can be caused by pre-eruptive and post-eruptive disruptions. in addition, several elements can cause further changes figure 5. nance appliance modification with hook around the palatal area of the canine. figure 6. patient’s cast during treatment. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 178 sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 figure 7. patient’s extraoral and intraoral photographs post treatment. figure 8. patient’s post-treatment panoramic cephalogram, which was superimposed pre and post-treatment. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 179sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 in post-eruptive tooth angulation, such as trauma, extraction, hypodontia, ectopic eruption and periodontitis in neighbouring teeth. the prevalence of tooth rotation is quite high in the population, and orthodontists should be aware of indications of dental anomalies, so they can determine the appropriate treatment.6 tooth rotation is an intra-alveolar displacement in a mesiolingual or distobuccal direction of the tooth around a longitudinal axis.4 gupta et al.3 categorised the rotation into three groups: <45°, 45° to 90° and >90°. in this case, a patient with complete (180°) rotations of the bilateral maxillary canines was treated with a modified nance appliance. the modification involved adding a hook around the palatal area of the canine, which served the purpose of simultaneously controlling the posterior anchorage and derotation of the severely rotated teeth. since the derotation that needed to be achieved was bilateral, the hook was soldered on both the right and left side. in relation to this, the preferred method when derotating teeth is generally to use couple force on the adjacent teeth. iatrogenic effects may occur if there was poor control of the adjacent teeth; thus, they may delay further treatment. consequently, in some cases where proper anchoring was required, controlling the anchorage can help to avoid unwanted tooth movement.7 in this case, in addition to using a molar band that is connected to the nance appliance, a clinician joined the premolar and both molars using a ligature wire to strengthen the posterior anchorage. a couple is a form of moment that is created by two forces in oppositional directions, which moves on an axis with equal magnitudes. this force system has the net potential to translate the teeth on which the action is nil, meaning only pure rotation occurs; this is due to the forces having the same magnitude but oppositional directions. the sum of the moments created by the two forces is called a moment couple, and the magnitude of the force and the distance between the two forces can affect its magnitude. the moment created was additive if there were two forces acting on the couple system in oppositional directions from a centre of resistance (cres), whereas the moment created was subtractive if two forces were on the same side as the cres.8 in different types of malocclusions, extraction of certain teeth may be required. oral hygiene, a high caries rate, tooth quality, attitude to treatment and the general health of the patient will influence the decision to have a tooth extracted. there are several conditions that cause teeth to be extracted during orthodontic treatment, such as crowding (increased tooth size associated with the arch size), hypodontia (need for extraction if set to close the space), supernumeraries, malformed teeth, caries teeth, open bite cases, increased overjet, tooth impaction, correction of the buccal segment, camouflage orthodontic treatment, periodontal disease involving the teeth, cleft lip and palate and orthognathic surgery.9 one of the main reasons for choosing extraction or non-extraction is an arch discrepancy.10 in this case, the patient had a significant discrepancy of eight mm in relation to the upper and lower arch. meanwhile, in instances of crowding teeth with a discrepancy of five to nine mm, arch expansion can be performed after carrying out a comprehensive diagnosis and treatment planning takes place. permanent tooth extraction is frequently performed in most of these cases to maintain facial aesthetics and the integrity of the soft tissue profile.11 since this patient had several problems, extracting the four premolars was chosen as the most suitable treatment. if there is moderate to severe crowding in the anterior segment, it is usually necessary to extract all of the first premolars to create space. however, in this case, a clinician decided to extract the upper first premolar on the right side and perform an odontectomy on the second premolar on the left side. the extraction and odontectomy were undertaken because of the poor prognosis of the premolar on each side, which would have prolonged the treatment period if the teeth were retained. the mandibular second premolars on the right and left side were the teeth selected for extraction, because their removal adjusted the upper jaw and would help correct the relation to a class i standard. there are two main retraction techniques to close extraction spaces: en masse retraction (er) and two-step retraction (tsr). with regard to the former, the incisors and canines are retracted together in just one step; meanwhile, during the latter, the first step is to retract the canines until they are in contact with the premolar. the canines will then be joined to the premolar and first and second molars as posterior anchorage units. in the second step, these units are used to hold the posterior region while retracting the incisors.12 in this case, we used the nance appliance and two-step retraction to lower the risk of anchorage loss. after the aforementioned orthodontic treatment, class i canine and molar relationships had been achieved in both sides (figure 7). the inter-incisal angles were normalised after the midline deviation and inclination of the upper and lower anterior teeth were corrected; the soft tissue profile has also been corrected in the patient. the severe rotations of the canines and crowding were corrected; thus, one of the treatment goals was satisfied (figure 8). in this instance, the clinician had suggested an odontectomy for the third molars, but the patient refused to go through this again because they had previously had an extraction and an odontectomy. the total amount of time taken to complete this orthodontic treatment was 26 months. from this case, we can conclude that successful orthodontic treatment can be influenced by specific dental and medical histories, extraoral and intraoral examinations, diagnoses and treatment planning, which are followed by a systematic approach to being treated. the selection of an appropriate appliance and an assessment are needed for each case; for example, a bracket prescription and the type of wire as well as techniques for levelling-aligning, derotation, space closure and anchorage all need to be chosen, and they must be prepared to obtain optimal orthodontic treatment results. here, the nance appliance modification helped to reduce the total treatment time by achieving controlled anchorage and derotation of the canines. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 180 sitasari et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 174–180 acknowledgements our gratitude goes primarily to the patient and to rsgm-p, faculty of dental medicine, universitas airlangga for their willingness to present us with photos of the patient and their treatment. references 1. massaro c, janson g, yatabe m, calil lr, oliveira tm, gariba d. dental anomaly pattern and multiple ectopic teeth. am j orthod dentofacial orthop. 2020; 158(1): 102-13. 2. cakan dg, ulkur f, taner t. the genetic basis of dental anomalies and its relation to orthodontics. eur j dent. 2013; 7(1): 143-7. 3. gupta sk, saxena p, jain s, jain d. prevalence and distribution of selected developmental dental anomalies in an indian population. j oral sci. 2011; 53(2): 231-8. 4. parisay i, boskabady m, abdollahi m, sufiani m. treatment of severe rotations of maxillary central incisors with whip appliance: report of three cases. dent res j. 2014; 11(1): 133-9. 5. kaur p. hypodontia, microdontia and tooth rotation: a rare clinical triad. international journal of dental research. 2016; 4(2): 57-61. 6. vani nv, saleh sm, tubaigy fm, idris am. prevalence of developmental dental anomalies among adult population of jazan, saudi arabia. 2016; 7: 29-33. 7. hasan sh, kolemen a, elkolaly m, marya a, gujjar s, venugopal a. tad’s for the derotation of 90° rotated maxillary bicuspids. hindawi case reports in dentistry. 2021; 1-8. 8. upadhyay m, nanda r. biomechanics principles in mini-implant driven orthodontics. greece: vasiliadis books; 2020. p 1-20. 9. al-ani mh, mageet ao. extraction planning in orthodontics. j contemp dent pract. 2018; 19(5): 619-23. 10. khanum a, prashantha gs, mathew s, naidu m, kumar a. extraction vs non extraction controversy: a review. j dent orofac res. 2018; 14(1): 41-8. 11. sharma ns, shrivastav ss, hazarey pv. mastering interproximal stripping: with innovations in slenderization. int j clin pediatr dent. 2012; 5(2): 163-6. 12. schneider pp, gandini-júnior lg, monini adc, pinto ads, k im k b. compa r ison of anter ior retraction and anchorage control between en masse retraction and two-step retraction: a randomized prospective clinical trial. angle orthod. 2019; 89(2): 190-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p174–180 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p174-180 p-issn: 1978-3728 e-issn: 2442-9740 volume 49, number 2, june 2016 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2016 january 2nd – december 31st, 2016 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga) editorial boards: roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); harry huiz peeters (laser research center, bandung, indonesia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); fajar hamonangan nasution (department of orthodontics faculty of dentistry, universitas trisakti, indonesia); pinandi sri pudyani (department of orthodontics faculty of dentistry, universitas gadjah mada, indonesia); boedi oetomo roeslan (department of biochemistry faculty of dentistry, universitas trisakti); rahmi amtha (department of oral medicine faculty of dentistry, universitas trisakti, indonesia); anita yuliati (department of dental material faculty of dental medicine, universitas airlangga, indonesia); darmawan setijanto (department of dental public health faculty of dental medicine, universitas airlangga, indonesia); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); boy muchlis bachtiar (department of oral biology faculty of dentistry, universitas indonesia, indonesia) managing editors: priyawan rachmadi (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); rostiny (department of prosthodontic dentistry, faculty of dental medicine, universitas airlangga, indonesia); markus budi rahardjo (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty vocational, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry faculty of dental medicine, universitas airlangga, indonesia); an’nisaa chusida (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia) assistant editors saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia) peer-reviewers harmas yazid yusuf (department of oral and maxillofacial surgery, faculty of dentistry, universitas padjadjaran); mei syafriadi (department of oral pathology, faculty of dentistry, universitas jember, indonesia); eky s. soeria soemantri (department of orthodontics dentistry, faculty of dentistry, universitas padjadjaran, indonesia); achmad gunadi (department of prosthodontics, faculty of dentistry, universitas jember, indonesia); widowati siswomihardjo (department of dental biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); erik idrus (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r.m. coen pramono d (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); i gusti aju wahju ardani (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); kus harijanti (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia) administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 641/11.16/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 49, number 2, june 2016 p-issn: 1978-3728 e-issn: 2442-9740 1. the correlation between rood and shehab’s radiographic features and the incidence of inferior alveolar nerve paraesthesia following odontectomy of lower third molars david b. kamadjaja, djodi asmara, and gita khairana ............................................................ 59–62 2. histological changes during orthodontic tooth movement due to hyperbaric oxygen therapy arya brahmanta, soetjipto, and ib narmada ............................................................................... 63–66 3. differences of streptococcus mutans adhesion between artificial mouth systems: a dinamic and static methods aryan morita, h. dedy kusuma yulianto, susmira d. kusdina, and nunuk purwanti .......... 67–70 4. synergistic effect of the combination of cinnamomum burmanii, vigna unguiculata, and papain exracts derived from carica papaya latex against c. albicans biofilms degradation muhammad luthfi, indah listiana kriswandini, and fitriah hasan zaba .............................. 71–75 5. correlation between estrogen and alkaline phosphatase expression in osteoporotic rat model sherman salim ................................................................................................................................ 76–80 6. the differences of effectiveness of β -1,3-glukanase vigna unguiculata and papain carica papaya enzymes in hydrolysis of denture plaque retno indrawati, muhammad lutfi, and erina fatmala yuli andari ...................................... 81–86 7. transforming growth factor beta 1 expression and inflammatory cells in tooth extraction socket after x-ray irradiation ramadhan hardani putra, eha renwi astuti, and rini devijanti .............................................. 87–92 8. antibacterial effects of pluchea indica less leaf extract on e. faecalis and fusobacterium nucleatum (in vitro) agni febrina pargaputri, elly munadziroh, and retno indrawati ............................................. 93–98 9. antibacterial ability of arabica (coffea arabica) and robusta (coffea canephora) coffee extract on lactobacillus acidophilus willy wijaya, rini devijanti ridwan, and hendrik setia budi .................................................. 99–103 10. antibacterial effect of 70% ethanol and water extract of cacao beans (theobroma cacao l.) on aggregatibacter actinomycetemcomitans ayu rafania atikah, hendrik setia budi, and tuti kusumaningsih ......................................... 104–109 11. changes in taste sensation of sour, salty, sweet, bitter, umami, and spicy, as well as levels of malondialdehyde serum in radiographers agniz nur aulia, jenny sunariani, and ester arijani r .............................................................. 110–114 196 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 196–200 research report acceleration of post-tooth extraction socket healing after continuous aerobic and anaerobic physical exercise in wistar rats (rattus norvegicus) aqsa sjuhada oki,1 moch febi alviansyah,1 christian khoswanto,1 retno pudji rahayu2 and muhammad luthfi1 1department of oral biology 2department of oral and maxillofacial pathology faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: physical exercise has been proven to accelerate wound healing. physical training itself consists of aerobic (continuous training) and anaerobic (interval training) exercise. the effectiveness of continuous physical exercise on post-tooth extraction wound healing is the focus of this study. purpose: this study aims to investigate the differences in post-tooth extraction wound healing in wistar rats (rattus norvegicus) after aerobic and anaerobic exercise based on the number of fibroblasts and neovascularisation. methods: wistar rats were divided into three groups: the control group (k1); k2 undertook continuous aerobic exercise, swimming at 50% maximum swimming capacity (msc) with an additional 3% bodyweight load; k3 undertook anaerobic continuous exercise, swimming at 65% msc with a 6% load. the rats swam three times per week for six weeks. the number of fibroblasts and neovascularisation were examined three days after tooth extraction. data was analysed using the one-way analysis of variance (anova) and least significant difference (lsd) tests (p<0.05). results: there was a significant difference in the number of fibroblasts between the k2 and k3 groups. there was no significant difference between k2 and k3 in the amount of neovascularisation. conclusion: there were differences in the number of fibroblasts but not neovascularisation after tooth extraction in wistar rats given aerobic and anaerobic continuous training. keywords: continuous aerobic physical exercise; continuous anaerobic physical exercise; fibroblasts; neovascularisation; post-tooth extraction wound healing correspondence: aqsa sjuhada oki, department of oral biology, faculty of dental medicine, universitas airlangga, jl mayjen prof dr moestopo no. 47 surabaya 60132 indonesia. email: aqsa@fkg.unair.ac.id introduction tooth extraction is an irreversible surgical procedure that sometimes causes complications if it is not handled properly.1 dental extraction complications can be divided into intraoperative complications, complications shortly after extraction and complications a long time after extraction. these occur because of microorganism infections, trauma, drugs or smoking. there have been efforts to reduce complications and accelerate wound healing using antibiotics, antifibrinolytics and topical drug applications.2 however, the administration of drugs can cause further problems, such as allergies, resistance and systemic complications. antibiotics can be used as life-saving drugs and treatments to prevent infection, but they are also a predisposing factor for superinfection due to resistance and toxicity.3,4 the effect of antibiotic resistance is called the eagle effect, which increases the minimum bactericidal concentration (mbc) of antibiotics so that the dose needed to kill the bacteria must be increased.5 efforts to improve wound healing with drugs are considered to have shortcomings, so an alternative form of therapy with exercise has been studied, but this is still not the primary choice. regular physical exercise produces positive health effects, such as reducing various cardiovascular diseases, disorders of metabolic syndrome and osteoporosis.6 it can supply nutrients and oxygen, which can accelerate the wound healing process, and it can dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p196–200 mailto:aqsa@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p196-200 197oki et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 196–200 increase the secretion of healing factors to help the wound healing process.7 previous studies have shown that physical exercise or sport is proven to heal wounds significantly in people who participate compared to those who do not.8 one study explored the effect of continuous moderate-intensity physical exercise on the number of fibroblasts and neovascular disease in wistar rat extract scars. the study found that participating in exercise increased the number of fibroblasts and neovascularisation in tooth extraction scars.9 physical exercise even accelerated wound healing in the inflammatory phase after tooth extraction. the study showed that physical exercise is one of the factors that can accelerate the wound healing process.10 the proliferation phase occurs at the end of the inflammatory phase for up to 14–21 days after the injury. this phase’s main objective is to repair injured tissue by fibroplasia, which includes wound closure, angiogenesis, re-epithelialisation and fibroplasia. fibroblasts play a huge role in the repair process responsible for product preparation and protein structure that is used during the tissue reconstruction process.11 the response made by fibroblast cells in fibroplasia is proliferation, migration, formation of the extracellular matrix and wound contraction; angiogenesis or neovascularisation also occur in this stage. angiogenesis is the process of forming new capillaries in a wound, and it is significant in the proliferation stage of the wound healing process because it generates new blood vessels to access nutrients, oxygen and other components in the wound healing process.12 at this stage, extracellular matrix biosynthesis occurs, which is the temporary matrix formed mainly from fibrin and fibronectin tissue and replaced by collagen matrix enriched in proteoglycans, glycosaminoglycans and non-collagen glycoproteins, which, in turn, causes the restoration of proper tissue structure and function. cells that play an essential role in ecm biosynthesis are fibroblasts that secrete extracellular matrix products and components and the formation of granulation tissue. first, components and growth factors previously secreted by macrophages will stimulate the formation of a scaffold in the form of a granulation tissue matrix, such as collagenase, fibronectin and extracellular matrix components. the scaffold formed by these components will function as a facility for the collagen fibrogenesis process. furthermore, within two weeks after injury, collagen increases and fibroblasts decrease due to apoptosis.13 based on the above explanation, this study aims to investigate the differences in the effectiveness of continuous aerobic and anaerobic exercise on the acceleration of wound healing after tooth extraction in wistar rats (rattus norvegicus) by observing the number of fibroblasts and neovascularisation. materials and methods this research was conducted after obtaining an ethical eligibility certification issued by the research ethics commission under 190/hrecc.fodm/iv/2019. this study involves a post-test only control group with in vivo true experimental design. thirty wistar rats (r. norvegicus) were used with the criteria being 8–12 weeks-old, male and 250–300 grams in body weight. the animals were kept in a plastic cage during the day with enough air and light for acclimatisation. the cages were divided by groups and labelled to distinguish each cage. after the acclimatisation process, each rat’s body weight was measured to determine the load given. the rats were divided into three groups: the control group (k1) did not perform any physical activity; k2 participated in continuous aerobic exercise (swimming) with an additional load of 3% of the rats’ body weight; k3 participated in continuous anaerobic exercise (swimming) with an additional load of 6%. the load was in the form of a paper clip tied to a rope one-third of the way from the base of the rat’s tail. the control group was not given a load. after being given a load, the rats’ maximum swimming capacity (msc) was calculated to distinguish between the types of physical activity in each treatment group. this was obtained using the rats’ weights and the maximum time they could swim for (until they started to sink, marked by the emergence of air bubbles) or when they stopped swimming. the msc calculation was not carried out for the control group because the rats were placed in containers of water that came up to their feet so that the body temperatures of the control group and treatment groups were equalised. k2 swam at 50% msc with a load of 3% body weight, and k3 swam at 65% msc with an additional load of 6% body weight.14,15 all three groups were treated three times a week for six weeks. in the seventh week, each rat was anaesthetised with a ketamine injection to extract the mandibular left incisor. this was performed with modified pliers then irrigated with distilled water to remove debris and the remnants of the extraction. on the third day after the extraction, euthanasia was carried out on all rats so their mandibles could be removed. histology tissue was taken to check the number of fibroblasts and neovascularisation in the posttooth extraction socket.10 the tissue was extracted using a microtome with a thickness of 4–6 μm then it was attached to a glass object. xylol was used for deparaffination and 90% alcohol for rehydration. hematoxylin-eosin (he) staining (merck chemical, darmstadt, germany) was used, which, in acidic conditions, will attract alkaline substances/solutions so they will turn blue, and the cytoplasm is alkaline, which will attract acidic substances/solutions so they turn red. to colour the nucleus and cytoplasm, 0.6% hcl was stained for differentiation using 0.5% lithium carbonate to give the nucleus a blue colour, and eosin stain was used to give the cytoplasm a red colour. the process ended with dehydration and mounting. preparations were read by counting the number of fibroblast cells and neovascularisation in five fields using a light microscope with 400x magnification. there were three observers.15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p196–200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p196-200 198 oki et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 196–200 after histological readings to find the number of fibroblasts and neovascularisation, data was collected for statistical tests using statistical package of social science (spss) version 16.0 for windows (ibm, new york, usa) with normality and homogeneity tests conducted in advance to determine the different tests performed. the normality test was conducted using the shapiro-wilk’s test and levene’s test was used to test homogeneity (p>0.05); subsequently, a parametric comparison test was performed with one-way anova along with a non-parametric comparison using the kruskal-wallis test (p<0.05). the lsd posthoc test was used to analyse the difference between groups after the anova test was performed (p<0.05). results the observation of fibroblast cells in the mandibular incisor socket of the wistar rat can be seen in figure 1. the average results of the number of fibroblasts in the wound healing process after the extraction of the wistar rat tooth and the results of normality and homogeneity tests are available in table 1. the highest mean value of the number of fibroblasts was found in the anaerobic continuous training group (k3), while the lowest fibroblast mean values were found in the control group that did not swim (k1). in the normality test results using the shapiro-wilk’s test, all groups had normal distribution values (p> 0.05), whereas the homogeneity test results using levene’s test had a significance of 0.101, which provides homogeneous data. table 2 displays the results from the anova test, which obtained a significance value of 0.001 (p<0.05); this indicates a significant difference between the groups. the results of the lsd posthoc tests, in table 3, shows significant differences between all three groups (p<0.05). the observation of neovascularisation in the mandibular incisor socket of the wistar rat can be seen in figure 2. a b c figure 1. fibroblast in the socket of a maxillary incisor tooth extraction with he staining, indicated by the yellow arrows. groups k1 (a), k2 (b) and k3 (c). a b c table 1. the mean, normality test and homogeneity test of the number of fibroblasts in each group groups mean ± sd normality test homogeneity test k1 15.00 ± 0.8165 0.144* 0.101*k2 17.86 ± 1.9518 0.200* k3 20.71 ± 3.3523 0.119* *significant at p>0.05 table 2. overall difference test of fibroblast groups one-way anova p-value 0.001* α 0.05 *significant at p<0.05 table 3. difference test between fibroblast groups using lsd posthoc test groups k k1 k2 k1 k2 0.031* k3 0.000* 0.031* *significant at p<0.05 figure 2. neovascularisation in the socket of a mandibular incisor tooth extraction with he staining, indicated by the yellow arrows. groups k1 (a), k2 (b) and k3 (c). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p196–200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p196-200 199oki et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 196–200 table 4 shows the neovascularisation mean values along with the results of the normality and homogeneity tests. in the table, the highest neovascularisation mean is in the continuous anaerobic exercise group (k3), and the lowest average is in the control group (k1). the normality test for each group shows that the distribution data is normal because p> 0.05, whereas the homogeneity test results show 0.335, which means homogeneous data continued with the one-way anova parametric test. table 5 shows that the different test results for the whole neovascularisation group gave a result of 0.098 (p>0.05), which means there were no significant differences between the groups on neovascularisation variables. discussion the wound healing process has several healing phases, including haemostasis, inflammation, proliferation and remodelling. as one of the factors that influences wound healing is oxygen, participating in physical exercise is expected to increase tissue oxygen consumption (vo2 max) so that the wound healing process is faster due to the stimulation of cells. cells that play a role in wound healing include pmn cells, macrophages, fibroblasts and neovascularisation.16 as experimental animals, wistar rats have several advantages: they are cheap, easy to obtain, breed and keep, and they have a physiological body that is almost the same as humans. during the experiment, several rats became sick and died, so they had to be removed from the study. the swim test was used because the research tools and materials could be easily prepared and have been proven by other researchers to obtain reasonably good results.17 the groups for aerobic and anaerobic continuous exercise training were determined by calculating the results of the msc and body weight of the experimental animals. aerobic exercise intensity was 50% of the msc and a weighting of 3% (medium intensity), while 65% of the msc and a load of 6% were chosen as anaerobic physical exercise (high intensity). an obstacle in determining anaerobic physical exercise occurred because all of the literature suggests that methods of high-intensity anaerobic exercise involve interval training. however, the focus of this study was on continuous training. therefore, as a preliminary study, we experimented with continuous anaerobic exercise at 65% msc and a 7% load. however, several wistar rats experienced fatigue and then death, so the researchers lowered the msc and load without reducing the essence of the high-intensity physical exercise.18,19 wound healing occurs in the proliferation phase, after haemostasis and inflammation but before remodelling. in the proliferation phase, several vital processes are related to studying the number of fibroblasts and neovascularisation. macrophage cells found in the inflammation phase secrete matrices and growth factors to stimulate fibroplasia and angiogenesis. these include metalloproteinase, plateletderived growth factor (pdgf), fibroblast growth factor (fgf), epidermal growth factor (egf), transforming growth factor-beta and alpha (tgf-β/α) and vascular endothelial growth factor (vegf). the process of angiogenesis and the proliferation of fibroblasts occur in tandem. when a wound occurs, branches of capillary blood vessels form around the edge resulting in bleeding for blood clot formation. the proliferation phase occurs after the fibroplasia process.12 fibroplasia is the process of stimulating fibroblast cells to actively proliferate, migrate and form an extracellular matrix (ecm). the matrix will function as a scaffold for the next wound healing process. after fibroplasia comes angiogenesis, which is the process of forming neovascularisation. this process restores blood circulation to the injured area and prevents the development of necrotic tissue. the process is stimulated by factors and growth components, such as basic fgf, tgf-β, tumour necrosis factor-α (tnf-α), vegf, angiogenin and angiotrofin. the cells that perform neovascularisation are stimulated and migrate to the temporary matrix (scaffold) that has been formed by fibroblast cells. subsequently, cells develop and form a new network of blood vessels into tubular structures. the processes of fibroplasia and angiogenesis go hand-in-hand and synergise to form a layer of collagen and epithelium, which is a sign of the final phase of wound healing or remodelling.20,21 this statement supports the proven research results on fibroblasts in the treatment group rather than the control group. the process of fibroplasia in wound tissue after tooth extraction in wistar rats is mediated by fibroblast cells stimulating the formation of the extracellular matrix so that fibroblasts can be seen in histological preparations. in this process, angiogenesis will be followed by neovascularisation. however, on the histopathological readings, the neovascularisation results were insignificant. this is likely to occur because the angiogenesis process occurs on the second and third day when the fibroplasia process has already taken place.22 another factor affecting the results is the determination of the training intensity method, which has almost the table 4. the mean and standard deviation of neovascularisation amounts groups mean ± sd normality test homogeneity test k1 8.57 ± 1.1339 0.262* 0.335*k2 9.28 ± 1.7043 0.140* k3 10.71 ± 2.2887 0.518* *significant at p>0.05 table 5. overall difference test of the neovascularisation groups one-way anova p-value 0.098 α 0.05 p-value: one-way anova significance value of neovascularisation variable; α: significance value of the different tests. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p196–200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p196-200 200 oki et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 196–200 same effect. in the continuous anaerobic exercise group, researchers have not used a standard method for determining the magnitude of the msc and the load that is given, so the results can be biased if there is no standardisation. additionally, it proves that interval-type training is better than continuous training because there is a resting phase that improves the effectiveness of the exercise.20 nonstandardised tooth extraction techniques can also affect the results due to the occurrence of root fractures and bleeding during high extractions, which will disrupt the wound healing process.15 theoretically, physical exercise will affect the wound healing process due to the appearance of free radicals in the body, which will disturb or delay wound healing. this happens if the period between the process of injury and participation in physical activity is close together, or even physical activity is only carried out after the injury. the healing process will be disrupted due to a lack of oxygen to the wound tissue and the build-up of lactic acid during sports activities. at a molecular level, the build-up of lactic acid causes reactive oxidative stress (ros) and free radicals and inhibits scaffold formation in fibroplasia.9 in this study, physical exercise was performed before tooth extraction in wistar rats. the training involved either continuous aerobic training or continuous anaerobic training. the results of this study indicated a higher number of fibroblasts and neovascularisation in the anaerobic training group (k3) because the heart and body adapt after 7–10 days after exercise by thickening the heart muscle and increasing lung capacity.21 at the beginning of exercise (<7 days), lactic acid increases and vo2 max decreases, which is proven to be significant, and this was demonstrated at the highest level in the anaerobic exercise group.22 anaerobically, vo2 max increases and the heart supplies blood to the body more efficiently and effectively due to the body’s adaptation processes.23 these conditions accelerate the wound healing process. statements and research by flora et al. and shi et al. support the results of this study, which showed the number of fibroblasts in the continuous anaerobic exercise group (k3) was higher than the continuous aerobic exercise group (k2).22,24 based on the results of this study, we concluded that anaerobic exercise provides a wound-healing acceleration effect on increasing the number of fibroblasts compared to aerobic exercise. acknowledgements we are particularly grateful to the head of the department of oral biology, faculty of dental medicine, universitas airlangga for technical and organisational support. references 1. fakhrurrazi, hakim rf, ulfa l. the differences blood glucose levels at random before and after tooth extraction of the patients at dental installation rsudza banda aceh. cakradonya dent j. 2017; 9(2): 96–100. 2. preetha s. an overview of dry socket and its management. iosr j dent med sci. 2014; 13(5): 32–5. 3. wright j, paauw ds. complications of antibiotic therapy. med clin north am. 2013; 97(4): 667–79. 4. blumenthal kg, peter jg, trubiano ja, phillips ej. antibiotic allergy. lancet. 2019; 393(10167): 183–98. 5. prasetyoputri a, jarrad am, cooper ma, blaskovich mat. the eagle effect and antibiotic-induced persistence: two sides of the same coin? trends microbiol. 2019; 27(4): 339–54. 6. harun l. perbandingan kadar interleukin-6 dan jumlah limfosit setelah latihan aerobik ringan dan sedang pada remaja. heal j. 2018; 1(2): 64–8. 7. keylock kt, vieira vj, wallig ma, dipietro la, schrementi m, woods ja. exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice. am j physiol regul integr comp physiol. 2008; 294(1): r179–84. 8. pence bd, woods ja. exercise, obesity, and cutaneous wound healing: evidence from rodent and human studies. adv wound care. 2014; 3(1): 71–9. 9. oki as, bimarahmanda me, rahardjo mb. increased number of fibroblasts and neovascularization after tooth extraction in wistar rats with moderate-intensity continuous exercise. j int dent med res. 2018; 11(3): 840–5. 10. oki as, amalia n, tantiana. wound healing acceleration in inflammation phase of post-tooth extraction after aerobic and anaerobic exercise. sci sport. 2020; 35(3): 168.e1-168.e6. 11. gonzalez acdo, andrade zda, costa tf, medrado arap. wound healing a literature review. an bras dermatol. 2016; 91(5): 614–20. 12. olczyk p, mencner ł, komosinska-vassev k. the role of the extracellular matrix components in cutaneous wound healing. biomed res int. 2014; 2014: 747584. 13. ercan e. the effect of platelet-rich fibrin and titanium prepared platelet-rich fibrin on early soft tissue healing of extraction sites. cumhur dent j. 2018; 21(4): 304–10. 14. way kl, sultana rn, sabag a, baker mk, johnson na. the effect of high intensity interval training versus moderate intensity continuous training on arterial stiffness and 24 h blood pressure responses: a systematic review and meta-analysis. j sci med sport. 2019; 22(4): 385–91. 15. khoswanto c. a new technique for research on wound healing through extraction of mandibular lower incisors in wistar rats. eur j dent. 2019; 13(2): 235–7. 16. oki as, farhana n, yuliati. the effect of aerobic and anaerobic interval exercise on the proliferation phase of wound healing in tooth extraction of rattus novergicus. acta med philipp. 2019; 53(5): 417–22. 17. setyadewi w, oki as, sunariani j. moderate intensity physical exercise effect on pmn and macrophage expression in rattus norvegicus post tooth extraction. j int dent med res. 2017; 10(2): 364–7. 18. goh j, ladiges wc. exercise enhances wound healing and prevents cancer progression during aging by targeting macrophage polarity. mech ageing dev. 2014; 139(1): 41–8. 19. rashidi m, salehian o, vaezi g. the effect of high intensity anaerobic training on the blood lactate levels after active recovery. eur j exp biol. 2013; 3(6): 346–50. 20. irmawati a, giffari fz, oki as. the effect of moderate exercise on vascular endothelial growth factor expression during tooth socket wound healing after tooth extraction. j postgrad med inst. 2018; 32(1): 19–23. 21. ding j, tredget ee. the role of chemokines in fibrotic wound healing. adv wound care. 2015; 4(11): 673–86. 22. shi m, wang x, yamanaka t, ogita f, nakatani k, takeuchi t. effects of anaerobic exercise and aerobic exercise on biomarkers of oxidative stress. environ health prev med. 2007; 12(5): 202–8. 23. tatullo m. the regenerative dentistry: current approaches and future insights. cumhur dent j. 2020; 23(1): 1–3. 24. flora r. pengaruh latihan fisik anaerobik terhadap kadar laktat plasma dan kadar laktat jaringan otot jantung tikus wistar. biomed j indones. 2015; 1(1): 40–2. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p196–200 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p196-200 68 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 68–73 original article bone remodeling using a three-dimensional chitosan hydroxyapatite scaffold seeded with hypoxic conditioned human amnion mesenchymal stem cells michael josef kridanto kamadjaja department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: bone regeneration studies involving the use of chitosan–hydroxyapatite (ch-ha) scaffold seeded with human amnion mesenchymal stem cells (hamscs) have largely incorporated tissue engineering experiments. however, at the time of writing, the results of such investigations remain unclear. purpose: the aim of this study was to determine the osteogenic differentiation of the scaffold ch-ha that is seeded with hamscs in the regeneration of calvaria bone defect. methods: ch-ha scaffold of 5 mm diameter and 2 mm height was created by lyophilisation and desalination method. hamscs were cultured in hypoxia environment (5% oxygen, 10% carbon dioxide, 15% nitrogen) and seeded on the scaffold. twenty male wistar rat subjects (8 – 10 weeks, 200 250 grams) were randomly divided into two groups: control and hydroxyapatite scaffold (has). defects (similar size to scaffold size) were created in the calvaria bone of the all-group subjects, but a scaffold was subsequently implanted only in the treatment group members. control group left without treatment. after observation lasting 1 and 8 weeks, the subjects were examined histologically and immunohistochemically. statistical analysis was done using anova test. results: angiogenesis; expression of vascular endothelial growth factor; bone morphogenetic protein; runx-2; alkaline phosphatase; type-1 collagen; osteocalcin and the area of new trabecular bone were all significantly greater in the has group compared to the control group. conclusion: the three-dimensional ch-ha scaffold seeded with hypoxic hamscs induced bone remodeling in calvaria defect according to the expression of the osteogenic and angiogenic marker. keywords: bone tissue engineering; chitosan-hydroxyapatite scaffold; human amniotic mesenchymal stem cells; hypoxia correspondence: michael josef kridanto kamadjaja, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: michael‑j‑k‑k@fkg.unair.ac.id introduction tissue engineering has been undertaken to remedy many medical conditions, for instance: complications arising from wound healing, bone defects, immune system responses, and donor‑transmitted disease. three dimensional scaffolds were created to provide adequate support forming an extracellular matrix that enables cells to proliferate and differentiate. chitosan alone as a scaffold suffers from its mechanical strength. chitosan could easily break and therefore not able to create a suitable matrix for cell delivery.1 carbonate apatite also faces the similar problem, its brittle nature has limited its application as a scaffold. therefore, combining both materials are predicted to create stronger scaffold.2 scaffold made from chitosan – carbonate apatite (ch‑ca) has been reported as producing a robust, interconnected three‑dimensional (3d) porous structure which could support the proliferation and differentiation of osteoblast during osteogenic differentiation.3,4 hydroxyapatite has chemical structure that similar to human bone, therefore it has good affinity towards the bone and subsequently form chemical bond directly to the hard tissue. 5,6 by combining chitosan and carbonate apatite into scaffold, this material was expected to increase its mechanical strength and reduce the degradation time. the biocompatibility of hydroxyapatite (ha) and the resemblance of its mineral composition to bone has rendered it an ideal material for bone tissue engineering dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 mailto:k@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 69kamadjaja/dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 68–73 (bte). the development of ha into a 3‑dimensional (3d) scaffold or a support to mesenchymal stem cells (msc) in vitro has also been extensively explored. ha scaffolds offer massive advantages within the field of bte.7,8 human amniotic mesenchymal stem cells (hamscs) derived from human placentas are known for their pluripotent properties; ability to differentiate into three forms of germ layer; and efficacy in reducing both potential inflammation and immune reaction.9,10 chitosan‑ hydroxyapatite scaffold seeded with hamscs was expected to intensify osteogenesis. the aim of this study was to observe the effect of ch‑ha scaffold seeded with hamscs within tissue engineering techniques. materials and methods the isolation and culture procedure were performed following the securing of approval from the faculty’s research ethics committee (no. 378/panke. kke/vii/2015). material from a newly‑formed amnion was peeled from the chorion and rinsed using phosphate‑buffered saline (pbs). the amnion was then soaked in ringer’s lactate (rl) containing 2.5 µg/ml gentamycin and 1000 u/ml amphotericin which had been obtained from gibco tm amphotericin b, new york, usa. the isolation and culture of hamscs using a modified soncini’s protocol. small, fine pieces of amniotic membrane were treated with 0.25% trypsin in order to remove the epithelial cells. centrifugation of five minutes duration at 2,000 rpm was carried with the supernatant subsequently being removed. this procedure was then repeated. the supernatant was washed using pbs containing 0.075 mg/ ml dnase 1 (takara bio, shiga, japan) and 0.75 mb/ml type iv collagenase (sigma‑aldrich, st. louis, mo, usa). incubation of the amnion was performed at 37°c for 60 minutes. filtration and centrifugation lasting five minutes were performed to obtain cells. single cells culture was created then using collagen–coated discs. the medium for the cells consisted of dulbecco’s modified eagle’s medium/nutrient mixture f‑12 (dmem/f12) at a ratio of 1:1, added to fetal bovine serum and 10 ng/ml human leukemia inhibitory factor (gibco brl, gaithersburg, md, usa). this medium was replaced every three days. once the cell growth had reached a confluent stage (80%), the cell was split using trypsin. the laboratory stem cell protocol was implemented as the isolation procedure. hamscs were cultured in hypoxia chamber (1% oxygen, 5% carbon dioxide, and 94% nitrogen). chitosan‑hydroxy apatite scaffold was prepared by dissolving 200mg of medium‑molecular weight ch powder (sigma‑aldrich, st. louis, mo, usa) into 5ml of ethanoic acid at room temperature and mixing them for 15 minutes. 15ml of sodium hydroxide solution was used for neutralising purposes in obtaining chitosan gel. furthermore, samples of the chitosan gel were mixed homogenously with 200mg of ha prior to centrifuging at 1,500 rpm for ten minutes. after extraction of excess water, the solution gel was placed into the specific mold to produce scaffolds (5 mm diameter and 2 mm height). before being transferred to a drying machine, the gel was frozen for two hours at ‑80°c.3,4 human amniotic mesenchymal stem cells were deposited onto a 96‑well cell culture plate (m96) at a density of 5 x 104 cell/well and incubated at 37°c for 24 hours with co2 5% concentration. once the cell proliferation population had reached 80%, ch‑ha scaffold was added together with 100 µl of growth medium. the cells underwent a second incubation at 37°c for 20 hours with 5% co2. after the addition of 5 mg/ml mtt reagent (25 µl/well), the cells were incubated a second time for four hours before being observed under an inverted microscope. the scaffold and medium were removed and added to 200 µl/well dmso. a 595 nm wave length elisa reader was employed to read the absorbance, while the living cells were counted by means of a cell counting kit.3,4 2% glutaraldehyde was used to fixate hamscs‑seeded on ch‑ha scaffold at 40°c for 2‑3 hours. the subsequent stage in the procedure consisted of washing with pbs solution three times every five minutes. after exposure to osmic acid 1% for 1‑2 hours, the cells were washed again with pbs. a 15‑minute dehydration procedure using alcohol at varying concentrations (30‑100%) was also completed for each concentration. the scaffold was dehydrated using a critical point drying (cpd) device, attached to a stud pad with specific adhesive, and coated with pure gold. the scaffold was examined under a scanning microscope and photographed by means of a scanning electron microscope (jeol jsm‑t100, japan).3,4 20 male wistar rats were used as the animal subjects of the experiment. the inclusion criteria applied were as follows: aged 8‑12 weeks old and weighing 100‑150 grams. the subjects were randomly divided into a control group and a treatment group of equal size which were observed during weeks 1 and 8. an anaesthetic procedure was performed 4‑6 hours after the subjects were denied further food and water. 20mg of ketamin hcl (ketalar, ireland) per kg of body weight and 3mg of xylazine (xyla,ireland) per kg of body weight were injected intramuscularly. a mid‑longitudinal skin incision was then made on the cranium dorsal surface after an aseptic procedure had been completed. the periosteum of the cranium was separated from the surface in order to produce a flap. a 2 mm diameter, circular, low speed bur (nsk, japan) was used to create the bone defect 5 mm in diameter. the scaffold was implanted and sutured in order to re‑attach the wound area but only in the treatment group.3,4 the defect was subsequently sutured with blue nylon 5‑0 mono suture (ailee co. ltd, busan, korea). the subjects were sacrificed during weeks 1 and 8 in order to obtain the required specimens. the implantation region was decalcified and embedded in paraffin to produce microscopic specimens. in order to highlight the angiogenesis and trabecular bone area, the specimens were stained with hematoxylin and eosin, while post‑scaffold implantation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 70 kamadjaja/dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 68–73 figure 1. ch‑ha scaffold. figure 2. sem image of cells attached and proliferated into the scaffold pores (sem, 1000x magnification). has k k k k k k k k has has has has has has has b c d e f g h i j k l m a n o p figure 3. angiogenesis (a and b), vegf (c and d), bmp2 (e and f), runx‑2 (g and h), akaline phosphatase (i and j), type‑1 collagen (k and l), osteocalcin (m and n) and trabecular bone area (o and p) with 1000x magnification. has: hydroxyapatite‑chitosan scaffold group seeded with hamscs. k: control group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 71kamadjaja/dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 68–73 0 200 400 600 800 k has angiogenesis * 0 5 10 15 k has vegf 0 5 10 15 k has bmp2 0 2 4 6 8 k has runx-2 * 0 2 4 6 8 k has alp * 0 5 10 15 k has type-1 collagen * 0 2 4 6 8 10 k has osteocalcin * 0 50000 100000 150000 k has trabecular bone area * immunohistochemical staining (using mouse anti‑human monoclonal (novus biological, usa) and polyclonal (thermo scientific, usa) antibody: bmp2, runx‑2, alkaline phosphatase, type‑1 collagen, osteocalcin, and vegf of the specimens of cranium calvaria preparations was carried out. the remmele scale index was used to measure the raw data. a nikon h600l (tokyo, japan) light microscope with 1000x magnification and a ds fi2 300‑ megapixel digital camera with image processing software (nikon image system) were respectively employed to examine the specimens and observe the tissue.4 the data were presented as mean values, and standard deviation. statistical package for social sciences (spss) software version 15.0 (spss inc., chicago, il, usa) was used to analyse the data by means of an anova test and p < 0.05 was considered statistically significant. results the chitosan–hydroxyapatite was a solid 3d scaffold 5mm in diameter and 2mm thick (figure 1). toxicity tests incorporating the use of mtt assay indicated that ch‑ ha scaffold was not harmful to the hamscs culture. the percentage of viable cells found in the ch‑ha scaffold was 79.42 %. sem imaging showed that cells were able to attach themselves to the ch‑ha scaffold’s porous surface that was embedded in the calvaria bone defect (figure 2). histological image of seeded cells in the scaffold are shown on figure 3. all groups were examined for expression of runx2, bmp2, vegf, alkaline phosphatase (alp), type 1 collagen, osteocalcin, angiogenesis and trabecular bone area after 8 weeks. all treatment groups possessed a higher mean value than the control group (figure 4). figure 4. the mean value and standard deviation for several parameters observed after 8 weeks. k : control group; has: hidroxyapatite – chitosan scaffold. *: p < 0.05 showed statistically significant. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 72 kamadjaja/dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 68–73 discussion in this study, an attempt was made to combine chitosan with hidroxy‑apatite in order to create ch‑ha scaffold. chitosan was combined with hydroxyapatite to increase the mechanical strength of the scaffold and decrease the degradation time of the material. similar previous study shown that chitosan addition on a scaffold immersed in synthetic body fluid yielded a stronger mechanical strengh, greater strain, and more stable characteristic.11 another similar study also shown thatchitosan‑hydroxyapatite scaffold had good biocompatibility and provided enhanced strength.12 hamscs could attach to and proliferate effectively within the scaffold’s porosity. the interconnected interstices of the scaffold were highly retentive and expected to be an excellent niche for osteoblast proliferation and differentiation. the ideal pore structure for tissue engineering scaffold ranged from 40‑300 µm since this enabled vascular tissue migration and tissue growth.4,13 the potential of hamscs to act as a form of xenogenic mscs during bone tissue engineering procedures has been thoroughly investigated. several studies utilising xenogenic hamscs transplantation in various organs of rats confirmed a less intense immune reaction that could affect the tissue healing process.14–16 the first three days post scaffold‑implantation consists the inflammatory phase, that is, the initial bone healing stage. during this phase, the hypoxic condition of the ch‑ ha scaffold and the degranulation of platelets resulting from hematoma trigger increased vegf expression that, in turn, induces angiogenesis which is essential in early healing processes. functional capillary tissues provide nutritional intake, essential bioactive molecules, and adequate oxygen tension. 17 angiogenesis plays an important role in the healing process in bone defects because it ensures cell survival in the scaffold.18 mesenchymal stem cells placed in hypoxic conditions enhance the expression of angiogenic factors, mainly vegf.19 during the early stages of the regeneration process, the proliferation of msc was followed by the differentiation of osteoblast. external signals produced by msc and osteoblasts, particularly bmp2 protein, influence this regeneration process. in later stages, activation of transcription factor runx‑2 led by bmp2 helped induce msc differentiation of preosteoblast and osteoprogenitors, which, in turn, continued to form a collagen and non‑ collagen bone matrix.20 the bone matrix maturation level was shown by the expression of type 1 collagen fibers. mineralisation within the bone matrix maturation process will be influenced by type 1 collagen in previous stages. if the maturation level of bone matrix increases, type 1 collagen fibers will also be thicker.21 in this experiment, matured osteoblast marker was identified by osteocalcin. osteoblast specifically expressed osteocalcin that is a non‑collagen protein present in bone matrix.22,23 in the treatment group the area of trabecular bone at the end of eight weeks was significantly higher compared to that of the control group, leading to the conclusion that new bone formation in the treatment group rate was higher than that in the control group. the process of osteogenesis indicated by the expressions of alp, type‑1 collagen, and osteocalcin produced a better result in the treatment group compared to the control group. therefore, the maturation level of bone matrix in the treatment group at the end of eight weeks was higher when compared to that of the control group. mesenchymal stem cells could undergo differentiation to become osteoblasts, thereby producing the apropriate environment or stimulus. during osteogenic differentiation, several markers such as alp, type 1 collagen, and osteocalcin were expressed by mscs. at the time, when osteoblasts turn into osteocytes, alp activity decreases. the latest marker of mature osteoblasts expressed by osteocytes was osteocalcin. in this study, certain limitations occurred, including lack of systematic complication. the purpose of this research was to focus on regeneration of calvaria bone defects using hamscs and chitosan–hidroxyapatite scaffold. the study reported here should be continued to include research on their clinical application for bone augmentation. in conclusion, combining ch‑ha scaffold and hamscs could be used as an alternative bone tissue engineering method in order to escalate the clinical use of bone formation. references 1. wahba mi. enhancement of the mechanical properties of chitosan. j biomater sci polym ed. 2020; 31(3): 350–75. 2. darus f, jaafar m. enhancement of carbonate apatite scafold properties with surface treatment and alginate and gelatine coating. j porous mater. 2020; 27: 831–42. 3. ariani md, matsuura a, hirata i, kubo t, kato k, akagawa y. new development of carbonate apatite‑chitosan scaffold based on lyophilization technique for bone tissue engineering. dent mater j. 2013; 32(2): 317–25. 4. kamadjaja mjk, salim s, rantam fa. osteogenic potential differentiation of human amnion mesenchymal stem cell with chitosan‑carbonate apatite scaffold (in vitro study). bali med j. 2016; 5(3): 71–8. 5. ardhiyanto hb. peran hidroksiapatit sebagai bone graft dalam proses penyembuhan tulang. stomatognatic. 2011; 8(2): 118–21. 6. samarawickrama. a review on bone grafting, bone substitutes and bone tissue engineering. in: icmhi ’18: proceedings of the 2nd international conference on medical and health informatics. tsukuba: association for computing machinery; 2018. p. 244–51. 7. dasgupta s. hydroxyapatite scaffolds for bone tissue engineering. bioceram dev appl. 2017; 7(2): 1000e110. 8. budiraharjo r, neoh kg, kang et. hydroxyapatite‑coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation. j colloid interface sci. 2012; 366(1): 224–32. 9. kim j, kang hm, kim h, kim mr, kwon hc, gye mc, kang sg, yang hs, you j. ex vivo characteristics of human amniotic membrane‑derived stem cells. cloning stem cells. 2007; 9(4): 581–94. 10. miki t, lehmann t, cai h, stolz db, strom sc. stem cell characteristics of amniotic epithelial cells. stem cells. 2005; 23(10): 1549–59. 11. sari n, indrani d, johan c, corputty j. evaluation of chitosan‑ hydroxyapatite‑collagen composite strength as scaffold material dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 73kamadjaja/dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 68–73 by immersion in simulated body fluid. j phys conf ser. 2017; 884: 012116. 12. zhao h, liao j, wu f, shi j. mechanical strength improvement of chitosan/hydroxyapatite scaffolds by coating and cross‑linking. j mech behav biomed mater. 2021; 114: 104169. 13. madihally s v., matthew hwt. porous chitosan scaffolds for tissue engineering. biomaterials. 1999; 20: 1133–42. 14. kadam ss, sudhakar m, nair pd, bhonde rr. reversal of experimental diabetes in mice by transplantation of neo‑islets generated from human amnion‑derived mesenchymal stromal cells using immuno‑isolatory macrocapsules. cytotherapy. 2010; 12(8): 982–91. 15. tsuji h, miyoshi s, ikegami y, hida n, asada h, togashi i, suzuki j, satake m, nakamizo h, tanaka m, mori t, segawa k, nishiyama n, inoue j, makino h, miyado k, ogawa s, yoshimura y, umezawa a. xenografted human amniotic membrane‑derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes. circ res. 2010; 106(10): 1613–23. 16. zhang d, jiang m, miao d. transplanted human am niotic membrane‑derived mesenchymal stem cells ameliorate carbon tetrachloride‑induced liver ccirrhosis in mouse. plos one. 2011; 6(2): e16789. 17. kanczler jm, oreffo roc. osteogenesis and angiogenesis: the potential for engineering bone. eur cells mater. 2008; 15: 100– 14. 18. hankenson kd, dishowitz m, gray c, schenker m. angiogenesis in bone regeneration. injury. 2011; 42(6): 556–61. 19. shi y, su j, roberts ai, shou p, rabson ab, ren g. how mesenchymal stem cells interact with tissue immune responses. trends immunol. 2012; 33(3): 136–43. 20. chen g, deng c, li y. tgf‑ β and bmp signaling in osteoblast differentiation and bone formation. int j biol sci. 2012; 8(3): 272–88. 21. kamadjaja db, purwati, rantam fa, ferdiansyah, pramono c. the osteogenic capacity of human amniotic membrane mesenchymal stem cell (hamsc) and potential for application in maxillofacial bone reconstruction in vitro study. j biomed sci eng. 2014; 7(8): 497–503. 22. nakamura h. morphology, function, and differentiation of bone cells. j hard tissue biol. 2007; 16(1): 15–22. 23. tanaka s, matsuzaka k, sato d, inoue t. characteristics of newly formed bone during guided bone regeneration: analysis of cbfa‑1, osteocalcin, and vegf expression. j oral implantol. 2007; 33(6): 321–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p68–73 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p68-73 6363 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 63–66 histological changes during orthodontic tooth movement due to hyperbaric oxygen therapy arya brahmanta,1 soetjipto,2 and ib narmada3 1departemen of orthodontic, faculty of dentistry, universitas hang tuah 2departemen biochemistry, faculty of medicine, universitas airlangga 3departemen of orthodontic, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: mechanical force of orthodontics causes changes in periodontal ligament vascularization and blood flow, resulting in biochemical and cellular changes as well as changes in the contour of the alveolar bone and in the thickness of the periodontal ligaments. hyperbaric oxygen (hbo) therapy is one of many solutions stimulating the growth of new blood vessels and increasing tissue oxygenation. thus, hbo plays a role in recovery of periodontal ligament and osteoblasts. purpose: this study aimed to determine the effects of hbo therapy for seven days on periodontal ligament size and osteoblast number in the tension site during bone remodeling in tooth movement. method: the study was true experimental laboratories with completely randomized control group post test only design. twenty-four males guinea pigs were randomly divided into three groups. k0 was the control group without any treatment, k1 was the group given a mechanical orthodontic pressure, and k2 was the group treated with the addition of hyperbaric oxygen therapy. the maxillary incisors were moved distally by elastic separator. after hbo therapy on day 7, all of the groups were sacrificed, and then periodontal ligament size and osteoblast number were analyzed by one-way anova and lsd statistical tests. result: the results showed significant differences in the size of the periodontal ligament and the number of osteoblasts in the tension site among the groups (p<0.05). conclusion: hbo therapy at 2.4 ata for 7 days is effective in recovery of periodontal ligament and increased osteoblast number during bone remodeling in tension area of orthodontic tooth movement. keywords: periodontal ligament; osteoblast; tooth movement; hyperbaric oxygen; bone remodeling correspondence: arya brahmanta, department of orthodontic, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim 150 surabaya, indonesia. e-mail: arya.brahmanta@hangtuah.ac.id, phone: 031-5945864, fax: 031-591219. introduction orthodontic tooth movement is indicated by remodeling changes in dental and paradental tissues, including periodontal ligament, alveolar bone, dental pulp and gingiva. periodontal ligament (pdl) is a connective tissue that attaches tooth to the alveolar bone, it plays a crucial role in the adaptation process of teeth to physiological forces as well as to orthodontic forces.1 these forces induced by strains would alter pdl vascularity and blood flow.2 during orthodontic tooth movement, the periodontal vasculature is severely impaired by chronic inflammation or excessive mechanical force. this leads to a hypoxic microenvironment of the periodontal cells and enhances the expression of various cytokines and growth factors that may regulate angiogenesis and alveolar bone remodeling.3 orthodontic forces are known to occlude periodontal ligament vessels on the pressure side of the dental root, decreasing the blood perfusion of the tissue.4 in other words, orthodontic tooth movement is stimulated by the force exerted on the periodontal tissue that affect the remodeling of the alveolar bone as a result. when a force greater than capillary blood pressure applied to a tooth, hyaline zone might occur in the direction of the force. this hyaline zone, free of cells, is a necrotic area created by osteoclast activity originated from the tension site. in the tension site, osteoblasts are produced during bone apposition process.5 during orthodontic tooth movement, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p63-66 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p63-66 64 brahmanta, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 63–66 therefore, osteoblasts in the tension side are generated from osteoprogenitor cells, which reside within the pdl.6 the bone remodeling is characterized by activation, resorption, reversal, and formation, in both tension and compression tooth sites during orthodontic tooth movement.2,7 orthodontic force application induces several biological process leading to resorption in the pressure sites and apposition in the tension site. histological changing occur for 5 days followed by its reversal in next 5 to 7 days and a late wave of bone formation between 7 and 14 days.8 hyperbaric oxygen (hbo) therapy increases the amount of oxygen dissolved in blood (oxygen tension) which can then increase the amount of oxygen delivered to hypoxic tissues reducing the effects of hypoxia. vascular endothelial growth factor (vegf) has been identified as one of the primary growth factors responsible for neovascularization during wound healing and embryonic development. oxygen tension is a key regulator of vegf expression in vitro and in vivo.9 hbo therapy is the inhalation of 100% oxygen inside a hyperbaric chamber pressurized to greater than 1 atmosphere absolute (ata; 760 mm hg). hbo therapy, consequently, is potential because it stimulates the growth of new blood vessels and results in a substantial increase in tissue oxygenation that can enhance wound healing. hbo therapy can also promote collagen and adenosinetriphosphate (atp) synthesis, capillary ingrowth, and osteoblastic and osteoclastic activities. the stimulation of osteogenesis by hbo has been reported applied in animal experiments and clinical treatments. oxygen tension has a triggerinrole in bone remodeling. increased oxygen tension causes cellular differentiation to osseous tissue, whereas decreased oxygen tension results in cartilage formation. there is a parallelism between the increasing of oxygen tension and the increasing of osteoblast and osteoclast activities.10 this study aimed to evaluate periodontal ligament size and osteoblast number in the tension site due to 2.4 ata hyperbaric oxygen therapy during bone remodeling in tooth movement. materials and methods the study was true experimental laboratories with completely randomized control group post test only design. ethical permission was obtained from ethics and scientific research committee of experimental animal use in faculty of dental medicine, universitas airlangga. twenty-four male guinea pigs (cavia cobaya) aged three to four months and weighed 300-400 grams. the guinea pigs, fed with a standard pellet diet and tap water ad libitum, were randomly divided into three equal groups. the materials used were 100% pure oxygen in hyperbaric animal chamber, 10% ketamine injection as anesthetic drug, a dose of 0.1-0.2 ml/kg for acepromazine 0.5 ml, 10% buffered formalin, betadine solution, and cotton. t h e p r o c e d u r e o f t h i s s t u d y w a s b e g a n w i t h acclimatization of animals for 48 hours. guinea pigs were divided into three groups, namely (k0) as the control group, (k1) as the orthodontic group and (k2) as the hbo group. an orthodontic force triggering orthodontic tooth movement on the maxilla by using elastic separator was administered to groups k1 and k2. the force (reciprocal) was measured with a gauge during the experiment. in group k2, treated with orthodontic force day for 14 days and then with daily hbo therapy at 2.4 ata for 90 minutes in 7 days, from day 8 to day 14. hbo therapy was conducted by using a veterinary hyperbaric chamber model. the guinea pigs were monitored during the experiment, and all of the groups were sacrificed on the fourteenth day of the experiment. the maxillary teeth were dissected and placed in 10% buffered formalin. afterwards, histological section were prepared with he, and then observed by using a microscope. the photos were taken to measure the number of osteoblasts seen on the microscope with an enlargement 400x. meanwhile, the size of the periodontal ligament on 1/3 apical in the tension site was observed with an enlargement 40x. each histological section was observed and calculated as many as three times in the field of view. the data were statistically measured by using statistical package for the social science (spss) version 20 program. the statistically significant differences among the hbo group, the orthodontic group and the control group were determined and evaluated by using one way anova and lsd tests (p<0.05). results the data obtained showed that there were differences of the periodontal ligament size in the tension site in each group. in the control group, the mean was 6.886 mm, the mean of the orthodontic group was 11.563 mm, and the mean of the hbo group was 5.030 mm (table 1). the data also showed that the number of osteoblasts increased in all of those treatment groups. the highest number found in the group treated with hbo 14.571 (cell/field of view). meanwhile, in the control group, the mean was 13.142, and in the orthodontic group the mean was 4.833 (table 2). the statistical hypothesis was conducted with a standard analytic significance of 95 percent (p=0.05) by using spss. the statistical results showed that there were significant differences among all of the groups. the statistical results of one way anova test and lsd test showed that there were significant differences of the periodontal ligament size in the tension site (figure 1) between the orthodontic group and the hbo group (p<0.005). the periodontal ligament size in the tension site of treatment group decreased with 1.856, thickness decreases, approaching normal size to control group (figure 2). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p63-66 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p63-66 6565brahmanta, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 63–66 there were also significant differences of the number of osteoblasts among all of the groups (figure 3). the number of osteoblast in the tension site increase with 9.74 mm, compared to k0 group (figure 4). discussion the results showed that group k2 given the orthodontic force + therapy at 2.4 ata for 90 minutes in 7 days had wider periodontal ligament size in the tension site than group k0 as the control group and group k1 as the orthodontic group. 7 table 1. the size of periodontal ligament observed in the control, orthodontic and hbo groups (mm) table 2. the number of osteoblasts found in the control, orthodontic and hbo groups (cell/field of view) (a) (b) (c) figure 1. histological section of ligament periodontal in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 40x. groups mean ± sd control 6.886 ± 2.572 orthodontic 11.563 ± 8.831 orthodontic + hbo 5.030 ± 3.411 groups mean ± sd control 13.142 ± 5.573 orthodontic 4.833 ± 1.602 orthodontic +hbo 14.571 ± 6.320 a 7 table 1. the size of periodontal ligament observed in the control, orthodontic and hbo groups (mm) table 2. the number of osteoblasts found in the control, orthodontic and hbo groups (cell/field of view) (a) (b) (c) figure 1. histological section of ligament periodontal in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 40x. groups mean ± sd control 6.886 ± 2.572 orthodontic 11.563 ± 8.831 orthodontic + hbo 5.030 ± 3.411 groups mean ± sd control 13.142 ± 5.573 orthodontic 4.833 ± 1.602 orthodontic +hbo 14.571 ± 6.320 b 7 table 1. the size of periodontal ligament observed in the control, orthodontic and hbo groups (mm) table 2. the number of osteoblasts found in the control, orthodontic and hbo groups (cell/field of view) (a) (b) (c) figure 1. histological section of ligament periodontal in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 40x. groups mean ± sd control 6.886 ± 2.572 orthodontic 11.563 ± 8.831 orthodontic + hbo 5.030 ± 3.411 groups mean ± sd control 13.142 ± 5.573 orthodontic 4.833 ± 1.602 orthodontic +hbo 14.571 ± 6.320 c figure 1. histological section of ligament periodontal in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 40x. 8 figure 2. the size of ligament periodontal observed in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). (a) (b) (c) figure 3. histological section of osteoblasts in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 400x. 6,886 11,563 5,030 0 2 4 6 8 10 12 control (a) ortho (b) ortho+hbo (c ) mm control ortho ortho +hbo figure 2. the size of ligament periodontal observed in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). table 1. the size of periodontal ligament observed in the control, orthodontic and hbo groups (mm) groups mean ± sd control 6.886 ± 2.572 orthodontic 11.563 ± 8.831 orthodontic + hbo 5.030 ± 3.411 table 2. the number of osteoblasts found in the control, orthodontic and hbo groups (cell/field of view) groups mean ± sd control 13.142 ± 5.573 orthodontic 4.833 ± 1.602 orthodontic +hbo 14.571 ± 6.320 8 figure 2. the size of ligament periodontal observed in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). (a) (b) (c) figure 3. histological section of osteoblasts in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 400x. 6,886 11,563 5,030 0 2 4 6 8 10 12 control (a) ortho (b) ortho+hbo (c ) mm control ortho ortho +hbo a 8 figure 2. the size of ligament periodontal observed in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). (a) (b) (c) figure 3. histological section of osteoblasts in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 400x. 6,886 11,563 5,030 0 2 4 6 8 10 12 control (a) ortho (b) ortho+hbo (c ) mm control ortho ortho +hbo c 8 figure 2. the size of ligament periodontal observed in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). (a) (b) (c) figure 3. histological section of osteoblasts in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 400x. 6,886 11,563 5,030 0 2 4 6 8 10 12 control (a) ortho (b) ortho+hbo (c ) mm control ortho ortho +hbo b figure 3. histological section of osteoblasts in the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c) with magnification 400x. 9 figure 4. the number of osteoblasts in the tension site of the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). 13.142 4.833 14.571 0 2 4 6 8 10 12 14 16 18 control (a) ortho (b) ortho+hbo (c ) cell/field of view control ortho ortho +hbo figure 4. the number of osteoblasts in the tension site of the control group (a), in the orthodontic group (b), and in the orthodontic + hbo group (c). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p63-66 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p63-66 66 brahmanta, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 63–66 hbo therapy, involving the administration of 100% oxygen at atmospheric pressures greater than 1 ata, has been proposed as an adjunctive therapy to improve the outcomes of patients suffering from bone fractures, osteoradionecrosis, and distraction osteogenesis, as well as of patients with bone grafts and dental implants.11 hbo therapy can increase hemoglobin saturation with oxygen from 97% to 100%, and hbo can also increase plasma saturation with oxygen. when breathing 100% oxygen, the arterial oxygen tension is raised 6 fold when under 1 ata, 14 fold under 2 ata, and 22 fold under 3 ata. it can raise tissue oxygen tension to a maximum of 500 mmhg at 3 ata, and can also increase oxygen delivery to up to 60 ml per liter of blood. this condition is enough for the basic metabolic tissue needs of reparative tissues in the human body without hemoglobin contribution. this represents the main path of the mechanism of hbo action.12 hbo therapy has been known as a process depended on oxygen to influence cellular proliferation positively. according to broussard et al., fibroblasts cannot proliferate in tissue cultures in the absence of oxygen.10,12 thus, hbo may promote angiogenesis, which is vital for bone healing. hbo therapy might increase expression of vegf, which is one of the key factors that stimulates angiogenesis.9 hbo therapy can increase nodules bone formation and activity in alkaline phosphatase osteoblast. the surface of alkaline phosphatase is a protein that can participate in proliferation of regulations, migration, and cell differentiation of osteoblast.10 in conclusion, periodontal ligament size thickness decreases, approaching normal size and osteoblast number were increased during tooth movement in the tension site due to the provision of hbo therapy 2.4 ata for 90 minutes in 7 days. references 1. alfaqeeh sa, anil s. lactate dehydrogenase activity in gingival crevicular fuid as a marker in orthodontic tooth movement. open dent j 2011; 5: 105 9. 2. alghamdi moh ym. the effect of hyperbaric therapy on bone distant from sites of surgery. thesis. toronto: department of dentistry university of toronto; 2011. 3. bildt mm, bloemen m, kuijpers-jagtman am, von den hoff jw. matrix metalloproteinase inhibitors reduce collagen gel contraction and a-smooth muscle actin expression by periodontal ligament cells. j periodontal res 2009; 44(2): 266–74. 4. wu d, malda j, crawford r, xiao y. effects of hyperbaric oxygen on proliferation and differentiation of osteoblasts from human alveolar bone. connect tissue res 2007; 48(4): 206-13. 5. flavio u, zhana k, john b, ravindra n, christopher o, david r, sunil w. early effects of orthodontic forces on osteoblast differentiation in a novel mouse organ culture model. angle orthodontist 2011; 81(2): 284–91. 6. gokce s, bengi o, akin e, karacay s, sagdic d, kurcu m, gocke hs. effects of hyperbaric oxygen during experimental tooth movement. angle orthod 2007; 78(2): 306-8. 7. kaya fa, hamamci n, basaran g, dogru m, yildirim tt. tnf-a, il-1 and il-8 levels in tooth early leveling movement orthodontic treatment. journal of international dental and medical research 2010; 3: 116-21. 8. khrisnan v, davidovitch z. cellular, molecular and tissue-level reaction to orthodontic force. am j orthod dentofacial orthop 2006; 129(4): 469.e1-32. 9. niklas a, proff p, gosau m, römer p. the role of hypoxia in orthodontic tooth movement. int j dent 2013; 2013: 841840. 10. wise ge, king gj. mechanisms of tooth eruption and orthodontic tooth movement. j dent res 2008; 87(5): 414-34. 11. wu y, cao h, yang y, zhou y, gu y, zhao x, zhang y, zhao z, zhang l, yin j. effects of vascular endothelial cells on osteogenic differentiation of noncontact co-cultured periodontal ligament stem cells under hypoxia. j periodontal res 2013; 48(1): 52-65. 12. fok tco, jan a, peel saf, evans aw, clokie cml, sándor gkb. hyperbaric oxygen results in increased vascular endothelial growth factor (vegf) protein expression in rabbit calvarial critical-sized defects. oral surg oral med oral pathol oral radiol endod 2008; 105(4): 417-22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p63-66 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p63-66 202 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 202–207 case report the diagnostic challenges and two-step surgical approach to an infected dentigerous cyst resembling a unicystic ameloblastoma: a case report cokorda gde suryabharata1, andra rizqiawan2,3, indra mulyawan2,3, sisca meida wati4, mohammad zeshaan rahman5 1resident of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2staff of oral and maxillofacial surgery, universitas airlangga academic dental hospital, surabaya, indonesia 3department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, surabaya, indonesia 5department of oral and maxillofacial surgery, pioneer dental college and hospital, dhaka, bangladesh abstract background: a dentigerous cyst is the second-most frequently observed odontogenic cyst, and an ameloblastoma is one of the most frequently observed odontogenic tumors. both are mostly associated with an impacted mandibular third molar and have similar characteristics. diagnostic difficulties often result in misdiagnosis and remain a challenge to overcome. comprehensive clinical, radiographic, and histopathological views are essential to correctly diagnose the problem and formulate the most suitable treatment plan. purpose: this case report aims to present a thorough approach to the diagnostic and surgical procedures involved in treating a dentigerous cyst that resembles an ameloblastoma by using marsupialization followed by enucleation. case: this article presents the case report of a 27-year-old male patient with an infected dentigerous cyst resembling a unicystic ameloblastoma associated with a totally impacted lower mandibular left third molar. the patient had a history of swelling, pain, and pus drainage in the retromolar area. symptoms subsided after antibiotic and analgesic prescriptions, but the lesion remained and was slowly progressing. the diagnostic approach began with a fine-needle aspiration biopsy, and the result confirmed a benign cystic lesion that was suspected to be an odontogenic tumor. then an incisional biopsy was conducted under local anesthesia, diagnosing an infected dentigerous cyst. case management: marsupialization and decompression using an obturator was preferred, followed by enucleation. nine months later, a radiographic examination revealed satisfactory bone regeneration without recurrence. conclusion: an incisional biopsy plays a vital role in establishing a definitive diagnosis. marsupialization followed by enucleation offers an excellent combination of treatments achieving complete cyst removal, anatomical structure preservation, and bone regeneration with minimal complications. keywords: dentigerous cyst; marsupialization; enucleation; obturator; medicine article history: received 10 january 2023; revised 3 february 2023; accepted 1 march 2023; published 1 september 2023 correspondence: andra rizqiawan, department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya, 60132 indonesia. email: andra-r@fkg.unair.ac.id introduction the remaining odontogenic epithelium involved in the process of odontogenesis associated with impacted teeth has the potential to transform into various odontogenic cysts and tumors.1 according to classification by the world health organization (2017), a dentigerous cyst is a developmental cyst that surrounds and envelops the crown of an unerupted tooth and is attached at cervical areas or the cemento-enamel junction of the tooth.2,3 clinically and radiographically, there are similarities between a dentigerous cyst and a unicystic ameloblastoma.4 in a number of cases it was found that an ameloblastoma was associated with dentigerous cysts and was common in patients younger than 30 years old.5 early diagnosis of jaw lesions such as odontogenic cysts and tumors is very important in order to determine the appropriate treatment plan and prevent misdiagnosis and overtreatment that can cause morbidity. a fine-needle aspiration biopsy (fnab) is a diagnostic modality that is copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 mailto:andra-r@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 203suryabharata et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 202–207 frequently used, but it has several drawbacks so it must be supported by other examinations such as an incisional biopsy or excision. a biopsy provides a high degree of accuracy because it can detect the morphology of the cells required for the correct diagnosis.6–8 surgical management and treatment of a dentigerous cyst consist of enucleation and extraction of the affected tooth.9 marsupialization and decompression release the pressure on the cyst and allow the bone cavity to progressively decrease in volume with the gradual apposition of bone.10,11 an obturator is used after marsupialization for decompression and to keep the cyst lumen open. its smooth surface can prevent the formed blood clot from lifting and avoid the formation of scar tissue. in marsupialization treatment, the cyst is left open and connected to the oral mucosa.12–15 the aim of this case report is to present and discuss a comprehensive management plan in terms of establishing a definitive diagnosis due to the clinical and radiological similarities between a dentigerous cyst and a unicystic ameloblastoma. in this case, a fnab and an incisional biopsy were performed to confirm the diagnosis. the operative treatments performed were an odontectomy of the affected bony impacted third molar, marsupialization and decompression using an obturator, and enucleation followed by curettage. case a 27-year-old male patient visited the oral and maxillofacial surgery clinic at the universitas airlangga teaching hospital chiefly complaining of a painful swelling on the left cheek that had been gradually increasing over the past year. the swelling had reduced in size due to an intraoral fluid discharge a week before the patient visited the hospital. there was no numbness in the lower left jaw, no significant weight loss in the past three months, no history of accidents or blows to the lower left jaw, and no lumps in other areas of the body. no history of systemic disease or allergies was confirmed. a physical examination revealed the patient was in good general condition. the results of an extraoral examination by inspection did not uncover facial asymmetry, oedema, hyperemia, or ulcers and fistulas in the buccal sinistra region. on extraoral palpation, the lump was evident on the buccal sinistra corpus region of the mandible around teeth 36 and 37. it was well-defined and was of a firm and solid consistency, and its temperature was equal to that of the surrounding tissue. there was minimal tenderness, no palpable paresthesia, and an intact inferior border of the sinistra mandible. there was no palpable enlargement nor pain in the regional lymph nodes of the head and neck. an intraoral examination revealed a lump with oedema on the buccal area from the retromolar pad to the region of teeth 36 and 37. the vestibulum was shallower and diffused. it was the same color as the surrounding tissue, and there was no expansion to the lingual, no ulcer, and visible pus drainage on the distal of tooth 37 when palpated. there was a palpable lump on the retromolar pad 38 to 36 in region 37, a cystic consistency originating from retromolar pad 38 to region 37, a hard solid consistency on region 36, no palpable fluctuation, a temperature equal to the surrounding tissue, and an intact lingual and buccal plate. there was no mobility of the surrounding tooth, and the vitality test on 36 and 37 was positive. several follow-up examinations were performed, including a hematological examination, panoramic radiography (figure 1), and anatomic pathology. the hematological examination was normal. radiography revealed a well-defined radiolucent lesion resembling a cyst in the region of 37 and 38 as well as an impacted third molar in both side of mandibula. an anatomic pathology examination was conducted using the fnab method first, revealing a benign cystic lesion with suspicions of an ameloblastoma. then an incisional biopsy was performed under local anesthesia to confirm a definitive diagnosis (figure 2). figure 1. pre-operative radiograph, location of lesion, and specimen collection point. blue dot is an ameloblastoma. red dot is the impacted third molar (#36). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 204 suryabharata et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 202–207 intraoral and extraoral asepsis was performed using a sterile drape before the surgical procedure. the patient was anesthetized using the local anesthetics lidocaine and adrenaline at the ratio 1:80,000. the incision was made according to the planned diagram on the retromolar pad area to the distal of 37, extended along the gingival margin of 37, and followed by a vertical oblique incision on the mesial of 37 to the anterior. the surgeon retracted the mucoperiosteal flap until the cyst wall was lined with epithelium and no solid mass was visible in the area. the cyst was punctured, and a cloudy red-black-brown fluid was obtained. the defect on the left mandible revealed the presence of a cavity with no septum. the walls of the buccal cortex, the lingual, and the inferior border of the mandible were intact. specimens were collected and then subjected to an anatomic pathology examination that demonstrated that the tissue section included cyst walls that were partially lined with squamous epithelium. the stroma revealed a dense infiltration of inflammatory cells including lymphocytes, histiocytes, and plasma cells, and no signs of malignancy were observed, confirming an infected dentigerous cyst. a b figure 3. obturator (a). obturator insertion (b), in the region of the left mandible. a b figure 2. incisional biopsy (a). specimen (b). figure 4. post-operative panoramic radiograph, in the region of the left mandible. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 205suryabharata et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 202–207 case management based on the histopathological result, the treatment plan for this case featured marsupialization, decompression using an obturator, and an odontectomy of the affected impacted left mandibular third molar under local anesthesia. the mucoperiosteal flap was lifted using the same procedure as the earlier incisional biopsy. exploration of the third molar was conducted that confirmed it was mobile and involved in the cyst. a further split technique without bone reduction was performed to atraumatically extract the third molar and preserve the adjacent anatomical structure. marsupialization was conducted using silk 3.0 surgical suture to suture the cyst epithelium to the oral mucosa to adequately drain the cystic fluid and convert the cystic mucosa into normal oral mucosa. kalmicetine gauze was inserted in the cystic lumen to prevent dead space and infection. an inspection was performed on the third day postoperative to remove the kalmicetine gauze and create an impression for the obturator. the marsupialization window was still maintained, and measurements of the canal were taken to manufacture the obturator. an impression was generated using hydrocolloid impression material with a silicone tube inserted in the lumen. subsequently, a decompression obturator was created with acrylic, featuring clasps for retention and a silicone tube to adequately drain the cystic fluid and facilitate irrigation. the patient returned for an examination on the seventh post-operative day. irrigation was performed through the marsupialization canal using 0.9% nacl with a 10cc syringe and fill the syringe with 20cc of the fluid until the cavity appeared clean. irrigation then continued using 1% or 3% hydrogen peroxide dissolved in 0.9% nacl at a ratio of 3:1. irrigation was conducted through the marsupialization canal with a 10cc syringe and as much as 10cc of fluid, and the foam was left for ten seconds before the patient was instructed to spit. the final stage of irrigation used 0.1% or 0.2% chlorhexidine digluconate dissolved in 0.9% nacl at a ratio of 1:1. irrigation was performed through the marsupialization canal using a 10cc syringe and as much as 20cc of fluid until it was clean, and then the patient was instructed to spit. after the marsupialization window was clear, the insertion of the obturator could be attempted. after the insertion of the obturator, the patient returned for examinations after one week, one month, three months, and ten months (figure 3). in the third month and the ten months, a panoramic radiograph was taken to evaluate the post-operative condition and readiness for the second surgery (figure 4). at every examination, the cystic lumen was irrigated using the same irrigation protocol explained above. in the tenth month, enucleation and curettage were performed to retrieve the remaining cyst, and then an evaluation was performed at eight days post-operative. the results revealed that there was adequate bone formation without any sign of recurrence. discussion establishing a definitive diagnosis and formulating the most appropriate treatment plan to manage odontogenic cysts remains a challenge for oral and maxillofacial surgeons.16 in this case, many similarities between a dentigerous cyst and a unicystic ameloblastoma were found that, if not correctly diagnosed and managed, could lead to a clinical misdiagnosis resulting in the patient’s morbidity.17–19 numerous diagnostic modalities, such as oral radiography and anatomic pathology, exist. radiographic images such as panoramic radiographs could highlight the anatomical position of the pathologic lesion, but they also present disadvantages because many lesions have a similar radiographic appearance.20 a biopsy is the gold standard for obtaining a microscopic image through a histopathologic examination, as conducted in this case. to determine the diagnosis, a triple analysis should be performed based on clinical signs, radiographic images, and histopathology.21 before conducting a histopathologic examination with an incisional biopsy, a procedure called fnab must be performed to ensure there is no malignancy in the lesion. after ensuring the absence of malignancy, an incisional biopsy can then be conducted if the fnab result is inconclusive and requires a more definitive result.22 the most frequently occurring jaw lesions that require a biopsy for an anatomic pathology examination are dentigerous cysts, odontogenic keratocysts, and unicystic ameloblastomas. these lesions display similarities in radiographic images, which can be a well-defined unilocular radiolucent lesion with a sclerotic border or a multilocular radiolucent with a scalloped border and involving an impacted tooth.14,16,17 the management of these three kinds of conditions necessitates different approaches, so a proper diagnosis is required to determine an adequate treatment plan. when the result reveals an ameloblastoma, resection is the treatment choice. however, when the histopathologic examination uncovers a cyst, the treatment plan involves enucleation or marsupialization.23,24 this case report highlighted an extensive dentigerous cyst related to an impacted left mandibular third molar. the patient presented with a pus-filled lump that could be diagnosed as a neoplasm, cyst, or infection. a dentigerous cyst is an odontogenic cyst that is generally associated with an unerupted or impacted tooth. this cyst forms around the crown of the unerupted tooth. it begins when there is an accumulation of fluid in the rest of the enamel epithelium and extends around the crown of the unerupted tooth.25–27 a dentigerous cyst can be treated by enucleation, marsupialization, or both of these surgical treatments in two steps. a decision about the management of a dentigerous cyst depends on several considerations, such as the size and location of the cyst, the removal of an unerupted tooth, and the possibility for follow-up with the patient.19 enucleation and extraction of the tooth directly risk weakening the jaw and causing a pathological fracture.28 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 206 suryabharata et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 202–207 marsupialization and decompression were performed in this case to adequately decompress the cyst and prevent injury to adjacent vital anatomical structures. this surgical step is a decompression stage to decrease intracystic and internal hydrostatic pressure.29,30 decompression eliminates tension in the surrounding bone surface that inhibits proliferation and differentiation of the osteogenic precursor, so this procedure induces a signaling pathway that promotes osteogenesis.31 the goals of marsupialization are to decrease the size of the lesion, promote bone growth, and convert cystic epithelium to normal oral epithelium.32 there are several disadvantages of marsupialization, including the duration of treatment, dependency on the patient’s cooperation, discomfort, and the risk of recurrence and transformation into neoplasia and malignancy.33 irrigation is a mandatory protocol during marsupialization to eliminate the pathologic tissues. in this case, the irrigation protocol was performed two to three times per day with chlorhexidine 0.12%. using a syringe, 5ml of chlorhexidine was inserted, and the liquid was left behind to recede after irrigating the cavity. irrigation was conducted for at least six months to support the bonehealing process.30,31 chlorhexidine is a broad-spectrum antimicrobial agent. concentrations of chlorhexidine that are greater than 0.1% can cause leakage of intracellular components out of the cell and undertake a bactericidal effect that prevents bacterial contamination that could hinder bone regeneration.32 in this case, the use of two-step surgery was based on research by marin et al.26 that demonstrated that enucleation after marsupialization is necessary in 54.4% of cases, especially in the posterior region. in this case, the odontectomy of the mandibular third molar was also performed to eliminate the possible main cause of the cyst.27 another case reported by irimia et al.34 revealed that six months after marsupialization, a stable decompression was achieved and enucleation was performed at that time. panoramic radiography is one of the parameters for assessing the improvement in the lesion after the first surgery. the area and ratio of lesion regression can be measured through a panoramic radiograph. in this case, an assessment of the bone regeneration and the size of the cyst was performed by comparing the preoperative panoramic radiograph and those taken three months and nine months after marsupialization. in the tenth month, after regression and bone regeneration had been achieved, the second stage of surgery could be performed. in conclusion, an incisional biopsy plays a vital role in establishing a definitive diagnosis. marsupialization using an obturator followed by enucleation offer an excellent combination of treatments to achieve complete cyst removal, anatomical structure preservation, and bone regeneration with minimal complications. routine long-term follow-up is required to monitor recurrence. references 1. caruso d, lee c, peacock zs. what factors differentiate dentigerous cysts from other pathologic entities in pericoronal radiolucent lesions? j oral maxillofac surg. 2021; 79(10): e57–8. 2. soluk-tekkesin m, wright jm. the world health organization classification of odontogenic lesions: a summary of the changes of the 2017 (4th) edition. turkish j pathol. 2018; 34(1). 3. thompson ldr. dentigerous cyst. ear, nose throat j. 2018; 97(3): 57. 4. rajendra santosh ab. odontogenic cysts. dent clin north am. 2020; 64(1): 105–19. 5. abu-mostafa n, abbasi a. marsupialization of a large dentigerous cyst in the mandible with orthodontic extrusion of three impacted teeth. a case report. j clin exp dent. 2017; 9(9): e1162–6. 6. mortazavi h, baharvand m. jaw lesions associated with impacted tooth: a radiographic diagnostic guide. imaging sci dent. 2016; 46(3): 147–57. 7. martin lhc, speight pm. odontogenic cysts: an update. diagnostic histopathol. 2017; 23(6): 260–5. 8. rioux-forker d, deziel ac, williams ls, muzaffar ar. odontogenic cysts and tumors. ann plast surg. 2019; 82(4): 469–77. 9. hallikeri k, babu b, sudhakaran a, monteiro r. cell block and its impact in the diagnosis of jaw lesions over fine needle aspiration cytology. acta cytol. 2021; 65(5): 361–7. 10. riachi f, khairallah cm, ghosn n, berberi an. cyst volume changes measured with a 3d reconstruction after decompression of a mandibular dentigerous cyst with an impacted third molar. clin pract. 2019; 9(1): 1132. 11. patil m, patil ru, panse am, patil as, jathar pn, bahutule sr. infected dentigerous cyst and its conservative management: a report of two cases. int j clin pediatr dent. 2019; 12(1): 68–72. 12. salama aa, abou-elfetouh a. marsupialization and functional obturator placement for treatment of dentigerous cyst in child: a successful blend. oral maxillofac surg cases. 2020; 6(4): 100200. 13. chouchene f, ameur w ben, hamdi h, bouenba m, masmoudi f, baaziz a, maatouk f, ghedira h. conservative approach of a dentigerous cyst. radnai m, editor. case rep dent. 2021; 2021: 5514923. 14. robinson ra. diagnosing the most common odontogenic cystic and osseous lesions of the jaws for the practicing pathologist. mod pathol. 2017; 30: s96–103. 15. kamadjaja db, satriyo h, setyawan a, lesmaya yd, safril jw, sumarta npm, rizqiawan a, danudiningrat cp, tran tt. analyses of bone regeneration capacity of freeze-dried bovine bone and combined deproteinized–demineralized bovine bone particles in mandibular defects: the potential application of biological forms of bovine-bone filler. eur j dent. 2022; 16(02): 403–13. 16. emamverdizadeh p, arta sa, ghanizadeh m, negahdari r, ghavimi ma, ghoreishizadeh a, tojih mr. compatibility of clinical and histopathological diagnosis of oral lesions in iranian patients. pesqui bras odontopediatria clin integr. 2019; 19(1): 1–7. 17. siregar m, sitam s, lita ya, hadikrishna i. analysis of dentigerous cyst, ameloblastoma, and odontogenic keratocyst panoramic radiograph and cbct: a scoping review. odonto dent j. 2022; 9: 115–30. 18. wulandari p, widkaja d, nasution ah, syahputra a, gabrina g. association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients. dent j. 2022; 55(1): 16–20. 19. vyas t. biopsy of oral lesion -a review article. j adv med dent sci res. 2018; 6(1): 27–35. 20. nugraha ap, kamadjaja db, suma r ta npm, r izqiawan a, pramono c, yuliati a, hendrianto e, rahman mz. osteoinductive and osteogenic capacity of freeze-dried bovine bone compared to deproteinized bovine bone mineral scaffold in human umbilical cord mesenchymal stem cell culture: an in vitro study. eur j dent. 2023; 21. bose ds, sur dj, khan df, dewangan dd, roul da. diagnostic dilemma of dentigerous cyst mimicking a radicular cyst: a case report. saudi j oral dent res. 2021; 6(3): 112–5. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 207suryabharata et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 202–207 22. r aja e e g, k a r i m a e h. d ent ige r ous cyst: enucle at ion or marsupialization? (a case report). pan afr med j. 2021; 40: 149. 23. soesilawati p, rachmat ea, arundina i, naomi n. the possibility of polymorphonuclear leukocyte activation in dental socket healing by freeze-dried aloe vera induction. dent j. 2021; 54(3): 124–7. 24. abu-mostafa n. marsupialization of dentigerous cysts followed by enucleation and extraction of deeply impacted third molars: two case reports. cureus. 2022; 14(4): e23772. 25. de moraes atl, soares ha, viana pinheiro j de j, ribeiro ribeiro al. marsupialization before enucleation as a treatment strategy for a large calcifying odontogenic cyst: case report. int j surg case rep. 2020; 67: 239–44. 26. marin s, kirnbauer b, rugani p, mellacher a, payer m, jakse n. the effectiveness of decompression as initial treatment for jaw cysts: a 10-year retrospective study. med oral patol oral y cir bucal. 2018; 24(1): e47–52. 27. consolo u, bellini p, melini gm, ferri a, lizio g. analysis of marsupialization of mandibular cysts in improving the healing of related bone defects. j oral maxillofac surg. 2020; 78(8): 1355. e1-1355.e11. 28. kivovics m, pénzes d, moldvai j, mijiritsky e, németh o. a custommade removable appliance for the decompression of odontogenic cysts fabricated using a digital workflow. j dent. 2022; 126: 104295. 29. fathi al-omar a, a. elmorsy k. treatment of a large maxillary cyst with marsupialization (case report). oral heal care. 2017; 3(1): 1–4. 30. oliveros-lopez l, fernandez-olavarria a, torres-lagares d, serrera-figallo m, castillo-oyague r, segura-egea j, gutierrezperez j. reduction rate by decompression as a treatment of odontogenic cysts. med oral patol oral y cir bucal. 2017; 22(5): e643–50. 31. aboulhosn m, noujeim z, nader n, berberi a. decompression and enucleation of a mandibular radicular cyst, followed by bone regeneration and implant-supported dental restoration. case rep dent. 2019; 2019: 9584235. 32. brookes zls, bescos r, belfield la, ali k, roberts a. current uses of chlorhexidine for management of oral disease: a narrative review. j dent. 2020; 103: 103497. 33. demir e, günhan ö. treatment results of dentigerous cysts managed by marsupialisation, enucleation or enucleation with platelet rich plasma-a retrospective study. meandros med dent j. 2021; 22(2): 116–24. 34. irimia a, moraru l, ciubotaru da, caruntu c, farcasiu a-t, ca r unt u a. m in ima lly invasive two -st aged su rger y in t he treatment of large cystic lesions of the jaw. healthcare. 2021; 9(11): 1531. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p202–207 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p202-207 mkgs vol 44 no 2 april-juni 2011.indd 67 vol. 44. no. 2 june 2011 endothelial cell cultured on ha/tcp/chitosan scaffold for bone tissue engineering bachtiar ew1, amir lr1, abbas b2, and utami s1 1 department of oral biology, faculty of dentistry, university of indonesia, jakarta-indonesia 2 center of education and isotope application technology, batan abstract background: angiogenesis is crucial for the success of bone reconstruction through tissue engineering. currently, is still not known the activity of endothelial cells that is responsible for blood vessel formation, cultured in ha/tcp/chitosan scaffold. the ability of the scaffold to facilitate the proliferation and migration of endothelial cell to form blood vessel is essential for cell survival especially in the inner area of the scaffold that is susceptible for cell death if adequate vascularization is not occurred. purpose: the purpose of this study was to evaluate the porosity of ha/tcp/chitosan scaffold and the biocompatibility of ha/tcp/chitosan scaffold to endothelial cells. methods: endothelial cells were isolated from umbilical vein (human umbilical vein endothelial cells/ huvec). ha/ tcp/chitosan scaffold was made from two gelling agents and various basic washing solutions. the characteristic of scaffold was examined by scanning electron microscopy. the activity of huvec was evaluated by mtt assay. results: initial average scaffold porosity size range from 68 μm and increased up to 134 μm after 7 days incubation with 10 mg/l lysozyme. there was no significant difference in the viability of huvec incubated with the scaffold compared to control. conclusion: ha/tcp/chitosan has a good biocompatibility for huvec. this condition supports the activity of huvec in the scaffold for angiogenesis process, to provide oxygen and nutrient necessary for osteoblast. key words: endothelial cells, scaffold, hydroxyapatite, tri calcium phosphate, chitosan abstrak latar belakang: angiogenesis merupakan proses yang penting untuk keberhasilan rekonstruksi tulang melalui rekayasa jaringan. saat ini, aktifitas sel endotel pembentuk dinding pembuluh darah pada ha/tcp/chitosan scaffold belum diketahui. kemampuan scaffold sebagai tempat proliferasi dan migrasi sel endotel untuk membentuk pembuluh darah penting untuk kelangsungan hidup sel osteoblast terutama di bagian dalam scaffold. tujuan: mengevaluasi porositas ha/tcp/chitosan scaffold serta sifat biokompatibilitas scaffold terhadap sel endotel. metode: sel endotel diisolasi dari vena tali pusat (human umbilical vein endothelial cells/huvec). ha/tcp/ chitosan scaffold dibuat dengan variasi gelling agents dan dicuci dengan berbagai larutan basa. karakter scaffold dievaluasi dengan scanning electron microscope. aktifitas huvec dievaluasi dengan mtt assay. hasil: pada tahap awal, rata-rata ukuran porus 68 μm dan meningkat menjadi 134 μm setelah inkubasi dengan 10 mg/ml lysosyme selama 7 hari. kultur huvec pada scaffold selama 24 jam tidak menunjukkan tingkat viabilitas yang berbeda dibandingkan dengan kontrol. kesimpulan: ha/tcp/chitosan memiliki sifat biokompatibilitas yang baik terhadap sel huvec. kondisi ini memberikan dukungan terhadap aktifitas sel huvec pada scaffold untuk proses angiogenesis yang akan memberikan oksigen dan nutrisi untuk osteoblas. kata kunci: sel endotel, scaffold, hydroxyapatite, tri calcium phosphate, chitosan correspondence: lisa r amir, c/o: departemen biologi oral, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat, indonesia. e-mail: lisa.amir@gmail.com. phone: 021-31930355 research report 68 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 67–71 introduction tissue engineering is a process of tissue regeneration and remodelling to repair, replace, maintain or increase organ function. this technique relies on the role of stem cells that have the potential to proliferate and differentiate in the scaffold with specific stimuli to form the desired tissue.1 there are three important factors involved in tissue engineering technique: stem/progenitor cell; biomaterial (scaffold) to give structural support for organisation, growth and differentiation of cells in the process of tissue formation; and chemical and mechanical signals required to increase the proliferation and differentiation of cells. these three components can be developed in vitro and transplanted to the defect area in vivo.1,2 tissue engineering technique has been developed as an alternative treatment for large-size bone defect reconstruction. recently, the development of a new biodegradable scaffold based on the biopolymer chitosan has been initiated by national atomic energy agency of indonesia in collaboration with the laboratory of oral biology, faculty of dentistry, unversity of indonesia. the new development to manufacture this material in indonesia is important to build-up scientific and clinical expertise, to become independent from high cost-import of this material and to reduce the high treatment costs accordingly. the scaffold made form hydroxyapatite (ha), tri calcium phosphate (tcp) and chitosan.3-6 the rationale of this scaffold design is to have materials mixed in different proportions with different resorption rates. the polymer which can be degraded to a certain extent thus leaving behind macropores for colonization of osteoblasts. the calcium phosphate could then be resorbed by osteoclasts and replaced with new bone. blood vessel formation (angiogenesis) is prerequisite for bone formation (osteogenesis) therefore angiogenesis is crucial for the success of bone reconstruction through tissue engineering.7-10 angiogenesis include migration, proliferation and differentiation of endothelial cells for new capillary from the existing vessels. firstly, vascularization delivers oxygen for osteoblasts metabolic activity.11 previous study shown a decrease in alkaline phosphatase activity and collagen synthesis in vitro in low oxygen tension environment. this condition inhibits the differentiation of osteoblast cells possibly through the downregulation of runx2.11 runx2 is the essential transcription factor for osteoblasts differentation and for maintaining their differentiated state as runx2 is needed for gene expression of collagen type i and most of bone non-collageous proteins. secondly, blood vessels carry variety of cells including progenitor cells from bone marrow and peripheral blood that are able to differentiate to osteoblasts under the appropriate signals. evidences showed the possibility of pericytes, the cells on the blood vessel wall to differentiate to osteoblasts.12 third, blood vessel cells secrete paracrine factors that regulate bone cells metabolism. a number of angiogenic factors have also their osteogenic potential such as vascular endothelial growth factor (vegf).13 vegf stimulate osteoblasts differentiation, showed by the increase in bone nodule formation and the increase in alkaline phosphatase activity in osteoblast cell line.13 currently, it is not known how is the activity of endothelial cells that is responsible for blood vessel formation, culture in ha/tcp/chitosan scaffold is still not known. the ability of the scaffold to facilitate the proliferation and migration of endothelial cell to form blood vessel is essential for cell survival especially in the inner area of the scaffold that is susceptible for cell death if adequate vascularization is not occurred. the purpose of this study was to evaluate the porosity of ha/tcp/chitosan scaffold and the biocompatibility of ha/tcp/chitosan scaffold to endothelial cells. the urgency of this research that this ha/tcp/chitosan scaffold is expected to facilitate the growth of endothelial and osteoblasts cells to form new bone matrix for large bone defect reconstruction such as cleft palate and rehabilitation of post tumor resection, cases that are frequently found in indonesia. the development of new scaffold material in indonesia is important to reduce the dependency to imported materials so as to promote a more affordable treatment with local product. materials and methods chitosan is obtained by deacetylation of chitin, the structural element in the exoskeleton of shrimp with sodium hydroxide solution and precipitated by hydrochloric acid. ha and tcp are prepared by wet chemical method of calcium hydroxide and phosphoric acid sodium phosphate and calcium nitrate, respectively. the paste is exposed by radiation sterilization of 25 kgy radiation. a scaffold premix liquid is prepared by incorporating 6 gr chitosan in 10 ml 3% acetic acid with gelling agent of 1% hydroxyprop ylmethylcellulose (hpmc) or 1% carboxymethyl cellulose (cmc) and 0.2m phosphate solution for 2 hours under ambient conditions. to reduce the acid residue derived from chitosan dilution solution, the scaffolds were incubated with na2so3 or naoh for 24 hours. the porosity of the scaffold was analyzed by scanning electron microscopy from three randomly regions of interest. porosity was measured before and 1 week after enzymatic degradation with 10 mg/l lysozyme.14 endothelial cells were isolated from umbilical cord human umbilical vein endothelial cells (huvec). only breach deliveries with caesarean section were included in this study. informed consents were signed by all patients. umbilical cords of approximately 15 cm were stored in cord buffer transport medium containing 2 g/ml glucose (sigma, st louis, usa) in pbs with 1% penicillin-streptomycin and processed for culture within 4 hours. isolation of huvec was performed according to baudin et al.,15 with some modification. umbilical vein were washed with sterile pbs with 2 cannulae fixed in both cord extremities to remove the remained blood clot. 0.2% collagenase i 69bachtiar, et al.: endothelial cell cultured on ha/tcp/chitosan scaffold (gibco, california, usa) was injected into the umbilical vein and incubated briefly for 7 minutes in 37° c incubator to avoid any contamination of fibroblast or smooth muscle cells (figure 1a). the cords were massaged gently to facilitate cell detachment and were washed with complete m200 supplement with lsgs kit containing fetal bovine serum, 2% v/v; hydrocortisone, 1 mg/ml; human epidermal growth factor, 10 ng/ml; basic fibroblast growth factor, 3 ng/ml; and heparin, 10 mg/ml (gibco, california, usa). huvec were cultured 6 well-plate (nunc, roskilde, denmark) for approximately 6 days until it reached 80% confluency (figure 1b). huvec were then incubated with scaffold for 24 hour and tested for their viability by means of mtt assay. huvec was cultured with various gelling agents (hpmc or cmc) and basic washing solutions (na2so3 or naoh) of ha/tcp/chitosan after 24 days of incubation mtt assay was performed and and as control huvec cultured in normal dmem medium. results evaluation of characteristic of ha/tcp/chitosan was performed by scanning electron microscopy (jeol, jsm 6510, japan) (figure 2). porous size was measured in three regions of interest of each sample with a magnification of 200–500×. before enzymatic degradation, it showed the initial porous size of the scaffold range from 19 μm to 122 μm (table 1). after enzymatic degradation with lysozyme the size of the porous increased range from 50 μm to 160 μm (table 1). the highest increase was found the hmpc-na2so3 and hpmc-naoh scaffold, where approximately 4.5-fold higher porous size was detected than the initial porous size. huvec cells were cultured until 80% confluence (figure 1b) and harvested using trypsin. huvec cells (5 × 103 cells) were plated in 96 well-plate with medium 200, lsgs kit and scaffold and incubated for 24 hours. experiments were in duplo and repeated twice. mtt data showed no significant difference in the huvec incubated with scaffold compared to huvec incubated in normal medium. a comparable viability compared to the group where huvec was cultured with normal medium (figure 3). the data showed the biocompatibility of the scaffold as huvec viability was unchanged with scaffold incubation compared to the control group. figure 2. porosity of the scaffold. scanning electron microscope photograph of scaffold after 7 days of enzymatic degradation with 10 mg/l lysozyme. hpmc-na2so3 hpmc-naoh cmc-na2so3 cmc-naoh kontrol scaffold 400 300 200 100 0 v ia b il it y (% ) figure 3. viability of huvec after 24 hours incubation with various scaffold materials. figure 1. isolation of human umbilical vein endothelial cells (huvec). a) endothelial cells were isolated from the vein of an umbilical cord. collagenase type i was injected into the vein, secured by surgical clamps and incubated for 7 minutes in 37° c incubator, b) huvec with 80% confluency after 5 days of culture. a b 70 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 67–71 discussion for tissue engineering, the ideal scaffold should have the characteristic: biocompatible, biodegradable, high surface area/and volume ratio that could support the attachment, proliferation and differentiation of cells.16 the design of our scaffold is materials mixed in different resorption rates. natural polymer of chitosan which can be degraded to a certain extent thus leaving behind macropores for colonization of osteoblasts. the calcium phosphate could then be resorbed by osteoclasts and replaced with new bone. data presented in the literature demonstrated the ability of chitosan to be degraded by enzyme to become oligosaccharide that is easy to degrade.14 it can also form a complex with connective tissue such as collagen and glycosaminoglycan to develop interconnected three dimensions structure.14 research works on enzymatic degradation of chitosan for human use have been carried out mostly in lysozyme14 since it is found in various human body fluids, including serum (concentration 4–13 mg/l) and tears. we studied porosity of the scaffold in its initial form and after in vitro enzymatic degradation with lysozyme. the data of the present study showed the immersion of scaffold in lysozyme in pbs solution for 7 days increased the porous size of the scaffold as high as 5-fold. the increase in porosity size gives a positive effect as it allows the infiltration of osteoblast and endothelial cells to the inner core of the scaffold. the migration of endothelial cells inside the scaffold to form the vascular beds has a crucial role for the survival of cell and scaffold complex. initially, cell and scaffold complex relies on diffusion of nutrition supply from the surrounding existing vascular until the vascularized bone is developed. this phase is a critical period, particularly in the large defect reconstruction where the diffusion of nutrition effectively ranged from 150-200 μm from the existing vessels. the deficiency of nutrition for cells in the inner core of the scaffold might prevent the formation of new bone and result in tissue necrosis.17 the data of the present study showed the potential of ha/tcp/chitosan as the ideal scaffold for bone tissue engineering. it revealed that the locally made scaffold composed of ha/tcp/chitosan do not have the negative effect on the viability of endothelial cells. metabolic activity of huvec incubated with ha/tcp/chitosan was comparable to the activity of huvec in normal medium. in contrast, metabolic activity of dental pulp cells incubated with ha/tcp/chitosan scaffold was significantly increased (data not shown). the differences could be explained by the osteoconductive property of the scaffold that could induce the proliferation of dental pulp cells and their differentiation to the osteoblastic lineage. the results indicated the good biocompatibility property of scaffold toward endothelial cells. as the blood vessel formation is prerequisite for bone formation, angiogenesis is therefore crucial for the success bone reconstruction through tissue engineering technique. in light of the critical role of endothelial cells (ecs) in the angiogenic process, a necessary step to evaluate and properly predict the vascularization potential of biomaterials is to assess the interaction of ecs with the respective substrate. huvec represent a model for any research on general properties of human ecs as huvec is the most simple and available human ec type, accurate for the preparation of large quantities of cells. the present study indicated a good biocompatibility property of scaffold material to huvec. the new development to manufacture this material in indonesia is important to build-up scientific and clinical expertise, to become independent from high cost-import of this material and to reduce the high treatment costs accordingly. the scaffold made form hydroxyapatite, tri calcium phosphate and chitosan for bone tissue engineering purpose. the current study evaluated the biocompatibility of ha/tcp/chitosan scaffold to huvec. proliferation of mammalian cells on a particular matrix takes place in three stages: first the cells on the matrix, then the spread and finally they divide in the presence of nutrients. attachment of huvec to the scaffold, migration of the scaffold to facilitate the blood vessel formation in the inner core of the scaffold will be studied in the near future. the conclusion of this study is ha/tcp/chitosan has a good biocompatibility for huvec. this condition supports the activity of huvec in the scaffold for angiogenesis process for oxygen and nutrient supply necessary for osteoblasts, successful bone tissue engineering could be achieved accordingly. table 1. porosity of the scaffold before and 7 days after enzymatic degradation with 10 mg/l lysozyme at 37° c scaffold porosity (μm)* increased of porosity (%)before enzymatic degradation (mean ± sd) after 7 days of enzymatic degradation (mean ± sd) hpmc-na2so3 19 ± 12 160 ± 60 534 hpmc-naoh 33 ± 28 134 ± 55 402 hpmc-natpp 68 ± 39 50 ± 48 73 cmcna2so3 95 ± 69 59 ± 29 62 cmc-naoh 122 ± 40 93 ± 45 77 cmc-natpp 72 ± 33 89 ± 47 125 71bachtiar, et al.: endothelial cell cultured on ha/tcp/chitosan scaffold acknoledgement the present study was financially supported by university of indonesia research grant. grant #2505/ h2.r12/ppm.00.01. references 1. yang s, leong kf, du z, chua ck. the design of scaffolds for use in tissue engineering. part i. traditional factors. tissue eng j 2001; 7: 679–89. 2. park dh, borlongan cv, eve dj, sanberg pr. the emerging field of cell and tissue engineering. med sci monit 2008; 14: ra206-10. 3. mi fl, huang ct, liang hf, chen mc, chiu yl, chen ch, sung hw. physicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film crosslinked by naturally occurring cross-linking agent, aglycone geniopocidic acid. j agric food chem 2006; 54: 3290–6. 4. hoemann cd, sun j, mckee md, chevrier a, rossomacha e, rivard ge, hurtig m, buschmann m. chitosan-glycerol phosphate/ blood implants elicit hyaline cartilage repair integrated with porous subchondral bone in micro drilled rabbit defects. osteoarthr cartil 2007; 15: 78–89. 5. khanal dr, choontanom p, okamoto y, minami s, rakshit sk, chandrakrachang s, steven w. management of fracture with chitosan in dogs. indian vet j 2000; 77: 1085–9. 6. seol y, lee j, park y, lee y, ku y, rhyu i, lee s, han s, chung c. chitosan sponges as tissue engineering scaffolds for bone formation. biotechnol lett 2004; 26: 1037–41. 7. amir l, becking a, jovanovic a, perdijk f, everts v, bronckers a. formation of new bone during vertical distraction osteogenesis of the human mandible is related to the presence of blood vessels. clin oral impl res 2006; 17: 410–6. 8. street j, lenehan b. vascular endothelial growth factor regulates osteoblast survival-evidence for an autocrine feedback mechanism. j orthop surg res 2009; 16(4): 19. 9. keramaris n, calori g, nikolaou v, schemitsch e, giannoudis p. fracture vascularity and bone healing: a systematic review of the role of vegf. injury. 2008; 2(suppl): s45–57. 10. schipani e, maes c, carmeliet g, semenza gl. regulation of osteogenesis-angiogenesis coupling by hifs and vegf. j bone miner res 2009; 24(8): 1347–53. 11. salim a, nacamuli rp, morgan ef, giaccia aj, longaker mt. transient changes in oxygen tension inhibit osteogenic differentiation and runx2 expression in osteoblasts. j biol chem 2004; 17: 40007–16. 12. dore-duffy, p. pericytes: pluricytes cells of the blood brain barrier. curr pharm des 2008; 14(16): 1581–93. 13. yao z, lafage-proust m, plouet j, bloomfoeld s, alexandre c, vico l. increase of both angiongenesis and bone mass in response to exercise dépends of vegf. j bone min res 2004; 19: 1471–80. 14. mi fl, huang ct. physicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film cross-linked by naturally occurring cross-linking agent, aglycone geniopocidic acid. j agric food chem 2006; 54: 3290–6. 15. baudin b, bruneel a, bosselut n, vaubourdolle m. a protocol for isolation and culture of human umbilical vein endothelial cells. nature prot 2007; 2(3): 481–5. 16. howard d, buttery l, shakesheff k, roberts s. tissue engineering: strategies, stem cells and scaffold. j anat 2008; 213: 66–72. 17. santos m, reis r. vascularization in bone tissue engineering, physiology, current stratégies, major hurdles and future challenges. macromol biosci 2010; 10: 12–27. vol 38-no4-2005-isi.pmd 194 root canal overfilling as an influencing factor for the success of endodontic treatment ardo sabir department of conservative dentistry faculty of dentistry hasanuddin university makassar – indonesia abstract the goal of endodontic treatment is to keep the teeth as long as possible in the mouth. the obturation process in the root canal is one of the most important processes in endodontic treatment. the purpose of this article is to explain that overfilling is an influencing factor to the success of endodontic treatment. it has been widely known that overfilling should be avoided during an obturation process. overfilling of the root canal is indicated only in cases which will be followed by apicoectomy, when the foreign material is removed. accidental overfilling may occur with soft material (for example, certain pastes and cements) or with solid material (such as gutta-percha or silver cones). such overfilling may cause an unnecessary mechanical and chemical irritation, which hinders the repair of periapical tissue, and thus, diminishes the probability of a successful endodontic treatment prognosis. many things could cause overfilling, which makes overfilling unpleasant feeling for the patient such as severe pain, periapical lesion, gingival discoloration, periodontal ligament breakage, or even paresthesia. there are several ways to overcome this root canal overfilling, from the conventional endodontic treatment up to endodontic surgery. the main conclusion from this article is to avoid overfilling and the importance of clinicians’ compliance to the right procedures. key words: overfilling, endodontic treatment correspondence: ardo sabir, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas hasanuddin. jln. kandea 5 makassar, indonesia. introduction the general purpose of endodontic treatment is to maintain teeth duration as long as possible in the mouth cavity. the treatment undergoes three stages: the pulp space biomechanic preparation, root canal sterilization, and root canal filling.1,2 the root canal filling can not be hindered if the stage prior to filling is done correctly and adequately.1 the purpose of root canal filling is to fill up or block all root canal and to form a fluid-tight seal on the apical foramen of the tooth, so that any possibility of a secondary infection occurrence due to the mouth cavity or periradicular tissue leakage into the root canal system can be avoided.1,3,4 clinically, the success of an endodontic treatment can be determined byan x-ray photo, clinical sign and/or symptom, histologic and immunopathologic examinations.5-7 ideally, the root canal filling stage has to achieve a hermetic filling which is the crucial stage to obtain a successful endodontic treatment.2 it is frequent to find accidents of endodontic treatment failure due to bacterial infection or because of wrong procedures during endodontic treatment process.6,8,9 the mistake can be classified into two natures.2,6,10 the first mistake is inadequate arsenic use during tooth pulp devitalization, that cause a gingival necrosis or periodontitis. the necrotic gingival after arsenic use is caused by the lost of temporary filling substance. the periodontitis is, infact, influenced by excessive arsenic use, where the arsen is too long inside the tooth. the second mistake is a complication during endodontic treatment process, a perforation in bifurcation area or trifurcation of the posterior tooth during root canal exploration, root canal broken instrument, or an overextension of filling substance overpassing the apical foramen (overfilling). if overfilling occurs, two filling incidents may happen i.e. root canal solidly filled or unsolidly filled conditions by filling substance.11 according to ingle et al.,1 60% of endodontic treatment failure iscaused by the failure of root canal filling . furthermore grossman et al.2 state that endodontic treatment failure is due to non-hermetic filling up to 67%. the aim of this article is to give information about the cause of overfilling and the involved consequences, the way to manage overfilling, and all precautions to prevent overfilling. overfilling cause and effect it is already known that the success rate of endodontic treatments decreases when overfilling occurs. there are several factors to cause overfilling: too much cement in the root canal, unprecise working length measurement, no x-ray photo prior, during, and after the treatment process, over-instrumentation, excessive pressing to filling 195sadamori: comparison of recognition about denture adhesive substance, resistency disappearance due to inadequate root canal preparation, the use of injection technique to fill the root canal, improper apical seal and operator’s lack of skill.1,2,9,12 the effect of overfilling is varied, determined by13 the type of filling substance, filling substance’s quantity which passes the apical foramen, and how far the filling substance surpasses the apical foramen. based on these three factors, the most common effects of overfilling are inflammation reaction of the periapical tissue causing severe pain accompanied by swollen tissue, periodontal ligament breakage, and a periapical lesion.2,13,15 other effects are necrosis of alveolar bone in the periapical area of the discoloration mucosa membrane covering tooth apex, or even a neurological complication such as paresthesia. managing overfilling there are several alternatives to overcome overfilling during an endodontic treatment. they are by taking no treatment, conventional endodontic treatment, or performing an endodontic surgery.2,3,6,14 in specific overfilling cases, one can do no correction measure depending upon the used type of filling substance and how far the filling substance overpasses the apical foramen. should the filling substance be biocompatible, e.g. gutta percha or titanium cones, and it does not pass the apical foramen more than 1 mm, no action or correction measures needed. the same approach is applied if the filling substance can be re-absorbed by our body in the form of paste or cement form substance (figure 1).3,6,10,11,14,16 if the filling substance which passes the apical foramen cannot be re-absorbed, or it is biocompatible but it passes the apical foramen not more than 1 mm, the conservative measure such as a conventional endodontic treatment is applicable.2,3,10,16 this treatment consists of x-ray photo of the overfilled tooth and expelling all filling substance from the root canal. if the filling substance is a gutta percha cone, one can use a root canal spreader, k type file, or a hedstroem type file. but if the guttapercha cone is still difficult to take out, one can help by giving chloroform in the root canal.2,17,18 the filling substance in the form of silver or titanium cones can be taken out from the root canal using a pincet or a hemostat,3,16 whereas for filling substance from paste or cement, one can use a reamer or k type file.10,18 consecutively, a root canal re-preparation with precise and adequate procedures must be done. it is then followed by re-filling the root canal using a new filling substance, and the last measure is an x-ray photo to see the precision of the filling.2,3,7,10,16 overfilling management can be done by performing an endodontic surgery. this is a radical measure to be chosen as operator’s last choice whenever the conservative treatment has fails. there are two indicators of conventional endodontic treatment failure. those are non-vanishable or heavier symptom on patients and operator’s failure to expel the filling substance irritating root canal’s periapical tissue.10 the endodontic surgery can be a curettage (apicocurettage) or apicoectomy. apicocurettage is a measure to take out tissues at the apical area, and apicoectomy is a surgery measure to cut dental apex and take out some surrounding tissue. the root canal filling can be done before or right after the dental apex is cut, or before or right after the dental apex is cut (figure 2).2,3,10 preventing overfilling to prevent an overfilling during endodontic treatment, operators should take cautious actions by emphasizing several matters, such as work with lege artis attitude during treatment process including calculating worklength precisely, proper usage of intracanal apparatus, maintaining resistency form during the root canal preparation, no excessive pressure towards intracanal tool during the preparation process and towards filling substance during root canal filling, as well as utilizing sealer only as much as it is needed. morerover, the applicated sealer must have a minimal toxicity level to prevent more damage when an figure 1. an x-ray photo of overfilling with paste form filling substance: zinc oxide eugenol, inflicting severe pain to patients. a) immediately after an endodontic reatment; b) one year after overfilling, 50% resorption of zinc oxide eugenol took place.14 a b 196 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 194–197 overfilling occurs. the use of a filling substance containing formaldehyde or paraformaldehyde should be avoided because this substance can produce damaging effects to the patient. endodontic treatment of the first molar distal dental root, and on both mandibles, as well as the dental root of premolar both mandibles should be carefully done because the anatomical form of those roots are close to canalis mandibula. the operator should be careful to choose a filling substance from paste texture, because it is really difficult to control while pouring it to the root canal. discussion the occurrence of overfilling caused by a filling substance with a soft constancy/texture such as paste or cement e.g. paraformaldehyde, ah26, hydron, diaket-a and zync oxide eugenol or a solid constancy such as gutta percha or silver cones can present bad reactions/effects to patients. sometimes, the reaction is minor or in light signs such as temporary inflammation, but eventually it can also give damaging effects of overfilling, by unavoidably deforming the surrounding tissue permanently.13 tschamer cit. kaufman and rosenberg19 found a periodontal ligament damage as an effect of overfilling using root canal filling substance containing paraformaldehyde. on the other hand, heling et al. and tal et al. cit kaufman and rosenberg19 reported several cases with alveolar bone necrosis as a result of overfilling with sealer containing paraformaldehyde (endomethasone ®). numerous writers also found serious damages in the periapical tissue including nerve fibers due to n2.20,21 several writers have reported overfilling cases from ah-26 substance and zinc oxide eugenol resulting inflammation reactions on patients.14,22,23 lindner24 found an incident of overfilling from diaket-a substance producing severe pain of overfilling area, followed by paresthesia five days post overfilling. some other writers reported that two important mechanisms were responsible for the nerve fibers damage after an overfilling causing paresthesia. the first mechanism was a chemical neurotoxic as a result of either one or more components of overfilling substance. the second mechanism was a damage and degenerated nerve fibers due to a mechanical effect after the pressed or smashed filling substance. it was then pushed to canalis mandibula.13,14 brodin et al.25 described quite a few overfilling cases with n2 or paraformaldehyde containing filling substance which displayed strong neurotoxic activity to generate permanent nerve damages. while several other writers wrote about the appearance of paresthesia of interior alveolaris nerves and a long stinging pain caused by root canal cement with paraformaldehyde.19,21 if the root canal filling substance (n2 or paraformaldehyde) makes overfilling, a quickest possible radical measure must be performed (endodontic surgery) to prevent the occurrence of nerve injury, since a nerve damage has no restoring process.13 it was reported that if a contact was established between phenol (or its derivatives) and the nerve for several weeks period, a permanent nerve damage would occurr.26 even if a healing process happened later, it would take a long period of time.14,26 besides, the healing process could take place only if the scarred tissue of the nerves did not obstruct the regeneration process of nerve fibers.13 eugenol is a phenol derivate used for a long duration of time in the dentistry world. it has a local anesthetic effect and is able to penetrate tissues yielding protein coagulation. with enough concentration, eugenol’s ability to coagulate protein can generate permanent nerve damage if the contact happens for several weeks.13,26 yet, it was reported that a spontaneous nerve healing process occured in 4-12 months in several cases of overfilling with eugenol contained filling substance.14,27 a b figure 2. an x-ray photo of overfilling with gutta percha cone in the second premolar of the lower jaw, causing abscess and paresthesia. a) immediately after an endodontic treatment; b) after apicoectomy.14 197sadamori: comparison of recognition about denture adhesive the nerve fibers damage is categorized into three types. the first one is a direct damage to the nerves. the second is a direct pressure from the filling substance and the last one is an indirect pressure.14 the direct damage to the nerves can be caused by gutta percha or silver cone overfilling. although these two substances are non-toxic,28 the human body itself can react with a rejection as a defence mechanism towards foreign substances. if this happens, the overfilling filling substance must directly be expelled to stop nerve irritation. the damage is caused by direct pressure from the overfilling filling substance e.g.ah-26 or diaket-a which has the capacity to spread and occupy spaces surrounding the nerves. this damage can be predicted by measuring the volume of the overfilling substance and human body’s ability to re-absorb the foreign material. if the volume is too high, it can generate nerve or blood vessel strangulation. even so, if the human body can re-absorb, nerve damage will not happen. should the operator find this condition, it would be best to lay off any action for the first month, unless there were complaints. then, a conservative measure in the form of conventional endodontic treatment can be applied. a nerve damage originated from an indirect mechanical pressure to the nerves can be caused by hematomes or edema which is a part of the inflammation stage of the wound healing. the longer the inflammation stage takes place, the more possible to have a complication in the form of scarred tissue resulting in permanent impairment nerve fibers or blood vessels.14 based on the explanation above, it can be concluded that the chemical neurotoxic factor of the overfilling filling substance is more influential to nerve damage (paresthesia) than the mechanical factor.25 from the article, the researcher draws conclusions that overfilling is one of local factors to produce an endodontic treatment failure. apart from the skill of the operator, overfilling can also caused by lack of standard treatment procedure. overfilling can show light sign up to heavy symptoms inflicting painful sufferings to patients. if a sign or complaint occurs, patient must be given a conservative measure in the form of conventional endodontic treatment, and if necessary a radical measure as endodontic surgery. the prognosis of an overfilling tooth very much depends on the toxicity level and the amount/volume of the overfilling substance, and how fast the human body can re-absorb that filling substance. references 1. ingle ji, newton cw, west jd, et al. obturation of the radicular space. in: ingle ji, bakland lk, editors. endodontics. 5th ed. hamilton: bc decker inc; 2002. p. 571-4. 2. grossman li, oliet s, del río ce. endodontic practice. 11th ed. philadelphia: lea and febiger; 1988. p. 179. 3. dummer pmh. root canal filling. in: ford trp, editor. harty’s endodontics in clinical practice. 5th ed. edinburgh: wright; 2004. p. 113. 4. sjörgen u, hagglund b, sundqvist g, wing k. factors affecting the long-term results of endodontic treatment. j endod 1990; 16:498-504. 5. frank al, simon jhs, abou-rass m, glick dh. clinical and surgical endodontics concepts in practice. philadelphia: jb lippincott; 1983. p. 88. 6. manisali y, yücel t, eri•en r. overfilling of the root. oral surg oral med oral path. 1989; 68(4):773-5. 7. selden hs. the endo-antral syndrome: an endodontic complication. j am dent assoc 1989; 119:397-402. 8. lin lm, skribner je, gaengler p. factors associated with endodontic treatment failures. j endod 1992; 18(12):625-7. 9. siqueira jr jf. aetiology of root canal treatment failure: why welltreated teeth can fail. int endod j 2001; 34:1-10. 10. frank rj. endodontic mishaps: their detection, correction, and prevention. in: ingle ji, bakland lk, editor. endodontics. 5th ed. hamilton: bc decker inc; 2002. p. 769-89. 11. bence r. 1976. buku pedoman endodontik klinik. sundoro eh. jakarta: ui-press; 1990. h. 199-200. 12. siswadi yls. pengisian saluran akar, masalah dan penanggulangannya. majalah ilmiah kedokteran gigi fkg usakti 2001; 16(46):181-6. 13. neaverth ej. disabling complications following inadvertent overextension of a root canal filling material. j endod 1989; 15(3):135-9. 14. nitzan dw, stabholz a, azaz b. concepts of accidental overfilling and over instrumentation in the mandibular canal during root canal treatment. j endod 1983; 9(2):81-5. 15. yaltirik m, berberoglu hk, koray m, dulger o, yildirim s, aydil ba. orbital pain and headache secondary to overfilling of a root canal. j endod 2003; 29(11):771-2. 16. nicholls e. endodontics. 3th ed. bristol: wright; 1984. p. 123, 17981. 17. gerstein h. techniques in clinical endodontics. philadelphia: wb saunders co; 1983. p. 259-310. 18. seltzer s. endodontology biologic considerations in endodontic procedures. 2nd ed. philadelphia: lea and febiger; 1988. p. 44853. 19. kaufman ay, rosenberg l. paresthesia caused by endomethasone. j endod 1980; 6(4):529-31. 20. montgomery s. paresthesia following endodontic treatment. j endod 1976; 2:345-7. 21. grossman li. paresthesia from n2 or n2 substitute. oral surg oral med oral path. 1978; 45(1):114-5. 22. speilman a, gutman d, laufer d. anesthesia following endodontic overfilling with ah-26. oral surg oral med oral path 1981; 52:554-6. 23. tamse a, kaffe i, littner mm, kozlovsky a. paresthesia following overextension of ah-26: report of two cases and review of the literature. j endod 1982; 8:88-90. 24. lindner g. irritation of mental nerve after root canal therapy on the left mandibular second premolar. quintessence int 1972; 3:312. 25. brodin p, roed a, aars h, orstavek d. neurotoxic effects of rootfilling materials on rat phrenic nerve in vitro. j dent res 1982; 61:1020-3. 26. formin gh, rood jp. successful retrieval of endodontic material from the inferior alveolar nerve. j dent res 1977; 5:47-50. 27. ørstavik d, brodin p, ass e. paresthesia following endodontic treatment: survey of the literature and report of a case. int endod j 1983; 16:167-72. 28. spangberg l, langeland k. biologic effects of dental materials. toxicity of root canal filling materials on hela cells in vitro. oral surg oral med oral path 1973; 35: 402-14. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 51 no 4 okt-des 2018.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 51, number 4, december 2018 editorial boards of dental journal (majalah kedokteran gigi) sk: 02/un3.1.2/2018 january 2nd – december 31st, 2018 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (department of dental health techniques, faculty of vocational studies, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); an’nissa chusida (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); retno palupi (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers harmas yazid yusuf (department of oral and maxillofacial surgery, faculty of dentistry, universitas padjadjaran, indonesia); eky s. soeria soemantri (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); siti sunarintyas (department of dental biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); retno widayati (department of orthodontics, faculty of dentistry, universitas indonesia, indonesia); mieke sylvia m.a.r. (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontology, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); i. b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); retno puji rahayu (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); david kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); titien hary agustantina (department of dental material science and technology, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); desiana radithia (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); irma josefina savitri (department of periodontology, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk. 406/09.18/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 51, number 4, desember 2018 p-issn: 1978-3728 e-issn: 2442-9740 1. variations of gelatin percentages in ha-tcp scaffolds as the result of 6and 12-hour sintering processes of blood cockle (anadara granosa) shells against porosity desak putu sudarmi ari, firda dean yonatasya, gita saftiarini, and widyasri prananingrum ........................................................................................................... 158–163 2. antioxidant activity test of ethyl acetate fraction of binjai (mangifera caesia) leaf ethanol extract k. khairiah, irham taufiqurrahman, and deby kania tri putri .............................................. 164–168 3. the difference between residual monomer dentin bonding hema and udma with acetone and ethanol solvent after binding to type i collagen normayanti, adioro soetojo, and nirawati pribadi ..................................................................... 169–172 4. a comparison between orthodontic model analysis using conventional methods and imodelanalysis vita previa indirayana, gita gayatri, and n. r. yuliawati zenab ............................................. 173–178 5. a study of cytotoxicity and proliferation of cosmos caudatus kunth leaf extract in human gingival fibroblast culture zhafira nur shabrina, ni putu mira sumarta, and coen pramono ........................................... 179–184 6. considerations in performing odontectomy under general anesthesia: case series anindita zahratur rasyida and andra rizqiawan ....................................................................... 185–188 7. application of pomegranate (punica granatum linn.) fruit extract for accelerating post tooth extraction wound healing intan nirwana .................................................................................................................................. 189–193 8. cytotoxicity test and characteristics of demineralized dentin matrix scaffolds in adipose-derived mesenchymal stem cells of rats desi sandra sari, ernie maduratna, f. ferdiansyah, i ketut sudiana, and fedik abdul rantam ................................................................................................................. 194–199 9. contrasting perceptions of male and female dental students regarding smile aesthetics based on their gingival display yessy josephine sijabat, c. christnawati, and dyah karunia .................................................... 200–204 10. the effects of acanthus ilicifolius chloroform extract on tlr-2 expression of macrophages in oral candidiasis dwi andriani and agni febrina pargaputri .................................................................................. 205–209 11. the evaluation of mandibular bone density in chronic periodontitis models yuliana mahdiyah da’at arina, f. ferdiansyah, and mohamad rubianto ............................... 210–215 12. horizontal transmission streptococcus mutans of dental caries in kindergarten student retno indrawati ............................................................................................................................... 216–221 186 the clinical potential and limits of the all-ceramic fixed partial denture restorations harry laksono department of prosthodontic faculty of dentistry airlangga university surabaya indonesia abstract high-strength all-ceramic systems for fixed partial dentures (fpds) is gaining popularity as an alternative to the well established metal-ceramic fpds. several new framework materials and technique such as lithium disilicate, aluminum oxide and yttrium tetragonal zirconia polycrystal have been developed with improved strength, marginal discrepancy and esthetics. since not every all-ceramic system can be used for a variety application, proper selection of the materials is an important for the success of all-ceramic fpds. the longevity of dental restorations is an important health concern and the clinician placed great emphasis on mechanical properties to define the clinical indication of the ceramic materials because of their brittleness and low fracture toughness. the stronger and tougher framework material would improve the reliability and the longevity of dental restoration. to fabricated of an all-ceramic fpds, material would be required with a flexural strength in excess of 300 mpa and fracture toughness 3 mpa/m½. zirconium has a better mechanical properties than alumina and lithium disilicate glass-ceramic, result from the transformation toughening, free of glass phase and minimal flaws. whereas lithium disilicate glass-ceramic has a better translucency than alumina and zirconium based ceramic, result from the higher content of glass phase than that two materials. the purpose of this article is to present the information that can guide the practioner in the decision making process about all-ceramic fpds systems. it can be concluded that the all-ceramic fpds are seems to be an acceptable clinically prosthodontic treatment according to the short-term studies and the lithium disilicate and alumina-based ceramic materials are acceptable for 3 units anterior fpds, whereas zirconia-based ceramic are acceptable for 3–5 units anterior and posterior fpds with 2 pontics. however, further investigation and more clinical long-term follow-up studies are needed. key words: all-ceramic, fixed partial dentures, framework correspondence: harry laksono, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction increased patients and clinicians for more esthetics with biocompatible properties for fabricating fixed partial dentures (fpds) and public scare about allegedly adverse side effects of dental metals and alloys has accelerated the development of alternatives to metallic dental restoration.1 it have led to widespread use of all-ceramic systems for full-coverage restoration use ceramic framework materials for fabrication and processing of infrastructure that are then veneered with porcelain,2,3 and the acceptance of this restorations because of their inherent esthetics, excellent biocompatibility, durability and the ability to withstand oral conditions for a long time without significant deteriorations.4 however, the strength of the ceramic remains a problem for a restoration longetivity,5 because they are brittle and weak when placed under tensile and torsional stress2,6 and the potential of catastrophic fracture is one of the disadvantage.7 recent progress in technology and research of new dental materials has resulted in an increased number of materials available for all-ceramic restorations. due to the successful of all-ceramic crowns both in anterior and posterior regions and with the introduction of high-strength ceramic framework materials, all ceramic systems for fpds may became a viable treatment option to the established metal-ceramic fpds.2,8 as an alternatives, this restoration must fulfill biomechanical requirement and provide longevity similar to metal-ceramic restorations while providing enhanced esthetic.9–11 several high-strength ceramic framework materials have been developed for fabricating fpds with several types of technologies applied for the fabrication.8 not all of these materials are alike, and as such they present with different properties that may affect their indication and limitation, the laboratory procedure used for their processing and their clinical handling.3 the benefits of the materials include a substantial improvement mechanical properties and longevity12 and long-term survival of prosthetic cases are the important factor in rehabilitation.8 the longevity of restoration is dependent upon many different factors including materials. proper selection of the materials and its properties if of utmost importance, since not every allceramic can be used for a variety of applications without restrictions and its influence the load-bearing capabilities of restoration.11 mechanical properties such as strength and 187laksono: the clinical potential and limits of the all-ceramic fixed partial denture restorations fracture toughness are the parameter assessed to understand the clinical potential and limits of the materials because allceramic fpds are submitted to intermittent forces during fabrication and mastication.13 although this seems to be very promising, but long-term clinical data on the success of all-ceramic fpds are limited.14 the purpose of this article is present the information that can guide the practioner in the decision making process about all-ceramic fpds system. lithium disilicate glass ceramic the empress 2® system (ivoclar vivadent) uses a lithium disilicate glass framework material. the framework is fabricated with the lost wax and heat-pressure technique or cad/cam technique with milled out of prefabricated blanks.8,15 lost wax and heat-pressure technique employs wax models that are invested and after preheating the investment ring, the ceramic material is pressed into the investment ring in the press furnace.16,17 cad/cam technique generally consist of computer integrated imaging and milling system that allow the user to design various types of restorations using computer technology (cad software) and the data is transferred to a milling unit (cam) for fabricating the framework.8 the fracture toughness of the framework material between 2.8–3.5 mpa/m1/2 and flexural strength a range of 300–400 mpa.8,15 the system is confined to fabricating 3–unit fpds that replace a missing tooth anterior to the second premolar. the minimal critical dimensions for the connector are 4–5 mm occlusogingivally and 3–4 mm bucco-lingually.16,17 new development of lithium disilicate glass–ceramic was introduced to supplement the product range with high strength and highly esthetics materials for the press technique. this system known as ips e. max press®. ips e.max press are lithium disilicate glass-ceramic ingots for the press technique. it available in two degrees of opacity are medium opacity (mo) are used to fabricate frameworks for vital or slightly discolored teeth and high opacity (ho) are used for non-vital teeth as well as metal core build-ups. the flexural strength of this material is 400 ± 40 mpa and the fracture toughness between 2.5–3.0 mpa/m1/2. this material suitable for crown and fpds in the anterior to premolar region. this material consist of lithium disilicate needle-like crystals (approx. 70%) which are embedded in a glassy matrix.18 glass–infiltrated aluminum oxide ceramic the in-ceram® alumina system (vita zahnfabrik) is a kind of glass-infiltrated aluminum oxide ceramic in corporate a porous, dry-sintered aluminum oxide substructure that is infiltrated with a low-viscosity lanthanum alumino silica glass to develop a ceramic coping with enhanced strength.8,19,20 it was the first restorative system introduced for the fabrication of 3–unit anterior fpds.8,21 the flexural strength of the framework material ranges from 236–600 mpa and the fracture toughness ranges between 3.1–4.6 mpa/m1/2.8 to fabricate the framework, can use either the slip–casting technique or be formed by milling from a dry press presintered block using a cad/cam system or with a cam system and help of the cerec inlab® (sirona) technology.2,8,22 the minimal critical dimension for the connectors are 4 mm occluso–gingival and 3 mm bucco–lingually.8 slip cast technique is a suspension of fine, insoluble particles in a liquid (zirconia/alumina powder is mixed with deionized water and a dispersing agent), then built up with a brush in the special porous gypsum dies and placed in an in-ceram furnace and the framework fired overnight.2 glass infiltrated aluminum oxide with 33% partially stabilized zirconia ceramic the in–ceram® zirconia system (vita zahnfabrik) is a kind of glass-infiltrated aluminum oxide with 33% partially stabilized zirconia ceramic uses combines glass– infiltrated aluminum with 33% partially stabilized zirconium dioxide to the split composition (33% zro2 stabilized by 16% ceo2).8,22,23 the addition of this material in order to provide a stronger and tougher framework material.2,22 the fracture toughness of the framework material ranges between 6–8 mpa/m½ and the flexural strength ranges form 600–800 mpa.2,8 to fabricate the framework is as same as in the in-ceram alumina system.8,24 the minimal critical dimension for the connectors are 4–5 mm occluso– gingival and 3–4 mm bucco–lingual. this material is confined to fabricating 3 unit anterior and posterior fpds,2,8 but is not recommended for fabricating anterior all-ceramic fpds where the translucency is a major factor in enhancing an esthetic result.8 densely sintered high – purity aluminum oxide ceramic the procera® all-ceram system (nobel biocare) is a kind of densely sintered high-purity aluminum oxide ceramic uses densely sintered high – purity aluminum oxide as the framework material, consisting of more than 99.9% aluminum oxide particles of 5 mm grain sizes with a dry pressing technique against the enlarged die of a prepared tooth.19,25–27 the framework are fabricated with cad/cam technique help of the procera system, which consists of a computer–controlled scanning and design station located in a dental laboratory that connected via a modem to procera sandvik ab in stockholm, sweden.27 the flexural strength of the framework material ranges from 487–699 mpa and the fracture toughness ranges between 4.48–6 mpa/m½. this material suitable for crown and 3 unit anterior and posterior fpds.25 yttrium tetragonal zirconia polycrystals (y-tzp) based ceramic there are several system uses yttrium tetragonal zirconium polycrystals (y-tzp) as the framework materials, such as procera®all-zirkon system (nobel biocare),26–28 in-ceram ® yz cubes (vita zahnfabrik),29,30 ips e.max® zir cad (ivoclar vivadent),31 lava™ system (3m espe),26,32,33 cercon® system (dentsply ceramco),26 dcs–precident® sytem (dcs dental)32,34,35 etc. dental restorations using prefabricated y-tzp ceramic blanks are 188 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 186-192 manufactured in two ways, the first is by milling enlarged restorations out of homogenous ceramic green body blanks of zirconia which are then sintered and shrunk to the desired final dimension, and the second is by milling the restorations directly with the final dimensions or complete sintering of highly densed sintered prefabricated zirconia blanks and known as hot isostatic pressure (hip).1,32,35 laboratory–based systems incorporate a variation of the digital scanning technique to custom framework design using virtual waxing on the computer monitor, such as lava system, procera system and cerec system or using information obtained from a wax model of the framework produced by the technician and known as cam system (semi cad/cam) such as cercon system.26 the fracture toughness of the framework material ranges between 9–10 mpa/m½ and the flexural strength ranges from 900–1200 mpa.8 recommended connector surface is 7–11 mm2 for cercon system,26 9 mm2 for lava system and 16 mm2 for dcs– precident system.8 the indication for ips e.max zir cad are for crown and 3–4 unit fpds frameworks for anterior and posterior design,31 procera all-zirkon system for crown and 2–4 unit fpds for anterior and posterior,36 lava system for crown and 4 unit fpds with 2 pontics for the anterior and posterior,32 in-ceram yz for crown and 5 unit fpds with 2 pontics for the anterior and posterior.37 discussion indication for all-ceramic restorations have been extended as their mechanical properties have been developed and now it is possible to use high-strength ceramic materials for the anterior and posterior fpds, as their strength seem to be sufficient enough to resist the occlusal forces.26 the longevity of dental restorations is an important health concern. a prosthetic restorative system can be considered successful if it demonstrates a survival rate of 95% after 5 years and 85% after 10 years.27 for interim all-ceramic fpds, an adequate clinical fracture resistance is required to avoid the fracture of the fpds under function and dr. mclean’s warning that all-ceramic system age, and that all data regarding their performance should at least provide for a 5 year period before they become routine modalities of therapy.19 the clinical failure of all-ceramic restorations is very often associated with their brittleness and low fracture toughness.38 the lack of sufficient clinical studies regarding the latest generation of materials has led the clinician to place great emphasis on mechanical properties to define the clinical indication of these materials. in this regard, the most relevant mechanical properties are flexural strength and fracture toughness.13,38 the strength is related to the flaw-size distribution and toughening mechanism.39 the toughening mechanism have been describe by swain and subsequently by evans, can be classified by: crack deflection, zone shielding, contact shielding and crack bridging.38,40 according to davenport and lawn et al., it has recently been advocated that stronger and tougher framework material would improve the reliability and therefore the lifetime of an all-ceramic fpds.33,41 some authors indicated that, in order to fabricated an all-ceramic fpds, a material would be required with fracture toughness 3 mpa/m½ and flexural strength in excess of 300 mpa.15 shiratsuchi et al.42 indicated that marginal adaptation is one of the most important elements for long-term clinical success of restorations, because poor marginal adaptation increases the potential for micro leakage and plaque retention, which in turn raises the risk of recurrent caries and periodontal disease. marginal discrepancies in the range of 100 mm have been reported to be clinically acceptable with regard to longevity of a restoration. the ips empress 2 in composed of densely arranged lithium disilicate crystals (± 70% volume) with a length of 4 mm and a diameter of 0.5 mm uniformly bounded in a glassy matrix. the interlocking structure of the ceramic hinders crack propagation and elevates flexural strength to 300–400 mpa.27,43 the evolution of ips technology continued with the introduction of ips e.max® press technique which used lithium disilicate glass-ceramic ingots for the press technique. this material as same as with the ips empress 2, but stronger and tougher because the ingots are produced by bulk casting. a continuous manufacturing process based on glass technology (casting/ pressing procedure) is utilized in the manufacture of the ingots. this new technology, which largely differs from sintering process employed in the production of empress 2 ingots. uses optimized processing parameters, which prevent the formation of defects (pores, pigments etc) in the bulk of ingots.18 this is corroborated by the study of guazzato et al.38 who found that the porosity, grain size, shape and orientation are important in determining the mechanical properties of glass-ceramic. the advantages of the ips e.max press is more widely clinical use than ips empress 2 because it available in two different levels of opacity are used to fabricate frameworks for vital, non-vital or discoloration teeth31 and the material has the flexural strength 400 ± 40 mpa.18,44 this flexural strength is similar with the in-ceram alumina (446 mpa),20,45 a strength that exceeds maximal occlusal loads recorded intraorally on anterior teeth.46,47 this is show clearly about the clinical indication of that two all-ceramic systems only for the use of anterior fpds as the manufacture’s suggestion. the ips e.max press and in-ceram alumina have similar strength, but different binders crack propagation. mclaren and white revealed that the strength of in-ceram system used the reinforcing compound form a continuous skeleton–like meshwork capable of stopping crack growth. this differed from glass-ceramic where each reinforcing particle is completely surround by their glassy matrices.2 it is corroborated by the revealed of guazzato et al.38 that the major difference between the pressable and the infiltrated ceramics is that the latter consist of two penetrating networks that are both the ceramic and the glass phase, whereas in the press sable materials only the glass phase 189laksono: the clinical potential and limits of the all-ceramic fixed partial denture restorations is continuous. the toughening mechanism of the in-ceram alumina is the crack bridging mechanism wherein the crack propagation is deflected along the grain boundaries, causing friction between the separated fragments. the longer path of the crack and the friction between the parts are responsible for dissipating the initial energy.48 the ips e.max press system used the interlocking structure of multielongated needle-like crystals and the technology of bulk casting in the manufacture of the ingots capable of stopping crack growth.18 the toughening mechanism for lithium disilicate glass-ceramic are thermally induced micro cracking and crack deflection.38 for the marginal discrepancy, kelly et al. and sorensen reported that vertical marginal discrepancy of fpds inceram alumina is 58 ± 38 mm,32,49 and stappert50 showed that marginal discrepancy of fpds empress 2 is 58–68 mm, and e.max press is similar with the empress 2. this is suggestion that in-ceram alumina and ips e.max press are within the range of clinically acceptable value. for the survival rate, vult von steyern et al. reported a 90% success rate after 5 years in an treatment for three unit fpds in-ceram alumina,51 marquardt and strub52 reported that survival rate after 5 years of the three unit fpds empress 2 was 70% and the 5 year clinical performance failure rate of ips e.max press is 3.3% if the manufacture’s direction were followed.18 this is suggestion that in-ceram alumina and ips e.max press are within the range of successful longevity of dental restorations, and need more attention if use ips empress 2. for the flexural strength of the two forms material of in-ceram alumina, in uniaxial flexural test by guazzato et al. shown the contradictory result with the manufacturer’s suggestion that dry-pressed material is thought to possess better mechanical properties on the basis of a more consistent sintering process. this suggestion is corroborated by the suggest of sailer et al. that the stability of ceramic is highly dependent on the quality (density) of the material and this in turn is dependent on the production technology.53 the study of guazzato et al. showed that however the toughening mechanism such as crack deflection, contact shielding and micro crack toughening operate in in-ceram alumina dry-press and slip, the microstructure of the two materials is somewhat diverse. in-ceram alumina drypress consist of equi-axed particles embedded in a glassy phase and the crack pattern is constantly intergranular. conversely, in-ceram alumina slip mainly consist of elongated grains and induced the crack propagate through (transgranular crack pattern) and/or around (intergranular crack pattern) the alumina grains according to their orientation, generating asymmetric cracks and dissipating a greater amount of energy.38 other study by tan et al. found that a material with greater fracture toughness should be expected when the crack is perpendicular oriented to the elongated grains.54 another aluminum oxide ceramic is procera all-ceram system. the difference with in-ceram alumina are about the composition, crystal volume and fabrication technique. the framework of procera all-ceram manufactured by densely sintered high-purity aluminum oxide and does not contain any silica,20,27 while in-ceram alumina is not a dense aluminum oxide because it used infiltrated with a special lanthanum glass and the resultant interpenetratingphase composite ceramic contained 85 % alumina and 15% glass.2 so, the strength of in-ceram alumina depends on the strength of the fired bond between the aluminum oxide particles and the complete wetting of the openpore microstructure by lanthanum glass infiltration.55 different framework meshwork materials result in different properties,2 and an increase in crystalline content to achieve greater strength.56 however, this differed from glass ceramic, where each reinforcing particle is completely surround by their glassy matrices and glasses undergo brittle fracture by rapid crack proportion at low critical strains.2 these condition can explain about the mechanical properties of procera all-ceram higher than in-ceram alumina and empress 2. this is corroborated by the study of pallis et al.12 who found that procera allceram had higher weibull modulus than empress 2, and wagner and chu who found procera all-ceram to have higher flexural strength than in-ceram alumina.57 weibull modulus is related to the flaw-size distribution and reported to relate to the probability of failure.39 the enlarged die of a prepared tooth must be done because the problem with aluminum oxide was large amount of sintering shrinkage during processing.19 the marginal discrepancy of procera all-ceram is 50–60 µm is within the range of clinically acceptable value.19 several authors reported that in-ceram zirconia has a better mechanical properties than in-ceram alumina, because attributed to the phase transformation toughening mechanism that takes place in the mass of the material.2,8 transformation toughening can occur when zirconia particles are in the metastable tetragonal form, and on the verge of transformation the metastability of the transformation is dependent on the composition, size, shape of the zirconia particles, the type and amount of the stabilizing oxides, the interaction of zirconia with other phases and the processing.22 when an internal stress is applied to the tetragonal zirconia, it can undergo a phase transformation to a different monoclinic crystals configuration. the monoclinic crystal is 3% to 5% larger than the tetragonal crystal it replaced. this phase transformation increases local compressive stresses, which increase the resistance to crack propagation.2 a different result reported by guazzato et al. in a uniaxial flexural strength test, however the toughening mechanism operating in in-ceram zirconia as a combination of several mechanism such as crack deflection and contact shielding attributed to the alumina grains and the phase transformation and micro crack nucleation mainly related to the zirconia particles, there’s no statistically significant difference was found between the strength of in-ceram zirconia and inceram alumina disks.22 the similarities in strength values between in-ceram alumina slip with in-ceram zirconia 190 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 186-192 slip and in-ceram alumina dry-pressed with in-ceram zirconia dry-pressed blocks seem not to be related to the processing, but more likely to the coincident effect of the porosity. the porosity in in-ceram zirconia was greater than in-ceram alumina. this may be explained by the poor distribution of alumina and zirconia particles and by their poor solubility with each other and the glass phase.22 other study found that the poor solubility of coarse grain aluminazirconia-glass compound was due to the low coefficients of diffusion of al2o3 and zro2 within the glassy phase. 58 guazzato et al. also suggested that the fracture behavior of in-ceram zirconia slip and dry-pressed is comparable, where the crack propagation is generally transgranular for the zirconia particles and intergranular or occasionally transgranular, depending on the orientation of the crack in respect of the elongated alumina grains.22 however, sailer et al. suggested that zirconia framework demonstrated sufficient stability for replacement of posterior teeth.53 study by luthy et al. in a static load bearing capacity test of four-unit frameworks, showed that in-ceram zirconia are not recommended for four-unit fpds in the molar region, it’s confirmed the indication of in-ceram zirconia as manufacturer’s suggestion.59 the recent framework material are y-tzp – based materials. conversely to in-ceram zirconia, y-tzp are fully sintered zirconia, therefore better mechanical properties.22,32 yttrium oxide is a stabilizing oxide added to pure zirconia to stabilize it at room temperature and to generate a multiphase material known as partially stabilized zirconia. the high flexural strength and fracture toughness of y-tzp result from the physical property (transformation toughening) of partially stabilized zirconia,8 free of glass phase and polycrystalline microstructure with minimal voids, flaws and cracks,60 they do not exhibit the phenomenon of sub critical crack propagation and stress corrosion.8 the longterm stability of ceramic is closely related to sub critical crack propagation and stress corrosion caused by water in the saliva reacting with the glass, resulting in decomposition of glass structure and increased crack propagation in glasscontaining system.8,61 an in-vitro study evaluating y-tzp fpds under static load demonstrated fracture resistance of more than 2000 mpa,23 and other investigators showed that strength ranging from 1000–1500 mpa.62 the framework can be fabricated mainly with the help of a cad/cam system by means of milling of a zro2 block. 27 sailer et al. showed that zirconia framework exhibit sufficient stability to be used for the replacement of molars and premolars.53 in biaxial disk flexural strength of ceramics under different storage condition, sorensen revealed that zirconia ceramics particularly attractive for posterior fpds.32 other study by luthy et al. showed that y-tzp recommended for four unit posterior fpds and the connector size recommended to be larger than 7.3 mm2 for clinical application.59 in a recent in-vitro study, the failure probability of fpds with zirconia framework after a simulated 10 year clinical service was nearly zero and 100% after 3 years. furthermore, the fpds included not only 3 unit but also longer span, as large as 5 units.53 however, more clinical long-term follow-up studies are needed. the marginal discrepancy of fpds y-tzp with cad/cam system, sorensen reported that lava was 87 ± 43 m,32 and riech et al. reported that was 65 m.64 for cercon system, tsumita et al. reported that was 86,9 m.65 tinscherd et al. reported that dcs-precident was 61–74 m.63 it is seems that all of the cad/cam system which are a clinically acceptable value and had similar value with the slip cast fabricated in-ceram alumina system as the most accurate all-ceramic fpds with a mean marginal discrepancy 58 ± 38 m.32,49 another important factor for the framework material is the translucency. the framework’s translucency as one of the primary factors in controlling esthetics and a critical consideration in the selection of materials.56 some investigators reported that an increase in crystalline content to achieve greater strength generally results in greater opacity, such as empress 2 have a lower crystal content within the matrix than in-ceram and procera materials.17 in-vitro study by heffernan et al. showed that procera and empress 2 more translucent than in-ceram alumina and in-ceram alumina more translucent than in-ceram zirconia.56 however, in-ceram alumina and procera may be exception. the crystal content of procera is higher than in-ceram alumina but the translucency of procera is higher than in-ceram alumina.56 it can explain by the suggestion from van noort that the pure alumina framework has a better translucency than the glass-alumina composite structure.21 zirconium-based ceramic is more whitish than aluminumbased ceramic and lithium disilicate glass-ceramic because zirconium oxide has refractive index higher than that two materials.56 it is concluded that the all-ceramic fpds are seems to be an acceptable clinically prosthodontic treatment according to the short-term studies and the lithium disilicate and alumina-based ceramic materials are acceptable for 3 units anterior fpds, whereas zirconia-based ceramic are acceptable for 3–5 units anterior and posterior fpds with 2 pontics. however, further investigation and more clinical long-term follow-up studies are needed. references 1. sundh a, molin m, sjogren g. fracture resistance of yttrium oxide partially–stabilized zirconia all-ceramic bridges after veneering and mechanical fatigue testing. dent mater 2004; 21:476–82. 2. mclaren ea, white sh. glass–infiltrated zirconia/alumina–based ceramic for crown and fixed partial dentures, clinical and laboratory guidelines. quintessence dent technol 2000; 23:63–76. 3. raigrodski aj. all-ceramic full-coverage restorations: concepts and guidelines for material selection. pract proced aesthet dent 2005; 17(4):249–56. 4. attia a, kern m. influence of cyclic loading and luting agents on the fracture load of two all-ceramic crown systems. j prosthet dent 2004; 92:551–6. 5. isgro g, kleverlaan cj, wang h, feilzer aj. the influence of ceramic materials used for the fabrication of layered all-ceramic dental restorations. dent mater 2004; 21:557–64. 191laksono: the clinical potential and limits of the all-ceramic fixed partial denture restorations 6. komine f, tomic m, gerds t, strub jr. influence of different adhesive resin cements on the fracture strength of aluminium oxide ceramic posterior crown. j prosthet dent 2004; 92(4): 359–64. 7. attia a, kern m. fracture strength of all-ceramic crown luted using two bonding method. j prosthet dent 2004; 91:247–52. 8. raigrodski aj. contemporary materials and technologies for allceramic fixed partial dentures: a review of literature. j prosthet dent 2004; 92(6):557–62. 9. da fonseca dm, ankli r. use of vita vm7 porcelain. quintessenceuse of vita vm7 porcelain. quintessence dent technol 2004; 27:31–42. 10. raigrodski aj, chiche gj, potiket n, hochstedler jl, mohamed se, billiot s, mercante de. the efficacy of posterior three–unit zirconium–oxide–based ceramic fixed partial dental prostheses: a prospective clinical pilot study. j prosthet dent 2006; 96(4):237–44. 11. clelland nl, ramirez a, katsube n, seghi rr. influence of bond quality on failure load of leucite and lithia disilicate–based ceramic. j prosthet dent 2007; 97(1):18–24. 12. pallis k, griggs ja, woody rd, guillen ge, miller aw. fracture resistance of three all-ceramic restorative system for posterior applications. j prosthet dent 2004; 91(6):561–9. 13. albakry m, guazzato m, swain mv. biaxial flexural strength and microstructure changes of two recycled press able glass ceramic. j prosthet dent 2004; 13(3):141–9. 14. sadowsky sj. an overview of treatment considerations for esthetic restorations: a review of literature. j prosthet dent 2006; 96(6):433–42. 15. filser f, luthy h, kocher p, scharer p, gaukler lj. posterior allceramic bridge work. assessment of fracture load and reliability of materials. quintessence dent technol 2003; 1(1):28–41. 16. sorensen ja. the ips empress 2 system: defining the possibilities. quintessence dent technol 1999; 22:153–63. 17. sorensen ja, cruz m, mito wt. research evaluation of a lithiumresearch evaluation of a lithium disilicate restorative system: ips empress 2. signature int 1999; 4(1):4–10. 18. buhler, zemp p, volkel t. scientifique documentation ips e.maxscientifique documentation ips e.max ® press. ivoclar vivadent research and development scientificivoclar vivadent research and development scientific services. bendererstrasse 2 fl-9494 schaan. liechtenstein; 2005. p. 4–24. 19. garber da, adar p, goldstein re, salama h. the quest for the allceramic restoration. quintessence dent technol 2000; 23:27–37. 20. blatz mb, sadan a, kern m. adhesive cementation of high – strength ceramic restorations: clinical and laboratory guidelines. quintessence dent technol 2003; 26:47–55. 21. van noort r. introduction to dental material 2nd ed. edinburgh, london, new york, oxford, philadelphia, st louis, sydney, toronto: mosby; 2002. p. 240. 22. guazzato m, albakry m, ringer sp, swain mv. strength, fracture toughness and microstructure of a selection of all-ceramic materials. part ii. zirconia–based dental ceramic. dent mater 2004; 20:449–56. 23. tinschert j, natt g, mantsch w, augthun m, spiekermann h. fracture resistance of lithium disilicate, alumina and zirconia–based three–unit fixed partial dentures: a laboratory study. int j prostho dent 2001; 14:231–8. 24. blatz m. long-term clinical success of all-ceramic posterior restoration. quintessence int 2002; 33(6):415–26. 25. rosenstiel sf, land mf, fujimoto j. contemporary fixed prosthodontics. 3rd ed. mosby inc; 2001. p. 643–69. 26. culp l. coping material selection for development of natural esthetics: cad/cam technologies 2003; 3(2):3–7. 27. strub jr. fracture resistance of three–unit posterior zirconia dioxide fixed partial dentures. an in-vitro study. inaugural dissertation; 2006. p. 6–21, 33–6. 28. blatz mb, sadan a, martin j, long b. in vitro evaluation of shear bond strength of resin to densely–sintered high purity zirconium– oxide after long-term storage and thermal cycling. j prosthet dent 2004; 91(4):356–62. 29. bindl a, thiel h, stephan m, mormann. in-ceram yz–cubes zirconium oxide ceramics: cad/cam–abutments for all ceramic bridges. acta stomatologica naissi 2003; 19(41):1–4. available from: http : // www.medfak. ni.ac.yu/asn/ broj-41/2-full%20 text. htm. accessed july 20, 2007. 30. mc laren ea, giordano ra. zirconia based ceramics, material properties, esthetics and layering technique of a new veneering porcelain vm9. quintessence dent technol 2005; 29:1–13. 31. ivoclar vivadent. ips e.max®. all-ceramic all you need. ivoclarall-ceramic all you need. ivoclarivoclar vivadent ag, schaan/liechtenstein 595194/0106/e/w, 2006. 32. sorensen ja. the lava system for cad/cam production of highstrength precision fixed prosthodontics. quintessence dent technol 2003; 26:57–67. 33. davenport m. predictable esthetic results from the placement of an anterior 3 -unit all-ceramic cad/cam restoration. synergy in dentistry 2005; 26(1):3–8. 34. fritzche j. zirconium oxide restorations with the dcs precident® system. int j comput dentist 2003; 6:193–201. 35. sadan a, blatz mb, lang b. clinical considerations for densely sintered alumina and zirconia restorations: part 1. int j perio & rest dent 2005; 25(3):213–9. 36. nobel biocare. product catalog nobel estheticstm including procera®. printed in sweden. nobel biocare services ag 2005; 25–32. 37. biendl a, thriel n, stephan m, mormann w. vita in-ceram® 2000 yz cubes zirconium oxide ceramic: cad/cam frameworks for all-ceramic bridges. technical and clinical reliability; 2005. p. 1–6. 38. guazzato m, albakry m, ringer sp, swain mv. strength, fracture toughness and microstructure of a selection of all-ceramic materials. part i. pressable and alumina glass-infiltrated ceramic. dent mater 2004; 20:441–8. 39. oblak c, jevnikar p, kosmak t, funduk n, marion l. fracture resistance and reliability of new zirconia post. j prosthet dent 2004; 91:342–8. 40. swain mv. toughening mechanism for ceramics. mater forum 1989; 13:237– 53. 41. lawn br, deng y, thompson. use of contact testing in the characterization and design of all-ceramic crown like layer structure: a review. j prosthet dent 2001; 86:495–50. 42. shiratsuchi h, komine f, kakehashi y, matsumura h. influence of finish line design on marginal adaptation of electroformed metalceramic crown. j prosthet dent 2006; 95(3):237–42. 43. quinn jb, sundar v, lloyd ik. influence of microstructure and chemistry on the fracture toughness of dental ceramics. dent mater 2003; 19:603–11. 44. albakry m, guazzato m, swain mv. biaxial flexural strength, elastic moduli and x-ray diffraction characterisation of three pressable allceramic materials. j prosthet dent 2003; 89:374–80. 45. seghi rr, sorensen ja. relative flexural strength of six new ceramic materials. j prosthodont 1995; 8:239–46. 46. suarez mj, lozano jfl, paz salido mp, martinez f. three year clinical evaluation of in-ceram zirconia posterior fpds. int j prosthodont 2004; 17:35– 8. 47. kern m, knode h, strub jr. the all-porcelain resin bonded bridge.the all-porcelain resin bonded bridge. quintessence int 1991; 22:257–62. 48. guazzato m, albakry m, swain mv, ironside j. mechanical properties of in-ceram alumina and in-ceram zirconia. int j prosthodont 2002; 15:39–46. 49. kelly jr, nishimura i, campbell sd. ceramics in dentistry: historical roots and current perspectives. j prosthet dent 1996; 75(1):18–32. 50. stappert cf, dai m, chitmongkolsuk s, gerds t, strub jr. marginal adaptation of three unit fixed partial dentures constructed from pressed ceramic system. br dent j 2004; 196:766–70. 51. vult von steyern p, jonsson o, nilner k. five-year evaluation of posterior all-ceramic three unit ( in-ceram ) fpds. int j prosthodont 2001; 14:379–84. 52. marquardt p , strub jr. survival rates of ips empress 2 all-ceramic crowns and fixed partial dentures: result of a 5-year prospective clinical study. quintessence int 2006; 37:253–9. 53. sailer is, feher a, filse f, luthy h, gauckler lj, scharer p, hammerle chf. prospective clinical study of zirconia posterior fixed partial denture: 3-year follow up. quintessence int 2006; 37:685–93. 54. tan sc, chai j, wosniak wt, takahashi y. flexural strength of a glass-infiltrated alumina dental ceramic incorporated with silicon carbide whiskers. int j prosthodont 2001; 14:350–4. 192 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 186-192 55. rizkalla as, jones dw. mechanical properties of commercial high strength ceramic core materials. dent mater 2004; 20:207–12. 56. heffernan mj, aquilino sa, diaz-arnold am, haselton dr, stanford cm, vargas ma. relative translucency of six all-ceramic system. part i: core materials. j prosthet dent 2002; 88:4–9. 57. wagner wc, chu tm. biaxial flexural strength and indentation fracture toughness of three new dental core ceramics. j prosthet dent 1996; 76:140– 4. 58. cassellas d, rafots i, llanes l, anglada m. fracture toughness of zirconia-alumina composites. int j refract met hard mater 1999; 17:11–20. 59. luthy h, filser f, loeffel o, schumacher m, gauckler lj, hammerle chf. strength and reliability of four-unit all-ceramic posterior bridges. dent mater 2005; 21:930–7. 60. papanagiotou hp, morgano sm, giordano ra, pober r. in-vitro evaluation of low-temperature aging effects and finishing procedures on the flexural strength and structural stability of y-tzp dental ceramics. j prosthet dent 2006; 96:154–64. 61. oh sw, delong r, anusavice kj. factors affecting enamel and ceramic wear: a literature review. j prothet dent 2002; 87(4): 451–9. 62. samaras c. a new options for single or multi-unit all-ceramic anterior restoration. 2005 vident. all rights reserved: 1–5. available from: http://www.vident.comp/general.php?id-pages=189. accessed august 1, 2007. 63. buhler-zemp p / volkel t. scientific docomentation ips e.max® zircad. ivoclar vivadent research and development scientific services. bendererstrasse 2 fl-9494 schaan, liechtenstein; 2005. p. 1–5. 64. reich s, wichmann m, nkenke e, proeschel p. clinical fit of all-ceramic three-unit fixed partial dentures, generated with three different cad/cam system. eur j oral sciences 2005; 113:174–9. 65. tsumita m, kokubo y, ohkubo c, nagayama y, sakurai s, fukushima s. clinical evaluation of marginal and internal gaps of zirconia-based 3-unit cad/cam fixed partial dentures. prosthodontic society 2007; 6(2):114–9. 16 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 16–20 original article the enhancement of type 1 collagen expression after 10% propolis-carbonated hydroxyapatite application in periodontitisinduced rabbits indi kusumawati1, suryono2 and ahmad syaify2 1clinical dentistry program, 2department of periodontology, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: alveolar bone defects caused by periodontitis may require regenerative therapy to restore bone structure. propolis possesses antibacterial, anti-inflammatory and antioxidant properties, which can stimulate bone regeneration. propolis-carbonated hydroxyapatite (cha) material was applied during open flap debridement (ofd) in periodontitis therapy. purpose: to analyse the effect of the application of 10% propolis-cha after ofd on type 1 collagen expression on periodontitis-induced rabbits. methods: six male rabbits, aged 5–8 months, weight 1500–2000 grams, were ligated with wire and injected with porphyromonas gingivalis lipopolysaccharide (lps) for six weeks to induce periodontitis. the samples were divided into three groups: group a (ofd only), group b (ofd+cha) and group c (ofd+10% propolis-cha). to acquire 10% propolis-cha, the cha block was cut into 10mg and then immersed in 1ml of 10% propolis solution for 24 hours at room temperature. decapitation was performed on the seventh and 14th day after ofd was performed on each group and microscopic slides were prepared for type 1 collagen examination. the data was analysed using a two-way anova with a 95% confidence interval followed by a post hoc lsd test. results: the type 1 collagen expression in group c (61.36±1.88 on day seven and 70.25±3.89 on day 14) was significantly different from group a (42.91±1.78 on day seven and 45.18±2.48 on day 14) and group b (43.91±5.31 on day seven and 59.63±3.27 on day 14) on both the seventh day and the 14th day (p=0.000). conclusion: the administration of 10% propolis-cha during ofd can increase the type 1 collagen expression in the alveolar bone of rabbits on the seventh and 14th day. keywords: carbonated hydroxyapatite; periodontitis; propolis; type 1 collagen correspondence: suryono, department of periodontology, faculty of dentistry, universitas gadjah mada, jl. denta 1, sekip utara, yogyakarta 55281, indonesia. email: suryonodent@mail.ugm.ac.id introduction periodontitis is a chronic inflammatory disease in periodontal tissues which is caused by particular microorganisms and characterized by the formation of pockets, recessions, or both.1 the main cause of periodontitis is polymicrobial periodontal pathogens, most of which are gram-negative anaerobic bacteria.2 periodontitis can result in damage to the periodontal ligament, cementum and alveolar bone, which then leads to the formation of defects in the alveolar bone.3 alveolar bone defects due to periodontitis require adequate treatment. a treatment used to obtain the biological conditions needed for the regeneration of periodontal tissues is open flap debridement (ofd), which has been proven to be successful in treating infrabony defect.4 ofd is a surgical procedure that aims to get direct access to the involved periodontal tissues and to remove necrotic tissues, yet it results in minimal periodontal tissue regeneration.5 the addition of bone graft material in the ofd treatment has been proven to increase the filling of bone defects.6 carbonated hydroxyapatite (cha) is one dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p16–20 mailto:suryonodent@mail.ugm.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p16-20 17kusumawati et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 16–20 example of alloplastic bone graft materials of which the composition is similar to that of human bone. besides, it is biocompatible, has excellent osteoconductivity and can be resorbed by osteoclasts.7 propolis is a natural resin substance produced by bees that possesses antibacterial, anti-inflammatory, antioxidant, anticancer, antiviral, antifungal and immunestimulant properties.8 in addition, propolis also has the ability to regenerate bone tissue by increasing osteoblast activity and decreasing osteoclast activity.9 an in vivo study showed that 10% propolis gel could stimulate the healing process of gingivitis in experimental animals by reducing the number of polymorphonuclear (pmn) cells, increasing the number of fibroblasts, and stimulating the formation of angiogenesis.10 a previous study showed that cha immersed in 10% propolis solution had an excellent mechanical and chemical bond between the two materials,11 and this new material showed excellent capability in stimulating fibroblast cell lines.12 devitaningtyas et al.13 proved the inhibition properties of cha-10% propolis against porphyromonas gingivalis. another study of propolis-coated allograft bone graft material showed that propolis also has osteoinductive properties by stimulating the proliferation and differentiation of bone cells – thus accelerating the bone regeneration processes in experimental animals.14 bone regeneration processes can be measured by the production of bone formation biomarkers: one of which is type 1 collagen. type 1 collagen is produced by osteoblasts during the reparative phase in the bone healing process.15 osteoblasts are mononucleated cells derived from mesenchymal stem cells (mscs) which are responsible for bone synthesis and mineralization in the early stage of bone formation and final stage of bone remodeling.16 expression of type 1 collagen can be observed on day seven, which increases on day 14.17 another study showed that the expression of type 1 collagen reached its peak on day 14 after treatment in experimental animals.18 the objective of this study was to analyse the effect of the application of 10% propolis-cha in ofd on the expression of type 1 collagen on periodontitis-induced rabbits. materials and methods all the procedures for this research were approved by the ethical committee of the faculty of dentistry, universitas gadjah mada, no. 00208/kkep/fkg-ugm/ec/2019. this was an experimental study with a randomised post-test only control group design. the cha (gama-cha®, pt. swayasa prakarsa, yogyakarta) was cut into pieces, each of which weighed 10mg. pure propolis solution (propolis brazilian®, minas gerais, brazil) was diluted using sterile water with a dilution ratio of 1:9, thus resulting in a 10% concentration propolis solution. to acquire 10% propolischa, the 10mg cha was then immersed in 1ml of 10% propolis solution for 24 hours at room temperature.11 the experimental animals that were used were six male rabbits (oryctolagus cuniculus) aged 5–8 months, weight 1500–2000g and induced with periodontitis. periodontitis induction on the rabbits was performed by the ligation technique and injection of lipopolysaccharide (lps) from porphyromonas gingivalis atcc 33277 from thermo scientific, usa. the rabbits were first allowed to acclimatization for one week. periodontitis induction was then started by anesthetizing the rabbits using ketamine hcl 40mg/kg of body weight (bw) and xylazine 3mg/ kg bw by intramuscular injection, and then the cervical region in the mandibular anterior teeth was ligated using silk 3.0 for six weeks. injection of 0.05ml lps from porphyromonas gingivalis was also performed three times a week by injecting the ligated mandibular anterior teeth.19 the rabbits as periodontitis animal models were randomly divided into three groups. clinical signs of the induced periodontitis that were observed in the rabbits were tooth mobility, gingival recession and redness of the gingiva. the rabbits in group a were treated with ofd; those in group b were treated with ofd and application of cha; and those in group c were treated with ofd and application of 10% propolis-cha. prior to the surgery, the rabbits were anesthetized by injecting ketamine 40mg/ kg bw and xylazine 3mg/kg bw intramuscularly. a full thickness envelope flap was performed in the buccal side of the anterior mandible and then the inflamed tissue was debrided using a universal scaler. the predetermined material was applied to each group using dental tweezers while pressed gently onto the bone. the flap repositioning was done using 4.0 nylon thread. all the rabbits were then returned to their cages at room temperature and administered a soft diet for the first 24 hours, followed by feeding ad libitum during the research. rabbits from each group were randomly assigned to be decapitated on the seventh day and the 14th day after the ofd. four microscopic slides were made from each rabbit. the decapitation procedure was carried out using a sodium pentobarbital overdose (i.e. 120mg/kg bw) injected intramuscularly. the defect area in the alveolar bone of the rabbits was fixed in 10% buffered formalin for 24 hours and then decalcified. samples were embedded in paraffin wax and then sliced into 5µm thick sections using a microtome for the immunohistochemistry (ihc) staining. this study used polyclonal antibody type 1 collagen (1:100, bs10423r, bioss, beijing, china). the observation of type 1 collagen expression was carried out using a light microscope in 400x magnification looking at three visual fields for each slide (reviewed by two independent observers). the observed osteoblasts were cuboidal in shape and were located on the peripheral side of the alveolar bone trabeculae. osteoblasts that positively expressed type 1 collagen were dark brown in colour, while those which negatively expressed collagen were a purplish-blue colour. the data was presented in the form of a percentage of osteoblasts which were positive to type 1 collagen ihc staining. the formula was as follows:20 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p16-20 18 kusumawati et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 16–20 the shapiro-wilk test was used as the data normality test and levene’s test of homogeneity of variance was also used. the data was then analysed using the two-way anova test, with a confidence interval of 95%, and followed by the post hoc lsd test. results figure 1 shows the histological view of the alveolar bone of oryctolagus cuniculus on the seventh and 14th days for each group. the type 1 collagen, which was expressed by osteoblasts in the peripheral of the bone trabecula, is shown in dark brown. the percentages of type 1 collagen expression found in all groups is presented in table 1. the highest number of osteoblasts was observed in group c on day 14 (70.25%), whilst the lowest number was observed in group a on day seven (42.91%). the shapiro-wilk normality test showed that the type 1 collagen expression in each treatment group on each decapitation day were normally distributed with p>0.05. levene’s homogeneity test showed that all data were homogeneous with p>0.05. based on the normality and homogeneity test results, a parametric statistical test two-way anova was performed. the two-way anova test showed significant differences in the type 1 collagen expression on days seven and 14 between the treatment groups with p=0.000 (p<0.05). the post hoc lsd test presented in table 2 shows that the type 1 collagen expression on day seven in group a was a b c day 7 day 14 figure 1. type 1 collagen expression in (a) ofd group, (b) ofd+cha group, (c) ofd+cha+10% propolis group on days 7 and 14 at 400x magnification. the red arrows show a positive expression of type 1 collagen. table 1. mean and standard deviation of type 1 collagen expression on days 7 and 14 groups n mean ± sd p day 7 day 14 a 4 42.91±1.78 45.18±2.48 0.000*b 4 43.91±5.31 59.63±3.27 c 4 61.36±1.88 70.25±3.89 * significant differences between groups (p<0.05) table 2. lsd test results of type 1 collagen expression on days 7 and 14 days groups a b c 7 a 0.687 0.000* b 0.000* c 14 a 0.000* 0.000* b 0.001* c * significant differences between groups (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p16-20 19kusumawati et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 16–20 not significantly different from that in group b (p>0.05), while that of group a was significantly different from group c and the group b expression was significantly different from group c also (p<0.05). on day 14, group a was significantly different from group b and group c. in addition, group b was significantly different from group c (p<0.05). thus, it can be concluded that the expression of type 1 collagen in the group treated with 10% propolischa was higher than the other groups: both on day seven and day 14. discussion type 1 collagen is the most abundant collagen found in bones, making up more than 90% of the organic bone mass.21 type 1 collagen is produced by osteoblasts during the reparative phase of bone healing processes.16 osteoblasts are mononucleated bone cells derived from mscs.22 the results of this study showed that the highest expression of type 1 collagen could be observed in the group given ofd+10% propolis-cha: both on day seven and day 14. an increase in the expression of type 1 collagen is affected by the addition of propolis into cha. these results prove that propolis has the ability to stimulate bone regeneration. this is in line with the findings of a previous study, showing that propolis has the ability to stimulate bone regeneration by stimulating the osteoblast differentiation process.9 according to srivastava et al.23 the ability of propolis to stimulate osteoblast differentiation can be attributed to flavonoids that increase the expression of runt-related transcription factor 2 (runx2). runx2 is a transcription factor needed by mscs to differentiate into preosteoblasts, which express type 1 collagen at a low level. the preosteoblasts then require runx2 transcription factors to differentiate into osteoblasts, which express type 1 collagen at a high level.24 the expression of type 1 collagen on day seven in the ofd group was not significantly different from that of the group given ofd+cha. this is probably because cha has lower osteoinductivity, thus resulting in insignificant new bone formation. this concurs with the results of a previous study showing that it is necessary to add growth factors into cha to increase the osteoinductivity of the material.25 the results of this study have proved that the addition of propolis into cha can increase the osteoinductivity of bone graft materials, which is evident from the increase in the expression of type 1 collagen in the group treated with ofd+10% propolis-cha. this is in agreement with the results from a previous study, showing that propoliscoated allograft bone graft material has osteoinductivity by stimulating the proliferation and differentiation of bone cells, thus accelerating bone regeneration processes in experimental animals.14 this study was limited by the number of subjects and the observation time. from the data, it can be concluded that the application of 10% propolischa in the ofd procedure could increase the expression of type 1 collagen in the alveolar bone of oryctolagus cuniculus on both the seventh and 14th days. a suggestion for future research is to investigate the variables over a longer time period with a larger sample size. acknowledgement this study was supported by the grant penelitian tesis magister simlitabmas in the fiscal year 2020 under contract no. 2024/un1/ditlit/dit-lit/pt/2020. references 1. carranzza, f.a., newman, m.g., takei, h.h. dan kpr. carranza’s clinical periodontology. 11th ed. st louis missouri: saunders elsevier; 2012. p. 62–3. 2. nishihara t, kosek i t. microbial etiology of periodontitis. periodontol 2000. 2004; 36: 14–26. 3. graves dt, kang j, andriankaja o, wada k, rossa c. animal models to study host-bacteria interactions involved in periodontitis. front oral biol. 2012; 15: 117–32. 4. crea a, deli g, littarru c, lajolo c, orgeas gv, tatakis dn. intrabony defects, open-flap debridement, and decortication: a randomized clinical trial. j periodontol. 2014; 85: 34–42. 5. muthu j, namasivayam a, perumalsamy r, pushparajan s, bose b, kannan a. efficacy of combination therapy using anorganic bovine bone graft with resorbable gtr membrane vs. open flap debridement alone in the management of grade ii furcation defects in mandibular molars a comparative study. j int soc prev community dent. 2014; 4(4): 38. 6. kaushal s. evaluation of ossifi ® as alloplastic bone graft material in treatment of periodontal infrabony defects. j clin diagnostic res. 2014; 8(10): 61–5. 7. landi e, celotti g, logroscino g, tampieri a. carbonated hydroxyapatite as bone substitute. j eur ceram soc. 2003; 23(15): 2931–7. 8. lara pereira yc, mardegan issa jp, watanabe e, nascimento gc, iyomasa mm, del ciampo jo, ervolino e. the therapeutic use of propolis extract in alveolar bone contaminated with bacterial endotoxin. dentistry. 2018; 8(3): 1–9. 9. zohery aa, meshri sm, madi mi, abd el rehim ss, nour zm. egyptian propolis compared to nanohydroxyapatite graft in the treatment of class ii furcation defects in dogs. j periodontol. 2018; 89(11): 1340–50. 10. suryono s, hasmy ns, pertiwi tl, benyamin b. propolis 10%-gel as a topical drug candidate on gingivitis. int j med pharm. 2017; 5: 12–7. 11. kusumawati i, suryono, syaify a. loading and release profile assay of carbonated hydroxyapatite incorporated with propolis as bone graft material. maj obat tradis. 2020; 25(2): 121–5. 12. wijayanti p, lastianny sp, suryono s. growth of nih 3t3 fibroblast cells exposed to carbonated hydroxyapatite with incorporated propolis. indones j cancer chemoprevention. 2020; 11(2): 54–9. 13. devitaningtyas n, syaify a, herawati d, suryono s. evaluation of antibacterial potential of carbonated hydroxyapatite combined with propolis on porphyromonas gingivalis. maj obat tradis. 2020; 25(1): 55–8. 14. abdellatif b, mohamed h, karim a, asma b. radiography monitoring of osteoconduction and osteoinduction of orthotopic allograft autoclaved covered with propolis. int j adv life sci technol. 2014; 1(1): 25–31. 15. burr db, allen mr. basic and applied bone biology. oxford: elsevier inc.; 2014. p. 55–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p16-20 20 kusumawati et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 16–20 16. neve a, corrado a, cantatore fp. osteoblast physiology in normal and pathological conditions. cell tissue res. 2011; 343(2): 289–302. 17. vieira ae, repeke ce, de barros ferreira s, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. 18. itagaki t, honma t, takahashi i, echigo s, sasano y. quantitative analysis and localization of mrna transcripts of type i collagen, osteocalcin, mmp 2, mmp 8, and mmp 13 during bone healing in a rat calvarial experimental defect model. anat rec adv integr anat evol biol. 2008; 291(8): 1038–46. 19. zenobia c, hasturk h, nguyen d, van dyke te, kantarci a, darveau rp. porphyromonas gingivalis lipid a phosphatase activity is critical for colonization and increasing the commensal load in the rabbit ligature model. bäumler aj, editor. infect immun. 2014; 82(2): 650–9. 20. zhang w, ju j, rigney t, tribble g. porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model. bmc oral health. 2014; 14: 1–9. 21. k ruger te, miller ah, wang j. collagen scaffolds in bone sialoprotein-mediated bone regeneration. sci world j. 2013; 2013: 1–6. 22. fawcett dw. buku ajar histologi (terj). 12th ed. jakarta: egc; 2002. p. 174–195. 23. srivastava s, bankar r, roy p. assessment of the role of flavonoids for inducing osteoblast differentiation in isolated mouse bone marrow derived mesenchymal stem cells. phytomedicine. 2013; 20(8–9): 683–90. 24. nakashima k, de crombrugghe b. transcriptional mechanisms in osteoblast differentiation and bone formation. trends genet. 2003; 19(8): 458–66. 25. torre e. molecular signaling mechanisms behind polyphenolinduced bone anabolism. phytochem rev. 2017; 16(6): 1183–226. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p16–20 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p16-20 p-issn: 1978-3728 e-issn: 2442-9740 volume 52, number 3, september 2019 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2019 january 2nd – december 31st, 2019 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers sri oktawati (department of periodontology, faculty of dentistry, universitas hasanuddin, indonesia); irna sufiawati (department of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); i. b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); tania saskianti (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 52, number 3, september 2019 p-issn: 1978-3728 e-issn: 2442-9740 1. a comparison of the severity of oral candidiasis between gestational and type 1 diabetes mellitus ayu ragil destrian pangestu, siti nosya rachmawati, leni rokhma dewi and mei syafriadi ............................................................................................................................. 110–116 2. effectiveness of applied behavior analysis (aba) with regard to tooth brushing in autistic children felicia melati, ratna indriyanti and arlette suzy setiawan ........................................................ 117–121 3. the differences in root canal smear layer removal between 6,25% pineapple (ananas comocus l. merr.) peel extract and 17% ethylene diamine tetra-acetic acid nirawati pribadi, karlina samadi, meliavita n. k. astuti, hendy j. kurniawan, adelina k. tandadjaja and ratna puspita hadi ........................................................................... 122–125 4. the effect of a combination of propolis extract and bovine bone graft on the quantity of fibroblasts, osteoblasts and osteoclasts in tooth extraction sockets louisa christy lunardhi, utari kresnoadi and bambang agustono ......................................... 126–132 5. hyperplasia of wistar rat tongue mucosa due to exposure to cigarette side-stream smoke nurina febriyanti ayuningtyas, grahania octaviono mahardika, bagus soebadi, adiastuti endah parmadiati, saka winias, hening tuti hendarti and rosnah binti zain ...................... 133–137 6. effects of sidestream tobacco smoke on p53 expressions in rattus novergicus tongue epithelial mucosa dian angriany, diah savitri ernawati, adiastuti endah parmadiati, hening tuti hendarti and rosnah binti zain ..................................................................................................................... 138–141 7. the effects of different 650 nm laser diode irradiation times on the viability and proliferation of human periodontal ligament fibroblast cells kun ismiyatin, ari subiyanto, ika tangdan, rahmi nawawi, reinold c lina, rizky ernawati and hendy jaya kurniawan ............................................................................... 142–146 8. closed reduction in the treatment of neglected mandibular fractures at the department of oral and maxillofacial surgery, universitas airlangga olivia jennifer gunardi, riska diana, david buntoro kamadjaja and ni putu mira sumarta .............................................................................................................. 147–153 9. applicability of moyers and tanaka-johnston analyses for the arab population of pekalongan, indonesia fani tuti handayani and rizki amalia hidayah .......................................................................... 154–158 10. contrasting efficacy of cocoa pod husk extract and 8% propolis extract in maintaining of root canal wall cleanliness tamara yuanita, uli sasi andari, mandojo rukmo, s. sukaton and deavita dinari .............. 159–162 11. reccurent trauma-induced aphthous stomatitis in adjustment disorder patients y. yuliana, saka winias, hening tuti hendarti and bagus soebadi .......................................... 163–167 105 dental journal (majalah kedokteran gigi) 2022 june; 55(2): 105–108 case report clinical appearance of acute pseudomembranous candidiasis in children and the importance of good communication, information and education to patients: a case report afryla femilian1, winda dwi malinda masuku1, nurina febriyanti ayuningtyas2, diah savitri ernawati2, fatma yasmin mahdani2, meircurius dwi condro surboyo2 1oral medicine specialist degree, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: oral candidiasis is a common opportunistic infection of the oral cavity caused by an overgrowth of the candida species, in particular, candida albicans. the incidence varies depending on age and certain predisposing factors. in the practice of dentistry, doctor-patient communication is an important component. effective communication between doctor and patient is needed to convey information and educate patients so that treatment can be administered appropriately. purpose: this case aimed to discuss the clinical appearance of acute pseudomembranous candidiasis (apc) in children and the importance of good communication, information and education of patients. case: a five-year-old male patient came with his mother on november 6, 2020 complaining of white deposits on the mucosa of the upper and lower lips that had been present for a week. case management: the diagnosis was defined as a typical apc lesion although the potassium hydroxide (koh) test showed negative results. characteristic lesions found in apc are often seen clearly in some cases and treatment can begin immediately. patients receive the empirical therapy, nystatin oral suspension 100.000 i.u, and the patient is instructed to maintain optimal oral hygiene care, maintain nutrient intake and book a follow-up consultation. conclusion: mistakes in patient preparation procedures in taking supporting examinations will result in false negative/positive results, so communication and education information regarding the preparation of supporting examinations for patients is important to note. keyword: acute pseudomembranous candidiasis; children; candida albicans correspondence: nurina febriyanti ayuningtyas, department of oral medicine, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: nurina-ayu@fkg.unair.ac.id introduction oral candidiasis is an opportunistic infection that mostly affects the oral mucosa. candida albicans (c. albicans) is the most common cause of lesions and is a parasitic stage organism developed from normal commensal flora (saprophytic stage). candida albicans are typically weak pathogens, and candidiasis affects the very young, the very old and the extremely ill. the use of broad-spectrum antibiotics, oral corticosteroids and an underdeveloped immune system are all common causes of fungal infections in infants and neonates.1 candida albicans infection is an opportunistic infection that occurs when the host’s immune system is impaired and the natural flora gets infected. exposure to causal agents and the likelihood of infection are two critical variables in opportunistic infections. c. albicans has virulence characteristics that can contribute to its potential to induce infection and can affect hosts who are immunocompromised. the role of candida virulence factors in infection is determined by the type of infection, its location, the infection stage and the host response. many variables, including virulence factors, phenotypic switching, morphological dimorphism, adhesion and production of hydrolytic enzymes play a role in the pathogenesis of c. albicans infection.2 subjective assessment, objective examination and supportive examination can all be used to confirm a candidiasis diagnosis. a characteristic clinical indication that suggests the existence of candidiasis infection dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p105–108 mailto:nurina-ayu@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p105-108 106femilian et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 105–108 might arise in some circumstances. direct and indirect examinations are examples of supporting examinations that can be used to confirm a candidiasis diagnosis. colouring with potassium hydroxide and parkertm ink (koh) is frequently used as a supportive examination to aid in the direct detection of candida infection and indirect examination with fungal culture. 3 it is important to keep track of the steps that the patient must undertake in order for the supportive examination to succeed and produce the desired outcomes. communication between the dentist and the patient is crucial in dentistry. in addition to being a scientific expert who is skilled in their speciality, a doctor must effectively communicate with the patient to establish a therapeutic doctor-patient connection.4 for doctors, poor doctorpatient communication can hinder therapeutic success. a doctor’s responsibility is to provide as much information as possible about a treatment technique in order for it to be effective. the success of the process of getting a diagnosis and choosing a treatment plan might be harmed by poor information transmission throughout treatment operations. in order for therapy to operate well, effective doctor-patient communication is required to convey information and educate patients. the goal of this case is to explore what constitutes clinical evidence. case a five-year-old male patient came with his mother on november 6, 2020 complaining of white deposits on the mucosa of the upper and lower lips that had been present for a week. there were no reports of discomfort, itching or burning. white plaques had appeared on the patient’s tongue, cheeks and palate during the preceding few days. there was no past history of recurring canker sores and the patient was active as normal with an adequate diet and plenty of rest. the patient had not experienced this before. there were no other lesions. when the white plaques formed on the patient’s lips, the patient’s mother purchased echinacea purpurea from the pharmacy and administered it to the patient once a day. the patient had not had this condition examined by a doctor. according to his mother, neither the patient nor his parents had a history of allergies or systemic disorders. there are no similar lesions elsewhere. the application of echinacea purpurea had no effect. case management objective inspection of the upper and lower labial mucosa, palate durum, dextra and sinistra buccal mucosa and lateral dextra and sinistra of the tongue revealed white, multiple, clear bordered, irregular edge pseudo membranes that could be scraped away, leaving a reddish region (figure 1). acute pseudomembranous candidiasis (apc) was the temporary diagnosis given to the patient. the patient was referred for a koh examination as well as a fungal culture test and given aloe vera extract gel. on the third day, the patient returned with the results of the laboratory examinations. the patient’s mother reported that in the morning before going to the laboratory, the patient’s father cleaned up the white plaques using sterile gauze and warm water. the evidence of white plaques was reduced. an objective examination on the upper and lower lips found macula, redness, multiple clear-bordered, irregular edge desquamation and white, clear bordered, irregularedged rough surfaces (figure 2). the laboratory examinations showed the results of direct fungal examinations but not fungal cultures. the examination of fungal cultures is not done by the laboratory. results of laboratory tests obtained from koh examinations that are negative can be influenced by a variety of factors and false negatives can be produced. anamnesis and clinical examination revealed that the most convincing diagnosis was acute pseudomembranous candidiasis and the patient was subsequently prescribed with the antifungal drug nystatin oral suspension of 100,000 iu/ml at the second visit. when the patient came ten days later for his third visit, the problem had cleared up and the patient reported that the nystatin oral suspension had been used regularly. an objective examination found that the mucosa and lesions had disappeared, which indicated that there had been a good healing process (figure 3). the patient was advised to use nystatin for two weeks afterwards. a) b) c) d) figure 1. the condition of the patient’s oral cavity on initial examination; a) pseudo membrane on upper and lower labial mucosa; b) pseudo membrane in dextra buccal mucosa; c) pseudo membrane in sinistra buccal mucosa; d) pseudo membrane in palatum durum and dextra-sinistra of the tongue. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p105–108 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p105-108 107 femilian et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 105–108 in this case, the operator provided information and education to the patient in relation to the preparation required before taking the supporting examination specimen. the operator instructed the patient not to use nystatin before taking the specimen. however, the operator did not inform the patient not to rub the lesion before taking a supporting examination specimen. the patient’s father cleaned the lesions using gauze and warm water before taking the specimen, thus causing a false negative result. discussion oral candidiasis in children can be caused by a number of different reasons, both local and systemic. saliva, topical medicine and food are examples of local variables that might affect children. candidiasis can be caused by a malfunction of the salivary glands, which might be a predisposing factor. candida overgrowth is inhibited by salivary constituents, such as histidine-rich polypeptides, lactoferrin, lysozyme and sialo peroxidase.5 topical medications, such as corticosteroid inhalers and antiseptics might disrupt the natural flora balance in the mouth cavity. unbalanced dietary consumption of refined sugars, carbohydrates and dairy products (rich in lactose) may act as growth promoters by lowering ph levels and so promoting candida development.6 systemic factors in children that may occur as predisposing factors of the appearance of candidiasis include age, a set of nutrients, systemic drugs, immune disorders and congenital conditions. c. albicans are usually weak pathogens and candidiasis occurs in very young, very old and very sick patients.1 transferrin and other iron-dependent enzymes lose their fungistatic ability when iron levels are low.6 the use of broad-spectrum antibiotic drugs, corticosteroids and antineoplasma over a long period of time causes many normal flora bacteria to die. this causes pressure on c. albicans growth to decrease, resulting in excessive proliferation and more growth of c. albicans.7 in research conducted by williams, oral candidiasis in hiv-uninfected paediatric patients was most likely due to long-term antibiotic use.8 the most known immune disorder condition closely related to candidiasis is acquired immunodeficiency syndrome (aids). candida infections are frequent in people with congenital diseases linked with a faulty immune system, such as di george’s syndrome, hereditary myeloperoxidase deficiency and chediak–higashi syndrome.5 diagnosis can be achieved with several examinations; firstly, from subjective examination or anamnesis; secondly, by objective or clinical examination; and thirdly, from supporting examination.9 diagnosis of oral candidiasis is basically clinically enforceable. when a clinical diagnosis has to be verified to rule out other diseases as a differential diagnosis, or when antifungal treatment resistance is suspected, a supporting evaluation using microbiological methods is performed. the commonly used method of supporting examination for diagnosing primary candidiasis a) b) c) figure 2. condition of the patient’s oral cavity at the first control; a) pseudo membrane in dextra-sinistra lateral of the tongue; b) macula in upper and lower lips; c) pseudo membrane in dextra buccal mucosa. a) b) c) figure 3. condition of the patient’s oral cavity at the second control; a) pseudo membrane disappears in upper and lower of lips mucosa; b) pseudo membrane disappears in palate durum; c) pseudo membrane disappears in dextra-sinistra lateral of the tongue. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p105–108 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p105-108 108femilian et al./dent. j. (majalah kedokteran gigi) 2022 june; 55(2): 105–108 is by staining koh and culture (sabouraud dextrose agar) or a biopsy performed on suspicion of hyperplastic candidiasis.3 some cases of acute pseudomembranous candidiasis will give a typical clinical appearance in the form of a pseudomembranous layer that can be scraped, and a reddish area left in the trace tissue scraped apc is a superficial infection of the epithelium’s outer layers that causes patchy white plaques or flecks to appear on the mucosal surface. when plaques are gently rubbed or scraped away, an area of erythema or even superficial ulceration is generally seen. the lesions of thrush or apc are easily identified because of their ubiquity, distinctive appearance and simplicity of removal, and a diagnosis of thrush is commonly established based on the appearance of the lesion.1 in this case, a specific clinical view of the apc was indicated with the presence of pseudo membrane that was easily rubbed and left a reddish area. nystatin is drug of choice for fungus infections. nystatin is a membrane-active polyene macrolide generated by streptomyces noursei strains that come in a variety of forms, including oral suspension, topical cream and oral pastille.10 nystatin binds to ergosterol membranes and plasma forms on pore fungus, producing intracellular potassium loss and fungicide action. in addition, autooxidation caused by nystatin induces additional cell damage. nystatin has a wide range of anti-candida properties.11 previous research revealed that nystatin’s effectiveness can be increased by giving it to patients for up to four weeks.12 appropriate information for the patient is important in the preparation of supporting examinations. specimen collection should be done before the application of any drug to the lesions. specimen collection should be done as soon as the patient arrives and no interventional measures should be taken on the lesion so that the specimen obtained can represent the actual condition. interventional measures such as wiping lesions using sterile kassa and warm water can remove the pseudomembranous layer containing hyphae so that false negative results are obtained. doctors have a role also in the success of supporting examinations with the right results. doctors must provide communication, information and education relating to diseases that may be suffered by patients, including causal factors, predisposing factors, conveying the stages of care that will be undergone by patients and supporting examination measures that must be carried out to enforce the diagnosis and prognosis of a disease. effective doctorpatient communication is a critical clinical function in developing a therapeutic doctor-patient relationship, which is the heart and soul of medicine.13 in the above case, the patient and the doctor experienced miscommunication about the actions to be taken before specimen collection. the patient’s parents had cleaned the pseudomembranous layer before the specimen was taken and it affected the results of the examination. poor information delivery creates ineffective communication, can lead to misconceptions and can result in a failure of therapy.4 the clinical appearance of the patient in this case was the presence of pseudo membrane that was easily rubbed and left an area of redness, which indicates a typical clinical appearance of acute pseudomembranous candidiasis. even although the koh painting test showed negative results and the patient was still treated using oral nystatin suspension, miscommunication and misinformation resulted in a false negative test. diagnosis will affect the success of the treatment. supportive examination is required to determine the definitive diagnosis. in conclusion, mistakes in patient preparation procedures in taking supporting examinations will result in false negative/positive results, so communication and education information regarding the preparation of supporting examinations for patients is extremely important. the delivery of good communication, information and education is the key to the success in the treatment of patients. references 1. lu s-y. oral candidosis: pathophysiology and best practice for diagnosis, classification, and successful management. j fungi. 2021; 7(7): 555. 2. lestari pe. peran faktor virulensi pada patogenesis infeksi candida albicans. stomatognaic j kedokt gigi. 2010; 7(2): 113–7. 3. corona do ca st el lot e l , ji ménezsor ia no y. cl i n ica l a nd microbiological diagnosis of oral candidiasis. j clin exp dent. 2013; 5(5): e279-86. 4. kee jwy, khoo hs, lim i, koh myh. communication skills in patient-doctor interactions: learning from patient complaints. heal prof educ. 2018; 4(2): 97–106. 5. patil s, rao rs, majumdar b, anil s. clinical appearance of oral candida infection and therapeutic strategies. front microbiol. 2015; 6: 1391. 6. martins n, ferreira icfr, barros l, silva s, henriques m. candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. mycopathologia. 2014; 177(5–6): 223–40. 7. williams d, lewis m. pathogenesis and treatment of oral candidosis. j oral microbiol. 2011; 3: 1–11. 8. mushi mf, loi n, mshana se. oral candidiasis in hiv-uninfected pediatric population in areas with limited fungal diagnosis: a case study from a tertiary hospital, tanzania. ther adv infect dis. 2021; 8: 20499361211016964. 9. national academies of sciences, engineering and m. improving diagnosis in health care. balogh ep, miller bt, ball jr, editors. washington, d.c.: the national academies press; 2015. p. 444. 10. kaur ip, kakkar s. topical delivery of antifungal agents. expert opin drug deliv. 2010; 7(11): 1303–27. 11. quindós g, gil-alonso s, marcos-arias c, sevillano e, mateo e, jauregizar n, eraso e. therapeutic tools for oral candidiasis: current and new antifungal drugs. med oral patol oral cir bucal. 2019; 24(2): e172–80. 12. lyu x, zhao c, hua h, yan z. efficacy of nystatin for the treatment of oral candidiasis: a systematic review and meta-analysis. drug des devel ther. 2016; 10: 1161–71. 13. ha jf, longnecker n. doctor-patient communication: a review. ochsner j. 2010; 10(1): 38–43. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i2.p105–108 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i2.p105-108 107107 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 107–110 research report introduction oral cavity cancer is a cancer that can be found throughout the world. according to the world health organization (who), there are an estimated 657,000 new cases of oral and pharyngeal cancer each year and more than 330,000 deaths. oral cancer can occur due to mutations of the p53 protein, which are triggered by a build-up of carcinogenic substances in the human body.1 the p53 protein will continue to mutate along with the proliferation of oral cancer cells, and this can be followed by the occurrence of unfolding proteins caused by an unstable cell microenvironment. to stabilise the protein, protein protectors called heat shock proteins (hsps) are needed. hsps are activated by a group of transcription factors known as heat shock factors (hsfs).1 the hsfs that play the leading role in regulating the chaperones transcription process are hsf1. hsf1 protects the proteome homeostasis of cancer cells through the activation of hsps so that the stabilization of oncoproteins is maintained and the cancer cells continue to grow and develop.2 due to its important role in carcinogenesis, hsf1 is targeted as a consideration in cancer therapy. flavonoid and isothiocyanate bioactive compounds are known to reduce the expression of hsf1.3 nagai et al.4 reported that the compound quercetin, which is a class of flavonoid, can reduce hsf1 expression in hela cells. yang et al.5 also stated that quercetin can significantly reduce hsf1 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p107–110 the potential of ethanolic extract of moringa oleifera leaves on hsf1 expression in oral cancer induced by benzo[a]pyrene vania syahputri, theresia indah budhy and bambang sumaryono department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: oral cancer is the sixth most common malignancy that occurs in the world, with more than 330,000 deaths a year. in cancer, mutations occur in proteins, accompanied by unfolding proteins, caused by the unstable micro-environment in cells. to stabilise this condition, protein protectors called heat shock proteins (hsps) are needed. hsps are activated by a group of transcription factors known as heat shock factor 1 (hsf1). hsf1 is a considered target in cancer therapy. moringa oleifera leaves are known to have anti-cancer properties because of bioactive compounds called flavonoid and isothiocyanate and are used as herbal therapy for cancer. purpose: to investigate the potential effect of ethanolic extract of moringa oleifera on hsf1 expression in oral cancer induced by benzo[a]pyrene. methods: this study used 25 male wistar rats divided into five groups consisting of the negative control group (k-), which was only given aquadest; the positive control group (k+), which was induced with benzo[a]pyrene and given aquadest; and treatment groups that were induced with benzo[a]pyrene and given moringa oleifera leaf extract at concentrations of 3.125% (p1), 6.25% (p2), and 9.375% (p3). examination of hsf1 expression was carried out by immunohistochemistry staining. data were analysed using the kruskal–wallis test and post-hoc tukey hsd. results: hsf1 expression in the p1, p2, and p3 groups decreased significantly compared to the k+ group. there were no significant differences between the p1, p2, and p3 groups (p > 0.005). conclusion: ethanolic extract of moringa oleifera leaves in three concentrations can decrease expression of hsf1 in oral cancer induced by benzo[a]pyrene. keywords: ethanolic extract of moringa oleifera; flavonoid; hsf1; isothiocyanate; oral cancer correspondence: theresia indah budhy, department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, jl. mayjen. prof. dr moestopo 47, surabaya 60132, indonesia. email: theresia-i-b-s@fkg.unair.ac.id mailto:theresia-i-b-s@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p107-110 108 syahputri et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 107–110 expression. sarkars et al.6 stated that isothiocyanates can reduce hsf1 and hsps expression so that the apoptosis of breast cancer cells can be induced. the gold standard for treating cancer nowadays is chemotherapy and surgery. but those therapies are known to have a negative impact on the patient due to their side effects, which can cause damage to normal cells and organs.7–9 therefore, there are several alternative treatments, one of which uses herbal plants, namely moringa oleifera. moringa oleifera leaves are a source of flavonoid and isothiocyanate components. the leaves have antioxidant, anti-inflammatory and anticancer benefits. several studies have shown these leaves can be used as an anti-neoproliferative agent that can inhibit the growth of cancer cells. moringa oleifera leaves have also been shown to be effective as safe anticancer agents at certain concentrations.10,11 based on this theory, further research is needed to see the potential of moringa oleifera leaf ethanol extract on hsf1 expression in oral cancer cells. the purpose of this study was to determine the potential of moringa oleifera leaf ethanol extract against hsf1 expression in oral cancer cells induced by benzo[a]pyrene. materials and methods this research was an experimental laboratory study with a posttest only group design that was approved by the committee of ethical clearance of health research, faculty of dentistry, universitas airlangga (592 / hrecc. fodm / ix / 2019). the experimental animals used were 25 male wistar rats (rattus norvegicus), with the addition of four male wistar rats examined histopathologically, weighing 160 grams, aged three months, and acclimated for seven days in a cage measuring 60 x 65 x 80 cm according to the laboratory standards in the animal laboratory unit of biochemistry laboratory, faculty of medicine, universitas airlangga. twenty-five male wistar rats were randomly divided into 5 groups consisting of a negative control group (k-), a positive control group (k+), treatment group 1 (p1), treatment group 2 (p2), and treatment group 3 (p3). in addition, four mice were sacrificed and examined clinically and histopathologically to establish whether cancer had formed or not. the kgroup of rats were not injected with benzo[a]pyrene and moringa oleifera leaf ethanol extract therapy. the k+ group was injected with benzo[a]pyrene but not given moringa oleifera leaf ethanol extract therapy. the p1, p2, and p3 groups were groups of rats induced with benzo[a]pyrene and injected with moringa oleifera leaf ethanol extract at concentrations of 3.125%, 6.25%, and 9.375%, respectively. five hundred grams of moringa oleifera leaves were obtained from kebun kelor lawang, malang, east java and dried for three months. the dried leaves were incubated at 600°c for 24 hours and then soaked in 96% ethanol solvent to soften them. the macerated mixture was put into a container, which was sealed and left for two days. after that, the mixture was filtered to obtain a clear liquid. this was then evaporated using a vacuum rotary evaporator at a temperature of 400°c. the moringa oleifera leaf extract was then subjected to phytochemical screening to detect the active compounds of the plant. the making of the phytochemical extracts and the screening were carried out at the surabaya laboratory of industry research and consultation. cancers in the k+, p1, p2 and p3 sample groups were induced by benzo[a]pyrene (sigma aldrich, saint louis, usa). benzo[a]pyrene induction was carried out in the biochemical laboratory experimental unit of the faculty of medicine, universitas airlangga. benzo[a]pyrene was used in the form of a solid powder with a dose of 8 mg / kgbb dissolved in the olivary oleum at a ratio of 2:1. induction was done by the injection of 0.2 ml of benzo[a] pyrene, using a syringe, 2–3-mm deep into the buccal mucosa of the wistar rats, twice a week for one month.12 then, a clinical examination was performed to check the success of the induction, by looking for signs of bumps on the buccal site.13 an hpa examination was also carried out by sacrificing four additional mice. histologically, cancer cells were characterized by anaplastic cell signatures, which are enlarged cell nuclei, varying core shapes, abnormal mitosis, nucleus:cytoplasmic ratio (1:1), hyperchromatic, irregular shape, and chromatin appear coarse and lumpy, that found in malignancy cases.14 moringa oleifera leaf ethanol extract was given to the treatment sample group in the following concentrations, 3.125% (p1), 6.25% (p2), and 9.375% (p3), and the rats were given ad libitum using 2 ml of insulin sonde every day for one month. after one month, the wistar rats were sacrificed to make histopathological preparations. the buccal mucosa tissue of the male wistar rats was fixed with 10% neutral buffered formalin (nbf) for further processing of the tissue by the paraffin method through several stages, namely dehydration, clearing, impregnation, and embedding. then the tissue was cut 4-mm thick with a rotary microtome. the network processing procedure was carried out at the anatomy pathology laboratory of the faculty of medicine, universitas airlangga, according to its standard operating procedures. immunohistochemical staining was carried out in the electron microscope laboratory of the faculty of medicine, universitas airlangga and was carried out in accordance with applicable procedures. immunohistochemical staining was used to check hsf1 expression with hsf monoclonal antibodies (santa cruz biotechnology, inc., usa) as a binder, which was then diluted (1:100) the chromogen used was dab chromogen and the staining was done with meyer’s hematoxylin. data collection was carried out by the counting technique, which observed hsf1 expression in five different fields, using a microscope (olympus microscope, tokyo, japan) with 400-times magnification. data analysis dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p107–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p107-110 109syahputri et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 107–110 used the kruskal–wallis test and the post-hoc tukey hsd test with the help of the statistical package for the social sciences (spss) (ibm spss, new york, usa) to find out significant differences between groups of variables. results figure 1 shows the preparations using a light microscope with a magnification of 400 to count the number of hsf1 expressions from each group on the cytoplasm designated by the black arrows. based on the research data, the positive control group (k+) showed high hsf1 expression compared to the negative control group (k-) and the treatment groups (p1, p2, and p3), while the treatment group 3 (p3) showed lower hsf1 expression than the other treatment groups (p1 and p2) (table 1). all the data were tested for normality (shapiro–wilk) and homogeneity (levene), but the data obtained were not normally distributed and not homogeneous because p was significantly less than 0.05. furthermore, a non-parametric test was performed using the kruskal-wallis test. the results showed that there was a significant difference of p = 0.000 (p < 0.05) between the sample groups, with hsf1 expression. after non-parametric tests using the kruskal–wallis test, a multiple comparison test was performed using the post-hoc tukey hsd test to identify significant differences between each sample group. table 2 shows that there were significant results concerning the k+ group and kgroup. the k+ group was significant for the p1, p2, and p3 groups and vice versa. the kgroup did not show significant results with the p2 and p3 groups, nor did the p1, p2, and p3 groups show significant differences from each other. discussion based on the results of the study, the mean expression of hsf1 in the k+ group was significantly higher compared to the kgroup. this contrast was due to the fact that hsf1 is over-expressed in an abnormal cell (e.g. cancer) compared to a normal cell. hsf1 is activated when there are stressors, such as changes in temperature, chemistry, or nutrition, that affect the cell environment. hsf1 in the form of monomers will be released under these circumstances and translocated to the nucleus. hsf1 monomers will be phosphorylated by protein kinase c, so hsf1 will be in active trimer form. trimer hsf1 will induce hsps that act as chaperones so that cellular homeostasis can be maintained.15,16 a b c e d table 2. post hoc tukey hsd test for hsf1 expression in each group groups p value kk+ p1 p2 p3 k0.000 0.013 0.085 0.179 k+ 0.000 0.000 0.000 p1 0.899 0.702 p2 0.994 p3 note: *p<0.05 table 1. mean and standard deviation of hsf1 expression in the five groups group sample mean sd k5 0.480 0.109 k+ 5 9.400 3.016 p1 5 3.712 0.326 p2 5 2.920 0.729 p3 5 2.560 0.517 figure 1. immunohistochemistry staining to see the expression of hsf1 in cytoplasma (indicated by arrows) (a) kgroup; (b) k+ group; (c) p1 group; (d) p2 group; (e) p3 group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p107–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p107-110 110 syahputri et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 107–110 when the cancer cell is still growing rapidly, there are many stressors that can affect its survival. in this case, hsf1 is widely expressed with the aim of maintaining the stability of cancer proteostasis, so that cancer growth can continue. this is in accordance with research showing that hsf1 expression is over-expressed in oral cancer cells compared to normal cells.17 hsf1 expression in the treatment groups (p1, p2, p3) was significantly lower compared to the k+ group. this is because the treatment groups were given moringa oleifera leaf ethanol extract while the k+ group was not given it. moringa oleifera leaf ethanol extract has isothiocyanates and flavonoids, which can inhibit hsf1 expression5,18, as its natural compounds. these can inhibit the phosphorylation of protein kinase c (pkc). this is a serine/threonine kinase that plays a role in several cellular activities, including cell proliferation, survival and apoptosis. pkc is an important mediator for cell survival in solid tumors.19 in cancer cells, pkc also regulates protein transcription, one of which is hsf1. if pkc is inhibited, the activation of hsf1 is inhibited and its expression decreases.5 the results of the treatment groups, p1, p2, and p3, showed no significant differences between them. there was no significant difference in the mean expression of hsf1 between the treatment groups presumably because moringa oleifera leaf ethanol extract has a very strong impact on cancer cells, so most of the concentration used can cause the death of cancer cells. the mean expression of hsf1 in group p1 (3.125%) compared with p2 (6.25%) and p3 (9.375%) showed that with high concentrations of moringa oleifera leaf ethanol extract, the expressions of hsf1 were lower. this was probably because many cancer cells had died. nararya20 stated that concentrations above 3.125% are toxic. it is suspected that in the p2 and p3 groups the expression of hsf1 reached lower amounts than the p1 group as a result of cancer cell death, along with other normal cell death. the conclusion of this study is that ethanolic extracts of moringa oleifera leaf concentration 3.125%, 6.25%, and 9.375% can reduce hsf1 expression in cancer cells in oral cancer. further studies are required to find the optimal concentration of moringa oleifera leaf ethanol extract that can decrease the expression of hsf1 in oral cancer. references 1. li d, yallowitz a, ozog l, marchenko n. a gain-of-function mutant p53-hsf1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress. cell death dis. 2014; 5(4): 1–11. 2. dai c, sampson sb. hsf1: guardian of proteostasis in cancer. trends cell biol. 2016; 26(1): 17–28. 3. chatterjee s, burns tf. targeting heat shock proteins in cancer: a promising therapeutic approach. int j mol sci. 2017; 18(9): 1–39. 4. nagai n, nakai a, nagata k. quercetin suppresses heat shock response by down regulation of hsf1. biochem biophys res commun. 1995; 208(3): 1099–105. 5. yang w, cui m, lee j, gong w, wang s, fu j, wu g, yan k. heat shock protein inhibitor, quercetin, as a novel adjuvant agent to improve radiofrequency ablation-induced tumor destruction and its molecular mechanism. chinese j cancer res. 2016; 28(1): 19–28. 6. sarkars r, mukherjee s, roy m. targeting heat shock proteins by phenethyl isothiocyanate results in cell-cycle arrest and apoptosis of human breast cancer cells. nutr cancer. 2013; 65(3): 480–93. 7. huang sh, o’sullivan b. oral cancer: current role of radiotherapy and chemotherapy. med oral patol oral cir bucal. 2013; 18(2): e233–40. 8. pearce a, haas m, viney r, pearson sa, haywood p, brown c, ward r. incidence and severity of self-reported chemotherapy side effects in routine care: a prospective cohort study. plos one. 2017; 12(10): 1–12. 9. kolokythas a. long-term surgical complications in the oral cancer patient: a comprehensive review. part i. j oral maxillofac res. 2010; 1(3): 1–10. 10. leone a, spada a, battezzati a, schiraldi a, aristil j, bertoli s. cultivation, genetic, ethnopharmacology, phytochemistry and pharmacology of moringa oleifera leaves: an overview. int j mol sci. 2015; 16(6): 12791–835. 11. gopalakrishnan l, doriya k, kumar ds. moringa oleifera: a review on nutritive importance and its medicinal application. food sci hum wellness. 2016; 5(2): 49–56. 12. budhy ti, istiati, sumaryono b, arundia i, khrisnanthi rs. hedyotiscorymbosa (l.) lamk the potential inhibitor extract of oral cancer cell progressivity in benzopyrene induced rattus novergicus. j int dent med res. 2018; 11(1): 312–7. 13. thalia a. potensi ekstrak daun kelor (moringa oliefera) terhadap ekspresi ki-67 pada sel kanker rongga mulut tikus wistar. thesis. surabaya: universitas indonesia; 2018. p. 33. 14. neville bw, damm dd, allen cm, chi ac. oral and maxillofacial pathology. 4th ed. st. louis: saunders; 2015. p. 286–7. 15. voisine c, orton k, morimoto ri. protein misfolding, chaperone networks, and the heat shock response in the nervous system. in: molecular neurology. elsevier; 2007. p. 59–76. 16. kim jy, yenari m. heat shock proteins and the stress response. in: primer on cerebrovascular diseases. 2nd ed. elsevier; 2017. p. 273–5. 17. wang q, zhang yc, zhu lf, pan l, yu m, shen wl, li b, zhang w, liu lk. heat shock factor 1 in cancer-associated fibroblasts is a potential prognostic factor and drives progression of oral squamous cell carcinoma. cancer sci. 2019; 110(5): 1790–803. 18. dayalan naidu s, suzuki t, yamamoto m, fahey jw, dinkovakostova at. phenethyl isoth iocya nate, a dua l activator of transcription factors nrf2 and hsf1. mol nutr food res. 2018; 62(18): 1–9. 19. chen z, forman lw, williams rm, faller d v. protein kinase c-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo. bmc cancer. 2014; 14: 1–15. 20. nararya sa. uji toksisitas daun kelor (moringa oleifera) terhadap sel fibroblas gingiva menggunakan uji mtt assay. thesis. surabaya: universitas airlangga; 2018. p. 41–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p107–110 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p107-110 144 the effect of acidulated phosphate fluoride application on dental enamel surfaces hardness edhie arief p and sri kunarti department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract enamel demineralization by acid is the first step of caries process. it has recently been shown that acidulated phosphate fluoride (apf) can maintain the hardness of enamel surface. the aim of this study was examine the effect of apf application in the hardest of enamel surface. fifty extracted teeth were cut at their crown, 40 teeth were taken randomly then divided into 4 groups, group 1 as the control, group 2 was treated with apf for 1 minute, group 3 for 4 minutes and group 4 for 7 minutes, then all the samples were washed with demineralized water. to see the effect of apf, all of the samples were soaked in lactic acid demineralization solution with ph 4,5 for 72 hours., the hardness of the surfaces of those samples before and after the treatment was measured by micro vickers hardness tester. the data were analyzed using one-way anova and lsd tests. in conclusion, 1.23% apf gel can reduce higher enamel demineralization. key words: acidulated phosphate fluoride, surface hardness, enamel demineralization correspondence: edhie arief p, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction dental caries is a multifactorial disease which is caused by some factors such as microorganism substrate, dental surface and time.1 organic acid resulted from carbohydrate glycolisis by microorganism decreases ph plaque and ph liquid around the teeth. along the next decades, many researches showing that there are acidogenic bacteria which can produce acid at ph lower than 12. bacteria which mostly have the role in dental caries is streptococcus, especially streptococcus mutans.2,3 streptococcus mutans metabolize carbohydrate to lactic acid. extra cellular bacterial enzyme such as glucocyltransferase and fructocyltransferase break the carbohydrat. than through glycolisis process, glucose is broke by bacteria and the results are two private molecules. this molecule is broke to lactit acid molecule. this lactit acid is strong acid which causes dental structure demineralization.4 many methods were done to prevent or to reduce caries risks, one of them is by making dental structure less dissolved towards acid through fluoride use.5 fluoride is effective anticaries agent when released into several forms or concentrations. fluoride necessity can be well fulfill both by systemically through water, food, vegetables, or supplements and topically through toothpaste, mouth-wash and topical application.6 topical fluoride is the prime protection agent after dental erosion. topical fluoride application is recommended for the children after permanent dental eruption and the patients with high caries risk.7 topical fluoride application which is done to the children with dental permanent in non-fluoride area for six months reduce caries risks at permanent teeth for 26%.8 there are several agents used as topical application, one of them is acidulated phosphate fluoride (apf). apf has been used at the countries with high caries activities, low consciousness level in prevention, and the system of dental health treatment that is not well organized yet.9 apf is sodium fluoride derivate in the form of solution, gel or powder which characterizes acid at ph 3 to 4 and characterizes buffer if it interacts with phosphate. apf has been tested in its use, both in the form of solution or gel, but gel is the form which is most frequently used. this agent has better ability in bonding to enamel calcium. it also non irritating and non staining that can be tolerated by adding the taste as well as easily accepted by patients. the affectivity of apf can be various, depends on the methods and frequency of its application.10 this agent is a mineral which can strengthen the enamel surfaces and prevent root caries as well as inhibit caries risks as the effect of saliva product which is less because of radiation therapy or chemotherapy. apf usually contains 2% sodium fluoride, 0.34% hydrogen fluoride and 0.98% phosphate acid. apf is in the form of solution, gel or foam.11 apf can be used for children 6 years up and adults with high caries risks but has contraindication to the patients who have hypersensitive reaction, the patients who have dental implant, patients with composite restoration, porcelain, compomer, and ionomer glass.12 the objectives of this study is to know the ability of apf in maintaining dental enamel surfaces hardness and 145arief p and kunarti:the effect of acidulated phosphate fluoride give the information about the use apf in reducing enamel demineralization so that it can be used as the model for dentists in choosing topical fluoride agents. materials and methods the materials that were used: 1.23% acidulated phosphate fluoride, 90% extra pure lactic acid (merck), demineralization solution which concise of 0,1 m lactic acid and 0.1% thymol at ph 4.513 the instruments that were used: ph meter, micro vickers hardness (osaka, japan). all samples from extracted teeth, which were intact in labial side. ten samples of group 1 (x1) were treated with apf agent by using cotton swab for 1 minute, 10 samples of group 2 (x2) for 4 minutes and group 3 for 7 minutes (x3). the teeth were cleaned using aquadest and dried, meanwhile 10 samples of controlling group (x4) are not put in to apf agent. forty samples were put in reaction tubes containing 5 ml of demineralization solution and closed tightly to avoid dehydration. then the tube was put into incubator with temperature 37 °c for 72 hours. after 3 days, the teeth are taken from the solution, then washed with water and measured back for their enamel surfaces (last hardness). the measurement of enamel surfaces hardness uses micro surface vickers hardness tester. indentation result can be seen at projector screen in the form of shadow shaping rhomb, the diagonal length is measured with micrometer. the measurement result, it could be known the hardness of enamel surfaces using the formula: 1.854 × p hvn = × d 2 description: hvn = sample hardness (kg/mm2) d = measurement result (mm) p = weight is given (kg) the measurement of enamel surfaces hardness was done for 3 times, at buccal side which was situated around the cut off vertical and horizontal line. data collected was gotten from the measurement of surfaces hardness before and after the treatment so was the control sample. the data were tested statically by using anova test with significance standard/ level p = 0.05. results the dental enamel surface hardness reductions after the application of apf were shown in table 1. from table 1, it can be seen that there are the reduction of enamel hardness at the groups with apf application for 1 minute, 4 minute, and 7 minute, as well as group without apf application. by using kolmogorov–smirnov test, it shown that all of the research groups have bigger value than 0.05 or at 0.152 (p > 0.05) that means all of the research groups data are normal, and homogeneity test with levene’s test shows the value 0,081 (> 0.05), then continued with one-way anova test to know the significance among the research groups. the highest average of enamel hardness reduction is found at 7 minutes apf application, then using one-way anova test it can be seen that there are significance differences for comparison among the four groups (p < 0.05). at table 2 there are significance differences for comparison among the groups, group 1, 4 and 7 minutes apf application as well as without apf application (p < 0.05). there are no significance differences between groups 4 and 7 minutes apf application. discussion the measurement of enamel surface hardness in this research is used as the evaluation towards chemical table 1. mean and standard deviation dental enamel surfaces hardness reduction after apf application for 1, 4, 7 minutes and without apf application in kg/mm2 groups n mean standard deviation 1 minute apf 4 minute apf 7 minute apf without apf 10 10 10 10 72.0875 19.4375 10.2461 202.4375 8.31821 3.66136 7.48215 9.01775 table 2. test of the decreased differences at enamel surfaces hardness using one–way anova and lsd 1 minutes apf 4 minutes apf 7 minutes apf without apf 1 minute apf 4 minute apf 7 minute apf 0.000* 0.000* 0.000* 0.000* 0.000** = significance difference (p < 0.05) 146 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 145-147 treatment methods. when fluoride concentration on enamel is above 100 ppm, caf2 is formed with thaw mechanism below: ca10(po4)6 (oh)2 + 2 of – + 8h+  10 caf2 + 6 hpo4 2– + 2 h2o. the above reaction is the reaction which is formed when the teeth is being applicated with topical fluoride. the higher fluoride concentration will produce bigger caf2 formation. furthermore, low ph in solution has strong influence in caf2 formation. it is because of the low solution ph can increase enamel delve a little bit of enamel and settles caf2 in big number on dental surfaces. 17 another research which is done using block enamel (5 × 5 × 2 mm) from incisive teeth which are soaked in 0.5 ml 1 m koh in room temperature for 24 hours shows that is found significance differences at the number of caf2 which is formed on enamel among grasps of apf gel application and controlling group. at enamel groups with apf application, the number of the formed caf2 is 31.72 ± 22.18 mg f/cm2 meanwhile at controlling group is 0.17 ± 0.04 mg f/cm2. these show that minerals in dental groups of apf application are greater than dental group without apf application. a b o u t t h e i r r e l a t i o n s h i p i n c l i n i c a l p r a c t i c e , demineralization of enamel as the result of acid which contacts with the teeth does not happen continuously and undoes for along time, it usually happens in 1 to 2 minutes. bitter taste usually stimulates the flow of saliva which is very effective as buffer system, but in permanent hyposalivation condition, neutralization from acid reflux will require longer time. effectiveness of apf application in caf2 formation which is useful in reducing enamel demineralization depends on the ph and duration of agent application so that can maintain the hardness of enamel surfaces. it concluded that apf gel can reduce high enamel demineralization as the consequence of acid, so it can maintain the hardness of enamel surface. references 1. kidd eam, bechal sj. dasar-dasar karies penyakit dan penanggulangannya. jakarta: egc; 1992. p. 58–93. 2. roth gi, calmes r. oral biology. st louis, baltimore, boston: the mosby company; 1981. p. 354–7. 3. bisla s. dental caries–a student project. 2000. available at: http: //www.sciweb.hfcc.com. accessed january, 2007. 4. burt ba, eklund sa. dentistry dental practice and the community. 5th ed. philadelphia: wb saunders company; 1999. p. 319–20. 5. samanarayake lp. essential microbiology for dentistry. 2nd ed. london: churchill livingstone; 2002. p. 217–23. 6. samuel smw, rubinstein c. microhardness of enamel restored with floride and non–flouride raleasing dental materials. braz dent j 2001; 12(1):35 8. 7. hayacibara mf, paes leme af, lima ybo, goncalves nclav, queiroz cs, gomes mj, kozlowski fc. alkali-soluble flouride deposition on enamel after professional application of topical fluoride in vitro. j appl oral sci 2004; 12(1):18–21. 8. robinson ds, bird dl. essential of dental assisting. 3rd ed. wb: saunders; 2001. p. 274–8. 9. hiratsuka k. regulation of sucrose–6–phosphate hydrolase activity in streptococcus mutans: characterization of the scr-rgene. 1998. available at: http:// rudyct.tripot.co.id. accessed january, 2007. bondings in enamel treatment procedures. the surfaces hardness measurement is used to evaluate demineralization, because it is found the positive correlation between enamel surfaces hardness with the lost of minerals from the teeth.13,14 apf gel has been used widely and its ability as anticariogenic agent has been evaluated through laboratory, clinically, and epidemiology studies. this in vitro study shows that apf is able to decrease the reduction of enamel surfaces hardness. the control group (without apf application) decrease enamel hardness higher than the other three groups. in control group, demineralization solution releases its hydrogen ion that reacts with apatite crystal teeth so that the apatite crystal becomes unstable. furthermore, water and soluble phosphate are formed which finally destroy enamel membrane. the reaction can be written as follows: ca10 + (po4)6(oh)2 + 8 h +  10 ca2+ + 6 hpo4 2– + 2 h2o. because of the destruction of enamel membrane, demineralization solution will penetrate deeper and dissolve apatite crystal. if acid atmosphere goes for long, demineralization will keep processing. in clinical use, application fluoride gel for 1 minute is more general in use than 4 or 7 minutes application. the biggest absorbance happens in first minute of application but the maximum result is by treatment for 4 minutes then decrease at 7 minutes. this is probably because of dental satiation in absorbing apf. apf is formulated in order the concentration of fluoride ion is sufficient to protect the teeth against the agents causing dental minerals lost. apf is used to increase fluoride absorbance in dental surfaces.15 many researches show that fluoride is more effective to protect enamel when the fluoride is in acid atmosphere than joining into enamel spaces.16 apf reacts with hydroxyapatite enamel forming fluoropatite which is more resistant towards acid if compared with the change of carbonate hydroxyapatite calcium. in short, apf helps to reduce the formation of cavity and increase of fluoride concentration in saliva quickly. beside that, apf leaves calcium fluoride layer which characterizes temporary on enamel surface. the research shows that flour with high concentration that is found in apf can protect caf2 on enamel surfaces so fluor reservoir along the remineralization process is formed when ph under goes the decrease. caf2 is the main product reaction that is formed along the application of topical fluoride on dental hard tissues. caf2 formation is important because caf2 roles in demineralization and remineralization phase on caries process. caf2 acts as the control of ph from ions f which is released along against the caries on enamel or in dental plaque. fluoride on caf2 is produced when ph in oral cavity goes down as the consequence of acid production. beside that the formation of caf2 on enamel surfaces can be gone quickly but this causes the formation of fluor apatite by fluor ion in enamel minerals. the formation of product is when fluor reacts with enamel depends on fluor concentration duration, ph, frequency and 147arief p and kunarti:the effect of acidulated phosphate fluoride 10. delbem acb, brighenti fl, mello vieira ae, cury ja. in vitro comparison of the cariostatic effect between topical application of fluoride gels and fluoride toothpaste. j appl oral sci 2004; 12(2):54–58. 11. craig rg. dental materials properties and manipulation. 7th ed. st. louis: mosby, inc; 2000. p. 34. 12. barnes cm, hlava gl. review of cdc recommendations for fluoride use. the preventive angle 2001; 2(2):22–4. 13. groenveld a, arends j. influence of ph and demineralization time on mineral content, thickness of surface layer and depth of artificial caries lesions. caries res. 9 st louis: mosby inc; 1975. p. 36–44. 14. featherstone jd. the caries balance: contributing factors and early detection. j california dent assoc 2003 february; 31(2):17–20. 15. delbem acb, brighenti fl, mello vieira ae, cury ja. in vitro comparison of the cariostatic effect between topical application of fluoride gels and fluoride toothpaste. j appl oral sci 2004; 12:20. 16. jones l, lekkas d, hunt d, mcintyre j, rafir w. studies on dental erosion: an in vivo-in vitro model of endogenous dental erosion–its application to testing protection by fluoride gel application. australian den j 2002; 47(4):304–8. 17. fejerskov, kidd e. dentistry dental practice and the community. 5th ed. philadelphia: wb saunders company; 2003. p. 252–60. 5252 research report dental journal (majalah kedokteran gigi) 2018 june; 51(2): 52–56 differences in mucin expression in the submandibular glands of rats during peridontitis induction nunuk purwanti,1 banun kusumawardhani,2 and kwartarini murdiastuti3 1department of biomedical dental science, faculty of dentistry, universitas gadjah mada, yogyakarta indonesia 2department of biomedical sciences, faculty of dentistry, universitas jember, jember indonesia 3department of periodontology, faculty of dentistry, universitas gadjah mada, yogyakarta indonesia abstract background: porphyromonas gingivalis (pg) produces lipopolysacharide (lps) which acts as a stimulator of inflammation in periodontal tissues. periodontitis-induced apoptosis and vacuolation of the salivary gland, therefore, causes hyposalivation. mucin secretion is produced by the submandibular gland under stimulation by the cholinergic and adrenergic receptors. both forms of stimulation influence the volume of mucin secretion. mucin saliva plays an important role in the early stages of pg colonization in the oral cavity. on the other hand, it serves to protect against bacterial invasion. purpose: the aim of this research was to identify differences in mucin expression in the submandibular gland during periodontitis induction. methods: 32 male wistar rats were assigned to either a sham periodontitis or a periodontitis group. the former group received a daily injection of a vehicle solution (n = 16), while members of the periodontitis induction group (n=16) were injected each day with 500 µl of pg 108 into the mesial area of the upper molar. mucin in the submandibular gland was analyzed at the 7th, 14th, 21th and 28th days after injection by means of periodic acid schiff (pas) staining. results: 28 days after injection mild gingivitis was developed in the periodontitis experiment group. junctional epithelium (je) thickness decreased gradually following the increase of pg injection periods (p<0.05). however, mucin expression increased prominently at 7th, 14th, and 21th days after injection and decreased on day 28th after pg injection. mucin was expressed in the duct cells of the submandibular gland. conclusion: the result of this study suggests that there are different levels of mucin expression in the submandibular gland during periodontitis induction. keywords: mucin; submandibular gland; periodontitis; porphorymonas gingivalis (pg) correspondence: nunuk purwanti, department of biomedical dental science, faculty of dentistry, universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: n_purwanti@mail.ugm.ac.id introduction periodontal disease has been renowed to cause inflammation in the gingiva, followed by connective tissue damage, alveolar bone resorption and systemic disease, including: atherosclerosis, rheumatoid arthritis, preterm-low birth weight, chronic kidney disease, diabetes and respiratory problems.1–5 one of the bacteria causing periodontitis is porphyromonas gingivalis (pg), an anaerobic gram negative oral bacteria that induces inflammation in periodontal tissue.2,3 pg fimbria activates the innate and adaptive human immune system to induce monocytes and poorly activated epithelial cells to produce interleukin il-6, il-8, macrophage colony stimulating factor (m-csf) and tumor necrosis factor (tnf).6,7 this pg virulence factor in periodontal areas will spread throughout the human body as one etiology of systemic disease. saliva plays an important role in the early-entry stages of pg bacteria in the oral cavity. furthermore, it helps the attachment and initiation of pg bacterial colonies on tooth surfaces and soft oral tissues. in the latest stages of infection, pg bacteria begins to form biofilm binding to other bacteria through saliva mediation.8 saliva performs multiple functions including: antibacterial, antimicrobial, antifungal, antiviral, immunity, clearance, wound healing and tissue repair9 due to its significant protein, carbohydrate and mineral content. saliva components have been used widely as a biomarkers dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p52–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p52-56 53 purwanti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 52–56 of systemic disease.10,11 mucin, as one component of saliva, is produced in large quantities in the submandibular gland. 20 types of mucin have been identified, five of them found in saliva, namely: muc5b, muc7, muc1, muc4, and muc16.11,12 mucin forms a slimy, viscoelastic film which coats all surfaces of the oral cavity. this layer acts as an important lubricant of opposing surfaces during mastication, swallowing and speech.9 the secreted salivary mucins, muc5b and muc7 modulate oral microorganisms.13 mucin enables bacteria to adhere to oral surfaces.11,14 other research shows that adolescents presenting a very high incidence of dental caries disease had increased levels of muc1 and muc5b, but decreased muc7 protein levels.13 saliva secretion is dynamic and susceptible to the effects of aging, systemic disease, medicine intake and radiotherapy treatment of the head and neck. rat models have been used in cases of experimental periodontitis in order to analyze changes in saliva secretion. these have produced similar results to those observed in humans.15 however, changes in the mucin production of the salivary glands during the periodontitis process have not been welldocumented in the literature on the subject. previous studies have demonstrated that proinflammatory il-1, il-6 tnf-α, ifn-δ and pg infection increase muc1 genes in human oral epithelial cells. however, no effect was observed after pg lps treatment.16 on the other hand, incubation of acinar cells in the sublingual gland with lps from pg was shown to produce a decrease in salivary mucin synthesis.17 the purpose of this study was to determine differences in mucin expression in the submandibular gland during periodontitis induction. mucin was used as a parameter considering its important role as one of the protective components of saliva. the results of this study will be used to develop mucin saliva as a biomarker of diagnostic and control tools in periodontitis therapy. materials and methods the experimental protocol of this study was approved by the research ethics committee, faculty of dentistry, universitas gadjah mada (no.00377/kkep/fkg-ugm/ ec/2015). 32 male, 8-week old sprague dawley rats with an average body weight of 150-200gms were involved. housed in standard conditions with ad libitum access to food and water, the subjects were randomly divided into two main groups, those receiving periodontitis induction and others subjected to sham induction. periodontitis induction was performed by means of a slightly modified kusumawardhani procedure .18 periodontitis induction groups received a daily 500 µl injection of pg 108 in the mesial area of the upper first molar, while the sham periodontitis group received a daily injection of a placebo solution. the peridontitis and sham periodontitis groups were subsequently divided into subgroups according to the duration of the injection period, i.e., 7, 14, 21 and 28 days. each group consisted of four models. during the injection period, the severity of experimentally induced periodontitis was monitored using a periodontal probe to ascertain the extent of pocket periodontal manifestation.19 the subjects were sacrificed one day after the last induction during their injection period by means of an overdose of intramuscular anesthesia. their maxillary and submandibular glands were removed and separated from muscle and soft tissue for the histological process. the submandibular gland and maxillary area were immersed in 10% buffer formalin for 24 hours. mandibles were incubated in 10% formic acid solution to promote decalcification and subsequently embedded in paraffin. they were then subjected to hematoxycilin eosin (he) staining, while the submandibular glands were processed for periodic acid shift (pas) staining.20,21 all tissues were examined in three areas under a light microscope. periodontal nuclei cell were stained blue by hematocylin, whereas eosin stains cytoplasm and extracellular matrix varying degrees of pink. the thickness of je was measured by counting the number of epithel layers from the cemento–enamel junction (cej) to the most coronal and apical sections of je.22 the pas positive gland staining produced a magenta-purple colour in the duct cell. the mucin level was measured based on the density of the mucin cell expression in the submandibular gland. since there is no standard cellular density calculation for mucin expression cells, the standard based on the results of the research reported here was arranged and presented in figure 1. 1 figure. 1. mucin expression-cell density standard, 1=less than 25% of cells express mucin; 2 = 25%50% of cells express mucin; 3 = 50%-75% cell expressed mucin; 4 = more than 75% of cells express mucin. magenta color indicates the presence of mucin (black arrowhead). 7 14 21 28 number of days elapsed after induction figure 2. periodontal pocket manifestation was examined in the molar region by means of a periodontal probe. increasing probe depth occurred after 28 days of induction in contrast to days 21, 14, and 7. p: periodontitis induction, s: sham induction. 1 2 3 4 s p figure 1. mucin expression-cell density standard, 1=less than 25% of cells express mucin; 2 = 25%-50% of cells express mucin; 3 = 50%-75% cell expressed mucin; 4 = more than 75% of cells express mucin. magenta color indicates the presence of mucin (black arrowhead). 2 3 41 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p52–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p52-56 54purwanti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 52–56 while periodontitis was confirmed by examining the thickness of the mesio buccal je of the upper molar,23 a kruskal wallis test was used to analyze differences between the four groups followed by a mann whitney test. the statistical significance level was set at p<0.05. results gingival inflammation was detected in the periodontitis induction group due to the increased probing depth on 1 figure. 1. mucin expression-cell density standard, 1=less than 25% of cells express mucin; 2 = 25%50% of cells express mucin; 3 = 50%-75% cell expressed mucin; 4 = more than 75% of cells express mucin. magenta color indicates the presence of mucin (black arrowhead). 7 14 21 28 number of days elapsed after induction figure 2. periodontal pocket manifestation was examined in the molar region by means of a periodontal probe. increasing probe depth occurred after 28 days of induction in contrast to days 21, 14, and 7. p: periodontitis induction, s: sham induction. 1 2 3 4 s p figure 2. periodontal pocket manifestation was examined in the molar region by means of a periodontal probe. increasing probe depth occurred after 28 days of induction in contrast to days 21, 14, and 7. p: periodontitis induction, s: sham induction. day 28 of treatment which had not occurred in the other groups (figure 2). je conversion to pocket epithelium is regarded as a hallmark in the development of gingivitis into periodontitis. therefore, to confirm whether periodontitis induction was associated with inflammation in the periodontal tissues in this research, the thickness of the je molar was examined by means of he staining.23 the results are contained in figures 3 and 4. as shown in figures 3 and 4, the number of je layers decreased in parallel with the number of days of 2 7 14 21 28 number of days elapsed after induction figure 3. changes in je thickness after periodontitis induction (p). data compared to sham (s) periodontitis induction on the same days (day 7, day 14, day 21 and day 28). the arrowhead ( ) indicated je. picture was taken at 200x magnification. figure 4. thickness of je molar teeth during periods of treatment. p s figure 3. changes in je thickness after periodontitis induction (p). data compared to sham (s) periodontitis induction on the same days (day 7, day 14, day 21 and day 28). the yellow arrowhead ( 2 7 14 21 28 number of days elapsed after induction figure 3. changes in je thickness after periodontitis induction (p). data compared to sham (s) periodontitis induction on the same days (day 7, day 14, day 21 and day 28). the arrowhead ( ) indicated je. picture was taken at 200x magnification. figure 4. thickness of je molar teeth during periods of treatment. p s 2 7 14 21 28 number of days elapsed after induction figure 3. changes in je thickness after periodontitis induction (p). data compared to sham (s) periodontitis induction on the same days (day 7, day 14, day 21 and day 28). the arrowhead ( ) indicated je. picture was taken at 200x magnification. figure 4. thickness of je molar teeth during periods of treatment. p s ) indicated je. picture was taken at 200x magnification. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p52–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p52-56 55 purwanti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 52–56 periodontitis induction. reduction in je thickness began to occur on day 14 in the periodontitis induction groups. this je thinning continued on days 21 and 28. the lowest thickness of je was occurred in the periodontitis induction after 28 days. while the thickest was observed in the sham group on day 14. mucin saliva was detected by the presence of a magenta coloring in the cytoplasm of the duct cells. as seen in figure 5, cell density which expressed mucin tended to increase up to days 21, but then decreased on day 28. the highest density was seen in rat periodontitis on day 21. the density of cells expressing mucin was analyzed statistically using a kruskal wallis test whose results indicated significant difference (p=0.0001). subsequent statistical analysis was performed by means of a mann whitney test to investigate differences in cell expressing mucin density in the periodontitis and sham groups of the same duration. the results showed there to be significant difference between the periodontitis and sham periodontitis groups on day 7 (p=0.008), day 14 (p=0.013), day 21 (p=0.008) and day 28 (p=0.004). discussion according to the results of this study, injections of pg decreased je thickness. depletion of the epithelial layer which facilitates the penetration of bacterial products into the deeper periodontal tissue was found on day 14. previous studies have shown that a third lps application induced the destruction of je22 which was composed of an epithelium layer with no keratin in the cell surface.24 this condition causes a decrease in the microscopic defensive system against injury. a previous study reported that proteinase from pg reduces epithelial cell adhesion to extracellular matrices, morphology changes and apoptosis.24 high expression of tunel and m30cytodeath as apoptosis markers results in the reduction of oral epithelial thickness.25 this study found that je thinning continued on days 21 and 28 and was confirmed by histology analysis that the method of injection of pg induced periodontitis, salivary glands will respond to oral inflammation by increasing the molecular synthesis of acinar cells to improve salivary protection function. mucin is one source of molecular protection in saliva which is produced in large quantities in the submandibular gland. it has been established that lps pg influences mucin salivary secretion in the sublingual gland cells of rats.17 it can be seen from this study that pas is strongly expressed in the duct cell, but very weakly in acinar cells. a previous study reported that mucin is expressed in the acinar duct and excretory duct cells.26 the weakness of mucin expression in acinar cells is probably due to the continuing presence of mucin in the form of secretory granules which cannot be detected by means of pas staining.27 saliva in patients suffering from chronic and aggressive periodontitis showed an increase in mucin and salivary amylase concentration.28 as a part of secretory salivary protein, mucin is produced by the acinar cells under the control of the autonomic nervous system, while secretion of muc5b is controlled by the parasympathetic nerve.29 it has been reported that the submandibular gland produces mucin regulated by adrenergic and cholinergic signaling.30 changes in diseases and the environment regulate sympathetic nervous system responses. this pathway may be involved in systemic responses included modulation pain responses and inflammation.31 this study showed that mucin expression tends to increase with the duration of treatment involving injections. it has been firmly established that during the initial stages of bacterial infection, goblet and epithelial cells produce more mucin to prevent bacteria colonization. on the other hand, mucin facilitates bacteria adherence in the epithelia, showing that pathogenic bacteria compete with the protective function of mucin.12,27 an increased mucin expression between days 7 and 21 would produce a double function of mucin. an increase in mucin production in saliva has been reported in oral disease.13,28 it has been established that the mucin submandibular gland is produced by the cholinergic and adrenergic system. lps pg induces inflammation through several mechanisms. lps binds tlr receptor to induce secretion inflammatory cytokine, nos and cox2. evoked nos 2 7 14 21 28 number of days elapsed after induction figure 3. changes in je thickness after periodontitis induction (p). data compared to sham (s) periodontitis induction on the same days (day 7, day 14, day 21 and day 28). the arrowhead ( ) indicated je. picture was taken at 200x magnification. figure 4. thickness of je molar teeth during periods of treatment. p s figure 4. thickness of je molar teeth during periods of treatment. 3 figure 5. mucin expression-cell density of submandibular gland on specific days of periodontitis induction. data is presented as the median, *p<0.05; significantly different in both groups on the same day. * * * * figure 5. mucin expression-cell density of submandibular gland on specific days of periodontitis induction. data is presented as the median, *p<0.05; significantly different in both groups on the same day. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p52–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p52-56 56purwanti, et al./dent. j. (majalah kedokteran gigi) 2018 june; 51(2): 52–56 induces cholinergic system.32 the cholinergic system increases calcium concentration in the acinar cells of the submandibular glands. furthermore, calcium evokes mucin secretion.30 on the other hand, lps pg generates cytosolic phospholipase a2 (cpla2) through up-regulation in the mapk/erk signaling pathway in the sublingual gland cell of rats resulting in cpla2 activated endhothelin-1 mucin secretion.17 a decrease in mucin expression occurred on day 28 when periodontitis conditions were more severe than on day 21, possibly due to different signaling pathways being involved on each occasion. further research is required to confirm which signaling pathways are involved in mucin secretion in the submandibular gland. it can be concluded from this study that mucin expression in the submandibular glands of rats differed during periodontitis induction. the expression of mucin increased gradually following the day of periodontitis induction with the highest level occurring on day 21 and decreasing on day 28. acknowledgements this research was funded by dana masyarakat 2015 of the faculty of dentistry, universitas gadjah mada. references 1. yu km, inoue y, umeda m, terasaki h, chen zy, iwai t. the peridontal anaerobe porphyromonas gingivalis induced platelet activation and increased aggregation in whole blood by rat model. thromb res. 2011; 127(5): 418–25. 2. kim d, lee g, huh yh, lee sy, park kh, kim s, kim j, koh j, ryu j. nampt is an essential regulator of ra-mediated periodontal inflammation. j dent res. 2017; 96(6): 703–11. 3. puertas a, magan-fernandez a, blanc v, revelles l, o’valle f, pozo e, león r, mesa f. association of periodontitis with preterm birth and low birth weight: a comprehensive review. j matern neonatal med. 2018; 31(5): 597–602. 4. han ss, shin n, lee sm, lee h, kim dk, kim ys. correlation between periodontitis and chronic kidney disease in korean adults. kidney res clin pract. 2013; 32(4): 164–70. 5. otomo-corgel j, pucher jj, rethman mp, reynolds ma. state of the science: chronic periodontitis and systemic health. j evid based dent pract. 2012; 12(3 suppl.): 20–8. 6. mysak j, podzimek s, sommerova p, lyuya-mi y, bartova j, janatova t, prochazkova j, duskova j. porphyromonas gingivalis: major periodontopathic pathogen overview. j immunol res. 2014; 2014: 1–8. 7. liu j, tang x, li c, pan c, li q, geng f, pan y. porphyromonas gingivalis promotes the cell cycle and inflammatory cytokine production in periodontal ligament fibroblasts. arch oral biol. 2015; 60(8): 1153–61. 8. enersen m, nakano k, amano a. porphyromonas gingivalis fimbriae. j oral microbiol. 2013; 5: 1–10. 9. dawes c, pedersen aml, villa a, ekström j, proctor gb, vissink a, aframian d, mcgowan r, aliko a, narayana n, sia yw, joshi rk, jensen sb, kerr ar, wolff a. the functions of human saliva: a review sponsored by the world workshop on oral medicine vi. arch oral biol. 2015; 60(6): 863–74. 10. kawas s al, rahim zha, ferguson db. potential uses of human salivary protein and peptide analysis in the diagnosis of disease. arch oral biol. 2012; 57: 1–9. 11. frenkel es, ribbeck k. salivary mucins in host defense and disease prevention. j oral microbiol. 2015; 7: 1–9. 12. hannig c, hannig m, kensche a, carpenter g. the mucosal pellicle – an underestimated factor in oral physiology. arch oral biol. 2017; 80: 144–52. 13. gabryel-porowska h, gornowicz a, bielawska a, wójcicka a, maciorkowska e, grabowska sz, bielawski k. mucin levels in saliva of adolescents with dental caries. med sci monit. 2014; 20: 72–7. 14. corfield ap. mucins: a biologically relevant glycan barrier in mucosal protection. biochim biophys acta. 2015; 1850: 236–52. 15. nakamura-kiyama m, ono k, masuda w, hitomi s, matsuo k, usui m, nakashima k, yokota m, inenaga k. changes of salivary functions in experimental periodontitis model rats. arch oral biol. 2014; 59(2): 125–32. 16. li x, wang l, nunes dp, troxler rf, offner gd. pro-inflammatory cytokines expression in oral epithelial cells. j dent res. 2003; 82(11): 883–7. 17. s l o m i a ny bl , s l o m i a ny a . p o r p hy r o m o n a s g i n g iva l i s lipopolysaccharide-induced cytosolic phospholipase a2 activation interferes with salivary mucin synthesis via platelet activating factor generation. inflammopharmacology. 2006; 14(3–4): 144–9. 18. kusumawardani b, soesatyo mh, dasuki d, asmara w. maternal endotoxin-induced fetal growth restriction in rats: fetal responses in toll-like receptor. dent j (maj ked gigi). 2012; 45(3): 144. 19. huang s, lu f, zhang z, yang x, chen y. the role of psychologic stress-induced hypoxia-inducible factor-1α in rat experimental periodontitis. j periodontol. 2011; 82(6): 934–41. 20. cao x, liu x, hao z , ma c, mao z. establishment of rat submandibular gland squamous cell carcinoma induced by dmba. hua xi kou qiang yi xue za zhi =. 2000; 18(3): 156–8. 21. nonose r, spadari app, priolli dg, máximo fr, pereira ja, martinez car. tissue quantification of neutral and acid mucins in the mucosa of the colon with and without fecal stream in rats. acta cir bras. 2009; 24(4): 267–75. 22. noguchi s, ukai t, kuramoto a, yoshinaga y, nakamura h, takamori y, yamashita y, hara y. the histopathological comparison on the destruction of the periodontal tissue between normal junctional epithelium and long junctional epithelium. j periodontal res. 2017; 52: 74–82. 23. mohamed a. the effect of diabetes mellitus on the thickness of gingival junctional epithelium. thesis. yogyakarta: universitas gadjah mada; 2011. p. 1-72. 24. bosshardt dd, lang np. the junctional epithelium: from health to disease. j dent res. 2005; 84: 9–20. 25. brant jmc, vasconcelos ac, rodrigues l v. role of apoptosis in erosive and reticular oral lichen planus exhibiting variable epithelial thickness. braz dent j. 2008; 19(3): 179–85. 26. lombaert ima, brunsting jf, weirenga pk, faber h, stokman ma, kok t, visser wh, kampinga hh, de haan g, coppes rp. rescue of salivary gland function after stem cell transplantation in irradiated glands. plos one. 2008; 3(4): 1–13. 27. dhanisha ss, guruvayoorappan c, drishya s, abeesh p. mucins: structural diversity, biosynthesis, its role in pathogenesis and as possible therapeutic targets. crit rev oncol hematol. 2018; 122: 98–122. 28. acquier ab, busch l, pita akdc, sanchez ga. comparison of salivary levels of mucin and amylase and their relation with clinical parameters obtained from patients with aggressive and chronic periodontal disease. j appl oral sci. 2015; 23(3): 288–94. 29. proctor gb, carpenter gh. regulation of salivary gland function by autonomic nerves. auton neurosci basic clin. 2007; 133: 3–18. 30. busch l, borda e. signaling pathways involved in pilocarpineinduced mucin secretion in rat submandibular glands. life sci. 2007; 80(9): 842–51. 31. morris ke, st. laurent cd, hoeve rs, forsythe p, suresh mr, mathison rd, befus ad. autonomic nervous system regulates secretion of anti-inflammatory prohormone smr1 from rat salivary glands. am j physiol cell physiol. 2008; 296(3): c514–24. 32. correia pn, carpenter gh, paterson kl, proctor gb. inducible nitric oxide synthase increases secretion from inflamed salivary glands. rheumatology. 2010; 49: 48–56. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i2.p52–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i2.p52-56 � vol. 43. no. 1 march 2010 n o m a m a n a g e m e n t i n a c h i l d w i t h s y s t e m i c l u p u s erythematosus irna sufiawati1, asri arum sari2, budi setiabudiawan3, and rahmat gunadi4 1 department of oral medicine, faculty of dentistry, padjadjaran university 2 department of oral surgery, faculty of dentistry, padjadjaran university 3 department of pediatric, faculty of medicine, padjadjaran university 4 department of allergy and immunology, faculty of medicine, padjadjaran university bandung indonesia abstract background: n��a, als� kn���n as cancr�� �ris, is an �r��acial gangrene, ���ic� d�ring its ��l�inating stage ca�ses �r�gressive and ��tilating destr�cti�n �� t�e in�ected tiss�es. ��e disease �cc�rs �ainly in c�ildren ��it� �aln�triti�n, ���r �ral �ygiene and de�ilitating c�nc�rrent illness. purpose: ��e ai� �� t�is �a�er ��as t� re��rt a �niq�e case �� n��a ass�ciated ��it� syste�ic l���s eryt�e�at�s�s in an 8-year-�ld ��y. case: an 8-year-�ld ��y re�erred t� oral �edicine de�art�ent c���laining a���t an �lcer at t�e le�t c�rner �� �is ���t� ��r 1 ��nt�, �ain��l and di��ic�lty in ��ening t�e ���t�. ��e �atient ��as diagn�sed syste�ic l���s eryt�e�at�s�s since 14 ��nt�s �e��re and �ad �een given i���n�s���ressive t�era�y. ��e �atient ��as als� diagn�sed severe �aln�triti�n. hae�at�l�gic investigati�ns revealed ane�ia. case management: pan�ra�ic radi�gra��y ��as �er��r�ed t� c�eck ��r dental �r �eri�d�ntal ��ci �� in�ecti�n, ��t n� a�n�r�alities ��ere �resent. ��e �icr��i�l�gy e�a�inati�n revelaed f�s��acteri�� necr����r��, sta��yl�c�cc�s a�re�s, and kla�siella. ��e �atient �as �een treated ��it� �ral irrigati�n �sing �ydr�gen �er��ide, saline and 0.2% c�l�r�e�idine, t��s �el�ed t� sl��g� t�e necr�tic tiss�e. oral anti�i�tics and analgesics ��ere �rescri�ed. ��e �atient ��as ad�itted t� ��s�ital �nder t�e care �� a �ediatrician, allergy and i���n�l�gy s�ecialist, and a n�triti�nist. ��e res�lt �� t�e c���re�ensive disease �anage�ent s����ed t�at t�e lesi�n �ealed c���letely, ��t leaving a scar �n �is c�rner �� t�e ���t�. �ts ��ysical e��ects are �er�anent and �ay req�ire rec�nstr�ctive s�rgery t� �e re�aired �y �ral s�rge�n. conclusion: n��a is n�t a �ri�ary disease, t�ere are vari��s �redis��sing �act�rs �s�ally �recede its �cc�rrence. ��e �anage�ent �� n��a req�ires a ��ltidisci�linary a��r�ac�. key words: n��a, syste�ic l���s eryt�e�at�s�s, �aln�triti�n, �anage�ent abstrak latar belakang: n��a, dikenal se�agai cancr�� �ris, adala� gangren �ada daera� �r��asial, yang �enye�a�kan ker�sakan �r�gresi� dari jaringan yang terin�eksi. penyakit ini terjadi ter�ta�a �ada anak dengan gizi ��r�k, kese�atan ��l�t yang ��r�k dan �enyakit yang �ele�a�kan. tujuan: �akala� ini �ert�j�an �nt�k �ela��rkan se��a� kas�s n��a yang �nik �ada se�rang anak laki-laki �er�sia 8 ta��n yang �enderita l���s erite�at�s�s siste�ik. kasus: se�rang anak laki-laki 8 ta��n dir�j�k ke bagian �l�� penyakit ��l�t, �engel�� adanya l�ka di s�d�t ��l�t se�ela� kiri yang tela� diderita sela�a 1 ��lan, terasa sangat sakit dan s�lit �e���ka ��l�t. pasien didiagn�sis l���s erite�at�s�s siste�ik sejak 14 ��lan se�el��nya dan tela� di�erikan tera�i i��n�s��resi�. pasien j�ga didiagn�sis �enderita �aln�trisi yang �erat. pe�eriksaan �e�at�l�gi �en�nj�kkan �asien �enderita ane�ia. tatalaksana kasus: radi�gra�i �an�ra�ik dilak�kan �nt�k �e�eriksa ��k�s in�eksi dental ata� �eri�d�ntal, teta�i tidak dite��kan adanya kelainan. pada �e�eriksaan �ikr��i�l�gi dite��kan adanya f�s��acteri�� necr����r��, sta��yl�c�cc�s a�re�s, dan kla�siella. pera��atan �ada �asien �eli��ti irigasi �ada daera� gangren dengan �idr�gen �er�ksida, lar�tan salin dan kl�r�eksidin 0,2% �nt�k �e��ersi�kan jaringan nekr�tik. pasien j�ga di�erikan anti�i�tik dan analgesik. pasien dira��at di r��a� sakit di �a��a� �era��atan d�kter s�esialis anak, d�kter s�esialis alergi i��n�l�gi, dan a�li gizi. hasil �enatalaksanaan �enyakit secara k���re�ensi� �e��erli�atkan adanya �enye����an, teta�i �eninggalkan jaringan �ar�t �ada s�d�t ��l�tnya. kelainan �isik terse��t �ersi�at �er�anen dan �e�erl�kan �e��eda�an rek�nstr�kti� �le� d�kter gigi s�esialis �eda� ��l�t. kesimpulan: n��a ��kanla� �enyakit �ri�er, terda�at �er�agai �akt�r �redis��sisi yang �iasanya �enda��l�i terjadinya �enyakit terse��t. pengel�laan n��a �e�erl�kan �endekatan ��ltidisi�lin. kata kunci: n��a, l���s erite�at�s�s siste�ik, �aln�trisi, �enatalaksanaan c�rres��ndence: irna sufiawati, c/o: bagian ilmu penyakit mulut, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i no. 1 bandung 40132. e-mail: irnasufiawati@yahoo.com case report �sufiawati, et al.: noma management in a child with systemic lupus erythematosus his mouth and difficulty in opening the mouth. the patient felt painful while he was eating. he had intermittent fever for the last one month. for the next 4 months, the patient kept complaining about his stomachache. he just ate 3 spoons of food daily, drank 5 glasses of milk, and seldom ate at night. the patient has been suffering from sle since 14 months ago before being admitted to the hospital and he was treated out of the hospital at department of allergy and immunology hasan sadikin hospital regularly. the result of systemic lupus erythematosus activity index (sledai) examination showed number 12 meaning that he was in moderate of sle disease activity. hematologic investigations showed anemia (haemoglobin 9.3 g/dl, hematocrit 28 %). the patient was diagnosed moderate malnutrition. the patient had also aphthous stomatitis and oral candidiasis histories. some medicines that he consumed were prednisone 3 × 2.5 mg daily, rocatrol caps 1 × 0.25 mcg daily, ca carbonate 3 × 250 mg daily, ranitidine tablet 150 mg 2 × 1/3 tablet daily, supplements and 1300 kcal diet. the patient appeared malnourished and weighed only 14 kg. his temperature was 38,5° c at the time, but all other vital signs were within normal limits. extra oral examination revealed necrotizing tissue at the left corner of his mouth about 2 × 2 cm in size surrounded by oedematous tissue showing a mild to moderate erythema (figure 1). the left submandibular lymph nodes was mildly painful, palpable, tender, and mobile. submental, right mandibular, and cervical lymph nodes were unpalpable. intraoral examination couldn’t be applied because of restricted mouth opening. it was also not possible to obtain intraoral photographs. figure 1. day 1. extraoral photographs showed the swelling and crust from the corner of the mouth. case management the patient was referred to microbiology laboratory to identify the microorganism of the necrotizing tissue. orthopantomogram (opg) was performed to check for dental or periodontal foci of infection, but no abnormalities were evident. based on the clinical and radiographic results, suspect of noma diagnosis was made. therefore, the treatment on the first day was irrigation with hydrogen peroxide, saline, and 0.2% chlorhexidine. furthermore, the patient was given oral antibiotics (amoxicillin 250 mg three times daily and metronidazole 200 mg three times daily) for introduction noma, also known as cancrum oris, is a destructive gangrenous stomatitis of the mouth, soft and hard tissues of the face.1 it may lead to devastating facial deformity, circumferential scarring, stenosis of the mouth, and in many cases death.2 “noma” derives from the greek word ne��, meaning “to graze” or “to devour”.3 the disease typically affects mostly children between 2 to 16 years of age.1 the epidemiology of noma has not changed much over the years, except that there has been a reduction in the mortality rate from 90% to about 8% to 10%, mainly because of modern antibiotics.3,4 the who estimated that 500,000 people are affected with 100,000 new cases each year. noma has disappeared from the industrialized countries since the 20th century, but is common in the third world especially in africa.5 this disease occurs almost exclusively among poor malnutrition children in developing countries.6,7 the exact cause of the disease is still not known.1 it is postulated that the disease is triggered by a consortium of micro organisms of which f�s��acteri�� necr����r�� is a key component.2,8 symbiotic relationship between fusiform bacilli and non-hemolytic streptococci and staphylococci has been considered a significant factor in the development of noma. anaerobic bacteria may be present in rapidly progressing disease.9-11 noma is considered to represent the “face of poverty” because factors connected with poverty, such as chronic malnutrition, poor oral hygiene, poor environmental sanitation, exposure to animal and human fecal material, and exposure to viral and bacterial infections, contribute to disease progression.3,12,13 weak immune system, past history of measles, scarlet fever, typhoid, bacillary dysentery, malaria, whooping cough, tuberculosis, malignancy and hiv are also predisposing factors of noma.3,13,14 this case report describes the risk factors and management of noma in 8-year-old boy suffering from systemic lupus erythematosus (sle). it is expected that this report will provide information for the clinician that a team approach is needed in better managing this devastating disease. case on 19th december 2008, an 8-year-old boy with systemic lupus erythematosus (sle) who was under treatment at department of pediatric hasan sadikin hospital was referred to department of oral medicine for evaluation and management for his oral disease. systemic lupus erythematosus is a multisystem autoimmune disease characterized by general autoantibody production and a wide range of mucocutaneous, renal, neuropsychiatric, cardiovascular, infectious, and hematologic manifestations. his mother reported that there was an ulcer at the left side of lips 1 month earlier. there was no history of trauma. she also reported discharge of pus from the left corner of � dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 6-10 15 days, analgesics (paracetamol 500 mg 3 × ½ tablet daily), 0.2% chlorhexidine gluconate gargles were prescribes. oral hygiene instructions was also given to his mother. on december, 30th, 2007 (11 days later), the patient came to the hospital for medical control. the pain of the mouth has decreased. there was no improvement healing of the noma. the necrotizing tissue on his mouth was enlarging. there was discharge of pus from the corner of the mouth (figure 2). the result of culture testing showed f�s��acteri�� necr����r��, sta��yl�c�cc�s a�re�s, and kla�siella. at this time, a definitive diagnosis of noma was made. the patient did not consume the medicine regularly because he didn’t feel convenient. the patient was referred again to microbiology laboratory for further examination (sensitivity test) because the previous treatment did not show recovery. hydrogen peroxide, saline, and 0.2% chlorhexidine were applied again to the wound. the dressing was also done to remove sloughed tissue. the same oral antibiotics and nutritional supplements were still given. at that day, the patient was admitted to the hospital under paediatrician and allergy and immunology specialist for evaluation of sle disease because his condition was decreasing. he was also referred to department of nutrition for malnutrition evaluation. parenteral fluid supplement replacement was also provided to maintain electrolyte balance. figure 2. day 7. discharge of pus from the corner of the mouth. figure 3. day 16. (a) the patient was diagnosed marasmus (severe malnutrition). (b). extensive necrosis affecting soft tissues. on january, 5th, 2008 (16 days later), the necrotizing tissue at the corner of the mouth became larger (figure 3). the patient was diagnosed marasmus (severe malnutrition) by his nutritionist. the result of sensitivity test showed that the patient has still sensitive to amoxicillin, but resistant to ampicillin, ceftazidime, chlorampenicol, ciprofloxacin, erythromycin, and metronidazole, intermediate to oxacilline, ceftriaxone dan cotrimoxazole. after this testing available, high dose antibiotic therapy was instituted to halt the spread of noma. parenteral antibiotics were started. the drug dosages were adjusted according to the patient’s age to prevent toxic effects. in addition, adequate hydration, correction of electrolytes and vitamin deficiencies with provision of sufficient nutritional support were also given by nutritionist. local debridement of necrotic tissue was still performed throughout the course of the treatment. figure 4. day 60. (a) moon face caused by corticosteroids therapy. (b) healing lesion. the lesion healed completely after 2 months, at the last visit on february 12th, 2008 (figure 4). but there was leaved scar of his corner of the mouth. moon face as a sideeffect of corticosteroids was seen in this patient (figure 4-a). the proposed treatment was polishing of all teeth and reconstructive surgery in the left corner of his mouth. the patient was then referred to department of oral surgery to have reconstruction done. until this report was made it has not been done yet because of his poor health condition. discussion in this case, the diagnosis of noma was made based on clinical features supported by microbiology examination. initially, the patients had an ulcer on the left corner of his mouth and then necrotizing tissue and discharge of pus from the corner of the mouth appeared rapidly. clinical feature of noma was began at the mucous membranes lining of cheeks which become inflamed and develop as an ulcer. the infection spreads from the mucous membranes to the skin thus causing necrosis of the tissues of lips and cheeks.15 foul smelling, purulent oral discharge were associated with profuse salivation, anorexia and palpable cervical lymphadenopathy. noma causes sudden, rapidly progressive tissue destruction.1 definitive diagnosis of noma with sle was based on the result of culture testing which showed f�s��acteri�� necr����r�� (f. necr����r��), sta��yl�c�cc�s a�re�s, and kla�siella. it is difficult to pinpoint the specific trigger agent in the complex microbiota of a noma lesion. it has been speculated that b�rrelia vincentii and f�s��acteri�� are prominent bacteria in such lesions.2,8 recent reports suggest that besides fusiform bacilli and spirochetes, �sufiawati, et al.: noma management in a child with systemic lupus erythematosus other anaerobic bacteria are present in a relatively high proportion of noma lesions.8 f. necr����r�� is considered a key component. this organism produces dermatotoxins, which could explain the rapid progression of the disease.9 f. necr����r�� elaborates several dermonecrotic toxic metabolites and is acquired by the impoverished children via fecal contamination, resulting from shared residential facilities with animals and very poor environmental sanitation. anaerobic bacteria may be present in rapidly progressing disease.9,10 prev�tella inter�edia has the ability to break down lipid structures, which contributes to tissue destruction. it also produces proteolytic enzymes capable of breaking down immunoglobulin g, which impedes elimination of microorganisms.12 it was supposed that there were various factor stimulating susceptibility of the disease in the patient of this case. the patient was diagnosed severe malnutrition called marasmus (a condition primarily caused by a deficiency in calories and energy) by nutritionist. malnutrition leads to alteration in cell-mediated immune function and early breakdown of the epithelial tissues, alterations in the oral mucosa facilitate invasion by pathogens.12 eating difficulties due to infection further was aggravated by any existing malnutrition. therefore, malnutrition can be considered to have been a major predisposing factor for development of noma in this patient. there was a complex three-way relationship existed between malnutrition, immune dysfunctions in the host, and increased susceptibility to infections.9 studies of the effect of malnutrition on oral microbial ecology in the village of nigerian children have demonstrated prominently increased recovery of spirochetes and anaerobic rods compared to control groups of well-fed children from the same ethnic background.16 cellular depletion of key nutrients such as zinc, retinol, ascorbic acid, and the essential amino acids, will impair the structural integrity of the oral mucosa, thus creating easy portals of entry for the pathogenic microorganisms and their products.17-19 another predisposing factor that could lead noma in the patient was the use of long term corticosteroids for his systemic disease. systemic lupus erythematosus (sle) is an autoimmune disease characterized by typical involvement of many different organ systems and by immunological abnormalities, such as hyperactive b cells producing various autoantibodies.20 the aetiology of sle is unspecific. it is likely that a combination of genetic, environmental, and possibly hormonal factors worked together to cause the disease. this led to inflammation and damage to various body tissues.20,21 the diverse presentations of lupus range from rash and arthritis through anemia and thrombocytopenia to serositis, nephritis, seizures, and psychosis. sle is considered a predominantly female disease. lupus should be part of the differential diagnosis in virtually any patient presenting with one of these clinical problems, especially in female patients between 15 and 50 years of age.22 approximately 20% of all patients who have sle are diagnosed in childhood. the diagnosis of sle often is considered in children who have prolonged unexplained complaints.23 the mainstay of lupus treatment involves the use of corticosteroid hormones, such as prednisone. they work by rapidly suppressing inflammation. persons who have been on l�ng-ter� oral c�rtic�ster�ids may become immunosuppressed and s�sce�ti�le t� infection. increased steroid levels in the mouth could also serve as a rich nutrient source for anaerobes.24 thus, the patient has high risk for developing secondary infection such as noma. lupus can affect many parts of the body, including the gastrointestinal (gi) disorders. william osler, in 1895, was the first to emphasize that the gi manifestations may overshadow other aspects of the disease and mimic any type of abdominal condition. gi manifestations of sle include mouth ulcers, dysphagia, anorexia, nausea, vomiting, haemorrhage and abdominal pain. anorexia, nausea and vomiting are seen in up to 50% of patients with sle. however, they may be due to the disease, represent intercurrent processes (e.g. secondary to uraemia) or sideeffects of medication.25 these complications may lead malnutrition in patient with sle. in the early stages, the child need oral irrigation with hydrogen peroxide, saline and 0.2% chlorhexidine, thus helps to slough the necrotic tissue. amoxicillin and metronidazole were given to the patient. as there is no clear consensus but most authors recommend penicillin plus metronidazole to cover predominant organisms. medication needs to be continued for at least 14 days. after the result of sensitivity testing, other antibiotics were given parenterally. the use of antibiotic may cause candida al�icans overgrowth, thus requires antifungal coverage with nystatin.1 after being treated together at the same time with the systemic disease, finally noma showed recovery. usually in such cases, maxillofacial and plastic surgeons repair the defect once the infection subsides. patients who have risk factors, such this child, have to be prevented from noma, by increasing the nutritional status of children and decreasing the animal-fecal contamination of the environment. proper and early treatment of oral lesions, and also maintenance of oral health would seem to be the means of control for preventing noma.8 it was concluded that noma is not a primary disease, there are various predisposing factors usually precede its occurrence, such as severe malnutrition and immune system disease with intensive immunosuppressive therapy. this case report may guide the clinician in better managing this devastating disease. management of noma requires a multidisciplinary team approach. references 1. wazir sm, khan su. cancrum oris. journal of pakistan associationjournal of pakistan association of dermatologists 2008; 18: 110–2. 2. falkler wa, enwonwu co, idigbe eo. isolation of fusobacterium necrophorum from cancum oris (noma). am j trop med hyg 1999; 60(1): 150–6. �0 dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 6-10 3. mark kw. a history of noma, the “face of poverty”. plast reconstr surg 2003; 111(5): 1702–7. 4. oji c. cancrum oris: its incidence and treatment in enugu, nigeria. br j oral maxillofac surg 2002; 40(5): 406–9. 5. adolph hp, yugueros p, woods je. noma: a review. ann plast surg 1996; 37(6): 657–68. 6. enwonwu co. epidemiological and biochemical studies of necrotizing ulcerative gingivitis and noma (cancrum oris) in nigerian children. archs oral biol 1972; 17: 1357–71. 7. enwonwu co. infectious oral necrosis (cancrum oris) in nigerian children: a review. commun dent oral epidemiol 1985; 13: 190–4. 8. falkler wa jr, enwonwu co, idigbe eo. microbial understanding and mysteries of noma (cancrum oris). oral dis 1999; 5: 150–5. 9. enwonwu co, falkler wa jr, idigbe, afolabi bm, ibrahim m, onwujkwe d, savage ko, meeks vi. pathogenesis of cancrum oris (noma): confounding interactions of malnutrition with infection. am j trop med hyg. 1999; 60(2): 223–32. 10. paster bj, falkler wa jr, enwonwu co, idigbe eo, savage ko, levanos va, tamer ma, ericson rl, lau cn, dewhirst fe. prevalent bacterial species and novel phylotypes in advanced noma lesions. j clin microbiol. 2002; 40(6): 2187–91. 11. brady-west dc, richards l, thame j, moosdeen f, nicholson a. cancrum oris (noma) in a patient with acute lymphoblastic leukemia. a complication of chemotherapy induced neutropenia. west indian med j 1998; 47(1): 33–4. 12. berthold p. noma: a forgotten disease. dent clin north am 2003; 47(3): 559–74. 13. enwowu co, falkler wa jr, idigbe eo, savage ko. noma (cancrum oris): questions and answers. oral dis 1999; 5(2): 144–9. 14. yuca k, yuea sa, cankaya h, caksen h, calka o, kiriş m. report of an infant with noma (cancrum oris). j dermatol 2004; 31(6): 488–91. 15. valadas g, leal mj. cancrum oris (noma) in children. eur j paed surg 1998; 8: 47–51. 16. sawyer dr, nwoku al, rotimi vo, hagen jc. comparison of oral microflora between well-nourished and malnourished nigerian children. j dent child 1986; 439–43. 17. beisel wr. nutrition and immune function: overview. j nutr 1996; 126 (10suppl): 2611s–5s. 18. thurnham di. impact of disease on markers of micronutrient status. proc nutr society uk 1997; 56: 421–31. 19. enwonwu co. cellular and molecular effects of malnutrition and their relevance to periodontal disease. j clin periodont 1994; 21: 643–57. 20. bengtsson aa, rylander l, hagmar l, nived o, sturfelt g. risk factors for developing systemic lupus erythematosus: a case–control study in southern sweden. rheumatology 2002; 41(5): 563–71. 21. mok cc, lau cs. pathogenesis of systemic lupus erythematosus. j clin pathol 2003; 56(7): 481–90. 22. rahman a, isenberg da. systemic lupus erythematosus. n engl j med 2008; 358(9): 929–39. 23. gottlieb bs, ilowite nt. systemic lupus erythematosus in children and adolescents. pediatr rev 2006; 27(9): 323–30. 24. loesche wj, syed sa, laughton be, stoll j. the bacteriology of acute necrotizing ulcerative gingivitis. j periodontol 1982; 53(4): 223–30. 25. sultan sm, ioannou y, isenberg da. a review of gastrointestinal manifestations of systemic lupus erythematosus. rheumatology 1999; 38: 917–32. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base mkgs vol 44 no 2 april-juni 2011.indd 111 vol. 44. no. 2 june 2011 research report effect of soybean extract after tooth extraction on osteoblast numbers rosa sharon suhono1, coen pramono2, and djodi asmara2 1 dental student 2 department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract background: many researches were done to find natural materials that may increase and promote bone healing processes after trauma and surgery. one of natural material that had been studied was soybean extract which contains phytoestrogen, a non-steroidal compounds found in plants that may binds to estrogen receptors and have estrogen-like activity. purpose: the aim of this study was to investigate the effect of soybean extract feeding on the number of osteoblast cells in alveolar bone socket after mandibular tooth extraction. methods: this study was studied on male rattus norvegicus strain wistar. seventeen rats divided into three groups were used in this study. group 1 fed with carboxy methyl cellulose (cmc) solution 0,2% for seven days, and the left mandibular central incisivus was extracted; group 2 fed with soybean extract for seven days and the left mandibular central incisives was extracted; group 3 received the left mandibular central incisives extraction followed by soybean extract feeding for seven days after the extraction. all groups were sacrificed on the seventh day post-extraction, and the alveolar bone sockets were taken for histopathological observation. the tissues were processed and stained using hematoxylin and eosin to identify the amount of osteoblast cells. the number of osteoblast cells was counted using an image tool program. the data was analyzed statistically using the one-way anova test. results: significant differences were found on the number of osteoblast cells in alveolar bone after tooth extraction between groups. group 2 (fed with soybean extract) is higher than group 1 (fed with cmc) and group 3 (fed with soybean extract after extraction). conclusion: soybean extract feeding that given for seven days pre-tooth extraction can increase the number of osteoblast cells compared with the group that were not given soybean extract feeding and also with the group that were given soybean extract feeding for seven days post-tooth extraction. key words: tooth extraction, soybean extract, phytoestrogen, osteoblast abstrak latar belakang: pada masa sekarang, banyak dilakukan penelitian-penelitian untuk menemukan bahan-bahan alami yang dapat mendukung dan meningkatkan proses remodeling tulang untuk mengembangkan perawatan penyakit osteoporosis dan juga untuk mengembangkan penyembuhan tulang pasca trauma dan pasca pembedahan. salah satu bahan alami yang banyak diteliti adalah ekstrak kedelai yang mengandung fitoestrogen, suatu senyawa non-steroid yang terdapat dalam tumbuhan, yang dapat berikatan dengan reseptor estrogen dan memiliki bioaktivitas yang sejenis dengan hormon estrogen. tujuan: tujuan dari penelitian ini adalah untuk meneliti pengaruh ekstrak kedelai yang mengandung fitoestrogen terhadap jumlah sel osteoblas pada tulang alveolar pasca pencabutan gigi. metode: penelitian ini dilakukan pada tikus wistar jantan. tujuh belas ekor tikus dibagi menjadi tiga kelompok sampel dalam penelitian ini. kelompok sampel tersebut mendapatkan perlakuan yang berbeda-beda. kelompok 1 dilakukan feeding larutan cmc 0,2% selama tujuh hari, kemudian dilakukan pencabutan satu gigi insisivus sentral kiri rahang bawah, kelompok 2 dilakukan feeding ekstrak kedelai selama tujuh hari, kemudian dilakukan pencabutan satu gigi insisivus sentral kiri rahang bawah, kelompok 3 kelompok yang dilakukan pencabutan satu gigi insisivus sentral kiri rahang bawah, kemudian diberikan feeding ekstrak kedelai selama tujuh hari pasca pencabutan gigi. semua kelompok dikorbankan pada hari ketujuh pasca pencabutan gigi, dan soket bekas pencabutan gigi tersebut diambil untuk dibuat sediaan histopatologis. jaringan tersebut diproses dan dilakukan pengecatan dengan hematoxylin and eosin untuk melihat sel osteoblas. setiap preparat diperiksa di bawah mikroskop cahaya dan sel osteoblas dihitung 112 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 111–116 dengan menggunakan program image tool. data hasil penelitian kemudian dianalisa dengan uji statistik one-way anova. hasil: terdapat perbedaan yang signifikan pada jumlah sel osteoblas pada tulang alveolar pasca pencabutan gigi, antara kelompok sampel yang mendapatkan feeding ekstrak kedelai sebelum pencabutan gigi (kelompok 2) dibandingkan dengan (kelompok 1) dan (kelompok 3). kesimpulan: pemberian ekstrak kedelai selama tujuh hari sebelum pencabutan gigi dapat meningkatkan jumlah osteoblas. kata kunci: pencabutan gigi, ekstrak kedelai, fitoestrogen, osteoblas correspondence: rosa sharon suhono, c/o: mahasiswa, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ros.sharon@yahoo.com equipments needed are commonly found in the household, except for the stripper, grinder, and mould.4 soybean contained some chemical substances, one of them is known as phytoestrogen, a non-steroidal compounds found in plants, which have in part some structural similarities to estrogen hormone, that is 17β-estradiol.5 currently, four different families of phenolic compounds produced by plants are concidered as phytoestrogen: the isoflavonoid, stilbenes, lignans and coumestans.6 soybean is the main source of phytoestrogen isoflavones group, namely as genistein and daidzein.7 endogenous estrogen levels are associated with human bone homeostasis. estrogen is known to play an important role in maintaining bone mass. lack of estrogen levels may causes excessive bone resorption, and the estrogen deficiency results in bone loss.8 bone volume is maintained by two phases of bone remodelling: one is bone resorption by osteoclasts, and the other one is bone formation by osteoblasts. it is known that bone remodelling is controlled by various cytokines and hormones, such as interleukin1 (il-1), interleukin-6 (il-6), parathyroid hormone, glucocorticoids, and estrogen hormone.9 many researches were done to find some natural materials that may increase and promote bone remodeling in order to improve treatment in osteoporosis, and bone healing process after trauma and after surgery. phytoestrogen such as genistein and daidzein found in soybean were investigated and showed its estrogen-like activity because of its structural similarities to 17βestradiol. natural phytoestrogen found in soybean extract can increase osteoblast cells proliferation in cells mc3t3e1, the osteoblast-like cell line from rat.10 phytoestrogen may as influence in increasing alkaline phosphatase activity in mc3t3-e1 cells in vitro and found that phytoestrogen can also be used as one of the osteogenic local agents in vivo.5 the aim of this study was to investigate the effect of soybean extract feeding on the number of osteoblast in the alveolar bone socket after mandibular tooth extraction histopathologically. materials and methods this study was done in healthy male wistar strain rats, aged 2-3 months and weighed between 150–200 introduction tooth extraction is a treatment that should be done for severely damaged tooth. the process of wound healing after tooth extraction generally consists of several phases, begins with the formation of blood clots in the socket, continued to inflammation process, infiltration of leukocytes, formation of connective tissue, formation of granulation tissue, epithelialization and regeneration of new bone to close the remaining empty socket. when a tooth is removed, the socket is filled with blood, which coagulates and form blood clot. the inflammatory stage occurs during the first week of post extraction. white blood cells enter the socket to remove contaminating bacteria and any debris from the area, such as bone fragments that are left in the socket. fibroplasia also begins during the first week, with the growth of fibroblasts and capillaries. finally, in the end of the first week of healing, osteoclasts accumulate along the crystal bone to start bone resorption, followed by osteoblasts that responsible to the process of bone formation. the cortical bone was resorbed from the socket and new trabecular bone is laid down accross the socket. as bones fill the socket, the epithelium moves toward the crest and eventually becomes level with adjacent gingival crest. this processes continue and usually take sometime before a socket heals and becomes the edentulous alveolar ridge.1,2 factors that can be related to the healing process is generally divided into common factors and local factors. the common factors includes: age, nutrition, vitamins, hormones, metabolic status, and circulation status. local factors includes: blood supply, foreign bodies, tissue movement and damage, type of tissue, infections, mechanical factors, size, location, and type of wound. those factos can also be related to the healing process of tooth extraction.3 nuts and seeds such as soybean, peanut, winged bean seeds, coconut and others are the sources of proteins and fats which are important in human life. soybean is mostly found in indonesia and has protein content of 35%. the improved varieties of soybean may even contain protein levels of 40–43%. compared to rice, maize, cassava flour, green bean, meat, fresh fish, and egg, soybean has higher protein content, nearly as high as the protein content found in dried skim milk. soybean can be processed into tempeh, tofu, soybean sauce, soybean milk, etc. the processing process of soybean into many kind of food is simple, and the 113suhono, et al.: effect of soybean extract after tooth extraction grams. samples were divided into three groups: control group who received feeding of carboxy methyl cellulose (cmc) solution concentration of 0.2% equivalent to 9 cc/200 gram of rat’s body weight per day for seven days before-tooth extraction; the treatment group 1 (p1) which fed with soybean extract feeding using a dose of 9 cc/ 200 gram of rat’s body weight/day for seven days beforetooth extraction; treatment group 2 (p2) which fed with soybean extract feeding 9 cc/200 gram of rat’s body weight per day for seven days after-tooth extraction. “surya” white soybean available in local market in madiun, east java was used in this study. the soybean extract was made using akebono brand soybean extractor machine and sixty grams of dried soybeans was soaked for 12 hours and inserted into the soybean extractor machine. 1500 cc of warm water was added and 1500 cc of soybean extract liquid obtained. the control group was fed with 0.2% cmc solution in a dose of 9 cc/200 gram of rat’s body weight per day for seven days. a feeding tube was connected into a syringe was the feeding instrument used in this purpose. seven days after fed with cmc 0.2% solution, the experimental animals in the control group were anesthetized for tooth extraction. general anesthesia with ether solution was used and the process was done by putting the experimental animal into a glass jar contained of cotton wool soaked with ether solution. the experimental animals were inserted and the jar was closed for several minutes until the animal was unconscious. the left mandibular central incisives was removed using a modified forceps which was made for rat’s tooth rat extraction. tooth extraction bleeding was stopped by pressing the socket wound using sterile cotton. soybean and cmc feeding in the control groups were not given for seven days but only received a drinking water. the same procedure was performed on the p1 group and this group was fed with soybean extract feeding with the dose of 9 cc/200 gram of rat’s body weight per day for seven days pre-tooth extraction. tooth extraction was performed in the experimental animals in p2 group. the same procedure as in the control group and p1 group was used. soybean extract feeding of 9 cc/200 gram of rat’s body weight per day was given for seven days post-tooth extraction in this group. table 1. number of osteoblast in control group (k) no. slides number of osteoblast mean of osteoblast cell number each slidebottom region middle region top region 1 k.4 40 32 36 36 2 k.5 18 23 22 21 3 k.6 34 78 123 78.33 4 k.7 44 40 48 44 5 k.9 41 27 30 32.67 table 2. number of osteoblast in treatment group 1 (p1) no. slides number of osteoblast mean of osteoblat number each slidebottom region middle region top region 1 p1.1 91 80 95 88.67 2 p1.2 172 181 134 161.33 3 p1.3 181 232 159 190.67 4 p1.4 172 180 138 163.33 5 p1.5 241 225 76 180.67 6 p1.6 79 93 43 71.67 table 3. number of osteoblast in treatment group 2 (p2) no. slides number of osteoblast mean of osteoblast number each slidebottom region middle region top region 1 p2.1 133 69 86 96 2 p2.2 159 125 117 133.67 3 p2.3 48 85 66 66.33 4 p2.4 28 97 60 61.67 5 p2.7 77 50 77 68 6 p2.8 80 49 35 54.67 114 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 111–116 after all experiment procedures were completed, all animal samples were sacrificed using an overdosed ether solution on the seventh day post-extraction to examine the osteoblast cell that has begun to accumulate along the alveolar bone lining the socket.1 the mandible bone of the experimental animals were taken from the skull and inserted into the fixative solution of 10% formalin solution. the alveolar bones were taken for further histopathological slide preparation procedure. the tissues were cutting in serial in rotary microtome with 6μ of thickness followed by tissue staining using haematoxylin and eosin (he). all slides fields were observed for the present of osteoblast cells in 400x of magnification. three different areas were observed as those are the top, bottom and the middle regions of the tooth extracted socket which are adjacent to the post extraction alveolar bone. the number of osteoblast cells were counted using the computer program “image tool”, a program used to analyze and process free images. analysis function of images in this program can be done in variety including the dimensional measurement, i.e. measurement of distance, angle, area, and also a certain scale of measurement in the form of points, lines, or areas and it is completed with a counting of its standard deviation.11 the statistical difference in the number of osteoblast cells during bone remodeling processes after tooth extraction among those three sample groups were statistical analyzed using the one-way anova test. results the results on osteoblast cells observation on those three areas of tooth extraction socket on the seventh day after-extraction is shown in table 1, 2, 3. based on data, it is shown that the difference number of osteoblast cells are found higher in the sample group who received soybean extract feeding (p1, p2 groups), compared to the control group (k) in which soybean extract feeding was not given (figure 1). the data of the number of osteoblast cells in each group were analyzed using kolmogorov-smirnov test for normality and homogeneity test results in each group. the result of this test showed that distribution of data in each groups were normal, means that the data in each groups were homogenous. the analysis was done using oneway anova test (p < 0.05, α < 0.05), indicating that there are significant difference in osteoblast cell numbers between in the control group (k) and in the treatment group 1 (p1). the same results also showed in treatment group 1 (p1) compared to those in the treatment group 2 (p2). no significant difference was found between the control group (k) and in the treatment group 2 (p2) (p > 0.05). discussion the soybean extracts and its proteins, carbohydrates, fats, calcium, phosphorus, iron, provitamin a, vitamin b complex (except b12) and water contained showed statistically able to enhance the regeneration process of bone healing after tooth extraction.4 the calcium and phosphorus contain found in soybean extract will increase the intake of calcium and phosphorus, which are needed for the bone regeneration process. soybeans also have low sulfur amino acid content, which known as acids can inhibit calcium resorption through kidneys, and therefore may cause considerable loss of calcium in the urine.7 these facts supported the result of this study that overall there are a significant difference in bone regeneration process, marked by the different number of osteoblast cells that found higher in the treatment group who received a soybean extract feeding, compared to the control group who did not fed with the soybean extract. statistical result of tests using the one-way anova showed a significant difference in the number of osteoblast cells between the control group and group p1 (p < 0.05). these results showed that soybean extract feeding on experimental animals seven days preextraction can affect the total amount of alveolar bone osteoblast cells compared to those the control group. phytoestrogens has a capability to bind to estrogen receptors in the body competitively with estrogen hormone, so the number of osteoblast cells production can be found higher.12,13 thus occured because the binding process between estrogen receptor and phytoestrogens may influence the osteoblast cells production by improving the process of synthesis, activation, receptor binding, and local growth factor binding protein in osteoblast, either directly or indirectly. transforming growth factor-β (tgf-β) and insulin-like growth factor-1 (igf-1) which may support the process of cell proliferation from osteoblast precursor cells that is preosteoblas into osteoblasts also activated during the presence of phytoestrogen. the synthesis of bone matrix by osteoblast cells thereby affect in increasing of osteoid deposition during bone regeneration process. soybean extracts containing phytoestrogens will increase the anabolic function of osteoblasts cells.13 the o st eo bl as t nu m be r 160 140 120 100 80 60 40 20 0 control group (k) treatment group 1 (p1) treatment group 2 (p3) figure 1. osteoblast cell number in alveolar bone at seventh days post-extraction, in control group, treatment group 1, and treatment group 2. 115suhono, et al.: effect of soybean extract after tooth extraction study on soybean extracts which contain phytoestrogens was reported can stimulate the protein synthesis in osteoblast cells in vitro.13 synthesis of these proteins will support the process of new osteoblast cell proliferation by accelerating the process of cell differentiation from preosteoblast into osteoblasts. binding of phytoestrogens with estrogen receptors on osteoblast cells will improve the synthesis of igf1 and tgf-β.13,14 increase of tgf-β as the effect of estrogen receptor binding to the phytoestrogens found in soybean extract on the process of osteogenesis has been demonstrated histochemically, and the results showed that mrna expression of tgf-β in cell culture of human bone marrow containing mesenchymal stem cells that is also called stem cells which can differentiate into osteoblast precursor cells, was observed at day 3 after phytoestrogens treatment in vitro, and continued to increase until day 21, then disappeared.15 increased growth factors will accelerate the process of differentiation and maturation of preosteoblas into osteoblasts. tgf-β and igf-1 also supports a mature osteoblast cells in bone matrix synthesis process for forming osteoid deposition of cartilage tissue that has not been calcified.16 in addition, it is also reported that soybean extracts containing phytoestrogens can increase the viability of osteoblast cells and increases the activity of alkaline phosphatase and osteocalsin synthesis by osteoblast cells.13 osteocalcin is a non-collagen protein bone matrix, which synthesis stimulated by 1.25-dihydroxicolecalsiferol.16 soybean extracts containing phytoestrogens can increase the synthesis of 1.25-dihydroxikolcalciferol, which receptors are also present in the cell membrane of osteoblast.13 soybean extracts containing phytoestrogen will stimulate 1.25-dihydroxicolecalciferol, which will provide a direct influence on the synthesis and stabilization of osteocalsin at the transcriptional and post-transcriptional phase, so that the process of osteocalsin synthesis by osteoblast cells will increase. osteocalsin is an important component in the process of bone regeneration because osteocalsin is a non-collagen bone protein that functions in the process of calcium ions binding in bone regeneration, assisted by vitamin k1.16 those reported facts can be used to explain our study in the present of osteoblast cells that produced in group p1 is found more mature, so that the new bone trabeculae also more in the experiment group of p1 than shown in the control group. in addition, alkaline phosphatase and osteocalsin produced by osteoblast cells indicates that osteoblast cells are already mature and can produce an optimal bone matrix.16 the one-way anova test results also indicates a significant difference in osteoblas cells contained shown in groups p1 and p2 (p < 0.05), where the mean of osteoblast cells number in groups p1 is higher than in group p2. this event can be explained that the soybean extract were given in different sequences in all experiment animals in groups p1 and groups p2. the removal of tooth initiates some sequences of inflammatory process, fibroplasia, formation of granulation tissue, ephitelialization. this pathophysiological sequence can be found during the formation of new bone to close the remaining empty post-extraction socket. the inflammatory stages occurs during the first week of healing process, and feeding with soybean extracts which containing of phytoestrogen during the first week post-extraction to the treatment group 2 (p2) will affect to those inflammatory process. it was reported that phytoestrogens contained in the soybean has an anti-inflammatory effect by inhibiting proinflammatory cytokine production, that is: il-6, through inhibition of gene transcription of il-6 process, which occur because of estrogen receptor binding with phytoestrogen.13 il-6 are proinflammatory cytokines that have a significant role in the inflammatory process by improving the accumulation and formation of osteoclast.16 il-1 and il-6 would increase the process of bone resorption, and inhibition of cytokines production will also decrease the number of osteoclasts accumulation, this event would affect in decreasing of bone resorption. therefore, soybean extracts which contain phytoestrogens given in group p2 might affects not only in the increasing number of osteoblast cells, but also decrease osteoclasts cells accumulation. although the process of new bone regeneration in tooth extracted socket is present, but the observation showed that the increased number of osteoblast cells in groups p2 is significantly found less as seen in group p1. the result of this study on the amount of osteoblast cells found in treatment group (p2) and in the control group (k) showed different mean value of osteoblast cells number although this evidence was not statistically significant. it is occurred because soybean extracts feeding which contain phytoestrogens given in group p2 might affects not only in the increasing number of osteoblast cells, but also related to the whole inflammatory process, so the number of osteoblast cells in group p2 was not significantly found higher than in control group which were not given soybean extract. control group did not receive soybean extract as in group p1 and group p2, so the process of bone regeneration occurred normally with normal amount of osteoblast cells proliferation. the 0.2% cmc solution feeding was given to the control group with purpose to achieve the same physiological conditions in their intestinal tract both the animal control group and treatment group. although the process of new bone regeneration in tooth extracted socket is present, but the observation showed that the increased number of osteoblast cells in groups p2 was not significantly different than the control group. it is concluded that soybean extract feeding for seven days before-tooth extraction can increase the number of osteoblast. 116 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 111–116 references 1. peterson lj, ellis e, hupp jr, tucker mr. contemporary oral and maxilofacial surgery. 4th ed. st. louis: mosby; 2003. p. 49–74. 2. sudiono j, kurniadhi b. ilmu patologi. jakarta: egc; 2003. p. 112–6. 3. kumar v, abbas ak, fausto n. robbins and cotran pathologic basis of disease. 7th ed. philadelphia: elsevier saunders; 2005. p. 107–14. 4. esti, sediadi a. buku panduan teknologi pangan, pusat informasi wanita dalam pembangunan pdii-lipi bekerjasama dengan swiss development cooperation. jakarta. 2000. available at: http://www. warintek.ristek.go.id/pangan_kesehatan/pangan/piwp/susu_kedelai. pdf. accessed march 30, 2010. 5. wong rwk, rabie abm.. effect of quercetin on preosteoblast and bone defects. the open orthopaedics j 2008(2): 27–32. 6. cornwell t, wendie c, ilya r. review: dietary phytoestrogens and health. phytochemistry. 2004; (65): 995–016. available online at: www.sciencedirect.com. accessed march 30, 2010. 7. koswara, sutrisno. isoflavon: senyawa multi manfaat dalam kedelai. 2006. available at: http://www.ebookpangan.com. accessed march 30, 2010. 8. arnita. fitoestrogen untuk wanita menopause. majalah farmacia 2007 juni; 6(11): 22. 9. ganong wf, 2001. buku ajar fisiologi kedokteran. edisi 20. widjajakusumah d, editor. jakarta: egc; 2003. p. 367–80, 423-6. 10. kanno s, hirano s, kayama f. effect of phytoestrogen and environmental estrogen on osteoblastic differentiation in mc3t3e1 cells. toxicology 2004; 196: 137–45. available at: www. sciencedirect.com. accessed april 5, 2010. 11. dove, brent s. uthscsa imagetool version 3.0 final is here. 2002. available at: http://ddsdx.uthscsa.edu/dig/itdesc.html. accessed december 13, 2010. 12. george nn, hopkins ne, boue s, alworth wl. interactions of dietary estrogen with human estrogen receptors and the effect of estrogen receptor-estrogen response element complex formation. environmental health prespective. 2000; 108(9): 867–72. 13. hassan, nahed m, hassan ra, setta lma, el-moinem mma, ahmed hh, hammouda fm. potent role of dietary phytoestrogen plants cultivated in egypt against osteoporosis in ovariectomized rats. australian j basic and applied sciences 2010; 4(2): 359–69. 14. kim h, xu j, su y, xia h, li l, peterson g, murphy-ullrich j, barnes s. actions of the soybean phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growh factor β. biochemical society transactions 2001; 29(2): 216–21. 15. heim m, frank o, kampmann g, sochocky n, pennimpede t, fuchs p, huznicker w, weber p, martin i, bendik i. the phytoestrogen genistein enhanced osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells. endocrinology 2004; 145(2): 848–59. 16. favus mj. primer on the metabolic bone diseases and disorders of mineral metabolism. 2nd ed. new york: raven press; 1993. p. 3–9, 15–37. �� molecular study of the dentin-pulp complex responses to caries progression yani corvianindya rahayu department of oral biology university of jember faculty of dentistry jember indonesia abstract the dentin-pulp complex exhibits various responses to caries, including events of injury, defense, and repair. the overall responses dependent on pulp cell activity and the signaling processes, which regulate the behavior of these cells. the signals for tissue repair are thought to be mediated by dentin-bound growth factors released during caries progression. growth factors are a key of molecules responsible for signaling a variety of cellular process following dental injury. the endogenous proteolytic enzymes (matrix metalloproteinases, mmps) present in dentin matrix might also participate in releasing bioactive molecule. several members of the mmp family are found in the soft and hard tissue compartment of dentin-pulp complex. their presumed role in many physiological process during the development and maintenance of the dentin-pulp complex, they may also contribute to the pathogenesis of dentin caries and the responses elicited by caries. key words: dentin-pulp complex, molecular events, caries progression correspondence: yani corvianindya rahayu, c/o: bagian biologi oral, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. introduction the human dentition is indispensable for nutrition and physiology. the teeth have evolved for mastication of food. caries is a common dental problem in which the dentin matrix is damaged. when the caries is deep and the dental pulp is exposed, the pulp has to be removed in many cases, resulting ultimately in loss of the tooth.1 the dentin-pulp complex comprises mineralized dentin and the vital soft tissues encased inside dentin, i.e. odontoblasts and pulp tissue. during caries progression, the dentinal minerals are dissolved and eventually the collagenous organic matrix is degraded. however, the exact mechanisms and enzymes responsible for the organic matrix breakdown remain unknown.2 the vitality of the dentin-pulp complex, both during tissue homeostasis and after injury, is dependent on pulp cell activity and the signaling processes, which regulate the behavior of these cells. research, particularly over the last ten to fifteen years, has led to a better understanding of the molecular control of cellular behavior. growth factors play a pivotal role in signaling the events of tissue formation and repair in the dentin-pulp complex. sequestration of growth factors in the dentin matrix during tissue formation provides a pool of these molecules, which may be released during injury and contribute to signaling of reparative events.3 it is obvious that much information is still lacking in our understanding of how the immune system and other components of the dentin/pulp complex communicate to mount defense, damage, and repair of the dentin/pulp complex. most reports in this area are descriptive, and information on molecular mechanisms regulating cellular responses is limited. future research should be extended to delineate the profile of bioactive molecules such as cytokines that sequentially regulate the immunological reactions of the dental pulp.2,4 this article will review current knowledge of the understanding the molecular events underlying the dentinpulp complex responses to carious progression. it will also describe the roles of some enzymes and bioactive molecules in dentin matrix might participate in degradative events of caries processes. biology of dentin-pulp complex teeth are composed principally of dentin, and the pulp is its formative tissue. biologically the two are inseparable, although mature teeth can functions adequately in the absence of the pulp, such as when the dental endodontic and restorations procedures and formed and successfully concluded. normally, however the pulp of the tooth is vital, active, and responsive to its environment, a responsiveness that is reflected not only as an inherent change in its structure, but also as an alterations in the form and structure of the dentin it produces. the presence of the pulp also imparts a sensitively to the dentin, a sensitivity that in physiologic terms may be distinctive to the body. because of this intimate relationship the dentin and the pulp are generally considered as a complex, the dentinpulp complex.5 biologically and developmentally, pulp and dentin function as a complex and may be regarded as one tissue. �2 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 11–15 dentinal fluid movement, resulting in hydrodynamic activation of pulpal a-delta nerve fibers and causing dentin sensitivity, is a common example of functional coupling of the tissues. both tissue are derived from the dental papilla, and development of two tissues is closely related. the structure and composition of dentin matrix, and of the dentinal tubules, are key influences in the process of bacterial invasion of dentinal tubules.5,6 the dentin extra cellular framework, into and on which the minerals are embedded, is also called organic matrix, with reference to its role as a template for mineralization and its presumable regulative activity on mineralization. collagen comprise over 90% of the dentin organic matrix, type i collagen being the most abundant collagen. type iii collagen expressed and synthesized by human odontoblast, it has been observed in intratubular dentin and predentin as well as inside the dentinal tubules.7 many non-collagenous ecm protein are considered important in actively promoting and controlling yhe mineralization of collagen fibrils and crystal growth during dentin formation. dentin contains large amounts of dentin sialoprotein and dentin phosphoproteins compared to bone. dentin non-collagenous ecm also contains plasma protein and several growth factors.2,8 responses of dentin-pulp complex to caries the dentin-pulp complex reacts to a variety of bacterial and nonbacterial stimuli. most common and perhaps most typical of the bacterial–related reactions is the manner in which the pulp responds to dental caries. microscopically the reaction is identified in the pulp by a disruption of the odontoblastic layer and an edema and inflammatory round cell infiltration of the pulp core. since the bacteria have not as yet entered the pulp, it thought that these changes represent an immunologic response to bacterial by products or components that diffuse into the pulp from carious lesion via dentinal tubules.4,7 the overall response of the tooth to injury, such as dental caries, represents the complex interplay between injury, defense, and regenerative process (figure 1). it is important to recognize that the interplay and relative balance among these processes will be primary determinant of tissue vitality and tooth survival.3 bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following a breach in the integrity of the overlying enamel or cementum. bacterial products diffuse through the dentinal tubule toward the pulp and evoke inflammatory changes in the dentin-pulp complex.6 defense encompassed a variety of local tissue and systemic responses to counteract the injury. these will include local inflammation within the tissues as well as local activation of immune defense reactions, which may trigger broader systemic immune responses. t lymphocytes and macrophages have been proposed to be important in the immunosurveillance of the pulp, and may be important in the initiation of pulpal specific immunity following exposure to protein antigens. b and t lymphocytes are increases in numbers of these cells have been correlated with increasing lesion depth in caries.4,6 macrophages, lymphocytes and antibody-producing plasma cells (immunocompetent cells) are predominant cell types notes in the pulp prior to its actual exposure (direct invasion by microorganism), polymorphouclear cells are also present. the immunocompetent cells within the pulp appear tobe enganged in antibody production. various antibodies (igg, ige, iga) have been identified in pulps affected by caries.4,6,7 dental injury perhaps represents a special situation in that the cellular pulp is enclosed by a rigid, mineralized, tissue shell and thus significant enamel and dentin matrix degradation can ensue before the disease process reaches the pulp. the tubular structure of dentin confers permeability properties on the tissue, and bacterial metabolic products from the carious process and dental tissue matrix degradation products may diffuse down the dentinal tubules and invoke cellular responses.3 dentin permeability, influenced by both the density and the diameter of the dentinal tubules, has a major effect on the extent of injury to the dentin-pulp complex cells by the noxious component. dentin permeability may be decrease by tertiary dentin formation and by obliteration of the tubules, through either physiological dentin sclerosis that is evenly distributed in dentin or reactive dentin sclerosis observed in carious teeth beneath and next to the caries lesion. reactive dentin sclerosis may result from the acceleration of intratubular dentinogenesis, but also by precipitation of dissolved minerals.9,10 deposition of tertiary dentin at the site of injury is generally intermittent, reflecting the intermittent nature of the caries attact. the processes of reactionary and reparative dentinogenesis at dental caries for secretion of tertiary dentin matrices, which either increase the dentin barrier between the site of injury and the underlying cells in the unexposed pulp.3,9 growth factors as key mediators growth factors are peptide molecules that transmit signals between cell functioning as stimulators and/or inhibitors of growth as well as modulators of differentiation state amongst other roles. as such, they play a central role figure 1. the overall response of the tooth to injury, such dental caries, represents the complex interplay among injury, defense and regeneration.3 injuri defense regeneration �3rahayu: molecular study of the dentin-pulp in controlling cell behavior and activity. growth factors are a key of molecules responsible for signaling a variety of cellular process following dental injury. they play a central role in signaling various aspects of tooth morphogenesis, differentiation, tissue regeneration, and events associated with the initial injury to the tissue and subsequent defense reactions also impact on their activities. importantly, they provide the basis to understand the biological mechanisms of tissue regeneration in dentin-pulp complex.3 growth factors may be released from the dentin matrix as a result of injury events. during caries, diffusion of acidic plaque bacterial metabolites into the tissue will lead to demineralization and release of soluble extracelluler matrix component, including growth factors (figure 2). bioactive molecules, which have potential to signal many of defense and repair events in the tooth. the endogenous proteolytic enzymes (matrix metalloproteinases, mmps) present in dentin matrix might also participate in releasing bioactive molecule, even in the absence of bacteria, if their normal regulatory control becomes compromised in pathologic conditions.3 the roles of mmps in carious progressions matrix metalloproteinases (mmps) have been detected in both soft and hard tissue compartment of the dentinpulp complex, and their activity has been suggested to be involved in various physiological processes of the formation and maintenance of the dentin-pulp complex. mmps, a family of endopeptidases capable of degrading in concert virtually all extra cellular matrix components, are expressed during normal dentin-pulp complex formation and maintenance. mmp activity has also been suggested to contribute to the organic matrix degradation during dentin caries progression and to the repair and defense reactions elicited by caries in the dentin-pulp complex cells.2 during early tooth development, dental mesenchymal cells are observed to express at least mmp-1, -2, -3 and -9. mmp-3 suggested to be involved in the organization of the dentin matrix constituents before mineralization inside the dentinal tubules. after the completion of tooth development, odontoblast synthesize mmp-2, -8, -9 and20. these mmps are also synthesized in human pulp tissue. some of the mmps involved in dentin formation become embedded into mineralized dentin, since a latent enzyme with collagenolytic activity as well as gelatinolytic metalloproteinases, specificallymmp-2.12 the mmps of the dentin-pulp complex have been suggested to be involved in pathological processes, including dentin ecm destruction during caries progression and activation of dentinal growth factors due to external non-physiological stimuli, the formation of tertiary dentin, tissue destruction in pulpal inflammation, and breakdown of demineralized collagen in the adhesive layer of dental composite restorations.2,9 discussion the dentin-pulp complex exhibits various responses to caries, including events of injury, defense and repair. these responses are reactions in the vital tissues of the tooth in combination with physico-chemical processes involving the mineral phase of the tooth.2,9 bacterial acids and other metabolites, even though diluted and mixed with dentinal constituents, are noxious to the cells of the dentin-pulp complex, and due to the tubular structure of dentin. caries induce changes in the morphology and enzymatic activity of odontoblast cells as well as a decrease in predentin width and accumulation of cells into the cell-free zone. the first sign of pulpal defense is a local inflammation under the site of irritation. it has now been possible to identify a number of growth factors in dentin matrix, which while quantitatively minor component, may nave potent biological effects. a key family of growth factors, which have been identified in dentin, are members of the tgf-b (transforming growth factor-b) family. it have been implicated in signaling of odontoblast differentiation. odontoblast express all three isoforms of tgf (tgf-1, -2, and -3), but only tgf-b becomes sequestrated within the matrix and may help to dampen the inflammatory response in the pulp.11 a variety of cytokines (cell-signaling molecules associated with inflammatory and immune reactions) recognized to be associated with pulp responses to dental caries. in caries lesion progression, the initial diffusion of acid into the tissue will be followed by diffusion of plaque bacteria-derived proteolytic enzyme, which will degrade the insoluble dentin matrix. release of the associated growth factors will further supplement the soluble pools of growth factors from the tissue. thus, the degradative events of caries may be important in releasing and unmasking figure 2. mehanisms of release of growth factors (grey stars) from dentin matrix and their subsequent diffusion along the dentinal tubules to interact with the odontoblast and pulp cells.3 bacterial acids caries �� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 11–15 the inflammatory cells infiltrate consist mainly of t-lymphocytes, macrophage, neutrophils, b-lymphocytes and plasma cells.13,14 proteolytic enzyme of host and/or microbial origin have been detected in carious dentin, especially in the dentinal tubules. cariogenic microbes are required for caries development, but evidence for their collagenolytic or even proteolytic activity is inconclusive.2 the matrix metalloproteinase family is mainly responsible for the degradation of the structural macromolecules of the interstitial connective tissue in the extracelluler milieu during physiological ecm turnover and reorganization in human body, presumably including the dentin-pulp complex. as in other tissues, mmp activity may also be altered in pathological conditions of the dentinpulp complex. mmps may contribute to the degradation of the dentin organic matrix during caries progression, but also to the response reactions to caries in the dentin-pulp complex.2,9 kamal, 1997 studied that the response of ia antigenexpressing cells to carious irritants triggers the defense reactions of the pulp. the intensity of the defense reaction may be correlated with the permeability of carious dentin.15 the signals for tissue repair are thought to be mediated by dentin-bound growth factors released during caries progression. the processes of reactionary and reparative dentinogenesis at sites of dental injury are responsible for secretion of tertiary dentin matrices, which either increase the dentin barrier between the site of injury and the underlying cells in the unexposed pulp or provide a dentin bridge across the exposed pulp. growth factors may be a key molecules in the signaling of the biological even responsible for these processes.13 a number of reports of the transdentinal or direct application of tgf-β-1 and bmp-7 to the odontoblast of unexposed pulp in cultured tooth shown ability of these gf to signal reactionary dentinogenesis. these findings suggest exciting opportunities for biologically based therapeutic approaches to dental tissue repair as well as providing valuable insights into how natural regenerative processes may be operating in the tooth.3,16 experimental investigations in mature teeth have shown that a network of extra cellular matrix molecules and growth factors signal tertiary dentinogenesis. application of dentin matrix components or growth factors in deep dentinal cavities stimulated up-regulation of biosynthetic activity of primary odontoblasts (reactionary dentin formation).16 lee et al.,17 analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries to understand the molecular events underlying the dentin-pulp complex responses to carious progression. dentin matrix proteins analyzed included collagen type i, phosphophoryn (pp) and dentin sialoprotein (dsp), all of which play decisive roles in the dentin mineralization process. the carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type i, pp and dsp proteins. this response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation. recent advances in stem cell biology and gene therapy technology have provided the great potential of adult stem cells for therapeutic use in regeneration of lost tissue due to diseases including cancer, trauma, and even caries. dental pulp tissues harbor mesenchymal stem/progenitor cells and have potential to regenerate and/or repair dentin-pulp complex after injury such as caries.18 the vitality of the dentin-pulp complex, both during tissue homeostasis and after injury, is dependent on pulp cell activity and the signaling processes, which regulate the behavior of these cells. the processes of reactionary and reparative dentinogenesis at sites of dental injury are responsible for secretion of tertiary dentin matrices, which either increase the dentin barrier between the site of injury and the underlying cells in the unexposed pulp or provide a dentin bridge across the exposed pulp. growth factors may be a key molecules in the signaling of the biological even responsible for these processes. the degradation of the collagenous organic matrix of dentin during caries progression is an enzymatic process. several members of the mmp family are found in the soft and hard tissue compartment of dentin-pulp complex. their presumed role in many physiological process during the development and maintenance of the dentin-pulp complex, they may also contribute to the pathogenesis of dentin caries and the responses elicited by caries. references 1. nakhasima m. bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy. cytokine growth factor rev 2005 jun;16:369–76. 2. sulkala, m. matrix metalloproteinase (mmps) in dentin-pulp complex of healthy and carious teeth. 2004. available from http://hercules. oulu.fi/isbn9514274598.htm. accessed november 28, 2006. 3. smith, aj. vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. j dent educ 2003 jun; 67:678–89 4. jontell m, okiji t. immune defense mechanisms of the dental pulp. crit rev oral biol med. 1998; 9(2):179–200. 5. roth gi, calmes r. oral biology. 1st ed. london: the cv mosby company; 1981. p. 119–37. 6. love rm, jenkinson hf. invasion of dentinal tubules by oral bacteria. crit rev oral biol med 2002; 13(2):171–83. 7. waltimo j, risteli l, risteli j. altered collagen expression in human dentin : increased reactivity of type iii and presence of type vi in dentinogenesis imperfecta as revealed by immunoelectron microscopy. j histochem cytochem 1994; 42:1553–63. 8. linde a, goldberg m. dentinogenesis. crit rev oral biol med 1993; 4:679–708. 9. pashley dh. dynamic of the pulpo-dentin complex. crit rev oral biol med 1996; 7:104–33. 10. arnold wh, konopka s, gaengler p. qualitative and quantitative assessment of intratubular dentin formation in human carious lesion. calsif tissue int 2001; 69:268–73. 11. cassidy n. comparative analysis of tgf-b1-3 in human and rabbit dentine matrices. arch oral biol 1997; 4(2):19–23. ��rahayu: molecular study of the dentin-pulp 12. palosaari h, permington cj, larmas m, edwards dr. expression profile of mmps and tissue inhibitors of mmps in mature human odontoblast and pulp tissue. eur j oral sci. 2003; 111:117–27. 13. magloire h, bouvier m, joffre a. odontoblast response under carious lesions. proc finn dent soc 1992; 88(suppl i):257–74. 14. izumi t, kobayashi i, okamura k, sakai h. immunohistochemical study on the immunocompetent cells on the pulp in human non-carious & carious teeth. arch oral biol 1995; 40:609–14. 15. kamal am, okiji t, kawashima n, suda h. defense responses of dentin/pulp complex to experimentally induced caries in rat molars: an immunohistochemical study on kinetics of pulpal ia antigen-expressing cells and macrophages. j endod 1997 feb; 23(2):115–20. 16. tziafas d. the future role of molecular approach to pulp-dentinal regeneration. caries research 2004; 38:314–20. 17. lee yl, liu j, clarkson bh, lin cp, godovikova v, ritchie hh. dentin-pulp complex responses to carious lesions. caries research 2006; 40:256–64. 18. nakhasima m, iohara k, zheng l. gene therapy for dentin regeneration with bone morphogenetic proteins. curr gene ther 2006 oct; 6(5):551–60. isi vol 39 no 2 april 2006 file pertama.pmd 68 oral and dental aspects of child abuse arlétte suzy puspa pertiwi and inne suherna sasmita pedodontics department faculty of dentistry padjajaran university bandung indonesia abstract child abuse is defined as those acts or omissions of care that deprive a child from the opportunity to fully develop his or her unique potentials as a person either physically, socially or emotionally. the overall incidence of child abuse is not really clear. statistical data do not show the actual rate because of the unreported cases. dentists are in a strategic position to recognize and report the children being abused because they often see the child and parents interacting during multiple visits and over a long period of time. the orofacial region is commonly traumatized during episodes of child abuse. the characteristics and diagnostic finding of child abuse, and the protocol of reporting such cases, should be familiar to the dentist so that appropriate notification, treatment and prevention of further injury can be instituted. dentists with experience or expertise in child abuse and neglect will strengthen their ability to prevent and detect child abuse and neglect and enhance the ability to care for and protect children. this paper discusses the oral and dental aspects of child abuse and the dentist role in evaluating this situation including prevention of child abuse. key words: child abuse, oral and dental aspects correspondence: arlétte suzy puspa pertiwi, c/o: bagian pedodonsia, fakultas kedokteran gigi universitas padjadjaran. jln. sekeloa selatan i bandung 40135, indonesia. e-mail: arlettesuzy@yahoo.com introduction recently, the cases of child abuse often appear either the ones with the family member as the actor or other party. in medical field, child abuse (ca) was reported at the first time in 1860 in france when 320 children died of the causes suspected as abuse.1 child abuse is defined as those acts or omissions of care that deprive a child from the opportunity to fully develop his or her unique potentials as a person either physically, socially or emotionally.2 child abuse is not limited to certain social class in a society. in the middleto lower-class society, many ca are often caused by poverty. meanwhile, in the middleto upperclass society, ca is often caused by the parents’ ambition to shape the children according to their ideals about the best thing for them. generally there are five factors that trigger ca, namely moral degradation, abnormal nurturing pattern, media exposure, low level of economy, and low level of education.3 there are 4 major types of ca, physical abuse, sexual abuse, emotional abuse, and neglect. there are also other types of ca including child exploitation or employing children under age for commercial purpose while pushing aside the child’s physical, mental, and social development.4 there is no well-known data on the incidence of ca. the statistical data does not show the actual numbers of cases because there are a lot of cases go unnoticed.5 in indonesia, based on the data of children national commission, there are 544 cases of ca in 2004 and increasing into 736 of cases in 2005.3,6 until mid-january of 2006, there have been 4 cases found on child abuse.7 some countries have legal regulation that defines legal obligation to report to the local child protection institution if there is any suspicion towards ca. the persons who have the authority to report the case includes doctors and dentists.1 dentist has a strategic position to identify and report children who experienced ca since he/she often observes interaction between children and parents/caregiver during treatment visit and in a long period of time.2 this paper describes oral and dental aspects of ca and the dentist role in evaluating the situation. physical abuse physical abuse is a physical injury experienced by children, which is not caused by accidents.4 craniofacial, facial, head and neck injuries are found in more than a half of ca cases. careful intraoral and perioral examination is needed in every suspect of ca.8 oral cavity and orofacial region often becomes the main focus of physical abuse because of their functions in communication and nutrition.2,8 a complete examination on oropharynx must include examination on frenulum, gingival, soft and hard palate, tongue, sublingual region, buccal mucosa and posterior pharynx to observe every signs of trauma.9 dental practioners are able to identify physical evidence of injuries to children, especially in the regions of the head and neck and oro-facial structures. when such injuries are of suspicious origin, the dentist has an obligation to document and report his/her findings to appropriate 69pertiwi and sasmita: oral and dental aspects of child abuse authorities so that proper action may be taken to protect the child while the problem is investigated.10 able to play the role of forensic odontologist in order to detect and evaluate bite marks. the lesion, besides being caused by sexual abuse, can also be caused by physical abuse. bite marks should be suspected as the signs of echymoses, abrasion, or laceration in elliptical or ovoid patterns. bite marks can be in the form of echymoses in the middle area that can be caused by 2 possibilities, positive pressure of teeth grinding with disturbance in the small vessels and negative pressure caused by tongue sucking.8 we should be able to tell the different between bite marks caused by a human being or caused by animal bites. animal bites, such as dog, tend to create an open wound while human bite marks tend to be wound caused by pressure, such as abrasion, contusion, and laceration. the spacing in human bites is normally 2.5 to 4 cm, which is the distance between the upper canines. if the intercanines distance is less than 2.5 cm, the big chance is that the bite is caused by children. if the distance is 2.5 to 3 cm, the bite is caused by a teenager, and if it’s more than 3 cm, it is caused by an adult bite.8 emotional abuse emotional abuse is defined as every attitudes or behaviors that can disturb mental health or social development of a child. other names for emotional abuse are verbal abuse, mental abuse, or psychological maltreatment. emotional abuse almost always happens together with other forms of abuse.3 emotional abuse does not leave trauma signs on the child’s body, but it leaves psychological trauma on the child. the children become scared of everything, apathetic, injury in oral cavity is commonly caused by blunt trauma, such as trauma caused by tableware, hand, fingers, blunt instruments or caustic substance. the violence can caused contusion, laceration and bruise on the tongue, buccal mucosa, palate, gingival mucosa or frenulum, dental or avulsion fracture, facial and jaw fracture, burn, and other kinds of injuries (figure 1). besides, trauma in oral cavity, tongue, palate and frenulum, blunt trauma on the teeth and facial bone and jaw bone fracture can be caused by repetitive trauma.9 discoloration of the teeth, besides the abovementioned injuries, also shows the existence of pulp necrosis that can be caused by previous trauma.8 wound, bleeding, erythema, or swollen outer ear canal, broken lips, unstable teeth or fractured teeth, tongue laceration and blue eyes without trauma on the nose indicates physical abuse.1 sexual abuse sexual abuse is every sexual act between older person and the child. the acts can be called sexual abuse if the one who does the act is the caregiver, family members, father, mother, nanny, or teacher, either at home or outside the child’s house. if a stranger performs that kind of action, the action is called sexual assault.4 oral cavity is one of the locations of sexual abuse often found in children. the existence of gonorrhea or syphilis in the oral or perioral region in prepubertal children is one of the signs of sexual abuse. cement detection in the child’s oral cavity can be performed in a few days period after the abuse. therefore, during an investigation towards a child who is suspected as a victim of sexual abuse, cotton swab should be performed to get the buccal mucosa and tongue smear.8 erythema or petechiae palatum, especially in the junction between soft and hard palate can become a proof of forced oral sex action (figure 2). another oral lesion can be found in the form of condylomata acuminata.8 bite marks can be a lesion that leads to abuse acts (figure 3-a and b). bite marks are one of several visual expressions of active child abuse.9 the dentist should be figure 1. bruise on frenulum caused by blows on the face2 figure 2. injuries caused by sexual assault2 figure 3. a) bite marks on 18 month old child and b) three month old baby2 a b 70 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 68–71 depressed, experience concentration disorders, or rebellions. if this situation keeps going on, it can create serious behavior disorders, cognitive disorders, emotional or mental disorders for the children. dentist should be able to recognize signs in children who is the victim of emotional abuse and apply appropriate new approaches.3 neglect neglect is an ignorance towards the child either in the form of nurturing, supervising, care, and education that can caused destructive effect on the child’s physical condition and his/her psychological development.1 there are 3 types of neglects; physically neglect, educationally neglect, and emotionally neglect.3 in the dentistry area, a dental neglect is also recognized as a part of physically neglect. 8 dental neglect, as defined by american academy of pediatric dentistry, is intentional neglect by the parents or caregiver towards the child’s oral cavity and dental health or a neglect that prevent the child to get dental and oral treatment needed by the child in order to achieve oral health level needed for adequate function.8 dental neglect can be seen as the appearance of caries, periodontal disease, and other oral cavity diseases.8,10 caries, periodontal diseases and other oral condition, if not treated, can lead to pain, infection, and loss of oral function that can affect communication, nutrition, learning activities and other children activities needed for normal growth and development.8 failure to get good dental treatment can be caused by various factors such as family isolation, poor financial status, parent's neglect and lack of appreciation towards oral health value. the point at which to consider a parent negligent and to begin intervention occurs after the parent has been properly alerted by a health care professional about the nature and extent of the child’s condition, the specific treatment needed, and the mechanism of accessing that treatment.8 the dentist should be certain that the caregivers understand the explanation of the disease and its implications and, when barriers to the needed care exist, attempt to assist the families in finding financial aid, transportation, or public facilities for needed service. parent is should be reassured that appropriate analgesic and anesthetic procedures will be used to assure the child’s comfort during dental procedures. if, despite these efforts the parent is fail to obtain therapy, the case should be reported to appropriate child protective services.11 prevention of child abuse children are a nation’s next generation. therefore, ca should be prevented because it can affect the long life process of a child.1 in indonesia, dentist’s authority to report ca has not been regulated in a law or regulation. however, prevention and management of abuse and violence towards children is a serious matter that should be acted on immediately by all related parties, including the parents, educators, law force, security force, mass media and health service.1 discussion child abuse is both shocking and commonplace. child abuses inflict physical, sexual, and emotional trauma on defenseless children every day. the scars can be deep and long lasting. unfortunately, the more subtle forms of child abuse such as neglect and emotional, it can traumatize the child as deep as violent physical abuse. focused support can help both victims of ca and the abusers. the effects of abuse and neglect depend on some factors. first, the age of the child when the abuse was happen. the younger child usually the harmful effect will occur, but different effects are associated with different developmental periods. second, who committed the abuse? effects are generally worse when it was the parent, stepparent or trusted adult than a stranger. third, has the child told anyone, and if so, the response of the person on the child’s story will give effects. doubting, ignoring, blaming and shaming responses can be extremely harmful in some cases even more than the abuse itself. fourth, was violence involved, and if so, how severe. fifth, how long the abuse went on. child abuse and dentistry have a close correlation because of three possible reasons. sixty five percent of injuries resulting from child abuse are on the head, neck, or mouth. the victims may avoid the same physician, but return to the same dentist. children are most likely to have regular preventive care in the same dental office.8,9 according to needleman, 1986, studies demonstrate that trauma to the head and associated areas occurs approximately 50% of the cases of physical abuse to children. soft tissue injuries, most frequent bruises, are the most common injury sustained to the head and face and are the single most common injury sustained in child abuse. injuries to the upper lip and maxillary labial frenum may be characteristic lesion in the severely abused young child.12 suspected abuse can be documented because of the trauma associated with burning, slapping, hitting, choking, pulling or pinching. broken teeth, burns, lacerations, bruises and broken bones could alert the dentist that something wrong had happened on the child. neglect, however, is more subtle. the dentist should look at overall hygiene as well as dental hygiene and adequacy of clothing. suspicion of poor nutrition, apparent lack of medical care, and absence of previous dental care are situations that should alert the dentist to consider neglect. the american academy of pediatric dentistry defines dental neglect as failure of the parent or guardian to seek treatment for caries, oral infection, or oral pain, or failure of the parent or guardian to follow through with treatment once he or she is informed that the aforementioned conditions exist. thus the guidelines of dental neglect are untreated rampant caries; untreated pain, infection, bleeding, or trauma; and lack of continuity of care once informed that above conditions exist. in foreign countries, physicians and dentists realize their responsibility to report suspected cases of ca. in us, they 71pertiwi and sasmita: oral and dental aspects of child abuse are required to report it to social service or law enforcement agencies.13 physicians receive minimal training in oral health and dental injury and dental disease, thus may not detect dental aspects of ca like they do on ca involving other areas of the body. therefore, physicians and dentists should collaborate to increase the prevention, detection, and treatment of this conditions.14, 15, 16 in indonesia, there is no such action yet. the dentist knowledge and skill in managing ca has not been fully gained. therefore, ca and forensic dentistry curriculum should get a place in the education program. child abuse (ca) consists of all bad treatments acted on a child by the parents, caregivers or other person/s who should be giving care and security for the children. ca can be found in all social classes and can be caused by various triggers. the long-term effects of chronic exposure to domestic violence and ca may affect a child’s overall development. social, cognitive, and emotional development may be adversely affected. the study suggest that dentist has a role in identifying, managing, helping and treating ca victims since the injuries are often found in the oral and facial areas. besides managing such cases, dentist also has a role in preventing ca because the ca can affect the future life of the children. such efforts will enhance the ability to care and protect children. references 1. sitohang na. asuhan keperawatan pada anak child abuse. medan: usu digital library; 2004. h. 1–6. available at: www.library.usu.ac.id. accessed february 23, 2006. 2. cameron a. trauma management. in: cameron ac, editor. handbook of pediatric dentistry. 2nd ed. sydney: mosby; 2003. p. 88–90. 3. sucahyani bd. kekerasan pada anak. batam: batam pos, 22nd january 2006. available at: www.batampos.com. accessed february 19, 2006. 4. de benedictis t, jaffe j, segal j. child abuse: types, signs, symptoms, causes and help. available at www.helpguide.org. accessed february 25, 2006. 5. hopper j. child abuse, statistic, research, and resources. available at: www.jimhopper.or.id. accessed february 19, 2006. 6. sofiyan i. pkk harus mengajak masyarakat terus berkiprah. available at: www.kotabogoronline.htm. accessed february 19, 2006. 7. data statistik kekerasan pada anak. available at: www.pdpersi.co.id. accessed february 19, 2006. 8. kellog n. oral and dental aspects of child abuse and neglect. pediatrics 2005; 116:1565–8. 9. jessee s. recognition of bite marks in child abuse cases. aapd journal 1994; 16:336–9. 10. sirotnak ap, grigsby t. physical abuse of children. pediatrics in review 2004; 25: 264–77. 11. croll tp, menna vj, evans ca. primary identification of an abused child in a dental office. american academy of pediatric dentistry journal 1981; 3:339–42. 12. needleman hl. orofacial trauma in child abuse: types, prevalence, management and the dental prefession’s involvement. pediatr dent 1986; 8:71–80. 13. sanger rg, bross dc. clinical management of child abuse and neglect: a guide for the dental professional. chicago: quintessence publishing co, inc; 1984. p. 37–46. 14. mouden ld, bross dc. legal issues affecting dentistry’s role in preventing child abuse and neglect. journal of american dentistry association 1995; 126:1173–80. 15. schwartz s, woolridge e, stege d. the role of the dentist in child abuse. quintessence int 1997; 7:79–81. 16. donly kj, nowak aj. maxillofacial, neck, and dental lesions of child abuse. in: reece, rm, editor. child abuse: medical diagnosis and management. philadelphia: lea & febiger; 1994. p. 211–51. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 33 figure 1. panoramic x-ray showed two dental implants over the region of upper first and second molars. they were not in good parallelism. no adequate amount of bone was seen around the implant fixtures and both of the apical part of fixtures seemed to lie in the antrum. closure of oroantral fistula with rotational palatal flap technique david b. kamadjaja department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract oroantral fistula is one of the common complications following dentoalveolar surgeries in the maxilla. closure of oroantral fistula should be done as early as possible to eliminate the risk of infection of the antrum. palatal flap is one of the commonly used methods in the closure of oroantral fistula. a case is reported of a male patient who had two oroantral communication after having his two dental implants removed. buccal flap was used to close the defects, but one of them remained open and resulted in oroantral fistula. second correction was performed to close the defect using buccal fat pad, but the fistula still persisted. finally, palatal rotational flap was used to close up the fistula. the result was good, as the defect was successfully closed and the donor site healed uneventfully. key words: oroantral fistula, rotational palatal flap correspondence: david b. kamadjaja, c/o: bagian bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction oroantral communication is any communication which occur between the oral cavity and the antrum. it is a common complication after extraction of upper posterior teeth because there is a close relationship between the root apices and the antrum. basically, oroantral communication should be closed immediately in order to prevent sinusitis. an oroantral communication which is less than 2 mm in diameter will usually close spontaneously, but when there is more than a 3 mm defect, or there is inflammation in the antrum or in the periodontal region, the opening often persists and may become oroantral fistula.1 variable methods to close oroantral fistula (oaf) have been reported in the literature, such as buccal sliding flap,1,2 pedicled graft of buccal fat pad,3,4 and palatal flap1,2,5 with varying clinical results. they have their own advantages and disadvantages depending on the cases and the size of the defects occured. a case of a oroantral fistula which failed to close despite a couple of surgery using buccal approach, but could eventually be closed with rotational palatal flap technique is presented. case a 61-year-old male patient came to our clinic with chief complaint of discomfort associated with two dental implants over his right upper jaw. the implants were inserted around 6 months previously by his dentist. artificial bone graft was used during the operation and it might be used to enhance the stability of the implants. a few weeks after implants placement, however, those two implants became loose and small granules were noted by the patient to keep coming out from the gingiva around the implant site. some efforts had been made by the dentist to keep the implants firm in his position but resulted in failure and he complained that the loose implants had given him a lot of discomfort especially during eating. clinical examination revealed the following. two implants abutments were seen protruding from the alveolar mucosa over the right edentulous maxilla. the surrounding mucosa was normal in color, no swelling nor signs of soft tissue inflammation was detected. on palpation, the abutments were loose in all directions, but only slight tenderness exhibited by the patietnt. panoramic x-ray showed two dental implants over the region of upper first and second molars and were not in good parallelism. no adequate amount of bone was seen to support the implant fixtures and both of the apical part of the fixtures seemed to lie in the antrum (figure 1). water’s x-ray, showed normal maxillary sinus on both sides. the case was assessed as 3� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 33–36 figure 2. opening of the oroantral fistula over the upper first molar region (anterior defect) seen after the second surgery. an implant failure. it was primarily due to lack of primary implant stability most likely caused by inadequate amount of healthy bone between the antrum and oral mucosa. the bone graft used, was unable to help achieve the stability of the implants. case management a three-stage surgical plans were made to overcome the existing problem. the first surgery was planed to remove the implant fixtures close the defect, followed by sinus lift procedure, approximately 4 weeks after the implant removal, to increase the bone height. the last planned surgery was to insert two implants in the region with adequate amount of bone which was expected to provide primary implant stability. in the first surgery, the following were done. after disinfection of the oral mucosa with chlorhexidine solution, local infiltration injection was done using 2% lidocaine and adrenalin 1:200.000. the removal of the two implants was done with case. an oroantral communication was seen clearly after removal of the implants. a simple buccal flap was then made to close up the defects. a clinical review one week after the surgery, most of the wound healed well except in the area of anterior defect where a small dehiscence was noted. antral wash-out with normal saline was done and clear return seen indicating that there was no antral infection. a second surgical step to close the defect which was not planned previously, was offered to the patient. upon the patient consent at that visit the second surgery to close the defect was done using buccal fat pad graft. during the surgery, the amount of fat tissue was noted to be insufficient therefore it had to be pulled downward with tension to cover the defect. in addition, the graft was seen to be light in colour and relatively softer in consistency indicating a poor quality of the fat graft. the fat pad graft was stitched in to the surrounding mucosa which had been freshened up with a sharp blade. the buccal flap overlying the graft could not be pulled loosely towards palatal mucosa because of some fibrotic tissue secondary to the first surgery, therefore only the fat tissue covered the defect. post operative reviews showed that the result of the second operation was unsatisfactory. most of the fat graft overlying the defect was lost in the first clinical review of 3 days post operatively. seven days after surgery the graft was found to be completely absent and the anterior defect remained open. a thorough examination through the opening of the defect showed that the oral and antral mucosa met directly with the antral mucosa because of complete bone loss in that area (figure 2). wash out of the antrum was done and showed clear return indicating that there was no antral infection. the patient was informed about the problem which still existed and about the impending infection of the antrum if the defect was not closed immediately. another surgery using palatal flap was offered to close up the defect. since the surgery was scheduled on a later date an upper jaw impression was taken to prepare for an obturator construction. the obturator which was aimed to cover the opening of the fistula. it was inserted on the following day and worn by the patient until the day of the surgery. the palatal flap operation was done about 3 weeks later. the following were done during the procedure. local anesthesia was done using 2% lidocaine solution with adrenaline (1:80,000). the surgery was initiated by making circular incision around the fistula opening until the underlying bony defect was exposed. a palatal flap design was visualized and planned according to the site and size of the defect. a partial thickness flap was raised based on its blood supply and repositioned laterally to cover the defect. the flap was then secured in its new place using 3/0 black silk sutures. the donor area which was left covered by the periosteum of the palatal bone was left open for a secondary epithelization. some adjustment had been made to the obturator which then reinserted in order to protect the donor as well as the defect area (figure 3). one week post operative review showed that the wound in the defect area healed without dehiscence, and the donor site was covered with normal fibrin. the patient did not complain of any pain and there was no sign of infection. fourteen days after surgery the wound in the defect area had healed well and the donor site was fully covered with granulation tissue. clinical review three months later showed healthy pink epithelium covering the donor site on the hard palate. the bulky palatal flap had shrunk considerably so that the hard palate had retained its normal shape (figure 4). 3�kamadjaja: closure of oroantral fistula is sufficient alveolar bone height, i.e. at least 5 mm, to stabilize the implants during healing. immediate placement of the implants is contraindicated if the available host bone is less than 5 mm since the implants will not be maintained mechanically.8 in the case presented here, the two implants were inserted at the same time as the bone grafting procedure of the antral floor. the simultaneous placement of implants and sinus lift grafting in this case were actually contraindicated as the primary mechanical stability was unlikely to be achieved, because only approximately 2 mm and 4 mm of bone height were available respectively. it was predicted that removing the implants would cause an oroantral communication especially in the anterior discussion the placement of dental implants in patients who are edentulous in the posterior maxilla can be difficult because of deficient posterior alveolus, increased pneumatization of the maxillary sinus, and close approximation of the sinus to crestal bone.6 in many cases, this problem can be overcome by increasing the alveolar height with bone grafting of the maxillary antral floors which will provide a sufficient quantity and quality of bone for the placement of osseointegrated implants.7 sinus lift grafting and implant placement are accomplished as either one-step or two-step procedure. one-step procedure can be performed if there figure 3. (a) preparation of the donor site, circular incision was made around the defect until healthy surrounding bone was exposed; (b) partial thickness pedicle flap was raised over the right hard palate adjacent to the defect; (c) the flap was rotated laterally to cover the defect and stitched to the mucosa surrounding the defect with 3/0 black silk suture, periosteum layer was noted over the donor site; (d) obturator was inserted to cover the donor and defect site. a ba b c d figure 4. (a) at one week post operative review, granulation tissue covered by normal fibrin layer was noted over the donor site; (b) three month post operatively, the palate resumed its normal shape and color. a b 3� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 33–36 region, therefore the surgery was also planned to close the defect immediately with buccal sliding flap. one week post operatively a small area of dehiscence was noted over the anterior defect. it may be caused by two reasons: the flap was too tense, or there was no bony support underneath the flap. the decision to close the anterior defect with buccal fat pad graft was based on several factors. this procedure was relatively simple and reliable with minimal incidence of failure when properly performed.9 the other reason was that the previous operation has made the buccal mucosa less flexible and caused obliteration of the buccal sulcus and these would make another buccal sliding flap impossible. the buccal flap in this second surgery could not cover the entire fat graft overlying the defect but this should not have been a problem. the fat pad will actually be able to epithelialize even if it is left unlined by the mucosa.10 the superficial layer of fat tissue will be replaced by granulation tissue, and is finally covered with stratified squamous epithelium migrating from the margin of the gingival.9,11 the most likely cause of the failure in the second surgery was the poor quantity and quality of the fat pad graft. since there was inadequate amount of fat tissue that was obtained, some degree of tension might have occurred during the reposition of the graft towards the defect. this might have compromised its blood supply. therefore necrosis of the graft was expected to occur. the light yellow in color and the low consistency of the fat tissue might indicated a poor fat quality in terms of its strength and survival capacity when used as a graft material. the decision to close the oroantral fistula with palatal flap was finally made because we could no longer use the fibrotic buccal mucosa as the consequence of the previous surgeries. the hard palate has a relatively thick mucoperiosteum layer and good vascularization from greater palatine artery.12 flaps raised from hard palate will, therefore, have high viability. this is the reason why palatal flap can be used either as pedicle flap or random flap.13 the other advantages of palatal flap are: it has good vascularity, similarity in thickness with that of gingiva, good esthetic, good accessibility, and does not cause obliteration of the vestibular sulcus. the donor site at the palate can be left to granulate and recover within 2-3 weeks with minimal deformity.5 the disadvantages of palatal flap include: rotation of the flap may compromise its vascularization, the operation take longer time compared to buccal fat surgery, temporary speech impairment, the risk of bone necrosis, unsuitable for large defect.14 in this case a rotation palatal flap was used based on the location of the defect which was situated at the region of tooth 17 where the flap can be easily rotated without any risk of compromising its blood supply. the circular incision made around the fistula opening was important in this procedure as it was the site at which the flap would be attached to. the incision was made in such a way that all of the healthy bone underlying the defect was exposed. this was to ensure a good bony support for the palatal flap which would be placed and stitched over the defect. this would prevent wound dehiscence around the flap.1 a partial thickness flap was used in this case, instead of full thickness one, in order to avoid post operative pain and infection of the palatal bone. as the periosteum at the donor site was not detached the healing would take shorter time if compared with full thickness flap. the design of palatal flap is important in that it influence the overall result of this procedure, the most critical factor of which being the length and width ratio of the flap. a clinical study showed that for the best result, the flap length and width should be approximately 2.3 in ratio.15 it can be concluded that an oroantral fistula can be closed with various methods using buccal or palatal tissue depending on the size and location of the defect. the first choice of surgery is usually the buccal advancement flap which is the easiest method. if the defect is larger or when the buccal advancement flap has failed, a buccal fat pad technique can be used with high success rate. in case of failure of buccal approach or if the defect cannot be closed using buccal tissue, a palatal flap should be used as the treatment of choice. the successful result of palatal flap depends on the appropriate choice of the type and the design of the flap. references 1. howe gl. minor oral surgery. 3rd ed.. bristol: john wright & sons ltd; 1985. p. 207–23. 2. kruger go. textbook of oral surgery. 4th ed. cv mosby co; 1974. p. 255–60. 3. yih wy, merril rg, howerton dw. secondary closure of oroantral s oronasal fistulas. j oral maxillofac surg 1988; 46:359. 4. egyedi p. utilization of the buccal fat pad for closure of oroantral and/or oronasal communication. j maxillofac surg 1977; 5:241. 5. anavi. palatal rotation-advancement flap for delayed repair of oroantral fistula: a restropective evaluation of 63 cases. oral surg oral med oral pathol oral radiol endod 2003; 96:527–34. 6. smiler dg, johnson pw, losada jl. sinus lift graft and endosseous implants. dent clin north am 1992; 36:151–86. 7. arun kg. practical implant dentistry. taylor publishing co; 1999. p. 89. 8. kent jn, block ms. simultaneous maxillary sinus floor bone grafting and placement of hydroxylapatite-coated implants: a preliminary report. j oral maxillofac surg 1990; 48:27–32. 9. hanazawa y, itoh k, mabashi t, sato k. closure of oroantral communications using a pedicled buccal fat pad graft. j oral maxillofac surg 1995; 53:771–5. 10. tideman h, bosanquet a, scott j. use of the buccal fat pad as a pedicled graft. j oral maxillofac surg 1986; 14:435. 11. chao ck, chang lc, liu sy, wan jj. histologic examination of pedicled buccal fat pad graft in oral submucous fibrosis. j oral maxillofac surg 2002; 60:1132–3. 12. snell rs. anatomy klinik untuk mahasiswa kedokteran. 5th ed. jakarta: egc; 1997. p. 156. 13. aston sj, beasley rw, thorne chm. grabb & smith’s plastic surgery. 4th ed. philadelphia: lippinvott-raven publisher; 1991. p. 20–35. 14. tambini kn. the maxillary sinus: a general review and closure of acute oroantral communication. chicago: university of illinois; 2000. p. 1-11. available in: http://www.uic.edu/dept/ doms/grand-index. html. accessed may 15th, 2000. 15. lee jj, kok sh, chang hh, yang pj, hahn lj, ko ys. repair of oroantral communicatiuon in the third molar region by random palatal flap. int j of oral & maxillofac surg 2002; 31:677–80. 59 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 59 the role of ubiquinone supplementation on osteogenesis of nonvascularized autogenous bone graft irham taufiqurrahman,1 achmad harijadi,2 roberto m. simanjuntak,3 coen pramono d,4 and istiati5 1department of oral and maxillofacial surgery, dentistry programme, faculty of medicine, universitas lambung mangkurat, banjarmasinindonesia 2,3,4department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, surabaya-indonesia 5department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: ubiquinone is one of food supplement which is known have positive effect in wound healing. however the study to evaluate the possible role of ubiquinone in bone healing in autogenous bone grafting after mandibular resection has not been studied. an in vitro study is required to evaluate whether ubiquinone or coenzyme q-10 (coq10) has a positive effect on osteogenesis. viability test of coq10 and a model of osteogenic-induced and hypoxic-condition mesenchymal stem cell culture were established to support the study. purpose: the study was made to evaluate the role of ubiquinone in osteogenesis by analyzing the toxicity effect and the optimal dose of coq10 that might interfere in bone marrow derived mesenchymal stem cell (bm-msc) that was dose in cell culture medium. the bm-msc culture under hypoxia condition were also observed. method: the toxicity and the optimum viability concentration of ubiquinone were observed using mtt assay. the osteogenic differentiation under hypoxic condition was done on bm-msc in osteogenic medium that composed of ascorbic acid, glycerolphosphate and dexamethasone in hypoxia chamber for 21 days. osteogenic differentiation and cellular hypoxia features were tested with immunocytochemical staining using anti-runx2 and anti-hif1α monoclonal antibody, respectively. result: the maximum density value of 1.826 was found in the group of ubiquinone concentration of 75μm/ml, increasing of in concentration of ubiquinone resulted with the decrease of optical density of coq10. statistic analysis using anova showed with no significant difference among groups with various concentration. immunocytochemical staining showed that runx2 expression in 3% hypoxia group (p<0.05). conclusion: ubiquinone was found non toxic in its optimum dose of 75μm/ml, showed by optimum result in the expression of runx2 and hif1α further study is necessary to evaluate the angiogenic and osteogenic effect of ubiquione. keywords: osteogenesis; ubiquinone; toxicity; hypoxia correspondence: achmad harijadi, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airalngga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: oesodo.hariadi@gmail.com research report introduction mandibular defects after tumor resection needs reconstruction to restore mastication, speech and facial aesthetic. gold standard of mandibular reconstruction is non-vascularized autogenous bone graft because of its osteogenic, osteoconductive, and osteoinductive properties. however, the result of bone healing with autogenous bone grafting depend largely on the integrity of surrounding periosteum since it is the source of mesenchymal stem cells (msc) and blood vessels which is critical in initial graft healing.1 periosteum, however, is often defective in patients affliced with extramural tumors in which case the graft failed to grow or incorporate with the surrounding bone.2 increasing angiogenesis in such cases can be theoretically achieved by supplementing the patients with micronutrient that increase the expression of angiogenic factor, such as vascular endothelial growth factors (vegf) by osteogeniccommitted mesenchymal stem cell. study showed that vegf expression was regulated by hypoxia-inducible dental journal (majalah kedokteran gigi) 2015 june; 48(2): 59–63 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 60 taufiqurrahman, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 59–63 factor 1 alpha (hif-1α), a trancription factor responding to hypoxic condition.3 ubiquinone, or coenzyme q-10 (coq10), is one of essential co-enzymes which has antioxidant effect to protect cell membrane from oxidation by the reactiveoxygen species (ros) and provides a regenerative effect, sustaining effective levels of antioxidants in damaged tissues.4 with regards to that, a viability test of ubiquinone and a model of osteogenic and hypoxic-induced cell culture is required prior to the study. the expression of runtrelated transcription factor-2 (runx2), the most important osteogenic trancriptional factor, was frequently used as a marker for osteogenic-committed msc. the aim of this study was to evaluate the role of ubiquinone in osteogenesis. the objectives of this current study are to analyze the toxicity and optimal dose of ubiquinone in bone-marrow derived mesenchymal stem cell (bm-msc); osteogenic differentiation of bm-msc culture under osteogenic medium; and hypoxia characterization of bm-msc culture under hypoxic condition. material and methods the study used stock of bm-msc culture which was isolated and expanded from human bone-marrow aspirates from iliac crestal bone. the processing and storage of bmmsc culture were performed in centre for biomaterial tissue bank and stem cell, dr. soetomo general hospital, surabaya. the thawing procedure was as follows. the vial of cells was removed from dry ice, defrosted in 370 c water bath until ice in the vial was no longer visible. the vial was opened and content transferred to a sterile 15 ml tube then slowly added 10 ml of alpha mem medium, and centrifuged for 10 minutes. the pellet was resuspended gently in the medium and then transferred into a coated culture flask and incubated at 370c and 5% co2. culture of bm-msc of 80-90% confluence was harvested using typsination, the cells were replaced in wells containing 2.5 x 104 cells per well and incubate under 370 c, 5% co2 for 24 hours. samples of ubiquinone with dose of 10, 20, 50, 75, 100µm were added into the wells and incubate for 20 hours. tetrazolium dyes composed of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2h-tetrazolium (mtt) were then added to the wells and incubate for 4 hours and finally dimethyl sulfoxide (dmso) was supplemented to increase the solubilizaton of formazan formed. the color changes in the well was analyzed with elisa reader using 595 nm wave length. bm-msc from passages 4 were detached with trypsin solution and seeded into 24-well microplate, with density of 2 x 107 cell/cm2, which were divided into experimental and control group consisting of 10 wells per group. the wells in experimental group was given osteogenic medium, composed of minimum essential medium eagle alpha modification, or α-mem, supplemented with ascorbic acid, glycerol phosphate and dexamethasone for osteogenic differentiation study. the wells from control groups were given normal medium (α-mem). both of culture groups were incubated at 370 c, 5% co2 and 95% air for 21 days. the medium in both groups were changed every 2 to 3 days. after 21 days, 5 culture wells from each group were kept in specific hypoxia chamber (modular incubator chamber) for 3 days at 370 c, 5% co2 and 3% oxygen concentration, while the remainders were maintained in normal oxygen (normoxic) condition. after 3 days, the cells in the osteogenic medium as well as normal medium, were detached from petri disk and seeded into 24-well plate containing 10% formaldehyde for cell fixation. osteogenic differentiation was confirmed using anti human runx2 monoclonal antibody, while hypoxic conditioned culture was evaluated using anti human hif1α monoclonal antibody. the methods for immunocytochemical staining was as follows. slides was washed with phosphate buffered saline (pbs) with ph 7.4 for 5 minutes, then dipped in h2o2 3% to block the endogenous peroxides and washed 3 times with pbs. blocking unspecific protein done using 10% fetal bovine serum (fbs) containing 1% triton x-100 and 0.02% nan3, then washing with pbs 3 times. the slides were incubated in anti-runx2 monoclonal antibody overnight at 400c. the slides were washed with pbs 3 times, incubated with universal link secondary antibody for 2 hours at room temperature and washed again 3 times, and incubated with streptavidine horse radish peroxidase (hrp) for 40 minutes at room temperature, after washing with pbs 3 times the slides were given diamino benzidine (dab) and incubated for 10 minutes and washed with distilled water for 5 minutes. counterstaining was done using mayer haematoxylin followed by incubation for 10 minutes, and washing with pbs and rinsing with distilled water, the slides were then let to dry at room temperature for microscope reading. the cells positive for runx2 expression were confirmed by the brown colour in the cell cytoplasm. the cells positive for hif1α were also confirmed by brownish cell cytoplasm. the cell counting were done manually by two investigators using zigzag method under inverted light microscope with x400 magnification. the result of mtt assay, and immunocytochemical staining of runx2 and hif1α expression were analyzed statistically using one-way anova (p<0.05). assumption of distribution normality and homogenity of the variances was made prior to anova testing. result the result of mtt assay showed that the mean of optical density in the evaluated doses of coq10 increased along with the increase in its dose. the maximum density value of 1.826 was found in the group of dose of 75 µm/ml. further increase in the dose of coq10 resulted in decrease in the optical density (figure 1). distribution normality test using kolmogorov smirnov showed that all the groups had the value above 0.05 61 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 61taufiqurrahman, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 59–63 10 figure 2. morphology observation of osteoblast-like cells culture in osteogenic medium at 21 days, showing higher density in culture under normal condition (a) compared to hypoxic condition (b)(red arrow pointing to osteoblast-like cells, inverted microscope, x100 magnification). figure 3. distribution of osteoblas-like cells showing expression of hif-1α (yellow arrow head) and runx-2 (red arrow head) under normoxic (a) and hypoxic condition (b) (inverted microscope, 400x). table 2. desriptive data of runx-2 expression in 3% hypoxia and normoxia groups gene expression groups mean ± std. deviation sig. (2-tailed) runx2 normoxia 83.20 3.768 0.000 hypoxia 45.60 4.561 hif1α normoxia 10.20 2.280 0.000 hypoxia 31.60 1.817 figure 3. distribution of osteoblas-like cells showing expression of hif-1α (yellow arrow head) and runx-2 (red arrow head) under normoxic (a) and hypoxic condition (b) (inverted microscope, 400x). a b 9 table 1. optic density of mtt assay result based on various concentratiion of co-enzyme q10 coq10 10µm/ml coq10 20 µm/ml coq10 50 µm/ml coq10 75 µm/ml coq10 100 µm/ml (control) without coq10 1 0. 241 0.252 0.282 0.344 0.297 0.280 2 0.295 0.299 0.305 0.259 0.290 0.292 3 0.358 0.368 0.288 0.279 0.290 0.353 4 0.257 0.261 0.306 0.298 0.331 0.279 5 0.232 0.226 0.222 0.265 0.278 0.331 6 0.259 0.263 0.290 0.381 0.250 0.257 n 1.642 1.669 1.693 1.826 1.736 1.792 π 0.273666 0.278166 0.282166 0.304333 0.289333 0.298666 note: the optimum optical density number of 1.826 was demonstrated in coq10 concentration of 75 µm/ml group. figure 1. the result of mtt assay. the mean of optical density of co-enzyme q10 with various doses. m ea n of o d control without figure 1. the result of mtt assay. the mean of optical density of co-enzyme q10 with various doses. 10 figure 2. morphology observation of osteoblast-like cells culture in osteogenic medium at 21 days, showing higher density in culture under normal condition (a) compared to hypoxic condition (b)(red arrow pointing to osteoblast-like cells, inverted microscope, x100 magnification). figure 3. distribution of osteoblas-like cells showing expression of hif-1α (yellow arrow head) and runx-2 (red arrow head) under normoxic (a) and hypoxic condition (b) (inverted microscope, 400x). table 2. desriptive data of runx-2 expression in 3% hypoxia and normoxia groups gene expression groups mean ± std. deviation sig. (2-tailed) runx2 normoxia 83.20 3.768 0.000 hypoxia 45.60 4.561 hif1α normoxia 10.20 2.280 0.000 hypoxia 31.60 1.817 figure 2. morphology observation of osteoblast-like cells culture in osteogenic medium at 21 days, showing higher density in culture under normal condition (a) compared to hypoxic condition (b)(red arrow pointing to osteoblast-like cells, inverted microscope, x100 magnification). (p>0.05) confirming the normal distribution of the data. homogenity of the variance test showed that significance was above 0.05 (p = 0.779; p>0.05) indicating the use of one-way anova. anova test showed that there was no significant difference among the groups which mean that coq10 was not toxic and the optimum viability dose for further studies was 75 µm/ml. culture of bm-msc in osteogenic medium under normal and 3% hypoxic condition for three days showed that the cell density and number of osteoblast-like cells were higher in those under normal condition compared to hypoxic condition (figure 2). expression of runx2 and hif-1α were evaluated under normoxia and 3% hypoxia within 24 hours. positive expression of hif-1α was identified by brown cytoplasm while expresion of runx2 by blue cell nucleus, positive double staining indicated osteoblast-like cells under hypoxia (figure 3). data in table 2 showed that there were significant differences both in runx2 and hif1α expression between normoxia and hypoxia group (p<0.05). a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 62 taufiqurrahman, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 59–63 table 1. optic density of mtt assay result based on various concentratiion of co-enzyme q10 coq10 10µm/ml coq10 20 µm/ml coq10 50 µm/ml coq10 75 µm/ml coq10 100 µm/ml (control) without coq10 1 0. 241 0.252 0.282 0.344 0.297 0.280 2 0.295 0.299 0.305 0.259 0.290 0.292 3 0.358 0.368 0.288 0.279 0.290 0.353 4 0.257 0.261 0.306 0.298 0.331 0.279 5 0.232 0.226 0.222 0.265 0.278 0.331 6 0.259 0.263 0.290 0.381 0.250 0.257 n 1.642 1.669 1.693 1.826 1.736 1.792 π 0.273666 0.278166 0.282166 0.304333 0.289333  0.298666 note: the optimum optical density number of 1.826 was demonstrated in coq10 concentration of 75 µm/ml group. table 2. desriptive data of runx-2 expression in 3% hypoxia and normoxia groups gene  expression groups  mean ± std.  deviation sig. (2tailed) runx2 normoxia 83.20 3.768 0.000 hypoxia 45.60 4.561 hif1α normoxia 10.20 2.280 0.000 hypoxia 31.60 1.817 discussion ubiquinone is one of essential co-enzymes which can be detected in mitochondria of every type of cell especially in heart, kidney and liver.5 it has antioxidant effect to protect cell membrane from oxidation by the reactive oxygen species (ros) produced in response to cutanoeus injuries such as burn wound. it also provides a regenerative effect, sustaining effective levels of antioxidants in damaged tissues 4 although its concentration in the body tends to decrease with age, the peak of which is at 20 and drop dramatically as much as 68% at 40.6 health articles reported that was generally used as food supplements to increase immune system in immune-deficiency patients, alzheimer and parkinson, cardiovascular diseases, endocrine disease, and anti-aging. the positive effect of ubiquinone in wound healing may, logically, be attributable to induction of angiogenesis. this in vitro study was made to prove this rationale. bone healing after reconstruction of mandibular bone defects with autogenous bone graft according to remodelling cycle were divided into three stages, i.e. activation, resorption, and new bone phase.7 the most important step in early activation phase was new blood vessels formation or angiogenesis in order to provide means for transportion of oxygen, nutrition, and biomolecules which were necessary for further healing into the implanted graft.8 the process of angiogenesis was induced by expression of angiogenic factors, such as vegf, by endothelial cells, osteoblast, and msc either in the graft or surrounding tissue. studies had proved that vegf expression was induced by regional hypoxic condition.9 hif-1α is transcription factor that regulates genes involved in the response to hypoxia, some of which promote neovascularity. vegf is one of the genes upregulated by hif-1α and is the primary cytokine related to angiogenesis. study showed that hif-1α expression resulted in increased vegf expression.3 the activation phase in bone healing was characterized by osteogenic differentiation of msc residing at the bone graft and periosteum overlying the graft. the osteogenic differentiation was usually induced by bone morphogenic proteins (bmp). the osteogenic committed msc were characterized by increased expression of trancriptional factor (runx2).10 in view of the above, this study used angiogenic and osteogenic parameters, i.e. hif-1α and runx2, respectively and used msc as research subjects of an in vitro model of osteogenic and hypoxic-induced cell culture. bm-msc was used in this study because msc derived from bone marrow was found to have the highest osteogenic capacity therefore was strongly suggested for such studies.11 the result of the study showed that expression of runx2 by osteoblast-like cells was significantly higher in normoxic than hypoxic osteogenic medium, as opposed to hif-1α expression which was significantly lower in normoxic compared to hypoxic condition. this result was in accordance with in vitro study which demonstrated that mesenchymal stem cell under hypoxic condition showed up-regulation of vegf and down-regulation of core binding factor-alpha 1 or cbfa-1/runx2.9 some studies also exhibited that over-expression of vegf inhibited osteogenesis in vitro and ectopic bone formation in vivo12 and inhibited in vitro osteogenic differentiation of msc induced by bmp2.13 the result of these studies suggested that there was inverse relationship between angiogenic and osteogenic signalling either in vitro or in vivo. the result of the study also showed that hypoxic condition in this procedure could induce expression of 63 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 63taufiqurrahman, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 59–63 hypoxic transcription factor hif-1α. the result confirmed that the culture of osteoblast-like cells obtained from the hypoxic procedure in this study were suitable for further in vitro angiogenesis study because it could mimic the hypoxic condition to which the osteoblast in non-vascularized autogenous bone graft might be exposed. the conclusion of the study was that ubiquinone was not toxic with the optimum viability concentration for further studies being 75 µm/ml, bm-msc culture in the osteogenic medium was osteoblast-like cells as they overexpressed runx2 and exhibited hypoxic feature as they overexpressed hif1α and underexpressed runx2. it was also suggested that the osteoblast-like cells culture in this study was appropriate for further in vitro study to evaluate the angiogenic and osteogenic effects of ubiquinone. references 1. crha m, nečhas a, srnec r, jonevec j, stehlik l, raušher p, urbanová l, planáka l, jarčár j, amler e. mesenchymal stem cells in bone tissue regeneration and application to bone healing. acta vet brno 2009; 78: 635-42. 2. pramono c. mandibular reconstruction using non-vascularized autogenous bone graft applied in decorticated cortical bone. int j maxillofac surg 2011; 22(1): 47-56. 3. lin c, mcgough r, aswad b, block ja, terek r. hypoxia induces hif-1alpha and vegf expression in chondrosarcoma cells and chondrocytes. j orthop res 2004; 22(6): 1175-81. 4. choi bs, song hs, kim hr, park tw, kim td, cho bj, kim cj, si m ss. effect of coenzyme q10 on cutaneous heal ing in skin-incised mice. archives of pharmacal research 2009; 32(6): 907-13. 5. murray rk, granner dk, mayes pa, rodwell vw. harper’s illustrated biochemestry. 26th ed. mcgraw-hill; 2003. p. 92. 6. kalen a, appelkvist el, dallner g. age related changes in the lipid compositions of rat and human tissues. lipid 1989; 24(7): 579-584, in: lee bj, lin yc, huang yc, ko yw, hsia s, lin pt; 2012, the relationship between coenzyme q10, oxidativ stress and antioxidant enzymes activities and coronary arteri disease. scientific world journal, 2012; 792756. 7. friedlaender g. current concept reviews: bone grafts-basic science rationale for clinical applications. j bone joint surg am; 69(5): 786-90. 8. kanczler jm, oreffo roc. osteogenesis and angiogenesis: the potential for engineering bone. eur cell mater 2008; 15: 100-14. 9. potier e, ferreira e, andriamanalijaona r, pujol jp, oudina k, logeart-avramoglou d, petite h. hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. bone 2007; 40(4): 1078-87. 10. lian jb, stein gs, aubin je. bone formation: maturation and functional activities of osteoblast lineage cells. in: favus mj, editor. primers on the metabolic bone diseases and disorders of mineral metabolism. washington dc: american society for bone and mineral research; 2003. p. 13-28. 11. sakaguchi y, sekiya i, yagishita k, muneta t. comparison of human stem cells derived from various mesenchymal tissues. superiority of synovium as a cell source. arthr & rheum 2005; 52(8): 2521-9. 12. schönmeyr bh, soares m, avraham t, clavin nw, gewalli f, mehrara bj. vascular endothelial growth factor inhibit bone morphogenetic protein 2 expression in rat mesenchymal stem cells. tissue eng part a 2010; 16(2): 653-62. 13. lin zw, wang js, lin lj, zhang jw, liu yl, ming shuai, qi li. effects of bmp2 and vegf165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. exp ther med 2014; 7(3): 625-9. 191191 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 191–195 case report orthodontic camouflage treatment using a passive self-ligating system in skeletal class iii malocclusion fransiska monika and retno widayati department of orthodontics, faculty of dentistry, universitas indonesia, jakarta – indonesia abstract background: the treatment options for adults with skeletal class iii malocclusion can be dentoalveolar compensation, also known as orthodontic camouflage, or orthognathic surgery. camouflage treatment can be carried out with teeth extractions, distalisation of the mandibular dentition, and use of class iii intermaxillary elastics. however, intermaxillary elastics as anchorage has its own risk–benefit. purpose: to explain that camouflage treatment with teeth extractions can be performed in a mild to moderate skeletal class iii malocclusion using intermaxillary anchorage with elastics, while minimising the deleterious effects and achieving a satisfactory treatment outcome. case: our patient was a 25-year-old female who had a skeletal class iii pattern, with normal maxilla and a protruded mandible. she had a straight facial profile with a class iii canine and molar relationship on her right and left sides. anterior crossbite was also present with crowding on both the maxilla and the mandible. case management: the treatment plan was carried out with dentoalveolar compensation by extracting teeth. extraction of the lower first premolars was conducted to eliminate the crowding and correct the anterior crossbite. the mandibular incisors were retroclined and the maxillary incisors were proclined with dentoalveolar compensation. passive self-ligating system was used with standard torque prescription, intermaxillary anchorage, and no additional appliances for anchorage control. class i canine and incisor relationship were both achieved at the end of the treatment, while maintaining the class iii molar relationship. conclusion: orthodontic camouflage treatment in an adult patient using a passive self-ligating system and intermaxillary anchorage can improve facial profile and improve dental occlusion. keywords: class iii malocclusion; orthodontic camouflage treatment; passive self-ligating system correspondence: retno widayati, department of orthodontics, faculty of dentistry, universitas indonesia, jl. salemba raya no. 4 jakarta, 10430, indonesia. email: widayati22@yahoo.com introduction edward angle described class iii malocclusion as a lower molar which is mesially positioned relative to an upper molar, with no specification of the line of occlusion. nonetheless, class iii malocclusion can be constituted from skeletal and dental irregularities. skeletal class iii malocclusion can be a result of maxilla deficiency, mandible excessiveness , or a combination of both. dental features include retroclined mandibular incisors, proclined maxillary incisors, edge-toedge incisor relationship and negative overjet.1,2 studies have showed that the prevalence of class iii malocclusion affects a great variety of different populations. it has been documented that there is a greater prevalence of it in asian races compared to other races.2–4 the treatment of choice for skeletal class iii malocclusion in adult patients often requires a combination of orthodontic and surgical procedures. however, with camouflage treatment it is also possible to correct skeletal class iii malocclusion, depending on the level of severity. adult patients who have a mild to moderate skeletal class iii malocclusion and a fairly good facial profile can be treated with camouflage treatment. camouflage treatment can be conducted by extracting teeth, distalising the mandibular dentition, and using class iii intermaxillary elastics. strategies in skeletal class iii malocclusion camouflage treatment are to procline the upper incisors and retrocline the lower incisors. acceptable occlusion, function, and facial aesthetics with dentoalveolar compensation are the objectives from camouflage treatment.5–8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p191–195 mailto:widayati22@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p191-195 192 monika and widayati/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 191–195 intermaxillary elastics have been used as intermaxillary anchorage and are available in many sizes and strengths.9 however, class iii intermaxillary elastics can promote extrusion of upper molars, proclination of upper incisors, distal tipping of lower molars, and extrusion of lower incisors.9,10 in some studies, a combination of skeletal anchorage and intermaxillary elastics were used to minimise the unwanted effects of intermaxillary elastics alone.11,12 this case report demonstrates a camouflage treatment in an adult patient with skeletal class iii malocclusion by the extraction of lower first premolars with the use of intermaxillary elastics and no additional appliances. case the patient was a 25-year-old woman, who came to the orthodontic clinic at the faculty of dentistry in the universitas indonesia dental and oral hospital. she was concerned about her crowded and crossbite of the anterior teeth; hence, she did not feel confident when smiling. the photographs taken before treatment showed a symmetric face and a dolichofacial appearance. her facial profile was straight and her lips were competent (figure 1). the intraoral examination showed anterior crossbite with –3 mm overjet, +4 mm overbite, single posterior crossbite of the upper right second premolar, and class iii canine and molar relationships. the degree of crowding on her maxilla was mild, while on the mandible it was moderate. there was no deviation on her maxillary dental midline with her facial axis, but there was a deviation in the mandibular dental midline by as much as 1mm to the right. there was premature contact on the upper right first incisor with lower right first incisor causing functional displacement to the anterior when closing the jaw. her oral hygiene and periodontal tissues were good, and all teeth were present (figure 2). the lateral cephalometric analysis revealed a pattern of skeletal class iii malocclusion with normal maxilla and prognathic mandible, concave skeletal profile, proclined maxillary incisors, and a normal interincisal angle. a panoramic radiograph showed impacted maxillary thirdmolars and partially erupted mandibular third-molars (figure 3). (a) (b) (c) figure 1. pre-treatment extraoral photographs. facial photos of (a) frontal view at rest, (b) during smiling, and (c) lateral view. (a) (b) (c) (d) (e) figure 2. pre-treatment intraoral photographs. intraoral view of (a) upper occlusal, (b) lower occlusal, (c) right lateral, (d) frontal, and (e) left lateral. (a) (b) figure 3. initial (a) lateral cephalometric and (b) panoramic radiographs. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p191–195 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p191-195 193monika and widayati/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 191–195 according to the patient, she knew of no relatives in her family that had skeletal class iii malocclusion features. meanwhile, the etiologic possibility for the single posterior crossbite of the upper right second premolar could be the retention of the upper right second deciduous molar. the treatment objectives were to improve the occlusion, including correction of the anterior and posterior crossbites and to achieve ideal overjet and overbite. the ideal treatment for skeletal malocclusion was a combination of orthodontic and surgical procedures to improve the facial profile. however, as the patient refused to have surgery, she chose to have the camouflage treatment, involving the extraction of the lower first premolars with fixed orthodontic appliances. case management clinicians should be able to make a proper diagnosis and establish realistic treatment objectives with the patient, in order to prevent undesirable outcomes when performing a camouflage treatment in a mild to moderate skeletal class iii malocclusion. it has been suggested that changes in the three aspects, such as skeletal, dental, and soft tissue, can be successfully camouflaged without damaging the periodontal tissue.7 in this case, the camouflage treatment was conducted by extracting lower first premolars. the patient had a well-formed maxillary arch with mild crowding, while the mandibular arch was prognathic with moderate anterior crowding. by extracting the lower first premolars, the extraction space was used to relieve crowding and retract the lower incisors. the lower first premolars were extracted before bracket bonding. a damon q passive selfligating system (0.022×x0.028-inch slot; ormco, glendora, california) with standard torque prescription was bonded on the upper and lower teeth. bite raisers were used on the mandibular posterior teeth and the patient was given an instruction to use the early class iii elastics (2 oz, 5/16-inch ormco). open coil springs were used between upper lateral incisors and canines to protract the upper incisors. power chain and class iii elastics were simultaneously used for retracting the mandibular anterior teeth. after six months of treatment, the overjet became positive and crowding was resolved. as we progressed to 0.018x×0.025-inch coppernickel-titanium archwire, we inverted the brackets of the four upper incisors, so that the upper incisors with labial root torque were inclined labio lingually. aligning and levelling with sequential copper-nickel-titanium archwires was achieved in 12 months. then, 0.019x0.025-inch stainless steel archwires were put into the upper and lower arches and elastics were also constructed to be used for improving interdigitation and detailing occlusion. after 22 months of treatment, the brackets and molar tubes were debonded and vacuum-formed essix retainers were used for stability on both upper and lower arches. a straighter soft tissue profile and a pleasant smile were obtained at the end of the treatment (figure 4). an ideal overjet and overbite were also attained with a class i canine relationship, while the molars are maintained in class iii relationship. crowding on both arches were relieved and the crossbite on the second upper premolar was also corrected (figure 5). after 20 months of treatment, a lateral cephalometric radiograph showed changes in skeletal, dental, and soft tissue parameters (figure 6). analysis from lateral cephalometric radiograph were shown in table 1. the anb angle showed improvement from –2° to 0° and the angle of convexity was also improved from –5° to 0°, while the lower facial height was maintained. dental parameters (a) (b) (c) figure 4. post-treatment extraoral photographs. facial photos of (a) frontal view at rest, (b) during smiling, and (c) lateral view. (a) (b) (c) (d) (e) figure 5. post-treatment intraoral photographs. intraoral view of (a) upper occlusal, (b) lower occlusal, (c) right lateral, (d) frontal, and (e) left lateral. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p191–195 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p191-195 194 monika and widayati/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 191–195 showed that the upper incisor to maxillary plane angle and lower incisor to mandibular plane angle decreased from 120° to 115° and 86° to 81°, respectively. soft tissue parameters showed that the positions of the upper and lower lip positions were also improved and confirmed in the lateral cephalometrics superimposition (figure 7). discussion in this case, class iii molar relationship was maintained with a class i canine relationship and an ideal overjet and overbite. this type of occlusion is also known as therapeutic class iii occlusion.13 previous study found that good occlusal stability and periodontal health were observed in patients with class iii molar relationship after 13–14 years of treatment.14 the alternative treatment by extraction of maxillary second premolars and mesialisation of the first molars to achieve a class i molar relationship could risk to depress the face, as the patient already had a fairly straight profile.15 the patient’s facial profile was improved as there were several skeletal and dental changes that affected the position of the upper and lower lip. increase in anb angle and the angle of convexity might be attributed to the protraction of the upper incisors and also the retraction of the lower incisors (table 1). the inclination of upper incisors were initially proclined as a common feature of dental compensation in skeletal class iii malocclusion, while the lower incisors have normal inclination. it has been suggested that using class iii elastics can cause some unwanted tooth movements. therefore, we used and prescribed the patient with light-force elastics and a bigger wire in the maxillary arch, so that the whole maxillary arch became an anchorage for mandibular anterior teeth retraction and also to minimise any unwanted effects. light force in class iii elastics was also used to prevent the maxillary posterior teeth from extruding, as this can cause (a) (b) figure 6. pre-debonding (a) lateral cephalometric and (b) panoramic radiographs. figure 7. superimposition of lateral cephalometrics on before (black) and after treatment (red). note there was changes in maxillary and mandibular incisor angulation, and also in the lip position. table 1. comparison of skeletal, dental, and soft tissue values of preand post-treatment lateral cephalometric radiographs measurement mean sd pretreatment posttreatment horizontal skeletal sna (o) 82 2 84 84 snb (o) 80 2 86 84 anb (o) 3 2 –2 0 the wits (mm) 1 2 –12 –5 angle of convexity (o) 0 10 –5 0 vertical skeletal y-axis (o) 60 6 59 58 go-angle (o) 123 7 134 134 sn-mandibular plane (o) 32 3 32 32 mmpa (o) 27 4 26 26 lafh (%) 55 2 55 55 anterior dental interincisal angle (o) 135 10 130 135 u1-palatal plane (o) 109 6 120 115 l1-mandibular plane (o) 90 4 86 84 soft tissue upper lip –e line (mm) 1 2 –6 –5 lower lip – e line (mm) 0 2 1 0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p191–195 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p191-195 195monika and widayati/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 191–195 the mandible to rotate backwards, therefore increasing the lower facial height.16 we also inverted the maxillary incisor bracket position to prevent more proclination on maxillary incisor teeth. the torque value for damon q brackets with standard prescription are +15° on the upper central incisors and +6° on the upper lateral incisors. when the brackets were inverted, the torque values were changed to –15° and –6° for the upper central and lateral incisors, respectively. therefore, the upper incisors had a higher labial root torque placed on them. the same effects were also obtained in previous study with damon 3 brackets by inverting the bracket position.17 the inclination of lower incisors was retroclined by the end of the treatment. a meta-analysis study found that self-ligating brackets could promote inclination of incisors become 1.5° less than the conventional brackets.18 another study also reported acceptable facial aesthetics and good dental occlusion when camouflaging skeletal class iii malocclusion with a passive self-ligating system without the use of auxiliary appliances.19 retention is needed after an active phase of orthodontic treatment because there is tendency to relapse. a vacuumformed retainer was used for this patient to maintain the tooth alignment and arch width stability. a previous study suggested that vacuum-formed retainers were more effective than the hawley retainer at holding the incisors in alignment.20 however, a recent systematic review also suggested that there are no differences between the hawley retainer and the vacuum-formed retainer in terms of cost, time, maintaining the arch width, occlusal contacts, and patient satisfaction.21 there is also limited evidence that suggests fixed retainers are better than vacuum-formed retainers. further studies are needed to make some recommendations about retention after orthodontic treatment.22 in conclusion, orthodontic camouflage treatment with passive self-ligating and intermaxillary anchorage can improve facial profile and dental occlusion. references 1. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st. louis: mosby elsevier; 2013. p. 220–75. 2. nanda r, kapila s. current therapy in orthodontics. st. louis: mosby elsevier; 2010. p. 143–58. 3. hardy dk, cubas yp, orellana mf. prevalence of angle class iii malocclusion: a systematic review and meta-analysis. open j epidemiol. 2012; 02(04): 75–82. 4. alhammadi ms, halboub e, fayed ms, labib a, el-saaidi c. global distribution of malocclusion traits: a systematic review. dental press j orthod. 2018; 23(6): e1–10. 5. rabie a-bm, wong rwk, min gu. treatment in borderline class iii malocclusion: orthodontic camouf lage (extraction) versus orthognathic surgery. open dent j. 2008; 2(1): 38–48. 6. janson g, de freitas mr, araki j, franco ej, barros sec. class iii subdivision malocclusion corrected with asymmetric intermaxillary elastics. am j orthod dentofac orthop. 2010; 138(2): 221–30. 7. troy ba, shanker s, fields hw, vig k, johnston w. comparison of incisor inclination in patients with class iii malocclusion treated with orthognathic surgery or orthodontic camouflage. am j orthod dentofac orthop. 2009; 135(2): 146.e1-146.e9. 8. burns nr, musich dr, martin c, razmus t, gunel e, ngan p. class iii camouflage treatment: what are the limits? am j orthod dentofac orthop. 2010; 137(1): 9.e1-9.e13. 9. mitchell l. an introduction to orthodontics. 4th ed. oxford: oxford university press; 2013. p. 179–92. 10. bellini-pereira s, pinzan a, castillo aa-d, janson g, sakoda kl, cury sen, bellini-pereira s. class iii malocclusion camouflage treatment in adults: a systematic review. j dent open access. 2019; 2019(1): 1–12. 11. fakharian m, bardideh e, abtahi m. skeletal class iii malocclusion treatment using mandibular and maxillary skeletal anchorage and intermaxillary elastics: a case report. dental press j orthod. 2019; 24(5): 52–9. 12. manni a, lupini d, cozzani m. combining skeletal anchorage and intermaxillary elastics in class ii treatment. j clin orthod. 2018; 52(4): 227–34. 13. k l ineberg i, e cker t s. functiona l occlusion in restorative dentistry and prosthodontics. st. louis: mosby elsevier; 2016. p. 201–13. 14. farret mmb, farret mm, farret am. strategies to finish orthodontic treatment with a class iii molar relationship: three patient reports. world j orthod. 2009; 10(4): 323–33. 15. philip-alliez c, le gall m, deroze d, orthlieb jd, canal p. therapeutic class iii molar occlusion. j dentofac anomalies orthod. 2009; 12(4): 169–81. 16. cai b, zhao xg, xiang ls. orthodontic decompensation and cor rection of skeleta l class i i i ma locclusion with gradua l dentoalveolar remodeling in a growing patient. am j orthod dentofac orthop. 2014; 145(3): 367–80. 17. jing y, han x, guo y, li j, bai d. nonsurgical correction of a class iii malocclusion in an adult by miniscrew-assisted mandibular dentition distalization. am j orthod dentofac orthop. 2013; 143(6): 877–87. 18. chen ssh, greenlee gm, kim je, smith cl, huang gj. systematic review of self-ligating brackets. am j orthod dentofac orthop. 2010; 137(6): 726.e1-726.e18. 19. deswita y, soegiharto bm, tarman ke. camouflage treatment of skeletal class iii malocclusion in an adult cleft-palate patient using passive self-ligating system. am j orthod dentofac orthop. 2019; 155: 117–26. 20. rowland h, hichens l, williams a, hills d, killingback n, ewings p, clark s, ireland aj, sandy jr. the effectiveness of hawley and vacuum-formed retainers: a single-center randomized controlled trial. am j orthod dentofac orthop. 2007; 132(6): 730–7. 21. mai w, he j, meng h, jiang y, huang c, li m, yuan k, kang n. comparison of vacuum-formed and hawley retainers: a systematic review. am j orthod dentofac orthop. 2014; 145(6): 720 –7. 22. littlewood sj, millett dt, doubleday b, bearn dr, worthington h v. retention procedures for stabilising tooth position after treatment with orthodontic braces. cochrane database syst rev. 2016; 2016: cd002283. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p191–195 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p191-195 189189 research report dental journal (majalah kedokteran gigi) 2016 december; 49(4): 189–194 effects of silane application on the shear bond strength of ceramic orthodontic brackets to enamel surface pinandi sri pudyani and setiarini widiarsanti department of orthodontic faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: fixed orthodontic appliances with ceramic brackets are used frequently to fulfill the aesthetic demand of patient through orthodontic treatment. ceramic brackets have some weaknesses such as bond strength and enamel surface damage. in high bond strength the risk of damage in enamel surfaces increases after debonding. purpose: this study aimed to determine the effect of silane on base of bracket and adhesive to shear bond strength and enamel structure of ceramic bracket. method: sixteen extracted upper premolars were randomly divided into four groups based on silane or no silane on the bracket base and on the adhesive surface. design of the base on ceramic bracket in this research was microcrystalline to manage the influence of mechanical interlocking. samples were tested in shear mode on a universal testing machine after attachment. following it, adhesive remnant index (ari) scores were used to assess bond failure site. statistical analysis was performed using a two-way anova and the mann-whitney test. a scanning electron microscope (sem) with a magnification of 2000x was used to observe enamel structure after debonding. result: shear bond strength was increased between group without silane and group with silane on the base of bracket (p<0,05). there was no significance different between group without silane and group with silane on adhesive (p<0,05). conclusion: application of silane on base of bracket increases shear bond strength, however, application of silane on adhesive site does not increase shear bond strength of ceramic bracket. most bonding failure occurred at the enamel adhesive interface and damage occurred on enamel structure in group contains silane of ceramic bracket. keywords: silane; shear bond strength; ceramic bracket; enamel structure correspondence: pinandi sri pudyani, department of orthodontic, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: pinandi@yahoo.com introduction orthodontic treatment is a treatment that aims to improve the aesthetics and function of orofacial region. tools used in the orthodontic treatment are divided into two, namely removable and fixed orthodontic appliances.1 the fixed orthodontic appliance with ceramic brackets is widely used to meet the demands of patients related to aesthetic needs. many researches have already been conducted on the clinical characteristics of the ceramic bracket materials.2 there are some kinds of brackets based on basic materials used, such as acrylic, polycarbonate, and ceramic bracket. the advantages of using ceramic bracket are highstrength material, resistance to change in shape, and good color stability. meanwhile, the disadvantages of using ceramic bracket are in terms of adhesion strength and to enamel surface damage. ceramic bracket also cannot be chemically bound to the acrylic adhesive bonding material due to aluminum oxide contained. silane is used to improve a chemical bond between the adhesive resin and the ceramic material resulting in a maximum strength.2 silica element contained in ceramic will be bounded to acrylic derived from composite resin through silanization.3 this chemical element also generates a strong bond between the bracket and the adhesive resin that can trigger a tension on the bond between the enamel and the adhesive resin, leading to enamel surface damage.2 self adhesive system is the seventh generation of adhesive material, categorized into the group of self etching.4 self adhesive system is also considered as dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 190 pudyani and widiarsanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 189–194 an adhesive technology with preparations consisted of two pasta, namely basic pasta and catalyst pasta. self adhesive system is composed of alkaline (basic) fillers, silanated fillers, phosphoric acid modified methacrylate monomers, methacrylate monomers, and initiators. self adhesive system also contains acidic monomer generating demineralization and infiltration effects on the enamel surface to form a micromechanical retention and a chemical bond with the tooth enamel. silanated fillers contained then will form a chemical bond between matrix and filler during polymerization.5 therefore, the addition of filler particles in the matrix resin can improve physical and mechanical strength of the composite resin, such as barium glass, silica, apatite, and silane coupling agent.6 the minimum strength of the adhesion is 6 to 8 mpa quite capable of holding clinical orthodontic needs. the adhesion strength is considered to be able to tolerate to the mastication and orthodontic forces.10 adhesive remnant index (ari) is used to both assess the rest of the adhesive materials on the surface of the teeth after the release of the ceramic bracket bonding,11, and determine the location of the failure of the bonding between the enamel and the adhesive and basic bracket materials.12 bracket can be detached from the tooth surface when receiving a force greater than the adhesive force. the detachement of the ceramic brackets mostly occurs on the bond between enamel and adhesive material. in contrast, the detachment of the metal brackets mostly occurs on the bond between the bracket and the adhesive materials resulting in damage enamel surface.11 scanning electron microscope (sem) is a tool that can be used to observe the surface of enamel.11 this study aimed to analyze effects of silanes contained in the bracket base and adhesive materials on the shear bond strength and the enamel surface scars caused by the attachment. materials and method this research was a laboratory experimental study. this research was conducted on 16 premolars randomly divided into four groups, namely group ia (bracket base and adhesive material), group ib (bracket base and adhesive material coated by silane agent), group iia (bracket base coated silanes agent and adhesive materials), and group iib (bracket base and adhesive materials coated by silane agents). the classification of a and b was based on the variable of adhesive materials. group a was a group using only adhesive materials, while group b was a group using adhesive materials coated by silane agent. on the other hand, the classification of i and ii was based on the variable of bracket base. group i was a group using bracket base without silane agent, while group ii was a group using bracket base containing silanes. the design of bracket base used in this research, moreover, was in the form of microcrystalline in order to control the effects of mechanical retention on the base of the brackets. adhesive materials containing no silanes used in this research, on the other hand, were transbond plus and pasta z250 composed of methacrylated phosphoric acid ester, tri ethylene glycol dimethacrylate (udma), bisphenol-polyethylene glycol dimethacrylate (bis-ema), and silica/ zirconia.13,14 meanwhile, adhesive materials containing silanes used in this research was relyx 200 consisted of two preparations, namely basic pasta and catalyst pasta, composed of methacrylated phosphoric acid esters, methacrylated monomer, silanated fillers, alkaline basic fillers, initiators, stabilizers, and staining substance.5 shear bond strength of ceramic brackets in each group then was measured, and both the failure of the bonding as well as the surface of the tooth enamel after debonding were measured. this research was conducted in several places. this research was performed at the research laboratory of the faculty of dentistry, universitas gadjah mada from the first phase to soaking the teeth in saliva in an incubator. to measure the shear bond strength using a universal testing machine (utm), this research was carried out at the laboratory of materials of the faculty of mechanical engineering, universitas gadjah mada. after that, to observe ari using a stereo-microscope with a magnification of 10x, this research was conducted at the laboratory of structural animal development of faculty of biology, universitas gadjah mada. to observe the structure of enamel using a sem with a magnification of 2,000x, this research was performed at lppt unit i, universitas gadjah mada. anova analysis test was carried out to compare the shear bond strengths between the groups with a significance α level of 0.05. the failure of the bracket ceramic attachment based on ari then was analyzed with nonparametric mannwhitney test. the structure of enamel was observed using a sem presented descriptively. results in this research, the effects of silane agents on the shear bond strengths of the ceramic brackets were observed on 16 samples divided into four groups, namely group ia (bracket base and adhesive materials), group ib (bracket base containing no silanes, while adhesive materials containing silanes), group iia (bracket base containing silanes, while adhesive materials containing no silanes), and group iib (bracket base and adhesive materials containing silanes). the results then showed the following means and standard deviations of the shear bond strengths of the four groups as shown in table 1. the mean values of the shear bond strength of the ceramic brackets were shown in table 2. moreover, the means and standard deviations of the shear bond strengths of the ceramic brackets based on the variable of bracket base between the groups using the bracket base containing no silanes and the groups using adhesive material, either containing silanes or not (group dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 191191pudyani and widiarsanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 189–194 table 1. means and standard deviations of the shear bond strengths of the ceramic brackets on the groups ia, ib, iia, and iib group number of samples mean (mpa) standard deviation ia: bracket base and adhesive materials containing no silanes 4 12.0175 1.44126 ib: bracket base containing no silanes, while adhesive materials containing silanes 4 5.6775 .35762 iia: bracket base containing silanes, while adhesive materials containing no silanes 4 15.5275 .98290 iib: bracket base and adhesive materials containing silanes 4 7.7300 .76123 table 2. mean values of the shear bond strengths of the ceramic brackets in a unit of mpa bracket base containing no silanes bracket base containing silanes mean adhesive materials containing no silanes 12.0175 15.5275 13.7725 adhesive materials containing silanes 5.6775 7.7300 6.7038 mean 8.8475 11.6287 table 3. means and standard deviations of the shear bond strengths of the ceramic brackets based on the variable of bracket base (group i and group ii) group bracket base mean (mpa) standard deviation i containing no silanes 8.8475 3.5625 ii containing silanes 11.6287 4.2466 table 4. means and standard deviations of the shear bond strengths of the ceramic brackets based on the variable of adhesive materials (group a and group b) group adhesive materials mean (mpa) standard deviation a containing no silanes 13.7725 2.1694 b containing silanes 6.7038 1.2275 table 5. results of the two-way anova test on the shear bond strengths of the ceramic brackets with the variables of bracket base and adhesive materials variables total of multiplication results df multiplication results of mean f p bracket base 30.941 1 30.941 30.071 .000* adhesive materials 199.868 1 199.868 194.246 .000* note: *: significatly different (p<0.05) table 6. distribution of data on the failure of the bracket ceramic attachments based on the variable of bracket base (group i and group ii) group ari values value 0 value 1 value 2 value 3 n % n % n % n % i 0 0 5 62.5% 2 25% 1 12.5 ii 4 50% 4 50% 0 0 0 0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 192 pudyani and widiarsanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 189–194 i), as well as between the groups using the bracket base containing silanes and the groups using adhesive material, either containing silanes or not (group ii) were shown in table 3. furthermore, the means and standard deviations of the shear bond strengths of the ceramic brackets based on the variable of adhesive materials between the groups using the adhesive materials containing no silanes and the groups using bracket base, either containing silanes or not (group a), as well as between the groups using the adhesive materials containing silanes and the groups using bracket base, either containing silanes or not (group b) were shown in table 4. normality and homogeneity tests were performed on the entire data of the shear bond strengths as seen in appendix 1 before a two-way anova test was performed. results of the normality test using the shapiro-wilk test showed that the data of all groups were normally distributed (p> 0.05). meanwhile, results of the homogeneity test using the levene test indicated that the data obtained were table 7. distribution of data on the failure of the bracket ceramic attachments based on the variable of adhesive materials (group a and group b) group ari values value 0 value 1 value 2 value 3 n % n % n % n % a 2 25% 3 37.5% 2 25% 1 12.5% b 2 25% 6 75% 0 0 0 0 12 a figure 5. photomicrographs of enamels in group ia and group iia using sem with a magnification of 2,000x. a) after debonding on group ia; b) after debonding on group iia. figure 6. photomicrographs of enamels in group ib and group iib using sem with a magnification of 2,000x. a) after debonding on group ib; b) after debonding on group iib. a b a b figure 5. photomicrographs of enamels in group ia and group iia using sem with a magnification of 2,000x. a) after debonding on group ia; b) after debonding on group iia. 12 a figure 5. photomicrographs of enamels in group ia and group iia using sem with a magnification of 2,000x. a) after debonding on group ia; b) after debonding on group iia. figure 6. photomicrographs of enamels in group ib and group iib using sem with a magnification of 2,000x. a) after debonding on group ib; b) after debonding on group iib. a b a b figure 6. photomicrographs of enamels in group ib and group iib using sem with a magnification of 2,000x. a) after debonding on group ib; b) after debonding on group iib. homogeneous (p>0.05) as shown in appendix 2. therefore, the two-way anova test then was performed. the results of the two-way anova test showed that there were significant differences in the shear bond strengths on the variables of bracket base and adhesive material (p<0.05). the failure of the bracket ceramic attachment was examined based on the ari using the stereo-microscope, 10x magnification. distribution of the data based on the bracket base used, namely group i and group ii was shown in table 6, while the data distribution based on the adhesive materials used, namely group a and group b, was demonstrated in table 7. the nonparametric mann-whitney test was performed to compare between group i and group ii based on the variable of bracket base and between group a and group b based on the variable of adhesive materials. the results showed that there was no significant difference between the groups (p<0.05). nevertheless, the results showed that there was a statistically significant difference in the values of ari (p=0.01) between group i and group ii based on the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 193193pudyani and widiarsanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 189–194 variable of bracket base. there was no significant difference in the value of ari (p=0.293) between group a and group b based on the variable of adhesive material. discussion ceramic bracket is mostly used for orthodontic treatment due to aesthetic demand of patients.15 ceramic bracket, nevertheless, has some weaknesses. for instance, ceramic bracket cannot chemically bind to acrylic adhesive materials due to aluminum oxide contained. therefore, silanes are added as a coupling agent to overcome this weakness.2 silanes are chemical elements that will make a bond between two materials, inorganic and organic ones.15 one part of silane molecules on the base of ceramic brackets will bind to an inorganic cluster, aluminum oxide, while the other part of the molecules will bind to an organic group, namely acrylic resin contained in the adhesive materials. the results of this research showed that the mean value of the shear bond strengths of the ceramic brackets in group ii (the bracket base containing silanes) increased significantly compared to group i (the bracket base containing no silanes) as shown in table 1 and table 3 (p<0.05). therefore, it can be said that silane contained in the bracket base could increase the shear bond strengths of the ceramic brackets. ceramic brackets actually have some weaknesses in terms of adhesion strength and damage to enamel surface.2 the optimal shear bond strength required in clinical orthodontic treatment is from 6 mpa to 10 mpa.20 meanwhile, the mean shear bond strength of the ceramic brackets containing silanes on the brackets base (group ii) was 11.6287 mpa. on the other hand, the mean shear bond strength of the ceramic brackets containing no silanes on the bracket bases (group i) was 8.8475 mpa. therefore, it can be said that the shear bond strengths in group i and group ii were in line with the shear bond strength required. the shear bond strength in group i containing no silanes on the bracket base even still was in line with the shear bond strength recommended. this indicates that the mechanical retention of microcrystalline without chemical retention is still able to produce good shear bond strength. group b, moreover, was a group using the adhesive materials containing silanes. silanes contained in the adhesive materials were in the form of silanated fillers. silanated fillers are derived from the addition of silanes as coupling agents in inorganic fillers of the adhesive materials that binds chemically to monomer as organic group, and then forms a matrix when polymerized.5 adhesive materials containing no silanes used in this research were transbond plus and z250 paste composed of methacrylated phosphoric acid ester, tri ethylene glycol dimethacrylate (udma), bisphenol-polyethylene glycol dimethacrylate (bis-ema), and silica/zirconia.13,14 meanwhile, adhesive material containing silanes used in this research was relyx 200 consisted of two preparations, namely basic pasta and catalyst paste, composed of methacrylated phosphoric acid esters, methacrylated monomer, silanated fillers, alkaline basic fillers, initiators, stabilizers, and staining substance.5 the mean shear bond strength of the ceramic brackets in group b (using adhesive materials containing silanes) significantly decreased compared to group a (using adhesive materials containing no silanes) (table 1 and table 3). therefore, the hypothesis stating that the adhesive materials containing silanes will increase the shear bond strength of the bracket ceramics was rejected. de munck et al.16 suggested that in an experimental study, a decrease in the shear bond strength of relyx unicem as an adhesive material is due to high viscosity factor and short penetration time, resulting in reducing of the adaptation ability of the materials on the surface after apllied. the morphological examination, furthermore, will show the porosity of enamel surface and dentin after the use of relyx unicem, illustrating superficial interactions.16 the lowest shear bond strength then may occur at the thinnest area of the adhesive materials due to the loss of homogeneity of the material and the increased pressure on the thinnest area.17 the failure of the ceramic bracket attachment, can be examined using the ari of the residual adhesive materials on the surface of the enamel using the 4-point scale.11 the location of the failure the ceramic bracket attachment actualy then can provide important information about the shear bond.18 table 6 illustrates that the value of ari was 0. it means that there were no residual adhesive materials on the surface of the enamels in group ii compared to group i. there was even a significant difference (table 8). this finding indicates that the location of the failure of the ceramic bracket attachment using the bracket base applied silanes occurred between the enamel surface and the adhesive materials. in addition, results of the observation on the values of the ari in group a and group b are shown in table 7. the results demonstrate that there was no significant difference in the location of the failure of the ceramic bracket attachment between the groups based on the variable of the adhesive materials (table 8). this is likely due to air ingestion in group b that affects bonding in the adhesive materials. this finding is in line with a research conducted by setiarini 21 showing that air trap formed will lead to the formation of the thinnest area of the bond between the teeth, the adhesive materials, and the bracket. consequently, the location of the failure of the bracket attachment cannot be predicted, depending on the location of the air trap. ideally, the strong shear bond strength will have a better failure of the attachment if located between the enamel surface and the adhesive materials since it will simplify the polishing process of the surface of the teeth after debonding.18 the shear bond strength above 13 mpa actually can cause damage in the form of tear out.20 the morphology of the enamels in group ia and group iia then was observed using a sem (figure 5). figure 5-a illustrates the morphology of enamels in group ia with the mean shear dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 194 pudyani and widiarsanti/dent. j. (majalah kedokteran gigi) 2016 december; 49(4): 189–194 bond strength of 12.0175 mpa after debonding, indicating the adhesive materials remained on the surface of the enamels. figure 5-b, on the other hand, demonstrates the morphology of the enamels in group iia with the mean shear bond strength of 15.5275 mpa, showing damages in the enamel surfaces, such as cracks and open dentinal tubules. enamels on preparations in group ib and group iib were observed using sem as shown in figure 6. figure 6-a illustrates enamel morphology in group ib (the mean shear bond strength of 5.6775 mpa) after debonding. this picture indicates porosity in the enamel surface not filled by the adhesive materials. figure 6-b, on the other hand, demonstrates enamel morphology in group iib (the mean shear bond strength of 7.7300 mpa) after debonding. this picture also shows porosity in the enamel surfaces not filled by the adhesive materials. the ceramic bracket containing silanes on the base and the adhesive materials, therefore, is hypothesized to damage the surface of the enamel structure on which the bracket attached. finally, it can be concluded that there are some effects of silanes contained in ceramic bracket on shear bond strength and enamel structure. for instance, silane applied on the base of a ceramic bracket can increase the shear bond strength of the bracket. silane applied on the ceramic bracket, nevertheless, can also make a failure of the bonding between enamel surface and adhesive material. silane application can influence the structure of enamel surface on which the ceramic bracket is attached. references 1. iyyer bs. orthodontics: the art and science. 3rd ed. new delhi: arya (medi) publishing house; 2004. p. 1, 271, 273. 2. russel js. aesthetic orthodontic brackets. j orthod 2005; 32(2): 146-63. 3. basaran g, veli i. principles in contemporary orthodontics. turkey: in tech, dicle university; 2011. p. 181-212. 4. merbeek b, van munck j, de yoshida y, inoue s, vargas m, vijay p, landuyt k, van lambrechts v, vanherle g. adhesion to enamel and dentin: current status and future challenges. operative dentistry 2003; 28(3): 215-35. 5. anonim. technical product profile relyxtm unicem. 3m espe; 2007. p. 1-60. 6. lung cyk, matinlinna jp. silane coupling agents and conditioning in dentistry. dental tribune indian 2013; 01: 5-6. 7. brantley wa, eliades t. orthodontics materials scientific and clinical aspects. new york: thieme stuugart; 2001. p. 107-12. 8. bishara se. text book of orthodontics. new york: wb saunders company; 2001. p. 186-231. 9. anusavice kj. philips: science of dental material. 11th ed. philadelpia: elsevier; 2003. p. 78. 10. cacciafesta v, bosch c, melsen b. clinical comparison between a resin-reinforced self-cured glass ionomer cement and a composit resin for direct bonding of orthodontics brackets. part 1: wetting with water. clin orth res j 1998; 1(5): 29-36. 11. kitahara-ceia fmf, mucha jn, santos pamd. assessment of enamel damage after removal of ceramic brackets. am j orthod dentofac orthop 2008; 134: 548-55. 12. olsen me, bishara se, jakobsen jr. evaluation of the shear bond strength of different ceramic bracket base designs. angle orthodontist 1997; 67: 179-82. 13. chu ch, ou kl, dong dr, huang hm, tsai hh, wang wn. orthodontic bonding with self-etching primer and self-adhesive systems. eur j orthod 2011; 33(10): 276-81. 14. obici ac, sinhoreti mac, correr-sobrinho l, de goes mf, cosani s. evaluation of mechannical properties of z250 composite resin light-cured by different methods. j appl oral sci 2005; 13(4): 393-8. 15. reddy yg, sharma r, singh a, agrawal v, agrawal v, chaturvedi s. the shear bond strenght of metal and ceramic brackets: an in-vitro comparative study. j clin diagn res 2013; 7: 1495-7. 16. de munck j, vargas m, van landuyt k, hikita k, lambrechts p, van meerbeek b. bonding of an auto-adhesive luting material to enamel and dentin. j dental material 2004; 01-09. 17. gittner r, muller-hartwich r, engel s, jost-brinkmann pg. shear bond strength and enamel fracture behavior of ceramic brackets fascination and fascination 2. j orofac orthop 2012; 1: 49-57. 18. al-saleh m, el-mowafy o. bond strenght og orthodontics brackets with new self-adhesive resin cements. am j orthod dentofacial orthop 2010; 137: 528-33. 19. eslamian l, ghassemi a, amini f, jafari a, afrand m. should silane coupling agent be used when bonding brackets to composite restoration? an in vitro study. european journal of orthodontics 2009; 31: 266-70. 20. naini fb, gill dis. tooth fracture associated with debonding a metal orthodontic bracket: a case report. world journal orthodontic 2008; 9(3): 32-6. 21. setiarini w, soekarsono h, pinandi sp. pengaruh silane terhadap kekuatan tarik pada perlekatan braket seramik. jurnal kedokteran gigi ugm 2016; dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i4.p189-194 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v49.i4.p189-194 �0 vol. 43. no. 1 march 2010 research report various curing methods on transverse strength of acrylic resin sherman salim department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: acrylic resin ��ere �irst �sed in dentistry ��r dent�re �ases. ��e �asic kn���ledge �� acrylic resin c�ring �et��d s���ld �e kn���n �y t�e dentist, �eca�se t�e i��r�ved �er��r�ance �� acrylic resin de�ends �n t�e c�ring �et��d. purpose: ��is st�dy ��as ai�ed t� �ind t�e ��st e��ective c�ring �et��d t� �r�d�ce t�e �ig�est transverse strengt� �� acrylic resin. method: 18 rectag�lar acrylic resin sa��les ��it� 65 × 10 × 2.5 �� size, divided int� 3 gr���s �ased �n di��erence c�ring �et��d (ja�an �nd�strial standard, 24 ���r in 70° c ��iling ��ater, and �icr���ave). ��ere ��ere tested ��r t�eir transverse strengt�. result: ��e res�lt �� t�is st�dy s����ed t�at c�nventi�nal j�s �et��d �as t�e �ig�est �ean and deviati�n sc�res (60.85 �pa ± 2.10) c���ared t� t��se �� 24 ���r in 70° c ��iling ��ater �et��d (55.77 �pa ± 2.09) and �� �icr���ave �et��d (56.60 �pa ± 1.45). conclusion: ��e �ig�est transverse strengt� is derived �r�� t�e c�nventi�nal j�s c�ring �et��d. key words: acrylic resin, c�ring �et��d, transverse strengt� abstrak latar belakang: resin akrilik �erta�a kali di�akai dala� �idang ked�kteran gigi �nt�k �asis �r�tesa gigi. pengeta��an dasar �er�agai �et�de ��li�erisasi resin akrilik �ar�s diketa��i �le� d�kter gigi karena �enye���rnaan �ena��ilan resin akrilik tergant�ng dari �er�agi �et�de ��li�erisasi. tujuan: penelitian ini �ert�j�an �nt�k �enda�atkan �et�de ��li�erisasi yang �eng�asilkan resin akrilik dengan kek�atan transversal yang �aling tinggi. metode: 18 sa��el dari resin akrilik �er�ent�k �al�k dengan �k�ran 65 × 10 × 2,5 �� di�agi dala� 3 kel����k �erdasarkan �et�de ��li�erisasi yang �er�eda (j�s, 24 ja� dala� air 70° c, dan �icr���ave). hasil: hasil dari �enelitian ini �en�nj�kkan �a���a rerata dan angka deviasi �et�de ��li�erisasi j�s k�nvensi�nal adala� yang ter�esar kek�atan transversa (60,85 �pa ± 2,10) di�andingkan dengan �et�de air 70° c 24 ja� (55,77 �pa ± 2,09) dan �et�de �icr���ave (56,60 �pa ± 1,45). kesimpulan: kek�atan transversa yang �aling �esar di�er�le� dari �et�de ��li�erisasi j�s k�nvensi�nal. kata kunci: akrilik resin, �et�de ��li�erisasi, kek�atan transversa c�rres��ndence: sherman salim, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction there are many kinds of denture materials used for denture base, such as cellulose, phenol formaldehyde, resin vinyl, and vulcanite. however, those materials have many weaknesses, only acrylic resin which has been used since mid 1940 is still commonly used as denture base material. the reason is because this material meet certain criteria such as: possessing natural character, high strength, stable dimension, good thermal conductivity, radiopaque, relatively not expensive, resistance from oral liquid or saliva, and bacterial growth, relatively easy to manipulate and clean.1–4 acrylic resin used for removable denture base are available in powder and liquid. the powder consists of prepolymerized spheres derived from poly (methyl ��salim: various curing methods on transverse strength methacrylate) and little benzoyl peroxide. benzoyl peroxide is used as initiator in the early curing process. the liquid consists of nonpolymerized methyl methacrylate with little hydroquinone. hydroquinon is used as inhibitor that can prevent both unexpected curing and setting derived from liquid during storing process. furthermore, cross-linking material, glycol dimethacrylate, is also added into the liquid. in this case, glycol dimethacrylate is usually used as crosslinking material in poly (methyl methacrylate) acrylic resin denture base. actually, glycol dimethacrylate is chemically and structurally the same as methyl methacrylate that can be combined with polymer bond growth.4 acrylic resin curing method has developed due to the development of science, technology, and dental materials. the commonly curing method technique are evaporation, pressure dry-heating, dry air oven, infrared heating, induction technique, and microwave radiation. each of those curing techniques has both advantages and disadvantage. this acrylic resin curing methods for denture base can also be conducted by some techniques such as activated heating, compression molding, chemically activated, and light activated.4,5 many studies even showed that those curing techniques had the similar result as conventional curing method in evaluating transverse strength. nevertheless, choosing the right curing method of acrylic resin that is still important in order to obtain acrylic resin suitable with physical and biocompatible characters of mouth cavity tissue.6 the acrylic resin with microwave curing method has actually been evaluated by many researchers and produces electromagnetic wave from electric generator known as magnetron. the microwave used has 2450 mhz and 12 cm wave length. methyl methacrylate molecule facing the microwave electromagnetic area even can be changed its direction, about 5 billions/second. as a result, this condition causes many intermolecular collisions which can produce heat quickly. the curing method with microwave has some advantages such as saving time, producing cleaner result, and causing more similar dimension change than conventional curing method.7 as a denture base, acrylic resin must meet a mechanical character, which is about transverse strength. the denture base resistance during chewing process actually is analogous with that of test bar during receiving transverse load. in the usa, acrylic resin used as denture base should be evaluated for its transverse strength.8 the acrylic resin transverse strength actually depends on the curing method. unfortunately, the curing method is influenced by certain factors such as temperature, mass, humidity, and environment like air or water is still discussed in many studies. therefore, it is assumed that all kinds of curing methods with many kinds of variables cause different transverse strength. this study was aimed to analyze how the different curing methods can influence the acrylic resin transverse strength. the significance of this study is to find the right acrylic resin curing method that can possibly produce the highest transverse strength. material and method samples were made through the following procedures: first, master model made of brass was polished with vaseline, put in the middle of the denture flask filled with hard gypsum, and then left over in press until the gypsum was set. the denture flask was opened, and the master model was taken but from the gypsum. brush the gypsum's surface with cold mould seal, and left until dry. then mixing the powder and liquid of acrylic resin bioresin (shovu, japan) based on the direction (10 grams powder: 4.5 ml liquid). after around 10 to 15 minutes, the mixing of the powder and liquid entering to the dough stage then it is ready for pouring to the flask (yoshida, japan). before the denture flask was closed, the dough stage acrylic covered with plastic cellophane and pressed slowly with hydraulic press (yoshida, japan). after that, the denture flask was reopened and the over dough of acrylic resin was cut. afterwards, it was closed again and pressed with 2200 psi pressure or 50 kg/cm2. those procedures must be redone approximately two to three times. then, it was moved into flask press, and left over for about 24 hours before curing process was conducted. meanwhile, samples cured with oven microwave were made of 10 grams of powder: 4.3 ml of liquid (based on the direction from the producer) and cured in special denture flask for microwave. the sample was divided into three groups. the first group, acrylic resin was cured with 70° celsius boiling water for about 2 hours, which then was continued into 100° celsius boiling water at the temperature of 100° celsius for about 30 minutes (japan industrial standard). the second group, acrylic resin was cured with a heating technique using 70° celsius boiling water for about 24 hours. and, the third group, acrylic resin was cured with a heating technique using microwave oven (500 watt) for about 3 minutes. after all the curing process was conducted, those treatment groups were being left until they were cold. each of those treatment groups consisted of six samples. before being tested, those samples were immersed into distilled water at 37° celsius for about 48 hours.9 the transverse strength testing method of samples was conducted with ada no. 12 specification. first, samples were put in the palate center of autograph tool, 50 mm between both buffers. afterwards, the mass was reduced with cross head speed 1/10 mm/minute until the sample was broken. the result number from the test tool then was noted and analyzed with transverse strength formula.6 �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 40-43 3 pi s = 2 bd 2 note: s = stress; p = load; l = length; b = width; d = thickness the data were analysed by using kolmogorov-smirnov and lsd test for the difference of acrylic resin transferse strength and among those treatments respectivity. and as for the different curing methods were analysed by oneway anova. result mean and standard deviation of transverse strength of acrylic resin cured with different methods can be seen in table 1. table 1. mean and standard deviation of transverse strength of acrylic resin cured with different methods (mpa) treatment group sample number mean + standard deviation conventional jis 6 60.85 + 2.10 24 hour boiling water 6 55.77 + 2.09 microwave 6 56.60 + 1.45 since the data result shown in table 1 are homogenous and has normal distribution, the data then were tested with one sample kolmogorov-smirnov test. furthermore, in order to find the difference of acrylic resin transverse strength processed by different curing method, one-way anova test was conducted. the result then showed that there was significant difference with score p < 0.05. the result showed that acrylic resin cured with conventional jis method had the highest mean score of transverse strength, meanwhile the cured with 24 hour boiling water method had the lowest mean score on the transverse strength. moreover, in order to find the difference among those treatments, lsd test was conducted, as shown in table 2. finally, the result showed that there was significant difference between transverse strength of acrylic resin cured with conventional jis method, compared to 24 hour in 70° c boiling water method, and microwave method. but, there was no significant difference between transverse strength of acrylic resin cured with 24 hour in 70° c boiling water method and microwave method. discussion poly (methyl methacrylate) used as denture base material actually can be produced through some curing methods. thus, all of those curing methods evaluated in this study were aimed to find an easier technique, to reduce denture manufacturing time, and to achieve better acrylic resin character in the terms of hardness, porosities, and monomer discharge.5,10 due to the development of technology, curing method with water-bath and pressure is still used until now even though the use of microwave energy usually used in food industry has more advantages in dentistry since it can be used for acrylic resin curing process for denture base. the study on the activation system of acrylic resin with microwave energy was conducted not only for laboratorial needs, but also for clinical needs.10 in supporting the treatment success, the resistance of the acrylic resin denture base must be examined through the height of transverse strength. it means that in this examination, all of three pressure points working together on the denture base during chewing process will be compared.11 in this study the acrylic resin transverse strength obtained through those three different kinds of curing process were examined. this study found that the acrylic resin transverse strength obtained from 24-hour boiling water curing method at the temperature of 70° celsius, for instance, was lower than that obtained from the conventional jis curing method and the microwave curing method (table 1). this condition was caused by the fact that there was residual monomer which did not reacted since acrylic resin cannot reach the boiling point of monomer at the temperature of 100.8° celsius. the residual monomer then will make acrylic resin more plastic because of the reduced transverse strength. as a result, the residual monomer will play potential tissue irritant which can make acrylic resin biocompatible.4 if the curing process were conducted in water-bath at low temperature for long period without increasing the temperature at the end of the process, the residual monomer will be three times as high as that with the increasing temperature at the end of the process. the residual monomer then can cause plasticizing effect, reduced acrylic resin strength, and cause many alteration because of pressure.12 similarly, another researcher also found that acrylic resin cured in water-bath for long period can cause acrylic resin to react as plasticizer, can reduced glass transition temperature table 2. lsd test on transverse strength of acrylic resin cured with different methods (mpa) curing method conventional jis 24 hour boiling water microwave conventional jis * * 24 hour boiling water – microwave note: * : significant, – : not significant ��salim: various curing methods on transverse strength (tg), and can make its mechanical character decreased.13 moreover, the cycle of curing process was successfully conducted for making the acrylic resin denture base in many sizes, forms, and depths when the curing process was conducted at 70° c for less than two hours and then continued at the temperature of 100° celsius for more than one hour. the result obtained will show the strongest transverse strength.4 acrylic resin curing method this microwave does not depend on thermal conduction, this is one of the advantage compared with the conventional boiling method. some researchers have concluded that the physical character of acrylic resin cured with microwave was the same as cured with conventional curing method. nevertheless, the acrylic resin denture base cured with microwave energy has more positive effects on the strength and resistance of the acrylic resin denture base since the microwave energy can potentially save time during the curing process of acrylic resin.10 as a result, it is important to choose the right curing method in order to acquire high physical and mechanical characters of acrylic resin. based on the result of this study, it can be concluded that different curing methods of acrylic resin can influence acrylic resin transverse strength. the strongest transverse strength was derived from japan industrial standard curing method. references 1. phoenix rd, mansueto ma, ackerman na, jones re. evaluation of mechanical and thermal properties of commonly used denture base resins. j prostodont 2004; 13: 17–27. 2. van noort r. introduction to dental materials. 3rd ed. edinburg, london, new york, oxford, philadelphia, st. louis, sydney, toronto: mosby elsevier; 2007. p. 216–27. 3. gladwin m, bagby m. clinical aspects of dental materials. theory, practice and cases. walters kluwer health. lippincot williams & wilkins; 2009. p. 145–58. 4. anusavice kj. phillips science of dental materials 11th ed. usa: elsevier science; 2003. p. 12–94. 5. botega dm, de sauza machado t, de melo jan, garcia rcmr, cury aadb. polymerization time for a microwave acrylic resin with multiple flasks. braz oral res 2004; 18(1): 23–8. 6. craig rg, powers jm. restorative dental materials. 11th ed. st louis: mosby; 2002. p. 636–89. 7. salim s, sadamori s, hamada t. the dimensional accuracy of rectangular acrylic resin specimen cured by three denture base processing methods. j prosthet dent 1992; 67(6): 879–81. 8. dogan om, bolayir g, keskin s, dogan a, bek b. the evaluation of some flexureal properties of a denture base resin reinforced with various aesthetic fibers. j mater sci: mater med 2008; 19: 2343–9. 9. american dental association. guide to dental materials and devices. 7th ed. chicago: illinois; 1974. p. 97–102, 203–8. 10. barbosa db, de sauza rf, pero ac, marra j, compagnoni ma. flexural strength of acrylic resin polymerized by different cycles. j appl oral sci. 2007; 15(5): 424–8. 11. yunus n, rasid aa, azmi ll, abu-hasan mi. some flexural properties of a nylon denture base polymer. j oral rehab 2005; 32: 65–71. 12. harrison a, huggett r. effect of the curing cycle on residual monomer levels of acrylic resin denture base polymers. j dent 1992; 20: 370–4. 13. smith lsa, schimitz v. the effect of water sorption on the glass transition temperature of poly (methyl methacrylate). polymer 1988; 29: 1871–8. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base 172 vol. 43. no. 4 december 2010 case report minor modification of millard's surgical technique for correction of complete unilateral cleft lip coen pramono d department of oral and maxillofacial surgery faculty of dentistry, airlangga university/dr. soetomo public and teaching hospital surabaya indonesia abstract background: a surgical technique for correction of complete unilateral cleft lip was done using a minor modification of millard’s surgical technique. the purpose of this modification is to achieve a good anatomical form of columelia, nostril cill and the position of nasal tip. purpose: this article presents the correction of the complete cleft lip which was done initially by correction of the slanted columella followed by correction of the nostril sill which was done before the sequence of closing the lip crevice. case: correction of a case with complete unilateral cleft lip on a fifteen year old girl using modification of millard’s surgical technique is presented. case management: rotation incision in the philtrum region was made as introduced by millard to make a triangular flap. the triangular flap was contralaterally rotated and pulled into the direction of cleft to achieve a normal position of the columella and nasal tip. the lateral side of the ala was incised in circular form down to the alar base and straight through to the direction of cleft formed an alar flap which consisted of ala, clefted and slanted nasal base tissue. the tip of the triangular flap was trimmed and approximated to the alar flap to form a new the nostril sill. adjustment of the size of the nostrill sill size was achieved during the approximation of those two flaps. the nasal base was built during approximation of the lateral and the medial segment flap or philtrum region and the base of new nostril sill. surgical correction of the complete unilateral cleft lip including correction of the nostrill sill using approximation of triangular flap and the alar flap was achieved. conclusion: this surgical technique with minor modification of millard’s surgical technique can be used for correction of the complete unilateral cleft lip with extremely slanted columella and nasal tip to form the nostril sill. key words: complete unilateral cleft lip, millard’s surgical technique, nostril sill abstrak latar belakang: koreksi celah bibir komplit satu sisi telah dilakukan menggunakan metode operasi millard yang dimodifikasi. modifikasi dimaksudkan untuk memperoleh bentuk anatomis yang baik dari columella, nostril dan letak ujung hidung yang baik. tujuan: melaporkan koreksi suatu celah bibir komplit satu sisi menggunakan metode operasi millard yang dimodifikasi yang dimulai dengan melakukan perbaikan pada columella yang miring pada bentuk nostril sebelum tahapan penutupan celah bibir dilakukan. kasus: dilaporkan satu kasus koreksi celah bibir komplit satu sisi pada pasien wanita usia 15 tahun menggunakan metode operasi millard yang dimodifikasi. tatalaksana kasus: dilakukan insisi berbentuk rotasi di regio seperti pada metode millard untuk membuat flap berbentuk trianguler. flap trianguler dirotasikan ke arah kontra lateral dan ditarik menuju arah celah bibir untuk memperoleh posisi columella yang normal, bentuk nostril yang simetris dan letak ujung hidung yang baik. dilakukan insisi sirkuler ke bawah ke arah dasar ala pada regio lateral ala menuju ke bagian celah bibir, selanjutnya akan diperoleh bentuk flap yang disebut alar flap yang terdiri atas bagian ala nasi, bagian dari jaringan yang berada pada sisi celah dan bagian dari dasar hidung di sisi celah bibir. bagian ujung dari trianguler flap dipotong dan selanjutnya ditautkan pada flap alar untuk membentuk bentukan nostril baru. penyesuaian ukuran nostril dilakukan pada tahap penautan kedua flap tersebut. bagian dari dasar hidung dibentuk pada saat tahapan penautan antara flap segmen lateral dan medial atau bagian dari philtrum dan bagian dasar dari nostril atau cuping hidung. koreksi bedah suatu celah bibir komplit satu sisi termasuk koreksi pada bentuk nostril diperoleh dengan menautkan antara flap trianguler dan flap alar. 173pramono: minor modification of millard's surgical technique kesimpulan: teknik bedah millard dengan modifikasi kecil dapat dipakai untuk melakukan koreksi pada kasus celah bibir komplit satu sisi yang disertai kemiringan columella dan ujung hidung yang ekstrim. kata kunci: celah bibir sebagian komplit, teknik bedah millard, nostril sill correspondence: coen pramono d, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: coen_pram@yahoo.com gaps of millimeters or centimeters. no exact size can be defined in complete clefts. in unilateral cleft, the alar base usually found rotated outwardly in a flare and the middle nasal base rotated in the direction of healthy lip side. the base of the ala in the cleft side usually connected with part of clefted nostril sill. numerous methods have been described to repair the cleft lip deformity. in 1960 millard described the concept of advancing a lateral flap into the upper portion of the lip combined with downward rotation of the medial segment. this technique preserves both cupid’s bow and the philtral dimple, and it has the additional advantage of placing the tension of closure under the alar base, thereby reducing flair and promoting better moulding of the underlying alveolar process.1 the fundamental actions of millard’s surgical technique are rotation and advancement. the lip on the noncleft side has two thirds to three quarters of cupid’s bow, median tubercle in the vermillion, and one column of philtrum and its associated dimple and this all is rotated down into normal position. the gap is closed using the advancement flap and corrects the alar flare and the wide nostril.2 millard also performed wide radical undermining the soft tissue supra periosteally of the lateral ala bellows the infraorbital foramen to avoid tension of lateral flap to achieve a good harmony during gap closure. the typical nasal deformity associated with congenital unilateral cleft lip presents both discrepancy and a displacement of parts persists without great improvement during growth. the distortion, being confined to the cleft side only, is emphasized by the constant comparison with the normal opposite. minor modification of millard’s technique was done based on the idea that correction of the nostril sill and nasal base should be simultaneously taken as an important step besides closing the gap using two flaps, ie. the lateral flap and medial segment flap. case a fifteen year old girl with complete unilateral cleft lip seek for treatment for her cleft lip and presented with complete unilateral cleft lip and extreme protruded of tooth 11. my primary concerns were the nostril sill defect, slanted columella and nasal tip and widening the right nostril. millard’s rotation-advancement surgical technique of lip closure with minor modification was used. introduction complete unilateral cleft lip is occasionally found with depressed nasal tip, displaced ala, widening of the nostril floor, slanted columella and dropping of the alar and lower lateral cartilage. complete surgical corrections of the orbicularis oris muscle continuity including straighten of the columella and the alar position, correction of the nostril sill and alar base regions are necessary to be made. the nasal floor is cleft not only in the skin and muscle but the bone can be also involved, and the position of the maxillary elements can be found varies from overlap to abutment to figure �. minor modification of millard’s surgical technique in schematic drawing: a) complete unilateral cleft lip, b) rotation incision as proposed by millard created a triangular flap (c) and flap a, c) the tip of the triangular flap incised followed by incision on the left perialar region to the direction of the inferior border straight forward through the crevice created an alar flap (d) and flap b, d) approximation of flap c and d built a new nostril sill, e) wide radical undermining to release tension of flap b facilitated an ease approximation of flap a and flap b during closing the crevice. the nasal base is closing by approximation of flap a and b to flap c and d, f) the crevice is closed by four flaps. c e d f ba 174 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 172–175 case of complete unilateral cleft lip was operated using a minor modification of millard’s surgical technique. the triangular flap was used to build a new nostril sill in approximation with the alar flap. the alar flap was made by a perialar incision down into the nasal base and was resulted a flap which consisted of alar rim and alar base and clefted nostril sill and nasal base tissues. correction of the slanted columella and nasal tip was done by rotating and pulling the triangular flap to achieve a straight columella and a good nasal tip position followed by a lock suture to maintain its new position. the tip of the triangular flap was trimmed and approximated into the alar flap to build a new nostril sill (figure 1a-f). case management as described by millard, the curved incision in the medial cleft element, in the lower part a mirror image of the contra lateral normal philtrum column, crossed the midline under the columella to allow the downward rotation of the cupid’s bow by 2 to 10 mm and to allow the philtrum dimple components to come into normal position. at the same time the incision lengthtens the short side of the columella by freeing it from the lip.1 the tooth 11 was first extracted followed by circular incision to make a triangular flap. alar flap was made by a circular incision in the perialar region down to the alar base to the direction of the crevice followed by releasing of the slanted nostril sill and the alar base region in the cleft region which attached to the lateral ala. the triangular flap was rotated into a cleft side and brought the philtrum dimple component into normal position followed by maintaining the flap with a lock suture to maintain in its new position. the tip of the triangular flap was than trimmed and approximated and then sutured into the alar flap to build a new nostril. the approximation of those two flaps built a new nostril sill and simultaneously corrected the position of the alar base and widening nostril. after the nostril sill, the surgical step was continued to close the cleft which initially by advancing the medial segment and lateral flaps to avoid of tension during approximation (figure 2a-d). as noted by nicolau that the orbicularis muscle consisted of two muscles layers, one layer is inserted into the philtral ridges and skin, the other layer is located in a deep layer that has little or no attachment to the skin,3 therefore in cleft cases that layers should be sutured by layers according to those layers. the skin and mucosa along each site were undermined several millimeters to facilitate separate and individual suturing of the muscle, mucosa, and skin during approximation. wide undermining at the medial side is contraindicated as it may destroy the philtral dimple and column. according to fara4 and pennisi et al.5 in complete cleft, the superficial fibers of the orbicularis oris muscle may insert into columella and septum medially and into alar base laterally. minor correction of the nose was done by a marginal incision to free the cartilage and followed by correction of the alar cartilage position and then joining the medial crura through vertical mattress sutures that catch the cranial edges together to form one unit. the anterior suture is placed at the height of the transition between the medial and middle crus, a b c d figures �. a) complete unilateral cleft lip shows the slanted columella and nasal tip; b) rotation incision as introduced by millard; c) the triangular flap rotated into the cleft side and approximated into the alar flap built a new nostril sill; d) seven days postoperative situation: the columella and nasal tip region corrected into a proper position toward midline. the right nostril sill is well corrected. 175pramono: minor modification of millard's surgical technique which known as medial crural fixation. the insufficient tip projection due to the great divergence between crura intermedia was corrected by inserting mattress suture close to the dome known as dome spanning suture. discussion the millard unilateral cleft lip repair has become the most popular single procedure in cleft treatment. the flexibility of the technique is reflected in the additive to “cut-as-you-go” and the adaptability to the individual pathomorphology of the affected area. the fundamental of the surgical technique are two folds: rotation and advancement. the scar is manuvered into hidden crevice, under columella, and the lower part stimulates a natural landmark, the philtrum column.2 in complete cleft, the superficial fibers of the orbicularis oris muscle may insert into columella and septum medially and into alar base laterally therefore releasing of these segments are necessary to repair the muscle continuity. the millard’s incision technique designed to create three flaps. the medial segment flap (flap a) is made to allow the downward rotation of cupid’s bow and to allow the philtrum dimple component to come into normal position. the lateral flap (flap b) is the lateral cleft lip element that provides advancement. the third is the triangular flap (flap c) that was made to absorb a part of the pull at the thigh test point in closure and pulls the deviated columella and the anterior septum into straight position. the additionally flap of alar flap (flap d) was made with primary destination to form the nostril sill after approximated of flap d to flap c (figure 1). the role of flap c was important in this case report as this flaps was used not only to pull the deviated columella, but also to build a new nostril sill by approximation it with the alar flap which consisted of cleft nostril sill and nasal base tissue. the size of the nostril was adjusted during this step. the crevice was closed by the approximation of advancement of the flap b and flap a and followed by sutured it into the base of the new nostrill sill. to reduce tension of the flap b during flaps a and b approximation, the “cut-as-you-go” tissue desection technique as presented by millard’s was used. wide radical supraperiosteal undermining as far as the infraorbital foramen was also used as proposed by millard.1, 6–9 in conclusion, surgery has successfully brought the columella and nasal tip region into a proper position toward midline as well as to build a new right side nostril sill which was corrected in the first surgical step during the crevice closure sequence. minor modification of millard’s technique was presented. this technique was applied in over 50’s patients presented with a good result therefore this technique can be use as an alternative surgical technique to treat a complete unilateral cleft lip. acknowledgement i would like to thank drg. herdi eko pranjoto, ms., sp.bm for drawing the schematic pictures. references 1. millard dr jr. complete unilateral clefts of the lip. plast recons sur 1960; 25: 595–605. 2. mommaerts my. the millard phylosophy of cleft lip and palate repair. in: booth pw, schendel sa, hausamen je, editors. maxillofacial surgery. 2nd ed. vol 2. churchil livingston, elsevier; 2007; p. 1048–61. 3. nicolau pj. the orbicularis oris muscle: a functional approach to its repair in the cleft lip. br j plast surg 1983; 36: 141–53. 4. fara m. the important of folding down muscle stumps in the operation of unilateral cleft lip. acta chir plast 1971; 13: 162–9. 5. pennisi vr, shadish wr, klabunde eh. orbicularis oris muscle in the cleft lip repair. am cleft palate j 1969; 6: 141–53. 6. millard dr jr. rotation-advancement in the repair of unilateral cleft lip. in: grabb wc, editor: cleft lip and palate. boston: little brown; 1971. p. 195–202. 7. millard dr jr. a radical rotation in single harelip. am j surg 1958; 95: 318–22. 8. millard dr jr. how to rotate and advance in a complete cleft. in: cleft craft. vol 1. boston: little brown; 1976. p. 449–85. 9. millard dr jr. example of complete unilateral cleft cases. in: cleft craft. vol 1. boston: little brown; 1976. 487–524. 2121 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 21–24 original article adjunctive radiograph diagnostic in vertical mandibular asymmetry kirubanandan sathya moorthy1, ervina sofyanti2, trelia boel3, jesslyn okto govanny1 and aditya rachmawati2 1 undergraduate dentistry program 2 department of orthodontics 3 department of dentomaxillofacial radiography faculty of dentistry, universitas sumatera utara, medan – indonesia abstract background: the development of radio diagnostics in orthodontics is still a challenge in treating skeletal anomaly with facial asymmetry. the assessment of skeletal symmetry, which can be obtained by frontal radiographs such as panoramic radiograph and posteroanterior cephalograph, is still limited. purpose: the aim of this study is to evaluate panoramic radiograph and posteroanterior cephalograph in measuring the vertical mandibular asymmetry based on kjellberg technique. methods: this study was a cross-sectional study of 43 pre-treatment panoramic radiographs and posteroanterior cephalographs from dental faculty students at universitas sumatera utara between 18–25 years old. the subjects have fully erupted permanent teeth until the second molar and complained about facial asymmetry. the validity and reliability of vertical mandibular asymmetry of kjellberg technique with cliniview software in both radiographs used cohen-k analysis. results: the measurement of vertical mandibular asymmetry showed no significant differences using panoramic radiograph and posteroanterior cephalograph (0.073-0.321 > 0.05). conclusion: the vertical mandibular asymmetry analysis with kjellberg technique in panoramic radiograph is potent as an adjunctive diagnostic tool in vertical mandibular asymmetry. keywords: asymmetry; digital radiograph; mandibular; vertical correspondence: ervina sofyanti, department of orthodontics, faculty of dentistry, universitas sumatera utara, jl. alumni no. 2, medan 20155, indonesia. e-mail: ervina.sofyanti@usu.ac.id introduction establishing an improved level of reliability in mandibular asymmetry analysis has been a challenge in dentistry. mandibular asymmetry is one of the common craniofacial abnormalities due to the lateral displacement of the mandible’s midline, beginning with the growth of mandibular asymmetry or certain diseases that affect facial growth.1,2 previous study explained that lateral deviations occur more frequently in the lower third of the face.3 eighty-five per cent of the abnormalities show a tendency towards lateral displacement to the left side – which is inherited – so that excessive growth is seen on the right side, or less growth on the left side, of mandible.3 the skeletal deviation in facial asymmetry is equal to or greater than 2 mm.4,5 however, dimensional differences reported as equal to or greater than 4 mm are considered to be mandibular asymmetry.6,7 according to lin’s study reporting the association between asymmetrical jaw function and joint remodelling in mandibular asymmetry patients, the 3-d morphology and bone density of the condyle on the deviated side differs from the non-deviated side.6 a previous study conducted on mixed dentition in patients between 8to 12-years-old reported that more than half had moderate to severe mandibular asymmetry.5 mandibular asymmetry can be diagnosed through clinical, photographic and radiographic examinations, which include frontal and lateral views, including lateral cephalograph, postero-anterior cephalograph (pa), panoramic radiography, cone-beamed computed tomography (cbct), submentovertex and single-positron emission computed tomography (spect).8–10 in order to achieve a proper analysis, the measuring procedure is performed in order to obtain the qualified or quantified dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p21–24 mailto:ervina.sofyanti@usu.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p21-24 22 moorthy et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 21–24 value of the characteristics of a research subject. mandibular asymmetry diagnosis is an important step in orthodontic treatment and a complicating factor in some malocclusion. even though the pa cephalograph is ideal in the frontal assessment of the skeletal aspect of facial asymmetry, some limitations are associated with the use of cephalometric radiographs, such as standardization, reproducing head position and maintaining film–object distance. since panoramic radiography allows the dental professional to view a large area of the maxilla and mandible on a single film, it is provided as an initial diagnostic image in dentistry.3,5,7 variable measurement produces a set of values or attributes from individuals called data. data are analysed to provide information, which will be interpreted in the results. errors in measurement, or measurement bias, can be anticipated or minimised with validity, reliability and generalisability in qualitative research. the concept of validity becomes an important matter when questioning the quality of the results of a qualitative study.the concept of reliability often becomes another consideration in assessing the scientific findings of qualitative research and also shows the consistency of findings when conducted by different studies.11,12 thus, this study aims to measure the validity and reliability of panoramic radiograph and posteroanterior cephalograph in measuring the vertical mandibular asymmetry based on the kjellberg technique. materials and methods this study was approved by the research ethics committee of the universitas sumatera utara medical faculty and was conducted in universitas sumatera utara dental hospital (number: 114/tgl/kepk fk usu-rsup ham/2018). this cross-sectional study commenced in september 2017, and continued until april 2018, and included 43 dental faculty students who complained about facial asymmetry, aged 18to 25-years-old. the participants had fully complete teeth until the second molar and absence of caries and/or radix. they also had no orthodontic treatment or facial trauma history. the panoramic and pa digital cephalograph of all the volunteers was taken under standard conditions and processed in the same x-ray machine (oc200d1-4-1 with digital sensor by a single operator). the initial measurement of vertical mandibular asymmetry was performed by a single operator using cliniview software (version 10.1.2) under a dentomaxillofacial radiograph specialist’s supervision. using this software, the parameter points to measure the condyle asymmetry index (iak) are as follows: the condylar (co) point is the most superior point of the condyle, whilst the mandibular notch (mn) is the lowest point between the coronoid process and the condyle process. the ramus line (rl) is drawn from the most lateral point in the condyle to the mandible angle. the gonion point (go) is located on the tangent of the intersection of the ramus line (rl) and mandibular line (ml). co, mn and go points are reflected in the rl line, forming a 90-degree angle, as shown in figure 1 and 2. evaluation was carried using the kjellberg symmetry index (si), of which more than 93.7 per cent is asymmetry (cit. kjellberg).9 the initial measurement of inter-observer vertical mandibular asymmetry category on ten selected radiographs were analysed with cohen’s kappa and showed fair agreement (r≥0.4). the final measurement, begun four weeks after the initial measurement, was measured by the same operator using a specific schedule three samples per day. ����������� ���� ����� ����������� ���� ����� x 100%kjellberg�s symmetry index/condylar ratio = pearson correlation was performed to analyse the validity and reliability of condylar and ramus height in vertical mandibular asymmetry for both sides between panoramic radiograph and pa cephalograph. since the data distribution was abnormal, a chi-squared test was used to obtain the difference between panoramic radiograph and pa cephalograph. results from 43 subjects of this study, the contribution of female subjects (62.8%) was higher than male subjects (37.2%), figure 1. condylar height asymmetry on panoramic radiography using cliniview software. figure 2. condylar height asymmetry on posteroanterior cephalometry using cliniview software. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p21–24 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p21-24 23moorthy et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 21–24 and the mean age was 20.93 ± 2.21 years. table 1 shows the results of the pearson test in determining the validity and reliability of vertical mandibular asymmetry of 43 students using panoramic radiograph and pa cephalograph. the r-count value (0.938–0.978) was greater than r table (0.301, n = 43) and, based on the significance level of 0.05, was valid and reliable. the difference in measuring the vertical mandibular asymmetry with kjellberg technique in panoramic radiograph and pa cephalograph with chi-squared (table 2) showed no significant difference (0.073–0.321) > 0.05. therefore, the measurement of mandibular asymmetry in the vertical direction on panoramic radiography and pa cephalograph are valid and reliable. discussion as one of common craniofacial deformities, mandibular asymmetry is related to mandibular displacement and lateral shift in the mandibular midline. the asymmetric growth of the mandible or other certain diseases can affect facial growth. some mandibular asymmetries are idiopathic, and non-syndromic asymmetry that develops gradually over the years after birth may become prominent during adolescence.3,6 panoramic radiography is widely used as initial d i a g n o s t i c r a d i o g r a p h y b e c a u s e i t d e m o n s t r a t e s mandibular anatomy bilaterally by providing sufficient information for vertical measurements. pa cephalographs, as well as panoramic radiographs, provide valuable mediolateral information that is not only useful for facial asymmetric evaluation but also evaluates skeletal craniofacial and dentoalveolar structures in the horizontal direction of view. however, the diagnostic accuracy and clinical efficacy of 3-d cbct (cone-beam computed tomography) in the maxillofacial region is better than pa cephalographs.13 there are several methods and techniques in analysing vertical mandibular asymmetry, such as habets and kjellberg, based on panoramic radiograph. in this study, the kjellberg technique is offered as acceptable clinical information in analysing condylar asymmetry within the limitations of these techniques in panoramic radiograph and pa cephalograph.9,14 based on previous studies, the panoramic and pa cephalograph did not affect the condyle asymmetry index on both sides due to the accuracy and reproducible method in predicting asymmetry of mandibular anatomy with digital imaging, providing similar analysis in anatomical points of interest on the skull that are common in the two-dimensional radiographic images: correction of the magnification as well as possible tilt of the skull used the vertical marker, and adjusting the contrast and brightness of images.15 previous studies also support the validity and reliability in the assessment of mandibular asymmetry in vertical directions using panoramic radiograph and pa cephalography, and they reported qualitative measurement in mandibular asymmetry differences for both sides.15–17 van eslande also identified the linear dimensions, especially in radiography, with some errors: distortion, magnification (either because of the projection geometry or because of the patient’s position) and image accuracy in determining left–right differences or asymmetry. according to kjellberg, the ratio is not affected by malposition, distortion or enlargement in the panoramic image.9,16 table 1. the results of the validity and reliability of mandibular asymmetry measurements in vertical direction on panoramic radiographs and postero-anterior cephalograph using pearson test measurements r count value* panoramic pa cephalograph right condylar height 0.956 0.942 left condylar height 0.938 0.925 right ramus height 0.975 0.975 left ramus height 0.978 0.978 index symmetry (is) 0.949 0.945 table 2. the results of condylar asymmetry between panoramic radiography and posteroanterior cephalograph using the chi-square test measurements radiograph mean ± sd p-value * right condylar height (ch1) panoramic 19.08 ± 3.23 0.073pa cephalograph 20.83 ± 2.83 left condylar height (ch2) panoramic 18.79 ± 3.24 0.299pa cephalograph 20.11 ± 2.94 right ramus height (rh1) panoramic 57.70 ± 5.74 0.266pa cephalograph 60.29 ± 5.72 left ramus height (rh2) panoramic 57.08 ± 5.75 0.287pa cephalograph 60.16 ± 5.64 index symmetry (is) panoramic 89.67 ± 3.12 0.321pa cephalograph 89.39 ± 2.99 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p21–24 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p21-24 24 moorthy et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 21–24 the complexity of mandibular asymmetry is also related to the presence of temporomandibular disorder (tmd). in mandibular asymmetry subjects with tmd, there are no significant differences in unilateral and bilateral side of tmd patients when comparing the asymmetry between panoramic radiograph and pa cephalographs.18 however, there are significant differences of horizontal mandibular asymmetry based on menton deviation.10 the complexity of mandibular asymmetry can be simplified by early identification of the characteristic of mandibular asymmetry, whether vertical or horizontal, if there is no 3-d radiography. based on this study, the early mandibular asymmetry can be detected by panoramic radiography with digital radiography improvement. the limitations of radiographic retrieval are restricted to radiation side effects, particularly in the treatment of malocclusions with developing mandibular asymmetry. in addition, pa analysis, which usually requires a special level of expertise, is not fully understood by general dental practitioners. thus, the panoramic radiographs is widely used to observe the eruption and growth patterns of the teeth and jaw fracture, evaluate the maxillofacial and/or dentoalveolar complex and mandibular asymmetry, which may be associated with temporomandibular disorder (tmd). the digital panoramic radiograph is potent as an adjunctive diagnostic tool in vertical mandibular asymmetry for early detection of complex mandibular asymmetry. distortion in both radiographs can happen in linear measurements and magnification of some image areas in different regions because vertical measurements on radiograph panoramic are relatively more reliable if patient positioning is accurate and has good radiography quality. due to some limitations associated with the use of pa radiographs, such as standardization, reproducing head position, maintaining film-object distance and requirement of special interpretation skill, the standardised radiography procedure and competence of digital panoramic radiograph analysis will help clinicians to obtain earlier asymmetry detection and minimise the radiographic exposure in analysing mandibular asymmetry development, treating skeletal anomaly with facial asymmetry, especially in the mandible. acknowledgement we want to thank the participants in this study and partially from talenta grant universitas sumatera utara (4167/ un5.1.r/ppm/2019). references 1. anison j, rajasekar l, ragavendra b. understanding asymmetry-a review. biomed pharmacol j. 2015; 8(special edition): 659–68. 2. thiesen g, gr ibel bf, freitas mpm, oliver dr, k im k b. mandibular asymmetries and associated factors in orthodontic and orthognathic surgery patients. angle orthod. 2018; 88(5): 545–51. 3. brionne c, cadre b, laroche y, lhotellier j, maze m, raffre a, sorel o. the diagnosis of mandibular assymmetries. j dentofac anomalies orthod. 2013; 16(3): 302. 4. kim j-y, jung h-d, jung y-s, hwang c-j, park h-s. a simple classification of facial asymmetry by tml system. j craniomaxillofacial surg. 2014; 42(4): 313–20. 5. ramirez-yanez go, stewart a, franken e, campos k. prevalence of mandibular asymmetries in growing patients. eur j orthod. 2011; 33(3): 236–42. 6. lin h, zhu p, lin y, wan s, shu x, xu y, zheng y. mandibular asymmetry: a three-dimensional quantification of bilateral condyles. head face med. 2013; 9(1): 42. 7. cheong y-w, lo l-j. facial asymmetry: etiology, evaluation, and management. chang gung med j. 2011; 34(4): 341–51. 8. yanez-vico r, iglesias-linares a, torres-lagares d, gutierrezperez j, solano-reina e. association between condylar asymmetry and temporomandibular disorders using 3d-ct. med oral patol oral y cir bucal. 2012; 17(5): e852–8. 9. hirpara n, jain s, hirpara vs, punyani pr. comparative assessment of vertical facial asymmetry using posteroanterior cephalogram and orthopantomogram. j biomed sci. 2017; 06(01): 1–7. 10. boel t, sofyanti e, sufarnap e. analyzing menton deviation in posteroanterior cephalogram in early detection of temporomandibular disorder. int j dent. 2017; 2017: 1–5. 11. maxwell j. understanding and validity in qualitative research. harv educ rev. 1992; 62(3): 279–301. 12. leung l. validity, reliability, and generalizability in qualitative research. j fam med prim care. 2015; 4(3): 324. 13. yousefi f, rafiei e, mahdian m, mollabashi v, saboonchi s, hosseini s. comparison efficiency of posteroanterior cephalometry and cone-beam computed tomography in detecting craniofacial asymmetry: a systematic review. contemp clin dent. 2019; 10(2): 358–71. 14. fuentes r, engelke w, bustos l, oporto g, borie e, sandoval p, garay i, bizama m, borquez p. reliability of two techniques for measuring condylar asymmetry with x-rays. int j morphol. 2011; 29(3): 694–701. 15. ludlow jb, laster ws, see m, bailey l ’tany. j, hershey hg. accuracy of measurements of mandibular anatomy in cone beam computed tomography images. oral surgery, oral med oral pathol oral radiol endodontology. 2007; 103(4): 534–42. 16. agrawal a, bagga dk, agrawal p, bhutani rk. an evaluation of panoramic radiograph to assess mandibular asymmetry as compared to posteroanterior cephalogram. apos trends orthod. 2015; 5(february 2016): 197–201. 17. gupta s, jain s. orthopantomographic analysis for assessment of mandibular asymmetry. j indian orthod soc. 2012; 46(1): 33–7. 18. cavalcante-leao bl, kusdra pm, francio la, cristoff k, sampaio r, santos ahl, stechman-neto j. mandibular asymmetry in patients with temporomandibular disorder: comparative study between posteroanterior teleradiographs (pa) and panoramic radiography. int j dev res. 2019; 09(11): 31145–50. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p21–24 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p21-24 vol 51 no 1 jan-mrt 2018.indd 3737 surface roughness of nanofilled and nanohybrid composite resins exposed to kretek cigarette smoke laksmiari setyowati, s. setyabudi, and johanna chandra department of conservative dentistry faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: cigarette smoking is a public health issue that may influence the physical properties of dental composites. surface roughness is one of the physical properties of restorative materials potentially influencing their success. the use of nanofilled and nanohybrid composites in dentistry has increased substantially over the past few years. purpose: the purpose of this study was to evaluate the surface roughness of nanofilled and nanohybrid composite resins exposed to kretek cigarette smoke. methods: twelve cylindrical specimens of each material were prepared and divided into two groups (n=6). in the control groups, the specimens were immersed in distilled water for 24 hours at 37°c, with the water being renewed daily. for the experimental groups, the specimens were exposed to kretek cigarette smoke on a daily basis, then washed and soaked in distilled water at 37°c. after 21 days, the specimens were measured using a surface roughness tester and the data was then statistically analyzed. results: an independent-t test revealed that there were statistically significant differences in the surface roughness between the control and experimental groups of both nanofilled and nanohybrid composites, as well as between the nanofilled experimental group and the nanohybrid experimental group. conclusion: exposure to kretek cigarette smoke can increase the surface roughness of nanohybrid composites to a significantly greater extent than nanofilled composites. keywords: composite resin; nanofilled; nanohybrid; surface roughness; kretek cigarette smoke correspondence: laksmiari setyowati, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: laksmi_dentist@yahoo.com research report introduction composite resin is one of the most widely used restorative materials in dentistry. nano composite resin is the newest composite resin with smaller filler sizes (1-100 nanometers) and increased filler concentration. thus, its physical, mechanical and esthetic properties are greatly enhanced.1 there are two kinds of nano composite resin, namely nanofilled composite (all fillers with nano size) and nanohybrid composite (partially nano fillers and micro fillers).2 nevertheless, the properties of composite resin as a restorative material may still be affected by several factors, including: matrix composition, filler, coupling agent and bonding techniques, among others.3 individual lifestyles, such as a smoking habit, can also affect the properties of the restorative material.4 smoking is a public health problem commonly found within communities. according to who data, after china and india, indonesia has the largest number of smokers in the world. a statistic which, unfortunately, is increasing from year to year. according to riskesdas (national basic health research) data from 2013, 24.3% of indonesians were active smokers, with the daily average number of cigarettes smoked being 12.3.5 in fact, there are many toxic materials contained in tobacco or produced through smoking which lead to specific diseases.6 for instance, smoking can increase the risk of dental caries.3 therefore, dental restoration, predominantly using composite resin, is required. according to research dental journal (majalah kedokteran gigi) 2018 march; 51(1): 37–41 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p37–41 mailto:laksmi_dentist@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p37-41 38 setyowati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 37–41 conducted by mathias et al., when composite resin is exposed to cigarette smoke there will be an increase in water absorption.3 although the strongest effect in the oral cavity still remains unclear, high temperature (55° c) can increase kinetic water diffusion, water absorption, and resin solubility.4 kretek cigarettes constitute a typical indonesian tobacco product dominating 90% of the domestic cigarette market.7 according to the top brand award index, the most famous non-filter kretek cigarette between 2012 and 2014 was dji sam soe. in contrast to white cigarettes, kretek cigarettes use heavy tobacco derived from chopped tobacco mixed with cloves.8 consequently, when ignited they release a high concentration of eugenol derived from cloves, approximately 28,700–30,200 μg per cigarette (dji sam soe brand).7 the processes of water absorption and eugenol release can then be used as plasticizers of the composite matrix, causing swelling which alters the dimensions of the restorative material.9 water absorption may be affected by the concentration of filler and rate of polymerization, as well as the type and number of monomers. water absorption may subsequently result in the release of unreacted monomers, as well as the process of hydrolysis so that the chemical bond between the filler and the resin matrix is broken. in addition, polymer degradation can occur due to sudden temperature changes, resulting in damage to the silane coating resulting in the bond between the filler and the resin matrix being compromised. degradation of the matrix and the release of filler particles onto the outer surface of the composite can lead to an increase in its surface roughness.10,11 the increased surface roughness of the restorative material can, in turn, trigger plaque and biofilm formation, thus increasing the risk of caries and periodontal inflammation. surface roughness can also affect aesthetics, i.e. reducing the brightness of restoration, increasing susceptibility to discoloration and shortening the age of restoration.12,13 as a result, this research aimed to evaluate the surface roughness of nanofilled and nanohybrid composite resins exposed to kretek cigarette smoke. materials and methods this research represented a laboratory experimental study with post test-only control group design. the research samples constituted 12 nanofilled composite resins (filtek z350 xt) and 12 nanohybrid composite resins (filtek z250 xt) cylindrical in shape, 5 mm in diameter and 2 mm in thickness. the samples were then divided into four groups (n=6), namely: a nanofilled control group, a nanohybrid control group, a nanofilled experimental group and a nanohybrid experimental group. the preparation of each sample commenced with the manufacture of an insulin syringe mold 5 mm in diameter and 2 mm thick which was given a celluloid strip base and placed on a plate glass. the composite resin was subsequently inserted into the sample mold until it was full and then covered with a celluloid strip. a 1 kg glass plate was placed on top for 30 seconds in order to make the sample surface flat and solid.13 the scales and glass plate were lifted and the composite irradiated with a light curing unit for 20 seconds at an intensity of 600–700 mw/cm2 (as per factory rule: 400–1000 mw/cm2) and a distance of 0.5–1 mm between the tip of the unit and the composite.2 hardened composite resin was subsequently removed from the mold. both of the composite control groups were immersed in distilled water and incubated at 37° c for 21 days. meanwhile, both of the experimental groups were exposed to smoke derived from 12 cigarette bars placed in a smoking machine at an exposure temperature of 55° c inside the tube (using a water bath) as illustrated in figure 1.4 smoke from each cigarette was introduced into the tube for 10 minutes14 before being removed. after the exposure of the 12 cigarettes had been completed, the sample was immersed in distilled water and incubated at 37° c for ± 21 hours. thereafter, both composite experimental groups were removed from the distilled water and dried using absorbant paper. all of the procedures described above were then repeated for 21 days. however, the distilled water had to be replaced daily for all groups. after 21 days, the control and experimental group samples were removed from the distilled water and dried using absorbant paper. both composite experimental groups were then immersed in acetone and agitated for ± 1 minute15 in order to dissolve any cigarette tar attached to the sample surfaces.16 later, the surface roughness of each sample was measured using a surface roughness tester (mitutoyo sj-201) tool with a standard stylus in three different areas (figure 2), with the mean values being calculated.13 the rokok tempat sampel pompa vakum water bath figure 1. the smoking machine used in this research.14 figure 2. the pattern of sample measurement areas.13 sample places cigarette vacuum pump dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p37–41 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p37-41 39setyowati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 37–41 results of the composite surface roughness measurements were then statistically analyzed using an independent-t test with a confidence level of 95%. results in this research, four sample groups were analysed, namely: the nanofilled control group, the nanohybrid control group, the nanofilled experimental group exposed to kretek cigarette smoke and the nanohybrid experimental group exposed to kretek cigarette smoke. the research was conducted over 21 days, at the end of which period the surface roughness of each sample was measured using a surface roughness tester (mitutoyo sj-201). the parameters used in this research consisted of a mean roughness value (ra) with a micrometer unit (μm). the mean and standard deviation values of the surface roughness of the composite resins can be seen in table 1. the results of the one sample kolmogorov smirnov normality test confirmed the data to be normally distributed with a p value of >0.05, while those of the homogeneity test (a levene’s test) revealed it to be homogenous with a p value of >0.05. in order to observe the significance of the differences between the research groups, an independent-t test was subsequently, performed whose results confirmed important differences in the surface roughness of the composite resin between the nanofilled control group and the nanofilled experimental group, the nanohybrid control group and the nanohybrid experimental group, and the nanofilled experimental group and the nanohybrid experimental group (p<0.05). in contrast, there was no significant difference between the nanofilled control group and the nanohybrid control group (p>0.05) in terms of surface roughness. discussion this research was conducted to evaluate the effect of exposure to cigarette smoke on the surface roughness of nanofilled and nanohybrid composite resins. this research focused on kretek cigarettes since they are considered to be the most famous indonesian tobacco product dominating 90% of the domestic cigarette market.7 the investigation did not use artificial saliva, but distilled water, since the former has not been clinically proven to be a more relevant storage medium. in previous research focusing on the effect of storage media on composite resin micromorphology, the same results were obtained using distilled water and artificial saliva.1 the samples of both experimental groups were soaked in acetone to dissolve the tar layer of cigarettes attached to the sample surfaces.16 the administration of acetone to the surface of the nano composite for ± 1 minute, according to research conducted by hamano et al., produced no effect on surface roughness since the nano composite resin has a strong crosslinking bond. thus, it is unlikely that acetone can dissolve the composite surface.15 moreover, the results of this research showed that there were significant differences in the surface roughness of the composite resins between the nanofilled control group and the nanofilled experimental group exposed to kretek cigarette smoke, as well as between the nanohybrid control group and the nanohybrid experimental group exposed to such smoke. this corresponds to the theory of composite degradation due to increased water absorption and acid exposure. the high temperature (approximately 55o c) of cigarette smoke in the oral cavity can increase the kinetic energy of water diffusion so that water absorption into the composite resin increases.4 water absorption is also affected by filler concentration. therefore, an increased amount of filler can decrease water absorption.17 the composite resins used in this research were nanofilled composite with a filler concentration of 59.5% (filtek z350 xt) and nanohybrid composite (filter z250 xt) with a filler concentration of 68%. consequently, the absorption of water in nanofilled composites is greater than that in in nanohybrid composites. water absorption can cause hydrolysis reaction, resulting in the water decomposing to h+ and oh-. due to the presence of element o in the resin matrix, ohderived from the water is then absorbed into the matrix and attacks the siloxane bond (si-o-si), a bond linking the matrix and filler particles. subsequently, this condition results in the breakdown of siloxane bonds to form silanol compounds, table 1. the mean and standard deviation values of the surface roughness of nanofilled and nanohybrid composite resins. mean (sample group n μ standard deviationm) 0.0247630.165006nanofilled control group 0.0367790.274336nanofilled experimental group 0.0329120.180006nanohybrid control group 0.0541200.401836nanohybrid experimental group table 2 the values of p in independent t-test results relating. to the surface roughness of composite resins nanofilled control group nanohybrid experimental group 0.000*0.395**nanohybrid control group nanofilled experimental group 0.001*0.000* note: * significant difference ** no significant difference dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p37–41 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p37-41 40 setyowati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 37–41 si-oh and si-o. in si-o, disorientation of its electrons results in a reaction when its contact with water produces si-oh and oh-. the ohwill subsequently break the siloxane bond again so that the reaction occurs continuously as long as the composite resin remains immersed in water. the longer these reactions occur, the greater the number of filler particles detached from the surface of the composite resin so that it becomes rougher.18 this process occurs until the composite reaches saturation point.19 furthermore, the burning of kretek cigarettes also releases a high concentration of eugenol derived from cloves, about 28,700–30,200 μg per cigarette compared to the average daily consumption of eugenol derived from food of about 70 μg.7 eugenol belongs to the phenol group and tends to be acidic. as a result, it can release h+ ions from its hydroxyl groups which also contribute to the degradation of the composite resin because of a potential break in the siloxane bond.18 moreover, the free h+ ion can react with the double bond carbon (c =) in the polymer chain of the resin matrix resulting in the polymer chain being disconnected which, in turn, triggers composite resin matrix degradation causing the filler particles on the surface to loosen easily. the release of the matrix and filler particles then results in many small cracks in the composite so that the surface roughness increases.20 in addition, cigarette smoke contains numerous other chemical components, as many as 4,800,21 which may also affect the surface roughness of the composite resin. within this research, the chemical component contained in kretek cigarette smoke with the highest concentration compared to others was found to be eugenol. there may also be other components working synergistically or in opposition to eugenol. based on research using scanning electron microscope (sem) images, there are also matrixes and fillers found in the composite resin surface of the control group.12 nevertheless, the results of this research revealed that the surface roughness of the nanohybrid control group was slightly greater than the surface roughness of the nanofilled control group, but statistically not significantly different. this is due to the fact that the surface roughness of the composite resin can be affected by the size and volume of the filler.22 composites with larger filler particles have rougher surfaces than those with small fillers.23 in nanofilled composites, all filler particles are round with a nano-size of 1–100 nm. meanwhile, nanohybrid composites have irregular particle fillers with partial nano fillers (1-100 nm) and micro fillers (0.4–5 μm).2,24 therefore, nanohybrid control composites have a slightly larger surface roughness than composite nanofilled controls. furthermore, the results of this research also found that the surface roughness of the nanohybrid experimental group was significantly greater than that of the nanofilled experimental group. this is because filler particles of the nanohybrid composite (≤5 μm) are larger in size than those of the nanofilled composite (≤100 nm), resulting in increased surface roughness in the nanohybrid composite greater than that in the nanofilled composite.23 thus, the mean the surface roughness value of the nanohybrid experimental groups in this research was 0.402 μm, suggesting that the fillers released may be small or medium-sized, whereas the siloxane bond on the large filler particles may be only partially discontinued so that the filler particles are not released. however, this point requires further research. in conclusion, kretek cigarette smoke can increase the surface roughness of nanohybrid composite resin to a greater extent than that of nanofilled composite resin. however, further research needs to focus on other chemical components that may affect composite surface roughness. in addition, such research is also expected to evaluate the surface roughness of composite resin with a confocal laser scanning microscopic (clsm) tool in order to analyze the topography of surface roughness in detail. references 1. erdemir u, yildiz e, eren mm, ozel s. surface hardness evaluation of different composite resin materials: influence of sports and energy drinks immersion after a short-term period. j appl oral sci. 2013; 21(2): 124–31. 2. sakaguchi r, powers j. craig’s restorative dental materials. 13th ed. st. louis: mosby elsevier; 2012. p. 143, 165–9, 179. 3. mathias p, santos srb, aguiar tr, santos prb, cavalcanti an. cigarette smoke: effects on water sorption and solubility of restorative dental composites. gen dent. 2014; 62(2): 54–7. 4. aguiar tr, gaglianone la, mathias p. an overview of the impact of lifestyle behaviors on the operative dentistry. jbr j interdiscip med dent sci. 2014; 2(4): 1–6. 5. infodatin kemenkes ri. perilaku merokok masyarakat indonesia berdasarkan riskesdas 2007 dan 2013: hari tanpa tembakau sedunia. kementerian kesehatan republik indonesia. 2015. p. 2–4. 6. bertold ce dos s, miranda d de a, souza-junior ej, aguiar fhb, lima danl, ferreira rl, claes ir, lovadino jr. surface hardness and color change of dental enamel exposed to cigarette smoke. int j dent clin. 2011; 3(4): 1–4. 7. polzin gm, stanfill sb, brown cr, ashley dl, watson ch. determination of eugenol, anethole, and coumarin in the mainstream cigarette smoke of indonesian clove cigarettes. food chem toxicol. 2007; 45(10): 1948–53. 8. jonatan s. launching for marketer and entrepreneur. jakarta: pt gramedia pustaka utama; 2007. p. 173. 9. darvell bw. materials science for dentistry. 9th ed. cambridge: woodhead publishing limited; 2009. p. 218. 10. tanthanuch s, kukiattrakoon b, siriporananon c, ornprasert n, mettasitthikorn w, likhitpreeda s, waewsanga s. the effect of different beverages on surface hardness of nanohybrid resin composite and giomer. j conserv dent. 2014; 17(3): 261–5. 11. soetojo a. penggunaan resin komposit dalam bidang konservasi gigi. surabaya: revka petra media; 2013. p. 1, 30, 34. 12. hossam ae, rafi at, ahmed as, sumanth pc. surface topography of composite restorative materials following ultrasonic scaling and its impact on bacterial plaque accumulation. an in-vitro sem study. j int oral health. 2013; 5(3): 13–9. 13. oliveira albm de, garcia ppns, dos santos pa, campos jádb. surface roughness and hardness of a composite resin: influence of finishing and polishing and immersion methods. mater res. 2010; 13(3): 409–15. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p37–41 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p37-41 41setyowati, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 37–41 14. wasilewski m de sa, takahashi mk, kirsten ga, de souza em. effect of cigarette smoke and whiskey on the color stability of dental composites. am j dent. 2010; 23(1): 4–8. 15. hamano n, chiang y-c, nyamaa i, yamaguchi h, ino s, hickel r, kunzelmann k-h. effect of different surface treatments on the repair strength of a nanofilled resin-based composite. dent mater j. 2011; 30(4): 537–45. 16. boyle p, gray n, henningfield j, seffrin j, zatonski w. tobacco and public health: science and policy. oxford: oxford university press; 2004. p. 53, 62. 17. anusavice kj, shen c, rawls hr. phillips’ science of dental materials. 12th ed. st. louis: elsevier saunders; 2012. p. 278–87. 18. aisya rkn. pengaruh perendaman obat kumur mengandung eugenia caryophyllata oil terhadap kekerasan resin komposit tipe hibrid. thesis. jakarta: universitas indonesia; 2008. p. 8–9. 19. noort r van. introduction to dental materials. 2nd ed. london: mosby elsevier; 2002. p. 96–7, 109–13. 20. maghfiroh h, nugroho r, probosari n. the effect of carbonated beverage to the discoloration of polished and unpolished nanohybrid composite resin. j dentomaxillofacial sci. 2016; 1: 16–9. 21. tirtosastro s, murdiyati as. kandungan kimia tembakau dan rokok. buletin tanaman tembakau, serat & minyak industri. 2010; 2: 33–44. 22. alandia-roman cc, cruvinel dr, sousa abs, pires-de-souza fcp, panzeri h. effect of cigarette smoke on color stability and surface roughness of dental composites. j dent. 2013; 41: e73–9. 23. tantanuch s, kukiattrakoon b, peerasukprasert t, chanmanee n, chaisomboonphun p, rodklai a. surface roughness and erosion of nanohybrid and nanofilled resin composites after immersion in red and white wine. j conserv dent. 2016; 19: 51–5. 24. moraes rr, gonçalves ls, lancellotti ac, consani s, corrersobrinho l, sinhoreti ma. nanohybrid resin composites: nanofiller loaded materials or traditional microhybrid resins? oper dent. 2009; 34(5): 551–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p37–41 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p37-41 mkgs vol 44 no 1 jan-mar 2011.indd 35 vol. 44. no. 1 march 2011 changes of the sweet taste sensitivity due to aerobic physical exercise ni luh putu ayu wardhani1, anis irmawati2, and jenny sunariani2 1dental student 2department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: sweet taste is a pleasant sensation. sweet taste is mostly consumed and fancied by many people. physiologically, glucose is body's source of energy, but if over used it can be affected to the body's metabolism. this can be worsen if the person's not doing a healthy lifestyle. one way to implement a healthy lifestyle is by doing physical exercises. purpose: the aim of this study was to determine changes in sensory sensitivity of sweet taste due to aerobic physical exercise. methods: this study was conducted on subjects aged 20 to 30 years. the subjects did aerobic exercise using 80% load of mhr. the measurement sensitivity of the senses of the sweet taste was done for three times before the subject take aerobic physical exercise, four weeks after doing aerobic physical exercise, and eight weeks after doing aerobic physical exercise. results: there was significant difference towards sensitivity of sweet taste sense before doing aerobic physical exercise, 4 week after doing the aerobic physical exercise, and 8 week after doing aerobic physical exercise. conclusion: aerobic physical exercise during eight weeks increase sweet taste sensitivity. key words: sense of taste sensitivity, sweet taste, aerobic physical exercise abstrak latar belakang: rasa manis memberikan sensasi yang menyenangkan. rasa manis merupakan jenis rasa yang paling banyak dikonsumsi dan disukai oleh sekelompok orang. secara fisiologis, glukosa bisa berperan sebagai sumber energi, namun apabila dikonsumsi secara berlebihan dapat menimbulkan efek patologis. hal ini dihubungkan dengan individu yang mempunyai gaya hidup yang tidak sehat. salah satu cara yang bisa dilakukan untuk membiasakan gaya hidup sehat adalah dengan latihan fisik (olah raga). tujuan: penelitian ini bertujuan untuk membuktikan adanya perubahan sensitivitas indera kecap rasa manis setelah melakukan latihan fisik aerobik. metode: penelitian ini melibatkan subyek laki-laki, berusia 20–30 tahun. subjek melakukan latihan fisik aerobik dengan intensitas sebesar 80% maximal heart rate. sensitivitas indera kecap rasa manis diukur 3 kali, yaitu sebelum melakukan latihan fisik aerobik, dan 4 serta 8 minggu setelah latihan fisik aerobik. hasil: terdapat perbedaan yang signifikan pada sensitivitas indera kecap rasa manis sebelum 4 dan 8 minggu sesudah latihan fisik aerobik. kesimpulan: latihan fisik aerobik selama 8 minggu menyebabkan peningkatan sensitivitas indera kecap rasa manis. kata kunci: sensitivitas indera kecap rasa manis, rasa manis, latihan fisik aerobik correspondence: ni luh putu ayu wardhani, c/o: pendidikan dokter gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. research report introduction sweet taste is one of the five basic tastes that are considered as a pleasant experience. supartono research states that sweet is a taste with more popularity and more consumed by the indonesians.1 physiologically, the sweet taste derived from glucose which is the body's main source of energy, but when consumed in excess will have 36 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 35–38 an impact on the body such as pathological obesity and diabetes mellitus.2 the major problem that often occurs in the oral cavity is dental caries. the prevalence of dental caries in developed countries continues to decline, while in developing countries like indonesia there is a tendency to increase. the data showed about 80% of indonesia's population has broken teeth caused by different factors, but the most common is dental caries. statistics showed that there is an average of two to three caries lesions in indonesia population.3 household health survey (nhhs) in 2004 showed that the prevalence of dental caries in indonesia reached 90.05%. data from the ministry of health also showed that the number of complaints of a toothache because caries is high, which is 1.3% or 2620 people every month.4 caries prevalence is quite high in indonesia leading to an alternative measures of prevention which is a priority attempts to curb the prevalence of teeth. the prevention of dental caries that has been done, such as improving nutrition, reducing the consumption of cariogenic diet, improving oral hygiene, the provision of systemic or topical fluoride and fissure sealant with adhesive materials. yet those efforts still do not provide optimal results.5,6 one way to implement a healthy lifestyle is to exercise regularly. in general, physical exercise is an activity that a person intentionally done by taking the time to train the body, not just physically, but also spirituality which is focused to maintain a balanced mind. so with physical exercise, a healthy physical condition as well as psychological condines.7 there are two types of physical exercises there are aerobic exercise and anaerobic exercise. aerobic exercise is an intense physical exercise, which can speed heart rate and is done for long periods of time, for about 20 minutes. this exercise strengthens the cardiovascular system and can burn glucose and fat stored in the body. activities such as jogging, swimming, dancing, brisk walking and cycling are examples of aerobic exercise. this type of anaerobic exercise performed for a short period of time, useful to help strengthen the muscles and joints. activities like weight lifting and running are examples of anaerobic exercises.7 in one exercise, various systems in the body are involved and work together. muscular system has a more prominent role, the blood circulation as a means of transport will run more smoothly, the nervous system as a "relay system" which coordinates the body's system can work faster, so it is with the hormone could help in an optimal metabolism. by doing aerobic physical exercise is expected to serve local circulation of the oral cavity, including the tongue that contain many taste buds become more optimal.8 the purpose of this study was to determine whether aerobic physical exercise can increase the sweet taste sensitivity. it expected that exercise can be used as one more effort to dental caries prevention research due to decreased consumption of cariogenic materials. materials and methods the research is quasi experimental research conducted in the physiology laboratory faculty of medicine, airlangga university, surabaya. this study used 10 student subjects people faculty of dentistry airlangga university, male, aged 20-30 years who had the criteria of good general health condition (no history of systemic disease), oral cavity healthy condition and there are no lesions or abnormalities in the tongue and oral cavity, not smoking and drinking alcohol, height 165-170 cm with an ideal body weight. age 20-30 years sample set on the grounds of cardiovascular endurance can be increased to maximum. the male sample was chosen to eliminate hormonal influences on taste sensitivity. the subjects were measured the sweet taste sensitivity before doing aerobic physical exercise for the first time, then the subject using a belt heart rate and did aerobic physical exercise (up and down the bench) using intervals of 1:1, i.e. the subject up and down the bench for five minutes to the beat 80 times per minutes, followed by rest 5 minutes. activities carried out for 20 minutes, 3 times a week, for 8 weeks. at week 4 and 8 measured the sweet taste sensitivity. to measure the sweet taste sensitivity, subjects were instructed to use aquades rinse, then dried with a tissue of the tongue subjects. at the tip of the tongue of subjects, using a pipette drops of sucrose solution starting from the lowest concentration, i.e. 0.003 m. at every turn of the concentration of sucrose solution, subjects were instructed to rinse using aquades three times, then rested for 2 minutes.10 after that the tongue is dried using a tissue, and begin again drops a sucrose solution with a greater concentration (0.01 m: 0.013 m: 0.017 m: 0.022 m; 0.029 m).9 when the subject has felt the sweet taste, they were told to sign by raising their hand. measurement of the sweet taste sensitivity was conducted in the morning at 07.00 am according to circadian rhythms and hormonal changes of cortisol11 with instructions to the subject to sleep and last consumption after 22.00 pm, and the samples were told not take breakfast. the goal is for homogenization and to minimize the retrieval of psycoadaptation factors, such as people who drank sweet tea before will be less sensitive to sweet taste than people who previously drank water. results anova test was performed among groups which showed significant differences among groups before treatment, after 4 weeks, 8 weeks of physical exercise (figure 1). first data distribution was tested using kolmogorovsmirnov one sample test. the results of the test was that data distribution obtained were normal. then anova 37wardhani, et al.: changes of the sweet taste sense of taste sensitivity in humans, can be influenced by age, sex, food temperatures, local and systemic disease and the number of taste receptor cells. increasing age led to decreased taste sensitivity. this is due to the decreasing number of taste buds on the papillae fungiformis per square centimeter of surface area on the tongue and on the circumpalatinal papillae. the sensitivity of taste declines with degeneration of taste buds after 45 years old. in addition, the influence of age can cause decrease tone of muscle tongue, lining and tongue papillae disappear into atrophy. the gender difference of taste sensitivity indicates the sensitivity of taste in women is higher than men because women have more taste receptors.13 based on the results of research which has been conducted on 10 subjects, at a time before doing aerobic physical exercise, the subjects had a sweet taste sensitivity varies (above the threshold value of sweetness). at fourth week after doing aerobic physical exercise increased the sweet taste sensitivity than before doing aerobic exercise. at the eight week after doing aerobic exercise, the increased sweet taste sensitivity is more significant. physical exercise can provide a change in the function of body systems. increased heart rate during exercise is a response from the heart, but after a long practice then slowly the heart rate becomes stable because of the strength of heart muscle to pump blood increases. this is an adaptation of the heart to undertaken the physical exercise. the more heavy the physical activity performed during exercise, the greater the need for oxygen in the body. to compensate for this, the heart and circulatory system must work hard to give the needs of oxygen and nutrients that are increasing in the tissue, starting with the physiological changes and in a relatively long time will change the morphology consistently.14 in the blood circulation system, physical exercise can improve the use of capillary blood vessels. the impact of this situation will cause the increase of blood into the tissues that are active. increased blood volume and the number of red blood cells will occur anyway, which means increasing the capacity of the blood to bring oxygen.15 in the muscular system, regular physical exercise can increase muscle mass, because the exercise will stimulate muscle cells to grow larger and the muscle cells that initially breaks will become active again. supply of food and oxygen grow well too. disposal of co2 and lactic acid becomes more fluently. thick muscle fibers also increased due to energy reserves in the form of atp, phosphocreatine, and glycogen.16 in the respiratory system, people who exercise have a high endurance because the lungs have the ability to accommodate air 1ω times more than ordinary people (vo2 max). when doing training, the lungs can take in more oxygen, which means better blood circulation, and muscle cells get more oxygen from the capillary blood vessels.17 during exercise, the body requires fuel/ energy. the energy generated from glucose is then metabolized by the mitochondria produce atp. when the body requires test was performed between groups. the results obtained was significant between groups before treatment with 4 weeks after treatment and 8 weeks after treatment (table 2). table 1. result of different test of the sweet taste sensitivity between groups group group significance 1 2 .000 3 .000 2 1 .000 3 .015 3 1 .000 2 .015 description: group 1: the sweet taste sensitivity before physical exercise; group 2: the sweet taste sensitivity 4 weeks after physical exercise; group 3: the sweet taste sensitivity 8 weeks after physical exercise. there are signofocant differences in sweet taste sensitivity among groups before physical exercise, after 4 weeks, and 8 weeks of physical exercise (table 1). discussion taste is the main function of taste buds in the oral cavity. the function of taste allows humans to choose food according to his/her wishes and in accordance with the needs of the tissue about the substance of certain nutrients. taste cells continuously replaced by mitosis of the cells around it every 10–11 days. the cellular mechanism how human feel the sweet taste is as follows: the transmission of impulses of the senses of sweet taste through g protein complex which activates second messengers or the adenylyl cyclase to trigger the conversion of adenosine triphosphate (atp) to cyclic adenosine 3'5' monophosphate (camp). the available camp activates protein kinase a so there are phosphorylation of k ion channels. k ion channels close and the depolarization occurs so that neurotransmitter is released and the stimulation of sensory neurons occurs so that the sweetness be felt.11,12 before treatment after week 4th after week 8th 0.018 0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 v al ue figure 1. the average and standard deviation of sweet taste sensitivity before and after the fourth week, the eight week of exercise. 38 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 35–38 energy, atp will be disconnected and removed a single molecule to be adenosine diphosphate (adp). adp is still able to take off again into a single molecule phosphate adenosine mono phosphate (amp). amp then transduced by camp thus stimulating the entry of glucose into the muscle. because the body transfer of glucose from the blood into muscle cells, this process will reduce the amount of glucose in blood.18 at the time of exercise, glucose uptake for cell metabolism is not affected by insulin. after doing aerobic physical exercise, insulin plays a role in facilitating the re-entry of glucose into the cells. another thing that happens is that these cells become more sensitive to insulin because while doing physical exercise, glucose can enter on its own without the help of insulin. with the increase in insulin sensitivity, blood glucose uptake is increased and will decreased blood glucose levels automatically. the impact, regular exercise can improve the body's response to insulin and help insulin work more efficiently.14 with frequent exercise, 80% of maximal heart rate constantly and continuously will automatically adapt the heart muscle so that the strength of the heart in pumping blood will be more improved than before exercise.7 aerobic physical exercise can cause increased heart rate through two pathways, the first its through autonomic nervous system which then stimulates the sympathetic nervous system so that release of neurotransmitters nor epinephrine and the second pass through the hypothalamus which is the central receptacle of all information, then continue through the stimulation and release of anterior pituitary cortico releasing hormone (crh). crh affects the adrenal cortex to release adeno cortico tropic hormone (acth) which then affects the medulla of the adrenal glands to release adrenaline. increased heart rate will affect the capacity of blood that bring nutrients and oxygen so that oxygen will quickly get to the tissue. because the heart's performance and microcirculation is better so nutrients and oxygen supply to the cells of the taste bud adequate, by itself taste bud cells may be working according to their function well.11 the long-term effects of aerobic physical exercise can lead to increased regeneration of taste receptor cells. synthesis of receptor obtained from protein material. growth hormone (somatotropic hormone) can assist protein synthesis by increasing amino acid transport and stimulates the synthesis of ribosomal proteins. growth hormone increases amino acid transport across the cell membrane to the inside of the cell. this situation increases the concentration of amino acids in the cell and contribute to increased protein synthesis. growth hormone also stimulates the transcription of dna in the nucleus, thereby increasing the amount of rna formation. increased translation of rna led to the synthesis of protein by ribosomes. even when amino acids are not increased in the cell, growth hormone still stimulate increased translation of rna, causing enhance the amount of protein synthesized by ribosomes in the cytoplasm.18 increasing number of receptors followed by increased expression of taste receptor cells so that cells will make more responsive and can increase the sweet taste sensitivity. the process of formation of new taste receptor cells that normally takes place every 11 days will be happened faster, because it receives the supply of nutrients and oxygen from the blood optimally. this will increase the sweet taste sensitivity.11 when the senses of sweet taste is more sensitive due to aerobic physical exercise in a long time, this will decreased the consumption of glucose/ sucrose (cariogenic material). decrease in glucose consumption coupled with improving nutrition, improving oral hygiene, the provision of systemic or topical fluoride and fissure sealant is expected to reduce the incidence of caries. in addition to caries, of course, longterm physical exercise can also lower the risk of diabetes mellitus, but this certainly requires further research. it is concluded that erobic physical exercise for 8 weeks could led to increase the sweet taste sensitivity. references 1. supartono. healthy food menu. new york: scholastic press; 2005. p. 7. 2. mahardika. healthy mouth in the age of 30, 40, 50. available from: http://cyberman.cbn. net.id. accessed february 2, 2010. 3. kawuryan u. relationship awareness dental and oral health dental caries in children's genesis ii sdn kleco class v and vi district laweyan surakarta. available from: http://www.etd.eprints.ums. ac.id/897/j210040006.pdf. accessed may 10th, 2010. 4. bsmi-ydsf. healthy kartini title. available at: http://bsmi-surabaya. or.id. accessed may 10, 2010. 5. sundoro eh. new concept of caries treatment. faculty of dentistry, university of indonesia. available from: http://www.pdpersi.co.id/ show=detailnews&kode=107&tbl=artikel.jni. accesssed 18, 2010. 6. roeslan bo. barriers to dental caries occurrence after immunization with streptococcus mutans glucosyltransferase ina99 are applied in oral mucosa: study on types of wistar rats. dental caries research. available from: http://jrnal.dikti.go.id. accesed may 1st, 2010. 7. winarno f. benefits of exercise. available from: http://dunia olahraga.com. accessed january 10, 2010. 8. soerjodibroto w. sports nutrition. new york: pjo koni-fkui; 1999. p. 35–8. 9. g a n o n g w f . r e v i e w o f m e d i c a l p h y s i o l o g i c a l . 2 1 t h e d . s a n f r a n c i s c o : l a n g e m e d b o o k s / m c g r a w h i l l m e d publ div; 2005; p. 344-8, 514–27. 10. ariesty ds. sensitivity difference to the sweet taste sense taste smoker and non-smokers. thesis. surabaya: faculty of dentistry airlangga university; 2008. p. 38. 11. guyton ac, hall je. textbook of medical physiology. 11th ed. new york: wb saunders co; 2006. p. 419-29, 699–707. 12. ramos e, johanningsmeier sd, mcfeeters rf. the chemistry and physiology of taste. concise and hypothesis in food science. 2007; p. 36–8. 13. bowen r. taste sensation. available from: http://www.doctorsaloe. com/ oftaste physiology. accesced january 14, 2010. 14. willmore jh. athletic training and physical fitness. boston: allyn and bacon inc; 1997. p. 20–6. 15. vander a, sherman j, luciano d. human physiology, the mechanism of body function. san francisco: mcgraw hill; 2001. p. 219-21, 380, 452–60. 16. lersten, k. physiology and physical conditioning. california: peek publications; 1997. p. 56. 17. moeloek d. health and sports. faculty of medicine, university of indonesia. new york: publishing center of faculty of medicine; 1999. p. 7–23. 18. sherwood l. human physiology, from cell to system. 5th ed. united states: thomson brooks/ cole; 2004. p. 709–19. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2323 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 23–29 original article molecular docking study of zingiber officinale roscoe compounds as a mumps virus nucleoprotein inhibitor viol dhea kharisma1, santika lusia utami2, wahyu choirur rizky3, tim godefridus antonius dings4, md emdad ullah5, vikash jakhmola6, alexander patera nugraha7,8 1department of biology, faculty of mathematics and natural sciences, universitas brawijaya, malang, indonesia 2department of biology, faculty of biology, universitas gadjah mada, yogyakarta, indonesia 3college of medicine, sulaiman alrajhi university, albukayriyah, qassim, kingdom of saudi arabia 4college of medicine, maastricht university, maastricht, the netherlands 5department of chemistry, mississippi state university, mississippi, united states 6uttaranchal institute of pharmaceutical sciences, uttaranchal university, dehradun, india 7dental regenerative and biomaterial research group, faculty of dental medicine, universitas airlangga, surabaya, indonesia 8department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: mumps virus (muv) can trigger severe infections, such as parotitis, epididymo-orchitis, and meningitis. the effectiveness of muv vaccine administration has been proven, but current outbreaks warrant the development of antivirals against muv. zingiber officinale var. roscoe or ginger is often used as an alternative remedy. currently, there are no known in vitro or in vivo studies that investigate ginger as an muv antiviral. purpose: this study aims to evaluate the antiviral potency of the bioactive compounds in zingiber officinale var. roscoe against muv. methods: antiviral activity screening was conducted by druglikeness analysis, antiviral probability, molecular docking, and molecular dynamic simulation. results: as an antiviral, 6-shogaol from zingiber officinale var. roscoe has potency against muv. it has a good binding affinity and can establish interactions with the binding domain of the target protein by forming hydrogen, van der waals, and alkyl bonds. conclusion: the complex of 6-shogaol_np was predicted to be volatile but stable for triggering inhibitory activity. however, these results must be proved by in vivo and in vitro approaches to strengthen the scientific evidence. keywords: communicable disease; medicine; mumps; nucleoprotein; zingiber officinale article history: received 16 april 2022, revised 7 june 2022, accepted 25 july 2022 correspondence: alexander patera nugraha, department of orthodontics, faculty of dental medicine, universitas airlangga. jl. mayjen prof dr. moestopo no. 47 surabaya, 60132 indonesia. email: alexander.patera.nugraha@fkg.unair.ac.id introduction mumps virus (muv) belongs to the paramyxoviridae family. this virus causes acute generalized viral infection that is often prominently manifested as parotitis, a nonsuppurative swelling and tenderness of the salivary (parotid) glands, with unilateral or bilateral involvement of the glands. meningitis and epidydymo-orchitis represent the two most important extra-salivary manifestations of this infection.1 the administration of muv vaccine in children has been proven highly effective in suppressing the incidence of mumps. however, recent global outbreaks that especially affect the adult population warrant the discovery of an antiviral against muv.1 muv became an outbreak in america in june 2017 and in japan in july 2015 and affected 25,000 people.2,3 in the past, this virus was introduced by hippocrates (circa fifth century bc) and was referred to in his first book, book of epidemics. however, the mechanism of muv infection was only discovered in 1930 through experimental animal studies by johnson and goodpasture with koch’s postulate approach.4,5 muv contains replication enzymes, such as rnadependent rna polymerase (vrdrp) with large protein (l), nucleocapsid protein (np) or genome virus sheath, and phosphoprotein (p). these proteins have important roles as transcription and replication machines in muv.6 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 mailto:alexander.patera.nugraha@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 24 kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 the template that initiates the replication and synthesis of muv comes from the rna genome virus (vrna). subsequently, vrna can establish a complex with np that generates helix-shaped proteins or ribonucleoproteins (rnps) to avoid degradation.7 rnp can associate with p and l proteins to make vrdrp. the role of the l protein in this complex is to trigger the rna synthesis process, which consists of initiation, elongation, and termination.8 the np protein in muv plays a role in the formation of the vrna complex to avoid degradation and initiate the replication process. inhibition of the np protein’s activity will disturb the initiation of the replication process by increasing the degradation of vrna in muv. zingiber officinale var. roscoe or ginger is often used as an alternative medication for inflammation and diabetes and is also used as an antibacterial and antioxidant.9 some chromatography research studies show that zingiber officinale var. roscoe contains the bioactive compounds 6-shogaol, 12-gingediol, 4-gingerol, gingerdione, 6-gingediol, 8-gingerol, methyl-6-shogaol, zingerone, 10-gingerol, methyl-6-gingerol, and 6-gingerol.10 in vitro research shows that zingiber officinale var. roscoe has potency as an antiviral on vero cell-lines infected with chikungunya virus. the research shows that there is an increase in the viability of cells of about 51.0%.11 in vitro and in vivo research show that gingerone/gingerol can affect the replication of influenza-a virus by inhibiting the overexpression of type 1 and type 2 of the janus kinase protein. previous research has not used zingiber officinale var. roscoe to combat mumps infections. likewise, antiviral drugs derived from natural ingredients for mumps have not yet been discovered. hence, this research is essential to reveal the potency of the bioactive compounds in zingiber officinale var. roscoe that inhibit muv replication. materials and methods bioactive compounds from zingiber officinale var. roscoe that were used in this research consisted of 6-shogaol, 12gingediol, 4-gingerol, gingerdione, 6-gingerdiol, 8-gingerol, methyl-6-shogaol, zingerone, 10-gingerol, methyl-6gingerol, and 6-gingerol.10 the three-dimensional (3d) structure in .sdf file format, collision-induced dissociation (cid), formula, and the canonical simplified molecularinput line-entry system (smile) of each compound were retrieved from pubchem (https://pubchem.ncbi.nlm.nih. gov/). the 3d structures in .pdb format were retrieved under “ligand minimization” from openbabel v2.3.1. at the same time, the target protein in muv, which is an np (7ewq), was retrieved from rcsb pdb (https://www. rcsb.org/) (figure 1). the canonical smile from the bioactive compounds 6-shogaol, 12-gingediol, 4-gingerol, gingerdione, 6-gingediol, 8-gingerol, methyl-6-shogaol, zingerone, 10gingerol, methyl-6-gingerol, and 6-gingerol were used for druglikeness analysis with the swissadme web server (http://www.swissadme.ch/). lipinski’s “rule of five” plus the rules of ghose, veber, egan, and muege and a bioavailability score must be fulfilled by the compounds in order for them to be categorized as a drug-like molecule. this prediction aimed to determine the resemblance and the ability of the chemical compounds query with the drug that refers to the rules of druglikeness and shows general activity.12 bioactive compounds from zingiber officinale var. roscoe that were categorized as a drug-like molecule were identified as a likely antiviral with the pass web server (http://way2drug.com/passonline/). query compounds are considered to have a positive prediction if the probability figure 1. molecular docking visualization: (a) 6-shogaol (b) 12-gingediol (c) 4-gingerol (d) gingerdione (e) 6-gingediol (f) 8-gingerol (g) methyl-6-shogaol (h) zingerone (i) 10-gingerol (j) methyl-6-gingerol (k) 6-gingerol (l) muv np. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 https://pubchem.ncbi.nlm.nih https://www http://www.swissadme.ch/ http://way2drug.com/passonline/ https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 25kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 activation value (pa) is greater than 0.3, denoting that they are a good candidate for being an antiviral agent.13 molecular docking simulation between the bioactive compounds from zingiber officinale var. roscoe with muv np were conducted by pyrx v0.9.9 (scripps research, usa) with an academic license. the docking aims to determine the ligand activity with the target protein, referring to the binding affinity value (kcal/mol). the ligand with the most negative binding affinity is predicted to trigger specific biological activity on the target protein.14,15 three-dimensional visualization of the molecules from the docking results was conducted by pymol v2.5.2 (schrodinger inc., usa) with an academic license.16,17 the molecule complexes from the docking results with the most negative binding-affinity value were then identified by using discovery studio visualizer™ v16.1 (dassault systèmes se, france) for the chemical interactions and bonds. the types of chemical bonds that can be identified from the docking results are hydrogen, hydrophobic, alkyl, electrostatic, and van der waals.18 the molecular dynamic simulation in the molecule complex with the most negative binding-affinity value was conducted by the cabs-flex v2.0 web server (http:// biocomp.chem.uw.edu.pl/cabsflex2). the molecular dynamic analysis aimed to identify the interaction stability in the molecule complex by assigning a root-mean-squarefluctuation (rmsf) value. this interaction is considered stable if the molecule has an rmsf value below 3 å.19 results druglikeness acts as an important factor to identify the query compounds’ activity resemblance to drug molecules. the druglikeness prediction refers to rules proposed by lipinski, ghose, veber, egan, and muege and also a bioavailability score. in effect, the rules explain some physicochemistry that has to be satisfied by the query compounds, such as hydrogen-donor bonds, acceptor, molar refractivity, partition coefficient (log p), molecular weight, topological polar surface area (tpsa), atomic number, and rotatable bonds.20 the query compounds should have a minimum bioavailability value of 0.55 to be categorized as a drug-like molecule. compounds with that value will easily be absorbed by the body because of their good pharmacokinetics value.21 the results from this research show that all the bioactive compounds from zingiber officinale var. roscoe are considered collectively as a drug-like molecule because the compounds fit the druglikeness parameters (table 1). the activity prediction as an antiviral was conducted from the bioactive compounds that were considered as a drug-like molecule. the antiviral activity was predicted by the probability activation (pa) having a value above 0.3 (medium confidence) and probability inhibition (pi) that was not greater than pa. however, the prediction was general and was only a theoretical result and should be proved by further experimentation.13,22 this research shows table 1. druglikeness prediction results compounds druglikeness parameters lipinski ghose veber egan muege bioavailability score 6-shogaol yes yes yes yes yes 0.55 12-gingediol yes yes no yes no 0.55 4-gingerol yes yes yes yes yes 0.55 gingerdione yes yes yes yes yes 0.55 6-gingediol yes yes yes yes yes 0.55 8-gingerol yes yes no yes yes 0.55 methyl-6-shogaol yes yes yes yes yes 0.55 zingerone yes yes yes yes no 0.55 10-gingerol yes yes no yes no 0.55 methyl-6-gingerol yes yes no yes yes 0.55 6-gingerol yes yes yes yes yes 0.55 table 2. antiviral probability score compounds cid formula antiviral probability prediction resultpa pi 6-shogaol 5281794 c17h24o3 0.477 0.034 positive 12-gingediol 86196540 c23h40o4 0.644 0.004 positive 4-gingerol 5317596 c15h22o4 0.543 0.013 positive gingerdione 162952 c17h24o4 0.402 0.089 positive 6-gingediol 101660275 c17h28o4 0.644 0.004 positive 8-gingerol 168114 c19h30o4 0.553 0.012 positive methyl-6-shogaol 91721066 c18h26o3 0.498 0.025 positive zingerone 31211 c11h14o3 0.384 0.052 positive 10-gingerol 168115 c21h34o4 0.553 0.012 positive methyl-6-gingerol 70697235 c18h28o4 0.574 0.009 positive 6-gingerol 442793 c17h26o4 0.553 0.012 positive copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 http://biocomp.chem.uw.edu.pl/cabsflex2 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 26 kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 table 3. binding affinity from molecular docking simulation compounds cid molecular weight (g/mol) target binding affinity (kcal/mol) 6-shogaol 5281794 276.4 muv np -6.5 12-gingediol 86196540 380.6 muv np -6.3 4-gingerol 5317596 266.33 muv np -6.1 gingerdione 162952 292.4 muv np -6.0 6-gingediol 101660275 296.4 muv np -5.8 8-gingerol 168114 322.4 muv np -5.8 methyl-6-shogaol 91721066 290.4 muv np -5.6 zingerone 31211 194.23 muv np -5.5 10-gingerol 168115 350.5 muv np -5.3 methyl-6-gingerol 70697235 308.4 muv np -5.3 6-gingerol 442793 294.4 muv np -4.9 figure 2. molecular docking visualization: (a) 6-shogaol_np (b) 12-gingediol_np (c) 4-gingerol_np (d) gingerdione_np (e) 6-gingediol_np (f) 8-gingerol_np (g) methyl-6-shogaol_np (h) zingerone_np (i) 10-gingerol_np (j) methyl-6-gingerol_ np (k) 6-gingerol_np. figure 3. structural visualization and ligand–protein interactions of 6-shogaol_np—the three-dimensional structure was displayed by pymol v2.5.2 (schrödinger inc., usa) with an academic license and visualizer™ v16.1 for molecular interaction copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 27kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 that all bioactive compounds from zingiber officinale var. roscoe have positive prediction as an antiviral with a pa value above 0.3 (table 2). molecular docking aims to predict the binding activity of the ligand with the target protein, pattern of interaction at the domain protein, and ligand activity.17 the determination of the inhibition activity of a ligand on the target protein can be predicted from the molecular docking method. grids from the docking method are used to direct the ligand to the specific domain on the target protein, especially on the aimed domain.23 this research used the bioactive compounds from zingiber officinale var. roscoe as the ligand and muv np as the target protein. the autogrid positions for the molecular docking simulation were center (å) x: 132.028 y: 224.440 z: 160.519 and dimensions (å) x: 76.725 y: 48.911 z: 71.149 with the grid covering the entire target-protein domain. the molecular-docking result shows that 6-shogaol had a binding affinity value that was more negative than the other compounds, that is, 6.5 kcal/ mol at muv np (table 3). hence, 6-shogaol is predicted to have stronger bonds than the other compounds and therefore trigger inhibitory activity at muv np. the 3d visualization of the molecular-docking result is shown as the transparent surface structure, corkscrew-like objects, lines, and color selection (figure 2). the molecular-docking result of 6-shogaol_np complex was then analyzed based on the position and types of the chemical bonds that were formed on that complex. a weak molecular interaction is formed when the ligand molecule interacts with the specific domain of the target protein.24 the interaction consists of van der waals, hydrogen, alkyl, hydrophobic, and electrostatic effects that act to trigger the biological response of some proteins. the hydrogen, hydrophobic, van der waals, and alkyl bonds that are formed in drug molecules lead to increased stability and bond strength.18 when 6-shogaol interacts with the muv np’s domain, 6-shogaol can form hydrogen, van der waals, and alkyl interactions (figure 3). the chemical interaction stability that was formed on the 6-shogaol-np complex in this research can be predicted with molecular dynamic (md) simulation. md analysis aims to identify the interaction stability in the molecule complex with reference to the root-mean-square-fluctuation value (rmsf). the chemical interaction in this molecule complex that is formed can be considered stable because the interactions should have an rmsf value below 3 å. the rmsf value of the 6-shogaol binding domain in muv np consisted of phe107 (0.434 å), thr111 (1.546 å), glu108 (0.722 å), pro109 (1.385 å), pro156 (2.354 å), tyr49 (0.219 å), arg57 (0.227 å), glu153 (1.320 å), asn53 (0.254 å), gly110 (1.830 å), gln50 (0.295 å), cys157 (2.671 å), thr46 (0.541 å), and tyr112 (0.898 å) according to the cabs-flex 2.0 server (http://biocomp. chem.uw.edu.pl/cabsflex2/job/20ed7c1748559f0/). the molecule complex of 6-shogaol_np is considered stable because it has an rmsf value below 3 å (figure 4). discussion muv belongs to the paramyxoviridae family. this virus causes an acute generalized viral infection that is often prominently manifested as parotitis, a nonsuppurative swelling and tenderness of salivary (parotid) glands, figure 4. molecular dynamic simulation results—the values of rmsf have fluctuations and are plotted against the 6-shogaol_np complex-residue index copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 http://biocomp https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 28 kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 with unilateral or bilateral involvement of the glands. meningitis and epidydymo-orchitis represent the two most important extra-salivary manifestations of this infection.1 muv has replication enzymes, such as rna-dependent rna polymerase (vrdrp) with large protein (l), np or genome virus sheath, and phosphoprotein (p). these proteins have important roles as transcription and replication machines in muv.6 np protein in muv plays a role in the formation of the vrna complex to avoid degradation and initiate the replication process. inhibition of the np protein’s activity will disturb the initiation of the replication process through an increase of degradation of vrna in muv. druglikeness prediction refers to rules used by lipinski, ghose, veber, egan, and muege as well as a bioavailability score. essentially, the rules explain some physicochemistry that has to be fulfilled by the query compounds, such as hydrogen donor bonds, acceptor, molar refractivity, partition coefficient (log p), molecular weight, topological polar surface area (tpsa), atomic number, and rotatable bonds.20 the results from this research showed that all the bioactive compounds from zingiber officinale var. roscoe could collectively be considered a drug-like molecule. molecular docking aims to predict the binding activity of the ligand with the target protein, the pattern of interaction at the domain protein, and ligand activity.17 this research used bioactive compounds from zingiber officinale var. roscoe as the ligand and muv np as the target protein. the molecular-docking results show that 6-shogaol is predicted to have stronger bonds than the other compounds and could trigger inhibitory activity at muv np. a weak molecular interaction is formed when the ligand molecule interacts with the specific domain of the target protein.24 when 6-shogaol interacts with the muv np’s domain, 6-shogaol can form hydrogen, van der waals, and alkyl interactions. the chemical interactions in this molecule complex can be considered stable if they have an rmsf value below 3 å.19 because it has an rmsf value below 3 å, 6-shogaol-np is considered stable. the compound from zingiber officinale var. roscoe, 6-shogaol, has potency as an antiviral for muv because it has a binding affinity that is more negative than the others and can form hydrogen, van der waals, and alkyl bonds in the target protein’s binding domain. the complex of 6-shogaol_np fluctuates but is stable and can trigger inhibitory activity at the target. however, this research must be proved and further explored by in vivo and in vitro approaches to strengthen the scientific evidence. acknowledgement the authors would like to thank publication center of faculty of dental medicine, universitas airlangga for the support. references 1. zumla a. mandell, douglas, and bennett’s principles and practice of infectious diseases. lancet infect dis. 2010; 10(5): 303–4. 2. matsui y, kamiya h, kamenosono a, matsui t, oishi k, kidokoro m, sunagawa t. first mumps outbreak in a decade: measuring impact of mumps among naïve population—tokunoshima island, japan. open forum infect dis. 2017; 4(suppl_1): s243–s243. 3. marlow m, even s, hoban mt, moore k, patel m, marin m. universities’ experience with mumps outbreak response and use of a third dose of mmr vaccine. j am coll health. 2021; 69(1): 53–8. 4. rubin s, eckhaus m, rennick lj, bamford cgg, duprex wp. molecular biology, pathogenesis and pathology of mumps virus. j pathol. 2015; 235(2): 242–52. 5. kaur rj, charan j, dutta s, sharma p, bhardwaj p, sharma p, lugova h, krishnapillai a, islam s, haque m, misra s. favipiravir use in covid-19: analysis of suspected adverse drug events reported in the who database. infect drug resist. 2020; 13: 4427–38. 6. briggs k, wang l, nagashima k, zengel j, tripp ra, he b. regulation of mumps virus replication and transcription by kinase rps6kb1. j virol. 2020; 94(12): e00387-20. 7. cox r, green tj, purushotham s, deivanayagam c, bedwell gj, prevelige pe, luo m. structural and functional characterization of the mumps virus phosphoprotein. j virol. 2013; 87(13): 7558–68. 8. kubota m, hashiguchi t. unique tropism and entry mechanism of mumps virus. viruses. 2021; 13(9): 1746. 9. mao q-q, xu x-y, cao s-y, gan r-y, corke h, beta t, li h-b. bioactive compounds and bioactivities of ginger (zingiber officinale roscoe). foods (basel, switzerland). 2019; 8(6): 185. 10. dibha a f, wa hy un ingsi h s, k ha r isma v d, a nsor i a n m, widyananda mh, pa r ikesit aa, rebezov m, matrosova y, artyukhova s, kenijz n, kiseleva m, jakhmola v, zainul r. biological activity of kencur (kaempferia galanga l.) against sars-cov-2 main protease. int j health sci (qassim). 2022; 6(s1): 468–80. 11. kaushik s, jangra g, kundu v, yadav jp, kaushik s. anti-viral activity of zingiber officinale (ginger) ingredients against the chikungunya virus. virusdisease. 2020; 31(3): 270–6. 12. banerjee r, perera l, tillekeratne lmv. potential sars-cov-2 main protease inhibitors. drug discov today. 2021; 26(3): 804– 16. 13. ramadhani nf, nugraha ap, rahmadhani d, puspitaningrum ms, rizqianti y, kharisma vd, noor tnebta, ridwan rd, ernawati ds, nugraha ap. anthocyanin, tartaric acid, ascorbic acid of roselle flower (hibiscus sabdariffa l.) for immunomodulatory adjuvant therapy in oral manifestation coronavirus disease-19: an immunoinformatic approach. j pharm pharmacogn res. 2022; 10(3): 418–28. 14. wijaya rm, hafidzhah ma, kharisma vd, ansori anm, parikesit aa. covid-19 in silico drug with zingiber officinale natural product compound library targeting the mpro protein. makara j sci. 2021; 25(3): 162–71. 15. luqman a, kharisma vd, ruiz ra, götz f. in silico and in vitro study of trace amines (ta) and dopamine (dop) interaction with human alpha 1-adrenergic receptor and the bacterial adrenergic receptor qsec. cell physiol biochem. 2020; 54(5): 888–98. 16. proboningrat a, kharisma vd, muhammad ansori an, rahmawati r, fadholly a, ann villar posa g, sudjarwo sa, abdul rantam f, budianto achmad a. in silico study of matural inhibitors for human papillomavirus-18 e6 protein. res j pharm technol. 2022; 15(3): 1251–6. 17. husen sa, ansori anm, hayaza s, susilo rjk, zuraidah aa, winarni d, punnapayak h, darmanto w. therapeutic effect of okra ( abelmoschus esculentus moench) pods extract on streptozotocininduced type-2 diabetic mice. res j pharm technol. 2019; 12(8): 3703. 18. nugraha ap, rahmadhani d, puspitaningrum ms, rizqianti y, kharisma vd, ernawati ds. molecular docking of anthocyanins and ternatin in clitoria ternatea as coronavirus disease oral manifestation therapy. j adv pharm technol res. 2021; 12(4): 362–7. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 29kharisma et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 23–29 19. susanto h, kharisma vd, listyorini d, taufiq a, sunaryono, aulanni a. effectivity of black tea polyphenol in adipogenesis related igf-1 and its receptor pathway through in silico based study. j phys conf ser. 2018; 1093: 012037. 20. ansori a, kharisma v, parikesit a, dian f, rebezov m, scherbakov p, burkov p, zhdanova g, mikhalev a, antonius y, pratama m, sumantri n, sucipto t, zainul r. bioactive compounds from mangosteen (garcinia mangostana l.) as an antiviral agent via dual inhibitor mechanism against sarscov2: an in silico approach. pharmacogn j. 2022; 14(1): 85–90. 21. cabrera n, cuesta sa, mora jr, calle l, márquez ea, kaunas r, paz jl. in silico searching for alternative lead compounds to treat type 2 diabetes through a qsar and molecular dynamics study. pharmaceutics. 2022; 14(2): 232. 22. prahasanti c, nugraha ap, kharisma vd, ansori anm, ridwan rd, putri tps, ramadhani nf, narmada ib, ardani igaw, noor tneba. a bioinformatic approach of hydroxyapatite and polymethylmethacrylate composite exploration as dental implant biomaterial. j pharm pharmacogn res. 2021; 9(5): 746–54. 23. h a r t a t i f k , d ja u h a r i a b, viol d h e a k . eva lu a t io n of pharmacokinetic properties, toxicity, and bioactive cytotoxic activity of black rice (oryza sativa l.) as candidates for diabetes mellitus drugs by in silico. biointerface res appl chem. 2021; 11(4): 12301–11. 24. kharisma vd, ansori anm, fadholly a, sucipto th. molecular mechanism of caffeine-aspirin interaction in kopi balur 1 as antiinflammatory agent: a computational study. indian j forensic med toxicol. 2020; 14(4): 4040–6. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p23–29 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p23-29 3535 original article silicone loop alternative for posterior bitewing radiography shinta amini prativi, shanty chairani and tyas hestingsih department of oral and maxillofacial radiology, faculty of medicine, universitas sriwijaya, palembang – indonesia abstract background: bitewing radiography is a technique that depicts the crown of the maxillary and mandibular teeth and the crest of the alveolar bone in the same receptor. the use of film holders and paper loops in bitewing techniques is very helpful in standardising radiographs, but it has some disadvantages, including the lack of efficiency and discomfort. therefore, silicone has been widely used in the medical field as a replacement for paper loops. purpose: this study was conducted to describe the compatibility of the silicone material as an alternative for bitewing radiography. methods: this research is experimental and a one-shot case study. it used the mann–whitney (p < 0.05) test for statistical analysis to compare the results of the radiographs using silicone loops and paper loops and to analyse the quality of each radiograph: object coverage, density, contrast, sharpness, geometry, and overlapping. results: the images where silicone loops were used show adequate results in six radiograph quality assessments. there was no significant difference between the radiographs that were obtained using the silicon loop and the paper loop (p > 0.05). conclusion: silicone loops can be an alternative tool for bitewing radiography because they result in optimal image quality. keywords: bitewing radiographs; quality images; silicone correspondence: shinta amini prativi, department of oral and maxillofacial radiology, faculty of medicine, universtias sriwijaya. jalan palembang-prabumulih, km 32 inderalaya 30662, indonesia. email: shintaaminiprativi@fk.unsri.ac.id introduction bitewing radiography depicts the crowns of the maxillary and mandibular teeth and the crest of the alveolar bone in the same receptor.1 this technique is particularly used to detect interproximal caries in their early stages of development before they become clinically visible, sees secondary caries under restoration, assesses this restoration, evaluates periodontal conditions and detects the calculus in the interproximal area.1–4 the procedure for taking bitewing radiographs requires the patient to bite a small wing that is placed on an intraoral film, film holder, wing tab or paper loop.1,5 however, the use of film holders has disadvantages: it is expensive and less convenient than film holder.1,2 the use of paper loops has several disadvantages too, as the movement of the tongue can cause the film to move, which negatively affects the quality of the obtained images. this results in repeated radiographs, which are very detrimental to the patient. in addition, using the paper loop only once makes it inefficient.2,3,6,7 based on kositbowornchai et al.’s research, the loop technique was 1.11 times more likely to cause overlapping than the holder technique.8 the use of silicone is common in the medical field. silicone is biocompatible, durable and hydrophobic, it has low surface pressure and toxicity, as well as good chemical and thermal stabilities. silicone can be used at high temperatures, so it can be sterilised using repeated autoclaves without any changes.9 the purpose of this study is to provide an alternative tool, especially to bitewing radiography, that can be used in the field of dental radiology and that can produce optimal radiographic images. materials and methods this research is experimental and a one-shot case study. the research design consisted of a group of people who were given a treatment; the results were then observed. the first procedure included making the loop using wax dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p35–38 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 35–38 mailto:shintaaminiprativi@fk.unsri.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p35-38 36 prativi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 35–38 and the mould using a dental stone. once the mould was ready, and after boiling off the wax, a silicone loop was made by mixing the silicone and the catalyst, then putting the mix in the mould and letting it stand until the silicone had set (figure 1).10 the next step was to do the bitewing radiography using silicone and paper loops. based on the results of the sample size calculation using the federer sample calculation, the researcher used six samples of each bitewing radiographs using a silicone loop and a paper loop.11 the film in a silicone or paper loop was placed on the lingual side of the phantom that was implanted with ten natural teeth, and the anterior edge of the film was distal to the mandibular canine. the bite portion of the loop was in the interocclusal space where the phantoms were occluded. the x-ray tube positioned towards the film passed through the interproximal space of the premolar and molars (figure 2).1–3 the researchers used aprons, and the x-rays were exposed using the phot x ii 303-h (belmont, usa) x-ray machine with 70 kvp and 7 ma and a time of 0.35 second. after being exposed, the film was processed. if the assessment of the bitewing radiograph quality with the silicone loop is said to be good, the next step will table 1. the mann–whitney test results. parameter silicone loop paper loop total p-value coverage object no 1 (100.0%) 0 (0.0%) 1 (100.0%) 0.317 good 5 (45.5%) 6 (54.5%) 11 (100.0%) density no 1 (2.0%) 4 (80.0%) 5 (100.0%) 0.093 good 5 (71.4%) 2 (28.6%) 7 (100.0%) contrast good 6 (50.0%) 6 (50.0%) 12 (100.0%) 1.000 no 0 (0.0%) 0 (0.0%) 0 (0.0%) sharpness good 4 (40.0%) 6 (60.0%) 10 (100.0%) 0.138 no 2 (100.0%) 0 (0.0%) 0 (0.0%) geometry good 6 (50.0%) 6 (50.0%) 12 (100.0%) 1.000 no 0 (0.0%) 0 (0.0%) 0 (0.0%) overlapping yes 0 (0.0%) 2 (100.0%) 2 (100.0%) 0.138 no 6 (60.0%) 4 (40.0%) 10 (100.0%) (a) (b) figure 3. the resulting bitewing radiographs with the silicone loop (a) and paper loop (b). (a) (b) (c) (d) figure 1. the process of making silicone loops: (a) making a loop wax; (b) the mould stone after boiling out the wax; (c) the silicone paste injected into the mould; (d) the silicone loop. figure 2. the process of making bitewing radiography with the silicone loop. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p35–38 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p35-38 37prativi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 35–38 be to compare the quality of the radiograph using a paper loop. the resulting bitewing radiographs were analysed for quality: object coverage, density, contrast, sharpness, geometry and overlapping.1–3 in this study, a comparative test analysis was used to calculate whether there were significant differences in the results obtained by using these different tools. the mann–whitney test (p < 0.05) was used because the data was not normally distributed.12 results the test was carried out to determine whether the tool that has been made is appropriate to be used as an aid in taking bitewing radiographs, which were reviewed based on the resulting images (figure 3). the parameters to be tested included object coverage, density, contrast, sharpness, geometry and overlapping. the following are the calculation results of the mann–whitney test for the six tested parameters. based on the comparison test recapitulation in table 1, it can be seen that all the parameters show no significant differences between the radiographs that were obtained using the silicon loop and the paper loop. this can be seen from the p-values of all the parameters, which are greater than 0.05. this means that these two tools have almost the same capacity in taking bitewing radiographs, which were reviewed based on the resulting images. discussion the silicone material can be used as an alternative for making bitewing loops to solve the shortage of paper loops. it is a supported silicone material that has been used in medical practices for over 60 years. silicone elastomer bases can meet the food-grade standards. they constitute soft and durable material for medical devices, medical adhesives (sealants), mould making and prototyping and on-site healing gaskets.13,14 in addition to the tested silicone material, the results of the images from using this silicone loop can be considered positive based on the six assessments that have been made. the radiograph quality assessments show that the resulting image when using a silicone loop based on the first assessment has a poor coverage object. the image should include the crown of canines, premolars, both maxillary and mandible molars and no cone cutting. however, the results show that the canine teeth are not in the image; this is because the canines are located in the arch, which makes it difficult to place the film. this is supported by emanuel’s research, which stated that 71% of the errors in bitewing radiographs were related to film placement.15 table 1 shows that the resulting image when using a silicone loop produces a brighter density than the paper loop. the density of a radiograph is influenced by the exposure, thickness and density of the object. the presence of a silicone loop will cause the x-rays to be slightly absorbed by the silicone and the object, resulting in a change in the density of the image.16 both images provide equal contrast and geometry qualities. kositbowornchai et al.’s research compared the in vivo bitewing film quality using the holder versus the paper loop techniques.8 the results showed that the qualities of the bitewing films taken by the loop and holder techniques were not significantly different.8 the contrast quality is influenced by the contrast of the object and film, x-rays and scattered radiation. this can be controlled by using the same object and film and setting the same kvp, ma and time.15 the distance of the x-rays from the objects and films when taking radiographs and the vertical and horizontal angulations were carried out correctly.2 the use of silicone loop tools did not affect the radiograph’s geometric accuracy, which is one of the most important assessments of the quality of a radiograph. the size of the natural teeth is the same as the teeth in the radiograph.1,2,16 sinpitaksakui et al. compared the quality of bitewing radiographs using paper wings and loops and the xcp instrument (rinn).17 the results showed that there were no differences in the distortion and blurred images between the two techniques.17 apart from the results of the adequate radiograph quality, the design of the silicone loop has other advantages: the silicone material is elastic and soft, and silicone is environmentally friendly. this study has two limitations: the experiment was not performed in an actual oral cavity and the assessment of the radiograph quality was visual. further research is needed in order to test silicone loops on humans to prove that silicone loops are comfortable and easily placed in the oral cavity. in conclusion, based on the radiograph quality analysis (object coverage, density, contrast, sharpness, geometry and overlapping), silicone loops can be used as an alternative tool for taking bitewing radiographs because they result in optimal image quality. acknowledgement this work is supported by the fund of dipa, faculty of medicine, universitas sriwijaya, no. 023.17.2.677515/2020. references 1. white sc, pharoah mj. oral radiology: principles and interpretation. 7th ed. st. louis: mosby; 2013. p. 154. 2. whaites e, drage n. essentials of dental radiography and radiology. 5th ed. philadelphia: churchill livingstone; 2013. p. 120. 3. iannucci j, howerton lj. dental radiography: principles and techniques. 4th ed. st. louis: saunders; 2011. p. 197. 4. dixon d, hildebolt c. an overview of radiographic film holders. dentomaxillofacial radiol. 2005; 34(2): 67–73. 5. thomson em, johnson on. essentials of dental radiography for dental assistants and hygienists. 10th ed. london: pearson; 2018. p. 169. 6. safi y, esmaeelinejad m, vasegh z, valizadeh s, aghdasi mm, sarani o, afsahi m. utility of a newly designed film holder for dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p35–38 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p35-38 38 prativi et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 35–38 premolar bitewing radiography. j clin diagnostic res. 2015; 9(11): tc04-7. 7. jørgensen pm, wenzel a. patient discomfort in bitewing examination with film and four digital receptors. dentomaxillofac radiol. 2012; 41(4): 323–7. 8. kositb owor ncha i s, pha d a n norg t, pe r mp o on si n so ok m, thinkhamrop b. bitewing film quality: a clinical comparison of the loop vs. holder techniques. quintessence int (berl). 2004; 35(4): 321–5. 9. rahimi a, mashak a. review on rubbers in medicine: natural, silicone and polyurethane rubbers. plast rubber compos. 2013; 42(6): 223–30. 10. apriantoro nh, mayarani m, karmawati iaka. product design of film dental holder “bitewing” for anterior dental radiography. sanitas j teknol dan seni kesehat. 2017; 8(2): 123–33. 11. dahlan ms. besar sampel dan cara pengambilan sampel dalam penelitian kedokteran dan kesehatan. 3rd ed. jakarta: salemba medika; 2013. p. 68–9. 12. dahlan ms. statistik untuk kedokteran dan kesehatan. 6th ed. jakarta: epidemiologi indonesia; 2014. p. 83. 13. wacker. solid and liquid silicone rubber material and processing guidelines. munich: wacker chemie ag; 2014. p. 14. 14. sardar vb, rajhans nr, pathak a, prabhu t. development in silicone mater ial for biomedical applicationsa review. in: 14th international conference on humanizing work and work environment. punjab, india; 2016. p. 14. 15. emanuel rj. a retrospective audit on the quality of periapical and bitewing radiographs taken in a primary care setting. qual prim care. 2003; 11: 305–8. 16. mallya sm, lam ewn. white and pharoah’s oral radiology: principles and interpretation. 8th ed. st. louis: mosby; 2018. p. 21. 17. sinpitaksakul p, tantisiriwat a, chongcharoen n. bitewing quality using paper wing and loop and xcp instrument. in: southeast asian division meeting. koh samui, thailand; 2004. p. 1. id95. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p35–38 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p35-38 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods 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... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dental medicine, universitas airlangga editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (+6231) 5039478 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg i wish to subscribe dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) name/nama: .......................................................................... date of birth/tanggal lahir: .................................................... job title/pekerjaan: ................................................................ institution/institusi: 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surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " mkgs vol 44 no 2 april-juni 2011.indd 72 vol. 44. no. 2 june 2011 elderly nutritional status effection salivary anticandidal capacity against candida albicans ria puspitawati, nurtami soedarsono, elisabeth a putri, anissha d putri, and boy m bachtiar department of oral biology faculty of dentistry, university of indonesia jakarta indonesia abstract background: elderly often suffer malnutrition and oral candidiasis. candida albicans (c. albicans) which is the most prominent cause of oral candidiasis, is one of commensal oral micro-flora. nutritional status affect the characteristic of saliva. saliva is the regulator in the development of c. albicans from comensal into pathogen. purpose: the purpose of this study was to determining the correlation between elderly nutritional status with salivary total protein and its activity in inhibiting c. albicans growth and biofilm formation. methods: using mini nutritional assessment 30 elderly were classified into normal and malnutrition groups. total protein of unstimulated saliva was measured using bradford protein assay. the colony forming unit (cfu) of c. albicans was counted on 72 hours on sda cultures without (control) or with 2 hour saliva exposure. biofilm formation was analyzed from the optical density of 10 –5 c. albicans suspension without saliva exposure (control) or with exposure of 10.000 μg/ml saliva and incubated in 37° c for 2 days. the suspension was put into 96 well plates, stained with crystal-violet dye, and analyzed using microplate reader. differences between groups were analyzed using independent t-test or kruskall-wallis. correlation between variables was analyzed using spearman test. results: salivary total protein of normal elderly (1.113.5 ± 1.1143.3) was higher than those of malnutrition (613.6 ± 253.6) but not statistically significant (p > 0.05). the cfu of c. albicans exposed to saliva of normal samples (2.060 cfu/ml) was significantly lower than control (24.100 cfu/ml) and those exposed to malnutrition saliva (5.513.3 cfu/ml). c. albicans biofilm formation is highest in controls (0.177), lower in those exposed to malnourished saliva (0.151) and lowest in those exposed to saliva of good nourished elderly (0.133). conclusion: although does not cause significant decrease of salivary total protein, malnutrition in elderly results in lower capacity of saliva in inhibiting the growth and declining the virulence of c. albicans. key words: candida albicans, elderly, malnutrition, saliva abstrak latar belakang: lansia sering menderita malnutrisi dan kandidiasis oral. candida albicans yang merupakan penyebab utama terjadinya candidiasis, adalah salah satu mikroflora rongga mulut yang bersifat konvensional. malnutrisi memengaruhi karakteristik saliva. saliva merupakan regulator utama perkembangan candida albicans (c. albicans) dari sifat konvensional menjadi bersifat patogen.tujuan: tujuan dari penelitian ini adalah untuk menentukan korelasi antara status gizi lansia dengan total protein dan aktivitas saliva dalam menghambat pertumbuhan dan pembentukan biofilm c. albicans. metode: menggunakan mini nutritional assessment, 30 lansia diklasifikasikan menjadi kelompok gizi baik dan gizi buruk. total protein unstimulated saliva diukur dengan metode bradford protein assay. colony forming unit (cfu) dihitung pada kultur c. albicans pada saburaud dextrose agar (sda) berusia 72 jam yang sebelumnya telah dipaparkan saliva selama 2 jam. kontrol adalah kultur c. albicans tanpa paparan saliva. pembentukan biofilm adalah pengukuran optical density suspensi 10 –5 c. albicans tanpa paparan saliva (kontrol) atau dengan paparan saliva 10.000 μg/ml dan diinkubasi pada suhu 37° c selama 2 hari. suspensi tersebut kemudian dimasukkan ke dalam 96 well plates, diberi pewarna crystal violet, dan diukur menggunakan microplate reader. analisis data menggunakan uji beda t independen atau kruskall-wallis, dan uji korelasi spearman. hasil: total protein saliva lansia gizi baik (1.113,5 ± 1.1143,3) lebih tinggi dari lansia gizi buruk (613,6 ± 253,6) tetapi tidak bermakna secara statistik (p > 0,05). pembentukan koloni c. albicans yang terpapar saliva lansia gizi baik (2.060 cfu/ml) secara signifikan lebih rendah dari kontrol (24.100 cfu/ml) dan daripada yang terpapar saliva lansia gizi buruk (5.513,3 cfu/ml). pembentukan biofilm c. albicans tetinggi pada kontrol (0,177), lebih rendah pada research report 73puspitawati et al.: elderly nutritional status affect yang terpapar saliva gizi buruk (0,151) dan terrendah pada yang terpapar saliva gizi baik (0,133). kesimpulan: meskipun malnutrisi tidak menyebabkan penurunan total protein saliva lansia, tetapi menurunkan kapasitasnya dalam menghambat pertumbuhan dan mengurangi virulensi c. albicans. kata kunci: candida albicans, lansia, malnutrisi, saliva corespondence: ria puspitawati, c/o: departemen biologi oral, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat, indonesia. e-mail: riapuspitawati@yahoo.com introduction elderly is prone to malnutrition mainly due to three ethiological factors. firstly, medical factors such as decreased apatite, malabsorbtion, disturbances of oro-dental tissue, taste, and smell sensation, presence of infection, systemic disturbances, and medicine side effects. second factor includes social condition such as poverty, social isolation, and decreased ability to provide meal. third factor is the psychosocial factors such as depression and dementia.1,2 disturbances of nutrition intake will eventually lead to alteration of nutritional status. poor nutrition in elderly could result in decreased ability in responding to any physiological challenge and decreased imunity which will be reflected as susceptibility in healing process and in resistence to infection.2 malnutrition results in decreased ability of immune system in protecting oral tissue from infection. oral infection commonly suffered by elderly is candidiasis. common predisposing factors of oral candidiasis in elderly include decreased immunity, presence of chronic disease, long term antibiotic intake, poor oral hygiene, decreased salivary flow rate, and malnutrition.3,4 presence of denture is another common condition in elderly which could trigger the occurence of oral candidiasis. a significant correlation between malnutrition and oral candidiasis was reported.5 the presence of oral candidiasis is usually causes annoying pain which disturbs mastication function and aggravate the existed malnutrition.6,7 candida albicans (c. albicans) which is the most prominent cause of oral candidiasis, is one of commensal oral micro-flora. the behaviour and expression of this yeast is correlated to decreased immune system of the host, as found in elderly and malnutrition subject.5,8,9 besides the equilibrium between the yeast and other oral microbes, c. albicans distribution and virulence in oral cavity are also influenced by its adhession capability, proteolytic enzymes production, and hyphal formation.10,11 one defence mechanism of candida spp. against the anti candida components of immune system in saliva is biofilm formation. the development of biofilm is related to the aggrevation of clinical infection on the host cell. in oral cavity, c. albicans could adhere and form biofilm on the surfaces of dental and oral mucous tissue or on denture base.11,12 in oral cavity, saliva has role in affecting the existence, composition, and behaviour of the microflora via clearence effect mechanism. in such mechanism, saliva mediating the activity of molecules responsible as the anti-microorganism or anti fungal of oral immune system.13 saliva is the main regulator in the development of c. albicans from comensal into pathogen. such regulatory function is especially determined by the salivary protein composition. salivary proteins known to have anti-candida capacity are siga, defensin, histatin, lactoferin, lisozim, and mucin.10,14,15 adequate nutrion intake influencing the quantity and the biological activity of saliva and affect its effectivity as ecological equilibrium regulator in oral cavity.16 various previous studies reported decreased protein components concentration of saliva in protein-calorie malnutrition cases.2,9,15-17 among two methods commonly used in measuring protein concentration, bradford assay is considered to be the simpler (could be read in 5 minutes) and more sensitive method compared to lowry method. in bradford protein assay, the concentration of protein measured is based on the formation of complex formed by brilliant blue dye and protein in the sample. the presence of protein-dye complex results in alteration of maximum absorption of the dye when read using 465–595 nm absorbance length. the determination of total protein concentration of sample is based on the comparison with the concentration of standard protein (bovine serum albumin).18 the objective of this study was to determine the correlation between elderly nutritional status with salivary total protein and its activity in inhibiting c. albicans growth and biofilm formation. materials and methods subject of this study were elderly aged 65–80 who did not consume anitibiotic/anti virus during the last 3 months, not having history of dm, hiv infection/aids, or malignancy, able to communicate, and willing to participate in the project by signing the informed consent. nutritional status of the samples were determined by using mini nutritional assessment (mna) developed by nestle. from this assessment subjects were classified into poor nutrition (< 18.5) and good nutrition (18.5–25) groups. fifteen mililiters unstimulated saliva was collected from each subject, placed in cooled closed tubes, centrifuged at 10.000 rpm for 5 minutes. the supernatant was kept in 74 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 72–76 –20° c until used. total protein of sample’s saliva was analyzed using bradford protein assay and read using microplate reader at 490 nm absorbance wave length.18 c. albicans strain atcc 10231 obtained from dept. microbiology, faculty of medicine, universitas indonesia was used in this study. c. albicans was cultured in sda at room temperature for 72 hours, and diluted into 10–3 suspension. the inhibition effect of saliva against c. albicans growth was analyzed by counting the colony forming unit (cfu) of 10–3 c. albicans suspension cultured on saburaud dextrose agar (sda) for 72 hours at room temperature. comparison were made between the cfu of c. albicans which previously been exposed to saliva of good-nutrition elderly, or to saliva of poor-nutrition elderly, or not exposed to saliva (control). saliva exposure on c. albicans was conducted by mixing 10 μl of 10–3 c. albicans suspension with 50 μl of 10.000 μg/ml diluted saliva, and incubated in 37° c waterbath. after 2 hours the mixture was diluted to make 10–3 solution from which 10 μl was cultured on sda at room temperature for 72 hours. the inhibition effect of saliva against c. albicans biofilm formation was analyzed by measuring the optical density of 10–5 c. albicans suspension which previously been exposed to 10.000 μg/ml saliva and incubated in 37° c for 2 days. the suspension was put into 96 well plates, stained with 10% crystal violet, and read using microplate reader with 655nm wave-length, following method used by paramanova10 modified by method used by bastiaan.19 differences between groups were analyzed using independent t-test or kruskall-wallis, while correlation between variables was analyzed using spearman test. degree of confidence was 0.05. results from 30 samples used in this study, the mna score of 15 subjects in good nutrition group were 20–24.5, while the mna score of 15 subjects in poor nutrition group were 15.5–18.5. nine out of 15 subjects in poor nutrition group have mna score between 17–18.5. comparison of salivary total protein concentration between groups was analyzed using independent t test while correlation between elderly nutritional status and salivary total protein was analyzed using pearson test. as could be seen in table 1, mean of saliva total protein concentration for good nutrition group is higher than those for poor nutrition group. however, this difference is not statistically significant (p > 0.05). pearson correlation test revealed that there is a weak (r = 0.323) positive correlation between elderly nutritional status and salivary total protein. this means that higher nutritional status relevance to higher salivary total protein concentration. however, this correlation is not statistically significant (p > 0.05). the parameter for c. albicans growth in this study is the cfu of the yeast on sda medium. as could be seen in graph 1, the cfu of control c. albicans (not exposed to saliva) is prominently higher than those exposed to saliva, either of those exposed to saliva from good or from poor nutrition group. the cfu of c. albicans exposed to saliva of good nutrition elderly was also lower than those exposed to saliva of poor nutrition elderly (figure 2a,b). kruskall-wallis test revealed that all these differences were significant, p < 0.05. spearman correlation test showed there is a weak (r = 0.395) negative correlation between elderly nutritional status and c. albicans growth. this means that higher nutritional status o p ti ca l d en si ty 0,25 0,2 0,15 0,1 0,05 0 figure 3. c. albicans biofilm formation of cultures not exposed to saliva (control), exposed to saliva from poor nutrition elderly, and from good nutrition elderly. blue = control, green = poor nutrition, red = good nutrition. figure 1. c. albicans cfu after exposed to elderly saliva with different nutritional status on the growth of c. albicans. blue = control, red = poor nutrition, green = good nutrition. 30000 25000 20000 15000 10000 5000 0 c f u /m l figure 2. c. albicans cultures on medium exposed to saliva of elderly with poor nutritional status (a) and good nutritional status (b). a b 75puspitawati et al.: elderly nutritional status affect is relevant to lower growth of c. albicans. figure 1 shows the pronounced difference of c. albicans cfu between those exposed to good nutrition and those exposed to poor nutrition elderly. kruskall-wallis test confirmed that the formation of c. albicans biofilm on c. albicans culture not exposed to saliva is significantly higher than those exposed to saliva (either from good or poor-nutrition elderly). conversely, the biofilm formation on medium exposed to saliva from poor nutrition elderly is not significantly (p > 0.05) higher than those exposed to saliva from good nutrition elderly (figure 3). pearson correlation test confirmed that a weak (r = 0.243) negative correlation between elderly nutritional status and c. albicans biofilm formation is not significant (p > 0.05). there is a tendention that the lower nutritional status relevance to higher c. albicans biofilm formation. discussion relevance to previous studies that decreased concentration of salivary total protein could be due to aging, poor nutrition, and disturbances of salivary gland,20 result of this study also showed pattern of lower salivary total protein concentration on poor nutrition elderly compared to those with good nutritional status. however, such differences were not statistically significant. such results could be due to composition of the samples where 60% subjects (9 out of 15) in poor nutrition group were actually in borderline nutritional status. mna is simple, has 98% accuracy, and frequently used to determine the nutritional status of elderly. in mna method developed by nestle, nutritional status is classified as good (> 18.5), moderate (17–18.5), and poor (< 17). the borderline status indicates condition with high risk to suffer malnutrition and not yet actually in malnutrition condition.21 another possible reason for results of this study was that slight difference of nutritional status of elderly participated in this study, does not lead to different concentration of their salivary total protein. such result is similar to a previous study by johansson et al.,22 who reported indifferent secreted total protein per minute between control and sample with protein-energy-malnutrition (pem). this study provide evidence of the efficacy of saliva in inhibitting the growth of c. albicans. as could be seen in figure 1, even saliva from elderly still has the capacity to inhibit the colony formation of c. albicans indicating inhibition of the yeast growth provided that the nutritional status of the subject is adequate. the pattern of decreased total protein concentration following decreased nutritional status, is relevant with the pattern of weaken inhibitting effect of saliva on the growth of c. albicans following decreased nutritional status. saliva contains many proteins with anti candidal capacity. malnutrition might lead to either lower secretion23 or weaker activity of these proteins.24 it is known that not all salivary proteins are secreted less in response to malnutrition. lisozim, lactoferrin and siga are three among various salivary proteins which were reported to decrease in children with pem.7–9,15,16 further study is required to determine the correlation between elderly nutritional status with concentration of each salivary antifungal protein. however, insignificant correlation between salivary total protein and elderly nutritional status found in this study indicating that nutritional status affects the salivary effectivity against c. albicans stronger than it affects the salivary protein concentration. this study also confirmed the efficacy of saliva, even the saliva of elderly with poor nutritional status, in inhibitting c. albicans biofilm formation. saliva has double roles in host-candida interaction. not only providing water, nutrition, and anti fungal components for the host, saliva also provides support for candida adhesion on the oral tissue surfaces.25 however, results of this study revealed that the role of saliva as anti fungal should be stronger than its regulatory role in supporting the adhesion of candida. the insignificant efectivity of saliva in ihibitting c. albicans biofilm formation between elderly with different nutritional status might be due to the composition of nutritional status of samples used in this study. although elderly nutritional status does not lead to significant decreased salivary total protein concentration but it is significantly correlated to salivary anti candidal acitvity against c. albicans. poor nutritional status which has not affected the quantity of salivary total protein might already lead to weaken effectivity of saliva against c. albicans. in conclusion, nutritional status of elderly affect the capacity of saliva both in inhibiting the growth and in declining the virulence of c. albicans. acknowledgement this study was granted by hibah riset awal universitas indonesia 2010. table 1. saliva total protein concentration difference between groups and correlation between elderly nutritional status and saliva total protein concentration groups n saliva total protein mean (mg/ml) ± sd p (t test) p (correlation) r correlation direction good nutrition 14 1113.55 ± 1143.3 0.111 0.093 0.323 positive poor nutrition 15 613.60 ± 253.60 76 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 72–76 references 1. darmojo b. ilmu kesehatan usia lanjut. jakarta: balai penerbit fk ui; 2010. p. 124–31. 2. tenovuo j. antimicrobial agents in saliva-protection for the whole body. j dent res 2002; 81: 807. 3. abbeele av, meel hind, ahariz m, perraudin jp, beyer i, courtois p. denture contamination by yeast in the elderly. gerodontology 2008; 222–8. 4. weindl g, wagner j, schaller m. epithelial cell and innate antifungal defense. j dent res 2010; 89(7): 666–75. 5. psoter wj, spielman al, gebrian b, rudolph j, katz rv. effect of childhood malnutrition on salivary flow and ph. arch oral biol 2008; 53(3): 231–7. 6. mardegan rdc, klein mi, golvea mb, rodrigues jao, goncalves rb, hofling jf. biotyping and genotypic diversity among oral candida albicans strain from caries-free and caries active healthy children. brazillian j microbiology 2006; 37: 26–32. 7. paillaud e, merlier i, dupeyron c, scherman e, poupon j, bories pn. oral candidiasis and nutritional deficiencies in elderly hospitalized patients. br j nutr 2004; 92: 861–7. 8. andrade fbd, jr adfa c, kitoko pm. relationship between oral health, nutrient intake and nutritional status in a sample of brazilian elderly people. gerodontology 2009; 26: 40–5. 9. tanida t, ueta e, aya t, toshihiro h, rao f, osaki t. influence of aging on candidal growth and adhesion regulatory agent in saliva. j oral pathol med 2001; 30: 328–5. 10. paramonova e, bastiaan p krom, van der mei hc, busscher hj, sharma pk. hyphal content determines the compression strength of candida albicans biofilms. microbiology 2009; 155: 1997–2003. 11. douglas lj. candida biofilms and their role in infection. trends microbiol 2003; 11: 30–6. 12. qin c, li h, xiao q, liu y, du y. water solubility of chitosan and its microbial activity. carbohydrate polymers 2006; 63: 367–74. 13. boyd, lampi. importance of nutrition for optimum health of periodontium. j contemp dent pract 2001 may; 2(2): 36–45. 14. elguelzabal n, maza jl, dorronsoro s, ponton j. whole saliva has a dual role on the adherence of candida albicans to polymethylmetacrylate. the open dentistry j 2008; 2: 1–4. 15. ueta e, tanida t, doi s, osaki t. regulation of candida albicans growth and adhesion by saliva. j lab clin med 2000; 136: 66–72. 16. dar-odeh ns, al-kayed ma. variations of some salivary antimicrobial factors in different disease states: a review. saudi dental j 2002; 14(2): 99–102. 17. li l, redding s, dongari-bagtzougloul a. candida glabrata, an emerging oral opportunistic pathogen. j dent res. 2007; 86(3): 204–15. 18. bradford protein concentration assay. 2001. available from: http://ww2.chemistry.gatech.edu/lw26/bcourse_information/4581/ techniques/bradford/bradford.html. accessed december 7, 2010. 19. bastiaan pk, cohen jb, mc-elhaney-feser g, busscher hj, conditions for optimal candida biofilm development in microtiter plates. methods in molecular biology 2008; 499(sep 1 2008): 55–62. 20. hajjar rr, kamel hk, denson k. malnutrition in aging. the internet j geriatrics and gerontology 2004; 1(1). 21. chernoff r. geriatric nutrition: the health professional’s handbook. 3rd ed. canada: jones and bartlett publishers, inc; 2006. p. 146–8. 22. johansson i, lenander-lumikaril m, saellstrom a-k. saliva composition in indian children with chronic protein-energymalnutrition. j dent res 1994; 73(1): 11–9. 23. al-drees am. oral and perioral physiological changes with ageing. pakistan oral & dental journal 2010; 30(26–30). 24. samaranayake yh, samaranayake lp, pow ehn, beena vt, yeung kws. antifungal effects of lysozime and lactoferrin against genetically similar sequential candida albicans isolates from a human immunodeficiency virus-infected southern chinese cohort. j clin microbiol 2001; 39: 3296–302. 25. almstahl a, wikstrom m, groenink j. lactoferrin, amylase and mucin muc5b and their relation to the oral microflora in hyposalivation of different origins. oral microbiol immunol 2001; 16: 345–52. 193 closed mouth method with dynamic and muco compressive impression on upper and lower jaw flat ridges for aid full denture retention utari kresnoadi and rostiny department of prosthodontic faculty of dentistry airlangga university surabaya indonesia abstract a patient with flat ridge difficult to have retentive complete denture. the aim of this paper is to describe the combination of impression using closed mouth technique with dynamic and muco compressive material. in this case, the combination technique of dynamic impressive material and muco compressive material with closed mouth method on patient with upper and lower jaw flat ridges. the patient has made complete denture 10 times but not satisfied. the treatment of upper and lower flat ridges using this technique resulted retentive, stable and comfortable denture. key words: closed mouth method, dynamic and muco compressive impression, flat ridge correspondence: utari kresnoadi, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction patient with flat ridge really needs full or complete denture for the purpose of chewing, speaking and improving the appearance. a dentist is considered successful to make a complete denture if the result is stable, retentive and comfortable to be used. hamada et al.1 suggested that the number of complete denture users would increase due to the increasing number of elderly people. left ridge resorption would occur in patient with prolonged tooth which are left untreated and are not replaced by denture and the ridge has functioned to chew the food. ridge resorption would also occur in patient with prolonged denture due to continuous pressure on the ridge while patient is chewing the food and it could cause resorption on the alveolar ridge bone as a result the bones becomes flat and, further, the mandible would be anthropy. it is also suggested by hamada et al.1 that after using prolonged complete denture, the space between denture base and soft tissue would be larger because of the increase resorption of alveolar bone. the early stage of resorption of residual ridge is initiated by the loss of the tooth and periodontal membrane which is capable to form the bone . the disappearance of alveoli could occur in labio lingual and vertical direction, so the ridge would be narrower. in some cases the ridge could be sharp like a knife or knife edge and shortening. further, procesus alveolaris would below, rounded or flat. if the resorption process continues resulting in disappearance of basal bone and followed by shortening ridge in oral cavity.2 accurate impression is needed to make complete denture if it is followed by flat alveolar ridge. denture impression were once made without regard to the muscular function involved. plaster, wax or gutta percha was used without muscle trimming in order to gain an impression of the basal seat.3 further, mucustatic impressive material which could record the jaw without distortion and procedure mucosal impression in detail. in twentieth century, impression with compression and involving functional muscle trimming is started to be applied. 3 to make a complete denture, impression with “making impression” mold is needed by preparing two impressions: mucostatic and muco compressive impressions. to impress using mucostatic, fabricated stock tray with hole with mucostatic impressive material is used.4 this material must be mixed with water, before it is used.3 the anatomical model of patients jaw is obtained from the impression in which. further, would be made for patients individual tray.4 individual tray is made before making impression using muco compressive material then continued by border molding which gives compound material on the edge of individual tray to get the form of pheripheal seal which is useful for denture’s retention. some experts3,5 made impression using muco compressive impression material to achieve accurate impression in case of lower jaw flat ridge. in case of lower jaw alveolar flat ridge, retention could be achieved by making additional retention on mandible that is the extension of retromylohyoid region by scarping the part of anatomy model so that individual tray in the region could be longer, it is also possible by adding compound material during molding.4 molding in retromyloyoid region, when it is seen laterally, it forms the letter “s” but it is seen from above, it would form butterfly wing.6 194 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 193-197 the principe of impression by using compression is to achieve well basis mucos on ridge, with light compression on the upper most ridge and to cover submucosal tissue.3 there are two methods of impression with compression those are: open and closed mouth. mostly open mouth method is more preferred because the operator can easyly trim the muscle and see the movement. while in close mouth method first the bite occlusion should be determine in wax.3 the tongue movement is stronger during occlusion when the mouth is closed simultaneously, and also there is no other power to disturb ridge when the jaw is closed in occlusal centric condition. 3 some experts1,7 suggested that closed moth technique with tissue conditioner/soft liner as dynamic impression material could make the same mouth movement producing good impression because the material could fungsionally distribute the movement on the surface of basal tissue on elderly patient. impression with closed mouth could develop physiological strength of muscle trimming during border molding the impression could record the compressed soft tissue to achieve good outcome, in this technique, patient’s cooperation is really needed.8 the purpose of this paper is to combine between dynamic and muco compressive impression material with closed mouth technique in patient with lower and upper jaw flat ridges as an effort to did retention of complete denture of upper and lower jaw. case a 70 year old female patient, a mandarin tutor, lost her teeth due to caries and extracted. she also suffered from diabetes mellitus and used a removable denture since she was 35 years old. the patient used ten times of dentures in which five times made by dental technician and five times made by a dentist. the denture was replaced due to pain and unfit and alveolectomy was ever done. the condition of mouth cavity: the conditions which were found: edentulous flat ridge in upper and lower jaw, shallow vestibulum, the height of ridge less than 1 mm in lower jaw (figure 1) and 2 mm in upper jaw (figure 2). flat torus mandibularis, low frenulum ridge relation either the transversal or the front was normal. the treatment plan for this patien was: complete denture of upper and lower jaws could be made using closed mouth technique. case management impression with closed mouth technique was done because the patient had resorption/upper and lower jaw flat. mucostatic impression with alginate material was initially done on the patient, using this impression model anatomy was obtained, then denture outline was made and it is very essential part in this stage in order to avoid “over extension” (figure 3). in this case wax spacer was not necessarily done due to the condition of flat ridge then, individual tray of self cured acrylic material was made. further, muco compressive impression using silicon rubber base impressive material was done on the patient. the result of mucopressive impression of upper and lower jaws could seen in figure 5. from the working cast acrylic base with bite wax made the height and bite position were searched and fixated (figure 6). the next step, dynamic tissue conditioner/softliner impressive material was placed on acrylic base and impression with closed mouth method was carried out, by returning fixated bite wax into the mouth using tissue figure 3. outline process of individual tray ra & rb border moulding was done individual tray with compound material to get peripheal seal (figure 4). figure 1. lower jaw flat ridge. figure 2. upper jaw flat ridge. 195kresnoadi and rostiny:closed mouth method with dynamic and muco compressive impression conditioner material as functional impression material which would functional distribute stress on the basal tissue surface. the result of compressive with closed mouth method using tissue conditioner/soft lining material could seen in figure 7. then the muco compressive/elastomer impression material was given on tissue conditioner impression. impressing process with closed mouth method using compressive material by returning bite wax fixated into the mouth (figure 8). and the result could seen in figure 9. the process was continued by filling with hard gypsum and working model was formed and put on articulator. the following step, teeth arrangement was done and adjusted on the patient. teeth arrangement should be on the tip of ridge netral zone (figure 10). over bite and overjet of anterior teeth should be paid. closed attention as well as curve of spee should be seen from sagital side and curve of monson of transversal side of posterior tooth arrangement. figure 4. giving compound material for border moulding.5 figure 6. searching the height and bite position. figure 7. the result of impression with closed mouth method using tissue conditioner/softliner material. figure 8. impressing process with closed mouth method using muco compressive material. figure 5. the result of muco compressive impression of upper (b) and lower (a) jaws.5 a b 196 dent. j. (maj. ked. gigi), vol. 40. no. 4 october-december 2007: 193-197 the adjustment was tried on the patient in the condition that wax was still used. if the patient was satisfied with denture wax, then, countour would be done. followed by acrylic processing and polishing. the next step, acrylic denture was adjusted on the patient. occlusal record was done for occlusal correction (figure 11), then continued by selective grinding, polishing and the last, it would be applied on the patient. the instructed given to the patient after the denture was insertion that was: denture was allowed only for drinking and speaking, but not eating. the denture was recommended used at night and followed up on the one day. the first day of follow up control, the patient complained of pain in mylohyoid region and lingual region of anterior lower jaw. in fact, the pain was caused by excessive pressure of denture, therefore, grinding was done in retromylohyoid region and lingual part of anterior denture. the upper jaw seemed retentive and no complaint presented by the patient. next, the patient was advised to use the denture to eat something soft, to drink and to speak . the denture should be removed at night and soaked in water, with the purpose that the tissue would rest. the patient was instructed to have follow up control three days later. on the second day followed up, the condition of upper and lower jaw denture was stable and retentive, but, the patient still complained of pain in lingual region of anterior lower jaw, because of excessive pressure from denture. to reduce the pain, anterior lingual region was grinded, but not excessively in order not to reduce denture retention. the patient was advised to have follow up control the following week. the instruction was similar to first control. on the third follow up control, the patient still complained of pain in anterior lingual lower jaw, but the complete denture was retentive and stable so grinding was done, slightly reducing part of lingual anterior and polishing, the instruction was still similar to the second control if there was any complaint, the patient was advised to have regular control. the patient came to control two months after insertion, without complaint, the denture was retentive and stable. the patient felt comfortable to use complete denture either for speaking or chewing the food, the patient felt satisfied. the patient was suggested to come for control periodically six months after the usage of denture. discussion in this case, as the procedure of complete denture can not be done by increasing other retention such as: tooth implantation, therefore, accurate method of impression must be done so the space between denture and basal seat would be vacuum, air pressure would be less than 1 atm, denture would become retentive and stable. de franco and sallustio8 also confirmed that if another treatment such as: implant denture could be not be applied in the case of atrophied mandible, therefore, supporting denture is only on the residual tissue such as: mucosa and ridge, so procedure of impression is made for atrophied mandible. hamada et al.1 suggested that it is not easy to produce good jaw impression in elderly patient who is toothless, dynamic impression would produce better outcome. in impression with closed mouth method, impression with soft liner/tissue conditioner material is initially done figure 9. the result of closed mouth technique with muco compressive material. figure 11. the result of intermaxilary record seen frontally. figure 10. netral zone.9 netral zone cheek tongue 197kresnoadi and rostiny:closed mouth method with dynamic and muco compressive impression because by using this material good pheriperal seal could be achieved and could balance mucosa reciliancy. in this case, it is similar to the opinion of hamada et al.1 that impression using closed mouth technique with dynamic impressive material is conducted in order to be able to make equal mouth movement. in other words that the result of impression is obtained from patient’s mouth movement with tissue conditioner material. the some opinion also showed by chase and starcke cit. abdul razek,8 that tissue conditioner material is functional impression material. functional impressive material is one of materials which is used on the surface of basal seat of denture which makes functional stress distribution or this material makes the surface of basal seat tissue and border tissue of denture recorded when it is functional. the patient had really flat ridge due to prolonged use of denture for more or less thirty five years. to increase retention, impression with muco compressive material is required to be able to compress the mucosa so it could produce accurate impression. in this way, vacuum space between mucosa and denture would be achieved. itjingsih9 and zarb et al.10 indicated the same opinion that impression with muco compression is needed to make the compression more equal, impression material could flow and fill complicated part, so it could impress accurately. in this case, after using complete denture, the patient felt her denture was retentive, stable and comfortable to be used comparing with her ten denture ago. even though she felt pessimistic at the beginning to have complete denture in mouth considering the ridge was flat. after using the new complete denture, the patient felt satisfied and she could chew the food, speak normally and she has better performance. it is concluded that to make a complete denture in the management of that flat ridge case on upper and lower jaw, it is needed to apply impression using closed mouth technique with dynamic (tissue conditioner/soft liner) and muco compressive to get retentive and stable. references 1. hamada t, murata h, razak a. pelapisan gigi tiruan, denture lining. cetakan i. surabaya: airlangga university press; 2003. p. 48–52. 2. nishimura i, hosokawa r, attwood da. the knife edge tendency in mandibular ridge in women. j prosthet dent 1992; 67:820–6. 3. sharry j. complete denture prosthodontic. 3rd ed. new york, st louis, toronto: mcgraw-hill book co; 1974. p. 200–3. 4. kresnoadi u. cara menanggulangi goyangnya gigi tiruan pada waktu mengunyah. buku ceramah ilmiah, surabaya dentistry 2003; 2003. p. 8. 5. rita iu, widyana h. disain dan tehnik mencetak pada pembuatan geligi tiruan lengkap. ceatakan 1. jakarta: hipokrates; 1994. p. 43. 6. heartwell cm, rhan ao. syllabus of complete denture. 4th ed. philadelphia: lea & febiger; 1986. p. 178–83. 7. abdel razek ma. assesment of tissue conditioning materials for functional impression. j prosth dent 1978. 8. de franco rl, sallustio a. an impression procedure for severely atrophied mandible. j prosthet dent 1995; 73:574–7. 9. itjiningsih wh. geligi tiruan lengkap lepas. cetakan ke-3. jakarta: penerbit buku kedokteran/egc; 1996. p. 26, 39. 10. zarb ga, bolender cl, hickey jc, carlson ge. bouchers prosthodontic treatment for edentulous patients. 7th ed. st louis, baltimore, philadelphia. 1990. p. 197–210. 100 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 100–104 research report permanent tooth eruption based on chronological age and gender in 6-12-year old children on madura agus marjianto,1 mieke sylvia,2 and soegeng wahluyo3 1department of dental nursing, health polytechnic of health ministry 2department of forensic odontology, faculty of dental medicine, universitas airlangga 3department of pediatric dentistry, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: tooth eruption, the movement of teeth toward the oral cavity clinically marked by the emergence of the cusp or incisal edge, is very important in determining the chronological age of a child. unfortunately, tooth eruption in 6-12-year olds on the island of madura has yet to be investigated. purpose: this study aimed to analyze differences in permanent tooth eruption between boys and girls aged 6 to 12 years old on madura. methods: this study employed an observational analytic design in combination with a cross-sectional approach. the samples used in this study were selected by means of simple random sampling technique. post-selection informed consent of the child subjects was obtained with their chronological age being assessed and determined prior to tooth eruption. the normality of the data was subsequently analyzed by application of a one sample non-parametric kolmogorov smirnov test. thereafter, repeated anova tests were conducted to determine differences in the permanent tooth eruption of the subjects. results: based on the normality test results, the significance value of the permanent maxillary teeth in the male subjects was 0.993, while that of their permanent mandibular teeth was 0.695. in contrast, the significance value of the permanent maxillary teeth in the female subjects was 0.970, while that of their permanent mandibular teeth was 0.918. according to the results of the repeated measure anova test, differences existed in the eruption of the permanent maxillary and mandibular teeth between the males and females with a significance value (ρ) of 0.020. the mean value of permanent mandibular tooth eruption in the females was 56.59 ± sd 33.403, while that of their permanent maxillary tooth eruption was 50.77 ± sd 34.201. the mean value of the permanent mandibular tooth eruption in the males was 55.31 ± 33.024, while that of their permanent maxillary tooth eruption was 48.77 ± sd 34.201. conclusion: on madura, the permanent teeth of chronological 6-12-year old females, particularly their permanent mandibular canine teeth, erupt earlier than those of their male counterparts. keywords: chronological age; permanent tooth eruption; sex correspondence: agus marjianto, department of dental nursing, health polytechnic of health ministry. jl. pucang jajar selatan 24, kertajaya, surabaya 60282, indonesia. e-mail: marjiantoagus@gmail.com introduction estimation of the age of an individual, conducted, for example, in the field of forensic odontology, is based on the sequence of tooth eruption within the oral cavity.1 the eruption of human teeth is divided into three periods, namely: deciduous tooth eruption between the ages of 6 months and 6 years, mixed tooth eruption between the ages of 6 years and 12 years, and permanent tooth eruption between the age of 12 years and the point at which an individual loses all of his/her teeth.1,2 forensic examination is conducted to estimate the chronological age of children for a variety of purposes, including: determining inheritance rights, child adoption cases, and crimes perpetrated against children.3 age estimation can be conducted by observing growth stages and body structure development in the form of consistent physical changes. each stage of the developmental process can be directly related to age of an individual.4 the parts of the body usually analyzed to determine age are the skeleton and teeth. unfortunately, skeletal maturity as a means of estimating age is subject to certain limitations dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p100–104 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p100-104 101marjianto, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 100–104 since it can only predict age within a specific age range with a large standard deviation. meanwhile, the use of teeth have several advantages as a means of age estimation, one of which is that it can predict age in individuals ranging from prenatal to adult.5 the accuracy of tooth-based age estimation depends on the selection of the method best suited to the circumstances of the individual case. tooth-based age estimation generally employs a non-invasive method that involves clinical and radiographic examinations without resort to tooth extraction. the number of deciduous and permanent teeth that have erupted in children, adolescents or adults can be clinically examined employing a method of assessing the number of permanent teeth suitable for individuals between the age of 6 and adulthood.5,6 the number of erupted teeth indicates the maturity level of an individual. thus, observing the number and condition of the teeth in the oral cavity can determine his/her age. this clinical observation involves no risk due to the absence of exposure to x-ray radiation.7 eruption is the process by which developing teeth move from the location of the tooth seed through the alveolar ridge into the oral cavity and experience occlusion with the antagonistic tooth. eruption occurs if all clinical crowns are above the gingiva and the level is then calculated from the tooth cusp of the posterior teeth or from the incisal edge of the anterior teeth.8 the timing of the eruption of permanent teeth between the ages of 6 and 12 depending on the age and sex of the individual is also considered important for archeology and forensic studies involving human remains. teeth have specific features useful in determining maturation which can predict the order of development, including: crown formation, roots, calcification, and eruption.9 the characteristics of distinct races, ethnic groups and populations differ, together with the timing of permanent tooth eruption.10 the population of indonesia is diverse, consisting of various ethnic groups including the peutero and deutero malay sub-races. one such deutero malay sub-racial group is the madurese who inhabit the island of madura in the province of east java and whose day-to-day life and dietary habits are both challenging. to date, no research on permanent tooth eruption among the madurese has been conducted. consequently, this research aims to analyze the differing permanent tooth eruption of males and females chronologically aged 6-12 years on madura. materials and methods this study focused on differences in permanent tooth eruption between males and females chronologically aged 6-12 years on madura and received ethical approval from the health research ethics commission, faculty of dentistry, universitas airlangga. it employed an analytic observational design with a cross-sectional approach. samples of this study were selected by means of a simple random sampling technique. the criteria for the study sample comprised: healthy males and females aged between 6 and 12 classified as third-generation native-born madurese based on their mothers/fathers/grandmothers/ grandfathers all having been born on madura. the subjects also had to be free of growth disorders, mental illness or congenital anomalies and did not suffer from dental caries or use orthodontia. tooth eruption measurement or scoring was subsequently conducted. if the tooth had not erupted, that is, it had not penetrated the gingiva with one part, with the entire incisal edge for incisors or with one or two cups for posterior teeth, its score would be 0. if the incisal edge or peak cups were visible, with a quarter of the anatomically crown erupted, a score of 0.25 was assigned. if half of the anatomical crown had erupted, the recorded score was 0.5. if more than half or almost all of the anatomically crown had erupted, its score would be 1. the resulting data was analyzed using a non-parametric one sample kolmogorov smirnov test to determine its normality. a repeated measure anova test was then performed in order to determine differences in tooth eruption between males and females with a significance value (α) of 0.05. (spss version 21). results the research samples comprised 117 children, 64 male and 53 female, aged between 6 and 12.99 years old. moreover, based on the results of a normality test using a non-parametric one sample kolmogorov smirnov test, the significance value of the maxillary permanent teeth in the males was 0.993, while that of their mandibular permanent teeth was 0.695. in contrast, the significance value of the maxillary permanent teeth in the females chronologically aged 6-12 years was 0.970, while that of their mandibular table 1. the mean and standard deviation values of normality test and repeated measures anova results tooth eruption mean ± sd kolmogorov smirnov one sample test repeated measure anova male maxilary tooth 48.68 33.608 0.993 0.02 male mandibular tooth 55.31 33.024 0.695 female maxilary tooth 50.77 34.201 0.970 female mandibular tooth 56.59 33.403 0.918 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p100–104 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p100-104 102 marjianto, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 100–104 table 2. permanent tooth eruption sequence in the males subjects based on average chronological age tooth 17 16 15 14 13 12 11 21 22 23 24 25 26 27 ± sd 0.98 0.113 0.63 0.897 0.422 0.516 0.612 0.612 0.708 0.517 0.922 0.589 0.113 0.859 mean 11.8 6.69 11 9.98 11.18 9.46 7.87 7.87 9.3 11.01 10.04 11.09 6.69 11.56 mean 11.68 6.69 10.82 10.94 10.53 7.56 6.69 6.69 7.63 10.61 10.87 10.98 6.69 11.74 ± sd 0.765 0.113 0.946 0.86 0.372 0.283 0.113 0.113 0.25 0.316 0.848 0.799 0.113 0.675 tooth 47 46 45 44 43 42 41 31 32 33 34 35 36 37 table 3. permanent tooth eruption sequence in the female subjects based on average chronological age tooth 17 16 15 14 13 12 11 21 22 23 24 25 26 27 ± sd 1.371 0.367 1.241 0.932 1.203 0.43 0.581 0.632 0.433 0.925 0.937 1.302 0.367 1.212 mean 11.77 6.64 10.29 9.6 10.42 8.15 7.11 7.14 7.99 10.46 9.57 10.18 6.64 11.61 mean 11.55 6.57 10.44 10.39 9.15 7.24 6.64 6.64 7.24 9.08 10.26 10.6 6.49 11.23 ± sd 1.135 0.359 1.116 1.117 0.7 0.647 0.367 0.367 0.647 0.701 1.078 0.897 0.368 1.447 tooth 47 46 45 44 43 42 41 31 32 33 34 35 36 37 b a c d e figure 1. a) a female maxillary and mandibular chronologically aged 7.74 years old when occlusion occurred; b) a female maxillary; c) a female mandibular; d) a female maxillary cast result; e) a female mandibular cast result. b a c d e figure 2. a) a male maxillary and mandibular chronologically aged 7.76-years-old when occlusion occurred; b) a male maxillary; c) a male mandibular; d) a male maxillary cast result; e) a male mandibular cast result. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p100–104 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p100-104 103marjianto, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 100–104 permanent teeth was 0.918. this signified differences in permanent tooth eruption between the males and females (table 1). the results of the repeated measure anova test indicated differences in permanent tooth eruption between the male and female samples of the madurese population with a significance value of 0.020 (ρ<0.05). moreover, the order of tooth eruption in the mandibular canine teeth of the females differed significantly from that in the males. the order of tooth eruption in the males in terms of chronological age was 16, 26, 31, 36, 41, 46, 42, 32, 11, 21, 22, 12, 14, 24, 43, 33, 45, 34, 44, 35, 15, 23, 25, 13, 27, 47, 37, 17 (table 2). meanwhile, the tooth eruption sequence in the females based on chronological age was 46, 36, 16, 26, 31, 41, 11, 21, 32, 42, 22, 12, 43, 33, 24, 14, 25, 44, 15, 34, 13, 35, 23, 45, 47, 37, 27, 17 (table 3). in addition, the permanent maxillary and mandibular canine teeth of the females chronologically aged 7.74 years erupted more rapidly (figure 1). meanwhile, the permanent maxillary and mandibular canine teeth in the males chronologically aged 7.76 years had not yet erupted (figure 2). discussion the results of this study revealed that the eruption of permanent teeth in the females of the madurese population occurred earlier than in the males. similarly, a study conducted at the prof. sudomo oral and dental hospital of the dentistry faculty at gadjah mada university found that the maturation of permanent teeth in females was higher than in males.11 another previous study conducted in surakarta also revealed that permanent tooth eruption in females occurs two months earlier than in males.12 similar to previous studies, one carried out in lithuania found that permanent tooth eruption in females was more rapid than in males.13 however, the results of this study were not in line with those of one focusing on permanent tooth eruption in children aged 4-15 years in kampala, uganda. generally, tooth eruption in females was found to more protracted than in males with the exception of teeth 25, 32 and 42, while the average tooth eruption between the two genders was known to differ by as much as 0.8 (with a range of 0-1.5 years).14 similarly, a study conducted in karachi, pakistan indicated there to be neither difference in tooth eruption between males and females, nor in tooth eruption compared to body weight and height.10 such findings are consistent with those of a study carried out in india showing that in the several populations studied, mandibular teeth erupted more rapidly than maxillary teeth, with a concomitant difference in tooth eruption time between the right and left regions of the maxilla and mandible.15 eruption is the process within which developing teeth move from the location of the tooth seed through the alveolar ridge into the oral cavity, experiencing occlusion with the antagonistic tooth. eruption occurs if all clinical crowns are above the gingiva with the level being calculated from the tooth cusp or the incisal edge. tooth eruption occurrence varies in each child. variations can even occur within each growth and development period of the teeth, being influenced by genetic and environmental factors.9 the contrasting findings of the above studies result from nutritional intake and status factors relating to children which largely determine tooth eruption. moreover, permanent tooth eruption time varies more than that of its deciduous counterpart.13 differences in permanent tooth eruption time in several regions are influenced by numerous factors, including; genetic and environmental factors involving socioeconomic and nutritional status, as well as climate. it is also known that permanent tooth eruption time varies between racial groups.10 tooth eruption is influenced by several factors such as socioeconomic, nutrition, sex, race, hormonal and genetic factors.13 socio-economic conditions represent one of the factors affecting tooth eruption. however, a study conducted in brazil posited that, in fact, no correlation exists.16 it has also been argued that there is no relationship between nutritional status and anthropometric measurements, such as height and weight, in addition to tooth eruption.14 the above statement accords with the 2015 assertion of the pasongsongan public health center in the sumenep regency that the environmental conditions found in the coastal pasongsongan region of sumenep regency where the majority of residents are fishermen, were such that nutrient intake, especially protein and calcium, was extremely abundant. eruption of permanent teeth is of considerable significance. in addition to determining the age of a person, it can also support certain diagnoses, for example, massive hard tooth tissue damage, since teeth are resistant to environmental changes and post-mortem decomposition and can be maintained without distortion. just as with fingerprints, the morphology and arrangement of teeth is unique to each individual and are, therefore, extremely helpful in identifying a specific individual.17 normal standards must be applied to the clinical assessment of the growth of a child in order that the physiological age of the tissue system can be compared with its chronological age.18 despite being of the same chronological age, children can present different stages of biological development and the dental care of those who still have mixed teeth requires special attention. therefore, it is essential for a pediatric dentist to have knowledge about the growth of a child, particularly the development of teeth and its variations.19 tooth eruption is frequently analyzed forensically in order to estimate the age of a child and to assess dental maturation or clinical age. dental age is used by dentists to determine the appropriate timing of certain orthodontic treatments, including that for deciduous teeth. establishing dental age can be conducted in two ways, namely; examining the status of those teeth that have erupted in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p100–104 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p100-104 104 marjianto, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 100–104 oral cavity, or clinical methods such as non-x-ray methods considered to be more practical and involving no use of radiation on patients.6,20 the effects of tooth eruption and the number of abnormalities that may be caused by resulting disorders in the oral cavity should enhance the awareness of dentists regarding the precise timing of tooth eruption. such knowledge is important for dentists, especially those active in the fields of pedodontics and orthodontics, in helping to determine diagnosis and the appropriate type of treatment. finally, it can be concluded that the permanent teeth of females within the population of madura chronologically aged 6-12 years erupt earlier than those of males. the earliest eruption occurs in the permanent mandibular canine teeth of females. references 1. scheid rc, weiss g. woelfel’s dental anatomy. 8th ed. philadelphia: lippincott williams & wilkins; 2012. p. 179-81,366-84. 2. nelson sj, ash mm. wheeler’s dental anatomy, physiology, and occlusion. 9th ed. st. louis: saunders; 2010. p. 27–31. 3. baghdadi zd. dental maturity in saudi children using the demirjian method: a comparative study and new prediction models. isrn dent. 2013; 2013: 1–9. 4. shah rk. estimation of age by eruption of permanent canine tooth. int j pharm biol sci. 2014; 4(1): 169–72. 5. panchbhai a. dental radiographic indicators, a key to age estimation. dentomaxillofacial radiol. 2011; 40(4): 199–212. 6. putri as, nehemia b, soedarsono n. prakiraan usia individu melalui pemeriksaan gigi untuk kepentingan forensik kedokteran gigi. j pdgi. 2013; 62(3): 55–63. 7. alshihri am, kruger e, tennant m. dental age assessment of 4 –16 year old western saudi children and adolescents using demirjian’s method for forensic dentistry. egypt j forensic sci. 2016; 6(2): 152–6. 8. australian human rights commission. inquiry into the treatment of individuals suspected of people smuggling offences who say that they are children. inquiry into age assessment in people smuggling cases. sydney: australian human rights commission; 2011. p. 7. 9. ogodescu ae, tudor a, szabo k, daescu c, bratu e, ogodescu a. up-to-date standards of permanent tooth eruption in romanian children. jurnalul pediatrului. 2011; 14(53): 10–6. 10. khan n. eruption time of permanent teeth in pakistani children. iran j public health. 2011; 40(4): 63–73. 11. kuswandari s. maturasi dan erupsi gigi permanen pada anak periode gigi pergantian (the maturition and eruption of permanent teeth in mixed dentition children). dent j (majalah kedokt gigi). 2014; 47(2): 72–6. 12. ahmad p. perbandingan waktu erupsi gigi m1 permanen mandibulla antara anak laki-laki dan perempuan di ta’mirul islam surakarta tahun 2014. thesis. surakar ta: universitas muhammadiyah surakarta; 2014. p. 1–10. 13. almonaitiene r, balciuniene i, tutkuviene j. standards for permanent teeth emergence time and sequence in lithuanian children, residents of vilnius city. stomatologija. 2012; 14(3): 93–100. 14. kutesa a, nkamba em, muwazi l, buwembo w, rwenyonyi cm. weight, height and eruption times of permanent teeth of children aged 4–15 years in kampala, uganda. bmc oral health. 2013; 13(1): 15. 15. gaur r, saini k, boparai g, kumar s, airi r. growth, oral hygiene and emergence of permanent dentition among 5-14 year old rajput children of solan district of himachal pradesh. hum biol rev. 2012; 1(1): 84–99. 16. frazão p. emergence of the first permanent molar in 5-6-year-old children: implications from a longitudinal analysis for occlusal caries prevention. rev bras epidemiol. 2011; 14(2): 338–46. 17. bérgamo al, de queiroz cl, sakamoto he, da silva rha. dental age estimation methods in forensic dentistry: literature review. forensic sci today. 2016; 2(1): 4–9. 18. smith bh. standards of human tooth formation and dental age assessment. in: kelley ma, larsen cs, editors. advances in dental anthropology. new york: wiley-liss inc.; 1991. p. 143–68. 19. peña gutiérrez ce. estimación de la edad dental usando el método de demirjian en niños peruanos. thesis. lima: universidad nacional mayor de san marcos; 2010. p. 1–75. 20. ambarkova v, galić i, vodanović m, biočina-lukenda d, brkić h. dental age estimation using demirjian and willems methods: cross sectional study on children from the former yugoslav republic of macedonia. forensic sci int. 2014; 234: 187.e1-187.e7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p100–104 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p100-104 210 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 210–215 original article effectiveness of capsaicin nanoparticle gel of capsicum frutescens l. on oral squamous cell carcinoma in rattus norvegicus fitri aniowati1, cantika nadrotan naim1, nova dwi anggraeni2 and pratiwi nur widyaningsih3 1student of dentistry, faculty of medicine, jenderal soedirman university, purwokerto, indonesia 2student of pharmacy, faculty of health sciences, jenderal soedirman university, purwokerto, indonesia 3departement of dentistry, faculty of medicine, jenderal soedirman university, purwokerto, indonesia abstract background: oral squamous cell carcinoma (oscc) is an oral cancer with a low life expectancy, less than five years after diagnosis. the drug therapy often used for oscc patients is cisplatin, but it is considered to cause tumour persistence, drug resistance, and high toxicity. therefore, it is important to test the development of alternative drugs from natural ingredients. one potential ingredient is green chilli pepper (capsicum frutescens l.). it contains capsaicin that functions as an anticancer agent by suppressing bca tumorigenesis so that proliferation is inhibited, as well as increasing and preventing p53 antibody mutations that play a role in cancer cell apoptosis. purpose: this study aimed to compare effectiveness using capsaicin nanoparticle gel from green chilli pepper extract levels of 1% and 3.3% to reduce oscc nodules. methods: this study used 20 rattus norvegicus that were randomly divided into five groups; c(rat without treatment), c+ (rat induced to 7,12-dimethylbenz(a)anthracene (dmba)), e1 (dmba exposed and given cisplatin, e2 (rat induced to dmba and capsaicin extract nanoparticle gel with a concentration of 1%), and e3 (rat induced to dmba and capsaicin extract nanoparticle gel with a concentration of 3.3%). the data were analysed statistically with the one-way anova and least significance difference (lsd) test. results: the comparison of mean nodule volume between c+ (5.834 ± 2.77 mm3) with e1 (1.75 ± 0.37 mm3), e2 (1.747 ± 0.36 mm3), and e3 (1.812 ± 0.11 mm3) had a significant difference (p = 0.00, p ≤ 0.05). conclusion: capsaicin nanoparticle gel with green chilli pepper extract at levels of 1% (e2) reduces oscc nodules by more than gel with green chilli pepper extract at 3.3% (e3) concentration. keywords: anticancer; capsaicin; oscc correspondence: fitri aniowati, student of dentistry, faculty of medicine, jenderal soedirman university. jl. dr. gumbreg no.1, mersi purwokerto 53112, indonesia. email: fitrianiowati12@gmail.com introduction cancer is one of the leading causes of death in the world. the prevalence of cancer in indonesia has increased from 1.4% in 2013 to 1.8% in 2018.1 of the different types of oral cancer, 95% is oral squamous cell carcinoma (oscc). oscc is an oral cancer with a low life expectancy, less than 5 years after diagnosis.2 oscc occurs due to a multitude of factors such as smoking, betel chewing, and alcohol consumption.3 the drug therapy often used for oscc patients is cisplatin, but cisplatin is considered to cause tumour persistence, drug resistance, and high toxicity.4 it is important to test the development of alternative drugs from natural ingredients, such as fruits and plants, to create anticancer benefits of price and applications that are more affordable for oscc patients. one such potential ingredient is green chilli pepper (capsicum frutescens l.) because it contains anticancer, high-antioxidant, pain-reliever, and anti-inflammatory benefits. these benefits are mainly due to the content of capsaicin. capsaicin (8-methyl-n-vanillyl6-noneamide) is an alkaloid compound that is responsible for the spicy taste of green chilli peppers.4 green chilli pepper (capsicum frutescens l.) has the highest capsaicin content of all the chilli varieties at 2.11%. capsaicin functions as an anticancer agent by suppressing bca dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 mailto:fitrianiowati12@gmail.com https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 211aniowati et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 210–215 tumorigenesis so that proliferation is inhibited, as well as increasing and preventing p53 antibody mutations that play a role in cancer cell apoptosis.6,7 green chilli pepper (capsicum frutescens l.) will be applied using a nanoparticle gel on the object of research, wistar rats (rattus norvegicus). wistar rats were chosen as research material because they have an anatomy similar to humans.8 nanoparticle gel preparations were selected because the particles are small, making it easier to reach the target. in addition, the use of nanoparticles will achieve higher bioavailability than oral preparations and is accepted by patients because it is practical, safe, and painless.9 with the low life expectancy of oscc patients, drugs treatment such as cetuximab and cisplatin has high resistance and toxicity. utilization of natural resources as the latest treatment developments is needed to overcome this problem. this study aimed to compare the effectiveness of using capsaicin nanoparticle gel from green chilli pepper extract at levels of 1% and 3.3% to reduce oscc nodules. the hope is that the presence of green chilli pepper extract nanoparticles will inhibit the growth rate of oscc with the right concentration of nanoparticle gel and facilitate the curing of oscc. materials and methods the research was performed in an experimental laboratory using the method of randomized post-test only control group design for four months at jenderal soedirman university. the research had ethical clearance agreement number 125/ kepk/vii/2021 and applied strict health protocols. wistar rats aged two months with an initial body weight of 150–200 grams were the test animals obtained from the gadjah mada university, medical faculty research laboratory. the rats were divided into five groups. capsicum frustescens was obtained from kemutug kidul, baturraden, banyumas, with 22 grams of urea fertilization in the plantation and got determination tests in the environmental laboratory of the faculty of biology, jenderal soedirman university.10 capsaicin extraction was performed using 96% ethanol solvent, at a stirring speed of 200 rpm, a temperature of 50ºc, and 4 hours by maceration method. the extract evaporated to form a thicker extract and filtrate. the sample, formed as capsicum frustescens powder, was dissolved in a single solvent of n-hexane and methanol.11 then, 5 grams of the capsicum frustescens powder was dissolved in 100 ml of methanol, with 10 ml taken for isolation. the solution had 10 ml of distilled water and n-hexane added to form a fraction containing purer capsaicin. after shaking the solution, the fraction that formed was placed in a vial. it was then placed in a desiccator for 24 hours in the refrigerator until crystals formed that proved it contained purer capsaicin. 11 the nanoparticles were prepared in 100-gram extract concentrations of 3.3% and 1%. the concentration used was based on research that stated that the capsaicin content of 333 µg/ml has a significant effect and the highest cytotoxic activity on the oscc cell line.12 materials were all weighed according to the calculation. the gel was prepared by mixing 0.5 grams of a gelling agent (carbopol) with heated water that was then allowed to form and expand the gel mass.13 then 0.3 grams of methylparaben was dissolved in 15 grams of propylene glycol, and 1 gram of sodium metabisulfite was added. this was stirring until it was homogeneous. the solution was placed on a carbopol base and homogenized. extracts of 1 gram and 3 grams were mixed into the resultant base and homogenized. the remaining water was added to this base and again homogenized.14 the manufacture of nanoparticles was carried out by stirring on a magnetic stirrer for 4 hours. carcinogen induction in rats used a formula of 96 mg 7,12-dimethylbenz(a)anthracene (dmba) dissolved in 24 ml corn oil, vortexed for ± 15 minutes until homogeneous. carcinogen induction was performed two times a week for two weeks by the right buccal mucosa injection route. the induction dose given was 20 mg/kg of a rat’s weight.15 rats were anaesthetized using ketamine intramuscularly at a dose of 0.2 ml/200 gr of a rat’s weight and observed by the method of palpation of the buccal mucosa four times a week for two weeks. rats were diagnosed with cancer if there were palpable nodules on the buccal mucosa.16 after observation and rats were diagnosed with cancer, treatment commenced and was carried out once a day for seven days. each test group of wistar rats received a marker, and the rats were acclimatized for one week. the rats then received an induced dmba solution by injection of the right buccal mucosa. the rats were sampled using the method random sampling. they were divided into five treatment groups, and the treatments were carried out once a day for seven days (table 1). the wistar rats were weighed after being divided into the five groups. weighing occurred three times: after the acclimatization process, before dmba was induced, and before treatment. these weights were recorded to help table 1. experiment group experiment group treatment control – (c -) rat without treatment control + (c+) rat induced to dmba experimental control 1 (e1) rat induced to dmba and cisplatin experimental control 2 (e2) rat induced to dmba and capsaicin extract nanoparticle gel with a concentration of 1% experimental control 3 (e3) rat induced to dmba and capsaicin extract nanoparticle gel with a concentration of 3.3% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 212 aniowati et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 210–215 track the symptoms of cancer.16 a surgical procedure was performed on each rat three months after it was diagnosed with cancer. the rat was given a capsaicin extract nanoparticle gel using the cervical dislocation method. the rat’s head was cut off to observe the tumour tissue, then stained using toluidine blue, cleaned using saline, and stored in a sample pot containing 10% neutral buffered formalin (nbf). the variables obtained were nodule volume, length and width. the volume of the nodules was calculated by the formula (p x l2), and the length and width were measured in millimetres using callipers.17 the analysis used in this research are the normality and homogeneity tests, the oneway anova test, and post-hoc lsd. the anova test and post-hoc lsd are completed if the results reach p ≤ 0.05. results the rats’ weight results in table 2 showed that there was a considerable weight loss before the inducement of dmba and before treatment (p ≤ 0.05) in 3 weeks, with a difference in body weight between the two periods of 27.23 g (table 3). cancer patients can experience the condition cachexia, and one of the symptoms of this is weight loss. observation of oscc nodules was carried out for two weeks after dmba induction through palpation of the buccal mucosa four times a week. the results of clinical observations showed the formation of oscc nodules on the right buccal of the rats in the form of nodules that extended to the outer cheek. the round nodules felt rubbery and contained tissue. they appeared purplish-red to black and were of exophytic, endophytic, or ulcerative types. the extension was on the extraoral, to the infraorbital, to the buccal anterior. group 1 (c-) had no buccal nodules. group 2 (c+) had exophytic-typical nodules with the largest volume, but some rats also had ulcerative-type lesions. lesions in this group appeared to contain tissue in some parts that were necrotic and purplish-red in colour, spreading to the infraorbital and buccal areas of the front. clinically, oscc had degraded the tissue to the extent it table 2. weight change comparison weight measurement time n mean weight (gr) ± sd weight before acclimatization 20 200.29 ± 24.3 weight before dmba induction 20 214.05 ± 24.7 weight before treatment 20 186.81 ± 40.6 table 3. comparison of weight change between groups weight measurement time mean difference p weight before acclimatization vs weight before dmba induction -13.762 0.174 weight before acclimatization vs weight before treatment 13.476 0.513 weight before dmba induction vs weight before acclimatization 13.762 0.174 weight before dmba induction vs weight before treatment 27.238 0.012* weight before treatment vs weight before acclimatization -13.476 0.513 weight before treatment vs weight before dmba induction -27.238 0.012* *anova, significant difference (p ≤ 0.05) a b c d e figure 1. intergroup rat head morphology; a. negative control group (c-); b. positive control group (c+); c. the cisplatin treatment group (e1); d. 1% nanoparticle gel treatment group (e2); e. 3.3% nanoparticle gel treatment group (e3) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 213aniowati et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 210–215 could be seen externally on the rat’s head. group 3 (e1) had exophytic nodules with volumes that were not as large as the other group and felt supple and filled with tissue, but externally there was no sign of redness. a difference in size and texture could be felt on both sides of the buccal. group 4 (e2) had an exophytic type of nodule, almost the same nodule volume as group 3, felt supple, and external inspection showed no redness on the outer buccal skin of the rats. group 5 (e3) had an exophytic type of nodule, with a larger nodule volume than the other two treatment groups. external inspection revealed a reddish colour on the buccal outer skin of the rats, which indicates the progression of oscc to almost the external structure of the rat (figure 1). another indicator in determining the development of cancer cells is the measurement of nodule volume. the volume of the nodule was calculated by the formula (p x l2), and the length and width were measured in millimetres using callipers. the measurement of nodule volume occurred in all groups. a one-way anova test performed on the nodule volume data of the five groups showed a significant difference in nodule volume between the five groups during the two-week observation (p ≤ 0.05). this difference indicates the effect of dmba administration in inducing oscc (c+) and the effect of capsaicin nanoparticle gel administration (in treatment groups e2 and e3). the lsd post hoc test that compared between groups showed that the positive control group (c+) and the treatment groups (groups e1, e2, and e3) had a significant difference (p ≤ 0.05). comparison between treatment groups (groups e1, e2, and e3) showed no significantly difference, but clinically there was a difference mean nodule volume in the measurement results (table 4). comparison indicated the difference in the volume of nodules between the groups that were not given any treatment (c+), with the groups treated with cisplatin (e1) and capsaicin nanoparticle gel separately (groups e2 and e3) (table 5). discussion patients with oscc have several manifestations in the development of cancer cells, two of which are weight loss and the formation of oscc nodules. there was considerable weight loss in the rats used in this study, before dmba was induced and before treatment. this shows that weight loss is one of the symptoms of pain in cancer patients. generally, cancer patients experience a decrease in their quality of life, and major weight loss is one of the most prominent signs of this reduction.18 inducing dmba in rats to cause breast cancer showed a decrease in body weight in rats, especially in the dmba-induced group who died in the middle of the study. in addition to the weight loss of rats, this group also showed symptoms in the form of decreased appetite, then reduced movement, until their death.19 there was a substantial difference in body weight after the inducement of dmba between the control group and the treatment group. this is because cachexia, a cancerinduced weight loss condition, is initiated by cachectin, a tumour necrosis factor that breaks down fat and reduces fat-storing enzymes. in addition, a decrease in the rats’ feed intake could be a factor in their weight loss. this decrease table 4. comparison of mean volume of nodules groups n mean volume nodule ± sd sig. negative control 4 0 0.000* positive control 4 5.834 ± 2.77 0.000* cisplatin 4 1.75 ± 0.37 0.000* 1% capsaicin nanoparticle gel 4 1.747 ± 0.36 0.000* 3.3% capsaicin nanoparticle gel 4 1.812 ± 0.11 0.000* *anova significant difference (p ≤ 0.05) table 5. comparison of mean volume of nodules each group groups negative control positive control cisplatin 1% capsaicin nanoparticle gel 3.3% capsaicin nanoparticle gel negative control positive control 0.000* cisplatin 0.097 0.001* 1% capsaicin nanoparticle gel 0.098 0.001* 0.998 3.3% capsaicin nanoparticle gel 0.087 0.001* 0.951 0.949 *post hoc lsd, a significant difference (p ≤ 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 214 aniowati et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 210–215 could be due to difficulty in accessing food due to lumps in the rats’ buccals.20 the mechanism of action of capsaicin on cachexia is not fully understood, but some literature states that the relationship between giving capsaicin to patients with cachexia affects the amount of food intake. capsaicin is thought to increase food intake by induced vagal afferent signalling, which results in weight gain and decreased metabolism.21,22 the oscc nodule formation that extends extra-orally looks purplish-red to blackish and reaches the infraorbital to the anterior buccal area. this supports the description of cancerous lesions that oscc can be exophytic, endophytic or ulcerative. lesions can also spread to several parts of the oral cavity and tend to be more common in areas with non-keratinized mucosa because it has a thinner barrier than other mucosa.23 measurement of nodule volume is one indicator to detect the progression and spread of cancer. changes in the size of cancer tissue indicate the development of cancer. this study measured the administration of the extract to cancer and noted whether a change occurred in the nodule volume.17 in general, cancer develops constantly, and the nodule volume increases gradually from day to day. this characteristic shows that the measurement of nodule volume can be an indicator of cancer development.17 development of nodule volume also supports differences in post-dmba induction in rats. observations were made post-induction by palpation of the buccal mucosa. nodules with an average size of 1.9 mm3 that developed after two weeks of induction were found in the dmba-induced group, and no nodules were found in the control group. the results of the nodule volume measurement also showed a notable difference between the control group and the treatment group (p ≤ 0.05). this difference indicates that the nodule volume in the treatment group was smaller than the positive control group. comparison between the three treatment groups showed no major difference, but in the mean volume of nodules, there were differences in numbers between groups. there were differences of 0.01 between groups e1 and e2, and 0.1 between groups e2 and e3. groups e1 and e2 had no real difference in their nodule volume measurements, indicating there is potential for a study to be developed on 1% capsaicin nanoparticle gel, used in e2, due to its anticancer effect, which was comparable with the standard oscc treatment with cisplatin used in e1. the comparison between groups e2 and e3, namely the 1% and 3.3% capsaicin nanoparticle gel treatment groups, showed that the 1% group had better results. this better result in the 1% group is because the use of capsaicin at a dose of 3% can cause saturation of the capsaicin concentration, and so the absorption power of rats is not optimal.24 previous in vivo studies state that capsaicin has mutagenic potential, depending on the dosage and the treatment period, which is why lower doses were non-mutagenic when given for several days. the 1% concentrate showed the same anticancer mechanism as in previous studies, with action induced g0/g1 phase cell arrest, inhibited tumour growth and promoted apoptosis.25,26 therefore, administering anticancer agents to a nodule can slow the progression and development of cancer.17 generally, normal cells have apoptotic ability with different triggers. cancer cells form due to dna damage that means cell division and replication cannot be controlled also do not have regulation in apoptosis. however, in preventing uncontrolled replication that causes cancer, the body naturally has tumour suppressor genes, one of which is the p53 gene in the p53 protein. the concentration of p53 protein in normal cells is low, and if the cell is damaged by dna causing uncontrolled replication, activation of p53 protein is required to prevent further cell division. the p53 protein can inhibit the g1 phase in cell division. the inhibited g1 phase can provide an opportunity for cells to repair dna or carry out apoptosis to suppress the further development of cancer cells.27 capsaicin is thought to be able to phosphorylate p53 to inhibit the growth of existing tumours. the cytotoxic effect appears by giving various doses of capsaicin from the extraction of capsicum annuum l. var. angulosum against human squamous cell carcinoma and human submandibular gland carcinoma. the administration of capsaicin can activate p53 protein that will increase apoptosis in cancer cells.28 dmba induction could lead to serious differences in body weight between the groups and the growth of cancerous nodules on the buccal mucosa. administration of the capsaicin nanoparticle gel with 1% and 3.3% concentrations, along with cisplatin, can suppress the growth of cancer cells when viewed from the morphology of the nodules and the measurement of the nodule volumes between groups. nevertheless, capsaicin nanoparticle gel from green chilli pepper extract at levels of 1% reduces oscc nodule more than 3.3% concentration. we suggest further research using the same green chilli pepper (capsicum frutescens l.) or another variety of chilli pepper. we also suggest that future research use various concentrations and testing using histopathology and other apoptosis cell activities. acknowledgements the author is grateful to unsoed medical pharmacy laboratory for technical support for this research and the lecturers for input and revisions to the article. there were no conflicts of interest while conducting the research and write up. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 1–384. 2. salian v, dinakar c, shetty p, ajila v. etiological trends in oral squamous cell carcinoma: a retrospective institutional study. cancer transl med. 2016; 2(2): 33–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 215aniowati et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 210–215 3. dikova v, jantus-lewintre e, bagan j. potential non-invasive biomarkers for early diagnosis of oral squamous cell carcinoma. j clin med. 2021; 10(8): 1658. 4. mehanna h, robinson m, hartley a, kong a, foran b, fultonlieuw t, dalby m, mistry p, sen m, o’toole l, al booz h, dyker k, moleron r, whitaker s, brennan s, cook a, griffin m, aynsley e, rolles m, de winton e, chan a, srinivasan d, nixon i, grumett j, leemans cr, buter j, henderson j, harrington k, mcconkey c, gray a, dunn j, de-escalate hpv trial group. radiotherapy plus cisplatin or cetuximab in low-risk human papillomaviruspositive oropharyngeal cancer (de-escalate hpv): an open-label randomised controlled phase 3 trial. lancet (london, england). 2019; 393(10166): 51–60. 5. lavorgna m, orlo e, nugnes r, piscitelli c, russo c, isidori m. capsaicin in hot chili peppers: in vitro evaluation of its antiradical, antiproliferative and apoptotic activities. plant foods hum nutr. 2019; 74(2): 164–70. 6. al-samydai a, al-mamoori f, abdelnabi h, aburjai t. an updated review on anticancer activity of capsaicin. int j sci technol res. 2019; 8(12): 2625–30. 7. chang c-f, islam a, liu p-f, zhan j-h, chueh pj. capsaicin acts through tnox (enox2) to induce autophagic apoptosis in p53mutated hsc-3 cells but autophagy in p53-functional sas oral cancer cells. am j cancer res. 2020; 10(10): 3230–47. 8. silva-santana g, aguiar-alves f, silva le, barreto ml, silva jfr, gonçalves a, mattos-guaraldi al, lenzi-almeida kc. compared anatomy and histology between mus musculus mice (swiss) and rattus norvegicus rats (wistar). preprints. 2019; : 2019070306. 9. macedo as, castro pm, roque l, thomé ng, reis cp, pintado me, fonte p. novel and revisited approaches in nanoparticle systems for buccal drug delivery. j control release. 2020; 320: 125–41. 10. mubarokah n, setyawan hb, sholikhah u. kadar capsaicin dua varietas cabai rawit (capsicum frutescens l.) sebagai respon pengaruh dosis pupuk nitrogen. berk ilm pertan. 2015; 1(1): 1–5. 11. ghozaly mr, elfahmi. isolasi dan karakterisasi senyawa golongan kapsaisinoid dengan metode ekstraksi fluida superkritik dan metode konvensional dari tanaman cabai rawit (capsicum frutescens l). arch pharm. 2020; 2(1): 17–32. 12. raybaudi-massilia r, suárez ai, arvelo f, zambrano a, sojo f, calderón-gabaldón mi, mosqueda-melgar j. cytotoxic, antioxidant and antimicrobial properties of red sweet pepper (capsicum annuum l. var. llanerón) extracts: in vitro study. int j food stud. 2017; 6(2): 222–31. 13. singh m, mittal v. formulation and evaluation of herbal gel containing ethanolic extract of ipomoea fistulosa. int j sci res. 2014; 3(7): 1862–6. 14. megawati, roosevelt a, akhir lo. formulasi dan uji stabilitas fisik sediaan gel ekstrak kulit buah rambutan (nephelium lappaceum l.) sebagai obat sariawan menggunakan variasi konsentrasi carbopol. j farm sandi karsa. 2019; 5(1): 5–10. 15. cahyati m, rahmawati paa, kusuma n, adam sa. pemanfaatan antioksidan (glutathione) teripang emas laut (golden stichoupus variegatus) berbasis nanoteknologi dalam apoptosis sel skuamosa kanker mulut. e-prodenta j dent. 2018; 2(2): 149–54. 16. martínez b da, barato gómez pa, iregui castro ca, rosas pérez je. dmba-induced oral carcinoma in syrian hamster: increased carcinogenic effect by dexamethasone coexposition. biomed res int. 2020; 2020: 1470868. 17. nugrahaningsih, yuniastuti a. identifikasi apoptosis dengan metode tunel pasca pemberian ekstrak sambiloto dan pengaruhnya terhadap volume tumor. sainteknol. 2014; 12(2): 139–46. 18. kusnul z, suryono s, tamsuri a. ekstrak propolis memperbaiki profil berat badan tikus model kanker payudara yang diinduksi dengan 7,12-dymethyilbenz(a) antracene (dmba). media penelit dan pengemb kesehat. 2019; 29(2): 135–42. 19. normakiyah, nurani lh. pengaruh pemberian ekstrak etanol akar pasak bumi (eurycoma longifolia jack) terhadap ekspresi protein p53 pada kanker payudara tikus betina sprague dawley (sd) yang diinduksi 7,12-dimetilbenz[α]anthrasen (dmba). pharmacon. 2010; 11(1): 13–8. 20. peixoto da silva s, santos jmo, mestre vf, medeiros-fonseca b, oliveira pa, m s m bastos m, gil da costa rm, medeiros r. human papillomavirus 16-transgenic mice as a model to study cancer-associated cachexia. int j mol sci. 2020; 21(14): 5020. 21. szolcsányi j. effect of capsaicin on thermoregulation: an update with new aspects. temp (austin, tex). 2015; 2(2): 277–96. 22. ricardo de brito bello s, naliwaiko k, vicentini ms, rossetti fx, claudio fernandes l, messias-reason ij de. nutrition and cancercapsaicin treatment reduces tumor growth, tumor cell proliferation ex vivo and partially reverses cancer cachexia in walker 256 tumor-bearing rats. nutr cancer. 2019; 71(1): 111–7. 23. li q, dong h, yang g, song y, mou y, ni y. mouse tumor-bearing models as preclinical study platforms for oral squamous cell carcinoma. front oncol. 2020; 10: 212. 24. arnold jt, stewart sb, sammut l. oral capsaicin ingestion: a brief update-dose, tolerance and side effects. res rev j herb sci. 2017; 5(2): 1–5. 25. chapa-oliver am, mejía-teniente l. capsaicin: from plants to a cancer-suppressing agent. molecules. 2016; 21(8): 931. 26. clark r, lee s-h. anticancer properties of capsaicin against human cancer. anticancer res. 2016; 36(3): 837–43. 27. chen j. the cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. cold spring harb perspect med. 2016; 6(3): a026104. 28. sukmana bi, budhy ti, ardani igaw. the potentiation of mangifera casturi bark extract on interleukin1β and bone morphogenic protein-2 expressions during bone remodeling after tooth extraction. dent j (majalah kedokt gigi). 2017; 50(1): 36–42. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p210–215 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p210-215 vol 51 no 4 okt-des 2018.indd acetone, 170 achanthus ilicifolius, 206 acid fast bacilli, 91 adipose-derived mesenchymal stem cells, 195 age group, 81 alfafa chlorophy, 47 alveolar bone, 86 anadara granosa shell, 158 anemia, 29 angiogenesis, 1 anti-bacterial activities, 104 antimicrobial photodynamic therapy, 47 antioxidant, 164 applied behavior analysis, 71 autism spectrum disorder, 71 avocado leaf extract, 129 bacterial adherence, 33 behavior management,71 binjai leaf, 164 body mass index, 10 breadfruit leaves, 143 buccal mucosa, 91 calcium hydroxide, 20 calibration, 42 candida, 62 albicans, 206 caries,10 caspase-3, 138 cephalometric radiograph, 81 children, 216 chlorhexidine mouthwash, 5 chronic periodontitis, 99, 211 traumatic ulcer, 76 cockle shells, 114 composite resin, 37 compression strength, 57 consideration, 186 conventional, 174 cosmos caudatus leaf extract, 180 cytotoxicity, 108, 133 test, 195 demineralized dentin matrix, 195 dental caries, 216 dental health component, 119 scaries, 216 x-ray radiation, 99 dfbbx, 86 digital panoramic, 25 diode laser 405 nm, 47 early detection, 147 enterococcus faecalis, 20, 47 atcc 29212, 104 subject index volume 51 ethanol extract of binjai (mangifera caesia) leaves, 108 ethanol, 170 ethyl acetate fraction, 164 exposure time, 95 expression, 67 fgf-2, 190 fiber position, 57 fibroblast, 129, 143 flavonoid, 108 flexural strength, 57 gastrointestinal polyps, 29 gelatin, 114r general anesthesia, 186 gingival crevicular fluid, 25 display, 201 hema, 170 herbal medicine, 153 hiv management, 153 horizontal transmission, 216 hpdlfc, 133 human gingival fibroblasts culture, 180 hyperpigmentation, 29 ic50, 164 igy, 33 immunosupressed, 206 imodelanalysis, 174 inter-rater reliability, 42 intracanal medicament, 20 ionic silver (ag+), 138 junior high school students, 119 kindergarten, 216 kretek cigarette smoke, 37 lymphocytes, 99 macrophage, 99, 124 malabsorption, 29 malocclusion, 119 management, 76 mandibular bone density, 211 morphology, 81 mangosteen peel extract, 133 mauli banana stem extract, 67 mechanical force, 14 medicinal plant, 124 micronucleus, 25 moringa oleifera leaf extract, 86 mtt assay, 108 mucin, 52 mutant p53 expression, 138 mycobacterium tuberculosis, 91 nanofilled, 37 nanohybrid, 37 naocl, 133 nf-kb, 67 nickel ion release, 5 obturator, 62 ocimum sanctum, 104 odontectomy, 186 oral, 91 and maxillofacial surgeon, 186 bacteria, 124 candidiasis, 206 epithelium, 138 health survey, 42 lesions, 147 manifestation, 62 squamous cell carcinoma,76 orthodontic treatment need, 119 osteoclast, 14 palatal perforation, 62 percentage of gelatin, 158 perception of aesthetic, 201 periodontitis, 52 peutz-jeghers syndrome, 29 peutz-jeghers syndrome, 29 phyllanthus niruri,124 pineapple peel, 20 piper betle linn. mouthwash, 5 porosity, 114, 158 porphorymonas gingivalis (pg), 52, 211 post extraction wound, 190 pufa/pufa index, 10 punica granatum linn, 190 rankl expression, 14 rat diabetic model, 14 residual monomer, 170 result of analysis, 174 scaffold, 114, 158, 195 silk fiber reinforced composite, 57 worm fiber, 57 smile aesthetics, 201 socioeconomic status, 119 socket healing, 1 preservation, 86 sponge amnion, 1 stainless steel bracket, 5 staphylococcus aureus, 95 stevens-johnson syndrome, 153 streptococcus sanguinis, 33, 124 mutans. 216 submandibular gland, 52 surface roughness, 37 systemic lupus erythematosus, 62, 147 tgf-ß, 190 time period of analysis, 174 tlr-2, 206 tooth extraction, 143 training, 42 transmission streptococcus mutans, 216 traumatic ulcer, 67 tuberculosis (tb), 91 type-1 collagen, 86 udma, 170 vegf expressions, 1 vero cell, 108 wound healing, 67, 129, 143, 180 andriani, dwi, 206 apriasari, maharani laillyza, 29, 67 ari, desak putu sudarmi, 158 arina, yuliana mahdiyah da’at, 211 arsyada, intan fajrin, 20 astuti, suryani dyah, 47 asymal, alhidayati, 99 bakti, reiska kumala, 91 dwidhanti, fifi, 108 faizah, ariyani, 57 ferdynanto, reyhan alvaryan, 114 hidayatullah, taufiqi, 71 hikmah, nuzulul, 14 hutomo, suryani, 33, 124 indirayana, vita previa, 174 indrawati, retno, 216 k. khairiah, 164 khoswanto, christian, 129 lubis, hilda fitria, 119 muharram, r. aries, 138 munthe, eliza kristina m., 147 nelonda, revi, 76 nirwana, intan, 190 normayanti, 170 prisinda, diani, 104 purwanti, nunuk, 52 rasyida, anindita zahratur, 186 rinaldi, darin hulwani, 143 rizal, moh. basroni, 1 s. suniti, 153 salmiah, siti, 10 sari, desi sandra, 195 setianingtyas, dwi, 62 setyowati, laksmiari, 37 shabrina, zhafira nur, 180 shantiningsih, rurie ratna, 25 sijabat, yessy josephine, 201 sitanggang, maria, 81 soekobagiono, 86 susilawati, sri, 42 tambayong, adeline jovita, 95 tanti deriaty,5 yuanita, tamara, 133 authors index volume 51 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english. • abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english. • correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal nbkbmbi!lfeplufsbo!hjhj faculty of dental medicine, universitas airlangga editorial address c/o: jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone/fax: +6231 5039478 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg i want to subscribe the dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) ................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... e-mail: ..................................................................................... date/tanggal: ......................................................................... order description/ keterangan pesanan: ................................ ................................................................................................. 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..................................................................................... fax: .......................................................................................... e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick ( )] country issue* 1 year6 month surabaya rp 200.000,00 rp 400.000,00 java island (pulau jawa) rp 250.000,00 rp 500.000,00 outside java island (luar pulau jawa) rp 300.000,00 rp 600.000,00 other countries (negara lain) us $ 30 us $ 60 * quarterly publication (terbit 4 kali setahun) name/nama: .......................................................................... i am paying this journal by: [please tick ( )] institution/institusi: ................................................................... saya membayar jurnal ini dengan: [beri tanda ( )] address/alamat surat: ............................................................ bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... : 142-00-1613621-9account no. bank : bank mandiri account holder : drg. udijanto tedjosasongko, ph.d., sp.kga ................................................................................................. ................................................................................................. guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all research reports, case reports, and literature reviews must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur – malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima – japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya – indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne – australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in research reports should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in literature reviews should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted. introductions to literature reviews should be followed by clearly headline topics and the main points to be discussed.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. all research reports should include at least ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all 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no. : 988.01010.00000.135 bank : bank bni account holder : fkg dental journal ................................................................................................. ................................................................................................. vol 38-no4-2005-isi.pmd 198 post core restoration after an endodontic treatment slamet soetanto department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract disorder after an endodontic treatment of the crown portion of a tooth, needs efforts to restore its function and aesthetic. to support the intention for a restoration, a strengthener and an optimal retention in the root canal become necessary. a strengthener in the root canal is a fabricated dowel which can be directly applied. this technique has been developed by using a fiber substance. a restoration as an effort to return the function of mastication, can also suffer a failure. with a right design, among others the posthole preparation, post-choosing, and the core design, any unfavorable situation can be limited. key words: endodontic treatment, post core restoration correspondence: slamet soetanto, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction at first, the coverage of restoration was emphasized upon returning the mastication function and dental anatomical repair. in this advanced era, people give more attention to function and aesthetic. supported now by more sophisticated materials and dental restoration equipments, the restoration covers emergency care, even after trauma or post endodontic treatment.1 the crown portion disorder caused by caries or trauma can result in loosing teeth before its due time. the extent of the hard tissue defect, determines the dental measures to be taken. the severity of the damage has prohibited the operator to have enough retention to do the restoration. these cases can be handled by optimizing retention in the root canal.2,3 it is necessary to improve the quality of dental service, particularly post endodontic treatment by using a fabricated dowel as a root canal retention. that way, an aesthetic restoration with optimal expected retention can be achieved. mastication load and rotation force a restoration measure, with a sole objective to improve the mastication function of the existing crown portion, or simply for an aesthetic purpose, can diminish more dental structures during the preparation. for instance, during endodontic treatment, making access to the cavity entrance or the existence of a caries will weaken the dentin in the middle of the crown portion. should the operator restore it directly, there is a possibility that it will not be strong enough to receive a mastication load.1,2 the anterior upper teeth receive a mastication load with a movement outside the curve towards the labial side. the load is concentrated to the labial marginal gingiva. while the anterior lower teeth bring the load to the dental apex and towards the inside of the dental curve. for the posterior teeth, the load is more concentrated to the center of the crown, causing a crack or fissure at the rest of the buccal or lingual crown portion.4 the rotation point has formed a rotating force at the bucco gingival crown portion or at linguo gingival part away from the crown. the crown’s movement is caused by the load hitting the dental cusp, especially for the posterior teeth i.e. at the fossa fissure of buccal cusp. up to a certain limit, the load can cause a crack at the remaining crown portion. to delete the fracture point, the heavy load should be spread along the dental root till the dental apex. thus, the remaining crown portion support -in a fabricated dowel form as a strengtheneralthough it is not a compulsory usage, can be greatly considered.1 the risk of a dowel placement in the root canal a dowel is essentially required for a restoration with a root canal retention to form the strength and integrity of the remaining crown portion and the dental root, to stay firm at its position. a mispositioned dowel placement will give an unexpected result. an excessive canal preparation is a dowel with a huge size weakening the remaining of dental root structures.5 molded dowel in flat root canal, will automatically form a flat dowel with less strength. on the other hand, a cylindrical or parallel dowel with unsuitable size to a small dental root, will eventually cause a fracture at the tip of the dowel. a fabricated cylindrical dowel can easily rotate after a masticating load, where an anti rotation preparation is required. a short dowel performs risks such as easily levered, small retention, easily detached and a crack at the cervical part of the remaining root.3,4 a successful treatment is backed up by more cautious measures during the root canal filling, taking out the guttap 199sadamori: comparison of recognition about denture adhesive and during the preparation of widening the canal for the dowel. principles for the preparation of the canal for the dowel restoration a dowel restoration is gravely needed to maintain optimally the remaining structure of the crown portion, particularly one third of the crown gingivally. the canal hole for the dowel should be made conservatively, sufficient and adequate. attention should be given to one third of the dental root plane at the cervical side especially when a molded dowel is going to be placed. the length of the canal must be in accordance with the requirement and a good obturation at the dental apex. part of the dowel which enters the canal, minimally must be the same length with the crown, whereas the height of the dowel outside the root is two third of the cervico incisally. figure 1. the proportion of the planned dowel. explanation: a: core length, b: dowel length, c: remaining guttap, d: root length, e: alveolar bone at best, the border of the preparation at the cervical part should be well formed for the placement of the artificial crown. the possibility of lateral perforation should be avoided, especially at flat dental roots. for flat roots, it is best to choose a fabricated dowel. careful choices of the right dowel, must be done. an xray photo will help to choose the right size, the exact dowel length to enter the preparation, and the size of the dowel against the dental root plane. a good dowel placement will obtain an optimal physical characteristics.4,5 it is important to prepare thoroughly the canal for the dowel, prior to preparation by differentiating the types of the dowels. for an active dowel or a graved dowel, the preparation of the canal must be smaller than the dowel plane. while for a passive dowel, the canal must be bigger than the dowel itself. the preparation should be done according to the sequence of the numbers to avoid broken equipments inside the root canal.3 canal widening for the dowel is ± one third of the dental root plane. for anti rotation the preparation should be made ovally. the minimal length of the dowel is the same with the clinical crown length. during the process of taking out the guttapercha, one can use pessoreamer, glade glidden drill, or with a heated reamer. it is hoped that during this widening process, no diverted preparation-course take place. what must be prepared is the anatomical dental root problem towards the preparation of the canal. in general, the anatomical shape of the dental root is conical. thus, should the dowel to be placed is cylindrical with a broader plane, risk will emerge in the form of a weak dental root, exactly at the tip of the dowel. a good dowel can form a precise, initial fit, with an adequate plane matching to the restorated dental dowel hole. besides, the distance of the teeth with their antagonist, the curvature of the coreand its paralelly, all should be examined meticulously.2 the failure of the dowel restoration several reasons of the failure are due to reckless preparation plan, the choice of the dowel and the crown, all contributes to the catastrophe.1 the failure can be: 1) a dowel detachment as a result of the incompatibility of dowel and dowel hole; 2) a dowel fracture, if the dowel size is too small, without a site; 3) a root fracture, due to a too big dowel size; 4) a crown detachment as a result of nonforming parallelity, and 5) a crown fracture, if the load is too big and the planning for the crown is not right.1 the utilization of dowel can provide beneficial services to the patient with a shorter length of treatment and a direct application technique is also an advantage for the operator. the direct dowel restoration technique has been developed with fiber materials. the latest development of the dowel, is the use of fiber reinforce composite as a replacement of metal dowels. the fiber post system is a root canal dowel using a carbon fiber material, strengthened with an epoxy matrix at the exterior. because the lateral part and the core of the dowel is made of composite resin, it is generally named as composite post. this dowel type is already fabricated with a cylindrical form, sized 1.4 to 2 mm. the application of this type is directly, initiated by choosing the right size. after the preparation of the root canal, a test was performed, followed by primary covering of bonding a and b that mixed together. the covering formed a thin layer, done up to the crown’s roof. the carbon fiber dowel was also bonded by using a paper point. the next stage was filling the dowel hole preparation, with bis-core-build-up composite (core flo composite) by mixing base and catalyst and put them in a syringe. before setting, the carbon fiber post was inserted as soon as possible. the excess composite can be used to form the core.6,7 another post fiber system is a product of nulite f. australia. the dowel is self-made using a fiberspan or a poly alkane fiber. several fibers are taken in accordance to the dowel hole plane, and then strengthener by covering with bis.gma urethane dimethacrylate or nsi.resist. show that it can be prooved in the canal according to its length. the core was formed by adding resin composite nsi f.2 microrod reinforced, and the crown can be made at once. after finishing, the crown is covered with microglacing, resulting in a smooth, shining crown surface (nulit system international information). 200 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 198–200 discussion the dental crown disorder can be caused by a fracture or dental caries. if the damage is more than two third of the crown, an endodontic treatment is a necessity. this is due to the lessening of the inner dental tissue after a root canal treatment, where the tooth is not vital and turns to be more fragile. this condition requires an effort to prevent more severe impairments by placing a dowel in the root canal and making the restoration. the management of placing a dental restoration with a dowel is to form resistance and an optimal retention in the root canal. this is the importance of the proportion of the dowel’s length in the root canal towards the core’s length, so as to prevent the failure of the dowel’s restoration caused by a changed form of the dowel, or a broken dowel. the alveolar bone as a support of the dental restoration must not be destructed at the apical part by having not less than two third of the dental root length. choosing the dowel, must be done carefully under certain conditions i.e. the size of the dental root plane, and the expected aesthetic goal. as an example, for improving position or crown angulation, the operator can select a molded dowel. for a small/flat root canal, a prefabricated dowel from metal or fiber composite post can be chosen. the above matter was explained by shillingburg.4 a molded dowel restoration needed a preparation of a wideenough root canal, that was two third of the dental plane. if the dental root was flat as the anterior lower molar, a preparation could weaken the dental root, even a perforation to the lateral side. for flat root teeth, there must be adequate, never an excessive preparation. the best dowel is a metal fabricated one. from the discussion, a conclusion was derived that to be able to maintain the remaining tooth post endodontic treatment, a strengthener inside the root canal in a dowel form was required. to have an optimal endurance, the dowel should be retentive, stable, and proportional, with a prerequisite of the dowel’s length inside the root canal minimally the same length with the crown’s restoration. the dental root length receiving a dowel, should be supported by two third of the alveolar bone. to obtain the expected optimal quality of the restoration, all clinicians should comply to proportional principles of the restoration plan. references 1. smith h. restoration of endodontically treated of teeth, a guide for the restorative dentistry. quintessence int 1997 ; v(28): 457-9. 2. shillingburg ht, yacobi r. fundamental of tooth preparation. 1st ed. chicago, berlin, rio de jeneiro, tokyo: quintessence publishing co inc; 1991. p. 355-7. 3. mount gj. preservation and restoration of tooth structure. 1st ed. philadelphia, st. louis, sydney, tokyo: cv mosby; 1998. p. 21823. 4. shillingburg ht, kessler jc. restoration of endodontically treated tooth. 1st ed. chicago, berlin, rio de jeneiro, tokyo: quintessence publishing co inc; 1982. p. 123-7. 5. abou-rass m. preparation of space for posting, effect on thickness of canal wall and incidence of perforation in molar. amer dent assoc 1982; v(104): 834-6. 6. bruce w. clinical application and use of the carbon fiber post. restorative quarterly bisco. 1998. 7. ferrari m. special issue laboratory and clinical data. new developments in fiber post system 2000; 3-19. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 49 no 1 jan-mrt 2016.indd 55 research report dental journal (majalah kedokteran gigi) 2016 march; 49(1): 5–9 the difference of saline and sterile water for tetracycline hydrochloride solvents in cementum demineralization shinta ferronika, ahmad syaify, and dahlia herawati department of periodontics, faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: the root cementum demineralization is an important step in regenerative periodontal therapy to smear layer removal on the root surface. smear layer on the root surface becomes a barrier of the new attachment between periodontal tissues with the root surface. the use of tetracycline capsules as root surface demineralizing agent cannot be applied directly on the root surface and solvents such as saline or sterile water are needed. purpose: the aim of this study was to determine differences between sterile water and saline solvent for tetracycline hcl (tetra hcl) as a cementum demineralization. method: in this study the specimens were divided into three groups: a control, tetra hcl dissolved in saline, and tetra hcl dissolved in sterile water. application using burnishing method for 3 minutes. samples were dehydrated with ethanol series of 30% to 100%. results of the root demineralization observed by scanning electron microscopy (sem). statistical analysis was performed using the kruskal-wallis followed by a mann-whitney nonparametric test. result: upon statistical analysis showed that the sterile water as a solvent of tetra hcl is more effective in smear layer removal and collagen structure exposure in the cementum. conclusion: tetra hcl dissolved in sterile water was found to be the best root cementum demineralization agent. keywords: root demineralization; cementum; tetracycline hcl; sterile water; saline correspondence: shinta ferronika, department of periodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: drg.ferronika@gmail.com introduction the root cementum demineralization is one important step in periodontal regenerative therapy. the aim of root surface debridement is to reduce the amount of bacteria and endotoxins on the root surface, treatment of the root surface with demineralizing agents such as acids, edta, and tetracycline primarily aims to expose collagen fi brils. to achieve this, the smear layer must be removed and the mineralized component of the supefi cial layer of cementum needs to be decalcifi ed.1 cementum is a mineralized tissue with primary function to insert the ligament fibers on the root surface.2 biological concept of demineralization of the root surface is eliminating the smear layer and helps blood clot adhesion to collagen exposed, thus playing a supporting role in the formation of connective tissue new attachment.3 the creation of a biologically acceptable root surface basically means that the root surface debridement should not hinder resolution of inflamation and without causing intentional removal of cementum.4 the structure of root cementum are multiloculated for the insertion area of sharpey fibers.5 failure of connective tissue regeneration characterized by long junctional epithelium extending between the root surface and the gingival connective tissue.6 some research has been suggested that endotoxin present in the cementum could impair periodontal healing and should be removed to promote a more biologically acceptable surface than the one obtained only after scaling and root planing. previous studies on tissue regeneration have used tetracycline hydrochloride (ph 1-2) to clean the root surface because it’s bactericidal and demineralizing effect better than citric acids and edta. however, it is not clear the real performance of this protocol on the root dentin, mainly the use of tetracycline capsules, regarding product residues left after use and the smear layer removal capacity.7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p5-9 6 ferronika, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 5–9 the use of tetracycline capsules cannot be applicated directly on the root surface. tetracycline hydrochloride (tetra hcl) is soluble in water, but dibart and karima explained that tetra hcl are dissolved in saline for root demineralization.8,9 the tetra hcl capsule dissolved in saline showed a severe demineralization of root dentin substrate with the presence of a high amount of residues on the surface, but there is no residue appear when dissolved in sterile water with same concentration.10,11 the solubility is dependent on the solvent intrinsic properties and solutesolvent interactions. it suggest that sodium chloride (nacl) are soluble in water (h2o), and less soluble in acid (hcl).12 the use of water in pharmaceutical industry is indispensable, especially in pharmaceutical liquid preparations. it serves many purposes such as an ingredient, solvent, excipients, for reconstitution of product, during synthesis, cleaning agents and other purposes in the production, processing and formulation of pharmaceutical products. water as a universal solvent is able to dissolve, adsorb or suspend many different compounds.13 all that is fine for pure water, but in saline solution some other factors need to be considered especially with the ions in solution.14 saline solution containing sodium (na) and chloride (cl), which tetracycline hcl has the same chloride ions. it suggest that further study needed to observed the root demineralization outcome based on pharmacological characteristic of saline or sterile water and its use for tetra hcl application. the aim of this study was to determine differences between sterile water and saline solvent for tetra hcl as a root cementum demineralization. research about the difference between sterile water and saline for tetra hcl solvent as a root cementum demineralization need to be established for science information in dentistry, especially regenerative periodontal therapy. materials and methods the design of this study was a laboratory experimental research and assessed by scanning electron microscopy (sem). samples were obtained from fresh extracted tooth, with no caries or restoration on cemento enamel junction (cej), and no periodontal treatment for last 6 months. a total of 14 single-rooted human teeth were used in this study after approval by the research ethics committee of fakultas kedokteran gigi, universitas gadjah mada, yogyakarta, indonesia (protocol #00243/kkep/fkgugm/ec/2015). samples were scaled with ultrasonic scaler and root planed with gracey currete for remove calculus and macro debris. two parallel grooves with approximately 2 mm deep were made using a high speed cylindrical bur under copious irrigation. one groove was made at the cej and another one approximately 4 mm distant from the first, in the apical direction. the mesial and distal root surfaces of each tooth were used in sample preparation. the specimens randomly divided into 3 groups (n=9/ group): control root surfaces were scaled with a ultrasonic scaler to remove calculus deposits, thus exposing visual clean dentin (this was the first step for all the others groups); saline + tetra hcl after scaling the specimens was etched for 3 min with a solution obtained by dissolving 500 mg capsule of tetracycline hcl in 6.7 ml of saline solution; and sterile water + tetra hcl after scaling the specimens was etched for 3 min with a solution obtained by dissolving 500 mg capsule of tetracycline hcl in 6.7 ml of sterile water solution. this method based on ishi et al.3 study that using concentration of tetra hcl 75mg/ml with stirring the capsule with solvents. application of the respective agents on the sample was done by burnishing method with cotton pellets saturated with the agent that were changed every 30 seconds for a total period of 3 minutes based on vandana et al.15 study. following treatment, samples were rinsed with 10 ml sterile water and air dried. samples were dehydrated in an increasingly graded series of ethanol: 30, 50, 70, 80, 95 and 100%. then, the samples were dried overnight in a table 1. mean and sd smear layer percentage and collagen structure scoring on cementum after treatment no. group mean and sd smear layer collagen structure 1. control 3.00 ± 0.00 1.00 ± 0.00 2. saline + tetra hcl 1.67 ± 0.50 2.00 ± 0.00 3. sterile water + tetra hcl 1.00 ± 0.00 2.67 ± 0.50 figure 1. cementum root surface with multilocular form and mineralized in the edge (magnif. 250x). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p5-9 77ferronika, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 5–9 dehydration jar, mounted on metallic stubs, sputter-coated with a thin titanium in a sputter coating machine (jeol jec3000fc), and examined with a sem (jeol jsm-6510la) at laboratorium penelitian dan pengujian terpadu, universitas gadjah mada, yogyakarta, indonesia. the surfaces of root specimens were scanned and observed on the computer screen fitted with the sem at 100x and 1000x magnification. each root surface was scanned in its entirety to obtain an overview of the general surface topography. representative areas which were characteristic of the general surface topography were selected on each specimen and photographed. the cemental sem photographs were scored for smear layer based on vandana et al.15 using following scoring criteria: 0 = none; 1 = smear layer involving random areas of surface that totals between 1-32% of total surface area; 2 = smear layer involving random areas of surface that totals between 33-65% of total surface area; 3 = smear layer involving > 66% of total surface area.15 the sem micrographs were scored according to ranking system by houshmand et al.21 for collagen structure exposure from cementum. grade 3: collagen structure could be seen and no debris present. grade 2: some collagen structure could be seen, although some debris is present. grade 1: significant debris may be seen and no collagen structure. the data were analyzed using the kruskal wallis statistical test. results normality and homogeneity test were done for both groups with the results are non-homogen distribution and not normal data. based on this results, data analyzed with kruskal wallis test. kruskal wallis test were done for the comparison of score percentage of smear layer per unit area and cementum collagen structure between the three study groups showed significance difference results (p<0.05). mann-whitney test were done for the mean difference of within groups showed that between saline + tetra hcl and sterile water + tetra hcl group are significantly difference (p< 0.05). data was conducted using the sem photomicrograph with the results as shown on figure 2 and figure 3. discussion the traditional treatment of pathologically altered root surfaces has relied on mechanical removal of plaque and calculus and contaminated cementum. but it is not possible to decontaminate a periodontitis affected root surface completely by mechanical means alone. root surface conditioning by topical application of acidic solutions has been demonstrated to remove not only root instrumentation smear layer but also any remaining root surface contaminants.16 morphologic alterations and in vitro demineralization seem to be dependent on the nature figure 2. surface morphology of smear layer cementum specimen (magnif. x100): (a) control; (b) saline + tetra hcl; (c) sterile water + tetra hcl. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p5-9 8 ferronika, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 5–9 of the treated root surface, the method of application, the optimal concentration and application interval of tetracycline. these root surface characteristics which may support periodontal reconstructive therapy are important considerations.17 in the present study, the concentration of tetra hcl was fixed as 75 mg/ml in sterile water or saline solution keeping into consideration the observation of various studies. previous study used different tetracycline hcl concentrations of 0, 10, 25, 50, 75, 100, 125 and 150 mg/ ml for root demineralization and found that concentration between 50 mg/ml and 150 mg/ml showed a statistically significant opening of dentinal tubules. the solution was applied using “burnishing technique” in the present study. it has been observed by various studies that a burnishing technique resulted in a chemical/mechanical action that enhances the removal of chemically loosened inorganic material and surface debris, exposing the underlying root surface to the demineralization action of fresh acid solution. this may ultimately achieve an optimal degree of demineralization within a short period of time, in comparison to other application modes.18 results of this study are discussed below on the percentage of smear layer and the collagen structure exposed cementum. control group showed there is no apparent effect on the smear layer after scaling and water irrigation. this is in accordance with findings of bhavikatti et al.19 who found that water irrigation appeared to remove only the superficial portion of the smear layer, leaving root surface occluded with debris. the results of test groups showed the average percentage of smear layer on the root cementum with sterile water + tetra hcl group was lowest and significantly different than saline + tetra hcl group. this means that sterile water solvent at tetra hcl was more effective in lowering the percentage of smear layer compared with saline solvent. tetra hcl well dissolved in sterile water (h2o), while the solvent is saline (nacl) in the tetra hcl produce a crystal residue as seen on figure 3b. the crystalline residue derived from excess chloride ion (cl-), which binds between hydrochloric acid and sodium chloride (nacl), causing the precipitate in solution and create the effect of demineralization ineffective. our results were consistent with the findings of soares et al. who found similar results of crystalline residue from dissolving saline with tetra hcl. sodium chloride less soluble in hydrochloric acid as compared to water, due to the high concentration of chloride ions in a solution of hydrochloric acid. the solubility of the studied antibiotics in water is due to the presence of the hydrochloride group, which, in water, becomes cl-, which leads to the formation of ionic species and, thus, promotes an enhancement in the solubility.20 results of this study showed that the means of amount of collagen structures exposed cementum on sterile water solvent tetra hcl group at most numerous and significant than saline solvent tetra hcl group. this means sterile water solvent at tetra hcl more exposed cementum collagen structure compared with saline solvent. the amount figure 3. surface morphology of cementum collagen structure (magnif. x1.000): (a) control; (b) saline + tetra hcl; (c) sterile water + tetra hcl. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p5-9 99ferronika, et al./dent. j. (majalah kedokteran gigi) 2016 march; 49(1): 5–9 of collagen structure more exposed to the sterile water solvent at tetra hcl group due to the effect smear layer removal better than saline solvent, so that the collagen structure that was originally coated by the smear layer can be exposed. this showed that better smear layer removal, the cementum collagen structure will be more visible.21 the result is supported by the action of chelating agents in tetra hcl as demineralization process in dissolving metals and other mineral.22 this study used same concentrations of tetra hcl (75mg/ml), application times (3 minutes), application method (burnishing technique), but with different solvents (saline or sterile water) for both test groups. the effect of demineralization with sterile water solvent for tetra hcl better than saline solvent, this means that sterile water as a solvent of tetra hcl is more effective for root cementum demineralization. this results was contrast with the findings of the silva et al.23 study where used saline as the solvent of tetra hcl, who found the residues showed after application on the root surface are probably related to the components present into the capsules, which were not completely dissolved. this study presents some limitations such as the minimum samples were used. further investigations needed for larger samples. other relative limitation of this study is the difference on the time application for each substance. the difference time on the performance of the different products on the root dentin may impair on the results on this. however it is a relative limitation, because it is not possible to adjust the same time application for the different samples. hence, additional studies of this variables are needed to validate the present findings. within the limits of this study, it can be concluded that tetra hcl dissolved in sterile water was better than in saline solution as a root cementum demineralization agent. in view of the present findings, further studies are necessary to establish the in-vivo importance of tetra hcl application as root demineralization agent as an additional step during periodontal therapy, especially in regenerative procedures. acknowledgement the authors are indebted to dr. ir. harini sosiati, m. eng for technical support in scanning electron microscopy. references 1. bosshardt dd, sculean a. does periodontal tissue regeneration really work?. periodontol 2000 2009; 51(1): 208-19. 2. newman mg, takei h, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. canada: elsevier saunders; 2015. p. 28-31. 3. ishi ep, dantas aa, batista lh, onofre ma, sampaio je. smear layer removal and collagen fiber exposure using tetracycline hydrochloride conditioning. j contemp dent pract 2008; 9(5): 2533. 4. george md, donley tg, preshaw pm. ultrasonic periodontal debridement: theory and technique. 1st ed. united kingdom: john wiley & sons; 2014. p. 3-22. 5. kumar gs. orban’s oral histology & embriology. 13th ed. new delhi: elsevier; 2011. p. 162. 6. polimeni g, xiropaidis av, wikesjo um. biology and principles of periodontal wound healing/regeneration. periodontol 2000 2006; 41: 30-47. 7. cavassim r, leite fr, zandim dl, dantas aa, rached rs, sampaio je. influence of concentration, time and method of application of citric acid and sodium citrate in root conditioning. j appl oral sci 2012; 20(3): 376–83. 8. berkovitz b, moxham b, linden r, sloan a. master dentistry volume three: oral biology. 1st ed. oxford: elsevier; 2011. p. 176. 9. dibart s, karima m. practical periodontal plastic surgery. united kingdom: blackwell publishing company; 2006. p. 37. 10. soares pbf, castro cg, branco ca, magalhães d, neto ajf, soares cj. mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy. braz j oral sci 2010; 9(2): 128-32. 11. chahal gs, chhina k, chhabra v, bhatnagar r, chahal a. effect of citric acid, tetracycline, and doxycycline on instrumented periodontally involved root surfaces: a sem study. j indian soc periodontol 2014; 18(1): 32-7. 12. lagowski jj, sorum ch. analisis kualitatif semimikro. 8th ed. jakarta: egc; 2012. p. 75-79. 13. ukwueze se, okpaleke cg, shorinwa oa. physicochemical and microbiological assessment of some commercially available sterile water for injection brands in nigeria. world j pharm res 2015; 4(6):186-96. 14. reddi ba. why is saline so acidic (and does it really matter?). int j med sci 2013; 10(6): 747-50. 15. va nda na k l, sada na nd k, cobb cm, desa i r. effects of tetracycline, edta and citric acid application on fluorosed dentin and cementum surfaces: an in vitro study. open corros j 2009; 2(1): 88-95. 16. grover hs, yadav a, nanda p. a comparative evaluation of the efficacy of citric acid, ethylene diamine tetra acetic acid (edta) and tetracycline hydrochloride as root biomodification agents: an in vitro sem study. j periodontol implant dent 2011; 3(2): 73-8. 17. mittal m, vashisth p, chaubey kk, dwivedi s, arora s. comparative evaluation of root surface morphology after planing and root conditioning with tetracycline hydrochloride--an in vitro sem study. j tenn dent assoc 2014; 94(1): 21-6; quiz 26-7. 18. nanda t, jain s, kaur h, kapoor d, nanda s, jain r. root conditioning in periodontology revisited. j nat sci biol med 2014; 5(2): 356-8. 19. bhavikatti sk, karthikeyan bv, prabhuji mlv. comparative sem study on tetracycline hydrochloride root conditioning: the effects of different concentrations and application times. int res j pharm 2015; 6(7): 423-30. 20. varanda f, pratas de melo mj, caco ai, dohrn r, makrydaki fa, epaminondas v, solubility of antibiotics in different solvents. 1. hydrochloride forms of tetracycline, moxifloxacin, and ciprofloxacin. ind eng chem res 2006; 45(18): 6368-74. 21. houshmand b, ghandi m, nekoofar m, gholamii ga, tabor rk, dummer pm. sem analysis of mtad efficacy for smear layer removal from periodontally affected root surfaces. j dent (tehran) 2011; 8(4): 157-64. 22. tripathi kd. essentials of medical pharmacology. 6th ed. new delhi: jaypee brothers medical publisher; 2008. p. 733-42. 23. silva ac, moura cc, ferreira ja, magalhães dd, dechichi p, soares pb. biological effects of a root conditioning treatment on periodontally affected teeth an in vitro analysis. braz dent j 2016; 27(2): 160-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i1.p5-9 102 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 102–107 case report space maintainer ‘y model’ as a preventive orthodontic treatment for paediatric patients: a case report laelia dwi anggraini,1 sunarno2, rinaldi budi utomo3 and dibyo pramono4 1department of pediatric dentistry, school of dentistry, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta 2department of nuclear engineering and physical engineering, faculty of engineering, universitas gadjah mada 3department of pediatric dentistry, faculty of dentistry, universitas gadjah mada 4department of dental health community and preventive dentistry, faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: caries is one of the most common oral diseases that occur among children. caries and dental trauma in children may cause early tooth loss, also known as premature loss, and result in occlusion abnormalities caused by the dental arch narrowing. a space maintainer is a preventive orthodontic appliance designed to maintain a narrow arch to prevent premature loss. purpose: this study aims to describe the treatment of a case of space management in a patient with premature loss by using the space maintainer ‘y model’. case: an eight-year-old boy was accompanied by his mother, complaining that the lower posterior right tooth had been extracted. the mother was worried that the new tooth would have an overlapping growth. case management: the diagnosis was mandibular primary molar loss. the study cast was analysed based on moyers 2.62 cm, huckaba 2.24 mm, and curve determination 2.40 mm. the mandibular removable space maintainer treatment was performed on the patient and was followed by nine control visits every week. the outcome was a successful treatment from the use of the space maintainer ‘y model’. conclusion: the space maintainer treatment with the y model in the paediatric patient showed a good result, evidenced by the tube opening of 1.2 mm, showing that the appliance followed lateral jaw growth. keywords: paediatric; premature loss; preventive; space maintainer; space management correspondence: laelia dwi anggraini, department of pediatric dentistry, school of dentistry, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta. jl. brawijaya, kasihan, bantul, yogyakarta 55183 indonesia. email: laelia.dwi@umy.ac.id introduction a primary dentition tooth is a tooth that commonly grows in a child at the age of 6 months and is replaced by the time the child is 6 years old.1 the majority of posterior primary dentition tooth losses are caused by dental caries. in addition, it may also be caused by tooth-related accidents.2 the definition of premature loss is to lose primary dentition teeth too early, meaning that the primary dentition teeth have fallen out but the new teeth have not grown yet.3 this condition is frequently found in children and increases in frequency with age.4 according to mc donald, the prevalence of premature loss of primary dentition teeth, as reported in a study, ranges from 4.30% to 42.60%.5 as seen recently, one issue is that the primary dentition molar teeth are extracted or fall out earlier, and both sides of the mesial or distal areas tend to shift or move in the direction of the open space. this shift hinders the permanent teeth that have not grown yet.5 preventive orthodontic treatment on children’s mixed dentition age is necessary, as losing primary dentition teeth hampers jaw growth.6 a space maintainer (sm) is a preventive orthodontic appliance that maintains the resultant space in the case of primary dentition tooth loss. a diagnosis for paediatric patients is important to decide if an sm is needed. sms are vital for cases of premature loss of primary dentition teeth to prevent malposition, supraeruption, impaction, or permanent dentition crowd.7 an sm can be used if there is a lack of space on one side of the jaw of 2–4 mm.5 removable sm devices, also known as preventive orthodontics, can be used to maintain the space for paediatric patients for the prevention of dental crowding problems.2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 mailto:laelia.dwi@umy.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 103anggraini et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 102–107 a removable sm is disadvantageous because of its interference with lateral jaw growth and stoppage of the growth of the intercanine jaw arch. this is because conventional sms do not split in the middle of the acrylic plate1. in addition, it is common for sms to fail, resulting in the shifting and movement of adjacent teeth. it can also lead to drifting, resulting in a more complicated treatment and appliance.2 furthermore, the aim of this case report describes the space maintainer ‘model y’, which uses a double tube in the middle of the appliance, and can follow growth and develop the mandibular jaw in line with lateral and anteroposterior angles. case the patient was an eight-year-old boy in the dental hospital. the patient came after being motivated by the operator and his mother to take care of the tooth that had been extracted. the patient’s mother complained that her child’s teeth were crowded and that she was worried that there was not enough space for the new teeth to grow. the unique part of this case was the installation of a modified preventive orthodontic appliance that would follow the growth of the jaw laterally and anteriorly, without the need to change tools every month. it is shown in figure 1 and figure 2. dental history showed that the patient admitted that there was empty space in the lower right molar after the extraction was done. before it was extracted (about a year ago) the patient felt pain that interfered with his eating, but the patient did not go to the dentist for examination. about two months ago, the patient came to the oral and dental hospital universitas muhammadiyah yogyakarta to have the tooth filled. the last oral medical record indicates that the patient went to the dental hospital for tooth filling and tooth extraction. the patient was instructed to brush his teeth regularly (2–3 times every day); however, the patient’s way of brushing his teeth was incorrect. the patient chewed food on both sides. there was no bad habit related to the patient’s complaints in terms of type of habit, duration, frequency, or intensity. the patient’s oral hygiene was good. the family’s medical record showed that the patient has a father with moderate jaw size, neat teeth, and no apparent history of systemic disease. the patient’s mother also has moderate jaw size, neat teeth, and no suspicion of having a history of systemic disease. the patient was quite cooperative and lives in his home with his parents. the patient’s parents run a laundry business, which is crowded with customers every day; thus, they don’t have much time to care for their children’s teeth. the patient has never been hospitalised. currently, the patient is in good health. the dental analysis showed that the dental age was early-mixed dentition. the curved shape of the teeth of the maxilla was parabolic and the mandible was parabolic. the malposition of individual teeth showed that in the upper jaw there were 12 and 21 (mesiopalatotorsiversions). the lower jaw showed 31 and 74 (mesiolinguotorsiversions), and 83 distolinguotorsiversions. the relation of occluded teeth was in the centric occlusion. from the anterior view, there was an overjet of 3.8 mm (distal tooth 21 and mesial 32) and an overbite of 2.10 mm (distal tooth 21 and mesial 32). the posterior view (permanent molar relation) on the left showed a class ii angle malocclusion and on the right showed a class i angle malocclusion. the occlusal view of the lower jaw is shown in figure 1 and the space maintainer appliance can be seen in figure 2. the maxillary midline and the lower jaw were aligned. this condition is shown in figures 3a, 3b, 3c, 3d, and 3e. figure 4 shows the schem space maintainer appliance with triple tube junction. case management the treatment for this case started with the implementation of the sm case based on the completed calculations (table 1). the measurement of the mesio-distal width gained from the study model resulted in the measurement of available spaces 63, 64, and 65, using callipers and measuring from the lateral incisivus distal surface to the mesial of the first permanent molar in each quadrant. the following data was obtained: the right lower jaw was 20.80 mm, and the left lower jaw was 21.80 mm. first, a measurement of the size of the mesio distal of the canines and permanent premolars was taken. it was determined that all lower premolars and canines were 7.00 mm and the upper canines 8.00 mm, respectively. the upper jaw (canines, premolar 1, and premolar 2 [cpp]) right molar relation: normal occlusion left molar relation: normal occlusion figure 1. occlusal view of lower jaw (source: author’s document). doble tube follows the jaw figure 2. space maintainer appliance. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 104 anggraini et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 102–107 was 22.00 mm, while the lower jaw’s cpp was 21.00 mm. in order to find the either adequacy or lack of space for cpp teeth in each quadrant, the space available in each quadrant was compared to the mean distal mesio cpp. based on this method, it was apparent that the right (20.80 mm–21.00 mm) = 0.20 mm and the left (21.80 mm–21,00 mm) = 0.80 mm. second, a measurement of the size of the mesio distal of the four lower incisors (moyers) was taken. with the moyers method, the teeth are used as predictors of the four lower incisors. the measurement of the mesio distal width of the four lower incisivus teeth was done in a straight line. the result was 24.30 mm. the predicted mesio distal width of canines and premolars number is determined using a moyers table. in the moyers table, it is shown that the mesio distal width of the lower incisivus was 4.3 mm, the mesio distal width of teeth c, p1, p2 was 23,42 mm for the right lower jaw and 22.42 mm for the left lower jaw. the measurement of the available space in the arch for the canines and premolars, which have not yet erupted, was completed and the results were compared. the right lower jaw was 20.80 mm–23.42 mm = 2.62 mm and the left lower jaw was 21.80 mm–22.42 mm = 0.62 mm. the third measurement used panoramic rongten and was a measurement of the teeth that have not yet erupted, a b c d e figure 3. (a) anterior; (b) molar relation; (c) maxilla-occlusal; (d) mandibular-occlusal; (e) panoramic radiograph. 1 2 2 4 3 figure 4. schem space maintainer appliance: (1) labial arch; (2) acrylic plate; (3) triple tube junction; (4) adam’s claps (source: author’s document). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 105anggraini et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 102–107 based on the huckaba method, as shown in table 2. the extension was calculated due to a radiographical error by measuring the erupted teeth in the radiograph and the same teeth in the mouth or in the study model. here, the tooth used was the upper left molar (m1). the mesio distal width of the teeth were calculated, which have not yet erupted in each quadrant. the sum of the radiograph width x y and was reduced by 10% of the mesio distal width of teeth, which have also not yet erupted. the 10% here was for the refraction found in the radiograph. the calculation was completed by comparing the ro photo of the lower right jaw. the prediction of cpp teeth size, which will still erupt, was (7.6 + 7.43 + 7.01) = 22.04 mm. the available space for eruption is 20.8 mm. thus, the right upper jaw is 20.80 mm–23.04 mm = -2.24 mm (lack of space). based on the previous arch determination, it could be concluded that there was an excess and a lack of space in the lower right jaw, which was 2.40 mm (table 3). three calculations show the lack of space, ranging from 2.00 mm to 4.00 mm. thus, the patient needed the space maintainer treatment (refer to the standard of the lack of space to determine the use of orthodontic preventive appliance). furthermore, based on the results of the calculations from the various above methods, it can be concluded that the patient’s right lower jaw had a lack of space for the growth of cpp teeth. therefore, after considering the jaw growth and development and the patient’s age, it could be concluded that the appliance to be used was a space maintainer. the complementary examination that supports the periapical radiograph showed that tooth 44 had not yet erupted. tooth 45, which is a tooth that will replace tooth 85, was estimated to grow at the age of 11 or 12 years. the patient’s age at the time of this study was 10 years. the growth direction of tooth 25 on x-ray showed normal growth. tooth 23, which is a tooth that will replace tooth 83, was estimated to grow at the age of 9 or 10 years. the growth direction of tooth 23 on x-rays showed normal growth. in order to replace the tooth and acquire space, the maintenance, namely the space maintainer, was implemented to maintain that space. table 1. the width of the mesiodistal teeth (mm) tooth upper jaw lower jaw right left right left primary dentition permanent primary dentition permanent primary dentition permanent primary dentition permanent 1 9.4 6 6.1 2 2.6 5.2 7.1 5.1 6.8 3 7.7 7.6 6.1 6.3 4 8.1 8.2 6.2 5 9.6 9.6 10.4 10.1 6 10.2 12.1 table 3. the results of various analytical calculations method lower jaw (mm)right average method 0.20 moyers 2.62 huckaba 2.24 arch -2.40 table 2. huckaba measurement huckaba description mesio distal width of tooth 43 x = (x’ – 10% x’) . y (y’ – 10% y’) = (10.7 – 1.07) x 9.2 mm = 7.59 mm (14.3 – 1.43) thus, the mesio distal width of tooth 43 is 7.60 mm. y : mesio distal width of upper left m1 tooth in the study model = 9.20 mm y’ : mesio distal width of upper left m1 tooth in the ro photo = 14.30 mm x : mesio distal width of teeth that are looked for x’ : mesio distal width of teeth that are looked for in ro photo 10% : refraction in radiographmesio distal width of tooth 44 x = (x’ – 10% x’) . y (y’ – 10% y’) = (11.5 – 1.15) x 9.2 = 7.40 mm (14.3 – 1.43) thus, the mesio distal width of tooth 44 is 7.40 mm. mesio distal width of tooth 45 x = (x’ – 10% x’) . y (y’ – 10% y’) = (10.9 – 1.09) x 9.2 = 7.01 mm (14.3 – 1.43) thus, the mesio distal width of tooth 45 is 7.01 mm dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 106 anggraini et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 102–107 discussion the steps for treatment include motivating the patient to take care of his teeth and to continue to take care of space management for the permanent teeth on the lower right. the assessment of this case was premature loss of tooth number 84, and the prognose was good. there was an edentulous area in tooth number 84. the patient came to perform space maintainer insertion. there were no complaints from the patient. in his second aftercare (6 february 2019) the patient came over to manage the space for his permanent teeth. the primary teeth were lost and had no replacement yet. in his eighth aftercare (3 july 2019) the patient again came over for continued care of the space management for his permanent teeth on the lower right. the retentive space maintainer appliance did not suppress the surrounding soft tissue. the measurement of the intertube was 1.20 mm, and the space for permanent teeth 64 and 65 was 15.60 mm. the conclusion of the treatment was a 1.20 mm tube opening, meaning that the appliance followed the growth and development of the jaws. severe caries conditions are unable to be treated and the teeth eventually have to be extracted.1 premature loss is often found in children and increases in frequency with age.4 this can also happen due to premature loss of primary teeth, which commonly happens with children. permanent teeth are more often disrupted in the eruption process when compared to the primary teeth. disruption of the growth process of both the baby and their permanent teeth can affect the time of eruption.8 this patient needed space maintainer treatment because his permanent replacement teeth still needed time to grow. a space maintainer is an appliance that is installed to maintain the space of primary teeth that undergo premature loss or premature extraction. this appliance aims to avoid narrowing space from the shifting of neighbouring teeth and also the extrusion or elongation of the patient’s teeth.5 this patient had a deficiency of space between 2.20 mm to 2.40 mm (according to the calculation of moyers, huckaba, and curved determination, it is an indication that a space maintainer is needed for the treatment. if the deficiency is >4 mm, the treatment is a space regainer). indications and counter indications about the use of space maintainers must be thoroughly considered in order for the treatment to be as successful as expected, without causing negative effects to the surrounding tissues.9 the advantages of a removable space maintainer are that it is easy to make, requires little time, is easy to widen, exerts little pressure on the remaining teeth because it does not hurt the soft tissue, is more aesthetic, is easily to clean, and can be made as a space maintainer.9 the drawbacks of using a removable space maintainer are that it can be easily lost, patients may not use it regularly, it is easy to brake, it can limit growth in the lateral direction of the jaw if the grip is not suitable, and can irritate soft tissues. the patient in this case required treatment and was approved for treatment with a removable space maintainer appliance.1 in addition, other disadvantages of the use of a removable space maintainer include when the patient has an allergy to resin materials used for making the appliance, when the patient is less cooperative the use of a removable space maintainer is not recommended, and when permanent teeth are expected to erupt as soon as the device is paired in the mouth.9 this patient had no allergies based on anamnesis and general examination. related to the material used in the space maintainer, toxicologically, there is no evidence to prove that commonly used dental resins produce systemic toxic effects in humans.10 this patient was treated with acrylic resin material, as this material is often used and recommended in the field of dentistry. in addition to being caused by dental caries, that the majority of posterior primary dentition tooth loss can also be caused by trauma (a collision or accident) that occurs in the teeth.2 in such a case, it is important that the initial condition that affects the development of the permanent teeth is followed by early treatment interventions and that orthodontic preventive measures are carried out to prevent the occurrence of severe dental malocclusion.11 the premature loss of primary dentition teeth can result in mesial-distal (mesial drifting) and vertical tooth migration causing the loss of jaw arch width, a deficit in dento-alveolar, dento-alveolar-maxillary development, permanent teeth growth disorders, inter maxilla relationship disorders, or dynamic occlusion.12 the installation of a space maintainer appliance in the patient aims to prevent the occurrence of mesial drifting of the surrounding teeth. this treatment needs to be done early. the space maintainer treatment for teenagers is done to keep the space from narrowing.13 space reduction increases when premature extraction is done two months too early.14 this patient came for the treatment after performing an extraction on his primary teeth. caring for the treatment is important. individual concern for the appearance and health of the teeth will increase with age. thus, awareness to perform treatment for teeth that have aesthetic and functional abnormalities will increase.15 this patient, supported by his parents, understood and knew that dental health was important for preventive care. the successful use of a space maintainer appliance was as a result of good cooperation between the dentist and the patient.16 the patient’s parents followed the instructions given and the purpose of using and installing the preventive orthodontic device was understood.17 in addition, it is important that there is parental support to motivate the child to use the space maintainer.18 the parents of this child patient were very supportive of this treatment. another supporting factor is related to the presence of dental health facilities, which affects severe malocclusion and orthodontic prevention treatment needs.19,20 the patient lived in yogyakarta, which is relatively close to the dental hospital. in addition, the effects of being far from dental clinics is a discouraging factor for people when utilising their chosen health services.21 the patient lived close to the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 107anggraini et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 102–107 dental health centre; this supported his treatments, which required multiple aftercare visits. the treatment was done in accordance with the space maintainer care standard, which states that if there is a space loss of about 2.00 mm to 4.00 mm then the treatment using a space maintainer must be done. if the loss of space is more than 2.00 mm to 4.00 mm and is accompanied by permanent m1 mesial drifting, then the treatment must be carried out using a space regainer. the patient meets the standards of space maintainer care.5 the space maintainer had a modified centre split plate with a double tube, a labial arch was used to maintain the arch of the teeth, and a clasp was on its right and left sides as retention. the presence of the double tube was expected to be more stable in the function of orthodontic prevention appliance. this space maintainer can follow development in a lateral direction.18,22 the patient was treated using a modified space maintainer, aimed at following the growth and development of the jaw. the patient underwent routine aftercare visits and records were completed about the changes in his jaw’s growth and his teeth condition, as the patient was still in his growing age.23 one problem encountered by the operator while treating the patient was that the patient needed a large amount of motivation from the parents and the operator for him to diligently use the appliance. the treatment plan is to continue regular examination and aftercare visits to see how the child’s teeth are progressing, as tooth number 44 has not erupted. it is recommended that the treatment proceeds, considering the age of the patient and that he is still in a development phase as the patient’s teeth have not yet erupted. thus, the treatment by means of the space maintainer should be continued and will require good cooperation between the patient, the operator, and the patient’s parents. finally, it can be concluded that space maintainer treatment for this patient has had good results, as indicated by the existence of intermolar and intercaninus growth. their growth can be monitored from the initial model and the final model. both can also be seen from the middle tube opening of 1.20 mm. it was apparent that there was development and growth of the mandibular jaw. a suggestion for further study is that further research could use more patients, with different ages and different genders. acknowledgements the authors thank the dental hospital universitas muhammadiyah yogyakarta, yogyakarta, for helping the patient in this case report. references 1. finn sb. clinical pedodontics. 4th ed. philadelphia: w.b. saunders; 1973. p. 342–69. 2. pinkham jr, casamassimo ps, mctigue dj, fields hw, nowak aj. pediatric dentistry. infancy through adolescence. 4th ed. philadelphia: elsevier saunders; 2005. p. 242–56. 3. kidd eam, joyston-bechal s. dasar-dasar karies: penyakit dan penanggulangannya. 5, editor. jakarta: egc; 2011. p. 18–76. 4. cameron a, widmer r. handbook of pediatric dentistry. 4th ed. st. louis: mosby; 2013. p. 5–10. 5. dean ja, mcdonald re, avery dr. mcdonald and avery dentistry for the child and adolescent. 9th ed. st. louis: mosby; 2010. p. 221–3. 6. dean ja. mcdonald and avery dentistry for the child and adolescent. 10th ed. st. louis: mosby; 2015. p. 721–6. 7. albati m, showlag r, akili a, hanafiyyah h, alnashri h, aladwani w, alfarsi g, alharbi m, almutairi a. space maintainers application, indication and complications. int j community med public heal. 2018; 5(11): 4970. 8. moyers re. handbook of orthodontics . 4th ed. london: year book medical publishers; 1988. p. 159–63. 9. snawder kd. handbook of clinical pedodontics. st louis: mosby; 1980. p. 242–75. 10. anusavice k, shen c, ralph h. phillips’ science of dental materials . 12th ed. philadephia: saunders ; 2012. p. 122–45. 11. ferguson dj. growth of the face and dental arches. in: mcdonald and avery dentistry for the child and adolescent. 9th ed. st. louis: mosby; 2010. p. 230–5. 12. peţcu a, bălan a, haba d, mârţu ştefanache am, savin c. implication of remature loss of primary molars. int j med dent. 2016; 6(20): 130–4. 13. rusdiana e, goenharto s, asdika rg. variation of fixed tongue crib for correcting tongue thrusting habit. j vocat heal stud. 2018; 1(3): 126–33. 14. andreeva rs, arnautska h, belcheva ab, georgieva mt, dimitrov ev. loss of space according to the time and the type of the premature extracted decidous teeth. j imab annu proceeding (scientific pap. 2016; 22(2): 1169–71. 15. arikan v, kizilci e, ozalp n, ozcelik b. effects of fixed and removable space maintainers on plaque accumulation, periodontal health, candidal and enterococcus faecalis carriage. med princ pract. 2015; 24(4): 311–7. 16. suri s, prasad c, tompson b, lou w. longitudinal comparison of skeletal age determined by the greulich and pyle method and chronologic age in normally growing children, and clinical interpretations for orthodontics. am j orthod dentofacial orthop. 2013; 143(1): 50–60. 17. costello bj, r ivera r d, shand j, mooney m. growth and development considerations for craniomaxillofacial surgery. oral maxillofac surg clin north am. 2012; 24(3): 377–96. 18. sharma p, arora a, valiathan a. age changes of jaws and soft tissue profile. sci world j. 2014; 2014: 1–7. 19. de carvalho tm, franco ma. preventive orthodontics: space maintainers in the early loss of deciduous tooth clinical case report. ec dent sci. 2017; 10(5): 143–8. 20. rao j. removable partial denture for children. in: finn sb, editor. clinical pedodontics . 4th ed. st louis: mosby; 1973. p. 165–70. 21. chandra hs, krishnamoorthy sh, johnson js, prabhu s. ill effects of conventional band and loop space maintainers: time to revolutionise. int dent med j adv res. 2018; 4: 1–3. 22. mitchell l. an introduction to orthodontics. 4th ed. oxford: oxford university press; 2013. p. 179–92. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p102–107 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p102-107 50 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 50–56 review article simple smartphone applications for superimposing 3d imagery in forensic dentistry haryono utomo,1 mieke sylvia margaretha amiatun ruth,1 levina gita wangsa,1 rodrigo ernesto salazar-gamarra2 and luciano lauria dib3 1department of forensic odontology, faculty of dental medicine, universitas airlangga, surabaya – indonesia 2maxillofacial prosthodontist, postgraduation programme, paulista university (unip), são paulo – brazil 3maxillofacial surgeon, paulista university (unip), são paulo – brazil abstract background: forensic dentistry identification commonly involves using dental cast models as ante-mortem data. here, dentists generally send the pictures as well as the dental records. however, in recent times, dentists – especially orthodontists and prosthodontists – are using 3d scanners in view of reducing the space for cast model storage as well as sending the 3d imaging for fabricating clear aligners and other items such as crowns and bridges. this new trend means data transmission and viewing has become more complicated since sophisticated laptops or personal computers are generally required. for more practical use, smartphones would be a better option, meaning various simple ideas for viewing 3d data must be explored. furthermore, the conclusions must be evaluated in terms of the validity for forensic dentistry use. purpose: to evaluate a number of smartphone applications that are simple, user friendly, scalable and capable of the measurement and superimposition of 3d imaging data. review: standard tessellation language (stl) is one of the 3d scan file formats that is also useful for 3d printing. recently, several applications for 3d viewing have been made available for iphones (ios) and android-based devices, which are able to view stl files. however, they have all received both positive and negative reviews in terms of various applications, including forensic dentistry, and they thus require further evaluation by forensic odontologists. conclusion: each application has advantages and disadvantages; however, in our experience as forensic odontologists, the cad assistant, exocad and adobe photoshop mix, which are available for ios and android devices, are preferable for forensic dentistry needs. keywords: 3d imaging; superimposition; smartphone; forensic dentistry correspondence: haryono utomo, department of forensic odontology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: haryono.utomo@fkg.unair.ac.id introduction the implementation of advanced computer-aided dentistry and computer-aided manufacturing technology (cad/ cam) has been widespread over the past thirty years since the field of modern clinical dentistry demands that patients must receive the highest quality treatment.1 cad has been used to digitise oral and dental structures for the virtual design of both simple and complex prosthetic units.2 meanwhile, forensic dentistry, a new field in the arena, requires individual unique identification when confirming the identity of individuals involved in a crime or death. the field also requires the highest possible accuracy to satisfy the logistical and emotional needs of all parties involved. however, a number of confounding factors can come into play here, including the relative experience and expertise of the individual performing the comparison, the availability, quality and age of the records, and the postmortem damage.3 frequently, post-mortem (pm) identification involves comparing the before death (ante-mortem, am) dental records (radiographs and dental cast models) with the teeth of the deceased individual, while injury analysis, or thanatology, involves examining the bone fragments or contusions in relation to various weapons or known injuries. a scientific and objective comparison using dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 51utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 advanced technology would strengthen the validity of forensic dentistry. within the forensic sciences, cad/cam technology has numerous benefits for procedures such as age estimation, bite-mark or lip-print analysis, injury analysis and pm identification. moreover, cad/cam technology can also be used to create three-dimensional (3d) crime scene reconstructions. 4,5 since the 1700s, dental impression techniques using specific impression materials have been used to record the 3d morphology and measurements of both hard and soft dental tissues. however, changes in the volume of the impression materials and the expansion of the dental stone can lead to distortion, which can affect the precision of prosthetic procedures.6 in order to resolve these issues, digital impressions using intraoral scanning (ios) have been developed in the field of dentistry.7 the application of the ios devices in dentistry falls in line with the development of cad/cam technology. currently, ios and cad/cam technology make treatment planning, case consultation and the communication with dental laboratories far easier while reducing chair-time and the amount of storage required.7,8 however, the widespread use of digital data and data transmission requires good access to the internet, especially during the disaster victim identification (dvi) process in victim identification centres, which is not always possible in developing countries or remote locations. the field of dvi must consider this issue when the centres plan their strategy for handling am data.9 exporting 3d data for viewing on other devices such as laptops or pcs, as well as small portable devices such as tablets or smartphones, can be an issue since the apps required to view the real 3d data often cannot be installed on these devices or perhaps require payment. in addition, these apps may involve lower quality, can be non-user-friendly and can include too much advertising as well as other unexpected disadvantages. moreover, there are different apps for the ios and android systems, which may vary in terms of picture quality depending on the system used.10 however, there do exist a number of smartphone apps that may useful in forensic dentistry, including cad assistanttm and exocadtm for viewing, and adobe photoshop mixtm for superimposing. nonetheless, there is a lack of reviews or reports on their use in forensic dentistry within the relevant body of literature. in view of this, obtaining the individual experiences of forensic odontologists who have already used these apps would undoubtedly be beneficial. with this in mind, this review is aimed at evaluating specific apps for both iphones and android devices that are deemed as simple, user friendly and scalable and which are appropriate for the measurement and superimposition of 3d imaging data for forensic dentistry. the evaluation is based on the operational procedure and involves direct comparisons with the actual images captured by a 3d intraoral scanner (3shape triostm). digital imaging in forensic odontology the forensic sciences are multidisciplinary fields that require both cooperation and coordination among police officers and other law-related officers, forensics experts such as forensic pathologists, forensic anthropologists and forensic odontologists, etc.11 meanwhile, forensic dentistry, or forensic odontology, is the implementation of dental science within the field of criminal and civil law, working in compliance with police organisations operating under the criminal justice system. forensic odontology plays a significant role in identifying criminals since, while the status of an individual’s teeth may change during their lifetime, the combination of decayed, missing and filled teeth can still be effectively measured and compared at any time.5,11 forensic dentistry encompasses a number of areas, including human remains identification, mass disaster body identification, the assessment of bite marks and lip prints, and age estimation, while it is often used in childabuse cases.5 the four types of violence outlined by the world health organization, namely, physical, sexual and psychological violence and neglect, can be manifest in the orofacial region, which is the domain of dentists and forensic odontologists.11 today, while advanced photography and scanning techniques are used in forensic odontology, the invention of 3d printing has signalled huge advances in areas such as bite-mark analysis.12 the benefits of digital imaging technologies for forensic odontology include computerised dental record systems, which cover both dental treatments and odontograms and which have replaced the physical dental record systems. conventional x-ray images, which include orthopantomography (opg) images, are captured as digital images in developed countries via computed tomography (ct), cone beam computed tomography (cbct) and intraoral 3d scanning.13 pm data should also be digitalised wherever possible, while processing x-ray images using chemicals should be avoided since the imagery must be directly digitised for storage to prevent any possible quality issues.13 according to javaid et al. (2019),14 errors may occur when the dentist labels the x-ray film with the patient’s name or when the forensic odontologist labels a film with the victim’s identification number after processing. the major benefits of digital data include that they can be stored for long periods of time on mass storage technologies such as cloud storage systems, which can be routinely backed up.14 other advantages of digital data include that it can be rapidly transmitted and received with identical characteristics and no loss of resolution or quality, meaning, unlike with x-ray film, no subsequent scanning is required.13,15,16 neither the original films nor the dental models are sent to the dvi centres, with the original copies remaining at the dental office to ensure the records cannot be lost during the dvi process. the use of digital dental pm data will ensure it can be inputted directly into computer dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 52 utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 applications such as dvisys, which will help prevent any transcription error. as forest17 declared, one of the most sophisticated aspects of 3d laser scanning in forensic dentistry is the am/pm superimposition (figure 1). 3d laser scanning the 3d laser scanning equipment senses the shape of an object and accumulates the data that describes the location of the object’s outer surface. such technology is useful for a large number of industries, including separate and process manufacturing, construction, utilities, law enforcement, archaeology, governance and entertainment. in the past two decades, laser scanning technology has evolved into being an important surveying technology for the acquirement of 3d information.14 3d laser scanners operate in terms of three main methods: time-of-flight, phase shifting and triangulation. meanwhile, there are three main types of 3d scanners: airborne scanners, terrestrial scanners and hand-held scanners. in addition, advanced technologies in the areas of software processing, data acquisition and user-friendly apps for 3d laser scanning are also being invented on a yearly basis.10,13 intraoral scanner technologies an intraoral scanner (ios) is a dental and medical device consisting of a hand-held camera (hardware) attached to a computer with 3d processing software. an ios is able to capture and record the 3d geometry of an object with high precision. the most widely used digital formats are the open standard tessellation language (stl) models or lock stl file models. stl, which is commonly used in many industrial areas, describes a series of triangulated surfaces where each triangle is defined by three points and a normal surface.9 however, the format has drawbacks in that it cannot capture the colour, transparency or texture of dental tissues, which can be captured by other file formats such as the polygon file format (ply). as with any form of image capturing, the cameras require the projection of light to record objects as individual images or videos, which are then collected by the software after the recognition of the points of interest (poi). the first and second coordinates (x and y) of each point are assessed on the image and the third coordinate (z) is then calculated depending on the object’s distance (figure 2).9,10 in order to acquire the shape of a 3d object digitally, 3d scanners are divided into two types: contact and noncontact, while the latter can be further divided into two main categories: passive scanners and active scanners.10,13 the 3d contact scanners are generally operated on a fixed platform and often contain a probe positioned at the end of a movable mechanical arm.13 in forensic dentistry, intraoral scanners such as the 3shape trios are categorised as active scanners that release or emit light or some form of radiation and then detect its reflection to probe an object or environment. the possible types of emissions include x-ray, ultrasound or light.9 other categories of 3d laser scanners operate in figure 2. determining the distance to the object. (a) triangulation: the bc can be determined according to the formula bc = ac x sin(a)/sin(a+c), (b) confocal: distance to the object is determined according to the focal distance, (c) aws requiring a camera and an off-axis that moves on a circular path around the optical axis and produces a rotation of interest points, (d) stereophotogrammetry is a technology that generates files via algorithm-based analysis.9 figure 1. three-dimensional (3d) superimposition. the pm surface is superimposed on the ante am surface. (adapted from forrest a: forensic odontology in dvi: current practice and recent advances17). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 53utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 terms of time-of-flight, phase shift or laser triangulation, methods that are generally used separately but can also be used together to create a multipurpose scanning system.13 standard tessellation language files in 3d scanning the stl file format was developed by charles hall in 1987 to support his stereolithographic 3d printer. the file extension (.stl) is an abbreviation of ‘stereolithography’ or an acronym for standard triangulation language or standard tessellation language. at present, it remains the leading file format used to transmit a 3d model to a 3d printer via a computer13 and is the most widely used file type for ios. the stl file defines the 3d model’s surface using a group of linked triangles to recreate the surface geometry. these small triangulations construct a surface that results in the faceting of the 3d model. while more recent file types can create more detailed data, the main advantage of stl is its simplicity. in fact, stl is based on open-source coding and is widely available, which means anyone can review, improve or share an stl file.17 other advantages include the universal format that allows for manipulation using almost every type of cad software program and 3d printer. in addition, its vectorbased (triangulation) graphics provide scalability without any loss of resolution. thus, the stl file format is arguably the most important aspect of the 3d printing procedure; however, it only transmits geometric structures and not colours.18 furthermore, a digital model requires some form of preparation such as smoothing prior to the 3d printing, since the software supplied with the 3d printer may not perform all manipulations completely, meaning other cad/ cam software program are required.19,20 3d intraoral scanner comparison cad/cam technologies in dentistry were introduced in 1971 to obtain ‘optical impressions’. following its continuous development, cad/cam technology has been applied in prosthetic and orthodontic procedures since the 1980s. the first ios that was commercialised within the dental market was the cerec system (sirona dental system gmbh, bensheim, germany).21 various manufacturers have subsequently developed ios systems with different performance properties for generating digital images (e.g. itero, align technologies, san jose, ca, 2007 and 3shape trios, copenhagen, denmark, 2010).10 ios and android according to asokan,22 mobile phones have become a major part of human life. indeed, almost every daily activity would appear to involve some form of smartphone technology, which has gradually replaced many of the functions of pcs. two of the most widely adopted operating systems in the current era are the ios and android systems, the former developed by apple and the latter by google. however, sahani23 noted various differences while using both operation systems in terms of both the system and the attendant security. elsewhere, mohamed24 noted the benefits of using the android system, an open sourcetype system, which include the prices, the wide variety of available devices, the customisability and the widget features. for certain, it allows for greater customisation than the ios system. however, ios provides more functionality, that is, higher speeds, less delay, and less buffering, since the chance of filling apple phones with unnecessary thirdparty launchers or customisation apps is reduced.22,23 applications for 3d viewing and superimposition with ios and android the minimum requirements for smartphone applications that can be used in forensic dentistry include the capacity for 3d image viewing, measuring and superimposition, while scalability is also a factor. several stl-based applications are available for ios and android smartphone systems. in terms of ios, the application can be downloaded via an app store, while in terms of android, google play is the required platform.22 in this article, we, as forensic odontologists, choose several applications that best suit our daily needs, that is, they are free and user friendly (i.e. it is easy to search for and upload files). in addition, we also choose applications that can superimpose am/pm stl data images. the chosen applications are cad assistant, exocad, and adobe photoshop mix. in order to allow for storing file data obtained via an ios (3shape trios) from a patient on a pc, the file has to be saved three times, in dcm, stl, and 3ox formats.5 subsequently, the stl data is transmitted to the android and ios smartphones – which, in this case, are the samsung galaxy s8+ and the iphone xr – via usb on the go (otg), before they are opened via cad assistant and exocad for 3d viewing. cad assistant the open cascade cad assistant is an offline 3d viewer and converter application for both ios and android system operations. the supported file formats are cad and mesh data formats (stl, step, and obj). it has several advantages over its competitors, including its simplicity, its measurement ability and its file size handling ability (up to 50 mb). however, devices with a low-range graphic processor can take some time to open the files.25 an illustration of an ios-exported stl file using the 3shape trios is presented in figure 3. in order to have an accurate view, a true horizontal position of the occlusal plane of the dental model 3d image makes it easier to achieve perfect superimposition, which can be achieved by tapping the view option buttons at the front and bottom right. the view option button located at the bottom right (figure 4) is used for zooming in and out using the thumb and index finger. adobe photoshop mix adobe photoshop mix is a creative photo editor. it is able to cut out, combine and create images. the functions of this app include combining, enhancing, sharing, blending, upright viewing, filling, and many more. it is available for pcs, tablets, and smartphones. the benefits of this application include that it is a freeware, powerful photo dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 54 utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 (a) (b) figure 4. flat shaded display mode on (a) android samsung s8+ and (b) ios iphone xr. figure 5. blending of two screenshots from cad assistant. (a) (b) (c) (d) (b) (a) figure 3. (a) cad assistant viewing intermolar distance of 3d stl data = 50.912 mm on a smartphone (b) 3shape trios dental scanner viewing intermolar distance = 50.80 mm on a laptop. figure 6. 3d stl data viewing on exocad smartphone app. (a) maxillary scan on ios, (b) maxillary scan on android, (c) combined two-file occlusion on ios, (d) combined two-file occlusion on android. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 55utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 editor (user can add more than one photo) and that it has a blending ability.26 blending is a technique that allows users to combine more than one picture as well as adjust the contrast, transparency and visibility. the app is also capable of superimposing two images of, for example, teeth model scans (figure 5).27 exocad while some applications are not compatible with pcs, exocad is available for pc users. this application was built by exocad gmbh and was specifically designed for the digital field of dentistry. according to the exocad website, users are able to design implants, bars, models, full denture models, virtual articulators and jaw-motion models. while this cannot all be done using a smartphone, the benefit of using a smartphone is that users can add more than one file to combine each fragment into an occluded model for matching jaw fragments (figure 6).28 discussion regarding the accuracy of ios for duplicating tooth preparations for prosthetics and clear aligners in orthodontics, several in-vitro and in-vivo studies have evaluated the system based on various best-fit software configurations of single units to full dental arches.10,21 here, muller et al. (2016),29 found that the difference in scanning approach among the ios systems resulted in statistically significant differences in accuracy. elsewhere, gimenez et al.30 revealed certain differences that resulted in bias when comparing the results obtained by both expert and non-expert users when scanning dental implant models. meanwhile, other research reported that certain varieties of material (e.g. gypsum and polyurethane) are almost perfect diffusers, while metals, which have a higher level of specular reflection, can affect the ios accuracy.31 however, these conditions are not the concern of forensic odontologists since the differences were only in µm (micro metres). the benefits of using ios as the operating system include, according to asokan,22 the battery life, the security and the quality of the apps. while apple systems do not provide as good multitasking efficiency as android systems, the battery life is shorter in the latter, while multitasking using mobile devices is not as easy as when using a desktop. according to sahani,23 ios has a more stable and secure system since it is more private unless it has been hacked. the apps provided in ios are among the best in terms of quality and quantity. in addition, according to mohamed,24 the ios operating system is so polished that when the same app is running on both operating systems, the quality is much higher using the ios system. in fact, the differences in the ios and android working systems result in the differentiation between the applications that can run on each system. here, while some can be run on either system, many can only be run on one or the other.22,23 the ios and android operating systems have their own applications, with some able to be operated on both systems and others not. the cad assistant, adobe photoshop mix and exocad are three 3d viewer apps that can be used on both systems. linear measurement, superimposition and occlusion viewing are arguably the most important aspects in forensic dentistry. cad assistant is capable of measuring linear distances. here, the important point is the precision, which depends on the screen itself (zoom and sensitivity) and the operator. park et al. (2015)32 used a stylus pencil to click the smartphone’s screen in order to increase the precision of the measurement. however, the difference in measurement between smartphones and pc is very slight.32 in our experience, this program can be considered as accurate for forensic dentistry needs because the difference in the app imagery compared with the 3d scanning imagery is negligible (trios = 50.8 mm, cad assistant = 50.912 mm in). in order to more accurately compare two models, we can use a program called cloudcompare, which is 3d point cloud processing software. it can compare two stl data formats, but is only available for pcs.33 the benefit of adobe photoshop mix relates to superimposition, which involves matching two stl images. this is achieved by manipulating the second dataset to match the size of the first before superimposing both sets. first, both stl datasets should be set up perpendicularly in cad assistant by pressing the front view option button before taking a screenshot (figure 4). then, the first and second screenshots are added and then blended (figure 5). while both cad assistant and exocad are able to view stl file types, the occlusion viewing in the latter allows for combining several datasets into a occlusion model. meanwhile, adobe photoshop mix is more advantageous in terms of its superimposition capability for matching am and pm scan results. therefore, all the applications are good and are useful for the forensic dentistry field. based on our experience of the superimposition of 3d imagery using the 3shape trios, the sequence of data processing should be as follows: converting the original 3d image scan file to a stl file, setting the cad assistant to perpendicular view by tapping on the front view option, ensuring the size comparison with the original by choosing the measurement points using cad assistant (shown on the screen), using the screen shots tool to capture the last view on the screen, and using adobe photoshop mix to compare the am/pm image by zooming in or out and rotating the image. overall, it can be concluded that the stl files of am and pm images can be rapidly transmitted with ease, accuracy and detail. in the future, the forensic odontology undertaken in dvi centres will increasingly depend on 3d imaging data captured via ct and 3d scanning, and newer forensic odontologists should be more experienced in using this digital imaging technology and will be quickly become accustomed to using it in practice on a daily basis. based on our experience in forensic odontology, the cad dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 56 utomo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 50–56 assistant, exocad and adobe photoshop mix apps, which are available for both ios and android systems, are the best options. however, further research should be conducted to ensure global acceptance. references 1. kalman l. utilization of an in-office cad/cam e.max maryland bridge a long-term anterior provisional. oral health. 2012; 102(8): 27–34. 2. crespi r, vinci r, capparé p, romanos ge, gherlone e. a clinical study of edentulous patients rehabilitated according to the “all on four” immediate function protocol. int j oral maxillofac implants. 2012; 27(2): 428–34. 3. ramesh g. cad/cam: a new revolution in forensics. forensic res criminol int j. 2018; 6(1): 1–3. 4. gibson i (ian), rosen dw (david w., stucker b (brent). additive manufacturing technologies: 3d printing, rapid prototyping and direct digital manufacturing. 2nd ed. new york: springer; 2015. p. 35. 5. khanna s, dhaimade p. exploring the 3rd dimension: application of 3d printing in forensic odontology. j forensic sci crim investig. 2017; 3(3): 1–3. 6. park hs, shah c. development of high speed and high accuracy 3d dental intra oral scanner. in: procedia engineering. elsevier ltd; 2015. p. 1174–81. 7. ali ao. accuracy of digital impressions achieved from five different digital impression systems. dentistry. 2015; 5(5): 1–6. 8. baheti mj, soni un, mahagaonkar p, khokhani r, dash s. intraoral scanners: a new eye in dentistry. austin j orthop rheumatol. 2015; 2(3): 1–7. 9. richert r, goujat a, venet l, viguie g, viennot s, robinson p, farges j-c, fages m, ducret m. intraoral scanner technologies: a review to make a successful impression. j healthc eng. 2017; 2017: 1–9. 10. gyorödi r, zmaranda d, georgian v, gyorödi c. a comparative study between applications developed for android and ios. int j adv comput sci appl. 2017; 8(11): 176–82. 11. dhingra r, munjal d. role of odontology in forensic medicine: an update. indian j forensic med toxicol. 2013; 7(2): 227–31. 12. ebert lc, thali mj, ross s. getting in touch-3d printing in forensic imaging. forensic sci int. 2011; 211(1–3): e1-6. 13. kalman l. role for cad/cam in forensics? dent today. 2013; 32(9): 120–3. 14. javaid m, haleem a, kumar l. current status and applications of 3d scanning in dentistry. clin epidemiol glob heal. 2019; 7(2): 228–33. 15. arthanari a, doggalli n, patil k, shankar hpj, vidhya a. bite mark: is it still valid?? int j forensic odontol. 2019; 4: 14–20. 16. makki l, ferguson dj, stapelberg r. measuring irregularity index: comparing study cast caliper method with 2d dimensional imagej photogrammetry and 3d stl image measurement abstract. apos trends orthod. 2017; 7(6): 260–6. 17. forrest a. forensic odontology in dvi: current practice and recent advances. forensic sci res. 2019; 4(4): 316–30. 18. logozzo s, zanetti em, franceschini g, kilpelä a, mäkynen a. recent advances in dental optics part i: 3d intraoral scanners for restorative dentistry. opt lasers eng. 2014; 54: 203–21. 19. neal kd, groth c, shannon t. cad/cam software for threedimensional printing. j clin orthod. 2018; 52: 22–7. 20. park hn, lim yj, yi wj, han js, lee sp. a comparison of the accuracy of intraoral scanners using an intraoral environment simulator. j adv prosthodont. 2018; 10(1): 58–64. 21. hack gd, patzelt sbm. evaluation of the accuracy of six intraoral scanning devices: an in-vitro investigation. ada prof prod rev. 2015; 10(4): 1–5. 22. asokan m. android vs ios – an analysis. int j comput eng technol. 2013; 4: 377–82. 23. sahani a. android v/s ios – the unceasing battle. int j comput appl. 2017; 180(3): 23–6. 24. mohamed i, patel d. android vs ios security: a comparative study. in: 12th international conference on information technology new generations. 2015. p. 725–30. 25. cad assistant | open cascade. available from: https://www. opencascade.com/content/cad-assistant. accessed 2020 apr 24. 26. pavlov v. the adobe mobile apps book: your complete guide to adobe’s creative mobile apps. 2016. p. 47–52. 27. image editing & composition app for ios, android | adobe photoshop mix. available from: https://www.adobe.com/products/ mix.html. accessed 2020 apr 24. 28. for dentists exocad. available from: https://exocad.com/youexocad/for-dentists. accessed 2020 apr 24. 29. müller p, ender a, joda t, katsoulis j. impact of digital intraoral scan strategies on the impression accuracy using the trios pod scanner. quintessence int (berl). 2016; 47(4): 343–9. 30. giménez b, özcan m, martínez-rus f, pradíes g. accuracy of a digital impression system based on parallel confocal laser technology for implants with consideration of operator experience and implant angulation and depth. int j oral maxillofac implants. 2014; 29(4): 853–62. 31. nedelcu rg, persson ask. scanning accuracy and precision in 4 intraoral scanners: an in vitro comparison based on 3-dimensional analysis. j prosthet dent. 2014; 112(6): 1461–71. 32. park e, del pobil ap, kwon sj. usability of the stylus pen in mobile electronic documentation. electron . 2015; 4(4): 922–32. 33. girardeau-montaut d. cloudcompare user’s manual for version 2.1. 2018. p. 1–68. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p50–56 https://www https://www.adobe.com/products/ https://exocad.com/you-exocad/for-dentists http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p50-56 170 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 170–174 research report the effect of fixed orthodontic treatment with edgewise and straightwire techniques on white spot lesions incidence and accumulation of streptococcus mutans bacteria elfira maharani, dyah karunia and pinandi sri pudyani department of orthodontics, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: fixed orthodontic appliances, such as edgewise and straightwire techniques, can increase the amount of plaque retention containing streptococcus mutans (s. mutans), which can lead to white spot lesions. purpose: the aim of this study is to analyse the correlation of fixed orthodontic treatment with edgewise and straightwire techniques on the incidence of white spot lesions and accumulation of s. mutans. methods: the samples consisted of three groups: control group (n=8), edgewise technique group, and straightwire technique group. we observed the samples at the sixth month and eighth month of the treatment, after the installation of the fixed orthodontic appliances. the observation of white spot lesions with caries detector was applied in all regions. bacterial swabs were acquired in the lateral incisor region, then a bacterial culture procedure was carried out on selective media of s. mutans, and then a bacterial count was performed. the data was analysed using two-way anova, the post-hoc least square differences test, and the pearson’s correlation test. results: the number of white spot lesions in the edgewise group was higher than in the straightwire group in the sixth and eighth month of treatment with insignificant difference (p>0.05). the number of s. mutans bacteria increased in all groups, but there were no significant differences (p>0.05). there was no significant relationship between the number of white spot lesions with the accumulation of s. mutans between groups (p>0.05). conclusion: the edgewise and straightwire techniques increase the incidence of white spot lesions but accumulation of s. mutans with the incidence of white spot lesions has no relationship. keywords: fixed orthodontic treatment; edgewise technique; straightwire technique; white spot lesions; s. mutans bacteria correspondence: elfira maharani, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta i, sekip utara, yogyakarta 55281, indonesia. email: elfiramaharanidrg@gmail.com introduction there are various bracket designs that have been developed.1 the fixed appliance technique, which has been widely used, was initiated with the discovery of a bracket with a square slot with a size that is adjusted to each individual tooth, namely the edgewise.2 technique appliance and the development of orthodontic appliance from the edgewise technique brackets – known as the straightwire technique – have surface contours to make it easier to insert on long axis points so that they can be placed precisely on the contours of the tooth surface.3 there are several stages at the beginning of treatment in the edgewise bracket technique: levelling and aligning, a correction of individual teeth malposition (such as correction of rotation), crowding and uprighting by requiring archwire or wire bending. archwire bending causes part of the tooth surface to be closed, making it difficult to clean the oral cavity and teeth, which poses a risk of causing plaque retention when compared to the straightwire bracket technique – with minimal use of archwire bending.2,4 orthodontic treatment with fixed appliances makes it difficult to clean plaque around the bracket using conventional oral hygiene methods. the ability to selfcleanse plaque with saliva is also reduced. orthodontic treatment with fixed appliances increases the risk of plaque retention and can increase the risk of caries and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p170–174 mailto:elfiramaharanidrg@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p170-174 171maharani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 170–174 periodontitis.5 early development of caries is characterized by white spot lesions (wsls) on the labial surfaces of teeth, which are quite serious side effects in orthodontic treatment with fixed appliances.6 after completion of active orthodontic treatment, the demineralization process will generally be reduced. some wsls can remineralize and return to normal, especially visually. however, wsls can be persistent, which makes the appearance less aesthetic.7 the emergence of an increase in white lesions in patients, on average, occurs from one month to thirty-six months during treatment, mostly appearing on the surface close to the gingival in premolar teeth because the placement of the bracket in some cases also causes gingival enlargement.8,9 a study conducted by van der veen et al.10 explained that the severity of wsls observed using light induced fluorescence after fixed orthodontic treatment with the straightwire technique was more than 60% – it appeared that there were still wsls. patients with fixed orthodontic treatment experience ecological changes in the oral environment, which cause an increase in the number of streptococcus mutans (s. mutans) bacteria in saliva and dental plaque.11 the increase in bacteria in the oral cavity is a major factor in increasing the accumulation of plaque on teeth, and the inflammatory response appears in new areas around the retention area of orthodontic fixed appliance components, such as bands, wires, ligature, or brackets.12 research conducted by kanaya et al.13 explains that the accumulation of s. mutans bacteria can increase up to three months after orthodontic bracket insertion. this study aims to analyse the difference and correlation of fixed orthodontic treatment using edgewise and straightwire techniques on the incidence of wsls and the accumulation of s. mutans bacteria. materials and methods an ethics permit was obtained from the research ethics commission of the faculty of dentistry, universitas gadjah mada, ref: 00299/kkep/fkg-ugm/ec/2019. the study type is a clinical laboratory experiment. the sampling of study subjects was conducted by documenting the number of patients in the orthodontics resident clinic of the orthodontics department, prof. soedomo dental and oral hospital, and faculty of dentistry, universitas gadjah mada, yogyakarta in 2019. the selection of subjects was based on research criteria via selected sampling methods in patients with fixed orthodontic appliances, such as edgewise techniques and straightwire techniques, and also patients who had not used fixed orthodontic appliances that were or would be treated by orthodontic residents during the 2018–2019 periods. prior to the study, patients were asked for informed consent as study participants. we classified the malocclusion cases subjects into angle class i malocclusion, accompanied by severe crowding, being treated at an early stage of at least six months (t1), and the second stage of eight months (t2), with 16–25 year olds. after meals but before brushing, subjects that were selected before observation of wsls were swabbed for bacterial sampling in the maxillary lateral incisors with sterile cotton swabs, which were then put into the 0.98% nacl solution for further processing in the integrated microbiology laboratory of the faculty of dentistry, universitas gadjah mada.14 subjects were observed to determine whether there were any wsls based on the modified wsl index of gorelick, on the first molars, premolars, canines, lateral incisors, and central incisors of the upper and lower, right and left regions. the examination was followed by these stages: archwire removal, tooth brushing to remove debris, drying the tooth element with a syringe, applying seek® on all buccal and labial surfaces of the teeth, waiting ten seconds, rinsing all surfaces with a water syringe and brushing or cleaning up the remaining seek® application material. the surface of the teeth that still appear to have staining from the seek® application material was the scoring observation area (enamel demineralization) with modified observation and gorelick scoring records. the gorelick15 modification index (figure 1) was recorded from the first molars, premolars, canines, lateral incisors and central incisors in the upper and lower jaws of the right and left regions.16 the results of the bacterial swab were examined and counted at the microbiology laboratory of the faculty of dentistry, universitas gadjah mada. the first stage of the individual sample immersed in 0.98% nacl solution was mixed. if there were debris, then centrifugation was carried out and followed with a five-stage dilution of the standard optimization series by inserting 0.2 ml of the solution into test tube 1 containing 0.8 ml of distilled sterile water. after dilution optimization, swab or streaking was performed on the selective media (mitis and bacitracin agar). the sample was stored at 37°c over a 24-hour period. we observed the colony in the media – if it did not appear, we proceeded to check again 24 hours later. in the subsequent observations, when a colony had formed, we counted it with cfu/ml (colony forming unit) = 1 bacterial cell = 1 colony using a colony counter. the number of s. mutans bacteria was obtained from the number of colonies, multiplying the dilution factor divided by the calculated solution volume.17 figure 1. detection of wsls based on the gorelick index.15 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p170–174 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p170-174 172 maharani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 170–174 we performed statistical analysis using two-way anova tests, followed by the post-hoc least square differences (lsd) test, and continued with the pearson’s correlation test to analyse the relationship of the number of bacteria with the incidence of wsls. all analyses were made using dedicated statistical software spss 16.0 (ibm, chicago, illinois, us) at a significance level of 5% (p <0.05). results the results of observations of the number of wsls and accumulation of the sixth and eighth month of s. mutans bacteria in the control, edgewise and straightwire groups can be seen in table 1. the lowest mean number of wsls are in the control group, followed by the straightwire group and the highest in the edgewise group. the mean number of wsls changed in all groups. the highest number of wsls increased in the edgewise group compared to the control group. the average number of wsls in the edgewise group in the sixth month was 3.50 and increased in the eighth month to 5.88. the mean number of s. mutans accumulations was lowest in straightwire compared to the control group, and highest in the edgewise group. the mean number of lesions accumulated by s. mutans changed in all groups. the highest accumulation of s. mutans bacteria increased in the edgewise group compared to the control group of 23.38 x105 cfu/ml in the sixth month and increased to 54.50 x 105 cfu/ml in the eighth month. data that had been tested for normality and homogeneity was eligible for parametric tests. the data was then analysed using the two-way anova test to determine differences between groups in table 2. the two-way anova test results (table 2) showed a significant difference in wsl scores in orthodontic treatment types, between observations (p<0.05) but not significant in the interaction of treatment types with observation time (p>0.05). the test results showed no significant difference in the number of bacteria in the type of treatment and the interaction of the type of treatment with the time of observation (p> 0.05), but there were significant differences in the number of bacteria at the time of observation (p<0.05). differences between groups in the two-way anova test can be seen through the post-hoc lsd test (table 3 and 4). table 1. mean and standard deviations in the number of wsls and accumulation of the sixth and eighth month s. mutans bacteria in the control, edgewise, and straightwire groups variables group 6th month 8th month mean sd mean sd number of lesions control 2.50 1.69 2.63 1.85 edgewise 3.50 2.00 5.88 1.64 straightwire 3.37 1.19 5.63 1.41 bacteria accumulation* control 24.50* 16.41* 49.75* 31.49* edgewise 23.38* 20.60* 54.50* 32.44* straightwire 14.13* 2.17* 39.88* 7.85* * bacterial unit in x105 cfu/ml table 2. two-way anova test for difference in increase in the number of wsls and the accumulation of s. mutans between the three groups of treatment variables white spot lesion s. mutans f p-value f p-value treatment 8.123 0.001* 0.888 0.419 observation time 11.035 0.002* 32.553 0.000* treatment*observation time 2.346 0.108 0.354 0.704 * significant difference (p <0.05) table 3. post-hoc lsd test results difference in number of wsls in the control group, edgewise technique, straightwire sixth and eighth month of treatment groups control 6 edgewise 6 straight 6 control 8 edgewise 8 straight 8 control 6 0.233 0.295 0.880 0.000* 0.000* edgewise 6 0.880 0.295 0.006* 0.014* straight 6 0.369 0.004* 0.009* control 8 0.000* 0.001* edgewise 8 0.764 * significant difference (p <0.05) table 4. post-hoc lsd test results on differences in the number of s. mutans bacterial colonies in the control group, edgewise technique, straightwire sixth and eighth month of treatment groups control6 edgewise 6 straight 6 control 8 edgewise 8 straight 8 control 6 0.696 0.164 0.011* 0.005* 0.020* edgewise 6 0.311 0.004* 0.002* 0.007* straight 6 0.000* 0.000* 0.000* control 8 0.734 0.812 edgewise 8 0.564 * significant difference (p <0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p170–174 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p170-174 173maharani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 170–174 the post-hoc lsd test results (table 3) on the incidence of wsls found significant differences between the control group with edgewise and straightwire at the eighth month (p <0.05) but not significant at the sixth month (p> 0.05). there was no significant difference between the edgewise and straightwire groups for the incidence of wsls at six and eight months (p> 0.05). the results of the post-hoc lsd bacterial test revealed no significant differences in the number of bacteria between the control, edgewise and straightwire groups in the sixth or eighth months (p> 0.05). the number of bacteria between times showed a significant difference between the sixth and eighth months in all control groups, edgewise and straightwire (p <0.05). the highest in the eighth month edgewise group compared to the sixth month edgewise group. the pearson’s correlation test results found no significant relationship between the number of wsls with the accumulation of s. mutans bacteria that occurred in the control group, the edgewise technique group or the straightwire technique group (p> 0.05). discussion the results of this study showed an increase in the number of wsls occurred in the edgewise and straightwire groups compared to the control group. this indicated the influence of orthodontic treatment techniques on the accumulation of wsls, where the control group had a lower risk of wsl growth compared to the edgewise and straightwire techniques. the increase in the number of wsls was greater in the group treated with edgewise and straightwire techniques compared to the control group in line with the theory that risk factors were widely reported in fixed orthodontic appliance users – namely the increase in the amount of plaque retention due to the difficulty of cleaning and the limited self-cleansing mechanism in each individual.18 the differences in the number of wsls grew over time in the edgewise technique group more than in the straightwire technique group although not significant at the sixth and eighth months. the edgewise technique has a bracket design with a zero degree angulation angle that requires the use of wire bending to be able to produce resilience forces that can correct malpositioned teeth.19 the bending of the wire to help tooth movement in the early stages and in several stages will affect the patient in maintaining oral hygiene, which results in food leftovers trapped in the bending of the wire, causing plaque retention and resulting in the formation of wsls.1 research compared the sample of patients with orthodontic treatment using the straightwire technique, which minimised the use of wire bending in the early stages of tooth correction so that it was easier for patients to clean the area of plaque retention from the effects of enamel demineralization that would become wsls.4 research conducted by mayne et al.20 which explained that the average increase in white lesions in patients occurs between four weeks to the first two months up to thirty-six months during treatment, which means within a period of two months during orthodontic treatment, the risk factor for wsls can occur. this research began to be observed in the sixth month in the edgewise and straightwire groups because each technique contained a correction stage for dental malposition – namely the levelling and unravelling stages. the number of wsls showed no significant difference between the edgewise technique and the straightwire technique in the sixth and eighth months, influenced by several important factors that cannot be avoided due to uncontrolled variables, such as dietary influence in each individual sample that cannot be equated – the type of food and the absence of precautions given to patients before orthodontic treatment was a separate concern that can be a possible trigger factor for the occurrence of wsls. there were differences in the amount of bacterial accumulation between the edgewise technique group more than the straightwire technique group, although not significant in the sixth and eighth months. this is in accordance with research conducted by kanaya et al.13 which explained that the accumulation of s. mutans bacteria can increase after the installation of orthodontic brackets at the initial stage, three months later. this situation is consistent with the results of research that with increased plaque retention in the oral cavity, bacterial accumulation increases and presents a risk of demineralization with the appearance of wsls in the same time span. in this study, we found no significant relationship between the number of wsls with the accumulation of s. mutans bacteria that occurred in the control group, the edgewise technique group or the straightwire technique group. this is explained in a study conducted by ranganath et al.21 that wsls can be reversible or irreversible, depending on the environmental conditions of the oral cavity of each individual related to the remineralization process involving salivary ph conditions, the ability of the host to regenerate cells and diet patterns. based on the results of this study, it can be concluded that there was no significant difference in the number of wsls and s. mutans between the edgewise technique and the straightwire technique and increasing the accumulation number of s. mutans bacteria. the number of s. mutans bacteria does not affect the relationship of the number of wsls in the orthodontic treatment of the edgewise and straightwire techniques. references 1. ribeiro glu, regis s, da cunha tdma, sabatoski ma, guarizafilho o, tanaka om. multiloop edgewise archwire in the treatment of a patient with an anterior open bite and a long face. am j orthod dentofac orthop. 2010; 138(1): 89–95. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p170–174 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p170-174 174 maharani et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 170–174 2. cobourne mt, dibiase at. handbook of orthodontics. london: mosby elsevier; 2010. p. 235–60. 3. filho lc, machado fm de c, ozawa to, cavassan a de o, cardoso m de a. bracket/wire play: what to expect from tipping prescription on pre-adjusted appliancese. dental press j orthod. 2012; 17(4): 85–95. 4. spena r. upper and lower incisor torque and straight-wire appliance. sido. 2014; : 21–32. 5. mattousch tjh, van der veen mh, zentner a. caries lesions after orthodontic treatment followed by quantitative light-induced f luorescence: a 2-year follow-up. eur j orthod. 2007; 29(3): 294–8. 6. boersma jg, van der veen mh, lagerweij md, bokhout b, prahlandersen b. caries prevalence measured with qlf after treatment with fixed orthodontic appliances: influencing factors. caries res. 2005; 39(1): 41–7. 7. ahmed i, saif-ul-haque, nazir r. carious lesions in patients undergoing orthodontic treatment. j pak med assoc. 2011; 61(12): 1176–9. 8. tufekci e, dixon js, gunsolley jc, lindauer sj. prevalence of white spot lesions during orthodontic treatment with fixed appliances. angle orthod. 2011; 81(2): 206–10. 9. sarver dm, yanosky m. principles of cosmetic dentistry in orthodontics: part 3. laser treatments for tooth eruption and soft tissue problems. am j orthod dentofac orthop. 2005; 127(2): 262–4. 10. van der veen mh, mattousch t, boersma jg. longitudinal development of caries lesions after orthodontic treatment evaluated by quantitative light-induced fluorescence. am j orthod dentofac orthop. 2007; 131(2): 223–8. 11. parahitiyawa nb, jin lj, leung wk, yam wc, samaranayake lp. microbiology of odontogenic bacteremia: beyond endocarditis. clin microbiol rev. 2009; 22(1): 46–64. 12. do nascimento leag, de souza mmg, azevedo arp, maia lc. are self-ligating brackets related to less formation of streptococcus mutans colonies? a systematic review. dental press j orthod. 2014; 19(1): 60–8. 13. kanaya t, kaneko n, amaike c, fukushima m, morita s, miyazaki h, saito i. a study on changes in caries risk and microbial flora with the placement of edgewise appliance. orthod waves. 2007; 66(2): 27–32. 14. politangeli r, sabatini s, nardi gm, di giorgio r, galluccio b. the filing of bacterial plaque on orthodontic appliances: type of bracket an motivational strengthening. prev res. 2015; 4(2): 70–4. 15. gorelick l, geiger am, gwinnett aj. incidence of white spot formation after bonding and banding. am j orthod. 1982; 81(2): 93–8. 16. yılmaz h, keleş s. recent methods for diagnosis of dental caries in dentistry. meandros med dent j. 2018; 19: 1–8. 17. pratiwi st. mikrobiologi farmasi. jakarta: erlangga; 2008. p. 108–9. 18. sudjalim tr, woods mg, manton dj. prevention of white spot lesions in orthodontic practice: a contemporary review. aust dent j. 2006; 51(4): 284–9. 19. graber l, vanarsdall r, vig k. orthodontics: current principles and techniques. 5th ed. philadelphia: mosby; 2011. p. 19–22, 517–34, 561–4. 20. mayne rj, cochrane nj, cai f, woods mg, reynolds ec. invitro study of the effect of casein phosphopeptide amorphous calcium fluoride phosphate on iatrogenic damage to enamel during orthodontic adhesive removal. am j orthod dentofac orthop. 2011; 139(6): e543–51. 21. ranganath lm, shet rgk, rajesh ag. saliva: a powerful diagnostic tool for minimal intervention dentistry. j contemp dent pract. 2012; 13(2): 240–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p170–174 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p170-174 212 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 212–216 research report combining 10% propolis with carbonated hydroxyapatite to observe the rankl expression in a rabbit’s alveolar bone nungky devitaningtyas,1 ahmad syaify2 and dahlia herawati2 1clinical dentistry programme, 2department of periodontics, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: periodontitis causes an increased receptor activator level in the nuclear factor-κβ ligand (rankl), which is one of the inflammatory mediators that plays a role in osteoclastogenesis. the open flap debridement (ofd) technique is the preferred treatment when accompanied by regenerative periodontal treatment using guided tissue regeneration (gtr) and guided bone regeneration (gbr). carbonated hydroxyapatite is a gbr material that serves as a scaffold and has strong osteoconductive properties for bone regeneration. propolis is natural product that can decrease osteoclastogenesis in periodontitis by decreasing the rankl expression. purpose: to investigate the rankl expression after open flap debridement by applying carbonated hydroxyapatite to 10% propolis in the alveolar bone of rabbits. methods: nine induced-periodontitis rabbits (oryctolagus cuniculus) were divided into three treatment groups of group a ofd, group b ofd followed by the application of carbonated hydroxyapatite, and group c ofd followed by application of 10% propolis-carbonated hydroxyapatite. each group was selected one to euthanised on the seventh, 14th and 28th day, respectively, and prepared using histology slides. the data was analysed using a two-way anova followed by a post-hoc lsd test (p<0.05). results: the rankl expression in each group showed significant differences (p=0.00; p<0.05) on the seventh, 14th and 28th day. the post-hoc lsd test showed that the rankl expression in the treatment group with carbonated hydroxyapatite-10% propolis had significant differences (p<0.05) in the intergroup analysis at different time points. conclusion: combining 10% propolis with carbonated hydroxyapatite in ofd treatment can decrease the rankl expression in a rabbit’s alveolar bone. keywords: 10% propolis; carbonated hydroxyapatite; periodontitis; rankl expression correspondence: ahmad syaify, department of periodontics, faculty of dentistry, universitas gadjah mada. jl. sekip utara, bulaksumur, yogyakarta, 55281 indonesia. email: ahmad.syaify@ugm.ac.id introduction periodontal disease is caused by porphyromonas gingivalis (p. gingivalis) and increases inflammatory cell infiltration, i.e. t lymphocytes, b lymphocytes and neutrophils in the connective tissues of the periodontium.1 these inflammatory cells may lead to an increase in inflammatory mediators, such as prostaglandin e2 (pge2), interleukin-1 (il-1) and the receptor activator of nuclear factor-κβ ligand (rankl). rankl is an inflammatory mediator that plays a role in osteoclastogenesis. t lymphocytes can activate rankl during inflammation in periodontal tissues, which causes the rankl expression to increase during periodontitis.2 periodontal treatment using the open flap debridement (ofd) technique is the preferred treatment when it is accompanied by regenerative periodontal treatment using guided tissue regeneration (gtr) and guided bone regeneration (gbr).3 carbonated hydroxyapatite is a gbr material that serves as a scaffold. it has strong osteoconductive properties for bone regeneration, can be well-resorbed by osteoclasts in the body and has good solubility in weak acidic conditions, i.e. when osteoclasts resorb bone by releasing h+ ions.4 the addition of 10% propolis in carbonated hydroxyapatite can stimulate the growth of fibroblast cells, and it is a good candidate for alveolar bone regeneration.5 a study by kusumawati dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p212–216 mailto:ahmad.syaify@ugm.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p212-216 213devitaningtyas et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 212–216 et al.6, which loaded the propolis into the carbonated hydroxyapatite using the immersion method, found that the 10% propolis solution had the strongest chemical bond with carbonated hydroxyapatite. this experiment is in line with the study by devitaningtyas et al.7, which found that 10% propolis with carbonated hydroxyapatite had a strong antibacterial inhibition against p. gingivalis bacteria. the largest active compounds in propolis are flavonoids and phenols. the phenolic content in propolis is usually called caffeic acid phenethyl ester (cape).8 the cape compound can increase osteoprotegerin (opg) in tissues and prevent osteoclastogenesis, as opg works by preventing rankl from binding to the receptor activator of nuclear factor-κβ (rank).9 bone regeneration processes can be observed from bone formation biomarkers, one of which is rank.10 carbonated hydroxyapatite can be combined with 10% propolis, which has anti-inflammation, antitumour and immunomodulator properties, to increase the bone graft material ability for regeneration by decreasing the rankl expression pathway. this study aimed to determine the effect of the application of carbonated hydroxyapatite-10% propolis in an open flap debridement on rankl expression. materials and methods this study was an experimental study with a randomised control group design. the carbonated hydroxyapatite that was used in this study was gama-cha (pt. swayasa prakarsa, yogyakarta, indonesia). to acquired carbonated hydroxyapatite-10% propolis, the gama-cha block was divided into 10mg and immersed in 1ml of 10% propolis solution for 24 hours at room temperature.6 the experimental animals that were used were nine male rabbits (oryctolagus cunicullus) aged 5–8 months that weighed 1500–2000g. periodontitis was induced in the experimental animals in the mandibular incisors using the ligation method with silk 3-0 and an injection of 0.05ml lps p. gingivalis using a tuberculin needle into the interdental area three times a week for six weeks.11 clinical signs of the induced periodontitis that were observed in the rabbits were tooth mobility, gingival recession and redness of the gingiva. the sampling method was stratified random sampling. the experimental animals were divided into three groups after ligation. group a had open flap debridement treatment, group b had open flap debridement treatment with the application of carbonated hydroxyapatite and group c had open flap debridement treatment with the application of carbonated hydroxyapatite-10% propolis. open flap debridement was done under anaesthesia using ketamine 40mg/kgbw and xylazine 5mg/kg bw. a sulcular incision was performed using scalpel no. 15 in the buccal sulcus of the mandibular central incisors, and the flap incision was then reflected using a small raspatorium. debridement was performed on both soft tissues and hard tissues. once ofd was done, irrigation with distilled water and flap repositioning was performed, followed by a suture using 4-0 nylon thread.12 groups b and c were given the same treatment; however, carbonated hydroxyapatite material was added to group b and carbonated hydroxyapatite-10% propolis was added to group c. after the treatment, the laboratory animals were administered soft food for 24 hours, tramadol at dose of 0.2–0.5mg/kgbw and one interflox antibiotic at a dose of 0.1 mg/kgbw after the treatment through intramuscular injection. one rabbit from each group was randomly selected to be decapitated on the seventh day after surgery, and the remaining rabbits were taken on the 14th day and 28th day after surgery. they were then euthanised using an intermuscular injection of an overdose of sodium pentobarbital, i.e. 120mg/kgbw. mandibular decapitation was carried out and fixed with formalin before the mandible was cut to obtain mandibular incisor specimens and placed onto four microscopic slides. the immunohistochemistry examination was conducted using antibody polyclonal rankl from bioss usa to measure the rankl expressions in the alveolar bone, which were viewed using a light microscope in 400x magnification on three different fields of view by two observers. each field of view showed both positive and negative cells. the calculation used the following formula:13 the data was analysed using the software spss version 21 for windows (ibm, chicago, usa). the data analysis was performed using the normality test with the shapiro– wilk test. a homogeneous variation test was conducted to discover the data variation in the groups with levene’s test (p>0.05) with a two way-anova and multiple comparison lsd test (p<0.05). results figure 1 shows the alveolar bone of oryctolagus cuniculus with a magnification of 400x at the seventh, 14th and 28th day in each group. osteoblasts were found in the sides of the bone. osteoblasts that positively expressed rankl were marked by dark brown cytoplasm, whereas those that negatively expressed rankl were marked by bright blue cytoplasm. the results from this experiment are shown in table 1. the carbonated hydroxyapatite-10% propolis group had the lowest expression of rankl. the normality test showed that the rankl expression in each treatment group and on each decapitation day had a significance greater than 0.05 (p>0.05). the homogeneity test also showed a significance greater than 0.05 (p>0.05). based on the normality and homogeneity tests results, it could be concluded that the rankl expression data was normally distributed and homogeneous. therefore, a statistical test using the parametric test – two-way anova – was carried out. the results of the two-way anova dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p212–216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p212-216 214 devitaningtyas et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 212–216 b a i h g f e d c figure 1. the expression of rankl in the alveolar bone in each group on the 7th, 14th and 28th day of examination shows the following using a black arrow (osteoblasts which positively expressed rankl were marked by dark brown cytoplasms): a. group ofd 7th day; b. group ofd 14th day; c. group ofd 28th day; d. group ofdcha 7th day; e. group ofdcha 14th day; f. group ofdcha 28th day; g. group ofdcha-10% propolis 7th day; h. group ofdcha-10% propolis 14th day; i. group ofdcha-10% propolis 28th day. table 1. mean and standard deviation of the rankl expression groups x±sd p 7th day 14th day 28th day ofd 43.21±8.72 30.79±2.76 46.77±6.35 0.00*ofdcha 42.90±4.52 26.08±4.66 37.49±6.81 ofdcha-10% propolis 22.60±6.77 15.670±6.42 14.42±2.97 *significant (p<0.005) table 2. lsd test of the rankl expression time point group treatment ofd ofdcha ofdcha-10% propolis 7th day ofd 0.952 0.002* ofdcha 0.002* ofdcha-10% propolis 14th day ofd 0.203 0.002* ofdcha 0.002* ofdcha-10% propolis 28th day ofd 0.045* 0.000* ofdcha 0.000* ofdcha-10% propolis * significant (p<0.005) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p212–216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p212-216 215devitaningtyas et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 212–216 showed that there were significant differences (p<0.05) (table 1). the result of the post-hoc lsd test showed that the ofd followed by application of 10% propoliscarbonated hydroxyapatite (ofdcha-10% propolis) group had a significant value (p<0.05) compared to the ofd and ofd followed by application of carbonated hydroxyapatite (ofdcha) groups at all time points (table 2), as well as the ofdcha to the ofd group at the 28th day (p<0.05). the ofdcha group compared to the ofd group at the seventh and 14th day was not significant (table 2). discussion rankl is a cytokine that regulates bone remodelling and is expressed by osteoblast cells.2 the rankl expression is stimulated by cytokines that bind to gp130 signal transducers, such as il-6, il-1 and tnf-α. with periodontitis, increasing il-6, il-1 and tnf-α will stimulate activators of transcription (stat) dan mitogenactivated protein kinase (mapk) in osteoblast cells, so that the rankl expression will increase and stimulate ostoclastogenesis.14,15 carbonated hydroxyapatite is a strong drug delivery system because it has a uniform pore size, high pore volume, mesoporous (2–5nm) and a large surface area. the –oh group in carbonated hydroxyapatite is an active compound that binds with the bioactive molecule on propolis and creates a hydrogen bond. these hydrogen bonds make the carbonated hydroxyapatite easier to load and release the propolis molecules.16 the results of the study showed that the rankl expression in the group that was treated with ofd and had the application of carbonated hydroxyapatite-10% propolis was the lowest compared to the ofd and ofdcha groups at all observation time points on day seven, 14 and 28. this indicates that propolis that is incorporated into carbonated hydroxyapatite decreases the rankl expression in the alveolar bone of rabbits until day 28. the results of this study are in line with the study by andrade et al.17, which showed that propolis-incorporated alloplastic bone graft material has good porosity and is able to release active substances until the 30th day. the addition of propolis to a carbonated hydroxyapatite graft material aims to boost the performance of the graft material by reducing the inflammatory response and providing osteoinductivity. propolis has antibacterial, antiviral, antifungal, antitumour and immunomodulatory properties.18 propolis is able to reduce the rankl expression by activating wnt signalling. activation of canonical wnt signalling leads to β-catenin over-expression in cytoplasms, which is translocated to the nucleus of osteoblasts. an increase in the β-catetnin expression suppresses the rankl expression.19 furthermore, propolis is anti-inflammatory as it decreases pro-inflammatory cytokines, such as il1β, il-1, il-8 and tnf-α. cytokine cause periodontal destruction by increasing the rankl expression and inducing osteclastogenesis.20 the rankl expression of the ofdcha-10% propolis group decreased from day seven to day 28. this indicates that propolis addition can reduce the rankl expression from the seventh day, whereas the reduction in the other groups began to take place on the 14th day. day seven is the end of the inflammatory phase and the start of the proliferation phase. at the end of the inflammatory phase, the rankl expression declines because the osteoblasts are preparing to secrete bone matrix. this is in line with research by steen et al.21, which showed that propolis could suppress the rankl expression at the beginning of inflammatory phase between days three to six. this is in line with the study by tang et al.22, which showed that propolis has the ability to suppress and regulate rankl in three phases of osteoblast development. although there was an increase during the proliferation phase, this was not significantly different to the mineralisation and maturation phases. this condition allows osteoblasts to secrete more bone formation matrices, which optimises tissue regeneration. in addition, the caffeic acid phenethyl ester (cape) in propolis triggers the osteoclastogenesis process by inhibiting osteoclastogenesis at the early stage of differentiation by suppressing ranklinduced nf-kb activation.23 the limitations of this research are that there were no baseline conditions and the duration of the experiment was limited. the conclusion from the study was that combining 10% propolis with carbonated hydroxyapatite in ofd treatment can decrease the rankl expression in a rabbit’s alveolar bone. it is necessary to conduct further research on the effect of the application of carbonate hydroxyapatite-10% propolis in open flap debridement with a longer period of observation. acknowledgements this study was supported by the grant penelitian tesis magister simlitabmas in the fiscal year 2020 under contract no. 3024/un1/ditlit/dit-lit/pt/2020. references 1. how ky, song kp, chan kg. porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. front microbiol. 2016; 7: 1–14. 2. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015; 2015: 1–10. 3. crea a, deli g, littarru c, lajolo c, orgeas gv, tatakis dn. intrabony defects, open-flap debridement, and decortication: a randomized clinical trial. j periodontol. 2014; 85(1): 34–42. 4. alhasyim i a a, p udyani ps, asma ra w, ana id. effect of carbonated hydroxyapatite incorporated advanced platelet rich fibrin intrasulcular injection on the alkaline phosphatase level during orthodontic relapse. in: aip conference proceedings. 2018. p. 030006. 5. wijayanti p, lastianny sp, suryono s. growth of nih 3t3 fibroblast cells exposed to carbonated hydroxyapatite with incorporated propolis. indones j cancer chemoprevention. 2020; 11(2): 54–9. 6. kusumawati i, suryono, syaify a. loading and release profile assay of carbonated hydroxyapatite incorporated with propolis as bone graft material. maj obat tradis. 2020; 25(2): 121–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p212–216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p212-216 216 devitaningtyas et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 212–216 7. devitaningtyas n, syaify a, herawati d, suryono s. evaluation of antibacterial potential of carbonated hydroxyapatite combined with propolis on porphyromonas gingivalis. maj obat tradis. 2020; 25(1): 55–8. 8. bittencourt mlf, ribeiro pr, franco rlp, hilhorst hwm, de castro rd, fernandez lg. metabolite profiling, antioxidant and antibacterial activities of brazilian propolis: use of correlation and multivariate analyses to identify potential bioactive compounds. food res int. 2015; 76: 449–57. 9. agustina s, swantara imd, suartha in. isolasi kitin, karakterisasi, dan sintesis kitosan dari kulit udang. j kim. 2015; 9(2): 271–8. 10. baharuddin na, coates de, cullinan m, seymour g, duncan w. localization of rank, rankl and osteoprotegerin during healing of surgically created periodontal defects in sheep. j periodontal res. 2015; 50(2): 211–9. 11. zenobia c, hasturk h, nguyen d, van dyke te, kantarci a, darveau rp. porphyromonas gingivalis lipid a phosphatase activity is critical for colonization and increasing the commensal load in the rabbit ligature model. bäumler aj, editor. infect immun. 2014; 82(2): 650–9. 12. z o h e r y a a , n o u r z m , a b d e l r e h i m s s , m a d y m i . histomorphometric analysis of bone regeneration after use of propolis versus nanobone graft materials for the management of class ii furcation defects in dogs. alexandria dent j. 2017; 42(2): 198–203. 13. zhang w, ju j, rigney t, tribble g. porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model. bmc oral health. 2014; 14: 1–9. 14. o’brien ca. control of rankl gene expression. bone. 2010; 46(4): 911–9. 15. meimandi-parizi a, oryan a, sayahi e, bigham-sadegh a. propolis extract a new reinforcement material in improving bone healing: an in vivo study. int j surg. 2018; 56(october 2017): 94–101. 16. šupová m. substituted hydroxyapatites for biomedical applications: a review. ceram int. 2015; 41(8): 9203–31. 17. andrade âl, manzi d, domingues rz. tetracycline and propolis incorporation and release by bioactive glassy compounds. j non cryst solids. 2006; 352(32–35): 3502–7. 18. kitamura h, saito n, fujimoto j, nakashima k, fujikura d. brazilian propolis ethanol extract and its component kaempferol induce myeloid-derived suppressor cells from macrophages of mice in vivo and in vitro. bmc complement altern med. 2018; 18(1): 138. 19. torre e. molecular signaling mechanisms behind polyphenolinduced bone anabolism. phytochem rev. 2017; 16(6): 1183–226. 20. franchin m, freires ia, lazarini jg, nani bd, da cunha mg, colón df, de alencar sm, rosalen pl. the use of brazilian propolis for discovery and development of novel anti-inflammatory drugs. eur j med chem. 2018; 153: 49–55. 21. steen bm, gerstenfeld lc, einhorn ta. the role of the immune system in fracture healing. in: osteoimmunology. elsevier; 2016. p. 297–310. 22. tang x, han j, meng h, zhao y, wang h, liu j, lin l, zhang d, li c, ma c. downregulation of rankl and rankl/osteoprotegerin ratio in human periodontal ligament cells during their osteogenic differentiation. j periodontal res. 2016; 51: 125–32. 23. ang esm, pavlos nj, chai ly, qi m, cheng ts, steer jh, joyce da, zheng mh, xu j. caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of rankl-induced nf-κb and nfat activity. j cell physiol. 2009; 221(3): 642–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p212–216 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p212-216 37 the effect of hegu acupoint stimulation in dental acupuncture analgesia fransiskus andrianto*, jenny sunariani** and theresia indah budhy s** ** student ** department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract in daily life, dental treatments are often related with oral pain sensation which needs anesthesia procedures. sometimes local anesthetics can not be used because patients have hypersensitive reaction or systemic diseases which may lead to complications. stimulating acupoint, such as hegu activates hypothalamus and pituitary gland to release endogenous opioid peptide substances that reduce pain sensitivity. the aim of the study was to determine hegu acupoint stimulation effect on the pain sensitivity reduction in maxillary central incisor gingiva. the laboratory experimental research was conducted on 12 healthy male wistar rats (3 months old, weights 150–200 grams). all rat samples received the same treatments and adapted within 1 month. the research was done in pre and post test control group design. 40-volt electro-stimulation was done once on the maxillary central incisor gingiva prior to the bilateral hegu acupoint stimulation, then followed by 3 times electro-stimulation with 3 minutes intervals. the pain scores were obtained based on the samples’ contraction in each electro-stimulation. the responses were categorized into 5 pain scores and statistically analyzed using wilcoxon test. the results showed that hegu acupoint stimulation lowered the pain scores significantly (p < 0.05). hegu acupoint stimulation could reduce the pain sensitivity in maxillary central incisor gingiva. therefore, the use of acupuncture analgesia in dental pain management can be considered in the future. key words: hegu acupoint, dental, acupuncture analgesia correspondence: theresia indah budhy s, c/o: bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction in daily life, dental treatments are often related to oral pain sensation. pain is a protective mechanism that occurs whenever any tissues are being damaged. oral surgery treatments, such as tooth extraction needs anesthesia procedures to relieve the pain. in certain conditions, local anesthetics can not be used because patients suffered systemic diseases or hypersensitive reaction.1,2 pain impulses are transmitted to the central nervous system (cns) by two fibers system. one nociceptor system is made up of myelinated a fiber that secrete glutamate and transmit fast sharp pain. the other consists of unmyelinated c fibers that release substance p and glutamate. these latter fiobers transmit slow-chronic pain. dental pulpal nociceptor system is made up of a fibers that secrete glutamate and transmit fast-sharp pain. both fiber group end at the dorsal horn of the spinal pain. both fibers groups end at the dorsal horn of the spinal cord.2–4 pain impulses from oral cavity are transmitted to trigeminal nerves by mandible and maxillary nerves.5,6 furthermore, these impulses are transmitted to cns through somatic sensory pathway from spinothalamic tract at medula pons, and thalamus midbrain. pain perception is processed at cerebral cortex to perceive pain location, intensity, and quality.7 acupuncture is a traditional chinese medicine (tcm) that uses specific needles insertion in various certain acupuncture points (acupoints). insertion of the needles in skin surface and muscle causes therapeutical effect and disease prevention.8 this technique can be used as an alternative in anesthesia procedure.9,10 hegu acupoint is often used in pain management because analgesia occurs by stimulating it.11 hegu acupoint is a major point in large intestine meridian that has a pathway to orofacial area.12 meridian is a specific pathway which interconnects acupoints to form a network with organs. because of this specific relationship, meridian can be defined as specific cellular pathway.13 stimulating acupoints results in yin-yang equilibrium by accelerating qi (bio-energy) flow. tcm’s yin-yang equilibrium phenomena is in accordance with western medicine’s homeostasis.11,14,15 acupuncture theory is based on cellular and molecular systems. stimulus of bioenergy from acupoint is transmitted through both systems to target organ.13 hegu acupoint is located between the first and the second metacarpal bones, approximately in the middle of the second metacarpal bone. anatomically, this point passes through skin and subcutaneous tissues, penetrates musculus interosseous dorsalis i, and then reaches musculus 3� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 37–41 adductor pollicis.16 stimulating this acupoint with various levels of stimulation activates hypothalamus and pituitary gland to release endogenous opioid peptide substances (b-endorphin, enkephalin, and dynorphin) that reduce pain sensitivity.11 based on the above background, the research was conducted to determine hegu acupoint stimulation effect on the pain sensitivity reduction in maxillary central incisor gingiva. the research was conducted on male wistar rats with medical bioenergy approach. by carrying out this research, anesthesia method using acupuncture is expected to be developed in dental science. materials and methods the laboratory experimental research was done in pre and post test control group design. a total of 12 male wistar rats were used in this research. all rats were healthy, 2–3 months old, weighed 150 to 200 grams, adapted within 1 month, and raised with same treatments. all rats were fixated with band aid on the wooden fixation board at their chest, stomach, upper legs, and lower legs (figure 2). hegu acupoint (figure 3) was located with acupuncture point detector ying di kwd-808-i, 6 channel output and 9 volt dc voltage (figure 1), then marked with board marker. the marked points were smeared with cotton bud soaked in 75% alcohol. figure 1. electro stimulator, acupuncture point detector, wooden fixation board, bekker glass with 75% alcohol, band aid, stopwatch, board marker, cotton bud, scissors, acupuncture needles. figure 2. rat fixated on wooden fixation board. figure 3. hegu acupoint located with acupuncture point detector. figure 4. needle insertion in hegu acupoint. figure 5. rat with acupuncture needles inserted. figure 6. electro-stimulation on maxillary central incisor gingiva with electro stimulator. 3�andrianto: the effect of hegu acupoint stimulation all rats were once stimulated on maxillary central incisor gingiva with 40-volt electro stimulator (figure 6), then followed by bilateral needle insertion in hegu acupoints with disposable stainless steel acupuncture needles 0.2 mm diameter and 13 mm long (figure 5). the needles were manipulated by twirling approximately 90° counter-clockwise twice. electo-stimulations were done three times with 3 minutes intervals after hegu acupoints stimulation. pain scores obtained in each electro-stimulation were categorized into 5 scores based on contraction and extremities movement caused by spinal nerve stimulation. the pain scores were:17 0 = no surrounding tissues contraction and no extremities movement, 1 = local mucosa contraction, 2 = mucosa and lip contractions, 3 = mucosa and lip contractions, superior extremities movements, 4 = mucosa and lip contractions, superior and inferior extremities movements. results pain scores at all rats were obtained by observation. the pain scores at the 3rd minute, 6th minute, and 9th minute were obtained after hegu acupoint stimulation (table 1). table 1. pain scores based on time intervals no. scores initial 3rd minute 6th minute 9th minute 1 2 3 4 5 6 7 8 9 10 11 12 4 4 4 3 3 3 4 4 3 3 4 4 3 4 3 3 3 3 2 2 3 3 3 3 0 3 3 3 2 2 0 2 3 3 3 3 0 2 0 2 2 0 0 2 3 2 0 3 based on above data, calculations were done to get mean and standard deviation (table 2). these calculation showed that pain scores decreased from the initial (initial = 3.58), then the 3rd minute (x 3 rd_minute = 2.92), the 6th minute (x 6 th_minute = 2.25), and the 9th minute (x 9 th_minute = 1.33). table 2. mean and standard deviation of pain scores time group mean std. deviation n initial_score 3.58 .515 12 3_minute_score 2.92 .515 12 6_minute_score 2.25 1.138 12 9_minute_score 1.33 1.231 12 the data calculated in table 2 was statistically analyzed using wilcoxon test to get the significance on differences of all time groups (table 3). table 3. wilcoxon test 3_minute – initial 6_minute – 3_minute 9_minute – 6_minute z -2.271a -2.060 a -2.232 a asymp. sig. (2-tailed) .023 .039 .026 wilcoxon test showed that there was a significant difference between 3rd minute group and initial group (p = 0.023, p < 0.05). there was a significant difference between 6th minute group and 3rd minute group (p = 0.039, p < 0.05). significant difference also occurred between 9th minute group and 6th minutes group (p = 0.026, p < 0.05). these results mean that the pain scores decreased significantly from initial to the 9th minute after hegu acupoint stimulation. discussion pain occurs whenever any tissues are being damaged, and it causes individual reacts to remove the pain stimulus. pain can be elicited by multiple types of stimuli. they are classified as mechanical, thermal, chemical, and electrical pain stimuli.2–4 the pain receptors in the skin and other tissues are all free nerve endings. pain stimuli on receptors cause sensitization and release of neurotransmitters. some of the neurotransmitters that were excited are histamine, serotonin, bradykinin, prostaglandins, and leucotrien. besides these chemicals, pain stimuli also cause the release of neuropeptide substance p that induce the release of histamine.2–4,7 pain is associated with behavioral response, motor reflexes, and alterations in autonomic output.18 the lateral and anterior corticospinal tracts are descending tracts that convey voluntary motor impulses from the brain (cerebrum) to spinal nerves at various levels in the cord.19 rats' extremities movements in this research are based on the level of spinal nerves stimulation.17 acupuncture technique used in this research was hand manipulated by twirling approximately 90° counterclockwise twice. this technique is in accordance with tcm theory which is xie, known as sedation and reduction method. based on tcm theory, toothache is caused by heat accumulation from the intestine accompanied with pathogenic factor. hegu acupoint is located in large intestine meridian so that sedation method reduced pathogenic qi and heat accumulation. it is showed that the pain in rat’s maxillary central incisor gingiva was relieved.20–22 in tcm, the natural world develops and constantly varies under the interaction of yin and yang. the philosophers and doctors in ancient china explained all the �0 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 37–41 phenomena and the nature of the universe and life with the theory yin-yang. ying and yang are opposite to each other in nature, they constantly repel and restrain each other. if one side is weak and the other side is strong, the general equilibrium is not maintained. thus, this abnormality will result in disorders, such as pain. results in table 2 showed mean of pain scores decrement from initial to 9th minute after hegu acupoint stimulation. the decrement occurred because there was an equilibrium between yin and yang by stimulating hegu acupoint which is defined as homeostasis in western medicine.11,14,15,23 acupoint stimulation induced bio-energy circulation through meridian to pain site. this stimulation also controlled the circulation physiologically. bio-energy disruption in the form of excess energy is the cause of acute pain.24 hegu acupoint is located in large intestine meridian which has a pathway to orofacial. hegu acupoint stimulation can reduce the pain by releasing the bio-energy obstruction at the area passed through meridian. the stimulation also relieved the pain by reducing pathogenic heat from large intestine pathway.22 hegu acupoint stimulation activated hypothalamus and pituitary gland to release endogenous opioid peptide substances to periaqueductal gray matter (pag) and nucleus raphe magnus (nrm). thus, analgesia occurred because all pain impulses were inhibited at dorsal horn of the spinal cord. these endogenous opioid peptide substances inhibited the pain by opioid mechanism.25–29 based on tcm, opiate receptor is categorized in yin because of its opioidreceive characteristic, while endorphin and enkephalin are categorized in yang because of their analgesic and therapeutic characteristic. hegu acupoint stimulation resulted in equilibrium between yin (opiate receptor) and yang (endorphin and enkephalin) which reduced the pain sensitivity.30 based on table 1, the most pain scores observed in initial group were 3 and 4. this data showed that pain occurred in initial group was in high intensity before hegu acupoint stimulation. score 3 happened because pain impulses inhibition was inadequate so that impulses were transmitted to the brain and spinal nerves at the brachial plexus area (c5 to t1). the electro-stimulation resulted in motor muscles movement and the response was superior extremities movements. score 4 was made based on superior extremities, inferior extremities, and tail flexion movements. these responses occurred because there was no inhibitory mechanism resulted in pain impulses transmission to brain and inferior spinal nerves at the sacral plexus (l5 to s1) and coccygeal plexus (s4 to co1) area.19 results showed that adequate analgesia occurred at 9th minute. the pain scores in this group were mostly 2 and 0. score 0 occurred because pain impulses were inhibited by b-endorphin at peripheral afferent terminal with the result that the impulses could not be transmitted to nerve ending. pain impulses transmitted to nerve ending were inadequate to induce stimulus at the area surrounding the nerve ending. score 2 was the result of pain impulses transmitted to peripheral afferent terminal. the pain impulses transmission reached nerve vii through dorsal horn of the spinal cord. there was a lip contraction caused by nerve vii which innervates lip’s motor muscles. score 1 occurred because of inadequate pain impulses inhibition at peripheral afferent terminal. thus, the impulses were transmitted to nerve vii without involving higher innervation system that caused local mucosa contraction.17,19 some of the pain scores at the 9th minute slightly decreased or did not decrease. the pain scores were both score 3 and 4. these high scores might be caused by bio-energy flow deficiency or disorder in some rats which were anxious, stress, angry, and fearful.31 bioenergy flow disorder caused imbalance between yin and yang, meridian function disorder, and disruption of bioenergy circulation. when acupuncture was done at hegu acupoint, bio-energy flow did not circulate well. thus, pain mechanism remained because obstruction or stagnation of bio-energy caused pain.14,24 high pain scores at the 9th minute happened because endorphin secretion was not enough. lack of endorphin resulted in pain because of inadequate analgesia. acupuncture analgesia needs a considerable amount of time to secrete enough endorphin. some rats have opiate receptor deficiency and different ability to secrete endorphin. inadequate analgesia could be happened to these rats. electro-stimulation on stress rats showed high pain score because neurotransmitters were released at peripheral nerve end resulted in sensitization. acupuncture analgesia also did not occur at rats which have pituitary suppression or ablation.25 table 2 showed that standard deviation at both 6th minute and 9th minute were above 1 (sd6_minute = 1.138 and sd9_minute = 1.231). this condition might be caused by individual characteristic of rats which some of them showed excessive responses. stress rats have higher pain sensitivity so that electro-stimulation could induce highest pain scores, while pain scores decrement in other rats occurred at these time groups. therefore, this difference resulted in higher standard deviation compared to the other time groups. the results concluded that hegu acupoint stimulation could effectively reduce the pain sensitivity in maxillary central incisor gingiva. clinical research can be conducted so that this research becomes applicable in dental science. references 1. idayanti a. akupunktur analgesia pada kasus pengobatan gigi. meridian 1999; vi(3):128–32. 2. guyton ac, hall je. textbook of medical physiology. 11th ed. philadelphia: elsevier saunders; 2006. p. 57–71, 598–609. 3. boron wf, boulpaep el. medical physiology. updated ed. philadelphia: elsevier saunders; 2005. p. 172–203. 4. ganong wf. review of medical physiology. 22nd ed. singapore: mcgraw-hill co. inc; 2005. p. 51–64, 85–120, 142–7. ��andrianto: the effect of hegu acupoint stimulation 5. sunariani j, mooduto l. peran kortisol terhadap persepsi rasa nyeri pulpitis. majalah kedokteran gigi (dental journal) 2002; 35(4):157–60. 6. roth gi, calmes r. oral biology. st. louis: the cv mosby co. 1981; p. 13–20. 7. putra ad. manajemen nyeri. ethical digest 2006; iv (26):70–2. 8. wardani n. akupunktur dalam bidang kedokteran gigi. meridian 1994; i(2):122–34. 9. saputra k. akupunktur analgesia. meridian 1997; iv(3):142–9. 10. rosted p. introduction to acupuncture in dentistry. br dent j 2000; 189(3):136–40. 11. saputra k. akupunktur klinik. surabaya: airlangga university press; 2002; p. 1–7, 71–5. 12. wu hg, luo dc. modern chinese medicine. vol. 2. beijing: people’s medical publ. house; 1984. p. 281, 555. 13. adikara rts. profil keseimbangan (taoisme) dalam era iptek modern untuk pembangunan bangsa indonesia. meridian 1995; ii (2):118–23. 14. wu cg, zhu zb. basic theory of traditional chinese medicine. shanghai, china: publishing house of shanghai university of traditional chinese medicine; 2000. p. 11–7, 102–9, 182–3, 194–209. 15. saputra k. akupunktur dalam pendekatan ilmu kedokteran. surabaya: airlangga university press; 2000. p. 42–52. 16. jing c. anatomical atlas of chinese acupuncture points. china: shandong science and technology press; 1990. p. 170, 184, 189. 17. sunariani j, soedarjanto h, soetjipto h, sari gm, chusaida a. pengaruh alkohol terhadap rasa nyeri pada tikus putih. majalah ilmiah kedokteran gigi trisakti desember 2000; 42:119–24. 18. woolf cj. pain: moving from symptom control toward mechanismspecific pharmacologic management. ann intern med 2004; 140:441–51. 19. solomon ep, schmidt rr, andragna pj. human anatomy and physiology. 2nd ed. san antonio: saunders coll. publ; 1990. p. 124, 333–51, 462–70, 482–94, 654, 794. 20. liu gw. techniques of acupuncture and moxibustion. china: huaxia publ. house; 1998. p. 47, 54-7. 21. pomeranz b, stux g. acupuncture textbook and atlas. berlin: springer-verlag. 1987; p. 223-6. 22. indian gyan home. traditional chinese therapeutics. 2000. available from url: http://www. indiangyan.com/books/therapybooks/ clinical_acupunture/traditional_chinese_therapeutic.shtml. accessed june 7, 2006. 23. san tc, wangsasaputera e, wiran s, budi h, kiswojo. ilmu akupunktur. edisi ke-2. jakarta: unit akupunktur rumah sakit dr. cipto mangunkusumo; 1985. p. 6–9. 24. hendromartono. akupunktur untuk nyeri kanker. meridian 1997; iv(3):195–201. 25. lee by, lariccia pj, newberg ab. acupuncture in theory and practice part i: theoretical basis and physiologic effects. hospital physician 2004 april; l:11–6. 26. ahadian fm. acupuncture in pain medicine: an integrated approach to the management of refractory pain. current pain and headache reports 2002; 6:444–51. 27. filshie j, white a. medical acupuncture: a western scientific approach. 5th ed. london: churchill livingstone; 1998. p. 69–79. 28. wu mt, hsieh jc, xiong j, yang cf, pan hb, chen yci, tsai g, rosen br, kwong kk. central nervous pathway for acupuncture stimulation: localization of processing with functional mr imaging of the brain-preliminary experience. radiology 1999; 212:133–41. 29. napadow v, makris n, liu j, kettner nw, kwong kk, hui kks. effects of electroacupuncture versus manual acupuncture on the human brain as measured by fmri. human brain mapping 2005; 24:193–205. 30. calehr h. pedoman akupunktur medis. jilid ii. jakarta: pt. gramedia pustaka utama; 1993. p. 171–2. 31. zijlstra fj, lange ivdb, huygen fjpm, klein j. anti-inflammatory actions of acupuncture. mediators of infl 2003; 12(2):59–69. 148 the determination of phenazone in blood plasma for obtained sistem suitable test of monitoring drug level mochamad lazuardi veterinary pharmacy subdivision, veterinary basic science veterinary faculty, airlangga university surabaya indonesia abstract the determining of phenazone to human blood plasma from healthy man after separated by solid phase extraction (spe) and spectroscopic measurements has been investigated. the objective of that research was to obtain system suitable test for determine the phenazone level in biological fluids (human blood plasma), for new performed dosage regimented in clinical dentistry. the method can be divided into the following four steps. 1. centrifugation the blood sample, 2. extraction from blood plasma and, 3. separation by spe with manual pressured, 4. elution to spe followed by the measurement on a spectrophotometer in the ultra violet region. the critical value of t at the 5% confidence level indicates that there is no systematic error in the linearity proposed method. recoveries for this research were obtained at ranging 93.460 to 95.598%. the coefficient variation precision of this procedure was clearly good at smallest than 2%. the analytical procedure can be carried out in one working operation as a monitored therapeutic activity. key words: phenazone, solid phase extraction, spectrophotometer uv-vis correspondence: mochamad lazuardi, c/o: veterinary faculty, airlangga university. jln. mulyorejo (kampus c) universitas airlangga surabaya, 60115. e-mail: ardiunair@hotmail.com introduction phenazone (as an analgesic and antipyretic properties) has been given by mouth.1 topically, solution containing 5 % of phenazone have been used locally as ulcers drops in disorders such as acute gingivitis.2 the dentist was usually approved with that dosage form as an analgesic and antiinflammatory agent. in clinical cases, the re-arrangement of regimentation dosage of phenazone for treated pediatrics patient was usually problems. those problems were appeared after showed that phenazone at long period’s usage can be induced reye syndrome of red blood cell. that problem would be reduced if the dentistry were using re-designing dosage form concepts by monitored drug level during the therapeutic periods of pediatric patients for obtained satisfaction dosage form. for obtained a suitable drug levels, the method of drug level determination was usually researching. during recent years several papers have been published on the phenazone.3-7 however, the results obtained are often in poor agreement. the accuracy and the precision of the analytical method used may give the contribution on the variation of the result. the new method of nuraini et al.,8 was reported that recoveries was obtained at ranging 86.010 to 88.069% and their coefficient variation of precision at ranging 0.433 to 3.871%. the recovery of new method at above was not satisfaction yet, although their precision was nearly exactly. therefore, the purpose of this work was to develop an accurate method for the determination of phenazone in human blood plasma using fourth-step sample preparation method. material and methods the analytical method used in the present work incorporates a partition-adsorption of drug-biological matrix separation concepts.9 the separation apparatus of that equipment was used solid phase extraction ods (ch17-ch3) reverse phase at 3 ml capacity (sigma chem. corp.,). plastic pestil 0f 3 ml spe ods-c18 was used as a manual mobile phase pressure. a pure phenazone from sigma corp. chem., was used as reference drug material. the spectronic hitachi 1100 uv-vis was used as a measurement drug concentration. the healthy human blood plasma (indonesian man) was used as an artificial human blood plasma. the human blood plasma was obtained from indonesia red cross unit. the research protocol was used four step procedures as follows; the 1st step was analyzed linearity of phenazone in distillated water from stock solution. the standard stock solutions were made up as 1000 µg.ml-1 from 100 mg of phenazone pure. the standard solutions of 0.5 to 100 µg.ml-1 were prepared by sequential dilution of the standard stock solution. both standard and stock solutions were placed in test tube. the linearity was analysis during two hours from 0.5 to 100 µg.ml-1 of the standard solution vs. absorbance (å) on 230 nm at five times replications. the result data of the step one on graphic illustration of the concentration solution series vs. absorbance unit full scale (auf) will be used as an additional standard solution data. 10 the 2nd step was produced artificial samples in human blood plasma. the pure of phenazone were weighing of 100 mg and dissolved 149lazuardi: the determination of phenazone with human blood plasma to produce samples stock at 1000 ppm. the stock samples were prepared at ranging series additional standard concentration of 0.5-100 ppm.10-12 the 3rd step was prepared artificial sample. the samples were extracted by spe as described akira et al.,13 and nuraini et al.,8 as follows; one ml plasma samples were added 1 ml chloroform and shake up well at 10 minutes. their solutions were added 2 ml with distillated water and centrifuge 3000 rpm (15 minutes). the supernatant were removed sample vials and keep on dark room about 5 minutes. the spe were activated with inserting 1 ml methanol and 1 ml distillated water consists of 5% glacial acetic acid. the supernatant were inserted to spe and vacuum drying the spe at about 30 minutes. the spe were added with 3 ml elution solution at rate 0.05 ml.second-1 and pressured gentle with plastic pestil. filtrate from elution were drying by n2 gas and added 3 ml distillated water and analyzing to obtained absorbance values at maximum wave length (230 nm). the graphic of the additional standard solution series vs. absorbance was using for calculation drug concentration on human blood plasma after plotted their absorbance values to the graph. the vx0 equation was used for measurement of linearity at series 0,5 to 100 ppm. the vx0 (vervahrenvariationk oeffizient) equation was described at equation 1 with sx0 (vervahrenstandardabweichung) and sy (mean residual deviation of the function) refereed to equation 2.14 equation 1. 100%• × =× x s v 0 0 equation 2, where 2 2 − ∑ − − =       n iyiy ys for yi = a + bxi b sy s =× 0 in order to investigate accuracy and precision of the method the samples were spiked with standard solutions and then analyzed by the mentioned method at below. the accuracy expressed as percent recovery was obtained by comparing the results between the phenazone found and the phenazone standard.15 the precision expressed as percent coefficient variation was obtained by divided the values of standard deviation with the mean of concentration at five times replicates.16 result the result procedures at the first step was appeared good correlation at ranging of the mean concentrations 0.5 to 100 mg.ml–1 (p < 0.05, table 1, figure 1). 0 0,5 1 1,5 2 2,5 3 0 10 20 30 40 50 60 concentration (ug/ml) a bs or ba n ce (a u f) series1 series2 figure 1. the linearity analysis of phenazone dissolved in distillated water (¨) at mean of 1.002 to 50.002 mg.ml–1 of 0,502 to 100 mg.ml–1. the linearity analysis of phenazone series at 1.002 to 50.002 mg.ml–1 in human blood plasma (n). but the best purposed for additional standard was obtained at 1 to 50 mg.ml–1 (cv of absorbency < 5%, table 2 and table 3). the v × 0 analysis of serial concentration (0.5–100 ppm) was not necessary linear (various) as referred to table 2 at below. the best serial concentration was obtained at 1.002 mg.ml–1 to 50.001 mg.ml–1. the result research of accuracy and precision of that procedures were table 1. the result analysis of linearity phenazone in distillated water concentration in mg.ml–1 absorbance (å) in auf mean ± (%cv) n–1 n–2 n–3 n–4 n–5 mean± (%cv) n–1 n–2 n–3 n–4 n–5 0.502 0.501 0.501 0.504 0.505 0.503 ± 0.36 0.002 0.003 0.002 0.003 0.002 0.002 ± 22.83 1.002 1.002 1.003 1.002 1.002 1.002 ± 0.045 0.048 0.049 0.051 0.052 0.051 0.050 ± 3.273 2.504 2.503 2.503 2.504 2.503 2.503 ± 0.023 0.136 0.137 0.136 0.137 0.135 0.136 ± 0.615 5.103 5.102 5.103 5.102 5.102 5.102 ± 0.022 0.271 0.270 0.269 0.270 0.271 0.270 ± 0.310 10.003 10.002 10.002 10.003 10.002 10.002 ± 0.005 0.545 0.546 0.544 0.545 0.543 0.545 ± 0.209 20.002 20.002 20.001 20.002 20.002 20.002 ± 0.002 1.091 1.091 1.090 1.091 1.091 1.091 ± 0.041 30.002 30.001 30.001 30.002 30.001 30.001 ± 0.002 1.638 1.637 1.638 1.637 1.638 1.638 ± 0.033 40.001 40.001 40.001 40.002 40.001 40.001 ± 0.001 2.180 2.181 2.180 2.182 2.181 2.181 ± 0.038 50.001 50.002 50.001 50.002 50.001 50.001 ± 0.001 2.728 2.729 2.726 2.727 2.726 2.727 ± 0.048 60.001 60.001 60.001 59.999 60.002 60.001 ± 0.002 3.268 3.272 3.269 3.888 2.865 3.112 ± 6.293 70.001 70.001 70.001 70.001 70.000 70.001 ± 0.001 3.789 3.798 3.811 2.989 3.790 3.635 ± 9.945 80.000 80.001 80.000 80.000 80.002 80.001 ± 0.001 4.365 4.333 3.994 3.345 3.911 3.990 ± 10.336 90.000 90.002 90.000 90.001 90.002 90.001 ± 0.001 4.911 4.912 4.231 3.881 4.001 4.387 ± 11.280 100.000 100.001 100.002 100.001 100.000 100.000 ± 0.001 5.214 5.111 4.895 4.021 4.233 4.695 ± 11.421 150 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 149-151 table 2. the linear analysis of phenazone in distillated water the mean of serial conct. (mg.ml–1) sy (mg.ml–1) coefficient correlation* (r) vx0** (%) xp*** (mg.ml–1) 0.503-100.00 0.446 0.971, p < 0.05 (good) 22.431 (not good) 33.534 (not good) 0.503-90.001 0.099 0.998, p < 0.05 (good) 5.585 (not good) 7.622 (not good) 0.503-80.001 0.076 0.999, p < 0.05 (good) 4.841 (moderate) 5.834 (not good) 0.5 to 70.001 0.050 0.999, p < 0.05 (good) 3.627 (moderate) 3.802 (not good) 0.5 to 60.001 0.045 0.999, p < 0.05 (good) 3.830 (moderate) 3.412 (not good) 0.5 to 50.001 0.008 0.999, p < 0.05 (good) 0.809 (good) 0.599 (not good) 0.5 to 40.001 0.008 0.999, p < 0.05 (good) 1.102 (good) 0.650 (not good) 0.5 to 30.001 0.008 0.999, p < 0.05 (good) 1.579 (good) 0.706 (not good) 1.002-100.00 0.132 0.997, p < 0.05 (good) 6.372 (not good) 10.656 (not good) 1.002-90.001 0.100 0.998, p < 0.05 (good) 5.241 (not good) 7.917 (not good) 1.002-80.001 0.077 0.999, p < 0.05 (good) 4.528 (moderate) 6.107 (not good) 1.002-70.001 0.051 0.999, p < 0.05 (good) 3.355 (moderate) 3.989 (not good) 1.002-60.001 0.046 0.999, p < 0.05 (good) 3.557 (moderate) 3.651 (not good) 1.002-50.001 0.003 0.999, p < 0.05 (good) 0.272 (good) 0.238 (good) * good: r observation >r table at significance 5% ** not good: at > 5%, moderate: at ranging 2–5%, good: at < 5%. *** not good: at more than the lowest conc. of their series, good: at least than the lowest conc. of their series. table 3. analysis of accuracy and precision of procedure determination of phenazone in human blood plasma phenazone in distillated water absorbance (å) phenazone in blood plasma (auf) replication-1 replication-2 replication-3 replication-4 replication-5 intraday precision (% cv) drug conc. (mg.ml–1) absorbance (auf) (å) auf recovery % (å) auf recovery % (å) auf recovery % (å) auf recovery % (å) auf recovery % 1.002 0.050 0.043 86.000 0.047 94.000 0.041 82.000 0.042 84.000 0.046 92.000 5.913 2.503 0.136 0.127 92.029 0.125 91.912 0.123 90.441 0.128 94.118 0.126 92.647 1.508 5.102 0.270 0.259 95.926 0.257 95.185 0.255 94.444 0.258 95.555 0.256 94.815 0.615 10.002 0.545 0.528 96.887 0.527 96.697 0.525 96.330 0.524 96.147 0.521 95.596 0.522 20.002 1.091 1.067 97.800 1.066 97.708 1.063 97.433 1.061 97.250 1.062 97.342 0.243 30.012 1.638 1.601 97.741 1.600 97.680 1.597 97.497 1.598 97.558 1.596 97.436 0.116 50.002 2.727 2.616 95.929 2.618 96.003 2.620 96.076 2.622 96.150 2.630 96.443 0.206 recovery (mean ± % cv) 94.616 ± 4.514 95.598 ± 2.197 93.460 ± 5.998 94.397 ± 5.002 95.183 ± 2.278 0.862 151lazuardi: the determination of phenazone apparently good at mean 94.651 percent of recoveries and 0.862 percent of coefficient variation as illustrated at table 3. discussion the method described here is based partly on the method development by nuraini et al.,8 but by pressuring of spe during the elution process, it was make a good recovery in determination of phenazone in human blood plasma.17 our recovery was apparently 1.5 to 3% highest than nuraini et al.,8 method at ranging 93.460 to 95.598%. the bias of precision described here was shown lowest than their protocol, although we saw both of them still satisfaction (the cv < 2%). each step in the assay method has been examined to give optimal results, provided the following precautions are observed. separation by spe should be carried out at room temperature by methanol: glacial acetic acid 0.5% (v/v) dissolve in distillated water 2 : 80 for at least 0.05 ml.second–1 rate pressure to ensure complete separation of phenazone. after drying by n2, all samples must be added by distillated water not more than 15 minutes. that process was doing to kept the chance of ph solution from acid to basic condition. the wavelength of 230 nm will be maximum detecting to aqueous sample in acid condition (5 to 6 ph solution) at not more than 2 hours.4,8 accurate timing and adequate mixing between the addition of each reagent is essential. if these precautions are taken, then reproducibility between experiments is 2%, and it is possible to determine accurately of 1 mg.ml–1 to 50 mg.ml–1 samples (figure 1). the sensitivity of the method is limited at least 1 to 50 mg.ml–1 sample concentration, because of a decrease in the yield of phenazone during the sample preparation especially in ph factor. our method could be of use in monitoring drug concentrations in human undergoing treatment of phenazone. phenazone as an analgesic and anti-inflammatory purposed or for dentistry cases was available effective in human blood bodies at about 250 mg.ml–1 to 500 mg.ml–1. that ranging concentration at above can be monitored by our procedures after diluted 10 times for reduced their concentration up to 50 mg.ml–1 as mentioned at additional standard procedure (table 1). these procedures could be of use for monitored therapeutics of phenazone for obtained suitable drug level at ranging 1 to 50 mg.ml–1. at 1 to 50 mg.ml–1 of phenazone have small deviation values of accuracy-precise and also still remain on the right tract of the recommended values of accuracy (80–120%) and precise (<10–20%). recommended of this research was follows; at more than 50 mg.ml–1, the series concentrations must be diluted to 10 times or 100 times. the monitored therapeutic activities may be important contributors to the re-arrangement regimentation dosage, especially for dentistry in children cases or elderly cases. acknowledgement this work is following research of nuraini farida, ms., pharm, bambang hermanto dr., ms and lazuardi m, phd., msc., dvm at mru research project 2005/2006. sponsorship: mru medical faculty airlangga university. the author would like to thank dean of medical faculty airlangga university as sponsorship of this research by medical research unit, medical faculty airlangga university at program 2005/2006. references 1. kenakin tp, editor. a pharmacology primer: theory, application and methods. 2nd ed. california, usa: academic press; 2006. p. 200–5. 2. reynolds fef, editor. martindale: the extract pharmacopoeia (reference material). london: the pharmaceutical press; 2004. p. 28–29. 3. miti ss, ∞ivanovi vv. a kinetic method for the determination of phenol. j serb chem soc 2002; 67(10):661–7. 4. moffat ac, osselton md, widdop b, editor. clarke’s analysis of drugs and poisons. vol 2. 1 lambert high street, london, uk: the pharmaceutical press (php); 2004. p. 555–8. 5. palette c, cordomier p, naline e, advenier c, pays m. highperformance liquid chromatographic method for determination of the three main oxidative and 3-carboxylic antipyrine metabolites in human urine. j chromatogr 1992; 563:103–13. 6. mikati ma, szabo gk, pylilo rj. improved high-performance liquid chromatographic assay of antipyrine, hydroxymethyl antipyrine and norantipiryne in urine. j chromatogr 1988; 433:305–11. 7. danhof m, de-groot-van der vis e, breimer dd. assay of antipyrine and its primary metabolites in plasma, saliva and urine by high performance liquid chromatography and some preliminary results in man. pharmacology 1979; 18:210–23. 8. nuraini f, bambang h, lazuardi m. the determination of antipyrine in plasma by spectrophotometer uv-vis. surabaya: research report medical research unit, medical faculty airlangga university, 2006 september. p. 5–20. 9. supelco chromatography. chromatography (product for analysis and purification). singapore (south asia region): sigma-aldrich 2001; p. 257–97. 10. nuraini f, lazuardi m, ratna sm. the determination of trypamidium in blood plasma by spectrophotometer uv-vis. medika eksakta 2001; 2(2):92–8. 11. lazuardi m. the determination of sulfamethazine in blood plasma by spectrophotometer uv-vis. in: aulanni’am, editor. basic science workshop mathematic and natural science brawijaya university, 2006 february 16–18, malang, jawa timur; 2006. p. 19. 12. eichllbaum m, somntag b, dengler hj. hplc determination of antipyrine metabolites. pharmacology 1981; 23:192–02. 13. akira k, negishi e, sakuma c, hashimoto t. direct detection of antipyrine metabolits in rat urine by 13c labeling and nmr spectroscopy. drug metabolism and disposition 1999; 27(1): 1248–53. 14. funk w, dammann v, donnevert g. qualitätsicherung in der analytischen chemie. london, new york: vch; 1992. p. 8–200. 15. ellison slr, king b, rösslein m, william a, editors. eurachem/ citac guide: quantifying uncertainty in analytical measurement. 2nd ed. uk: eurachem/citac/aoac/iaea/ea working groups; 2000. p. 4–120. 16. lazuardi m. a rapid isocratic hplc assay of diminazene aceturatein blood plasma. jurnal kedokteran yarsi 2005; 13(3):298-05 17. aoac-international. volume i: official method of analysis of aoac international. (agriculture; chemicals; contaminants drugs). 17th eds. usa: the scientific association aoac international press; 2002. p. 29–30. 4949 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 49–55 case report management of patients with aphthous-like ulcers related to aplastic anaemia in the covid-19 pandemic era through teledentistry: a case report lani berlina talahatu1, bima ewando kaban1, nurina febriyanti ayuningtyas2, intan noha brilyanti3, adiastuti endah parmadiati2, desiana radithia2, aulya setyo pratiwi2 1oral medicine specialist degree, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3internist of internal medicine clinic, dr. soebandi general hospital, jember, indonesia abstract background: aplastic anaemia is a disease that occurs when the body fails to produce enough blood cells. oral lesions, such as canker sores, are often used as an early marker of this disease condition. in the covid-19 pandemic era, a teledentistry method is expected to assist in the management of patients with various diseases including aplastic anaemia. purpose: this case report discusses the management of patients with aphthous-like ulcers related to aplastic anaemia in a 34-year-old woman, based on history, clinical examination, and blood tests through teledentistry. case: the patient complained of a bleeding wound on the inner left cheek and corner of the left lip that appeared one month ago. there was an ulcer lesion that was bleeding easily in the buccal mucosa sinistra and the angular sinistra, as well as macules lesion on her hand and foot. the complete blood count test exhibits a low platelet level. case management: the diagnosis was an aphthous-like ulcer related to aplastic anaemia. patient was treated with topical antiseptic and anti-inflammatory mouthwashes for the oral lesions, as well as collaboration with the medical team. the lesions improved after the patient received a blood transfusion and drug therapy from the internist while she was hospitalized, but the lesions reappeared in other locations in the oral cavity when the platelet level dropped. conclusion: management of oral manifestations in patients with aphthous-like ulcers related to aplastic anaemia in the covid-19 pandemic era can be done through teledentistry. however, this requires sensitivity from the dentist, patient compliance in following instructions, and the involvement of a multidisciplinary approach, such as collaboration with internists to achieve recovery. delay in detecting and treating aplastic anaemia will lead to death, as in this case. keywords: aphthous-like ulcer; aplastic anaemia; oral ulcer; teledentistry correspondence: nurina febriyanti ayuningtyas, department of oral medicine, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no 47, surabaya, 60132 indonesia. email: nurina-ayu@fkg.unair.ac.id introduction aplastic anaemia is a disease rarely encountered in everyday life. this disease can be life-threatening for the sufferer, but 90% of cases can be successfully treated. the condition of bone marrow-associated pancytopenia with persistent hypocellularity in the absence of major dysplastic signs and fibrosis can be defined as aplastic anaemia. with this disease, the body fails to produce enough blood cells. blood cells are produced in the bone marrow, and stem cells play a role in the production of blood cells. to confirm the diagnosis of aplastic anaemia, it is necessary to involve at least two components of the peripheral blood cell examination. aplastic anaemia causes low levels of all components of blood cells, be it erythrocytes, leucocytes or platelets. values should be lower for haemoglobin (lower than 10 gr/dl), neutrophils (lower than 1.5x109/l) and platelets (lower than 50x109/l).1–3 aplastic anaemia often occurs in people in their teens between 15 and 25 years old, but can also occur in people over 60 years old with less incidence. it is caused by genetic factors, autoimmune conditions, exposure to chemicals or radiation and drug use. but in some cases, the aetiology of aplastic anaemia is unknown.4,5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 mailto:nurina-ayu@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 50 talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 based on the cause, there are two types of aplastic anaemia, acquired aplastic anaemia and inherited aplastic anaemia. acquired aplastic anaemia is a t-cell-mediated autoimmune disease. hematopoietic stem/progenitor cells (hspcs) destroyed by immune cells have a vital role in the pathophysiological process of acquired aplastic anaemia. dysregulated cd81 cytotoxic t cells, cd41 t cells namely th1 (t helper type 1), th2, regulatory t cells, th17 cells, nk (natural killer) cells, nk t cells, ifn (interferon)-γ, tnf (tumour) necrosis factor)-α and tgf (transforming growth factor)-β, induce continuous apoptosis of hspcs and characterize severe acquired aplastic anaemia.6 the unsurvival rate of patients with aplastic anaemia can be affected by infection from parvovirus. parvovirus b19 is often associated with aplastic crises involving only erythrocytes, whereas aplastic anaemia involves all the different components of blood cells. in addition to parvovirus b19, viruses that are frequently associated with the development of aplastic anaemia are hiv, hepatitis, cytomegalovirus and epstein-barr.7 in several studies with animals (ferrets), aplastic anaemia has another possible cause, namely caused by the toxic nature of oestrogen. in female ferrets treated with ovulation induction and not mated, the oestrogen levels will be high. when oestrogen levels are higher than normal limits, the bone marrow will stop producing erythrocytes.7 in aplastic anaemia condition, oral manifestations are often found and this is directly related to pancytopenia. common oral manifestations involve petechial or spontaneous bleeding, gingival swelling, pallor, ulceration and severe periodontal disease. an aphthous-like ulcer is a diagnosis for recurrent oral ulceration associated with a systemic condition. in aplastic anaemia, these manifestations, as well as petechial haemorrhagic lesions and oral trauma are associated with decreased platelet levels. these lesions are most likely due to a clotting disorder-induced thrombocytopenia, which causes bleeding after minor trauma such as friction with the tongue or teeth.8 the world health organization (who) has declared the covid-19 outbreak a global pandemic. overall, this condition has resulted in crises in various fields, including public health. in the field of dentistry, it is known that the potential for cross-infection in the dental clinic is very high because most dental procedures produce aerosols and droplets that can be contaminated with the sars-cov-2 virus. therefore, the indonesian government issued a policy calling for the use of online health services known as telemedicine through circular letter no. hk.02.01/ menkes/303/2020 concerning the implementation of health services through the utilization of information and communication technology in the context of preventing the spread of corona virus disease 2019 (covid-19). the letter explained that telemedicine is a health service facility in the form of consultation to establish a diagnosis, therapy and/or disease prevention.9 in the field of dentistry, telemedicine is referred to as teledentistry, which is defined as a combination of telecommunications and dental practice that involves the exchange of clinical information in the form of electronic medical records and digital images remotely for dental consultations treatment planning. teledentistry services are best used for early detection of disease, providing pretreatment systemically (orally) to treat disease emergencies and can be a means to monitor a disease condition. in the field of oral disease, the role of teledentistry is an effective alternative in diagnosing oral lesions by sending digital images through electronic media.9 this case report discusses the management of patients with aphthouslike ulcers related to aplastic anaemia in a 34-year-old woman during the covid-19 pandemic era, based on history, clinical examination and blood tests through teledentistry. case a 32-year-old female patient was consulted via teledentistry on december 26, 2020, complaining of a spontaneous bleeding wound and sore on her inner left cheek and corner of the left lip. the wound appeared one month ago. at first it was a scratch and one week later the wound broke open. if the patient opens her mouth, blood comes from the wound. the patient treated her complaint by drinking cool juice, refreshing solution, and getting injections from a health worker, but her condition did not improve. the patient also stated that her body feels weak. therefore, 22 days after the initial complaint, the patient was hospitalized for one day at a health worker’s house. two days later the patient went to the doctor and underwent blood tests. the results of the examination showed haemoglobin 5.3 g/ dl, erythrocytes 1.8 million/ul, haematocrit 16%, mcv 86 fl, mch 28 pg, mchc 33 g/dl, leukocytes 410/ul (type count cannot be done because the leukocyte level is too low), and platelets 150000/ul. the patient received medication, namely: cefadroxil, benostamin, etabion, alphamol, and becom-z. this condition had been experienced by the patient several months before with a bleeding and painful wound on the left upper back gum, and was treated by drinking refreshing solution and getting injection therapy from the nurse. but the patient did not know what kind of medication was injected at that time, and then the wound healed within 10 days. two months before, bruises appeared on the patient’s feet and hands. this condition existed until the time the patient’s teledentistry consultation, without any new bruises reported. no one in the patient’s family has had a similar experience. this patient has no history of food or drug allergies. she eats vegetables every day, but rarely eats fruit and has a consumption of mineral water more than 1500 ml. the patient’s 52 kg in weight and 154 cm in height. the body mass index (bmi) is 22, which is normal. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 51talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 in the family medical history, her mother had hypertension. on extraoral examination (figure 1) there are multiple desquamations in the upper and lower lips, clear irregular margin, 1 mm in size, normal surrounding tissue, painless. there is also a solitary crust on the corner of the lip sinistra (figure 2), 2-3 mm in size, clear irregular margin, rough surface texture, spontaneous bleeding, painful. on her legs and arm (figure 3), there are multiple macules, various shapes and sizes, a b figure 1. ab. upper and lower lips: multiple desquamations, clear irregular margin, 1 mm in size, normal surrounding tissue, painless. figure 2. lip corners sinistra: solitary crust, 2-3 mm in size, clear irregular margin, rough surface texture, spontaneous bleeding, pain. a b blackish-red in colour, smooth surface texture, clear irregular margin, normal surrounding tissue, and painless. on intraoral examination (figure 4) there is a solitary ulcer on the buccal mucosa sinistra, 1 x 1.5 cm in size, clear irregular margin, rough surface texture, spontaneous bleeding. the diagnosis of this disease is aphthous-like ulcer related to aplastic anaemia, with a differential diagnosis of aphthous-like ulcer et causa thalassemia and systemic lupus erythematosus. figure 3. the legs (a) and arm (b): multiple macules, varying in shape and size, red-black in colour, smooth surface texture, clear irregular margin, normal surrounding tissue, painless. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 52 talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 case management the oral lesions were treated with 10% povidone-iodine and 1% feracrylum mouthwash. communicating with the patient about the disease and its possible cause. instructing the patient in the use of oral topical medications that have been given with the following instructions, eating high nutrition food, not touching the lesions with their tongue and fingers, and using a mask to avoid secondary infection, as well as maintaining oral hygiene. after consulting through teledentistry, the first action taken was to refer the patient to the emergency department to get therapy for her low platelets level. during hospitalisation, the patient was diagnosed with aplastic anaemia by the internist and received blood transfusion therapy. the patient was hospitalized for four days and received outpatient medication consisting of methylprednisolone, cotrimoxazole, and cefixime. ten days later (january 5, 2021), the patient had controlled the condition through teledentistry, and she explained that the bleeding wound on the inside of the left cheek has decreased, and was painless. but new painful and bleeding lesions appeared in other areas of her mouth, namely on the upper right gums. the patient explained that she had checked with the internist four days after discharge from the hospital and underwent another blood test and was given outpatient medication in the form of tranexamic acid, phytomenadione, and cefixime. the results of this second blood test were 11.4 g/dl of haemoglobin, 150/ ul of leukocytes, 33.1% of haematocrit, and 7000/ul of platelets. on extraoral examination (figure 5) there are multiple macules on her feet, with varying shapes and sizes, blackish-red colour, smooth surface texture, clear irregular margin, normal surrounding tissue, and painless. on the lip corner sinistra there are multiple crusts, 2-3 mm in size, blackish-red colour, clear irregular margin, rough surface texture, spontaneous bleeding, and pain. while on intraoral examination, there was a solitary ulcer on buccal mucosa sinistra, 1x1.5 cm in size, clear irregular margin, rough surface texture, spontaneous bleeding, painless. on the posterior gingival maxilla dextra, there figure 4. buccal mucosa sinistra: solitary ulcer, 1x1.5 cm in size, clear irregular margin, rough surface texture, spontaneous bleeding. figure 5. the leg at the first control: multiple maculae, varying in shape and size, blackish-red, smooth surface texture, clear irregular margin, normal surrounding tissue colour, painless. a b c figure 6. intraoral examination at first control (a) corner of the lips sinistra: multiple crusts, 2-3 mm in size, blackish-red, clear irregular margin, rough surface texture, spontaneous bleeding, painless; (b) the buccal mucosa dextra: solitary ulcer, 1x1.5cm in size, clear irregular margin, rough surface texture, spontaneous bleeding, painless; (c) gingival of the posterior maxilla (dextra): spontaneous gingival bleeding with a recession, painless. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 53talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 was spontaneous bleeding with gingival recession, and painless (figure 6). the diagnosis was aphthous-like ulcer related to aplastic anaemia, with a differential diagnosis of aphthous-like ulcer et causa thalassemia and systemic lupus erythematosus. the lesions in the oral cavity are being treated with 10% povidone-iodine solution and aloe vera extract gel. the use of topical drugs as instructed. discussion aplastic anaemia is described as a severe haematological syndrome, which has the potential to be life-threatening. a hypo or aplastic bone marrow condition indicates aplastic anaemia, which is associated with peripheral pancytopenia. this condition is a rare, sporadic, and non-communicable condition.10 based on the severity, there are classifications of aplastic anaemia: moderate, severe, and very severe. in moderate aplastic anaemia, the bone marrow cellularity and levels of two of the three components of hematopoietic cells decreased. in severe aplastic anaemia, they found bone marrow cellularity <30%, a decrease in two of the three components of hematopoietic cells, absolute neutrophil count (anc) <0.5x109/l, and the need for transfusion. very severe aplastic anaemia showed the same symptoms as severe aplastic anaemia, but the anc in very severe aplastic anaemia was <0.2x109/l.11,12 in this case, there is a decrease in haemoglobin, erythrocytes, haematocrit, mcv, mch, mchc, and leukocytes. the anc cannot be done because it is too low. so, the aplastic anaemia in this patient is very severe aplastic anaemia. the oral manifestations of aplastic anaemia may be the very first clinical symptoms of the disease and are directly associated with pancytopenia. the common features include petechial purpuric spots or oral mucosal hematomas, while gingival haemorrhage may be seen in some cases; these findings are seen because of platelet deficiency. ulcerative lesions of oral mucosa and pharynx are seen due to neutropenia.13 in this case, the patient had oral manifestations in the form of oral ulceration and spontaneous bleeding in the gingiva and oral mucosa. there are multiple crusted lesions in the upper and lower lip. oral ulcers in patients with aplastic anaemia have been reported to be associated with deficiencies of vitamins b (include b1, b2, b6, b12), folic acid, iron, and serum ferritin. aplastic anaemia associated with iron deficiency results in painful angular cheilitis and depapillation of the tongue, as well as erosive and crusty lesions in the perioral area and lip commissures.14 an aphthous-like ulcer is similar to recurrent aphthous stomatitis (ras). if the occurrence of aphthae is associated with a compromised systemic condition, the term aphthouslike ulcer is more appropriate, since aphthae are considered a secondary manifestation of systemic disease and have a different clinical history, as well as require different management strategies from ras. clinically, aphthouslike ulcers are shallow, round/oval-shaped lesions, granular surface texture, and pain. usually, the lesions appear suddenly and are accompanied by systemic symptoms.14 the aphthous-like ulcer can be determined based on the patient’s medical history, the presence of lesions affecting the skin, mucosa other than the oral cavity (eye mucosa or genital mucosa), and the digestive and respiratory tract. in addition, a complete blood count can also confirm the diagnosis of an aphthous-like ulcer and relate it to the systemic condition.15 in this case, the ulcer lesion was found in the patient’s oral cavity, as well as spontaneous bleeding in the gingiva, that took one month to heal. extraoral lesions were also found; there are multiple blackish macules in the extremities. on supportive examination, several blood component levels were decreased, namely haemoglobin, erythrocytes, haematocrit, mcv, mch, mchc, leukocytes, and platelets. based on anamnesis and objectives examination, this case was diagnosed as an aphthous-like ulcer related to aplastic anaemia. in the covid-19 pandemic era, many patients with various diseases were unable to obtain health services as they should have. therefore, to facilitate patient access so that they can continue receiving health services, dentists provide consulting services and disease therapy as first aid efforts through the teledentistry method. this case was handled during the covid-19 pandemic era, so their medical history, objective examination, treatment planning, and controlling the patient’s oral complaints were all carried out through teledentistry. management of patients through teledentistry considers the sensitivity of a dentist in detecting disease, patient compliance in carrying out dentist’s instructions, as well as the selection of medications that are easy to obtain and safe. management of oral lesions in patients with aplastic anaemia requires treatment in conjunction with other disciplines, such as internists or haematology. immediate referral of patients for special care is undoubtedly an important step in the management of this disease.8 so in this case, before treating the oral lesions, first refer her to the emergency department for first aid. as for the treatment of teeth or lesions in the oral cavity, it should be done at the same time as the platelet transfusion. the effective management of aplastic anaemia will lead to improving oral lesions caused by systemic conditions. according to theory, the management of aplastic anaemia consists of general management and specific management. general management can include insertion of a nasogastric tube, platelet transfusion or packed red cell depending on need, antifibrinolytic, antibiotics, and antipyretics. specific management may include giving immunosuppressants, such as cyclosporine/antithymocyte, corticosteroids, granulocyte colony-stimulating factor, and stem cell transplantation.15 general management is carried out in the form of platelet transfusions, cotrimoxazole, cefixime, tranexamic acid, and phytomenadione. platelet transfusions are given to patients due to platelet levels being too low (150000/ ul). antibiotics are given to prevent secondary infection. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 54 talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 the phytomenadione and tranexamic acid were intended as an anticoagulant and antifibrinolytic due to the patient’s severe thrombocytopenia. specific management performed on the patient is the administration of immunosuppressants (methylprednisolone). treatment of patients with aphthous-like ulcers depends on the aetiology, i.e., therapy for the underlying cause. if there is a medical history that is the cause of an aphthouslike ulcer (haematological, infectious, gastrointestinal, or skin disease), it should be considered to determine the type of therapy that is needed. it is also important to pay attention to local factors, including sharp tooth surfaces, wearing of dentures and other devices, and the occurrence of biting the oral mucosa during chewing. for aphthous ulcers such as aphthous-like ulcers, topical corticosteroids or tacrolimus can be given.15 during primary dental care, patients should take antifibrinolytics to avoid excessive bleeding. these agents can reduce the occurrence of bleeding, especially in the oral mucosa. in patients with thrombocytopenia, this can be done by stabilizing the thrombus. there are case reports explaining that aplastic anaemia can be treated by platelet transfusion at the start of treatment, instructions for maintaining oral hygiene, prophylaxis, and systemic administration of aminocaproic acid. infections often occur in susceptible aplastic anaemia sufferers, therefore dental treatment should be postponed until the patient’s condition is stable, that is until the patient’s leukocyte level is within normal limits. dentists should consider prescribing antibacterial mouthwashes and oral antibiotics before dental procedures to prevent foci of infection. consultation with a haematologist also needs to be done before receiving dental and oral treatment, so that the potential for systemic infection during treatment can be avoided.8 in this patient, the overall management of oral lesions was carried out by teledentistry and assisted by an internist who cared for her during hospitalisation. the patient had received oral corticosteroid medication from an internist, namely methylprednisolone, so the patient did not need to be given any more topical corticosteroid medication for her oral lesions. to prevent secondary infection in the oral mucosa, the patient was given an antiseptic mouthwash in the form of 10% povidone-iodine solution. also, a 1% feracrylum mouthwash was given to avoid spontaneous bleeding in the oral mucosa. in the current study, the 1% feracrylum citrate was used as a novel haemostatic agent. it is an effective, safe, reliable topical agent.16 as an antiinflammatory topical drug, aloe vera extract gel was used to inhibit the inflammation in the oral lesions. the patient was instructed not to touch the wound in her mouth carelessly because the wound bleeds easily even with a light touch. the prognosis of patients with aphthous-like ulcers related to aplastic anaemia will be better if the doctor detects early systemic abnormalities suffered by the patient. prompt, appropriate, and adequate therapy will also improve the prognosis of this disease. if there is a delay in detecting aplastic anaemia that is the cause of the aphthouslike ulcer, so that appropriate therapy is delayed, it can lead to death. the patient in this case experienced delays in early detection of the disease, resulting in delays in treatment. this was fatal, and the patient died 46 days after the first complaint appeared and 21 days after her consultation through teledentistry, despite receiving multidisciplinary therapy thereafter. management of oral manifestations in patients with aplastic anaemia requires a multidisciplinary approach to achieve complete recovery. in conclusion, during the covid-19 pandemic, patient management in the field of oral medicine was carried out through teledentistry. the management of this aphthous like ulcer et causa aplastic anaemia, including history taking, diagnosis, and treatment planning was done online. an aphthous-like ulcer is an intra-oral condition that can occur in patients with haematological disorders. an aphthouslike ulcer in haematological disorders like aplastic anaemia needs to be treated quickly and appropriately. in this case, the management requires a multidisciplinary approach, so that the patients can achieve complete recovery. acknowledgement the author wishes to thank the internal medicine clinic of dr. soebandi general hospital, jember, east java, indonesia. references 1. miano m, dufour c. the diagnosis and treatment of aplastic anemia: a review. int j hematol. 2015; 101(6): 527–35. 2. young ns. aplastic anemia. longo dl, editor. n engl j med. 2018; 379(17): 1643–56. 3. djusad s, yoarva malano. management of aplastic anemia in pregnancy: a case report. indones j obstet gynecol. 2020; 8(2): 125–8. 4. brodsky ra, jones rj. aplastic anaemia. lancet. 2005; 365(9471): 1647–56. 5. pei nema n n f, ba r t el c, g rouven u. fi r st-l i ne a l logeneic hematopoietic stem cell transplantation of hla-matched sibling donors compared with first-line ciclosporin and/or antithymocyte or antilymphocyte globulin for acquired severe aplastic anemia. cochrane database syst rev. 2013; 2013(7): cd006407. 6. zeng y, katsanis e. the complex pathophysiology of acquired aplastic anaemia. clin exp immunol. 2015; 180(3): 361–70. 7. wang l, liu h. pathogenesis of aplastic anemia. hematology. 2019; 24(1): 559–66. 8. rai a, vaishali v, naikmasur vg, kumar a, sattur a. aplastic anemia presenting as bleeding of gingiva: case report and dental considerations. saudi j dent res. 2016; 7(1): 69–72. 9. amtha r, gunardi i, astoeti te, roeslan mo. characteristic of oral medicine patient using teledentistry during covid-19 pandemic. odonto dent j. 2021; 8(1): 18–27. 10. dinca al, marginean oc, melit le, damian r, chincesan m. aplastic anaemia: therapeutic and deontological aspects. rom j pediatr. 2016; 65(1): 56–9. 11. singh p, sinha a, kamath a, malhotra s, chandra ab. aplastic anemiaa quick review. j cancer prev curr res. 2017; 7(5): 1–6. 12. guinan ec. diagnosis and management of aplastic anemia. hematol am soc hematol educ progr. 2011; 2011: 76–81. 13. anitha n, appadurai p. anemia and it’s oral manifestation. eur j mol clin med. 2020; 7(8): 1715–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 55talahatu et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 49–55 14. cappello f, rappa f, canepa f, carini f, mazzola m, tomasello g, bonaventura g, giuliana g, leone a, saguto d, scalia f, bucchieri f, fucarino a, campisi g. probiotics can cure oral aphthous-like ulcers in inflammatory bowel disease patients: a review of the literature and a working hypothesis. int j mol sci. 2019; 20(20): 5026. 15. putra m, aprijadi h. anemia aplastik berat dengan komplikasi febril neutropenia dan perdarahan pada perempuan usia 20 tahun. j agromedicine. 2019; 6(1): 226–30. 16. valse d, hosalli kumaraswamy n. to evaluate the role of feracrylum (1%) as hemostatic agent in tonsillectomy. indian j otolaryngol head neck surg. 2021; 73(2): 240–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p49–55 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p49-55 vol 49 no 3 juli-sept 2016.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 49, number 3, september 2016 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2016 january 2nd – december 31st, 2016 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga editorial boards: roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); harry huiz peeters (laser research center, bandung, indonesia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); fajar hamonangan nasution (department of orthodontics faculty of dentistry, universitas trisakti, indonesia); pinandi sri pudyani (department of orthodontics faculty of dentistry, universitas gadjah mada, indonesia); boedi oetomo roeslan (department of biochemistry faculty of dentistry, universitas trisakti); rahmi amtha (department of oral medicine faculty of dentistry, universitas trisakti, indonesia); anita yuliati (department of dental material faculty of dental medicine, universitas airlangga, indonesia); darmawan setijanto (department of dental public health faculty of dental medicine, universitas airlangga, indonesia); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); boy muchlis bachtiar (department of oral biology faculty of dentistry, universitas indonesia, indonesia). managing editors: priyawan rachmadi (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); markus budi rahardjo (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); rostiny (department of prostodontic dentistry, faculty of dental medicine, universitas airlangga, indonesia); elly munadziroh (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty vocational, universitas airlangga, indonesia); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia) assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers rasmidar samad (department of epidemiology, faculty of dentistry, universitas hasanuddin, indonesia); melanie sadono djamil (department of biomedic, faculty of dentistry, universitas trisakti, indonesia); siti sunarintyas (dental biomaterial, faculty of dentistry, universitas gadjah mada, indonesia); dewi priandini (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); thalca agusni (department of orthodontics dentistry, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); kus harijanti (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); titiek berniyanti (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); retno indrawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ernie maduratna setiawatie (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health faculty of dental medicine, universitas airlangga, indonesia). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 086/01.17/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 49, number 3, september 2016 p-issn: 1978-3728 e-issn: 2442-9740 1. correlation between predictions to get a new dental caries with residence area and parental socioeconomic conditions in adolescents in sleman diy bambang priyono, hari kusnanto, al. supartinah, and dibyo pramono .................................. 115–119 2. corellation between p53 expressions and histopathological grading in oral cavity squamous cell carcinoma silvi kintawati ................................................................................................................................. 120–124 3. acceleration of fibroblast number and fgf-2 expression using channa striata extract induction during wound healing process: in vivo studies in wistar rats gunawan oentaryo, istiati, and pratiwi soesilawati .................................................................... 125–132 4. topical application of 1% znso4 on oral ulcers increases the number of macrophages in normal or diabetic conditions of wistar rats rochman mujayanto, kus harijanti, and iwan hernawan ......................................................... 133–136 5. effect of cassave leaf flavonoid extract on tnf-α expressions in rat models suffering from periodontitis zahara meilawaty and banun kusumawardani ........................................................................... 137–142 6. effects of robusta coffee (coffea canephora) brewing on levels of rankl and tgfβ1 in orthodontic tooth movement herniyati, ida bagus narmada, and soetjipto ............................................................................... 143–147 7. inhibitory effect of jengkol leaf (pithecellobium jiringa) extract to inhibit candida albicans biofilm muhammad luthfi, ira arundina, and nizamiar hanmi ............................................................ 148–152 8. compressive strength and porosity tests on bovine hydroxyapatite-gelatin-chitosan scaffolds nadia kartikasari, anita yuliati, and indah listiana ................................................................. 153–157 9. inhibitory effects of siwak (salvadora persica. l) extract on the growth of enterococcus faecalis planktonics and biofilms in vitro ika rhisty cendana sari, rini devijanti ridwan, and diah savitri ernawati ......................... 158–162 10. oral health status of elementary-school children varied according to school they attended sri widiati, al. supartinah santosa, yayi suryo prabandari, and johana endang prawitasari ............................................................................................................ 163–167 11. effects of soy isoflavone genistein on orthodontic tooth movement in guinea pigs sri suparwitri, pinandi sri pudiyani, sofia mubarika haryana, and dewi agustina .............. 168–174 vol 51 no 4 okt-des 2018.indd 210210 the evaluation of mandibular bone density in chronic periodontitis models yuliana mahdiyah da’at arina,1 f. ferdiansyah,2 and mohamad rubianto3 1department of periodontics, faculty of dentistry, universitas jember, jember – indonesia 2department of orthopedics and traumatology, dr. soetomo general hospital, surabaya – indonesia 3department of periodontology, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: bone density, an important factor in functional bone quality, can affect the success of implant osteointegration or orthodontic treatment. a number of studies report that chonic periodontitis constitutes one risk factor of osteoporosis characterized by low bone mineral density and that the mandible is susceptible to osteoporosis. purpose: the purpose of this study was to evaluate mandibular bone density in animal subjects suffering from chronic periodontitis. methods: 40 male wistar rats were divided into four chronic periodontitis groups and four control groups (each group n=5). as chronic periodontitis models, the subjects were injected with 2×109 cfu/ml of porphyromonas gingivalis in the sulcular gingiva, whereas control group members were injected with normal saline. after 2, 3, 4 and 6-week injection periods, the subjects were sacrificed and radiographic examination of the mandibular bone subsequently performed. mandibular bone density was evaluated by histometric analysis. results: the mandibular bone density in members of the chronic periodontitis group was significantly lower than those of the control group (p<0.05). the reduced mandibular bone density in the chronic periodontitis group was in line with the protracted bouts of periodontitis. conclusion: reduced mandibular bone density was found in the chronic periodontitis model. the longer the duration of a bout of chronic periodontitis, the greater the reduction in mandibular bone density. keywords: chronic periodontitis; mandibular bone density; porphyromonas gingivalis correspondence: yuliana mahdiyah da’at arina, department of periodontology, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. email: yulianamahdiyah@gmail.com research report introduction bone density is an important factor determining functional bone quality, with lower mandibular bone density impairing both implant osteointegration and orthodontic treatment. during dental implant therapy, bone quality should be evaluated because of its importance as a predictor of implant osseointegration. there are many factors that influence successful implant osseointegration, including bone mineral density. poor bone quality, characterised by a thin cortex, sparse trabeculae or altered trabecular architecture, eventually results in poor implant stabilization and longer osseointegration.1 osteoporosis, a form of bone disturbance characterised by low bone mineral density, constitutes a condition negatively affecting bone quality. the jawbones, particularly the mandible, can be affected by osteoporosis which decreases mandibular bone density.2 in addition to bone mineral density, trabecular bone structure can be evaluated as a means of determining bone quality. a dense mandibular alveolar trabecular pattern in a dentate subject represents a reliable indicator of normal bone mineral density, while a sparse trabecular pattern confirms low bone mineral density.3 chonic periodontitis has been reported as one of the risk factors relating to osteoporosis since the increasing severity of periodontitis exacerbates susceptibility to the condition.4 dental journal (majalah kedokteran gigi) 2018 december; 51(4): 210–215 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 mailto:yulianamahdiyah@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 211 arina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 210–215 chronic periodontitis constitutes a severe infectious disease resulting in inflammed supporting tissues of the teeth, progressive attachment loss and bone loss.5 considerable research has been conducted into the relationship between periodontitis and osteoporosis. however, to date, the results remain inconclusive. several studies found a positive correlation between chronic periodontitis and osteoporosis, while others did not. the investigation conducted by lafzi et al.6 indicated that the bone mineral density of patients suffering from chronic periodontitis is lower than those afflicted with gingivitis. the mandibular bone density found in individuals with chronic periodontitis was significantly lower than that of healthy individuals.7 however, another study reported that no correlation existed between chronic periodontitis and osteoporosis.8,9 therefore, understanding the nature of mandibular bone density in cases of chronic periodontitis remains a crucial research topic. consequently, the aim of this study was to evaluate the mandibular bone density in a subject suffering from chronic periodontitis that had been induced by a local injection of porphyromonas gingivalis (p. gingivalis). materials and methods this study constituted a true experiment incorporating a completely randomized design. its experimental procedures were all performed under a protocol approved by ethical clearance certificate number 017/hrecc.fodm /ii/ 2017, issued by the ethical committee of the faculty of dental medicine at universitas airlangga. the protocol to develop a model of chronic periodontitis was based on previous study with minimal modification.10 the bacterial suspension was prepared by means of the following procedure. p. gingivalis (atcc 33277, medimark, france) was cultured at 37°c on trypton soya agar (tsa) plates with 10% sheep blood, supplemented with 0.4 μl/ml vitamin k and hemin in an oxygen-free atmosphere (80% nitrogen, 10% carbon dioxide and 10% hydrogen). after 14 days of growth on tsa, certain p. gingivalis colonies were selected and a solution of 2x109 cfu/ml in 100 μl of 0,9% sodium chloride subsequently prepared for immediate use. gram’s method was applied to confirm the purity of the colonies. chronic periodontitis was induced in wistar-strain rattus novergicus subjects by injection of p. gingivalis into the gingiva. 3-4 month-old male subjects (n = 40), each 200-220 grams in weight, were randomly divided into four periodontitis groups and four control groups, each containing five members. the periodontitis group subjects were injected three times a week at 2-day intervals with 0.05 ml live p. gingivalis at a concentration of 2109 cfu/ml into the buccal and lingual subgingival sulcus area between the first and second left mandibular molar. the injection involved the use of a tuberculin disposable syringe (onemed, indonesia) with a 30 gauge needle (bd precisionglidetm needle, usa). control group members were injected with normal saline in accordance with the periodontitis group protocol. for the duration of the experiment, the subjects were housed in plastic cages, fed a standard laboratory diet and provided with water ad libitum. the periodontitis and control group subjects were sacrificed during the 2nd., 3rd., 4th. and 6th. weeks following injection. the mandibular specimens were harvested and fixed in normal buffer formalin. radiographic examination was performed to evaluate the alveolar bone resorption as a clinical symptom of chronic periodontitis. the sample was confirmed as a case of chronic periodontitis if alveolar bone resorption was present in the interproximal area between the first and second molar. furthermore, the mandibular specimens were decalcified in 10% ethylenediamine-tetra-acetic acid (edta) at ph 7, which was replaced twice a week during a period of 6-8 weeks until the process was completed. the decalcified specimens were subsequently submitted to routine histologic processing and embedded in paraffin. in order to facilitate histologic descriptive analysis, each sample was sliced into 5 mm continuous sections in the mesial-distal direction and prepared for haematoxylin and eosin (h&e) staining. histologic analysis was performed on mandibular bone from the mesial section of the first molar mesial root and the distal section of the second molar distal root. the mandibular bone density was considered to be the percentage of trabecular bone volume calculated by dividing the trabecular bone area by the total bone area, including the trabecular bone and bone marrow. all examination procedures involved the use of a nikon h600l light microscope at 100x magnification fully equipped with a ds fi2 300 megapixel digital camera and calibrated image processor using nikkon image system software. two examiners unsighted as to the allocation of samples and subjects to specific groups performed this analysis with the average of two sets of results being calculated as the data of each sample. data was presented in the form of a mean ± sd. the differences between the mandibular bone density of groups were analysed using one-way anova followed by an lsd post hoc test. the data was considered statistically significant when the p value was less than 0.05. results the result of radiographic examination showed there to be alveolar bone resorption in the interproximal area between the first and second molar in all periodontitis group members which was confirmed as a chronic periodontitis model (figure 1). the alveolar bone crest resorption was found in the 2-week periodontitis group and the alveolar bone resorption was greater in line with the injection period. the mandibular bone density of all control groups was not significantly different. the average mandibular bone density of the periodontitis groups was lower than that of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 212arina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 210–215 the control groups (table 1), but the difference between the 2-week periodontitis group and control group was not significant. the mandibular bone density between the 3, 4 and 6-week periodontitis and control groups was significantly different (p<0.05). the mandibular bone density in the periodontitis groups was more reduced than that in the control groups. the greatest reduction occurred in the 6-week periodontitis group (table 1). the reduced mandibular bone density coincided with the injection periods (figure 2). the longer the period during which injections were administered, the lower the mandibular bone density. the mandibular bone density of the 4 and 6-week periodontitis groups was significantly lower than that of the 2-week group (p<0.05). a significant difference existed between the respective mandibular bone density of the 3 and 6-week periodontitis groups (p<0.05). however, no significant differences were observed between the 3 and 4 -week periodontitis groups or between the 4 and 6 -week periodontitis groups. the histological feature of the mandibular bone density in the periodontitis and control groups is presented in figure 3. the histological feature of the control groups was a dense and compact trabecular bone, whereas the bone marrow was narrow. this result was similar to the histological feature of the 2-week periodontitis group. in the 3, 4 and 6-week periodontitis groups, the bone marrow was wider and greater in quantity, while in the trabecular bone it was narrow. this sparse trabecular bone in the periodontitis groups is considered to have a lower mandibular bone density than that of the control groups. discussion bone density is an important factor influencing functional bone quality. reduced mandibular bone density will impair implant osteointegration or orthodontic treatment. certain diseases are known to reduce bone density, including osteoporosis which can affect the mandibular bone. an association exists between osteoporosis and periodontitis4,6,7 and it is assumed that chronic periodontitis not only causes reduced alveolar bone height, but also reduced bone density. therefore, in this study, the mandibular bone density of a subject which had suffered from chronic periodontitis for a protracted period was evaluated. animal subjects have long been used to investigate the pathogenesis of periodontitis, because studies involving humans are limited due to the diffi culty of controlling the pathogenesis of periodontal disease.11 rats were employed as research subjects because the periodontal anatomy of their molar region shares several similarities with that of humans.12 an study of periodontal disease in animals plays an important role in facilitating the study of defi ned aspects of periodontitis, including: pathogenesis, the development and progress of disease.11 there are certain methods of initiating experimental periodontitis, including: ligature, injection of a bacterial or pathogenic component, and oral gavage.12 in this study, a model of chronic periodontitis as occurs naturally in humans was induced by localized injection of live p. gingivalis. p. gingivalis has been identifi ed as one major periodontal pathogen of chronic periodontitis.13 about 40–100% of adults affl icted by a b c d e f g h figure 1. representative radiographic images of the mandibular bone in the chronic periodontitis model. a greater degree of alveolar bone resorption occurs in the periodontitis groups compared to the control groups. the 2-week periodontitis group presented resorption in the alveolar bone crest (red arrow) (e), a greater degree of resorption occurring in the 3-week periodontitis group (f), increased resorption at 4 weeks (g) and the highest level in the 6-week periodontitis group (h). the control group at 2, 3, 4 and 6 weeks is shown in images a,b,c and d respectively. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 213 arina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 210–215 figure 2. the mandibular bone density of the chronic periodontitis model at various injection times. a b c d e f g h figure 3. a histological section of mandibular bone in the periodontitis and control groups (x100). the trabecular bone of the control group after 2 weeks (a), 3 weeks (b), 4 weeks (c) and 6 weeks (d) showed significant density. the 2-week periodontitis group demonstrated the same pattern as the 2-week control group (a). the sparse trabecular bone with wider bone marrow (black arrow) was present in periodontitis groups at 3 weeks (f), 4 weeks (g) and 6 weeks (h). table 1. the percentage of mandibular bone density in a chronic periodontitis model average (ngroup ±sd) p 2 week 58.43 (±9.18)5control d 0.000* 5periodontitis 55.41 (±9.84)cd control3 week 57.06 (±3.17)5 d 47.64 (±4.37)5periodontitis bc control4 week 55.97(±5.55)5 d 42.22 (±6.66)5periodontitis ab 6 week control 54.60 (±5.03)5 cd periodontitis 5 36.25 (±4.37)a *significant at α=0.05 a,b,c,d the same superscript showed no difference between groups dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 214arina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 210–215 periodontitis have been infected by these opportunistic bacteria.14 this research confi rmed a reduction in alveolar bone height of the periodontitis groups compared with that of the control group following injection of live p. gingivalis (figure1). alveolar bone crest resorption was detected in the 2-week periodontitis groups and increased over time. the results reported here were similar to those of the research conducted by han et al.15 and zhang et al.16 indicating that p. gingivalis infection results in increased bone resorption. numerous reviews explain the bone resorption mechanism in periodontitis. in short, the lipopolysaccharide (lps) and other bacterial toxins can stimulate the immune cells and osteoblast to release proinfl ammatory cytokines il-1α, il-1β, il-6, tnf-α, prostaglandin e2, komponen mmps and to increase the rankl/opg ratio that regulates the proliferation, differentiation and activation of osteoclast resulting in bone resorption.17 p. gingivalis lps, lipids and metabolic products have been reported as possessing the ability to modulate rankl and/or opg expression in osteoblasts to stimulate osteoclastogenesis.15 in many reports, alveolar bone loss can be detected as early as two weeks after the fi nal oral bacterial infection or three weeks after initiating infection,18 although the study conducted by de molon et al.11 found that oral infection through p. gingivalis was ineffective at inducing alveolar bone resorption compared with the ligature model. the different strains, concentration of p.gingivalis used in the study and the length of the experimental period may explain the variability of the research results. in this study, subjects were injected with 100μl live-p.gingivalis at a concentration of 2×109 cfu/ml a total of eighteen times, while in the research conducted by molon et al. 11 subjects were infected fi ve times with 1×109 cfu/ml. this investigation demonstrated that chronic periodontitis resulted in both reduced alveolar bone height and bone density, while a reduction in mandibular bone density was observed in the periodontitis group (see table 1). the sparse trabecular bone was observed in the 3-week, 4-week and 6-week periodontitis groups (figure 3) and indicated lower mandibular bone density.3 the lowest bone density was that of the 6-week periodontitis group. this reduced mandibular bone density increased progessively during the period of the experiment (figure 2). this study indicated that chronic periodontitis not only reduces alveolar height, but also bone density, thereby paralleling the fi ndings of a clinical study completed by tonguc et al.7 that the mandibular bone mineral density of subjects suffering from periodontitis was signifi cantly lower than that of the periodontally healthy subjects. a study of animal subjects conducted by zhang et al.16 demonstrated signifi cantly decreased residual alveolar bone volume and mineral density in p. gingivalis-infected animals compared with sham infected controls. similar to the alveolar bone resorption which appeared in radiographic images, the reduction in mandibular bone density among periodontitis group members was found to be greater in more prolonged periods. the 6-week periodontitis group presented the largest reduction in mandibular bone density. however, although reduced mandibular bone density was detected in the 2-week periodontitis group, a signifi cant difference was found early in the 3-week periodontitis group. from this result, it can be assumed that in chronic periodontitis, alveolar crest resorption occurs earlier than reduced bone density. nevertheless, further study is required to confi rm the validity of this assumption. the mechanism of reduced mandibular bone density in chronic periodontitis has yet to be clearly explained, although there is theoretical uncoupling between bone destruction and remodelling. increased osteoclastic destruction, decreased osteoblastic formation, or a combination of the two will reduce bone density.19 in addition to p. gingivalis possessing the ability to stimulate osteoclastogenesis, the components of p. gingivalis can inhibit the differentiation and osteogenesis of osteoblasts, thereby impeding alveolar bone formation.20 several potential questions emerge from the results of this study, for example, whether the mechanism of reduced bone density is the same as that of alveolar resorption, which bone cells play a dominant role in that mechanism and whether a specific cytokine determining the timing of reduced bone density exists. this study successfully showed that chronic periodontitis reduced mandibular bone density which was also found to occur during orthodontic tooth movement. however, in the latter case, the reduced bone density recovered to its pre-orthodontic treatment level.21 it was interesting to investigate whether the reduced mandibular bone density caused by chronic periodontitis can be repaired following periodontal treatment, or whether it will be continue even if bacterial infection has been cured. however, based on result of this study, mandibular bone density examination is recommended for chronic periodontitis patients, especially those due to receive a dental implant or planning orthodontic treatment. the role of mandibular bone density in chronic periodontitis remains a critical topic for investigation. futher studies should explore the mechanism that promotes a reduction in mandibular bone density as a result of chronic periodontitis, particularly in order to discover the key molecule or cytokine in the pathogenesis of reduced mandibular bone density. such investigation would support the prevention and treatment of reduced mandibular bone density caused by chronic periodontitis. in conclusion, reduced mandibular bone density was found to have occurred in a chronic periodontitis model which suggests that the condition constitutes a risk factor in osteoporosis. the longer the periods of chronic periodontitis, the greater the reduction in mandibular bone density. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 215 arina, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 210–215 acknowledgement the authors express their gratitude to the directorate of research and community service, directorate general of research and development enhancement, ministry of research, technology and higher education, republic of indonesia (research contract number: 058/sp2h/lt/ drpm/2018) for its financial support of this research. references 1. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. philadelphia: saunders elsevier; 2015. p. 706-22. 2. yuce hb, toker h, ozdemir h, goze f. effects of two experimental models of osteoporosis on alveolar bone: histopathologic and densitometr ic study. oral health dent manag. 2014; 13(4): 915– 20. 3. jonasson g, billhult a. mandibular bone structure, bone mineral density, and clinical variables as fracture predictors: a 15-year follow-up of female patients in a dental clinic. oral surg oral med oral pathol oral radiol. 2013; 116(3): 362–8. 4. lohana m, suragimath g, abbayya k, varma s, zope s, kale v. a study to assess and correlate osteoporosis and periodontitis in selected population of maharashtra. j clin diagn res. 2015; 9(6): zc46-50. 5. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. philadelphia: saunders elsevier; 2015. p. 309-20. 6. lafzi a, amid r, kadkhodazadeh m, ahrara f. is there any association between systemic bone mineral density and clinical manifestations of periodontal disease? j periodontol implant dent. 2012; 4(2): 49–55. 7. tonguç mö, büyükkaplan uş, fentoğlu ö, gümüş ba, çerçi ss, kırzıoğlu fy. comparison of bone mineral density in the jaws of patients with and without chronic periodontitis. dentomaxillofacial radiol. 2012; 41(6): 509–14. 8. brennan-calanan r m, genco r j, wilding ge, hovey k m, trevisan m, wactawski-wende j. osteoporosis and oral infection: independent risk factors for oral bone loss. j dent res. 2008; 87(4): 323–7. 9. guiglia r, di fede o, lo russo l, sprini d, rini g-b, campisi g. osteoporosis, jawbones and periodontal disease. med oral patol oral cir bucal. 2013; 18(1): e93–9. 10. kusumawardani b, arina ymd, purwandhono a. evaluation of the placental development and fetal growth in a pregnant rat model induced by periodontal disease. bjog an int j obstet gynaecol. 2015; 122(s1): 9–10. 11. de molon rs, de avila ed, boas nogueira av, chaves de souza ja, avila-campos mj, de andrade cr, cirelli ja. evaluation of the host response in various models of induced periodontal disease in mice. j periodontol. 2014; 85(3): 465–77. 12. oz hs, puleo da. animal models for periodontal disease. j biomed biotechnol. 2011; 2011: 1–8. 13. how ky, song kp, chan kg. porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. front microbiol. 2016; 7: 1–14. 14. mysak j, podzimek s, sommerova p, lyuya-mi y, bartova j, janatova t, prochazkova j, duskova j. porphyromonas gingivalis: major periodontopathic pathogen overview. j immunol res. 2014; 2014: 1–8. 15. han x, lin x, yu x, lin j, kawai t, larosa kb, taubman ma. porphyromonas gingivalis infection-associated periodontal bone resorption is dependent on receptor activator of nf-kb ligand. infect immun. 2013; 81(5): 1502–9. 16. zhang w, ju j, rigney t, tribble g. porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model. bmc oral health. 2014; 14: 1–9. 17. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015; 2015: 1–10. 18. graves dt, li j, cochran dl. inflammation and uncoupling as mechanisms of periodontal bone loss. j dent res. 2011; 90(2): 143–53. 19. sims na, martin tj. coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. bonekey rep. 2014; 3: 1–10. 20. di benedetto a, gigante i, colucci s, grano m. periodontal disease: linking the primary inflammation to bone loss. clin dev immunol. 2013; 2013: 1–7. 21. yu j-h, huang h-l, liu c-f, wu j, li y-f, tsai m-t, hsu j-t. does orthodontic treatment affect the alveolar bone density? medicine (baltimore). 2016; 95(10): 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p210–215 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p210-215 206 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 206–211 research report electronic application for oral health school programme enhances the quality of the information in dental health data records nurul fatikhah1, gilang yubiliana2 and fedri ruluwedrata rinawan3 1 department of dental nursing, poltekkes kemenkes bandung 2 department of public dental health, faculty of dentistry, universitas padjadjaran 3 department of public health, faculty of medicine, universitas padjadjaran bandung – indonesia abstract background: dental and oral health problems among elementary students can be resolved through an oral health programme in schools. the main factor that inhibited this scheme was that the recording and reporting still employed a manual system, making it less effective and efficient. the electronic application of this programme can help managers to complete both of these tasks. purpose: this study’s aim is to assess the effectiveness of the electronic application that is utilised in the oral health school programme to increase the quality of the information relating to the recording of dental health data in schools. methods: this study used a pre-test and post-test one-group design, and the sample consisted of 37 oral health programme managers in schools who were chosen via simple random sampling. the data in this study was analysed using the wilcoxon signed-rank test. results: the results showed that the use of the electronic application as part of the oral health scheme influenced the quality of the information when details were recorded and reported. this can be seen in the rise in the standard of the information that was noted and disclosed when comparing data before and after using the electronic application; the average value of 1.54 (standard deviation=1.45) increased to an average value of 3.58 (standard deviation=2.84) with a significance level of 0.000 (p<0.05). conclusion: the electronic application used in conjunction with the oral health programme was effective in raising the quality of dental health records in schools. keywords: electronic application; oral health school programme; quality information; recording and reporting correspondence: nurul fatikhah, department of dental nursing, poltekkes kemenkes bandung, jl. prof. eyckman, no. 40 bandung 40161, indonesia. email: uul.fatikhah@gmail.com introduction oral and dental health are a part of the overall well-being of the body, but they have often been neglected by some people. keeping our mouths and teeth healthy is not only useful for chewing, talking and our appearance, it also supports our general welfare as living beings. world health organization (who) data for 2005–2015 showed that 90% of dental caries ware found in school-age children.1 the fit for school programme research conducted in cambodia, indonesia and laos stated that 94.4% of children had caries in their primary teeth and 73.2% were shown to have odontogenic infections, which can cause disruptions to eating and sleeping, poor quality of life, school absence and growth retardation.2 the results of the 2018 basic health research, or riset kesehatan dasar (riskesdas), disclosed that 57.6% of indonesia’s population had dental and oral problems and only 10.2% received dental medical treatment. meanwhile, in west java, 58% of the populace had dental and oral health issues and only 11.9% had received treatment.3 however, often neglected dental and oral health problems in children can be overcome through a full integration of oral and dental health into public health promotion strategies and school health programmes. such schemes in places of education teach children to maintain their oral and dental health and to comprehensively and continuously care for their mouths and teeth; thus, improving their health status in this regard. collaboration between schools and health workers was an important dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 mailto:uul.fatikhah@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 207fatikhah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 206–211 element in maximising this oral-health-based school programme. nevertheless, there were obstacles in terms of accessing information, such as difficulties in providing information on the condition of children’s oral health and the availability of details related to the programme with regards to schools and parents.4 the development of information and communication technology provided convenience when strengthening and developing health information systems. currently, there is a need to utilise information and communication technology in the health information system to improve the management and implementation of the field’s progression. these types of systems could be used as a tool to help oral health school programme managers provide data quickly and easily to schools, simplifying the process of monitoring performance and reporting activities online in a way that is both integrated and easily accessible by everyone.5 recording and reporting were part of the health information system, which was an indicator of success when assessing a programme, and the details generated from the processed data could be used for planning subsequent schemes. indeed, the information had an important role in the decision-making process. at the time, the recording and reporting of data in relation to the oral health school programme were still done manually, and the management team continued to use a book to record the results of the scheme’s oral health activities in schools. doing so was less effective and efficient because the person in charge of the scheme had to rewrite the results of said activities and had subsequent difficulties finding the relevant data; therefore, they took a relatively long time to compile the reports. electronic recording systems in the health sector have emerged as a result of the development of information and communication technology, which has been utilised to improve the quality and efficiency of health services.6 this kind of system could be easily accessed, reduce paper costs and diminish the number of problems when sharing information between patients and health workers. in addition, it could provide accurate and consistent care record details that are required for further maintenance,7 which can lead to the sustainability and success of a programme.8 furthermore, an electronic recording system could integrate data into one place that allows analysis to be done easily and quickly when making decisions. the use of electronic health records also leads to more comprehensive data input by patients, and can make examinations more accurate if a more detailed previous medical history has been given.9 the electronic recording system makes it easy to find patient data and their histories, so it saves time and is more effective. additionally, data pertaining to those being treated is stored more safely, meaning it is not easily lost and confusing patient data can be avoided. android is an operating system for smartphones based on linux, which is often used in the health sector as an innovation in the use of information technology. the advantage of android compared to other smartphone operating systems is that it employs an open-source code, making it easier for developers to create and modify applications or features that do not already exist within the operating system according to their wishes.10 the west java province school’s health programme advisory team has created a medisis website application, which is a student health management information system. its purpose is to store a history of the health and physical growth of students obtained through several measurements and examinations that can be done as part of the institution’s healthcare scheme. moreover, pupils can see the state of their well-being by using the aforementioned androidbased application. the aim is to use it to collate medical histories after they have been entered through the medisis website by the teacher or the medisis operator. the faculty of dentistry at airlangga university has also developed the simdagilut application, which is an onlinebased information system website and a tool for data management for dental and oral health surveys. dental health records on the medisis website and simdagilut used an odontogram, which recorded the dental health condition of school children, but did not explain in detail their dental and oral hygiene or the condition of any dental caries. the follow-up plan and the treatment carried out were also not included in the medisis and simdagilut applications. the simdagilut version of this digital tool only displays the status of immediate care needs without presenting the type of treatment needed. the results of a preliminary study conducted at public health centres, or pusat pelayanan kesehatan masyarakat (puskesmas), in bandung city show that the recording and reporting of oral health school programmes still utilises a manual system that involves the assist book. indeed, medisis and simdagilut applications have not been used at all by these places in the city, and manual notation and reporting make it difficult to find dental health data records; consequently, dental health statuses and the related care required for each child have not been included in the oral health programme report in schools. in addition, data from the scheme as reported to the health office is still being combined with information from visits to puskesmas and details from the community-based dental health programme. while the report on the oral health school programme is very important for determining policies or resolving problems related to oral health in elementary school children, the aim of this study is to investigate the effectiveness of the scheme’s electronic application in increasing the quality of the information held in dental health data records in schools. materials and methods this research has received approval from the research ethics commission of padjadjaran university (1505/ un6.kep/ec/2019) and has met the basic principles of ethics based on the belmont report, namely respect for dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 208 fatikhah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 206–211 persons (respect for human dignity), beneficence and nonmaleficence (principle benefit and not harming anyone) and justice. this research was conducted in bandung city from december 2019 to april 2020. the sample used in this study consisted of the managers of oral health school programmes in elementary schools within the working boundaries of the public health centre in bandung city, and they were chosen via a simple and randomised sampling method. the researchers compiled a list of the names of public health centres, and made a paper containing the number of notes that corresponded to this list. the paper was rolled up for the purpose of a draw to select a sample with a predetermined number, and the numbers on the paper that were picked were selected as the research sample. the inclusion criteria for this study were managers of oral health school programmes in elementary schools within the working parameters of health centres in bandung city; having oral health schemes in the elementary schools within the aforementioned area; the ability to use an android-based mobile phone that supports the programme’s electronic application; and the propensity to operate said application. concurrently, the exclusion criteria for this study was oral school programme managers who were not present at the time of the research. meanwhile, the conditions for dropping out were the programme managers who resigned in the middle of the research. this study did not use a control group, and the treatment group alone was given a pre-test and post-test. the sample formula used pairwise numerical analytic research (two-average hypothesis testing with a single sample) as follows: n = � (𝑍� + 𝑍�) 𝑆 𝑋� − 𝑋� � � where: n = minimum sample size, zα = standard deviation of alpha / z value to a certain degree of significance, zβ = standard deviate beta / z value at a given power test, s = standard deviation of the difference in value between groups, x1 x2 = the difference between the minimum mean which is considered significant. this study also used mean and standard deviation values based on the research by hoogerheide et al.11 because it was considered to be similar in nature, with a mean difference of 8.84 and a standard deviation of 0.12. estimation was done with a 5% degree of significance and 90% test strength. through the calculation of the sample size, the minimum number of subjects was 33 respondents. anticipating contributors dropping out, the number of subjects was increased by 10%, meaning the total number of respondents was 37.11 this research was conducted using a pre-test and posttest one-group design. respondents were asked to do a pre-test by filling out a questionnaire on the quality of the recording and reporting of information that is currently used, which still employs a manual system that involves the use of a book. the subject was then given an explanation of the uses of the oral health school programme’s electronic application. after that, the researcher asked scheme managers to enter existing dental and oral health screening data, specifically in relation to first graders based on last year’s screenings. the decision behind choosing this information in relation to this group of pupils was based on the programme’s guidelines that dental and oral health screening should be carried out on first-grade students. after 30 days, those in charge of the oral health schemes in schools were asked to fill out a questionnaire (the posttest) that quizzed them in the same manner as the pre-test to assess the quality of recording and reporting after using the scheme’s android-based electronic application. this was completed on the google form sent by the researchers. during the applicable trial period of 30 days, the scholars involved monitored the activities of the oral health school programme managers when collecting apposite data using the aforementioned application. the instrument used in this study was a questionnaire for assessing the quality of information recording and reporting on the oral health programme in schools, which consisted of 22 questions divided into four dimensions, namely the intrinsic, contextual, accessibility and representation elements. the assessment was carried out by the researcher and was assisted by the dental nurse. the independent variable in this study is the use of the electronic application, and its dependent counterpart is the quality of the recording and reporting information as part of the same scheme. the quality of the information can be measured from a person’s point of view by using a product and service that is in accordance with users’ expectations, and it can be quantified via the quality dimension method, which is a determining factor in the degree to which the benefits it contains have been achieved. wang and strong12 categorised the dimensions of information quality as intrinsic, contextual, representation and accessibility. the intrinsic dimension is a basic assessment that appears in relation to individuals that are using a product or service. this includes evaluating the accuracy, objectivity, content validation and the reputation of the information’s source. the contextual dimension scrutinises the content of an interrelated detail to make it easier for users to receive information. meanwhile, the contextual dimension relates to the relevance, added value, timeliness, reliability and completeness of information, and the representation dimension is a form of conveying information to users. the parameters of the presentation include the ability to interpret, ease of understanding and the conciseness/ consistency of the way the data is presented. lastly, the accessibility dimension assesses the ease of accessing information and the security system for guaranteed information or the existence of an arrangement for the sake of privacy. the instrument used has been tested for its validity and reliability on 30 oral health school programme managers and was found to have a cronbach’s alpha value of 0.92 (very good), person reliability of 0.90 (good) and item dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 209fatikhah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 206–211 reliability of 0.82 (good). the univariate analysis of the frequency distribution of general data, such as age, gender and education, can be utilised to describe the characteristics of each variable studied as well. in the meantime, bivariate analysis was used to determine the influence of the oral health programme’s electronic application on the quality of recording and reporting in the scheme. the data was examined using ibm statistical analysis for social science (spss) software version 20 (ibm corporation, illinois, chicago, us), and the shapiro-wilk test was employed to assess its normality. based on the shapiro-wilk test, it was found that the data was not normally distributed, so the next form of analysis used the wilcoxon test and found a significant value of 0.000 (p< 0.05), which indicates a significant change. results in terms of the characteristics of the respondents in this study, 83.78% of them were female, 51.36% were aged 26– 45 years, those with a diii education accounted for 75.66% of contributors and 86.47% of the sample group worked as dental nurses. the quality of information recorded and reported during the activities completed by android-based electronic application for oral health school programme were assessed based on users’ perceptions regarding the standard of the data provided, and were assessed using the following scores: one (very poor), two (poor), three (sufficient), four (good) and five (very good). table 1 highlights the fact that the average value of the recording and reporting quality of the oral health school programme increased after using the associated electronic application; the overall improvement in the standard of notation and delineation after using the application went from an average value of 1.54 (standard deviation=1.45) to an average value of 3.58 (standard deviation=2.84) where a significant value of 0.000 (<0.05) indicates a noteworthy change. when viewed based on the dimensions of information quality, only the intrinsic, contextual and accessibility dimensions show significant changes with a significant value <0.05. discussion a high calibre of information was needed to support the success of the system’s development. the results showed that the android-based electronic application for oral health school programme gave influenced to the quality of electronic application for oral health school programme recording and reporting. this was in accordance with salsabila’s13 research, which states that the use of an android-based smartphone can be a ready-made means of shortening the time spent on data collection and automatic data processing. this android-based recording and reporting application can also minimise paper requirements; therefore, reducing the risk of lost and corrupted data and facilitating the creation of fast and accurate reports based on the data needed at the highest level.13 android applications are mobile devices that provide a good platform to develop from, and they can be used as data collection systems. the relatively low cost, its portable nature and the ease of access to wireless networks where there is network range make these implements very attractive to users.14 the results of asgari’s15 research in iran showed that electronic dental health records could provide benefits with regards to storage, processing and data utilisation, which were all major components in delivering effective and efficient service based on the available evidence. digital versions of these records are more effective and efficient because they reduce paperwork, lead to the fast processing of data, facilitate ease of analysis and increase the accuracy, precision and quality of data. in addition, dental and oral health data records can be used in conjunction with programme planning and intervention.15 in relation to this, petersen et al.16 found in their research that in constructive health-related decision-making and planning, the information system must cover aspects of risk factors associated with oral health and the quality of information related to it, services and interventions, administrative processes and the quality of clinical care outcomes as well as epidemiological details. using the school programme’s android-based application minimises errors when entering data, makes it easier to create reports and offers information about the status of oral table 1. analysis of pre and post changes in the quality of electronic application for oral health school programme recording and reporting variable n mean sd min max significant value pre-all 37 1.54 1.45 -0.97 5.48 0.000 post-all 37 3.58 2.84 -3.46 9.52 pre-intrinsic 37 1.15 2.30 -3.06 5.50 0.021 post-intrinsic 37 -0.53 4.37 -8.50 8.29 pre-contextual 37 1.68 2.09 -2.50 6.73 0.044 post-contextual 37 0.66 3.29 -7.08 6.90 pre-representation 37 1.74 2.05 -2.02 7.07 0.056 post-representation 37 0.19 5.03 -9.65 9.64 pre-accessibility 37 1.33 2.64 -3.90 7.45 0.004 post-accessibility 37 2.89 2.76 -3.14 8.19 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 210 fatikhah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 206–211 health that can be used when making choices that bolster dental and oral health schemes. this was in keeping with what detsomboonrat’s et al.17 discovered in 2019, which was that android applications can be used to assist health care professionals in carrying out many important tasks, including time management, maintenance of and access to medical records, consultation, referrals, information gathering, clinical decision-making and educational health. the application was seen to be capable of reducing the number of errors and the time spent on data recording, and it could provide a good system for collecting information that can be used to assess the dental and oral health status of a population to determine policies and strategies that support dental and oral health programmes.17 the success of the information system was assessed by ascertaining the level of usefulness of the information when making reports and settling on related decisions. its degree of accomplishment could also be scrutinised from the perspective of the information generated as its quality was indispensable when the validity of the system’s development needed to be verified. the dimension of information quality was an approach used to measure the standard of data from the aforementioned electronic application based on users’ perceptions as user perception has become the most important factor in determining the value of the information supplied because everyone’s needs are different in this respect.18 in this study, measurements of the information in relation to the programme’s recording and reporting were undertaken using wang and strong’s theory,12 which divides the data into four strata: intrinsic, contextual, presentation and accessibility. the results of the influence analysis when using the school scheme’s electronic application to affect the quality of recording and reporting on oral health from each of these subdivisions showed that the ones that had a meaningful influence were the intrinsic, contextual and accessibility dimensions; meanwhile, the representation dimension was shown to have an insignificant bearing. the intrinsic dimension assessed the accuracy, objectivity and validity of the informational content as well as the reputation of the source of the data. the electronic application used in conjunction with the oral health programme in schools provided details that were free from the errors that may usually occur in the implementation of data processing, because it can automatically provide the results of the calculation of the dental hygiene index. the presentation of accurate information was vital as what was provided from the source to the recipient can cause interruptions and mistakes that can damage the data collated from the information. the material gathered by the programme’s application reflected the actual conditions and was in accordance with the requirements of the oral health manager in the specified schools. furthermore, the application presented information in an objective, complete and reliable manner, and the users’ trust in the details it offered showed that the information has a high level of credibility. this is supported by hazen’s et al.19 who asserted that the quality of the information depends on the accuracy of the data in line with the actual situation. the contextual dimension assessed the relevance, added value, timeliness, reliability and completeness of the information. the application linked to the aforementioned school programme provided details that were relevant to the oral health scheme, meaning they complete and useful for recording and reporting on it. complete information was imperative for both the provider and the recipient of the information, so that it can be understood clearly. the electronic application also helped the programme managers to create reports on time as they no longer need to make repetitive notes. information must be given in a timely manner so that its recipient is not too late to act, or to make decisions based on what has been brought to light. moreover, the scheme’s application provided added value, because it could automatically calculate the status of dental and oral health and generate reports that can be downloaded directly onto it. this was consistent with the findings of a study by lee et al.20, which stated that in the contextual dimension, users must make relevant, timely, complete and accepted considerations, which has added worth when providing changes to existing information, making it superior in terms of how it can be utilised. the representation dimension assessed the ability of interpretation, ease of understanding and the conciseness and consistency of presentation. the electronic application that was employed during the oral health scheme provided information that was easy to understand, systemic, consistent, in accordance with the reported needs and that could facilitate the production of reports on time. the use of clear and succinct language was also an added value when assessing the quality of the information. good recording and reporting produced statistics that did not conflict with previous information and did not vary. the results also underlined that the electronic application had no significant effect on the dimensions of the presentation. this was because the oral health program managers in schools had only just recently been introduced to it, meaning that they were not familiar with its employment. consequently, those in charge of the above oral health schemes were confused by application, so they had to ask about how to use it once again. this is supported by research by mcgill et al.21, which proposes that the utilisation of developed information systems should refer to how often users employ them; the more often they use information systems, the more they learn about them.21 the accessibility dimension assessed the ease of accessing information as well as the standard of the security system for guaranteed information or the existence of a system for privacy. the aforementioned application can be accessed without difficulty to get the information needed when the programme leader wants to make important decisions, which can in turn increase the success of such a scheme. the quality of the information was determined by its availability; in other words, the extent to which it can be retrieved whenever and wherever it is necessary. failure to dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 211fatikhah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 206–211 access the school programme’s electronic application was found to occur if the internet network was interrupted or if it was in the process of being maintained. the application was also equipped with a data security system, and the information that was presented had a privacy system, which meant that the data stored in the application could not be easily corrupted by means of duplication, dissemination or by being changed by irresponsible people.22 information quality was often measured as a major component of user satisfaction. indeed, the quality of the information was a determining factor in increasing the intention to use information technology. if users believed in the quality of the system they were utilising and felt that it was not difficult to use, they would then believe it would provide greater benefits and could optimise their performance. if the information generated by the system was more accurate, timely and exhibited a good degree of reliability, it will further increase user confidence in the system. rai et al.23 came to similar conclusions, stating that an increase in the trust of users of information systems was expected to further improve one’s performance. nevertheless, with regards to the limitations of this study, the electronic application for oral health school programme that was used is still a prototype, so suggestions are needed for further improvement and development. additionally, the details entered into the application were secondary data due to circumstances that make it impossible to carry out direct dental health checks on elementary school students. based on the results of the research on the influence of the android-based electronic oral health school programme application on the quality of recording/reporting in elementary schools around the working parameters of the public health centre in bandung city, it can be concluded that the tool was effective in increasing the calibre of the information contained in dental health records. the above application was utilised in accordance with the needs of the scheme’s managers and the existing conditions, so that it could be a solution to the problems associated with the manual recording and reporting system. it made these dual tasks effortless, prevented the necessity for repetitive work and could be accessed at any time. this research has contributed to enriching studies in the field of public dental health and producing new innovations regarding the electronic application for oral health school programme, which is used for noting down and delineating dental health in schools. it was hoped that the android-based electronic application relating to the oral health school programme can be used as a medium for recording and reporting in order to monitor the dental and oral healthcare of elementary school students. references 1. nguyen tt, nguyen bbt, nguyen ms, olak j, saag m. effect of school oral health promotion programme on dental health and health behaviour in vietnamese schoolchildren. pediatr dent j. 2016; 26(3): 115–21. 2. duijster d, monse b, dimaisip-nabuab j, djuharnoko p, heinrichweltzien r, hobdell m, kromeyer-hauschild k, kunthearith y, mijares-majini mc, siegmund n, soukhanouvong p, benzian h. ‘fit for school’ a school-based water, sanitation and hygiene programme to improve child health: results from a longitudinal study in cambodia, indonesia and lao pdr. bmc public health. 2017; 17: 302. 3. badan penelitian dan pengembangan kesehatan. hasil utama r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 66–71. 4. carpino r, walker mp, liu y, simmer-beck m. assessing the effectiveness of a school-based dental clinic on the oral health of children who lack access to dental care: a program evaluation. j sch nurs. 2017; 33(3): 181–8. 5. qureshi q, shah b, khan n, miankhel k, nawaz a. determining the users’ willingness to adopt electronic health eecords (ehr) in developing countries. gomal univ j res. 2013; 28(2): 140–8. 6. kruse cs, stein a, thomas h, kaur h. the use of electronic health records to support population health: a systematic review of the literature. j med syst. 2018; 42: 214. 7. jha ak. meaningful use of electronic health records: the road ahead. jama j am med assoc. 2010; 304(15): 1709–10. 8. fowler tt, matthews g, black c, kowal hc, vodicka p, edgerton e. evaluation of a comprehensive oral health services program in school-based health centers. matern child health j. 2018; 22(7): 998–1007. 9. nguyen l, bellucci e, nguyen lt. electronic health records implementation: an evaluation of information system impact and contingency factors. int j med inform. 2014; 83(11): 779–96. 10. hanrahan c, aungst t, cole s. evaluating mobile medical applications: an ashp ereport. usa: ashp publications; 2014. p. 1–23. 11. hoogerheide v, loyens smm, van gog t. effects of creating videobased modeling examples on learning and transfer. learn instr. 2014; 33: 108–19. 12. wang ry, strong dm. beyond accuracy: what data quality means to data consumers. j manag inf syst. 1996; 12(4): 5–34. 13. salsabila s. sistem informasi pencatatan dan pelaporan pelayanan kesehatan berbasis android di kawasan terpencil dan sangat terpencil. j rekam medis dan inf kesehat. 2019; 2: 39–47. 14. champanis m, rivett u. reporting water quality a case study of a mobile phone application for collecting data in developing countries. acm int conf proceeding ser. 2012; 12: 105–13. 15. asgari i. development an electronic oral health record application for educational dental setting. j educ health promot. 2018; 7: 124. 16. petersen pe, bourgeois d, bratthall d, ogawa h. oral health information systems towards measuring progress in oral health promotion and disease prevention. bull world health organ. 2005; 83(9): 686–93. 17. detsomboonrat p, pisarnturakit pp. development and evaluation: the satisfaction of using an oral health survey mobile application. telemed e-health. 2019; 25: 55–9. 18. delone wh, mclean er. the delone and mclean model of information systems success: a ten-year update. j manag inf syst. 2003; 19(4): 9–30. 19. hazen bt, boone ca, ezell jd, jones-farmer la. data quality for data science, predictive analytics, and big data in supply chain management: an introduction to the problem and suggestions for research and applications. int j prod econ. 2014; 154: 72–80. 20. lee yw, strong dm, kahn bk, wang ry. aimq: a methodology for information quality assessment. inf manag. 2002; 40(2): 133–46. 21. mcgill t, hobbs v, klobas j. user developed applications and information systems succes: a test of delone and mclean’s models. inf resour manag j. 2003; 16: 24–45. 22. michnik j, lo mc. the assessment of the information quality with the aid of multiple criteria analysis. eur j oper res. 2009; 195(3): 850–6. 23. rai a, lang ss, welker rb. assessing the validity of is success models: an empirical test and theoretical analysis. inf syst res. 2002; 13: 50–69. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p206–211 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p206-211 149149 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 149–152 research report howe’s dental cast analysis of students at the university of sumatera utara hilda fitria lubis and tiopan beltsazar sinurat department of orthodontics, faculty of dentistry, universitas sumatera utara, medan – indonesia abstract background: cast analysis is the measurement of the upper and lower arch in a 3-dimensional occlusal relationship. howe’s analysis can be used to observe total tooth material, arch length, and basal arch width. howe’s analysis was conducted to analyse abnormalities of both teeth and the jaw to help diagnose and plan dental care. purpose: to analyse the difference in total tooth material, arch length, basal arch width, and howe’s index between male and female undergraduate students from the university of sumatera utara who are of proto-malay origin. methods: ninety-six samples comprised of 48 male and 48 female students were recruited through the purposive sampling method. all samples were undergraduate students at the university of sumatera utara and of proto-malay origin, aged between 18 to 26, and have met the exclusion and inclusion criteria. we measured the variables using a digital caliper to measure basal arch width and arch length and moorrees’ method to measure total tooth material. data were analysed statistically using an independent t-test to compare means between groups (p<0.05). results: the male samples’ average value of total tooth material 98.49 mm±3.79, arch length 45.55 mm±2.28, basal arch width 41.07 mm±2.83, and howe’s index were 41.71 %±2.63, respectively. the female samples had a lower value compared to the male samples, 94.51 mm, 43.27 mm, 36.89 mm, and 39.05 % respectively. there was a significant difference in the total tooth material, arch length, basal arch width, and howe’s index between male and female undergraduate students from the university of sumatera utara of proto-malay origin (p=0.001; p<0.05). conclusion: the average measurement of total tooth material, arch length, basal arch width, and howe’s index was higher in male than female undergraduate students from the university of sumatera utara of proto-malay origin. keywords: howe’s analysis; howe’s index; orthodontics; proto-malay correspondence: hilda fitria lubis, department of orthodontics, faculty of dentistry, university of sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: hildadrgusu@gmail.com introduction recently, there has been an increase in interest for orthodontic treatment not only in children, but also in adolescents and adults. in general, a patient’s main request is to fix the alignment and arrangement of the dentition to provide a better facial appearance and to positive selfimage. orthodontic treatment is a long-term procedure that aims to reach optimum stability, function, and aesthetic. an abnormal alignment will become malocclusion.1–3 one of the most common types of malocclusion is crowding, which can be found in 2/3 of the human population. crowding is defined as the discrepancy between tooth size and available space on the arch, so that the dentitions become crowded and rotated.4 treatments of crowding are proximal stripping, expansion plates, proclination of anterior teeth, distalization, and extraction. according to graber, the goal of orthodontic treatment is to prevent and fix malocclusion, provide a harmonious facial shape, and provide good mastication. to achieve that, a cast analysis can be used as a guide to determine the diagnosis and proper planning in an orthodontic treatment.5,6 cast analysis or study model analysis is the measurement of the upper and lower arch in a 3-dimensional occlusal relationship. the correlation between the total tooth dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p149–152 mailto:hildadrgusu@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p149-152 150 lubis and sinurat/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 149–152 material, arch length, and basal arch width can be observed using cast analysis. one of analysis methods that is often used in orthodontic treatment is howe’s analysis proposed by ashley howe.2,7 based on expert opinions, every race has its own distinctive features, especially in the head and face regions. measurements using howe’s analysis have been done in individuals of caucasoid origin by howe, however it is not yet known whether the same result applies to other races. because of that, there is a need to measure the dentitions in the upper jaws of patients of different origins and compare them with howe’s findings.2 indonesia has a variety of ethnic groups and cultures that divide the ethnic groups in indonesia into five major groups: negrito, proto-malayid, dayakid, deuteromalayid, and madagassians. the percentage of the protomalay ethnic group in medan city is 24.46% from a total of 2,210,624 people and has become the biggest ethnic group in medan. the researchers chose the proto-malay ethnic group because proto-malay is the biggest ethnic group in medan.8,9 thus, the aim of this study is to analyse the difference of total tooth material, arch length, basal arch width, and howe’s index between male and female undergraduate students from the university of sumatera utara of proto-malay origin. materials and methods this study is a descriptive study on the measurement of the total tooth material, arch length, basal arch width, and howe’s index. this study was conducted at the university of sumatera utara. the samples were recruited using a purposive sampling method based on the inclusion and exclusion criteria which consisted of 96 samples. the population in this study are the male and female undergraduate students at the university of sumatera utara of proto-malay origin aged between 18 and 26. the inclusion criteria in this study were patients that were willing to participate in the research, signed their informed consent, were of proto-melayu race, 18 to 26 years old, of class i molar malocclusion, of class i canine malocclusion, had a complete number of teeth, had no history of orthodontic treatment, had crowding < 2-3 mm, and an ideal face profile. the exclusion criteria in this study were the patients that refused to participate, had caries, lesions or broken contacts, poor oral hygiene and a previous history of orthodontic treatment. measurements were taken after receiving consent from the participants. we took both maxillary and mandibular impressions of the samples chosen based on the purposive sampling method. we measured the basal arch width with a digital caliper and ruler according to howe’s method, total tooth material, and arch length. after obtaining the measurements, howe’s index was calculated using the following formula6: description: total tooth material = sum of the mesio-distal width of 16-26 (mm)6 basal arch width = measurement of canine fossa from one side to another or between 14 to 24 (mm)6 figure 1. measurement of the basal arch width which is measured from the root tips of the first premolar teeth to the fossa of the right and left canines. figure 2. measurement of total tooth material which is obtained from the total width of the mesio-distal teeth of the right maxillary permanent first molar to the left maxillary permanent first molar. figure 3. measurement of premolar diameter. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p149–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p149-152 151lubis and sinurat/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 149–152 data collection were done with the consent of the samples in this study. upper and lower alginate dental impressions were taken (mixing alginate powder with distilled water, which is the ratio following the manufacturer’s recommendation) from the samples who were undergraduates from the university of sumatera utara obtained through purposive sampling method and who met the inclusion and exclusion criteria. the researcher collected the model to be analysed and prepared the tools necessary for the cast analysis. the variables measured were basal arch width with a digital caliper and ruler using howe’s method (figure 1), tooth material according to moorrees’ method (figure 2), and premolar diameter (figure 3).10,11 each day only 10 cast measurements were taken to avoid eye fatigue and the obtained data were further analysed. results in this study, we found that the average value of total tooth material, the arch length, the average basal arch width, and the average ratio of howe’s index (table 1) were all higher in male students compared to female students. statistical analysis using the shapiro-wilkanalysis showed that the data were normally distributed (p>0.05) with p-value result of total tooth material (p=0.68), arch length (p=0.68), basal arch width (p=0.102), and howe’s index (p=0.051). the results of the independent t-test showed a significant difference in the total tooth material, arch length, basal arch width, and howe’s index between male and female undergraduate students from the university of sumatera utara of proto-malay origin (p=0.001; p<0.05). discussion the orthodontic treatment of crowded teeth usually involves proximal stripping or grinding, expansion, proclination of the anterior teeth, distalization, and extraction. for that reason, we need an analysis of the study model or cast analysis. cast analysis serves as a guide and has been the gold standard to determine proper diagnosis and treatment planning in orthodontic treatment for many years.5–7,12 various methods have been used for measuring and analysing the study models, including calipers, rulers, and other measuring tools. the data obtained from the measurement results were calculated with the relevant formulas.12 howe’s analysis is one of the cast analysis developed by ashley howe. howe discovered that the crowding of the teeth is generally due to a lack of width at the apical base, so that conditions where the width of the apical base is narrow can lead to irregular tooth arrangement. it was also found that there was a close relationship between the amount of tooth material and the width of the apical base, thus creating a formula for measuring howe’s index.5–7,13 in this study, we found that the average value of total tooth material, the arch length, the average basal arch width, and the average ratio of howe’s index were all higher in male students compared to female students. this study had the same results as the study conducted by pawar and jayade in karnataka utara. pawar and jayade found that the male samples had a higher average measurement in total tooth material, arch length, basal arch width, and howe’s index (89.46 mm±3.20, 41.78 mm ±1.60, 42.28 mm ± 1.49, 47.26 %±1.59 respectively).13 however, the values in pawar and jayade’s study had a lower value than the results we found in this study. the variations of the measurement results were due to the differences in the ethnic groups, hereditary traits, geographic locations, and cultural involvement between the two populations.13,14 there was a significant difference in the total tooth material, arch length, basal arch width, and howe’s index between male and female undergraduate students from the university of sumatera utara of proto-malay origin that indicated that there was a difference in the amount of dental material, the width of the dental arch, the width of the apical base, and the howe’s index of the students at the university of sumatera utara of proto-malay origin which is significant between women and men. this study had a similar result as the study by pawar and jayade’s and burris and harris’s study in america that showed a different result between male and female samples.13,15 studies of different ethnic groups also had been done by eunike et al. in 30 patients in maranatha dental and mouth hospital which showed that there were significant differences in the measurements of the length and width of the dental arch and howe’s index, whereas the measurement of the apical base showed no significant difference between the results of the study and the reference to the caucasian race.2 this study also differs from the study conducted by govindaraj table 1. mean and standard deviation of total tooth material, arch length, basal arch width, and howe’s index for male and female students from the university of sumatera utara’s undergraduate students of protomalay origin variables gender mean sd p-value total tooth material males (n=48) 98.49 mm 3.79 0.001 females (n=48) 94.51 mm 3.46 arch length males (n=48) 45.55 mm 2.28 0.001 females (n=48) 43.27 mm 2.37 basal arch width males (n=48) 41.07 mm 2.79 0.001 females (n=48) 36.89 mm 2.80 howe’s index males (n=48) 41.71 % 2.54 0.001 females (n=48) 39.05 % 2.85 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p149–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p149-152 152 lubis and sinurat/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 149–152 et al. on 30 diagnostic models of patients at saveetha hospital in cheennai city in a south indian population.14 a variation value was found in these ethnic groups due to influences in the size and shape of the dentition and the jaw, different culture, environment, and lifestyle in different places. 2,13,14,16 anthropology experts believe that each race holds a distinctive feature in the head and neck regions. howe’s cast analysis was commonly evaluated in caucasoid samples and the difference in the results with different ethnic groups is not yet known.2 this study found that the average measurement of total tooth material, arch length, basal arch width, and howe’s index was higher in males compared with female undergraduate students from the university of sumatera utara of proto-malay origin. the results in this study of the proto-malay race of students at the university of sumatera utara showed that the amount of dental material in males had a greater value than females, which indicated that males had a wider tooth shape than females. the arch width of the teeth and the width of the apical base in men had a greater value than women which indicated that the upper jaw in men was wider than in women, and the howe’s index results obtained by men showed a greater value than in women. this difference was caused by hormonal factors and lifestyle. these results were similar to the results of a study conducted by azlan et al. that showed a significant difference in intermolar width in indonesia, where the intermolar width in men was greater than in women.17 from this study result, it can be concluded that the average measurement of total tooth material, arch length, basal arch width, and howe’s index is higher in male than female undergraduate students from the university of sumatera utara of proto-malay origin. indonesia consists of many races (many of which are mixed) which have various shapes and tooth sizes, thus the use of howe’s analysis for diagnosing abnormalities of maxillary teeth in indonesian people needs to be studied further for its importance in diagnosis and treatment planning. further research is needed with a larger number of samples and it is necessary to conduct the study on other races, for example the deutromelayu and mixed races. it is also necessary to do further research using samples with class i and class ii malocclusion and samples with different age groups. references 1. patel d, mehta f, patel n, mehta n, trivedi i, mehta a. evaluation of arch width among class i normal occlusion, class ii division 1, class ii division 2, and class iii malocclusion in indian population. contemp clin dent. 2015; 6: s202–9. 2. eunike e. howes’ analysis measurement of rumah sakit gigi dan mulut maranatha bandung patients. j med heal. 2017; 1(6): 540–7. 3. goenharto s, rusdiana e, khairyyah in. comparison between removable and fixed orthodontic retainers. j vocat heal stud. 2017; 1(2): 87. 4. erliera, alamsyah rm, harahap nz. the relationship between nutritional status and crowding of junior high school students in medan baru. dentika dent j. 2015; 18(3): 242–6. 5. wahyuningsih s, hardjono s, suparwitri s. perawatan maloklusi angle klas i dengan gigi depan crowding berat dan cross bite menggunakan teknik begg pada pasien dengan kebersihan mulut buruk. maj kedokt gigi indones. 2014; 21(2): 2014–211. 6. graber l, vanarsdall r, vig k. orthodontics: current principles and techniques. 5th ed. philadelphia: mosby; 2012. p. 4–5. 7. kurniawan i, soeria soemantri es, evangelina ia. dental arch symmetry analysis in orthodontic treatment. padjadjaran j dent. 2008; 20(2): 89–94. 8. gupitasari a, heriniyati, putri lsda. the prevalence of bad habits as the etiology of angle’s class i malocclusion in orthodontic clinic dental hospital jember university 2015-2016. e-jurnal pustaka kesehat. 2018; 6(2): 365–70. 9. moyers re. handbook of orthodontics. 4th ed. chicago: year book medical publishers; 1988. p. 228–33. 10. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publisher; 2007. p. 84–8, 162–4. 11. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st louis-missouri: mosby elsevier; 2013. p. 1–10. 12. indirayana vp, gayatri g, zenab nry. a comparison between orthodontic model analysis using conventional methods and imodelanalysis. dent j (majalah kedokt gigi). 2018; 51(4): 173–8. 13. pawar rl, jayade vp. reliability of various study model indices in an adult population of north karnataka. j indian orthod soc. 2013; 47(4): 443–51. 14. govindaraj a, kumar sa, srirengalakshmi m. reliability of ashley howe’s analysis in south indian population. drug invent today. 2019; 11(2): 413–8. 15. burris bg, harris ef. maxillary arch size and shape in american blacks and whites. angle orthod. 2000; 70(4): 297–302. 16. rieuwpassa ie, toppo s, haerawati sd. difference of size and shape of dental arch between male and female of buginese, makassarese, and toraja. j dentomaxillofacial sci. 2012; 11(3): 156–60. 17. azlan a, mardiati e, evangelina ia. a gender-based comparison of intermolar width conducted at padjajaran university dental hospital, bandung, indonesia. dent j (majalah kedokt gigi). 2019; 52(4): 168–71. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p149–152 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p149-152 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 64 expression of cd133 in various premalignant and proliferative lesions rahmi amtha, 1 indrayadi gunardi,2 ferry sandra,3 and diah savitri ernawati4 1,2department of oral medicine, faculty of dentistry, universitas trisakti, jakarta indonesia 3laboratory of biocore, faculty of dentistry, universitas trisakti, jakarta indonesia 4department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: in jakarta, oral squamous cell carcinoma (oscc) usually detected in late stage with very low survival rate of about 1.1 years. oscc may be preceded by premalignant lesion, so that early detection of the lesion may decrease the mortality rate due to oral malignancy. cd133 is a hematopoietic stem cell that play role in tissue regeneration, inflammation and tumor. upregulated of cd133 was reported on tumor progression. purpose: the aim of study is to determine circulating cd133 expression on premalignant (pml) and proliferative (pl) lesion. method: observational research was carried out on patients who seek treatment of pml and pl at oral medicine clinic. cd133 was taken from peripheral blood serum, examined using pcr. data was analyzed by chi square test. result: 15 subjects (each of five subjects for pml, pl and control) consist of 40% male and 60% female. age group of above 41 years old was most affected pml and pl (66.7%). tongue is common site for oral lesion (40%). there is a significant different of circulating cd133 rate among all groups lesion (p=0.039). conclusion: cd133 express differently in premalignant and proliferative lesions. keywords: cd133; premalignant; proliferative correspondence: rahmi amtha, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa no. 1 grogol, jakarta barat, indonesia. e-mail: rahmi.amtha@gmail.com research report introduction squamous cell carcinoma (scc) is one of the most common cancers found in the oral cavity. it occurs almost 90% of all other types of cancer in the body.1 the number of oral cancer in the world increases annually, including in indonesia. the risk factors of oral scc are multifactorial. one of them are tobacco use, alcohol drinking and betel quid chewing habit.2 each country has different number of prevalence of scc, depend on their most risk factors applied in their society. it is also found that clinically scc can be developed from lesions in the mouth called premalignant lesion. oral lesions can be found either in the form of a normal variant or pathologic lesions. variants of the normal oral lesions such as torus, fissure tongue, geographic tongue, linea alba, frictional keratosis, fordyce spots, pigmentation physiological, lingual tonsils, and median rhomboid glossitis. the pathological lesions can be as benign lesion of the oral cavity is including benign tissue growth (such as papilloma, fibroma, epulis, cysts etc.) and pathologic lesions such as premalignant lesions, infections, hypersensitivity, autoimmune, systemic disease manifestations, as well as lesions caused by the treatment of systemic manifestations.2 premalignant lesions that usually found are leukoplakia, erythroplakia, oral lichen planus, lichenoid reaction, and submucous fibrosis. most of scc patients in jakarta population who come and need treatment was already in advanced/ late stage. its survival rate was very short, that was only about 1.1 years. this is likely due to lacking of early detection system of premalignant lesions that are clinically symptomless but microscopically already have dysplasia cell that potentially turn to malignant transformation.3 one of the factors that can affect changes in cells, which end up to uncontrolled cells, was involving the environment. in the human body, there are known as stem cells that can be transformed into the desired cell. in its dental journal (majalah kedokteran gigi) 2015 june; 48(2): 64–68 65 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 65amtha, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 64–68 development stem cell itself is also strongly influenced by external factors and internal cells. external factors such as the environment or habits that lead individuals exposed to substances that are carcinogenic, systemic disease, drugs, etc.4 whereas internal factors are including the factors, which are found in the body such as the genetic, hereditary carried by each individual, the cancer stem cell (csc) and growth factors that exist in the surrounding environment such as cells and other hormone.5 csc defines as a small subpopulation of cancer cells that form a pool of autonomous cell with the exclusive ability to make the heterogeneous lineages of cancer cells that comprehend the tumor.6 there are three main characteristic of cscs. first, the cell must show ability of initiation of tumor formation and it can revive the tumor that it was derived from a limited number of cells. the second, cells should show self-renewal in-vivo, which is practically observed through the regrowth of phenotypically indistinguishable and heterogeneous tumor following serial transplantation of relocated cscs in secondary and tertiary recipients. the third, cells must show a differentiation capacity allowing them to give rise to a heterogeneous progeny which represent a phenocopy of the original tumour.7 recently, csc study is widely done, because a lot of indications and prognostic value can be generated and useful to the clinician in determining the management of malignancy. the existence of csc is considered to be one of the factors which led to a pathologic lesion may become more aggressive, more resistant to cancer treatment and the possibility of metastasis. the prognostic value of the finding can change the paradigm of cancer management by controlling the csc population. csc can be found in the area where the tumor lesions were obtained and in the bloodstream that are called circulating cancer stem cell (ccsc). ccsc have been found in some cancer in the human body such as breast cancer, brain and blood cancer. the types of ccsc are cd133, cd24, cd44, cd26, aldh, and others.8,9 cd133 is a hematopoietic stem cell that is cholesterol interacting penta-span transmembrane glycoprotein (120kd), play a role in tissue regeneration, inflammation and tumors.10,11 the function of cd133 is unknown, but is expressed in biological stress. cd133 can be found on cancer stem cells.12 disorders of the cell mitosis that causes asymmetric cell tumors associated with the expression of cd133 positive. cd133 may enrich the csc to 200 times in human tumor tissues.13 by evaluating cd133 in the peripheral blood, the clinical output can be predicted and the success of the treatment can be improved. although the function of cd133 is unknown but previous study demonstrated that cd133 is a marker of cscs in various tumors.14-18 research on the expression of one of the csc in premalignant lesions and benign lesions in the oral cavity is still very rare, especially that drawn from the population of indonesia. therefore, it is deemed to know how the presence of circulating cd133 in premalignant lesions and benign lesions of the oral cavity. it can be useful as a baseline data to look for the correlation of csc expression in the tumor tissue. it is finally useful to predict the prognostic of lesions and treatment planning to prevent malignancy earlier. cd133 (prominin-1) is derived from prominin family found on hematopoietic stem cells, called ac133.19 therefore, cd133 is cholesterol interacting penta-span transmembrane glycoprotein (120kd), then carried by circulating cd133 endothelial progenitors (cep) and plays a role in tissue regeneration, inflammation and tumors.10,11 until now, there are three isoforms, namely cd1331, cd133-2 and cd133-3. the function of cd133 is unknown, but it is expressed in the change process including biological stress. it is known that by eliminating cd133 from the tissue, there will be no formation of new cd133, but this condition will stimulate the mammary glands to form new branching. while cd133 mutation (r733c) will cause retinal disorders.12 cd133 can be found on cancer stem cells.12 disturbances in embryo cell division that causes asymmetric cell associated with the expression of the cd133 (+) tumor cells. the cancer that have a high concentration of cd133 (+), associated with cancer resistant to radiation and chemotherapy treatments. cd133 high concentration is also associated with poor prognosis of some solid tumors such as lung, colon, and prostate.12,17,18 although the function of cd133 is unknown but it can be demonstrated that cd133 is a marker of cancer stem cells in various tumors.14-18 relative to cd133, a number of studies have demonstrated the expression of cd133 on a subpopulation of cancer cells from the brain, colon, lung, melanoma and other tumors. this indicates that the assumption that these cells have the ability of stem cells or progenitors and cd133 is considered as a marker of cancer stem cells.18,20-22 premalignant lesions are oral lesions that have a potential to turn to malignant or oral cancer. these lesions are including: leukoplakia, erytroplakia, lichen planus and submucous fibrosis. many literature reported that the premalignant lesions has tendency to become oral cancer in about 1-5.6 % depend on the country and risk factors applied in their community.2 besides the premalignant lesions, it is also known benign lesion that have no potencies to turn to malignant or oral cancer. these lesions including: mucocel, papilloma, fibroepitelial polyp and epulis.4,25 materials and methods a cross sectional observational research was carried out on patients who have suffered premalignant lesion (pml: leukoplakia, erythroplakia, oral lichen planus, lichenoid reaction, and submucous fibrosis.) and proliferative lesion (pl) such as papilloma, fibroma, epulis, and cysts that seek treatment to oral medicine clinic under informed consent. based on sample size calculation, fifteen of patients are needed in this research that will represent each of group of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 66 amtha, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 64–68 table 1. socio-demographic characteristics of the sample   n % sd age     20-30 3 20   31-40 2 13.3 0.843   >41 10 66.7 sex   male 6 40 0.507   female 9 60 oral lesions   premalignant 5 33.3 0.845   proliferative 5 33.4   normal 5 33.5 location tongue 6 40 0.516   buccal mucosa 4 26.7 o  thers 5 33.3 pml, pl and normal as a control. cd133 was taken from peripheral blood serum of 15 patients. the expression of cd133 was examined by using pcr and data was analyzed by chi square test. results the study was conducted at the dental hospital of faculty of dentistry universitas trisakti from september 2013 to july 2014, obtained sample of 15 subjects were then divided into 3 groups of premalignant lesions (33.3%), proliferative lesions (33.4%) and normal lesions (33.5%). table 1 showed that most of the sample was female (60%), older than 41 years (66.7%) and 40% most of the location is in the area of the tongue. from each sample, 5 ml of blood were taken and cd133 expression examined using pcr technique with followed by glyceraldehyde 3-phosphate dehydrogenase (gapdh) as a guardian gene to confirm the presence of cd133 genes (figure 1). cd133 was express at 120 bp and gapdh was expressed at 240 bp. cd133 and gapdh were found expressed at all samples with different quantity (figure 2). there was a significant different (p= 0.039) of cd133 expression of three groups (pml, pl and normal samples) as shown at table 2. discussion based on the literature, premalignant lesions are lesions that have the potential to malignant transformation or squamous cell carcinoma. the changes varied from 0 to 5.6% within 1 to 10 years. socio-demographic characteristics of this study showed that premalignant lesions are more common in women than men, aged over 40 years and this is in accordance with many other studies.24,26 tongue is the most common predilection of pml compared to other area. this result is also the same with many other studies.27 tongue as the most predilection site of pml and pl may be due to the nature of the tongue that prone to get trauma during mastication, speaking and other activities. besides that tongue has different texture of epithelium compared to buccal mucosa and other area in the mouth. the papillae of the tongue makes specific texture that predisposes the trap of food and increases the exposure of microorganism to the epithelium that may induce the inflammation. cd133 is a transmembrane glycoprotein that has 865 amino acids with a total molecular weight of 120kda.28 cd133 gene expression seen in conjunction with the guardian gene called gapdh on 120 bp and 240 bp respectively. cd133 ratio was obtained from a comparison between cd133 and gapdh quantitative calculations. the ratio helped to assay the consistency of pcr mix reaction. the quantity of expression (ratio) of cd 133 was calculated from the mean of both of them (gapdh and cd 133). this study found an increase in cd133 expression (figure 1) that circulate in the peripheral blood sample with premalignant lesions and proliferative lesions compared to normal samples (no lesions). cd133 ratio increased on premalignant lesions and proliferative lesions (figure 2) compared to normal. it demonstrates that cd133 is a cell that circulates in the peripheral blood circulation and the amount increase when one part of oral tissue stimulated/ irritated or undergoing cells proliferation. this is consistent with the function of progenitor cells that will adjust depending on the environment they entered. the abovedental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 66 amtha, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 64–68 figure 2. cd133 ratio on different lesions. table 1. socio-demographic characteristics of the sample n % sd age     20-30 3 20 31-40 2 13.3 0.843 >41 10 66.7 sex male 6 40 0.507 female 9 60 oral lesions premalignant 5 33.3 0.845 proliferative 5 33.4 normal 5 33.5 location tongue 6 40 0.516 buccal mucosa 4 26.7 others 5 33.3 pml, pl and normal as a control. cd133 was taken from peripheral blood serum of 15 patients. the expression of cd133 was examined by using pcr and data was analyzed by chi square test. results the study was conducted at the dental hospital of faculty of dentistry universitas trisakti from september 2013 to july 2014, obtained sample of 15 subjects were then divided into 3 groups of premalignant lesions (33.3%), proliferative lesions (33.4%) and normal lesions (33.5%). table 1 showed that most of the sample was female (60%), older than 41 years (66.7%) and 40% most of the location is in the area of the tongue. from each sample, 5 ml of blood were taken and cd133 expression examined using pcr technique with followed by glyceraldehyde 3-phosphate dehydrogenase (gapdh) as a guardian gene to confirm the presence of cd133 genes (figure 1). cd133 was express at 120 bp and gapdh was expressed at 240 bp. cd133 and gapdh were found expressed at all samples with different quantity (figure 2). there was a significant different (p= 0.039) of cd133 expression of three groups (pml, pl and normal samples) as shown at table 2. dna ladder [+] [-] pml 1 pml 2 pml 3 pl 1 pl 2 n 1 n 2 figure 1. expression of cd133 and gapdh (control gene) is positive in all samples of the study. note: pml: premalignant lesions, pl: proliferative lesions, n: no lesions. gapdh 240bp cd133 120bp figure 1. expression of cd133 and gapdh (control gene) is positive in all samples of the study. note: pml: premalignant lesions, pl: proliferative lesions, n: no lesions. 4.56 4.66 5.33 0.75 0.99 0.46 4.41 2.42 3.28 2.96 1.28 0.87 1.07 1.16 0.73 cd133 figure 2. cd133 ratio on different lesions. gapdh 240bp cd133 120bp 67 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 67amtha, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 64–68 table 2. cd133 expression on different lesions   low n(%) cd133  middle n(%) high n(%) p   lesion pml 2(40) 0(0) 3(60) 0.039* proliferative 1(40) 0(0) 3(60) normal 2(40) 3(60) 0(0) note: p<0.05 mentioned conditions are categorized that the increased cd133 is normal cell or not a cancer stem cell. however, if the cd133 increased in premalignant lesions, which later histopathologically proved the presence of dysplasia, then the type of this cd133 is most probably a cancer stem cell. it is suitable with the characteristic of cd133 that expression more biological stress conditions.10 cd133 was found to increase up to 200 times of the amount of csc in human tumor tissues.11 therefore, ideally the histopathological examination of cd133 on the tissue is required to be observed to see the correlation and also confirm by other marker of cds groups such as cd44. the results of this preliminary study was also supported by several other studies that showed a significant increase (p=0.002) of cd133 in lichen planus lesions that turn into squamous cell carcinoma.28 the existence of a variety of gene expression and chemical mediators/cytokines in premalignant lesions have been studied and proved a meaningful relationship. the discovery of chronic inflammatory cells in all premalignant lesions is assumed to contribute significantly to changes in these lesions into oral cancer. inflammatory process is able to create a microenvironment that can affect the growth, proliferation and differentiation of cells that end up on the condition of genetic mutations.26 mutated cells will lead to uncontrolled proliferation and known malignancy (cancer). stem cells are also influenced by micro-environmental conditions surrounding and under certain conditions can be transformed into stem cells, called cancer stem cells.6 the existence of cancer stem cells have been widely demonstrated to see the prognosis of a disease, effects of therapy and even help to see the prognosis of malignant transformation in some diseases.10,11 until now there is absence of study discuss about the association of expression of cd133 in above lesions. this study found that cd133 is expressed in premalignant and proliferative lesions as well as normal conditions differently. so that it may be useful as basic information for further study to see their correlation in carcinogenesis. references 1. thompson p. oral precancer: diagnosis and management of potentially malignant disorders. first edition. oxford: john wiley & sons, john wiley; 2012. p. 1-12. 2. scully c. oral and maxillofacial medicine the bases of diagnosis and treatment. 2nd ed. edinburgh: churchill livingstone; 2008. p. 289-90. 3. amtha r. risk factor and genetic polymorphism of oral cancer in jakarta population. case control studies. dissertation. kuala lumpur: university of malaya; 2007. 4. bourseau-guilmain e, griveau a, benoit jp, garcion e. the importance of the stem cell marker prominin-1/cd133 in the uptake of transferrin and in iron metabolism in human colon cancer caco-2 cells. plos one 2011; 6(9): e25515. 5. yunoue s, arita k, kawano h, uchida h, tokimura h, hirano h. ident i f icat ion of cd133+ cel ls i n pit u it a r y adenoma s. neuroendocrinology 2011; 94(4): 302-12. 6. major ag, pitty lp, farah cs. cancer stem cell markers in head and neck squamous cell carcinoma. stem cells int 2013; 2013: 319489. 7. chen z. the cancer stem cell concept in progression of head and neck cancer. j oncol 2009; 2009: 894064. 8. greaves m. cancer stem cells as ‘units of selection’. evol appl 2013; 6(1): 102-8. 9. ewald pw, swain ewald ha. toward a general evolutionary theory of oncogenesis. evol appl 2013; 6(1): 70-81. 10. asahara t, murohara t, sullivan a, silver m, van der zee r, li t, witzenbichler b, schatteman g, isner jm. isolation of putative progenitor endhothelial cells for angiogenesis. science 1997; 275(5302): 964-7. 11. shaked y, ciarrocchi a, franco m, lee cr, man s, cheung am, hicklin dj, chaplin d, foster fs, benezra r, kerbel rs. therapyinduced acute recruitment of circulating endhothelial progenitor cell to tumors. science 2006; 313(5794): 1785-7. 12. yu cc, lo wl, chen yw, huang pi, hsu hs, tseng lm, hung sc, kao sy, chang cj, chiou sh. bmi-1 regulates snail expression and promotes metastasis ability in head and neck squamous cancerderived aldh1 positive cells. j oncol 2011; 2011. pii: 609259. 13. ricci-vitiani l, lombardi dg, pilozzi e, biffoni m, todaro m, peschle c, de maria r. identivication and expansion of human colon-cancer-initiating cells. nature 2007; 445(7123): 111-5. 14. collins at, berry pa, hyde c, stower mj, maitland nj. prospective identivication of tumorigenic prostate cancer stem cells. cancer res 2005; 65(23): 10946-51. 15. eramo a, lotti f, sette g, pilozzi e, biffoni m, di virgilio a, conticello c, ruco l, peschle c, de maria r. identification and expansion of the tumorigenic lung cancer stem cell population. cell death differ 2008; 15(3): 504–14. 16. ma s, chan kw, hul, lee tk, wo jy, ng io, zheng bj, guan xy. identification and characterization of tumorigenic liver cancer stem/progenitor cells. gastroenterology 2007; 132(7): 2542–56. 17. o’brien ca, pollett a, gallinger s, dick je. a human colon cancer cell capable of initiating tumour growth in immunodeficient mice. nature 2007; 445(7123): 106–10. 18. singh sk, hawkins c, clarke id, squire ja, bayani j, hide t, henkelman rm, cusimano md, dirks pb. identivication of human brain tumour-initiating cells. nature 2004; 432(7015): 396-401. 19. yin ah, miraglia s, zanjani ed, almeida-porada g, ogawa m, leary ag, olweus j, kearney j, buck dw. ac133, a novel marker for human hematopoetik stem and progenitor cells. blood 1997; 90(12): 5002-12. 20. harper lj, piper k, common j, fortune f, mackenzie ic. stem cell patterns in cell lines derived from head and neck squamous cell carcinoma. j oral pathol med 2007; 36(10): 594–603. 21. ieta k, tanaka f, haraguchi n, kita y, sakashita h, mimori k, matsumoto t, inoue h, kuwano h, mori m. biological and genetic characteristics of tumor-initiating cells in colon cancer. ann surg oncol 2008; 15(2): 636–48. 22. bertolini g, roz l, perego p, tortoreto m, fontanella e, gatti l, pratesi g, fabbri a, andriani f, tinelli s, roz e, caserini r, lo vullo s, camerini t, mariani l, delia d, calabrò e, pastorino u, sozzi g. highly tumorigenic lung cancer cd133+ cells display stem-like features and are spared by cisplatin treatment. proc natl acad sci 2009; 106(38): 16281–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 68 amtha, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 64–68 23. lavanya n, jayanthi p, rao uk, ranganathan k. oral lichen planus: an update on pathogenesis and treatment. j oral maxillofac pathol 2011; 15(2): 127–132. 24. sun li, feng j, ma l, liu w, zhou z. cd133 expression in oral lichen planus correlated with the risk for progression to oral squamous cell carcinoma. ann diagn pathol 2013; 17(6): 486-9. 25. scully c, felix dh. oral medicine–update for dental practicioner: red and pigmented lesions. br dent j 2005; 199(10): 639-45. 26. otero-rey em1, suarez-alen f, peñamaria-mallon m, lopez-lopez j, blanco-carrion a. malignant transformation of oral lichen planus by a chronic inflammatory process. use of topical corticosteroids to prevent this progression?. acta odontol scand 2014; 22: 1-8. 27. k r ish na rao sv, mejia g, rober ts-t homson k, loga n r. epidemiology of oral cancer in asia in the past decade--an update (2000-2012). asian pac j cancer prev 2013; 14(10): 5567-77. 28. li z. cd133: a stem cell biomarker and beyond. exp hematol oncol 2013; 2(1): 17. 177177 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 177–182 dental student’s satisfaction towards orthodontic laboratory work from rsgm dental laboratory sianiwati goenharto,1 dini setyowati,2 and elly rusdiana1 1department of health, faculty of vocation, universitas airlangga 2department of dental public health, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: customer satisfaction plays an important role in the success and continuity of a business. dental laboratory of rsgm faculty of dental medicine universitas airlangga provides services for faculty of dental medicine, universitas airlangga students enrolled in a dental profession program. one of services offered is making of removable orthodontic appliances. until now there has been no research on customer satisfaction after the appliances made in the laboratory. purpose: this study was conducted to determine whether universitas airlangga dental students as customers were satisfied with the work of the rsgm dental laboratory, particularly in orthodontics. method: an analytic observational study was carried out on 48 faculty of dental medicine universitas airlangga students who were enrolled in a dental profession program and made removable orthodontic appliances in rsgm dental laboratory. the students were asked to fill out a questionnaire about satisfaction regarding the timeliness, the suitability of results, the services provided, the quality of both active and passive components, the ease of insertion and activation. data were analyzed descriptively. the relationship between satisfaction towards the quality of clasp, spring, bow and satisfaction towards the quality of acrylic plate to the ease of insertion were analyzed with fischer exact test. the relationship between satisfaction towards the quality of clasp, spring, bow and satisfaction towards the quality of acrylic plate to the ease of activation were analyzed by chi square. result: respondents who were satisfied with the timeliness of work were 54.17%, the suitability of the results of the query were 79.17%, the services provided were 95.83%, the results of clasp, bow and spring were 72.92%, the results of acrylic plate were 77.08%, the ease of insertion were 54.17%, and the ease of activation were 89.58%. the results of the statistical analysis showed a significant correlation between the ease of insertion and the quality of clasp, spring and bow (p = 0.01) as well as acrylic plate made (p = 0.045 <0.05), however, there was no significant correlation between the ease of activation with quality of clasp, spring and bow (p = 0.08) and the acrylic plate made (p = 0.337 (p> 0.05)). conclusion: it was concluded that the majority of respondents are satisfied with the results of removable orthodontic appliances produced, but punctuality still needs to be improved. keywords: satisfaction; dental laboratory; orthodontic appliance correspondence: sianiwati goenharto, c/o: departemen kesehatan, fakultas vokasi universitas airlangga. jl. srikana 65 surabaya 60286, indonesia. e-mail: sianiwati.goenharto@yahoo.co.id introduction customer satisfaction has long been known as one of important roles in the success and continuity of a business.1 dental laboratory is one of services included in health care, which directly affects and relates to human health. dental laboratory of rsgm faculty of dental medicine universitas airlangga provide services to produce appliances for prosthodontics, orthodontics and conservative dentistry. services are provided for students of professional programs, so that the students of faculty of dental medicine do not need to make their own patient’s appliances. one of the facilities provided for students working in the orthodontic clinic is the making of removable orthodontic appliances. removable orthodontic appliances consists of clasps as retention components, labial bows and springs as the active components and acrylic plate as base and supporting other components.2 each component must be well made, because dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 178 goenharto, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 177–182 the effectiveness of the active component also relies on other component.3 if each component is well prepared, insertion of the appliances can be done easily and the activation of the springs and bow as the active component can move the teeth as expected. until now there has been no research on customer satisfaction after orthodontic appliances made in the dental laboratory of rsgm faculty of dental medicine universitas airlangga, whereas feedback is necessary for the progress of a services in this highly competitive era. customer satisfaction includes the difference between the expectations of the performance or results should be surveyed. customers are satisfied when the results (outcomes) equal or exceed customer expectations.4 in other words, customer satisfaction is a feeling obtained from the comparison between presentations or product perceived and expected. a study about the aspects of quality or quantity of products, including customer satisfaction is needed to evaluate the performance of a business. study of customer satisfaction is often done in survey method. survey can be done by post, telephone or personal interview such as directly reported satisfaction or direct measurement through questions. there is an agreement between rsgm dental laboratory with teknik kesehatan gigi (tkg) study program, whereas orthodontic work that available at the laboratory will be done by tkg students. making removable orthodontic appliances in rsgm dental laboratory is free of charge, nevertheless not all of the dental students willing to give the work to that laboratory. so, this study was conducted to determine whether dental students as customers were satisfied with the work of the dental laboratory of rsgm faculty of dental medicine universitas airlangga, particularly in orthodontics. it is hoped that this research can provide input to trigger corrective measures so that there will be more customers and students of tkg study program as technicians have enough practice before working in the community and dental student can have proper orthodontic appliances to treat orthodontic patients well. materials and method this research was conducted at orthodontic clinic of the faculty of dental medicine universitas airlangga, on july-august 2015. this study was analytic observational with cross sectional approach. samples were dental students of faculty of dental medicine universitas airlangga which made orthodontic appliances in the dental laboratory of rsgm faculty of dental medicine universitas airlangga. samples was taken by total sampling technique. the number of samples that meet the criteria of this research was 48 respondents. measurement of respondent’s satisfaction towards orthodontic appliances made in the dental laboratory was done by questionnaire survey. respondents were asked to fill out questionnaires after receiving removable orthodontic appliances, after insertion and activation of the appliances in patients. after receiving orthodontic appliances, respondents were asked to provide an assessment of satisfaction towards orthodontic appliances made in the dental laboratory, including satisfaction with the timeliness of completion of removable orthodontic appliances, satisfaction with the suitability of conformity orthodontic appliances received with the ordered, satisfaction with the service provided, satisfaction with the result of making clasp, bow and spring as well as satisfaction with the acrylic plate produced. after respondents perform the insertion of the appliances to the patient, they were also asked to assess satisfaction with the ease of appliance insertion and activation to the patient. this questionnaire answers used the guttman scale, where there were only two possible answers to each question, which were satisfied and not satisfied.5 data were analyzed descriptively and analytically by using spss 16. in descriptive, the results of this study will provide an overview of student satisfaction on the results of orthodontic appliances made in the dental laboratory of rsgm faculty of dental medicine, universitas airlangga. while the analytical analysis was used to examine the correlation between variables, including the relationship between student satisfaction on the results of making clasp, spring, and bows and student satisfaction for ease of insertion and activation, as well as the relationship between student satisfaction on the results of the making of acrylic plate with student satisfaction for ease of insertion and activation. chi-square and fischer correlation test were used in this study with significance p value ≤ 0.05. result of the 49 students who made orthodontic appliances in dental laboratory of rsgm faculty of dental medicine universitas airlangga, a student has not done the insertion and activation yet. so that the number of samples that are eligible in this study were 48 students. results of the analysis of questionnaire data from this study showed a picture of student satisfaction on the results of orthodontic appliances made in the dental laboratory of rsgm faculty of dental medicine universitas airlangga (table 1). from table 1 it can be seen that most respondents are satisfied with the results of orthodontic appliances made in dental laboratory of rsgm faculty of dental medicine universitas airlangga. after receiving orthodontic appliance from the dental laboratory, respondents felt the greatest satisfaction in terms of services provided (95.83%). in the second place was satisfaction towards result suitability with the request (72.92%). in terms of timeliness, almost half of the respondents (45.83%) still feel unsatisfied with the timeliness of orthodontic appliances completion. most respondents have been satisfied with the result of making clasp, spring and bow (72.92%) and the result of making the acrylic plate (77.08%). after insertion and activation, almost half of the respondents (45.83%) still dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 179179goenharto, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 177–182 table 2 shows the results of cross tabulation between satisfaction towards the result of making clasp, spring and bow and satisfaction towards the ease of insertion. the cross-tabulation of the results showed that the largest percentage of respondents who feel unsatisfied with the result of making clasp, bow and spring were also dissatisfied in terms of ease of insertion (76.92%). likewise, respondents who were satisfied with the result of making clasp, bow and spring were also satisfied towards the ease of insertion (65.71%). statistical analysis with fischer ‘exact test in table 2 was obtained p = 0.01 (p <0.05), which means there was a significant correlation between satisfaction towards the result of making clasp, bow and spring and the ease of insertion. the odds ratio for 6.389 showed that the respondents were satisfied with the result of making clasp, bow and spring had the possibility satisfied with the ease of insertion 6.389 times greater than the respondents who were not satisfied with the result of making clasp, bow and spring. table 3 shows the results of cross tabulation between satisfaction towards the result of making clasp, spring and bow and satisfaction towards the ease of activation. the cross-tabulation of the results indicates that most respondents who satisfied with the result of making clasp, bow and spring will also satisfied towards the ease of activation (94.29%). however, the results of the cross tabulation also indicates that there was a large majority of respondents who were not satisfied with the result of making clasp, bow and spring but were satisfied towards the ease of activation (76.92%). statistical analysis using chi square in table 3 was obtained p = 0.08 (p> 0.05), which means there was no significant correlation between the ease of activation to the quality of clasp, spring and bows were made. the odds ratio of 4.95 indicates that students who satisfied with the result of making clasp, bow and spring had the possibility satisfied with the ease of activation 4.95 times greater than students who were unsatisfied with the result of making clasp, bow and spring. table 4 shows the results of cross tabulation between satisfaction towards the results of the making of acrylic plate and satisfaction towards the ease of insertion. the cross-tabulation of the results showed that the largest percentage of respondents who feel dissatisfied with the result of making acrylic plate were also unsatisfied with table 1. satisfaction of students against the results of orthodontic appliances made in the dental laboratory of rsgm faculty of dental medicine universitas airlangga satisfaction assessment satisfied unsatisfied punctuality 26 (54.17%) 22 ( 45.83%) suitability with the order 38 (79.17%) 10 (20.83%) service provided 46 (95.83%) 2 (4.17%) quality results a. making the clasps, springs and bows 35 (72.92%) 13 (27.08%) b. making the acrylic plate 37 (77.08%) 11 (22.92%) ease of insertion 26 (54.17%) 22 (45.83%) ease of activation 43 (89.58%) 5 (10.42%) table 2. relationship of satisfaction towards the result of making clasp, spring and bow and satisfaction towards the ease of insertion ease of insertion satisfied unsatisfied result of making clasps, spring and bows satisfied 65.71% 34.29% unsatisfied 23.08% 76.92% table 3. relationship of satisfaction towards the result of making clasp, bow and spring and satisfaction towards the ease of activation ease of activation satisfied unsatisfied result of making clasps, spring and bows satisfied 94.29% 5.71% unsatisfied 76.92% 23.08% table 4. relationship of satisfaction towards the results of making the acrylic plate and satisfaction towards the ease of insertion ease of insertion satisfied unsatisfied result of making the acrylic plate satisfied 62.16% 37.84% unsatisfied 27.27% 72.73% feel unsatisfied with the ease of insertion, but the majority of students (89.58%) were satisfied with the ease of activation of the appliances. table 5. relationship of satisfaction towards the results of making the acrylic plate and satisfaction towards the ease of activation ease of activation satisfied unsatisfied result of making clasp, spring and bow satisfied 91.89% 8.11% unsatisfied 81.82% 18.18% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 180 goenharto, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 177–182 the ease of insertion (72.73%). likewise, respondents who were satisfied with the results of acrylic plate were also satisfied with the ease of insertion (62.16%). statistical analysis with fischer’s exact test in table 4 was obtained p = 0.045 (p <0.05), which means there was a significant correlation between satisfaction towards the result of making the acrylic plate and satisfaction towards the ease of insertion. the odds ratio for 4.381 showed that the respondents were satisfied with the results of making the acrylic plate has the possibility satisfied with the ease of insertion 4.381 times greater than the respondents who were unsatisfied towards the results of making the acrylic plate. table 5 shows the results of cross tabulation between satisfaction towards the results of making the acrylic plate and satisfaction towards the ease of activation. the crosstabulation of the results indicates that most respondents who satisfied with result of making the acrylic plate will also satisfied towards the ease of activation (91.89%). however, the cross-tabulation of the results showed that there was a majority of respondents who were not satisfied with results of making the acrylic plate but was satisfied towards the ease of activation (81.82%). statistical analysis using chi square in table 5 obtained p = 0.337 (p>0.05), which means there was no significant relationship between satisfaction towards result of making the acrylic plate and satisfaction towards the ease of activation. the odds ratio for 2.519 showed that the respondents were satisfied with results of making the acrylic plate had the possibility satisfied towards the ease of activation 2.519 times greater than the respondents who were not satisfied with results of making the acrylic plate. discussion during research, the number of dental students who work in the orthodontic clinic approximately 120 people. students who participated in this study only 49 students (total sampling) although only 48 students who met the criteria as samples. this is because students are not required to make orthodontic appliances at dental laboratory of rsgm faculty of dental medicine universitas airlangga. they can give the work to another dental laboratory outside faculty of dental medicine universitas airlangga. from these results it can be seen that the satisfaction of students to timeliness of work were 54.17% satisfied and 45.83% dissatisfied. although there was a higher percentage of satisfied, but not much different from dissatisfied. timeliness of work is one of the important factors customers want. in practice, a high quality of service does not guarantee high customer satisfaction or positive behavior intention. customers may be satisfied with the results of the work but be dissatisfied because it was not completed on time. likewise, advanced/modern equipment owned is interesting or in accordance with customer expectations, but may not be able to compensate for the dissatisfaction due to long waiting time.6,7 the appliance that is not completed on time will have a negative impact, such as schedule changing for both operators and patients. it will make some difficulties because everybody has already had something else to do. students generally also work in other clinics, and the patients as pupils also have test schedules in the school. so, a skilled laboratory technician is needed, not only doing well, but also can meet the given deadline. maybe this timeliness factor also make many students chose another dental laboratory outside faculty of dental medicine universitas airlangga. towards the question about result suitability with the request, 79.17% of respondents were satisfied and only 20.83% were unsatisfied. at the time of giving the job, respondents must fill out the forms of orders and describe the appliance design and write the information very clearly. the design was also drawn to the working model as well, in order to clarify what is required and minimize mismatch. good communication, reading accuracy and the ability to interpret the contents of the work order were important to produce proper orthodontic appliance. dissatisfaction can occur due to false appliance’s components that are not in accordance with the order, or mistakes in interpreting the content of the work order. this will lead to the need for revision or even making a new appliance that would require additional time. sometimes operators must make a new impression to get a new working model. a quality control is needed prior delivering the work to the customer. pasuraman8 revealed 10 dimensions of service quality, ie: tangibles, reliability, responsiveness, competence, courtesy, credibility, security, access, communication, and the ability to understand customer needs. many studies showed that the quality of service and satisfaction are different things. quality of service is represented by cognitive judgment, while satisfaction is an affect-laden evaluation.9 quality of service is not only based on technical matters, but also the complex relationship between all personnel involved. job description and responsibilities sharing should be given clearly.10 results showed that 95.83% of respondents were satisfied with the services provided. this showed that the personnel in charge is quite capable of implementing some of the 10 dimensions of service quality, such as: responsiveness (willingness to help customers and deliver services rapidly), courtesy (politeness, respect, attention and friendliness of the front line staff), credibility (nature honest and trustworthy), access (ease to be contacted and met), communication (giving information to customers in a language that can be understood and always listen to their complaints), and the ability to understand customer needs. other dimensions are: reliability (ability to provide the promised services accurately and reliably), competence (mastery of skills and knowledge required in order to provide services required), safety (assurance), and physical evidence (reality/tangibles) that highly correlated with the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 181181goenharto, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 177–182 quality of the work produced. good process is needed to achieve good quality. quality assessment is a dynamically process.11 quality of orthodontic appliance can be seen from result of making clasp, spring and bow and acrylic plate. most respondents (72.92%) satisfied with the result of making clasp, spring and bows, as well as to the resulting acrylic plate (77.08%). towards the question about the ease of insertion, only 54.17% respondents were satisfied, while the remaining 45.83% were not satisfied. nonetheless, the majority (89.58%) were satisfied with the ease of activation. statistical analysis showed that there was a significant correlation between the ease of insertion with the quality of clasp, spring and bow (p=0.01) as well as acrylic plate made (p=0.045<0.05). when active and passive components have been made properly, it is expected both insertion and activation can be done easily. however, even though the orthodontic appliance has been well made, it is still required operator’s skills to do the adjustment, so that the appliance can be inserted properly in the patient. these skills are needed especially for the mandibular appliances, because patients are generally children during mixed dentition. the first permanent molars are often not fully eruption, so it is not easy to make clasp on these teeth. this factor may contribute to the decline in the percentage of respondents’ satisfaction towards making clasp, spring and bow in the amount of 72.92% to only 54.17% were satisfied with the ease of insertion. statistical analysis using chi square showed there was no significant correlation between the ease of activation to the quality of clasp, spring and bow (p=0.08) as well as acrylic plate made (p 0.337 (p>0.05). activation is strongly influenced by the skills of the operator. skilled operators who are capable with wire bending techniques will be easy to do the adjustment and activation. although the appliances have been well made, unskilled operators will also have difficulty in doing the activation. excessive activation can make the appliances become unstable. it seems that most of the respondents already had enough skill in doing so, despite only 77.08% respondents were satisfied with the acrylic plate produced, 89.58% are satisfied with the ease of activation customer satisfaction leads to customer loyalty.12 a satisfied customer will come back with more jobs and even can interested to other products.13 the fact that 39 of 48 (81.25%) of respondents made orthodontic appliances more than 1x, indicates that most respondents are satisfied and want to make another orthodontic appliance in the dental laboratory of rsgm faculty of dental medicine universitas airlangga. dissatisfaction occurs because the performance of the product is lower than the expectations of customers. dissatisfaction can lead customers to switch services to another dental laboratory. students can switch to other dental laboratories that generally provide services without additional delivery charge. dissatisfied customers usually become spreaders of the bad image that will be distributed by mouth, and give negative impacts on previous laboratory. satisfied customer will tell 4-5 another customers. dissatisfied customers will tell 9-10 people and 13% of unsatisfied customers will tell to more than 20 people.14 strategy to increase service laboratory work can be done by monitoring and measuring customer satisfaction with administration of the questionnaire such as in this study. dissatisfaction should be overcome with an efficient strategy for handling complaints. if the complaint is handled properly, unsatisfied customers can be changed to satisfied customers. problems should be identified quickly and precisely to demonstrate concern towards unsatisfied customers. other factor such as after sales service is important to provide. if there is any complaint about the orthodontic appliance, it should be well overcome by repairing or even making a new one without any charge. it is concluded that the majority of respondents were satisfied with the results of removable orthodontic appliances produced, but dental laboratory of rsgm faculty of dental medicine universitas airlangga still need to improve the timeliness, so there will be more dental students become customers and they will be facilitated enough in treating patients. acknowledgement thanks for the supporting funds provided by rkat faculty of vocation universitas airlangga 2015 no. 001/ un3.1.15.lt/2015 references 1. athanassopoulos a. behavioral responses to customer satisfaction: an empirical study. european journal of marketing 2001; 35(5/6): 687-707. 2. alam mk. a to z orthodontics. volume 10: removable appliance. kota bharu: ppsp publication; 2012. p. 6-7. 3. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publishers (p) ltd; 2007. p. 387-409. 4. kotler p, armstrong g. principles of marketing. 14th ed. boston: pearson prentice hall; 2012. p. 6-7. 5. martono m. metode penelitian sosial: konsep-konsep kunci. depok: pt rajagrafindo persada; 2015. p. 280-1. 6. li sj, huang yy, yang mm. how satisfaction modifies the strength of the influence of perceived service quality on behavioral intentions. leadership in health services 2011; 24(2): 91-105. 7. iliyasu z, abubakar is, abubakar s, lawan um, gadija au. patients’ satisfaction with services obtained from aminu kano teaching hospital, kano, northern nigeria. niger j clin pract 2010; 13(4): 371-8. 8. parasuraman a, zeithaml v, berry l. a conceptual model of service quality and its implications for future research. journal of marketing 1985; 49(4): 41-50. 9. wu hl, liu ch, hsu wh. an integrative model of customers’ perceptions of health care services in taiwan. the service industries journal 2008; 28(9): 1307-19. 10. vakani f, fatmi z, naqvi k. three-level quality assessment of a dental hospital usingj efqm. int j health care qual assur 2011; 24(8): 582-91. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 182 goenharto, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 177–182 11. jones ml, hobson rs, plasschaert ajm, gundersen s, dummer p, roger-leroi v, sidlauskas a, hamlin j. quality assurance and benchmarking: an approach for european dental schools. eur j dent educ 2007; 11(3): 137-43. 12. kotler p, keller k. marketing management. 14th ed. new jersey: prentice hall; 2012. p. 27. 13. depaire b, vanhoof k, wets g. a decision support tool for evaluating customer intentions. expert syst appl 2012; 39(8): 6903-10. 14. pakdil f, harwood tn. patient satisfaction in a preoperative assessment clinic: an analysis using servqual dimensions. total quality management 2005; 16(1): 15-30. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p177-182 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p177-182 10 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 10–15 research report the effect of propolis extract and bovine bone graft combination on the number of osteoclast and osteoblast as an effort to preserve post-extraction socket (on cavia cobaya) much nizar, utari kresnoadi and soekobagiono department prosthodontics faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: post-extraction alveolar bone height and width never reach the same dimensions as before extraction. a combination of propolis extracts and bovine bone graft (bbg) that are anti-inflammatory, antioxidant, osteoinductive and osteconductive is expected to improve bone regeneration. purpose: knowing the effect of the combination of propolis extracts and bbg on the number of osteoclast and osteoblast as an effort to preserve the socket after extraction of cavia cobaya teeth. methods: 56 cavia cobaya was divided into eight groups: the lower left incisor was extracted, and the socket was filled with 2% poly ethylene glycol (peg), propolis extract+peg, bbg+peg, and a combination of propolis extract+bbg+peg. the incisors socket of animals models were executed on the 14th and 30th days. using he for histopathological examination, the number of osteoclasts and osteoblasts were counted with a 400x magnification light microscope with nine visual fields. the data were analysed via one-way anova and tukey hsd tests. results: the highest mean number of osteoclasts occurred in the bbg+peg 14th day group and the lowest occurred in the propolis extract+bbg+peg 14th day group. the highest mean number of osteoblasts occurred in the propolis + bbg + peg combination 30th day group, the lowest occurred in the control group (peg) on the 14th day. conclusion: the 2% combination of propolis extracts and bbg effectively reduces the osteoclast number and increases the osteoblast number in preserving the socket after extracting cavia cobaya teeth. keywords: bovine bone graft; osteoblast; osteoclast; propolis extract; socket preservation correspondence: utari kresnoadi, department of prosthodontics, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: utari-k@fkg.unair.ac.id introduction tooth extraction is a common procedure in the field of dentistry. alveolar ridge bone resorption is a condition that always accompanies tooth extraction. after healing, the height and width dimensions of the alveolar bone never reach their original dimensions. the process of alveolar bone resorption begins with the bond between the receptor activator nuclear kappa-b ligand (rankl) produced by the osteoblast and the receptor activator nuclear kappa-b (rank) presented by pre-osteoclasts. the binding between rankl and rank will activate osteoclasts. when osteoclast growth increases, alveolar bone resorption will occur. without socket preservation after extraction, there will be a 50% reduction in alveolar ridge volume within 12 months. therefore, socket preservation is needed to maintain the alveolar bone dimensions.1–3 autogenous bone graft is the best bone-grafting material/gold standard compared to allograft, xenograft and other synthetic materials. however, its application has some disadvantages because it requires a surgical procedure that presents difficulties regarding level of action, risk of infectious complications, pain, morbidity in donor area, hematoma, limited donor availability, the need for general anaesthesia, longer operative time, bleeding and the need for further surgery.4 over the past few decades, researchers have focused on the field of bone regenerative materials to improve such characteristics as mechanical strength, molecular composition, biocompatibility and degradation capacity to determine bone’s natural features. xenograft, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg 11nizar, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1):10–15 using material originating from different species donors, is an alternative choice, as it offers osteoconductive features with limited resorptive potential and can be combined with growth factors or other sources of bone graft material; moreover, it can be mass produced at a relatively affordable cost.5 however, in the application of xenograft, postsurgery inflammation on a live host is inevitable. surgical injuries are followed by inflammation, blood-biomaterial interactions and tissue fibrosis. because xenograft is only osteoconductive and has no anti-inflammatory properties, inflammation can be prolonged and become chronic inflammation. bone substitution by bone graft material can significantly increase the inflammatory response.6 some researchers say that propolis extract has antiinflammatory, antioxidant, anti-bacterial, anti-fungal, anti-viral and anti-cancer properties, as well as the ability to accelerate wound healing.7 the content of caffeic acid phenetyl ester (cape) in propolis, as an anti-inflammatory and antioxidant, can increase growth factors, increase extra cellular matrix (ecm) remodelling and increase re-epithelialisation so that it can improve socket healing after tooth extraction.8 bovine bone graft (bbg) and propolis extract is a combination of materials that are osteoconductive and osteoinductive and have antiinflammatory activity which is expected to be an alternative bone-grafting material. this research focused on cavia cobaya experimental animals which were treated by inserting a gel containing peg, propolis extract, bbg, and a combination of propolis extract with bbg into the post-tooth extraction socket of cavia cobaya with 2% active substance concentration for 14 days and 30 days, intended to determine the effectiveness of the administration of a combination of propolis and bbg extracts to decrease the number of osteoclasts and increase osteoblasts in the post-extraction tooth socket of cavia cobaya. materials and methods this study was approved by the ethics commission of the faculty of dental medicine of universitas airlangga, as stated in the ethical clearance certificate number: 587/ hre.ccfodm/ix/2019. the experimental animal used was a healthy and active male cavia cobaya, weighing around 300-350 gr, aged 3-3.5 months. propolis extract was obtained from bees (apis mellifera) from beekeeping in lawang, malang, east java, which was carried out at the surabaya industrial research and consultation center. bbg comes from good bovine bones, has passed screening and is free of infectious diseases. at the tissue bank of dr. soetomo general hospital-surabaya, bbg from good bovine processed through a dissection process to remove soft tissue, followed by cutting, washing, drying, deepfreezing, freeze drying, packing, sterilisation with x-ray gamma radiation to remove immunogenic properties and packaging in 150-355 µm powder. poly ethylene glycol (peg) obtained from mixing peg 400 and peg 4000 in a ratio of 1: 1 is used as a carrier so that the mixture becomes a gel to facilitate its application into the socket.9 a 2% concentration of active substance is derived from 0.5 gr propolis and 0.5 gr bbg mixed with 24 gr peg, so we get 25 gr propolis + bbg + peg gel. the gel was filled as much as 0.1 cc according to the tooth extraction socket volume, then sewn with a sterile polyamide monofilament yarn (braun aesculap, ds 12 3 / 8c, 12 mm, 6/10 metric, 0.7). a total of 56 cavia cobaya were divided into eight groups, seven in each. they were then separated into groups for 14 days (i, iii, v, vii) and groups for 30 days (ii, iv, vi, viii). each group received four treatments. the left lower incisor was extracted and the socket was given peg (control), propolis extract + peg, bbg + peg, and a combination of propolis extract + bbg + peg. groups i and ii, after extraction, had the socket filled with peg only. for groups iii and iv, after extraction, the socket was filled with propolis extract + peg. for groups v and vi, after extraction, the socket was filled with bbg + peg. for groups vii and viii, after extraction, the socket was filled with a combination of propolis extract + bbg + peg. the lower jaw in the tooth extraction area was removed, softened with edta for ± 3 months, and then a paraffin block preparation was made. the paraffin block was then cut using a rotary microtome with a thickness of ± 4µ, after which it was placed on a glass object wrapped in polylycin. deparafinisation was achieved by dissolving the specimen in xylol for 2×x3 minutes. the remainder of xylol was washed with absolute alcohol, 99%, 95%, 90%, 80%, 70%, for each of them for 2×x1 min, followed by staining he.9 then, a histopathological examination (hpa) was performed to count the number of osteoclasts and osteoblasts with a 400x magnification light microscope with nine visual fields. the data tabulation was statistically analysed via one-way anova and tukey hsd tests. results the highest mean number of osteoclasts occurred in group v, with bbg + peg 14th day treatment at 13.71 ± sd 3.20, and the lowest occurred in group vii with propolis + bbg + peg 14th day treatment at 6.29 ± sd 1.38. meanwhile, the highest mean number of osteoblasts occurred in group viii with a combination treatment of propolis extract + bbg + peg 30th day at 25.86 ± sd 3.18 and the lowest occurred in the i/control group (peg) 14th day at 5.14 ± sd 0.9. a block diagram of the average number of osteoclasts and osteoblasts can be seen in figures 1 and 2. histological features can be seen in figures 3 and 4. the results of one-way anova analyses in groups of 14 days and 30 days show a significant difference in the number of osteoclasts and the number of osteoblasts between the control group, propolis extract + peg, bbg + peg, and propolis extract + bbg + peg, with a value dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg 12 nizar, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1):10–15 table 1. tukey hsd test results on osteoclasts on the 14th and 30th days group i ii iii iv v vi vii viii i 0.996 0.756 0.049* 0.023* 1.000 0.004* 0.049* ii 0.311 0.007* 0.136 1.000 0.000* 0.007* iii 0.756 0.000* 0.480 0.241 0.756 iv 0.000* 0.016* 0.988 1.000 v 0.070 0.000* 1.000 vi 0.001* 0.016* vii 0.988 viii * = significant (p< 0.05) table 2. tukey hsd test results on osteoblasts on the 14th and 30th days group i ii iii iv v vi vii viii i 0.000* 0.000* 0.000* 0.032* 0.000* 0.000* 0.000* ii 0.968 0.000* 0.307 0.781 0.539 0.000* iii 0.000* 0.032* 1.000 0.985 0.000* iv 0.000* 0.001* 0.004* 0.000* v 0.008* 0.002* 0.000* vi 1.000 0.000* vii 0.000* viii * = significant (p< 0.05) 10.29 ±2.21 8.71 13.71 6.29 11.00 7.14 10.71 7.14 0 2 4 6 8 10 12 14 16 18 control propolis bone graft propolis+bone graft osteoclast day14 osteoclast day30 ±1.63 ±1.70 ±1.11 ±1.67 ±3.20 ±1.38 ±0.90 figure 1. the mean and standard deviation of osteoclast number on the 14th and 30th days. 5.14 12.57 8.86 13.5711.43 18.14 13.14 25.86 0 5 10 15 20 25 30 35 control propolis bone graft propolis+bone graft osteoblast day14 osteoblast day30 ±0.90 ±1.27 ±0.78 ±2.85 ±1.34 ±2.26 ±2.50 ±3.18 figure 2. the mean and standard deviation of osteoblast number on the 14th and 30th days. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg 13nizar, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1):10–15 a b c d figure 3. osteoclasts (black arrows) and osteoblasts (red arrows) on the 14th day; new bone tissue density has not yet been seen. a. group i (control/peg), with the lowest number of osteoblast (5.14 ± sd 0.9); b. group iii (propolis extract + peg); c. group v (bbg + peg), with the highest number of osteoclasts (13.71 ± sd 3.2); d. group vii (propolis + bbg + peg extract), with the lowest number of osteoclasts (6.29 ± sd 1.38). a b c d figure 4. osteoclasts (black arrows) and osteoblasts (red arrows) on day 30th, beginning to show the density of new bone tissue that has formed. a. group ii (control / peg); b. group iv (propolis extract + peg); c. group vi (bbg + peg); d. group viii (propolis + bbg + peg extract), with the highest number of osteoblast (25.86 ± sd 3.18). of p = 0.000 (p<0.05). tukey hsd test results can be seen in tables 1 and 2. the microscopic images of osteoclasts and osteoblasts can be seen in figure 3 (day 14) and figure 4 (day 30). discussion the height and width of the alveolar ridge bone naturally heals after tooth extraction without socket preservation, albeit never reaching the same height and width as before extraction. therefore, it is important to develop socketpreservation procedures to maintain the height and width of the alveolar bone. the adequate height and width of the alveolar bone are very important in supporting the successful application of implants and conventional prostheses.1,2 based on research data, significant differences exist between treatment groups. the highest mean of osteoclasts number occurred in group v (bbg + peg on day 14) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg 14 nizar, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1):10–15 and the lowest occurred in group vii (propolis extract + peg on day 14). while the highest mean of osteoblasts number occurred in group viii (propolis extract + bbg + peg on day 30), and the lowest mean occurred in group i (control on day 14). this was reinforced by the results of the one-way anova test that showed a significant difference in the number of osteoclasts and osteoblasts between each treatment group. the highest number of osteoclasts occurred on day 14 (group v), indicating that osteoclastogenesis activity was still ongoing and thus suggesting a possibility that bone-grafting material can increase inflammatory response, thereby triggering an increased number of osteoclasts. markel et al. (2012),6 revealed that bone-graft material significantly triggers an increase in the inflammatory response. inflammation can increase the number of osteoclasts, pro-inflammatory cytokines (tumor necrosis factor-α/tnf-α and interleukin 1/il-1), receptor activator nuclear factor kappa-β ligand (rankl) and receptor activator nuclear factor kappa-β (rank).10 these results are in line with vieira’s study which states that, on days 10 to 14, osteoclasts begin to absorb cortical margins and smooth sharp bones.11 this did not occur in group viii on day 30, other than a decrease in inflammation, which is also likely due to the role of propolis content that is anti-inflammatory, depressing osteoclastogenesis and decreasing the number of osteoclasts; on the other hand, osteoblastogenesis activity is increasing. the number of osteoclasts in the group not treated with propolis extract (i, ii, v, and vi) is higher than the group treated with propolis extract (iii, iv, vii, and viii). the decrease in the number of osteoclasts in this group is probably caused by the content of propolis extract, which is able to reduce the inflammatory response. the number of osteoblasts in the group treated with propolis extract (iii, iv, vii, and viii) is higher than the group not treated with propolis extract (i, ii, v, and vi). the increased number of osteoblasts in this group is likely due to the propolis extract’s ability to induce mscs to differentiate into osteoblasts. this is in line with research by darmadi and mustamsir (2016)12 and by altan et al. (2013),13 which focused on the application of propolis extract on femoral fractured bones of wistar rats. in these studies, propolis extract appeared to inhibit osteoclastic activity and stimulate osteoblastic activity on bone metabolism, thus decreasing the number of osteoclasts and increasing the number of osteoblasts and chondrocytes. the examination of the surabaya industrial research and consultation institute showed that the ethanol extract of propolis from lawang-malang contained 2.5% caffeic acid (cape), 1.05% apigenin, 1.28% flavonoids, 0.82% saponin, 1.03% qiersetin and 1.15% terpenoid. propolis is a natural ingredient from bee products that has antiinflammatory, anti-oxidant, anti-microbial, anti-cancer and anti-fungal properties, and is able to accelerate wound healing. these properties are closely related to the inherent flavonoid, phenolic acids, terpenoid and aromatic acid compounds. flavonoids and hydroxycinnamic acid, as the main components, are bioactive substances that act as antioxidants, preventing the negative effects of free radicals by binding to anion peroxide and hydroxide radicals, thereby reducing oxidative pressure. the decrease of oxidative pressure level suppresses the activation of nuclear factor kappa beta (nf-κb) that acts as a transcription factor for the coding of pro-inflammatory cytokine genes, including tnf-α and ifn-γ so as to reduce inflammation.7,14 caffeic acid phenetyl ester (cape) is an antioxidant that can inhibit excessive oxidative reactions caused by inflammatory reactions and metabolic processes of cell injury. as an anti-inflammatory, cape acts to inhibit phospholipase in the arachidonic acid cascade, so that it does not release prostaglandins and leukotrin and inhibits the process of lipoxygenase (lox) and cycloxygenase (cox), which play a role in inflammatory metabolic pathways. cape is lipophilic and facilitates cell infiltration, releases anti-inflammatory cytokines (tgf-β, il-10, il-4), is a specific inhibitor of nf-κb transcription, inhibits the release of pro-inflammatory cytokines (tnf-α, il-1, il-8, and il-6) and increases the proliferation of fibroblasts, so as to accelerate the healing of the socket after tooth extraction.15,16 the combination of propolis extracts and bbg, in addition to being anti-inflammatory and anti-oxidant, also has osteoconduction and osteoinduction characteristics in the bone-regeneration process. bbg acts as a scaffold and as a medium for stem cells and osteoblasts to attach, live and develop properly in bone defects. new blood vessels can attach to the scaffold and stimulate the apposition of new bone cells. inorganic material from bbg is able to support the attachment and proliferation of osteoblasts, which is the first step in the process of new bone formation. the material can support the bone matrix for regulation through three mechanisms: (a) spacing out strong fillers; (b) providing osteoblast attachment and proliferation media; and (c) as a means for stimulating bone formation. osteoblasts play important role in the process of bone remodelling. bone graft material in support of new bone formation occurs through several biological processes: osteoinduction, osteoconduction, osteopromosi and osteogenesis. osteoinduction occurs when osteoprogenitor cells are stimulated to differentiate into osteoblast cells and begin new bone formation. osteoconduction occurs when the material acts as a scaffold or framework for osteoblast cells in expanding the framework in new bone growth. osteopromosi occurs when bone graft material is able to strengthen the osteoinduction process, whereas osteogenesis occurs when there are osteoblast cells derived from bone graft material that play a role in the growth of new bone during bone formation.17 in conclusion, the combination of propolis extracts and bbg effectively reduced the number of osteoclasts and increased the number of osteoblasts in the socket preservation of a post-extraction tooth with 2% active substance concentration. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg 15nizar, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1):10–15 references 1. pagni g, pellegrini g, giannobile w v., rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 1–13. 2. kresnoadi u, hadisoesanto y, prabowo h. effect of mangosteen peel extract combined with demineralized freezed-dried bovine bone xenograft on osteoblast and osteoclast formation in post tooth extraction socket. dent j (majalah kedokt gigi). 2016; 49(1): 43–8. 3. soekobagiono s, alfiandy a, dahlan a. rankl expressions in preservation of surgical tooh extraction treated with moringa (moringa oleifera) leaf extract and demineralized freeze-dried bovine bone xenograft. dent j (majalah kedokt gigi). 2018; 50(3): 149–53. 4. periya sn, hammad hgh. bone grafting in dentistry: biomaterial degradation and tissue reaction: a review. ec dent sci. 2017; 9(6): 239–44. 5. titsinides s, agrogiannis g, karatzas t. bone grafting materials in dentoalveolar reconstruction: a comprehensive review. jpn dent sci rev. 2019; 55(1): 26–32. 6. markel dc, guthrie st, wu b, song z, wooley ph. characterization of the inf lammatory response to four commercial bone graft substitutes using a murine biocompatibility model. j inflamm res. 2012; 5(1): 13–8. 7. khurshid z, naseem m, zafar ms, najeeb s, zohaib s. propolis: a natural biomaterial for dental and oral healthcare. j dent res dent clin dent prospects. 2017; 11(4): 265–74. 8. ernawati ds, puspa a. expression of vascular endothelial growth factor and matrix metalloproteinase-9 in apis mellifera lawang propolis extract gel-treated traumatic ulcers in diabetic rats. vet world. 2018; 11(3): 304–9. 9. kresnoadi u, rahayu rp, rubianto m, sudarmo sm, budi hs. tlr2 signaling pathway in alveolar bone osteogenesis induced by aloe vera and xenograft (xcb). braz dent j. 2017; 28(3): 281–6. 10. kresnoadi u, ariani md, djulaeha e, hendrijantini n. the potential of mangosteen (garcinia mangostana) peel extract, combined with demineralized freeze-dried bovine bone xenograft, to reduce ridge resorption and alveolar bone regeneration in preserving the tooth extraction socket. j indian prosthodont soc. 2017; 17(3): 282–8. 11. vieira ae, repeke ce, de barros ferreira s, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. 12. darmadi d, mustamsir e. the effect of propolis on increasing the number of osteoblasts and chondrocytes , and decreasing the number of osteoclasts in wistar rats (rattusnovergicus) with femoral bone fracture. j dent med sci. 2016; 15(12): 90–5. 13. altan ba, kara im, nalcaci r, ozan f, erdogan sm, ozkut mm, inang s. systemic propolis stimulates new bone formation at the expanded suture a histomorphometric study. angle orthod. 2013; 83(2): 286–91. 14. ningsih fn, rifa’i m. propolis action in controlling activated t cell producing tnf-α and ifn-γ in diabetic mice. turkish j immunol. 2017; 5(2): 36–44. 15. puspasari a, harijanti k, soebadi b, hendarti ht, radithia d, ernawati ds. effects of topical application of propolis extract on fibroblast growth factor-2 and fibroblast expression in the traumatic ulcers of diabetic rattus norvegicus. j oral maxillofac pathol. 2018; 22(1): 54–8. 16. günay a, arpağ of, atilgan s, yaman f, atalay y, acikan i̇z. effects of caffeic acid phenethyl ester on palatal mucosal defects and tooth extraction sockets. drug des devel ther. 2014; 8: 2069–74. 17. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p10–15 http://dx.doi.org/10.20473/j.djmkg.v53.i1.p10-15 http://e-journal.unair.ac.id/index.php/mkg mkgs vol 44 no 1 jan-mar 2011.indd 39 vol. 44. no. 1 march 2011 research report plaque index between blind and deaf children after dental health education cynthia carissa, jakobus runkat, and yetty herdiyati department of pediatric dentistry faculty of dentistry, padjadjaran university bandung indonesia abstract background: difficulty in mobility and motor coordination could affect the health at teeth and mouth. dental health education of the blind and deaf children differs according their limitation. blind and deaf children need a particular guidance in dental health education to promote oral hygiene as normal children do. purpose: the objective of this study was to observe the difference of plaque index between blind and deaf children before and after dental health education. methods: this research used purposive sampling technique. twenty-three blind children were taken as samples from slb-a negeri bandung and 31 deaf children from slb-b cicendo bandung. the data were then collected through plaque index examination using modified patient hygiene performance (php) test. results: the result descriptively showed that plaque index average value of 23 blind children before dental health education was 3.0725 and after, was 1.7970. on the other hand, the plaque index average of deaf children before dental health education was 2.7474 and after was 1.5. conclusion: it is concluded that plaque index of deaf children is better than blind children before and after dental health education. key words: plaque index, blind children, deaf children, dental health education abstrak latar belakang: kesulitan dalam pergerakan dan koordinasi motorik akan memengaruhi kesehatan gigi dan mulut. pendidikan kesehatan gigi dan mulut anak buta dan tuli akan berbeda tergantung tingkat kekurangan mereka. anak tunanetra dan anak tunarungu membutuhkan pendidikan khusus berupa pendidikan kesehatan gigi untuk meningkatkan kebersihan gigi dan mulut serupa dengan anak normal. tujuan: untuk mengetahui perbedaan indeks plak antara anak-anak buta dan tuli sebelum dan sesudah pendidikan kesehatan gigi. metode: penelitian ini menggunakan teknik purposive sampling. dua puluh tiga anak tunanetra diambil sebagai sampel dari slb-a negeri bandung dan 31 anak tunarungu dari slb-b cicendo bandung. data tersebut kemudian dikumpulkan melalui pemeriksaan indeks plak menggunakan indeks patient hygiene performance (php) modifikasi. hasil: hasil penelitian secara deskriptif menunjukkan bahwa nilai indeks plak rata-rata 23 anak tunanetra sebelum pendidikan kesehatan gigi adalah 3,0725 dan sesudah pendidikan kesehatan gigi adalah 1,7970. sedangkan, indeks plak rata-rata anak tunarungu sebelum pendidikan kesehatan gigi adalah 2,7474 dan sesudah pendidikan kesehatan gigi adalah 1,5. kesimpulan: indeks plak anak tunarungu lebih baik dibandingkan dengan anak tunanetra sebelum dan sesudah pendidikan kesehatan gigi. kata kunci: indeks plak, anak tunanetra, anak tunarungu, pendidikan kesehatan gigi correspondence: cynthia carissa, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: cynthia.carissa88@yahoo.com 40 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 39–42 introduction the disabled form a substantial section of the community. it is estimated that there are about 500 million people with disabilities worldwide. prevalence of disabled people vary from country to country, the variance in prevalence may be attributed to ascertainment basis, the standardization methods employed from study to study.1 number of persons with disabilities in indonesia in 2005 reached 6.7 million people (3.11% of the total population). blind population numbered was 309.146 (4.6%), deaf population was 192.207 (2.8%), mental retarded was 178.870 (2.6%), and 94.423 persons with another disability. indonesia is the second country in the world that has the greatest amount of visual impairment. this amount could increase because of earthquakes, disasters, accidents, and so forth. knowledge is the main factor in shaping a person’s behavior and occurs after a person does a sense of particular object. sensing occurs through human senses i.e. seeing, hearing, smelling, tasting, and touching. however, most of the knowledge is acquired through our eyes and ears.2 blind and deaf children are considered as people with specific needs due to physical and mental impairment that limit their daily activities such as walking, seeing, listening, speaking, working, and studying.3 difficulty in mobility and motor coordination could affect the health of teeth and mouth.4 therefore, the blind and deaf children need special dental health education and guidance to improve their ability as normal children do. dental health education of the blind and deaf children differs according to their limitations. dental health education of blind children can be done by recording oral hygiene instructions using tape recordings, performing tooth brushing demonstration with big tooth model. dental health education of deaf children was done by pantomime and demonstration. although individuals who are disabled are entitled to the same standards of health and care as the general population, there is evidence that they experience poorer general and oral health. the oral health of the disabled may be neglected because of the disability condition, a demanding disease or limited access to oral health care. moreover, because of their level of function and their limited ability to undergo an oral examination, the disabled persons present specific challenges when their oral health is assessed. however, with appropriate planning, clear communication and careful limitations to the service provided, the dramatic dental neglect experienced by the majority of these individuals can be successfully alleviated.2 their oral health care needs are compounded by their disorders, medications and lack of oral hygiene at home. gingival hyperplasia, periodontal disease, and caries are prevalent. eighty percent of the children are residential and rely on dorm personnel, teachers and must be helped for oral hygiene care.5 plaque control is important in oral health program because dental plaque is the main cause of dental caries and periodontal disease.6 maintaining healthy teeth and mouth should be done since the age of primary school because it was the right time to train a child’s motor skills including brushing teeth.7 in this study, the plaque index between deaf and blind children were compared before and after dental health education. the one who can receive more information should be able to repeat what has been taught in dental health education even with their limited capabilities. materials and methods the research was a descriptive study and using purposive sampling method. samples were taken from 23 blind children in slb-a negeri bandung and 31 deaf children in slb-b cicendo bandung with the criteria as follows: ages from 6–12 years old, with total sensory impairment, and still active attending their school activities. the procedure was begun by filling the questionnaire about dental hygiene habits accompanied by each guardian and examiner. the plaque index was examined by putting disclosing solution at the tip of the tongue then spreading out to the entire surface of the teeth. the result was written on the examination sheet. then, the children were taught about dental health education based on their capabilities followed by conducting a joint tooth brushing. dental health education for children with visual impairment can be done by recording the oral hygiene instruction using a cassette tape, do show the correct way to brush teeth by using big teeth model. deaf children given dental health education by using a special method such as pantomime and demonstration performing.6 plaque index examination was done after dental health education and the result was written once again on the examination sheet. modified php test was used to examine plaque index on six surfaces of teeth (facial surface 11, 31, 16, and 26 and lingual surface 36 and 46) with criteria, if there was no plaque on tooth surface, the value was 0, but if there is a plaque on tooth surface, the value was 1. plaque index of each person was calculated by total value of all the subdivisions of dental plaque divided number of teeth examined.8 independent t test was used in this study. /b b t s n = b = average of plaque index sb = standard deviation √n = number of children 41carissa, et al.: plaque index between blind and deaf children results data was obtained from 23 blind chidren from slb-a negeri bandung and 31 deaf children from slb-b cicendo bandung. plaque index average values between blind and deaf children can be seen at table 1. modified php index was used to measure the plaque index. plaque index of the blind children from slb-a negeri bandung before dental health education was 3.07 and after was 1.79, both were included in average category. plaque index of the deaf children from slb-b cicendo bandung before dental health education was 2.75 (average) and after, was 1.5 (good). that plaque index of deaf children was lower than blind children, it could be said that deaf children's oral hygiene was better than blind children which was caused by various factors (figure 2). 3,0725 1,797 2,75 1,5 0 0,5 1 1,5 2 2,5 3 3,5 before after blind children deaf children before after 3,5 3 2,5 2 1,5 1 0,5 0 figure 2. plaque index of blind children and deaf children. discussion the result showed that there were differences of plaque index between blind children and deaf children before and after the dental health education. plaque index of blind children before dental health education was 3.07 and after, was 1.79. on the other hand, the plaque index of the deaf children before dental health education was 2.75 and after, was 1.5. according to these data, the plaque index of deaf children was better than the blind children. this was due to different information delivery, daily habits such as tooth brushing, eating sweet and sticky foods as well as social and family factors, and the last but not the least, the appreciation of dental health education. changes in sugar consumption, preventive dental treatment and improvements in oral hygiene care are other possible reasons for the reduction of plaque accumulation.9 information delivery of the blind children and the deaf children were different because of their limitations. based on the research conducted by brydon lamb, people learn 83% through seeing, 11% through hearing, 3.5% through smelling, 1.5% through touching, and 1% through the tasting.10 deaf children use their vision, which is the main line in receiving information and blind children use their hearing and feeling senses in receiving information. general sense of hearing and sense of tasting in the blind child is more sensitive than deaf children. both senses are working to replace the function of sight as the primary senses. habitual factor also plays an important role in plaque formation. parents play a vital role in filtering the interaction between children and their environment through the feeding habits, oral hygiene care, and other preventive practices and services they make available to their children. predisposing, enabling, and reinforcing factors affect parents' ability to install healthy oral habits into a child's daily routine.11 research conducted in india showed that the education of the mother was the single best predictor for oral hygiene status and explained 92% of the variance. these findings show that children with hearing and visual impairment have poor oral hygiene and high levels of periodontal disease. this may be due to a lack of communication; hence, appropriate oral health education should be tailored to the needs of these students with the support of their teachers and their parents.13 tooth brushing and other mechanical measures are the most practical and effective means of achieving and maintaining adequate oral hygiene. although tooth brushing is a simple and effective means of removing plaque, the high prevalence of periodontal disease in the general population indicates that tooth brushing performance is inadequate.10 from the results of the questionnaire conducted showed that the habit of blind and deaf children have similarities and also differences. t h i s i s e v i d e n t f r o m t h e f o l l o w i n g s u r v e y . habit of brushing teeth every day on blind children as many as 21 people (91.30%) and who never brush their teeth as much as 2 people (8.70%) while on blind children as many as 29 people (93.55%), brushing teeth every day and 2 people (6.45%) never brush their teeth. a total of 19 children with visual impairment (66.67%) and 20 children with hearing impairment (68.97%), brush their teeth twice a day at breakfast and before sleeping at night. according to carranza12 and manson and eley (1993), frequency of figure 1. five subdivision of dental surfaces in plaque index php.8 note: m = mesial; d = distal; i = incisal; o = oclusal; c = central; g = gingival m m i c g d d g c o 42 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 39–42 brushing is correct twice a day e.g. at breakfast and before bed time. difference in way of tooth brushing in blind children and deaf children can be seen from the auxiliary brushing equipment. a total of 16 blind children (76.19%) never use the auxiliary equipment and 18 deaf children (58.06%) use auxiliary equipment like toothpicks (40%). in this study, blind children have never used the auxiliary equipment whereas deaf children use the auxiliary equipment such as toothpick. brushing teeth by using auxiliary equipment such as dental floss, toothpicks are an effective way to remove dental plaque.12 this difference shows that deaf children have a higher awareness of oral hygiene compared with the blinds. candy and chocolate are examples of sweet foods that can damage the tooth surface. high sugar content in sweet food provides good environment to bacteria in the oral cavity. most blind children and deaf children like sweet foods. this can be seen from the percentage of blind children and deaf children who like sweet foods. after eating foods sweet blind children and deaf children more often drink water than rinsing and brushing teeth. according to survey, a total of 14 blind children (60.87%) never get a dental health education while 20 deaf children (64.52%) get a dental health education. therefore, deaf children more understand and know how to keep and maintain oral hygiene. decrease in plaque index of blind children was higher than deaf children. this was caused by more sensitivity in the sense of taste and sense of hearing compared to the blind children so that their function can replace the primary senses of sight. however, in this study differences in plaque index were not significant because the frequency of dental health education was only done once. plaque index of blind children before and after dental health education was in the average category. this could occur because the blind children still did not fully understand how to brush their teeth in the correct way and also dental health education was not given effectively. on the other hand, plaque index of the deaf children before dental health education was in average category and after was in a good category. this could happen because the particular deaf children have already had dental health education before so that they were more understanding, in maintaining oral hygiene. deaf children can receive information better due to the normal function of sight. it is concluded that plaque index of deaf children was better than blind children before and after dental health education. references 1. kumar s, sharma j, duraiswamy p, kulkarni s. determinants for oral hygiene and periodontal status among mentally disabled children and adolescents. j indian of society 2009; 27: 151–7. 2. notoatmojo s. metodologi penelitian kesehatan. jakarta: rineka cipta; 2002. p. 26–27, 88–9. 3. barbara vs. population with special needs. tennese: oak ridge national laboratory; 2006. 559: 2-6. 4. nowak aj. dentistry for handicapped patient. st louis: cv mosby co; 1976. 121–31. 5. valerie lc, eileen w. blind children and oral health. available at: www.nidcr.nih.gov/nr/rdonlyres/.../children_special_needs.pdf. accessed march 30, 2011. 6. harris n. primary preventive dentistry. 4th ed. connecticut: appleton and lange; 1995. p. 4–5. 7. kim hs. evaluation of dental hygiene status of 6–7 year old children.2009. available at : http://iadr.confex.com/iadr/2009miami/ webprogram/paper.html. accessed november 12, 2009. 8. andlaw rj. a manual of pediatric dentistry. 4th ed. edinburg: churchill livingstone; 1996. p. 31. 9. abdullah ma. oral health status of primary dentition among 551 children aged 6 to 8 years in jazan, saudi arabia. sds journal 2000; 12: 67–71. 10. sunanto j. potensi anak berkelainan penglihatan. jakarta: departemen pendidikan dan kebudayaan ri; 2005. p. 7. 11. das um, singhal p. tooth brushing skills for the children aged 3–11 years. j indian of society 2009; 27: 104–7. 12. carranza fa. clinical periodontology. 10th ed. philadelphia: wb saunders; 2006. 10: 735–6. 13. kumar s, dagli rj. oral hygiene status in relation to sociodemographic factors of children and adults who are hearing impaired, attending a special school. pubmed 2008; 28: 258–64. 14. manson jd. outline of periodontic. philadelphia: wright; 1995. p. 114–7. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 126 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 126–132 case report telescopic overdenture as an alternative rehabilitation for the loss of several anterior teeth due to traffic accidents birgitta dwitya swastyayana subiakto and utari kresnoadi department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: telescopic overdenture is a prosthesis consisting of a primary coping, or an inner crown, that is attached to a supporting tooth in the oral cavity, and a secondary coping, or an outer crown, attached to a denture, which must be compatible with the primary coping. purpose: the purpose of this study was to restore function and aesthetics and uplift the psychological status of the patient by fabrication of a fixed removable prosthesis using the existing abutment teeth as a telescopic overdenture. case: a 36-year-old female came to the prosthodontic rsgm unair speciality clinic on her own volition to make front dentures for her upper and lower jaws after a traffic accident one and a half years ago. the patient had an arch bar installed two months after the accident, which was removed after two months. the patient wanted new dentures to improve both her ability to eat and her appearance. case management: preliminary treatments performed were; maxillary and mandibular scaling and root planing; crown lengthening of tooth 15; extraction of teeth 16, 41, 42, and 43; and alveolectomy of the mandibular anterior region. before the definitive treatment to improve the aesthetic appearance could be carried out, the first step was to make maxillary and mandibular transitional dentures. these were to be used while waiting for healing to occur, following the socket preservation in the mandibular area. then, for the definitive restoration, we used telescopic overdenture for the maxilla and a removable partial denture for the mandible. conclusion: telescopic overdenture is recommended for patients who need good aesthetics for anterior tooth loss. telescopic overdenture uses double crowns as the retentive elements, which give better aesthetic results compared to clasps, thereby improving the psychological status of the patient. in addition, they have better retention and stability compared to conventional complete dentures. keywords: telescopic denture; overdenture; double crown; inner and outer coping; aesthetic correspondence: utari kresnoadi, department of prosthodontics, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: utari-k@fkg.unir.ac.id introduction telescopic dentures are prostheses consisting of primary coping cemented into the abutment in the patient’s mouth, and secondary coping attached to the prosthesis, suitable for primary coping. this increases retention and enlarges the prosthesis. according to the glossary of prosthodontic terms,1 telescopic dentures are also referred to as overdentures, which are defined as removable tooth prostheses that are connected to and rest on one or more teeth that grow, on natural teeth, and/or on implanted teeth. this is also referred to as denture overlay, prosthetic overlay, and superimposed prosthesis. the double crown systems are typically distinguished from each other by their differing retention mechanisms. there are four different types of double crown systems: cylindrical crowns, conical crowns, resilient designs, and modified designs. cylindrical crowns, or telescopic crowns, achieve retention by using friction between the inner and the outer crowns. conical crowns, or tapered telescope crowns, exhibit friction only when they are completely seated by using a “wedging effect”. the magnitude of the wedging effect is mainly determined by the convergence angle of the inner crown: the smaller the convergence angle, the greater the retentive force. resilient designs are non-rigid designs, as they allow some freedom in the vertical and rotational dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 mailto:utari-k@fkg.unir.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 127subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 movements between the inner and the outer crowns. this may be achieved by some modifications in the inner crown, the outer crown, or both. these modifications result in reduction of the intimate contact and creation of a space between the inner and the outer crowns. modified designs were developed by considerable modifications in the double crown concept. they mostly depend on the merging of a telescopic system with another type of attachment. in this case, we used conical crown designs as a guidance for manufacturing a double crown system. one of the advantages of using telescopic overdenture is aesthetic; using double crowns as retentive elements allows better aesthetics than clasps. good aesthetics can be provided by using ceramide on the labial surface and a suitable colour selection. telescopic overdenture also has good retention and stabilization properties due to the double crown system, secondary splinting action, transference of occlusal forces through the long axes of abutments, creation of a common path of insertion, and improved hygienic properties.2 case a 36-year-old female came to the prosthodontic specialty clinic at the dental and oral hospital of universitas airlangga on her own volition to make front dentures missing in her upper and lower jaw after a traffic accident one and a half years ago. the patient reported having been involved in a traffic accident, which caused several avulsions of the anterior maxillary teeth and increased mobility on several lower-jaw teeth. the first treatment was an arch bar placed on the lower jaw by an oral surgery specialist in gresik, indonesia. this was removed after two months, when an evaluation revealed no mobility. the patient had no systemic disease, and wanted new dentures to be made to improve her eating and appearance (figure 1). on extraoral examination, the temporomandibular joint (tmj) had no issues: the face was oval in shape; eyes, nose and lips were symmetrical; and there were no abnormalities. intraoral examination revealed multiple missing teeth at 18, 14, 12, 11, 21, 22, 28, 31, 44, and 48; tooth mobility 3° at 31, 41, 42; and gangrene radix at 16 (figure 2). on radiographic examination, there was a visible decrease in alveolar bone at the apical third of teeth 41, 42, and 43. radiopaque appearance was seen around teeth 23, 24, and 25 (susp: ligature wire). radiopaque features extended from region 36 to region 46 (susp: arch bar) (figure 1). case management preliminary impressions of the maxillary and mandibular residual ridges by the alginate were taken on the first visit. preliminary casts were made by pouring the gypsum into a preliminary impression; then, diagnosis, survey and blockout were conducted. preliminary treatments performed were: maxillary and mandibular scaling and root planing; crown lengthening of tooth 15; extraction of teeth 16, 41, 42, 43; and alveolectomy of the mandibular anterior region (figure 3). then, the first treatment was to make maxillary and mandibular transitional dentures before the definitive aesthetic treatment while waiting for healing after socket preservation in the mandibular area (figure 4). the next treatment, which is the definitive denture manufacturing, was the telescopic partial overdenture of the maxilla and removable partial denture of the mandible. abutment preparation was carried out for 15, 13, 23, and rest seat preparation at 17. gingival retraction of teeth 15, 13, and 23 using ultrapack® retraction thread size 000 (ultradent, south jordan, utah) (figure 5) was done next, followed by maxillary functional impressions using the double-step technique with polyvinylsiloxane putty and light body elastomer (3m espe, minnesota, united states), and mounting of the cast. figure 1. panoramic radiographic examination. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 128 subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 a b c d e figure 2. intraoral condition: (a) right side view, (b) labial view, (c) left side view, (d) occlusal maxillary appearance, (e) occlusal mandibular appearance. figure 3. intraoral condition after extraction and alveolectomy in regions 31 to 44. figure 4. transitional denture for maxilla and mandible. figure 5. results of abutment teeth preparation 15, 13, 23, and after being given gingival retraction thread before functional impression. figure 6. try-in of inner coping of abutment teeth on 15, 13, and 23. a b figure 7. try-in of outer crown to the patient along with the inner crown attachment to the teeth, viewed from the front (a) and occlusal (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 129subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 a b c figure 8. denture teeth setup view from the right side (a), labial (b), and left side (c). a b c figure 9. telescopic partial overdenture in patient, viewed from the right side (a), labial (b), and occlusal maxillary (c). a b c figure 10. try-in of metal frame mandibular on the patient, view from the right side (a), labial (b), and left side (c). figure 11. denture teeth setup on 31,41,42,43,44. a b c figure 12. intraoral condition after insertion of removable partial mandibular denture, view from the right side (a), labial (b), and left side (c). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 130 subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 inner copings of 15, 13, 23 were customized in the dental laboratory. we did a try-in of the inner coping of 15, 13, and 23 in the patient with attention to the cervical so as to close the cervical teeth (figure 6). then, we made a final functional impression of the maxilla with a one-step technique using polyvinylsiloxane putty elastomer and light body with inner coping attached to the teeth. meanwhile, the inner crown settings took part inside the impression. manufacture of outer coping and metal frames took place in dental laboratories. then, we did a try-in of the outer crown and metal frame while the inner crown was attached to the abutment tooth (figure 7). the wax occlusal rim was made and placed on the metal frame. then, a bite registration was done with polyvinylsiloxane medium body o-bite® (dmg, hamburg, germany). a denture tooth setup was performed on the articulator (shofu, kyoto, japan) and fitted to the patient (figure 8) with acrylic denture processing. fixed cementing inner coping, fitting and delivering the denture on patient (figure 9). we next continued treatment for the lower jaw, performing rest seat preparation in the distal areas 35 and 45, and in the mesial areas 36 and 46. functional impressions for making metal frames were done using a stock tray with polyvinylsiloxane putty elastomer and light body. the cast was poured from the final functional impression and mounted. try-in of the metal frame and making the occlusion rim wax occurred next (figure 10), followed by denture tooth setup for 31, 41, 42, 43, and 44 (figure 11). next steps included acrylic denture processing, then fitting and delivering the removable partial mandibular denture on the patient (figure 12). next visit for control was done 24 hours after insertion of the denture. discussion in this case, the patient came to us to make dentures for the upper and lower jaw. the patient reported that she had been involved in a traffic accident, which had caused several avulsions of anterior maxillary teeth and mobility of several teeth on the mandible. the first treatment was by the oral surgery specialist in gresik, indonesia, who placed an arch bar on the mandible. the arch bar was removed after two months, when evaluation revealed there was no mobility. the first treatment was to make maxillary and mandibular transitional dentures, while waiting for healing after socket preservation in the mandibular area. transitional dentures were used until definitive dentures could be inserted.3 transitional dentures are designed to transition a patient from one oral condition to the next. transitional dentures are temporary appliances that will ultimately be replaced and discarded. the need for this type of treatment arises when a patient is faced with the extraction of some or all of their remaining dentition but does not want to be toothless while they heal and recover post-surgery. one of the options patients can consider is an immediate denture. this is a denture that is fabricated prior to extractions and placed immediately after the natural dentition has been removed.4 tooth conditions on 15, 13 and 23 had a good prognosis, so they could be used as abutment teeth. this was due to the good condition of the alveolar bone; pocket depth on the mesial, distal, labial and palatal teeth (2mm); no bleeding on probing examination; and no bleeding, inflammation, or tooth mobility. this examination was carried out in accordance with carranza’s opinion that a tooth’s supporting tissue is declared healthy if the normal gingival sulcus is ± 2 mm deep, the gingiva is pink, the size of the gingiva is not enlarged, and there is no bleeding. additionally, the tissue should be supported by healthy bones and the absence of tooth mobility.5 the condition of being partially edentulous can be treated with various treatment options, including both fixed and removable prostheses. the periodontal status of abutment teeth present in such cases dictates the prognosis of the treatment option chosen.6,7 a limited number of available abutments and decreased crown-to-root ratio of the present teeth pose a great prosthetic challenge.8 removable prosthodontics was the only answer to cases before the advent of fixed removable prosthesis in the form of telescopic overdenture.9 in the case of the maxillary tooth loss at 16, 14, 12, 11, 21, 22, teeth 15, 13, and 23 with good periodontal tissue conditions were selected as abutments. the anterior location required a good aesthetic. based on these considerations, the treatment that was chosen for the maxilla was a telescopic partial denture. telescopic denture has the advantage of a double crown that can directly transfer occlusal loads to the axial axis of the abutment teeth.2 in telescopic partial denture restoration, aesthetics and good retention can be achieved because the attachment is based on mechanical frictional resistance. patients who present with multiple periodontally and endodontically compromised abutment teeth pose a great prosthodontic challenge. opting for a fixed prosthodontic treatment for such patients does not ensure prognosis and longevity.6,7 however, apart from the cripples of a removable prosthesis, compromised support and stability are added disadvantages to such a treatment plan.7 fabrication of a fixed removable prosthesis combines advantages of both fixed and removable dental prostheses.8 undue leverage forces due to parafunction are avoided as the superstructure is removable. the rigid splinting action, support, and retention provided by abutment teeth, as well as better distribution of forces, are some of the advantages of such a prosthesis. furthermore, a better prognosis of abutment teeth can be predicted due to improvement in the crown-to-root ratio, better hygiene maintenance, and reinforcement provided by the primary metal copings. disadvantages associated with such a prosthesis are the extra time, effort, and cost involved.10,11 vertical space requirement is another limitation to planning treatment with such a prosthesis. a minimum of 9 mm vertical space is required to accommodate the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 131subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 metal copings (1.5–2 mm) and ceramide superstructure (3 mm) on abutments prepared to an adequate height (4–5 mm). furthermore, the procedure is technique-sensitive.11 a satisfactory periodontal and endodontic status of the abutment teeth dictates a fair prognosis with a removable cast partial denture if the design of the components satisfies the mechanical requirements. in the current case, a removable cast partial denture was fabricated to rehabilitate the lower arch due to the favourable prognosis of the abutment teeth. however, a telescopic overdenture prosthesis was fabricated for the upper arch due to the guarded prognosis of abutment teeth present. according to the glossary of prosthodontic terms,1 telescopic dentures are also referred to as overdentures, in the form of removable dentures that cover and ride on one or more natural teeth, natural tooth roots, or dental implants. the retention and stability of the telescopic denture depends on the number and distribution of the supporting teeth along the dental arch, and the taper wall of the inner crown. the smaller the degree of taper, the greater the friction retention of the retainer.12 tooth preparation in all mesial-distallabial-palatal-incisal sections is around 1.8-2 mm. the inner crown requires a thickness of about 0.3-0.5 mm (the mesial/distal part that uses friction elements requires more thickness), while the outer crown requires a thickness of about 0.3-0.5 mm, and the ceramide requires a thickness of 0.81 mm.13 in this case, the double crown system used was the conical crown. braces were prepared subgingivally, and walls were tapered (2°–5°) to increase retention. the inner crown was made by a laboratory with a slope of about 2º so that a spring tension arises from the outer crown to the inner crown. the main requirements for durable telescopic dentures were vertical wall height (around 4mm), sufficient thickness of coping (minimum 0.7mm), and degree of inclination of around 6º.12 in this case, the patient was also advised to use a removable partial denture (metal frame) design for the mandible. the removable partial denture (metal frame) compared to other materials has many advantages: it is more comfortable to wear because it can be made thinner than acrylic resin; all parts of artificial teeth are one unit and homogeneous; and the occlusal load can be transferred evenly. additionally, there is better thermally conductivity and better hygiene because porosity on metal surfaces is lower than acrylic resins, thereby reducing food and plaque accumulation, and maintaining healthy tissue.4 according to carr and brown4 in mccracken’s removable partial prosthodontics, the type of tooth loss and design of the mandibular denture in this case include the classification of kennedy class iv, so that the support obtained is from the teeth. after a survey on the work model, retention in lingual teeth 45 and 46, and 35 and 36 was obtained. therefore, the selected clasp design used a tooth-borne clasp, namely double akers' clasps at 35 and 36, and 45 and 46, as a direct retainer. there was plate expansion above the cingulum on teeth 32 and 33 as an indirect retainer. the direct retainer was placed on teeth 35 and 36, and 45 and 46, so that the clasp was not too visible from the front. therefore, it looks good aesthetically.4 patients are advised to remove dentures at night, because according to watt and gregor, this is an efficient way to control caries and the development of periodontal disease.14 patients are also instructed to always maintain the cleanliness of the oral cavity by brushing teeth and using mouthwash so that there is no accumulation of food debris on the supporting teeth,. according to ai and shiau, maintenance of periodontal tissue health is a critical success factor in overdenture treatment, which also depends on the effectiveness of plaque control by the patient.15 denture treatment can be done using an antibacterial denture cleanser by brushing the denture with a soft bristled toothbrush and liquid soap without strong pressure. cleaning this way is in accordance with the opinion of zarb; it is not recommended to use toothpaste when cleaning dentures because most contain abrasive materials that can erode the surface of acrylic resin.16 telescopic overdenture has aesthetic advantages; using double crowns as retentive elements allows better aesthetics than clasps. good aesthetics can be provided by using ceramic faces and a suitable colour selection. telescopic overdenture also has good retention and stabilization properties due to the double crown system and secondary splinting action. it also transfers occlusal forces through the long axes of abutments, creates a common path of insertion, and offers improved hygiene. it can be concluded that telescopic overdenture is recommended for patients who need good aesthetics for anterior tooth loss; it uses double crowns as the retentive elements, which give a better aesthetic result compared to clasps, thereby increasing the psychological status of the patient. telescopic overdenture has better retention and stability compared to conventional complete dentures. references 1. the glossary of prosthodontic terms: ninth edition. j prosthet dent. 2017; 117(5): e1–105. 2. hakkoum ma, wazir g. telescopic denture. open dent j. 2018; 12: 246–54. 3. payne sh. a transitional denture. j prosthet dent. 2014; 14(2): 221–30. 4. carr ab, brown dt. mccracken’s removable partial prosthodontics. 12th ed. saint louis: mosby elsevier; 2011. p. 385. 5. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. st. louis: saunders elsevier; 2015. p. 16, 336, 475. 6. bukleta ms, bukleta d, selmani m, kuhar m. frequency of complete and removable partial denture treatment in the primary health centres in three different regions of kosovo from 2002 to 2013. slov j public heal. 2019; 58(3): 104–11. 7. nemcovsky ce. evidence-based decision making in periodontal tooth prognosis. clin dent rev. 2017; 1: 3. 8. breitman jb, nakamura s, freedman al, yalisove il. telescopic retainers: an old or new solution? a second chance to have normal dental function. j prosthodont. 2012; 21: 79–83. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 132 subiakto and kresnoadi/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 126–132 9. kaira l, mishra r. telescopic denture a treatment modalitity of preventive prosthodontics. int j oral heal sci. 2013; 3(2): 121–4. 10. shruthi cs, poojya r, ram s, anupama. telescopic overdenture: a case report. int j biomed sci. 2017; 13: 43–7. 11. schwindling fs, dittmann b, rammelsberg p. double-crown– retained removable dental prostheses: a retrospective study of survival and complications. j prosthet dent. 2014; 112(3): 488–93. 12. singh k, gupta n. telescopic denture a treatment modality for minimizing the conventional removable complete denture problems: a case report. j clin diagnostic res. 2012; 6(6): 1112–6. 13. wulfes h. precision milling and partial denture constructions – modern design and efficient production. bremen: academia dental, international school bego germany; 2012. p. 151–152. 14. milward p, katechia d, morgan mz. knowledge of removable partial denture wearers on denture hygiene. br dent j. 2013; 215(10): e20. 15. zarb g, hobkirk j, eckert s, jacob r. prosthodontic treatment for edentulous patients: complete dentures and implant-supported prostheses. 13th ed. saint louis: mosby elsevier; 2012. p. 464. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p126–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p126-132 176 vol. 43. no. 4 december 2010 biocompatibility and osteoconductivity of injectable bone xenograft, hydroxyapatite and hydroxyapatite-chitosan on osteoblast culture bachtiar ew�, bachtiar bm�, abas b�, harsas na�, sadaqah nf�, and aprilia r� 1department of oral biology, faculty of dentistry, university of indonesia 2badan tenaga nuklir nasional (batan) jakarta indonesia abstract background: bone graft in the form of injectable paste gives several advantages over the powder form as it could be placed in the defect area that has limited accessibility. purpose: the purpose of this study was to assess biocompatibility and osteoconductivity of an injectable bone xenograft (ibx), injectable hydroxyapatite (iha) and injectable hydroxyapatite-chitosan (iha-c) on osteoblastic cell line (mg-63). methods: three concentrations (0.25%, 0.5% and 1.0%) of ibx, iha and iha-c were supplemented with dmem culture medium. the viability cells were measured by mtt assay 4 hour after incubation. alp activity was measured at day 1, 3, 5 and 7. calcium deposition was tested at day 3 and day 7 by means of von kossa staining. results: mtt assay showed that the viability cells of all the test groups were above 100% compared to the control group. the cell viability of the 0.25% iha paste was significantly higher (115.02% ± 4.37%, p < 0.05) compared with ibx paste and iha-c in all concentrations tested. the highest level of alp secretion of all test groups was found on the fifth day of exposure. the highest level of alp in the ibx paste group was 0.25% concentration while the highest level of alp in the iha-c and iha paste group was 1% and 0.25%, respectively. in addition, the highest calcium deposition was shown on iha 1% at day 7 (p > 0.05). conclusion: it was suggested that adequate biocompatibility and osteoconductivity was evident for all injectable pastes tested. key words: injectable bone xenograft, injectable hydroxyapatite, injectable hydroxyapatite-chitosan, osteoblast abstrak latar belakang: bahan tandur tulang dalam bentuk pasta injeksi memiliki kelebihan dibandingkan bila bahan tersebut berupa bubuk, karena lebih mudah diaplikasikan pada daerah yang sulit dijangkau. tujuan: penelitian ini bertujuan untuk mengamati sifat biokompatibilitas dan osteokonduktifitas biomaterial tandur tulang dalam bentuk injectable bone xenograft (ibx), injectable hydroxyapatite (iha) dan injectable hydroxyapatite-chitosan (iha-c) pada galur sel osteoblas (mg-63). metode: bahan tandur tulang ibx, iha and iha-c masing-masing dengan konsentrasi 0,25%, 0,5% dan 1,0% dipaparkan dalam larutan medium kultur sel dmem yang telah disebari sel mg 63. selanjutnya setelah 4 jam inkubasi maka viabilitas sel diukur dengan cara uji mtt, sedangkan aktifitas fosfatase alkali (alp) diukur pada hari ke-1 (24 jam), hari ke-3, 5 dan 7. deposisi kalsium diukur pada hari ke-3 dan ke-7 dengan metoda pewarnaan von kossa. hasil: uji mtt menunjukkan bahwa pemberian semua jenis bahan pasta injeksi tandur tulang meningkat di atas 100% dibandingkan kontrol. viabilitas sel pada pemberian 0,25% pasta iha tampak paling tinggi dibandingkan pasta ibx dan iha-c pada semua konsentrasi yang diuji. sekresi alp tertinggi pada semua kelompok eksperimen terjadi pada hari ke lima setelah paparan bahan injeksi tandur tulang. sekresi alp tertinggi pada tiap jenis pasta terjadi pada pemberian ibx 0,25%, iha-c 1% dan iha 0.25%. sedangkan deposisi kalsium tertinggi terjadi pada pemberian 1% iha setelah 7 hari kultur sel. kesimpulan: semua bahan injeksi tandur tulang yang diuji pada kultur osteoblas bersifat biokompatibel dan berpotensi osteokonduktif. kata kunci: injectable bone xenograft, injectable hydroxyapatite, injectable hydroxyapatite-chitosan, osteoblas correspondence: bachtiar ew, c/o: bagian biologi oral, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat 10430, indonesia. email: endang04@ui.ac.id/endangwiniati08@gmail.com research report 177bachtiar, et al.: biocompatibility and osteoconductivity of injectable bone xenograft introduction bone defect is a common finding in oral maxillofacial and orthopedic surgeries. this condition resulted in impair bone function as a structural support. the primary causes of bone defect in the oral maxillofacial region are periodontal disease and tooth loss. alveolar bone loss in periodontal disease is triggered through immune responses, resulting from inflammatory reaction directed against periodontopathic bacteria.1 when tooth is extracted and no dental implant is placed, there is no more direct loading that is normally carried by the dentition and transferred through the periodontal ligament to the alveolar bone. this condition leads to a decrease in osteoblasts activity and an increase in osteoclasts activity as mechanical loading play a major role on maintaining bone mass.2,3 the increase in osteoclasts activity will results in bone resorbtion and subsequently the formation of bone defect that impair the structure as well as the function of bone. treatment of bone reconstruction is therefore necessary to reform the volume and density of the bone, thus maintaining normal bone function. bone reconstruction technique is normally carried out using bone graft.4 bone grafts are extensively used for oral and maxillofacial applications including treatment of fractures and nonunions, replenishment of bone loss resulting from tooth extraction, periodontal diseases or tumor.5 which material is the most appropriate to restore bone volume is a subject of debate. autogenous bone graft whereby the bone donor derives from patient’s own body is considered the gold standard due to its biological characteristic, providing both organic and inorganic matrices, biological signals and viable bone cells.5,6 however, in a case of large bone defect, a large quantity of autogenous bone is required to reconstruct adequate bone volume. the procedure gives rise to patient’s morbidity. several alternative materials have been used to overcome the problem such as allograft, xenograft and alloplast.7-9 the advantage of using bone substitute material is the absence of additional surgery. the utilization of these materials as bone grafts may reduce the need for autogenous bone graft, which mostly available in a limited volume. one of strategies for tissue engineering is the transplantation of cells that have been expanded in vitro by biodegradable scaffold. ideal scaffold should possess characteristics such as; biocompatible, biodegradable, high surface area/volume ratio that could support the attachment, proliferation and differentiation of cells to develop the desired tissue.10 a variety of carriers and matrices have been used for bone regeneration including bone autograft, bone allograft, natural component such as collagen membrane and synthetic carrier such as bioglass, b-tcp.10,11 chitosan is a compound that consists of co-polymer glucosamine and n-acetylglucosamine that is currently used for food industry. chitosan has a good biocompatibility property, degradable by enzyme to become oligosaccharide that can be easily absorbed, forming an insoluble complex with connective tissue such as collagen and glycosaminoglycans to become porous scaffold, film or particle.12 chitosan has a porous interconnected structure that makes this biomolecule suitable for scaffolding material especially for tissue engineering.11 xenograft is a graft of tissue taken from a donor of one species and grafted into a recipient of another species. the most common sources of xenogenic grafts are bovine.7-9 this graft acts as scaffold to support the growth of the new tissue and will be replaced with the tissue from host with some reported its minor osteoinductive property. hydroxyapatite (ha) is often used as a bone substitute material due to its osteoconductive and biocompatible properties allowing the integration with the host bone.13 the combination of ha and chitosan in the form of injectable, porous and biodegradable structures seem to be an interesting route to promote localized bone regeneration.14 biocompatibility and the absence of contagious substance in the graft are the importants characteristic for ideal bone graft. bone graft in the form of injectable paste gives several advantages as it could be placed in the defect area that had limited accessibility, allowing the cavity filled with the biomaterial in a homogenous manner accordingly. this research focus on assessing the biocompatibility and osteoconductivity of three different pastes, namely injectable hydroxyapatite paste (iha), injectable hydroxyapatite chitosan (iha-c) paste, and injectable bone xenograft paste (ibx) as prospective scaffolding materials to be used for bone tissue engineering. all tested were performed in the osteoblastic cell line (mg-63). the testing of the toxic effect was carried out with the mtt assay, whereas osteoconductivity property was evaluated with the alkaline phosphatase and von kossa staining for calcium deposition. materials and methods the study was conducted at oral biology laboratory, faculty of dentistry university of indonesia. and it was design as an in vitro experiment. the experiments were divided into 4 main groups: one control group that did not received any materials as well as three treatment groups that received a) 0.25%, 0.5% and 1.0% of ibx; b) 0.25%, 0.5% and 1.0% of iha and c) 0.25%, 0.5% and 1.0% iha-c. each experiment was perform in 6 replicates. osteoblastlike mg-63 cells (attc no. crl-1427; a kind give from prof. suttatip kalmolmatyakul, prince of sonkla university, thailand). the cells were cultured up to near-confluence in 75 cm2 flasks (nunc) using dmem, dulbecco’s modified eagle medium (invitrogen) supplemented with 10% fetal bovine serum (fbs, sigma), penicillin, 100 iu/ml and 100 pg/streptomycin. cells were then harvested and seeded at 1 × 105 cells per well (incubated overnight in dmem with 10% fbs to promote cell attachment at 37° c with 5% co2 in air. the cells were then divided into two groups, 178 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 176–180 a test group, incubated with media containing various concentrations of injectable grafts tested and a control group, incubated only with the media. ibx, iha-c, and iha (batan, indonesia) were diluted in the culture medium until 1%, 0.5%, and 0.25% concentration were reached. to estimate the density of viable cells, mtt (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay was conducted.15 the samples were divided into 1 × 106 cells/ml/well in a 96-well culture plate and were incubated for 24 h. injectable paste grafts were then added to the media for 4h. the samples were washed by pbs and mtt dye agent (sigma) was mixed to each well and incubated for another 4h. the absorbance was measured using a microplate reader at the wavelength of 490 nm (biorad). to obtain the percent of cells viability, the optical density (od490) of treatment group was devided to the control group. to measure early osteoblast differentiation, alkaline phosphatase (alp) activity test was carried out from culture medium by colorimetry.16 the samples were divided into 1 × 105 cells/ml/well in a 24-well culture plate for 24 h and followed by incubation with injectable paste grafts for 1, 3, 5 and 7 days. alp substrate solution of p-nitrophenyl phosphate (sigma) was added to each solution at room temperature for 30 m. the absorbance was measured using a microplate reader at the wave length of 405 nm. to measure calcium deposition by mg63, van kossa staining was performed. the samples were divided into 1x105 cells/ml/well in a 6-well culture plate for 24 h and cultured in the media containing various concentrations of bone graft tested for 3 and 7 days. mineralized nodules were stained with silver nitrate solution according to the von kossa method as described.17 the results were statistically analyzed by anova for normal data and mann whitney for abnormal data. p value of < 0.05 represented a significant difference. results viability cells measurement using mtt assay showed that all of the test groups compared to control group was above 100% (table 1). the highest cell viability was found in the group of cells incubated with 0.25% iha paste (115.02% ± 4.37%, p < 0.05). of all concentrations tested in three different injectable graft pastes, lower concentration of 0.25% demonstrated higher cell viability compared to high concentrations of 0.5% and 1.0% (p < 0.05). the highest level of alp secretion of all test groups occurred on the fifth day of exposure (table 2). the highest level of alp was found in the 0.25% concentration of ibx paste group (optical density = 1.8895); whilst the highest level of alp in the iha-c and iha paste group was 1% and 0.25% concentration respectively (optical density = 1.8465; 1.7475). however, the increase was not statistically significant (p > 0.05) when compared with the control. in addition, the highest calcium deposition was shown on iha 1% at day 7 (p > 0.05). all injectable pastes increase calcium deposisition compare to the control at day 7 of culture, whereas at day 3 these initial mineralization have occured in lower level (above 1040 spots), (table 3). discussion in vitro culture system was one of the methods commonly used to evaluate the biological responses of biomaterial prior its to be tested on animal model. this system has some avantages over the animal study, as we can directly asses the appropriate cell responses against material to be tested. here we use osteoblast cell line to evaluate the effects of bone graft pastes on the cells viability, osteogenic biomarker secreted by the cells as well as calcium deposisition. the outcome of the study is expected to be the foundation for the development of bone tissue engineering. table �. effect of ibx, iha-c, ha pastes. the viability of mg63 osteblastic cell line according to the mtt assay concentration of paste mg63 cell viability ibx iha-c iha 1% 104.42% 107.52% 102.37% 0.5% 108.54% 112.82% 100.38% 0.25% 109.21% 115.02%* 109.32% *the cell viability of the 0.25% iha paste was significantly higher (115.02% ± 4.37%, p < 0.05) compared with ibx paste and iha-c in all concentrations tested. table �. the effect of injectable pastes in alkaline phosphatase level of mg63 osteblastic cell line culture medium day ibx (concentration) iha-c (concentration) iha (concentration) control 1% 0.5% 0.25% 1% 0.5% 0.25% 1% 0.5% 0.25% 1 1.1095 1.103 1.5845 1.427 0.7725 1.641 1.4325 0.868 1.651 1.2215 3 1.57 1.621 1.1345 1.295 1.6655 1.232 1.274 1.555 1.253 1.497667 5 1.865 1.4755 1.8895 1.8465 1.269 1.749 1.734 1.448 1.7475 1.515833 7 1.2065 1.6065 0.8325 1.3545 1.287 1.072 1.1535 1.5915 0.9865 1.454 179bachtiar, et al.: biocompatibility and osteoconductivity of injectable bone xenograft the viability of the test group of ha based pastes (iha and iha-c) did not have toxic effect rather they increased cell proliferation and alp secretion. the result in line with a report from karaj et al.,7 which found that ha is able to increase cells proliferation. however, the iha 0.25% group was the lowest in producing alp at fifth day of exposure, compared with ibx 0.25% and iha-c 1%. these results probably because iha paste only contained ha. serre et al.,8 reported that biomaterial which contained ha and collagen can increase protein matrix synthesis more than a biomaterial with ha only. the addition of chitosan in ha as bone substitute material was expected to improve the biocompatibility of the biomaterial. chitosan with degree of deacetylation (dda) greater than 99% could improve bone regeneration process at a defect in the femurs of sheep.9 chitosan also had an positive effect on cartilage regeneration.10–12 however, the result in this study showed the cells viability percentage of iha-c group was lower than the iha group. one argument to explain this finding is the dda of the chitosan in iha-c paste. the range of dda is commonly 70–90% and the ability to induce cell proliferation increases with the rise of dda.5 unlike the cells proliferation, the alp secretion of iha-c group is higher than iha group. this result shows that alkaline phosphatase expression as an initial marker of bone regeneration process in in-vitro. the result of this study indicates that all injectable pastes increasing osteoblast cell differentiation, showed by the height of alkaline phosphatase expression. this was also reported in previous in-vitro studies in cell culture by takamori et al.,13 and joss et al.15 based on the data presented in this study, cell viability percentage of ibx group was the lowest compared with the other paste groups. this phenomenon probably because the ibx paste contain bovine xenograft which taken from bovine bones loosed some of their organic components and pathogens during the chemical extraction.3,13 besides that, the size of the particles can also influence the viability percentage. it is believed that smaller particle size can improve the properties of synthetic bone substitute, due to its higher surface area.7 the size of bone xenograft in ibx is 60 mesh, and the ha particle in iha and iha-c is 100 mesh. the alp secretion research showed that the ibx paste groups produced more alp than the other two pastes groups. this is probably because the bovine xenogaft still have a small amount of bmp which can stimulate cells differentiation.14 the discrepancies of the results in the cell proliferation and alp secretion also appropriated with the report of joss et al.15 which stated that when there is an increment in alp production during the exposure of bovine extract on osteoblast cells, the amount of cell proliferation decreased. in this study von kossa staining showed the increment of calcium deposition in human osteoblast cell culture exposed with ibx, iha and iha-c compared to control group. the highest calcium deposition was shown on 1% of iha at day 7 followed by 1% iha-c and 1% of ibx (p > 0.05). similar finding reported by carnes et al.17 and shen et al.,18 showing that early calcium deposition in osteoblast cell culture reached at day 7. therefore, this result proved that all form of injectable pastes have the ability to increase mineralization of osteoblast cell in bone regeneration process. biocompatibility and osteoconductive properties of ibx, iha-c and iha were evaluated in this study and resulted no toxic effect of these materials as indicated by cell viability that was assayed by mtt test. in addition, the secretion alp as early biomarker of osteogenesis occured from day 3 and increase at day 7, whereas mineralization were detected at day 7 of culture. this study showed the biological activities of osteoblast as its responses to the injectable pastes tested. after 24 hours, we have observed an increment in the percentage of cellular viability in the treatment group as compared to that of the control group. this result suggests the possibility of observing biocompatibility property of bone graft pastes 24 hours post treatment period. in addition, we have observed an alp secretion in the osteblasts culture. this observation and in addition to the observed alp 7 days turnover rate indicates a possible role of this molecule on calcium deposition.8 in almost all of the experimental groups, the alp were secreted in small amount in day 3. in this experiment, alp secretion rate rose to its peak on day 5 and subsequently dropped in day 7 of culture and at this time calcium deposition was detected. in conclusion, all bone graft pastes seem to be biocompatible as indicated by the mtt assay and have an osteoconductivity capability based on the alp secretion and calcium deposition. evaluation of the osteogenic property of pastes on macaca’s mandibular bone is currently ongoing. the results of this work is significant because of the potential usage of the injectable scaffold tested here, which can be considered as an alternative source of effective and affordable biomaterials for tissue engineering. the outcome table ��. level of calcium deposition of mg63 osteoblastic cell culture after 3 and 7 day of injectable pastes application day ibx (concentration) iha-c (concentration) iha (concentration) control 1% 0.50% 0.25% 1% 0.50% 0.25% 1% 0.50% 0.25% 3 860 900 940 900 960 980 1.02 1010 1.04 230 7 1.340 1.380 1.440 1.340 1.360 1.460 1.600 1.540 1.460 660 180 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 176–180 of this research could help to reduce our dependency on imported scaffolding biomaterials for an affordable bone reconstructive treatment. acknowledgement this study was supported by the grant of ruui 2008 provided by universitas indonesia. references 1. garg ak. in: lynch se, robert g, robert em, editors. tissue engineering: applications in maxillofacial surgery and periodontics. illnois: quintessence public inc; 1999. p. 83–9. 2. laschke mw, witt k, pohlemann t, menger md. injectable nanocrystalline hydroxyapatite paste for bone subtitution: in vivo analysis of biocompatibility and vascularization. j biomed mater res b appl biomater 2007; 82(2): 494–505. 3. yamamoto s, massuda h, shibukawa y, yamada s. combination of bovine derived xenografts and enamel matrix derived in the treatment of intrabony periodontal defects in dogs. int j periodontics restorative dent 2007; 27(5): 471–9. 4. camilli ja, da cunha mr, bertran ca, kawachi ey. subperiosteal hydroxyapatite implants in rats submitted to ethanol ingestion. arch oral biol 2004; 49(9): 747–53. 5. hu q, li b, wang m, shen j. preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture. biomaterials 2004; 25(5): 779–85. 6. abarrategi a, lópiz-morales y, ramos v, civantos a, lópez-durán l, marco f, lópez-lacomba jl. chitosan scaffolds for osteochondral tissue regeneration. j biomed mater res a 2010; 95(4): 1132–41. 7. kasaj a, willershausen b, reichert c, röhrig b, smeets r, schmidt m. ability of nanocrystalline hydroxyapatite paste to promote human periodontal ligament cell proliferation. j oral sci 2008; 50(3): 279–85. 8. serre, cm, papillard m, chavassieux p, boivin g. in vitro induction of a calcifying matrix by biomaterials constituted of collagen and/or hydroxyapatite: an ultrastructural comparison of three types of biomaterials. biomaterials 1993; 14(2): 97–106. 9. muzzarelli ra, mattioli-belmonte m, tietz c, biagini r, ferioli g, brunelli ma, fini m, giardino r, ilari p, biagini g. stimulatory effect on bone formation exerted by a modified chitosan. biomaterials 1994; 15(13): 1075–81. 10. alini m, roughley pj, antoniou j, stoll t, aebi ma. biological approach to treating disc degeneration: not for today, but maybe for tomorrow. eur spine j 2002; 11 (suppl 2): s215–20. 11. lu jx, prudhommeaux f, meunier a, sedel l, guillemin g. effects of chitosan on rat knee cartilages. biomaterials 1999; 20(20): 1937–44. 12. nettles dl, elder sh, gilbert ja. potential use of chitosan as a cell scaffold material for cartilage tissue engineering. tissue eng 2002; 8(6): 1009–16. 13. takamori er, figueira ea, taga r, sogayar mc, granjeiro jm. evaluation of the cytocompability of mixed bovine bone. branz dent j 2007; 18(3): 179–84. 14. ragetly gr, slavik gj, cunningham bt, schaeffer dj, griffon dj. cartilage tissue engineering on fibrous chitosan scaffolds produced by a replica molding technique. j biomed mater res a 2010; 93(1): 46–55. 15. joos ue, fehrenbach e, hogh-janovsky k, wimmer fm, schneider em, schmidt kh. effect of a new bone-inducing biomaterial on mesenchymal cells in vitro. artif organs 2008; 16(4): 354–60. 16. li h, zhai w, chang j. in vitro biocompatibility assessment of phbv/wollastonite composites. j mater sci mater med 2008; 19(1): 67–73. 17. shen b, bhargav d, wei aq, williams la, tao h, ma dd, diwan ad. bmp-13 emerges as a potential inhibitor of bone formation. int j biol sci 2009; 5(2): 192–200. 18. carnes dl jr, de la fontaine j, cochran dl, mellonig jt, keogh b, harris se, ghosh-choudhury n, dean dd, boyan bd, schwartz z. evaluation of 2 novel approaches for assessing the ability of demineralized freeze-dried bone to induce new bone formation. j periodontol 1999; 70(4): 353–63. isi vol 39 no 2 april 2006 file pertama.pmd 72 the business of dental practice niken widyanti sriyono department of community and preventive dentistry faculty of dentistry gadjah mada university yogyakarta indonesia abstract globalization including general agreement on trade in services (gats) and asia fair trade agreement (afta) are a new free trade system. in globalization era, there will be an intense and free competition in looking for jobs throughout the world. this new system will affect the health services system in which health services tend to follow an industrial model. meaning that dentistry or dental health services tend to be part of a business system, and this system has caused controversy among the community and the profession itself. the results of the discussion revealed that professional and business of dentistry is compatible and complementary. the tendency of increasing number of legal form of practice (group and a professional corporation) and the worldwide advertisement of these practices supported the premise that delivering dental practice tends to follow the industrial model. dentists should not only more focus on achieving financial success in running the business of practice but profession should have the most concern for the people who seek their services. delivering quality of dental care depends on the high skill of the dentist and on the satisfactory income for the survival of the practice in the long run, and this make the practice will be viewed by the public and profession as being appropriate and of high quality. facing the globalization, besides possessing high clinical skill, dentists must have a firm understanding of management concepts and apply them in their practice. in conclusion: the profession and the business of dentistry are compatible and complementary. the delivery of the dental services tends to follow the industrial model, which is a current reality. dentist should concern more on the delivering high quality of dental services, not only focus on the business of the practice, although the satisfactory income is important for the survival growth of the practice in the long run. it is suggested for dentists to follow as much business training or dental practice management training as the dentist can. it is also suggested to give the undergraduate students the basic concepts of dental practice management by putting this in the curriculum. key words: business, dental practice correspondence: niken widyanti sriyono, c/o: bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii, sekip utara yogyakarta 55281, indonesia. e-mail: bu_nikensri@yahoo.com. telp. (0274) 515307. introduction globalization including general agreement on trade in services (gats) and asia fair trade agreement (afta) will be implemented soon. globalization and afta are a new free trade system, and this system will have great impact on health services,1 specifically dental health service systems. people can work in whatever country they choose, since theoretically there are no borderlines among countries in the world. therefore, there will be an intense and free competition in looking for jobs throughout the world. skilled and professional people will have the greatest advantage in competing for these jobs. consequently, health workers should prepare themselves for globalization and afta. moreover, this new order will also affect the health services system in which health services tend to follow an industrial model.1,2 meaning that dentistry or dental health services tend to be part of a business system, and this system has caused controversy among the community and the profession itself.2 as is mentioned above, dentists will face the tough/ intense competition in finding a job. taking this into consideration therefore, dentists should aware of and know about the dental health status of the community, the projected growth of the dental health manpower, and the demand of the people in buying or using a dental service.3 for example, by evaluating the condition of the dental health status of indonesians and the current dental manpower; in which the dental health status of indonesian increases for the last 15 years, while the ratio between dentists and population is about 1 : 13.000.4 added by the big population of indonesia (220 million), it means that indonesia will be considered as a big “market” for dental health personnel not only for the indonesian but also for regional or may be international dental personnel. as the consequences therefore, there will be more tough/intense competition in carrying out dental health services in indonesia. indonesian dentists should aware and prepare themselves in facing globalization and the current reality of the business system in delivering dental services. although the big majority of dental practice is still dominated by solo practice but from the field observation showed that there is a tendency that dental practices are growing to be a group of practice/partnership or 73sriyono: the business of dental practice a professional corporation especially in some big cities in indonesia. partnership/group practice and a professional corporation is the legal form of business organization, and these practices tend to be managed using a business system. therefore, this paper will discuss about the professional and the business of dentistry, will introduce the legal forms of dental practice with their advantages and disadvantages as the current reality of the business system in delivering dental services. profession wikipedia,5 the free encyclopedia defines that a profession is an occupation that requires extensive training and the study and mastery of specialized knowledge, and usually has a professional association, ethical code and process of certification or licensing. for examples are law, medicine, finance, the military, nursing, the clergy and engineering. a member of a profession is termed professional. a profession is always held by a person, and it is generally that person’s way of generating income. membership in the profession is usually restricted and regulated by a professional association.5,6 in modern usage, professions tend to have certain qualities in common. however, there is no standard definition of a modern professional. beyond the classical examples (lawyers, doctors, etc.) there are many groups that claim status as a profession, and many who dispute the status. for example, schoolteachers often refer to their occupation as a profession; even though it is not exclusive, nor is entrance competitive, nor are they self-regulating.5,7 contemporary professions arose during the middle ages and are represented by fields such as the military, medicine and law. all these held a specific code of ethics, and members were almost universally required to swear some form of oath to uphold those ethics. each profession also provided and required extensive training in the meaning, value and importance of that oath in the practice of profession.6 freidson8 suggests that there are six characteristics that can be used to determine whether a group qualifies as a true profession: 1) members of a profession posses abstract knowledge in their area of expertise, 2) entry into a profession requires prolonged specialized training, 3) a profession regulates itself through licensure and through determining its own standards of education, 4) members of a profession are relatively free of lay control, 5) the services provided by a profession are necessary for the ongoing functioning of society and 6) members of a profession has a service orientation rather than a profit orientation in line with freidson’s qualification of a profession, jong6 stated that all professionals have 4 common requirements: 1) a distinct body of knowledge generally requiring education beyond the usual level, 2) a component of service to society, 3) the right and responsibility to be self-governing, and 4) a code of ethics. professions, such as dentistry, determine their own standards for licensure and control the numbers of entering professionals, the length and conditions of education, the distribution of services, and to great extend the cost of services. it is society’s belief that professionals place the welfare of the patient above their own welfare, which helps support the independence of the professions in regulated society.6,7 characteristic of business a business can be defined as any individual or group effort to produce and distribute goods or services for a profit.9 in most of the economic systems, four groups play important roles in a business: 1) owners, 2) managers, 3) producers, and 4) consumers. sometimes, individuals could belong to more than one group. for example, an owner may also be a manager and a producer, and a producer could also be a consumer. owners are those who use their own capital or obtain capital to begin a business. a business can be owned by a single individual in the form of a sole proprietorship or by a group of individuals in the form of partnership or corporation. managers are those who have the responsibility for conducting and supervising the operation of a business. producers perform the actual physical and mental labor necessary to produce the goods or services. consumers are the buyers of the goods and services produced by the business.10 domer et al.10 stated that there are five criteria that can be used to determine if someone is engaged in business: 1) the capital obtained, 2) the goods or service are produced and distributed to consumers, 3) willingness of the consumers to buy the goods or services, 4) the production, distribution and sale of goods or services results in profit and 5) the owner of a business is confronted with risks. legal forms of practice in the management literature, the legal form of practice is the synonymous of the form of business organizations. there are three types of legal forms of practice: 1) sole proprietorship, 2) partnership and 3) professional corporation young11 divided the mode of practices into 3 basic types: 1) independent solo practice (including associateships), 2) solo with facilities sharing, and 3) partnership-group of practice. but, domer et al.10 divided the mode of practices into 2 basic types: alone (including solo practice and expense sharing), and with others (including one other, general group and multispecialty group). the legal forms of practice are separate and distinct from the modes of practice. for example, the legal form of solo practice could be a sole proprietorship or cooperation with 2 dentists as stockholders even though one is not actively practicing dentistry. and the legal form of the group practice could be a partnership or corporation.10 the majority of dentists seem to practice alone (solo practice), and the predominant legal forms of their practice are the sole proprietorship.7,11 74 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 72–76 partnership in general, a partnership is 2 or more dentists agree to share the profit and loss of an enterprise in an unincorporated business. they pool their capital and labors, they share their patients, and they split the practice income and expenses according to a predetermined agreement.10,11 young11 stated that this type of legal practice is the most radical departures from the traditional practice. there have been a variety of definitions of this type of practice, but sarner cit. young11 has pointed out that the crucial characteristic of this method of organization is that the participants share income, facilities and expenses. some of the advantages of this partnership are the increase of the capital in beginning of the practice and the control is shared with the partner. moreover, the division of income often results in higher income than an individual proprietorship because of less downtime of the practice. one of the disadvantages is that the actions of one partner can influence on all other partners. for example, in a malpractice claims against one partner, the other partners could also be influenced by the public opinion and also could be held liability.10,11 corporation or a professional corporation this is a true business in which members of the corporation are members by virtue of their purchase of stocks. in this type of legal practice, members are usually opened for dentists, but sometimes it is also opened for someone who wants to invest his money on stocks. a professional corporation is a means to give a legal form of business to the controller of the business, which is separated from the owner with the controller of the business. therefore the difference between corporate and partnership is in the controller of the business.10 as was the partnership, a professional corporation has some advantages, for example: more capital available, the death or withdrawal of one of the members does not affect the life of the organization. while of the disadvantages of a corporate is that one must divide the profits of a corporation with the other stockholders.10 discussion as was mentioned above, dentistry or dental service delivery is becoming a business system or industrial model. this makes controversial opinions among dental profession and community, but the professional and business aspects of dentistry are a current reality of dental practice.2 regarding these various opinions, domer10 proposed four premises that can be used as the basic knowledge to discuss the professional and business aspects of dentistry in which health services tend to follow an industrial model. premise i: dentistry is a profession, premise ii: dentistry is a business, premise iii: professional and business aspects of dentistry are compatible and complementary, and premise iv: management is important to both professional and business aspects of dentistry. the first premise is that dentistry is a profession. referring to the 1st and 2nd professional characteristic of freidson8 mentioned above, any dentist as a profession should fulfill the 1st and 2nd qualifications of freidson’s. in this regard, the length of dental school curriculum and their focus on both scientific knowledge and skills indicate that dentistry fulfills the first two criteria of a true profession of freidson’s qualifications and also the wikipedia‘s definition of a profession as mentioned above. the existing mechanism for licensure and for the accreditation of dental schools reflects that dentistry should regulate itself and be free of lay control.6 woodall,12 freidson8 and jong6 stated that the criterion of autonomy differentiates a profession from a non-profession. professionals have the special privilege of freedom from the control of outside. however, the autonomy that is granted to the professions is a question that is continually raised by the consumers, the government and the third party such as insurance companies.5 the degree of self-regulation in the licensure of individual dentists and in the accreditation of dental schools reveals that dentistry also satisfies the 3rd and 4th criteria of a true profession of freidson’s qualifications and wikipedia’s definition of a profession. the services provided by individual should be professional reflects that services provided must be necessary to society. maintaining and improving oral health is critical to the maintenance and improvement of overall health and the quality of life.3,13 therefore, dentistry also satisfies the 5th criterion of a true profession of freidson’s qualifications and wikipedia’s definition of a profession. some people interpreted the 6th criterion literally that a professional should be wholly unconcerned with the business aspects and the financial rewards of providing services. the service criterion should not be interpreted literally.7 however, if dentists are unconcerned with the business aspects of dentistry they will be unable to provide necessary dental services to the public at a reasonable and affordable cost.7,14 the service orientation should be interpreted as meaning that individuals in profession have the most concern for the people who seek their services.12 from the discussion above it can be concluded therefore that dentistry is a profession, because it has been proved that dentistry fulfill all criteria of a profession. the 2nd premise proposed by domer10 is that dentistry is a business. the first criterion of business is the capitals obtain. any dental practice needs capital to purchase equipment and supplies, lease or buy an office, and to cover overhead costs for the first several months of its initial operation. although some dentists work for a salary and do not invest capital in the practice that employs them, the employers should invest capital at when the practice was first established.7 therefore, all dental practice fulfills this first criterion of business. the 2nd criterion of a business is that services are produced and distributed to consumers. it can happen that individuals could belong to more than one group. an owner 75sriyono: the business of dental practice may also be a manager and a producer, and a producer could also be a consumer.10 usually dentists are being the owner of the dental practice, and also they are in most being the manager and producers in the business of the dental practice. it was supported by young11 that most dental students chose dentistry because they want to “be one’s owns boss” (owner and manager) and to work with their hands (producers).15 therefore, dentists and their staffs therefore fulfill the 2nd criterion of a business willingness of consumers to buy the services or willingness of consumers to exchange money for goods or services is the 3rd criterion of a business. money is exchanged for the dental care received either directly from patients (out of pocket) or through the third party.14,16 it is clearly therefore, that dentistry fulfills the 3rd criterion of a business. the 4th criterion of a business is that the production, distribution and sales of goods or services result in a profit. profit is defined as the excess of revenue over the costs of operating a business.10, 16 although most dentists use the term net income rather than profit, for all practical purposes they do make a profit. the operation of most of dental practices does in fact result in a profit or net income for the dentists.10 dentists who invest in and operate a private practice certainly assume the risk of associated with the less of successful of the practice or it may completely fail or they face the risk of a business. or in other words, dentists should aware of the risk of a dental practice.6,10 the dental practice therefore fulfills the 5th criterion of a business that the owners of a business are confronted with financial risks. in addition to the five criterion of business mentioned above, silker14 makes a clear statement that private practice dentistry is a business just as banking or mcdonald’s or the grocery store. a dentist must efficiently run the practice as a business. however, silker14 warned that the more focus on achieving financial success (net income or profit); the more financial success evades the dentists. possibly the patients can feel when a dentist is sincerely and totally interested in what is best for them. if the dentist is more concerned with the business of dentistry than with the patient’s well being, the successful business of dental practice will fail. moreover, silker14 said that having a dental practice or starting a dental practice, a dentist should get as much business training and marketing courses as soon as the dentist can. from the presentation above it can be concluded that a dental practice is a profession and also a business. the professional and business aspects of dentistry are compatible and complementary is the 3rd premise of freidson’s.8 domer et al.10 compared the professional and business objectives of a dental practice. examination the professional objectives of a dentist and the commonly accepted objectives of a business showed that they are quite similar. professional and business objectives both include the following: 1) delivery of services that are needed and valued by the public, 2) continued survival and growth, 3) improvement of services through experimentation, the acquisition of new knowledge, and the development of a new process, 4) provision of a means for individuals and groups (customers, employers, suppliers, etc.) to satisfy their needs, 5) the provision of an economic service that results in equitable profit or income. if a dentist were to assume that the professional and business objectives of a practice were not identical, it would be impossible to achieve the objective of professional satisfactorily without achieving the objectives of business as well. for example, delivering high quality of dental care not only depends on the high skill of the dentist, but also depends on a cash flow and sufficient income to buy equipment and other supplies. on the other hand, it would be impossible to get satisfactory income in the long run if the public and profession didn’t not viewed the practice as being appropriate and of high quality.10,14 therefore, it is clear that the 3rd premise is fulfilled. however, many dentists feel uneasy to accept dentistry as both a profession and a business. traditionally, most dentists have not had sufficient training in running a business for a dental practice; they usually learn from their seniors how to run a practice. they usually prefer clinical aspects of dental works. young11 and domer et al.10 stated that many dentists and dental school do not value that dentistry as profession and as business is compatible. this value resulting in the dental curriculum of some dental schools, in which traditionally the dental curriculums have not included business management. premise iv is that management is important to both the professional and business aspects of dentistry. to operate a dental practice requires many resources, such as personnel, dental supplies, money, etc. obtaining and utilizing resources to achieve objectives is the universal goals of management.16 the experts of dental practice management stated that poor management might cause the practice objectives are not achieved well. possession of clinical knowledge and skills on the part of dentist and staff is not sufficient for the practice to achieve success. to create a successful practice, besides possessing highly clinical skill, a dentist must have a firm understanding of management concepts and apply them in their practice.10,14 indeed, the dental practice management can be learned through experiences in running or working in a private practice, however this approach is time consuming. sometimes it results in making unnecessary and costly mistakes. it is suggested therefore, to follow continuing education on dental practice management or spend some amount of money for consulting services relating to practice management.14 therefore, it can be concluded from the discussion above that management is important to the both professional and business aspect of dentistry. the business aspects of dentistry are a current reality.1,2 actually, young7 has identified the trend of the business of dentistry two decades ago. young7 stated that there have been an increasing number of the legal practice (group practice and a professional corporation) in last decade, or the number of “department store” or “shopping mall” of 76 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 72–76 dental practices, which are often franchised or operated on a contract with a parent company doing business in one or more states in america. meaning that the dental practice is managed using a business system. it was mentioned above that the professional corporation is a true business. the development of the professional corporation of the dental practice can be identified by the great number of a professional corporation that is advertised worldwide through internet. for example the medical tourism: globalization.17 advertised that the group of medical and dental practices in some thailand’s international hospitals provides complete services for international patient clients with 26 languages. it is also interesting to note, that the advertisement also provides information that the services have 20% lower cost for some medical and dental care compare with the service cost in usa. the underlying of this advertisement is that the medical and dental services have a similarity in selling the services just like a department store or a shopping mall in giving the price discount in selling their products. regarding the trend of the development of a corporate practice, molin and molin18 stated that dentist must know about the secret agenda of the managed care industry, insurance companies and corporate dental conglomerates in future. steele19 mentioned that dentists are currently in an unprecedented age of prosperity in the dental industry, many practices are enjoying heightened profit. however, steele19 said that prosperity can be dangerous, if it leads to complacency, because dentists should always concern with the patient’s well being not only concern with the business of dentistry14. regarding the discussion above and the current reality that the delivery of dental practice to follow a business system, it is suggested therefore to give the undergraduate students the basic concepts of dental practice management by putting this in the curriculum. it is also important to stress dental students and dentists to understand the basic criteria that enable dentistry to be considered a profession and continually to fulfill that criteria mentioned above. dentistry have to continue the service orientation and to continue developing appropriate regulatory mechanisms, otherwise, outsider groups will increase their effort to modify the dental care delivery systems. as a result of this, dentistry as profession might possibly be destroyed.10,12 in addition to these, although the jobs for dentists in indonesia are still promising, dentists and also the students of dentistry should aware and prepare them in facing these globalization and afta. it can be concluded that the profession and the business of dentistry are compatible and complementary. the delivery of the dental services tends to follow the industrial model, which is a current reality. dentist should concern more on the delivering high quality of dental services, not only focus on the business of the practice, although the satisfactory income is important for the survival growth of the practice in the long run. facing the globalization, besides possessing high clinical skill, dentists must have a firm understanding of management concepts and apply them in their practice. it is suggested for dentists to follow as much business training or dental practice management training as the dentist can. it is also suggested to give the undergraduate students the basic concepts of dental practice management by putting this in the curriculum. dental students and dentists should always realize the importance of continually to fulfill the basic criteria of a profession that enable dentistry always to be considered as a profession, otherwise outsider groups will increase their effort to modify the dental care delivery systems. as a result of this, dentistry as profession might possibly be destroyed. references 1. who. general agreement on trade in services (gats). available at: www.who.int.entity/trade/glosary/story033/en/index.html. accessed april 24, 2006. 2. hamid a. implications of globalization and multilateral on healthcare services. berita akademi 2001; 10(4):1–4. 3. who. oral health for the 21st century, who/orh/oral 21.94.1. dist. general english only, geneva. 1994. p. 11–12. 4. sriyono nw. dental practice management, establishing a dental practice. kumpulan makalah ceramah ilmiah dan poster ilmiah, peringatan 6th pendidikan dokter gigi universitas jember; 2001. h. 320–9. 5. wikipedia. profession. available at: http://en.wikipedia.org/wiki/ profession. accessed april 4, 2006. 6. jong aw. community dental health. 3rd ed. st louis: mosby-year book, inc; 1993. p. 299, 305, 307. 7. young wo. the social setting of dental health practice. in: striffler, df, young wo, burt ba. dentistry, dental practice & the community. 3rd ed. sydney: wb saunders company; 1983. p. 8–19, 28–29. 8. freidson e. profession of medicine: a study of the sociology of applied knowledge. new york: dodd, mead co; 1975. p. 135–8. 9. broom hn, longenecker jg. small business management. cincinati: south-western publishing co; 1971. p. 20. 10. domer lr, synder tl, heid dw. dental practice management, concept and application. st louis: the cv mosby company; 1980. p. 3–12, 64–65. 11. young wo. career perspectives. in: striffler df, young wo, burt b. dentistry, dental practice and the community. 3rd ed. sydney: wb saunders company; 1983. p. 45–52. 12. woodall ir. legal, ethical, and management aspects of the dental care system. 2nd ed. st louis: the cv mosby co; 1983. p. 40–42, 48–49, 81–98. 13. sheiham a. oral health, general health and quality of life. bulletin of the who, past issues, 2005; 83(9):641–720. 14. silker el. dentistry, building your million dollar solo practice. minnesota: silk pages publishing; 1995. p. 13–19, 288–9, 296–8. 15. kress gc. the impact of professional education on the performance of dentists. in: cohen lk, bryant ps. social sciences and dentistry, a critical bibliography. volume ii. london: quintessence publishing company, ltd; 1984. pp. 323, 328. 16. azwar a. pengantar admisistrasi kesehatan. cetakan pertama. jakarta: binarupa aksara; 1996. h. 15–16, 124–5. 17. anonym. medical tourism: globalization. available at: http:// www.csmngt.co/medical2.htm. accessed april 23, 2006. 18. molin dj, molin sd. the future of dentistry. available at: http:// proquest.com/pqdweb. accessed may 8, 2006. 19. steele t. the gold watch. available at: http://www.tysonsteele.com/ n-articles.phtml. accessed may 8, 2006. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 150 volume 45 number 3 september 2012 novel development of carbonate apatite-chitosan scaffolds based on lyophilization technique for bone tissue engineering maretaningtias dwi ariani department of prosthodontics faculty of dentistry, universitas airlangga surabaya indonesia abstract background: the natural biopolymer chitosan (ch) is currently regarded as a candidate for bone tissue engineering. however, ch is poor for cell adhesion and low bone formation ability. in order to enhance cell adhesion and bone formation ability, combination of ch with carbonate apatite (ca) was developed. purpose: the aim of this study was to make carbonate apatite-chitosan scaffolds (cachss) and evaluate its osteoconductivity in terms of cell proliferation. methods: chitosan scaffolds (chss) were made by the following procedure. twenty-five, 50, 100, 200 and 400 mg ch was dissolved into 5 ml of 2% acetic acid (ch3cooh), shaked for 15 min and neutralized with 15 ml of 0.1 m sodium hydroxide (naoh) solution. after centrifugation, ch gel was packed into the molds then frozen at -80°c for 2h and dried in a freeze dry machine for 24h. the sponges were subjected to uv radiation for 2h. to make ca-chss, 200 mg ch was selected. after neutralization, 50 mg of 0.06 m ca were added into the 200 mg ch gel. the structure of ca-chss was observed by scanning electron microscope (sem). mouse osteoblast-like cell (mc3t3-e1) proliferation in these scaffolds was investigated at 1, 7, 14 and 21 days. results: three dimensional porous structures of ca-chss were clearly observed by sem. proliferated cell numbers in ca-chss was significantly higher than those in chss (control) at each stage (p<0.05). conclusion: it can be concluded that newly developed ca-chss had three-dimensional interconnected porous structure, good handling property and supporting ability of proliferation of osteoblasts. it is suggested that newly developed ca-chss could be considered as a scaffolds material for bone tissue enginearing. key words: carbonate apatite, chitosan, scaffolds, bone tissue engineering abstrak latar belakang: kitosan yang merupakan biopolimer alami dianggap sebagai salah satu kandidat untuk rekayasa jaringan tulang. namun, kitosan memiliki kelemahan terhadap adhesi sel dan kurang mampu membentuk tulang yang cukup. untuk meningkatkan adhesi sel dan kemampuan pembentukan tulang, telah dikembangkan suatu scaffolds yang menggabungkan kitosan dengan carbonate apatite (ca). tujuan: penelitian ini bertujuan untuk membuat carbonate apatite-chitosan scaffolds (ca-chss) serta mengevaluasi osteokonduktivitas ca-chss dari sudut pandang proliferasi sel. metode: chitosan scaffolds (chss) dibuat dengan prosedur berikut ini. dua puluh lima, 50, 100, 200 dan 400 mg bubuk kitosan dilarutkan dalam 5 ml asam asetat (ch3cooh) 2%, dikocok selama 15 menit dan dinetralkan dengan 15 ml 0,1 m larutan sodium hidroksia (naoh). setelah sentrifugasi, gel kitosan dikemas ke dalam cetakan teflon kemudian dibekukan pada suhu -80°c selama 2 jam dan dikeringkan dalam mesin beku kering pada suhu -54°c selama 24 jam. selanjutnya dilakukan radiasi ultraviolet pada chss selama 2 jam. untuk membuat ca-chss, dipilih chss yang berisi 200 mg bubuk kitosan. setelah dinetralisir, 50 mg dari 0.06 m ca ditambahkan ke dalam kitosan gel yang berisi 200 mg bubuk kitosan. struktur ca-chss diamati dengan scanning electron microscope (sem). proliferasi mouse osteoblast-like cell (mc3t3-e1) dalam chss dan ca-chss dievaluasi pada hari ke-1, 7 dan 14. hasil: ca-chss dengan struktur tiga dimensi yang berpori dapat diamati dengan jelas menggunakan sem. jumlah pertumbuhan dan perkembangan sel pada ca-chss secara signifikan lebih banyak dibandingkan pada chss (kontrol) pada setiap tahap pengamatan di hari ke-1, 7, 14 dan 21 (p<0.05). kesimpulan: dapat disimpulkan bahwa caresearch report 151ariani: novel development of carbonate apatite-chitosan scaffolds introduction in order to reconstruct damaged bone tissue because of trauma or pathologic disease, tissue engineering is needed as an emerging technology.1 various kinds of bone substitutes have been developed to replace bone defect. autogenous bone graft (autograft) is the most effective bone substitute. however, autograft sometimes has significant limitations coming from donor site morbidity, a limited donor bone supply and an inadequate resorption rate during the healing process.2,3 these limitations have prompted increasing interest in alternative bone substitute. allograft as an alternative offers the same characteristics as an autograft, but have several problems such as the risk of disease transmission, immunogenicity, loss of biologic and mechanical properties, and religious concerns.4,5 consequently, significant efforts are being made to develop an ideal bone substitute. i d e a l l y s y n t h e t i c b o n e s u b s t i t u t e s s h o u l d b e biocompatible, show minimal fibrotic reaction, undergo remodeling and support new bone formation.4 scaffolds play an essential role in supporting bone substitute. recently, significant attention is being given to threedimensional polymer scaffolds for in vitro study of cellscaffolds interaction and in vivo study of bone substitute.6-8 in recent years, one promising tissue engineering strategy has been given to natural polymer chitosan (ch) because of its properties. ch, an amino polysaccharide (poly-1,4-d-glucossamine), is the alkaline deacetylated product of chitin that can be extracted from crustacean. it is biocompatible, biodegradable, renewable, non-toxic, can be fabricated into various forms, has been shown to support the attachment and growth of osteoblasts in vitro and has been widely applied in biomedicine.9-11 furthermore, as a foreign object, ch is not rejected from the body.9 however, ch is relatively weak and unstable and swells in solution. pure ch scaffolds (chss) is easily absorbed and difficult to control absorbance time period. to make suitable chss for bone tissue engineering, current attempts are focused on improving the mechanical strength and biological properties through the incorporation of bio ceramics, such as carbonate apatite (ca). in the field of hard tissue repair and regeneration, ca has been used as a biocompatible and osteoconductive material because of its similarity to inorganic component of hard tissue.12 it has been reported that the main inorganic content of bone is ca, which contains about 7% carbonate by weight. ca is easier to dissolve because the solubility increased as the content of carbonate in ca increased, and thermodynamically under neutral and basic condition. therefore ca is expected to become an ideal bone replacement material, which possesses both osseoconductivity and bioresorbability.13,14 in this study, ca was chose to combine with ch as a biodegradable material in order to overcome their mechanical limitation and maximize the beneficial properties of each and create a biodegradable scaffolds.15 therefore, the purpose of this study was to make carbonate apatite-chitosan scaffolds (ca-chss) based on lyophilization technique and evaluate their microstructure and cell proliferation-conductivity for bone tissue engineering. materials and methods cylinder chss were made by using various amounts of ch powder provided by ysk (98.7% deacetylation, yaizu suisankagaku industry co., ltd., japan). the procedures were based on previous study.16 acetic acid (ch3cooh) was selected as the solvent for ch powder and sodium hydroxide (naoh) was used for neutralization. chss were fabricated by the following procedure. first, 25, 50, 100, 200 and 400 mg of ch powder was dissolved in 5 ml of ch3cooh at room temperature, shaken for 15 min, neutralized with 5 ml of naoh solution, and then centrifuged at 1500 rpm for 10 min to prepare ch gels. the ch gels were obtained after the removal of excess water and were packed into cylindrical molds (diameter: 5 mm, height: 2 mm). the molds were frozen at -80°c for 2h and transferred into a freeze-drying machine (fd 700d, tokyo rikakikai co., ltd, tokyo, japan). cylindrical chss were obtained after drying at -54°c for 24hr and exposure to uv radiation for 2hr. the production procedures of ca-chss were based on previous study.16 firstly, 200 mg ch powder was dissolved in 5 ml ch3cooh at room temperature, shaken for 15 min and neutralized with 15 ml naoh solution to obtain ch gels. 50 mg of ca was then homogenously mixed with ch gels and centrifuged at 1500 rpm for 10 min to prepare ca-ch gels. after the removal of excess water, ca-ch gels were transferred into cylindrical molds (diameter: 5 mm, height: 2 mm), frozen at -80°c for 2hr and transferred into the freeze-drying machine. cylindrical ca-chss were obtained after drying at -54°c for 24hr and exposure to uv radiation for 2hr. chss mempunyai struktur tiga dimensi dengan pori-pori yang saling berhubungan satu sama lain dan dapat meningkatkan proliferasi osteoblast. hal ini menunjukkan bahwa ca-chss adalah kandidat untuk rekayasa jaringan tulang. kata kunci: carbonate apatite, kitosan, scaffolds, rekayasa jaringan tulang correspondence: maretaningtias dwi ariani, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: etaprosto@yahoo.com 152 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 150–155 macroscopically images of chss and ca-chss were obtained by a digital camera (60d, canon, japan). the microscopic structure and porosity of the chss and cachss were analyzed using a scanning electron microscope (sem) (3d microscope ve-8800, keyence, japan). mc3t3-e1 cells were cultivated on a tissue culture polystyrene dish using dmem supplemented with 10% fetal bovine serum (fbs), 100 u penicillin, 0.1 mg/ml streptomycin and 2 mm l-glutamine. the cells were sub cultured every 7 days for two times. mc3t3-e1 cells were harvested by trypsinization and suspended in dmem containing 10% fbs, 100 u penicillin, 0.1 mg/ml streptomycin and 2 mm l-glutamine. then, cell suspensions (20 µl) were added to chss and ca-chss placed in each well of a 24-well tissue culture plate to the density of 2 √ 104 cells. after 2hr, 980 µl of the medium was added to each well. the cells were incubated in an incubator under 5% co2 at 37°c for 1, 7, 14 and 21 days. the culture medium was changed every 3 days. the number of living cells was determined by mtt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays using cell counting kit-8 (dojindo molecular technologies, inc., kumamoto, japan). chss and cachss were washed with pbs and incubated for 2h in 500 µl of culture medium containing 50 µl mtt reagents in an incubator under 5% co2 at 37 oc. the supernatant medium (110 µl) was transferred into a 96-well culture plate and the absorbance was measured using a micro plate reader (550, biorad) at 450 nm wavelength. data of cell proliferation were statistically analyzed using spss® software (version 12.0). to evaluate the differences in cell proliferation among experimental groups, a b c d e figure 1. macroscopical shape of chss prepared with different amount of ch powders (ø: 5.0 mm). ch powder used (mg) a: 25; b: 50; c: 100; d: 200; e: 400. a, b and c were brittle. on the other hand, d and e showed good handling property. a b 100 µm 100 µm figure 2. sem images of chss prepared with different amount of ch powders. ch powder used (mg) a: 200; b: 400. (a) shows three-dimensional structure with many pores. on the other hand, (b) shows three-dimensional structure with few pores. 153ariani: novel development of carbonate apatite-chitosan scaffolds one-way analysis of variance (anova) with tukey's post hoc test (p<0.05) was performed and data are presented as the mean ± standard deviation. results macroscopic images of chss are shown in figure 1. chss prepared with 25, 50 or 100 mg ch powders were brittle and could not maintain their own shapes (figure 1a, b, c). in contrast, chss prepared with 200 and 400 mg ch powders could maintain their shapes with good handling properties (figure 1d, e). as shown by sem images (figure 2a), chss prepared with 200 mg ch powder showed three-dimensional structure with many pores. on the other hand, chss prepared with 400 mg ch powder exhibited three-dimensional structure with few pores (figure 2b). this result suggests chss prepared with 200 mg ch powder was a possible candidate. however, pure chss was easily absorbed and difficult to control absorbance time period. as shown in fig. 3 we could observe threedimensional structure with many pores both of chss and ca-chss. the pore size of the scaffolds was approximately 60 µm. in general, ca-chss prepared with 50 mg ca showed three-dimensional structure and attachment of ca powder (figure 3b). the results of cell proliferation assay on the chss and ca-chss are shown in figure 4. the absorbance values for ca-chss containing 50 mg of ca were significantly higher than those of chss at all-time points (day 1: chss: 0.364 ± 0.02, ca-chss: 0.378 ± 0.04; day 7: chss: 0.548 ± 0.06, ca-chss: 0.594 ± 0.07; day 14: chss: 0.503 ± 0.05, ca-chss: 0.631 ± 0.08; day 21: chss: 0.720 ± 0.09, ca-chss: 0.814 ± 1.01) (p < 0.05). chss ca-chss 100 µm 100 µm chss ca-chss 100 µm 100 µm figure 3. sem images of chss prepared with 200 mg ch powder and ca-chss prepared with 50 mg ca powder. days 0,364 0,378 0,548 0,594 0,503 0,631 0,720 0,814 o p ti ca l d en si ty ( o d ) figure 4. cell proliferation on chss and ca-chss, analyzed by the mtt assay. ca-chss prepared with 50 mg ca powder showed an increasing tendency in the absorbance values on the first day of observation until day 21 (p<0.05) (n=6). 154 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 150–155 discussion in bone tissue engineering, scaffolds play an important role in supporting bone regeneration. to date, many attempts have been made to develop three-dimensional scaffolds that provide necessary support as artificial extracellular matrices, allowing cells to proliferate, differentiate and maintain their functions.1,10 prerequisites of scaffolds include non-toxicity, controllable biodegradability, suitable microstructure and appropriate mechanical properties.1,2,8 additionally, they must be capable of promoting cell adhesion and retaining their functions.5 in the present study, combination of ch with ca was developed. firstly, to decide optimal concentration of ch powder. chss with several amounts of ch powder were evaluated by sem in terms of porosity. based on sem images, chss prepared with 200 mg ch powder had a good handling property and three-dimensional porous structure with pore size was approximately 50–200 µm. these findings may mean space-making ability of this chss. it was suggested that an optimal content of ch powder was 200 mg. current attempts are focused on improving the osteoconductivity and bone formation ability of chss through the incorporation of calcium phosphate, such as hydroxyapatite (ha), b-tricalcium phosphate (b-tcp) and carbonate apatite (ca).11,16 ha and b-tcp have drawbacks such as their degradation or dissolution rates are difficult to be predicted. highly sintered ha is non-degradable. however, it has been reported that interconnected porous calcium hydroxyapatite (ip/cha) composites had a systematic arrangement of uniform pores and almost all pores were interconnected and it had a good bone formation ability in vivo study. on the other hand, crystallinity of b-tcp is too low, so it degrades too fast. in some cases, this material absorbs before obtaining enough new bone formation. among these calcium phosphates, ca has been reported to have a proper absorbance time and good bone formation ability. however, ca also has limitations for use as a scaffold because of difficulties in shaping and designing for bone tissue engineering.12-14,17,18 in this study, ch as a biodegradable material and ca was chose to combine with ch in order to get a proper absorbance time period and enhance bone formation ability. the characteristics of scaffolds depend on the fabrication method. several techniques have been developed to fabricate scaffolds, including solvent-casting technique, solgel technique and lyophilization technique.19,20 however, it has been reported that in the solvent-casting technique, the existence of a pyrogen salt harmfully affects cells because of the loss of water-soluble biomolecules and the induction of non-uniform deformation. 21,22 in the sol-gel technique, difficulties have been reported for the control of the precise pore structure and the separate completion of hydrolysis and condensation.19,21 in this study, a lyophilization technique was adopted to fabricate scaffolds, because this technique is simpler and easier than other methods previously described and by using lyophilization technique, porous properties of ca-chss were provided by ice removal and varied freezing rate.16 nowdays, one promising tissue engineering strategy is to use three-dimensional porous structure and biodegradable scaffolds to facilitate bone tissue regeneration. in this study, ca-chss were fabricated with favorable three-dimensional porous structures and had a good handling property with approximately 50–200 µm pore sizes. as scaffolds for tissue engineering should have porous structures with pore size ranging from 40 to 300 µm to allow tissue ingrowth and migration of vascular tissues.23,24 these three-dimensional materials are suitable for cell and vascularization including growth factors. when considering to use ca-chss for clinical applications, such as bone augmentation at buccal defect of implant treatment or to fill bone socket after tooth extraction, ca-chss must be sterilized prior to use. regarding sterilization, gamma irradiation, steam autoclaving, ethylene oxide and radio frequency glow discharge plasma sterilization methods have been used. because, ca-chss is an organic polymer composite, it is considered that gamma irradiation is appropriate for cachss sterilization in order to maintain chemical structure of this material. after the development of an adequate porous structure, the choice of reliable source of cells has an influence on the success of tissue engineering. in this study, mc3t3e1 cells were cultured on ca-chss for cell proliferation measurement. for bone tissue engineering applications, osteoblasts are commonly used to confirm responsibility for the bone formation.25,26 from the findings of cell proliferation assays, it was shown that by combining chss with ca powder, higher cell proliferation ability was observed compared with that of only chss. it might be suggested that the addition of ca powder into chss serves to increase surface area of the materials which is available for cells to adhere. furthermore, ca-chss prepared from 200 mg ch powder with 50 mg ca powder showed significantly higher cell proliferation ability than those of other groups. it was suggested that it had a favorable three-dimensional porous structure and adequate surface area of the material for bone tissue engineering. further histological study in bone regeneration on ca-chss should be warranted to ensure the possibility of scaffolds of ca-chss. it can be concluded that newly developed ca-chss had three-dimensional interconnected porous structure, good handling property and supporting ability of proliferation of osteoblasts. based on the limited results of this study, it is suggested that newly developed ca-chss may be a possible scaffolds material for bone tissue engineering. 155ariani: novel development of carbonate apatite-chitosan scaffolds references 1. chuna hj, kimb gw, kimb ch. fabrication of porous chitosan scaffold in order to improve biocompatibility. j phys chem of solids 2008; 69: 1573–6. 2. ikada y. challenges in tissue engineering. j r soc interface 2006; 3: 589–601. 3. kenar h, kose gt, hasirci v. tissue engineering of bone on micro patterned biodegradable polyester films. biomaterials 2006; 27: 885–95. 4. pa r i k h sn. bone gra ft substit utes: past, present, f ut ure. j postgraduate master 2002; 48: 142–8. 5. chiara g, letizia f, lorenzo f, edoardo s, diego s, stefano s, eribeto b. nanostructured biomaterials for tissue engineered bone tissue reconstruction. int j mol sci 2012; 13: 737–57. 6. muzzarelli c, muzzarelli ra. natural and artificial chitosaninorganic composites. j inorg biochem 2002; 92(2): 89–94. 7. giannoudis pv, dinopoulos h, tsiridis e. bone substitutes: an update. int j care injured 2005; 36: 20–7. 8. venkatesan j, se-kwon k. chitosan composites for bone tissue engineering-an overview. mar drugs 2010; 8: 2252–66. 9. li z, ramay hr, hauch kd, xiao d, zhang m. chitosan-alginate hybrid scaffolds for bone tissue engineering. biomaterials 2005; 26: 3919–25. 10. kong l, gao y, cao w, gong y, zhao n, zhang x. preparation and characterization of nano-hydroxyapatite/chitosan composite scaffolds. j biomed mater res 2005; 75a: 275–80. 11. chesnutt bm, viano am, yuan y, yang y, guda t, appleford m, ong jl, haggard wo, bumgardner jd. design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration. j biomed mater res 2009; 88a: 491–6. 12. william lm, david jm. bioinspired growth of crystalline carbonate apatite on biodegradable polymer substrata. j am chem soc 2002; 124: 1910–7. 13. legeros rz. calcium phosphate-based osteoinductive materials. chem rev 2008; 108: 4742–53. 14. daitou f, marutai m, kawachi g, tsuru k, matsuya s, terada y, ishikawa k. fabrication of carbonate apatite block based on internal dissolution precipitation reaction of dicalcium phosphate and calcium carbonate. dent mater j 2010; 29(3): 303–8. 15. shen x, tong h, jiang t, zhu z, wan p, hu j. homogeneous chitosan/carbonate apatite/citric acid nanocomposites prepared through a novel in situ precipitation method. composites science and technology 2007; 67: 2238–45. 16. matsuura a, kubo t, doi k, hayashi k, morita k, yokota r, hayashi h, akagawa y. bone formation ability of carbonate apatite-collagen scaffolds with different carbonate contents. dent mater j 2009; 28: 234–42. 17. doi k, kubo t, hayashi k, imura k, akagawa y. development of cell-hybrid artificial bone: effect of osteogenic differentiation of bone marrow stromal stem cells on bone formation with newly developed interconnected porous calcium hydroxyapatite. dent mater j 2007; 26: 162–9. 18. saikia kc, bhattacharya td, bhuyan sk, talukdar dj, saikia sp, jitesh p. calcium phosphate ceramics as bone graft substitutes in filling bone tumor defects. indian j orthop 2008; 42: 169–72. 19. sachlos e, czernuszka jt. making tissue engineering scaffolds work: review on the application of solid free form fabrication technology to the production of tissue engineering scaffolds. eur cell mater 2003; 5: 29–40. 20. khang g, kim ms, lee hb. a manual for biomaterials/scaffold fabrication technology. tissue eng reg med 2006; 3: 376–95. 21. jones jr, hench lr. factors affecting the structure and properties of bioactive foam scaffolds for tissue engineering. j biomed mater res 2004; 68: 36–44. 22. ma px. biomimetic materials for tissue engineering. adv drug deliv rev 2008; 60: 184–98. 23. arpornmaeklong p, suwatwirote n, pripatnanont p, oungbho k. growth and differentiation of mouse osteoblasts on chitosan collagen sponges. int j oral maxillofac surg 2007; 36: 328–37. 24. wang j, de boer j, de groot k. proliferation and differentiation of mc3t3-e1 cells on calcium phosphate/chitosan coatings. j dent res 2008; 87: 650–4. 25. bacakova l, filova e, rypacek, svorcik v, stary v. cell adhesion on artificial materials for tissue engineering. physiol res 2004; 53: 35–45. 26. costa-pinto ar, reis rl, neves nm. scaffolds based bone tissue engineering: the role of chitosan. tissue eng 2011; 5: 331–47. mkgs vol 44 no 1 jan-mar 2011.indd 1 vol. 44. no. 1 march 2011 research report posterior transverse interarch discrepancy on hbe β thalassemia patients yuniar zen1 and loes d. sjahruddin2 1department of orthodontic 2department of pediatric dentistry faculty of dentistry, trisakti university jakarta indonesia abstract background: one of the symptoms that often arises on thalassemia patients is disharmony dentofacial, class ii skeletal malocclusion, as a result of the malrelation of maxilla and mandible. this malrelation can be affected by either maxillary bone position, dentoalveolar maxillary position, mandibular bone position, dentoalveolar mandibular position, or combinations of those components. purpose: the study was aimed to examine whether there is posterior transverse interarch discrepancy on the hbe β thalassemia patients or not. methods: this study is an observational research with cross-sectional design. the sample consisted of 33 hbe β thalassemia patients and 33 non-thalassemia patients as a control group aged 12–14 years. lateral cephalogram was carried out and dental casts of maxillary and mandibular dental arches were also taken in all of those patients. results: there was no difference between the maxillary intermolar width of the hbe β thalassemia patients and that of the normal ones, but the mandibular intermolar width of the hbe β thalassemia patients was significantly smaller than that of the normal ones. beside that, posterior transverse interarch discrepancy of of the hbe β thalassemia patients was significantly greater than that of the normal ones, which showed great difference between maxillary and mandibular intermolar widths. conclusion: posterior transverse interarch discrepancy of the hbe β thalassemia patients was different from that of the normal ones. the dentofacial abnormalities on the hbe β thalassemia patients aged 12–14 years primarily was due to disporposional dentofacial growth in the vertical, sagittal, and transversal directions, especially in the posterior region. key words: hbe β thalassemia, dentofacial disharmony, interarch discrepancy abstrak latar belakang: salah satu akibat yang sering timbul pada penderita talasemia adalah disharmoni dentofasial berupa maloklusi skeletal kelas ii yang merupakan kelainan hubungan maksila dan mandibula. malrelasi ini dapat dipengaruhi oleh posisi maksila, posisi dentoalveolar maksila, posisi mandibula, dan posisi dentoalveolar mandibula atau kombinasi komponen ini dalam banyak variasi. tujuan: penelitian ini adalah untuk melihat apakah ada diskrepansi antar rahang arah tranversal di regio posterior pada penderita talasemia beta hbe. metode: penelitian ini adalah penelitian observasional dengan disain potong lintang. sampel terdiri atas 33 penderita talasemia beta hbe dan 33 subjek normal (non talasemia) usia 12–14 tahun. dilakukan pengambilan foto sefalogram lateral dan pembuatan model studi gigi ra dan rb pada semua subjek penelitian. hasil: jarak intermolar maksila tidak berbeda dengan subjek normal, namun jarak intermolar mandibula lebih kecil secara bermakna dibandingkan dengan subjek normal. selain itu, diskrepansi antar rahang dalam arah transversal di regio posterior lebih besar secara bermakna dibanding subjek normal, yang menunjukkan besarnya selisih jarak antara jarak intermolar maksila dan jarak intermolar mandibula. kesimpulan: diskrepansi antar rahang arah transversal di regio posterior antara penderita talasemia beta hbe dengan subjek normal usia 12–14 tahun. kelainan 2 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 1–6 dentofasial pada penderita talasemia beta hbe usia 12–14 tahun disebabkan oleh pertumbuhan disporposional dentofasial berbeda arah vertikal, sagital dan transversal terutama di regio posterior. kata kunci: talasemia beta hbe, disharmoni dentofasial, diskrepansi antar rahang correspondence: yuniar zen, c/o: bagian ortodonti, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa grogol jakarta 11440, indonesia. e-mail: zenyuniar@yahoo.co.id. introduction disharmony of dentofacial component growth may cause chewing function problem and disharmony. one of the diseases which can cause the dentofacial disharmony is thalassemia.1 based on data derived from a variety of major hospitals and educational centers, indonesia is a country with quite high frequency of thalassemia cases, namely between 3–8%, which means that 3 to 8 people out of 100 indonesia has thalassemia genes.2 patients with thalassemia often get spacing/diastema and protrusion of maxillary anterior teeth, as a result, it may be an indication of orthodontic treatment.3 in a study of hbe β thalassemia patients in jakarta, it was known that the rate of dentofacial disharmony or class ii skeletal malocclusion was quite high, which is about 90.6%.2,4 hbe β thalassemia is a disease of genetically inherited blood disorder caused by disturbances of hemoglobin formation. on hbe β thalassemia patients, it was found that there was growth problems, one of which was skeletal growth retardation. in severe cases, the growth problems that occur particularly on dentofacial bone even lead to distinctive facial abnormalities, called as facies cooley. in severe circumstances, besides causing distinctive facial abnormalities, it will also cause the disruption of chewing and talking functions. later, it can lead to feeling of inferiority, which eventually becomes a psychological burden on a thalassemia patients.4 the result of cephalometric research, shows that dentoskeletal deformity in patients with thalassemia is generally caused by the retardation and disproportion of dentoskeletal components.5 it then leads to class ii skeletal malocclusion caused by the retardation of mandibular growth and the retrognati of mandibular position.2,4 class ii skeletal malocclusion is actually a malocclusion with maxillary and mandibular malrelations. more than 60% of this malocclusion case are caused by mandibular discrepancy in the sagittal direction, which was inclined more to the distal mandibular position against maxilla.6 this malrelation can also be caused by either the maxillary bone position, maxillary dentoalveolar position, mandibular bone position, mandibular dentoalveolar position, or combinations of these components in many varieties.7 class ii malocclusion is usually characterized by a convex facial profile and a large overjet, even not rarely accompanied also with deepbite. in such condition, the pressure of the facial muscles and tongue become abnormal, as a result, there is often deep mentolabial groove, often called as lip trap. this such description of class ii malocclusion, thus, usually encourage patients or their parents to obtain orthodontic treatment.8 in orthodontic treatment, a complete examination and accurate data are required to diagnose, including clinical examination, modeling study analysis, and cephalometric analysis.10 an examination of jaw in transversal direction must be conducted in class ii malocclusion since disporposional jaw growth in the sagittal direction will affect the growth of jaw in the transversal direction.11 the size of tranversal interarch discrepancy in the posterior region then will assist both in establishing the diagnosis and in determining the class ii malocclusion treatment.12 untreated class ii malocclusion without posterior transverse interarch discrepancy in mixed dentition will become class ii malocclusion with posterior transverse interarch discrepancy in its development.11,12 therefore, this study is aimed to examine if there is posterior transverse interarch discrepancy on the hbe β thalassemia patients aged 12–14 years old compared to that on non thalassemia patients. the result of this study is then expected to be able to provide information about posterior transverse interarch discrepancy, as a result, a diagnosis of class ii malocclusion in hbe β thalassemia patients will be more easily conducted. thus, the determination of treatment plan and treatment time do not only become more accurate, but the determination of class ii malocclusion prognosis on hbe β thalassemia patients will also become easier. materials and methods this study is an observational research with crosssectional design. the samples of this study were hbe β thalassemia patients aged 12–14 years old who were routinely treated in the thalassemia clinical center of child health, medical faculty of ui/rscm jakarta. however, those children must also meet several inclusion criteria and must be willing to join the study by signing an informed consent. the inclusion criteria are that both 12–14 year old men and women suffer with hbe β thalassemia (defined by a pediatrician) and have no interdental caries. on the other side, the selection of the control group (non-thalassemia) in this study is adjusted to its equivalent aged 12–14 years. similarly, non-thalassemia patients classified into a control group must also meet some criteria,which are; with first molar teeth that are still intact, no interdental caries, with 3zen and sjahruddin: posterior transverse interarch discrepancy class i molar relationship with distobuccal cusp of the first mandibular molar located on the central fossa of the first maxillary molar.11 figure 1. maxillary intermolar distance.11 figure 2. mandibular intermolar distance.11 in this study, parameters were measured by using study model and cephalogram. in the study model, maxillary intermolar distance (figure 1) and mandibular intermolar distance (figure 2) were measured. the difference between maxillary intermolar distance and mandibular intermolar distance was then defined as posterior transverse interarch discrepancy (ptid).11 figure 3. cephalometric analysis.11 cephalometric analysis (figure 3) was conducted, in which anterior-posterior relations were measured, such as maxilla in the sagittal direction (sna angle), mandible in the sagittal direction (snb angle), maxillary-mandibular discrepancy in the sagittal direction (anb angle), glenoid fossa position in the sagittal direction (ns-tm angle), the vertical relations of the maxillary inclination relative to the cranial base (nl/nsl angle), mandibular inclination relative to the cranial base (ml/nsl angle), vertical maxillary-mandibular relations (nl/ml angle) and gonial angle (ar-go-me angle). mandibular dimension involves mandibular length (go-pg), mandibular ramus length (cogo), and total mandibular length (co-pg). results there are thirty-three samples of hbe β thalassemia aged 12–14 years and 33 samples of non-thalassemia individuals aged 12–14 years who met the inclusion criteria distributed (table 1). table 1. number of sample based on sex sex thalassemia normal men 16 16 women 17 17 besides lateral cephalogram was carried out for each subject of this study, both dental casts of maxillary and mandibular dental arches as well as study model were also taken in all of those patients. statistical test was also conducted in order to know the difference between variables of the thalassemia group and those of the non thalassemia group (table 2). the results of the test then showed that there was significant difference on sna angle, angle ns-tm, mandibular ramus length, and maxilla intermolar distance (p < 0.05). discussion the results of observing dentofasial components with cephalometric analysis showed that there was disproportional growth in the vertical direction on the hbe β thalassemia patients indicated by the width of nl/nsl angle, ml/nsl angle, nl/ml angle, and ar-go-me angle. the width of nl/nsl angle indicated that the relative maxillary inclination on the hbe β thalassemia patients towards the cranial base was significantly smaller than that on the normal ones, while the width of ml/nsl angle showed that the relative mandibular inclination towards the cranial base was significantly larger than that on the normal ones. similarly, the width of nl/ml angle also showed that vertical relation between maxilla-mandibula is significantly greater than that on the normal ones. besides that, the width of ar-go-me angle showed that the gonial angle was significantly greater than that on the normal ones. 4 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 1–6 these results also completed the results of sjahruddin’s observation that there was facial abnormality in the vertical direction on the hbe β thalassemia patients. these results were also in accordance with rothstein’s and the pan’s13 ideas that in class ii malocclusion, small and retrognative mandible as well as mandibular plane angle (mpa) were obtained. the same observation results obtained from dentofacial components on the hbe β thalassemia patients also showed that there was no disproportional growth in the sagittal (anteroposterior) direction. this condition could be seen from the fact that anb angle was significantly greater than that on the normal group, so class ii skeletal malocclusion occured on the hbe β thalassemia patients. the fact that snb angle was significantly smaller than that on the normal group indicated that the position of the mandible towards the cranial base on the hbe β thalassemia patients was inclined more to the posterior region or retrognati than that on the normal subjects. nevertheless, the width of sna angle on the hbe β thalassemia patients must not be different from that on the normal ones. it means that the position of maxilla towards cranial base on the hbe β thalassemia patients is the same as that in the normal ones. in addition, mandibular dimension consited of mandibular length which total was smaller than that on the normal ones although the length of the mandibular ramus was not different from that of the normal ones. on the other side, ns-tm angle showed that the anteroposterior position of the glenoid fossa on the hbe β thalassemia patients was not different from that on the normal ones. this finding is the same as that of sjahruddi’s4 and retno hayati’s5 researches in which it was known that class ii skeletal malocclusion on the hbe β thalassemia patients occured due to the retardation of mandibular growth and retrognative mandibular position. these results of observing dentofacial components then showed that the transversal direction on the hbe β thalassemia patients measured from the maxillary intermolar distance was not different from that on the normal ones, but the mandibular intermolar distance was significantly smaller than that of the normal ones. similarly, it was also known that the posterior transverse interarch discrepancy on the hbe β thalassemia patients significantly greater than that on the normal ones which indicated the difference distance between the maxillary intermolar distance and the mandibular intermolar distance. this finding was different from that in wahadni’s and omarii’s14 research among youth and adult of jordanian which stated that the maxillary intermolar distance on the hbe β thalassemia patients was smaller than that on the normal ones. in contrast to this study, the maxillary intermolar distance on the hbe β thalassemia patients was the same as that on the normal ones. the posterior transverse interarch discrepancy of the hbe β thalassemia patients also indicated that class ii malocclusion was caused by the narrowing of the mandibular arch since the posterior teeth were inclined more to the lingual, and the basal spinal was also narrowing.15 several other researchers even said that the position of the first mandibular molars were normal, but the position was inclined more to the mesial of the maxillary teeth.13,15-17 this condition was contrast to the finding of this study showing that the position of the first mandibular molars on the hbe β thalassemia patients was more distally than that on the first maxillary molars or the first angle class ii molar relation. table 2. mean, standard deviation, and t-test result between variables of the thalassemia group and those of the normal group with a sample (n) of each group=33 variable thalassemia normal ttest result mean sd mean sd t p sna angle 82.77 2.88 83.23 2.19 0.69 0.490 snb angle 76.65 3.42 79.10 2.55 3.20 0.002 anb angle 6.13 1.67 4.10 1.60 4.90 0.000 n-s-tm angle 132.55 8.54 128.84 6.57 1.92 0.060 nl/nsl angle 6.55 3.55 10.26 5.39 3.20 0.002 ml/nsl angle 38.29 6.27 32.90 7.57 3.05 0.003 nl/ml angle 32.87 7.89 25.52 5.67 4.22 0.000 ar-go-me angle 127.45 7.94 121.74 4.65 3.45 0.001 mandibular length 65.48 5.34 71.42 3.20 5.31 0.000 mandibular ramus length 52.87 4.92 54.58 5.14 1.34 0.186 mandibular length total 105.84 5.83 111.03 4.93 3.79 0.000 maxillary intermolar distance 45.40 2.56 46.39 2.31 1.60 0.114 mandibular intermolar distance 40.07 2.54 44.97 1.26 0.17 0.005 ptid 5.33 1.75 1.24 1.03 1.92 0.001 5zen and sjahruddin: posterior transverse interarch discrepancy mandibular retrusion or maxillary protrusion is considered as a cause of skeletal abnormalities in class ii malocclusion which is more dominant disorder and still a problem for researchers. 56.3% of class ii malocclusion case were caused by the maxillary protrusion, meanwhile the mandibula was normal either in size, shape and position.16 class ii malocclusion due to mandibular retrusion can only be obtained about 27%,16 but on the hbe β thalassemia patients class ii malocclusion can mostly be obtained due to mandibular retrusion. dr. e.h. angle distinguished class ii malocclusion into two types, namely class ii division 1 malocclusion with the typical signs of maxillary arch constriction and class ii division 2 with normal maxillary arch,17 so that the maxillary intermolar distance was usually smaller in class ii division 1 malocclusion than that in class ii division 2. this condition was caused by class ii division 1 skeletal malocclusion often accompanied by anomalous teeth compared with class ii division 2.18 that maxillary intermolar distance on the hbe β thalassemia patients, furthermore, is not different from that on the normal ones which indicated that there is no narrowing of the maxillary arch. this condition can be caused due to the compensation of the teeth towards the skeletal discrepancy.19 these results, however, are different from those of tollaro et al.,12 research stating that all the class ii malocclusion samples with or without the posterior tranverse discrepancy had a narrower maxillary arch. various opinions about the cause of the absence of maxillary arch constriction indicated by the maxillary intermolar distance, for example, is that in class ii division 2 malocclusion, there will not be any the disharmony of maxillary bone, and the characteristics of typical stereotypes. the malocclusion is not only caused by the disharmony of tooth eruption, the pressure of the muscles, and/or some variation of its compesantion.20 another researcher even says that in class ii division 2 malocclusion, the reduction of mesiodistal and labiolingual of maxillary and mandibular incisors occurs. the reduction of incisor width then will reduce the need of space in the dental arch, as a result, it has positive effects since dentoalveolar arch does not only become sufficient enough to accommodate all of the teeth, but will also have spacing in anterior teeth.15, 21 according to litt and nielsen, in class ii division 2 malocclusion, there is normal jaw transverse dimension.21 in this study, mandibular intermolar distance on the hbe β thalassemia patients was significantly smaller compared to that of the normal ones. actually, the reduction of mandibular arch width measured from the mandibular intermolar distance actually will show the characteristics of class ii division 2 malocclusion, particularly looked in the region of the mandibular interkanina.15 this condition can also be caused by excessive overbite so that the maxillary incisor teeth cover the crown of mandibular incisor teeth (deep bite). mandibular incisor teeth that are blocked then will inhibit the development of the mandibular dentoalveolar to the anterior. the teeth, as a result, will be depressed by the lack of space caused by the rotation of mandibula.15,21 it means that the deficiency of transversal direction on the maxilla has been an abnormal characteristics of occlusal pattern in class ii malocclusion since the early development. after the first permanent molar eruption, transversal discrepancy is still ongoing, and becomes a typical condition of class ii malocclusion in mixed tooth period.22 in this study, moreover, the mandibular intermolar distance of the hbe β thalassemia patients was very different from that of the normal ones. the small mandibular intermolar distance and the normal intermolar distance caused posterior transverse interarch discrepancy. this result is the same as what sjahruddin explained4 that the hbe β thalassemia patients with retardation of mandibular growth and anterior cranial base growth have the position of the mandible against the anterior cranial base that was inclined more to retrognati compared to the normal ones. posture, activity, and behavior of orofacial muscles and mastication may also cause the narrowing of the dental arch and basal arch. this condition can also be triggered by several factors, such as habit factor or the abnormal pulling of muscles that causes the inclination plain distal locked. other factors are the lack of muscle pressure against the labial surface of maxillary incisors, the error coordination function of the muscles, and the abnormal pulling of the mandibular corpus by muscles attached to the buccal side of the mandible. there is also mechanical force factor that causes mandibular incisor teeth pushed into the posterior and inhibits the growth of the mandible to the anterior direction and toward the transversal direction.19 finally, it can be concluded that the posterior transverse interarch discrepancy of the hbe β thalassemia patients aged 12–14 years was different from that of the normal ones in the same ages. the dentofacial abnormalities in hbe β thalassemia patients primarily was due to disporposional dentofacial growth in the vertical, sagittal, and transversal directions, especially in the posterior region. references 1. sassouni v, forrest ed. orthodontics in dental practice. st. louis: cv mosby co; 1971. p. 82–118, 121–66. 2. hayati r. pertumbuhan lengkung rahang anak thalasemia di jakarta. (tinjauan terhadap oklusi gigi dan relasi rahang arah sagital). seminar laporan penelitian lpui, jakarta; 1993. p. 6–8. 3. scully c, cawson ra. medical problems in dentistry. 3rd ed. oxford: wright; 1998. p. 119. 4. sjahruddin ld. indeks kelainan dentofasial dan maturasi tulang vertebra servikal pada penderita talasemia beta hemoglobin e serta hubungannya dengan beberapa faktor risiko. disertation. jakarta: universitas indonesia; 2004. p. 4–12. 5. hayati r. pola deformitas dentoskeletal pada anak talasemia dan faktor determinannya. disertation. jakarta: universitas indonesia; 1998. p. 7–10. 6. mcnamara ja jr. components of class ii malocclusion in children 8–10 years of age. angle orthod 1981; 51: 177–202. 7. mcnamara ja jr, brudon wl. orthodontics and dentofacial orthopedics. ann arbor, michigan: needam press, inc; 2001. p. 63–82, 319–30. 6 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 1–6 8. moyers re. handbook of orthodontics. chicago: year book publisher inc; 1988. p. 187–93. 9. mcnamara ja jr, brudon wl. orthodontic and orthopedic treatment in the mixed dentition. 4th ed. ann arbor, michigan: needam press, inc; 1994. p. 243–57. 10. rakosi t, jonas i, graber tm. color atlas of dental medicine. orthodontic–diagnosis. georg thieme verlag. new york: thieme medical publisher inc; 1993. p. 46. 11. staley rn, stuntz wr, peterson lc. a comparison of arch widths in adults with normal occlusion and adults with class ii division 1 malocclusion. am j orthod 1985; 88(2): 163–9. 12. tollaro i, baccetti t, franchi l, camellia dt. role of posterior transverse interarch discrepancy in class ii, division 1 malocclusion during the mixed dentition phase. am j orthod dentofac orthop 1996; 110: 417–22. 13. rothstein t, phan xl. dental and facial skeletal characteristics and growth of females and males with class ii division 1 malocclusion between the ages of 10 and 14 (revisited). part ii. anteroposterior and vertical circumpubertal growth. am j orthod dentofac orthop 2001; 120(5): 542–55. 14. wahadnii ma, qudemat, omarii m. dental arch morphological and dimensional characteristics in jordanian children and young adults with betathalassaemia major. int j of paediatric dent 2005; 15: 98–104. 15. rothstein t, tarlie cy. dental and facial skeletal characteristics and growth males and females with class ii, division malocclusion between the ages of 10 and 14 (revisited)-part l: characteristics of size, form and position. am j orthod dentofac orthop 2000; 117(3): 320–32. 16. walkow tm, peck s. dental arch width in class ii division 2 deepbite malocclusion. am j orthod dentofac orthop 2002; 122(6): 608–13. 17. rosenblum re. class ii malocclusion: mandibular retrusion or maxillary protrusion. angle orthod 1995; 65(1): 49–62. 18. bacceti t, franchi l, mcnamara j, tollaro i. early dentofacial class ii malocclusion longitudinal study from the deciduous through the mixed dentition. am j orthod dentofac orthop 1997; 111(5): 502–9. 19. spalding p. treatment of class ii malocclusion. in: rudolph p, editor. textbook of orthodontics. philadelphia, pennsylvania: wb saunders co; 2001. p. 324–35. 20. hershcopf sa. class ii division 2 malocclusion-non extraction. am j orthod dentofac orthop 1990; 97(5): 374–80. 21. litt ra, nielsen l. class ii division 2 malocclusion, to extract or not extract. angle orthod 1984; 54(2): 123–38. 22. peck s, peck l, kataja m. class ii division 2 malocclusion: a heritable pattern of small teeth in welldeveloped jaws. angle orthod 1998; 68(1): 9–20. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 161 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 161–164 original article estimation of children’s age based on dentition via panoramic radiography in surabaya, indonesia agung sosiawan1, an’nisaa chusida2, beshlina fitri widayanti roosyanto prakoeswa2, arofi kurniawan2, maria istiqomah marini2, beta novia rizky2, tito krisna gianosa3, najminoor ramadhani ridlo3, mumtaz ramadhani putra pesat gatra3, aspalilah alias4 1 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2 department of forensic odontology, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3 undergraduate student of dental medicine education study program, faculty of dental medicine, airlangga university, surabaya, indonesia 4 department of basic sciences and oral biology, faculty of dentistry, universiti sains islam malaysia, negeri sembilan, malaysia abstract background: age may be estimated using tooth eruption sequence and/or calcification stage. because many factors may affect the time of eruption, the tooth calcification stage shows higher accuracy than the time of tooth eruption. demirjian’s and willems’ methods have been most commonly used for dental age estimation. both willems and demirjian use the calcification stage as an indicator. studies comparing these methods have shown varied results, as they have been performed on different populations. purpose: this paper aims to analyse the estimation of children's age based on dentition via panoramic radiography in surabaya, indonesia. methods: this is a cross-sectional study using a purposive sampling method. one hundred digital panoramic radiographs of children between 6 and 15 years that match the inclusion criteria from the airlangga university dental hospital in surabaya, indonesia were evaluated. one researcher analysed estimated dental age (eda) three times in a one-week time-lapse using demirjian’s and willems’ methods. statistical analysis was carried out using a paired t-test and wilcoxon signed-rank test. eda was calculated using both demirjian’s and willems’ methods. results: the mean chronological age (ca) was 10.57 ± 2.70 for males and 10.73 ± 2.84 for females. the mean difference between ca and eda using the demirjian and willems methods was -0.57 ± 1.17 and 0.10 ± 0.96 for males and 0.58 ± 1.40 and 0.44 ± 0.94 for females. conclusion: the results suggest that willems’ method is more precise than demirjian’s method in males and females and more suitable for children in surabaya, indonesia. keywords: demirjian; dental age estimation; dentistry; forensic odontology; willems correspondence: an’nisaa chusida, department of forensic odontology, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: an-nisaa-c@fkg.unair.ac.id introduction forensic odontology is a branch of forensic science that plays a role in identifying victims of natural and non-natural disasters.1,2 the scope of forensic odontology includes identification using dental records, bitemark analysis, and age estimation.3 age estimation has various advantages, including identifying unknown victims, determining age at death, and determining the chronological age (ca) of children with unknown birth documents.4 in general, the objects used in age estimation are teeth and bones. teeth have the advantage of making it possible to estimate an individual’s age from prenatal to adult age, whereas bones can only be used in a certain age range. in addition, teeth are the strongest parts of the body, so they can be used for identification even if the body has been burned, mutilated, or decomposed.5 there are several methods of estimating dental age, including radiographic, morphological, and biochemical methods. the selection of a method must consider the individual’s status (alive or dead), age range, dental condition, and also the availability of facilities.5 radiography is one of the most common methods for age estimation. it also provides nearly exact estimations for both living and deceased victims.1,5 in this study, willems’ and demirjian’s radiographic methods were used because they are considered simple and non-invasive. these methods were also chosen because they use the stage of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p161–164 mailto:an-nisaa-c@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p161-164 162sosiawan et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 161–164 tooth calcification as an indicator of their assessment and are, therefore, more precise than the tooth eruption sequence.4 in research on age estimation, some researchers compare the demirjian’s and willems’ methods to analyse which method is more precise in determining dental age. in the research of yang et al.,6 the demirjian method was shown to be more precise than willems’ in the south china population, where the estimated dental age (eda) of the demirjian method was underestimated by -0.03 ± 1.20 years for males and overestimated by 0.03 ± 1.05 years in females. the willems method, meanwhile, overestimated by 0.44 ± 1.15 years for males and 0.54 ± 1.08 years for females. contrarily, in ozveren et al.’s research on the turkish population, willems’ method was shown to be more precise than demirjian’s method, with the eda of the demirjian method being an underestimation of -1.04 ± 0.95 years for males and an underestimation of -0.87 ± 0.92 years for females.7 the willems method underestimated by -0.40 ± 0.85 years for males and -0.17 ± 1.02 years for females. according to research conducted by esan et al.8 regarding the differences in the results of the demirjian’s and willems’ methods, different results were achieved because they were conducted on different populations. this study aims to analyse the estimation of children’s age based on dentition via panoramic radiography in surabaya, indonesia. materials and methods this study’s sample was 100 digital panoramic radiographs from 50 males and 50 females aged 6 to 15 years who matched the inclusion criteria. the inclusion criteria were clear panoramic radiographs, seven permanent mandibular left teeth shown on the panoramic radiographs, and confirmation of both birth date and panoramic radiograph date. the exclusion criteria were: panoramic radiographs showing any pathological condition and/or tooth extraction, systemic diseases or genetic disorders that could impair skeletal and dental development, orthodontic appliances, and congenital or developmental anomalies. demirjian’s and willems’ methods were used to score all digital panoramic radiographs. the seven left mandibular teeth were evaluated based on the stage of tooth calcification. each tooth score was turned into a gender-specific chronological table. radiographic pictures were used to categorise tooth development into eight stages ranked on a scale of ‘a’ to ‘h,’ and schematic diagrams were used to explain the specific parameters required for each step in both uniradicular and multiradicular teeth.9 ca is determined based on the date, month and year of birth. it is calculated from the date the panoramic radiograph was taken minus the date of birth. eda is the age obtained through calculations using the demirjian’s and willems’ methods from panoramic radiographs. each method was calculated separately.9 data calculations were carried out three times with an interval of one week with one observer to eliminate bias in the results. the sample was analysed by statistical tests using ibm® spss® statistics version 26.0 (ibm, armonk, ny, usa). a cronbach’s alpha test was used to determine the level of reliability of the variables. the demirjian method variables were 0.96 and the willems method was 0.98, which means that both methods are feasible to use. shapiro wilk and levene’s tests were used to perform normality and homogeneity tests. variables that have a p-value > 0.05 were elaborated using the paired t-test, while variables that have a p-value < 0.05 were elaborated using the wilcoxon signed-rank test. results a cronbach’s alpha test was conducted to examine the inter-examiner agreement of tooth development stage scoring, with a coefficient of 0.6. the kolmogorov-smirnov test result indicated that the data were normally distributed and suitable for further statistical analysis, with a p-value > 0.05. to determine the significance of differences between ca and eda, a paired t-test was used. table 1 compares ca and eda based on the demirjian method for males and females. in both sexes, the overall mean difference between ca and eda was -0.57 ± 1.17 and 0.58 ± 1.40, respectively. the age categories were classified for further analysis. in ages 6 to 10, the mean difference between ca and eda was -0.14 ± 0.80 and 0.22 ± 0.94 for males and females, respectively. in the age group of 10 to 15, the mean difference between ca and eda was -1.07 ± 1.34 and 0.95 ± 1.68 for males and females, respectively. table 1. comparison of chronological age (ca) and estimated dental age (eda) based on demirjian’s method gender age group ca x ± sd eda x ± sd age difference x ± sd p-value remarks male 6–10 8.42 ± 1.40 8.57 ± 1.35 -0.14 ± 0.80 0.35 underestimate 11–15 13.10 ± 1.27’ 14.17 ± 1.63 -1.07 ± 1.34 0.00* underestimate total 10.57 ± 2.70’ 11.15 ± 3.18’ -0.57 ± 1.17 0.00* underestimate female 6–10 8.27 ± 1.43 8.05 ± 1.04’ 0.22 ± 0.94 0.07 overestimate 11–15 13.18 ± 1.37 12.23 ± 1.95 0.95 ± 1.68 0.00* overestimate total 10.73 ± 2.84’ 10.14 ± 2.61’ 0.58 ± 1.40 0.00* overestimate paired t-test, wilcoxon signed-rank test (*p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p161–164 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p161-164 163 sosiawan et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 161–164 table 2 compares ca and eda based on willems’ method for males and females. in both sexes, the overall mean difference between ca and eda was 0.10 ± 0.96 and 0.44 ± 0.94, respectively. the age categories were classified for further analysis. in the 6 to 10 years old group, the mean difference between ca and eda was 0.02 ± 0.93 and 0.27 ± 0.68 for males and females, respectively. in the age group of 10 to 15, the mean difference between ca and eda was 0.18 ± 1.02 and 0.52 ± 1.16 for males and females, respectively. discussion a person’s age can be established by a number of factors, including bones and teeth. teeth reflect a wide range of ages, from intrauterine to adult. dental age estimation methods such as demirjian’s and willems’ methods are used for children. demirjian (1973) introduced a new system of calculating dental age based on seven teeth on the left side of the mandible (central incisor to second molar teeth) to calculate dental maturity scores. in 2001, willems improved demirjian’s dental age estimation approach. willems’ method showed increased accuracy in determining ca.10 demirjian’s and willems’ techniques estimate children’s ca using teeth calcification sequence as an indicator. researchers continue to discuss the demirjian and willems dental age estimation methods for various populations, as different results are achieved for each researcher with different populations.8 based on the findings of the current study, it can be stated that the willems method shows more precise results than the demirjian method. these findings are consistent with several similar studies, namely nik-hussein et al.11, grover et al.12, ye et al.13, and kumaresan et al.14, who also concluded that the willems method was more precise than the demirjian. nik-hussein et al.’s study11 was carried out on the malaysian population. the demirjian method in the male and female groups overestimated the ages by 0.7 ± 1.3 and 0.5 ± 1.2 years, respectively. meanwhile, the willems method overestimated the ages by 0.3 ± 1.3 years for the male group and 0.05 ± 1.1 years for the female group.11 grover et al.12, who conducted their study on the population of south india, showed that the demirjian method in the male and female groups underestimated the ages by -0.66 ± 0.38 and -0.56 ± 0.36 years, respectively. the willems method overestimated by 0.36 ± 0.41 years in the male group and 0.23 ± 0.43 in the female group.12 in a study by ye et al.13 on the chinese population, the demirjian method in the male and female groups overestimated the ages by 1.68 ± 1.29 and 1.28 ± 1.17 years, respectively. the willems method overestimated the ages by 0.36 ± 1.19 years in the male group and underestimated by -0.02 ± 1.18 in the female group.12 in kumaresan et al.’s study,14 which was carried out on the malaysian population, the demirjian method in the male and female groups overestimated the ages by 0.98 ± 1.29 and 0.97 ± 1.12 years, respectively. meanwhile, the willems method overestimated the ages by 0.55 ± 1.40 years for the male group and 0.53 ± 1.20 for the female group.14 however, the study on the south china population by yang et al.6 discovered that the demirjian method was more precise than the willems method. demirjian’s method underestimated the ages by -0.03 ± 1.20 in the male group and overestimated by 0.03 ± 1.05 years in the female group. the willems method overestimated the ages by 0.44 ± 1.15 years for the male group and 0.54 ± 1.08 years for the female group.6 the disparity in results between this study and other studies could be attributed to biological variations due to ethnic differences. furthermore, sample size and statistical approach of the age range could also lead to a difference in results.6 these differences may also occur due to differences in the level of dental development in different populations.10 this can be attributed to differences in genetic factors in each population that will affect the growth and development of teeth.5 according to mohammed et al.,15 sample size, different age groups, statistical methodologies, and the accuracy of the methods tested can affect the results of age estimation. in addition, differences in results can also occur due to the influence of lifestyle, nutrition, and dietary habits as an example of environmental influences.7 based on this study, willems’ dental age estimation method was found to be more precise than the demirjian method in both sexes, and it may be applicable to children in surabaya, indonesia. in forensic science, dental age estimation research is very important in order to determine the most reliable method to be applied to a certain population. however, we propose additional research to achieve more precise estimates of different demographic groupings and ethnicities. table 2. comparison of chronological age (ca) and estimated dental age (eda) based on willems’ method gender age group ca x ± sd eda x ± sd age difference x ± sd p-value remarks male 6–10 8.42 ± 1.40 8.39 ± 1.71 0.02 ± 0.93 0.87 overestimate 11–15 13.10 ± 1.27 12.91 ± 1.40 0.18 ± 1.02 0.39 overestimate total 10.57 ± 2.70’ 10.47 ± 2.75 0.10 ± 0.96 0.27 overestimate female 6–10 8.27 ± 1.43 7.90 ± 1.77 0.27 ± 0.68 0.01* overestimate 11–15 13.18 ± 1.37 12.66 ± 1.76 0.52 ± 1.16 0.03* overestimate total 10.73 ± 2.84’ 10.28 ± 2.97 0.44 ± 0.94 0.00* overestimate paired t-test, wilcoxon signed-rank test (*p < 0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p161–164 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p161-164 164sosiawan et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 161–164 references 1. singh nn, gowhar o, ain ts, sultan s. exploring trends in forensic odontology. j clin diagn res. 2014; 8(12): zc28-30. 2. prakoeswa bfwr, kurniawan a, alias a, chusida a, marini mi, rizky bn. palatal rugoscopy as an aid for sex determination in tengger population, indonesia. bull int assoc paleodont. 2021; 15(2): 77–82. 3. lewis jm, senn dr. dental age estimation utilizing third molar development : a review of principles , methods , and population studies used in the united states. forensic sci int. 2010; 201(1–3): 79–83. 4. macha m, lamba b, avula jss, muthineni s, margana pgjs, chitoori p. estimation of correlation between chronological age, skeletal age and dental age in childrena cross-sectional study. j clin diagnostic res. 2017; 11(9): zc01–4. 5. putri as, nehemia b, soedarsono n. prakiraan usia individu melalui pemeriksaan gigi untuk kepentingan forensik kedokteran gigi (age estimation through dental examination in forensic denstistry). j pdgi. 2013; 62(3): 55–63. 6. yang z, geng k, liu y, sun s, wen d, xiao j, zheng y, cai j, zha l, liu y. accuracy of the demirjian and willems methods of dental age estimation for children from central southern china. int j legal med. 2019; 133(2): 593–601. 7. ozveren n, serindere g. comparison of the applicability of demirjian and willems methods for dental age estimation in children from the thrace region, turkey. forensic sci int. 2018; 285: 38–43. 8. esan ta, yengopal v, schepartz la. the demirjian versus the willems method for dental age estimation in different populations: a meta-analysis of published studies. plos one. 2017; 12(11): e0186682. 9. demirjian a, goldstein h, tanner jm. a new system of dental age assessment. hum biol. 1973; 45(2): 211–27. 10. willems g, van olmen a, spiessens b, carels c. dental age estimation in belgian children: demirjian’s technique revisited. j forensic sci. 2001; 46(4): 893–5. 11. nik-hussein nn, kee km, gan p. validity of demirjian and willems methods for dental age estimation for malaysian children aged 5-15 years old. forensic sci int. 2011; 204(1–3): 208.e1-6. 12. grover s, marya cm, avinash j, pruthi n. estimation of dental age and its comparison with chronological age: accuracy of two radiographic methods. med sci law. 2012; 52(1): 32–5. 13. ye x, jiang f, sheng x, huang h, shen x. dental age assessment in 7–14-year-old chinese children: comparison of demirjian and willems methods. forensic sci int. 2014; 244: 36–41. 14. kumaresan r, cugati n, chandrasekaran b, karthikeyan p. reliability and validity of five radiographic dental-age estimation methods in a population of malaysian children. j investig clin dent. 2016; 7(1): 102–9. 15. mohammed rb. accuracy of four dental age estimation methods in southern indian children. j clin diagnostic res. 2015; 9(1): 1–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p161–164 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p161-164 101 introduction root canal obturation consists of placing an inert filling material in the space previously occupied by pulp tissue. to achieve successful endodontic therapy, it is important to obturate the root canal system completely. gutta-percha is used with various techniques for obturation of the root canal system. throughout the years, a variety of techniques using gutta-percha have been developed for root canal fillings. these techniques include lateral condensation, warm vertical condensation, and injectable thermoplasticized. investigators have evaluated the apical seals obtained by these various gutta-percha filling techniques. 1 lateral condensation remains the most widely accepted and used obturation technique.2 almost all other techniques are compared to it to evaluate success. it seems that lateral condensation is a gold standard for obturation. based on that reasons, this study will use lateral condensation for root canal obturation. in root canal obturation various materials have been used. the most frequent material used is gutta percha in combined with a root canal sealer. it must have ideal properties to be used for root canal obturation, as biocompatibility and sealing ability fundamental to promote apical and periapical tissue repair.3 many materials are used for root canal sealer, but none of the available sealer consistency prevents leakage.4. the hermetic sealing of the root canal space is one of the objectives in root canal therapy.5 the most common cause of failure involving endodontic therapy can be attributed to the lack of an apical seal leading to leakage at the apex. effective endodontic obturation thus, must provide a dimensionally stable, inert fluid tight apical seal that will eliminate any portal of communication between the canal space and the surrounding periapical tissues through the apical foramen.6 hydroxyapatite is the most thermodynamically stable synthetic calcium phosphate cement.7 a calcium phosphate cement has indicated that is useful as a sealer because can seal a furcation perforation, is shown to be biocompatible and also has potential to promote the healing of bone in endodontic treatment.8 recently, most of the sealers commonly used contains zinc oxide or calcium hydroxide as a base ingredient of the powder.9 sealing ability of hydroxyapatite as a root canal sealer: in vitro study widowati witjaksono,1,6 lin naing,2 ema mulyawati,3 ar. samsudin,4 and mon mon tin oo5 1 department of restorative dentistry school of dental sciences universiti sains malaysia 2 institute of medicine national university of brunei darussalam 3 department of conservative dentistry, gadjah mada university, indoensia 4 department of oral surgery and 5 department of community dentistry school of dental sciences universiti sains malaysia 6 department of periodontic, faculty of dentistry airlangga university, indonesia abstract hydroxyapatite (ha) is the most thermodynamically synthetic calcium phosphate cement, and has indicated useful as a sealer because can seal a furcation perforation, is shown to be biocompatible and also has potential to promote the healing of bone in endodontic therapy. the objective of this study is to determine the sealing ability of ha produced by school of engineering, universiti sains malaysia (usm) when used as a sealer in root canal obturation, compare with tubli-seal (zinc-oxide base) and sealapax (calcium hydroxyde base) sealers. forty five single rooted human anterior teeth were instrumented and randomly divided into three experimental groups of 15 teeth each. all teeth in the experimental groups were obturated with laterally condensed gutta percha technique. teeth in the first group were sealed using zinc-oxide (zno) based sealer and those of second group using calcium hydroxide (caoh) based root canal sealer. third experimental group was sealed using ha from school of engineering usm. teeth were then suspended in 2% methylene blue. after this, teeth were demineralized dehydrated and cleared. linear dye penetration was determined under magnifying lense with calibrated eye piece. statistical analyses of the linear dye penetration were performed with kruskal wallis test. the intergroup comparison between ha and zno groups and caoh groups were analyzed by mann-whitney test. the dye penetration for group which were sealed with ha exhibited the lowest penetration and it showed that there was a statistically significant difference both between ha and zno groups and also between ha and caoh groups (p < 0.001).in conclusion, it was found that value added ha based endodontic material which were produced by usm can be used as a root canal sealing materials when it used in combination with epoxy resin since it leaked comparatively less as compared to zno and caoh sealers. before reaching a definitive conclusion, this material requires further extensive exploration both clinically and in vitro. key words: apical seal, endodontic sealers, micro leakage correspondence: widowati, department of restorative dentistry, school of dental sciences universiti sains malaysia, health campus 16150 k.kerian, malaysia. e-mail: widowati@kb.usm.my 102 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 101-105 the present study was thereby designed to determine the sealing ability of hydroxyapatite when used as a sealer in root canal obturation, compare with zinc-oxide base and calcium hydroxide base. therefore, it would be interesting to examine whether or not hydroxyapatite is able to act as a root canal sealer. the rationale is that if adequate sealing is obtained, this material has the potential to be clinically useful. materials and method this study was designed to evaluate the in vitro sealing abilities of endodontic materials. the following materials were selected and grouping for the study. materials: tubli_seal tm sealer (sybronendo, kerr/ usa). a zinc-oxide based root canal sealer (zno) sealapex tm sealer (sybronendo, kerr/usa). a calcium hydroxide based root canal sealer (ca(oh)2) hydroxyapatite (ha) (from school of engineering, universiti sains malaysia/ usm) and were mixed with epoxy resin (dentsply, detrey). this type of ha were used as part of a larger study (not yet published) in many fields and clinical trials. instruments: ultrasonic scaler, bone’s cutting, contra angle hand piece, burs, endodontic box, glass lab, cement spatula, plastic filling instruments, tweezer, explorer, paper point, magnifying lense 10×, le crown mesh knife. the study was carried out in vitro on forty-five extracted human single rooted, noncarious anterior teeth which were collected from the outpatient department of oral and maxillofacial surgery, school of dental sciences, universiti sains malaysia. all external debris were removed with an ultrasonic scaler. all teeth randomly divided into 3 groups each is 15 teeth. the crown were separated from the root until the length of the roots were 14 mm and store in saline. the pulps were broch and the root canals were prepared by step-back technique with working length 13 mm until no:80 file with master apical file (maf) no 50. after the use of each instrument (file), the root canals were irrigated with 1ml h2o2 3% and 1 ml naocl 2.5% and dry with paper point and ready for root canal filling. the sealer were mixed according the manufacture’s directions. hydroxyapatite granules were mixed up with epoxy resin liquid for hardener. the sealer was put along the lentulo plugger and coated to the inner walls of the canal by moving lentulo plugger clockwise according the groups, group a (zno based root canal sealer), group b (ca (oh)2) based root canal sealer and group c (ha from school of engineering usm). one third apical of gutta percha master cone (no. 50) was coated with sealer and seat in the canal to the full working length. the canal were obturated with lateral condensation technique. a finger spreader was inserted into the root canal to a level that was –1 mm short of the working length. the root canal was filled with accessory cones until the entire canal was obturated. the access cavities of teeth in all groups were then filled with zinc polycarboxylate cement and hydraulic temporary restorative, then the root were immersed in saline solution for 4 weeks at 37 °c. after storage, the roots were double coated with nail polish, with the exception of the apical 2 mm. specimens used for the dye leakage test were placed in 2% methylene blue solution (37 c, ph &) for 48 hours. the roots were then taken from the dye solution, remove the nail polish with le crown mesh knife, washed and dried with compressed air. the depth of dye penetration were evaluated with clearing method.10 all the teeth were immersed in hno3 5% for 72 hours for teeth demineralized. hno3 was changed with the fresh one every 24 hours. the teeth were then placed in alcohol 96% for 48 hours for teeth dehydration, and every 24 hours alcohol was changed with the fresh one. the final stage was clearing.10 all the teeth were placed in methyl salicylate, until dye penetration were able measured visually. apical leakage was measured from the apex to the most coronal extent of dye penetration (figure 1, 2, and 3). linear dye penetration was measured under magnifying lense with calibrated eye piece and analyse by kruskal wallis test. the intergroup comparison between hydroxyapatite and zinc oxide groups and calcium hydroxide groups data analyzed by mann-whitney test. results in the present study, measurements of maximum linear dye penetration were made to quantify the relative leakage (figure 1, 2, 3). dye penetration data for all the three groups are summarized in table 1. in group c teeth which were filled with laterally-condensed gutta-percha and hydroxyapatite (ha) sealer exhibited the lowest minimum-maximum (min-max) value of dye penetration. the min-max of dye penetration for group c(ha) was between 0-1mm. the min-max of dye penetration for group a teeth which were filled with laterally-condensed guttapercha and zinc oxide (zno) base sealer was between 1–3 mm. the corresponding values for group b teeth which were filled with laterally condensed gutta-percha and calcium hydroxide (ca(oh)2) base sealer was also between 1–3 mm. ten samples in group c (ha) showed no dye penetration whereas all samples in group a and b showed dye penetration. the comparison of the three study groups using kruskal wallis test (table 2) revealed that there were at least one significant difference among the study groups (p < 0.001). further mann-whitney test (post-hoe multiple comparisons between two groups) (table 3) revealed that ha group has significance lesser penetration compared to zno or ca(oh)2 groups (p < 0.001). 103witjaksono et al.: sealing ability of hydroxyapatite discussion ha is one of the ceramic materials commercially used for orthopaedic and dental implants, and it forms the principal mineral component of bone and comprises 60% to 70% of the calcified skeleton. it’s chemical composition is ca 10(po4)6 (oh)2 and it has been produced synthetically since the early 1970’s and used clinically for the last 20 years.11,12 it has received considerable attention over the past two decades primarily because of it’s excellent biocompatibility with hard tissues.13,14 and when placed in contact with viable bone, result in osteoconduction figure 1. measurement of apical leakage in zno base (group a). figure 2. measurement of apical leakage in ca(oh)2 base (group b). figure 3. measurement of apical leakage in hydroxyapatite. table 1. amount of dye penetration in group a, b and c (mm) specimen no of each group amount of dye penetration (in mm) group a zinc oxide base (zno) group b calcium hydroxide (ca(oh)2) group c hydroxyapatite (ha) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 min–max median (iqr) 2 1.5 2.5 2 2 1 2 3 1.5 2 3 2 1 2 2 1–3 2.0 (0.5) 2 1 1.5 2.5 3 1.5 3 2 3 2 2 3 3 2 3 1–3 2.0 (1.0) 0 0 0 1.0 0 0 0 0 0 0.5 0.5 0 0.5 0 0.3 0–1 0.0 (0.5) 104 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 101-105 table 3. comparison of dye penetration between hydroxyapatite (ha) and others comparison z stat. p valuea zno vs ha ca(oh)2 vs ha –4.76 –4.77 < 0.001 < 0.001 a mann-whitney test; p value is adjusted using bonferroni procedure for multiple comparison and osteointegration.15,16 ha does not cause a chronic inflammatory respons, toxic reactions or a foreign body giant cell reaction.12 although ha is a promising implant material, the greatest stumbling block to it’s wider application and utilization is the brittleness of the material and it’s low strength for load-bearing applications.17 thus the material used in this study is the value added ha based material which were produced by a group of scientist at the school of engineering universiti sains malaysia (usm). the pure ha has been added with zirconia and other additional components, hot pressed and then sintered at a temperature of 1300 °c, to increase the toughness and strength of ha ceramic. composites formed by ha ceramic in combination with zirconia have been proven not to produce any local or systemic adverse reactions or any cytotoxix effects in various in vivo studies.18 this material showed no decrease in strength after ageing up to 1 year, which is in agreement with the study done by some investigators in 1993.19 it has been recognized for decades that the ideal end result of root canal therapy would be a closure of the apical foramen with reparative cementum. the goals for stability of successful endodontic therapy are total obliteration of the canal and perfect sealing of the apical foramen at the dentino-cemental junction and accessory canals at locations other than the root apex with an inert, dimensionally stable and biologically compatible material.20 according to other researchers,21 endodontic sealers are used to eliminate the interface between the gutta-percha and the dentinal walls. thus, the quality of the filling depends largely on the sealing capacity offered by sealers.22,23 from this study an average leakage values of zn o base sealer and caoh base sealer both were minimum of 1 mm to maximum of 3 mm. the lesser value of dye penetration shown by value added ha sealer in the present study may be because of the better sealing abilities of ha. one possible explanation for this observed difference may be that ha has ability to bind strongly with natural bone tissue,24 and synthetic ha has the same chemical composition as biological ha and thus mimics many properties of natural bone.25as for epoxy-resin based endodontic sealers to the human dentin showed a higher capacity to attach to the dentinal walls than other endodontic sealers and provide bonding between it and gutta-percha points.26 however the exact mechanism by which ha is incorporation with epoxy resin, then can be function as a good root canal sealer in the present study remain far from clear. it would be necessary to carry out further studies in order to make a larger evaluation of these value added ha based endodontic materials as well as their potential benefits. the in vivo evaluation should be done to assess the reaction to this value added ha as compared to the pure ha. it can be concluded from this study that the value added ha based endodontic material can be used as a root canal sealing materials when it used in combination with epoxy resin since it leaked comparatively less as compared to zno and caoh sealers. before reaching a definitive conclusion this material requires further extensive exploration both clinically and in vitro. acknowledgements the authors acknowledge the universiti sains malaysia for funding this research as a short term grant in the year 2006–2007. special thanks to ir. endro for help with the special work. references 1. goodman a, schilder h, burg, kjl, porter s. the thermo mechanical properties of gutta-percha, ii: the history and molecular structure of gutta-percha. oral surg oral med oral path oral radiol endod 1974; 37:954–95. 2. dummer pmh. comparison of undergraduate endodontic technique programs in the united kingdom and in some schools in europe and the united states. int endod j 1991; 24:169–77. 3. duarte mah, demarchi acco, giaxa mh, kuga mc, fraga sc, souza lcd. evaluation of ph and calcium ion release of three root canal sealers. j endo 2000; 26(7):389–90. 4. mannocci f, ferrari m. apical seal of root obturated with laterally condensed gutta-percha, epoxy resin cement and dentin bonding agent. j endod 1998; 24(1):41–4. table 2. comparison of dye penetration between study groups (mm) group n dye penetration x2 stat. (df) p valuea median (iqr) min.-max. zn o ca (oh)2 ha 15 15 15 2.0 (0.5) 2.0 (1.0) 0.0 (0.5) 1–3 1–3 0–1 31.00 (2) <0.001 a kruskal wallis test iqr = interquartile range; min. = minimum; max. = maximum 105witjaksono et al.: sealing ability of hydroxyapatite 5. goldberg f, massone ej, artaza lp. comparison of the sealing capacity of three endodontic filling techniques. j endod 1995; 21(1):1–3. 6. hovland ej, dumsha tl. leakage evaluation of in vitro of the root canal cement seal apex. int endodon j 1985; 18:179–92. 7. hayashi y, imai m, yanagiguchi k, viloria il, ikeda t. hydroxyapatite applied as direct pulp capping medicine substituted for osteodentin. j endod 1999; 25(4):225–9. 8. cheng am, chow lc, takagi s. in vitro evaluation of calcium phosphate cement root canal filler/sealer. j endod 2001; 27(10):6133–5. 9. ingle ji, west jd. obturation of the radicular space. in: ingle ji, bakland lk, editors. endodontics. 4th ed. baltimore: lea and febiger; 1994. p. 229–57. 10. robertson d, leeb ij, mc kee m, brewer e. a clearing technique for the study of root canal systems. j endod 1980; 6:421–4. 11. jarcho m. calcium phosphate ceramics as hard tissue prosthetics. clin orthop rel res 1981; 157(9):259–78. 12. constantino pd, friedman cd, lane a. synthetic biomaterial in facial plastic and reconstructive surgery. facial plast surg 1993; 9(1):1–15. 13. wozney jm, rozen v. bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. clin orthop rel res 1998; 346:26–37. 14. suchanek w, yoshimura m. processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. j mater res 1998; 13(1):94–117. 15. burg kjl, porter s, kellam jf. biomaterial developments for bone tissue engineering. biomaterials 2000; 21:2347–59. 16. green d, walsh d, mann s, oreffo roc. the potential of biomimesis in bone tissue engineering: lessons from the design and synthesis of invertebrate skeletons. bone 2002; 30(6):810–15. 17. muralithran g, ramesh s. the effects of sintering temperature on the properties of hydroxyapatite. ceram int 2000; (26):221–30. 18. piconi c, maccauro g. zirconia as a ceramic biomaterial. biomaterials 1999; 20:1–25. 19. shimizu k, oka m, kumar p. time-dependent changes in the mechanical properties of zirconia ceramic. j biomed mater res 1993; 27:729–34. 20. paul wesselink. root filling techniques. in: bergenholtz. g, bindslev ph, reit claes, editors. text book of endodontology. oxford: blackwell publishing company; 2003. p. 286–90. 21. timpawat s, amornchat c, trisuwan wr. bacterial coronal leakage after obturation with three root canal sealers. j endod 2001; 37:36–9. 22. oliver cm, abbot pv. an in vitro study of apical and coronal microleakage of laterally condensed gutta-percha with ketac-endo and ah-26. aust dent j 1998; 43:262–8. 23. cobankara fk, adanir n, belli s, pashley dh. a quantitative evaluation of apical leakage of four root-canal sealers. int endod j 2002; 35:979–84. 24. dalby mj, di silvio l, harper ej, boneld w. initial interaction of osteoblasts with the surface of a hydroxyapatite-poly (methyl methacrylate) cement. biomaterials 2001; 22:1739–47. 25. jarcho m. rertrospective analysis of hydroxyapatite development for oral implant applications. dent clin north am 1992; 36:19–26. 26. pecora jd, cussioli al, gurisoli dmz, marchesan ma, sousa-neto md, brugnera-junior a. evaluation of er: yag laser and edtac on dentin adhesion of six endodontic sealers. braz dent j 2001; 12(1):27–30. 2929 angular cheilitis and oral pigmentation as early detection of peutz-jeghers syndrome maharani laillyza apriasari1 and amy nindia carabelly2 1 departement of oral medicine 2 departement of oral pathology faculty of dentistry, universitas lambung mangkurat banjarmasin indonesia abstract background: peutz-jeghers syndrome (pjs) is an inherited autosomal dominant disease determined by a mutation localized at 19p13.3 characterized by the occurrence of gastrointestinal hamartomatous polyps in association with mucocutaneous hyperpigmentation. the manifestation of pjs may first be encountered by a dentist during routine examination due to the presence of pigmented spots in the oral cavity. purpose: to prevent a high risk of pjs, the dentist must establish its oral manifestation through early detection. case: a 14-year-old male patient attended complaining of a week-long pain at the corners of the lips. an extra-oral exam revealed fissure lesions, redness, white crust and pain. the patient had experienced bleeding in his bowel movements, abdominal pain, nausea and vomiting since childhood. a number of black, painless, macular lesions, some 1-3 mm in diameter, were present on the upper lips, lower lips, fingers and palms. case management: the patient was referred for a complete blood count check. the results obtained confirmed him to be suffering from severe anemia and he was, therefore, referred to an internist for treatment for pjs. conclusion: it can be concluded that the early detection of pjs is crucial in order that the patient receives prompt treatment. keywords: anemia; gastrointestinal polyps; hyperpigmentation; malabsorption; peutz-jeghers syndrome correspondence: maharani laillyza apriasari, departement of oral medicine, faculty of dentistry, universitas lambung mangkurat. jl. veteran 128 b, banjarmasin 70232, indonesia. e-mail: maharaniroxy@gmail.com case report introduction peutz-jeghers syndrome (pjs) is an inherited autosomal dominant disease determined by a mutation localized at 19p13.3. pjs results in polyps and mucocutaneeous pigmentation evident since childhood or early adulthood.1,2 in the united states, pjs is a rare disease with an incidence rate of between one case per 60,000 people and one case per 300,000 people. pjs has a prevalence of 1 in 120,000 live births, irrespective of race or gender. the mutant gene stk11 (also known as lkb1) is located at 19p13.3. stk11 is a tumor-suppressing, germline mutation gene which is documented in up to 70–80% of patients with pjs and as many as 15% of cases show complete or partial eradication of stk11.3–5 diagnosis of pjs is based on clinical findings and the histopathological patterns of polyps. histologically, these lesions show increased basiler melanin without a rise in the number of melanocytes.5 the manifestation of pjs may first be encountered by a dentist during routine examination in the form of pigmented spots in the oral cavity. round, oval or irregular, 1-5 mm diameter patches of brown or almost black pigmentation, irregularly distributed throughout the oral mucosa, gums, hard palate and lips are observed. the pigmented facial maculae, particularly encountered around the nose and mouth, are smaller.1,6 melanotic macules may be present in other body parts including the extremities, rectum, intranasal mucosa and conjunctiva.6 the intensity of macular pigment is unaffected by exposure to sunlight. fading or disappearance of the spots is usually observed in older age.5,7 dental journal (majalah kedokteran gigi) 2018 march; 51(1): 29–32 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p29–32 mailto:maharaniroxy@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p29-32 30 apriasari and carabelly/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 29–32 pjs is characterized by the occurrence of gastrointestinal hamartomatous polyps in association with mucocutaneous hyperpigmentation.1 this condition is usually accompanied by bowel obstruction and severe abdominal pain. acute upper gastrointestinal bleeding and chronic fecal blood loss may be present during the course of the disease.2,8 pjs is associated with significant morbidity, variable clinical causes and considerable predisposition to gastrointestinal and non-gastrointestinal malignancies. there is also an increased risk of malignant transformation of internal organs such as the gastrointestinal tract, pancreas, breast and thyroid.6 the purpose of this article is to prevent the high risk condition of pjs whose oral manifestation dentists must recognise through early detection. case a 14-year-old male patient sought medical treatment having experienced week-long pain at the corners of the lips. an extra-oral examination revealed fissure lesions, redness, a white crust and pain. the patient had suffered a history of frequent abdominal pain, nausea and vomiting since childhood, while bleeding had accompanied his bowel movements for a week. the lesions at both corners of the lips were diagnosed as angular cheilitis. there were a number of black, painless, macular lesions, 1-3 mm in diameter on the upper and lower lips, as well as on the fingers and palms and the buccal and labial mucosa. these lesions had first appeared in infancy and had subsequently increased considerably in size. in terms of anamnesis, the patient complained of often having experienced dizziness and light-headedness. according to the patient’s account, during his infancy, his father had passed away due to unknown causes. an extra oral examination showed paleness on the face, palms and conjunctiva. case management the definitive diagnosis of lesions at both corners of the lips was angular cheilitis which constituted the main reason for the patient having consulted a dentist. the patient was prescribed miconazole gel to be applied topically four times a day for two weeks. it was suspected that the patient was suffering from angular cheilitis due to anemia, a diagnosis confirmed by a history of frequent, week-long bouts of abdominal and bowel pain accompanied by bleeding. the results of an examination of alleged extra-oral melanotic lesions on the surface of the lips, hands, and labial and buccal mucosa represented the clinical manifestations of pjs. the patient was referred for a complete blood count, the results of which revealed the patient as suffering from severe anemia. therefore, the patient was referred to an internist for treatment for pjs. figure 3 and 4. peutz-jeghers syndrome manifestations in the form of multiple, black, 1-3 mm diameter, painless, macula lesions to the right and left of the buccal mucosa. discussion the diagnosis of pigmented oral lesions and perioral tissues is challenging. even though epidemiology can assist in orientating the clinician and certain lesions may be diagnosed on the basis of clinical manifestation, but the definitive diagnosis is usually based on histopathologic evaluation.7 pjs lesions within the oral mucosa and perioral are accompanied by several symptoms of gastrointestinal diseases. oral lesions may occasionally occur before the onset of gi disease, be present during the development of figure 1 and 2. peutz-jeghers syndrome symptoms in the form of multiple, painless, black, 1-3 mm diameter macula lesions on the lower and upper lips. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p29–32 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p29-32 31apriasari and carabelly/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 29–32 the disease or persist in a worsening form after the disease has been cured.9 patients with pjs often have a history of intermittent abdominal pain due to small bowel intussusceptions caused by polyps usually found in the gastrointestinal tract. on occasion, the oral lesions are similar to gastrointestinal lesions while, at other times, oral changes are caused by gastrointestinal disease which results in malabsorption disorders.9 lesions can also occur in other extraintestinal tissue such that in the kidneys, ureter, gall bladder, bronchial tree and nasal passages.1 the cause of pjs is related to the mutation of the stk11/ lkb1 (serine/threonine kinase 11) tumor suppressor gene located in chromosome 19p13. stk11 is a tumor suppressing gene. over-expression induces the arrest of cell growth at the g1 phase of the cell cycle and somatic inactivation of the allele of stk11. it is often observed in polyps or cancers in pjs patients.3 the stk11/lkb1 encodes a 433 amino acid, ubiquitously expressed protein with a central catalytic domain, and regulatory nand c-terminal domains. the function of lkb1 is to regulate downstream kinases. it includes adenosine monophosphate–activated protein kinase (ampk), together with the related kinases mark1 through mark4 and brain-specific kinases of the amphiddefective kinase sad. these are involved in cellular metabolic regulation–stress response and cellular polarity through tubulin stabilization, tight junction formation and e-cadherin localization. this occurs between the lkb1 pathway along with other tumor suppressor p53 and tensin homologue pten. the abnormalities in lkb1 function causes polyposis together with a loss of heterozygosity that influences tumorigenesis. the gene mutation is variable, resulting in a spectrum of phenotypic manifestations among patients with peutz-jeghers syndrome (localization of polyps and differing presentation of the macules) and a variable presentation of cancer.10,11 the clinical diagnostic criteria of pjs included histopathologically proven pjs polyps, the classic mucocutaneous pigmentation and a positive family history.4 some intussusceptions spontaneously reduce, while others lead to the development of small bowel obstruction. the pjs polyps can also ulcerate resulting in acute blood loss or chronic anemia.9 the patient consulted a dentist because of the degree of pain caused by the lesions at the corners of his mouth and the accompanying difficulty in eating and talking. angular cheilitis, as a side-effect of chronic anemia, was caused by pjs. the results of anamnesis confirmed that the patient complained of dizziness and fatigue, symptoms similar to those of anemia. other symptoms included: blood in the bowel movements, frequent abdominal pain, nausea and loss of appetite. the extra oral examination result showed paleness on the conjunctiva, palms and face with hyperpigmentation on the lips, lips and buccal mucosa. this supported the provisional diagnosis of the patient’s condition, namely; angular cheilitis resulting from pjsinduced anemia. angular cheilitis is one symptom of anemia evident through clinical oral manifestations. thus, the patient had to be referred for a complete blood count in order to confirm the presence or otherwise of anemia. this constituted the first action undertaken by the dentist. the result of a complete blood count test confirmed that the patient was suffering from anemia, given the low hemoglobin/hb 5.8 dl (normal 13 to 17.5 dl), erythrocytes 4.26 million/ml (normal 4,5-6 million/ml), hematocrit/ pcv 22.3% (normal 40-50%), thrombocyte 772,000/μl (normal 150000-350000), mcv 52.3 fl (normal 80-97 fl), mch 13.6 pg (normal 27-32 fl), and mchc 26% (normal 32-40%). in this phase, latent anemia often leads to abnormalities in the oral mucosa, including: glossitis, glossodynia, angular cheilitis, recurrent aphthous stomatitis and burning mouth syndrome.5 a diagnosis of anemia was supported by the patient’s clinical symptoms such as fatigue and dizziness, as well as angular cheilitis and tongue depapilation. anemia causes disruption to the circulation of oxygen around the body whose tissues receive it from red blood cells. if the number of such cells decreases, this will cause reduced hemoglobin resulting in a lack of oxygen. chronic anemia produces clinical manifestations in patients, including: fatigue, weakness and palpitations.5,12 anemia pathophysiology which leads to angular cheilitis is a form of anemia that causes enzyme activity in the mitochondria in the cell to decrease by disrupting the transport of oxygen and nutrients. this impairs cellular immunity, reduces the activity of bactericidal polymorphonuclear leukocytes, resulting in an inadequate antibody response and abnormalities in the epithelial tissues. anemia causes the activity of enzymes in cell mitochondria to decrease by disrupting the transport of oxygen and nutrients, thus inhibiting the differentiation and growth of epithelial cells. as a result, the process of terminal differentiation of epithelial cells toward the stratum corneum will be impeded, and subsequent oral mucosa will be thinner because of the absence of normal keratinization, atrophy and greater susceptibility to ulceration. this causes depapilation of the tongue in patients, a condition often occurring in individuals suffering from a deficiency of vitamin b12, folate, and iron.12–14 the therapy used to treat angular cheilitis consists of the topical application of miconazole gel four times a day for two weeks. supplements are administered orally as a maintenance dose once a day for a month. the supplements contain 250 mg of fe gluconate, 0.2 mg of manganese sulfate, 0.2 mg of copper sulfate, 50 mg of vitamin c, 1 mg of folic acid and vitamin b12 is prescribed for nutritional deficiency-related anemia. these supplements contain ferrous gluconate which is iron essential for energy metabolism. manganese sulfate and copper sulfate are both substances that support the absorption of iron by the intestines and subsequent introduction into the bloodstream through blood serum. vitamin c supports the liquidising of iron facilitating its easy absorption by the intestines. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p29–32 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p29-32 32 apriasari and carabelly/dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 29–32 vitamin b12 and folic acid are important cofactors for blood cell dna synthesis. the patient was referred to an internist for treatment of digestive disorders.13,14 patients with pjs are at greater risk of developing gastrointestinal and non-gastrointestinal malignancies. other non-gastrointestinal sites of malignancy include: the pancreas, lung, breast, uterus, cervix, ovary, testis and thyroid.1 a common recommendation for pjs patients is that they undergo not only gastrointestinal multiple polyp examination, but also regular lifelong cancer screening. early detection and proper observation are vital in order to minimize the risk of carcinoma.1 the management of pjs has to be undertaken by an interdisciplinary team and aids in the early detection and monitoring of this disease.2 in the final stage of the management of pjs, after oral lesion treatment, the patient was referred to an internist for treatment of this systemic condition. it can be concluded that the early detection of pjs is important to enable dentists to provide patients with appropriate therapy. references 1. parikh sb, parikh bj, prajapati ha, shah ck, shah nr. peutzjeghers syndrome. gujarat med j. 2013; 68(2): 106–8. 2. suresh kv, shenai p, chatra l. peutz-jeghers syndrome: in siblings with palmer-plantar pigmentation. j indian acad oral med radiol. 2011; 23(1): 68–72. 3. to bat, cagir b. peutz-jeghers syndrom. 2017. p. 1–5. available from: https://emedicine.medscape.com/article/182006-overview. accessed 2017 dec 20. 4. linhart h, bormann f, hutter b, brors b, lyko f. genetic and epigenetic profiling of a solitary peutz-jeghers colon polyp. cold spring harb mol case stud. 2017; 3(3): 1–9. 5. glick m. burket’s oral medicine. 12th ed. usa: people’s medical publishing house; 2014. p. 194–201. 6. patil s, raj t, rao rs, warnakulasuriya s. pigmentary disorders of oral mucosa. j pigment disord. 2015; 2(11): 1–9. 7. gondak ro, da silva-jorge r, jorge j, lopes ma, vargas pa. oral pigmented lesions: clinicopathologic features and review of the literature. med oral patol oral cir bucal. 2012; 17(6): e919–24. 8. bentley bs, hal hm. obstructing hamartomatous polyp in peutzjeghers syndrome. case rep radiol. 2013; 2013: 1–3. 9. jaja m m, bozzolo p, ni k la nder s. o ra l ma n ifest at ions of gastrointestinal disorders. j clin exp dent. 2017; 9(10): e1242–8. 10. katajisto p, vaahtomeri k, ekman n, ventelä e, ristimäki a, bardeesy n, feil r, depinho ra, mäkelä tp. lkb1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis. nat genet. 2008; 40(4): 455–9. 11. rosner m, hanneder m, siegel n, valli a, fuchs c, hengstschlager m. the mtor pathway and its role in human genetic diseases. mutat res. 2008; 659(3): 284–92. 12. regezi ja, sciubba jj, jordan rck. oral pathology: clinical pathologic correlations. 7th ed. st. louis: saunders; 2016. p. 23–6. 13. gammon a, jasperson k, kohlmann w, burt rw. hamartomatous polyposis syndromes. best pract res clin gastroenterol. 2009; 23(2): 219–31. 14. apriasari ml, tuti h. stomatitis aftosa rekuren oleh karena anemia. j dentofasial. 2010; 9(1): 39–46. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p29–32 https://emedicine.medscape.com/article/182006-overview http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p29-32 �� vol. 43. no. 1 march 2010 research report the frequency of bottle feeding as the main factor of baby bottle tooth decay syndrome mochamad fahlevi rizal1, heriandi sutadi1, boy m bachtiar2, and endang w bachtiar2 1department of pediatric dentistry 2department of oral biology faculty of dentistry, university of indonesia jakarta indonesia abstract background: dental caries re�ains as �ain �r��le� in �nd�nesia and its �revalence is �ig� (90.05%). h���ever, t�ere is n� a��r��riate data t�at can �e �sed t� analyze dental caries in t�ddlers, es�ecially �a�y ��ttle t��t� decay syndr��e (bb�d), t���g� t�e n���er �� bb�d cases is �ig� in s��e �ediatric dental clinics (90% �� �atients visiting t�e clinics). even t���g� s��e �act�rs �ave already �een c�nsidered t� �e t�e risk �act�r �� bb�d, t�e �ain risk �act�r �� bb�d is still �nkn���n, es�ecially bb�d in �nd�nesia. purpose: ��is researc� ��as ai�ed t� ��tain data relating ��it� ��ttle-�eeding �a�it in 3–5 year �ld c�ildren in �nd�nesia and its caries risk. method: ��e st�dy ��as an ��servati�nal researc� c�nd�cted ��it� clinical e�a�inati�n t�r��g� caries stat�s (de�t) �� eac� c�ild deserved �y �ediatric dentists and t�r��g� q�esti�nnaire distri��ted t� �arents t� e�a�ine t�e risk �act�r �� bb�d. o�servati�n ��as c�nd�cted �n 62 c�ildren in t�e range �� age 3 t� 5 years �ld ��it� ��ttle-�eeding �a�it. result: ��e res�lts revealed t�at stat�s �� caries ��as vari��s. ��e data s����ed t�at t�e �req�ency �� ��ttle �eeding ��re t�an t��ice c��ld trigger bb�d 2.27 ti�es �ig�er t�an �t�er �act�rs s�c� as t�e �se �� ��ttle �eeding as a �aci�ier �ri�r slee�ing, t�e �eri�d �� ��ttle-�eeding, and t�e �reast-�eeding e��erience. conclusion: t���g� �ilk as s��tract can ��ssi�ly �ec��e a �act�r triggering caries, t�e �req�ency �� ��ttle-�eeding is �ig�ly c�nsidered as �ain �act�r. since it c��ld ��d�lated t�e �acterial c�l�nizati�n �n dental s�r�ace, ���ic� a��ects its vir�lence. key words: t�e �req�ency �� ��ttle-�eeding, risk �act�r, �a�y ��ttle t��t� decay syndr��e abstrak latar belakang: karies �asi� �enjadi �asala� �ta�a di �nd�nesia. dala� �raktek se�ari-�ari �revalensi karies �asi� sangat tinggi (90.05%). bel�� ada data yang �e�adai dala� �enelaa�an karies yang s�esi�ik �ada anak �alita sela�a ini k��s�snya kas�s sindr��a karies ��t�l (skb) se�entara it� kas�s skb dite��kan sangat tinggi di �e�era�a klinik gigi anak (90% dari j��la� �asien yang datang ke klinik). be�era�a �akt�r �enjadi resik� kejadian skb dan �el�� diketa��i �akt�r resik� �ta�a kejadian karies k��s�snya di �nd�nesia. tujuan: penelitian ini dilak�kan g�na �enda�atkan data yang �er����ngan dengan ke�iasaan �in�� s�s� ��t�l �ada anak �sia 3–5 ta��n di �nd�nesia serta resik� kejadian karies yang diti���lkannya. metode: penelitian ini �er��akan �enelitian ��servasi�nal yang dilak�kan dengan �et�de �e�eriksaan klinis �elal�i �encatatan stat�s karies (de�t) setia� anak �le� d�kter gigi anak serta �engisian k�esi�ner yang dilak�kan �le� �rang t�a �nt�k �enent�kan �akt�r resik� kejadian skb. penga�atan dilak�kan �ada 62 �rang anak �sia 3–5 ta��n yang �e���nyai ke�iasaan �in�� s�s� ��t�l ses�ai dengan kriteria inkl�si. hasil: hasil �e�eriksaan klinis dan k�esi�ner �e��erikan ga��aran stat�s karies yang �ervariasi. data yang dida�at dari �enelitian ini �enjelaskan, �a���a �rek�ensi �in�� s�s� ��t�l le�i� dari d�a kali �enye�a�kan skb 2.27 kali le�i� �esar di�andingkan dengan �e�era�a �akt�r lain, se�erti �enjadikannya �engantar tid�r, la�anya �eng�ns��si, dan ri��ayat �in�� as�. kesimpulan: s�s� se�agai s��trat ��ngkin da�at dijadikan alasan kejadian karies akan teta�i yang �enjadi resik� �ta�a kejadian adala� �rek�ensi ��rizal et al.: frequency of bottle-feeding introduction dental caries remains as a dental health problem among children in indonesia. the prevalence of caries among toddlers in indonesia is approximately 85%, while caries prevalence in general is approximately 90.05%.1-3 the data indicated that the public health improvement programs conducted by the government for long time is considered to be failed. however, this epidemiologic description is not only found in developing countries, but also found in industrial countries.4-12 world health organization in 2003 reported that the prevalence of caries among children reached 60–90%.13 as a result dental caries, especially nursing bottle syndrome (nbs) or baby bottle tooth decay (bbtd) syndrome, becomes one of major concerns for medical experts, and many research have already been conducted.6 since baby bottle tooth decay (bbtd) syndrome usually attacks toddlers, it is also known as nursing caries, baby bottle tooth decay, rampant caries, labial caries, and maxillary anterior caries.14 in addition, clinical phenomena found in jakarta and surrounding areas indicate that the number of bbtd patients in pediatric dental clinics is high. the preliminary research conducted in three hospitals (two in urban area and one in rural area of jakarta) showed that the proportion of children who suffer dental caries was about 95%. based on that result, we knew that even though milk is considered as the source of nutrients for those children, not all of children with bottle-feeding habit are suffering caries. the number of tooth decay cases in children who suffer bbtd is various. nevertheless, as reported by some researcher, the patients of bbtd are dominated by toddlers who consume milk.16, 17 however, there is a controversy that nursing bottle feeding could cause bbtd. milk reported could prevent demineralization process in enamels.18,19 on the other hand bowen20 reported that the sugar additional (2% minimum) can increase the cariogenity of milk. it proved in the experiment using mice. those differences then consider as an obstacle in determining whether milk could cause bbtd in toddlers who have bottle-feeding habit. most of children in urban areas rely on bottle milk as the source of nutrients.21 therefore, the further analysis of the condition in indonesia, especially in jakarta, is needed to study baby bottle tooth decay (bbtd) syndrome that mostly attack toddlers. the process of tooth decay which started in early ages could affect the growth and development of children.15 the study was aimed to examine the bottle-feeding habit in 3-5 year old children in indonesia and its caries risk. material and method this research was a cross sectional study. the subject of study was obtained from non-probability sampling method with consecutive sampling technique. the subject were 62 children in the range of age 3 to 5 years old, with primary teeth have erupted and have bottle-feeding habit. clinical examination was conducted in children and to obtain further information, questionnaire was distributed to their parents. the questionnaire searched the information of the breast feeding experience (period of breast feeding), and the bottle-feeding habit (the usage, period, and frequency of bottle-feeding). clinical examination was done under sufficient light. afterwards, the status of caries was noted based on world health organization standard.22,23 the analysis of prevalence ratio then was conducted to examine any risk factors of bottle-feeding habit. the study had been approved by the research ethic commission of faculty of dentistry, university of indonesia, and the subjects’ parents had been informed and asked for their written approval prior to the study. result based on consecutive method, 62 children were considered as the subjects of the research since they met some inclusion criteria. the distribution of the subjects as seen in table 1, showed that the distribution of boys (61.3%) was bigger than that of girls (38.7%). the age interval of subjects was in between 3 to 5 years old with the various numbers of teeth erupted (20 to 24 teeth), but most of subject had 20 teeth erupted (85.5%). most of subjects (72.6%) suffer caries which was similar to baby bottle tooth decay (bbtd) syndrome, while the rest (27.4%) was free from caries though they had bottlefeeding habit. the de�t score of all subjects approximately 4.66 teeth. the teeth were classified into some groups based on the number of decayed tooth, it revealed that 27.4% were free from caries, 40.3% one to five teeth had caries, and 32.3% had caries in more than five teeth. the questionnaire result showed that 83.9% subjects stopped breast feeding since their first year. most of them (66.1%) still continue drinking milk with bottle. the children who drinking 4 times or more a day reach 46.8% and 53.2% subjects had bottle-feeding once a night, while 32.3% of them had bottle-feeding twice a night. moreover, 9.7% of them had bottle-feeding three times a night, while k�ns��si s�s� ��t�l it� sendiri. k�ndisi ini da�at di����ngkan dengan ��d�lasi s��strat ter�ada� �erke��angan k�l�nisasi �akteri di �er��kaan gigi, se�ingga secara tidak langs�ng j�ga �e��engar��i vir�lensinya. kata kunci: frek�ensi �in�� s�s� ��t�l, �akt�r resik�, sindr��a karies ��t�l c�rres��ndence: mochamad fahlevi rizal, c/o: bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta, indonesia. e-mail: levi_pedo@yahoo.com �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 44-481 only 3.2% of them had bottle-feeding four times a night. it revealed that most of subjects (72.6%) rely on bottlefeeding as pacifier prior sleeping, while the rest (27.4%) did not. in addition, for profiling those children with bottlefeeding habit as shown in table 1, the analysis of prevalence ratio (pr) was then conducted as seen in table 2. it showed that nursing bottle-feeding prior sleep could not always increase the risk of bbtd since the score of pr was 1.03 times with the interval of reliability was in between 0.91 to 1.15. in details, it showed that the frequency of bottlefeeding ≥ 2 times a day could increase the risk of bbtd 2.27 times with the interval of reliability between 2.17 to 2.37, while the frequency of bottle-feeding ≥ 2 times a night could increase the risk of bbtd 1.16 times with the interval of reliability in between 1.04 to 1.28. moreover, analysis based on the age when they stopped bottle feeding, the prevalence of caries was almost 1. it means that this factor did not affect the number of caries occurred. nevertheless, it appeared that breast feeding could give protection against bbtd. table 2. the estimation of prevalence ratio (pr) as the risk estimation of bbtd in children who have bottlefeeding habit analyzed group n (subject) prevalence ratio (pr) interval of reliability 95% the risk of bbtd in children having bottlefeeding as pacifier the risk of bbtd in children having bottlefeeding until in the age of 3 years old or more the risk of bbtd in children whose frequency of bottle-feeding is  twice per day** the risk of bbdt in children whose frequency of bottle-feeding is  twice per night** the risk of bbtd in children having breastfeeding 62 62 62 62 62 1.03 0.9++ 2.27 1.16 0.716* 0.91–1.15 0.48–1.02 2.,17–2.37 1.04–1.28 0.6–0.83 * < 1: causing protection effect ++ 1: causing neutral effect ** once of bottle-feeding equals to 200 cc discussion the history of bbtd is needed to be examined in this study in order to categorize kinds of caries occurred in those children which can possibly be caused by bottlefeeding habit or by other factors. the categorization is also needed in this study to analyze further the specific damage occurred in their primary teeth. therefore, if the tooth decay has already attacked their lower anterior teeth, they could not involve as the subjects of this study since this condition can cause bias result, which indicates caries with other pattern. moreover, only children in the range of age of 3 to 5 years old were chosen to be the subjects of this study since their primary teeth were still in the phase of growth,24 thus, it means that only those whose primary teeth are still persisted table 1. profile of children, as the subjects of the research, who have bottle-feeding habit n (person) % sex male female the total number of teeth 20 21 23 24 diagnosed bbtd syndrome bbdt syndrome free of caries number of caries teeth free of caries 1–5 teeth > 5 teeth the length period of breast-feeding until 1 year old until 2 years old until 3 years old never the length period of bottle-feeding still on going until 2 years old until 3 years old frequency of bottle-feeding per day* once twice 3 times 4 times or more the role of bottle feeding as pacifier not as pacifier frequency of bottle-feeding per night* once twice 3 times 4 times or more none 38 24 53 3 2 4 45 17 17 25 20 52 7 1 2 41 9 12 3 9 21 29 45 17 33 20 6 2 1 61.3 38.7 85.5 4.8 3.2 6.5 72,6 27,4 27.4 40.3 32.3 83.9 11.3 1.6 3,2 66.1 14.5 19.4 4.8 14.5 33.9 46.8 72.6 27.4 53.2 32.3 9.7 3.2 1.6 *once of bottle-feeding equals to 200 cc ��rizal et al.: frequency of bottle-feeding can involve in this study. it is aimed to obtain the description of caries caused by bottle-feeding habit. healthy tooth reflect sufficient remineralization and demineralization processes. so we assumed that the unbalancing condition can be considered as a trigger factor of caries. caries free can reflect the balance condition between remineralization and demineralization processes. however, the similar condition cannot be expected to be found in children under three years old. this statement is also supported by the research using cariostat as the predictor of caries which found that the increasing of caries in children around 3–5 years old is not as progressive as that in children under 3 years old.25 the succession of anterior teeth in children older than five years old has generally been occurred,24 thus, the possibility of the involvement of anterior teeth becomes bias. based on the analysis result of prevalence ratio in table 2, it showed that the period of bottle-feeding habit was only 0.96. it means that the period of bottle-feeding is not considered as the only factor causing bbtd. furthermore, based on the distribution of the data, it appeared that the number of boys who had bottle-feeding habit is not the same as that of girls. the result make sense since caries is not an infectious diseases which affected by gender.26-29 even though there is a research that distinguishes the dental health treatment based on gender, there is still no significant difference in result.25,30 actually, as an infectious disease caused by bacteria, the incubation and colonization of bacteria tends to be determined by micro environment inside oral cavity. but, even though this micro condition is also influenced by internal factors like hormonal factor, it will not affect too much on children in the age of 3 to 5 years old. eighty five point five percents of the subjects have met the criteria to become the samples of the study since the total number of their teeth was 20 (table 1). the rest of them (14.5%) had more than 20 teeth. however, this condition did not affect the study since the erupted teeth were permanent molars, and it did not affect the diagnosis of baby bottle tooth decay syndrome. the examination conducted on children who have bottle-feeding habit showed that 72.6% of them suffer baby bottle tooth decay with various numbers of caries (1–15) teeth and with the mean score of deft 4.66 teeth. it means that every child has caries in their four teeth. pattern of baby bottle tooth decay syndrome which is linier with the order of dental eruption, concluded that most of children with baby bottle tooth decay syndrome get caries in their four anterior teeth of upper jaw, and it is not different from what happens with non caucasian children in the age of 4–5 years old in countries with good health program.31 however, caries in the upper incisive can not only cause infection for those children, but can also affect their aesthetics, especially concerning with their self-esteem. if compared with the research involving five year old children in china which population is similar to indonesia’s in the term of biosocioculture, the mean score of de�t is almost similar (the mean of de�t 3 from 780 children).26 similarly, the mean score of de�t in riyadh, saudi arabia is 5.27 nevertheless, the mean score of de�t in 0–5 year old children is different as shown in the research conducted in brazil, which is 1.53. this condition can possibly occur since based there is a correlation between the length of age interval and caries occurred, which means that the increasing of caries occurs as the increasing of age.32 it is also supported by the result of a research involving toddlers in depok in 1992 which mean score of deft is 4.67 teeth.25 it indicates that there has not been significant change in the mean of de�t for the last two decades. actually, tooth decay suffered by most of children (72.6%) is possibly caused by many factors. one of them is related with the activity of parents in urban areas that tends to be busy and have not enough time to meet their children. like in indonesia, this phenomenon also occurs in some countries, except in developed countries in which promotion and prevention programs that have already been developed can reduce the unawareness of parents toward their children.33 therefore, it can be concluded that bottle-feeding habit is considered to be an important factor triggering caries. the result showed that 66.1% of the subjects in this study have bottle-feeding habit. the proportion of children who use bottle-feeding prior sleep is the same with the proportion of children who suffer caries. in general, it is known that 45 children (72.6%) have this bottle-feeding habit, but, not all of the children who have bottle-feeding habit prior sleep get caries. based on the result of prevalence ratio in table 2 it is known that those who rely on bottlefeeding as pacifier when falling asleep do not always get caries since the interval of reliability is between 0.91–1.15. nevertheless, based on the observation of 55 kindergarten students in italia, it is known that bottle-feeding habit can cause caries.28 similarly, researches conducted in north brisbane and montreal is also showed that bbtd emerged in children who rely on bottle-feeding when falling asleep.34,35 moreover, based on the research conducted in australia, it is known that the frequency of bottle-feeding twice or more a day can increase the risk of caries for about 2.27 times, while the frequency of bottle-feeding twice or more a night can increase the risk of caries for about 1.16 times.31 the occurence of bbtd can be examined from the experience of bottle-feeding habit. most of the children got breast feeding until they were one year old (83.9%). it means that the weaning of breast feeding start after first primary tooth erupted or just before the eruption of primary tooth. therefore, the correlation between breast feeding and bbtd can be eliminated. it is also supported by the score result of the prevalence ratio, which is about 0.716. it means that 60–83% of subjects have breast feeding as a protection against caries or breast feeding did not affect caries occurrence during the study. this result is similar to the result of the cross sectional research conducted on children in the age of 2–5 years old in the united states of �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 44-481 america in 1576 which concludes that the length period of breast feeding cannot be considered as the single risk factor of caries.4 therefore, it can be concluded that though milk as subtract can possibly become a factor triggering caries, the frequency of bottle-feeding is highly considered as main factor. since it could modulated the bacterial colonization on dental surface, which affects its virulence. acknowledgement the grand research of universitas indonesia. references 1. yuyus r, magdarina d, sintawati f. karies gigi pada anak balita di 5 wilayah dki tahun 1993 cermin dunia kedokteran 2002; 134: 39–42. 2. soemantri s, pradono j, bachroen c. survey kesehatan nasional: badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia; 2004. p. 215–16. 3. sutadi h. aktifitas karies gigi anak sekolah di jakarta. jurnaljurnal kedokteran gigi universitas indonesia 1993; 1(1): 23–5. 4. iida h, auinger p, billings rj, weitzman m. association between infant breastfeeding and early childhood caries in the united states. pediatrics 2007; 120: e944–e952. 5. robert y, sheiham a. the burden of restorative dental treatment for children in third world countries. int dent j 2002; 52(1): 1–9. 6. ferro r, besostri a, meneghetti b, beghetto m. comparison of data on early childhood caries (ecc) with previous data for baby bottle tooth decay (bbtd) in an italian kindergarten population. eur j paediatr dent 2004; 5(2): 71–5. 7. pitts nb, boyles j, nugent zj, thomas n, pine cm. the dental caries experience of 5-year-old children in england and wales (2003/4) and in scotland (2002/3). surveys co-ordinated by the british association for the study of community dentistry. community dent health 2005; 22(1): 46–56. 8. livny a, assali r, sgan-cohen hd. early childhood caries among a bedouin community residing in the eastern outskirts of jerusalem. bmc public health 2007; 24(7): 167. 9. helderman whvp, soe w, hof mavt. risk faktors of early childhood caries in a southeast asian population. j dent res 2006; 85(1): 85–88. 10. schroth rj, smith pj, whalen jc, lekic c, moffatt mek. prevalence of caries among preschool-aged children in a northern manitoba community. j can dent assoc 2005; 71(1): 27. 11. stadtler p, bodenwinkler a, sax g. prevalence of caries in 6-year-old austrian children. oral health prev dent 2003; 1(3): 179–83. 12. seow wk. biological mechanisms of early childhood caries. community dent oral epidemiol 1998; 26(1 suppl): 8–27. 13. edelstein b. the dental caries pandemic and disparities problem. bmc oral health 2006; 6(suppl 1): s2. 14. ismail ai, sohn w. a systematic review of clinical diagnostic criteria of early childhood caries. j public health dent 1999; 59(3): 171–91. 15. sheiham a. dental caries affects body weight, growth and quality of life in pre-school children. british dental journal 2006; 201(10 ): 625–26. 16. tiberia mj, milnes ar, feigal rj, morley kr, richardson ds, croft wg, cheung ws. risk faktors for early childhood caries in canadian preschool children seeking care. pediatr dent 2007; 29(3): 201–8. 17. smith pj, moffatt me. baby-bottle tooth decay: are we on the right track? int j circumpolar health 1998; 57 suppl 1: 155–62. 18. levine rs. milk, flavoured milk products and caries. br dent j 2001; 191(1): 20. 19. merritt j, qi f, shi w. milk helps build strong teeth and promotes oral health. j calif dent assoc 2006; 34(5): 361–6. 20. bowen wh, pearson sk. effect of milk on cariogenesis. caries res 1993; 27(6): 461–6. 21. wiley as. does milk make children grow? relationships between milk consumption and height in nhanes 1999-2002. am j hum biol 2005; 17(4): 425–41. 22. cypriano s, hoffmann rhs, sousa mdlrd, wada rs. dental caries experience in 12-year-old schoolchildren in southeastern brazil. j appl oral sci 2008; 16(4): 286–92. 23. world health organization, programme ohcap. world health organization, oral health country/ area profile programme caries prevalence: dmft and dmfs. in: world health organization; 2008, http://www.whocollab.od.mah.se/expl/orhdmft.html. accesssed june 20, 2008. 24. tooth development in: american dental association; 2005, www. ada.org/public/topics/tooth_eruption.asp. accessed may 14, 2008. 25. sutadi h. the determination of the predictive value of a caries activity test and its suitability for mass screening and clinical use in indonesia. disertation. okayama: okayama university dental school; 1992. p. 115-25. 26. li kz, li x, hu dy, fan x, nie l. prevalence of deciduous tooth caries in 780 children aged 5 years. hua xi kou qiang yi xue za zhi 2008; 26(1): 70–72. 27. wyne ah. caries prevalence, severity, and pattern in preschool children. j contemp dent pract 2008; 9(3): 24–31. 28. campus g, solinas g, sanna a, maida c, castiglia p. determinants of ecc in sardinian preschool children. community dent health 2007; 24(4): 253–6. 29. kalsbeek h, truin gj, poorterman jh. oral health and gender. ned tijdschr tandheelkd 1998; 105(11): 408–11. 30. dagli rj, tadakamadla s, dhanni c, duraiswamy p, kulkarni s. self reported dental health attitude and behavior of dental students in india. j oral sci 2008; 50(3): 267–72. 31. hallett kb, o'rourke pk. pattern and severity of early childhood caries. community dent oral epidemiol 2006; 34(1): 25–35. 32. ferreira sh, béria ju, kramer pf, feldens eg, feldens ca. dental caries in 0to 5-year-old brazilian children: prevalence, severity, and associated faktors. int j paediatr dent. 2007; 17(4): 289–96. 33. tickle m, milsom km, humphris gm, blinkhorn as. parental attitudes to the care of the carious primary dentition. br dent j 2003; 195(8): 451–5. discussion 449. 34. hallett kb, o'rourke pk. social and behavioural determinants of early childhood caries. aust dent j 2003; 48(1): 27–33. 35. galarneau c, brodeur j-m, gauvin l. the cariogenic nature of childhood bedtime rituals. journal de l’ordre des dentistes du québec 2006; supplement: 17–19. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base �� vol. 43. no. 1 march 2010 tissue engineered bone as an alternative for repairing bone defects evy eida vitria and benny s. latif department of oral & maxillofacial surgery faculty of dentistry, universitas indonesia jakarta indonesia abstract background: dentist es�ecially �ral s�rge�n, are �req�ently �aced ��it� de�ect in ��ne res�lting �r�� disease �r tra��a. �� t�e de�ect is s�all, it ��ill �req�ently �as a g��d �ealing, ����ever, i� t�e de�ect is larger, inc���lete regenerati�n ��ten �cc�rs and a �i�r��s scar res�lts. �rans�lantati�n �� a�t�gen��s ��ne �as �een �ne �� t�e ��st �req�ent �r�ced�res �� rec�nstr�ctive �ral and �a�ill��acial s�rgery �eca�se it �as s����n e�cellent clinical s�ccess; ����ever, a�t�gen��s ��ne gra�ting is ��ten related t� disadvantages like li�ited availa�ility, and d�n�r ��r�idity. purpose: ��e ��r��se �� t�is revie�� is t� e��lain t�e �asic �rinci�les �� tiss�e engineering, �ackgr��nd �� regenerati�n �r�cess, als� advantages and disadvantages �� tiss�e engineered ��ne c���ared t� a�t�gen��s ��ne gra�t. review: recently, tiss�e engineered ��ne �r�vides a �r��ising strategic inn�vati�n and �ec��es a ne�� alternative ��r ��ne regenerati�n �r�cess. �iss�e engineering is a ter� �riginally �sed t� descri�e tiss�e �r�d�ced in is�lati�n and c�lt�re �y cells seeded in vari��s ��r��s a�s�r�a�le �atrices. �iss�e engineering generally c���ines t�ree key ele�ents (�iss�e engineering �riad) i.e: sca���lds (�atrices), signaling ��lec�les (gr���t� �act�rs), and cells (�ste��last, �i�r��last, etc). conclusion: �iss�e engineering ��ill �acilitate initial ��ne �ealing in �rder t� acc���lis� tiss�e regenerati�n �r�cess. key words: �iss�e engineering, a�t�gen��s ��ne gra�t, ��ne de�ects abstrak latar belakang: se�rang d�kter gigi k��s�snya d�kter gigi �eda� ��l�t, seringkali di�ada�kan dengan keadaan de�ek t�lang aki�at dari s�at� �enyakit ata� tra��a. jika de�eknya kecil ��ngkin da�at se���� dengan �aik, teta�i �ila de�eknya �esar, ke��ngkinan regenerasi t�lang tidak se���rna dan �eng�asilkan scar/ jaringan �ar�t. �rans�lantasi dengan �engg�nakan a�t�gen��s ��ne gra�t �eski��n sa��ai saat ini �asi� �anyak dig�nakan �nt�k ��erasi rek�nstr�ksi di �idang �eda� ��l�t dan �aksil��asial karena tela� �en�nj�kkan ke�er�asilan klinik yang c�k�� �aik, na��n cara ini �e���nyai �anyak kek�rangan, diantaranya ��r�iditas dari sisi d�n�r. tujuan: ��j�an dari �en�lisan ini adala� �nt�k �enjelaskan tentang �rinsi�-�rinsi� dasar tiss�e engineering, �al-�al yang �er�eran dala� �r�ses regenerasi serta ke�nt�ngan dan ker�gian tiss�e engineering di�andingkan dengan a�t�gen��s ��ne gra�t. tinjauan pustaka: saat ini �engg�naan tiss�e engineered ��ne �er��akan s�at� strategi in�vati� yang tela� dike��angkan dan �e��erikan s�at� alternati� dala� �r�ses regenerasi t�lang. �iss�e engineering ata� rekayasa jaringan �er��akan s�at� istila� yang dig�nakan �nt�k �enjelaskan �agai�ana s�at� jaringan di�asilkan dengan cara is�lasi dan k�lt�r sel dala� �er�agai �atriks ��r��s a�s�r�le. �iss�e engineering akan �eli�atkan tiga ele�en k�nci (tiss�e engineering triad) yait� sca���ld (�atriks), ��lek�l-��lek�l signal (gr���t� �act�rs) dan sel-sel (�ste��last, �i�r��last, dll). kesimpulan: �eknik tiss�e engineering akan �e��asilitasi �r�ses a��al �enye����an t�lang se�ingga �r�ses regenerasi jaringan akan terca�ai. kata kunci: �iss�e engineering, a�t�gen��s ��ne gra�t, de�ek t�lang c�rres��ndence: evy eida vitria, c/o: bagian bedah mulut, fakultas kedokteran gigi universitas indonesia. jl. salemba raya no. 4 jakarta pusat, indonesia. e-mail: evy_eida@yahoo.com review article �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 11-16 introduction the need of bone regenerating is gradually increasing as the quality of life is improving and the consequence the life expectancy is also increasing. now, regeneration of bone tissue is still a challenging in cranio-maxillofacial surgery. the surgical treatment is commonly conducted for repairing bone defect caused by trauma, tumor, infection or any abnormal bone growth.1, 2 transplantation is a procedure to anticipate the above problems. in order to repair the bone defect, transplantation can be conducted by using many grafts, such as autogenous bone graft (graft derived from the patient’s body), allogeneic bone graft (graft obtained from donor), and bone matrix that has been demineralized (demineralized bone matrices) or synthetic biomaterial, like metal, ceramics, polymer, and composites.1–3 until now, the use of autogenous bone graft still becomes the first option for repairing the bone defect and regenerating, and is also commonly used for reconstruction in oromaxillofacial surgery. the advantages of autogenous bone graft are that there is no immunogenic reaction and that it has good osteogenicity and osteoinductivity. besides, autogenous bone graft can recruit mesenchim cells and then induce them to differentiate into osteogenic cells through osteoinductive growth factors.1–3 though the use of autogenous bone graft has many advantages, there are still many main weaknesses, such as morbidity of donor, continual pain after the surgery, hypersensitivity, infection, and paresthesia. the complication can occur in 10–30% patients. besides that, bone obtained is also limited.2,3 another alternative is by using allograft. the use of allograft can eliminate the weaknesses of autogenous bone graft, but the quality of bone obtained from allograft is worse than that is from autogenous bone graft. allograft has worse cell cellularity degree, worse revascularization, bigger resorbsion level, and slower bone formation than those in autogenous bone graft. the most serious disadvantages is that there is immunogenic reaction potency and viral transmission risk for the patients.3 though processing technique like demineralization, freeze-drying method, and irradiation can eliminate the immune response of patients, the processing can also disturb the graft structure and reduce the potency of inducing the bone recovery process (osteoinductivity) while there is still possibility of disease transmission.3 in order to anticipate those weaknesses, the new alternative technology for reconstructing the bone defect through tissue engineering technique by using bone marrow stem cells is developing now.3–6 many studies on animals have shown that tissue engineering can produce bone, either in non-bone environment (ectopic bone formation) or in bone environment (orthotopic bone formation).3–6 tissue engineering is actually a new multidisciplinary in medical, surgery, molecular and cellular biology, polymer and physiology chemistry. therefore, the objective of this study is to analyze the principles of tissue engineering as well as the advantages and disadvantages of tissue engineering compared with the transplantation of autograph or allograft. tissue engineering tissue engineering is tissue regenerating in body involving cells, biologic mediators, such as growth factors of synthetic or biologic matrix that can be implanted into the patient’s body in order to regenerate certain tissue.1 tissue engineering is multidisciplinary field using biologic principles and engineering technique for improving a substitute material that can repair and maintain the function of bone tissue.1 it involves the use of synthetic polymers in order to facilitate the regenerating process of tissue. these polymers then will be absorbed and substituted by natural and physiologic tissues.1 many studies of tissue engineering have actually been conducted either in vitro or in vivo, for instance: caplan7 who said that mitotic isolation and expansion from autologous stem cells can cause faster and more specific reparation of bone tissue. friedenstein et al.5 moreover, shows that a specific cell group, which is a colony forming fibroblast unit or mesenchim cells located in bone marrow can differentiate into many different cell types, including osteoblast. quarto et al.8 published the first clinic paper that reports the repairment of bone defect by using autologous bone marrow stromal cells. next, schimming & schmelzeisen9 conducted the first study on human beings showing that periosteum-derived osteoblast can form lamellar bone in 3 months after transplantation. urist10,11 then showed that bone tissue contains specific growth factors that can induce the bone formation in ectopic sites (non bone environment). tissue engineering actually involves 3 key elements (tissue engineering triad) which are: scaffolds (matrix), signalling molecules (growth factors), and cells (osteoblast, fibroblast). by combining those three elements, the process of tissue engineering can be conducted (figure 1).2 scaffolds (collagen, bone mineral, synthetics) cells (osteoblast, firoblast, chondrocytes) signalling molecules (growth factors, morphogens, adhecins) figure 1. tissue engineering triad.2 the brief procedure of tissue engineering involves the following stages: first, cells (osteoblast, fibroblast) and signaling molecules (protein growth factors) are ��vitria and latif: tissue engineered bone as an alternative for repairing bone defects induced into scaffolds or highly biodegradable matrix, and then those are cultured in vitro. after being cultured, those scaffolds are induced or implanted into a defected bone in order to induce the growing of new bone in vivo. those cells then will adhere into scaffolds, multiply or regenerate themselves, differentiate from non-specific or primitive cells into specific cells that have bone function, and continually organize into normal and health bone cells. finally, after engineering health new bone, those scaffolds then will degrade (figure 2).3 figure 2. the role of sca���ld as g�idance in the process of tissue engineering.3 however, it must be remembered and understood that we cannot harvest some cells like osteoblast, and then culture them for forming a complete or whole bone. in tissue engineering, there are three important components: matrix, cell and soluble regulator.1-3 matrix (porous structure) matrix in bone tissue engineering is involving many biomaterial groups, such as synthetic polymers, natural polymers, ceramic, and composites. synthetic polymer is an organic or inorganic structure . this material is widely used in biomedical field. its characteristics are degradable/ absorbable and non degradable/non absorbable. for instance, degradable synthetic polymer is polylactic acid and polyglycolic acid that have got hydrolysis into lactate acid and glicolate acid. nowadays, degradable synthetic polymers that are being improved are polycaprolactone, polyanhydrides, and polyphosphazenes. meanwhile, non degradable synthetic polymers that are being improved are polytetrafluoroethylene (ptfe), polymethylmethacrylate (pmma), and polyhydroxyethylmethacrylate (phema). these materials are commonly used for making dentures, arthroplasty, and cranioplasty, as well as used as cements in orthopedic prosthesis. ptfe, moreover, is commonly used for subcutan augment material and guide bone regeneration in order to regenerate bone by making line for osteoblast cells.1, 2,12-14 ceramics are materials that have osteoinductive porous structur. these materials are widely used in dentistry and in tissue engineering. ceramics commonly used in dentistry are alumina (al2o3) and hydroxyapatite (ha). alumina is very resistant to corrosion, and its biocompatibility is very good and strong. meanwhile, hydroxyapatite is ceramics with calcium phosphate as the basic materials and has been used more than 20 years in medical field and dentistry. hydroxyapatite is a main inorganic component of bone that is osteoinductive, biocompatible, and biodegradable, but has low mechanical power. degradation of hydroxyapatite is controlled by many chemical structures. besides hidroxyapatite, materials of ceramics commonly used are tricalcium phosphate (tcp). tricalcium phosphate can be degraded faster than hydroxyapatite.1,2, 12-15 natural polymer is extracellular protein which is often used as bone graft. natural polymer includes collagen (type i, ii, iii, iv), glycosaminoglycans copolymer, polysaccharide hyaluronic acid (hy) and chondroitin sulfat. polysaccharide hyaluronic acid is glycosaminoglycans found in synovial liquid and kartilago which can induce chondrogenesis and angiogenesis. if it is combined with collagen, it acts as matrix in bone regeneration. chondroitin sulfate is glycosaminoglycans found in kartilago functioning as scaffolds in tissue engineering. the mechanical strength of collagen matrix is little and its size is not enough to cover defect. collagen can be osteoinductive especially if it is combined with bone marrow.1, 2,5,8,13-15 composites is a combination between ceramics and polymer. for example, collagraft is a combination between collagen type i (95%) and collagen type iii (5%) taken from bovine and mixed with ha. collagraft is mostly used in orthopedic surgery. in craniomaxillofacial, bio-oss is often used and it is combination between collagen bovine and deorganified bovine bone. combination between collagen and ceramics made from calcium functions as osteoinductive i.e. a function of matrix found in bone that supports adhesion, migration, growth, and cell differentiation.1, 2, 5,8, 12-16 a matrix has some roles during the tissue regeneration in vivo. structurally, matrix can support the defect so that it can sustain its shape from defect and keep distortion away from the tissue. it can function as barrier for the tissue growth. it also functions as regulator of insoluble cell function through its interaction with other receptor cells. it can function as scaffolds to migrate and proliferate the cells in vivo or implant the cells in vitro.1, 3,6 cells dynamics of bone metabolism is a remodeling process that continually occurs through 3 main cells: osteoblast, osteocyte and osteoclast. osteoblast is a cell that has a role to synthesize and organize deposition and mineralize extracellular matrix of bone. the activity and differentiation of osteoblastic are organized by either systemic or local hormones, growth factors, ions, lipid and steroid. osteoblast, pre-osteoblast and osteoblastic work to investigate transduction signal. proliferation and differentiation of osteoblast cells are modulated by transforming growth factor beta (tgf-b) and bone morphogenetic proteins (bmps) that are very important in bone homeostasis.1,3,5, 17–19 osteocytes is a cell that has high differentiation with alkaline phosphatase activity, pth receptor and functions as mechanosensory cell. the mechanical stimulus can interfere the bone structure and the bone mass. osteocytes has lacuno-canalicular in bone porosity that mediates �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 11-16 mechanosensory system. mechanosensory system of osteocytes in bone responds any changes. consequently, there is a flow of interstitial liquid through osteostitic canalicular tissue. this flow will initiate the electrokinetic and mechanic signal. then, the secretion of molecule signals will take place, for examples, insulin-like growth factor, igf-1, prostaglandin g/h synthase, pge2 and nitrit oxide which contributes to coordinate metabolic response from adjacent cells: osteoblast, osteoclast. osteocytes has a role in cellular organization of bone that responds the changes of mechanics by augmenting and reducing from bone apposition. osteocytes do not resorb dentine surface in vitro. this indicates that osteocytes do not have a role in calcium homeostasis.1,3,6, 20-22 osteoclast is multinuclear cell from hemopoietic cell. it’s function is to to resorb bone. the bone resorbsion by osteoclast is the result of blend from acid intravesical cytoplasm and plasma membrane.1,3,6 soluble regulators soluble regulator is soluble molecule either used with or without another biomaterial as delivery system. there are some examples of soluble regulators such as growth factors–polypeptide mitogens, and differentiation factors (e.g.bone morphogenetic protein).2, 11 some functions of soluble regulators are stimulate cell diffusion and infiltrate in the defect, stimulate particular differentiation cell, stimulate angiogenesis process and act as chemoattractan for certain cells.2, 6, 11 in dentistry, platelet-derived growth factor has shown significant roles in tissue healing in which the role of growth factor in periodontal tissue engineering has shown mitosis effect, migration, matrix synthesis, and differentiation of periodontal ligament cells and osteoblast. in addition, bmp is frequently used with biomaterials like collagen, tricalcium phosphate or ha to surpass the bone defect.1,11, 18, 21-24 discussion bone tissue engineering has important role to overcome clinical problems especially dealing with bone defect retrieval by requiring 3 important elements: matrix, cell and soluble regulator/signaling molecules. in tissue engineering there are various approaches depending on the cell source e.g. autologous (taken from the patients), allogeneic (taken from donors) or xenograph (taken from animal); whether the scaffolds are used or not, such as the use of growth factor in the defective tissue found in small defect area. in larger defect area, matrix as structural factor is more needed; whether the scaffolds are implanted with cultured cells before the surgery or those cells are embedded in matrix and implanted when the surgery takes place.1-3 in bone tissue engineering there are two approaches: growth factor like bone morphogenic protein (bmp) and transforming growth factor (tgf), and osteogenic cells like stem mesenchim cells (mesenchymal stem cell). bone marrow is the source of osteogenic cells that has high proliferation and large capacity to differentiate.on the first approach (growth factor based), bone morphogenic proteins from tgfa are used. the weakness of this approach is that it needs high concentration to obtain osteoinductive effect. besides, its side effect is greater and the cost is expensive. on the second approach (cell-based approach) which is considered to be more interesting, combination between osteogenic cells and biomaterial scaffolds through ex vivo may trigger the growth of tissue structures in three dimensions.3, 9, 22–27 in bone tissue engineering, osteogenic potential and mesenchymal stem cells (msc) have widely been studied. these cells can easily be isolated from various tissues like fat tissue (adipose), muscle from the edge blood and bone marrow. msc does not only have ability to proliferate in a culture but also to change immature progenitor cells through several ways, for examples, osteogenic, chondrogenic or adipogenic.7, 25–27 previous studies are conducted on some animals as specimen/invitro concerning tissue engineering on jaw bone/alveolus. one of them is conducted by li et al.27 that studied repairing process on mandibula defect by applying bone tissue engineering on rabbit. osteoblast cells taken from the rabbit’s bone morrow are cultured and implanted in scaffolds in the form of allogeneic demineralized bone in order to form tissue engineering bone graft through in vitro which is used to repair bone defect in mandibula. vesala et al.,28 evaluated a variety of absorbable materials in order to direct bone regeneration on cranium bone defect by applying self reinforce poly-l, d-lactide 96/4 (sr-pla96) implanted on the rabbit’s cranium bone defect. from the study, it is obtained that on the 48th week the defect on cranium bone is perfectly covered. a study by weng et al.,29 involved human’s condyle tmj as model by applying a mixture between synthetic non woven mesh poly-glycolic acid fibers and polylactic acid in methylene chloride as scaffolds implanted with osteoblast cells from periosteum bovine for 12 weeks. after that, it is evaluated in two ways: macroscopic and microscopic. the result of the study shows that bone forming and cartilage take place and the bone tissue or cartilago found in condyle is normal.29 similar study reconstructing mandibula in human by applying titanium mesh filled with hydroxyapatite, rhbmp7 and bone marrow stromal cell in order to stimulate osteogenesis process on mandibula bone. in the follow-up process, repairing the defect on mandibula shows good result so that, as consequence, the quality of patient’s life will be increased.30 another study by weng29 involved dog’s alveolar mongrel bone which has resorbtion due to periodontal disorder. the study applies bone marrow stromal cell (bmsc) mixed with calcium alginate that is used to form gel which functions as scaffolds in bone tissue engineering. after it is evaluated for 4 weeks of post-surgery, mature ��vitria and latif: tissue engineered bone as an alternative for repairing bone defects bone has been formed and on the 12th weeks the forming of similar bone has normally taken place. since friedenstein et al.5 published the similar study, it has been known that mesenchymal stem cells (mscs) can be used to engineer mesenchim tissue like bone and cartilago therefore, researchers around the world work hard to obtain proper carrier for those cells. bone transplantation is conducted so that the bone regeneration will occur.5, 18 bone marrow is the source of msc. in addition, it is the source of osteogenic cells taken by simple aspiration procedure. this method is more minimal invasive than method which assembles osteogenic cells by biopsy from calvarium. besides bone marrow, periosteum, bone trabeculae taken from fat tissue and stem cell taken from dental pulp show osteogenic potentials.31–32 caplan,7 have combined mscs with scaffolds to produce bone matrix after being implanted. to gain success in tissue engineering, four conditions are required: number of cells with adequate osteogenic capacity, proper scaffolds to implant cells, factors to stimulate osteogenic differentiation in vivo, and sufficient supply of blood vessels. the first three conditions can be applied by tissue engineering while the fourth condition depends on patients like defect size. the lack of supply in blood vessels leads to the cell death after being implanted. this may cause the bone tissue engineering on the patients failed.3,8, 27, 31 the use of mscs in tissue engineering can be the best solution for regeneration in medical future in the near future. dental and maxillofacial surgeon often deal with large bone defect which is difficult to reconstruct so that they optimally need either bone tissue and biomaterial to restore structure and tissue function. hence, reconstructive maxillofacial needs an innovation in the form of studies or researches to seek biocompatible material which can be used in tissue engineering. the use of autogenous bone has become the main option to repair the bone defect, but difficulty in gaining enough amount of bone often appears. procedure to gain autogenous bone will bring some pain, anatomical restraint, and morbidity on donor domain. therefore, bone tissue engineering has important role to solve problems in clinics especially problems in bone defect repairing.18, 26, 32 it is concluded that tissue engineering will facilitate the healing process of bone so that the tissue regeneration will be obtained. the biggest challenge in tissue engineering is how to ensure that angiogenesis has important role in tissue regeneration where cells without sufficient supply will die and the regeneration will not be obtained. new biomaterials are needed to give response for unknown object and degrade perfectly in expected time. knowledge about bone tissue engineering should be developed more. thus, it needs further research, better materials of analysis, more realistic in vitro studies, better tissue developing through in vivo and non invasive approach. references 1. lynch se, genco rj, marx re. tissue engineering: applications in maxillofacial surgery and periodontics. illinois: quintessence publishing co; 1999. p. 3–13. 2. habibovic p, groot k. osteoinductive biomaterials-properties and relevance in bone repair. j tissue eng regen med 2007; 1: 25–32. 3. gert jm, joost db, ron k, clemens, ab. cell-based bone tissuecell-based bone tissue engineering. plos med 2007; 4(2): 9–10. 4. puelacher wc, vacanti jp, ferraro nf, schloo b, vacanti ca. femoral shaft reconstruction using tissue-engineered growth of bone. int j oral maxillofac surg 1996; 25: 223–8. 5. friedenstein aj, chailakhyan rk, gerasimov uv. bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. cell tissue kinet 1987; 20: 263–72. 6. anderson jm, davies je, toronto em. the cellular cascades of wound healing. bone engineering 2000; 81–93 7. caplan ai. mesenchymal stem cells. j orthop res.1991; 9: 641–50. 8. quarto r, mastrogiacomo m, cancedda r, kutepov sm, mukhachev v, lavroukov a, elizaveta k, maurilio m. repair of large bone defects with the use of autologous bone marrow stromal cells. n engl j med 2001; 344: 385–6. 9. schimming r, schmelzeisen r. tissue-engineered bone for maxillary sinus augmentation. j oral maxillofac surg 2004; 62: 724–29. 10. urist mr. bone: formation by autoinduction. science 2007; 150: 893–9. 11. urist mr, delange rj, finerman ga. bone cell differentiation andbone cell differentiation and growth factors. science 1983; 220: 680–6. 12. ichijima k, yoshikawa t, ohgushi h, nakajima h, yamada e, okumura n, jin iida. in vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution. transplantation 2000; 69: 128–34. 13. ohgushi h, goldberg vm, caplan ai. repair of bone defects withrepair of bone defects with marrow cells and porous ceramic. experiments in rats. acta orthop scand 1989; 60: 334–9. 14. ohgushi h, goldberg vm, caplan ai. heterotopic osteogenesisheterotopic osteogenesis in porous ceramics induced by marrow cells . j orthop res 1989; 7: 568–78. 15. pelissier p, villars f, mathoulin-pelissier s, bareille rinfluences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats. plast reconstr surg 2003; 111: 1932–41 16. kon e, muraglia a, corsi a, bianco p, maracci m, martin i, boyde a, ruspantini i, chistolini p, rocca m, giardino r, cancedda r, quarto r: autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. j biomed mater res 2000; 49: 328–7. 17 . schliephake h, knebel jw, aufderheide m, tauscher m. use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep. int j oral maxillofac surg 2001; 30: 531–7. 18. kruyt mc, dhert wj, yuan h, wilson ce, van blitterswijk ca, verbout aj, de bruijn jd. bone tissue engineering in a critical size defect compared to ectopic implantations in the goat. j orthop res 2004; 22: 544–51. 19. h a y n e s w o r t h s e , g o s h i m a j , g o l d b e r g v m , c a p l a n a . characterization of cells with osteogenic potential from human marrow. bone 1992 ;13: 81–8. 20. schliephake h, knebel jw, aufderheide m, tauscher m. the role of osteoprogenitor cells in bone formation: int j oral maxillofac surg 2004; 25: 51–8. 21. shang q, wang z, liu w, shi y, cui l, cao y. tissue-engineered bone repair of sheep cranial defects with autologous bone marrow stromal cells. j craniofac surg 2001; 12: 586–93. 22. drosse e, volkmer r, capanna p, biase w, mutschler m. schieker. tissue engineering for bone defect healing: an update on a multicomponent approach injury..eur j trauma emerg surg 2008; 39: 9–20 23 . blum js, barry ma, mikos ag, jansen ja. in vivo evaluation of gene therapy vectors in ex vivo-derived marrow stromal cells for bone regeneration in a rat critical-size calvarial defect model. humhum gene ther 2003; 14: 1689–701. �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 11-16 24. laino g, graziano a, d’aquino r, pirozzi g, lanza v, valiante s, de rosa a, naro f, vivarelli e, papaccio g. an approachable humanan approachable human adult stem cell source for hard-tissue engineering. j cell physiol 2006; 206: 693–701. 25. peptan ia, hong l, mao jj. comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits. plast rec�nstr s�rg. 2006; 15: 1462–70. 26. petite h, viateau v, bensaid w, meunier a, de pollak c, bourguignon m, oudina k. tissue-engineered bone regeneration. nat biotechnol 2000; 18: 959–63. 27. li z, li zb. repair of mandible defect with tissue engineering bonerepair of mandible defect with tissue engineering bone in rabbits. anz journal of surgery 2005; 75(11): 1017–21. 28. vesala, anna-liisa, kallioinen, matti, tomala, pertti, kellomaki, minna, waris, timo, ashammakhi, nureddin. bone tissuebone tissue engineering: treatment of cranial bone defects in rabbits using selfreinforced poly-l, d-lactide 96/4 sheets. journal of craniofacial surgery 2002; 13(5): 607–13. 29. weng y, wang m, liu w, hu x, chai g, yan q, zhu l, cui l, cao y. repair of experimental alveolar bone defects by tissueengineered bone. tissue engineering 2006; 2(6): 1503–13. 30. warnke ph, springer in, wiltfang j, acil y, eufinger h, russo pa, bolte h, sherry e, behrens e, terheyden h. growth and transplantation of a custom vascularised bone graft in a man. lancetlancet 2004; 364: 766–70. 31. cancedda r, mastrogiacomo m, bianchi g, derubeis a, muraglia a, quarto r. bone marrow stromal cells and their use in regenerating bone. novartis found symp 2003; 249: 133–43. 32. levenberg s, rouwkema j, macdonald m, garfein es, kohane ds, darland dc, marini r, van blitterswijk ca, mulligan rc, d’amore pa, langer r engineering vascularized skeletal muscle tissue. nat biotechnol 2005; 23: 879–84. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dentistry airlangga university editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (062-31) 5039478 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id, www.dentj.fkg.unair.ac.id, i wish to subscribe dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) name/nama: .......................................................................... date of birth/tanggal lahir: .................................................... job title/pekerjaan: ................................................................ institution/institusi: .................................................................. address/alamat surat: ............................................................ ................................................................................................. ................................................................................................. country/negara: ................................................ telp.: 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...................................................................... telp.: ....................................................................................... fax.: ........................................................................................ e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month 1 year 2 years surabaya q rp80.000,00 q rp160.000,00 q rp320.000,00 java island (pulau jawa) q rp90.000,00 q rp180.000,00 q rp360.000,00 outside java island (luar pulau jawa) q rp100.000,00 q rp200.000,00 q rp400.000,00 other countries (negara lain) q us $ 27 q us $ 54 q us $ 108 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 033-01-11343-16-0 name of bank : bank niaga cabang dharmahusada name of beneficiary : drg. sianiwati goenharto " 142 the effects of different 650 nm laser diode irradiation times on the viability and proliferation of human periodontal ligament fibroblast cells kun ismiyatin, ari subiyanto, ika tangdan, rahmi nawawi, reinold c lina, rizky ernawati and hendy jaya kurniawan department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: endo-perio lesions are clinical manifestations of inflammation in the periodontal and pulp tissue. damage to the periodontal ligament can inhibit its ability to regenerate. therefore, laser therapy use is expected to improve the prognosis with regard to healing lesions. unfortunately, the duration of irradiation during laser diode therapy can influence the viability and proliferation of human periodontal ligament fibroblast (hpdlf) cells. purpose: this study aims to determine the effects of different irradiation exposure times of the 650 nm laser diode of the pulsed mode type on the viability and proliferation of human periodontal ligament fibroblast cells. methods: this study constituted a laboratory experiment on hpdlf cells using 650 nm laser diode irradiation. six groups formed the research subjects in this study, namely; two control groups, two radiation groups respectively subjected to irradiation exposure of 15 seconds and 35 seconds duration followed by 24-hour incubation, and two radiation groups exposed to irradiation for 15 and 35 seconds respectively followed by 72-hour incubation period. the viability and proliferation of those cells were subsequently calculated by elisa reader, while the data was analyzed by means of one-way anova and tukey tests. results: the significance value of the viability scores between the 15-second irradiation group and the 35-second irradiation group was less than 0.05, indicating that there was a significant difference between these treatment groups. similarly, the significance value of proliferation scores between the 15-second irradiation group and the 35-second irradiation group was less than 0.05, again indicating a significant difference between these treatment groups. conclusion: irradiation using a 650 nm laser diode 15 seconds and 35 seconds in duration can induce an increase in the viability and proliferation of hpdlf cells. keywords: cell proliferation; cell viability; human periodontal ligament fibroblast cells; laser diode correspondence: kun ismiyatin, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: kun-is@fkg.unair.ac.id dental journal (majalah kedokteran gigi) 2019 september; 52(3): 142–146 research report introduction periodontal tissue and dental pulp actually have a very close relationship since they are derived from ectomesenchymal tissue. endo-perio lesions are clinical manifestations of inflammation that occur due to the relationship between periodontal and pulp tissues.1 moreover, endo-perio lesions can undergo root canal treatment.2 unsuccessful conventional root canal treatment is usually due to complex root canal anatomy rendering the removal of all healthy pulp tissue and bacterial residue difficult.3 since damage to the periodontal ligament limits its regenerative ability, root canal treatment and laser therapy can improve the prognosis of the lesion healing process.4 progenitor cells are identified in periodontal structures including periodontal ligaments. these cells can differentiate into fibroblasts, osteoblasts, and cementoblasts which play a crucial role in healing periodontal tissue.5 as dominant cells, fibroblasts are found in connective tissue where they secrete collagen fibers and extracellular substances. fibroblast cells can also be used to see viability and proliferation. human primary fibroblasts derived from periodontal ligaments also play an important role in the development, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p142–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p142-146 143ismiyatin, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 142–146 functioning, and regeneration of periodontal tissue.6,7 human periodontal ligament fibroblasts (hpdlf) are the main cell types in the tooth-supporting tissue. fibroblasts, the most common connective tissue cells found in the pulp and periodontal ligaments, produce collagen fibers that actively participate in the healing process.8 fibroblasts also function as defense cells because of their capacity to differentiate into odontoblasts and osteoblasts during the healing process. the ability of fibroblast cells to develop rapidly into wound tissue or survive independently accounts for the ease with which they can be cultured for use in biological research.9 light amplification by stimulated emission of radiation (laser) is a device that produces coherent electromagnetic radiation that has been employed in the field of dentistry since 1960.10 lasers are known to induce biphasic dose responses (bdr), namely; biostimulation and bioinhibition responses.11 this effect is related to an increase in adenosine triphosphate (atp) in the mitochondria that produce deoxyribonucleic acid (dna) and ribose nucleic acid (rna) and whose synthesis increases after laser diode irradiation. this increase can cause cellular responses to injury through the production of proteins associated with repair and healing processes.12 low-level lasers operate within several parameters, including: wavelength, energy source, energy density (fluence), potential density (irradiance), irradiation time, and laser light emission area. a previous study stated that laser light plays a role in increasing fibroblast proliferation.13 furthermore, the wavelength of red light emitting diodes (leds) light is 600-700 nm. the wavelength of laser therapy is known to have an influence on cell culture, with one previous study even stating that wavelengths of 600-700 nm can stimulate an increase in cell proliferation and differentiation.5 another investigation using low-level laser therapy (lllt) at a wavelength of 650 nm argued that the light of photons received by a cell chromophore can serve to regenerate tissue, reduce inflammation, and reduce pain.14 similarly, a proliferation test on fibroblast cells in periodontal ligaments with irradiation times of 16 seconds and 33 seconds reportedly produced an increase in the number of fibroblast cells.5 in addition, laser irradiation is divided into two types, namely; continuous mode and pulsed mode. the latter has the advantage that a delay in the irradiation time of the “quench period” causes a decrease in temperature on the surface of the tissue during laser exposure. the use of pulsed mode also allows higher energy consumption than the use of continuous mode.15 consequently, this study aims to determine the effects of irradiation by a 650 nm laser diode using pulsed modes of 15 seconds and 35 seconds’ duration on the viability and proliferation of hpdlf cells. the significance of this study is to determine the method and irradiation time of laser diode therapy at a wavelength of 650 nm in root canal treatment in order to achieve optimal improvement in the treatment of periapical lesions. materials and methods the hpdlf cell culture was produced at the integrated research and testing laboratory, universitas gadjah mada, yogyakarta. this study was approved by the faculty of dentistry, airlangga university through the issuing of ethical clearence certificate no. 135/hrecc.fodm/ iv/2019. the production of the hpdlf cell culture was subsequently approved by patients who had undergone dental extraction for orthodontic reasons at professor soedomo dental and oral hospital, yogyakarta. following initiation of the cell harvesting phase, the first maxillary premolar extracted was inserted into a 10ml tube containing 10% dulbecco’s modified eagle’s medium (dmem, d16171, sigma-aldrich pte. ltd, singapore) to which 0.5µg/ml fungizone (amphotericin b, gibcotm fungizone antimycotic, fisher scientific gsa) and penstrep 2% (gibcotm streptomicin, fisher scientific gsa) was added.16 these teeth were removed from the tube, washed three times with buffered saline phosphate (pbs, p7059 sigma-aldrich pte. ltd, singapore) and placed on a petri dish containing a fetal bovine serum medium (fbs 10%, f4135, sigma-aldrich pte. ltd, singapore). the teeth were subsequently scalped in the 1/3 apical section, placed on a small petri dish covered with a sterile glass deck, added to a complete medium of 3ml of dmem 10%, and placed in a large petri dish to be incubated in a co2 incubator (memmert, inc108med, germany). the medium in the petri dish was removed and replaced once every three days.17 the cells were observed until 80% confluent had been achieved, the periodontal tissues were then removed, and the medium was washed with pbs.18 during the secondary culture stage, 2ml of trypsinedta 0.25% (sm-2003-c sigma-aldrich pte. ltd, singapore) was added to the medium. if the cells appeared to have been released, the medium would be deposited by pipette into a 5ml tube. centrifuging was carried out for ten minutes at a speed of 1500 rpm at which point the supernatant was discarded and 1ml of complete medium of dmem 10% was added until it was homogenized. the cells were then placed in several petri dishes and incubated in a co2 incubator. the medium was discarded and replaced with new medium once every three days. during subsequent observation under a microscope, if cell confluent reached 80%, calculation of the number of cells would be undertaken.18 during the cell treatment stage, the cells were divided into 96 well plates. 100 µl of cell suspension at a density of 2 x 104 cells/well was added to each plate and allowed to stand for two hours. the laser diode light (fnrdentolaser 650 nm) was then irradiated for 15 seconds and 35 seconds at a distance of 1 cm withan output power of 20 mw.5 in this study, three research groups were formed for viability, namely; a control group, a 15-second irradiation group (incubated for 24 hours) and a 35-second irradiation group (incubated for 24 hours). there were also three research groups for proliferation, namely; a control group, a 15dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p142–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p142-146 144 ismiyatin, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 142–146 second irradiation group (incubated for 72 hours), and a 35-second irradiation group (incubated for 72 hours). these cells were incubated in a co2 incubator for four hours in order that they were reattached after harvest. four hours later, observations were carried out under a microscope and photographs were taken. the medium on the plate was then discarded by means of a pipette. 100µl of pbs was added to all well cells before itself being disposed of. at this point, complete media was added to each plate containing cells and incubated in a co2 incubator with a co2 level of 5% at a temperature of 37˚ c and a humidity level of 98% for a period of 24 hours (for the purposes of viability) and another period of 72 hours (to induce proliferation). observation was conducted under a microscope and photographs taken to determine the viability and proliferation of hpdlf cells. the cell complete culture medium was removed and washed with pbs which was then disposed of. 100µl of mtt was added [5 mg mtt (ct01-5 sigma-aldrich pte. ltd, singapore) to each well, together with 1ml of pbs and 9ml of complete dmem medium. it was then incubated for four hours until formazan was formed. 100µl sds of 10% stopper solution (rabstop1 sigma-aldrich pte. ltd, singapore) was added to 0.01 nhcl (titripur® sigma-aldreich pte. ltd, singapore) in each well and incubated overnight.18 finally, the number of hpdlf cells was calculated with elisa reader (reader type: model 680 xr, benchmark) at a wavelength of 550nm and a temperature of 25.1°c using endpoint (fast read) reading type with a mix time of 0 sec. the six plates were alternately inserted into the elisa reader, in other words; plates 1-3 for viability (flat-shaped cells with oval nuclei) and plates 4-6 for proliferation (large, flat-branched cells). the living hpdlf cells were colored purplish blue, in contrast to the dead cells which did not display this color. the data was subsequently analyzed statistically with one-way anova and brown-forsythe tests to compare more than two groups. the data was also subjected to analysis by a tukey hsd test in order to compare all treatment group pairs. the statistical analysis undertaken employed spss version 20 (ibm, armonk, new york, usa) statistical software. results the results of a brown-forsythe test, as illustrated by table 1, showed a significant difference in the average viability scores of the 15-second irradiation group and the 35second irradiation group with a p-value of <0.05. similarly, based on the anova test results, as shown in table 1, a significant difference existed in the average proliferation scores between the 15-second irradiation group and the 35second irradiation group with a p-value of <0.05. moreover, the tukey hsd test results, as shown in table 2, indicated a significant difference in the average viability scores between the 15-second irradiation group and the 35-second irradiation group with a p-value of <0.05. similarly, as shown in table 2, according to the tukey hsd test results, there was a significant difference in the average proliferation scores between the 15-second irradiation group and the 35-second irradiation group with a p-value of <0.05. table 1. the mean and standard deviation scores of the viability and proliferation of hpdlf (od) cells treatment n x ± sd p viability proliferation control 5 0.712 ± 0.013 0.699 ± 0.009 p 15’ 5 0.822 ± 0.03 0.815 ± 0.019 0.000 < 0.05 p 35’ 5 0.936 ± 0.009 0.921 ± 0.02 0.000 < 0.05 note: x = mean, p = probability, sd = standard deviation, p15’= 15 second irradiation, n = replication, p35’= 35 second irradiation table 2. the results of tukey hsd test of the viability and proliferation of hpdlf cells (l) treatment groups (j) treatment groups p viability proliferation control p 15’ p 35’ 0.000 0.000 0.000 0.000 p 15’ control p 35’ 0.000 0.000 0.000 0.000 p 35’ control p 15’ 0.000 0.000 0.000 0.000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p142–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p142-146 145ismiyatin, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 142–146 discussion in this research, hpdlf cells were employed since in several previous studies the use of such cells produced significant results. in addition to fibroblast cells, osteoblast cells were also involved in bone formation and regulation. in a previous study, a 940 nm laser diode used on osteoblast cells was shown to be capable of triggering the release of autocrine factors, such as tgf-β1 in response to irradiation, but the results were insignificant.19 furthermore, the viability of hpdlf cells indicated their number living in a culture medium, where viability after irradiation with a 570 nm low-level laser was observed at six hours and 24 hours.13 low-level laser therapy (lllt) on stem cells from human exfoliated deciduous teeth (shed) at a wavelength of 660nm generated the highest cell viability 24 hours after irradiation. on the other hand, the proliferation of hpdlf cells indicated the number of such cells that grow and divide in a culture medium. lllt at a wavelength of 660 nm on shed teeth generated the highest cell proliferation which occurred 72 hours after irradiation.20 a previous study of a fibroblast cell proliferation test conducted on periodontal ligaments with irradiation times of 16 seconds and 33 seconds and a wavelength of 660 nm showed a significant increase in the number of fibroblast cells.5 another previous study using low-level laser diode (λ = 680) suggested that diode lasers be used as an alternative to biomodulation. this effect is related to the increase in atp in the mitochondria which causes an increase in dna and rna synthesis after irradiation with a laser diode. this increase can cause cellular responses to injury through the production of proteins associated with processes of repair and healing.12 in addition, pulsed mode has the advantage of there being a delay in irradiation time during the “quench period” which causes a decrease in the surface temperature of the tissue during laser exposure. moreover, six out of nine previous studies using lllt show that the use of pulsed mode is more effective than continuous mode.15 the laser light power dose per laser area (density, unit j/ cm2) is the total laser power (laser power multiplied by the length of exposure time) divided by the total area of the laser. this determines the duration of the laser light exposure which is adjusted to the power dose and the quantum yield.21 therefore, this study employed the pulsed mode irradiation method. a previous study using 650nm lllt argued that released photon light can be absorbed by the cell chromophore to regenerate tissue, reduce inflammation, and ease pain.14 low-level lasers at a wavelength of 650nm are also used in the treatment of oral-facial pain, such as mucositis pain.22 therefore, this study employed a 650nm laser diode light. in conclusion, the results for 650nm laser diode light showed that 35-second irradiation produced higher scores for viability and proliferation caused by the energy absorbed by the cells which is sufficient to stimulate their biological activity. laser light exposed to hpdlf cells can trigger absorption of laser-emitted photons by the cell chromophore. conversely, a biostimulation response ensues due to accelerated electron transport reactions which can, in turn, cause an increase in atp production. increasing atp synthesis in mitochondria then accelerates the speed of cell mitosis. the effects of laser biostimulation can increase the secretion of growth factors, such as tgf-β, which are responsible for inducing collagen synthesis from fibroblasts. tgf-β is involved in cell proliferation, which can extend the lifespan of fibroblast cells.10 in conclusion, irradiation using a 650nm laser diode with irradiation times of 15 seconds and 35 seconds can cause an increase in hpdlf cell viability and proliferation. references 1. kumar r, patil s, hoshing u, medha a, mahaparale r. non-surgical endodontic management of the combined endo-perio lesion. int j dent cl. 2011; 3(2): 82–4. 2. hegde mn, bhat r, shetty p. semiconductor diode laser in disinfection of the root canal system in endodontics. endodontology. 2015; 27(2): 158–61. 3. siqueira junior jf, rôças i das n, marceliano-alves mf, pérez ar, ricucci d. unprepared root canal surface areas: causes, clinical implications, and therapeutic strategies. braz oral res. 2018; 32: 1–19. 4. asnaashari m, asnaashari n. clinical application of 810nm diode laser and low level laser therapy for treating an endodontic problem a case presentation. j lasers med sci. 2011; 2(2): 82–6. 5. soares dm, ginani f, henriques ág, barboza cag. effects of laser therapy on the proliferation of human periodontal ligament stem cells. lasers med sci. 2015; 30(3): 1171–4. 6. souto-lopes m, azevedo á, teixeira a, bastos-aires d, lordelo j, pérez-mongiovi d. cytotoxicity of acrylic based resin compounds in a human gingival fibroblast cell line. rev port estomatol med dent e cir maxilofac. 2013; 54(2): 87–90. 7. ok e, adanir n, hakki s. comparison of cytotoxicity of various concentrations origanum extract solution with 2% chlorhexidine gluconate and 5.25% sodium hypochlorite. eur j dent. 2015; 9(1): 6–10. 8. mescher al. junqueira’s basic histology. 14th ed. usa: mcgrawhill; 2015. p. 96–100. 9. gutiér rez-venegas g, cardoso-jiménez p. lipoteichoic acid promotes nuclear accumulation of β-catenin via akt in human gingival fibroblasts. int immunopharmacol. 2011; 11(9): 1278–84. 10. saquib s, jadhav v, priyanka n, perla n. low-level laser therapy in dentistry: a review. int j contemp dent med rev. 2014; 2014: 1–3. 11. gagnon d, gibson twg, singh a, zur linden ar, kazienko je, lamarre j. an in vitro method to test the safety and efficacy of lowlevel laser therapy (lllt) in the healing of a canine skin model. bmc vet res. 2016; 12: 1–10. 12. ferriello v, faria mr, cavalcanti bn. the effects of low-level diode laser treatment and dental pulp-capping materials on the proliferation of l-929 fibroblasts. j oral sci. 2010; 52(1): 33–8. 13. de souza lm, rinco ugr, aguiar dat, de almeida junior la, cosme-silva l, oliveira tm, teixeira marques nc, sakai vt. effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions. laser phys. 2018; 28(2): 1–5. 14. farivar s, malekshahabi t, shiari r. biological effects of low level laser therapy. j lasers med sci. 2014; 5(2): 58–62. 15. hashmi jt, huang yy, sharma sk, kurup db, de taboada l, carroll jd, hamblin mr. effect of pulsing in low-level light therapy. lasers surg med. 2010; 42(6): 450–66. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p142–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p142-146 146 ismiyatin, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 142–146 16. freshney ri. culture of animal cells: a manual of basic technique and specialized applications. 6th ed. new jersey: john wiley & sons; 2010. p. 1–8. 17. tucci-viegas vm, hochman b, frana jp, ferreira lm. keloid explant culture: a model for keloid fibroblasts isolation and cultivation based on the biological differences of its specific regions. int wound j. 2010; 7(5): 339–48. 18. zare jahromi m, ranjbarian p, shiravi s. cytotoxicity evaluation of iranian propolis and calcium hydroxide on dental pulp fibroblasts. j dent res dent clin dent prospects. 2014; 8(3): 130–3. 19. illescas-montes r, melguizo-rodríguez l, manzano-moreno fj, garcía-martínez o, ruiz c, ramos-torrecillas j. cultured human fibroblast biostimulation using a 940 nm diode laser. materials (basel). 2017; 10(7): 1–10. 20. fernandes ap, junqueira mda, marques nct, machado maam, santos cf, oliveira tm, sakai vt. effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth. j appl oral sci. 2016; 24(4): 332–7. 21. astuti sd. an in-vitro antimicrobial effect of 405 nm laser diode combined with chlorophylls of alfalfa (medicago sativa l.) on enterococcus faecalis. dent j (majalah kedokt gigi). 2018; 51(1): 47–51. 22. khalighi hr, anbari f, beygom taheri j, bakhtiari s, namazi z, pouralibaba f. effect of low-power laser on treatment of orofacial pain. j dent res dent clin dent prospects. 2010; 4(3): 75–758. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p142–146 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p142-146 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 104 research report dental journal (majalah kedokteran gigi) 2015 june; 48(2): 104–107 the cleanliness differences of root canal irrigated with 0.002% saponin of mangosteen peel extract and 2.5% naocl anis sakinah, laksmiari setyowati, and devi eka juniarti department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: root canal treatment consists of preparation, sterilization, and obturation. during root canal preparation, debris is smeared over the dentinal surface forming a smear layer. smear layer will reduce the attachment of root canal filling materials. organic material in smear layer can be substrated for microorganism. preparation of root canal should be followed by irrigation. naocl is common irrigation solution in endodontics. it has been very effective for their disinfecting and tissue-dissolving properties, but it is incapable of removing the smear layer. on the other hand, saponin of mangosteen peel extract has an ability as a surfactant to lower the surface tension, and it can dissolve debris containing of anorganic and organic materials. purpose: this study aims to know the differences between 2.5% naocl and 0.002% saponin of mangosteen peel extract in removing the debris in the root canal after the preparation procedure. method: three groups of teeth (7 teeth in each) were instrumented with k-file and irrigated as follow: group 1 (control) with aquadest; group 2 with 2.5% naocl; and group 3 with 0.002% saponin of mangosteen peel extract. furthermore, those teeth were split horizontally and longitudinally 4mm above the apical. the apical third of root canal walls was observed by a scanning electron microscope (sem). result: there were significant differences between each group (p<0.05). median value of the group 3 was score 1 considered as the smallest value. it indicates that group 3 with 0.002% saponin of mangosteen peel extract was the cleanest group. conclusion: it can be concluded that 0.002% saponin of mangosteen peel extract can clean the smear layer of the root canal better than 2.5% naocl. keywords: saponin; mangosteen peel extract; naocl; debris correspondence: laksmiari setyowati, c/o: departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: laksmi_dentist@yahoo.com introduction endodontic treatment is a procedure to maintain teeth for long time, consisting of three main steps of preparation, sterilization, and obturation. one of the primary reasons for irrigating root canal is to ensure the cleanliness of the canals prior to obturation. this cleanliness involves both elimination of microorganisms and removal of organic matter.1 the procedure of root canal preparation can produce smear layer, involving organic material, odontoblastic processes, bacteria and blood cells. the presence of an infected smear layer may prevent antimicrobial agents from gaining access to the infected dentinal tubules. increased penetration of smear material into dentinal tubules may cause the reduction of surface tension of irrigants during instrumentation. removal of the smear layer may enhance the penetration of sealers into dentinal tubules and the adaptation of obturation materials to the root canal walls.2,3 several irrigants and irrigant delivery systems are available, which behave differently and have relative advantages and disadvantages. common root-canal irrigants consist of sodium hypochlorite (naocl), chlorhexidine gluconate, alcohol, hydrogen peroxide and ethylene diamine tetra acetic acid (edta).4 however, the most effective and commonly used is naocl. they have a unique ability to dissolve necrotic tissue and organic components of the smear layer. in general, the concentration 105 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 105sakinah, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 104–107 of naocl commonly used is 2.5%, since this concentration has the ability to dissolve the tissue and has the power as antibiofilm.5 nevertheless, naocl also has some negative effects. naocl has a toxic effect on the tissue, and can cause allergic reactions.6 naocl is alkaline and corrosive to metals that can damage the instrument used. thus, naocl cannot dissolve inorganic dentin particles and cannot prevent the formation of smear layer during instrumentation process.4 naocl solution can cause pain to the periapical tissues and swelling spontaneously.5 mangosteen fruit has the latin name garcinia mangostana l. mangosteen is being used to heal diarrhea, tonsillitis, whitish, dysentery and toothache. mangosteen peel can be used as a medicine for ulcers, dysentery, diarrhea and uric acid.8 methanol extract of mangosteen peel (garcinia mangostana l.) contain saponin, triterpenoids, tannins, polyphenols, flavonoids and alkaloid.9 mangosteen peel consists 1.82% of saponin.10 due to the presence of a lipid-soluble aglycone and water-soluble sugar chain in their structure (amphiphilic nature), saponin is surface active compound with detergent, wetting, emulsifying, and foaming properties, so saponin can dissolve the organic and inorganic debris of dentin.11 therefore, this study aims to study the effects of saponin extract of mangosteen peel on the cleanliness of the root canal walls. materials and methods this study is a laboratory experimental research with post-test only control group design. twenty-one mandibular premolars were extracted for orthodontic treatment with the provisions of a single root canal with apical tip that has grown perfectly by inserting files no. 15 due to the working length. it means that the root canal had to be straight without any caries, restoration, and obstruction. the teeth were stored in isotonic saline solution to avoid any effect that fixative might have on the dissolution of organic tissue then randomly selected and divided in the three groups. access opening was done with endo access bur, and working length was determined. afterwards, root canals were prepared using a needle k-file no. 15 to no. 60 using standard techniques. each sample was irrigated by 3 ml of treatment solution with a pressure of 1 atm using irrigation needle 27 g. after irrigated, root canals were rinsed with aquadest to stop the chemical process. root canal wall was dried with paper points 3 times, and then cotton pellets were put at the orifice and closed with temporary seals. all roots were grooved longitudinally on the buccal and lingual surfaces, and horizontally at the apical third (4 mm from apex) with a small round diamond bur, avoiding penetration into the cavity. the roots were then split longitudinally with a small chisel into two halves. roots that have been cut were placed on the sample holder, and coating process was performed. all specimens were examined using hitachi tm 3000 scanning electron microscope (sem) with 1000x magnification at the apical third. scoring was performed by three observers. assessment of the cleanliness of the root canal was then conducted with transparent plastic tools divided into 12 fields. transparent plastic was attached to a picture, and an assessment was determined by the percentage for each field. the percentage results were averaged and converted into a score. superficial debris was independently subjected to a standardized semiquantitative evaluation in four grades based on the classification of gutmann et al. (1994). criteria for the scoring were as follow: score 1: little or no superficial debris covering up to 25% of the specimen; score 2: little to moderate debris covering between 25 and 50% of the specimen; score 3: moderate to heavy debris covering between 50 and 75% of the specimen; and score 4: heavy amounts of aggregated or scattered debris over 75% of the specimen. the data were processed and analyzed using a frequency test to determine the median of each treatment group, and also a non-parametric test, kruskal-wallis test, to determine differences between groups of aquadest (control), saponins, and naocl. mann-whitney test was conducted to determine the differences between the treatment groups. results there were 7 pieces of tooth samples with 0.002% saponin extract of mangosteen peel median with median score 1. it indicates that there was little or no superficial debris covering up to 25% of the specimen. meanwhile, the score of the group with 2.5% naocl was 2. it means that there was little to moderate debris covering between 25% and 50% of the specimen. and the score of the group with aquadest (control) was 3. it indicates that there was moderate to heavy debris covering between 50 and 75% of the specimen (table 1). table 1. the results of frequency test in each group no. group n median sd 1. aquadest (control) 7 3 0.48795 2. 2.5% naocl 7 2 0.53452 3. 0.002% saponin extract of mangosteen peel 7 1 0.53452 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 106 sakinah, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 104–107 furthermore, the results of kruskal-wallis test showed that there were significant differences between the root canal wall cleanliness irrigated with aquadest water group (control), 2.5% naocl and 0.002% saponin extract of mangosteen peel (p<0.05). mann-whitney test showed that there was a significant difference between the root canal wall cleanliness of the two groups, aquadest (control) and 2.5% naocl, about 0.015 (p<0.05). there was also a significant difference between the root canal wall cleanliness of the two groups, aquadest (control) and 0.002% saponin extract of mangosteen peel, about 0.001 (p <0.05). and, there was a significant difference between the root canal wall cleanliness of the two groups, 2.5% naocl and 0.002% mangosteen peel extract, about p=0.010 (p<0.05). finally, further analyzing of the data was conducted using the scanning electron microscope photomicrograph (sem) with the results as shown on figure 1. discussion the main purpose of irrigation is generallly to clean root canal prior to obturation and also to eliminate microorganisms and organic components. based on previous researches, it can be said that 41.5% of researchers support the cleaning process because the smear layer can block the filling materials contacting with the root canal wall. organic debris of the smear layer even can became the media for growth of bacteria.12 the most effective irrigation material often used is naocl. naocl has a good antibacterial ability. as a lubricant, naocl can remove the organic debris and smear layer on root canal. however, naocl has some disadvantages because it has a toxic effect on the periapical tissue and can cause allergic reaction.4 saponins in mangosteen peel contains approximately 1.82% surfactant properties, often referred to as natural detergent. surfactant properties due to the non-sugar group can be called sapogenin. sapogenin which have both hydrophilic and lipophilic groups are able to lower surface tension to function as detergents, emulsifiers, wetting and foaming, consequently, it can dissolve impurities, such as organic and inorganic debris dentin.10 thus, this study was conducted to analyze the differences of 0.002% saponin extract of mangosteen peel and 2.5% naocl in cleaning debris on the surface of the root canal walls. the examination of debris was conducted through a laboratory research using sem. the results of sem were evaluated with the photomicrograph using the criteria of how much debris covering the walls of the root canal. the apical third was then selected since this section is smaller than the other parts, so the root canal debris can be more easily buried in this part.13 the results of sem photomicrograph assessment showed a little debris on the surface of the root canal walls and plenty of opened dentin tubules on the canal walls irrigated by 0.002% saponin extract of mangosteen peel. meanwhile, the surface of the root canal wall and dentin tubules irrigated with 2.5% naocl were covered with debris, and opened dentin tubules were rarely seen. in addition, 0.002% saponin extract of mangosteen peel was better in cleaning debris on the canal wall because it has surfactant effects able to bind impurities, such as organic and inorganic debris. surfactants can clean up by lowering the surface tension of the root canal wall covered with debris, consequently, the surface was wetted. saponins particles, as a result, can penetrate into the dentin tubules and bind with debris to form an emulsion in water, and then the emulsion will be held in suspension to be carried out by the water, so root canal debris will be removed and the dentin tubules will be opened.14 saponins can remove organic debris because it has a lipophilic group which can bind impurities, such as fat. saponins then form a stable emulsion that can be dispersed into water, so the debris will be carried out by irrigant. saponins also can remove inorganic debris because its hydrophilic group can bind ca2+ ions in the root canal wall that contains of hydroxyapatite, so saponins can remove organic and inorganic debris of root canal wall.3 the group irrigated with 2.5% naocl showed poor results compared to the group irrigated with 0.002% saponin extract of mangosteen peel. this is because naocl does not have any ability to clean up inorganic debris. this irrigant actually has a chemical reaction stage only with organic materials, namely saponification reaction. dissolved organic will be formed when the saponification reaction 7 table 1. the results of frequency test in each group no. group n median sd 1. aquadest (control) 7 3 0.48795 2. 2.5% naocl 7 2 0.53452 3. 0.002% saponin extract of mangosteen peel 7 1 0.53452 figure 1. the results of sem with 1000x magnification on the root canal walls irrigated with (a) 0.002% saponin extract of mangosteen peel (b) 2.5% naocl; (c) aquadest (control). 7 table 1. the results of frequency test in each group no. group n median sd 1. aquadest (control) 7 3 0.48795 2. 2.5% naocl 7 2 0.53452 3. 0.002% saponin extract of mangosteen peel 7 1 0.53452 figure 1. the results of sem with 1000x magnification on the root canal walls irrigated with (a) 0.002% saponin extract of mangosteen peel (b) 2.5% naocl; (c) aquadest (control). figure 1. the results of sem with 1000x magnification on the root canal walls irrigated with (a) 0.002% saponin extract of mangosteen peel; (b) 2.5% naocl; (c) aquadest (control). a b c 107 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 107sakinah, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 104–107 breaks down organic matter and fats into fatty acids (soaps) and glycerol (alcohol), so naocl only can clean debris of organic materials and does not have any ability to clean the inorganic debris materials.4 finally, the control group irrigated with aquadest was the dirtiest group with a lot of debris since aquadest does not have any ability to remove debris, but merely as a rinse. in conclusion, 0.002% saponin of mangosteen peel extract can clean the smear layer of the root canal better than 2.5% naocl. references 1. torabinejad m, walton re. endodontics principles and practice. 4th ed. singapore: elsevier; 2012. p. 259. 2. drukteinis s, balciuneine i. a scanning electron microscopic study of debris and smear layer remaining following use of aet instruments and k-flexofiles. stomatologij a, baltic dental and maxillofacial j 2006; 8(3): 70-5. 3. akcay i, sen bh. the effect of surfactant addition to edta on microhardness of root dentin. j endod 2012; 38(5): 704-7. 4. glassman g. endodontic irrigants and irrigant delivery system. review endodontic irrigation; 2013. p. 30-7. 5. chong bs. harty’s endodontics in clinical practice. 6th ed. london: churchill livingstone, elsevier; 2010. p. 101-15. 6. estrela c, estrela cr, barbin el, spanó jc, marchesan ma, pécora jd. mechanism of action of sodium hypochlorite. braz dent j 8 2002; 13(2): 113-7. 7. clarkson rm, moule aj. sodium hypochlorite and its use as an endodontic irrigant. aust dent j 1998; 43(4): 250-6. 8. anjelita n. khasiat fantastis kulit manggis. jakarta: gramedia widiasarana; 2011. p. 20. 9. suwanmanee s, thitinan k, nathane jp. in vitro screening of 10 edibles thai plants for potential antifugal properties. evidence based complementary and alternative med; 214(2): 38587 10. servy ap, rulianto m, nirawati p. toxicity assay of mangosteen pericarp extract on bhk-21 fibroblast cell culture. conservative dentistry journal 2014; 4(2): 15-21. 11. güçlü-ustündağ o, mazza g. saponins: properties, applications and processing. 2007; 47(3): 231-58. 12. torabinejad m, walton re. endodontics priniples and practice. 4th ed. singapore: saunders elsevier; 2012. p. 260. 13. nevi y. perbedaan sitotoksisitas dan kebersihan dinding saluran akar gigi antara larutan saponin dari buah sapindus rarak dc dengan larutan naocl 5%. tesis. surabaya: universitas airlangga; 1998. 14. ramayanti fe, sudirman a, prasetyo ep. the effectiveness of mangosteen peel extracts (garcinia mangostana l.) againts root canal cleanliness. conservative dentistry journal 2014; 4(1): 12-7. 46 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 46–51 original article introduction oral disease, especially dental caries, is one of the most common diseases worldwide. dental caries in permanent teeth was found in about 30% of the world’s population.1 it can be caused by various factors, such as host factors, oral microbes, food intake, oral hygiene and other environmental factors.2 food intake is a critical factor that plays a role in caries’ incidence.3 previous studies have shown that obese individuals often have a higher dental caries index and a higher periodontal index than normal individuals.4–6 according to the results of basic health research (riskesdas), the prevalence of obesity among adults in indonesia was 10.5% in 2007, increasing to 15.4% in 2013 and 21.8% in 2018.7–9 obesity is defined as an abnormal or excessive accumulation of fat, which can damage health. it is a condition occurring due to an imbalance between energy intake and energy expenditure.10 excessive fat accumulation can occur if we over-consume fat-producing foods, such as carbohydrates or sugar. excessive carbohydrate intake can cause an increase in body weight, which can then develop into obesity.11 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 correlation between carbohydrate intake and dental caries in obese individuals ignatius setiawan1,2, ananda sagita3, ibnu suryatmojo4, dewi marhaeni diah herawati5, irna sufiawati6 and sunardhi widyaputra7 1department of dental public health, faculty of dentistry, maranatha christian university 2postgraduate student, faculty of medicine, padjadjaran university 3undergraduate student, faculty of dentistry, maranatha christian university 4department of conservative dentistry, faculty of dentistry, maranatha christian university 5department of public health, faculty of medicine, padjadjaran university 6department of oral medicine, faculty of dentistry, padjadjaran university 7department of oral biology, faculty of dentistry, padjadjaran university bandung – indonesia abstract background: nowadays, obesity is one of the biggest public health problems. obesity is an excessive accumulation of fat that can occur when fat-producing foods, such as carbohydrates or sugar, are over-consumed. sucrose is a type of carbohydrate contained in food and is a medium for bacterial growth. therefore, the consumption of sucrose can increase the risk of dental caries. purpose: this study aims to analyse the correlation between carbohydrate intake and dental caries in obese individuals. methods: this study was an observational analytic study with a cross-sectional design. in this study, 50 participants aged 18–40 were selected from an obese community in jakarta using a quota sampling technique. the carbohydrate intake was assessed using the food frequency questionnaire (ffq), the body fat percentage was measured with the bioelectrical impedance analysis method, and the dental caries index was assessed using the decayed, missing and filled teeth (dmf-t) index. the data obtained were tested with a simple linear regression statistical test at a significance level of 5%. results: the results showed that the average carbohydrate daily intake value of obese individuals was 1209.84 g, while the average value of the dmf-t index for obese individuals was 7.98. the results of the statistical tests revealed that there was a strong and positive correlation between carbohydrate intake and the dmf-t index. the effect of carbohydrate intake on the dmf-t index was 50.98%. conclusion: a positive correlation means that the larger the carbohydrate intake, the higher the dmf-t index. hence, controlling carbohydrate intake can prevent dental caries. keywords: carbohydrate intake; dmf-t index; ffq; obesity correspondence: ignatius setiawan, department of dental public health, faculty of dentistry, maranatha christian university, jl. surya sumantri no. 65 bandung, indonesia. email: ignatius.setiawan@dent.maranatha.edu mailto:ignatius.setiawan@dent.maranatha.edu https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 47setiawan et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 46–51 the foods consumed the most by indonesians are grains (41.11%), processed foods and beverages (21.09%), and oil and coconuts (12.24%).12 sugar or glucose is classified as a simple carbohydrate composed of carbon, hydrogen and oxygen elements. simple carbohydrates are easily absorbed by the intestines for energy use.13 excess glucose is then stored in the liver and muscle cells in the form of glycogen. when the body needs glucose, the liver will release glucose into the bloodstream to be distributed to the body parts that need it, including the brain, nervous system, heart and other organs. when glucose enters the cells, enzymes will break it down into small pieces to produce energy, carbon dioxide and water. excess carbohydrates are converted into fat and then stored in fat tissue.14 carbohydrates are also cariogenic ingredients. processed sugars, such as glucose and sucrose, will cause a drastic drop in the oral ph to below 5.5, which increases the occurrence of demineralisation.15 if demineralisation is higher than remineralisation, dental caries will develop.16 high carbohydrate intake in obese individuals may be associated with the prevalence of dental caries.3 the increasing prevalence of caries in obese individuals can also be caused by a decreased salivary flow rate, which decreases the anti-bacterial effect of saliva.17 the expansion of adipose tissue in obese individuals significantly influences the physiological response and can interfere with salivary gland function.18 the food frequency questionnaire (ffq) can be used to conduct a nutritional study to assess individuals’ nutrition and energy intake habits. the ffq is designed to elicit information on some specific aspects of the diet, such as carbohydrate intake. since obesity is related to the type and amount of nutritional intake, the appropriate ffq to use is the semi-quantitative ffq, which adds estimated food serving sizes.19,20 as far as the authors know, there are limited studies analysing carbohydrate intake and dental caries in obese individuals in indonesia. by understanding the correlation between carbohydrate intake and dental caries in obese individuals, we can prevent the incidence of dental caries and obesity by controlling carbohydrate intake. therefore, this study aims to analyse the correlation between carbohydrate intake and dental caries in obese individuals. materials and methods this study is an observational analytic study with a crosssectional study design. since the population’s proportion is unknown, the minimum sample size was calculated using the modified lemeshow et al. formula.21 according to the study conducted by mathur et al. in 2011, the prevalence of obese people who also experience periodontal disease is 88%, and in people who are not obese, the prevalence of periodontal disease is 74.4%.22 based on these data using a significance level of 95%, it can be calculated that the minimum sample size is 30 people. in this study, 50 obese individuals were selected as participants using the quota sampling technique. all participants were members of an obese community in jakarta. the body mass index (bmi) does not differentiate between body weight and muscle or body fat, so two individuals with the same bmi score could have a significant difference in fat mass and free fat mass.19 therefore, in this study, obesity was determined based on the bmi and body fat percentage measured using a body composition monitor (bc-601, tanita corp, japan). bioelectrical impedance analysis (bia) is a method that can be used to measure a person’s body fat percentage. the bia method measures electrical conductivity by circulating electrical signals in human body fluids. thus, the muscle mass, body fat mass, water content of the body and even individual bone mass can be measured.20 the participants’ inclusion criteria are 18–40 years old, bmi score >30 and body fat percentage >25% for men and >33% for women.23 on the other hand, the exclusion criteria are individuals who have conditions that may affect the result of the study (such as mental disorders, illnesses with a high risk of infection and conditions that interfere with the bmi) and the body fat percentage (such as pregnant women, sportsmen and people who have run a weight loss programme in the last one year). dental caries was measured using the decayed, missing and filled teeth (dmf-t) index. the dmf-t index is the most widely used and universally accepted index of dental caries. it can be used for individuals or groups. the dmf-t index is based on the fact that damage done to the tooth’s hard tissue cannot heal on its own and will leave lasting marks.16,24 d stands for a tooth affected by caries, m stands for a tooth that has been lost or extracted due to caries, and f stands for a tooth that is affected by caries but has been filled. all teeth are examined except for the third molars. the dmf-t index has rules that apply to scoring an individual tooth. each tooth may be counted only once.24 the dmf-t scores were divided into five categories: very low (0.0– 1.1), low (1.2–2.6), medium (2.7–4.4), high (4.5–6.5) and very high (> 6.6).25 the dental caries examination was carried out using an intraoral clinical examination by four examiners with equal competence and knowledge. carbohydrate intake was assessed using the semiquantitative ffq. the ffq is a self-reporting method of measuring food intake by filling out a questionnaire. each study participant reports how often each type of food and drink is consumed during a specific period.23,26 in the semi-quantitative ffq, the amount of nutrient present in each type of food consumed can be calculated based on a questionnaire’s result. the list of foods used in the ffq is obtained from a preliminary study using a food record.19 all participants filled in the frequency and portion of consumption of each food on the food list. the ffq assessment procedure was as follows: participants were required to fill in the list provided in the questionnaire regarding the frequency of their intake. there were five categories of intake frequency: daily (d), dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 48 setiawan et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 46–51 weekly (w), monthly (m), yearly (y) and rarely/never (n). the participants wrote the number of times each food was consumed in the column for the most appropriate category. the participants were also required to mark the list provided in the questionnaire regarding the intake portion. there were three categories of intake portion, which indicated the quantity of food usually consumed: small (s), medium (m) and large (l).19 the food book photo from the ministry of health was used as the standard measure of food portion.27 to measure the daily nutrient intake, all categories of frequency were converted into a daily basis, which meant once a day (1). the daily frequency was multiplied by the selected portion (in grams) to obtain the weight in grams consumed each day. the carbohydrate content in the weight of the food consumed was determined based on the indonesian food composition table (tkpi).28 the data obtained in this study were analysed using several statistical tests. the pearson correlation coefficient is used to measure the strength and direction of correlation between variables. both variables were measured on an interval scale. a linear regression analysis was used to identify the strength of the effect that the carbohydrate intake had on dental caries in obese individuals. the statistic was analysed using spss version 19 for windows (ibm, chicago, usa). first, the t-count value and t-table value were determined. the t-count was first determined using this formula: with df = 48 and the level of significance of 5%, the t-table value was 1.677. then, the t-count value was compared with the t-table value. if the t-count value was bigger than the t-table value, it would mean that carbohydrate intake had a significant effect on the incidence of dental caries. the coefficient of determination was indicated by the r2 value of the regression model. the percentage value of the coefficient of determination showed the amount of variability of the prevalence of dental caries, which could be explained with the carbohydrate intake in the study, while the rest is explained by other variables outside the study model.29 this study was conducted according to the guidelines laid down in the declaration of helsinki and was approved by the health research ethics committee, faculty of medicine, maranatha christian university – immanuel hospital, bandung, indonesia (no.008/.kep/ii/2019). written informed consent was obtained from all participants. results this study’s participants were 39 women (78%) and 11 men (22%). the average bmi score of the participants was 35.84. the largest age group, consisting of 21 people, was 25–29 years old. table 1 shows the characteristics of the participants based on body weight. a total of 16 participants weighed 81–90 kg (32%), and 15 participants weighed 91–100 kg (30%). as a result of this study, 12% of obese individuals had a dmf-t index in the high category, and 66% of obese individuals had a dmf-t index in the very high category. only 4% of participants had a dmf-t index in the very low category (table 2). the average dmf-t index score of participants in this study was 7.98. the highest dmf-t score in this study was 15, while the lowest dmf-t score was 1 (table 3). the average body fat percentage of all participants was 45.5%. the average value of daily carbohydrate intake assessed using the ffq was 302.46 g or 1209.84 kcal. the highest value of daily carbohydrate intake in this study was 2408.2 kcal, while the lowest value of carbohydrate intake was 291.7 kcal. table 3 displays the values of bmi scores, body fat percentage, carbohydrate intake and dmf-t index in this study. regarding each dmf-t index element (d, m and f elements), d had the highest average score at 4.62. on the other hand, m and f had similar scores. in this study, the median and mean values of the dmf-t index were similar – the median value was 8.00, and the mean value was 7.98. this showed that there was no extreme value in the data set. the result of the pearson correlation coefficient test showed that the correlation coefficient value of carbohydrate table 3. the values of bmi scores, body fat percentage, carbohydrate intake and dmf-t index value bmi body fat percentage (%) carbohydrate intake (kcal) d m f dmf-t mean 35.84 45.59 1209.84 4.62 1.70 1.66 7.98 median 35.64 45.95 1150.05 4.50 1.00 1.00 8.00 range 30.36–46.71 31.80–58.70 291.7–2408.20 1–11 0–7 0–10 1–15 table 2. the category of the dmf-t index of participants category of dmf-t index f (n = 50) percentage very low (0.0–1.1) 2 4% low (1.2–2.6) 2 4% medium (2.7–4.4) 7 14% high (4.5–6.5) 6 12% very high (> 6.6) 33 66% table 1. characteristics of participants based on body weight30 body weight (kg) f (n = 50) percentage (%) 70–80 5 10% 81–90 16 32% 91–100 15 30% 101–110 9 18% >110 5 10% dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 49setiawan et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 46–51 intake was 0.714, which is between 0.60–0.799. this shows a strong and positive correlation between the carbohydrate intake and the dmf-t index of obese individuals. a positive correlation shows that the higher the carbohydrate intake, the higher the dmf-t index. for the calculations’ result, the t-count value was 7.065, and the t-table value was 1.677. from these results, it could be seen that the t-count value was bigger than the t-table value. in addition, the significance level was 0.000 (<0.1). the result showed that carbohydrate intake has a significant effect on the dmf-t index. the value of the coefficient of determination, which is indicated by the r2 value of the regression model, was used to determine the amount of variability of the dmf-t index, which can be explained with the carbohydrate intake. in this study, r = 0.714, and r2 = 50.98%. this means that the variability of the dmf-t index in this study, which could be explained with the carbohydrate intake, was 50.98%. the remaining 49.02% was explained with other variables outside the study model. discussion in this study, most of the participants had a bmi score >30, which is categorised as level ii obesity by the who.19 all participants had a body weight beyond the indonesian health ministry’s recommendation, which is 60 kg for men aged 18–29 and 55 kg for women of the same age.31 bmi measurement is the most frequently used indicator to identify individuals’ nutritional status. however, the bmi, which assesses the ratio of body weight to height, cannot always reflect whether a person is overweight or obese or not. one of the more specific methods of determining obesity is by calculating the percentage of body fat.20 the average body fat percentage of all participants was 45.5%. this figure far exceeds the limit for the percentage of normal body fat and is even much higher than the limit for the percentage of body fat in obesity. the normal body fat percentage is 8–15% for men and 13–23% for women.19 a total of 78% of the dmf-t index of obese individuals in this study were in the high and very high categories. similar results were obtained in several studies looking for a correlation between caries incidence and bmi scores.6,32 a high d score of the dmf-t index showed that most of the dental caries of participants in this study had not been treated. many factors influenced this condition, some of which are knowledge, attitude and behaviour.33 dental caries is caused by the association of several factors over a period. it is related to several factors that are categorised into host susceptibility, microorganism and substrate. all these factors must intersect during a defined period, along a continuum, for caries to occur.24 dental caries is caused by a decrease in the oral cavity’s ph associated with food intake, oral bacteria, oral hygiene and saliva. social factors, such as education level and income level, also affect knowledge, attitudes and behaviour related to dental caries.16 the recommended nutritional adequacy value for indonesian people can be found in the regulation of the minister of health of the republic of indonesia no. 28, 2019.31 according to this regulation, men aged 19–29 need 430 g of carbohydrates, 65 g of protein, 75 g of fat and 2650 kcal of total energy. women aged 19–29 need 360 g of carbohydrates, 60 g of protein, 65 g of fat and 2250 kcal of total energy.31 however, these values are higher than the recommendation of the dietary guidelines for americans 2015–2020.2 the average carbohydrate intake of participants in this study far exceeded the recommended daily carbohydrate intake for adults, which is 130 g/day or 520 kcal.34 large nutritional intake of obese individuals can also be influenced by biological factors, where the control of food intake by the brain is an integral part of nutritional intake. the hormone that plays a role in regulating food intake by controlling appetite is leptin. leptin is produced by adipose tissue and provides signals to the brain centre via leptin receptors to regulate food intake and increase energy metabolism.35,36 fermented carbohydrate and dental plaque should not remain on the tooth surface for a long time because it will increase the demineralisation process of tooth enamel. these carbohydrates help oral bacteria produce acids that cause the demineralisation of enamel. complex carbohydrates are less harmful because they are not completely digested in the oral cavity, but simple carbohydrates, such as sugar, dissolve easily into dental plaque and are then metabolised by oral bacteria, thereby rapidly lowering the oral cavity’s ph.16 based on the statistical analysis in this study, the high dmf-t index in obese individuals is influenced by carbohydrate intake. obesity can occur if we excessively consume fat-producing foods, one of which is carbohydrates or sugar.12 carbohydrates consist of simple carbohydrates (sugar) and complex carbohydrates (starch and fibre). simple carbohydrates are chemically divided into monosaccharides, consisting of glucose, fructose and galactose, and disaccharides, consisting of maltose, sucrose and lactose. complex carbohydrates consist of polysaccharides (large molecules composed of monosaccharide chains). the recommended carbohydrate intake for adults is 130 g per day, based on the average amount of glucose used by the brain.37 the amount of complex carbohydrates intake is 50–70% of the total carbohydrate intake, while the amount of processed sugars allowed is only 0–10% of the total carbohydrate intake.37 the carbohydrates we consume are then metabolised by bacteria, which are involved in the formation of biofilms. the bacteria then produce acids that decrease the oral cavity’s ph to <5 within 2–5 minutes. meanwhile, the time required for the oral cavity to neutralise acids is 60 minutes. if sugary foods are continuously consumed, the oral cavity’s condition will remain acidic, leading to the demineralisation of the tooth structure, which can lead to dental caries.6,16 overweight and obese individuals also experience changes in size, distribution, cell composition and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 50 setiawan et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 46–51 adipose tissue function. the expansion of adipose tissue significantly influences physiological responses and can impair the function of this tissue.38 adipose tissue experiences hypertrophy, ectopic fat deposition, hypoxia and chronic stress in this obese state.38 enlargement of the parotid glands, caused by increased adipocyte storage, was found in overweight individuals.32 one of the endocrine organs that play a role in the physiological regulation of the body is adipocytes. enlarged adipocytes in obese individuals activate macrophages, which then secrete proinflammatory mediators, resulting in an imbalance between anti-inflammatory adipokines and pro-inflammatory adipokines.18,39 the presence of these inflammatory cells impairs the function of the salivary glands, which results in decreased salivary flow.32 the decreased salivary flow rate can be one of the factors causing dental caries.17 however, this study has several limitations. the participants in this study do not represent the obesity community in indonesia. there are other factors besides carbohydrate consumption that can influence the occurrence of dental caries, which were not discussed in this study. the limitations of this study can be addressed in future studies. based on the study’s results and the discussion above, it is concluded that there is a strong and positive correlation between carbohydrate intake and the dmf-t index. a positive correlation shows that the higher the carbohydrate intake, the higher the dmf-t index. the statistical test results also show that carbohydrate intake affects the dmf-t index as much as 50.98%. controlling carbohydrate intake can prevent dental caries and obesity. education and monitoring of carbohydrate intake in the community are small prevention steps that can have a significant impact on the community’s health, especially community oral health. acknowledgements we thank the faculty of dentistry, maranatha christian university and the faculty of dentistry and faculty of medicine, padjadjaran university, for their technical support during the study. references 1. gbd 2 017 d ise a se a nd i nju r y i ncidenc e a nd p r eva lenc e collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study 2017. lancet. 2018; 392(10159): 1789–858. 2. rathee m, sapra a. dental caries. treasure island (fl): statpearls publishing; 2020. p. nbk551699. 3. zero d, moynihan p, lingstrom p, birkhed d. the role of dietary control. in: fejerskov o, kidd e, editors. dental caries: the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p. 223–44. 4. setiawan i, lathifah gg, fikri sn, herawati dmd, sufiawati i, widyaputra s. the correlation between body mass index with dmf-t index and cpi modified. in: the 4th bali dental science & exhibition balidence 2019. denpasar: universitas mahasaraswati; 2019. p. 843–9. 5. hidayatullah, adhani r, triawanti. hubungan tingkat keparahan karies dengan status gizi kurang dan gizi baik: tinjauan pada anak balita di tk kecamatan kertak hanyar kabupaten banjar. dentino j kedokt gigijurnal kedokt gigi. 2016; 1(1): 104–7. 6. alswat k, mohamed ws, wahab ma, aboelil aa. the association between body mass index and dental caries: cross-sectional study. j clin med res. 2016; 8(2): 147–52. 7. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2007. jakarta: kementerian kesehatan republik indonesia; 2007. p. 48–53. 8. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2013. jakarta: kementerian kesehatan ri; 2013. p. 223–7. 9. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 95–8. 10. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https:// www.who.int/news-room/fact-sheets/detail/obesity-and-overweight. accessed 2020 nov 10. 11. te morenga l, mallard s, mann j. dietary sugars and body weight: systematic review and meta-analyses of randomised controlled trials and cohort studies. bmj. 2012; 346: e7492. 12. budijanto d, kurniawan r, kurniasih n, indah is, habibi h. konsumsi makanan penduduk indonesia. jakarta: infodatin pusat data dan informasi kementrian kesehatan republik indonesia; 2017. p. 1–7. 13. delli bovi ap, di michele l, laino g, vajro p. obesity and obesity related diseases, sugar consumption and bad oral health: a fatal epidemic mixtures: the pediatric and odontologist point of view. transl med @ unisa. 2017; 16: 11–6. 14. burhan fz, sirajuddin s, indriasari r. pola konsumsi terhadap kejadian obesitas sentral pada pegawai pemerintahan di kantor bupati kabupaten jeneponto. j media kesehat masy indones. 2013; 9(1): 1–14. 15. ramayanti s, purnakarya i. peran makanan terhadap kejadian karies gigi. j kesehat masy. 2013; 7(2): 89–93. 16. fejerskov o, kidd e. dental caries: the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p. 141–61. 17. setiawan i, putri y, damayanti a, herawati dmd, sufiawati i, widyaputra s. the comparison of salivary flow rate and the dmf-t index in obese and normal-weight individuals. j int dent med res. 2020; 13(4): 1488–93. 18. fasshauer m, blüher m. adipokines in health and disease. trends pharmacol sci. 2015; 36(7): 461–70. 19. fahmida u, dillon dhs. food frequency questionnaires (ffq). in: handbook nutritional assessment. 2nd ed. jakarta: seameotropmed rccn university of indonesia; 2011. p. 21–8. 20. soegih rr, wiramihardja kk. obesitas: permasalahan dan terapi praktis. jakarta: sagung seto; 2009. p. 29–38. 21. lemeshow s, hosmer dw, klar j, lwanga sk. adequacy of sample size in health studies. new york: john wiley & sons; 1990. p. 1–239. 22. mathur lk, manohar b, shankarapillai r, pandya d. obesity and periodontitis: a clinical study. j indian soc periodontol. 2011; 15(3): 240–4. 23. fahmida u, dillon dhs. anthropometry assessment. in: handbook nutritional assessment. 2nd ed. jakarta: seameo-tropmed rccn university of indonesia; 2011. p. 92–122. 24. shulman jd, cappelli dp. epidemiology of dental caries. in: cappelli dp, mobley cc, editors. prevention in clinical oral health care. st. louis: mosby elsevier; 2008. p. 2–13. 25. moreno-quispe la, espinoza-espinoza la, bedon-pajuelo ls, guzmán-avalos m. dental caries in the peruvian police population. j clin exp dent. 2018; 10(2): e134–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 51setiawan et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 46–51 26. kowa l kowska j, slowi nska m a, slowi nsk i d, dlugosz a, niedzwiedzka e, wadolowska l. comparison of a full foodfrequency questionnaire with the three-day unweighted food records in young polish adult women: implications for dietary assessment. nutrients. 2013; 5(7): 2747–76. 27. badan penelitian dan pengembangan kesehatan. buku foto makanan: survei konsumsi makanan individu (skmi 2014). jakar ta: kementerian kesehatan republik indonesia; 2014. p. 1–257. 28. direktorat gizi masyarakat. tabel komposisi pangan indonesia 2017. jakarta: kementerian kesehatan republik indonesia; 2018. p. 1–135. 29. dahlan ms. statistik untuk kedokteran dan kesehatan. 6th ed. jakarta: epidemiologi indonesia; 2014. p. 223–58. 30. beaglehole r, benzian h, crail j, mackay j. the oral health atlas. switzerland: fdi world dental federation; 2009. p. 1–120. 31. menteri kesehatan republik indonesia. peraturan menteri kesehatan tentang angka kecukupan gizi yang dianjurkan untuk masyarakat indonesia. 28 indonesia; 2019 p. 33. 32. modéer t, blomberg cc, wondimu b, julihn a, marcus c. association between obesity, f low rate of whole saliva, and dental caries in adolescents. obesity (silver spring). 2010; 18(12): 2367–73. 33. dixit lp, shakya a, shrestha m, shrestha a. dental caries p r eva l e n c e , o r a l h e a lt h k n owl e d ge a n d p r a c t ic e a m o ng indigenous chepang school children of nepal. bmc oral health. 2013; 13: 20. 34. u.s. department of health and human services and u.s. department of agriculture. 2015–2020 dietary guidelines for americans. 8th ed. washington: usda; 2015. p. 1–144. 35. cahyaningrum a. leptin sebagai indikator obesitas. j kesehat prima. 2015; 9(1): 1364–71. 36. friedman j. the long road to leptin. j clin invest. 2016; 126(12): 4727–34. 37. whitney en, rolfes sr. understanding nutrition. 12th ed. belmont: wadsworth; 2011. p. 97–129. 38. greenberg as, obin ms. obesity and the role of adipose tissue in inflammation and metabolism. am j clin nutr. 2006; 83(2): 461–5. 39. wardhana imw, wangko s. interaksi antara makrofag dan jaringan adiposa dan obesitas. j biomedik. 2011; 3(2): 111–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p46–51 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p46-51 p-issn: 1978-3728 e-issn: 2442-9740 volume 53, number 3, september 2020 editorial team of dental journal (majalah kedokteran gigi) sk: 07/un3.1.2/2020 january 2nd – december 31st, 2020 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: saka winias, drg., m.kes., sp.pm (department of oral medicine, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); nastiti faradilla (department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia); beta novia rizky (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); aulia ramadhani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers siti sunarintyas (department of biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); arlette suzy setiawan (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); kurnia dwi artanti (department of epidemiology, faculty of public health, universitas airlangga, indonesia); ida ayu evangelina (deparment of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); rosa amalia (department of of preventive and community dentistry, faculty of dentistry, universitas gadjah mada, indonesia); retno widayati (deparment of orthodontics, faculty of dentistry, universitas indonesia, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); dini setyowati (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); maretaningtyas dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); ratri maya sitalaksmi (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); desiana radithia (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); nurina febriyanti ayuningtyas (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 53, number 3, september 2020 p-issn: 1978-3728 e-issn: 2442-9740 1. the pore size of chitosan-aloe vera scaffold and its effect on vegf expressions and woven alveolar bone healing of tooth extraction of cavia cobaya sularsih ............................................................................................................................................. 115–121 2. the correlation analysis of dental caries, general health conditions and daily performance in children aged 2–5 years darmawan setijanto, taufan bramantoro, nanissa dyah anggraini, ardhyana dea maharani, dwita angesti, dani susanto hidayat and aulia ramadhani .................................. 122–125 3. telescopic overdenture as an alternative rehabilitation for the loss of several anterior teeth due to traffic accidents birgitta dwitya swastyayana subiakto and utari kresnoadi ..................................................... 126–132 4. the changing of occlusal plane inclination in class ii malocclusion nelvi yohana, siti bahirrah and nazruddin ................................................................................. 133–139 5. the effect of body mass index on tooth eruption and dental caries mohamed salim younus, karam ahmed and duran kala ......................................................... 140–143 6. the influence of functional tooth units on body mass index in the elderly of the jember regency jevina sicilia ahliawan, zahreni hamzah and mei syafriadi ..................................................... 144–148 7. howe’s dental cast analysis of students at the university of sumatera utara hilda fitria lubis and tiopan beltsazar sinurat ......................................................................... 149–152 8. the correlation between dental caries and serum iron (fe) levels in female students of mamba’ul khoiriyatil islamiyah (mhi) madrasa in jember, east java, indonesia ristya widi endah yani, taufan bramantoro, farida wahyuningtyas and talitha zulis islaamy .................................................................................................................................... 153–158 9. leukoplakia in hiv patients and risk of malignancy: a case report yuli fatzia ossa, anandina irmagita soegyanto, diah rini handjari and endah ayu tri wulandari .................................................................................................................................. 159–163 10. effect of caffeine in chocolate (theobroma cacao) on the alveolar bone mineral density in guinea pigs (cavia cobaya) with orthodontic tooth movement bramita beta arnanda, sri suparwitri and pinandi sri pudyani ............................................... 164–169 11. the effect of fixed orthodontic treatment with edgewise and straightwire techniques on white spot lesions incidence and accumulation of streptococcus mutans bacteria elfira maharani, dyah karunia and pinandi sri pudyani .......................................................... 170–174 contents page �2 oral and dental management in children with tetralogy of fallot arlette suzy puspa pertiwi, inne suherna sasmita and yetty herdiati nonong department of pediatric dentistry faculty of dentistry, padjadjaran university bandung indonesia abstract tetralogy of fallot is one of the congenital cyanotic heart disease that is often found in children. the disorder has four features, a ventricular septal defect, aortic overriding, infundibulary stenotic, and hypertrophy right ventricular. like other congenital heart disease, tetralogy of fallot sometimes related to fatal complications, such as bacterial endocarditis which was related to dental infections. the correct management of oral and dental conditions is important to prevent bacterial endocarditis. this paper discussed the oral and dental conditions and its management in two cases of tetralogy of fallot children. key words: oral and dental condition, antibiotic, cyanotic heart disease correspondence: arlétte suzy puspa pertiwi, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung 40132 indonesia. e-mail: arlettesuzy@yahoo.com, telp/fax. 022 253 3031. introduction tetralogy of fallot (tof) is a congenital cyanotic heart disease that is often found in children, approximately around of 7–10% from overall congenital heart disease in children. the disorder includes ventricular septal defect, aortic overriding, infundibullary stenotic, and hypertrophy right ventricular.1 defects of tof describe as a hole in the wall between the ventricles (ventricular septal defect) which lets oxygen-poor blood mix with oxygen-rich blood. an obstruction from ventricle to the lungs (pulmonal stenotic) is an important feature cause by the blockade of blood flow from the right ventricle into the lungs due to a narrowed outlet to the pulmonary artery, usually along with an abnormal pulmonary valve. be aside of all, an aorta that straddles the septum (overriding) between the ventricles will lets oxygen-poor blood flow into the aorta and also due to a thickened and enlarged heart muscle tissue in the right ventricle.2 overriding aorta above the ventricular defect and pulmonal stenotic allows the blood from the ventricles to be pump into the body. together, these defects make the level of oxygen in the blood low. these allow a bluish appearance (cyanotic) when oxygen-poor blood is pumped into the body, the fingers, toes, and lip.2 child with tof may feel easily exhausted, short winded, and hyperpnoea caused by hypoxia. sometimes the cyanotic does not clearly appear. tip of fingers and toes showed a clubbing shape and cyanotic. patient often do a squatting position after a certain distance walk or even cry. this position is necessary to enhance periphery resistance and restrain the blood flow from the extremities into the heart.1 further on, the increase of systemic resistance from a squatting position may repair the lungs oxygenation.3–5 like any other cyanotic congenital heart disease, in tof sometimes lead to a fatal complication such as bacterial endocarditis. incidence of bacterial endocarditis in tof is 15%.1 bacterial endocarditis is caused by a bacterial infection in heart valve or endocardium. in the field of dentistry, bacterial endocarditis is closely related to dental infection. poor dental condition mostly found in a child with tof is due to a disturbance in enamel development. gingival hypertrophy may be present and the tongue shows a map appearance of geographic tongue.2 dentist’s role is highly needed in an attempt to prevent this complication.5 invasive dental treatment, such as extraction, scaling, oral surgery, and endodontic treatment may cause bacteriemia.6 dental procedures that allow trauma in soft tissue or bone may lead to a bleeding which can cause transient bacteriemia. the condition in a certain patient may cause endocarditis. minor dental treatment, such as placing matrix band may lead to transient bacteriemia which further lead to bacterial endocarditis.5,6 the attempt to prevent bacterial endocarditis is the administration of prophylactic antibiotic. oral streptococcus such as s. sanguis and s. mitis are the organism which mostly cause bacterial endocarditis.6 children with tof are in a high risk of caries, especially in primary dentition. these are due to a certain medication for heart disease contains a high concentrate of sugar and further on, there is an increase in the prevalence of enamel mineralization disturbance. preventive efforts include the dietary counseling, fluoride administration, pit and fissure sealant, and oral hygiene instruction.6,7 these clinical reports discuss two cases of dental management in children with tof. �3pertiwi: oral and dental management cases first case: r, nine year old boy was referred to special dental care clinic in hasan sadikin hospital from his pediatric cardiologist to search for focal infection prior heart surgery. patient was diagnosed as tof since the age of eight, but the clinical signs such as fatigue, short winded, and often make a squatting position was already exist since toddler. at the moment, the patient is unable to walk and have to be carried by his parents. diagnoses from the pediatric cardiologist were diagnosis of function (df): compensated heart disease, diagnosis of anatomy (da): tof, and diagnosis of etiology (de): congenital heart disease cyanotic type. physical examination showed a delayed growth and development compare to a normal child at his age. patient looked fatigue and bluish. fingers and toes showed clubbing shape (figure 1). intra oral examination showed cyanotic in lip, buccal, and gingival mucosa. diagnosis of necrotic pulp in teeth no 75, 74, 84, 85, 64, 65 and pulp hyperemia in teeth no 54, 53, 62, 63. oral hygiene was in mild condition. second case: rz, four year-old boy also referred to special dental care clinic by his pediatric cardiologist to search for focal infection and it’s management prior to heart surgery. patient showed the sign of heart disease since the age of 5 months. diagnosis from pediatric cardiologist is diagnosis of function (df): compensated heart disease, diagnosis of anatomy (da): variant tof + bacterial endocarditis, and diagnosis of etiology (de): congenital heart disease cyanotic type. physical examination showed failure to thrive. patient was unable to walk. skin was pale and bluish with clubbing fingers and toes (figure 3). intra oral examination showed poor oral hygiene and generally chronic marginal gingivitis. lips, tongue, gingival and buccal mucosa were cyanotic. dentine caries at teeth no 55, 53, 63, 65, 73, 72, 71, 81, 82, 83. caries that reach the pulp in teeth no 54, 64, 75, 74, 84. radix in teeth no 52, 51, 61, 62. figure 1. clubbing and cyanotic fingers and toes. figure 2. patient was monitored by pulse oxymetry and administred oxygen inhalation. �� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 42–45 cases management dental treatments for the first case consist of preventive and curative efforts. preventive treatments were carried out by oral hygiene instruction and topical fluoride application. curative treatments are extraction of the teeth with necrotic pulp and glass ionomer cement fillings in hyperemia pulp teeth. to prevent bacterial endocarditis, patient was given a prophylactic antibiotic 50mg/kg body weight one hour prior before treatment. during the dental procedure, patient was monitored by pulse oximetry to monitor the oxygen saturation and heart pulse. patient was also inhale oxygen (figure 2). patient was very cooperative, so the dental treatment was done after two months periods. as for the second case, dental management was different than the first case. patient was extremely uncooperative. treatment plan were extraction of teeth no 52, 51, 61, 62, 54, 64, 75, 74, 84 and filling in teeth no 55, 53, 63, 65, 73, 72, 71, 81, 82, 83. antibiotic prophylactic (amoxicillin) was considered to be given 50mg/kg body weight 1 hour before the procedure. in the first visit, patient was very uncooperative, he revolted and cried so the oxygen saturation drop to 10 percent (normally, he have oxygen saturation of 60–70 percent, normal child should have the value of 100 percent). considering the dental treatment will harm the patient, so he was scheduled to the dental treatment under general anesthesia. laboratory and radiographic examination were carried out and the case was discussed in a join conference with pediatricians and anesthesiologist. thorax photo showed an enlarged heart of increase vascular marking, no sign of active specific process, and an opaque round shadow in the right par tracheal. the join conference concluded that the dental treatment under general anesthesia can not be carried out at hasan sadikin hospital because this patient is in a high risk due to a severe condition of tof. patient was referred to undergo mouth preparation under general anesthesia at harapan kita hospital. discussion oral and dental treatment in a child with congenital heart disease is very important. main point which have to be noticed is that patient with this disorder is highly susceptible figure 3. clubbing and cyanotic fingers and toes. figure 4. patient’s profile. ��pertiwi: oral and dental management to bacterial endocarditis. in the first case, the history of bacterial endocarditis was not yet exists. but in the second case, the bacterial endocarditis was already exists. this was due to poor oral hygiene in the second patient. every dental procedures which may lead to a damaged of soft tissue can cause transient bacteriemia and thus in a certain condition cause to bacterial endocarditis.5 transient bacteriemia may exists after physiological activities which involves the mouth, such as mastication and tooth brushing. according to guntheroth, transient bacteriemia that happens after physiological activities is 1000 fold higher than after dental procedures. however, the risk of bacteriemia in a patient with a clean and healthy oral cavity is lower than patient with poor oral hygiene.5 bacterial endocarditis that caused from oral bacteria has to be prevented with the administration of prophylactic antibiotic. antibiotic can prevent endocarditis by killing the bacteria or damage it so it can be destroyed by host defense. the effect may occur in oral cavity, blood flow, or after the organism adherence in the heart.5 both cases was administered prophylactic antibiotic (amoxicillin) in a dose of 50 mg/kg body weight 1 hour prior to each visit. the important point to be remembered in the administration of prophylactic antibiotic is to be carried out at every treatment in every visit, considering patient’s emotional state.6 in every visit, the first patient was extracted 1–2 teeth depend on the patient’s state of health. beside prophylactic antibiotic, the risk of developing bacterial endocarditis can be minimized by preventive dental procedure. these contain of routine dental check up, fluoridation, dietary modification to minimize the risk of dental caries and periodontal disease, and day care maintenance of oral hygiene (tooth brushing).5,9,10 topical fluoride application in the first case is aimed as preventive treatment while curative treatment is in the form of extraction of a tooth with pulp involved caries and filling in dentine caries diagnosed tooth. in a normal patient, pulp involved tooth can be treated with pulp therapy, but in patient with tof can not be carried out due to the risk of bacterial endocarditis.11 patient in the first case is very cooperative, thus the dental treatment can be carried out through psychological approach. during the procedure, patient’s status was monitored by pulse oximetry. oxygen saturation of this patient was below 70 percent, thus patient was administered oxygen inhalation during the treatment. the patient have the risk of respiratory arrest due to disorders in pulmonary value. 12,13,14 in the second case, conventional dental treatment can not be carried out due to patient uncooperativeness. patient was scheduled to undergo mouth preparation under general anesthesia, but considering that this patient has severe tof, the procedure can not be carried out in hasan sadikin hospital. oral and dental treatment is important to be carried out in children with the risk of bacterial endocarditis. patient in a good general condition can receive every dental treatment as long as covered by prophylactic antibiotic. the important aspects of dental treatment are dental health education, preventive dental treatment, and maintenance after dental treatment. dental health education can be delivered since the child showed heart disorder considering congenital heart disease mostly showed the clinical signs in infancy. early dental health education can minimize oral and dental disease which may risk the existence of bacterial endocarditis. references 1. baraas f. penyakit jantung pada anak. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 1995. p. 140–51. 2. tetralogy of fallot. texas: texas heart institute. available from url: www.americanheart.org accessed nopember 28, 2005. 3. markum ah. buku ajar ilmu kesehatan anak. jilid 1. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 1991. p. 577–81. 4. gordon ib. sistem kardiovaskuler. in: behrman re, voughan vc, nelson, editors. ilmu kesehatan anak. bagian 2. jakarta: egc; 1992. p. 726–34. 5. sinaga le, wahab as. kardiologi anak tetralogi fallot. yogyakarta: bagian ilmu kesehatan anak, fakultas kedokteran universitas gadjah mada; 2004. p. 26. 6. little jw, falace da. dental management of the medically compromised patient. 6th ed. st louis: mosby inc; 2002. p. 21–63. 7. koch g, poulsen s. pediatric dentistry, a clinical approach. copenhagen: munksgaard; 2001. p. 428. 8. cameron ac, widmer rp. handbook of pediatric dentistry. 2nd ed. sydney: mosby inc; 2003. p. 234–6. 9. welburry rr. pediatric dentistry. 2nd ed. new york: oxford university press; 2001. p. 374–8. 10. mccallum ca. oral surgery for children. in: finn sb, editor. clinical pedodontics. 4th ed. philadelphia: wb saunders co; 1991. p. 392, 438, 581–2. 11. mathewson rj. fundamentals of pediatric dentistry. 3rd ed. chicago: quintessence books; 1995. p. 89–104. 12. van der wall ee, mulder jm. pulmonary valve in patient with tetralogy of fallot. european heart journal 2006; 26(24):2624–5. 13. saygih a, aytekin c, boyvat f, barutçu ö, mercan , tokel k. endobronchial stenting in a two-,onth-old infant with bronchial compression secondary to tetralogy of fallot and absentpulmonary valve. the turkish journal of pediatric 2004; 46(3):268–71. 14. rowland dg, caserta t, foy p, wheller j, allen h. congenital absence of the pulmonary valve with stenosis and patent ductus arteriosus: a prenatal diagnosis. american heart journal 1996; 132(5):1075–7. isi vol 39 no 3 juli-september 2006.pmd 133 the copper concentration variation to physical properties of high copper amalgam alloy aminatun department of physics faculty of mathematics and physics airlangga university surabaya indonesia abstract the function of copper (cu) inside amalgam is to increase hardness and impact force and to decrease thermal expansion coefficient. in general, amalgam which is used in dentistry and available in the market is contain cu 22%, while the maximum cu concentration is 30%. it is necessary to determine the concentration cu does generate the best physical properties to be used as dental restorative agent. amalgam is made by mixing blended-metal ag-sn-cu (with cu concentration of 13%, 21%, 22%, and 29%) and hg, stirred manually in a bowl for 15 minutes,leave it in temperature 27°c for 24 hours to become hardened. the result of x-ray diffractometer (xrd), analyzed by rietveld method and rietica program, shows amalgam with cu 29% concentration for cu3sn compound density is 31.790 sma/å3, for ag2hg3 compound is 41.733 sma/ å3, a cu3sn relative weight percentage of 43.23%, ag2hg3 of 54.54%, cu7hg6 of 2.23% and hardness of cu 29% is 90.700 ± 0.005 kgf/mm2. these numbers are the highest values on cu 29% concentrations compared to other copper concentration variants. whereas amalgam thermal expansion coefficient on cu 29% is (2.17 ± 0.91)10-3 mm/°c is the lowest value compared to other cu concentration. the conclution is that adding cu concentration into amalgam will increase density value, cu3sn relative weight percentage, hardness level and will decrease amalgam thermal expansion coefficient. amalgam 29% cu concentration has better physical properties compared to amalgam cu 22% concentration. key words: amalgam, cu, density, hardness level, thermal expansion coefficient correspondence: aminatun, c/o: jurusan fisika – fmipa universitas airlangga. jln. mulyorejo, sukolilo surabaya 60115, indonesia. factories or there is also another presentation as a separate particle to be added (add-mixed) according to one’s desired concentration. in practical use, amalgam in dentistry is amalgam with ag-sn-cu elements formed by dental factories and the circulating amalgam in the market is the cu 22% concentration. in high copper amalgam alloy, the permissible cu concentration is between 12–30%. for that reason, this research applies constant concentration of ag-sn compound added with varying cu concentration of 13%, 21%, and 29% with one hope to generate a better quality amalgam. this research expects to give information on what cu concentration in amalgam which will produce better physical properties compared to amalgam in the market. materials and methods this is an experimental laboratory research done in the material physical laboratory of the faculty of mathematics and physics of both airlangga university and the institute of sepuluh november and also at the research center of the institute of sepuluh november. material were used powder of ag-sn-cu compound, ag powder, sn and cu powders with 98% purity, thick paper, the softest sand paper, liquid hg, aquadest, acetone and nacl of 98% purity. introduction amalgam is an alloy of mercury (hg), silver (ag), tin (sn), copper (cu) and zync (zn) metal compounds.1 based on the concentration of cu in the alloy, amalgam is divided into two types, conventional and high copper amalgam alloy.2 the conventional amalgam contains lower cu concentration (0–6%) and high copper amalgam alloy contains higher cu concentration (12% or more).2,3 based on the concentration of zync (zn), amalgam is divided into 2 types i.e. the blended alloy type with 1% zync content and composition alloy with 0% zync content. amalgam high copper alloy has contained more than 12% copper (cu) concentration and 0% zync (zn) concentration (no mixture of zn inside amalgam). the presence of cu in amalgam will form cu3sn compound replacing sn7hg which has a corrosive nature and weakens amalgam’s hardness. cu inside amalgam can perform a reaction with sn creating a compound of cu3sn, so that the newly formed amalgam is not easily corroded and its marginal strength is better than the conventional amalgam (amalgam without cu). furthermore, cu increases amalgam’s hardness and its impact force and decreases amalgam thermal coefficient.4 the more cu inside amalgam, the more amalgam’s impact force and hardness will be. copper (cu) inside amalgam is presented as part of ag-sn compound with definite concentration from dental 134 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:133–137 utilized tools were a small-sized bowl for the ag-sn compound powder, amalgam stirrer, plastic cast fitting to sample, scissor, tray and plastic pipette, x-ray diffractometer (xrd), microhardness vickers test future tech fm 7, and thermal expansion coefficient test tool. sample was blended of 5 grams ag-sn-cu powder in weight comparison 3:1:1, along with dental factory composition which is mixed with hg liquid equal to the weight of ag-sn-cu compound (1:1). stirred manually in the bowl using amalgam stirrer for 15 minutes (until the sample homogenous) and leave for 24 hours in room temperature to harden, will produce sample with cu concentration similar to the dental factory (22%).3 five grams ag-sn powder of dental factory composition is added separately (add-mixed) with varying cu concentration of 13%, 21%, and 29%. then, it is stirred manually for ±15 minutes until homogenous and leave it to harden in room temperature (27 °c) for 24 hours.3 the four results of the above process (with cu concentration of 13%, 21%, 22%, and 29%) were printed in plate forms, each with 3 different sizes i.e. 2 cm × 1.5 cm for measuring the level of hardness, 5 cm × 5 cm for measuring x-ray diffractometer, and 2 cm × 0.2 cm for measuring thermal expansion coefficient. the samples were pressed with the tip of stirrer to obtain samples in plate forms and a soft sand paper were used to smooth their surfaces. afterwards, the four samples were characterized with xrd test, with level of hardness measurement and with thermal expansion coefficient. xrd was used to obtain information pertaining to amalgam composing compound and impurity phases. amalgam samples of 5 cm × 5 cm were put on xrd holder and x-rayed. the diffraction results are recorded for 20 certain angles acquiring a curve of the correlation of x-ray diffraction intensity towards 20 diffraction angles. the curve was analyzed qualitatively and quantitatively. qualitative way is to determine the amalgam composing compound by comparing to joint committee on powder diffraction standard data (jcpds). furthermore, to find out the density and the relative weight percentage of each element is done by rietveld method and rietica program.5,6,7 the xrd test was performed on all four samples. measuring the level of hardness was used micro vickers hardness (mvh) test tool with procedures as follows: the 2 × 1.5 cm 2 amalgam sample was pressed with a pyramidal shape diamond in 136° slanted angle. the penetration on sample surface leaves diagonal bundles which can be observed by mvh test tool as d1,d2, p values. vhn (vickers hardness number or sample hardness level) can be calculated with formula )(kgf/mm d p 1.854vhn 2 2 = .8 level of hardness test is performed on all four samples with similar procedure. this is the procedure to measure thermal expansion coefficient: the 2 × 0.2 cm2 amalgam sample is immersed in water, the water is heated until sample’s temperature reaches 37° (t1), then the amalgam is taken out and measured. the measurement at that time is the initial amalgam length (l1). afterwards, amalgam is re-immersed in water, the water is heated until sample’s temperature reaches 60 °c (t2), then amalgam is taken out and remeasured, that is the final length (l2).1,9 grounded on t1,t2, l1, and l2, the thermal expansion coefficient can be counted with formula: t 1 l l δ δ =α the thermal expansion coefficient test is performed to all four samples. result x-ray diffractometer test result of the four samples is shown in figure 1(a), (b), (c), and (d). based on xrd test result, two analyses can be conducted i.e. qualitative and quantitative analyses. (a) a : ag2hg3 c : cu3sn counts 02 theta (b) a : ag2hg3 c : cu3sn counts 02 theta 135aminatun: the copper concentration variation qualitative analysis is done by search matching the spectrum of xrd test result with joint committee on powder diffraction standard (jcpds) data to find out the phases inside amalgam (identification phase). quantitative analysis is meant to know the relative weight percentage of each phase inside amalgam and it is conducted with rietveld analysis method. rietveld method is an analysis method using search-match of calculated diffraction pattern (model data) and measured diffraction pattern (data from experiment’s result).10 one of the soft-wares of this method is rietica program. assissted by rietica program and supported by international crystallography standard database (icsd) crystallographic data , one can calculate the relative weight percentage of each amalgam composing compound. the compounds composing amalgam are cu3sn and ag2hg3. special condition on amalgam cu 29%, besides having cu3sn and ag2hg3, it has another compound, i.e. cu7hg6 (table 1 and figure 2) based on crystallography data (icsd), the structure of crystal cu3sn is hexagonal with lattice parameter a = b ≠ c; the structure of crystal ag2hg3 is cubical with figure 1. xrd spectrum of four samples. (a) xrd spectrum of amalgam cu 13%; (b) xrd spectrum of amalgam cu 21%; (c) xrd spectrum of amalgam cu 22% (factory); (d) xrd spectrum of amalgam cu 29%. figure 2. graph of amalgam composing compound relative weight percentage on cu concentration. table 1. relative weight percentage of amalgam composing compound high copper type single composition alloy in cu concentration variation relative weight (%) no. cu concentration (%) cu3sn ag2hg3 cu7hg6 1. 2. 3. 4. 13 21 factory (22%) 29 7.74 40.52 42.91 43.23 92.26 59.48 57.09 54.54 2.23 (c) (d) a : ag2hg3 c : cu3sn b : cu7hg6 counts 02 theta a : ag2hg3 c : cu2sn counts 02 theta amalgam + cu 22% (pabrik) 0 10 20 30 40 50 60 70 80 weight percentage (%) 13 21 22 29 cu concentration (%) cu sn3 ag hg2 3 cu hg7 6 136 dent. j. (maj. ked. gigi), vol. 39. no. 3 july–september 2006:133–137 lattice parameter a = b = c; and the structure of crystal cu7hg6 is trigonal with lattice parameter a = b ≠ c. the value of each lattice parameter can be seen in table 2. based on the output result of rietveld analysis method, the density of each sample can be found (table 3). grounded on table 3, a diagram of each density of amalgam composing compound in cu concentration variation, can be made (figure 3). adding cu concentration will decrease amalgam volume size and increase the density value. this is in accordance with the theory: density is in reverse proportion with its volume size. amalgam cu 29% concentration has the smallest volume size so that it has the highest density of each composing compound. the measuring result of amalgam high copper type single composition alloy sample thermal towards varying cu concentrations with an initial temperature of 37 ºc and final temperature 60% is presented in table 4. the result of level of hardness test using vickers hardness is presented in table 5. table 2. lattice parameter for high copper amalgam alloy with cu concentration variation latice parameter no. cu concentration (%) cu 3sn (å) ag 2hg3(å) cu7hg6 (å) 1. 2. 3. 4. 13 21 factory (22%) 29 a = b = 7.1 ; c = 8.4 a = b = 7.3 ; c = 7.8 a = b = 7.3 ; c = 7.8 a = b = 5.3 ; c = 5.8 a = b = c = 9.9 a = b = c = 9.9 a = b = c = 10.0 a = b = c = 6.9 a = b = 5.3 ; c = 5.1 the addition of cu concentration has elevated amalgam level of hardness. amalgam high copper type single composition alloy with cu 29% concentration possesses the highest level of hardness, i.e. 90.70 ± 0.05 kgf/mm2. discussion as seen in table 1 and figure 2, amalgam relative weight percentage, particularly cu3sn, has continually increased table 3. density and volume size of composing high copper amalgam alloy in cu concentration variation density (sma/ å3) volume size (å3) no. cu concentrate 9%0 cu 3sn ag 2hg3 cu 3sn ag 2hg3 1. 2. 3. 4. 13 21 factory (22%) 29 9.074 11.876 12.455 31.792 14.235 14.335 14.641 41.733 369.3 365.9 364.9 143.3 999.990 999.999 998.8 341.0 figure 3. graph of the correlation between each density of amalgam composing compound towards cu concentration. 0 10 20 30 40 50 13 21 22 29 cu concentration (%) cu sn3 ag hg2 3 cu hg7 6 density (sma/a )3 table 4. thermal expansion coefficient of amalgam high copper type single composition alloy in cu concentration variation no. cu concentration (%) thermal expansion coeffisient (°c) 1. 2. 3. 4. 13 21 factory (22) 29 [(3.48 ± 0.17) 10-3] [(3.04 ± 0.13) 10-3] [(2.61 ± 0.16) 10-3] [(2.17 ± 0.91) 10-3] 137aminatun: the copper concentration variation amalgam to yield a higher level of hardness. it is deducted that amalgam with cu 29% is the most durable to receive permanent pressure i.e. mastication load. based on physical properties analyses i.e. density, weight percentage, level of hardness and thermal expansion coefficient which are already done, the added cu in amalgam had increased relative weight percentage of amalgam composing compound, density and level of hardness. whereas adding cu percentage can cause a smaller thermal expansion coefficient. this condition fits the physical properties of cu i.e. that adding cu concentration into amalgam will increase hardness leveland amalgam strength, and will decrease amalgam thermal expansion coefficient.12 amalgam with the best physical properties in this research is amalgam with 29% cu concentration, so that this type can be considered as restorative agent which is inexpensive, durable to bear the mastication load and has a small thermal expansion coefficient. the conclution of this research that adding cu concentration into amalgam will increase amalgam density value, cu3sn relative weight percentage, hardness level and will decrease amalgam thermal expansion coefficient. amalgam cu 29% concentration has better physical properties compared to amalgam produced by factories (cu 22%), fits to be used as dental restorative agent. it is necessary to do further studies to complete the basis of amalgam quality determination by observing corrosive durability and impact force. references 1. craig rg, power jm. restorative dental materials. 11th ed. st lois: mosby; 2002. p. 53–4, 288–300. 2. combe ec. notes on dental materials. 6th ed. edinburgh, london: churchill livingstone; 1992. p. 191–204. 3. craig rg, power jm, wataha jc. dental materials properties and manipulation. 7th ed. st louis: mosby. inc; 2000. p. 79–85. 4. ford ptr. restorasi gigi. edisi kedua. jakarta: penerbit buku kedokteran egc; 1993. h. 61–6. 5. pratapa s. prinsip-prinsip dan implementasi metode rietvelt untuk analisis data difraksi sinar-x. angkatan ii. surabaya: lembaga penelitian its; 2004. h.1–20. 6. hill rj. data collections strategis: fitting the experiment to the need intensification the rietveld method. young ra, edited. oxford; oxford university press; 1993. p. 61–101. 7. hill rj, howard cj. quantitative phase analysis form neutron powder data using the rietveld method. journal appl crystallography 1986; 20: 467–74. 8. vlack vlh, djaprie s. ilmu dan teknologi bahan (ilmu logam dan bukan logam). edisi kelima. jakarta: erlangga; 1985. h. 136-9. 9. tippler pa. physics for scientist and engineers. new york: worth publisher; inc. 1991. p. 568–70. 10. hunter ba. rietica in newsletter of international union of crystallography commission powder diffraction. sydney 1998; 20:21. 11. mml medical. tambal gigi yang mengandung merkuri, amankah? available from: http://www. kalbe.co.id. accessed april 19, 2006. 12. noort vr. introduction to dental materials. 2nd ed. london: mosley; 2002. p. 81–93. table 5. level of hardness of high copper amalgam type single composition alloy in cu concentration variation no. cu concentration (%) level of hardness (kgf/mm2) 1. 2. 3. 4. 13 21 factory (22) 29 (60.30 ± 0.05) (66.90 ± 0.05) (82.70 ± 0.05) (90.70 ± 0.05) along with the addition of cu concentration. the extent of relative weight percentage of each compound is closely related with mixing and stirring phase, although in amalgam cu 29% occurs cu7hg6 (2.23%), it still does not decrease cu3sn relative weight percentage. the occurrence of impurity factor is assumedly due to non-optimum mixing and stirring process producing non-homogenous sample. the copper is supposedly bonding with sn instead of hg. based on table 3 and figure 3, there is an increase of amalgam density caused by the increase of cu concentration after cu is being added into agsn + hg. the reason of this occurrence is the granule of amalgam composing particles are not the same size, where any cu and ag-sn-hg particles are mutually complementing during the mixing and stirring phase. ag2hg3 compound density is higher than cu3sn compound, so that the micro structure of ag2hg3 is more dense than cu3sn. thus, the most dense micro structure of the sample is amalgam with 29% cu concentration. mercury (hg) is a neurotoxic agent. on one of previous researches, the amount of ejected hg from amalgam which caused negative effect on cognitive (especially on children) was observed. numerous new studies published in the weekly news of the american medical association have stated the safety assurance for amalgam use as dental filling.11 based on the measurement of thermal expansion coefficient (table 4) one can see that more concentration of cu will result in the lower value of thermal expansion coefficient. accordingly, the cu concentration has a significant impact on thermal expansion coefficient. this physical property is related to the density of amalgam composing compound. if the amalgam becomes more dense, the amalgam composing compound inter-atom bond, also becomes stronger that makes thermal expansion coefficient decreases. high thermal expansion coefficient in amalgam will cause amalgam to be easily cracked, thus the smaller thermal expansion coefficient, the better amalgam quality is to be used as a restorative agent.12 based on thermal expansion coefficient in this research, the best quality sample for dental restorative is cu with 29% concentration. the density of amalgam composing compound becomes bigger corresponding to more added cu. amalgam with 29% cu has more solid micro-structures giving impact on << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 6363 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 63–67 original article prevalence of dental anomalies in pediatric patients at dental and oral hospital of universitas muhammadiyah yogyakarta laelia dwi anggraini, denna idryareza augustyana, nurrofi sekarjati department of pediatric dentistry, school of dentistry, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta, yogyakarta, indonesia abstract background: a dental abnormality is a deviation from the normal shape and structure of the teeth due to interference during growth and development. various kinds of abnormalities occur, such as anomalies in the size, shape, position, number, and structure of the teeth. these conditions cause problems in the arch length and occlusion of the maxilla and mandible. purpose: this study aimed to describe the prevalence of developmental dental anomalies in pediatric patients at the dental and oral hospital of universitas muhammadiyah yogyakarta (umy) and its networks (qatrunnada kindergarten, budi mulia dua taman siswa kindergarten, and muhammadiyah sapen pusat primary school). methods: a descriptive observational study with a cross-sectional design was conducted. there were 10,714 pediatric patients included. results: the prevalence of developmental dental anomalies in pediatric patients at dental and oral hospital, umy and its network was 0.30%. the prevalence of mesiodens, hypodontia, and fusion dental anomalies were 0.14%, 0.056%, and 0.028%, respectively. there was a 0.019% prevalence of microdontia, peg tooth, and amelogenesis imperfecta. the prevalence of taurodontia and gemination was 0.009%. conclusion: dental anomalies occurred more frequently in male pediatric patients, and mesiodens was the most prevalent. keywords: developmental dental anomaly; hypodontia; mesiodens; prevalence article history: received 26 january 2022, revised 20 june 2022, accepted 16 july 2022 correspondence: laelia dwi anggraini, department of pediatric dentistry, school of dentistry, faculty of medicine and health sciences, universitas muhammadiyah yogyakarta. jl. lingkar selatan, bantul, special region of yogyakarta 55183, indonesia. email: laelia.dwi@umy.ac.id introduction a dental anomaly is a deviation from the normal tooth shape due to a disruption in growth and development.1 dental anomalies are caused by growth disorders, but not all growth disorders produce abnormalities. dental anomalies can occur in both deciduous teeth and permanent teeth. both types of teeth are found in childhood.2 genes determine the variations in a child’s dental anomalies, as well as the patient’s habits, nutrition, and multivitamin intake. genetic and environmental factors play an essential role in determining variations.3 anomalies in the size, shape, position, number, and structure of teeth cause problems in the length of the arch and occlusion of the maxilla and mandible. dental abnormalities cause dysfunction, decreased activity, and lowered work productivity, affecting the quality of life.4 identifying the prevalence of dental anomalies can provide important information about the anthropology and clinical management of patients. the incidence and distribution of these anomalies are also important for understanding differences in and between populations. early diagnosis of dental anomalies is needed to foresee the possibility of complications and to minimize the risks associated with dental procedures such as orthodontic treatment for children. epidemiological studies have been carried out in various parts of the world; the prevalence of deciduous dental anomalies was 2.27% in south india, and other studies identified a range of 0.4–8.1%.5 however, this issue has not been reported in indonesia. the most common dental anomaly in children, according to the study, is hypodontia, followed by fusion and gemination (double tooth). the third most common dental anomaly is supernumerary teeth.6 this study copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p63–67 mailto:laelia.dwi@umy.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p63-67 64 anggraini et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 63–67 aimed to determine the prevalence of developmental dental anomalies in pediatrics, to ensure that patients with anomalies receive proper treatment. dental and oral hospital, umy was selected due to its central location and the high number of people that seek treatment there. materials and methods this study used a descriptive observational method with a retrospective study that used the medical records of the patients at the dental and oral hospital of universitas muhammadiyah yogyakarta (umy) and its networks (qatrunnada kindergarten, budi mulia dua kindergarten, and muhammadiyah sapen pusat primary school). this study was approved by the ethical research committee of the medical and health science faculty, universitas muhammadiyah yogyakarta, indonesia (number: 456/ ep-fkik-umy/x/2018). there were 10,714 medical records reviewed. this study analyzed primary and secondary data. the primary data were obtained from pediatric patients who participated in national dental health month or bulan kesehatan gigi nasional (bkgn) 2018. the secondary data was obtained from the pediatric patients’ medical records at dental and oral hospital, umy from 2013–2018. all who participated in bkgn 2018 were given and signed informed consent. the inclusion criteria were orthopantomogram (opg) or panoramic dental roentgen, medical records, data from pediatric patients aged 0–18 years at dental and oral hospital, umy and its networks in indonesia, and agreement with informed consent. the exclusion criteria were incomplete medical records of pediatric patients in the dental and oral hospital, umy and its network. incomplete medical records included those that did not have opg or panoramic dental roentgen and patients that refused to give informed consent. the disease diagnosis, age, and address data of patients were obtained from the medical records. the data were analyzed by frequency distribution. all data were analyzed by the descriptive statistical method of cross-tabulation using statistical analysis for social science (spss) version 21 (ibm, chicago, us). results thirty-two patients had dental anomalies with a prevalence of 0.30 %. the highest dental anomalies occurred in the number of teeth, namely mesiodens (0.14%) followed by hypodontia (0.056%). dental shape anomalies were less common. the prevalence of fusion was 0.028%, the prevalence of microdontia and peg shape abnormalities was 0.019%, while taurodontia and gemination were only found in one patient with the prevalence of each anomaly being 0.009%. additional dental anomalies were found as shown in figure 1. a structural anomaly, amelogenesis imperfecta, was found in one patient with a prevalence of 0.009% (table 1). figure 1. various dental anomalies found in children, from left to right: fusion, gemination, and mesiodens. table 1. distribution of dental anomalies based on gender dental anomalies frequency prevalence gender male female mesiodens 15 0.140% 9 6 hypodontia 6 0.056% 4 2 fusion 3 0.028% 1 2 microdontia 2 0.019% 2 0 peg shaped 2 0.019% 1 1 amelogenesis imperfecta 2 0.019% 1 1 taurodonsia 1 0.009% 0 1 gemination 1 0.009% 1 0 total 19 13 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p63–67 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p63-67 65anggraini et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 63–67 table 2 shows the distribution of dental anomalies based on the age of the patients; the highest frequency was found in pediatric patients 6–12 years old. table 3 shows the distribution based on address with the highest frequency in jogja city (15), bantul regency (9), and sleman regency (8). discussion our study showed that mesiodens was the most prevalent anomaly identified at dental and oral hospital, umy and its network. the prevalence of mesiodens was 0.14%. the results of this prevalence were lower than the previous studies in various countries. gündüz et al.7 mentions the prevalence of mesiodens dental anomalies in the caucasian population is 0.45%, in the finnish population it is 0.4%, in the norwegian population it is 1.43%, and in the hispanic population it is 2.2%. mesiodens is a dental anomaly in the form of canine-like teeth in the middle of the arch of the jaw. hypodontia is the absence of one or several teeth. microdontia is a tooth that looks smaller than normal size. peg shapes are pointed teeth while amelogenesis imperfecta is a structural dental anomaly.8 hereditary patterns found in dental anomalies include autosomal dominant, recessive, and x-linked characteristics, so the number of individuals affected in one family can vary. the most common form is x-linked, and it is this gene that regulates the size and shape of human teeth.9 systemic conditions and syndromes associated with macrodontia are otodental syndromes, 47 xyy syndromes, facial hemihyperplasia, and insulin-resistant diabetes. these anomalies can also arise due to developmental disturbances during the morpho-differentiation stage.10 in general, dental anomalies are caused by complex interactions between genetic, epigenetic, and environmental factors during the long process of tooth development.11 as shown in table 2, mesiodens was more prevalent in male pediatric patients. our results were in line with the research conducted on the indian population by peediayil et al.12 and khandelwal et al.13. mesiodens is the most common dental anomaly in permanent teeth and is rarely found in deciduous teeth.14 this anomaly commonly irritates the tip of the tongue and interferes with appearance.15 mesiodens is primarily found between 6–12 years, coinciding with the time of eruption of the maxillary central incisors. radiographs are performed to screen for congenital hypodontia, cysts, and tumours in late tooth eruptions or malposition.16 the next most prevalent anomaly in the number of teeth was hypodontia with a prevalence of 0.056%. the prevalence is very low compared to research conducted by altug-atac and erdem,17 who found the prevalence to be 0.56%. larger results were reported in india, where hypodontia had the highest prevalence at 16.3%.5 in this study, and a study of 4,180 children in india by shilpa et al.18, hypodontia was predominantly found in male patients. the cause of hypodontia is strongly related to genetic factors, where there is a developmental disturbance of the teeth and disturbances in eruption.19 among dental shape anomalies, fusion showed the highest prevalence at 0.028%. fusion is an abnormality that occurs at the developmental stage and can cause two adjacent teeth to become one tooth. in general, the crown of fused teeth is larger in size.1 the high prevalence of fusion obtained in this study is close to the results of studies conducted in turkey, which showed a prevalence of 0.09%.18 a higher prevalence of fusion, 4.85%, was demonstrated in a study conducted in india by guttal et al.20 fusion and gemination anomalies can be referred to as double teeth that appear larger than normal size teeth. in gemination, the number of teeth is normal due to a single tooth that enlarges or connected teeth (double) being counted as one tooth.21 the levita classification is a practical method for distinguishing between cases of fusion and gemination.22 in this study, gemination was only found in one male patient with a prevalence of 0.009%. this is similar to the research conducted by guttal et al.20 on 20,182 patients who found only one male patient with a gemination anomaly. other dental anomalies also showed a low prevalence; the prevalence of microdontia was 0.019%, and taurodontia was found in only one patient with a prevalence of 0.009%. these results are lower than previous studies conducted in various countries.5,14,23 the position of the teeth is influenced by race and hereditary tendencies.8 in this study, anomalies in tooth size were found. microdontia was found in two patients (0.019%). table 2. distribution of dental anomalies based on age dental anomalies age (years old) 0–5 6–12 13–18 mesiodens 1 11 3 hypodontia 4 2 0 fusion 3 0 0 microdontia 0 0 2 peg shaped 0 0 2 amelogenesis imperfecta 0 0 2 taurodontia 0 1 0 gemination 0 1 0 total 8 15 9 table 3. distribution of dental anomalies based on address dental anomalies location jogja city sleman bantul mesiodens 5 3 7 hypodontia 5 1 0 fusion 2 1 0 microdontia 0 1 1 peg shape 0 1 1 amelogenesis imperfecta 1 1 0 taurodontia 1 0 0 gemination 1 0 0 total 15 8 9 copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p63–67 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p63-67 66 anggraini et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 63–67 macrodontia was not found. a study carried out in india showed the prevalence of microdontia was 1.0%. studies in pakistan found the prevalence of microdontia was higher, at 4%.6,24 microdontia in this study was found in male patients, in contrast to the study by ezoddini et al.25, where microdontia was more common in females. race may be one of the causes of these differences. a disturbance can cause the occurrence of dental anomalies in the form, size and structure at the stage of morpho differentiation. this study also found structural anomalies, namely amelogenesis imperfecta. in this study, there were two patients with a prevalence of 0.019%. this is in accordance with the research conducted in india where the prevalence of amelogenesis imperfecta was 0.02%.26 while this is the rarest dental anomaly encountered, in a turkish study conducted by altug-atac and erdem,17 the prevalence of amelogenesis imperfecta was 0.43%. this difference in the prevalence can be caused by hereditary factors and the grouping of patients affected by this dental anomaly in certain geographical areas which increases the prevalence of disorders in the region. in addition, rigorous diagnostic criteria can affect the reported prevalence in various studies.27 this study also describes the results of the distribution of pediatric dental anomalies based on address. as shown in table 3, there were no patients with dental anomalies living in gunungkidul and kulonprogo regencies, and the highest number was found living in jogja city. the high frequency of patients residing in jogja city and the absence of those who reside in gunungkidul regency and kulonprogo regency may be due to the location of the dental and oral hospital, umy. in general, rural areas are often associated with lower education levels and are related to lower levels of health literacy and the poor use of available healthcare services.28 this could account for the absence of children who have dental anomalies in the gunungkidul regency and kulonprogo regency. urban residents are more aware of the importance of health, the importance of maintaining and identifying abnormalities, and the importance of early detection. awareness of dental and oral health programs needs to be improved, especially for rural regions. since this study was limited to the dental and oral hospital, umy, further research will be improved by widening the area and population included. in conclusion, most dental anomalies are found in pediatric patients aged 0–18 years old in the dental and oral hospital, umy. the prevalence of mesiodens, hypodontia, and fusion was 0.14%, 0.056% and 0.028%. dental anomalies in pediatric patients were more prevalent in male children. the age range of pediatric patients who had the most dental anomalies was 6–12 years old. the greatest distribution of anomalies occurred in jogja city, sleman and bantul. for further research, a larger area and population should be studied. acknowledgement authors would like to thank all patients of dental and oral hospital, umy who has participated in this study. patients as subjects has agreed with informed consent. this research was conducted independently (independent funds) while research, publication, and community service institutions or lembaga penelitian, publikasi, dan pengabdian masyarakat (lp3m) umy for funding this research. references 1. white sc, pharoah mj. oral radiology: principles and interpretation. 7th ed. st. louis: mosby; 2013. p. 9, 16,17, 18, 41, 43. 2. welbury r, duggal ms, hosey mt. paediatric dentistry. 3rd ed. oxford: oxford university press; 2005. p. 234–61. 3. afify ar, zawawi kh. the prevalence of dental anomalies in the western region of saudi arabia. isrn dent. 2012; 2012: 837270. 4. pay m n, widiati s, sr iyono n w. identif i kasi fa ktor ya ng mempengaruhi perilaku anak dalam pemeliharaan kebersihan gigi dan mulut: studi pada pusat pengembangan anak agape sikumana kota kupang, nusa tenggara timur, indonesia. maj kedokt gigi indones. 2017; 2(1): 27–34. 5. almaz me, sönmez is, oba aa. prevalence and distribution of developmental dental anomalies in pediatric patients. meandros med dent j. 2017; 18(2): 130–3. 6. patil s, doni b, kaswan s, rahman f. prevalence of dental anomalies in indian population. j clin exp dent. 2013; 5(4): e183-6. 7. gündüz k, celenk p, zengin z, sümer p. mesiodens: a radiographic study in children. j oral sci. 2008; 50(3): 287–91. 8. jahanimoghadam f. dental anomalies: an update. adv hum biol. 2016; 6(3): 112–8. 9. bailleul-forestier i, molla m, verloes a, berdal a. the genetic basis of inherited anomalies of the teeth. part 1: clinical and molecular aspects of non-syndromic dental disorders. eur j med genet. 2008; 51(4): 273–91. 10. krishnan ar, jayakrishnan, raj s v., s s, kamal ss, rajan a. bilateral mandibular second premolar macrodontia: an enigmatous anomaly. int j dent sci res. 2014; 2(6a): 12–4. 11. brook ah. multilevel complex interactions between genetic, epigenetic and environmental factors in the aetiology of anomalies of dental development. a rch oral biol. 20 09; 54(suppl 1): s3–17. 12. peediayil f, kottayi s, jose d, sreenivasan p, babu a, hashim a, khader da. prevalence of mesiodens among 6-14 year school children. j res dent. 2014; 2(3): 243–50. 13. khandelwal v, nayak au, naveen rb, ninawe n, nayak pa, sai prasad s v. prevalence of mesiodens among sixto seventeen-yearold school going children of indore. j indian soc pedod prev dent. 2011; 29(4): 288–93. 14. rehan qamar c, bajwa ji, rahbar mi. mesiodens etiology, prevalence, diagnosis and management. pakistan orthod journal. 2013; 5(2): 73–6. 15. sujlana a, pannu p, bhangu j. double mesiodens: a review and report of 2 cases. gen dent. 65(5): 61–5. 16. tyrologou s, koch g, kurol j. location, complications and treatment of mesiodentes--a retrospective study in children. swed dent j. 2005; 29(1): 1–9. 17. altug-atac at, erdem d. prevalence and distribution of dental anomalies in orthodontic patients. am j orthod dentofacial orthop. 2007; 131(4): 510–4. 18. shilpa g, gokhale n, mallineni sk, nuvvula s. prevalence of dental anomalies in deciduous dentition and its association with succedaneous dentition: a cross-sectional study of 4180 south indian children. j indian soc pedod prev dent. 2017; 35(1): 56–62. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p63–67 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p63-67 67anggraini et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 63–67 19. vastardis h. the genetics of human tooth agenesis: new discoveries for understanding dental anomalies. am j orthod dentofacial orthop. 2000; 117(6): 650–6. 20. guttal ks, naikmasur vg, bhargava p, bathi rj. frequency of developmental dental anomalies in the indian population. eur j dent. 2010; 4(3): 263–9. 21. vasudev sk, goel br. endodontic management of dens evaginatus of maxillary central incisors: a rare case report. j endod. 2005; 31(1): 67–70. 22. neves aa, neves mla, farinhas ja. bilateral connation of permanent mandibular incisors: a case report. int j paediatr dent. 2002; 12(1): 61–5. 23. anitha, roopashri g, david mp. prevalence of developmental dental anomalies – a clinical study. int j contemp med res. 2018; 5(3): 22–4. 24. khan sq, ashraf b, khan nq, hussain b. prevalence of dental anomalies among orthodontic patients. pakistan oral dent j. 2015; 35(2): 224–7. 25. ezoddini af, sheikhha mh, ahmadi h. prevalence of dental developmental anomalies: a radiographic study. community dent health. 2007; 24(3):140–4. 26. banda r u bk, than kappan p, kuma r nandan sr, amudala r , a n nem sk , r ajend r a sa ntosh a b. t he p r eva lenc e of developmental anomalies among school children in southern district of andhra pradesh, india. j oral maxillofac pathol. 2019; 23(1): 160. 27. proffit wr, fields hw, sarver dm. contemporary orthodontics. 5th ed. st. louis: mosby elsevier; 2013. p. 3–114. 28. fitri ab, zubaedah c, wardani r. hubungan pengetahuan dengan sikap pemeliharaan kesehatan gigi dan mulut siswa pondok pesantren salafiyah al-majidiyah relationship of knowledge and attitude in maintaining oral health of the salafiyah al-majidiyah islamic boarding school students. j kedokt gigi univ padjadjaran. 2017; 29(2): 145–50. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p63–67 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p63-67 mkgs vol 44 no 2 april-juni 2011.indd 77 vol. 44. no. 2 june 2011 saliva as a future potential predictor for various periodontal diseases zahreni-hamzah laboratory of biomedic faculty of dentistry, jember university jember indonesia abstract background: there are many diagnostic biomarkers have been found in saliva. saliva contains a wide variety of proteins, including bacteria and products, enzymes, inflammatory mediators and host response modifiers, products of tissue breakdown. purpose: the purpose of the study was studied current development of diagnostic biomarkers in saliva that will lead to the development of simple and accurate diagnostic tools for periodental disease. reviews: specifically, the salivary biomarkers divided for three aspects of periodontitis i.e. inflammation, collagen degradation and bone turnover, correlated with clinical features of periodontal disease. the diagnostic biomarkers is in saliva, such as enzyme, immunoglobulin, cytokines, bacteria and bacterial products, hormones. for the past two decades, oral health researchers have been developing salivary diagnostic tools to monitor oral diseases. conclusion: the indicators of acute periodontitis can detect with ß-glucuronidase and ast, il-1β, and mmp-8, whereas indicators for chronic periodontitis can detect with alp. the indicators for collagen degradation and bone turnover suggest ictp, fibronectin fragments, and osteonectin. the indicators of severity of periodontitis especially can be predict by b. forsythus. key words: saliva, diagnostic biomarker, periodontal disease abstrak latar belakang: banyak biomarker telah ditemukan dalam saliva. saliva terdiri dari berbagai protein unik meliputi bakteri dan produk bakteri, enzim, mediator inflamasi dan modifikasi respon host (immunoglobulin, sitokin), produk kerusakan jaringan (telopeptida kolagen, osteokalsin, proteoglikan, fragmen fibronectin). tujuan: mengkaji biomarker dalam saliva untuk pengembangan metode diagnostik sederhana dan akurat untuk penyakit periodontal. tinjauan pustaka: secara khusus, biomarker saliva pada periodontitis dibagi dalam tiga aspek yaitu inflamasi, dan degradasi kolagen serta pergantian tulang. biomarker diagnostik dalam saliva, meliputi enzim, imunoglobulin, sitokin, bakteri dan produk-produk bakteri, hormon. selama dua dekade terakhir, para peneliti kesehatan mulut telah mengembangkan alat diagnostik melalui saliva yang tepat untuk memonitor beberapa penyakit periodontal. kesimpulan: indikator periodontitis akut dapat dideteksi dengan β-glucuronidase dan ast, il-1β,dan mmp-8, whereas indikator untuk periodontitis kronis dapat dideteksi dengan alp. indikator untuk degradasi kolagen dan penggantian tulang dideteksi melalui ictp, fibronectin fragments, dan osteonectin. sedang indikator untuk keparahan periodontitis terutama dapat diprediksi melalui b. forsythus. kata kunci: saliva, biomarker diagnostik, penyakit periodontal correspondence: zahreni-hamzah, c/o: laboratorium biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: reni61_hamzah@yahoo.co.id literature review introduction periodontitis is a chronic destructive category of periodontal disease that progresses to the resorption of alveolar bone, which leads to progressive bone destruction and tooth loss. as a consequence of resorption, breakdown of products are released into periodontal tissues, migrating toward the gingival sulcus and gathering from the 78 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 77–81 surrounding site in whole saliva, where several of them have been identified. several important components of saliva that can be used as indicators of periodontal disease are microbial factors, host derived enzymes, inflammatory mediators and host response modifiers, and products of tissue breakdown.1–8 recently, saliva has been studied as an important biological marker for introducing new diagnostic tests that can contribute to making the accurate diagnosis to explain the pathogenesis of a disease. there was many biomarkers of periodontal diseases have found in saliva.2–5 over the last two decades, researchers in the oral health have been trying to develop a diagnostic tool to use saliva to monitor various periodontal diseases.6,8 the development of salivary diagnostic technology currently leads the development of diagnostic tools that simply, quickly and accurately to facilitate the dentists in making clinical decisions and predict the outcome of treatment in the oral cavity, including the diseases related to the general health. considering the exchange activity of extra cellular fluid and saliva is high, so its allow the compounds that important as an indicators of disease associated with general health can be found also in saliva.1,8,9 furthermore, the dentist expected to be the first person to recognize the oral disease and the other general disease. the salivary biomarkers specific that correlated with clinical features of periodontal disease can determine by three aspects: inflammation, collagen degradation, and bone turnover.3,10 the purpose of the study was to studied current development of diagnostic biomarkers in saliva that will lead to the development of simple and accurate diagnostic tools for periodontal disease. it can use to make clinical decisions and predict the outcome of treatment in the oral cavity, including diseases related to the general health. periodontal inflammation periodontal diseases are further divided into reversible and nonreversible categories. gingivitis is a reversible infl ammatory reaction of the marginal gingiva to dental plaque biofi lms. gingivitis is characterized by an initial increase in blood fl ow, enhanced vascular permeability, and infl ux of cells (polymorphonuclear leukocytes [pmns] and monocyte-macrophages) from the peripheral blood into the periodontal connective tissue. overt soft tissue alterations during the state of gingivitis include redness, edema, bleeding, and tenderness. whereas, periodontitis is the destructive category of periodontal disease, is a nonreversible inflammatory state of the supporting structures. after its initiation, the disease progresses with the loss of collagen fi bers and attachment to the cemental surface, apical migration of the pocket epithelium, formation of deepened periodontal pockets, and the resorption of alveolar bone.11 recent studies have indicated that host response factors to infl ammation and periodontal diseases includes a mixture of molecules from blood, host tissue, plaque films, such as electrolytes, small molecules, proteins, cytokines, antibodies, bacterial and product bacterial antigens and enzymes. the number and various of the materials that present in saliva differ in different types of periodontal disease. until now, the number of markers analyzed was limited. edentulous persons and chronic periodontitis persons have reduced salivary concentrations of host infl ammatory proteins. these fi ndings suggest that a reduction in host responsiveness might play a role in the pathogenesis of chronic periodontitis.3,10,12 salivary indicator of periodontal inflammation is cytokines. the concentrations of interleukine/il-1α, il-6 and il-8 were found in whole saliva at signifi cantly higher than in major salivary gland secretions. in another study, transforming growth factor (tgf)-β, interleukin (il)-1α and tumour necrosis factor (tnf)-α were statistically signifi cantly higher in whole saliva compared to parotid saliva. although not statistically signifi cant, il-8 and il-6 also displayed a trend towards higher levels in whole saliva. the other study examined the relationship between clinical parameters of periodontal disease and the levels of il-1β, matrix metalloproteinase (mmp)-8, and osteoprotegerin (opg) in whole saliva. they reported that the mean levels of il-1β and mmp-8 in saliva were signifi cantly higher in periodontitis subjects than in periodontally healthy controls. combined elevated salivary levels of mmp-8 and il-1β increased the risk of experiencing periodontal disease 45-fold. among the salivary cytokines (il-1β, il-6, and tnf-α,), il-1β was the only biomarker associated with periodontitis. they suggest that salivary il-1β in saliva more thoroughly as markers of periodontitis.3,12-17 opg is glycoprotein that inhibits osteoclast differentiation an activity competitively by preventing osteoclast differentiatin factor receptor activator of nuclear factor kappa-beta ligand (rankl) from binding to osteoclast precursor and promoting the formation of bone-resorbing osteoclast. opg used as a marker of bone turnover.4,5 oncostatin m (osm), a member of the il-6 family of cytokines, is multifunctional unique cytokine that plays an important role in various biological systems such as infl ammatory response, haematopoiesis, tissue remodelling and development.13 in periodontitis, osm alone may stimulate the production of il-6, or it may act synergistically with il-6 or tnf-α to upregulate the production of mmps or augment il-6 production. il-6 may act on both the osteoblasts and osteoclasts through autocrine and paracrine rankl regulation causing bone resorption. osm as an infl ammatory and bone resorptive biomarker of periodontal disease.15 salivary indicators such as intracellular enzymes are increasingly released from the damaged cells of periodontal tissues into the gingival crevicular fl uid (gcf) and saliva.2 the activities several enzymes of periodontal disease such as creatine kinase (ck), lactate dehydrogenase (ldh), aspartate aminotransferase (ast), alanine aminotransferase (alt), gamma glutamil transferase (ggt), alkaline phosphatase (alp) and acid phosphatase (acp) enzymes were signifi cantly increased in the saliva of patients with periodontal disease when compared to healthy subjects. 79hamzah: saliva as a future potential predictor meanwhile, other investigators reported that enzymes elastase, ldh was the only biomarker associated with periodontitis. ldh activity increased proportionally with the advance of periodontitis.12,18–21 the other marker of the acute infl ammatory response is immunoglobulin. levels of immunoglobulin g (igg) have been found not to have a consistent association with periodontal disease, although subtypes of igg may be found in subjects at higher risk for periodontal disease progression. levels of immunoglobulin have been found not to have a consistent association with risk for periodontal disease progression.4,5 there are relationship of periodontal parameters to the presence of these periodontopathic bacteria. most of bacteria can produce tissue destruction by two ways: directly, through invasion of the tissue and production of harmful substances that induce cell death and tissue necrosis; and indirectly, through activation of inflammatory cells that can produce and release mediators that act on effector, with potent inflammatory and catabolic activity. the pathogenesis of periodontal destruction involves the sequential activation of different components of the host immune and inflammatory response.22 in chronic gingivitis, gram-negative bacteria constitute ± 45% and anaerobic organisms ± 45% of the total recoverable subgingival flora. predominant isolates include various species of actinomyces, streptococcus, fusobacterium, and bacteroides, as well as eikenella corrodens and capnocytophaga gingivalis. acute necrotizing ulcerative gingivitis (anug) has been associated with high proportions of bacteroides intermedius and treponema. advanced adult periodontitis lesions are characterized by approximately 75% gram-negative and 90% anaerobic organisms. common isolates include bacteroides gingivalis, b. intermedius, actinobacillus actinomycetemcomitans, prevotella intermedia (pi), and tannerella forsythensis (tf) and various species of fusobacterium, wolinella, capnocytophaga species, campylobacter rectus, and non-pigmenting bacteroides. in recent studies of the analysis microbiota associated with identify specific periodontal diseases, identify antibiotic susceptibility of infecting organisms colonizing diseases sites, and predict diseases activity.2–4,22–26 collagen degradation and bone turnover initially, tissue degradation is limited to ephitelial cells and collagen fibers from the connective tissue. later on, the inflammatory process may reach periodontal supportive tissue and leading to bone resorption.27 collagen degradation products is markers of bone turnover in multitude of bone resorptive and metabolic diseases. the collagen degradative molecules is in periodontal such as pyridoline cross-links (including pyridinoline, deoxypyridinoline, n-telopeptides, and c-telopeptides). pyridoline cross-links considered as specific biomarkers for bone resorption. the pyridoline cross-linked carboxyterminal telopeptide of type i collagen (ictp) have been shown to be correlated with bone resorptive rate in several bone metabolic diseases. in a cross-sectional study evaluated the markers of bone turnover are ictp, osteocalcin and osteonectin in stimulated whole saliva collected from untreated dental patients. increased levels of salivary osteonectin were associated with less bone loss.4,5,28–30 discussion a biomarker or biologic biomarker is substance that is objectively measured and evaluated as indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to therapeutic intervention.3 whole saliva is important physiologic fluid that contain a highly complex mixture of substances, locally and systemically, derived markers of periodontal and systemic diseases that easily to collected. so, it may offer the basis for a patient-specific biomarker assessment for periodontitis and other systemic diseases.1 generally, there are many clinicians reported that clinical parameters of periodontal disease, such as probing depth and bleeding on probing were limited in their ability to predict the activity of periodontal disease. therefore, saliva can be used as a non-invasive diagnostic fluid to measure biomarkers released during disease initiation and progression (tabel 1).4–8 various assays have been used to detect the presence of specific periodontal pathogens, such as culture techniques, immunoflourescence, and dna probe technology. using culture techniques requires that a viable bacterial sample be obtained from the patient and transferred to the laboratory. the sampling technique for these assays is relatively noninvasive, using either endodontic paper points or a sterile curette to obtain bacteria from the subgingival environment, include b. forsythus, p. gingivalis, t. denticola, and a. actinomycetemcomitans as periodontal pathogen.22–26 the prevalence of bacteroides forsythus in severe periodontitis patients was reported significantly higher than table 1. potential predictors of periodontal disease identified from whole saliva3,4 category mediator biomarkers microbial factors b. forsythus, p. gingivalis, a. actino-mycetemcomitans, treponema denticola host-derived enzyme alp, ast, elastase inflammatory mediators & host response modifier il-1β; il-6, tnf-α, mmp-8 connective tissue breakdown products collagen telopeptides (itcp), osteoprotegerin (opg), osteocalsin, osteonectin, fibronectin fragments. 80 dent. j. (maj. ked. gigi), vol. 44. no. 2 june 2011: 77–81 in those with healthy gingiva or gingivitis. this suggests that the presence of bacteroides forsythus in whole saliva are possible to be risk indicators for periodontal disease, especially in elderly patients.1,22–26 evaluating utility of new laboratory-based diagnostic methods must present either the sensitivity and specifi city of test or as a measure of risk periodontal disease. sensitivity refers to the ability of a diagnostic test to detect the presence of the disease. specifi city refers to the ability of a diagnostic test to detect the absence of a disease. sensitivity and specifi city tend to be inversely related. the goal of ideal test for periodontal disease that would be able to predict which patients will experience attachment loss (“active” periodontal disease) in the near future and to predict which teeth or which sites will experience attachment loss in the near future. in addition to predicting which patients or sites are at higher risk of becoming active, diagnostic tests can be used to categorize patients into different disease categories; ie, aggressive or chronic periodontitis, or response or no response to treatment. besides, it can also be used to determine the prognoses of treatment.27 in addition, an ideal periodontal diagnostic test would have to be economically feasible and easy to use. analysis of the host responses to periodontal disease for diagnostic purposes has involved the quantifi cation of specifi c host-derived molecules within gingival crevicular fl uid, serum, or saliva. in whole saliva, ast, alt, ggt, ldh, ck, alp, acp are intra cellular enzymes that have enzymatic activity refl ect metabolic changes in the gingiva and periodontium infl amation.1 those enzyme used to measure a death cell. the elevation of ast levels was associated with a 9 to 16 times greater risk of experiencing active periodontal tissue destruction. whereas, alp shown a remarkably increased activity in acute phase of periodontal disease, and restored to the value in healthy person.18,19 the other site, elastase found in the lysosomal granules of polymorphonuclear leukocytes (pmns). also elevated in patients with active periodontal disease.9,18–21 alp is a membrane-bound glycoprotein produced by many cells, such as pmns leukocytes during infl ammation, osteoblasts, macrophages, and periodontal ligament fibroblasts, during bone formation and periodontal regeneration respectively, within the area of the periodontium and gingival crevice. the alp activity was found highest in osteoblasts, moderate in periodontal ligament (pdl) fi broblasts, and lowest in gingival fi broblasts. the enzyme alp plays a role in bone metabolism and bone mineralization by releasing an organic phosphate that contributes to the deposition of calcium phosphate complexes into the osteoid matrix. alp might also promote mineralization by hydrolyzing inorganic pyrophosphate, a potent inhibitor of hydroxyapatite crystal formation and dissolution, within the extra cellular calcifying matrix vesicles. in the periodontium, alp is very important enzyme as it is part of normal turnover of periodontal ligament, root cementum, and maintenance of bone homeostasis. some studies have shown a remarkably increased activity of alp in the acute and chronic phase of periodontal disease, and after the periodontal and orthodontic therapy. the periodontal destruction such as alveolar bone loss, periodontal pockets, gingival bleeding and suppuration are related to higher alp and ast levels in saliva. ast is a tissue destruction biomarker released from necrotic cells in gcf, is associated with periodontitis severity. 8,18–21 cytokines are molecules that modulate the function of a wide variety of cells and are involved in regulating the immune and infl ammatory response. pro-infl ammatory cytokine with the most promise for diagnostic testing is known as il-1β, il-6 and tnf-α. il-1β and tnf-α are is produced by a wide variety of cell types (macrophages, fibroblasts, keratinocytes, and pmns leucocytes), but the primary producer of il-1β in the gingival tissues is the macrophage.4,5,13–17 il-1β has a number of biologic effects, including initiating the acute phase response to infection. however, in the periodontium, it likely functions to mediate connective tissue destruction and osteoclastic bone resorption. levels of il-1β have been found to be consistently associated with the severity of periodontal disease.23 the acute infl ammatory response in periodontal disease is mmps. it released by inflammatory cells, polymorphonuclear leukocytes and osteoclasts, leading to the degradation of connective tissue collagen and alveolar bone that have also been shown to aid in the diagnosis of periodontal disease. the mmp-8 in gcf is very effective in evaluating the outcome of periodontal treatment. salivary levels of il-1β, mmp-8, opg, and mip-1α refl ected disease severity and response to therapy suggesting their potential utility for monitoring periodontal disease status.13–17 opg used as a marker of bone turnover.3–5,17 it can be concluded that the indicators of acute periodontitis can detect with β-glucuronidase and ast, il-1β, and mmp-8, whereas indicators for chronic periodontitis can detect with alp. the indicators for collagen degradation and bone turnover suggest ictp, fibronectin fragments, and osteonectin. the indicators of severity of periodontitis especially can be predict by b. forsythus. references 1. keles gc, acikgoz g, ayas b, sakallioglu e, firatli e. determination of systemically and locally induce periodontal defect in rats. indiana j med res 2005; 121: 176–84. 2. khiste sv, ranganath v, nichani as, rajani v. critical analysis of biomarkers in the current periodontal practice. j indian soc periodontol 2011; 15: 104–10. 3. taba m, kinney j, kim as, giannobile v. diagnostic biomarkers for oral and periodontal diseases. dent clin north am 2005; 49(3): 551–6. 4. ali j, pramod k, tahir ma, ansari sh, autoimmune responses in periodontal disease. autoimmunity reviews 2011 may; 10 (issue 7): 426–31. 5. miller cs, king cp, langub mc, kryscio rj, thomas mv. salivary biomarkers of existing periodontal disease: a cross-sectional study. j am dent assoc 2006; 137: 322–9. 81hamzah: saliva as a future potential predictor 6. grbic jt, steven p, engebretson sp. the future of periodontal diagnostic testing. dentistry today. 2010. available at: http://www. dentistrytoday.com/periodontics/1617. accessed october 30th, 2010. 7. giannobile wv, beikler t, kinney js, ramseier ca, morelli t, wong dt. saliva as a diagnostic tool for periodontal disease: current state and future directions. periodontology 2000, 2009; 50: 52–64. 8. pfaffe t, cooper-white j, beyerlein p, kostner k, punyadeera c. diagnostic potential of saliva: current state and future applications. clinical chemistry 2011; 57: 675–87. 9. reddy s, kaul s, prasad mgs, agnihotri j, asutkar h, bhowmik n. biomarkers in periodontal diagnosis: “what the future holds...”. int j of clin dent sci 2011; 2(1): 76–83. 10. ansai t, soh i, ishisaka a, yoshida a, awano s, hamasaki t, sonoki k, takata y, takehara t. determination of cortisol and dehydroepiandrosterone levels in saliva for screening of periodontitis in older japanese adults. int j dent 2009; 2009: 280737. 11. kinney js., ramseier ca, giannobile wv. oral fluid–based biomarkers of alveolar bone loss in periodontitis. ann ny acad sci 2007; 1098: 230–51. 12. desai s, shinde h, mudda j, patil v. levels of alkaline phosphatase (alp) in saliva of patients with chronic periodontitis; a clinical and biochemical study. the internet j dental science 2009; 8(1): 1–19. 13. gursoy uk, könönen e, uitto vj, pirkko j, pussinen pj, kati hyvärinen k, liisa suominen-taipale l, knuuttila m. salivary interleukin-1β concentration and the presence of multiple pathogens in periodontitis. j clin periodontol 2009; 36(11): 922–7. 14. aurer a, jorgic-srdjak k, plancak d, stavljenic-rukavina a, aurerkozelj j. proinflammatory factors in saliva as possible markers for periodontal disease. coll antropol 2005; 29(2): 435–9. 15. pradeep ar, manojkumar st, garima g, raju a. serum levels of oncostatin m (a gp 130 cytokine): an inflammatory biomarker in periodontal disease. biomarkers 2010; 15(3): 277–82. 16. sexton wm, lin y, kryscio rj, dawson dr, ebersole jl, miller cs. salivary biomarkers of periodontal disease in response to treatment. j clin periodontol 2011 may; 38(5): 434–41. 17. rai b, kharb s, jain r, anand sc. biomarkers of periodontal disease in oral fluids. j oral sci 2008; 50: 53–6. 18. totan a, greabu m, totan c, spinu t. salivary aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase: possible markers in periodontal diseases? clin chemistry and laboratory med 2006; 44(5): 612–5. 19. todorovic t, dozic i, vicente-barrero m, ljuskovic b, pejovic j, marjanovic m. salivary enzymes and periodontal disease. med oral patol oral cir bucal 2006; 11: e115–9. 20. zappacosta b, manni a, persichilli s, boari a, scribano d, minucci a, raffaelli l, giardina b, sole pd. salivary thiols and enzyme markers of cell damage in periodontal disease. clinical biochemistry 2007; 40(9–10): 661–5. 21. kumar r, sharma g. salivary alkaline phosphatase level as diagnostic marker for periodontal disease. j int oral health 2011; 3(issue 5): 81–6. 22. ruhl s, hamberger s, betz r, sukkar t, schmalz g, seymour ra, hiller ka, thomason jm. salivary composition in gingival overgrowth. j dent res 2004; 83(4): 322–6. 23. bascones-martinez a, munoz-corcuera m, noronha s, mota p, basconez-ilundain c, campo-trapero j. host defence mechanisms against bacterial aggression in periodontal disease: basic mechanisms. med oral pathol oral cir bucal 2009; 14(12): e680–5. 24. bascones martínez a, figuero ruiz e. periodontal diseases as bacterial infection. av periodon implantol. 2005; 17(3): 111–8. 25. kesic l, milasin j, igic m, obradovic r. microbial etiology of periodontal diseases–mini review. medicine and biology 2008; 15(1): 1–6. 26. ramseier ca, kinney js, herr ae, thomas braun t, sugai jv, shelburne ca, rayburn la, tran hm, singh ak, giannobile wv. identification of pathogen and host-response markers correlated with periodontal disease. j periodontol 2009; 80: 436–46. 27. zia a, khan s, bey a, gupta nd, mukhtar-un-nisar s. oral biomarkers in the diagnosis and progression of periodontal diseases. biology and medicine 2011 march; 3(2): 45–52. 28. seibel mj. clinical application of biochemical markers of bone turnover. arq bras endocrinol metabol 2006; 50: 603–20. 29. mcgehee jr. jw, johnson rb. biomarkers of bone turnover can be assayed from human saliva. the journals of gerontology: series a 2003; 59 (issue 3): b196–200. 30. frodge bd, ebersole jl, kryscio rj, thomas mv, miller cs. bone remodeling biomarkers of periodontal disease in saliva. j periodontol 2008; 79: 1913–9. 5757 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 57–61 research report effects of alkalisation and volume fraction reinforcement of bombyx mori silk fibre on the flexural strength of dental composite resins dyah anindya widyasrini and siti sunarintyas department of dental biomaterial, faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: composite resins are widely used in dentistry to restore dental caries. recently, short fibre-reinforced composite (frc) resins have been widely used for high-stress areas, especially in posterior teeth. bombyx mori silk fibre is under research to reinforce dental composite resin as it has good mechanical properties. purpose: this study aims to obtain the effects of alkalisation and silk fibre volume fraction on the flexural strength of frc. methods: bombyx mori silk fibres were obtained from perhutani, pati, indonesia. samples were divided into two alkalisation groups (4% and 8%). alkalisation of the silk fibres was conducted through the scouring process in naoh, hydrolysis (30% h2so4) and drying. silk fibres were then reinforced in a resin matrix. the samples were subdivided based on the fibre volume fraction reinforcements, which were 0%, 5%, 10% and 15%. each group of samples consisted of three specimens (n = 3). flexural strength was measured using a universal testing machine. data were analysed by two-way anova (p < 0.05) and post-hoc least significant difference test (p < 0.05). results: the results showed the flexural strength (mpa) means of the 4% alkalisation group were 169.31 ± 54.28 (0%), 76.08 ± 43.69 (5%), 107.86 ± 40.61 (10%) and 101.99 ± 10.61 (15%). the flexural strength (mpa) means of the 8% alkalisation group were 169.31 ± 54.28 (0%), 82.62 ± 22.41 (5%), 111.07 ± 32.89 (10%) and 153.23 ± 23.80 (15%). statistical analysis by anova indicated that the fibre volume fraction affected the flexural strength of composite resins. conclusion: it can be concluded that the volume fraction of silk fibre increases the flexural strength of composite resins, although the strength is not as high as a composite resin without fibres. however, the alkalisation percentage did not affect the flexural strength of composite resins, and there was no interaction between alkalisation percentage and fibre volume fraction with the flexural strength of composite resins. keywords: alkalisation; composite resins; flexural strength; silk fibre; volume fraction correspondence: siti sunarintyas, dental biomaterial department, faculty of dentistry, universitas gadjah mada, sekip utara yogyakarta 55281 indonesia, e-mail: sunarintyassiti@ugm.ac.id introduction oral and dental health are inseparable parts of overall body health. to date, caries pose a major problem in dental health throughout the world and cause significant tooth decay. dental restorative materials are needed to prevent or repair tooth decay.1 composite resins are the most preferred restorative material due to their good biocompatibility, aesthetic properties, affordable costs and mechanical bonding to the tooth structure.2 however, there are some problems concerning composite resin restorations, such as inadequate mechanical properties, high water absorption, shrinkage after polymerisation and poor wear resistance when used.3,4 recently, short fibre-reinforced composite (frc) has been introduced as a restorative material of composite resins for bearing high-stress areas, especially in molars. the results of laboratory mechanical tests show that there are substantial improvements in load-bearing capacity, flexural strength and fracture toughness in composite resins that are reinforced with short e-glass fibre fillers compared to conventional fillers.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p57-61 58 widyasrini and sunarintyas/dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 57–61 the availability of synthetic dental frc resin for restorations in indonesia is still limited and more expensive than regular composite resin because it must be imported from another country. as an alternative to using synthetic fibres to reinforce composite resin as a restorative material, silk fibre, taken from the bombyx mori silkworm cocoon, is used. bombyx mori silk fibre is one of the strongest natural fibres.6 previous studies have shown that the silk fibre of the bombyx mori silkworm cocoon has a higher tensile strength than glass fibres or other organic synthetic fibres and has good resilience and elasticity.7 widely used in biomedical applications, bombyx mori silk fibres have shown not only good mechanical properties but also excellent biocompatibility and low immunogenicity.6 another study shows that up to 100 µg/ml of extract from the silkworm cocoon of cricula triphenestrata is not cytotoxic to gingival fibroblast cells.8 a study conducted by fransiska et al.9 shows that an increase in the bombyx mori silk fibre volume fraction in composite resins increased water absorption and decreased flexural strength. the study found that a small gap was allegedly due to weak interfacial bonds between the resin matrix and the bombyx mori fibre. this induced the water to penetrate easily, reducing flexural strength. sericin, which was still attached to the surface of the bombyx mori fibre, was ostensibly the cause of the bad bond between the resin matrix and the fibre.9 in addition, sericin has been shown to induce allergic and immunological reactions, so it must be removed before use in biological applications.10 sericin can dissolve in water and can be removed by a thermochemical process known as degumming.11 during the degumming process, sericin is hydrolysed, and amide bonds with long protein molecules are broken into smaller fractions, then dispersed and dissolved in solutions containing alkali, soap or synthetic detergents.12 in order to overcome the damage that occurs during the degumming process, it is necessary to research the effects of alkalisation, which aims to remove sericin and impurities in the flexural strength of composite resins. this study aims to reveal the effects of alkalisation and the addition of a volume fraction of bombyx mori silk fibre on the flexural strength of composite resins as dental filling material. materials and methods bombyx mori silk fibres were obtained from perhutani, pati, central java, indonesia; the silk fibres had been reeled and spun from the bombyx mori silkworm cocoon. udma was obtained from esstech inc. (essington, pennsylvania). bisgma, tegdma, champorquinone (cq) and cema were obtained from sigma-aldrich chemical (darmstadt, germany). naoh, ch3cooh and other chemicals used at analysis levels were obtained from sigma-aldrich chemical (darmstadt, germany). bombyx mori silk fibres were alkalised based on the method developed by mohammed and dauda,13 with modifications. bombyx mori silk fibres were alkalised by cutting silk fibres to 2 mm long using stainless steel scissors and grouping them into two groups based on the alkalisation percentage. afterwards, the scouring process was performed by soaking the silk fibres in 250 ml of naoh solution in a beaker glass at 65ºc for three hours on a hotplate stirrer. the concentrations of naoh solution used were 4% and 8%. the silk fibres were rinsed with distilled water, then neutralised with 500 ml of 2% ch3cooh solution in a beaker glass for two hours at 65ºc on a hotplate stirrer. the fibres were then rinsed using distilled water until the litmus paper showed a neutral ph. the fibres were left to dry at room temperature.13 drying was continued until it reached a constant fibre weight. the results of the 4% and 8% silk fibre alkalisation were mixed with a composite resin matrix mixture until they were homogeneous, according to sunarintyas et al.14 (bisgma 67%, tegdma 30%, udma 1%, cq 1%, cema 1%). the results of each percentage of silk fibre alkalisation were then subdivided into four groups based on the silk fibre volume fraction. the volume fractions of the silk fibres used were 0%, 5%, 10% and 15%.9 the samples of the silk frc for flexural strength tests were made according to iso 4049 (2009). three (n = 3) samples were made for each volume, so there were eight groups of samples. afterwards, the composite was put into a mould measuring 25 mm x 2 mm x 2 mm until it was fully loaded. the composite surface was covered with a glass slide, and the top of the sample was illuminated using a light-curing unit perpendicular to the sample from a distance of 2 mm. the tip diameter of the light-curing unit was 8 mm, while the sample length was 25 mm. the illumination was divided into three parts, each of which was illuminated for 20 seconds; non-illuminated parts were covered with aluminium foil. after the illumination was complete, the sample was removed from the mould. flexural strength was tested with a three-point bending test using a universal testing machine (tokyo testing machine, japan), according to iso 4049.15 the test was performed by putting the sample on a supporting board with a support distance of 20 mm (l), then the sample was loaded in the middle until it broke. afterwards, the monitor screen displayed a number (f), which was the maximum pressure that the sample received. furthermore, the measurement data obtained were entered into the following equation:15 σ = (3f.l) / 2 b.h2 where: σ = flexural/transverse strength (n/mm2 or mpa) f = maximum load (n) l = distance between the two supports b = width of the sample (mm) h = thickness of the sample (mm) normality (shapiro–wilk) and homogeneity (levene’s test) tests were performed on the data obtained from the results of the flexural strength test. furthermore, to test dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p57-61 59widyasrini and sunarintyas/dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 57–61 the hypothesis, a two-way anova test was performed to reveal the effects of alkalisation and the addition of the volume fraction of bombyx mori silk fibre on the flexural strength of composite resins with a significance level of p < 0.05. furthermore, the data were analysed using the post-hoc least significant difference (lsd) test to see the average difference between each group. results figure 1 presents the results of the research into the effects of alkalisation and volume fraction of bombyx mori silk fibre on the flexural strength of composite resins. figure 1 shows that the lowest flexural strength was seen in composite resins with a 5% silk fibre volume fraction, and the highest value was in composite resins without fibre additions (0% silk fibre volume fraction) in both the 4% and 8% alkalisation groups. in composite resins with the addition of silk fibre, there was an increase in the flexural strength value along with the addition of the fibre volume fraction in the two alkalisation groups. the results of the two-way anova test indicated the effects of adding fibre volume fraction on flexural strength (p = 0.007), but the percentage of fibre alkalisation did not significantly affect the flexural strength of silk fibre composite resins (p = 0.344). based on the statistical analysis, there was no interaction between the percentage of alkalisation and fibre volume fraction and the flexural strength of composite resins (p = 0.626). the results of observations with a scanning electron microscope (figure 2) showed a small gap between the fibre and the matrix in the 8% alkalisation group specimen. furthermore, the data obtained were tested post-hoc using the lsd test table 1). the post-hoc lsd test results indicated that there were significant differences between the flexural strength values of the composite resins with 0% and 5% volume fractions in both alkalisation groups. meanwhile, the flexural strength values of composite resins with 0%, 10% and 15% fibre volume fractions were not significantly different. 0 50 100 150 200 250 0% 5% 10% 15% fl ex ur al s tr en gt h (m pa ) fiber volumetric fraction 4% alkalisation 8% alkalisation figure 1. mean and standard deviation of flexural strength of silk fibre composite resins. figure 2. the results of an sem photograph on a silk fibre composite resin in 8% alkalisation with a magnification of 500x. there are small gaps between the fibre and the matrix (white arrow). table 1. summary of lsd test of fibre volume fraction of 4% alkalisation group on the flexural strength of silk fibre composite resin volume fibre fraction 0% 5% 10% 15% 0% 93.23000* 61.45333 67.32000 5% 31.77667 25.91000 10% 5.86667 15% *= significantly different (p <0.05) table 2. summary of lsd test of fibre volume fraction of 8% alkalisation on the flexural strength of silk fibre composite resin volume fibre fraction 0% 5% 10% 15% 0% 86.69333* 58.24000 16.07333 5% 28.45333 70.62000* 10% 42.16667 15% *= significantly different (p <0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p57-61 60 widyasrini and sunarintyas/dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 57–61 discussion the results of the research indicated that the greatest flexural strength in the two alkalisation groups was in the 0% volume fraction treatment, while the lowest flexural strength was shown in the 5% silk fibre volume fraction. this may be due to the absence of fibre additions (0% volume fraction), causing no gaps to form in the matrix (homogeneous matrix without fibre additives). in composite resins with the addition of fibres, especially fibres that are not perfectly clean, there is a gap between the matrix and fibres, which happens because there is still a layer of sericin and other impurities that have not been completely removed from the silk fibres through the alkalisation process. the presence of a sericin layer can prevent physical contact or chemical attachment between the fibres and the matrix, reducing the mechanical properties of the silk fibre composite resins.16 the small gap between the fibres and the matrix shown in figure 2 might be due to imperfect adhesion due to sericin remaining in the fibres. fibrematrix attachment is another important factor affecting the mechanical properties of short-frc. the gap formed between the fibre and the matrix is like a cavity without reinforcement, reducing the composite’s overall strength. poor interfacial attachment between the silk fibre and polymer matrix dominates other factors that might influence the decrease in mechanical strength.16 this shows that a better sericin cleaning process is needed so that the sericin does not interfere with the fibre’s attachment to the matrix. in addition, a coupling agent can be added to improve the interfacial attachment between silk fibres and the matrix. the addition of coupling agents to composites made from natural fibres can improve mechanical properties by up to 61% because they can increase the bond between organic and inorganic materials.17 with the addition of a coupling agent, it is expected that the load transfer to the fibre will be more effective, increasing mechanical strengths, one of which is flexural strength. in the 8% alkalisation group, the flexural strength increased from the 5% to the 15% volume fraction. meanwhile, in the 4% alkalisation group, the flexural strength increased from the 5% to the 10% volume fraction but slightly decreased in the 15% volume fraction. the increased value in the flexural strength of the composite resins might be due to the addition of silk fibre as a reinforcement material. bombyx mori silk fibre is a natural fibre that has good mechanical strength. silk fibre has high tensile strength, good flexibility and resistance to compression strength, making it suitable to be used as a composite material to bear the load.4 the mechanical strength of bombyx mori silk fibre is equal to e-glass fibre and is greater than that of other synthetic fibres, including kevlar.18 the mechanical strength of bombyx mori silk fibre is obtained from the β-sheet structure, which is formed by a repeated sequence of the amino acids glycine, alanine and serine (g-a-g-a-g-s) in the crystalline phase of fibroin.10 the addition of a volume fraction of silk fibres increases the amount of silk fibre in the composite composition, thereby increasing the mechanical strength of the composite resins. the results of the two-way anova indicated the effects of fibre volume fraction on flexural strength (p = 0.007). the effectiveness of fibre reinforcement is affected by the amount of fibre in the composite resin. an increase in fibre content increases mechanical strength.19 this is consistent with the results of this research; the flexural strength of composite resins generally increased in line with the addition of silk fibre in composite resins. however, statistically, the difference in the percentage of fibre alkalisation indicated that there was no significant effect on the flexural strength of the composite resin (p = 0.344). this might be due to the degumming procedure using alkalisation being poorly performed so that the results of the two alkalisation percentages did not show any differences. in the degumming process with alkali, the non-covalent fibroin bond in a silk fibre is modified, making the fibre swell. the swelling effect of the fibre is due to the difference in osmotic pressure arising between the fibre and the solution, forming protein salts. furthermore, sericin degrades into sericin peptides or hydrolysed sericin.12,20 the degumming process of silk fibres using naoh is widely used, but this process results in strong irritation to the fibroin content of silk fibre.12 degumming using alkali may result in a damaged structure of the silk fibres, poor stretch quality and lack of shine.21 therefore, it is necessary to use a more optimal and safer degumming procedure when removing sericin to maintain the physical and mechanical properties of fibroin. in addition, proper degumming procedures are needed to increase fibre roughness, thus increasing attachment strength. this is consistent to ho et al.’s16 statement on the interaction between the fibre and matrix being supported by the existence of the mechanical interlocking obtained from fibre surface roughness, providing weak adhesion. based on the results of the post-hoc test, it can be seen that there was an increased value in the flexural strength of composite resin with 5%, 10% and 15% fibre volume fractions in the 8% alkalisation group. these results indicate that fibre has the potential to increase the mechanical strength of composite resins, although the results are not as good as the composite resins with a 0% fibre volume fraction. also, the results of the post-hoc lsd test indicated that the flexural strength of composite resins with 0%, 10% and 15% fibre volume fractions did not differ significantly (tables 1 and 2), especially in the 8% alkalisation group. this indicates that degumming using 8% naoh has better cleaning potential than using 4% naoh, although it was not perfect. in addition, according to iso 404915 restorative material is deemed to have met the initial requirements if the flexural strength of the test sample is more than 80 mpa. all samples in the 8% alkalisation group had an average flexural strength greater than 80 mpa. based on the results of the research, it can be concluded that the fibre volume fraction increases the flexural strength dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p57-61 61widyasrini and sunarintyas/dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 57–61 of composite resins, although they are not as strong as the composite resin without fibres. silk fibre has the potential to increase the mechanical strength of composite resins; however, the optimum volume fraction of silk fibre to be added in the composite resins still needs to be found. meanwhile, the percentage of silk fibre alkalisation does not affect the flexural strength of composite resins acknowledgement this research was financially supported by the faculty of dentistry, universitas gadjah mada, under contract number 4310/un1/fkg1/set.kg1/pt/2019. references 1. powers j, wataha j. dental materials foundation and applications. 11th ed. missouri: elsevier; 2017. p. 272. 2. liu y, sun y, zeng f, xie w, liu y, geng l. effect of nano sio2 particles on the morphology and mechanical properties of poss nanocomposite dental resins. j nanoparticle res. 2014; 16(12): 1–8. 3. foroutan f, javadpour j, k havandi a, atai m, rezaie hr. mechanical properties of dental composite materials reinforced with micro and nano-size al2o3 filler particles. iran j mater sci eng. 2011; 8(2): 25–33. 4. kundie f, azhari ch, muchtar a, ahmad za. effects of filler size on the mechanical proper ties of polymer-filled dental composites: a review of recent developments. j phys sci. 2018; 29(1): 141–65. 5. garoushi s, vallittu pk, lassila l v. fracture toughness, compressive strength and load-bearing capacity of short glass fibre-reinforced composite resin. chinese j dent res. 2011; 14(1): 15–9. 6. ude au, eshkoor ra, zulkifili r, ariffin ak, dzuraidah aw, azhari ch. bombyx mori silk fibre and its composite: a review of contemporary developments. mater des. 2014; 57: 298–305. 7. kundu b, kurland ne, bano s, patra c, engel fb, yadavalli vk, kundu sc. silk proteins for biomedical applications: bioengineering perspectives. vol. 39, progress in polymer science. pergamon; 2014. p. 251–67. 8. sunarintyas s, siswomihardjo w, tontowi ae. cytotoxicity of cricula triphenestrata cocoon extract on human fibroblasts. int j biomater. 2012; 2012: 1–5. 9. fransiska a, sunarintyas s, dharmastiti r. effect of bombyx mori silk-fiber volume on flexural strength of fiber-reinforced composite. maj kedokt gigi indones. 2018; 4(2): 75. 10. zafar ms, al-samadani kh. potential use of natural silk for biodental applications. j taibah univ med sci. 2014; 9(3): 171–7. 11. qi y, wang h, wei k, yang y, zheng ry, kim is, zhang kq. a review of structure construction of silk fibroin biomaterials from single structures to multi-level structures. int j mol sci. 2017; 18(3): 1–21. 12. ho mp, wang h, lau kt, lee jh, hui d. interfacial bonding and degumming effects on silk fibre/polymer biocomposites. compos part b eng. 2012; 43(7): 2801–12. 13. mustafa hauwa mohammed, dauda b. unsaturated polyester resin reinforced with chemically modified natural fibre\n. iosr j polym text eng. 2014; 1(4): 31–8. 14. sunarintyas s, siswomihardjo w, irnawati d, matinlinna jp. biomechanical effects of new resin matrix system on dental fiberreinforced composites. asian j chem. 2016; 28(7): 1617–20. 15. iso iso 4049:2000 dentistry — polymer-based filling, restorative and luting materials. geneva: international organization for standardization; 2009. p. 4, 15. 16. ho mp, lau kt, wang h, bhattacharyya d. characteristics of a silk fibre reinforced biodegradable plastic. compos part b eng. 2011; 42(2): 117–22. 17. kim jg, choi i, lee dg, seo is. flame and silane treatments for improving the adhesive bonding characteristics of aramid/epoxy composites. compos struct. 2011; 93(11): 2696–705. 18. omenetto fg, kaplan dl. new opportunities for an ancient material. science (80). 2010; 329(5991): 528–31. 19. swapn i l as, sat hesa nd ip b, chaud ha r ibapu p, visha l sj. experimental investigation of mechanical properties of glass fibre/ epoxy composites with variable volume fraction. in: materials today: proceedings. elsevier ltd; 2017. p. 9487–90. 20. talebpour f, veysian sm, ebrahim m, golfazani h. degumming of silk yarn using alkali, enzyme and seidlitzia rosmarinus. j text polym. 2013; 1(2): 60–4. 21. zusfahair z, fatoni a, ningsih dr. pemanfaatan protease dari kulit nanas (ananas comosus, l.) dalam degumming benang sutera. j kim ris. 2016; 1(1): 22–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p57–61 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p57-61 vol 49 no 3 juli-sept 2016.indd guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages 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duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, 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355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all 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........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita 38 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 38–43 original article the effects of unilateral posterior crossbite toward the superficial masseter and anterior temporalis on muscle activity during mastication: a surface electromyographic study agnes imelda izach1, christnawati 2 , darmawan sutantyo 2 1orthodontics specialty education program, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of orthodontics, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: adapted patterns of mastication caused by unilateral posterior crossbite require early orthodontic treatment to prevent permanent muscle change. stable orthodontic results depend on the harmonious contraction of the occlusion and masticatory muscles. purpose: using surface electromyography, this study aimed to analyse the effects of unilateral posterior crossbite on the superficial masseter as well as anterior temporalis muscle activity on the crossbite and non-crossbite sides during chewing soft and hard foods. methods: a cross-sectional study was conducted on 20 subjects with at least two posterior teeth who had a unilateral posterior crossbite without mandible shifting. surface electromyography was used to measure activity amplitudes for the superficial masseter and the anterior temporalis muscles while chewing soft and hard foods. an independent t-test was used to determine the mean difference between chewing soft and hard foods through the superficial masseter and anterior temporalis muscles. results: results showed a significant difference in amplitude mean between crossbite and non-crossbite sides of the superficial masseter and anterior temporalis muscles with both soft and hard food chewing (p < 0.05). the study also revealed a decrease in the activities of superficial masseter and anterior temporalis muscles when masticating soft and hard foods on the crossbite sides as compared to the non-crossbite sides. conclusion: a unilateral posterior crossbite results in a decrease in the superficial masseter and the anterior temporalis muscle activity when masticating both soft and hard foods on the crossbite side. keywords: anterior temporalis muscle activity; surface electromyography; superficial masseter muscle activity; unilateral posterior crossbite. correspondence: agnes imelda izach, orthodontics specialty education program, faculty of dentistry, gadjah mada university. jl. denta 1, sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: agnes.imelda.i@mail.ugm.ac.id introduction mandibular deviation in normal occlusion generally occurs due to genetic and environmental factors such as oral bad habits, premature loss, deciduous teeth persistency or broad caries that evolve into a malocclusion.1 transversal malocclusion causes a posterior crossbite with a higher prevalence of the unilateral side than the bilateral. the onset of a posterior crossbite occurs during the eruption of the deciduous teeth, involving permanent teeth at a later stage of development and affecting the masticatory function through the growth stage.2 a unilateral posterior crossbite causes dentoalveolar asymmetry, mandible deviation and imbalanced muscle function.3 early mixed dentition has a variable prevalence of unilateral posterior crossbite. early orthodontic correction is crucial to prevent abnormal transversal growth in the intermolar region by developing an advanced upper jaw.4 the effectiveness of mastication in a unilateral posterior crossbite is different between each side of the jaw, changing the mastication pattern via a preferred chewing side that is considered more effective and comfortable. occlusal interference in mastication leads to excessive stimulation of mechanic sensors at the periodontal ligament and continuous afferent impulses to the receptor that stimulates motoneuron behaviour, increasing the mastication muscles’ activity.5,6 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 mailto:agnes.imelda.i@mail.ugm.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 39izach et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 38–43 electromyography (emg) can identify changes in muscle activity and kinematic patterns with regard to chewing load capacity adjustment. surface emg (semg) is performed by attaching electrodes to the skin’s surface to detect potential electric action of a muscle movement or rest period.7 many studies have focused on the relation between malocclusion and the muscle activity of mastication using emg. some research into class i malocclusion has shown more increased semg activity than class ii or class iii. as elevator muscles, the superficial masseter and anterior temporalis are responsible as anti-gravity extensors, maintaining muscle posture during the rest position and mandible movement during mastication.8,9 assessments of semg in superficial masseter and anterior temporalis muscle activity during clenching and chewing gum among post-orthodontic treatment patients showed more balanced results than normal occlusion in patients without orthodontic treatment. 9 research on children aged 10–14 years revealed that superficial masseter muscles were less active among crossbite subjects than normal occlusion subjects with bilateral mastication patterns.9,10 the aim of this study was to investigate muscle activity of the superficial masseter and anterior temporalis during mastication soft and hard foods in subjects 19–21 years old with unilateral posterior crossbite using surface electromyography. materials and methods cross-sectional analysis was performed to investigate the influence of risk factors and effects of this study. a total of 20 subjects were involved in this study, aged between 19–21 years and consisting of 15 woman and 5 men, with unilateral posterior crossbite. subjects included students in the dentistry and dental hygiene program of dentistry faculty, universitas gadjah mada, selected via the following inclusion criteria: (a) a unilateral posterior crossbite involving at least two teeth; (b) unilateral posterior crossbite identified during centric relation and centric occlusion position; (c) no mandible displacement when opening and closing; (d) complete teeth except third molars; (e) never had facial trauma; (f) no temporomandibular joint clicking, crepitus or pain when mouth opening; (g) no dentures or occlusal splint; (h) no history of orthodontic or orthognathic care; and (i) not diagnosed with a systemic disease. the exclusion criteria included (a) subjects with single tooth crossbite and (b) bilateral posterior crossbite. this study used a questionnaire as the instrument to measure oral condition and history of malocclusion before examination. the subjects were measured on crossbite and non-crossbite sides. the non-crossbite is defined as the area with normal occlusion, and the control group contains the angle class i molars. the sampling technique performed was purposive sampling. the subjects underwent an initial assessment regarding health status and crossbite history. they received an explanatory sheet describing the study objectives, benefits, evaluation, process and informed consent. subjects were approved to join this research protocol after reading the assigned informed consent form. this research received a feasibility permit from the faculty of dentistry, universitas gadjah mada research ethics commission 00367/kkep/fkg-ug/ec/2020. the research instrument used in this study was an electromyograph (nihon kohden series emb 2306, us) with a 10khz/10hz filter and 2 mv calibration, consisting of three electrodes. the active electrode was positioned over the muscle, a reference electrode was positioned on the nose, and a ground electrode was positioned on the forehead. forty grams of roasted peanuts (dua kelinci, indonesia) and 42 grams soft cakes (nextar, indonesia) were used in this study. the measured object in this study were the superficial masseter and the anterior temporalis muscles (figure 1). preceding electrode placement, the superficial masseter muscle was measured three millimetres from the line of the inferior mandible to each side. the electrodes of anterior temporalis muscle were attached at two-thirds inferior arcus zygomaticus of both the crossbite and the non-crossbite sides. the subjects were initially instructed to initiate a clenching movement to detect the muscle. the electrode on the superficial masseter muscle was attached parallel to the muscle, and the electrodes of anterior temporalis were placed perpendicular to the muscles. the activities of the superficial masseter and the anterior temporalis muscle were not measured simultaneously. the surface electrode applied with electrolyte gel and tape over skin cleaned before application. subjects were instructed to remain in the rest position for two minutes without any measurements, and then chew soft cakes for 20 seconds for the evaluation. afterwards, they were to chew roasted peanuts for 20 seconds, and measurements were taken. evaluations began with the right superficial masseter, right anterior temporalis, left superficial masseter and left anterior temporalis. subjects had to rinse their mouths to remove food debris when measuring each muscle was complete. electrode removal was performed after all measurements were completed. a b figure 1. (a) electrode placement during temporalis muscle examination. (b) electrode placement during superior masseter muscle examination. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 40 izach et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 38–43 0 0.5 1 1.5 2 a m pl itu do e m g (m v ) crossbite non-crossbite soft food sm soft food at hard food sm hard food at figure 2. mean and standard deviation superficial masseter and anterior temporalis muscle on the crossbite and non-crossbite sides. note: sm: superficial masseter; at: anterior temporalis. table 1. normality test of superficial masseter and anterior temporalis muscle activity on soft and hard foods between crossbite and non-crossbite side variable sig. (p) sm soft food sm hard food at soft food at hard food crossbite 0.055* 0.626 0.68 0.99 non-crossbite 0.179 0.274 0.38 0.55 note: lowest significant p > 0.05; sm: superficial masseter; at: anterior temporalis table 2. independent t-test results of superficial masseter and anterior temporalis muscle activity on soft and hard foods between crossbite and non-crossbite side muscle activity mean ± standard deviation (mv) sig. (p) crossbite non-crossbite superficial masseter (soft food) 0.529 ± 0.103 1.254 ± 0.275 0.001* anterior temporalis (soft food) 0.639 ± 0.080 1.478 ± 0.235 0.001* superficial masseter (hard food) 0.301 ± 0.102 0.604 ± 0.229 0.001* anterior temporalis (hard food) 0.403 ± 0.104 0.981 ± 0.401 0.001* the measurement results are interpreted based on the synchronisation of the observation time at intervals of 0–5 seconds; 5–10 seconds; 10–15 seconds and 15–20 seconds by marking the highest amplitude in each period. intervals were determined by manual motor unit potential (mup) recording muscle action potentials through four scoring panels on the emg screen monitor. the evaluation not only measured the highest amplitude of each interval, but also the arithmetic process on one screen per interval time measurement. the saphiro-wilk was used for data analysis in this study for the normality test and an independent t-test to compare the mean of each group. results the difference in activity of the superficial masticatory muscle and the anterior temporal muscles during chewing can be seen in figure 2. the results of the normality test (shapiro-wilk) have a significance value (p > 0.005) showing that the data of both muscles are normally distributed in soft and hard chewing (table 1). the independent t-test (table 2) showed that a unilateral posterior crossbite affected the activity of the superficial masseter and anterior temporal muscles during the chewing of soft and hard foods (p<0.05). muscle activity on the crossbite side was lower than on the non-crossbite side in both the superficial masseter muscle and the anterior temporomandibular muscle during chewing of soft and hard foods. activity of the superficial masseter muscle on the crossbite side is least when chewing soft foods, followed by the anterior temporalis muscle. the highest activity was observed by the anterior temporalis muscle during hard food chewing, followed by the superficial masseter muscle of the noncrossbite side. differences in amplitude of superficial emg activity between superficial masseter muscle and anterior temporalis muscle from the crossbite and note: *) significant p <0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 41izach et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 38–43 non-crossbite side (figure 3) revealed that the amplitudes of the superficial masseter and anterior temporalis muscle are lower on the crossbite side than the non-crossbite when chewing soft food. similar to soft food, the amplitude was increased in hard food chewing than on the crossbite side, characterised by more narrow and steep waves than the non-crossbite side. discussion muscle activity decreased in the crossbite side of the superficial masseter and anterior temporal during mastication of soft and hard foods. there was a decrease in elevator muscle activity in crossbite side when chewing soft and hard food, influenced by the broad surface areas of chewing, duration and individual strength. occlusal disturbances caused narrow chewing areas, contributing to the lower duration and reduced strength of individual mastication.1 in this study, surface emg indicated a narrower peak amplitude with amplitude frequency caused by similar food characteristics at the non-crossbite side. shapes and heights of amplitudes differed between the crossbite and non-crossbite sides were a result of the type of muscle contraction during mastication.2 this study revealed decreasing anterior temporalis muscle activity on the crossbite side during the soft and hard food mastication compared to the non-crossbite side. it also showed the peak of amplitude was lower on the crossbite side than the noncrossbite for both types of food (figure 3). at the same time, occlusal disturbances decreased masticatory strength on the crossbite side, which affected the activity of the superficial side crossbite non-crossbite soft food superficial masseter hard food superficial masseter soft food anterior temporalis hard food anterior temporalis figure 3. amplitude difference among crossbite and non-crossbite side of superficial masseter and anterior temporalis muscle during mastication soft and hard food mastication. blue arrows indicate the highest amplitude in the area of the electromyograph. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 42 izach et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 38–43 masseter and anterior temporalis muscle contraction during jaw closure.3 the emg evaluation revealed that the distance between the amplitudes on the crossbite side is larger than on the non-crossbite side. magnitude gap refers to the decreasing frequency of amplitude as the muscle on the crossbite side contracts. this study’s results differ from those reached by piancino et al.3 who found that masseter muscle activity on the non-crossbite side did not increase during chewing based on the assessment of food density. emerging potential action that occurred due to contractions are influenced by food variant, area and duration of chewing.4 the electromyogram in this study revealed frequent low amplitude on the side of the crossbite, whereas, in the non-crossbite side, several peak amplitudes arose with a narrow form and approached the limits (figure 3). during the hard food mastication, the contraction of the superficial masseter muscle on the non-crossbite side occurs more rapidly due to the texture of the food that require greater mastication power. muscle contraction acceleration during the hard food mastication is influenced by the linear work of chewing duration and surface areas.5,6 these findings suggest that the non-crossbite side has more substantial occlusal stability, resulting in the mastication force experienced by mastication muscles compared to the crossbite side.6,7 gungor et al.8 confirmed that the activity and strength of the superficial masseter and anterior temporalis muscles on the crossbite side were less active than the non-crossbite side in 6–9 year age group. the chewing area in this study was represented by molars and premolars that had a crossbite. mastication efficiency was thus also affected by the occlusal surface which received the mechanical load.9 the size of teeth varied among the participants, which also affected the chewing area during mastication.10 despite tooth morphology, ardani et al.11 did not find significant difference in class i and ii malocclusions among javanese ethnic patients. these were measured by semg temporalis, masseter and suprahyoid muscles. rahmawati et al.12 also investigated the relationship between muscle activity in class i and class iii malocclusion among subjects of javanese ethnicity. results showed a greater value in the class iii temporalis muscles during clenching than in class i malocclusion.12 komino et al.13 concluded that the pathway, rhythm, and velocity of masticatory movements are altered by varying the muscular activity of the masseter muscles responsible for the hardness of test foods. tomonari et al.14 suggested that the use of standardised food is the best choice for measurements with emg given the homogeneous shape and size and a texture that is not easily decomposed; in contrast, raw foods can change shape, colour and texture according to temperature and conditions. controlled variables in this study, including age, seating position with natural head position, type and size of food, and electrode position that were more than 2 cm apart, are known to affect the measurement results. one of the inclusion criteria in this study defined crossbite as involving at least two teeth, so it is not merely involving the first dental molar. shimada et al.15 found that efficiency of chewing on the premolar area was less than the molar area. this difference is caused by difference surfaces and the anatomy of those teeth. value improvements of the amplitude side without a crossbite are caused by maximal muscle contraction occurring simultaneously without occlusal interference, while decreased muscle activity on the crossbite side was influenced by the number of chewing teeth and muscle thickness.16,17 in a study focusing on gender, koç et al.18 found that masticatory strength among males is more remarkable than females due to differences in muscle volume. the limitations of this study included sample size and sex distribution, which affected the results. the large standard deviation value is due to the research being conducted in a study with a cross-sectional approach that aims to identify a relationship of exposure to risk factors. an unequal sex distribution influences the chewing force on both sides when chewing soft and hard foods. contributory factors such as poor oral habits, crossbite molar involvement and muscle thickness had not been previously controlled for. according to this study, reducing the activity of the crossbite side in superficial masseter and anterior temporalis muscles by using surface emg had a significant effect on mastication. future studies should differentiate between mastication muscle activity and controlled oral bad habits, first molar involvement, gender and muscle thickness. evaluation of mastication muscle activity using surface emg can provide accurate data to make better orthodontic treatment. references 1. kusnoto j, nasution fh, gunadi ha. ortodonti. jakarta: egc; 2015. p. 62–76; 129. 2. zamanlu m, khamnei s, salarilak s, oskoee ss, shakouri sk, houshyar y, salekzamani y. chewing side preference in first and all mastication cycles for hard and soft morsels. int j clin exp med. 2012; 5(4): 326–31. 3. p ia nc i no mg, ky rk a n id e s s. und e r st a nd i ng m a st icat or y function in unilateral crossbites. oxford: wiley-blackwell; 2016. p. 98–112. 4. sciote jj, horton mj, rowlerson am, ferri j, close jm, raoul g. human masseter muscle fiber type properties, skeletal malocclusions, and muscle growth factor expression. j oral maxillofac surg. 2012; 70(2): 440–8. 5. premkumar s. text book of orthodontics. 1st ed. new delhi: reed elsevier india pvt. ltd; 2015. p. 338, 435, 436, 481. 6. adhikari h, kapoor a, prakash u, srivastava a. electromyographic pattern of masticatory muscles in altered dentition. part ii. j conserv dent. 2011; 14(2): 120–7. 7. merletti r, farina d. surface electromyography: physiology, engineering, and applications. canada: wiley-ieee press; 2016. p. 54–8; 89, 100–23. 8. gungor k, taner l, kaygisiz e. prevalence of posterior crossbite for orthodontic treatment timing. j clin pediatr dent. 2016; 40(5): 422–4. 9. nishi se, basri r, alam mk, komatsu s, komori a, sugita y, maeda h. evaluation of masticatory muscles function in different malocclusion cases using surface electromyography. j hard tissue biol. 2017; 26(1): 23–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 43izach et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 38–43 10. shim j, ho kcj, shim bc, metaxas a, somogyi-ganss e, di sipio r, cioffi i. impact of post-orthodontic dental occlusion on masticatory performance and chewing efficiency. eur j orthod. 2020; 42(6): 587–595. 11. ardani igaw, rahmawati d, narmada ib, nugraha ap, nadia s, taftazani h, kusumawardani mk. surface electromyography unveil the relationship between masticatory muscle tone and maloclusion class i & ii in javanese ethnic patient. j int dent med res. 2020; 13(4): 1447–54. 12. rahmawati d, ardani igaw, hamid t, fardhani i, taftazani h, nugraha ap, kusumawardani mk. surface electromyography reveal association between masticatory muscles with malocclusion class i and class iii skeletal in javanese ethnic patient. j int dent med res. 2021; 14(4): 1542–6. 13. komino m, shiga h. changes in mandibular movement during chewing of different hardness foods. odontology. 2017; 105(4): 418–25. 14. tomonari h, seong c, kwon s, miyawaki s. electromyographic activity of superficial masseter and anterior temporal muscles during unilateral mastication of artificial test foods with different textures in healthy subjects. clin oral investig. 2019; 23(9): 3445–55. 15. shimada a, yamabe y, torisu t, baad-hansen l, murata h, svensson p. measurement of dynamic bite force during mastication. j oral rehabil. 2012; 39(5): 349–56. 16. singh sp, kumar v, narboo p. prevalence of malocclusion among children and adolescents in various school of leh region. j orthod endod. 2015; 1(2): 1–6. 17. woźn i a k k , p iąt kowsk a d, l ip sk i m , me h r k . su r fa c e electromyography in orthodontics a literature review. med sci monit. 2013; 19(3): 416–23. 18. koç d, doğan a, bek b. effect of gender, facial dimensions, body mass index and type of functional occlusion on bite force. j appl oral sci. 2011; 19(3): 274–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p38–43 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p38-43 �� effect of combination antiretroviral therapy on the frequency of oral candidiasis in hiv/aids patient sayuti hasibuan department of oral medicine dentistry faculty of university of north sumatera medan indonesia abstract oral candidiasis is one of the most common opportunistic infections in hiv/aids patients. it serves as important markers of hiv infection, viral load, and cd4 cells count in the blood and predict disease progression to aids. the development of oral candidiasis in hiv/aids patients associated by imbalances between candida and impaired host immune defenses that caused by decreased of cd4 cell counts and the increased of plasma hiv-viral load. since the introduction of antiretroviral therapy combination, commonly known as highly active antiretroviral therapy (haart), it has been observed that certain oral lesions, such as oral candidiasis as declined. the aim of this paper is to review the mechanism of combination antiretroviral therapy influenced the frequency of oral candidiasis in hiv/aids patient. we conclude that combination antiretroviral therapy generally reduced the frequency and severity of oral candidiasis in hiv/aids patient. key words: hiv/aids, oral candidiasis, antiretroviral, haart correspondence: sayuti hasibuan, c/o: bagian penyakit mulut. fakultas kedokteran gigi universitas sumatera utara. jl. alumni no. 2, kampus usu medan, indonesia. introduction acquired immune deficiency syndrome (aids) is caused by human immunodeficiency virus (hiv). this virus will be binding with the surface of cd4 cells on the helper t lymphocytes (t4 lymphocyte), subsequently interacts and infects healthy cells. furthermore, the population of helper t lymphocytes/cd4 cells decreased and viral load increased, causing impaired of immune system.1,2 once a retrovirus hiv had been identified as an agent caused of aids, there has been a concerted effort on the part of physicians, scientists and pharmaceutical industry to discover and develop compound that are effective in inhibiting hiv replication. initially, were introduced in 1997, treatment for hiv-infected patients relied on nucleoside reverse transcriptase inhibitor (nrti), namely zidovudine (azt). similar to nrti, were introduced non-nucleoside reverse transcriptase inhibitor (nnrti). furthermore, protease inhibitor (pi) were introduced as a new drug in 1996. at present, the recommended treatment for hiv infection is highly active antiretroviral therapy (haart) which consists of a combination of nrti, nnrti or pi.3,4,5 since the early reported cases of aids in the early 1980s, a number of oral lesions, including oral candidiasis have been associated with hiv infection. oral candidiasis are reported to have an increased prevalence among patients with hiv-infected or aids and has been used as diagnostic and prognostic markers for the disease.6,7,8 the introduction of combination therapies, called haart as a treatment for hiv-infected patients in the mid-1990s, however has been accompanied by multiple report of reduction in the incidence and prevalence of oral mucosal diseases including oral candidiasis.6 for instance, patton et al from united states (1995–1999) have reported significant decrease in the prevalence of oral candidiasis from 20.3% to 16.7%.9 in this paper, will be review about combination antiretroviral therapy and its effect on oral candidiasis in hiv-infected and aids patients. hiv/aids and oral candidiasis from the earliest periods of aids epidemic, oral candidiasis was recognized as an important sign of disease process and its progression. the frequency of candida isolation and clinical signs of oral candidiasis increases with advancing hiv infection.10,11 the prevalence ranges between 30 and 60% among hiv-infected subjects and reaches 90% in patients with aids.12,13 oral candidiasis in hiv-infected and aids patients may present as pseudomembranous, erythematous, hyperplastic and angular cheilitis variants.10,11,12 the presence of oral candidiasis in hiv-infected and aids patients was associated with a number factors including low cd4 counts, increased viral load, xerostomia, age and secretory aspartyl proteinase (sap), extracellular hydrolytic enzyme was product by candida.7,10,13,14 oral candidiasis occurs more frequently in hivinfected patients with low cd4 counts. imam et al found a statistically significant increase in the frequency of hivrelated oral candidiaisis in patients with cd4 counts of less than 300 cells/mm3.7 this condition made candida, �7hasibuan: effect of combination antiretroviral therapi normally flora normal of oral cavity was easy develop and inadequate immune system will made resistance to candida decreased and facilitated candida to invasion of tissue.10 viral load has important role in the pathogenesis of oral candidiasis in the hiv-infected and aids patients. several investigations had shown that associated of oral lesions with elevated viral load levels because immune system disorders.15 migliorati et al.11 demonstrated that the risk of developing oral candidiasis in hiv patients is higher when the viral load is above 3,000 copies/ml. there are reports that salivary anticandidal activities are compromised in aids patients and give contribution to increased incidence of candida oral infection. a pilot study of 12 aids patients showed that salivary anticandidal activity was decreased as compared with healthy controls. this decrease in salivary anticandidal activity in aids patients has been attributed to a decrease in the concentration of salivary histatines and/or to dysfunction of these proteins.13 histatine are a family of histidine-rich polypeptides and are the major antifungal proteins present in human saliva. in addition, many of the specific proteins secreted by the salivary glands have antifungal activities like ig a, lactoferin and lysozyme were decreased too.13 neutropenia is a frequent hematological complication in hiv infection, detected in 20% to 50% of symptomatic patients, it is usually accompanied by functional alterations of neutrophils. as we know, neutrophils play a pivotal role in the defense mechanism against candida. dios et al.16 had shown in their study, 6 neutropenic patients with aids and neutrophils counts below 1 × 109 per liter have greatest number of episodes of oral candidiasis. secretory aspartyl proteinase (sap) have been the most comprehensively studied has an important key in the presence of oral candidiasis. sap is an extracellular hydrolytic enzymes produced by candida albicans.17 one investigation had reported a comparable increase in sap activity in candida albicans strains isolated from the oral cavities of 44 hiv-positive patients with oral candidiasis compared with that in 30 hiv-negative candida albicans carriers. apparently, candida albicans isolates from hivpositive subjects produced significantly more proteinase than did isolates from hiv-negative individuals.17 antiretroviral therapy for the hiv/aids patients until this day, there are three classes of drugs that used treatment hiv-infected patients. they are nucleoside reverse transcriptase inhibitor (nrti), non-nucleoside reverse transcriptase inhibitor (nnrti) and protease inhibitor (pi).4,18,19 nucleoside reverse transcriptase inhibitor (nrti): nrti was the first drugs made available for the treatment of hiv-infected patients. the reverse transcriptase inhibitor is phosphorylated in the cell to the tryphosphate, which inhibits the hiv reverse transcriptase and leads to premature termination of the hiv dna chain. include in this class of dugs are zidovudine (azt), didanosine (ddi), zalcitabine (ddc), stavudine (d4t) and lamivudine (3tc).4,19 non nucleoside reverse transcriptase inhibitor (nrti): non nucleoside reverse transcriptase inhibitor (nrti) are a group of structurally diverse agents which bind to reverse transcriptase at a site distant to the active site resulting in conformational changes at the active and inhibition of enzyme activity. nrti consists of nevirapine (nev) and delavirdine (dlv).4,18,19 protease inhibitor (pi): protease inhibitor (pi) was introduced in 1996 and has different targeted with the reverse transcriptase inhibitor.4,5 the protease inhibitor bind competitively to the substrate site of the viral protease. this enzyme is responsible for the post-translational processing and cleavage of a large structural core protein during budding from the infected cell. inhibition results in the production of immature virus particles.4,18,19 protease inhibitor consists of saquinavir (sqv), indinavir (idv), ritonavir (rtv) and amprenavir.19,20 in the mid-1990s, were introduced combination antiretroviral therapy, so called highly active antiretroviral therapy (haart) as a standard treatment for hivinfected and aids patients.3 these therapies consists of a combination of at least 3 antiviral drugs2 nrti with either a protease inhibitor or an nnrti.3,5,18 the aim of this treatment is to reduce the plasma viral load as much as possible and for as long as possible by attacking the virus at different stages of its replication cycle (figure 1), inhibiting the multiplication rate of the virus and preventing the development of drug resistance.3 figure 1. targets for antiretroviral therapy.4 thus, haart decreases hiv viral load and leads to the increase of cd4 lymphocytes counts, causing an improvement of immunity and a decrease in the incidence of opportunistic infections.11,21 the presence of oral candidiasis in hiv/aids patients after combination antiretroviral therapy combination antiretroviral therapy appears have an impact on the oral opportunistic infections. since the introduction of haart, there have been striking changes in the frequency and character of the oral complication of hiv disease. antiretroviral drugs significantly lessen hiv viral �� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 16–19 load, increase cd4 cell counts and lessen the frequency and severity of oral opportunistic infections. for instance, salobrena et al.,6 from spain had evaluated the oral lesions from 1995–2000 and described a decrese in the prevalence of oral lesions from 22.4% to 5.2%. similarly, patton et al.9 reported significant decreases in the prevalence of hiv related oral diseases with haart (from 47.6% to 37.5%) in us patients. some studies specifically demonstrated the decrease prevalence of oral candidiasis with the widespread used of haart. a study in united kingdom by tappuni and fleming comparing subjects on haart group and non haart group. from this study showed that subjects on haart group had significantly less oral candidiasis than did subjects on non haart group, that is pseudomembranous candidiasis variant from 12% to 8% and erythematous variant from 25% to 8%.3 a study on 807 hiv/aids patients in spain, found a declining rate of oral candidiasis following the introduction of haart with stabilization or improvement in the immune status of patients.6 cattopadhyay et al.22 also reported decreases in oral candidiasis in 283 patients in north carolina. the european community clearinghouse on oral problems related to hiv infection and who collaborating centre showed the greatest reduction in the prevalence of oral candidiasis (from 51.4% to 17.1%), especially of the pseudomembranous variant.23 discussion at present, the recommended treatment for hiv infection is haart, which consists of a combination of at least 3 antiviral drugs, 2 nrti with either a pi or an nnrti. this combination attacking the virus at different stages of its replication cycle. combination therapies appears to have a critical role in the prevention of oral manifestations of hiv, probably because of its role in the reconstitution of the immune system.3 as we know, oral lesions strongly associated with hiv, such as oral candidiasis, hairy leukoplakia, kaposi’s sarcoma and necrotizing periodontal diseases, are also strongly associated with reduced of immune system.9 in the reduction of frequency of oral candidiasis, combination therapies have several mechanisms. one of them, with these regimens demonstrated increases cd4 cells counts, decreases viral load as much as possible (below 3,000 copies/ml). such a thing related an improving immunity thereby reduces the potential risk of opportunistic infections such as candida species.3,6,9,14 in addition, combination therapies has been significantly associated with increases in neutrophils count. neutrophils plays a pivotal role in the defense mechanisms against candida. neutrophils produce granulocyte-colony stimulating factors (g-csf) and granulocyte macrophagecolony stimulating factors (gm-csf) that induce improvement of their functionality thereby might inhibit oral candidiasis.9,16,23 on the other hand, decreased in prevalence of oral candidiasis in hiv-infected patients might also attributed to the direct effect of antiretroviral protease inhibitor over secretory aspartyl protease, a potent virulence factor of candida species.11 protease inhibitor therapy has capacity inhibited production and activity of secretory aspartyl protease, which are involved in candida adherence.14,22 some believe that the antifungal effect of antiretroviral protease inhibitor is equivalent to fluconazole.11 however, there were systemic and oral adverse effects can arise with the used of combination therapies. systemic effects include dry skin, headache, nausea, neuropathy and liver disorder. on the orofacial regions, haart seems to increase the incidence of hiv-related salivary gland disease, especially enlargement of parotid gland. the other oral side effects are mouth ulcer and oral warts.24 in conclusion, oral candidal infection in hiv-infected patients have shown a decrease in the era of haart. haart decreases hiv viral load and lead to increases of cd4 lymphocytes count, causing an improvement of immunity. references 1. muma rd, lyons ab, borweki mj. hiv manual untuk tenaga kesehatan. shinta prawitasari. jakarta: egc; 1997. p. 9–21. 2. yeh ck, puttaiah r, cottone ja. human immunodeficiency virus infection, acquired immune deficiency syndrome, and related infection. in: cottone ja, terezhalmy gt, molinary ja, editors. practical infection control in dentistry. 2nd ed. baltimore: williams & wilkins; 1996. p. 48–55. 3. tappuni ar, fleming gjp. the effect of antiretroviral therapy on the prevalence of oral manifestations in hiv-infected patients: a uk study. oral surg oral med oral pathol radiol endod 2001; 92:623-8. 4. weller ivd, williams ig. antiretroviral drugs. bmj 2001; 322:1410–12. 5. sepkowitz ka. aidsthe first 20 years. n engl j med 2001; 344:1764–72. 6. salobrena ac, cepeda lg, garria lc, samaranayaka lp. the effect of antiretroviral therapy on the prevalence of hiv-associated oral candidiasis in a spanish cohort. oral surg oral med oral pathol radiol endod 2004; 97:345–50. 7. mc carthy gm. host factors associated with hiv-related oral candidiasis. oral surg oral med oral pathol 1992; 73:181–6. 8. patton ll, mckaig rg, strauss rp, eron jr jj. oral manifestations of hiv in a southeast usa population. oral dis 1998; 4:164–9. 9. patton ll, mccaig rg, strauss rp, rogers d, eron jr jj. changing prevalence of oral manifestations of human immunodeficiency virus in the era of protease inhibitior therapy. oral surg oral med oral pathol oral radiol endod 2000; 89:299–304. 10. samaranayake lp. oral mycosis in hiv infection. oral surg oral med oral pathol 1992; 73:171–80. 11. migliorati ca, birman eg, cury ae. oropharyngeal candidiasis in hiv-infected patients under treatment with protease inhibitors. oral surg oral med oral pathol oral radiol endod 2004; 98:301–10. 12. romagnoli p, pimpinelli n, reichart pa, eversole lr, ficarra g. immunocompetent cells in oral candidiasis of hiv-infected patients: an immunohistochemical and electron microscopical study. oral dis 1997; 3:99–105. 13. lin al, johnson da, paterson tf, wu y, lu dl, shi q, yeh ck. salivary anticandidal activity and saliva composition in an hivinfected cohort. oral microbiol immunol 2001; 16:270–8. ��hasibuan: effect of combination antiretroviral therapi 14. cassone a, tacconelli e, debernardis f, tumbarello m, torosanfucci a, chiani p, cauda r. antiretroviral therapy with protease inhibitors has an early, immune reconstitutionindependent beneficial effects on candida virulence and oral candidiasis in human immunodeficiency virus infected subjects. j infec dis 2002; 185:188–95. 15. baqui aama, meiler tf, jaber-rizk ma, zhang m, kelley jf, falkler wa. association on hiv viral load with oral diseases. oral dis 1999; 5:294–8. 16. dios pd, ocampo a, miralles c, otero i, iglesias i, rayo n. frequency of oropharyngeal candidiasis in hiv-infected patients on protease inhibitor therapy. oral surg oral med oral pathol oral radiol endod 1999; 87:437–41. 17. naglick jr, challombe sj, hube b. candida albican secreted aspartyl proteinasses in virulence and pathogenesis. microbiology and molecular biology virus. 2003. available at: http://www.mmbr. a5m.org/cgi/content/full/67/3/400. accessed march 27, 2005. 18. montaner jsg, hogg r, raboud j, harigan r, o’shaughnessy. antiretroviral treatment in 1998. the lancet 1998; 352:1919–22. 19. lewin sr, crowe s, chambers de, cooper da. antiretroviral therapies for hiv. melborne: pubmed 2000; p. 45–54. 20. bangsberg d, tulshy jp, hecht fm, moss ar. protease inhibitor in the homeless. jama 1997; 278:63–5. 21. aquirre jm, echebarria ma, ocina e, ribacoba l, montejo m. reduction of hiv-associated oral lesions ahter highly active antiretroviral therapy. oral surg oral med oral pathol oral radiol endod 1999; 88:1–3. 22. chattopadhyay a, jouen d, caplan dj, slade gd, shugars dc, tien hc, patton ll. incidence of oral candidiasis and oral hairy leukoplakia in hiv-infected adults in north carolina. oral surg oral med oral pathol oral radiol endod 2001; 92:623–8. 23. dios pd, ocampo a, miralles c, limeres j, tomas i. changing prevalence of human immunodeficiency virus-associated oral lesions. oral surg oral med oral pathol oral radiol endod 2000; 90:403–5. 24. frezzini c, leao jc, porter s. current trends of hiv disease of the mouth. j oral pathol med 2005; 34:513–31. 55 p53-protein over-expression and gene mutational of oral carcinoma in-situ mei syafriadi* and takashi saku** ** division of pathology, department of biomedical sciences, faculty of dentistry, jember university ** division of oral pathology, department oral life sciences, graduates school of medical and dental sciences, niigata university, japan abstract we had been reported histological types of oral carcinoma in-situ (oral cis), such as basaloid, verrucous, and acanthothic/ atrophic types. we considered that they have different histological appearance influenced by molecular behavior. to understand the molecular behavior of them we examined p53 exon 4–8 gene mutation and their protein expression. using 35 cases formalin-fixed paraffin sections of oral cis and 10 cases of mild and moderate squamous epithelial displasia (sed) as a control were subjected to p53 immunohistochemistry. in the next step all cases were subjected to p53 gene mutations analysis by laser capturing microdissection and direct sequencing of pcr product for exon 4–8. showed that p53-protein over-expression were found in basal layer of sed and the p53 protein over-expression were confined in the whole layer of cis-basaloid type, basal and parabasal layers of cis-verrucous type, and sporadically in the basal layer of cis-acanthothic type. mutational analysis for p53 gene showed 43% of total cases of cis had p53 gene mutation therefore cis-basaloid type had mutations more frequently than the other types and mutation in exon 8 more dominant than other exons, which had some common mutation at codons 196, 248, 282, 291, and 306, while no particular mutations were found in control (sed).our criteria to diagnose several types of oral carcinoma in-situ by p53 protein expression and mutational analysis could be used to understand molecular behavior of cis. key words: p53, carcinoma in-situ, oral, mutation correspondence: mei syafriadi, c/o: laboratorium patologi, bagian biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: mei_syafriadi@hotmail.com introduction it is known that carcinoma in-situ (cis) composed of dysplastic change of squamous epithelium does not have keratinization, but it is different to dysplastic change arising in the oral cavity, which has prominent keratinization.1 according to carcinoma which arise only limited in intra epithelial, many authors using different term such as severe dysplasia,2 or squamous intra neoplasia (sin) high grade.3 whereas who classified oral cis as one classification that separate to squamous epithelial dysplasia (sed) classification.4 the concept of oral cis is the most important aspect in the present criteria, because sed and invasive squamous cell carcinoma (scc) can be easily discriminated however when and what stage could be called as cis is still unclear and controversial because there has not any fixed criteria yet. cis has defined as a true and not invasive neoplasm yet lying within an epithelial layer. the neoplastic cells are different from sed, because they have already been proliferated and subsequently differentiated. in epithelial dysplasia, the constituent cells are only proliferated but not differentiated yet.5 as we reported before, we have noticed that oral cis histologically has any mimicking which classified to cisbasaloid type, cis-verrucous type and cis-acanthotic type,6 which all of them are not invasive yet.7 based on this histological appearance, we speculated that those cis types are several molecular behaviors. the p53 gene structure is consist of eleven exon which is exon 2–3 is transactivation domain, exon 5–8 is dna binding domain and exon 10 is oligomerization domain that they have multifunctional such as transcription factor, which regulates cell cycle progression and interacts with several key proteins which involved in dna replication, transcription and reparation. nearly 60% of human cancers are accompanied by mutation in p53 gene.8 the p53 protein over-expression is frequently found in both malignant and dysplastic lesions which increase due to grades of dysplasia, and may be an early event in multistage carcinogenesis of head and neck cancer.8–13 the abnormal p53 protein expression reported in 10 to 80% of oral sed,14–19 that some cases related to p53 gene mutation.8,9,19,20 however, in some cases it was possible to observe protein over expression but did not show any mutation gene or p53 gene mutation without p53 protein expression20 but chiang et al.,21 reported p53 over expression were correlated to decreasing of survival rate of patient with squamous cell carcinoma. based on it we observed p53 gene exon 4–8 and its protein expression in several types of oral cis. the purpose is to understand their molecular behavior chances by protein expression pattern and gene mutation. 56 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 55–60 materials and methods specimen were formalin paraffin-fixed block selected from the surgical pathology files in the division of oral pathology, niigata university graduate school of medical and dental sciences, japan, during sixth year period from 1999 to 2004 and eleven biopsy specimens from department of oral pathology, sichuan university school of stomatology, china, during 2000–2002, after critical reviewing of hematoxylin and eosin (he) stained sections. these researches consisted of ten cases of oral sed (mild and moderate cases) as a control and 35 of cis (15 cases basaloid, 10 cases verrucous and 10 cases acanthothic type). two oral pathologists with the japanese society of pathology board certification and one pathologist from china screened all the specimens, when the diagnoses of grading of cis were not identical those cases would be reevaluated together. all of the specimens were routinely fixed in 10% formalin and embedded in paraffin. serial 4 µm sections were cut from paraffin blocks. one set of the sections was stained with hematoxylin and eosin and used for reevaluation of histological diagnosis, and the other sets were toluidine blue staining used for microdissection as well as immunohistochemistry for p53 protein. the antibodies against p53-protein used in this study were mouse monoclonal antibody clone bp53-11, (igg2a) (progen, progen biotechnik gmbh, heidelberg, germany), that reacted to wild-type and mutant forms of human p53 antigen within n-terminal region epitope aa20–31. for immunohistochemistry staining were tissue sections 4 µm in thickness were taken from tissue blocks. after deparaffinization and dehydration, sections were washed in 0.01 m phosphate buffered saline (pbs). to restore the antigenic sites, sections were autoclaved in 0.01 m citrate buffer (ph 6.0) for 15 min at 121°c and then kept standing for 20 min at room temperature. to block endogenous peroxidase activities, all the sections were quenched with 0.001% h2o2 in 100% methanol for 30 min at room temperature and rinsed with pbs containing 0.5% skim milk and 0.05% triton x-100 (pbst). after rinsing in pbst, the sections were incubated in 5% skim milk in pbs containing 0.05% tritonx-100 for 1 hr at 37°c to block non-specific protein bindings. the sections were then incubated with monoclonal primary antibodies against p53-protein were p53-protein/clone p53 abbp53-11 (1: 100, progen biotechnik gmbh, heidelberg, germany), for overnight at 4°c. after incubations with the primary antibodies, the sections were rinsed in pbst and then treated with polymer-immune complexes (envision+peroxides, rabbit/mouse, dako, 1:1) for 1 hr at room temperature. the peroxidase reaction products were visualized by incubation with 0.02% 3, 3-diaminobenzidine (dab, dohjin laboratories, kumamoto, japan) in 0.05 m tris-hcl solution (ph 7.6) containing 0.005% h2o2. the sections were counterstained with hematoxylin. cells were regarded as positive for p53 protein if nuclear staining was intense and could be readily visualized at 10 times magnification. positive staining for p53 protein staining were calculated quantitatively using a micrometer scale 1mm/square at 10 times magnification. calculation of positive cells had been done three times and the average was taken. for control experiments, the primary antibodies were replaced with pre-immune mouse igg2a (dako). after evaluation of immunostaining pattern for p53, each sample was stained by toluidine blue for laser microdissection (lmd). appropriate ten of slides each case were cutted due to appropriate area and collected in collection tube for extracted dna using proteinase k 10% for one night at 37°c. the dna solution was purified using phenol/chloroform/isoamyl alcohol mixture (25:24:1). pcr amplification was carried out for p53 exons 4–8. all primer sets were designed on intron sequences adjacent to each exon as follow: [exon 4] sense, 5’-tgc tct ttt cac cca tct ac-3’, antisense, 5’-ata cgg cca ggc att gaa gt-3’ spanning 353 bps; [exon 5] sense, 5’-gtt tct ttg ctg ccg tgt tc-3’, antisense, 5’-agg cct ggg gac cct ggg ca-3’ spanning 323 bps; [exon 6] sense, 5’-tgg ttg ccc agg gtc ccc ag-3’, antisense 5’-gga ggg cca ctg aca acc a-3’ spanning 223 bps; [exon 7] sense, 5’-ctt gcc aca ggt ctc ccc aa-3’, antisense 5’-tgt gca ggg tgg caa gtg gc3’ spanning 196 bps; [exon 8] sense, 5’-ttc ctt act gcc tct tgc tt-3’, antisense 5’cgc ttc ttg tcc tgc ttg ct-3’ spanning 201 bps. pcr were performed on a thermal cycler (pc-800, astec co., ltd., fukuoka japan), after a pre-denaturation at 94°c for 5 minutes. the amplification step was carried out for 35 cycles in 100 µl of a pcr reaction mixture containing 5 units of takara ex taq polymerase (takara biotechnology, co., ltd., otsu, japan), 10× ex taq buffer, 2.5 mm each of dntp mixtures, and 20 pmol of each sense and antisense primers. the thermal cycling condition was as follow: denaturation at 94°c for 1 min, annealing at 63°c for exon 5, at 60°c for exon 4, 6, and 8, and 62°c for exon 7 for 0.30 sec each, and extension at 72°c for 1 min. the last extension was prolonged by additional 7 min. amplification products were analyzed by electrophoresis on 3% agarose gel (nusieve 3:1 agarose, cambrex bioscience rockland inc., rockland, me usa) and the band were visualized by ethidium bromide upon exposure to an ultraviolet transilluminator. all pcr products were subjected to cycle sequencing by using thermo sequenase primer cycle sequencing kit with 7-deaza -dgtp (amersham biosciences corp., piscataway, usa). the sequence primers were synthesized based on the published data (lehman ta et al.) and labeled with texas red 5’-end. the labeled primers were as follow: 5’tgc tct ttt cac cca tct ac-3’ for exon 4; 5’-gtt tct ttg ctg ccg tgt tc-3’ for exon 5; 5’-gcc tct gat tcc tca ctg at-3’ for exon 6; 5’-ctt gcc aca ggt ctc ccc aa-3’ for exon 7; 5’-ttc ctt act gcc tct tgc tt-3’ for exon 8. one tube sequencing reaction contained 3 ml of master mixes (appropriate nucleotides/ reaction buffer/thermo sequenase dna polymerase), 2 ml of the template pcr products, which were purified with 57syafriadi: p53-protein over-expression and gene mutational gfx pcr dna and gel band purification kits (amersham biosciences corp., piscataway, usa), 8 ml of distillated water and 2 ml (2pm) of texas red-labeled primers. each sequencing reaction added 3 ml of a, c, g, t reagent. after denaturation at 95 °c for 30 sec and annealing at 55 °c for 30 sec. the reaction products were dissolved in 3 ml loading dye by vortexing and concentrated with vacuum desiccators. then 3 ml of samples for each lane were loaded on a gel (7% long ranger/6.1 m urea/1.2 × tbe buffer (10 mm tris, 10 mm boric acid, and 2 mm edta). the electrophoresis was performed in a fluorescent dna sequencer (sq-5500-s, hitachi ltd., tokyo, japan), and the sequencing data were analyzed by using the sq-5500 analysis software ver.3.03 (hitachi). for statistical analysis, the numbers of p53 and ki-67 positive cells in a square unit 1 mm2 were counted on a microscope equipped with a micrometer. ten fields were randomly counted per section at ×100 magnification. oneway anova was used for statistical comparison of cell numbers between each group by using the spss software program (spss inc., chicago, il, usa). result oral sed-mild is regarded to lesions with low proliferation and considered as low risk malignant transformation, but it could continue proliferate to moderate dysplasia and cis which classified to basaloid, acanthotic and verrucous type. in oral cis-basaloid there is basaloid cells proliferation that replaces whole layer of epithelium, but it still shows differentiation and keratinization in the surface layer. the verrucous type is characterized by round shape of rete processes, gradual differentiation, and enhanced keratinization in the surface. the keratinization creates keratin plugs formation. the acanthothic type is the most difficult subtype to be diagnosed, because it looks like sed due to cells proliferation and fairly extent of keratinocytic differentiation. although this type has distinctive keratinization often on the surface, their rete processus are sharp, finger looks and tends to infiltrate to lamina propria (figure 1). immunohistochemical staining for p53 protein, when observed, was found exclusively in the nuclei of epithelial cells. in sed-mild, p53-protein was over-expressed in basal and parabasal layer (figure 2-a), in basaloid type it was expressed in whole layer (figure 2-b), sporadically until one-third of epithelium of cis-acanthothic (figure 2-c), and over-expressed in two-third of epithelium in verrucous type. in some cases, both in basaloid types or verrucous types showed no p53 protein expression (figure 2-d). when dysplastic cells were transformed to cisacanthothic, basaloid and verrucous type, it increased of p53-protein expression in number statistically significant (p < 0.05) (figure 3). figure 1. histological appearance of several types of oral cis (a), sed-mild (b), cis-basaloid type (c), cisacanthothic type (d), cis-verrucous type. a b c d figure 2. immunostaining of p53 protein over expressed in sed-mild (a), cis-basaloid (b), cis-acanthothic type (c), and cis-verrucous type. in some cases either basaloid type or verrucous type showed no p53 protein expression (d). a b c d 0 50 100 150 200 250 300 350 dysplasia cisbasaloid cisacanthothic cisverrucous figure 3. p53-protein expression was increased significantly from sed to cis-basaloid, acanthothic and verrucous type. 58 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 55–60 all of dysplasia cases and cis were sequencing for p53 gene analyses of exon 4–8. 40% of cis-acanthothic; 20% of cis-verrucous, 60% of cis-basaloid, had shown p53 gene mutation. therefore, exon 4 had not showed any mutation but several cases showed pleomorphism in codon 72. all of p53 gene mutation in ciss were point mutations (100%) of which result were 66% missense, and nonsense were 33%. meanwhile no mutation were found in dysplasia cases (table 1). discussion oral cis histologically showed irregular stratification, lost of epithelial cell cohesion, increasing of n/c ratio hyperchromatic, nuclear polymorphism, and preserved basement membrane.4,5 the surface layer covered by keratin and hyperparakeratin layer, therefore, clinically showed white lesion. some parts of this histological appearance were similar to dysplastic change of sed, but cis showed more dysplastic changed than sed and not invasive yet which we considered to be an advanced stage of moderate dysplasia (figure 1). according to who, squamous epithelial dysplasia showed 13 representative appearances and classified to sed-mild, moderate and table 1. p53 gene mutational analysis in sed and ciss no. cases p53 gene mutation no. cases p53 gene mutation 1 sed-mild – 14 cis-basaloid e8: 263, aat-aag 282,cgg-tgg 2 sed moderate – 15 cis-basaloid – 3 sed-mild – 1 cis-acanthothic – 4 sed-moderate – 2 cis-acanthothic – 5 sed-mild – 3 cis-acanthothic – 6 sed-moderate – 4 cis-acanthothic – 7 sed-moderate – 5 cis-acanthothic e8:282, cgg-tgg 8 sed-moderate – 6 cis-acanthothic e7:243, atg-aag 9 sed-moderate – 7 cis-acanthothic e7:231, acc-gcc 10 sed-mild – 8 cis-acanthothic e8:263, aat-aag 1 cis-basaloid e8:282, cgg-tgg 9 cis-acanthothic – 2 cis-basaloid e7:248,cgg-cag 10 cis-acanthothic – 3 cis-basaloid e6:196, cga-tga 1 cis-verrucous – 4 cis-basaloid – 2 cis-verrucous – 5 cis-basaloid e8:276, gcc-acc 3 cis-verrucous – 6 cis-basaloid e7:237, atg-ata 4 cis-verrucous – 7 cis-basaloid e8:306, cga-tga 5 cis-verrucous – 8 cis-basaloid – 6 cis-verrucous e8:306, cga-tga 9 cis-basaloid – 7 cis-verrucous – 10 cis-basaloid e8:282, cgg-tgg 8 cis-verrucous e7:242, tgc-ttc 11 cis-basaloid – 9 cis-verrucous – 12 cis-basaloid e8:291, aag-tag 10 cis-verrucous – 13 cis-basaloid – severe dysplasia, however, the borderline between mild, moderate, and severe dysplasia was not clear.4 meanwhile many pathologists called severe dysplasia as cis. recently we introduced several histological subtypes of oral cis such as cis-basaloid, verrucous and acanthotic type.6 the verrucous type was characterized by round shape of rete processes, gradual differentiation, and enhanced keratinization in the surface. the keratinization created keratin plugs formation. the acanthotic type was the most difficult subtype to be diagnosed, because it looked like sed due to cells proliferation and fairly extent of keratinocytic differentiation. although this type often has distinctive keratinization often on the surface, their rete processus were sharp, finger looked and tended to infiltrate to lamina propria (figure 1-c). all of cis subtypes showed preservation of basement membrane and no stromal induction, but it should get more intention for clinician because not only oral cis different from cis cervix uteri but also they were potential transform to scc which showed destruction of basement membrane and some small carcinomatous foci formation that could invade close to muscle layer and also followed by stromal formation. the present study demonstrated distinction of localization and number of positive cells of p53-protein over-expression 59syafriadi: p53-protein over-expression and gene mutational of cis-acanthothic, verrucous, and basaloid type and sed. the location of p53-protein expression started from basal layer of sed-mild then involving parabasal cells due to severity of dysplasia,11,17 in this study it was highly overexpressed in whole layer of cis-basaloid type (figure 3). cruz et al.17 reported that p53 protein expression above basal cell layer is as early even of malignant transformation, but why their over-expression pattern different in those cis subtypes and sed were not documented yet. the over-expression of protein 53 started from basal layer of sed-mild and then involved two or three parabasaloid layer of sed-moderate, and their expression more enhanced in cis-verrucous which was expressed whole of basaloid cells but not in middle layer because of keratinization there were present seemed-like keratin plug. it means that type some cells are showed differentiation, contrast to cis-basaloid type which basaloid cells replaced whole layer and all basaloid cells showed p53 protein over-expression, meanwhile, p53 protein over-expression in cis-acanthothic type were sporadic which seemed the same to moderate dysplasia (figure 2). p53 protein over-expression in head and neck carcinoma were related to gene mutation or none,8, 9, 19, 20 similar with this present study, those cis-verrucous, acanthothic, and basaloid, showed p53 protein over-expression but only 43% of cis cases found any p53 mutation, 57% of cases found no p53 gene mutation. in sed some cells showed p53 protein expression but had not shown any mutation. several cases showed no p53 protein immunopositivities, because p53 gene mutation had stop codons mutation, and the antibody could not detect p53 protein expression because protein had not been produced yet. cis-basaloid showed more dominantly p53 gene mutation than other types which, histologically, increased basaloid cell proliferation and decreased keratinization. cis-basaloid type could be considered as malignant transformation, meanwhile, cis-verrucous and acanthothic type, even though it showed few p53 gene mutations but it should also be considered as high risk malignant transformation because several cases found p53 gene mutation. involving other onco-supressor gene should be thought as possible factor to p53-protein over-expression even no p53 gene mutation in sed and cis, because it is known that p53 gene produce protein interacts to other gene such as p21 and p63 or p73.8 p53 gene mutation in cis occurred in varies exon but exon 8 more frequently (53% of total mutation cases) and was followed by exon 7 (40% of total mutation cases) and codon 248, 282 in exon 7 and 8 known as “hot spot”.8 in addition, other point mutation in exon 6 and exon 8 codon 196 (cga→tga) resulted from arginine to stop codon; codon 291 (aag tag) resulted from lysine to stop codon and codon 306 (cga→tga) resulted from arginine to stop codon also found. these mutations were denoted as hot spots of p53 mutations. several study showed that arginine residue function is involving in dna repair,22 so if there is mutation in this protein it could consider that dna failed to repair their dna damage. many reports demonstrated p53 gene mutation of head and neck carcinoma and showed similarity to cis p53 gene mutation in this present study, it suggested oral squamous cell carcinoma was starting from carcinoma in-situ but it needed more study to understand the malignancy occurring in head and neck cancer or in oral carcinoma. from this research could be concluded that histologically oral cis has three subtypes such as cis-basaloid, verrucous and acanthothic/atrophic type. the differences of those types not only in histological appearance but also it showed different p53 protein over-expression pattern. p53 gene mutation analysis showed cis-basaloid type that frequently had point mutation than other types. it considered that cis-basaloid type is potentially transform to scc because of some mutation was involving hot spot of p53 gene mutation as reported in head and neck carcinoma. p53 gene mutation in cis had stop codon mutation, showing no protein immunopositivities could be detected by antibody against p53 protein. p53 protein over-expression could be detected in basal and parabasal of sed but no p53 gene exon 4–8 mutations were found. from this present study it could suggested that oral cis subtypes could be clearly distinguished from their subtypes and sed by p53 protein expression and mutational gene analysis but it still need further study by analysis the other exon of p53 gene and other oncogenes that involved in head and neck carcinoma. references 1. syafriadi m, ida-yanemochi h, ikarashi t, maruyama s, jen ky, cheng j, hoshina h, takagi r, saku t. carcinoma in-situ of the oral mucosa has a definite tendency towards keratinization. oral med pathol 2003; 8:43–44. 2. coltrera md, zarbo rj, sakr wa, gown am. markers for dysplasia of the upper autodigestive tract. suprabasal expression of pcna, p53 and ck 19 in alcohol-fixed, embedded tissue. am j pathol 1992; 41:817–25. 3. sakr wa, crissman jd, gnepp dr. squamous intraepithelial neoplasia the upper aero digestive tract. in diagnostic surgical pathology of the head and neck. wb saunders company; 2001. p. 1–9. 4. world health organization international histological classification of tumors: histological typing of cancer and precancer of the oral mucosa. 2nd ed. springer, 1997; p. 26. 5. the japanese society for oral pathology. guidelines for histopathological diagnosis of borderline malignancies of the oral mucosa: a preliminary proposal, 2005; 7–11. 6. syafriadi m and takashi s. histological types of oral carcinoma in-situ. indonesian journal of dentistry 2006; 13(1):12–5. 7. pindborg jj, reichart pa, smith cj, van der wall i. histological typing of cancer and precancer of the oral mucosa, 2nd ed. world health organization international histological classification of the tumors, springer-verlag, berlin, 2005. 8. glazko gv, koonin ev, rogozin ib. mutation hotspots in the p53 gene in tumors of different origin: correlation with evolutionary conservation and signs of positive selevtion. biochimica et biophysica acta 2004; 1679:95–106. 9. shahnavaz sa, regezi ja, bradley g, dube id, jordan rck. p53 gene mutation in sequential oral epithelial dysplasias and squamous cell carcinomas. j pathol 2000; 190:417–22. 60 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 55–60 10. pindzola ja, palazzo jp, kovatich aj, tuma b, nobel m. expression of p21 waf1/cip1 in soft tissue sarcomas: a comparative immunohistochemical study with p53 and ki-67. pathol res pract. 1998; 194:685–91. 11. ogden gr, kiddie ra, lunny dp, lane dp. assessment of p53 protein expression in normal, benign, and malignant oral mucosa. j pathol 1992; 166:389–94. 12. regezie ja, zarbo rj, regev e, pisanty s, silverman s, gazit d. p53 expression in sequential oral dysplasias and in situ carcinomas. j oral pathol med 1995; 24: 18–22. 13. kushner j, bradley g, jordan rck. patterns of p53 and ki-67 protein expression in epithelial dysplasia from the floor of the mouth. j pathol 1997; 183:418–23. 14. alves fa, pires fr, de almeida op, lopes ma, kowalski lp. pcna, ki-67 and p53 expressions in submandibular salivary gland tumours. int j oral maxillofac surg 2004; 33:593–97. 15. kovesi g, szende b. changes in apoptosis and mitotic index, p53 and ki-67 expression in various types of oral leukoplakia. oncology 2003; 65:331–6. 16. farrar m, sandison a, peston d, gailani m. immunocytochemical analysis of ae1/ae3, ck 14, ki-67 and p53 expression in benign, premalignant and malignant oral tissue to establish putative markers of oral carcinoma. br j biomed sci 2004; 61:117–24. 17. cruz ib, snijder pjf, meijer cj, braakhuis bj, snow gb, walboomers jm, van der waal i. p53 expression above the basal cell layer in oral mucosa is an early event of malignant transformation and has predictive value for developing oral squamous cell carcinoma. j pathol 1998; 184:360–68. 18. mallofre c, castillo m, morente v, sole m. immunohistochemical expression of ck20, p53, and ki-67 as a objective markers of uroepithelial dysplasia. mod pathol 2003; 16:187–91. 19. taylor d, koch wm, zahurak m, shah k, sidransky d, westra wh. immunohistochemical detection of p53 protein accumulation in head and neck cancer: correlation with p53 gene alteration. hum pathol 1999; 30:1221–5. 20. cruz i, snijders pj, van houten v, vosjan m, van der waal i, meijer cj. specific p53 immunostaining patterns are associated with smoking habits in patients with oral squamous cell carcinomas. j clin pathol 2002; 55:834–40. 21. chiang cp, huang js, wang jt, liu by, kuo ys, hahn lj, kuo my. expression of p53 protein correlates with decreased survival in patients with areca quid chewing and smoking-associated oral squamous cells carcinomas in taiwan. j oral pathol med 1999; 28:72–6. 22. blencowe bj. exonic splicing enhancer: mechanism of action, diversity and role in human genetic diseases. tibs 2000; 25: 106–10. vol 49 no 3 juli-sept 2016.indd 133133 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 133–136 topical application of 1% znso4 on oral ulcers increases the number of macrophages in normal or diabetic conditions of wistar rats rochman mujayanto,1 kus harijanti,2 and iwan hernawan2 1department of oral medicine, faculty of dentistry, universitas islam sultan agung, semarang-indonesia 2department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: therapy for chronic ulcer in diabetic patient is by modifying local inflammation response using drugs that acts as immunomodulator, neuromodulator and growth factors stimulator. topical zinc is one of drug that can modifiy local inflammation response, immunostimulation or immunosuppresion. purpose: this study was to prove about the number of macrophage in oral ulcer between normal and diabetes microscopically and the difference if treated by 1% znso4 gel topically. method: ulcer in lower labial mucosa was made in normal and diabetic wistar rats (induced by stz), then applied 1% znso4 gel and cmc-na gel as control. they were decapitated in third and fifth day and specimen was made by processing lower labial mucosa result: microscopically, the result showed the number of macrophages in oral ulcer in diabetic condition was significantly higher than normal and the application of 1% znso4 increased the number of macrophages in fifth day. conclusion: the number of macrophages was higher in diabetic than normal condition, and was proven that topical application of 1% znso4 increased the number macrophages of oral ulcer diabetic and normal condition. keywords: macrophage; diabetes; znso4 1% gel correspondence: rochman mujayanto, department of oral medicine, faculty of dentistry, universitas islam sultan agung. jl. kaligawe km 4,5 semarang 50164, indonesia. e-mail: rochman.mujayanto@unissula.ac.id. introduction chronic ulcers is one of complications experienced by diabetic patients. it is caused by a deviating local inflammatory response which is marked by the number of persistent inflammatory cells. this condition is caused by a decreased chemokine expression which, in turn, leads to a slower production of growth factor and a slower inflammatory cell infiltration.1,2 the amount of persistent macrophages in a diabetic condition influences the healing process of ulcers, resulting in a continuous cell deterioration and increasing expressions of interleukin-1β (il-1β), tumor necrosis factor-α (tnf-α) and matrix metalloproteinases (mmps).3-5 a study proposed a calculation of macrophages in ulcers both in normal and diabetic conditions using a mathematical formula, concluding that the amount of macrophages in the diabetic condition keeps increasing and thus becomes more persistent than the normal condition’s. the model study, however, the formula has not been proven.6 polyuria in diabetic patients causes excessive zn excretion (hyperzincuria),5,7-11 resulting in zn deficiency.12,13 the topical zn application on ulcer of a diabetic patient helps the healing process more than the systemic zn application.11 the topical zn application on ulcer will increase the concentration bioavailability of zn approximately 1.000-3.000 μmol/l. zn may help autodebridement process, influencing mmp to eliminate any necrotic tissues and facilitate keratinocyte migration in the re-epitelisation process.8 zinc sulphate (znso4) is one of active substances that can be topically absorbed by oral mucosa and recommended to heal oral ulcers and prevent recurrent ones.14-16 the topical application of 0,5% znso4 for oral suspension is dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p133-136 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p133-136 134 mujayanto, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 133–136 effective in reducing oral lesions in patients with oral herpes simplex.17 the gels of 1% znso4 may reduce perioral lesions and pains of any patients who infected by herpes simplex virus (hsv),18,19 and speed up the healing process of post dental extraction scars by reducing the amount of macrophages and increasing fibroblast poliferation as well as capillaries.20 this study aimed to microscopically observe differences in the amount of macrophages in both normal and diabetic conditions and the influences of topical 1% znso4 on the amount of macrophages in the diabetic rats’ oral ulcers. materials and method the method employed in this research was the true experimental post test only control group design, using wistar rats as the laboratory animals. the rats were divided into two groups, 24 normal wistar rats and 24 normal wistar rats which later were made diabetic. the preparation of the laboratory animals, treatment application and tissues retrieval were performed in the animal house of the biochemistry laboratory in the faculty of medicine, universitas airlangga. the tissues processing and the creation of histological preparation were performed in the laboratory of anatomic pathology at the regional public hospital of dr. soetomo, surabaya. the identification and the calculation of the amount of the macrophages were performed in the laboratory of anatomic pathology at the hospital of islam sultan agung semarang. the wistar rats which were made diabetic had been fasting for 4 hours before being induced with streptozotocin (stz) (bioworld, dublin, ohio, usa) dissolved into citric buffer at ph 4.5, in order to empty their stomachs and reduce the risks of aspiration. the amount of stz needed was 150mg/kg of the weight of a wistar rat, dissolved with a concentration of 22.5mg/ ml stz in citrate buffer solutions. the stz solutions then were injected through intraperitoneal according to the appropriate dose for each rat. the induction process was only performed once. in order to avoid sudden hypoglycemic post injection, the rats received 10% sucrose or dextrose during the first night. the blood sugar level of the rats during fasting hours was observed every morning. the fasting rats did not receive food and the cages were devoid of husks for six hours. any meaningful hyperglicemia condition would be found two days post induction.21 a scar was made in the lower labial mucosa using the tip of round burniser in 2 mm diameter which had been heated for ±15 seconds with a bunsen burner. the tip of burnisher then was touched the lower labial mucosa of each wistar rat for one second in 2 mm deep. we spread some gels of 20% benzocaine as a topical anesthesia in the lower labial mucosa of each rat 5 minutes before and after ulcer was made. the observations were performed at the 24th and 48th hours after the scars being made. at the 24th hour observation, a decay in the lower labial mucosa with a thin, white base with 3 mm diameter was visible. in the 48th hours post-trauma, a deep ulcer with a yellowish base in the lower labial mucosa was visible (figure 1). the applications of gels of sodium carboxymethyl cellulose (cmc na) and 1% znso4 were performed after the ulcers were formed in the oral mucosa of the rats. the gels were applied using cotton buds in the morning and the evening until the days when the animals’ tissues would be retrieved, which were the 3rd dan 5th days. the rats did not receive food or drink for 30 minutes after the gel applications. several wistar rats were randomly selected from each group according to the predetermined amount of samples. the selected rats were then terminated on the 3rd and 5th days. their lower lips were cut and used in the tissues processing to produce histological preparations and calculated their macrophages cells. the microscopic preparations were observed using a microscope with a camera ds fi2 300 megapixel (nikon h600l, konan, minota-ku, tokyo, japan), while the tissues were calculated using software (optilab.image raster v21, sleman, yogyakarta, indoneisa). a b figure 1. the illustration of a macroscopic ulcer in the oral mucose of a rat with a scar made by the tip of burnisher in 2mm diameter. in the 24th hours after trauma, a scar of the mucose with a thin, white base in 3mm diameter was observed (a). in the 48th hour post-trauma, a deep ulcer in the mucose with a yellowish base was observed (b). table 1. the average amount of macrophages in the treatment groups on the 3rd and 5th days 3rd day 5th day normal + cmc na 464 144 diabetes + cmc na 352 183 normal + zn so4 414 303 diabetes + zn so4 194 324 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p133-136 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p133-136 135135mujayanto, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 133–136 results the kolmogrov smirnov (one-sample k-s) test showed that the data from each variable of each treatment group on the 3rd and 4th days were normally distributed. the next data testing employed the independent sample t test by comparing the data on the 3rd and 5th days from the normal and diabetic groups with cmc na therapy and from the normal and diabetic groups with znso4 therapy. on the 3rd day, the amount of macrophages of the diabetic group applied with cmc showed a less meaningful distinction from the normal one’s, while on the 5th day, the amount of macrophages showed a meaningful distinction although it was larger than the normal group’s. in the comparison between the amount of macrophages with znso4 and omitted the one with cmc na both in the normal and diabetic conditions, the amount of macrophages on the 5th day increased after the application of znso4. discussion the macrophages in the healing process of ulcers manage inflammatory and angiogenesis processes. the macrophage proliferation responds to pathogen, hemostasis process in tissues, inflammation, resolution, and repair processes. the macrophages remove cytokines and growth factor which influence various cells and induce cytokines (anti-inflammation), glucocorticood, and glucose metabolism and lipid.5,22-24 during an inflammation in normal condition, the macrophages increase shortly between 3rd and 5th days.25 the macrophages coming from monocytes that migrate to the tissues will increase in numbers for two days (48 hours) around an ulcer and stay there for the next five days. once the inflammation can be controlled by the macrophages, the healing process will begin from the 3rd day until the 5th day, marked by the migration and increase of fibroblast, proliferation of endothelial cells and then the emergence of granulation tissue. 2,5,11,26,27 diabetes reduces the amount or quality of insulin receptor or makes it resistant to peripheral tissue, resulting in insulin binding, or afinity, or insulin’s decreased sensitivity.5,7,28 the reduced quality of insulin receptor will disturb the functionality of mitochondria in producing atp as the source of cell energy. atp is a product of glucose metabolism in the intercells which enters through flut-4 in the cell membranes. glut-4 is activated by the signal transduction sent by the insulin receptor which is linked to insulin.28 the disturbed mitochondria may cause disturbances in the neutrophil apoptosis during the autophagocytosis process which is supported by the macrophages, disturbances in the macrophages migration to the tissue, and increase pro-inflammatory cytokine (il-6 dan tnf-α) which is produced by pro-inflammatory macrophages (m1).1,2,4,5,22-24,28,29 a normal person’s body contains 2-3 grams of zinc which influences cell metabolisms. in the cell membranes, there is a transporter in which ionic zinc may enter (zip=zirt-irt-protein) and exit (znt=zinc transport) to maintain the amount of ionic zinc in cytoplasm. ionic zinc inside the cytoplasm serves as a pro-antioxidant which helps zinc superoxide dismutase enzyme to scavenge ros which is a sideline product of glucose metabolism (in the cells).30,31 in a normal condition, the amount of zinc will increase 15-20% in the edges of ulcer on the 24th hour after inflammation phase. during a formation of granulation tissue and epitherial proliferation, the amount of zn will increase up to 30%.8 in diabetic patients, the amount of zn in blood (hypozincemia) decrease which in turn will reduce the amount of zn in the tissues.8,9,11,13,16 during an inflammation in diabetic patients, there is an increase in the amount of neutrophil that results in an imbalance between the amount of neutrophil and of macrophages. in fact, the balance between the two are necessary in the ulcer healing process.2 neutrophil will increase dramatically on the 48th hour of the inflammation26 and increasingly stimulate the amount of macrophages. after that, neutrophil will experience apoptosis.2 deficit figure 2. the average amount of macrophages in the treatment groups on the 3rd and 5th days. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p133-136 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p133-136 136 mujayanto, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 133–136 zinc will influence the amount and function of leukocyte (neutrophil-granulocyte/pmn and monosit) and reduce neutrophil chemotaxis which, in turn, will reduce the performance of phagocytosis.30 therapy on chronic ulcers towards diabetic patients may be performed by modifying the deviating local inflammatory response2 with the use of several drugs which serve as imunomodulator, neuromodulator, and growth factors stimulator, during one or more phases of ulcer healing.32 zinc is one of the topical immunomodulators used to modify local immune responses which may stimulate immune response or suppress immune response.31,33 zinc serves to manage proliferation process, differentiation and cell apoptosis. during an inflammation phase, zinc helps the functions of neutrophil and macrophages. zinc affects the production of pro-inflammatory cytokine (il-1 β, il-6 and tnf-α) which helps chemotaxis process and phagocytosis of neutrophil.8,16,18,19,31,34 ions of topical zinc applied to ulcers will pass epitelium after an hour, enter the sub-epitelium and then be absorbed in the blood circulation. the deeper the damage in epitelium is, the more zinc will enter blood circulation.8 zinc influnces chemokine phorbol myristate acetate (pma) which serves a role in the attachment of monocyte in the endothelial cells and monocyte chemoattractant protein-1 (mcp-1) which serves in the monocyte migration to the tissue, becoming macrophages. zinc in the monocyte-macrophages serves as a pro-inflammation or anti-inflammation, depending on its concentration in the cells,34,35 but not an antioxidant and does not take part in phagocytosis.31 diabetic patients are recommended to use topical zinc application to heal their ulcers.8,11 in conclusion, the amount of macrophages in the diabetic condition was greater than the one in the normal condition microscopically. the application of 1% znso4 increased the amount of macrophages in both normal and diabetic conditions, and clinically the application of 1% znso4 showed a faster ulcer recovery process in the oral mucosa. references 1. le n, rose m, levinson h, klitzman b. implant healing in experimental animal models of diabetes. journal of diabetes science and technology 2011; 5(3): 605-18. 2. larjava h. oral wound healing. chichester, west sussex: john wiley & sons; 2012; p. 39-56. 3. guo s, dipietro l. factors affecting wound healing. journal of dental research 2010; 89(3): 219-29. 4. brancato s, albina j. wound macrophages as key regulators of repair. the american journal of pathology 2011; 178(1): 19-25. 5. cotran r, kumar v, robbins s. pathologic basis of disease. 9th ed. philadelphia: saunders elsevier; 2015. p. 69-120. 6. waugh h, sherratt j. macrophage dynamics in diabetic wound dealing. bulletin of mathematical biology 2006; 68(1): 197-207. 7. tjokroprawiro a, setiawan p, soegiarto g, santoso d. buku ajar ilmu penyakit dalam fakultas kedokteran universitas airlangga rumah sakit pendidikan dr. soetomo. 2nd ed. surabaya: airlangga university press; 2015. p. 29-76. 8. lansdown a, mirastschijski u, stubbs n, scanlon e, ågren m. zinc in wound healing: theoretical, experimental, and clinical aspects. wound repair and regeneration 2007; 15(1): 2-16. 9. rungby j. zinc, zinc transporters and diabetes. diabetologia 2010; 53(8): 1549-51. 10. singh u. zinc in relation to type 1 and type 2 diabetes: an overview. journal of applied and natural science 2014; 6(2): 898-903. 11. chow o, barbul a. immunonutrition: role in wound healing and tissue regeneration. advances in wound care 2014; 3(1): 46-53. 12. khopkar u, pande s, nischal k. handbook of dermatological drug therapy. new delhi: reed elsevier india publications; 2007. p. 198200. 13. shekokar p, kaundinya p. study of serum zinc in diabetes melitus. indian journal of basic & applied medical research 2013; 2(8): 977-83. 14. gupta singh s, pal singh r, kumar gupta s, kalyanwat r. buccal mucosa as a route for drug delivery: mechanism, design and evaluation. research journal of pharmaceutical, biological and chemical sciences 2011; 2(3): 371. 15. derakhshandeh k, abdollahipour r. oral mucoadhesive paste of triamcinolone acetonide and zinc sulfate: preparation and in vitro physicochemical characterization. journal of reports in pharmaceutical sciences 2014; 3(2): 115-25. 16. gupta m, mahajan v, mehta k, chauhan p. zinc therapy in dermatology: a review. dermatology research and practice 2014; 2014: 1-11. 17. altei t. treatment of herpes simplex by zinc sulphate. j college dentistry 2005; 17(1): 54-6. 18. godfrey h, godfrey n, riley d. a randomized clinical trial on the treatment of oral herpes with topical zinc oxide/glycine. altern ther health med 2001; 7(3): 49-56. 19. opstelten w, neven a, eekhof j. treatment and prevention of herpes labialis. canadian family physician 2016; 54(12): 1683-7. 20. akbar m, nuawati d, tantiana. efek pemberian znso4 (zinc sulfat) terhadap percepatan penyembuhan luka pencabutan gigi (penelitian eksperimental laboratorik pada tikus wistar. skripsi. universitas airlangga; 2011. 40. 21. purwanto b, liben p. model hewan coba untuk penelitian diabetes. surabaya: pt. revka putra media; 2015. p. 4-20. 22. koh t, dipietro l. inflammation and wound healing: the role of the macrophage. expert rev mol med 2011; 13. 23. martinez f. regulators of macrophage activation. european journal of immunology. 2011; 41(6): 1531-4. 24. ferrante c, leibovich s. regulation of macrophage polarization and wound healing. advances in wound care 2012; 1(1): 10-6. 25. liddiard k, rosas m, davies l, jones s, taylor p. macrophage heterogeneity and acute inf lammation. european journal of immunology 2011; 41(9): 2503-8. 26. nanci a, ten cate a. ten cate’s oral histology. 8th ed. st. louis: mosby inc; 2013. 27. nauta t, van hinsbergh v, koolwijk p. hypoxic signaling during tissue repair and regenerative medicine. ijms 2014; 15(11): 19791815. 28. gilkerson r, materon l. two roads converging: mitochondria and inflammatory signaling. j clin immunol immunother 2014; 1(1): 1-7. 29. xu f, zhang c, graves d. abnormal cell responses and role of tnf in impaired diabetic wound healing. biomed research international 2013; 2013: 1-9. 30. haase h, rink l. zinc signals and immune function. biofactors 2013; 40(1): 27-40. 31. bonaventura p, benedetti g, albarède f, miossec p. zinc and its role in immunity and inflammation. autoimmunity reviews 2015; 14(4): 277-85. 32. pradhan l, andersen n, logerfo f, veves a. molecular targets for promoting wound healing in diabetes. recent patents on endocrine, metabolic & immune drug discovery 2007; 1(1): 1-13. 33. khandpur s, sharma v, sumanth k. topical immunomodulators in dermatology. j postgrad med june 2004; 50(2): 131-9. 34. kaplanski g. il-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. trends in immunology 2003; 24(1): 25-9. 35. haase h, rink l. signal transduction in monocytes: the role of zinc ions. biometals 2007; 20(3-4): 579-85. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p133-136 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p133-136 148148 dental journal (majalah kedokteran gigi) 2022 september; 55(3): 148–153 original article labial and palatal alveolar bone changes during maxillary incisor retraction at the universitas sumatera utara dental hospital suci purnama sari, mimi marina lubis, muslim yusuf department of orthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: the fundamental concept of tooth movement during orthodontic treatment is the occurrence of bone remodelling accompanied by tooth movement in equal proportions. the thickness of the alveolar bone, which supports incisors, is important in estimating the direction of tooth movement. purpose: the study aimed to measure labial and palatal alveolar bone thickness changes after maxillary incisor retraction using lateral cephalograms. methods: cephalograms of 40 patients (18.58 ± 4.2 years) with skeletal class i bimaxillary protrusion after maxillary first premolar extraction for insisivus retraction had been taken before (t0) and after (t1) orthodontic treatment. changes in alveolar bone thickness were measured in linear and angular directions and then analysed with spearman correlative analysis. then the samples were separated into two groups based on the type of tooth movement (tipping and torque), and then the data were analysed using wilcoxon analysis to see differences in the bone thickness (p<0.05). results: there was a significant difference in the apical palate (p<0.05) and a relationship between retraction and alveolar bone thickness in the midroot area. in the angular direction, there was no significant difference and relationship; however, there was a significant difference in the labial crestal in the tipping group. in the torque group, the difference in bone thickness occurred in the crestal and apical palatal areas. conclusion: the retraction and the type of tooth movement difference influence the alveolar bone thickness. keywords: alveolar bone thickness; retraction; tipping; torque correspondence: suci purnama sari, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: drgsuciig@gmail.com introduction the reaction of periodontal tissue to orthodontic tooth movement is influenced by several factors: bone thickness, root height and morphology, bone dimensions, tooth angulation, and tooth position. orthodontic treatment not only produces an esthetic facial and dental profile but also carries the risk of complications in the periodontal. the incisors may move labio-lingually/palatally due to compensatory or decompensated dental forces during orthodontic treatment. the thickness of the alveolar bone which supports the incisors is an important consideration in estimating the direction of tooth movement. 1–3 several previous studies stated alveolar bone loss was more common in extraction cases.3–9 sarikaya et al.2 investigated changes in alveolar bone thickness in retracted anterior teeth. on the labial side, there was no significant change in bone thickness, while on the palatal side, there was a reduction in bone thickness at the boundary between the cej to the middle of the tooth root.2 yodthong et al.3 investigated the factors influencing alveolar bone thickness in the maxillary incisor retraction. after retraction, the thickness of the labial and apical bones showed a critical increment of remodelling. the massive contrast in the tipping group was in the crestal labial and apical palatal; the torque group obtained the same results. the outcomes confirmed the thickness of the alveolar in the incisors during retraction could be influenced by various tipping and torque movement of the teeth and the intrusion or extrusion of the teeth.3 nayak et al.9 investigated the thickness of the alveolar bone during anterior tooth retraction with premolar extraction for the presence of dehiscence and fenestration. there was no significant remodelling in the maxillary incisor labial area, while significant changes in the palatal area occurred in the crestal and apical regions.9 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 mailto:drgsuciig@gmail.com https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 149 sari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 148–153 several methods are used to detect bone thickness. using three-dimensional ct is more accurate in measuring levels of bone thickness. however, two-dimensional radiography is more practical and is most often used in daily practice with lower radiation levels despite some drawbacks such as superimposition or distortion.10,11 the objectives of this research were: 1. to assess changes of alveolar bone in linear and angular direction after maxillary incisors retraction; 2. to determine the correlation between alveolar bone thickness and the average of retraction on maxillary incisors; 3. to determine the differences in changes of bone thickness based on the type of tooth movement after the retraction of maxillary incisors. material and methods this cross-sectional review was supported by the ethical committee of universitas sumatera utara number 10/ kep/usu/2022. lateral cephalograms were obtained from the patient’s records in the orthodontics department, faculty of dentistry, universitas sumatera utara, medan, indonesia. the 40 subjects (21 females and 19 males) were selected based on the inclusion criteria. subjects aged ≥18-40 years with skeletal class i bimaxillary protrusion (anb = 2° ± 2; mean age = 18.58 ± 4.2 years; treatment period = 28.81 ± 5.77 months; anb = 2.23 ± 1.03°), medical records, and cephalograms before and after treatment were complete. excluded patients included those with a crowding discrepancy over 3 mm, those under the influence of non-steroidal, anti-inflammatory and metabolic drugs before or during orthodontic treatment, and those with periodontal or gingival disease. after the extraction of two maxillary premolars, patients were treated with the edgewise technique using closed helical loops for anterior retraction. cephalogram measurements were performed on three labial and palatal areas. ‘a single examiner re-examined cephalogram measurements at four weeks. image data of lateral cephalograms were taken at the pretreatment (t0), and posttreatment (t1) were imported into imagej software 1.52a (2018) for analysis (figure 1). the measurement variables used in this study were adjusted from a previous review.3,5 first, the amount of incisor retraction pre-and posttreatment was calculated by the distance tip of the central maxillary incisor (u1) to the n-perpendicular line (mm) of the frankfurt horizontal plane (figure 2). second, the linear measurements were taken on the crestal (3 mm), mid-root (6 mm), and apical (9 mm) from cej to apex labial and palatal maxillary incisor. they were categorised as labial pretreatment (l1a, l2a, l3a), labial posttreatment (l1b, l2b, l3b), palatal pretreatment (p1a, p2a, p3a), and palatal posttreatment (p1b, p2b, p3b). for accuracy, the distance was measured using imagej software by triple magnification (figure 3). third, the angular measurements used the point between a. u1the superficial labial line of maxillary central incisor and alveolar bone; b. u1the palatal superficial line of maxillary central incisor and alveolar bone (figure 4). a paired hypothesis test formula separated the samples into two groups (tipping and torque), including the 20 samples in each group. referring to the previous study,3 in the tipping group, the apex of the maxillary incisor moved anteriorly. in contrast, in the torque group, the apex of the maxillary incisor moved posteriorly in a superimposed pre-and posttreatment position. the data will be analysed using the shapiro wilk test to see its normality. the wilcoxon test used comparative analysis to examine differences in bone thickness preand post-treatment. spearman’s correlative analysis was used to investigate the relationship of the alveolar bone remodelling with other related variables. statistical analysis was conducted using spss version 26 (ibm, usa). figure 1. landmarks of reference lines point to a measured amount of retraction. figure 2. l a t e r a l c e p h a l o g r a m a n a l y s e d w i t h i m a g e j software. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 150sari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 148–153 table 1. comparative analysis of labial and palatal alveolar bone in a linear direction (mm) pre-and post-treatment (paired t-test) variable pretreatment t0 (n=40) mean±sd posttreatment t1(n=40) mean±sd mean change (∆) p l1 l1a 1.352 ± 0.392 l1b 1.477 ± 0.459 ∆l1 0.125 ± 0.070 0.591 l2 l2a 1.283 ± 0.436 l2b 1.327 ± 0.555 ∆l2 0.044 ± 0.119 0.681 l3 l3a 1.506 ± 0.646 l3b 1.477 ± 0.885 ∆l3 -0.029 ± 0.239 0.681 p1 p1a 2.572 ± 0.864 p1b 2.492 ± 0.665 ∆p1 -0.008 ± 0.199 0.621 p2 p2a 3.446 ± 1.012 p2b 3.578 ± 0.934 ∆p2 0.123 ± 0.078 0.480 p3 p3a 4.434 ±1.145 p3b 4.963 ± 1.391 ∆p3 0.529 ± 0.246 0.032* *wilcoxon; p<0.05; (-) in terms of reduced alveolar bone table 2. comparative analysis of labial and palatal alveolar bone in angular direction (mm) pre-and post-treatment (paired t-test) variable pretreatment t0(n=40) mean±sd posttreatment t1(n=40) mean±sd mean change (∆) p lab (⁰) θla 9.304 ± 3.882 θlb 11.362 ± 6.118 ∆θl 2.058 ± 2.336 0.081 pal (⁰) θpa 28.939 ± 7.606 θlb 28.563 ± 3.901 ∆θp 4.806 ± 0.253 0.882 *wilcoxon; p<0.05 table 3. correlation analysis of the changes in maxillary alveolar bone and average retraction variable n average of retraction (mm) r p l1 40 0.222 0.168 l2 0.394 0.012* l3 0.284 0.075 labial (⁰) 0.044 0.786 p1 0.167 0.303 p2 -0.050 0.761 p3 -0.153 0.345 palatal (⁰) -0.213 0.188 *spearman; p<0.05 table 4. comparative analysis of the changes in maxillary alveolar bone in a linear direction (mm) pre-and post-treatment (paired t-test) variable tipping (n = 20) (∆) mean ±sd p torque (n = 20) (∆) mean ±sd p l1 0.082 ± 0.521 0.023* 0.021 ± 0.535 0.717 l2 -0.469 ± 0.566 0.715 -0.042 ± 0.799 0.984 l3 -0.281 ± 0.996 0.235 1.500 ± 0.506 0.194 p1 -0.461 ± 1.031 0.600 -0.295 ± 0.950 0.021* p2 0.103 ± 1.163 0.696 0.367 ± 1.157 0.208 p3 0.545 ± 1.510 0.123 0.512 ± 1.527 0.045* *wilcoxon; p<0.05; (-) in terms of reduced alveolar bone figure 3. measurement of alveolar bone thickness at 3 mm intervals from the cementoenamel junction. figure 4. the angle between a) u1 (maxillary central incisor) – superficial labial line of maxillary central incisor and alveolar bone; b. u1 – palatal superficial line of maxillary central incisor and alveolar bone. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 151 sari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 148–153 results the average retraction pre-and post-treatment was 4.806 ± 0.253 mm. table 1 showed statistically significant differences in alveolar bone thickness in a linear direction at the p3 position pre-and post-treatment. the lowest mean labial alveolar bone thickness was found at l2a (1.283 ± 0.436 mm) and p1b palate (2.492 ± 0.665 mm), while the highest mean labial and palatal alveolar bone thickness were found at l3a (1.506 ± 0.646 mm) and p3b (4.956 ± 1.391 mm). the changes in the thickness of the labial and palatal alveolar bones in a linear direction were lowest at positions l3 (0.029 ± 0.246 mm) and p1 (0.008 ± 0.199 mm), while the greatest thickness changes were found at positions l1 (0.125 ± 0.070 mm) and p3 (0.529 mm). ± 0.246 mm). table 2 shows the differences in the thickness of the labial and palatal alveolar bones in the angular direction. there was no significant difference in the retraction of maxillary incisors teeth pre-and post-treatment on the thickness of the labial and palatal alveolar bone in the angular direction. the average increase in the alveolar bone’s thickness occurred in the labial area at 2.058 ± 2.336, while the decrease in the alveolar bone’s thickness occurred on the palate, which was 4.806 ± 0.253. table 3 shows a correlation between the thickness of the alveolar bone in a linear direction at the l2 position. however, no correlation was found between the alveolar bone’s thickness and the magnitude of retraction in an angular direction. table 4 shows the results of statistical calculations in the tipping and torque group. there was a reduction in alveolar bone thickness, the lowest was at l3 (-0.281 ± 0.996 mm), and the highest was at l2 (-0.469 ± 0.566 mm). the highest increase in bone thickness was at the p3 level (0.545 ± 1.510 mm) and the lowest at l1 (0.082 ± 0.521 mm), while a significant difference in mean bone thickness was found at l1 (crestal) (p < 0.05) in the tipping group. as a result of changes in bone thickness in the torque group, there was a decrease in the average bone thickness with the lowest l2 (-0.042 ± 0.799 mm) and the highest result in the p1 area (-0.295 ± 0.950 mm), the lowest increase in bone thickness was at l1 (0.021 ± 0.535 mm), and the highest was at l3 (1.500 ± 0.506 mm) and a significant difference in p1 (crestal) and p3 (apical) bone thickness pre-and post-treatment (p < 0.05). discussion the occurrence of bone resorption on the stress side and bone apposition on the strain side is a process in tooth movement during orthodontic treatment. tooth movement is directly proportional to bone remodelling, but in some studies, this has not been proven, especially in the retraction of anterior maxillary teeth. many studies show the factors affecting the thickness of alveolar bone, especially those related to anterior tooth retraction.3–9 previous studies have generally described the thickness of the alveolar bone with varying results.2–6,11–17 however, none of the earlier studies used a sample of a skeletal class i malocclusion and a comparison based on the type of tooth movement tipping and torque – if any. previous studies used different techniques and mechanics from this research and generally used a skeletal class ii sample, which requires a larger number of retractions.2–6,11,14,18,19 in addition, the mechanics of retraction using closing loops in the edgewise technique is still common and is often used for incisor retraction cases. as we have seen, the type of retraction mechanics and the treatment technique can also affect the thickness of the alveolar bone. this study can also increase clinician awareness of the direction of anterior retraction to reduce the risk of fenestration or dehiscence in the alveolar bone.16,20,21 tables 1 and 2 show the difference and mean alveolar bone thickness of the maxillary labial and palatal incisors in linear and angular directions pre-and post-treatment. the maxillary incisor labial alveolar bone thickness increased in the crestal and mid-root areas after anterior retraction, while the apical labial bone thickness decreased. the maxillary mid-root and apical palatal alveolar bone thickness increased after anterior retraction, while the palatal crestal thickness decreased. the tables shows the results of statistical tests with a significant difference (p<0.05) at the apical palatal level (9 mm from the cej), namely a decrease in bone thickness of 0.5 mm in the apical palatal area, while bone thickness in the angular direction in labial and palatal before and after treatment (θ, p<0.05) were not significantly different. this study’s results agree with sarikaya et al.2, who investigated linear changes in alveolar bone thickness in the maxillary incisors’ crestal, mid-root, and apical regions. this study was conducted on 19 patient cases of premolar extraction in bimaxillary protrusion. after canine distalisation and continued incisor retraction for three months, the bone thickness significantly decreased in the mid-root area, and the bone thickness reduction was also greater in the apical palatal region (9 mm from the cej).2 the results of this study differ from aakash et al.16, who investigated the effect of retraction on changes in alveolar bone thickness with sliding mechanics using a mini-implant in the case of bimaxillary protrusion. the number of retractions before and after treatment was measured in 15 samples, and the results showed a significant increase in alveolar bone thickness (p > 0.05) in the maxilla’s crestal and apical labial areas. alveolar bone thickness changes are significantly affected by retraction.16 the decrease in bone thickness in the palatal crestal area in this study did not occur in nayak et al.9, which studied ten samples with bimaxillary protrusion cases. after three months of retraction using sliding mechanics, there was a decrease in bone thickness in the labial crestal area, which was due to the concentrated force on the alveolar crest.9 a study on changes in palatal bone thickness linearly and angularly with different results was conducted by son et al.5. the study included 33 samples with sliding mechanics dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 152sari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 148–153 using mini implants. linear measurement of palatal alveolar bone thickness was performed at a distance of 2,4,8,10 mm from the cej boundary, showing a significant reduction in bone thickness in the entire palatal area of the central incisor (p<0.001), except at a distance of 10 mm from the cej (apical). the labial side showed a significant difference from the palatal area in the angular direction, which showed a close relationship to the inclination of the tooth after retraction. differences in alveolar bone thickness pre-and post-treatment were also significantly correlated with maxillary incisor retraction. changes in bone thickness in the crestal region indicated excessive resorption in the cervical region of the teeth, but a significant ratio to the number of retractions in this study was not found.5 different results in the study by mao et al.14 showed a significant increase in the thickness of the labial alveolar bone (p<0.05) in the mid-root area. whereas in the palate, it occurred in the crestal area. the results of this study explain excessive incisor retraction can increase the thickness of the alveolar bone in the crestal and mid-root sections. alveolar bone thickness can be maintained if the tooth movement is not too fast, and the force is not excessive. the labial area has a resorption rate which tends to be lower than the palatal area, so the tendency for bone prominence in the labial area can occur more easily.14 table 4 shows a significant correlation between changes in bone thickness and the number of retractions in the mid-root area (l2 r = 0.394, p = 0.012). this result differs from the study by yodthong et al.3 and aakash et al.9, which showed a significant relationship in the crestal area. the difference in the results of this study may be due to the varying magnitude of the incisor retraction force. differences in treatment mechanics, changes in inclination, and intrusion size are also the cause of the incompatibility of bone remodelling and retraction processes.3,16 changes in alveolar bone thickness in the apical region were associated with changes in the inclination and intrusion of the incisors. the inclination position of the anterior teeth plays a vital role in the function and stability of the treatment. based on several previous studies, orthodontic treatment with extraction has the effect of root resorption, and bone loss is greater than the case without extraction.3 significant changes in bone thickness were found in the labial crestal area of the tipping group after treatment (table 4) (l1 p = 0.023; p<0.05), while in the torque group, the alveolar bone thickness in the crestal area was not significantly different. significant differences in bone thickness in the torque group occurred in the mid-root and apical palatal regions (table 4) (p1 = 0.021 and p3 = 0.045; p<0.05). these results may be due to differences in the type of movement, the direction of angulation, and the magnitude of the retraction force applied to the alveolar bone.3 alveolar bone loss visible in this study was in the apical labial and palatal crest regions. this bone loss could be due to changes in the angulation of tooth movement. the retraction force applied to the incisors concentrates more on the alveolar crest, increasing the cervical region’s force. the significant decrease in bone thickness was due to the periodontal tissues’ reaction centred in the anterior teeth’ cervical area. periodontal tissue reaction is also a factor causing variations in bone reaction to orthodontic forces. it depends on the width, height and morphology of the roots, angulation, and position of the teeth, dimensions of the teeth to the alveolar bone, bone anatomy, physiology and adaptability of the patient. the average decrease in bone thickness in the labial aspect in this study was statistically higher than in the palatal aspect. it could be due to a slower bone deposition process in the strain area compared to the stress area’s resorption process. the results of this study are from research by sarikaya et al.2 and ahn et al.22 this study concluded a significant difference in the apical palate (p<0.05) and a relationship between retraction and alveolar bone thickness in the mid-root area, while in the angular direction, there was no significant difference and relationship. there was a significant difference in bone thickness in the labial crestal on tipping. in the torque group, the difference in bone thickness occurred in the crestal and apical palatal areas. the retraction and the type of tooth movement influence the difference in the thickness of the alveolar bone. increased awareness of the direction of anterior retraction in the tipping type of tooth movement can reduce the risk of fenestration and dehiscence of the root tip in a labial direction. in contrast, the type of torque movement must be aware of the direction of movement of 2/3 of the tooth root against the palatal cortical plate for treatment stability. references 1. lin jc-y, yeh c-l, liou ej-w, bowman sj. treatment of skeletalorigin gummy smiles with miniscrew anchorage. j clin orthod. 2008; 42(5): 285–96. 2. sarikaya s, haydar b, ciğer s, ariyürek m. changes in alveolar bone thickness due to retraction of anterior teeth. am j orthod dentofacial orthop. 2002; 122(1): 15–26. 3. yodthong n, charoemratrote c, leethanakul c. factors related to alveolar bone thickness during upper incisor retraction. angle orthod. 2013; 83(3): 394–401. 4. tian y-l, liu f, sun h-j, lv p, cao y-m, yu m, yue y. alveolar bone thickness around maxillary central incisors of different inclination assessed with cone-beam computed tomography. korean j orthod. 2015; 45(5): 245–52. 5. son ej, kim sj, hong c, chan v, sim hy, ji s, hong sy, baik u-b, shin jw, kim yh, chae hs. a study on the morphologic change of palatal alveolar bone shape after intrusion and retraction of maxillary incisors. sci rep. 2020; 10(1): 14454. 6. zhang f, lee s-c, lee j-b, lee k-m. geometric analysis of alveolar bone around the incisors after anterior retraction following premolar extraction. angle orthod. 2020; 90(2): 173–80. 7. domingo-clérigues m, montiel-company j-m, almerich-silla j-m, garcía-sanz v, paredes-gallardo v, bellot-arcís c. changes in the alveolar bone thickness of maxillary incisors after orthodontic treatment involving extractions a systematic review and metaanalysis. j clin exp dent. 2019; 11(1): e76–84. 8. morais jf, melsen b, de freitas kms, castello branco n, garib dg, cattaneo pm. evaluation of maxillary buccal alveolar bone before dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 153 sari et al./dent. j. (majalah kedokteran gigi) 2022 september; 55(3): 148–153 and after orthodontic alignment without extractions: a cone beam computed tomographic study. angle orthod. 2018; 88(6): 748–56. 9. nayak krishna us, shetty a, girija mp, nayak r. changes in alveolar bone thickness due to retraction of anterior teeth during orthodontic treatment: a cephalometric and computed tomography comparative study. indian j dent res. 2013; 24(6): 736–41. 10. helal nm, basri oa, baeshen ha. significance of cephalometric radiograph in orthodontic treatment plan decision. j contemp dent pract. 2019; 20(7): 789–93. 11. picanço prb, valarelli fp, cançado rh, de freitas kms, picanço gv. comparison of the changes of alveolar bone thickness in maxillary incisor area in extraction and non-extraction cases: computerized tomography evaluation. dental press j orthod. 2013; 18(5): 91–8. 12. wei d, zhang l, li w, jia y. quantitative comparison of cephalogram and cone-beam computed tomography in the evaluation of alveolar bone thickness of maxillary incisors. turkish j orthod. 2020; 33(2): 85–91. 13. pudyani ps, sutantyo d, suparwitri s. morphological changes of alveolar bone due to orthodontic movement of maxillary and mandibulary incisors. dent j (majalah kedokt gigi). 2008; 41(1): 21. 14. mao h, yang a, pan y, li h, lei l. displacement in root apex and changes in incisor inclination affect alveolar bone remodeling in adult bimaxillary protrusion patients: a retrospective study. head face med. 2020; 16(1): 29. 15. hong sy, shin jw, hong c, chan v, baik u-b, kim yh, chae hs. alveolar bone remodeling during maxillary incisor intrusion and retraction. prog orthod. 2019; 20(1): 47. 16. aakash s, purvesh s, kumar gs, romina k, romil s, bhumi m. changes in alveolar bone thickness during upper incisor retraction. rev latinoam ortod y odontopediatría. 2017; : 19. 17. chen ssh, greenlee gm, kim je, smith cl, huang gj. systematic review of self-ligating brackets. am j orthod dentofac orthop. 2010; 137(6): 726.e1-726.e18. 18. oliveira tmf, claudino lv, mattos ct, sant’anna ef. maxillary dentoalveolar assessment following retraction of maxillary incisors: a preliminary study. dental press j orthod. 2016; 21(5): 82–9. 19. thongudomporn u, charoemratrote c, jearapongpakorn s. changes of anterior maxillary alveolar bone thickness following incisor proclination and extrusion. angle orthod. 2015; 85(4): 549–54. 20. masumoto t, hayashi i, kawamura a, tanaka k, kasai k. relationships among facial type, buccolingual molar inclination, and cortical bone thickness of the mandible. eur j orthod. 2001; 23(1): 15–23. 21. al-nimri ks, hazza’a am, al-omari rm. maxillary incisor proclination effect on the position of point a in class ii division 2 malocclusion. angle orthod. 2009; 79(5): 880–4. 22. ahn hw, moon sc, baek sh. morphometric evaluation of changes in the alveolar bone and roots of the maxillary anterior teeth before and after en masse retraction using cone-beam computed tomography. angle orthod. 2013; 83(2): 212–21. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i3.p148–153 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i3.p148-153 40 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 40–44 research report effective dose of propolis extract combined with bovine bone graft on the number of osteoblasts and osteoclasts in tooth extraction socket preservation teguh setio yuli prabowo, utari kresnoadi and hanoem eka hidayati department of prosthodontics, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: maintaining a good ridge is required during prosthodontic treatment. hence, adequate alveolar bone support is considered an important factor in pursuing successful dentures. propolis extract combined with bovine bone graft is a recent and innovative material in the process of socket preservation, as the caffeic acid phenethyl ester (cape) it contains can suppress the inflammatory process. purpose: this study aims to determine the effective dose of propolis extract combined with bovine bone graft on the number of osteoblasts and osteoclasts in socket preservation. methods: twenty-eight cavia cobaya animals were divided into four groups of seven. group i was given 25 grams of peg, while group ii were given a propolis extract at a dose of 0.5% combined with bovine bone graft. group iii were given a propolis extract at a dose of 1% combined with bovine bone graft and group iv were given a propolis extract at a dose of 2% combined with bovine bone graft. on day 30, the lower incisor of each subject was extracted and induced with peg and propolis (dose 0.5%, 1%, 2%). histopathological examinations of osteoblasts and osteoclasts were measured with a 400x magnification light microscope. one-way anova and tukey hsd tests were performed to analyse data statistically. results: the propolis extract combined with bovine bone graft not only increased the number of osteoblasts but also reduced the number of osteoclasts. the most effective dose for the propolis extract combined with bovine bone graft was 2%. conclusion: the propolis extract combined with bovine bone graft could be effective in tooth extraction socket preservation at a dose of 2%. keywords: bovine bone graft; effective dose; osteoblasts; osteoclasts; propolis extract correspondence: utari kresnoadi, department prosthodontics, faculty of dental medicine universitas airlangga. jl. mayjend. prof. dr. moestopo, 47 surabaya 60132, indonesia. e-mail: utari-k@fkg.unair.ac.id introduction dental and oral diseases are among the top-ten diseases in indonesia. the significant number of dental disease cases also has an impact on tooth decay, which is a major cause of tooth-loss in the indonesian population.1 moreover, this also leads to problems in subsequent dental treatment and has an impact on the tooth-supporting tissue and alveolar bone. alveolar bone plays an important role in obtaining ideal prosthetic reconstruction.2 damage to bone tissue, as a result of tooth extraction, can cause atrophy of alveolar bone and the healing process can result in bone deformity. in other words, alveolar bone needs to be maintained during the healing process.3 maintaining alveolar bone has been a focus of recent studies and one of the leading materials used to maintain alveolar bone is graft. graft is a material used to support bone regeneration, reconstructing alveolar bone by filling the tooth extraction socket to maintain the height and width of the alveolar ridge.4 the formation of new bone from graft material is very time-dependent, therefore, it requires a certain level of material innovation to stimulate the graft and accelerate bone formation.5 in addition to bone graft, there are also several other studies arguing that propolis can accelerate the bone remodelling process. propolis possesses anti-inflammatory activity containing caffeic acid phenethyl ester (cape). cape inhibits receptor activator of nuclear factor kappa-b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p40–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p40-44 41prabowo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 40–44 ligand (rankl) induced through the activity of nuclear factor kappa beta (nf-κb) during the osteoclast formation process.6 hence, this study aims to reveal the effects of propolis extract combined with bovine bone graft on tooth extraction socket preservation, through experimental animal subjects (cavia cobaya), to accelerate bone formation. materials and methods this study is experimental research with a randomized post-test control group. research design has been approved by the ethical clearance team, number: 595/hrecc. fodm/ix/2019. the research population of 28 male cavia cobaya, aged 3-3.5 months and between 300-350 g in weight, were divided into four groups of seven subjects. the cavia cobaya were anesthetized intravenously with ketamine, at a dose of 0.1 cc/300 g bb. afterwards, their tooth was extracted with a needle holder and given a propolis extract combined with bovine bone graft, as much as 0.1 cc, according to the volume of the tooth extraction socket, and sewn. subsequently, they were divided into 4 groups.7 in group i, the extracted tooth sockets of those cavia cobaya animals were given 24 g of peg, as much as 0.1 cc (as control group). in group ii, the extracted tooth sockets were given 0.1 cc of the combination of 0.5 g of propolis, 0.5 g of bovine bone graft, and 99 g of peg, at a dose of 0.5%. in group iii, the extracted tooth sockets were given 0.1 cc of the combination of 0.5 g of propolis, 0.5 g of bovine bone graft, and 49 g of peg, at a dose of 1%. in the final group, group iv, the extracted tooth sockets were given 0.1 cc of the combination of 0.5 g of propolis, 0.5 g of bovine bone graft, and 24 g of peg, at a dose of 2%. after 30 days, the cavia cobaya test subjects were sacrificed, and their jaw decalcified with edta for a month. next, a paraffin block was prepared for each one, manufactured and cut to a thickness of 4μ with a rotary microtome, before being deparaffinized through dissolution in xylol for two intervals of 3 minutes. the residual xylol was respectively washed with 99%, 95%, 90%, 80%, and 70% absolute alcohol for two intervals of 1 minute. any residual alcohol was removed with running water. at this point, haematoxylin eosin (he) staining was performed for 30 seconds before rinsing with water. staining with he was conducted for 1–2 minutes prior to washing with 70%, 80%, 90%, 95%, and 99% absolute alcohol for two intervals of 1 minutes. observation was subsequently carried out under a light microscope; each slide being examined at 400x magnification and a maximum of 8 fields of view (fov). the calculation results were recorded on a worksheet with a mean value per fov. at this point, the quantity of osteoblasts and osteoclasts was calculated.7 the data obtained was statistically analysed using the one-way anova test to observe differences in each group. if the data in each group proved significantly different, the data was then analysed using the hsd test. results the results of this study showed an increase in the number of osteoblast cells in all the treatment groups (figure 1 and 2). the results also showed a decrease in the number of osteoclast cells in all the treatment groups (figure 1 and 3). a kolmogorov-smirnov normality test was conducted on the results of the statistical analysis on the number of osteoblasts. in this research, all the research groups had a p value greater than 0.05, signifying that the data derived from all the test subjects (table 1). moreover, based on 0 5 10 15 20 25 30 35 group i group ii group iii group iv osteoblast osteoclast figure 1. the diagram of the average number of osteoblasts and osteoclasts at a dose of 0.5%, 1%, and 2%. note: group i: peg only (control group); group ii: the combination of propolis extract and bovine bone graft at a dose of 0.5%; group iii: the combination of propolis extract and bovine bone graft at a dose of 1%; group iv: the combination of propolis extract and bovine bone graft at a dose of 2%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p40–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p40-44 42 prabowo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 40–44 table 1. statistical analysis data (means, standard deviation, and normality) of the quantity of osteoblasts and osteoclasts in each group group mean standard deviation normality test osteoblast i 11.86 1.345 0.508 ii 15.03 1.254 0.674 iii 19.00 2.380 0.900 iv 26.56 1.345 0.789 osteoclast i 15.71 1.799 0.680 ii 12.36 1.345 0.508 iii 11.14 1.952 0.738 iv 7.18 1.799 0.680 note: normality test score of p>0.05 means the data follows normal distribution; group i: peg only (control group); group ii: the combination of propolis extract and bovine bone graft at a dose of 0.5%; group iii : the combination of propolis extract and bovine bone graft at a dose of 1%; group iv: the combination of propolis extract and bovine bone graft at a dose of 2%. group i group ii group iii group iv figure 2. black arrows indicate histopathological staining identifying osteoblasts (he staining observed through a light microscope at a magnification of 400x). group i: control group (peg); group ii: the combination propolis extract and bovine bone graft at a dose of 0.5%; group iii: the combination propolis extract and bovine bone graft at a dose of 1%; group iv: the combination propolis extract and bovine bone graft at a dose of 2%. group i group ii group iii group iv figure 3. black arrows indicate histopathological staining identifying osteoclasts (he staining observed through a light microscope at a magnification of 400x). group i: control group (peg); group ii: the combination propolis extract and bovine bone graft at a dose of 0.5%; group iii: the combination propolis extract and bovine bone graft at a dose of 1%; group iv: the combination propolis extract and bovine bone graft at a dose of 2%. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p40–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p40-44 43prabowo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 40–44 table 2. statistical analysis data tukey hsd test relating to the quantity of osteoblasts group tukey hsd test i ii iii iv osteoblast i * * * ii * * iii * iv note*: significant table 3. statistical analysis data tukey hsd test relating to the quantity of osteoclasts group tukey hsd test i ii iii iv osteoclast i * * * ii .868 * iii * iv note *: significant the results of the one-way anova test, conducted on both the between groups as well as within the treatment groups, the data obtained was significant, with a value of 0.000 (α<0.05). the results of the hsd test (table 2) showed that there was a significant difference between groups i and ii, with α value of 0.017 (α<0.05). similarly, there were also significant differences between groups i and iii, with α value of 0.000 (α<0.05); groups i and iv, with α value of 0.000 (α<0.05); group ii and group iii, with α value of 0.000 (α <0.05); groups ii and iv, with α value of 0.000 (α <0.05); and group iii and group iv with α value of 0.000 (α<0.05). next, the results of the statistical analysis on the number of osteoclasts. a kolmogorov-smirnov normality test was conducted. in this research, all the research groups had p value greater than 0.05 signifying that data derived from all (table 1). moreover, based on the results of the oneway anova test conducted on both between groups as well as within the treatment groups, the data obtained were significant with value of 0.000 (α<0.05). based on the results of the hsd test (table 3), there was a significant difference between group i and group ii, with α value of 0.002 (α<0.05), and between group i and group iii, with α value of 0.000 (α<0.05). there were also significant differences between groups i and iv, with α value of 0.000 (α<0.05), as well as between group ii and group iv, with α value of 0.000 (α<0.05), and between group iii and group iv, with α value of 0.000 (α <0.05). however, unlike the previous results, there was no significant difference between groups ii and iii, with α value of 0.868 (α<0.05), as the difference concentration was slightly than the others. dicussion the purpose of this study is to determine the effective dose of propolis extract combined with bovine bone graft in socket preservation after 30 days. the results revealed that the combination of propolis extract (a dose of 0.5%, 1%, and 2%) and bovine bone graft can effectively affect the formation of alveolar bone during tooth extraction socket preservation after 30 days. moreover, the results from this study also reveal that the combination of propolis extract (with dose of 5%, 1%, and 2%) and bovine bone graft also generates more osteoblasts than osteoclasts. we found the highest increased number of osteoblasts was in the treatment group treated with the propolis extract combined with bovine bone graft at a dose of 2% (group iv), compared to the other test groups (group i as the control, group ii at a dose of 0.5%, and group iii at a dose of 1%). meanwhile, the lowest number of osteoclasts was also found in the treatment group treated with the propolis extract combined with bovine bone graft at the dose of 2% (group iv), compared to the other test groups (group i as the control, group ii at a dose of 0.5%, and group iii at a dose of 1%). we discovered that the effective dose of propolis extract combined with bovine bone graft to increase the number of osteoblasts and decrease the number of osteoclasts in socket preservation is 2% (group iv). this endorses a previous study which argues that propolis combined with graft can potentially be used for alveolar bone regeneration during socket preservation.8 propolis extract is known to contain antioxidants that can increase alveolar bone density, as well as accelerate the bone formation process. moreover, cape contained in propolis extract is also known to have strong properties that support the growth and development of human bones, as well as activating osteoblast progenitor cells to increase collagen formation. consequently, propolis extract can also inhibit the formation and maturation of osteoclasts.6,9 cape is a natural nf-κb inhibitor derived from propolis; the inhibition of nf-κb and nuclear factor from t-cell activation (nfat), triggered by cape, can result in weakening osteoclastogenesis. in other words, cape can suppress osteoclastogenesis and bone loss through the inhibition of mitogen-activated protein kinase (mapk), induced by mitogen-rankl.10,11 moreover, the mechanism of inhibiting osteoclastogenesis through cape involves the inhibition of dna binding activity and nf-κb transcription, therefore, cape can directly inhibit the binding of nf-κb dna in osteoclast precursors by breaking down its mechanism. cape reduces the expression of nfat and c-fos after stimulation of rankl, derived from osteoclast precursors.12 in inhibiting the binding activity of nf-κb dna, cape can not only reduce the transcription of c-fos by nf-κb, but can also interfere with or damage the induction of nfat by c-fos during osteoclastogenesis. cape also inhibits m-csf and rankl, which induce osteoclast differentiation. thus, cape is considered a potential therapeutic agent for the inhibition of bone resorption triggered by osteoclasts, as well as in the prevention of bone resorption.13 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p40–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p40-44 44 prabowo, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 40–44 cape, inhibits the release of inflammatory cytokines and increases stimulant production of anti-inflammatory cytokines, such as il-10 and il-4.14,15 il-10 stimulants have an anti-inflammatory function, which can reduce regulation of il-5 production by t cells, while il-5 plays a role in differentiation and activation of eosinophil function, by controlling eosinophil accumulation in inflamed tissue. il-10 has main function, such as inhibiting tnf-α, il-1, chemokines, and il-12 produced by macrophages. il-4 has an inhibitory effect on the expression and release of proinflammatory cytokines. these cytokines inhibit or suppress cytokines derived from monocytes, including il-1 tnf-a, il-6, il-8, and the macrophage inflammatory protein (mip). il-4 is also known to suppress macrophage cytotoxic activity, kill parasites, and produce nitric oxide derived from macrophages, so that the role of il-1, il-6, and tnf-α in osteoclast mitogenesis is inhibited, causing osteoclastogenesis to be disrupted.14 bovine bone graft is the main type of bone graft. the main function of bone graft is to stimulate osteogenesis. the biologic mechanisms that provide a rationale for bone graft are osteoconduction, osteoinduction, and osteogenesis. osteoconduction is a graft resorption process, that replaces new bone from the margin. graft material is used as a framework upon which to spread and generate new bone from the margin of defect. as in osteoinduction, it stimulates osteoprogenitor cells to differentiate into osteoblasts and begins the formation of new bone.16,17 osteoinduction is a process of attracting pluripotential cells from recipients around graft and bone, because the material of bone graft and bone contain osteoinduction mediators, such as bone morphogenic protein (bmp). material from bovine bone graft is capable of supporting the attach and proliferate of osteoblast cells, which represents the first step in the process of osteogenesis.16 consequently, since propolis extract reduces proinflammatory cytokines (il-1, tnf-α), inhibits nf-κb, and increases certain osteoblast by tgf-β, it can be argued an inverse relationship exists between osteoblasts and osteoclasts. bovine bone graft can stimulate the osteoblast on the biological mechanism to accelerate new bone growth. similarly, the results of this research indicate that a combination of propolis extract (at a dose of 0.5%, 1% and 2%) and bovine bone graft increases the quantity of osteoblasts and reduces that of osteoclasts. however, the combination of propolis extract at a dose of 2% and bovine bone graft is more effective than the other doses. this research demonstrates that propolis extract combined with bovine bone graft at a dose of 2% effectively affects the number of osteoblasts and osteoclasts during tooth extraction socket preservation. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 110. 2. masaki c, nakamoto t, mukaibo t, kondo y, hosokawa r. strategies for alveolar ridge reconstruction and preservation for implant therapy. j prosthodont res. 2015; 59(4): 220–8. 3. faverani lp, ramalho-ferreira g, santos ph dos, rocha ep, garcia júnior ir, pastori cm, assunção wg. surgical techniques for maxillary bone grafting literature review. rev col bras cir. 2014; 41(1): 61–7. 4. lupovici ja. histologic and clinical results of dfdba with lecithin carrier used in dental implant applications: three case reports. pract proced aesthetic dent. 2009; 21(4): 223–30. 5. elo ja, herford as, boyne pj. implant success in distracted bone versus autogenous bone-grafted sites. j oral implantol. 2009; 35(4): 181–4. 6. guney a, karaman i, oner m, yerer mb. effects of propolis on fracture healing: an experimental study. phyther res. 2011; 25(11): 1648–52. 7. kresnoadi u, hadisoesanto y, prabowo h. effect of mangosteen peel extract combined with demineralized freezed-dried bovine bone xenograft on osteoblast and osteoclast formation in post tooth extraction socket. dent j (majalah kedokt gigi). 2016; 49(1): 43–8. 8. lunardhi lc, kresnoadi u, agustono b. the effect of a combination of propolis extract and bovine bone graft on the quantity of fibroblasts, osteoblasts and osteoclasts in tooth extraction sockets. dent j (majalah kedokt gigi). 2019; 52(3): 126–32. 9. pileggi r, antony k, johnson k, zuo j, shannon holliday l. propolis inhibits osteoclast maturation. dent traumatol. 2009; 25(6): 584–8. 10. ha j, choi hs, lee y, lee zh, kim hh. caffeic acid phenethyl ester inhibits osteoclastogenesis by suppressing nfκb and downregulating nfatc1 and c-fos. int immunopharmacol. 2009; 9(6): 774–80. 11. ang esm, pavlos nj, chai ly, qi m, cheng ts, steer jh, joyce da, zheng mh, xu j. caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of rankl-induced nf-κb and nfat activity. j cell physiol. 2009; 221(3): 642–9. 12. jules j, zhang p, ashley jw, wei s, shi z, liu j, michalek sm, feng x. molecular basis of requirement of receptor activator of nuclear factor κb signaling for interleukin 1-mediated osteoclastogenesis. j biol chem. 2012; 287(19): 15728–38. 13. al-molla bh, al-ghaban n, taher a. immunohistochemical evaluation: the effects of propolis on osseointegration of dental implants in rabbit’s tibia. j dent res rev. 2014; 1(3): 123–31. 14. duan w, wang q, li f, xiang c, zhou l, xu j, feng h, wei x. anticatabolic effect of caffeic acid phenethyl ester, an active component of honeybee propolis on bone loss in ovariectomized mice: a microcomputed tomography study and histological analysis. chin med j (engl). 2014; 127(22): 3932–6. 15. al-saeed hf, mohamed ny. the possible therapeutic effects of propolis on osteoporosis in diabetic male rats. nat sci. 2015; 13(3): 136–40. 16. kumar p, vinitha b, fathima g. bone grafts in dentistry. j pharm bioallied sci. 2013; 5(suppl 1): s125–7. 17. periya sn, hammad hgh. bone grafting in dentistry: biomaterial degradation and tissue reaction: a review. ec dent sci. 2017; 9(6): 239–44. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p40–44 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p40-44 69 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 69 the effects of curcuma zedoaria oil on high blood sugar level and gingivitis juni handajani and dhinintya hyta narissi department of oral biology faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: hyperglycemia is a condition when blood sugar level is higher than normal. hyperglycemia is also one of diabetes mellitus (dm) symptoms. hyperglycemia has a correlation with the occurrence of periodontal disease. curcuma zedoaria oil is known to decrease concentration of serum glucose. purpose: this study was aimed to determine the effects of curcuma zedoaria oil on high blood sugar level and gingivitis in rats. method: this study used twenty-five male wistar rats, divided into two groups, namely the treatment group and the control group. in the treatment group, fifteen rats were divided into three subgroups (each of which was induced with 10 μl/ml, 30 μl/ml and 50 μl/ml of curcuma zedoaria oil). the control group was consisted of ten rats, divided into two subgroups, as the positive control group (induced with 10 mg/kg of glibenclamide) and the negative control group (induced with propylene glycol). streptozotocin (stz) (naclai tesque, kyoto japan) with a dose of 40 mg/kg was used to create hyperglycemia condition in those rats. gingivitis was then made by using silk ligature in those hyperglycemia rats. silk ligature was twisted at the margin of gingiva anterior mandibular incisors for seven days. after the rats had gingivitis, curcuma zedoaria oil, glibenclamide and propylene glycol were orally administered for seven days. their gingivitis condition was observed, and their blood sugar level was measured before and after the induction of stz and during the treatment. the data obtained were analyzed by using manova. result: there were significant differences of blood sugar levels between the treatment group before and after the administration of curcuma zedoaria oil and the positive control group (p<0.05). healthy gingiva was then found in the treatment group and the positive control group. conclusion: curcuma zedoaria oil can decrease blood sugar level and gingivitis. keywords: curcuma zedoaria oil; gingivitis; blood sugar level correspondence: juni handajani, c/o: departemen biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: junihandajani@yahoo.com research report introduction hyperglycemia is a condition when blood sugar level is higher than normal. hyperglycemia is also one of diabetes mellitus (dm) symptoms, and it is considered as a metabolic disorder. in metabolic disorder condition characterized by the presence of oxidative stress, the balance between production and inactivation of reactive oxygen species (ros) is disrupted. ros has a very important role in various physiological systems, especially for celluler metabolism.1,2 hyperglycemia condition will cause changes in glucose homeostasis metabolism. pathological mechanism of hyperglycemia involving β cells of the pancreas, for instance, can cause its inability of to produce insulin. hyperglycemia is also known to be associated with periodontal disease, especially related to the mechanism of glycation end-product (age) formation. organism physiologically produce age, but the production of age under hyperglycemia condition or augmented oxidative stress can be excessive.2,3 age is a substance that is able to increase cytokines produced by macrophages, such as tumor necrosis factor-α (tnf-α) and interleukin-6 (il-6), as well as to stimulate the secretion of hepatic protein acute-phase, including c-reactive protein (crp), fibrinogen, plasminogen dental journal (majalah kedokteran gigi) 2015 june; 48(2): 69–73 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 70 handajani and narissi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 69–73 activator/inhibitor and amyloid a serum. age is also known to correlate with the occurrence of oral infections and cardiovascular disease, especially in patients with periodontal disease. in addition, age can increase the activities of monocyte migration, endothelial permeability, fibroblast permeability and muscular cells, and can also bind to collagen. age substance can cause a rapid expansion in the respiratory activity of polymorphonuclear neutrophils (pmn), leading to an increased severity of periodontal tissue destruction and some changes in bone metabolism, especially during healing process, and can also reduce the production of extracellular matrix.3,4 periodontal disease is likely caused by neutrophilic basic immune response as a result of excessive and uncontrolled production of ros. this condition then leads to local and peripheral oxidative damage directly and indirectly caused by genes redox-sensitive transcription factors. nuclear factor kappa b and activator protein-1 can trigger inflammatory mediator of cascade and accelerate cellular aging. hyperglycemia, thus, has associated with the changes in oral mucosa barrier marked with pmn accumulation, increased matrix metalloproteinase (mmp) and ros in periodontal tissues. the changes then may trigger inflammation in the periodontium tissue, so the gingival crevice fluid can be detected from the increased level of prostaglandin e 2 (pge 2) and il-1β.2,3,4 on the other hand, curcuma zedoaria has been studied for oral health maintenance. in dentistry, curcuma zedoaria oil even has been used for its potential therapeutic effects as antimicrobial and anti-inflammatory herbs and for lowering blood sugar levels.5,6 the chemical composition of herbal extracts is actually depends on several variables, such as growing conditions, soil quality, season and post-harvest handling. the pharmacological effects of the extracts in both liquid and powder are expected to be related to the interaction of chemical substances contained, not only to isolated molecules. after measured with gas chromatography and mass spectrometry (gc-ms), curcuma zedoaria oil is known to contain several compounds, namely camphene; 1-betapinene; mycrene; 1,8-cineole; camphor; beta-elemene; bicyclo [2.2.1] heptane-2-ol, 1,7,7-trimethyl-, exo; borneol l; eremophilene; (+) calarene; valencene; beta-elemenone; germacrone and 2-ethoxy-6-ethyl-4,4,5-trimethyl-1,3dioxa-4-sila-2-boracyclohex-5-ene.7 some previous studies even indicated that curcuma zedoaria oil can be used as an anti-inflammatory for artificial edema at a dose of 400 mg / kg, 8 increase phagocytic activity of neutrophils 9 and reduce gingival inflammation by decreasing cd4+ expression. 10 potential phytochemistry of curcuma zedoaria powder can also decrease glucose serum.11 however, the effects of curcuma zedoaria oil on hyperglycemia accompanied by gingivitis in rats have not been known yet. therefore, this study was aimed to determine the potential therapeutic effects of curcuma zedoaria oil on the high blood glucose level and gingivitis. materials and methods this study was an experimental study to analyze the biological activities of curcuma zedoaria oil against hyperglycemia and gingivitis in the oral cavity of rats. determination of curcuma zedoaria was conducted at the laboratory of biology pharmacy, faculty of pharmacy, universitas gadjah mada. curcuma zedoaria oil was prepared in unit ii of integrated research and testing laboratory (lppt) universitas gadjah mada by using water vapor distillation method. the procedures of this study were approved by the ethics committee of dentistry faculty of dentistry, universitas gadjah mada. this study used twenty-five male wistar rats aged 2 months old obtained from unit iv of lppt, universitas gadjah mada. to adapt to the laboratory environment, those wistar rats were put in individual cages for one week, feed with standard pellets as much as 360 grams per day and administered with mineral water ad libitum. the procedures of this study were conducted in several stages. to create hyperglycemic condition in those rats, they were fasted for 18 hours before treated. intraperitoneal injection of streptozotocin (stz) (nacalai tesque, kyoto japan) was administrated in the abdomen of those rats with a dose of 40 mg/kg in 0.1 m citrate buffer. those rats were drunk with 5% glucose solution to protect the effects of stz.12 those rats were intramuscularly anesthetized into their right thigh by ketamine hcl with a dose of 0.2 ml/200 grams. gingivitis was made by using silk ligature with size 3.0. ligation was placed on margin of anterior gingiva mandibular incisors for six days. silk ligature was checked every day to observe the possibility of the release of ligature. the changes of gingiva were observed, such as reddish color, shiny look, and bleeding easily. those hyperglycemia and gingivitis rats were divided into two groups randomly, namely the treatment group (fifteen rats) and the control group (ten rats). the treatment group was divided into three subgroups, each of which was consisted of five rats and administered orally with 10 µl/ml, 30 µl/ml and 50 µl/ml of curcuma zedoaria oil. meanwhile, the control group was divided into two subgroups. five rats as the positive control group induced with 10 mg/kg of glibenclamide and five rats as the negative control group induced orally with propylene glycol. the administration of materials in the treatment and control groups was conducted every day for seven days by using oral gavage. their gingiva condition was then observed, and their blood sugar level were measured before and after the induction of stz and after the treatment. the measurement of the blood sugar levels was conducted 24 hours after the injection of stz. totally, three rats of the treatment group induced with 10 µl/ml of curcuma zedoaria oil, of the positive control and of the negative control died after 24 hours of stz injection. finally, data obtained were analyzed by using manova statistical test. 71 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 71handajani and narissi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 69–73 9 figure 1. the mean and standard deviation of the blood sugar level (mg/dl) of those rats in the treatment and control groups. the mean of the blood sugar level increased after the stz injection. the mean of the blood sugar level decreased after the administration of curcuma zedoaria oil and glibenclamide. figure 1. the mean and standard deviation of the blood sugar level (mg/dl) of those rats in the treatment and control groups. the mean of the blood sugar level increased after the stz injection. the mean of the blood sugar level decreased after the administration of curcuma zedoaria oil and glibenclamide. table 2. the results of lsd post hoc test on the effects of curcuma zedoaria oil on the blood sugar level of those hyperglycemia and gingivitis rats group curcuma zedoaria oil 10 µl/ml curcuma zedoaria oil 30 µl/m curcuma zedoaria oil 50 µl/m control + control curcuma zedoaria oil 10 µl/ml 20.300 (0.291) 57.700* (0.003) 62.250* (0.004) -24.250 (0.270) curcuma zedoaria oil 30 µl/ml 20.300 (0.291) 37.400* (0.004) 41.950* (0.001) -44.550* (0.013) curcuma zedoaria oil 50 µl/m 57.700* (0.003) 37.400* (0.004) -4.550 (0.999) 81.950* (0.000) control + 62.250* (0.004) 41.950* (0.001) -4.550 (0.999) 86.500* (0.001) control -24.250 (0.270) -44.550* (0.013) 81.950* (0.000) 86.500* (0.001) results the mean of their fasting blood sugar level before the induction of stz was 89.12 mg/dl, while the mean of their blood sugar level 24 hours after the induction of stz was 192.24 mg/dl. the blood sugar level of those rats in the treatment and control groups was then observed every day for seven days (figure 1). the observation of gingiva after seven days of the ligation showed that all of those rats had gingivitis, characterized by reddish color, shiny look, and bleeding easily. the results of the gingiva observation in the treatment and control groups can be seen in table 1. meanwhile, the results of gingivitis and healthy gingiva were showed in figure 2. the statistical results of manova and lsd post hoc tests, moreover, showed that the data obtained were normal and homogen (p>0.05). the statistical results of manova test showed that there was a significant difference of blood sugar level between in the positive control and in the treatment group (p<0.05). it indicated that curcuma zedoaria oil had a significant effect on the decreasing of the fasting blood sugar level. the results of lsd post hoc test also showed that curcuma zedoaria oil had a significant effect on the blood sugar level of those hyperglycemia and gingivitis rats as seen in table 2. table 1. the results of the gingiva observation in the treatment and control groups. during the observations, the number of rats with healthy gingiva or gingivitis was measured group number of rats with gingivitis number of rats with healthy gingiva treatment : a. curcuma zedoaria oil 10 µl/ml b. curcuma zedoaria 30 µl/ml c. curcuma zedoaria 50 µl/ml 4 5 5 control : a. positif (glibenclamide) b. negatif (prophylene glycol) 4 4 10 table 1. the results of the gingiva observation in the treatment and control groups. during the observations, the number of rats with healthy gingiva or gingivitis was measured group number of rats with gingivitis number of rats with healthy gingiva treatment : a. curcuma zedoaria oil 10 µl/ml b. curcuma zedoaria 30 µl/ml c. curcuma zedoaria 50 µl/ml 4 5 5 control : a. positif (glibenclamide) b. negatif (prophylene glycol) 4 4 figure 2. normal gingiva (a) and gingivitis (b). gingivitis looked reddish and shiny. table 2. the results of lsd post hoc test on the effects of curcuma zedoaria oil on the blood sugar level of those hyperglycemia and gingivitis rats group curcuma zedoaria oil 10 µl/ml curcuma zedoaria oil 30 µl/m curcuma zedoaria oil 50 µl/m control + control curcuma zedoaria oil 10 µl/ml 20.300 (0.291) 57.700* (0.003) 62.250* (0.004) -24.250 (0.270) curcuma zedoaria oil 30 20.300 (0.291) 37.400* (0.004) 41.950* (0.001) -44.550* (0.013) 10 table 1. the results of the gingiva observation in the treatment and control groups. during the observations, the number of rats with healthy gingiva or gingivitis was measured group number of rats with gingivitis number of rats with healthy gingiva treatment : a. curcuma zedoaria oil 10 µl/ml b. curcuma zedoaria 30 µl/ml c. curcuma zedoaria 50 µl/ml 4 5 5 control : a. positif (glibenclamide) b. negatif (prophylene glycol) 4 4 figure 2. normal gingiva (a) and gingivitis (b). gingivitis looked reddish and shiny. table 2. the results of lsd post hoc test on the effects of curcuma zedoaria oil on the blood sugar level of those hyperglycemia and gingivitis rats group curcuma zedoaria oil 10 µl/ml curcuma zedoaria oil 30 µl/m curcuma zedoaria oil 50 µl/m control + control curcuma zedoaria oil 10 µl/ml 20.300 (0.291) 57.700* (0.003) 62.250* (0.004) -24.250 (0.270) curcuma zedoaria oil 30 20.300 (0.291) 37.400* (0.004) 41.950* (0.001) -44.550* (0.013) figure 2. normal gingiva (a) and gingivitis (b). gingivitis looked reddish and shiny. a b dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 72 handajani and narissi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 69–73 table 2 showed the significant effect of curcuma zedoaria oil and glibenclamide on reducing blood sugar level. there were no significant differences of the effects of curcuma zedoaria oil 10 µl/ml and 30 µl/ml, and between curcuma zedoaria oil 10 µl/ml and propylene glycol. that there was no significant difference of the effects of curcuma zedoaria oil 10 µl/ml and 30 µl/ml on blood sugar indicated that both doses could decrease blood sugar level almost at the same statistic level. insignificant difference of the effects of curcuma zedoaria oil 10 µl/ml and propylene glycol indicated that both could have almost the same results in decreasing blood sugar level . discussion after having the stz induction with a dose of 40 mg/kg in 0.1 m citrate buffer intraperitoneally, all of those rats got hyperglycemia. the measurement of their blood sugar level before the induction was 89.12 mg/dl, while after the induction it was 192.24 mg /dl. the blood sugar level of those rats increased around 215%. stz compound known as glucosamine-nitrosurea can cause toxic to cells since it can trigger dna damage. dna damage causes the activation of poly adp-ribosylation, allegedly playing an important role in increasing blood sugar level. stz similar to glucose can be transported to cells by a glucose-transporting protein, glucose transporter type 2 (glut2). stz mechanism to induce an increase in blood sugar level occurs in three phases. however, temporary hypoglycemic effect only occurrs a few minutes up to 30 minutes after stz injection. in the first phase, blood sugar concentration is increased one hour after stz administration, and hypoinsulinaemia occurs as a result of inhibition of insulin secretion in the next 2-4 hours. at this stage, the morphological characteristics of beta cells were characterized by intracellular vacuolisasi, dilated rough endoplasmic reticulum, reduced golgi area, decreased secretory granules and insulin, as well as swollen mitochondria.13,14 in the second phase, hypoglycemia condition occurs for approximately 4-8 hours after stz injection and lasts up to several hours. nevertheless, this condition is usually looked bad in experimental animals experienced convulsions (seizures) since it can even cause death if not given with glucose soon, particularly when their liver glycogen storage is depleted due to fasting. in the last phase, permanent hyperglycemia morphologically characterized by degranulation and beta cell integrity loss occurs for approximately 12-48 hours. that non-beta cells are still intact shows the characters of selective beta-cells against the action of toxic substance.13,15 table 1 showed that gingivitis occured after the ligation using a silk ligature. the results of the study also showed that after the administration of curcuma zedoaria oil, gingiva appeared normal (healthy). these results could be predicted since curcuma zedoaria oil contained curcumin and sesquiterpenoid, which can change gingivitis to be healthy gingiva. similarly, a study conducted by kaushik et al.8 also showed that extracts of curcuma zedoaria had anti-inflammatory effects in rats induced with carrageenan. the ability of curcumin contained in curcuma zedoaria oil as an anti-inflammatory can reduce prostaglandins by inhibiting cyclooxygenase mechanism. sesquiterpenoid contained in curcuma zedoaria oil can also allegedly inhibit the production of pge and nitric oxide. pge is one of inflammation mediators. sesquiterpenoid contained in curcuma zedoaria oil even can inhibit the release of tnf-α from macrophages. cytokine tnf-α is an inducer of the inflammatory response due to bacterial infection. in addition, like the results of this study, the results of the previous study also showed that curcuma zedoaria oil has anti-inflammatory against artificial edema in female wistar rats at a dose of 400 mg/kg. 8 curcuma zedoaria oil administered daily at a dose of 30.6 µl/ml orally for 14 days can also effectively increase the phagocytic activities of neutrophils in rats induced with aggregatibacter actinomycetemcomitans.9 curcuma zedoaria oil can reduce gingiva inflammation characterized by a decrease in cd4+ expression.10 similarly, in this study curcuma zedoaria oil could decrease blood sugar level in hyperglycemia rats (figure 1). this result is also supported by a previous study conducted by rahmatullah et al.11 showing that curcuma zedoaria extract at a dose of 50 mg, 100 mg, 200 mg and 400 mg per kg of body mass can decrease serum glucose concentrations respectively of 36.9%; 39.4%; 41.1% and 55.1% compared to controls. the result of the previous study also suggested that the methanol extract of curcuma zedoaria leaves can decrease blood sugar levels in swiss albino mice. luteolin and luteolin 7-o-glucoside of flavonoids contained in curcuma zedoaria leaves have abilities to decrese serum glucose level, cholesterol, triglyceride and low lipoprotein density (ldl) level.16 rhizome curcuma zedoaria has an ability to decrease blood sugar levels because of α-pinene and curcumin.17 curcuma zedoaria extract can decrease blood sugar level allegedly through several mechanisms since curcuma zedoaria extract has many abilities to amplify insulin secretion, to increase glucose uptake from the serum, or to decrease the absorption of glucose from intestinum.11 in this study, curcuma zedoaria oil was easily absorbed in the digestive tract of those rats, and rapidly reached their bloodstream within 15 minutes. their blood sugar level, as a result, decreased since curcuma zedoaria oil has an ability to improve the immune system of those rats which blood sugar level was increased due to the stz induction. this was probably due to the mechanism of curcuma zedoaria oil that could protect beta pancreatic cells so that the damage of beta pancreatic cells induced by stz was not permanent or worse. another possibility was that curcuma zedoaria oil has an ability to improve beta pancreatic cells to secrete insulin. this condition could be detected by a decrease in the blood sugar level of those rats after the 73 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 73handajani and narissi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 69–73 administration of curcuma zedoaria oil. in conclusion, the oral administration of curcuma zedoaria oil could decrease blood sugar level and gingivitis in hyperglycemia rats. acknowledgement this study was supported by community service grants from faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia. references 1. ceriello a, motz e. is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? the common soil hypothesis revisited. arterioscler thromb vasc biol 2004; 24(suppl5): 816–23. 2. iain lc c, matthews jb. the role of reactive oxygen and antioxidant species in periodontal tissue destruction. periodontol 2007; 43:160– 232. 3. pietropaoli d, tatone c, d’alessandro, monaco a. possible involment of advanced glycation end products in periodontal diseases. b int j immunopathol and pharmacol 2010; 23(suppl 3): 683–91. 4. bohlender jm, franke s, stein g, wolf g. advanced glycation end products and the kidney. am j physiol renal physiol 2005; 289: f645–f59. 5. chen in, chang cc, wang cy, shyu yt, chang tl. antioxidant and antimicrobial activity of zingiberaceae plants in taiwan. plant foods hum nutr 2008; 63: 15–20. 6. marinho bvs, araújo acs. use mouthwash in gingivitis and dental biofilm. intern j dentistry 2007; 6: 124–31. 7. hartono m, nurlaila, batubara i. potensi temu putih (curcuma zedoaria) sebagai antibakteri dan kandungan senyawa kimia. prosiding pengembangan pulau-pulau kecil, 2011. p. 203-12. 8. kaushik ml, jalalpure ss. antiiflammatory efficacy of curcuma zedoaria rosc root extract. asian j pharm clin res 2011; 4(3): 902. 9. handajani j. temu putih (curcuma zedoaria rosc., zingiberaceae) volatile oil increased phagocytic activity of neutrophils exposed to a. actinomycetemcomitans. proceeding the international symposium on oral and dental sciences, 17-18 januari 2013. p. 51-7. 10. handajani j. minyak atsiri temu putih (curcuma zedoaria rosc., zingiberaceae) menurunkan ekspresi cd4+ pada gingiva terpapar a. actinomycetemcomitans. majalah kedokteran gigi 2013; 20(1): 9-12. 11. rahmatullah m, azam nk, pramanik n, sania rahman n, jahan r. antihyperglycemic activity evaluation of rhizomes of curcuma zedoaria (christm.) roscoe and fruits of sonneratia caseolaris (l.) engl. int j pharm tech res 2012; 4(1): 125-29. 12. sunmonu to, afolayan aj. evaluation of antidiabetic activity and associated toxicity of artemisia afra aqueous extract in wistar rats. evidence-based compl alt med 2013; 1-8. 13. lenzen s. the mechanism of alloxanand streptozotocin-induced diabetes. diabetologia 2008; 51: 216-6. 14. konrad rj, mikolaenka i, tolar jf, liu k, kudlow je. the potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic β-cell o-glcnac-selective n-acetyl-β-dglucosaminidase. biochem j 2001; 356: 31-41. 15. akbarzadeh a, norouzian d, mehrabi mr, jamshidi sh, farhangi a, verdi aa, mofidian sma, rad l. induction of diabetes by streptozotocin in rats. ind j clin biochem 2007; 22(2): 60-4. 16. shoha j, jahan h, mamun aa, hossain mt, ahmed s, hosain mm, rahman s, jahan r, rahmatullah m. antihyperglycemic and antinociceptive effects of curcuma zedoaria (christm.) roscoe leaf extract in swiss albino mice. adv nat appl sci 2010; 5: 6-8. 17. el-moselhy ma, taye a, sharkawi ss, el-sisi s, ahmed a. the antihyperglycemic effect of curcumin in high fat diet fed rats. role of tnf-α and free fatty acids. food chem toxicol 2011; 49: 1129-40. 140 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 140–143 research report the effect of body mass index on tooth eruption and dental caries mohamed salim younus, karam ahmed and duran kala faculty of dentistry, tishk international university, erbil, iraq abstract background: children were compared to their siblings, cousins or peers regarding the eruption of their permanent teeth. genetic and environmental factors can affect dental development and, therefore, the body mass index (bmi) could be considered as a factor that may influence dental development. purpose: to determine any possible association between bmi and either dental caries or the eruption of permanent teeth (central incisor and molar). methods: a cross-sectional study was completed for six-year-old school children. a total of 218 children (116 boys, 102 girls) from public elementary schools in erbil city were entered into the study. dental caries assessments were carried out using the who criteria for decayed, missing and filled primary teeth and indices (dmft). bmi was used to classify obesity status. results: overall, 27.98% of the children were classified as overweight, 59.17% as normal and 12.84% as underweight. the dmft was 5.247, while 12.39% of the children were caries-free. conclusions: children of normal weight had most permanent teeth erupted and a low caries index. underweight children had fewer erupted teeth and a higher caries index. the complex relationship between body composition and oral health should be considered in paediatric patients. keywords: body mass index; dmft; eruption of central incisors and molars correspondence: mohamed salim younus, faculty of dentistry, tishk international university, erbil, kurdistan region, iraq. email: mohamed.salim@tiu.edu.iq introduction eruption of the teeth is positively related to the somatic growth (height and weight) of an individual. many studies worldwide describe how poor nutrition during the growth period adversely influences aspects of dental development, including delaying the eruption of deciduous and permanent teeth. the prevalence of overweight and obese children is increasing worldwide, including in europe.1 this marked increase in body weight was described by the world health organisation (2003) as a ‘global epidemic disease’. obesity can be defined as a condition in which the energy intake becomes higher than the required energy, leading to deposition of body fat. this accumulation of extra fat within the body may have either environmental or genetic causes.2 being overweight causes health problems in children, both directly and over time, including: type ii diabetes, metabolic problems, high blood pressure, hypercholesterolaemia, hyperandrogenism, orthopaedic complications, sleep disorders, cardiovascular disease and behavioural issues.3 the obesity trend in children and adolescents between six and seventeen years old in the us is characterised by differences in relation to age, gender, race-ethnicity, income and level of education. representative surveys performed from 1963–1994 to measure weight and height showed 11% of the population to be obese in the period from 1988–1994. this level of obesity was not related to race-ethnicity, income or education. obesity levels became higher over time, with the largest increase in the period from 1976–1994. the reasons for this fast increase in obesity in the us population is unclear but could be a sign of societal influence.4 according to the iotf (international obesity task force), the level of obesity in italian children between eight and nine years of age varies from 16.6% in the south of italy to about 7.5% in the north.5 in france, 15.8% of children from seven to nine years old are overweight, with nearly 2.8% being obese.6 this study presents the distribution of children according to bmi (categorised as underweight, normal weight or overweight) in erbil city primary schools, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p140–143 mailto:mohamed.salim@tiu.edu.iq http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p140-143 141younus et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 140–143 the relationship between bmi and eruption of the teeth (central incisors and molars), and the association between bmi and the level of dental caries (dmft). materials and methods the study was carried out from january 2019 until the end of may 2019 including diagnosis, sample collection and data analysis. approval was obtained from the ministry of education to diagnose the students (no. 08/18, on 23-12-2018). before starting the study, the aims of the research were explained to each school manager and written approval was obtained from parents for participation of their child in the study. the study sample included 218 six-year-old children from five governmental primary schools in erbil city in iraq, including 116 males and 102 females. all six-yearolds were invited to participate. students with systemic diseases were excluded from the study. bmi is calculated in the same manner for adults and children, although there are differences in the recommended amounts of body fat based on age and gender.7 the bmi represents the ratio of weight (kg) to height (m), i.e. bmi = weight / height2.8 weight status was defined by gender-related bmi according to the centres for disease control and prevention (cdc) guidelines as follows:9 underweight (bmi ≤5th percentile), normal weight (bmi >5th and <85th percentile), at risk of overweight (bmi ≥85th and <95th percentile), overweight (bmi ≥95th percentile) (see table 1). instruments and supplies used during sample collection included gloves and masks, dental mirrors, dental probes, height measuring tape (yishen measuring tape, china) and bathroom scales (tianshan brand penguin electronic scales, china). before each examination the procedure and instructions were explained to the children. height and weight were measured for each child, after which the teeth were examined while the students were seated in their classroom. the examiner stood in front of each student’s chair for diagnosis. data analysis and processing were carried out using the statistical package for social science (spss), version 20 (ibm, new york, usa). results the eruption of central incisors and molars was calculated in relation to bmi. table 2 shows the number and percentage of erupted and non-erupted central incisors and molars for the sample group. the data shows that 12% of children were free of caries and therefore the dental caries prevalence was 88%. the dmft was 5.2, with the highest percentage in the underweight group (table 3). no significant difference was observed in bmi between genders. more central incisors and molars had erupted in the overweight group and fewest in the underweight group, but this was not statistically significant. most caries-free children were present in the normal weight group and fewest in the underweight group, again with no significant difference. the dmft was highest in underweight children and lowest in those of normal weight but, as before, this difference was not significant (table 4). table 1. categories according to bmi variables categories no. % gender male 116 53.2 female 102 46.8 bmi under weight 28 12.8 normal weight 129 59.2 over weight 61 28.0 table 3. caries free and dmft in bmi categories bmi underweight normal weight overweight total (all types) caries free 2(7.14%) 18(13.95%) 7(11.48%) 27(12.39%) dmft 6.142 5.116 5.114 5.247 table 2. the eruption of permanent centrals and molars variables categories no. % eruption of central incisors not erupted 49 22.5 erupted 169 77.5 eruption of molars not erupted 37 17.0 erupted 181 83.0 dmft categories caries free 20 9.2 dmft 198 90.8 total 218 100 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p140–143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p140-143 142 younus et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 140–143 discussion in this study, most children were of normal weight and the lowest proportion was underweight, a similar distribution to that found by najmeh et al.10 however, different results were recorded by lobstein et al.11 many studies have shown that the proportion of overweight children in both developing and developed countries is increasing, making it a significant public health concern.12 a higher percentage of females were evident in the underweight category, while the proportion of males was higher in the normal and overweight groups. this may be related to males having a greater acceptance of different food types than females. this finding is in agreement with the findings of the 2015 national health and nutrition examination survey.13 however, in contrast to our study obesity rates were higher in girls than in boys, potentially because most of the boys were significantly more active and more likely to meet physical activity guidelines than the girls.14 tooth eruption is a growth process and so is related to other body factors such as height and weight.15 the relationship between bmi and eruption of the central incisors and molars was strongest in the overweight category and weakest in the underweight category. this could be due to effects of good nutrition on the eruption of the teeth, as previously reported by must et al.16 who found that obese children were more likely to have a high number of erupted teeth than non-obese children. other studies have also reported comparable results.17,18 in contrast, elamin et al.19 studied tooth development in malnourished sudanese children and found that sustained malnutrition during childhood had little effect on dental development in this population. moreover, eid et al.20 found no significant correlation between dental maturation and bmi in brazilian children aged between six and fourteen. this study found that 12.39% of the children were caries-free. in comparison, costacurta et al.21 found a higher proportion to be caries-free (16.82%). the normal weight group had the highest proportion of caries-free children, whilst the underweight group showed the lowest. malnutrition in children could also increase the risk of caries. it was found that there is an association between dental caries (dmft) and nutrition, as this data shows the highest proportion of dental caries in the underweight category but comparable rates in the normal and overweight categories. in contrast to our study, dental caries and being overweight are concomitant situations in many communities due to risk factors such as high intake of calories and carbohydrate. surveys by werner et al.22, kantovitz et al.23 and gatta et al.15 showed that cariogenic foods may play a significant role in dental caries, suggesting a relationship between caries and body weight after the age of six.24 however, other studies showed no association between childhood weight and dental caries.12,25–29 from the results of this study, we can conclude that underweight children may experience delays in tooth eruption and higher dental caries than normal weight and overweight children. this could be attributed to effects of diet on the eruption of teeth, with adequate nutrition protecting against dental caries. we conclude that children of normal weight and overweight children have more erupted permanent teeth and lower dental caries. the complicated association between oral health and body composition must be taken into consideration when treating paediatric patients. references 1. lobstein t, frelut ml. prevalence of overweight among children in europe. obes rev. 2003; 4(4): 195–200. 2. marshall ta, eichenberger-gilmore jm, broffitt ba, warren jj, levy sm. dental caries and childhood obesity: roles of diet and socioeconomic status. community dent oral epidemiol. 2007; 35(6): 449–58. 3. wyatt sb, winters kp, dubbert pm. overweight and obesity: prevalence, consequences, and causes of a growing public health problem. am j med sci. 2006; 331(4): 166–74. table 4. the relation between bmi and the other variables variables categories bmi p under weight normal weight over weight gender male 13 (46.4%) 70 (54.3%) 33 (54.1%) 0.743 female 15 (53.6%) 59 (45.7%) 28 (45.9%) eruption of central incisors not erupted 8 (28.6%) 33 (25.6%) 8 (13.1%) 0.112 erupted 20 (71.4%) 96 (74.4%) 53 (86.9%) eruption of molars not erupted 7 (25%) 22 (17.1%) 8 (13.1%) 0.401 erupted 21 (75%) 107 (82.9%) 53 (86.9%) dmft categories caries free 2 (7.1%) 13 (10.1%) 5 (8.2%) 0.944 dmft 26 (92.9%) 116 (89.9%) 56 (91.8%) total 28 (100%) 129 (100%) 61 (100%) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p140–143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p140-143 143younus et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 140–143 4. nhanes. national health examination survey (nhes) and the national health and nutrition examination surveys (nhanes i, 1971 to 1974; nhanes ii, 1976 to 1980; and nhanes iii, 1988 to 1994. centers for disease control and prevention. available from: https://wwwn.cdc.gov/nchs/nhanes/. 5. binkin n, fontana g, lamberti a, cattaneo c, baglio g, perra a, spinelli a. a national survey of the prevalence of childhood overweight and obesity in italy: national prevalence of obesity. obes rev. 2010; 11(1): 2–10. 6. salanave b, peneau s, rolland-cachera mf, hercberg s, castetbon k. stabilization of overweight prevalence in french children between 2000 and 2007. int j pediatr obes. 2009; 4(2): 66–72. 7. bhadoria a, sahoo k, sahoo b, choudhury a, sufi n, kumar r. childhood obesity: causes and consequences. j fam med prim care. 2015; 4(2): 187. 8. hilgers kk, kinane df, scheetz jp. association between childhood obesity and smooth-surface caries in posterior teeth: a preliminary study. pediatr dent. 2006; 28(1): 23–8. 9. kuczmarski rj, ogden cl, guo ss, grummer-strawn lm, flegal km, mei z, wei r, curtin lr, roche af, johnson cl. 2000 cdc growth charts for the united states: methods and development. vital health stat 11. 2002; (246): 1–190. 10. najmeh a, anousheh rm, ali b. the relationship between body mass index and dental development by demirjian’s method in 4to 15-year-old children in mashhad. j dent mater tech. 2013; 2(3): 82–5. 11. lobstein t, baur l, uauy r. obesity in children and young people: a crisis in public health. obes rev. 2004; 5(s1): 4–85. 12. gokhale, sivakumar n, nirmala s, abinash m. dental caries and body mass index in children of nellore. j orofac sci. 2020; 2(2): 4–6. 13. hales cm, carroll md, fryar cd, ogden cl. prevalence of obesity among adults and youth: united states, 2015-2016. nchs data brief. 2017; (288): 1–8. 14. borraccino a, lemma p, iannotti rj, zambon a, dalmasso p, lazzeri g, giacchi m, cavallo f. socioeconomic effects on meeting physical activity guidelines: comparisons among 32 countries. med sci sports exerc. 2009; 41(4): 749–56. 15. gatta ea, al-alousi ws, diab bs. primary teeth emergence in relation to nutritional status among 4-48 months old children in baghdad city. mustansiria dent j. 2008; 5(1): 62–70. 16. must a, phillips sm, tybor dj, lividini k, hayes c. the association between childhood obesity and tooth eruption. obesity. 2012; 20(10): 2070–4. 17. hedayati z, khalafinejad f. relationship between body mass index, skeletal maturation and dental development in 6to 15year old orthodontic patients in a sample of iranian population. j dent (shiraz, iran). 2014; 15(4): 180–6. 18. psoter w, gebrian b, prophete s, reid b, katz r. effect of early childhood malnutrition on tooth eruption in haitian adolescents. community dent oral epidemiol. 2008; 36(2): 179–89. 19. elamin f, liversidge hm. malnutrition has no effect on the timing of human tooth formation. plos one. 2013; 8(8): 72274. 20. eid rmr, simi r, friggi mnp, fisberg m. assessment of dental maturity of brazilian children aged 6 to 14 years using demirjian’s method. int j paediatr dent. 2002; 12(6): 423–8. 21. costacurta m, di renzo l, bianchi a, fabiocchi f, de lorenzo a, docimo r. obesity and dental caries in paediatric patients. a cross-sectional study. eur j paediatr dent. 2011; 12(2): 112–6. 22. werner sl, phillips c, koroluk ld. association between childhood obesity and dental caries. pediatr dent. 2012; 34(1): 23–7. 23. kantovitz kr, pascon fm, rontani rmp, gavião mbd. obesity and dental caries--a systematic review. oral health prev dent. 2006; 4(2): 137–44. 24. tramini p, molinari n, tentscher m, demattei c, schulte ag. association between caries experience and body mass index in 12year-old french children. caries res. 2009; 43(6): 468–73. 25. hong l, ahmed a, mccunniff m, overman p, mathew m. obesity and dental caries in children aged 2-6 years in the united states: national health and nutrition examination survey 1999-2002. j public health dent. 2008; 68(4): 227–33. 26. kopycka-kedzierawski dt, auinger p, billings rj, weitzman m. caries status and overweight in 2to 18-year-old us children: findings from national surveys. community dent oral epidemiol. 2008; 36(2): 157–67. 27. macek md, mitola dj. exploring the association between overweight and dental caries among us children. pediatr dent. 2006; 28(4): 375–80. 28. jamelli sr, rodrigues cs, de lira pi. nutritional status and prevalence of dental caries among 12-year-old children at public schools: a case-control study. oral health prev dent. 2010; 8(1): 77–84. 29. sudhakar, rsharath p, shanthi m, fareed n, sudhir k. relationship between dentition status and body mass index among 5 to 15 years old age group children of an orphanage in nellore city. j indian assoc public heal dent. 2010; 8(15): 45–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p140–143 https://wwwn.cdc.gov/nchs/nhanes/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p140-143 isi vol 39 no 2 april 2006 file pertama.pmd 77 the change of temperature on the shear strength of permanent soft-liner on acrylic resin waloejo noegroho*, utari kresnoadi**, and adi subianto** * staff of bhayangkara hospital, polda jatim ** department of prostodontic, faculty of dentistry airlangga university surabaya – indonesia abstract the characteristics of dental material such as solubility and water resorption, the use of adhesive, storage condition or used as thermo cycling or elevated-temperature are factors that can effect bond strength. the purpose of this study was to investigate the change of temperature on the shear strength of permanent soft liner on acrylic resin. twenty-four specimens were divided into 3 groups and immersed in water at: 5 °c, 37 °c and 55 °c. autograph ag 10 te shimadzu was used to determine the shear strength. the statistical test (anova and lsd); showed that there were significant differences between temperature groups. the shear strength of 37 °c was higher than the temperature of 5 °c and 55 °c. key words: temperature, shear strength, permanent soft liner, acrylic resin correspondence: utari kresnoadi, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. intoduction making acrylic removable denture requires soft liner material to overcome post-denture insertion problem such as denture unsteadiness due to the resorption of alveolar bone, and relief the traumatic pain. it is a cushion between the denture base and the gingival underneath. the aim of using soft liner is to decrease the denture mastication force and distribute it to the underneath tissue.1,2 soft liner is a soft, elastic, and spongy material which is used to line all or part of denture surface.3 it could avoid the pressure on one spot which could traumatized the underneath tissues.2–4 this material has been widely used for more than a century and the first soft liner was made of natural rubber. in 1945, polyvinyl plastic, a synthetic resin was utilized as soft liners and in 1958 silicon elastomer was introduced.4 hamada et al.5 divided soft liner materials as follows (figure 1). the main problem often encountered in permanent soft liner usage was the change or increased temperature during utilization which affected the bonding of soft liner and acrylic denture.3 there is a possibility that the patients had drinking habit with specific temperatures either cold or hot. the aim of the research was to examine the effect of different immersed temperature on the shear strength of permanent soft-liner self-cured on the acrylic resin denture base. figure 1. denture liners material scheme. 5 acrylic vinyl acrylic cold curing heat curing light curing cold curing heat curing heat curing heat adhesive light curing silicone fluoric polyolephin isoprene denture liners hard lining material soft lining material resilient denture liners light curing cold curing single paste single paste powder/liquid powder/liquid direct reline resins periphery resins for border molding tissue conditioner + dynamic impression material 78 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 77–79 materials and methods the research was done at prosthodontics department of airlangga university faculty of dentistry and at basic laboratory of airlangga university. materials used were heat-cured acrylic (adm brand, america) permanent soft liner (gc reline extra soft, japan), sterile aquadest, red paraffin (cavex, holland), hard gypsum (moldano, bayer, jerman), soft gypsum (super france) cellophane plastic, and couls mould seal. instruments used were a refrigerator for the 5 ºc temperature (toshiba, japan); waterbath for the 55 ºc (memert, japan); autograph ag 10 te. (shimadzu, japan) to measure shear strength with its modification apparatus; porcelain pot to mix acrylic, metal cuvette, bench press hydraulic, vibrator (yoshida, japan), digital stick thermometers, and becker glass. the specimen was made from a master model of a metal plate which was specifically designed, cylinder formed, with 8 mm diameter, 2 mm thickness, and it had a hole in the middle of the cylinder with the diameter of 1 mm (figure 2). each group consisted of 8 samples and each was immersed in aquadest with the temperatures of 5 ºc, 37 ºc and 55 ºc. the sample immersion was done for two days which was equal to 1 year utilization, assuming that the patients drank twice a day, every time they drank was in 4 minutes duration, in one year (365 days) = 365 × 8 minutes = 2920 minutes = 2920 : 1440 = 2 days in body temperature 37 ºc, cold 5 ºc or warm 55 ºc, the immerse aquadest was replaced every day.6 using autograph ag 10 te shimadzu, the shear strength test was examined by way of: adapted-to-template specimen was precisely positioned so that the assisting instrument could be sealed without any gap. a pair of assisting instruments was put into holder tube, until the fixated dowel could lock the hook at the upper end, while the metal holder at the lower end could be clamped to the measuring machine. the machine was revived; there was a pulling ties/bonding on assisting instrument made of metal brass. the magnitude of shear strength was calculated with shear strength formula: (π) = f/dh. f = unloaded strength to release specimen d = specimen diameter h = specimen thickness (mm) the results data was tabulated and analyzed with twoway anova, after a normal distribution test with kolmogorov smirnov was done. multiple comparisons were carried out with lsd. result shear strength mean score was presented in table 1. afterwards, specimen as a result of curing process was cleaned and dried to remove dirt or fat, and assisting instrument was installed. permanent soft liner mixed according to manufacturer’s manual was inserted to other pair’s assisting apparatus. then, both surfaces were bonded and fixated for 5 minutes (following manufacturer’s manual) (figure 3). specimen was released by pushing their stalk holders. figure 2. blueprint of specimen final form. note: 1. permanent soft liner, 2. acrylic resin 1 2 figure 3. profile plane of an assisting instrument. note: 1. grooved and hooked stalk 2. holder tube 3. specimen location 4. fixating dowel 5. pair tube 6. metal hook table 1. the mean score ( x ) and standard deviation (sd) of a statistical test of permanent soft liner shear strength on the basis of acrylic dentures, after immersion in sterile aquadest with temperatures 5 ºc, 55 ºc, 37 ºc observed for 2 days (kg/mm2) temperature n mean standard deviation 5 ± 1 ºc 55 ± 1 ºc 37 ± 1 ºc 8 8 8 47.01 50.71 54.43 1.96 1.86 2.51 prior to statistical test, a normal distribution test kolmogorov smirnov was carried out with p = > 0.05 and the result showed that samples were in normal distribution. afterwards, two-way anova test gave result p = 0.01 < 0.05 showed a significant difference. later, an lsd test was done as presented in table 2. 79noegroho et al.: the change of temperature table 2. result of lsd test of temperature groups during two days immersion (p < 0.05) immersion temperature 5 ºc 55 ºc 37 ºc 5 ºc 55 ºc 37 ºc 0.01* 0.01* 0.01* 0.01* 0.01* 0.01* note: *: significant difference was present table 2 showed the differences between immersion groups 5 ºc and 55 ºc where p = 0.01 < 0.05, meaning that there was a significant difference. also, in immersion group with 5 ºc and 37 ºc, p = 0.01 < 0.05 there was a significant difference. and in immersion group with 37 ºc and 55 ºc, p = 0.01 showed a significant difference. discussion the statistical analysis (table 2) revealed a significant difference of shear strength (p < 0.05) in groups of 5 ºc, 55 ºc and 37 ºc. the result of shear strength of group 5 ºc showed the lowest value compared to group 55 ºc and 37 ºc. several possible reasons for the low shear strength from group 5 ± 1 ºc were: 1) the difference of heat expansion coefficient between acrylic denture basis material (pmma) and silicon elastomer. acrylic heat expansion coefficient was 81 × 10-6/ ºc, 7 while the heat expansion coefficient of silicon elastomer was 190 × 10-6/ ºc.8 this was in accordance with combe7 and al-athel and jagger’s 9 opinions that having a different heat expansion coefficient would result in making temperature enable to influence the dimension change of a material; 2) sudden material shrinkage, causing micro leakage in the interface area.9-10 for the 55 ± 1 ºc grou p (table 1), in general it had lower shear strength compared to control group (37 ± 1 ºc). besides the difference of two bonded materials with different heat expansion factors, another possibility might be caused by silicon elastomer which experienced expansion in high temperature. this was in accordance with wright’s11 that although silicon‘s transition temperature was very low, in sensitive to temperature’s change, yet at 40 ºc it already decreased the bonding strength because of simultaneous expansion in which cause water resorption would occur to filler contents. thus, water could directly enter the bonding area, causing swelling and pressure to the interface that decreased the bonding strength.12,13 the material swelled and the interface was pressured, or it could also caused by elasticity change of the resilient lining, which produced material stiffness and external load flow towards the bonding.12,13 the research out come should be informed to the patients that low (5 ± 1 ºc) and high (55 ± 1 ºc) temperatures could decrease permanent soft liner self-cured shear strength. soft liner material low shear strength could bring about a failure in bonding and shorter duration of utilization. this condition was likely to generate bacterial growth, plaque, and calculus forming, so that evaluation of soft liner changes must be done correctly or even periodical soft liner changes.12 from the result of the influence of temperature to the shear strength of permanent soft liner on acrylic resin, a conclusion was derived that there was a significant difference of shear strength among immersion groups in sterile aquadest of 5 ºc, 55 ºc and 37 ºc, whereas the shear strength of the low temperature (5 ºc) and high temperature groups (55 ºc) were lower than the control group (37 ºc). references 1. huggett r. soft lining materials in prosthetic dentistry: a review. j prosthet dent 1990; 3(5): 477–83. 2. anusavice kj. 1996. phillip’s ilmu bahan kedokteran gigi. budiman ja, purwoko s. jakarta: egc; 2004. h. 197–233. 3. hadary ae, drummond jl. comparative study of water sorption, solubility, and tensile bond strength of soft lining material. j prosthet dent 2000; 83(3): 356–61. 4. baysan a, parker s, wright ps. adhesion and tear energy of a longterm soft lining material activated by rapid microwave energy. j prosthet dent 1998; 79(2): 182–7. 5. hamada t, murata a, razak a. pelapisan gigi tiruan. cetakan i. surabaya: airlangga university press; 2003. h. 54–62. 6. inayati e. perbedaan jumlah candida albicans pada permukaan resin akrilik heat cured setelah perendaman dalam larutan kopi dan teh hijau. majalah kedokteran gigi (dental j) 2001; 34(1): 10–2. 7. combe ec. notes on dental materials. 5th ed. edinburgh: churchill livingstone; 1986. p. 255–73. 8. mc cabe jf. anderson’s applied dental materials. 7th ed. oxford, london, edinburg: blackwell scientific publication. 1990. p. 78–103. 9. al-athel ms, jagger rg. effect of test method on the bond strength of a silicone resilient denture lining material. j prosthet dent 1996; 76: 535–40. 10. anil n, canan h, nesrin b, meral te. microleakage study of various soft denture liners by autoradiography effect of accelerated aging. j phosthet dent 2000; 84(4): 394–9. 11. wright ps. characterization of the rupture properties of denture soft lining materials. j dent res 1980; 59(3): 614–24. 12. pinto jrr, mesquita mf, henriques gep, nobilo maa. effect of thermocycling on bond strength and elasticity of 4 long-term soft denture liners. j prosthet dent 2002; 88(5): 516–21. 13. ozkan yk, sertgoz a, gedik h. effect of thermocyling on tensile bond stregth of six silicone-based, resilient denture liners. j prosthet dent 2003; 89(3): 303–10. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 56 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 56–61 review article exercise as a method to reduce the risk of oral cancer: a narrative review anis irmawati1, lia aulia rachma2, sidarningsih1, muhammad naufal hatta2, ira arundina1, mohammed aljunaid3 1department of oral biology, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3department of dental medicine, faculty of medicine, university of taiz, taiz, yemen abstract background: cancer is a major cause of death worldwide. one of the most common forms of cancer is oral cancer, which can occur due to exposure to carcinogenic factors, such as tobacco cigarettes, alcohol, betel-nut chewing, ultraviolet rays or human papillomavirus infection. physical exercise is known to have many benefits and can contribute to reducing the risk of cancer, minimising the side-effects of treatment and increasing the curative effect of cancer treatment. purpose: this study aimed to explain the role of exercise as a method to reduce oral cancer risk. reviews: studies examining the impact of exercise on reducing oral cancer risk are currently limited due to a lack of research on this subject. however, according to several laboratory experimental research studies on mus musculus test subjects, moderate-intensity exercise contributes to suppressing the proliferation and development of oral squamous epithelial cells, which can subsequently become cancer cells. exercise can also increase intracellular proteins that can induce apoptosis in cells (e.g. wild protein p53, the ratio of bax/bcl-2, and caspase-3), and can also decrease p53 mutant expression and transformed cells that can trigger cancer. exercise must be optimally performed to prevent or control cancer symptoms, although the exact duration and intensity of exercise required to reduce cancer risk in humans have not been established. conclusion: exercise plays a role in reducing oral cancer risk by inducing apoptosis and preventing the development of transformed cells that can lead to developing cancer. keywords: apoptosis; exercise; oral cancer; oral squamous cell carcinoma; physical activity; moderate-intensity exercise; transform cells; wild p53 correspondence: anis irmawati, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: anis-m@fkg.unair.ac.id introduction cancer is a major cause of death worldwide. according to the global cancer observatory, 19.3 million new cases of cancer and roughly 10 million deaths are estimated to have occurred worldwide in 2020.1 in 2018, riset kesehatan dasar reported that indonesia had a cancer prevalence of 1.79 per 1000 people, an increase compared with 2013 at 1.4 per 1000 people. as a result, indonesia has risen to the eighth position in the list of countries in southeast asia with the most cancer cases.2 head and neck cancer cases include approximately 60%–70% of cases occurring in the oral cavity and larynx.3 in 2020, oral and lip cancers included 377,713 new cases and 177,757 deaths.1 in indonesia, the five-year prevalence for oral and lip cancer cases is 5.19 per 100,000 population.4 squamous cell carcinoma accounts for more than 90 malignancies that occur in the oral cavity.5 most malignancies arise from a complex and multifactorial aetiology that includes genetic, environmental and lifestyle factors, as well as the interconnections between them.6 the excessive use of tobacco, alcohol and betel nut chewing are among the primary causes of oral cancer.7 if the body’s cells are exposed to these substances, cell mutations will occur. the mutated cells will grow abnormally and develop uncontrollably, forming tumours and giving rise to cancer. many studies have discussed exercise as an effective treatment and rehabilitation option for oral cancer. in addition to the various existing medical treatments, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 mailto:anis-m@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 57irmawati et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 56–61 discussing prevention measures is also essential. ample evidence suggests that exercise provides many health benefits; hence, exercise is a common method that is used in health promotion settings because it is not cost-prohibitive and is safe and feasible.8 exercise has been proven to improve both physical and mental health. in addition to improving cardiovascular function and supporting weight loss, as well as strengthening skeletal muscles, regular exercise also inhibits the formation of transformed cells commonly found in the presence of cancer.9 transformed cells are normal cells that undergo behavioural changes due to dna mutations; as a result, these transformed cells cannot die and proliferate abnormally, subsequently forming tumour tissue.10,11 exercise has been found to lower the risk of breast, colorectal, endometrial, bladder, oesophageal, and kidney cancers. moderate evidence is associated with lung cancer risk, with relative risk rates decreasing by 10%–20%. due to a lack of research on head and neck malignancies, the evidence supporting a reduced risk of cancer remains limited.6 this paper aims to explain the role of exercise as a method for reducing oral cancer risk. oral cancer oral cancer is a malignancy that occurs in the lips, oral cavity, tongue, gingiva, oropharynx, hypopharynx and other oral mucosa but does not include the nasopharynx or the major salivary glands.12 men are much more likely than women to develop oral cancer, as represented by a 5.5–2.5 ratio for every 10,000 people worldwide in 2020.13 the incidence of oral cancer increases with age. according to the american cancer society (2021), the average age of patients diagnosed with oral cancer is 63. however, this type of cancer can also develop among young people, with 1 in 5 cases (20%) occurring in patients younger than 55.14 oral cancer is a disease with multifactorial causes. some of the risk factors for oral cancer include smoking, consuming alcohol, viral infections, e.g. the human papillomavirus or hepatitis b, betel-nut chewing, ultraviolet radiation, the presence of pre-cancerous lesions, including leukoplakia, erythroplakia and lichen planus, immunosuppressed conditions, and a genetic predisposition. tobacco cigarettes contain benzopyrene and nitrosamines, which are carcinogenic and result in dna mutations when exposed to human cells.13,15,16 oral cancer, also known as oral squamous cell carcinoma (oscc), is a malignant tumour that develops on the lips and oral cavity. oscc cancers, which target the epithelial cells of the oral cavity and oropharynx, contribute to 90% of all oral cancers.17 in the early stages, oscc often presents as very small, painless or asymptomatic. pain is a typical symptom in people with oral cancer; however, it generally develops only when the tumour has grown to a significant size. notably, in slower-growing and more widespread lesions on the tongue, symptoms may range from slight discomfort to a severe ache. ear discomfort, bleeding, tooth movement, difficulty breathing, dysphagia, trismus and numbness are other symptoms. the clinical features of oscc vary. in the early stages, lesions are typically leukoplakia, erythroplakia or erythroleukoplakia. lesions present as slightly rough and coarsely circumscribed, defined red or red and white areas. in advanced oscc lesions, lesions may be ulcerated, nodular (exophytic) or fixed to the underlying tissue (endophytic) (figure 1).18 the ulcer form is the most common type of oscc.17,19,20 oral cancer can impact the patient’s quality of life by affecting their physical, mental and economic well-being. oral cancer patients may experience speech and swallowing dysfunction, facial and vocal changes, sensory disturbances, and prolonged pain, leading to poor mental health.21 tumour suppressor gene p53 the p53 gene is a tumour suppressor gene. cell-cycle arrest, cell ageing and dna repair are all controlled by the p53 gene. deoxyribonucleic acid damage will activate ataxiatelangiectasia mutated, which phosphorylates and releases p53 from mouse double minute 2 homolog (mdm2) binding, causing the amount of p53 proteins to increase. increased p53 proteins will activate the p21 gene, which works as a cyclin-dependent kinase inhibitor to stop the cell cycle in damaged cells. after cell cycle arrest, damaged figure 1. an exophytic form with a central depression and widespread keratosis on the left lateral side of the tongue characterises a well-differentiated squamous cell carcinoma lesion.18 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 58 irmawati et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 56–61 cells can be repaired.22,23 if dna damage is extensive and irreversible, p53 can initiate apoptosis. additionally, p53 stimulates cytochrome-c from mitochondria by activating apoptotic genes, e.g. bax, and by inhibiting anti-apoptotic genes, such as bcl-2. cytochrome-c triggers caspases, which subsequently activate deoxyribonuclease (dnase), an enzyme which penetrates the cell nuclear membrane and damages dna, causing cell death.22,24 therefore, cells with mutations in the p53 gene can turn into cancerous cells. a mutated p53 gene is associated with 50% of cancer cases.25 one study noted that the expression of the p53 gene increased in 63% of oscc cases.26 the altered p53 gene, also known as mutant p53, caused a reduction or loss of wild p53 gene activity, resulting in the build-up of dna damage and the cell’s inability to repair dna and undergo apoptosis.27 apoptosis apoptosis is a form of programmed cell death that aims to preserve a balance between living and dead cells, both physiologically and pathologically. apoptosis can be triggered by dna damage, uncontrolled cell proliferation, stressful or toxic conditions, and several diseases.28 where cancer is present, cell division and death are out of balance. because the p53 gene is downregulated or inactivated, cells that should undergo apoptosis do not receive an apoptotic signal. the cells cannot experience death, and the growth and development of the cells lead to increased tumours.24 pro-apoptotic and antiapoptotic genes influence the incidence of apoptosis. pro-apoptotic genes stimulate the release of mitochondrial-derived cytochrome-c enzymes (e.g. bax and bak). antiapoptotic genes, on the other hand, act to prevent the release of cytochrome-c enzymes (e.g. bcl-2).29 there are several pathways for initiating apoptosis (figure 2). when a cell recognises harm from within via various intracellular signals, known as the intrinsic pathway, apoptosis can be triggered. in contrast, apoptosis can occur from interactions with immune system cells or other damaged cells, known as the extrinsic pathway.30 the two pathways can meet to activate caspases. there are two groups of caspases, i.e. initiators and executors. if cell damage is detected, and apoptosis is stimulated, initiator caspases (caspases 8 and 9) will be activated by procaspases. initiator caspases will then activate the executor caspases (caspases 3 and 6). the disintegration of dna, the destruction of the cell nucleus and the cytoskeleton, protein cross-linking, the production of ligands for phagocytic immune cells and the development of apoptotic bodies, which are eventually phagocytised by macrophages, are all caused by the activation of the executor caspase.24,29,31 exercise exercise is a structured, systematic and persistent physical activity that is engaged in to improve or maintain physical fitness. exercise should consider the co-called fitt principles, i.e. frequency, intensity, time and type. the frequency is the number of times exercise is completed within a certain period. intensity is the degree to which the exercise or physical activity is completed. the intensity is determined according to the heart rate (hr) percentage, i.e. the maximum oxygen volume (vo2max) and the maximum work capacity (mc) of the muscles. based on hr, vo2max, and mc, exercise intensity is classified as mild (0%–50%), moderate (50%–70%), submaximal (70%–85%) and maximal (above 85%).32 the time aspect refers to the duration of the exercise. there are figure 2. the apoptotic mechanism. two pathways can initiate apoptosis, i.e. the intrinsic and extrinsic pathways.28 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 59irmawati et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 56–61 two types of exercise times, i.e. continuous and interval times. continuous time refers to a period longer than 30 minutes, with mild to moderate intensity, and the interval is performed alternately between activity and rest, with submaximal and maximum intensity. type refers to the type of exercise that is performed, such as walking, jogging, cycling, swimming and aerobics.32,33 exercise is divided into aerobic and anaerobic types, based on the amount of oxygen consumption or the dominant energy system used. aerobic exercise is a type of continuous, mild-to-moderate-intensity exercise, such as walking, jogging, running, swimming and cycling. conversely, anaerobic exercise is a form of high-intensity exercise that requires energy quickly within a short time and cannot be done continuously for an extended period.34 regular exercise can help to relieve stress, anxiety and depression, as well as lower blood pressure and the risks of cardiovascular disease, stroke, certain malignancies and diabetes.35 many studies have underscored the role of exercise in lowering the risk of various malignancies. exercise has been shown to lower the risk of cancer in the breast, colon, endometrium, and urinary bladder, as well as adenocarcinoma in the oesophagus and kidney.6 exercise activities can open ca2+ channels in cell membranes, then activate mitogen-activated protein kinase (mapk), which can enter the cell nucleus and act as transcription factors, one of which is wild p53. increased wild p53 expression activates bax messenger ribonucleic acid (mrna) transcription, allowing bax protein production as a proapoptosis factor for increasing and triggering apoptosis in cells.32,36,37 discussion exercise is a physical activity that has been shown to widely affect the quality of life and physical health of people. programmed exercise can be used as a cancer prevention method, to improve physical fitness and prevent several chronic diseases.38 exercise helps lower the risk of cancer, reduces treatment-related adverse effects and can improve cancer treatment’s curative impact. according to hojman et al., several systemic factors, such as sympathetic stimulation, increased blood flow, tissue stress and elevated body temperature are experienced during exercise, which can put direct pressure on tumour homeostasis and metabolism.39 by decreasing cancer cell growth, signalling apoptosis, controlling cancer metabolism and modulating immunological conditions, exercise provides benefits for preventing cancer. when followed by long-term exercise, the acute effects on the body during exercise cause adaptations in intratumoral tissue, i.e. increased blood perfusion, immunity and metabolic adjustments that slow tumour progression.39 exercise has many benefits for people with cancer, such as an improved quality of life, an increased chance of recovering from disease and improved physical abilities.40 however, due to a lack of research on this issue, studies concerning the benefits of exercise for lowering oral cancer risk is currently limited. exercise can induce apoptosis in cancer cells because of the increased mobilisation of natural killer cells towards tumour tissue. natural killer cells are immune cells that are particularly sensitive to exercise and play a role in destroying infected cells or cells that have been converted into cancer cells.41,42 several studies have discussed the effect of exercise in preventing the transformation of cells in the mucosal cells in the mouth, i.e. squamous epithelial cells. in mus musculus test subjects, moderate-intensity exercise (swimming) significantly increased the ratio of bax/bcl-2 (n = 6, p = 0.00) and caspase-3 expression (n = 18, p = 0.00) in oral squamous epithelial cells that had been injected with benzopyrene, a carcinogenic substance commonly found in cigarettes.43,44 the bax/bcl-2 ratio, as well as caspase-3, are components in cells that, when their expression increases, receive a signal to perform apoptosis. exercise can open ca2+ channels in cell membranes, thus increasing ca2+ concentration. increased intracellular ca2+ activates signal transduction ras-gap (gtpase activating protein) and src, thus activating mapk. activated mapk will enter the cell nucleus and initiate the wild p53.36,37,45 wild p53 expression will activate bax mrna transcription to increase the bax expression (bax/bcl-2 increases). the permeability of the outer mitochondrial membrane increases when bax/bcl-2 levels rise. this causes the release of cytochrome-c, smac/diablo, an apoptotic-inducing factor, and htra2/omi, a group of pro-apoptotic proteins from the mitochondria into the cytoplasm. these released proteins will activate caspase-9, thus conforming to the apoptotic pathway and activating caspase-3, which will act as an executor for inducing cells to undergo apoptosis.24,31,46 another study stated that moderate-intensity exercise could significantly increase the expression of wild p53 (n = 18, p = 0.611), decrease the expression of the p53 mutant (n = 18, p = 0.00) and reduce the number of transformed cells (n =18, p = 0.0874) in the oral squamous epithelial cells of mus musculus test subjects that had been injected with benzopyrene.11,47,48 the p53 mutant is a p53 gene that undergoes mutation; the p53 mutant is capable of causing the loss of function of the wild p53 gene. the absence of the wild p53 gene due to mutant p53 causes genetic instability and stops the apoptotic process from occurring.49 if the apoptotic process cannot be activated, cells that have been mutated due to exposure to carcinogenic agents continue to proliferate. the mutated cells will pass on the genetic trait to the successor cell. transformed cells are normal cells that undergo behavioural changes caused by the transcription of an oncogene. normal cells that transform into cancer cells result from a disrupted cell cycle or regulatory system, causing cancer cells to multiply uncontrollably.11,48 according to the present studies, moderate-intensity exercise inhibits the development of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 60 irmawati et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 56–61 oral squamous epithelial cells that have the potential to become cancer cells. exercise appears to have a role in lowering oral cancer risk, according to the studies described above. oral cancer risk can be considerably reduced by moderate-intensity exercise, which can potentially cause apoptosis by elevating pro-apoptotic expressions, such as the ratio of bax/bcl-2 and caspase-3. in addition, moderate-intensity exercise is beneficial for inhibiting the development of transformed cells that can turn into cancer. the duration, type and intensity of exercise are essential for cancer prevention. the exact duration and intensity of exercise required to reduce oral cancer risk in humans have not been established. however, experts recommend that to prevent the occurrence of cancer, at least 150–300 minutes of moderate-intensity exercise per week, or the equivalent of 75–150 minutes of vigorous-intensity exercise should be completed; exceeding more than 300 minutes of vigorousintensity exercise per week is not recommended.50 in conclusion, exercise can help to reduce the risk of cancer, treatment-related adverse effects and improve the cancer treatment’s curative impact. however, research on exercise’s efficacy in reducing the risk of oral cancer remains limited due to a lack of research on the subject. although many studies have shown the effect of moderateintensity exercise in inhibiting the development of cells that can lead to cancer and induce apoptosis in mus musculus study subjects, no research has indicated that exercise reduces the incidence of oral cancer in humans. in addition, further research must be conducted regarding the exact amount of exercise required to prevent oral cancer in humans specifically. references 1. sung h, ferlay j, siegel rl, laversanne m, soerjomataram i, jemal a, bray f. global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2021; 71(3): 209–49. 2. badan penelitian dan pengembangan kesehatan. hasil utama r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 66–71. 3. heroiu cataloiu a-d, danciu ce, popescu cr. multiple cancers of the head and neck. maedica (buchar). 2013; 8(1): 80–5. 4. ferlay j, ervik m, lam f, colombet m, mery l, piñeros m, znaor a, soerjomataram i, bray f. global cancer observatory: cancer today. lyon, france: international agency for research on cancer. 2020. available from: https://gco.iarc.fr/today. accessed 2021 sep 19. 5. subramani k, ahmed w. nanoparticulate drug-delivery systems for oral cancer treatment. in: emerging nanotechnologies in dentistry. elsevier; 2018. p. 355–70. 6. mctiernan a, friedenreich cm, katzmarzyk pt, powell ke, macko r, buchner d, pescatello ls, bloodgood b, tennant b, vaux-bjerke a, george sm, troiano rp, piercy kl, 2018 physical activity guidelines advisory committee*. physical activity in cancer prevention and survival: a systematic review. med sci sports exerc. 2019; 51(6): 1252–61. 7. scully c, robinson na. oral cancer. in: international encyclopedia of public health. 2nd ed. elsevier; 2017. p. 348–58. 8. warburton der, bredin ssd. health benefits of physical activity: a systematic review of current systematic reviews. curr opin cardiol. 2017; 32(5): 541–56. 9. shadap a. physical activity to stay fit. j heal allied sci nu. 2021; 11(1): 8–11. 10. wang-michelitsch j, michelitsch tm. cell transformation in tumor-development: a result of accumulation of misrepairs of dna through many generations of cells. arxiv: 150501375. 2015; : 1–14. 11. irmawati a, hadi sr, haris a, rahaju rp, budhy ti, balqis nf. the role of continuous moderate exercise on hsp70 expression and the transform cell number on oral squamous cell mus musculus injected by benzopyrene. pollut res. 2019; 38: s91–7. 12. permasutha mb. tinjauan atas kanker rongga mulut. cdk-293. 2021; 48(3): 133–7. 13. salehiniya h, raei m. oral cavity and lip cancer in the world: an epidemiological review. biomed res ther. 2020; 7(8): 3898–905. 14. american cancer society. key statistics for oral cavity and oropharyngeal cancers. oral cavity (mouth) and oropharyngeal (throat) cancer. 2021. p. 7–10. 15. ogbureke kue, bingham c. overview of oral cancer. in: ogbureke kue, editor. oral cancer. rijeka: intech; 2012. p. 1–12. 16. kumar m, nanavati r, modi tg, dobariya c. oral cancer: etiology and risk factors: a review. j cancer res ther. 2016; 12(2): 458–63. 17. rivera c. essentials of oral cancer. int j clin exp pathol. 2015; 8(9): 11884–94. 18. shah jp, johnson nw. oral and oropharyngeal cancer. 2nd ed. shah m, johnson c, editors. oral and oropharyngeal cancer. boca raton: crc press; 2018. p. 112. 19. bagan j, sarrion g, jimenez y. oral cancer: clinical features. oral oncol. 2010; 46(6): 414–7. 20. rosebush ms, rao sk, samant s, gu w, handorf cr, pfeffer lm, nosrat ca. oral cancer: enduring characteristics and emerging trends. j tenn dent assoc. 2011; 91(2): 24–7; quiz 28–9. 21. valdez ja, brennan mt. impact of oral cancer on quality of life. dent clin north am. 2018; 62(1): 143–54. 22. yunani r. kajian peran gen p53 dalam tumorigenesis. vitek bid kedokt hewan. 2018; 6: 11–6. 23. joyce c, rayi a, kasi a. tumor-suppressor genes. statpearls. statpearls publishing; 2022. 24. wong rsy. apoptosis in cancer: from pathogenesis to treatment. j exp clin cancer res. 2011; 30: 1–14. 25. sun z. the general information of the tumor suppressor gene p53 and the protein p53. j cancer prev curr res. 2015; 3(1): 119–22. 26. ghanghoria s, ghanghoria a, shukla a. p53 expression in oral cancer: a study of 50 cases. j pathol nepal. 2015; 5(9): 747–51. 27. muller paj, vousden kh. mutant p53 in cancer: new functions and therapeutic opportunities. cancer cell. 2014; 25(3): 304–17. 28. kumar v, abbas ak, aster jc. robbins and cotran pathologic basis of disease. 9th ed. philadelphia: elsevier saunders; 2015. p. 73–91. 29. mohan h. textbook of pathology. 7th ed. new delhi: jaypee brothers medical publishers; 2014. p. 39–41. 30. d’arcy ms. cell death: a review of the major forms of apoptosis, necrosis and autophagy. cell biol int. 2019; 43(6): 582–92. 31. fernald k, kurokawa m. evading apoptosis in cancer. trends cell biol. 2013; 23(12): 620–33. 32. irmawati a. mekanisme hambatan pembentukan sel transform pada sel epitel skuamosa rongga mulut akibat latihan renang intensitas sedang (pada mus musculus strain swiss webster yang dipapar benzopyrene). [surabaya]: universitas airlangga; 2018. 33. yasirin a, rahayu s, junaidi s. latihan senam aerobik dan peningkatan limfosit cd4 (kekebalan tubuh) pada penderita hiv. j sport sci fit. 2014; 3(3): 1–6. 34. palar cm, wongkar d, ticoalu shr. manfaat latihan olahraga aerobik terhadap kebugaran fisik manusia. j e-biomedik. 2015; 3(1): 316–21. 35. abou elmagd m. benefits, need and importance of daily exercise. int j phys educ sport heal. 2016; 3(5): 22–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 https://gco.iarc.fr/today https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 61irmawati et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 56–61 36. cheng w, zhu y, wang h. the mapk pathway is involved in the regulation of rapid pacing-induced ionic channel remodeling in rat atrial myocytes. mol med rep. 2016; 13(3): 2677–82. 37. hawley ja, hargreaves m, joyner mj, zierath jr. integrative biology of exercise. cell. 2014; 159(4): 738–49. 38. kesting s, weeber p, schönfelder m, renz bw, wackerhage h, von luettichau i. exercise as a potential intervention to modulate cancer outcomes in children and adults? front oncol. 2020; 10: 196. 39. hojman p, gehl j, christensen jf, pedersen bk. molecular mechanisms linking exercise to cancer prevention and treatment. cell metab. 2018; 27(1): 10–21. 40. lanje ah, misurya r, vaid s, sargaiyan v, purohit m, purohit n. oral cancer and physical activity. kumar a, editor. int j oral care res. 2017; 5(3): 249–51. 41. wang q, zhou w. roles and molecular mechanisms of physical exercise in cancer prevention and treatment. j sport heal sci. 2021; 10(2): 201–10. 42. idorn m, hojman p. exercise-dependent regulation of nk cells in cancer protection. trends mol med. 2016; 22(7): 565–77. 43. irmawati a, jasmin n, sidarningsih. the effect of moderate exercise on the elevation of bax/bcl-2 ratio in oral squamous epithelial cells induced by benzopyrene. vet world. 2018; 11(2): 177–80. 44. irmawati a, pamita bg, soesilawati p. the influence of moderate exercise on caspase-3 expression in inhibiting transformation of oral squamous epithelial cells. j int dent med res. 2018; 11: 285–8. 45. combes a, dekerle j, webborn n, watt p, bougault v, daussin fn. exercise-induced metabolic fluctuations influence ampk, p38mapk and camkii phosphorylation in human skeletal muscle. physiol rep. 2015; 3(9): e12462. 46. sari lm. apoptosis: mekanisme molekuler kematian sel. cakradonya dent j. 2018; 10(2): 65–70. 47. irmawati a, muljono hj, ketut sudiana i. the gadd45 and wild p53 expressions resulting from moderate swimming exercise on mus musculus injected by benzopyrene. j int dent med res. 2019; 12(3): 964–8. 48. irmawati a, handayani atw, balqis nf, surboyo mdc. the decreased of p53 mutant expression on squamous cell epithelial of oral in mus musculus by moderate intensity of exercise. malaysian j med heal sci. 2020; 16(supp 4): 1–5. 49. zhu g, pan c, bei j-x, li b, liang c, xu y, fu x. mutant p53 in cancer progression and targeted therapies. front oncol. 2020; 10: 595187. 50. patel a v., friedenreich cm, moore sc, hayes sc, silver jk, campbell kl, winters-stone k, gerber lh, george sm, fulton je, denlinger c, morris gs, hue t, schmitz kh, matthews ce. american college of sports medicine roundtable report on physical activity, sedentary behavior, and cancer prevention and control. med sci sports exerc. 2019; 51(11): 2391–402. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p56–61 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p56-61 229229 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 229–234 research report lemuru fish oil gel as host modulation therapy in periodontal ligaments induced with porphyromonas gingivalis widyastuti,1 dian widya damaiyanti,2 dian mulawarmanti,2 cindy aprilia sari2 and diah ayu siwi1 1department of periodontics, 2department of oral biology, faculty of dentistry, universitas hang tuah, surabaya – indonesia abstract background: periodontitis affects approximately 20%–50% of the global population and is caused by gram-negative bacteria, such as porphyromonas gingivalis (p. gingivalis). host modulation therapy (hmt) is part of a periodontal therapy that is used as an adjunct to conventional periodontal treatment to reduce tissue damage. lemuru fish oil containing eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) can reduce the formation of matrix metalloproteinase species (mmps) and will further increase the number of fibroblasts thereby stimulating collagen formation. purpose: to determine the effect of lemuru fish oil gel on the collagen density and width of the periodontal tissue induced by p. gingivalis and the correlation between these parameters. methods: thirty male wistar rats were divided into five groups. induction of p. gingivalis was carried out first, then lemuru fish oil gel was applied to the gingival sulcus for 14 days, according to collagen scores in histological preparations using masson’s trichrome (mt). the width of the periodontal ligament was measured with an image raster program in µm. the data were analysed using statistics to test hypotheses using statistical product and service solutions (spss) version 24. results: significant differences in the results of the collagen density were observed between groups kand k+ and groups k+ and p2. meanwhile, no significant difference was observed between groups kand p2, p3, p2 and p3 and k+ and p1. the mean values of the periodontal ligament widths were 299.61 ± 51.82µm (k-), 425.85 ± 61.54µm (k+), 346.93 ± 33.53µm (p1), 370.15 ± 49.42µm (p2) and 379.6 ± 49.26µm (p3). conclusion: lemuru fish oil can affect the width of the ligament and the collagen density with an optimal concentration of 20%. the correlation between the collagen density and the periodontal ligament width was negative and not significant. keywords: collagen density; lemuru fish oil; periodontitis-width of the periodontal ligament correspondence: dian widya damaiyanti, department of oral biology, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim 150, surabaya 60111, indonesia. email: damaiyanti@hangtuah.ac.id introduction periodontal disease is a common issue, as seen in the basic health research data published in 2018, which recorded the prevalence of periodontal disease in indonesia reaching 73.1%–75%. chronic periodontitis has been found to be one of the most widespread diseases among indonesians.1 the main factor causing periodontitis is anaerobic negative plaque bacteria. one of the most dominant bacteria found in chronic periodontitis is porphyromonas gingivalis (p. gingivalis).2 p . g i n g i v a l i s b a c t e r i a s e c r e t e e n d o t o x i n lipopolysaccharides (lps), which can stimulate the activation of lymphocyte b cells to produce antibodies and stimulate the excretion of mediators by macrophages, including the tumour necrosis factor alpha (tnf-α), interleukin 1 (il-1), interleukin 6 (il-6), prostaglandin e2 (pge2) and matrix metalloproteinase species (mmps).3 in pathological conditions such as inflammation, tnf-α prevents macrophages from excreting intermediates such as tnf-α, il-1, il-6, pge2, and mmps.4 in pathological conditions such as inflammation, tnf-α dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 mailto:damaiyanti@hangtuah.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 230 widyastuti et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 229–234 inhibits macrophage activity. the inhibition of collagen synthesis and the presence of mmps increase collagen destruction. collagen is absorbed continuously and replaced by inflammatory cells so that damage occurs in the periodontal tissue and the collagen density decreases.2,5 untreated periodontitis can result in tooth loss. the goal of the treatment in periodontitis is to control bacteria as a local factor to minimise the systemic effects as a form of non-surgical treatment for periodontal disease.6,7 host modulation therapy (hmt) is part of a periodontal therapy that is used as an adjunct to conventional periodontal treatments such as scaling and root planning, and aims to reduce damage and regenerate the periodontal tissue by reducing the destructive aspects of the host response.2,8 the hmt properties can be found naturally in lemuru fish oil. lemuru fish oil contains n-3 polyunsaturated fatty acids (pufa), namely eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). this has the potential as an anti-inflammatory that works by degrading potent eicosanoids in the form of pge2 and leukotriene b4, so that the production of proinflammatory cytokines is inhibited. lemuru fish oil will also affect growth factors by increasing the fibroblast growth factor (fgf), which plays a role in the proliferation of fibroblasts by stimulating collagen formation.9,10 research on the topical application of catfish oil with content similar to epa and dha to tooth extraction sockets, in concentrations of 5% and 10%, showed an increase in the inactivation and amount of the bone morphogenetic protein-2 (bmp-2), which was most effective at a concentration of 10%. however, in the available research on lemuru fish oil with topical administration, there are no references proving which concentration is effective in curing periodontitis. available research investigated the effect of applying lemuru fish oil with modified concentrations of 10%, 20% and 40%.11 given these limitations in the current research, the aim of this paper is to investigate the effect of lemuru fish oil gel on collagen density and on the width of the ligament in the periodontal tissue of wistar rats induced by p. gingivalis. materials and methods this research received the approval of the ethics commission of the faculty of dentistry, hang tuah university, with the number s.ket / 068 / kepk-fkguht / xii / 2019. the type of research was a true experimental laboratory investigation with a post-test only design. the samples used in this study were male wistar rats (n = 30) aged 3–4 months, weighing 250–300g and divided into five groups, namely normal (k-) group, p. gingivalis bacterial induction without therapy (k+) group, p. gingivalis bacterial induction with 10% lemuru fish oil gel therapy (p1) group, p. gingivalis induction with 20% lemuru fish oil gel treatment (p2) group and p. gingivalis induction with 40% lemuru fish oil gel treatment (p3) group.11 periodontitis was obtained by bacterial induction using p. gingivalis (pg) american type culture cell (atcc) 33277. the application of 2 ml of a 1 × 109 cfu / ml solution of p. gingivalis bacteria, with 1.5 ml of a 1 × 109 cfu / ml solution of live bacteria in phosphate buffered saline (pbs) with 2% of carboxymethylcellulose was carried out orally. in addition, 0.5 ml of bacteria was smeared using a cotton swab along the gingival groove on all teeth and anus in the colorectal area. the frequency of the p. gingivalis induction was three times in four days (0h, 48h and 96h). periodontal tissue damage takes as long as four weeks counting from when the first bacterial induction is given.12 rats with periodontitis presented bleeding, redder than normal gingiva and a decrease in bone height, according to criteria described in a previous study.4 lemuru fish oil was obtained from a caning waste factory from banyuwangi, east java (cv. biji sesawi, banyuwangi, indonesia). the induction of p. gingivalis was carried out first, then lemuru fish oil gel with concentrations of 10%, 20% and 40% was applied topically using a micro brush, once a day for 14 days. approximately 1 ml/day of lemuru fish oil gel was applied topically each day on the lower jaw gingival sulcus of the wistar rats using a micro brush (cotisen, china). therapy was carried out for 14 days in order to follow the period of the angiogenesis process, the process of osteoblast formation and to overcome the operator error process.4 the rats were euthanised one day after administration of the therapy using ketamine (sigma, germany) for termination at a dose of 100 mg/kg body weight (bw) intraperitoneally. diazepam at a dose of 5 mg/kg bw was administered by inhalation. the rat was put into a container containing ether, and once it was in a sedative state, its neck was dislocated. then the whole mandible was taken, and the rat was buried. the mandible was transferred to disposable polypropylene tubes (gp, china) containing 10% formalin liquid. then the mandible was decalcified using 10% formic acid and cut sagittally between the mandibular left molar and the jawbone in the interdental area of the posterior mandible of the rattus norvegicus, which was then stained using mayer’s haematoxylin (orsatech, germany). the histometric analysis was carried out using an olympus cx-22 (olympus, germany) and the optilab program (miconos, indonesia), with 400×x magnification. histometric was performed with modification methods from the damaiyanti and mulawarmanti experiment, where the sample slides were divided into five fields of view and scored as +0 (no collagen fibres found in the wound area), +1 (density of collagen fibres in the low wound area (25%)), +2 (density of collagen fibres in moderate wound area (50%)), +3 (density of collagen fibres in tightly wound areas (75%)) and +4 (the density of collagen in the wound area is very tight (100%)).13,14 the periodontal ligament width was measured in the cross section using a 400x× magnification microscope, the olympus cx22. the measurements were carried out using a raster image program at three locations in one field of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 231widyastuti et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 229–234 view with a micrometre unit. the reading was carried out by two observers.15 the data obtained were analysed using descriptive statistics to obtain a description of the data distribution and ranking in relation to the dependent variable (collagen density and periodontal ligament width), before proceeding with the analysis test. the kruskal–wallis test was used to compare the collagen density data between groups and the one-way analysis of variance (anova) parametric statistical test was performed to analyse the periodontal ligament width. all tests were carried out using statistical product and service solutions (spss) version 24 (ibm, armonk, us). results the data obtained from the results of the study were tabulated and analysed using statistics to test hypotheses using spss version 24. because the collagen density data were measured as interval scores, nonparametric analysis was used. the mode of collagen density is shown in table 1. based on table 1 and figure 1, the lowest collagen density mode was observed for the k+ and p1 groups and the highest for kand p2. a nonparametric test was performed using the kruskal–wallis test with a significance level of p < 0.05. the result of the significance between groups was p = 0.001 (p < 0.05). this shows that there are significant differences in the negative group, positive group and treatment group. subsequently, the mann–whitney analysis was carried out. significantly different results were obtained between groups, except between kand p2, p3, p2 and p3, and k+ and p1, where no significant results were obtained, with p > 0.05 (table 2). based on the results of the histological examination and the statistical calculations, the results of the width of the periodontal ligament obtained between groups are shown in table 3 and figure 2. the histopathology of the periodontal ligament width shown in figure 2 was measured in three areas, with every field of view slide stained with haematoxylin eosin. table 3 shows that the highest periodontal ligament width measured from bone and tooth was observed in the control positive group (k+ = 425.85µm) and the narrowest periodontal ligament width was observed in the control negative group (k= 299.87µm). table 1. c o l l a g e n d e n s i t y s c o r e m o d e v a l u e i n t h e experiments group number of samples mode k6 4 k+ 6 1 p1 6 1 p2 6 4 p3 6 3 p1 p2 p3 kk+ table 2. significance test between groups using the mann– whitney test groups k+ p1 p2 p3 k.002* 0.004* 0.589 0.132 k+ 0.589 0.004* 0.004* p1 0.015* 0.026* p2 0.485 *significant (p < 0.05) figure 1. histopathology of collagen density with mt staining at 400x× magnification (collagen = blue colour) for the groups control negative (k-), control positive (k+), lemuru fish oil gel 10% (p1), lemuru fish oil gel 20% (p2) and lemuru fish oil gel 40% (p3). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 232 widyastuti et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 229–234 the data collected on the periodontal ligament width were analysed using the shapiro–wilk normality test and resulted in a normal distribution because of p > 0.05. the one-way anova parametric statistical test resulted in a significance of p = 0.004 (p < 0.05), which means that there is a significant difference in the periodontal ligament width. subsequently, the post hoc least significant differences (lsd) test was performed, as shown in table 4, to determine the value of the difference between groups. the results of this test show that there are significant differences between the groups kand k+, kand p2, kand p3, and k+ and p1. the spearman correlation analysis was used to determine the correlation between the parametric data, namely the width of the periodontal ligament, and the nonparametric data on collagen density scores. the correlation value obtained was -0.292, which indicates an opposite relationship, i.e., the higher the collagen score, the smaller the periodontal ligament width. this shows that the higher the density value, the smaller the periodontal ligament widening, but the significance result obtained was p = 0.11, which means that the relationship between the collagen density and the periodontal ligament width is not significant (p > 0.05) as shown in table 5. table 3. mean and standard deviations of the estimation of the periodontal ligament width at each group experiment groups replication average (µm) standard deviation k6 299.612 51.82 k+ 6 425.850 61.54 p1 6 346.932 33.53 p2 6 370.159 49.42 p3 6 379.671 49.26 figure 2. image of the histopathological preparations showing the periodontal ligament width with haematoxylin eosin (he) staining and 400×x magnification (arrow between bone and tooth). table 4. significance test of the periodontal ligament width k+ p1 p2 p3 k.000* 0.113 0.022* 0.01* k+ 0.011* 0.065 0.122 p1 0.428 0.267 p2 0.744 table 5. correlation between collagen density and periodontal ligament width correlations spearman’s rho collagen density score periodontal ligament width correlation coefficient -0.292750482 sig. (2-tailed) 0.116427602 n 30 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 233widyastuti et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 229–234 discussion in this study, the induction of p. gingivalis bacteria causes an inflammatory response in the periodontal tissue because they secrete biologically active endotoxins or lps and cause the activation of macrophages. this plays an important role in the synthesis of pro-inflammatory cytokines such as il-1 and tnf-alpha, pge2 and hydrolytic enzymes.4,16 secretion of inflammatory mediators such as cytokines and prostaglandins responds to produce mmps, proteolytic enzymes that affect the degradation of extracellular matrix macromolecules, namely collagen. in pathological conditions such as inflammation, tnf-α inhibits the activity of fibroblasts thereby inhibiting collagen synthesis as well as the presence of mmps, which triggers collagen destruction.5,17 the induction of p. gingivalis bacteria was shown to cause periodontal ligament damage characterised by an increase of the periodontal ligament width, which is more visible in the positive control group compared to negative control group. according to the periodontal regeneration model conducted by montevecchi et al.,18 a sign of periodontal ligament repair is a shorter width of the periodontal ligament compared to when the periodontal tissue damage occurred. this is because the formation of a good alveolar bone during the healing process causes the periodontal width to decrease.18 the groups induced by p. gingivalis bacteria and the group treated with 20% lemuru fish oil gel therapy had a significant difference in collagen density. this shows the positive effect of 20% lemuru fish oil gel on the increase of the collagen density. eicosapentaenoic acid and dha act as anti-inflammatory agents because of their ability to bind eicosanoids. they are contained in lemuru fish and compete with arachidonic acid, which can reduce the formation of mmps and stimulating fibroblast cell regeneration. fibroblasts, active inflammatory cells such as macrophages and neutrophils, epithelial cells and vascular endothelial cells stimulate the formation of mmps. with the help of mmps, fibroblasts digest the fibrin matrix and replace it with glycosaminoglycan. over time, this extracellular matrix is replaced by type iii collagen, which is also produced by fibroblasts. furthermore, type iii collagen is subsequently replaced by type i collagen during the maturation phase. lemuru fish oil also affect growth factors, namely fgf, which plays a role in the proliferation of fibroblasts so that it stimulates collagen formation. when extracellular matrix deposition occurs, collagen synthesis is augmented by growth factors, such as platelet-derived growth factor (pdgf), fgf and transforming growth factor beta (tgf-β), so the tissue remodelling process modulates the synthesis and the activation of metalloproteinases, an enzyme that functions to degrade the extracellular matrix. the result of the synthesis and degradation of the extracellular matrix is the remodelling of the connective tissue framework. this structure is the main feature of tissue healing in chronic inflammation.4,19 the sample group with the induction of p. gingivalis bacteria and 10% lemuru fish oil gel did not result in a significant difference from the induced groups that did not receive therapy. this shows that lemuru fish oil gel with a concentration of 10% did not have a significant effect on collagen density and periontal ligament-width. this is because the low doses were unable to stimulate growth factors such as fgf2. this mediator is needed in the healing process to trigger cell healing and differentiation and to initiate the recovery of damaged tissue.20,21 compared to the smaller concentration, the 20% concentration of lemuru fish oil gel showed better repair of the collagen density and the periodontal ligament. however, there was no significant difference in the results compared to the 40% concentration. this shows that if the gel preparation has a high concentration or excessive molecular weight, it will produce a thick gel layer when applied. the penetration of the gel through the hypodermic layer becomes less effective, thus making the therapeutic effect last longer.15,22 the results showed that the negative control group did not have a significant difference in collagen density when compared with the study group treated with lemuru fish oil gel therapy at a concentration of 20%. omega-3 fatty acids, especially epa, have been shown to increase the number of fibroblasts and stimulate the formation of collagen. eicosapentaenoic acid plays a role in increasing the amount of il-6 cytokines, which consequently increases collagen production by fibroblasts and stimulates endothelial cells to form neovascular tissue through the process of angiogenesis and lead to the healing process.9 lemuru fish oil contains unsaturated fatty acids consisting of omega-3 and omega-6. omega-3 has been shown to regulate various proteins in periodontal tissue, such as mmp-8, mmp-13, mmp-14 and tissue inhibitor of metalloproteinases (timp). omega-6 can affect the production of eicosanoids, pge2, leukotriene and lipoxin. the ability of lemuru fish oil to influence eicosanoid metabolism is related to the long-chain structure of epa and dha, which has similarities to the long-chain structure of arachidonic acid (aa). because of this, epa and dha can become aa competitor substrates to blend with the phospholipid membrane and directly inhibit the enzymes cyclooxygenase-2 (cox-2) and lipoxygenase. inhibition of the cyclooxygenase pathway results in inhibited prostaglandins so that there is no monocyte activation mechanism to produce tnf-α and interleukin 1β, which can inhibit collagen synthesis.9,23 the limitation in this study was the difficulty in ensuring that the whole 1ml of lemuru fish oil gel was fully absorbed in the sulcus of the periodontal ligament. therefore, further research on the absorption capacity of lemuru oil is required, to ensure the effect of lemuru as a topical drug in the treatment of periodontitis. based on this research, lemuru fish oil gel influences the increase of collagen density in the periodontal tissue of wistar rats induced by p. gingivalis. the concentration dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 234 widyastuti et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 229–234 of lemuru fish oil gel that had the most profound effect on the increase of the collagen density and the width of the ligament of the periodontal tissue of wistar rats induced by p. gingivalis was 20%. the correlation between collagen density and the periodontal ligament width was negative and not significant. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 198. 2. newman mg, takei hh, klokkevold pr, carranza fa. newman and carranza’s clinical periodontology. 13th ed. philadelphia: elsevier saunders; 2018. p. 30–1, 41–7, 101, 107, 164, 198–9, 484–5. 3. kristanti ra. penggunaan doksisiklin hyclate sebagai inhibitor matriks metalloproteinase pada terapi tambahan periodontitis. sainstis. 2012; 1(2): 65–73. 4. damaiyanti dw, widyastuti w, paramita a, megantara a, ibrohim m. lemuru fish oil (sardinella longiceps) therapy on periodontal wistar rats induced with phorphyromonas gingivalis bacteria: osteoblast and osteoclast. j biotechnol strateg heal res. 2019; 3(2): 128–34. 5. damaiyanti dw, mulawarmanti d, parisihni k. protection against periodontal destruction in diabetic condition with sardinella longiceps fish oil: expression of matrix-metalloproteinase 8 and tissue inhibitor of metalloproteinase 1. dent j (majalah kedokt gigi). 2019; 52(1): 51–6. 6. yıldırım tt, kaya fa. the effects of menopause on periodontal tissue. int dent res. 2011; 1(3): 81–6. 7. zulfa l, mustaqimah dn. terapi periodontal non-bedah nonsurgical periodontal therapy. j dentomaxillofacial sci. 2011; 10(1): 36–41. 8. shinwari ms, tanwir f, hyder pr, bin saeed mh. host modulation therapeutics in periodontics: role as an adjunctive periodontal therapy. j coll physicians surg pakistan. 2014; 24(9): 676–84. 9. calder pc. omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology? br j clin pharmacol. 2013; 75(3): 645–62. 10. radisky es, raeeszadeh-sarmazdeh m, radisky dc. therapeutic potential of matrix metalloproteinase inhibition in breast cancer. j cell biochem. 2017; 118(11): 3531–48. 11. budhy ti, sumaryono b, suardita k, rizkita ap. bmp-2 expression of post tooth extraction that catfish oil application. in: the veterinary medicine international conference 2017. kne life sciences; 2017. p. 69–76. 12. garlet gp, avila-campos mj, milanezi cm, ferreira br, silva js. actinobacillus actinomycetemcomitans-induced periodontal disease in mice: patterns of cytokine, chemokine, and chemokine receptor expression and leukocyte migration. microbes infect. 2005; 7(4): 738–47. 13. mulawarmanti d, andriani d, damaiyanti dw, khoirunnisa fp, juliatin an. the effects of shark liver oil on fibroblasts and collagen density in the periodontal ligaments of wistar rats induced with porphyromonas gingivalis. dent j (majalah kedokt gigi). 2019; 52(4): 209–14. 14. damaiyanti dw, soesilowati p, arundina i, sari rp. effectiveness of gold sea cucumber (stichopus hermanii) extracts in accelerating the healing process of oral traumatic ulcer in rats. padjadjaran j dent. 2019; 31(3): 208–14. 15. sri pradnyani iga. tetrasiklin hcl gel 0,7% meningkatkan jumlah sel fibroblas dan mempertebal ligamen periodontal pada sulkus gingiva tikus yang mengalami periodontitis. intisari sains medis. 2017; 8(1): 14–8. 16. cheng r, liu w, zhang r, feng y, bhowm ick na, hu t. porphyromonas gingivalis-derived lipopolysaccharide combines hypoxia to induce caspase-1 activation in periodontitis. front cell infect microbiol. 2017; 7: 474. 17. almeida t, valverde t, martins-júnior p, ribeiro h, kitten g, carvalhaes l. morphological and quantitative study of collagen fibers in healthy and diseased human gingival tissues. rom j morphol embryol. 2015; 56(1): 33–40. 18. montevecchi m, parrilli a, fini m, gatto mr, muttini a, checchi l. the influence of root surface distance to alveolar bone and periodontal ligament on periodontal wound healing. j periodontal implant sci. 2016; 46(5): 303–19. 19. christina bbh, fransisca c, kristin k, caroline, sudiono j. peran monosit (makrofag) pada proses angiogenesis dan fibrosis. in: seminar nasional cendekiawan. jakarta: universitas trisakti; 2015. p. 254–9. 20. momose t, miyaji h, kato a, ogawa k, yoshida t, nishida e, murakami s, kosen y, sugaya t, kawanami m. collagen hydrogel scaffold and fibroblast growth factor-2 accelerate periodontal healing of class ii furcation defects in dog. open dent j. 2016; 10(1): 347–59. 21. ogawa k, miyaji h, kato a, kosen y, momose t, yoshida t, nishida e, miyata s, murakami s, takita h, fugetsu b, sugaya t, kawanami m. periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs. j periodontal res. 2016; 51(6): 758–67. 22. septiningsih e. efek penyembuhan luka bakar ekstrak etanol 70% daun pepaya (carica papaya l.) dalam sediaan gel pada kulit punggung kelinci new zealand. thesis. surakarta: universitas muhammadiyah surakarta; 2008. p. 1–23. 23. burger b, kühl cmc, candreva t, cardoso r da s, silva jr, castelucci bg, consonni sr, fisk hl, calder pc, vinolo mar, rodrigues hg. oral administration of epa-rich oil impairs collagen reorganization due to elevated production of il-10 during skin wound healing in mice. sci rep. 2019; 9(1): 9119. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p229–234 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p229-234 p-issn: 1978-3728 e-issn: 2442-9740 volume 48, number 4, december 2015 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2015 january 2nd – december 31st, 2015 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga) editorial boards: rahmi amtha, drg., mds., ph.d (department of oral medicine – faculty of dentistry, universitas trisakti); prof. thalca, drg., mhped., ph.d., sp.ort(k) (department of orthodontics – faculty of dental medicine, universitas airlangga); prof. dr. anita yuliati, drg., m.kes (department of dental material – faculty of dental medicine, universitas airlangga); prof. dr. sri kunarti, drg., ms., sp.kg(k) (department of conservative dentistryfaculty of dental medicine, universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (department of oral medicine faculty of dental medicine, universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (department of pediatric dentistry – faculty of dental medicine, universitas airlangga); adi hapsoro, drg., ms (department of dental public health – faculty of dental medicine, universitas airlangga); dr. intan nirwana, drg., m.kes. (department of dental material – faculty of dental medicine, universitas airlangga); dr. retno pudji rahayu, drg., m.kes (department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga); dr. rini devijanti ridwan, drg., m.kes. (department of oral biology – faculty of dental medicine, universitas airlangga); dr. theresia indah budhy, drg., m.kes (department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga); dr. indah listiana kriswandini, drg., m.kes (department of oral biologyfaculty of dental medicine, universitas airlangga); wisnu setyari, drg., m.kes (department of oral biologyfaculty of dental medicine, universitas airlangga); dr. haryono utomo, drg., sp.ort (dental hospital – faculty of dental medicine, universitas airlangga); maretaningtias dwi ariani, drg., ph.d., m.kes., sp.pros (department of prosthodontics – faculty of dental medicine, universitas airlangga). managing editors: rostiny, drg., m.kes., sp.pros(k) (department of prosthodontics – faculty of dental medicine, universitas airlangga); sianiwati goenharto, drg., ms (department of health – faculty of vocation, universitas airlangga); dr. hendrik setia budi, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); dr. anis irmawati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); yuliati, drg., m.kes (department of oral biology – faculty of dental medicine, universitas airlangga); eric priyo prasetyo, drg., m.kes., sp.kg (department of conservative dentistry – faculty of dental medicine, universitas airlangga); saka winias, drg., m.kes. (department of oral medicine – faculty of dental medicine, universitas airlangga). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 contents page printed by: airlangga university press. (rk 237/05.16/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 48, number 4, december 2015 p-issn: 1978-3728 e-issn: 2442-9740 1. challenges in the management of oral ulceration in elderly patients nanan nur’aeny ............................................................................................................................... 165–169 2. purple sweet potato (ipomea batatas p.) as dentin hypersensitivity desensitization gel chariza hanum mayvita iskandar, hardita bicevani mulya, windy pretyani kusumawati, and andina rizkia putri kusuma ................................................................................................... 170–172 3. allergic contact cheilitis due to lipstick yatty ravitasari, desiana radithia, and priyo hadi .................................................................... 173–176 4. dental student’s satisfaction towards orthodontic laboratory work from rsgm dental laboratory sianiwati goenharto, dini setyowati, and elly rusdiana ............................................................ 177–182 5. levels of crystalline silica dust in dental laboratorium of dental health technology study program of vocational faculty, universitas airlangga eny inayati, sherman salim, sonya harwasih, and sri redjeki indiani ................................... 183–187 6. antioxidant activity test on ambonese banana stem sap (musa parasidiaca var. sapientum) hendrik setia budi, indah listiana kriswandini, and aditya dana iswara ............................. 188–192 7. toll-like receptor–4 gene polymorphisms in javanese aggressive and chronic periodontitis patients chiquita prahasanti ......................................................................................................................... 193–196 8. the development of early childhood caries impact on quality of life-indonesia instrument as assessment instrument of dental caries impact on quality of life of children aged 3-5 years based on indonesian community characteristics taufan bramantoro, yayi suryo prabandari, djauhar ismail, and udijanto tedjosasongko 197–203 9. hydroxyapatite combined with hyaluronic acid metronidazole gel increased the quantity of osteoblasts in the alveolar bone wistar rat ernie maduratna setiawatie .......................................................................................................... 204–208 10. the amount of macrophages and activated plasma cells on wound healing process affected by spirulina regina purnama dewi iskandar, retno indrawati, ira arundina, and retno pudji rahayu . 209–212 11. application of chitosan scaffolds on vascular endothelial growth factor and fibroblast growth factor 2 expressions in tissue engineering principles ariyati retno pratiwi, anita yuliati, istiati soepribadi, and maretaningtias dwi ariani ....... 213–216 181 vol. 43. no. 4 december 2010 alveolar ridge rehabilitation to increase full denture retention and stability mefina kuntjoro, rostiny, and wahjuni widajati department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: atrophic mandibular alveolar ridge generally complicates prostetic restoration expecially full denture. low residual alveolar ridge and basal seat can cause unstable denture, permanent ulcer, pain, neuralgia, and mastication difficulty. pre-proshetic surgery is needed to improve denture retention and stability. augmentation is a major surgery to increase vertical height of the atrophic mandible while vestibuloplasty is aimed to increase the denture bearing area. purpose: the augmentation and vestibuloplasty was aimed to provide stability and retentive denture atrophic mandibular alveolar ridge. case: a 65 years old woman patient complained about uncomfortable denture. clinical evaluate showed flat ridge in the anterior mandible, flabby tissue and candidiasis, while residual ridge height was classified into class iv. case management: augmentation using autograph was conducted as the mandible vertical height is less than 15 mm. autograph was used to achieve better bone quantity and quality. separated alveolar ridge was conducted from left to right canine region and was elevated 0.5 mm from the previous position to get new ridge in the anterior region. the separated alveolar ridge was fixated by using t-plate and ligature wire. three months after augmentation fixation appliances was removed vestibuloplasty was performed to increase denture bearing area that can make a stable and retentive denture. conclusion: augmentation and vestibuloplasty can improve flat ridge to become prominent. key words: augmentation, vestibuloplasty, athropic ridge, mandible abstrak latar belakang: ridge mandibula yang atrofi pada umumnya mempersulit pembuatan restorasi prostetik terutama gigi tiruan lengkap (gtl). residual alveolar ridge dan basal seat yang rendah menyebabkan gigi tiruan menjadi tidak stabil, menimbulkan ulser permanen, nyeri, neuralgia, dan kesulitan mengunyah. tujuan: augmentasi dan vestibuloplasti pada ridge mandibula yang atrofi dilakukan untuk menciptakan gigi tiruan yang stabil dan retentive. kasus: pasien wanita usia 65 tahun datang dengan keluhan gigi tiruan yang tidak nyaman. pemeriksaan klinis menunjukkan ridge flat pada anterior mandibula, jaringan flabby dan kandidiasis, sedangkan residual ridge digolongkan menjadi kelas iv. tatalaksana kasus: augmentasi dilakukan karena ketinggian vertikal mandibula kurang dari 15 mm. autograf digunakan untuk mendapatkan kuantitas dan kualitas tulang yang lebih baik. alveolar ridge diambil dari sisi kiri dan kanan region kaninus dan digunakan 0,5 mm dari posisi awalnya untuk mendapatkan ridge baru pada region anterior. alveolar ridge telah diseparasi difiksasi menggunakan t-plate dan ligature kawat. tiga bulan setelah fiksasi dilepas, dilakukan vestibuloplasti untuk meningkatkan denture bearing area sehingga gigi tiruan lebih stabil dan retentive. kesimpulan: augmentasi dan vestibuloplasti dapat memperbaiki ridge atrofi sehingga menjadi tinggi kembali. kata kunci: augmentasi, vestibuloplasti, ridge atrofi, mandibula correspondence: mefina kuntjoro, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: mefina_kuntjoro@yahoo.com. case report 182 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 181–185 introduction full denture is a denture used for replacing all missing teeth on the maxilla and mandible, supported by mucosa, connective tissue and bone. full denture can only be used well if it is stable and retentive.1 full denture could be stable if during its function the denture only moves slightly above the bone. meanwhile, the denture could be retentive if the power coming on the denture can not release the adhesion between denture and underneath mucosa. thus, retention can be defines as the resistance of the denture to its disposition in the mouth.2–5 after tooth extraction, the alveolar bone in the jaw would be atrophic which is called as residual ridge resorbtion. this condition can cause reduction of supporting tissue and bone to make full denture stable and retentive, however this problems will become worse if it occurs in the mandible.6 in order to estimate alveolar ridge resorbtion, radiographic examination could be conducted through cephalometry and panoramic. cephalometry can be used to examine dimensions of the jaw vertically and horizontally, but the lack of this radiography is the superimposed alveolar ridge. panoramic is more often used to conduct the evaluation.7 the alveolar ridge height can be calculated from top of the ridge to the anatomical landmark, such as the maxillary sinus or mandibular canal in the posterior region. in the maxillary anterior region, the base of the nose is used, while in the mandible, the inferior border of the mandible is used.8 the initial phase of the residual ridge resorbtion is actually started immediately after the tooth is extracted that can cause lost of periodontal membrane that has the ability to regenerate bone. the lost of the alveolar bone occurs in the labiolingual region and vertical height causing the ridge become narrower, and in some cases also causing it to shape like sharp knife (knife edge). next, processus alveolaris becomes low, round or flat. as the resorbtion continues, alveolar bone and basal bone can become small, and ridge become shortened. if it occurs in the mandible, it will cause problems for prosthodontics to make full denture.9 the alveolar ridge height can be classified into four classes; class i, the alveolar ridge height is adequate, but less wide and usually accompanied by the deficiency of the lateral or undercut regions; class ii, the height and width of the ridge is less, there is sharp ridge like a knife; class iii, alveolar ridge resorbtion until basiliar bone that can cause sharp configuration on the ridge; class iv, resorbtion of the basiliar bone occurs, so the mandible become as thin as pencil with ridge in the maxilla become flat.10 there are several techniques that can be conducted to rehabilitate atrophic ridge in the mandible before prostodontics treatment is conducted. the technique is divided into three categories; augmentation with bone or with bone graft continued with vestibuloplasty, using skin or mucosal graft to add surface area; and implant (subperiosteal, transosteal and endosteal implants).11 augmentation is a major surgery to increased the vertical height of the atrophic ridge, continued with vestibuloplasty to increase denture bearing area. augmentation is usually performed on the mandibular ridge crest region or on the inferior border. if the vertical height of the mandible less than 15 mm, augmentation is absolutely needed to raised alveolar ridge and make enough retention to make full denture in the mandible. if the height is more that 15 mm, augmentation will not be required, but only vestibuloplasty is needed.12–14 vestibuloplasty is an additional procedure to repair ridge for achieving additional vertical ridge height. the aim is to improve the height of the atrophic ridge, so the wide basal seat will be enough to make full denture stable.15,16 case the patient a 65 years old woman, came to prosthodontics clinic, faculty of dentistry, airlangga university to make a new denture. she had been using full denture since five years ago. since two years ago her lower denture has become loose and caused pain so she complained difficulty to chew food. she also complained her gums in the front lower denture were often bloody and got bumps. as a result, she want to make a new denture that can make her comfortable and chew food well. on extra oral examination, the temporomandibular joint, eyes, nose and lips were normal with oval face. gangrene radix on 12, 14, 17, 22, flabby tissue and candidiasis in the anterior mandibular region were found on intra oral examination. vestibulum of the maxilla were all quite deep except on the left posterior. maxillary ridge was ovoid while mandibular ridge was flat. the anterior ridge of mandible was very flat. torus palatine and mandibula were flat. palatum was ovoid with large tuber maxilla. no exostosis on the maxilla and mandible. retromylohyoid on the left and right sides were shallow (figure 1). in the radiological examination, gangrene radix on 12, 14, 17, 22 and resorbed processus alveolaris until 1/3 length of the root were found. residual ridge height in the mandible was measured from the edentulous region to the opposite anatomical landmarks, in this case ridge height was less than 15 mm in all regions of the mandible (figure 2). case management in this case, gangrene radix on 12, 14, 17, 22 were extracted. residual ridge height was classified into class iv, since it was below 15 mm, so it was essential to conduct augmentation in the anterior region and followed by vestibuloplasty. after this was done, full denture was made using acrylic base and acrylic artificial teeth. augmentation procedure was begun by doing anesthesia in the anterior mandible with infiltration technique, while extra oral anesthesia was conducted in the chin of the mandibular region by using 2 cc lidocaine. then, horizontal 183kuntjoro, et al.: alveolar ridge rehabilitation a b figure �. the intra-oral condition of the patient: a) maxilla and mandible; b) flabby tissue and mandibular candidiasis in the anterior mandible. (figure 1a, 3, and 4 published under the courtesy of prof. coen p., department of oral surgeon, faculty of dentistry, airlangga university) ba figure �. a) panoramic photo of the patient at 12, 14, 17, 22; b) the height of the available alveolar ridge measured from the edentulous region to the opposite anatomical landmarks. a) maxillary canine, b) nose base, c) maxillary sinus, d) tuberosity, e) inferior mandibular canal, f) anterior mandible, g) mandibular canine.17 incision was conducted in the anterior mandibular region until the alveolar ridge was exposed by using scalpel. the alveolar ridge from left canine region to the right was cut by using low speed handpiece and irrigated with saline solution. the cutting process was done until mandibular base. the alveolar ridge was separated from mandibular bone by using chisel. the separated alveolar ridge was heightened 0.5 mm from the previous position to get a new ridge in the anterior mandibular region. this was done to get retention and stability for the new full denture. then the separated alveolar ridge was fixated by using t-plate and screwed in four locations, 2 in mandibular regions and 2 in the separated alveolar ridge, then tied by using ligature wire for making it stable and unmovable. suturing procedure was done by using polyglycolide thread until there was no opened tissues so that food could not be trapped and disturb healing process (figure 3). control i was done 2 weeks after augmentation, the wound was recovering. the surrounding tissue was under normal condition. there was even no visible swelling. then the suture was opened and evaluated 3 months later to see the augmentation result. control ii was done 3 months later, the wound was very well, asymptomatic and the newly formed ridge was seen in the anterior mandible. fixation appliances t plate, screws, and ligature wire was removed by making incision on the newly formed ridge, then ligature wire, retrieving four screws, and finally removed t plate. as a result of bone reposition, the formation of the new bone joint has occurred in the area below the ridge (figure 4). vestibuloplasty according to gordon was conducted by making split thickness flap and secondary epithelialization technique in the anterior mandible. vestibuloplasty was done by moving muscle attachment more inferior in order to obtain the required depth of vestibule. then suturing procedure was conducted in mucosa and gengigel containing hyaluronic acid was given to accelerate epithelialization. the patient was instructed to rinse her mouth with antiseptics to maintain oral hygiene. one week after vestibuloplasty, primary impression was done to make temporary full denture (transitional denture) to prevent relapse after vestibuloplasty. control was done four weeks after vestibuloplasty. the mandibular ridge was recovering very well, so impression was done by using alginate and impression tray to make anatomical cast. the anatomical cast on the maxilla was given a layer of wax used as a spacer to place impression material, meanwhile on the mandible a spacer was not given since the ridge was flat. then by using this model, individual tray for maxilla and mandible was done. border moulding was done on maxilla and mandible to get good peripheral seals. for the mandible, moulding in the retromylohyoid region must be done maximally in order 184 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 181–185 to improve retention for the full denture. then impression was done by using polyvinyl siloxane (figure 6). after recovery time about 30 minutes the impression was filled with dental stone type iii to make master cast. using master cast, bite rim was done to gain vertical and horizontal jaw relation, then the master cast was put on articulator. the artificial teeth were set in the bite rim and located in the neutral zone to improve stability. the heat cured acrylic was packed and cured. selective grinding i was done to make sure that the vertical and horizontal jaw relation was correct. intermaxillary record was done and continued with selective grinding ii. insertion was done, with consideration on the retention and stability of the full denture (figure 5). control i was done one day after insertion. patient complained pain on the left posterior mandible. on intra oral examination, left retromylohyoid region looked reddish, then grinding on that area was done. control ii was done three days after control i, patient had no complaint and feel comfortable with the new full denture. control iii was done one week after control ii. patient had no complaint and was able to chew food well. afterwards, the next controls would be periodically every six months. six months after insertionl, the anterior ridge in the mandible was looked quite high with no abnormality. the patient was very satisfied with the present condition, the denture was comfortable and was able to chew food properly. discussion in this case, there was a problem with the anterior mandibular ridge which was atrophic, as a result, there was not enough basal seat as a support for retention and stability for full denture. this condition could even lead to many problems in the setting of full denture. patient with flat alveolar ridge could usually have uncomfortable denture because of less retentiton and stability, permanent ulcer on the soft tissues, neuralgia and minimum chewing ability.14 as a consequence, preprosthetics surgery was needed to establish higher anterior mandibular ridge in order to make full denture more stable and retentive. in this case, augmentation was conducted because the height of the mandibular ridge is less than 15 mm in the premolar region and then continued with vestibuloplasty.11 bone graft is also required in augmentation, its selection must be selective to obtain maximum result. the disadvantage of bone graft is that graft can be resorbed after two to five years. to prevent excessive resorbtion in using bone graft, hydroxyapatite could be used or miced with autogenous bone to reconstruct higher ridge in the flat ridge mandible.12 the use of autograft is more usefull than allograft in augmentation. the reason is the result of using autograft is predictable because better bone quantity and quality figure 5. the insertion of complete denture in maxilla and mandible. figure 6. the condition of the mandibular ridge 6 months after insertion of the complete denture. figure ��. the seperated alveolar ridge was fixated with t-plate screwed and tied with a ligature wire. figure �. three months after the augmentation, the new ridge was started to form. 185kuntjoro, et al.: alveolar ridge rehabilitation could be obtained. allograft is still usefull because it does not need invasive surgery, but the result is not as better as autograft because of poor bone quality and quantity. autograft is used in situations of losing many bones, while as allograft is used for small fenestration, labial dehiscence or socket after extraction.18,19 the augmentation in this case was conducted using autograft. the autograft was obtained by separating alveolar ridge and then placing it higher based on the required height. and was fixated using t-plate, screw and ligature wire in order to make the separated ridge unmovable. by using autograft, resorbtion of the ridge was expected to be minimal since the graft was from the patient and in the sama area. after the new bone was formed, vestibuloplasty was conducted.11 vestibuloplasty was done to remove the muscle lower and to provide connective tissue for the remained ridge. to conduct succesfull vestibuloplasty, mucosal flap should be free from any tension to avoid relapse, this can be minimized by making an incision at the base of the sulcus. in this case one week after vestibuloplasty, impression was done to make transitional denture to prevent relaps. it is very important that the full denture should not irritate granulation tissue, the length of the denture should be deep enough to form new sulcus. then four weeks after vestibuloplasty, final impression was done to make new full denture.20 to make the full denture it is important to make sure high accuracy in each procedure, especially in the case of flat ridge in the mandible. the cause of lack stability and retentive is several factors such as less accurate impression, improper horizontal and vertical jaw relation, setting tooth acrylic not in the neutral zone.21 in the case of extremely flat ridge, the final impression must be conducted properly to get accurate master cast that could support retention and stability of the full denture. the accuracy of the impression could also be obtained by using individual tray and border moulding to get peripheral seal. then final impression was done by using mucocompresive material to make pressure to the mucosa and the materials could flow to fill complicated part.22 additional retentiton could be obtained at retromylohyoid region by digging cast so individual tray at that area could be longer, otherwise compound stick could also be used at the time of border moulding. impression result for retromylohyoid region should have “s” form from lateral part and butterfly wing when seen from above.22 horizontal and vertical jaw relation was done and continued with adjust cast with bite rim in the articulator. all of those three phases must be done properly in order to make the new full denture stable, mastication muscle should not get tired easily, create good esthetics, support patient face not to look older, phonetics not disturbed and prevent costen syndrome.22 it can be concluded that augmentation and vestibuloplasty could give good result in forming new prominent ridge on atrophic mandibular ridge. as a consequence, it could provide a better prognosis in making full denture. references 1. henderson d, mc. givey glen, castleberry d, mc. cracken’s. removable partial prosthodontics. st. louis, toronto, princeton: cv mosby company; 1985. p. 1–7. 2. watt david, mac gregor ar. designing complete denture. philadelphia: wb saunders company; 1986. p. 1–6. 3. findlay a. introduction to physical chemistry. 3rd ed. london, longmans: green & company; 1960. p. 534. 4. john s. complete denture prosthodontics. new york, toronto, sydney, london: mc.graw-hill book company inc; 1974. p. 147–52. 5. baat ca, albert, mulder. prosthetics condition and patient judgement of complete denture. j prosthet dent 1997; 78: 472–8. 6. tina mb, brian lm. histological analysis of healing after tooth extraction with ridge preservation using mineralized human bone allograft. j periodontology 2010; 81(12): 1765–72. 7. toshiho h. diagnostic imaging by panoramic radiograph in edentulous patient. j japan prosthodontics society 1999; 43(1): 13–9. 8. nishimura i, hosokawa r, atwood da. the knife edge tendency in mandibular residual ridges in women. j prosthet dent 1992; 67: 820–6. 9. misch ce. contemporary implant dentistry. 2nd ed. st. louis: mosbyyear book, inc; 1993. p. 123–8. 10. stafne ec. oral roentgenographic diagnosis. 3rd ed. philadelphia: wb saunders; 1969. p. 2. 11. pramono cd. mandible vertical height correction using lingual bonesplit pedicle onlay graft technique. dent j 2006; 39(3): 93–7. 12. robert em, thomas s, james w. severely resorbed mandible: predictable reconstruction with soft tissue matric expansion (tent pole) grafts. j oral maxillofac surg 2002; 60: 878–88. 13. findlay a. introduction to physical chemistry. 3rd ed. london, longmans: green & company; 1960. p. 534. 14. joseph e, michael t. review of surgical ridge augmentation procedures for the atrophied mandible. j prosthet dent 1984; 51(1): 5–10. 15. hisham fn, aichelmann-reidy, yukna r. bone and bone subtitutes. periodontology 2000; 19(2): 74–83. 16. gordon wp. buku ajar praktis medah mulut. cetakan i. philadelphia:cetakan i. philadelphia: wb saunders; 1996. p. 132–7. 17. carl em. contemporary implant dentistry. st. louis: mosby-year book, inc; 1993. p. 123–8. 18. abdel se, pipco dj. autogenous and allogenous bone grafting techniques to maximize esthetic: a clinical report. j prosthet dent 2000; 83: 153–7. 19. zhdanov e. the innovative approach to the treatment of total edentulism and advance alveolar atrophy. smile dental journal 2009; 4 (issued 3): 36–9. 20. starshak. preprosthetic oral surgery. united states of america:starshak. preprosthetic oral surgery. united states of america: cv mosby; 1971. p. 157–8. 21. heartwell cm, rahn ao. syllabus of complete denture. 4th ed. philadelphia: lea & febiger; 2004. p. 200–10, 277–305, 327–37. 22. itjiningsih wh. geligi tiruan lengkap lepas. cetakan ke-3. jakarta: penerbit buku kedokteran egc; 1996. p. 26, 39, 70, 95. 183183 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 183–187 levels of crystalline silica dust in dental laboratorium of dental health technology study program of vocational faculty, universitas airlangga eny inayati,1 sherman salim,2 sonya harwasih,1 and sri redjeki indiani1 1dental health technology diploma study program, faculty of vocational, universitas airlangga 2department of prosthodontics, faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: silicosis is an occupational lung disease caused by inhaling particles of crystalline silica in a long time. the disease then results in inflammation and defects in lung tissue. prosthesis construction is usually conducted in dental laboratory using a lot of materials containing crystalline silica, such as gypsum, ceramics, planting material, sandblast and others. purpose: this research aims to determine levels of crystalline silica dust in the dental laboratory of dental health technology diploma study program, vocational faculty, universitas airlangga. method: three measurement points was determined, namely point 1, point 2 and point 3 in each dental laboratory space (i and ii). suctioning dust was performed at those points using low volume dust sampler (lvds). samples taken were divided into two groups, namely x and y. taking dust samples were carried out for 30 minutes. elements of crystalline silica contained in the dust were quantitatively measured using xr defractometry tool, while size and morphology of silica were measured using sem edx tool. data obtained were statistically analyzed by paired t test. result: the results showed significant differences in the levels of the total dust measured and crystalline silica in the form of quartz and cristobalite among those two dental laboratory spaces. conclusion: it can be concluded that the levels of the total dust and silica quartz dust in the dental laboratory spaces i and ii were greater than the threshold limit value (tlv) determined. keywords: dust; silica; crystalline; dental laboratory correspondence: eny inayati, c/o: program studi d3 teknik kesehatan gigi, fakultas vokasi universitas airlangga. jl. srikana 65 surabaya 60286, indonesia. e-mail: enysyamsul@gmail.com introduction workers in dental laboratory, often known as dental technicians, during their work are always exposed to a variety of factors that can affect their health condition, such as physical exposure (noise, vibration, illumination, electrics), chemical exposure (gas, dust), radiation exposure (microwave, infrared, ultraviolet) and biological exposure (bacteria, fungi, viruses). however, pathogenic effects still depend on the concentration of toxins in the air and the duration of the exposure. there are three ways how toxic agents penetrate into the organism, namely inhalation (gas, vapor, dust), direct contact with the skin or mucous membranes and gastrointestinal process (when the norms of hygiene and labor protection are ignored).1 various kinds of prosthesis construction, such as crowns and bridges, dentures with cobalt chromium metal frame, acrylic denture and other dental products are manufactured in the dental laboratory. the use of materials to make a wide range of these products may cause health problems for dental technicians or people who work in dental laboratory, such as respiratory disease, skin problems and neurotoxicity. as reported, some cases of breathing problems are found in dental technicians, and an epidemiological study show there is a relation between a high prevalence of pneumoconiosis and exposure duration.2 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p183-187 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p183-187 184 inayati, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 183–187 silicosis is an occupational lung disease caused by inhaling crystalline silica mostly common used in denture manufacture. silicosis is a respiratory illness that causes inflammation and defects in lung tissue. crystalline silica dust inhaled will be stored in the respiratory system. deposition point, however, depends on particle size. the largest size of the particle will be saved in the nasopharynx (the upper respiratory tract, nose and throat) and eliminated by the organisms. meanwhile, the smallest particles (respirable) will be penetrated into the trachea, bronchi and alveolar duct (throat, lung area above and below). this condition is considered as the early development of silicosis.3 during 1994-2000, the center for disease control and prevention (cdc) in atlanta had conducted a research reporting nine cases of silicosis suffered by dental technicians.4 the cdc claims that dental technicians have a high risk for exposure to silicosis dust through air.5 dental health technology diploma study program of vocational faculty, universitas airlangga is an educational institution that graduates dental technicians. students take this study program for 3 years or more. during their study, students learn how to make prosthesis of teeth in the dental laboratory using materials containing crystalline silica, such as gypsum, ceramics, planting material, sandblast and others. as a result, those students are exposed to crystalline silica through breathing during denture making process causing dust in the air, such as stirring the powder, removing the results of mold casting, cutting, polishing casting and ceramics, as well as using sandblaster. therefore, students, dental technicians and teaching staffs who daily work in the lab will have a high risk for exposure to crystalline silica material. nevertheless, levels of total dust and silica in the laboratory of dental health technology diploma study program of vocational faculty, universitas airlangga had never been measured. therefore, this research aims to determine the levels of crystalline silica in the dust in the dental laboratory of dental health technology diploma study program, vocational faculty, universitas airlangga. finally, the significance of this research is to control and prevent air pollution optimally against harmful silica material impact on those students, dental technicians and teaching staffs working in there. materials and method samples used were dust aspirated or collected from the dental laboratory spaces i and ii using a lvds (low volume dust sampler, hitachi type 35 rc-20sc5). filtration technique was used in this research to collect dust particles. before collecting the dust, measurement points were determined. there were three measurement points, namely point 1, point 2 and point 3 in the dental laboratory spaces i and ii. suctioning dust was conducted on such points, each of which was measured 2 times, and the pump speed used was 10-30 lpm.6 this technique used a circular filter (round) with a porosity of 0.3-0.45 µm. the samples were divided into two groups, namely group x and group y. filters for both x and y were previously weighed before stored in petri discs, and then put in a desiccator for 24 hours. there are two filters used in group x, namely x1 set on holder before practicum and x2 set on holder during practicum. meanwhile, in group y as a control group, there were also two filters used, namely y1 put in petri disc and y2 placed in the open space in the dental laboratory. afterwards, the pump was turned on, and the speed of air flow was checked again. collecting dust was performed for 30 minutes. the indoor air temperature and pressure then were recorded when dust sampler was turned on. the indoor air temperature and air pressure were also measured when the pump was turned off. the filters were removed from the holders by using tweezers, put in petri disc, and put back into the desiccator for 24 hours. level of total dust collected then was weighed using an analytical balance (with a minimum sensitivity of 0.01mg). data obtained were put into the following formula:7 c = (x2-x1) – (y2-y1) × 1000 f x t note: c = total dust concentration (mg/m3) x1 = the weight of the filter prior to exposure x2 = the weight of the filter after exposure y1 = the weight of filter without treatment before exposure y2 = the weight of filter without treatment after exposure t = time (minutes) f = flowrate (liters per minute). elements of crystalline silica in dust were measured quantitatively using x-ray defractometry tool (xpertpro panalytical), while the size and morphology of silica were measured using a scanning electron microscope, energy dispersive x-ray (edx sem, inspect s50). the result data obtained in the scale ratio were tabulated and statistically analyzed by t test. results the results of the statistical test using paired t-test, moreover, showed that there were significant differences in the total dust found at those three points in the dental laboratory space i between before and during practicum with p: 0.003<α 0:05. similarly, there were significant differences in the total dust found at those three points in the dental laboratory space ii between before and during practicum with p: 0.000 <α 0:05. the results of the statistical test using independent t-test, furthermore, showed that there were significant differences in the total dust between the dental laboratory dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p183-187 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p183-187 185185inayati, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 183–187 space i and the dental laboratory space ii during the practicum with p: 0.015<α 0:05. the mean results indicate that the total dust in the dental laboratory space i during practicum was currently greater than in the dental laboratory space ii. the focus of this research was crystalline silica dust in the form of quartz and cristobalite. 5 table 3. levels of crystalline silica dust (quartz and cristobalite) in the dental laboratory spaces i and ii in dental health technology diploma study program, vocational faculty, universitas airlangga ___________________________________________________________________________ activities quartz (mg/m3) cristobalit (mg/m3) ___________________________________________________________________________ the dental laboratory space i point 1 0.12 0.40 point 2 0.11 0.38 point 3 0.11 0.48 the dental laboratory space ii point 1 0.21 0 point 2 0.16 0 point 3 0 0 figure 1. the distribution of si (purple color) in the total dust found in the dental laboratory space ii at point 2 (with 40.000x magnification). figure 2. si with various morphology and size found in the dental laboratory space ii at point 2 (with 40.000x magnification). figure 1. the distribution of si (purple color) in the total dust found in the dental laboratory space ii at point 2 (with 40.000x magnification). 5 table 3. levels of crystalline silica dust (quartz and cristobalite) in the dental laboratory spaces i and ii in dental health technology diploma study program, vocational faculty, universitas airlangga ___________________________________________________________________________ activities quartz (mg/m3) cristobalit (mg/m3) ___________________________________________________________________________ the dental laboratory space i point 1 0.12 0.40 point 2 0.11 0.38 point 3 0.11 0.48 the dental laboratory space ii point 1 0.21 0 point 2 0.16 0 point 3 0 0 figure 1. the distribution of si (purple color) in the total dust found in the dental laboratory space ii at point 2 (with 40.000x magnification). figure 2. si with various morphology and size found in the dental laboratory space ii at point 2 (with 40.000x magnification). figure 2. si with various morphology and size found in the dental laboratory space ii at point 2 (with 40.000x magnification). discussion the results showed that the total dust in both dental laboratory spaces was higher than the threshold limit value (tlv) about 10 mg/m3.8 as a result, it indicates that the condition of those two dental laboratory spaces was not good and healthy for both dental technicians and students working there since the total dust can make them discomfort and trigger several potential diseases. for instance, it can reduce vision, cause unpleasant sediment on eyes, nose and ear as well as lead to skin damage. edx of sem examination results obtained on the dust, moreover, indicated that the level of silica (si) contained was quite high (figure 1). this was due to the use of lab materials in the form of planting materials, gypsum, as well as ceramic materials for the manufacture of dental crowns, permanent dentures and removable dentures. materials contained in dental ceramics are kaolin and feldspar table 1. the mean and standard deviations of the total dust measured before and during the practicum in the dental laboratory spaces i and ii in dental health technology diploma study program, vocational faculty, universitas airlangga activities mean standard deviations (mg/m3) p the dental laboratory space i before the practicum 0.90 0.70 during the practicum 22.02 2.53 0.003 the dental laboratory space ii before the practicum 0.58 0.02 during the practicum 10.42 0.28 0.000 p = probability table 2. the mean and standard deviations of the total dust measured during the practicum in the dental laboratory spaces i and ii in dental health technology diploma study program, vocational faculty, universitas airlangga activities mean (mg/m3) standard deviations p the dental laboratory space i 22.02 2.53 0.015 the dental laboratory space ii 10.42 0.28 table 3. levels of crystalline silica dust (quartz and cristobalite) in the dental laboratory spaces i and ii in dental health technology diploma study program, vocational faculty, universitas airlangga activities quartz (mg/m3) cristobalit (mg/m3) the dental laboratory space i point 1 0.12 0.40 point 2 0.11 0.38 point 3 0.11 0.48 the dental laboratory space ii point 1 0.21 0 point 2 0.16 0 point 3 0 0 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p183-187 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p183-187 186 inayati, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 183–187 (k2o-al2o3.sio2) on build-up, polishing and finishing processes, which can be a source of spread of silica dust in room.9 similarly, figure 1 depicts how silica spread at all points 1, 2 and 3 in the dental laboratory spaces i and ii. the results of xray defractometry examination, furthermore, showed that there was quartz dust found at all sampling points in the dental laboratory space i. meanwhile, in the dental laboratory space ii, quartz dust was only found at point i and ii. on the other hand, cristobalite dust was found at all sampling points in the dental laboratory space i. nevertheless, there was no cristobalite dust found at all sampling points in the dental laboratory space ii. these results indicate that both the dental laboratory spaces i and ii had been exposed to silica dust, either quartz or cristobalite although quartz dust was only found at point i and ii in the dental laboratory space ii, while both quartz and cristobalite were not found at point 3 in the dental laboratory space ii. in addition, levels of silica found in this research were greater than the specified threshold limit value determined, about 0.1 mg/m3 for quartz and 0.05 mg/m3 for cristobalit.10,11 this situation was triggered by a condition that during practicum there were no specific rooms divided based on kinds of laboratorial work. all of the rooms are interconnected and not separated by a partition. the rooms are also cramped and lack of exhaust systems and ventilation (especially dental laboratory i). as a result, students get a high risk of being exposed or contaminated with air containing hazardous materials, such as metal, resin or silica causing pneumoconiosis. some cases of pneumoconiosis due to silica actually have been found.12 silica dust can cause silicosis. silica contained in dental ceramics can be spread when stirring the ceramic powder or during grinding or polishing. consequently, the surrounding environment can be exposed to it. exposure can also occur when stirring planting material and divesting (demolishing casting results). planting materials often contain cristobalite, silica crystalline material that is the most toxic. during 19942000, occupational disease surveillance program in five countries had identified nine cases of silicosis in people working in dental laboratories.4 silicosis arising from inhalation of fine dust containing crystalline silica through the respiratory tract. silica particles larger than 0.6 µ will be retained in the upper respiratory tract, while silica particles between 0.3 µ s/d 0.6 µ will arrive at the alveoli. silica particles below 0.3 µ will follow brown movement, which is that dust particles can be inhaled and exhaled again.13 size of silica dust particle found in this research was in a variety of sizes ranging from 0.5 s/ d 7.3 µ (figure 2). the size and morphology of silica particles found were various due to grinding, divesting, polishing, sandblasting processes during practicum. as a result, dust can enter through the upper respiratory tract to pulmonary alveoli parts, and this can cause silicosis. silica dust found in the dental laboratory spaces, however, was not primarily lead to health problems of the students, dental technicians and teaching staff since there are many factors influencing health problems caused by silica dust exposure. the health problems may arise when there is an interaction of several factors, such as high level of silica dust, frequency of exposure time, condition and endurance of those students, dental technicians and teaching staffs. a health problem caused by silica dust is known as silicosis disease. symptoms of silicosis can be started by short breath, mid cough and chest tightness. silicosis can get worse even though the cause has been terminated since silicosis cannot be cured, but the severity can only be prevented by avoiding silica dust exposure. silicosis disease is often associated with other diseases, such as tuberculosis, kidney disease, lung cancer, fever and weight loss, even leading to death. if exposed to organisms that cause tuberculosis, mycobacterium tuberculosis, silicosis patients will have a risk three times more likely to suffer tuberculosis.14 the level of silica in the dental laboratory spaces i and ii was quite high since materials used in the laboratory contained a lot of silica. besides that, a range of laboratory processes, such as investing, sandblasting, grinding, polishing and others, can cause silica dust flying in the laboratory. laboratory conditions that are not too wide with the big number of students and dental technicians and inadequate ventilation have significantly made silica dust exposure high enough. fortunately, students and dental technicians work in the laboratory less than 8 hours and not every day. this is in accordance with regulation of minister of labor and transmigration no. 1311 stating that based on threshold limit values (tlv) as the standards of working environmental factors recommended in workplace, workers can still tolerate bad condition of the workplace causing disease or illness for not more of 8 hours a day or 40 hours a week. nevertheless, those students, dental technicians and teaching staffs working in the dental laboratory still need to be protected form silicosis disease. prevention of silicosis disease in the dental laboratory spaces i actually can be conducted by organizing the spaces of the laboratory to prevent dust produced from flying anywhere. for example, adequate ventilation needs to be set by installing exhaust fan or vacuum cleaner connected to a pipe, which ends can get into the water reservoir so that dust does not fly, but directly go into the water. besides, students, dental technicians and teaching staffs must wear personal protective equipment (ppe), such as gloves, goggles, gowns and masks during in the laboratory in order to maintain health. personal protective equipment used should meet the standards of osha, namely: can protect against hazards, can be worn comfortably, cannot restrict movement, as well must be durable and can easily be cleaned.15 finally, in conclusion the levels of total dust and silica quartz dust in both the dental laboratory spaces i and ii were greater than tlv determined. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p183-187 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p183-187 187187inayati, et al/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 183–187 references 1. kartaloglu z, ilvan a, aydilek r. dental technician’s pneumoconiosis: mineralogical analysis of two cases. yonsei med j 2003; 44: 169 73. 2. hu sw, lin yy, wu tc, hong cc, lung sc. workplace air quality and lung function among dental laboratory technician. am j ind med 2006; 49(2): 85-92. 3. maynard ad, kuempel ed. airbone nanostructured particle and occupational health. j of nanoparticle researach 2005; 7: 587 614. 4. rosenman kd, petcher e, schill dp, valiante dj, bresnitz ea, cummings kr, socie e, filios ms. silicosis in dental laboratory technicians – five states, 1994-2000. mortality & morbidity weekly report 2004; 53(9): 195-7. 5. alavi a, shakiba m, nejad at, massahnia s, shiari a. resporatory fendings in dental laboratory technicians in rasht (north of iran). tannafos 2011; 10(2): 44-9. 6. badan standarisasi nasional. pengukuran kadaar debu total di udara tempat kerja. sni 16-7058-2004: 3. 7. aditya sa, denny a. identifikasi kadar debu di lingkungan kerja dan keluhan subyektif pernafasan tenaga kerja bagian finish mill. jurnal kesehatan lingkungan 2007; 3(2): 161-72. 8. menteri kesehatan ri. persyaratan kesehatan lingkungan kerja perkantoran dan industri. kemenkes no. 1405/men/xi/2002. 9. kim ts, kim ha, heo y, park y, park cy, roh ym. level of silica in the respirable dust inhaled by dental technicians with demonstration of respirable symptoms. ind health 2002; 40(3): 260-5. 10. badan standarisasi nasional. nilai ambang batas (nab) zat kimia di udara tempat kerja. sni19-0232-2005: 18. 11. permernakertrans ri no 13.2011. nilai ambang batas bahan fisika dan kimia di tempat kerja. 12. morgen roth k, k ronenberger h, michalke g, sck nabel r. mor phology and pathogenesis of pneumoconiosis in dental technicians. pathol res pract 1985; 179(4-5): 528-36. 13. teguh p, susanto jp. kualitas debu dalam udara sebagai dampak industri pengecoran logam ceper. jurnal teknologi lingkungan 2001; 2(2): 168-74. 14. farazi a, jabbariasi m. silico tuberculosis and associated risk factors in central province of iran. pan afr med j 2015; 20: 333. 15. occuptional safety and health administration (osha). personal protective equipment. available from: www.osha.gov. 2003. accesed june 11, 2015. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p183-187 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p183-187 156 volume 45 number 3 september 2012 research report analgesic effect of coconut shell (cocos nucifera l) liquid smoke on mice meircurius dwi c.s, tantiana and ira arundina department of oral biology faculty of dentistry, universitas airlangga surabaya – indonesia abstract background: drugs can be used to eliminate pain by inhibiting the activity of conversing arachidonic acid into prostaglandin. the chemical compositions of coconut shell are cellulose, pentosan, lignin, solvent extraction, uronat anhydrous, nitrogen, and water. one active ingredient in coconut shell is phenyl propanoid (consisting in lignin structure) and guaicol. phenyl propanoid and guaicol are phenolic compounds that can be used as antioxidant, antiseptic, anti-inflammatory, anesthetic and analgesic. liquid smoke of coconut shell (cocos nucifera l) contains phenolic compound is believed able to bind a component conversing arachidonic acid into prostaglandin. purpose: the study was aimed to examine the analgesic effect of liquid smoke of coconut shell (cocos nucifera l). methods: the study was a laboratory experimental research, conducted on 2-3 months old male mice (mus musculus) with 20-30 grams of weight. there were control group and treatment groups each of which had seven mice. control group was orally given 0.01 ml/weight (ml/gr) of distilled water, after 30 minutes 0.01 ml/weight (ml/gr) of acetic acid 0.6% was delivered via intraperitoneal injection. the treatment groups were given liquid smoke of coconut shell (cocos nucifera l) with the concentrations of 25%, 50%, and 100% respectively. the analgesic effect was then determined by decreasing of writhing reflex on mice recorded every 5 minutes for 30 minutes. results: there were significant differences of writhing reflexes in the treatment groups given liquid smoke of coconut shell with the concentrations of 25%, 50%, and 100%. the higher concentration of liquid smoke the higher its analgesic effect. conclusion: liquid smoke of coconut shell (cocos nucifera l) has analgesic effect. key words: analgesic effect, liquid smoke of coconut shell, acetic acid abstrak latar belakang: salah satu mekanisme obat yang digunakan untuk menghilangkan rasa nyeri adalah menghambat aktivitas konversi asam arakhidonat menjadi prostaglandin. komposisi kimia tempurung kelapa terdiri dari selulosa, petosan, lignin, solvent, uronat unhidrat, nitrogen dan air. salah satu bahan aktif dalam tempurung kelapa adalah phenyl propanoid (terdapat dalam struktur lignin) dan guaiakol. phenyl propanoid dan guaiakol adalah suatu senyawa fenol yang mempunyai sifat sebagai antioksidan, antiseptic, anti-inflamasi, anastesi dan analgesik. liquid smoke tempurung kelapa (cocos nucifera l)mengandung senyawa fenol yang dapat mengikat komponen dalam konversi asam arakhidonat menjadi prostaglandin. tujuan: studi ini bertujuan untuk meneliti efek analgesik dari liquid smoke tempurung kelapa (cocos nucifera l). metode: penelitian ini adalah eksperimental laboratoris dengan jenis post test only control group design pada mencit (mus musculus) jantan usia 2-3 bulan dengan berat badan 20-30gram. kelompok kontrol dan perlakuan terdiri dari 7 hewan coba. kelompok kontrol diberi aquades 0.01ml/bb (ml/gr) (po) dan setelah 30 menit diberi asam asetat 0.6% 0.01ml/bb (ml/gr) (ip). kelompok perlakuan diberi liquid smoke tempurung kelapa (cocos nucifera l) dengan konsentrasi 25%, 50% and 100%. efek analgesik ditentukan dengan melihat penurunan writhing reflex (liukan atau geliat tubuh hewan coba) yang dihitung setiap 5 menit selama 30 menit. hasil: terdapat perbedaan yang bermakna jumlah writhing reflex pada pemberian 157dwi, et al.: analgesic effect of coconut shell (cocos nucifera l) introduction nociception is a neural tissue response towards unpleasant stimuli while pain is a perception that arises as a result of nociception.1 inflammation is a complex biological response which include vascular tissue response to both damaged tissue and irritation.2 tissue damage from excessive stimulus are associated with unpleasant and uncomfortable sensory called pain.1,3 pain management that can be conducted involves reducing variety of factors both peripheral and central. there are three classes of drugs that can be used to relieve pain, namely non-opioid analgesics, opioid analgesics, and local anesthetics.4 coconuts in indonesia is produced about 15.5 billion/ year.5 unfortunately, the processing industry of coconut in general is still focused on the processing of it as fruit, as the primary outcome, while the by-products processing industry such as coconut husk and coconut shell is still be conducted traditionally.6 coconut shell is one part of the agricultural products which has high economic value and high functional value.7 chemical composition of coconut shell consists of cellulose, pentosan, lignin, ash, solvent extraction, uronat anhydrous, nitrogen, and water.8 one of active components in the coconut shell is phenylpropanoid compound contained in lignin. phenylpropanoid compound is a phenol compound that can be used as an antiseptic, antioxidant, anti-inflammatory, anesthetics, and analgesics.9,10 liquid smoke is available from condensation of coconut shell through pyrolysis process at 400 °c. liquid smoke contains many chemical components, such as phenols, aldehydes, ketones, organic acids, alcohols, and ester.11 those various chemical components may act as an antioxidant and antimicrobial effect, as well as give color and distinctive flavor to food products.12 currently, liquid smoke of coconut shell has been widely used by food industry to give flavor and texture of food products, such as meat, fish, and cheese.13 in indonesia, liquid smoke is also used for making smoked fish,14 as well as for preservatives of tuna, eel, and fresh noodles.15 in addition, liquid smoke can also be used as a substance that can reduce pain in wound, and can remove the scar. knowledge about medicinal plants, based on experience and skills, has been passing from generation to another. however, the role of medicinal plants still needs to be justified medically through analysis and scientific experiments. efforts towards a rational scientific evidence must be done through the analysis of substances contained as well as their therapeutic effects.16 therefore, it is necessary to examine the analgesic effect of liquid smoke of coconut shell (cocos nucifera l) as the by-products. materials and methods this research was laboratory experimental with post test only control group design. materials used in this research were shells of 6–8 months old coconut (cocos nucifera l) obtained and identified in plant conservation and botanical garden, purwodadi, pasuruan. experimental animals used were healthy male mice (mus musculus) in the age of 2–3 months with the weight of 20–30 grams. those animals were obtained from the unit of experimental animals in biochemistry laboratory of medical faculty, airlangga university. liquid smoke of coconut shells (cocos nucifera l) was obtained through pyrolysis process. pyrolysis was a thermochemical decomposition of organic materials at above 430 °c without oxygen.17 the raw materials needed were 5 pounds of coconut shells producing 20 ml of liquid smoke through pyrolysis process. the making process of liquid smoke of those coconut shells (cocos nucifera l) was then conducted in research laboratory and industry consultant, surabaya. this research was considered as a preliminary study on the analgesic effect of liquid smoke of coconut shells (cocos nucifera l). liquid smoke used in this research was at concentration of 100% obtained through pyrolysis. to obtain liquid smoke of coconut shells with lower concentrations (50% and 25%), dilution was conducted by distilled water. the use of the distilled water, as a result, could change the concentration and enlarge the volume, but still stable.18 afterwards, analgesic test on liquid smoke of coconut shells (cocos nucifera l) was determined by acetic acidinduced writhing reflex. this method is used for screening peripheral acting analgesics, local peritoneal cell response, and prostaglandin pathway.19 mice used as experiment were divided into 4 groups, each group consisted of 7 mice (n = 7) and treated as follows: control group was given orally 0.01 ml/gr of distilled water (bb.po); group i was given 0.01 ml/g of 100% liquid smoke of coconut shells (cocos nucifera l) (bb.po), group ii was given 0.01 ml/g of 50% liquid smoke of coconut shells (cocos nucifera l) (bb.po); and group iii was given 0.01 ml/g of 25% liquid smoke of coconut shells (cocos nucifera l) (bb.po). after liquid smoke tempurung kelapa konsentrasi 25%, 50% dan 100%. semakin tinggi konsentrasi liquid smoke, semakin tinggi pula efek analgesic yang ditimbulkan. kesimpulan: liquid smoke tempurung kelapa mempunyai efek analgesik. kata kunci: efek analgesik, liquid smoke tempurung kelapa, asam asetat correspondences: ira arundina, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: arundinafkg@yahoo.com. 158 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 156–160 thirty minutes, those mice were given 0.6% acetic acid at a dose of 0.01 ml/g intra-peritoneal (bb.ip) to induce pain. after 5 minutes, writhing reflex (abdominal constriction) of those experimental animals was measured every 5 minutes for 30 minutes.20,21 the effect of analgesics was determined by comparing the percentage of analgesic power to writhing reflex (abdominal constriction) between the control group and the treatment groups given with liquid smoke of coconut shells (cocos nucifera l)22 with formula:23 percentage of inhibition = control group mean – test group mean x 100% control group mean the number of writhing reflex (canting or stretching) was then analyzed by using one-way anova with 95% level of significance. if the results showed no difference, then least significant difference test (lsd test) would be conducted. results injection of acetic acid through intraperitoneal in experimental animals can cause pain response described as writhing reflex (stretching or canting of experimental animals' body). but, with liquid smoke of coconut shell (cocos nucifera l) at three different concentrations of 100%, 50% and 25% could decrease the writhing reflex. the data obtained were analyzed by kolmogorov smirnov test. the result showed that all groups had probability value greater than 0.05 (p>0.05), indicating that the data were normally distributed. afterwards, levene test was conducted to indicate whether the data are homogeneous or not. the result then showed that the significance value was greater than 0.05, p = 0.608 (p > 0.05) which indicates the data were homogenous. since the data were normally distributed and homogenous, one-way anova was conduced to analyze the data. concentration of 100% liquid smoke had 40.29% inhibition. it is also known that 50% liquid smoke of coconut shells had 25.28% inhibition. meanwhile, 25% liquid smoke had 19.70% inhibition. data analysis using one-way anova showed the greater level of significance (p < 0.05), which means that there was significant difference of stretching or canting of experimental animals' body among the groups. thus, lsd test was then conducted to know the difference among experimental groups (table 2). there were significant differences between the control group given with distilled water and the treatment group given with 100% liquid smoke, between the treatment group given with 50% liquid smoke and the treatment group given with 25% liquid smoke, between the treatment group given with 50% liquid smoke and the control group as well as the treatment group given with 100% liquid smoke, and between the treatment group given with 25% liquid smoke and the control group as well as the treatment group given with liquid smoke 100% (p < 0.05), which means that there was a reduction of stretching or canting of experimental animals' body. discussion liquid smoke (liquid of evaporation result) is a result of condensation of vapor derived from burning process of materials containing a lot of lignin, cellulose, hemicellulose, and carbon compounds.24 moreover, liquid smoke can also be considered as a complex system consisted of both dispersed liquid phase and gas phase as dispersant. smoke is actually produced by incomplete combustion involving constituent decomposition reaction of polymers into organic compounds with low molecular mass because of heat including oxidation reaction, polymerization reaction, and condensation reaction. the nature of liquid smoke is affected by main materials, namely cellulose, hemicellulose, table 1. mean of canting and stretching in animals' body, standard deviations and the percentage of inhibition (power analgesics) group treatments x + sd percentage of inhibition control distilled water 48.57 + 4.79086 – 1 25% liquid smoke of coconut shell liquid smoke of coconut shelliquid smoke of coconut shell 39.00 + 2.70801 19.70% 2 50% liquid smoke of coconut shell liquid smoke of coconut shelliquid smoke of coconut shell 36.29 + 3.30224 25.28% 3 100% liquid smoke of coconut shell liquid smoke of coconut shelliquid smoke of coconut shell 29.00 + 4.54606 40.29% table 2. lsd test between the control group and the treatment groups a b c d control group (a) * * * 100% liquid smoke of coconut shell (b) liquid smoke of coconut shell (b)iquid smoke of coconut shell (b) * * * 50% liquid smoke of coconut shell (c) liquid smoke of coconut shell (c)iquid smoke of coconut shell (c) * * 25% liquid smoke of coconut shell (d) liquid smoke of coconut shell (d)iquid smoke of coconut shell (d) * * * : there was significant difference among the groups 159dwi, et al.: analgesic effect of coconut shell (cocos nucifera l) and lignin with various proportions depended on types of the materials in pyrolysis.25 furthermore, one of materials used in the process of making liquid smoke is coconut shell which is part of the coconut fruit. coconut shell is widely used to produce liquid smoke because it consists of lignin, cellulose, and metoksil causing good organoleptic nature.13 there are actually twelve identified components in liquid smoke of coconut shell, mainly from thermal degradation of wood carbohydrates, such as ketones about 6.53%, carbonyl and acid about 2.98%, as well as furan and pyran derivatives about 3.02%. in addition, liquid smoke of coconut shell also contains 28 components derived from degradation of lignin thermal, such as phenol about 24.11%, guaiacol and its derivatives about 36.58%, and syringol and its derivatives about 18.26 %, and alkyl aryl ether about 8.5%.15 a compound that can cause irritation on tissue (irritant substance), such as acetic acid, can stimulate the release of prostaglandins through nociceptive neurons that are sensitive to non-steroidal anti-inflammatory drug. in other words, irritant substance can cause the release of endogenous substances, such as prostaglandins, which can stimulate peripheral nociceptor and neurons that are sensitive to non-steroidal anti-inflammatory drug.26 writhing reflex induced significantly decreased due to the provision of liquid smoke of coconut shell (cocos nucifera l). it means that the provision of liquid smoke of coconut shell (cocos nucifera l) could reduce response to pain. this is because active ingredient contained in the liquid smoke of coconut shell is phenolic compound that can inhibit pain. phenolic compound contained in the liquid smoke is a compound of phenol and guaiacol, which is a potent radical trapping compound. this compound is also considered as an antioxidant compound that has the ability as a redox compound, which serve as a reductant, hydrogen supplier, and inhibitor of the initiation stage of the lipid oxidation reaction.27,28 antioxidant is actually considered as an anti-inflammatory agent that works through the capture of free radical oxygen released by peroxide. phenol can inhibit prostaglandin production and reduce oxidized cyclooxygenase enzyme that plays a role in the metabolism of arachidonic acid altered into prostaglandin h2 (pgh2), an unstable molecule that can turn into a variety of proinflammatory compounds.29 the mechanism of liquid smoke of coconut shell to inhibit pain response was due to the role of phenolic compound that inhibits cyclooxygenase of tissue by reducing the synthesis of prostaglandin e2 (pge2). 30 the decreasing of prostaglandin e2 (pge2) is caused by the binding of prostaglandin compound, g2 (pgg2), and prostaglandin compound, h2 (pgh2), when arachidonic acid is converted into prostaglandin compound, e2 (pge2), by phenolic compound. both compounds can actually be considered as endoperoxide, a compound produced during the conversion of arachidonic acid to prostaglandins. as a consequence, the provision of liquid smoke of coconut shell (cocos nucifera l) is related to the mechanism of cyclooxygenase inhibiting in peripheral tissues, so the synthesis of prostaglandins can be reduced, and the main transduction mechanisms of afferent nociceptor can be disrupted.31 the mechanism of analgesic effect caused by liquid smoke of coconut shell then works by inhibiting the production and function of prostaglandins. therefore, it can be said that the liquid smoke of coconut shell is a non-steroidal anti-inflammatory like drug. the percentage of inhibition is the ability of liquid smoke of coconut shell (in %) in reducing the number of writhing reflex (stretching or canting of experimental animals' body) due to acetic acid. in other words, percentage of inhibition will increase as the concentration of liquid smoke of coconut shell increases. there was a significant difference between the percentage of inhibition of liquid smoke of coconut shell with concentration of 100% and that with concentrations of 50% and 25%. the difference is caused by the fact that the amount of phenolic compound contained in liquid smoke of coconut shell with concentration of 100% is considered as a minimum amount that can inhibit the production of prostaglandins. in other words, the concentration of phenolic compound in each liquid smoke concentration is proportional to the concentration of liquid smoke itself. the study showed that liquid smoke of coconut shell (cocos nucifera l) has analgesic effect. references 1. kidd bl, urban la. mechanism of inf lammatory pain. br j anaesthesia 2001; 87(1): 3–11. 2. divya ts, latha pg, usha k, anuja gi, suja sr, shyamal s, shine vj, sini s, shikha p, rajasekharan s. anti-inflammatory, analgesic and anti lipid peroxidative properties of wattakaka volubilis (linn. f.) stapf. natural product radiance 2009; 8(2): 137–41. 3. guyton ac, hall je. textbook of medical physiology. 11th ed. st. louis: elsevier inc; 2006. p. 598. 4. jackson kc. pharmacotherapy for neuropathic pain. j world institute of pain 2006; 6(1): 27–33. 5. agustian as, supadi friyatno, askin a. analisis pengembangan agroindustri komoditas perkebunan rakyat (kopi dan kelapa) dalam mendukung peningkatan daya saing sektor pertanian. makalah seminar hasil penelitian pusat penelitian dan pengembangan sosial ekonomi pertanian bogor. 2003. p. 38. 6. mahmud z, ferry y. prospek pengolahan hasil samping buah kelapa. perspektif 2005; 4(2): 55–63. 7. uwubanmwen io, nwawe cn, okere ra, dada m, eseigbe e. harnessing the potentials of the coconut palm in the nigerian economy. world j agricultural sciences 2011; 7(6): 684–91. 8. okafor pc, okon pu, daniel ef, ebenso ee. adsorption capacity of coconut (cocos nucifera l.) shell for lead, copper, cadmium and arsenic from aqueous solutions. int j electrochem sci 2012; 7: 12354–69. 9. singla rk. review on the pharmacological properties of cocos nucifera endocarp. webmed central pharmaceutical sciences. 2012; 3(5): wmc003413. 10. taguri t, tanaka t, kouno i. antibacterial spectrum of plant polyphenols and extracts depending upon hydroxyphenyl structure. biological & pharmaceutical bulletin 2006; 29(11): 2226–35. 11. guiilenmd, manzanos mj, ibargoitia ml. carbohydrate and nitrogenated compounds in liquid smoke flavorings. j agric food chem 2001; 49: 2395–403. 160 dent. j. (maj. ked. gigi), volume 45 number 3 september 2012: 156–160 12. karseno, darmadji p, rahayu k. daya hambat asap cair kayu karet terhadap bakteri pengkontaminan lateks dan ribbed smoke sheet. agritech 2002; 21(1): 10–15. 13. soldera s, sebastianutto n, bortolomeazzi r. composition of phenolic compounds and antioxidant activity of commercial aqueous smoke flavorings. j agric food chem 2008; 56: 2727–34. 14. hadiwiyoto s, darmadji p, purwasari sr. perbandingan pengasapan panas dan penggunaan asap cair pada pengolahan ikan; tinjauan kandungan benzopiren, fenol, dan sifat organoleptik ikan asap. agritech 2000; 20: 1–9. 15. budijanto s, hasbullah r, prabawati s, setyadjit, sukarno, zuraida i. identifikasi dan uji keamanan asap cair tempurung kelapa untuk produk pangan. j pascapanen 2008; 5(1): 32–40. 16. lusia orkm. pemanfaatan obat tradisional dengan pertimbangan manfaat dan keamanannya. majalah ilmu kefarmasian 2006; 3(1): 1–7. 17. tsamba aj, yang w, blasiak w, wojtowicz ma. cashew nut shells pyrolysis: individual gas evolution rates and yields. energy & fuels 2007; 21(4): 2357–62. 18. jan su, khan gm, khan h, rehman a, khan ka, shah su, shah ku, badshah a, hussain i. release pattern of three new polymers in ketoprofen controlled-release tablets. african j pharm and pharmacol 2012; 6(9): 601–7. 19. ezeja mi, ezeigbo ii, madubuike kg. analgesic activity of the methanolic seed extract of buchholzia coriacea. research j pharmaceutical, biological and chemical sciense 2011; 2(1): 187–93. 20. afolayan aj, aboyade om, adedapo aa, sofidiya mo. antiinflammatory and analgesic activity of the methanol extract of malva parviflora linn (malvaceae) in rats. african j biotechnology 2010; 9(8): 1225-9. 21. ra k h ms, chaud ha r i sr. eva luation of a na lgesic activity of momordica dioica roxb. willd fruit pupl. international j pharmaceutical science and research 2010; 1(9): 53–6. 22. debprasad c, hemanta m, paromita b, durbadal o, kumar ka, shanta d, kumar hp, tapan c, ashoke s, sekhar c. inhibition of no2, pge2, tnf-α, and inos expression by shorea robusta l.: an ethnomedicine used for anti-inflammatory and analgesic activity. evidence-based complementary and alternative medicine 2012. 1–14. 23. akuodor gc, usman mi, i brahim ja, chilaka kc, akpan jl, dzarma s, muazzam, osunko ua. anti-nociceptive, antiinf lamatory and antipyretic effect of the methanolic extract of bombax buonopozense leaves in rats and mice. american j biotechnology 2011; 10(16): 3191–6. 24. darmadji p. optimasi pemurnian asap cair dengan metoda redistilasi. jurnal teknologi dan industri pangan 2002; 13(3): 267–71. 25. girrard jp. smoking in technology of meat products. new york: clermont ferrand. ellis horwood; 1992. p. 165, 205. 26. bukhari ia, khan ra, gilani ah, ahmed s, saeed sa. analgesic, anti-inflamatory and anti-platelet activity of the methanolic exctract of acacia modesta leaves. inf lammopharmacolgy 2010; 18: 187–96. 27. duganath n, kumar sr, kumanan r, jayaveera kn. evaluation of anti-denaturation property and anti-oxidant activity of traditionally used medicinal plants. international j pharm and bio science 2010; v1(2): 2. 28. purwantini i, purwatiningsih, puspita oe. afek analgesik fraksi etanol dari ekstrak etanol daun midi (melia azedarach l) pada mencit jantan. skripsi. yogyakarta: fakultas farmasi universitas gadjah mada. 2009. p.100. 29. roberts lj, marrow jd. analgesic-antipyretic and antiinflammatory agent and drugs employed in the treatment of gout. in "goodman & gilman's the pharmalogical basis of therapeutics 10th edition". new york: mcgraw hill; 2001. p. 687. 30. sighn s, sheora ss. evaluation of the antinociceptive activity of ama ranthus hybr idus linn, root extract. acta poloniae pharmaceutica-drug researche 2011; 68(2): 255–9. 31. prabhu vv, nalini n, chidambarathan n, kisan ss. evaluation of anti inflammatory and analgesic activity of tridax procumber linn againts formalin, acetic acid and cfa induced pain models. int j pharm pharm sci 2011; 3(2): 126–30. mkgs vol 44 no 1 jan-mar 2011.indd 43 vol. 44. no. 1 march 2011 cost effectiveness and quality of life assessment on dental filling and tooth extraction in balongsari public health center taufan bramantoro1 and thinni nurul r2 1department of dental public helath, faculty of dentistry, airlangga university 2faculty of public health, airlangga university surabaya indonesia abstract background: dental health services program implementation in balongsari public health center during three years, 2006 until 2008, have a high average ratio of filling treatment compared to tooth extraction treatment (1:1.79) as compared to the standard set by the ministry of health (1:1). cost effectiveness analysis and quality of life is needed as a form of economic evaluation of costs incurred by the consequences or impacts of health care programs, especially dental filling and tooth extraction, use to help in supporting the process of policy making in health care. the objective of this study was to assess cost effectiveness analysis (cea) and quality of life (qol) on dental filling and extraction treatment in public health center. methods: the study was conducted on 31 respondents who received filling treatment and 38 respondents who received tooth extraction. all of the respondents carried out to evaluate the total costs incurred in obtaining treatment and qol between before and after treatment, which consist of the physical aspects, psychological, social, and economic. results: the average total cost of dental filling treatment of the 31 respondents was rp. 27,934.45, and in tooth extraction of the 38 respondents at rp. 22,406.83. the average difference in the qol, before and after dental filling treatment amounted to 121.25. in extractions, qol difference in value before and after treatment at 132.36. cost effectiveness ratio value in dental filling treatment amounted to 230.37, and in tooth extraction at 169.63. conclusion: it is concluded that cost effectiveness ratio in the filling treatment is higher than the extraction, that the tooth extraction treatment is considered more cost effective than filling treatment. key words: cost effectiveness analysis, quality of life, dental filling, tooth extraction abstrak latar belakang: pelaksanaan program pelayanan kesehatan gigi di puskesmas balongsari selama tiga tahun, yaitu tahun 2006 hingga 2008, memiliki rata-rata rasio perbandingan perawatan tumpatan dengan pencabutan gigi (1:1,79) yang lebih tinggi dibandingkan dengan standar rasio yang ditetapkan oleh kementerian kesehatan (1:1). analisis efektifitas biaya dan kualitas hidup, dibutuhkan sebagai bentuk dari evaluasi secara ekonomi, dilihat dari biaya yang dibandingkan dengan dampak program pelayanan kesehatan, khususnya perawatan tumpatan dan pencabutan gigi, untuk mendukung proses pengambilan kebijakan dalam pelayanan kesehatan. tujuan: tujuan penelitian ini adalah untuk menilai cost effectiveness analysis (cea) dan quality of life (qol) pada perawatan tumpatan dan pencabutan gigi di puskesmas balongsari. metode: penelitian ini dilakukan terhadap 31 orang pasien yang mendapatkan perawatan tumpatan dan 38 orang pasien yang mendapatkan pencabutan gigi. pada seluruh responden dilakukan evaluasi total biaya yang dikeluarkan untuk mendapatkan perawatan dan pengukuran kualitas hidup sebelum dan sesudah perawatan, yang terdiri dari aspek fisik, psikologis, sosial, dan ekonomi. hasil: rata-rata total biaya perawatan tumpatan gigi dari 31 responden adalah rp. 27,934.45, dan pada pencabutan gigi sejumlah 38 responden sebesar rp. 22,406.83. rata-rata nilai selisih qol, sebelum dengan sesudah perawatan tumpatan gigi sebesar 121.25. pada pencabutan gigi, nilai selisih qol sebelum dengan sesudah perawatan sebesar 132.36. nilai cost effectiveness ratio pada perawatan tumpatan gigi adalah sebesar 230.37, dan pada pencabutan gigi adalah sebesar 169.63. kesimpulan: dapat disimpulkan bahwa cost effectiveness ratio pada perawatan tumpatan gigi lebih tinggi dibandingkan pada pencabutan gigi, sehingga pencabutan gigi dinilai lebih cost effective atau efektif secara biaya, dibandingkan dengan perawatan tumpatan gigi. research report 44 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 43–48 kata kunci: analisis efektifitas biaya, kualitas hidup, tumpatan gigi, pencabutan gigi correspondence: taufan bramantoro, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: tbramantoro@yahoo.com introduction done, it has a big role in shaping the thinking of person's psychology, involving the assessment or perception and motivation based on the felt experience. quality of life used in health care field to analyze a person's emotional, social, and physical abilities are normal, including the ability to meet the demands of the activities in daily life as normal, and the impact of illness, can potentially degrade the quality of health-related life.3 the oral health impact profile (ohip) is a good option for identifying dimensions in oral health related quality of life (ohrqol), since it is one of the most sophisticated and most popular instruments for measuring ohrqol. the scientific advisory committee of the medical outcomes trust defined a set of attributes and criteria for the assessment of health status and quality-of-life measurement. the conceptual model is particularly wellsuited, this instrument is developed by slade and spencer, based on a conceptual framework of oral disease and its functional and psychological consequences. the ohip is grounded on a theoretical framework based on the world health organization's international classification of impairments, disabilities, and handicaps and an accordingly derived multidimensional model of oral health. the ohip constituted of 49 lengthy questions and partly for this reason there was a need to develop a shorter derivative, the ohip-14. the ohip-14 proves to have good statistical properties and validity in the cross-sectional setting. the benefit of using the ohip-14 is that data can be collected using less fieldwork and respondent burden.3 cost effectiveness analysis was used to help in supporting the process of policy making in health care, because it is a form of economic evaluation of costs incurred by the consequences or impacts of health care programs or performed as a settlement of existing health problems.4,5 cost effectiveness analysis as a method that can be applied in the health field to explain the comparative economic impact of spending on the intervention of the health measures is obtained. cost effectiveness analysis can be used also as a consideration in the decision making process in support of resource management activities related to health care.4,5 this study used the method of assess cost effectiveness analysis, the objective of this study was to assess cost effectiveness analysis (cea) and quality of life (qol) on dental filling and extraction treatment in public health center. oral health as part of the whole human health have an important role associated with the presence of oral stomatognatic functional, in improving quality and productivity of human resources. implementation of oral health efforts is one of the public health center’s main activities, that are comprehensive, integrated and include the improvement, prevention, healing and rehabilitation of oral health as a basic service unit directly needed to achieve improved quality of oral health services to society and realize the optimal degree of public health according to the vision and mission of the ministry of health of the republic of indonesia.1 dental health services include dental fillings, tooth extraction and scaling. one of the indicators of program level assessment efforts basic oral health is by looking at the comparison between permanent dental fillings and permanent tooth extraction according to the standard 1:1 ratio set by the ministry of health of the republic of indonesia. over the past 3 years i.e. in 2006-2008, the average ratio of permanent dental fillings with permanent tooth extraction in balongsari public health center is higher (1:1.79) than the standard ratio set by the ministry of health (1:1). loss of teeth in the oral cavity system, will bring the impact of the occurrence of bone support, which will continue on the issue of masticatory function and balance of facial proportions in general, can occur due to changes in the position of teeth with dental tendencies that still exist have a tendency to fill the empty space left by extracted teeth. dental filling treatment with tooth preparation before filling process with less precision, would allow the occurrence of secondary infections from bacteria found under filling material. that may lead to the occurrence of secondary caries that sometimes can only be detected when the severity is in high level or when a similar pain in teeth that have been previously filled. problems arise in post-treatment, will have an impact on patient time and cost that must be paid back for retreatment or continuing the treatment which has been done before.2,3 quality of life related to oral health describes the state of teeth and mouth in social functioning, physical and psychological as well as economically to the activity of a person's life. assessment of successful treatment later was observed from the loss of the pathological effects of the existing dental problems, assessment of life quality as an analysis of the impact of dental treatment that has been 45bramantoro: cost effectiveness and quality material and methods this was an observational descriptive study, namely the analysis of the cea related to changes of oral health quality of life by using observation approach results obtained from analysis of changes in qol of patients before and after dental filling treatment and permanent tooth extraction. quality of life was used to analyze the achievement of treatment results, generated by the patient's care, as a form of post-evaluation of patient’s decision making in dental care. the study uses a prospective study design, by conducting observations and analysis at the time before getting treatment and observation carried back to see the impact through a period of 2 months after post-treatment, to obtain changes in the value of qol based on ohip instrument and cea.3 the questionnaire used to refer from the ohip questionnaire by slade,6,7 that questionnaire is translated into indonesian to see the essence of the question of health-related quality of life of the oral and dental. based on the concept of essence is then compiled 12 questions related purposes questions and the meaning understood by the respondents, that are divided into four aspects of qol analysis, i.e. physical aspects, psychological aspects, social aspects and economic aspects.3,6 qol questionnaire using a likert scale, response categories for the four scales, was: "very often", "often", "very rare" and "never". physical aspect, consisting of, assessment of how often before treatment and 2 months after treatment, experiencing the impact of the presence of oral and dental before and after treatment by patients, assessed from the onset of pain, chewing, bad breath, sleeping conditions and circumstances of the patient's headache. psychological aspects, consists of, how often assessment before and 2 months after treatment, experiencing the psychological effects are uncomfortable with the situation by the patient's oral and dental, which was considered of anger or irritability and the depression. social aspects, consists of, how often the patient do assessment before and 2 months after treatment, experiencing social impacts related to social relations and confidence. economic aspects, consists of, how often the patient assessment before and 2 months after treatment have been affected by oral and dental conditions of activity or employment and financial circumstances.6,7 the populations in this study were patients in dental clinic of balongsari public health center, who provide dental filling treatment and permanent tooth extraction was observed for 1 month, on 15th april to 15th may of 2010. the sample used in this research uses total sampling method or using the whole sample is calculated dental filling treatment and permanent tooth extraction on 15th april to 15th may of 2010 that meet the criteria of the sample, in which patients indicated for dental filling treatment and willing to do interviews, and domiciled in the territory of balongsari public health center. analysis of the total cost referred to in this research is the sum of direct and indirect costs incurred by patients in getting treatment. direct costs in this study are counter tariffs and treatment tariff, while the indirect costs derived from the sum of the following expenses: cost of transportation, which cost the patient to go or come to a place of health services; the lost of opportunity costs, i.e. costs incurred patients related expenditure of time working or productive activity to obtain health services; cost of losses, i.e. costs related to treatment of patients post-treatment problems that been done before; consumption costs, i.e. costs related to patient activities of eating and drinking in the process of getting health care.4,5 analysis of changes qol of patients, which is obtained from the patient, the result of questionnaire responses oral health-related qol before and after getting dental filling or tooth extraction. measurements using a qol instrument that consists of 4 aspects of analysis, namely: physical aspects, psychological aspects, social aspects, and economic aspects. analysis of cer calculation of dental filling and permanent tooth extraction, with qol approach, obtained from the calculation of total cost divided by the change in qol of patients.4,6,7 results on the characteristics of gender, dental filling and tooth extraction patients, that most (69.6%) had a female gender. most respondents in both men and women get a permanent tooth extraction. distribution of respondent’s age, dental filling and tooth extraction patients, that most (52.2%) with age range between 26-35 years. most respondents in the age range of 26-35 years and 36-45 years old get a permanent tooth extraction. while the range of 15-25 years most of the respondents receive dental filling treatment. job characteristics on the observation showed that most respondents (50.7%) had a job as employees. most respondents, who worked as employees, had dental filling treatment, and the housewives or who does not work, mostly had permanent tooth extraction. the condition of dental caries was found in dental filling treatment or tooth extraction, the majority (75.4%) obtained a large caries condition. in medium and large caries, most respondents have a permanent tooth extraction care. value of direct costs for dental filling treatment and tooth extraction issued by the respondents in this study is equal to the rate paid by the patient at a counter at rp. 2,500.and rates dental filling treatment and tooth extraction for rp.7,500.-. the direct costs incurred by patients for dental filling treatment and tooth extraction is the same, which is rp 10,000.-. in assessing the types of indirect costs, the average value of opportunity cost, transportation, and losses, dental toward filling treatment as higher than tooth extraction, while the average value of the cost of consumption at the extractions is higher than dental filling treatment. in the assessment of 46 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 43–48 indirect costs, which is the sum of the cost of transportation, consumption, the opportunity is lost, and losses, the average value of indirect costs incurred by respondents that had dental filling treatment was higher than tooth extraction. total cost assessment is shown in the table 1, which is the sum of direct and indirect costs, the average total cost incurred by respondents who had dental filling treatment were greater than permanent tooth extraction. table 1. total cost (in rupiah) value of dental filling treatment and permanent tooth extraction by the respondents treatment minimum maximum average standard deviation dental filling 13.000,00 75.000,00 27.934,45 19.560,02 tooth extraction 13.000,00 66.575,00 22.406,83 16.019,93 the sum value describes the quality of life of patients qol related to oral and dental condition, measured before and after dental filling treatment, is shown in the table 2. the average value of qol before treatment respondents experienced an increase in the measurement after treatment. the sum value describes the quality of life of patients qol related to oral and dental condition, measured before and after tooth extraction treatment, is shown in the table 3. the average value of qol before treatment respondents experienced an increase in the measurement after treatment. the average difference value of qol of respondents before and after dental filling treatment lower than the permanent tooth extraction, except on social aspects (table 4). table 2. the average of qol value in before and after dental filling treatment aspect dental filling before after average standard deviation average standard deviation physical 86.93 16.25 135.89 11.48 psychological 28.17 7.14 39.03 2.46 social 47.93 11.35 74.29 6.88 economic 77.94 16.48 113.01 10.34 total value of qol 240.96 33.12 362.22 23.19 the total cost value is obtained from the sum of the total value of the total cost, i.e. the number of direct and indirect costs, of each respondent in the group of dental filling treatment and tooth extraction. the total value of the excess of the sum obtained qol overall qol difference value of each respondent in each group, dental filling treatment and tooth extraction. table 5 shows the information that the cer of respondents who receive dental filling treatment higher than the tooth extraction. tooth extraction is more cost effective when compared with dental filling treatment. discussion through observation of patient characteristics, it was found that through gender, most patients who receive tooth extraction and dental filling treatment are female. dental filling treatment and tooth extraction is performed mostly to patients with age between 26–35 years. in observation of job characteristics of dental filling treatment and tooth extraction, which most have a job as employees. condition of dental caries is found in dental filling treatment and tooth extraction, most of the condition obtained a large caries. the condition of large caries can be associated with understanding of the caries process and its implications for treatment stages who impact less in the public perception table 3. the average of qol value in before and after tooth extraction treatment aspect tooth extraction before after average standard deviation average standard deviation physical 85.15 16.88 146.37 6.71 psychological 27.63 6.00 39.91 0.54 social 51.95 9.20 68.19 5.76 economic 76.97 15.62 119.32 2.78 total value of qol 241.44 30.61 373.79 10.01 table 4. the average difference in qol value, before and after dental filling treatment and permanent tooth extraction by the respondents aspect difference value of qol dental filling tooth extraction average standard deviation average standard deviation physical 48.96 16.83 61.21 18.36 psychological 10.86 6.14 12.27 5.85 social 26.35 13.06 16.24 8.84 economic 35.07 17.65 42.35 16.11 total value of qol 121.25 35.18 132.36 31.23 table 5. cost effectiveness ratio (cer) value of dental filling treatment and permanent tooth extraction by the respondents treatment n variable total cost (rupiah) total the differences value of qol cer dental filling 31 865,967.95 3,759.02 230.37 tooth extraction 38 851,459.62 5,019.41 169.63 47bramantoro: cost effectiveness and quality because the caries process itself is running without causing serious symptoms in general health, so that then raises the idea to delay and perform treatment until they feel sick or interfere in a condition of caries that involve most of the tooth.2,9,10 in this research, the results of measuring qol that has a higher value on the measurement after treatment compared with the measurement of time before getting treatment. this applies to both types of treatment, namely dental filling treatment and permanent tooth extraction, in an increase also in the four aspects of qol analysis, but the difference value of qol on dental filling is smaller when compared with qol value on tooth extraction increasing the value of quality of life that occurred on both types of treatment is to give an analysis, that dental filling treatment and tooth extraction in balongsari public health center produce positive impacts on solving problems of patients attending dental health. quality of life has a purpose in an effort to bring the assessment of health care. viewpoint of clinical conditions, quality of life has become subject in connection with the use of instruments that measure health-related condition of patient satisfaction and physiological benefits. total concept of human health combines both physical and mental factors.10,11 efforts to improve the quality of dental treatment need to pay attention to the characteristics of the patient-related conditions that use to conform to the approach to be used by the health center to raise public awareness about dental caries and the implications for dental treatment stages. result analysis of the characteristics of obtained treatment, showed that there is a link, providing information those efforts to increase dental filling treatment not limited to activities involving women and children, but also employees in the industrial environment.10–12 the difference value of qol on dental filling is smaller than qol value on tooth extraction. that is probably associated with the process of dental filling treatment which consists of several stages, with adjustments to the state of dental caries activity. this can be caused by active caries process then filling or cavity cleanup before dental filling process that has not reached an optimal cleaning, so caries activity is still possible to occur. in the treatment of tooth extraction, more minimal post-treatment problems occur because vital tooth extraction was performed or not in gangrene state, so the likelihood of infection is lower, with lower level of difficulty in the removal process against the low possibility of post-treatment problems of tooth extraction.9,13 in this research, the results of calculating the costeffectiveness ratio are lower in the permanent tooth extraction compared with dental filling treatment. that is related to the higher amount of the total cost and the smaller difference value of qol when compared with the number of total costs and excess qol value in the treatment of permanent tooth extraction. process of dental filling treatment and permanent tooth extraction consists of several steps that must be passed in accordance with the theory of applied dentistry, which aims to achieve treatment success, such as to eliminate the potency for secondary caries in dental filling, preventing damage of dental filling, the occurrence of infection and excessive bleeding on tooth extraction scar. small size and the location of tooth, create the potential barriers related to internal factors associated with patient conditions, and external factors that can reduce effectiveness of treatment results, including resource constraints factor, related to the ability of the service, completeness of equipment and facilities, the quality and ability of the materials used, and the sterility of the dental treatment process factor.14,15 psychological factors which are formed from the impact of quality of life, and it can potentially influence the selection of types and care to do next. consideration of the analytical quality of life experienced by a person giving a strong influence, it can be a solid basis for decisions affecting assessment and relatives in his neighborhood. dental health care carried out on the basis of eliminating pathological impact, regardless of the impact of quality of life of patients on treatment, then it affects the patient's quality of life, it will create a negative analysis or dissatisfaction in the completion of dental problems they have experienced.16 discussion of the quality of life and cost effectiveness becomes increasingly important for health topic related costs and values complexity of the relationship of health care services are obtained. health care providers are expected to make economic policy as an intermediary that connects between the health care needs.16,17 quality of life that describes the patient groups or regions is also relevant in the assessment of population health needs. conventional health indicators did not include analysis about the state of healthy or distortion by the clinical demand and supply factors. evaluation of effectiveness and assessment of health needs are often necessary to cut the program area and an extensive treatment is associated with the allocation of resources.4,16 it is concluded that the total costs incurred by patients to obtain filling treatment are higher than tooth extraction. differences of qol values in filling treatment are lower than the extraction. cost effectiveness ratio in the filling treatment is higher than the extraction, that the tooth extraction treatment is considered more cost effective than filling treatment. references 1. departemen kesehatan ri. pedoman upaya pelayanan kesehatan gigi dan mulut di puskesmas. jakarta: ditjen yanmed depkes ri; 2000. p. 2–7. 2. chestnutt ig, gibson j. clinical dentistry. sydney: churchill livingstone; 2002. p. 15–78. 3. adam rz. do complete dentures improve the quality of life of patients?, thesis. university of the western cape. 2006. available at: http://etd.uwc.ac.za/usrfiles/modules/etd/docs/etd_ init_ 5933_ 1173097639.pdf. accessed december 10th, 2009. 48 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 43–48 4. sumawan iw. cost effectiveness analysis (cea) metode kontrasepsi iud, suntik dan pil dengan pendekatan quality of life. thesis. surabaya: universitas airlangga; 2007. p. 35–74. 5. kumar s, williams ac, sandy jr. how do we evaluate the economics of health care?. european j of orthodontics 2006; 28: 513–9. 6. john mt, hujoel p, miglioretti dl, leresche l, koepsell td, micheelis w. dimensions of oral health related quality of life. j dent res 2004; 83: 956. available at: http://jdr.sagepub.com. accessed december 19th, 2009. 7. allen pf. assessment of oral health related quality of life. health and quality of life outcomes 2003; 1: 40. available at: http://www. hqlo.com/content/1/1/40. accessed december 25th, 2009. 8. brennan ds, singh ka, spencer aj, thomson kfr. positive and negative affect and oral health-related quality of life. health and quality of life outcomes 2006; 4: 83. 9. kent gg, blinkhorn as. pengelolaan tingkah laku pasien pada praktik dokter gigi. edisi 2. jakarta: egc; 2005. p. 5–21. 10. kumar s, bhargav p, patel a, bhati m, balasubramanyam g, duraiswamy p, kulkarni s. does dental anxiety influence oral health-related quality of life? observations from a cross-sectional study among adults in udaipur district, india. j of oral sci 2009; 51: 245–54. 11. klages u, bruckner a, zentner a. dental aesthetics, self awareness, and oral health related quality of life in young adults. european j of orthodontics 2004; 26: 507–14. 12. hanafi. manajemen mutu pelayanan kesehatan. surabaya: airlangga university press; 2004. p. 8–19. 13. naito m, yuasa h, nomura y, nakayama t, hamajima n, hanada n. oral health status and health related quality of life: a systematic review. j of oral sci 2006; 48: 1–7. 14. tjiptono f. total quality managementyogyakarta: penerbit andi; 2005. p. 27–64. 15. saroso. sistem manajemen kinerja. jakarta: gramedia pustaka; 2003. p. 12–23. 16. nuca c, amariei c, rusu dl, arendt c. oral health-related quality of life evaluation. ohdmbsc 2007; 6: 3. 17. lee jy, bouwens tj, savage ms, vann wf. examining the cost effectiveness of early dental visits. pediatric dentistry 2006; 28: 2. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 159 vol. 42. no. 4 october–december 2009 immunopathological aspects of oral erythema multiforme maharani laillyza apriasari1 and retno pudji rahayu2 1 oral medicine resident 2 department of oral biology faculty of dentistry, university of airlangga surabaya indonesia abstract background: erythema multiforme is an acute disease on the skin and mucous membrane. this lesion can erupt in mucous membranes of the oral cavity. improper and late treatment may cause stevens johnson syndrome which may cause patient mortality, therefore proper and accurate diagnosis are needed. purpose: the immunopathological aspect of oral erythema multiforme through literature study can help us to find the definite diagnosis and to know the differential diagnosis. review: in immunopathology, minor type of erythema multiforme is vasculitis caused by the immune complex hypersensitivity reaction among antigen antibodies. the mayor type of erythema multiforme may appeared from autoimmune reaction and from untreated minor type of erythema multiforme. conclusion: immunopathological approach of erythema multiforme is important beside the clinical manifestation, histology, and the differential diagnosis to find the definitive diagnosis. key words: eythema multiforme, hypersensitivity, autoimmune correspondence: maharani laillyza apriasari, c/o: ppdgs departemen ilmu penyakit mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: rany_rakey@yahoo.com review article introduction erythema multiforme is an acute disease on skin and mucous membrane which may cause several skin lesions, therefore named multiforme.1 this disease is an immunologic reaction of hypersensitivity reaction. erythema multiforme is characterized with an ulcerated lesion on mucous or target lesion on skin in the form of reddish macula surrounding vesicular or bulla in the center. target lesions are also called iris lesion.2 severe cases of erythema multiforme are also called as stevens johnson syndrome. this syndrome involves the mouth, eyes, genitalia, and skin. twenty to thirty percent of erythema multiforme cases occur in oral mucous membrane, in the form of multiple vesicular lesion which burst and leave wide eroded area covered by white pseudomembrane.3,4 this disease often happen in young adults and children, especially in males and seldom occur in elderly.1–5 there are two types of erythema multiforme: minor and mayor erythema multiforme. mayor type of erythema multiforme has higher degree of severity which is called as stevens johnson syndrome. this disease may worsen and cause extensive skin peeling, and often cause mortality through secondary infection and electrolyte liquid imbalance.1,3,6 the etiology of erythema multiforme is still unclear to this day. erythema multiforme is considered as an immunologic disease.4 this is probably happen because of predisposing factors as side effect reaction against certain microorganisms, radiotherapy, systemic diseases, malignancy, and food or drug allergy.1-7 common drugs which often cause erythema multiforme are antibiotics, barbiturates, phenylbutazone, and carbamazepin.1 immune system is a body mechanism which is used to maintain homeostasis condition against diseases.8 the body’s ability to eliminate foreign bodies depend on the ability of the immune system in recognizing strange molecules or called immunogens. response against foreign bodies is done by immunocompetent cells in the body which has three characteristics: specific recognition on certain immunogens, function to differentiate non-self bodies, retaining memory and amplification by remembering known previous immunogens by differentiation.9 160 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 159-163 today, the development of science and technology is focused on increasing disease diagnosis more accurately. this literature review describes the immunopathogenesis of erythema multiforme and its identification through immunopathological approach not only to get accurate diagnosis but also to give proper therapy. erythema multiforme the clinical manifestation of this disease are acute, begin with general symptom of fever, dizziness, and malaise. in less than 24 hours, an explosive lesion will occur in the skin and mucous membranes, where the lightest manifestation is formed as macula and papula in 0.5–2 cm diameter, whereas in the oral cavity it is started with easily ruptured vesicular and bulla. this condition is a distinctive clinical description which happened in 20–30% of cases (figure 1).1 figure 1. lesion as crusts, bleeding, and desquamation.10 minor erythema multiforme often occur in the oral mucous membrane and skin, seldom happen in oral mucous membrane only. multiple vesicular and easily rupture lesions leave an eroded area which hurt and covered with white pseudo membrane. minor erythema multiforme also happen on other mucous membrane in genitalia mucosa but seldom happen in conjunctiva, while on skin usually appeared as reddish macula papula. this lesions are often occur as target lesion (figure 2).4 figure 2. lesion as bulla and target lesion.10 mayor erythema multiforme occurs more often on oral mucosa. at the beginning it seemed as a reddish area which turn quickly into vesicle and soon rupture leaving a reddish eroded area covered by white pseudomembrane and crust from bleeding. other mucous parts could be found on the eyes, genitalia, pharynx, larynx, esophagus, and bronchial; especially in severe cases. on skin, this lesion often found as redness edematous lesion, forming a target lesion.4 diagnosis was based on characterized clinical manifestation, as broad and quick lesion, easily ruptured bulla, bleeding, and crusts on lips. 4,6 biopsy for histopathology examination was done to bring ultimate diagnosis of erythema multiforme. morphological changes usually show hyperplastic and spongiosis epithelial cells. apoptosis on basal and parabasal layers are always seeen. vesicular could be found on superficial epithel on supporting tissues and sometimes in intraepithel. necrotic epithel could always be found. changes on supporting tissues showed lymphocyte and macrophage infiltration on perivascular and papilla supporting tissue area.3 even though histopathological features are unspecific, the presence of perivascular lymphocyte infiltrates, epithelial edema and hyperplasia, were enough to considered the suspect of erythema multiforme.1 histopathology examination on mucosa affected by erythema multiforme showed specific characteristic, which is not always present. inflammations with inflammatory cell infiltration such as lymphocyte, neutrophil, and eusinophil are more often seen. these cells are organized parallel with perivascular. immunopathologic feature on erythema multiforme is less specific, therefore it is not grouped into vesicobulous diseases. ultimate diagnosis of erythema multiforme usually can be set through anamnesis and clinical examination (figure 3).11 systemic therapy given on erythema multiform is antihistamine if there is hypersensitivity reaction of drug and to avoid predisposing factors.12 administration of oral corticosteroids, especially after the second to fourth days will decrease the eruption period of acute symptoms. on minor type, oral corticosteroids were given 20–40 mg/day for 4–6 days in tapering dose not more than two weeks.1,4 on major type corticosteroid therapy is needed orally 40–80 mg/day for 2–3 weeks and antibiotic to avoid secondary infection risk, and high calorie and protein soft diet. topical therapy can be done by using oral rinse with topical anesthesia, oral rinse containing antibiotic and topical corticosteroids to reduce patient discomfort.4 hypersensitivity reaction specific immune system is like a two sided knife, in one side it is a body defense system, but in other side it can promote tissue destruction.13 all kind of immune system trauma is a hypersensitivity reaction.14 hypersensitivity is a disease caused by over reaction of immune system. this reaction happen on second contact with antigen, which sensitized previously.5 coombs and bell classified hypersensitivity reaction into 4 groups. type i, ii, and iii are reactions which 161apriasari and rahayu: immunopathological aspects depend on antigen and antibody interactions, while type iv depend on increasing receptor expression on the surface of lymphocyte.13 type i hypersensitivity reaction is called as anaphylactic type which is quickly set, where allergen bound ige antibody in releasing vasoactive amine, other mediators from basophil and mast cell, which lead to other inflammatory cells recruitment, prototype distraction as anaphylactic and other type of bronchial asthma.9 type ii hypersensitivity reaction is a cytotoxic hypersensitivity reaction which depend on antibody. this reaction happened because of free antibody interaction with antigen from tissue or cell surfaces. this antigen can originate from part of cell or host tissue and can also be absorbed from the outside which stick to cell or tissue, such as in hemolytic anemia autoimmune and erythroblastosis foetalis.13 figure 3. vasculitis on immune complex process.14 figure 4. stages of immune complex formation.14 type iii hypersensitivity reaction is called as immune complex disease (figure 3). the mechanism begun with the formation of antigen antibody complex which activate complement to attract neutrophil, lysosim enzyme release, oxygen free radicals, etc., as in arthus reaction, serum sickness, and systemic lupus erythematosus (figure 4).14 type iv is a cellular hypersensitivity, where t lymphocyte with receptor on its surface, will be activated from macrophage contact which bound with antigen. this will sensitize t cell and make it release cytokine as mediator in slow type of hypersensitivity reaction, as in tuberculosis, contact dermatitis, and transplant rejection.9,13 hypersensitivity reactions often occur in the oral cavity, involving part or all of oral mucosa. angioderma alergica which is a type i hypersensitivity reaction, stomatitis contacta of toothpaste or topical anesthesia are examples of hypersensitivity reaction in part of the oral cavity, while stomatitis alergica, erythema multiforme, and lichen planus are examples which involve all part of the oral cavity.5 autoimmune autoimmune is an immune response which considers host tissue as antigen. these processes involve cellular and humoral reaction. the process or mechanism inside the host that lead to tissue destruction is not yet known precisely (figure 5). in this problem there are many theories which still in debate.5 in every autoimmune disease there can be more than one defect and this defect may vary from one disturbance to other disturbances. disturbances on tolerance and autoimmune initiation involve interaction between immunologic factors, genetics, and microbial infections.9,13,14 figure 5. infection activate autoimmune disease which lead to tissue destruction.14 162 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 159-163 immunology factor mechanism from the tolerance failure such as: the failure of cell death induced by t cell activation which is auto reactive persistently, the activation of b cell without initiation stage which will form autoantibody, the failure of suppression mediated by t cell because of the reduced function of t cell, activation of polyclonal lymphocyte (non specific antigen) b cell because of microorganism production, and foreign antigen release because of tissue inflammation which arrange the induction of immune response and epitop spreading.15 the phenomenon of autoimmune is often connected with genetics. there are genetic components which are identical to hla specificity, as in systemic lupus erythematosus (sle) with class ii hla gene especially on hla-dq locus.13,14 in relation with microbes, some bacteria, microplasm, and virus are able to trigger the autoimmune reaction through several mechanisms, such as: epitop recognition which cross linked with host antigen, microbial antigen and auto antigen combined forming an immunogenic unit to activate the tolerance of t cell, some virus and bacteria as a mitogen to t cell or b cell non specific polyclonal which can induce the formation of antibody, and microbial infection with tissue necrosis and inflammation which can activate antigen presenting cell (apc) stimulation in tissues.14 discussion erythema multiforme is an acute disease which occur on skin and mucosa with many manifestations, there are papula, bulla, or vesicular and target lesion on skin, whereas ulceration with white pseudomembrane on mucosa and specific desquamation with bleeding and crusts on lips.1,3,4 the etiology is often triggered by drugs side effects and can also caused by infection from herpes simplex virus and mycoplasma pneumonia.4 this can result the type iii hypersensitivity reaction and lead to minor type of erythema multiforme. this minor type of erythema multiforme may worsen, affected by body autoimmune factor and become major type which usually called stevens johnson syndrome. from immunopathological aspect, minor erythema multiforme is caused by type iii hypersensitivity reaction, which involves immune complex reaction of antigen and antibody. biopsy on blood vessel wall of erythema multiforme patient found the increasing level of igm, complement and fibrin deposits.3 this vasculitis caused by immune complex reaction of antigen and antibody. the pathogenesis is divided into three steps: the formation of antigen-antibody complex in circulation, the deposit of immune complex in numerous tissues, and the appearance of inflammatory reaction in many parts of the body.14 on the first stage, when antigen enters the body, specific antibody will be produced. and then in the circulation system these two form antigen-antibody complex. if the antigen could not be eliminated or phagocyte cells fail to do its function, antigen will be in the circulation for a longer time. this situation can also be caused by malfunctioned macrophage, leading to deposition of immune complex in many parts and causing vasculitis.13 immune complex which leave circulation and deposit inside or outside blood vessel wall, will cause the increase of blood vessel permeability. this condition is marked by immune complex which bound with inflammatory cells through fc and c3b receptors and trigger the release of vasoactive and cytokine mediators.14 on condition when immune complex deposit within the tissues, third inflammatory reaction occurs. in this stage appears symptoms such as fever, urticaria, arthralgia, and lymphoid gland expansion.1,15 this can happen in the beginning of erythema multiforme to quickly appear its clinical manifestation, but the prodromal symptoms which precede it may not as severe as in diseases of viral infections. severe erythema multiforme (major type) is mostly caused by autoimmune process. in histopathology examination, autoantibody on desmoplakins 1 and 2 are found, which show the involvement of humoral immune system.3 the formation of autoantibody could happen through several mechanisms. they are cross reaction, virus, drugs, synthetic error or abnormal lysosome which modify body constituent molecule into autoantigen.9 drug administration is considered as foreign antigen which will be absorbed by cell surface and trigger chemical reaction with hapten which could change the immunogenicity. drugs like nsaid, carbamazepin, antibiotic and barbiturates can trigger autoimmune reaction which can cause erythema multiforme both major and minor type. the involvement of microbial infection between simplex herpes virus and mycoplasma pneumonia will cause microbial endotoxin release. this microbial endotoxin stimulates b cell through non specific second induction signal without t cell help (t cell independent), and later produce autoantibody detectable in serum.14,16 other cause of autoimmune is the failure of autoregulation in antigen presentation, infection which increase major histocompatibility complex (mhc) response and low level of cytokines. the surveillance of several autoreactive cells is predicted dependent to t suppressor (ts) cell. if the t supressor (ts) cells fail, t helper (th) cells can be induced and therefore promoting autoimmune.9 the immunopathology of major erythema multiforme is characterized by t cell with autoreactive potential when meeting with autoantigen without constimulation. this situation is caused by infection or tissue necrosis and local inflammation.14 this might occur if therapy is not done immediately on minor erythema multiforme or not well maintained and worsen, leading to major type of erythema multiforme. until now, the diagnosis of erythema multiforme is still based on specific clinical manifestation and histopathology examination result. for accuracy, immunology test is 163apriasari and rahayu: immunopathological aspects needed to support more precisely and adequate therapy. immunity system repair is needed to achieve homeostasis condition for successful disease treatment of immunity disturbance. on minor erythema multiforme, immunology diagnosis is based on vasculitis as immune complex reaction caused by type iii hypersensitivity reaction, while on major type of erythema multiforme is often found desmoplakins autoantibody 1 and 2 which involve humoral immune system, showing an autoimmune reaction. this can be used as a way to help the diagnosis of erythema multiforme, to be differentiated by its differential diagnosis. the differential diagnosis of erythema multiforme in the oral cavity is primary herpetic stomatitis. both often occur acutely as ulceration, begin with prodromal symptoms such as malaise, fever, arthralgia, and occur in the oral cavity and lips. the difference between them is the appearance of erythema multiforme as ulceration with white pseudomembrane on oral mucosa. this white pseudomembrane is fibrin formed by vasculitis bleeding and the crusts on lips with bleeding, while these are not occurred in primary herpetic stomatitis. the location of ulceration differs, where erythema multiforme do not always occur on gingival, while primary herpetic stomatitis often occur on gingival. prodromal symptoms starting erythema multiforme are not as severe as in primary herpetic stomatitis, therefore to establish the diagnosis, deep anamnesis must be carried out. corticosteroid is used to avoid the causing factor. mouth wash with topical anesthesia and antibiotic is aimed to avoid secondary infection. t h r o u g h t h i s l i t e r a t u r e r e v i e w , i t c a n b e concluded that erythema multiforme is classified into 2 types, the minor type which is caused by hypersensitivity reaction by vasculitis on antigen antibody complex reaction, and the major type which is caused by autoimmune process. immunopathological approach on oral erythema multiforme could help in taking the definitive diagnosis and providing adequate therapy. references 1. glick m, greenberg sm. burket’s: oral diagnosis and treatment. 10burket’s: oral diagnosis and treatment. 10th ed. hamilton, ontario: bc decker inc; 2003. p. 208–11. 2. sen p, chua sh. a case of recurrent erythema multiforme and its therapeutic complication. case report. j an acad med singapore 2004; 33: 793–6. 3. regezi aj, sciubba jj, jordan ckr. oral pathology: clinical pathologic correlations. 4th ed. missouri: saunders elsevier; 2003. p. 44–6. 4. laskaris g. treatment of oral disease: a concise textbook. stuttgart germany: thieme; 2005. p. 66–7. 5. field a, longman l. tyldesley’s oral medicine. 5th ed. new york: oxford university press; 2004. p. 135–6. 6. wray d, lowe dog, dagg hj, felix hd, scully c. textbook of general and oral medicine. london: churchill livingstone; 2001. p. 238–9. 7. cawson ra, odell ew. cawson’s essentials of oral pathology and oral medicine, 7th ed . london: churchill livingstone; 2002. p. 205–7. 8. roitt im. essensial immmunology. 8th ed. oxford: blackwell science limited; 2003. p. 1–31. 9. baratawidjaja kg. imunologi dasar. edisi 5. jakarta: fakultasedisi 5. jakarta: fakultasjakarta: fakultas kedokteran universitas indonesia; 2006. p. 3, 220–1. 10. hunter jaa, salvin ja, dahl mv. clinical dermatology. 3clinical dermatology. 3rd ed. usa: blackwell science publishing; 2003. p. 100. 11. neville wb, damm dd, allen mc, bouquot ej. oral andoral and maxillofacial pathology. 2nd ed. philadelphia, pennsylvania: saunders; 2002. p. 676. 12. shah kn, honig jp, yan ca. a case series and review of acute annular urticarial hypersensitivity syndrome in children. journal watch dermatology: ”urticaria multiforme”. pediatrics 2007; 119(5): 117–83. 13. roeslan bo. imunologi kelainan di dalam rongga mulut. jakarta: abadi dhaya insani; 2000. p. 69–76, 86–9. 14. kumar v, cotran sr, robbins ls. pathologic basis of disease. 7pathologic basis of disease. 7th ed. philadelphia, pennsylvania: elsevier saunders; 2005. p. 128–30. 15. zlotoff b, leggott j, doherty, segal. cutaneous reactions to drugs in children. journal of the american academy of pediatrics 2007; 120: 1082–96. 16. horn a, girschick, latsch k. stevens johnson syndrome without skin lesions. journal of medical microbiology 2007; 52: 49–56. mkgs vol 44 no 1 jan-mar 2011.indd 7 vol. 44. no. 1 march 2011 research report cytotoxicity difference of 316l stainless steel and titanium reconstruction plate ni putu mira sumarta1, coen pramono danudiningrat1, ester arijani rachmat2, and pratiwi soesilawati2 1department of oral and maxillofacial surgery 2department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract background: pure titanium is the most biocompatible material today and used as a gold standard for metallic implants. however, stainless steel is still being used as implants because of its strength, ductility, lower price, corrosion resistant and biocompatibility. purpose: this study was done to revealed the cytotoxicity difference between reconstruction plate made of 316l stainless steel and of commercially pure (cp) titanium in baby hamster kidney-21 (bhk-21) fibroblast culture through mtt assay. methods: eight samples were prepared from reconstruction plates made of stainless steel type 316l grade 2 (coen’s reconstruction plate®) that had been cut into cylindrical form of 2 mm in diameter and 3 mm long. the other one were made of cp titanium (stema gmbh®)) of 2 mm in diameter and 2,2 mm long; and had been cleaned with silica paper and ultrasonic cleaner, and sterilized in autoclave at 121° c for 20 minutes.9 both samples were bathed into microplate well containing 50 μl of fibroblast cells with 2 x 105 density in rosewell park memorial institute-1640 (rpmi-1640) media, spinned at 30 rpm for 5 minutes. microplate well was incubated for 24 and 48 hours in 37° c. after 24 hours, each well that will be read at 24 hour were added with 50 μl solution containing 5mg/ml mtt reagent in phosphate buffer saline (pbs) solutions, then reincubated for 4 hours in co2 10% and 37° c. colorometric assay with mtt was used to evaluate viability of the cells population after 24 hours. then, each well were added with 50 μl dimethyl sulfoxide (dmso) and reincubated for 5 minutes in 37° c. the wells were read using elisa reader in 620 nm wave length. same steps were done for the wells that will be read in 48 hours. each data were tabulated and analyzed using independent t-test with significance of 5%. results: this study showed that the percentage of living fibroblast after exposure to 316l stainless steel reconstruction plate was 61.58% after 24 hours and 62.33% after 48 hours. and after exposure to titanium reconstruction plate, the percentage of living fibroblast was 98.69% after 24 hours and 82.24% after 48 hours. based on cytotoxicity parameter (cd50%), both reconstruction plate made of 316l stainless steel or titanium showed as a non-toxic materials to fibroblast. conclusion: both reconstruction plate made of stainless steel and cp titanium were non-toxic to fibroblast, although the stainless steel plate showed lower cytotoxicity level compared to titanium. therefore a reconstruction plate made from stainless steel type 316l can be used as a safe material for mandibular reconstruction. key words: 316l stainless steel plate, titanium plate, cyototoxicity, mtt assay abstrak latar belakang: titanium murni adalah bahan yang paling biokompatibel saat ini dan digunakan sebagai standar emas implan logam. saat ini stainless steel masih digunakan karena kekuatan, ductility, harganya yang murah, tahan terhadap korosi dan cukup biokompatibel. tujuan: penelitian ini dilakukan untuk mengetahui perbedaan sitotoksisitas antara plat rekonstruksi yang terbuat dari titanium murni komersial dan plat rekonstruksi yang terbuat dari stainless steel pada kultur sel fibroblas baby hamster kidney21 (bhk-21) menggunakan mtt assay. metode: delapan sampel yang masing-masing tipe 316l terbuat dari stainless steel 316l grade 2 (coen’s reconstruction plate®) yang dipotong berbentuk silinder diameter 2 mm dan panjang 3 mm, serta yang terbuat dari titanium murni komersial (stema gmbh®) diameter 2 mm dan panjang 2,2 mm; dan dibersihkan dengan kertas silika dan pembersih ultrasonik serta disterilkan dengan autoclave pada suhu 121° c selama 20 menit. kedua sampel dimasukkan ke dalam sumur mikroplat yang mengandung 50 μl sel fibroblas dengan kepadatan 2 × 105 dalam media rosewell park memorial institute-1640 (rpmi-1640), diputar dengan kecepatan 30 rpm selama 5 menit. sumur mikroplat diinkubasi selama 24 dan 48 jam pada suhu 37° c. setelah 24 8 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 7–11 jam, pada tiap sumur yang akan dibaca pada jam ke 24 ditambahkan 50 μl cairan yang mengandung 5mg/ml mtt dalam phosphat buffer saline (pbs), kemudian diinkubasi kembali selama 4 jam dalam co2 10% pada suhu 37° c. assay kolorimetri dengan mtt digunakan untuk mengetahui viabilitas populasi sel setelah 24 jam. setiap sumur ditambahkan pelarut dimetil sulfoksida (dmso) dan diinkubasi kembali selama 5 menit pada suhu 37° c. sumur-sumur tersebut kemudian dibaca dengan elisa reader dengan panjang gelombang 620 nm. langkah yang sama dilakukan pada sumur-sumur yang akan dibaca pada jam ke 48. data kemudian ditabulasi dan dianalisis dengan menggunakan independent t-test dengan signifikansi 5%. hasil: penelitian ini menunjukkan presentase fibroblas hidup setelah terpapar plat rekonstruksi yang terbuat dari stainless steel adalah 61,58% setelah 24 jam dan 62,33% setelah 48 jam. dan setelah paparan dengan plat rekonstruksi yang terbuat dari titanium murni adalah 98,69% setelah 24 jam dan 82,24% setelah 48 jam. berdasarkan pada parameter sitotoksisitas (cd50%) kedua plat rekonstruksi baik yang terbuat dari titanium murni maupun yang terbuat dari stainless steel tipe 316l merupakan bahan yang tidak bersifat toksik terhadap fibroblas. kesimpulan: kedua plat rekonstruksi baik yang terbuat dari stainless steel maupun cp titanium tidak bersifat toksik terhadap fibroblas, walaupun plat stainless steel menunjukkan level sitotoksisitas yang lebih rendah daripada titanium murni. dengan demikian plat rekonstruksi yang terbuat dari stainless steel 316 l aman digunakan sebagai bahan untuk rekonstruksi mandibula. kata kunci: plat stainless steel, plat titanium, sitotoksisitas, mtt assay correspondence: ni putu mira sumarta, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: putumira_omfs@yahoo.co.id introduction implant materials can be classified into biotolerant, bioinert, and bioactive materials. stainless steel is a biotolerant implant material, that characterized by the presence of a thin fibrous layer overlying implant surface in contact to bone. titanium is a bioinert material with a characteristic of direct contact to bone or osseointegration, osseointegration can be achieved because there is no chemical reaction between material surface to surrounding tissue or to body fluid.3 metallic materials implanted in the human body rarely induce serious conditions. metallic materials conventionally used in medicine and dentistry does not show toxicity. however, some elements of the alloys show toxicity. the toxicity of a metallic material is governed not only by the elements content of the material but also by its corrosion and wear resistance.4 before implantation, every materials should pass compatibility study. in vitro citotoxicity study is the primary study to determined a material or material component’s biocompatibility. in this study, an unprocessed material or material component’s are placed directly into tissue cell culture.5,6 cell culture can be used to evaluate material’s citotoxicity through microscopic examination or quantitatif analysis. cell morphology and characteristics in the adhesion process are evaluated to determine the effect of cellular citotoxicity, adhesion mechanism changes, cell atypia, or cell damage. fibroblast and osteoblast are the common cell type used in the study of implant biocompatibility.7 one of the citotoxicity study that commonly used is the mtt assay or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. this study are based on the reduction of yellow tetrazolium salt into purple formazan crystal by hidrogenase enzyme secreted from the mitochondria of the metabolically active cells. amounts of purple formazan crystal define amounts of living cells.8 until these days, stainless steel and titanium alloys commonly used as implant material in orthopedics and dentistry. since 2003 until 2008, there were 51 patients with benign mandibular tumor at the department of oral and maxillofacial surgery, airlangga university/dr. soetomo hospital surabaya were being treated with mandibular resection and immediate reconstruction wether using stainless steel reconstruction plate, bone graft, and combined bone graft and stainless steel plate as stabilizer. airlangga university dental hospital use stainless steel plate because it’s cheaper than titanium plate but have a good strength, ductility, resistant to corrosion, and compatible. whereas pure titanium is considered as the most biocompatible material, so commonly used as a gold standard in metallic implant. inexpensive reconstruction material are still needed in indonesia because of the social economic level of the indonesian people which mayoritas could not afford expensive materials. the aim of this study was to evaluate the cytotoxicity of mandibular reconstruction plates made from stainless steel type 316l and the one made from pure titanium in bhk-21 cell culture through mtt assay in 24 and 28 hours measurements. materials and methods this was an experimental laboratory study conducted in december 2009 at pusat veterenaria farma (pusvetma) surabaya. eight samples were prepared from reconstruction plates made stainless steel type 316l grade 2 (coen’s reconstruction plate®) that had been cut with wire cutter into cyllindrical form of 2 mm in diameter and 3 mm long; and from one made of commercially pure (cp) titanium (stema gmbh®)) that had been cut into cylindrical form of 2 mm in diameter and 2.2 mm long. after cutting, all 9sumarta, et al.: cytotoxicity difference of 316l stainless steel samples were cleaned with silica paper to remove cutting debris, soaked into ethanol 70% in ultrasonic cleaner for 60 minutes, and sterilized in autoclave at 121° c for 20 minutes.9 fibroblast cells from bhk-21 cell line were cultivated in a roux bottle until confluent, and harvested with trypsine versene solution. harvested fibroblast were cultured in rosewell park memorial institute-1640 (rpmi-1640) media which contain 10% fetal bovine serum albumin, incubated for 24 hours in 37° c, cells were transported into small roux botle dan cultured in the density of 2 × 105 into each 96 well microplate until confluent. each microplate well contain 50 μl of cells with 2 × 105 density in rpmi media, spanned at 30 rpm for 5 minutes. there was also cell control contains cells in culture media as a positive control which assumed as 100% living cells, and media control contains culture media without cells which assumed as 0% living cells. each microplate well was examined under light microscope to ensure that the incubation time was enough to form crystals. both samples made from stainless steel and titanium bathed into the well containing fibroblast cells and rpmi. microplate well was incubated for 24 and 48 hours in 37° c. after 24 hours, each well that will be read at 24 hour were added with 50 μl solution containing 5 mg/ml mtt reagent in pbs, then reincubated for 4 hours in co2 10% and 37° c. colorimetric assay with mtt was used to evaluate viability of the cells population after 24 hours. then, each well were added with 50 μl dmso and reincubated for 5 minutes in 37° c. the wells were read at elisa reader in 620 nm wave length. same steps were done for the wells that will be read in 48 hours. each data were tabulated and analyzed using independent t-test with significance of 95%. percentage of the living fibroblast cells were calculated with the following formula, according to experimental study by meizarini et al:10 percentage of the living cells = treatment + media × 100% cell + media results cell in control group had the highest mean value of optical density and media control group had the lowest mean value of optical density (table 1). normality study with nonparametric test using kolmogorof smirnov test, showed that all study groups had p > 0.05 which mean that all study groups had a normal distribution that mean values of the data lies between standard deviation and significance test was done with independent t-test, showed in table 2 and 3. table 1. average of optical density and standard deviation of each study groups in 24 and 48 hours measurements groups n x + sd 24 hours 48 hours titanium 8 0.42 ± 0.10 0.55 ± 0.05 stainless steel 8 0.23 ± 0.05 0.39 ± 0.05 cell control 8 0.42 ± 0.14 0.69 ± 0.12 media control 8 0.09 ± 0.02 0.10 ± 0.02 table 2. difference test of the optical density from each study groups in 24 hours measurement using independent t-test titanium stainless steel cell control media control titanium 0.001** 0.915 0.001** stainless steel 0.002** 0.001** cell control 0.001** media control table 3. difference test of the optical density from each study groups in 48 hours measurement using independent t-test titanium stainless steel cell control media control titanium 0,001** 0,013** 0,001** stainless steel 0,001** 0,001** cell control 0,001** media control there was a significant difference of the formazan density in the 24 and 48 hours measurement, showing there was a significant increased number of living fibroblast following exposure time to titanium and stainless steel plate (table 2 and 3). there was no significant difference between the titanium group and the cell control group in the 24 hours measurement with p > 0.05 (table 2). table 4. percentage of the living fibroblast cells in each study groups in 24 and 48 hours measurement cell group n 24 hours (%) 48 hours (%) titanium 8 98.69 82.24 stainless steel 8 61.58 62.33 10 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 7–11 the percentage of the living fibroblast cells in each study groups were more than 50% (table 4). this results showed both plate made of stainless steel and cp titanium were non-toxic to fibroblast based on cd50%. 11 discussion biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local and systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimizing the clinically relevant performance of that therapy.12 biocompatibility is important because implant surface in contact with the tissue can undergo corrosion in vivo. implant corrosion can cause lost of load bearing strength and can undergo degradation into toxic substances in the tissue.13 main criteria in choosing metallic implants is its biocompatibility.14 materials can be classified as a biocompatible material if culture cells remain living and metabolically active in long term culture. there are 2 quick and simple quantitative assay to test biocompatibility in vitro, these are cell viability based on physical uptake of neutral red (nr) and based on cell’s metabolic activity through mtt assay, which based on cellular enzyme activity. both test are widely accepted as a biocompatibility and cytotoxicity study to evaluate cell viability and growth.15 in this study mtt assay was performed to evaluate the biocompatibility of stainless steel 316l and titanium reconstruction plates, because this method is a simple, accurate measure, and can be done in a large scale study.16–18 this study showed that there was a significant optical density difference of treatment group exposed to stainless steel in 24 and 48 hours measurement, and in treatment group exposed to titanium in 48 hours measurement. whereas in 24 hours measurement, there was no significant optical density difference between group exposed to titanium and cell control group. this was consistent with the reference that stated titanium is a bioinert material because there is no chemical reaction between material surface to surrounding tissue or to body fluid.3 there were increased optical density of the cell control group from 0.4223 in 24 hours into 0.6860 in 48 hours measurement, optical density of the group exposed to titanium only increased from 0.4156 in 24 hours into 0.5470 in 48 hours measurement. this study showed that reconstruction plates made from stainless steel 316l and cp titanium had a good in vitro biocompatibility to fibroblast cell which indicated from there was no cytotoxic effect to bhk-21 cells. that was concluded from the percentage of living fibroblast cell after exposure to stainless steel 316l in 24 hours was 61.58%; after 48 hours was 62.33%; after exposure to cp titanium was 98.69% in 24 hours; and 82.24% after 48 hours. all measures showed there was no cytotoxicity based on cd50%, the cytotoxicity parameter.11 the difference caused by increasing number of living fibroblast cell from both exposure time, showing increasing living fibroblast cell, because fibroblast were able to anchor and adapt accordingly to metal particle,9 so that cells can replicate. this study showed that cp titanium plates had a higher biocompatibility level compared to stainless steel 316l plates, this consistent with the classification of material which categorize cp titanium into bioinert material, whereas stainless steel 316l is a biotolerant material.19 biocompatibility of implant material also affected by their resistance and their alloy to corrosion process in body fluid that was known as electrolyte. after implantation there was changes in neutral ph that depend on implantation time and appropriate healing process, tissue ph were between 6.8–7.4.20 titanium and its alloy are materials with the best corrosion resistance, because of their passive nature and an unreactive passive film formed on titanium surface. stainless steel 316l had an acceptable corrosion resistance. stainless steel 316l implant was mostly manufactured in india to be used for orthopedic application because of its lower price, easier welding and processing compared to cobalt-chromium alloy and titanium or titanium alloy. other properties of stainless steel 316l are biocompatible, good tensile strength, fatigue resistance, with appropriate density for weight bearing, made it a preferable surgical implant material. the disadvantage of titanium are expensive, easily wear off, and brittle.21 it can be concluded that both reconstruction plate made of stainless steel and cp titanium were non-toxic to fibroblast, although the stainless steel plate showed lower cytotoxicity level compared to titanium. therefore a reconstruction plate made from stainless steel type 316l can be used as a safe material for mandibular reconstruction. references 1. disa jj, hidalgo da. mandibular reconstruction in: thorne ca, beasley rw, aston sj, bartlett sp, gurtner gc, spear sl, editors. grabb and smith’s plastic surgery. 6th ed. philadelphia: lippincott williams and wilkins; 2007. p. 428. 2. doty jm, pienkowski d, goltz m, haug rh, valentino j, arosarena oa. biomechanical evaluation of fixation techniques for bridging segmental mandibular defects. arch otolaryngol head and neck surg 2004; 130: 1388–92. 3. gupta r, caiozzo vj, skinner hb. basic science in orthopedic surgery. in: skinner hb, editor. current diagnosis and treatment in orthopedics. 4th ed. new york: the mcgraw-hill companies, inc; 2006. p. 31–3. 4. hanawa t. evaluation techniques of metallic biomaterials in vitro. sci and tech of adv mater 2002; 3: 289–95. 5. wataha jc. biocompatibility of dental materials. in: anusavice kj, editor. science of dental materials. 11st ed. philadelphia: elsevier science; 2003. p. 173–92. 6. kao ct, ding sj, min y, hsu tc, chou my, huang th. the cytotoxicity of orthodontic metal bracket immersion media. europ j orthodont 2007; 29: 198–203. 7. an yh, martin kl, editors. handbook of histology methods for bone and cartilage. new jersey: humana press inc; 2003. p. 36, 443–5. 8. vannet vb, hanssens jl, wehrbein h. the use of three-dimensional oral mucosa cell cultures to assess the toxicity of soldered and welded wires. european j orthodont 2007; 29: 60–6. 11sumarta, et al.: cytotoxicity difference of 316l stainless steel 9. cheung s, gauthier m, lefebvre lp, dunbar m, filliaggi m. fibroblastic interactions with high porosity ti-6al-4v metal foam. j biomed mater res part b: apll biomater 2007; 82b: 440–9. 10. meizarini a, munadziroh e, rachmadi p. sitotoksisitas bahan restorasi cyanoacrylate dengan variasi perbandingan powder dan liquid menggunakan mtt assay. jurnal penelitian medika eksakta 2005; 6(1): 16–25. 11. telli c, serper a, dogan al, guc d. evaluation of the cytotoxicity of calcium phosphate root canal sealers by mtt assay. j endodon 1999; 25: 811–3. 12. williams df. on the mechanisms of biocompatibility. biomaterials 2008; 30: 1–13. 13. desai s, bidanda b, bártolo p. metallic and ceramic biomaterials: current and future developments. in: bártolo p, bidanda b, editors. bio-materials and prototyping applications in medicine. new york: springer-science; 2008: 1–2. 14. mudali uk, sridhar tm, raj b. corrosion of bio implants. sadhana 2003; 28(3–4): 601–37. 15. schmutz p, quach-vu nc, gerber i. metallic medical implants: electrochemical characterization of corrosion processes. the electrochemical society interface. summer 2008; 35–40. 16. wutticharoenmongkol p, sanchavanakit v, pavasant p, supaphol p. preparation and characterization of novel bone scafolds based on electrospun polycaprolactone fibers filled with nanoparticles. macromol biosci 2006; 6: 70–7. 17. sjogren g, sletten g, dahl je. cytotoxicity of dental alloys metals, and ceramics assessed by millipore filter, agar overlay, and mtt tests. j prosthet dent 2000; 84: 229–36. 18. tomadiki p. assessment of acute cytoand genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes. j orofac orthop 2000; 61: 2–19. 19. beloti mm, rollo jmda, filho ai, rosa al. in vitro biocompatibility of duplex stainless steel with and without 0.2% niobium. j app biomat & biomech 2004; 2: 162–8. 20. kiel m, marciniak ak. corrosion resistance of metallic implants used in bone surgery. arch mater sci and engin 2008; 30(2): 77–80. 21. mudali uk, sridhar tm, raj b. corrosion of bio implants. sadhana 2003; 28(3-4): 601–37. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 106 jaw locking after maxillofacial trauma david b. kamadjaja and r. soesanto department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract the purpose of this report is to present two cases of jaw locking with two different etiologies. in case #1, jaw locking occured 5.5 months after a surgical reduction and internal fixation on the fractured maxilla and mandible. some plain radiographic x-ray were made but failed to give adequate information in establishing the cause of trismus. the three dimensional computed tomography (3d-ct) was finally made and able to help guide the pre-operative diagnosis and treatment. two-steps gap arthroplasty were done comprising a gap arthroplasty leading to acceptable outcome. an adult patient in case #2 with a history of trauma at his childhood and bird-like face apprearance clinically, was unable to open the mouth since the time of accident. the patient was diagnosed with bilateral ankylosis of temporomandibular joints. one side (right) gap arthroplasty was done and resulted in normal mouth opening. key words: trismus, zygoma fracture, gap atrthroplasty, three dimensional computed tomography (3d-ct), bird face appearance correspondence: david b. kamadjaja, c/o: bagian ilmu bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: davidbk@sby.dnet.net.id introduction facial injuries are most commonly associated with falls, violence, motor vehicle accidents and sport-related trauma. mandible, middle third area of the face, and temporomandibular region are frequently involved in such facial injuries. the complexity of the temporomandibular joint (tmj), and its anatomical proximity to other craniofacial structures makes its diagnosis and treatment especially challenging.1 facial fractures in children are relatively uncommon, it is probably due to the elasticity of pediatric skeleton. with respect to facial fractures, mandible is the second most frequently fractured bone, after nasal bone fracture. hall in 19722 reported 20.7% incidence of mandible fracture in 495 patients younger than 14 years of age. carroll et al.2 in 1987 studied 268 facial fractures, of which 26.5% were mandibular fracture. the involvement of condylar process is found higher than seen in the adult, ranging from 40% to 60%.3,4 the actual growth of the mandible occurs at the mandibular condyle and along the posterior surface of the ramus.5 therefore it is understandable that fracture of the head of the condyle in the growing period may lead to joint ankylosis and might interfere with the mandibular growth and development. in delayed treatment of condylar fracture some potential complications might occur including the followings: 1) malocclusion; 2) temporomandibular joint dysfunction; 3) ankylosis, and 4) mandibular growth disturbances, the middle third of the face consists of nasoethmoidal complex, maxilla and smaller bones attached to it together with malar complex. it lies between the mandible and the cranial cavity and calvarium, in particular the frontal and sphenoid bones.1 treatment of the midface and mandible fractures might result in unsatisfactory outcome in terms of aesthetic and function such as facial deformity and jaw movement restriction. fracture of the zygoma complex has a specific clinical symptom i.e. facial bone flattening. flattening of the side of the face was noted in 57% of isolated zygomatic arch fractures in a study by ellis et al.2 accompanying the zygomatic arch fractures might be trismus due to impingement of fractured segment on temporal muscle. this finding was noted in 45% of isolated zygomatic arch fractures.2 post-fracture trismus, known as limitation of mouth opening which presented clinically in various degrees, from difficulty to inability in opening the mouth, may be associated with several conditions. the reason often cited for post-fracture trismus is impingement of the translating coronoid process of the mandible by the displaced zygomatic fragments.3 trismus can also be caused by fractures of facial or mandible in which bone fragments have displaced and rotated and restricted the mandible movements. problem of joint movements might also arise from bony ankylosis which is usually formed between the distal end of the fractured mandibular condyle and the surrounding bones. fractures of zygomatic compartments or complex and those involving temporo-mandibular joint are the most common types of fracture which are much related to the jaw movement problem, especially in severe trauma, in 107kamadjaja and soesanto: jaw locking after maxillofacial trauma which the fractured zygomatic complex is depressed in the direction of the coronoid process of the mandible and impairs the jaw movements. in patients suspected with condylar fracture, a series of plain x-ray projections consisting of posteroanterior skull view, two lateral oblique, and towne’s view are usually sufficient in supporting the diagnosis, but in other cases it might be difficult to adequately evaluate the cause of trismus. chayra et al.5 found that panoramic radiograph had a higher accuracy in detecting all types of mandible fractures in 92%. in mid facial injury, the panoramic film gives little value, therefore a maxillofacial radiograph series become necessary.2 in case of trismus presenting in complex fractures of maxilla and mandible, plains radiographic projection might be difficult in finding the causes of the trismus. with the advent of newer imaging techniques, such as computed tomography, identification of complex maxillofacial fractures can now be done with ease. computed tomography (ct) has largely been supplanted in diagnosis of maxillofacial as it yields excellent bony detail of the facial skeleton in multiple views.2 revolutionary progress of the ct technique which has been developed from two into three-dimension image, called 3d-ct, may represent the section of the patient that is scanned. this leads to performing profile to profile for the entire image reconstruction process. it can be used as the best means for establishing the diagnosis and surgical guidance in maxillofacial traumas. cases case #1: a-49 year old woman visited the department of oral and maxillofacial surgery at dr. soetomo general hospital surabaya asking for a treatment due to her trismus. the patient had a history of trauma caused by car traffic accident. the patient was seated just behind the driver and she had been thrown forward by the force to the driver chair. she underwent her first surgical treatment in a district hospital shortly after the accident. she also had a history of wearing intermaxillary fixation for 2 months post-operatively. clinically she appeared with midfacial flattening which was a characteristic sign of post-bilateral zygomatic bone fractures. multiple fractures in the mandible, including the condylar fracture, were also suspected. intraoral examination showed severe trismus, the maxillary and mandibular teeth were seen in occlusion but not in the proper alignment. the mandibular and maxillary arches were not in good relationship (figure 1). malalignment lengkung mandibula dan maksila figure 1. the patient presented with midface flattening and severe trismus (left); intraorally, both mandible and maxilla arches collapsed medially. figure 2. preoperative posteroanterior skull x-ray (left); water’s projection taken postoperatively (right). 108 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 106-113 figure 3. panoramic view showing fixed paramedian fracture and right condylar fracture with the condylar head being displaced medially. figure 4. submentovertex radiograph showing the coronoid process free from bone obstacles (left); pa skull x-ray showing right condylar neck fracture, but no obvious bony ankylosis seen (center); lateral skull view presenting unclear joint situation (right). a b dc figure 5. three d (ct) in series: (a) occipital view shows clearly the position both mandible joints and coronoid processes. both coronoid processes free from bone obstacles, the right condyle fractured and displaced, ankylosis is present. the left condyle slightly displaced laterally. (b) anterior view: fractured of the left zygoma and median site of the mandible and fixed with miniplates. the mandible arch is repositioned in wider form. (c) the right joint displaced and attached to the arcus zygoma and (d) the left joint displaced outside from the glenoid fossae. 109kamadjaja and soesanto: jaw locking after maxillofacial trauma figure 6. mouth opening ten days after surgery of right joint a gap arthroplasty and relapse 3 month afterward (left); five days after left joint gap arthroplasty, the patient was able to open her mouth more easily. figure 7. panoramic radiograph showing an abnormal bone growth in both condyles areas and ankylosis on the right joint; the left condyles showing a gap between fossae and condyle indicating a pseudo arthrosis. figure 8. preoperative trismus (left); normal mouth opening three days after surgery. 110 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 106-113 some pre and post-operative plain x-ray films were available at her presentation in our clinic, but they were not able to give adequate information as to point out the primary cause of the trismus. the gross bone fractures patterns could be seen in the pre-operative antero-posterior skull film showing multiple mandibular fractures of comminutive type between the midline and the right paramedian region. water’s projection made after the surgery showed a bilateral zygomatic fracture involving the left fronto-zygoma suture. this multiple facial bone fractures had been treated surgically, internal fixation using miniplates osteosynthesis were applied. the left zygoma was fixed with 2 miniplates over the left zygomatico-frontal and zygomatico-maxilla regions. the comminutive type of fracture on the mandible at the right paramedian region had been fixed with x-shape miniplate osteosynthesis (figure 2). few plain x-rays were subsequently made which are: orthopantomogram (figure 3), submentovertex, posteroanterior and lateral skull film (figure 4) and yet unable to detect the primary cause of the existing trismus. in panoramic film the right condylar head of the mandible was clearly seen to displace out from the glenoid fossa and figure 10. schematic drawing of: preauricular incision as an approach to have access to the temporomandibular joint (left), the location of bone cutting in gap arthroplasty and the amount of bone to be excised (right).8 figure 9. post-operative situation: facial appearance with bird face and pre-auricular approach for tmj surgery. 111kamadjaja and soesanto: jaw locking after maxillofacial trauma was trapped medial to the ascending ramus of the mandible, but it did not seem to interfere with the joint movements. the pre-operative schenario was made based on the clinical situation and the results of the plain imagings. the patient has sustained multiple trauma on her left body, shown by the fracture of the left fibula and radius bones, and some facial scars over the region of left lateral upper eye lid and lower lip. these clinical findings might indicate that the left site of the body including the face was subjected to blunt force during the accident. the chronology of the facial trauma suggested that before the face hit the object she had in reflex turned her head slightly to the left, therefore the mandible sustained fractures in the region of midline and right paramedian. the comminutive type of fracture, the lateral displacement of both ascending ramus, and the alteration of mandibular arch indicated that a powerful force must have struck this part of the mandible. the mandible including the alveolar process and the teeth on both sides seemed to have displaced medially resulting in a narrowed mandible arch. the possible rationale to explain this condition is as follows. the fracture on the right parasymphysis region of the mandible may have caused the mandible to displace laterally. as some bone loss may have occurred on that part of the mandible, the effort to forcedly reposition the two segments in an end to end approximation have resulted in the mandible assuming an awkwardly narrow arch and the upper part of the mandible on both sides collapsing toward median line. a series of three dimensional-computed tomogram was finally made to help establish the diagnosis of trismus. the result of 3d-ct showed that both condylar processes were fractured at the level of condylar neck in which the condylar heads were displaced laterally out from the glenoid fossa. it was clearly seen that bony ankylosis was formed in the region of the posterior part of the right zygomatic arch (figure 5). case #2: a 24 year old man visited department of oral and maxillofacial surgery at dr. soetomo general hospital surabaya asking for a treatment to his trismus. patient was unable to open his mouth since his childhood. the parent explained a history of trauma when he was 7 year old. clinical evaluation showed that the patient had a birdface profile indicating a hypoplastic mandible and an old scar was noted on his chin. intraorally, the patient present with severe trismus, whereas the occlusion was normal (figure 8). panoramic showed old bilateral condylar fractures, the joints were seen to be covered with a massive bone which had grown irregulary replacing the curvature of both condylar articular surfaces. the right joint was seen to be more severely ankylotic compared to the left site as a thin radiolucency was seen surrounding the enlarged left joint (figure 7). the surgery of gap arthroplasty was planned initially on the right joint as the suspected side of the cause of the trismus keeping in view of left gap arthroplasty if the mouth opening was not adequately obtained after the right arthroplasty. case management case #1: the surgery was performed under general anesthesia. as the patient was unable to open her mouth, two options of intubation techniques were planned, fiber opticguided intubation or tracheostomy. the fiber optic guided intubation was chosen and performed successfully. two step surgical treatments were planned. a gap arthroplasty of the right joint would be done first, if this surgery and post-operative physiotherapy cannot achieve the normal mouth opening, a gap arthroplasty of the left condyle is considered. surgery for jaws realignment was also offered to the patient, but the patient refused this option as the primary concern was only the jaw locking. after the right gap arthroplasty had been done, refracturing in the median part of the mandible was done to realign the mandible arch. unfortunately, this part of the mandible had healed up well and the two fragments were fixed and united in new position, therefore surgical reconstruction for mandible realignment was ceased. the mandible was repositioned and fixed with 2 miniplates. two heister’s mouth opener were used to force the mouth to open and maintained for 24 hours with rubber mouth gag to avoid relapse of jaw locking. post-operative physiotherapy exercise was done to help the patient achieve a maximum mouth opening. until one month after right joint gap arthroplasty, the patient was able to open her mouth, but 3 months after the surgery the jaw locking recurred (figure 6). left joint clicking and slight pain during mouth opening were reported. reevaluation of the 3 d-ct brought us to the decision to do a left joint gap arthroplasty. intra-operative direct visualization showed the left condylar neck had displaced laterally out from its fossae and formed ankylosis with the surrounding bony structure. condylectomy was done and a normal mouth opening was directly achieved. immediately after the left joint surgery the patient was able to open her mouth much wider compared to before the surgery, allowing for a direct normal jaw function (figure 6). physiotherapy exercise was continued to overcome the masticatory muscles problem due to jaw locking which had been lasting since almost 1 year previously. case #2: the general anesthesia was performed via tracheostomy cannulation. a pre-auricular approach was used. the surgery of gap arthroplasty was done initially on the right joint as the suspected side of the cause of the trismus. after the joint had been osteotomized, using a mouth spreader the mouth was successfully forced to open, therefore the gap arthroplasty on the left joint was not done. an allograft material of premilen mesh graft was interposed into the gap to prevent it from developing re-ankylosis. a rubber mouth gag was placed between upper and lower teeth for 48 hours to avoid recurrence of the trismus due to masticatory muscle contraction. five days after the surgery the patient was able to open his mouth normally (figure 8 and 9). 112 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 106-113 discussion in the treatment of facial trauma detailed observation of the facial skeleton situation is considerably important. the facial bones and mandible should be clearly observed from part to part to determine their involvement in the fracture. occlusion should be used as primary guidance in establishing the diagnosis of facial and mandibular bones displacement as well as in the surgical reduction and fixation of the bones. besides occlusion, the form of jaw arch can also be used as an important guidance during the procedure of jaws alignment if both jaws are found to be mal-aligned. in severe mandible fracture, reconstruction can be done by holding such anatomical landmarks as: the form of mandible arch, tooth to tooth contact, the curve of spee, and the alignment of the bony structure in the body of the mandible.6 in severe maxillofacial trauma, post-operative complications may be in the form of two major problems, i.e. aesthetic and functions. the aesthetic problem is the most common complication especially when the middle third of the face is involved in. the most frequent aesthetic problem is flattening of the face due to the depression of malar bones or the whole middle third of the face as a result of le fort iii fracture. malocclusion and jaw locking or trismus are two major functional problems which may occur after surgical correction of maxillofacial fractures. case #1 presented unsatisfactory result of surgical correction of maxillofacial fractures where both postoperative aesthetic and functional problems existed. it is considered a difficult clinical situation in that both the maxilla and the mandible, as well as both the zygomatic bones, were involved in the injuries. in addition to flattening of the patient’s midface, the abnormal arch of the mandible has contributed to the altered appearance of the lower third of the face. the mandible has not been repositioned to its anatomical arch, the normal tubular form. this may be caused by the loss of anatomical landmarks due to some bone loss in the anterior region of the mandible. the effort to reposition the mandible using the occlusion guidance has made the mandible to assume an abnormally wider form on its inferior part and narrower arch on its superior or alveolar part (figure 1). in single jaw fracture, the healthy jaw can be used as a template during the surgical reconstruction. simultaneous surgical treatment of facial and mandibular fractures might cause a mal-alignment due to the loss of anatomical landmark orientations as the normal mandible can no longer be used as a template for guiding the reconstruction procedure. in a fresh jaw bone fracture, anatomical reposition of the bone fragments can usually be achieved but in other cases the bone ends may not be well aligned. nevertheless, non-anatomical reduction of bone approximation is usually still acceptable so long as the occlusion is obtained. such a situation was encountered in the first case. in our opinion the surgical approach of bottom to top as suggested by fonseca7 is the advisable method which should be used when both jaws are involved in a severe facial trauma. mandible reconstruction can be achieved relatively more easily than maxilla therefore mandible reconstruction should be done first. the maxilla can then be corrected following the template given by the mandible arch. in the treatment of mandible fracture, application of arch bars is usually very helpful in achieving bone alignment, the displaced bone fragments will return to its anatomical alignment relatively easily after the arch bar has been applied. if some difficulty is encountered in the effort to realign the bone segments, bone chips or foreign body materials in the fracture line should be suspected. in consolidated fracture lines, removal of the fragments should be done to achieve an anatomical alignment of the mandible. the jaw locking presented in this case was initially suspected to be caused either by: the fractured zygoma bone being rotated in counter clockwise in left zygoma or clockwise in the right zygoma restricting the movement of the mandible in the region of coronoid processes, or the fractured right condyle having restricted normal mandibular movement. according to the series of plain imagings both considerations were not supported in determining the primary cause of the existing trismus. a series of three dimensions computed tomography (3d-ct) could eventually determine the cause of jaw locking. it was found that the trismus is caused by ankylosis over the right joint which was formed by the displaced condylar neck laterally consolidating with the surrounding bone in the area of the right zygoma bone. both coronoid processes were seen to be free from impingement by the displaced zygomas. a gap arthroplasty at the right side was done as the initial step. the preauricular incision (figure 10) was chosen in this case because it: provides good access to the temporomandibular joint area, is cosmetically acceptable, and minimal post-operative complications. in the procedure of gap arthroplasty some amount of bone at the condylar neck region was removed to create a gap between the ankylotic condyle and the distal segment of the mandible. this was done by making two horizontal bone cuts 1-1.5 cm apart just below the joint and the piece of bone was removed (figure 10). care should be taken not to damage the internal maxillary artery which runs below the neck of the conydle. since a gap is created the procedure is known as gap arthroplasty. the left gap arthroplasty alone resulted in an acceptable mouth opening. the recurrence of jaw locking three months post operatively indicated that there existed some restriction of the movement at the left joint as well which may not be as severe as the right side. the left gap arthroplasty showed some bony ankylosis formed by the displaced right condylar neck and the surrounding bone. the unpleasant thing was that the patient had to go through two surgeries. if the right gap arthroplasty had not been able to open the mouth then we would have performed the 113kamadjaja and soesanto: jaw locking after maxillofacial trauma left arhtroplasty. the reason why the right gap arthroplasty alone could attain acceptable mouth opening in the first surgery remains unclear. in case #2 the jaw locking problem was focused on the temporomandibular joint which was found to have ankylosis due to trauma during the period of childhood. gap arthroplasty on the right side was done with good result. an allograft material inserted interpositionally between two segments was necessary to prevent the temporomandibular joint from re-ankylosing secondary to bone re-growing. the bird face appearance presented clinically as the result of bilateral temporomandibular joint during the childhood period supported the initial theory about condylar cartilage as the primary center for mandibular growth. it was believed that the cartilage cap provided the driving force forward and downward for mandibular growth. limitation of normal growth is related to an alteration in the normal condylar growth center.7 the theory described by moss in year 1968,7 which is taken to be believed by some authors, giving a new understanding of mandibular growth described that the mandible being pushed down and forward as the result of changes in the growing soft tissue envelope, or functional matrix, surrounding it. the growth at the condylar center is secondary and compensatory to these primary changes. these 2 theories of condylar cartilage as the center of growth and functional matrix theory are 2 theories that cannot be separated. the case #2 presented a good example of bilateral condylar fractures which was followed by mandible growth disturbances. this supported the first theory as well as that of johnson and moore in 19979 which explained that mandible growth and development began with the meckel’s cartilage through the process of intramembranous development. references 1. rombach dm, quinn pd. trauma to the temporomandibular joint. in: fonseca rj, walker rv, betts nj, editors. oral and maxillofacial trauma. 2nd ed. vol 1. philadelphia. 1997. p. 527–70. 2. carroll mj, hill cm, mason da. facial fractures in children. br dent j 1987; 163:23. 3. hall rk. injuries of the face and jaw in children. int j oral surg 1972; 1:65. 4. amaratunga na des. the relation of age to the immobilization period required for healing in mandibular fractures. j oral maxillofac surg 1987; 45:111. 5. chayra ga, meador lr, laskin dm. comparison of panoramic and standard radiographs for the diagnosis of mandibular fractures. j oral maxillofac surg 1988; 44:677. 6. profifit wr, field hw, ackerman jl, bailey l’tnya, tulloch jfc. contemporary orthodontics. 3rd ed. st louis: mosby an imprint of elsevier; 2000. p. 41. 7. fonseca rj, walker vw. bett nj, barber hd. oral and maxillofacial trauma. 2nd ed. vol. 1. philadelphia: 1991. p. 571–652. 8. temporomandibular joint investigation and surgery. available at: www.fleshandbones.com/ readingroom/pdf/760.pdf. accessed july 7th, 2007. 9. johnson dr, moore wj. anatomy for dental student. hongkong: oxford university press; 1997. p. 144. guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as research reports, case reports or literature reviews that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted 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ten references. citation format for journal articles: 1. thesleff i. the genetic basis of tooth development and dental defects. am j med genet. 2006; 140(23): 2530-5. 2. fekonja a. hypodontia in orthodontically treated children. eur j orthod. 2005; 27: 457-60. citation format for textbooks: 1. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: elsevier; 2003. p. 205-9, 231-48. 2. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: mosby co; 2002. p. 330-40. citation format for proceedings: 1. syafiar l. titanium as metallic implant material. in: timnas v & lustrum xvi. surabaya; 2009. p. 16-20. 2. sutowijoyo a, suardita k, prasetyo ep. restoring mastication by one visit endodontic as a preliminary treatment for immediate overdenture. in: temu ilmiah nasional ikorgi i. surabaya; 2010. p. 131-4. citation format for thesis and dissertations: 1. munadziroh e. karakterisasi, ekspresi dan kloning gen penyandi protein secretory leukocyte protease inhibitor membrana emnion sebagai kandidat untuk mempercepat penyembuhan luka gingiva. dissertation. surabaya: universitas airlangga; 2008. p. 8-21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (jpeg, png, or tiff format). non-file photos should be printed on clear glossy paper with minimum dimensions of 125mm x 195mm. the maximum number of figures, illustrations, photos and tables contained in the research report and literature review is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as 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... ... ... ... ... ... ... ... ... ... ... . d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal nbkbmbi!lfeplufsbo!hjhj faculty of dental medicine, universitas airlangga editorial address c/o: jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone/fax: +6231 5039478 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/mkg/index i want to subscribe the dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) ................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... e-mail: ..................................................................................... date/tanggal: 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.................................................................................................................. country/negara: ...................................................................... phone: ..................................................................................... fax: .......................................................................................... e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick ( )] address issue* 1 year (four copies)6 month (two copies) surabaya idr 200,000 idr 400,000 java island (pulau jawa) idr 250,000 idr 500,000 outside java island (luar pulau jawa) idr 300,000 idr 600,000 other countries (negara lain) usd 30 usd 60 * quarterly publication (terbit 4 kali setahun) name/nama: .......................................................................... i am paying this journal by: [please tick ( )] institution/institusi: ................................................................... saya membayar jurnal ini dengan: [beri tanda ( )] address/alamat surat: ............................................................ bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... account no. : 988.01010.00000.135 bank : bank bni account holder : fkg dental journal ................................................................................................. ................................................................................................. �� vol. 43. no. 1 march 2010 mozart effect on dental anxiety in �–�� year old children arlette suzy setiawan, hilma zidnia, and inne suherna sasmita department of pediatric dentistry faculty of dentistry, university of padjadjaran bandung indonesia abstract background: c�ildren an�iety in dental treat�ent ��ten �ec��es a �arrier ��r dentist t� �er��r� ��ti��� dental treat�ent �r�ced�re. vari��s �et��ds t� �anage an�iety and �ear in c�ildren �ave �een a��lied incl�ding listening t� classical ��sic d�ring dental treat�ent. one �� t�e classical ��sic �s�ally �sed is ��sic �y ��zart. purpose: ��is st�dy is ai�ed t� disc�ver t�e r�le �� classical ��sic �y ��zart in dental an�iety c�anges. method: ��is st�dy ��as a q�asi e��eri�ental st�dy �sing ��r��sive sa��ling �et��d. ��e sa��les c�nsist �� 30 c�ildren �et��een 6-12 years �ld gr��� ���� ��ere treated at t�e pediatric dentistry clinic, dental h�s�ital, fac�lty �� dentistry padjadjaran university. ��e an�iety data ��as c�llected �sing vis�al anal�g�e scale (vas) �e��re and a�ter listening �n t�e classical ��sic d�ring treat�ent. result: ��e res�lt �� t�is st�dy s����ed t�at t�ere ��ere 23 c�ildren (76.67% �� s��jects) ���� �resent decreased an�iety, 7 c�ildren (23.33% �� s��jects) did n�t �resent decreased an�iety and n�ne �� �� s��jects s����ed increased an�iety. conclusion: �t ��as c�ncl�ded t�at listening t� ��sic �y ��zart d�ring dental treat�ent can red�ce an�iety in 6–12 year �ld c�ildren. key words: ��zart e��ect, dental an�iety, c�ildren abstrak latar belakang: kece�asan �ada anak saat �era��atan gigi seringkali �er��akan �eng�alang �agi d�kter gigi �nt�k �elaksanakan �r�sed�r �era��atan gigi yang ��ti�al. ber�agai �et�de �nt�k �engatasi kece�asan dan rasa tak�t �ada anak tela� dilak�kan ter�as�kber�agai �et�de �nt�k �engatasi kece�asan dan rasa tak�t �ada anak tela� dilak�kan ter�as�k �endengarkan ��sik klasik sela�a �era��atan gigi. sala� sat� ��sik klasik yang �anyak dig�nakan adala� ��sic �le� ��zart. tujuan: penelitian ini dit�j�kan �nt�k �ene��kan �eran ��sic klasik ��zart dala� �er��a�an kece�asan �ada �era��atan gigi. metode: penelitian ini adala� k�asi eks�eri�ental �engg�nakan �et�de �enga��ilan sa��el ��r��si� . sa��el terdiri dari 30 anak antara 6–12 ta��n yang dira��at di klinik ked�kteran gigi anak, r��a� sakit gigi dan ��l�t fak�ltas ked�kteran gigi universitas padjadjaran. data kece�asan dia��il dengan vis�al anal�g�e scale (vas) se�el�� dan setela� �endengarkan ��sic klasik sela�a �era��atan. hasil: hasil �en�nj�kkan �a���a terda�at 23 anak (76,67% dari s��jek) yang �en�nj�kkan �en�r�nan kece�asan, 7 anak (23,33%) tidak �en�nj�kan �er��a�an kece�asan dan tidak ada (0 anak ata� 0%) yang �en�nj�kkan �eningkatan kece�asan. kesimpulan: disi���lkan �a���a �endengarkan ��sik ��zart sela�a �era��atan gigi da�at �en�r�nkan kece�asan di antara anakanak �sia 6–12 ta��n. kata kunci: e�ek ��zart, kece�asan, anak-anak c�rres��ndence: arlette suzy setiawan, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: arlettesuzy@yahoo.com research report introduction anxiety related to dental treatment is frequently found, especially in children. this often becomes a direct reason why a child reluctantly goes to a dentist.1,2 a study of milgrom and weinstein in 1993 has proven that anxiety during dental treatment occupies the fourth rank among other anxieties.1 anxiety is an individual normal �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 17-20 emotional reaction that appears when facing dangerous or life threatening situation because basically each person wants comfort in his or her life. dental treatment in clinics can create anxiety leading to non cooperative behavior of the patient during the treatment that may interfere the medical procedures causing longer chair time. uncooperative patient will cause difficulties in treatment leading to non-optimum treatment result.1 according to a study performed by kartono and sartono in 1992 at sutadi,3 the factors of anxiety include the sound of a bur, 81.46%, sitting on dental chair 50.72%, needle 39.13%, dental instruments 39.13%, and negative story on dental treatment 33.33%. anxiety and fear in children during dental treatment is not a new phenomenon in dentistry. the child anxiety in dental treatment often becomes a barrier for the dentist to perform optimum dental treatment procedure.1,2 anxiety is become severe by unpleasant experiences during the previous visit that make the child refuses to continue dental treatment. one of the factors that cause anxiety during dental treatment is the sound of dental bur. based on the study done by wardle 1982 as mentioned by budiman,4 anxiety during dental preparation using bur occupies the second place after dental extraction. the sound of bur instrument often creates anxiety in children.3 one of the methods to deal with anxiety and fear in children during dental treatment is by using distraction technique, i.e. distracting the child’s attention away from the source of anxiety.4 one of the ways to distract patient’s attention is by listening to the music.5 pediatric patients who are afraid of the sound and the vibration of handpiece can be managed by simple audio technology, i.e. installing stereo music instrument in the dental clinic. this can reduce and cover unpleasant sounds. therefore, music can help reducing stress and make the patient relax.6 a soft and gentle music can reduce fear and anxiety in children. a soft classical music can neutralize the sharp noise of bur used by the dentist. a study by campbell5 has proven that listening to classical music during treatment is very effective for relaxation, especially in reducing discomfort in pediatric patients. six to twelve year is a transitional age from the childhood to teenage which is also often called the end period of childhood. in this period the child’s attention to the surrounding environment increases. in terms of dental treatment, the children in 6–12 years old group are generally cooperative and have the ability to accept the treatment. with better cognitive development the child will enable to absorb abstract explanation. however, some still cannot accept it, which will leading to be anxious.7 the aim of this study is describing the effect of the classical music on anxiety changes in children aged 6 to 12 years old before and after listening to classical music at the pediatric dentistry clinic, dental hospital, faculty of dentistry padjadjaran university by using the visual analogue scale (vas).7 material and method the type of this study is a quasi-experimental method. the study population were pediatric patients which will receive dental treatment at pediatric dentistry clinic, dental hospital, faculty of dentistry padjadjaran university. the purposive sampling technique is used with the following criteria: 30 children age 6–12 years old, male and female, come for a class one cavity preparation for dentin caries in lower first molar and with a good general health condition. a walkman and a mozart’s song (andantino grazioso from symphony no.18) was used as an instrument to listen. visual analogue scale sheet is used to assess the anxiety level with a scale from 1 to 7 which shows the expression degree of increased anxiety (figure 1). the anxiety changes are measured from vas pictures. the anxiety was stable when there was no change in vas scale. the anxiety was reduced when there was reduction in vas scale and it was increased when there was increase in vas scale. figure 1. visual analogue scale.7 figure 2. distribution of vas scale before treatment and during treatment without and with listening to mozart music. the study was started by asking the child to select a picture in vas that represented his/her emotion before the treatment procedure was applied. next, the child’s tooth was prepared using round diamond bur for two minutes. during the first two minutes the child is asked to mark the picture in vas that he or she considered as representing his or her anxiety. after that, the child listened to the music using walkman through an earphone. the preparation work using bur is continued while the child was listening to the classical music. after the preparation was finished the ��setiawan, et al.: mozart effect on dental anxiety child was asked to mark the picture in vas that he or she considered as representing his or her anxiety. result based on the study results of 30 children, data on the anxiety during treatment is represented in the following tables. the child anxiety change before the treatment and after the treatment without listening to mozart music showing 40% of the children reduced anxiety, 23.33% increased anxiety and 36.67% showed no decrease or increase in anxiety (table 1). table 1. anxiety change before dental treatment and duringanxiety change before dental treatment and during dental treatment without mozart music condition f % reduced anxiety increased anxiety no change 12 7 11 40.00 23.33 36.67 the child’s anxiety change before the treatment and during the treatment by listening to mozart music. it showed that there was 70% of the children reduced the anxiety, 10.00% increased the anxiety and 20.00% with no anxiety increase or decrease (table 2). table 2. anxiety changes before dental treatment and during treatment while listening to mozart music condition f % reduced anxiety increased anxiety no change 21 3 6 70.00 10.00 20.00 the child’s anxiety change before listening to the classical music during treatment and after listening to mozart music. a reduction in anxiety was found in 76.67% and 23.3% of the samples do not show increase or decrease in anxiety. no sample shows any increase in anxiety (table 3). table 3. anxiety change during dental treatment while listening to mozart music after the previous condition i and ii condition f % reduced anxiety increased anxiety no change 23 0 7 76.67 0.00 23.33 overall, the data collected is grouped based on the three conditions when the anxiety is measured and the vas scale selected by the respondents. table 4 show the scale distribution in the visual analogue scale marked by respondents in each treatment, i.e. before treatment (condition i), during preparation without listening to mozart music (condition ii) and during preparation while listening to mozart music (condition iii). table 4. distribution of vas scale before treatment and during treatment without and with listening to mozart music. vas scale condition i % ii % iii % 1 2 3 4 5 6 7 6 8 5 10 1 0 0 20.00 26.67 16.67 33.33 3.33 0.00 0.00 4 9 9 8 0 0 0 13.33 30.00 30.00 26.67 0.00 0.00 0.00 14 11 3 2 0 0 0 46.67 36.67 10.00 6.67 0.00 0.00 0.00 note: i: before treatment ii: during treatment, before listening to classical music iii: during treatment while listening to classical music discussion anxiety is a manifestation of fear without clear cause.8 the anxiety that is related to the dentist and dental treatment is generally triggered by the lack of knowledge of the patient on the treatment that he or she is going to receive. information from friends about unpleasant dental treatments can increase the anxiety. the condition that triggers anxiety during dental treatment is various, including dental preparation using bur. another factor that may affect patient’s anxiety level is the dentist is skill to communicate with the patient.9 generally anxiety experiences by patient who visit the dental practices for the first time because the patient could not predict what the dentist will do on him/she. the study sample generally includes patients who have visited the dentist before to treat his or her teeth periodically at pediatric dentistry clinic, dental hospital, faculty of dentistry padjadjaran university. patients tend to be cooperative because they are used to the instruments and equipments used by the operator. the communication between the operator and the patient is good that patient tends to be not anxious. the patient knows about the treatment to be because it has informed by the operator beforehand. they also have heard stories from their school friends who have received dental treatment that the dental treatment at this clinic is quite pleasant. music is a universal language that encompasses status, age, religion, and race borders. classical music can calm, soothe and heal. it can also give joy and joy is a kind of therapy. calm classical music can function as emotional barrier. in the medical field, music is used for treatment �0 dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 17-20 just like medicine.5 it has been reported that the pulse, respiration, electromyogram and electroenchepalogram are changed when the patient listens to music.11 changes in the form of increased anxiety level in this study are seen mostly during before treatment and during treatment without music (table 1). reduced anxiety level in this study is mostly seen during before and after listening to the classical music during dental treatment (table 2). subjects who did not experience increased or reduced anxiety are mostly found during before and during treatment without music (table 1). increased anxiety during listening to the classical music in the treatment may be triggered by the loud volume of the walkman or the volume was too weak that the patients unable to hear the music. no changes in anxiety level may be caused by the condition where the children have never listened to the classical music before or because the child’s psychological nature showed no response to situation change or uneasiness in using headphones. reduced anxiety during dental treatment by listening to classical music was caused by the distraction ability of the classical music or because listening to classical music may trigger relaxation effect and analgesia. the study shows that the anxiety experienced by the subject during dental drilling was reduced if the drilling is performed while the patient listens to the classical music. this is according to previous study showing positive response in dental patients who receive treatment while listening to the music.5 reduced anxiety during dental treatment with classical music is caused by the fact that children like and able to enjoy music that make the children feel more relax. according to marzuky,6 music for relaxation can reduce hart beat up to beat per second that the heartbeat becomes calmer. the slower heartbeat and pulse create lower stress and physical tension level. one of the efforts of the dentist to reduce anxiety and making the patient relax is by providing relaxing music. classical music has sedative effect that anxiety and tension can be lowered making the patients feel calmer. slow classical music makes the patient’s emotion low and relax that the anxiety level during dental treatment will be reduced. not all classical music can be used for anxiety therapy. classical music used for therapy includes music with 60 beats per minute or less tempo that will affect physiological and psychological aspects. according to campbell5 slow music such as andantino grazioso from symphony no. 18 of wolfgang amadeus mozart can be used as musical therapy for children who experience stress to make them more relax. gentle sound and soft beat will be perceived by the brain that a person becomes relax. in relax individual, there is reduced sympathetic nerve activity in autonomic nervous system part that will lead to reduced epinephrine (adrenaline) secretion. adrenalin plays a role in one of the anxiety clinical symptom, i.e. pulse and blood pressure. reduced adrenalin release will reduce pulse and blood pressure.12 based on this study, patiens who listen to classical music while recovering dental treatment feel more relax than those who did not listen to the music. it can be concluded that classical music reduces anxiety in dental treatment of children aged 6–12 years old at this clinic. references 1. al-madi em, hoda al. assesment of dental fear and anxiety among adolescent females in riyadh saudi arabia. saudi dental journalsaudi dental journal 2002; 14(2): 77–80. 2. engkling n, marwinski g, johren p. dental anxiety in a representative sample of residents of a large german city. clin oral invest 2006; 10: 84–91. 3. sutadi h. rasa takut dan cemas terhadap perawatan gigi. kumpulan makalah ilmiah kongres pdgi xviii 1992; 135–42. 4. budiman ja. pengelolaan tingkah laku pasien pada praktek dokter gigi. edisi ke-2. jakarta: egc; 1994. p. 63–86, 106–7. 5. campbell d. 2000. efek mozart bagi anak-anak. widodo atk, editor. jakarta: pt gramedia pustaka utama; 2001. p. 331. 6. fung hc. medicine and music. lifestyle 2006: 11(6): 24–5. 7. latifa w, soemartono sh, sutadi h. pengaruh musik terhadap perubahan kecemasan dalam perawatan gigi pada anak usia 8-10 tahun. jitekgi fkg updm(b) 2006; 3: 125–8. 8. chaplin jp. 1999. kamus psikologi. kartono k, editor. 10th ed. jakarta: pt raja grafindo persada; 2005. p. 32–3. 9. peretz b, katz j, elda a. behavior of dental phobic residents of large and small communities. braz dent j 2000; 10(1): 1–3. 10. murabayashi n. psychosomatic medicine and music therapy. asian med j 2000; 43(11): 538–42. 11. marwah n, prabhakar ar, raju os. music distraction-its efficacy in management anxious pediatric dental patient. j indian soc pedod prev 2005; december: 168–70. 12. chafin s, roy m, gerinw, christenfeld n. music can facilitate blood pressure recovery from stress. british journal of health and pscycology 2004; 9: 393–403. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base 11 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 1–6 review article a case study of informed consent in indonesian law number 29, 2004 agung sosiawan1,2,3, vera rimbawani sushanty4, dian agustin wahjuningrum5, fery setiawan6,7 1undergraduate student, faculty of law, bhayangkara university, surabaya, indonesia 2department of forensic odontology, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4law department, faculty of law, bhayangkara university, surabaya, indonesia 5department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia 6doctoral program of medical science, faculty of medicine, universitas airlangga, surabaya, indonesia 7department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: informed consent is an agreement between the doctor/the provider of medical services and the patient/the recipient of medical services. this relationship between these parties has changed from a paternalistic to a contractual relationship due to technological shifts. doctors are obliged to notify the patient of all the risks and benefits of a procedure while respecting their autonomy by not intervening the decision-making process. this article will look at three government and academic hospitals in surabaya, as informed consent has to be practiced in all medical settings. purpose: this study aims to review the role of informed consent according to law number 29, 2004. review: this study aims to discuss the characteristics of informed consent under law number 29, 2004, because there are too few articles addressing this issue. it also explains the roles of the patient and the doctor/dentist in informed consent according to this piece of legislation. conclusion: according to article 184, informed consent provides vital evidence that can be used to hold doctors and dentists legally accountable because it contains information about standard operating procedures (sops) that medical professionals are legally required to follow. guidelines for informed consent are given in law number 29, 2004, article 45, paragraph 2. keywords: medical action agreement; doctor-patient relationship; paternalistic-contractual; medical information; medicine article history: received 19 april 2022; revised 11 july 2022; accepted 15 september 2022 correspondence: agung sosiawan, forensic odontology and dental public health department, faculty of dental medicine, universitas airlangga. jl. mayjen prof dr. moestopo no. 47 surabaya, 60132, indonesia. email: agung-s@fkg.unair.ac.id introduction the fourth paragraph in the introduction of the 1945 constitution of the republic of indonesia and the body of the 1945 constitution in article 28a states that the national goal of the indonesian people is to protect the entire indonesian nation and fellow indonesian citizens by making contributions in advancing public welfare and education while protecting freedoms and maintaining peace and social justice. article 28a explains the right of every citizen to access health services because health is key to protecting life in general.1,2 as providers and recipients of health services, doctors and patients share a unique relationship that has become the object of lengthy legal study about the responsibilities of, and protections for, both parties. one of issues that makes these concerns necessary is that of medical malpractice. indictments for malpractice can be submitted by the public against a medical professional who is deemed to have abused their power to harm a patient, sometimes causing pain, injury, physical disability, or death.3 disputes can occur if doctors are negligent in carrying out their legal responsibilities, as this may lead to a violation of the patient’s rights, causing them to demand justice. justice must be proportional whenever there is a dispute between the two parties.4,5 to avoid disputes around healthcare, doctors must be responsible, as outlined in law no. 29, 2004. accountability copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 mailto:agung-s@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 2 sosiawan et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 1–6 must be based on the principle of social justice for patients whose rights are violated and doctors who must be held responsible for the patient’s condition. the principle of social justice discussed here is the concept of proportional justice laid out in the introduction of the 1945 constitution of the republic of indonesia mentioned above.6 the objective of this article is to explain the characteristics of informed consent under law number 29, 2004, concerning medical practice and the responsibilities of medical professionals by analyzing a combination of case studies and providing a narrative review of published articles. the aim of reviewing literature already published on this topic is to provide an overview of informed consent and the legal nature of the doctor–patient relationship. this article will look at three informed consent procedures from three different hospitals (referred to as hospitals a, b, and c) in surabaya to assess how strictly they conform to the guidelines laid out in paragraph 2 in article 45 of law number 29, 2004. there have been a number of examples of improper implementation of informed consent in dental care in indonesia since the implementation of this law. the first case discussed here occurred in 2010, when a dentist mistakenly extracted a patient’s tooth, causing them to be charged with malpractice. the malpractice charge levelled against the dentist was negligence. another case in 2016 involved a dentist’s failure to inform their patient of the relevant medical risks before failing to successfully extract a tooth, leaving fragments of the tooth in the gum, the removal of which later required a further operation. a third case of dental malpractice happened in 2020, when a dentist left an open wound in a patient’s mouth during an operation. the dentist was reported to the police and the dental discipline council before being given a five-year prison sentence. the purpose of the narrative review is to see how informed consent under law number 29, 2004, is enforced in indonesian dental practice. giving informed consent under this law involves respecting the patient’s autonomy and requires the doctor or dentist to carry out their duties in line with the sop. this is necessary to avoid the improper implementation of informed consent in indonesia by forming a contractual relationship, made possible by technological developments that change patient perceptions. review law number 29, 2004 according to law number 36, 2009, certain pieces of information must be given to the patient for their consent to qualify as informed: details regarding the diagnosis and suggested medical procedure, the purpose of the proposed procedure, details of any other medical action to be carried out that may affect the patient, an account of the risks associated with the procedure, and a prognosis. informed consent informed consent emerged to establish a change in the relationship between doctor and patient from a vertical, paternalistic relationship to a horizontal, contractual relationship. informed consent is essentially a therapeutic agreement between doctors and patients based on the patient’s health status. this can take two forms: implied consent (considered as given without being stated explicitly) and expressed consent (stated by the patient to the doctor). with treatments that pose a high risk of harm, informed consent must be given in written form. doctors must prioritize the implementation of informed consent in their daily activities unless they believe that there are other people who are more competent and can provide assistance. for emergency care, informed consent does not need to be given, but if the patient or a family member is capable of receiving the necessary information and giving their consent, this must be carried out. the therapeutic agreement of informed consent between doctor and patient is binding as soon as the agreement is signed. once signed, it remains in effect until both parties consent to terminate the agreement. there are several principles that guide the implementation of informed consent, the most important of which is the principle of good faith. this is the principle that underlies the pre-negotiation stage before the contract or therapeutic agreement can be implemented. therapeutic agreements between doctors and patients are based on mutual trust, but with that trust, there is responsibility and accountability that must be carefully considered by doctors when agreeing to perform medical interventions. responsibility in law has two facets, namely responsibility (verantwoordelijkheid) and liability (aansprakelijkheid). liability refers to the position of a person or legal entity who must pay some form of compensation after a legal battle due to malpractice (liability with fault) or error (liability without fault), also known as risk responsibility (strict liability). the application of responsibility and liability requires a clear awareness of the relationship between the professional who has committed the crime and their employer. article 2 of the criminal code states that criminal provisions in indonesian legislation apply to anyone who commits an offense in indonesia, including medical professionals. criminal law recognizes that crime in the health services can be justified and forgiven as outlined in jurisprudence, but this does not necessarily mean that justification and forgiveness can overturn criminal proceedings. the science of criminal law and jurisprudence gives specific reasons for the abolition of unwritten crimes based on justification and forgiving. reasons for justification include when an unlawful act was regarded as lawful by the defendant. regardless, the defendant’s actions may still be unlawful, even if they are not criminal. informed consent acts as a piece of evidence that can be brought forth if ever there is a lawsuit filed by a patient against a doctor based on article 184 of the criminal procedure code, allowing the court to assess whether the copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 3sosiawan et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 1–6 doctor’s actions can be considered negligent. hence, it is important that informed consent is as thorough and accurate as possible, especially in matters relating to diagnosis, the patient’s treatment plan, and the prognosis. also, the informed consent form must be signed by a witness. literature search strategy a search for studies on informed consent between doctor and patient was conducted from january to february 2022. a librarian with knowledge of medical referencing developed individual search strategies and retrieved citations from sciencedirect, pubmed and google scholar. a mix of terms were used together to find the relevant literature (“informed consent and therapeutic contract and doctor-patient relationship”). the search strategies used in each database will be explained in the next paragraph. criteria the narrative review includes studies that examine indonesian doctors’ awareness of their legal responsibilities under law number 29, 2004. the following inclusion and exclusion criteria were used. the articles had to include a discussion of doctors and their patients, the therapeutic relationship they share, the contractual relationship they share, informed consent from the point of view of indonesian law, and specific reference to paragraph 2 in article 45 of law number 29, 2004. articles were excluded if they had an abstract only or belonged to obscure, protected, or unassessed journals or papers. these criteria were based on technical issues and reliability. data extraction this study is based on descriptive data, including the legal points of view of the doctor and the patient. table 1 shows how data was extracted from the 22 sources used in the narrative review. there are two kinds of study analyzed here: those that focus on the practice of dentists and doctors and those that focus on legal responsibility in the doctor–patient relationship. the former could be seen as being based on law number 29, 2004, and the application of informed consent. the latter could be seen as being based on the theory, legality, therapeutic agreement, legal liability theory, and medical treatment risks that guide the dentist/ doctor’s daily practice. the description criteria were formulated by analyzing the contents of the informed consent contract for hospitals a, b and c (concerning labelling, the consent body, level of detail, ease of interpretation, the purpose of medical action, alternatives and risks, the prognosis, details of the contract between the operator and patient, personal data, the number and details of the witnesses’ present, and the full names of the operator and the patient; see table 2). the extraction of component data from the informed consent contracts in these three hospitals is detailed in table 3. the author has categorized each of these requirements as very clear, clear enough, unclear, and very unclear based on how well they convey the criteria covered in paragraph 2 in article 45 of law number 29, 2004. the data was extracted after reading and analyzing the informed consent forms of the three hospitals. discussion a description of the gold standard for informed consent can be found in paragraph 2 in article 45 of law number 29, 2004, which discusses the issue of seeking approval for medical or dental interventions. paragraph 1 of article 45 states that every medical or dental action to be carried out by a doctor or dentist on a patient requires their approval. paragraph 2 states that this consent must be given after the patient has received a complete explanation of the proposed medical procedure in line with the gold standard of informed consent, covering the diagnosis, the nature of the procedure, its purpose, its risks, any other alternative actions and associated risks, and a prognosis.7,8 article 45, paragraph 4, explains that the approval mentioned in paragraph 2 can be given in writing or verbally. paragraph 5 goes on to state that any medical or dental procedure that comes with a high risk must be given written approval and signed by the person permitted to give consent. paragraph 6 explains the provisions relevant to the approval of medical or dental procedures as referred to in paragraphs 1–5, which are regulated by ministerial regulation. a number of additional requirements were also identified in the published literature, namely: the category of risk that comes with the given medical action (high, medium, and low); the time, place, and date of the signing of the informed consent agreement; the number of witnesses who were present and participated in signing the informed consent form; a column with the full names of doctors, patients, and witnesses who were present at the time of signing.9–11 based on the data in table 1, it is clear that the informed consent procedure at hospital a comes closest to the gold standard of informed consent outlined in law number 29, 2004. the informed consent procedures at hospitals b and c are less clear and do not follow these guidelines as closely as hospital a. the four comparisons made in table 2 address the fundamental issues that make informed consent one of the best ways to ensure proportional justice between doctors and patients. these comparisons are based on cases in dental treatment. here, the dentists had to ensure that their medical treatment would not endanger their patients or involve medical malpractice. at hospital b, the majority of labels and informed consent bodies do not clearly reflect a sufficiently rigorous informed consent procedure. the label section of hospital b does not clearly state the location as given by the surabaya city government because the font is too small. this is important because the label must clearly state where the informed consent was given so that copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 4 sosiawan et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 1–6 table 1. data extraction process from 22 references used in narrative review reference % sign1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 related to practice of both dentist/ doctor in medicine                 72.7 law no. 29 of 2004                    86.3 informed consent                     90.9 related to the position of a doctorpatient relationship                    86.3 covenant theory                   legal aspect                  77.2 therapeutic agreement                    86.3 legal liability theory                   81.8 medical treatment risks                       100 table 2. informed consent analysis of three hospitals (hospital a, hospital b, and hospital c) in surabaya description hospital a hospital b hospital c informed consent label very clear very unclear very clear informed consent body detailed information on the action (diagnosis and medical procedures) that will be carried out very clear very unclear clear enough ease of interpretation of the purpose of medical action in detail info on the action to be taken very clear very unclear very unclear details of other alternative actions and the risks of medical treatment that can occur after the procedure is carried out very unclear very clear very unclear prognosis of the medical action to be taken very clear very unclear very unclear the contract between the operator and the patient on the points to be held very clear very unclear very unclear informed consent person data very clear unclear very clear closing informed consent detail contract time very clear very clear unclear number of witnesses involved in the informed consent signing process very clear very clear very clear the full name of the operator and patient in the informed consent very clear very clear very clear table 3. extracting data of informed consent taken from three hospitals based on component mentioned in paragraph 2 article 45 law number 29, 2004 component mentioned in paragraph 2 article 45 law number 29, 2004 informed consent of hospital a informed consent of hospital b informed consent of hospital c medical diagnosis and procedures   the purpose of the medical action taken   alternative courses of action and their risks   risks and complications that may occur  the prognosis for the action taken  copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 81.8 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 5sosiawan et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 1–6 the necessary documentation can be found in the patient’s medical record if a legal issue arises.12–14 the informed consent body of hospital b is also unclear, except for the detailed description of the relevant risks of a procedure. in the detail section of action info, information on relevant risks, the prognosis, and the contract between the operator and the patient lacks clarity. as such, some of the important parts of the informed consent form are not clear enough to meet the gold standard of informed consent as required by law number 29.15 these sections are important because the purpose of informed consent is to inform the patient about all of the proposed medical actions and to ensure that the patient is protected by law and can refuse any medical procedure they do not feel comfortable with. doctors are not allowed to perform medical actions in any form without the consent of the patient. according to sk pb idi number 319/pb/ a4/88, all relevant information must be given to the patient in its entirety, and the doctor must not withhold any such information. this must include all the potential advantages and disadvantages of planned medical treatments.16,17 the signatures are an important part of the contract because they confirm that all the necessary information has been exchanged, including that which relates to competence, the delivery of information, the patient’s understanding of the information they have received, and the patient’s right to approve of or reject proposed medical procedures.18,19 these checks must be made in an informed consent form because there is never a guarantee that medical treatment will come without side effects or risk. even the most seemingly benign medical treatment, such as the administration of a commonly used drug, can pose a risk that may result in the patient suffering from an unforeseen reaction. in addition, the signatures on the contract help guarantee the patient’s autonomy, as they can refuse to sign the document if they do not want to take the treatment. if the patient is later dissatisfied with their treatment, the doctor may be subject to charges under the kuhp, articles 359 and 360, which include reference to legal proceedings for negligence (culpa).20 the informed consent procedure at hospital c is equally unclear and lacking in rigor compared with the gold standard of informed consent. in particular, there is a lack of clarity regarding the ease of interpretation, the information provided on the proposed treatment, the level of risk, and the agreement between the doctor and the patient stating that the doctor has fully informed the patient regarding their condition and the potential effects of the treatment.8,21 this lack of clarity on such important medical issues can endanger not just the patient, but the reputation of the medical professionals involved by undermining the effectiveness of the agreement. correctly and clearly established informed consent that adheres to the principle of proportional justice is needed for the student medical professionals at these facilities to protect themselves and their patients in the future.20,21 moreover, hospital c’s informed consent form only contains reference to the place and date it was signed by the patient, omitting the exact time. the signing of the informed consent agreement indicates that the five requirements of informed consent have been fulfilled and that the patient’s right to autonomy in giving their consent for medical action without coercion from other parties has been granted.19,20 hospital c’s procedure is also unclear in laying out the information that should be conveyed to the patient regarding the proposed treatment, the prognostic risks of the treatment, and the contract between the doctors and the patient. these are important matters in adhering to the gold standard of informed consent and ensuring proportional justice between doctors and patients.21 in contrast, hospital a provides a good example of informed consent that is clear, safe, and adheres to the gold standard of informed consent. as such, it acts as an effective therapeutic agreement between the doctor and the patient, thereby allowing both parties access to proportional justice. however, hospital a’s form is lacking when it comes to details about the informed consent body, precisely in the absence of a detailed section on the risks associated with proposed treatments. it is crucial that patients are given a detailed account of the relevant risks because they allow the patient to consider all possible advantages and disadvantages of treatment before consenting to it. this also helps to avoid conflict between patients and doctors if patients are later dissatisfied with the outcome of the treatment.19 the missing pieces of information in the informed consent forms from hospitals b and c could lead to acts of negligence (culpa) because they fail to set out the professional standards and code of ethics necessary to ensure that the patient is properly informed when they give their consent.8,18,22,23 though important, informed consent procedures should not prevent doctors from acting quickly to save someone’s life in an emergency, and doctors should always provide first aid when necessary. they may carry out the informed consent procedure later on with the patient or a family member for any further treatments. nonetheless, a doctor or dentist providing emergency care must still comply with the applicable sops.22,24–27 the gold standard of informed consent is an important starting point in evaluating the informed consent procedures of health facilities (faskes) on a large scale. this can help improve patient and doctor safety and the quality of health services in indonesia while minimizing the occurrence of patient–doctor lawsuits and conflicts.28–30 in conclusion, informed consent, based on guidelines given in paragraph 2 in article 45 of law number 29, 2004, provides crucial evidence in medical cases, as stated in article 184. it is used to ensure the legal accountability of doctors and dentists because it contains information about their adherence to sops when providing diagnoses, treatments, and relevant treatment information. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 6 sosiawan et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 1–6 references 1. ad i p. kebija k a n for mu la si hu k u m pid a na d a la m r a ng k a penangulangan tindak pidana malpraktik kedokteran. law reform. 2010; 6(1): 83–96. 2. amri sr. peran informed consent sebagai aspek legal dalam pelayanan kesehatan the role of informed consent as the legal aspects in health care. artik telaahan. 2020; (april): 1–6. 3. aprilyansyah ms. kajian yuridis persetujuan tindakan medik (informed consent) dalam perspektif undang-undang nomor 29 tahun 2004 tentang praktik kedokteran. j ilmu huk tambun bungai. 2020; 5(2): 755–74. 4. busro a. aspek hukum persetujuan tindakan medis (inform consent) dalam pelayanan kesehatan. law, dev justice rev. 2018; 1(1): 1–18. 5. eutheriana rra. informed consent as a legal protection for patients. dentika dent j. 2016; 19(2): 168–73. 6. felenditi d. penegakan otonomi pasien melalui persetujuan tindakan medis (informed consent). j biomedik. 2009; 1(1): 29–40. 7. gizela ba. studi evaluasi pelaksanaan informed consent pada tindakan invasif berisiko tinggi. in: prosiding pertemuan ilmiah tahunan 2017. pekanbaru: universitas riau; 2017. p. 40–9. 8. herwanda, rahmayani l, fadhilla s. gambaran penggunaan persetujuan tindakan medis oleh dokter gigi muda di rsgm unsyiah. cakradonya dent j. 2016; 8(2): 123–31. 9. nasichin m. pelaksanaan persetujuan tindakan medis (informed consent) antara pihak rumah sakit muhammadiyah gresik dengan pasien operasi caesar berdasarkan pasal 45 undang-undang no 29 tahun 2004 tentang praktik kedokteran. j pro huk. 2017; 4(1): 1–19. 10. nurhasannah. tinjauan yuridis pertanggungjawaban pidana terhadap dokter dan rumah sakit tidak memberikan informed consent kepada pasien dalam hal terjadinya kematian atau luka bagi pasien. jom fak huk. 2018; 5(2): 1–13. 11. octaria h, trisna wv. pelaksanaan pemberian informasi dan kelengkapan informed consent di rumah sakit umum daerah bangkinang (rsud bangkinang). j kesehat komunitas. 2016; 3(2): 59–64. 12. oktarina. kebijakan informed consent dalam pelayanan gigi di indonesia dental care informed consent policy in indonesia. j manaj pelayanan kesehat. 2010; 13(1): 3–8. 13. pakendek a. informed consent dalam pelayanan kesehatan. alihkam j huk pranata sos. 2010; 5(2): 309–18. 14. patel jy. a study on evaluation of patient satisfaction with dental health care services. int j sci res publ. 2014; 4(8): 9–12. 15. pongantung i, pangkerego oa, pinangkaan n. kedudukan alat bukti elektronik dalam pembuktian tindak pidana informasi dan transaksi elektronik berdasarkan undang-undang nomor 19 tahun 2016. lex crim. 2021; 10(7): 147–56. 16. pontoh mr. penegakan hukum pidana terhadap resiko medik dan malpraktek dalam pelaksanan tugas dokter. lex crim. 2013; 2(7): 74–83. 17. pribadi i. legalitas alat bukti elektronik dalam sistem peradilan pidana. lex renaiss. 2018; 3(1): 109–24. 18. purnama sg. modul etika dan hukum kesehatan. universitas udayana; 2017. p. 1–11. 19. soekanto s. pendidikan hukum, penelitian dan profesi yang berhubungan dengan hukum. maj huk dan keadilan tahun ke iv. 2010; (5–6): 32. 20. suntama w. kajian tentang informed consent (persetujuan tindakan medik) menurut uu no. 36 tahun 2009 tentang kesehatan. lex priv. 2017; 5(5): 90–8. 21. supr iyatin u. aspek hukum dalam penyelengga ran pra ktik kedokteran dihubungkan dengan undang-undang nomor 29 tahun 2004 tentang praktik kedokteran. j ilm galuh justisi. 2018; 6(1): 117–24. 22. rohman a, syafruddin s. model perlindungan dan pemenuhan hak pasien terhadap pelaksanaan informed consent di indonesia. mimb huk. 2019; 31(2): 222–36. 23. judi. penerapan informed consent pada pasien gawat darurat di rumah sakit umum daerah ambarawa. in: seminar rekam medis dan manajemen informasi kesehatan. semarang: pormiki; 2017. p. 62–5. 24. wagiu cg, kristanto eg, lumunon t. informed consent di instalasi gawat darurat rsup prof. dr. r. d. kandou manado. j biomedik. 2017; 9(1): 58–61. 25. pratiwi i, atmadja ibp, bagiastra in. pelaksanaan persetujuan tindakan medis (informed consent) pada proses persalinan yang dilakukan oleh bidan di klinik citra asri yogyakarta. kertha semaya. 2014; 2(1): 1–15. 26. realita f, widanti a, wibowo db. implementasi persetujuan tindakan medis (informed consent) pada kegiatan bakti sosial kesehatan di rumah sakit islam sultan agung semarang. soepra j huk kesehat. 2017; 2(1): 30–41. 27. sulistyaningrum hp. informed consent: persetujuan tindakan kedokteran dalam pelayanan kesehatan bagi pasien covid-19. simbur cahaya. 2021; 28(1): 166–86. 28. arini ldd, ifalahma d, sumarna a. studi literatur pelaksanaan informed consent atas tindakan kedokteran di rumah sakit. i n: si kesnas. su ra ka r t a: un iversit as dut a ba ngsa; 2021. p. 1–5. 29. mayasari de. informed consent on therapeutic transaction as a protection of legal relationship between a doctor and patient. mimb huk. 2017; 29(1): 176–88. 30. sinaga na. perjanjian terapeutik kaitannya dengan informed consent dalam praktik kedokteran di indonesia. j ilm huk dirgant. 2021; 12(1): 876. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p1–6 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p1-6 58 vol. 43. no. 2 june 2010 research report the efficacy of honey solution as plaque reducing agent dewi nurul m�, indria rizki s��, indriani s��, masyitoh��, and auerkari ei�� 1 department of periodontology faculty of dentistry, university of indonesia 2 general dental practitioners, jakarta 3 department of oral biology faculty of dentistry, university of indonesia jakarta indonesia abstract background: periodontal care is an important step of periodontal health management. some chemically active substances have been studied as an adjunct to mechanical plaque control. honey is a traditional topical treatment for infected wounds and have inhibitory effect to around 60 species of bacteria including aerobes and anaerobes, gram-positives and gram-negatives. purpose: to compare the efficacy of 5% and 25% honey solution and aquadest as mouth-rinses to control dental plaque during 4 days period. method: after a thorough prophylaxis, during 4 days period of no oral hygiene all subjects were rinsed with 10 ml mouth-rinse they received 3 times a day after meal. group i rinse with 5% honey solution, group ii with 25%, and group iii with aquadest as control. results: there were significant increases of plaque index within each group, but no differences between all three groups in every experimental day. the fact that the probability value from day 1 (0.766) were gradually decreased to day 4 (0.076). conclusion: anti-microbial properties of honey solution as mouth-rinse did not show any inhibition effect on plaque formation until day 4. key words: periodontal care, plaque control, honey solution, mouth-rinse abstrak latar belakang: menjaga kesehatan periodontal merupakan tahap penting dalam pemeliharaan kesehatan periodontal. beberapa substansi kimiawi aktif telah diteliti untuk membantu dalam kontrol plak gigi secara mekanik. madu merupakan obat tradisional untuk luka terinfeksi dan dinyatakan mempunyai pengaruh menghambat sekitar 60 spesies termasuk bakteri aerob dan anaerob gram positif dan gram negatif. tujuan: membandingkan manfaat larutan madu 5% dan 25% terhadap akuades sebagai obat kumur untuk mengontrol pembentukan plak gigi selama 4 hari penelitian. metode: setelah tindakan profilaksis pembersihan sempurna, semua subjek penelitian dipersilahkan berkumur dengan 10 ml larutan yang telah diterima, 3 kali sehari setelah makan. kelompok 1 berkumur dengan larutan madu 5%, kelompok 2 dengan 25%, dan kelompok 3 dengan akuades sebagai kontrol. hasil: didapatkan peningkatan bermakna indeks plak dalam setiap kelompok penelitian, tetapi tidak berbeda antara ketiga kelompok pada setiap hari dalam waktu penelitian. namun ditemukan nilai p sejak hari 1 (0,766) menurun secara bertahap ke hari ke 4 (0,076). kesimpulan: sifat antimikroba larutan madu sebagai obat kumur belum menunjukkan pengaruh bermanfaat untuk menghambat pembentukan plak gigi hingga hari ke 4 penelitian. kata kunci: penjagaan kesehatan periodontal, kontrol plak gigi, larutan madu, obat kumur correspondence: dewi nurul m, c/o: bagian periodonsia, fakultas kedokteran gigi universitas indonesia. jl. salemba raya 4 jakarta 10430, indonesia. e-mail: dewi_nurul_m@yahoo.co.id introduction many studies from developed countries estimated that over 90% of the general population have some form of periodontal diseases.1 epidemiologic study revealed a peculiarly high correlation between supragingival plaque levels and chronic gingivitis, and clinical research led to the proof that plaque was the primary etiologic factor in 59nurul, et al.: the efficacy of honey solution gingival inflammation.2 it has been reported that dental plaque is a biofilm containing approximately 500–700 different microbial species3,4 which capable of colonizing the oral cavity.5 this biofilm adheres to the tooth surfaces in close vicinity to the periodontal tissue, and to the root surfaces in the subgingival micro-environtment.6 the aim of controlling dental plaque is to prevent biofilm-associated diseases like caries and periodontitis.7 the subgingival position of biofilm lies in the specific defense strategies that biofilm has evolved to overcome both the natural and the standard antibacterial defensive mechanism. these strategies compromise the efficacy of treatment regimes.6 while the tongue and oral mucosa serve as reservoirs of pathogenic bacteria which are able to relocate and colonize on the teeth and in sulci.8 once exposed to a bacterial stimulus, the gingival epithelial cells can elicit a wide array of responses including cytokines and chemokines that recruit inflammatory and immune cells.5 an antiseptic mouth-rinse produces an antimicrobial effect throughout the entire mouth, including areas easily missed during tooth-brushing and interdental cleaning.8 chemically active substances can be a valuable aid to mechanical plaque removal if manual measures are not performed long enough or on a regular daily basis.7 for many years, many studies has been done about chemical agents to remove plaque. many vehicles for the delivery of these agents2 for example as mouthwashes as of value in reducing bacterial plaque and gingivitis and are useful adjuncts to mechanical methods of plaque removal.9 honey has been used as a medicine since ancient times in many cultures. the popular literature on health and selftreatment of ailments gives the impression that honey can be taken to cure almost anything. since the last century it has been known that many ailments are the result of infection by microorganisms.10 there are many reports of honey having bactericidal as well as bacteriostatic activity against a broad spectrum of bacteria, and antifungal activity.10,11 the purpose of this study was to compare the efficacy of 5% and 25% honey solution and aquadest as mouth rinses to control dental plaque during 4 experimental days. if honey is proved beneficial to inhibit plaque formation, we can suggest patients to use it as it is cheap and easy to make at home. material and method the volunteers were 44 healthy dental students from the university of indonesia, ranging from 18 to 22 years old. the reasons for this study were explained and consent forms signed by all participants. all participants were randomly taken as sample and distributed into three groups. group i (n=20) rinsed with the 5% honey solution, group ii (n=20) with the 25% honey solution, and group iii (n=20) with aquadest (placebo). the 5% honey solution was made by 5 ml honey diluted in 95 ml aquadest, and the 25% honey solution was made by 25 ml honey which is diluted in 75 ml aquadest. the honey was produced from the nectar of the flowers of randu tree (ceiba petandra), to be bought from pusat perlebahan nasional (pusbahnas), desa ciomas, parung panjang bogor, west java. because of the double-blind design, all solutions were kept in the same kind, size and colour of bottle. in the pretreatment phase, the individuals were subjected to a thorough prophylaxis. at day 0, they were asked to refrain from all oral hygiene measures for a period of 4 days, during which they rinsed three times daily for 30 seconds with 10 ml of one of the mouth rinses. at the end of the investigation period, all participants were again subjected to a thorough prophylaxis. results the differences of mean data between and within study groups in every investigation day were shown in table 2. there were significant differences within every group (p<0.000) but no differences between groups in every investigation days. discussion it is well-known fact that plaque is formed immediately after meticulous tooth brushing. by the end of 24 hours the plaque is well on its way towards maturation and table �. demographic data of the volunteers group total i ii iii gender & age range f 12 19-22 f 15 18-22 f 10 18-22 37 m 4 19-22 m 1 19 m 2 18-22 7 total 16 16 12 44 group i : rinse with 5% honey ii : rinse with 25% honey iii : rinse with aquadest 60 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 58–61 hence starts its deleterious effects on the gingival.12 more severe disease will be progressed because of the anatomy of posterior teeth that impedes accessibility for individual oral hygiene, and offer less favorably to tooth brushing.13 it is also widely recognized that specific microorganisms in sub-gingival micro-flora are determinant agents for periodontitis.14 periodontal maintenance or supportive periodontal therapy is a key part of periodontal treatment. the goals of periodontal maintenance include prevention or minimization of disease recurrence, prevention or reduction of tooth loss, and diagnostic and treatment of recurrent disease and or other oral diseases in a timely basis. this therapy must be scheduled at regular intervals, usually ranging from 2 to 6 months, depending on the clinical needs of the individual patient continuing for the life of the dentition.15 periodontal care at each recall visit comprises three parts. the first part is concerned with examination and evaluation of the patient's current oral health. the second part includes the necessary maintenance treatment and oral hygiene reinforcement. because the amount of supra-gingival plaque affects the number of sub-gingival anaerobic organisms, and incomplete sub-gingival plaque eradication usually caused the recurrence of periodontal disease16, it is needed to use chemically active substances as a valuable aid to mechanical plaque removal. there are numerous studies about the effect of antimicrobial agents on oral micro-organisms.7 three known systems are responsible for the major anti-microbial activity found in honey. these are the acidity, the osmotic pressure, and the presence of inhibine.10,17 several chemicals with antibacterial activity have been identified in honey by various researchers, e.g. pinocembrine, terpenes, benzyl alcohol etc. however, the quantities of these phytochemical factors present were far too low to account for any significant amount of activity.10 the most anti-microbial activity of honey is its inhibine number. the inhibine number of honey is the degree of dilution to which a honey will retain its antibacterial activity. the inhibine number is presented as hydrogen peroxide. the number rises only when the honey is diluted. on dilution of honey, the activity increases by a factor of 2.500–50.000; thus giving a "slow release" antiseptic as a level which is antibacterial but not tissue-damaging.10 in this experiment, two concentrations were used, 5% and 25%. indriani et al.17 in their study used 5% and 25% honey solution. they stated that in 5% solution the inhibine value was higher than that in 25%, because the inhibine number is formed by enzymatic effect of glucose oxidase in honey itself when it is referred from glucosa to gluconic acid. in higher concentration, much more inhibine will be formed; therefore this experiment only used the 5% and 25% to find how honey dilution works against the plaque bacteria of some indonesian oral hygiene condition. the inhibine number depends on its dilution to which still have its antibacterial activity.10 h2o2 is an active germicide, and the effervescence of oxygen affords a mechanical debridement for healing of infected post surgical wounds.17 the on that is released from h2o2 can inhibit the anaerob bacteria in the plaque.18 it is also well known that anaerob bacteria for example porphyromonas gingivalis, tannerella forsythensis, treponema denticola as well as aggregatibacter actinomycetemcomitans could be found even in not deep periodontal pocket and between papillae of dorsal part of the tongue.19 molan18 wrote on how the method of honey antibacterial activity works is on oxidizing the protein of bacteria by inactivating bacterial enzymes (catalase, peroxidase, superoxide dismutase) as ametabolic product. in this study, there were significant increases of plaque index from day 1 to day 4 within every groups. but there were no differences of plaque index in every experiment days between study groups. the probability of its differences was decreased from 0.766 in day 1 until 0.076 in day 4. significant differences can be found in day 5 or day 6 or more. but the fact that 4 days period of investigation is not enough to show the efficacy of honey solution as a good rinsing agent to inhibit plaque formation in periodontal care. plaque index in day 1 of group i (38.38) is lower than group ii (42.88) or group iii (41.67). the same results also found in day 3 (84.19; 91.44; 91.08) and day 4 (103.38; 111.38; 116.33). these results showed that rinsing with honey solution has more inhibiting effect of plaque formation than with aquadest only, and more water in dilution (honey 5%) raised the anti-microbial activity than less water (honey 25%). this phenomenon showed its inhibine effect as stated by molan10,11 and indriani et al.17 from the results of this study it can be concluded that honey solution as mouth rinse did not show any plaque reducing effect on biofilm until day 4 investigation. further research in longer time to prove antibacterial effect is needed. it is hoped this honey as mouth rinse is sufficient to reduce plaque formation. if it is proved, it should be suggested that someone gargling with honey solution then with water only as periodontal care to control supragingival plaque. table ��. plaque score rate within and between the study groups day group p i ii iii 1 38.38 42.88 41.67 0.766 2 69.69 65.75 72.33 0.712 3 84.19 91.44 91.08 0.510 4 103.38 111.38 116.33 0.076 p 0.000 0.000 0.000 group i : rinse with 5% honey ii : rinse with 25% honey iii : rinse with aquadest 61nurul, et al.: the efficacy of honey solution references 1. needleman i, mcgrath c, floyd p, biddle a. impact of oral health on the life quality of periodontal patients. j clin periodontol 2004; 31: 454–7. 2. addy m, moran j. chemical supragingival plaque control. in: lang np, lindhe j, eds. clinical periodontology and implant dentistry. 5th ed. singapore: blackwell munksgaard; 2008. p. 734–65. 3. al-otaibi m, al-harthy m, gustafsson a, johansson a, claesson r, angmar-mansson b. subgingival plaque microbiota in saudi arabians after use of miswak chewing stick and toothbrush. j clin periodontol 2004; 31: 1048–53. 4. schacher b, baron f, robberg m, wohlfeil m, arndt r, eickholtz p. aggregatibacter actinomycetemcomitans as indicator for aggressive periodontitis by two analyzing strategies. j clin periodontol 2007; 34: 566–73. 5. stathopoulou pg, benakanakere mr, galicia jc, kinane df. epithelial cell pro-inflammatory cytokine response differs across dental plaque bacterial species. j clin periodontol 2010; 37: 24–9. 6. ohrn k, sanz m. prevention and therapeutic approaches to gingival inflammation. j clin periodontol 2009; 36(suppl.10): 20–6. 7. auschill tm, hein n, hellwig e, follo m, sculean a, arweiler nb. effect of two antimicrobial agents on early in situ biofilm formation. j clin periodontol 2005; 32: 147–52. 8. darby ml. changing perspectives on the use of antimicrobial mouth rinses. j dent hyg 2007; spec suppl: 3. 9. de a werner cw, seymour ra. are alcohol-containing mouthwashes safe? british dent j 2009; 207: e19. (abdj taster for aadr 2010). 10. molan pc. honey as an antimicrobial agent. 2002. available from http://honey.bio.waikato.ac.nz accessed april 4, 2002. 11. molan pc. honey as topical agent. 2001. available from http://www. worldwidewounds.com accessed august 23, 2004. 12. el-mostehy r, al-jassem aa, al-yassin ia, el-gindy ar, shoukry e. siwak as oral health device (preliminary chemical and clinical evaluation). 2003. available in http://www.islamset.com/sc/plant/ siwak.htm. accessed on february 9, 2004. 13. dannewitz b, lippert k, lang np, tonetti ms, eickholz p. supportive periodontal therapy for furcation sites: non-surgical instrumentation with or without topical doxycycline. j clin periodontol 2009; 36: 514–22. 14. vettore mv, leao att, monteiro da silva am, quintanilha rs, lamarca ga. the relationship of stress and anxiety with chronicthe relationship of stress and anxiety with chronic periodontitis. j clin periodontol 2003; 30: 394–402. 15. preshaw pm, heasman pa. periodontal maintenance in a specialist periodontal clinic and in general dental practice. j clin periodontol 2005; 32: 280–6. 16. merin rl. supportive periodontal treatment. in: newman mg, takei hh, klokkevold pr, carranza fa, eds. carranza's clinical periodontology. 10th ed. philadelphia: saunders; 2006. p. 1194–205. 17. indriani s, indria rs, masyitoh, dewi nm. perbedaan manfaat larutan madu 5% dan 25% sebagai bahan kumur terhadap pembentukan plak. scient j dent 2005;20(61): 259–64. 18. molan pc. 2001. honey as a topical antibacterial agent for treatment of infected wounds. available in http://www.worldwidewounds/ com/2001/november/molan/honey-as-topical-agent.html. accessed on march 9, 2005. 19. dewi-nurul m. evaluation of alkaline phosphatase, lymphocyte, immunoglobulin g against porphyromonas gingivalis conditions of juvenile periodontitis and rapidly progressive periodontitis in confirming the clinical and radiographical diagnosis. dissertation. jakarta: faculty of dentistry, university of indonesia; 2001. p. 230. mkg vol 39 no 1 jan 2006 isi.pmd 12 antimicrobial effects of coleus amboinicus, lour folium infusum towards candida albicans and streptococcus mutans devi rianti and sri yogyarti department of dental material and technology faculty of dentistry airlangga university surabaya indonesia abstract a laboratory experimental study conducted on antimicrobial effects of coleus amboinicus, lour folium infusum towards candida albicans and streptococcus mutans (s. mutans). effective concentration of coleus amboinicus, lour to decrease the quantities candida albicans and s. mutans colonies is expected to be found out in this study. this study was using coleus amboinicus, lour folium infusum with 12.5%, 15%, 17.5%, 20%, and 22.5% concentrations. sterilized aquadest used as a control. candida albicans and s. mutans quantities was enumerated by counting the amount of candida albicans and s. mutans growth in the sabouraud ,s dextrose agar and tryptone and yeast agar media, using colony forming unit per milliliter (cfu/ ml) unit. data analysis was using a one-way anova and lsd with 5% degree of significance. the result showed 22.5% concentration of cal folium infusum was the most effective in decreasing the quantity candida albicans and s. mutans colonies. key words: antimicrobial, coleus amboinicus, lour, candida albicans, streptococcus mutans correspondence: devi rianti, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction according to national health system guidance, the traditional medication which can successfully, will be lead and used for research and examination literally to indonesian herbs so it is safe to be used. indonesian government supports the use of traditional materials as one of alternative medication, since indonesia is enriched with medical herbs. one of the herbs usually plant in the garden and easily growth is jinten leaf. according to wijayakusuma et al.1 jinten leaf in latin called coleus amboinicus, lour and simplified as plectranthi amboinicus folium, is a plant which peculiar for stomatitis and antifungal medication. the chemical contents of coleus amboinicus, lour are calium, essential oils which contains carvacrol, isoprophylo-cresol, phenol and sineol. weehuizen2 stated, that from 120 kg fresh leaf of coleus amboinicus, lour (cal) will be resulted essential oils such 25 ml, and 25 ml essential oils equal to 0.2% essential oils which contains phenol generations, i.e. isoprophyl-o-cresol, which has high antiseptic effect.2 preliminary examination of cal showed, that from tawangmangu central java chemical content, extracted with alcohol 96%, it contains alkaloids, saponin, cardenolids, and bufadienolids together with polyphenol.3 devi’s4 experiment stated that extract solution of cal is effective to decrease candida albicans (c. albicans) in acrylic resin after 2 hours soaking with 15% concentration. chemical component sineol has antifungal effects to c. albicans, trichiphyton metagrophytes and cryptococcus neoformans.5 wisterich and lechtman stated that phenol and cresol can kill fungus vegetative cell and bacteria by protein denaturation and surface tension reduction, so that increases bacteria permeability.6 streptococcus mutans (s. mutans) is a normal occupant bacteria in oral, however if the atmosphere fits, and population increases it becomes pathogen.7 s. mutans have been admitted in the dentistry as the main cause of caries.7 based on that, the growth of s. mutans should be obstructed to become pathogen. according to bahalwan and sjahbana8 the use of traditional medication in indonesia is as a prevention. some of herbal traditional medication can be used as a mouthwash and they also work for antiseptic as if as a disinfectant are semanggi leaf, jinten leaf (cal), sirih leaf, gambir, saga leaf and also kaca piring leaf. usually, extract materials are hard to find, the alternative ways to process the herbs are needed. in one of research, infusum technique for herbs is better than boiled.9 the advantage of infusum, is easier to use and it has been socialized, cheaper because there is no special treatment and complicated instrument is needed as well. in some of literatures said, that cal has an antiseptic character because of it chemical contents. some of researches stated that it could decrease the growth of c. albicans, trichiphyton metagrophytes and cryptococcus neoformans, either ways there are some possibilities of cal to have antibacterial character towards s. mutans. the best technique to use and to, reduce the cost and to be more socialized, the infusum technique is chosen. the minimum concentration of cal folium infusum to decrease s. mutans and c. albicans colonies is unknown yet. the aims of this 13rianti and yogyarti: antimicrobial effects of coleus amboinicus article is to investigate if cal folium infusum concentration variations effect towards c. albicans and s. mutans colonies and to find the concentration of cal folium infusum that has an effective antimicrobial character towards c. albicans and s. mutans. materials and methods the research is an experimental laboratories and the research design is post test only control group. independent variable is cal folium infusum with 12.5%, 15%, 17.5%, 20%, 22.5% concentration. dependent variable is the amount of c. albicans and s. mutans colonies. the control variables are the similarity of planting area, crops time, drying time, temperature and time of c. albicans and s. mutans proliferation, growth media, counting c. albicans and s. mutans, and cal folium infusum process, in accordance with farmakope indonesia.10 this research has been done in phytochemical laboratory faculty of pharmacy airlangga university, oral microbiology laboratory faculty of dentistry airlangga university, surabaya. materials those being used in these research are c. albicans suspension, s. mutans, sabouraud’s dextrose media and sabouraud’s broth, tryptone and yeast agar (tyc) media and brain hearth infusion (bhi) and cal leaf from central traditional medication research garden. instruments to count c. albicans and s. mutans are: incubator (precision, japan), 0,2 μm milipore filter unit (millex-ha, bedford), reaction tube, stopwatch, petridish (anumbra), autoclave (foundry), ose, spreader, pinset, vibrator (vortex), 5 cc injection syringe (terumo), 1 cc tuberculine syringe (terumo), ependorff micropipette (titertek, england), anaerobic jar, mechanic counter instrument (hand tally model h-102, japan). the research methods are making of cal folium infusum in accordance with farmakope indonesia, and counting of s. mutans and c. albicans colonies. counting s. mutans colonies is by preparing 7 reaction tubes and giving numbers by order 1 until 7. reaction tubes 1 until 5 are filled with test materials i.e. 1 ml cal folium infusum in 2 ml brain hearth infusion broth (bhib) media and s. mutans with a density of 3 × 108 that already liquefied by 10–7 as much as 0.1 ml. cal folium infusum concentrations in reaction tubes number 1 until 5 are 22.5%, 20%, 17.5%, 15%, 12. 5%. reaction tube number 6 is not filled with test material, but it is added with s. mutans with 3 × 108 densities that already liquefied as positive control. reaction tube number 7 filled with only bhib media without s. mutans as negative control. ten times of replication are done. all of reaction tubes are placed in an anaerobic jar, in anaerobic environment and incubated as long as 24 hours on 37 °c. the result is read by observing whether there is a deposit or turbid. to count the s. mutans colonies, all test materials are planted in tyc media in petridish. each of petridish is filled with the liquid in the tube (cal folium infusum + bhib media + s. mutans) using pipette which have the same number as the reaction tube, then spread using spreader at the surface of solid tyc media. all the petridish to put in anaerobic jar incubated in 37 °c degree, as long as 48 hours. result reading is by observing whether there is a colony proliferation at the surface of tyc, and counting the colonies (cfu/ml). to count c. albicans colonies is by preparing 7 reaction tubes and giving numbers 1 until 7. reaction tubes number 1 until 5 are filled with 1 ml cal folium infusum in 2 ml sabouraud’s broth media and with c. albicans density of 3 × 108 that already liquefied by 10–7 as much as 0,1 ml. cal folium infusum concentrations in reaction tubes 1 until 5 are 22.5%, 20%, 17.5%, 15%, 12.5%. reaction tube number 6 is not filled with test material, but it is added with c. albicans which has 3 × 108 densities that already liquefied as positive control. reaction tube number 7 filled with sabouraud’s broth media without c. albicans as negative control. replications are done in ten times. the result is read by observing whether there is a deposit or turbid. to count the c. albicans colonies, all test materials are planted in sabouraud’s dextrose media in petridish. each of petridish is filled with the liquid in the tube (cal folium infusum + sabouraud’s broth media + c. albicans) using pipette which have the same number as the reaction tube, then spread using spreader at the surface of sabouraud’s dextrose media media. all the petridish to put in incubator, incubated in 37 °c degree, as long as 48 hours. result reading is by observing whether there is a colony proliferation at the surface of sabouraud’s dextrose media, and counting the colonies (cfu/ml). the result is being tabulated according to each group then statistic test is held using one-way anova, with deviation degrees 5%. however, if there is a significant result, it will be continued by lsd test. results the average result and standard deviation c. albicans and s. mutans colonies after contacted with cal folium infusum in concentration 12.5%, 15%, 17.5%, 20%, 22.5% can be seen at table 1 and 2. table 1. average and standard deviation from total c. albicans colony (cfu/ml) after contact with cal folium infusum concentration n x sd control 12,5% 15% 17,5% 20% 22,5% 10 10 10 10 10 10 479.50 244.60 183.30 114.30 55.20 18.60 18.91 36.04 10.79 10.66 10.43 9.43 14 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 12–15 table 2. average and standard deviation from total s. mutans colony (cfu/ml) after contacted with cal folium infusum concentration n x sd control 12,5% 15% 17,5% 20% 22,5% 10 10 10 10 10 10 495 286.80 233.40 165 120.30 55.70 42.52 30.36 26.66 11.24 13 12.09 notes: n: samples amount x: average sd: standard deviation before parametric test is done, to know the significant difference result, a normality test using kolmogorovsmirnov test should be done. the result is, 5 treatment group and control are normally distributed (p > 0.05), moreover, comparison test to 5 treatment group and control is done by one-way anova test. from one-way anova test, it appears that there is a significant result among treatments with p = 0.001 (p < 0.05), to find out further the differences among treatments, the analysis is continued with least significant difference (lsd) test. lsd test shows that there is significant result in each concentration groups; 12.5%, 15%, 17.5%, 20%, 22.5% and control. in other words the increase of cal folium infusum concentration will affect c. albicans and s. mutans colonies. discussion the use of natural herbs medication has already been socialized and barely a new thing, but it has been exercised long time ago to fulfill the need of medication in solving health problem, and the experience has been inherited. the benefit is admitted by a lot of people, but still there is not lot of literatures. guided by national health system, it is stated that a useful traditional medication will be lead and used for some scientific research and test to indonesian plants. one of traditional medication herbs that usually planted in the garden and easily to grow is jinten leaf, in latin it is called coleus amboinicus, lour (cal), which have chemical content, phenol which is as an antiseptic.1,2 preliminary examination on chemical content of cal material from tawangmangu central java filtered with alcohol 96%, it is appears containing alkaloids, saponin, kardenolids, bufadienolids also polyphenol.3 base on that, there are some possibilities those materials can be used for oral antimicrobial as antiseptic in dentistry. result from the research shows the average c. albicans and s. mutans colonies after contact with cal folium infusum in increasing infusum concentration, i.e. 12.5%, 15%, 17.5%, 20%, 22.5% will decrease. on the contrary, in lower concentration the total c. albicans and s. mutans colonies increased constantly. this is caused by coleus amboinicus, lour contains phenol and phenol generations which are useful antiseptic.1,2 supported from devi research’s, stated that, in dry cal leaf extract contains 5.15% phenol and 3.65% sineol.4 increasing cal folium infusum concentration will cause decreasing c. albicans and s. mutans colonies, since the increased concentration will increase the phenol and sineol in infusum. the increased phenol and sineol, will increase the antimicrobial effect of infusum towards c. albicans and s. mutans. it is further explained by regezi and sciubba11 that c. albicans is a very sensitive species towards phenol. besides phenol, hammerschimdt et al.5 stated that the other chemical materials, sineol, also have antifungus activity towards c. albicans, trichiphyton metagrophytes and cryptococcus neoformans. hugo and rusell,12 stated that phenol generally can be used as disinfectant which have antimicrobial activity and fast bactericide, the activity usually decreased by watering. it is also supported by siswandono and soekarjo,13 who stated that materials effectiveness affected by concentration, time and temperature. in these research, infusum ph is approximately between 4.78–4.80, which is possibly has good effect in antimicrobial activities of cal folium infusum, since phenol appears more effective in acid ph and moreover, it is explained that disinfection process influenced by several factors such as, concentration, temperature, and ph.12 other factor which possibly the cause is the presence of alkaloid contents. alkaloid is a chemical compound contains nitrogen, and this group can be diverged from other plant component based on its alkali characteristic. this compound is found in plant as salt of various organic acids, which is functioned to protect plant from parasites and predator, and also effective as antimicrobial.14 until now, there still no references for antimicrobial mechanism of this alkaloid. based on antimicrobial mechanism, phenol can eliminate fungus vegetative and bacteria by protein denaturation and surface tension reduction which caused the bacteria and fungus permeability are increased.6 it is explained by melville and russel15 that reaction with cell protein, is an obstruction or elimination process by ruining the colloid system using protein coagulation and precipitation. microbial cell protein coagulation will cause metabolism distraction and membrane cell permeability change is by decreasing the surface tension which increase the membrane cell permeability causing liquid enters and eliminates the microbe. renner et al.16 proved that people using denture without pathologic change of its supporting mukosa orally, as if the c. albicans colonies are not more than 100 cfu/ml. candida species experiment will be decided as positive, if the amount is more than 100 colonies of subject samples with sabouraud’s dextrose breeding, or if the amount is more than 500 cfu/ml saliva. according to brightman & greenberg17 c. albicans is assumed as a normal flora part in oral, as it can be found in some healthy individuals and 15rianti and yogyarti: antimicrobial effects of coleus amboinicus carriers if it not more than 200 cfu/ml, detected by oral swapping inoculation of sabouraud’s dextrose agar breeding. average value of c. albicans colonies in this research are 244.60 cfu/ml, 183.30 cfu/ml, 114.30 cfu/ ml, 55.20 cfu/ml, 18.60 cfu/ml (tabel 1), from contact with cal infusion in 12.5%, 15%, 17.5%, 20%, 22.5% concentrations. c. albicans colonies which are contacted with cal folium infusum with a 15% concentrate are less than total colonies of a healthy individual. from those scholars’ research and opinion comparison, the use of 15% concentrate of cal folium infusum is found has been effective to decrease the amount of c. albicans colonies. in conclusion, increasing of cal folium infusum concentration, i.e. 12.5%, 15%, 17.5%, 20%, 22.5% will decrease the total c. albicans and s. mutans colonies. the most effective cal folium infusum to decrease c. albicans and s. mutans colonies is 22.5%. more experiments are needed to find out the biocompatibility of coleus amboinicus, lour folium infusum to be used as an alternative mouth wash. references 1. wijayakusuma h, dalimartha s, wirian ag, tanaman obat berkhasiat indonesia iv. edisi 1. jakarta: pustaka kartini; 1996. p. 38–41. 2. heyne k. tumbuhan berguna indonesia. jilid iii. jakarta: badan litbang kehutanan. yayasan sarana wana jaya; 1987. p. 1698. 3. hutapea jr. pemeriksaan pendahuluan golongan kandungan kimia tanaman coleus amboinicus, lour (labiatae) asal tawangmangu. balai penelitian tanaman obat tawangmangu, pusat penelitian farmasi, badan penelitian dan pengembangan kesehatan, departemen kesehatan ri; 1982. p. 389–94. 4. devi r. daya antimikroba ekstrak coleus amboinicus, lour terhadap candida albicans pada resin akrilik. j kedokteran gigi indonesia 2003; 10(edisi khusus):845–51. 5. hammerschmidt fj, clark am, soliman fm, el-kashoury es, kawy mm, fishawy am. chemical composition and antimicrobial activity of essential oil of jasonia candicans and jasonia montana. planta med 1993; 59:68–70. 6. rahardjo mb. perbedaan daya antibakteri allium sativum linn dan kaempferia galanga terhadap streptococcus mutans dan bermacammacam bakteri yang berasal dari saluran akar gigi gangraena pulpae. thesis. surabaya: universitas airlangga; 1993. p. 13. 7. kidd eam, bechal sj. dasar-dasar karies penyakit dan penanggulangannya. essentials of dental caries. new york: the desease and its management. 1-8. company; 1992. p. 676, 688–9. 8. bahalwan r, sjabana d. mengkudu seri referensi herbal. jakarta: salemba medika; 2002. p. 3. 9. eha d. khasiat obat kumur infusa daun kacapiring terhadap perubahan mikroorganisme rongga mulut pemakai gigi tiruan lepasan. majalah ilmiah kedokteran gigi fakultas kedokteran gigi usakti 1999;edisi khusus foril vi:497–501. 10. dep kes ri. farmakope indonesia. edisi 3. jakarta: dep kes ri; 1979. p. 12–13. 11. regezi ja, sciubba jj. oral pathology: clinical pathologic correlation. philadelphia: wb saunders company; 1989. p. 110–6. 12. hugo wb, russel ad. pharmaceutical microbiology. 4th ed. oxford, london, edinburgh, boston, melbourne: blackwell scientific publications; 1989. p. 226–33. 13. siswandono, soekarjo b. kimia medisinal. cetakan i. surabaya: airlangga university press; 1995. p. 247–8. 14. robinson t. kandungan organik tumbuhan tinggi. padmawinata k. edisi ke-6. bandung: penerbit fmipa itb; 1995. p. 191–222. 15. melville ph, russel c. microbiology for dental student. 3rd ed. london: williem heinewmann medical book ltd; 1981. p. 155–76. 16. boedi s. aspek klinis dan penetapan diagnosis kandidiasis mulut. majalah ilmiah kedokteran. gigi fakultas kedokteran gigi usakti 2001; 16(44):86–95. 17. brightman vj, greenberg ms. candidiasis. in: lynch ma, editor. burket’s oral medicine diagnosis and treatmen. 8th ed. philadelphia: lippincott; 1984. p. 223–7. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 20 u bow activator, an alternative functional orthodontic appliance anita budihardja* and jusuf sjamsuddin** *orthodontics adaptation program at the department of orthodontic **department of orthodontic faculty of dentistry, airlangga university surabaya indonesia abstract functional orthodontic treatment has been proved to be one of the most effective and successful treatment modality in orthodontics. functional orthodontic appliance can only be applied in growing young patients. since andresen’s activator, there are a lot of other functional appliances that have been developed and introduced. u bow activator, introduced by prof. karwetzky from wilhelms university of muenster, is one of the appliances that can be chosen. in this case report, u bow activator type 1 is used to treat class ii malocclusion and proved to give satisfying result. patient’s cooperation is the most important factors in achieving success. key words: functional orthodontic appliance, u bow activator, karwetzky correspondence: anita budihardja, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction daskalogiannakis1 in glossary of orthodontics terms defines functional appliance as a removable or fixed appliance that alters the posture of the mandible. this appliance transmits the forces created by the resulting stretch of the muscles and soft tissues and by the chance of the neuromuscular environment to the dental and skeletal tissues to produce movement of teeth and modification of growth. in 1880, kingsley introduced the term and concept of “jumping the bite” for patients with mandible retrusion. he inserted a vulcanite palatal plate consisting of an anterior incline that guided the mandible to a forward position when the patient close it.2 later in 1902, pierre robin published an article describing an appliance, called the monobloc, because it was a single block of vulcanite. he used this appliance to position the mandible forward in patients with glossoptosis and severe mandible retrognathism who risked occluding their airways with their tongues. this problem usually associated with cleft palate and known as pierre robin syndrome. robin noted that forward mandible posture reduced this hazard and also led to significant improvement in the jaw relationship.2,3 inspired by kingsley appliance, without first knowing robin’s monobloc, andresen developed a mobile, loose fitting appliance modification that transferred functioning muscle stimuli to the jaws, teeth and supporting tissues. at first, andresen used his appliance as a retainer over a summer vacation for his own daughter after removal of fixed appliances used to correct a distocclusion. this appliance was a modification from kingsley appliance, to which he added lateral extensions to cover the lingual aspects of the mandibular teeth. the “biomechanical working retainer” was also preventing mouth breathing. further use of the “retention activator”, as he later called the device, brought encouraging result. he found the results of the previous treatment were not only preserved but in many cases actually improved during the vacation period.2,3 later, andresen worked together with häupl, developing the concept of the appliance, which they both called activator. they believed that it has ability to activate the muscle force. they regarded “functional jaw orthopedics” as vastly superior to all previous methods in bringing about growth changes in an entirely physiological manner. they believed that this appliance induced growth changes in physiological manner and stimulated or transformed the natural forces with an intermittent functional action transmitted to the jaw, teeth and investing tissue.2,3 the introduction of andresen’s activator was a milestone in the history of orthodontics. since then the development of removable appliances and modification of activator grew so fast, especially in europe. in 1964, rudolf karwetzky from wilhelms university münster, with his article “ein neues funktions-kieferorthopädisches gerät” at the deutsche zahnärzteblatzt, introduced a new functional appliance, which he called u bow activator or u bügel activator (uba). u bow activator from karwetzky (figure 1) consists of maxillary and mandibular active plate, joined by a u bow in the region of the first permanent molars. in addition to acrylic covering the lingual tissue aspects, gingiva and the teeth, the plates also extend over the occlusal aspects of all teeth. each plate has labial bow and protrusion bow (closed spring), and the upper plate has expansion screw.4 labial bow, made from 0.9 mm stainless steel wire, extends from 2�budihardja: u bow activator canine to canine. protrusion bow extends from middle of canine to canine at the palatal region, made from 0.7 mm stainless steel wire. the height of these two components depends on front teeth movements that want to be achieved. expansion screw is placed in the upper plate, at the height of p1 or dm1. u bow made from 1.2 mm wire, placed at both sides at the height of m1. u bow activation will define the mandible reposition.4 there are three types of u bow activator developed by karwetzky: a) uba type 1. in type 1, the u bows are placed downward and this activator is used to correct class ii malocclusion; b) uba type 2. in type 2, the u bows are placed upward and this activator is used to correct class iii malocclusion; c) uba type 3a and 3b. the placements of the u bows are different between the right side and the left side. this type is usually used to correct asymmetry and functional midline shifting (figure 2). from these three types of uba, the one that used most is uba type 1 to correct class ii malocclusion. ehmer, with the dysgnathy classification, said that indication of using uba type 1 are mandible retrognathy, maxilla prognathy, upper front teeth protrusion and or lower front teeth retrusion, deck bite (angle class ii div 2), and skeletal or functional asymmetry that accompany class ii malocclusion.4,5 just as other functional appliance, the optimal time of using uba are during growth, between 8–11 years old. uba can also be used earlier (4–7 years), usually in patients with class ii div 1 malocclusion accompanied by extreme over jet. this kind of malocclusion can enhance the risk of front teeth trauma, caused negative functional pattern (lower lip is trapped behind upper front teeth and incompetence lip closure) and usually caused psychological stress to the patient (being mocked about appearance).4,5 figure 1. u bow activator from karwetzky. figure 2. three types of uba developed by karwetzky. 22 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 20–26 modes of actions of uba are splinting of dental elements, sequential anterior reposition of the mandible (stepwise forward positioning of the mandible), selective transversal expansion of the maxilla and incorporation of active elements for desired tooth movement. acrylic relief on occlusal and palatal (lingual) region will give splinting effect to the teeth. acrylic is usually grinded when permanent teeth are erupting, or when elongation of posterior teeth is wanted.4–6 u bow that join the upper and lower plates can be activated (figure 3) and this activation will caused sequential anterior reposition of the mandible. the construction bite is made 3–4 mm sagittally to anterior. after using the uba for 3–5 months, activation can be started. the activation is usually 2 mm every 2–3 months.4–6 in class ii div 1 malocclusions with mandible retrognathy, the upper jaw is usually transversal underdeveloped. by using uba, it will be possible to do anterior reposition of the mandible together with transversal maxillary expansion. this expansion can be started after 2 months using the uba (adaptation time) and can be done 1–2 times a week.4–6 tooth movement, even limited, can also be achieved with labial bow and protrusion bow at upper and lower jaw. torque control at upper front teeth (not active torque movement) can be achieved if labial bow placed passive more gingivally and the protrusion bow active and more incisal. inclination and position of lower front teeth can also be corrected by using labial bow and protrusions bow correctly.4-6 case nine years old male patient came to department of orthodontics at wilhelms university muenster with chief complaint that his upper front teeth was too protruded. he has difficulties to close his mouth and his upper fronts teeth were exposed at relax position. his self confidence was low because he was often mocked by his brothers and friends as bugs bunny. anamnesis showed that some people at his family (his elder and younger brother) have the same problem (protruding upper teeth/jaw). he also has incompetence lip closure and he breath oft through the mouth. extra oral photos before and after treatment can be seen in figure 4 and 5. molar and canine relations on both sides were class two. he has deep curve of spee, spacing on upper jaw, overjet is 11 mm and overbite is 6,5 mm. panoramic radiograph showed that all permanent teeth exist, except m3 that can not yet be seen at this age (figure 6). cephalometry analysis shows class ii malocclusion with normal maxilla and mandible retrognathy, the face was mesofacial with normal growth pattern, upper and lower front teeth were proclined and the profile was convex (figure 7). figure 3. u bow activation by making the “u” smaller. figure 4. extra oral photos before treatment. 23budihardja: u bow activator figure 5. intra oral photos before treatment. figure 6. panoramic radiograph before treatment. figure 7. cephalometry radiograph before treatment. figure 8. intra oral photos after 15 months using uba. 2� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 20–26 case management our treatment plan was using functional appliance to achieve anterior reposition of the mandible and to influence the growth of the mandible to reach its maximum. functional appliance used was u bow activator type 1 from karwetzky. the first construction bite made was 4 mm sagittal to anterior, vertical 4 mm and transversally lower midline was adjusted to midline of the face. transversal expansion in upper jaw was also needed in this case. during the first six months, patient’s cooperation was not so good. he lost his uba once that a new one has to be made. after that incident, patient was motivated during his visits to the clinic, and since that his motivation became better. he wore his uba regularly, all night and during day time as well. the uba was activated 2 mm every 2–3 months and the expansion screw once in a week. after 15 months using uba (figure 8), his over jet was reduced to 7 mm and he had no difficulties wearing the uba at all. after using uba for 25 months (figure 9 and 10), over jet was reduced to 2–3 mm and the profile changed significantly. molar relationships on both sides were class 1. both patient and parents were happy and satisfied with the treatment result and they did not want any further orthodontic treatment with fixed appliance. it was decided to go to retention phase and during this stage the patient is told to wear uba only at nights. after 15 months using uba as retainer (figure 11 and 12), the treatment result was stabile. patient has stabile static and dynamic occlusion, nice profile, beautiful smile and his self confidence become better after orthodontic treatment. patient and his parents were highly satisfied with treatment result. figure 9. extra oral photos after 25 months using uba. figure 10. intra oral photos after 25 months using uba. 2�budihardja: u bow activator figure 11. extra oral photos after 15 months using uba as retainer. figure 12. extra oral photos after 15 months using uba as retainer. discussion u bow activator is one of functional appliance that can be use to place mandible forward and to modify the growth in class ii malocclusion. benefit of using uba is that the operator can do forward positioning of the mandible gradually. karwetzky, in 1964, postulated that gradually forward positioning of the mandible is more effective than a direct anterior reposition.3–5 his postulation is then proved with the experiment done by petrovic in 1975. the experiment did by petrovic et.al on rats showed that gradual mandible reposition will give result that is more stable and can enhance the growth in condyle effectively.4,7 gradual forward mandible positioning was done in this patient. at the beginning of the treatment, construction bite was made only 4 mm to anterior (patient’s over jet was 12 mm) and after 6 months u bow was activated 1–2 mm every 2 months. after 25 months using uba, the over jet was reduced to 2–3 mm. the maxilla was transversally expanded by opening the expansion screw once in a week. skeletal changes that can be achieved with uba are growth restriction of the maxilla (when combined with head gear), unwanted reaction of the maxilla can be minimized (such as anterior-inferior rotation of the maxilla), enhance the growth in condyle area so that optimal growth border for each individual can be reached, and enhance the growth in temporal and dentoalveolar region which can stabilize treatment result.4,5 uba can also cause dental changes which are torque control in anterior front teeth (by placing labial bow and protrusion bow correctly) and avoiding unwanted dental changes such as proclination of lower front teeth. beside skeletal and dental changes, uba can also cause neuromuscular adaptation which is need for stabilization of treatment result.4,6 significant changes was seen in this patient, class i relation in molar and canine region was achieved, with overbite and over jet 2–3 mm. upper and lower front teeth inclination were in normal and treatment result in stabile even until 15 months after retention time. optimal result can be achieved with correct diagnosis and treatment plan, at the right time regarding the age of the patient. ehmer5 said that the optimal time to start treatment with functional appliance is before patient reached the peak of the growth (stadium mp3 = in carpal radiograph). after 2� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 20–26 that, functional appliance can still be used even though the treatment started at the late stage.5 when the patient is still in stadium mp3 = according to the carpal radiograph (epiphyse and diaphyse are the same width, usually 9,7 years old in girls and 11,2 years old in boys), it is optimal to start orthodontic treatment with functional appliance. when the patient is in stadium mp3 cap (epiphyse is capping the diaphyse, usually 12,4 years old in girls and 14,0 years old in boys), treatment success is limited and will not be as optimal when it is started before. beginning of the treatment time is considered late, and can only be successful with good patient cooperation and favorable growth pattern.5 being in mp3 unit stadium (epiphyse and diaphyse are joined, usually 14,3 years old in girls and 16,0 years old in boys), the patient is considered too old for orthodontic treatment with functional appliance. treatment can still be done with the knowledge that success rate is not high and must be support with patients cooperation and favorable growth pattern as well.5 patient’s cooperation, as said before, is a very important factor in determining treatment success and stability.5 during the first 6 months, patients cooperation was bad, he did not wear his appliance as told and he even lost his uba. a new uba was made and the patient was motivated. after his cooperation was better, he wore his appliance all night and for several hours during the days as told, he came to the clinic for control regularly and always had good oral hygiene. these points really contribute a lot in achieving good treatment results. it concluded that u bow activator from karwetzky is one of functional appliance that can be used in treating class ii malocclusion, both skeletal or dentoalveolar. the indications and contra indications for the use of an u-bow activator have to be considered within the context of the indications dentofacial orthopedics and functional orthodontics. there is a broad area of clinical application during the period fo growth for different forms of class ii malocclusion. there are some effects that can be achieved by using u-bow activator: splinting of the teeth, stepwise forward positioning of the mandible, selective maxillary expansion, and incorporation of active elements for desired tooth movement. this appliance is proved to work well and is easy to be used or made. references 1. daskalogiannakis j. glossary of orthodontic terms. 1st ed. berlin: quintessence publishing; 2000. p. 18, 123. 2. graber tm, neumann b. removable orthodontic appliance. 2nd ed. philladelphia: wb. saunders co; 1984. p. 175–243. 3. graber tm, rakosi t, petrovic ag. dentofacial orthopedics with functional appliance. 2nd ed. st louis: mosby; 1997. p. 161–222 4. ehmer u. u bügel aktivator. in: miethke rr, drescher d, editors. kleines lehrbuch der angle kalsse ii,1 unter besondere berücksichtigung der behandlung. berlin: quintessence bibliothek; 1996. p. 161–82. 5. ehmer u. indikatiobshinweise für den u bügel aktivator typ 1 nach karwetzky. prakt kieferorthop 1988; (2):75–84 6. ehmer u. indikatiobshinweise für den u bügel aktivator typ 1 nach karwetzky, wirkungsprinzipien – indikationsbereiche – klinische handabung. prakt kieferorthop 1994; (8):11–22. 7. petrovic a, gasson n, oudet c. wirkung der übertriebenen posturalen vorschubsteilung des unterkiefers auf das kondylenwachstum der normalen und der mit wachstumhormonen behandelten ratte. fortsch kierorthop 1975; (36):86–97. 5555 research report dental journal (majalah kedokteran gigi) 2017 june; 50(2): 55–60 the efficacy of sarang semut extract (myrmecodia pendens merr & perry) in inhibiting porphyromonas gingivalis biofilm formation zulfan m. alibasyah,1 ambrosius purba,2 budi setiabudiawan,3 hendra dian adhita,4 dikdik kurnia,5 and mieke h. satari6 1department of periodontology, faculty of dentistry, universitas syiah kuala, banda acehindonesia 2department of physiology, faculty of medicine, universitas padjadjaran, bandung-indonesia 3department of child health, faculty of medicine, universitas padjadjaran, bandung-indonesia 4department of conservative dentistry, faculty of dentistry, universitas padjadjaran, bandung-indonesia 5department of chemistry, faculty of mathematics and natural science, universitas padjadjaran, bandungindonesia 4department of microbiology, faculty of dentistry, universitas padjadjaran, bandung-indonesia abstract background: porphyromonas gingivalis (p. gingivalis) is a pathogenic bacteria present in the oral cavity involved in the pathogenesis of chronic periodontitis and biofilm. this mass of microorganisms represents one of the virulent factors of p. gingivalis which plays an important role as an attachment initiator in host cells. sarang semut is a natural material possessing the ability to inhibit the growth of p. gingivalis. purpose: this study aims to analyze the effect of sarang semut extract on the formation of p. gingivalis biofilm. methods: the study used methanol sarang semut extract and p. gingivalis atcc 33277 and phosphomycin as a positive control. treatment was initiated by means of culturing. biofilm test and p. gingivalis biofilm formation observation were subsequently performed by means of a light microscope at a magnification of 400x. results: the formation of p. gingivalis biofilms tended to increase at 3, 6, and 9 hours. results of the violet crystal test showed that concentrations of 100% and 75% of the sarang semut extract successfully inhibited the formation of p. gingivalis biofilm according to the incubation time. meanwhile, the sarang semut extracts at concentrations of 50%, 25%, 12.5%, and 6.125% resulted in weak inhibition of the formation of p. gingivalis biofilm. the biofilm mass profile observed by a microscope tended to decrease as an indicator of the effects of the sarang semut extract. conclusion: sarang semut extract can inhibit the formation of p. gingivalis biofilm, especially at concentrations of 100% and 75%. nevertheless, phosphomycin has stronger antibiofilm of p. gingivalis effects than those of the sarang semut extract at all of the concentrations listed above. keywords: porphyromonas gingivalis; biofilm; sarang semut extract, phosphomycin correspondence: zulfan m. alibasyah, department of periodontology, faculty of dentistry, universitas syiah kuala. jln. teuku nyak arief darussalam, banda aceh, aceh, 23111, indonesia. e-mail: zulfanmalibasyah@gmail.com introduction in indonesia, the prevalence of periodontal disease across all age groups has been estimated at 96.58%.1 the main cause are gram-negative bacteria, such as porphyromonas gingivalis (p. gingivalis), also known as biofilm-forming bacteria, which demonstrate the ability to grow into a biofilm mass.2 this is positively correlated with their phenotypic characteristics as a cause of periodontitis.3 these anaerobic bacteria have been identified as the main orbital microbiota of biofilm formation in sub-gingiva, and contribute to the pathogenesis of root canal infection together with other bacteria facilitated by co-aggregation proteins.4 interactions of both proteins derived from these different pathogens with type 1 collagen of the host cell are considered to stimulate the pathogens to adhere, invade, and infect.5 there are a number of different antibiotics that have been used to eliminate the development of pathogens associated with periodontal disease, including that caused dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 mailto:zulfanmalibasyah@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 56 alibasyah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 55–60 by p. gingivalis. ripamfisin and phosphomycin are the most commonly used antibiotics to prevent infection of anaerobic group bacteria. however, the use of synthetic drugs and antibiotics tends to increase host antibody immunetolerance against pathogens.6 unfortunately, the long-term use of these drugs may result in bacteria becoming resistant. their consequent ability to grow within the mass of biofilms can protect these bacteria from detection by the body’s defense system, both specific and adaptive.7 on the other hand, polyphenol plants containing several active components (flavonoids, tannins, anthocyanins, phenolic acids, stilbenes, coumarins, lignans, and lignins) as antibacterial and antioxidant agents are greatly preferred as anti-bacterial materials by some pharmacologists. in addition to their antibacterial properties, polyphenols also act as antioxidants protecting a host’s defense system against pathogens, as well as non-toxic active components for mammalian cells to protect them from oxidative stress.8 moreover, the content of prenyl flavonoids may also inhibit expression or function of gingipains adherence, thus preventing p. gingivalis biofilm formation.9 our previous research even indicates that sarang semut extract proves highly effective in inhibiting the growth of enteroccus faecalis atcc 29212.10 unfortunately, the application of sarang semut extract on p. gingivalis biofilm formation has not been found in any of the references consulted. therefore, the study reported here aimed to determine the effectiveness of sarang semut extract against p. gingivalis biofilm ormation through comparison with phosphomycin. materials and methods this research was approved by the ethics review committee of the faculty of medicine, universitas padjadjaran, bandung, no. 557/un6.c1.3.2/kepk/ pn/2016. methanol sarang semut extract was obtained from the natural organic chemistry laboratory, department of chemistry, faculty of mathematics and natural sciences, universitas padjadjaran, bandung, indonesia. p. gingivalis atcc 33277 bacteria were drawn from the stock in the laboratory. the sarang semut extract was subsequently tested for its anti-biofilm potential against p. gingivalis, while phosphomycin (meiji, japan) was used as a positive control. sarang semut extract was analyzed in order to predict its bioactive component by means of a prediction of activity spectra for substances (pass) (pharmaexpert, moscow, russia) approach with a positive pass value ≥ 0.70.11 in general, the sarang semut extract had a pass value in excess of 0.70, confirming that flavonoids contained in the sarang semut both possessed a complexity value and met the standard value of phytochemical analysis.12 p. gingivalis atcc 33277 bacteria taken from the stock of glycerol-800c were, thereafter, cultured within a mueller-hinton agar (mha) medium (thermo fisher scientific inc, oxoid, uk) and incubated in an anaerobic atmosphere at 37 °c for 48 hours using an anaerocult® gaspack (merck, darmstadt, germany) aerobic jar. a colony of p. gingivalis bacteria was re-cultured in 5 ml of mueller-hinton broth (mhb) medium (thermo fisher scientific inc, oxoid, uk) at an anaerobic temperature of 37 °c, for 48 hours. p. gingivalis bacteria grown in the aqueous medium were further compared with mcfarland 0.5 (-1 x 108 cfu/ml) and used for biofilm testing. at that point, a biofilm test was performed on the basis of peeters’ working principle, modified by the use of violet crystals at several stages.13,14 each well on the micro plate (96-well plate) was coated with 100 μl of mhb for 15 minutes before being re-suspended. 100 μl of p. gingivalis bacteria was added and incubated for 5 minutes at room temperature. the supernatant then having been removed, 100 μl of the test material was added to each well on the micro plate at concentrations of 100%, 75%, 50%, 25%, 12.5%, and 6.125% (μg/ml). they were further cultured in an anaerobic atmosphere with incubation time periods of 3, 6, and 9 hours using an anaerocult® gaspack (merck, darmstadt, germany). 100 μl of phosphate buffer saline (pbs) (merck, darmstadt, germany) was added to each well twice and then agitated for 10 minutes at 300 rpm. 100 μl of 2% violet crystals was introduced into each well and then incubated for 10 min at 250 rpm. the crystals were washed with pbs twice for 10 minutes above the sheker. the extraction of violet crystals from microorganisms was effected by adding 100 μl of 98% ethanol prior to agitating the solution for 5 min at 300 rpm.15 optical density (od) of serial duplo biofilm was then measured with an elisa reader (bio-rad laboratories inc., ca, usa) using a wavelength of 590 nm. the resultant biofilm mass of p. gingivalis bacteria formed on each well base after interaction with the sarang semut extract was prepared with 100 μl of glycerol for 24 hours to maintain its moisture level. biofilm mass visualization was then performed by adding 10 μl of emersile oil (thermo fisher scientific inc, oxoid, uk) to each cell well plate before observation was carried out under a light microscope (olympus, shinjuku, tokyo, japan) at a magnification of 400x.16 results p. gingivalis has been ability a biofilm formation that is strongly in 9 hours and will be decreased in 6 hours (figure 1). while, the extract of sarang semut has the capability to inhibitory the biofilm formation of p. gingivalis in 9 hours compared 3 hours and 6 hours, specifically in concentration of 100%, 75%, and 50% (μg/ml), nonetheless the fosfomycin has been strongest biofilm inhibit of e-faecalis compared sarang semut extract (figure 2). figure 3 had shown the mass of biofilm formation of p. gingivalis dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 5757alibasyah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 55–60 on the micro-plate 96-well before and after treated by the material of assay. discussion p. gingivalis bacteria represent anaerobic gram-negative bacteria contributing to the pathogenesis of periodontitis with biofilm as one virulent factor determinant.2 anaerobic bacteria contribute to colonization, adhesion, and penetration activities in host cells.17 the formation of p. gingivalis biofilm in this research was evaluated by the use of violet crystals.18 the results of the biofilm test, illustrated in figures 1 and 2 showing p. gingivalis biofilms which had formed, were observed at the end of 3, 6, and 9-hours incubation periods and analyzed on the basis of an absorbance value at a wavelength of 590 nm. as shown in figure 2, the inhibitory effects of the biofilm formation in all threetreatment groups were equally effective. during the initial 6-hours incubation period, the biofilm mass of p. gingivalis diminished, although it increased during the following incubation period lasting 9 hours (figure 1). similarly, the inhibitory effects of the biofilm formation in the sarang semut extract at the highest to lowest concentration tended to increase after the 9-hours incubation period. fluctuation in such effects is closely related to the biofilm formation phases (initiation, adhesion, and maturation) which indicate gradation in the intensity of the biofilm formation.19 the high or low expression of biofilm proteins by each pathogen is highly dependent upon environmental influences and communal formation intensity with other bacteria.20 although this research evaluated only the formation of p. gingivalis biofilm as monospesies, a decrease in potential biofilm formation of p. gingivalis was assumed to be related to environmental changes (in wells), such as changes in the ph of the medium due to the introduction of test material that may have affected the metabolism of p. gingivalis biofilm mass.21 environmental factors, such as ph, temperature, cytokines, hormones, and oxidative stress have an effect on the formation of bacterial biofilms, including p. gingivalis, in the pathogenesis of periodontal infection. specifically, changes in temperature could improve attachment, coaggregation, and production of protease.22 alkaline ph (8.2) may increase hydrophobicity potentially inducing co-adhesion and biofilm formation of p. gingivalis.23 from the results of this research (figure 1), it could be assumed that during this 6-hours period bacteria would pass through the adaptation phase (the first maturation stage) with the o pt ic al d en si ty o f b io fil m ( 59 0 nm ) concentration of sarang semut extract (µg/ml) 3 hours 6 hours 9 hours 100% 75% 50% 25% 12,50% 6,125% phosphomycin 2,500 2,000 1,500 1,000 0,500 0,000 figure 2. the inhibitory effect of the sarang semut extract on p. gingivalis biofilm formation compared to that of phosphomycin. figure 1. the biofilm formation activity of p gingivalis based on the incubation periods of 3, 6, and 9 hours. 3 hours 1,191 0,057 3 hours 0,629 0,045 3 hours 2,076 0,058 2,500 1,500 2,000 1,000 0,500 0,000 porphyromonas gingivalis (cfu/ml) p. gingivalis medium 6 hours 9 hourso pt ic al d en si ty o f b io fil m ( 59 0 nm ) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 58 alibasyah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 55–60 environment so that the biofilm protein expression activity might be interrupted before entering the second maturation phase.24 the time frame used to observe the tendency of biofilm formation activity is related to the initiation, adaptation, and maturation phases.25 the results showed that during the initiation phase (the first observation period of 3 hours) od value increased, indicating the presence of p. gingivalis activity associated with potential biofilm formation. on the elapsing of the second observation period of 6 hours (the adaptation phase), the od value had decreased, although the intensity then increased again after the biofilm had reached the maturation phase (the third observation period of 9 hours). the adaptation phase is included in the initial facula of biofilm formation by a number of pathogens or bacteria.26 the decrease in p. gingivalis biofilm formation in this phase is related to the change in properties to interact with the target (host cell) or communication properties with other pathogens to form co-adhesion and co-aggregation.27 as a result, it is possible that the production activity of the biofilm protein ceases before passing through the maturation phase. therefore, the increased biofilm formation in the maturation phase (9 hours) can potentially be triggered by the increase in co-adhesion and co-aggregation activities of bacteria which is in line with the augmented biofilm formation as a link between the two activities.28,29 unlike these previous researchs, research conducted by davey et al. used the period of 48 hours as the maximum indicator of p. gingivalis biofilm formation.25 similarly, research conducted by martin observed p. gingivalis biofilm formation over varying time periods of 3, 24, 48, and 72 hours. the study identified an increasing trend in the biofilm formation of p. gingivalis between 3 hours to 20 hours, followed by a decrease after 24, 48, and 72 hours.30 meanwhile, research conducted by yamamoto used longer periods (3, 6, 9 and 14 days) to model visually observed biofilm formation of p. gingivalis with a confocal laser scanning microscopy (clsm). this study revealed that the maximum activity of biofilm formation occurred at 14 days, confirming that time becomes a determinant factor in the development of p. gingivalis biofilm formation.3 based on figures 1 and 2, p. gingivalis bacteria were shown to be capable of forming a stronger biofilm. however, the formation of this bacterial biofilm was inhibited by about 14% after the administration of the sarang semut extract at a concentration of 100% during an incubation period lasting nine hours. in general, figure 2 shows that phosphomycin had a considerably stronger inhibitory effect on p. gingivalis biofilm formation than the sarang semut extract at all concentrations. however, compared to concentrations of 50%, 25%, 12.5% and 6.125%, the sarang semut extract at concentrations of 75% and 100% produced better inhibitory effects, especially at 9 hours. it can be argued that the active component of the sarang semut extract (flavonoid) interacts with nitrogenfixing bacteria facilitated by plant bind. nodd protein also binds to nod-factor receptors (nr), thus damaging the bacterial cell flagella31 where several biofilm proteins and gram-positive and negative bacterial adhesion proteins are located. meanwhile, phosphomycin is able to destroy the bacterial cell surface proteins and also prevents the interaction between bacterial cell fimbriae proteins and extra cellular cell matrix proteins.32 the advantage of phosphomycin is that it disrupts cytoplasmic activity in peptidoglycan biosynthesis as well as inhibits the synthesis of the mura enzyme that attaches to host cells.33 therefore, phosphomycin may act as a potent anti-biofilm to the micro plate since they are 5 figure 2. the inhibitory effect of the sarang semut extract on p. gingivalis biofilm formation compared to that of phosphomycin. figure 3. p. gingivalis biofilm mass without the administration of the sarang semut extract as control groups (on paths a, e, i). p. gingivalis biofilm mass with the administration of the sarang semut extract on paths b, f, j (with a 3-hour incubation period), on paths c, g, k (with a 6-hour incubation period), and on paths d, h, l (with a 9-hour incubation period). in this research, the sarang semut extract was at concentrations of 100% (on path b, c, d) and 75% (on path f, g, h, i). meanwhile, phosphomycin was used on path j, k, l. discussion p. gingivalis bacteria represent anaerobic gram-negative bacteria contributing to the pathogenesis of periodontitis with biofilm as one virulent factor determinant.2 anaerobic bacteria contribute to colonization, adhesion, and penetration activities in host cells.17 the formation of p. gingivalis biofilm in this research was evaluated by the use of violet crystals as performed by saito18 and bachtiar.15 the results of the biofilm test, illustrated in figures 1 and 2 showing p. gingivalis biofilms which had formed, were observed at the end of 3, 6, and 9-hour incubation periods and, then, analyzed on the basis of an absorbance value at a wavelength of 590 nm. as shown in figure 2, the inhibitory effects of the biofilm formation in all three-treatment groups was equally effective. during the initial 6-hour incubation period, the biofilm mass of p. gingivalis diminished, although it increased during the following incubation period lasting 9 hours (figure 1). similarly, the inhibitory effects of the biofilm formation in the sarang semut extract at the highest to lowest concentration tended to increase after the 9-hour incubation period. fluctuation in such effects is figure 3. p. gingivalis biofilm mass without the administration of the sarang semut extract as control groups (on paths a, e, i). p. gingivalis biofilm mass with the administration of the sarang semut extract on paths b, f, j (with a 3-hours incubation period), on paths c, g, k (with a 6-hoursincubation period), and on paths d, h, l (with a 9-hours incubation period). in this research, the sarang semut extract was at concentrations of 100% (on path b, c, d) and 75% (on path f, g, h, i). meanwhile, phosphomycin was used on path j, k, l. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 5959alibasyah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 55–60 capable of forming a covalent bond to activate cysteine residue of bacterial cells which, in turn, activate udpn-acetyl glucosamine to form hydrogen bonds to inhibit peptidoglycan synthesis constituting an antibacterial defense site.34 figure 2 shows that the ability of the sarang semut extract as an anti-biofilm of p. gingivalis remained limited. moreover, as shown in figure 3, the sarang semut extract at the two highest concentrations (100% and 75%) was not yet effective in inhibiting the biofilm mass on the base of the micro plate. this indicates that the sarang semut extract components were still less effective as an anti-biofilm of p. gingivalis. it may also be assumed that bacteria (p. gingivalis) growing in the mass of biofilm demonstrate metabolic activity triggering resistance to antibiofilm35 which, in case of this research, is the sarang semut extract. in other words, this extract was not able to penetrate the mass of biofilms while continuously forming during the 3, 6, and 9-hour incubation periods. in general, the results showed that sarang semut extract possessed the ability to inhibit p. gingivalis biofilm formation since some of its active components, such as flavonoids and bioflavonoid, are assumed to be able to inhibit bacterial cell protein synthesis. secreted cysteine proteases and some proteins classified into the gingipains protein group,3 for example, fima protein facilitate bonding with epithelial cells through interactions with extra cellular matrix proteins such as fibrinogen, fibronectin, and collagen type i.36 finally, it can be concluded that sarang semut extract (myrmecodia pendens merr & perry) exerts an inhibitory effect on p. gingivalis biofilm formation, especially at high concentrations (100% and 75%), such effect remains far lower compared to that of phosphomycin. acknowledgement we would like to extend our sincere gratitude to the laboratory of natural organic chemistry, department of chemistry, faculty of mathematics and natural sciences, universitas padjadjaran, bandung for providing sarang semut extract (myrmecodia pendens merr & perry). references 1. casanova l, hughes fj, preshaw pm. diabetes and periodontal disease: a two-way relationship. br dent j. 2014; 217(8): 433–7. 2. bostanci n, belibasakis gn. porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen. fems microbiol lett. 2012; 333(1): 1–9. 3. darveau rp. periodontitis: a polymicrobial disruption of host homeostasis. nat rev microbiol. 2010; 8(7): 481–90. 4. ma rsh pd, moter a, devi ne da. dent a l plaque biof il ms: communities, conflict and control. periodontol 2000. 2011; 55(1): 16–35. 5. amano a. bacterial adhesins to host components in periodontitis. periodontol 2000. 2010; 52(1): 12–37. 6. becattini s, taur y, pamer eg. antibiotic-induced changes in the intestinal microbiota and disease. trends mol med. 2016; 22(6): 458–78. 7. costalonga m, herzberg mc. the oral microbiome and the immunobiology of periodontal disease and caries. immunol lett. 2014; 162(2): 22–38. 8. gülçin i̇. antioxidant activity of food constituents: an overview. arch toxicol. 2012; 86(3): 345–91. 9. kariu t, nakao r, ikeda t, nakashima k, potempa j, imamura t. inhibition of gingipains and porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids. j periodontal res. 2017; 52(1): 89–96. 10. soraya c, dharsono hda, aripin d, satari mh, kurnia d, hilmanto d. effects of sarang semut (myrmecodia pendens merr. & perry) extracts on enterococcus faecalis sensitivity. dent j (maj ked gigi). 2016; 49(4): 175–80. 11. clsi. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 9th ed. wayne pa, editor. vol. 32, m07-a9. usa: clinical and laboratory standards institute; 2012. p. 1-5. 12. aliyu mm, musa ai, kamal mj, mohammed mg. phytochemical screening and anticonvulsant studies of ethyl acetate fraction of globimetula braunii on laboratory animals. asian pac j trop biomed. 2014; 4(4): 285–9. 13. brackman g, cos p, maes l, nelis hj, coenye t. quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. antimicrob agents chemother. 2011; 55(6): 2655–61. 14. bridier a, dubois-brissonnet f, boubetra a, thomas v, briandet r. the biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput clsm method. j microbiol methods. 2010; 82(1): 64–70. 15. bachtiar ew, bachtiar bm, dewiyani s, surono akbar sm. enterococcus faecalis with capsule polysaccharides type 2 and biofilm-forming capacity in indonesians requiring endodontic treatment. j investig clin dent. 2015; 6(3): 197–205. 16. heuker m, gomes a, van dijl jm, van dam gm, friedrich aw, sinha b, van oosten m. preclinical studies and prospective clinical applications for bacteria-targeted imaging: the future is bright. clin transl imaging. 2016; 4(4): 253–64. 17. ashida h, ogawa m, kim m, mimuro h, sasakawa c. bacteria and host interactions in the gut epithelial barrier. nat chem biol. 2011; 8(1): 36–45. 18. sabrah aha, yassen gh, gregory rl. effectiveness of antibiotic medicaments against biofilm formation of enterococcus faecalis and porphyromonas gingivalis. j endod. 2013; 39(11): 1385–9. 19. kuboniwa m, lamont rj. subgingival biofilm formation. periodontol 2000. 2010; 52(1): 38–52. 20. kreth j, herzberg mc. molecular principles of adhesion and biofilm formation. in: chávez de paz le, sedgley cm, kishen a, editors. the root canal biofilm. berlin, heidelberg: springer; 2015. p. 23–53. 21. schlafer s, ibsen cjs, birkedal h, nyvad b. calcium-phosphateosteopontin particles reduce biofilm formation and ph drops in in situ grown dental biofilms. caries res. 2017; 51(1): 26–33. 22. pöllänen m, paino a, ihalin r. environmental stimuli shape biofilm formation and the virulence of periodontal pathogens. int j mol sci. 2013; 14(8): 17221–37. 23. park jh, lee j-k, um h-s, chang b-s, lee s-y. a periodontitisassociated multispecies model of an oral biofilm. j periodontal implant sci. 2014; 44(2): 79–84. 24. shrout jd, chopp dl, just cl, hentzer m, givskov m, parsek mr. the impact of quorum sensing and swarming motility on pseudomonas aeruginosa biofilm formation is nutritionally conditional. mol microbiol. 2006; 62(5): 1264–77. 25. davey me. techniques for the growth of porphyromonas gingivalis biofilms. periodontol 2000. 2006; 42(1): 27–35. 26. van acker h, coenye t. the role of efflux and physiological adaptation in biofilm tolerance and resistance. j biol chem. 2016; 291(24): 12565–72. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 60 alibasyah, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 55–60 27. marsh pd. dental plaque as a microbial biofilm. caries res. 2004; 38(3): 204–11. 28. bao k, belibasakis gn, thurnheer t, aduse-opoku j, curtis m a, bostanci n. role of porphyromonas gingivalis gingipains in multispecies biofilm formation. bmc microbiol. 2014; 14(1): 1–8. 29. teles fr, teles rp, uzel ng, song xq, torresyap g, socransky ss, haffajee ad. early microbial succession in redeveloping dental biofilms in periodontal health and disease. j periodontal res. 2012; 47(1): 95–104. 30. martin b, tamanai-shacoori z, bronsard j, ginguené f, meuric v, mahé f, bonnaure-mallet m. a new mathematical model of bacterial interactions in two-species oral biofilms. amar s, editor. plos one. 2017; 12(3): 1–24. 31. coll rc, o’neill laj. new insights into the regulation of signalling by toll-like receptors and nod-like receptors. j innate immun. 2010; 2(5): 406–21. 32. wakabayashi h, kondo i, kobayashi t, yamauchi k, toida t, iwatsuki k, yoshie h. periodontitis, periodontopathic bacteria and lactoferrin. biometals. 2010; 23(3): 419–24. 33. olesen sh, ingles dj, yang y, schönbr unn e. differential antibacterial properties of the mura inhibitors terreic acid and fosfomycin. j basic microbiol. 2014; 54(4): 322–6. 34. furchtgott l, wingreen ns, huang kc. mechanisms for maintaining cell shape in rod-shaped gram-negative bacteria. mol microbiol. 2011; 81(2): 340–53. 35. jakubovics ns, kolenbrander pe. the road to ruin: the formation of disease-associated oral biofilms. oral dis. 2010; 16(8): 729–39. 36. chagnot c, listrat a, astruc t, desvaux m. bacterial adhesion to animal tissues: protein determinants for recognition of extracellular matrix components. cell microbiol. 2012; 14(11): 1687–96. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p55-60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p55-60 6363 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 63–67 original article the effectiveness of the combination of moringa oleifera extract and propolis on porphyromonas gingivalis biofilms compared to 0.7% tetracycline hansen kurniawan, widyastuti and mery esterlita hutapea department of periodontics, faculty of dentistry, universitas hang tuah, surabaya – indonesia abstract background: periodontitis is an inflammatory disease that occurs in periodontal tissues. porphyromonas gingivalis is also known as a bacterium commonly associated with the pathogenesis of periodontitis. tetracycline is one of the antibiotics often used in periodontal tissue treatment. propolis and moringa oleifera are also known to have certain compounds assumed to be able to inhibit biofilm growth. purpose: this study aims to understand the effectiveness of the combination of moringa oleifera and propolis on porphyomonas gingivalis biofilms compared to 0.7% tetracycline. methods: a biofilm inhibition activity test was performed using the broth micro dilution method. first, bacteria were prepared by making a suspension in brain heart infusion media and adjusting it to 0.5 mcfarland i standard. second, fifteen samples were divided into five groups; group k as control group (0.1% sodium carboxymethyl cellulose), t (0.7% tetracycline), and treatment groups with the combination of propolis and moringa oleifera in various concentrations, such as p1(10%+20%), p2(10%+40%), and p3(10%+80%). third, the result data obtained in the form of optical density (od) was read by using an elisa reader. next, statistical analysis using analysis of the variance test was conducted (p<0.05. results: there was no significant difference between group t and group p1 (0.075). nevertheless, there were significant differences between group t and group p2 as well as between group t and group p3 (0.00) (p=< 0.05). conclusion: the combination of 10% propolis and 40% moringa oleifera as well as the combination of 10% propolis and 80% moringa oleifera have better antibacterial effectiveness against porphyromonas gingivalis biofilm than 0.7% tetracycline. keywords: biofilm; moringa oleifera; p. gingivalis; propolis; tetracycline correspondence: hansen kurniawan, department of periodontics, faculty of dentistry, universitas hang tuah. jl. arif rahman hakim no. 150 surabaya, 60111 indonesia. email: hansen.kurniawan@hangtuah.ac.id introduction periodontitis is an inflammatory disease that occurs in the supporting tissues of teeth caused by specific microorganisms triggering inflammation on periodontal ligament and alveolar bone.1 plaque and calculus bacteria are considered to be the main causes of periodontal disease. plaque containing pathogenic microorganisms plays an important role in causing damage to the periodontal tissue.2 porphyromonas gingivalis (p. gingivalis), moreover, is a bacterium commonly associated with periodontitis pathogenesis. almost 40–100% of periodontitis cases are caused by the opportunistic bacterial antigen of p. gingivalis. the presence of these bacteria in chronic periodontitis patients, according to a previous research on subgingival plaque as much as 85.75%.3 furthermore, biofilm is a collection of microorganisms in which microbial cells attach to each other on a living or non-living surface, producing their own extracellular polymer matrix.4 biofilm consists of bacterial cells (5–25%) and glycocalyx matrix (75–95%).5 in addition, biofilm is known as a microbial cell encased in a matrix of extracellular polymeric substances, such as polysaccharides, proteins and nucleic acids.6 in the lower layer of biofilm, microbes are bound together in a polysaccharide matrix with other dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p63–67 mailto:hansen.kurniawan@hangtuah.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p63-67 64 kurniawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 63–67 organic and inorganic materials, while on the top layer, there is an amorphous layer extending to the medium around it. the fluid layer adjacent to the biofilm has a stationary and dynamic sub-layer.7 biofilm is also known to have an extracellular polymeric substance (eps) matrix that is able to prevent the occurrence of antimicrobial diffusion, and, as a result, bacterial resistance to antibiotics increases while cell metabolic activity on the biofilm decreases.8,9 on the other hand, moringa oleifera, also known as moringa, is a plant widely used as a traditional medicine or for mystical therapy in indonesia.10 moringa oleifera is also known to have anti-cancer, antibacterial and hypotensive properties that can inhibit bacterial and fungal activities.11 according to septiyani et al.12 moringa oleifera extract can be effective in inhibiting the growth of p. gingivalis bacteria at doses of 80% and 40%. similarly, propolis, according to previous research, has natural antibacterial and antibiotic properties.13 propolis, one of the natural products produced by honey bees, has been widely used not only as a medicine or supplement, mouth wash, anti-inflammatory and disease therapy, but also for accelerating the wound-healing process. in addition, propolis has many special benefits and potential since it has antibacterial and antiviral properties, so it can inhibit cancer growth.14,15 according to suryono et al.16 10% propolis even has an ability to inhibit p. gingivalis bacterial activities optimally. tetracycline is one of the oldest classes of broadspectrum antibiotics in use. in addition to its antibacterial activity, tetracyclines are also known to have antiinflammatory, anti-collagenase and wound healing properties, as well as the ability to reduce bone loss.17 tetracyclines, consequently, can be used as antibiotics for periodontitis cases. the biocompatibility of tetracyclines, mostly studied in the form of tetracycline gel with a concentration of 0.7%, is acceptable for tissue and can remove the smir layer and open dentinal tubules and the collagen matrix.18 finally, it can be said that propolis and moringa oleifera contain different secondary metabolites that have anti-bacterial activity. meanwhile, tetracycline is one of the antibiotics that has been widely used for the therapy of periodontal disease. however, whether the combination of propolis and moringa oleifera will be as effective as tetracyclines and whether propolis and moringa oleifera have side effects is still debatable. therefore, this research aims to explore the antibacterial effectiveness of the combination of propolis extract and moringa oleifera leaf extract against p. gingivalis biofilms compared to 0.7% tetracycline. materials and methods this research is a true laboratory experimental research with the post only control group design. in this research, moringa oleifera extract was prepared with a maceration technique, in which 100 g of moringa oleifera simplisa powder was put into erlernmeyer, immersed in 500 ml of 96% ethanol solution and covered with aluminium foil for five days, while being stirred occasionally. after five days, the sample was filtered using filter paper to produce filtrate 1 and residue 1. the residue was then added to 250 ml of 96% ethanol solution, covered with aluminium foil for two days, being stirred occasionally.11 after two days, the sample was filtered using filter paper to produce filtrate 2 and residue 2, then mixed into one and evaporated using a rotary evaporator to obtain moringa oleifera extract. the moringa oleifera extract obtained was kept in a water bath until all the ethanol solvent evaporated. the extract obtained was then weighed and stored in a closed glass container before used for testing. in total there were three test solutions made, namely moringa oleifera extract at a concentration of 20% (0.2:0.8), moringa oleifera extract at a concentration of 40% (0.4:0.8), and moringa oleifera extract at a concentration of 80% (0.8:0.2).11 afterwards, 800 g of propolis was cooled in a refrigerator, then put in an oven at 40°c for three days, and 2l of 70% ethanol liquid was added. to speed up the dissolving process, propolis was crushed with a stirrer and allowed to stand for a moment.18 after that, the propolis was stirred every day and filtered, but the obtained filtrate was left to stand to precipitate substances that were not needed, but not dissolved in ethanol. the remaining filtrate was then put into a 90% ethanol solution. all of these steps were repeated three to five times, and the filtering process was also repeated three times until a thick propolis extract was obtained. next, 0.1 g of the thick propolis extract was dissolved in 1 ml of 0.1% sodium carboxymethyl cellulose (na cmc) so that the concentration of the propolis extract obtained became 10%.19 subsequently, 0.7% tetracycline was prepared. first, tetracycline tablets were crushed. afterwards, 175 mg of the crushed tetracycline was dissolved in 25 ml of aquadest.20 next, moringa oleifera extract and propolis at various concentrations were mixed in a ratio of 1:1. meanwhile, 0.1% na cmc solution was divided into several groups. first, in the control group (k), 0.1 ml of p. gingivalis biofilm mixed with 0.1 ml of 0.1% na cmc was put on a microtiter plate. second, in the tetracycline (t) group, 0.1 ml of p. gingivalis biofilm mixed with 0.1 ml of 0.7% tetracycline was put on a microtiter plate. third, in treatment group 1 (p1), 10% propolis extract mixed with 0.1 ml of 20% moringa oleifera leaf extract was put on a microtiter plate. fourth, in treatment group 2 (p2), 10% propolis extract mixed with 0.1 ml of 40% moringa oleifera leaf extract was put on a microtiter plate. and, in treatment group 3 (p3), 10% propolis mixed with 0.1 ml of 80% moringa oleifera leaf extract was put on a microtiter plate. after that, a detection test for the formation of p. gingivalis bacterial biofilm was carried out using the congo red agar (cra) method. first, p. gingivalis bacteria were inoculated on congo red agar and incubated for 48 hours at dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p63–67 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p63-67 65kurniawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 63–67 37°c under anaerobic conditions. congo red agar that can interact directly with polysacharida intercellular adhesion formed a colour complex. after 48 hours, the plates were examined. if black colonies were formed, this indicated that the bacterial strain had produced biofilms. meanwhile, if the colony showed a red colour, this illustrated that the strain had not formed any biofilm.21 in addition, a biofilm inhibition test was conducted by using the broth micro dilution method. first, bacteria were prepared by making a suspension in a brain heart infusion (bhi) media and adjusted to 0.5 mcfarland i standard. second, the equalized suspension of p. gingivalis bacteria was then diluted to a ratio of 1:100. third, the bacterial suspension was inserted into a 96-well round bottomed plastic tissue culture plate (microtiter plate) with a total volume of 0.1 ml (100 µl)/ in each well using a micropipette. fourth, observation was conducted on each test plate and each blank plate. fifth, the test plate filled with a bacterial suspension was put on microtiter plate and then incubated at 37°c for 24 hours. sixth, after 24 hours, the microtiter plate was removed from the incubator, and then the test extract solution was inserted into the microtiter plate filled with bacterial suspension, while the blank plate was filled with the test extract solution without bacteria. seventh, the microtiter plate was put back into the incubator for 24 hours.22 eighth, the microtiter plate was removed from the incubator, and then the test solution was discarded and washed three times with 0.2 ml of phosphate buffer saline. ninth, the microtiter plate was dried, and then 0.2 ml (200 µl) of 1% crystal violet was added to each well and left to stand for 15 minutes. tenth, it was rinsed using distilled water and then dried for 15 minutes in an incubator at 37°c. in the last step, 0.2 ml of 2% 80 tween was added to each microtiter plate.22 next, an optical density (od) test was performed. the values of density (od) values obtained and were read using an elisa reader at a wavelength of 515 nm. afterwards, the inhibitory power of biofilm formation generated from the test solution was measured by the following formula: note: ods : optical density (515 nm) of tested sample odbs : optical density of blank sample odp : (od of test solvent od of blank solvent) the reading results of the optical density values obtained with the elisa reader were in the form of quantitative data. the data were then tested using the one-way analysis of variance (anova) test. results the research data were analysed descriptively to depict the distribution of data as well as summarise the characteristics of data in order to clarify the presentation of the data results. the data shown in table 1 illustrates that the highest mean percentage of p. gingivalis biofilm inhibition was in the p3 group, with a value of 72.4, while the lowest mean percentage was in the k group, with a value of 0. moreover, it also demonstrates that the mean percentage of p. gingivalis biofilm inhibition in the t group was 31.8, 36.4 in the p1 group, and 57.8 in the p2 group. based on table 2, the results of the lsd test showed that there was a significant difference in the percentage value of biofilm inhibition (p <0.05) in all groups, except between t and p1 (0.075> 0.05). discussion the prevalence and severity of periodontal disease can be reduced by scaling root planning and antibacterial therapy. the antibacterial therapy often used to treat periodontal disease is tetracycline. hence, this study aims to see the effect of moringa oleifera extract at concentrations of 20%, 40% and 80% combined with 10% propolis on the biofilm formation of p. gingivalis bacteria strain atcc 33277, compared with 0.7% tetracycline. moringa oleifera is known to have antibacterial ability in p. gingivalis at concentrations of 40% and 80%.12 similarly, propolis is also known to have antibacterial ability against p. gingivalis bacteria.23 the inhibition ability of moringa oleifera is known to be caused by a variety of active metabolic compounds it contains. not only does moringa oleifera contain great antibacterial properties consisting of saponins, triterpenoids and tannins, which work by damaging cells from bacteria, but also flavonoids as antioxidants playing a role in damaging the permeability of bacterial cell walls, microsomes and lysosomes as a result of the interaction between flavonoids and bacterial dna.24 table 1. the average (%) of p. gingivalis bacterial biofilm inhibition group mean (%) std. deviation k 0 0 t 31.8 3.76051 p1 36.4 0.71531 p2 57.8 3.40274 p3 72.4 2.10350 table 2. the results of lsd post-hoc test group t p1 p2 p3 k 0.000* 0.000* 0.000* 0.000* t 0.075 0.000* 0.000* p1 0.000* 0.000* p2 0.000* * p <0.05 (there are differences) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p63–67 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p63-67 66 kurniawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 63–67 propolis, on the other hand, is a natural material collected by honey bees from various plants, such as poplar, palm, pine, coniferous secretions, sap, resin, mucus and leaf buds. it is collected and carried by honey bees and used to seal the gaps in their hives. initially, it acts as an antiseptic that prevents the beehive from microbial infection as well as the decomposition of intruders.25 propolis is known to have antibacterial, anti-viral, anti-fungal, antioxidant and antiinflammatory functions that enhance the body’s system.26 propolis is also known to contain amino acids, terpenoids and polyphenols (phenolic acids, esters and flavonoids).27 flavonoid is one of the important ingredients in propolis, and has antioxidant, anti-cancer, anti-inflammatory, allergy, antiviral and antibacterial effects.28–30 hence, flavonoids can eliminate the permeability of p. gingivalis bacterial cells.31 flavonoids can damage bacterial cells by lysing bacteria through protein binding so that bacteria will die.32 according to the study of asdar and cindrakori, propolis contains apigenin and tt-farnesol which can decrease the amount of polysaccharides in the biofilm of microorganisms, so the biofilm formation can be stopped.33 previous research conducted by septiyani et al.12 shows that moringa oleifera extract at concentrations of 40% and 80% was able to reduce the biofilm formation of p. gingivalis bacteria. this previous research also reveals that 20% moringa oleifera extract combined with 10% propolis even can inhibit the biofilm formation of p. gingivalis bacteria. therefore, it can be said that based on this previous research, the combination of 10% propolis extract and 20% moringa oleifera extract can be used for an alternative antimicrobial treatment as effective as 0.7% tetracycline. meanwhile, the combination of 10% propolis and 40% moringa oleifera as well as the combination of 10% propolis and 80% moringa oleifera can be more effective than tetracycline 0.7%. hence, two materials can be used synergistically to inhibit the biofilm formation of p. gingivalis bacteria. finally, it can be concluded that the combination of propolis extract and moringa oleifera can generate antibacterial power to inhibit the biofilm formation of p. gingivalis bacteria. it is also known that 10% propolis and 20% moringa oleifera have antibacterial power as great as 0.7% tetracycline. nevertheless, the combination of 10% propolis and moringa oleifera at concentrations of 40% and 80% has greater antibacterial power than 0.7% tetracycline. besides, propolis and moringa oleifera contain natural ingredients, so they have lower cytotoxicity than tetracyclines. thus, it can be said the combination of propolis and moringa oleifera is better than tetracyclines. references 1. newman m, takei h, klokkevold p, carranza f. newman and carranza’s clinical periodontology. 13th ed. philadelphia: elsevier saunders; 2018. p. 346–7. 2. quamilla n. stres dan kejadian periodontitis (kajian literatur). j syiah kuala dent soc. 2016; 1(2): 161–8. 3. putri cf, bachtiar ew. porphyromonas gingivalis and pathogenesis of cognitive disfunction: role of neuroinf lammation cytokine (literature review). cakradonya dent j. 2020; 12(1): 15–23. 4. jamal m, tasneem u, hussain t, andleeb s. bacterial biofilm: its composition, formation and role in human infections. res rev j microbiol biotechnol. 2015; 4(3): 1–14. 5. huang r, li m, gregory rl. bacterial interactions in dental biofilm. virulence. 2011; 2(5): 435–44. 6. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. philadelphia: elsevier saunders; 2015. p. 1779. 7. chandki r, banthia p, banthia r. biofilms: a microbial home. j indian soc periodontol. 2011; 15(2): 111–4. 8. gerits e, verstraeten n, michiels j. new approaches to combat porphyromonas gingivalis biofilms. j oral microbiol. 2017; 9(1): 1300366. 9. chadha t. bacterial biofilms: survival mechanisms and antibiotic resistance. j bacteriol parasitol. 2014; 5(3): 190. 10. rockwood jl, anderson bg, casamatta da. potential uses of moringa oleifera and an examination of antibiotic efficacy conferred by m. oleifera seed and leaf extracts using crude extraction techniques available to underserved indigenous populations. int j phytothearpy res. 2013; 3(2): 61–71. 11. dima llrh, fatimawali f, lolo wa. uji aktivitas antibakteri ekst ra k daun kelor ( mor inga oleifera l.) terhadap ba k ter i escherichia coli dan staphylococcus aureus. pharmacon. 2016; 5(2): 282–9. 12. septiyani ri. efektivitas ekstrak daun kelor (moringa oleifera l.) dalam menghambat pertumbuhan bakteri porphyromonas gingivalis. thesis. universitas muhammadiyah semarang: semarang; 2019. p. 70–1. 13. kurniawati d. uji aktivitas antibakteri propolis trigona spp. asal bukit tinggi pada tikus putih sprague-dawle. j prog kim sains. 2011; 1(1): 25–31. 14. salatino a, fernandes-silva cc, righi aa, salatino mlf. propolis research and the chemistry of plant products. nat prod rep. 2011; 28(5): 925–36. 15. milah n, bintari sh, mustikaningtyas d. pengaruh konsentrasi antibakteri propolis terhadap pertumbuhan bakteri streptococcus pyogenes secara in vitro. life sci. 2016; 5(2): 95–9. 16. suryono s, kusumawati i, devitaningtyas n, sukmawati a, wijayanti p. characteristic assay of incorporation of carbonated hydroxyapatite–propolis as an alternative for alveolar bone loss therapy on periodontitis: an in vitro study. j int oral heal. 2020; 12(5): 463–9. 17. vagish kumar ls. pharmaceuticals: tetracyclines and periodontal disease. br dent j. 2015; 218: 213. 18. sri pradnyani iga. tetrasiklin hcl gel 0,7% meningkatkan jumlah sel fibroblas dan mempertebal ligamen periodontal pada sulkus gingiva tikus yang mengalami periodontitis. intisari sains medis. 2017; 8(1): 14–8. 19. gani a, sawal nw. daya hambat ekstrak propolis trigona sp terhadap pertumbuhan bakteri aggregatibacter actinomycetemcomitans. thesis. makassar: universitas hasanuddin; 2013. p. 1–8. 20. perayil j, menon ks, biswas r, fenol a, vyloppillil r. comparison of the efficacy of subgingival irrigation with 2% povidone-iodine and tetracycline hcl in subjects with chronic moderate periodontitis: a clinico microbiological study. dent res j (isfahan). 2016; 13(2): 98–109. 21. purbowati r, devi e, rianti d, ama f. kemampuan pembentukan slime pada staphylococcus epidermidis, staphylococcus aureus, mrsa dan escherichia coli. j florea. 2017; 4(2): 1–9. 22. o’toole ga. microtiter dish biofilm formation assay. j vis exp. 2011; (47): 2437. 23. amanda ea, oktiani bw, panjaitan fua. efektivitas antibakteri ekstrak flavonoid propolis trigona sp (trigona thorasica) terhadap pertumbuhan bakteri porphyromonas gingivalis. dentin j kedokt gigi. 2019; 3(1): 23–8. 24. widowati i, efiyati s, wahyuningtyas s. uji aktivitas antibakteri e k st ra k dau n kelor ( mor i nga olei fera) terhad ap ba k ter i pembusukan ikan segar. pelita. 2014; ix(2): 146–57. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p63–67 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i2.p63-67 67kurniawan et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 63–67 25. anjum si, ullah a, khan ka, attaullah m, khan h, ali h, bashir ma, tahir m, ansari mj, ghramh ha, adgaba n, dash ck. composition and functional properties of propolis (bee glue): a review. saudi j biol sci. 2019; 26(7): 1695–703. 26. kaihena m. propolis sebagai imunostimultor terhadap infeksi micobacterium tuberculosis. in: prosiding fmipa universitas pattimura. ambon: universitas pattimura; 2013. p. 71–2. 27. pujirahayu n, ritonga h, uslinawaty z. properties and flavonoids content in propolis of some extraction method of raw propolis. int j pharm pharm sci. 2014; 6(6): 338–40. 28. rismawati sn, kimia jt, jakarta u. pengaruh variasi ph terhadap kadar flavonoid pda ekstrasi propolis dan karakteristiknya sebagai antimikroba. j konversi. 2017; 6(2): 89–94. 29. gr umezescu a m, holban a m. t herapeutic, probiotic, and unconventional foods. st. louis: elsevier; 2018. p. 141–50. 30. hermalinda r, taufiqurrahman i, helmi zn. total flavonoid content analysis flavonoid content analysis of ramania leaves’ extract using ethanol, methanol and n-hexane as solvents. dentino j kedokt gigi. 2019; 4(1): 60–3. 31. karlina cy, ibrahim m, trimulyono g. aktivitas antibakteri ekstrak herba krokot (portulaca oleracea l.) terhadap staphylococcus aureus dan escherichia coli. lenterabio berk ilm biol. 2013; 2(1): 87–93. 32. christianto cw, nurwati d, istiati. efek antibakteri ekstrak biji alpukat (persea americana mill) terhadap pertumbuhan streptococcus mutans. oral biol dent j. 2012; 4(2): 40–4. 33. asdar a, cindrakori hn. daya hambat gel propolis dari sulawesi selatan terhadap pertumbuhan bakteri porphyromonas gingivalis. b-dent. 2015; 2(2): 101–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p63–67 https://e-journal.unair.ac.id/mkg/index 122 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 122–125 research report the correlation analysis of dental caries, general health conditions and daily performance in children aged 2–5 years darmawan setijanto,1 taufan bramantoro,1 nanissa dyah anggraini,2 ardhyana dea maharani,2 dwita angesti,2 dani susanto hidayat3 and aulia ramadhani1 1 department of dental public health, 2 dental profession program, 3 postgraduate program, faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: oral health is important for general health and quality of life. one of the oral diseases with a high prevalence in indonesia is dental caries. dental caries can cause limiting disturbances of daily activities such as biting, chewing, smiling and talking, and of psychosocial well-being, including development and general health of children. purpose: this study aims to analyse the correlation of dental caries incidence rate with general health conditions and daily performance of children aged 2–5 years. methods: this was an analytical observational cross-sectional study. the study sample was 103 pairs of children and their mothers, selected using cluster random sampling technique. intra-oral examination was conducted on the children to obtain decay, missing, filled-teeth (dmf-t) index score. information about oral impacts on daily performance (oidp) of the children was collected through a questionnaire distributed to the mothers. the data obtained were statistically analysed with a regression test (p < 0.05). results: it was found that dental caries had a significant correlation with general health (p = 0.00) and daily performance, including chewing function disorder (p = 0.00), difficulties in maintaining oral health (p = 0.039), sleep disorders (p = 0.00), and emotional instability (p = 0.00). conclusion: the incidence rate of dental caries has a significant effect on the general health conditions and daily performance of children aged 2–5 years. keywords: child health; daily performance; dental caries; oral impact on daily performance correspondence: taufan bramantoro, department of dental public health, faculty of dental medicine, universitas airlangga, jl. mayjen prof. dr. moestopo no. 47, surabaya 60132, indonesia. email: taufan-b@fkg.unair.ac.id introduction oral and dental health is important for general health. unfortunately, children seem to be vulnerable to oral and dental diseases because they generally have poor oral and dental care habits. eating sweet food and drinking sweet drinks are some examples of their bad habits.1 based on the 2018 basic health research data, dental caries prevalence in indonesia was 81.5% in children aged 3–4 years and as high as 92.6% in those aged 5–9 years. indonesia’s decay, missing, filled-teeth (dmf-t) index in 2018 was 7.1, an increase of 54% from riskesdas 2018.2 oral health is also considered fundamental to public health since a healthy mouth allows individuals to talk, eat, and socialise without experiencing pain, discomfort, or embarrassment.3 however, without adequate care, dental caries may occur and eventually lead to tooth decay. dental caries is the main cause of toothache and tooth loss. everyone is susceptible to dental caries throughout their lives.4 nonetheless, dental caries is one of the many childhood diseases that can be prevented. dental caries can also interfere with the chewing system in general or become a focal infection, thus affecting the health and development of children.5 for instance, dental caries greatly affects the quality of life of children in america, canada and england. in aboriginal children in western australia, dental caries is the fifth most common disease causing preschool children to be hospitalised (ages dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p122–125 mailto:taufan-b@fkg.unair.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p122-125 123setijanto et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 122–125 1–4 years).6 toothache caused by dental caries causes a loss of 50 million school hours per year, affecting school attendance and future adult life. in indonesia, toothache has caused 62.4% of the population to experience discomfort at work/in school for an average of 3.86 days per year. this condition indicates that dental disease, although not fatal, reduces work productivity. a research in medan even reveals the impact of dental caries on four dimensions of quality of life, namely, limited function, pain, psychological discomfort and physical disability. in addition, sheiham7 highlights three effects of untreated dental caries on the growth and development of preschool children. first, pain caused by dental caries can interfere with children’s food intake. second, pain caused by dental caries can trigger sleep disturbances and subsequently leads to glucocorticoid production and growth disturbances. third, chronic inflammation caused by dental caries can suppress haemoglobin and lead to anaemia, since the production of erythrocytes in the bone marrow is reduced. thus, it is essential to treat dental caries in preschool children to improve not only their growth and development but also their quality of life.8–10 a preliminary survey of 30 respondents conducted in pre-kindegarten schools in kenjeran health center working area found a 50% incidence rate of caries severity. for the problems outlined earlier, this study is focused on the dental caries incidence rate and the general health conditions of children aged 2–5 years in some pre-kindergarten and kindergarten schools around the kenjeran health center, surabaya city. this study aims to analyse the correlation of the dental caries incidence rate with the general health conditions and the daily performance of children aged 2–5 years. materials and methods this was an analytical observational cross-sectional study. the dmf-t index of children aged 2–5 years was collected along with questionnaire results distributed to their mothers. the study sample was 103 pairs of pre-kindergarten and kindergarten school children and their mothers, around kenjeran health center, surabaya, selected using the cluster random sampling method. this research has received a certificate (628/hrecc.fodm/x/2019) from the ethics commission of the faculty of dental medicine, universitas airlangga. each respondent’s parent was asked to provide informed consent before participating in this study. the severity of children’s caries was observed through direct primary tooth examination (intra oral). next, dmf-t index measurement was conducted to observe the dental health conditions of children by observing cavities (decay), teeth lost due to caries (missed), and teeth that had been filled. based on the data collected, the dmf-t score was obtained and analysed statistically to find any correlation with the general health conditions and the daily performance of the children collected through questionnaires distributed to their mothers. the questionnaire used in this study was concerned with oral impact on daily performance (oidp) of the children involving a) eating and enjoying food, b) talking and pronouncing clearly, c) cleaning teeth, d) sleeping and relaxing, e) smiling, laughing and showing teeth without embarrassment, f) keeping emotions so as not to be easily offended, g) performing main work or social roles, and h) being able to understand conversations with people around them. in addition, a question instrument was added to analyse the general health conditions of the participants. the data obtained were statistically analysed using a regression test with the statistical package for social science (spss, ibm corporation, illinois, us) software version 22, with a p-value of 0.05%. results this research was conducted in pre-kindergarten and kindergarten schools in surabaya on 103 pairs of children aged 2–5 years and their mothers. the dependent variable in this study is caries, while the independent variable is the general health conditions of children aged 2–5 years. the data were statistically analysed with a regression test to find correlation between these variables. the regression test results showed a significant correlation between the incidence rate of dental caries and the general health conditions of those children. the distribution of respondents in this study can be seen in table 1. based on table 1, the results show that 53.4% of the respondents had a high caries index of >6.6. similarly, the data of the children’s general health conditions indicate that 72.8% of the respondents had experienced illness in the last two months. the correlation of dental caries incidence rate with the children’s daily performance was statistically analysed using the results of the oidp questionnaire. the results of this analysis can be seen in table 2. table 2 also shows the correlation of the incidence rate of dental caries with oidp of each respondent. the table also illustrates that there was a significant correlation between the incidence rate of dental caries and chewing function disorders, with a p-value of 0.000 (p < 0.05). however, there was no significant correlation between the incidence rate of dental caries and speech difficulties, with a p-value of 0.195 (p > 0.05). the oidp index scores indicate a significant correlation between the incidence rate of dental caries and difficulties in maintaining oral hygiene, with a p-value of 0.039 (p < 0.05). there was also a significant correlation between the incidence rate of dental caries and sleep disorders due to oral and dental health problems, with a p-value of 0.000(p < 0.05). there was a significant correlation between the incidence rate of dental caries and emotional instability, with a p-value of 0.000 (p < 0.05). there was no significant correlation between the incidence rate of dental caries and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p122-125 124 setijanto et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 122–125 difficulties in smiling due to oral health problems, with a p-value of 0.078 (p > 0.05). in other words, the incidence rate of dental caries had a significant correlation with the general health conditions of children aged 2–5 years, related to chewing function disorders, difficulties in maintaining oral hygiene, sleep disorders due to oral and dental health problems, and emotional instability due to oral and dental health problems. however, the incidence rate of dental caries had no significant correlation with speech difficulties, avoiding meeting people, and difficulties in smiling. however, the statistical test using a regression test shows that the incidence rate of dental caries has a significant effect on the general health conditions of children aged <5 years, with a p-value of 0.000 (p < 0.05). discussion this study shows a significant correlation between the incidence rate of dental caries and the general health conditions of children aged 2–5 years. this may be due to several factors that can increase the severity of dental caries, such as education level; thus, the higher the education level, the higher the awareness of maintaining one’s own general health.5 in addition, it can also be assessed from how dental and oral health is maintained, such as not eating carcinogenic foods, use of toothbrushes, brushing teeth frequently, and using proper brushing technique.11 similarly, wening et al.8 argue that although there is no significant correlation between the severity of dental caries and the nutritional status of children, a decrease in desire table 1. distribution of respondents variables n* percentage (%) sex male 49 47.6% female 54 52.4% age 2–3 years old 18 17.5% 4–5 years old 85 82.5 % caries severity low (0-6.6) 48 46.6% high (>6.6) 55 53.4% toothache never experienced 53 51.5% had experience 50 48.5% daily brushing habit 1x a day 17 16.5% 2x a day 79 76.7% 3x a day 7 68% general health conditions in the last two months never sick 28 27.2% ever sick 75 72.8% having toothache often 62 60.2% never 41 39.8% chewing function disorders no 38 36.9% yes 65 63.1% sleep disorders no 71 68.9% yes 32 31.1% difficulties in maintaining oral hygiene no 71 68.9% yes 32 31.1% avoid meeting people due to oral and dental health problems no 93 90.3% yes 10 9.7% emotional instability due to oral and dental health problems no 80 77.7% yes 23 22.3% difficulties in smiling due to oral and dental health problems no 93 90.3% yes 10 9.7% * number of respondents table 2. regression test results on the correlation of the dental caries incidence rate with the children’s daily performance using the oral impact on daily performance (oidp) questionnaire risk factor n sig. chewing function disorders 103 *0.000 speech difficulties 103 0.195 difficulties in maintaining of oral hygiene 103 *0.039 avoiding meeting people due to oral and dental health problems 103 0.077 sleep disorders due to oral and dental health problems 103 *0.000 difficulties in smiling due to oral and dental health problems 103 0.078 emotional instability due to oral and dental health problems 103 *0.000 * significant at p-value <0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p122-125 125setijanto et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 122–125 to eat still can be triggered by discomfort felt when eating when having a toothache. decreased appetite can also have an impact on children’s general health as nutrient intake decreases and causes decreased endurance. ramayani et al.9 state that children suffering from dental caries are lighter in weight than those without dental caries. the findings of the previous studies strengthen the results of this study, which revealed a significant correlation between dental caries and children’s general health. this study also evaluates the severity of dental caries in children aged <5 years, using the dmf-t index and oidp. it is known that severe dental caries can affect quality of life intrinsically and extrinsically. intrinsically, a severe dental cavity can penetrate the pulp chamber and cause inflammation of the pulp tissue, causing pain and discomfort leading to sleep disorders, which can also reduce immune function. extrinsically, dental caries can cause poor oral hygiene (oh) and tooth morphology, which can interfere with chewing function, leading to reduced nutrient intake, which can also reduce immune function. the decline in immune function can cause general health problems in toddlers. dental health is one of several oral health factors that are important for child development especially. dental caries is the most common dental health problem found in children, which is caused by food residue that sticks to the teeth. calcification of teeth causes teeth to become porous, hollow, and even fractured (broken). dental caries can also cause children to experience loss of chewing power and disruption of digestion, which results in less optimal growth.12,13 this study found that age and gender of children aged <5 years had no significant effect on the incidence rate of dental caries. the oidp questionnaire results consists of eight items evaluating the impacts of oral health on children’s ability to perform their daily activities, including the measurement of physical, psychological and social dimensions. this questionnaire instrument focuses on ten basic daily activities, namely, eating, talking, cleaning the mouth, performing light physical activities, sleeping, relaxing, smiling, having emotional states, going out, and enjoying interacting with others.14–16 in addition, the influence of daily life performance on oral caries aims to provide alternative sociodental indicators, focusing on measuring a person’s ability to carry out the indicated daily activities with the condition of the oral caries. the results of the oidp questions concerned with the general health conditions of children aged <5 years who often have toothache and dental caries indicated a significant correlation between the incidence rate of dental caries and the frequency of having toothache. there was a significant correlation between the incidence rate of dental caries and the behaviour of avoiding meeting people due to oral and dental health problems. there was also a significant correlation between the incidence rate of dental caries and emotional instability due to oral and dental health problems. in other words, the incidence rate of dental caries had a significant effect on the emotions of children. while the oidp focuses on ten basic daily activities, it does not mean that all of those ten basic daily activities necessarily affect dental caries in children.17–19 finally, it can be concluded that the incidence rate of dental caries has a significant effect on the general health conditions and the daily performance of children aged 2–5 years. references 1. berwulo h. gambaran tingkat karies berdasarkan status kebersihan gigi dan mulut pada siswa sekolah dasar di desa ranowangko ii kecamatan kombi. thesis. manado: universitas sam ratulangi; 2011. p. 23–24. 2. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 110. 3. alrmaly b, assery m. need of oral health promotion through schools among developing countries. j int oral heal. 2018; 10: 1–3. 4. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10: 7. 5. smith l, blinkhorn fa, blinkhorn as, hawke f. prevention of dental caries in indigenous children from world health organization–listed high-income countries: a systematic review. health educ j. 2018; 77(3): 332–48. 6. boy h, khairullah a. hubungan karies gigi dengan kualitas hidup remaja sma di kota jambi. j kesehat gigi. 2019; 6: 10–3. 7. sheiham a. dental caries affects body weight, growth and quality of life in pre-school children. br dent j. 2006; 201(10): 625–6. 8. wening gs, bramantoro t, palupi r, ramadhani a, alvita d. overview of dental caries severity and body mass index (bmi) on elementary school children. j int oral heal. 2019; 11(7): 48–55. 9. ramayani mp, nadhiroh sr. relationship between dental caries and the level of consumption and nutritional status of primary school age children. media gizi indones. 2012; 7(2): 1492. 10. nagarajappa r, batra m, sanadhya s, daryani h, ramesh g. relationship between oral clinical conditions and daily performances among young adults in india a cross sectional study. j epidemiol glob health. 2015; 5(4): 347–57. 11. berniyanti t, bramantoro t, palupi r, wening gs, kusumo a. epidemiological investigation of caries level in 2nd and 3rd grader primary school student. j int oral heal. 2019; 11(7): 44–7. 12. bönecker m, abanto j, tello g, oliveira lb. impact of dental caries on preschool children’s quality of life: an update. braz oral res. 2012; 26(spl. iss.1): 103–7. 13. akbar fh, pratiwi r, multazam a. hubungan status karies gigi dengan kualitas hidup terkait kesehatan mulut anak usia 8-10 tahun (studi kasus sdn 3 dan sdn 5 kota parepare). thesis. makassar: universitas hasanuddin; 2014. p. 1–15. 14. rebouças ap, bendo cb, abreu lg, lages emb, flores-mir c, paiva sm. cross-cultural adaptation and validation of the impact of fixed appliances measure questionnaire in brazil. braz oral res. 2018; 32: e14. 15. nasia aa, arumrahayu w, rosalien r, maharani a, adiatman m. child-oral impacts on daily performances index in indonesia: cross-cultural adaptation and initial validation. malaysian j public heal med. 2019; 19(2): 68–77. 16. duarte-rodrigues l, ramos-jorge j, drumond cl, diniz pb, marques ls, ramos-jorge ml. correlation and comparative analysis of the cpq8-10 and child-oidp indexes for dental caries and malocclusion. braz oral res. 2017; 31: e111. 17. gilchrist f, rodd h, deery c, marshman z. assessment of the quality of measures of child oral health-related quality of life. bmc oral health. 2014; 14: 40. 18. yusof zym, jaafar n. a malay version of the child oral impacts on daily performances (child-oidp) index: assessing validity and reliability. health qual life outcomes. 2012; 10: 63. 19. nurelhuda nm, ahmed mf, trovik ta, åstrøm an. evaluation of oral health-related quality of life among sudanese schoolchildren using child-oidp inventory. health qual life outcomes. 2010; 8: 152. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p122–125 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p122-125 177 volume 47, number 4, december 2014 research report the differences of orthodontic tooth movement on menstrual and ovulation cycle sonya grecila susilo,1 rahmi amtha,2 boedi oetomo roeslan3 and joko kusnoto1 1department of orthodontics 2 department of oral medicine 3department biochemistry faculty of dentistry, universitas trisakti jakarta indonesia abstract background: estrogens are sex hormon that play an important role in bone metabolism, including in bone remodeling during orthodontic treatment. women has a monthly cycle which is affected by fluctuations of estrogen that is menstruation and ovulation. purpose: the study was aimed to determine the differences of orthodontic tooth movement during menstrual an ovulation cycle. methods: five women were given ± 100 g orthodontic force using fixed orthodontic appliance with straight wire technique at the time of menstruation and ovulation with an interval of 1.5 months. orthodontic tooth movement and levels of estrogen were measured during menstruation and ovulation. results: statistical results showed a significant differences between estrogen levels and orthodontic tooth movement (p < 0.05). when estrogen levels decline as in menstruation, tooth movement as results of orthodontic force would increase, whereas when estrogen levels increase as the time of ovulation, tooth movement would decrease. conclusion: the estrogen level in menstruation and ovulation cycle may affect the tooth movement on ortodontic treatment. key words: estrogen level, tooth movement, orthodontic force, menstruation, ovulation abstrak latar belakang: estrogen merupakan hormon seks yang mempunyai peran penting dalam metabolisme tulang termasuk dalam remodeling tulang selama perawatan ortodonti. wanita memiliki siklus bulanan yang dipengaruhi oleh fluktuasi estrogen yaitu menstruasi dan ovulasi. tujuan: penelitian ini bertujuan untuk mengetahui perbedaan pergerakan gigi akibat gaya ortodonti pada siklus menstruasi dan ovulasi. metode: lima orang wanita diberikan gaya ortodonti ±100 gr menggunakan alat orthodontik cekat dengan teknik straight wire pada saat menstruasi dan ovulasi dengan selang waktu 1,5 bulan. selain itu subyek juga diukur kadar estrogennya saat menstruasi dan ovulasi. hasil: hasil statistik menunjukan kadar estrogen berbanding terbalik dengan pergerakan gigi ortodonti (p < 0,05). saat kadar estrogen menurun seperti pada menstruasi, maka pergerakan gigi akan meningkat, sedangkan pada saat kadar estrogen meningkat seperti saat ovulasi, maka pergerakan gigi akan menurun. simpulan: kadar estrogen pada siklus menstruasi dan ovulasi dapat mempengaruhi pergerakan gigi pada perawatan ortodonti. kata kunci: kadar estrogen, pergerakan gigi, gaya ortodonti, menstruasi, ovulasi correspondence: sonya grecila susilo, c/o: departemen ortodonsia, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa no. 1 grogol jakarta 11440, indonesia. e-mai: sonya.grecila@yahoo.com 178 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 177–180 introduction orthodontic tooth movement begin when the remodeling occurs on alveolar bone. on the pressure side, activity of osteoclasts will produce a resorption, whereas on the tension side, activity of osteoblasts will make the apposition.1,2 one of the hormones which can affect bone remodeling is estrogen. estrogen influences the composition and degradation of collagen fibers in the periodontal ligaments and the remodeling of the alveolar bone.3 currently, patients who come to the orthodontic clinic is dominate by teenage girls and adult womens.4 women has a monthly cycle that is influenced by estrogen fluctuations called menstruation and ovulation. estrogen will increase when women had ovulation cycle and will decrease when had the menstrual cycle. the menstrual cycle is characterized by periodic vaginal bleeding that had occurred because release of the uterine mucosa.5 menstruation cycle averages 28 days in length.6 ovulation is the rupture of mature follicle and the release of ovum.7 in average 28-day cycle, ovulation occurs on day 14 and is followed 2 weeks later by start of the menstrual flow. although the role of estrogen on bone remodeling has been known, but until now the effect of menstruation and ovulation to orthodontic tooth movement is still questionable. to ensure the differences orthodontic tooth movement during the menstrual and ovulation cycle should be done further research. the purpose of this study was to determine the differences of orthodontic tooth movement during menstrual and ovulation cycle. materials and methods five female subjects in range of age 18-25 years old were selected to participated in this study. the local research biomedic and ethics committee (no: 73/ke/fkg/12/2012) approved the research protocol. the entire of subjects are patients who come and seek orthodontic treatment that performed by resident of department ortodontic trisakti university. woman who present good general and oral health with regular menstrual cycle of 26-32 days each month with skeletal maloclussion class i that needs bilateral first premolar extractions will fulfill the inclusion criteria. women who were pregnant, breast-feeding, treated with contraseptive drugs or estrogen therapy, diagnosed with systemic disease or periodontal disease were excluded from the study. the study was conducted at universitas trisakti and biomedical laboratory. the study began by giving an explanation to the subject about study procedures and they were asked to signed an informed consent as an agreement to be participating in this study. after that the subjects were asked about their history of menstrual cycle to estimate when will the next menstruation and ovulation. menstrual phase is determined at the time the subject were experiencing the first day of bleeding, while the ovulation phase is determined approximately 14 days after first day menstruation and proved by ovulation test kits. in addition, subjects also performed fixed orthodontic treatment by using a straight wire technique. bracket used is roth perscription 0.018 slots (forestadent, germany). molar bands was cemented on the first molar, and brackets were bonded from left to right second premolars. leveling and aligning phase begins with 0.014 nickel-titanium (niti) wire on the upper and lower arches. after leveling and aligning phase was completed, then started the canine retraction using 0.016 stainless steel wire. retraction canines performed using niti closed coiled spring with a force of approximately 100 grams were measured using dontrix gauge. the data was collected in three sessions. first, when the subject was having first day period of menstruation, a ± 100 gr of orthodontic force was given on the canine with niti closed coiled spring. the level of estrogen menstruation were measured and also impression was taken to determine the range of tooth movement. second session was collected in 1.5 month later when subject were having period of ovulation, a ± 100 gr of orthodontic force was given on the canine with niti closed coiled spring and the level of estrogen ovulation were measured. also performed imprression to determine the range of tooth. the third session was collected when the subject had menstrual cycle next 1.5 month later. this session only performed impression to determine the range of tooth. the work flow can be seen on figure 1.. determination of estrogen level was done by taking 10 ml blood with venous puncture, then blood was put into vacutainer tubes. blood samples were immediately sent to the laboratory for processing using the elisa method. whereas determination range of tooth movement was done by taking impression for study model. the range of menstruation tooth movement was calculated with digital calliper from the difference between the linear distance (mm) from right distal canine to the second mesial figure 1. work flow diagram on differences orthodontic tooth movement on menstrual and ovulation cycle. subject → female 18-25 yrs old statistical analysis 179susilo, et al.: the differences of orthodontic tooth movement on menstrual and ovulation cycle premolar on right between session i and ii, while the range of ovulation tooth movement was calculated from the difference between the linear distance (mm) of right distal canine to second right mesial premolar between session ii and iii. this measurement was also performed on the left side in the same way. kolmogorov smirnov test was used to analyze the normality of range of tooth movement and level of estrogen. if the sample distribution was normal, then t-test was used to analyze differences between level of estrogen and range of tooth movement. the pearson’s correlation test was used to assess the correlations between range of tooth movement and estrogen level during menstruation and ovulation. results this study collected sample from 5 subject with average age of subjects was 22±2.44 years and body mass index (bmi) was 21.28 ± 1.29. the outcome data were tested for normality with kolmogorov-smirnov test and showed normal data distribution (p > 0.05). the results measurement of estrogen level during menstruation and ovulation can be seen in table 1. the measurement showed that estrogen levels was lower during menstruation (28.66 ± 11.61 pg/ml) than ovulation (143.9 ± 62.68 pg/ml) cycle. the mean and standard deviation of the range of tooth during menstruation was 1.71 ± 0.324 mm and when ovulation was 0.66 ± 0.2 mm (table 1). the mean range of tooth increase during the menstruation cycle than ovulation. the t-test was used to identify differences between level of estrogen and range of tooth movement. t-test result showed there were significant differences between two groups (t-value = 7.66, p-value = 0,000). pearson’s correlation test result showed a negative correlation between estrogen levels and orthodontic tooth movement (r = -0.823, p < 0.05) (table 1). it means when estrogen level decline, it will increase the tooth movement and conversely. discussion this study was done by activating orthodontic force during peak of estrogen level on ovulation and during the lowest estrogen level during menstruation. the force that applied to canine distalization was approximately 100 grams to generate translation movement. type of force is a continuous force that generated from the ni-ti closed coil spring.8 the result showed that fluctuations in estrogen levels during menstruation and ovulation cycle can affect tooth movement due to ortodontic force. when estrogen levels decline as in menstruation, tooth movement will increase, whereas when estrogen levels increase as the time of ovulation, tooth movement will decrease. this opinion is in accordance with sirisoontorn et al.3 research which revealed that lack of estrogen may increase the orthodontic tooth movement. that results is same as some previous studies such as haruyama et al.,9 ghajar et al.,10 and olyaee et al.,11 which said that estrogen level inversely related to orthodontic tooth movement. orthodontic tooth movement occurs because the presence of osteoblast and osteoclasts that stimulate remodeling of alveolar bone. estrogen is a hormone that is known to inhibit the activity of osteoclasts in a direct or indirect manner. estrogen directly inhibits bone remodeling by decrease osteogenesis and chondrogenesis. estrogen can also work indirectly on osteoclasts by increasing the production of calcitonin. increased secretion of calcitonin by estrogen would inhibit the action of osteoclasts in bone resorption.12 limited amount of estrogen will increased remodeling process so that bone density will be increased too.12 this hormone also inhibits cytokine products such as interleukin 1β (il-1β), interleukin 6 (il-6), and tumor necrosis factoralpha (tnf-α), a macrophage colony-stimulating factor (m-csf) that is involved in the activity of osteoclasts that makes the process of bone remodeling will be also inhibited.4,13 during menstruation estrogen levels will decrease, thus when orthodontic force was given, the activity of osteoclasts will increase. the remodeling process will also be faster. this is what causes the orthodontic tooth movement became more rapid. in contrast during ovulation, estrogen levels will rise that makes osteoblast activity is inhibited and tooth movement will be slower. effects of estrogen to orthodontic tooth movement can be used in orthodontic treatment especially in order to shorten the duration of treatment. the duration of treatment is not only often complained by the patient but also can cause negative effects on teeth.14 prolonged orthodontic treatment often causes demineralization email, root resorption and periodontal disease. several methods have been developed to accelerate ortodontic tooth movement such as selective alveolar decortication, gingival fiberotomy, reduce friction between bracket and table 1. summary of statistical analysis rate standart deviation t-test pearson correlation t p-value r p-value menstruation estrogen level 28.66 11.61 7.66 0.000* -0.823 0.003* ovulation estrogen level 143.9 62.88 mentrustion range of tooth 1.71 0.324 ovulation range of tooth 0.66 0.2 *significant p < 0.05 180 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 177–180 wire, physical or mechanical stimulation such as by using a low-energy laser and injection several drugs both locally and systemically such as prostaglandin, corticosteroid and vitamin d.4,15 but there is no single method that is actually received by the experts. this study may suggest an option for orthodontists to accelerate the tooth movement by performing activation of orthodontic force during menstruation, this method will be safer and more comfortable for the patient and the orthodontic treatment will be also more effective and efficient, even though this option is only can be done by female patients and sometimes there are psychological barries that women feel embarrassed to tell their sexual cycle. therefore futher studies with bigger sample and longer observation period should be performed to prove the validity and consistency. the study at 5 women showed that there was a significant differences in orthodontic tooth movement during the menstrual and ovulation cycle. estrogen levels were tend to inversely related to orthodontic tooth movement, which means decreased estrogen levels such as during menstruation would increase tooth movement. meanwhile, when estrogen levels increase as at the time of ovulation, tooth movement would decrease; orthodontic tooth movement would be faster if during menstruation the orthodontic force was activated. the estrogen level in menstruation and ovulation cycle may affect tooth movement on orthodontic treatment. references 1. qing z, zhen t, jie g, yang-xi c. influences of applying force during the different stages of estrous cycle on orthodontic tooth movement of rats. hua xi kou qiang yi xue za zhi 2005; 23(6): 480-2. 2. henneman s, von den hoff jw, maltha jc. mechanobiology of tooth movement. eur j orthod 2008; 30(3): 299-306. 3. sir isoontor n i, hotokeza ka h, hashimoto m, gonzales c, luppanapornlarp s, darendeliler ma, yoshida n. tooth movement and root resorption: the effect of ovariectomy on orthodontic force application in rats. angle orthod 2011; 81(4): 570-7. 4. xu x, zhao q, yang s, fu g, chen y. a new approach to accelerate orthodontic tooth movement in women: orthodontic force application after ovulation. med hypotheses 2010; 75(4): 405-7. 5. benson r, pernoll m. buku saku obstetri dan ginekologi. 9th ed. wijaya s, editor. jakarta: penerbit buku kedokteran egc; 2010. h. 46-47, 56. 6. cunningham f, gant n, leveno k, gilstrap l, hauth j, wenstrom k. obstetri williams. 21th ed. andri h, editor. jakarta: penerbit buku kedokteran egc; 2012. h. 68. 7. saladin k. anatomy and physiology: the unity of form and function. 5th ed. new york: mc graw hill; 2010. p. 1092-4. 8. proffit w, fields h, sarver d. compemporary orthodontics. 4th ed. st. louis: mosby elsevier; 2007. p. 332-5. 9. haruyama n, igarashi k, saeki s, otsuka-isoya m, shinoda h, mitani h. estrous-cycle-dependent variation in orthodontic tooth movement. j dent res 2002; 81(6): 406-10. 10. ghajar k, olyaee p, mirzakouchaki b, ghahremani l, garjani a, dadgar e. the effect of pregnancy on orthodontic tooth movement in rats. med oral patol cir bucal 2013; 18: e 351-5. 11. olyaee p, mirzakouchaki b, ghajar k, seyyedi s, shalchi m, garjani a. the effect of oral contraseptives on orthodontic tooth movement in rat. med oral patol cir bucal 2013; 18: 146-50. 12. derek s, kalangi s, wangko s. kerja osteoklas pada perombakan tulang. bik biomed 2007; 3: 97-107. 13. ames ms, hong s, lee hr, fields hw, johnston w, kim d. estrogen deficiency increases variability of tissue mineral density of alveolar bone surrounding teeth. arch oral biol 2010; 55(8): 599-605. 14. krishnan v, davidovitch z. biological mechanisms of tooth movement. oxford: blackwell publishing ltd; 2009. p. 9, 155, 1734. 15. baloul s, gerstenfeld l, morgan e, carvalho r, van dyke t, kantarci a. mechanism of action and morphologic changes in the alveolar bone in response to selective alveolar decortication–facilitated tooth movement. am j orthod dentofacial orthop 2011; 139: s83-101. vol 51 no 1 jan-mrt 2018.indd 10 the correlation between untreated caries and the nutritional status of 6–12 years old children in the medan maimun and medan marelan sub-district siti salmiah, l. luthfiani, zulfi amalia, and deandini kusumah department of pediatric dentistry, faculty of dentistry, universitas sumatera utara medan indonesia abstract background: in indonesia, dental caries constitute one of the most common dental health problems in children. untreated dental caries will cause both pain and inconvenience when eating, resulting in a reduced appetite which can negatively affect the body mass index (bmi). purpose: this study aimed to investigate the correlation between untreated caries and nutritional status in children aged 6-12 years old in the medan maimun and medan marelan sub-districts. methods: an analytical observation study with cross-sectional design was adopted. the number of child subjects totaled 350, divided into two groups, namely; the pufa/pufa group (n=172) and the non-pufa/pufa group (n=178). samples were selected on the basis of purposive sampling. oral examination was subsequently performed using the pufa/pufa index. the height and weight of the subjects were assessed according to the indonesian ministry of health’s bmi criteria of 2011. thereafter, chi square, spearman and mann-whitney tests were all performed as analytical tests. results: the results of this research revealed a significant correlation between caries status and bmi (p<0.001) in both the pufa/pufa and non pufa/pufa groups (r=-0.515), as well as between the mean pufa/pufa score and age. however, there was no significant correlation between the mean pufa/pufa score and gender. conclusion: it can be concluded that a correlation exists between untreated caries and the nutritional condition of children aged 6-12 years old in the medan maimun and medan marelan sub-districts. keywords: pufa/pufa index; body mass index; caries correspondence: siti salmiah, department of pediatric dentistry, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2 medan 20155, indonesia. e-mail: salmiadentist@yahoo.co.id research report introduction the global oral health program of the world health organization (who) emphasizes that dental and oral health is integrated with general health.1 caries represent one of the most common dental health problems affecting the world’s population. caries are formed through a process of demineralization of hard tooth tissue, followed by damage to organic matter caused by various mutually-influencing factors (host, substrate, bacterium, time factor and immune response). caries can affect individuals within most populations around the world at various ages, in a range of cultures and ethnicities and socioeconomic situations.2–4 according to the who, 60–90% of cases of caries were found in school-aged children and a certain number of adults worldwide.5 based on statistics from the riskesdas of north sumatra, in 2007 the prevalence of dental and oral problems among the medan municipal population amounted to 18.8%, increasing to approximately 19.4% in 2013 data indicating an increase in such problems. these conditions also suggested that dental and oral problems, especially caries, should receive greater attention.6,7 the continuous caries process, if untreated, can make various microbes in the oral cavity expand into the pulp tissue via open dentine tubules, leading to acute and chronic inflammatory responses in the pulp possibly accompanied by lesions around the soft tissue.8–10 children with caries are likely to tolerate their condition as long as the caries do not interfere with their activities. moreover, the limited access to hospital and high cost of oral health services lead dental journal (majalah kedokteran gigi) 2018 march; 51(1): 10–13 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p10–13 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p10-13 11salmiah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 10–13 to a lack of consciousness on the part of parents regarding dental and oral care for their children.11 these conditions are exacerbated by health policies that consider caries a low priority health issue since they are rarely reported as causing death.12 children with severe caries are reported as experiencing frequent toothache and problems when eating certain foods, feeling embarrassed about smiling, as well as no longer playing with other children.12 another piece of research showed that 88.7% of the children studied were experiencing dental problems, affecting at least one of their eight daily activities. activities that have the highest impact are eating (81.3%), keeping the mouth clean (40.5%) and smiling (32.2).13 the pain and discomfort experienced by children when eating can even lead to a decrease in their appetite and life quality affecting their growth and development.3,9,14 decreased food intake due to the pain experienced by children when chewing can reduced nutritional intake compromising their nutritional status. the nutritional status of children is usually indicated by their body mass index (bmi).12 several pieces of research have actually revealed there to be a correlation between the severity of caries (pufa/ pufa) and bmi. a recent investigation even suggested that children with odontogenic infections are at increased risk of weight loss compared to those without odontogenic infections.3 thus, preventing severe caries is important since it can affect general health, life quality, productivity and growth in children. this study aimed to analyze the correlation of untreated caries and nutritional status in children aged 6–12 years old in the medan maimun and medan marelan sub-districts. materials and methods this research was conducted from march to august 2016 and representedan observational analytic study with cross-sectional design. the research population consisted of elementary-school children aged 6–12 years resident in the medan maimun and medan marelan sub-districts. sampling was then performed using a purposive sampling technique. the child samples selected for this research numbered 350, consisting of 178 (50.9%) males and 172 (49.1%) females. these children were drawn from the students of sdn 060788 and sds al-falah in medan maimun sub-district, as well as from those attending sdn 064007 and sds mandiri in medan marelan sub-district. they were then divided into two groups, namely: a nonpufa/pufa group consisting of 172 children and a pufa/ pufa group comprising 178 children. oral examination was performed using a pufa/pufa index, assessing oral cavity conditions caused by untreated caries, such as: infection with pulp involvement (p/p), ulceration of the oral mucosa due to root residual tooth fragments (u/u), fistula (f/f) and abscesses (a/a). lesions around tissue without pulp involvement due to untreated caries were not recorded in this research. one score was given for each tooth. uppercase (pufa) was used to assess permanent teeth, while lowercase (pufa) was used to assess decidual teeth. one score was given if there was a permanent tooth has problematic. however, if decidual and permanent teeth proved to be problematic, both would be given a score. non pufa/pufa criteria consisted of dental caries that did not involve pulp.9 the nutritional status of children was subsequently assessed by calculating the bmi, dividing body weight (in kilograms) by height (in meters) squared. the bmi criteria used in this research related to age and sex in children aged 5–18 years and was based on those of kemenkes ri 2011 which assigned them to one of three nutritional status categories, namely: underweight, normal and overweight. results the total number of research samples amounted to 350 children aged 6–12 years old in medan maimun and medan marelan sub-districts. the samples consisted of 178 (50.9%) male children and 172 (49.1%) female children (table 1). moreover, the research results indicated that the largest percentage of the underweight category, as many as 90 samples, was found in groups of children with pufa/pufa (table 2). furthermore, based on the chi-square test results, there was a significant correlation between the pufa/pufa group and the non-pufa/pufa group with bmi (p<0.001). the results revealed that the highest mean of pufa/pufa score was in the underweight category (table 3). a normality test was subsequently performed by means of a kolmogrov-smirnov test the results of which showed that the variables of pufa/pufa score and bmi were not normally distributed (p value <0.001). consequently, a spearman’s correlation test was conducted whose results confirmed a significant correlation with moderate correlation strength between the mean pufa/pufa score and the mean bmi score (r=-0.515) with a p value of less than 0.001. according to sopiyudin, the mean correlation table 1. characteristics of the research samples n (%)femalesmalesage 46 (13.2)29176 49 (14.0)27227 50 (14.3)18328 48 (13.7)24249 53 (15.1)272610 49 (14.0)262611 55 (15.7)243112 350 (100)172178total dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p10–13 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p10-13 12 salmiah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 10–13 strength is supposedly in the range of 0.40–0.599. the negative sign indicates the direction of correlation, which means the result is in the opposite direction. in other words, the higher the pufa/pufa score, the smaller the bmi score in the samples. in addition, the results of this research also demonstrated that there was no correlation between the severity of caries, as indicated by the pufa/pufa score, and gender (p=0.606). however, the mean pufa + pufa score was higher in males than in females. moreover, the results of this research also revealed that the mean pufa + pufa score in the 6-8 years age group was 4.50 ± 2.255, while that in the 9-12 years age group was 3.08 ± 1.869 with p value <0.001. this suggests a correlation between caries severity status based on pufa/ pufa score and age group (table 4). discussion the results of this research found that there was a significant correlation (p <0.001) between the pufa/ pufa group and the non-pufa/pufa group with bmi. the underweight children, numbering as many as 90 individuals, tended to be more commonly found in the pufa/pufa group. nevertheless, in the non-pufa/pufa group there were evidently still a number of children within the underweight category. this indicates that caries may affect bmi since the growth of children depends on their diet and metabolism factors that can be affected by caries.15 thus, children with many caries that do not extend to the pulp can, nonetheless, experience inefficient mastication with the result that the food is not processed perfectly, eventually resulting in a challenge for the body in absorbing food efficiently (table 2). the results of this research also indicated a negative correlation between the mean pufa + pufa score and bmi (r=-0.515) as illustrated in table 4. this suggests a close correlation between pufa/pufa and bmi. therefore, the higher a child’s pufa/pufa score, the lower the value of his/ her bmi. in other words, a child with a significant amount of pufa/pufa in his/her oral cavity will lose weight. similarly, research conducted by benzian in 2011 revealed that children with caries extending to their dental pulp have a higher risk of decreased bmi than children with caries that do not affect the pulp in their teeth.3 as with previous investigations, in this research the mean pufa/pufa score in children falling within the underweight category was 4.68 ± 2.27, higher than with that in children constituting the overweight category (1.66 ± 0.81) as shown in table 3. this is because caries has a number of impacts on the daily activities of children, one of which is a reduced appetite. toothache renders mastication difficult for them, so they will be fastidious in selecting food. in the long term, this condition can impede their nutritional intake, with the resulting lack of nutrient intake, ultimately, having an impact on their bmi. however, unlike the results reported here, research conducted in surabaya argued that there is no correlation between pufa or pufa and bmi in children aged 6–12 years.16 according to hooley,17 there are several factors causing the absence of correlation between caries and bmi. one of these is a failure to categorize samples into various categories of bmi when assessing samples with lower weight, average weight and higher weight. in this research, the distribution of variables based on body weight was not normal. age also affects the occurrence rate of caries. the results of this research indicated that the mean pufa score was higher in children aged 6–8 years, while the mean pufa score was higher in children aged 9–12 years (table 4). similarly, research conducted by jain in 2014 indicated that the highest mean pufa score, as much as 2.63, is found in children aged 5–8, while the mean pufa score increases at age 9–12 and 13–16, to 0.18 and 0.99 respectively.18 this condition may occur since at the age of 6–8 years the number of decidual teeth exceeds that of permanent teeth and these decidual teeth have been exposed far longer to caries factors in the oral cavity. table 2. correlation of pufa/pufa and bmi in children aged 6–12 years group of children body mass index ptotalunderweight n (%) normal n (%) overweight n (%) <0.00117234 (19.8)129 (75.0)9 (5.2)non pufa/pufa 1786 (3.4)82 (46.1)90 (50.6)pufa/pufa 35040 (11.4)total 99 (28.3) 211 (60.3) * p value <0.05 = the result of the chi-square statistical test was meaningful table 3. distribution of the mean pufa/pufa score to body mass index in children aged 6-12 years body mass index mean pufa score mean pufa score mean pufa + pufa score i. 4.68±2.270.64±1.424.04±2.65underweight ii. 2.89±1.620.14±0.412.75±1.73normal iii. 1.66±0.810.33±0.511.33±1.21overweight dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p10–13 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p10-13 13salmiah, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 10–13 the mean pufa + pufa score in this research was higher in the 6–8 years age group than in that of 9–12 years. this means that the mean pufa + pufa score will decrease with age. this finding is consistent with a theory suggesting that the risk of caries decreases with age since, as children mature, their knowledge of dental health will expand, causing them to strive to maintain healthy teeth.11 as a result, it can be concluded that there is a correlation between untreated caries and nutritional status in children aged 6–12 years old in the medan maimun and medan marelan sub-districts. references 1. petersen pe. world hea lt h o rga n ization globa l pol icy for improvement of oral health--world health assembly 2007. int dent j. 2008; 58(3): 115–21. 2. tarigan r. karies gigi. 2nd ed. jakarta: egc; 2014. p. 1–2. 3. benzian h, monse b, heinrich-weltzien r, hobdell m, mulder j, van palenstein helderman w. untreated severe dental decay: a neglected determinant of low body mass index in 12-year-old filipino children. bmc public health. 2011; 11(1): 558. 4. pintauli s, hamada t. menuju gigi & mulut sehat : pencegahan dan pemeliharaan. medan: usu press; 2008. p. 4–9, 17–8. 5. petersen pe. priorities for research for oral health in the 21st century--the approach of the who global oral health programme. community dent health. 2005; 22: 71–4. 6. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar provinsi sulawesi utara tahun 2007. medan: kementerian kesehatan ri; 2007. p. 105. 7. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar provinsi sulawesi utara tahun 2013. medan: kementerian kesehatan ri; 2013. p. 129. 8. tarigan r. perawatan pulpa gigi (endodonti). 3rd ed. jakarta: egc; 2013. p. 23–4. 9. monse b, heinrich-weltzien r, benzian h, holmgren c, van palenstein helderman w. pufa--an index of clinical consequences of untreated dental caries. community dent oral epidemiol. 2010; 38(1): 77–82. 10. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cjl, marcenes w. global burden of untreated caries: a systematic review and metaregression. j dent res. 2015; 94(5): 650–8. 11. tambayong aj, heroesoebekti r, hapsoro a. the overview of primary school children caries severity in sdn klakahrejo i-ii district benowo surabaya. dent public heal j. 2014; 5(1): 25–33. 12. feitosa s, colares v, pinkham j. the psychosocial effects of severe caries in 4-year-old children in recife, pernambuco, brazil. cad saude publica. 2005; 21(5): 1550–6. 13. castro r de al, portela mc, leão at, de vasconcellos mtl. oral health-related quality of life of 11and 12-year-old public school children in rio de janeiro. community dent oral epidemiol. 2011; 39(4): 336–44. 14. mishu mp, hobdell m, khan mh, hubbard rm, sabbah w. relationship between untreated dental caries and weight and height of 6-to-12-year-old primary school children in bangladesh. int j dent. 2013; 2013(2013): 1–5. 15. mohammadi tm, hossienian z, bakhteyar m. the association of body mass index with dental caries in an iranian sample of children. j oral heal oral epidemiol. 2012; 1(1): 29–35. 16. nabila a, setijanto d, santosa lm. the relationship between caries severity with nutritional status on children aged 6-12 years old. dent public heal j. 2015; 6(1): 9–15. 17. hooley m, skouteris h, boganin c, satur j, kilpatrick n. body mass index and dental caries in children and adolescents: a systematic review of literature published 2004 to 2011. syst rev. 2012; 1: 57. 18. jain k, singh b, dubey a, avinash a. clinical assessment of effects of untreated dental caries in school-going children using pufa index. chettinad heal city med j. 2015; 3(3): 105–8. table 4. correlation of the mean pufa/pufa score and the age group age (years) pmean pufapmean pufan mean pufa+pufa p <0.0014.5 ± 2.255<0.0016–8 85 4.48 ± 2.25 <0.001 0.02 ± 0.152 3.08 ± 1.8699–12 93 2.33 ± 1,936 0.75 ± 1.404 * p value <0.05 = the result of the mann-whitney statistical test was meaningful dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p10–13 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p10-13 160 dental care for children with autism spectrum disorder amrita widyagarini and margaretha suharsini department of pediatric dentistry faculty of dentistry, university of indonesia jakarta indonesia abstract background: providing dental treatment for children with autism spectrum disorder (asd) represents a challenge for dentists. in the dental care of such children, the treatment plans implemented are usually determined by several factors, including: the type of autism spectrum disorder, the degree of patient cooperation, dentist/patient communication, the required treatment, self-care skills and parental/dentist support. purpose: the purpose of this case report was to report the dental care delivered in the cases of two pediatric patients with asd. case 1: a 10.7 year-old boy with a nonverbal form of asd who was experiencing recurrent pain in his lower left posterior tooth and also presented a blackened tooth. case 2: a 9.6 year-old boy with a nonverbal form of asd suffering from numerous painful cavities. case management 1: on the day of the first visit, the boy was the subject of several behavioral observations. during the day of the second visit, he underwent a brief intraoral examination at a dental unit in order to arrive at a temporary diagnosis before appropriate was decided upon treatment in consultation with his parents. the implemented treatment plans comprised dental extraction and preventive restoration under general anesthesia. case management 2: on the first visit, the boy underwent behavioral observations followed by early intraoral examination involving physical restraint approach. during the second visit, several treatment plans such as: general anesthesia, tooth extraction, restoration, and pulp-capping treatment were formulated. conclusion: it can be concluded that general anesthesia was considered an appropriate dental treatment plan since the two patients in question were extremely co-operative during the necessary procedures. in other words, pediatric dental care treatment plans in cases of asd should be determined by clearly-defined criteria, specifically the benefits and risks of the treatment plans for the safety of both patient and dental care team. keywords: autism spectrum disorder; dental care; children correspondence: amrita widyagarini, department of pediatric dentistry, faculty of dentistry, university of indonesia. jl. salemba raya 4, jakarta 10430, indonesia. e-mail: amrita.widyagarini02@ui.ac.id dental journal (majalah kedokteran gigi) 2017 september; 50(3): 160–165 case report introduction autism spectrum disorder (asd) constitutes a group of developmental disorders characterized by impaired social interaction and communication, as well as behavioral limitations or repetition.1,2 most children with asd suffer several disabilities, for example: learning disabilities, attention deficiency and sensation and stimulation reaction disabilities. autism spectrum disorder, usually diagnosed before a child celebrates its third birthday, represents a life-long condition and affects members of all racial, ethnic and socioeconomic groups.3 the prevalence of asd is estimated to be 1% worldwide.2 the etiology of asd remains undefined, although it is believed to be a result of genetic and environmental factors.1,2 gene mutation, mitochondrial defects, cytosine regulatory disturbances, intrauterine androgen concentrations and the relatively advanced age of the mother during pregnancy are probable contributory factors in autistic pathophysiology.1 another theory is that children with autism have abnormal levels of serotonin and neurotransmitters in their brains.4,5 other potential factors comprise: infection, metabolic disorders, immunological disorders, poisoning, and fetal alcohol syndrome.4 children with asd tend to demonstrate behavioral problems which present a challenge for dentists when implementing routine treatment plans.6 as a result, a knowledge and understanding of the behaviorial patterns of children suffering from asd are key to successfully treating such individuals.1 those afflicted with asd also dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 mailto:amrita.widyagarini02@ui.ac.id 161161widyagarini and suharsini/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 160–165 tend to demonstrate limitations in verbal and/or non-verbal communication, often repeating words that do not refer to a specific context, while sudden or delayed echolalia can also occur.7 children with asd usually have little interest and engage in limited activity, while also demonstrating repetitive behavior often triggered by stress, pleasant feelings or certain stimuli (such as noise). they also tend to be routine-obsessed and show both a lack of motor coordination and repetitive body movements. one of the main characteristics of such children is a low frustration threshold often culminating in temper tantrums. the nature of agitation, aggression, and self-harming behavior can intensify as children with asd age. sensory perception may also be affected by audio and tactile hypersensitivity, excessive reaction to light and odor and an increased pain threshold.7,8 several studies have compared the health or otherwise of oral cavities in children suffering from asd with those of individuals free of the condition. these studies found that asd children tend to have low levels of oral hygiene with an elevated plaque index as well as poor gingival and periodontal conditions.9–11 oral hygiene is the most influential risk indicator associated with the occurrence of new caries and lesions in children with asd.12 consequently, their prevalence among asdafflicted children is higher than in those without asd, although the difference is not significant.10,11 moreover, there is also an increase in the incidence of malocclusion, as well as parafunction or self-harming habits in children with asd.10 various approaches and methods should be attempted to render such children co-operative during dental care procedures. a number of previous studies have already shown that children with asd have a tendency to be more less cooperative during dental examinations, for example, being reluctant to open their mouths or rejecting instruments inserted into their oral cavity9,11 there are actually several behavioural approaches that can be used for asd children during dental care. in general, more than one approach should be used for a patient with asd during his/her dental treatment.7 in other words, a modification of approaches in dental care is necessary.1 for instance, a commonly used tell-show-do approach is often ineffective with asd children because of their limited ability to focus. moreover, voice control technique accompanied by facial expressions are also ineffective because of the inability of asd children to understand the language and interpret the emotional expressions of others.6 most asd children have difficulty in understanding abstract contexts so communication should use short, clear, and simple language.1,7 as a result, a visual pedagogical approach combined with sensory adaptation in the clinical environment, applied behaviour analyses, and pharmacological techniques under general anaesthesia are considered to be useful.1 nevertheless, children with asd demonstrate a range of characteristics that vary, to a greater or lesser degree, between individuals. thus, there is no single behavioural approach that can be generalized across all asd-affected children.7 for these reasons, a pair of dental care cases conducted with two children presenting nonverbal varieties of asd are reported here. case case 1: a 10.7 year-old boy with asd weighing 45 kg and 130 cm tall attended a consultation accompanied by his mother and his caregiver. the mother reported that, during the previous week, her son had been shouting continuously, while pointing to the mandibular teeth on the left-hand side of his jaw. she also complained that the incisors of her son’s upper jaw had become blackened. the boy had been undergoing routine therapy with a psychiatrist as well as medical rehabilitation in an attempt to train his motorics. several drugs prescribed by the psychiatrist, such as persidal, prohiper, piracetam, carbamazepine and folic acid were being consumed by the boy on an ongoing basis. however, he had never consulted a dentist. significantly, the mother acknowledged that her son had been diagnosed with autism when he was three years old and had recently attended a school providing additional teacher support. based on the results of observations made during the first visit, the boy was known to be able to mimic words spoken by another person, although with imperfect articulation, responding to a conversation with a nod or a shake of the head. unfortunately, two-way communication proved impossible since, during the consultation at the clinic, the boy was unable to calm himself, his eyes perpetually moving, while he continually produced incomprehensible language amounting to little more than sounds. case 2: a 9.6 year-old boy weighing 28 kg, and 137 cm. in height was referred by a previous dentist for management of his teeth. the patient had attended the clinic on two previous occasions, but had never undergone treatment. two weeks before the visit, his mother had phoned the reception to make an appointment and explain her son’s condition. following an anamnesis with the patient’s mother held in the dental examination room, the resulting information confirmed that the child had been diagnosed with both asd and speech delay at the age of 3 years old. the patient did not attend school, but went to therapy sessions three times a week. he was not receiving therapy from a psychiatry and had not been prescribed any course of drugs. several bruises on his left hand were clearly evident, the result of selfinjury. both parents observed that their child appeared to suffer from toothache because of his frequent pointing at his cavitied teeth. they also commented that their child hardly ever brushed his teeth. the child had been to a dentist, but had never received any form of treatment. on attending the clinic, the child could not speak and behaved aggressively. however, he obeyed the father’s instructions. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 162 widyagarini and suharsini/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 160–165 case management case 1: during the first visit, an extraoral examination was performed, the results of which revealed no facial asymmetry. from the outset of the consultation, the child would sit on the dental chair, although only briefly, but did not want to open his mouth. the patient simply pointed with a finger to his left cheek. education on oral hygiene was delivered during the first visit. the patient’s mother was also instructed to train the child at home before the following visit, while the patient himself was asked to practice opening his mouth for ten seconds before brushing his teeth every day. the second visit took place one month later. the mother complained that the child was experiencing pain in his left rear mandibular teeth. unfortunately, the response of the child himself was consistent with that of his previous consultation. while the child remained agitated, tending to make sudden movements, he opened his mouth, even if only briefly, to be inspected. the examination was, therefore, limited to one using a dental mouth mirror. the results of the initial examination indicated the widespread presence of caries in the occlusal portion of tooth 36, the persistence of the residual roots of teeth 54, 52, 51, and 75, as well as general plaque without staining. however, a radiographic examination could not be performed. several treatment plans, including oral prophylactics, tooth extractions, and tooth restorations were to be carried out under general anesthetic by a pediatrician. in other words, the patient was scheduled for several dental treatments by a dentist and along with circumcision by a plastic surgeon on the same day under general anesthesia. during the third consultation, a full examination was performed after the patient had been placed under anaesthetic in the operating theatre. the examination led to a diagnosis of pulp caries on tooth 36 with widespread clinical conditions in the occlusal and proximal mesials followed by: loss of tooth marks, persistence of residual roots on teeth 54, 52, 51, 75, and 84, as well as email caries on teeth 16, 15, 14, 24, 25, 26, 36, 35, 34, 44, 45, and 46 (figure 1). several treatments were performed, namely: oral prophylaxis, dental restorative restorations on teeth 16, 15, 14, 24, 26, 36, 35, 34, 44, 45 and 46, tooth extractions on 54, 52, 51, 75, 84, and 36 as well as fluoride application. after surgery, several instructions were issued to maintain oral hygiene, including: brushing of the teeth, compressing the wound area with gauze soaked in chlorhexidine and following a soft food diet for two days. management of post-action pain and fluid therapy was performed and monitored by a pediatrician with a 7-hour post-operative control subsequently being conducted. the control results consisted of subjective data indicating the absence of both pain in the extraction sites and clinical bleeding, but blood clots already covering the extraction sockets. during the fourth visit, there was no subjective complaint when the patient came to the control two weeks after surgery. again, attempts were made to motivate him to maintain oral hygiene at a high level, while his mother was taught, on this occasion, the most effective technique for brushing her son’s teeth. the fifth visit occurred when the patient came to the control after a year. he initially resisted, having no memory of the atmosphere at the clinic. however, a brief examination was conducted by means of a mouth glass, the results of which confirmed a high level of oral hygiene and the absence of new caries. case 2: during the first visit, an initial examination was performed with the assistance of the subject’s father and an assistant, while the subject himself was restrained in the dentist’s chair. the examination resulted in a provisional diagnosis of residual roots on tooth 84, pulp caries in teeth 55, 65, 74, and 85, as well as dentine caries in teeth 16, 12, 21, 26, 36, and 46 (figure 2). the patient’s parents were then instructed during the initial visit on how to brush their child’s teeth. the patient’s apparent greater obedience to his father rendered the latter more motivated to help the child brush his teeth. the patient was subsequently scheduled for several treatment sessions under general anaesthetic in accordance with the parents’ wishes following a discussion involving the dentist and both the mother and father. radiographic examination could not be performed since the child could remain calm. during the second visit, a complete diagnosis was made when the patient was in the operating room, indicating the persistence of residual roots on tooth 84, pulp caries in teeth 55, 65, 74, and 85, dentine caries in teeth 16, 12, 21, 22, 26, and 36, and rocking movement in tooth 64. several dental treatments were subsequently performed, namely: oral prophylaxis, pulpcapping with mta, restoration of composite resins on tooth 21, restoration of composite resin on tooth 22, restoration of glass ionomer cement on teeth 16, 12, 26, 36, and 46, as well as tooth extractions of 55, 65, 74, 84, and 85 followed 3.0 vicryl yarn stitches between teeth 84 and 85. management of post-action pain and fluid therapy were performed and monitored by a pediatrician. a 19-hour post-operative control was then conducted which produced clinical data indicating controlled bleeding and no swelling. figure 1. pretreatment intraoral examination in case 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 163163widyagarini and suharsini/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 160–165 the patient was able to eat 15 hours after surgery. the patient’s mother was asked to compress the extraction area with chlorhexidine and maintain the oral hygiene of the child by regular brushing. she also received an explanation about the prognosis of care. the patient then returned for a further consultation three weeks later during which he made no complaint of pain. during the third visit, the patient’s behavior was more positive than during the first. for example, he proved able to briefly sit alone in the dentist’s chair without being forced to. moreover, the father confirmed that his son had started to brush his teeth regularly. the patient was then refered to the previous dentist for regular check-ups. discussion the initial examinations of both patients showed signs of asd confirmed by anamneses from the patients’ mother. there are actually several indicators of autism disorders in children, namely; a failure to communicate effectively due to their ignorance about the environment as well as their inability to provide information using verbal language, gestures and eye contact.1 a number of previous studies have also revealed that most children with asd demonstrate a range of self-harming behavior as a means of expressing their feelings of stress, anger and discomfort.11,13 similarly, both patients also suffered from a similar inability to express their emotions verbally, to engage in two-way communication, to concentrate on others, to make eye contact, or to avoid aggressive behaviour. for instance, several bruises on the second patient’s left hand were caused by self-harming, a fact corresponding to the findings of previous studies. against this background, obtaining as much information as possible from parents/caregivers is essential before treating asd children.7 anamneses involving parents should focus on those children’s positive points, the things they love, appropriate types of gift for them, whether they can talk or not, as well as the best means of communicating with them. in addition, it is necessary to explore information about what those children are afraid of.14 some experts even suggest pre-visit interviews with parents in order to establish the potential for their children to show cooperative behavior and to plan a behavioral approach.7 in general, certain risk factors cause asd children to be uncooperative during dental care, namely: age (4-7 years or >7 years), reading ability (strong or weak), accompanying systemic diagnosis (present or absent) and speaking ability (evident or not evident). the presence of two or more risk factors indicates a tendency towards uncooperative behavior. by knowing these facts in advance, the dental care team is then expected to be able to predict the cooperativeness of such children. 7 in case 1, information about the patient was obtained during anamnesis on the first visit. meanwhile, in case 2 this was elicited from a combination of the pre-visit history and his first consultation at the clinic. however, the patient’s parents in case 2 were more prepared to provide information than were their case 1 counterparts since the patient in case 2 had previously visited a dentist. the resulting information then served as the dental team’s guidance in preparing its members and in managing the duration of the visit. actually, the presence of parents/caregivers in the clinic can also help to increase the trust and cooperativeness of patients.7 in both cases, this had even proved extremely helpful to the dental team in treating each of the two patients with nonverbal forms of asd. such parental involvement also provided benefits in delivering dental health education to patients. in these cases, operators were able to motivate parents to maintain dental and oral hygiene at home as a preventive measure since they faced challenges in delivering education directly to those children. patients with asd are very easily distracted. consequently, certain steps, such as making physical contact with them during dental treatment, should be avoided. in addition, sensory stimuli (sound, odor, etc.) should also be reduced in intensity.7 thus, prior to the initial examination of both patients, the examination chair had been adjusted in order to minimize potential distractions. sudden movements of the chair in question were also avoided, as was the use of dental handpiece swivel instruments during the initial visit. unfortunately, the intraoral examination was neither completely nor successfully performed during the first visit of either patient. individuals with asd generally experience hypersensitivity in the intraoral and perioral areas and tend to be sensitive to even a light touch during dental examination. thus, refusal to cooperate or physical resistance during a dental examination is a distinct possibility with young asd patients.1,13 similarly, previous research has already shown that most asd children will demonstrate unhelpful behavior during dental examination.11,13 therefore, it is advisable that such patients be introduced pre-visit to clinical situations through pictorial stories or home exercises in order to familiarize them with tools and procedures, including the type of orders issued by dentists.1,7 certain literature suggests that the use of a visual-based pedagogical approach, including the use of photographs, figure 2. pretreatment intraoral examination in case 2. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 164 widyagarini and suharsini/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 160–165 during dental examinations is more effective than oral explanations given to children with asd.1,14 in other words, before attending a dental clinic, such asd patients should be introduced to what a consultation involves.14 visits to a dental clinic should also be repeated, if possible, and scheduled for the same time and day on each occasion. similarly, they should, ideally, involve the same medical team and the same examination chair. both waiting time and total duration of treatment should be minimized. in addition, in order to render patients calmer during an examination, it is advisable that they bring their favorite objects and/or music as a “distractor” from stress or panic.1,7 the patient’s mother in case 1 was asked to help the dentist train the child to open his mouth by pretending to be a dentist herself. the patient’s appointment was then rescheduled. however, obstacles occurred during the oral examinations of both children. for instance, the results of a provisional clinical examination indicated that invasive action in relation to certain teeth was required. however, the patients proved uncooperative and it was thus necessary to administer a pre-treatment general anesthetic. according to certain literature, the behavior of asd children in the clinic can actually determine appropriate approaches.7 a number of experts also argue that a restraining approach may prevent the possibility of sudden aggressive behavior.7 this method was then adopted in treating the patient in case 2 during the oral examination. however, such an approach is not considered a good option for asd patients during dental treatment due to their relatively advanced age and specific physical factors. during dental treatment, asd children also require certain pharmacological support. consequently, both their medical history and medication intake need to be reviewed.7 the administering of nitrous oxide to asd patients represents a challenge, given the prerequisite level of communication. therefore, if the patients are unable to respond to a form of sedation using nitrous oxide, dental treatment involving the administering of a general anesthetic should be undertaken. moreover, the need for extensive treatment (involving four quadrants) and/or complex treatment also triggers the use of general anesthesia.7 according to other literature, endodontic treatment in children with growth and developmental disorders depends on the patient’s behavioral aspects. if radiographic examination is not possible, endodontic treatment may still be possible although the success of the treatment may be seriously compromised. therefore, tooth extraction is considered to constitute a better option.3 in case 1, extraction was performed on tooth 36 following a diagnosis of pulp caries since the success of previous endodontic treatment remained uncertain. the patient could not be subjected to radiographic photograph examination. similarly, definitive restoration after endodontic treatment of the molars in question could not be performed since it requires several visits to complete onlay restoration. meanwhile, if direct plastic restoration had been performed, it would not have endured for long due to a lack of support from the remaining healthy dental tissues resulting in the failure of endodontic treatment. in case 2, extraction was also performed on the first molars with pulp caries since the patient could not undergo radiographic photograph examination. as a result, the prognosis remained provisional and definitive restoration, using stainless steel crowns, was questionable since his teeth were almost exfoliated. however, while direct restoration could still be performed, restoration resistance was open to doubt since the teeth in question had already lost a considerable amount of structure. consequently, restoration was required to protect the entire crown of each tooth. direct restoration, however, would play a role as temporary restoration that later needed to be replaced. partial pulpotomy treatment was also performed on tooth 21 after the pulp was opened during caries excavation, even though it was not preceded by the taking of radiographic photographs. extraction was not carried out on tooth 21 due to aesthetic reasons. the patient’s parents were then given information about the prognosis of dental care (pulp treatment and post-treatment restoration). in addition, previous research suggests that dentists should educate parents of asd children receiving dental care under general anesthesia about the risk of restoration failure.15 actually, while there is no limit to the frequency with which an individual can be placed under general anesthetic, it is advisable to reduce the risk of anesthesia by minimizing the need for revisits.3 as a result, restoration given to patients undergoing general anesthesia should be considered for its prognosis. another point to consider in planning dental care for a patient with impaired growth and development is the assessment of that individual’s understands of the role and importance of oral care. therefore, aggressive patients who refuse caregivers’ assistance in maintaining oral hygiene will not achieve promising restoration results. similarly, patients who are physically incapable of brushing or flossing cannot achieve any complex dental restoration since cooperation and careful attention play an important role in its success.3 finally, it can be concluded that dental care for children with asd should take account of the safety benefits and risks to both patient and dental teams. the dental care provided to children with asd should also be supported by preventive efforts on the part of parents/caregivers and children. moreover, general anesthesia may be considered to be a valid solution if other behavioral management options have been implemented. ultimately, dental treatment under general anesthesia will have greater benefits than risks. references 1. delli k, reichart pa, bornstein mm, livas c. management of children with autism spectrum disorder in the dental setting: concerns, behavioural approaches and recommendations. med oral patol oral cir bucal. 2013; 18(6): e862–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 165165widyagarini and suharsini/dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 160–165 2. american psychiatric association. diagnostic and statistical manual of mental disorders. 5th ed. arlington: american psychiatric publishing; 2013. p. 5-25. 3. raposa k a, perlman sp. treating the dental patient with a developmental disorder. 1st ed. iowa: wiley-blackwell; 2012. p. 1–31, 155–67, 195–206. 4. nagendra j, jayachandra s. autism spectrum disorders: dental treatment considerations. j int dent med res. 2012; 5(2): 118–21. 5. gabriele s, sacco r, persico am. blood serotonin levels in autism spectrum disorder: a systematic review and meta-analysis. eur neuropsychopharmacol. 2014; 24(6): 919–29. 6. hernandez p, ikkanda z. applied behavior analysis: behavior management of children with autism spectrum disorders in dental environments. jada. 2011; 142(3): 281–7. 7. limeres-posse j, castaño-novoa p, abeleira-pazos m, ramosbarbosa i. behavioural aspects of patients with autism spectrum disorders (asd) that affect their dental management. med oral patol oral cir bucal. 2014; 19(5): e467–72. 8. spence sj, sharifi p, wiznitzer m. autism spectrum disorder: screening, diagnosis, and medical evaluation. semin pediatr neurology. 2004; 11(3): 186–95. 9. demattei r, cuvo a, maurizio s. oral assessment of children with an autism spectrum disorder. j dent hyg. 2007; 81(3): 1-11. 10. bartolomé-villar b, mourelle-martínez mr, diéguez-pérez m, de nova-garcía m. incidence of oral health in paediatric patients with disabilities: sensory disorders and autism spectrum disorder. systematic review ii. j clin exp dent. 2016; 8(3): e344–51. 11. el khatib aa, el tekeya mm, el tantawi ma, omar t. oral health status and behaviours of children with autism spectrum disorder: a case-control study. int j paediatr dent. 2014; 24(4): 314–23. 12. marshall j, sheller b, mancl l. caries-risk assessment and caries status of children with autism. pediatr dent. 2010; 32(1): 69–75. 13. murshid ez. oral health status, dental needs, habits and behavioral attitude towards dental treatment of a group of autistic children in riyadh, saudi arabia. saudi dent j. 2005; 17: 132–9. 14. wright gz, kupietzky a. behavior management in dentistry for children. 2nd ed. oxford: wiley blackwell; 2014. p. 93–105. 15. tate ar, ng mw, needleman hl, acs g. failure rates of restorative procedures following dental rehabilitation under general anesthesia. pediatr dent. 2002; 24(1): 69–71. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p160-165 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p160-165 209209 dental journal (majalah kedokteran gigi) 2019 december; 52(4): 209–214 research report the effects of shark liver oil on fibroblasts and collagen density in the periodontal ligaments of wistar rats induced with porphyromonas gingivalis dian mulawarmanti, dwi andriani, dian widya damaiyanti, farizia putri khoirunnisa and alifati nita juliatin department of oral biology, faculty of dentistry, universitas hang tuah, surabaya – indonesia abstract background: periodontitis is an infection in tooth-supporting tissues caused by a specific microorganism, porphyromonas gingivalis (pg), which can trigger collagen destruction. generally, periodontal therapy employs a combination of mechanical (scaling root planning/srp) and chemical (antibiotics) remedies, the latter of which can cause bacterial resistance. on the other hand, shark liver oil contains active natural ingredients such as alkylglycerols, squalene, squalamine, and omega-3, which have antibacterial and antioxidant effects. purpose: this study aims to determine the impact of shark liver oil on fibroblasts and collagen density in the periodontal ligament of wistar rats induced with pg. methods: this study represents a laboratory experiment with post-test only control group design. the research subjects consisted of 35 wistar rats divided into five groups, namely; a negative control group (k-); a positive control group with pg induction (k+); and three treatment groups induced with pg and shark liver oil once a day for seven days at varying doses of 0.2 g/gbb (p1), 0.3 g/gbb (p2), and 0.4 g/gbb (p3). following treatment, the subjects were euthanized. the number of fibroblasts was then histologically examined with hematoxylin eosin (he). meanwhile, the collagen density was histologically analyzed with masson’s tricrome. fibroblast cells were observed through a microscope at 400x magnification. data was statistically analyzed with a one-way anova and post hoc lsd. collagen density scoring was then performed. the results were analyzed with a non-parametric kruskal-wallis test (p=0.05), and subsequently with a mann-whitney u test (p<0.05). results: the number of fibroblasts in the periodontal ligament areas of each group were 18.6 ± 1.21 for k-; 12 ± 1.26 for k;16.8 ± 1.72 for p1; 17.1 ± 1.94 for p2; and 23.16 ± 2.78 for p3. the results also indicated that there were significant differences between kwith k+ and p3, k+ with p1, p2, and p3, as well as p3 with p1 and p2. however, there was no significant difference between kand p1 and p2 or p1 and p2. the results showed that collagen density in the negative control group did not significantly decrease compared to that in the positive control group in which pg was induced. meanwhile, collagen density in all three treatment groups following doses of 0.2 g/gbb, 0.3 g/gbb, and 0.4 g/gbb being administered significantly increased compared to that in the negative control group and the positive control group subjected to pg induction. conclusion: shark liver oil can significantly increase fibroblast cells and collagen density in the periodontal ligament of wistar rats induced with pg. keywords: collagen density; fibroblast; periodontitis induced with porphyromonas gingivalis; shark liver oil correspondence: dian widya damaiyanti, department of oral biology, faculty of dentistry, universitas hang tuah, jl. arif rahman hakim no. 150, surabaya, indonesia. e-mail: damaiyanti@hangtuah.ac.id introduction various oral and dental diseases have recently been found to be widespread among indonesians. based on national basic health research statistics issued by the indonesian ministry of health in 2018, the prevalence of such diseases within the country increased from 25.9% in 2013 to 57.6% in 2018.1 periodontitis is an infection that occurs in tooth supporting tissues caused by a specific microorganism which triggers progressive damage in periodontal ligaments and alveolar bone resorption.2 the dominant bacteria found in chronic periodontitis are gram-negative anaerobic varieties such as porphyromonas gingivalis.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 210 mulawarmanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 209–214 porphyromonas gingivalis produces various pathogenic virulence factors such as lipopolysaccharide (lps).3 lps are then recognized by toll-like receptors-4 (tlr-4) present in adjacent cells, namely; junctional epithelium (je), macrophages, and dendritic cells.4 macrophages function to secrete anti-inflammatory mediators, proinflammatory mediators, and growth factor. the activated tlr 4 pathway can subsequently affect the release of pro-inflammatory mediators including il-1, il-8, il-12, and tnf-α.5 tnf-α and il-1 causing neutrophils and monocytes to be attracted to the site of bacterial invasion.4 furthermore, untreated periodontitis can cause periodontal pocket formation, damage to periodontal ligaments, and alveolar bone density changes.6 certain types of collagen responsible for maintaining tissue structure1 are present in the periodontal ligaments, the most common of which are type i collagen (80%) and type iii collagen (20%).7,8 current periodontitis therapy usually employs a combination of mechanics, including scaling root planning, and chemicals such as antimicrobials.2 unfortunately, at the specified dose, the use of antimicrobials, especially antibacterials (antibiotics), is ineffective, while the passage of time can cause resistance. as a result, several experts conducted various studies to identify an antibacterial in order to overcome the problem.9,10 however, the wound healing process is influenced by local and systemic factors. hence, periodontitis therapy is expected to not only eliminate disease-causing bacteria (local factors), but also to suppress damage to the host cell inflammatory response components (systemic factors).11 sharks can be found in almost all indonesian waters, be they territorial, oceanic, or the country’s exclusive economic zone (eez).12 centrophorus moluccensis is a type of shark whose liver oil is commonly extracted for subsequent use for medicinal purposes.13 previous research conducted by agustina (2015)14 indicated that shark liver oil has inhibitory properties in relation to porphyromonas gingivalis bacteria. the alkylglycerol contained in sharks can even protect the structure and function of white blood cell membranes and macrophages.15 macrophages play a role in the phagocytosis of bacteria and secrete anti-inflammatory cytokines, thus triggering fibroblast proliferation and collagen synthesis.16 in addition, another investigation conducted by hafez et al. (2011),17 argued that the safe dose of orally-administered shark liver oil for rats is one of 1,000-2,000 mg/kg/day. therefore, this study aims to reveal the effects of shark liver oil on fibroblast cells and collagen density in the periodontal ligaments of rats induced with porphyromonas gingivalis at certain doses, namely; 0.2 g/g bw, 0.3 g/g bw, and 0.4 g/g bw, as an supplementary therapy for seven days. materials and methods this study was approved by the dental research ethics commission of hang tuah university (certificate number: ec/008/kepk-fkguht/vii/2019). its conduct followed the guidelines of the dental research ethics commission, universitas hang tuah and constituted a true laboratory experiment incorporating post-only group design. the research subjects comprised 35 rats aged 4-6 months and weighing 200-250 grams which were divided into five groups, namely; a negative control group (k-) which received no treatment; a positive control group (k+) induced with porphyromonas gingivalis, but without shark liver oil; and three treatment groups induced with porphyromonas gingivalis and shark liver oil at specific doses, namely; 0.2 g / g bw (p1); 0.3 g / g bw (p2); and 0.4g / g bw (p3). wistar rats were acclimatized to the experimental laboratory in cages with sufficient air and light for seven days.18 on the eighth day, all groups of subjects were given 20 mg of kanamycin and 20 mg of ampicillin in drinking water. their oral mucosa was then smeared with chlorhexidin gluconate 0.12% once daily for four days.19 on day 12, groups 2, 3, 4, and 5 were orally induced with up to 2 ml of 1 x 109 cfu/ml of porphyromonas gingivalis bacteria using feeding tubes three times in four days at 32hour intervals, before being applied topically to their oral cavity and anus in the colorectal section using a cotton bud or microbrush. the subjects were subsequently incubated for three weeks following the first induction. periodontitis is characterized by a change in the periodontal tissue during which blood cells from the vasa migrate into the extravagant tissue as part of the healing process.20,21 after three weeks, the wistar rats in groups 3, 4, and 5 were orally fed shark liver oil (centrophorus sp obtained from factory x) at various concentrations for seven days. on the 41st day, all the subjects were sacrificed and their mandibles extracted. these were then fixed in a formalin buffer solution (pbs ph 7.0 and formalin 10%). the number of fibroblasts was observed with he staining technique, while collagen density was examined by means of mt painting technique. fibroblast cells in the periodontal ligament were examined with a microscope from three fields of view and their number calculated based on an average magnification of 400x. fibroblast cells appeared with a large chromatic figure 1. histopathological picture of fibroblasts in a periodontal ligament at 400x magnification. fibroblast cells are indicated by black arrows. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 211mulawarmanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 209–214 nucleus and eosinophilic or spindle-shaped cytoplasm. meanwhile, the assessment of collagen density in periodontal ligaments was observed from five fields of view using an olympus cx 22 light no.5 microscope at 400x magnification. mt staining produced a light blue or greenish blue colour in type 1 collagen and red in the core, keratin, and cytoplasm. all research data was then tabulated and analyzed statistically with a one-way anova test to calculate the number of fibroblasts, followed by a kruskal wallis test. meanwhile, a mann-whitney test was performed to assess collagen fiber density using 2016 spss version 23. results the examination results of fibroblasts with he painting were examined by two observers with their mean values being subsequently calculated. figure 1 shows the results of he painting on the mandibular after decalcification. moreover, based on the calculation results of the mean number of fibroblast cells, the highest number of fibroblasts was found in group p3 which had been administered a 0.4 g / g bw dose of shark liver oil (figure 2). the data was analyzed with a one-way anova test the results of which revealed that there were differences (p<0.05) within each group. a post-hoc lsd test was then performed to analyze differences between the groups. based on the results of the lsd post-hoc test (table 1), there were no significant differences between kwith p1 and p2, or between p1 and p2 (p>0.05). the observation results of collagen density based on the data in figure 3 were as follows: the highest collagen density value occurred in group p3 which had received shark liver oil at a concentration of 0.4 g / g bw. histochemistry result of collagen density was shown in figure 4. collagen density was assessed by two observers with a value mode to identify the highest score. the second and third highest collagen density values were found in group p2 and group k, while the lowest was that in group k+ which had been induced only with pg bacteria. the data obtained was then statistically analyzed with a kruskal wallis test. since the significance value obtained was 0.001 (p<0.05), a mann whitney analysis was completed to identify the differences between the groups. 18.6 12 16.8 17.1 23.1 5 10 15 20 25 30 k(-) k(+) p1 p2 p3 the numbers of fibroblast figure 2. the graph of the mean number of fibroblast cells in periodontal ligament suffering from periodontitis in each group. kk+ p1 p2 p3 figure 4. the results of collagen fibrous density preparations in periodontal ligaments at 400x magnification using mt staining (collagen fibers are indicated by black arrows). 0,5164 0,54772 0,54772 0,75277 0,83666 0 1 2 3 4 5 6 kk+ p1 p2 p3 modus figure 3. a graph of collagen density. 18.6 12 16.8 17.1 23.1 5 10 15 20 25 30 k(-) k(+) p1 p2 p3 the numbers of fibroblast dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 212 mulawarmanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 209–214 based on the results of mann-whitney (table 2), no significant difference existed between group kand group k+ (p=0.575). however, there was a significant difference between group kand group p1(0.030), group p2(0.016), and group p3(0.007). similarly, a significant difference was identified between group k+ and group p1 (0.019), group p2 (0.011), and group p3 (0.006). in contrast, there was no significant difference between group p1 and group p2 (0.423). while a significant difference existed between group p1 and group p3 (0.042), there was none between group p2 and group p3 (0.147). discussion the bacterial induction procedure for porphyromonas gingivalis in this study was conducted for three weeks based on previous research by praptiwi (2008)22 and mulawarmanti et al. (2014).23 the condition of periodontitis is detected by an anatomic pathological examination of periodontal tissue that shows the existence of extravation. periodontitis is characterized by changes in periodontal tissue that indicate the migration of blood cells from the vasa to the extravagant tissue as part of the healing effort.24 shark liver oil was applied orally to the wistar rats (rattus novergicus) in the treatment groups which had been induced with porphyromonas gingivalis bacteria potentially triggering collagen destruction. the incidence of such destruction was found in group k+ induced with porphyromonas gingivalis bacteria since the results of collagen density in this group confirmed that the number of fibroblast cells was the lowest. the lps component in the outer membrane layer of the porphyromonas gingivalis bacterium will usually bind to the periodontal tissues, thereby causing the release of trl-4.5,25 the activation of trl-4, in turn, triggers the release of pro-inflammatory mediators, such as il-1 and tnf-α, with the result that it influences the attempts of the host to fight infection by activating immune cells, for example; pmns, macrophages, and lymphocytes.25,26 excessive pmn activity can increase the ros to levels that damage various cell mechanisms. such harm can cause the destruction of gingival tissue, periodontal ligaments, and alveolar bone.25,27 the results of this study show that the number of fibroblast cells in the group k members, i.e normal rats, was not significantly different to that in the treatment groups. this means that the administration of shark liver oil can render the condition of the subjects induced with porphyromonas gingivalis bacteria normal again, as those in group k-. furthermore, this also indicates that its provision can prevent further damage by stimulating fibroblast cell regeneration. the results of this study also reveal that a significant increase in the number of fibroblast cells occurred in those subjects induced with porphyromonas gingivalis and subsequently treated with shark liver oil at doses of 0.2 g / g bw, 0.3 g / g bw, and 0.4 g / g bw. the strongest antibacterial properties were found in the treatment group following the administration of shark liver oil at a dose of 0.4 g / g bw compared to those in the other treatment groups whose members received doses of 0.2 g / g bw and 0.3 g / g bw. this indicates that the high antibacterial content of shark liver oil at a dose of 0.4 g / g bw enables it to inhibit inflammatory cytokines and also increase fibroblast proliferation in the periodontal ligaments. an additional finding of this study is the significant difference in the density of collagen between group k-, i.e. normal mice, and the treatment groups which had received shark liver oil at doses of 0.2 g / g bw, 0.3 g / g bw, and 0.4 g / g bw. the density of collagen in group k was lower than that in the treatment groups following the administration of shark liver oil at doses of 0.2 g / g bw, 0.3 g / g bw, and 0.4 g / g bw. this indicates that the provision of this oil can inhibit further collagen damage by stimulating the formation of new collagen. in general, collagen begins to form on the third day and increases until the 21st day at the end of the proliferation process.16 high-speed collagen synthesis returns the wound to normal tissue within a period of six to twelve months. early in the proliferation process, fibroblasts will proliferate binding to extracellular matrix to form scar tissue and accelerate wound healing. collagen accumulation caused by excessive fibroblast synthesis will reorganize causing regular tissue to form along the wound. synthesis of collagen by fibroblasts is controlled by collagenase and other factors that damage new collagen.28 collagen remodeling during the maturation phase actually depends on collagen synthesis by fibroblasts and collagen degradation in the maturation phase (remodeling).39 the maturation phase is the longest within the healing process and lasts approximately one year. table 1. results of post-hoc lsd test on the mean number of fibroblasts in periodontal ligament suffering from periodontitis groups/mean k+ p1 p2 p3 k(18.6) .000* .103 .178 .000* k+ (12) .000* .000* .000* p1 (16.8) .761 .000* p2 (17.16) .000* note *: p<0.05 table 2. the results of a mann-whitney test k+ p1 p2 p3 k.575 .030* .016* .007* k+ .019* .011* .006* p1 .423 .042* p2 .147 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 213mulawarmanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 209–214 the excessive production of collagen by fibroblast cells will be degraded by collagenase and metalloproteinase enzymes resulting in collagen being more organized.16 fibronectin gradually disappears and both hyaluronic acid and glycosaminoglycans are replaced by proteoglycans. at this time, collagen fibers close together causing collagen cross-linking and ultimately degrading excess collagen.28 type iii collagen will then be replaced by type i collagen, thereby increasing the strength of collagen fibers (tensile strength). however, the strength of collagen fibers can only recover up to 80% of the strength of normal collagen fibers which prevailed before wound formation.16 this study also proved that subjects induced with porphyromonas gingivalis bacteria had a significant difference from the treatment groups induced with porphyromonas gingivalis bacteria and then administered specific doses of shark liver oil, namely; 0.2 g / g bb0.3 g / g bw, and 0.4 g / g bb up to a maximum of 2 ml orally to subjects once a day for seven days. the shark liver oil therapy provided was expected to increase fibroblast cells and restore collagen density leading to inhibition of further damage. previous research conducted by alhanout et al. (2010)10 indicated that squalene and squalamine can act as antibacterials. they were both tested on both gramnegative bacteria, for example; escherichia coli and pseudomonas aeruginosa, in addition to gram-positive bacteria such as staphylococcus aureus and streptococcus pneumoniae. similarly, another investigation undertaken by agustina (2015)14 showed that shark liver oil has inhibitory properties in relation to porphyromonas gingivalis bacteria. alkylglycerol can bind to phospholipid cell membrane changing its structure to membrane fluidity and antioxidants and protecting the structure and function of membranes in white blood cells and macrophages.15 moreover, squalen and omega 3 can be considered as scavengers of radical peroxide (antioxidant) capable of inhibiting ros, thereby preventing oxidative stress.29,30 this intensive antioxidant activity can, in turn, reduce ros and increase collagen synthesis.31 the group of subjects given shark liver oil therapy through a 0.2 g / g dose of bw experienced a significant difference to that administered a 0.4 g / g dose of bw. however, there was no significant difference between the group of subjects given shark liver oil therapy at a dose of 0.2 g / g bw to that administered a 0.3 g dose of / g bw. this might be due to subject susceptibility to small doses. in other words, the higher the dose administered, the more active the ingredients of the drug compound in the connective tissue will be. hence, the inhibition of inflammation and destruction progression is higher.32 the group of subjects given shark liver oil therapy through a 0.2 g / g dose of bw did not demonstrate a significant difference from the group given one of 0.3 g / g bw. however, there was a significant difference in collagen density between the group of subjects given shark liver oil therapy at the dose of 0.2 g / g bw and the group administered one of 0.4g / g bw. the highest number of fibroblasts was also found in the group of subjects which received shark liver oil therapy at a dose of 0.4g / g bw. this indicates that this form of therapy at a dose of 0.4g / g bw is effective in treating porphyromonas gingivalis bacteria since it possesses the ability to increase the number of fibroblasts and stimulate the formation of new collagen. shark liver oil also contains squalene with a high level of oxygen carried throughout cell membrane.28 the nature of squalene is contrary to the that of porphyromonas gingivalis bacteria which are anaerobic gram-negative bacteria. as a result, it can render unfavourable the environmental conditions of the porphyromonas gingivalis bacteria. the mechanism of squalamine contained in shark liver oil can even change the integrity of the bacterial cell membrane by increasing its permeability characterized by the release of atp and also directly encountering gram negative bacteria which cause damage to the outer cell membrane. shark liver oil contains alkylglycerol that can release proteases which transform autolytic enzymes from an inactive to an active state. this compound can even inhibit the synthesis of peptidoglycan in the bacterial cell wall causing it to experience lysis, leading to bacterial cell death.33,34 changes to the cell membrane will be evident from the wrinkled membrane structure of empty cells. stunted bacterial growth can then reduce the activity of macrophages in the phagocytic process.16 shark liver oil contains omega 3 composed of epa and dha which share the same physical and structural properties.35 squalene and omega 3 can also be considered scavengers of radical peroxide (antioxidants) which can inhibit ros, thereby preventing oxidative stress.29,30 moreover, omega 3 plays an important role as a useful anti-inflammatory in the wound healing process.35 in addition, alkylglycerol can also protect the structure and function of white blood cell membranes and macrophages.15 macrophages are cells that play the most important role in the wound healing process since they promote the phagocytosis of bacteria, thereby assuming the role of pmn. macrophages will turn into macrophage efferocytosis (m2) which secrete anti-inflammatory cytokines such as il-4, il-10, and il-13. il-4 plays a role in fibroblast proliferation and collagen synthesis.16 macrophages also produce growth factors, for example pdgf, fgf and tgf-β, which induce fibroblasts to proliferate, migrate, and form an extracellular matrix.16,36 over time, this extracellular matrix will be replaced by type iii collagen which is also produced by fibroblasts. the type iii collagen will be subsequently replaced by type i collagen during the maturation phase.19 increased collagen fibers can signal the process of wound healing.37 ultimately, the wound healing process is influenced by both local and systemic factors with the result that therapy not only removes disease-causing bacteria (local factors), but also suppresses damage to the host cell inflammatory response component (systemic factors).11 in other words, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 214 mulawarmanti, et al./dent. j. (majalah kedokteran gigi) 2019 december; 52(4): 209–214 given its antibacterial properties, antioxidants, and immune cell abilities, shark liver oil promotes the healing process of chronic periodontitis at doses of 0.2 g / g bw; 0.3 g / g bw; and 0.4 g / g bw. in conclusion, shark liver oil at doses of 0.2 g / g bw; 0.3 g / g bw; and 0.4 g / g bw can increase fibroblasts and collagen density in the periodontal ligaments of wistar rats induced with porphyromonas gingivalis bacteria. nevertheless, shark liver oil at a dose of 0.4 g / g bw has the most profound effect on increasing fibroblasts and collagen density in the periodontal ligaments of wistar rats induced with porphyromonas gingivalis bacteria. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2018. jakarta: kementerian kesehatan republik indonesia; 2018. p. 627. 2. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 12th ed. st. louis: saunders elsevier; 2015. p. 50–1. 3. alibasyah zm, ningsih ds, ananda sf. daya hambat minumann probiotik yoghurt susu sapi terhadap porphyromonas gingivalis secara in vitro. j syiah kuala dent soc. 2018; 2(3): 65–75. 4. geh r ig js (jill s, shin de, willmann de. foundations of periodontics for the dental hygienist. 3rd ed. philadelphia: wolters kluwer; 2012. p. 879. 5. hikmah n, dewanti idar. peran toll like receptors (tlrs) pada innate immunity (the role of toll like receptors to innate immunity). stomatognatic. 2011; 8(1): 21–6. 6. holtfreter b, albandar jm, dietrich t, dye ba, eaton ka, eke pi, papapa nou pn, kocher t. sta nda rds for repor ting chronic periodontitis prevalence and severity in epidemiologic studies: proposed standards from the joint eu/usa periodontal epidemiology working group. j clin periodontol. 2015; 42(5): 407–12. 7. bartold pm, narayanan as. biology of the periodontal connective tissues. usa: quintessence publishing; 1998. p. 203–5, 211. 8. berkovitz bkb, moxham bj, newman hn. the periodontal ligament in health and disease. 24th ed. london: mosby-wolfe; 1995. p. 55–6, 71–2. 9. kusumawardani b, pujiastuti p, sari s. uji biokimiawi sistem api 20 a mendeteksi porphyromonas gingivalis isolat klinik dari plak subgingiva pasien periodontitis kronis. j pdgi. 2010; 59(3): 110–4. 10. alhanout k, malesinki s, vidal n, peyrot v, rolain jm, brunel jm. new insights into the antibacterial mechanism of action of squalamine. j antimicrob chemother. 2010; 65(8): 1688–93. 11. newman mg, takei hh, klokkevold pr, carranza fa. carranza’s clinical periodontology. 11th ed. st. louis: saunders elsevier; 2012. p. 13, 33–54, 230–60, 387–8. 12. alaydrus is, fitriana n, jamu y. jenis dan status konservasi ikan hiu yang tertangkap di tempat pelelangan ikan (tpi) labuan bajo, manggarai barat, flores. al-kauniyah j biol. 2014; 7(2): 83–8. 13. guyton ac, hall je. guyton dan hall buku ajar fisiologi kedokteran. 12th ed. widjajakusumah m, tanzil a, editors. singapore: elsevier saunders; 2014. p. 1151. 14. agustina fm, mulawarmanti d, wedarti yr. daya hambat minyak hati ikan hiu terhadap pertumbuhan bakteri porphyromonas gingivalis. denta. 2015; 9(2): 1–5. 15. deniau al, mosset p, pédrono f, mitre r, le bot d, legrand ab. multiple beneficial health effects of natural alkylglycerols from shark liver oil. mar drugs. 2010; 8(7): 2175–84. 16. primadina n, basori a, perdanakusuma ds. proses penyembuhan luka ditinjau dari aspek mekanisme seluler dan molekuler. qanun med med j fac med muhammadiyah surabaya. 2019; 3(1): 31–43. 17. abdel-hafez amm, othman ma, seleim maa. effect of shark liver oil on renal cortical structure in hypercholesterolemic rats. egypt j histol. 2011; 34(2): 391–402. 18. kusumawati d. bersahabat dengan hewan coba. yogyakarta: gadjah mada university press; 2004. p. 5–22. 19. kesavalu l, sathishkumar s, bakthavatchalu v, matthews c, dawson d, steffen m, ebersole jl. rat model of polymicrobial infection, immunity, and alveolar bone resorption in periodontal disease. infect immun. 2007; 75(4): 1704–12. 20. rukmini a. regenerasi minyak goreng bekas dengan arang sekam menekan kerusakan organ tubuh. in: seminar nasioanal dosen universitas amikon yogyakarata. yogyakarta: universitas amikom yogyakarta; 2007. p. 1–5. 21. setyawati a. pengaruh pemberian senyawa crcl3●6h2o terhadap kadar gula darah tikus jantan galur wistar yang diinduksi dengan streptozotocin-nicotinamide. thesis. yogyakarta: universitas negeri yogyakatya; 2016. p. 1–62. 22. praptiwi h. inokulasi bakteri dan pemasangan cincin atau ligature untuk induksi periodontitis pada tikus. maj kedokt gigi. 2008; 15(1): 81–4. 23. mulawarmanti d, parisihni k, wedarti yr. catalase activitty of sea cucumber extract (stichopus hermanii) to periodontitis induced by porphyromonas gingivalis. in: regional oral biology scientific meeting 2014. depok: central library universitas indonesia; 2014. p. 26–30. 24. praptiwi, sulistyowati e, kustiyono. pola makan dan pertumbuhan bobot tubuh tikus yang diinokulasi porphyromonas gingivalis sebelum dan sesudah terjadinya periodontitis. media med indones. 2009; 43(5): 229–34. 25. dahiya p, kamal r, luthra r, mishra r, saini g. miswak: a periodontist′s perspective. j ayurveda integr med. 2012; 3(4): 184–7. 26. quamilla n. stres dan kejadian periodontitis (kajian literatur). j syiah kuala dent soc. 2016; 1(2): 161–8. 27. kim s-c, kim o-s, kim o-j, kim y-j, chung h-j. antioxidant profile of whole saliva after scaling and root planing in periodontal disease. j periodontal implant sci. 2010; 40(4): 164–71. 28. kusyati e. pengaruh suplementasi vitamin c terhadap jumlah fibroblas disekitar luka insisi pada tikus usia tua. thesis. semarang: universitas diponegoro; 2010. p. 1–80. 29. gupta p, singhal k, jangra ak, nautiyal v, pandey a. shark liver oil: a review. asian j pharm educ res. 2012; 1(2): 1–15. 30. güneş fe. medical use of squalene as a natural antioxidant. j marmara univ inst heal sci. 2013; 3(4): 220–8. 31. andarina r, djauhari t. antioksidan dalam dermatologi. j kedokt dan kesehat. 2017; 4(1): 39–48. 32. i nda hya n i de. minya k i ka n l emur u (sa rdinella longicep) menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (fish oil of lemuru (sardinella longicep) reduced the osteoblast apoptosis in wistar rat alveolar bone). dent j (majalah kedokt gigi). 2013; 46(4): 185–8. 33. iannitti t, palmieri b. an update on the therapeutic role of alkylglycerols. mar drugs. 2010; 8(8): 2267–300. 34. lavigne jp, brunel jm, chevalier j, pagès jm. squalamine, an original chemosensitizer to combat antibiotic-resistant gramnegative bacteria. j antimicrob chemother. 2010; 65(4): 799–801. 35. ngginak j, semangun h, mangimbulude jc, rondonuwu fs. komponen senyawa aktif pada udang serta aplikasinya dalam pangan. sains med. 2013; 5(2): 128–45. 36. futamura a, higashiguchi t, ito a, kodama y, chihara t, kaneko t, tomatsu a, shimpo k. experimental research on stimulation of wound healing by n-3 fatty acids. wounds a compend clin res pract. 2013; 25(7): 186–92. 37. novitasari aim, indraswary r, pratiwi r. pengaruh aplikasi gel ekstrak membran kulit telur bebek 10% terhadap kepadatan serabut kolagen pada proses penyembuhan luka gingiva. odonto dent j. 2017; 4(1): 13–20. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i4.p209–214 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i4.p209-214 61 dimensional change of acrylic resin plate after the reinforcement of glass fibre dwiyanti feriana ratwita and rinda mahalistiyani department of dental material and technology faculty of dentistry airlangga university surabaya indonesia abstract the effect of fibre reinforcement of polymethyl methacrylate was investigated. glass fibres have been studied as strengthening material added to polymethyl methacrylate. the purpose of this study was to evaluate dimensional change of acrylic resin plate after glass fibre reinforcement. as a research subject is an acrylic resin plate of 65 × 10 × 2.5 mm with the number of 32 samples were distributed randomly in 4 experimental groups. each group consisted of 8 samples and control groups. group 1: acrylic resin plate and 1 sheet glass fibre; group 2: acrylic resin plate and 2 sheet glass fibre; group 3: acrylic resin plate and 3 sheet glass fibre. control group which was not given treatment. dimensional change was measured by profile projector. the data was analyzed by one-way anova and lsd test showed that there was significant difference in dimensional change (p < 0.005). the conclusion suggested that dimensional change of the acrylic resin plates after glass fibre reinforcement minimally done 1 sheet glass fibre. correspondence: dwiyanti feriana ratwita, c/o: bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction acrylic resin especially polymethyl methacrylate (pmma) is frequently used as denture base in dentistry field. the use of acrylic resin is due to the advantageous property such as fulfilling aesthetic requirement, either the color or the texture could be made similar to gingival so the aesthetic performance in oral cavity is good, the absorption is relatively low, the dimensional change is small. easily repaired and processed when it is broken and the most important thing is relatively cheap.1,2,3 despite the advantageous property there are some disadvantageous properties of acrylic resin denture base i.e. fragile against hard surface or fatigue due to repeated flexure of a load,4 being broken due to unequal chewing pressure, especially, if the pressure is stronger than flexural power of the dental material, so that, repeated broken of denture base would occur.5 the most fragile part of the acrylic resin denture base is the frenulum. the acrylic resin dental material is continually developed in order to obtain better physical and mechanical property, one of the methods to make it better by adding strengthening material into acrylic resin material are: polymethyl fibre, composite glass carbon and aramid.6,7 in indonesia, not all of strengthening acrylic resin base is available on the market. the use of glass fibre is based on beneficial consideration i.e. being able to increase physical and mechanical property of acrylic resin.6,7 one of widely used polymer materials in dentistry field is pmma of crosslink type, due to the additional material: ethylenglyco methacrylate which can increase the mechanical strength of acrylic resin.8,9 to overcome the shortcomings, a study has been performed on various types of glass fibre as strengthening material: pmma. giving glass fibre into acrylic resin functions as strengthening material is possible to be done since glass fibre can adhere to polymer matrix so that it might increase the strength of acrylic resin.8,9 the other factors which correlate with the strength are the quantity of glass fibre on polymer matrix and the adhesion of fibre. the effect of glass fibre adhesion on polymer is the important aspect due to the strong effect of the attachment.10 the accurate placement of glass fibre in acrylic resin is the most important requirement. there are two kinds of placement i.e. partial fibre reinforcement (pfr) which is placed on the weakest part and total fibre reinforcement (tfr) which is placed on the whole part.10 filling glass fibre as strengthening material will increase the transverse strength of denture.11,12 glass fibre which is used in the form of cloth and placed in the middle of the acrylic resin will perform the most effective strength to the load force of acrylic resin. glass fibre as reinforcement should have longer size than the diameter size in adding the fibre into the polymer, the polymer would attach mechanically on the fibre and continue the polymerization process on the surrounding fibres. the polymer will distribute the load to the fibres which are the stronger component and also will protect the fibres from the moisturizing effect13 another study suggested that the use of 2 sheet glass fibre might increase the transverse strength, the quantity of the fibre in polymer matrix and the adhesion of fibre would really affect the adhesion strength.9,14 62 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 61–64 dimensional stability of denture case during the processing period is the most important, because it would affect on the accuracy of denture in the oral cavity. the dimensional change of denture case would possibility occurred during the processing period and curing process which correlates with water absorption, this case is due to the excessive thickness so that the polymerization process would be hamped. dimensional change of acrylic resin is < 1%, the frequently part in which the most dimensional change would occur is in the frontal area: 0.2–8.1% meanwhile in the lateral area is 0.2–9%. based on the above background, so it is necessary to perform the study on the effect of glass fibre reinforcement to dimensional change of acrylic resin plate.15 the purpose of this study was to know dimensional change of acrylic resin plate after glass fibre reinforcement. the benefit of this study is to stimulate innovation on acrylic resin with glass fibre inforcement to dimensional change. material and methods research materials which were used in this study were: acrylic resin heat cured crosslink (vertex–dentimex), separator material (cms, qc), hard gypsum, vaseline, aquadest, glass fibre of woven type (yakasu, japan). the tools which were used were: model master (size 65 × 10 × 2.5 mm),16 cuvet, digital analytic (vertling, jerman), hydraulic press (bego, jerman), vacum mixer, profile projector (nikon, japan). the process of making sample is hard gypsum with the ratio between the powder and aquadest: 50 g : 15 ml (according to manufacture recommendation ), mixed using vacuum mixer for 30 seconds, then gypsum dough put into the curvet placed on vibrator. master model made of metal size 65 × 10 × 2.5 mm placed in the middle of cuvet on hard gypsum dough. two specimen of master model was planted and arranged in parallel direction and made the gypsum hardening for about 15 minutes, after hardening the surface of gypsum was polished by vaseline. the antagonistic cuvet was planted and filled completely by hard gypsum was dough on the vibrator. after the gypsum was hardened then curvet was opened, the master model was taken out, finally the mould was obtained. next, heat cured acrylic resin was filled into the mould, the ratio between powder and acrylic resin liquid 4,6 g : 3 ml (according to manufacture recommendation) mixed and stirred in porcelain pot, mould surface was dry. the sample was made by adding glass fibre with the following method: polymer and monomer of heat cured acrylic resin which had been mixed until it reached dough stage, then, divided into two: half was put into the mould on the upper part of curvet and the other half was put into the mould on the lower part of curvet, the next step, mould on the lower part of cuvet was added 1 sheet glass fibre (group 1). 2 sheet glass fibre (group 2) and 3 sheet (group 3). on the control group without being added glass fibre, finally cuvet was closed and pressed using hydraulic bench press, with pressure 2200 psi or 50 kg/mm2. the procedure was repeated three times and let them dry for 15 minutes. curing process was done in cuvet with acrylic resin, through boiling process for1 hour in the pot containing 3 liter water, temperature 100 °c, until curing process was completed and it became cold then it was opened. sample of acrylic was taken out and fined using abrasive paper no 1000 afterward, it was kept in the bottle glass and soaked into the water for 2 × 24 hour in order not to be dry. in this study, the number of samples was 32 and every group consisted of 8 samples in 4 experimental groups. the test of dimensional change on acrylic resin plate was done using profile projector (nikon), acrylic resin was placed on plate steel infront of the lens. the reflection of the acrylic resin could be seen on the screen. the number stated on the monitor would be noted and calculated using the equation.15 dimensional change which studied was vertical and horizontal dimension. dimension change = l1 –l0 note: l0 = initial length (mm) l1 = end length (mm) result the mean result and standard deviation of the study on the effect of reinforcement of glass fibre toward table 1. the mean and standard deviation resulted from strengthening material of glass fibre toward dimensional change of acrylic plate (mm) no. control mean of length change and standard deviation mean of width change and standard deviation mean of thickness change and standard deviation 1. 2. 3. 4. group 1 group 2 group 3 control 1,757 ± 0,066 1,829 ± 0,084 2,276 ± 0,097 1,503 ± 0,055 1,186 ± 0,090 1,554 ± 0,096 2,002 ± 0,099 0,791 ± 0,063 0.390 ± 0,042 0.516 ± 0,094 0.563 ± 0,040 0.300 ± 0,046 note: group 1: acrylic resin plate + 1 sheet glass fibre; group 2: acrylic resin plate + 2 sheet glass fibre; group 3: acrylic resin plate + 3 sheet glass fibre; control: uncontrol group. 63ratwita: dimensional change of acrylic resin plate dimensional change of acrylic resin plate would be shown on table 1. the data on the effect of strengthening material of glass fibre toward dimensional change of acrylic plate was analyzed using one-way anova test. anova test was preceded by homogeneity data test using levene test. the result of homogeneity test found: length change p = 0.746 (p > 0.05), width change p = 0.103 (p > 0.05), this means all the data on length width thickness change of acrylic resin plate derivate from homogeneous source. the result of anova test p = 0.001 (p < 0.05) means there is significant difference on length width, and thickness of acrylic resin plate. in order to know significant between treatment, followed by least significant different (lsd) shows on table 2. table 2. lsd test, the effect of strengthening material of glass fibre toward length change of acrylic resin plate (mm) control group 1 group 2 group 3 control group 1 group 2 group 3 s s ns s s s note: s: significant (p < 0.05); ns: non significant table show that in every control group there is significant difference p = 0.01 (p < 0.05), except on length change between group 1 compared with group 2 p = 0.076 (p > 0.05). table 3 shows in every control there is significant difference p = 0.01 (p < 0.05). table 4 shows in every control group there is significant difference p = 0.01 (p < 0.05), except the thickness change between group 1 compared with group 3 (p > 0.05). tabel 3. lsd test, the effect of strengthening material of glass fibre to width change of acrylic resin plate (mm) control group 1 group 2 group 3 control group 1 group 2 group 3 s s s s s s note: s: significant (p < 0.05) tabel 4. lsd test, the effect of strengthening material of glass fibre toward thickness change of acrylic resin plate (mm) control group 1 group 2 group 3 control group 1 group 2 group 3 s s s s ns s note: s: significant (p < 0.05); ns: non significant discussion some of literary consideration which indicate the dimensional accuracy of acrylic resin plate are important to obtain accurate molding. in this study, a great number of variables are controlled in order to gain homogeneous samples, but some factors are completely beyond the capability such as: the placement of glass fibre in the middle of acrylic resulting of un similarity among one and the others, in which it might influence the outcome of the study. however the short coming has been minimalized. dimensional change closely related by the stability and the retention of removable denture therefore dimensional change would be correlated with comfort or discomfort of denture.17,18 some factors induce dimensional changes are the between monomer and polymer, the type of acrylic resin, the curing process and storing factors. furthermore, the monomer absorption of acrylic resin after curing process is also one of the causes of dimensional change of denture.15 the most dimensional change is caused by polymerization shrinkage of acrylic in strengthening material of glass fibre. the high ratio of the difference between monomer and polymer might cause higher dimensional change.13,19 the outcome of study suggest that the influence of strengthening material of glass fibre on the dimensional change of acrylic resin plate found the highest mean of change: 2.276 mm, the width change: 2.002 mm and the thickness change: 0.563 mm, occurring in the addition of 3 sheet glass fibre. this case might be due to the use of excessive glass fibre which could contribute the biggest absorption of monomer during curing process. the more glass fibres are used resulting higher dimensional change.14 it is advisable to be well considered the use of strengthening material of glass fibre because glass fibres have monomer absorption property. therefore, fluid absorption of acrylic resin is an important phenomenon to be considered due to the direct correlation to dimensional change.20 different curing process also contributes different dimensional change, acrylic of heat-cured type has worse dimensional accuracy than acrylic of cold-cured type because acrylic of cold-cured type has lower tensile surface, smaller dimensional change and better adaptation property, linear shrinkage value or shrinkage of acrylic cold–cured type is about 0.01–0.43%, meanwhile shrinkage value of acrylic heat-cured type is 0.5%.21 one-way anova test, significant difference was found with p = 0.001 (p < 0.05 ) for the whole change of least, width and thickness. lsd was done in order to understand the effect of the difference, the result showed significant difference with p = 0.001 (p < 0.005) the significant change of length, width and thickness found in acrylic resin plate added by 1 sheet glass fibre with p = 0.001 (p < 0.005), the difference is due to the thickness of glass fibre which would affect the thickness of acrylic plate. the plate was due to the fibre tensility which is 64 dent. j. (maj. ked. gigi), vol. 40. no. 2 april–june 2007: 61–64 important property to strengthen fragile material such as: acrylic resin, therefore, strong adhesion of fibre attached to polymer is an essential factor to obtain the strength of acrylic resin. proper adhesion could make better fluid absorption of acrylic resin polymer into the glass fibre so it would affect the dimensional change.13 the use of glass fibre as ideal strengthening material should have higher length comparing to the diameter. the in for cement of glass fibre into polymer, the polymer would mechanically attach to the glass fibre and, further, polymerization would continue at the surrounding part of glass fibre. polymer would distribute the load into the fibre in a stronger component and also protect the acrylic resin from the moisture so the process might minimalize the dimensional change.9 in this study, it could be concluded that acrylic resin shows minimal dimensional change after reinforcement of 1 sheet of glass fibre. references 1. combe ec. notes on dental materials. 6th ed. edinburg, london, melbourne, new york: churchill livingstone; 1992. p. 79–120. 2. craig rg. restorative dental material. 11th ed. st louis missouri: mosby company; 2002. p. 655–8. 3. taner b, dogan a, tincer t, akinay ae. a study on impact and tensile strength of acrylic resin filled with short ultra-high molecular weight polyethylene fihers. j oral science 1999; 41:15–8. 4. billmeyer fw. textbook of polymer science. 3rd ed. new york, singapore: a wiley inter science pub, john wiley & sons; 1984. p. 11–6. 5. mitchell l, mitchell da. oxford handbook of clinical dentistry. 3rd ed. oxford: oxford university press; 1999. p. 369, 700. 6. vallitu pk. glass fibre reinforcement in repaired acrylic resin removable denture preliminary results of clinical study. quintessence int 1997; 28:39–44. 7. vallitu pk. curing of silane coupling agent and its effect on the transverse strength of autopolymerizing polymethylmethacrylate glass fibre composite. j oral rehabil 1997; 24:124–30. 8. vallitu pk, lassila vp, lappalaine rm. acrylic resin-fibre composite. part i. the effect of fibre concentration on fracture resistance. j prosthet dent 1994; 71:607–12. 9. vallitu pk. acrylic resin fibre composite. part ii. the effect of polymerization shrinkage of polymethylmethacrylate applied to fibre roving on transverse strength. j prosthet dent 1994; 71:613–7. 10. vallitu pk. flexural properties of acrylic resin polymers reinforced with undirectional and woven glass fibre. j prosthet dent 1999; 8:318–25. 11. dixon dl, fincher m., breeding lc. mechanical properties of light polymerizing provisional restorative material with and without reinforcement fibres. j prosthet dent 1995; 73:510–4. 12. dixon dl, fincher m, breeding lc. the transverse strength of three denture base resins reinforced with polyethylene fibres. j prosthet dent 1995; 67:417–9. 13. v a l l i t u p k . d i m e n s i o n a l a c c u r a c y a n d s t a b i l i t y o f polymethylmethacrylate reinforced with metal wire or with continuous glass fibre. j prosthet dent 1996; 75:617–21. 14. vallitu pk, lassila up. reinforcement of acrylic resin denture base material with metal or fibre strengthenes. j oral rehabil 1992; 19:225–30. 15. craig rg, o’brien w, powers jm. dental materials properties and manipulation. 4th ed. st louis, washington dc, toronto: the cv mosby company; 2004. p. 264–80. 16. american dental association, ada spesifikasi no. 12. revised american national standard/ american dental association specification. denture base polymers. chicago: american dent association 1975; 203–8. 17. vallitu pk. a review of fibre-reinforced denture base resins. j prosthet dent 1996; 5:270–6. 18. stipho hd. repair of acrylic resin denture base reinforced with glass fibre. j prosthet dent 1998; 546–50. 19. vallitu pk, ruyter ie, ekstrand k. effect of water storage on the flexural properties of e-glass and silica fibre acrylic resin composite. int j prosthodont 1998; 11:340–50. 20. reitz pv, sanders jl, levin b. the curing acrylic resins by microwave energy, physical properties. quintessence international 1985; 8:547–51. 21. salim s. pengaruh humiditas dan lama penyimpanan serta cara kuring terhadap sifat fisik, kimia dan mekanik resin akrilik basis gigi tiruan. dissertation. surabaya: universitas airlangga; 1995. p. 5–45. editorial team of dental journal (majalah kedokteran gigi) sk: 15/un3.1.2/2022 january 4 – december 31, 2022 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga editor in chief: muhammad dimas aditya ari department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200578006] editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478, 5030255. fax. +62 31 5039478, 5026288 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg/index accredited no. 158/e/kpt/2021 cover photo purchased from: https://stock.adobe.com order number: ae00770304146cid volume 55, issue 1, march 2022 p-issn: 1978-3728 e-issn: 2442-9740 editorial boards roeland jozef gentil de moor, department of restorative dentistry and endodontology, dental school, ghent university, belgium [scopus id: 7005928380] cortino sukotjo, department of restorative dentistry, university of illinois at chicago college of dentistry, united states [scopus id: 6508194317] guang hong, liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan [scopus id: 7203031334] kenji yoshida, department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, japan [scopus id: 57080640700] miguel rodrigues martins, co-worker aachen dental laser center, rwth aachen university, germany [scopus id: 55993479000] sajee sattayut, department of oral surgery, faculty of dentistry, khon kaen university, thailand [scopus id: 55431381300] samir nammour, department of dental science, faculty of medicine, university of liege, belgium [scopus id: 6602922393] reza fekrazad, laser reseach center in medical science, dental faculty, aja university of medical science, iran [scopus id: 22952665700] hong sai loh, department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore [scopus id: 7202491277] hamid nurrohman, missouri school of dentistry & oral health, a.t. still university, united states [scopus id: 52564067000] harry huiz peeters, laser research center, bandung, indonesia [scopus id: 51864447300] rahmi amtha, department of oral medicine, faculty of dentistry, universitas trisakti, indonesia [scopus id: 26031894400] elza ibrahim auerkari, department of oral biology, faculty of dentistry, universitas indonesia, indonesia [scopus id: 10139113000] r. darmawan setijanto, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 55212583700] anita yuliati, department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 43462222100] udijanto tedjosasongko, department of pediatric dentistry, faculty of dental medicine, universitas airlangga [scopus id: 6508026751] managing editors ketut suardita, department of conservative dentistry, faculty of dentistry, iik bhakti wiyata, indonesia [scopus id: 6506788956] alexander patera nugraha, department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57194112535] astari puteri, department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57200385443] nastiti faradilla ramadhani, department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57191881659] associate editors beshlina fitri widayanti roosyanto prakoeswa, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57467259800] saka winias, department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57211330310] aulia ramadhani, department of dental public health, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57205630113] beta novia rizqy, department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia [scopus id: 57214805206] administrative assistant novi dian prastiwi, faculty of dental medicine, universitas airlangga; abdullah mas’udy, faculty of dental medicine, universitas airlangga. printed by: airlangga university press. campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 55, issue 1, march 2022 p-issn: 1978-3728 e-issn: 2442-9740 1. progressivity analysis of pleomorphic adenoma toward carcinoma ex pleomorphic adenoma mei syafriadi, dina zakiyatul ummah, aisyah izzatul muna, maria evata krismawati surya ................................................................................................................................................. 1–6 2. the effect of an 8% cocoa bean extract gel on the healing of alveolar osteitis following tooth extraction in wistar rats isnandar, olivia avriyanti hanafiah, muhammad fauzan lubis, lokot donna lubis, adzimatinur pratiwi, yeheskiel satria yoga erlangga ............................................................... 7–12 3. a comparison of the accuracy of the cervical vertebrae maturation stage method and demirjian’s method on mandibular length growth alfira putriana dewi, seno pradopo, sindy cornelia nelwan .................................................... 13–15 4. association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients pitu wulandari, dody widkaja, aini hariyani nasution, armia syahputra, gebby gabrina ............................................................................................................................................. 16–20 5. the effect of brotowali (tinospora crispa l.) stem ethanolic extract on the inhibition of candida albicans biofilm formation suryani hutomo, christiane marlene sooai, maria silvia merry, ceny gloria larope, haryo dimasto kristiyanto ............................................................................................................. 21–25 6. tumor necrosis factor-α and osterix expression after the transplantation of a hydroxyapatite scaffold from crab shell (portunus pelagicus) in the post-extraction socket of cavia cobaya irvan salim, michael josef kridanto kamadjaja, agus dahlan ................................................. 26–32 7. addition of gourami (osphronemus goramy) fish scale powder on porosity of glass ionomer cement erawati wulandari, farah rachmah aulia wardani, nadie fatimattuzahro, i dewa ayu ratna dewanti .................................................................................................................................. 33–37 8. the effects of unilateral posterior crossbite toward the superficial masseter and anterior temporalis on muscle activity during mastication: a surface electromyographic study agnes imelda izach, christnawati, darmawan sutantyo ............................................................ 38–43 contents original articles page case reports 9. the aesthetic management of a midline diastema with direct composite using digital smile design, putty index and button shade technique: a case report nirawati pribadi, sukaton, galih sampoerno, sylvia, hendy jaya kurniawan, maya safitri, rahmadanty mustika ...................................................................................................................... 44–48 10. management of patients with aphthous-like ulcers related to aplastic anaemia in the covid-19 pandemic era through teledentistry: a case report lani berlina talahatu, bima ewando kaban, nurina febriyanti ayuningtyas, intan noha brilyanti, adiastuti endah parmadiati, desiana radithia, aulya setyo pratiwi ...................... 49–55 review article 11. exercise as a method to reduce the risk of oral cancer: a narrative review anis irmawati, lia aulia rachma, sidarningsih, muhammad naufal hatta, ira arundina, mohammed aljunaid ....................................................................................................................... 56–61 vol 50 no 4 desember 2017.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 50, number 4, december 2017 editorial boards of dental journal (majalah kedokteran gigi) sk: 275/un3.1.2/2017 may 5th – december 31st, 2017 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (faculty of vocation, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers roosje rosita oewen (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); harmas yazid yusuf (department of oral surgery, faculty of dentistry, universitas padjadjaran, indonesia); eky s. soeria soemantri (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); mei syafriadi (department of oral pathology, faculty of dentistry, universitas jember, indonesia); muslita indrasari (department of prosthodontics, faculty of dentistry, universitas indonesia, indonesia); adioro soetojo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); els sunarsih budipramana (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) i.b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); ari triwadhani (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); intan nirwana (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); david kamadjaja (department of oral surgery and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontic, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 557/12.17/aup-a7e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 50, number 4, december 2017 p-issn: 1978-3728 e-issn: 2442-9740 1. effects of herbal medicine components on the physical properties of trial denture adhesives kenichiro nakai, takeshi maeda, guang hong, tadafumi kurogi, and joji okazaki ............ 171–177 2. composite resin shear bond strength on bleached dentin increased by 35% sodium ascorbate application tunjung nugraheni, n. nuryono, siti sunarintyas, and ema mulyawati ................................... 178–182 3. effects of filler volume of nano-sisal in compressive strength of composite resin dwi aji nugroho, w. widjijono, n. nuryono, widya asmara, wijayanti dwi astuti, and dana ardianata ................................................................................................................................ 183–187 4. the role of active ingredients nanopowder stichopus hermanii gel to bone resorption in tension area of orthodontic tooth movement noengki prameswari and arya brahmanta .................................................................................. 188–193 5. the effects of anadara granosa shell-stichopus hermanni on bfgf expressions and blood vessel counts in the bone defect healing process of wistar rats rima parwati sari, sri agus sudjarwo, retno pudji rahayu, widyasri prananingrum, syamsulina revianti, hansen kurniawan, and aisah faiz bachmid .......................................... 194–198 6. antioxidant potency of mangosteen peel extract topical application in reversing reduced orthodontic brackets tensile strength after bleaching ananto ali alhasyimi ...................................................................................................................... 199–204 7. analysis of ki-67 expression as clinicopathological parameters in predicting the prognosis of adenoid cystic carcinoma silvi kintawati, murnisari darjan, and winny yohana .............................................................. 205–210 8. dentoalveolar changes in post-twin block appliance orthodontic treatment class ii dentoskeletal malocclusion y. yoana, eka chemiawan, and arlette suzy setiawan ............................................................... 211–215 9. comparison of salivary alpha-amylase levels as stress markers in gingivitis and periodontitis dyah nindita carolina, yanti rusyanti, and agus susanto ......................................................... 216–219 10 the role of cervical vertebrae maturation in defining the chronological age of down syndrome children anggiani dewi rahmawati, iwan ahmad, and arlette suzy setiawan ....................................... 220–225 11. a comparison of the adhesive strength of zinc phosphate and self-adhesive resin cement as fiber post cementation materials setyawan bonifacius, deddy firman, and hasna djiab ............................................................... 226–229 6868 dental journal (majalah kedokteran gigi) 2023 june; 56(2): 68–72 case report multiple impacted third molars with pre-eruptive intracoronal resorption in geriatric patients: two case reports emel olga onay1, cemre koc2, mete ungor3 1department of endodontics, faculty of dentistry, baskent university, ankara, turkey 2department of endodontics, faculty of dentistry, aydin adnan menderes university, aydin, turkey 3department of endodontics, faculty of dentistry, istanbul medipol university, istanbul, turkey abstract background: pre-eruptive intracoronal resorption (peir) is a rare condition usually detected through an incidental radiographic finding. the etiology and pathogenesis of this phenomenon are not fully understood. purpose: to describe two cases in which multiple impacted third molars with peir defects were identified. cases: female patients aged 77 and 82 years, respectively, were presented with dental issues. radiolucencies in the dental crown areas of the impacted maxillary and mandibular third molars were initially detected on the panoramic radiographs. cone-beam computed tomography (cbct) was performed to better evaluate the impacted teeth. the results showed that the intracoronal defects extended through more than two-thirds of the thickness of the coronal dentin. case managements: considering the patients’ age and their asymptomatic status, a conservative approach with radiographic followup was considered most appropriate. four-year follow-up checks revealed that the teeth remained asymptomatic in both patients. conclusion: this case report confirms that peir can affect impacted third molars, even in elderly patients. cbct images are preferred for diagnosing peir defects because this method provides an accurate assessment of internal tooth anatomy. with an accurate diagnosis of asymptomatic peir, the lesion can be monitored. keywords: cone-beam computed tomography; diagnostic imaging; geriatric dentistry; impacted tooth; tooth resorption article history: received 4 september 2022; revised 28 september 2022; accepted 5 oktober 2022 correspondence: emel olga onay, department of endodontics, faculty of dentistry, baskent university, 82. sok. no: 26, 06490, bahcelievler, ankara, turkey. e-mail: eonay@baskent.edu.tr introduction pre-eruptive intracoronal resorption (peir) is an anomaly that most often occurs within the dentin of unerupted teeth, although the enamel may also be involved in advanced cases.1,2 this finding is uncommon, and most peir lesions are identified incidentally during routine radiography.3 studies have revealed the prevalence of peir to be 0.7% to 9.5% by subject and 0.5% to 2% by tooth, depending on the tooth type and radiographic technique employed.2,4,5 one study that used cone-beam computed tomography (cbct) revealed peir in 15% of a turkish population.6 of the 48 intracoronal resorption lesions investigated, 30 (63%) were in third molars. the etiology of peir remains unclear, but histologically it is a resorptive process that is driven by activated osteoclast-like giant cells on the pulpal wall of the dentin7 or originates from undifferentiated cells in the developing dental follicle.8 predisposing factors, such as ectopic positioning of the tooth or the adjacent teeth, may create local pressure that induces the resorption process to invade the dentin along enamel fissures or through the cementoenamel junction.1 the treatment options for these lesions are monitoring without treatment until the tooth erupts, surgical exposure with treatment of the lesion, including endodontic treatment as needed, or extraction.9 in the case of unerupted teeth, peir lesions have often been considered aseptic since there is no bacterial invasion.2 most static cases have been addressed using a preventative, non-invasive approach of monitoring without intervention.7,10 this report presents two cases of peir involving bony impacted third molars in elderly patients treated at baskent university, faculty of dentistry, department copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p68–72 mailto:eonay@baskent.edu.tr https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p68-72 69 onay et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 68–72 of endodontics. our intent was to describe the clinical and radiographic features of this condition to help guide clinicians in diagnosis and to suggest a preventative, noninvasive approach of monitoring. the ethics committee of baskent university, ankara, turkey, waived the ethical approval of this study because it was a case report. informed consent was obtained from the patients for publication of this case report and any accompanying images. case 1 a 77-year-old caucasian female patient was referred for treatment with the primary complaint of discomfort on the right side of her mandible. an informed consent was obtained for further investigation. her medical history included osteoporosis, rheumatic mitral stenosis, and a kyphoplasty procedure for a lumbar compression fracture. her regular medications were triamterene 50mg daily, atenolol 75mg daily, acetylsalicylic acid 100mg daily, and furosemide 40mg on alternate days. a panoramic radiograph revealed secondary caries in the patient’s mandibular right second molar and second premolar teeth, which had been restored with a fixed partial denture. the same radiograph also showed radiolucencies in the dental crown areas of the maxillary right and left third molars, which were impacted (figure 1). the affected teeth were asymptomatic, and no sinus tract or communication was identified between them and the oral cavity. cbct was performed to better evaluate the impacted teeth. all cbct images were taken using a morita 3d accuitomo 170 (j morita, japan) according to the following parameters: 90 kvp, 5 ma, voxel size: 0.08 mm, field of view (fov) size: 40x40 mm. the i-dixel software (v.2.2.1.6, morita, japan) was used to analyze the images on a medical monitor (eizo radiforce mx270w, eizo corporation, japan). this showed that the defects extended through the full dentin thickness of the crowns (figures 2 and 3). a connection between the surrounding bone and the defect was noted in the distal intracoronal area of the maxillary right third molar (figure 2). the enamel of the maxillary left third molar was intact (figure 3). case management 1 the positions of the lesions, the patient’s clinical history, and her historical and current radiographic findings supported a diagnosis of peir. because of her age and asymptomatic status, monitoring the maxillary right and left third molars was considered the most appropriate treatment. four years after presentation, a telephone followup was carried out due to the covid-19 pandemic and the patient’s poor health. no radiographs could be taken, but it was verbally confirmed that her affected teeth remained asymptomatic. it was decided that follow-ups would be conducted periodically in order to monitor the patient’s affected teeth. case 2 an 82-year-old caucasian female patient had a primary complaint of discomfort in the right posterior area of her maxilla. an informed consent was obtained for further investigation. her medical history included osteoporosis, hypertension, hyperlipidemia, iron deficiency anemia, right and left temporal meningioma, and surgery for a right femur fracture. her regular medications included nifedipine 30mg daily, atorvastatin 40mg daily, acetylsalicylic acid 100mg daily, pantoprazole 40mg daily, and zoledronic acid 5mg once yearly. a panoramic radiograph revealed that her first molar and second premolar teeth had been treated endodontically (figure 4). the same radiograph showed radiolucencies in the dental crown areas of the maxillary and mandibular right third molars, which were impacted and ectopically positioned (figure 4). cbct was performed exclusively on the maxillary right third molar in accordance with the patient’s request; however, the imaging also encompassed the mandibular right third molar. all cbct images were taken using a morita 3d accuitomo 170 (j morita, japan) according to the following parameters: 90 kvp, 5 ma, voxel size: 0.08 mm, fov size: 40x40 mm. the i-dixel software (v.2.2.1.6, morita, japan) was used to analyze the images on a medical monitor (eizo radiforce mx270w, eizo corporation, japan). the results showed that the intracoronal defect in the maxillary right third molar extended through more than two-thirds of the thickness of the coronal dentin (figure 5). additionally, a small part of the adjacent enamel was found to be affected by the resorption, and a connection between the surrounding bone and the defect was noted on the distal and occlusal aspects of the maxillary right third molar (figure 5). the cbct analyses also showed that the defect extended through the full thickness of the dentin of the crown of the mandibular right third molar, and the occlusal portion of the defect was in contact with the surrounding bone (figure 5). one of the images also revealed a hypodense area associated with the roots of the second molar (figure 5). case management 2 the patient’s complaints diminished after root canal therapy was performed on the maxillary right second molar. due to her age and asymptomatic status, monitoring the maxillary and mandibular right third molars was considered the most appropriate treatment. a four-year follow-up examination revealed that the affected teeth remained asymptomatic, and there was no sinus tract or communication between the impacted teeth and the oral cavity. radiographic examination showed no changes in the extent of the lesions (figure 6). cbct was not performed in accordance with the patient’s request, and it was also desired to comply with the alara (as low as reasonably achievable) principle.11 copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p68–72 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p68-72 70onay et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 68–72 figure 1. a panoramic radiograph of patient 1 reveals radiolucencies in the dental crown areas of the maxillary right and left third molars, which were impacted (arrows). figure 2 cbct images of patient 1: coronal (a) and sagittal (b) images of the maxillary right third molar show the defect extending through the full dentin thickness of the crown (arrows); an axial view (c) reveals a connection (arrow) between the surrounding bone and the defect in the distal intracoronal area. figure 3. cbct images of patient 1: coronal (a) and sagittal (b) views of the maxillary left third molar show the defect extending through the full dentin thickness of the crown (arrows); an axial view (c) shows the enamel intact (arrow). figure 4. a panoramic radiograph of patient 2 reveals radiolucencies in the dental crown areas of the maxillary and mandibular right third molars, which were both impacted (arrows). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p68–72 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p68-72 71 onay et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 68–72 discussion in both cases, and as described previously,1,12 all lesions were below the dentinoenamel junction and extended from this part to various depths through the dentin. a previous report noted that 40% of 57 defects extended through more than two-thirds of the thickness of the coronal dentin.1 we also observed this with the lesions in our two patients. a previous study also found no association between peir lesions and medical conditions.1 interestingly, both our patients with peir had a history of osteoporosis. estrogen withdrawal has a critical role in the pathogenesis of postmenopausal osteoporosis, inducing bone remodeling with a negative calcium balance.13 decreased levels of estrogen are due to the unbalanced production of the receptor activator known as nuclear factor kb ligand (rankl), which both induces differentiation of hemopoietic precursor cells into osteoclasts and stimulates bone resorption activities.14,15 nishida et al.16 reported a common regulatory mechanism for resorptions from mineralized tissues in bone and teeth, as their study revealed rankl expression in odontoclasts located on resorbing root dentin. in addition, rankl has been shown to play a key role in the differentiation of odontoblast-like cells into odontoclast-like cells or the function of odontoclasts.17 further investigations with larger study groups are needed to identify whether peir defects are associated with osteoporosis. cbct enables high-resolution images and threedimensional evaluation and has been demonstrated to be an accurate technique for diagnosing and assessing peir. the sizes and locations of peir defects may not be accurately visualized using two-dimensional radiography techniques, such as bitewing, periapical, and panoramic radiographs.5 this suggests that in cases where peir defects are identified or suspected, conventional twodimensional radiographs should be analyzed in detail, and cbct should be performed when available.8 it is highly recommended that cbct be utilized to diagnose and evaluate the resorptive lesions to determine the treatment needed, as specified in position statements by the american association of endodontists/american association of oral and maxillofacial radiology18 and the european society of endodontology19 concerning the use of cbct in endodontics. it has been widely reported in the literature that the quality of cbct images changes when different fovs or voxel sizes are selected.20 cbct with a limited fov, which is typically used for endodontic diagnosis of peir, figure 5. cbct images of patient 2: coronal (a), sagittal (b), and axial (c) views of the maxillary right third molar show the intracoronal defect extending through more than two-thirds of the thickness of the coronal dentin (arrows). additionally, a small portion of the adjacent enamel was affected by the resorption, and a connection between the surrounding bone and the defect was noted on the distal (b) and occlusal (c) sides of the maxillary right third molar (arrows). a hypodense area (asterisk) related to the roots of the maxillary right second molar was also identified (d). the cbct image (d) of mandibular right third molar shows the defect extending through the full dentin thickness of the crown and the occlusal portion of the defect in contact with the surrounding bone (arrow). figure 6. a panoramic radiograph taken during patient 2’s four-year follow-up exam revealed no changes to the extent of the lesions in her maxillary and mandibular right third molars (arrows). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p68–72 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p68-72 72onay et al. dent. j. (majalah kedokteran gigi) 2023 june; 56(2): 68–72 ranges in diameter from 40 to 100 mm. the voxel size is generally smaller for a limited fov (0.08–0.2 mm), thus providing higher resolution and greater utility for detecting and monitoring peir lesions. various factors can clinically affect detection of peir on cbct images, such as the observer’s experience level, different cbct parameters, viewing conditions, and artefacts.21 in this case report, an invasive treatment method was not preferred due to the age of the patients, the aseptic conditions of the teeth, and their asymptomatic nature. a conservative perspective with meticulous clinical and radiographic follow-up is usually preferred in these cases. an invasive approach, such as restorative treatment, endodontic treatment, and extraction, can be postponed until the teeth erupt and/or get infected.8,9 the rate of peir defects affecting the third molar teeth can be different in the same individual. generally, only a single tooth has been reported to be affected,1,3 although some studies have shown more than one affected tooth in the same individual.5,6 no explanation has been offered for this finding, but it is conceivable that teeth with longstanding or large peir defects would have been reported less frequently, especially in geriatric patients, because such teeth in these patients are more likely to have been extracted.8 in conclusion, our two cases confirm that peir can affect impacted third molars, even in elderly patients. cbct is preferred for diagnosing peir defects because this modality enables the accurate assessment of internal tooth anatomy. if asymptomatic peir is accurately diagnosed, the lesion can be monitored. references 1. umansky m, tickotsky n, friedlander-barenboim s, faibis s, moskovitz m. age related prevalence of pre-eruptive intracoronal radiolucent defects in the permanent dentition. j clin pediatr dent. 2016; 40(2): 103–6. 2. uzu n i, gu nduz k , ca n itezer g, avsever h, o rha n k. a retrospective analysis of prevalence and characteristics of preeruptive intracoronal resorption in unerupted teeth of the permanent dentition: a multicentre study. int endod j. 2015; 48(11): 1069–76. 3. lenzi r, marceliano-alves mf, alves f, pires fr, fidel s. preeruptive intracoronal resorption in a third upper molar: clinical, tomographic and histological analysis. aust dent j. 2017; 62(2): 223–7. 4. le vnt, kim j-g, yang y-m, lee d-w. treatment of pre-eruptive intracoronal resorption: a systematic review and case report. j dent sci. 2020; 15(3): 373–82. 5. demirtas o, dane a, yildirim e. a comparison of the use of conebeam computed tomography and panoramic radiography in the assessment of pre-eruptive intracoronal resorption. acta odontol scand. 2016; 74(8): 636–41. 6. demirtas o, tarim ertas e, dane a, kalabalik f, sozen e. evaluation of pre-eruptive intracoronal resorption on cone-beam computed tomography: a retrospective study. scanning. 2016; 38(5): 442–7. 7. counihan kp, o’connell ac. case report: pre-eruptive intra-coronal radiolucencies revisited. eur arch paediatr dent. 2012; 13(4): 221–6. 8. al-batayneh ob, altawashi ek. pre-eruptive intra-coronal resor ption of dentine: a review of aetiology, diagnosis, and management. eur arch paediatr dent. 2020; 21(1): 1–11. 9. chouchene f, hammami w, ghedira a, masmoudi f, baaziz a, fethi m, ghedira h. treatment of pre-eruptive intracoronal resorption: a scoping review. eur j paediatr dent. 2020; 21(3): 227–34. 10. spierer wa, fuks ab. pre-eruptive intra-coronal resorption: controversies and treatment options. j clin pediatr dent. 2014; 38(4): 326–8. 11. vaz de souza d, schirru e, mannocci f, foschi f, patel s. external cervical resorption: a comparison of the diagnostic efficacy using 2 different cone-beam computed tomographic units and periapical radiographs. j endod. 2017; 43(1): 121–5. 12. manmontri c, mahasantipiya pm, chompu-inwai p. preeruptive intracoronal radiolucencies: detection and nine years monitoring with a series of dental radiographs. case rep dent. 2017; 2017: 6261407. 13. manolagas sc, o’brien ca, almeida m. the role of estrogen and androgen receptors in bone health and disease. nat rev endocrinol. 2013; 9(12): 699–712. 14. udagawa n, koide m, nakamura m, nakamichi y, yamashita t, uehara s, kobayashi y, furuya y, yasuda h, fukuda c, tsuda e. osteoclast differentiation by rankl and opg signaling pathways. j bone miner metab. 2021; 39(1): 19–26. 15. macari s, duffles lf, queiroz-junior cm, madeira mfm, dias gj, teixeira mm, szawka re, silva ta. oestrogen regulates bone resorption and cytokine production in the maxillae of female mice. arch oral biol. 2015; 60(2): 333–41. 16. nishida d, arai a, zhao l, yang m, nakamichi y, horibe k, hosoya a, kobayashi y, udagawa n, mizoguchi t. rankl/opg ratio regulates odontoclastogenesis in damaged dental pulp. sci rep. 2021; 11(1): 4575. 17. duan x, yang t, zhang y, wen x, xue y, zhou m. odontoblastlike mdpc-23 cells function as odontoclasts with rankl/m-csf induction. arch oral biol. 2013; 58(3): 272–8. 18. american association of endodontics. aae and aaomr joint position statement—use of cone beam computed tomography in endodontics—2015/2016 update. cone beam computed tomography. 2016. p. 1–6. available from: https://www.aae.org/specialty/wpcontent/uploads/sites/2/2017/06/conebeamstatement.pdf. accessed 2022 jun 21. 19. patel s, brown j, semper m, abella f, mannocci f. european society of endodontology position statement: use of cone beam computed tomography in endodontics. int endod j. 2019; 52(12): 1675–8. 20. da silveira pf, fontana mp, oliveira hw, vizzotto mb, montagner f, silveira hl, silveira he. cbct-based volume of simulated root resorption influence of fov and voxel size. int endod j. 2015; 48(10): 959–65. 21. pauwels r, araki k, siewerdsen jh, thongvigitmanee ss. technical aspects of dental cbct: state of the art. dentomaxillofac radiol. 2015; 44(1): 20140224. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i2.p68–72 https://www.aae.org/specialty/wp-content/uploads/sites/2/2017/06/conebeamstatement.pdf https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i2.p68-72 vol 49 no 3 juli-sept 2016.indd 168 effects of soy isoflavone genistein on orthodontic tooth movement in guinea pigs sri suparwitri,1 pinandi sri pudiyani,1 sofia mubarika haryana,2 and dewi agustina3 1department of orthodontics, faculty of dentistry, universitas gadjah mada 2department of histology & cell biology, faculty of medicine, universitas gadjah mada 3department of oral pathology, faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: osteoblast and osteoclast are the important factor in periodontal tissue remodeling for the orthodontic treatment success. resorption process takes place in compression area by osteoclast and apposition in the tension area by osteoblast. in general hormone condition and age affect remodeling process. estrogen has a high contribution in remodelling process and decreased in elderly individual such as menopausal women. soybean contains isoflavone genistein which has similar structure and activity to estrogen. many researchers indicate that isoflavone genistein not only has an inhibitor effect in osteoporosis but also has estrogenic and antiestrogenic effect as well. purpose: the study aimed to investigate the effect of soybean isoflavone genistein administration on osteoblast and osteoclast cells number in orthodontic tooth movement of young and old guinea pigs. method: the research was quasi-experimental study with post test only with control design. the experimental animals were 24 male guinea pigs that divided into: young guinea pigs (±4 months old) and old guinea pigs (±2.5 years old). each group was divided into 4 subgroups for receiving the treatment namely; control, orthodontic treatment, genistein treatment and orthodontic+genistein treatment. all of the subjects were sacrificed at day 7 and the specimens were histologically analyzed using tartrate resistance acid phosphatase (trap) and hematoxylin eosin (he) staining and observed using microscope that connected to obtilab and an image raster program. result: u mann-whitney statistical analysis showed there were significant differences in osteoblast cell numbers; between orthodontic treatment and orthodontic+genistein treatment in the old guinea pigs (p=0.004); between orthodontic treatment in the young guinea pig and orthodontic+genistein treatment in the old guinea pig (p=0.016); between orthodontics treatment and orthodontic+genistein treatment in the young guinea pigs (p=0.025). u mann-whitney statistical analysis showed there were significant differences in osteoclast cell numbers: between the orthodontic treatment in the old guinea pig and orthodontics+genistein treatment in the young guinea pigs (p=0.007); between orthodontic treatment group in the young guinea pigs and orthodontics+genistein treatment in the old guinea pigs; between orthodontic treatment and orthodontic+ genistein treatment in the young guinea pigs (p=0.007). all groups administered by genistein the numbers of osteoblast in the surrounding of the tension sites increased, while in the surrounding of the compression sites had less osteoclasts; even, there were no osteoclasts found in some samples. conclusion: soybean isoflavone genistein administration on orthodontic tooth movement increased osteoblast numbers in the tension sides and decreased osteoclast numbers in the compression sides. keywords : isoflavone genistein of soybean; guinea pig; orthodontic tooth movement correspondence: sri suparwitri, department of orthodontics, faculty of dentistry universitas gadjah mada. jl. denta1, sekip utara yogyakarta 55281, indonesia. e-mail: mbak_loki@yahoo.com research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 168–174 introduction orthodontic treatment aims to improve the functionality and aesthetics of teeth, either in children, in adults, or in elderly (seniors). one of the main problems that encourage the orthodontic treatment usually is the presence of malocclusion, especially related with the function, aesthetics, and physiological function of teeth . this means that the orthodontic treatment is performed not only for the aesthetic needs of patients, but also for some functional dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 169169suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 reasons.1,2 one way of handling the orthodontic treatment of malocclusion is by moving the teeth and jaws into right position.3 based on data obtained from the ministry of health in 2004, 60% of indonesia’s population suffers from oral and dental diseases, and malocclusion disorder ranked the second position after dental caries.4 the prevalence of malocclusion in adolescents in indonesia is quite high, and the number is relatively still stable, about 80-90%.5 in 2014, the percentage of malocclusion patients in indonesia amounted to 80% of the total population.6 malocclusion can also be considered as a oral and dental abnormality associated with occlusion. occlusion can be defined as a contact between under teeth and upper teeth when mouth is closed.7 the success of orthodontic treatment depends on the process of periodontium tissue remodeling during the treatment. periodontium tissue surrounding and supporting the teeth, which main components are osteoblasts, osteoclasts, sementoblas, and a series of collagen fibers with a basis of proteoglycans and glycoprotein.8 bone remodeling process that involves apposition and resorption processes is closely associated with the number of osteoclasts and osteoblasts. bone is a dynamic tissue constantly experiencing apposition and resorption. the peak of the human ossification occurs around the age of 35 years, and will decrease slowly.9 mundy also states that the bone mass decreases progressively with age in both men and women starting at the age of 30 years.10 bone formation or apposition in children who are developing is greater than bone resorption, whereas in healthy adults bone apposition and bone resorption are balanced. menopause and aging process both in men and in women will trigger a decrease in bone formation or apposition compared to bone resorption.11 the imbalance between bone formation by osteoblasts and bone resorption by osteoclasts in menopausal women is due to hormonal changes, leading to the high rate of osteoporosis-related fractures.12 osteoblasts and osteoclasts are two important cells involved in orthodontic tooth movement, so a lot of researches have focused on these cells. osteoblasts involved in bone formation will appear 40-48 hours after the provision of orthodontic power.13 a study was done by boulpaep and boron cited by kini and nandeesh states that after several hours under the pressure, osteoclasts on the side of the periodontal membrane will multiply and begin to resorb bone surface, whereas in areas exposed to traction, osteoblasts will multiply and begin to form new bone layers on the bone walls.14 based on the circumstances, therefore, the success of orthodontic treatment is determined by the success of the remodeling process that depends on the quantity and quality of osteoclasts and osteoblasts.15 some phytochemical compounds also known as phytonutrients including phytoestrogens are plant compounds that exist in the daily diet. phytoestrogens can positively regulate a number of physiological functions in mammalian systems involved in chronic diseases, such as osteoporosis. however, their effects on tooth movement still have not been known certainly.13 natural products containing phytoestrogens are soy isoflavone genistein that can stimulate osteoblast differentiation and new bone formation in osteoporosis treatment. phytoestrogens are natural estrogenic compounds found in many plants and seeds. this compound has a structure similar to mammalian estrogen.16 this compound also has estrogenic or antiestrogenic effects.16,17 soy isoflavone genistein is bioactive compounds that have estrogen-like activity. according to a lot of researches, soy isoflavone genistein also plays a role in the prevention and treatment of osteoporosis associated with a decrease in estrogen levels.12,17,19 isoflavones, thus, may inhibit bone osteoporosis in women after the menopause.18 some derivatives of the soybean have been identified to have a positive effect on bone without triggering side effects.19 for those reasons, this research aimed to analyze the effects of soy isoflavone genistein on remodeling of tissues supporting teeth in old and young guinea pigs which their teeth were moved orthodontically. materials and methods this research was a quasi-experimental study with posttest control group design. the subjects included 24 male guinea pigs, consisted of 12 young males (age ± 4 months) with an average weight of ± 300 g and 12 old males (age ± 2.5 years) with an average weight of ± 800 grams obtained from lppt unit 4 universitas gadjah mada. guinea pigs were adapted for 5 days by giving a standard diet in the form of pellets and green vegetables as a source of vitamin c. they were kept in a cage at room temperature. those young and old samples were randomly divided into four (groups), namely: 1) control group; 2) treatment group given orthodontic treatment; 3) treatment group given isoflavone genistein (genistein treatment); 4) treatment group given both orthodontic treatment and isoflavone genistein treatment. each tooth moved was lower incisor with an open coil spring with a strength of 35 cn (figure 1). genistein was derived from genistein tempe formula (produced by prof. mien karmini from ipb) composed of soybean, tape, flour, sugar, creamer, salt, and baking powder. genistein orally given to guinea pigs was at doses of 1.2 mg to 0.6 mg daily. on the 7th day, they were sacrificed, and then their mandible was cut. histological tests on osteoclasts for all groups using tartrate resistance acid phosphatase (trap) staining similar to the method of a research conducted by kalajzic et al.20 and histological tests for all groups on osteoblasts using hematoxylin eosin staining (he) corresponding to the method of a research conducted by mirhashemi et al.21 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 170 suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 osteoclasts and osteoblasts were observed by a light microscope (olympus cx21fsi, tokyo, japan)) with attachement of obtilab advance. a raster program software was used to calculate them. results based on the results of both histological observation using optilab, as well as osteoclast and osteoblast calculations in both pressure and tension sites using raster image program to 5 visual fields on each subject research, means and standard deviations of osteoclasts can be seen in table 1, while means and standard deviations of osteoblasts can be seen in table 3. osteoclasts appeared with many red nucleated cells after using trap staining, while osteoblasts attached to bone after using he staining (figure 2, figure 3, figure 4 and figure 5) figure 1. orthodontic appliance installation of an open coil spring mounted with round wire between two brackets on the incisors of the guinea pigs. tension site was next to a mesial area of incisors, both right and left. pressure site was next to a a distal area of incisors, both right and left. a) open coil springs; b) bracket. a b c d figure 2. the results of histology test on periodontal tissues of the old guinea pigs, on the 7th day, at 400x magnification, with trap staining. a) the control group; b) the orthodontic treatment group; c) the genistein treatment group; d) the orthodontic + genistein treatment group. a b c d figure 3. the results of histology test on periodontal tissues of the young guinea pigs, on the 7th day, at 400x magnification, with trap staining. a) the control group; b) the orthodontic treatment group; c) the genistein treatment group; d) the orthodontic + genistein treatment group. a b c d figure 4. the results of histology test on periodontal tissues of the old guinea pigs, on the 7th day, at 400x magnification, with he staining. a) the control group; b) the orthodontic treatment group; c) the genistein treatment group; d) the orthodontic + genistein treatment group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 171171suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 the results of kruskal wallis test on all four treatments (the control group, the orthodontic treatment group, the genistein treatment group, and the orthodontic + genistein treatment group) showed that there was no significant difference in the number of osteoclasts with a p value of 0.0000. based on the results, the highest number of osteoclasts in the surrounding of the pressure sites of those old guinea pigs was in the orthodontic treatment, followed with the orthodontic + genistein treatment group, the control group (osteoclasts seemed a bit), and the genistein treatment group (osteoclasts did not appear). it means that the lowest number of osteoclasts in the surrounding of the pressure a b c d figure 5. the results of histology test on periodontal tissues of the young guinea pigs, on the 7th day, at 400x magnification, with he staining. a) the control group; b) the orthodontic treatment group; c) the genistein treatment group; d) the orthodontic + genistein treatment group. table 1. means and standard deviations of osteoclasts on the pressure sites based on treatment and age age treatment control orthodontic genistein orthodontic + genistein old 1.00± 1.26 10.67± 8.66 0 4.00± 6.20 young 12.00± 9.59 20.17± 16.46 1.50± 2.34 0 table 2. results of the mann-whitney u test on the number of osteoclasts in the pressure sites based on treatment and age treatment control orthodontic genistein ortho+genistein old young old young old young old young control old 1.00 0.872 0.028 0.059 1.00 1.00 0.059 young 0.872 0.470 0.007 0.037 0.096 0.007 orthodontic old 0.297 0.007 0.02 0.242 0.007* young 0.007 0.02 0.046* 0.007* genistein old 0.14 0.138 1.00 young 0.702 0.14 ortho+ genistein old 0.138 young note: * there was a significant difference (p<0.05) table 3. means and standard deviations of osteoblasts on the tension sites based on treatment and age age treatment control orthodontic genistein orthodontic + gensitein tension tension tension tension old 43.33± 19.46 55.33± 23.58 42.17± 33.24 104.17± 57.87 young 78.83± 36.60 69.83± 39.10 135.17± 87.25 145.67± 44.88 sites of those old guinea pigs was in the genistein treatment group. on the other hand, the highest number of osteoclasts in the surrounding of the pressure sites of those young guinea pigs was in the orthodontic treatment, followed with the control group, the genistein treatment group, and the orthodontic + genistein treatment group (osteoclasts did not appear). it can be said that the lowest number of osteoclasts in the pressure sites of those young guinea pigs was in the orthodontic + genistein treatment group. moreover, the results of mann-whitney u test showed that there was a significant difference in the number of osteoclasts in the surrounding of the pressure site (p = 0.007) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 172 suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 between the orthodontic + genistein treatment group of the old guinea pigs and the orthodontic + genistein treatment group of the young guinea pigs. there was also a significant difference in the number of osteoclasts in the surrounding of the pressure site between the orthodontic treatment group of the young guinea pigs and the orthodontic + genistein treatment group of the young guinea pigs (p = 0.007). the number of osteoclasts in the orthodontic + genistein treatment group of the young guinea pigs was higher than the orthodontic treatment group of the young guinea pigs. similarly, there was also a significant difference in the number of osteoclasts in the surrounding of the pressure site between the orthodontic treatment group of the young guinea pigs and the orthodontic + genistein treatment group of the old guinea pigs (p = 0.046) (table 2). based on the results of the kruskal wallis test, furthermore, it is also known that there was a significant difference on the number of osteoblasts in the surrounding of the tension sites between all four groups (p=0.0000). based on the results, the highest number of osteoblasts on the tension sites of those old guinea pigs was in the orthodontic+genistein treatment group, followed with the genistein treatment group, the control group, and the orthodontic treatment group. this means that the lowest number of osteoblasts in the surrounding of the tension sites of those old guinea pigs was in the orthodontic treatment group. on the other hand, the highest number of osteoblasts in the surrounding of the tension sites of those young guinea pigs was in the orthodontic+genistein treatment group, followed with the control group, the genistein treatment, and the orthodontic treatment group. this indicates that the lowest number of osteoblasts in the surrounding of the tension sites of those young guinea pigs was in the orthodontic treatment group. in addition, the results of the mann-whitney u test showed that there was a significant difference (p = 0.004) in the number of osteoblasts in the surrounding of the tension sites between the orthodontic treatment group of the old guinea pigs and the orthodontic+genistein treatment group of the old guinea pigs. the number of osteoblasts in the (s el ) treatment group age group old young figure 6. mean number of osteoclasts on the pressure sites of young and old guinea pigs. treatment group age group old young figure 7. mean number of osteoblasts on the tension sites of young and old guinea pigs. table 4. results of the mann-whitney u test on the number of osteoblasts in the tension sites based on treatment and age treatment control orthodontic genistein ortho+genistein old young old young old young old young control old 0.423 0.419 0.688 0.297 0.037 0.01 0.02 young 1.00 0.336 0.149 0.261 0.006 0.037 orthodontic old 0.376 0.335 0.054 0.004* 0.054 young 0.631 0.078 0.016* 0.025* genistein old 0.016 0.004 0.016 young 0.748 0.81 old 0.748 young note: *there was a significant difference (p<0.05) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 173173suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 orthodontic+genistein treatment group of the old guinea pigs was higher than in the orthodontic treatment group of the old guinea pigs. there was also a significant difference in the number of osteoblasts in the surrounding of the tension sites between the orthodontic treatment group of the young guinea pigs and the orthodontic+genistein treatment group of the old guinea pigs (p = 0.016). the number of osteoblasts in the orthodontic+genistein treatment group of the old guinea pigs was higher than in the orthodontic treatment group of the young guinea pigs. similarly, there was a significant difference in the number of osteoblasts in the surrounding of the tension sites between the orthodontic treatment group of the young guinea pigs and the orthodontic+genistein treatment group of the young guinea pigs (p = 0.025). the number of osteoblasts in the orthodontic+genistein treatment group of the young guinea pigs was higher than in the orthodontic treatment group of the young guinea pigs (table 4). discussion the results of kruskal wallis test showed that there were no significant differences in the number of osteoclasts (p<0.05) and in the number of osteoblasts (p<0.05) between the orthodontic treatment and the orthodontic+genistein treatment groups. similarly, the results of the mannwhitney u showed that there was no significant difference (0.242) between the orthodontic treatment of the old guinea pigs and the orthodontic + genistein treatment of the old guinea pigs groups. this condition is probably caused by the fact that the observation was carried out on the 7th day. osteoblasts and osteoclasts involved in bone formation will appear 40-48 hours after the provision of the orthodontic force.13 therefore, on the 7th day, the osteoclasts had been replaced with bone-forming cells, known as osteoblasts. based on the results, the highest number of osteoclasts in the surrounding of the pressure sites of those old guinea pigs was on the orthodontic treatment, followed with the orthodontic + genistein treatment, the control (osteoclasts seemed a bit), and the genistein treatment groups (osteoclasts did not appear). this condition is in accordance with a statement of dang et al.22 arguing that the estrogenic effects of isoflavone genistein involve a mechanism mediated by estrogen receptors. isoflavone in this case acts as an estrogen stimulating osteogenesis, forming osteoblasts and inhibiting osteoclasts. as a result, in the orthodontic + genistein treatment group, the number of osteoclasts reduced, and even in the genistein treatment group, there was no any osteoclast. this is likely also due to the administration of genistein, considered as phytonutrients or phytoestrogens, have a chemical composition and function similar to estrogen. some phytochemical compounds are also known as phytonutrients, including phytoestrogens considered as plant compounds that exist in daily diet. phytoestrogens can positively regulate a number of physiological functions in mammalian systems, involved in chronic diseases such as osteoporosis. on the other hand, the highest number of osteoclasts in the surrounding of the pressure sites of those young guinea pigs was in the orthodontic treatment, followed with the control group, the genistein treatment group, and the orthodontic + genistein treatment group. osteoclasts even did not appear in the orthodontic + genistein treatment group of the young guinea pigs. meanwhile, osteoclasts did not appear on the genistein treatment group of the old guinea pigs. this is in accordance with a statement of dang and lowik23 arguing that the anti-estrogenic effect of isoflavones on micro molar concentrations cannot be explained only through its role when mediated by estrogen receptors, but also can be related to the results of the competition between estrogen and isoflavones to bind to the estrogen receptors. in addition, the results of the statistical analysis on osteoblasts showed that there were significant differences (p<0.05) between the orthodontic treatment group of the old guinea pigs and the orthodontic + genistein treatment group of the old guinea pigs, between the orthodontic treatment group of the young guinea pigs and the orthodontic + genistein treatment group of the old guinea pigs, and also between the orthodontic treatment group of the young guinea pigs and the orthodontic + genistein treatment group of the young guinea pigs. on the other hand, there were not statistically significant differences in the number of osteoblasts between the orthodontic treatment group of the old guinea pigs and the orthodontic + genistein treatment group of the young guinea pigs. in overall, the highest number of osteoblasts was on the orthodontic + genistein treatment group. it is in line with a research conducted by tsuda and ushiroyama showing that isflavones have a variety of effects, such as increasing osteoblast activity and inhibiting osteoclast activity.24,25 the effects of isoflavones on the number of osteoblasts in vitro actually have already been known.12,19,22,27 genistein is able to bind to β estrogen receptors in osteoblasts. dna contained in osteoblasts, as a result, will increase significantly due to the addition of genistein or daidzein, triggering isoflavones to stimulate the proliferation of osteoblasts. isoflavones can also increase the activity of alkaline phosphatase enzyme as a marker in the differentiation of osteoblasts.26,27 it can be concluded that the administration of soy isoflavone genistein can increase the number of osteoblasts in the surrounding of tension sites. the administration of soy isoflavone genistein also can decrease the number of osteoclasts, or can make osteoclasts not appear at all in the surrounding of pressure sites in both genistein treatment group of old guinea pigs and orthodontics + genistein group of young guinea pigs. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 174 suparwitri, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 168–174 references 1. dewanto h. aspek-aspek epidemiologi maloklusi. yogyakarta: gadjah mada university press; 1993. p. 1-77. 2. graber rl, vanarsdall jr. orthodontics, current principles and techniques. 4th ed. mosby, st. louis: vig publisher; 2000. p. 557646. 3. english jd, buschang ph, throckmorton gs. does malocclusion affect masticatory performance?. angle orthod 2002; 72(1): 21-7. 4. dewi o. analisis hubungan maloklusi dengan kualitas hidup pada remaja smu kota medan tahun 2007. tesis. medan: universitas sumatera utara; 2008. 5. atashi mha. prevalence of malocclusion in 13-15 year-old adolescents in tabriz. joddd 2007; 1(1): 1-6. 6. laguhi va, anindita ps, gunawan pn. gambaran maloklusi dengan menggunakan hmar pada pasien di rumah sakit gigi dan mulut universitas sam ratulangi manado. jurnal e-gigi 2014; 2(2): 1-7. 7. lew kk, foong wc, loh e. malocclusion prevalence in an ethnic chinese population. aust dent j 1993; 38(5): 442-9. 8. roberts-harry d, sandy j. orthodontics. part 10: impacted teeth. br dent j 2004; 196: 319-27. 9. ren y, maltha jc, van‘t hof ma, kuijpers-jagtman, am. age effect on orthodontic tooth movement in rats. j dent res 2003; 82(1): 3842. 10. mundy gr. nutritional modulators of bone remodelling during aging. am j clin nutr 2006; 83(suppl): 427s-30s. 11. parfitt am. bone remodeling. relationship to the amount and structure of bone and pathogenesis and prevention of fractures. in: riggs bl, melton lj, editors. osteoporosis etiology, diagnosis and management. new york: raven press; 1988. 12. ma df, qin lq, wang py, katoh r. soy isoflavone intake inhibits bone resorption and stimulates bone formation in menopausal women: meta-analysis of randomized controlled trials. eur j clin nutr 2008; 62(2): 155-61. 13. cornwell t, cohick w, raskin i. dietary phytoestrogens and health. phytochemistry 2004; 65(99): 5-1016. 14. kini u, nandeesh bn. physiology of bone formation, remodeling, and metabolism. in: fogelman i, gnanasegaran g, van der wall h, editors. radionuclide and hybrid bone imaging. springer-verlag berlin heidelberg; 2012. p. 29-55. 15. ariffin, shz, yamamoto, abidin lzz, wahab rma, ariffin zz. cellular and molecular changes in orthodontic tooth movement, review article. scientific world j 2011; 11: 1788–803. 16. s e t c h el l k dr , a d l e r c r e u t z h . m a m m a l i a n l ig n a n s a n d phytoestrogens. recent studies on their formation, metabolism and biological role in health and disease. in: rowland ir, editor. role of the gut flora in toxicity and cancer. london, uk: academic press; 1988. p. 315-45. 17. tepavčević v, cvejić j, poša m, popović j. isoflavone content and composition in soybean. in: tzi bun ng, editor. soybean biochemistry, chemistry, and physiology. croatia: intech; 2011. p. 281-94. 18. price k, fenwick g. naturally occurring oestrogens in foods-a review. food addit contam 1985; 2: 73 -106. 19. turhan no, bolkan f, duvan ci, ardicoglu y. the effect of isof lavones on bone mass and bone remodelling markers in postmenopausal women. turk j med sci 2008; 38(2): 145-52. 20. kalajzic z, peluso eb, utreja a, dyment n, nihara j, xu m, chen j, uribe f, wadhwa s. effect of cyclical forces on the periodontal ligament and alveolar bone remodeling during orthodontic tooth movement. angle orthod 2014; 84: 297-303. 21. mirhashemi ah, afshari m, alaeddini m, etemad-moghadam s, dehpour a, sheikhzade s, akhoundi msa. effect of atorvastatin on orthodontic tooth movement in male wistar rats. j dent (tehran) 2013; 10(6): 532-9. 22. dang zc, audinot v, papapoulos se, boutin ja, lowik cwgm. peroxisome proliferator-activated receptor γ (pparγ) as a molecular target for the soy phytoestrogen genistein. j biol chem 2003; 278(2): 962-7. 23. dang zc, lowik c. dose-dependent effects of phytoestrogens on bone. trends endocrinol metab 2005; 25(5): 208-13. 24. tsuda m, kitazaki t, ito t, fujita t. the effect of ipriflavone (tc80) on bone resorption in tissue culture. j bone miner res 1986; 1: 207-11. 25. ushiroyama t, okamura s, ikeda a, ueki m. efficacy of ipriflavone and 1 alpha vitamin d therapy for the cession of vertebral bone loss. int j gynaecol obstet 1995; 48: 283-8. 26. kuiper gjm, lemmen jg, carlsson bo, corton jc, safe sh, van der saag pt, van der burg b, gudtafsson j-a. interaction of estrogenic chemicals and phytoestrogen with estrogen receptor β. endocrinology 1998; 139(10): 4252-62. 27 yamaguchi m. isof lavone and bone metabolism: its cellular mechanism and preventive role in bone loss. j health sci 2002; 48(3): 209-22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p168-174 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p168-174 36 dental journal (majalah kedokteran gigi) 2020 march; 53(1): 36–39 research report the distribution of streptococcus mutans and streptococcus sobrinus in children with dental caries severity level nur dianawati,1 wahyu setyarini,2 ira widjiastuti,3 rini devijanti ridwan4 and k. kuntaman5 1post-graduate program of basic medical science, faculty of medicine, universitas airlangga 2institute of tropical disease, universitas airlangga 3department of conservative dentistry, faculty of dental medicine, universitas airlangga 4department of oral biology, faculty of dental medicine, universitas airlangga 5department of clinical microbiology, faculty of medicine, universitas airlangga and dr. soetomo hospital surabaya – indonesia abstract background: the prevalence of dental caries is high worldwide and specifically in indonesia, especially in children. cariogenic bacteria are the major cause of dental caries. streptococcus mutans (s. mutans) is one of the bacteria often associated with caries, due to its ability in producing acid and forming the biofilm for bacterial colonisation on the surface of oral cavities. in addition to s. mutans, streptococcus sobrinus (s. sobrinus) bacteria are also thought to play an important role in the process of caries. purpose: this study aims to analyse the distribution of s. mutans and s. sobrinus in children with seriously high dental caries levels. methods: this study was an observational analytical study. bacterial isolation was conducted in carious lesions of 50 paediatric patients 6-12 years old with superficial dental caries. samples of caries lesions were put directly into a tube containing the brain heart infusion broth (bhi-b) and incubated at 37o c for 24 hours. the samples were sub-cultured on selective tryptone yeast cystine sucrose bacitracin (tycsbhimedia) agar, and then incubated for two days. bacterial identification was then performed using the polymerase chain reaction (pcr) multiplex method. statistical analysis with chi-square. results: the total number of children with dental caries included in this study was 50. among these, 94% showed positive for s. mutans and 30% positive for s. sobrinus. the analysis of the prevalence of bacterial colonisation (s. mutans and s. sobrinus) based on caries severity and the simplified oral hygiene index (ohi-s), showed there was no significant difference (p> 0.05). conclusion: this study showed that among 50 caries noted in the children, 94% were colonised s. mutans and 30% s. sobrinus. there was no significant difference between the colonisation of s. mutans and s. sobrinus among children from the severe to mild decayed exfoliated filling teeth (deft) category, and between bad and good ohi-s. keywords: caries severity; dental caries; ohi-s; streptococcus mutans; streptococcus sobrinus correspondence: k. kuntaman, department of clinical microbiology, faculty of medicine, universitas airlangga. jl mayjend. prof. dr moestopo 47 surabaya 60132, indonesia. email: kuntaman@fk.unair.ac.id introduction dental caries is a serious oral health problem in indonesia and the rest of the world. based on the 2018 basic health research (riskesdas) data, the prevalence of caries in indonesia was significantly high, above the world health organization (who) target.1,2 according to the riskesdas data, the prevalence of caries reached 93% in children aged between five and six years, while who and federation dentaire internationale (fdi) had a target to make 50% of children free of dental caries. the decay missing filling teeth (dmft) index for primary teeth in children at these ages was 8.43, indicating severe early childhood caries were found in roughly nine teeth per child. moreover, dental caries is mostly caused by cariogenic bacterial infections. streptococcus mutans (s. mutans) is the main cariogenic bacterium in the pathogenesis process of caries.3 streptococcus sobrinus (s. sobrinus) is also thought to play a role in the production of caries.4 the pathogenesis process for dental caries involving s. mutans usually starts with bacterial colonisation. s. mutans biofilm then produces dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p36–39 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p36-39 37dianawati, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 36–39 organic acids as a by-product of fermentable carbohydrate metabolism. this acid can cause the local ph to fall below the critical value, resulting in the demineralisation of dental tissue. one of the results of s. mutans producing high cariogenicity levels is its ability to adhere to the surface of a tooth. this attachment is successfully performed by extracellular polysaccharides (eps) derived from sucrose. this process also involves the microbiological characteristics of the bacterial cell wall structure.5 in most cases, s. mutans is the main cause of caries. nevertheless, the role of acidogenic and other aciduric bacteria, such as s. sobrinus, is also assumed to be important. based on several epidemiological and in vitro studies, s. sobrinus can be more cariogenic than s. mutans.6 the virulence of the s. mutans group is related to its ability to colonise and develop on the tooth surface during acidic conditions. these properties include the production and regulation of adhesion proteins, glucosyltransferases (gtf), and extracellular polysaccharides, such as glucans which allow bacteria to attach firmly to the surface of teeth in biofilms. the two species (s. sobrinus and s. mutans), however, have different strategies in their attachment mechanism. s. mutans uses pellicles and specific surface antigens directly, while s. sobrinus uses glucans.7 previous research in mongolia also showed that children aged between five and seven with both s. mutans and s. sobrinus in their saliva had significantly more dental caries than those who had only s. mutans or s. sobrinus.8 in test animals, s. sobrinus can produce more acids than other species in the s. mutans group. the prevalence and level of s. mutans and s. sobrinus colonies, as a result, have been used as biological markers for caries prediction.9 this study aims to analyse the distribution of s. mutans and s. sobrinus in children with severe levels of dental caries. materials and methods this study was approved by the ethics committee number: 328/hrecc.fodm/vi/2019 of the faculty of dental medicine, universitas airlangga. this study was an observational analysis research. samples were collected october 2nd-10th, 2019. the dental and oral hospital, faculty of dental medicine, universitas airlangga, surabaya, supplied samples from 50 paediatric patients. the patients were aged 6-12 years. parents of these paediatric patients signed informed consent. the teeth examined were deciduous molars. the types of caries examined were superficial caries or caries media. next, the severity of the caries was analysed based on decayed exfoliated filling teeth (deft) and the simplified oral hygiene index (ohi-s). s. mutans and s. sobrinus were then identified with multiplex polymerase chain reaction (pcr). the caries lesions were taken from the first molar teeth using a sterile excavator and placed directly into the bottom of a tube containing brain heart infusion broth (bhi-b) (merck, darmstadt, germany). next, the tube was put into an incubator at 37oc for 24 hours. on the second day, the lesion was sub-cultured on selective tryptone yeast cystine sucrose bacitracin (tycsb) (himedia, himedia laboratories pvt ltd, india), and then incubated for two days. the growth of the colonies was indicated by the macroscopic characteristics of the colony of s. mutans, such as the hardened and sticky crystal form on the media, which were then examined with pcr. the pcr multiplex method was used to detect s. mutans and s. sobrinus bacteria. the results of the amplification were then visualised using an electrophoresis method. dna extraction was performed using the boiling method in te buffer. a suspected three to five colonies on tycsb media were taken and inoculated in eppendorf tubes containing 100 μl of te buffer, and homogenised by vortex mixer. the suspension was heated at a thermostat temperature of 95oc for ten minutes (eppendorf, north america). after the samples reached room temperature, they were centrifuged at 10,000 rpm for ten minutes. the extracted dna in the supernatant was stored at -20oc before use as the dna template for pcr.10 pcr was run 25 μl prc mixture, as follows: 12.5 μl of dntpmix (dream taq green, thermo scientific, usa), 0.5 μl (50pmol), 3.5 μl of bacterial dna template, 1ul tag pol, and then adding 17 μl of distilled water. the primers sequence of s. mutans used gtf-b f :act aca ctt tgc ggt ggc ttgg as forward and gtf-b r :cag tat aag cgc cag ttt cact as reverse in 517 bp.11 primers sequence of s. sobrinus used gtf-i f : gat aac tac ctg aca gct gac t as forward and gtf-i r : aag ctg cct taa ggt aat cac t as reverse in 712 bp.12 dna amplification was then performed using a thermal cycler pcr machine (icyler, biorad thermal cycler).13 the pcr was first run using a hot initial temperature of 95oc for one minute and amplified for 35 cycles with denaturations at 94oc for 30 seconds, annealing at 53oc for one minute, elongation at 72oc for two minutes, and ending at 72oc for seven minutes. pcr results were visualised using electrophoresis in 2% agarose gel (spectronics corporation, usa), with 100 bp marker ladder. electrophoresis was run at 100 volts for 30 minutes. next, the agarose gel was stained with ethidium bromide solution for 20 minutes. the amplicons were visualised using geldoc (digibox 7000, mbiotech, korea). positive results were shown by the presenting of amplicon 517 bp for s. mutans and 712 bp for s. sobrinus. the results were studied using descriptive analysis of the distribution of s. mutans and s. sobrinus with caries severity, and then statistically analysed with the chi-square test. results after conducting research, bacterial isolates obtained in this study were identified by multiplex pcr to confirm s. mutans and s. sobrinus (figure 1). figure 1 showed results of multiplex pcr positive s. mutans (gtf-b) in the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p36–39 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p36-39 38 dianawati, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 36–39 number 7038, 7039, 7040, 7041, 7042, 7043, 7044, 7045, 7046, 7048, 7049 and positive s. sobrinus (gtf-i) in the number 7039, 7043, 7048, 7049. the results were then grouped based on deft severity and ohi-s, then analysed by frequency distribution and chi-square. the prevalence of the colonisation of s. mutans was higher compared to s. sobrinus, 94% vs 30% (table 1). based on the level of caries using deft scores, severe scores for s. mutans were higher than mild scores. statistically, there was no significant difference using the chi-square test (p value >0.05). as with s. mutans, s. sobrinus was higher in severe scores than mild scores of deft, but statistically, there was no significant difference. based on ohi-s scores, s. mutans good scores were higher than bad scores. statistical tests using chi-square showed there was no significant difference (p value >0.05). as with s. mutans, the good deft s. sobrinus scores were higher than the mild scores, but again, statistically, there was no significant difference. discussion several studies have shown that preventive efforts are effective in deterring early s. mutans colonisation from causing dental caries in children.8 hence, this study aims to reveal the incidence pattern of s. mutans and s. sobrinus in children based on the deft and ohi-s. next, the results of this study found that the highest incidence of bacteria causing dental caries was s. mutans (94%). meanwhile, the incidence of s. sobrinus was 30%. these findings indicate the existence of s. mutans is considered not only as a microflora of the oral cavity but also as a pathogenic bacterium causing caries. both s. mutans and s. sobrinus can proliferate in dental biofilm plaque. their virulence is mainly due to their high adhesion ability, acidity, and their properties. moreover, dental biofilm containing cariogenic bacteria (caries-related micro-organisms) is one of the most harmful factors associated with the development of tooth decay. dental biofilms can be found on hard surfaces in the oral cavity, such as on surfaces, implants, orthodontic devices, or restorative materials. the development of biofilm processes involves several progressive stages. the formation of initial biofilm accumulation involves specific processes. variations in the biofilm coat in the oral cavity have a significant impact on oral ecology and dental caries development.14 the higher colonisation rate of s. mutans and s. sobrinus, as demonstrated in this study would be ruled table 1. the isolation rate of s. mutans and s. sobrinus among children (n=50) with various dental caries severity level from patients visiting dental and oral hospital universitas airlangga severity level children with dental caries (n=50) s. mutans (n=47=94%) p value s. sobrinus (n=15=30%) p value not identified s. mutans and s. sobrinus (n=3=6%) deft mild 12 (24%) 0.124 3 (6%) 0.409 2 severe 35 (70%) 12 (24%) 1 ohi-s good 35 (70%) 0.315 9 (18%) 0.083 3 bad 12 (24%) 6 (12%) 0 figure 1. the results of multiplex pcr on s. mutans (gtf-b) and s. sobrinus (gtf-i). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p36–39 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p36-39 39dianawati, et al./dent. j. (majalah kedokteran gigi) 2020 march; 53(1): 36–39 by antigen i/ii protein that strengthens the adherence to the tooth surface. it was also facilitated by glycoprotein receptors present in saliva, called salivary agglutinin.15 the other factors are cell-to-cell adherence and development of cohesive and pathogenic biofilms via the expression of gtfs. these enzymes (140 to 160 kda) produce extracellular adhesive glucans that vary in chain length, contain α-1,3 and α-1,6 glucosyl linkages, and have a degree of branching and solubility. s. mutans comprises three genes for gtf: gtf-b, responsible for insoluble glucan synthesis; gtf-c, for soluble and insoluble glucan synthesis; and gtf-d, for soluble glucan synthesis. s. sobrinus expresses gtf-i and gtf-s, encoding enzymes that produce insoluble and soluble glucans, respectively.15 the prevalence of s. mutans and s. sobrinus is widely associated with caries. in several epidemiological studies, there was a correlation between the existence of s. sobrinus and the high incidence of dental caries.16 the results of this study show that there was no statistically significant difference in the incidence of s. mutans and s. sobrinus between the high and low caries severity level. this can be caused by several factors, such as host factors, bacterial virulence, diet, environment, and time. risk factors, such as sociodemographic factors, socioeconomic factors, knowledge levels, as well as behaviour, also affect the incidence of caries. dental caries occurs because of an imbalance between demineralisation and remineralisation. when demineralisation is higher than remineralisation, caries can occur. oral hygiene also has a role in this balance.17 nevertheless, in this study, there was no significant difference in the incidence pattern of s. mutans, s. sobrinus based on high or low deft and ohi-s. finally, this study interestingly reveals that there was no significant difference in the incidence pattern of s. mutans and s. sobrinus bacteria based on the severity of caries and ohi-s. this means that although these two bacteria are considered the main factors that cause dental caries, other factors may have an equally important role in caries. hence, further research is expected to focus on more in-depth studies of s. mutans and s. sobrinus bacteria with other risk factors. in conclusion, this study showed that among carious teeth, 94% were colonised by s. mutans, and 30% of cases demonstrated co-colonisation of s. mutans and s. sobrinus. there were no significant differences in these bacterial colonisations between various levels of dental caries. references 1. esberg a, sheng n, mårell l, claesson r, persson k, borén t, strömberg n. streptococcus mutans adhesin biotypes that match and predict individual caries development. ebiomedicine. 2017; 24: 205–15. 2. badan penelitian dan pengembangan kesehatan. hasil utama r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 66–71. 3. shimomura-kuroki j, nashida t, miyagawa y, sekimoto t. the role of genetic factors in the outbreak mechanism of dental caries. j clin pediatr dent. 2018; 42(1): 32–6. 4. fontana m, zero dt. assessing patients’ caries risk. j am dent assoc. 2006; 137(9): 1231–9. 5. selwitz rh, ismail ai, pitts nb. dental caries. lancet. 2007; 369(9555): 51–9. 6. okada m, taniguchi y, hayashi f, doi t, suzuki j, sugai m, kozai k. late established mutans streptococci in children over 3 years old. int j dent. 2010; 2010: 1–5. 7. conrads g, de soet jj, song l, henne k, sztajer h, wagner-döbler i, zeng ap. comparing the cariogenic species streptococcus sobrinus and s. mutans on whole genome level. j oral microbiol. 2014; 6(1): 1–13. 8. soyolmaa m, munguntsetseg l, sharkhuu mo, hulan1 u, nishino m. pcr detection of streptococcus mutans and streptococcus sobrinus in plaque samples from mongolian mother-child pairs. pediatr dent j. 2011; 21(2): 154–9. 9. li y, cauf ield pw, red mo ema nuelsson i, t hor nqvist e. differentiation of streptococcus mutans and streptococcus sobrinus via genotypic and phenotypic profiles from three different populations. oral microbiol immunol. 2001; 16(1): 16–23. 10. kuntaman k, hadi u, setiawan f, koendor i eb, rusli m, santosaningsih d, severin j, verbrugh ha. prevalence of methicillin resistant staphylococcus aureus from nose and throat of patients on admission to medical wards of dr soetomo hospital, surabaya, indonesia. southeast asian j trop med public health. 2016; 47(1): 66–70. 11. widyaga r ini a, sutadi h, budia rdjo sb. serotype c and e streptococcus mutans from dental plaque of child-mother pairs with dental caries. j int dent med res. 2016; 9(specialissue): 339–44. 12. oho t, yamashita y, shimazaki y, kushiyama m, koga t. simple and rapid detection of streptococcus mutans and streptococcus sobrinus in human saliva by polymerase chain reaction. oral microbiol immunol. 2000; 15(4): 258–62. 13. hakimi alni r, mohammadzadeh a, mahmoodi p, alikhani my. detection of toxic shock syndrome (tst) gene among staphylococcus aureus isolated from patients and healthy carriers. avicenna j clin microbiol infect. 2018; 5(1): 1–5. 14. steinberg d, eyal s. early formation of streptococcus sobrinus biofilm on various dental restorative materials. j dent. 2002; 30(1): 47–51. 15. lamont rj, hajishengallis gn, jenkinson hf. oral microbiology and immunology. 2nd ed. washington: asm; 2013. p. 242, 403. 16. okada m, soda y, hayashi f, doi t, suzuki j. pcr detection of streptococcus mutans and s. sobrinus in dental plaque samples from japanese pre-school children. j med micro. 2002; 51(5): 443–7. 17. koch g, poulsen s. pediatric dentistry: a clinical approach. 2nd ed. london: wiley-blackwell; 2013. p. 105. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i1.p36–39 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i1.p36-39 subject index volume 48 1,25-dihydroxyvitamin d3, 43 a. actinomycetemcomitans adhesin,39 aggressive periodontitis, 39, 193 alcohol-containing mouthwash, 109 alkaline phosphatase, 130 allergic contact cheilitis, 173 alveolar bone, 43 angiogenesis, 159 antioxidant, 188 apoptotic, 135 autogenous tooth transplantation, 139 bahasa indonesia, 1 blood sugar level, 69 bone morphogenetic protein-2, 53 resorption, 80 ca(oh)2, 16 candida albicans, 26, 84 carbamide peroxide, 7 caries, 31 caspace-3, 48, 135 cd133, 64 cfss-ds, 1 children, 1 chitosan; 53, 213 solution, 154 chronic inflammation, 12 periodontitis, 193 citrus limon, 84, 144 cpp-acp, 7 crp, 113 crystalline, 183 curcuma zedoaria oil, 69 curcumin, 35 cvd, 113 debris, 104 delayed-type hypersensitivity, 173 dental caries, 197 laboratory, 177, 183 dentin hypersensitivity, 170 desensitization, 170 duration, 48 dust; 183 early childhood, 197 elderly, 109, 165 epithel, 150 essential oil, 84 extracoronal bleaching, 7 extracted tooth, 139 fgf2, 213 fiber reinforced composite, 22 fibroblast, 94 growth factor-2, 94 finite element method, 154 flexural strength, 7 fracture resistance, 154 gene polymorphisms, 193 genome, 74 genotoxicity, 16 gingival mesenchymal cell,144 gingiviti, 69 glicosaminoglycans, 100 hairdresser, 74 healing, 12 herbal medicine, 188 hiv/ aids, 84 hyaluronic acid metronidazole, 204 hydrogen peroxide, 7 hydroxyapatite, 204 hypoxia, 59 il-8, 39 impregnation, 22 incompletely formed root, 139 inflammation, 188 irrigation solution, 154 jatropha multifidi, 119 junctional epithelium gingiva, 35 l. reuteri, 31 lactobacillus casei strain shirota, 126 reuteri, 126 level of bd-2, 31 lipstick allergen, 173 lstr 3mix-mp, 12 lymphocytes, 100 macrophages; 209 magnesium; 130 mangosteen peel extract, 104 mauli banana stem extract, 150 micronuclei, 74 mineral trioxide aggregate, 16 mmp-8, 39 msx2, 43 nanochitosan, 26 nano-particles, 135 naocl, 104 nfκb, 35 nickel ion, 26 non alcohol-containing mouthwash, 109 non-growing, 80 oral candidiasis, 84 oral mucosa, 119, 150, 165 orthodontic, 126 appliance, 177 tooth movement, 80 oosteoblast, 204 osteogenesis, 59 palm polybacteria, 144 periodontal disease, 130 periodontitis, 113 plasma cells, 209 polymorphism, 113 potassium ion, 170 premalignan, 64 probiotic, 31, 126 proliferative, 64 propolis extract, 16 pulp cells, 48 purple sweet potato, 170 quality of life, 197 reliability, 1 replication, 48 s. mutans, 31 salivary neutrophils, 89 saponin, 104 satisfaction, 177 s-ecc, 89 silane, 22 silica, 183 silver, 135 single-cell gel electrophoresis, 16 smoking, 113 socket healing, 159 spirulina, 209 stichopus hermanii’s extract, 100 streptococcus mutans, 26, 89 tensile strength, 22 tgf-β2, 80 tissue engineering, 213 tlr4, 193 tooth, 43 extractions, 94 toxicity, 59, 144 traumatic ulcer, 100, 119 ubiquinone, 59 ulceration, 165 ultrasonic scaling, 48 validity, 1 vegf, 159, 213 wound healing, 53, 94, 100, 150, 188 xerostomia severity, 109 x-rays, 94 irradiation, 159 authors index volume 48 adianti, 80 amtha, rahmi, 64 apriasari, maharani laillyza, 150 archadian nuryanti, 48 arundina, ira, 100 bramantoro, taufan, 197 budi, hendrik setia, 188 ernani, 154 gani, basri a, 119 goenharto, sianiwati, 177 handajani, juni, 69 hernawan, iwan, 84 inayati, eny, 183 iskandar, chariza hanum mayvita, 170 iskandar, regina purnama dewi, 209 kamadjaja, david b, 139 kartika w.p, ceples dian, 16 kasuma, nila, 130 kohar, natasia melita, 126 krismariono, agung, 35 kusumaningsih, tuti, 31 luthfi, muhammad, 89 mahdani, fatma yasmin, 94 marinna, astrid, 144 nugroho, raditya, 12 nur’aeny, nanan, 165 prahasanti, chiquita, 193 pratiwi, ariyati retno, 213 ratih, diatri nari, 7 ravitasari, yatty, 173 ridwan, rini devijanti, 39 rosyida, niswati fathmah, 16 ruspita, intan, 43 sakinah, anis, 104 setiawatie, ernie maduratna, 204 suhartono, antonius winoto, 113 sularsih, 53 susanto, hendri, 109 suzy, arlette, 1 taufiqurrahman, irham, 59 triawan, andi, 26 widowati, kharinna, 135 woroprobosari, niluh ringga, 159 yee, koh hui, 74 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dental medicine, universitas airlangga editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (+6231) 5039478 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg i wish to subscribe dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) name/nama: .......................................................................... date of birth/tanggal lahir: .................................................... job title/pekerjaan: ................................................................ institution/institusi: .................................................................. address/alamat surat: 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........................................................................................... address/alamat: ...................................................................................... .................................................................................................................. country/negara: ...................................................................... telp.: ....................................................................................... fax.: ........................................................................................ e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month 1 year surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " mkg vol 41 no 4 oct-dec 2008.indd 190 vol. 41. no. 4 october–december 2008 research report bootstrap study to estimate linear regression parameter (application in the study on the effect of oral hygiene on dental caries) ristya widi endah yani department of dental public health faculty of dentistry jember university jember indonesia abstract background: bootstrap is a computer simulation-based method that provides estimation accuracy in estimating inferential statistical parameters. purpose: this article describes a research using secondary data (n = 30) aimed to elucidate bootstrap method as the estimator of linear regression test based on the computer programs minitab 13, spss 13, and macrominitab. methods: bootstrap regression methods determine β̂ and ŷ value from ols (ordinary least square), iii yy ˆ−=ε value, determine how many repetition for bootstrap (b), take n sample by replacement from iε to )(iε , )(ˆ iii yy ε+= value, β̂ value from sample bootstrap at i vector. if the amount of repetition less than, b a recalculation should be back to take n sample by using replacement from iε . otherwise, determine β̂ from “bootstrap” methods as the average β̂ value from the result of b times sample taken. result: the result has similar result compared to linear regression equation with ols method (α = 5%). the resulting regression equation for caries was = 1.90 + 2.02 (ohi-s), indicating that every one increase of ohi-s unit will result in caries increase of 2.02 units. conclusion: this was conducted with b as many as 10,500 with 10 times iterations. key words: bootstrap, caries correspondence: ristya widi endah yani, c/o: departemen ilmu kedokteran gigi masyarakat dan pencegahan, fakultas kedokteran gigi universitas jember. jl. kalimantan no.37 jember 68121, indonesia. telp. (0331) 333536. e-mail: ristya-widi@yahoo.com introduction bootstrap is a computer simulation-based method that provides estimation accuracy in estimating inferential statistical parameters. to solve problems in insufficient statistical samples (small number of samples), computerbased method such as bootstrap has rarely been employed although its use is quite simple.1 the simplicity of application can be observed in the use of the media and more advanced method, which is an implementation of the basic concept in statistics. being computer-based, this method does not use classical statistical method anymore, which application use a relatively complex formulation.2 multiple linear regression analysis is an extension of simple linear regression analysis. simple linear regression analysis of two variables correlation analysis is made between one dependent variable (y) and one independent variable (x). in multiple linear regression analysis, there are one dependent variable (y) and more than one independent variables (xi), in which i = 1, 2, 3 ... p, with an aim to predict y value (dependent variable) based on x values (independent variables). the correlation between one independent variable and one dependent variable is discussed in simple linear regression, and correlation between more than one independent variables in multiple linear regression analysis.3 as an application in this study, we used data on the effect of oral hygiene on dental caries. the dependent variable was dental caries and the independent variable was oral hygiene. the problem of this study addressed the process of bootstrap method application to assess linear regression parameter. the objective of this study was to evaluate bootstrap method as an estimation of 191yani: bootstrap study to estimate linear linear regression parameter. the benefit of this study was to find the solution using bootstrap method in estimating linear regression parameter. materials and methods the research using secondary data,4 with independent variable (oral hygiene) and dependent variable (dental caries). data source was secondary data entitled “permanent teeth eruption and oral hygiene among elementary school children in goiter endemic area, district of jember”.5 data analysis was undertaken using computer (minitab 13, spss 13, macrominitab). the algorithm of data regression method of bootstrap result (figure 1). this program begins to determine β̂ and ŷ values from ols, iii yy ˆ−=ε , the number of repetition bootstrap (b), then taking n sample from the return of iε , which is regarded as )(iε . determine )(ˆ iii yy ε+= values, and then determine β̂ values in ith sample. if the number of repetition bootstrap < b consequently taking n sample from the return of iε , which is regarded as )(iε . if the number of repetition < b consequently determining β̂ from “bootstrap” method as the average of β̂ of sample taking in b times. result thirty out of 100 secondary data were randomized. the data were tried to be firstly subjected to linear regression determine β̂ and ŷ values from ols determine iii yy ˆ determine the number of repetition bootstrap (b) taking n sample from the return of i , which is regarded as )(i determine )( ˆ iii yy values determine β̂ values in ith sample is the number of repetition <b yes no determining β̂ from “bootstrap” method as the average of β̂ of sample taking in b times start finish figure 1. the algorithim of data regression method of bootstrap result.6 192 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 190−193 analysis, and after regression equation was obtained, they were subjected to linear regression analysis of the bootstrap result using macrominitab program. determining regression coefficient parameter using linear regression analysis analysis regression: caries vs. ohi-s the regression equation is karies = 1.90 + 2.02 ohi-s predictor coef se coef t p constant 1.9028 0.4557 4.18 0.000 ohi-s 2.0228 0.2660 7.61 0.000 s = 1.167 r-sq = 67.4% r-sq(adj) = 66.2% analysis of variance source df ss m f p regression 1 78.818 78.818 57.85 0.000 residual error 28 38.149 1.362 total 29 116.967 unusual observations obs ohi-s karies fit se fit residua st resid 6 1.90 10.000 5.746 0.236 4.254 3.72r r denotes an observation with a large standardized residual durbin-watson statistic = 2.00 using enter method on the result of t test above, the p value for ohi-s variable was significant (p-value = 0.000). in variance test the significance was p = 0.000. the regression parameter of b0 = 1.9028 and b1 = 2.0228. the regression equation : caries = 1.9028 + 2.0228 ohi-s or caries = 1.90 + 2.02 ohi-s. several assumption tests: ει was normally distributed and ε was a randomized variable with (σει) = 0 . the kolmogorov-smirnov test revealed “approximate p -value” of > 0.15. residual had normal distribution. the result of spearman’s correlation test revealed insignificant correlation between residual and ohi-s variable (“sig” = 0.685), indicating no heteroscedacity. no correlation assumption was observed by comparing the values in durbin watson table to the values of durbin watson values from the estimation. the value of d was > du or 4-d > du, h0 was accepted, indicating no correlation between residuals. the independent variable was only one, multicolinearity assumption test could not be performed. plot dots were distributed around the value 0, indicating the presence of linearity. determining regression coefficient parameter of bootstrap result data bootstrap method applied was performed by resampling the residuals. the call of bootstrap command in the form of macrominitab with 10 iterations was %d:\bootstrap_baru. txt c1 c2 c3-c12 c13-c22 c23 c24 c25 c26 c27 c28, which was subsequently entered into minitab program. description: %d:\bootstrap_baru.txt is formula bootstrap, c1 is column variable dependen, c2 is column variable independent, c3-c12 is column regression parameter b0 . c13-c22 is column regression parameter b1, c23 is column b0 bootstrap, c24 is column b1 bootstrap, c25 is column lowb1, c26 is column up b0, c27 is column low b1, c28 is column up b1. first, we used b of 1000 as many as 1000 times iteration, and the b was augmented with the addition of 500 until reaching convergent (constant) regression parameter, with an agreement that resulted regression coefficient parameter is using two decimal places. it was found that in b = 10.500 in 10 times iteration the regression coefficient parameter value of b0 was convergent/constant. table 1 shows that b0 was 1.90, and b1 was between 2.02 –2.03 (two decimal places). mean of b0 = 1.90 and mean of b1 = 2.02. subsequently, the variance of each bootstrap was estimated. the variance of b0 and b1 of b = 10.500 can be seen in table 2. table 2. variance values of b0 and b1 in b = 10.500 b parameter b0 b1 10.500 0.000002072 0.000002813 table 2 shows that in b = 10.500 the variance of b0 is 0.000002072, b1 = 0.000002813. b = 10.500 with 10 times iteration revealed the least variance (minimum) compared to other b. discussion regression equation produced using bootstrap method (with b = 10,500 and 10 times iteration) is not far different from simple linear regression equation. the resulted regression equation was caries = 1.90 + 2.02 table 1. parameters of b0 and b1 in b = 10.500 in 10 times iteration b= 10.500 parameter i = 1 i =2 i = 3 i = 4 i = 5 i = 6 i = 7 i = 8 i = 9 i = 10 b0 1.8 982 5 1.9 021 6 1.9 006 5 1.9 022 6 1.9 012 1 1.8 990 5 1.8 995 5 1.8 995 2 1.9 014 4 1.9 020 8 b1 2.0 220 6 2.0 250 6 2.0 223 8 2.0 237 3 2.0 2 608 2.0 238 5 2.0 251 5 2.0 256 2 2.0 215 5 2.0 219 8 193yani: bootstrap study to estimate linear ohi-s, indicating that every increase of one ohi-s unit will increase 2.02 unit of the caries. linear regression analysis with bootstrap method requires a longer time, because repetition will be done until required convergent (constant) regression coefficient and minimum variance are obtained. prior to performing linear regression analysis using bootstrap method, it should be considered first that not all data can be bootstrapped. bootstrap method is used only in highly necessary conditions, such as insufficient (small) number of samples, unknown data distribution, and in the measurement of parameter estimation accuracy. from b = 10.500 the convergent (constant) regression parameter values (b0 1.90, b1 2.02) were obtained. the estimation of regression parameter (b) was obtained by adding the beta (b0, b1) in each resampling, and divided with b value. thus, it presents as the mean of beta estimation in each resampling process.7 there was no explanation in the literature that determines the amount of bootstrap that should be used in a study. it is apparent that bootstrap recommended in various literatures today is increasing along with the advanced capability in computerization. in b = 100,000 in an increase of 500 in each bootstrap would quickly produce more centralized (more convergent) parameter. in this study the bootstrap was started in 1000.8 a general guidelines, b = 1000 is the most frequently used bootstrap for the first bootstrapping. iteration was performed 10 times to produce convergent (constant) regression coefficient parameter.9 iteration process is performed until obtaining convergent (constant) regression coefficient parameter.10 in b = 10.500 with 10 times iteration, the least (minimum) variance were produced, i.e., b0 = 0.000002072 and b1 = 0.000002813. the more convergent the data, the less the variance produced. however, this was not supported by walpole and sudjana11 who found that the best estimator was the one with minimum variance (estimator with the least variance among all other estimators for the same parameter).12 ohi-s variable has effect on dental caries (p-value = 0.000). poor dental hygiene is one cause of dental caries, either milk or permanent teeth, particularly in children who are mostly unable to brush their teeth appropriately. the better the oral hygiene, the lower the severity of the caries. in contrast, the worse the oral hygiene, the higher the severity of the caries. this confirms the assumption that oral hygiene is one of the factors that influence dental caries. the prevalence of dental caries increased in children with poor dental hygiene compared to those with good dental hygiene.13 there was a strong correlation between poor oral hygiene, the presence of plaque, and the prevalence and severity of periodontal diseases and dental caries.14 regression equation produced by using simple linear regression is not far different from bootstrap method with b = 10,500 and 10 times iteration. linear regression analysis with the data resulting from bootstrap should be employed in highly required conditions, such as insufficient (small) number of samples, unknown data distribution, and in the measurement of parameter estimation accuracy. references 1. longini, halloran a. resampling-based test to detect person to person transmission of infectious disease. journal of the annals of applied statistics 2007; 1(1). available at: http://www.litbang.depkes.go.id/ download/artikel/artikel-ai/article-ai-statistics-2007.pdf. accessed february 22, 2008. 2. efron b, thibshirani rj. an introduction to the bootstrap. new york: chapman and hall; 1993. p. 5, 6, 10. 3. djarwanto. mengenal beberapa uji statistika dalam penelitian. yogjakarta: liberty; 1996. p. 175–6. 4. neuman w. lawrence, social research methods qualitative and quantitative approach. 6th ed. boston: pearson; 2006. p. 17. 5. handayani atw. erupsi gigi permanen dan kesehatan rongga mulut pada anak sekolah dasar di daerah endemik gondok kabupaten jember. tesis. surabaya: fakultas kesehatan masyarakat universitas airlangga; 2007. p. 48–63. 6. atmono d. manajemen data. surabaya: institut sepuluh nopember; 2005. p. 48. 7. sahinler, topuz. bootstrap and jackknife resampling algorithms for estimation of regression parameters. journal of applied quantitative methods 2007; 2(2). available at: http://jaqm.ro/issues/volume2,issue-2/pdfs/sahinler_topuz.pdf. accessed february 22, 2008. 8. lange k. statistics and computing, numerical analysis for statisticians. new york: springer; 1999. p. 309–10. 9. anonim. bootstrapping (statistics). available at: http://en.wikipedia. org/wiki/bootstrapping_(statistics). accessed may 15, 2008. 10. atkinson k. elementary numerical analysis. new york: john wiley & sons; 1985. p. 17–9. 11. sudjana. metoda statistika. bandung: penerbit tarsito; 1996. p. 198–9. 12. ronald we, raymond mh. ilmu peluang dan statistik untuk insinyur dan ilmuwan. edisi keempat. bandung: itb; 1995. p. 363–4. 13. kuntari s. hubungan antara kebersihan gigi dan karies gigi pada anak usia 4–6 tahun di kotamadya surabaya. majalah kedokteran gigi 1996; 29(1): 13–5. 14. boedihardjo. hubungan antara kerusakan jaringan periodontal yang disebabkan oleh plak dengan kebetuhan perawatan periodontal. disertation. surabaya. 1996. p. 25–32. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isi vol 39 no 2 april 2006 file pertama.pmd 80 candidosis on oral lichen planus kus harijanti and mintarsih department of oral medicine faculty of dentistry airlangga university surabaya indonesia abtract oral lichen planus (olp) is a chronic inflammatory disease of the oral mucous membrane which is characterized by unpredictable exacerbation and remission. the pathognomonic of oral features of olp are hyperkeratotic striation surrounded white patches of mucosal erythema are called wickham’s striae. chronic inflammation or epithelial damage of the mucous membrane often followed by candidosis as secondary infection. candida species are commensal microorganism, its population in oral cavity reach 70% of the oral microorganism. it is harmless, but it could become opportunistic pathogen when the condition of oral environment support, i.e. decrease of oral immune response or the oral microorganism ecosystem change. this purpose of the paper was to report the case of a female patient (49 years old) who came to the clinic of oral medicine faculty of dentistry airlangga university surabaya with clinical and mycological evidence of thrush (oral acute pseudomembrane candidosis). the patient not only suffered thrush, but also chronic cervicitis vaginalis. so the patient also consumed the antibiotic which was given by gynecologist. the used of the antibiotic for chronic servicitis vaginalis was the contrary treatment of oral thrush, after treated with nystatin oral suspention, clinical examination showed clearly hyperkeratotic lesion (wickham’s striae), and hystopathological test result showed that it was olp. the chronic oral inflammation and epithelial damage (olp) or antibiotic consumption could inhibited the candidosis treatment. the case report suggested that the first treatment should be given antimicotic if the mycological test of candidosis showed positive result. key words: oral lichen planus, thrush correspondence: kus harijanti, c/o: bagian oral medicine, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. epithelium and the connective tissues, focal areas of hyperkeratinized epithelium (which give rise to the clinically apparent wickham’s striae), and atrophic epithelium where the rete pegs may be shortened and pointed (as sawtooth rete pegs).7 degeneration of the basal keratinocytes and disruption of the anchoring elements of the epithelium on basement membrane and basal keratinocyte (eg. hemidesmosomes, filaments, fibrils) weakened epithelial connective tissue interface.2 candida albicans (c. albicans) are commensal organism, present in more than 70% of the population.2 it is harmless, but it could become opportunistic pathogen when the condition of oral environment support, i.e. decrease of oral immune response or the oral microorganism ecosystem change. candidosis is an infection cause by candida, mainly c. albicans. clinical features of oral candidosis could be divided into 4 types as follow: acute pseudomembrane candidosis (thrush); acute atrophic candidosis; chronic atrophic candidosis and chronis hyperplastic candidosis. oral candidosis has many predisposition factors for example treatment with broad spectrum antibiotics particularly high dose, prolonged administration of topical or systemic corticosteroids, and immunologic disorders or chronic inflammatory/damage on the oral mucous membrane.8 introduction lichen planus (lp) is inflammatory disease that manifests on skin and oral mucous membrane and lp in oral mucous membrane is called oral lichen planus (olp).1 the genitals are involved in as many as 25% of women with olp, compared with only 2–4% of men with olp.2 the ethiopathogenesis of olp is still unknown, but recent studies revealed that cellular immune mechanisms might have a significant role.2–5 in many patients, the onset of olp is insidious and the patients are unaware of their oral condition. in such instances, the referring medical or dental practitioner identifies the clinical changes in the oral mucosa. some patients report a roughness on the lining of the mouth, sensitivity of the oral mucosa to hot/spicy foods or oral hygiene products.2 oral lichen planus (olp) could persist in years as a chronic disease, but it could often disappeared spontaneously. the incidence of olp associated with emotional stress.1 it appears in the oral mucosa in various lesions, i.e. white striation (wickham’s striae), white papula, erythema (mucosal atrophy), erosions/ shallow ulcers or blisters. the predominant side of the lesion in oral mucosa are on the bilateral buccal mucosa, it also could be found in the other side of oral mucosa.6 histological finding of olp are densed bandlike lymphocytic infiltrate (t cells) at interface between the 81harijanti and mintarsih: candidosis on oral lichen planus this case appeared to be a problem for dentists if they found oral candidosis (thrush), which clinically characterized milky white patch, and under the thrush could be a more serious problem as olp. at the clinic of oral medicine faculty of dentistry airlangga university surabaya, only 10 cases of symptomatic olp had been found since 1997 to 2002. the patients complained of pain sensation and the oral mucosa was very sensitive to hot/spicy food. duration of complaint was in the range of one to two years.9 oral lichen planus (olp) rarely found at clinic but the patients really suffered because the disease could not be totally recovered. the paper reported a case of olp with thrush and chronic cervicitis vaginalis. case visit 1: a 49-year-old javanese female patient, was referred to clinic of oral medicine faculty of dentistry airlangga university surabaya by the dentist from madiun. the complaint of patient was the presence of painful white-plaques bilateral buccal mucosa, sensitive of the spicy foods and difficult to open the mouth. she had those complaints for one year. for the last one month, the patient had more severe complaint than before. afterward she went to a physician and was treated with topical-paste, but the lesions did not recover. later, the patient was referred to a dentist. firstly, the patient was treated with extract sanguine 5% + polidocanol 0.1% topically-gel 2 tubes @ 5 g. since the patient still complaint of pain sensation, she was then treated with 0.1% triamcinolone acetonid in-orabase and tetracycline tablets. few days later, the patient could open her mouth, but the pain and the lesions in the oral mucosa still exist. during the oral treatment, the patient also visited the oncologist since three months before the oral treatment, she experienced intercourse pain followed by post coital bleeding pervaginam. from the clinical examination revealed that general condition of the patient was not bad, but extra oral examination showed acute lymphadenitis on the right and left submandibular gland. no skin lesion was found. in intra oral appeared multiple thick-white-plaques on the left of buccal mucosa of 34, 35 and 36, which could not be scraped off. its size was approximately 10 x 20 mm, painful, surrounded by erosion area (figure 1). it also found on the right buccal mucosa. the pain sensation on the right side was not as severe as the pain on the left side. white-irregular patch and erythemathous base were found on attached gingiva of maxilla and mandibula (figure 2). there were multiple ulcers on the mucosa of lower lips. the diameters of multiple ulcers were approximately 10 mm, 8 mm, and 6 mm wich covered by yellowish surface (fibrinous exudate), surrounded by erythematous base and painful. irregular erosions with the size approximately 10 x 2 mm were found on the mucosa of upper lips. the rontgenographic showed the presence of resorption of the processus alveolaris and no signs of malignancy on the left mandibula and maxilla. the lesion was suspected as thrush and candidal leukoplakia as the differential diagnose. the first treatment was 250 ml oral rinsing containing1.5% h2o2 and recalled visit on the next day. visit 2: on the following day, the patient could open her mouth wider than before but the symptoms and intra oral lesions still persist. the patient was referred for mycological examination and as the result a lot of yeast were found. patient was treated with nystatin oral suspention and instructed to apply a thin layer 4 times a day after meals and at bedtime; 10 caplet of 500 mg vitamineral (multivitamin + mineral) for 10 days and oral rinsing with 1.5% h2o2. visit 3: according to anamnesis, three days later after the second visit, the the patient was transferred by oncologist to gynecologist and received 250 mg etmasilat and 500 mg tiamphenicol (antibiotic). after patient took the drugs, white patch in oral mucosa became thicker so the patient stopped to take etamsilat and tiamphenicol, and continued to use nystatin oral suspension, vitamineral and 1.5% h2o2 rinse. the patient was suggested to control 3 days later. figure 2. white irregular patch and erythematous base were found on attached gingiva of maxilla and mandibula. figure 1. multiple thick white plaques, suspected acute pseudomembrane candidosis. 82 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 80–84 visit 4: eight days later, after had taken nystatin oral suspension, vitamineral and 1.5% h2o2 rinse from oral medicine department for 6 days, patient felt better. intra oral examination showed better improvement and then treatment was repeated. patient was suggested for recalled visit the next 7 days. visit 5: according to anamnesis, vaginal-biopsy was taken by a gynecologist and tiamphenicol, etamsilat, mefenamic acid, carbozokrom natrium sulfonat were given to the patient. after taking the drugs, she felt very painful in the mouth. from intra oral examination, white patch became thicker, painful, with ulcers on buccal mucosa of 46 and on the left-mucobuccalfold, its length approximately 1 cm. the use of medicine from the gynecologist and oral medicine’s clinic were continued. the patient was then transferred to oral surgery’s clinic for oral-mucosal biopsy. visit 6 (22nd day) seven days later after the fifth visit, according to anamnesis, after oral-mucosal biopsy, patient received antibiotic and anti-inflammation. later, patient felt generally painful. on extra oral examination, right and left submandibular glands were larger, massive and painful. intra orally, ulcers were on the left and right buccal mucosa, diameter approximately 1 cm, surrounded erytemathous area and large white patch which could be scraped off. attached gingival of maxilla and mandibula were diffuse, red and bright erythematous. the white patch on the right buccal mucosa was thinner than the one on the left. clinical diagnosis suspected as acute pseudomembrane candidosis. the 1% topical gentian violet and 1.5% h2o2 mouthwash were given and patient suggested for recalled 7 days later. visit 7 (29th day) on the seventh visit, the patient felt better. the hystopathological features proved that the lesion was oral lichen planus (figure 3). intra oral examination, white patch was thinner and presented lace-like white striations on an erythematous background which unscrappable. fiery red erythema involving attached gingival appeared on left and right gingiva, this condition called desquamative gingivitis. yellow surface ulcer surrounded by erythematous area presented on the left buccal mucosa. a final-diagnosis was an erosive oral lichen planus disease. triamcinolone acetonid 0.1% in ora base and mycostatin oral suspension (applied 4 times a day), vitamineral tablets were given and the patient was suggested to consume various-fruit juice. vaginal hystopathological test showed chronic cervicitis vaginalis, so the patient was treated with isoprenosine, dalfarol and noxogin vaginal tablets. visit 8 (35th day) the patient felt better and could eat some various diet, only slight soreness when she ate chilli. the unscrappable lace-like white striae, surrounded by erythematous area and painless was found on the left and right buccal mucosa. visit 9: on the third month after the eighth visit, the patient felt much improvement with normal masticatory function. intra oral examination, erythematous area and surrounded by radier white striae showed on left and right buccal mucosa. nystatin and 0.1% triamcenolone acetonide were continuously applied turning 4 times a day and consumed carrot/tomato juice. the patient was referred to the clinic of periodontia, oral surgery and conservative dentistry for scaling, extracting the gangrene radix and teeth filling. the patient was suggested to maintain the oral hygiene. three months later the patient did not totally recover, but the treatment reduced severity of the symptoms. the treatment was aimed primarily at reducing the duration and severity of the symptomatic outbreaks. discussion on the first visit, the complaint of patient was the presence of white-plaques bilateral buccal mucosa, painful, sensitive to the spicy foods and was difficult to open her mouth for one year. one month later, the severity increased. an extra oral examination showed acute lymphadenitis on the right and left submandibular gland, there was evidence of a chronic inflammation with acute exacerbation. due to responseless to 5% sanguine extract + 0.1% polidocanol and 0.1% triamcenolone acetonide, the lesion was not only suspected chronic inflammation with acute exacerbation, but also there were other potential factors. general condition of the patient was normal, but intra orally, there were thick irregular massive, painful white plaque 10 × 20 mm, with erythematous mucosa which difficult to be scrapped off; on the left and right buccal mucosa. it suspected hyperkeratotic lesion or malignant lesion, and simultaneously correlated complaint of the lesion on vagina. irregular white patch with erythematous mucosa and difficult to be wiped away, were on the left mucobuccal figure 3. lace-like white striations on an erythematous background which unscrappable. 83harijanti and mintarsih: candidosis on oral lichen planus fold and attached gingiva on maxilla and mandibula. the clinical diagnosis was suspected as acute pseudomembrane candidosis with the differential diagnosis of chronic hyperplastic candidosis (candidal leukoplakia).8 h2o2 1.5% mouthwash and vitaminerals were given and the aim of the treatment was to avoid false negative mycological result. aerob intra oral condition was made by giving on (o nasen) of 1.5% h2o2 in order to weaken anaerob microorganism. the foam of h2o2 acted as an active ingredient to clean all oral debris, thus oral hygiene could be improved. excellent oral hygiene is believed to be able to reduce severity of the symptoms.7 vitaminerals were given to increase the immune system and to support the recovery process. while waiting for mycological result, 1.5% h2o2 was given and observed the present of positive response in improving the lesions. the following day on visit 2, the lesions showed slight improvement, the patient could opened her mouth a little wider than before, this might be caused by 1.5% h2o2. in this experiment 1.5% h2o2 acted as antiseptic agent instead of antimycotic agent. direct mycological test has shown a lot of candida hyphae type. that was evidence of candida infection even the result of mycological culture test had not known yet. thus, 10 drops of nystatin oral suspension was applied 4 times a day after meal and bedtime and as long as possible in the mouth before swallowing. five days later on third visit, a lot of yeast was found in the result of the culture mycological test. acute pseudomembranous candidosis was decided as a definitive diagnosis. on clinical examination of the intra oral showed white patch on the right buccal mucosa became thinner but there was no improvement on the other side. the lesions showed responsless to antimycotic drugs. according to anamesis, the patient felt better after nystatin oral suspension was given. white patch in the intra oral became thinner, width of the lesions became smaller and painless. for three days the patient was referred to a gynecologist by an oncologist. vaginal biopsy was taken and etamsilat 250mg 3 times a day was given to stop bleeding and tiamphenikol 500mg (antibiotic) was given 3 times a day to manage the infection. disorder recurred after the patient took both of the drugs once. the patient stopped taking etamsilat and tiamphenicol, but nystatin oral suspension still continued. therefore, it could be understood that responsless to antimycotic showed 5 days later due to antibiotic drug. the same episode was repeated at visit 6 (22nd day), after left buccal mucosal biopsy was taken by an oral surgeon. the antibiotic treatment was assumed to be one of the predisposition factors to acute pseudomembrane candidosis.7 the commensal flora in the mouth is a stabile population, the balance of different species can be disturbed by drugs which resulted selective growth of a certain microorganism in the intra oral and followed by excessive growth of resistant organism. if it happened, c. albicans can develop to be oral candidosis.10 acute pseudomembrane candidosis either clinically or symptomatically, will gradually improve 2-3 days after antimicotic treatment and get recovery for 10-14 days. if the lesion is persistent, there is possibility that acute pseudomembrane candidosis simultaneously occur with other chronic mucositis such as olp, pemphigus vulgaris or leukoplakia.11 the mucous membrane and skin are psychochemical barrier to cover the connective tissue.12 erosion and ulceration are epithelial damage of the mucous membrane which reach basal membrane or even deeper.13 principally, type hyphae of candida never penetrates to stratum corneum except in epithelial damage mainly chronic process as seen on olp.11 on the fourth visit (8th day) after taking antimicotic for 7 days, the patient felt much better. on intra oral examination, white plaque showed to be thinner, width of the lesion was smaller and painless, ulceration and erosion disappeared. in this case, the lesion clearly had positive response to antimycotic. on the 5th visit (15th day) the patient had oral mucosa complaint and the pain re-occurred. on intra oral examination white patch showed to be thicker, painful with ulcer on buccal mucosa of 46 and the left-mucobuccalfold, its length approximately 1cm. according to anamnesis, vaginal biopsy was done and given tiamphenicol, etamsilat, mefenamat acid and carbozocrom natrium sulfonat were given. it seems clearer that the effect of antibiotic (etamsilat) to severity of oral candidosis was adequate. however, based on suspection of tissue damage under pseudomembrane of oral candidosis, histopathologycal examination was done to find out the underlying disease, such as a malignant lesion. on visit 6 (22nd day) the post biopsy area was swelling and painful, to lead difficult to open her mouth, 1 cm diameter of the lesion on left buccal mucosa was still found. almost the entire buccal mucosa and attached gingival showed white patch which could be difficult scraped off, diffuse of the border and surrounded erythemathous area. the white patch on right buccal mucosa was thinner than left side, surrounded erythematous, painless and could be scrapped off. the lesion were found on the left and the right side of maxilla and mandibula. according to anamnesis, 250mg amoxillin was given by oral surgeon post biopsy. the use of antibiotic might influence the recurrency or severity of oral candidosis. so 1% gentian violet was given to prevent candida resistention to nystatin. on visit 7th ( 29th day) in intra oral examination showed white plaque was thinner and presented unscrappable lace like white striations. on left and right gingival appeared a fiery red erythema covered the attached gingival, a condition called desquamative gingivitis. after candida infection decreased, clinical examination showed erosive oral lichen planus (olp) and supported by histopathologycal examination. thus, topical corticosteroid was given as the drug of choice on olp and also nystatin oral suspension consecutively, because corticosteroid is predisposition factor of the occurrence of candida infection. if the result of mycological examination showed positive to c. albicans, it is suggested to give antimicotic earlier until showed negative to c. albicans.4 84 maj. ked. gigi. (dent. j.), vol. 39. no. 2 april–june 2006: 80–84 olp was rarely found, but the patient really suffered by the disease, mainly on erosive/ ulcerative olp. precise ethiopathogenesis of olp is unknown, therefore the aim of the treatment is palliative.6 medical treatment of olp is essential for the management of painful, the resolution of mucosal lesions, the reduction of the risk of oral cancer and maintenance of oral hygiene. in patients with recurrent painful disease, another goal is the prolongation of their symptom-free intervals. the agent is used to treat painful of olp (symptomatis) is topical/systemic corticosteroids. no treatment of olp is curative.2,6 in this case, it was concluded that the triggers of oral candidosis are 1) chronic damaged/chronic inflammatory in oral mucous membrane as olp, 2) the used of antibiotic, 3) the used of topical corticosteroid. if mycological examination showed positive to candidosis, it is suggested to use the antimicotic earlier than the antibiotic until mycological examination showed negative. references 1. cawson ra, odell ew. essential of oral pathology and oral medicine. 6th ed. toronto: churchill livingstone; 2000; p. 187–93. 2. sugerman p. oral lichen planus. available at: http://www.emedicine.com/derm /topic663.htm. accessed january 10, 2002. 3. eisenberg e. lichen planus and oral cancer is there a connection between the two. jada 1992 may; 123: 187–93. 4. vinncent sd. diagnosis and managing oral lichen planus. jada 1991; 22: 93–96. 5. axell t. the oral mucosa as a mirror of general health or diseases. scand j dent res 1992; 100: 9–16. 6. dorta rg, souza jb, oliveira dt. ginggival erosive lichen planus: case report. braz dent j 2001; 12(1): 63–66. 7. edward pc, kelsch r. oral lichen planus: clinical presentation and management. j canadian dental association 2002 september; 68(8): 494–9. 8. scully c. candidiasis, mucosal. copyright 2002, emedicine.com, inc. accessed january 24, 2002. 9. harijanti k. penggunaan kortikosteroid pada perawatan olp (penelitian retrospektif mulai tahun 1997–2002). tugas akhir pendidikan dokter gigi spesialis program studi oral medicine pada fakultas kedokteran gigi unair; 2003. 10. duxburry aj. systemic pharmacotherapy. in: jones jh, mason dk. oral manifestation of systemic diseases. 2nd ed. london: bailliere tindall; 1990. p. 443–6. 11. allen cm. diagnosing and managing oral candidosis. jada 1992 january; 123: 77–82. 12. abbas ak, lichtma ah, pober js. cellular and molleculer immunology. 1st ed. philadelphia: wb saunders co; 1991. p. 4–5. 13. sonis st, fazio rc, fang l. principle and practice of oral medicine. 2nd ed. philadelphia: wb saunders; 1995. p. 361443. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 193193 toll-like receptor–4 gene polymorphisms in javanese aggressive and chronic periodontitis patients chiquita prahasanti department of periodontic faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: tool-like receptor-4 (tlr4) gene polymorphisms affect the ability of the host in response to pathogenic bacteria, and can also be associated with the severity of periodontitis. tlr4 gene polymorphisms (asp299gly and thr399ile) are ones of gene mutations that occur in patients with aggressive periodontitis. purpose: to investigate the involvement of tlr4 gene polymorphism as a risk factor of aggressive and chronic periodontitis of javanese population in surabaya. method: this research can be considered as an analytic observational study, with a case-control study design in patients with aggressive periodontitis and chronic periodontitis. dna samples were derived from peripheral blood. tlr4 gene polymorphisms (asp299gly and thr399ile) were then observed by pcr-rflp. result: there was no tlr4 gene polymorphism (asp299gly) in the whole samples. and, based on the results of simple logistic regression analysis on tlr4 gene polymorphisms (thr399ile), mutants heterozygote and homozygote obtained had or value about 0.25. conclusion: in surabaya, there was no heterozygote and homozygote mutant in tlr4 gene polymorphisms, (asp299gly) and (thr399ile), that can be considered as risk factors of chronic periodontitis. keywords: gene polymorphisms; tlr4; aggressive periodontitis; chronic periodontitis correspondence: chiquita prahasanti, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: chiquita_prahasanti@yahoo.com research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 193–196 introduction there is a close relationship in periodontal disorder between genetic, environmental, patient age factor when exposed to the severity of the disorder, and the course of their illness. individual responses to infection, however, vary widely, and genetic factor plays an important role in triggering abnormality, such as periodontitis. host genotype may influence the composition of subgingival bacteria, and its data even state that host gene polymorphism affects the host response to infection. changes in gene structure can have an impact on both response quality and polymorphism setting, and can also alter the patient’s response mechanism.1 aggressive periodontitis, a specific type of periodontitis due to its own character, is characterized by a progressive course of the disease with severe periodontal tissue damage. in many cases, the disease occurred in apparently healthy individuals can be considered as a multifactorial disease caused by periodontal pathogenic bacteria. the incidence and severity of the disease, moreover, is determined by the host response to infection. inflammation that occurs in periodontitis is a complex process begun with tissue damage and continued with repair process.2,3 genetic and environmental factors have implications for the cause of periodontal disease. patients with aggressive periodontitis seem to have abnormal immunological factors, expected to be influenced by genetic factors.4 patients with aggressive periodontitis will have inadequate host response to periodontal pathogenic bacteria seen in the increased expression of a wide variety of immunological factors and genetic risk factors. actinobacillus actinomycetemcomitans (a. actinomycetemcomitans) bacteria were commonly found dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p193-196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p193-196 194 prahasanti/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 193–196 in aggressive periodontitis. yang et al.5 even stated that 84.3% of aggressive periodontitis was caused by a. actinomycetemcomitans serotype b bacteria found in all regions of aggressive periodontitis, especially in patients with aggressive periodontitis not treated, as well as in the domain which has a pocket inside (> 5mm). lipopolisakarida (lps) is a product of a. actinomycetemcomitans bacteria that will trigger tlr4. the initiation of lps and tlr4 signal was caused by a complex process, which involves several additional proteins.6,7 toll-like receptors (tlrs) is suspected as the starting point of immunity in which extracellular environment factors continuously give information to the cells to respond to infection and to facilitate cellular responses through the top of signaling pathways in new gene transcription.2 the mechanism of innate immunity has always responded quickly to infection that occurs in the patients’ body and to the spread of bacterial pathogens in order to evolve an effective and adaptive immunity well.8 tlr4 located on chromosome 9 (9q32-33) has complex regulation, involving specific different tissues and cells, and the regulation largely determines innate immune system.9 polymorphisms that occur in tlr4 gene will disrupt the function of tlr4 receptor against lps germs in bodies, causing interference to the cell membrane transport and also to both ligand binding and protein interaction, as a result, the expression of tlr4 protein will be affected.10,11 tlr4 actually has an ability to protect from inflammation, and there is a significant relationship between tlr4 gene polymorphism, asp299gly, and aggressive periodontitis in adult caucasian population. previous studies reported that tlr4 gen polymorphism in caucasian was 5 percent.12,13 amino acid changes that occur in the extracellular domain of tlr4 are related to lps that is hypo-responsive in human epithelial cells and alveolar macrophages in vitro. functionally, it will make tlr4 hypo-responsive against lipopolysaccharide of periodontopathogen germs, a. actinomycetemcomitans, so it becomes less susceptible to infections caused by gram-negative bacteria. tlr4 can also be related to the severity of aggressive periodontitis. tlr4 polymorphism will disrupt the function of tlr4 receptor against lps, then causing transport disorder to the cell membrane, ligand binding disruption, and also protein interaction interference, so the expression of tlr4 protein will be affected.11,14 thus, genetic aggressive periodontitis, especially genetic pattern of the host needs to be studied to determine the location of specific polymorphism gene. it is important that the number and type of modifying disease genes for the same disease may not be same in different ethnic population. therefore, some studies should be done to reveal the etiologies that may involved. thus, the aim of the study to investigate the involvement of tlr4 gene polymorphismas a risk factor of aggressive and chronic periodontitis of javanese population in surabaya. material and methods this research was an observasional analytic study with case control study design in patients with aggressive periodontitis and chronic periodontitis. thus, polymorphism test must be conducted on tlr4 gene (asp299gly and thr399ile) by pcr-rflp. the subject for this study were 40 cases of aggressive, and 40 cases of chronic periodontitis as control group who visited the periodontic clinic, faculty of dental medicine airlangga university. all subject were javanese and subject with a history of systemic disease or smoking were excluded from the study. the nature of the study was explained to all subject verbally and in writing and all signed a content form. genomic dna derived from peripheral blood of patients was taken about 2ml. dna isolation principle was conducted by lysing leukocyte cell membrane using lysis buffer solution, cell membrane lysis buffer (cmlb), dna extraction was conducted by using lysis buffer solution, nmlb (nuclear membrane lysis buffer). p r i m e r s e q u e n c e u s e d i n t h e r e a c t i o n w a s t l r 4 ( a s p 2 9 9 g l y ) u s e d w e r e f o r w a r d 5 ’ a g c a t a c t t a g a c t a c t a c c t c c a t g 3 a n d reverse 5’-gagagatttgagtttcaatgtggg-3’. pcr condition had to be with pre-denaturation 950c for 3 minutes, and then 35 cycles were performed at a temperature of 940 c for 30 seconds, at a temperature of 620 c for 30 seconds, and at a temperature of 720 c for 30 seconds. enzyme restriction endonucleases used were ncoi, which will produce two fragments 80bp and 22bp (g allele) and 102bp (a allele). tlr4 (thr399ile) used were forward 5’-ggttgctgttctcaaagtgatttt gggagaa-3’ and reverse 5’-ggaaatccagatgtt ctagttgttctaagcc-3’. pcr condition had to be be with pre-denaturation at 950 c for 3 minutes, and then a total of 35 cycles was performed at a temperature of 940 c for 30 seconds, at a temperature of 600 c for 30 seconds, and at a temperature of 720 c for 30 seconds. restriction endonuclease enzyme used was hinfi, which will produce two fragments 121bp and 25bp (t allele) and 146bp (c allele). finally, visualization of pcr products for tlr4 was conducted by agarose gel electrophoresis using 4% agar at 120v, 70 mamp, for 40 minutes. results based on the entire samples of this research, there was no tlr4 gene polymorphism (asp299gly) found in patients with aggressive periodontitis and chronic periodontitis. in the other hand, there was an tlr4 gene polymorphism (thr399ile) found in the whole samples as seen in the following figure 1. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p193-196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p193-196 195195prahasanti/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 193–196 as variable of tlr4 gene polymorphisms (thr399ile), especially allele 1+ 2 and allele 2, was not a risk factor for aggressive periodontitis process. furthermore, the frequencies of tlr4 genotype (thr399ile) in patients with chronic periodontitis were 20% cc (allele 1 = wild type), 80% ct (allele 1 + 2 = mutant heterozygote) and 0% tt (not found 2 = mutant allele homozygote). the frequency distribution shows that the frequency of mutant heterozygote of tlr4 polymorphism (thr399ile) was 80%, 4 times higher than that of the wild-type genotype (20%). and, the frequency of the mutant allele (t) in this research was 40% (table 2), so the variables of tlr4 gene polymorphisms (thr399ile) in cp, especially allele 1+ 2 and allele 2, can be considered as risk factors (4x normal) of cp abnormalities. discussion tlr4 gene has two missense polymorphisms, namely asp299gly and thr399ile, affecting extra cellular proteins and essential to reduce both lps signal strength and inflammation. in this research, polymorphism occurs only in tlr4 gene (thr399ile). tlr4 gene polymorphism (asp299gly/thr399ile) can cause susceptibility to aggressive periodontitis. it means that polymorphisms occurred will increase the severity of disorder, and will also cause variations in the structure of tissues (innate immunity), antibody response (adaptive immunity), as well as inflammatory mediators (non-specific inflammation).16 the occurrence of polymorphism, moreover, is associated with response to endotoxin exposure. in other words, tlr4 polymorphism (asp299gly and thr399ile) can affect the extracellular domain of tlr4 protein in term of its expression and function causing a failure to respond to lps since tlr4 will fail to capture lps signal. changes in the extracellular domain and the amino acid, playing a role in receptor function to capture the lps signal, then will be disrupted and will result in a reduced ability to inhibit the figure 1. results of pcr-rflp in tlr4 gene (thr399ile). amplicon line 1 and 11 at 124bp (normal homozygote); amplicon line 2, 3, 4, 6, 7, 8, 9, 10 at 124 bp + 98 bp + 26 bp (not visible) (heterozygote mutant); amplicon line 5 hydrolyzed at 98bp + 26bp (not shown) (homozygote mutant); line m = dna marker at 20bp. table 1. distribution of tlr4 polymorphism genotype (thr399ile) in patients with aggressive periodontitis (ap) and chronic periodontitis (cp) genotype periodontitis ap cp n % n % cc (allele 1) 20 (50%) 8 (20%) ct (allele 2) 1 (2.5%) 0 (0%) tt (allele 1+2) 19 (47.5%) 32 (80%) total 40 (100%) 40 (100%) table 2. distribution of tlr4 gene alleles (thr399ile) in patients with aggressive periodontitis (ap) and chronic periodontitis (cp) allele periodontitis ap cp n % n % c 59 (73.75%) 48 (60%) t 21 (26.25%) 32 (40%) total 80 (100%) 80 (100%) distribution of genetic sequences of tlr4 gene (thr399ile) (genotype distribution and allele frequency) on the ap and cp can be seen in table 1 and table 2. table 1 shows the frequencies of tlr4 genotype polymorphism (thr399ile) in patients with ap and cp. based on the table, the frequencies of tlr4 genotype (thr399ile) were 50% cc (1 = wild-type allele), 47.5% ct (allele 1 + 2 = mutant heterozygote), and 2.5% tt (2 mutant allele homozygote). the frequency distribution, moreover, shows that the frequency of mutant heterozygote of tlr4 polymorphism (thr399ile) was 47.5% almost equivalent to the wild-type genotype (50%). meanwhile, the frequency of the mutant allele (t) in patients with ap was 26.25% (table 2). based on these results, it can be said that aggressive periodontitis 1 figure 1 table 1. distribution of tlr4 polymorphism genotype (thr399ile) in patients with aggressive periodontitis (ap) and chronic periodontitis (cp) genotype periodontitis ap cp n % n % cc (allele 1) 20 (50%) 8 (20%) ct (allele 2) 1 (2.5%) 0 (0%) tt (allele 1+2) 19 (47.5%) 32 (80%) total 40 (100%) 40 (100%) table 2. distribution of tlr4 gene alleles (thr399ile) in patients with aggressive periodontitis (ap) and chronic periodontitis (cp) allele periodontitis ap cp n % n % c 59 (73.75%) 48 (60%) t 21 (26.25%) 32 (40%) total 80 (100%) 80 (100%) 200bp wildtype 98bp homo hetero 100bp dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p193-196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p193-196 196 prahasanti/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 193–196 occurrence of inflammation.10,13 mutations that occur will facilitate the occurrence of sepsis, leading to the failure of lps signaling in individuals with mutant homozygote and heterozygote. the type and number of genetic abnormalities in the same disease manifestations that occur are not similar to different ethnic populations ranging from 0.1% to 15%.12 therefore, various polymorphisms that occur are likely to have contributed and considered as one of the risks to a person’s susceptibility to the severity of aggressive periodontitis. it means that genetic and environmental factors have implications in the etiology of periodontitis.17,18 based on the data in the previous researches, it is known that the allele frequency of tlr4 polymorphisms, especially asp299gly and thr399ile, is very different between the populations of asia, africa and caucasia. a research on population related to geography even shows that the frequency of tlr4 polymorphism, especially asp299gly haplotypes, in african populations was 10-20 times different from the frequency of tlr4 polymorphisms, especially asp299gly, thr399ile and tlr4 asp299gly/ thr399ile haploptype in asian populations.16 a research conducted by laine et al.19 aimed to see the relationship of tlr4 polymorphism and periodontitis severity shows that the prevalence of polymorphisms, especially asp299gly and thr339ile, was 5% in dutch citizens belonging to the caucasoid race. the results of studies analyzing the correlation of gene polymorphism in chronic periodontitis and aggressive periodontitis, unfortunately, are less clear although tlr4 is biologically an important gene considered as the etiology of periodontal disease.4 genetic factors influencing the immune response to bacterial infection play an important role in individual susceptibility to inflammation caused by periodontal pathogens. it is because tlr4 plays a role as a receptor in responding to lps signal from gram-negative bacteria. polymorphisms that occur will increase the severity of abnormality. according to genetic variants that occur can produce variations in the structure of tissue (innate immunity), antibody response (adaptive immunity), and inflammatory mediators (non-specific inflammation). 20 in other words, susceptibility to periodontitis is influenced by genetic and environmental factors, such as the influence of periodontopathogen bacteria. it means that genetic factors influencing the immune response to bacterial infections play an important role and affect a person’s vulnerability reflected in the increased expression of tlr4 protein. therefore, impaired function of tlr4 will disrupt homeostasis in a person’s body so that the body is easy to be exposed to a disease. conclusion, in surabaya there was no heterozygote and homozygote mutant in tlr4 gene polymorphisms, (asp299gly) and (thr399ile) in javanese population, that can be considered as risk factors of chronic periodontitis. therefore, others factors that may involved in the etiology of chronic periodontitis should be investigated. references 1. nibali l, ready dr, parkar m, brett pm, tonetti ms, griffiths gs. gene polymorphisms and the prevalence of key periodontal pathogens. j dent res 2007; 86(5): 416-20. 2. armitage gc, cullinan mp, seymour gj. comparative biology of chronic and aggressive periodontitis: introduction. periodontology 2000 2010; 53: 7-11. 3. kinane df, shiba h, stathopoulou pg, zhao h, lappin df, singh a, eskan ma, beckers s, waigel s, alpert b, knudsen tb. gingival epithelial cells heterozygous for toll-like receptor 4 polymorphisms asp299gly and thr399ile are hyporesponsive to porphyromonas gingivalis. gene and immunity 2006b; 7: 190-200. 4. takashiba s, naruishi k. gene polymorphisms in periodontal health and disease periodontol 2000 2006; 40: 94–106. 5. ya ng h w, hua ng y f, cha n y, chou my. relationsh ip of actinobacillus actinomycetemcomitans serotypes to periodontal condition: prevalence and proportions in subgingival plaque. eur j oral sci 2005; 113(1): 28–33. 6. koraha j, tsuneyoshi n, kimoto m, gauchat jf, nakatake h, fukudome k. comparison of lipopolisakarida-binding functions of cd14 and md-2. clin diag lab immunol 2005; 12(11): 129297. 7. carpenter s, o’neill la. how important are toll-like receptor for antimicrobial respon?. cell microbiol 2007; 9(8): 1891-901. 8. whitestt ja, bachurski cj, barnes kc, bunn jr pa. genetic regulation of innate immunity lessons learned from tlr4. am j respiratory cell and molecular biology 2004; 31(2): 48–51. 9. kaisho t, akira s. toll-like receptor function and signaling. j allergy clin immunol 2006; 117(5): 97687. 10. rallabhandi p, bell j, boukhvalova ms, medvedev a, lorenz e, arditi m, hemming vg, blanco jcg, segal dm, vogel sn. analysis of tlr4 polymorphic variants: new insights into tlr4/md-2/ cd14 stoichiometry, structure and signaling. j immunol 2006; 177(1): 32232. 11. folwaczny m, glas j, torok hp, limbersky o, folwaczny c. toll like receptor (tlr) 2 and 4 mutation in periodontal disease. clin exp immunol 2004; 135(2): 330– 35. 12. arbour nc, lorenz e, schutte bc, zabner j, kline jn, jones m, frees k, watt jl, schwartz da. tlr4 mutations are associated with endotoksin hiporesponsifness in human. nat genet 2000; 25(2): 18791. 13. james ja, poulton kv, haworth se, payne d, mckay ij, clarke fm, hughes fj, linden gj. polimorfismes of tlr4 but not cd14 are associated with a decreased risk of aggressive periodontitis. j clin periodontol 2007; 34(2): 111–17. 14. azuma m. fundamental mechanisms of host immune responses to infection. j periodontal res 2006; 41(5): 361-73. 15. ya ng h w, hua ng y f, cha n y, chou my. relationsh ip of actinobacillus actinomycetemcomitans serotypes to periodontal condition: prevalence and proportions in subgingival plaque. eur j oral sci 2005; 113: 28–33. 16. ferwerda b, mccall mb, verheijen k, kullberg bj, van der ven aja, van der meer jwm, netea mg. functional consequences of toll-like receptor 4 polymorphisms. molmed 2008; 14: 346 – 52. 17. loos bg, john rp, laine ml. identification of genetik risk factors for periodontitis and possible mechanisms of action. j clin periodontol 2005; 32(suppl. 6): 159–79. 18. ozturk a, vieira ar. tlr4 as a risk factor for periodontal disease: a reappraisal. j clin periodontol 2009; 36(4): 279–86. 19. laine ml, morre sa, murillo ls, van winkelhoff aj, pena as. cd 14 and tlr4 gene polymorphisms in adult periodontitis. j dent res 2005; 84(11): 1042-46. 20. takahashi n, kobayashi m, takaki t, takano k, miyata m, okamatsu y, hasegawa k, nishihara t, yamamoto m. actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblast. oral microbiol immunol 2008; 23: 259-64. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p193-196 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p193-196 128 vol. 41. no. 3 july–september 2008 the role of proper treatment of maxillary sinusitis in the healing of persistent oroantral fistula david b. kamadjaja department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya – indonesia abstract background: oroantral communication (oac) is one of the possible complications after extraction of the upper teeth. if not identifiedoroantral communication (oac) is one of the possible complications after extraction of the upper teeth. if not identified and treated properly, a large oac may develop into oroantral fistula (oaf) which means that there is a permanent epithelium-lined communication between antrum and oral cavity. such fistulas may cause ingress of microorganism from oral cavity into the antrum leading to maxillary sinusitis. oroantral fistula usually persists if the infection in the maxillary antrum is not eliminated. therefore, treatment of oroantral fistula should include management of maxillary sinusitis in which surgical closure of oroantral fistula should be done only when the sinusitis has been cured. purpose: this case report emphasizes on the importance of proper management ofthis case report emphasizes on the importance of proper management of maxillary sinusitis in the healing of oroantral fistula. case: a case of an oroantral fistula following removal of upper left third molara case of an oroantral fistula following removal of upper left third molar is presented. as the maxillary sinusitis was not identified pre-operatively, two surgical procedures to close the fistula had ended up in dehiscence. case management: the diagnosis of maxillary sinusitis was finally made and the sinusitis subsequently treated withthe diagnosis of maxillary sinusitis was finally made and the sinusitis subsequently treated with combination of trans-alveolar sinus wash out, insertion of an acrylic splint, and two series of nasal and sinus physiotherapy procedures. the size of the defect decreased gradually during the treatment of the sinusitis and finally closed up without any further surgical intervention. conclusion: this case report points out that it is important to detect intraoperatively an antral perforation after anythis case report points out that it is important to detect intraoperatively an antral perforation after any surgery of the maxillary teeth and to close any oroantral communication as early as possible and that it is important to treat properly any pre-existing maxillary sinusitis before any surgical method is done to close the fistula. key words: persistent oroantral fistula, treatment of maxillary sinusitis correspondence: david b. kamadjaja, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132. email: davidbk@sby.dnet.net.id introduction perforation of maxillary sinus, referred to as oroantral communication, which may lead to formation of oroantral fistula is a relatively uncommon condition. it may occur as a complication of trauma, surgery, irradiation, infection, cyst or neoplasm. one of the common causes of oroantral fistula is extraction of maxillary molars especially in cases where the roots of the teeth are in close relationship with a large antrum.1 although extraction of upper second premolars, first and second molars are the procedures most frequently associated with antrum perforation removal of upper third molars may also cause oroantral communication especially when surgical intervention, either with or without ostectomy, is performed.2 once the diagnosis of oroantral fistula is confirmed it should be closed surgically. however, successful closure of chronic oroantral fistula can only be achieved if there is no antral infection. therefore, the first aim of the treatment of oroantral fistula is to eliminate any coexisting maxillary sinus infection.3 this paper presents a case of oroantral fistula following surgical removal of impacted upper left third molar. as maxillary sinusitis was not suspected and treated accordingly, a couple of surgical methods to close the fistula ended up in dehiscence. after maxillary sinusitis had been case report 129kamadjaja: the role of proper treatment of maxillary sinusitis diagnosed and treated properly the oroantral fistula closed up gradually and uneventfully. this case report emphasizes on the importance of proper management of maxillary sinusitis in the healing of oroantral fistula. case a 36-year-old female patient came to private clinic to have her all wisdom teeth removed as was suggested by her physician because of chronic gastric problem. panoramic x-ray showed all of her four third molars were impacted. it was also noted from the x-ray film that the upper third molars on both sides were closely related to the maxillary sinuses (figure 1). as requested by the patient, all of the impacted teeth were surgically removed in one visit under local anesthesia. upon removal of the upper right third molar an oroantral communication was confirmed clinically and was subsequently closed with pedicle buccal fat pad graft as it was readily available during the excision of the tooth. the left upper third molar, on the other hand, was removed with less difficulty and clinically no oroantral communication was suspected, therefore the surgical wound was closed primarily with interrupted suturing. the patient was instructed to avoid strong gargling and nose blowing for one week and she was put on a course of amoxicillin/clavulanic acid 500 mg 3 times daily for 5 days. seven days after the surgery the patient complained of fluid leakage into the left nose during drinking and tooth brushing. she admitted having history of chronic nasal discharge through her left nostril particularly in the morning way before the surgery but no foul smell was noted by the patient. intra orally, the surgical wound on the left upper third molar was still open and the socket measuring 8 mm in diameter was not filled with adequate amount of granulation tissue. irrigation through the socket with normal saline solution indicated that there was minimal fluid leakage into the left maxillary sinus but did not reveal any sign of infection. diagnosis of oroantral communication was made and two days later the patient was operated on to close the communication with primary closure using buccal flap method. one week after the surgery the patient came back with a complaint of fluid leakage from mouth to her left nose but it was still considered as minimal by the patient. on clinical examination dehiscence of the previous surgical wound was again noted showing some pus discharge from the socket. post operative panoramic x-ray confirmed that there was no root fragment left in the socket and both antrum seemed to have similar appearance (figure 2) which might indicate that the left antrum was not infected. diagnosis of suppurative infection of the surgical wound was made. under local anesthesia the wound was debrided and irrigated with 3% hydrogen peroxide solution and normal saline. iodoform gauze was subsequently inserted to the wound and the patient informed that she would need another surgery to close the wound in order to prevent infection of the left antrum. figure 1. panoramic x-ray showing four impacted third molars and the close relationship between maxillary sinuses and the upper third molars on both sides. figure 2. post operative panoramic x-ray showing that all post excision socket are clear of root fragments and the antral floor in the area of post excision sockets seem to be partly missing which might indicate antral perforations on both sides. figure 3. water’s film showing reduced radiolucency of the left antrum with obvious thickening of the antral lining indicating chronic inflammation of the left maxillary sinus. the right antrum appears normal. 130 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 128-131 wound. the patient was subsequently referred to an ent doctor for management of the maxillary sinusitis. from clinical examination and water’s x-ray (figure 3) the ent doctor confirmed the diagnosis of left maxillary sinusitis. the patient was given oral medications consisting of levofloxacine 500 mg once daily for five days, antihistamine, mucolytic agent, and nasal decongestant. she was also treated with two series of nasal and sinus physiotherapy using short wave diathermy and nasal nebulizing therapy using budesonide solution. during the above therapy trans-alveolar sinus wash out was done twice a week for consecutive two weeks and the patient was kept wearing a unilateral palatal acrylic splint (figure 4) to cover the oral opening of the fistula and chlorhexidine mouth wash was prescribed to be used twice daily. the treatment of the maxillary sinusitis took a total of three weeks and at the end of the treatment the size of the fistula had shrunk considerably. the patient was instructed to wear the splint at all times, to be removed only during application of mouthwash and cleaning of the splint, and she was reviewed on a regular basis. any surgery was postponed and any progress observed during the healing period. about two months after the initial non-surgical treatment the fistula was found to close up completely which was confirmed visually (figure 5) and by the absence of fluid leakage into the left nose when intra socket saline irrigation was performed. discussion extraction of maxillary molars may advertently create an oroantral communication especially in cases where the roots of the teeth are in close relationship with a large antrum. the most frequent cause underlying oroantral communication is surgical extraction of the second premolar and of the first and second molars of the upper jaw, the latter also being referred to as antral teeth4 which is primarily due to the proximity between the apexes of these teeth and the figure 4. unilateral palatal acrylic splint, the palatal plate is extended buccally at the region of upper left third molar to cover the fistula opening (left); the acrylic splint is inserted in the patient’s mouth (right). figure 5. intra oral examination showing that the fistula opening has closed completely, confirmed by the absence of fluid leakage into the nose upon saline irrigation of the post extraction socket. three days later, the wound was surgically closed applying buccal fat pad graft with the overlying mucosa being approximated and held in place with stay sutures to avoid compromising the graft. nine days after the second closure surgery, however, almost all of the fat pad graft overlying the socket disappeared and the wound opened up again showing some pus coming out from the socket. irrigation with normal saline to the socket indicated that there was a more obvious communication between the tooth socket and the antrum. case management diagnosis of oroantral fistula and coexisting maxillary sinusitis was made. the wound was debrided and washed out with normal saline solution and packed up with iodoform gauze. impression of upper left dentition was taken for construction of an acrylic splint to cover the 131kamadjaja: the role of proper treatment of maxillary sinusitis maxillary sinus.5,6 the complication may also arise in the case of upper third molar extractions especially when an aggressive surgical technique or excessive post-extraction alveolar curettage are performed, or when the patient in the immediate post-operative period performs maneuvers that tend to increase intra antral pressure.2 in the current case, the intimate relation of the upper left third molar to the inferior antral wall definitely contributed to the increased likelihood of the incidence of oroantral communication. the risk of such communication became higher as ostectomy was performed to surgically remove the bone overlying the impacted upper left third molar. the intraoperative diagnosis of oroantral communication can be made with valsalva maneuver7 which offers a sensitivity of 52% or by the use of a blunt-edged bowman probe to assess perforations of the maxillary sinus floor with a sensitivity of 98%.8 however, since it is not a routine procedure to check for such communication after every tooth extraction the operator may miss its occurrence so that no specific treatment is done to close the defect. in the case presented here, following removal of the left upper third molar oroantral communication was not suspected thus no specific measure is performed to close it. it is a good practice, therefore, to implement diagnostic procedure mentioned above on a routine basis in cases where the risk for oroantral communication is considered high. an oroantral communication which is less than 5 mm in diameter usually heals spontaneously. however, a sinus perforation of more than 5 mm in diameter frequently fails to close spontaneously and therefore requires proper surgical closure.1,9 if the oroantral communication is left untreated and remains open or if infection persists for a long period of time, chronic inflammation of the antral membrane may result with permanent epithelization of the oral-sinus fistula – a situation that further increases the risk of sinusitis.5 the left maxillary sinusitis in this patient was initially thought to be solely caused by the oroantral fistula, however the ent doctor who examined her confirmed that it was actually of nasal origin as the patient had history of chronic rhinitis of the left nose. in my opinion, the sound judgment would be that the pre-existing maxillary sinusitis of nasal origin has impaired the healing capacity of the postexcision wound of upper left third molar and this gradually has led to the formation of oroantral fistula. the fistula has, in turn, exacerbated the infection of the left antrum due to invasion of microorganisms from the oral cavity. this may become the reason why the two surgical procedures has failed to close the defect and ended up in dehiscence. this is in accordance with howe3 who mentioned that successful closure of chronic oroantral fistula can only be achieved if there is no antral infection and therefore the first aim of treatment is to eliminate any coexisting maxillary sinus infection. it is interesting to note, however, that after conservative treatment with combined oral medication and nasal and antral physiotherapies and covering the wound with splint for nearly two months the oroantral fistula finally closed up without further surgical intervention. this is in accordance with a case reported by logan and coates10 in which complete healing of an oroantral fistula was evident following eight weeks of wearing a surgical splint. sokler et al.11 suggested that with permanent wearing of a palatinal plate, occasional rinsing of the sinus with physiological solution, and enteral application of antibiotic, it is possible to cure an inflamed sinus and achieve spontaneous closure of the fistula, even in cases which have existed for more than a month. this case report points out two important things. first, it is a good practice that after removal of an impacted upper third molar which is closely related to maxillary sinus a thorough examination should be done to confirm any existence of oroantral communication and primary closure of the wound be performed accordingly if it exists. second, it is important that the pre-existing antral infection be properly treated in every case of oroantral fistula before deciding to close the fistula surgically no matter what surgical methods are used. references 1. awang mn. closure of oroantral fistula. int j oral maxillofac surgint j oral maxillofac surg 1988; 17:110–5. 2. del rey-santamaría m, valmaseda-castellón e, berini-aytés l, gay-escoda c. incidence of oral sinus communications in 389 upper thirmolar extraction. med oral patol oral cir bucal 2006; 11:med oral patol oral cir bucal 2006; 11: e334–8. 3. howe gl. minor oral surgery. 3rd ed. bristol: john wright & sons ltd; 1985. p. 207–23. 4. güven o. a clinical study on oroantral fistulae. j cranio-maxillofac surg 1998; 26:267–71. 5. skoglund la, pedersen ss, holst e. surgical management of 85 perforations to the maxillary sinus. int j oral surg 1983; 12:1–5. 6. punwutikorn j, waikakul a, pairuchvej v. clinically significant oroantral communicationsa study of incidence and site. int j oral maxillofac surg 1994; 23:19–21. 7. kretzschmar dp, kretzschmar cjl, salem w. rhinosinusitis. review from a dental perspective. oral surg oral med oral pathol oral radiol endod 2003; 96:128–35. 8. ehrl pa. oroantral communication. int j oral surg 1980; 9:351–8. 9. kruger go. textbook of oral surgery. 4th ed. cv mosby co; 1974. p. 255–60. 10. logan rm, coates ea. non-surgical management of an oro-antral fistula in a patient with hiv infection. australian dental journal 2003; 48(4):255–8. 11. sokler k, vuksan v, lauc t. treatment of oroantral fistula. acta stomatol croat 2002; 36(1): 135–40. 1717 dental journal (majalah kedokteran gigi) 2023 march; 56(1): 17–22 case report management of bimaxillary protrusion with missing molar using t-loop and couple force ida bagus narmada1, shali wikynikta purnomo2, putri intan sitasari3, nabilla vidyazti rishandari prasetyo,1 aldila rahma1 1department of orthodontics, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2postgraduate of orthodontics, faculty of dental medicine,universitas airlangga, surabaya, indonesia 3faculty of dentistry, universitas mahasaraswati, denpasar, bali, indonesia abstract background: management of bimaxillary protrusion can be challenging and should be used with maximum anchorage to prevent loss of anchorage and improve the facial profile. in addition, a patient with a missing molar is often found in a dental clinic. space closure can cause tipping movement rather than bodily, so couple force should be used. purpose: this case report aims to manage the bimaxillary protrusion with a missing molar using a t-loop and a transpalatal arch (tpa) as maximum anchorage for correction of the facial profile and couple force to create bodily movement for the space closure of a missing first molar. case: a 21-year-old female patient complained about her protruding teeth. an intraoral examination indicated angle’s class i malocclusion on the left molar relation, with the lower-right first molar missing, mild crowding maxilla and mandible, 6 mm of overjet and 5 mm of overbite, and midline shift at the maxilla and mandible. case management: the treatment plan was the extraction of teeth 14, 24, 34; alignment with pre-adjusted mclaughlin bennett trevisi (mbt) 0.022; retraction of the anterior segment with a t-loop, tpa, and close spacing of the missing first molar with couple force on the buccal and lingual side and tip back. retention was done with removable retainers. at the end of the treatment, normal incisive inclination and closed space of the missing first molar were achieved, along with an improvement of the facial profile. conclusion: bimaxillary protrusion can be successfully treated by means of extraction of the premolar(s), space closure for correction of the profile with t-loop and tpa, and closing the space of the missing molar with couple force on the buccal and lingual sides and tip back. keywords: bimaxillary protrusion; medicine; missing molar; malocclusion; orthodontics article history: received 27 october 2021, revised 16 july 2022, accepted 15 september 2022 correspondence: ida bagus narmada, orthodontics department, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47 surabaya, 60132 indonesia. email: ida-b-n@fkg.unair.ac.id introduction bimaxillary dental protrusion or bimaxillary protrusion is a condition where the anterior teeth of the maxilla and mandible are protruded relative to the maxilla and mandible basal bones.1–3 the characteristics seen are an incompetent lip in a resting position, an excessive effort to close the lips completely, thus creating lip strain and prominent lips.1–3 bimaxillary dental protrusion is affected by race and ethnicity.1,3 this condition is often found in asian, african, and american patients.1,3 bimaxillary protrusion etiology is multi-factorial. there are genetic factors, as well as environmental factors, such as mouth breathing, habitual movement of the lips and tongue, and tongue volume.4 the main goals of treatment of bimaxillary protrusion are to reduce the inclination of the maxilla and mandible incisors with extraction of the premolar(s) by using maximum anchorage, so that the dentofacial aesthetics and smile can be improved.5 from the patient’s perspective, they seek orthodontic treatment to reduce their protrusive profile because they have psychosocial problems.3 bimaxillary protrusion correction includes incisive retraction using the first premolar extraction at both jawbones (depending on the case) and retraction of the anterior segment at the extraction site.1 anchorage control is important for obtaining the treatment goals and correcting the profile.6 in this case, anchorage is the resistance of the posterior segment to the force of anterior retraction. if there copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 mailto:ida-b-n@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 18 narmada et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 17–22 were anchorage loss, when the posterior segment slides to the anterior segment because of the reciprocal force, the treatment goals cannot be achieved. another indicator of anchorage loss is mesial tipping of the maxillary molars, which leads to changes to the occlusal plane.6 patients with missing molars can find orthodontic treatment in a clinic. the treatment choice is closing the space or inserting a dental prosthesis into the space.7,8 in some cases, replacement of the edentulous area with neighboring teeth has proven to be an excellent treatment outcome. however, moving the neighboring teeth must be performed by bodily movement without any inclination because it could change the vertical dimension.8 a missing lower molar is more complex than an upper molar because the mandible has a thick cortical bone and a small trabecular bone, and the roots of the lower molar are wider.8 therefore, this case aims to report on the management of bimaxillary protrusion with a missing molar using a t-loop and transpalatal arch (tpa) as the maximum anchorage to correct the profile and couple force to create bodily movement for close spacing of the missing first molar. case a 21-year-old female patient came to the dental hospital of universitas airlangga with a chief complaint of teeth protrusion. an extra-oral examination showed a convextype profile with incompetent lips (figure 1). an intraoral examination indicated angle’s class i on the left molar, with the lower-right first molar missing, mild crowding maxilla and mandible, 6 mm of overjet, and 5 mm of overbite. in addition, there was a shift at the maxilla median 1 mm to the left and at the mandible 2 mm to the left. oral hygiene and periodontal tissues were good (figure 2). the arch length discrepancy was presented in both the maxillary arch (10 mm) and the mandibular arch (4 mm). according to the patient, her father has a similar convex profile with protruding teeth. there were no clinical signs of clicking or discomfort in temporomandibular joints, and there was no restriction or deviation in jaw movement. the lateral cephalometric analysis indicates that ∠ sna 84º, ∠ snb 76º, and anb 8° refer to the skeletal pattern class ii malocclusion. dental measurements indicated that upper and lower incisors were proclined, with ∠ i-na 35º, ∠ i-nb 32º, and ∠ inter incisal 100º. the patient has a convex skeletal profile (fh–np 81.5º, nap 15º), with mandible clockwise rotation with ∠ mp–fh 36° and mandible retrognathic with ∠ nap 15°. rickett’s lip analysis and steiner's lip analysis indicated the lips were far in front of the e and s lines (table 1). the intraoral photograph shown in the figure 3. a panoramic radiograph indicated teeth 28, 38, and 48 were impacted (figure 4). case management the treatment’s objectives were to improve the occlusion, including correcting the profile and the crowding protrusion maxilla and mandible, correcting the midline shift, and correcting the lower-right posterior diastema due to the absence of 46. based on the clinical examination, diagnostic records, and cephalometric analysis, it was planned to use a a b figure 1. (a) pre-treatment and (b) post-treatment extraoral photographs. facial photos of the frontal view at rest, smiling, and lateral view. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 19narmada et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 17–22 table 1. pre and post treatment cephalogram’s measurements measurement surabayan subject pre post ∠sna 84.3 84 83 ∠snb 81.4 76 77 ∠anb 3 8 6 ∠op–sn 15–32 24 23 ∠mp–sn 20–40 36 35 ∠1-na 26 35 17 1-na (mm) 6.3 mm 10 mm 3 mm ∠1-nb 29 32 28 1-nb (mm) 7.9 mm 12 mm 8 mm nasolabial angle 110, 120o 118o 120o upper lips – e line -2–3 mm +4 mm +1 mm lower lips e line -1–2 mm +10 mm +2 mm upper lips – s line 0 +4 mm +4 mm lower lips – s line 0 +11 mm +4 mm figure 2. pre-treatment intraoral photographs. intraoral view of upper-occlusal, lower occlusal, right lateral, frontal, and left lateral. figure 3. during treatment, intraoral photographs. intraoral view of upper-occlusal, lower occlusal, right lateral, frontal, and left lateral. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 20 narmada et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 17–22 a b c d e figure 5. post-treatment intraoral photographs. intraoral view of (a) upper occlusal, (b) lower occlusal, (c) right lateral, (d) frontal, and (e) left lateral. a b figure 4. (a) pre-treatment and (b) post-treatment lateral cephalometric and panoramic radiographs. figure 6. superimposition of lateral cephalometric on pre(black) and post-treatment (red). note: there were changes in maxilla and mandibular incisor angulation and the lip position. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 21narmada et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 17–22 fixed appliance along with the extraction of the premolars. the treatment plan was suggested as follows: (1) extraction of teeth 14, 24, 34; (2) alignment of the upper and lower teeth with pre-adjusted mclaughlin bennett trevisi (mbt) 0.022; and (3) retention with the upper and lower hawley removable retainers. after extraction of the premolars, the treatment was started by bonding the brackets and buccal tubes using 0.022 slots with pre-adjusted edgewise brackets, mbt. the patient was treated with the conventional anchorage system consisting of the tpa to limit the anchorage loss risk for the upper jaw. the first step was processing alignment and leveling with 0.012, 0.014, 0.016, and 0.016 x 0.016 nickel– titanium wire. the second step was retracting the canine using stainless steel wire 0.016 x 0.22 and a power chain until the relation of the canine became class i. the third step was retracting the anterior teeth using a t-loop (0.016 x 0.022 tma wire), and then 47 was mesialized with a power chain on the buccal and lingual sides by using a tip back. the last step was to arch compatibility by using a stainless-steel wire 0.017 x 0.025. after 49 months of treatment, the brackets and buccal tubes were debonded and hawley retainers were used for stability on both the upper and lower arches. soft-tissue analysis indicated that the position of the upper and lower lips was more backward in the end of the treatment. the crowding on the maxilla and mandible was corrected, an ideal overjet and overbite were obtained, the midline shift was corrected, the posterior diastema due to the absence of 46 was corrected, and a clockwise mandible rotation was obtained. the incisive inclination of the maxilla and mandible was normal, but the relation between the maxilla and mandible was still indicated as skeletal class ii (figures 5 and 6). discussion teeth extraction in orthodontic treatment is still being debated in the orthodontic field. however, it has become a fundamental consideration in some cases. the affected factors in an extraction decision are features of malocclusion, the purpose of the treatment, and the techniques used to provide the desired outcomes. the discrepancy of dental arches also affects the extraction decision. the first extraction selected is of the premolar because it will provide good long-term stability. patients with more proclined incisors and more protrusive lips may be better with an extraction treatment plan to help compensate.9 incisive retraction needs a space in the jaw arches to reduce prominent lips.1 in this case, there was a 10-mm maxilla and 4 mm mandible discrepancy and an incisive protrusion of the maxilla and mandible. therefore, the first treatment selected was premolar extraction. there was no extraction in the fourth region due to a gap from the absence of the lower-right molar, so a closed space was conducted. during leveling and aligning, a tpa was used as an additional tool to create maximum anchorage. tpa is economical, easy to fabricate, and the most reliable method.2 although tpa cannot be used as an absolute anchor, it can be used as an additional tool during orthodontic treatment to control vertical, transversal, and sagittal dimension anchoring (anteroposterior).10 anterior retraction is divided into one-step (en-masse) and two-step retractions (single canine retraction).1,11,12 the canine retraction is separated and followed by incisive retraction so it can keep posterior anchorage.11 this step can prevent mesialization of the posterior segment due to retraction of the anterior segment because lighter force had been used.2 an indication for single canine retraction is a case of crowding and midline shift.11 in this case, single canine retraction is conducted due to midline shift and anterior crowding. after leveling and aligning are successful, retraction canine is conducted by using the “sliding mechanics” technique. this technique applies force between two teeth or a segment of teeth, and a straight wire is inserted into the respective brackets. therefore, there will be friction between the wire and the bracket surface. sliding mechanics is selected because it is more controlled during space closure (reduced rotation effect and tipping), increasing patient comfort and avoiding excessive force.12 after canine retraction is completed, anterior retraction with the “segmental mechanics” method is conducted by using t-loop and step-up. step-up is used to correct a deep bite. the second premolar, canine, and first molar comprise a segment that serves as an anchor or passive segment. four incisor teeth serve as an active segment. therefore, both segments are not connected by a wire (the teeth are not moving on the wire) so this technique can be called “frictionless mechanics.”12 during anterior retraction, a midline shift correction is also conducted. while performing a space closure, the distance between the force and the center of resistance must be considered to provide moments. this is often called the moment-to-force (m/f) ratio.13 in this case, anterior retraction requires bodily movement; therefore, a high m/f ratio on the posterior segment is required. if using a t-loop, the m/f ratio can be increased by increasing the height of the t-loop because the wire is more flexible and releases less force.13 the height of the t-loop varies between 6 mm and 10.45 mm.13 the m/f ratio can also be increased by adding apical length, but this is never ideal for controlled inclination and translation due to anatomy limitations.13 thus, it is recommended to make preactivation bends.13 the preactivation bend can sometimes reach 180° from the horizontal, according to the anchorage needs of the case. a 0.016 x 0.022-inch tma t-loop preactivated to 180° and activated 6 mm horizontally delivers approximately less than 243 g.12 apical lengths vary from 10 mm to 16 mm.13 in this case, although protraction of the second molar was time-consuming and relatively difficult, the decision copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 22 narmada et al. dent. j. (majalah kedokteran gigi) 2023 march; 56(1): 17–22 was still to protract 47 because the periodontal health was good, and the protraction of 47 can control the wisdom tooth (48) positioning. space reopening for dental prosthesis would be indicated if the periodontal health of the second molar were not good or if the wisdom tooth were absent.7 close spacing of the missing 46 was conducted by using sliding mechanics. protraction of 47 used couple force with a power chain at the buccal and lingual (using the lingual button) and tip back. couple force or balancing lingual force was used to prevent mesial rotation, tipping, and buccal sweep of the molar.7,8 an effective tip back angle was 20°–30° and slightly in to prevent mesiolingual rotation.14 at the end of this case, the molar was slightly tipped distally during protraction (figure 5). to make a bodily movement, the tip back angle should be 10°.14 after orthodontic treatment, canine relation class i was achieved on both sides. the overjet and overbite were normal, the crowding of the maxilla and mandible was corrected, the incisive inclination was normal, the shift midline was corrected, and the posterior diastema was corrected. at the end of the treatment, the soft tissue of the patient improved, and the goals of the treatment were achieved (figure 5). however, the limitation of treatment for this case is that the relation between the maxilla and mandible was still indicated as skeletal class ii. this might be due to the use of an anchorage that should have been an absolute anchorage to prevent zero anchorage loss or movement of the anchorage unit. the alternative treatment is to use temporary anchorage devices as an absolute anchorage to prevent the distal movement of the anterior teeth or posterior teeth (or both) without anchorage loss.5 in conclusion, an orthodontic patient was treated with a bimaxillary protrusion case that included maxilla and mandible incisive retraction. the treatment was conducted by extraction of the first premolars. a retraction can be conducted by using sliding mechanics or segmental mechanics. closing the space of the missing molar was achieved with couple force on the buccal and lingual sides and the tip back. at the end of the treatment, there was normal incisive inclination and improvement of the smile and facial profile. references 1. proffit w, fields h, larson b, sarver d. contemporary orthodontics. 6th ed. philadelphia: mosby; 2018. p. 528, 533. 2. halwa hk, yadav sk, dutta k, gupta sk, shrestha r, shah ak. bimaxillary protrusion a case report. j univers coll med sci. 2019; 7(1): 70–3. 3. hoyte t, ali a, bearn d. prevalence of bimaxillary protrusion: a systematic review. open j epidemiol. 2021; 11(01): 37–46. 4. qa ma r y, feh m i m, ta r iq m, ver ma sk. ma nagement of mimaxillary protrusion in hyperdivergent case; a case report. int j contemp med res. 2018; 5(3): c1–3. 5. novianti s, siregar e. treatment of bimaxillary protrusion case with asymmetric extraction pattern manage by the use of tads as anchorage. in: indonesian association of orthodontists (iao). medan: usu press; 2017. p. 203–7. 6. chandra p, kulshrestha rs, tandon r, singh a, kakadiya a, wajid m. horizontal and vertical changes in anchor molars after extractions in bimaxillary protrusion cases. apos trends orthod. 2016; 6: 154–9. 7. aghoutan h, alami s, el aouame a, el quars f. orthodontic management of residual spaces of missing molars: decision factors. in: human teeth key skills and clinical illustrations. intechopen; 2020. p. 1–15. 8. raveli tb, shintcovsk rl, knop lah, sampaio lp, raveli db. orthodontic replacement of lost permanent molar with neighbor molar: a six-year follow-up. case rep dent. 2017; 2017: 4206435. 9. ganguly r, suri l, patel f. a literature review of t extraction decision and outcomes in orthodontic treatment. j mass dent soc. 2016; 65(2): 28–31. 10. almuzian m, alharbi f, chung ll-k, mcintyre g. transpalatal, nance and lingual arch appliances: clinical tips and applications. orthod updat. 2015; 8(3): 92–100. 11. ribeiro glu, jacob hb. understanding the basis of space closure in orthodontics for a more efficient orthodontic treatment. dental press j orthod. 2016; 21(2): 115–25. 12. nanda r. esthetics and biomechanics in orthodontics. 2nd ed. philadelphia: saunders; 2014. p. 115. 13. viecilli af, freitas mpm. the t-loop in details. dental press j orthod. 2018; 23(1): 108–17. 14. ryu w-k, park jh, tai k, kojima y, lee y, chae j-m. prediction of optimal bending angles of a running loop to achieve bodily protraction of a molar using the finite element method. korean j orthod. 2018; 48(1): 3–10. copyrigrt © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i1.p17–22 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i1.p17-22 mkg vol 39 no 1 jan 2006 isi.pmd 16 manufacturing hollow obturator with resilient denture liner on post hemimaxillectomy michael josef kridanto kamadjaja department of prosthodontia faculty of dentistry airlangga university surabaya indonesia abstract a resilient denture liner is placed in the part of the hollow obturator base that contacts to post hemimaxillectomy mucosa. replacing the resilient denture liner can makes the hollow obturator has an intimate contact with the mucosa, so it can prevents the mouth liquid enter to the cavum nasi and sinus, also eliminates painful because of using the hollow obturator. resilient denture liner is a soft and resilient material that applied to the fitting surface of a denture in order to allow a more distribution of load. a case was reported about using the hollow obturator with resilient denture liner on post hemimaxillectomy to overcome these problems. key words: hollow obturator, resilient denture liner correspondence: michael josef kridanto kamadjaja, c/o: bagian prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction maxillofacial prosthetics is the prosthetic rehabilitation of regions of the head and neck that are missing or defective by means of non-living tissue substitutes. these deficiencies may be due to surgical treatment, trauma, pathology, or congenital malformation. intraoral prostheses or obturators are used to reconstruct defects associated with the oral cavity. an obturator prosthesis is used for reconstructing part of the maxilla (upper jaw) and will close oral-nasal openings in the palate, aids breathing/speech and supports oral prosthesis. some advantages of obturation are have access to defect area, supports facial profile, no donor site required, restores feelings of normality, builds confidence, to overcome sense of loss and less socially vulnerable.1,2 base of the obturator made of acrylic or metal, and acrylic is usually used as a base of obturator, because it can be adjusted easilly. obturator usually does not have enough stability, because of lack support and retention, and the weight of the obturator exerts dislodging and rotational forces on the abutment teeth.3,4 the forces exerted on the artificial teeth, especially during mastication, frequently cause a cantilever effect on the abutment teeth. greater attention must therefore be paid to the preservation of the abutment teeth. brown5 and desjardins6 have suggested hollow obturator to minimize the weight of the obturator. the hollow obturator showed rapid damping of vibration and minimal displacement of the retainers. obturator needs to have maximum support, retention and stability. for this purpose it is necessary to use soft material, e.g resilient denture liner placing in the part of the hollow obturator base that contacts to post hemimaxillectomy mucosa. replacing the resilient denture liner can makes the hollow obturator has an intimate contact with the mucosa, so it can prevents the liquid enter the nasal cavum and sinus, also eliminates painful using the hollow obturator. resilient denture liner is a soft and resilient material that applied to the fitting surface of a denture in order to allow a more equal distribution of load, to permit the mucosal tissues to assume a more normal position.7 resilient denture liner must have general properties such as permanent resiliency, adequate torn resistance, low water sorption and minimal solubility in saliva, aesthetically acceptable, good compatibility with mucosa, odourless and tasteless, high bond strength with denture base and high dimensional stability.8 the purpose of this case report is to give information about using hollow obturator with resilient denture liner on post hemimaxillectomy. case a 40-years old male patient came to the prosthodontic clinic due to opening in the left upper jaw, because of post hemimaxillectomy. he was sent by dr. soetomo surgery department for an obturator. teeth in the left upper jaw were extracted during the operation. the remaining teeth in the right upper jaw were from lateral incisor up to second molar. the resection was performed along the left side of the upper jaw and extended across the midline of the maxilla. the right incisive central was also extracted during the maxillectomy. radiotherapy and chemotherapy were not performed after the operation. food and liquid penetrated to nasal 17kamadjaja: manufacturing hollow obturator cavum through defect area during mastication and gave problems to the patient. case management impressions were taken to the upper and lower jaw used alginate impression material in order to make a diagnostic cast. individual tray was fabricated and final impression was performed for the upper jaw. preparing base and bite wall on the working cast and bite registration was done in the patient’s mouth. working cast and bite registration wax were mounted in the articulator. artificial teeth were arranged and hollow obturator was made in wax. the hollow obturator was inserted for "try in" the patient’s mouth, position and occlusion checked with the natural teeth. placing back the hollow obturator in the working cast and claps were made in teeth 12, 13, 15 and 17. final contour was done and processed with acrylic heat cured. after polishing the hollow obturator was tried in the mouth patient and adjust was done if needed. intermaxillary record was performed and the hollow obturator was mounted in figure 1. defect area in the left upper jaw post hemimaxillectomy. figure 2. hollow obturator with upper partial denture. a: the lid and hollow obturator, b: the lid fixed with self curing acrylic, c: the bulb of the obturator ba c figure 3 (a, b, c). replacing hollow obturator with resilient denture liner. ba c figure 4 (a, b). hollow obturator was inserted in the mouth. 18 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 16–18 the articulator for selective grinding. the lid of the hollow obturator was fixed with self cured acrylic. base the hollow obturator that contact to mucosa was roughened with bur and covered with primer solution and waited until it dried. replacing the resilient denture liner to that area and put the hollow obturator back in the mouth. patient was ordered to close the mouth and performed muscle trimming. the patient kept closing the mouth until the resilient denture liner hardened. the hollow obturator was taken out and excess material was tidied up with knife and bur. borderline between acrylic denture liner and hollow obturator base was coated by layer material and dried up. hollow obturator was inserted in the mouth and ready to use. discussion the resection in aramany’s class iv was performed across the midline of the maxilla and the right incisive central was extracted during the maxillectomy. retention and stability of the obturator were obtained from the remaining teeth and mucosa around defect area. lack of support and retention made the hollow obturator lost its stability, especially during mastication, so frequently cause a cantilever effect on the abutment teeth.2,3 cantilever effect is an rotational and dislodging effect on the abutment teeth, due to of support in the defect area. on the defect area we could also set up artificial teeth, but mastication on this side must be avoided, because lack of tissue support. in this case four abutment teeth were used as direct retainers and the lateral wall of the hollow obturator was extended to maximize support, retention and stability. silicone resilient denture liner was placed on the hollow obturator that contacted with the mucosa, so there was an intimate contact between the hollow obturator and the mucosa. the hollow obturator showed rapid damping of vibration and minimal displacement of the retainers. intimate contact between the two parts could prevent food and liquid enter the nasal cavum and sinus, also eliminate painful.4 when the patient came back for control, he reported that there was no pain, the hollow obturator had enough retention and stability, food and liquid did not enter the nasal cavum anymore. hollow type obturator was fabricated in this case, so the weight of the obturator could be reduced. the obturator must have minimal weight, stability and good retention. the lid of the hollow obturator was processed with heatpolymerizing acrylic resin and fixed to the bulb with autopolymerizing acrylic resin. replacing hollow obturator with resilient denture liner by direct technique method was more simple than indirect technique method, because it does not need complicated procedure in laboratorium. thickness of the resilient denture liner have to be 2 or 3 mm, that it could have effective function. resilient denture liner was a temporary or semipermanent material, because it has some disadvantages such as lost of softness and resiliency, poor bond strenght with denture base, dimensional changes, difficult in cleaning, polishing and finishing.10 denture cleanser had to be used and chosen properly, because denture cleanser had to keep the resilient denture liner in a good condition, so it could be used longer.10 it can be concluded that replacing the resilient denture liner can make the hollow obturator has good seal to the mucosa, that it could prevent the mouth liquid entered to the cavum nasi and sinus, also eliminated painful due to the used of the hollow obturator. references 1. laney wr. maxillofacial applications of removable partial prosthodontics. mc cracken’s removable partial prosthodontics. the cv mosby company 1985; 23:443–59. 2. aramany ma. basic principles of obturator design for partially edentulous patients. part i: clasification. j prosthet dent 1978; 40:554–7. 3. aramany ma. basic principles of obturator design for partially edentulous patients. part ii: design principles. j prosthet dent 1978; 40:656–82. 4. kobayashi m, oki m, ozawa s, inoue t, mukohyama h, takato t, ohyama t, taniguchi h. vibration analysis of obturator prostheses with different bulb height designs. j med dent sci 2002;49:121–8. 5. brown ke. peripheral consideration in improving obturator retention. j prosthet dent 1969; 20:176–81. 6. desjardins rp. obturator prosthesis design for acquired maxillary defects. j prosthet dent 1978; 39:424–35. 7. huggett r. soft lining materials in prosthetic dentistry: a review. the international journal of prosthodontics 1990. p. 477–82. 8. pesun ij, hodges j, lai jh. effect of finishing and polishing procedures on the gap width between a denture base resin and two long-term, resilient denture liners. j prosthet dent 2002. p. 311–8. 9. lammie ga, storer r. a preliminary respon on the resilient denture plastics. j prosthet dent 1958; 8:411–24. 10. hamada t, murata h, razak a. pelapisan gigi tiruan denture lining. airlangga university press; 2003. p. 32–44. 11. schmidt wf, smith de. a six-year retrospective study of molloplastb lined dentures. part ii: liner serviceability. j prosthet dent 1980; 50:459–65. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 133133 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 133–139 research report the changing of occlusal plane inclination in class ii malocclusion nelvi yohana, siti bahirrah and nazruddin department of orthodontics, faculty of dentistry, universitas sumatera utara, medan – indonesia abstract background: camouflage treatment of skeletal class ii malocclusion can be performed using extraction or non-extraction techniques. these treatments can cause changes in occlusal plane. steep occlusal plane during corrective treatment generally relapses after active orthodontic treatment, resulting in unstable interdigitation. purpose: this study aims to determine and evaluate changes in occlusal plane inclination in skeletal class ii malocclusion cases using extraction or non-extraction techniques of the permanent maxillary first premolar. methods: the samples consisted of initial and final cephalometry of 40 adult patients with skeletal class ii malocclusion divided into two groups, namely extraction of the permanent maxillary first premolar and non-extraction group. the inclination of occlusal planes in both groups was measured using the imagej software, then the factors associated with these changes were observed. furthermore, the occlusal plane inclination was compared between the extraction and non-extraction groups by using t-test. results: the occlusal plane inclination in the non-extraction group increased slightly, while the inclination in the extraction group increased significantly (p = 0.017, p-value < 0.05). however, there was no correlation found in the occlusal plane inclination between the extraction and non-extraction groups (p = 0.07, p-value < 0.05). conclusion: class ii malocclusion correction with either extraction or non-extraction of the maxillary first premolar increased the inclination of the occlusal plane. this study indicated that control of the occlusal plane inclination is highly essential. keywords: cephalometry; class ii malocclusion; extraction; inclination; occlusal plane correspondence: nelvi yohana, department of orthodontics, faculty of dentistry, universitas sumatera utara, jl. alumni no. 2, medan 20155, indonesia. email: nelviyohana@yahoo.com introduction skeletal class ii malocclusion is the most common problem in orthodontics, and about one-third of these patients are treated by orthodontists.1 this type of malocclusion is not a single diagnosis, but is produced from various dentoalveolar skeletal components. skeletal class ii patterns can be caused by protrusive maxilla with normal mandible, normal maxilla with retrusive mandible, or a combination of protrusive maxilla and retrusive mandible.2,3 sridharan et al.4 state that about 10% of the tumkur population have class ii malocclusion. ardani et al.5 in their study of 65 lateral cephalometric radiographs from the adult javanese (deutero malay) population, have found that the highest frequency of class ii malocclusion variations was in the combination of normal maxilla with mandibular retrusion. the disturbance can be in the form of size, position or relation between the jaws.3,6,7 clinical manifestations of patients with skeletal class ii malocclusion include maxillary and anterior teeth protrusion, deep bites, clockwise rotation of the mandibular growth, incompetent lips and an aesthetic face.8,9 this appearance could affect the patient’s confidence, mental health and daily communication. successful orthodontic treatment not only forms a balanced and stable occlusion relation, but also achieves an aesthetic facial appearance.10,11 comprehensive orthodontic treatment using fixed orthodontic appliances usually consists of two treatment modalities, namely extraction and non-extraction. 10,11 orthodontic treatment with tooth extraction is performed dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 mailto:nelviyohana@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 134 yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 to treat moderately to severely crowded teeth, and/or to reduce dental or dentoalveolar protrusions. in contrast, non-extraction orthodontic treatment is conducted in cases with minor skeletal discrepancies and moderate dental discrepancies.11 the orthodontic treatment affects various parameters, such as vertical dimensions, treatment stability, arch width, perioral soft tissue, facial convexity and occlusal plane.11,12 occlusal plane (op) is a line following the bite of the teeth and is considered an important reference plane to achieve functional balance.8 the shape and inclination of the occlusal plane depend on each person’s characteristics and are related not only to the stomatognathic system, but also dentofacial aesthetics.13 the occlusal plane inclination is an important factor in dentofacial morphology and is one of the standards for reconstructing occlusion.14 the inclination of the occlusal plane is obtained through angular measurements between the occlusal plane (op) relative to the reference plane, such as sella-nasion (sn) plane, basion-nasion plane (ban) or horizontal frankfort plane (fh).8,15 there are various ways to determine the occlusal plane, including bisected occlusal plane (bop), functional occlusal plane (fop) and lower incisor occlusal plane (liop).15–17 downs has defined bop (a line that connects two points dividing the overlapping distobuccal first molar and overlapping overbite incisors) as the most commonly used method. fop is a plane that divides the first premolar intercuspation and the first molar cuspid intercuspation. 15,16 changes in the occlusal inclination can be caused by molar movement to the mesial (loss of anchoring) or due to extrusion and intrusion (molars and incisors).17 factors affecting the morphology and function of the occlusal plane include growth, head and neck muscles, mandibular rotation during growth, tooth eruption and bad habits.13 class ii malocclusion has a relatively steep occlusal plane.15 bawman and johnston state that increased occlusal plane inclination during treatment indicates reduced vertical control and tends to become unstable, because the occlusal plane angle is determined by the muscle balance, especially masticatory muscles.12 orthodontic treatment changes the position and angulation of the teeth and moves them to the ideal aesthetic and functional position. a slight angular change in orthodontic treatment will cause a significant occlusion change, so it can lead to functional disharmony and relapse.16 a study conducted by li et al.16 has shown that the occlusal plane inclination (bop-sn and fop-sn) in skeletal class ii samples was steeper than the skeletal class i and iii samples. there was a significant increase in the average bop-sn angle of 1.51° after orthodontic treatment without premolar extraction in growing and developing patients.16 contrary to this, a study by zenab et al.18 examining changes in the occlusal plane inclination before and after the extraction of four premolars in bimaxillary protrusion cases has shown that the occlusal plane inclination after treatment became smaller than before treatment. according to zimmer and nischwitz,19 there was no significant change in bop inclination to the anterior cranial base before and after treatment in skeletal class ii patients treated with elastic. furthermore, there are still limited studies investigating the changes in occlusal plane inclination in skeletal class ii malocclusion treated with extraction of the first maxillary premolars and nonextraction in adult patients. therefore, the present study aims to evaluate changes in the occlusal plane inclination in skeletal class ii malocclusions treated with maxillary premolar extraction and non-extraction at the department of orthodontics, faculty of dentistry, universitas sumatera utara, indonesia. materials and methods ethical clearance was obtained from the health research ethics committee of the faculty of medicine, universitas sumatera utara, number: 900/tgl/kepk fk-rsup ham/2019. this study was retrospective analytical and sample selection method was applied by purposive sampling method adhering to fulfilled inclusion and exclusion criteria. the number of samples was determined using the formula of sample size for two-means-dependent samples. the research samples were taken from lateral cephalometric radiographs of mongoloid race before and after orthodontic treatment using fixed orthodontic devices from 40 class ii skeletal patients with anb > 4° at the department of orthodontics, faculty of dentistry, university of sumatra utara between august 2019 and january 2020. the patients were willing to become research participants and gave written informed consent. the samples were divided into two groups, namely the group treated with non-extraction and the group treated with permanent maxillary first premolar extraction. the average age of the samples was 22.5 years. the samples were selected based on the following inclusion criteria: (1) cephalometric radiographs before and after treatment showed a good condition; (2) male and female patients aged 18-35 years; (3) a complete number of permanent teeth in patients before treatment regardless of the presence or absence of third molars; (4) no congenitally missing teeth/ agenesis; (5) no supernumerary or anomalous form teeth; and (6) no history of oral cavity trauma and bad habits. the exclusion criteria in this study were a history of cleft lip and/or palate and patients who underwent functional or orthognathic surgery before and after treatment. photographs were taken from lateral cephalogram radiographs that were placed on the tracing box using a nikon d90 camera (12.3 megapixel digital single-lens reflex camera made in thailand) with a nikon dx af-s nikkor 18-105 mm lens mounted on a tripod in zero tilting position (seen waterpass on a tripod) and 50 cm away from the lateral cephalogram radiograph. the images were then traced and measured using imagej software version 1.53c (figure 1 and 2).20 imagej is a java-based image dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 135yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 figure 1. occlusal plane inclination bop-sn (°) and fop-sn (°). figure 2. determination angulation and position of central incisors and first molar. (1) angulation of the maxillary central incisor (u1-sn (°)), (2) maxillary central incisor position (u1-pp (mm)), (3) maxillary permanent first molar position (u6-pp (mm)), (4) angulation of the mandibular central incisor (l1-mp (°)), (5) mandibular central incisor position (l1-mp (mm)), (6) mandibular permanent first molar position (l6-mp (mm)). table 1. the cephalometric variables used in this study variable definition used plane bop the bisected occlusal plane is a region formed by connecting points that bisect the overlapping distobuccal cuspid of the maxillary and mandibular first molars with points that bisect the overlapping overbite incisors (downs). fop the functional occlusal plane is a plane that divides the intercuspation of the first premolar with the first molar cuspid intercuspation (jacobson). sn the line that runs through the mid sella turcica and nasion. pp the palatal plane is a line connecting the anterior nasal spine and posterior nasal spine. mp the mandible plane is a line connecting gonion and menton. measurements bop-sn (°) inclination of occlusal plane formed between the sella turcica-nasion plane and bop. fop-sn (°) inclination of occlusal plane formed between the sella turcica-nasion plane and fop. u1-sn (°) angulation of the maxillary central incisor, which is a posterior-inferior angle that is formed from the long axis of the maxillary central incisor (u1) and sn plane. u1-pp (mm) the position of the maxillary central incisor, which is the perpendicular distance from the maxillary central incisor (u1) to the palatal plane (pp). u6-pp (mm) the position of the maxillary first molar, which is the perpendicular distance from the distobuccal cusps of the maxillary first molar (u6) to the palatal plane (pp). l1-mp (°) angulation of the mandibular central incisors, i.e. angles formed from the long axis of the mandibular central incisors (l1) and mp plane. l1-mp (mm) the position of the mandibular central incisors, which is the perpendicular distance from the mandibular central incisors (l1) and the mandibular plane (mp). l6-mp (mm) the position of the mandibular first molar, which is the perpendicular distance from the mandibular first molar distobuccal cusps (l6) to the mandibular plane (mp) before and after treatment. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 136 yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 processing programme developed at the national institutes of health and the laboratory for optical and computational instrumentation (university of wisconsin, united states). determination of landmark points, lines, and reference angles in the extraction and non-extraction groups can be seen in table 1. the tracing and measurement process was carried out by one operator and repeated twice. the study statistics use statistical package for social science (spss) software version 22 (new york, united states). p-value of this study was <0.05 using bivariate test, pearson test and t-test. results analysis of the mean values of variables in patients treated with non-extraction using the bivariate test showed a significant change in l1-mp (°) and l6-mp (mm) (p <0.05) before and after treatment, where the inclination of the mandibular incisors was more anterior, and extrusion occurred from the permanent mandibular first molars. there was also an increase in fop-sn (°) and bop-sn (°) with an insignificant value. the fop-sn (°) value was greater than that of bop-sn (°) in this group. analysis table 2. average changes on fop-sn (°), bop-sn (°), u1-sn (°), u1-pp (mm), u6-pp (mm), l1-mp (°), l1-mp (mm) and l6-mp (mm) before and after treatment in patients with maxillary premolar extraction and non-extraction variable non-extraction extraction before x±sd after x±sd p-value before x±sd after x±sd p-value fop-sn (°) 16.67±5.29 16.88±4.19 0.818 bop-sn (°) 15.86±4.58 16.37±5.05 0.549 16.78±6.21 18.72±4.11 0.017* u1-sn (°) 108.08±8.19 105.21±9.54 0.295 108.02±6.71 97.39±6.12 0.0001* u1-pp (mm) 31.28±2.85 32.09±3.36 0.078 32.99±3.47 33.94±3.28 0.036* u6-pp (mm) 23.15±2.54 23.73±2.79 0.066 25.27±3.48 25.92±3.38 0.083 l1-mp (°) 101.39±7.47 109.05±6.91 0.0001* 101.40±6.36 101.58±6.82 0.892 l1-mp (mm) 44.27±3.31 44.61±3.46 0.437 46.48±3.91 45.35±4.05 0.019* l6-mp (mm) 32.74±2.73 33.53±3.11 0.039* 33.09±2.75 33.97±3.31 0.019* *p<0.05= significant table 3. the relationship between the inclination of the fop-sn (°) and bop-sn (°) occlusal plane in patients given non-extraction treatment variable x±sd r p-value bop-sn (before) fop-sn (before) 15.86±4.58 16.67±5.29 0.780 0.0001* bop-sn (after) fop-sn (after) 16.37±5.05 16.88±4.19 0.892 0.0001* *p<0.05= significant table 4. the relationship between the average changes in u1-sn (°), u1-pp (mm), u6-pp (mm), l1-mp (°), l1-mp (mm) and l6mp (mm) to bop-sn (°) in the non-extraction and extraction treatment groups variable x±sd r p-value x±sd r p-value u1-sn (°) bop-sn (°) 2.88±11.95 0.50±3.70 -0.598 0.005* 10.07±13.85 2.97±4.52 -0.230 0.329 u1-pp (mm) bop-sn (°) 0.81±1.94 0.50±3.70 0.435 0.056 0.51±3.09 2.97±4.52 0.376 0.102 u6-pp (mm) bop-sn (°) 0.57±1.31 0.50±3.70 -0.267 0.254 0.86±2.84 2.97±4.52 -0.341 0.141 l1-mp (°) bop-sn (°) 7.66±6.46 0.50±3.70 0.173 0.464 0.23±9.78 2.97±4.52 0.143 0.548 l1-mp (mm) bop-sn (°) 0.34±1.91 0.50±3.70 -0.226 0.338 0.92±3.23 2.97±4.52 -0.215 0.363 l6-mp (mm) bop-sn (°) 0.79±1.59 0.50±3.70 0.743 0.0001* 0.92±2.38 2.97±4.52 0.287 0.220 *p<0.05= significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 137yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 of the mean values of variables in patients treated with maxillary premolar extraction using the bivariate test showed that the changes in bop-sn (°), u1-sn (°), u1-pp (mm), l1-mp (mm), and l6-mp (mm) were significant (p < 0.05) before and after treatment with maxillary premolar extraction (table 2). the pearson test was used to investigate the relationship between fop-sn (°) and bopsn (°) in patients treated with non-extraction orthodontic treatment (table 3). the statistical results showed a strong correlation between bop-sn (°) and fop-sn (°) before and after treatment (p = 0.0001). the pearson test used to analyse the relationship between u1-sn (°), u1-pp (mm), u6pp (mm), l1-mp (°), l1-mp (mm) and l6-mp (mm) to bop-sn (°) in the nonextraction treatment group showed a significant relationship between the maxillary central incisor angulation (p = 0.005) and mandibular first molar position (p = 0.0001) to the occlusal plane inclination. correlation analysis of changes between u1-sn (°), u1-pp (mm), u6-pp (mm), l1-mp (°), l1-mp (mm) and l6-mp (mm) to bop-sn ( °) in orthodontic treatment patients with maxillary first premolar extraction showed that there was no significant relationship between the variables with p > 0.05 (table 4). the correlation between the maxillary premolar extraction and non-extraction groups was analysed using the t-test. the comparison analysis of the non-extraction and extraction groups showed that there was a significant correlation in the maxillary central inclination u1-sn (°), mandibular incisors inclination l1-mp (°) and position of the central mandibular incisors l1-mp (mm) between the two groups. however, there was no significant difference between the occlusal plane inclination in the extraction and non-extraction of the maxillary first premolar groups, but there was a greater change in the occlusal plane inclination in the extraction group than the non-extraction group (table 5). discussion this study has found that the mean occlusal plane was steeper after orthodontic treatment, both fop-sn (°) and bop-sn (°) with insignificant values. this correlates with research conducted by li et al.16 who had found an increase in bop-sn (°) and fop-sn (°) after nonextraction orthodontic treatment. these changes can occur due to the extrusion of molars and incisors from orthodontic treatment mechanics. class ii elastic is often used in treating patients with skeletal class ii malocclusion, which can cause rotation of the occlusal plane downward and backward.16,18,21 there was a significant increase in the mandibular incisor inclination (l1-mp/°) and vertical position of mandibular first molars (l6-mp (mm)) before and after non-extraction treatment in this study. these results correlate with the findings of janson et al.22 which have shown that there was a change in the mandibular incisor inclinations to the anterior and extrusion of the mandibular first molar. this change can be caused by the use of elastic class ii.22 according to braun and legan,23 the use of elastic can also lead to extrusion of posterior teeth, in this case extruding mandibular posterior teeth in skeletal class ii cases. increased mandibular incisor inclination can also be associated with skeletal class ii camouflage treatment, by protruding mandibular anterior teeth so that overjet is reduced.24 singh et al.25 state that the lower second molar should be used to extend the elastic if used for more than two months of treatment. this treatment regimen minimises the side effects from the use of elastics (extrusion of the lower posterior teeth and labial tipping of the lower anterior teeth, lowering of the anterior occlusal plane and the creation of gummy smile).22,25 this study has found a significant increase in the mean of occlusal plane inclination after orthodontic treatment with the extraction of two maxillary first premolars. demir et al.26 have found similar results in a study conducted on 53 table 5. differences in the changes of bop-sn (°), u1-sn (°), u1-pp (mm), u6-pp (mm), l1-mp (°), l1-mp (mm) and l6-mp (mm) between the extraction and non-extraction groups variable group x±sd p-value bop-sn (°) non-extraction 0.50±3.70 0.07 extraction 1.93±5.16 u1-sn (°) non-extraction 2.88±11.96 0.022* extraction 10.62±8.10 u1-pp (mm) non-extraction 0.81±1.94 0.808 extraction 0.95±1.88 u6-pp (mm) non-extraction 0.57±1.31 0.877 extraction 0.64±1.57 l1-mp (°) non-extraction 7.66±6.46 0.001* extraction 0.18±5.96 l1 mp (mm) non-extraction 0.34±1.91 0.022* extraction 1.13±1.97 l6-mp (mm) non-extraction 0.79±1.59 0.870 extraction 0.87±1.53 *p<0.05=significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 138 yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 class ii malocclusion patients with mandibular retrognathia with insignificant values. elih et al.27 report that posterior anchorage must be considered so that the position does not change during retraction. changes in occlusal plane inclination can also be caused by extrusion and intrusion. changes in the position of the posterior teeth can change the vertical dimensions, leading to increased occlusal plane inclination. it can be concluded that maximum anchorage can prevent posterior tooth changes.26 if the anterior teeth are retracted to prevent the posterior teeth from moving forward, posterior anchorage can be added by involving the second molar.18 in this study, we have found that there was a significant reduction of maxillary central incisor inclination, maxillary central incisor and mandibular first molar extrusions, and mandibular central incisor intrusions. this finding correlates with demir et al.26 who have found a significant reduction in the maxillary incisor inclination and an increased mandibular incisor inclination. adverse effects from the use of intermaxillary elastics may contribute to an increased height of the anterior upper face and also to tip the incisors.23,26 class ii elastic adverse effects include retroclination of the maxillary incisors and proclination of the mandibular incisors. the vertical vector of elastic class ii causes the anterior part of the maxilla to rotate downwards.26 according to a study by li et al.16 there was a very strong correlation between bop-sn (°) and fop-sn (°) in patients treated with non-extraction. the study also reports that the bop and fop occlusal plane inclinations were statistically steeper in class ii malocclusion groups compared to class i and class iii before and after treatment.16 similarly, the present study has found a strong relationship between bop-sn (°) and fop-sn (°) before and after orthodontic treatment without maxillary premolar extraction. the inclination of fop-sn (°) was greater than that of bop-sn (°). moreover, bop was found to be a more reproducible reference plane compared to fop during the cephalometric imaging process. determination of the point that bisects the maxillary and mandibular first premolar intercuspation is difficult to determine, especially in malpositioned teeth. the present study has shown a significant correlation between the angulation of the maxillary central incisor, and the vertical position of the mandibular first molar, to occlusal plane inclination. maxillary central incisor angulation was negatively correlated, whereas the vertical position of the mandibular first molar was positively correlated to the changes in occlusal plane inclination. this change may be due to the use of intermaxillary elastics.22 according to lamarque,28 changing and maintaining the occlusal plane during orthodontic treatment depends on molar movement to the mesial, vertical control of the maxillary and mandibular molars, and extrusion and intrusion of the incisors. a molar movement to the mesial is usually less in non-extraction treatment cases, so these are only affected by two factors.29 the present study has not shown a relationship between the changes in maxillary central incisor angulation, and mandibular central incisor angulation with changes in the occlusal plane inclination (bop-sn (°)), in patients treated with maxillary first premolar extraction. changes in the position of maxillary central incisors, mandibular central incisors, and permanent mandibular first molars also did not have a significant relationship to changes in the occlusal plane inclination. the group comparison analysis with and without maxillary first premolar extraction showed a significant relationship between maxillary central incisor inclination u1-sn (°), mandibular incisor inclination l1-mp (°) and central mandibular position l1-mp (mm). the extraction group showed changes in maxillary central incisor inclination u1-sn (°), which was greater than that of the non-extraction group. this may be due to the fact that when the anterior teeth were retracted in the extraction group, there was a greater change in the inclination of the maxillary incisors to the lingual, than to bodily movements due to lack of thirdorder bend control. these results are in line with a study conducted by saelens which concludes that the extraction group produced more retroclined teeth, which were generally caused by the use of intramaxillary elastic.26 there was a greater change in mandibular incisor inclination (l1-mp (°)) in the non-extraction group, where the mandibular incisor inclination became more anterior. similarly, saelens reported that teeth inclination was more proclined in the non-extraction group, especially on mandibular incisors. this result may be due to the use of elastic class ii.26 the position of the central mandibular incisor l1-mp (mm) in the two groups was also significantly different, possibly due to the relatively protracted use of class ii elastic in the non-extraction cases. from this present study, it can be concluded that there was not a significant increase in changes to occlusal plane inclination treated with non-extraction in skeletal class ii malocclusion. changes in maxillary central incisor inclinations and vertical position of the mandibular first molar have a significant correlation to changes to occlusal plane inclination before and after non-extraction treatment. occlusal plane inclination increased significantly in the group treated with extraction of the maxillary first premolar in skeletal class ii malocclusion. changes in position and angulation of the molar and incisor are not correlated with changes in occlusal plane inclination after orthodontic treatment with maxillary first premolar extraction. the increase in occlusal plane inclination was greater in the extraction group compared to the non-extraction group. there were no significant differences in the changes to occlusal plane inclination between the two groups. further studies are expected to be conducted with larger sample sizes. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 139yohana et al./dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 133–139 references 1. aslam a, naeem a, jan h, bukhari ga, abbas q, amjad m. prevalence of class ii malocclusions in pakistani sample a study. pakistan oral dent j. 2010; 30: 96–100. 2. kapoor d, garg d. cephalometric characteristics of class ii division 1 malocclusion in a population living in the chitwan distric of nepal. int j contemp med res. 2017; 4(4): 947–9. 3. sivaraj a. essentials of orthodontics. new delhi: jaypee brother medical publishers; 2013. p. 83–100. 4. sridharan k, udupa v, srinivas h, kumar s, sandbhor s. prevalence of class ii malocclusion in tumkur population. j dent scinces res. 2011; 2(2): 14–7. 5. ardani igaw, anandamaya d, alida. the relationship between skeletal and dental characteristics in patients with class ii malocclusion. j int dent med res. 2019; 12(4): 1421–5. 6. premkumar s. textbook of orthodontics. new delhi: elsevier; 2015. p. 265–98. 7. bishara se. class ii malocclusions: diagnostic and clinical considerations with and without treatment. semin orthod. 2006; 12: 11–24. 8. hassan r, rahimah ak. occlusion, malocclusion and method of measurements-an overview. arch orofac sci. 2007; 2: 3–9. 9. xuan j, bing l, li s-f, ma y-n, kwon t-g, wu x-p. morphological characteristics of soft tissue profile of angle’s class ii division i malocclusion before and after orthodontic treatment. int j morphol. 2018; 36: 26–30. 10. hosseinzadeh-nik t, eftekhari a, shahroudi as, kharrazifard mj. changes of the mandible after orthodontic treatment with and without extraction of four premolars. j dent (tehran). 2016; 13(3): 199–206. 11. konstantonis d, vasileiou d, papageorgiou sn, eliades t. soft tissue changes following extraction vs. nonextraction orthodontic fixed appliance treatment: a systematic review and meta-analysis. eur j oral sci. 2018; 126(3): 167–79. 12. bowman sj, johnston le. the esthetic impact of extraction and nonextraction treatments on caucasian patients. angle orthod. 2000; 70: 3–10. 13. vukušić n, lapter m, muretić ž. change in the inclination of the occlusal plane during craniofacial growth and development. coll antropol. 2000; 24: 145–50. 14. ogawa t, koyano k, suetsugu t. characteristics of masticatory movement in relation to inclination of occlusal plane. j oral rehabil. 1997; 24(9): 652–7. 15. jacobson a, jacobson rl. radiographic cephalometry: from basics to 3-d imaging. canada: quintessence publishing; 2006. p. 63–151. 16. li j, kau ch, wang m. changes of occlusal plane inclination after orthodontic treatment in different dentoskeletal frames. prog orthod. 2014; 15: 41. 17. thayer ta. effects of functional versus bisected occlusal planes on the wits appraisal. am j orthod dentofac orthop. 1990; 97(5): 422–6. 18. zenab ny, hambali ts, salim j, mardiati e. changes of occlusal plane inclination after orthodontic treatment with four premolars ext raction in dento -a lveola r bima x il la r y prot r usion cases. padjadjaran j dent. 2009; 21(2): 100–3. 19. zimmer b, nischwitz d. therapeutic changes in the occlusal plane inclination using intermaxillary elastics. j orofac orthop. 2012; 73(5): 377–86. 20. lemos ad, katz crt, heimer mv, rosenblatt a. mandibular asymmetry: a proposal of radiographic analysis with public domain software. dental press j orthod. 2014; 19(3): 52–8. 21. kawamura j, park jh, kojima y, kook ya, kyung hm, chae jm. biomechanical analysis for total mesialization of the mandibular dentition: a finite element study. orthod craniofacial res. 2019; 22(4): 329–36. 22. janson g, sathler r, fernandes tmf, branco ncc, de freitas mr. correction of class ii malocclusion with class ii elastics: a systematic review. am j orthod dentofac orthop. 2013; 143(3): 383–92. 23. braun s, legan hl. changes in occlusion related to the cant of the occlusal plane. am j orthod dentofacial orthop. 1997; 111(2): 184–8. 24. suresh r, priya k. orthodontic camouflage treatment of skeletal class ii malocclusion with severe maxillary dentoalveolar protrusion. j pierre fauchard acad (india sect. 2013; 27(4): 118–23. 25. singh v, pokharel p, pariekh k, roy d, singla a, biswas k. elastics in orthodontics: a review. heal renaiss. 1970; 10(1): 49–56. 26. demir a, uysal t, sari z, basciftci fa. effects of camouflage treatment on dentofacial structures in class ii division 1 mandibular retrognathic patients. eur j orthod. 2005; 27(5): 524–31. 27. elih, thahar b, soemantri ess, rasyid hn. evaluation of dento facial vertical dimension in class ii division 1 malocclusion after premolar extraction. int j sci res. 2016; 5(6): 1396–9. 28. lamarque s. the importance of occlusal plane control during orthodontic mechanotherapy. am j orthod dentofac orthop. 1995; 107(5): 548–58. 29. fushima k, kitamura y, mita h, sato s, suzuki y, kim yh. significance of the cant of the posterior occlusal plane in class ii division 1 malocclusions. eur j orthod. 1996; 18(1): 27–40. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p133–139 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p133-139 62 vol. 43. no. 2 june 2010 research report optimum dose of 2-hydroxyethyl methacrylate based bonding material on pulp cells toxicity widya saraswati department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: 2-hydroxyethyl methacrylate (hema), one type of resins commonly used as bonding base material, is commonly used due to its advantageous chemical characteristics. several preliminary studies indicated that resin is a material capable to induce damage in dentin-pulp complex. it is necessary to perform further investigation related with its biological safety for hard and soft tissues in oral cavity. purpose: the author performed an in vitro test to find optimum dose of hema resin monomer that may induce toxicity in pulp fibroblast cells. method: the method of this study was experimental laboratory with post test control group design. primary cell culture was made from dental pulp fibroblast cells, and was given with hema resin bonding material in various concentrations (5 µg/ml–2560 µg/ml), and then subjected to toxicity test (mtt assay). result: hema optimum concentration was 320 µg/ml to induce cytotoxicity in pulp fibroblast cells. conclusion: the used of hema base bonding material with the concentration of 200 µg/ml may induced pulp fibroblas cell toxicity. key words: citotoxicity, hema, resin, bonding material, fibroblast cells abstrak latar belakang: keberhasilan suatu bahan bonding secara klinis tergantung pada kandungan fisik, kimia dan keamanan secara biologis. hema (2-hydroxyethyl methacrylate) adalah bahan resin yang paling banyak digunakan karena memiliki sifat fisik-kimia yang baik. beberapa penelitian pendahuluan menyebutkan bahwa resin merupakan bahan yang mampu menyebabkan gangguan pada kompleks dentin pulpa sehingga perlu dilakukan penelitian lebih lanjut menyangkut segi keamanan secara biologis bagi jaringan keras dan jaringan lunak di rongga mulut. tujuan: penelitian ini akan menguji secara in vitro (pada kultur sel fibroblas pulpa gigi) untuk mengetahui dosis optimal monomer resin hema yang dapat menyebabkan toksisitas pada sel fibroblas pulpa. metode: metode penelitian ini adalah eksperimental laboratoris dengan rancangan penelitian post test control group design. kultur sel primer dibuat dari sel fibroblas pulpa gigi, dan diberi bahan bonding resin hema dengan berbagai konsentrasi (5 µg/ml–2560 µg/ml) kemudian dilakukan uji toksisitas (mtt assay) hasil: didapatkan konsentrasi optimal hema adalah 320µg/ml untuk dapat menginduksi terjadinya sitotoksisitas pada sel fibroblas pulpa. kesimpulan: penggunaan bahan dasar bonding hema dengan konsentrasi mulai 320 µg/ml dapat menyebabkan toksisitas pada sel fibrosis pulpa. kata kunci: sitotoksisitas, hema, resin, bahan bonding, sel fibroblas correspondence: widya saraswati, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend.jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: widya.saraswati@yahoo.com introduction bonding material, one of the materials commonly applied in dentistry, particularly in the field of dental conservation, has an important role. the application of adhesive resin material in dentistry may enforce the adherence retention to the teeth and reduce micro leakage.1 bonding materials marketed in public are mostly using resin 63saraswati: optimum dose of 2-hydroxyethyl methacrylate as the basic material. resin monomer commonly used as the primary composition of bonding material is 2-hydroxyethyl dimethacrylate (hema).2 hema (figure 1) is known to have satisfactory biocompatibility as bonding base material. maximum binding strength may also be obtained from this material base.2 hema has several advantages, such as relatively easy production, relatively longer endurance since it is generally added with preservative substances such as hydroquinone and bytylated hidroxytoluene (bht), relatively lower viscosity, and good physical as well as chemical characteristics.3,4 hema is regarded to have capacity to wet (wettability) dentin surface since hema has a character as a hydrophilic humidifier material.2 hema is a resin that combines composite restoration material with tooth, both in enamel and dentin. the hema group has a function either as hydrophilic or hydrophobic group.2 hydrophilic means possessing a predisposition to have strong binding with water or, in other words, having capability to wet the dentin surface. hydrophobic indicate a predisposition not to bind with water. hydrophilic group adheres to the dental dentin, while the hydrophobics adhere to upperlying composite resin.5 ch3 h2c = c c = o och2ch2oh figure �. molecular structure of hema (c6h10o3). 14 several authors remark that hema is ideal as dentin bonding base material due to its reliable penetration into demineralized dentin.4 it was also reported that hema had good adherence to caries dentin.5 in addition to its ideal characteristics as dentin bonding material, several studies also reported that hema have potential to kill cells in vitro.6 one requirement of material in dentistry, particularly when the material is going to be applied in oral mouth, is that it should be biocompatible, implying that it can be accepted by the body, non-toxic, no irrigative, not carcinogenic, and not inducing allergic reactions.2 toxicity test is conducted to find whether the material can be accepted by the tissue, containing systemic response-inducing substances when it is diffused and absorbed into circulatory system, free from sensitizing agents that may induce allergy, and not carcinogenic.2 toxicity test of a material is related to the viability of living cells. quantitative determination of viable cells can be performed using mtt assay.7 the frequently employed toxicity test is using in vitro method by means of cell culture.7 the source of cultured material is generally a primary cell culture, a culture from materials directly taken from animals or humans, which may present as organs, tissues, or cells. cell culture is the growth of cells within a media after being removed from an organ or tissue. primary cell culture has a short life. therefore, subculture can be performed only several times and thereafter the cells will die.11 compared to cell lines, primary cell culture is more sensitive against the toxicity of any chemical materials. cell lines has an advantage of being able for 50–70 times passage, having rapid cell growth, maintained cell integrity, and able to multiply in suspension.11 one of cell types that can be used for material toxicity test in dentistry is the pulp fibroblast cells. the cells are predominant in dental connective tissue, having a shape like spindle with oval nuclei and a long cytoplasmic process. fibroblasts are cells that mostly found in the pulp and they originate from undeveloped mesenchymal pulp cells or from the part of fibroblast. the cells are present in the whole pulp, but they tend to be present in cell-dense area, particularly in the corona. fibroblasts may produce denticle and are able to develop for replacing dead odontoblasts to create reparative dentin.8 the toxicity test can be applied as the early stage of a series of deeper studies on resin monomer, the basic of bonding material, and can be used as the preliminary test on biocompatibility of resin monomer material. therefore, it is necessary to carry out cytotoxicity test on the basic material of dentin bonding, the hema monomer, to identify the optimum concentration that may lead to cell death. it was expected that it can be applied as a consideration to choose safe bonding material for the patient. materials and methods this was a laboratory experimental study using post test only controlled group design. samples in this study were pulp fibroblast cells isolated from the pulp of intactextracted third molar. this study was performed in lembaga pengembangan dan penelitian terpadu (lppt) universitas gadjah mada yogyakarta. material used in this study was pulp fibroblast culture. cells were isolated from intactextracted third molar. the resin was pure hema (sigma) in concentration of 95%. pulp fibroblast cell was isolated from pulp tissue of the third molar through odontectomy operation. the pulp tissue was put on petri dish and added with 10% rosewellpark memorial institute-1640 (rpmi-1640) media in a volume of 2 ml, then penicillinstreptomycin and gentamycin were added. cell culture was kept within 5% cc>2 incubator in 37° c for 4 days. media replacement (followed with penicillin-streptomycin and gentamycin administration) was done every 4 days. after the cells became confluent, tripsination and centrifugation were performed, and divided into several flasks (cellnumber about 800,000–1 million in one flask). cells were put within 96 culture wells, each containing 2 ml solution, with hema concentrations in 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560 ug/ml. control group was media culture given with stop solution without the addition of hema. samples were incubated in coa incubator in 64 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 62-66 37 degree c for 24 hours. subsequently, toxicity test was carried out to the samples to obtain lethal concentration (lc) of cell culture that had been given with hema monomer in various concentrations. toxicity test was performed according to standard procedure for mtt assay. prior to toxicity test, the samples were counted and entered into plate as many as 2×104 per well. starvation was done for 2–4 hours in 37° c, and then fetal bovine serum (fbs) was added in concentration of 0.5%. the media were replaced and added with fbs solution in 10% concentration. hema was added in concentration of 5–2560 ug/ml. the sample was incubated for 24 hours with cc>2 incubator in 37° c. the reactant mtt {3-(4,5 dimethylthiazol-2yl) 2-5 diphenyltetrazolin bromide} was added as much as 5 mg/ml in phosphat buffer saline (pbs) as much as 20 ul in each well and re-incubated for 4-6 hours in 37° c. furthermore, stop solution (sds) was added in each well and incubated overnight (12–18 hours). reading was done with elisa reader (wavelength of 550 nm). the result was presented in opticaldensity/absorbent and the magnitude of absorbent in each well indicated viable cell count in media culture. result toxicity levels measurable from the application of hema bonding material in fibroblast cell culture can be seen in table 1. prior to performing toxicity comparative test between hema monomer concentration groups, we performed data distribution test using kolmogorovsmirnov test, and followed by variance homogeneity test with levene test. data distribution test with kolmogorov smirnov revealed p>0.05, indicating that sample groups in various hema concentrations had normal data distribution. data homogeneity test using levene statistical test revealed p<0.05, indicating that in sample groups (hema and control) the variance was homogeneous. to analyze the difference of hema concentration in one group, we performed one-way anova statistical test, which revealed p=0.374 (p<0.05), indicating the influence of different concentration on cell death between cell culture groups receiving hema in concentrations varied from 5 mg/ml to 2560 µg/ml. subsequently, tukey's hsd test was performed to identify the different group. the result of tukey's hsd test revealed p<0.05, indicating significant difference between sample groups, showing that hema administration had effect on pulp fibroblast cells apoptosis. discussion the use of resin monomer as bonding material has been significant in dentistry. a reliability of dental material cannot be evaluated from their physical and chemical characteristics only, but its biological safety also becomes a primary consideration.13 it is reported that resin monomer may induce various biological damages.14 it was also disclosed that there are cellular metabolism alterations due to induction of resin material.15 resin monomer with imperfect polymerization may produce reactive chemical compounds that impairs cell balance within dental tissues.16 toxicity test using mtt assay in this study used samples of pulp fibroblast culture provided with resin material 2 hydroxyethyl dimethacrylate (hema) in various concentrations, which was determined serially from 5 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 180 mg/ml, 320 mg/ml, 640 mg/ml, 1280 mg/ml and 2560 mg/ml. statistical test using one-way anova with significance of 5% indicated that hema addition concentrations from 5 mg/ml–180 mg/ml did not induce toxicity in pulp fibroblast cell culture, since hema concentrations in that range did not influence cluster function and structure that may result in toxicity. this study shows that in a concentration as much as 320 mg/ml, the comparison between living and dead cells was each 50%. mtt assay was performed based on the capability of living cells to reduce mtt salt. the principle of this examination was to breakdown mtt tetrazolium ring (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide) that has a yellow color due to dehydrogenation in active mitochondria and produces insoluble purplish blue formazan crystal.8 reduction mechanism of yellow tetrazolium salt occurs in cells with metabolism activity. in this mechanism, mitochondria in viable cells have an important role in producing dehydrogenation. when the dehydrogenation process does not take place actively due to cytotoxic effect, formazan may not be formed. formazan production can be measured by solving it and the optic density of produced solution is assessed. the lower the optic density percentage, the lower the number of active metabolic cells that are able to reduce mtt. detected cell count from mtt reduction can be measured using spectrophotometer or elisa reader. the darker the color, the higher the absorption value, indicating that the number of living cells are also higher.12 this toxicity test method showed difference in living and dead cells due to damaged cell wall marked by the entry of tryphan blue into the cells. living cells is round and have bright color, while dead cells look enlarged and have blue color before they finally break down.11 one-way anova statistical test with significance level of 50% demonstrates that increased concentration affects formazan crystal optic density. whereas, the result of tukey's hsd value in this study showed the effect of different concentrations on cell death between cell cultures receiving hema in concentrations varied between 5 mg/ml to 2560 mg/ml. in the addition of hema monomer in concentrations of 640 mg/ml, 320 mg/ml, 160 mg/ml and 80 mg/ml. significant difference was found with p<0.05. the effect of concentration on toxicity indicates that the higher the hema monomer concentration, the higher the toxicity. a study by thimbrell12 revealed that all materials are toxic, but it is the difference of concentration level in one material that differentiates the degree of toxicity of that 65saraswati: optimum dose of 2-hydroxyethyl methacrylate material. toxicity test in this study showed the occurrence of optimum cell death with hema administration in a concentration of 320 mg/ml. the cause of the cell death may be the presence of free radicals possessed by the hema. hema has two hydroxyl groups in each of its carbon chains. hydroxyl group is a free radical that has potentials to damage cell membrane.17 cell membrane contains fatty acid elements that are responsive to free radicals. cell membrane damage may alter osmotic balance, which relates to the damage of protein, enzyme, co-enzyme and ribonucleic acid that induce cell death. therefore, when there is reaction of the fatty acid in membrane cell due to the presence of free radicals, the damage of function and structure of cell membrane will lead to cell death. table �. mean and standard deviation of pulp fibroblast cells in toxicity test with hema addition (mg/ml) monomer concentrations x sd hema 320 175.600 6.465 160 166.400 7.765 80 63.400 8.561 40 42.000 8.944 20 17.000 4.159 control 2.000 1.870 one of the requirements for a reliable material in dentistry is the presence of physical and biological safety.18 to obtain such reliability, one of the early actions that should be taken is testing material toxicity. based on the result of this study, it can be concluded that pure hema material (95%) in concentration of 320 mg/ml is an optimum concentration that may induce toxicity in pulp fibroblast cells. the result of this study can be used as the basis for further research on hema material, one of the materials applied in dentistry. references 1. mantellini mg, botera tm. adhesive resin induces apoptosis and cell-cycle arrest of pulp cells. j dent res 2003; 82(8): 592–6. 2. anusavice kj. phillip's science of dental materials. 11th ed. philadelphia, london, toronto: wb saunders co; 2003. p. 21–395. 3. pashley dh, zhang y, agee ka, rouse cj, carvalho rm, russel cm. permeability of demineralized dentin to hema. dent mater 2000; 16: 7–14. 4. craig rg, powers jm. restorative dental materials. 11th ed. st louis: mosby co; 2002. p. 330–40. 5. summit jb, rubbins jw, schwartz rs. fundamentals of operative dentistry. a contemporary approach. 2nd ed. illinois: quintessence publishing co inc; 2001. p. 178–81. 6. kitamura c, ogawa y, morotomi t, terashita m. differential induction of apoptosis by capping agents during pulp wound healing. journal of endodontic 2006; 29(1):41–3. 7. walton re, torabinejad m. prinsip dan praktek ilmu endodonsi. edisi 2. jakarta: egc; 1997. p. 360–78. 8. mc cabe jf. applied dental material. 7th ed. london: four dragons edition, blackwell scientific publications; 1992. p. 231–3. 9. soeprapto m. kultur jaringan (hewan) (tissue culture). surabaya:surabaya: program pascasarjana universitas airlangga; 1999. p. 15–63 10. freshney ri. culture of animal cells. a manual of basic technique. 5th ed. new york. willey -liss; 2005. p. 366–81. 11. telli c, serper a, dogan al, guc d. evaluation of the cytotoxicity of calcium phosphate root canal sealer by mtt. j endodontic 1999; 25: 811-3. 12. timbrell aj. principles of biochemical toxicology. 2nd ed. london: taylor & francis ltd; 1994. p. 33–5, 216–27. 13. hume wr, wang my. resin monomer 2-hydroxyethyl methacrylate (hema) is potent inducer of apoptotic cell death in human. 2002. j dent res 2005; 84(2): 172–7. figure ��. pulp fibroblast primary cell culture before treatment. note: (-->) confluent pulp fibroblast cells figure ��. profile of pulp fibroblast cell culture after hema administration in 320 mg/ml, cells appear to be round with bluish nuclei. figure �. profile of pulp fibroblast cell culture after hema administration in 1280 mg/ml, cells appear to be irregular with the absence of nuclei. 66 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 62-66 14. rune b, hilde mk, ronald h, samuelsen jt, else m, dahlman hj, lilleaas em, jon ed. pattern of cell death in vitro exposure to gdma tegdma, hema and two compomer extract. j den mat 2006; 22: 630–45. 15. chang hh, guo mk, kasten fh, chang mc, huang gf, wang yl. stimulation of gluthathione depletion, ros production and cell cycle arrest of dental pulp cell and gingival epithelial cell by hema. j biomat 2004; 26: 745–53. 16. noort rv. introduction to dental materials. 2nd ed. cv edinburgh, london, new york, oxford: mosby co; 2002. p. 11–78. 17. suryohusodo p. kapita selekta ilmu kedokteran molekuler. cetakan ke-2. surabaya: cv agung seto; 2007. p. 31–7. 18. baum l, philips rw, lund mr. buku ajar ilmu konservasi gigi. edisi ke-3. tarigan r, editor. jakarta: egc; 1997. mkgs vol 44 no 1 jan-mar 2011.indd 12 vol. 44. no. 1 march 2011 case report calcium hydroxide as intracanal dressing for teeth with apical periodontitis sari dewiyani department of conservative dentistry faculty of dentistry prof. dr. moestopo (b) university surabaya indonesia abstract background: root canal infection and periapical diseases are caused by bacteria and their products. long term infection may spread bacteria throughout the root canal system. apical periodontitis caused by infectious microbe that persistent in root canals can cause radiographic and histopathology periapical changes. chemomechanical preparation and intracanal dressing then are recommended to be conducted and used in between visits to eliminate microbes in root canals. calcium hydroxide (ca(oh)2) can be used as intracanal dressing since it can be used as musical physical defense barrier to eliminate re-infection in root canal and to disturb nutrition supply for bacterial development. purpose: the aim of this study is observe the effectiveness of ca(oh)2 in treating endodontic teeth with apical periodontitis. cases: case 1 and 3 are about patients whose left posterior mandibular teeth had spontaneous intermittent pain. case 2 is about a patient whose left posterior maxillary teeth had gingival abscess and fracture history. based on the radiographic examination, it was known that the filling of root canal was incomplete and there was radiolucency in the apical area. case management: the cases were treated with triad endodontics, which involves preparation, disinfection by using 2.5% naocl as irrigation substance and calcium hydroxide as intracanal dressing, and then the filling of root canal with gutta percha and endomethasone root canal cement. evaluations were conducted one month, 12 months, and 24 months after the treatment. conclusion: calcium hydroxide is effective to be used as intracanal dressing in apical periodontitis cases. key words: apical periodontitis, triad endodontic, intracanal dressing, calcium hydroxide abstrak latar belakang: infeksi saluran akar dan penyakit periapeks disebabkan oleh mikroba dan produknya. infeksi yang berlangsung lama memungkinkan bakteri masuk ke dalam seluruh sistem saluran akar. periodontitis apikal disebabkan oleh infeksi persisten mikroba di dalam sistem saluran akar disertai perubahan radiografik dan histopatologik periapeks. preparasi kemomekanis dan penggunaan obat saluran akar antar kunjungan sangat dianjurkan untuk mengeleminasi mikroba dalam saluran akar. kalsium hidroksida merupakan obat saluran akar karena dapat berperan sebagai barrier pertahanan fisik, mencegah infeksi ulang dan mengganggu suplai nutrisi untuk perkembangan bakteri. tujuan: untuk membuktikan efektifitas kalsium hidroksida pada perawatan gigi dengan periodontitis apical yang disertai dengan keluhan sakit dan adanya gambaran radiolusensi di periapikal. kasus: kasus 1 dan 3 adalah penderita dengan keluhan gigi belakang bawah kiri sakit dengan nyeri spontan hilang timbul, kasus 2 adalah penderita yang gigi depan atas terdapat benjolan di gusi dan riwayat fraktur. pada pemeriksaan radiografis terlihat pengisian saluran akar yang tidak sempurna dan gambaran radiolusensi di apikal. tatalaksana kasus: pada kasus ini dilakukan tindakan triad endodontik, yaitu preparasi saluran akar, desinfeksi dengan larutan irigasi naocl 2,5% dan obat intrakanal kalsium hidroksida disertai dengan pengisian saluran akar dengan gutta percha dan semen saluran akar endomethason. evaluasi dilakukan 1 bulan, 12 bulan, dan 24 bulan. kesimpulan: kalsium hidroksida efektif sebagai obat intrakanal pada kasus periodontitis apikal. kata kunci: periodontitis apikal, triad endodontik, obat intrakanal, kalsium hidroksida correspondence: sari dewiyani, bagian konservasi gigi, fakultas kedokteran gigi universitas prof. dr. moestopo (b). jl. bintaro permai raya 3 jakarta 12330, indonesia. e-mail: sari.drg@gmail.com 13dewiyani: the use of calcium hydroxide as intracanal dressing introduction root canal infections and periapical diseases are actually caused by microbes and their products. long term infection can not only make bacteria spread through root canal system, either within ramifications, isthmus, or dentin tubules, but also can make microbes remain alive through chemomechanical preparation.1 it means that trough chemomechanical preparation can reduce the number of microbes inside the root canal, about 50-70%, the microbes can still survive, especially in the dentin tubules. thus, the use of root canal dressing in between visits is highly recommended in order not only to prevent microbes from breeding, but also to kill them simultaneously inside the root canal.2,3 apical periodontitis is a periapical disease caused by persistent microbiology in root canals followed with radiographic and histopathology periapical changes which can cause bone damage.4 according to another expert’s opinion, apical periodontitis is periapical defense response that can be treated by localizing the infection in order not only to prevent microbes from spreading, but also to eliminate microbes from necrotic root canal caused by microorganisms and their biofilm.3,5 calcium hydroxide, furthermore, can be used as a intracanal dressing since it has the ability to kill bacteria.6 it is because of the constant releasing of hydroxyl ions which can inhibit microbial lipopolysaccharide, as a result, it can reduce inflammatory process, dissolve the remaining necrotic tissue, and make an acidic environment become alkali that can stimulate the growth of bone.7 therefore, calcium hydroxide as intracanal dressing has alkaline character which is considered as effective disinfectants and has useful osteogenic ability to heal periapical tissue.8,9 for those reasons, the purpose of this treatment is to prove the effectiveness of calcium hydroxide as intracanal dressing on teeth with apical periodontitis. case case 1: a 65 year old male patient came with complaint of spontaneous intermittent left posterior mandibular tooth pain suffered since one month before. actually, the tooth was treated six months before. the pain sometimes also occurred on tooth next to it. based on clinical examination, it was known that tooth 35 was restored with porcelain crown, and not sensitive with thermal and palpation tests, but still sensitive with percussion test. based on radiographic examination, it was also known that the root canal of tooth 35 restored with porcelain crown was filled with gutta percha, but not in accordance with its length. furthermore, it was known that the periodontium was widened; lamina dura was thickened; and there was no radiolucency in the apical area. based on diagnosis, it was known that tooth 35 was pulpless with chronic apical periodontitis. case 2: a 27 year old male patient came with complaint of intermittent anterior maxillary tooth pain. the patient also complained about gingival abscess which tasted salty. moreover, it was knownn that the tooth was fractured ten years before because of motorcycle accident, and then was treated in medan, but not completed. four years ago, the tooth then got dental crown restoration, but it still has been painful until now. based on clinical examination, it was known that there was dental fistula in the gingival/buccal mucosa of tooth 21. it was also known that the tooth did not react with thermal tests, but still reacted with percussion and palpation tests. based on radiographic examination, it was known that dental caries in tooth 21 had already reached the pulp; the periodontium was widened; lamina dura was thick and broken; and there was radiolucency in periapical area. based on diagnosis, it was known that tooth 21 got chronic apical periodontitis with acute exacerbation from pulp necrosis. on the other hand, the dental crown of tooth 22 was acrylic. moreover, it did not react with thermal and palpation tests, but still reacted with percussion test. based on the radiographic examination, it was known that dental caries of tooth 22 had already reached the pulp; periodontium widened; lamina dura was thick and broken; and there was also radiolucency in the periapical area. based on diagnosis it was known that tooth 22 got chronic apical periodontitis because of untreated pulp necrosis. case 3: a 61 year old male patient came with complaint of spontaneous intermittent left lower back tooth pain suffered since 2 weeks ago sick. moreover, it was known that the tooth was treated 5 years before. it was also known that the crown of tooth next to it was lost, but without any complaints. thus, the patient had his teeth treated. based on clinical examination, furthermore, it was known that teeth 34, 35 had already got porcelain crown restorations, and did not react with thermal and palpation tests, but still reacted with percussion test. besides that based on radiographic examination, it was known that tooth 34 got post-core restoration with porcelain crown. it was also known that the periodontium was widened; lamina dura was lost; and there was no radiolucency in periradicular area. based on diagnosis, finally, it was known that tooth 34 got chronic apical periodontitis because of pulp necrosis. on the other hand, based on radiographic examination, it was known that the root canal of tooth 35 was filled less than its length; periodontium widened; lamina dura was thick and broken; and there was no radiolucency in its periapical area. based on diagnosis, finally, tooth 35 no pulp tissue with chronic apical periodontitis. therefore, tooth 35 needed root canal re-treatment with post-core restoration by using porcelain crown. case management case 1: based on the case management procedures, the root canal retreatment of tooth 35 was conducted with 14 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 12–16 porcelain crown restoration (figure 1a). from the first to the third visit, the porcelain crown was demolished, and the root canal preparation was also conducted, in which it was irrigated with 2.5% naocl, dried, then filled with calcium hydroxide, and finally casted temporarily. a week later, the measurement of working length was conducted with x-ray images; master apical cone (mac) 30/25 was also determined; step-back was prepared and irrigated with 2.5% naocl; then the root canal was dried and filled with intracanal dressing, calcium hydroxide, and finally the cavity was closed with a temporary cast. in the fourth visit, moreover, the root canals of tooth 35 were filled. besides that, it was known that dental caries on oclusal and proximal next to distal of tooth 34 reacted with thermal and percussion tests, but not reacted with palpation test. based on the radiographic image of tooth 34, furthermore, it was also known that dental caries had already reached to the pulp chamber, and periodontium was dilated, but the lamina dura was still normal. therefore, tooth 34 was diagnosed with chronic dental pulpitis. based on the case management procedure of tooth 34, root canal treatment was conducted with postcore restoration by using porcelain crown (figure 1a). in the first visit, the root canal of tooth 34 was prepared and filled with calcium hydroxide as intracanal dressing. in the second visit, the root canal of tooth 3.4 was filled with gutta percha and root canal cement. after the root canal of both teeth was filled, in the third visit teeth 34 and 35 was casted with porcelain crowns. the re-examination was then conducted 2 years later in which it was known that there was no complaint of pain; it also did not reacted with percussion and palpation test; and finally there was also no radiolucency in periradicular area (figures 1a, b, and c). case 2: based on the case management procedures, the root canal treatment of tooth 21 was conducted with prophylactic post restoration with composite resin cast. from the first visit to the fourth one, the root canal preparation of tooth 21 was conducted with if 45/21 mm. figure 1. case 1 teeth 34, 35. a) in the first visit, b) 6 months after treatment, c) 2 years after treatment. a b c figure 2. case 2 teeth 21, 22. a) one month after treatment, b) 6 months after treatment, c) 2 years after treatment. cba figure 3. case 3 teeth 34, 35. a) in the first visit, b) one year after treatment, c) 2 years after treatment. cba 15dewiyani: the use of calcium hydroxide as intracanal dressing it then was irrigated with 2.5% naocl, dried, and finally filled with calcium hydroxide as intracanal dressing. after that, the cavity was casted with temporary cast. in the next visit, based on x-ray images, mac was 60/21 mm, and it was irrigated with 2.5% naocl, dried, and filled with calcium hydroxide medicine. afterwards, the cavity was covered with temporary cast. in the fifth visit, finally, the root canal of tooth 21 was filled with gutta percha and root canal cement. on the other hand, the root canal treatment was also conducted on tooth 22 with post-core restoration by using porcelain crown (figure 2a). in the first visit, the root canal preparation of tooth 22 was conducted. then, unlike the root canal of tooth 21 which got prophylactic preparation and composite cast, the root canal of tooth 22 was irrigated with 2.5% naocl, dried, filled with calcium hydroxide, and finally casted with temporary cast. in the third visit, the root canal of tooth 22 then was filled with mac 35/24 mm. in the next visit, the porcelain crown insertions of teeth 21 and 22 were conducted. in the control visit of teeth 21, 22, two months after the treatment, there were no reaction with percussion and palpation tests, and there was also no subjective complaint. finally, the re-examination of teeth 21, 22 was conducted two years later in which it was known that there was no subjective complaints; it did not react with percussion and palpation tests; and the image of radiolucency in the apex area was smaller. (figure 2-a, b, and c). case 3: based on the case management procedure of tooth 34, the root canal treatment was conducted with postcore restoration by using porcelain crown. from the first to the fourth visit, the previous post-core crown restoration of the tooth 34 was demolished in order not only to prepare access for root canal preparation, but also to be irrigated with 2.5% naocl, then dried and filled with intracanal calcium hydroxide dressing, and temporarily casted. in the next visit, the measurement of working length was conducted with x-ray images. mac the was determined, 30/18 mm, and step back was conducted and irrigated with 2.5% naocl, then dried, and filled with calcium hydroxide as intracanal medicine. afterwards, the cavity was finally closed with a temporary cast. the root canal of tooth 34 then was filled with root canal sealer and gutta percha. in the next visit, the preparation was conducted on tooth 34 in order to make post-core. in the first control visit, the prophylactic post of the tooth 3 was demolished in order to repair root canal preparation through irrigation with 2.5% naocl, and filled with calcium hydroxide as intracanal medicine. in the fourth control visit, the root canal of the tooth 35 was filled by using endomethasone root canal sealer. in the sixth visit, the teeth 34 and 35 were restored with post-core cast, by using porcelain crowns. in control visits conducted 1 and 2 years after the treatment, there was no subjective complaints; there were no reaction with percussion and palpation tests; and there was no radiolucency in periradicular area (tooth 34) (figure 3a, b, and c). discussion microorganisms can reach dental pulp through caries, inappropriate clinical procedures (leak restoration, excessive instrumentation, and chemical or endodontic material irritation), fracture, and traumatic occlusion. as a result, it can cause a continuous response followed with clinical symptoms, such as aches and pains. and, when inflammatory process almost attacks pulp-periapical junction tissue, periodontitis will occur in apical.10 apical periodontitis is a periapical defense response against pulp tissue damage because of microbial infection in root canal system which is aimed both to localize the infection in the root canal system and to prevent its spreading. although this defensive reaction can minimize the spread of infection, this reaction still cannot eliminate the microbes that infiltrated into the necrotic root canal because it is protected by biofilm.11 this apical periodontitis treatment can conventionally be conducted with endodontic treatment. the purpose of this treatment, moreover, is not only to eliminate or reduce microbial populations in the root canal system, but also to reduce and prevent re-infection by blocking the root canal. the combination of mechanical instruments, root canal irrigation, and anti-microbial drug application in root canal can reduce the number of microbes. calcium hydroxide as a intracanal dressing, furthermore, can create a good environment for healing the periapical tissues, stimulating the hard tissue formation, eliminating exudation in the periapical area, and dissolving necrotic tissue.12 the success of this pulp treatment, however, is determined by the examination and ability to eliminate causative factor and to cut infected tissue so that biological recovery process on the tissue can occur.12,13 in addition, diagnosis in case 1, 2 and 3 were established based on the patients’ complaints and their radiographic images. their complaints of intermittent pain involving discomfort feeling, severe pain in those teeth, and infection spreading into the periapical tissue. the pain can also possibly caused by the filling of the root canal that is not appropriate with the working length (case 1, 3). besides that, the use of improper instruments in root canal because of wrong measurement can also push dentin debris, irrigation solutions, toxic components of necrotic tissue into the periapical, and the excessive use of intracanal dressing (in case 2). for example, necrotic tissue still remained in one-third apical of the root canal (in case 1 and 3), and the use of inappropriate mechanical instruments and intracanal medicine causing irritations (case 2) become the focus of infection that causes the inflammation of periapical, which is apical periodontitis.14 the root canal re-treatment was then conducted in those three cases which aims not only to reduce or eliminate microbes in the root canal system, but also to prevent reinfection. the re-use of calcium hydroxide, thus, is essential in the process of mineralization and its antimicrobial effect. it is because the increasing of concentration caused by 16 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 12–16 calcium hydroxide can initiate the mineralization process since high concentration with a combination of the ability of ca2+ ions and ohcan cause an effect on the metabolic enzymes and mineralization processes. therefore, it means that calcium hydroxide can increase the metabolic processes of the periapical tissue, including collagen synthesis, glicosaminoglicans, and protein synthesis.15 furthermore, another reason of using calcium hydroxide as intracanal medicine is because it has qualities in both hydrolyzing some fats derived from bacterial lipopolysaccharide and eliminating its biological action.7 calcium hydroxide is also used to control the exudate (in case 2) with persistent periapical abnormalities. the high concentration of ca ions then can cause pre-capillary contraction so that the flowing of blood to capillaries is reduced. this condition then can affect the reduction of fluid amount that comes out of plasma into the tissue as a result of inflammatory reaction. with the reduction of plasma fluid that comes out, the tissue can possibly get the healing and calcification processes.12 as a result, the healing process then occurred in case 1, 2 and 3, which was about two years after the root canal treatment. the filling of calcium hydroxide dressing into the pulp tissue can stimulate mesenchyme cells to form fibroblasts to arrange new odontoblast cells that can both replace the damaged cells and support the smooth vascularization. ca2 + ions in high concentrations will not only improve the role of pyrophosphatase enzyme, activate adenosine, but also activate triphosphatase so that they can force defense through dentin mineralization.16-18 it can be concluded that calcium hydroxide can effectively be used as intracanal medicine in apical periodontitis cases. references 1. walton re, riviera em. cleaning and shaping. in: walton re, torabinejad m, eds. principles and practice of endodontic. 3rd ed. philadelphia: wb saunders; 2002. p. 206–38. 2. schafer e, bossmann k. antimicrobial effifacy of chlorhexidine and two calcium hydroxide formulation against enterococcus faecalis. j endod 2004; 31: 53–6. 3. basrani b, pascon e, friedman s. effiacy of chlorhexidine and calcium hydroxide containing medicament against enterococcus faecalis in vitro. j oral surg oral med oral path 2003; 96: 618–2. 4. jiang j, zoo j, chen sh, holliday ls. calcium hydroxide reduces lipopolysaccharide stimulated osteclast formation. oral surg oral med oral pathol 2003; 95: 348–54. 5. ingle jl, simon jh. endodontic. 5th ed. california: elsevier; 2002. p. 179–884. 6. podbielski a, spahr a, haller b. additive microbial activity of calcium hydroxide and chlorhexidine on common endodontic bacterial pathogens. j endod 2003; 29: 340–5. 7. cwikla s, bellanger m, giguere s, fox a, verticci f. dentinal tubules desinfection using thre calcium hydroxide formulation. j endod 2000; 31: 50–2. 8. o’brien, willian j. dental material and their selection. 3th ed. chicago: quintessence publishing co, inc; 2004. p. 141. 9. weine fs. histophysiology and diseases of the dental pulp. endodontic therapy. 5th ed. toronto: mosby; 2004. p. 161–73. 10. amanda l, harrod m. an evidence base analysis of the antibacterial effectiveness intracanal medicaments. j endod 2004; 30: 689–94. 11. distel jw, hatton jf, gillerspie mj. biofilm formation in medicated root canals. j endod 2002; 28: 689–93. 12. akbar sms. perawatan endodontik. jakarta: fkg ui; 2009. p. 1–29. 13. teixeira, fabricio b. investigation of ph at different dentinal sites after placement of calcium hydroxide dressing by two method. oral surg oral med oral pathol endodontology 2005; 99–516. 14. hommez gm, giard jc, hartke a, flahaut s. the relation between apical periodontitis and root–filled teeth in patient with periodontal treatment need. j endod 2006; 39: 299–304. 15. jaunberzins a, gutmann jl, witherspoon de, harper hp. effects of calcium hydroxide and tumor growth factor–ß on colagen synthesis in subcultures i and of osteoblast. j endod 2000; 26: 494–9. 16. sidharta w. penggunaan kalsium hidroksida di bidang konservasi gigi. jkgui 2000; 7 (edisi khusus): 435–43. 17. berkitten m, okar i, berkitten r. in vitro of penetration of sanguish and prevotella intermedia strain into human dentinal tubulus. j endod 2000; 26: 236–9. 18. ferreira fb, vale ms, granjeirob jm. evaluation of ph levels and calcium ion release in various calcium hydroxide endodontic dressing. oral surg oral med oral pathol 2003; 97: 388–92. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 181 volume 47, number 4, december 2014 research report penambahan xylitol dalam glukosa, sukrosa terhadap pertumbuhan streptococcus mutans (in vitro) (the additional xylitol in glucose and sucrose on growth of mutans streptococci (in vitro) susilowati, udijanto tedjosasongko, dan fx suhariadji departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya-indonesia abstract background: xylitol is a sugar alcohol group consisting of five-carbon chain and the sugar substitutes are recommended to prevent caries. dietary sugars known as a good substrate for the growth of streptococcus mutans (s. mutans). two types of sugar, xylitol and dietary sugars have different effects on the growth of s.mutans. purpose: the objective of this study were to determine the minimal inhibitory concentration (mic) of xylitol on the growth of s.mutans and to determine the addition of xylitol in glucose and sucrose in the growth of s. mutans in vitro. methods: the samples were divided into 3 groups: xylitol group, xylitol and sucrose combination group, and xylitol and glucose combination group . in all groups were tested against s.mutans growth in various concentrations. results: the minimum inhibitory concentration against s.mutans xylitol was equal to 0.625%. the addition of xylitol in sucrose the inhibition of s.mutans growth occurred at concentrations of 0.625 % and 2.5%. the addition of xylitol in glucose inhibited the growth of s.mutans at all concentrations. conclusion: this study showed that the combination of xylitol with dietary sugars could inhibit the growth of s.mutans. key words: xylitol, glucose, sucrose, streptococcus mutans, dental caries abstrak latar belakang: xylitol adalah golongan gula alkohol yang terdiri dari lima rantai karbon dan merupakan sugar substitutes yang dianjurkan untuk mencegah terjadinya karies. dietary sugars diketahui sebagai substrat yang baik untuk pertumbuhan bakteri rongga mulut salah satunya streptococcus mutans (s. mutans). dua jenis gula yaitu xylitol dan dietary sugars memiliki pengaruh yang berbeda pada pertumbuhan s. mutans. tujuan: tujuan dari penelitian ini adalah meneliti konsentrasi hambat minimal (minimal inhibitory concentration/ mic) xylitol terhadap pertumbuhan s mutans dan meneliti pengaruh penambahan xylitol dalam glukosa dan dalam sukrosa terhadap pertumbuhan s. mutans secara in vitro. metode: sampel dibagi dalam 3 kelompok: kelompok xylitol, kelompok kombinasi xylitol dan sukrosa, dan kelompok kombinasi xylitol dan glukosa. pada ketiga kelompok tersebut dilakukan pengujian terhadap pertumbuhan s.mutans dalam berbagai konsentrasi. hasil: konsentrasi hambat minimum xylitol terhadap s. mutans adalah sebesar 0,625%. pada penambahan xylitol dalam sukrosa terjadi penghambatan s. mutans pada konsentrasi 0,625% dan 2,5%. pada penambahan xylitol dalam glukosa terjadi penghambatan s. mutans pada semua konsentrasi. simpulan: penelitian ini menunjukkan bahwa kombinasi xylitol dengan glukosa dan dengan sukrosa dapat menghambat pertumbuhan s.mutans. kata kunci: xylitol, glukosa, sukrosa, streptococcus mutans, karies gigi korespondensi (correspondence): udijanto tedjosasongko, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: udijanto@gmail.com 182 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 181–185 pendahuluan xylitol adalah golongan gula alkohol yang terdiri dari 5 rantai karbon yang banyak ditemukan pada beberapa tanaman, buah-buahan dan diproduksi dalam jumlah kecil di dalam tubuh manusia. seorang ahli kimia dari jerman bernama fischer adalah orang yang pertama kali menemukan jenis gula ini pada tanaman odorless putih. sejak tahun 1960an xylitol mulai dikenal luas sebagai terapi cairan pada pasien post operative, luka bakar, shock, diet terapi pada pasien diabetes millitus dan akhir-akhir ini diperkenalkan sebagai pemanis dalam beberapa produk yang dapat meningkatkan kesehatan rongga mulut, seperti dalam pasta gigi dan obat kumur.1 xylitol direkomendasikan oleh american academy of pediatric dentistry sebagai sugar substitutes untuk meningkatkan kesehatan rongga mulut pada anak-anak, dewasa, atau pasien dengan special health care. diet makanan merupakan salah satu faktor dalam terbentuknya karies gigi. peningkatan oral hygiene dan tindakan pencegahan seperti penggunaan fluoride, serta kontrol diet yang tepat merupakan beberapa strategi yang baik untuk mencegah karies gigi. penggunaan xylitol sebagai sugar substitutes dianjurkan sebagai tindakan pencegahan karies, namun penggunaan xylitol sepenuhnya dalam mengganti dietary sugars juga tidak dianjurkan. hal ini dikarenakan xylitol diserap lambat oleh lambung sehingga dalam pemakaian berlebih dapat menyebabkan gangguan pada saluran pencernaan.2 glukosa merupakan bagian utama diet penduduk di indonesia. selain sebagai makanan pokok, gula juga dikonsumsi sebagai makanan ringan atau camilan seperti yang terdapat dalam permen, wafer, kue, biskuit, dan dalam minuan ringan. manifestasi konsumsi sukrosa dalam kehidupan sehari-hari adalah dalam bentuk gula putih. konsumsi sukrosa dalam jumlah besar dapat menurunkan kapasitas buffer saliva sehingga mampu meningkatkan insiden terjadinya karies.3 bakteri rongga mulut seperti s. mutans sebagai salah satu penyebab karies gigi dalam pertumbuhannya sangat membutuhkan karbohidrat sebagai media pertumbuhannya. hasil fermentasi dari karbohidrat dapat menimbulkan suasana asam yang sangat membantu acidogenic mikroflora berkolonisasi pada permukaan gigi dan dengan lamanya waktu akan dapat menyebabkan karies gigi. makanan yang mengandung sukrosa, fruktosa, laktosa ataupun polisakarida yang lain kita konsumsi sebagai makanan sehari-hari atau biasa disebut sebagai dietary sugars termasuk jenis karbohidrat dengan dasar 6 rantai karbon. jenis karbohidrat ini diketahui sebagai subtrat yang baik dalam pertumbuhan bakteri rongga mulut. beberapa isu tentang diet mengatakan bahwa dietary sugars adalah yang bertanggung jawab dalam timbulnya karies.4 konsumsi makanan atau diet memang sangat berpengaruh pada efek kerja dari xylitol dalam mencegah karies. salah satu kemungkinan yang dapat dihubungkan efek xylitol dalam menghambat pertumbuhan dari bakteri adalah karena xylitol mempunyai 5 rantai karbon. hal ini dapat dijelaskan pada “rule 5/6” bahwa metabolisme karbohidrat yang mengandung 6 rantai karbon dihambat oleh karbohidrat dengan 5 rantai karbon. nutrisi yang banyak digunakan sebagai sumber energi pada kehidupan manusia adalah karbohidrat dengan 6 rantai karbon dan derivatnya. xylitol mencegah proses glikolisis karbohidrat 6 rantai karbon. hal ini merugikan bagi mikroba yang ada pada tubuh manusia dalam hal ini bakteri rongga mulut.1 dari beberapa uraian diatas terlihat bahwa dua jenis gula yaitu xylitol dan dietary sugars memiliki pengaruh yang berbeda pada pertumbuhan s. mutans. penggunaan dua jenis gula ini dalam waktu bersamaan kemungkinan akan terjadi penghambatan metabolisme sehingga akan berpengaruh pada pertumbuhan bakteri rongga mulut. untuk itu kami ingin meneliti efek penambahan xylitol dalam glukosa dan dalam sukrosa yang termasuk dietary sugars terhadap pertumbuhan s. mutans yang merupakan salah satu jenis bakteri yang bertanggung jawab dalam timbulnya karies gigi. tujuan penelitian ini adalah untuk meneliti pengaruh penambahan xylitol dalam glukosa dan dalam sukrosa terhadap pertumbuhan s mutans, serta mengetahui konsentrasi hambat minimalnya secara in vitro. pada akhir penelitian ini diharapkan dapat memberikan wawasan dalam penentuan diet jenis karbohidrat yang tepat dalam pengkombinasiaannya dengan xylitol sebagai pemanis yang non cariogenic dalam upaya pencegahan karies gigi. bahan dan metode penelitian ini merupakan penelitian eksperimental laboratoris dengan desain penelitian post test only control group. lokasi penelitian ini di laboratorium mikrobiologi fakultas kedokteran gigi universitas airlangga surabaya dan laboratorium fakultas matematika dan ilmu pengetahuan alam kimia bagian kimia analitik universitas airlangga surabaya. pada penelitian ini digunakan 7 sampel pada masing-masing konsentrasi kelompok perlakuan dan 7 sampel untuk kelompok kontrol. isolat s. mutans stock laboratorium mikrobiologi fakultas kedokteran gigi universitas airlangga tersimpan dalam media bhib-gliserol dalam bentuk beku. isolat yang masih beku dicairkan pada suhu kamar kemudian 2 ml dimasukkan dalam media 5 ml bhi-b dan selanjutnya diinkubasi dalam incubator aerob selama (24 jam, 37o c) untuk mengaktifkan s. mutans. prosedur kultur ulang ini kemudian diulang sekali atau dua kali untuk memastikan bahwa kuman sudah benar-benar aktif dan bisa digunakan dalam penelitian. setelah diaktifkan, isolat s mutans siap untuk digunakan dalam penelitian dan disesuaikan kekeruhannya dengan standard mc farland.5 pembuatan larutan xylitol dalam aquadest steril dan dilakukan pengenceran seri dengan aquadest steril hingga didapatkan konsentrasi 10%, 5%, 2,5%, 1,25%, 0,62%. persiapan suspensi bakteri dalam media bhi-b dengan 183susilowati, et al.: penambahan xylitol dalam glukosa, sukrosa terhadap pertumbuhan streptococcus mutans menyamakan kekeruhan suspensi bakteri dengan standard 0,5 mc farland hingga didapatkan suspensi bakteri 1,5 x 108 cfu/ml dan diencerkan lagi hingga mencapai 5 x 105 cfu/ml. kemudian dari masing-masing konsentrasi xylitol diambil 1 ml dan ditambah 0,03 ml inokulum bakteri dan keduanya dimasukkan dalam media bhi-b dan selanjutnya dilakukan inkubasi dalam incubator aerob selama (24 jam, 37o c). tahap selanjutnya dilakukan pengukuran ph dengan lakmus universal dan dilakukan pengamatan dengan spectrofotometer dan dengan melihat angka kekeruhan (od) yang terlihat pada alat spectrofotometer akan menunjukkan semakin besar angka berarti semakin keruh dan semakin banyak jumlah kuman. dengan metode yang sama dilakukan pengujian larutan sukrosa dalam konsentrasi 2,5%; 1,25%; 0,625%; 0,325%; 0,175%. konsentrasi tersebut diperoleh dari konsentrasi terendah sukrosa dalam suatu media biakan yang dapat menumbuhkan bakteri. prosedur yang sama dilakukan pada larutan glukosa dalam aquadest steril dengan konsentrasi 0,8%; 0,4%; 0,2%; 0,1%; 0,05% yang merupakan konsentrasi terendah glukosa dalam suatu media biakan yang dapat menumbuhkan bakteri. hasil hasil perhitungan koloni bakteri pada pemberian xylitol untuk melihat pertumbuhan s. mutans tampak pada gambar 1. pada konsentrasi 0,625% xylitol memperlihatkan penurunan optical density (od) jumlah koloni yang berarti pada konsentrasi tersebut xylitol dapat menghambat pertumbuhan koloni s. mutans. selain itu pada konsentrasi 0,625% terlihat kenaikan ph dibanding kelompok kontrol. minimal inhibitory concentration (mic) xylitol terhadap s. mutans adalah 0,625%. dari tabel 1 dapat diketahui, signifikansi pada konsentrasi 0,625% dan 2,5% dengan kelompok kontrol <0,05 sehingga dapat dikatakan ada perbedaan yang bermakna pada konsentrasi 0,625% dan 2,5% dengan kelompok kontrol. selain itu terlihat kenaikan ph pada kelompok perlakuan dibandingkan dengan kelompok kontrol. dari tabel 2 dapat diketahui, signifikansi pada semua konsentrasi dengan kelompok kontrol <0.05 sehingga dapat dikatakan ada perbedaan yang bermakna pada semua konsentrasi dengan kelompok kontrol. selain itu terlihat kenaikan ph pada kelompok perlakuan dibandingkan dengan kelompok kontrol. pembahasan hasil pengamatan dari pertumbuhan s. mutans menunjukkan xylitol pada konsentrasi 0,625% mulai menunjukkan penghambatan pertumbuhan s. mutans dan terjadi kenaikan ph. penghambatan xylitol terhadap gambar 1. jumlah koloni s.mutans setelah pemberian xylitol dalam berbagai konsentrasi tabel 1. hasil uji-t penambahan xylitol dalam sukrosa pada pertumbuhan s. mutans kelompok mean difference sd df uji-t sig 2.55% -0.01969 0.07005 6.574 -2.810 0.028 1.25% -0.0313 0.4334 12 -0.722 0.484 0.625% -0.0826 0.02350 12 -3.514 0.004 0.325% -0.0457 0.04067 12 -1.124 0.283 0.175% -0.0971 0.05208 12 -1.865 0.087 tabel 2. hasil uji-t penambahan xylitol dalam glukosa pada pertumbuhan s. mutans kelompok mean difference sd df uji-t sig 0.8% -0.0980 0.04236 12 -2.314 0.039 0.4% -0.1003 0.02882 12 -3.480 0.005 0.2% -0.1601 0.03302 7.509 -4.849 0.002 0.1% -0.1420 0.03755 12 -3.782 0.003 0.05% -0.1881 0.02020 12 -9.313 0.000 184 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 181–185 pertumbuhan s. mutans dan kenaikan ph berarti juga penghambatan karies. penjelasan mekanisme bagaimana xylitol dapat memberikan efek tersebut sangatlah komplek dan belum bisa dijelaskan secara detail, namun penghambatan enzim kemungkinan terlibat dalam hal ini.1 beberapa hal yang mungkin dapat menjelaskan hal tersebut adalah sebagai berikut, xylitol ditransport dan difosforilasi oleh phosphoenolpyruvate xylitol phosphotransferase system (pep-xylitol pts) kedalam sel bakteri dalam hal ini s. mutans. setelah masuk xylitol diubah menjadi xylitol 5 phosphate (x5p). penumpukan x5p dalam intrasel bakteri ini akan menghambat enzim yang ada dalam proses glikolisis dan hasilnya akan menyebabkan penurunan produksi asam dan pada akhirnya menghambat pertumbuhan dari bakteri tersebut. kemudian proses regulasi dari dalam bakteri melakukan dephosphorilasi x5p menjadi xylitol lagi dan kemudian mengeluarkan xylitol keluar dari sel. siklus phosphorilasi dan dephosphorilasi ini disebut futile-cycle dimana hasil dari proses ini berupa pep yang berfungsi sebagai donor phosphorilasi untuk pembentukan atp dan sumber energi untuk pep-sugar pts. keberadaan futile cycle ini membawa akibat penurunan produksi asam dan penurunan pertumbuhan bakteri.6 pada penelitian ini didapatkan konsentrasi hambat minimal xylitol terhadap s. mutans adalah sebesar 0,625%. modesto dan drake,7 mengatakan kombinasi antara chlorhexidine 0,12% dan xylitol 0,5% dapat menghambat kolonisasi dari s. mutans. pada penelitian lain yang dilakukan sahni et al.,8 menunjukkan konsentrasi hambat minimal xylitol terhadap s. mutans sebesar 1,56% sedangkan pada s. salivarius dan s. sanguis sebesar 12%. adanya perbedaan besar konsentrasi xylitol dalam menghambat bakteri rongga mulut tergantung kondisi bakteri. mekanisme kerja atau prosedur penelitian serta asal isolat dari s. mutans juga akan mempengaruhi hasil dari penelitian. pada penelitian ini isolat s. mutans yang digunakan berasal dari plak, karies pasien klinis. selain itu pada konsentrasi larutan tertentu yang terbanyak sekitar 10-50% bakteri mengalami kesulitan untuk homeostasis, banyak bakteri yang tidak bisa bertahan pada perubahan tekanan osmotik yang drastis ini.8 pada penelitian penambahan xylitol dalam sukrosa dan glukosa diperoleh bahwa pada penambahan xylitol dalam glukosa terdapat perbedaan yang bermakna pada semua konsentrasi sedangkan pada penambahan xylitol pada sukrosa hanya pada konsentrasi 0,625% dan 2,5% ada perbedaan yang bermakna. dalam pembagiannya karbohidarat diklasifikasikan dari yang sederhana (monosakarida, disakarida) sampai yang komplek (polisakarida). karbohidarat monosakarida terdiri dari glukosa, fruktosa dan galaktosa sedangkan disakarida terdiri dari sukrosa, maltosa dan laktosa. jenis polisakarida merupakan jenis karbohidrat yang lebih komplek diantaranya starch, cereal, nasi, crackers, snack dan sebagainya. pada penelitian ini jenis karbohidrat yang digunakan adalah glukosa yang termasuk monosakarida dan sukrosa yang termasuk disakarida. sukrosa tersusun dari satu molekul glukosa dan satu molekul fruktosa.4 menurut trahan,9 pada penambahan xylitol dalam beberapa dietary sugar, s. mutans juga mempunyai pepfruktosa pts yang fungsinya sama dengan pep-xylitol pts. penambahan xylitol dalam beberapa dietary sugar xylitol ditransport melalui pep-fruktosa pts, dan dalam penambahan xylitol dalam fruktosa jumlah x5p intraseluler lebih sedikit dibanding jenis dietary sugar lainnya. daya tarik pep-fruktosa pts pada fruktosa lebih kuat dibandingkan pada xylitol sehingga jika xylitol dan fruktosa digabungkan pembentukan x5p sedikit atau bahkan tidak terjadi. pada penambahan xylitol dalam beberapa dietary sugar, s mutans juga mempunyai pep-fruktosa pts yang fungsinya sama dengan pep-xylitol pts.6 pada penelitian ini penambahan xylitol dalam sukrosa hanya terdapat perbedaan pada konsentrasi 0,625% dan 2,5% sedangkan pada konsentrasi lain tidak didapatkan perbedaan yang bermakna. sukrosa merupakan disakarida yang terbentuk dari molekul glukosa dan molekul fruktosa. molekul fruktosa mempunyai daya tarik yang lebih kuat terhadap pep-fruktosa pts yang membuat xylitol masuk ke dalam tubuh bakteri melalui fruktosa phosphotransferase sistem sehingga pembentukan x5p sedikit dan hal ini menyebabkan tidak terjadinya penghambatan pertumbuhan s. mutans dan produksi asam pada beberapa konsentrasi penambahan xylitol dalam sukrosa. adanya keseimbangan glukosa dan fruktosa dalam sukrosa sangat mempengaruhi kerja pep-fruktosa pts dalam menghasilkan x5p yang akhirnya berakibat pada penghambatan pertumbuhan s. mutans. penelitian ini menunjukkan bahwa kombinasi xylitol dengan glukosa dan dengan sukrosa dapat menghambat pertumbuhan s. mutans. penambahan xylitol dalam sukrosa terjadi penghambatan s. mutans dan penurunan produksi asam pada konsentrasi 0,625% dan 2,5%. pada penambahan xylitol dalam glukosa terjadi penghambatan s. mutans dan penurunan produksi asam pada semua konsentrasi. perlu dilakukan penelitian lebih lanjut untuk menentukan pengkombinasian yang tepat antara xylitol dengan jenis dietary sugars yang lain. daftar pustaka 1. cronin jr. xylitol a sweet for healthy teeth anf more. alternative and complementary therapies 2003; 139-41. 2. american academy of pediatric dentistry. policy on the use xylitol on caries prevention. council on clinical affairs, 2006. 3. soesilo d. peranan sorbitol dalam mempertahankan kestabilan ph saliva pada proses pencegahan karies. maj ked gigi (dent j) 2005; 185susilowati, et al.: penambahan xylitol dalam glukosa, sukrosa terhadap pertumbuhan streptococcus mutans 38(1): 25-8. 4. carole ap. diet and nutritionin oral health. new jersey: pearson education inc; 2003. p. 59-70. 5. bailey s. diagnostic microbiology. 7th ed. st louis: mosby inc; 1986. p. 229-49. 6. kakuta h, iwami y, mayanagi h, takahashi n. xylitol inhibition of acid production and growth of mutans streptococci in the presence of various dietary sugars under strictly anaerobic conditions. caries res 2003; 37(6): 404-9. 7. modesto a, drake dr. multiple exposure to chlorhexidine and xylitol: adhesion and biofilm formation by streptococcus mutans curr microbiol 2006; 52(6): 418-23. 8. sahni ps, gillespie mj, botto rw, otsuka as. in vitro testing of xylitol as an anticariogenic agent. gen dent 2002; 50(4): 340-3. 9. trahan l, bareil m, gauthier l, vadeboncoeur c. transport and phosphorylation of xylitol by fructose phosphotransferase system in streptococcus mutans. caries res. 1985; 19(1): 53-63. 204204 dental journal (majalah kedokteran gigi) 2022 december; 55(4): 204–208 original article effectiveness of telemedicine approach as a treatment to reduce severity of temporomandibular disorders ricca chairunnisa, siti dyah fadilla department of prosthodontics, faculty of dentistry, universitas sumatera utara, medan, indonesia abstract background: temporomandibular disorder (tmd) is the most common pain in the maxillofacial area. overall prevalence of tmd was approximately 31% for adults/elderly and 11% for children/adolescents. tmd is considered a multifactorial disorder. there are various treatments for tmd, one of which is jaw exercises therapy which is the most widely used initial treatment to relieve signs and symptoms. currently, conventional therapy for tmd patients has been difficult to control patient compliance. hence, this therapy can be done at home independently through telemedicine as an alternative tool to rehabilitateate the patients that can be accessed via smartphone. purpose: to determine the severity of tmd before and after jaw exercises and the effect of telemedicine approach using jaw exercises via smartphone apps on dental students. methods: this study used a quasi-experimental method with a one-group pretest post-test design using fonseca anamnestic index (fai) questionnaire to measure the severity before and after two weeks of jaw exercises on 35 dental students using purposive sampling based on inclusion and exclusion criteria. wilcoxon signed-rank test was used to analyze the data (p<0.05). results: before jaw exercises, 21 people (60%) had mild tmd, 14 people (40%) had moderate tmd, and none had severe tmd. after jaw exercises, the severity of 9 people (42.8%) in the mild tmd group has decreased (p=0.007), and the severity of 11 people (78.5%) in the moderate tmd group has decreased (p=0.003). conclusion: there was positive effect that decreased the level of severity of tmd through telemedicine approach after jaw exercises via smartphone apps on dental students. keywords: fonseca anamnestic index; jaw exercises; telemedicine; smartphone apps correspondence: ricca chairunnisa, department of prosthodontics, faculty of dentistry, universitas sumatera utara, jl. alumni no. 2, medan 20155, indonesia. email: ricca@usu.ac.id introduction temporomandibular disorders (tmd) are recognized by the american academy of orofacial pain as a group of musculoskeletal and neuromuscular conditions involving the temporomandibular joint, muscles of mastication, and related tissues.1 prevalence of tmd was approximately 31% for adults/elderly and 11% for children/adolescents.2 tmd occurs more frequently in women, possibly due to biological, psychological, and/or social factors associated with female gender, increasing the risk of tmd.3 the etiology of tmd is multifactorial. the diagnosis of this disorder can be made by clinical history, physical examination and radiographic studies.4,5 clinical history can use the fonseca anamnestic index (fai), which is the most widely used because it is simple, low cost, and effective in describing the presence and severity of symptoms of tmd.6 signs and symptoms of this disorder are usually pain and range of motion that require treatment.7 jaw exercises are one of the treatments for tmd that aims to achieve relaxation in sore jaw muscles and optimize jaw function. this exercise can be done independently and is recommended because it is simple, cost-effective, and allows an easy approach to self-management.8 some of the recommended therapeutic procedures are coordination exercises, strengthening and resistance exercises, and stretching and relaxation exercises that can be done with varying duration, frequency and length of exercise. the duration and frequency of exercise are generally carried out for 3-4 minutes 2-3 times a day. however, the length of exercise, in some studies, was carried out for two to three months with periodic evaluations. in the evaluation carried out every week, it showed changes in the disturbances that occur. that way, even though the exercise is only done dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p204–208 mailto:ricca@usu.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p204-208 205 chairunnisa and fadilla/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 204–208 for a short period of time, if the patient does the exercise consistently it can reduce the signs and symptoms of tmd.9–12 due to the covid-19 pandemic that makes it difficult for patients to get treatment, telemedicine can be a major tool to self-care at home by the patient without loss of monitoring from the practitioner. telemedicine is an informationbased technology and communication system for crossdistance care services and has been shown to improve the consistency and quality of healthcare, sometimes cheaper than conventional practice.13 information can be sent through various media such as text, audio, images, or video using a device such as a smartphone or desktop computer.14,15 using telemedicine through smartphones is an efficient and effective way of remote consultation as it allows for increased triage, which ultimately provides better care to maxillofacial patients.16 in addition, salazarfernandez et al.13 said that telemedicine allows adequate diagnosis and treatment in most cases of tmd. because of the above idea, researchers are interested in seeing whether there is an effect of telemedicine approach using jaw exercises via smartphone apps on the severity of tmd in dental students. materials and methods this study used a quasi-experimental method with a one group pre-test post-test. thirty-five dental students of north sumatra university were selected using purposive sampling with inclusion criteria, which is students aged 18 – 23 years who had complete teeth up to m2, and had tmd which can be known through filling out the fai questionnaire. this questionnaire was proposed by fonseca in 1994, and is commonly used to classify tmd severity because it is good in obtaining relevant data and covers multi-dimensions and provides a full true picture of tmd. it was used for its simplicity and clearness of the questions.17 it is composed of 10 questions, includes checking for the presence of pain in the temporomandibular joint, head, and back, while chewing, para-functional habits, movement limitations, joint clicking, a perception of malocclusion and sensation of emotional stress. for each question there are three answer choices, no (score 0), sometimes (score 5), and yes (score 10). categories are determined by the sum of the scores of all question items and allow the following classification, absence of signs and symptoms of tmd (0-15 points), mild tmd (20-45 points), moderate tmd (50-65 points) and severe tmd (70-100 points) (figure 1). they were summed up and the total score was out of a 100 maximum. the exclusion criteria included history of trauma in the maxillofacial and tmd treatment, using prosthetics and orthodontics. this study had permission from the research ethics committee of universitas sumatera utara (number 1218/kep/usu/2021). there are three types of jaw exercises performed, d coordination exercises, strengthening and resistance exercises, and stretching and relaxation exercises (figure 2). coordination exercises were performed with the patient instructed to open the jaw in a straight line in front of a mirror. the open motion of the subject must be in a straight line according to the midline of the teeth and use a straight object such as a ruler as a comparison. strengthening and resistance exercises are performed by placing the thumb under the center of the patient’s chin. the patient is instructed to open the jaw slowly by lowering the jaw, while applying steady light pressure to the underside of the chin with the thumb. stretching and relaxation exercises were performed by hold the tongue on the palate for eight seconds while opening and closing the jaw slowly. each exercise is performed for 10 repetitions, of which one repetition is performed for eight seconds. the exercises will be carried out for two weeks, three times a day. the exercises will be carried out via a telemedicine application on a smartphone. in addition to jaw exercises, the telemedicine application also contains an initial screening in the form of an fai questionnaire, as well as an evaluation of care after doing jaw exercises. the fai questionnaire will be filled out when the patient completes two 2 weeks of jaw exercises to see the effect before and after exercise via telemedicine on the severity of tmd experienced by the patient. initially, patients who had been selected according to the inclusion criteria were invited to meet via zoom to be given instructions on how to use the application as well as an independent examination of signs and symptoms to answer the fai questionnaire. after that, the patient was instructed to download the telemedicine application by writing “tmd exercises” on their smartphone. once downloaded, the patient was confirmed to have a stable internet network to be able to access the telemedicine application, and register on the application by filling in personal identification such as name, gender, age, and telephone number. one patient filled it in, the application instructed them to enter the code sent to their phone number to log in. on the home page, the application instructed them to fill out the fai questionnaire according to the results of the previous examination. after that, the answers that have been saved were sent to the operator to be reviewed. if appropriate, the operator provided feedback in the form of instructions to perform jaw exercises for two weeks. patients who got feedback could access jaw exercise tutorial videos and the application directed them to record their jaw exercises. once recorded, the application asks the patient to upload the recording and send it to the operator as proof that the patient has performed the jaw exercises properly and according to the instructions. the application automatically provides jaw training notifications according to a predetermined time at 8 am, 2 pm, and 8 pm. patients who miss jaw exercises will be contacted periodically by the operator by telephone. after two weeks of doing jaw exercises, the application will direct them to fill out the fai questionnaire again. after filling out the questionnaire, the operator will provide education such as reducing bad habits that can increase tmd. to determine the effect of jaw exercise therapy dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p204–208 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p204-208 206chairunnisa and fadilla/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 204–208 figure 3. effect of telemedicine approach before and after jaw exercises therapy. table 1. results of statistical test using wilcoxon signed rank test jaw exercises mild severity level moderate severity level n x ± sd p n x ± sd p before 21 26.9 ± 5.1 0.007* 14 55.7 ± 7.3 0.003* after 21 22.1 ± 6.8 14 40.7 ± 16.5 *significant (p<0.05) figure 1. tmd exercises by telemedicine using a smartphone apps. a b c figure 2. three types of jaw exercises: coordination exercises (a); strengthening and resistance exercises (b); stretching and relaxation exercises. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p204–208 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p204-208 207 chairunnisa and fadilla/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 204–208 by telemedicine approach using smartphone apps on the severity of tmd, a statistical test was performed using the wilcoxon signed rank test (p<0.05)to find out the average between two related samples. the telemedicine application has been validated by two prosthodontic doctors, both from video tutorials to evaluating the success of treatments that are adapted to conventional methods that are often carried out in practice. telemedicine-tmd exercises have been registered to intellectual property right (ipr) from the ministry of law and human rights of indonesia (number 000341260). results the results of the questionnaire before jaw exercise therapy showed 21 people (60%) had mild tmd, 14 people (40%) had moderate tmd and none had severe tmd. the questionnaire result showed after jaw exercises therapy at mild severity, 9 (42.8%) people out of 21 experienced a decrease in the total score. meanwhile, at the moderate level based on the results of the fai questionnaire after jaw exercise therapy, 11 people (78.5%) out of 14 experienced a decrease in the total score (figure 3). the results of statistical tests using the wilcoxon signed rank test obtained significant results (p<0.05), at mild severity level p=0.007, and the moderate severity level p=0.003 (table 1). discussion this study showed significant results, which indicates there is an effect. this can be caused by patients who are dental students being familiar with the symptoms, signs and impacts of tmd. nomura et al.18 saw many dental students who unknowingly had a tmd. when aware of having a disorder, steps of prevention and treatment become easier to do because individuals knew the effects that will occur if the disorder is ignored.18,19 one of the initial treatments for tmd is jaw exercise therapy. in the delphi study, lindfors et al.20 showed that there is an international agreement among therapists for tmd that jaw exercise therapy is an effective treatment and is recommended for patients with tmd and pain. in addition, patients also feel safe and protected because they have the tools (jaw exercises) to deal with the problem on their own if the symptoms reappear. the patients also appeared to be more confident, stronger and in control when using jaw exercises. this process can be defined as empowerment and can play an important role in pain treatment and rehabilitation.20 jaw exercise therapy carried out in this study consisted of three types: (1) coordination exercises aimed to improve muscle coordination, relax tense muscles, overcome limitations in jaw movement, and restore normal muscle length and function by stimulating blood circulation to the temporomandibular joint muscles. (2) strengthening and resistance training aimed to overcome muscle spasms, limited movement, muscle weakness, and muscle in-coordination. this exercise involves contracting the mandibular muscles against resistance during jaw opening movements. this exercise increases reflex inhibition and relaxation of the corresponding antagonist muscles. (3) stretching and relaxation exercises aimed to improve the range of motion of the jaw and eliminate or reduce joint sounds.5,11 this study carried out jaw exercise therapy independently at home (home physical therapy) using telemedicine because it has been proven to be effective. telemedicine is a cross-distance care service so that patients can perform independent jaw exercise therapy anywhere but still under the supervision of expert practitioners. some research said that telemedicine enables adequate diagnosis and treatment in the majority of cases of tmd, shortens treatment time delays and reduces unnecessary costs to patients with tmd. treatment through telemedicine as a support application can be used as a means to send questionnaires, jaw exercise videos, reminders for exercises and to evaluate so that it can meet the goals of tmd treatment, self-management and self-efficacy.13,21 therefore, jaw exercise therapy is recommended to be carried out through telemedicine compared to conventional. besides that, telemedicine has proven to improve consistency and quality of healthcare and is less expensive than conventional practice.13 telemedicine via smartphone apps in this study is designed as a simulation video containing instructions for jaw exercise therapy, and a recording feature that is equipped with a timer along with its repetitions as a means for patients to perform jaw exercise therapy. the recording results will be sent to and stored by the operator so that the patient can be monitored and can be given direction if the exercise therapy is not appropriate. also, they have an automatic reminder three times a day which is installed automatically immediately after the application is downloaded. telemedicine approach via smartphone apps can be done anywhere so that it can improve patient compliance in doing jaw exercises. in addition, it also has supporting features, namely education in the form of articles and fai questionnaires as a means of measuring the severity of tmd before and after two weeks of jaw exercise therapy. patients who feel they still have symptoms and signs of tmd can continue therapy through the application with the frequency and duration according to their settings. jaw exercise therapy will be effective if the patient routinely undergoes the instructions and procedures given. according to kartika et al.,10 jaw exercise therapy should be done for 2-3 minutes with a frequency of 2-3 times a day, and in most cases, it takes about 2-3 months to get satisfactory benefits, both subjectively and objectively.10 however, research by sakuma et al.12 found that clinical symptoms such as maximum mouth opening distance, pain on movement, pain on mastication, and impact on daily activities in the second week were significantly increased.12 the following is in accordance with this study, where therapy was carried out for a duration of three minutes dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p204–208 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p204-208 208chairunnisa and fadilla/dent. j. (majalah kedokteran gigi) 2022 december; 55(4): 204–208 three times a day and for two weeks which showed a decrease in severity before and after jaw exercise therapy, that respondents no longer felt symptoms of tmd such as sound and pain in temporomandibular joint, and fatigue when chewing. likewise, in this study, as many as 15 people (42.9%) did not show any changes before and after jaw exercise therapy. this can be caused by the patient being uncooperative and not aware of the need for treatment for the tmd they are experiencing so they often miss the automatic jaw exercise therapy alarm on their smartphone. in addition, there are also external factors such as daily activities that make patients skip jaw exercise therapy. to conclude, there was significant positive effect of telemedicine approach using jaw exercises via smartphone apps on dental students. the weakness of this study is that the severity of tmd cannot be checked directly considering the covid-19 pandemic situation, the honesty of patients when filling out questionnaires cannot be measured, and the length of exercise carried out will show more satisfactory results if carried out in a longer period. references 1. ferreira clp, da silva mamr, de felício cm. signs and symptoms of temporomandibular disorders in women and men. codas. 2016; 28(1): 17–21. 2. valesan lf, da-cas cd, réus jc, denardin acs, garanhani r r, bonotto d, ja nuzzi e , de souza bdm. p reva lence of temporomandibular joint disorders: a systematic review and metaanalysis. clin oral investig. 2021; 25(2): 441–53. 3. akhter r. epidemiology of temporomandibular disorder in the general population: a systematic review. adv dent oral heal. 2019; 10(3): 1–13. 4. de leeuw r, klasser g. orofacial pain: guidelines for assessment, diagnosis, and management. 6th ed. quintessence publishing; 2018. p. 143–72. 5. okeson jp. management of temporomandibular disorders and occlusion. 8th ed. st. louis: elsevier; 2020. p. 2, 5–19, 108–9, 132, 174–5, 206–8, 262–71, 277–82. 6. alyessary as, yap au, almousawi a. the arabic fonseca anamnestic index: psychometric properties and use for screening temporomandibular disorders in prospective orthodontic patients. cranio. 2020; : 1–8. 7. de rossi ss, greenberg ms, liu f, steinkeler a. temporomandibular disorders: evaluation and management. med clin north am. 2014; 98(6): 1353–84. 8. lindfors e, magnusson t, ernberg m. patients’ experiences of therapeutic jaw exercises in the treatment of masticatory myofascial pain-a postal questionnaire study. j oral rehabil. 2019; 46(9): 800–6. 9. shimada a, ishigaki s, matsuka y, komiyama o, torisu t, oono y, sato h, naganawa t, mine a, yamazaki y, okura k, sakuma y, sasaki k. effects of exercise therapy on painful temporomandibular disorders. j oral rehabil. 2019; 46(5): 475–81. 10. kartika l, himawan ls. penatalaksanaan kasus gangguan sendi temporomandibula dengan latihan rahang (laporan kasus). indones j dent. 2007; 14(1): 12–7. 11. moraes a da r, sanches ml, r ibeiro ec, guima rães as. therapeutic exercises for the control of temporomandibular disorders. dental press j orthod. 2013; 18(5): 134–9. 12. sakuma s, yamaguchi y, taguchi n, ogi n, kurita k, ito y. pilot study of the short-term effects of range-of-motion exercise for the temporomandibular joint in patients with temporomandibular joint disc displacement without reduction. j phys ther sci. 2017; 29(2): 274–7. 13. salazar-fernandez ci, herce j, garcia-palma a, delgado j, martín jf, soto t. telemedicine as an effective tool for the management of temporomandibular joint disorders. j oral maxillofac surg. 2012; 70(2): 295–301. 14. jampani nd, nutalapati r, dontula bsk, boyapati r. applications of teledentistry: a literature review and update. j int soc prev community dent. 2011; 1(2): 37–44. 15. kadir ma. role of telemedicine in healthcare during covid-19 pandemic in developing countries. telehealth med today. 2020; : 1–5. 16. aziz sr, ziccardi vb. telemedicine using smartphones for oral and maxillofacial surgery consultation, communication, and treatment planning. j oral maxillofac surg. 2009; 67(11): 2505–9. 17. al moaleem mm, okshah as, al-shahrani aa, alshadidi aaf, shaabi fi, mobark ah, mattoo ka. prevalence and severity of temporomandibular disorders among undergraduate medical students in association with khat chewing. j contemp dent pract. 2017; 18(1): 23–8. 18. nomura k, vitti m, oliveira as de, chaves tc, semprini m, siéssere s, hallak jec, regalo sch. use of the fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders in brazilian dental undergraduates. braz dent j. 2007; 18(2): 163–7. 19. özdinç s, ata h, selçuk h, can hb, sermenli n, turan fn. temporoma ndibula r joint disorder deter m ined by fonseca anamnestic index and associated factors in 18to 27-year-old university students. cranio. 2020; 38(5): 327–32. 20. lindfors e, arima t, baad-hansen l, bakke m, de laat a, giannakopoulos nn, glaros a, guimarães as, johansson a, le bell y, lobbezoo f, michelotti a, müller f, ohrbach r, wänman a, magnusson t, ernberg m. jaw exercises in the treatment of temporomandibular disorders-an international modified delphi study. j oral facial pain headache. 2019; 33(4): 389–398. 21. van der meer ha, de pijper l, van bruxvoort t, visscher cm, nijhuis-van der sanden mwg, engelbert rhh, speksnijder cm. using e-health in the physical therapeutic care process for patients with temporomandibular disorders: a qualitative study on the perspective of physical therapists and patients. disabil rehabil. 2022; 44(4): 617–24. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i4.p204–208 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v55.i4.p204-208 mkgs vol 44 no 1 jan-mar 2011.indd 49 vol. 44. no. 1 march 2011 tnfα expression on rats after candida albicans inoculation and neem (azadirachta indica) extract feeding i dewa ayu ratna dewanti department of biomedic faculty of dentistry, jember university jember indonesia abstract background: neem is a known traditional medicine from trees which function as immunomodulator. candidiasis found in mouth is 80% caused by candida albicans (c. albicans). immunity is important to limit c. albicans since medicine price is relatively traditional medicine may become a good choice. in the other side the medicine price may not be reached by the citizen, cause citizen choose the traditional medicine. purpose: the research is aimed to explain of tnf-α expression on rats after inoculated by c. albicans and fed with neem extract (azadirachta indica). methods: there were 5 groups, the first group which was called as control group (ko) hadn’t been fed aqueous extract from neem leaves and was not inoculated by c. albicans, the other group (treatment) was classified into 4 groups. the first group was inoculated by c. albicans only (kp1), second group was fed with 50 mg/day/kg body weight aqueous extracts from neem leaves, then inoculated with c. albicans starting from day 8 until day 21 (kp2), third group was fed with 100 mg/day/kg body weight aqueous extract from neem leaves, then inoculated with c. albicans start from day 8 until day 21 (kp3), fourth group was fed with 200 mg/day/kg body weight aqueous extract from neem leaves, then inoculated by c. albicans start from day 8 until day 21 (kp4). the data was collected from by swabbing the rat’s tongue to calculate c. albicans colonies. the rats were acclimated and collected for immunohistochemistry measurement. results: the study showed that there were different result on anova, hsd test, and linier regression. anova showed significant difference (p < 0.01) between groups. the hsd test showed significant difference (p < 0.05) between each groups. tnf-α was the stimuli sensor from environment, and used as parameter to see the effect from the change of innate immunity component to c. albicans. conclusion: aqueous extract from neem leaves increased the macrophage tnf-α expression on in rat in oculated with c. albicans. key words: neem leaves aqueous extract, azadirachta indica, macrophage, phagocytosis, candida albicans abstrak latar belakang: mimba merupakan salah satu tanaman obat tradisional yang telah dikenal masyarakat dan berfungsi sebagai imunomodulator. penyakit infeksi yang paling banyak dijumpai di rongga mulut (80%) adalah kandidiasis dengan penyebab utama candida albicans (c. albicans). di mana peran imunitas sangat penting pada c. albicans. di sisi lain harga obat yang semakin mahal semakin tidak terjangkau masyarakat, menyebabkan masyarakat memilih obat tradisional. tujuan: riset ini untuk menjelaskan tentang ekspresi tnf-α makrofag pada tikus wistar yang diinokulasi c. albicans dan diberi konsumsi ekstrak cair daun mimba. metode: penelitian ini terbagi menjadi 5 kelompok, kelompok kontrol (ko) tidak diberi perlakuan, kelompok yang diinokulasi c. albicans (kp1), kelompok yang diberi konsumsi 50 mg/hari/kg dan diinokulasi c. albicans mulai dari hari 8 sampai hari 21 (kp2), kelompok yang diberi konsumsi 100 mg/hari/kg dan diinokulasi c. albicans mulai dari hari 8 sampai hari 21 (kp3), kelompok yang diberi konsumsi 200 mg/hari/kg dan diinokulasi c. albicans mulai dari hari 8 sampai hari 21 (kp4). data dikumpulkan dari swabbing lidah untuk dihitung koloni c. albicans dan jaringan lidah dengan metode immunohistochemistry untuk penghitungan sel makrofag yang mengekspresikantnf-α. hasil: studi menunjukkan terdapat perbedaan yang signifikan dari hasil anova, uji hsd, regresi linier. anova menunjukkan perbedaan (p < 0,01) antar kelompok. uji hsd menunjukkan perbedaan (p < 0,05) antar kelompok. hal ini dapat dikatakan bahwa tnf-α adalah sensor stimuli dari lingkungan, yang digunakan sebagai parameter untuk melihat pengaruh dari research report 50 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 49–53 perubahan dari komponen respons innate terhadap c. albicans. kesimpulan: ekstrak cair daun mimba dapa meningkatkan ekspresi makrofag tnf-α dari tikus yang diinokulasi c. albicans. kata kunci: ekstrak cair daun mimba, azadirachta indica, makrofag, fagositosis, candida albicans correspondence: i dewa ayu ratna dewanti, c/o: bagian biomedik, fakultas kedokteran gigi universitas jember. jl. kalimantan 37 jember 68121, indonesia. e-mail: dewadewanti@yahoo.co.id introduction neem (azadirachta indica) contains bioactive component such as azadirachtin, salanine, meliantriole, nimbin, nimbolide, gedunine, mahmodine, gallic acid, catechin, epicatechin, margolone, margolonone, isomargolonone, cyclictrisulphide, cyclictetrasulphide and polysaccharide. neem has been widely used by the community to treat diseases including worm infection, scabies, malaria, fungal infection, tumor, and allergy.1,2 researches had proven that neem modulates innate and adaptive immunity,3–6 while innate immunity (phagocytosis) especially macrophage, plays important role in fighting candida albicans (c. albicans) which was the main etiology for oral candidiasis.7,8 oral candidiasis is one of the most common infectious diseases found in oral cavity (80%).7 previous researches from the author had proved that aqueous extract from neem leaves could inhibit the growth of c. albicans in vitro.9 other than having antifungal effect, neem leaves could also function as immunomodulator. many antifungal drugs have no immunomodulator properties, while infection of c. albicans is highly depending on the state of immune system. destruction and elimination by phagocytic cells, could occur by both oxidative and non oxidative pathways. oxidative pathway including the production of superoxide and no by inos system, where both activities could be induced by tnf-α, while phagocytic activity and fungicidal uptake functions and intracellular fungal destruction. non oxidative measures including production of cytokines, such as tnf-α that may modulate the activity of phagocytosis.1012 other researches had proved that neem leaves could increase macrophage activity in vitro, so it was assumed that neem leaves might affect tnf-α which was a cytokine that play role in activating phagocytosis, but until today, the mechanism of the increasing activity of tnf-α to c. albicans had not yet fully explained.13 the aim of the research was to know tnf-α expression on rats were inoculated by c. albicans and fed with neem extract. materials and methods this research was an experimental laboratory research with sample of 25 male wistar rats that have fulfilled ”declaration of helsinki”. each rat was 100-200 grams in weight, age 2–3 months that received one week adaptation. there were five groups which were control group (ko) which were not provided with aqueous extract of neem leaves and not inoculated with c. albicans, treatment groups which consisted of the group that were inoculated with c. albicans only (kp1), a group which consumed aqueous extract of neem leaves with a dose of 50 mg/day/kg bodyweight, then were inoculated with c. albicans started from day 8 to day 21 (kp2), a group that were provided with aqueous extract of neem extract with a dose of 100 mg/day/kg body weight, and were inoculated with c. albicans from day 8 to day 21 (kp3), and a group that were provided with aqueous extract of neem leaves with a dose of 200 mg/day/kg body weight and were inoculated with c. albicans (kp4). all groups were observed in day 22 by conducting light swab with cotton bud on the rats’ dorsal tongue with one swab to count the number of c. albicans colony. the rats were collected and lingual tissues were obtained and prepared, then tnf-α was analyzed with immunohistochemistry methods, through: deparanization with ethanol started from absolute to 70%, water, phosphate buffer saline (pbs) ph 7.4 and were provided with tripsin. preparation was flooded within the solution of 3% h2o2, washed with pbs twice and were undergoing blocking process with 3% bsa. anti rat tnf-α was then reacted, and was incubated for 24 hours in 4° c temperature in a humidity chamber. the substances were then reacted with biotiyilized secondary anti rabbit (ab). washed three times with pbs, and were provided with peroxidase-labeled streptavidin and were incubated for 1 hour. substances were then re-washed three times with pbs, reacted with diamine benzidine (dab) substrate, and were added with ”meyer-he”. data obtained were analyzed with anova and continued with hsd test. results the result showed less tnf-α in macrophages on groups inoculated with c. albicans compared to control group. the higher d the dose of neem extract, the higher the tnf-α expression. anova test show of tnf-α expression showed that there was a significant difference (p<0.01), this is continued with hsd test which also gave significant difference. thus indicates that aqueous extract from neem leaves can increase tnf-α expression with dose 50, 100, 200 mg/weight/day, on the other side c. albicans reduces tnf-α expression. linear regression showed a strong positive correlation (0.985), meaning the higher the dose of neem leaves aqueous extract, the higher the number of tnf51dewanti: tnf-α expression on rats α expression macrophage (figure 3). expression of tnf-α (circles) were scattered around the rightward straight line with upward position, which indicated that the higher the dose of neem given, the higher the expression of tnf-α (figure 3). c. albicans colony was counted by colony counter after grown on saburound’s agar (figure 4). there are no c. albicans at control group. the highest number of colony was found at kpi, and the smallest at kp4 (figure 4). anova test showed there are a significant difference between groups, and using hsd test indicated that aqueous extract from neem leaves with dose 200mg/ weight/day can reduce c. albicans colony. figure 1. expression of tnf-α in macrophage with immunohistochemistry technique (400× magnification). brown color shows presence of tnf-α expression ( ) blue color shows presence of tnf-α expression ( ) a) staining control (macrophage appeared in blue staining); b) expression of tnf-α in macrophage of control group (k0); c) expression of tnf-α in macrophage of treatment group 1 (kp1); d) expression of tnf-α in macrophage of treatment group 2 (kp2); e) expression of tnf-α in macrophage of treatment group 3 (kp3); f) expression of tnf-α in macrophage of treatment group 4 (kp4) a b c d e f figure 2. macrophage expression of tnf-α. neem close (mg/kg weight) kp0 kp1 kp2 kp3 kp4 30 25 20 15 31 5 0 t n f -α 52 dent. j. (maj. ked. gigi), vol. 44. no. 1 march 2011: 49–53 discussion stimuli response was evaluated through oral mucosa immunity mucous membrane, oral lymphoid tissue, extraoral lymphoid, intraoral lymphoid tissue, lymphoid gland saliva, gingiva lymphoid tissue. in this research, the sampling was done on mouse tongue because candidiasis is most commonly found at tongue. oral mucosa immune system will influence systematical immune system through vascular immune system. aqueous extract of neem leaves containing galic acid, catechin, epicatechin which can influence macrophage response through two ways, one was directly influences c. albicans and indirectly by influencing macrophage. effect of c. albicans was anticipated to causes change at c. albicans cell membrane, so it is easier to be recognized by macrophage to do phagocytosis. indirect effect of aqueous extract from neem leaves by the way of changing macrophage activity to c. albicans, presentation by the way of immunemodulation. this research was conducted to solve the problem about aqueous extract from neem leaves to tnf-α activity related to macrophage phagocytosis activity to c. albicans. cd14, tlr2, tlr4, tnf-α, phagocytosis activity and number of c. albicans colonies, because cd14, tlr2, tlr4, tnf-α, phagocytosis activity were component at commissioned innate immune system as censor stimuli. while colony amounts of c. albicans was applied as parameter existence of effect from change of innate immune component to c. albicans. stimuli can be recognized by censor system cd14, tlr2, tlr4 and will be followed up with selection, organizational and interpretation so that response to stimuli can be adapted for requirement of host to survive candidiasis to cause cd14 suppression which is to functioning increase tlr activity, pursues tlr2 and tlr4 resulting degradation protein of transcription activity residing in macrophage cell. function of phagocytosis and tnf-α is playing important role at macrophage level which also influence the degradation of adaptive immune response on c. albicans on the other side with existence of aqueous extract from neem leaves situation of imunosuppresion resulted from c. albicans will be improve and repaired. macrophage as professional phagocyte function to break immunogen as antigen presenting cells (apc) which recognizes microbe through some receptors its to stimulate migration of cell to the place of infection and stimulates the production of substance. activation of macrophage at innate immunity through cd14 expressed to surface of cell and activates toll-like receptors (tlrs) showed that neem leaves with their immunomodulatory components (assumed as galic acid, catechin, epicatechin) built a new balance through immune system regulation in which we could recognize the outcomes of products from macrophage cells in facing stressors. improvement mechanism of tnf-α by neem to c. albicans started by the existence of improvement of cd14, tlr2 and tlr4 (c. albicans reduces all immune activities). tlr2, tlr4 affected figure 3. linear graphic of tnf-α expression. tabel 1. hsd test of tnf-α expression to macrophage groups n mean sd kpo kp1 kp2 kp3 kp4 total 5 5 5 5 5 25 2.6666 1.7334 11.2000 15.8000 26.6666 11.6133 .47146a 1.01113b .44721c 1.26073d .78181e 9.40825f description: different letter shows existence of difference significant tnf-α expression n um be r of c . a lb ic an s co lo ni es research sample (5 samples) ko kp1 kp2 kp3 kp4 120 100 80 60 40 20 0 1 2 3 4 5 figure 4. number of c. albicans colony between groups. mhc cd14 – tlr4 protein rac ikk il-10 nfb tlr2 ap-1 mr mr il-6, tnfp50, neem tnferk jun i b pp p65 p50 ifnfagositosis jnk, p38 neem leaves degrad asi i b sel t figure 5. the activity of neem leaves to immune response. 30 25 20 15 10 5 0 0 50 100 150 200 observed linear 53dewanti: tnf-α expression on rats phospatidilinositol in macrophage cell membrane and would then activate rac protein, which then activated nfκb and ap-1 through jun kinase via the mitogen-activated protein kinase (mapk) pathway. included within this pathways was extracellular signal-regulated kinase (erk), c-jun n-terminal kinase (jnk) and p38. erk affected jun activity, while p38 affected the production of il-6, il-8 and il-12. activity of p38 and erk could activate ap-1. these three mapk pathways could be activated simultaneously at the same time. nf-κb was a regulator from early response to pathogen and as an activator of immune system. nf-κb was p50–p65 from a family of heterodimer protein that transcribed many genes. activation of nf-κb require i-κb protein phosporilation, which was continued by degradation of p50–p65 within the nucleus and it would activate the gene. after the release of i-κb, an increase would occur in the activity of transcription factor nf-κb which stimulated gene expression that affected the production of tnf-α and phagocytic activity. stimulation of gene expression among others affected the production of tnf-α. in immune response to c. albicans, tnf-α played the role as primary immunity in immune system regulation. specifically to macrophage, this cytokine increased the activity in killing pathogens, in which this action became an important mediator in inflammation. activity of innate immune response may affected mhc, so it would give effect on the activity of adaptive immunity (t cell and b cell).14-16 briefly, the mechanism of the increased expression of tnfα induced by aqueous extract of neem was explained in figure 5. aqueous extract of neem with component galic acid, epicatechin, catechin, can reduce number of colonies c. albicans through improvement of tnf-α activity, though the number is not absolute to show the existence of infection, but applicable to show the existence of infection. amount of which more than control group serve the purpose of parameter the happening of infection. this research shows number of c. albicans which increasingly declines with aqueous extract from neem leaves dose excelsior mimba, where dose 200 mg/kg/weight seen number of least colonies. degradation of c. albicans colonies number is anticipated by immunomodulator content (galic acid, epicatechin, catechin) and nimbidin content, azadirachtin, gedunin, cyclic trisulphide, cyclic tetrasulphide which can function as antifungal. immunomodulator content can increase immune response to c. albicans, while antifungal content can kill candida directly and destructive to its cell membrane. it concluded that aqueous extract from neem leaves could increase the expression of tnf-α in rats inoculated with c. albicans. references 1. ganguli sj. neem: a therapeutic for all seasons. current science 2002; 82(11): 1304. 2. goel rk, sairam k. anti ulcer drugs from indigenous source with emphasis on musasapientum, tamrabhasma, asparagus racemous and zingiber officinale. indian j of pharmacology 2002; 34: 100–10. 3. upadhayay d, garg s, talwar gp. immunomodulation effects of neem (aza dirachta indica) oil. int j immunopharmacol 1992 october; 14(7): 1187–93. 4. sairam k, sharma sk, havazhagan g, kumar d, selavamurthy w. immunomodulatory effect of nim-76, a volatile fraction from neem oil. j ethnopharmacol 1997; 55(2): 133–9. 5. sastrodihardjo s. evaluasi daya insektisida dari ekstrak daun mimba (azadirachta indica a. juss). seminar hasil penelitian pangan dan gizi, ilmu hayati. jakarta: pau; 1988. p. 18. 6. sadekar d, kolte ay, barnase bs, desai vf. immunolpotentiating effects of azadirachta indica (neem) dry leaves powder in broilers, naturally infected with ibd virus. indian j exp biol 1998; 36(11): 1151–3. 7. lehner t. immunologi of oral disease. imunologi pada penyakit mulut. edisi 3. farida r, suryadhana ng, editors. jakarta: penerbit buku kedokteran egc; 1992. p. 112–5. 8. roeder a, kirschning cj, ropec ra, schaller m, weindl g, korting hc. toll-like receptors as key mediators in innate antifungal immunity. med mycol pub med 2004 december; 42(6): 485–98. 9. ratna d. daya hambat pertumbuhan c. albicans oleh perasan daun mimba (azadirachta indica juss). maj ked gigi (dent j) 2003 agustus; edisi khusus temu ilmiah nasional iii: 342–4. 10. diamond rd, caron a. lyman dr, wysong. disparate effects of interferon γ and tumor necrosis factor α on early neutrophil respiratory burst and fungicidal responses to candida albicans hyphae in vitro. j clin invest 1991; 87: 711–20. 11. newman sl, angela h. candida albicans is phagocy-tosed, killed, and processed for antigen presentation by human dendritic cells. infection and immunity 2001; 69(11): 6813–22. 12. ulmann bd, hadley m, wiriya c, anna ll, qiang z, luis av, jose l, lopez r, paul rg, michael cg. inducible defense mechanism against nitric oxide in candida albicans. eukaryotic cell 2004; 3(3): 715–23. 13. ray b, banerjee bd, sen pj, modulation of hmoral and cell-mediated immune responses by azadirachta indica (neem) in mice. indian j exp biol 1996; 34(7): 698–701. 14. tada h, eiji n, hidetoshi s, tshihiko w, takeshi m, tatsuji m, naohito o, hiroshi t, ken-ichiro s, sachiko a, kensuke m, shunji s, haruhiko t. sachoromyces cerevisiae and candida albicans derived mannan induced production of tumor necrosis factor alpha by human monocytes in a cd14 and toll-like receptor 4 dependent manner. mycrobiology and immunology, 2002; 46(7): 503–12. 15. wang je, warris a, ellingsen ea, fio t, espevick t, solberg r, verwe pe. involvement of cd14 and toll-like receptors in activation of human monocyte. infect immun pub med 2001; 69(4): 2402–6. 16. andor p, bodai l, rethi b, kenderessy-szaboa a, koreck a, szell m, beer z, bata-csorgo z, mafocsi m, rajnavolgyi e, dobozy a, kemeny l. expression and function of toll-like receptors 2 and 4 in human keratinocyte. international immunology 2003; 15(6): 721–30. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 62 students’ evaluation of preclinical simulation for all ceramic preparation (in faculty of dentistry universiti kebangsaan malaysia) natasya ahmad tarib and marlynda ahmad department of prosthodontics faculty of dentistry, universiti kebangsaan malaysia abstract the purpose of the study is to evaluate all ceramic crown (acc) preparations those were made by dental undergraduate students during the preclinical sessions. 104 plastic teeth were prepared by 4th year dental undergraduates during the preclinical session for acc crown examined. the teeth were placed on the frasaco arches and were mounted in the frasaco head. the preparations were examined for the tapering, presence of undercuts, incisal and cingulum reductions as well as preparation of shoulder margin. preparations were examined using hand instruments and visual. the sample size was 92 plastic teeth. most of the preparations were acceptable with acceptable placement and types of margins, adequate axial and incisal reductions and acceptable tapered of the axial walls. on the other hand, most of the teeth showed absence of cingulum wall. most of the crowns prepared by the students were acceptable. it showed that they understood the principles of crown preparation. cingulum wall preparation has to be given greater emphasis as it is important in the retention and resistance of the restoration. key words: crown preparation, all ceramic crown, preclinical simulation, skills correspondence: natasya ahmad tarib, department of prosthodontics, faculty of dentistry universiti kebangsaan malaysia, jalan raja muda abdul aziz, 50300 kuala lumpur, malaysia. e-mail: drtasyatarib@yahoo.com introduction significant changes in esthetics demand and dental materials significantly affect the dental education system. teaching conventional crown preparation cannot be considered adequate for modern dental practitioner. the purpose of every curriculum is to provide the graduating dental students with a well-rounded, balanced educational experience and the preclinical and clinical exposure necessary for competence.1 changes in dental materials significantly affect the treatment options a dentist could provide to the patients. the list of new procedures and materials includes bonded restorations and prostheses and new porcelains that are strong to resist high occlusal forces.2 dental students are responsible for the provision of invasive, irrevocable treatment of patients in their care. thus, they are required to develop the knowledge, skills and attitudes necessary to equip them to be competent and independent practitioners after their undergraduate years. dental educators can only give so much to the students; it is the students’ responsibility and effort to make the full use of it. the assessment of students’ knowledge and skills is important to educators as it is worth noting that their teaching methods are effective in producing a competent dental student. before dental students enter their clinical session, they have to undergo the preclinical session where they will perform the required tasks on frasaco or plastic teeth. the assessment of the preclinical performance is essential for patient’s safety, as well as to provide feedback on the teaching methods.3 fixed prosthodontic course in faculty of dentistry universiti kebangsaan malaysia (ukm) is introduced to the undergraduates during the 4th year of study. students are expected to complete certain tasks in the preclinical session before they are allowed to treat patients with fixed prosthodontic prostheses. the session usually runs for 5 weeks and it consists of 10 hours of lectures and 30 hours of simulation clinic together with 4 hours of video and live demonstrations. didactic part of the course is given, including case managements. the simulation clinic is performed with a system that consists of a manikin head connected to artificial jaws containing frasaco teeth. the head is attached to a torso which height can be adjusted. the system also includes a swiveling delivery unit, with dental handpieces and light. students are presented with video demonstrations on the particular task for the session. throughout the session, students are also shown the models, clinical and laboratory photos, as well as types of bur that they need for the preparation. at the end of the session, students will show the prepared teeth to their supervisors, and it then be evaluated using a validated criteria evaluation form (table 1). for the preparation of all ceramic crown (acc), students were given 1-hour of lecture and 6 hours of simulation clinic. they were shown a 45 minutes long video 63tarib and ahmad: students' evaluation of preclinical simulation for all ceramic preparation demonstration on how to prepare acc. the students were asked to evaluate their work as well, and the supervisor guided the student if there were major differences. the instructional outcome of this process was to develop selfassessment skills in a structured environment through a discriminative learning.4–6 it is believed that this process could help the students to be critical to their work. for the crown preparation, students were taught the ideal preparation, where 6° of tapered need to be achieved. tapers ranging from 0° to 16° have been suggested over the years as it will provide optimum retentive walls for the extracoronal restorations as the tapered and parallelism of the axial walls will contribute to retention and resistance of the restorations.7,8 the students were taught to hold the rotary cutting bur so the ideal taper and parallelism could be achieved. as for the margins, students were taught to prepare shoulder margin for the ceramic. the amount of other reductions such as incisal reduction was taught to the students and they had to prepare putty indexes to evaluate the amount of tooth reductions. guidelines of tooth preparation were followed from goodacre et al.9 the objective of this study is to evaluate the quality of all ceramic crown preparations done by dental undergraduate students during their preclinical sessions before entering clinical session. the hypothesis for this research is that the preparation done would be moderately acceptable as they had no experience in acc preparation, with close monitoring from the supervisors. materials and methods research information was given to 4th year (batch 2005/2006) ukm dental undergraduates before proceeding the preclinical session. written consent was obtained. a pilot study was conducted for calibration and the consistency was revealed using cohen’s kappa test. one hundred and four plastic teeth, upper right central incisor (11) that have been prepared by 4th year ukm dental undergraduates during the preclinical session for acc were collected. the formulated exclusion criteria are teeth with attached provisional restoration, teeth with fungus and teeth with damaged or fractured surfaces. the teeth were placed on the frasaco arches and then mounted in the frasaco head. the preparations were examined for tapering, presence of undercuts, reductions, location of margins and preparation of shoulder margin. a putty index was fabricated earlier using unprepared frasaco teeth to determine the amount of crown preparation reductions. these include incisal, buccal, palatal and table 1. criteria of assessment for crown preparation excellent 3 good 2 poor 1 unacceptable 0 path of insertion or withdrawal no undercut present in the preparation (s). slight undercut present in one of the preparation(s) wall. undercuts present in preparation(s), but the defect is correctable with additional preparation. undercut defect is major and must be corrected with root canal treatment or other means of treatment. resistance and retention optimum resistance and retention present in preparation(s). bridge preparation must draw. abutment(s) have sufficient resistance and retention. bridge may not draw. resistance and retention has been compromised because preparation(s) is over tapered. other form of resistance and retention is needed and can be attained with further preparations. resistance and retention has been compromised. other methods of treatment is needed to attain an acceptable situation i.e. root canal treatment. structural durability of preparation and restoration absolute structural durability has been produced. acceptable tooth preparation has been performed to permit functional restoration. conservative, no sharps or unsupported areas, and the surface of preparation is smooth. abutments need further preparation to be acceptable. adjacent tooth has been damaged. abutments are under or over prepared. preparation has sharp or unsupported areas and surface is rough. further preparation is needed. abutments are over prepared, tooth need root canal treatment or alternate preparation design is required. adjacent tooth has been severely damaged and need a restoration. finish line (margin) ideal margin placement, form, smoothness and dimension in all areas of the tooth. margin is adequately placed and identifiable, smooth and continuous with no steps. dimension of margin is not ideal but acceptable. margin is located on the sound tooth surface. incorrect placement of margin, with steps in some areas, dimension of margin is inadequate. margin is located on the existing restoration. further preparation is needed. incorrect placement of margin. dimension of margin is correctable with further periodontal surgery or orthodontic procedures. 64 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 62-66 interproximal reductions, together with biplanar reduction. students were reminded to preserve the cingulum anatomy while preparing for palatal and cingulum reductions. all margins should be shoulder margin. the location of the margin should be at the gingival margin. the preparations were examined using hand instruments and visual. evaluations were made based on the criteria of assessment for crown preparation. each preparation represented the effort of first time dental undergraduates preparing acc preparation. they worked utilizing their understanding from the lectures and the video demonstration, together with visual understanding of the models given throughout the preclinical session. all preparations were completed by the students under clinical setting in the simulation clinic, where the frasaco arches were mounted in the frasaco head and students used protective gloves, masks and glasses. students were also shown the correct body position for the preparation as well as how to angulate the bur while cutting the teeth to prevent creation of undercuts or over tapered of the axial walls. data collected were presented with descriptive statistics. frequencies analyses and cross tabulations were included in presenting the data obtained. results following the sample selection of 104 teeth, only 92 (88.46%) plastic teeth were qualified to be included (table 2). table 2. the result of samples selection criteria samples number percentage (%) attached provisional restoration teeth with fungus fractured or damaged teeth accepted teeth sample size 10 0 2 92 104 9.62 0 1.92 88.46 100.00 overall results were presented in table 3. majority of the teeth 77 (84%) had about right and acceptable tapered without the presence of undercuts. the remaining 15 teeth presented with poor and unacceptable tapered, either presented with undercut, or they were overly tapered. biplanar preparation was evidenced on 89 (97%) teeth. for axial and incisal reductions, majority of the teeth showed acceptable reductions, with 88 and 87 teeth respectively. the reductions would fulfill the structural durability component of the criteria. cingulum wall preparation only evidenced in 53% of the sample. margin of 90 teeth (98%) were found to be located either on the gingival margin or supragingival, while 2 of the teeth showed subgingival margin. margins located supragingivally and on gingival margin would be considered as acceptable. shoulder margin was expected to be prepared buccal-palatally. 90 (98%) teeth were prepared with shoulder margin buccally and 89 (97%) palatally. the remaining teeth were prepared with chamfer margin. examples of the teeth were presented in figure 1 to figure 4. table 3. overall results for crown evaluation based on criteria of assessment criteria sample excellent and good (acceptable) poor and unacceptable number (n) percentage (%) number (n) percentage (%) tapering of axial walls biplanar reduction structural durability •  axially •  incisally cingulum wall margin location types of margin •  buccally •  palatally 77 89 88 87 49 90 90 89 84 97 96 95 53 98 98 97 15 3 4 5 43 2 2 3 16 3 4 5 47 2 2 3 figure 1. margin located about 0.5 mm from the gingival margin. 65tarib and ahmad: students' evaluation of preclinical simulation for all ceramic preparation figure 4. acceptable tapered has been prepared in one of the sample. figure 2. shoulder margin were prepared around the tooth, continuously and smoothly. figure 3. acceptable cingulum wall height, contribute to retention and resistance, as well as structural durability. discussions cosmetic dentistry has becoming more and more demanding. patients nowadays opt for something that would improve their appearance. therefore, dental undergraduates have to be prepared with the competence in preparing ceramic restorations. preclinical training in ukm includes the preparation of all ceramic crown. the students must be competent and confident in treating patients with this type of crown. it is overwhelming to learn that most of the students were on the right track in terms of the overall preparation. however they need to be reminded of the importance of preservation of cingulum wall as it would contribute to the retention and resistance aspect of the extracoronal restoration. tapering of the axial walls ranging from 0° to 16° have been suggested over the years as it will provide optimum retentive walls for the extracoronal restorations.7,8 the tapered and parallelism of the axial walls will ensure that the retention and resistance of the restorations are not compromised. in the present study, 84% of the sample showed acceptable tapered. the presence of undercut or overtapered may be due to the angulation of the bur during tooth preparation. dental educators must stress out the importance of tapering to the undergraduates to fulfill one of the principles of tooth preparation. the presence of biplanar in tooth preparation would contribute to the structural durability of the extracoronal restoration. most of the teeth showed biplanar reduction in the preparation. other reductions such as axial and incisal are as important as the preparation of the margin. acceptable axial and incisal reductions were found in the present study, 96% and 95% respectively. such reductions are important as the optimum bulk of material must be present in the restoration to prevent any fracture or perforation after cementation. preservation of the cingulum would also contribute to the structural durability and prevention of pulpal exposure, as well as retention and resistance of the restoration. however, only 53% of the sample showed acceptable cingulum wall preparation. it may be due to the wrong angulation of the bur during preparation that lead to flat or unacceptable cingulum wall. the ideal location of the margin must be supragingival or on the gingival margin. these would prevent any plaque accumulation as oral hygiene is easy to maintain. it would also contribute to the maintenance of the periodontium and preservation of biologic width. almost all of the teeth presented with acceptable margin location, accounted for 98%. shoulder margin is expected to be prepared by the students buccal-palatally for all ceramic crown. shoulder margin would provide enough bulk of ceramic at the margin as to prevent any fracture that could lead to microleakage of the extracoronal restoration. only 2% of the sample did not present with shoulder margin buccally, and 3% palatally. most of the crowns prepared by the students were acceptable. it showed that they understood the principles of crown preparation. cingulum wall preparation has to 66 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 62-66 be given more stress as it is important in the retention and resistance of the restoration. acknowledgement we would like to thank those who helped and supported us with this project, including students and simulation laboratory staffs. special thanks to madam siti zubaidah anuar, celpad english language teacher of international islamic university of malaysia for proof reading this report. references 1. ferguson mb, sobel m, niederman r. preclinical restorative training. j dent educ 2002; 66(10):1159–63. 2. seals rr jr. restorative and prosthetic dental education: changes, challenges and opportunities. j prosthodont 1993; 2(2):73–4. 3. manogue m, brown g, foster h. clinical assessment of dental students: values and practices of teachers in restorative dentistry. medical education 2001; 35:364–70 . 4. knight gw, walcott am, guenzel pj. a paradigm for teaching a remedial preclinical course. j dent educ 1988; 52:558–61. 5. knight gw, guenzel pj. discrimination training and formative evaluation for remediating basic waxing skill. j dent educ 1990; 54:194–8. 6. feil ph, guenzel pj, knight gw. theoritical foundations of motor skill performance and their applications to dental education. j dent educ 1994; 58:806–12. 7. kent wa, shillingburg ht, duncanson mg. taper in clinical preparations for cast restorations. quintessence int 1988; 19:339–45. 8. shillingburg ht, hobo s, whitsett ld. fundamentals of fixed prosthodontics 3rd ed. chicago il: quintessence publishing; 1997. p. 119. 9. goodacre jc, campagni wv, aquilino sa. tooth preparations for complete crowns: and art form based on scientific principles. j prosthet dent 2001; 85:363–76.prosthet dent 2001; 85:363–76. 186 dental journal (majalah kedokteran gigi) 2021 december; 54(4): 186–189 original article an effective concentration of propolis extract to inhibit the activity of streptococcus mutans glucosyltransferase enzyme riyan iman marsetyo1, sagita putri andyningtyas1, chonny salsabilla zamrutizahra1, ivan nur fadela1, agus subiwahjudi2 and ira widjiastuti2 1undergraduate student, 2department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: according to riset kesehatan dasar (riskesdas) (2013) and the world health organisation (who), caries is still a global problem and highly prevalent in indonesia. caries is mainly caused by streptococcus mutans with virulence factors known as glucosyltransferase (gtf). the gtf enzyme contribute to the pathogenesis of caries by converting sucrose to fructose and glucan, which are then used in the formation of biofilms and dental plaques. natural propolis compounds containing flavonoids, terpenoids, saponins and tannins, can inhibit gtf enzyme activity. purpose: this study aimed to determine an effective concentration of propolis extract for inhibiting the s. mutans gtf enzyme activity. methods: this study used propolis extract at 14 μg/ ml, 16 μg/m and 1 μg/ ml to determine the inhibitory effect on s. mutans gtf enzyme activity. the gtf enzyme were obtained from the supernatant from s. mutans culture centrifugation. the gtf enzyme activity was measured using high-performance liquid chromatography (hplc) to calculate the fructose level. results: the mean fructose concentration at 14 μg/ml, 16 μg/ml, and 18μg/ml were 3.31%, 1.56%, and 0.29%, respectively. conclusion: the most effective concentration of propolis extract for inhibiting the effect of s. mutans gtf enzyme activity is 14 μg/ml. keywords: glucosyltransferase enzyme; medicine; propolis extract; streptococcus mutans correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47, surabaya 60132, indonesia. email: ira-w@fkg.unair.ac.id introduction dental caries is local damage to the teeth’s hard tissue from acid products produced by bacteria during carbohydrate fermentation. caries is generally chronic and progress slowly as a result of an imbalance between tooth minerals and biofilm.1 data from who show that 60–90% of children have dental caries, and almost 100% of adults have decay in their teeth.2 according to riskesdas (2013) , the national prevalence of dental and oral problems in indonesia was 25.9%, with the national dmf-t index reaching 4.6, which who categorises as ‘high’.3 streptococcus mutans (s. mutans) is the oral cavity’s normal flora and can be the main cause of caries formation and development due to the ability of biofilm formation by producing extracellular glucosyltransferase (gtf) enzyme, which are a virulence factor in the dental caries pathogenesis.4 the gtf enzyme convert sucrose into glucose and fructose and catalyses the formation of glucan from sucrose. glucan increases the adhesion and build-up of s. mutans bacteria on the enamel surface and is a crucial factor in forming dental plaque.5 inhibiting the activity of the gtf enzyme is one way to prevent caries using natural ingredients as an alternative. the development of natural ingredients is expected to have a more effective antibacterial ability and minimal side effects.6 gtf enzyme activity can be measured by the level of free fructose produced during the catalytic reaction. in contrast, glucose cannot be measured using high-performance liquid chromatography (hplc) because it is bound back by the gtf enzyme to form glucans. several methods can be used to measure the activity of the s. mutans gtf enzyme, such as using hplc or a fluorescence system to measure fructose levels.7,8 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p186–189 mailto:ira-w@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p186-189 187marsetyo et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 186–189 propolis is a natural resinous material collected by honeybees (apis mellifera) from various plants and mixed with their saliva and different other enzyme to build nests.9 there has been much research conducted on propolis. the outcomes show that propolis has good antibacterial and anti-inflammatory properties. it increases the body’s resistance to self-healing and phagocytic activity, stimulates immune cells, and inhibits growth prostaglandin synthesis.10 propolis has antimicrobial properties and can withstand more than 100 types of bacteria, viruses and fungi, including those that cause influenza, diphtheria, syphilis and tuberculosis.11 indonesia is considered to have a diverse range of local bee species, including the honey bee or apis mellifera that produce most of indonesia’s propolis.12 the properties of propolis compounds depend on the polyphenol and content of polyphenols, both of which are influenced by seasonal factors, local vegetation, place of origin, bee species and the condition of propolis in either fresh or preserved form.13 propolis is made up of resin and balsam (50–70%), wax and essential oils (30–50%), pollen (5–10%), and other components such as minerals, amino acids, vitamins a, b complex, e and biochemically active ingredients including bioflavonoids (vitamin p), phenols, and aromatic compounds.13 the active ingredients in propolis extract have an antibacterial ability that hinders the enzymatic activity of gtfs, such as flavonoids, tannins, terpenoids, and saponins. according to research conducted by achmad et al.,14 flavonoids can inhibit gtf activity from s. mutans bacteria. based on this research, further analysis is required to determine the effective concentration of propolis extract that can inhibit the activity of s. mutans gtf enzyme. materials and methods before conducting the experiment, ethical clearance was obtained from the health research ethical clearance commission universitas airlangga faculty of dental medicine (186/hrecc.fodm/ix/2017). the s. mutans bacteria used in this study were stock obtained from universitas airlangga faculty of dental medicine research centre. this s. mutans stock was inoculated into 7 ml of brain heart infusion broth (bhib) media, followed by a 24-hour incubation process at 37°c.8 the s. mutans culture in bhib was then centrifuged at 1500 rpm for 10 minutes at 4°c to produce the supernatant from which the gtf enzyme were extracted.11 meanwhile, propolis extract obtained from apis mellifera plantation beehives in lawang, malang regency, was extracted by a maceration method using 70% ethanol. it was then diluted with aquadest.12 in this research, 16 samples were split into three treatment groups and one control group. each group received four test tubes containing 0.875 ml of 0.25 m sucrose in ph 7, 0.2 m phosphate buffer, and 0.1 ml of gtf enzyme solution. in the control group, 0.025 ml of aqua dest was added, while in the treatment group, 0.025 ml propolis extract was added in 14 μg/ml, 16 μg/ml, and 18 μg/ml. an incubation process was carried out in all control and treatment groups at 37°c for 2 hours. enzyme activity testing was carried out at the faculty of pharmacy’s testing service unit, universitas airlangga. after incubation and a filtration process using 0.45 μm filter paper, the fructose level was determined using hplc by injecting 10 μl of treatment or control solution. before calculating the fructose level, the retention time of the fructose standard solution was measured. the retention time can be used as a guide for reading the chromatogram results. with knowledge of the solution’s area, a specific formula can be used to calculate the fructose level of the sample solution. furthermore, the fructose level is obtained by applying the following formula to read the area of fructose in the standard solution:8 concentration (%) = ��𝐴𝐶𝐴𝑆� 𝑥 � 𝑉𝐼𝑆 𝑉𝐼𝐶�𝑥 𝐹𝑃� 𝑥 100% 𝐾𝑆 notes: ac = sample area as = standard area vic = volume of sample injection vis = volume of standard injection ks = standard concentration fp = diluted factor the study measured means and standard deviations. data analysis was based on one-way anova testing, demonstrating a significance level of 0.05, followed by the post-hoc tukey hsd test. results the outcome of the hplc instrument’s calculations of the fructose levels revealed that the treatment group had lower fructose levels than the control group, as shown in table 1. the decrease in fructose levels occurred after the administration of propolis extract with concentrations of 14 μg/ml, 16 μg/ml, and 18 μg/ml. this indicates that the increase in the concentration of the propolis extract is inversely proportional to the decrease in fructose level material. the lowest fructose level compared to other treatment groups was found at 18 μg/ ml concentration. table 1. the mean and standard deviation of the tested groups’ fructose level tested groups n mean (%) sd control group (aqua dest) 4 6.09 0.67014 14 μg/ml propolis extract 4 3.31 0.30561 16 μg/ml propolis extract 4 1.56 0.21313 18 μg/ml propolis extract 4 0.29 0.06238 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p186–189 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p186-189 188 marsetyo et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 186–189 when performing the one-way anova analysis, there was a significant difference between treatment groups with a value of p = 0.00 (p < 0.05). in the post-hoc tukey hsd analysis, all treatment groups had a p-value < 0.05, suggesting significant differences between treatment groups, as shown in table 2. this indicated that propolis extracts at doses of 14 μg/ml, 16 μg/ml, and 18 μg/ml were found to suppress the activity of the s. mutans gtf enzyme. discussion the control group had the highest fructose levels, with an average of 6.09%. for the control group, the study used aqua dest, which tends to be neutral, so that the enzymatic reaction of the gtf was not inhibited. the increased activity of the gtf enzyme is related to the increased fructose levels. meanwhile, in the treatment groups, with 14 μg/ml, 16 μg/ml, and 18 μg/ml propolis extract, a decline in fructose level was detected. this was inversely associated with increased propolis extract concentration. this shows that gtf enzyme activity had been inhibited. the most significant level of inhibition was at a concentration of 18 μg/ml because, at this concentration, the lowest fructose level was obtained (0.29%). the most effective concentration of propolis extract in this experiment was 14 μg/ml, which was the lowest concentration level that could inhibit the s. mutans gtf enzyme activity. propolis extract has an inhibitory effect on s. mutans gtf enzyme activity because it contains active ingredients that inhibit enzymatic reactions. temperature, ph, the amount of enzyme and substrate present, and the presence of inhibitors and activators all seem to be factors that can alter enzyme activity level. enzymatic inhibitors are molecules that interact with enzyme and decrease enzyme activity. the decrease in enzyme activity occurs because of a metabolic imbalance that reduces enzyme reactions.15 this research was conducted using propolis extract as an inhibitor for gtf enzyme activity in s. mutans. at a concentration of 18 μg/ml, the lowest fructose level was obtained because the higher levels of active ingredients inhibited the gtf enzyme activity. the phytochemical tests showed that saponins were the active element with the highest concentration (2.48%). saponins have detergent-like properties and can increase cell membrane permeability without causing bacterial lysis. the increase of cell membrane permeability causes interference with the substances that pass through the cell membrane. saponins interact with sterols and form single ion channels that destabilise cell membranes. this results in the release of cellular proteins and enzyme, thereby inhibiting enzyme activity.16 furthermore, according to veloz et al.,17 flavonoids like pinocembrin and apigenin alter the arrangement of s. mutans biofilm structures. previous research has shown that polyphenols have an additive effect in small dosages, inhibiting biofilm production. the biofilm thickness was likewise reduced by pinocembrin and apigenin, which was linked to enzymatic gtf suppression. flavonoids have a and b rings that play a role in the hydroxylation of hydroxyl groups so that the basic arrangement of dna and trna is disrupted which results in the inhibition of nucleic acid and cell protein synthesis.18 the inhibition of propolis extract against the s. mutans gtf enzyme was also due to the terpenoid content as the active ingredient. however, the concentration of terpenoids was not as high as that of saponins and flavonoids (2.05%). these compounds were found to be effective inhibitors of gtf enzyme activity. terpenoids and saponins have the same target of action on cell membranes. terpenoids react with porins to form strong polymeric bonds, thus causing damage. porins act as transmembrane proteins, so that damage to this structure causes membrane permeability disorders. if protons enter the cell easily, it can trigger a decrease in ph and acid sensitisation. this condition can hinder the activity of gtf enzyme.19 in addition to saponins, flavonoids, and terpenoids, tannins are active components in propolis extract that inhibit gtf enzyme activity in s. mutans. the inhibition mechanism of these compounds is the result of taking over the substrate needed by bacteria, inhibiting energy metabolism, triggering redox reactions, and causing protein precipitation which can suppress the number of enzyme.6,20 tannin contains a hydroxyl group in its structure that can trigger a redox reaction, which is an oxidationreduction reaction in which electrons are exchanged between two chemical structures, and thus inhibits gtf enzyme activity.21 however, based on phytochemical tests, the concentration of tannins in propolis extract was only 0.24%. therefore tannin-inhibited gtf enzyme table 2. post-hoc tukey hsd analysis test results tested groups control group 14 μg/ml group 16 μg/ml group 18 μg/ml group control group 0.000* 0.000* 0.000* 14 μg/ml group 0.000* 0.000* 16 μg/ml group 0.003* 18 μg/ml group note: * indicates that the tested groups have meaningful differences dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p186–189 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p186-189 189marsetyo et al./dent. j. (majalah kedokteran gigi) 2021 december; 54(4): 186–189 activity is less significant when compared to other active ingredients. based on the research conducted, it was found that a decrease in fructose levels indicated inhibition of s. mutans gtf enzyme activity caused by the active ingredients in propolis extract as an inhibitor of enzymatic activity. therefore, it can be concluded that propolis extract at a concentration of 14 μg/ml effectively inhibits gtf enzyme activity in s. mutans. however, further research is needed into the role of each fraction of active ingredient in inhibiting the activity of the gtf enzyme. additionally, clinical research on propolis extract in the form of a topical agent or mouthwash for inhibiting the formation of dental plaque should be considered. references 1. yadav k, prakash s. dental caries: a review. asian j biomed pharm sci. 2016; 6(53): 1–7. 2. world health organization. oral health. 2012. available from: https://www.who.int/news-room/fact-sheets/detail/oral-health. accessed 2017 may 9. 3. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar 2013. jakarta: kementerian kesehatan republik indonesia; 2013. p. 110–8. 4. ozdemir d. dental caries and preventive strategies. j educ instr stud world. 2014; 4(4): 20–4. 5. nijampatnam b, casals l, zheng r, wu h, velu se. hydroxychalcone inhibitors of streptococcus mutans glucosyl transferases and biofilms as potential anticaries agents. bioorg med chem lett. 2016; 26(15): 3508–13. 6. isnarianti r, wahyudi ia, puspita rm. muntingia calabura l leaves extract inhibits glucosyltransferase activity of streptococcus mutans. j dent indones. 2013; 20(3): 59–63. 7. feng l, yan q, zhang b, tian x, wang c, yu z, cui j, guo d, ma x, james td. ratiometric fluorescent probe for sensing streptococcus mutans glucosyltransferase, a key factor in the formation of dental caries. chem commun (camb). 2019; 55(24): 3548–51. 8. amanda a, kunarti s, subiwahjudi a. daya hambat aktivitas enzim glukosiltransferase (gtf) streptococcus mutans oleh ekstrak temulawak (curcuma xanthorrhiza roxb.). conserv dent j. 2017; 7(1): 32–6. 9. dwiandhono i, effendy r, kunarti s. the thickness of odontoblastlike cell layer after induced by propolis extract and calcium hydroxide. dent j (majalah kedokt gigi). 2016; 49(1): 17–21. 10. kartika w.p cd, kunarti s, subiyanto a. genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast bhk-21 cell cultures. dent j (majalah kedokt gigi). 2015; 48(1): 16–21. 11. pribadi n, yonas y, saraswati w. the inhibition of streptococcus mutans glucosyltransferase enzyme activity by mangosteen pericarp extract. dent j (majalah kedokt gigi). 2017; 50(2): 97–101. 12. yuanita t. the cleanliness differences of root canal walls after irrigated with east java propolis extract and sodium hypoclorite solutions. dent j (majalah kedokt gigi). 2017; 50(1): 6–9. 13. huang s, zhang c-p, wang k, li gq, hu f-l. recent advances in the chemical composition of propolis. molecules. 2014; 19(12): 19610–32. 14. achmad mh, ramadhany s, suryajaya fe. streptococcus colonial growth of dental plaque inhibition using f lavonoid extract of ants nest (myrmecodia pendans): an in vitro study. pesqui bras odontopediatria clin integr. 2019; 19: 1–9. 15. balbaa m, el ashry esh. enzyme inhibitors as therapeutic tools. biochem physiol. 2012; 1(2): 103. 16. arabski m, węgierek-ciuk a, czerwonka g, lankoff a, kaca w. effects of saponins against clinical e. coli strains and eukaryotic cell line. j biomed biotechnol. 2012; 2012: 286216. 17. veloz jj, alvear m, salazar la. antimicrobial and antibiofilm activity against streptococcus mutans of individual and mixtures of the main polyphenolic compounds found in chilean propolis. biomed res int. 2019; 2019: 7602343. 18. slobodníková l, fialová s, rendeková k, kováč j, mučaji p. antibiofilm activity of plant polyphenols. molecules. 2016; 21(12): 1717. 19. jeon j-g, pandit s, xiao j, gregoire s, falsetta ml, klein mi, koo h. influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on streptococcus mutans physiology and survival within mixed-species oral biofilms. int j oral sci. 2011; 3(2): 98–106. 20. sendamangalam v, choi ok, kim d, seo y. the anti-biofouling effect of polyphenols against streptococcus mutans. biofouling. 2011; 27(1): 13–9. 21. wahjuningrum da, subijanto a. the antibiofilm activity of extract propolis against biofilm enterococcus faecalis as herbal medicine potential in root canal treatment. esp endod soc philipp. 2014; 8: 15–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i4.p186–189 https://www.who.int/news-room/fact-sheets/detail/oral-health https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i4.p186-189 �� vol. 43. no. 1 march 2010 the comparison of minocycline oral-rinse and gel on pocket depth eka fitria augustina department of periodontics faculty of dentistry airlangga university surabaya indonesia abstract background: �n�ecti�n disease is still c�nsidered as a �r��inent disease in �any devel��ing c��ntries, like �nd�nesia. ��e ��st �ral in�ecti�n disease is �eri�d�ntitis. des�ite scaling and r��t �lanning as t�e �ain t�era�y, �in�cycline as adj�nct t�era�y �as already �een �sed ��r �eri�d�ntitis. ��ere are a l�t �� �edia �sed, s�c� as �ral rinse and gel. �any researc�es even �ave als� s����n t�at t�e �se �� �in�cycline as adj�nct t�era�y can decrease in�la��ati�n in �eri�d�ntitis. like tetracycline, �in�cycline as an anti in�la��at�ry and antic�llagenase is als� c�nsidered t� �e very e��ective ��r t�e treat�ent �� �eri�d�ntitis. �edia �� �in�cycline t�at are availa�le are gel, �i�er, and �ral rinse, as t�e ne��est �ne. purpose: ��e ��r��se �� t�is researc� is t� e�a�ine t�e c���aris�n �� 0.2% �ral rinse �in�cycline and 2% �in�cycline gel t� red�ce t�e ��cket de�t�. method: ��e sa��les ��ere divided int� t��� gr���s, t�e �irst gr��� �sing �ral rinse and t�e sec�nd �ne �sing gel a�ter scaling. result: ��ere ��as n� statistically signi�icant di��erence �et��een t�e gr��� ��it� �in�cycline gel and �ral rinse. conclusion: ��e a��licati�n �� 2% �in�cycline gel �r 0.2% �in�cycline ���t� ��as� a�ter scaling and r��t �lanning �as t�e sa�e e��ect in red�cing ��cket de�t�.. key words: �yn�cycline gel, �ral rinse, ��cket de�t� abstrak latar belakang: penyakit in�eksi �asi� �er��akan kas�s yang �en�nj�l di �anyak negara �erke��ang, se�erti �nd�nesia. �n�eksi r�ngga ��l�t yang �anyak terjadi adala� �eri�d�ntitis. selain tera�i �ta�a yait� skeling dan r��t �lanning, �engg�nakan �in�siklin se�agai tera�i ta��a�an tela� �anyak dig�nakan, se�erti ��at k���r dan gel. banyak �enelitian �en�nj�kkan �a���a �engg�naan �in�siklin se�agai tera�i ta��a�an da�at �en�r�nkan keradangan �ada �asien �eri�d�ntitis. se�erti tetrasiklin, �in�siklin se�agai anti keradangan dan anti k�lagenase, sangat e�ekti� se�agai �era��atan �eri�d�ntitis. �edia �in�siklin yang �anyak dig�nakan di antaranya yait� gel, �i�er, dan ��at k���r yang ter�ar�. tujuan: ��j�an �enelitian adala� �engeta��i �er�andingan antara �engg�naan 0,2% ��at k���r �in�siklin dan 2% �in�siklin gel �nt�k �eng�rangi kedala�an ��ket �eri�d�ntal. metode: sa��el di�agi �enjadi d�a gr��, gr�� �erta�a �engg�nakan ��at k���r, dan kel����k ked�a �engg�nakan gel, setela� tera�i skeling. hasil: �idak ada �er�edaan yang signi�ikan antara kel����k �in�siklin ��at k���r dan gel. kesimpulan: �in�siklin gel dan ��at k���r sa�a-sa�a e�ekti� dala� �eng�rangi kedala�an ��ket. kata kunci: �in�siklin gel, ��at k���r, kedala�an ��ket c�rres��ndence: eka fitria augustina, c/o: department of periodontics dentistry, faculty of dentistry airlangga university, jln. prof mayjend moestopo no. 47 surabaya, indonesia. e-mail : firstyaugustina@yahoo.com research report introduction periodontitis actually can be defined as an infection disease attacking periodontal tissue caused by specific microorganism with many clinical manifestation, started from bleeding, inflammation, bone resorbtion, dental instability, to dental extraction. periodontitis, moreover, manifests in various infections with many clinical manifestation of medical history as well as with many treatment responses depended on many factors, including �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 21-25 bacteria, response of host immune and also environmental factor.1 periodontitis is usually related with the increasing of the number of bacteria pathogen, such as p�r��yr���nas gingivalis, prev�tella inter�edia, bacteri�des ��rsyt�s, and actin��acill�s actin��icete�c��itans that has already been published widely. periodontal disease can be classified into advanced chronic periodontitis, refractory periodontitis, and aggressive periodontitis.2 therefore, eliminating plaque and calculus is considered to be the most prominent stage in periodontitis therapy even though the common recurrence caused by periodontophatogen invading into gingival epithel, cementum, and dentin tubuli cannot be cleaned mechanically. many researches have already shown that the use of antibiotics either systemically or locally was very useful to support the early periodontal therapy, involving scaling and root planning. antibiotics can be given systemically per oral or locally like in gel, encapsulated, mouthwash, and others.3 this fact becomes the base of the critical framework that antibiotics is needed to support the success of the treatment for periodontal disease, either systemically or locally. in addition, the use of antibiotics locally is by inducing it directly into the pocket in order to make the level of medicine in pocket increasing, as a result, the medicine can also penetrate into root surface and periodontal smooth tissue, which then is expected to be more effective than systemically antibiotics.2 locally antibiotics can also prevent and minimize many side effects caused by the use of antibiotics systemically.4 minocycline, as antibiotics derived from the second generation of tetracycline, has already been improved for therapy of periodontal disease since it can effectively attack periodontal phatogen.4,5 minocycline, has wide spectrum that can actively attack negative and positive gram bacteria that cause chronic periodontitis. the character of minocycline actually is bacteriostatic which can constrain the protein synthesis from bacteria.6 minocycline also has anti-inflammatory character which can constrain apoptosis (cell death) by increasing tnf alpha and managing cytokine regulation. the effect of this anti-inflammatory charater is also influenced by direct action of t cell in microglia which can decrease the capability of t cell in connecting with microglia, and then can affect the producing of in signal mediator of cell t and microglia.6 there are many treatments for periodontal disease that have already used the application of minocycline in many media. the most available media of minocycline is gel, but the media of minocycline in mouthwash has been improved, especially for curing reccurent apthous stomatitis (ras).8 based on the results of many researchers, it is known that the use of minocycline locally can also reduce the pocket depth, bleeding during probing, and improve clinical adhesion.8 locally antibiotics is a local anti-microbe that is effective for periodontophatogen, as anti inflammation.2 based on the above explanation, the writer would like to analyze which kind of minocycline is more effective in reducing the pocket depth. material and method this research is a clinical research for measuring the depth of periodontal pocket before and after the application of minocycline. this experiment was done in periodonsia clinic of faculty of dentistry airlangga university, from november 2007 to januari 2008. samples are involving patients who must meet the following criteria: 30–50 years old, male or female, having no allergic history with minocycline (tetracycline), not having pregnancy or breastfeeding, not having period, having chronic periodontitis with 3–6 mm periodontal pocket depth, not systemically (per oral) using antibiotics or anti-inflammation and application of gel or mouthwash minimally about 30 days, in good condition or having no systemic abnormality from the beginning to the end of measuring. the samples are divided into two groups, the first group with the application of 2% minocycline gel (figure 1) and the second one with the application of 0.2% minocycline mouthwash. based on the previous study, 2% of minocycline is the best concentration for reducing the pocket depth compared with other concentrations.12 furthermore, 2% minocycline gel is made from 2 grams of pure minocycline powder mixed with 100 grams of unguintum gel (poly prophylene glycol). this mixing process must be done immediately before it is applied and then put into spuit used for inducing it into the pocket. minocycline mouthwash 0.2% is made by 0.2 grams of pure minocycline powder mixed with 100 ml sterile aquadest, and then is used for mouth-washing. figure 1. application of 2% minocycline gel in periodontal pocket. for both groups, the measuring of the pocket depth has been done before scaling. after scaling, the application of 2% minocycline gel is done in the first group and redone one week later.11 the measuring of the periodontal pocket depth is redone in the fourth and sixth weeks. meanwhile, the second group is instructed to wash their mouth with 0.2% minocycline twice a day for seven days.11 the measuring of the pocket depth was also done in the fourth and sixth ��augustina: the comparison of minocycline oral rinse and gel weeks. before the fourth week, the measuring of the pocket depth cannot be done since the condition of periodontal table 1. the t test result of the difference of the pocket depth in the group with the application of mouthwash n x sd se signif. 2 tailed t before the application of mouthwash after the application of mouthwash in the fourth week before the application of mouthwash after the application of mouthwash in the sixth week after the application of mouthwash in the fourth week after the application of mouthwash in the sixth week 15 15 15 15 15 15 3.6667 2.6000 3.6667 2.3333 2.6000 2.3333 0.61721 0.63246 0.61721 0.48795 0.63246 0.48795 0.15936 0.12599 0.16330 0.12599 0.000 0.000 0.104 5.172 8.367 1.174 table 2. the t test result of the difference of the pocket depth in the group with the application of gel n x sd se signif 2 tailed t before the application of gel after the application of gel in the fourth week before the application of gel after the application of gel in the sixth week after the application of gel in the fourth week after the application of gel in the sixth week 15 15 15 15 15 15 4.3333 3.1333 4.3333 2.8000 3.1333 2.8000 0.61721 0.74322 0.61721 0.56061 0.74323 0.56061 0.15936 0.19190 0.15936 0.14475 0.19190 0.14475 0.000 0.000 0.55 6.874 11.500 2.092 table 3. the t test result of the difference of the pocket depth between the group with the application of mouthwash and that with the application of gel n x sd se signif 2 tailed t after the application of mouthwash in the fourth week after the application of gel in the fourth week after the application of mouthwash in the sixth week after the application of gel in the sixth week 15 15 15 15 1.0667 1.2000 1.3333 1.5333 0.79881 0.67612 0.61721 0.57640 0.20625 0.174517 0.15936 0.13333 0.610 0.334 -0.521 -1.000 tissue is still weak. therefore, if probing is done before the fourth week, it will affect the process of recovery. �� dent. j. (maj. ked. gigi), vol. 43. no. 1 march 2010: 21-25 result the difference of the pocket depth before and after the application of 0.2% minocycline mouthwash can be seen in table 1. there was significant difference between before and after the application of mouthwash in the fourth week (p = 0.000). similarly, there was also significant difference between before and after the application of mouthwash in the sixth week (p = 0.000). however, there was no significant difference after the application of mouthwash in the fourth week and in the sixth week (p = 0.104). thus, it can be concluded that there was significant difference in the sample group before and after the application of mouthwash. the comparison of the difference of the pocket depth between before and after the application with minocycline 2% gel can be seen in table 2. there was significant difference between the group with the application of 2% gel in the fourth week compared with that before the application (p = 0.000). similarly, it is known that there was also significant difference between the group with the application of 2% gel in the sixth week compared with that before the application (p = 0.000). nevertheless, there was no significant difference between the group with the application of 2% gel in the fourth week compared with that in the sixth week (p = 0.55). therefore, it can be concluded that there was significant difference in the sample group before and after the application of gel. the comparison of the difference of the pocket depth between the group with the application of minocycline 2% gel and that with the application of minocycline mouthwash can be seen in following table 3. there was no significant difference between the group with the application of mouthwash and that with the application of gel in the fourth week, about p = 0.610 (p < 0.05). similarly, there was no significant difference between the group with the application of mouthwash and that with the application of gel in the sixth week, about p = 0.334. thus, there was no significant difference between the sample group with the application of mouthwash and that with the application of gel. discussion periodontal disease is marked by the inflammation and the dental supporting tissue damage. this inflammation is marked by the progressive damage of periodontal ligament, alveolar bone, followed by pocket forming (the gingival sulcus pathologically becomes deeper), and gingival recession occurred (the clinical decreasing of gums).2 this periodontal treatment is aimed to maintain the dental function as well as to prevent and reduce the severity of the disease. the success of this treatment can be obtained by decreasing or eliminating bacteria pathogen, and by repairing the capability of tissue in maintaining and repairing itself.2 many clinical researchs about the use of antibiotics for treating periodontal disease have been conducted. the use of antibiotics even can be done as the single method of the treatment or combined with scaling and root planing in periodontitis treatment. the use of antibiotics is aimed to eliminate bacteria pathogen in periodontal pocket, while scaling and root planing are aimed to repair the gingival health by eliminating all factors that can cause the inflammation on the dental surface. gel antibiotics in the low concentration can directly applied in the dental surface without causing side effects like in systemically using.2,10 in this research, scaling and root planning together with the measuring of the pocket depth was done in both groups, one group with the application of mouthwash and the other one with the application of gel. the result of the research showed that the pocket depth in the group with the application of mouthwash was decreasing in the fourth week after the application (table 1). this result is similar with the result of the previous study in which the use of minocycline as the additional therapy together with scaling and root planing can improve the recovery of periodontitis. one of the recovery processes is the decreasing of the pocket depth.11 meanwhile, unlike before the application of mouthwash, the pocket depth in the group was decreasing after the application in the sixth week (table 1). the result is similar with the result of the previous studies in which the additional therapy of minocycline with the application of mouthwash is proved to be able to decrease the inflammation.11 since it can cause the eliminating of bacteria, the recovery of periodontitis can become better than that only with scaling and root planing.3 however, after the application of mouthwash, there was no statistically significant difference between the pocket depth in the fourth week and that in the sixth week. but, based on the data, the bigger decreasing of the pocket depth occurred in the sixth week. unlike before the application, in the fourth week, the decreasing of the pocket depth was about 1.0667 mm. meanwhile, in the sixth week the decreasing was about 1.3334 mm (table 1). it is may be caused by the scaling before the application of mouthwash can eliminate the amount of subgingiva microorganism and can affect the health of periodontal tissue.2 from the fourth week to the sixth week the sample group is predicted to be able to maintain oral hygiene well, therefore, in the sixth week the decreasing of the pocket depth would be better than that in the fourth week. by improving the health of the tissue, the pocket depth would be decreasing because of the improving of the adhesion. the decreasing of the pocket depth actually is clinically important since if the pocket is deep, plaque control will be difficultly done, thus, the abnormality can possibly recurrent.2 there was pocket depth decreasing after the application of 2% minocycline gel in the fourth week (table 2). this result was similar with the result of the previous finding that minocycline gel is effective to be applied directly in the periodontal pocket in order to effectively attack microorganism relating with periodontitis, such as ��augustina: the comparison of minocycline oral rinse and gel p�r��yr��inas gingivalis, �. ��rsyt�ia, p. inter�edia, and a. c��itans.12 the significant difference of the decreasing of the pocket depth between before and after the application of 2% minocycline gel, moreover, occurred in the sixth week (table 2). it is also known that with the application of minocycline gel the reparation of periodontal tissue can involve the decreasing of bleeding on probing (bop) and the decreasing of the pocket depth in the sample group compared with that in the control group.12 based on the comparison of the sample group using the application of gel before and after the application in the sixth week, furthermore, there was the decreasing of the pocket depth (table 2). this result was similar with the result of the previous researches that minocycline gel is effectively used for the additional therapy of periodontal disease, especially for eliminating microorganism, decreasing the pocket depth, decreasing the score of bleeding index, and repairing the adhesion.3 it is also caused by the effective character of minocycline in eliminating the growth of negative gram periodontal pathogen, by the high concentration of gingival crevicular fluid (gcf), and by the slow release in the periodontal pocket.13 based on the comparison of the sample group using the application of gel in the fourth week and in the sixth week (table 2) there was no significant difference among them. however, the decreasing of the pocket depth was bigger in the sixth week. in the fourth week, the decreasing of the pocket depth was only about 1.200 mm compared with that before the application. in the sixth week, the decreasing of the pocket depth was about 1.533 mm compared with that before the application (table 1). the reason is possibly because the patients can maintain oral hygiene as instructed by operator, thus, the decreasing of the pocket depth was better in the sixth week. there was no significant different between the sample group using the application of mouthwash. nevertheless, it is also known that the decreasing of the pocket depth in the group with the application of gel was better, about 1.0667 mm, than the one with the application of mouthwash, about 1.200 mm (table 3). the reason is because with the application of gel, the medicine can directly penetrate into periodontal pocket and can eliminate bacteria causing periodontitis in the pocket. therefore, if the amount of bacteria causing the damage of periodontal tissue is decreasing, the recovery process will be better. besides that, minocycline also has capability in constraining the protein synthesis of bacteria, and in attacking periodontal pathogen, such as p. gingivalis, p. inter�edia, f. n�cleat��, and a. c��itans. 6 there was no decreasing of the pocket depth in the sample group with the application of mouthwash and the one with the application of gel in the sixth week (table 3). however, the decreasing of the pocket depth in the group using the application of gel was better, about 1.533mm, than the one using the application of mouthwash, about 1.337 mm. it indicates that scaling and root planing therapies together with the use of minocycline gel can reduce the pocket depth as same as the result of the previous researches. it is related with bacteriostatic effect and resistance activities of metaloproteinase matrix (mmp) derived from minocycline. minocycline considered as analog of tetracycline, actively attacks bacteria with wide spectrum from periodontal pathogen. the high concentration of local minocycline has anti-infection effect when penetrates into biofilm of plague compared with that used systemically.7 compared with the application of mouthwash, the application of gel was more useful, cause of directly penetrated into the pocket, and then the release occurred slowly. but, the decreasing of the pocket depth in both groups are the same. based on the result of this research, it can be concluded the application of 2% minocycline gel or 0.2% minocycline mouth wash often scaling and root planning has the same effects in reducing pocket depth. refferences 1. kamma jj, slots j. herpesviral-bacterial infection in aggressive periodontitis. j clin periodontol. 2003, 30(5): 420–6. 2. newman mg, takei hh, carranza fa. carranza�s clinicalcarranza�s clinical periodontology. 10th ed. philadelphia: wb saunders co; 2006. p. 798–802, 803–11. 3. niederman r. minocycline gel gives adjunctive improvement to scale and polish. summary evidence based dentistry 2000; 2(3): 654. 4. cortelli jr, rodrigues sm, aquino dr, ricardo lh, pallos d. longitudinal clinical evaluation of adjunct minocycline in the treatment of chronic periodontitis. j periodontol 2006; 77(2): 161–6. 5. giuliani f, hader w, wee yong vw. minocycline attenuates t cell and microglia activity to impair cytokine production in t cell-microglia interaction. journal of leucocyte biology 2005; 78: 135–43. 6. perno m. pharmacotherapy in periodontal therapy. 2007. p. 6–13. 7. oringer rj, al-shammari kf, aldredge wa, iacono vj, eber rm, wang hl, berwald b, nejat r, giannobile wv. effect of locally delivered minocycline microspheres on markers of bone resorption. j periodontol 2002; 73(8): 101–8. 8. paquette d, oringer r, lessem j, offenbacher s, genco r . l o c a l l y d e l i v e r e d m i n o c y c l i n e m i c r o s p h e r e s f o r t h e treatment of periodontitis in smokers. j clin periodontol 2003; 30(9): 787–94. 9. meinberg at, barnes cm, dunning dg, reinhardt ra. comparison of conventional periodontal maintenance versus scaling and root planning with subgingival minocycline. j periodontol 2002; 73(2): 167–73. 10. yaffe a., herman a, bahar h, binderman l. combined local application of tetracycline and biophosphonate reduces alveolar bone resorption in rats. j periodontol 2003; 74: 1038–42. 11. gorsky m, epstein j, rabenstein s, elishoov h, yarem n. topical minocycline and tetracycline rinses in treatment of recurrent aphthous stomatitis: a randomized cross over study. dermatology online journal 2007; 2. 12. mc coll e, patel k, dahlen g, tonneti m, graziani f. supportive periodontal therapy using mechanical instrumentation or 2% minocycline gel: a 12 month randomized, controlled, single masked pilot study. j clin periodontol 2006; 33(2): 141–50. 13. grenier d, huot mp, mayrand d. ion-chelating activity of tetracycline and its impact on the susceptibility of actinobacillus actinomycetemcomitans to these antibiotics. american society for microbiology 2000; 44(3): 763–6. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base guide for authors the dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental-related disciplines. articles are considered for publication on condition that they have not been previously published or submitted for publication by other academic journals. articles can be classified as original articles, case reports or review articles that inform readers about current issues, innovative cases and reviews in the field of dentistry. they should also promote scientific advancement, education and dental practice development. all manuscripts submitted to the journal must be written in english. since manuscripts will be published in english, it is the author’s responsibility to ensure that the language of submitted material is of appropriate clarity and quality. manuscripts must not exceed the maximum number of words, must not contain numbers in the form of figures and must be free of typing errors. articles must be between ten and twelve pages in length. manuscripts must be typed on a word processer and submitted in the form of a soft copy file. the obligatory times new roman font should be size 14 pt for the title and 12 pt for all other sections of text. headlines should be written in bold type with any latin names presented in italics. manuscripts must be of a4 format typed with one and a half space between lines and a 2.5 cm (1 inch)-wide margin. authors are strongly advised to follow the manuscript preparation guidelines provided below. all original articles, case reports, and review articles must contain:  title: brief, specific, informative and written in english. it must contain a maximum of ten words (not exceeding a total of 40 letters and spaces) with the first word starting with a capital letter.  name(s) of author(s): should include author(s)’ full name(s), mailing address(es) for proofs, name(s) and address(es) of the department(s) to which the work should be attributed listed sequentially using a number (1) symbol. example: jamal bin razak1, matsuo hamada2, ninuk hartati3 and harold whitfield4 1 department of oral and maxillofacial surgery, faculty of dentistry, university of malaya, kuala lumpur, malaysia 2 department of prosthodontics, school of dentistry, hiroshima university, hiroshima, japan 3 department of dental public health, faculty of dental medicine, universitas airlangga, surabaya, indonesia 4 department of endodontics, school of dental and health sciences, the university of melbourne, melbourne, australia  abstract: a concise (maximum 250 words), one-paragraph description in english with single space formatting. footnotes, references, and abbreviations are not to be included in the abstract.  the abstract in original articles should consist of a single paragraph containing background:, purpose:, methods:, results: and conclusion: written in bold type.  the abstracts in case reports should consist of background:, purpose:, case(s):, case management: and conclusion: typed in bold within one paragraph.  the abstracts in review articles should be divided into background:, purpose:, review:, and conclusion: typed in bold within one paragraph.  keywords: 3-5 words and/or a phrase must be provided below the abstract. key standard scientific phrases or words must be provided in english. each word/phrase in the keywords section should be separated by a semicolon (;).  correspondence: details of the lead author with complete mailing and e-mail addresses (consisting of full name, name of institution, mailing address, telephone number, fax number and email address). correspondence is followed by the following sections according to type of article (original articles, case reports, or review articles) as follows: i. contents in original articles: the original articles should contain the following sections: introduction, materials and methods, and results.  introduction: background to the problem, formulation and purpose of the work, case or review and prospects for future research. the rationale of the study is stated together with the main problem under investigation, any resulting findings and, finally, the references consulted.  materials and methods: clear description of materials consulted, experiments conducted and methods applied. these are deemed necessary to facilitate duplication of the research and re-assessment of its validity. reference should be made to any novel methods employed. research ethics relating to the use of animal and/or human subjects must also be outlined in accordance with academic convention.  results: presented accurately and concisely in a logical sequence with the minimum number of tables and illustrations necessary to summarize the most important observations. undue repetition of text and tables should be avoided. tables must be presented horizontally (without vertical line separation) to facilitate understanding of their content. calculation results should be reported in si units. mathematical equations should be clearly expressed. mathematical symbols unavailable on computer keyboards may be hand-written using a soft lead pencil. decimal numbers should be identifiable by the appropriate location of a decimal point (.). tables, illustrations, and photographs should be cited consecutively within, but presented separately to, the manuscript text. titles and detailed explanations of figures should appear in the legends corresponding to illustrations (figures, graphs) rather than within the illustrations themselves. all non-standard abbreviations used must be explained in the footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction: outlines the background and formulation of the problem, the purpose of the work, case or review and prospects for the future. the rationale for the study is stated, a number of references identified and the main problem and unusual clinical cases highlighted or the use of cutting-edge technology in a clinical case.  case(s): contains a clear and detailed description of the case(s) presented, including: anamnesis and clinical examinations. the specific system of tooth nomenclature: zygmondy, world health organization or universal must be clearly stated.  case management: presented accurately and concisely in chronological order supported with figures and a detailed description of the research methodology employed. iii. contents in review articles literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in review articles are followed by headline topics and overviews to be discussed. all original articles, case reports, and review articles must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver superscript no et al. style. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, books, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communication, are not acceptable as references. only those sources cited in the text should appear in the reference list. the names of authors must be written in a consistent manner throughout the text. the numbers and volumes of journals must be cited, with edition, publisher, city and page numbers of textbooks also included. references to downloaded internet sources must include the time of access and web address. any abbreviations of journal titles must comply with dental and medical index conventions. original articles and case report should include at least ten references. review articles should include more than 30 references. citation format for journal articles: 1. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. citation format for textbooks: 1. blom a, warwick d, whitehouse m. apley & solomon’s system of orthopaedics and trauma. 10th ed. oxford: crc press; 2018. p. 455–89. citation format for proceedings: 1. virbanescu ca. bone augumentations with autologous bone in oral implantology. in: 2nd international conference on dental health and oral hygiene. london, uk: allied academies; 2019. p. 45. citation format for thesis and dissertations: 1. alharbi i. study the effects of cigarette smoke on gingival epithelial cell growth and the expression of keratins. thesis. québec: université laval; 2015. p. 22–24, 42. citation format for electronic publications (web page): 1. world health organization. obesity and overweight. world health organization media centre fact sheet. 2020. available from: https://www.who.int/news-room/fact-sheets/ detail/obesity-and-overweight. accessed 2020 nov 10. citation format for patents: 1. zhang z, liu r, zou s, wu l, zeng y, deng x. digital integrated molding method for dental attachments. united states; us20210000575a1/2021. figures or illustration all figures, illustrations and photos must be concise, relevant, informative, referenced and contained in a file (high resolution jpeg, png or tiff format at least 300dpi). the maximum number of figures, illustrations, photos and tables contained in the original articles and review articles is 4 (four), while that for case reports is 8 (eight). all figures, illustrations and photos must be separated from the manuscript text. images should be referred to in the text and figure legends should be listed at the end of the manuscript, citing illustrations in numerical order (figure 1, figure 2, etc.) as they appear in the text. written permission must be obtained for the reproduction of content previously published in copyrighted material, including: tables, figures and quoted text exceeding 150 words in length. signed patient release forms are required in cases of photographs featuring identifiable persons. a copy of all written permission and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, tailor articles to the available space in order to ensure conciseness, clarity and stylistic consistency. all manuscripts accepted, together with their accompanying illustrations, become the permanent property of the publisher. as such, they may not be published elsewhere in full or in part, in print form or electronically, without the written permission of the publisher. all data presented and all opinions or statements expressed in the manuscript remain the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal (majalah kedokteran gigi) accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. tables tables should be submitted in the same format as the article and embedded in the document where the table should be cited. if table(s) are presented in excel format, they must be copied and pasted into the manuscript file. in extreme circumstances, excel files can be uploaded as supplementary files. however, this is not advised as they will not be accepted should the article subsequently be approved for publication. tables should be self explanatory, containing data that is not duplicated within the text and figures. online submission  the author should first register as author and/or offer to be a reviewer via the following address: https://e-journal.unair. ac.id/mkg/about/submissions#onlinesubmissions  the author can also submit the manuscript by sending email via the following account: dental_journal@fkg.unair.ac.id 115115 dental journal (majalah kedokteran gigi) 2020 september; 53(3): 115–121 research report the pore size of chitosan-aloe vera scaffold and its effect on vegf expressions and woven alveolar bone healing of tooth extraction of cavia cobaya sularsih department of dental materials, faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: pore size of scaffolds affects cellular activity, stimulates angiogenetic factors of vascular endothelial growth factor (vegf), synthesises new blood vessels to regulate migration and proliferation, and accelerates alveolar bone healing of tooth extraction. purpose: this study aims to analyse the pore size of chitosan-aloe vera scaffold and its effects on vegf expression and woven alveolar bone healing of tooth extraction of cavia cobaya. methods: 36 male cavia cobaya, aged 3-3.5 months were divided into six groups: negative control groups (without scaffold), positive control groups (chitosan scaffold), and treatment groups (chitosanaloe vera scaffold) on 7and 14-day observations. histopathological examination was performed to account the woven alveolar bone areas, and immunohistochemical examination was conducted to examine vegf expressions on endothelial cells. data was analysed using a one-way analysis of variance (anova) and least significant difference (lsd) test (p<0.05). scaffold pore size examination was performed with scanning electron microscope (sem) with 250x and 500x magnification. results: chitosan-aloe vera scaffold was found to have open pore interconnectivity, the largest pore size was 138.9 μm, while the smallest was 110.5 μm and average pore size was 134.85 μm. the highest expression of vegf was observed in the treatment group on days 7 (11.5 ±1.39) and 14 (15.28±1.78), while the largest woven alveolar bone was observed in the treatment group on days 7(17.83±1.47) and 14 (37.67±3.65). statistically, there was a significant difference between control groups and the treatment groups (p=0.000; p<0.05). conclusion: chitosan-aloe vera scaffold has pore characteristics increasing vegf expressions and woven alveolar bone areas. keywords: aloe vera; chitosan; scaffold pore size; vegf; woven alveolar bone correspondence: sularsih, department of dental materials, faculty of dentistry, universitas hang tuah, jl. arief rachman hakim no. 150 surabaya 60111, indonesia. email: sularsih@hangtuah.ac.id introduction alveolar ridge bone resorption often occurs after tooth extraction. vertical and horizontal dimensional changes occur during the first three months after tooth extraction.1 alveolar bone resorption will remain and even can cause more than 40 – 60% ridge volume loss during the first three years post tooth extraction.2–4 the damage of the alveolar bone, unfortunately, can cause failure or the instability of dentures or dental implant placement.4,5 ridge preservation and grafting materials can be used to prevent bone loss and to regenerate alveolar bones. although grafting materials do not completely prevent bone loss, it could reduce the severity of the loss.1,6 grafting materials currently used are autograft, bovine xenograft, allografts and alloplast. however, the use of bovine grafts to the tooth socket still cannot bring satisfactory results. there are developments of a combination of natural and synthetic graft materials or polymers used to achieve alveolar bone resorbtion.1,2 actually, bone tissue engineering innovation has recently developed scaffold that can be absorbed by the body, such as chitosan polymers material, in order to accelerate the replacement of damaged tissue as well as to proliferate, differentiate and maintain tissue function.7 the application of chitosan to the tooth extraction socket of rattus norvegicus can increase the number of osteoblasts, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 mailto:sularsih@hangtuah.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 116 sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 fibroblasts and type i collagen on 7 and 14 days of observation.8 one percent of chitosan gel is also known to be able to increase bone morphogenetic protein-2 (bmp-2) expressions of rattus norvegicus during bone formation after tooth extraction on 7, 14 and 21 days.9 aloe vera is a natural plant that can be used as a biogenic stimulator to stimulate and accelerate alveolar bone regeneration. aloe vera has active compounds that play a role in the bone healing process. it is a compound protein named alloktin that is synergistic with the other components like amino acids, enzymes, alkaloids, flavonoids, saponins, collagen, vitamins, calcium, potassium and polysaccharides mannan.10–12 hence, in the previous study, the use of aloe vera scaffold containing acemannan increased bone marrow stromal cells (bmscs), vegf, bmp-2 proliferations, alkaline phosphatase (alp) activity, bone sialoprotein, mineralisation and osteopontin expressions on bone healing of tooth extraction. aloe vera can be considered as a natural candidate for bone regeneration.13 scaffold made of the combination of chitosan and aloe vera is assumed to have a synergistic effect on tooth extraction sockets to regenerate the alveolar bone and prevent alveolar bone resorption. chitosan has osteoconductivity that can support the attachment of bone-forming cells. aloe vera was also shown to have high osteoinductivity and osteogenity that can stimulate the differentiation of osteoprogenitor cells into osteoblast cells. thus, it also can trigger new bone formation and bone regeneration.10,14 the characteristic porosity and pore size of scaffold are known to be able to affect cellular activities including stimulating new cell growth and cell adhesion as well as supporting cell proliferation and angiogenetic factors so that it will accelerate the bone healing process. vegf is the most dominant growth factor considered as an angiogenetic factor released by endothelial cells, which can synthesise new blood vessels to regulate the migration, proliferation and differentiation processes of endothelial cells and the formation of new bone.15,16 furthermore, this study aims to analyse the pore size of chitosan-aloe vera scaffold and its effects on vegf expression and woven alveolar bone healing of tooth extraction of cavia cobaya (c. cobaya) on 7and 14-day observations. materials and methods this study was an experimental study with a randomised post-test only control group design. the chitosan powder used in this study had a deacetylation degree of >75–85% and a molecular weight of 50,000–190,000 da (sigma, product number: 448869, lot number: mkbh7256v). 1% chitosan gel (w/p) was made by dissolving 1 gram of chitosan powder in 100 ml of 2% acetic acid (ch3cooh). it was stirred using a magnetic stirrer, neutralised with naoh solution, centrifuged at a speed of 2000 rpm for 30 minutes and filtered with filter paper. aloe vera extract gel was made by the maceration method. aloe vera was cleaned, and its thorns were removed. aloe vera was blended until smooth, dried with a freeze dry machine, dissolved with 70% ethanol for 48 hours and stirred for 30 minutes with a magnetic stirrer. the maceration results were filtered with whatman grade 1 filter paper and accommodated with erlenmeyer. the filtrate was evaporated with a vacuum rotary evaporator and dissolved using 3.5% sodium carboxymethyl cellulose (na-cmc). subsequently, chitosan-aloe vera scaffold was made by mixing the chitosan gel and the ethanol extract of aloe vera gel in a ratio of 1:1. the combination of chitosan and aloe vera gel was put into the scaffold mould after it was put in a freezer at a temperature of -80° c for 24 hours. afterwards, freeze drying was carried out at a temperature of 95°–103° c for 72 hours.17 the scaffold was removed from the mould and sterilised with a uv clean bench steriliser. scaffold pore size examination was performed with a scanning electron microscope (sem) tool (jcm-5700, jeol, tokyo, japan) with 250x and 500x magnification. ethical approval for this research was obtained from the ethical committee of airlangga university, faculty of dental medicine with number 012 / hrecc.fodm / iii / 2018. in this study, the experimental animals used were 36 male c. cobaya aged 3–3.5 months and weighed 300–375 grams. the sample size was determined by using lameshow’s minimum sample size formula. the sample was selected blind-randomly into control and treatment groups, which were divided into six groups. each group consisted of 6 c. cobaya divided into six groups: on day 7: negative control groups (without scaffold administration), positive control groups (chitosan scaffold administration), and treatment groups (chitosan-aloe vera scaffold administration). on day 14: negative control groups (without scaffold administration), positive control groups (chitosan scaffold administration), and treatment groups (chitosan-aloe vera scaffold administration). the left mandibular incisor of c. cobaya was extracted. the tooth socket was irrigated with sterile aquadest water. the scaffold was applicated in the tooth socket to the apical end of the tooth and sutured with non resorbable sutures. c. cobaya from each group were sacrificed after 7 and 14 days. the mandibular bone in the interdental region of the mandibular incisors was cut and soaked in a fixation solution using 10% formalin buffer. a decalcification process was carried out with 10% of ethylenediaminetetraacetic acid (edta) (onemed dental, medika industri, indonesia) for 4 weeks. subsequently, paraffin blocks were made and cut with microtome in a buccolingual plane parallel to the tooth vertical axis into sections of 4-micro thickness. histopathological examination was conducted with haematoxylin eosin (he) staining (sigma aldrich, merck kgaa, darmstadt, germany) to account the woven alveolar bone areas using image raster software version 3 (developed by pt miconos, yogyakarta, indonesia). light microscope (nikon e100, tokyo, japan) on 100x magnification on five different fields of view by two observers was performed. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 117sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 the immunohistochemical examination was conducted using a 3.3’-diaminobenzidine stain kit (dab) (sigma aldrich, merck kgaa, darmstadt, germany). the antibody monoclonal vegf (ab38909, abcam, united kingdom) was used to measure the vegf expressions in the apical third of teeth, which were viewed using a light microscope (nikon e100, tokyo, japan) in 400x magnification on five different fields of view by two observers. the data was analysed by statistical package for the social sciences 21.0 software (spss for windows, chicago, usa). data analysis was performed using the normality test with the shapiro wilk test. a homogeneous variation test was conducted to find out data variation in the groups with levene’s test (p>0.05) continued with oneway anova and multiple comparison lsd test (p<0.05) to determine the different pairs of the groups. results the results of pore size examination of chitosan-aloe vera scaffold using sem tool with 250x and 500x magnifications showed the largest scaffold pore size was 138.9 μm, the smallest scaffold pore size was 110.5 μm, and the average scaffold pore size was 134.85 μm. open pore interconnectivity of chitosan-aloe vera scaffold was found. there was interconnection between pores on the scaffold. the pore size and connectivity of scaffold can be seen in figure 1. based on figure 2, the vegf expression of endothelial cells in the treatment group with chitosan-aloe vera on 7 and 14 days increased more than negative control groups and positive control groups with chitosan scaffold. the vegf expression in the negative control group (without scaffold administration), positive control group with chitosan scaffold and treatment group with chitosan-aloe vera scaffold in 7 days can be seen in figures 2a, c, e. the vegf expression in negative control group (without scaffold administration), positive control group with chitosan scaffold and treatment group with chitosan-aloe vera scaffold in 14 days can be seen in figures 2b, d, f. the width of woven alveolar bone in the treatment group with chitosan-aloe vera in 7 and 14 days increased more, as pointed by red arrows, than the negative control group and positive control group with chitosan scaffold. figure 3a, c, e shows the width of woven alveolar bone in the negative control group (without scaffold administration), positive control group with chitosan scaffold and treatment group with chitosan-aloe vera scaffold in 7 days. figure 3b, d, f shows the width of woven alveolar bone in the negative control group (without scaffold administration), positive control group with chitosan scaffold and treatment group with chitosan-aloe vera scaffold in 14 days based on table 1 and figure 4, the results of the analysis showed that treatment groups with chitosan-aloe vera scaffold could significantly increase the vegf expressions and the width of woven alveolar bone areas in 7 and 14 days compared with the negative control group and positive control group with chitosan scaffold (p<0.05). the highest vegf expression and the largest woven alveolar bone were found in the treatment groups with chitosan-aloe vera scaffold. the vegf expression and width of woven alveolar bone in the positive control group with chitosan scaffold could increase more than negative control groups without scaffold administration. figure 1. the pore size of the chitosan-aloe vera scaffold using sem tool with the magnifications of 250x (a) and 500x (b): the red lines shows the pore size of scaffold. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 118 sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 a b c d e f figure 2. the vegf expression on endothelial cells. (a) k(-) group on day 7; (b) k(-)group on day 14; (c) k(+) groups with chitosan scaffold on day 7; (d) k(+) groups with chitosan scaffold on day 14; (e) the treatment group with chitosan-aloevera scaffold on day 7; (f) the treatment group with chitosan-aloe vera scaffold on day 14, with 400x magnification; the orange arrow shows vegf expression on endothelial cells. a b c d e f figure 3. the woven alveolar bone areas. (a) k(-) group on day 7; (b) k(-)group on day 14; (c) k(+) groups with chitosan scaffold on day 7; (d) k(+) groups with chitosan scaffold on day 14; (e) the treatment group with chitosan-aloe vera scaffold on day 7; (f) the treatment group with chitosan-aloe vera scaffold on day 14, with 100x magnification; the orange arrow shows the width of woven alveolar bone areas. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 119sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 discussion in the development of tissue engineering, the use of chitosan scaffold in medical applications has been mostly modified by many crosslinks with other ingredients such as collagen, gelatin, hydroxyapatite or growth factors to increase osteoinduction and osteointegration resulting in the acceleration of the bone healing process. the single use of chitosan as scaffold has inadequate pore size, poor porosity and close interconnectivity to facilitate the transportation of nutrients, growth factors and blood vessels.7,18,19 scaffold made of the combination of chitosan and aloe vera, based on the sem test results, has a mean pore size of 134.85 μm. the chitosan-aloe vera scaffold has a good pore interconnectivity or open pore interconnectivity. the recommended minimum pore size for scaffold is 100 μm, which enables the scaffold not only to provide a good and suitable microenvironment for the proliferation of osteoblasts and mesenchymal stem cells as well as the attachment and migration of cells, but also to be capable of nutrient diffusion. open pore interconnectivity can also increase tissue vascularisation and oxygenation, which support the bone healing process. pore size and pore interconnectivity of scaffold affect cellular activity, stimulate angiogenetic released by endothelial cells and also synthesise new blood vessels to regulate migration, proliferation and new bone formation.20,21 in our study, the use of chitosan-aloe vera scaffold could increase vegf expressions as well as woven alveolar bone areas in the 7and 14-day observation compared to the use of chitosan scaffold. moreover, the alveolar bone healing process of tooth extraction actually begins with a haemostasis phase, which activates platelets and blood clotting factors to form a blood clot that fills the socket. the cytoplasm of platelets contains α granules containing growth factors such as platelet derived growth factor (pdgf) and transforming growth factor-β (tgf-β). these molecules can activate and attract polymorphonuclear cells (pmns), macrophages and endothelial cells to the socket. macrophages are the main cells that play an important role in the healing process involving phagocytosis and secretion of cytokines and growth factors that modulate the bone healing process.22,23 in the final inflammatory phase, macrophage cells begin to stimulate the increase of induced growth factors such as pdgf, fibroblast growth factor (fgf), vegf, tgf-β and transforming growth factor-α (tgf-α).23,24 vegf is the most dominant angiogenetic factor released by endothelial cells to synthesise new blood vessels to regulate the migration, proliferation and differentiation processes.15,16 hence, the vegf expressions in the treatment groups with the administration of the chitosan-aloe vera scaffold in this study tended to increase. the increasing of vegf expressions in those treatment groups after day 7 was not even significantly different from that in the groups with the administration of the chitosan scaffold on day 14. it may be caused by the inflammatory phase still ongoing before the 7th day, so a time lag is needed to lead to the proliferation phase. as a result, the release of growth factors that induce vegf has not been maximised yet.22 differentiated osteoblasts on the apical third region of the tooth socket form a bone matrix, and immature or woven alveolar bones begin from the apical region of the socket to the lateral wall of the socket on day 7 and then extend to the centre of the socket leading to trabecular bones. along with the healing process of the alveolar bone after the complete table 1. the mean and standard deviation of vegf expressions and woven bone areas in all groups groups n vegf expression (cells/lp) woven bone areas (μm2) p  x ±sd x ±sd k (-) on day 7 6 6.50 ±1.64a 10.50±1.23a 0.000* k (-) on day 14 6 8.33±1.75a 17.83 ±2.99c k (+) chitosan on day 7 6 8.00±1.79a 12.67±1.63b k (+) chitosan on day 14 6 11.60±1.72b 27.17±3.98d chitosan+a.vera on day 7 6 11.50±1.39b 17.83±1.47c chitosan+a.vera on day 14 6 15.28±1.78c 37.67±3.65e note: * significant at α=0.05 (one-way anova) abc different superscripts show that there were differences between groups (multiple lsd comparisons) 0 10 20 h-7 h-14 ve g f ex pr es si on s the duration of treatment 0 20 40 60 h-7 h-14w ov en b on e ar ea s (µ m 2) the duration of treatment k (-) k (+) chitosan chitosan+a.vera figure 4. diagram of vegf expressions and woven bone area in k(-) groups, k(+) groups with chitosan scaffold and treatment groups with chitosan-aloe vera scaffold on 7 and 14 days. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 120 sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 tooth extraction, the area of woven alveolar bone will be greater.25 this can also be seen in the results of this study on the 7th day when the formation of woven alveolar bone had occurred in both the control groups and the treatment groups. the width of woven alveolar bone areas had even been getting greater in all groups from day 7 to day 14. furthermore, angiogenesis is a key component in the bone healing process. during the bone healing process the formation of new blood vessels is also needed in metabolic callus regeneration for the supply of nutrients, oxygen, growth factors, cytokines, osteoblast precursors and osteoclasts.16 in the proliferation phase, for instance, angiogenesis plays an important role during the migration of endothelial cells into proliferating new tissue. in a normal alveolar bone healing process post tooth extraction the proliferation phase is started with the onset of hypoxic conditions causing an increase in intracellular concentration of the active form of a gene regulating protein called hypoxia-inducible factor 1 (hif-1). this condition triggers endothelial cells and macrophages to release angiogenetic factors in response to inflammation and increased hif-1. subsequently, endothelial cells and macrophages will secrete angiogenetic factors such as basic fibroblast growth factor (bfgf or fgf-2) and acid fgf (afgf or fgf-1), pdgf, vegf, and tgf-β. bfgf then will produce mature endothelial cells and synthesise new blood vessels. afterwards, cell surface receptors will bind to vegf and fgf which are activated by kinase receptors so that they can regulate the migration, proliferation and differentiation processes of endothelial cells.15,16 thus, in the control groups of this study, the mean number of vegf expressions increased from day 7 to day 14 although there was no significant difference. this means that in post tooth extraction conditions the bone healing process without scaffold administration in tooth sockets that have tissue damage is a hypoxic condition triggering bfgf and vegf secreted by endothelial cells. in contrast, the number of vegf expressions in the groups with the administration of the chitosan scaffold and that in the groups with the administration of chitosan-aloe vera scaffold increased after day 7, and the increasing of vegf expressions in those groups was even significantly different between those on 7 and 14 days. this indicates that the process of angiogenesis in the treatment groups supports the process of alveolar mineralisation. c h i t o s a n a s a n a t u r a l b i o p o l y m e r c o n t a i n i n g glycosaminoglycans is known not only to have unique properties, biocompatible and biodegradable characteristics but also to be able to stimulate the release of important growth factors in bone healing such as fgf, pdgf, tgfβ1, vegf, bmp-2 and collagen type 1.8,9,26 hence, in this study the vegf expressions and the width of woven alveolar bone areas on 7 and 14 days in the groups with the administration of the chitosan scaffold and those in the groups with the administration of chitosan-aloe vera scaffold were increasing and significantly different from those in the control groups. the results of this study also revealed that vegf expressions and the width of woven alveolar bone areas on 7 and 14 days in the groups with the administration of chitosan-aloe vera scaffold were significantly different from those in the control groups and the groups with the administration of the chitosan scaffold. the highest average and increase of vegf expressions and the width of woven alveolar bone areas on 7 and 14 days were found in the groups with the administration of chitosan-aloe vera scaffold compared to the other groups. the increased vegf expressions in the use of aloe vera is known to be through the phosphatidylinositol 3-kinase (pi3k/akt), extracellular-signal-regulated kinase (erk 1/2) and endothelial nitric oxide synthase / nitric oxide (enos/no) pathways.27,28 hif-1 alpha binds to the hypoxic response element in the vegf gene promoter which stimulates transcription. vegf binds to two vegf receptors, vegfr-1 / flt (fms-like tyrosine kinase) and vegfr-2/kdr. vegfr-2 activation is linked to mechanisms that depend on the formation of multi-protein complexes including vegfr-2, pi3k, as well as ve-cadherin and β-catenin proteins. vegf binds to serine receptors on endothelial cells then initiates vegfr-2 autophosphorylation followed by activation of angiogenesis enzymes such as mapk and akt / kinase b protein (pkb) to induce cell migration. erk 1/2 pathway plays an important role in the growth and differentiation mechanisms of endothelial cells during the process of angiogenesis in wound healing.16,27 subsequently, through the erk 1/2 pathway and the c-jun n-terminal kinase (jnk) pathway, the chitosan-aloe vera scaffold will activate macrophages with m2 modulation more dominant than m1. in m2 modulation, macrophages will activate m2 which stimulates anti-inflammatory cytokines, il-2 and il-10. in addition, macrophages also induce cell migration and proliferation by activating activator protein-1 (ap-1), which then activates fgf, vegf and bmp-2 playing a role in stimulating osteoblast formation.27,29 bonding components of the lectin protein or aloktin with aloe vera polysaccharides will activate the complement system and increase coagulation to prevent loss of blood clots in bone healing.30,31 the interactions of the protein components, such as lectin, polysaccharides, anthraquinone and betasitosterol are then identified as angiogenetic factors in the healing process since they stimulate human umbilical vein endothelial cells (huvec).27,32 polysaccharides and flavonoids contained in aloe vera can also increase angiogenic factors in bmscs.33 the administration of aloe vera to tooth sockets and alveolar bone defects can increase the expression of runt-related transcription factor 2 (runx2) that plays a role in inducing pre-osteoblast differentiation into mature osteoblasts. as osteoblasts increase, the expression of osteoprotegerin (opg) released by osteoblasts increases as does alp activity. as a result, osteoclastogenesis can be prevented through receptor activation of nuclear factor kappa b ligand (rankl)/receptor activator of nuclear dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 121sularsih/dent. j. (majalah kedokteran gigi) 2020 september; 53(3): 115–121 factor kappa b (rank)/opg system signals. runx2 then induces osteoblasts to secrete osteopontin, osteocalcin and type 1 collagen, which influence the mineralisation and bone healing processes.32–34 therefore, it can be concluded that chitosan-aloe vera scaffold has pore characteristics increasing vegf expressions and woven alveolar bone areas on alveolar bone healing of tooth extraction on c. cobaya. references 1. jamjoom a, cohen r. grafts for ridge preservation. j funct biomater. 2015; 6(3): 833–48. 2. k resnoadi u. the increasing of fibroblast growth factor 2, osteocalcin, and osteoblast due to the induction of the combination of aloe vera and 2% xenograft concelous bovine. dent j (majalah kedokt gigi). 2012; 45(4): 228–33. 3. sheikh z, sima c, glogauer m. bone replacement materials and techniques used for achieving vertical alveolar bone augmentation. materials (basel). 2015; 8(6): 2953–93. 4. beck tm, mealey bl. histologic analysis of healing after tooth extraction with ridge preservation using mineralized human bone allograft. j periodontol. 2010; 81(12): 1765–72. 5. javed f, ahmed h, crespi r, romanos g. role of primary stability for successful osseointegration of dental implants: factors of influence and evaluation. interv med appl sci. 2013; 5(4): 162–7. 6. holzwarth jm, ma px. biomimetic nanofibrous scaffolds for bone tissue engineering. biomaterials. 2011; 32(36): 9622–9. 7. ariani md, matsuura a, hirata i, kubo t, kato k, akagawa y. new development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. dent mater j. 2013; 32(2): 317–25. 8. sularsih. type 1 collagen on the wound healing process of dental extraction with different molecular weight of chitosan. in: international seminar 2nd dentisphere, current concept in dentistry. surabaya: hang tuah university; 2013. p. 46–52. 9. sularsih s, wahjuningsih e. expression of bone morphogenetic protein-2 after using chitosan gel with different molecular weight on wound healing process of dental extraction. dent j (majalah kedokt gigi). 2015; 48(2): 53–8. 10. silva ss, popa eg, gomes me, cerqueira m, marques ap, caridade sg, teixeira p, sousa c, mano jf, reis rl. an investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine. acta biomater. 2013; 9(6): 6790–7. 11. sudarshan r, annigeri rg, sree vijayabala g. aloe vera in the treatment for oral submucous fibrosis a preliminary study. j oral pathol med. 2012; 41(10): 755–61. 12. salinas c, handford m, pauly m, dupree p, cardemil l. structural modifications of fructans in aloe barbadensis miller (aloe vera) grown under water stress. plos one. 2016; 11(7): 1–24. 13. boonyagul s, banlunara w, sangvanich p, thunyakitpisal p. effect of acemannan, an extracted polysaccharide from aloe vera, on bmscs proliferation, differentiation, extracellular matrix synthesis, mineralization, and bone formation in a tooth extraction model. odontology. 2014; 102(2): 310–7. 14. rahman s, carter p, bhattarai n. aloe vera for tissue engineering applications. j funct biomater. 2017; 8: 1–17. 15. yin s, ellis de. first-principles investigations of ti-substituted hydroxyapatite electronic structure. phys chem chem phys. 2010; 12(1): 156–63. 16. saran u, gemini piperni s, chatterjee s. role of angiogenesis in bone repair. arch biochem biophys. 2014; 561: 109–17. 17. sularsih, soetjipto, rahayu rp. the fabrication and characterization of chitosan-ethanol extracted aloe vera scaffold for alveolar bone healing application. j int dent med res. 2019; 12(4): 1376–81. 18. martínez a, blanco md, davidenko n, cameron re. tailoring chitosan/collagen scaffolds for tissue engineering: effect of composition and different crosslinking agents on scaffold properties. carbohydr polym. 2015; 132: 606–19. 19. yuliati a, kartikasari n, munadziroh e, rianti d. the profile of crosslinked bovine hydroxyapatite gelatin chitosan scaffolds with 0.25% glutaraldehyde. j int dent med res. 2017; 10(1): 151–5. 20. chiara g, letizia f, lorenzo f, edoardo s, diego s, stefano s, eriberto b, barbara z. nanostructured biomaterials for tissue engineered bone tissue reconstruction. int j mol sci. 2012; 13(1): 737–57. 21. holzapfel bm, reichert jc, schantz jt, gbureck u, rackwitz l, nöth u, jakob f, rudert m, groll j, hutmacher dw. how smart do biomaterials need to be? a translational science and clinical point of view. adv drug deliv rev. 2013; 65(4): 581–603. 22. la r java h. o ra l wound hea l ing: cell biology a nd cl in ica l management. singapore: wiley-blackwell; 2012. p. 195–9. 23. khullar s, a m, datta p. healing of tooth extraction socket. heal talk. 2012; 4(5): 37–9. 24. vo tn, kasper fk, mikos ag. strategies for controlled delivery of growth factors and cells for bone regeneration. adv drug deliv rev. 2012; 64(12): 1292–309. 25. vieira ae, repeke ce, de barros ferreira s, colavite pm, biguetti cc, oliveira rc, assis gf, taga r, trombone apf, garlet gp. intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. plos one. 2015; 10(5): 1–22. 26. kung s, devlin h, fu e, ho ky, liang sy, hsieh yd. the osteoinductive effect of chitosan-collagen composites around pure titanium implant surfaces in rats. j periodontal res. 2011; 46(1): 126–33. 27. majewska i, gendaszewska-darmach e. proangiogenic activity of plant extracts in accelerating wound healing a new face of old phytomedicines. acta biochim pol. 2011; 58(4): 449–60. 28. sargowo d, handaya ay, widodo ma, lyrawati d, tjokroprawiro a. aloe gel enhances angiogenesis in healing of diabetic wound. indones biomed j. 2011; 3(3): 204–15. 29. chantarawaratit p, sangvanich p, banlunara w, soontornvipart k, thunyakitpisal p. acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class ii furcation defect model. j periodontal res. 2014; 49(2): 164–78. 30. van der ende j, van baardewijk lj, sier cfm, schipper ib. bone healing and mannose-binding lectin. int j surg. 2013; 11(4): 296–300. 31. yagi a. putative prophylaxes of aloe vera latex and inner gel as immunomodulator. j gastroenterol hepatol res. 2015; 4(5): 1585–98. 32. akev n, can a, sütlüpmar n, çandöken e, özsoy n, özden ty, yanardaǧ r, üzen e. twenty years of research on aloe vera. j pharm istanbul univ. 2015; 45(2): 191–215. 33. wang xf, zhang yk, yu zs, zhou jl. the role of the serum rankl/opg ratio in the healing of intertrochanteric fractures in elderly patients. mol med rep. 2013; 7(4): 1169–72. 34. zhou y, wu y, jiang x, zhang x, xia l, lin k, xu y. the effect of quercetin on the osteogenesic differentiation and angiogenic factor expression of bone marrow-derived mesenchymal stem cells. plos one. 2015; 10(6): 1–21. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i3.p115–121 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i3.p115-121 114 new insight in pediatric dentistry: preventive dentistry in allergy management protocol seno pradopo1 and haryono utomo2 1 department of pediatric dentistry 2 department of oral biology faculty of dentistry airlangga university surabaya indonesia abstract the relationship between oral health and systemic diseases had been abundantly studied, however, mostly were related to adult such as cerebrovascular disease, cardiovascular disease, diabetes mellitus etc. nevertheless, it was still uncommon that oral health also related to allergic disease. the field of pediatric dentistry is mostly related to preventive dentistry (i.e. prophylactic procedures, preventive orthodontic etc., but rarely related to preventive medicine such allergy prevention in children. allergic diseases develop out of a close interaction between genetic predisposition and environmental triggers, and progress continuously since infancy regarding to the allergic march. concerning to the partially developed immunity in children, children are more susceptible to infection and allergic diseases than adults. unfortunately, infection and allergic diseases are interrelated; infection impaired allergy and vice versa. poor oral health is closely related to infection; however, improving oral health is not included in allergy management protocol. in order to anticipate the future, dentist or especially pediatric dentist should be able to review about basic children immunity and oral mucosal immunity. additionally, it is essential to explain to the parents and medical practitioners who are not familiar to this new paradigm. the objective of this study is to review articles related to children’s oral health and allergic symptoms. regarding to the successful oral management of allergic symptoms, the propensity that improving oral health could be included in children’s allergy management protocol is likely. key words: pediatric dentistry, oral health, allergy management protocol correspondence: seno pradopo, c/o: bagian ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: pradopo_seno@yahoo.com; dhoetomo.indo.net. telp. (031) 5030255. introduction allergic diseases develop out of a close interaction between genetic predisposition and environmental triggers. they are common in childhood, with at least 25% of children being affected by some atopic illness. in addition, the impacts of these disorders are disturbing regular activities such as absences children from schools, and parents from works, also increasing costs of health care and medications. moreover, there are unwanted side effects from allergy medications that impaired the quality of life such as drowsiness (i.e. antihistamines) and suppression of growth (i.e. systemic glucorticoids).1 nevertheless, with recent allergy management, food avoidance and immunotherapy, allergic diseases in children still continue until adult or even elderly. the connection between oral health and general health had been studied by abundant literatures.2–4 nevertheless, the relationship with allergy is still in controversy. evidence-based cases revealed that improving oral health diminished urticaria,2 rhinitis,5 and sinusitis symptoms;6,7 however, improving oral health is not included in allergy management protocol. chronic infections, either caused by virus or bacteria are able to stimulate hypothalamic-pituitary-adrenal axis (hpa axis), which released cortisol. excessive cortisol act as an immunosuppressive agent which also facilitate shift to t-helper 2 (th2) cytokines profile which related to allergy. 8 in addition, bacteria and their toxins are able to trigger mast cells and basophil degranulation, and macrophages stimulation via complement system and toll-like receptors (tlrs). inflammatory mediators released by these reactions may deteriorate allergic symptoms.9 general practitioner dentists and pediatric dentists may be the first health personnel who examine children’s oral health. consequently, they also have the responsibility to explain the importance of optimal oral health to general health, and also allergic diseases. in addition, it is mandatory to review basic medical sciences including microbiology and immunology to understand the connection between oral health and allergic diseases. the purpose of this study is to conduct a literature study regarding the connection of oral health and allergic disease. moreover, in the future, hopefully, improving oral health could be included in allergy management protocol; thus increase the contribution of pediatric dentistry in allergic 115pradopo and utomo: new insight in pediatric dentistry prevention and obtaining optimal general health, thus also promotes optimal growth and development in children. basic immune system there are two important part of immune system, the innate immunity and the acquired immunity. the simplest way to avoid infection is to prevent the microorganisms from gaining access to the body. the first line of defense which preventing from infection are skin, sweat, saliva, nasal secretions etc. microbial and other foreign particles trapped within mucus secreted by the lining membranes are removed by mechanical stratagem such as coughing and sneezing.9 if penetration still occurs, two further innate defensive operations come into play, those are bactericidal enzymes and phagocytes (i.e. polymorphonuclear neutrophil and macrophages). other parts of innate immunity are the complement system, natural killer and eosinophils. there are also other killing mechanisms which involved, for instance nitric oxide (no), defensins and cathepsin g.9,10 for faster recognition and facilitate phagocytosis, antibodies were synthesized in part of the acquired immunity. there are several antibodies in the immune system that termed as immunoglobulin, the immunoglobulin m (igm), igg, iga, igd and ige furthermore, there are two kinds of immunity mechanisms involved, the cell-mediated immunity and humoral immunity. antibodies are mostly produced in humoral immunity which needs the interactions of t-cells and b-cells lymphocytes.10 allergic diseases allergic diseases are a group of ailments that share common pathogenic mechanisms including ig-e mediated immediate hypersensitivity reactions to environmental allergens and a more fundamental pathogenic activation of th2 immune responses. included among these diseases are upper and lower allergic airway diseases (rhinitis, rhinosinusitis, asthma), ocular allergic diseases, eczema and food and drug allergies. they frequently cluster together in the same family and are transmitted vertically from generation to generation, consistent with a strong genetic component in disease pathogenesis.11 allergic diseases are related to the subpopulation of t-cells so called the t-helper 2 (th2) cells. these th2 lymphocytes release cytokines upon stimulations that are interleukin 4 (il-4) and il-13 which able to induce antibody production by b-cells. actually, in normal individuals, these cytokines stimulate immunoglobulin m (igm) and igg production which provide defense against bacteremia via opsonization. nevertheless, in allergic individuals, these cytokines convert igm and igg to ige through a mechanism so called “isotype switching”.10 the increase of allergen-specific ige in the circulation which then attached to the fcei receptors of immunocompetent cells such as mast cells and basophils make individuals more sensitive to allergens.1 immunoglobulin a and allergic disease antigens, that are microorganisms and allergens before attaching to the mucosa are “blanket” by iga, which is the first immunoglobulin involved.12 immunoglobulin a which also a constituent of saliva and termed as salivary iga (siga) is a part of the secretory iga (siga) of the human body.9 failure of iga to protect antigens from adherence to the mucosa leads to further penetration of microorganisms and allergens into the mucosa and may lead to bacteremia.9,10 in children, iga is the last immunoglobulin that reaches adult level.13 the atopic/allergic march the allergic or atopic march refers to the natural history of allergic or atopic manifestations characterized by a typical sequence of clinical symptoms and conditions appearing during a certain age period and persisting over a number of years. characteristic of the clinical signs is whereas others diminish or disappear completely. in general, the clinical features of atopic eczema occur first and precede the development of asthma and allergic rhinitis (figure 1).14 figure 1. incidence of atopic disease in early childhood. the hygiene hypothesis and contra hygiene hypothesis hygiene hypothesis was introduced by dp strachan in 1989,15 it stated that cleanliness make people more allergic; it was based on his epidemiological study that children who lived in rural area were more allergic resistant. this was resulted from abundant infection since childhood which exposed to these children. consequently, there was a tendency to shift into th1 cytokines profile that was not related to allergy.15,16 on the opposite, renz and henz; and prescott, showed that low dose of infections which involved lipopolysaccharides (lps) may lead to the persistence of allergy.15–18 balance of the th1/th2 immune system naturally, infants were born with the th2 type; in nonatopic infant, it gradually shift to th1 type thorough time upon exposed to environmental allergens; whereas, atopic infant has a further increase of th2 cells that were potentially primed in utero by transplacental exposure to allergens.1 however, according to prescott, the influence of inductive infection toward the th1 immune system could only happened if it occurred in the early event of 116 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 114-118 life, that was before 18 months of life.18 other literatures stated that not only the th1/th2 balance leads to allergic development, instead, the th2 cells which release tumor growth factor b (tgfb) were responsible for the regulatory or balancing system.15,17 infection and allergy bacterial lps from gram negative bacteria is not the only microbial component that can act as an immunomodulator. polymorphism in the toll receptor 2 (tlr-2) promoter has been associated with reduced allergic sensitization, asthma and hay fever.16,19 tlr-2 recognize peptidoglycans primarily produced by gram-positive bacteria, lipoprotein and zymosan, which is a component of yeast. furthermore, the level of muramic acid, a major component of peptidoglycan that can be considered a marker for exposure to gram-positive bacteria, was inversely correlated with wheezing and asthma.15 principles of treatment of allergic disease basic principles of the treatment of allergic disease or allergy management protocol include: a) environment control measures (i.e. dust, smoking); b) pharmacologic therapy (i.e. adrenergic agents, antihistamines, glucocorticoids); c) allergen immunotherapy and d) food avoidance (i.e. peanut, cow’s, milk eggs, fish, chocolate, fruits).1,11 the connection between oral health, systemic diseases and allergy there were abundant literatures related to systemic infection which originated from periodontal or pulpal infections. cerebrovascular and cardiovascular diseases were the most renowned; others are diabetes mellitus and pregnancy problems.2-4 nevertheless, there were only several studies which related to allergy and controversial. most literatures were epidemiological studies and concluded that allergic patients (i.e. rhinitis, asthma) had poor oral health;20 another allergic case reported were urticaria and sinusitis.2 it was interesting that according to friedrich et al., periodontitis made people allergy-resistant, which coincidence with the hygiene hypothesis.21 the connection between poor oral health and allergy in children since 1990, the evidence-based medicine (ebm) had been popularized and facilitates clinicians to conduct their own researches. it also seeks to empower clinicians so they can develop independent views regarding medical claims and controversies.22 concerning to ebm, it had been revealed that oral plaque control therapy in allergic rhinitis children resulted in disappearing of the symptoms.5 evidence-based cases related to sinusitis in children also successfully treated with dental procedures.7 oral microbiota in children according to tanner et al., since birth, abundant microorganisms colonized in oral mucosa and tongue of children. even after their sixth months of life gram negative bacteria such as prevotella intermedia and porphyromonas gingivalis could be found in children.22 furthermore, according to socransky, dental plaque may contain 100 million to one billion bacteria in a single tooth. one of harmful effect of dental plaque is droplet infections which cause respiratory infections.5 discussion allergic diseases are common in children and the prevalence is increasing, especially in western societies. environmental factors such as exposure to allergens and pollution play an important role in the development and sustenance of allergic diseases. however, the development of allergic diseases is also governed by strong genetic influences, when one of both parents have history of allergic diseases, the incidence climbing up to 40% and 70%.11 additionally, children with allergy have several disadvantageous characteristics against infection. first, allergic children have the predisposition of iga deficiency, iga is essential for the first body defense mechanism. second, they have a th2 cytokines profiles that inactivate macrophages which needed for phagocytosis. third, they usually consumed antihistamines which diminished histamine concentration; actually histamine is essential for vasodilatation of blood vessels which intended for recruitment of white blood cells into the area of infection. others are food avoidance which mostly consisted of proteins (i.e. eggs, fish and cow’s milk) that are essential for immunoglobulin synthesis and children’s growth and development.1,9,24 moreover, infections, especially low grade infections, may enhance the allergic symptoms. product of inflammation, such as bradykinins and prostaglandins were able to increase the sensitivity of nerve which involved in allergic mechanism, such as the maxillary nerve (cn v2) in rhinitis and sinusitis.25 moreover, according to literatures, tumor necrosis factor –a (tnfa) may loosen the epithelial junction which facilitates penetrations of antigens or allergens into the mucosa.26,27 thus, allergic patient may become more sensitive to allergens. it was also in concordance with zeldin et al.16 who stated that effective tlrs, thus an effective mechanism against infection reduced asthma symptoms. the possible correlation between infection and other allergy symptoms could be extrapolated via the development of allergy concept which related to the atopic march. for this reason, the idea to reduce infection for suppressing allergic symptoms is plausible. allergic patient may be more susceptible to infection, especially oral infection, because of their certain characteristics i.e. ineffective th3 cytokine profiles 15 which responsible to decrease siga and siga and the conversion of igm and igg to ige (isotype switching), which reduced opsonization effectivity of immunoglobulin; and macrophage inactivation by th2 cytokines, its activity 117pradopo and utomo: new insight in pediatric dentistry actually needed for phagocytosis. in children, it becomes more pronounced since children reached their igm and igg adult level after 5 years old, whether iga is around 10.13 allergic diseases consisted of symptoms ranging from mild (i.e. urticaria, rhinitis) to life-threatening (i.e. anaphylactic shock); whatsoever the characteristic of the symptoms it could affect the quality of life. additionally, it also affect the socioeconomic status of the patients and their parents. allergic children are supposed to be less confidence because they are prohibited to do heavy sports, eating their children’s favorite food (i.e. ice cream, food made from poultry products etc). since allergic diseases may continue for a long time. allergic diseases mostly had comorbidities, such as asthma with rhinitis and sinusitis; it is interesting that treatment between comorbidities was able to cure each other.28 infections which elicited by bacteria and their toxins are able to trigger mast cells degranulation which release an array of mediators (i.e. histamine, prostaglandins, leukotrienes), cytokines (i.e. tnf-a, il-3, il-4, il-5, il-13), enzymes and chemotactic factor for granulocytes; subsequently it leads to an influx of plasma igg, complements and neutrophils. infections also triggers degranulation of basophils (white blood cells which mimicking the characteristics of mast cells); moreover, it also responsible for macrophages stimulation that release il-1, tnf-a. reactions of these immunocompetent cells release an array of mediators and enzymes that if present in excessive amount are harmful for allergic patient.9,29 according to socransky, droplets infection originated from dental plaque may lead to respiratory infections.30 therefore, it is not surprising that children have chronic cough, which actually is a kind of defense mechanism. since it has recurrent and frequent episodes, this cough symptom is blamed to be caused by allergy. as the result, chronic cough may also be originated from droplets infection that adheres to the respiratory tract. since cough is a part of innate immunity,9 it become natural that chronic droplet infections, thus continuing irritations may stimulate persistent cough. the possible connection between oral health and allergic symptoms should also happen to chronic rhinitis or sinusitis. according to boyd, inflammation of the same distribution of nerve, that was the maxillary nerve, which caused nasal congestion may be originated from the inflammation in other maxillary nerve (cn v2) distribution.25 the possibility of the other distribution of maxillary nerve involvement in rhinitis and sinusitis had been revealed by several literatures. in those studies and some case reports were discussed about the neurogenic switching mechanism.6,7,31 through neurogenic switching mechanism, local inflammation was able to propagate to a distant organ (i.e. food allergy, sinusitis, asthma, urticaria) via mast cell–nerve interactions.31 moreover, this mechanism could be initiated from oral inflammation i.e. gingivitis.,32 referred to these literatures, propagation of oral inflammation to distant organs is likely. it was interesting that there were evidencebased cases which showed that removal of plaque and improving oral health were able to diminished rhinitis and sinusitis symptoms.5–7 according to these case reports, even though only preliminary studies; the idea how improving oral health may able to suppress allergic symptoms should be supported to be accepted in the dental and medical societies. food avoidance as a part of allergy management had several disadvantages for children growth and development. first, the prohibited food was essential for general health such as egg, milk, fruits. even chocolate, which mostly harmful for asthmatic patients, had a beneficial content, that was the flavonoid, an anti-oxidant. nevertheless, it was limited to the dark chocolate which contains 70% cocoa. according to literatures, flavonoids could be found alsoin fruits i.e. apple.33 additionally, since immunoglobulins were made from proteins, food avoidance made allergic children more prone to infections. consequently, according to above discussion, they became more allergic. second, food avoidance may also disturb children’s social life, i.e. they could not eat the same food with their friends in a birthday party. in order to improve oral health which considered advantageous to allergic children, dental practitioners have to check up and clean teeth, tongue and other mucosal area regularly. this procedure is a part in pediatric preventive dentistry. in addition, microorganisms which also exist in dental plaque may also accumulate on the tongue since infancy.23 consequently, removing dental plaque which attached on the teeth and tongue may prevent from bacteremia; thus preventing children from suffering more severe allergic symptoms. dental health education (including tongue brushing) should also be taught to their parents for daily oral health care maintenance. taken together, it could be summarized that dental practitioner, especially who is interested in pediatric dentistry, should be aware about the possible connection between children’s oral health and allergic symptoms, even though it was still in researches. for this reason, dental practitioner is suggested to improve their knowledge (i.e. basic immunology) in order to explain questions that may come to mind by patients, their parents or their physician. however, the collaboration with general practitioner, pediatrician, especially allergy experts, is mandatory. furthermore, in order to receive the acceptance of this hypothesis by the medical society, evidence-based cases should be collected and reported, and collaborated researches with medical practitioners should be conducted. moreover, after less consumption of allergic medication which may cause drowsiness and uncomfortable feeling, children become more active and cheerful. reduced allergic symptoms also stress-relieving, because children feel free to eat prohibited food, to play and to do many sports activities. relief of stress is advantageous for allergic patients because it may shift the th2 cytokine profile (the atopic type) to the th1 (the non-atopic type). the conclusion are that pediatric 118 dent. j. (maj. ked. gigi), vol. 40. no. 3 july-september 2007: 114-118 dentistry, via the preventive dentistry procedure has the propensity to improve general health, especially in allergic prevention; and minimizing the need of food avoidance which is considered disadvantageous for promoting growth and development. hopefully, in the future, after further researches pediatric dentistry could be included in allergy management protocol. references 1. leung dym. in: behrman re, kliegman rm, jenson hb, editors. nelson textbook of pediatrics. 17th ed. philadelphia: saunders; 2004. p. 743–77. 2. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microbiol rev 2000; 13(4):547–58. 3. lavigne se. your mouth–portal to your body. probe 2004; 38(3):114–34. 4. scannapieco fa. periodontal inflammation: from gingivitis to systemic disease. compendium 2004; 25(7):s17–s25. 5. utomo h, setijanto d. apakah terapi pengendalian plak gigi dapat menurunkan keparahan rinitis alergika pada anak. majalah kedokteran gigi (dental journal) 2005; 38(2):96–102. 6. utomo h. sensitization of sphenopalatine ganglion by periodontal inflammation: a possible etiology for sinusitis and headache in children. majalah kedokteran gigi (dental journal) 2006; 39(2):63–71. 7. utomo h, pradopo s. a practical dental approach in children’s rhinosinusitis management. indonesian j dent 2006; 13(3):133–6. 8. padgett da, glaser r. how stress influence the immune response. trends in immunology 2003; 24:444–8. 9. rabson a, roitt im, delves pj. really essential medical immunology. 2nd ed. carlton: blackwell publishing; 2004. p. 8–10. 10. abbas ak, lichtman ah. cellular and molecular immunology. 5th ed. philadelphia: saunders; 2005. p. 34–35. 11. chatila ta. in: rudolph cd, rudolph am, editors. rudolph’s pediatric. 21st ed. new york: mcgraw-hill; 2004. p. 809–11. 12. pilette c, durham sr, vaerman jp, sibille y. mucosal immunity in asthma and chronic obstructive pulmonary disease: a role for immunoglobulin a? proc am thorac soc 2004; 1:125–35. 13. mcdade tw. life history theory and the immune system: steps toward a human ecological immunology. yearbook of physical immunology 2003; 46:100–25. 14. weinberg eg. the atopic march. curr allerg clin immunol 2005; 18(1):4–5. 15. romagnani s. the increased prevalence of allergy and the hygiene hypothesis: missing immune deviation, reduce immune suppression, or both? j allergy clin immunol 2004; 112:352–63. 16. zeldin dc, eggleston p, chapman m, piedimonte g, renz h, peden d. how exposures to biologics influence the induction and incidence of asthma. environ health perspect 2006 april; 114(4):620–6. 17. renz h, herz u. the bidirectional capacity of bacterial antigens to modulate allergy and asthma. eur resp j 2002; 19:158–71. 18. prescott sl. bacteria and the allergy epidemic: the culprits and the cure. curr allerg clin immunol 2004; 17(3):108–14. 19. supajatura v, ushio h, nakao a, akira s. differential responses of mast cell toll like receptors 2 and 4 in allergy and innate immunity. clin invest 2002; 109(10):1351–9. 20. laurikainen k. asthma and oral health: a clinical and epidemiological study. academic dissertation. tampere: tampere university press; 2002. p. 1–182. 21. friedrich n, volzke h, schwahn c, kramer a, junger m, schafer t, et al. inverse association between periodontitis and respiratory allergies. clin exp allergy 2006; 36(4):495–502. 22. del mar, glasziou p, mayer d. teaching evidence based medicine. bmj 2004; 329:989–90. 23. tanner acr, milgrom pm, kent r. the microbiota or young children in tongue and tooth samples. j dent res 2002; 81(1):52–7. 24. palmer ca, boyd ld. nutrition, diet and oral condition. in: harris no, godoy fg, editors. primary preventive dentistry. 6th ed. new jersey: penson prentice hall; 2004. p. 449–64. 25. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http://www. migraineprevention.com/index/html. accessed february 15, 2006. 26. okeson jp. bell’s orofacial pain. 6th ed. carol stream: quintessence pub; 2005. p. 262. 27. go m, kojima t, takano k, murata m, ichimiya s, tsubota t. expression and function of tight junction in the crypt epithelium of human palatine tonsils. j histochem cytochem 2004; 52(12):1627–38. 28. serrano c, valero a, picado c. rhinitis and asthma: one airway, one disease. arch bronconeomol 2005; 41:569–78. 29. walsh l j. mast cells and oral inflammation. crit rev oral biol med 2003; 14(3):188–98. 30. socransky e. american medical network. oral bacteria from gum disease can cause ailments elsewhere in body. available at: url http://www.dental.am. accessed november 10, 2004. 31. cady rk, schreiber cp. sinus headache or migraine. neurology 2002; 58:s10–s14. 32. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 33. garcia v, arts icw, sterne jac, thompson rl, shaheen so. dietary intake of flavonoids and asthma in adults. eur respir j 2005; 26(3):449–52. 181181 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 181–186 research report effect of different final irrigation solutions on push-out bond strength of root canal filling material rahmatillah,1 isyana erlita1 and buyung maglenda2 1department of conservative dentistry, faculty of dentistry, universitas lambung mangkurat 2endodontist, sultan suriansyah general hospital banjarmasin – indonesia abstract background: the adhesion of root canal filling material to dentin is one of the crucial factors in determining the success of endodontic treatment. however, the smear layer that forms during instrumentation serves as an interface that impedes the bonding mechanism of the filling material. a proper irrigation solution is required to remove the smear layer and provide a dentin surface that supports the bonding mechanism of the filling material in establishing good adhesion. purpose: this study aims to evaluate and compare the bond strength of filling material with different final irrigation solutions. methods: mandibular premolars were prepared by a crown down, pressure-less technique and divided into three final irrigation groups (2.5% naocl, 17% edta and 20% citric acid). the root canal of each tooth was obturated using epoxy sealer and gutta-percha. a two-millimetre-thick section of the apical third portion of each group was arranged for the push-out assessment using a univer sal testing machine in an apical to coronal direction at 1 mm/ min crosshead speed. results: a one-way anova test indicated the difference in push-out bond strength among the groups (p<0.05). a post hoc bonferroni test presented a statistically significant difference in the bond-strength value between the 2.5% naocl group compared with the 20% ca group (p<0.05). conclusion: the push-out bond strength of root canal filling material is increased by applying a chelating agent as the final irrigation solution where 20% of ca presents the highest push-out bond strength. keywords: 20% citric acid; 17% edta; 2.5% naocl; push-out bond strength correspondence: rahmatillah, department of conservative dentistry, faculty of dentistry, universitas lambung mangkurat, jl. veteran 128b, banjarmasin 70122, indonesia. email: rahmatillahaz@gmail.com introduction the bonding ability to dentin is a crucial feature of the root canal filling material. the material, which is frequently used as a root canal filling, is gutta-percha. nevertheless, gutta-percha must be combined with a root canal sealer since gutta-percha does not adhere to root canal dentin.1 there are two main concerns regarding material adhesion to root canal dentin. in static situations, the adhesion would prevent fluid percolation between the filling material and the root canal dentin. in dynamic situations, it would prevent dislodgement of the filling material from the root canal during subsequent manipulations, thereby reducing the risk of contamination.2 the smear layer serves as an interface between the root canal filling material and the dentin.3 the removal of the smear layer advances sealer adhesion and affects the bond strength of the ah plus sealer.4,5 smear layer removal increases the contact area and the sealing ability of the sealer, so it produces better adaptation. smear layer removal enables the sealer tags extension to the dentin tubules, which results in the formation of mechanical locking and efficient micro-retention.5,6 the sealer contact to the dentin also becomes closer, so it optimises the adhesion due to the formation of chemical bonds.7 ethylenediaminetetraacetic acid (edta) is suggested as an irrigation solution because of its nature as a chelating agent. this irrigation solution has the ability to eliminate the inorganic portions of the smear layer. however, edta that is used as a single irrigation solution is not effective to eliminate the smear layer entirely.8 a proteolytic agent, one of which can be sodium hypochlorite (naocl), must dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 mailto:rahmatillahaz@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 182 rahmatillah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 181–186 be used to eliminate the organic portion of the smear layer. naocl and edta irrigation solutions can remove the inorganic portions of the smear layer and expose collagen fibres. moreover, the collagen fibres serve as a substrate for sealer infiltration and hybrid layer formation.8,9 a combination of naocl with a chelating agent or acidic material is required to eliminate both organic and inorganic portions of the smear layer. consequently, it has been recommended to apply naocl along with edta or citric acid for irrigation procedures.10 citric acid has been recommended as an alternative chelating agent. the effectiveness of 10%–50% citric acid in removing inorganic portions of the smear layer has been evaluated.11 olivieri et al.11 reported that citric acid has a more effective smear layer removal effect in the apical and middle third root canals compared to edta 17%. besides, prado et al.12 showed that both edta and citric acid are more effective in eliminating the smear layer at the apical third with a three-minute application. the push-out test has been described as one of the most reliable, accurate, effective, and easy methods to measure the bond value between the sealer, dentin, and core material. likewise, the push-out test can evaluate the bond strength to a low value at various depths of root canal dentin.8,13 the current study was performed to analyse the push-out bond strength between gutta-percha and epoxy resin sealer to dentin with the final irrigation solution 17% edta and 20% citric acid. materials and methods the ethics committee of the dentistry faculty, universitas lambung mangkurat no. 023 / kepkg-fkgulm / ec / i / 2020 approved this research and declared it to be clear from any ethical issues. this study used a post-test only with a control group design. the samples were 21 premolar teeth with the following inclusion criteria: mandibular premolar teeth extracted due to orthodontic treatment, straight and perfectly formed apex, and no root fractures. teeth with caries, root morphological anomalies, and more than one root canal were excluded. root canal treatment was carried out on the selected teeth. the teeth were cut through the cemento-enamel junction with a double-sided, diamond disk (suzhou syndent tools co., ltd, suzhou, china) to leave a 14 mm root section with a working length of 13 mm.13,14 preparation of the root canal was performed by a crown down, pressure-less technique with protaper hand-use instruments (dentsply maillefer, ballaigues, switzerland). preparation was initiated by k-file #10 (dentsply maillefer, ballaigues, switzerland) throughout 2/3 of the working length. thereafter, the preparation was carried out with s1 and s2 files according to the working length for widening the 2/3 coronal portion. furthermore, a 1/3 apical portion preparation was performed with f1, f2, and f3 files according to the working length (according to the manufacturer’s instructions). all of the root canals were irrigated with a 3 ml 2.5% naocl solution during instrumentation for each file size up to the f3 file (size 30, 0.09 taper). at the end of instrumentation, the root canals were randomly divided into three final irrigation groups, namely group i (5 ml 2.5% naocl), group ii (5 ml 17% edta), and group iii (5 ml 20% citric acid). the irrigation was carried out using a 30 g, close-end, single side, vent needle (onemed, sidoarjo, indonesia) for three minutes.15,16 irrigation was done by a manual, dynamic-agitation technique (hand-activated, wellfitting, #f2 gutta-percha (dentsply maillefer, ballaigues, switzerland)) with push-pull movement 100 times/30 seconds.17 thereafter, a paper point was inserted to dry up the root canal.18 the obturation was performed by manipulating the sealer (ah plus, dentsply, detrey gmbh, konstanz, germany) and protaper #f3 gutta-percha (dentsply maillefer, ballaigues, switzerland) and used a single-cone technique as stated by the manufacturer’s instructions. then, a plugger (cerkamed medical company, poland) was heated to cut off the remaining gutta-percha that exceeded the root canal. furthermore, the obturation was covered with zinc phosphate cement (elite cement 100, gc corporation tokyo, japan) and radiographed to ensure a hermetic obturation system. after that, the sample was conditioned in a plastic container that contained moist gauze for the incubation procedure of seven days at 37°c with 100% humidity.14 the sample was segmented in the transverse plane, perpendicular to the root canal’s long axis using a circular diamond disk (louyang penghao ceramic technology co., ltd, louyang, china). the apical third of the root was removed with a thickness of 2 mm. the procedure was continued to obtain an apical third sample that would be used in the test. furthermore, the sectioning was carried out to a thickness of 2 mm and measured with an electronic digital calliper (mitutoyo, kawasaki, japan). the coronal surface of each sample was marked and coded for each group.13 the sample was positioned on the surface of a custommade, cylindrical, resin fixture (20 mm diameter x 7 mm height) with a hole in the middle (2 mm diameter), which would accommodate material dislodgement during the push-out test.14 the push-out test was conducted with a universal testing machine (tn 20 md, france) with a 0.53 mm stainless-steel plunger (custom made) that pushed the filling material at a crosshead speed of 1 mm/min in apicalcoronal direction.19 the bond strength was calculated by the following formula:13 pbs = f / a where: pbs = push-out bond strength (mpa), f = maximum load (n), a = bonding area of root canal filling (mm2), calculated by the following formula:20 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 183rahmatillah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 181–186 a = π (r1 + r2) √ (r1 – r2) 2 + h2 where: π = 3.14, r1 = coronal radius, r2 = apical radius, h = sample thickness. the data were processed using ibm spss statistics for windows version 26.0 (ibm corp., armonk, ny, usa) for normality with the shapiro–wilk test and homogeneity was carried out with levene’s test. statistical analysis was conducted with a one-way anova test to compare each group. furthermore, the data were further tested with a post hoc test using the bonferroni method to determine the value of comparison between groups (significant level set as p<0.05). results the mean, standard deviation, and one-way anova significant value of filling-material bond strength are described in table 1. the one-way anova test implied that the bond strength value of the filling material was statistically different among final irrigation solution groups (p<0.05). according to the summary of the bonferroni test in table 2, there was a difference that was statistically significant between the 2.5% naocl group compared to the 20% citric acid group (p<0.05). otherwise, the differences between the final irrigation 2.5% naocl group compared to the 17% edta and between 17% edta group compared to 20% citric acid were not statistically significant. table 1. one-way anova test result: the push-out bond strength of root canal filling material with final irrigating solution 2.5% naocl, 17% edta, and 20% citric acid final irrigation group n mean ± standard deviation sig 2.5% naocl 7 2.05 ± 0.75 mpa 0.041*17% edta 7 2.75 ± 0.60 mpa 20% citric acid 7 2.98 ± 0.59 mpa n: number of specimens, *: value shows a significant difference at p<0.05. table 2. post hoc test result using the bonferroni methods: the push-out bond strength of root canal filling material with final irrigating solution 2.5% naocl, 17% edta, and 20% citric acid final irrigation group 2.5% naocl 17% edta 20% citric acid 2.5% naocl 0.182 0.048* 17% edta 1.000 20% citric acid *: value shows a significant difference at p<0.05. discussion the push-out test provides information about the material bonding properties and material resistance, and it is intended to assess the degree of material bonding to the dentin. as the push-out bond-strength value of the filling material gets higher, the adhesion of the material also gets better. in endodontics, the push-out test is conducted to study filling material resistance, perforation improvement, post retention, and sealer bonding to dentin.10,21 moreover, the push-out test provides better outcomes in assessing the bond strength of intra-canal materials than the conventional shear test method. this is due to the dislodgment of material that occurs parallel to the dentin and thus is more useful in representing the clinical setting.22 the current study complies with the research of alkhudhairy et al.23 and rocha et al.,24 which reported a deleterious effect on the filling material bond strength when 2.5% naocl was used as the final irrigation solution. this research confirmed that naocl as a single irrigation solution does not effectively eliminate the smear layer. the physicochemical properties of naocl only work on the organic portions of the smear layer.25 an naocl irrigation solution can degrade dentin collagen. consequently, it affects sealer bond strength.26 ah plus sealer is chemically bonded with collagen.27 collagen is the main component of dentin, which plays a critical role in the bonding between the resin sealer and dentin.28 the bonding mechanism of the epoxy resin sealer is the arrangement of covalent bonds from the open epoxide ring to the amino group of collagen dentin.29 thus, the removal of collagen fibrils from the dentin due to the use of an naocl irrigation solution leads to a decrease in the bond strength value of the adhesive system.28 the deproteinisation effect of naocl makes the amino group of the collagen become unstable and easily dissolve.30 this produces a less receptive dentin surface, provides weak micromechanical bonds, and decreases the bond strength of the epoxy resin sealer.27,30 the deproteinisation of dentin that is irrigated by naocl leads to hydrophilic surface properties that do not support the spreading of the hydrophobic ah plus sealer.31 the removal of dentin organic matrix (fragmentation between carbon atom bonds and degradation of the primary structure of collagen) potentially restrains the hybrid layer formation. after breaking down long collagen chains, naocl also chlorinates protein terminal groups. the presence of chloramine protein results in premature termination of the polymer chain and incomplete resin polymerisation.27 the release of oxygen from naocl may inhibit the polymerisation process and thereby reduce bond strength, especially in the apical third.23,24 besides, a negative correlation has been found between the exposure time of naocl and the material bond strength.26 a combination of naocl and the chelating agent has a positive effect on the push-out bond strength of dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 184 rahmatillah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 181–186 the epoxy resin sealer.6,32 the chelating agent that was used in this study, edta 17% (one med) and 20% citric acid (biochemistry laboratory, universitas lambung mangkurat), indicated similar bond-strength values. filling material that previously was irrigated with 2.5% naocl 17% edta and 2.5% naocl 20% citric acid indicated higher bond strength in comparison with the group that was irrigated with 2.5% naocl as a single irrigation solution. the current study is in accordance with the research by berástegui et al.25 that showed that a higher bond strength was obtained when naocl was combined with a chelating agent. however, statistical analysis of the current study is in line with farag et al.,13 who conducted a push-out test of filling material with different irrigation solutions. the study reported that the difference of filling material bond strength between the irrigation group with 2.5% naocl compared to the irrigation group with 2.5% naocl-17% edta was not statistically significant.13 alkhudhairy et al.23 and gündoğar et al.33 explained that smear layer removal became more effective when the root canal was irrigated with edta solution and provided the higher bond-strength value than irrigation with an naocl solution. irrigation with edta showed the higher bond-strength value was caused by its effectiveness in removing the smear layer, demineralising dentin, opening dentin tubules, and increasing dentin surface roughness.23,34 therefore, 17% edta, which was used as the final irrigation solution, facilitated collagen exposure, increased sealer spreading, and established a dentin substrate more conducive to ah plus adhesion.35 final irrigation with edta shows a higher bondstrength value because edta can significantly reduce dentin wetting, thereby resulting in a dentin substrate that has a favourable condition for hydrophobic properties of ah plus.5,36 a comparative evaluation of the contact angle suggests that the contact angle of the sealer is reduced after irrigation with edta 17%.37 the ah plus sealer shows better surface wetting in the application of edta and naocl irrigation solutions than using naocl irrigation solutions only. this is due to the intimate contact between the dentin surface and the sealer, possibly achieved by adequate smear layer removal, which enhances sealer infiltration into the dentinal tubules.38 the effect of edta on dentin is determined by its concentration and time of exposure.39 in the present study, the final irrigation was intended for three minutes as mentioned by mello et al.,15 who suggested that root canal irrigation with 5 ml of edta for three minutes could eliminate the smear layer effectively. besides, the dynamic manual agitation technique was also used during irrigation. this technique has been proven to be more capable of removing dentinal debris, the smear layer, and biofilm than the static irrigation technique.17 statistical analysis of the current study indicated that the bond strength of the final irrigation group edta 17% and the final irrigation group 20% were not significantly different. the current study is in line with ravikumar et al.36 who examined the bond strength of filling material with these chelating agents. the study showed that the difference of bond strength among the final irrigation group with edta and citric acid was not significant.36 different concentrations of citric acid (1–50%) have been widely used in removing the smear layer.7 citric acid with a concentration of 20% was used in the current study because of its biocompatibility and capacity to demineralise the inorganic portion of the smear layer. this was conformable with the study that stated that concentrations of 1–40% citric acid were adequate in eliminating the smear layer, dissolving dentin debris, and demineralising intra-tubular dentin to expose the dentinal tubules. besides, citric acid with a concentration of 20% does not have any detrimental effect on the dentin surface. based on this reasoning, the current study was carried out using a concentration of 20% to increase its capacity as a chelating agent.18 the highest bond strength of the final irrigation group with 20% citric acid can be attributed to the previous study, which showed that the root canals that were irrigated with citric acid showed more effectiveness than the 17% edta in eliminating the smear layer at the apical and middle third portion of the root canal. besides, the application of citric acid with a concentration of 20% also increases the chelating effect.12,25 several factors that determine the effect of the chelating agent are contact time, ph, concentration, and the volume of the solution. research conducted by berástegui et al.25 showed a similar capacity for the smear layer removal between 20% citric acid and 17% edta. the concentration of citric acid 20% does not have any damaging effect on the surrounding tissue because it is not highly ionised. another study that applied citric acid at a concentration of 20% showed that the chelating effect of citric acid became higher with increased concentrations. the application of 20% citric acid as a chelating agent results in an increase in the contact area and covalent bond, thereby resulting in a higher ah plus sealer bond to the dentin.25,40 however, due to its ability to disinfect and dissolve organic tissue, naocl irrigation remains an option in contemporary endodontics, although the current study showed the lowest filling material bond strength compared to other groups.13,41 naocl does not remove the smear layer that coats the dentin and occludes the orifice of the dentinal tubules, thereby restricting the sealer penetration into dentinal tubules.13,41,42 meanwhile, the use of edta or citric acid as final irrigation solution can remove the smear layer and open the dentinal tubules, which facilitates the collagen exposure in intratubular dentin, thereby providing a higher filling material bond strength as the adhesion mechanism of the epoxy resin sealer is an arrangement of covalent bonds by any exposed amino groups in dentin collagen to the open epoxide ring of ah plus sealer. 29,41 the bond strength of the epoxy resin sealer is also associated with creep capacity, low shrinkage levels during setting, flow-ability properties, low polymerisation shrinkage, sealer volumetric expansion, and long-term dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 185rahmatillah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 181–186 dimensional stability.1,23 several studies have found diversity on the sealer bond strength values when the samples were examined with different root canal depthlevel sections.13,19,23,29 nonetheless, the current research used samples in the apical one-third that tended to show the lowest value of bond strength compared to the middle and coronal third.3 the reduction in root canal diameter, anatomic variation, and vapour lock effect in the apical third interferes with the irrigation solution flow and makes removing the smear layer even more challenging.12 the decrease in dentinal tubule density, sclerotic dentin, and inhomogeneous hybridisation of dentin in the apical third also reduces the level of material adhesion to dentin.3,6,23,43 the results of the current study indicate that there are differences in the bond strength of root canal filling material with different final irrigation solutions. the push-out bond strength of root canal filling material is increased by applying a chelating agent as final irrigation. the final irrigation with 20% citric acid shows the highest bond strength value of filling material and implies a significant difference in bond strength compared to 2.5% naocl. references 1. gade vj, belsare ld, patil s, bhede r, gade jr. evaluation of push-out bond strength of endosequence bc sealer with lateral condensation and thermoplasticized technique: an in vitro study. j conserv dent. 2015; 18(2): 124–7. 2. madhuri gv, varri s, bolla n, mandava p, akkala ls, shaik j. comparison of bond strength of different endodontic sealers to root dentin: an in vitro push-out test. j conserv dent. 2016; 19(5): 461–4. 3. hargreaves km, berman lh, rotstein i. cohen’s pathways of the pulp. 11th ed. st. louis: mosby elsevier; 2015. p. 250–93. 4. hasan rk, al-hashimi mk. shear bond strength of endodontic sealers to dentin with and without smear layer and gutta percha: an in vitro study. j baghdad coll dent. 2014; 26(4): 86–9. 5. topçuoğlu hs, demirbuga s, tuncay o, arslan h, kesim b, yaşa b. the bond strength of endodontic sealers to root dentine exposed to different gutta-percha solvents. int endod j. 2014; 47(12): 1100–6. 6. buldur b, öznurhan f, kaptan a. the effect of different chelating agents on the push-out bond strength of proroot mta and endosequence root repair material. eur oral res. 2019; 53(2): 88–93. 7. haapasalo m, qian w, shen y. irrigation: beyond the smear layer. endod top. 2012; 27: 35–53. 8. yusnita r, yanuar ichrom mn, diana s. the effectiveness of dayak onion bulbs extract (eleutherine palmifolia (l) merr.) against root canal mixed bacterial (preface study as root canal irrigation materials). dentino j kedokt gigi. 2018; 3(2): 132–7. 9. al-askary ra, ismail sa, al-sabawi na. bond strength of endodontic sealers to gutta percha and dentin. al-rafidain dent j. 2013; 13(2): 282–9. 10. turk t, kaval me, şen bh. evaluation of the smear layer removal and erosive capacity of edta, boric acid, citric acid and desy clean solutions: an in vitro study. bmc oral health. 2015; 15: 104. 11. olivieri jg, garcía font m, stöber e, de ribot j, mercadé m, duransindreu f. effect of manual dynamic activation with citric acid solutions in smear layer removal: a scanning electron microscopic evaluation. j dent sci. 2016; 11(4): 360–4. 12. prado m, gusman h, gomes bpfa, simão ra. scanning electron microscopic investigation of the effectiveness of phosphoric acid in smear layer removal when compared with edta and citric acid. j endod. 2011; 37(2): 255–8. 13. farag ha, etman wm, alhadainy ha, darrag am. effect of different irrigating protocols on push out bond strength of resilon/ epiphany obturation system. tanta dent j. 2015; 12(4): 241–8. 14. widhihapsari s, nari ratri d, nugraheni t. perbedaan bahan irigasi akhir saluran akar terhadap kekuatan pelekatan push-out bahan pengisi saluran akar berbahan dasar resin pada dinding saluran akar. j ked gi. 2016; 7(2): 171–8. 15. m e l l o i , k a m m e r e r ba , yo s h i m o t o d, m a c e d o m c s , antoniazzi jh. influence of final rinse technique on ability of ethylenediaminetetraacetic acid of removing smear layer. j endod. 2010; 36(3): 512–4. 16. nurisawati im, muryani a, nurdin d. perbedaan kebersihan sepertiga apikal saluran akar yang diirigasi sodium hipoklorit 2,5% dengan teknik non agitasi dan agitasi manual dinamik. j kedokt gigi univ padjadjaran. 2017; 29(3): 184–8. 17. chatterjee r, venugopal p, jyothi k, jayashankar c, kumar sa, kumar ps. effect of sonic agitation, manual dynamic agitation on removal of enterococcus faecalis biofilm. saudi endod j. 2015; 5(2): 125–8. 18. hardhitari r, kamizar, sumawinata n. effects of 2.625% naocl 20% citric acid and 2.625% naocl 17% edta on cleanliness of smear layer on apical one third. j phys conf ser. 2018; 1073(6): 62023. 19. cakici f, cakici eb, ceyhanli kt, celik e, kucukekenci ff, gunseren ao. evaluation of bond strength of various epoxy resin based sealers in oval shaped root canals. bmc oral health. 2016; 16: 106. 20. verma d, taneja s, kumari m. efficacy of different irrigation regimes on the push-out bond strength of various resin-based sealers at different root levels: an in vitro study. j conserv dent. 2018; 21(2): 125–9. 21. sirisha k, rambabu t, shankar yr, ravikumar p. validity of bond strength tests: a critical review: part i. j conserv dent. 2014; 17(4): 305–11. 22. pasdar n, seraj b, fatemi m, taravati s. push-out bond strength of different intracanal posts in the anterior primary teeth according to root canal filling materials. dent res j (isfahan). 2017; 14(5): 336–43. 23. alkhudhairy fi, yaman p, dennison j, mcdonald n, herrero a, bin-shuwaish ms. the effects of different irrigation solutions on the bond strength of cemented fiber posts. clin cosmet investig dent. 2018; 10: 221–30. 24. rocha aw, de andrade cd, leitune vcb, collares fm, samuel smw, grecca fs, de figueiredo jap, dos santos rb. influence of endodontic irrigants on resin sealer bond strength to radicular dentin. bull tokyo dent coll. 2012; 53: 1–7. 25. berástegui e, molinos e, ortega j. to comparison of standard and new chelating solutions in endodontics. j dent sci. 2017; 2(3): 000131. 26. barreto ms, rosa ra, seballos vg, machado e, valandro lf, kaizer ob, so mvr, bier cas. effect of intracanal irrigants on bond strength of fiber posts cemented with a self-adhesive resin cement. oper dent. 2016; 41(6): e159–67. 27. abuhaimed ts, neel eaa. sodium hypochlorite irrigation and its effect on bond strength to dentin. biomed res int. 2017; 2017: 1–8. 28. gomes bpfa, vianna me, zaia aa, almeida jfa, souza-filho fj, ferraz ccr. chlorhexidine in endodontics. braz dent j. 2013; 24(2): 89–102. 29. abada hm, farag am, alhadainy ha, darrag am. push-out bond strength of different root canal obturation systems to root canal dentin. tanta dent j. 2015; 12(3): 185–91. 30. nugraheni t. pengaruh konsentrasi dan lama aplikasi sodium hipoklorit (naocl) sebagai bahan irigasi saluran akar terhadap kekuatan geser perlekatan siler berbahan dasar resin pada dentin saluran akar. maj kedokt gigi indones. 2012; 19(1): 21–4. 31. fahmy sh, el gendy aah, el ashry sh. dentin wettability enhancement for three irrigating solutions and their effect on push out bond strength of gutta percha / ah plus. j clin exp dent. 2015; 7(2): e237–42. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 186 rahmatillah et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 181–186 32. donnermeyer d, vahdat-pajouh n, schäfer e, dammaschke t. influence of the final irrigation solution on the push-out bond strength of calcium silicate-based, epoxy resin-based and siliconebased endodontic sealers. odontology. 2019; 107(2): 231–6. 33. gündoğar m, sezgin gp, erkan e, özyılmaz oy. the influence of the irrigant qmix on the push-out bond strength of a bioceramic endodontic sealer. eur oral res. 2019; 52(2): 64–8. 34. mohan r, pai ar. the comparison between two irrigation regimens on the dentine wettability for an epoxy resin based sealer by measuring its contact angle formed to the irrigated dentine. j conserv dent. 2015; 18(4): 275–8. 35. tuncel b, nagas e, cehreli z, uyanik o, vallittu p, lassila l. effect of endodontic chelating solutions on the bond strength of endodontic sealers. braz oral res. 2015; 29(1): 1–6. 36. ravikumar j, bhavana v, thatimatla c, gajjarapu s, reddy sgk, reddy br. the effect of four different irrigating solutions on the shear bond strength of endodontic sealer to dentin an in-vitro study. j int oral heal. 2014; 6: 85–8. 37. kaushik m, sheoran k, reddy p, roshni, narwal p. comparison of the effect of three different irrigants on the contact angle of an epoxy resin sealer with intraradicular dentin. saudi endod j. 2015; 5(3): 166–70. 38. tummala m, chandrasekhar v, shashi rashmi a, kundabala m, ballal v. assessment of the wetting behavior of three different root canal sealers on root canal dentin. j conserv dent. 2012; 15(2): 109–12. 39. abraham s, raj jd, venugopal m. endodontic ir rigants: a comprehensive review. j pharm sci res. 2015; 7(1): 5–9. 40. mohammadi z, jafarzadeh h, shalavi s, kinoshita ji. unusual root canal irrigation solutions. j contemp dent pract. 2017; 18(5): 415–20. 41. wright pp, kahler b, walsh lj. alkaline sodium hypochlorite irrigant and its chemical interactions. materials (basel). 2017; 10: 1147. 42. kuçi a, alaçam t, yavaş ö, ergul-ulger z, kayaoglu g. sealer penetration into dentinal tubules in the presence or absence of smear layer: a confocal laser scanning microscopic study. j endod. 2014; 40(10): 1627–31. 43. shahnaz, lone ma, masoodi a, farooq r, purra a, ahmad f. comparison of push out bond strength of two adhesive systems on fibre posts : an in vitro study. int j appl dent sci. 2019; 5(2): 76–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p181–186 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p181-186 144 dental journal (majalah kedokteran gigi) 2017 september; 50(3): 144–148 research report a comparison of class i malocclusion treatment outcomes with and without extractions using an abo grading system for dental casts and radiographs bunga ayub rukiah, amalia oeripto, and nurhayati harahap department of orthodontics faculty of dentistry, universitas sumatera utara medan indonesia abstract background: class i malocclusion can be treated with or without resort to extraction. however, despite the indications, a controversy is still ongoing as to whether one option is preferable to another. one of the most frequent controversies centers on whether treatment involving extractions will produce superior results than treatment not culminating in extraction. purpose: this study aimed to compare the results of treating class i malocclusion with extractions and those without extraction using an abo grading system. methods: comparing abo scores in patients’ dental casts and radiographs with class i malocclusion with and without extraction. observational research incorporating case control methods was conducted involving 40 patients with class i malocclusion. samples were divided into two groups, one treated with extraction (group e) and the other without extraction as the control group (k). the results of the treatment were measured and assessed using eight variables of the abo grading system. results: the total score for the group treated with extractions was 23.65±7.82, while that for group k was 26.50±7.02. there was no significant difference in the total score between the two groups. nevertheless, class i malocclusion treated with extraction had a lower score than without extraction. conclusions: there was no difference in the total score of the abo grading system for class i malocclusion patients treated with and without extractions. keywords: class i malocclusion; extraction; non extraction; grading system abo correspondence: bunga ayub rukiah, department of orthodontics, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, padang bulan, kampus usu, padang bulan, medan baru, kota medan, sumatera utara 20155, indonesia. e-mail: lie_bunga@yahoo.com introduction there has been an increase in the prevalence of malocclusion over the last few decades, constituting, in addition to dental caries, periodontal diseases and dental fluorosis, one of the most common dental problems. in tanzania, 93.6% of 1.601 children with an average age of between 12 and 14 years presented the class i molar relationship and 63.8% of the population had at least one anomaly.1 the patterns of skeletal and dental malocclusion in 602 orthodontic patients in saudi arabia revealed that the most common pattern of skeletal malocclusion was the class i variety at 51.7%.2 meanwhile, the prevalence of class i malocclusion in the deutro-melayu indonesian population was 48.8%, class ii 33.1% and class iii 18.1%.3 class i malocclusion treatment can be performed with or without extraction. controversies over the resort to extraction have been ongoing since the beginning of the 20th century. in attempting to arrive at treatment-related decisions as to whether or not to perform extractions, there several factors require consideration, including: occlusion stability, as well as the characteristics of dental arches and facial aesthetics. moreover, the resulting effects on the dentofacial complex also need to be explained.4,5 the extent to which orthodontic treatment is believed to prove successful still varies among clinicians. efforts to reduce the degree of subjective assessment of particular malocclusion include the use of a particular malocclusion dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p144-148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p144-148 145145bunga, et al. /dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 144–148 figure 2. measuring with abo measuring gauge. (a) measuring buccolingual inclination of mandibular molars (b) measuring alignment of molar (c) measuring overjet in posterior segment (d) measuring marginal ridge (e) measuring buccolingual inclinations of maxillary molar. index during the conducting of assessments. this can be employed to objectively assess the severity of the malocclusion and the degree to which the treatment will be successful.6 a grading system represents a parameter for the success of orthodontic treatment proposed by the american board of orthodontics (abo) in 1999. this parameter assesses the efficacy of treatment on the research model and the panoramic radiography. there are eight criteria to be assessed within this parameter, namely: alignment, marginal ridge, buccolingual inclination, occlusal relationship, occlusal contact, overjet, interproximal contact, and root angulation.7,8 this study aimed to compare the results of the treatment of class i malocclusion with extractions and control using the abo grading system. it involved comparing eight abo parameters and the total abo score for both treatment groups. materials and methods for the purposes of the retrospective research, measurements were performed on 20 samples of dental casts and radiographs for each group taken from dental hospital, faculty of dentistry, universitas sumatera utara. ethical approval was obtained from the ethical comittee faculty of dentistry universitas airlangga. the inclusion criteria comprised: class i malocclusion, a point–nasion–b point angle (anb = 2° ± 2°), complete teeth (except the third molars), the absence of growth abnormalities, treatment performed using standard edgewise brackets and no history of oral cavity trauma. the eight grading system parameters were measured in accordance with standard abo measurement using an abo measuring gauge (figure 1 and 2). measurement was performed on the research model whose treatment and panoramic radiography of each sample had been completed. the total measurement results were subsequently calculated, with the treatment being deemed successful if the total score of each sample was ≤27. the sample of each group was classified into one of two categories, namely; successful or unsuccessful. each parameter was measured by two raters and thereafter, the inter-rater reliability was examined. inter-rater reliability was determined by means of cohen’s kappa. differences between the groups were examined using the mannwhitney test with a confidence level of 0.05. figure 1. abo measuring gauge.8 a b c d e dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p144-148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p144-148 146 bunga, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 144–148 results cohen’s kappa as an inter-rater reliability test generated 100% similarity for the two observers. the success of the treatment given to the group subjected to extractions and the control group was equal at 60%. the mann-whitney test for the total score suggested that the group treated with extractions and the control group were not significantly different (p=0.15; p>0.05) (table 1). a mann-whitney test was conducted for each parameter to examine differences between the group treated with extractions and the group treated without extractions (table 2). the test results showed that the significant difference was found only in the parameter marginal ridge (p=0.03; p <0.05). nevertheless, overall, each parameter of group k had a higher mean than the parameters of group e (figure 3). discussion selection of treatment for patients with class i malocclusion involving the use or otherwise of extractions has raised controversies among clinicians. selection of extraction-based treatment requires careful consideration of various aspects, including: the facial profile and the crowding level prior to treatment.9 a considerable volume of research comparing extraction-based treatment to that without extractions has been conducted.10,11 one factor affecting the duration of orthodontic treatment is extractions. patients undergoing extractions will require orthodontic treatment of longer duration those whose treatment does not include this procedure. the length of the treatment is also associated with the number of teeth extracted. the treatment in patients requiring the extraction of four premolars is of longer duration than that of patients who need two premolars to be removed.10 treatment of malocclusion is successful if the abo score is 27 or less.7,8 overall, the mean of the results of the class i skeletal malocclusion treatment was equal to 23.65 ± 7.82 for group e and 26.50 ± 7.02 for group k. the results of both e and k groups suggest that the treatment of class i malocclusion among orthodontic patients treated in the orthodontic department of faculty of dentistry universitas sumatera utara proved successful. the results also indicate that, overall, treatment group k had a higher score than group e. table 1. differences in the total score between the group treated with extractions and the group treated without extractions variable treatment total score p-value mean sd score with extractions 23.65 7.82 0.15 without extractions 26.50 7.02 table 2. differences in the scores for the mean and the standard deviation of each variable receiving treatment with extractions and treatment without extractions variable treatment mean ± sd p-value alignment extractions 1.65 ± 1.69 0.12 non extractions 2.55 ± 1.93 marginal ridge extractions 3.30 ± 2.47 0.03* non extractions 4.35 ± 1.35 buccolingual inclination extractions 2.90 ± 1.89 0.37 non extractions 3.65 ± 2.41 overjet extractions 5.10 ± 3.06 0.41 non extractions 6.40 ± 3.97 occlusal contact extractions 1.70 ± 1.63 0.46 non extractions 2.45 ± 2.56 occlusal relationship extractions 4.85 ± 3.18 0.89 non extractions 4.80 ± 3.04 interproximal contact extractions 1.20 ± 1.67 0.34 non extractions 0.75 ± 1.29 root angulation extractions 2.20 ± 1.64 0.74 non extractions 2.30 ± 1.59 * significant according to the mann-whitney test. p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p144-148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p144-148 147147bunga, et al. /dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 144–148 long-term research to determine the respective effect of treatment with and without extractions argued that posttreatment changes in both groups were the same. the results of treatment involving the extraction of differing numbers of premolars suggest that patients having two premolars removed enjoyed superior results than their counterparts undergoing the extraction of four premolars.10,12 the research reported here revealed no significant difference between the two groups. it is consistent with the research conducted by anthopoulou, there are no significant differences in dental alignment, symmetry of the median line, overjet, overbite and posterior occlusion.12 in the present research, the marginal ridges of group e and group k were significantly different. this was because in group k the second molar was not included in the treatment. consequently, the marginal ridge between the first and second molars was not corrected. meanwhile, the variable overjet in both groups generated the highest score; 5.1 for group e and 6.4 for group k. this was because the treatment in both groups was performed using standard edgewise brackets which do not exert any torque control, especially on the posterior segment. treatment using brackets prescribed by roth results in better posterior tooth angulation than does the treatment using standard brackets. therefore, it is crucial for clinicians to pay more attention to torque, especially on the posterior segment, in order to avoid large overjet.8 within the present research, the interproximal contact generated the lowest scores, namely 1.2 and 0.7 for e and k groups respectively. this suggests that space closure was the most basic problem which orthodontists might overcome with little difficulty. the variable alignment, marginal ridge, occlusal contacts, buccolingual inclusion and overjet had higher values in group k than in group e, although the significant difference was present only in the variable marginal ridge. this may be because, in the case of group e, clinicians had more room to adjust the position of the teeth so as to create the better position and interdigitation than those of group k. in this research, group e produced better scores than group k. however, in the case of class i malocclusion, extractions are not a decisive factor for satisfactory treatment outcomes in the event that only the eight parameters of the grading system are taken into account. overall, group e generated lower scores than group k. the significant difference was only found in the variable marginal ridge. in conclusion, there was no difference in the total score of the abo grading system for class i malocclusion patients treated with and without extractions. references 1. mtaya m, brudvik p, astrom an. prevalence of malocclusion and its relationship with socio-demographic factors, dental caries, and oral hygiene in 12to 14-year-old tanzanian schoolchildren. eur j orthod. 2009; 31(5): 467–76. 2. aldrees am. pattern of skeletal and dental malocclusions in saudi orthodontic patients. saudi med j. 2012; 33(3): 315–20. 3. wahab rma, idris h, yacob h, ariffin shz. cephalometric and malocclusion analysis of kadazan dusun ethnic orthodontic patients. sains malaysiana. 2013; 42(1): 25–32. 4. konstantonis d, anthopoulou c, makou m. extraction decision and identification of treatment predictors in class i malocclusions. prog orthod. 2013; 14: 1–8. figure 3. differences in the mean of each variable for the group treated with extractions and the group treated without extractions. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p144-148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p144-148 148 bunga, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 144–148 5. konstantonis d. the impact of extraction vs nonextraction treatment on soft tissue changes in class i borderline malocclusions. angle orthod. 2012; 82(2): 209–17. 6. hong m, kook y, kim m, lee j, kim h, baek s. the improvement and completion of outcome index: a new assessment system for quality of orthodontic treatment. korean j orthod. 2016; 46(4): 199–211. 7. hong m, kook y, baek s, kim m. comparison of treatment outcome assessment for class i malocclusion patients: peer assessment rating versus american board of orthodontics-objective grading system. j korean dent sci. 2014; 7(1): 6–15. 8. jain m, varghese j, mascarenhas r, mogra s, shetty s, dhakar n. assessment of clinical outcomes of roth and mbt bracket prescription using the american board of orthodontics objective grading system. contemp clin dent. 2013; 4: 307–12. 9. premkumar s. orthodontics : preparatory manual for undergraduates. 2nd ed. new delhi: elsevier; 2008. p. 436-40. 10. cansunar ha, uysal t. comparison of orthodontic treatment outcomes in nonextraction, 2 maxillary premolar extraction, and 4 premolar extraction protocols with the american board of orthodontics objective grading system. am j orthod dentofac orthop. 2014; 145(5): 595–602. 11. anthopoulou c, konstantonis d, makou m. treatment outcomes after extraction and nonextraction treatment evaluated with the american board of orthodontics objective grading system. am j orthod dentofac orthop. 2014; 146(6): 717–23. 12. mavreas d, athanasiou ae. factors affecting the duration of orthodontic treatment: a systematic review. eur j orthod. 2008; 30(4): 386–95. d dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p144-148 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p144-148 164 vol. 42. no. 4 october–december 2009 case report cemento-ossifying fibroma of the jaw david b. kamadjaja department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract background: cemento-ossifying fibroma is a benign neoplasm characterized by replacement of normal bone by fibrous tissue and varying amounts of newly formed bone or cementum-like material, or both. the cemento-ossifying fibroma has caused considerable controversy because of confusion regarding terminology and the criteria for its diagnosis. in addition, the cemento-ossifying fibroma often shows variations in clinical, radiographic, and histopathologic features, hence require different treatment options. purpose: this paper attempts to elaborate the classification and terminology of cemento-ossifying fibroma of the jaw, the clinical characteristic, radiographic, and histopathologic features, the different tumor behaviors, and the surgical treatment modalities required. case: two patients diagnosed with cementifying fibroma and two patients with ossifying fibroma were reported, presenting their clinical presentation, diagnostic imaging, and histopathology reports, as well as their surgical treatments. classifications of fibro-osseous lesion of the jaws and characteristics as well as variations in several aspects of cemento-ossifying fibroma of the jaws are discussed. conclusion: the diagnosis of cemento-ossifying fibroma of the jaw can be established quite consistently based on clinical, radiographic, and microscopic features. however, these tumors may exhibit variations in their neoplastic behaviors. it is therefore important to take into account the individual tumor behavior when one is planning a proper surgical treatment. the behavior of the tumor governs the required surgical treatment which may range from simple curettage of the tumor to radical resection of the jaw. key words: cementifying fibroma, ossifying fibroma, tumor’s behaviors correspondence: david b. kamadjaja, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: davidbk@sby.dnet.ned.id introduction the ossifying fibroma is a benign neoplasm characterized by the replacement of normal bone by fibrous tissue and varying amounts of newly formed bone or cementum-like material, or both. as a result of histological similarities, ossifying fibroma, fibrous dysplasia, and cemento-osseous dysplasia are classified together as benign fibro-osseous lesions. the diagnosis of benign fibro-osseous lesions is based on clinical, radiographic, and histopathologic correlation.1 ossifying fibroma is a benign neoplasm usually presented as a painless, slow-growing, expansile lesion which is believed to be confined to the jaws and craniofacial complex.2 there are numerous similarities between this lesion and the cementifying fibroma, a fibroosseous lesion arising from the periodontal membrane, regarding predilection of age of occurrence, sex, location, roentgenographic appearance, and clinical behavior. therefore, the term cemento-ossifying fibroma is now more widely used. the cemento-ossifying fibroma is odontogenic in origin, whereas ossifying fibroma is of bony origin. cementoossifying fibroma is a fibro-osseous lesion that arises from the periodontal membrane.3 it contains multipotential cells that are capable of forming cementum, lamellar bone, and fibrous tissue.4,5 the cemento-ossifying fibroma has caused considerable controversy because of confusion regarding terminology and the criteria for its diagnosis.6 in addition the cementoossifying fibroma often shows variations in clinical, radiographic, and histopathologic features depending on the nature of the tumors. majority of the lesions grow slowly and unidentified by the patient until swelling of the face is noted while in other cases some tumor may grow rapidly and cause symptoms. inadequate surgical treatment may 165kamadjaja: cemento-ossifying fibroma cause recurrence of the lesions, therefore proper diagnosis and treatment plan are required to achieve good result in the management of this tumor. in this paper, four patients diagnosed with ossifying fibroma and cemento-ossifying fibroma of the jaw who subsequently underwent surgical treatment are presented. cases case #1: a 21-years-old male patient came to our clinic with chief complaint of large swelling of left mandible. it was first noted by the patient 8 months ago, has been growing slowly and was not associated with pain. clinically, a large swelling was noted on the left buccal region measuring approximately 8 cm in diameter, which was firm and non-tender on palpation (figure 1-a). intraorally, a large smooth-surface mass was found on the left mandible extending to the left buccal mucosa. it was firm and non-tender on palpation (figure 1-b). panoramic x-ray showed round shape radiolucency on the left mandible extending from the distal aspect of #34 towards the region of #37. it has sharp and sclerotic margin, showing multiloculation and spots of radiopacity in the centre of the lesion. the teeth #35, 36, and 37 were missing (figure 1-c). the incisional biopsy of the lesion revealed the microscopic diagnosis of cementifying fibroma. figure 1. (a) a large swelling on the left buccal region; (b) the intra oral mass on the left mandible extending to the left buccal mucosa); (c) on panoramic a round shape radiolucency noted in the left mandible showing sharp, sclerotic margin, multiloculation and spots of radiopacity in the centre of the lesion; (d) the tumor appeared as a round, encapsulated mass with multiple foci of whitish component in the centre of the lesion reflecting the tissue with calcified material; (e) microscopic view showing stroma of fibroblastic proliferation with foci of cementum-like material e c d ba 166 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 164-171 the tumor was removed together with the adjacent bone with segmental resection of the left mandible. upon removal of the bone segment it was found that the tumor was a round, encapsulated mass with multiple foci of whitish component in the centre of the lesion reflecting the tissue with calcified material (figure 1-d). the defect in the mandible was reconstructed by placement of bridging plate using coen’s stainless steel reconstruction plate. the histopathologic result shows that the tumor consists of fibroblast proliferation with foci of cementum-like material (figure 1-e) which confirm the diagnosis of cementifying fibroma. case #2: a 29-year-old female patient came to our clinic with complaint of painless swelling of the left maxilla. it started as a small mass six years ago which grew slowly and did not give any symptoms. the mass, however, grew relatively faster in the past six months causing noticeable facial deformity which rendered her to seek medical consult. clinically, a large swelling was noted over her left cheek and malar region which was firm and non-tender on palpation (figure 2-a). intra orally, a rounded, smooth-surface, well-circumscribed mass was found on the buccal side of maxilla from the region of #21 through #26. the mass was also found on the posterior part of the left hard palate extending nearly to the midline (figure 2-b). panoramic x-ray show a large mass in maxilla extending from #11 region towards that of #26 and involves the nasal cavity as well as the left maxillary sinus. the tumor consists of a blend of radiopaque and radiolucent components and figure 2. (a) a large swelling on the left cheek and malar region; (b) intra orally, a rounded, smooth-surface, well-circumscribed mass found on the buccal side of maxilla and over the posterior part of the left hard palate extending nearly to the midline; (c) panoramic x-ray showing a blend of radiopaque and radiolucent components with defined boundaries showing sclerotic margin; (d) ct scan showing the extension of the mass to nasal cavity as far as middle nasal conchae and towards medial part of the left maxillary sinus; (e) macroscopically, the tumor was well encapsulated on its buccal part which was easily shelled out from the surrounding bone whereas the medial part had relatively poor margin requiring maxillectomy; (f) the microscopic view of the lesion showing proliferation of spindle-shape cells with multiple formation of bony trabeculae showing osteoblastic rimming at the periphery of the trabeculae. a b c d e f 167kamadjaja: cemento-ossifying fibroma has defined boundaries showing sclerotic margin (figure 2-c). computed tomogram scan shows a round tumor mass at the buccal side of the left maxilla which extends to hard palate, nasal cavity as far as middle nasal conchae, and towards medial part of the left maxillary sinus (figure 2-d). incisional biopsy of the lesion revealed cementifying fibroma. the surgery was done using weber-fergusson approach, followed by complete removal of the whole tumor from the left maxilla. the tumor was found to be rounded and well encapsulated on its buccal part but has a relatively poor margin on its medial part, therefore resection of the left maxilla, or hemimaxillectomy, was done (figure 2-e). the defect in the maxilla after resection was reconstructed by packing it up with vinyl polysiloxane impression material (putty type, exaflex™, gc, japan) supported with acrylic surgical obturator serving as a base plate. the histopathology examination of the lesion shows proliferation of cellular fibrous connective tissue with multiple formations of bony trabeculae showing osteoblastic rimming at the periphery of the trabeculae (figure 2-f). these findings support the diagnosis of ossifying fibroma. case #3: a 17-year-old female patient came with a large swelling of the right mandible which had been growing from a small mass in the gum since 10 years previously. the mass never caused pain except that there had been episodes of bleeding from the tumor recently. clinically, a large swelling was noted over her right mandible which was firm and non-tender on palpation (figure 3-a). intra orally, the tumor was found on the buccal side of the mandible obliterating the mucobuccal fold over the region of #45 to the right retromolar area. the lower right molars were severely displaced lingually. the mass was firm and not tender on palpation. the lingual side of the mandible was normal (figure 3-b). figure 3. (a) a large swelling over the patient’s right cheek involving the lower border of the mandible. (b) intra orally, the tumor noted on the buccal side of the mandible obliterating the mucobuccal fold; (c) on panoramic the lesion noted as irregularlyshaped, intrabony lesion with ill-defined margins showing flecks of radiopacity within the radiolucent area indicating increased calcification of the tumor; (d) the resected mandible showing a well encapsulated mass involving the body and ascending ramus of the mandible; (e) microscopic view showing formation of bone trabeculation within fibrous connective tissue with osteoblastic rimming being strongly evident at periphery of the trabeculae. e b c d a b 168 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 164-171 panoramic x-ray showed irregularly-shaped intrabony lesion with ill-defined margins extending from the region of tooth #33 to the right ascending ramus; flecks of radiopacity were seen within the radiolucent area indicating increased calcification of the tumor (figure 3-c). the incisional biopsy of the tumor showed the microscopic features of ossifying fibroma. the surgical treatment performed in this case was hemimandibulectomy of the right mandible extended to the left parasymphysis distal to tooth #33. following resection of the mandible it was found that the tumor was a well encapsulated mass involving the body and ascending ramus of the mandible and causing destruction of the lower border of the mandible (figure 3-d) but the lingual part of the mandible was normal. reconstruction of the mandible was performed using autogenous rib graft which was supported with stainless steel reconstruction plate. the histopathologic examination shows formation of bone trabeculation within fibrous connective tissue with osteoblastic rimming being strongly evident at periphery of the trabeculae (figure 3-d). these findings confirmed the diagnosis of ossifying fibroma. case #4: a 30-year-old female patient came with a swelling on her right mandible of six months duration. there had been no pain or chewing problem except for facial deformity caused by the swelling. clinically, there was a marked swelling on the right parasymphyseal region and body of the mandible which was firm and non-tender on palpation (figure 4-a). intra orally, the tumor appeared as obliteration of the vestibulum over the region of #43 to #46, slightly displacing tooth #45 (figure 4-b). the mass was firm and not tender on palpation and the lingual side of the mandible was normal. c d a b figure 4. (a) an oval-shaped swelling on the right parasymphysis region and body of the mandible; (b) intra orally, the tumor appeared as obliteration of the vestibulum oris; (c) panoramic x-ray showing oval-shaped radiolucency at the body of the mandible which has distinct boundaries but lacks sclerotic margins, flecks of radiopacity is seen within the lesion; (d) the microscopic examination showing cellular fibrous connective tissue with multiple foci of mineralized component resembling the features of cementum. 169kamadjaja: cemento-ossifying fibroma panoramic x-ray showed that the tumor appeared as radiolucency at the body of the mandible from #42 to mesial of #46, which was somewhat oval in shape, had distinct boundaries but lacks sclerotic margins. the anterior extension of the lesion was not very clear in the panoramic x-ray due to its superimposition with the cervical bony structure. a small amount of radiopaque component was noted in the center of the lesion (figure 4-c). the incisional biopsy of the tumor revealed the microscopic features of cementifying fibroma. the surgical treatment performed in this case was en bloc resection of the right mandible followed by ostectomy of the surrounding bone using large round surgical bur, leaving the lingual plate of the mandible intact. coen’s stainless steel reconstruction plate was subsequently placed across the defect, serving as a stabilizing plate, to prevent the mandible from pathologic fracture (figure 4-d). the histopathology examination of the lesion shows cellular fibrous connective tissue with multiple foci of mineralized component without osteoblasting rimming, the feature resembling that of cementum (figure 4-d). these findings confirmed the diagnosis of cementifying fibroma. discussion maxillofacial fibro-osseous lesions comprise a group of face and jaw disorders characterized by the replacement of normal bone by a benign connective-tissue matrix with varying amounts of mineralized substances.7 the designation “fibro-osseous lesion” is not a specific diagnosis and describe only a process. fibro-osseous lesions of the jaws were initially classified by waldron8 into three main categories namely, fibrous dysplasia, fibro-osseous (cemental) lesions such as ossifying and cementifying fibroma, and fibro-osseous neoplasms such as juvenile active ossifying fibroma. the concept of ‘fibro-osseous lesions’ of bone has evolved over the last several decades and now includes two major entities: fibrous dysplasia and ossifying fibroma, as well as the other less common lesions such as florid osseous dysplasia, periapical osseous dysplasia, focal sclerosing osteomyelitis, proliferative periostitis of garre, and ostitis deformans.6 in recent years, these lesions were reclassified into fibrous dysplasia, reactive (dysplastic) lesions arising in the tooth-bearing area, and fibrous osseous neoplasms such as cementifying and ossifying or cemento-ossifying fibroma.9,10 in contrast, based on nomenclature by kramer et al.11 the cementoossifying fibroma is described as an osteogenic neoplasm and the fibrous dysplasia as a non-neoplastic bone lesion. a neoplastic etiology of ossifying fibroma is supported by examples of lesions that achieve a large size, exhibit aggressive behavior, and produce significant osseous destruction.2 additionally, recurrences, though rare, have been described in some studies of ossifying fibroma. chromosomal translocations have been identified in a few cases of ossifying fibroma, however, the molecular mechanisms that underlie the development of this tumor remain unknown.2 the cemento-ossifying fibroma has caused considerable controversy because of confusion regarding terminology and the criteria for its diagnosis. the cemento-ossifying fibroma is odontogenic in origin, whereas ossifying fibroma is of bony origin. cemento-ossifying fibroma is a fibro-osseous lesion that arises from the periodontal membrane.3 it contains multipotential cells that are capable of forming cementum, lamellar bone, and fibrous tissue.5 a close histogenetic relationship exists between the central ossifying fibroma and the central cementifying fibroma. it is based on the marked similarity between the two regarding predilection of age of occurrence, sex, race, location, roentgenographic appearance, and clinical behavior, these two lesions represent the same basic neoplastic process. the only difference between the two being in the type of cell involved and its end product-cementum in one case and bone in the other. this has prompted many to use the term cemento-ossifying fibroma.2 its occurrence in anatomical regions, not associated with periodontal membrane, is unexplained. it was supposed that pluripotential mesenchymal cells could differentiate, as does the periodontal ligament, to produce calcified material resembling bone and cementum, as well the presence of ectopic periodontal membrane has been hypothesized.12 despite its origin in the periodontal membrane, the factors that stimulate this structure to produce cementum in an aberrant anatomical site remain controversial. inflammation secondary to either infections or trauma has been proposed as a causative agent.13 clinically, the cemento-ossifying fibroma presents as a painless, slowly growing mass in the jaw where displacement of teeth may be the only early clinical feature.6 the lesion is therefore frequently ignored by the patient until the growth produces a noticeable swelling and facial deformity. the tumor is well-circumscribed from its surrounding bone and will continue to grow bigger, slowly or actively, until it is removed surgically.11 these seem to be the case in all of our patients considering that they have ignored the masses in their jaws as they had been asymptomatic and that the lesions had all grown considerably big when they first came to our clinic. all of the three cases in the mandible above showed only buccal bony extension with the lingual bone being normal. this is in contrast to sapp et al.14 who suggest that cementoossifying fibroma often exhibit marked buccal and lingual bony expansion. cemento-ossifying fibroma has a marked predilection for female sex, the female: male ratio being 2:1.6,14 central cemento-ossifying fibromas are more commonly found in the mandible than in the maxilla6 some reports indicate 90 percent of all cases are located in the mandible.10 in mandible, it occurs particularly in the premolar-molar region.1,2,6,15 these characteristic clinical features of cemento-ossifying fibroma support several facts in the our cases where three of the four cases occurred in females, 170 dent. j. (maj. ked. gigi), vol. 42. no. 4 october–december 2009: 164-171 three of the four cases were found in mandible, and all of the tumors in the mandible occurred in the premolar-molar region. regarding the age of onset, one case was found in the second decades of life, one case in the early third decades and the other two cases in their late third decades of life. this is in accordance with the majority of literatures3,11 which show that cemento-ossifying fibroma occur mainly in the second to the fourth decades of life. the radiographic appearance is of utmost importance in the diagnosis of cemento-ossifying fibroma because it is often needed to separate it from other fibro-osseous lesions. the lesions may be either unilocular or multilocular.14 all of the tumors in the mandible above radiographically appeared as multilocular lesion and the tumor in maxilla appeared as a unilocular lesion. in the early stages, the cementoossifying fibroma appears as a radiolucent lesion with no evidence of internal radiopacities. as the tumor matures, there is increasing calcification so that the radiolucent area becomes flecked with opacities until ultimately the lesion appears as an extremely radiopaque mass. the cementoossifying fibroma presents a radiolucent appearance in 53%, a sclerotic radio density in 7% and mixed or mottled appearance in 40% of the cases.12 variation in the amount of radiopacities and radiolucency are seen in our reported cases. the first and fourth case show small amount of radiopaque component whereas in the second and third case much larger amount of radiopacities are found in the centre of the lesions. interestingly, the differences in the amount of opacities within the lesion seem to have correlation with the duration of tumor themselves. the tumors in the first and fourth case were of eight and six months duration respectively, whereas those found in the second and third case were six and ten years old respectively. these facts strongly suggest that the older the tumor the larger the amount of the calcified material found within the lesion. o n e a d d i t i o n a l i m p o r t a n t d i a g n o s t i c f e a t u r e radiographically is that there is a centrifugal growth pattern rather than a linear one and therefore the lesions grow by expansion equally in all directions and present as a round tumor mass.6 this characteristic rounded-shape is reflected in three of the cases reported herein, only one case in the mandible exhibiting irregular shape. there are three different patterns of radiographic borders of cemento-ossifying fibroma which are: defined lesion without sclerotic border (40%); defined lesion with sclerotic border (45%); and lesion with ill-defined border (15%) indicating a rapidly growing tumor.12 these variations in the tumor borders are also evident in our cases. the first two cases showed defined margins with sclerotic border, the fourth case exhibited defined margin but lacked sclerotic border, while the third case showed ill-defined border. the case with ill-defined border has, actually, a history of ten year old tumor which seemingly does not support the theory which stated that ill-defined border indicated a rapidly growing tumor. however, since there was a history of bleeding episodes experienced by the patient over the past few months it is possible that the tumor may have grown rapidly within the bone so that it has made the border of the tumor became less distinct. the characteristic macroscopic features of this tumor is replacement of normal bone by a benign connective-tissue matrix with varying amounts of mineralized substances, however, there are some variations in microscopic features of this tumor. the microscopic findings mirror the radiographic findings. the more radiolucent lesions are composed of cellular fibrous connective tissue, frequently in a whorled pattern.14 collagen fibers are often arranged haphazardly, although a whorled, uniform pattern may be evident. calcified deposits are noted throughout the fibrous stroma. the nature of the hard tissue is generally quite variable within a given tumor as well as between lesions. irregular trabeculae of woven bone or lamellar bone are most consistently noted in these tumors. additional patterns of calcified material include small, ovoid to globular, basophilic depostis and anastomosing trabeculae of cementum-like material.2 these variations in hard tissue configuration make no difference to the clinical behaviour of the tumour. however, recognition of these structures is important in establishing its diagnosis.16 osteoblast may or may not be evident at the periphery of the bone deposits. a thin outer zone of fibrous connective tissue is usually present, separating the fibro-osseous tissue from the surrounding normal bone.14 the microscopic examination of all of the cases presented above show characteristic calcified material within fibrous connective tissue which are the indicative features of cemento-ossifying fibroma. in the first and fourth case the foci of calcified material observed show the characteristic feature of cementum hence the term of cementifying fibroma. in the second and third case, however, the foci of calcified material appear as bony trabeculae with evidence of osteoblastic rimming at the periphery of the trabeculae therefore the term ossifying fibroma is applied as the histopathologic diagnosis. treatment of cemento-ossifying fibroma generally has been by conservative enucleation or curettage or radical surgery1,2,11 depending on the size and location of the individual lesion.4 they are characterized by easy shell out from the surrounding bone.2 conservative surgery is therefore recommended even if the tumour is large with bowing and erosion of the inferior border of the mandible and radical treatment of the tumour such as an en bloc resection should only be considered if there are recurrences due to its aggressive nature.11 slootweg and muller18 reported that there was no difference in outcome between patients treated in a more limited way and those treated by major surgery. other authors, however, advocate more extensive surgery for more aggressive lesions and lesions involving craniofacial bones in light of the potential for recurrence.19,20 sakoda et al.17 described the procedure of a segmental resection of an extensive ossifying fibroma with the replacement of the excised segment with immediate reconstruction. eversole and his coworkers19 in a study of 64 cases of cemento-ossifying fibroma reported a recurrence rate of as high as 28 per cent following surgical curettage of these lesions. 171kamadjaja: cemento-ossifying fibroma it is interesting to note that all of our current cases were treated radically comprising en bloc and segmental resection of the mandible in the fourth and first case respectively, hemimandibulectomy with autogenous bone graft in the third case, and surgical excision combined with radical resection of the maxilla in the second case. in the author’s opinion they were reasonable sort of treatment since most of the patients came with relatively large tumors and had histories of rapid increase in size which might indicate increase aggressiveness of the lesion. moreover, it is almost impossible to accomplish complete excision of the tumor in cases when the size of the tumor is extensively large only with surgical curettage through intra oral approach, not to mention the higher risk for mandible fracture following curettage procedure especially if the inferior border of the mandible had been involved in the tumor. the radical surgical treatments in the above cases were, therefore, aimed at eliminating the risk of tumor recurrence as well as the risk of pathological fracture of the jaw following tumor exicision. it is useful to note, however, that since ossifying fibromas do not display infiltrative patterns into bone booth2 suggest smaller margins than the 1 cm typically required for ameloblastoma, odontogenic myxoma, or calcifying epithelial odontogenic tumor. en bloc resection followed by surgical ostectomy performed in the fourth case was the least aggressive surgical treatment in this case series since there was no history of rapid tumor growth, clinically and radiographically it was relatively not aggressive, and there still remained sufficient amount of bone in the inferior border of the mandible after excision of the tumor. as the conclusion of this paper, although it is relatively not difficult to establish the diagnosis of cemento-ossifying fibromas from clinical, radiographic, and microscopic features, these tumors may exhibit variations in their neoplastic behaviors. it is, therefore, important to take into account the individual tumor behavior when one is planning a proper surgical treatment in order to eliminate the tumor completely and avoid tumor recurrence and at the same time improve the patient’s cosmetic and functional problems. references 1. booth pw, schendel sa, hausamen je: maxillofacial surgery. 2nd ed. st. louis, missouri: churchill livingstone; 2007. p. 506–9. 2. regezi ja, sciubba jj. oral pathology-clinical pathologic correlations. 3rd ed. philadelphia: wb saunders co; 1999. p. 357–60. 3. waldron ca. fibro-osseous lesions of the jaws. j oral maxillofac surg 1993; 51:828–35. 4. brannon rb, fowler cb. benign fibro-osseous lesions: a review of current concepts. adv anat pathol 2001; 8: 126–43. 5. alawi f. benign fibro-osseous diseases of the maxillofacial bones. a review and differential diagnosis. am j clin pathol 2002; 118(suppl): s50–s70. 6. sarwar hg, jindal mk, ahmad ss. cemento-ossifying fibroma-a rare case. j ind soc pedo and prev dent 2008; 26: 128–31. 7. toyosawa s, yuki m, kishino m, ogawa y, ueda t, murakami s, et al. ossifying fibroma vs fibrous dysplasia of the jaw: molecular and immunological characterization. modern pathology 2007; 20: 389–96. 8. waldron ca. fibro-osseous lesions of the jaws. j oral maxillofac surg 1985; 43: 249–62. 9. kos m, luczak k, godzinski j, et al. treatment of monostotic fibrous dysplasia with pamidronate. j craniomaxillofac surg 2004; 32: 10–15. 10. neville bw, damm dd, allen cm, bouquot je. oral & maxillofacial pathology. philadelphia: wb saunders co; 1995. p. 469–70. 11. ong ahm, siar ch, cemento-ossifying fibroma with mandibular fracture. case report in a young patient. austrl dent j 1998; 43(4): 229–33. 12. barberi a, cappabianca s, colella g. bilateral cemento-ossifying fibroma of the maxillary sinus. br j radiol 2003; 76: 279–80. 13. brademann g, werner ja, jänig u, mehdorn hm, rudert h. cemento-ossifying fibroma of the petro-mastoid region: case report and review of the literature. j laryngol otol 1997; 111: 152–5. 14. sapp jp, eversole lr, wysocki gp: contemporary oral and maxillofacial pathology. 2nd ed. st louis, missouri: mosby; 2004. p. 116–7. 15. su l, weathers dr, waldrom ca. distinguishing features of focal cemento-ossifying dyplasia and cemento-ossifying fibromas (ii). a clinical and radiologic spectrum of 316 cases. oral surg oral med oral pathol oral radiol endod 1997; 84: 540–9. 16. neyaz z, gadodia a, gamanagatti s, mukhopadhyay s. radiographical approach to jaw lesions. sing med pict essay j 2008; 49(2): 165. 17. sakoda s, shiba r, irino s. immediate reconstruction of the mandible in a patient with ossifying fibroma by replantation of the resected segment after freezing. j oral maxillofac surg 1992; 50: 521–4. 18. slootweg pj, muller h. juvenile ossifying fibroma: report of four cases. j cranio-max-fac surg 1990; 18: 125–9. 19. eversole lr, leider as, nelson k. ossifying fibroma: a clinicopathologic study of sixty-four cases. oral surg oral med oral pathol 1985; 60(5): 505–11. 20. commins dj, tolley ns, milford ca: fibrous dysplasia and ossifying fibroma of the paranasal sinuses. j laryngol otol 1998; 112(10): 964–8. vol 49 no 1 jan-mrt 2016.indd p-issn: 1978-3728 e-issn: 2442-9740 volume 49, number 1, march 2016 editorial boards of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2016 january 2nd – december 31st, 2016 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii of faculty of dental medicine, universitas airlangga chief editor: ketut suardita (department of conservative dentistry faculty of dental medicine, universitas airlangga) editorial boards: roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); boy muchlis bachtiar (department of oral biology faculty of dentistry, universitas indonesia, indonesia); boedi oetomo roeslan (epartment of biochemistry faculty of dentistry, universitas trisakti); rahmi amtha (department of oral medicine faculty of dentistry, universitas trisakti, indonesia); fajar hamonangan nasution (department of orthodontics faculty of dentistry, universitas trisakti, indonesia); anita yuliati (department of dental material faculty of dental medicine, universitas airlangga, indonesia); darmawan setijanto (department of dental public health faculty of dental medicine, universitas airlangga, indonesia); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); pinandi sri pudyani (department of orthodontics faculty of dentistry, universitas gadjah mada, indonesia) managing editors: hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ira widjiastuti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); markus budi rahardjo (department of oral biology, faculty of dental medicine); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia) assistant editors saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia) peer-reviewers jenny sunariani (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); haslinda z tamin (department of periodontics, faculty of dentistry, universitas airlangga, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); tri murni abidin (department of conservative dentistry faculty of dentistry, universitas sumatera utara, indonesia); agung krismariono (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); istiati (department of oral patology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); adioro soetojo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); diah savitri ernawati (department of oral medicine faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral pathology, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuniardini septorini wimardhani (department of oral medicine, faculty of dentistry, universitas indonesia, indonesia) administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 237/05.16/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 49, number 1, march 2016 p-issn: 1978-3728 e-issn: 2442-9740 1. methisoprinol as an immunomodulator for treating infectious mononucleosis maharani laillyza apriasari .......................................................................................................... 1–4 2. the difference of saline and sterile water for tetracycline hydrochloride solvents in cementum demineralization shinta ferronika, ahmad syaify, and dahlia herawati ............................................................... 5–9 3. potency of stichopus hermanii extract as oral candidiasis treatment on epithelial rat tongue syamsulina revianti and kristanti parisihni ................................................................................ 10–16 4. the thickness of odontoblast-like cell layer after induced by propolis extract and calcium hydroxide irfan dwiandhono, ruslan effendy, and sri kunarti ................................................................... 17–21 5. the potential of chitosan combined with chicken shank collagen as scaffold on bone defect regeneration process in rattus norvegicus fitria rahmitasari, retno pudji rahayu, and elly munadziroh ................................................. 22–26 6. effects of application of anadara granosa shell combined with sardinella longiceps oil on oesteoblast proliferation in bone defect healing process rima parwati sari, eddy hermanto, dinda divilia, indira candra, wisnu kuncoro, and tantri liswanti .......................................................................................................................... 27–31 7. salivary neutrophils isolation of severe early childhood caries patients with flow cytometry analysis using magnetic beads and cd177 marker muhammad luthfi and tuti kusumaningsih ................................................................................ 32–36 8. the effect of combined moringa oleifera and demineralized freeze-dried bovine bone xenograft on the amount of osteoblast and osteoclast in the healing of tooth extraction socket of cavia cobaya rostiny, eha djulaeha, nike hendrijantini and agus pudijanto ............................................... 37–42 9. effect of mangosteen peel extract combined with demineralized freezed-dried bovine bone xenograft on osteoblast and osteoclast formation in post tooth extraction socket utari kresnoadi, yurike hadisoesanto, and harly prabowo ...................................................... 43–48 10. beta-defensins-2 expressions in gingival epithelium cells after probiotic lactobacillus reuteri induction tuti kusumaningsih ........................................................................................................................ 49–53 11. early detection and treatment of speckled leukoplakia selviana tampoma and iwan hernawan ....................................................................................... 54–58 vol 51 no 1 jan-mrt 2018.indd 11 research report dental journal (majalah kedokteran gigi) 2018 march; 51(1): 1–4 the increase of vegf expressions and new blood vessels formation in wistar rats induced with post-tooth extraction sponge amnion moh. basroni rizal,1 elly munadziroh,2 and indah listiana kriswandini3 1 department of dental material, faculty of dentistry, universitas hang tuah 2 department of dental material, faculty of dental medicine, universitas airlangga 3 department of oral biology, faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: tooth extraction is the process of removing a tooth from the oral cavity potentially triggering a wound healing response in the body. as a result, many methods have been applied to improve the wound healing process, especially in wounds resulting in complications. one such method involves the application of amniotic membrane which has anti-inflammatory, anti-bacterial, antifibrosis, anti-scarring properties with low immunogenicity, epithelialization effects, and secretory leukocyte protease inhibitor (slpi). it also contains collagen, various growth factors, transferrin, fibronectin, nidogen, proteoglycans, hyaluronan and laminin. purpose: this study aimed to determine the effects of sponge amnion on the number of vegf expressions and new blood vessels in post-tooth extraction wounds of wistar rats. methods: sponge amnion was produced by mixing freeze-dried amnion membrane from the tissue bank at rsud dr. soetomo with 1% gelatin before freeze drying the mixture. wistar rats were then divided into two groups. in group 1, referred to as the control group, the post-extraction wounds of the rats received no treatment. meanwhile, in group 2, the treatment group, the subjects’ post-extraction wounds were treated with sponge amnion. the rats of both groups were sacrificed on day 3 to allow observation of the number of vegf expressions and new blood vessels. a statistical analysis test, a t-test, was subsequently conducted. results: there was a significant difference in the number of new blood vessels in the control group and that of the treatment group with a p value of 0.018 (p<0.05). there was also a significant difference in vegf expression between the two groups with a p value of 0.000 (p <0.05). conclusion: sponge amnion can generate a number of vegf expressions and new blood vessels in the post-extraction wounds of wistar rats. keywords: sponge amnion; angiogenesis; socket healing; vegf expressions correspondence: elly munadziroh, department of dental material, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: emunadziroh@yahoo.com; m.basroni.rizal@gmail.com; introduction tooth extraction is a common procedure performed by dentists with a prevalence rate in indonesia of 38.5%.1 nevertheless, the process can lead to complications in 37.6% of cases. fractures are the most common complication at 30.4% prevalence. the tooth extraction socket may be considered as a form of bone fracture which can cause disruption to the wound healing process, possibly triggering a wound healing response from the body.2 in general, the wound healing process can be divided into three phases, namely; inflammatory, proliferative and remodeling.3 the inflammatory phase initiates a condition in which macrophages secret cytokines and regulatory factors. these consist of vascular endothelial growth factor (vegf), fibroblast growth factor (fgf) and transforming growth factor-beta1 (tgf-β1), all of which play an important role in the wound healing process.4 new blood vessels formed through a process of angiogenesis play a role in maintaining the continuous function of various tissues and organs affected by the wound5 by subsequently supplying oxygen and nutrients useful for the formation of new tissues.6 currently, various methods of improving wound healing have been implemented, one of which involves placing an amniotic membrane on the wound. amniotic membrane dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p1–4 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p1-4 2 rizal, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 1–4 is known to contain collagen types i, iii, iv, v and vi, fibronectin, nidogen, proteoglycans, hyaluronan, laminin and secretory leukocyte protease inhibitor (slpi).7,8 moreover, it also contains various growth factors, such as epidermal growth factor (egf), keratinocyte growth factor (kgf), hepatocyte growth factor (hgf) and transforming growth factor (tgf-α).9 in fact, amniotic membranes have widely been employed as biomaterials within various clinical applications due to the fact that it is flexible and semi-transparent, protects the wound, reduces pain and has a re-epithelization effect.7,10,11 therefore, this research aimed to assess the application of amnion sponge on postextraction wounds by observing the number of vegf expressions and new blood vessels formation as indicators of the angiogenesis process. materials and methods t h i s r e s e a r c h c o n s t i t u t e d a l a b o r a t o r y b a s e d experimental investigation using ten male wistar rats weighing 200–250 grams. the subjects were obtained from the biochemistry laboratory of the faculty of medicine, universitas airlangga. they were then divided into two groups of five. in group 1, referred to as the control group, the wistar rats’ post-tooth extraction wounds remained untreated. meanwhile, in group 2, known as the treatment group, such wounds were given sponge amnion. the wistar rats were anesthetized intramuscularly, their mandibular incisors subsequently being extracted using lower anterior forcep. in the control group, suturing was conducted post-tooth extraction. meanwhile, in the treatment group, after extraction, amnion sponge was applied to the extraction sockets, before suturing was performed. sponge amnion was produced by smoothing freezedried amnion membranes (<24 hours) from biomaterials center / tissue bank / dr. soetomo hospital with a sterile aquadest at a ratio of 1: 1. after becoming amnionic porridge, it was added to 1% gelatin at a ratio of 1:1, and then freeze dried (lyophilizer) for 2 x 24 hours to induce the formation of sponge. on the third day, both groups of wistar rats were sacrificed. their mandibular tissue, together with the extraction socket, was removed and soaked in fixation solution for 48 hours. the two were then decalcified until the bone became sufficiently tender to be cut. thereafter, paraffin blocks were prepared and cut to a thickness of 4–5μ. histologic preparations were made incorporating the use of both imunohistochemical imaging (ihc) to enable observation of vegf expression and hematoxylin-eosin (he) staining to highlight the number of new blood vessels. data calculations were then performed under a light microscope at 400x magnification on the 1/3 apical socket area of the tooth extraction. the vegf expressions measured were the number of endothel cells emitted a brownish color on immunohistochemical staining with anti-vegf polyclonal antibodies (abcam product, ab46154). the number of blood vessels, on the other hand, was determined by the prevalence of luminal formations surrounded by a layer of endothelial cells. the data obtained was then analyzed by means of a parametric test using an independent t-test with a significance level of 95% (0.05). results the results of this research confirmed that vegf expressions in one third of the tooth extraction apical sockets of the treatment group were higher than those in the control group. figure 1 illustrates vegf expressions after ihc staining on one third of the tooth extraction apical sockets at a magnification of 400x. moreover, the results indicated that the number of new blood vessels in one-third of the tooth extraction apical sockets of the treatment group were higher than those of the control group. figure 2 demonstrates that the appearance of a lumen-formed image surrounded by a layer of endothelial cells in one-third of the tooth extraction apical socket preparations after he staining at a magnification of 400x. a b figure 1. vegf expressions. (a) vegf expressions in the control group on day 3. (b) vegf expressions in the treatment group on day 3. arrows show the endothelial cells expressing vegf (brown color) after the ihc staining at a magnification of 400x. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p1–4 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p1-4 3rizal, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 1–4 with regard to normality test results, a kolmogorov smirnov test produced a p value of 0.634 (p>0.05) for vegf expressions and a p value of 704 (p>0.05) for the new blood vessels. these results indicated the data to be normally distributed (table 1). the mean of vegf expressions in the treatment group was also confirmed to be higher than that in the control group. there was a significant difference in vegf expressions between the control and treatment groups with a p value of 0.000 (p<0.05). the mean number of new blood vessels in the treatment group was also higher than that in the control group. similarly, there was a significant difference in the number of new blood vessels between the control group and the treatment group with a p value of 0.018 (p<0.05) (table 2). discussion bone tissue engineering innovation has focused on biomaterial applications, including scaffolds, being developed. sponge-shaped scaffolds with a porous structure are suitable for cell attachment, cell proliferation, cell differentiation and specific tissue formation.12 in this research, amniotic and gelatin membrane biomaterials were made of sponge with the objective of their being more easily applied to the post-extraction socket and of reducing post-extraction bleeding. amniotic membranes have anti-inflammatory, anti-bacterial, anti-fibrosis and anti-scarring properties with low immunogenicity, re-epithelialization effects, several growth factors, slpi, collagen, fibronectin, nidogen, proteoglycans, hyaluronan, and laminin.7–9,13 the availability of almost infinite amniotic membranes and the process of obtaining them easily and cheaply for therapeutic purposes cause them to be regarded as having greater potential as biomaterials.14 various growth factors are also found in amniotic membranes, including: egf, kgf, hgf, fgf, tgf-α and tgf-β.9 the results of this research showed that there was a significant difference in vegf expressions between the control and treatment groups. vegf expressions are generally influenced by stimulation of the host, such as estrogen, nitric oxide (no), various growth factors (bfgf, pdgf, tnf-α, tgf-β, egf, and igf1), as well as inflammatory cytokines (il-6).15,16 in the early phase, post-extraction inflammation will occur, in which lactate levels increase and oxygen concentration is between 0-10 mmhg. the increased activity of inflammatory cells then leads to a hypoxic atmosphere and elevated lactate levels. table 1. distribution of normality number of newvegf expressions blood vessels 1010number of samples kolmogorovsmirnov z 0.7040.634 b a figure 2. the number of new blood vessels. (a) the number of new blood vessels in the control group on day 3. (b) the number of new blood vessels in the treatment group on day 3. the arrows show the lumen of the new blood vessels after the he staining at a magnification of 400x. table 2. the mean, standard deviation, and p value of vegf expressions and new blood vessels in the control and treatment groups vegf expressionsgroups number of new blood vessels 7.60 ± 2.073.60 ± 0.89control 12.60 ± 3.1311.80 ± 2.05treatment 0.0180.000p value figure 3. the mean and standard deviation of vegf expressions and new blood vessels in the control and treatment groups. vegf control vegf treatment mean standard deviation blood vessels control blood vessels treatment dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p1–4 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p1-4 4 rizal, et al./dent. j. (majalah kedokteran gigi) 2018 march; 51(1): 1–4 the latter, together with oxygen concentration at 0-10 mmhg, will attract young macrophages and fibroblasts migrated to the extraction sockets. thereafter, macrophages in the wound will elaborate growth factors, known as macrophage-dependent angiogenic factors, resulting in chemotactic for endothelial cells. endothelial cells then lead to the injury, and then release vegf playing a role in angiogenesis and vasculogenesis.5 the results of this research also showed that the average number of new blood vessels in the treatment group was statistically higher than that in the control group. in other words, there was a significant difference in the number of new blood vessels between the control group and the treatment group. the results also revealed that the amniotic membrane was able to increase the number of new blood vessels since it contains fgf and tgf-β, two of the angiogenic factors.9 angiogenic factors comprising vegf, fgf, tnf-α, tgf-β, and pdgf bind to endothelial cell receptors around the site of the old blood vessels before activating, as well as generating, signals sent to the nucleus to produce protease enzymes which play an important role in the degradation of the extracellular matrix.5 migration of endothelial cells then occurs to strengthen the branching structures of blood vessels, followed by the formation of new ones. therefore, amniotic membranes can increase the number of new blood vessels.17 several studies have shown that tgf-β plays a role in inhibiting the proliferation of endothelial cells in vitro and in vivo since tgf-β induces angiogenesis and stimulates vegf expressions by attracting inflammatory cells.5 in infant rats, the administration of tgf-β at a dose of 1 μg even can stimulate the occurrence of increased production of macrophages, fibroblasts, and collagen, as well as the formation of new capillaries.5 tgf-β may also regulate vegf expressions through the apoptotic process of endothelial cells by activating tgf-β derived from vascular endothelial growth factor receptor-2 (vegfr-2). the apoptotic process of endothelial cells is required for the formation of lumen within blood vessels.18 fgf, on the other hand, is known not only to stimulate endothelial cell proliferation in vitro (at concentrations of 1 to 10 ng / ml), but also to stimulate an in vivo angiogenic process leading to new blood vessel growth during the wound healing process by increasing the reendothelialization process in damaged blood vessels.5 growth factors affecting vegf expressions and existing in the amniotic membrane are fgf and tgf-β. finally, it can be concluded that amnion sponge can increase the expressions of vegf and the number of new blood vessels in the post-tooth extraction wounds of wistar rats. references 1. kementerian kesehatan republik indonesia. riset kesehatan dasar (riskesdas). jakarta: badan penelitian dan pengembangan kesehatan republik indonesia; 2007. p. 135. 2. dani fr. potensi ekstrak umbi teki (cyperus rotundus l.) dalam menurunkan jumlah limfosit jaringan granulasi setelah sencabutan gigi tikus wistar jantan. scription. jember: unversitas jember; 2012. p. 14. 3. li j, chen j, kirsner r. pathophysiology of acute wound healing. clin dermatol. 2007; 25(1): 9–18. 4. la r java h. o ra l wou nd hea l i ng: cel l biolog y a nd cl i n ica l management. singapore: wiley-blackwell; 2012. p. 1–82. 5. frisca, sardjono ct, sandra f. angiogenesis: patofisiologi dan aplikasi klinis. j kedokteran maranatha. 2009; 8(2): 174–87. 6. de mendona rj. angiogenesis in wound healing. in: davis j, editor. tissue regeneration from basic biology to clinical application. intech; 2012. p. 93–108. 7. niknejad h, peirovi h, jorjani m, ahmadiani a, ghanavi j, seifalian am. properties of the amniotic membrane for potential use in tissue engineering. eur cell mater. 2008; 15: 88–99. 8. munadziroh e. isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene. dent j (maj ked gigi). 2008; 41(3): 123–7. 9. effendi rg, suhendro g, handojo i. perbedaan kadar epidermal growth factor pada selaput amnion tanpa preservasi dan dengan cr yopreser vat ion (penel it ia n ek sper i ment a l laborator is). j oftalmologi indonesia. 2009; 7(1): 1–5. 10. hennerbichler s, reichl b, pleiner d, gabriel c, eibl j, redl h. the influence of various storage conditions on cell viability in amniotic membrane. cell tissue bank. 2007; 8(1): 1–8. 11. parolini o, alviano f, bagnara gp, bilic g, bühring h-j, evangelista m, hennerbichler s, liu b, magatti m, mao n, miki t, marongiu f, nakajima h, nikaido t, portmann-lanz cb, sankar v, soncini m, stadler g, surbek d, takahashi ta, redl h, sakuragawa n, wolbank s, zeisberger s, zisch a, strom sc. concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells. stem cells. 2008; 26(2): 300–11. 12. niu x, fan y, liu x, li x, li p, wang j, sha z, feng q. repair of bone defect in femoral condyle using microencapsulated chitosan, nanohydroxyapatite/collagen and poly(l-lactide)-based microsphere-scaffold delivery system. artif organs. 2011; 35(7): e119–28. 13. andriani i, sudibyo. perbedaan efektivitas antara bedah flap posisi koronal dengan dan tanpa membran amnion pada perawatan resesi gingiva. thesis. yogyakarta: universitas gadjah mada; 2010. p. 18. 14. ihsan i, prijanto p. the difference of epidermal growth factor concentration between fresh and freeze-dried amniotic membranes. j oftalmologi indonesia. 2009; 7(2): 62–6. 15. obianime aw, uche fi. the phytochemical screening and the effects of methanolic extract of phyllanthus amarus leaf on the biochemical parameters of male guinea pigs. j appl sci environ manag. 2008; 12(4): 73–7. 16. istvan s. investigation of incident and development of spontaneous tumours typical of crl: cd br rat strain in two carcinogenicity studies performed under similar circumstance. diser tation. keszthely: keszthely; 2008. 17. kleinsmith lj. understanding cancer and related topics understanding angiogenesis. 2008. available from: http://cancer.gov/cancertopics/ understandingcancer. accessed 2008 oct 20. 18. fer ra r i g, cook bd, ter ush k in v, pintucci g, mignatti p. transforming growth factor-beta 1 (tgf-β1) induces angiogenesis through vascular endothelial growth factor (vegf)-mediated apoptosis. j cell physiol. 2009; 219(2): 449–58. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i1.p1–4 http://cancer.gov/cancertopics/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i1.p1-4 mkg vol 39 no 1 jan 2006 isi.pmd 39 dental root periapical resorption caused by orthodontic treatment pinandi sri pudyani department of orthodontia faculty of dentistry gadjah mada university yogyakarta indonesia abstract dental root resorption especially in maxillary incisive region almost always happens simultaneously with orthodontic treatment, and it gained researchers attention, in particular after the use of periapical radiography. however, the fundamental etiology of dental root resorption is still dubious. multifactoral causes are mentioned, among others are hormonal, nutritition, trauma, dental root form and dental root structure anomalies, genetic, while from treatment side are duration, types, strength scale and dental movement types. based on these findings, orthodontic treatment was proven to cause dental root resorption in maxillary incisive teeth. key words: dental root resorption, orthodontic treatment correspondence: pinandi sri pudyani, c/o: bagian ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii, sekip utara yogyakarta 55281, indonesia. introduction dental root resorption in maxillary incisive region, almost always happens simultaneously with orthodontic treatment and it became researcher’s concern after the availability of periapical radiography.1,2,3 dental root resorption case studies from 1856 until 1993 with 120 reported cases were still unable to conclude the fundamental etiology of dental root resorption caused by orthodontic treatment.3,4 it was stated that dental root resorption had multifactoral causes.5 epidemiologic studies revealed that dental root resorption post-orthodontic treatment was not a major problem. apical morphologic scoring by measuring directly the pre-treatment dental length, seldom demonstrated heavy resorption. recent studies showed that risk factor had an important role in dental root resorption. the risk factor constituted of genetic factor and the application of orthodontic equipment system. that research explained how risk factor only 20% influenced the dental root resorption due to orthodontic treatment, while many other factors were assumed as risk factors, such as dental agenesis, dental malformation, hormonal, pre-treatment trauma, number and dental movement types.2,6 the aim of this writing was to study dental root resorption in various type of orthodontic treatment along with its cause. dental root resorption dental root resorption is a related condition of physiologic and pathologic processes causing dentin, cementum or bone disappearance. the resorption process is a joint-work of inflammation cells, resorber, and hard tissue structure, which are difficult to predict, to diagnose and also to treat. dental root resorption is very similar to bone resorption. bone, dentin and cementum laceration and irritation will bring about chemical changes of those tissues causing the formation of multinucleotide giant cells or more commonly known as osteoclasts. this cell working along with macrophage and monocyte is responsible for hard tissue resorption. as a whole, this cell holds a role in molecular biologic complex involving cytokine, enzyme and hormones on the continuation of resrption process.7 resorption causal factors numerous studies found out the influence of orthodontic treatment did not cause heavy dental root resorption. but, several individual variations must be noticed. some studies revealed individual predisposing factor existed and turned into multifactoral causes. thus, the study result of the relation between post-orthodontic dental root resorption and risk factors was not clear.8 various causal factors were assumed to be the cause of dental root resorption due to orthodontic treatment, they were called risk factors, i.e.: 1) long, narrow and atypical dental root form, 2) pre-treatment traumatic laceration, 3) bad habit of fingers and tongue for a long period of time, 4) some dental agenesis, 5) peg-form teeth, dental invagination, taurodontism, severe dental root deviation; and 6) eruption pattern of decidual teeth and abnormal permanent and ectopic eruption.6,9 other researchers expressed that peg-formed and rudimentary teeth will not increase the risk factor of dental root resorption caused by orthodontic treatment.10 other influential factor was biologic factor i.e. endocrinologic gland deviations, nutrition, hormonal imbalance, dental root structure, and alveolar bone density. the causal factor was also from the treatment side i.e. 1) treatment, 2) the beginning age of orthodontic treatment, this was connected to dental root forming stage, and 40 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 39–42 3) variations of mechanical factor, such as the type of tools, its strength, treatment types, plaque retention on the application of orthodontic equipment, and the direction of dental movement.3,4 dental root resorption in various orthodontic treatment system treatment with edgewise system from 200 patient's post-treatment panoramic radiography using edgewise system, the dental root resorption was as follows: 40% was moderate resorption i.e. blunting teeth until ¼ dental root in one region that was central or lateral incisive; and 20% had root resorption level equal with incisive central mandibular teeth. severe dental root resorption was shown in 6 patients (3%) on both sides of central incisive teeth. moderate dental root resorption was found in premolar teeth (6%). in this study, the mean of root resorption on maxillary central incisive teeth was 1.17 mm, whereas on mandibular incisive was 0.87 mm.11 other study with edgewise system on division 1 class ii mal-occlusion treatment, without tooth extraction, added with maxillary oral extra equipment, and the use of class ii elastic, revealed 99.08% subject, suffered post-treatment dental root resorption. from 208 treated maxillary incisive teeth, 172 (82.69%) endured dental root resorption. male patients was 84.37% and female patients was 81.25%. the dental root resorption in male was more often on lateral incisivum (87.50%) compared to central incisivum (81.25%). ignoring gender, the resorption on lateral incisivum was 83.65%, while on central incisive was 81.73%. the percentage of right maxillary incisive dental root resorption was 84.62%. and the left side was 80.77%. ignoring gender, the frequency of dental root resorption on right maxillary incisive was bigger than the left side. the mean of central incisive was 2.19 mm, and the lateral incisivum was 2.31 mm.12 treatment with begg system the study was done on 42 patients treated with begg system. pre-treatment already showed dental root resorption on 4% cases, during treatment, before doing uprighting on tipping teeth, irregular dental root form was found 25% (score 1) and 31% score 3 root resorption (less than 2 mm). at the end of treatment, 48% was root resorption less than 2 mm (score 3) and score 5 (less than 2 mm up to 1/3 dental root was 3%). six months after treatment, the biggest dental root resorption was on mandibular central incisive (95%), maxillar central incisivum (90%), maxillar lateral incisivum (87%), mandibular canine (79%), and maxillar canine (72%). there was low incidence of mandibular premolar dental root resorption. it was affirmed that should dental root resorption already existed on pre-treatment, will cause more severe resorption during orthodontic treatment.13 dental root resorption in various dental movement orthodontic treatment with dental intrusion, acquired a significant root shortening. mandibular incisive dental root shortening was smaller than maxillary, probably due to smaller intrusion on mandibula or because of the different speed of bone turnover rates. several cases showed severe dental root resorption in upper and lower jaw with treatment-length 28.6 ± 4.3 months. the total number of intruded teeth in the lower jaw was significant with the total number of dental root shortening.14 other writer obtained insignificant correlation (r = 0.03) between dental root resorption number and intrusion achievement on both sides of incisivus. it was said that no correlation was found between dental root resorption and the span of time of teeth intrusion (r = 0.02).15 on treatment of anterior opened bite case, there was no significant difference between the length of dental root preand post-treatment. if compared with control group, i.e. inside bite, the dental root of anterior opened bite case was shorter before treatment. posttreatment showed dental root resorption and less significant facial bone support. the result of this study demonstrated orthodontic treatment had no big role in dental root resorption of anterior opened bite case treatment of adults.16 the research on the correlation of dental root resorption and dental root to labial side treatment with torque, displayed dental root torque to labial, did cause dental root resorption. observation was done towards incisive teeth, as the result of dental movement yielding a shortening of the central incisive teeth length by 0.9 mm per year. the disapperance of dental root structure was 1.4 mm per year or 1.56% per year. this happened because of dental extrusion with arch wire mechanical power.17 vardimon et al. 18 in his research on external dental root resorption with iatrogenic nature on treatment with palatal expansion, found a dental mesiobuccal root of tooth with more than 1 root, closest to buccal cortical plate(12%), while distobuccal root was external dental root resorption (49%). in canines, the dental root resorption was 12% lower than tooth abutment. it can be explained as follows, canine dental root moved farther from solid bone lamellae whereas dental root with more than one translation bodily into buccal cortical plate. dental root located close to buccal cortical plate, will have external root rersorption bigger than dental root located closer with less solid palatal cortical plate. discussion irreversible dental root resorption on apical dental root was caused by dental movement in orthodontic treatment and it was known as treatment phenomenon. the specific feature of hard tissue disappearance, was widely publicized on orthodontic library, although the causal factor and the real impact of orthodontic treatment was yet to be proven. dental root resorption caused by orthodontic treatment most often happened to maxillary incisive teeth.3,9,13,15,16,19,20 many possibilities were considered as the cause, such as high abnormality frequency from dental root and the distance passed through of dental movement during orthodontic treatment. 41pudyani: dental root periapical resorption another factor for incisive dental root resorption was due to the development of dental root closer with maxillary canine dental eruption. therefore, there was a possibility that lateral incisive dental root acted as the guide for caninus eruption teeth. erupted canine follicles often brought damage to lateral incisive apical, but the damage will not be seen in the radiography. caninus eruption teeth often resorbed lateral incisive dental root from the palatal side.9,19 maxillary dental root resorption was more severe than mandibular. incisive teeth had undergone more root resorption than canine teeth on its curve. lateral incisive dental root was the most severe resorption, followed by maxillary central incisivus, maxillary canine, mandibular canine, mandibular central incisivus and mandibular lateral incisivus. several possibilities to explain why maxillary lateral incisivus undertook the most severe resorption, was the maxillary lateral incisivus showed the highest percentage in abnormality of dental root form i.e. peg-form, barrel, crown. also in maxillary lateral incisive was ranked the 3rd of missing teeth after m3 and premolar of two mandibles. the root of lateral incisive dental root proned to be slimmer, so that often mislocated to messial direction should there be growth deficit on premaxillary. in this condition, if a bodily movement was needed at distal of anterior region, only lateral incisive teeth could move to 3 sectors significantly. the lateral incisive teeth often showed abnormalities i.e. dilacerated teeth, bottled form, and pointed teeth.9,19 as a rule, the sign of incisive dental root resorption had come out in the first stage of utilizing the orthodontic equipment, although only minimal change of periapical contour without significant root length shortening.20,21 in this research, 24% research subjects underwent root shortening, but only 3.6% experienced shortening more than 2 mm. more or less 4.1% of patients endured root resorption with mean of 1.5 mm on all four maxillary incisive teeth. minimal one maxillary incisive tooth underwent dental root shortening ≤ 2 mm. dental root resorption happened in 3–9 months after the application of orthodontic equipment. this research also revealed the influence of risk factor to dental root resorption caused by orthodontic treatment with less than 25% explained.20 the impact of history of trauma as risk factor in dental root resorption caused by orthodontic treatment was still debatable. previous studies demonstrated dental movement with severe lacerations often followed by moving of dental place without resorption and inflammation. malmgren22 in his study of light and moderate traumatic history showed no additional dental root resorption. pre-treatment observation of minimal 5 months gave the same result with observed cases more than 1 year. dental root resorption caused by trauma, was reported on all cases for 2–5 months after trauma causing luxation and subluxation. trauma on incisive teeth causing dental root resorption will continue without signs. this happened due to the damage of periodontal ligament and cementum which will yield to easier process of dental root resorption. orthodontists tried to minimize dental root resorption by avoiding big power on cases with history of trauma. treatment system used in this study was varied between fixed and removable including the use of activator. incidence and the degree of resorption without trauma history was 33% with score 2 (blunting dental root until ¼ of root length) and 10% with score 3 (blunting dental root more than ¼ root length) on treatment with edgewise system. in begg system treatment, 43% was with score 2 and 5% with score 3. the degree of dental root resorption in begg system treatment was lower than previously reported study.22 external dental root resorption was also connected to genetic predisposing. there was a correlation of the scale of dental root resorption during orthodontic treatment to il-bl gene. this gene was produced by cytokine il-1, believed to be able to decrease the risk of external dental root resorption.23 it was concluded that orthodontic treatment was proven to cause dental root resorption, in particular the maxillary incisivum. the dental root resorption caused by orthodontic treatment most often happened to maxillary incisive teeth, the most severe was on lateral incisive dental root. no conclusive data of the influence of risk factor to dental root resorption caused by orthodontic treatment. the result of this study was not yet able to demonstrate clearly the impact of treatment variables, i.e. treatment duration, system and the scale of power being used in orthodontic treatment towards dental root resorption. dental root resorption happened almost always to teeth undertaking orthodontic power. therefore, orthodontic strength must be adjusted to dental movement, avoiding to give extra power to dental with risk factor, among others history of trauma, hormonal imbalance, dental form deviation, and genetic abnormality. references 1. reitan k. initial tissue behavior during apical root resorption. angle orthod 1974; 44(1):68–82. 2. remington dn, joondeph dr, artun j, riedel ra, chapko mk. long term evaluation of root resorption occuring during orthodontic treatment. am j orthod dentofac orthop 1989; 96(1):43–6. 3. baumrid s, korn el, boyd rl. apical root resorption in orthodontically treated adults. am j orthod dentofac orthop 1996; 110(3):311–20. 4. lupi je, handelman cs, sadowsky c. prevalence and severity of apical root resorption and alveolar bone loss in orthodontically treated adults. am j orthod dentofac orthop 1996; 109(1):28–37. 5. linge l, linge bo. patient characteristics and treatment variables associated with apical root resorption during orthodontic treatment. am j orthod dentofac orthop 1991; 99(1): 35–43. 6. lee r, artun j, alonzo, ta. are dental anomalies risk factors for apical root resorption in orthodontic patients?. am j orthod dentofac orthop 1999; 116(2):187–95. 7. ne rf, witherspoon de, gutmann jl. tooth resorption. quintessence int 1999; 30(1):9–24. 8. mirabella d, artun j. risk factors for apical root resorption of maxillary anterior teeth in adult orthodontic patients. am j orthod dentofac orthop 1995; 108(1):48-55. 9. kook ta, park s, sameshima gt. peg-shaped and small lateral incisors not at higher risk for root resorption. am j orthod dentofac orthop 2003;123(3):253–8. 42 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 39–42 10. saidarriaga jr, patino cp. ectopic eruption and severe root resorption. am j orthod dentofac orthop 2003; 123(3):259–65. 11. kaley j, phillips c. factors related to root resorption in edgewise practice. angle orthod 1991; 61(2):125–32. 12. de shields r. a study of root resorption in treated class ii division 1 malocclusions. angle orthod 1969; 39(4):231–45. 13. goldson l, henrikson co. root resorption during begg treatment: a longitudinal roentgenologic study. am j orthod 1975; 68(1):55–66. 14. mc fadden wm, engstrom c, engstrom h, anholm m. a study of the relationship between incisor intrusion and root shortening. am j orthod dentofac orthop 1989; 96(5): 390–6. 15. dermaut lr, de munck a. apical root resorption of upper incisors caused by intrusive tooth movement: a radiographic study. am j orthod dentofac orthop 1986; 90(4):321–6. 16. harris ef, butler ml. patterns of incisor root resorption before and after orthodontic correction in cases with anterior open bites. am j orthod dentofac orthop 1992; 101(9): 112–9. 17. .goldin b. labial root torque: effect on the maxilla and incisor root apex. am j orthod dentofac orthop 1989; 95(3):208–19. 18. vardimon ad, graber tm, voss lr, lenke j. determinants controlling iatrogenic external root resorption and repair during and after palatal expansion. angle orthod 1990; 61(2):113–22. 19. sameshima g, sinclair p. predicting and preventing root resorption: part i. diagnosing factors. am j orthod dentofac orthop 2001; 119(5):505–10. 20. smale i, artun j, behbehani f, doppel d, van‘t hof m, jagtman amk. apical root resorption 6 months after initiation of fixed orthodontic appliance therapy. am j orthod dentofac orthop 2005; 128(1):57–67. 21. sameshima g, sinclair pm. predicting and preventing root resorption: part ii. treatment factors. am j orthod dentofac orthop 2001; 119(5):511–5. 22. malmgreen o, goldson l, hill c, orwin a, petrini l, lundberg m. root resorption after orthodontic treatment traumatized teeth. am j orthod 1992; 82(6):487–91. 23. al-qawasmi ra, hartsfield j, everett et, flury l, liu l, foroud tm, macri j, roberts we. genetic predisposition to external apical root resorption. am j orthod dentofac orthop 2003; 123(3):242–52. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 27 chronic periodontitis as an etiology of sleep disturbances and premenstrual syndrome (pms) haryono utomo department of oral biology faculty of dentistry airlangga university surabaya indonesia abstract it is obvious that sleep disturbances may induced by acute pulpal or periodontal pain. other causes of sleep disturbances which also termed as sleep dysfunction, or insomnia, according to the patient has to be treated by physician. nevertheless, in a case report, surprisingly, periodontal treatment relieved sleep disturbances and premenstrual syndrome (pms). coincidentally, women also more vulnerable to sleep disturbances and periodontal disease. it is also interesting that the exact etiology of pms is still unknown, and 80% women who suffered from pms also experience sleep disturbances. recently, there has been increasing numbers of literatures and evidence-based cases linking periodontal disease to systemic diseases. however, systemic effects of periodontal disease that lead to pms which associated with sleep disturbances are rarely discussed. several mechanisms had been proposed to involve in these symptoms: female sexual hormonal imbalance, stimulation of the hypothalamic-pituitary-adrenal axis (hpa-axis) and neurogenic switching mechanism. in addition, as estrogen makes women more susceptible to stress, it worsen the symptoms. the glucocorticoid hormones synthesized upon stimulation of the hpa-axis, either by stress or pro-inflammatory cytokines, may disrupt the sleep-wake cycle; and also create estrogen dominance. the aim of this study is to propose the etiopathogenesis of pms which associated with sleep disturbances that may be related to chronic periodontitis. since in this case report scaling and curettage resulted in the disappearing of pms and sleep disturbances; the conclusion is that chronic periodontal disease may act as one of the etiologies of pms and sleep disturbance. key words: chronic periodontitis, sleep disturbances, premenstrual syndrome correspondence: haryono utomo, c/o: bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id. telp. 5053195 introduction what is a good sleep? a study revealed that sleeping shorter that 6 hours and more than 9 hours a day for adults is considered unhealthy, one of the risk factor is diabetes. the best time to sleep is at night, if nighttime sleep disturbances exists and daytime sleep or take a nap is needed, it should be less than 30 minutes, otherwise the circadian rhythm will be altered.1 in daily general medicine practice, most frequently reported complaints which affecting the quality of life by women is insomnia, a kind of sleep disturbance. it is interesting that as many as 80% of women report premenstrual syndrome (pms) is associated with insomnia; in contrast, generally premenstrual insomnia disappears a few days after menstruation begins.2,3 nevertheless, recent publications revealed that the exact etiology of pms is still unknown; as the result, in insomnia related to pms patients, the drug of choice is anti insomniacs i.e., hypnotics, anti-depressant.3 if the sleep disturbances are predicted to be one of pms symptoms, the medications are mostly hormonal, i.e. gonadotropinreleasing hormone agonist; another drug of choice are cyclooxygenase-2 (cox2) inhibitors. other treatments are biofeedback, massage and chiropractic.4 unfortunately, dental and periodontal treatments are not included in treatment protocol for insomnia and pms. compare to men, women are twice as likely to have difficulties in falling asleep or maintaining sleep at night, so called sleep disturbance. sleep disturbance is also termed as sleep dysfunction, sleep disorders or insomnia. nevertheless, sleep is sounder and less prone to disturbances during young adulthood; however, some women are prone to sleep problems during their reproductive years (30-40 years). hormonal fluctuations associated with menstrual cycle and pregnancy may affect circadian rhythms and stress reactivity, thereby active women are more vulnerable to emotional stress and to concomitant sleep disturbances.2 it is also interesting that based on literatures, women are not only susceptible to stress,5 and debilitating symptoms (i.e. pain,6 sleep disturbances,2,3 chronic fatigue syndrome7), but also to periodontal disease.8 recently, the role of periodontal disease as a source of focal infection that may initiate, perpetuate or aggravate the symptoms of systemic diseases such as cerebrovascular disease, cardiovascular disease, pregnancy problems and respiratory problems had already been recognized.9 nevertheless, the relationship between sleep disturbances and chronic periodontitis is not clearly understood. 2� dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 27–32 a possible correlation between sleep disturbances and chronic periodontitis could be predicted regarding to a case report related to a female patient who suffered from sleep disturbance, symptoms mimicking pms and dysmenorrhoe. after periodontal treatment had been done, headache disappeared instantly and she had an undisturbed sleep at the same night. in her next menstrual period, pms and dysmenorrhoe did not reappear.10 however, the etiopathogenesis of the sleep disturbance associated with pms in women which may induce by chronic periodontitis is still unclear. researches should be done to find out an established explanation how untreated periodontitis may lead to sleep disturbances and pms. if periodontal treatments are included in the treatment protocols, it will give a positive value to the sufferers, at least by avoiding unnecessary medications. the objective of this study is to propose the possibility of chronic periodontitis as an etiology of pms which associated with sleep disturbances based on literatures review. female sexual hormones and stages of menstrual period since puberty, the role of sexual hormones is important in body system regulation. estrogen and progestin are the most important female hormones. there are three estrogens present in significant quantities in the plasma of human female: estrone (e1), 17-b estradiol (e2), and estriol (e3). the estrogenic potency of 17-b estradiol is 12 times that of estrone and 80 times of estriol. by far, the most important hormone of the progestins is progesterone.11 there are several stages of menstrual period after puberty: (1) child bearing age (until 45 years old), (2) peri-menopausal period (45–50 years) and (3) menopausal periods (50 years and beyond).12 during peri-menopause there is a steep decrease of progesterone with a gradual decrease in estrogen. at this period women usually experience several uncomfortable symptoms including sleep disturbance or insomnia that are attributed to vasomotor symptoms (e.g., hot flushes and night sweats) rather than to menopausal status.2 the premenstrual syndrome (pms) up to 85 percent of menstruating women report having pms, and 2 to 10 percent report disabling, incapacitating symptoms. symptoms typically begin between the ages of 25 and 35 years.12 premenstrual syndrome is a recurrent luteal phase condition characterized by physical, psychological, and behavioral changes of sufficient severity to result in deterioration of interpersonal relationships and normal activity.4 there are many symptoms related to pms such as: irritability, insomnia, depression, severe fatigue, headache, vertigo, syncope, visual disturbances, heart palpitation, numbness, hyperalgesia of arms and leg, allergy and infection.12,13 the exact etiology of pms is still unknown, nevertheless pms seems to be related to fluctuations in estrogen and progesterone.4,12 the following has been suggested as possible causes of pms: estrogen-progesterone imbalance; excessive aldosterone, or adh (hormone that functions in the regulation of the metabolism of sodium, chloride, and potassium); carbohydrate metabolism changes; hypoglycemia; allergy to progesterone; retention of sodium and water by the kidneys and psychogenic factors.13 other factor is the raise of body temperature during the luteal phase mediated by increasing progesterone level.14 effects of female sexual hormones to the periodontium estrogen and progesterone has disadvantageous effects to gingiva, as they may cause the susceptibility of women to periodontal disease estrogen decreases gingival keratinization, whereas progesterone increases vasodilatation and permeability. additionally, estrogen and progesterone may act as growth factors of prevotella intermedia that is gram negative periodontopathogenic bacteria.8 estrogen in inflammation and immunity estrogen has several role in inflammation, depends on the level. at low or physiological level, estrogen increases pro-inflammatory cytokine production interleukin-1 (il-1), tumor necrosis factor- (tnf-) and il-6 by monocytes.15 other effect of estrogen in low or physiological level is polarization of the cd4+ (t-helper, th) cells to the th1 cells expression, through the stimulation of cytokines il-12, tnf- and ifn-g upon lipopolysaccharides (lps) activation of antigen presenting cells (apc). interferon-g, a th1 cytokine, stimulates macrophages or monocytes proliferation and activity. conversely, high estrogen as in pregnancy, stimulate the th2 cytokines (il-4, il-10 and il-13) which inactivate macrophages.11,16 many stress-related mental illnesses, including depression and post-traumatic stress disorder (ptsd), occur at least twice as often in women as in men. depression and ptsd are characterized by the dysfunction of an area of the brain called the prefrontal cortex (pfc), which is known to govern higher cognitive abilities like level and shortterm memory.5 a better understanding of such processes may help to elucidate the reason why women are more susceptible to stress-related disorders. in an experiment, after exposure to higher levels of stress, both males and females made significant memory errors. however, after exposure to a moderate level of stress, females were impaired, but males were not, suggesting that females were more sensitive to the pfc-impairing effects of stress. in this study, ovariectomized females showed increased stress sensitivity only after estrogen replacement 8,17 the relationship between progesterone and cortisol cortisol, a glucocorticoid hormone is a derivate of progesterone (figure 1),16 and also able to compete between each other which termed the competitive binding mechanism. it is not surprising because progesterone binds to the human mineralocorticoid receptors (hmrs) with nearly the same affinity as cortisol.18 as the result, higher cortisol level which caused by stressor or pro-inflammatory cytokines may bind to the progesterone receptors which then lowers the progesterone level. one of the side effects 2�utomo: chronic periodontitis as an etiology of low progesterone level is high physiologic estrogen level which leads to estrogen dominance.19 estrogen dominance estrogen and progesterone work in synchronization with each other as checks and balances to achieve hormonal harmony in both sexes. if estrogen level is relatively higher than normal to progesterone or it is rather the relative dominance of estrogen and relative deficiency of progesterone, it creates the condition that termed as estrogen dominance. symptoms that exist in estrogen dominance are almost the same to pms 13,19 the sleep – wake cycle and sleep disturbances individuals differ considerably in their natural sleep patterns. children and adolescents sleep more than adults, and young adults sleep more than older ones. normal sleep consists of four to six behaviorally and electroence phalographically (eeg) defined cycles, including periods during which the brain is active (associated with rapid eye movements, called rem sleep), preceded by four progressively deeper, quieter sleep stages graded 1 to 4 on the basis of increasingly slow eeg patterns. stages 1 and 2 are considered as light sleep; stages 3 and 4 are deep sleep or slow wave sleep (sws) which gradually lessens with age and usually disappears in the elderly. rapid eye movement sleep occurs cyclically throughout the night at intervals of approximately 90 minutes in all age groups (figure 2).20,21 related to the sleep-wake cycle, cortisol and il-6 level fluctuates in a constant pattern which termed the circadian rhythm. it reaches the peak level at about 08.00 – 09.00 am and gradually decreases, the lowest level is around midnight; low cortisol during nighttime sleep is advantageous; the dysregulation of this circadian rhythm is able to disturb the sleep-wake cycle (figure 3).22,23 figure 3. cortisol level and the sleep – wake cycle over a 24-hour period.23 sleep disturbances, in general are the manifestation of disrupted sleep-wake cycle; as a clinical syndrome is defined as difficulty in falling or staying asleep, or non refreshing sleep.14 other criteria for sleep disturbances are: 1) insomnia: difficulty with falling asleep or staying asleep, 2) sleep-onset insomnia: difficulty with falling asleep, 3) sleep-maintenance insomnia: fragmented sleep, difficulty with maintaining sleep, 4) sleep-disordered breathing (sdb): some degree of sleep-related upper airway obstruction, ranging in severity from upper airway resistance syndrome (uars) to obstructive sleep apnea (osa).3 sleep disturbances may arise from several causes such as fear condition, aging, or disease, chronic fatigue figure 1. biosynthesis of steroid hormones.16 figure 2. stages of the sleep-wake cycle.22 30 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 27–32 syndrome; elevation of cortisol and interleukin-6 (il-6) during night time.7,21,24,25 periodontal disease and sleep disturbances according a case report, sleep disturbances may have a correlation with periodontal disease. a female patient, aged 37, had been suffered from sleep disturbances, headache, musculoskeletal pains, fatigue and symptoms mimicking pms and dysmenorrhoe for approximately one year. this patient also suffered from chronic periodontitis. after deep scaling and curettage had been done, the painful symptoms such as headache disappeared instantly; at the same night the patients also had an undisturbed sleep. before and during her next menstrual period, the female patient did not suffered from painful symptoms and symptoms mimicking pms. the patient was evaluated six month later and all symptoms did not reappear.10 discussion according to literatures, the interrelationship of migraine headache, stress, sleep disturbances, female sexual hormones, pms and dysmenorrhoe is exist. nevertheless, which symptom initiates the chain reaction is still in controversy. according to bolay et al.26 migraine headache is triggered by stress and sleep disturbance. conversely, according to baker et al.14 pains in dysmenorrhoe, pms and headache lead to sleep disturbances. in addition, sleep disturbances may be triggered by elevated body temperature in the luteal phase that is mediated by progesterone. in general, female sexual hormones play a role in the etiology of sleep disorders in women, either by having a direct effect on sleep processes or through their effect on mood and emotional state. sexual hormones influence eeg sleep during the luteal phase by increasing the eeg frequency and core body temperature. it is also suggested that lack of estrogen, later in life, contributes to vasomotor symptoms, including hot flashes that cause sleep disturbances. dysmenorrhoe is associated with significantly disturbed sleep quality prior to menses. when compared to control women, women with dysmenorrhoe had altered sex hormones, body temperature, and sleep throughout the menstrual cycle.2,3 despite the controversial theories about the relationship between sleep disturbances and pms; their treatment protocols are mostly indicated for anxiety, depression or hormonal regulation. as this condition could be worsen and lasting for months or years, another solution should be investigated to avoid unwanted effects of the medications. fortunately, some case reports revealed that periodontal treatments that were scaling and curettage, which considered to be noninvasive, were able to relief pain, such as migraine, musculoskeletal pains and dysmenorrhoe; fatigue, sleep disturbances and other symptoms mimicking pms.10,27 periodontal disease is a potential source of bacterial endotoxins (lipopolysaccharides, lps) from gram-negative bacteria and pro-inflammatory cytokines interleukin-1 (il-1), tumor necrosis factor- (tnf-) and il-6. the increase level of these pro-inflammatory cytokines could also be detected in blood serum.9 current proposed mechanism that could elucidate how periodontal disease is able to initiate inflammation in distant site is the neurogenic switching mechanism. it is the interplay between immunogenic and neurogenic inflammation; through this mechanism, the maxillary nerve (cn v2) is able to elicit sinusitis and migraine symptoms via the sphenopalatine ganglion (spg).28,29 in the reviewed case report, the first symptom that instantly disappeared after scaling procedure was headache, followed by sleep disturbance at the same night. it was relevant to articles by boyd28 and bolay et al.26 about the involvement of the trigeminal nerve in headache stimulation. concurrently, it is also important that according to bolay et al.26 migraine headache acts as an etiology of sleep disturbances. the possible explanation of the stimulation and the resolution of migraine headache which initiated by periodontal disease is as follows. first, the sensitization mechanism, headache in this case was primarily stimulated by pro-inflammatory mediators involved in chronic periodontal disease (i.e. il-1, tnf-, il-6 and pge2), bradykinin and no via the neurogenic switching mechanism.28–30 neuropeptides which released by maxillary periodontal primary afferent sensory nerve fibers are able to sensitize the spg which subsequently stimulate the vasodilatation of arteries in the meninges and elicits pain in the surrounding primary afferent sensory fibers.28 secondly, the resolution or the cut off mechanism; as periodontal disease in this case report was considered as chronic periodontitis, deep scaling mostly followed by bleeding; this bleeding was considered advantageous because it facilitates drainage of pro-inflammatory mediators from the inflamed tissue. as the result, it reduced or cut off the neurogenic switching mechanism.30 concerning about the susceptibility of women in inflammatory diseases, according to nalbandian, low estrogen and physiological estrogen (17- estradiol, e2) levels are stimulators of th1 immune response that activate macrophages or monocytes.11 the activated macrophages by e2 are able to release additional pro-inflammatory cytokines i.e. il-1, tnf- and il-6 upon lps challenge which stimulate the hpa-axis that leads to increase cortisol synthesis. interleukin-6 is also renowned as a potential stimulator of hpa-axis an has often been called as stressinducible cytokine.31,32 the proposed mechanism of the relationship between chronic periodontitis and sleep disturbance, especially cortisol and il-6 dependent sleep, could be explained as follows: chronic periodontitis leads to increase serum il-6 level, therefore stimulates cortisol synthesis which in turn inhibits il-6 level. if stress also involved, there will be additional increase of il-6, which also followed by cortisol. stages 3 and 4 that are deep sleep stages of the sws during normal sleep-wake cycle in nighttime are associated with low il-6 level and declining cortisol level.21 consequently, 3�utomo: chronic periodontitis as an etiology high level of il-6 and cortisol have adverse effects in the sleep-wake cycle because they lead to wakefulness and sleep disturbance. the repetition of il-6 and cortisol stimulation-inhibition mechanism causes disturbance of sleep-wake cycle, as the result, the patient could not reach the deep sleep stage.9,21,32 sleep disturbances also related to estrogen dominance which has nearly the same characteristics with pms.3,19 while stimulated hpa-axis increase of cortisol synthesis, it may also decrease progesterone level in several ways: 1) as cortisol itself is a derivate of progesterone, increase of cortisol synthesis may lead to decrease of progesterone level, 2) by competitive binding; as cortisol and progesterone has the same affinity both to corticoid and progesterone receptors, cortisol may binds to progesterone receptors. these mechanisms may reduce the circulating progesterone level, thus increasing the estrogen level and cause estrogen dominance.16,18,19 in contrast to baker et al. findings about the connection of the luteal phase, or the high progesterone phase, with sleep disturbances; the female patient in the reviewed case report suffered from headache and sleep disturbances everyday, albeit she was not having her premenstrual or menstrual period. so far, according to literatures in this review, women are more susceptible to stress, sexual hormonal imbalance, and periodontal diseases. consequently, since periodontal disease is a potent source of lps; stress and low and physiologic e2 which exposed to lps challenged macrophages are able to produce additional pro-inflammatory cytokines, especially il-6 that stimulate the hpa-axis.5,10,11,14,31 these mechanism is proposed to answer the question, why women also more vulnerable to sleep disturbances, especially which are cortisol and il-6 dependent. since periodontal treatment was able to relief headache instantly and sleep disturbance at the same night, therefore it was strongly suggested that periodontal treatment eliminated the symptoms. how periodontal treatment, in this case deep scaling procedure and curettage, resulting in the disappearance of sleep disturbance could be explained in two ways: 1) deep scaling and curettage reduces the source of infection by removing subgingival biofilms, a potential source of lps; 2) they also stimulate bleeding of the inflamed periodontal tissue, thus resulting in drainage of the pro-inflammatory mediators and cut off the neurogenic switching mechanism. resolution of periodontal inflammation which is the source of pro-inflammatory cytokines that responsible in sleep disturbances, especially il-6, subsequently reduces stimulation of the hpa-axis, hence also regulate the cortisol circadian rhythm. concerning to the disappearance of sleep disturbances and symptoms mimicking pms before the next menstrual period after periodontal treatment in the reviewed case report; the conclusion is that chronic periodontitis may act as an etiology of sleep disturbances associated with pms. nevertheless, further researches are needed to include periodontal treatments in the treatment protocols of sleep disturbance and pms. references 1. kemp g, segal r. tips for a good night’s sleep. available online at url http://www.helpguide.org/life/sleep_tips.htm. accessed december 3, 2005. 2. national center of sleep disorder research. sleep, sex differences, and women’s health. available online at url http://www.nhlbi.nih. gov/ health/prof/sleep/res_plan/section4/section4a.html. accessed nov 22, 2006. 3. hertz g, cataletto me. sleep dysfunction in women. available online at url http://www.emedicine.com/neuro/topic656.htm accessed november 2, 2006. 4. moreno ma, giesel a. premenstrual syndrome (pms). updated may, 2006. available online at url http://www.emedicine.com/ ped/topic1890.htm. accessed december 5, 2006. 5. shansky rm, bloom cg, lerman d, mcrae p, benson c, miller k, et al. estrogen mediates sex differences in stress-induced prefrontal cortex dysfunction. mol psychiatry. 2004; 9(5):531–8. 6. gremillion ha. multidisciplinary diagnosis and management of orofacial pain. general dentistry 2000; 2(1):178–84. 7. afari n, buchwald d. chronic fatigue syndrome: a review. am j psychiatry 2003; 160(2):221–6 8. güncü cn, tözüm tf, çalayan f. effects of endogenous sex hormones on the periodontium. aus dent jour 2005; 50(3):138–45. 9. lavigne se. your mouth–portal to your body. probe 2004; 38(3): 114–34. 10. utomo h, prahasanti c. periodontal disease as an etiology of orofacial and musculoskeletal pains in women. indonesian journal of dentistry special edition for kppikg ui xiv 2006; 13(2):202–5. 11. nalbandian g, kovats s. estrogen, immunity and autoimmune disease. curr med chem immun endocrinol metab agents. 2005; 5(1):85–91. 12. dickerson lm, mazyck pj, hunter mh. premenstrual syndrome. am fam phys 2003; 67(8):1743–52. 13. university of maryland medical center. premenstrual syndrome. available online at url http://www.umm.edu/women/pms.htm. accessed december 5, 2006. 14. baker cf, driver hs, paiker j, rogers gg, mitchell d. acetaminophen does not affect 24-h body temperature of sleep in the luteal phase of the menstrual cycle. j appl physiol 2002; 92(4):1684–91. 15. tostes rc, nigro d, fortes zb, carvalho mhc. effects of estrogen on the vascular system. braz j med biol res 2003; 36(2):1143–58. 16. van den broek hh, damoiseaux jg, de baets mh, hupperts rm. the influence of sex hormones on cytokines in multiple sclerosis and experimental autoimmune encephalomyelitis: a review. multiple sclerosis 2005; 11(3):349–59. 17. goldman n, glei da, seplaki c, liu iw, weinstein m. perceived stress and physiological dysregulation in older adults. stress 2005; 8(2):95–105. 18. quinckler m, meyer b, vogt cb, grossmann c, gruber u, et al. agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor. eur j endocrinol 2002; 146:789–800. 19. lam m. estrogen dominance: the silent epidemic available online at url. http://www.lammd.com. accessed november 12, 2005. 20. sherwood l. fundamentals of physiology : a human perspective. 3rd ed. belmont: thomson brooks/cole; 2005. p. 133–35. 21. vgontzas an, chrousos gp. chapter 31. hpa axis and sleep. endotext. 2005. available at url. http: // www.endotext.com. accessed november 2, 2005. 22. lemoyne college website. stages of sleep and brain mechanisms. available online at url http://web.lemoyne.edu/~hevern/psy340/ graphics/sleep_cycle.jpg. accessed. 23. become healthy now. cortisol circadian rhythm in the sleep and awake period. available online at url http://www.becomehealthynow. com/popups/circadian_rhythm.htm. accessed november 20, 2006. 32 dent. j. (maj. ked. gigi), vol. 40. no. 1 january-march 2007: 27–32 24. redwine l, hauger rl, gillin jc, irwin m. effects of sleep and sleep deprivation on interleukin-6, growth hormone, cortisol, and melatonin levels in humans. j clin endocrinol metab 2000; 85(10):3597–603. 25. alesci s, martinez pe, kelkar s, ilias i, ronsaville ds, listwak sj, et al. major depression is associated with significant diurnal elevation in plasma interleukin6 levels, a shift of its circadian rhythm and loss of physiological in its secretion: clinical implications. j clin endocrinol metabolism 2005; 90(5):2522–30. 26. bolay h, reuter u, dunn ak, huang z, boas d. intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. nature med 2002; 8(2):136–42. 27. utomo h, prahasanti c. penyakit periodontal pada nyeri kepala dan nyeri haid. edisi khusus lustrum fkg universitas gadjah mada, 2005; 14(4):101–6. 28. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http://www. migraineprevention.com/index/ html. accessed february 15, 2006. 29. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2):82–98. 30. utomo h. the sphenopalatine ganglion sensitization by periodontal inflammation: a possible etiology for headache and sinusitis in children. majalah kedokteran gigi (dental journal) 2006; 39(2): 66–71. 31. padgett da, glaser r. how stress influence the immune response. trends in immunol 2003 aug; 24(8):4–8. 32. kamma jj, giannopoulou c, vasdekis vds, mombelli a. cytokine profile in gingival crevicular fluid of aggressive periodontitis: influence of smoking and stress. j clin periodontol 2004 october; 31(10):894–902. 5757 dental journal (majalah kedokteran gigi) 2021 june; 54(2): 57–62 original article the effect of 3% binahong leaf extract gel on the wound healing process of post tooth extraction olivia avriyanti hanafiah1, diana sofia hanafiah2 and rahmi syaflida1 1department of oral surgery, faculty of dentistry, universitas sumatera utara 2department of agroecotechnology, faculty of agriculture, universitas sumatera utara medan – indonesia abstract background: one of the common frequently performed procedures in dentistry is tooth extraction. after tooth extraction, a series of biological events occurs in the alveolar socket that ultimately results in healing of the socket. binahong (anredera cordifolia) is an herbaceous plant that contains active compounds that may accelerate the wound healing process. purpose: the aim of this study is to investigate the effect of 3% binahong leaf extract gel on the healing process of the postextraction wound. methods: 3% binahong leaf extract gel was prepared with the composition of 1 g carbopol, 1 g hydroxypropilmethylcellulose (hpmc), 4 g glycerin, 3 g triethanolamine (tea), 0.04 g nipagin, 0.04 g nipasol, 1.2 g binahong leaf extract and aq ad 40 g. a total of 18 people were subjected to tooth extraction and were then divided into two groups, with nine people in the binahong group and nine in the control group. following that, residual socket volume (rsv) was examined on days 3, 7 and 14 after the extraction in both the binahong and control groups. the rsv value was obtained by calculating the mesiodistal x bucolingual width x the depth probing and measured using a calliper and probe. repeated analysis of variance (anova) and friedman test followed by wilcoxon and dependent t-test (p<0.05) were used to analyse data. results: significant difference in the mean rsv between the control and binahong groups was observed on days 3, 7 and 14 post tooth extraction. the rsv value in the binahong group was lower when compared to the control group (p <0.05). conclusion: 3% binahong leaf extract gel can accelerate the process of healing socket wounds. keywords: 3% binahong extract gel; binahong; residual socket volume; socket healing; tooth extraction correspondence: olivia avriyanti hanafiah, department of oral surgery, faculty of dentistry, universitas sumatera utara. jl. alumni no. 2, medan 20155, indonesia. email: olivia.hanafiah@usu.ac.id introduction one of the more frequent procedures in dentistry is tooth extraction.1 after tooth extraction, a series of biological events occurs in the alveolar socket that ultimately results in socket healing. the series of biological events that occur during healing are vascular changes; inflammation; cells migration, proliferation and differentiation; maturation of the extracellular matrix and bone formation. these biological events combine to restore the lost tissue.1 when the tooth is extracted, in the empty socket remains the cortical bone (lamina dura), which is covered by the torn periodontal ligament, with a rim of gingiva epithelium left at the coronal portion.3 blood will fill the socket then coagulate to seal the socket from the oral cavity. during the first week of healing, inflammation occurs.3–5 contaminated bacteria and debris left in the socket are destroyed by white blood cells.3 the growth of fibroblasts and capillaries indicates the onset of fibroplasia in the first week of healing. under the blood clot, the epithelium migrates down the socket wall until it comes into contact with the epithelium on the other side of the wall socket or encounters the granulation tissue. there is accumulation of osteoclasts along the crest bone during the first week of healing.3,4 an abundance of granulation tissue fills the socket by the second week.5 there has been osteoid deposition along the alveolar socket bone. in the smaller socket, the epithelium may be completely intact by this time.3,4,6 the process that started during the second week continues into the third and fourth weeks of healing. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 mailto:olivia.hanafiah@usu.ac.id https://e-journal.unair.ac.id/mkg/index 58 hanafiah et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 57–62 by this time, the epithelialization of most of the socket has already finished.3,5 cortical bone resorption in crest and socket walls continues, and there has been deposition of new trabecular bone.3,4,6 the cortical socket bone is completely resorbed in less than four to six months. on radiographs, it is characterised by the absence of the lamina dura. as the bone fills the socket, the epithelium moves towards the cristae and becomes aligned with the adjacent gingival crest. after a year, fibrous tissue or scar is the only remaining visible sign at the alveolar margins of the edentulous area.3,6 the use of medication can reduce the chance of complications and accelerate the postextraction wound healing process. to reduce the inflammatory process and postoperative pain, oral surgeons usually prescribe non-steroidal anti-inflammatory drugs (nsaids), but the intake of nsaids has several adverse effects, mainly gastrointestinal and haematological problems, renal alterations and mucous and skin reactions.7,8 due to their minimal adverse effects, herbal ingredients are starting to be used as a substitute for chemical drugs. anredera cordifolia, commonly known as binahong, is a herbal plant and is believed to bring various health benefits due to its active compound.9 binahong leaf contains flavonoids, terpenoids, oleanolic acid, ascorbic acid, tannins and saponins.10 as an anti-inflammatory, flavonoids inhibit the metabolic pathway of arachidonic acid, prostaglandin formation and histamine release in inflammation so that in this phase, macrophages can easily carry out their function as phagocytes for debris cells and microorganisms present in the wound.11,12 the antimicrobial effect is obtained from triterpenoids and saponins. to inhibit microbial growth, terpenoids interfere with membrane and cell wall formation so that the membrane or cell wall is not completely formed or is formed but incomplete. saponins can interfere with the permeability of the bacterial cell membrane, which results in the destruction of cell membrane, causing the release of proteins, nucleic acids and nucleotides, which are important components of bacteria. saponins also have the ability to act as an antiseptic, which kills or suppresses the growth of microorganisms in the wound to prevent more severe infection.11 according to hanafiah et al.,13 3% binahong leaf extract gel promotes better wound healing in palatal mucosa than 5% and 7% binahong leaf extract gel. based on the above description, the researchers are interested in identifying the effect of 3% binahong leaf extract gel in accelerating the healing process of soft and hard tissues in postextraction wounds in humans. the main objective of this study is to investigate the effect of 3% binahong leaf extract gel on the postextraction wound healing process. materials and methods the study type was clinical experimental with posttest study design with a control group. this study was carried out at the oral and dental hospital, universitas sumatera utara between june and july 2020. the research ethics commission universitas sumatera utara approved this study (no. 55/kep/usu/2020), according to the declaration of helsinki on medical protocols and ethics. the population used in this study were all oral surgery patients at oral and dental hospital, universitas sumatera utara who had teeth extracted corresponding with inclusion and exclusion criteria. the inclusion criteria were patients aged 20–30 years, not currently taking drugs, no history of systemic disease that could affect wound healing, undergoing extraction of lower molar without complications and generally good oral hygiene. the exclusion criteria were patients who were not willing to be treated and patients who withdrew during the follow-up period. the sampling technique in this study was simple random sampling in which samples that corresponded with inclusion and exclusion criteria were taken randomly from a population. the sample size in this study was calculated using the sample size formula for hypothesis test of the mean difference between two unpaired groups, referring to a study conducted by yüce et al.14 the number of samples in this study was 18 people, consisting of nine control groups and nine treatment groups. the binahong leaf came from a family medicinal plant in simpang pergendangan village, tiga binanga village, karo regency, north sumatra province. the selected binahong leaves were those that were completely opened and at least 12 weeks old. the planting did not use pesticides. the binahong leaves that were collected were washed under running water, drained, then weighed as wet weight. this material was then dried in a drying cabinet at a temperature of ± 35°c, until the consistency of the binahong leaf was dry and dark brown. it was then extracted with 70% ethanol. furthermore, the 3% binahong leaf extract gel was made with the composition of 1 g carbopol, 1 g hydroxypropilmethylcellulose (hpmc), 4 g glycerin, 3 g triethanolamine (tea), 0.04 g nipagin, 0.04 g nipasol, 1.2g binahong leaf extract and aq ad 40. g.13 subjects that corresponded to the inclusion criteria were asked to sign a written informed consent form to participate in this study after understanding the aims and procedures of the study and their benefits and rights as a subjects. all subjects received full mouth scaling before the tooth extraction, so oral hygiene was optimal. the tooth extraction was carried out at the oral and dental hospital, universitas sumatera utara. before the tooth extraction procedure, patients’ vital signs were checked by the operator. subsequently, patients were instructed to rinse their mouths with a 0.2% chlorhexidine solution as an asepsis measure. to extract the lower first molar, the operator performed mandibular block anaesthesia and submucous infiltration with articaine 4% (septanest) 1:100,000 in the region of the tooth to be extracted. after the clinical signs of successful anaesthesia were observed, the operator began an extraction procedure, including loosening of the gingiva using a raspatorium, loosening dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 https://e-journal.unair.ac.id/mkg/index 59hanafiah et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 57–62 and lifting of the tooth from the socket using an elevator, extracting the tooth using forceps and grinding the sharp bone using a bone file. after extraction was complete, the socket was irrigated with sterile saline.15 in the treatment group, 0.3 cc binahong leaf extract gel was smeared directly in the post-extraction socket, once a day, after brushing teeth at night before sleep, for 14 days, while in the control group, no gel application was done. patients were given postextraction instructions, such as to bite the tampon for 30–60 minutes, not to consume hot food and drinks, not to rinse too vigorously, not to play with the postextraction wound area with the tongue and not to suck the postextraction wound area.16,17 clinical evaluation of wound healing was performed by measuring the residual socket volume (rsv). rsv is the ratio of the socket volume. the rsv value was obtained by calculating the mesiodistal width x bucolingual width x the probing depth. mesidostal and bucolingual width was measured with a calliper. probing depth was measured with a unc-15 probe (figure 1).18 rsv examination was carried out on the 3rd, 7th and 14th day after extraction. the measurement results were recorded, and the data were processed statistically to determine the differences between the two groups. the shapiro-wilk test was used to examine the data’s normality. data was presented in tabular form and processed using ibm spss statistics for windows, version 25.0 (new york, usa). the data were a b c figure 1. rsv measurement (a) bucolingual width, (b) mesiodistal width, and (c) probing depth. analysed using repeated analysis of variance (anova) to identify the differences in each group and the friedman test followed by wilcoxon’s and a dependent t-test to identify the differences between groups (p<0.05). results based on the clinical examination of the postextraction socket, it was shown that the application of 3% binahong leaf extract gel did not cause any irritation or inflammation symptoms. the clinical conditions of the postextraction socket in the binahong group were better when compared to the control group, in terms of the colour and consistency of surrounding tissue and bleeding, as shown in figure 2. in the control group, the rsv mean decreased at each measurement interval. in the control group, the highest mean rsv value was on the 3rd day of examination, and the lowest was on the 14th day of examination. the results of repeated anova tests showed that there was a significant change in the size of the rsv in the control group (p = 0.001). the results of a paired wise comparison test showed a significant difference in the rsv for each measurement (p <0.05) (figure 3). in the binahong group, the mean of rsv decreased at each measurement interval. similarly to the control group, day 0 day 3 day 7 day 14 a b figure 2. clinical picture of post-extraction socket groups (a) control and (b) binahong. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 https://e-journal.unair.ac.id/mkg/index 60 hanafiah et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 57–62 the highest mean rsv value in the binahong group was found on the 3rd day of examination, and the lowest was on the 14th day. the post hoc result using a wilcoxon test (for data that were not normally distributed) and a dependent t-test (for data that were normally distributed) showed a significant difference in rsv for each measurement. those were between the 3rd and 7th day (p = 0.008), the 3rd and 14th day (p = 0.008), and the 7th and 14th day (p = 0.004) (figure 4). 0.5295 0.2903 0.1123 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 day 3 day 7 day 14 m ea n r sv figure 3. repeated anova test result rsv in control group. 0.2292 0.1111 0.0536 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 day 3 day 7 day 14 m ed ia n r sv figure 4. friedman test result rsv in the binahong group. 0.5295 0.2903 0.1123 0.3029 0.123 0.0576 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 day 3 day 7 day 14 m ea n r sv control binahong figure 5. test results for the difference in the mean rsv between the control and binahong groups. observed at each measurement interval, the mean of rsv in the binahong group was lower than the control (figure 5). the results of the mann-whitney test showed that there was a significant difference in the mean of rsv between the control and binahong groups on the 3rd day after extraction. the results of the dependent t-test showed a significant difference in the mean of rsv between the control and binahong groups on the 7th day (p = 0.001) and 14th day (p = 0.015) after extraction. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 https://e-journal.unair.ac.id/mkg/index 61hanafiah et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 57–62 discussion this study was preceded by preliminary in vivo and in vitro studies by hanafiah et al.13 and hanafiah et al.19 regarding binahong leaf extract gel formulation and evaluation on wound healing of the wistar rat palatal mucosa and its effect on fibroblast proliferation on palatal mucosa wound healing. based on this study’s results, the rsv value, both in the control group and the binahong group, decreased on the 7th and 14th day. this can be caused by the occurrence of fibroplasia which begins during the first week. fibroplasia is characterised by the growth of fibroblasts and capillaries to migrating epithelium.3 the 7th day of postextraction was the peak of angiogenesis. in the second week postextraction, the socket is filled with granulation tissue and osteoid deposition begins to occur in the alveolar bone.3 the 14th day is the peak of fibroblasts in the wound area.20 the results showed that there was a significant difference in the mean rsv between the control and binahong groups on the 3rd, 7th and 14th days after extraction, while the binahong group showed lower rsv values than the control group. this shows that the wound healing process in the binahong group was better than the control group, which was indicated by the size of the clinical wound shrinking faster. as well as its anti-inflammatory effect, binahong leaf gel can also increase the expression of various growth factors involved in wound healing, increase the proliferation and migration of fibroblasts and have an antibacterial effect. the study conducted by hanafiah21 regarding the transforming growth factor beta 1 (tgf-β1) and plateletderived growth factor two b subunits (pdgf-bb) expression test with immunohistochemistry showed that there was a significant difference between the binahong leaf extract gel group and the control group and the aloclair® gel group with the control group, where administration of 3% binahong leaf extract gel could increase the expression of pdgf and tgf-β.21 stimulating angiogenesis (mitosis of endothelial cells in functional capillaries), mitogenesis (an increase in cell population for wound healing) and macrophages activation (cleaning the wound area and triggering secondary growth factors for bone regeneration and tissue repair) are the specific functions of pdgf.22 regulating the proliferation, differentiation, migration, invasion and chemotaxis of epithelial cells, fibroblasts and immune cells (inflammatory phase) of the tissue, as well as the proliferation, migration, invasion and maturation of endothelial cells (to produce functional blood vessels) during angiogenesis are the functions of tgf-β.23 one of the compounds contained in binahong leaf extract, saponins, can affect the activation and synthesis of tgf-β1 and is able to modify the tgf-β1 and tgf-β2 receptors on fibroblasts. in the remodelling phase, this is an important process for matrix collagen formation. in the proliferative phase, saponins, as angiogenetic agents, can increase the mitogenic activity of endothelial cells in the formation of blood vessels by regulating vegf. based on this, binahong is considered to be able to accelerate wound healing.24 the previous study, conducted by ardiana et al.,25 showed that giving 5% binahong leaf extract gel was also known to increase the number of fibroblast cells in the socket after tooth extraction. hanafiah et al.’s19 in vitro study showed binahong leaf extract gel with a concentration of 62.5 ppm can stimulate fibroblast proliferation and shows a greater potential for wound contraction than aloclair gel with a concentration of 250 ppm in wounds on the palatal mucosa of rats. the results of the scratch wound healing assay in vitro also showed that giving binahong leaf extract gel resulted in a better 3t3 fibroblast cell migration acceleration value compared to aloclair® gel.19 the increase of fibroblasts stimulates the synthesis of the formation of collagen and extracellular matrix so that new tissue can be formed.26,27 in addition, the alkaloid compound from binahong leaf plays a role as an antioxidant and antimicrobial that can help the wound healing process by preventing and protecting the wound area from damage from free radicals and inhibiting the growth of pathogenic bacteria in the wound.28 a study by leliqia et al.29 showed that binahong leaf extract contains oleanolic acid and ursolic acid. oleanolic acid and ursolic acid are triterpenoids that are found in many plants. from the results of examination through a scanning electron microscope (sem), it is known that oleanolic acid can damage bacterial cell membranes.30 meanwhile, ursolic acid works by destroying the integrity of the bacterial cell membrane at an early stage, which is then followed by inhibition of protein synthesis and various bacterial metabolic pathways.31 the limitation of this study was the small sample size because it was conducted during the covid-19 pandemic era. further study with a larger number of samples with detailed examination is still needed. in this study, it was concluded that there was a significant difference in the mean rsv between the control and binahong groups on the 3rd, 7th and 14th day after extraction, where the binahong group showed a lower rsv value than the control group (p <0.05). a 3% binahong leaf extract gel can accelerate the process of post tooth extraction wound healing. further study is needed regarding the effectiveness of 3% binahong leaf extract gel to post tooth extraction socket healing. acknowledgement financial support for this research was provided by a grant from universitas sumatera utara through the applied research contract, talenta usu 2020 (442/un5.2.3.1/ ppm/spp-talenta usu/2020). the authors wish to thank dr., apt. denny satria, m.si for the work on making a 3% binahong leaf extract gel. the authors also express their dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 https://e-journal.unair.ac.id/mkg/index 62 hanafiah et al./dent. j. (majalah kedokteran gigi) 2021 june; 54(2): 57–62 deepest gratitude to rahma, s. kg, gian mubarani, s. kg and rianda dwi m. lubis, s. kg who have been incredibly supportive and reached out during the research. references 1. araújo mg, silva co, misawa m, sukekava f. alveolar socket healing: what can we learn? periodontol 2000. 2015; 68(1): 122– 34. 2. pagni g, pellegrini g, giannobile w v., rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 1–13. 3. hupp jr, ellis e, tucker mr. contemporary oral and maxillofacial surgery. 6th ed. st louis missouri: mosby elsevier; 2014. p. 703. 4. khullar s, a m, datta p. healing of tooth extraction socket. heal talk. 2012; 4(5): 37–9. 5. de sousa gomes p, daugela p, poskevicius l, mariano l, fernandes mh. molecular and cellular aspects of socket healing in the absence and presence of graft materials and autologous platelet concentrates: a focused review. j oral maxillofac res. 2019; 10(3): e2. 6. farina r, trombelli l. wound healing of extraction sockets. endod top. 2011; 25(1): 16–43. 7. herrera-briones fj, prados sánchez e, reyes botella c, vallecillo capilla m. update on the use of corticosteroids in third molar surgery: systematic review of the literature. oral surg oral med oral pathol oral radiol. 2013; 116(5): e342–51. 8. hernández-viana s, silva-gómez n, gálvis-pareja da, martínezpabón mc. the use of analgesics and anti-inflammatories in an oral surgery service in medellín, colombia, 2013-2015. rev fac odontol. 2019; 30(2): 154–68. 9. utami p, puspaningtyas de. the miracle of herbs. jakarta: agromedia pustaka; 2013. p. 38–41. 10. hariana a. 262 tumbuhan obat & khasiatnya. jakarta: penebar swadaya; 2013. p. 60–1. 11. mutiara g, nurdiana, utami yw. efektifitas hidrogel binahong (anredera cordifolia (ten.) steenis) terhadap penurunan jumlah makrofag pada penyembuhan luka fase proliferasi tikus putih (rattus norvegicus) galur wistar kondisi hiperglikemia. maj kesehat fkub. 2015; 2(1): 29–40. 12. rathee p, chaudhary h, rathee s, rathee d, kumar v, kohli k. mechanism of action of flavonoids as anti-inflammatory agents: a review. inflamm allergy drug targets. 2009; 8(3): 229–35. 13. hanafiah oa, trimurni a, ilyas s, nainggolan m, syamsudin e. formulation and evaluation of binahong leaves extract gel on wound healing of the palatal mucosa. j biomimetics, biomater biomed eng. 2020; 48: 85–91. 14. yüce e, kömerik n. potential effects of advanced platelet rich fibrin as a wound-healing accelerator in the management of alveolar osteitis: a randomized clinical trial. niger j clin pract. 2019; 22(9): 1189–95. 15. srinivas b, das p, rana mm, qureshi aq, vaidya kc, ahmed raziuddin sj. wound healing and bone regeneration in postextraction sockets with and without platelet-rich fibrin. ann maxillofac surg. 2018; 8(1): 28–34. 16. faheem, samra. patients compliance and follow-up rate after tooth extraction. iosr j dent med sci. 2017; 16(5): 115–20. 17. motamedi mhk, navi f, koushki es, rouhipour r, jafari sm. hemostatic tampon to reduce bleeding following tooth extraction. iran red crescent med j. 2012; 14(6): 386–8. 18. mozzati m, gallesio g, di romana s, bergamasco l, pol r. efficacy of plasma-rich growth factor in the healing of postextraction sockets in patients affected by insulin-dependent diabetes mellitus. j oral maxillofac surg. 2014; 72(3): 456–62. 19. hanafiah oa, abidin t, ilyas s, nainggolan m, syamsudin e. wound healing activity of binahong (anredera cordifolia (ten.) steenis) leaves extract towards nih-3t3 fibroblast cells. j int dent med res. 2019; 12(3): 854–8. 20. supriatno. antitumor activity of papua’s myrmecodia pendans in human oral tongue squamous cell carcinoma cell line through induction of cyclin-dependent kinase inhibitor p27kip1 and suppression of cyclin e. j cancer res ther. 2014; 2(3): 48–53. 21. hanafiah oa. pengaruh pemberian gel ekstrak daun binahong (anredera cordifolia (ten.) steenis) dan gel aloclair® terhadap ekspresi tgfβ1 dan pdgf-bb sebagai penanda proliferasi sel fibroblas pada penyembuhan luka di mukosa palatal tikus wistar. dissertation. medan: universitas sumatera utara; 2019. p. 100–3. 22. shah p, keppler l, rutkowski j. a review of platelet derived growth factor playing pivotal role in bone regeneration. j oral implantol. 2014; 40(3): 330–40. 23. gilbert rwd, vickaryous mk, viloria-petit am. signalling by transforming growth factor beta isoforms in wound healing and tissue regeneration. j dev biol. 2016; 4(2): 21. 24. gurcharan singh gk a/p, utami nv, usman ha. effect of topical application of binahong [anredera cordifolia (ten.) steenis] leaf paste in wound healing process in mice. althea med j. 2014; 1(1): 6–11. 25. ardiana t, kusuma apk, firdausy md. efektivitas pemberian gel binahong (adredera cordifolia) 5% terhadap jumlah sel fibroblast pada soket pasca pencabutan gigi marmut (cavia cobaya). odonto dent j. 2015; 2: 64–70. 26. kumar v, abbas ak, aster jc. robbins and cotran pathologic basis of disease. 9th ed. philadelphia: elsevier saunders; 2015. p. 73–91. 27. ågren m. wound healing biomaterials volume 1: therapies and regeneration. duxford: elsevier; 2016. p. 7–8. 28. agyare c, dwobeng as, agyepong n, boakye yd, mensah kb, ayande pg, adarkwa-yiadom m. antimicrobial, antioxidant, and wound healing properties of kigelia africana (lam.) beneth. and strophanthus hispidus dc. adv pharmacol sci. 2013; 2013: 692613. 29. leliqia npe, sukandar ey, fidrianny i. antibacterial activities of anredera cordifolia (ten.) v. steenis leaf extracts and fractions. asian j pharm clin res. 2017; 10(12): 175–8. 30. kim s, lee h, lee s, yoon y, choi k-h. antimicrobial action of oleanolic acid on listeria monocytogenes, enterococcus faecium, and enterococcus faecalis. plos one. 2015; 10(3): e0118800. 31. wang c-m, jhan y-l, tsai s-j, chou c-h. the pleiotropic antibacterial mechanisms of ursolic acid against methicillin-resistant staphylococcus aureus (mrsa). molecules. 2016; 21(7): 884. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i2.p57–62 http://dx.doi.org/10.20473/j.djmkg.v54.i2.p57-62 https://e-journal.unair.ac.id/mkg/index vol 50 no 4 desember 2017.indd 35% hydrogen peroxide, 178 35% sodium ascorbate, 178 60% bay leaf aqueous decoction, 1 acute myeloid leukemia, 154 adenoid cystic carcinoma, 205 adhesion strength, 171 adhesive strength, 226 aggressive periodontitis, 10 alpha-amylase-level, 216 alveolar bone, 149, 166 anadara granosa, 138 shell, 194 artificial intelligence, 116 attitude, 66 audio video, 66 autism spectrum disorder, 160 automation, 116 basic fibroblast growth factor, 194 biofilm, 55 bite force, 76 blast cells, 154 bleached dentin, 178 bleaching, 199 blood vessel, 194 bonding, 199 bone graft, 138 healing, 86, 194 morphogenic protein-2, 36 remodeling, 36 brackets, 199 buccal corridors, 127 candida albicans, 43 canines, 116 capillary, 91 cariogram 23 cephalometric, 211 chicken feet, 86 children, 160 chitosan, 86, 106 cytotoxicity, 171 chronic periodontitis, 10 chronological age, 220 citric acid, 102 citrus limon, 43 class i malocclusion, 144 clinical parameters, 205 cluster mallow seed, 171 collagen, 71, 86 composite, 102 resin, 183 compressive strength, 183 cvm, 220 subject index volume 50 cytomorphometric changes, 43 deep dentin 14 dental age, 220 care, 160 technician, 19 dentoalveolar alterations, 211 dentoskeletal malocclusion, 211 denture adhesive, 171 installation, 49 deoxypyridinoline, 131 dfdbbx, 149, 166 diabetes mellitus, 71 down syndrome, 220 elderly, 111 electrolyzed reduced water, 10 extraction, 144 facial vertical dimension, 76 fgf-2, 121 fiber post, 226 fibroblast cells, 121 fixed ortodontic appliance, 1 gingival crevicular fliud,131 enlargement, 154 gingivitis, 216 glucosyltransferase enzyme, 97 grading system abo, 144 hema-based, 14 hema-free based, 14 herbal medicine, 171 histopathological grading, 205 hoelen, 171 hypertension, 111 ideal smile, 127 il-1β expression, 166 immunohistochemistry, 205 inflammation, 106 inhibitory power, 97 initial viscosity, 171 insoluble glucan, 28 intensity of smoking, 61 interleukin-1β, 36 ki-67, 205 knowledge, 66 lactobacillus acidophilus, 28 liquid smoke coconut shell, 71 lymphocyte, 106 macrophage, 106 malondialdehyde, 32 level, 10 mangifera casturi (kosterm.), 36 mangosteen peel extract, 166, 199 pericarp extract, 97 mauli banana stem, 121 menopause, 131 mineral, 131 moringa leaf extract, 149 musculoskeletal complaint, 32 nanofiller, 183 nanohybrid, 102 nanopowder, 188 nanosisal, 183 naocl, 6 necrosis, 43 ni ions release, 80 nicotine levels, 61 niti archwires, 80 non extraction, 144 non-professional dentists, 49 ophiogon, 171 oral condition, 111 orthodontic tooth movement, 91, 188 osteoblasts, 91, 138, 166 osteoclasts, 138, 166 p53 wild 19 pembarong, 76 perception, 127 periodontal disease, 131 systemic disease related, 154 periodontitis, 216 personal protective equipment, 19 ph, 102 phosphomycin, 55 plaque accumulation, 1 pmn, 106 porphyromonas gingivalis, 55 propolis extract, 6 ethanol, 28 public interest, 49 rankl, 149 reactive oxygen species, 111 resorption, 188 retrognathic mandible, 211 robusta coffee, 91 root canal walls, 6 saliva, 216 salivary flow rate, 61 ph, 61, 80 sarang semut extract, 55 sardinella longiceps, 138 scaffold, 86 self-adhesive resin, 226 sexual dimorphism, 116 shear bond strength, 178 skeletal age, 220 socioeconomic, 23 socket preservation, 149 spirulina, 106 ss brackets, 80 stichopus hermanii, 188, 194 streptococcus mutans, 97 stress, 216 superficial dentine, 14 surface roughness, 102 tensile bond strength, 199 the risk prediction of early childhood caries, 23 tooth extraction, 36 trap-6, 188 traumatic ulcer, 71 twin block, 211 vegf, 91 wistar rat, 10 working position, 32 wound healing, 121 young slow learners, 66 zinc phosphate, 226 alhasyimi, ananto ali, 192 alibasyah, zulfan m., 55 alifen, gabriela kevina, 103 aspriyanto, didit, 121 avriliyanti, fitria, 1 azhar, imam safari, 166 bonifacius, setyawan, 226 carolina, dyah nindita, 216 christina, 77 dharmayanti, agustin wulan suci, 131 fidya, 116 hendrijantini, nike, 107 hermanto, eddy, 138 herniyati 92 kintawati, silvi, 198 kuswandani, sandra olivia, 154 narmada, ida bagus, 81 nugraheni, tunjung, 171 nugroho, dwi aji, 176 nur’aeny, nanan, 111 nurfitrah, astriana, 127 prabajati, rina, 43 prameswari, noengki, 181 pribadi, nirawati, 98 rahmawati, anggiani dewi, 220 ridwan, rini devijanti, 10 rohmaniar, puspa dila, 19 rukiah, bunga ayub, 144 saputri, dewi, 61 sari, rima parwati, 187 soekobagiono, 149 sopianah, yayah, 49, 66 sukmana, bayu indra, 36 surboyo, meircurius dwi condro, 71 wibowo, hari, 32 wibowo, wahyu aji, 23 widjiastuti, ira, 28 widyagarini, amrita, 160 winias, saka, 87 wurdani, eresha melati kusuma, 14 yoana, 204 yuanita, tamara, 6 authors index volume 50 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the 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decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in 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300.000,00 rp 600.000,00 other countries (negara lain) us $ 30 us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick ( )] saya membayar majalah ini dengan: [beri tanda ( ] bank draft/cheque money-order/wesel transfer to: others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1613621-9 name of bank : bank mandiri name of beneficiary : drg. udijanto tedjosasongko, ph.d., sp.kga vol 49 no 3 juli-sept 2016.indd 115115 research report dental journal (majalah kedokteran gigi) 2016 september; 49(3): 115–119 correlation between predictions to get a new dental caries with residence area and parental socio-economic conditions in adolescents in sleman diy bambang priyono,1 hari kusnanto,2 al. supartinah,3 and dibyo pramono1 1department of preventive dentistry and dental public health, faculty of dentistry, universitas gadjah mada 2department of public health sciences, faculty of medicine, universitas gadjah mada 3department of pediatric dentistry, faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: adolescence is a period when an individual experiences physical and psychological growth, thus requiring higher energy intake. as a result, they have a high appetite, but at the same time the supervision of parents on their oral hygiene behavior is decreases. they become free to choose their preferred food, sometimes containing high carbohydrates that may increase risks of dental caries and overweight. sleman is one of districs in yogyakarta, also considered as an agglomeration area of yogyakarta town, which still has urban and rural areas. purpose: this study aimed to examine the correlation between residence area and parental socioeconomic conditions with prediction to get a new dental caries. method: this study was an analytic survey study conducted on 275 adolescents in sleman. samples were selected by using stratified cluster random sampling technique. prediction to get a new dental caries was measured by using cariogram, involving 10 variables. residence area was observed based on territorial characteristics, such as urban and rural areas matched to their id card. meanwhile, parental socio-economic condition was measured on daily expenses of their parents. a multiple regression analysis with dummy variables was used to analyses the correlation between the independent and dependent variables at a confidence level of 95%. result: the results showed that the prevalence of caries in those adolescents in urban areas was 70.7%, while 81.95% in rural areas. the dmft index in urban areas was 2.27, while 2.65 in rural areas. the mean percentage of prediction to get a new dental caries in urban areas was 47.83 ± 23.63, while 53.61 ± 24.68 in rural areas. the results of the statistical analysis then showed that there was no significant correlation of residence area and parental sosio-econimic conditions with prediction to get a new dental caries. conclusion: in conclusion, residence areas, rural and urban areas, and parental socio-economic conditions, from low to high economic status were not correlated with prediction to get a new dental caries in adolescents. keywords: prediction to get a new dental caries; residence area; parental socio-economic conditions correspondence: bambang priyono, department of preventive dentistry and dental public health, faculty of dentistry, universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: iyenkg@yahoo.com introduction adolescents are very specific individuals because of their physical and psychological growth states. their body, including their face and jaw, changes, and their dental occlusion reaches perfection, except their last molars. as a result, in this phase their lower face profile will reach the final development. they also have biological changes, known as puberty, a human reproduction development marked with changes in genital organ.1 along with the physical growth, there are also enormous cognitive, psyhological, and and emotional changes. consequently, those changes often trigger overreactions in them, such as complaining, arguing, and being prejudiced dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p115-119 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p115-119 116 priyono, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 115–119 on new information received. in late adolescence, those probleme are slowly coming to a close, thus, they are more sociable and enthusiastic in communication.2 the growth of their body requires higher intake of carbohydrates and protein, thus increasing their appetite. as a result, the frequency of their meals and snacks will indirectly increase. unfortunately, at the same time the direct supervision of their parents on their oral hygiene pattern is getting low. they become free to choose their preferred food, sometimes containing high carbohydrates that may increase risks of dental caries and overweight.3 the prevalence of caries in adolescents aged 12 years in indonesia 29.8%, and the average of dmft at age 12 and 15 years when new permanent teeth begin to function was 1.4 and 1.5 respectively whereas thus permanent teeth begin to function. 4 dental caries commonly occurs in adolescents and other age groups due to the activity of microorganisms fermenting food debris, especially carbohydrates in dental plaque, producing lactic acid, which makes a drop in ph of plaque on tooth surface leading to demineralization of tooth enamel.5 susceptibility to dental and oral diseases is generally due to inadequate dental health maintenance and lots of sugary food consumption. individuals with poor dental and oral health are likely to have problems in their activities, including being absent from school, suffering from pain, having difficulties in sleeping and eating, and experiencing weight loss ultimately affecting their quality life.5,6 dental caries is a multifactorial disease in hard tissue of teeth caused by several factors, such as low salivary flow, the number and composition of cariogenic bacteria, inadequate utilization of fluoride, gingival recession, immunological factors, and genetic factors. the disease is also influenced by lifestyle behaviors, including oral hygiene maintainance as well as eating habits that can increase the risk of caries, such as high consumption of refined carbohydrates and sweet snacks.7,8 there are also other risk factors indirectly contributing to the occurrence of caries, namely poverty or social status, education level, health insurance participation, orthodontic appliance use, and poor or unfit conditions of partial denture.7-9 those risk factors of dental caries will also affect oral health in adolescents since the frequency of their meal intake and their habit of eating snacks increase. if this condition is not accompanied with good knowledge about health, it can trigger obesity and dental caries. in other words, behavioral factors play an important role in the occurrence of dental caries in adolescents. 8 urban adolescents generally have easier access to reach places of entertainment providing meals/ snacks than rural ones. support from their family-owned good economic condition also trigger the urban adolescents to reach the required entertainment more easily. socio-economic conditions can usually be observed on their phases of life, namely the transition from childhood to adulthood and later at the time they start to work.1 some study have shown that oral health of adolescents and people in rural areas is lower than those in urban areas.10 some researchers even have developed a computer software used to quantify risk of caries, for example cambra and cariogram.11,12 cariogram is used to measure the contribution of the risk factors playing a direct role in the occurrence of dental caries in the future. the result is in the form of percentage of prediction to avoid dental caries. therefore, to gain a percentage of prediction to get a new dental caries is subtarction of 100% with the result of cariogram output. cariogram can also be used to analyze the indirect influence of socio-economic conditions in dental caries. sleman is one of regencies in yogyakarta located in the northern part of yogyakarta with a wide range of geographical areas. sleman is located on the slopes of mount merapi, an area of water resources and eco-tourism oriented to activities of mount merapi and its ecosystem. the eastern area of sleman includes prambanan, kalasan, and berbah districts, where ancient heritages are located as the center of cultural tourism. the central area of sleman is an agglomeration area of yogyakarta city, a center of education, trade and services. meanwhile, the western area of sleman is an agricultural area of wetlands providing enough water and sources of raw materials used for craft industry activities, such as rushes, bamboo, and pottery.13 in other words, sleman is a developing place, but still have urban and rural areas in accordance with the research design. the attitudes of adolescents still unstable, on the other hand, they have a high appetite due to their growth. this study aimed to analyze the correlation of residence area, urban and rural areas, and low-high parental socioeconomic conditions with prediction to get a new dental caries in adolescents in sleman. materials and method this research was an analytic survey study with cross sectional design. the subjects in this research were junior high school students aged 13-15 years, who live in both of urban and rural areas in sleman in yogyakarta. the samples in this research then were selected by using stratified cluster random sampling technique14 (census block) on some districts in those urban and rural areas. of 216 clusters (census block) in sleman, there were 874 teenagers (736 people in the urban areas and 138 people in the rural areas).15 the proportion of those adolescents who live in the urban and rural areas was 5.33: 1. the total of samples in this research was determined through a calculation using the results of a previous research conducted by amalia et al.16 on children aged over 12 years in yogyakarta in 2011. based on the results of this previous research, the index of dental caries in children aged over 12 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p115-119 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p115-119 117117priyono, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 115–119 years is 3.4 ± 3.0. the sample size used for single adolescent population in sleman was determined as follows: n = minimum sample size z1-α/22 = standard normal distribution value (table z) at α = 0.05 s2 = population variant values approximated by the sample variance (= 3) d = expected difference from the real mean (0.5) n= 3.7416 x 32 = 134.697 0.52 n = 135 people due to design effects derived from the cluster sampling type, the number of samples became 2 x n.17 as a result, the minimum number of samples in this research was 270. however, the number of samples used in this research was 275. measurement of the prediction to get a new dental caries as a dependent variable was performed by using cariogram program, involving 10 variables measured from each sample, namely caries experience (the number of caries in the mouth of a subject as measured by dmft index), diseases inhibiting tooth cleaning e.g paralysis observed in the examination of samples, frequency and composition of the diet measured by food diary for 3 days, the amount of plaque as measured by loe and silness’s plaque index, the number of plaque bacterial colonies measured by cariostat, saliva volume and buffer saliva capacity by measuring the ph of saliva 1-2 hours after eating, exposure to fluoride orally or through toothpastes, as well as clinic assessment when checking the subject’s condition. on the other hand, independent variables in this research were parental socioeconomic conditions of the samples measured by the costs of daily expenses, as well as residence areas obtained based on residential address inhabited for at least 2 years and then adapted to the characteristics of the subdistrict areas. chi-square test, was carried out to differentiate between the prediction to get a new dental caries and the parental socio-economic conditions in those adolescents in both urban and rural areas. a multiple regression analysis with dummy variables at a confidence level of 95% then was performed to analyze the correlation between the parental socio-economic conditions and the residence areas. results in this research, the number of samples used was 275 people selected by using random stratified cluster technique on 6 districts out of 17 districts (table 1). based on table 1, the number of samples in the urban area was 180 people, while 95 people in the rural areas. dental caries prevalence in the urban areas was lower than that in the rural areas. similarly, the dmft in the urban areas was lower than that in the rural areas. the results of the correlation measurement between the prediction to get a new dental caries and the residence areas are shown in table 2. criteria used in the prediction to get a new dental caries was modified from the criteria set by bratthall, then adopted in a research performed by kemparaj et al.18 table 2 shows that the percentage in the low category of the prediction to get a new dental caries in the urban areas (24.4%) was higher than that in the rural areas (18.9%). in the high and very high categories, the percentages of the prediction to get a new dental caries were higher in the rural areas than those in the urban areas. these results table 1. results of research variable measurement the number of samples urban areas rural areas males females 69 individuals 111 individuals 34 individuals 61 individuals prevalence of dental caries dmft/individual 70.7% 2.27 81.05% 2.65 table 2. distribution of samples based on the prediction to get a new dental caries and the residence areas prediction to get a new dental caries total low 7 – 28.5 medium 29 – 50.5 high 51 – 72.5 very high 73 94 urban total 44 60 38 38 180 % 24.4 33.3 21.1 21.1 100 rural total 18 26 24 27 95 % 18.9 27.4 25.3 28.4 100 total 62 86 62 65 275 % 22.5 31.3 22.5 23.6 100 x2= 3.422 p = 0.331 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p115-119 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p115-119 118 priyono, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 115–119 suggest that adolescents in the urban areas had lower value of the prediction to get a new dental caries than those in the rural teens. however, the results of the chi square analysis showed that there was no significant difference (p = 0.331) between those urban and rural adolescents. the measurement of the prediction to get a new dental caries correlated with the parental socio-economic conditions are shown in table 3. table 3 shows that the majority of the samples had a relatively low parental socio-economic conditions. in the group of samples with the low parental socio-economic conditions, the highest percentage of the prediction to get a new dental caries was in the category of medium (33.7%). the next highest number of samples was in the group of samples with the medium parental socio-economic conditions. in this group, the percentages of the prediction to get a new dental caries in the categories of low, medium, and high were the same. nevertheless, the results of the chi square analysis showed that there was no significant difference. the results of the multiple regression analysis also showed that there was no significant correlation of the parental socio-economic conditions and the residence areas as independent variables with the prediction to get a new dental caries (f = 1.898 at p: 0.152). discussion prediction to get a new dental caries is a percentage reflecting that a person will suffer from dental caries in the future if their oral hygiene condition is still poor. the predictive value is obtained by using a computer program, called cariogram.11 this program is used to analyze risk factors that play a direct role in the occurrence of dental caries, such as dental caries experience, saliva volume, saliva buffer ability, and so forth. the results of the analysis using this program illustrate the interaction of risk factors causing dental caries in individuals examined.12 in this research, the percentage values of the prediction to get a new dental caries in adolescents in sleman were between 7-93%. the value of 7% means that there was only a small chance (7%) for the adolescents to suffer from dental caries in the future. it was probably due to small caries risk factors, such as good oral hygiene patterns, suffering no disease that weakens their teeth, and having no snack between meal times. moreover, the results of this research also showed that the prediction to get a new dental caries in the urban areas was almost comparable in every category with the one in the rural areas. in the low category, the percentage of the prediction to get a new dental caries in the urban areas (24.4%) was higher than the one in the rural areas (18.9%). meanwhile, in the high and very high categories, the percentages of the prediction to get a new dental caries in the rural areas were higher than the ones in the urban areas. these results suggest that the adolescents in the urban areas had lower prediction to get a new dental caries than those in the rural areas. in other words, it demonstrates that the risk factors, such as dental caries experience, eating habit, snack consumption, and oral health maintainance, in those adolescents in the urban areas were better than those in the adolescents in the rural areas in sleman. furthermore, the percentages obtained above 20% in the high and very high categories indicate high risk factors that could endanger their dental health.11 the amount of plaque on the tooth surface is a problem that needs more attention in caries prevention. biofilm behavior on the tooth surfaces that is difficult to be reached by purifier, will develop into plaques damaging the teeth with the presence of bacteria and food waste, derived from carbohydrates, especially if the subject does not use fluorine, playing a major role in preventing dental caries.8,9 table 3. distribution of samples based on the prediction to get a new dental caries and the parental socio-economic conditions prediction to get a new dental caries total low 7 – 28.5 medium 29 – 50.5 high 51 – 72.5 very high 73 94 parental socioeconomic conditions low 300 hundreds -2.8 38 20.7 62 33.7 38 20.7 46 25 184 100. medium 2.9 – 5.4 19 20 19 12 70 27.1 28.6 27.1 17.1 100 high 5.5 – 7.2 3 2 3 4 12 25 16.7 25 33.3 100 very high 7.3 – 9.5 2 2 2 3 9 22.2 22.2 22.2 33.3 100 total 62 86 62 65 275 22.5 31.3 22.5 23.6 100 x2= 5.755 p = 0.764 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p115-119 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p115-119 119119priyono, et al./dent. j. (majalah kedokteran gigi) 2016 september; 49(3): 115–119 the results of this research indicate that adolescents in both urban and rural areas had the same access to food centers since yogyakarta is considered as both of a tourist town and a student city. yogyakarta has many food centers easily reached, also providing internet access, therefore making them very attractive for young people.13 in addition, the results of this research could not show how the parental socio-economic conditions influenced the prediction to get a new dental caries since all the categories of the parental socio-economic conditions, either low, medium, or high, were evenly distributed on all the categories of the prediction to get a new dental caries. the results of the multiple regression analysis even also showed that there was no significant correlation of the residence areas and the parental socio-economic conditions with the prediction to get a new dental caries in those adolescents in sleman. in contrast, a research conducted in ibadan nigeria shows that adolescents with higher parental socio-economic conditions have poor oral hygiene since they often consume biscuits. 18 based on the results of this research, the prediction to get a new dental caries could be caused by several risk factors. it means that the parental socio-economic conditions of the families do not directly affect the prediction to get a new dental caries. but, it more directly plays a role in nutrition intake strengthening their teeth, the use of toothpastes containing fluoride, and their health care behavior in visiting to dentist. this finding is in line with a research conducted in pennsylvania demonstrating that the parental socio-economic conditions do not directly affect oral health, but through oral health care behavior and preventive intervention.20 a previous study on rural areas shows that rural communities with limited funds they still have to spend money for health services and for transportation to reach health care facilities, so the health conditions of those communities are bad. nevertheless, the effect of access to health care in this research was not significant since those adolescents in both urban and rural areas still had the same prediction to get a new dental caries.19 many factors must be involved in measuring health behaviors that affect prediction to get a new dental caries, in addition to socio-economic conditions, such as knowledge, lifestyle, access to health care, and health insurance.5 it may be concluded that there was no correlation of residence area, namely urban and rural areas, as well as low and high parental socio-economic conditions with prediction to get a new dental caries in adolescents. acknowledgement we would like to express our gratitude to people in six sub-districts of sleman, the head of the team of researchers, staffs and field officers in demography health surveillance system (hdss) of faculty of medicine, as well as the research team of lecturers and students of dental health education and dental hygienis programs of faculty of dentistry, universitas gadjah mada for collecting and managing research data. references 1. who. maternal, newborn, child and adolescent health: adolescent development. 2016. available from: http://www.who.int/maternal_ child_adolescent/topics/adolescence/dev/en/. 2. stang j, story m, (eds). guidelines for adolescent nutrition services. 2012. available from: http://www.epi.umn.edu/let/pubs/adol_book. shtm. 3. american academy of pediatric dentistry. guideline on adolescent oral health care. clinical practice guidelines 2015; 37(6): 151-8. 4. kemenkes. riset kesehatan dasar 2013. jakarta: badan penelitian dan pengembangan kesehatan depkes, 2013. 5. selwitz rh, ismail ai, pitts nb. dental caries. lancet 2007; 369(9555): 51-9. 6. baginska j, rodakowska e, borawska m, jamiolkowski j. index of clinical cosequences of untreated dental caries (pufa) in primary dentition of children from north-east poland, advances in medical sciences 2012; 58(2): 442-7. 7. ajayi dm, abiodun-solanke if. sociobehavioural risk faktors of dental caries among selected adolescents in ibadan, nigeria. pediatric dental j 2014; 24(1): 1-6. 8. featherstone jdb. caries prevention and reversal based on the caries balance. pediatr dent 2006; 28(2): 128-32. 9. young da, featherstone j db. ca r ies ma nagement by r isk assessment. community dent oral epidemiol 2013; 41: 1-12. 10. varenne b, petersen pe, fournet f, msellati p, gary j, ouattara s, harang m, salem g. illness-related behaviour and utilization of oral health services among adult city-dwellers in burkina faso: evidence from a household survey. bmc health services research 2006; 6: 164. 11. bratthall d, petersson gh, stjernswärd jr. cariogram. internet version 2004; 1-51. 12. jenson l, bundenz aw, featherstone jdb, ramos-gomez fj, spolsky vw, young da. clinical protocols for caries management by risk ssessment. j calif dent assoc 2007; 35(10): 714-23. 13. pemerintah kabupaten sleman. 2015 http: www. slemankab, go,id 14. pratiknya. dasar-dasar metodologi penelitian kedokteram dan kesehatan. jakarta: pt. raja grafindo perkasa; 2008. 15. fakultas kedokteran universitas gadjah mada . health and demography surveillance system (hdss) 2015 htp: // f k.ugm.ac.id //02/ hdss-penelitian-berbasis-populasi-sa ranapengabdian-kepada-masyarakat/ 16. amalia r, schaub rmh, widyanti n, stewart r, groothoff jw. the role of school-based dental programme on dental caries experience in yogyakarta province, indonesia. int j paediatr dent 2012; 22(3): 203-10. 17. lemeshow s, hosmer jr, dw, klar j, lwanga sk. besar sampel dalam penelitian kesehatan. yogyakarta: gadjah mada university press; 2008. p. 5. 18. kemparaj u, chavan s, shetty nl. caries risk assessment among school children in davangere city using cariogram. int j of prev med 2014; 5(5): 664-71. 19. polk dj, weyant r j, manz mc. socio economic factors in adolescents’ oral health: are they mediated by oral hygiene behaviors or preventive interventions?. community dent oral epidemiol 2010; 38: 1-9 20. hoeven m. van der, kruger a, greeff m. differences in health care seeking behaviour between rural and urban communities in south africa. int j equity health 2012; 11: 31. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i3.p115-119 http://dx.doi.org/10.20473/j.djmkg.v49.i3.p115-119 81 vol. 43. no. 2 june 2010 research report dental modifications: a perspective of indonesian chronology and the current applications rusyad adi suriyanto� and toetik koesbardiati�� 1 laboratory of bioanthropology and paleoanthropology, gadjah mada university faculty of medicine 2 faculty of social and political sciences department of anthropology, and department of anatomy and histology section/ laboratory of physical anthropology, faculty of medicine airlangga university surabaya indonesia abstract background: dental modifications are one of the forms of initiation rite. thus tradition can be found in all of indonesian regions, even in south east asia, in previous era. modes and dentistry, as a culture product, including its modifications or decorations toward body and teeth have appeared in present day, such as tattoo and decorations on teeth which are taken particular model and superimpose or inculcate ornament of jewel, diamond, gold and others. the first research aim is to describe how functions of modifications on teeth represent cultural affinity and population of biologic affinity that accompany it from time to time; starting from prehistoric period to present day, especially in indonesian region. the second aim is to submit applicable proposal that is useful for medical area, particularly in dentistry. method: the research materials include teeth of the adult human skulls of java, balinese, and east nusa tenggara from some paleoanthropological–archaeological sites, as well as isolated permanent dental sample from modern balinese population. the methods used are visual comparative descriptive method, and browsed through ethnographic and archaeological classic literatures. result: chronologically, dental modifications as reference to the pattern of migration and the domination of the culture in the past, and these facts indicate to the biological affinity and indicate to how the culture influences other culture. conclusion: some effects of the practices of dental modifications are the emergence of some diseases. therefore, it is necessary for the agent of health and the traditional practitioner to be aware when they practice the dental modifications. nevertheless, on the other side, based on explanations the research results, it is clear that dental modifications provide broader knowledge, because it has a very long journey of migration history, occupancy, and culture in this indonesian archipelago, which stretches from the period of about ten thousand years ago until now. this knowledge can be used for either practical purposes of medicine and dentistry even forensic. thus, it is also useful in forensic identification, as guidance with cultural background such as certain patterns of dental modifications cannot be disregarded. in the same way, patterns of modifications either intentional or unintentional can give a guidance to strengthen identification. key words: dental modifications, indonesian chronology, medicine and dentistry applications, australomelanesoid, mongoloid abstrak latar belakang: modifikasi gigi geligi adalah salah satu bentuk ritus inisiasi. tradisi ini dapat ditemukan di seluruh wilayah indonesia, bahkan di asia tenggara pada masa lalu. persoalan-persoalan kecantikan dan dentistri sebagai produk budaya termasuk modifikasi dan dekorasi tubuh dan gigi geligi telah muncul pada masa kini, contohnya tatto tubuh atau dekorasi gigi geligi dengan ornament hiasan dari emas, intan berlian dan juga bahan lainnya. tujuan awal dari penelitian ini adalah untuk mendeskripsikan bagaimana fungsi modifikasi gigi geligi mewakili afinitas kultural dan afinitas biologis dari suatu populasi yang saling berkaitan dari waktu ke waktu, dimulai dari masa prasejarah hingga masa kini, terutama di wilayah indonesia. tujuan penelitian yang kedua adalah untuk menyumbangkan pemikiran yang aplikatif yang berguna untuk bidang kesehatan terutama dentistri. metode: bahan penelitian adalah gigi geligi dari tengkorak dewasa jawa, bali dan nusa tenggara yang berasal dari situs-situs paleoantropologis-arkeologis, demikian pula sampel gigi geligi permanen individual dari populasi bali saat ini/modern. metode yang digunakan adalah deskriptif komparatif visual, dan penelusuran literatur etnografi dan arkeologi klasik. hasil: secara kronologis, modifikasi gigi geligi adalah rujukan bagi migrasi pada masa lalu dan dominasi budaya masa lalu. kenyataan ini menunjukkan adanya afinitas biologis dan menunjukkan bagaimana budaya yang satu dapat mempengaruhi budaya yang lain. simpulan: beberapa efek dari praktek modifikasi 82 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 81-90 introduction dental modifications are a form of initiation rite that was once very common, and in some places and certain ethnic groups still ongoing; which the human teeth was mutilated in some way so as to form a certain patterns according to their culture.1-5 this event symbolizes that somebody has entered his adult phase.6 it also means that this person has entered a marriage phase. the tradition can be found in all of indonesian regions, even in south east asia, especially in previous period. there are a few researches, which concern on morphology of teeth from indonesia, especially from ancient period to present period, which has various population groups. these conditions make comparison study on dental aspects very useful. this paper will discuss about dental modifications by using prehistoric and ethnographic evidences. dental modifications will also be related to current studies and its application for today's medicine and dentistry. in another word, chronological dental modifications can provide knowledge and benefaction for present day life, which can be used to anticipate dental problems, especially dental modifications in our future life. modes, as a culture product, including in it's modifications or decorations toward body and teeth have appeared in present day, such as tattoo and decorations on teeth which is taken particular model and superimpose or inculcate ornament of jewel, diamond, gold and others. tiesler7 has shown that cultivation jadeite, hematite, pyrite, turquoise and different organic substances were used as obturation material in mayan society (guatemala, south america) in classic period is an a adulthood sign of its member (that somebody has entered 15 years old age). dentistry also as a culture product, had emerged on prehisctoric periods, according to white et al.8 of his finding of a prehistoric native american mandible from a fremont site (circa ad 1025) in colorado; it has a conical pit in the worn occlusal surface of the lower right canine. natural causes for this modification are ruled out by the presence of internal striae, a finding confirmed by experimental replication. the canine was artificially drilled before the individual's death and is associated with a periapical abscess. this is one of a very few examples of prehistoric dentistry in the world, and the first from the american southwest teritory. the first research aim is to describe how functions of modifications on teeth represent cultural affinity and population biologic affinity that accompany it from time to time; starting from prehistoric period to present day, especially in indonesian region. the second aim is to submit applicable proposal that is useful for medical area, particularly in denstistry. materials and methods the research materials are permanent teeth of adult skull of java, bali and east nusa tenggara prehistoric population from some paleoanthropological–archaeological sites. some permanent isolation teeth of modern balinese population are also used as a comparison, although it is not being presented explicitly. the anatomical identification is based on standardization of physical anthropology and anatomy. sequence of its antiquity, chronology of settlement and culture, and its biologic affinity related to jacob,5,9 sukadana10-13 and boedhisampoerno.14,15 environmental and cultural context can give a broader inference.16 distinction of environment and cultural practices will result in physical distinctions that manifested on its bone and teeth.17 the first method is visual comparative descriptive research.17,18 these selected material are observed, classified and compared, i.e. unmodified teeth were compared with modified teeth. then, these teeth were compared by the modification treatment patterns. at this phase, we used modified teeth of modern population which its practice still being done in bali. here it is needed to emphasize that paleoanthropological–archeological materials which come from prehistoric human remains is limited in quality and in quantity, which needed special treatment in handling, analyzing, and interpretating.5,13,19,20 the second method is browsed chronologically in previous sources such as archaeological reports and the first ethnographic reports which reported about practices of dental modifications, particularly in nusantara/ indonesian ethnic groups. gigi geligi adalah timbulnya beberapa penyakit. oleh karena itu, hal ini sangat penting diketahui oleh praktisi kesehatan modern dan praktisi kesehatan tradisional untuk mempertimbangkan kenyataan ini dalam melakukan praktek modifikasi gigi geligi. di sisi lain, berdasarkan hasil penelitian ini, modifikasi gigi geligi memberikan pengetahuan dan wawasan yang sangat luas, karena modifikasi gigi geligi telah lama dilakukan melalui perjalanan yang panjang dalam sejarah migrasi, penghunian dan budaya di kepulauan indonesia, dengan rentang waktu sekitar 10.000 tahun yang lalu hingga saat ini. pengetahuan tentang hal ini dapat digunakan untuk kepentingan studi kesehatan dan dentistry, bahkan forensik. khususnya untuk identifikasi forensic, modifikasi gigi geligi adalah petunjuk latar belakang budaya seseorang, dimana pola tertentu modifikasi gigi geligi tidak dapat diabaikan. dengan demikian, pola modifikasi gigidengan demikian, pola modifikasi gigi geligi baik sengaja (intentional) maupun yang tidak disengaja (unintentional) merupakan penguat dari identifikasi individual. kata kunci: modifikasi gigi geligi, kronologi indonesia, aplikasi untuk kedokteran dan dentistri, australomelanesoid dan mongoloid correspondence: rusyad adi suriyanto, c/o: laboratorium bioantropologi dan paleoantropologi, fakultas kedokteran universitas gadjah mada, yogyakarta. e-mail: rusyat_suriyanto@yahoo.co.id. 83suriyanto: dental modifications: a perspective of indonesian chronology table �. paterns of dental modifications among prehistoric and early historic indonesian series series antiquity patterns of dental modifications racial affinity* sexextracting and its position filing and its position** sharpening and position blackening liang bua (lb) mesolithic i2 – c (r/ l) occlusal i – c √*** australomelanesoid/ mongoloid male liang x (lx) mesolithic labial i – c (r/ l) occlusal i – c labial and lingual and or median and distal i – c (r/ l) ? australomelnesoid/ mongoloid male gua alo (ga) mesolithic labial i – c (r/ l) labial and lingual and or median and distal i – c (r/ l) ? australomelanesoid/ mongoloid male liang toge (lt) mesolithic labial i – c (r/ l) occlusal i – c (r/ l) ? √ australomelanesoid/ /mongoloid male lewoleba (ll) early neolithic i2 – c (r/ l) occlusal i – c √ australomelanesoid/ mongoloid male melolo neolithic i2 – c (r/ l) occlusal i – c √ australomelanesoid/ mongoloid male puger (pgr) neolithic labial i – c (?) labial and lingual i – c australomelanesoid male ntodo leseh (nl) paleometalic mongoloid/ australomelanesoid gunung piring (gp) paleometallic occlusal i – c (r/ l) mongoloid/ australomelanesoid male semawang (smw) paleometallic labial i – c (r/ l) occlusal i – c (r/ l) labial and lingual i – c √ mongoloid/ australomelanesoid male female gilimanuk (glm) paleometallic labial i – c (r/ l) occlusal i – c (r/ l) labial and lingual i – c mongoloid kelor (kl) classic labial i – c occlusal i – c √ australomelanesoid male caruban (crb) classic – islam i – c (r/ l) √ mongoloid/ australomelanesoid male notes: * = the first mentioned race is the major one. ** = r is right, and l is left. *** = is found. results table 1 shows the summary of the patterns of dental modifications associated with antiquity, racial affinity, and sex. there are four patterns of dental modifications that are identified among prehistoric indonesian populations, i.e. dental extracting, sharpening, filing, and blackening (figure 2 and 3). the spread of prehistoric until recent dental modifications can be seen on figure 1. the first can be called as evulsion, while the next two could be called as ablation. discussion normally, dental modifications were done more among male than female and indicated the rite of initiation and passages, and social status. dental extraction is practiced among the mesolithic until neolithic populations such as liang bua, lewoleba and melolo in flores island. they were australomelanesoid but there were some degree of mongoloid. jacob5 also reported patterns of dental sharpening that some dwellers of this area had modificated lateral incisor and canine in sharp-pointedly form (the peg 84 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 81-90 shaped); even this practice opened its dentin, while the others were only on its enamel. dental filing on labial site was practiced among the populations from liang toge, liang x, gua alo, semawang, puger, and kelor. this kind of dental modification has a long of period i.e. from mesolithic until classic period. dental filing on the labial probably reemerged in the later period. this evidence is reinforced by the racial affinity that was dominated by australomelanesoid. further, based on the observation, it is found that the most common modification was occlusal filing. this occlusal filing is even found and is still practiced to day (e.g. among the balinese). based on the commonness of occlusal filing and period of use that is relatively found in the sample from younger period, this indicated that occlusal filing was a new influence (culture) at that time. related to the history of occupancy of indonesia, this kind a b c figure ��. a) extracting both maxillar right and left i2 and c and dental blackening (mesolithic liang bua, manggarai, flores); b) labial filing both maxillar right and left i–p and dental blackening (mesolithic liang toge, ngada, flores); and c) dental staining probably because of betel-nut chewing (mesolithic liang bua, manggarai, flores).liang bua, manggarai, flores). aa b c d figure ��. a) dental modifications (labial and occlusal dental filing and blackening) at liang toge's series; b) dental modificationdental modification (dental extract/ evulsion and blackening) at caruban's series; c) occlusal filing both maxillar right and left i – p (paleometallicocclusal filing both maxillar right and left i – p (paleometallic gilimanuk, bali); and d) dental blackening (paleometallic semawang, sanur, bali). figure �. the spread of dental modification in indonesia. note: acronym name of the location on this map should refer to table 1. 85suriyanto: dental modifications: a perspective of indonesian chronology of dental mutilation might be brought by the people of latest migration that so called mongoloid. in other words, the mongoloid people might bring the occlusal filing. dental blackening (dental coloring) is a primitive method of caries prevention in southeast asia.21 based on archaebotanical reports from areca nut (areca catechu l.) residues those were guessed as the ingredients of betel chewing aged 13000 bp (zumbroich, 2007/ 2008). dentitions of 31 individuals excavated from the bronze age site of nui nap (thanh hoa province, vietnam) were examined for the presence of areca catechu (betel nut).22 many of the teeth of the vat komnou cemetery (dated between 200 bc and ad 400 or the early historic period in the mekong delta, angkor borei, cambodia) show evidence of betel staining.23 dental coloring is also found in other areas such as polynesia and micronesia even in indonesian archipelago such as sumatra, nias, borneo, celebes, java, madura, bali, flores, timor, papua, and other remoted islands. the coloring uses chalk, pinang (areca catechu), gambir (uncaria gambir roxburgh) dan sirih (piper betle). papuan inhabitants made alternative several plant species i.e. openg (exocarpus latifollus), tawal (celastraceae sp.), sambiwal (erythroxylum ecarinatum), ntuo (cryptocaria nitida) and agya (endiandra montana).24 the mixture of sirih pinang with saliva generate brownish red on the teeth. to smooth all over the teeth, it is used tobacco (nicotiana sp., l.) stroked all over the surface of the teeth. once chewing sirih pinang, the stoke of the tobacco is done twice to five times. the intensity can leave carving on the teeth. other variant that is found in manggarai is using certain wood to black the teeth. tradition of coloring teeth is still existed until nowadays as honor sign for guest and part of tradition in manggarai.25 tayanin and bratthall26 reported that kammu women in laos and vietnam habitually paint their teeth black with cratoxylum formosum and croton cascarilloides wood that purposed to be beautiful and caries–free, and it is now known only among the elderly although this practice existed for many generations. suddhasthira et al.27 also reported their experiment to the woods that habitually practiced in thai people, this tradition was practiced around 5000 – 4000 years ago based on archeological remains. nguyen28 observed and reported the habit of applying black lacquer to teeth is widespread in vietnam and its effect on incidence of dental caries and its usefulness in caries treatment. dental modification practices had been done since last thousand years in america, several parts of east asia, oceania, and in africa.18,29,30 this pattern of teeth vary broadly, but basically it has only covered 7 patterns.18 uhle2 and has found 17 forms of dental modification which spread over among indonesia ethnic groups. lignitz29,30 depicted widely that african ethnic groups practiced tradition of dental modification, either etnographically or biologically; lignitz also figured in 25 dental modification patterns which were practiced. there is such an interesting explanation from wilken3 that areas which recognized tradition of dental modification has covered almost all indonesia archipelago, particularly the regions which are dwelled by the tribes who have mongoloid element; including philippines archipelago. von jhering1 previously elaborated the matter that this tradition had found in africa, in indonesian archipelago, and in area that has mongoloid elements and in indian ethnic groups in america. finucane et al.31 reported the earliest securely dated evidence for intentional dental modification in west africa from 11 individual human remains which were excavated from the sites of karkarichikat nord and karkarichikat sud in the lower tilemsi valley of eastern mali in late stone age (circa 4500–4200 bp). the dental modification involved the removal of the mesial and distal angles of the incisor, as well as the mesial angles of the canines. the modifications did not result from task–specific wear or trauma, but appear instead to have been produced for aesthetic purposes. all of the filed teeth belonged to probable females, suggesting the possibility of sex–specific cultural modification. haour and pearson32 reported prehistoric dental modification in the region comes from kufan karawa, niger (circa ad 1300 – 1600), that the modification takes the form of interproximal grooving of the maxillary incisors resulting from task–related wear. the examples of america area has been explained above particularly are reported by white et al.8 and tiesler.7 practices of dental modifications had been recognized by the end of prehistory period in indonesia as a habit pattern. this habit has been also practiced in several areas at early agriculture period (end period of mesolithic – early period of neolithic). some human remains from gilimanuk (specimen r.xxvii and r.xxxii),33 based on the dating of c14 have antiquity between 1486 – 2466 years ago and its charcoal have antiquity between 1805 – 1990 years ago, it have shown the filing at incisor, canine, and molar on its occlusal surface from maxilla and mandible. this dental modification pattern also can be found in minahasa.3 the teeth which were found in some caves of paleoanthropological–archaeological sites showed that these teeth had been modificated in simple manner, such as in gua alo and liang x in west manggarai (flores island), that is modification on dental labial surface without forming many patterns.5 other patterns were also reported by jacob5 that some dwellers of this area had modificated lateral incisor and canine in sharp-pointedly form (the peg shaped), even this practice opened its dentin, while the others were only on its enamel. these patterns can be found at the human remains of neolithic ban kao from thailand. this population had younger antiquity than the others cave dwellers in flores island that its antiquity reach about 4000 years ago or end period of mesolithic – early period of neolithic, which has australomelanesoid dominant elements. an interesting finding was also reported, it was concerned on dental modification by fracturing or repealing incisor and canine. wilken3 noted that this pattern represented mourning rite in polynesia, and von jhering1 also mentioned that it was found in melanesia and australia. 86 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 81-90 concerning to the peopling of indonesia, some schollars concluded that those dominant elements of indonesian population are australomelanesoid and mongoloid.5,3436 in the history of peopling of indonesia, since early holocene (about 10000 years ago) had been dwelled by australomelanesoid race and it covered almost all the continent and archipelago of southeast asia. the relics of them can be found in vietnam, cambodia, thailand, malacca cape, sumatera, north kalimantan, java, bali, sumba, flores, lembata, timor, sulawesi, palawan and luzon, both skull, bone fragment, and teeth in caves and in cockle shell hill in ashore. before early period of neolithic (about 4000 years ago) migration flow of mongoloid had shown its track sporadically, started from southeast asia through malacca cape to sumatera and java and through north philippine to sulawesi and selayar. polarization among its racial elements had become clearly during transition period of neolithic to early period of metal (about 4000–1500 years ago), in west and north sides of nusantara (indonesia) mongoloid element were stronger or as a single element. while in east and south sides of nusantara (indonesia) australomelanesoid element were stronger or as a single element.5,9,35 this condition is still taking place until now, and the process of mongoloidization tends to move to east.9,26,37–41 besides its function as initiation rites, dental modifications have shown as cultural affinity. related to its tradition, these races have its manner and its treatment pattern on dental modifications; even though in fact the first represented influence of migration motion (mongoloidization) that occurred earlier in indonesian region. both treatments of dental modifications can be seen in several morphology of dental in indonesia. racial determination (representing biologic affinity) is important in indonesian settlement history, paleoanthropology, human genetic and anthropology; because in the early period of present mongoloidization (about 15000–10000 years ago) the distribution of racial had changed gradually, this changing connected with several cultural aspects.34 richerson & boyd42 have shown that culture and human biology represent very subtle unity, and foley & donnelly43 furthermore affirmed that to study its fields are needed an integrative study from various sciences, both social science and scientific science. dental modification traditions are well-known among ethnic groups in indonesia archipelago.1,3 this tradition has been conducted as a rite during human life span, generally as a symbol of maturity and in marriage rite; these traditions has been found in several areas in indonesia. there are some evidences that dental modification also had been conducted as a mourning tradition; it was conducted when one of family member was death. these guidance can be found in kedu, bengkulu, sula archipelago, selayar island, and alfuru in minahasa.3 its inhabitants shall only conduct this tradition if their nuclear family member (their parents–one or both, sister or brother) have passed away; particularly in selayar island, a woman conducted dental modification tradition when her baby passed away (at that time or as soon as she lost her baby) and women also conducted it when her fiancée passed away. at this kind event, the tooth that was modificated is mandible's incisor. if they conduct this tradition when their nuclear family member is still alive, they believe that it will generate the death for their family. this fact has shown that there is a tight order of this tradition in that community. at the same time, dental modifications as a mourning rite can be parallel with tradition of dental's extraction in polynesia.3 extraction of two lateral incisors or canine proved that this tradition was conducted in indonesia; these evidences can be found in several ethnic groups in central sulawesi (tonapa, tobada and tokulabi). in these regions, adult women conducted this tradition, and in enggano women conducted this rite when they were marriage. wilken3 furthermore noted that dental modification tradition represents refiner action (een verzwakte) than dental fracturing. the aim of this action–which is broadened by dental modification–is to sacrifice a part of human body (hair, finger, dental and so on) as a sign of mourn or has a function to refuse the danger. this opinion differs from von jhering1, he emphasizes at aesthetic function. some findings have shown treatment patterns of dental modifications from mesolithic to modern period, there are filing, sharpening and extracting.40,41 the teeth are commonly modificated are incisor, canine, and first premolar. occasionally, only incisor to canine is modificated, but sometimes its modification also has been done at first premolar. dental filing has several variations including grinded on surface of mandibular occlusal and maxillar occlusal and labial. recorded variation of sharpening phenomenon is to sharpen the teeth, particularly at incisor, canine, and premolar. both dental filing and sharpening were done by attenuating and smoothing. recording of these events have shown that filing pattern at occlusal surface is the most common to be conducted, then followed by dental sharpening and extracting. both of these modification patterns are recorded at findings of semawang (sanur, bali island) and caruban (rembang, central java).40 this pattern of dental modification has been found in some population in polynesia, melanesia, australia, tonabo, tobada dan tokulabi (the last third–place is in central sulawesi) as a mourn rite, and in enggano this rite had been conducted by adult women as marriage rite.1-3 von jhering1 and wilken2 explained that the regions which practiced tradition of dental modifications cover almost all archipelago of indonesia, particularly the regions which has been dwelled by ethnic groups who have mongoloid characteristic or who had been mixed with mongoloid. this explanation has shown the broadness of mongoloid footstep region; it covers southeast asia, east asia, philippine archipelago, indonesia archipelago, polynesia, melanesia, australia, africa, and america. nowadays, this tradition is no longer conducted. this tradition is decreased for present generation relates to health problem and the increasing of education level.44 bali ethnic 87suriyanto: dental modifications: a perspective of indonesian chronology still continues this tradition because of their religio–cultural reason.45 balinese's dental filing practice at the maxillary incisor and canine very smoothly. the practice aim is to eliminate evil character such as passion, anger, greed, covet, rebellious and drunken. in fact, although this tradition is being subsided, but occasionally as a mode, certain group or certain generation will appear this tradition once more; and the mongoloid elements as biologic entity will continue to future generation. nowadays research about prehistoric human dental pithecanthropus erectus (homo erectus) from sangiran area (sragen, central java) has been accomplished by arif & kapid46 based on fifty-two isolated teeth hominid specimens of von koenigswald's collection. the result of study demonstrated the big size of upper third molar of the specimens from grenzbank/ sangiran (g/s) (near sragen, central java) assemblage which might be affected by the incident of carabelli's trait on their upper third molar. it is also reported that the findings of deciduous teeth (right i1, left m1, right m 2, and left m2) are assumed belongs to relatively same species and same period which have been found in this area.47 jacob5 also conducts a complete research that covers space and time. jacob examined human remains of mesolithic–late neolithic period which spread out from southeast asia to east nusa tenggara; even though it was not concerned on patterns of dental modifications. jacob48 examined anyar human remains' teeth, and concluded that this human antiquity is neolithic and its dominant element is australomelanesoid. jacob4 concluded that the glimanuk population has mongoloid element based on its teeth. boedhisampurno18 has examined the findings of human teeth from the ulu leang cave cemetery that refers early period of metal age (maros, south sulawesi) which has older antiquity than gilimanuk and has mongoloid element. sukadana10,12,14 has examined the findings of human teeth of mesolithic liang bua (flores island), neolithic lewoleba (lembata island), neolithic melolo (sumba island) and neolithic ntodo leseh (komodo island) in east nusa tenggara. he has concluded that inhabitants of this region have older antiquity with stronger australomelanesoid element and so does with the inhabitants of region that passes further wallacea line (more to the east of this region). on the contrary, more to west their regions have shown that mongoloid element start to present and have younger antiquity. some research has been done increasingly strengthen the argument of this research.5,10,34,37–41 patterns of dental modifications related to its biologic affinity that had been tried to examine based on human remain findings from a period around 4000 years ago to present day. koesbardiati and suriyanto40 have reported that dental filing represents the most common of findings which have mongoloid element, particularly from later period (neolithic – paleometallic). this fact indicates that there is much more mongoloid influence to indonesia. in other words, mongoloid migration from southeast asia mainland entered more intensively into nusantara archipelago (indonesia). moreover, koesbardiati and suriyanto41 also have reported that dental sharpening and extracting represents older or early tradition, this findings are supported by evidence from late mesolithic – neolithic (around 4000–3000 years ago). the last pattern is a tradition that is still practiced in ethnic groups in regions of eastern indonesia, polynesia, melanesia, and australia that have dominant australomelanesoid element. much more specific for flores island and the satellite islands, in a period around 5000–3000 years ago, represented two patterns of dental modification. this can be used as an indicator to determine whether flores inhabitants originated from northern or southern culture. some dental studies and its modification have also been conducted toward modern population in indonesia. lie44 surveyed javan regions (malang, sidoarjo, surabaya, jogjakarta, pacitan, tangerang, blitar, tengger and banyuwangi), madura, bali, sumba, flores, adonara and lomblen (lembata). research questions of this research that were exploitated are why dental modifications were conducted; when dental modifications were done; were the modification conducted before or after marriage; who were conducted dental modifications; what appliances were used; in what manner the dental sharpened and how long its process; how much it costs; where are the place to conduct dental modifications; which teeth will be modificated; there are a ceremony and abstention in conducting this tradition or not; and how long its pain after being modificated. there are some reasons of conducting dental modifications, for example aesthetic, initiation rite, sosiologic and sacrifice (belief, religion). some arguments are also figured in, that research of dental modification is useful for anthropological research (both cultural and physical anthropology), particularly to determine spreading, movement and influence of anthropological groups in indonesia, either in present day or previous period. moreover, there are assumptions that dental modifications have positive elements in preserving the teeth. the human remains and paleopathologic examination of ancient is useful for archaeologists, anthropologists and physicians (medicine, dentistry).49,50 the techniques can also be applied to any desiccated tissues, including recent remains, as in forensic settings. that is clear that human remains from previous period along with its aspects, its life history to refind, the methods to gain and to identify and the degree of its extrapolation contribute to knowledge and to applied science for present day life, particularly related to medical aspects. realizing that there are various ethnic and culture in indonesia and abandon of fossil finding in indonesia from the early period until present, soedomo51 suggested, it is important for the dentist to learn about dental evolution and variation, especially in indonesian region. mentawainese has a habit to mutilate their anterior tooth (sipiat sot or mapiat sot). according to them, the main reason in dental mutilating is for identity mentawai. they feel that they are not mentawai ethnic member if 88 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 81-90 they do not mutilate their anterior dental. anterior dental mutilation of mentawai can be seen from sharpening form of their maxillary and mandibulary teeth. besides as an ethnic identity, this is also meant to beauty purpose, style, and mastication so that they could bite as a wild animal. koerniati's research52 was found that relatively falls out at older age at cases of anterior dental mutilation, whereas falls out at younger age at cases of anterior unmutilation. this research also found a significant correlation between anterior mutilation and posterior attrition. in consequence of anterior mutilation, hence the burden of mastication activity has moved to posterior, thus part of posterior has a heavier burden, and it has caused the occurrence of posterior dental attrition. here posterior dental attrition has caused food mastication process disorder; therefore, this food is not masticated properly so that causes problems toward disgestion.52 cultural action that is influenced by cultural system can give guidance of disease and to find the possibility of disease causes that occurred on teeth and other aspect of health including periodontitis, antemortem tooth loss, mouth cancer, hypertension, diabetes, chronic kidney diseases, increasing the cardiovascular diseases, metabolism disorder and schizophrenia. hour and pearson32 found the indication of tooth usage as a tool; and they mentioned it as"the third hand." anterior dental is used to twist ropes that come from animal or plant. because of this activity, a gap beetween anterior tooth sometimes has unequal form because of different pressure of twist. if this activity is repeated, it will strengthen anterior dental abrasion so that it will form a gap beetwen two anterior teeth. some recheacher assumed that dental mutilations could cause alveolar bone pathology.53 in africa, the most common type of dental mutilation is inverted v-shaped. the effects of this dental mutilation in africa are pulp exposure and periapical ostitis or radicular cysts. the most common effect is general loss of alveolar bone with a marked loss of the maxillary anterior labial alveolar bone plate. coloring teeth can be caused by betel–nut chewing activities. betel nut chewing consists of betel leaves, areca nut (areca catechu), burned coral or shell and or tobacco for wiping the saliva at the end of the proses of betel nut chewing. the effect is the saliva will be red–brownish, and will be permanent on the teeth. the medical effects are dental pathologies such as periodontitis, antemortem tooth loss, mouth cancer, hypertention, diabetes, chronic kidney diseases, increasing the cardiovascular diseases, metabolism disorder and in all-cause mortality.54 betel nut chewing activities brings to skizophrenia in palau.55 another important application of dental modifications for forensic study is tracing ethnic affiliation, especially for the countries that their inhabitants practice dental modifications, for example indonesia, and african, american, oceania, southeast, and east asian countries. paleodontological research has proven that some form of cosmetic dentistry existed in ancient times.56 intentional dental mutilations, dental decorations and modifications on anterior teeth have been widespread occurrences in many cultures. the fact that there are various names for these phenomena indicates different interpretations of data gained from research into this type of intervention into human dentition. although archaeological specimens of modified teeth are usually isolated and damaged, they broaden our knowledge of ancient nations and human behavior in the past. these behaviors in some places continuing as a tradition, and its importance is that it can assist the efforts of forensic identification i.e., what their cultural and race background. as mentioned above, some effects of the practices of dental modifications are the emergence of some diseases. therefore, it is necessary for the agent of health and the traditional practitioner to be aware when they practice the dental modifications. nevertheless, on the other side, based on explanations above, it is clear that research on dental modifications provide broader knowledge because it has a very long journey of migration history, occupancy, and culture in this indonesian archipelago, which stretches from the period of about ten thousand years ago until now. this knowledge can be used for either practical purposes of medicine and dentistry even forensic. thus, it is also useful in forensic identification, as guidance with cultural background such as certain patterns of dental modifications cannot be disregarded. in the same way, patterns of modifications either intentional or unintentional can give a guidance to strengthen identification. acknowledgment 1. prof. dr. t. jacob, m.s., m.d., d.sc. (the late) who permitted us to examine frame collection in laboratory of bioanthroplogy and paleoanthropology, gadjah mada university faculty of medicine. 2. prof. drg. etty indriati, m.a., ph.d. as head of bioanthroplogy and paleoanthropology laboratory, gadjah mada university faculty of medicine. 3. dr. abdoel kamid iskandar, m.s. who permitted us to examine frame collection in section of physical anthropology, department of anatomy and histology, airlangga university faculty of medicine. 4. drg. susy kristiani, m.s. who bestowed her collection of sample on modern bali dental pangur. 5. drs. koeshardjono (the late) and sugiyo who assited in the process of this research. 6. hermann müller in biozentrum grindel, abteilung für humanbiologie, universität hamburg, who assisted to trace and to strive the old literature in stadt bibliothek universität hamburg, germany. this research is dedicated to: 1. dr. drg. a. adi sukadana (the late), who endowed the spirit of studying and discovering the science. much more learning from a little is equal to not stakes on limited research materials. 89suriyanto: dental modifications: a perspective of indonesian chronology 2. prof. dr. j. glinka, svd for his restlessness to motivate us to be more sensitive about surroundings. little learning from many is equal to make its science area is not higher than others are, but it will complete each other. references 1. von jhering h. die künstliche deformirung der zähne. zeitschrift für ethnologie 1882; xiv: 213–62. 2. uhle m. ueber die ethnologische bedeutung der malaiischen zahnfeilung. berlin: abhandlungen und berichte des königlichen zoologischen anthropologischen–ethnograpischen museums zu dresden, 1886/1887: 1–18. 3. wilken ga. de verspreide geshriften van dr. g.a. wilken, verzameld door f.d.e. van ossenbruggen, del iii-iv. semarang:semarang: gravenhage, von dorp. & co; 1912.ge, von dorp. & co; 1912. 4. jacob t. racial identification of the bronze age human dentitions from bali. journal of dental research 1967; xlvi(5), suppl.: 903–910. 5. jacob t. some problems pertaining to the racial history of the indonesia region. utrecht: drukkerij neerlandia; 1967. p. 114. 6. k o e n t j a r a n i n g r a t a s a s a s a s r i t u s , u p a c a r a d a n r e l i g i . in: koentjaraningrat, editor. ritus peralihan di indonesia. jakarta:jakarta: pn balai pustaka; 1985: 11–48. 7. tiesler v. head shaping and dental decoration among the ancient maya: archaeological and cultural aspects. paper presented at the 64 meeting of the society of american aechaeology. chicago: the society of american aechaeology; 1999. p. 1-11. 8. white td, degusta d, richards gd, baker sg. prehistoric dentistry in the american southwest: a drilled canine from sky aerie, colorado. american journal of physical anthropology 1997; 103: 409–14. 9. jacob t. studies on human variation in indonesia. journal of national medical association 1974; 66 (5): 389–99. 10. sukadana aa. persamaan mutilasi dentisi pada kerangka-kerangka prasejarah dari liang bua, lewoleba dan melolo, serta beberapa catatan anthropologis mengenai penemuan-penemuan itu. majalah kedokteran gigi surabaya 1970; 3 (2): 13–30. 11. sukadana aa. perubahan-perubahan pada tulang dan gigi subfosil manusia dan aplikasinya dalam penentuan kronologi peninggalan itu. berkala ilmu kedokteran 1979; xi (2): 57–68. 12. sukadana aa. peninggalan manusia di liang bua dan hubungannya dengan penemuan di lewoleba dan melolo. berkala bioanthropologi indonesia 1981; i (2): 53–72. 13. sukadana aa. studi politipisme dan polimorfisme populasi pada beberapa peninggalan di nusa tenggara timur. dissertation. surabaya: universitas airlangga; 1984. p. 100–1. 14. boedhisampoerno s. studi gigi geraham belakang subresen dari gua ulu leang 2, maros, sulawesi selatan. berkala bioanthropologi indonesia 1982; iii (1): 21 – 31. 15. boedhisampoerno s. kerangka manusia dari caruban, lasem, jawa tengah. rapat evaluasi hasil penelitian arkeologi ii. jakarta:jakarta: pusat penelitian arkeologi nasional, departemen pendidikan dan kebudayaan r.i; 1985. 16. schiffer mb. behavioral archaeology. new york: academic press; 1976. p. 109–79. 17. swedlund ac, wade wd. laboratory methods in physical anthropology. prescott: prescott college press; 1972. 18. hillson s. dental anthropology. cambridge: cambridge university press; 1996. 19. jacob t. garis-garis besar methodologi penelitian dan analysis paleoanthropogi. in: indriati e, editor. buku bacaan antropologi biologis. jakarta: direktorat jendral pendidikan tinggi departemenjakarta: direktorat jendral pendidikan tinggi departemen pendidikan tinggi departemen pendidikan nasional r.i; 2000. p. 205–16. 20. sukadana aa. metodologi sampling populasi berhubung dengan kekhususan konstelasi dan sejarah anthropologik indonesia. berkala bioanthropologi indonesia 1983; iv (1): 17–l27. 21. flynn m. black teeth: a primitive method of caries prevention in southeast asia. journal of american dentist association 1977; 95: 96–97. 22. zumbroich tj. the origin and diffusion of betel chewing: a synthesiszumbroich tj. the origin and diffusion of betel chewing: a synthesis of evidence from south asia, southeast asia and beyond. electronic journal of indian medicine 2007/ 2008; 1: 63–116. 23. oxenham mf, locher c, nguyen lc, nguyen kt. identification of areca catechu (betel nut) residues on the dentitions of bronze age inhabitants of nui nap, nothern vietnam. journal of archaeological science 2002; 29: 909–15. 24. pietrusewsky m, ikehara–quebral r. the bioarchaeology of the vat komnou cemetery, angkor borei, cambodia. indo–pacific prehistory association bulletin 2006; 26: 86–97. 25. susiarti s. jenis-jenis pengganti pinang dan gambir dalam budaya menginang masyarakat di kawasan taman nasional wasur, merauke, papua. biodiversitas 2005; 6: 217–9. 26. tayanin gl, bratthall d. black teeth: beauty or caries prevention? practice and beliefs of the kammu people. community dentistry & oral epidemiology 2006; 34: 81–86. 27. suddhasthira t, thaweboon s, dendoung n, thaweboon b, dechkunakorn s. antimicrobial activity of cratoxylum formosum on streptococcus mutans. southeast asian journal of tropical medicine public health 2006; 37: 1156–9. 28. nguyen vc. the habit of black lacquering of teeth and dental caries. czah stomatology 1990; 43: 600–603. 29. lignitz h. die künstlichen zahnverstümelungen in afrika im lichte der kulturkreisforschung. anthropos 1919/1920; xiv – xv: 891–943. 30. lignitz h. die künstlichen zahnverstümelungen in afrika im lichte der kulturkreisforschung. anthropos 1921/1922; xvi – xvii: 247 – 64, 866 – 89. 31. finucane bc, manning k, touré m. prehistoric dental modification in west africa – early evidence from karkarichinkat nord, mali. international journal of osteoarchaeology 2008; 18 (6): 632 – 40. 32. haour a, pearson ja. an instance of dental modification on human skeleton from niger, west africa. oxford journal of archaeology 2005; 24: 427–33. 33. soejono rp. sistim-sistim penguburan pada akhir masa prasejarah di bali. dissertation. jakarta: universitas indonesia; 1977. p. 55. 34. jacob t. manusia makhluk gelisah: melalui lensa bioantropologi. surakarta: muhammadiyah university press; 2006. p. 135–9. 35. jacob t. the problem of austronesian origin. in: simanjuntakproblem of austronesian origin. in: simanjuntak t, pojoh ihe, hisyam m, editors. austronesian diaspora and the ethnogeneses of people in indonesian archipelago. jakarta: lipi press; 2006. p. 7–13. 36. bellwood p. the early movements of austonesian–speaking peoples in the indonesian region. in: simanjutak t, pojoh ihe, hisyam m, editors. austronesian diaspora and the ethnogenesis of people in indonesian archipelago. jakarta: lipi press; 2006: 61 – 82. 37. glinka j. racial history of indonesia. in: schwidetzky i, editor.in: schwidetzky i, editor. rassengeschichte der menschheit. münchen: r. oldenbourg verlag;münchen: r. oldenbourg verlag; 1981. p. 79 – 133. 38. glinka j. reconstruction the past from present. paper of international conference on human paleocology: ecological context of the evolution of man. jakarta: lipi, 1993. p. 1–11. 39. suriyanto ra, koesbardiati t. perbandingan karakteristik epigenetis dan metris upper viscerocranium dari populasi tengkorak manusia yang berasal dari situs prasejarah liang bua, lewoleba, melolo dan ntodo leseh di nusa tenggara timur. jurnal anatomi indonesia 2005; 1 (2): 60–70. 40. koesbardiati t, suriyanto ra. menelusuri jejak populasi morfologi pangur gigi-geligi: kajian pendahuluan atas sampel gigi-geligi dari beberapa situs purbakala di jawa, bali dan nusa tenggara timur. humaniora 2007; 19 (1): 33–42.2007; 19 (1): 33–42.19 (1): 33–42. 41. koesbardiati t, suriyanto ra. dental modification in flores: a biocultural perspective. in: indriati e, editor. recent advances on southeast asian paleoanthropology and archaeology. yogyakarta:yogyakarta: laboratory of bioanthropology and paleoanthropology faculty of medicine gadjah mada university; 2007. p. 259–68. 42. richerson pj, boyd r. not by genes alone: how culture transformed human evolution. chicago: the university of chicago press; 2005. p. 191–224. 90 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 81-90 43. foley ra, donnelly p. towards in integrated approach to human evolution. in: donnelly p, folley ra, editors. genes, fossils and behavior: an integrated approach to human evolution. amsterdam: ios press; 2001. p. 1–2. 44. lie gl. beberapa aspek pengasahan gigi di indonesia terutama dari daerah djawa timur dan madura. majalah kedokteran gigi surabaya iii 1966; (1-4): 3–15. 45. jensen gd, suryani lk. orang bali: penelitian ulang tentang karakter. bandung: penerbit itb; 1996. p. 20.1996. p. 20. 46. arif j, kapid r. morphological trait of early hominid's molar from sangiran. in: indriati e, editor. recent advances on southeast asian paleoanthropology and archaeology. yogyakarta: laboratory of bioanthropology and paleoanthropology faculty of medicine gadjah mada university; 2007. p. 128–39. 47. arif j, kapid r, kaifu y, baba h, abdurrahman m. announcement of glom 2006.03: a four isolated deciduous teeth from sangiran, central java, indonesia. in: indriati e, editor. recent advances on southeast asian paleoanthropology and archaeology. yogyakarta: laboratory of bioanthropology and paleoanthropology faculty of medicine gadjah mada university; 2007. p. 140–50. 48. jacob t. a mandible from anyar urn-field, indonesia. journal of national medical association 1964; 56 (5): 421–6. 49. ubelaker dh. forensic anthropology. in: ember cr, ember m, editors. encyclopedia of medical anthroplogy: health and illness in the world's cultures, vol. i. new york: springer science+business media, inc; 2004. p. 37–42. 50. zimmerman mr. paleopathology and the study of ancient remains. in: ember cr, ember m, editors. encyclopedia of medical anthropology: health and illness in the world's cultures, vol. i. new york: springer science+business media, inc; 2004. p. 49–58. 51. soedomo. kemadjuan ilmu kedokteran gigi di indonesia sesudah th. 1950 sampai sekarang. in: sardjito, editor. perkembangan ilmu pengetahuan kedokteran di indonesia. jakarta: madjelis ilmu pengetahuan indonesia departemen urusan research nasional, 1965. p. 245–58. 52. koerniati i. mutilasi gigi anterior dengan terjadinya atrisi gigi posterior: suatu studi sosio-atnropologi kesehatan pada suku mentawai di pulau siberut. dissertation. surabaya: universitas airlangga; 2004. p. 128–69. 53. reichart pa, creutz u, scheifele c. dental mutilations and associated alveolar bone pathology in africa skulls of the anthropological skulls collection, charite, berlin. journal of oral pathology and medicine 2008; 37(1): 50–55. 54. lin wy, chiu ty, lee lt, lin, cc, huang cy, huang kc. betel nut chewing is associated with increased risk of cardiovascular disease and all-cause mortality in taiwanese men. american journal of clinical medicine 2008; 87: 1204–11. 55. sullivan rj, allen js, otto c, tiobech j, nero k. effects of chewing betel nut (areca catechu) on the symptoms of people with schizophrenia in palau, micronesia. british journal of psychiatry 2000; 177: 174–8. 56. vukovic a, bajsman a, zukic s, secic s. cosmetic dentistry in ancient times–a short review. bulletin of international association of paleodontology 2009; 3: 9–13. 166 dental journal (majalah kedokteran gigi) 2023 september; 56(3): 166–171 original article a m e l o g e n i n a n d a l k a l i n e p h o s p h a t a s e e x p r e s s i o n i n ameloblast after saltwater fish consumption in pregnant mice (mus musculus) sandy christiono1, seno pradopo2, i ketut sudiana3, islamy rahma hutami4 regilia shinta mayangsari5, yayun siti rochmah6, zurairah ibrahim7 1department of pediatric dentistry, faculty of dentistry, universitas islam sultan agung, semarang, indonesia 2department of pediatric dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3department of pathology anatomy, faculty of medicine, universitas airlangga, surabaya, indonesia 4department orthodontics, faculty of dentistry, universitas islam sultan agung, semarang, indonesia 5dental student, faculty of dentistry, universitas islam sultan agung, semarang, indonesia 6department of oral surgery, faculty of dentistry, universitas islam sultan agung, semarang, indonesia 7department orthodontics, faculty of dentistry, universiti sains islam malaysia, kuala lumpur, malaysia abstract background: the intricate process of tooth formation during embryonic development ensures sufficient nutrition for the growth of healthy dental tissues. amelogenin and alkaline phosphatase (alp) are serine proteinases secreted by the ameloblast during the transition and maturation phases of the amelogenesis process. consumption of saltwater fish is predicted to increase the expression of amelogenin and alp in ameloblast cells during tooth formation. only now have the function of each gene, tooth-forming cells, and the proteins they produce in the biomolecular amelogenesis of tooth enamel, which began during prenatal development, been clarified. purpose: this study aims to determine how saltwater fish powder affects the ability of mother mice to increase the expression of amelogenin and alp in cell ameloblast. methods: using a completely randomized design, this study was experimental and aimed to examine the effects of sardine (sardinella fimbriata), splendid ponyfish (leiognathus splendens), and tuna (euthynnus affinis) powder. as samples, twenty-four female mice (mus musculus) were used. two groups of mice were created: group 1 (2.14 mg/0.5 ml) and the control group. the expression of amelogenin and alp was determined using immunohistochemistry (ihc) and t-test (p<0.05). results: expression of ameloblast was significantly different between the treatment and control groups (p<0.05). conclusion: the consumption of saltwater fish reduces the amelogenin and alp expressions of mouse fetal ameloblast cells during tooth development in vivo. keywords: amelogenin; alkaline phosphatase; medicine; saltwater fish consumption; tooth development article history: received 7 september 2022; revised 18 january 2023; accepted 29 january 2023; published 1 september 2023 correspondence: seno pradopo, department of pediatric dentistry, faculty of dental medicine, universitas airlangga. jalan mayjen prof. dr. moestopo no. 47 surabaya, 60132, indonesia. email: seno-p@fkg.unair.ac.id introduction the growth and development of children’s teeth must be considered at an early age, specifically during the embryonic stage of tooth development. at the stage of tooth growth and development, abnormal teeth are found in many children. the development of teeth begins approximately 28 days after conception.1 the mineralization phase of primary teeth begins during the fourteenth intrauterine week, and all primary teeth will be fully mineralized after birth.2,3 calcium and phosphate comprise around 95% of enamel, making it the most complicated tissue in the human body. one of the proteins that become engaged in the enamel development stage is called amelogenin. ameloblasts, responsible for 80–90% of the total protein, are responsible for the production of this hydrophobic protein.4 this protein is expressed in secretions until the post-secretory ameloblast stage. amelogenin is also essential for the formation of hydroxyapatite prisms and the production of standard enamel thickness.5 the high alkaline phosphatase (alp) levels produced by differentiated cells in the stratum intermedium during the last stages of tooth formation are responsible for this. the epithelial cells that make up enamel are diverse, each differentiating to perform copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 mailto:seno-p@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 167christiono et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 166–171 a somewhat distinct set of tasks. these cells include the stratum intermedium cells, which have a role in ameloblast development and promote enamel mineralization via strong alp activity. alp expression is elevated in the stratum intermedium layer.6,7 tooth formation in the prenatal period occurs in the fourth intrauterine month, when the enamel and dentin deposit structures will begin to form. teratogens and poor nutrition during pregnancy significantly affect the development of the primary and young permanent teeth. nutritional deficiency during pregnancy impacts tooth size, time of tooth eruption, defects in tooth mineralization that can increase the risk of caries, and impaired salivary gland formation.8 the process of tooth development and growth, which is divided into several stages, allows the occurrence of abnormalities or growth abnormalities starting at the initiation stage of tooth bud formation, which, if disturbed, can cause hypoplasia, anodontia, oligodontia, or odontogenic tumors.1,9,10 disruption in the histodifferentiation stage can lead to amelogenesis imperfecta or dentinogenesis imperfecta. disturbances in the morph differentiation stage cause disturbances in the shape of the teeth.11 the hypoplastic/ hypomineralization effect can decrease tooth strength and delay tooth eruption time. exogenous and endogenous variables lead to the factors that cause the above disturbances in growth and development. endogenous factors include genetics. the role of each tooth-forming gene and cell, as well as the protein they produce in the biomolecular calcification of tooth enamel that begins in prenatal time, has not yet been elucidated.12 numerous studies have documented the impact of daily nutrition on the development of teeth. according to reports, providing proteins such as saltwater fish powder containing calcium and omega-3 optimizes enamel density.9 based on the preceding information, the purpose of this study is to determine how saltwater fish powder affects the ability of mother mice to increase the expression of amelogenin and alp in cell ameloblast. materials and methods following seven days of environmental adaption, female mice (mus musculus) were housed in plastic cages with wire mesh. the hardwood shavings at the bottom of the cages were changed every three days, and the animals had free access to food and water (ad libitum). on the third day, a pregnancy examination was performed. if the female mice’s vulva developed a visible vaginal plug on a given day, that day was recognized as day zero of pregnancy. the pregnant mice were subsequently housed in five-cage groups and provided with a regular diet. the ethics committee of the faculty of dental medicine at airlangga university had already assessed the ethical treatment of animals in this research. therefore, a certificate with the number 010/hreccfodm/ii/2018 was granted. this research used a completely random experimental design. as the unit of analysis, 10-week-old, 20–30 gram, healthy-appearing adult female mus musculus mice were employed in this investigation (agile, not lethargic, clean skin without wounds, bright eyes). both the treatment and control groups were comprised of 24 mice. the average intake of saltwater fish powder was converted into a dosage of 2.14 mg/0.5 ml. the dose is delivered thrice daily, every 6 to 8 hours. both experimental and control groups were slaughtered on the eighteenth day of gestation. material for this research was prepared at the faculty of chemistry, universitas gadjah mada, indonesia, by drying sardines (sardinella fimbriata), splendid pony fish (leiognathus splendens), and tuna (euthynnus affinis), reducing them to a powder, and adding the emulsifier carboxymethylcellulose (cmc). in the first process of making saltwater fish powder, the test material was dose-weighed and dissolved in hot water heated to 70 degrees celsius. using an ultra-turrax (ika, germany), sea fish powder was homogenized to crush and grind, and 1% cmc material was agitated for 15 minutes until homogeneous. after the fish powder and cmc had been combined, the mixture was deposited in the appropriate containers. the inductively coupled plasma technique revealed the calcium content of saltwater fish powder to be 5.56% w/w. at the same time, the gas chromatography method determined the omega-3 content to be 3.34% w/w. mice were euthanized with 10 to 20 cc of chloroform (henan haofei kimia co., ltd, indonesia). for dental histology preparations, formalin 10% alcohol was employed in concentrations of 70%, 80%, 95%, and 96% absolute alcohol, xylol, paraffin, and hematoxylin eosin.9 the surgical operation was done on mice while sedated with chloroform. mice were put in hermetically sealed jars, then 10 to 20 cc of chloroform was poured onto cotton and placed in the jars with the mice. after two to five minutes, breath and heart rate measurements were taken. if the mice were not breathing, the cover of the jar was removed. before surgery, the mice’s cervical spines were dislocated to ensure their demise. the mice were put on the surgical board using pins. surgical incisions were made in the abdomen or uterus using curved scissors. the embryo was removed from the uterus after examination of the right and left corpus luteum. to analyze prenatal dental tissue, they were simultaneously coated by an amniotic membrane and preserved in formalin. since the detected substance interacts with enzyme-labeled antibodies, immunohistochemical responses are specific. a color indication shows the existence of an enzymatic process (chromogen). among the chromogens available are naphthol (blue) and dab (brown). using mice from each housing arrangement, immunohistochemical preparations are produced in the same way as histopathological preparations up to the stage of tissue sectioning. 13 the preparations were examined using a leica dm 750 germany light microscope at 100x magnification to observe all fields of view, and then at copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 168 christiono et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 166–171 400x magnification for a more thorough study. under a microscope with a magnification of 400x, the edges of the peripherally organized ameloblast cells (amelogenin sc-33121 santa cruz biotechnologi, inc, europe, alp bs-1535r-hrp bioss, boston, massachusetts) may look brown. two researchers and analysts conducted computations with 400x magnification in particular visual fields, specifically on ameloblast cells, with 95% clinical agreement. the shapiro-wilk test from ibm statistical package for social sciences statistics 26 was used to assess the normality of distribution, continuing descriptive analysis to acquire mean and standard deviation, homogeneity, and significance of independent t-test at p<0.05. results the shapiro-wilk test was performed to determine the normality of the distribution between all research data, with a total of eight replications for each treatment group and seven replications in normal mice because one mouse died. the results of the shapiro-wilk test showed a normal distribution of amelogenin and alp expression data (p>0.05). for amelogenin and alp data, descriptive analysis was continued to get the independent t-test mean and standard deviation homogeneity (table 1). hpa of amelogenin in the treatment groups is shown in figure 1a-c, and control groups are shown in figure 1d-f. in ameloblast cells of the oral tissue treated with saltwater fish powder, amelogenin expression was reduced. the decrease in amelogenin in the dental tissue fed with saltwater fish powder appeared to be significant compared to the control group. according to the analysis’s findings, the control group had lower levels of alkaline phosphatase (alp) expression in dental cells than the group that received saltwater fish powder (p<0.05) (table 2). hpa of alp in the treatment groups is shown in figure 2a-c, and control groups are shown in figure 2d-f. there was an increase in the expression of alp in ameloblast cells in the dental tissue treated with saltwater fish powder. this increase was significant compared to the control group. table 1. mean expression of amelogenin from ameloblast cells in fetal tooth tissue of mice group n amelogenin expression p mean sd median minimum maximum control 7 4.64 0.66 4.40 4.10 5.80 0.004* treatment 8 3.13 0.77 2.95 2.30 4.90 description: * significant level p<0.05 table 2. mean number of ameloblast cells expressing alkaline phosphatase in fetal tooth tissue of mice group n alkaline phosphatase expressions p mean sd minimum maximum control 7 3.00 0.54 2.50 3.90 0.000* treatment 8 5.98 0.63 4.80 6.70 figure 1. hpa of amelogenin. a, b, and c in the treatment group. d, e, and f in the control group. a, d 100x magnification, b, e 400x magnification, c, f 1000x magnification. b and c showed amelogenin expression with weak brown staining of the cytoplasm. figures e and f show the expression of amelogenin with solid brown staining in the cytoplasm. information: am: ameloblast, od: odontoblast, pulp: dental pulp. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 169christiono et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 166–171 discussion ameloblast cells are responsible for forming dental enamel, a dense limb of the tooth. ameloblasts are a unique kind of tooth cell responsible for the secretion of three major enamel-specific matrix proteins. these enamel-specific matrix proteins include amelogenin, ameloblastin, and enamelin. this matrix protein is expressed by ameloblasts in the secretory stage, and its presence is necessary for enamel biomineralization.14 for mice that become pregnant when they eat fish powder with calcium and omega-3 polyunsaturated fatty acids, the calcium will bind to transient receptor potential cation channel subfamily v member 6 (trpv6) to get through the placental membrane. it will then be carried to the mouse fetus by a ca2+-binding protein with the help of a ca2+ pump. omega-3 polyunsaturated fatty acids in non-esterified fatty acids are natural ligands that signal ppars to tell fatps to break through the placental membrane. fatty acid binding protein will then transport the fatps. ca2+ will be absorbed by ameloblast cells through ca2+ sensing receptors. fatps make the insulin growth factor (igf) send out more signals. igf will send a transduction signal into the cell nucleus. this will turn on p38 mitogen-activated protein kinase (map) and p44/p42 map, which will increase heat shock protein 27 as a chaperone molecule to keep the cell’s structure intact. igf will help move the amelogenin protein secreted by ameloblast cells during the secretory stage.3,9,16 this study found a significant decrease in the expression of amelogenin with the provision of saltwater fish powder (figure 1). this is done by wu et al.,17 and c-terminal brushite-interaction amelogenin with afm at low concentrations is stated (1-10 nm). several clinical states cause ameloblasts to express varying amounts of amelogenin, and the amelogenin found in ameloblasts during enamel development may be a reaction to hypomineralization. consequently, in the hypomineralized state, amelogenin is up-regulated in ameloblasts.18,19 amelogenesis and the formation of the tooth structure are connected to the function of the tooth tissue. the mineralization of calcium phosphate is an essential component of this process. the modulation of calcium levels inside the cell will cause an increase in acidity, which in turn will cause amelogenin levels to decrease.20,21 during the secretory phase, the protein amelogenin is secreted and degraded by kallikrein-4. after 18 days of gestation, when the enamel is fully formed and matrix development has begun, amelogenin expression was assessed in these fetuses. mice begin developing dental enamel between 14.5 and 16.5 days when immunohistochemistry reveals a transition from the cap to the bell stage; at the bell stage, ameloblast cells have started to differentiate.22 according to wahluyo et al.,23 in the examination of the effect of sodium fluoride on the development of ameloblasts and kidney proximal tubular cells, the expression of amelogenin was shown to be significantly lower in the control group than in the treatment group. in addition to the liver and osteoblasts (new boneforming cells), the intestines, proximal renal tubules, placenta, and milk-producing mammary glands provide alp. a buildup of blockage in the bile ducts causes a rise in serum alp levels (cholestasis).24 liver (hepatobiliary) and bone (bone resorption) illnesses are two common conditions that can be diagnosed with the help of the alp test. in bone diseases like paget’s disease, an aberrant increase in osteoblastic activity (bone cell production) leads to high alp levels. in youngsters, elevated levels of a protein called alanine aminotransferase can be observed before and after puberty (physiological). bone remodeling can be accelerated in vitro by increased synthesis of bone figure 2. hpa of alkaline phosphatase. a, b, and c in the treatment group. d, e, and f in the control group. a, d 100x magnification, b, e 400x magnification, c, f 1000x magnification. b and c showed alkaline phosphatase expression in the cytoplasm with a solid brown color. e and f showed weak brown alkaline phosphatase expression in the cytoplasm. am: ameloblast, od: odontoblast, pulp: dental pulp. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 170 christiono et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 166–171 alkaline phosphatase from gingival mesenchymal stem cells cultured in platelet-rich fibrin.25–27 this study showed a significant increase in the treatment group given saltwater fish powder (figure 2). indeed, research conducted by lacruz et al.28 claimed that alp is crucial to the calcification process. a study on experimental mice increased the development of dental enamel and mineralization of the stratum intermedium by ameloblast cells showed decreased alp expression in tissue hypoxia after 24 and 48 hours.29 research by zhang et al.30 found that alp was lower in hypoxia, the mitochondrial count was lower, the endoplasmic reticulum was smaller, and mineralization was slowed when mitochondria were damaged. an elevation of alp in the treatment group did not correlate with decreased tissue oxygen saturation. as the number of osteoblasts involved in bone formation increases, so will the amount of the alp enzyme being expressed.31,32 during the histodifferentiation phase of the tooth creation process, the animals used in this research were put to sleep on day 18. as a result, the alp level was elevated in tooth enamel tissue, particularly in the stratum intermedium. because alp plays an important role in the mineralization process, the intermediate layer of tooth enamel contains a high concentration of the enzyme’s expression. one important disclaimer is that in vivo research can only provide an incomplete representation of the amelogenesis process. despite this, it was discovered that the expression of amelogenin and alp increased on day 18 of prenatal consumption of saltwater fish powder. this study did not look at mmp-20, known to be one of the progenitors of tooth enamel, nor did it look at any other proteins that can be tested.6,22 in conclusion, the consumption of saltwater fish powder can reduce the number of ameloblast cells expressing amelogenin and alp in mice. acknowledgement the authors appreciate the financial support (grant number: 3463/d.3/sa/iv/2021) from the islamic university of sultan agung. references 1. dean j. mcdonald and avery’s dentistry for the child and adolescent. 11th ed. st louis: mosby elsevier; 2021. p. 752. 2. hovorakova m, lesot h, peterka m, peterkova r. early development of the human dentition revisited. j anat. 2018; 233(2): 135–45. 3. putri wa, christiono s, fathurrahman h. the effect of consumption of marine fish nanoparticles on the hardness of teeth enamel in mice (mus musculus). j kesehat gigi. 2021; 8(2): 109–14. 4. bartlett jd, simmer jp. kallikrein-related peptidase-4 (klk4): role in enamel formation and revelations from ablated mice. front physiol. 2014; 5: 1–8. 5. gil-bona a, bidlack fb. tooth enamel and its dynamic protein matrix. int j mol sci. 2020; 21(12): 4458. 6. wang x, chiba y, jia l, yoshizaki k, saito k, yamada a, qin m, fukumoto s. expression patterns of claudin family members during tooth development and the role of claudin-10 (cldn10) in cytodifferentiation of stratum intermedium. front cell dev biol. 2020; 8: 595593. 7. liu j, saito k, maruya y, nakamura t, yamada a, fukumoto e, ishikawa m, iwamoto t, miyazaki k, yoshizaki k, ge l, fukumoto s. mutant gdf5 enhances ameloblast differentiation via accelerated bmp2-induced smad1/5/8 phosphorylation. sci rep. 2016; 6(1): 23670. 8. gondivkar sm, gadbail ar, gondivkar rs, sarode sc, sarode gs, patil s, awan kh. nutrition and oral health. disease-a-month. 2019; 65(6): 147–54. 9. christiono s, pradopo s, sudiana ik. the effect of saltwater fish consumption by female house mice (mus musculus) on the increasing teeth enamel density of their pups: microct analysis. j int dent med res. 2019; 12(3): 947–52. 10. wahluyo s. peran kalsium sebagai prevensi terjadinya hipoplasia enamel (the role of calcium on enamel hypoplasia prevention). dent j. 2013; 46(3): 113–8. 11. brookes sj, barron mj, boot-handford r, kirkham j, dixon mj. endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate. hum mol genet. 2014; 23(9): 2468–80. 12. poulter ja, brookes sj, shore rc, smith cel, abi farraj l, kirkham j, inglehearn cf, mighell aj. a missense mutation in itgb6 causes pitted hypomineralized amelogenesis imperfecta. hum mol genet. 2014; 23(8): 2189–97. 13. sudiana ik. teknologi ilmu jaringan dan imunohistokimia. jakarta: sagung seto; 2005. p. 36–44. 14. hu jc-c, hu y, lu y, smith ce, lertlam r, wright jt, suggs c, mckee md, beniash e, kabir me, simmer jp. enamelin is critical for ameloblast integrity and enamel ultrastructure formation. van wijnen a, editor. plos one. 2014; 9(3): e89303. 15. lokappa sb, chandrababu kb, moradian-oldak j. tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its n-terminus. biochem biophys res commun. 2015; 464(3): 956–61. 16. christiono s, pradopo s, sudiana ik. the effect of saltwater fish consumption by mother mice (mus musculus) on the expressions of fabps and type 1 collagen regarding increase in enamel density. j int dent med res. 2022; 15(4): 1535–40. 17. wu s, zhai h, zhang w, wang l. monomeric amelogenin’s c-terminus modulates biomineralization dynamics of calcium phosphate. cryst growth des. 2015; 15(9): 4490–7. 18. mitsiadis ta, filatova a, papaccio g, goldberg m, about i, papagerak is p. distribution of the amelogenin protein in developing, injured and carious human teeth. front physiol. 2014; 5: 00477. 19. dewi n, syaify a, wahyudi ia. effect of gestational diabetes mellitus on the expression of amelogenin in rat offspring tooth germ. dent j. 2013; 46(3): 135–9. 20. habelitz s. materials engineering by ameloblasts. j dent res. 2015; 94(6): 759–67. 21. bronckers aljj. ion transport by ameloblasts during amelogenesis. j dent res. 2017; 96(3): 243–53. 22. ida-yonemochi h, otsu k, ohshima h, harada h. the glycogen metabolism via akt signaling is important for the secretion of enamel matrix in tooth development. mech dev. 2016; 139: 18–30. 23. wahluyo s, ismiyatin k, purwanto b, mukono is. the influence of sodium fluoride on the growth of ameloblasts and kidney proximal tubular cells. folia biol (praha). 2017; 63(1): 31–4. 24. vimalraj s. alkaline phosphatase: structure, expression and its function in bone mineralization. gene. 2020; 754: 144855. 25. al nofal aa, altayar o, benkhadra k, qasim agha oq, asi n, nabhan m, prokop lj, tebben p, murad mh. bone turnover markers in paget’s disease of the bone: a systematic review and meta-analysis. osteoporos int. 2015; 26(7): 1875–91. 26. nugraha ap, narmada ib, ernawati ds, dinaryanti a, hendrianto e, r iawan w, rantam fa. bone alkaline phosphatase and osteocalcin expression of rat’s gingival mesenchymal stem cells cultured in platelet-rich fibrin for bone remodeling (in vitro study). copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 171christiono et al. dent. j. (majalah kedokteran gigi) 2023 september; 56(3): 166–171 eur j dent. 2018; 12(04): 566–73. 27. triwardhani a, tjandra rr, hamid t, ariani tn. correlations of alkaline phosphatase expression with osteoblast number during orthodontic tooth movement, in vivo. j int dent med res. 2022; 15(4): 1497–502. 28. lacruz rs, habelitz s, wright jt, paine ml. dental enamel formation and implications for oral health and disease. physiol rev. 2017; 97(3): 939–93. 29. sidaly r, landin ma, suo z, snead ml, lyngstadaas sp, reseland je. hypoxia increases the expression of enamel genes and cytokines in an ameloblast-derived cell line. eur j oral sci. 2015; 123(5): 335–40. 30. zhang h-y, liu r, xing y-j, xu p, li y, li c-j. effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts in vitro. exp ther med. 2013; 6(6): 1553–9. 31. pudyani ps, asmara w, ana id, utari tr. alkaline phosphatase expression during relapse after orthodontic tooth movement. dent j. 2014; 47(1): 25–30. 32. hasib a, wahjuningrum da, ibrahim mhr, kurniawan hj, e r nawat i r , ha d i noto m ek , mo o duto l . a l p (a l k a l i ne phosphatase) expression in simple fracture incident in rat (rattus norvegicus) femur bone supplemented by apis mellifera honey. j int dent med res. 2018; 11(3): 1636–9. copyright © 2023 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v56.i3.p166–171 https://e-journal.unair.ac.id/mkg/index https://doi.org/10.20473/j.djmkg.v56.i3.p166-171 95 effect of il-1 and gustducin expression change on bitter taste during fever jenny sunariani department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract homeostatic changes in the body, such as fever, cause inflammation, whose one of its impacts is the sense of bitterness inside the mouth. it implies in the reduction of appetite, which may finally result in the reduction of physical condition due to the inadequacy of food intake. it causes the inhibition of healing process, which reduces working productivity. the objective of this study was to identify the mechanism of bitterness due to inflammation, as proved locally in the taste buds of wistar rats. this study was carried out experimentally using post-test only control design in experimental animals of male wistar strain rattus norvegicus. the animals were divided into two groups. first group served as control, while the second group received treatment with salmonella typhimurium 0.5 ml/kg bw. blood sample and tongue incision were taken from the animals. il-1 was counted, and tongue incision was used for immunohistochemical staining for the variables of gustducin. data were analyzed using kolmogorov-smirnov test for data normality, and followed with comparative test. the discriminant analysis was also done to find the discriminant variable. it was found that there was an increase of biological response of signaling transduction of bitterness in taste buds, as indicated from the increase of gustducin in treatment group or in inflammatory fever condition as compared to control group (p < 0.05), but no change of concertation at il-1 significan whenever there was any change of concertation by unfolding its mechanism. further studies can be recommended to find the way to inhibit this sense of bitterness. the results are intended to overcome homeostatic disorder in the body to prevent loss of appetite, so that physical endurance can be maintained. it concluded that there is no increase of serum il-1 expression in fever, but there is a significanly increase of taste buds gustducin. further studies should focus on gustducin cellular role in other factors that play a role in taste buds signal transduction, either in homeostatic condition or in the condition of homeostatic disorder. key words: inflammation, bitter taste, gustducin, il-1 correspondence: jenny sunariani, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. telp. 62-31-5030255. e-mail: jennymdtc@yahoo.com introduction oral cavity is the early site of entrance of food and the taste of food is determined by the sense of taste or the receptor of the sensation of taste in oral cavity, particularly the tongue. the taste of food is also determined by individual perception on the food itself. nutrition is one of human basic needs required as the source of energy for the body. the fulfillment of nutritional requirement depends on the appetite, which is affected by central and peripheral nervous systems. in central nervous system, it is influenced by several factors, such as memory of the food, while in peripheral nervous system it is determined by the receptor of taste sensation.1,2 homeostatic disorder in the body, such as infection, may result in the reduction of appetite. the reduction of appetite may occur either at central level, which is in brain, or peripheral level, in the receptor of taste or taste buds. predominant change of taste into bitterness may reduce appetite, so that it may also reduce body endurance, which may finally result in the reduction of immunologic endurance of the body. the latter will then lead to aggravate infection.3 therefore, fever may result in the reduction of body endurance of working productivity of the patient.4 however, the mechanism of appetite reduction in taste receptor within taste buds through signal transduction in inflammatory fever has not been disclosed. there are several tastes in oral cavity: salty, sour, sweet, bitter, and umami.5,6 the occurrence of bitterness results from the binding of chemical substances as the stimulator of the sense of bitterness in the receptor.7-9 this reaction makes the gprotein to release alpha unit, which, in bitter receptor it is called as gustducin.10,11 gustducin activates enzyme, so that in such condition it results in blocked k+ channel, and stimulates plc (phospholipase c) to activate pip (phosphoinositol phosphate) to become ip3 (inositol triphosphate). ip3 (inositol triphosphate) causes ca2+ release from endoplasmic reticulum and mitochondria, resulting in depolarization.12,13,14 increased ca2+ expression within bitter taste receptor cells causes intensified bitterness and delivered further to the memory in brain.15,16 one manifestation of homeostatic disorders is inflammatory fever, either endogenous or exogenous. fever is one of clinical symptoms of infection resulting from bacteria, such as typhoid fever, in which there is a typical symptom in the tongue, called as typhoid 96 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 95-99 tongue. histopathological examination reveals an increase of polymorphonuclear (pmn) inflammatory cells, the neutrophil, which is also a typical sign of acute inflammation.15 in fever, the clinical manifestation of infection, there is also a predominant symptom of intensified bitterness in tongue. physiobiologically, the function of taste receptor is acted by taste buds. the mechanism of taste stimulation is commenced by the presence of primary taste in oral cavity by taste buds as the receptor of taste nerve cells, the part of the body that induces biological response.17 the response can be triggered by the exposure of bacteria as signaling initiation.18,19 in inflammatory fever, the mechanism of change at cellular level in taste receptor on intracellular molecular changes may occur through ion signal transducing, such as k+, ca2+, mg+2. until today, the mechanism of taste buds biological change due to bacteria infection modulation has not been investigated and clearly unfolded. since there is a clinical symptom of bitterness in inflammation and fever and also changes at cellular level, a detailed study is needed on unclear physiobiological dynamics in predominant changes of bitter taste at peripheral level. this study was conducted on wistar strain rats (rattus norvegicus) exposed to salmonella typhimurium. the observation was carried out on tongue incision for il-1 expression. elisa test was done to find gustducin count and histopathological examination was undertaken using immunohistochemical staining. we investigated whether there was an increase of serum il-1 expression and gustducin count in taste buds. materials and methods this was a true experimental study using post-test only control group design. materials used were serum and the incision of wistar strain rattus norvegicus rats tongue invaded with bacteria through injection with the bacteria salmonella typhimurium. serum il-1 expression was measured using elisa method,20 and tongue incision was stained immunohistochemistry using monoclonal antibody produced by santa cruz biotechnology, inc., to observe gustducin expression. this study was undertaken at the departments of biochemistry, microbiology, anatomic pathology, and gramik, airlangga university school of medicine, laboratory of veterinary immunology, airlangga university school of veterinary medicine, and regional health laboratory, surabaya. in this study, the experimental animals were divided into two groups. group i served as control group and group ii as treatment group that was rendered to have fever. the experimental animals were allocated randomly, treated adequately and ethically eligible. in group i, the animals were injected with distilled water, and in group ii the experimental animals received treatment (fever). their early temperature was measured and they were injected with salmonella typhimurium of 0.5 ml/kg bw. after 6 hours, the final body temperature was measured. they were sacrificed to take their blood and tongue. serum il-1 was measured in blood serum, while paraffin blocks were made on the tongue, which was followed with immunohistochemical staining to measure gustducin expression in taste buds. experimental animals that experienced inflammation were those rendered to have fever by administered with salmonella typhimurium and had temperature increase above normal (36o c). il-1 is cytokine produced by hypothalamus and measured in the serum of cardiac blood with elisa.20 gustducin was gprotein subunit alpha expressed by trcs of circumvalate papillae taste buds in posterior tongue in inflammation and control groups, measured based on the number of brownish trcs seen after immunohistochemical staining under light microscope at 400 times magnification. results prior to data analysis, statistical test was conducted using kolmogorov-smirnov test to find data normality. from the analysis it was found that the samples had normal distribution (p > 0.05). mean and standard deviation of the variable of inflammation in the examination of temperature and histopathological examination of the tongue in control and treatment groups can be seen in table 1. table 1. t-test on the variable of systemic inflammatory response temperature control treatment mean sd n 36.686 0.485 7 38.486 0.478 7 p 000 notes: mean : mean of control and treatment of temperature sd : standard deviation n : number of sample p : significantly table 1 shows that there is difference in control and treatment group. the mean in treatment group is higher than that in control group. this indicates that after injection or bacterial invasion, body temperature in those rats was increasing. statistical analysis revealed mean and standard deviation as displayed in table 2. 97sunariani: effect of il-1 and gustducin expression change on bitter taste during fever table 2. results of il-1 and taste buds gustducin measurement group n mean std. deviation il-1 control treatment gustducin control treatment 7 7 7 7 45.7275 49.6536 0.38095 0.69722 22.5943 29.9844 0.10516 0.12067 notes: n: sample number, mean: mean of il-1 and gustducin expression in treatment group, mean il-1 showed no significant difference, while comparative test between treatment and control group showed significant difference (p < 0.001) of gustducin in treatment group, while il-1 also showed difference, but not significant. figure 1. mean of il-1 expression the result of univariate analysis of each il-1 variable, the result of univariate analysis in treatment and control group using t-test showed no significant difference of the variables in treatment group. figure 2. gustducin expression the results of univariate analysis of the variables of il-1 and gustducin in treatment and control group using t-test revealed significant difference of the variables in treatment group. this indicated that those rats were in inflammatory condition after being exposed to salmonella typhimurium, which intensified the sense of bitterness. discussion various homeostatic disorders in the body may occur due to several factors, one of which is inflammation that may induce clinical symptoms, such as fever. the disorder may also result in changes of taste sensed by peripheral nerve in taste buds, and also result in biological changes of cells, which is presenting as the change of intracellular signaling. this condition may affect the appetite, a clinical manifestation of the presence of inflammation. reduced appetite may present as a change of the sense of taste, particularly the intensification of bitterness. the quality of food taste may change, resulting from two factors, the food itself and the presence of systemic change in the body beyond the taste cells. food taste perception depends on taste receptor in taste buds, on other co-stimuli that result from stimulation of somatic taste receptor of the food, on the presence of memory of the food in central nervous system, and the transmission of food perception through taste pathway. to support the idea in this study, a preliminary study had been undertaken using questionnaire to 63 respondents who suffered from fever. the results showed that 96.82% of the patients had intensified bitterness in the mouth, while only 3.18% (3 persons) felt the intensification figure 3. results of immunohistochemical staining with gustducin antibody, on circumvalate papillae taste buds in normal group, showing blue trcs ( ), magnification 400 times. figure 4. results of immunohistochemical staining with monoclonal antibody on gustducin, on circumvalate papillae taste buds in treatment group, showing brown trcs ( ), magnification 400 times. 98 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 95-99 of sour. based on the findings in this preliminary study, this study tried to unfold the mechanism of homeostatic disorder in inflammatory fever and sense of bitterness as observed from several molecular variables in the taste cells, such as il-1 and gustducin. this study observed biological response of taste buds based on appetite reduction in patients with infection and fever, by using wistar strain rattus norvegicus that were invaded with salmonella typhimurium to find the homogeneity of the cause of fever. the aim of salmonella administration was to provide condition similar to typhoid fever in human.21 this disease is commonly found in tropical population, such as that in indonesia, with symptoms of reduced appetite due to the intensification of bitterness. salmonella typhimurium was used because if those rats were given with salmonella that invades human, fever would not occur, since rats are animals that live in dirty places.22 to find whether the samples had normal distribution, we conducted analysis using kolmogorov smirnov test, and the result of analysis revealed that data distribution was normal (p > 0.05). this study was conducted to two groups. the first group was control to obtain data on samples in healthy condition, and the second group received salmonella typhimurium invasion to induce fever. various factors affect the occurrence of inflammatory fever, such as the formation of prostaglandin e2, whose ep3 receptor is related with heat, and nitric oxide (no) that has an important role in inflammation during the eosinophil infiltration.23,24,25 to empiricize the biologic response variable, we conducted statistical comparative analysis using t test. to prove the presence of inflammation, we undertook temperature measurement and blood sampling for serum il-1 expression. it was found that there was a significant increase of temperature, indicating that the rats were infected with salmonella typhimurium. to prove the change of gustducin expression and il-1 expression in both groups, two sample t-test was undertaken. results of examination in experimental animals injected with salmonella typhimurium of 0.5 cc/kg bw, which was equal to mcfaland iii solution, revealed the occurrence of fever in inflammation, presenting as the increase of acute inflammatory cells, i.e. polymorphonuclear cells, particularly the neutrophil (63%), as proved by histopathological test.26 by the presence of acute inflammation as indicated by clinical symptoms of inflammatory fever, further identification of other biological variables, i.e., serum il-1, was conducted using elisa and gustducin examinations through immunohistochemical staining. the results showed that during inflammation, clinical symptom that presents as fever is apparent, particularly acute inflammation. this was indicated by the increase of serum il-1 expression, which was an inflammatory cytokine produced by macrophage or other apc due to the invasion of salmonella typhimurium. although mean il-1 expression did increase in treatment group, the increase was not significant, but systemically it had showed the occurrence of temperature increase. this was likely due to the presence of other inhibiting factors or due to untimely sampling time. sampling was undertaken after the reduction of il-1 level, as il-1 is a sensitive cytokine, so when it had been measured at lower level, the result would appear to be less significant. in addition, il-1 is an inflammatory cytokine produced by macrophage, and it is the only cytokine that has natural inhibitor. the inhibitor is recognized as il-1 receptor antagonist (il-1ra), an endogenous regulator for il-1 activity. there are also other factors that may inhibit il-1 production in certain conditions, such as the activity of cd8+ cells.3 data analysis showed significant increase (p < 0.05) in biological marker, the gustducin (gprotein subunit alpha) in bitterness taste within treatment group. the increase of gustducin expression in trc is marked by the absorption of immunohistochemical materials that renders the taste buds color to become brownish. during inflammation, there is an increase of pge2.2,27 the inflammation activated ep3 receptor, which is the receptor of pge2, and ep3 receptor will receive heat information that mobilizes ca2+, resulting in the increase of receptor sensitivity to stimulate adenilate cyclase.23 the increase results in gprotein binding, which activates plc, and plc activation results in pip breaking to become ip3 and dag. ip3 binding with er receptor stimulates ca2+ release from mitochondria and endoplasmic reticulum and results in the opening of er membrane ca2+ channel, so that ca2+ is released toward cytosol.28 ca2+ is also released from endoplasmic reticulum when there is stimulation from pge2, which results in depolarization, and this condition leads to the transduction of bitter taste to the brain.23,29,30 structural and chemical diversity of the tastant leads to various transduction mechanisms. this was different from that occurs in the sense of vision and olfaction, whose stimulation only runs through general stimulation pathway by the presence of photon or small volatile molecules transduced through a basic mechanism.31 this study was only viewed the role of signaling cells in peripheral nerves by confirming the biological change in elements that run the complex sense of taste transduction. the element of gustducin presents within the taste buds. it has a role in the transmission of the sense of taste, and the mechanism of each taste has its own specification. the receptor 7-transmembrane helix has a role to commence signaling cascade by binding the gproteins.15,32,33 the sense of taste in oral cavity may change if there is homeostatic change in the body, such as in a condition of natrium ion loss, in which taste receptor related to natrium ion channel has sensitivity reduction against the taste of salt, and so does other receptors. transduction and coding depend on trcs input through afferent nerve fibers. the information codes the taste quality that depends on comparative pattern in the fibers. each nerve fiber has specification according to their sensitivity against particular taste. for example, in fiber sensitive against salt, the nerve fiber has a high sensitivity against the taste of salt, but it can also sense other tastes in a lower sensitivity.2,34 99sunariani: effect of il-1 and gustducin expression change on bitter taste during fever the entrance of ca2+ into cytosol in membrane damage generally occurs due to injury35,36 or intoxication. this results in the change of ca2+ expression within cytosol and results in cell death.37 most of cytosol ca2+ is preserved particularly within reticulum endoplasma and mitochondria, as well as other vesicles in lower amount. ca2+ atpase pumps the ca2+ in the cytosol to cross the plasma membrane outward or to the preservation site within reticulum endoplasma and mitochondria. minute ca2+ increase from normal within cytosol may result in various cellular responses, such as the stimulation of the sense of bitterness in gustducin. excessive and continuous increase of ca2+ within cytosol leads to cellular intoxication. in this case it was manifested as inflammatory fever.15 it concluded that there is no increase of serum il-1 expression in fever, but there is a significanly increase of taste buds gustducin. further studies should focus on gustducin cellular role in other factors that play a role in taste buds signal transduction, either in homeostatic condition or in the condition of homeostatic disorder. references 1. carlson nr. physiology of behavior. 5th ed. boston: allyn and bacon; 2000. p. 213–7. 2. guyton ac, hall je. textbook of medical physiology. 11th ed. philadelphia: elsevier saunders comp; 2006. p. 44-8, 54, 61, 87–9, 76–8, 567, 675–80, 725, 758, 771. 3. kresno sb. imunologi: diagnosis dan prosedur laboratorium. edisi ke 4. jakarta: fakultas kedokteran universitas indonesia; 2001. p. 3–58, 65–90. 4. mandel gl, bennett je, dolin r. principles and practice of infectious diseases. 4th ed. new york: churchill livingstone; 1995. p. 569–607. 5. adler e, hoon ma, mueller kl, chandrashekar j, ryba njp, zuker cs. a novel family of mammalian taste receptors. rev by john palma of cell 2000; 100:693–702. 6. jacob t. a brief tutorial. available: http://www.cf.ac.uk/biosi/ stsff/jacob/teaching sensory/ taste/ html. updates in 1st of february 2008. 7. ogura t, mackay-sim a, kinnamon sc. bitter taste transduction of denatonium in the mudpuppy necturus maculosus. j of neurosci 1997; 17(10):3580–7. 8. zald dh, hagen mc, pardo jv. neural correlates of tasting concentrated quinine and sugar solutions. j of neurophys 2002; 87(2):1068–75. 9. yan c, kim d, aizawa t, berk bc. functional interplay betweenfunctional interplay between angiotensin ii and nitric oxide cyclic gmp as a key mediator. arterioscler thromb vasc biol 2003; 23:26–36. 10. kusakabe y, miura h, hashimoto r, sugiyama c, ninomiya y, hino a. the neural differentiation gene mash-1 has a distinct pattern of expression from the taste reception-related genes gustducin and t1r2 in the taste buds chem. senses 2002; 27:445–51. 11. jansen g, weinkove d, plasterk rha. the g-protein subunit gpc-1 of the nematode c. elegans is involved in taste adaptation. embo 2002; 21(5):986–94. 12. ogura t, kinnamon sc. ip3-independent release of ca 2+ from intracellular stores: a novel mechanism for transduction of bitter stimuli. j of neurophys 1999; 82(5):2657–66. 13. akopian a, withowsky p. intracellular calcium reduces light-induced excitatory post-synaptic responses in salamander retinal ganglion cells. j of phys 2001; 532(1):43–53. 14. ando h, mizutami a, matsu-ura, mikoshiba k. irbit, a novel inositol 1,4,5-triphosphate (ip3) receptor-binding protein, is released from the ip3 receptor upon ip3 binding to the receptor. j of biol chem 2003; 278(12):10602–12.278(12):10602–12. 15. ganong wf. review of medical physiology. 19th ed. seoul: mcgraw hill publ; 2001. p. 37-46:59–60, 90–110, 180–1, 183–7, 242, 249, 382. 16. vander a, sherman j, luciano d. human physiology. the mechanism of body function. 8th ed. new york: mcgraw-hill; 2001. p. 262–70. 17. purves d, augustine gj, fitzpatrick, katz lc, lamanta as, mcnamara jo. neuroscie. massachussets: sinauer assc; 1997.neuroscie. massachussets: sinauer assc; 1997.1997. p. 273–97. 18. stewart re, desimone ja, hill dl. new perspectives in a gustatory physiology: transduction, development, and plasticity. ajp cell phys 1997; 272 (1):c1–26. 19. goldsby ra, kindt tj, osborne ba. kuby immunology. 4th ed. new york:. wh freeman and co; 2000. p. 351–5, 365–9. 20. arondel j, singer m, matsukawa a, zychlinsky a, sansonetti pj. increased interleukin-1 (il-1) and imbalance between il-1 and il-1 receptor antagonist during acute inflammation in experimental shigellosis infection and immunity. 1999. 67(11):6056–66. 21. faucher sp, porwollik s, dozois cm, mcclelland m, daigle f. transcriptome of salmonella enterica serovar typhi within macrophages revealed through the selective capture of transcribed sequences. medicine sci 2006; 22(10):792–3. 22. hill ch. effect of salmonella gallinarum infection on zinc metabolism in chicks. poultry science 2001; 68(2):297–305. 23. hatae n, sugimoto y, ichikawa a. prostaglandin receptor: advances in the study of ep3 receptor signaling. bioc soc 2002; 131(6):781–4. 24. hull ma, ko scw, hawcroft g, prostaglandin ep receptors: targets for treatment and prevention of colorectal cancer. cancer resr 2004; 3:1031–9. 25. nguyen mt, solle m, audoly lp, tilley sl, stock jl, mcneish jd, coffman tm, dombrowicz d, koller bh. receptor and signaling mechanisms required for prostaglandin e2-mediated regulation of mast cell degranulation and il-6 production. american assoc of immunologists 2002; 169:4586–93. 26. gronert k, colgan sp, serhan cn. characterization of human neutrophil and endothelial cell ligand-operated extracellular acidification rate by microphysiometry: impact of reoxygenation. j pharmacol exp ther 1998; 285(1):252–61. 27. gallin ji, snyderman r, fearon dt, haynes bf, nathan c. inflammation basic principles and clinical correlates. 3rd ed. philadelphia, usa: lippincott williams & wilkins; 1999. p. 433–6, 443–51, 837, 1207–11. 28. spielman ai. gustducin and its role in taste. j of dent res 1998; 77:539–44. 29. fagni l, chavis p, ango f, bockaert j. complexs interactions between mglurs, intracellular ca2+ stores and ion channels in neurons. trends in neurosc 2000; 23(2):280–8. 30. braunwald e, fauci as, kasper dl, hauser sl, longo dl, jameson jl. harrison’s. principles of internal medicine. 15th ed. washington dc: mcgraw-hill publ; 2001. p. 1125–30. 31. katz db, nicolelis mal, simon sa. nutrient tastingand signaling mechanisms in the gut iv. there is more to taste than meets the tongue. am j physiol gastrointest liver physiol 2000; 278:g6–g9. 32. ueda t, ugawa s, yamamura h, imaizumi y, shimada s. functional interaction between t2r taste receptors and g-protein a subunits expressed in taste receptor cells. j of neuroscie 2003;2003; 23(19):7376–80. 33. boron wf, boulpaep el. a cellular and molecular approach. med phys. update ed. philadelphia: elsevier saunders; 2005. p. 207, 208, 328–31. 34. kandel er, schwart jh, jessel tm. stamfort, a simon & schuster co.stamfort, a simon & schuster co. neuroscience. massachussets: sinauer assc publ; 2000. p. 273–97.–97.97. 35. repo ru, finlay jb. survival of articular cartilage after controlled impact. j bone joint surg (am) 1977; 59:1068–76.–76.76. 36. jeffrey s, fedan. nucleosides and nucleotides in the lung. am js, fedan. nucleosides and nucleotides in the lung. am j respir cell mol biol 1995; 21(1):7–9. 37. blanco fj, guitian r, vasquez me, de-toro fj, galdo f. a possible pathway for osteoarthritis pathology. arth rheum 1998; 41:284–9. issn 1978 3728volume 47, number 4, december 2014 editorial board of dental journal (majalah kedokteran gigi) sk: 059/un3.1.2/2014 january 2nd– december 31st, 2014 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 47, number 4, december 2014: dr. i. b. narmada, drg., sp.ort (k) (orthodontics – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 206708019-204225738 contents page printed by: airlangga university press. (rk 003/01.15/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 47, number 4, december 2014 issn 1978 3728 1. the differences of orthodontic tooth movement on menstrual and ovulation cycle sonya grecila susilo, rahmi amtha, boedi oetomo roeslan and joko kusnoto ...................... 177–180 2. penambahan xylitol dalam glukosa, sukrosa terhadap pertumbuhan streptococcus mutans (in vitro) (the additional xylitol in glucose and sucrose on growth of mutans streptococci (in vitro) susilowati, udijanto tedjosasongko, dan fx suhariadji ............................................................ 181–185 3. prevalensi early childhood caries dan severe early childhood caries pada anak prasekolah di gunung anyar surabaya (the prevalences of early childhood caries and severe early childhood caries in preschool children at gunung anyar surabaya) rahel wahjuni sutjipto, herawati, dan satiti kuntari ............................................................... 186–189 4. indirect pulp capping in primary molar using glass ionomer cements murtia metalita, udijanto tedjosasongko, and prawati nuraini ................................................ 190–193 5. microleakage of conventional, resin-modified, and nano-ionomer glass ionomer cement as primary teeth filling material dita madyarani, prawati nuraini, and irmawati ........................................................................ 194–197 6. daya antibakteri penambahan propolis pada zinc oxide eugenol dan zinc oxide terhadap kuman campur gigi molar sulung non vital (the antibacterial effect of propolis additional to zinc oxide eugenol and zinc oxide on polybacteria of necrotic primary molar) yemy ameliana, herawati, dan seno pradopo ............................................................................. 198–201 7. usia saat inisial akuisisi streptococcus mutans dan jumlah erupsi gigi sulung pada anak (initial acquisition age of mutans streptococci and number of erupted primary teeth in children) citra adinda, udijanto tedjosasongko, dan teguh budi wibowo ............................................ 202–205 8. kekerasan mikro enamel gigi permanen muda setelah aplikasi bahan pemutih gigi dan pasta remineralisasi (enamel micro hardness of young permanent tooth after bleaching and remineralization paste application) budianto liwang, irmawati, dan els budipramana .................................................................... 206–210 9. daya antibakteri obat kumur chlorhexidine, povidone iodine, fluoride suplementasi zinc terhadap streptococcus mutans dan porphyromonas gingivalis (antibacterial effect of mouth washes containing chlorhexidine, povidone iodine, fluoride plus zinc on streptococcus mutans and porphyromonas gingivalis) betadion rizki sinaredi, seno pradopo, dan teguh budi wibowo ............................................ 211–214 10. uji toksisitas ekstrak bawang putih (allium sativum) terhadap kultur sel fibroblast (garlic (allium sativum) extract toxicity test on fibroblast cell culture) yulie emilda, els budipramana, dan satiti kuntari .................................................................... 215–219 11. hubungan tweed triangle dan posisi bibir terhadap garis estetik (relationship between tweed triangle and the lips position to esthetic line) intan oktaviona, i.g.a. wahju ardani dan achmad sjafei ....................................................... 220–225 vol 51 no 4 okt-des 2018.indd 216216 research report horizontal transmission of streptococcus mutans in children attending kindergarten retno indrawati department of oral biology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: transmission of streptococcus mutans (s. mutans), the main pathogen found in dental caries, is particularly prevalent during the first two years of infancy. numerous children are reportedly infected with s. mutans by their mothers with early initial acquisition of the condition considered to carry a higher risk of subsequent dental caries. purpose: this research aimed to examine the possibility of horizontal transmission of s. mutans in children attending surabaya-based kindergartens. methods: the number of subjects who satisfied the inclusion criteria totaled 146. dental plaque was collected for one minute with a sterile toothbrush. after completion of an isolation process, 25 s. mutans colonies were identified in tripticase cysteine yeast (tyc) media by means of morphological, microscopic and biochemical tests using api 20 strept (biomerieux france). a polymerase chain reaction with opa-2 and 13 was subsequently used to determine the genotype of s. mutans. primary data collection was completed by the administering of a questionnaire intended to elicit information regarding the gender, age, diet and medical history of subjects. results: an arbitrarily primed polymerase chain reaction (ap-pcr) fingerprint profile of the strains isolated from the subjects indicated similarities in five genotypes of s. mutans and differences in 18 genotypes. conclusion: the study indicated that certain children are similarly infected by s mutans bacteria which might be due to horizontal transmission between classmates. keywords: children; dental caries; horizontal transmission; kindergarten; streptococcus mutans correspondence: retno indrawati, department of oral biology, faculty of dental medicine universitas airlangga. jl. mayjend prof. dr. moestopo 47 surabaya 60123, indonesia. e-mail: retnoindrawati@fkg.unair.ac.id; retno_in2007@yahoo.co.id introduction during the last 20 years, a considerable body of research has confirmed that the prevalence of childhood dental caries, especially in developing countries, has increased and is considered to represent a significant child health problem. streptococcus mutans (s. mutans) constitutes the main bacterium causing dental caries whose prevalence in indonesia increases year-on-year. according to basic health research (riskesdas), the national community based research which conducted by the indonesian ministry of health, the rise was one from 43.4% in 2007 to 53.2% (a total of 93 million individuals) in 2013.1 dental caries in children impede appetite, eating patterns and appropriate sleep regimes, can cause discomfort and negatively affect health. berkowitz (2006) reported that s. mutans can be transmitted from one person to another due to the similarity of its strains in children and parents, especially mothers.2 the transmission of s. mutans is frequently reported in the research conducted as a condition probably induced by altered social relationships leading to changes in the pattern of family life, especially in large cities.3 child care, paud (education program for infants), and kindergartens are all locations where children spend the majority of their time, sharing toys and eating/drinking utensils contaminated with s. mutans.4,5 global changes in behavior have been triggered by the boom in daycare centers (tpa) where children of pre-school age spend 5-10 hours on weekdays, leading to changes in s. mutans infection patterns. research conducted by klein showed that with regard to children as many as dental journal (majalah kedokteran gigi) 2018 december; 51(4): 216–221 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 217 indrawati, dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 216–221 71% in america, 60% in sweden and 45% in china are at high risk of dental caries.6 the emergence of molecular tools over the past few decades has substantially promoted an understanding of microbial dental caries pathogenicity, thereby improving the detection of bacteria and genotypes. in addition, the etiology of dental caries currently focuses on the host and physical behavior linking dental caries with changes in microbial ecology based on physiological imbalances.6 nowadays, the arbitrarily primed polymerase chain reaction (ap-pcr) molecular method can be employed to evaluate s. mutans transmission. characterization of the s. mutans genotype by means of ap-pcr can provide the practical and reproducible data necessary to compile a catalog comprising a large number of longitudinal s. mutans isolates. the genotype data collected is expected to provide an important basis for tracking s. mutans colonization in populations at risk of caries.7 this research was intended to examine horizontal transmission of s. mutans in children attending kindergarten in surabaya through the s. mutans genotype equation. materials and methods the research reported here constituted an observational laboratory study whose subjects were randomly selected kindergarten students in surabaya who met the inclusion criteria of belonging to either gender, aged 4-6 years old and with a dmf-t 4. subjects also had to be of sound health as confirmed by their responses to a questionnaire indicating their general medical condition. moreover, they could not have undergone any treatment which had suppressed their immune response. each subject was examined by a dentist using dental operatory lights, a dental mirror and an explorer. the def-t number (d = decay, e = exfoliation, f = restoration, t = teeth) used was based on who criteria.8 isolation of streptococcus mutans bacteria the dental caries of subjects were examined and recorded. plaque samples were taken with a sterile toothbrush which was subsequently inserted into a sterile tube containing 10ml of brain heart infusion (bhib) media and transferred to the laboratory within two hours. thereafter, the plaque samples were vortexed for 30 seconds to ensure that all plaque present on the surface of the toothbrush was dissolved in the bhib media. serial depletion was then carried out with liquid media yeast numbering between 1/10 and 1/10,000. ten μl of plaque was planted on selective tyc agar media before being incubated in an anaerobic jar at 37oc for 48 hours using an anaerobic kit (an aerogen thermo science, japan) in order to facilitate microscopic identification of s. mutans. one colony, suspected of being s. mutans, was extracted from the tyc culture, re-planted in bhi media and incubated at 37oc in an anaerobic jar for 24 hours using an anaerobic kit. the results of isolation were subsequently identified microscopically. colonies suspected of being s. mutans were stained with gram dyes and examined under a light microscope at 100x magnification. macroscopic and microscopic test results were further analyzed by means of an hemolysis test. one s mutans colony was planted in blood agar media and incubated in an anaerobic jar at 37oc for 48 hours using an anaerobic kit. a biochemical and enzymatic reaction test was then conducted with an api 20 strep test to determine the validity of the previous isolation results. an api 20 strep test (biomerieux, lyons, france) was conducted in successive stages. firstly, the results of an s. mutans culture on blood agar were harvested, placed in 2ml of aquadest, agitated until homogeneous and, finally, adjusted to mcfarland standard 4. secondly, the incubation box was prepared with the wells being filled with 5ml of aquadest to maintain moisture levels. thirdly, the test strip was placed on top of the wells and 100 μl of bacterial suspension including: vp (sodium pyruvate), hp (hippuric acid), esc (esculin), pyra (pyroglutamic acid), gal ( d-galactipyranoside), ßgur (naphthol asbi-glucoronic acid), ßgal (2-naphthyl ßd-galactopyranoside), pal (2-naphthyl phosphate), lap (l-leucine-ß-naphthylamide) and adh (l-arginine) placed into ten enzymatic test wells. the 2 ml of media available in the api 20 strep kit was added to 0.5 ml of bacterial suspension and mixed until homogeneous. one hundred ul of suspension, namely: rib (d-ribose), ara (l -arabinose), man (d-mannitol), sor (d-sorbitol), lac (d-lactose), tre (d-trehalose), inu (inulin), raf (d-raffinose), amd (starch (2) and glyg (glycogen) was then deposited in each of the ten fermentation test wells. in order to prevent penetration by air, mineral oil was deposited in the wells which were then incubated for 4.5 hours at 37oc (in accordance with the manufacturer’s instructions). a single drop of vp1 and vp2 reagent was introduced into the vp and nin wells, while two drops were also added to the hip well. the enzymatic results were then reviewed after an incubation period of 4.5 hours. the reaction caused by the addition of a drop of zym a and zym b reagent subsequently added to the pyra, gal, ßgur, ß gal, pal and lap wells lead to discoloration. color changes produced by the enzymatic test were recorded after ten minutes, while the fermentation test results were reviewed after 24 hours. the resulting color changes were identified with fire-web software and assigned a score according to their nature. the number of scores was then read with the analytical profile index software, the results confirming the bacteria as belonging to the streptococcus species. all identification procedures outlined above were carried out using the s. mutans comparison (atcc 25175) as a positive control. the s. mutans isolation results were taken from one colony and replanted in bhi 37oc for 24 hours, before 500ul was extracted and stored in 20% glycerol media at -80oc for later ap-pcr examination.9 the culture of s. mutans was incubated for 16 hours (logarithmic phase) in 5 ml bhib. the number of bacteria dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 218indrawati, dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 216–221 in culture tube was measured using the mcfarland standard. after the number of bacteria reaching standard 4, the culture was centrifuged at 7,000 rpm for five minutes. after discharge the culture liquid, 5ul of lysozyme and 200 ul of pbs were added to the remaining pellets and incubated for 15 minutes at 37oc. two hundred ul binding buffer was added and 40 ul proteinase k was agitated and incubated at 70oc for ten seconds. one hundred ul isopropanol was subsequently added and vortexed for ten seconds. the sample was then inserted into a filter tube placed above the collection tube and centrifuged at 12,000 rpm for one minute. at that point, the collection tube was replaced with a new one. the samples to be used were those in the filter tube which were washed twice, first with 500 ul washing buffer centrifuged at 12,000 rpm for one minute and, subsequently, with 500 ul washing buffer before being centrifuged at 12,000 rpm for 10 seconds to remove the remaining washing buffer. the filter tube was then inserted into a sterile micro centrifuge tube, 200 ul elution buffer (previously heated to 70oc) was added, centrifuged again in 12,000 rpm for one minute. microcentrifuge tubes contained isolated dna to be stored at -20oc until used (in accordance with the high pure template preparation kit roche-germany procedure).10 determination of bacterial dna up to 10ul of the purification results of dna samples were added to 100 ul of aquadest, homogenized, and deposited in quart cuvettes to measure its absorption with ultraviolet spectrophotometers at wavelengths of 260nm and 280nm. aquadest was used as a blank. the dna purity value obtained from the calculation of the ratio a260/ a280 dna was categorized as pure if the a260/a280 ratio ranged from 1.8-2.0. twenty five s. mutans strains resulting from isolation with api-20 strep were employed for genotyping.11 ap-pcr fingerprinting was performed with two random primers, opa-02 (5 -tgccgagctg-3 ) and opa-13 (5 -cagcacccac-3 ). pcr beads ready-to-go was added to dna template, primer and sterile water producing a final volume of 25ul. a pcr tube was then inserted into the master cycler machine with hotstart at 94oc for five minutes, denaturation at 94o c for one minute, turning at 35oc for two minutes, extension at 72oc for two minutes, and 35 cycles, followed by an extra extension at 72oc for five minutes. amplification products in 2% agarose gel were analyzed by electrophoresis. staining with ethidium bromide was performed for 30 minutes prior to the gel being photographed with a red filter under uv310 illuminators and polaroid 667 black and white film.11,12 electrophoresis results can be detected in the form of a band in different lanes and will appear after completion of the coloring process. a lane can be considered as the direction of movement of the sample from the “well” gel. bands that are equidistant from the gel well in electrophoresis contain molecules that move at the same speed indicating that they are of the same size. markers which are a mixture of molecules of different sizes can be used to determine the size of a molecule in a sample band by electrophoresing the markers in a strip in the gel parallel to the sample. the bands in the mark lanes can be compared with the sample bands to determine their size. the distance of the band from the gel well is inversely proportional to the logarithm of the molecular size. the molecular weight and mobility of electrophoresis in polyacrylamide gels can, consequently, be measured using the mobility rate (mr) or rf formula in which the distance traveled by the compound is divided by the distance traveled by the solvent to enable calculation of the band distance from the well as follows: y = 0.322 (x) + 4.753 (y = log bp. x = tape distance from the well).13,14 results twenty five samples were identified as s. mutans. the genotyping results of the 25 sample analyzed using the appcr method can be seen in figure 1. as positive control we use s mutans atcc 1275 as a guide (lane 15 and 16). in figure 3 samples 12 and 13 were exclude from the results due to lack of s. mutans detection accuracy (api strep 20 showing only 50%). twenty five s. mutans samples were isolated from students attending 15 kindergardens in surabaya. in figure 2, there were three types of s. mutans strains that are similar based on visual determination, type a (samples 3 and 8), b (samples 4, 6 and 9), and c (samples 12 and 13), while in figure 3 two types of s. mutans were shown based on visual determination, types d (samples 6 and 7) and e (samples 9, 10, and 11). based on the type of similarity, it is known that in type a, samples 3 and 8 came from kindergartens in one area of east surabaya, while type b sample 4 had similarities with samples 6 and 9, with the s. mutans isolated originating in the same area in the west of the city. s. mutans suspected was transmitted horizontally between children who were classmates. s. mutans also featured type c with which sample 12 demonstrated similarity to sample 13. s. mutans was isolated from kindergarten students in the same class in south surabaya. similarly, type d (samples 6 and 7) indicated that s. mutans was isolated from students in the same class in central surabaya. meanwhile, type e (samples 9, 10 and 11) showed that s. mutans was isolated from kindergarten students in the same class in north surabaya (figure 4). in addition, the bands 1, 2, 5, 7, 10, 11 and 14 in figure 1 and bands 1, 2, 3, 4, 5 and 8 in figure 3 are s. mutans derivative species which are not similar on the basis of visual comparison. thirteen s. mutans strains came from different kindergartens and classes. except for those in sample 11 and 14, kindergarten students based in south surabaya are shown in figure 1. samples 2 and 5, featuring students from the surabaya center, are shown in figure 2. the students in samples 11 and 14, and samples 2 and 5 were all members of the same class. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 219 indrawati, dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 216–221 figure 3. ap-pcr fingerprint profiles of the 11 s. mutans strains that demonstrated the same two s. mutans genotypes; d (samples 6 and 7), e (samples 9, 10 and 11). samples 12 and 13 were exclude from the results due to lack of s. mutans detection accuracy (api strep 20 showing only 50%). figure 1. ap-pcr fingerprint profile of the 14 s. mutans strains that demonstrated the same s. mutans genotype: a (samples 3 and 8), b (samples 4, 6 and 9) and c (samples 12 and 13). figure 2. grouping results of ap-pcr fingerprint profiles of the samples of s. mutans strains that demonstrated the same three genotypes of s. mutans: a (samples 3 and 8), b (samples 4, 6 and 9) and c (samples 12 and 13). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 220indrawati, dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 216–221 figure 4. grouping ap-pcr fingerprint profiles of the s. mutans strains demonstrating the same two s. mutans genotypes, d (samples 6 and 7), e (samples 9,10 and 11). discussion dental caries constitute a communicable disease. considerable research has been conducted into the initial acquisition of s. mutans and its role as the main cause of dental caries. mother to child transmission has been identified as the predominant means of early acquisition of s. mutans in infants. however, mothers are not the only source of s. mutans transmission. according to tedjosasongko, s. mutans can be transmitted to children through intraand extrafamilial transmission.5 in this research, 18 different strains of s. mutans were identified. the visual data depicting the comparison results of ap-pcr bands indicated horizontal transmission between non-genetically related 4 to 6-year-olds at kindergarten in surabaya. eighteen strains of s. mutans were identified. there were five similar strains in five pairs of children who were classmates at 15 schools. the discovery of five pairs of children who demonstrated the same s. mutans strain confirmed the occurrence of horizontal transmission. the kindergarten students from south surabaya forming samples 12 and 13 are shown in figure 1. the kindergarten students from central surabaya forming samples 6 and 7 who, it transpired, were classmates are shown in figure 3. based on visual comparison, there were no similar s. mutans strains present in the electrophoresis tape, strongly suggesting the absence of horizontal transmission of s. mutans. gamboa (2010) argues that low transmission rates can occur due to short contact time and less intimate contact between individuals.13,15 furthermore, research conducted by klein found that s. mutans transmission can be transitory or permanent, strongly influenced by various factors, including: behavior, the frequency of salivary contact, s. mutans levels present in individuals, culture, environmental conditions (socioeconomic and/or education), individual vulnerabilities (e.g. sucrose consumption) and the window for potential infection occurring at the age of 8.64 months.5,6 another piece of previous research conducted by berkowitz which involved 786 one-year olds also identified high caries risk factors for such children such as s. mutans infection, fluoride exposure, eating habits and oral hygiene (ohi).2 the results of this research can be detected from crosstabulation between the oral hygiene index (ohi) and age, indicating that the ohi is in poor category (no data shown). the ap-pcr method was then applied to investigate s. mutans transmission and genotypic determination. the primary choices (opa 05 and opa 13) were used in this study since these two ap-pcr primers, based on previous investigations, can increase sensitivity and specificity to identify various streptococci.13 s. mutans transmission will indirectly increase the prevalence of dental caries and other problems. cases of dental caries in children such as early childhood caries (ecc) will have an impact on various aspects of a child’s life, including: physical, psychological and social. consequently, ecc must be addressed immediately and requires cooperation from the child’s parents since this case requires comprehensive, high quality care. unfortunately, in this research, no data or samples were collected from mothers, teachers or caregivers. as a result, the possibility of s. mutans infection from these sources remains unknown. this study confirmed the possibility of horizontal transmission between kindergarten students in surabaya aged 4-6 years. however, further research is required to identify several risk factors strongly associated with horizontal transmission of cariogenic bacteria among children of different ages in order to break the infection chain in children as part of the prevention of dental caries in indonesia.5 the study showed that some children share similar s mutans bacteria possibly resulting from horizontal transmission between classmates. references 1. kementerian kesehatan republik indonesia. profil kesehatan indonesia tahun 2017. jakarta: kementerian kesehatan ri; 2018. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 221 indrawati, dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 216–221 2. berkowitz rj. mutans streptococci: acquisition and transmission. pediatr dent. 2006; 28(2): 106–9; discussion 192-8. 3. milgrom p, riedy ca, weinstein p, tanner ac, manibusan l, bruss j. dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6to 36-month-old children. community dent oral epidemiol. 2000; 28(4): 295–306. 4. doméjean s, zhan l, denbesten pk, stamper j, boyce wt, featherstone jd. horizontal transmission of mutans streptococci in children. j dent res. 2010; 89: 51–5. 5. tedjosasongko u, kozai k. initial acquisition and transmission of mutans streptococci in children at day nursery. asdc j dent child. 2002; 69(3): 284–8, 234–5. 6. klein mi, florio fm, pereira ac, hofling jf, goncalves rb. longitudinal study of transmission, diversity, and stability of streptococcus mutans and streptococcus sobrinus genotypes in brazilian nursery children. j clin microbiol. 2004; 42(10): 4620–6. 7. cheon k, moser sa, wiener hw, whiddon j, momeni ss, ruby jd, cutter gr, childers nk. characteristics of streptococcus mutans genotypes and dental caries in children. eur j oral sci. 2013; 121(3 pt 1): 148–55. 8. world health organization. oral health surveys: basic methods. 5th ed. france: world health organization; 2013. 9. arbique jc, poyart c, trieu-cuot p, quesne g, carvalho m d. gs, steigerwalt ag, morey re, jackson d, davidson rj, facklam rr. accuracy of phenotypic and genotypic testing for identification of streptococcus pneumoniae and description of streptococcus pseudopneumoniae sp. nov. j clin microbiol. 2004; 42(10): 4686–96. 10. soemantadiredja yh, satari mh. isolasi gen kariogenik gtf bc streptococcus mutans dari plak gigi anak (the isolation of streptococcus mutans cariogenic gtf bc gene from children’s tooth plaque). dent j (maj ked gigi). 2005; 38(3): 151–3. 11. fatchiyah, widyarti s, arumningtyas el, permana s. teknik analisis biologi molekuler. malang: universitas brawijaya; 2012. p. 1–38. 12. javed m, butt s, ijaz s, asad r, wahid a, khan aa. mother-child pair transmission of streptococcus mutans; pcr based study in urban population of pakistan. saudi j pathol microbiol. 2016; 1: 4–9. 13. gamboa f, chaves m, valdivieso c. genotypic profiles by ap-pcr of streptococcus mutans in caries-active and caries-free preschoolers. acta odontol latinoam. 2010; 23(2): 143–9. 14. napimoga mh, höfling jf, klein mi, kamiya ru, gonçalves rb. tansmission, diversity and virulence factors of sreptococcus mutans genotypes. j oral sci. 2005; 47(2): 59–64. 15. adinda c, tedjosasongko u, wibowo tb. usia saat inisial akuisisi streptococcus mutans dan jumlah erupsi gigi sulung pada anak (initial acquisition age of mutans streptococci and number of erupted primary teeth in children). dent j (maj ked gigi). 2014; 47(4): 202–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p216–221 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p216-221 issn 1978 3728volume 47, number 1, march 2014 editorial board of dental journal (majalah kedokteran gigi) sk: 059/un3.1.2/2014 january 2nd– december 31st, 2014 patron: dean of faculty of dental medicine universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 47, number 1, march 2014: prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry-universitas airlangga) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) prof. dr. jenny sunariani, drg., ms (oral biology – universitas airlangga) achmad gunadi, drg., ms., ph.d., sp.pros. (prosthodontics – universitas jember) dr. rini devijanti, drg., m.kes. (oral biology – universitas airlangga) kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) david buntoro k, drg., mds., sp.bm (maxillofacial surgery – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 206708019-204225738 contents page printed by: airlangga university press. (rk 231/07.14/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 47, number 1, march 2014 issn 1978 3728 1. combination of aloe vera and xenograft induction on decreasing of nf-kb of tooth extraction socket preservation in cavia cobaya utari kresnoadi and retno pudji rahayu ..................................................................................... 1–6 2. the role of hsp60, cd-8 and ifn-γ in immunopathobiogenesis of periapical granuloma in dental caries risya cilmiaty and mandojo rukmo ............................................................................................ 7–12 3. the expressions of nf-kb and tgfb-1 on odontoblast-like cells of human dental pulp injected with propolis extracts ira widjiastuti, ketut suardita and widya saraswati ................................................................. 13–18 4. spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket rostiny, mefina kuntjoro, ratri maya sitalaksmi and sherman salim .................................... 19–24 5. alkaline phosphatase expression during relapse after orthodontic tooth movement pinandi sri pudyani, widya asmara, ika dewi ana and tita ratya utari ............................... 25–30 6. antimicrobial proteins of snail mucus (achatina fulica) against streptococcus mutans and aggregatibacter actinomycetemcomitans herluinus mafranenda dn, indah listiana kriswandini and ester arijani r ......................... 31–36 7. the effect of immersion in soda on nano hybrid composite resin discoloration m. chair effendi, yuli nugraeni and rizki widya pratiwi ......................................................... 37–40 8. the role of heat shock protein 27 (hsp 27) as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture sri wigati mardi mulyani, ernie maduratna setiawati, erma safitri dan eha renwi astuti . 41–44 9. uji sensitivitas dan spesifisitas perangkat lunak “prediktor karies anak” (the sensitivity and specificity test of pediatric caries predictor software) quroti a’yun, julita hendrartini, al. supartinah santoso, dan lukito edi nugroho .............. 45–51 10. pengaruh posisi dan fraksi volumetrik fiber polyethylene terhadap kekuatan fleksural fiber reinforced composite (the effect of position and volumetric fraction polyethylene fiber on the flexural strength of fiber reinforced composite) catur septommy, widjijono dan rini dharmastiti ...................................................................... 52–56 11. oral hygiene and number of oral mucosal lesion correlate with oral health-related quality of life in elderly communities dewi agustina ................................................................................................................................... 57–61 p-issn: 1978-3728 e-issn: 2442-9740 volume 53, number 2, june 2020 editorial team of dental journal (majalah kedokteran gigi) sk: 07/un3.1.2/2020 january 2nd – december 31st, 2020 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: saka winias, drg., m.kes., sp.pm (department of oral medicine, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors eric priyo pasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); nastiti faradilla (department of oral and maxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia) . peer-reviewers irna sufiawati (department of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); masniari novita (deparment of dental forensic, faculty of dentistry, universitas jember, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); tuti kusumaningsih (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); priyawan rachmadi (department of dental materials, faculty of dental medicine, universitas airlangga, indonesia); maretaningtyas dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); indah listiana kriswandini (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); nurina febriyanti ayuningtyas (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). kampus c unair, mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. e-mail: adm@aup.unair.ac.id volume 53, number 2, june 2020 p-issn: 1978-3728 e-issn: 2442-9740 1. effects of alkalisation and volume fraction reinforcement of bombyx mori silk fibre on the flexural strength of dental composite resins dyah anindya widyasrini and siti sunarintyas ........................................................................... 57–61 2. a comparative study of the e. faecalis antibiofilm efficacy of photoactivated curcumin, chlorophyll and riboflavin riski setyo avianti, sri kunarti and ari subiyanto ..................................................................... 62–66 3. nickel release and the microstructure of stainless steel orthodontic archwire surfaces after immersion in detergent and non-detergent toothpaste: an in vitro study hilda fitria lubis, kholidina imanda harahap and dina hudiya nadana lubis .................... 67–70 4. effectiveness of light-emitting diode exposure on photodynamic therapy against enterococcus faecalis: in vitro study nanik zubaidah, agus subiwahjudi, dinda dewi artini and karina erda saninggar ............ 71–75 5. the combination of carbonate hydroxyapatite and human β-defensin 3 to enhance collagen fibre density in periodontitis sprague dawley rats ika andriani, edy meiyanto, s. suryono and ika dewi ana ....................................................... 76–80 6. during and post covid-19 pandemic: prevention of cross infection at dental practices in country with tropical climate rikko hudyono, taufan bramantoro, benni benyamin, irfan dwiandhono, pratiwi soesilawati, aloysius pantjanugraha hudyono, wahyuning ratih irmalia and nor azlida mohd nor ............................................................................................................... 81–87 7. efficacy of topical hydrogel epigallocatechin-3-gallate against neutrophil cells in perforated dental pulp kun ismiyatin, ari subiyanto, michelle suhartono, paramita tanjung sari, olivia vivian widjaja and ria puspita sari ............................................................................................. 88–92 8. correlation between age and dental arch dimension of javanese children atiek driana rahmawati,iwa sutardjo rus sudarso, dibyo pramono and eggi arguni ........ 93–98 9. effects of manufacturing methods of abalone gel as a desensitisation material on the closing of dentinal tubules sri budi barunawati, wayan tunas artama, suparyono saleh, siti sunarintyas and yosi bayu murti ........................................................................................................................ 99–106 10. the potential of ethanolic extract of moringa oleifera leaves on hsf1 expression in oral cancer induced by benzo[a]pyrene vania syahputri, theresia indah budhy and bambang sumaryono .......................................... 107–110 11. comparison of thrombocyte counts during the post-oral administration of aspirin and the holothuria scabra ethanol extract in wistar rats (rattus norvegicus) dian mulawarmanti and rima parwati sari ................................................................................. 111–114 contents page 186 volume 47, number 4, december 2014 prevalensi early childhood caries dan severe early childhood caries pada anak prasekolah di gunung anyar surabaya (the prevalences of early childhood caries and severe early childhood caries in preschool children at gunung anyar surabaya) rahel wahjuni sutjipto, herawati, dan satiti kuntari departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: early childhood caries (ecc) is a serious health problem especially in young children. frequent night time bottle feeding with milk and prolong breast-feeding are reported to be the possible cause. purpose: the purpose of study was to determine the prevalence of ecc and severe early childhood caries (secc) in children at preschool gunung anyar district surabaya. methods: the subjects were 65 children consist of children aged 6 months (5 children), 1 year (8 children), 2 years (24 children), and 3 years (28 children). all tooth surface is evaluated. if there was one or more dmfs, it was indicated as ecc, whereas if there was one or more dmfs on smooth surfaces, then indicated as secc. results: no caries has found in 6 months and 1 year old children. the higher prevalence of ecc and secc is in 3 years old children. the most caries was found on mesial maxillary central incisors. conclusion: this study shows that the prevalence of ecc in group of children aged 6 months 3 years at gunung anyar surabaya was 30.8 % , while the prevalence was 29.2 % secc . key words: early childhood caries, severe early childhood caries, bottle feeding, prevalence, preschool children abstrak latar belakang: karies anak usia dini merupakan masalah kesehatan yang serius terutama di kalangan anak-anak. sering mengkonsumsi susu melalui botol pada malam hari dan pemberian asi yang berkepanjangan dilaporkan sebagai faktor penyebab. tujuan: penelitian ini bertujuan untuk meneliti prevalensi ecc dan secc pada kelompok anak di paud gunung anyar surabaya. metode: subyek penelitian ini adalah 65 anak yang terdiri dari anak usia 6 bulan (5 anak), 1 tahun (8 anak), 2 tahun (24 anak), dan 3 tahun (28 anak). semua permukaan gigi dievaluasi. apabila terdapat 1 atau lebih dmfs, maka diindikasikan sebagai ecc, sedangkan apabila terdapat 1 atau lebih dmfs pada permukaan gigi yang halus, maka diindikasikan sebagai secc. hasil: tidak ditemukan karies pada kelompok anak usia 6 bulan hingga 1 tahun. prevalensi tinggi ecc dan secc ditemukan pada kelompok anak usia 3 tahun. area gigi yang paling sering terkena karies adalah bagian mesial geligi insisif sentral rahang atas. simpulan: penelitian ini menunjukkan bahwa prevalensi ecc pada kelompok anak usia 6 bulan-3 tahun dikawasan gunung anyar surabaya adalah 30,8%, sedangkan prevalensi secc adalah 29,2%. kata kunci: early childhood caries, severe early childhood caries, minum susu botol, prevalensi, anak prasekolah korespondensi (correspondence): rahel wahjuni sutjipto, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: wsrahel@yahoo.com research report 187sutjipto, et al.: prevalensi early childhood caries dan severe early childhood caries pendahuluan early childhood caries (ecc) merupakan masalah kesehatan gigi paling utama terjadi pada bayi dan anakanak balita, yang dapat mempengaruhi kesehatan dan perkembangan gigi anak. prevalensi dan keparahan karies gigi pada anak-anak di bawah usia 5 tahun di beberapa negara cukup tinggi. di indonesia, prevalensi karies pada anak usia 3-5 tahun terus meningkat. pada tahun 2001, prevalensi karies pada anak-anak usia 3-5 tahun di dki jakarta adalah 81,2%.1 prevalensi karies pada anakanak balita di indonesia adalah sekitar 90,05%. karena prevalensi tinggi tersebut dapat mempengaruhi kualitas hidup anak-anak serta memiliki potensi resiko karies gigi sulung yang tinggi, maka ecc merupakan kondisi yang paling serius yang dapat merugikan anak-anak. pengalaman karies pada bayi dan anak-anak di bawah usia 6 tahun (71 bulan ke bawah) disebut dengan ecc, yang merupakan adanya satu atau lebih kerusakan, kehilangan, dan tumpatan pada permukaan gigi sulung. pada usia 1 tahun, beberapa anak telah memiliki lesi karies, dan pada usia 3 tahun, sekitar 30% anak memiliki karies (termasuk lesi karies tanpa kavitas). early childhood caries biasanya pertamakali melibatkan permukaan labial dan palatal gigi insisif sulung rahang atas. sebagaimana kerusakan gigi berlanjut, maka karies tersebut dapat melibatkan gigi molar sulung rahang atas bahkan seluruh gigi sulung.2 gigi insisif rahang bawah jarang terkena karies, kecuali dalam kasus yang paling parah. anak – anak sering mengalami kerusakan berat pada gigi insisif rahang atasnya.3 pada usia di bawah 3 tahun, segala tanda karies pada permukaan gigi yang halus diindikasikan sebagai severe early childhood caries (secc). menurut american academy pediatricdentistry (aapd), 70% anak-anak usia 2-5 tahun ditemukan karies. selama bertahun-tahun telah diketahui bahwa setelah gigi sulung mulai erupsi, konsumsi susu dengan botol saat tidur pada malam maupun siang hari yang terlalu sering dapat menyebabkan karies anak usia dini. secara klinis, secc muncul pada anak usia 2, 3, atau 4 tahun dengan mengikuti pola dan bentuk tertentu yang khas. pengalaman karies ini berhubungan dengan faktor sosial dan perilaku lain yang ada di dalam keluarga. para orangtua sering memberikan pola makan yang tidak tepat, yaitu susu atau minuman yang mengandung gula diberikan saat anak berada di tempat tidur, sehingga ketika mereka tertidur, maka cairan minuman akan menggenang pada permukaan gigi rahang atas (gigi anterior rahang bawah biasanya terlindungi oleh lidah sehingga jarang terkena). dapat terlihat bahwa mikroorganisme kariogenik dapat berkembang biak di dalam rongga mulut akibat cairan minuman yang mengandung karbohidrat tersebut. aliran saliva menurun selama anak tidur, sehingga clearance saliva terhadap cairan minuman pada rongga mulut juga lambat.4 penelitian yang dilakukan ini bertujuan untuk meneliti prevalensi ecc dan secc pada anak prasekolah di wilayah kecamatan gunung anyar surabaya. bahan dan metode jenis penelitian yang digunakan adalah penelitian analitik observasional. penelitian ini dilakukan di sekolah pendidikan anak usia dini (paud) wilayah kecamatan gunung anyar surabaya. subyek penelitian ini adalah 65 anak yang terdiri dari anak usia 6 bulan (5 anak), 1 tahun (8 anak), 2 tahun (24 anak), dan 3 tahun (28 anak). metode yang digunakan dalam pemeriksaan gigi adalah indeks ecc dan secc. permukaan gigi yang dievaluasi adalah setiap permukaan (mesial, distal, facial, lingual, oklusal) dari setiap gigi yang tampak dalam rongga mulut anak. apabila terdapat 1 atau lebih dmfs, maka diindikasikan sebagai ecc, sedangkan apabila terdapat 1 atau lebih dmfs pada permukaan gigi yang halus, maka diindikasikan sebagai secc. hasil pemeriksaan dihubungkan dengan kuesioner orangtua. hasil berdasarkan data penelitian prevalensi ecc dan secc, maka didapatkan hasil sebagai berikut, pada kelompok anak usia 6 bulan dengan jumlah sampel 5 anak dan kelompok anak usia 1 tahun dengan jumlah sampel 8 anak tidak ditemukan karies. pada usia 2 tahun, dari jumlah sampel 24 anak, 11 diantaranya memiliki karies pada sisi mesial insisif sentral rahang atas, sedangkan pada usia3 tahun, dari jumlah sampel 28 anak, semua memiliki karies pada sisi mesial insisif sentral rahang atas (gambar 1 dan 2). dari 24 anak pada kelompok usia 2 tahun yang diteliti, 8 (33,3%) anak diantaranya mengalami ecc, sedangkan 3 (12,5%) anak mengalami secc, dan 13 (54,2%) anak tidak ditemukan karies. pada kelompok anak usia 3 tahun, 28 anak diteliti, 12 (42,9%) anak mengalami ecc, sedangkan 16 (57,1%) anak mengalami secc (gambar 3). prevalensi ecc pada kelompok anak usia 6 bulan-3 tahun adalah 30,8%; sedangkan prevalensi secc adalah 29,2%. pada anak usia 6 bulan, tidak terdapat anak-anak yang memiliki kebiasaan mengonsumsi makanan manis, namun semua anak minum susu saat tidur. pada anak usia 1 tahun, 2 (25%) anak memiliki kebiasaan mengonsumsi makanan manis, dan semua anak memiliki kebiasaan minum susu saat tidur. pada anak usia 2 tahun, 20 (83,3%) anak memiliki kebiasaan mengonsumsi makanan manis, dan 23 (95,8%) anak memiliki kebiasaan minum susu saat tidur. pada kelompok anak usia 3 tahun, 26 (92,9%) anak memiliki kebiasaan mengonsumsi makanan manis, dan 24 (87,5%) anak memiliki kebiasaan minum susu saat tidur. hasil kuesioner secara keseluruhan menunjukkan bahwa 77% orangtua berpendidikan terakhir perguruan tinggi (termasuk d3) dengan 15 (23%) orang memiliki anak dengan ecc dan 16 (25%) orang memiliki anak dengan secc, dan 23% berpendidikan terakhir sma dengan 5 (8%) orangtua memiliki anak dengan ecc dan 3 (5%) orangtua memiliki anak dengan secc. berdasarkan pekerjaan orangtua secara keseluruhan, 43% orangtua bekerja sebagai ibu rumah tangga, dengan 14 diantaranya memiliki anak dengan ecc, 188 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 186–189 gambar 2. jumlah anak kelompok usia 6 bulan 3 tahun dengan lokasi karies pada permukaan masing-masing gigi rahang bawah. gambar 1. jumlah anak kelompok usia 6 bulan-3 tahun dengan lokasi karies pada permukaan masing-masing gigi rahang atas. gambar 3. jumlah anak yang terkena ecc dan secc pada kelompok usia 6 bulan -3 tahun. dan 7 orang lainnya memiliki anak dengan secc; 21,5% bekerja sebagai wiraswasta engan 7 orang diantaranya memiliki anak dengan ecc dan 2 orang memiliki anak dengan secc, 20% bekerja sebagai pegawai swasta, 2 diantaranya memiliki anak dengan ecc, dan 1 orang memiliki anak dengan secc, dan 25,5% bekerja sebagai pegawai negeri, 4 diantaranya memiliki anak dengan secc, dan 2 orang memiliki anak dengan ecc. dengan demikian, keparahan ecc dan secc pada kelompok anak usia 6 bulan3 tahun cukup tinggi. pada usia 2 dan 3 tahun, permukaan gigi yang paling banyak ditemukan karies adalah sisi mesial gigi insisif sentral rahang atas. pembahasan pada anak-anak yang diteliti, permukaan gigi yang paling banyak ditemukan karies adalah sisi mesial gigi insisif sentral rahang atas. hal ini disebabkan oleh kebiasaan minum susu saat sedang tidur sehingga ketika mereka tertidur, maka cairan minuman akan menggenang pada permukaan gigi rahang atas. gigi anterior rahang bawah biasanya terlindungi oleh lidah sehingga jarang terkena. berdasarkan kuesioner orang tua, prosentase terbesar pekerjaan orangtua adalah sebagai ibu rumah tangga, dan berpendidikan akhir di perguruan tinggi. sehingga dapat dikatakan bahwa pekerjaan dan pendidikan dari orangtua tidak mempengaruhi ecc dan secc. berdasarkan kuesioner, 15% orangtua dengan berpendidikan rendah memiliki anak dengan ecc, dan 60% orangtua yang 189sutjipto, et al.: prevalensi early childhood caries dan severe early childhood caries berpendidikan tinggi memiliki anak dengan ecc maupun secc. hal ini tergantung dari perawatan dan pemeliharaan orangtua terhadap gigi anak. selain itu, pekerjaan orangtua sebagai ibu rumah tangga, di mana mereka memiliki waktu luang yang lebih banyak dengan anak-anak, memiliki anakanak yang juga terkena karies. hal ini mungkin disebabkan karena pemberian pola makan yang tidak tepat. sebanyak 92,3% orangtua membiarkan anaknya mengonsumsi minuman manis atau susu pada malam hari hingga tertidur pulas dan 74% orangtua memberikan makanan manis pada anak, sehingga pola makan (diet) anak kurang teratur. ecc merupakan hasil interaksi antar faktor yang terlibat dalam karies gigi (bakteri kariogenik, diet karbohidrat, dan faktor host). faktor diet mencakup seringnya mengkonsumsi minuman yang mengandung karbohidrat fermentasi (laktosa, fruktosa, dan lain-lain), khususnya dengan botol (dot) saat tidur. ketika botol susu diberikan pada bayi saat sedang tidur, cairan minuman tersebut akan menggenang di sekitar gigi insisif rahang atas dan dapat menyebabkan perkembangan kerusakan struktur gigi yang parah dan cepat. american academy of pediatric dentistry tidak merekomendasikan bayi mengonsumsi minuman saat tidur dengan botol dan pemberian asi pada malam hari harus dihindari setelah gigi sulung pertama erupsi. penggunaan botol susu harus dihentikan saat usia 12 hingga 14 bulan.5 makanan yang menempel pada gigi akan lebih mungkin untuk meningkatkan produksi asam dan memberikan lingkungan bagi pertumbuhan bakteri dan dekalsifikasi enamel. faktor kariogenik pada makanan yang dikonsumsi juga mencakup ph dari makanan tersebut. seringnya mengonsumsi makanan kariogenik memiliki hubungan erat dengan resiko perkembangan karies. hasil kuesioner juga menunjukkan bahwa anak-anak sering mengonsumsi makanan yang mengandung gula (manis). semakin sering kontak dengan gula saat waktu makan dan sering mengonsumsi makanan ringan, maka akan mengakibatkan gigi semakin rentan, dan memungkinkan untuk membersihkan mulut dalam waktu yang lama, sehingga akhirnya anak-anak kurang dapat membersihkan gigi secara maksimal. terdapat anak-anak yang tidak mengonsumsi makanan manis dan tidak minum susu atau minuman manis lainnya saat malam hari, namun memiliki karies. hal ini mungkin disebabkan faktor kerentanan gigi (host), di mana enamel belum matang setelah erupsi, dan adanya kerusakan enamel seperti hypoplasia.6 penelitian ini menunjukkan bahwa permukaan gigi yang paling banyak ditemukan karies adalah sisi mesial insisif sentral rahang atas, dengan jumlah 11 (45,8%) anak-anak usia 2 tahun, dan semua anak-anak usia 3 tahun. prevalensi ecc pada kelompok anak usia 6 bulan-3 tahun adalah 30,8%; sedangkan prevalensi secc adalah 29,2%. daftar pustaka 1. sugito fs, djoharnas h, darwita rr. breast feeding and early childhood caries (ecc) severity of children under three years old in dki jakarta. 2008. 12(2): 86-91. 2. saraf s. textbook of oral pathology. new delhi, india: jaypee brothers medical publishers; 2006. 3. crain ef, gershel j. clinical manual of emergency pediatrics. new york: cambridge university press; 2010. p. 75. 4. mcdonald re, avery dr, dean ja. dentistry for thechild and adolescence. st. louis: mosby; 2004. 5. pinkham jr, casamassimo ps, mctigue dj, fields hw, nowak aj. pediatric dentistry: infancy through adolescence. fourth edition. st. louis: elsevier saunders; 2005. p. 320-474. 6. muthu ms, sivakumar n. pediatric dentistry: principles and practice. india: elsevier; 2009. p-issn: 1978-3728 e-issn: 2442-9740 volume 54, number 3, september 2021 editorial team of dental journal (majalah kedokteran gigi) sk: 17/un3.1.2/2021 january 4 – december 31, 2021 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: muhammad dimas aditya ari, drg., m.kes (department of prosthodontics, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material sciences and technology, faculty of dental medicine, universitas airlangga, indonesia); udijanto tedjosasongko (department of pediatric dentistry, faculty of dental medicine, universitas airlangga). managing editors ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); alexander patera nugraha (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); beshlina fitri widayanti (department of forensic odontology, faculty of dental medicine, universitas airlangga, indonesia); astari puteri (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); aulia ramadhani (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); nastiti faradilla ramadhani (department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers ida ayu evangelina (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); arlette suzy setiawan (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); irna sufiawati (deparment of oral medicine, faculty of dentistry, universitas padjadjaran, indonesia); retno widayati (deparment of orthodontics, faculty of dentistry, universitas indonesia, indonesia); niswati fathmah rosyida (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); trimurni abidin (deparment of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); ricca chairunnisa (deparment of prostodontics, faculty of dentistry, universitas sumatera utara, indonesia); mei syafriadi (deparment of biomedical sciences, faculty of dentistry, universitas jember, indonesia); sianiwati goenharto (vocational faculty, universitas airlangga, indonesia); diah savitri ernawati (deparment of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); ida bagus narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); retno pudji rahayu (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral and maxillofacial pathology, faculty of dental medicine, universitas airlangga, indonesia); rini devijanti ridwan (department of oral biologyy, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira sumarta (department of oral an maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); ratri maya sitalaksmi (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); dini setyowati (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); tania saskianti (department of pediatric dentistry, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine, universitas airlangga jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia phone. +62 31 5039478/5030255. fax. +62 31 5039478/5020256 email: dental_journal@fkg.unair.ac.id; website: https://e-journal.unair.ac.id/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 printed by: airlangga university press. (rk. 310/07.19/aup-a5e). campus c unair mulyorejo surabaya 60115, indonesia. phone. +62 31 5992246, 5992247, fax. +62 31 5992248. email: adm@aup.unair.ac.id volume 54, number 3, september 2021 p-issn: 1978-3728 e-issn: 2442-9740 1. formula milk increases lactoferrin levels in 7–9 years old children luthfiani, dwi suryanto and suzanna sungkar ........................................................................... 113–118 2. evaluation of osteogenic properties after application of hydroxyapatite-based shells of portunus pelagicus michael josef kridanto kamadjaja, alya nisrina sajidah gatia, agtadilla novitananda, lintang maudina, harry laksono, agus dahlan, bambang agustono satmoko tumali and muhammad dimas aditya ari ................................................................................................ 119–123 3. the possibility of polymorphonuclear leukocyte activation in dental socket healing by freeze-dried aloe vera induction pratiwi soesilawati, ester arijani rachmat, ira arundina and nita naomi ............................. 124–127 4. the effect of x-ray irradiation to the formation of polychromatic erythrocyte cell micronucleus in wistar rats (rattus norvegicus) eha renwi astuti, hutojo djajakusuma, indeswati diyatri and nastiti faradilla ramadhani ........................................................................................................................................ 128–131 5. analysis of soft tissue facial profiles of chinese students at w.r. supratman 1 and 2 high schools in medan using linear and angular measurements hilda fitria lubis and maureen olivia ......................................................................................... 132–136 6. comparison of rat tooth eruption in rats born from diabetic mothers salsabila qotrunnada, dina z. ummah and mei syafriadi ......................................................... 137–142 7. malocclusion with posterior unilateral crossbite affects superficial masseter and anterior temporal muscle activity during mastication yona pricilia anggi siregar, christnawati and darmawan sutantyo ........................................ 143–149 8. ethanol extract of imperata cylindrica leaves inhibits proliferation and migration of hsc-3 cell lines moehamad orliando roeslan and gabriella tasha ..................................................................... 150–154 contents original articles page 9. the treatment of covid tongue in an isolation unit dwi setianingtyas, nafiah, cane lukisari, paulus budi teguh, felicia eda haryanto and erni marlina ............................................................................................................................. 155–159 10. oral rehabilitation using immediate implant placement in mandibular lateral incisors – a case report nila sari, abil kurdi, bambang agustono satmoko tumali and muhammad dimas aditya ari ......................................................................................................................................... 160–164 case reports 11. enamel remineralisation-inducing materials for caries prevention sri kunarti, widya saraswati, dur muhammad lashari, nadhifa salma and tasya nafatila ........................................................................................................................... 165–168 review article 44 dental journal (majalah kedokteran gigi) 2022 march; 55(1): 44–48 case report the aesthetic management of a midline diastema with direct composite using digital smile design, putty index and button shade technique: a case report nirawati pribadi1, sukaton1, galih sampoerno1, sylvia2, hendy jaya kurniawan2, maya safitri3, rahmadanty mustika3 1department of conservative dentistry, faculty of dental medicine, universitas airlangga, surabaya, indonesia 2resident of conservative dentistry department, faculty of dental medicine, universitas airlangga, surabaya, indonesia 3undergraduate student, faculty of dental medicine, universitas airlangga, surabaya, indonesia abstract background: a diastema is the distance or space between two or more adjacent teeth. this abnormality can interfere with the aesthetics of a patient, and 97% of diastemas occur in the maxilla. various treatments can be performed for diastema closure in patients, one of which is composite resin restoration. purpose: to explain the aesthetic procedure for diastema closure. case: a 20-year-old female patient presented with complaints of the distance between her anterior teeth (class i angle occlusion with normal overjet and overbite). the labial frenum associated with the diastema was normal in size and position. the patient was not amenable to invasive procedures. case management: management of midline diastema closure using the direct composite technique with dsd, the putty index method and button shade technique. conclusion: the closure of a midline diastema with direct composite using dsd, the putty index method and button shade technique provides aesthetic results with less cost and time due to the absence of laboratory procedures. keywords: anterior teeth; diastema closure; direct composite correspondence: nirawati pribadi, departement of conservative dentistry, faculty of dental medicine, universitas airlangga. jl. mayjen prof. dr. moestopo no. 47, surabaya, 60132, indonesia. email: nirawati-p@fkg.unair.ac.id introduction a diastema is the distance or space between two or more adjacent teeth. the midline diastema is the distance or space between the first incisors of the maxilla or mandible.1 the presence of a midline diastema can be of special concern to patients, particularly with regard to aesthetics. one study found that 97% of maxillary midline diastemas was more common than mandibular. the aesthetic appearance of the teeth is part of the overall profile that is closely related to facial aesthetics because facial symmetry and midline coordination are important criteria for achieving facial alignment and balance.2,3 the aetiology of a diastema is considered to be multifactorial and include various factors of influence, such as the labial frenulum, microdontia, mesiodens, post lateral incisors, agenesis and cysts in the midline area. in addition, a midline diastema can be caused by habits such as thumb sucking, tongue thrusting, and/or lip sucking, dental malformations, genetics, proclination in the maxillary incisor, jaw discrepancy and an imperfect fusion of the interdental septum. these spacings can cause an unaesthetic smile, phonetic disorders and hindrance in maintaining oral hygiene.4–6 various alternative treatment plans are available for a diastema closure, such as orthodontic appliances, restorative techniques, prosthodontic treatment or combination of procedures; among which include the use of conservative and more practical direct composite resin restorations.7 determining the appropriate treatment techniques and materials for patients are also based on time constraints and any physical, psychological and economic problems. in the case of a diastema closure, the composite resin makes it easier for dentists to control the patient’s natural smile.8 the latest aesthetic resin composite materials have physical and mechanical properties similar to natural teeth, i.e. similar in terms of tooth colour and structure with a compressive strength similar to enamel and dentine. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p44–48 mailto:nirawati-p@fkg.unair.ac.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p44-48 45pribadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 44–48 composite resins include a range of colours and opacities specifically designed for coating techniques.9,10 the use of index putty is an innovation in dentistry to restore the anterior teeth using resin composites. the index putty technique is used for a midline diastema closure because it can correct the anatomical contour of the tooth, thereby reducing the need for further adjustments and saving time in the restoration process.11 the colour of the composite resin is selected using the button shade technique, which involves selecting a slightly different colour on the facial surface of the tooth to be restored.12–14 this case report presents the aesthetic management of a midline diastema using the direct composite technique along with digital smile design (dsd), the putty index method and button shade technique. case a 20-year-old female patient presented to the department of conservative dentistry, universitas airlangga dental and oral hospital with the chief complaint of anterior teeth spacing, categorised as an angle’s class i occlusion with normal overjet and overbite. the labial frenum associated with the diastema was normal in size and position. various treatment modalities were discussed with the patient. the patient was not willing to undergo any invasive procedure. therefore, a minimally invasive approach using a direct composite resin restoration was chosen to restore the diastema. a treatment plan was explained to the patient, including the selection materials and treatment procedure. case management on the first visit, a subjective examination was acquired from the patient’s history. the patient complained of the distance between her anterior teeth and no complaints of pain, swelling or interfering with eating and speaking activities. subsequently, an objective examination identified a gap between teeth 11 and 21, no change in tooth colour, and negative results of both a percussion test and bite test (figure 1). the posterior occlusion was in a cusp to fossa state, while the anterior occlusion was characterised as an overbite and overjet of 1 mm; thus, the classification of the malocclusion in this case was class i angle. in this case, no radiographic examination was performed, and pulp was determined to be normal. furthermore, vitality tests were performed on teeth 11 and 21. upon electric pulp testing (ept) for tooth 11, both the control tooth and test tooth reacted to number 3, which indicated that tooth 11 was vital. upon ept for tooth 21, both the control tooth and test tooth reacted to number 3, which indicated that tooth 21 was vital. the saliva examination showed a satisfactory result; i.e. hydration within 12 seconds, watery viscosity, a ph of 7.8, quantity >5 ml/minutes, and a buffer capacity of 13. after several examinations, the dentist made a dsd, a diagnostic waxup, and silicone guides (figure 2). on the second visit, a rubber dam was placed on teeth 11 21 to isolate the work area. then, shade taking was performed using the button shade technique (figure 3a). the composite resin was placed slightly (like a button) on the facial surface of the tooth, whereby the dentin colour was placed in the cervical area and the enamel colour was a b figure 1. clinical smile of the patient showed a central diastema (a and b). a b figure 2. digital smile design (a), diagnostic wax-up and silicon guide (b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p44–48 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p44-48 46 pribadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 44–48 placed in the incisal area14. the next step involved tooth surface preparation using a sof-lex disc (coarse, 3m, usa; see figure 3b). after preparation, etching was applied for 15 seconds, and bonding was then applied via light-curing for 20 seconds (figure 3c). next, a palatal shell with an oa1 composite (palfique lx5, tokuyama, japan; see figure 3d) was constructed. then, metal matrix bands (gc new metal strips, gc corporation, tokyo, japan) were installed on the interproximal teeth 11 and 21 (figure 3e). finally, the a1 colour composite (palfique lx5, tokuyama, japan) was applied to the dentin and enamel. after all stages were completed, the composite resin was finished and polished using a fine finishing bur (komet dental, gebr. brasseler, germany), a disc (coarse, 3m, usa) and polishing system (diacomp plus, eve america inc., naples, fl, usa; see figure 3f). on the third visit, the patient returned to the department of conservative dentistry, universitas airlangga dental and oral hospital without any complaints or extraoral abnormalities. upon intraoral examination, the gingiva was normal, the composite was still satisfactory (and did not change colour) and the vitality of teeth 11 and 21 were normal. figure 4 show the images on teeth 11 and 21 after central diastema closure using digital smile design, putty index and button shade technique. discussion one of the advantages of using a direct composite resin is that it can be done in one visit, as they often do not require a pre-model or wax-up and also do not require a b c d e f figure 3. serial clinical treatment procedure begin with shade taking using the button shade technique (a), minimal preparation of the teeth (b), etch & bond (c), palatal shell with silicone index (d), matrix interdental placement & direct composite manipulation (e) and finishing & polishing (f). a b figure 4. clinical smile of the patient showed a central diastema closure after the treatment (a and b). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p44–48 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p44-48 47pribadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 44–48 expensive laboratory costs. in aesthetic dental treatment, this restoration has many advantages over other treatments such as ceramic veneers and orthodontic treatments: they are more compatible with antagonistic teeth than ceramic materials, and if there is a fracture, it will be easier to repair because it does not require additional time and cost.15–17 however, composite resins also have several disadvantages, including shrinkage during polymerisation, a low wear resistance, poor long-term durability, postoperative sensitivity and (in clinical use) direct composite resins have the potential to become secondary caries and fracture.18,19 digital smile design is a digital aesthetic planning tool in dentistry that is used to evaluate the aesthetic relationship between the teeth, gingiva, smile and face. the use of dsd tools provides a new perspective for diagnosis and treatment planning and facilitates communication between dentists, technicians and patients. using dsd design tools makes it easier to create and project a new smile design to get a pre-visualisation of the final treatment plan result.20,21 according to several studies, the putty index method can reduce shrinkage due to the dimensional stabilisation of the mould that results from polymerisation. the putty setting material and contraction of the wash impression material results in minimal dimensional changes. the putty material in the one-step putty/wash technique also tends to push the wash impression material away from the prepared tooth and from important areas such as the finish line, which can be obscured by the putty material and thus cannot record detail satisfactorily.22–24 the disadvantage of the putty index method is that some of the wash impression material can spread occlusally when the putty impression material is placed back or when the wash material is introduced. this can cause distortion that reduces dimensional accuracy.25 a diastema of the anterior teeth can interfere with aesthetics and reduce the patient’s self-confidence.26 closure of the diastema using a composite resin is the main treatment plan offered, but in the case of large spaces between the teeth, simple closure may not provide a natural and pleasant solution for the patient. direct composite restoration is the simplest of all procedures for diastema closure.27 closure of a wide midline diastema uses an index putty because it can form a detailed palatal contour specific to the shape, size and inclination that has been made previously.11 metal matrix bands were chosen in this case because they can provide a detailed contour area on the proximal teeth specific to the anatomical requirement not found on a transparent matrix.28 dentists and patients have complete control over the formation of a natural smile. digital smile design can make it easier for dentists to visualise a patient’s smile to form a treatment plan and provide knowledge about the procedure to patients.29 however, sometimes an interdisciplinary approach is needed to achieve better aesthetic results. the control treatment results were evaluated on the third visit, in which the patient returned after 1 month for an evaluation of the results of the direct resin restoration.30 the control results showed no abnormalities on extraoral and intraoral examination. restoration using the direct composite looked good and there was no discolouration; the colour around the gingival teeth was normal, the vitality of teeth 11 and 21 were normal. in this case, the diastema closure was successfully performed using the direct restorative technique and the patient was very satisfied with the results. in conclusion, closure of a diastema using the direct composite technique provides good aesthetic results with less cost and time due to the absence of laboratory procedures. a clinical evaluation at a 1-month follow-up showed a good restoration condition and no discoloration. the patient was satisfied with the results. references 1. hasan h, al azzawi a, kolemen a. pattern of distribution and etiologies of midline diastema among kurdistan-region population. j clin exp dent. 2020; 12(10): e938–43. 2. gupta r, miglani a, gandhi a, kapoor n. esthetic management of diastema closure: an innovative technique utilizing putty index method. int j oral care res. 2018; 6(1): 104–8. 3. korkut b, yanikoglu f, tagtekin d. direct midline diastema closure with composite layering technique: a one-year follow-up. case rep dent. 2016; 2016: 1–5. 4. sękowska a, chałas r. diastema size and type of upper lip midline frenulum attachment. folia morphol (warsz). 2017; 76(3): 501–5. 5. nuvvula s, ega s, mallineni sk, almulhim b, alassaf a, alghamdi sa, chen y, aldhuwayhi s. etiological factors of the midline diastema in children: a systematic review. int j gen med. 2021; 14: 2397–405. 6. biswas i, gupta sd, chowdhury d, mazumdar p. midline diastema closure using direct light cured composite resin a case series. int j adv res. 2019; 7(4): 1559–66. 7. k a m bl e a , sh a h p, vel a n i p, ja d h av g . l a s e ra s s i s t e d multidisciplinary approach for closure and prevention of relapse of midline diastema. indian j dent res. 2017; 28(4): 461–4. 8. tüter e, korkut b, yılmaz atalı p, türkmen c. prepless direct midline diastema closure in a single visit: 18 months follow-up report. eur j res dent. 2019; 1(3): 29–34. 9. abdulgani m, abdulgani a, muhamad a-h. closing diastemas with resin composite restorations; a case report. iosr j dent med sci. 2019; 18(3): 30–3. 10. vasques wf, sá ta, martins f v., fonseca em. composite resin cad-cam restorations for a midline diastema closure: a clinical report. j prosthet dent. 2020; : 1–4. 11. bansode p v, pathak sd, wavdhane mb, bhunne ss. anterior esthetic restorations , the putty index technique – a case report. j dent med sci. 2020; 19(9): 18–22. 12. varvară e-b, mesaroş aş, culic b, varvară am, dudea d. resin composite layering technique for direct anterior teeth restorations. palestrica third millenn civiliz sport. 2018; 19(4): 234–8. 13. soetojo a, widjaja ba. management of white spot and central diastema on anterior teeth with direct composites restoration (a case report). conserv dent j. 2020; 9(2): 66–9. 14. amaro i, saraiva j, gomes ac, araújo a, marto cm, coelho a, ferreira mm, paula a, carrilho e. direct restorations for anterior esthetic rehabilitation and smile symmetry recovery: two case reports. symmetry (basel). 2021; 13(10): 1848. 15. abu-hussein m, watted n. maxillary midline diastema – aetiology and orthodontic treatmentclinical review. iosr j dent med sci. 2016; 15(6): 116–30. 16. comba a, vergano ea, baldi a, alovisi m, pasqualini d, castroflorio t, stura i, migliaretti g, berutti e, scotti n. 5-year dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p44–48 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p44-48 48 pribadi et al./dent. j. (majalah kedokteran gigi) 2022 march; 55(1): 44–48 retrospective evaluation of direct composite restorations in orthodontically treated patients. j dent. 2021; 104: 103510. 17. septyarini be, dwiandhono i, imam dna. the different effects of preheating and heat treatment on the surface microhardness of nanohybrid resin composite. dent j (majalah kedokt gigi). 2020; 53(1): 6–9. 18. zhou x, huang x, li m, peng x, wang s, zhou x, cheng l. development and status of resin composite as dental restorative materials. j appl polym sci. 2019; 136(44): 48180. 19. soesilo d, hadinata y, pangabdian f, rochyani l. direct composite restoration using stamp technique and pizza technique: a case report. int j dent res. 2020; 5(1): 4–6. 20. garcia pp, da costa rg, calgaro m, ritter av, correr gm, da cunha lf, gonzaga cc. digital smile design and mock-up technique for esthetic treatment planning with porcelain laminate veneers. j conserv dent. 2018; 21(4): 455–8. 21. jafri z, ahmad n, sawai m, sultan n, bhardwaj a. digital smile design-an innovative tool in aesthetic dentistry. j oral biol craniofacial res. 2020; 10(2): 194–8. 22. martins f, branco p, reis j, barbero navarro i, maurício p. dimensional stability of two impression materials after a 6-month storage per iod. act a biomater odontol sca nd. 2017; 3(1): 84–91. 23. kabbach w, sampaio cs, hirata r. diastema closures: a novel technique to ensure dental proportion. j esthet restor dent. 2018; 30(4): 275–80. 24. pastoret m-h, krastl g, bühler j, weiger r, zitzmann nu. accuracy of a separating foil impression using a novel polyolefin foil compared to a custom tray and a stock tray technique. j adv prosthodont. 2017; 9(4): 287–93. 25. sherwood ia, rathakrishnan m, savadamaoorthi ks, bhargavi p, vignesh kumar v. modified putty index matrix technique with mylar strip and a new classification for selecting the type of matrix in anterior proximal/incisal composite restorations. clin case reports. 2017; 5(7): 1141–6. 26. durán g, vivar f, tisi j, henríquez i. the use of direct composite resin to close maxillary midline diastema complementary to orthodontic treatment. rev clínica periodoncia, implantol y rehabil oral. 2019; 12(2): 106–8. 27. goyal a, nikhil v, singh r. diastema closure in anterior teeth using a posterior matrix. case rep dent. 2016; 2016: 1–6. 28. chandrasekhar v, rudrapati l, badami v, tummala m. incremental techniques in direct composite restoration. j conserv dent. 2017; 20(6): 386. 29. pinzan-vercelino crm, pereira cc, lima lr, gurgel ja, bramante fs, pereira alp, lima dm, bandeca mc. two-year follow-up of multidisciplinary treatment using digital smile design as a planning tool for esthetic restorations on maxillary midline diastema. int j orthod milwaukee. 2017; 28(1): 67–70. 30. sajjanhar i, nikhil v, mishra p. diastema closure and aesthetic rehabilitation; an interdisciplinary approach. ip indian j conserv endod. 2019; 4(2): 66–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 158/e/kpt/2021. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v55.i1.p44–48 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v55.i1.p44-48 132 vol. 41. no. 3 july–september 2008 the management of oral candidosis in diabetic patient with maxillary herpes zoster kus harijanti1, dwi setyaningtyas2, and isidora ks2 1 departement of oral medicine faculty of dentistry airlangga university 2 oral medicine clinic of surabaya navy hospital surabaya indonesia abstract background: oral candidosis is an infection caused by mainly candida albicans. candida species are common normal flora in oral candidosis is an infection caused by mainly candida albicans. candida species are common normal flora in the oral cavity and have been reported to be present in 40% to 60% of the population. candida is predominantly an opportunistic infectious agent. infection frequency has increased because of the presence of both local and systemic risk factors. the elderly age and diabetes mellitus may decrease the amount of saliva (xerostomia) and potentially increase the risk of colonization and secondary infection by candida. herpes zoster (hz) is a manifestation of the reactivation of latent varicella zoster virus. it is characterized by unilateral, painful, vesicular rash with a dermatomal distribution. the clinical manifestations of this disease can erupt to the skin and mucous membrane. if maxillary nerve is involved, the lesion can appear on unilateral facial skin and oral mucous membrane. purpose: the purpose of this paper is to report and discuss the difficulties in managing the oral candidosis in elderly patient (57 the purpose of this paper is to report and discuss the difficulties in managing the oral candidosis in elderly patient (57 year old male) who suffered from maxillary herpes zoster and diabetes mellitus. case management: at first, the patient was treated at first, the patient was treated with 2% chlorhexidine gluconate and mycostatin oral suspension as topical antimycotic and reffered to dermathology clinic for viral infection treatment, however the oral candidosis did not improved. subsequently, ketokonazole tablet was given three times daily for three weeks and regulated blood glucose level. in systemic antifungi (ketokonazole) treatment the oral candidosis disappeared. conclusion: in this case, it is conclude that the management of oral candidosis are adequate, antiviral, blood glucose level regulating in this case, it is conclude that the management of oral candidosis are adequate, antiviral, blood glucose level regulating and systemic antifungal therapy. key words: herpes zoster, oral candidosis correspondence: kus harijanti, c/o: departemen ilmu penyakit mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: kus_oralmedair@yahoo.com introduction candida is a yeast-like fungus. candida species are common in the normal flora and have been reported to be present in 40% to 60% of the population.1 it is harmless, but it might become opportunistic pathogen when there is oral immune decreasing as a result of the oral microorganism ecosystem changes. candidosis is an infection caused by candida, mainly c. albicans. infection frequency by candida species has increased because of the presence of both local and systemic risk factors. the systemic risk factors are treatment with broad spectrum antibiotic, diabetes mellitus and immunosuppressive illness.1,2 the symptoms of candidosis may include burning sensation, sensitivity, altered taste and smell. clinical intra oral sign may vary from a red form (erythematous) to psedomembranous form (white, thrush); simultaneous red and white changes are common. oral candidal overgrowth is almost superficial and rarely penetrates deeper than epithelial cells surfaces. the pseudomembranous form is the colonies of organisms that attach to the surface and can be removed with rubbing, frequently leaving red or bleeding sites.1 varicella zoster virus (vzv) is dna virus that causes chicken pox in children as primary infection and herpes zoster (shingles) in adult as subsequent reactivation. this disease can affect the skin and oral mucous membrane. chicken pox is caused by droplet infection or direct contact with patient, usually benign illness that occurs in epidemic among susceptible children. although, the clinical case report 133kamadjaja and pramono: management of zygomatic-maxillary fracture symptom on skin or mucous membrane disappear, zoster virus still resides in dorsal-root ganglia and subsequent reactivation can occur.3,4 virus reactivation does not always give the clinical symptoms, because virus-cell mediated immunity can protect the host. the risk of shingle is increased by declining cell mediated immune responses, which occur naturally as a result of aging or induced by immunosuppressive illness or medical treatments.3,5 at an incidence of 2/1000, about 500,000 cases annually would occur in the united states and about half this number in the united kingdom. the incidence rises steeply with age, being less than 1 per 1000 person years in children and as many as 12 per 1000 person years in those over 65 years old.5 seventy percent of herpes zoster occurred on persons over 50 year old.4 the initial symptoms are pain and tenderness in the affected area and prodromal severe pain of herpes zoster. characteristic of herpes zoster are multiple vesicles, which distributed in appropriate sensory nerve region. when the maxillary nerve is involved, the lesion can appear on unilateral facial skin and oral mucous membrane, this is about 15% of the cases.4 the patient suffers from tooth-ache in the nerve region which is involved and frequently it can not be distinguished with trigeminal zoster.3,6 when this disease is not treated, vesicles on skin and ulcers in oral mucous membrane will disappear in 2-4 weeks or more. crusts appears on the skin and might form scarring. although there are several serious complications of zoster (ophthalmic, splanchnic, cerebral motor), in immunocompromise adult the most common case is post herpetic neuralgiapain. postherpetic neuralgia is a condition in which the patient suffers from anesthesia, paraesthesia and severe pain in the involved nerve region. the pain sensation is still persisted for prolong time until years after clinical sign disappears. both the incidence and the duration of postherpetic neuralgia are directly correlated with the patient’s age.3,4,5 this article reported a case of oral candidosis on herpes zoster which involved the maxillary nerve. the patient was 57 years old and also suffered from diabetes mellitus. case in october 17, 2006, a 57 years old male came to the dental clinic at dr. soetomo general hospital surabaya, the patient complained of severe left tooth-ache in 5 days. initially, the patient came to the public medical center for treating his teeth, but the pain didn’t reduce although the teeth was already filled, even the left facial skin was oedem and erythemathous, followed by fever, malaise and weakness. then, the patient went to general practitioner and was treated with antibiotic and analgesic/anti-inflammation. the patient still suffered from severe teeth-ache on the maxillary left-region. formerly, the patient suffered from localized abnormal skin sensations, ranging from itching or tingling on the left facial-skin and followed by the appearance of clusters of clear vesicles. extra oral examination showed pustulation, ulceration, crusting and oedema on the left facial skin (figure 1). several lesions on the left lower lid caused lid edema and ectropion, involved left nose and upper lip. left submandibular lymph nodes were enlarged and tender. on intra oral examination (figure 2 & 3), white plaque was revealed on hard palate, thick and soft as creamy milk with irregular border and form, surrounded by erythemathous and can be removed with rubbing. erythemathous and multiple ulcers with 2-4mm diameter were found on maxillary left buccal-fold and left buccal mucosa, the center of ulcers were yellowish and painful. erythemathous mucosa and white thin-plaque, irregular form and scrapable were found on the left upper lip. case management the patient previously came to the public medical center with chief complaint left upper teeth-ache. in fact, the figure 1. first visit: extra oral view. figure 2. first visit: intra oral view. 134 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 132-136 figure 3. first visit: palatal view. lesions on the skin had emerged but the patient neglected and only focused on his tooth-ache. in the dental clinic, the patient was treated with benzidamine-hcl rinse 4 times daily and multivitamin plus zn one caplet daily. because of weak general condition and chief complaint of severe pain on the facial skin, the patient referred to dermatology clinic and had to control a week later. control first (october 30, 2006): according to the anamnesis, the patient said that he had been hospitalized for 4 days in dermatology clinic and given erythromycin 3 × 500 mg/daily, acyclovir 5 × 800 mg/daily, amytriptilin 2 × 25 mg/daily, mefenamic acid 3 × 1/daily, bioneuron 1 × 1/daily and eye drops and skin ointment. the patient suffered from severe pain, liked tingling sensation on the left-facial skin, with pain-duration about 15 seconds continually almost everyday. extra oral examination showed left submandibular lymph nodes were enlarged, springy and pain. dark brown-crusting surrounded by erythemathous on left facial skin, and desquamation followed yellowish by crusting on upper lips. intra oral examination showed that ulcers and erythemathous on the left buccal mucosa and buccal-fold disappeared, but white plaque on the left palate still persisted. chlorhexidine gluconate 0, 2% oral rinse and multivitamin plus zn caplet one daily were administered. control second (november 07, 2006): pain sensation on the left facial skin and teeth-ache still persisted, as well as the left ear region although aulin tablet had been given. left submandibular lymph nodes did not change, left facial skin revealed hyper-pigmentation. white plaque on the left palate was thinner and difficult to be scraped (figure 4). oral rinse and multivitamin plus zn caplet were repeated and mycostatin oral suspention was added. therefore, the patient referred to mycological laboratory and ent (ear nose throat) clinic. control third (november 14, 2006): pain sensation on the left ear had gone, but still existed on the facial skin and teeth. left submandibular lymph nodes were normal; hyperpigmention on the facial skin was thinner. white plaque on the left palate changed to be smaller and thinner, left upper teeth region were unstable grade three. radiographic examination showed the resorbtion of alveolar bone. this data supported that the patient has diabetes mellitus. mycological finding showed positive candida infection, therefore final diagnosis on the palatum was oral candidosis (thrush). the mycostatin oral suspension and chlorhexidine gluconate 0.2% oral rinse treatment was continued. in fact, the third control (four weeks later) showed that oral candidosis did not change; therefore the patient was given ketokonazole tablets three times daily. then, the patient was referred to clinic of diabetes mellitus and neurology. control fourth (november 21, 2006): from neurology clinic, the patient was given amitriptilyn, carbamazepin, neurobion and pondex, that were regularly taken for reducing pain. actually, pain sensation on the left facial skin and left upper teeth region were reduced, but the patient complained of xerostomia. hyperpigmentation on the left facial skin still existed although it was thinner. white plaque on left palate was smaller and slighter, on upper and lower buccal mucosa were painful. there was white plaque on dorsal tongue, thick and scrapable, surrounded by erythematous. oral rinse was repeated and ketokonazole tablet was continued. control fifth (november 28, 2006): laboratory result (november 23, 2006) showed that fasting blood glucose level was 109mg/dl and 2 hours postprandial was 162mg/dl, it proved that the patient suffered from diabetes mellitus. according to the anamnesis, 5 days before control the patient got spontaneous loss of two of the upper left teeth (canine and first premolar). extra oral and intra oral examination showed that hyperpigmentation of the left facial skin was thinner and sometime less pain sensation (figure 5 and 6). the upper lip was dry, desquamative and fissured. dry socket was found in the region of post spontaneous teeth loss. white plaque on figure 4. third visit: white plaque on the left palate was thinner and difficult to be scraped 135kamadjaja and pramono: management of zygomatic-maxillary fracture the left palate disappeared, but on the dorsum of the tongue still existed. therefore, ketokonazole tablet was repeated, and vitamin c tablet as supporting treatment, mefenamic acid and benzidamine hcl oral rinse for reducing pain was added and alvogyl fibre was topically given in the dry socket region. the patient was referred to diabetes clinic for blood regulation controlling. if the blood regulation was normal, the left upper teeth which unstable grade three would be extracted. control sixth (december 05, 2006): although the patient was given medicines for diabetes mellitus, the blood glucose level was still higher than normal, consequently the patient’s pain-labile-teeth could not be extracted, therefore the patient had to wait for normal condition. the brownmacula presented on the left facial skin and there was no pain sensation, thinner white plaque was presented on the dorsum of the tongue. ketokonazole tablets was continued, and the patient was given vitamin c and b complex tablets for supporting treatment. the patient suggested to control one month later. control seventh (january 9, 2007): a week before control, the patient got spontaneous loss of the front of the upper teeth (left second incisor). the brown macula on the left facial skin was thinner, and intra oral examination showed white plaque disappeared. the fasting blood glucose level was 78 mg/dl and 2 hours postprandial blood glucose level was 136mg/dl, it indicated the normal condition was reached. then, the patient referred to oral surgery clinic for extracting of the front upper teeth which unstable grade three (the left upper central incisor and canine). blood glucose level decreasing would improve the patient’s general condition. discussion base on the general condition, anamnesis and extra oral examination, the clinical diagnosis of this case was herpes zoster which involved trigeminal nerve. the appearance of herpes zoster is sufficiently distinctive that a clinical diagnosis is usually accurate.7 this case involved left facial skin and limited on the left lower lid caused lid edema and ectropion, involved left nose and upper lip. the present unilateral ulceration or skin lesions limited in area supplied by one division of the trigeminal nerve suggests a diagnosis of zoster.3 the maxillary division of the trigeminal nerve is entirely sensory, supplying the skin of the middle portion of the face, lower eyelids, side of the nose, upper lip, mucous membranes of the nasopharynx, maxillary sinus, soft palate, and teeth. near its origin it branches off to form the middle meningeal nerve, which supplies the ipsilateral middle meningeal artery and branches to durameter. one of its terminal branches is the greater palatine nerve, which supplies the hard palate, a portion of the maxillary gingival, the uvula, and a portion of the soft palate. the other important branch is the superior alveolar nerve, which supplies the maxillary gingival, teeth, and mucous membranes of the cheek.8 if the maxillary or mandibulary division of the trigeminal nerve were involved, the patient may experience toothache-like pain for several days prior to the onset of more characteristic cutaneous lesions.7 the patient suffers from tooth-ache in the nerve region which is involved and frequently can not be distinguished with trigeminal zoster.3,6 on intra oral examination, erythemathous and multiple ulcers were found on maxillary left buccal-fold mucosa, left buccal and upper lip mucosa, uvula and left soft palate. on left hard palate there was white plaque, thick and soft as figure 5. post treatment: extra oral view. figure 6. post treatment: intra oral view. 136 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 132-136 creamy milk, irregular border and form, can be removed with rubbing and surrounded by erythemathous. based on intra oral examination, trigeminal nerve which involved in this case was maxillary division. thrush as secondary infection was suspected on the left hard palate and upper lip mucosa. it occurred because the patient took antibiotic from private physician and there were multiple ulcers caused by maxillary herpes zoster infection. acyclovir 800 mg five times daily will inhibit the replication of varicella zoster virus by shortening the duration of viral shedding, halting the formation of new lesions more quickly, accelerating the rate of healing, and reducing the severity of acute pain. attenuation to the severity of the acute infection and the neural damage will reduce the likelihood of post herpetic neuralgia.3,5 the pain of post herpetic neuralgia can be reduced by a number of medications. tricyclic antidepressant medications such as amitriptyline (elavil), as well as anti-seizure medications such as gabapentin (neurontin) and carbamazipine (tegretol), have been used to relieve the pain associated with herpetic neuralgia. finally, capsaicin cream (zostrix), a derivative of hot chili peppers, can be used topically on the area after all the blisters have healed, to reduce the pain.9 until the 13th days, the patient still suffered from severe pain on the left facial skin although a number of medications was given to reduce pain. it proved that, the patient suffered acute herpetic neuralgia. according to johnson and dworkin,5 acute herpetic neuralgia defines as pain within 30 days from rash onset. at the second control, white plaque at he left palate was thinner but difficult to be scrapped, therefore chlorhexidine gluconate 2% (and multivitamin plus zn caplet) were repeated and mycostatin oral suspension as antimycotic topical was added. intra oral examination at the third control revealed that left upper teeth region were unstable grade three. radiographic examination showed resorbtion of alveolar bone. this data supported that the patient suffered from diabetes mellitus. mycological finding showed positive candida infection, thus final diagnosis on the palatum was oral candidosis (thrush). antimycotic topical was continued and the patient was given antimycotic systemic (ketokonazole tablets three times daily). laboratory finding (november 23, 2006) showed that fasting blood glucose level was 109 mg/dl and 2 hours postprandial was 162 mg/dl, it is really proved that the patient suffered from diabetes mellitus. diabetes mellitus on this patient is caused by poor condition, xerostomia, leading to unstable upper left-teeth on the third degree and followed by spontaneous loss of teeth. this disease also caused oral candidosis which was difficult to treat. blood glucose level decreasing would improve the patient’s general condition. since the patient not only suffered from herpes zoster in the maxillary nerve but also oral candidosis and diabetes mellitus, he was classified as immunocompromised patient. oral candidosis of immuno-compromised patients should be treated with systemic antifungals.10 the involvement of oral candidosis, herpes zoster virus infection and diabetes mellitus, made this case complicated and multidisciplinary approached is needed. it is concluded that the management of in this case, it oral candidosis are adequate antiviral, blood glucose level regulating and systemic antifungal therapy. references 1. silverman s, epstein jb. oral fungal infection on burket’s of oral medicine. diagnosis and treatment. 10th ed. ontario: bc decker inc; 2003 p. 170–9. 2. scully c. candidosis, mucosal. available at: emedicine.com, inc. accessed january 24, 2002. 3. gnan jw, whitley rj. herpes zoster. the new england journal of medicine 2002; 347(5): 340–6. 4. field a, longman l. tyldesley’s oral medicine. 5th ed. new york: oxford university press; 2003. p. 42–43. 5. johnson rw, dworkin rh. treatment of herpes zoster and post herpetic neuralgia. br med j 2003; 326:748–50. 6. cawson ra, odel ew. oral pathology and oral medicine. 7th ed. churchill livingstone. london: churchill livingstone; 2005. p. 181-2, 185–90. 7. lamey pj, lewis mao. oral medicine and practice: viral infection. br dent j 1989;. 167:269–74. 8. okeson jp. orofacial pain. chicago: quintessence publishing co, inc; 1996. p. 3–5. 9. cunningham al, breuer j, dwyer de, gronow dw, helme rd, litt jc, et al. the prevention and management of herpes zoster. clinical update mja 2008 february; 188 (3): 171–6. 10. firriolo fj. oral candidosis. available at: www.dentalcare/intermed/ oralcan.htm. accessed may 5, 2003. 67 vol. 43. no. 2 june 2010 case report ankylosis of the temporomandibular joint and mandibular growth disturbance caused by neglected condylar fracture in childhood endrajana department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract background: fractures of the mandibular condyle may lead to complications such as disturbance of occlusal function, internal derangement of the joint, ankylosis and mandibular growth disturbance. when treating young patients with the history of mandible trauma, ankylosis of the temporomandibular joint and mandibular growth disturbance are two most important complications of condyle fracture that should be considered. purpose: this case report attempts to emphasize the long term complication of neglected condylar fracture in children i.e. ankylosis of the temporomandibular joint and subsequently lead to mandibular growth disturbance. case: a case of right temporo-mandibular joint ankylosis and mandibular growth disturbance in a 28 years old male patient is presented. he had a history of trauma to the mandible after a traffic accident when he was 8 years old. since then, he experienced difficulty in mouth opening which eventually developed into severe trismus. case management: the case was treated surgically with gap and interpositional arthroplasty using mersilen mesh™. conclusion: mandibular fractures involving temporomandibular joint in young children should be examined thoroughly and treated adequately in order to prevent ankylosis of the tmj and the subsequent mandibular growth disturbance. key words: condylar fracture, ankylosis, mandibular growth disturbance abstrak latar belakang: fraktur pada kondilus mandibula dapat menyebabkan beberapa komplikasi berupa: gangguan oklusi, internal derangement sendi, ankilosis serta gangguan pertumbuhan mandibula. pada perawatan penderita usia muda dengan riwayat trauma pada mandibula, perlu diwaspadai dua macam komplikasi akibat fraktur pada kondilus, yaitu ankilosis sendi temporo mandibula dan adanya gangguan pertumbuhan mandibula. tujuan: laporan kasus ini bertujuan untuk menekankan bahwa fraktur kondilus pada anak-anak yang tidak mendapatkan perawatan yang semestinya akan mengakibatkan komplikasi jangka panjang berupa ankilosis sendi temporomandibula yang diikuti dengan gangguan pertumbuhan tulang mandibula. kasus: penderita laki-laki umur 28 tahun dengan keadaan tidak dapat membuka mulut dan adanya gangguan pertumbuhan tulang mandibula. terdapat riwayat trauma pada mandibula akibat kecelakaan lalu lintas saat penderita berumur 8 tahun. semenjak kecelakaan tersebut penderita merasakan kesulitan membuka mulut dan akhirnya sama sekali tidak dapat membuka mulut. tatalaksana kasus: untuk merawat kasus ini dilakukan tindakan pembedahan dengan bius umum yaitu gap arthroplasty dengan memakai mersilen mesh™ sebagai interposisional graft. kesimpulan: fraktur kondilus mandibula pada penderita anak-anak memerlukan pemeriksaan dan perawatan yang adekuat untuk menghindari terjadinya ankilosis sendi temporomandibula dan gangguan pertumbuhan tulang mandibula. kata kunci: fraktur kondilus, ankilosis sendi temporomandibula, gangguan pertumbuhan tulang mandibula correspondence: endrajana, c/o: departemen bedah mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: bm_enjana@yahoo.co.id 68 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 67-71 introduction the mandible is different from other facial bones in some important respect. in addition to its contribution to facial dimension and asymmetry, the mandible has unique and important functional features. the mandible is the only bone in the face that moves in relation to the skull. additionally the mandible bears powerful muscular stresses and injury can make this bone functionally disabled.1 condylar neck is the weakest structural part of the mandible in relation to their resistance to mechanical force. the incidence of condylar fracture is the highest (35.6%) among those of other regions in mandible.2,3 the pattern of the fracture can vary greatly and may occur anywhere down a line from sigmoid notch to the mandibular angle. condylar neck fractures are clearly different from other mandibular fractures in as much as they are always located behind and above the lingual. they also differ from mandibular body fractures because they are more difficult to diagnose clinically and radiologically.3 condylar fracture may occur as a result of either direct trauma in the area of temporomandibular joint or indirect trauma in the chin region, mandibular angle, condyle and the subcondylar region. complications of condylar fracture may include occlusal disturbances, internal derangement of the articular disc, ankylosis of the joint, and mandibular growth disturbances. if the condylar fractures occur in children at the age of active mandibular growth it should be treated with caution as ankylosis of the temporomandibular joint may take place and lead to the mandibular growth disturbances which may eventually cause facial deformities and functional problem later in life. in this article a case of condylar fracture because of mechanical trauma during childhood is presented which has caused ankylosis of temporomandibular joint and mandibular growth disturbances. case a 28 years old male patient came to oral and maxillofacial surgery clinic, faculty of dentistry, airlangga university complaining that he was unable to open his mouth. according to the patient this condition has been lasting for 20 years which started not long after he got a traffic accident at the eight years of age. he was admitted to a hospital during which suturing to the right side of his head and chin were made. since then his mouth opening was restricted and the condition worsened with time until finally he was completely unable to open his mouth. he has been on liquid diet until today. clinical examination showed facial asymmetry with large area of scar tissue on his right zygomatic and preauricular region, mandibular retrognathism, and severe trismus (figure 1). intra oral examination was difficult to perform due to the trismus. panoramic x-ray showed ankylosis of the right temporomandibular joint whereas the left joint seemed somewhat normal with the presence of joint space (figure 2). ct scan of the head revealed loss of the normal structure of the right temporomandibular joint showing large ossification area over the region (figure 3). the diagnosis made was bony ankylosis of the right temporomandibular joint. figure �. the profile of the patient showing "bird face" appearance indicating a severe mandibular retrognathism (left) and severe trismus (right). 69endrajana: ankylosis of the temporomandibular joint case management the case was treated surgically with gap arthroplasty of the right temporomandibular joint under general anesthesia. tracheostomy was performed pre-operatively as blind intubation was difficult to do. preauricular incision was made and the wound deepened layer by layer until the ankylotic condylar bone was found. a bur was used to create a gap between body of the mandible and the bony ankylosis as wide as 1 cm. the resulting rough edges were smoothened out using rongeurs forceps and bone file. using heister mouth opener the mouth was able to open approximately 3 cm and maintained afterwards. mersilen meshtm was packed up in the gap and sutured to the muscles. a redon drain was placed and vicryl 4.0 sutures were use to close the wound in layers. a nylon 5.0 suture was used to close the skin. the patient had no specific complaints after surgical procedure. the heister were maintained for two days after the surgery to keep the mouth open for two days to prevent recurrence of the trismus caused by muscles spasm. three days post operative, the tracheostomy tube was taken out and redon drain removed at fourth day. the patient was discharged from the hospital after removal of the drain. post operative panoramic x-ray was taken on the day he left the hospital, the result of which clearly showing the gap created in the right subcondylar neck region (figure 4). the patient was instructed to continue mouth opening exercise using heister for 6 month. the skin sutures were removed at seventh day. three weeks after the surgery the patient came back in good general health condition and was able to open his mouth normally (figure 5). figure ��. pre-operative panoramic x-ray showing the absence of normal structure of temporomandibular joint on the right side, anatomical structure of the left joint seems normal. figure ��. axial projection of ct scan of the head showing large ossification area replacing the normal anatomical structure of right temporomandibular joint. 70 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 67-71 discussion the causes and treatment of tmj ankylosis have been well documented, with trauma and infection identified as the two leading causes.4–6 in children, tmj ankylosis can result in mandibular retrognathism with attendant esthetic and functional deficits. classification of ankylosis can be based on the tissues involved and extent of involvement: complete or partial, true or pseudo ankylosis, or bony, fibrous, or fibroosseus.4 in the case presented above it is most likely that the ankylosis of right temporomandibular joint and the mandibular retrognathism are associated with the history of trauma to the mandible when he was 8 years-old. condylar fractures are usually caused by indirect trauma in the chin. there are 3 types of kinetic energy that can cause fracture of the condyle: a) kinetic energy that comes from a moving object hitting a static individual, e.g. a punch, b) kinetic energy coming from a moving individual towards a static object e.g. fall from height and hitting the floor, c) combination of the two types above.2 in the above case there was no evidence of chin trauma but a large scar tissue was found over the skin of preauricular region. it is likely that there has been a direct, high energy trauma to the right tmj region causing condylar fracture, instead of indirect trauma on the chin through one of the three types of kinetic energy mentioned above. the ankylosis of the temporomandibular joint in this case was possibly caused by intra capsular hemorrhage which led to formation of bony union in the joint space. the predisposition to ankylosis in the joint may also be associated with the occurrence of intracapsular type of fracture which are common in children as their condylar heads are still immature, thinly covered and highly vascularized which tend to burst open and result in hemarthrosis filled with multiple comminuted fragments of bone with high osteogenic potential. it is suggested that direct contact between the articular fossa and the fractured fragments with torn joint meniscus are commonly found in intracapsular fractures of temporomandibular joint.2 the patient in this case exhibited severe growth disturbance of the mandible characterized by the obvious facial asymmetry, mandibular retrognathism, and radiographically by the presence of bony prominence at the angle of the mandible. how the ankylosis of the temporomandibular joint can affect mandibular growth can be explained with several theories of mandibular growth. brash in 1930 dictated that condylar cartilage was thought to provide the dominant growth impetus to the mandible and the term "condylar growth centre" was much used. the mandible was likened to a long bone, with the cartilage of the condyle acting as an epiphyseal growthplate cartilage. additional appositional bone formation was thought to be stimulated at the sites of insertion of the masseter at the angle, the temporalis to the coronoid, and the alveolar margin in response to the stimulus of mastication.2 the theory, proposed by mosh in 1968, also known as "functional matrix theory'' stated that the mandible develops in conjunction with the morphogenetic demands of the enveloping soft tissues, particularly muscles and ligaments, acting through their periosteal attachments. the mandibular condyle is not the site of primary growth but has secondary adaptive response which allows the condylar head to stay in the glenoid fossa as the mandible develops downwards and forwards with the demands of the functional matrix.2 the condylar growth centers are crucial in mandibular development. each centre consists of chondrogenic, cartilaginous, and osseous zones. a thin vascular layer covers the chondrogenic zone. bone is deposited at the posterior borders of the rami and condyles. trauma to the growth center just beneath the articular disc is important to be concerned. delayed growth on the affected side can cause facial asymmetry, mandibular deviation, and malocclution.1mandibular growth is principally attributed to intramembranous osteogenesis, augmented by focal figure �. one week post-operative panoramic x-ray showing the gap created in the right subcondylar neck. figure 5. three weeks post-operative condition showing normal mouth opening. 71endrajana: ankylosis of the temporomandibular joint endochondral ossification at the condylar head, mandibular angle, and coronoid process. the subsequent complex growth patterns including synchronized cortical drifting at the lateral and medial periosteal surfaces leading to forward and downward mandibular rotation and expantion. mandibular growth also depends on muscles attachment, i.e. temporalis, masseter, and pterygoid muscles, while tooth development and eruption influence alveolar development.7 regardless of which theory is used to explain the mandibular growth, it is clear that since the condylar fracture in the above case took place within the growing period of the mandible, the normal growth on the right side of the mandible has not ensued. the condyle, which plays important role in the elongation of the mandible, was structurally damaged. in addition, as there has been no mastication for a long time the apposition type of growth did not occur in the sites of masseter and temporal muscle insertion. this two masticatory muscles induce the mandibular growth. it is believed that temporomandibular ankylosis in growing child may alter the growth because of destruction of the growth area in the tmj cartilage as well as function limitation will decrease the influence of soft tissues on developing bone.8,9 it is clear that both panoramic and ct scan showed bony union between condyle and the fossa on the right side. these findings are consistent with the criteria for bony ankylosis.4,10 the left temporomandibular joint, on the other hand, seems to have normal structure with the articular space still being present. as the diagnosis of the patient was bony ankylosis of the temporomandibular joint the treatment of choice was surgery. the basic objective of tmj ankylosis surgery should be to restore mouth opening and joint function.3,11 regardless of variables, such as age and type of ankylosis, the basic principles of ankylosis release should be followed, these including gap arthroplasty for resection of the ankylosis mass and interpositioning of a material or structure of choice to prevent the recurrence of ankylosis. in this case gap arthoplasty was performed and an interpositional material, mersilen meshtm, was placed in the gap to prevent the recurrence of ankylosis. the created gap would then act as a new joint, also termed as pseudoarthrosis.12 this surgical procedure enables the patient to open his mouth normally. in conclusion, ankylosis of temporomandibular joint and disturbance of mandibular growth may occur as the complication of condylar fracture in children. the first sign of significant problems may be the increasing limitation of jaw opening. early diagnosis, good treatment and postoperative physiotherapy of the temporomandibular joint should be done to achieve the optimum mouth opening as well as to avoid the recurrence of the trismus. references 1. faust ra, younes aa. mandible fractures in children. available at: http://emidicine.medscope.com/ article/872662-overview. accessed june 16, 2010. 2. bradley p. injuries of the condylar and coronoid process. in: rowe nl, williams jli. editors. maxillofacial injuries. volume 1. edinburg, london, melbourne, new york: churchill, livingstone; 1985. p. 337–61. 3. gerlach kl, erle a, eckelt u, loukota ra, luhr hg. surgical management of mandibular, condylar neck, and atropic mandible fractures. in: booth pw, schendel sa, hausamen je, editors. maxillofacial surgery. volume 1. churchill, livingstone. 2007 p. 80–92. 4. nayak pk, nair sc, krishnan dg, perciaccante vj. ankylosis of theankylosis of the temporo mandibular joint. in: booth pw, schendel sa, hausamen je, editors. maxillofacial surgery. volume 2. churchill, livingstone. 2007. p. 1521–37. 5. rishiray b, mc. fadden lr. treatment of temporomandibular joint ankylosis: a case report. j can dent assoc 2001; 67(11): 659–63. 6. weteid aa, elkrish e, mutairi ka, foghm sa. temporomandibular joint ankylosis caused by mastoiditis: presentation of a rare case and literature review. saudi dent j 2000; 12(2): 103–5. 7. lavelle c. craniofacial development in development and anatomy relevant to the head, neck, and orofacial tissues. available at: http://www. abe.pl/html/samples/b/0198510969.pdf. accessed june 16, 2010. 8. tucker mr. correction of dentofacial deformities. in: peterson lj, ellis e iii, hupp jr, tucker mr, editors. contempory oral and maxillofacial surgery. 2nd ed. st. louis: mosby; 1993. p. 613-5. 9. li z, zhung w, li zb. induction of traumatic temporomandibular joint ankylosis in growing rats: a preliminary experimental study. dent traumatol 2009; 25(1): 136-41. 10. el-hakim ie, metwalli sa. imaging of temporomandibular jointimaging of temporomandibular joint ankylosis: a new radiographic classification. dentomaxillofac radiol 2002; 31(1):19-23. 11. bayat m, badri a, moharamnejad n. treatment of temporomandibular joint ankylosis: gap and interpositional arthroplasty with temporal muscle flap. j oral maxillofac surg 2009; 13(4): 207-232-212. 12. ortak t, ulusoy mg, sungur n. silicon in temporomandibular joint ankylosis surgery. j craniofac surg 2001; 12(3): 232-6. contents page printed by: airlangga university press. (043/03.09/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail:aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 42 number 1 january-march 2009 issn 1978 3728 dental journal majalah kedokteran gigi 1. the transformation of ordinal scale for parametric statistic analysis on dental health questionnaire adi hapsoro ...................................................................................................................................... 1–5 2. chronic gingivitis and aphthous stomatitis relationship hypothesis: a neuroimmunobiological approach chiquita prahasanti, nita margaretha, and haryono utomo ..................................................... 6–11 3. the role of microendodontics in treating mandibular second molar with five canals harry huiz peeters .......................................................................................................................... 12–14 4. toxicity testing of chitosan from tiger prawn shell waste on cell culture maretaningtias dwi ariani, anita yuliati, and tokok adiarto .................................................. 15–20 5. the effect of toothpaste containing kayu sugi extract on plaque formation widowati w, rar awang, nh ismail, and sh othman ............................................................. 21–24 6. simple replantation protocol to avoid ankylosis in teeth intended for orthodontic treatment yuli nugraeni, david buntoro kamadjaja, and haryono utomo ............................................... 25–30 7. treatment on temporomandibular disorder using occlusal splint agus dahlan ..................................................................................................................................... 31–36 8. inhibitory effect of n-hexane: ethyl acetate fraction from artemisia vulgaris l. on cell culture of oral epithelial carcinoma ira arundina ..................................................................................................................................... 37–40 9. antioxidant effect of minocycline in gingival epithelium induced by actinobacillus actinomycetemcomitans serotype b toxin ernie maduratna setiawati ............................................................................................................. 41–45 10. esthetic rehabilitation of crowded and protruded anterior dentition cecilia g. j. lunardhi and eric priyo prasetyo ............................................................................ 46–49 11. patient safety oriented to improve patient retention in oral health services tri erri astoeti ................................................................................................................................. 50–53 mkg vol 41 no 4 oct-dec 2008.indd 173 vol. 41. no. 4 october–december 2008 case report delayed bracket placement in orthodontic treatment chandra wigati dr. saiful anwar hospital, malang abstract background: beside bracket position, the timing of bracket placement is one of the most essential in orthodontic treatment with fixed appliances. even it seems simple the timing of bracket placement can be crucial and significantly influence the result of orthodontic treatment. however it is often found brackets are placed without complete understanding of its purpose and effects, which could be useless and even detrimental for the case. purpose: the aim of this case report is to show that the timing of bracket placement could be different depending on the cases. case: five different cases are presented here with different timing of bracket placement. case management: on these cases, brackets were placed on the upper arch first, on the lower arch first, or even only on some teeth first. good and efficient orthodontic treatment results were achieved. conclusion: for every orthodontic case, from the very beginning of treatment, bracket should be placed with the end result in mind. if brackets are correctly placed at a correct time, better treatment result could be achieved without unnecessary round tripping tooth movement. key words: bracket placement, various orthodontic cases, delayed placement correspondence: chandra wigati, c/o: buring 51 malang, indonesia. tel. 0341 363415. e-mail: chandrawigati@yahoo.com introduction setting up the case is the most important aspect of the treatment, after the correct diagnosis and treatment planning. banding and bonding should therefore not be delegated and should be managed by the orthodontist, to ensure accuracy of appliance placement.1 in bracket placement there are two things to be considered: the position and the timing of bracket placement. of these, only the timing of bracket placement will be discussed in this article. for many patients, it is correct to place all the brackets and bands at the start of treatment so that any discomfort is limited to one episode and all the teeth start to be corrected from the outset. however, in some situations, it may be beneficial to consider partially setting up the case, leaving individual teeth, and in some instances groups of teeth, without attachments. if individual teeth are vertically or horizontally displaced from the primary arch form (figure 1), it is often good technique to delay bracketing the displaced tooth until the other teeth are well aligned, and space has been made available.2 if bracket placement on individual teeth interferes with tooth movements of their antagonist, then it is better to left those teeth not bracketed.3 in deep-bite cases, when it has been decided not to use a bite plate or occlusal build-up, upper arch should be bonded first, later, after the overbite has started to correct, it will be possible to place the lower incisor brackets without discomfort to the patient or risk figure 1. bracket placement on upper laterals were delayed, until space became available.2 174 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 173−178 of damage to the enamel or the newly placed brackets. in cases where the incisors would inevitably procline if they are bracketed at the start of treatment, as in non-extraction case with triangular-shaped incisors, it is better not to bond the incisors until space become available. where a sliding jig is used to control or distalize molars, upper bicuspids and sometimes upper canines are normally not bracketed. in many mixed dentition treatments, only the permanent teeth are included in the set-up. primary teeth may be included in some cases, either to improve the strength and stability of the appliance, or to influence the position of the primary teeth.1 nowadays, with the increasing popularity of self ligating brackets, every system must be thoroughly understood, as in some cases the timing of bracket placement can be different depending on the system. with damon system, it is always advisable to bond and engage as many teeth as possible so lateral adaption can start as early as possible.4 it is clear that there are so many variations in the timing of bracket placement, which can greatly influence the treatment outcome and length of treatment. unfortunately it is often found brackets were placed without complete understanding of their purpose and effects, making undesirable side effects and lengthen treatment time. the aim of this case report is to show some alternatives of the correct timing of bracket placement, so that best orthodontic treatment outcome could be achieved without unnecessary round tripping tooth movement. hopefully more patients would get benefit from good orthodontic treatment, by correct timing of bracket placement with the most efficient time. cases five different cases are shown to illustrate the effect of different timing of bracket placement. besides the careful mechanics chosen, some bracket placements were delayed to get the best treatment outcome in an efficient time. case i: a 15 year-old male with class i malocclusion, severe crowding, 33 was impacted, and 36 had a severe cavity figure 2). case ii: a 15 year-old female with class iii malocclusion, anterior and posterior cross bite, severe crowding(figure 5). case iii: a 12 year-old male with class i malocclusion, moderate crowding, 21 protruded, and 15 missing (figure 9). case iv: an 11 year-old female with a class i malocclusion, moderate crowding and impacted upper left canine (figure 12). case v: 13 year-old female with class i malocclusion, constricted maxillary arch, and severe upper and lower crowding (figure 15). figure 2. case i: before treatment. figure 3. case i: eight months of treatment, brackets were about to be bonded on lower arch. 175wigati: delayed bracket placement case management case management i: 12, 24, 34, 36, and 44 were extracted. fixed appliance were placed in the upper dentition first while waiting for the 37 to erupt fully. brackets were bonded on the lower arch 8 months after bracket placement on the upper arch (figure 3). the case finished after 28 months of treatment (figure 4). case management ii: 14, 24, 34, 44 were extracted. brackets were bonded on upper and lower teeth, except on 31, 32, 41, 42 (figure 6). after 33 and 43 were uprighted, brackets were bonded on 31, 32, 41 and 42 (figure 7). the case was finished after 39 months of treatment (figure 8). case management iii: 24, 34, 44 were extracted. brackets were bonded on upper teeth except on 21 figure 4. case i: at the time of upper teeth debonding. figure 5. case ii: before treatment. figure 6. case ii: bracket were bonded on upper and lower teeth except on 31, 32, 41, 42. figure 7. case ii: brackets were bonded on 31, 32, 41, 42. figure 8. case ii: at the time of debonding. figure 9. case iii: before treatment. 176 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 173−178 (figure 10). the case was finished after twenty months of treatment (figure 11). case management iv: treated with extraction of 63, 14, 24, 35, 45. brackets were bonded on upper and lower dentition except on 22, bracket was bonded on 22 after 23 moved away from 22 (figure 13). the case was finished after 48 months of treatment (figure 14). case management v: treated with extraction of 14, 24, 34, 44. rapid palatal expander was placed on the maxillary arch to expand the constricted maxilla. brackets placement figure 10. case iii: (a) brackets were bonded on upper teeth except on 21, (b) 1 month after upper bracket bonding, (c) 2 months after upper bracket bonding, 21 was bonded, (d) 4 months after upper bracket bonding. figure 11. case iii: at the time of bracket debonding. figure 12. case iv: before treatment. a b dc figure 13. case iv: (a) 4 months after treatment, (b) 6 months after treatment, (c) 8 months after treatment, (d) 11 months after treatment, 22 was bonded. a b dc 177wigati: delayed bracket placement on upper teeth were delayed until expansion completed and upper first bicuspids were extracted (figure 16). the case was finished after 44 months of treatment (figure 17). discussion in case i, bracket placement in the lower arch was delayed 8 months after the brackets were placed on upper arch. the delay was mainly caused by the time needed for 37 to fully erupt so that a tube can be bonded on it. according to alexander,3 in an extraction case the mandibular anterior teeth have a tendency to drift distally, while the mandibular posterior teeth drift mesially, but much more slowly. the mandibular anterior crowding has a tendency to treat itself during the first few months of therapy. the late placement of mandibular appliances is referred to as “drifttodontics”. the treatment is usually completed in both arches at approximately the same time, despite the fact that the maxillary arch was bonded earlier in treatment. in this case delayed bracket placement on the lower arch made the crowding in the lower dentition less severe as the anterior lower teeth unraveled and moved distally, the impacted 33 could erupt spontaneously. careful attention must be considered in cases where maximum anchorage is needed. in case ii, the 33 and 43 were distally inclined, if lower incisors were bonded and archwire was tied, the effects would be bite deepening and proclination of the incisors, which were not favorable for this case. with this in mind, brackets placement on lower incisors were delayed until the position of canines were more upright and had moved figure 14. case iv: at the time of debonding. figure 15. case v: before treatment. figure 16. case v bracket placement on upper teeth were delayed until expansion completed. figure 17. case v at the time of bracket debonding. 178 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 173−178 distally, and more rooms for the crowded lower incisors were available. according to bennett5 when the cuspids were very upright, or distally inclined, the most effective way to manage the situation was to delay placing brackets on the incisors. because when the incisors brackets were placed, expression of the archwires caused extrusion of the incisors and undesirable bite deepening. lacebacks were then applied to the cuspids while waiting for the cuspid roots to distalize and the cuspid slot to become more parallel to the occlusal plane. in case iii, if 21 was bonded and engaged into the upper arch wire, it would only procline the other upper incisors and made the treatment more complicated. in this case it was better to leave the 21 without bracket, archwire passed on it and pressing it lingually, while other teeth moved distally occupying the extraction spaces. after there was enough space, and the 21 was not so protrude, then 21 was bonded. to avoid proclination of the other incisors in a case where upper central incisors are proclined in a non extraction class i case, bennett6 left the incisors not bonded, until space are available. for teeth which are significantly out of the arch form, should be left unbracketed until adequate space is provided for their movement and positioning.1 in case iv, the crown of the impacted 23 overlying the root of the 22, causing it to tilt labially. if 22 was bonded and engaged into the archwire, the crown of 22 would tend to tip palatally and the root labially, such movement could cause root resorption of 22. in this case it was better to delay bracket placement on the 22, until the crown of the 23 moved away from the root of the 22. according to bennett and mclaughlin,6 in cases where the unerupted cuspid crown is overlying the root of the lateral incisor and the lateral incisor crown is proclined, it is possible to cause damage to the lateral incisor root by attempting to align the incisor orthodontically before the cuspid crown has been exposed. if a bracket is placed on a lateral in this situation early in treatment, care should be taken not to change the torque and tip position of the lateral incisor. after the cuspids have been moved away from the lateral incisor area, normal incisor leveling and aligning can then be carried out (figure 18). in case v bracket placement on upper teeth was delayed until the expansion was stopped. according to alexander2 correction of the transverse dimension is performed during the early stages of treatment. if a patient has a posterior cross bite, and corrected with rapid palatal expander, it is done prior to upper brackets bonding. as central incisors separated during the separation of median palatine suture, the upper teeth should be free to move and not be bonded (figure 19).7 those five cases showed the timing of bracket placement could be different in different cases. beside these five cases shown here, there are still many variations in the timing of bracket placement depending on the cases. successful tooth alignment depends on recognizing that unwanted tooth movements can occur early in treatment, mainly owing to the bracket placement. these unwanted tooth movements need to be controlled, or the underlying malocclusion will worsen during tooth alignment, which will increase the time and effort needed to complete the case, later in treatment.1 if brackets are correctly placed at a correct time, better treatment outcome could be achieved without unnecessary round tripping tooth movement. therefore, at the first stage of treatment, bracket placement should be carried out with the final treatment goal in mind. in conclusion, in orthodontic treatment each case must be accessed individually and carefully, as the timing of bracket placement could be different and could affect the treatment outcome and length of treatment. references 1. bennett jc, mclaughlin rp, trevisi. systemized orthodontic treatment mechanics. new york: mosby; 2001. p. 57–8, 109. 2. smith pl, dyer f, sandler pj. alignment of blocket-out maxillary lateral incisors. j clin orthod 2000; 34(7): 434–7. 3. alexander rg. the alexander discipline. california: ormco corp; 1986. p. 183, 211. 4. jong lin jj. creative orthodontics. blending the damon system and tads to manage difficult malocclusions. taiwan: elitecolor repro & prints; 2007. p. 142, 144, 148, 155. 5. bennett jc, mclaughlin rp. orthodontic treatment mechanics and the preadjusted appliance. london: mosby-wolfe; 1993. p. 71. 6. bennett jc, mclaughlin rp. orthodontic management of the dentition with the preadjusted appliance. oxford: isis medical media ltd ; 1997. p. 86, 98, 171. 7. bishara se, staley rn. maxillary expansion: clinical implications. am j orthod dentofac orthop 1987; 91(1):3-14. figure 18. damage of the lateral incisor root may has been caused, or made worse, by attempt to correct lateral incisor, before the cuspid crown was moved out of the way.6 figure 19. central incisors were separated during the separation of median palatine suture.7 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 41 no 4 oct-dec 2008.indd 194 vol. 41. no. 4 october–december 2008 research report immunodetection of rasp21 and c-myc oncogenes in oral mucosal swab preparation from clove cigarette smokers silvi kintawati department of oral biology faculty of dentistry padjadjaran university bandung indonesia abstract background: smoking is the biggest factor for oral cavity malignancy. some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance and produce abnormal genes (oncogenes). oncogenes ras and myc are found on malignant tumor in oral cavity which are associated with smoking. purpose: this research is to find the expression of oncogenes rasp21 and c-myc in oral mucosa epithelial of smoker with immunocytochemistry reaction. methods: an oral mucosal swab was performed to 30 smokers categorized as light, moderate, and chain, and 10 non smokers which was followed by immunocytochemistry reaction using antibody towards oncogene rasp21 and c-myc is reacted to identify the influence of smoking towards malignant tumor in oral cavity. the result is statistically analyzed using kruskal-wallis test. result: based on the observation result of oncogene rasp21reaction, it shows that there is significant difference between non smoker group and light smoker, compared to moderate and chain smoker group (p < 0.01). on the other side, the observation result of oncogene c-myc indicates that there is no significant difference between the group of non smokers and the group of light, moderate, and chain smokers (p > 0.05). conclusion: the higher the possibility of oral cavity malignancy and that the antibody for rasp21 oncogene can be used as a marker for early detection of oral cavity malignancy caused by smoking. key words: smokers, rasp21, c-myc, oncogene, immunohistochemistry correspondence: silvi kintawati, c/o: bagian biologi oral, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: letisrei@bdg.centrin.net.id introduction today, malignant tumor is one of the most dangerous causes triggering death for human. tumor in oral cavity is one of malignant tumors that is rapidly increased. even though its cause is not definitely known, it has been claimed that there are multi-factors causing malignant tumor in oral cavity, for examples environmental predisposition factor and genetic factor. of various factors causing malignant tumor in oral cavity, smoking is the most common cause.1,2 some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance. the growth of malignant tumor is related to the epithel change that is triggered by the number of carcinogens and the length of carcinogen development. the longer the carcinogens are developed, the more the risk of getting malignant tumor.2,3 malignant tumor is clinically difficult to be detected in early stage, so it is mostly known at acute stadium. to socialize early diagnosis of malignant tumor, immunocytochemistry reaction is undergone by mucosal swab technique. on mucosal swab technique, it is known that in oral cavity there are changes of mucosal cells caused by development of irritation substance, like tobacco.4,5 carcinogen can produce abnormal genes (oncogene), yielding specific anti-gen. specific anti-gen examination is done by using antibody based on reaction of anti-gene– antibody known as immunocytochemistry reaction. besides oncogene egf-r and c-erbb, other oncogenes found on malignant tumor in oral cavity are myc and ras 195kintawati: immunodetection pf rasp21 and c-myc oncogenes in oral mucosal which are associated with smoking. it has been reported that rasp21 produced from lineage protein of gene ras is found on dysplasia and on early stage of tumor. meanwhile on malignant tumor, c-myc level is augmented.6,7 based on the background mentioned above, this study is conducted to detect the expression growth of oncogene myc (c-myc) and ras (rasp21) on the cells found in oral cavity of cigarette smokers whose mucosa has been swabbed. by considering that smoking is one of the greatest factors causing malignant tumor in oral cavity,2,8 increased the expression of these oncogenes–c-myc and rasp21–on cigarette smokers is possibly found. the reason is that those smokers have consumed many cigars. materials and methods the research conducted by the writer was laboratory research using ex post facto model. the samples of the research were 30 smoker employees of faculty of dentistry, padjadjaran university. the criteria for sample were 1) male; 2) aged above 40 years old; 3) they had been smokers for 5 years or more; 4) clinically there was no disorder of oral cavity. the smokers were classified into light smokers (less than 10 cigars in one day), moderate smokers (11-20 cigars in one day), chain smokers (more than 20 cigars in one day). as comparison, 10 non smokers were selected from employees. one-way swabbing from inner part to outer part in mucosa of oral cavity for three times was conducted for smokers and non smokers using wooden spatula. after the result of one-way swabbing was smeared on glasses, it was dried for 1 hour. then, ethyl-alcohol was reacted on it for 1 hour. reacting immunocytochemistry was the next step, using dako lsab (labeled streptavidin biotin/lsab method) kit peroxidase. antibody used in this research was antibody towards rasp21 and c-myc. monoclonal antibody rasp21was obtained from “oncogene science”, being dissolved 1 : 20. meanwhile, c-myc was taken from “oncogene science” clone 9e10; being dissolved 1 : 10. lsab kit peroxidase was gained from dako ko-681. the process: after the result of mucosal swab technique was dried in open air and reacted with ethylalcohol for one hour, it was incubated by methanol containing h2o2 2% for 20 minutes. next, it was rinsed using alcohol 90% for 5 minutes, alcohol 80% for 5 minutes, alcohol 70% for 5 minutes and put in “tri buffer saline” (tbs) for 3 × 5 minutes. then, it was incubated using blocking reagent for 5 minutes and later incubated in each primary antibody (rasp21 and c-myc) for 30 minutes; rinsed with tbs for 3 × 5 minutes; incubated by linking antibody for 10 minutes; rinsed with tbs for 3 × 5 minutes; incubated by strep-avidin biotin for 30 minutes; rinsed with tbs for 3 × 5 minutes; incubated by chromogene substance, diaminobenzidine (dab), for 5 minutes. after the color was formed, it was rinsed by flowing water, and counter-stain was given on it, using meyer’s hematoxylin for 2 minutes. the result of immunocytochemistry reaction was observed by light microscope using 400 times enlargement. the observation of oncogene rasp21 and c-myc level was based on percentage (the number of positive cells on each oncogene and the number of all cells on each result of swabbing mucosa in oral cavity), which was shown on the following equation: number of positive cells × 100% number of all cells (in 1 result) the result of reaction would be stated positive for rasp21 and c-myc if the color on cytoplasm cell turned brown. it would be stated negative if the color did not turn into brown. result the data were taken from 30 smoker-employees and 10 non smoker employees in faculty of dentistry padjajaran university, who had been treated with the mucosal swab technique for oral cavity, and then had been tested with immunocytochemistry reaction by using ras p-21 and c-myc antibodies, shown with the following table 1 and table 2. table 1. the estimation of the expression level of ras p-21 oncogenes (%) groups of smokers ras p-21 expression nonsmokers light smokers moderate smokers chain smokers subject 1 subject 2 subject 3 subject 4 subject 5 subject 6 subject 7 subject 8 subject 9 subject 10 0 0 0 0 0 0.200 0.394 1.122 2.621 4.860 0 0 0.381 0.852 1.131 1.142 2.385 2.842 4.110 5.054 2.624 3.921 4.230 6.245 6.786 10.600 12.170 19.372 23.410 23.951 2.901 3.010 4.067 7.425 9.197 11.573 12.197 21.171 30.072 30.870 x 0.9197 1.7897 11.3309 13.2483 by using kruskal-wallis test, the estimation of the expression level of ras p-21 oncogenes between nonsmoker group and the other three groups, light smoker groups, moderate smoker groups, and chain smoker 196 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 194−198 groups did show any significant difference (p < 0.01). in addition, mann-whitney test was also used in order to determine pairs of different groups. the result of mannwhitney test shows that there was no significant difference (p > 0.05) between light smoker group and non smoker group. however, there was significant difference (p < 0.01) between non-smoker group and other two groups, moderate smoker group and chain smoker group. there was also significant difference (p < 0.01) between light smoker group and other two groups, moderate smoker group and chain smoker group. nevertheless, there was no significant difference between moderate smoker group and chain smoker group (p > 0.05). table 2. the estimation of the expression level of c-myc oncogenes (%) groups of smokers c-myc expression nonsmokers light smokers moderate smokers chain smokers subject 1 subject 2 subject 3 subject 4 subject 5 subject 6 subject 7 subject 8 subject 9 subject 10 0 0 0 0 0 0 0 0 0.112 0.193 0 0 0 0 0 0 0 0 0.972 1.091 0 0 0 0 0 0 0 0 0.670 1.840 0 0 0 0 0 0 0 0.198 1.023 1.240 x 0.0305 0.2063 0.2510 0.2461 by using kruskal-wallis test, the estimation of the expression level of c-myc oncogenes between non-smoker group and the other three groups, light smoker groups, moderate smoker groups, and chain smoker groups did not show any significant difference. (p > 0.05). figure 1. positive cell towards ras p-21 oncogenes 200×. figure 2. positive cell towards ras p-21 oncogenes 400×. figure 3. positive cell towards c-myc oncogenes 200×. figure 4. positive cell towards c-myc oncogenes 400×. figure 5. negative cell towards ras p-21 and c-myc oncogenes 200×. 197kintawati: immunodetection pf rasp21 and c-myc oncogenes in oral mucosal figure 6. negative cell towards ras p-21 and c-myc oncogenes 400×. discussions it has been clearly analyzed that cigarette smoke consists of many harmful chemicals, as a result, smoking habit can cause many defects in oral cavity like malignant tumor.9 the malignant tumor in oral cavity, which is often suffered, is squamous cell carcinoma.8,10 based on many researches, there is a strong relation between smoking habit and the malignant tumor in oral cavity.9,11 according to a research in america, the risk of smokers in suffering the malignant tumor is even six times as high as that of nonsmokers.12 moreover, the risk of suffering the malignant tumor in oral cavity is higher for patients above 40 years old, as well as for chain smokers.10 many carcinogens contained in cigarette can stimulate epithel cells in oral cavity and cause trouble in the defense mechanism of tissues. the development of the malignant tumor is related to the change of epithel caused by the number and the period of the spread carcinogens. the longer the period of the spread carcinogens and the bigger the number of them, the higher the risk of getting the malignant tumor is.2,3 the mucosal swab technique for oral cavity can become a routine diagnostic examination for detecting the defects in oral cavity since this examination is sensitive, fast, and unpainful.4,13 in this research, the mucosal swab technique for oral cavity is taken in order to take and analyze mucosa cells of oral cavity. as we know that, carcinogenes of cigarette can create abnormal genes while oncogenes can produce specific antigens. thus, the examination of specific antigens can be done by using antibodies based on antigen-antibody reaction known as immunocytochemistry reaction. in this research, the immunocytochemistry reaction is used in order to know whether the level of ras p-21 oncogene expression and the level of c-myc oncogene expression in mucosal cells swabbed from oral cavity are related to the number of clove cigarettes consumed. the result of this immunocytochemistry reaction then shows that there is a relation between the level of ras p-21 oncogene expression and the number of clove cigarettes consumed. however, it does not show any relation between the level of c-myc oncogene expression and the number of clove cigarettes consumed. ras p-21 protein is a product of ras gene family, mammal gene, which is often related to the malignant tumor. ras p-21 is a phosphor protein bound by plasma membrane with 188–189 amino acid residues, and has intrinsic gtpase activities. ras p-21 also has the same biological characteristics as protein-g, known as “signal transducer” from membrane receptors to cytoplasm effectors. in other words, ras p-21 has a function as a controller of the information exchange from membrane to nucleus. if this ras p-21 genes is activated by mutation, its function can be disturbed since the p21-gtp complexity becomes ceaselessly active and then causes any possibilities of the neoplastic change. ras p-21 protein mutant has a character of transferring transduction signals from outside cell, especially the signals of developing extra cell into its effectors in nucleus. ras p-21 protein mutant can stimulate tumorgenesis process with its abilities of continuing transferring transduction signals of developing extra cell ceaselessly, so apoptosis can be inhibited. this condition then causes an early stimulation process of tumorgenesis.14 according to varghese et al.,6 ras p-21 can be found in the malignant tumor in oral cavity. based on the result of this study, it can be noticed that there is an increasing expression level of ras p-21, which is higher for moderate smoker group and chain smoker group than for light smoker group and non-smoker group (table 1). it indicates that the bigger the number of cigarettes consumed, the higher the level of rasp21 oncogenes, causing the inhibition of apoptosis and the possibility of the malignant tumor in oral cavity. furthermore, c-myc gene is located on chromosome 8. the level of c-myc expression can increase in malignant tumor in oral cavity, especially in an advanced stage or in relating to poor prognosis.7 based on the result of this study, it can be realized that there is no increasing expression level of c-myc oncogenes in mucosal cells swabbed from oral cavity either for light smoker group, moderate smoker group, chain smoker group, or for non-smoker group (table 2). the reason is because the level of c-myc expression can increase if the malignant tumor is aggressive, and in an advanced stage,7 meanwhile in this study the mucosal cell swabbed from oral cavity of light smoker group, moderate smoker group, and chain smoker group, which are clinically diagnosed without any symptoms of the malignant tumor in their oral cavity. based on all the results, it can be concluded that there is an activation of ras p-21 oncogenes in swab mucosal technique for smokers’ oral cavity. as a comparison, the result of this study is the same as the result of kintawati15study, showing that there is an activation of some oncogene and receptor of development factor, like egf-r and c-erbb oncogenes in swab mucosal technique for smokers’ oral cavity. according to some researchers, the oncogene activation which is related to the gene changes can stimulate the malignant tumor including the malignant 198 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 194−198 tumor of oral cavity.1,13 this study shows that moderate smoking and chain smoking can stimulate gene mutation which then cause the malignant tumor of oral cavity. thus, it may be concluded that ras p-21 oncogenes expression has increased in moderate smoker groups and in chain smoker ones. as a result, it can become an indicator for early detection of malignant tumor in oral cavity because of smoking. for this reason. moderate smokers and chain smokers are recommended to do routine screening in order to detect malignant tumor of mucosa in oral cavity. references 1. lippman sm, sudbo j, hong wk. oral cancer prevention and the evolution of molecular-targeted drug developement. j clin oncol 2005; 23(2): 346–56. 2. regezi ja, sciubba jj, jordan rck. oral pathology. 5th ed. saunders: elsevier; 2008. p. 48–52. 3. abeloff md, armitage jo, niederhuber je, kastan mb, mckenna wg. clinical oncology. 3rd ed. philadelphia: elsevier inc. 2004; p. 1498–1541. 4. koss lg. koss’ diagnostic cytology and its histopathologic bases. 5th ed. philadelphia: lippincott; 2006. p. 99. 5. sundbo j, samuelsson r, risberg b, heisten s, nyhus c, samuelsson m, et al. risk markers of oral cancer in clinically normal mucosa as an aid in smoking cessation counseling. j clin oncol 2005; 23(9): 1927–33. 6. varghese p, balaram p, das bc. ras p21 and its expression variations in the development of oral squamous cell carcinoma. ind j med resch 2005; 81. 7. vita m, henriksson m. the myc oncoprotein as a therapeutic target for human cancer. semin cancer biol 2006; 16(4): 318–30. 8. morse de, psoter wj, cleveland d, cohen d, mohit-tabatabai m, kosis dl, et al. smoking and drinking in relation to oral cancer and oral epithelialdysplasia. j cancer causes control. 2007; 18(9): 919–29. 9. study suggests nicotine may promote cancer development. ca cancer j clin 2003; (53):66-68. [update 2008]. available from: http:// www. caonline.amcancersoc.org/. accesed november, 2006. 10. slootweg pj, everson jw. tumours of the oral cavity and oropharynx. in: barners i, everson jw, reichart p, sindransky, eds. pathology & genetics of head and neck tumours. lyon: iarc press; 2005. p. 166–75. 11. lee ch, ko yc, huang hl, chao yy, tsai cc, shieh ty, et al. the precancer risk of betel quid chewing, tobacco use and alcohol consumption in oral leukoplakia and oral submucous fibrosis in southern taiwan. british j cancer 2003; 88: 366–72. 12. gosselin bj. malignant tumours of the mobile tongue. available from url:http//www.emedicine.com/ent/topic256.htm. accessed january, 2007. 13. kujan o, desai m, sargent a, bailey a, turner a, sloan p. potential applications of oral brush cytology with liquid-based technology: result from a cohort of normal oral mucosa. j oral oncology. 2006; 42: 810–18. 14. diaz r, lopez-barcons l, ahn d, garcia-espana a, yoon a, matthews j, et al. complex effects of ras proto-oncogenes in tumorigenesis. carcinogenesis. 2004; 25(4): 535–39. 15. kintawati s. immunodetection of egf-receptor, c-erbb oncogenes and hsv-1 antigenes in oral mucosal swab preparation from clove cigarette smokers. padjadjaran j dent 2008; 20(2): 116–22. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 41 no 4 oct-dec 2008.indd issn 1978 3728 dental journal ! nbkbmbi!lfeplufsbo!hjhj editorial board of dental journal (majalah kedokteran gigi) sk: 118/j03.1.21/kp/2008 january 2nd, 2008 − january 2nd, 2010 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: dr. elly munadziroh, drg, m.s. (dental material – airlangga university) editorial boards: prof. dr. m rubianto, drg, m.s., sp.perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d., fds. (conservative dentistry – university of guy’s dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery – university of saarland, homburg, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery – university science malaysia, malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic – university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry – university of hiroshima, japan); prof. kozai katsuyuki, d.d.s., ph.d. (pediatric – university of hiroshima, japan); dr. nugrohowati, drg, m.kes. (conservative dentistry – prof. dr. moestopo university); dr. m. suharsini, drg, m.s., sp.kga. (pediatric dentistry – indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic – jember university); widowati witjaksono, drg., ph.d. (periodontic – university science malaysia, malaysia); prof. dr. a.g.m. tielens (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands); kok van kessel (medical microbiology – university medical centre, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. lakshman samaranayake (oral microbiology – the university of hongkong). managing editors: dr. r. darmawan setijanto, drg, m.kes. (department of dental public health – airlangga university); prof. dr. arifzan razak, drg, msc, sp. pros. (prostodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp. kg. (conservative dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d.,sp.ort. (ortodontic – airlangga university); prof. dr. mieke sylvia m. a. r., drg, m.s.,sp.ort. (ortodontic – airlangga university); prof. dr. istiati soehardjo, drg, m.s. (oral biology – airlangga university); dr. anita yuliati, drg, m.kes. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); seno pradopo, drg, s.u., ph.d. sp. kga. (pediatric dentistry – airlangga university); udijanto tedjosasongko, drg, ph.d.,sp.kga. (pediatric dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg.,su.,sp.bm. (oral maxillofacial surgery – airlangga university); markus budi rahardjo, drg, m.kes. (oral biology – airlangga university); endang pudjirochani, drg, m.s., sp. pros. (prostodontic – airlangga university); ira widjiastuti, drg, m.kes.sp.kg. (consevative dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); bagus soebadi, drg, mhped. sp.pm. (oral medicine – airlangga university); ketut suardita, drg.,ph.d. (conservative dentistry – airlangga university); sianiwati goenharto, drg., m.s. (ortodontic – airlangga university); devi rianti, drg., m.kes. (dental material – airlangga university); chiquita prahasanti, drg.,sp.perio. (periodontic – airlangga university); dr. eha renwi astuti, drg., m.kes. (roentgen – airlangga university); dr. diah savitri ernawati, drg.,msi. (oral medicine – airlangga university); rostiny, drg., m.kes.,sp.pros. (prostodontic – airlangga university). administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 41 no. 4 october–december 2008: prof. lakshman samaranayake (oral microbiology – the university of hongkong) kok van kessel (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands) sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) endanus harijanto, drg., m.kes. (dental material – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478 / 5030255. fax. (031) 5039478 / 5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 48/dikti/kep/2006 volume 41 number 4 october-december 2008 contents page printed by: airlangga university press. (020/01.09/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail:aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 41 number 4 october-december 2008 issn 1978 3728 dental journal ! nbkbmbi!lfeplufsbo!hjhj 1. effect of oxygen hyperbaric therapy on malondialdehyde levels in saliva of periodontitis patients with type 2 diabetes mellitus dian mulawarmanti and widyastuti .............................................................................................. 151–154 2. reducing allergic symptoms through eliminating subgingival plaque haryono utomo, chiquita prahasanti, and iwan ruhadi ........................................................... 155–159 3. the role of dentists on medically compromised children’s oral and dental prophylaxis in hospital roosje rosita oewen ....................................................................................................................... 160–163 4. the surface roughness difference between microhybrid and polycrystalline composites after polishing eric priyo prasetyo, karlina samadi, and cecilia gerda juliani lunardhi .............................. 164–166 5. the relation of periodontal diseases to systemic diseases melanie sadono djamil and boedi oetomo roeslan .................................................................... 167–172 6. delayed bracket placement in orthodontic treatment chandra wigati ................................................................................................................................ 173–178 7. complex aesthetic treatment on anterior maxillary teeth with malposition febriastuti ......................................................................................................................................... 179–181 8. stimulation of type i collagen activity in healing of pulp perforation sri kunarti ........................................................................................................................................ 182–185 9. the combination of sodium perborate and water as intracoronal teeth bleaching agent ananta tantri budi .......................................................................................................................... 186–189 10. bootstrap study to estimate linear regression parameter(application in the study on the effect of oral hygiene on dental caries) ristya widi endah yani .................................................................................................................. 190–193 11. immunodetection of rasp21 and c-myc oncogenes in oral mucosal swab preparation from clove (oral cavity) of cigarette smokers silvi kintawati .................................................................................................................................. 194–198 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 80 the level’s changing of transforming growth factor β2 during canine retraction in non-growing age patient adianti and ameta primasari department of oral biology faculty of dentistry, universitas sumatera utara medan indonesia abstract background: orthodontic tooth movement occurred as a result of alveolar bone remodeling and collagen due to mechanical load. this mechanical load applied to the tooth will exert a number of cytokine and growth factors. one of the growth factors that are often associated with orthodontic tooth movement is transforming growth factor-β (tgf-β). it has 3 isoforms, tgf-β1, tgf-β2, and tgfβ3. it has been known that in adult patient, tooth movement rate was slower. purpose: the aim of this study was to investigate the changing level of tgf-β2 in non-growing patient due to mechanical load in canine retraction. method: gingival crevicular fluid from 6 subjects who undergo canine retraction was taken to investigate changing level of tgf-β2. distal site of each upper canine served as an experimental tooth. the gingival crevicular fluid from experimental tooth was taken just prior to mechanical load, at 24h and 72h after mechanical load. result: elisa reader showed that level of tgf-β2 was decreasing during experiment time. conclusion: it can be concluded that in non-growing patient, tgf-β2 has less role in alveolar bone resorption in orthodontic tooth movement. keywords: bone resorption; non-growing; orthodontic tooth movement; tgf-β2 correspondence: adianti, c/o: fakultas kedokteran gigi universitas sumatera utara. jl. alumni no. 2 kampus usu medan, indonesia. e-mail: adianti9090@gmail.com research report introduction malocclusion has a high impact in individual life quality. the prevalence of malocclusion in indonesia is around 80% and in third place after caries and periodontal disease. along with the improvement of the knowledge society and the desire to improve the quality of life, the demand for orthodontic needs in the community was also increased. the aims of orthodontic therapy were to corrected dental irregularities and disharmony in jaw relations. it utilizes the potential of the periodontal ligament and the alveolar bone to adapt to changing mechanical circumstances by tissue remodeling. by these adaptations teeth can be moved through the alveolar bone and also distant skeletal locations can be affected. by these two mechanisms a stable occlusion and a proper jaw relation can be established.1 there was an increase in the demand for adult orthodontic therapy in the past decades. however, our knowledge on efficiency of adult tooth movement is still incomplete. there is a common believe among the orthodontists that orthodontic procedure will be more timeconsuming in adult patient or non-growing age patient than in growing age patient. younger patient had a greater tooth movement than adults.2 another study also showed there was a faster initial tooth movement in juvenile than in adult animals.3 this finding corroborate another study by ren,4 who found that cellular response in crevicular fluid are less responsive to orthodontic force in old rat than in juvenile. orthodontic tooth movement is achieved by the remodeling of periodontal ligament and alveolar bone in response to mechanical loading. this remodeling is mediated by the activation of several bones remodeling marker. many studies were done in order to give us additional information about this molecular signalization, for example timp-1, col-1, rankl, il-6, sgp-130, etc.3-7 one of the growth factor that still has an unclear role in orthodontic tooth movement is transforming growth factor dental journal (majalah kedokteran gigi) 2015 june; 48(2): 80–83 81 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 81adianti and primasari/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 80–83 7 figure 1. gcf collection with filter paper strip. gcf was collected at distal site of canine represented resorption area. figure 2. graphic showed mean value of tgf-β2 at just prior to mechanical loading, 24 h after mechanical loading, and 72 h after mechanical loading. table 1. the changing level of tgf-β2 at just prior to application of mechanical load, 24 h and 72 h after mechanical load time x ± sd (pg/ l) p 0h – 24h 0.87864 ± 0.96999 0.077 0h – 72h 1.86711 ± 0.77038 0.002 24h – 72h 0.98847 ± 1.28459 0.118 time point * *p<0.05 figure 1. gcf collection with filter paper strip. gcf was collected at distal site of canine represented resorption area. 7 figure 1. gcf collection with filter paper strip. gcf was collected at distal site of canine represented resorption area. figure 2. graphic showed mean value of tgf-β2 at just prior to mechanical loading, 24 h after mechanical loading, and 72 h after mechanical loading. table 1. the changing level of tgf-β2 at just prior to application of mechanical load, 24 h and 72 h after mechanical load time x ± sd (pg/ l) p 0h – 24h 0.87864 ± 0.96999 0.077 0h – 72h 1.86711 ± 0.77038 0.002 24h – 72h 0.98847 ± 1.28459 0.118 time point * *p<0.05 figure 2. graphic showed mean value of tgf-β2 at just prior to mechanical loading, 24 h after mechanical loading, and 72 h after mechanical loading. table 1. the changing level of tgf-β2 at just prior to application of mechanical load, 24 h and 72 h after mechanical load time x ± sd (pg/µl) p 0h – 24h 0.87864 ± 0.96999 0.077 0h – 72h 1.86711 ± 0.77038 0.002* 24h – 72h 0.98847 ± 1.28459 0.118 *p<0.05 β (tgf-β). some author found it as mediator for suppressed osteoclast activity8,9, but in conversely, others found it may contribute to the induction of bone resorption.10,11 tgf-β has 3 isoforms, they are tgf-β1, tgf-β2, and tgf-β3. overexpression of tgf-β2 was found to give a result on an osteoporosis-like phenotype.12 consider there is a role of tgf-β2 in bone remodeling, the aim of this study was to investigate the changing level of tgf-β2 due to mechanical loading in orthodontic tooth movement. this changing level of tgf-β2 was investigated at just prior to mechanical load application, 24 hours after mechanical load and 72 hours after mechanical load. materials and methods subjects of this study were 6 patient age 30-35 years old who undergo canine retraction in private clinic. changing level of tgf-β2 was investigated in gingival crevicular fluid (gcf). ethical approval was obtained from ethic committee and research faculty of dentistry universitas indonesia. subjects of this research were selected according to these following criteria: (1) no history of bone metabolism disease; (2) pocket probing less than 4 mm; and (3) no clinical and radiographic sign of gingivitis and periodontitis. subjects were excluded if they were smoker, and use of antibiotics or non-steroid anti-inflammatory agents in the 6 months prior to the study. informed consent in written form was obtained from the subject before the beginning of the study. gcf samples were collected using filter paper strips (whatman, no. 1) (figure 1 and 2). samples were collected at distal side of upper right and left canine at just prior to mechanical load (0 hour), 24 hours after mechanical load, and 72 hours after mechanical load. teeth were gently washed with water, the site under study were isolated with cotton rolls, and dried gently with air-syringe before paper strips were applied. paper strips were inserted 1 mm subgingivally for 30 seconds. mechanical load applied to teeth was 100 g of force with elastomeric chain. force magnitude was measured with dontrix gauge. paper strip contained tgf-β2 was then diluted with phosphate buffer saline ph 7,7 (gibco) in 1,5 ml eppendorf tube and stored in -80°c in oral biology laboratory faculty of dentistry university of indonesia. elisa assay was done to determinate the level of tgfβ2 in gcf. eppendorf tube contained filter paper strip and phosphate buffer saline were centrifuged at 12000 x 9 rpm for 10 minutes to separate supernatant form. elisa assay was done under the standard procedure. results the changing level of tgf-β2 due to mechanical loading in canine retraction was shown in figure 2. this study showed that there is a changing level of tgf-β2 at different time point. mean value of tgf-β2 level before application of mechanical load is 24.06 ± 0.62 pg/µl. at 24 hour after mechanical load, level of tgf-β2 was slightly decreased (23.28 ± 1.05 pg/µl). 72 hours after mechanical load, it continued to decrease (22.19 ± 1.03 pg/µl). t-test dependent was used to evaluated the statistical significance (p<0.05) between 0 hour compare to 24 hours, 0 hours compare to 72 hours, and 24 hours compare to 72 hours. the results showed at table 1. there was a significance difference at 0 hour compare to 72 hours dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 82 adianti and primasari/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 80–83 (p=0.02; p<0.05). however, it was found there were no significances different at 0 hour compare to 24 hours and 24 hours compare to 72 hours. discussion orthodontic tooth movement is a good example to easily understanding bone remodeling due to mechanical load. in orthodontic treatment, tooth was moved as a result of applied force. this force, according to pressure and tension theory will cause bone resorption in compressed area, and bone formation in stretched area. this movement can be controlled by the magnitude of the applied force and the biological responses from the periodontal ligament (pdl). the force applied on the teeth will cause changes in the microenvironment around the pdl due to alterations of blood flow, leading to the secretion of different inflammatory mediators such as cytokines, growth factors, neurotransmitters, colony-stimulating factors and arachidonic acid metabolities. as a result of these secretions, remodeling of the bone occurs.13 monitoring the biological system in clinical situations would diminish the gap between basic research and clinical implications. it would be advantageous for the clinician and the patient if the reactions of the biological system could be monitoring during treatment. this would allow the adaptation of the treatment to the biological condition of the patient. tgf-β is a multifunction growth factor that played role in bone remodeling. tgf-β is a part of tgf-β super family along with activin, nodal, bone morphogenetic proteins (bmp) and so on. tgf-β have 3 isoforms, they are tgfβ1, tgf-β2, and tgf-β3. how its role in orthodontic tooth movement was remain unclear. this study was done as a pilot study to confirm the present of tgf-β2 due to mechanical load in orthodontic tooth movement. some authors found that level of tgf-β is higher in compression site than tension site.10,11 others found that it has the same level in both compression and tension site.5 during development, tgf-β2 expressed later than tgf-β1 and tgf-β3.14 however, 2 days after application of mechanical load, tooth movement begins as a result of osteoclast and osteoblast remodels the bony socket.13 this is the main reason for time interval choosing in this study. subjects of this study were 30-35 years old patient, because we need to be sure these subjects has no potential adolescent growth to be expected. so there will be no growth hormone affected the changing level of tgf-β2. distal site was choose as an experimental site because we believe this site will be more represent the bone resorptive response than in mesial site in this kind of experimental design. gcf sampling was taken from compression site because according to erlebacher et al.,12 overexpression of tgf-β2 will cause an osteoporosis-like phenotype. thus, it can be concluded that tgf-β2 may play role in bone resorption. we have detected a level of tgf-β2 before application of mechanical load, this might be because there still remain a force used for leveling and aligning. at 24 hours after mechanical load, there was a slight decrease level of tgf-β2. its decrease was continued until 72 hours after mechanical load. we found a statistical significance in comparison of 0 hour and 72 hours after mechanical load (p=0.02). this happen because level of tgf-β2 continued to decrease at 72 hours after mechanical load. this result was different from uematsu et al because they found that in 24 hours after mechanical loading, tgf-β1 was reached its peak level before continued to decrease at 7 days after mechanical loading.10 however, it remains unknown whether tgf-β2 exhibits activity similar to that of tgf-β1.15 it seemed like in non-growing patient, tgf-β2 had no or less effect on bone remodeling in orthodontic tooth movement. this might be an explanation why some treatment in orthodontic seems more time-consuming in adult than in younger patient, as our previous study showed that in juvenile, level of tgf-β2 were increased at 72 hours after mechanical loading. ren3 found that in early state of orthodontic tooth movement, mediator response in adult was slower than in juvenile. this is why there was a delay on initial tooth movement in adult rat.4 on the other hand, in a study experimental on rats, tgf-β showed a lack responsiveness in old than in young rats.16 this result might explain our finding, why in non-growing patient, tgf-β2 continued to decrease after mechanical loading. the result of this study might give the orthodontist additional information of how bone remodeling marker were reacted in adults. however, further study with prolonged time is needed to give information about changing level pattern of tgf-β2 due to mechanical load in orthodontic tooth movement in adult patient. there was a changing of level of tgf-β2 during application of mechanical load in orthodontic tooth movement. tgf-β2 was decreased at 24 h and 72 h after mechanical load. it can be concluded that in non-growing patient, tgf-β2 are less responsive to mechanical load in orthodontic tooth movement. references 1. proffit wr. contemporary orthodontics. 4th ed. st. louis: mosby inc; 2007. p. 296-304. 2. dudic a, giannopoulou c, kiliardis s. factors related to the rate of orthodontically induced tooth movement. am j orthod dentofacial orthop 2013; 143(5): 616-21. 3. ren y, maltha jc, van ‘t hof ma, kuijpers-jagtman am. age effect on orthodontic tooth movement in rats. j dent res 2003; 82(1): 38-42. 4. ren y. cytok ine changes in gcf during or thodontic tooth movement. j clin perio 2002; 29: 757-62. 83 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 83adianti and primasari/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 80–83 5. garlet tp, coelho u, silva js, garlet gp. cytokine expression pattern in compression and tension sides of the periodontal ligament during orthodontic tooth movement in humans. eur j oral sci 2007; 115(5): 355-62. 6. faulkner mg. gingival crevicular fluid (gcf) levels of interleukin-6 (il-6), soluble glycoprotein 130 (sgp), and soluble interleukin-6 r during orthodontic tooth movement. thesis. las vegas: university of nevada; 2011. 7. junior jc, kantarci a, haffajee a, teles rp, fidel r. matrix metalloproteinases and chemokines in the gingival crevicular fluid during orthodontic tooth movement. eur j orthod 2011; 1-7. 8. janssens k, ten djike p, janssens s, van hul w. transforming growth factor beta1 to the bone endocr rev 2005; 26(6): 743-74. 9. kanaan ra, kanaan la. transforming growth factor β1, bone connection. med sci monit 2006; 12(8): ra164-9. 10. uematsu s, mogi m, deguchi t. increase of transforming growth factor-β1 in gingival crevicular fluid during human orthodontic tooth movement. arch oral biol 1996; 41: 1091-95. 11. barbieri g, solano p, alarcón ja, vernal r, rios-lugo j, sanz m. biochemical markers of bone metabolism in gingival crevicular fluid during early orthodontic tooth movement. angle orthod 2013; 83(1): 63-9. 12. erlebacher a, derynck r. increased expression of tgf-β2 in osteoblast result in an osteoporosis-like phenotype. j cell biol 1996; 132(1-2): 195-210. 13. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publisher ltd; 2007. p. 216-20. 14. blobe gc, schiemann wp, lodish hf. role of transforming growth factor β in human disease. n engl j med 2000; 342(18): 1350-8. 15. nishimura r. a novel role for tgf-β1 in bone remodeling. ibms bonekey 2009; 6(11): 434-8. 16. davidson enb, scharstuhl a, vitters el, van der kraan pm, van den berg wb. reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity. arthritis research & therapy 2005; 7(6): r1338-47. 187187 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 187–190 case report burning mouth syndrome caused by xerostomia secondary to amlodipine tengku natasha eleena binti tengku ahmad noor dental officer of 609 armed forces dental clinic, kuching, sarawak, malaysia abstract background: xerostomia, generally referred to as dry mouth, has been identified as a side effect of more than 1,800 drugs from more than 80 groups. this condition is frequently unrecognised and untreated but may affect patients’ quality of life and cause problems with oral and medical health, including burning mouth syndrome (bms). purpose: the purpose of this case is to discuss how to manage a patient with bms caused by xerostomia secondary to medication that has been taken by the patient. case: we reported that a 45-year-old male military officer from the royal malaysian air force came to kuching armed forces dental clinic with dry mouth and a burning sensation since he started taking 10 mg of amlodipine due to his hypertension. after a thorough physical and history examination, we made a diagnosis of burning mouth syndrome (bms) caused by xerostomia secondary to amlodipine. case management: oral hygiene instructions, diet advice and prescription of oral7 mouthwash has been given to reduce the symptoms of bms. the patient has been referred to the general practitioner to reduce his amlodipine dosage from 10 mg to 5 mg (od) in order to prevent xerostomia, and oral hygiene instructions have been given. a review after two weeks showed significant changes in the oral cavity, and the patient was satisfied as he is no longer feeling the burning sensation and can enjoy his food without feeling difficulty in chewing and swallowing. conclusion: adverse drug events are normal in the oral cavity and may have a number of clinical presentations such as xerostomia. xerostomia can cause many implications as saliva helps in maintaining oral mucosa and has a protective function. the signs of adverse drug incidents in the oral cavity should be identified to oral health care professionals. keywords: amlodipine; burning; dry mouth; military officer; xerostomia correspondence: lieutenant (dr) tengku natasha eleena binti tengku ahmad noor, malaysian armed forces dental officer, 609 armed forces dental clinic, kem semenggo, kuching, sarawak, malaysia. email: tengkunatashaeleena@gmail.com introduction burning mouth syndrome (bms) is a benign disorder that presents as a burning sensation in the absence of any apparent findings in the mouth and in irregular blood tests. the underlying bms aetiology remains unclear and is typically characterised by a feeling of burning, itching, or tingling, preferentially at the tip and sides of the tongue, lips, and anterior palate.1 the manifestations of bms are usually bilateral but in some cases may prove unilateral.2 according to cerchiari et al.,3 bms can be classified according to the associated risk factors: idiopathic, psychogenic, local, and systemic. psychopathological processes such as anxiety, depression, and certain phobias are among the psychogenic risk factors. in essence, local causes include infectious processes, allergic reactions, and irritative phenomena, while the systemic aetiological factors include salivary secretion changes, endocrine disorders, neurological changes, dietary factors, and drug substance.2 it is estimated that more than 400 drugs affect the salivary gland function and contribute to hyposalivation.4,5 drugs with anticholinergic activity, including antihypertensive drugs, can cause hyposalivation by reducing the acetylcholine released by the parasympathetic nerves.6,7 amlodipine, which is an antihypertensive agent that acts as a calcium channel blocker, can cause xerostomia through the muscarinic m3 receptor, which results in reduced salivary flow.8,9 diagnosis is based on the history and chronology of oral adverse reactions that are usually identified within weeks or months of the drug being administered. xerostomia is a concomitant symptom in patients with burning mouth syndrome, with prevalence varying between dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p187–190 mailto:tengkunatashaeleena@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p187-190 188 noor/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 187–190 34 and 39%.8,9 saliva offers a protective function as well as an antimicrobial, buffering, and lubricating feature to help cleanse and eliminate food debris within the mouth. patients will begin to develop oral problems before experiencing dryness, such as a burning mouth sensation, when the protective environment created by saliva is altered. the purpose of this case is to discuss how to manage a patient with bms caused by xerostomia secondary to medication that has been taken by the patient. case a 45-year-old male military officer from the royal malaysian air force came to kuching armed forces dental clinic with complaints of a burning sensation and dryness throughout his mouth for the past two months. the patient claimed that he feels hot and sharp and has been on a cold food diet as he cannot swallow any spicy or hot food. a clinical examination was performed to rule out any pre-existing pathology other than dry mouth. the patient is fit, conscious and has no other underlying medical diseases besides hypertension. the patient admitted feeling the changes in his mouth since taking anti-hypertensive medication, which was 10 mg of amlodipine per day given by the general practitioner in the last two months. further inquiry into the patient’s social history revealed no tobacco or alcohol use. the patient is happily married with three children, and after a deeper investigation, the patient claimed that he feels neither stress nor any psychological disturbances related to his daily life. however, he had not been able to eat well during the past two months due to the burning sensation he was having, and since then he had started to drink lots of cold water and apply lip balm on his cracked lips (figure 1). an intraoral examination demonstrated that both the buccal and palatal sides of the mouth were reddish, dry, and inflamed, while the tongue appeared scalded and burnt (figure 2). the patient is not wearing any dentures and has relatively good oral hygiene, with no teeth restored. subsequently, the patient was referred to a medical for a full blood count (fbc) with differential analysis to check for any possible relationship with diabetes or sjögren’s syndrome and deficiencies of iron, folate, zinc, and group b vitamins. the results came out that the patient was fit and well without any abnormality or deficiencies. besides, the swab test showed the absence of candidiasis infection, although there is a severe decrease in unstimulated salivary flow rate (0.1 ml per minute) and stimulated salivary flow rate (0.5 ml per minute) according to the spit test that was done. the challacombe scale of clinical oral dryness shows a score of 6, which indicates moderate dryness of the patient’s mouth. based on the overall clinical and laboratory findings, the differential diagnosis of this patient is bms secondary to xerostomia due to amlodipine. figure 1. photograph showing the patient’s cracked and peeled lips. a b c d figure 2. pre-treatment photographs showing dry and inflamed palatal side (a), scalded tongue (b), and inflammation of both sides of the buccal region (c and d). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p187–190 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p187-190 189noor/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 187–190 a b c d figure 3. two weeks later, follow-up treatment shows improved mucosa appearance, which is not dry or sticky (a, c, and d) and includes the non-scalded appearance of the tongue (b). case management the patient was referred to the general practitioner for advice about lowering his dose of 10 mg of amlodipine or replacing it with other types of antihypertensive drug that can reduce xerostomia leading to burning mouth syndrome. besides prescribing a hydrating mouthwash, oral hygiene advice has been given to the patient, including on how to relieve his burning and dry mouth symptoms, such as frequent sucking on ice cubes, increasing the intake of water and watery fruits, avoiding hot and spicy food, and chewing gum. a follow-up appointment was given 14 days after the initial treatment. the patient claimed absence of burning sensation and dry mouth and said he can enjoy his food well without feeling difficulty in swallowing. intraoral examination revealed an improved mucosa appearance without dry and sticky appearance or scalded appearance of the tongue (figure 3). the dosage of 10 mg of amlodipine has been reduced to 5 mg, which has less effect in drying the mouth. discussion this clinical report described the bms caused by xerostomia secondary to amlodipine. initial management focused on eliminating the cause of xerostomia in order to treat the burning mouth syndrome. after establishing a diagnosis, a step-wise management approach should be implemented. this includes alleviating symptoms, the implementation of preventive measures, the treatment of oral diseases, the enhancement of salivary function, and the management of any underlying systemic condition.10 initial treatments of this case include changing or lowering the dose of 10 mg of amlodipine by referring the patient to the general practitioner. the dry mouth side effects of medications may be alleviated or reduced by substituting the problem medications with similar drugs that have lesser side effects.10 in addition, alteration in the timing or dosing schedule of medication doses at night-time, when salivary flow is usually at its lowest, can maximise the dry mouth effects. the diagnosis of burning mouth syndrome caused by dry mouth is based on clinical findings, with the characteristic of burning and itching sensation located bilaterally in the mouth, including the palate and tongue, with the absence of any oral mucosal pathology.11 according to millsop et al.,12 patients with dry mouth have clinical manifestations of difficulty in swallowing, chewing, and/or speaking and present with burning mouth, halitosis, dry buccal mucosa, cracked and peeled lips, and oral candidiasis. this patient encountered burning mouth sensation together with dry mouth, difficulty in swallowing, and cracked lips, without any presence of other oral pathology, after taking an antihypertensive drug, and he was diagnosed with burning mouth syndrome caused by dry mouth secondary to amlodipine. persistent hyposalivation leading to dry mouth can leads to infections, such as candidiasis and dental caries, as well as bacterial sialadenitis.13 lubrication loss can also result in erythema and mucosal susceptibility to frictional damage to the teeth, causing the patient to feel discomfort.14 the more commonly used agents for dry mouth can be categorised into chewing gums, salivary stimulants, and saliva substitutes.12 chewing gums or candies should be sugar-free to prevent dental caries. the patient has been prescribed with a hydrating mouthwash to minimise the dry dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p187–190 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p187-190 190 noor/dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 187–190 mouth effect as well as to stimulate the production of saliva, which helps to eliminate the burning sensation. saliva substitutes that commonly contain carboxymethylcellulose, xanthan gum, mucins, hydroxyethylcellulose, polyethylene oxide, or linseed oil may help to increase salivary viscosity by resembling natural saliva.15 very clearly, amlodipine can cause dry mouth, leading to burning mouth syndrome. antihypertensive agents are the drugs most often associated with the appearance of symptoms compatible with burning mouth syndrome because they can act upon the angiotensin-renin system.2 salivary secretion depends on parasympathetic and sympathetic signalling, and parasympathetic activation leads to increased ca2+ release and water fluxing out of the cell.16 thus, taking a calcium channel blocker such as amlodipine, which acts as a ca2+ antagonist, may cause dry mouth by inhibiting the voltage-dependent ca2+ channels that are activated by depolarisation to cause resting salivation.16 of the worldwide cases of medication-induced bms, 33% were known to be dose-dependent phenomena, as the burning sensation only occurred when the medication dose was increased in pursuit of therapeutic efficacy.2 bms can be managed easily with adequate diagnosis and treatment planning, or it can give discomfort to the patient psychologically; in the worst case scenario, other oral diseases might arise, such as dental caries and fungal infections. the first phase is an acute treatment to prevent the progression of burning mouth syndrome and to provide pain relief. the second phase is the amelioration of preexisting conditions such as dry mouth, where symptoms would be improved after the initial acute phase. this included intense oral hygiene instruction and control of systemic factors such as smoking, diet, and stress. the alteration of medication that causes the burning mouth syndrome comes in the third phase and is considered only if it is confirmed to be the cause of bms. the fourth and final treatment phase proceeds through supportive therapy to maintain oral hygiene and control of systemic and local factors.15 the patient discussed in this report went through the first and second phases of treatment, where we managed to treat the acute phase, and the patient was compliant with the treatment. for the third phase, of altering the patient’s medication, the general practitioner has been consulted, and the patient is under review every three months as part of oral condition maintenance. in this case, as discussed in this report, a proper diagnosis was followed by improvement of the patient’s condition. xerostomia or dry mouth is the result of reduced or absent salivary flow. it may occur as a result of ongoing drug use and happen concurrently with bms. there are over 500 widely used medications, including various antidepressants, antipsychotics, antihistamines, diuretics, and sedatives, which list xerostomia as a side effect. temporary relief options include oral mouthwashes, gels and sprays, lozenges, and change of dietary habit; however, more prolonged treatment options will be medication such as pilocarpine or saliva substitutes with longer mucosal surface retention. acknowledgement the author would like to thank lt col (dr) sophia ann murray for her guidance in managing this case and the dental service of the malaysian armed forces for this case write-up. references 1. lópez-jornet p, camacho-alonso f, andujar-mateos p, sánchezsiles m, gómez-garcía f. burning mouth syndrome: update. med oral patol oral cir bucal. 2010; 15(4). 2. jääskeläinen sk, woda a. burning mouth syndrome. cephalalgia. 2017; 37(7): 627–47. 3. cerchiari dp, de moricz rd, sanjar fa, rapoport pb, moretti g, guerra mm. burning mouth syndrome: etiology. rev bras otorrinolaringol. 2006; 72(3): 419–23. 4. thomson wm. dry mouth and older people. aust dent j. 2015; 60(s1): 54–63. 5. turner md. hyposalivation and xerostomia. etiology, complications, and medical management. dent clin north am. 2016; 60(2): 435–43. 6. saleh j, figueiredo maz, cherubini k, salum fg. salivary hypofunction: an update on aetiology, diagnosis and therapeutics. arch oral biol. 2015; 60(2): 242–55. 7. ristevska i, armata rs, d’ambrosio c, furtado m, anand l, katzman ma. xerostomia: understanding the diagnosis and the treatment of dry mouth. j fam med dis prev. 2015; 1(2): 1–5. 8. mayer t, haefeli we, seidling hm. different methods, different results how do available methods link a patient’s anticholinergic load with adverse outcomes? eur j clin pharmacol. 2015; 71(11): 1299–314. 9. nishtala ps, salahudeen ms, hilmer sn. anticholinergics: theoretical and clinical overview. expert opin drug saf. 2016; 15(6): 753–68. 10. femiano f, rullo r, di spirito f, lanza a, festa vm, cirillo n. a comparison of salivary substitutes versus a natural sialogogue (citric acid) in patients complaining of dry mouth as an adverse drug reaction: a clinical, randomized controlled study. oral surgery, oral med oral pathol oral radiol endodontology. 2011; 112: e15–20. 11. tiisanoja a, syrjälä amh, kullaa a, ylöstalo p. anticholinergic burden and dry mouth in middle-aged people. jdr clin transl res. 2020; 5(1): 62–70. 12. millsop j w, wa ng ea, fazel n. etiology, evaluation, a nd management of xerostomia. clin dermatol. 2017; 35(5): 468–76. 13. lavanya n, jayanthi p, rao u, ranganathan k. oral lichen planus: an update on pathogenesis and treatment. j oral maxillofac pathol. 2011; 15(2): 127–32. 14. yuan a, woo s bin. adverse drug events in the oral cavity. oral surg oral med oral pathol oral radiol. 2015; 119(1): 35–47. 15. vissink a, mitchell jb, baum bj, limesand kh, jensen sb, fox pc, elting ls, langendijk ja, coppes rp, reyland me. clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers. int j radiat oncol biol phys. 2010; 78(4): 983–91. 16. mizuhashi f, koide k, toya s, nashida t. oral dryness caused by calcium blocker -comparison with saliva of healthy elderly persons and patients with sjögren’s syndrome-. med res arch. 2017; 5(9): 1–12. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p187–190 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p187-190 197197 research report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 197–203 the development of early childhood caries impact on quality of life-indonesia instrument as assessment instrument of dental caries impact on quality of life of children aged 3-5 years based on indonesian community characteristics taufan bramantoro,1 yayi suryo prabandari,2 djauhar ismail,3 and udijanto tedjosasongko4 1department of dental public health, faculty of dental medicine, universitas airlangga, surabaya-indonesia 2department of public health, faculty of medicine, universitas gadjah mada, yogyakarta-indonesia 3department of pediatrics, faculty of medicine, universitas gadjah mada, yogyakarta-indonesia 4department of pediatric dentistry, faculty of dental medicine, universitas airlangga, surabaya-indoneia abstract background: improvement of quality of life is one of objectives in establishment of healthcare system according to world health organization policy. studies of dental health-related factors that influence quality of life of children aged 3-5 years have not been developed yet, particularly in indonesia. previous study which adopted international instruments has some limitations of inappropriate characteristics of question items with local community characteristics. quality of life assessment could describe sociodemographic status and community cultural background, as well as varies of special characteristics in a community group, related to oral and dental health features with sensitivity to the difference of age group. purpose: this study was aimed to develop early childhood caries impact on quality of life-indonesia (ecciqol-ina) as assessment instrument of dental caries impact on quality of life of children aged 3-5 years based on indonesian community characteristics. method: initially, this study was conducted qualitatively using focus group discussion method, validity and reliability test, subsequently the study was continued using quantitative method as a cross-sectional analytical study to analyze the utilization of instrument in 309 children aged 3-5 years enrolled in kindergarten and early childhood education programs (ecep) with their mothers at working area of community health centre wates in mojokerto. result: there were four question items as assessment instrument of dental health problems that impact on quality of life on children aged 3-5 years, i.e, “has your child ever been irritable/restless?” (“apakah anak ibu pernah merasa rewel/gelisah?”), “has your child ever refused to eat and drink or felt discomfort while eating and drinking?” (“apakah anak ibu pernah tidak mau atau tidak enak makan dan minum?”), “has your child ever been absent from school?” (“apakah anak anda pernah tidak masuk sekolah?”) “has your child ever been difficult or unable to sleep?” (“apakah anak ibu pernah tidak bisa tidur atau sulit tidur?”). conclusion: ecciqol-ina instrument had been successfully developed and could be utilized as assessment instrument of dental caries impact on quality of life on children aged 3-5 years based on indonesian community characteristics. keywords: quality of life; dental caries; early childhood correspondence: taufan bramantoro, c/o: departemen ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: tbramantoro@gmail.com introduction improvement of oral and dental health should be started as early as possible, because in toddlers and preschoolers it is such a crucial factor for the arrangement of further dentition. in addition, it also affects children’s ability to speak and their mastication. various attempts to develop dental caries prevention and management keep continuing in many countries. currently, dental caries prevention and management have shown a positive and dynamic dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 198 bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 development, however dental caries problem still shows a high prevalence, particularly in developing countries.1,2 some studies in northern philippines and china found that early childhood caries prevalence ranged from 52.9% to 90%. another study in southern brazil found that about 40% of preshoolers (0-5 years) has caries in their primary teeth. moreover, some studies in taiwan found a high prevalence of 57.69% in children aged 3 years.3 in some developed countries it also found high incidence of dental caries in children.1,2 in a review of early childhood caries in indonesia, according to study conducted in five urban communities of special capital region of jakarta in 2008, it had been found that prevalence of early childhood was 52.7%.4 another study conducted by the authors in mojosari, east java, found prevalence of dental caries in children aged 4-5 years was up to 91%, and 89% of them had untreated dental caries.5 untreated dental caries has negative impacts that could lead to experience of pain and then affect children’s appetite, sleep disturbance, which could impact upon children’s growth process. chronic inflammation process of dental caries also could affect the cycle of red blood cell production.6,7 attempt to obtain assessment of comprehensive oral and dental health by observation in individual and community group not only about the disease status, but also physical, psychological, and social function, as well as health satisfaction. in addition to that, it could be emphasized about the importance of concerning quality of life aspect in planning and assessing the effectiveness of oral and dental health programs. quality of life assesment could give an addition of strong dimension in planning and health promotion program. it is related to consideration of someone’s efforts in receiving explanation about oral and dental health program in order to improve quality of oral and dental health.8-10 improvement of quality of life is one of objectives in establishment of healthcare system according to world health organization policy. the plan of dental caries prevention and management is not only concern to clinical impact, but also to assessment and measurement of impact on quality of life. as the pattern of life continues to develop, chronic oral and dental problems and developing symptoms as well as some effects caused by those problems require assessment method of social and psychological chronic impacts.8 early prevention concept is still not facilitated yet by the development of assessment instrument of oral and dental health-related children’s quality of life, particularly children aged under 5 years compared to the development of instrument in the older children.11 children aged under 5 years have special characteristics, i.e considering psychological development status of early childhood, they still could not interpret the questions of assessment instrument of quality of life. therefore, parent is considered to be more effective to complete the assessment of their children’s quality of life.7,12 unlike with observation of factors related to dental caries incidence in children, studies about factors that influence dental health-related quality of life of children aged 3-5 years have not developed yet, particularly in indonesia. previously study employing adopted international instruments has some limitations of inappropriate characteristics of question items with local community characteristics. according to some studies about factors that influences the assessment of children’s quality of life, it had been found that majority of study population focused on school-age children. one thing that could potentially describe it is the lack of development of assessment instrument for dental health-related quality of life of children, especially in early childhood group.7,12 precently, study had developed oral health related quality of life-children 5 (ohrqol-c5) instrument as assessment instrument of quality of life of primary school-age children and had been tested in children aged 6-7 years.13 based on the discussion above, there is a need to develop assessment instrument of quality of life with consideration to cultural background of indonesian community, especially in children aged under 6 years. this is in accordance with observation by john et al. and al shamrany which stated that assessment of quality of life could describe sociodemographic status and community cultural backgorund, as well as varies of special characteristics in a community group, especially concerned with age group. it was related to oral and dental health features with sensitivity to the difference of age group.14-15 step in development of questionnaire to assess quality of life of children aged 3-5 years in accordance with characteristics of indonesian community is based on that of previously studies have been done in some countries.7,12,16 in order to fulfill such need, so this study was aimed to develop early childhood caries impact on quality of life-indonesia (ecciqol-ina) instrument as assessment instrument of dental caries impact on quality of life of children aged 3-5 years based on characteristics of indonesian community. materials and methods initially, this study was conducted qualitatively using focus group discussion method, validity and reliability test. subsequently the study was continued using quantitative method as a cross-sectional analytical study to analyze the utilization of instrument in 309 children aged 3-5 years enrolled in kindegarten and early childhood education programs (ecep) including their mothers at working area of community health centre wates in mojokerto. that area was chosen in relation with development of dental health promotion programs by community health center, because prevalence of early childhood caries was still found more than 70% although community health center had conducted dental health promotion program for children and mothers during 4 years. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 199199bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 mother’s attitude regarding children’s dental health was mother’s response about children’s dental health, shown by the mean score of mother’s chosen answers about aspect of knowledge in maintaining children’s dental health, children’s dietary habit, and children’s dental visit.17,18 the answer choices to each question item on mother’s attitude instrument regarding children’s dental health were “strongly disagree”, “disagree”, “agree”, and “strongly agree”, with score ranged from 1 to 4. measurement of dental caires on children was the number of carious primary teeth, missing teeth due to caries, and filled teeth. assessment of children’s quality of life was the assessment of mother’s perception about impaired quality of life of children as impact of dental health problems, shown by the mean score of children’s quality of life measurement. the assessment involved three aspects: physical symptoms aspect, functional aspect, and children’s psychological aspect. questionnaire about impact of children’s quality of life was adapted and developed based on synthesisof some assessment instrument of children’s quality of life that had been developed previously, i.e early childhood oral health impact scale (ecohis), self-reported scale of oral health outcomes for 5 year-old children (soho-5), and oral health related quality of life-children 5 (ohrqol-c5).7,12,13,19 assessment instrument of dental health problem impact on quality of life of children aged 3-5 years employing in this study had passed validity and reliability test series. assessment was obtained from parent’s perception, particularly mother, the one who had the major role in early childhood care, as the first social environment of children. parent is considered able to recognize and understand early childhood problems such as pain, sleep disturbance, and any discomfort feelings due to early childhood caries. it was thought due to psychological development status of early childhood which still could not interpret the questions in assessment instrument of quality of life. parent could be the source of good and valid information to obtain the assessment of quality of life in early childhood.7,12 development of questionnaire to assess quality of life of children aged 3-5 years in accordance with characteristics of indonesian community was based on that of previously had been done in some countries.7,12,16 the first step was through focus group discussion (fgd) to know and determine the topic of constructs in development of measurement instrument of dental caries status-related quality of life in early childhood. fgd was carried out in a group of mothers, whose children had dental caries. in that group, there were 7 purposively selected respondents. fgd was carried out according to instrument that had been used and published in some references. after determining the topic, then the draft of question items in measurement instrument was composed, followed by feasibility test of contents and the composition of instrument. feasibility test was conducted by interview or discussion with dentist, pedodontist, and public health dentist to know comprehensiveness, relevance ranking, and clarity. total number of respondents was 10 selected by purposive sampling. the draft of question items in instrument that had passed the feasibility test by dental experts, subsequently tested its feasibility by mothers whose children had early childhood caries. this test was aimed to know comprehensiveness, relevance ranking, clarity, addition, understanding, and readability. total number of respondents was 10 selected by purposive sampling. the draft of question items that had passed feasibility test both by experts and mothers of children with early childhood caries, then had to pass the validity and reliability test to get the final question items composition for measurement instrument of dental health problems impact on quality of life of children aged 3-5 years. this involved 55 respondents. prior to start the process of completing questionnaire and dental examination in children, the mothers who agreed to get involved in this study as study sample, were given informed consent to be filled and signed. this study had been through the application procedure of ethical clearance and approved by medical and health research ethics committee faculty of medicine universitas gadjah mada. results the result of fgd found that there were 4 aspects in frequently impaired daily life of children due to dental caries. they were the condition of irritable or restless children, discomfort or refuse to eat or drink, absent from school and difficult to sleep. these 4 aspects were in accordance with the previous sutdy, although there were some different aspects such as feelings of inferiority among their friends, difficulty in speaking and smiling.5,11,17 those 4 aspects then be arranged in a draft of question items consisted of: a) “has your child ever been irritable/restless?” (“apakah anak ibu pernah merasa rewel/gelisah?”); b) “has your child ever refused to eat and drink or felt discomfort while eating and drinking?” (“apakah anak ibu pernah tidak mau atau tidak enak makan dan minum?”); c) “has your child ever been absent from school?” (“apakah anak anda pernah tidak masuk sekolah?”); d) “has your child ever been difficult or unable to sleep?” (“apakah anak ibu pernah tidak bisa tidur atau sulit tidur?”). the draft of question items was considered relevant by those dental experts as respondents of feasibility test for that instrument, and also considered clear and readable by mothers of children aged 3-5 years with dental caries. the result of reliability test showed that assessment instrument of quality of life of children aged 3-5 years employing in this study had cronbach’s alpha score as 0,867 or more than critical value for two-tailed correlation coefficient with the number of sample by 55 subjects, i.e r=0,261. therefore, those question items were considered internally reliable. external reliability test was conducted by comparing respondent’s answer in the first and second assessment of question items. interval between the first and second assessment was 3 weeks. reliability test and dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 200 bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 re-test used wilcoxon test for ordinal and nominal data. from all question items in instrument, it had been found that the difference between the first answer and the second answer, 3 weeks after the former, were not significant with p>0,05. validity test was conducted by comparing (r)-value from each item with pearson’s critical value r in two-tailed. the result of validity test for each question items found that overall of critical value (r=0,261) was more that critical value (r) for 55 samples, and therefore those questien items were valid (table 1). question items in assessment instrument of dental health problems impact on quality of life of children aged 3-5 years that had passed validity and reliability test were as follows, “has your child ever been irritable/restless?” (“apakah anak ibu pernah merasa rewel/gelisah?”), “has your child ever refused to eat and drink or felt discomfort while eating and drinking?” (“apakah anak ibu pernah tidak mau atau tidak enak makan dan minum?”), “has your child ever been absent from school?” (“apakah anak anda pernah tidak masuk sekolah?”) “has your child ever been difficult or unable to sleep?” (“apakah anak ibu pernah tidak bisa tidur atau sulit tidur?”). answer choices to the question items in that instrument were “yes, he/she has” and “no, he/she hasn’t”. “yes, he/she has” answer had score 1 while “no, he/she hasn’t” had score 2. in this present study of developed instrument utilization, the gender distribution was likely similar between boys and girls, with total number of boys was 159 (51.5%), and girls was 150 (48.5%). table 2 shows that majority of mothers (88,7%) as respondents had been through formal education during more than 9 years. distribution of average monthly household expenses groups was higher in average monthly household expenses >rp1.485.000 group, that was two-fold higher than average of monthly household expenses in indonesia based on statistic indonesia 2014, rp. 1.485.000,-. majority of mothers (81.6%) had and lived in the same house with 1-2 children. table 3 shows that based on mothers’ assessment, being irritable or restless was the most common impact on quality of life of children with dental caries. in assessment of 4 aspects regarding impacts on quality of life, we found that children whose quality of life had ever been impaired, had the higher number of dental caries, and the poorer mother’s attitude on children’s dental health than children whose quality of life had never been impaired. overall in all aspects of dental caries impacts on children’s quality of life, we found that the number of dental caries had a significant influence on quality of life of children aged 3-5 years. in assessment of mother’s attitude on children’s dental health, overall it is also found a significant influence on aspects of dental caries impacts on children’s quality of life. overall significance of the differences in test result between ever and never being impaired in the number of dental caries variable and value of mother’s attitude on children’s dental health variable, i.e less than 0.05. in correlation test between the number of dental caries and the value of quality of life, it is found that the number of dental caries had a significant strong negative correlation with quality of life (p<0.05;r = -0.808), i.e the higher the number of dental caries in children, the poorer the quality of life they would have. this result also could be applied in correlation between mother’s attitude on children’s dental health and children’s quality of life, that we found both of them had a significant strong positive correlation (p<0.05;r = 0.626), between so the better mother’s attitude on children’s dental health, the better quality of life of their children. discussion dental caries in children has such negative impacts on their quality of life compared to those with no dental caries. this is related to pain as the result of inflammation process in dental caries, discomfort feelings, and limited function of teeth, and psychological impairment. children with early childhood caries more likely feel discomfort while table 1. validity coefficient in assessment of children’s dental caries impact on quality of life impacts on quality of life n validity coefficient has your child ever been irritable/ restless? (apakah anak ibu pernah merasa rewel/gelisah?) 55 0,819 has your child ever refused to eat and drink or felt discomfort while eating and drinking? (apakah anak ibu pernah tidak mau atau tidak enak makan dan minum?) 55 0,719 has your child ever been absent from school? (apakah anak ibu pernah tidak masuk sekolah?) 55 0,486 has your child ever been difficult or unable to sleep? (apakah anak ibu pernah tidak bisa tidur atau sulit tidur?) 55 0,872 table 2. distribution of study respondents’ characteristics at working area of community health centre wates in mojokerto socio-economic variable group frequency mother’s formal education ≤ 9 years 35 (11,3%) >9 years 274 (88,7%) average monthly household expenses ≤ 1.485.000 105 (34%) >1.485.000 204 (66%) number of children living in the same house >2 anak 57 (18,4%) ≤2 anak 252 (81,6%) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 201201bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 eating and has ever been absent from school.10,20 impaired children’s quality of life aspects due to dental caries are learning and playing activities, mastication process, social, and sleep activities. other observation also found phonetic impairment, and impaired growth and development in early childhood due to severe dental caries.7,11,20 impact on such functions due to oral and dental health impairment could occur regardless of age, it is just a few difference about the type of activities they had. in early childhood, oral and dental health impairment could lead to pain and discomfort in oral cavity, impaired mastication, phonetic impairment especially for certain words, impaired playing and learning activities, as well as sleep disturbance.21,22 the basic thing that should be fulfilled by an instrument is its substances should be able to find out information required in a study. it could be achieved by referring to empirical concept or determined indicators. the development process of measurement instrument of oral and dental health-related quality of life begins with determine the problem and variables as clear and spesific indicator to describe dimension of measurement concept. determination of indicator is based on basic review about who’s quality of life concept, i.e health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity.8,9,23 the next step is formulating the relevant criteria to each dimension in sort of question component in quality of life measurement instrument. this could be done based on review of experts group, supported by studies from literature reviews. question component formulation that has been composed as the result of review from experts and references, then is evaluated for its clarity and readability, relevance, and correlation between question component by subjects of study. the next step is a step to ascertain the reliability and validity of question component formula that has been evaluated for its clarity and readability, relevance, and correlation between question component.16 research instrument intends to function as measurement tool designed to obtain information data about varies of characteristics in study variables. research instrument and its quality have an important role in affecting quality of data table 3. distribution of children’s dental caries impacts on quality of life, the test result about the number of dental caries, and the value of mother’s attitude on children’s dental health impacts on quality of life event frequency dental caries significance of test in different dental caries value of mother’s attitude on children’s dental health significance of test in different mother’s attitude on children’s dental health mean sd mean sd has your child ever been irritable/restless? (apakah anak ibu pernah merasa rewel/ gelisah?) yes, he/she has 159 (51,5%) 10,566 4,576 0,000 2,546 0,604 0,000 no, he/she hasn’t 150 (48,5%) 4,967 3,726 2,991 0,532 has your child ever refused to eat and drink or felt discomfort while eating and drinking? (apakah anak ibu pernah tidak mau atau tidak enak makan dan minum?) yes, he/she has 130 (42,1%) 11,408 4,607 0,000 2,431 0,607 0,000 no,he/she hasn’t 179 (57,9%) 5,263 3,529 3,002 0,491 has your child ever been absent from school? (apakah anak ibu pernah tidak masuk sekolah?) yes, he/she has 73 (23,6%) 12,986 4,529 0,000 2,211 0,638 0,000 no, he/she hasn’t 236 (76,4%) 6,259 4,019 2,932 0,491 has you child ever been difficult or unable to sleep? (apakah anak ibu pernah tidak bisa tidur atau sulit tidur?) yes, he/she has 102 (33%) 11,726 4,672 0,000 2,365 0,625 0,000 no, he/she hasn’t 207 (67%) 5,937 4,005 2,958 0,501 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 202 bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 or study result, therefore an instrument must be accountable scientifically. moreover, research instrument must be met reliability and validity criteria to produce accurate and objective information or data.12,16,19 the study result of number of children’s dental caries and value of mother’s attitude on children’s dental health showed that there was a significant influence. it was confirmed by the result of correlation test that found number of dental caries and mother’s attitude on children’s dental health had a significant correlation with children’s quality of life. analysis result showed that instrument employing in this study could provide a consistent result with general analogy about correlation between number of caries and mother’s attitude on early childhood quality of life. problems in children’s daily activities due to dental caries is manifestation of oral and dental tissue in whole body tissue system. oral and dental tissue in body tissue system play a role in nutrition supply and social interaction. impairment in oral and dental health could impact the overall body function or in other words, that impairment could influences quality of life.6,10,21,22 the measurement of oral and dental health-related quality of life is an assessment of health impacts particularly on oral and dental health.this assessment is conducted by analyzing functional, social and psychological impacts as well as any discomfort feelings due to oral and dental problems. quality of life assessment as a multidimensional construct is found in oral and dental health report by united states surgeon general’s report, as quoted by al shamrany,15 that quality of life is a multidimensional construct that reflects (among other things) people’s comfort when eating, sleeping, and engaging in social interaction, their self-esteem, and their satisfaction with respect to their oral health. assessment of oral and dental health-related quality of life in addition to the current concept generally used, is sort of analysis of respondent’s answers to the questions in quality of life measurement instrument.15 that measurement is multidimensional and including physical, social, emotional, and cognitive well-being, correlation with their position and occupation, psychological aspect in relation to variety of disease symptoms, and financial impact. 15,23,24 quality of life concept is a sort of correlation between oral and dental status with social and psychological wellbeing affected by someone’s value and cultural background, then it is known the need to develop assessment instrument of quality of life based on any community’s cultural background, and any characteristics, particularly agebased characteristic.15 assessment of quality of life could describe sociodemographic status in a community group, especially concerned with age-based group. it is rela ted to oral and dental health features with sensitivity to the difference between age-based group.24 quality of life assessment instrument was developed in adult group, and also has been developed and adapted in children group. the development of quality of life assessment instrument for children had special characteristic, particularly children aged under five years.7,12,15 not all aspects in quality of life assessment for adult could be applicable in early childhood group. in the context of early childhood, there are aspects that more concerned with assessment of physically, socially, and psychologically impacts, interpreted in assessment of quality of life in early childhood regarding to eating and drinking activities, regular playing and school activities, sleep activity, and confidence to smile. 19 in conclusion, ecciqol-ina instrument had been successfully developed and could be utilized as assessment instrument of dental caries impact on quality of life of children aged 3-5 years based on indonesian community characteristics. references 1. ismail ai. determinants of health in children and the problem of early childhood caries. pediatr dent 2003; 25(4): 328-33. 2. bagramian ra, garcia-godoy f, volpe af. the global increase in dental caries. a pending public health crisis. am j dent 2009; 21(1): 3-8. 3. tsai ai, chen c, li l, hsiang c, hsu k. risk indicators for early childhood caries in taiwan. community dent oral epidemiol 2006; 34(6): 437-45. 4. sugito fs, djoharnas h, darwita rr. breastfeeding and early childhood caries (ecc) severity of children under three years old in dki jakarta. makara kesehatan 2008; 12(2): 86-91. 5. bramantoro t, hapsoro a, roesanto h, harumi rs, berniyanti t, hariyani n, lydia m. impacts of daily activities related dental caries on 4-6 years old children in pekukuhan mojosari. presentation on 7th asian conference of oral health promotion for school children. bali. 2013. 6. sheiham a. dental caries affects body weight, growth and quality of life in pre-school children. br dent j 2006; 201(10): 625-6. 7. filstrup sl, briskie d, marcio da f, lawrence l, wandera a, inglehart mr. early childhood caries and quality of life: child and parent perspectives. pediatr dent 2003; 25(5): 431-40. 8. petersen pe, kwan s. evaluation of community-based oral health promotion and oral disease prevention – who recommendations for improved evidence in public health practice. community dental health 2004; 21 (supplement): 319–29. 9. petersen pe. challenges to improvement of oral health in the 21st century–the approach of the who global oral health programme. int dent j 2004; 54(6 suppl 1): 329–43. 10. sischo l, broder hl. oral health-related quality of life: what, why, how, and future implications. j dent res 2011; 90(11): 1264-70. 11. abanto j, carvalho ts, mendes fm, wanderley mt, bönecker m, raggio dp. impact of oral diseases and disorders on oral healthrelated quality of life of preschool children. community dent oral epidemiol 2011; 39(2):105-14. 12. pahel bt, rozier rg, slade gd. parental perceptions of children’s oral health: the early childhood oral health impact scale (ecohis). health qual life outcomes 2007; 30(5): 6. 13. susilawati s, djuharnoko p, syaefullah a, kartini sari d. caries and quality of life – evidence of impacts from indonesia. presentation on 7th asian conference of oral health promotion for school children. bali. 2013. 14. john mt, hujoel p, miglioretti dl, leresche k, koepsell td, micheelis w. dimensions of oral-health-related quality of life. j dent res 2004; 83: 956. 15. al shamrany m. oral health-related quality of life: a broader perspective. east mediterr health j 2006; 12(6): 894-901. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 203203bramantoro, et al./dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 197–203 16. jokovic a, locker d, tompson b, guyatt g. questionnaire for measuring oral health-related quality of life in eight-to ten year-old children. pediatr dent 2004; 26(6): 512-8. 17. arrow p, raheb j, miller m. brief oral health promotion intervention among parents of young children to reduce early childhood dental decay. bmc public health 2013; 13: 245. 18. chen c, chiou s, ting c, lin y, hsu c, chen f, lee c, chen t, chang c, lin y, huang h. immigrant-native differences in cariesrelated knowledge, attitude, and oral health behaviors: a crosssectional study in taiwan. bmc oral health 2014; 14(3). 19. tsakos g, blair yi, yusuf h, wright w, watt rg, macpherson lmd. developing a new self-reported scale of oral health outcomes for 5-year-old children (soho-5). health and qual of life outcomes 2012; 10: 62. 20. feitosa s, colares v, pinkham j. the psychosocial effects of severe caries in 4-year-old children in recife, pernambuco, brazil. cad saude publica 2005; 21(5): 1550–6. 21. moynihan p, petersen pe. diet, nutrition and the prevention of dental diseases. public health nutr 2004; 7(1a): 201–26. 22. martins-junior pa, vieira-ansdrade rg, correa-faria p, oliveiraferreira f, marques ls, ramos-jorge ml. impact of early childhood caries on the oral health-related quality of life of preschool children and their parents. caries res 2013; 47: 211–8. 23. naito m, yuasa h, nomura y, nakayama t, hamajima n, hanada n. oral health status and health related quality of life:a systematic review. j oral sci 2006; 48(1): 1-7. 24. john mt, hujoel p, miglioretti dl, leresche k, koepsell td, micheelis w. dimensions of oral-health-related quality of life. journal dental research 2004; 83: 956. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p197-203 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p197-203 subject index volume 44 0.15% benzydamine hydrochloride , 88 10% povidone iodine, 159 35% piper betle linn extract, 159 acid, 63 acrylic resin, 196 acupuncture analgesia, 25 aerobic physical exercise , 35 aesthetic, 30 alginate impression, 187 alkali, 63 aloe vera, 200 anterior makeover, 150 anterior teeth, 30 apical periodontitis, 12 apoptosis, 210 azadirachta indica, 49 bacteriocid test, 159 betel leaf toothpaste, 169 bimaxillary protrusion, 101 blind children, 39 bond strengt, 17 calcium hydroxide, 12 candida albicans, 49, 72, 137 candida species, 177 carious lesion, 164 cell number, 192 children, 205 chitosan, 67 chlorine dioxine, 210 complex caries, 30 composite restoration, 150 cost effectiveness analysis, 43 crystal structure, 181 cyototoxicity, 7 deaf children, 39 dental, 25 anxiety degree, 205 ceramic, 17 erosion, 145 filling, 43 health education , 39 plaque, 169 pulp, 164 trauma, 154 visits frequency, 205 dentofacial disharmony, 1 diagnostic biomarker, 77 effectiveness, 59 elderly, 72 enamel demineralzation, 141 microhardness testing, 141 endorestoration treatment, 30, 154 endothelial cells, 67 fibroblast, 192 fixed orthodontic appliance, 54 patients, 169 fractured teeth, 150 fungal infections, 132 gas chromatograph, 196 glass ionomer cement, 173 hbe beta thalassemia, 1 heat cured acrylic resin, 137 hemophilia patient, 127 hiv/aids, 177 horizontal crown fracture, 154 hybrid composite resin, 63 hydroxyapatite, 67, 181 hypercholesterolemia, 106 idiopathic dental pain, 82 in vivo characterization, 173 interarch discrepancy, 1 intracanal medicine, 12 lactobacillus casei, 117 laser ablation, 181 macrophage, 49 malnutrition, 72 management, 127 mesio-distal, 122 methods for obtaining isolate, 177 microhardness, 181 mimba oil, 187 mineral solubility, 145 mmp-8, 54 mtt assay, 7 nasopharynx carcinoma, 88 nd:yag laser, 181 neem leaves aqueous extract, 49 nigella sativa, 137 nystatin, 132 odontoblast, 164 odontometry, 122 omega-3, 106 oral bleeding, 127 cancer, 215 candidiasis, 177 microorganism, 187 osteoblast, 111, 200 osteoclast, 117, 200 pain, 25 relief, 82 periodontal disease, 77 health, 59 periodontitis, 106, 210 phagocytosis, 49 subject index volume 44 phytoestrogen, 111 piperin, 215 piplartin, 215 plaque index, 39 plasma, 181 polycaboxylate cement, 173 porphyromonas gingivalis, 117 prevention, 88 probiotics, 117 quality of life, 43 radiation-induced oral mucositis, 88 reactivation time, 54 recurrent aphthous stomatitis, 159 residual monomer, 196 saliva, 72, 77 scaffold, 67 sense of taste sensitivity, 35 sex,122 sexual dimorphism, 122 skeletal anchorage system, 101 soft drinks, 145 soybean extract, 111 spirulina gel, 192 stainless steel plate, 7 stichopus hermanii extract, 106 streptococcus viridans, 159 surface roughness, 63 sweet taste, 35 tartrate-resistant acid phosphatase, 117 teeth, 122 threshold value, 145 titanium, 17 plate, 7 tooth extraction, 43, 111 socket, 200 toothpick tooth brushing method, 59 topical agents,141 traumatic ulcers, 132 tri calcium phosphate, 67 triad endodontic, 12 triglyceride, 106 unilateral free end ridge, 101 volatile sulphur compounds, 210 without drugs therapy, 82 wound healing, 192 xenograft cancelous bovine, 200 zinc oxide eugenol, 173 phospate cement, 173 abdurachman, 25 amalia, rosa, 93 apriasari, maharani laillyza, 159 apsari, retna, 181 artaria, myrtati dyah, 122 astri, mia giri, 205 bachtiar ew, 67 bidarisugma, berlian, 215 brahmanta, arya, 101 bramantoro, taufan, 43 carissa, cynthia, 39 desiana, radithia, 177 dewanti, i dewa ayu ratna, 49 dewiyani, sari, 12 goenharto, sianiwati, 169 hamzah, zahreni, 77 haniastuti, tetiana, 164 hanoem eh, 137, 187 hendrawan, priska lestari, 141 ilyas, muhammad, 145 laksono, harry, 17 authors index volume 44 prahasanti, chiquita 59 prasetyo, eric priyo, 150 prasetyo, remita adya, 88 pribadi, nirawati, 63 puspitawati, ria, 72 rahmitasari, fitri, 192 salim, sherman,196 sari, desi sandra, 117 sari, rima parwati, 106 sella, 132 setiawan, monica widyawati, 127 setiawatie, ernie maduratna, 210 suhono, rosa sharon, 111 sumarta, ni putu mira, 7 susilowati, 54 utari, kresnoadi, 200 utomo, haryono, 82 wardhani, ni luh putu ayu, 35 widiyanti p, 173 zen, yuniar, 1 zubaidah, nanik, 30, 154 thanks to editor dental journal (majalah kedokteran gigi) volume 44 number 1 march 2011: 1. prof. dr. regina titi christinawati tandelilin, drg., msc (oral biology – gadjah mada university) 2. armasastra, drg., ph.d (dental public health – indonesia university) 3. h. m. bernad ongki iskandar, drg., sp.kg., ficcpe, ficd (conservative dentistry – trisakti university) 4. sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) 5. dr. daniel haryono utomo, drg., sp.ort (dental clinic – faculty of dentistry, airlangga university) 6. retno palupi, drg., m.kes. (dental public health – airlangga university) 7. eka fitria, drg., sp.perio (perodontic – airlangga university) 8. hendrik setiabudi, drg., m.kes (oral biology – airlangga university) volume 44 number 2 june 2011: 1. dr. theresia indah budhy, drg., m.kes (oral biology – airlangga university) 2. adi hapsoro, drg., ms (dental public health – airlangga university) 3. dr. indah listiana kriswandini, drg., m.kes (oral biology – airlangga university) 4. dr. daniel haryono utomo, drg., sp.ort (dental clinic faculty of dentistry, airlangga university) 5. sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) 6. wisnu setyari juliastuti, drg., m.kes (oral biology – airlangga university) volume 44 number 3 september 2011: 1. prof. dr. drg. iwa sutardjo rus sudarso, s.u., sp.kga(k) (pediatric dentistry – gadjah mada university) 2. drg. h.m.bernad ongki iskandar, sp.kg (conservative dentistry – trisakti university) 3. prof. dr. trijoedani widodo, drg., ms., sp.kg (conservative dentistry – airlangga university) 4. prof. dr. diah savitri ernawati, drg., msi., sp.pm (oral medicine – airlangga university) 5. dr. retno indrawati r, drg., msi (oral biology – airlangga university) 6. dr. ernie maduratna setiawati, drg., m.kes., sp.perio (periodontic – airlangga university) 7. wisnu setyari juliastuti, drg., m.kes (oral biology – airlangga university) volume 44 number 4 december 2011: 1. sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university) 2. dr. theresia indah budhy, drg., m.kes (oral biology – airlangga university) 3. dr. retno indrawati r, drg., msi (oral biology – airlangga university) 4. dr. daniel haryono utomo, drg., sp.ort (dental clinic – faculty of dentistry, airlangga university) dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3–5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusionocclusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. p-issn: 1978-3728 e-issn: 2442-9740 volume 51, number 1, march 2018 editorial boards of dental journal (majalah kedokteran gigi) sk: 02/un3.1.2/2018 january 2nd – december 31st, 2018 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors sianiwati goenharto (faculty of vocation, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers roosje rosita oewen (department of pediatric dentistry, faculty of dentistry, universitas padjadjaran, indonesia); harmas yazid yusuf (depart-department of oral surgery, faculty of dentistry, universitas padjadjaran, indonesia); eky s. soeria soemantri (department of orthodontics, faculty of dentistry, universitas padjadjaran, indonesia); pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); mei syafriadi (department of oral pathology, faculty of dentistry, universitas jember, indonesia); muslita indrasari (department of prosthodontics, faculty of dentistry, universitas indonesia, indonesia); adioro soetojo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); els sunarsih budipramana (department of pediatric dentistry, faculty of dental medicine, universitas airlangga) i.b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); ari triwadhani (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); intan nirwana (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); david kamadjaja (department of oral surgery and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontic, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@fkg.unair.ac.id; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 144/04.18/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 51, number 1, march 2018 p-issn: 1978-3728 e-issn: 2442-9740 1. the increase of vegf expressions and new blood vessels formation in wistar rats induced with post-tooth extraction sponge amnion moh. basroni rizal, elly munadziroh, and indah listiana kriswandini ................................... 1–4 2. nickel ion release from stainless steel brackets in chlorhexidine and piper betle linn mouthwash tanti deriaty, indra nasution, and muslim yusuf ........................................................................ 5–9 3. the correlation between untreated caries and the nutritional status of 6–12 years old children in the medan maimun and medan marelan sub-district siti salmiah, luthfiani, zulfi amalia, and deandini kusumah .................................................. 10–13 4. the rankl expression and osteoclast in alveolar bone of rat diabetic model at different mechanical force application nuzulul hikmah, amandia dewi permana shita, and hafiedz maulana ................................... 14–19 5. antibacterial activity of mixed pineapple peel (ananas comosus) extract and calcium hydroxide paste against enterococcus faecalis intan fajrin arsyada, devi rianti, and elly munadziroh ........................................................... 20–24 6. biological changes after dental panoramic exposure: conventional versus digital rurie ratna shantiningsih and silviana farrah diba ................................................................. 25–28 7. angular cheilities and oral pigmentation as early detection of peutz-jeghers syndrome maharani laillyza apriasari and amy nindia carabelly ............................................................ 29–32 8. analysis of anti-streptococcus sanguinis igy ability to inhibit streptococcus sanguinis adherence suryani hutomo, heni susilowati, dewi agustina, and widya asmara ..................................... 33–36 9. surface roughness of nanofilled and nanohybrid composite resins exposed to kretek cigarette smoke laksmiari setyowati, s. setyabudi, and johanna chandra ........................................................ 37–41 10. building team agreement on large population surveys through inter-rater reliability among oral health survey examiners sri susilawati, grace monica, r. putri n. fadilah, taufan bramantoro, darmawan setijanto, gilang rasuna sadho, and retno palupi .................................................. 42–46 11. an in-vitro antimicrobial effect of 405 nm laser diode combined with chlorophylls of alfalfa (medicago sativa l.) on enterococcus faecalis suryani dyah astuti ........................................................................................................................ 47–51 67 differences in cytotoxicity between 5% tetracycline hydrochloride and 15% edta as root canal irrigant devi eka juniarti, karlina samadi, and achmad sudirman department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract 5% tetracycline hydrochloride and 15% edta as a root canal irrigant have been proven to be able to remove smear layer, open dentinal tubules and have antimicrobial activity. an effective root canal irrigation solution must be able to dissolve organic and anorganic debris, lubricate endodontic instruments, disinfect microorganism and non toxic. the purpose of this laboratory experimental study was to determine cytotoxicity differences between 5% tetracycline hydrochloride and 15% edta. 21 samples were used and classified into 3 groups: control, 5% tetracycline hydrochloride and 15% edta groups. cytotoxicity test was done using bhk21 cells. the data was analyzed using bird and forrester formula. it concluded that 5% tetracycline hydrochloride more toxic than 15% edta as a root canal irrigant. key words: cytotoxicity, 5% tetracycline hydrochloride, 15% edta, fibroblast correspondence: devi eka juniarti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction washing and forming root canal, though, other aspects could not be ignored. during the washing and forming rot canal should be followed by irrigation to remove fragment of pulp tissue and accumulation of dentinal fragment.1 podbeilski2 suggested that root canal preparation without root canal irrigation contributing about 70% debris left on apical region. several active chemical substances were used for irrigation with the purpose to wash, lubricate, kill microorganism, solve the tissue and remove smear layer.3 smear layer on dentinal tubule might inhibit diffusion of post root canal filling on the surface of root canal.4,5 removal of smear layer using demineralization material such as: citrate acid, edta and tetracycline hydrochloride solution. demineralization material (chelating agent) would remove smear layer due to the capability of forming complex binding (chelate) with calcium content of smear layer.6 further study on cytotoxicity of root canal irrigant material should be done to find irrigant with maximal anti microbial effect and capability to remove smear layer with minimal toxic effect. citrate acid 30% could be used as root canal irrigant material which has the capability as chelating agent but it would contribute color change (brownish/ burned like color) if it affects soft tissue.7 edta is know as most effective chelating agent in root canal treatment.8 edta has the capability to solve dentin, to remove smear layer, relative nontoxic effect as well as light irritation.1 edta has natrium saline content, which could solve calcium in 15% concentration.9 the previous study on cytotoxicity comparison between 0.2% edta as washing material of tooth cavity and 1% benzalconium chloride, the result suggested 0.2% edta 9–10% had higher toxicity comparing to 1% benzalconium chloride.10 ogundele11 had done in vitro study on edta cytotoxicity using human cell damaged cells taken from breast milk and the result suggested the presence of damaged cells in breast milk cells with edta and the absence of damaged cells without edta. the an other study on baby hamster’s kidney given edta showed vacuolization and degeneration of proximal tubule cells.12 another root canal irrigant material which has the capability as chelating agent is tetracycline hydrochloride. in in vitro study using tetracycline hydrochloride could remove smear layer, release debris and open dentinal tubule. the result was better comparing to aquadest irrigant, sodium hypochlorite and citrate acid.13 irrigation using tetra cycline hydrochloride 5–15% shows it could effectively remove smear layer and significantly open dentinal tubule.7,14,15 meanwhile there is no significant difference found in anti bacterial capability of 5–15% tetracycline hydrocloride.15 cytoxicity study suggested that all cells died after being given 10% tetracycline hydrochloride while the mean of live cells was 0.25% after being given 7.5% tetracycline hydrochloride. fifteen percent edta has capability to solve calcium,9 therefore smear layer is effectively washed and would soften. the dentin in obstructive root canal treatment. five percentage tetracycline hydrochloride have been proved to be able to wash smear layer, to open dentinal tubule as well as to have anti bacterial capability.15,16 68 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008:67-69 both 15% edta and 5% tetracycline hydrochloride have proved to have effectively in washing smear layer and binding calcium dentin in the treatment of root canal, however, difference of toxicity between both of them as irrigant material toward fibroblast tissue still requires further study. the purpose of the study is to know the difference of toxicity in 15% edta with 5% tetracycline hydrochloride. materials and methods the sample of study was fibroblast cell (baby hamster kidney-21) and the free variables were 5% tetracycline hydrochloride p.a. and 15% edta p.a., dependent variables was the number of alive and death fibroblast cells: control variables were temperature 37° c, volume, contact time between irrigant and fibroblast cells, tool sterility, the amount of irrigant, media, the number of fibroblast cells, time cell culture incubation. the study was performed at pusvetma, jl a. yani, surabaya. the material which were used in the study were sterile aquadest, 5% tetracycline hydrochloride p.a., 15% edta p.a, bhk2, 54 cell culture papase, 10% bovine serum, phosphat buffer saline, 0.25% trypsine versene, tryphan blue, eagle media. the tools which were used: petrdish diameter 5 cm, micropipet, laminar flow, light microscope, incubator, hemositometer, culture bottle (roux), balance. firstly, 5% tetracycline hydrochloride was made, weighing 5% tetracycline hydrochloride mixed with aquadest 100 ml, secondary 15% edta was made, weighing 17 gr pure natrium powder dinatrium edta. solved in 9,25 ml naoh and 100 ml sterile aquadest. sample preparation using fibroblast cell (bhk-21) kept in sustaining media containing 10% dimethyl sulfoxid (dmso) + 90% bovine serum, in freezing condition (–850 c). revival of fibroblast cell bhk-21, was done (revivaling freezing bhk-21 fibroblast cell) before the study was started, by placing into the incubator (37° c) for 10 minutes until media melted. melting bhk-21 fibroblast was balanced by adding some water, after the balance was obtained, bhk-21 fibroblast cell was put into centrifuge and was stirred for 10 minutes at speed 200 rpm. saving media of precipitation of fibroblast cell (bhk-21) was disposed and replaced by new growing media consisted of eagle + 10% bovine serum. bhk-21 fibroblast cells which was in the new growth media (media eagle + 10% bovine serum) were put into roux bottle and kept into incubator (37° c) (2 × 24 hour) until the cells completely grew (confluent 20x105cell/ml). roux bottle which was fully filled with bhk-21 fibroblast cell (100%) then washed twice using pbs followed by trypsination in 0,25% trypsin versene added by new eagle media, next it was classified into 4 bottles with initial confluent 5 × 105 cell/ml, put into incubator 37° c (48 hour) until cell confluent would reach 2105 cell/ml. this process was called 55th papase. in order to separate into individual cell which were initially cluster, therefore they should be placed on petridish. the making of bhk-21 fibroblast cell on the petridish required 21 petridish which were divided in 3 groups in which each group consisted of 7 samples. bhk-21 fibroblast cell mono layer fibroblast cell was left to contact 15% tetracycline hydrochloride and 15% edta for 3 minutes in which it was done according to control group, then, the irrigant was disposed an trypsination was repeated similarity to previously done. 21 samples were used in this study and divided into 3 control groups in which each group consisted of 7 samples. three minutes after fibroblast cell (bhk-21) processing was done in petridish, 0,1 cc of cell suspension was taken and added by 0,9 cc tryphan blue, stirred by spraying and sucking 3 times by pipet until the cell was homogeneous, when homogeneous condition was obtained than 0.1 cc was taken and dropped into hemocytometer and soon the calculation was done through light microscope, finally bird and forrester16 equation was applied: % = survice cell × 100% survice cell + dead cell the higher the percentage of survival cells (clear), the lower the percentage dead cells (dark), so the toxicity was lower meaning the test material would more bio compatible. to compare the mean among the groups on toxicity test of 5% tetracycline hydrochloride and 15% edta, independent t-test was done. result the result of the study on difference of cytotocity between 5% tetracycline hydrochloride and 15% edta has been shown on table 1. before the best for different percentage of alive fibroblast cell among control groups was done, in every control group, data distribution test was done for the percentage of alive cells, kolmogorov smirnov test was used for variant homogeneity and levene test was for statistical test. the result of data distribution test shows in all control groups the percentage of alive fibroblast cell has normal data distribution (p > 0.05). independent t-test is done to know the difference of percentage of survive fibroblast cells among control groups. i have been shown on table 2. table 1. the mean percentage of alive fibroblast cells n x sd control 15% edta tetracycline hcl 7 7 7 95,9671 80,5371 17,8186 1,1581 8,6092 6,3769 note: n: the number of samples; sd: standard deviation; x: mean (%) 69juniarti, et al.: differences in cytotoxity between 5% tetracycline hydrochloride and 15% edta table 2. p value of independent t-test on percentage of survive fibroblast cell among group control, 15%group control, 15% edta and 5% tetracycline hcl control 15% edta 5% tetracycline hcl control 15% edta 5% tetracycline p = 0.003 p = 0.001 p = 0.001 on the table 2 has been shown the test result of difference among all groups measuring the percentage of survive fibroblast cell (control, 15% edta, 5% tetracycline hcl) with p < 0.05. it is shows there is significant difference among 3 groups measuring the percentage of survive. discussion in high concentration, tetracycline could inhibit not only synthesis of bacterial protein but also mammalian cell protein.7,16 tetracycline is capable to diffuse passively through hydrophilic pores in outer part of cell membrane and through active transport passed inner part cell membrane and bind ribosome sub unit. this binding would inhibit the entry of t-rna and amino acid in peptide chain elongation process therefore protein synthesis is inhibited. tetracycline also tends to irritate tissue due to strong acid nature.16,17 15% edta would influence fibroblast cell metabolism through chelating effect i.e to have complex binding with ion of inorganic cell and outer surface of plasma of cell membrane so structure disturbance and permeability of cell membrane would occur.10,18 based on the previous study that 15% edta was less toxic due to chelating agent mechanism with limited reaction on calcified tissue and periapical tissue was not significantly influenced, in addition edta also has netral ph: 7.5 therefore tissue irritation will not occur.19,20 cytotoxicity test was done using cell culture method with the advantage of having more accurate result, quantitative toxicity measurement could be achieved and cell response could be observed.21 it is conclude that 5% tetracycline hydrochloride has higher toxicity comparing to 15% edta. references 1. grossman ll, olliet s, del rio ce. ilmu endodontik dalam praktek.ilmu endodontik dalam praktek. abyono r, editor. edisi ke-11. jakarta: egc; 1995. p. 47–8, 59, 205–11. 2. podbielski a, bolckh c, haller. growth inhibitory activity ofgrowth inhibitory activity of gutta percha points containing root canal medications on common endodontic bacterial pathologenis as determined by an optimized quantitative in vitro assay. j endodon 2000; 7:398–403. 3. cohen sc, burns rc. pathways of the pulp. 7th ed. st. louis: cv mosby co; 1998. p. 206–7. 4. torabinejad m, khademi aa, babagoli j, cho y, johnson wb, bozhilov k. a new solution for the removal of the smear layer. j endodon 2003; 29(3):170–5. 5. foster kh, kulild jc, weller n. effect of smear layer removal on the diffusion of calcium hydroxide through radicular dentin. j endodon 1994; 2:78. 6. maduratna e. kebersihan permukaan dentin akar setelah pemberian larutan tetrasiklin hidroklorida disbanding asam sitrat. penelitian dosen muda. surabaya: universitas airlangga; 1998. p. 9–10. 7. maduratna e. terlepasnya lapisan smir pada permukaan akar setelah pemberian larutan tetrasiklin hidroklorida. majalah kedokteran gigi (dental journal) 2000; 33(3):106–8. 8. serper a, calt s. the demineralizing effects of edta at different concentrations and ph. j endodon 2002; 28(7):501–2. 9. utami lm. perbedaan larutan edta 15% dan edtac 15% dalam menghilangkan smear layer pada dinding saluran akar. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 1998. p. 40. 10. subiyanto a. pengaruh pemakaian bahan edta 0,2% dan benzalkonium klorid 1% sebagai pembersih kavitas gigi terhadap kultur jaringan hidup. penelitian dosen muda. surabaya: universitas airlangga; 1996. p. 13–5. 11. ogundele m. cytotoxicity of edta used in biological samples: effect on some breastmilk studies. 2003. p. 1–9. available at: http://www. medfive.com/edta. accessed 2006. 12. kunardi l, setiabudi r. farmakologi dan terapi. jakarta: bagianjakarta: bagian farmakologi fakultas kedokteran, universitas indonesia; 1995. p. 651-60, 794–5.–5.5. 13. haznedaroglu, ersev h. tetracycline hcl solution as a root canal irrigant. j endodon 2001; 27(12):738–40.–40.40. 14. isik ag, tarim b, hafez aa, yalcin fs, onan u, cox cf. a comparative scanning electron microscope study on the characteristic of demineralized dentin root surface using different tetracycline hcl concentrations and application times. j periodontal 2000; 71(2):219–25. 15. rostyarini s. daya hambat mikroorganisme menggunakan larutan tetrasiklin hcl 5%, 10%, 15% sebagai bahan irigasi pada perawatan saluran akar. karya tulis akhir. surabaya: universitas airlangga 2005. p. 33. 16. maduratna e. sitotoksisitas larutan tetrasiklin hidroklorida terhadap kultur sel dibanding asam sitrat. majalah kedokteran gigi (dental journal) 2002; 35(4):164–6. 17. rianto s. farmakologi dan terapi. edisi ke-4. jakarta: bagianjakarta: bagian farmakologi fakultas kedokteran universitas indonesia; 2003. p. 651-60, 794–5.–5.5. 18. ogundele m. the role of divalent cations in the mechanism of edta cytotoxity. http://f: inabis`98 2006. p. 1. 19. maduratna e. biokompatibilitas gel tetrasiklin hidroklorida terhadap kultur jaringan. majalah kedokteran gigi (dental journal) 1999; 32(4):140–3. 20. canderasari nm. perbedaan sitotoksisitas larutan tetrasiklin hidroklorida 1% dengan natrium hipoklorit 2,5%. karya tulis akhir. surabaya: universitas airlangga; 2004. p. 1. 21. walton re, torabinejad m. prinsip dan ilmu praktek ilmu endodonsi. edisi ke-2. jakarta: egc; 1997. p. 360–78. 86 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 86–89 research report density of streptococcus mutans biofilm protein induced by glucose, lactose, soy protein and iron indah listiana kriswandini, indeswati diyatri, and intan amalia putri department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: caries constitute an infectious disease that result from the interaction of bacteria with the host and the oral environment. streptococcus mutans (s. mutans) represents the main bacterium that causes caries. the ability of s. mutans to form biofilms in the oral cavity is influenced by daily nutrient intake. this study of bacterial biofilm proteins can be used in the manufacture of kits for the detection of infectious diseases such as caries in the oral cavity. a biomarker is required for the manufacture of the detection kit. consequently, research must first be conducted to determine the molecular weight and density of s. mutans biofilm proteins induced by several different daily nutrients, namely; 5% glucose, 5% lactose, soy protein and 5% iron. purpose: this study aimed to analyse the density of s. mutans biofilm protein induced by 5% glucose, 5% lactose, soy protein, and 5% iron. methods: the density of the s. mutans biofilm protein bands induced were measured using ez imager gel doctm software. results: a band of biofilm protein (61.7 kda) was obtained from s. mutans induced by 5% glucose, four bands of biofilm protein (180 kda; 153,9 kda; 43,9 kda; 37,5 kda) from 5% lactose induction and seven bands of biofilm protein (157,9 kda; 86,6 kda; 66,5 kda; 50,1 kda; 37,9 kda; 32,3 kda; 29,4 kda) from soy protein induction. in contrast, s. mutans induced by 5% iron did not show any protein bands. the proteins that result from each inducer are of differing densities. conclusion: the protein bands from each inducer are of different densities which can be used in the further test to make a biomarker for dental caries detection kits. keywords: caries; nutrient intake; protein biofilm; protein density; streptococcus mutans correspondence: indah listiana kriswandini, department of oral biology, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: indah-l-k@fkg.unair.ac.id introduction caries constitute an infectious disease that results from the interaction of bacteria with the host and the oral environment. dental caries form when food waste adheres to the teeth producing a demineralization process involving bacterial interactions on the tooth surface. the etiologic factors of caries include: plaque microorganisms, diet and time.1 the predominant microorganisms playing a role in the occurrence of caries are streptococcus mutans (s. mutans) which demonstrate the ability to attach to enamel surfaces, produce acid metabolites and form damaging biofilms.2 biofilms are layers formed by colonies of microbial cells, slimy in texture, which attach themselves to the surface and are difficult to remove. plaque constitutes a biofilm that forms in the oral cavity3 and whose subsequent development is influenced by changes in the prevailing environmental conditions. one such change results from exposure to chemical nutrients present in food. examples of daily consumed food include glucose and lactose as sources of carbohydrate and soy protein, in addition to iron which is one of the minerals required by the body. various kinds of food can induce s. mutans biofilm formation in the oral cavity, the most basic example being glucose. a high concentration of glucose modulated by hydrogen ions can increase the metabolism of s. mutans resulting in the formation of extracellular polymeric substances (eps). these eps will help s. mutans adhere to the tooth surface and form a matrix as a means of self-defense.4 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p86–89 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p86-89 87kriswandini, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 86–89 s. mutans in a microenvironment will form biofilms in individual cells whose character is influenced by the nutrients around them5 and which will express the same or different proteins from the planktonic cell depending on the inducer. biofilms formed by individual s. mutans cells are controlled by specific genes that express biofilm formation.5 because the biofilm proteins formed from daily food intake have different characteristics, they can be used as a reference in the manufacture of detection kits which measure the severity of s. mutans-induced dental caries. for the manufacture of disease detection devices, an accurate biomarker is required. this renders necessary calculating both the molecular weight and expression strength (density) of each specific protein. therefore, research must be conducted on s. mutans biofilm protein candidates which have been induced by several variant nutrients in order to determine their density. the aim of this study was to analyse the expression strength of s. mutans biofilm proteins by measuring the density of protein bands using the gel doc ™ ez imager software after induction by 5% glucose, 5% lactose, soy protein and 5% iron. these inducing agents represent food nutrients which form part of a daily diet, are consumed during metabolism and contain microbes.6 this method of measuring protein density is intended to determine the volume of biofilm proteins produced after the inducing of specific materials characterized by their greater density. such materials are used to meet the minimum requirements of follow-up work to a western blot and also for manufacturing vaccines. materials and methods this research was conducted at the microbiology laboratory, faculty of medical and the biomedical laboratory at universitas brawijaya, malang. procedures for measuring the protein density strength of biofilm include: bacterial culture, biofilm growth, isolation of biofilm proteins, sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (page) and protein density analysis using gel doc ™ ez imager software. s. mutans were cultured anaerobically and replicated in luria bertani (lb) medium to ensure that bacterial growth occurred with an indication of turbidity equivalent to mcfarland 8 standard. gram staining was subsequently conducted and observed through a microscope to ensure that the culture results were not contaminated. following biofilm growth, s. mutans was inserted into 50 ml brain heart infusion (bhi) in an erlenmeyer tube supplemented with 5% glucose, 5% lactose, 5% iron (fecl2) in each tube and s. mutans inserted into 50 ml bhi in an erlenmeyer tube without an inducer (as a control group). for soy protein, s. mutans was inserted into a 50 ml tube with trypticase soy broth (tsb) before being incubated anaerobically overnight at 37°c in order to grow s. mutans biofilm. isolation of biofilm protein was performed by scraping biofilms formed from the base of each erlenmeyer tube, adding phosphate buffer saline (pbs) + tween 0.05% and transferring it to an eppendorf tube prior to centrifuging it at 12,000 rpm for 10 minutes. the supernatant was transferred to an eppendorf tube before being precipitated with alcohol at a ratio of 1:1, and incubated for one night. the concentrations of the proteins obtained were measured by means of nanodrop.7 the method of sds-page electrophoresis began with preparation of the gel. after the gel had been formed from a mixture of 12% separating gel and 4% stacking gel, the plate was mounted on an electrophoresis device and the buffer was poured into the electrophoresis vessel. injection of samples into the gel made by inserting 10 µl of isolate protein into each s. mutans biofilm in tsb medium, glucose-induced s. mutans biofilm in bhi media, lactoseinduced s. mutans biofilm in bhi media, and iron-induced s. mutans biofilm in bhi media was added to 10 µl tris-cl + 20 µl reducing sample buffer (rsb). it was then inserted into the micro tube, before being heated in a water heater at 100oc for five minutes. after being cooled, 20 µl of the sample was inserted into each of the gel wells. an electric current of 30 ma and 100 v for electrophoresis was activated. after the gel had been removed from the plate, staining and washing the gel was completed. staining was performed by soaking the gel in a staining solution for ± 4 hours–overnight in a shaker incubator.8 the sds-page electrophoresis gel was documented in the form of images. the results in the form of this image were analyzed to measure the protein molecular weight and the strength of the expression (density) of the protein bands using gel doc ™ ez imager software. the program read the thickness of the band per column selected as a fluctuating curve. results from the sds-page procedure that used 4% stacking gel and 12% separating gel, each protein fraction that appeared could be seen in its molecular weight expressed in units of kda and its density in units of int. the results produced using ez imager gel doc ™ software indicated the presence of several s. mutans protein bands that appeared after being induced with 5% glucose, 5% lactose and soy protein, while s. mutans induced with 5% iron did not produce any protein band. the marker protein used was jena bioscience blueray which contained ten standard proteins ranging from 11-180 kda (figure 1). as shown from the contents of table 1, within the procedure for calculating protein density using ez imager gel doc tm software each visible biofilm protein band possessed a different density. the density of each protein band was expressed in units of intensity (int). the manner in which the ez imager gel doc tm software functions dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p86–89 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p86-89 88 kriswandini, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 86–89 is simply to compare the peak of the protein band graphs which appear on the monitor screen. the thickness (density) of the protein band is illustrated by the graph above. the density of each protein band with a different inducer can be read in the data above (bio-rad laboratories, inc., 2014). from the table above, it can be seen that biofilms induced by 5% glucose contain one protein band of 61.7 kda with a density of 184.21 int. whereas biofilms induced by 5% lactose contain four protein bands, there are seven protein bands in biofilms induced by soy protein that appear with their respective molecular weight and density in table 1. each of these protein molecular weights can be said to be the biofilm protein candidates which require further tests to enable its use as a marker in detecting the severity of caries disease caused by s. mutans. in iron-induced biofilms, no protein bands appear which could be due to several factors preventing iron from forming protein bands. figure 1. the result of electrophoresis. kda = molecular weight in units of daltonkilo; lane 1 = marker; lane 2 = standard (planktonic); lane 3 = glucose-induced whole cell; lane 4 = glucose-induced biofilm; lane 5 = lactose-induced whole cell; lane 6 = lactoseinduced biofilm; lane 7 = soy protein-induced whole cell; lane 8 = soy protein-induced biofilm; lane 9 = iron-induced whole cell; lane 10 = iron-induced biofilm. table 1. the molecular weight and density of s. mutans biofilm protein corresponding to each inducer inducer band no. mol. wt. (kda) volume (int) planktonic (control) 1 180.0 339.388 2 122.8 401.676 3 86.6 340.612 4 66.5 702.712 5 39.4 434.180 6 34.2 356.184 7 31.4 155.244 8 27.3 138.108 9 24.5 188.156 10 18.8 137.904 11 16.3 475.728 12 15.5 380.392 13 14.0 276.488 14 12.0 767.992 15 11.0 1.548.972 glucose 1 61.7 184.21 lactose 1 180.0 174.76 2 153.9 76.64 3 43.9 143.62 4 37.5 155.72 soy protein 1 157.9 260.24 2 86.6 152.86 3 66.5 169.66 4 50.1 175.17 5 37.9 157.96 6 32.3 102.54 7 29.4 115.06 iron dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p86–89 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p86-89 89kriswandini, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 86–89 discussion the analysis in this study of s. mutans biofilm protein with sds-page electrophoresis aimed to determine s. mutans biofilm proteins that had been induced by 5% glucose, 5% lactose, soy protein and 5% iron. the density of the protein bands was subsequently analyzed using gel doc tm ez imager software, the results of which are shown in table 1. the density of each protein can be determined from the measurements taken in units of int. the density measurement in this study was taken to determine the amount of protein to be used in subsequent western blotting tests. s. mutans induced by 5% glucose had one band of biofilm protein of 61.7 kda and with a density of 184.21 int. because only one protein band appears, the protein can be considered to be a 5% glucose-induced s. mutans biofilm specific protein. further tests such as western blotting are required until the protein can finally be used as a marker in the identifiy of caries which result from glucose consumption. s. mutans induced by 5% lactose contains four bands of biofilm proteins ranging from 37.5 kda to180 kda, each of which is of a different density. protein with a molecular weight of 37.5 kda has a density of 155.72 int which is the highest protein density compared to that of the others. that means that protein content is dominant in lactoseinduced biofilms. s. mutans induced by soy protein contains seven biofilm protein bands which appear ranging from 29.4 kda to 157.9 kda. each protein band is of a different density, but there is one protein with a more dominant protein content than the others. this is a protein whose molecular weight is 157.9 kda and which has a density of 260.24 int. according to svensäter et al.9 the names of proteins in s. mutans, it can be said that those protein bands with a molecular weight of 29.4 kda are thought to be protein phosphoglycerate mutase. proteins with a molecular weight of 32.3 kda are regarded as grpe proteins since the gene that becomes the protein is the grpe gene. a protein with a molecular weight of 37.9 kda is considered to be a protein exopolyphosphatase.9 s. mutans induced with 5% iron did not demonstrate the development of any protein band. this is probably due to excessive iron concentration which inhibits biofilm formation. iron is a micronutrient crucial to the optimal growth of s. mutans. however, too high a concentration of this element can kill s. mutans bacteria. ribeiro et al.10 in their in situ study, stated that iron (fe) at a concentration of 100µg/ml was able to reduce the number of s. mutans cells present in dental biofilms.10 fe has an antibacterial effect not only in terms of killing s. mutans cells, but also by disrupting the ability of these bacteria to form biofilms. it has been shown in situ that dental biofilms formed in humans exposed to fe contain a lower number of s. mutans. in terms of its working mechanism, fe possesses the ability to inhibit f-atpase contained in s. mutans. as a result, fe can affect the acidogenicity and aciduricity of s. mutans. on the other hand, by interfering with sucrose metabolism, fe can reduce the production of eps.10 protein expression in biofilm formation inside the oral cavity is influenced by daily nutrient intake from the food consumed. the analysis above suggests that each inducer produces protein bands of differing molecular weights and densities which differ from those of plankton and from other inducers. protein bands that result from 5% glucose induction, 5% lactose, soy protein and 5% iron are ones that play a role in biofilm formation. high or low protein density affects the calculation of the amount of protein that will be used in subsequent western blot tests. the protein band with the highest density indicates the dominant protein content of each inducer. however, those proteins that have a dominant protein content are not necessarily a specific protein from each inducer. rather, all protein bands that appear after being induced by each inducer are only candidate proteins that can be further tested to identify s. mutans biofilm specific proteins. further analysis to determine the function of these specific biofilm proteins is a prerequisite to the protein being chosen as a marker in a dental caries detection kit and its association with daily consumption of glucose, lactose, soy protein and iron. in conclusion, s. mutans biofilm induced by 5% glucose has one protein band candidate, 5% lactose has four protein bands, while soy protein contains seven protein bands. all protein bands from each inducer possess different densities which can be used in the further test to make a biomarker for dental caries detection kits. references 1. mustika md, carabelly an. insidensi karies gigi pada anak usia prasekolah di tk merah mandiangin martapura periode 2012-2013. dentino j kedokt gigi. 2014; 2(2): 200–4. 2. forssten sd, björklund m, ouwehand ac. streptococcus mutans, caries and simulation models. nutrients. 2010; 2: 290–8. 3. fatmawati dwa. hubungan biofilm streptococcus mutans terhadap resiko terjadinya karies gigi. stomatognatic. 2011; 8(3): 127–30. 4. simon l. the role of streptococcus mutans and oral ecology in the formation of dental caries. lethbridge undergrad res j. 2007; 2(2): 1–6. 5. krzyściak w, jurczak a, kościelniak d, bystrowska b, skalniak a. the virulence of streptococcus mutans and the ability to form biofilms. eur j clin microbiol infect dis. 2014; 33(4): 499–515. 6. brooks gf, carroll kc, butel js, morse sa, mietzner ta. jawetz, melnick & adelberg’s medical microbiology. 26th ed. new york: mcgraw-hill medical; 2013. p. 313–21. 7. deddy k. karakterisasi dan pola distribusi antibodi hasil induksi protein membran spermatozoa manusia 20 kda. thesis. malang: fakultas mipa universitas brawijaya; 2009. p. 30–35. 8. fatchiyah, widyarti s, arumingtyas el. teknis analisis biologi molekuler. malang: fmipa universitas brawijaya; 2018. p. 6–37. 9. svensäter g, welin j, wilkins jc, beighton d, hamilton ir. protein expression by planktonic and biofilm cells of streptococcus mutans. fems microbiol lett. 2001; 205(1): 139–46. 10. ribeiro ccc, ccahuana-vásquez ra, carmo cds do, alves cmc, leitão tj, vidotti lr, cury ja. the effect of iron on streptococcus mutans biofilm and on enamel demineralization. braz oral res. 2012; 26(4): 300–5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p86–89 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p86-89 vol 38 no 2-2005 60 keasaman minuman ringan menurunkan kekerasan permukaan gigi (acidity of soft drink decrease the surface hardness of tooth) edhie arif prasetyo bagian ilmu konservasi gigi fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract acidity can bring about tooth erosion. a laboratory experiment about soft drink acidity to the hardness of tooth surface was done. the purpose of the study was to investigate the surface hardness of the tooth surface after immersion in some kinds of soft drinks. thirty maxillary premolars were randomly divided into three groups. the first group was immersed in aqua, ph 7.6, the second group in the tea, ph 6,7 and the last group in cola ph 2,5 for 30, 60 and 120 minutes. the surface hardness measurement was done before and after immersion using micro vickers hardness tester. the achieved data were analyzed using anova followed by hsd. it was concluded that the immersion in soft drink for 120 minutes could decrease the surface hardness of tooth. key words: acidity, surface hardness, soft drink korespondensi (correspondence): edhie arif prasetyo, bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan erosi gigi dan karies gigi mempunyai kesamaan dalam jenis kerusakannya yaitu terjadi demineralisasi jaringan keras yang disebabkan oleh asam. asam penyebab erosi berbeda dengan asam penyebab karies gigi. erosi gigi berasal dari asam yang bukan sebagai hasil fermentasi bakteri. karies gigi berasal dari asam yang merupakan hasil fermentasi karbohidrat dari sisa makanan oleh bakteri dalam mulut.1 erosi terjadi secara merata di permukaan gigi. hal ini mungkin karena terjadi suatu kelarutan dari elemen anorganik elemen gigi secara perlahan-lahan atau kronis. makanan yang menggunakan kuah atau cairan yang asam (ph < 7), misalnya acar, pempek, dapat menyebabkan erosi pada gigi.1 demikian juga dalam suasana atau media yang bersifat asam (ph < 7) dapat mengakibatkan erosi pada enamel gigi.2,3 minuman ringan merupakan minuman yang tidak mengandung alkohol (non-alkohol), merupakan minuman yang berkarbonat.4 minuman ringan mengandung bahan pemanis, asam dan bahan perasa alami maupun buatan. bahan alami dapat berupa kacang-kacangan, buah-buahan, sayur-sayuran. kopi, teh, susu serta coklat bukan merupakan minuman ringan, yang termasuk minuman ringan adalah cola, lemon, orange dan kopi bir serta anggur. demineralisasi dapat terjadi apabila enamel berada dalam suatu lingkungan ph di bawah 5,5, saat ini banyak minuman ringan dengan ph di bawah 5,5 yang dikonsumsi oleh masyarakat.5,6,7 ph berperan pada demineralisasi karena ph yang rendah akan meningkatkan konsentrasi ion hidrogen dan ion ini akan merusak hidroksiapatit enamel gigi.8 produksi berbagai jenis minuman ringan yang dipasarkan dan dikonsumsi secara global diketahui secara pasti dapat menyebabkan demineralisasi enamel yang secara langsung dikenal sebagai erosi. bila melalui fermentasi karbohidrat dalam hubungannya dengan aktivitas bakteri dikenal sebagai karies gigi. demineralisasi secara langsung yang diakibatkan oleh kandungan asam dalam suatu jenis minuman ringan, kemungkinan lebih bermakna dibanding kerugian yang diakibatkan kandungan gulanya.9 proses erosi gigi dimulai dari adanya pelepasan kalsium enamel gigi, bila hal ini berlanjut terus akan menyebabkan kehilangan sebagian elemen enamel, dan apabila telah sampai ke dentin maka penderita akan merasa ngilu.8 demikian juga air minum yang bersifat asam (ph < 7) ternyata dapat juga menyebabkan erosi pada gigi.9 minuman ringan yang berbahaya bagi enamel adalah minuman yang mengandung karbohidrat yang mudah difermentasi, sangat asam dan mempunyai adesi termodinamik yang sangat tinggi, sehingga minuman ini tidak mudah dihilangkan oleh saliva.5 hal ini disebabkan oleh beberapa faktor yang mempengaruhi proses demineralisasi, yaitu jenis dan konsentrasi asam minuman yang tidak berdisosiasi, kandungan karbohidrat dalam minuman, ph dan kapasitas dapar minuman serta kandungan fosfat dan f1uor yang ada dalam minuman.3,5,10 sebagaimana diketahui bahwa enamel sebagian besar terdiri dari hidroksiapatit (calo (po4)6 (oh)2) atau fluoroapatit (calo (po4)6 f2), kedua unsur tersebut dalam 61prasetyo: keasaman minuman ringan suasana asam akan larut menjadi ca2+; po4-9 dan f-, oh-. ion h+ akan beraksi dengan gugus po4-9, f-, atau ohmembentuk hso4-; h2so4 hf atau h2o, sedangkan yang kompleks terbentuk cahso4; capo4 dan cahpo4.3,5 kecepatan melarutnya enamel dipengaruhi oleh derajat keasaman (ph), konsentrasi asam, waktu melarut dan kehadiran ion sejenis kalsium, dan fosfat.1 minuman ringan yang menyebabkan demineralisasi enamel gigi adalah minuman yang mempunyai ph rendah dan kapasitas dapar tinggi. kapasitas dapar adalah jumlah basa yang diperlukan untuk menaikkan ph minuman ke ph netral.2 reaksi kimia pelepasan ion kalsium dari enamel gigi dalam medium yang bersifat asam, yaitu pada ph 4,5 sampai 6 merupakan reaksi orde nol. adapun pengaruh ph terhadap koefisien laju reaksi menunjukkan, bahwa semakin kecil atau semakin asam media, maka makin tinggi laju reaksi pelepasan ion kalsium dari enamel gigi.11 reaksi kimia pelepasan ion kalsium dari enamel gigi dalam suasana asam ditunjukkan dengan persamaan reaksi sebagai berikut: 2,11 calo (po4)6 f2 calo (po)6 f2 + 2n h+ padat terlarut n ca2+ + calo – n h20 – 2n (po4)6 f2 terlepas padat mengingat bahwa kalsium merupakan komponen utama dalam struktur gigi, dan demineralisasi enamel terjadi akibat lepasan ion kalsium dari enamel gigi, maka pengaruh asam pada enamel gigi merupakan reaksi penguraian. demineralisasi yang terus-menerus akan membentuk pori-pori kecil atau porositas pada permukaan enamel yang sebelumnya tidak ada. untuk mengetahui sampai seberapa jauh keasaman (ph) yang terdapat di dalam minuman ringan cola, dan teh botol dapat menyebabkan kelarutan dari elemen gigi, sehingga dapat menurunkan kekerasan permukaan enamel gigi, maka peneliti merasa perlu untuk melakukan penelitian. penelitian ini bertujuan untuk mengetahui sampai seberapa jauh penurunan kekerasan permukaan enamel gigi, oleh karena keasaman (ph) yang ada di dalam minuman ringan. manfaat penelitian ini memberi informasi pada masyarakat bahwa minuman yang terlalu asam dapat melarutkan elemen gigi sehingga dapat menurunkan kekerasan permukaan gigi. bahan dan metode jenis penelitian ini yaitu eksperimental laboratoris, tempat penelitian di laboratorium konservasi gigi univsersitas airlangga dan laboratorium teknik mesin institut teknologi 10 nopember surabaya, penelitian dilakukan pada bulan april 2004. dalam penelitian ini digunakan bahan: air mineral (aqua danone), teh botol (pt sosro), cola (the cocacola company), gigi premolar atas yang telah dicabut untuk perawatan ortodonsia, gips keras (moldano), malam perekat. pembuatan sampel dilakukan dengan cara: 50 gigi premolar rahang atas dipotong bagian mahkota dari akarnya. kemudian secara random diambil sebanyak 30, dibagi menjadi 3 kelompok yang masing-masing kelompok terdiri dari 10 sampel. setiap sampel diberi tanda (nomer urut) untuk setiap kelompok. selanjutnya sampel ditaman dalam balok gips dengan ukuran 2 × 3 cm. permukaan bagian bukal menghadap ke atas bagian tengah balok gips diberi tanda dengan garis guratan. kemudian dilakukan pengukuran kekerasan permukaan dan dicatat, yang merupakan kekerasan awal sebelum diberi perlakuan perendam dengan cara sebagai berikut: balok gips dijepit dengan permukaan gigi menghadap ke atas kemudian dijepit dengan alat penjepit pada meja alat mikro vickers hardness tester. selanjutnya sampel diatur supaya tepat di tengah lensa obyektif dan difokuskan dengan cara memutar pegangan yang ada pada kanan alat, searah dengan jarum jam, setelah pada lensa okuler terlihat gambar dalam keadaan fokus, sampel dipindah dengan cara menggeser ke arah kanan sehingga tepat berada di bawah diamond penetrator, lalu tombol penetrator ditekan, diamond penetrator akan turun, ini ditandai lampu hijau akan menyala, bila diamond penetrator telah menyentuh sampel, maka lampu merah akan menyala. setelah 30 detik diamond penetrator akan naik, lalu ditunggu sampai lampu merah dan hijau padam. sampel digeser kembali ke tempat lensa okuler dan difokuskan lagi, maka akan terlihat gambar bentukan belah ketupat, kemudian panjang diagonalnya diukur langsung dengan mikrometer yang ada pada lensa okuler. hasil pengukuran panjang diagonal kemudian diambil rataratanya. (d) dimasukkan ke dalam rumus: nvh = 1,854 × p d 2 nvh = kekerasan sampel (kg/mm2) p = berat beban (100 gram) d = panjang diagonal (1/1000 mm) sehingga didapatkan nilai kekerasan permukaan sampel, dan hal di atas dilakukan untuk semua kelompok. kelompoknya i direndam dalam air mineral (aqua) selama 30 menit kemudian dilakukan pengukuran kekerasan permukaan (p1), dan dicatat, sampel direndam lagi selama 30 menit dan dilakukan pengukuran kekerasan permukaan sampel yang merupakan pengukuran setelah 60 menit (p2), dan yang terakhir sampel direndam selama 60 menit lagi dan dilakukan pengukuran (p3). kelompok yang lain dilakukan seperti dengan kelompok 1, perbedaan terletak pada cairan perendam, pada kelompok 2 direndam dalam teh botol (ph 6,7) sedangkan kelompok 3 direndam dalam 62 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 60–63 cola (ph 2,5). setiap sampel dilakukan pengukuran 3 kali, kemudian diambil rata-ratanya yang merupakan kekerasan sampel. hasil hasil pengukuran rerata dan simpang baku kekerasan permukaan sampel setelah direndam dalam air mineral, teh botol dan cola, selama 30 menit, 60 menit, dan 120 menit serta setelah perendaman dapat dilihat pada tabel 1. sebelum dilakukan analisis dilakukan uji homogenitas varians, ternyata data yang diperoleh merupakan data yang variansnya homogen dan distribusi normal, maka dilakukan analisis dengan uji anova dilanjutkan dengan hsd. hasil perhitungan anova satu arah didapatkan hasil tidak ada perbedaan bermakna kekerasan permukaan gigi setelah perendaman dalam teh botol dan cola selama 30, 60 dan 120 menit. untuk mengetahui perbedaan antara kelompok lama perendaman digunakan uji hsd. tabel 2. uji hsd kekerasan permukaan setelah perendaman dalam air, teh botol dan cola selama 30, 60, dan 120 menit air teh botol cola 30 60 120 30 60 120 30 60 120 menit menit menit sebelum tb tb tb b b b b b b 30 menit tb tb b b b b 60 menit tb b b 120 menit keterangan: b = bermakna; tb = tidak bermakna pada tabel 2 menunjukkan tidak ada perbedaan bermakna setelah perendaman dalam air antara sebelum (kontrol) dengan setelah direndam selama 30, 60, dan 120 menit, didapatkan perbedaan yang bermakna setelah direndam dalam teh botol dan cola, antara sebelum perendaman dan setelah direndam selama 30, 60, dan 120 menit, ini menunjukkan terjadi penurunan kekerasan permukaan gigi setelah direndam dalam teh botol dan cola. pembahasan berdasarkan tabel 1, terlihat hasil rerata kekerasan permukaan sebelum dan sesudah dilakukan perendaman dalam aqua didapatkan hasil yang sama demikian juga dengan uji anova, didapatkan angka lebih besar dari 0 yang berarti tidak ada perbedaan yang bermakna, hal ini mungkin karena air (aqua) mempunyai ph 7,5 sehingga tidak menyebabkan perubahan kekerasan permukaan sampel atau tidak terjadi perubahan atau kelarutan dari enamel gigi. pada perendaman sampel dalam teh botol (ph 6,7) terjadi penurunan kekerasan permukaan meskipun hanya sedikit (tabel 1) ada perbedaan yang bermakna pada perlakuan lama perendaman yang diberikan. hal ini mungkin selain ph, molekul asam yang tidak berdisosiasi akan melarutkan enamel gigi. dalam penelitian, minuman ringan yang lebih mendalam, ternyata ada korelasi yang bermakna antara intensitas deminera1isasi enamel dengan ph, kapasitas dapar minuman ringan, jenis dan kadar asam yang tidak berdisosiasi, kandungan fosfor dan fluor dalam minuman. 3,10 pada perendaman da1am minuman ringan (cola) yang mempunyai ph 2,5, merupakan ph terendah, terjadi penurunan kekerasan yang sangat nyata dan ada perbedaan yang bermakna (tabel 2). hal ini kemungkinan disebabkan karena banyak terjadi kelarutan pada enamel, karena gigi yang digunakan sebagai sampel sebagian besar mengandung kalsium (dalam suasana asam ph 2,5) sehingga menyebabkan kekerasan permukaan gigi berkurang atau menurun. hasil ini sesuai dengan penelitian sebelumnya, bahwa air minum yang bersifat asam (ph < 7) dapat menyebabkan terjadinya kasus erosi gigi. peneliti lain menyatakan, enamel gigi dapat mengalami erosi, disebabkan oleh bahan makanan dan minuman yang bersifat asam (ph < 7) misalnya kuah pempek, buah sitrun dan jeruk manis.5,7,8 demineralisasi enamel adalah rusaknya hidroksi apatit gigi yang merupakan komponen utama enamel akibat proses kimia. kondisi deminera1isasi enamel terjadi bila ph larutan disekeliling permukaan enamel lebih rendah dari 5,5, (umumnya ph minuman ringan berkisar 2,3–3,6) dan konsentrasi asam yang tidak berdisosiasi itu lebih tabel 1. nilai rata-rata simpang baku kekerasan permukaan sampel setelah direndam dalam air mineral, teh botol, dan cola selama 30, 60, dan 120 menit.dan kelompok kontrol {kg/mm2 (vhn)} air mineral teh botol cola lama perlakuan n x ± sb x ± sb x ± sb 0 (kontrol) 30 menit 60 menit 120 menit 10 10 10 10 314,313 ± 2,631 314,218 ± 2,636 314,227 ± 2,647 314,245 ± 2,627 321,585 ± 3,465 313,170 ± 3,962 308,928 ± 4,480 293,537 ± 4,921 317,853 ± 1,797 315,464 ± 2,962 275,962 ± 4,719 218,700 ± 5,196 63prasetyo: keasaman minuman ringan tinggi di permukaan enamel, daripada di dalam enamel. demineralisasi enamel terjadi melalui proses difusi, yaitu proses perpindahan molekul atau ion yang larut dalam air ke atau dari dalam enamel ke saliva karena ada perbedaan konsentrasi dari keasaman minuman di permukaan dengan di dalam enamel gigi. keasaman minuman (hl) yang mempunyai konsentrasi tinggi, dan ph awal minuman yang rendah akan berdifusi ke dalam enamel, melalui kisi kristal dan prisma tubuli enamel yang mengandung air dan matriks organik atau protein. bagaimana minuman tersebut melekat pada permukaan enamel dan juga untuk menentukan kemampuan saliva menggantikan minuman tersebut, untuk mencegah terjadinya demineralisasi enamel, perlu dilakukan pengukuran termodinamika. minuman yang mempunyai adesi termodinamika yang lebih besar dari adesi termodinamika saliva, tidak dapat digantikan oleh saliva dari permukaan gigi. sebaliknya minuman yang mempunyai adesi termodinamika yang lebih rendah dari adesi termodinamika saliva, dapat digantikan oleh saliva sehingga mengurangi demineralisasi enamel.5 erosi gigi dimulai dari adanya pelepasan ion kalsium, dan jika hal ini berlanjut terus, maka akan menyebabkan kehilangan sebagian dari prisma enamel, apabila terus berlanjut akan terjadi porositas. porositas akan menyebabkan kekerasan permukaan enamel gigi akan berkurang. penurunan kekerasan permukaan enamel gigi yang besar akibat perendaman dalam cairan cola (ph 2,5) sesuai dengan penelitian terdahulu, mengatakan bahwa bila terjadi penurunan satu satuan ph, akan dapat menyebabkan laju pelepasan kalsium sebesar 19,5 kali, ini berarti semakin kecil ph atau semakin asam media, maka semakin tinggi laju reaksi pelepasan kalsium dari enamel gigi.11 uji kekerasan didapatkan enamel menjadi lunak, dan dapat hilang oleh karena tersikat, sesudah kontak dengan asam, terutama sari sitrun.8 hasil penelitian yang telah dilakukan dapat ditarik kesimpulan bahwa keasaman minuman (ph) yang kurang dari 7 atau bersifat asam dapat menurunkan kekerasan permukaan enamel gigi, selama perendaman 12 jam. saran dari penelitian ini adalah mencegah agar suasana di dalam rongga mulut tidak terlalu asam, baik yang dihasilkan oleh bakteri atau makanan atau minuman, sehingga dapat mencegah pelepasan ion kalsium dari enamel gigi. mengurangi proses demineralisasi dapat dilakukan dengan menghentikan difusi asam, yaitu mengurangi kontak asam dengan gigi, misal mengurangi intake asam atau minum minuman ringan dengan memakai sedotan, cara lain yaitu dengan menghentikan terbentuknya persenyawaan kompleks kalsium fosfat dengan meningkatkan ketahanan enamel melalui fluoridasi air minum atau topikal aplikasi dengan f1uor atau penambahan ion fluor dalam minuman. daftar pustaka 1. tri budi. hubungan erosi gigi dengan kebiasaan makan pempek di palembang sumatra selatan. disertasi. surabaya: pascasarjana univesitas airlangga; 1989. h. 190–3. 2. jarvinen vk, stabholz a, wilkinson g. in vitro determineralization of erosion. j dent rest 1990; 79(6):942–7. 3. grobler sr, senekal pjc, laubscher ja. in vitro demineralization of enamel by orange juice, apple juice, pepsi cola and diet pepsi cola. clin prevent dent 1990; 12: 5–9. 4. robert p. the new encyclopaedia britannica. 14th ed. chicago: encyclopaedia britanica inc; 1986. p. 14, 752–3. 5. ireland aj, guinness nm, sherriff m. an investigation into the ability of soft drink to adhere to enamel. caries res 1995; 29: 470–6. 6. heinzt sd, bastos jrm, tomita n. fluoride content and ph of beverages found on the bazilian market. j dent res 1996; 75: 192. 7. fung r, yaari am. fluoride levels in popular brands of soft drink. j dent res 1996; 12:1395. 8. schuurs afb, 1991. gebitspathologie, afwijkigen van de harge tendweefsels. sutatmi sutyo, rafiah abyono. dalam: potologi gigi-geligi, kelainan jaringan keras gigi. yogyakarta: gadjah mada university press; 1991. h. 163–75. 9. sri soebekti w. hubungan penggunaan air minum yang mengandung timah dan bersifat asam dengan erosi gigi. surabaya: pascasarjana universitas airlangga; 1993. h. 24–43. 10. lussi a, jaeggi t, ucharer uj. predition of the erosive potential of some beverages. caries res 1995; 29: 349–54. 11. zainuddin m. kinetika reaksi pelepasan kalsium dari enamel dalam medium yang bersifat asam. majalah kedokteran gigi surabaya 1999; 32(3):126–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 100 potency of probiotic therapy for dental caries prevention indah listiana kriswandini department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract probiotic therapy is one of the therapies to prevent dental caries prospectively. such therapy has been used in the medical area but not in dentistry. probiotic therapy is important to be done since this therapy study ecosystem in oral cavity which has many commensal bacteria more detail. the probiotic material used to prevent dental caries is the microorganism which counter microorganism causing dental caries and its virulent product (acid lactic). veillonella sp. use lactic acid as the end product of s. mutans which cause the dental caries. the principle of probiotic therapy is the comensalism symbiosis found in oral cavity ecosystem. veillonella sp could be added to anticipate the lactic acid which cause enamel demineralization. hopefully dentist will apply probiotic therapy, so there will be more study of veillonella sp. as probiotic material for dental caries prevention. further research on veillonella sp in probiotic therapy and immunology need to be done to achieve the balance of ecosystem. key words: probiotic therapy, dental caries, veillonella sp, preventive dentistry correspondence: indah listiana kriswandini, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya. introduction probiotic therapy has been used and improved medical treatment. its function is to neutralize pathogenic flora which attack intestinal organ. probiotic therapy is done by inserting normal flora which is needed to neutralize pathogenic bacteria. those normal flora could achieve organ target in alive condition. through this therapy it is hoped that the number of normal flora in the organ’s ecosystem could be balanced and the product of pathogen bacteria could be neutralized. probiotic flora for dental caries theraphy can be served as beverage, tooth paste or oral rinse. the probiotic therapy could potentially be used in oral cavity since the oral ecosystem has normal flora and pathogen flora. the normal floras live as commensal bacteria by using product of other bacteria in the same ecosystem. the example of normal flora in oral cavity is veillonella sp. which use lactic acid of end product of s. mutans as a metabolic energy.1 schonfeld2 also mentioned that lactic acid which produced by some microorganisms, such as streptococcus and actinomyces, could be used as the energy source of microorganism of veillonella and neisseria. the lactic acid could inhibit the growth of certain organism, like yeast. the objective of this paper is to review the potential use of probiotic acid from commensal bacteria and end product of s. mutans for dental caries prevention. oral probiotic the dental caries disease is characterized by local damage of tooth enamel and dentin, due to bacterial fermentation of carbohydrate diet.1 the main bacteria that damage the enamel are streptococcus mutans, which is virulent and has important role on caries process by lactic acid formation. dental caries problem occurs either in developed country or developing country. actually s.mutans is normal oral flora, in certain condition such as the excessive sucrose diet, bad oral hygiene and abnormalities of tooth structure; s.mutans would colonize very fast and produce lactic acid as end product. this acid product could decalcify enamel structure and cause irreversible caries.3 streptococcus mutans get the energy from glycolytic pathway which produces lactic acid. many kinds of organic acids caused by dental plaque bacteria, but lactic acid are the most dominant product which causes enamel decalcification. carbohydrate which metabolized by dental plaque bacteria produce lactic acid and other acid such as piruvic acid and formic acid, can cause oral ph decline until 5.5.1 the characteristics of veillonella are coccus shape, gram negative and anaerob condition is needed for its growth. three species of veillonella in oral cavity are veillonella parvula, veillonella atypica and veillonella dispar. the population of those species in oral cavity is approximately 3%.4 veillonella sp has the energy from the result of organic acid fermentation which are piruvic acid, lactic acid or malatic acid, fumarat and oxalo acetate. the end results of metabolism process are acetic acid, propionic acid, co2 and hydrogen which have weaker acid and less cariogenic than lactic acid. the results of lactic acid metabolism by veillonella are vitamin k and h2. vitamin k is needed for cell metabolism of the “black pigmented” and h2 is used for 101kriswandini: potency of probiotic therapy to prevent dental caries cell metabolism of wolinella.5 the bacteria growth needs additional energy such as lactic acid or piruvic acid on the base medium of py extract which contained vancomycin antibiotic. formerly veillonella parvula known as staphylococcus parvulus which means “small”. then it was found another strain, veillonella alcalescens. these two species have high similarity of dna.6,14 the form of veillonella is usually in a pair, single or group chain. a pair chain which has flat surface found in buccal mucosa, tongue surface, saliva and dental plaque.6,7 veillonella atypica almost like parvula, and it could be differentiated by using the protein profile and dna test which taken from buccal mucosa. on the other hand, veillonella dispar in biochemistry test could break hydrogen peroxide and its growth needs putrechine.8,13 normal floras in oral cavity have a lot of variety. oral flora could help to balance the oral ecosystem. in oral ecosystem, there are bacteria which have commensally life by using other bacteria product.1 the bacteria which produced lactic acid, such as streptococcus mutans and lactobacillus, have the energy from glycolytic pathway. there are a lot of organic acid which are made by the dental plaque bacteria, but lactic acid is the most dominant product which cause enamel decalcification. the carbohydrate is metabolized by plaque bacteria became lactic acid and other acid (piruvic acid and formic acid). lactic acid will be used by veillonella sp. for reducing acetic acid and propionic acid. the acetic acid reduction is done through metil-malonil co-a carboxil transferase. with complex-biotin carbon dioxide, lactic acid is changed into oxalo acetic and reducted to malat, fumarat and succinate. succinate is through to electron transport fosforilation and changed from co-a transferase (succinic co-a, propionic co-a transferase) into co-a derivat, then succinate will be activated. the intermediate product is divided by carbondioxide, so propionil co-a formed and propionic released from propionil-coa, while co-a transferase moved co-a to succinic. in the propionic formation, the two groups (co2 and co-a) are coming from the advanced product. it is moved into introduction phase without releasing that group. this process included three co-factors which are biotin, co-a and b12 coenzym.9 probiotic is a group of certain microorganism when it is inserted in digestive tract; it gives good effect to prevent and gives specific therapy for pathological condition. this group of microorganism has biological effect such as colonization resistance. it gives enzyme which needed by digestive tract.10 the use of probiotic can modulate immune system contain commensally microorganism to inhibit the progress of pathogenic microorganism in its ecosystem. the inhibition process could be done by bacterial antagonism, bacterial interference barrier effect, colonization resistance and competitive exclusion.11,12 discussion from the literature review revealing an idea to make an alternative plan for caries prevention by using probiotic therapy, since the oral cavity had specific ecosystem. the oral ecosystem has normal flora and pathogen flora which commensally live. it could use to balance the oral ecosystem. as we know s. mutans are the normal flora in oral cavity, and if its number is more than normal they would disturb the oral ecosystem and decalcified tooth enamel.1 veillonella sp is also oral normal flora. they live by using lactic acid. after metabolism process, they change from lactic acid to weaker acid: acetic acid, formic acid and propionic acid which is not cariogenic.5 through commensally symbiosis veillonella could reduce the potency of lactic acid causing enamel demineralization.1 the number of veillonella sp in oral cavity is only 3%. if the product of lactic acid produced by s. mutans is too much, the veillonella sp could not use the excessive acid.4 base on this concept the writer suggest that veillonella sp. can be use as probiotic material for prevent dental caries. the probiotic therapy use veillonella sp because they could inhibit pathogenic agent through competition with nutrient.10,11 this probiotic theraphy has harmless effect because the bacteria use for therapy is normal flora of oral ecosystem. veillonella sp. gives more advantage as probiotic material than lactobacillus sp. because it is normal oral flora and do not produce lactic acid. veillonella sp act as lactic acid user which assessed as enamel demineralization. nevertheless, much research on veillonella sp is needed for proper probiotic material to prevent dental caries; e.g. antibiotic susceptibility test. the antibiotic test is intended to determine veillonella sp survival on the patient which on antibiotic therapy. lysozyme tolerance test is purposed to examine the survival of veilonella sp. with lysozyme existence in oral cavity.15 hopefully there will be more study on veillonella sp which used as probiotic material for dental caries prevention. further research on number of veillonella sp needed for probiotic therapy and immunological study should be done to achieve balance oral ecosystem. references 1. marsh p, martin mv. acquisition, adherence, distribution and metabolism of the oral microflora. in oral microbiology. great britain: mpg books ltd; 2000. p. 34–57. 2. schonfeld se. oral microbiology ecology. contemporary oral microbiology and immunology. chapter 16. in: slots j, taubman ma, editors. st. louis, missouri: mosby year book; 1992. p. 267–74. 3. lehner t. immunology of oral diseases. 3rd ed. london: blackwell scientific publication; 1992. p. 70–1. 4. nisengard rj, newman mg. oral microbiology and immunology. 2nd ed. philadelphia : wb saunders co; 1994. p. 145–46. 102 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 100-102 5. harold. oral microbial ecology and the role of salivary immunoglobulin a. am soc for microbiology 1998; 62(1): 71–99. 6. taubman ma. immunological aspects of dental caries. chapter 29. in: slots j, taubman ma, editors. contemporary oral microbiology and immunology. st. louis, missouri : mosby year book; 1992. p. 533–41. 7. samaranayake l. bacterial phisiology and genetics in essential microbiology for dentistry. 3rd ed. toronto: harcourt publisher limited. churchill livingstone elsevier; 2006. 8. tanner a, lai ch, maiden m. characteristics of oral gram-negative species. chapter 19. in: slots j, taubman ma, editors. contemporary oral microbiology and immunology. st. louis, missouri : mosby year book; 1992. p. 299–341. 9. schlegel hg. mikrobiologi umum. 6th ed. yogyakarta: gadjah madayogyakarta: gadjah mada university press; 1994. p. 324–6. 10. chow j. probiotics and prebiotics: a brief overview. j ren nutr 2002 april; 12(2): 76–86. 11. kailasapathy k, chin j. survival and therapeutic potential of probiotic organisms with reference to lactobacillus acidophilus and bifidobacterium spp. immunol cell biol 2000 feb; 78(1): 80-8. 12. young rj, huffman s. probiotic use in children. j pediatr health care 2003 nov-dec; 17(6): 277–83. 13. maiden mfj, lai ch, tanner a. characteristics of oral gram-positive bacteria. chapter 20. in: slots j, taubman ma, editors. contemporary oral microbiology and immunology. st. louis, missouri: mosby year book; 1992. p. 342–72. 14. rosen s. dental caries. chapter 33. essential dental microbiology. in: willet np, white rr, rosen s, editors. new jersey: prentice hall, englewood cliffs, printed in the republic of singapore; 1991. p. 341–56. 15. koll p, mandar r, marcotte h, lelbur e, mikelsaar m, hammarstrom l. characterization of oral lactobacilli as potential probiotic for oral health. oral microbiology and immunology 2008; 23: 139–47. 86 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 86–90 research report scaffold combination of chitosan and collagen synthesized from chicken feet induces osteoblast and osteoprotegerin expression in bone healing process of mice saka winias,1 diah savitri ernawati,1 maretaningtias dwi ariani,2 and retno pudji rahayu3 1department of oral medicine 2department of prosthodontics 3 department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: over 500.000 of the 2,3 million surgical treatments requiring bone grafting procedures that are performed annually are likely to be necessitated by or will result in bone defects that will not regenerate. treatment to regenerate new tissues is needed, especially for hard tissue repair, which not only relies on a natural osseointegration process, but also requires a physical support to guide the differentiation and proliferation of cells into the targeted functional tissue. chitosan and collagen extracted from chicken feet combinations are expected to enhance the bioactive surface and provide mechanical strength as a bone graft scaffold. purpose: the aim of this study was to investigate the role of chitosan and collagen scaffold synthesized from chicken feet applications to increase the expression of osteoprotegerin (opg) and osteoblast cells on the fourteenth day of bone healing. methods: eighteen three-month old, adult, male, rattus novergicu strain rodents with a body weight ranging from 200-350 g were kept under controlled environmental conditions. the mice were randomly divided into three groups consisting of three subjects, each treated with collagen, chitosan, chitosancollagen combination (50:50) scaffolds. on the 14th post-treatment day, three members of each group were sacrificed. examination of osteoprotegerin (opg) expression was conducted by means of immunohistochemistry staining with anti-opg polyclonal antibodies. meanwhile, osteoblast cell examination was performed by means of hematoxilin-eosin (he) staining. results: the mice treated with collagen and a chitosan-collagen combination scaffold presented an increase in the expression of osteoprotegerin (opg) and the number of osteoblast cells respectively. conclusion: a combination of chitosan-collagen (50:50) scaffold extracted from chicken feet increased the expression of opg and the number of osteoblasts in the bone healing process. the combination scaffolds demonstrated the highest opg expression and number of osteoblasts compared to the other groups. keywords: collagen; chitosan; scaffold; chicken feet; bone healing correspondence: saka winias, department of oral medicine, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: saka.winias@gmail.com introduction debridement is a surgical procedure resulting in massive tissue loss. more than 2,300,000 operations have been recorded and over 500,000 bone replacements involving the use of grafts are performed annually as forms of health care.1–3 thus, a therapy to regenerate new tissues is required. treatments for tissue and bone defects incorporating tissue engineering methods, such as the use of bone graft and stem cells, have been developed as an alternative to conventional defect treatments.4 in recent decades, treatments involving the use of grafts have represented a novel approach to tissue and bone repair. tissue engineering methods primarily intended for hard tissue repair not only rely on natural osteointegrative processes, but also on a material promoting osteointegration which is the bone graft.5 in bone tissue engineering, a bone graft is formed into a scaffold for attachment, proliferation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p86–90 mailto:saka.winias@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p86-90 8787winias, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 86–90 and differentiation of bone tissue cells,6,7 to replace, repair and regenerate damaged tissue.8 currently, there are three kinds of natural bone graft widely used in the medical field, namely; autograft, a bone substitute derived from the patient him/herself, allograft, bone substitute provided by human donors and xenografts and bone substitutes derived from other species, such as cows. autograft has several disadvantages: the need for surgery to remove bones from the donor potentially resulting in clinical problems, the limited availability of bones and the risk of death. certain allograft materials and xenograft have the drawback of possibly inducing autoimmune reactions, while the nature of the osteoinduction of materials is less than optimal.9,10 chitosan constitutes a natural polymer alloplast and bone replacement material whose use in biomedical field applications has attracted considerable attention due to its biodegradability, biocompatibility, antibacterial and regenerative properties, all of which can accelerate tissue and bone healing.11 poly[-(1,4)-2-amino-2-deoxyd-glucopiranose] or chitosan is a natural biopolyaminoaccharide obtained from the stable deacetylation of chitin. however, the use of chitosan alone in tissue regeneration is less than optimal because it is incapable of entirely replacing the bone tissue.12 in addition to chitosan, another biomaterial renowned as a tissue substitute is collagen which constitutes a group of proteins with special characteristics, found in all multicellular animals, and secreted by connective tissue and various other cells. the synthesis of collagen was originally thought to be confined to fibroblasts, condroblasts, osteoblasts and odontoblasts. however, it later turned out that this material can be synthesized by various cells. most collagen is synthesized in fibroblasts, whereas bone collagen is produced by osteoblasts and cartilage collagen by condroblasts respectively. in experimental studies, collagen has been shown to reconstruct damaged tissue and, being one of the main components of bone, offers hope for positive tissue reaction.11,13 in this study, the synthesis of collagen scaffold from chicken feet was combined with chitosan in an attempt to analyze and identify the potential role of collagen combination scaffolding of chicken feet and chitosan in accelerating the bone healing process in mice. materials and methods this research was accepted by the ethics committee of the faculty of dental medicine of universitas airlangga, no. 45/kkepk.fkg/iv/2015. it represented an experimental in vivo laboratory research with post test-only control group design. three treatment groups were established, each treated with collagen, chitosan and chitosan-collagen (50:50) scaffolds. the research subjects were randomized and divided into three groups, namely; the collagen, chitosan and chitosan-collagen scaffold treatment groups respectively. they were subsequently adapted to the environment over seven days, with all receiving basal rations. basal ration composition, consisting of carbohydrates, proteins, fats, minerals, vitamins and water, was prepared according to american institute of nutrition (ain) standards.14 the collagen was synthesized from a broiler of chicken feet skins obtained from pt. wonokoyo. the chicken feet were cut into small pieces, mixed with trypsin enzyme and placed in an incubator at a temperature of 370 c for 24 hours. this mixture was added to glacial acetic acid and then agitated with a mixer until the formation of fiber was observed. the synthesized results were centrifuged at 9000 rpm with the supernatant being extracted to obtain collagen. the supernatant was subsequently added together with 5% nacl to the formation of fibers/collagen bands. the extraction by means of acetic acid and sodium hydroxide was analyzed using a cellophane membrane (sigma, 58188). the results of dialysis can be formed using a mold/ scaffold mold and then freeze dried. the preparation of a combination of chitosan collagen scaffold was based on a weight ratio of 50:50. the chitosan (sigma, smb00279) gel was obtained from sigma brand chitosan powder at 85% deacetylation that had been dissolved with an acid base and then added to collagen gel and acetic acid. the chitosan and collagen gel mixture was agitated and centrifuged at 9000 rpm. the resulting supernatant was subsequently inserted into the mould scaffold, enabling it to be frozen for 24 hours. prior to surgery, the three month-old, male rats were anesthetized. bone defects in two areas of smelting (one on the right and the other on the left) of 5 mm were produced using round burs angle (dentsply, 63503001) on their femur bones. after these defects had been made, they were administered the collagen scaffold, chitosan scaffold and 50:50 chitosan-collagen scaffold. thereafter, a suture was performed on the wound with 3/0 non-absorbable black silk (sinorgmed, china). on the 14th post-operative day, members of each group was sacrificed to enable observation of the degree of osteoblast cell and opg expression as an indicator of bone regeneration. the femoral bone tissue taken from the animal was tested with 10% formalin buffer solution before being decalcated by means of 2% nitric acid. the tissue processing continued involving dehydration, clearing, impregnation, embedding, tissue cutting and coloring. morphology and the number of osteoblast cells were investigated using a light microsope, while staining by means of hematoxylineosin was conducted. in order to observe the expression of osteoprotegerin, immunohistochemical imaging using anti-opg (bioss, bs-0431r) polyclonal antibodies was conducted. the data of this study were subsequently analyzed through the application of one-way anova and tukey hsd tests. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p86-90 88 winias, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 86–90 results on cellular examination involving hematoxilyn-eosin staining, the visible osteoblast cells were found to be single-core hexagonal-shaped cells often present at the edges of the bone matrix. inspection was carried out with a light microscope at 400x magnification. the results of the examination conducted on the fourteenth day can be seen in figure 1. the results show that a combination treatment involving collagen or chitosan scaffolds results in a more pronounced increase in osteoblasts than treatment without combination. from the results of the kolmogorov-smirnov test statistical analysis of data, the p value (2-tailed) amounted to 0.296> 0.05. thus, it could be argued that the data was normally distributed. a homogeneity test was subsequently administered by means of a lavene test which produced a p value of 0.15> 0.05, indicating that the data was homogeneous or demonstrated the same variance. therefore, the data was valid for the parametric test using one-way anova. from the results, it could be seen that the p value was 0.000 <0.05 meaning that there was a significant difference between treatments. consequently, a post-hoc tukey hsd test was administered which showed that chitosan scaffold treatment was not significantly different (p value 0.38>0.05) to collagen scaffold treatment, but chitosan scaffold was significantly different compared to 50:50 chitosan-collagen scaffold combination. immunohistochemistry examination incorporating the use of a polyclonal antibody against opg was conducted. positive results were characterized by the presence of brown spots on the cytoplasm of osteoblasts. checked with a light microscope at 400x magnification, the results of the 14th day observation and examination can be seen in figure 2. the arrows indicate a positive result as confirmed by the brownish color on the osteoblast cell cytoplasm. the results show that combination treatment produces increased osteoprotegerin expression in osteoblasts compared with non-combination treatment, i.e collagen or chitosan scaffolds. from the statistical analysis of the data, the kolmogorovsmirnov test recorded a p (2-tailed) value of 0.350> 0.05. hence, it can be said that the data was normally distributed. a homogenity test using lavene’s test produced the p value of 0.20> 0.05 which means that the data was homogeneous or presented the same variance. therefore, the data was valid for the parametric test using one-way anova. the results of several such tests using one-way anova confirmed the p value as 0.000 <0.05 which means that there was a considerable difference between treatments. the subsequent post-hoc tukey hsd confirmed that, while chitosan scaffold treatment was not significantly different (p value 0.06>0.05) from a collagen scaffold treatment, 50:50 chitosan-collagen combination scaffolds contrasted sharply with chitosan scaffold. discussion the regeneration of bone tissue requires an artificial structure, or so-called scaffold, as a location for tissue growth that maintains tissue mechanical stability, thereby 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. cba figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. 10 figure 1. image of osteoblast cell featuring the hematoxylin-eosin staining at 400x magnification under treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50 on the 14th day of observation. a cb figure 2. brownish images of the osteoblast cell cytoplasms of imunohistochemical imaging that show opg (400 × magnification) in treatment a. chitosan, b. collagen, c. chitosan-collagen 50:50, on the 14th day. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p86-90 8989winias, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 86–90 allowing bone defects to be restored to their original form.1 collagen is considered to be the most promising material for tissue engineering applications because of its excellent biocompatibility, degradability, low antigenicity and abundance in mammals. like collagen, chitosan has been utilised in a variety of biomedical fields. including skin tissue engineering. in addition to being antibacterial, chitosan has specific properties including; bioactivity, biocompatibility, and biodegradability. the quality of chitosan can be seen from its intrinsic properties, its purity, molecular mass, and deacetylation degree of 75100%. the degree of deacetylation of chitosan affects the physico-chemical properties of polysaccharides, such as the rheological nature of chitosan and the flexibility of the molecular chains. the ideal scaffold would consist of a biodegradable material possessing a pore structure that can provide a microenvironment for osteogenesis and osteoblast cell proliferation. scaffolds made from chitosan have been widely used as a biomedical material because of their non-toxicity and osteoconductivity. scaffolds made from collagen represent the most suitable material to repair damaged tissue because it is the main protein structure in bone. in this study, the average number of osteoblasts and opg in collagen and chitosan scaffolds was lower than in the 50:50 chitosan-collagen combination group. chitosancollagen combinations in the form of scaffolds are normally used for attaching and cell migration, delivering and maintaining the cells from biochemical factors, enabling the diffusion of vital cell nutrients and both producing and exerting mechanical and certain biologic influence in order to modify the behavior of the cell phase.15,16 in other studies revealed that osteoblasts increased significantly at the outset of the 20 days of addition of the scaffold in the experimental specimens,17 however, in our study blood vessels were formed on day 14,18,19 and osteogenesis started on the same day after the graft was implanted in the bone, so the observation of this study was on day 14.15 50:50 chitosan-collagen scaffold treatment is significantly different to collagen and chitosan scaffolds because the latter are porous ± 650μm-850μm.20 the pore size is too large for the scaffold whose mechanical properties it can influence. these properties are essential for tissue repair as they affect the function of certain tissue cells, as well as attachment, migration and cell proliferation in tissues.1,21 therefore, multiple or combination agents are rapidly provided to the wound through the normal bone healing process. the collagen treatment provides protein in the form of a matrix in which cells can proliferate and infiltrate. in addition to providing the cells with a matrix largely lost during wound creation, the collagen scaffold was observed to activate platelets within the chitosan combination. the greater the diameter of the pores, the less the extent to which mechanical stability of the tissue is maintained resulting in the healing process being the same as in the group without the addition of the scaffold.22,23 collagen and chitosan are good natural ingredients used as tissue engineering materials but when used separately they inhibit the growth of new blood vessels transporting new bone nutrients and decrease the mechanical properties of the scaffold. the chitosan-collagen combination scaffold is more stable because the chitosan content of the combination scaffold can serve as a bridge that increases the efficiency of the bonds between the amino acid of the chitosan-collagen chains in the tissue.24–26 when used separately, a scaffold of chitosan and collagen is less conducive to bone healing because one ingredient is too rapidly degraded by the body. therefore, the scaffold that serves as a cell infiltration site and guide for the differentiation and proliferation of cells into functional tissue does not function optimally.24 strong bonds between amino acid chains within the combination of chitosan-collagen scaffold cannot easily be degraded in the tissues, thus increasing the latters’ mechanical strength and structure.27 it can be concluded that therapy incorporating the application of a chitosan-collagen scaffold combination derived from chicken feet can increase the number of osteoblast cells and opg expression in the healing process of bone defects in mice. references 1. kretlow jd, mikos ag. from material to tissue: biomaterial development, scaffold fabrication, and tissue engineering. aiche j. 2008; 54(12): 3048–67. 2. mehta m, schmidt-bleek k, duda gn, mooney dj. biomaterial delivery of morphogens to mimic the natural healing cascade in bone. adv drug deliv rev. 2012; 64(12): 1257–76. 3. maraldi t, riccio m, pisciotta a, zavatti m, carnevale g, beretti f, la sala gb, motta a, de pol a. human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization. stem cell res ther. 2013; 4(3): 1–12. 4. huang y, niu x, wang l, fan j, li x, zhou g, feng q, fan y. effects of hydroxyapatite/collagen composite on osteogenic differentiation of rat bone marrow derived mesenchymal stem cells. j compos mater. 2014; 48(16): 1971–80. 5. depalle b, qin z, shefelbine sj, buehler mj. influence of crosslink structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils. j mech behav biomed mater. 2015; 52: 1–13. 6. lanza rp, langer r, vacanti j. principles of tissue engineering. 2nd ed. academic press; 2000. p. 995. figure 3. average osteoblast cell count and osteoprotegerin expression on day 14 60 50 40 30 20 10 0 chitosan collagen 50:50 osteoblast opg dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p86-90 90 winias, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 86–90 7. ma l, shi y, chen y, zhao h, gao c, han c. in vitro and in vivo biological performance of collagen-chitosan/silicone membrane bilayer dermal equivalent. j mater sci mater med. 2007; 18(11): 2185–91. 8. lópez-álvarez m, rodríguez-valencia c, serra j, gonzález p. bio-inspired ceramics: promising scaffolds for bone tissue engineering. procedia eng. 2013; 59: 51–8. 9. matsuno t, omata k, hashimoto y, tabata y, satoh t. alveolar bone tissue engineering using composite scaffolds for drug delivery. jpn dent sci rev. 2010; 46(2): 188–92. 10. trotta dr, gorny c, zielak jc, gonzaga cc, giovanini af, deliberador tm. bone repair of critical size defects treated with mussel powder associated or not with bovine bone graft: histologic and histomorphometric study in rat calvaria. j cranio-maxillofacial surg. 2014; 42(6): 738–43. 11. shin ja, choi jy, kim st, kim cs, lee yk, cho ks, chai jk, kim ck, choi sh. the effects of hydroxyapatite-chitosan membrane on bone regeneration in rat calvarial defects. j korean acad periodontol. 2009; 39: 213–22. 12. venkatesan j, kim sk. chitosan composites for bone tissue engineering--an overview. mar drugs. 2010; 8(8): 2252–66. 13. zhao h, ma l, gao c, shen j. fabrication and properties of mineralized collagen-chitosan/hydroxyapatite scaffolds. polym adv technol. 2008; 19(11): 1590–6. 14. reeves, nielsen fh, fahey gc. ain-93 purified diets for laboratory rodents: final report of the american institute of nutrition ad hoc writing committee on the reformulation of the ain-76a rodent diet. j nutr. 1993; 123(11): 1939–51. 15. keogh mb, o’brien fj, daly js. a novel collagen scaffold supports human osteogenesis--applications for bone tissue engineering. cell tissue res. 2010; 340(1): 169–77. 16. salgado aj, coutinho op, reis rl. bone tissue engineering: state of the art and future trends. macromol biosci. 2004; 4(8): 743–65. 17. muzio g, martinasso g, baino f, frairia r, vitale-brovarone c, canuto ra. key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering. j biomater appl. 2014; 29(5): 728–36. 18. kon t, cho tj, aizawa t, yamazaki m, nooh n, graves d, gerstenfeld lc, einhorn t a. expression of osteoprotegerin, receptor activator of nf-kappab ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. j bone miner res. 2001; 16(6): 1004–14. 19. cunniffe gm, o’brien fj. collagen scaffolds for orthopedic regenerative medicine. jom. 2011; 63(4): 66–73. 20. silalahi im, yuliati a, soebagio. chicken shank collagen synthesis as a candidate for tissue engineering biomaterials. mater dent j. 2015; 6(2): 53–7. 21. vial x, andreopoulos f. novel biomaterials for cartilage tissue engineering. curr rheumatol rev. 2009; 5(1): 51–7. 22. cui k, zhu y, wang xh, feng ql, cui fz. a porous scaffold from bone-like powder loaded in a collagen–chitosan matrix. j bioact compat polym. 2004; 19(1): 17–31. 23. kim se, cho yw, kang ej, kwon ic, lee eb, kim jh, chung h, jeong sy. three-dimensional porous collagen/chitosan complex sponge for tissue engineering. fibers polym. 2001; 2(2): 64–70. 24. liu y, ma l, gao c. facile fabrication of the glutaraldehyde crosslinked collagen/chitosan porous scaffold for skin tissue engineering. mater sci eng c. 2012; 32(8): 2361–6. 25. ma l, gao c, mao z, zhou j, shen j, hu x, han c. collagen/ chitosan porous scaffolds with improved biostability for skin tissue engineering. biomaterials. 2003; 24(26): 4833–41. 26. kung s, devlin h, fu e, ho ky, liang sy, hsieh yd. the osteoinductive effect of chitosan-collagen composites around pure titanium implant surfaces in rats. j periodontal res. 2011; 46(1): 126–33. 27. haifei s, xingang w, shoucheng w, zhengwei m, chuangang y, chunmao h. the effect of collagen-chitosan porous scaffold thickness on dermal regeneration in a one-stage grafting procedure. j mech behav biomed mater. 2014; 29: 114–25. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p86–90 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p86-90 8787 research report dental journal (majalah kedokteran gigi) 2016 june; 49(2): 87–92 transforming growth factor beta 1 expression and inflammatory cells in tooth extraction socket after x-ray irradiation ramadhan hardani putra,1 eha renwi astuti,1 and rini devijanti2 1department of dentomaxillofacial radiology 2department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: radiographic examination is often used in dentistry to evaluate tooth extraction complications. x-ray used in radiographic examination, however, has negative effects, including damage to dna and inflammatory response during wound healing process. purpose: this study aimed to analyze the effects of x-ray irradiation on transforming growth factor beta 1 (tgf-ß1) expression and number of inflammatory cells in tooth extraction sockets. method: thirty rats were divided into three groups, which consist of control group (with a radiation of 0 msv), treatment group 1 (with a radiation of 0.08 msv), and treatment group 2 (with a radiation of 0.16 msv). these rats in each group were sacrificed on days 3 and 5 after treatment. inflammatory cells which were observed in this research were pmn, macrophages, and lymphocytes. histopathological and immunohistochemical examinations were used to calculate the number of inflammatory cells and tgf-ß1 expression. obtained data were analyzed using spss 16.0 software with one way anova and tukey’s hsd tests. result: there was no significant decrease in the number of pmn. on the other hand, there were significant decreases in the number of macrophages and lymphocytes in the sacrificed group on day-5 with the radiation of 0.16 msv. similarly, the most significant decreased expression of tgf-ß1 was found in the group sacrificed on day 5 with the radiation of 0.16 msv. conclusion: x-ray irradiation with 0.08 msv and 0.16 msv doses can decrease tgf-ß1 expression and number of inflammatory cells in tooth extraction sockets on day 3 and 5 post extraction. keywords: x-ray irradiation; inflammatory cells; tgf-ß1; tooth extraction; socket healing correspondence: ramadhan hardani putra, department of dentomaxillofacial radiology, faculty of dental medicine, universitas airlangga. jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ramadhan.hardani@gmail.com introduction radiographic examination is often conducted in the field of dentistry. radiographic examination may assist dentists in establishing a diagnosis to determine a treatment plan and evaluation of treatment results.1 tooth extraction often requires radiographic examination. it means that if a fracture occurs during tooth extraction, it will be evaluated with radiographic examination to see the state of the remaining teeth and to determine further treatment plan.2 nevertheless, the use of dental x-ray to produce a radiograph has a negative impact on tooth extraction sockets since the body cannot be fully protected from the effects of x-ray irradiation. ionizing radiation in cells actually depends on many factors. in addition to physical factors, some cells are known to have certain characteristics which are sensitive to radiation, referred as radiosensitive. therefore, the effects of irradiation on an organism as a whole will depend on the size and type of cells affected. the cells, which are radiosensitive, are white blood cells or leukocytes.1,3 on tooth extraction sockets, various kinds of white blood cells will emerge as a response to the presence of injury, such as polymorphonuclear cells (pmn), lymphocytes, and macrophages that act as inflammatory cells. growth factors also play a role in regulation of cell proliferation, differentiation, and migration, in synthesizing dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 88 putra, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 87–92 extracellular matrix proteins, as well as in angiogenesis. a growth factor which plays a role and often expressed during wound healing process is transforming growth factor-β1 (tgf-β1). the role of tgf-β1 emerges on the second phase of the wound healing process, from inflammatory phase to the final phase, i.e tissue remodeling.4,5 low dose irradiation could cause biological effects on the body since ionization process of x-ray could cause damage to dna.6 variation of dna damage caused by ionization could be changes to the base, losing a nucleotide bases, breakage of hydrogen bonds between the chains, single strand fractures, double strand fractures, and cross linking in helix.7 dental x-ray irradiation at a dose of 0.08 msv, 0.16 msv, and 0, 24 msv in mice even can lead to increased apoptosis and necrosis of the oral mucosal cells.8 x-ray irradiation could also inhibit initial inflammatory response and decrease infiltration of macrophages and neutrophils, as a result, the wound healing process becomes longer.9 the effects of x-ray irradiation on inflammatory cells and tgf-ß1 expression in tooth extraction sockets are still unsolved. thus, this research was aimed to analyze the effects of x-ray irradiation on a decrease in both inflammatory cells during the inflammatory phase of wound healing process and tgf-β1 expression during the wound healing process. as a result, the results of this research are expected to reveal the effects of x-ray irradiation with a low dose during the wound healing process of tooth extraction based on molecular biology aspect. materials and methods thirty rats (rattus norvegicus) aged 8-11 weeks and weighed 250-500 grams were randomly divided into three groups, which consist of control group, treatment group i, and treatment group ii. each group consisted of ten rats. all of these rats were adapted in the laboratory of biochemistry, faculty of medicine, universitas airlangga in surabaya. tooth extraction was conducted on these thirty rats. the anterior mandibular incisor of those rats was extracted after administration of anesthesia using ketamine intramuscularly. before the extraction, cervical preparation was carried out first using a bur with low speed. the extraction then was performed using luxation technique until fractures occurred in the crown of the teeth. after the irradiation process in each study group, the rest of the teeth were taken, the wound was stitched, and the rats were returned to the cage for adaptation. x-ray irradiation on injured rat (tooth extraction) was performed using conventional radiographic dental instrument, belmont searcher model dx-068 70 kvp 8 ma. before the x-ray irradiation, those rats were fixed with a wire mesh so that the rats would not move around when exposed to radiation. the control group was not given x-ray irradiation. treatment group i was given radiation at a dose of 0.08 msv or one x-ray irradiation exposure. meanwhile, treatment group ii was given radiation at a dose of 0.16 msv or twice the x-ray irradiation exposure. the rats in each group then would be sacrificed on days 3 and 5 after the extraction process. retrieval and processing of tissues were started by cutting the mandibular tissue of the rats under anesthesia with 10% ether on day 3 and day 5. fixation of mandibular tissue then was performed using 10% neutral buffered formalin (nbf) and decalcified using 10% edta. after the bone tissues become soft, dehydration, clearing, impregnation, and embedding processes were performed on the tissues. the paraffin blocks then were cut. next, the results were embedded in solid paraffin. the results which obtained in this phase were preparation slides. h e m a t o x y l i n e o s i n s t a i n s w a s c o n d u c t e d t o observe the number of inflammatory cells. meanwhile, immunohistochemical method with monoclonal anti-tgfβ1 (t0438; sigma-aldrich) was used to observe tgf-β1 expression. inflammatory cells and tgf-ß1 expressions on the mandibular preparations then were observed using he staining under a light microscopy, a nikon h600l digital camera equipped with 300 megapixel ds fi2. after that, observations were made on the healing area, one-third of the apical incisor sockets. inflammatory cells observed in this research were pmn cells, macrophages, and lymphocytes. the mean number of the inflammatory cells was calculated by using a light microscope with a magnification of 1000x on five fields of view. pmn cells have segmented cell nucleus with 2-4 purple cores. meanwhile, the macrophage cells have oval nucleus located eccentrically, and the lymphocytes have a round and dark nucleus which almost fills the entire cell with little cytoplasm. tgf-β1 expressions were calculated by counting the number of cells expressing tgf-β1. the mean positive expressions of tgf-β1 were observed by counting the number of macrophages expressing tgf-β1 which characterized by a brownish color in the cytoplasm counted under a light microscope with a magnification of 400 times on five field of view. data obtained in this research were analyzed using spss 16.0 software and statistical tests, namely one way anova test followed by post hoc tukey’s hsd test. results based on the calculation results, the mean expressions of tgf-β1, pmn, macrophages, and lymphocytes in each sample group were presented in table 1 and figure 1. the results of histopathologic examination with ihc staining on tgf-β1 expression were presented in figure 2. meanwhile, the results of histopathologic examination with he staining on pmn cells, macrophages, and lymphocytes were presented in figure 3. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 8989putra, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 87–92 table 1. the mean and standard deviation of tgf-ß1 expression and inflammatory cells on days 3 and 5 control group treatment group i treatment group ii tgf-ß1 day 3 7.4 ± 1.14 5.8 ± 1.64 3.0 ± 0.70 day 5 7.6 ± 1.14 6.0 ± 1.00 2.2 ± 0.83 pmn day 3 271.4 ± 75.25 269.6 ± 63.89 235.2 ± 67.69 day 5 156.8 ± 64.91 152.4 ± 41.22 124.2 ± 48.47 macrophages day 3 64.6 ± 25.98 51.6 ± 21.98 25.4 ± 9.91 day 5 69.0 ± 26.63 57.6 ± 31.43 21.8 ± 6.76 lymphocytes day 3 37.2 ± 11.73 20.8 ± 1.92 19 ± 6.32 day 5 51.8 ± 21.54 26.6 ± 7.40 18.0 ± 7.58 table 2. the results of post-hoc tukey’s hsd test on tgf-ß1 expression and inflammatory cells group p value tgf-ß1 control, day 3 treatment i, day 3 0.139 treatment ii, day 3 0.000 treatment i, day 3 treatment i, day 3 0.009 control, day 5 treatment i,day 5 0.064 treatment ii, day 5 0.000 treatment i, day 5 treatment ii, day 5 0.000 macrophages control, day 3 treatment i,day 3 0.588 treatment ii, day 3 0.026 treatment i, day 3 treatment ii, day 3 0.149 control, day 5 treatment i, day 5 0.741 treatment ii, day 5 0.023 treatment i, day 5 treatment ii, day 5 0.087 lymphocytes control,day 3 treatment i, day 3 0.015 treatment ii, day 3 0.008 treatment i,day 3 treatment ii, day 3 0.929 control,day 5 treatment i, day 5 0.064 treatment ii, day 5 0.000 treatment i, day 5 treatment ii, day 5 0.000 note: p value<0.05 indicating a significant difference figure 1 revisi figure 1. the mean expression of tgf-ß1 and the mean number of inflammatory cells. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 90 putra, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 87–92 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f 10 figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). c d e a b f figure 2. the positive expression of tgf-ß1 observed under light microscope at 400x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. the lowest number of cells expressing tgf-ß1 was found in the treatment group ii both on day 3 and day 5 compared to the control group and the treatment group i (arrows indicating cells expressing tgf-ß1). 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d 11 figure 3. the results of hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a d figure 3. the hpa on pmn (red arrows), macrophages (yellow arrows), and lymphocytes (black arrows) with hematoxylin-eosin staining technique observed under a microscope at 1000x magnification. (a) control group on day 3; (b) treatment group i on day 3; (c) treatment group ii on day 3; (d) control group on day 5; (e) treatment group i on day 5; and (f) treatment group ii on day 5. a b c d e f dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 9191putra, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 87–92 according to the results of kolmogorov-smirnov and levene tests, the expression of tgf-ß1 on days 3 and 5 had a normal and homogeneous distribution. next, according to the results of one way anova test results, there was a significant difference in tgf-β1 expression between the research groups on days 3 and 5 since a value of p was less than 0.05. similarly, the results of post hoc tukey’s hsd test showed that there were significant differences (p<0.05) in tgf-β1 expression between the control group and the treatment group ii as well as between the treatment group i and the treatment group ii both on day 3 and day 5 as seen in table 2. nevertheless, there was no significant difference (p>0.05) in tgf-β1 expression between the control group and the treatment group i. in addition, according to the results of kolmogorovsmirnov and levene tests, the number of pmn, macrophages, and lymphocytes on day 3 and 5 had a normal and homogeneous distribution. thus, one way anova test then was performed. the results of one way anova test showed that there was no significant difference in the number of pmn between those research groups on days 3 and 5 (p>0.05). however, there were significant differences in the number of macrophages and lymphocytes between the research groups (p<0.05). post-hoc tukey’s hsd test was conducted to find out which groups that differed in the number of macrophages and lymphocytes. the results of post-hoc tukey’s hsd test showed that there was a significant difference (p<0.005) in the number of macrophage cells between the control group (without x-ray radiation) and the treatment group ii (with x-ray radiation at a dose of 0.16 msv), either on day 3 or on day 5 as seen in table 2. similarly, there was also a significant difference (p<0.005) in the number of lymphocytes between the control group, the treatment group i (with x-ray radiation at a dose of 0.08 msv), and the treatment group ii, either on day 3 or on day 5. discussion x-ray irradiation is a type of ionizing radiation which could cause ionization process in the media path, including human body. the radiation dose for dental x-ray is categorized as low dose in the range of 0.01-10 msv.10 however, ionizing radiation has been known to cause a varied effects associated with the occurrence of changes or damage in cells as a result of the consequences. sometimes, cell damage caused by interaction with the radiation can be recovered through the process of cell repair which possessed by every individual living cell, but it depends on the cell type and the radiation exposure dose.6 dna damage caused by x-ray irradiation could be either direct or indirect. irradiation can damage dna directly or through the mechanism of free radical formation. the damage to dna, which cannot be repaired, would activate apoptosis, which in this case, is the pathological apoptosis. effect of x-ray irradiation on cells of the body could be affected by the amount of the received dose and the type of cell. apotosis due to irradiation could occur to leukocytes since leukocytes are one of the radiationsensitive cells.3 leukocytes or white blood cells have a very important role in the inflammatory phase during wound healing revocation, which act as both acute and chronic inflammation cells. the results of this research showed that there were significant differences in tgf-β1 expression between the control group and the treatment group i and the treatment group ii, either on day 3 and day 5. decreased expression of tgf-β1 might be caused by a decrease in the number of macrophages due to x-ray irradiation. tgf-β1 is secreted by macrophages, platelets, and keratinosit.11 in this research, platelets and keratinocytes were not observed, but a significant decrease in the number of macrophages occurred in the treatment group ii, both on day 3 and day 5. decreased expression of tgf-β1, may disrupt the healing process of tooth extraction. tgf-β1 has a broad role in wound healing which plays an important role in inflammatory phase and formation of tissue granulation in proliferation phase.12 in addition, tgf-β1 also plays a role in angiogenesis, extracellular matrix formation, and bone formation in maturation phase.13 in formation bone, tgf-β1 has a role as chemoattractor and stimulates the proliferation and differentiation of osteoblast precursors. tgf-β1 may also increase bone formation by recruiting progenitor of osteoblasts and stimulating proliferation of osteoblasts.14 in this research, the studied inflammatory cells were pmn, macrophages, and lymphocytes. the inflammatory cells play an important role in wound healing, which can kill bacteria and prevent infection in a wound.15 pmn are cells which were very dominant in acute inflammatory phase. in this research, there was no significant difference in the number of pmn between the control group and the treatment groups. but, the number of pmn were the most in the control group, while the least number was found in the treatment group ii, either on day 3 and day 5. this indicates that the greater the radiation is given to the injured tooth extraction, the higher the number of pmn will decrease although not significant. this could happen because the given radiation dose can be categorized as low so that a decrease in the number of pmn did not occur significantly. damage to dna in the cell nucleus as a result pmn x-ray irradiation could actually be repaired by the body so that the occuring decrease was not significant. in addition, the results of this research also showed that there was a significant difference in the number of macrophages between the control group and the treatment group ii, either on day 3 or day 5. it has similarities with a research conducted by liu x et al.9 showing that the effects of x-ray radiation on wound healing incision in the skin of mice could reduce macrophage infiltration. macrophages are derived from monocytes that circulate in the blood to the tissues. x-ray irradiation on wound dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 92 putra, et al./dent. j. (majalah kedokteran gigi) 2016 june; 49(2): 87–92 healing can form reactive oxygen species (ros) that could cause oxidative damage to dna monocytes. monocytes are blood cells that are particularly sensitive to x-ray irradiation in which the expression of proteins playing a role in dna repair would be disturbed, thus influencing dna repair. monocytes which cannot be repaired by proteins of dna repair will activate caspase 8, caspase 3, and caspase 7, which can cause apoptosis of monocyte cells.3 the number of monocytes will be indirectly decreased as a result of apoptosis. in wound healing, monocyte will differentiate into macrophages. the decrease in the number of monocytes, consequently, will decrease the number of macrophages. although there was no significant difference between the control group and the treatment group i, but the number of macrophages was still decreased due to x-ray irradiation. in lymphocytes, moreover, there was also a significant difference between the control group and the treatment group i and the treatment group ii, either on day 3 or day 5. the decrease in the number of lymphocytes could occur because of the rapid mechanism of apoptosis after experiencing double strand break in dna before the dna is repaired.16 a research conducted by faraj et al. showed that the percentage of apoptosis in lymphocytes increases as the given dose of x-ray irradiation increases as well. apoptosis in lymphocyte cells could be detected by the activated caspase 3 since caspase 3 is a protease which is often activated in apotosis mechanism.17 in the treatment group ii (with a radiation dose of 0.16 msv), the number of macrophages and lymphocytes decreased from day 3 to day 5. this was different from what occured in the control group and the treatment group i in which there was an increase in the number of macrophages and lymphocytes on day 5. the decrease in the number of macrophages and lymphocytes from day 3 to day 5 might indicate a delay in the acute inflammatory phase because in normal wound healing, an increase in the number of macrophages and lymphocytes should occur on day 5. long inflammatory phase would inhibit the healing process since components in the inflammatory reaction that destroy and eliminate the microorganisms or tissue injury may also damage normal tissue.15 in the treatment group ii (with a radiation dose of 0.16 msv), the number of tgf-β1 expression also decreased from day 3 to day 5. this was different from what happened in the control group and the treatment group i in which there was an increase in the number of tgf-β1 on day 5. besides, there was also a significant decrease in the number of macrophages and lymphocytes in the treatment group ii (with a x-ray irradiation dose of 0.16 msv). these results can be taken into consideration before taking periapical radiograph. although there is no clinical evidence, a dentist or radiographer should be more cautious in the making of periapical radiograph in patients with fractures which caused by tooth extraction in order to avoid both a failure in radiograph and unnecessary repetition of x-ray irradiation exposure since the repeated process of making periapical radiograph on the wound of the tooth extraction may have an impact on the molecular aspects of inflammatory cells, especially macrophages and lymphocytes. finally, it can be concluded that the x-ray irradiation at a dose of 0.08 msv and 0.16 msv can disrupt the wound healing process of tooth extraction caused by a decrease in tgf-β1 expression and number of inflammatory cells in the tooth extraction sockets on day 3 and day 5. nevertheless, further researches on the effects of x-ray irradiation on cells or growth factors affecting the wound healing process still need to be conducted. references 1. white sc, pharoah mj. oral radiology: principles and interpretation. 6th edition. missouri: mosby; 2008. p. 175. 2. miloro m. peterson’s principle of oral and maxillofacial surgery. 2nd ed. london: bc decker inc; 2003. p. 151. 3. bauer m, goldstein m, christmann m, becker h, heylmann d, kaina b. human monocytes are secverely impaired in base dan dna double strand break repair that renders them vulnerable to oxidative stress. proc natl acad sci u s a 2011; 108(52): 21105-10. 4. barrientos s, stojadinovic o, golinko ms, brem h, tomic-canic m. growth factors and cytokines in wound healing. wound repair regen 2008; 16(5): 585-601. 5. reed mj, koike t, puolakkainen p. wound repair in aging: a review. methods in molecular medicine 2003:78: 217–37. 6. alatas z. efek kesehatan pajanan radiasi dosis rendah. cermin dunia kedokteran 2007; 154: 27-39. 7. minicucci em, kowalski lp, maia ma, pereira a, ribeiro lr, de camargo jl. cytogenetic damage in circulating lymphocytes and buccal mucosa cells of head and neck cancer patiens undergoing radiotherapy. j radiat res 2005; 46: 135-42. 8. saputra d. apoptosis dan nekrosis sel mukosa rongga mulut akibat radiasi sinar-x dental radiografik. surabaya: universitas airlangga; 2012. p. 61-78. 9. liu x, liu jz, zhang e, li p, zhou p, cheng tm, zhou yg. impaired wound healing after local soft x-ray irradiation in rat skin: time course study of pathology, proliferation, cell cycle, and apoptosis. j trauma 2005; 59(3): 682-90. 10. whaites e. essentials of dental radiography and radiology. 4th ed. philladelphia: churchill livingstone; 2007. p. 187. 11. rolfe kj, richardson j, vigor c, irvine lm, grobbelaar ao, linge c. a role for tgf-beta1-induced cellular responses during wound healing of the non-scarring early human fetus. j invest dermatol 2007; 127(11): 2656–67. 12. mohammadreza p, ali f, mohsen km, aziz g. critical role of transforming growth factor in different phases of wound healing. j adv wound care (new rochelle) 2013; 2(5): 215–24. 13. r icha rd w dg, mat t hew k v, a l icia m v p. sig na l l i ng by transforming growth factor beta isoforms in wound healing and tissue regeneration. j dev biol 2016; 4(2): 21. 14. maeda sm, hayashi m, komiya s, imamura t, miyazono k. endogemous tgf-ß1 signalling suppreses maturation of steoblastic mesenchymal cells. embo j 2004; 23(3): 552-63. 15. larjava h. oral wound healing: an overview of biological science. endodontic topics 2012; 24(1): 1-3. 16. fujikawa k, hasegawa y, matsuzawa s, fukunaga a, itoh t, kondo s. dose and dose-rate effects of x-ray and fission neutrons on lymphocyte apoptosis in p53(+/+) and p53(-/-) mice. j radiat res 2000; 41(2): 113-27. 17. faraj ka, elias mm, baaout s. effect of x and gamma rays on human lymphocytes. romanian j biophys2010; 20(4): 355-67. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v49.i2.p87-92 http://dx.doi.org/10.20473/j.djmkg.v49.i2.p87-92 110 dental journal (majalah kedokteran gigi) 2019 september; 52(3): 110–116 research report a comparison of the severity of oral candidiasis between gestational and type 1 diabetes mellitus ayu ragil destrian pangestu,1 siti nosya rachmawati,1 leni rokhma dewi,2 and mei syafriadi1 1 laboratorium of oral pathology, department of biomedical sciences 2 department of oral medicine faculty of dentistry, universitas jember, jember – indonesia abstract background: diabetes mellitus is a metabolic disorder caused by insufficient insulin production due to pancreatic β cell destruction, whereas in gestational diabetics an increase of hormone estrogen induces insulin resistance. oral candidiasis constitutes an opportunistic fungal infection due to a compromised immune system that is a medical condition reported by diabetics, including those suffering from gestational diabetes. purpose: the study aimed to determine the severity of oral candidiasis in female wistar rats with type 1 and gestational diabetes mellitus. methods: this research constituted a laboratory experiment incorporating a post test-only group control design whose subjects were female wistar rats divided along the following lines: group 1 consisted of diabetic non-pregnant rats, group 2 contained diabetic pregnant rats induced by streptozotocin and the control group members constituted normal female rats. diabetes induction was performed by means of 40 mg/kgbw streptozotocin administrated intraperitoneally. diabetes mellitus was confirmed when the blood glucose level ≥ 120 mg/dl. all groups were exposed to 0.2 ml candida albicans (c. albicans) suspension (5x108 cfu/ml) in the oral buccal vestibule between the distal incisors and mesial maxillary first molar for three days. a swab was performed on the third day after final exposure before the samples were observed under a light microscope. c. albicans cultivation and calculation of the resulting colonies was carried out on sabouraud dextrose agar after they had been identified by means of a germ tube test. results: the result confirmed the absence of hyphae in the control group, while in group 1 all samples contained hyphae. moreover, group 2 featured a dense hyphae population. a chi-square test indicated a statistical significance (p<0.05) between all groups. conclusion: oral candidiasis in gestational diabetes is more severe than that occurring during type 1 diabetes mellitus. keywords: candida; diabetes type 1; gestational correspondence: mei syafriadi, laboratorium of oral pathology, department of biomedical sciences, faculty of dentistry, universitas jember, jember – indonesia. email: didiriadihsb@gmail.com introduction diabetes mellitus is a chronic metabolic disorder with characteristics of hyperglycemia that occurs due to abnormal insulin secretion or the inability of the body to process insulin effectively. insulin is a hormone that regulates blood sugar levels the deficiency of which causes an increase in the concentration of glucose in the blood (hyperglycemia).1,2 gestational diabetes results from increased secretion of the hormone estrogen which has a metabolic effect on glucose tolerance, while pregnancy is a diabetogenic state. this hormonal factor results in insulin resistance and, ultimately, hyperglycemia.1 oral candidiasis constitutes one of the opportunistic fungal infections of the oral mucosa caused by candida albicans (c. albicans) whose symptoms include white patches that are confluent and adhere to the oral mucosa and pharynx, particularly affecting the mouth and tongue.3,4 the most prevalent form of lesion presented by sufferers of diabetes mellitus is white plaque of a pseudomembranous type (oral thrush) most commonly found on the dorsum of the tongue.5,6 diabetes mellitus is a predisposing factor in the onset of oral candidiasis especially in pregnant patients since hormonal changes occurring in the body of an expectant woman render her more susceptible to c. albicans infection. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 mailto:didiriadihsb@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 111pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 in particular, elevated levels of the hormone estrogen cause high levels of glycogen, thereby providing a sufficient source of carbon to support the growth of c. albicans.7 several studies have investigated the proportion of individuals within a specific population suffering from diabetes mellitus with oral candidiasis without establishing its severity in terms of the number of c. albicans spore colonies in diabetics who present candidiasis. contrastingly, in the field of gestational diabetics, a number of studies have only examined c. albicans infection in the vaginal region, while scant research has been conducted on the oral cavity. based on the background outlined above, an investigation of oral candidiasis infections under diabetic conditions compared the relative severity of diabetes mellitus and gestational diabetes mellitus infections by observing the growth of spora and hyphae after exposure to oral mucousal containing c. albicans. it is hoped that this study will demonstrate how the maintenance of stable blood sugar levels can provide protection against oral candidiasis infection, especially in diabetes mellitus patients. materials and methods the research constituted a laboratory experiment with a post test-only group control design involving a total of 12 female wistar rats (ratus norvegicus) divided into three groups of four samples in accordance with notoatmodjo’s theory.8 ethical approval of the research was granted through ethic commitee approval number 108/un25.8/ kepk/dl/2018, issued by the faculty of dentistry, university of jember. the sample group consisted of healthy, fully mobile, pregnant and non-pregnant female rattus norvegicus, weighing 150-300 grams which were free of eye disease. the control group contained only healthy rats, while the samples in treatment group 1 consisted of diabetic nonpregnant rats and treatment group 2 were diabetic pregnant rats. the diabetes mellitus samples in treatment groups 1 and 2 were induced by streptozotocin (stz) at a dose of 40 mg/kg bw dissolved in 50 mg / ml 0.1 m citric acid buffer (ph 4.5) administrated intraperitoneally.9,10 those samples with blood glucose levels ≥ 120 mg/dl were categorized as suffering from diabetes mellitus.10 all sample groups were exposed to 0.2 ml c. albicans suspension (5x108 cfu/ml) derived from the oral buccal vestibule between the distal incisors and the mesial maxillary 1 molar for three days.11 a swab was taken on the third day after final exposure. the c. albicans used during this research was derived from the candidal culture held by the microbiology laboratory at the faculty of dentistry, jember university. a swab procedure was performed on the buccal vestibule using a plastic filling instrument until the white plaque had been removed and the base of the lesion appeared reddish in colour. the resulting swabs were smeared on a glass slide before being observed under a light microscope (olympus cx 21 led) in order to view any hyphae and spores, while also establishing the density of the hyphae population. furthermore, the candidal on the glass slide was added to 0.5 ml of sterile aquadest and mixed to form candidal suspension.12 a total of 0.1 ml of c. albicans suspension was removed from the glass slide by means of a pipette and applied to sabouraud dextrose agar (sda) in order to culture c. albicans spore colonies for 24-48 hours at 37oc.12 the respective sizes of the resulting c. albicans colonies present on the media were then calculated using a colony counter.13 calculation of spore colonies on the sda media was undertaken three times by different observers. spore colonies grown in a petridish were placed on the colony counter and divided into four quadrants two of which contained seven boxes while the other two quadrants contained eight boxes, giving a total of 30 boxes. the results provided by the three observers were averaged and entered into the formula cfu/ml = number of colonies x 1/dilution. for the purposes of this research, dilution was set at 10-3.12,14 the identification of c. albicans involved the use of 2 ml of chicken egg white as the culturing medium which was incubated for 30 minutes at 37oc. c. albicans suspension from the swab was added and then incubated for 2-3 hours at 37ºc. positive c. albicans was found in the form of cells that germinate in the form of a germ tube when observed under a light microscopic.15 histopathological examination was perform from oral mucosa thas was stained with hematoxylin eosin (he). parametric qualitative data relating to the severity of oral candidiasis and morphology of c. albicans was analyzed descriptively. statistical analysis of the data was undertaken through the conducting of a chi-square test. if the p-value <0.05, this indicated the existence of a significant relationship between the rows and columns. results examination of candidal spores obtained by means of oral mucosal swab confirmed that all control group samples (100%) contained spores, but no hyphae growth occurred. in contrast, treatment group 1 contained spores and hyphae (100%), while in the treatment group 2 spores were found in all samples (100%) but hyphae growth only occurred in 75% of them (table 1). a chi-square test confirmed the significance (p-value) to be 0.012 (p<0.05), indicating that there was a significant correlation between all groups due to the presence of candidal hyphae. hyphae quantity scoring is based on both the presence and density of hyphae and can be undertaken to distinguish their severity. in the control group, there were no (negative/-) hyphae, while in treatment group 1 the hyphae scoring was positive (+1) (not dense population) leading to a classification of mild oral candidiasis. in treatment group 2, 50% of the samples contained a dense hyphae dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 112 pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 figure 1. microbiological pictures of spores (blue arrow) and hyphae (black arrow) obtained from an oral mucosal swab. control group (a-d), treatment group 1 (e-h), treatment group 2 (i-l). 400x magnification. b c f j g k d l h a e i figure 1. microbiological pictures of spores (blue arrow) and hyphae (black arrow) obtained from an oral mucosal swab. control group (a-d), treatment group 1 (e-h), treatment group 2 (i-l). 400x magnification. figure 2. control group (a-d), treatment group 1 (e-h), treatment group 2 (i -l). pictures of candida albicans spore colonies (blue arrow) on sda culture media. spore colonies are identified as candida albicans if they are circular with a slightly convex surface, smooth, slippery and yellowish white with a sour aroma similar to that of yeast. a b c d e f g h i j k l figure 2. control group (a-d), treatment group 1 (e-h), treatment group 2 (i-l). pictures of candida albicans spore colonies (blue arrow) on sda culture media. spore colonies are identified as candida albicans if they are circular with a slightly convex surface, smooth, slippery and yellowish white with a sour aroma similar to that of yeast. table 1. presence of candidal spores and hyphae in oral mucosal swabs group sample spore hyphae control group (normal subjects) 1 yes no 2 yes no 3 yes no 4 yes no treatment group 1 (type 1 diabetes mellitus subjects) 1 yes yes 2 yes yes 3 yes yes 4 yes yes treatment group 2 (gestational diabetes subjects) 1 yes yes 2 yes no 3 yes yes 4 yes yes dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 113pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 population (+2) leading to a classification of severe oral candidiasis, one sample indicated sporadic hyphae growth (+1), while in another sample no candidal hyphae were located (figure 1 and table 2). statistical tests produced a p-value of 0.006 (p<0.05), confirming a significant correlation between all groups due to hyphae growth or the quantity of hyphae. the calculation of spore colonies on the sda media in all groups was >400 cfu/ml, which meant that 100% of the subjects were at risk of developing oral candidiasis (table 3). the term tntc (too numerous to count) signifies a situation in which the number of colonies on the media is too high to be calculated. this can occur because of low dilution factor levels resulting in a high concentration or uneven distribution of c. albicans in the suspension, in turn causing an accumulation of c. albicans spore colonies which renders calculating them difficult.16 the calculations of the spore colonies present in the media can be seen in figure 2 and table 3. histopathological images confirmed the presence of mucosal epithelial hyperplasia and dense inflammatory cells in the lamina propria indicating a host cell immune response to the presence of c. albicans which resulted in inflammation (figure 3). table 2. the hyphae quantity scores group sample hyphae control group (normal subjects) 1 (–) 2 (–) 3 (–) 4 (–) treatment group 1 (type 1 diabetes mellitus subjects) 1 (+1) 2 (+1) 3 (+1) 4 (+1) treatment group 2 (gestational diabetes subjects) 1 (+2) 2 (–) 3 (+2) 4 (+1) (–): no hyphae; (+1): sporadic hyphae growth (mild candidiasis); (+2): dense hyphae (severe oral candidiasis) table 3. calculation of spore colonies in sda media group sample number of spore colonies (cfu/ml) control group (normal subjects) 1 tntc 2 tntc 3 2 x 105 4 1.9 x 105 treatment group 1 (type 1 diabetes mellitus subjects) 1 2.5 x 105 2 4.2 x 105 3 4.2 x 105 4 4.1 x 105 treatment group 2 (gestational diabetes subjects) 1 6.6 x 105 2 tntc 3 tntc 4 5.1 x 105 *tntc: too numerous to count normal: < 400 cfu/ml risk of oral candidiasis: >400 cfu/ml figure 3. histopathological appearance showed focal hyperplastic epithelium of oral mucousal (blue arrow) and dense inflammatory population in lamina propria (black arrow) due to the presence of spora >400 cfu/ml and hyphae. control group (a-b), treatment group 1 (c-d), treatment group 2 (e-f). (he staining, a-e: 100x and b-f: 400x magnification). a c b d e f figure 3. histopathological appearance showed focal hyperplastic epithelium of oral mucousal (blue arrow) and dense inflammatory population in lamina propria (black arrow) due to the presence of spora >400 cfu/ml and hyphae. control group (a-b), treatment group 1 (c-d), treatment group 2 (e-f). (he staining, a-e: 100x and b-f: 400x magnification). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 114 pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 discussion candida albicans constitutes a normal flora found in the human oral cavity in the form of spores. under certain conditions, for example diabetes, the growth of the c. albicans becomes excessive resulting in infection,17 as was the case in this study where all groups were infected after oral mucousal exposure to multiplying c. albicans spores. the research findings related to treatment groups 1 and 2, the members of which all suffered from diabetes mellitus whose spores may develop into hyphae. increasing the number of spores and degree of hyphae penetration can potentially produce an oral mucousal infection. histopathological observation confirmed dense inflammation infiltration and focal epithelial hyperplastic as a host immune response to lamina propria. given the nature of this infection, all samples could be classified as mild to severe cases of oral candidiasis. the severity of oral candidiasis was found to be in gestational diabetes mellitus compared to type 1 diabetes mellitus. the contents of table 1 indicate the c. albicans growth on the buccal mucosal surface of all samples after exposure for three days. observation confirmed both their spore form and hyphae-like fungal form. c. albicans can be easily detected because it is an organism that has two forms (dimorphic organisms), namely; spores that form a yeast-like substance (non-invasive) and hyphae-like fungal forms that produce root-like rhizoid structures capable of penetrating the mucosa (invasive) and causing infection (oral candidiasis).4 in this study, it was found that hyphae-like fungal forms invasive to oral mucosal tissue produced an infection which appeared histopathologically as an immune response such as dense inflammatory cells in the subepithelium (figure 3.) this infection affected both treatment groups 1 and 2 who suffered from diabetes. the contents of table 2 show that no hyphae were found in any samples contained in the control group. only spores numbering more than 400 cfu/ml were present. these were capable of producing an infection in the buccal mucosa which, histophatologically, appeared as dense inflammatory cell infiltration in the lamina propria (see figures 3a and 3b). meanwhile in treatment group 1 (type 1 diabetes mellitus group), all samples were found sporadically in both the spores and candidal hyphae (score +1). therefore, based on the scoring system utilised, they were classified as mild oral candidiasis and this condition also presents inflammatory cell infiltration in the lamina propria (figure 3c and 3d). in treatment group 2 (gestational diabetic group), 50% of the samples presented dense hyphae (score +2) that could be categorized as severe oral candidiasis. gestational diabetics often suffer severe oral candidiasis perhaps caused by a compromised immune system resulting from infiltration by t lymphocyte cells of the pancreatic gland that will destroy pancreatic beta cells.18 another factor determining the severity of oral candidiasis in this group was the presence of glucose in the saliva which was deposited in the mucosa thus providing the food necessary for candidal growth. on the other hand, salivary flow rate also plays a role in this condition because saliva flow decreases significantly in individuals suffering from diabetes mellitus, a condition known as xerostomia. frequent thirst (polydipsia), dry mouth and binge eating (polyphagia) are characteristics of individuals with diabetes mellitus who demonstrate poor glycemic control which can result in increased diuresis and fluid loss (polyuria). uncontrolled diabetes causes abnormal defense cell function. polymorphonuclear leukocytes (pmn) constitute the main defense cells; neutrophils, monocytes and macrophages, in the periodontium. diabetics typically exhibit the main defense against cell defects because of the imbalance between chemotaxis and phagocytosis which causes sufferers of diabetes mellitus to be more susceptible to infection by c. albicans.19 through the research reported here, it indicated that the oral candidiasis afflicting the members of treatment group 2 (gestational diabetes) was more severe than that affecting those in group 1 (diabetes type 1/dm 1). this can be attributed to elevated levels of estrogen during pregnancy which will, in turn, produced high levels of glycogen. the increase in glycogen provides a sufficient carbon source for the growth of c. albicans.7 on the other hand, in treatment group 2 (gestational diabetes) 25% of the sample suffered from mild oral candidiasis, while a further 25% were found to be uninfected. this contrast in infection was possibly due to differences in the respective resistance of members of the sample. the samples who suffered from oral candidiasis had >400 cfu/ml c. albicans whereas, in general, the normal number of c. albicans is <400 cfu/ml.20,21 table 3 contains the spore counts in sda culture media which indicated that all members of the sample groups were at risk of developing oral candidiasis. the results showed the number of colonies of c. albicans in all sample groups to be > 400 cfu/ml. the number of spore colonies indicated the risk of developing oral candidiasis, but could not quantify the severity of the condition. the potential risk could arise because the form of yeast-like c. albicans colony has a cell wall containing mannoprotein, chitin and glucan. mannoprotein possesses immunosuppressive properties which enhance the defense of c. albicans against the host’s immune system. c. albicans cells will decompose polysaccharides, proteins and glycoproteins which not only stimulate that system, but also facilitate attachment to the host cells.21–23 in addition to adhering to the surface of the epithelium, c. albicans penetrates deeply, especially in the cell junction, by forming an infective hyphae.21 under pathogenic conditions, the form of pseudohyphae and hyphae plays an important role in the penetration process compared to the form of the spores. indeed, the forms of pseudohyphae and hyphae demonstrates a higher penetrative ability than those of spore forms.4,21 candidal hyphae is known to be highly virulent due to the large shape of its hypae which renders it difficult to be engulfed by dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 115pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 macrophage cells (phagocytosis process). therefore, the body’s immune system requires other mechanisms in order to be able to eliminate candidal hyphae in infected tissue. disorders that result from phagocytosis reduce the capability of pmn. phagocystosis due to pmn and macrophages can be inhibited by peptides and acid production from the extracellular glycoprotein c. albicans.23 extracellular c. albicans proteins important to virulence include aspartyl proteinases and phospholipases. penetration by hyphae supported by aspartyl proteinase and phospholipase will cause reduced production of saliva. aspartyl proteinase suppresses the production of host proteins such as albumin, hemoglobin, keratin, and immonoglobulin a secretion (iga) which play a role in the immune system. the pathogenic effects of c. albicans will increase acid production followed by reduced siga (secretory iga) ultimately compromising the body’s immune response. aspartyl proteinase is keratolytic, thereby facilitating the penetration of the epidermis by candida. the phospholipase enzyme is one of the virulent factors that contributes to maintaining infection and also to hydrolyzing phospholipid epithelial cell membranes.21,22,24 the contents of table 3 show that the number of spore colonies in group 2 (gdm) was higher than that of group 1 (dm 1). therefore, subjects with gdm were considered to be at greater risk of developing oral candidiasis than those with dm 1, a conclusion strengthened by the results contained in table 2 which indicate that the hyphae scoring in gdm subjects was greater than that in dm 1 subjects. it can be affirmed that subjects with gdm presented more severe oral candidiasis than those with dm 1, perhaps due to hormonal changes in gdm that lead to an increase in the severity of oral candidiasis.1,7 the presence of hyphae could prove useful in predicting the severity of oral candidiasis because, under pathogenic conditions, the form of pseudohyphae and hyphae play an important role in tissue penetration. penetration by hyphae supported by aspartyl proteinase and phospholipase will result in reduced saliva production. the phospholipase enzyme is also one of the virulent factors that contributes to the severity of infection.23 from the results of this investigation, it can be supposed that future sufferers of diabetes mellitus should always maintain their oral hygiene and blood sugar levels in order to protect against oral candidiasis infection. further research could be undertaken to detect hyphae in oral candidiasis infection using periodic acid-schiff (pas) staining methods on oral tissue lesions. by employing an elisa technique, it could also identify the role of enzymes produced by c. albicans in the severity of oral candidiasis infection as a means of applying the most effective therapy. acknowledgements the writers would like to express their gratitude to the chancellor of jember university and the head of the research institute and community service, jember university for their support and to the healthy aging research group for the funding for this research which it provided in 2018. references 1. setiati s, alwi i, sudoyo aw, simadibrata m, setiyohadi b, syam af. buku ajar ilmu penyakit dalam. jilid ii edisi vi. jakarta: interna publishing; 2015. p. 616. 2. kementerian kesehatan republik indonesia. situasi dan a na l isis d iab et es. ja k a r t a: p usat dat a d a n i n for m a si, kementerian kesehatan republik indonesia; 2014. p. 1–8. 3. tarçın bg. oral candidosis: aetiology, clinical manifestations, diagnosis and management. müsbed j marmara univ inst heal sci. 2011; 1(2): 140–8. 4. mutiawati vk. pemeriksaan mik robiologi pada candida albicans. j kedokt syiah kuala. 2016; 16(1): 53–63. 5. krishnan p. fungal infections of the oral mucosa. indian j dent res. 2012; 23(5): 650–9. 6. nur’aeny n, hidayat w, dewi ts, herawati e, wahyuni is. profil oral candidiasis di bagian ilmu penyakit mulut rshs bandung periode 2010-2014. maj kedokt gigi indones. 2017; 3(1): 23. 7. tasik nl, kapantow gm, kandou rt. profil kandidiasis vulvovaginalis di poliklinik kulit dan kelamin rsup prof. dr. r. d. kandou manado periode januari-desember 2013. j e-clinic. 2016; 4(1): 207–14. 8. notoatmodjo s. metodologi penelitian kesehatan. jakarta: rineka cipta; 2015. p. 216. 9. abdelmeguid ne, fakhoury r, kamal sm, al wafai rj. effects of nigella sativa and thymoquinone on biochemical and subcellular changes in pancreatic β-cells of streptozotocininduced diabetic rats. j diabetes. 2010; 2(4): 256–66. 10. dewi n. lebar benih gigi anak tikus yang dilahirkan oleh induk tikus pengidap diabetes mellitus gestasional. dentino j kedokt gigi. 2014; 2(1): 46–50. 11. martins j da s, junqueira jc, faria rl, santiago nf, rossoni rd, colombo ced, jorge aoc. antimicrobial photodynamic t herapy i n rat exp er i ment a l ca nd id iasis: eva luat ion of pathogenicity factors of candida albicans. oral surgery, oral med oral pathol oral radiol endodontology. 2011; 111(1): 71–7. 12. indahyani d, izzata b, yani cr, atik k, yeni y. petunjuk praktikum biologi mulut ii: mikrof lora rongga mulut dan respon imun. jember: fakultas kedokteran gigi, universitas jember; 2009. 13. getas iw, wiadnya ibr, waguriani la. pengaruh penambahan glukosa dan waktu inkubasi pada media sda (sabaroud dextrose agar) terhadap pertumbuhan jamur candida albicans. media bina ilmiah. 2014; 8: 51–7. 14. nufus bn, tresnani g, faturrahman. populasi bakteri normal dan bakteri kitinolitik pada saluran pencernaan lobster pasir (panulirus homarus l.) yang diberi kitosan. j biol trop. 2016; 16: 15–23. 15. jayanti nks, jirna in. isolasi candida albicans dari swab mukosa mulut penderita diabetes melitus tipe 2. j teknol lab. 2018; 7: 1–7. 16. waluyo l. mikrobiologi umum. malang: umm press; 2012. p. 343. 17. saskia ti, mutiara h. infeksi jamur pada penderita diabetes mellitus. major (medical j lampung univ). 2015; 4(8): 69–74. 18. price sa, wilson lmc. patofisiologi: konsep klinis prosesproses penyakit. 6th ed. jakarta: egc; 2012. p. 812. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 116 pangestu, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 110–116 19. farizal j, dewa ears. identifikasi candida albican pada saliva wanita penderita diabetes melitus. j teknol lab. 2017; 6(2): 67–74. 20. singh a, verma r, murari a, agrawal a. oral candidiasis: an overview. j oral maxillofac pathol. 2014; 18: 81–5. 21. komariah rs. kolonisasi candida dalam rongga mulut. maj kedokt fk uki. 2012; 28: 39–47. 22. brooks gf, caroll kc, butel js, morse sa, mietzner ta. jawetz, melnick & adelberg’s medical microbiology. 26th ed. new york: mcgraw-hill education; 2013. p. 880. 23. nasution ai. virulence factor and pathogenicity of candida albicans in oral candidiasis. world j dent. 2013; 4(4): 267– 71. 24. adiguna ms. aspek kronisitas kandidiasis mukokutaneus. in: national symposium and workshop: skin infection and its complication. tangerang: perdoski; 2015. p. 1–17. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p110–116 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p110-116 2525 dental journal (majalah kedokteran gigi) 2021 march; 54(1): 25–30 original article comparison between school and home-based dental health promotion in improving knowledge, parental attitude and dental health of children with mild disabilities putri raisah, rosa amalia and bambang priyono department of preventive and community dentistry, faculty of dentistry, universitas gadjah mada, yogyakarta – indonesia abstract background: in general, children with physical disabilities have a lower level of oral hygiene compared to able-bodied children because their access to dental health care services is affected by their physical limitations. the level of oral hygiene available to children with disabilities can be improved with the involvement of parents/caregivers equipped with good knowledge and attitudes regarding oral health. purpose: determine the difference between the effectiveness of school and home visit-based dental health promotion in improving the knowledge and attitudes of parents/caregivers and students regarding oral hygiene as well as lowering the dental plaque scores of students with mild physical disabilities. methods: this study is quasi-experimental and uses the two-group pretest–posttest design. the study’s design was created with a model of one observation before intervention (o1), two interventions (x1-2) and two observations after intervention (o2-3). the study samples are students with mild physical disabilities aged 8–15 years old in special needs school, or sekolah luar biasa (slb), in sleman, yogyakarta, and their parents/caregivers. the locations of the study were slbs and the respondents’ homes. the study instrument was a knowledge test for students with mild physical disabilities and their parents/caregivers, along with a questionnaire on the parents/caregivers’ attitudes towards oral and dental health. plaque control examinations for students with mild physical disabilities were conducted using the o’leary index. results: school-based oral health promotion was better at improving the attitudes of parents/caregivers to oral hygiene than the home visits (p<0.05). both school-based and home visit-based oral health promotion was effective in furthering the oral hygiene knowledge of students with mild disabilities and their parents/caregivers as well as in improving the students’ dental plaque scores (p>0.05). conclusion: the school based-oral health promotion model was more effective in improving attitudes of the students’ parents/caregivers. both the school-based and the home visit-based oral health promotion models were equally effective in enhancing the knowledge of parents/caregivers, along with the knowledge and dental plaque scores of students with mild disabilities. keywords: effectiveness; home visits; mild disability; parents/caregivers; promotion of oral health; school. correspondence: putri raisah, department of preventive and community dentistry, faculty of dentistry, universitas gadjah mada, jl. denta 1, sekip utara, yogyakarta 55281, indonesia. email: putriraisah580@yahoo.co.id introduction children with special needs are one of the human resources whose quality must be improved so that it can participate in future development. the children’s quality-of-life improvement needs to be developed and implemented through health programs for children with special needs because their rights to quality services are equal to the rights of other children. according to the mandate of law no. 23 of 2002 regarding child protection and law no. 36 of 2009 regarding health, children with special needs require attention and protection from the government, society and their families. thus, health service needs to be developed to be accessible to children with special needs in accordance with their problems.1 ‘mild disability’ is one type of situation of a child with special needs who suffers from a form of abnormality or disability in the muscular system, bones and joints that may dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 mailto:putriraisah580@yahoo.co.id https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 26 raisah et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 25–30 cause impaired coordination, communication, adaptation, mobilisation and development of personal wholeness.2 the limbs of children with mild disabilities cannot carry out normal functions. this condition may be caused by a disease or accident, or it can also be congenital by nature and present from birth. hence, special services are needed in their learning processes.3 so far, children with mild physical disabilities have not received sufficient attention in terms of dental and oral health. barriers to dental and oral health services include: the presence/absence of access to dental health services, lack of information about dental and oral health for children with mild physical disabilities and obstacles for skilled dental health professionals. government support in this area is inadequate, which can be seen in the unavailability of special health facilities for children with mild physical disabilities at public health centres, or pusat pelayanan kesehatan masyarakat, and hospitals, the lack of public awareness about the importance of oral health for children with mild physical disabilities and the problem of quite expensive treatment costs.4 children with mild disabilities have lower levels of oral and dental hygiene compared to able-bodied children because they have limited limb function and, thus, limited access to dental health care services. children with mild physical disabilities require parental/caregiver assistance in maintaining oral and dental hygiene. these children are generally unable to make independent decisions and need to rely on their parents/caregivers to assist and monitor their daily activities. the level of dental and oral hygiene for children with mild disabilities will be better if their parents/caregivers have adequate knowledge, behaviour and attitudes regarding oral health. parents/caregivers play an important role in promoting these children’s dental health and are responsible for teaching them proper and effective hygiene.5 the dental health of both the children and parents/ caregivers can be improved through a school-based dental health promotion program. the school-based dental health promotion program is a community health service aimed at maintaining and improving the oral health of all students in the target schools, which is supported by individual health efforts in the form of curative efforts for students who need dental and oral health care.6 apart from school-based promotion, dental health promotion can be implemented on a home visit basis. home visit-based dental health promotion programs are health services provided to individuals and families in their homes, with the aim of improving health and maximising the level of independence.7 this study provides evidence regarding the differences between school-based and home visit-based dental health promotion for children with mild physical disabilities and their parents/caregivers. it aims to provide them with the best dental health promotion method and can affect their knowledge and the parents/caregivers’ attitudes as well as decrease the dental plaque scores of students with mild physical disabilities. the novelties of science in this study are: identifying the benefit of performing schoolbased and home visit-based dental health promotion on children with mild disabilities by involving their parents/ caregivers in indonesia (1) and modelling dental health promotion delivered in individual and small groups (3–4 people) by taking into account a comfortable and supportive environment in the learning processes of students with mild physical disabilities (2). the aim of this study is to determine the differences in the effectiveness of school and home visit-based dental health promotion in improving the knowledge and attitudes of the students and their parents/ caregivers in this area as well as lowering the students’ dental plaque scores. materials and methods this study is quasi-experimental and uses a two-group pretest–posttest design.8 the study design consists of one observation before intervention (o1), two interventions (x1-2) and two post-intervention observations (o2-3). 9 o1-3 were carried out by the students and their parents/caregivers completing the knowledge test and attitude questionnaire and the dental plaques of the students being examined. x1-2 for the students and their parents/caregivers took the form of counselling on oral health and demonstrations of how to brush their teeth properly and correctly. the study samples were students with mild disabilities aged 8–15 attending the slb of the sleman district, province of yogyakarta, and their parents/caregivers. based on the calculation of the sample size formula, ten parents/ caregivers and ten students with mild disabilities for group one (school-based) and ten parents/caregivers and ten students with mild disabilities for group two (home visitbased) were obtained. the study location of group one was at the slb and the location of group two was conducted at the respondent’s house. purposive sampling was used as the sampling method. the independent variables were school-based and home visit-based dental health promotions. the dependent variables were the knowledge and the dental plaques of students with mild disabilities and the knowledge and attitudes of their parents/caregivers regarding oral hygiene. the inclusion criteria of this study were students with mild disabilities in class d, based on data from the school, and cerebral system disorders. the respondents who attended slb in sleman district were cooperative and agreed to an informed consent signed by the students and their parents/caregivers. the parents/caregivers in this study were mothers or caregivers who accompany their children on a daily basis. the students were children in class d, where their intelligence can be developed and they can live together with other able-bodied children. even though they have disabilities, they do not interfere with their life and education. cerebral system disorders, such as cerebral palsy or cerebral disability, are located in the central nervous system dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 27raisah et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 25–30 and are characterised by movement, gesture or body shape and coordination disfunction, sometimes accompanied by psychological and sensory disfunction, which is caused by damage or defection during brain development. the exclusion criteria for this study were respondents who had systemic disease and/or paralysis of both hands.10 the instrument used in this study was a knowledge test for parents/caregivers about dental and oral diseases and how to maintain oral hygiene. the text consisted of 18 questions, with the highest score being 18 and the lowest 0. the questionnaire on the parents/caregiver’s attitudes focused on their knowledge (cognitive), attitude (affective) and behaviour regarding dental and oral health, with a total of 17 statements. a likert scale with the highest score of 68 and the lowest score of 17 was used. the knowledge test for the students focused on dental and oral diseases and how to maintain oral hygiene and had a a total of 14 questions. the highest score was 14 and the lowest 0. the assessment the students’ plaque control with the o’leary index used disclosing solutions, the assessment of dental plaque accumulation was performed on all existing teeth and missing teeth were marked with a thick line on the record form. the examination was carried out on four surfaces: the facial, lingual, mesial and distal. if dental plaque was observed on one surface, the score was 1, whereas if there was no dental plaque, the score was 0. the results of the dental plaque assessment were obtained by summing up the dental plaque scores of each tooth surface, so that the dental plaque score for each tooth ranged from 0 to 4. the outcome value was in percentages. dental plaque of the students was divided into two categories, namely: ≤10% as the good dental plaque score category and ≥10% as the bad dental plaque score category. the difference between school-based and home visitbased dental health promotions is that a school-based dental health promotion is health care service provided to the respondents at their school, with the aim to maintain and improve oral health in students at targeted schools that require dental and oral health care,11 while home visit-based dental health promotion is a health care service provided to individuals and families at their homes, with the aim to improve health and maximise the students’ level of independence.12 the general linear model repeated measure and a statistical package for the social sciences (spss) version 24 program (ibm, new york, usa) were used for data analysis. all statistical analyses were conducted with a significance of p < 0.05. approval of research ethics was obtained from the faculty of dentistry, gadjah mada university, with the number of 00400/kkep/fkg-ugm/ ec/2020. results this study was conducted during february–march 2020 and involved 20 students with mild disabilities and 20 parents/ caregivers who met the inclusion and exclusion criteria. figure 1 shows that there was a difference in the mean knowledge of parents/caregivers between school-based and home visit-based promotion in o1, o2 and o3. figure 2 shows that there was a difference in the mean attitude of parents/caregivers after school-based and home visit-based promotions in o1, o2 and o3. figure 3 shows that there was a difference in the mean knowledge of students with mild disabilities after school-based and home visit-based promotion in o1, o2 and o3. figure 4 shows that there was 11.7 17.8 17.9 9.5 16.7 16.9 0 5 10 15 20 observasi i observasi ii observasi iii parents' knowledge sekolah rumah + 6.1 + 0.1 + 7.2 + 0.2 observation i observation ii observation iii school home figure 1. mean knowledge of parents/caregivers in observation i, observation ii and observation iii. 55.1 67.6 67.6 52.2 62.9 63.2 0 10 20 30 40 50 60 70 80 observasi i observasi ii observasi iii parents' attitude sekolah rumah + 12.5 + 10.7 + 0 + 0.3 observation i observation ii observation iii school home figure 2. mean attitude of parents/caregivers in observation i, observation ii and observation iii. 5.2 2.21 12.3 7.3 13.8 13.8 0 2 4 6 8 10 12 14 16 observasi i observasi ii observasi iii knowledge of students with mild disabilites sekolah rumah + 6.5 + 7 + 0 + 0.1 observation i observation ii observation iii school home figure 3. mean knowledge of students with mild disabilities in observation i, observation ii and observation iii. 50.6 21.2 19.6 44.1 12 11.1 0 10 20 30 40 50 60 observasi i observasi ii observasi iii plaque of students with mild disabilites sekolah rumah 1.6 29.4 32.1 0.9 observation i observation ii observation iii school home figure 4. mean dental plaque score of students with mild disabilities in observation i, observation ii and observation iii. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 28 raisah et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 25–30 a difference in the mean plaque scores of students with mild disabilities between school-based and home visit-based promotions in o1, o2 and o3. table 1 shows that there was no difference in the knowledge of parents/caregivers in the school-based and home visit-based groups in o2–o2 (δ1) and o3–o2 (δ2) with a value of p = 0.11. there was a difference in the attitudes of parents/caregivers in the school-based and home visit-based groups in δ1 and δ2 with a value of p = 0.01. therefore, it can be said that school-based and home visitbased dental health promotion were both equally effective in improving the knowledge of parents/caregivers about oral hygiene and school-based dental health promotion was more effective in improving the attitudes of parents/ caregivers regarding oral hygiene than home visit-based promotion. table 2 shows the improvement of the parents/ caregivers’ knowledge regarding oral hygiene in δ1 in the school-based and home visit-based groups, with a value of p = 0.04 and an improvement in the parents/caregivers’ attitudes in δ1 in the school-based and home visit-based groups, with a value of p = 0.02. therefore, it can be said that there was a statistically significant improvement in the knowledge and attitudes of parents/caregivers regarding oral health in δ1. there were improvements of the parents/ caregivers’ knowledge regarding oral health in the schoolbased and home visit-based groups in δ2, with a value of p = 0.66 and in the attitudes of parents/caregivers in the schooltable 1. differences in knowledge and attitudes of parents/caregivers between school-based and home-based oral health promotion variable observation group mean sd sig. knowledge δ1 (o2 – o1) school 6.10 1.28 0.11 home 7.20 0.92 δ2 (o3 – o2) school 0.10 0.57home 0.20 0.422 attitude δ1 (o2 – o1) school 12.50 1.50 0.01 home 10.70 1.83 δ2 (o3 – o2) school 0.10 0.00home 0.30 0.48 table 2. improvements in knowledge and attitudes of parents/caregivers of school-based and home visit-based from observation i, observation ii and observation iii variable observation group mean sd sig. knowledge δ1 (o2 – o1) school 6.10 1.28 0.04home 7.20 0.92 δ2 (o3 – o2) school 0.10 0.57 0.66home 0.20 0.422 attitude δ1 (o2 – o1) school 12.50 1.50 0.02home 10.70 1.83 δ2 (o3 – o2) school 0.10 0.00 0.06home 0.30 0.48 table 3. differences in knowledge and dental plaques of students with mild physical disabilities in school-based and home-based oral health promotion variable observation group mean sd sig. knowledge δ1 (o2 – o1) school 7.00 1.24 0.49home 6.50 1.08 δ2 (o3 – o2) school 0.10 0.31home 0.10 0.47 dental plaque δ1 (o2 – o1) school -29.37 19.00 0.39home -32.07 11.90 δ2 (o3 – o2) school -5.66 14.29home -0.94 2.33 table 4. improvements in knowledge and the decrease of dental plaque scores of students with mild physical disabilities after schoolbased and home visit-based interventions in observation i, observation ii and observation iii variable observation group mean sd sig. knowledge δ1 (o2 – o1) school 7.00 1.24 0.35home 6.50 1.08 δ2 (o3 – o2) school 0.10 0.31 0.58home 0.10 0.47 dental plaque δ1 (o2 – o1) school -29.37 19.00 0.70home -32.07 11.90 δ2 (o3 – o2) school -5.66 14.29 0.31home -0.94 2.33 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 29raisah et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 25–30 based and home visit-based groups in δ2, with a value of p = 0.06. thus, it can be said that there was no statistically significant improvement in the knowledge and attitudes of parents/caregivers regarding oral health in δ2. table 3 shows that there was no difference in the knowledge of students with mild physical disabilities in the school-based and home visit-based groups in δ1 and δ2, with a value of p = 0.49. there was no difference in the dental plaque scores of students with mild physical disabilities in the school-based and home visit-based groups in δ1 and δ2, with a value of p = 0.39. therefore that it can be said that school-based and home visit-based dental health promotions were equally effective in improving knowledge and lowering dental plaque scores for students with mild physical disabilities. table 4 shows the improvement in knowledge of students with mild physical disabilities in the school-based and home visit-based groups in δ1, with a value of p = 0.35, and δ2, with a value of p = 0.58. there was a decrease in dental plaque scores for students with mild physical disabilities in the school-based and home visit-based groups in δ1, with a value of p = 0.70, and δ2, with a value of p = 0.31. hence, it can be said that there was no statistically significant difference in knowledge improvement and the decrease of dental plaque scores of students with mild physical disabilities in δ1 and δ2. discussion this study generally finds that after the end of the intervention, there was no statistically significant difference between the school-based and home visitbased dental health promotion groups in terms of improving parental knowledge of dental and oral health. it concludes that school-based and home visit-based dental health promotion were equally effective in improving the knowledge of parents/caregivers regarding dental and oral health. school-based and home visit-based dental health promotion were equally effective in improving knowledge of parents/caregivers regarding dental and oral health because the counselling sessions at school and at home were both relaxed and comfortable and the parent/caregivers were able to focus and not be distracted by other things. counselling activities at school and at home also facilitated discussions with questions and answers, allowing a direct approach to parents in order to solve cases or problems related to dental and oral health and provide opportunities and flexibility for respondents to help them in accordance with what was needed. counselling activities at school and at home were equally flexible according to the agreement of the counsellor and counselee regarding both the location and time of counselling. these results are in line with a study conducted by imazu et al., which compared individual and groupbased educational interventions to monitoring service providers for patients with type 2 diabetes mellitus. the results obtained from both individual and group-based interventions were equally effective in improving the patients’ knowledge about type 2 diabetes mellitus. the success of this study was based on several advantages. the individuals with type 2 diabetes were equally monitored and the intervention process was carried out deeply, providing optimal development services according to the problems needed. individual-based and group-based interventions allow dialogue, reflection, exchange of experiences and knowledge as well as shared responsibility for the patients’ own health.13 this study finds that generally after the end of the intervention, there was a statistically significant difference between the school-based and home visit-based dental health promotion groups in regard to the improvement of their parents/caregivers’ attitudes towards oral health, with the school-based group significantly better than the home visit-based group. it concludes that school-based dental health promotion was more effective than home visit-based promotion in terms of improving the parents/caregivers’ attitudes towards oral health. school-based dental health promotion was more effective in improving attitudes of parents/caregivers towards oral and dental health because counselling activities in schools were more formal and the parents/caregivers who attended counselling activities could focused more clearly and not be distracted by other activities. respondents were able and active in following the course of counselling, such as asking questions, listening and issuing their ideas/ insights and being able to respect the opinions of others. school-based counselling programs facilitate discussions, questions and answers between respondents. every question and answer can be listened to together. with such variations, the parents/caregivers’ knowledge can be further improved. the activeness of respondents in counselling activities can be seen in their activeness in group discussion activities. this result was in line with the study conducted by tugeman et al. about the effect of dental and oral health education programs in schools on improving caregiver attitudes. the results obtained were that dental and oral health education programs in schools are effective in improving caregiver attitudes. the success of this study was due to several advantages, namely the good response rate of parents/caregivers of deaf students during the study process, almost everyone participating in the study and the intervention being conducted by a professional dental health provider, which affected the results of the scores obtained by the parents/caregivers of deaf students.14 this study has generally obtained the results that after the end of the intervention, there was no statistically significant difference between the school-based and home visit-based dental health promotion groups in improving knowledge and decreasing dental plaque scores of students with mild physical disabilities. the conclusion is that school-based and home visit-based dental health promotion are equally effective in improving knowledge dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 30 raisah et al./dent. j. (majalah kedokteran gigi) 2021 march; 54(1): 25–30 and lowering dental plaque scores of students with mild physical disabilities. school-based and home visit-based dental health promotion are both effective in improving knowledge and lowering dental plaque scores of students with mild physical disabilities because both counselling events at school and at home have the effect of a comfortable and supportive environment in the students’ learning processes, stimulating more enthusiasm in the learning processes. this is because at school they can interact with friends, while at home the parents/caregivers are present to accompany the counselling activities, so students with mild disabilities can focus more on listening to counselling, helping them understand what was being said. both counselling activities at school and at home can improve knowledge and reduce dental plaque scores for students with mild physical disabilities because every explanation and all attention given by researchers can focus on students with mild physical disabilities. these results were in line with the study conducted by suliono et al. on the effectiveness of the implementation of group counselling services and individual counselling services as an effort to overcome student delinquency problems. the results obtained from both group counselling services and individual counselling services were equally effective in an effort to overcome student delinquency problems. the success in this study was due to several advantages, such as students feeling that they were equally cared for, the counselling process being carried out more deeply and to the point of problems faced by students. the teacher acted as a companion to the students in an effort to provide them with optimal development services in accordance with the problems faced by students, by not excluding the principles and functions of counselling guidance.15 in conclusion, school-based oral health promotion is better at improving attitudes of parents/caregivers towards oral health than home visitbased programs. school and home visit-based oral health promotion are both effective in improving knowledge of the students and their parents/caregivers and lowering the student’s dental plaque scores. acknowledgements the author would like to thank to (1) the principal and the entire board of special school teachers, who have given permission to conduct research and provided assistance during the research; (2) all students with mild disabilities and parents/caregivers who provided good cooperation and were willing to take the time to become respondents in this study. references 1. menter i kesehata n republik i ndonesia. peratura n menter i kesehatan republik indonesia nomor 9 tahun 2014 tentang klinik. jakarta; 2014 p. 12–3. 2. m isba ch d, c h r isna . selu k-b elu k t u na d a k sa d a n st r at eg i pembelajarannya. yogyakarta: javalitera; 2012. p. 15–24. 3. somantri ts. psikologi anak luar biasa. bandung: refika aditama; 2012. p. 121–3. 4. menter i kesehata n republik i ndonesia. peratura n menter i kesehatan republik indonesia nomor 89 tahun 2015 tentang upaya kesehatan gigi dan mulut. jakarta; 2016 p. 11–2. 5. liu h-y, chen j-r, hsiao s-y, huang s-t. caregivers’ oral health knowledge, attitude and behavior toward their children with disabilities. j dent sci. 2017; 12(4): 388–95. 6. amalia r, schaub rmh, stewart re, widyanti n, groothoff jw. impact of school-based dental program performance on the oral health-related quality of life in children. j investig clin dent. 2017; 8(1): e12179. 7. minihan pm, morgan jp, park a, yantsides ke, nobles cj, finkelman md, stark pc, must a. at-home oral care for adults with developmental disabilities. j am dent assoc. 2014; 145(10): 1018–25. 8. swarjana ik. metodologi penelitian kesehatan: tuntunan praktis pembuatan proposal penelitian. yogyakarta: andi offset; 2015. p. 68–73. 9. brown ra. training and assessment of toothbrushing skills among children with special needs. grad theses diss univ south florida. 2012; : 1–55. 10. atmaja jr. pendidikan dan bimbingan anak berkebutuhan khusus. bandung: rosdakarya; 2018. p. 127–37. 11. luksamijarulkul n, pongpanich s, panza a. protective factors for caries of a school-based oral health program in bangkok, thailand: a retrospective cohort study. public health. 2020; 187: 53–8. 12. huang j-c, lin m-s, chiu w-n, huang t-j, chen m-y. the effectiveness of an oral hygiene program combined with healthpromoting counseling for rural adults with cardiometabolic risks: a quasi-experimental design. appl nurs res. 2020; 55: 151333. 13. imazu mfm, faria bn, arruda go de, sales ca, marcon ss. effectiveness of individual and group interventions for people with type 2 diabetes. rev lat am enfermagem. 2015; 23(2): 200–7. 14. tugeman h, abd rahman n, daud mkm, yusoff a. effect of oral health education programme on oral health awareness and plaque maturity status among hearing-impaired children. arch orofac sci. 2018; 13: 22–35. 15. suliono, rufi’i, karyono h. penerapan layanan konseling kelompok dan layanan konseling individu dalam upaya mengatasi masalah kenakalan siswa di smp negeri 1 kebomas gresik. j educ dev. 2019; 7(2): 248–55. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at https://e-journal.unair.ac.id/mkg/index doi: 10.20473/j.djmkg.v54.i1.p25–30 https://e-journal.unair.ac.id/mkg/index http://dx.doi.org/10.20473/j.djmkg.v54.i1.p25-30 dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. editor 9191 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 91–96 research report the increased number of osteoblasts and capillaries in orthodontic tooth movement post-administration of robusta coffee extract herniyati department of orthodontics faculty of dentistry, universitas jember jember indonesia abstract background: the application of orthodontic forces subjects blood capillaries to considerable pressure, resulting in hypoxia on the pressure side. vascular endothelial growth factor (vegf), expressed in osteoblasts represents an important mitogen that induces angiogenesis. osteoblasts and blood capillaries play an important role in bone formation. robusta coffee contains chlorogenic acid and caffeic acid both of which produce antioxidant effects capable of reducing oxidative stress in osteoblasts. purpose: the aim of this study was to analyze the effects of robusta coffee extract on the number of osteoblasts and blood capillaries in orthodontic tooth movement. methods: this research constituted a laboratory-based experimental study involving the use of sixteen male rodents divided into two groups, namely; control group (c) consisting of eight mice given orthodontic mechanical force (omf) and a treatment group (t) containing eight mice administered omf and dried robusta coffee extract at a dose of 20mg/100 g bw. the omf was performed by installing a ligature wire on the maxillary right first molar and both maxillary incisors. in the following stage, the maxillary right first molar was moved to the mesial using tension gauze with a nickel titanium orthodontic closed coil spring. observation was subsequently undertaken on the 15th day by extracting the maxillary right first and second molar with their periodontal tissues. thereafter, histological examination was performed using hematoxylin-eosin (he) staining technique to measure the number of osteoblasts and blood capillaries on the mesial and distal periodontal ligaments of the maxillary right first molar. results: the administration of robusta coffee extract increases the number of blood capillaries and osteoblasts on both the pressure and tension sides were found to be significantly higher in the t group compared to the c group (p<0,05). conclusion: robusta coffee extract increase the number of osteoblasts and blood capillaries, thereby playing a role in improving the alveolar bone remodeling process in orthodontic tooth movement. keywords: orthodontic tooth movement; robusta coffee; vegf; capillary; osteoblasts correspondence: herniyati, department orthodontics, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. e-mail: herniyati@unej.ac.id introduction orthodontic tooth movement depends on the remodeling of periodontal ligament and alveolar bone associated with a number of biological and mechanical responses of the surrounding tissue. subjecting periodontal ligaments to pressure will result in bone resorption, whereas placing periodontal ligaments under tension will lead to bone formation.1 in other words, bone resorption is effected by osteoclasts on the pressure side, while a new bone formation is produced by osteoblasts on the tension side.2 osteoblasts are bone-forming cells that express parathyroid hormone receptors and execute several important roles in bone remodeling, namely; osteoclastogenic factor expression, bone matrix protein production, and bone mineralization.3 at the tension site, new bone is formed as a result of orthodontic force during treatment.4 osteoblast cells are involved in bone formation which commences some 40-48 hours after the initial application of orthodontic force. differentiation of osteoblasts subsequently begins with stem cells derived from bone marrow moving to the blood vessels. the migration of mesenchymal stem cells from the blood vessel wall or the activation of mesenchymal stem cell precursors and preosteoblasts formation occurs dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 92 herniyati, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 91–96 approximately 10 hours after the application of orthodontic force.5 mature osteoblasts form osteoid leading to a mineralization process,4 with endothelial nitric oxide synthase (enos) then facilitating bone formation at the tension site.6 furthermore, periodontal ligaments consist of blood capillaries, very small blood vessels, at the end of the arteries. the walls of blood capillaries consist of a thin endothelial layer and lymph fluid that can be secreted to form a tissue fluid capable of carrying nutrients, including water and minerals, to cells. through a process of gas exchange between the capillary blood vessels and cells in the tissues, the former can both provide oxygen and remove carbon dioxide. in this manner, blood capillaries play an important role in distributjng to the tissues important substances used for various processes in the body.7 the application of orthodontic force can narrow the blood vessels, thereby reducing the blood supply and causing hypoxia at the pressure site. hypoxia constitutes a deficiency of oxygen in the tissues due to decreased partial oxygen pressure beyond the physiological level.8 hypoxia can also compromise cellular energy levels by reducing glycolytic activity and adenosine tri phosphate (atp) production. cells then respond to hypoxia by expressing cellular mediators, especially hypoxia-inducible factor 1 (hif-1) that can promote angiogenesis, stimulate cell proliferation, and prevent cell death.9 moreover, hypoxia induces the formation of an active transcription factor hif-1 and activates genes that encode endhothelial vascular growth factor (vegf)9 known as one of the most important mitogens to induce angiogenesis.10 angiogenesis is the growth of new blood vessels from existing ones. by binding to endothelial cell receptors, vegf activates a signal cascade, resulting in various cellular and blood vessel reactions.11 when the vegf binds to its receptor a signal is generated on the surface of the activated endothelial cell which is then sent to the cell nucleus. new molecules are produced by endothelial cell organelles, namely; protease enzymes, which serve to destroy the extracellular matrix as a branching point for capillary vessels. angiopoietin growth factors and a matrix metalloproteinase enzyme produced by endothelial cells are required to initiate the formation of new blood vessels.12 matrix metalloproteinase (mmp) is also important for angiogenesis because of its role in extracellular matrix degradation, resulting in the migration of endothelial cells.9 mmp-2 is even considered a direct transcriptional target of hif-1α, mediating endothelial cell migration in response to hypoxia.13 in general, vegf expression is detected in osteoblasts, osteocytes, and fibroblasts at the tension site after 10 days of orthodontic tooth movement.14 unfortunately, relatively protracted orthodontic treatment constitutes a major problem for patients since it is often associated with a variety of conditions such as dental caries,15 external root resorption,16 and open gingival embrasures.17 therefore, orthodontic tooth movement needs to be accelerated in several ways, one of which is through the use of additional medicines.18 crucially, coffee represents one of the popular beverages consumed by societies.19 robusta coffee contains chlorogenic acid, phenylalanines formed during the roasting process,20 and caffeic acid, which produce antioxidant effects in reducing oxidative stress in osteoblasts21 and stimulate osteoblast activity.22 moreover, clorogenic and caffeic acids in robusta coffee produce antioxidant effects in enhancing angiogenesis through increased vegf.23 results of research on mice even showed that the administration of robusta coffee at a dose of 20 mg/100 g bm (equivalent to one cup of coffee in humans) increased the number of osteoclasts on the 15th day.24 the study reported here aimed to analyze the increased number of osteoblasts and blood capillaries in orthodontic tooth movement after the administration of robusta coffee extract materials and methods this research was a laboratory-based experimental study conducted over a period of three months at the department of biomedicine, faculty of dentistry, universitas jember. this research was approved by the ethics commission of the faculty of dental medicine, universitas airlangga, surabaya number: 8/kkepk.fkg/ii/2015. this research involved sixteen healthy, male mice (spraque dauwley) weighing 250-300 grams and aged between 3 and 4 months. they were divided into two groups, namely a control group (c) consisting of eight mice who were given orthodontic mechanical force (omf) and a treatment group (t) consisted of eight rats given omf and freezedried robusta coffee extract with a dose of 20 mg/100 g bw (equivalent to one cup of coffee in humans) dissolved in 2ml of aquades. coffee extract was given orally via a stomach sonde over a period of 14 consecutive days. subsequent to this, the mice were anesthetized with ketamine before an omf was applied. a ligature wire (3m unitek, germany) 0.20 mm in diameter was mounted by attaching it to the maxillary right first molar and both maxillary incisors afterwards, the maxillary right first molar was moved to mesial using a tension gauge (ormco, usa) to generate 10 g/cm2 strength with a nickel titanium orthodontic closed coil spring (3m unitek, germany) 6 mm in length. therefore, this closed coil spring extending from the maxillary right first molar to both maxillary incisors.25 observation was conducted by means of the rats being sacrificed on day 15 before their maxillary right first and second molar were extracted together with periodontal tissue. histological examination was then performed using hematoxylin-eosin (he) staining technique to observe the number of osteoblasts and blood capillaries on the mesial and distal areas of periodontal ligaments. observations were conducted using a microscope at 400x magnification. the mesial area represented the pressure side, while the distal dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 9393herniyati, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 91–96 area represented the tension side of the maxillary right first molar. the data obtained was subsequently analyzed by means of an independent t-test, mann whitney test, paired t-test and wilcoxon signed ranks test with a confidence level of 95% (α = 0.05). results the results of this research showed the mean ± standard deviation of osteoblasts at the pressure sides in c group and t group to be 2.88 ± 0.99 and 7.50 ± 1.69, respectively; whereas at the tension side it was 4.13 ± 1.64 and 11.50 ± 1.20, respectively. this confirmed that the number of osteoblasts in both the pressure and tension sides in group t was greater than those in group c. these results strongly suggest that the number of osteoblasts at the tension side was greater than at the pressure side in both of group c and t. in addition, it suggests that the number of osteoblasts at the tension side was greater than at the pressure side of both research groups. at this point, a difference test was performed on the number of osteoblasts at the pressure side of groups c and t using a mann-whitney test since the data was not normally distributed (p <0.05). the results showed that the number of osteoblasts in the pressure sides was found significantly higher in the t group compared to the c group (p<0.05). on the other hand, an independent t-test was conducted on the number of osteoblasts at the tension side of the c and t groups since the data was normally distributed (p>0.05). the results of the independent t-test indicated that the number of osteoblasts in the tension sides in the t group was found to be significantly higher than that of the c group (p<0.05). a wilcoxon signed rank test was then conducted to reveal the difference in osteoblasts between the pressure and the tension sides of both research groups since the data were not normally distributed (p<0.05). the results indicated that the number of osteoblasts in the c group was not found to be significantly higher in the tension side compared to the pressure side (p>0.05), while in the t group there was a significantly higher in the tension side compared to the pressure side (p<0.05). the results also showed that the mean ± standard deviation of blood capillaries at the pressure sides in c and t groups was 3.20 ± 0.11 and 4.30 ± 0.38, respectively; while on the tension sides it was 3.31 ± 0.13 and 5.14 ± 0.21, respectively. this indicated that the number of blood capillaries on both the pressure and the tension sides in t group was greater than in c group. furthermore, it suggests that the number of blood capillaries at the tension side was greater than at the pressure side in both research groups. an independent t-test was subsequently performed on the number of blood capillaries in both the pressure and the tension sides of c group and t group since the data were normally distributed (p>0.05). the results showed that the number of blood capillaries in both the pressure and the tension sides was found significantly higher in the t group compared to the c group (p<0.05). paired t-test then was conducted to reveal the difference of blood capillaries between the pressure and the tension sides of both research groups since the data were normally distributed (p>0.05). the paired t-test results indicated that the number of blood capillaries in the c group was not significantly higher in the tension side compared to the pressure side (p>0.05), while in t group it was significantly higher in the tension side compared to the pressure side (p<0.05) (table 1). a histological examination was carried out on the number of osteoblasts and blood capillaries in the periodontal ligaments. the results indicated that the number of osteoblasts and blood capillaries on both the pressure and the tension sides was higher in the mice treated with the robusta coffee extract than in the control group (figure 1). figure 1 illustrates the number of osteoblasts and capillaries on the pressure side in c group not treated with the robusta coffee extract (figure 1-a) and in t group treated with the robusta coffee extract (figure 1-b). in addition, the number of osteoblasts and blood capillaries in the tension side of c group not treated with the robusta coffee extract can be seen in figure 1-c. meanwhile, figure 1-d demonstrated the number of osteoblasts and blood capillaries in the tension side of t group treated with the robusta coffee extract. table 1. the mean ± standard deviation of osteoblasts and blood capillaries at the pressure and tension sides group n osteoblasts (mean ± standard deviation) blood capillaries (mean ± standard deviation) pressure tension p pressure tension p c 8 2.88 ± 0.99 4.13 ± 1.64 0.156 3.20 ± 0.11 3.31 ± 0.13 0.135 t 8 7.50 ± 1.69 11.50 ± 1.20 0.012 4.30 ± 0.38 5.14 ± 0.21 0.007 p 0.000 0.000 0.000 0.000 note: p<0.05 = significant difference; p>0.05 = no significant difference dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 94 herniyati, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 91–96 discussion orthodontic force can decrease blood flow on the pressure side as the blood vessels are depressed while, conversely, blood flow on the tension side increases. such changes in the blood flow will alter the blood chemistry.26 the reduced oxygen supply in the pressure zone will trigger a hypoxic condition in the periodontal capillaries. local hypoxia will then increase vegf expression in fibroblasts of the periodontal ligament, while also potentially inducing the formation of an active hif-1 transcription factor and activating genes encoding vegf.9 consequently, orthodontic tooth movement can accelerate osteoblast activity, resulting in increased vegf expression.27,28 the results of this research showed that the administration of robusta coffee extract enhanced the number of osteoblasts at the pressure and tension sides of the t group compared to those of the untreated c group. this was because the antioxidant effects of chlorogenic acid and caffeic acid contained in the coffee extract can reduce oxidative stress in osteoblasts, thereby increasing the differentiation and stability of osteoblasts.21,28 orthodontic tooth movement can also trigger the occurrence of reactive oxygen species (ros) which can then increase lipid peroxidation, the main cause of damage to the cell membranes, disrupting the structure and function of osteoblast cells. thus, chlorogenic acid and caffeic acid contained in the coffee extract will act as antioxidant agents to protect cells by converting free radicals into stable products, thereby preventing osteoblast cell membrane damage.28 the results of the research also indicated that the robusta coffee extract increased the number of blood capillaries at both the pressure and tension sides of t group compared to the c group that was not treated with the robusta coffee extract. this could be caused by osteoblasts expressing vegf which stimulated angiogenesis.10 in other words, the increased number and activity of osteoblasts due to the administration of robusta coffee extract can generate vegf expression.27,28 angiogenesis enhances the number of blood capillaries since the width of the major blood vessel plexus significantly increases because of branched capillaries which will then transform those branched capillaries into perfect vascular tissue.29 angiogenesis begins with the degradation of the preexisting blood vessel wall before activating the proliferation and migration of endothelial cells. the endothelial cells are arranged in a tubular structure around the walls of the blood vessels that will be formed. endothelial end cells then stimulate shoots to grow, with the result that new blood vessels will be developed and the dilation and replenishment of blood containing oxygen will occur.30 in this research, the administration of robusta coffee extract further increased the number of osteoblasts and blood figure 1. osteoblasts (red arrows) and capillaries (black arrows) on the pressure side of the control group (figure a), the pressure side of the treatment group (figure b), the tension side of the control group (figure c), and the tension side of the treatment group (figure d). alveolar bones (yellow arrows), while the roots of the tooth (blue arrows) (he staining, 100x magnification). a c d b figure 1. osteoblasts (red arrows) and capillaries (black arrows) on the pressure side of the control group (figure a), the pressure side of the treatment group (figure b), the tension side of the control group (figure c), and the tension side of the treatment group (figure d). alveolar bones (yellow arrows), while the roots of the tooth (blue arrows) (he staining, 100x magnification). a c d b figure 1. osteoblasts (red arrows) and capillaries (black arrows) on the pressure side of the control group (figure a), the pressure side of the treatment group (figure b), the tension side of the control group (figure c), and the tension side of the treatment group (figure d). alveolar bones (yellow arrows), while the roots of the tooth (blue arrows) (he staining, 100x magnification). a c d b figure 1. osteoblasts (red arrows) and capillaries (black arrows) on the pressure side of the control group (figure a), the pressure side of the treatment group (figure b), the tension side of the control group (figure c), and the tension side of the treatment group (figure d). alveolar bones (yellow arrows), while the roots of the tooth (blue arrows) (he staining, 100x magnification). a c d b figure 1. osteoblasts (red arrows) and capillaries (black arrows) on the pressure side of the control group (a), the pressure side of the treatment group (b), the tension side of the control group (c), and the tension side of the treatment group (d). alveolar bones (yellow arrows), while the roots of the tooth (blue arrows) (he staining, 100x magnification). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 9595herniyati, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 91–96 capillaries at the tension side compared to the pressure side. this scenario could be caused by the antioxidant effects of the chlorogenic acid and caffeic acid contained in the coffee extract not only increasing osteoblast differentiation, but also promoting osteoblast activity.22,28 consequently, both vegf expression and the number of capillaries increase. the area of the tension side is larger than that of the pressure side with the result that a higher number of osteoblasts and blood capillaries is required in bone formation. this finding is consistent with the results of the study showing that robusta coffee brewing increases the incidence of bone islands and vegf in tension areas to a greater extent than in pressure areas.24 blood vessels actually play a key role in the remodeling of bone growth and development.5 the process of bone formation is related to the development of new capillaries within the existing blood vessels.31 osteoblasts playing an important role in bone formation are required to remodel the resorption region on the pressure side and to form new bone on the tension side.32 osteoblasts are differentiated from mesenchymal cell cursors, while mature osteoblasts form osteoids, followed by bone mineralization process.4 in addition, vegf can modulate the recruitment, differentiation, and activation of osteoclast precursors, thereby increasing bone resorption. vegf also indirectly leads to bone resorption since it promotes angiogenesis in vitro, enabling new capillaries to assist in the recruitment of osteoclasts to the nearest bone surface and then to the resorption area.13 in vitro research has confirmed that vegf can stimulate osteoclast differentiation by increasing the rankl/opg ratio. rankl (a receptor activator of nuclear factor kb ligand) and opg (osteoprotegerin) are osteoblast-generated cytokines and vegf plays an important role in the proliferation, migration, and invasion ability of osteoblasts.33 finally, the active osteoclasts will result in bone resorption, with osteoblasts therefore also indirectly playing a role in bone resorption.34,35 in conclusion, robusta coffee extract can increase the number of osteoblasts and blood capillaries that play an important role in orthodontic tooth movement. references 1. domenico m di, d’apuzzo f, feola a, cito l, monsurrò a, pierantoni gm, berrino l, rosa a de, polimeni a, perillo l. cytokines and vegf induction in orthodontic movement in animal models. j biomed biotechnol. 2012; 2012: 1–4. 2. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofacial orthop. 2006; 129(4): 469.e1-32. 3. karsenty g. transcriptional control of skeletogenesis. annu rev genomics hum genet. 2008; 9: 183–96. 4. sprogar s, vaupotic t, cör a, drevensek m, drevensek g. the endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats. bone. 2008; 43(4): 740–7. 5. masella rs, meister m. cur rent concepts in the biology of orthodontic tooth movement. am j orthod dentofacial orthop. 2006; 129(4): 458–68. 6. tan sd, xie r, klein-nulend j, van rheden re, bronckers al, kuijpers-jagtman am, von den hoff jw, maltha jc. orthodontic force stimulates enos and inos in rat osteocytes. j dent res. 2009; 88(3): 255–60. 7. pearce e. anatomi dan fisiologi untuk paramedis. handoyo sy, editor. jakarta: gramedia pustaka utama; 2006. p. 102-20. 8. greijer ae, van der wall e. the role of hypoxia inducible factor 1 (hif-1) in hypoxia induced apoptosis. j clin pathol. 2004; 57(10): 1009–14. 9. niklas a, proff p, gosau m, römer p. the role of hypoxia in orthodontic tooth movement. int j dent. 2013; 2013: 1–7. 10. adair th, montani j-p. angiogenesis. san rafael (ca): morgan & claypool life sciences; 2010. p. 34. 11. dandajena tc, ihnat ma, disch b, thorpe j, currier gf. hypoxia triggers a hif-mediated differentiation of peripheral blood mononuclear cells into osteoclasts. orthod craniofacial res. 2012; 15(1): 1–9. 12. dai j, rabie ab. vegf: an essential mediator of both angiogenesis and endochondral ossification. j dent res. 2007; 86(10): 937–50. 13. krock bl, skuli n, simon mc. hypoxia-induced angiogenesis: good and evil. genes cancer. 2011; 2(12): 1117–33. 14. andrade i, taddei sra, souza pea. inflammation and tooth movement: the role of cytokines, chemokines, and growth factors. semin orthod. 2012; 18(4): 257–69. 15. richter ae, arruda ao, peters mc, sohn w. incidence of caries lesions among patients treated with comprehensive orthodontics. am j orthod dentofacial orthop. 2011; 139(5): 657–64. 16. jiang r, mcdonald jp, fu m. root resorption before and after orthodontic treatment: a clinical study of contributory factors. eur j orthod. 2010; 32(6): 693–7. 17. ikeda t, yamaguchi m, meguro d, kasai k. prediction and causes of open gingival embrasure spaces between the mandibular central incisors following orthodontic treatment. aust orthod j. 2004; 20(2): 87–92. 18. shenava s, nayak sk, bhaskar v, nayak a. accelerated orthodontics – a review. int j sci c study. 2014; 1(5): 35–9. 19. sukendro s. keajaiban dalam secangkir kopi. yogyakarta: media pressindo; 2013. p. 144. 20. baek kh, oh kw, lee wy, lee ss, kim mk, kwon hs, rhee ej, han jh, song kh, cha by, lee kw, kang m il. association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. calcif tissue int. 2010; 87(3): 226–35. 21. farah a, donangelo cm. phenolic compounds in coffee. brazilian j plant physiol. 2006; 18(1): 23–36. 22. banfi g, iorio el, corsi mm. oxidative stress, free radicals and bone remodeling. clin chem lab med. 2008; 46(11): 1550–5. 23. kenisa yp, istiati i, juliastuti ws. effect of robusta coffee beans ointment on full thickness wound healing. dent j (maj ked gigi). 2012; 45(1): 52–7. 24. herniyati. mekanisme pergerakan gigi ortodonti dan proses remodeling tulang alveolar yang diinduksi gaya mekanis ortodonti akibat pemberian seduhan kopi. universitas airlangga; 2016. p. 99-101. 25. d’apuzzo f, cappabianca s, ciavarella d, monsurrò a, silvestrinibiavati a, perillo l. biomarkers of periodontal tissue remodeling during orthodontic tooth movement in mice and men: overview and clinical relevance. sci world j. 2013; 2013: 1–8. 26. knop lah, shintcovsk rl, retamoso lb, grégio amt, tanaka o. the action of corticosteroids on orthodontic tooth movement: a literature review. dental press j orthod. 2012; 17(6): 20e1-5. 27. schipani e, maes c, carmeliet g, semenza gl. regulation of osteogenesis-angiogenesis coupling by hifs and vegf. j bone miner res. 2009; 24(8): 1347–53. 28. kont a s-a sk a r t, a lt ug m e , k a r ap eh l iva n m, at a k isi e , hismiogullari aa. is cape a therapeutic agent for wound healing? j anim vet adv. 2009; 8(1): 129–33. 29. coultas l, chawengsaksophak k, rossant j. endothelial cells and vegf in vascular development. nature. 2005; 438(7070): 937–45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 96 herniyati, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 91–96 30. karamysheva af. mechanisms of angiogenesis. biochemistry (mosc). 2008; 73(7): 751–62. 31. roberts we, hartsfield jk. bone development and function: genetic and environmental mechanisms. vol. 10, seminars in orthodontics. 2004. p. 100–22. 32. kawakami m, takano-yamamoto t. local injection of 1,25d i hyd roxy vit a m i n d3 en ha nce d bone for mat ion for toot h stabilization after experimental tooth movement in rats. j bone miner metab. 2004; 22(6): 541–6. 33. huang h, ma l, kyrkanides s. effects of vascular endothelial growth factor on osteoblasts and osteoclasts. am j orthod dentofac orthop. 2016; 149(3): 366–73. 34. meikle mc. the tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after carl sandstedt. eur j orthod. 2006; 28(3): 221–40. 35. yamaguchi m. rank/rankl/opg during orthodontic tooth movement. orthod craniofac res. 2009; 12(2): 113–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p91–96 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p91-96 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. subject index volume 47 discoloration, 37 early malocclusion, 92 early childhood caries, 186 elderly, 57 enamel, 206 esthetic line, 220 estrogen level, 177 eugenol, 198 excision, 77 expression, 135 extracellular polymeric substance, 103 fgf2, 126 fiber reinfoced composite, 52 fibroblasts, 130, 215 flexural, 52 fluoride, 211 garlic, 215 gingival crevicular fluid, 25, 141 glass ionomer, 194 cements, 121, 190 glass transitional temperature, 173 glucose, 181 guinea pigs, 25 hand-wrist maturation index, 67 high molecular chitosan nanoparticles (hmcn), 63 hsp 27, 41 hsp60, 7 hypoxia, 41 ifn-γ, 7 immersion time, 168 immunopathobiogenesis, 7 impacted, 158 maxillary, 77 indirect pulp capping, 190 indonesian deutero-malayid, 67 inhibitory concentration, 164 initial acquisition, 202 intermaxillary elastic, 98 iron, 103 isolated and culture techniques, 115 ki-67, 135 lactobacillus acidophillus, 13, 82 lactoferrin, 141 lower lip, 220 mass change, 173 menarche, 67 menstruation, 177 mesenchymal stem cells, 41 micro leakage, 194 hardness, 206 achasin, 31 achatina fulica, 31 acrylic resin, 173 adhesive interface, 121 aggregatibacter actinomycetemcomitans, 103 algyrogel, 130 alkaline phosphatase, 25 allium sativum, 215 aloe vera, 1 anterior posterior cross bite, 98 antimicrobial, 31 activity, 198 anxiety, 87 apoptosis, 41 aspirin, 135 australian wire, 168 autism, 146 band intensity, 141 bifidobacterium bifidum, 82 biofilm, 103 bleaching, 206 bone remodeling, 19 synthesis, 130 bottle feeding, 186 candida albicans, 164 caries, 202 cariogram, 45 casein phosphopeptide–amorphous calcium phosphate, 110 caspase-9, 41 cavia cobaya, 19 cd-8, 7 cell-culture, 215 central incisor, 77 characterization, 115 child, 146 children, 82, 87, 110, 190, 202 chitosan, 19 high molecule, 121 chlorhexidine, 211 chromium ion, 168 chronologic age, 72 computer software, 45 cpp-acp, 206 cytotoxicity, 130 dental caries, 7, 146, 181 maturity, 72 plaque, 153 treatment, 87 dermijian method, 72 mixed dentition period, 72 modifications, 115 mouthwash, 211 mutans streptococci, 82, 202 mytimacin-af, 31 naf, 206 nano hybrid composite, 37 nf-κβ, 1, 13 nickel ion, 168 nystatin, 164 occlusal vertical dimension, 92 odontoblast-like cells, 13 operative-rehabilitative treatment, 92 oral health, 57, 146 oral mucosal epithelial thickness, 135 orthodontic fixed appliance, 158 orthodontic force, 177 treatment, 77 ovulation, 177 parental knowledge, 146 pediatric caries predicator, 45 periapical granuloma, 7 periodontitis, 141 polyethylene, 52 porphyromonas gingivalis, 211 position, 52 povidone iodine, 211 premature loss, 77 preschool children, 186 prevalence, 186 primary molar, 190 teeth, 194 probiotic yoghurt, 82 propolis, 198 extracts, 13 quality of life, 57 rapid maxillary expansion, 98 relapse distance, 25 rice husk ash nanoparticles (rhan), 63 saliva, 110 scanning electron microscopy, 63, 121 severe early childhood caries, 92, 186 sensitivity, 45 shed, 115 siwak, 153 toothpaste, 153 social learning, 87 soda beverages, 37 sodium fluoride, 110 specific protein, 103 specificity, 45 spirulina, 19 stainless steel crown, 92 stainless steel, 168 stichopus hermanii gel, 126 streptococcus mutans, 110, 181, 211 sucrose, 164, 181 surgical exposure, 158 tgfβ-1, 13 tissue healing, 19 tooth eruption, 72, 202 extraction socket, 1 movement, 177 toxicity, 215 trauma, 77 traumatic ulcer, 126 tweed triangle, 220 upper lip, 220 vertebrae maturation index, 67 volumetric, 52 wistar mice, 135 xenograft, 1 xylitol, 164, 181 young permanent tooth, 206 zinc oxide, 198 authors index volume 47 adinda, citra, 202 agustina, dewi, 57 ameliana, yemy, 198 arinawati, dian yosi, 135 bibi, dewi anggreani, 82 bramanti, indra, 153 cilmiaty, risya, 7 dewati, retno, 98 djajusman, sarah kartimah, 164 effendi, m. chair, 37 emilda, yulie, 215 fajriani, 110 hendrayanti w, marchella, 103 herdiyati n., yetty, 146 kresnoadi, utari, 1 kuswandari, sri, 72 liwang, budianto, 206 madyarani, dita, 194 mafranenda dn, herluinus, 31 mardiati, endah, 67 melati, bingah fitri, 77 metalita, murtia, 190 mulyani, sri wigati mardi, 41 oktaviona, intan, 220 pudyani, pinandi sri, 25 puspitasari, tri wijayanti, 115 quroti a’yun, 45 rasyid, nolista indah, 168 rostiny, 19 saktiyawardani, stefany elan, 130 salim, sherman, 173 sari, rima parwati, 126 septommy, catur, 52 setiawan, arlette suzy, 87 silalahi, pretty farida sinta, 63 sinaredi, betadion rizki, 211 susilo, sonya grecila, 177 susilowati, 181 sutjipto, rahel wahjuni, 186 sutrisman, henny, 121 wati, sisca meida, 141 widjiastuti, ira, 13 widyagarini, amrita, 92 wijaya, syeh brata, 158 thanks to editor dental journal (majalah kedokteran gigi) volume 47 number 1 march 2014: 1. prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry-universitas airlangga) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) 3. prof. dr. jenny sunariani, drg., ms (oral biology – universitas airlangga) 4. achmad gunadi, drg., ms., ph.d., sp.pros. (prosthodontics – universitas jember) 5. dr. rini devijanti, drg., m.kes. (oral biology – universitas airlangga) 6. kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) 7. david buntoro k, drg., mds., sp.bm (oral and maxillofacial surgery – universitas airlangga) volume 47 number 2 june 2014: 1. prof. dr. hj. roosje rosita oewen, drg., sp.kga (pediatric dentistry – universitas padjadjaran) 2. prof. dr. al supartinah, drg., su., sp.kga (pediatric dentistry – universitas gadjah mada) 3. prof. dr. drg. regina titi christinawati, m.sc (oral biology – universitas gadjah mada) 4. maretaningtias dwi ariani, drg., m.kes., ph.d., sp.pros (prosthodontics – universitas airlangga) volume 47 number 3 september 2014: 1. endanus harijanto, drg., m.kes. (dental material – universitas airlangga) 2. dr. intan nirwana, drg., m.kes. (dental material – universitas airlangga) 3. dr. theresia indah budhy, drg., m.kes. (oral pathology and maxillofacial – universitas airlangga) 4. dr. retno indrawati, drg., m.si (oral biology – universitas airlangga) volume 47 number 4 december 2014: 1. dr. ib. narmada, drg., sp.ort(k) (orthodontics – universitas airlangga) guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. vol 38 no 3 2005 112 gigi tiruan sebagian lepasan sebagai benda asing dalam trakea (removable partial denture as foreign body in trachea) sri herawati jpb bagian ilmu kesehatan telinga hidung tenggorok (tht), rsu dr. soetomo surabaya indonesia abstract foreign bodies in the trachea are not uncommon. usually the patients come with dyspnea and a history of having aspirate something. this is an emergency case and needs extraction of the foreign body via bronchoscopy with general anesthesia as soon as possible. the optimal preparation is a controlled, well-equipped and well prepared operative setting. a case of removable partial denture as foreign body in trachea was reported and the extraction of the denture was done with difficulty due to the size of the denture compared with the width of the rima glottis and the trachea. key words: trachea, bronchoscopy, foreign body, denture korespondensi (correspondence): sri herawati jpb, bagian ilmu kesehatan telinga hidung tenggorok (tht), rsu dr. soetomo. jln. kertajaya indah tengah 6/14 (h-208), surabaya, indonesia. pendahuluan benda asing di jalan napas dapat terjadi pada orang dewasa maupun anak-anak. jenis benda asing pada orang dewasa berbeda dengan anak-anak. pada orang dewasa yang sering adalah jarum pentul, tulang dan gigi tiruan sebagian lepasan, sedangkan pada anak-anak adalah kacang.1-3 penderita dengan aspirasi benda asing di jalan napas merupakan kasus yang gawat darurat dan memerlukan tindakan yang segera yaitu ekstraksi benda asing dengan menggunakan forsep secara bronkoskopi dengan pembiusan umum.1–4 penderita biasanya datang sudah dalam keadaan sesak napas yang berat ringannya tergantung pada besar kecilnya benda asing.3,4 berikut ini akan dilaporkan seorang penderita dengan aspirasi gigi tiruan sebagian lepasan (gtsl) yang berhasil dilakukan ekstraksi melalui bronkoskopi segera (cito) dengan pembiusan umum. kasus seorang penderita laki-laki berumur 42 tahun asal dari surabaya, datang ke ird rsu dr. soetomo surabaya dengan keluhan tertelan gtsl 2 jam sebelum ke rumah sakit, pada waktu penderita tidur. pada anamnesis didapatkan penderita terbangun dari tidurnya karena batuk hebat. kemudian disadari oleh penderita bahwa gtslnya sudah tak ada dan penderita mengira gtsl tersebut tertelan waktu tidur. penderita mengeluh sesak napas dan ada yang tersangkut dalam jalan napasnya, makan, minum tak ada keluhan. gigi tiruan sebagian lepasan (gtsl) ini sudah 6 tahun dipakai, tidak pernah dilepas waktu tidur dan tidak pernah kontrol ke dokter gigi, karena merasa tidak pernah diberitahu. dua bulan yang lalu gtsl ini pernah patah dan kemudian setelah di lem dapat dipakai lagi. tiga hari yang lalu gtsl patah lagi tapi masih dapat dipakai walaupun terasa longgar. penderita bermaksud ke dokter gigi setelah punya biaya. pada pemeriksaan fisik didapatkan sesak napas, stridor, retraksi pada suprasternal, supraklavikular dan interkostal, tetapi tidak didapatkan sianosis atau suara parau. pada foto leher lateral (gambar 1) tampak ada bayangan benda di trakea, sedangkan pada foto leher postero-anterior tak jelas ada kelainan dan foto toraks tak ada kelainan. segera dipersiapkan bronkoskopi dengan anestesi umum. gambar 1. benda asing tampak pada foto leher lateral. 113herawati: gigi tiruan sebagian lepasan tatalaksana kasus bronkoskopi dikerjakan satu jam kemudian. pada bronkoskopi, yang menggunakan bronkoskop ukuran 6 × 24 cm, didapatkan korda vokalis sedikit edema dan kemerahan (hiperemi), sedangkan pada trakea tampak benda asing pada jarak ± 1,5 cm di bawah korda vokalis. ekstraksi gtsl dengan forsep gigi satu gagal karena ada bagian yang menyangkut pada rima glotis sehingga gtsl terlepas dari forsep yang memegangnya. usaha berikutnya dilakukan dengan menggunakan tipe forsep yang lain (aligator) dengan ukuran yang berbeda. usaha ini beberapa kali harus dihentikan karena keadaan penderita memburuk yaitu terjadi bradikardia, apnea atau sianosis, sehingga bronkoskop harus dikeluarkan dahulu dan jalan napas diserahkan kepada anestesi untuk memperbaiki keadaan umumnya. akhirnya pada usaha yang keempat, didapatkan ukuran bronkoskop dan forsep (gambar 2a) yang sesuai, sehingga benda asing dapat diekstraksi keluar dari trakea dan melewati rima glotis bersama dengan bronkoskop dan forsepnya. pada bronkoskopi ulangan didapatkan lesi pada trakea jam 6 dan jam 12 serta lesi minimal pada hipofaring dan uvula, ternyata benda asingnya (gambar 2b) berupa 1 buah anasir gigi insisivus kiri atas dengan basis akrilik tanpa kawat pengait sebesar 3 × 2 × 1,5 cm. perdarahan aktif tak ada. lamanya bronkoskopi adalah 55 menit. pasca operasi diberikan antibiotik dan kortikosteroid. penderita dipulangkan 2 hari kemudian dalam keadaan baik. pembahasan aspirasi dan tertelan benda asing lebih sering terjadi pada anak daripada orang dewasa.3,5 aspirasi atau tertelan benda asing perlu dibedakan mulai dari anamnesis, sebab aspirasi benda asing berarti benda asing masuk ke dalam jalan napas dan ini berarti perlu tindakan bronkoskopi yang segera (cito), sedangkan tertelan benda asing berarti benda asing masuk ke dalam jalan makanan yang memerlukan tindakan esofagoskopi yang bersifat urgent, yang berarti tidak perlu segera dikerjakan dan dapat ditunda dalam waktu 24 jam.1–4 gejala klinis yang tampakpun berbeda, yaitu pada aspirasi gejala klinisnya adalah sesak napas dan batuk hebat sedangkan tertelan benda asing gejala klinis lebih ke arah sulit makan dan minum.4 gejala klinis juga seringkali bervariasi, tetapi bila ada anamnesis tertelan atau aspirasi benda asing, perlu segera dilakukan pemeriksaan selanjutnya tanpa melihat usia atau ada tidaknya gejala klinis yang jelas untuk memastikan ada tidaknya dan letak benda asing yang tersangkut, di jalan napas atau di jalan makanan.2 pada penderita ini, pada waktu datang di ird rsu dr. soetomo mengatakan tertelan gtsl, tetapi pada pemeriksaan tampak penderita sesak napas, batuk hebat yang timbul segera setelah aspirasi dan pada foto leher lateral tampak bayangan radiopaque pada trakea, sehingga diagnosis menjadi dugaan benda asing pada trakea. diagnosis pasti ditegakkan setelah melakukan bronkoskopi.6 menurut literatur, benda asing di trakea jarang terjadi yaitu hanya sekitar 10–20% dari benda asing di jalan napas, sedangkan lokasi yang tersering adalah pada bronkus (80–90%).1,3,6 pada penderita ini, karena besar dan bentuknya, benda asing tertahan sekitar 1,5 cm di bawah pita suara. peranan radiografi dalam menentukan lokasi benda asing sangat tergantung pada sifat radiodensitas dari benda asingnya. bila benda asing bersifat radiopaque atau mempunyai bahan atau komponen yang radiopaque, foto biasa (plain radiograph) sangat berguna, tetapi bila benda asingnya bersifat radiolucent, tidak akan tampak pada foto biasa sehingga memerlukan ct scan.6 selain tergantung dari radiodensitas benda asingnya, pembuatan foto juga gambar 2. a) alat bronkoskopi; b) benda asing gtsl setelah ekstraksi. a b 114 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 112–114 tergantung dari keadaan penderita. bila penderita dalam keadaan sesak berat sampai sianosis, bronkoskopi tak perlu ditunda hanya karena menunggu pembuatan foto, sebab penyelamatan jiwa (life-saving) lebih penting. pada penderita ini, gambaran benda asing tak jelas tampak tetapi jelas ada sesuatu pada trakea. penanganan aspirasi benda asing adalah ekstraksi benda asing melalui bronkoskopi dengan anestesi umum. penggunaan bronkoskopi kaku (rigid), sampai sekarang masih lebih diunggulkan dibandingkan dengan bronkoskopi fleksibel, terutama bila benda asingnya besar.1,2,7 keberhasilan ekstraksi tergantung dari bentuk dan besar benda asing dan lamanya aspirasi yang akan menyebabkan terbentuknya granulasi di sekeliling benda asing.7 keberhasilan ekstraksi juga tergantung pada kelengkapan alat endoskopi yang tersedia antara lain bermacam ukuran bronkoskop dan bentuk, ukuran serta tipe forsep yang bervariasi.2 pada penderita ini, kesulitan ekstraksi disebabkan karena bentuk dan besar gtsl, sehingga walaupun benda asing segera dapat ditemukan dan dapat dengan mudah dipegang oleh forsep, tetapi pada waktu mengeluarkan atau melewati rima glotis yang merupakan celah yang berbentuk segitiga, beberapa kali terlepas. hal ini diperberat d e n g a n p i t a s u a r a y a n g s u d a h e d e m a, s e h i n g g a diperlukan ukuran bronkoskop dan forsep yang lebih kecil dari yang seharusnya. disamping itu karena adanya bagian yang tajam menyebabkan terjadinya lesi pada trakea dan hipofaring, sedangkan bradikardi, apnea dan sianosis yang terjadi selama operasi, oleh karena harus menunggu anestesi untuk memperbaiki keadaan umum penderita sehingga makin memperpanjang waktu lamanya proses ekstraksi. penyebab aspirasi gtsl ini tampaknya karena kelalaian penderita yang karena tidak mengerti akan bahaya serta kurangnya pengetahuan tentang perawatan gtsl yang memerlukan kontrol teratur ke dokter gigi dan dilepas waktu tidur, disamping itu juga karena faktor ekonomi, yang tampak dari usahanya menyatukan kembali akrilik yang patah dan memakainya kembali akrilik yang sudah patah tersebut. pada kasus ini, dapat disimpulkan bahwa gtsl sebagai benda asing dalam trakea merupakan kasus gawat darurat di bidang jalan napas yang memerlukan tindakan segera (cito) karena dapat berakibat fatal. hal tersebut dapat dicegah dengan pemberian pemahaman yang baik pada penderita untuk tetap kontrol yang teratur setelah pemasangan gtsl, dan melepas gtsl pada waktu malam atau tidur. daftar pustaka 1. giannoni cm. foreign body aspiration. 1994. available at: http:// www.bcm.edu/oto/ grand/31094.html. accessed march 18, 2005. 2. stroud rh. foreign bodies of the upper aerodigestive tract. 1997. available at: http://www.utmb.edu/otoref/grnds/forbody.html. accessed march 18, 2005. 3. munter dw, gelford b. foreign bodies, trachea. department of emergency medicine, united states naval hospital at okinawa, 2001 agust; available at: http://www.emedicine.com/emerg/ topic751.htm. accessed october 20, 2003. 4. toliver r. airway foreign body. 2004. available at: http:// www.emedicine.com/radio/ topic19.htm. accessed december 24, 2004. 5. murthy psn, ingle vs, edicula g, ramakrishna s, shah fa. sharp foreign bodies in the tracheobronchial tree. american journal of otolaryngology 2001; 22:154–6. 6. dibiase at, samuels rha, ozdiler e, akcam mo, turkkahraman h. hazards of orthodontics appliances and the oropharynx. journal of orthodontics 2000; december; 27(4):295–302. 7. yang tsung-lin, hsu mow-ming. occult foreign body aspiration in adults. journal otolaryngology-head and neck surgery 2003 january; 128(1). available at: http://www2.us.elsevierhealth.com/ scripts/om.dll. accessed may 28, 2004. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 91 vol. 43. no. 2 june 2010 research report enamel defect of deciduous teeth in small gestational age children willyanti s syarif�, roosje r. oewen�, sjarif h. effendi�� and bambang sutrisna�� 1 faculty of dentistry, padjajaran university 2 faculty of medicine, padjajaran university 3 faculty of medicine, pelita harapan university abstract background: enamel defect could be caused by genetic and environmental factors in prenatal period. meanwhile, prenatal malnutrition could also cause small gestational age (sga). small gestational age is the term used for a neonatal baby with birthweight below the -2sd normal value or 10th percentile on the intrauterine lubchenco curve. this condition is due to intra-uterine growth restriction, and eventually ends up with several developmental defects of organs, including teeth. in fact, deciduous tooth development has a critical phase within this development period. purpose: the aim of this study is not only to find out the incidence of enamel defect in sga children, but also to know the percentage of sga risk factor to develop enamel defect. method: this was a epidemiology research with consecutive admission technique. it consisted of 153 sga children aged 9–48 months. next, the ponderal index was used to assign sga types, symmetrical or asymmetrical one-in this study 59 and 94 respectively. on the other hand, three hundred and ninety appropriate for gestational age (aga) children aged 4–48 months were also included in the study as a control group. enamel defect then was determined by intraoral examination, classified into hypoplasia and hypocalcifications. chi-square test was finally used to determine the relative risk ratio between the sga and the control aga children. result: the result of this research showed that incidence of enamel defect in sga children was 86.92%, meanwhile, that in aga children was 23.08%, 66.00% of which were commonly suffered from hypocalcification. with p<0.05 it is also known that sga children has the risk of enamel defect with hypocalcification, about 79% higher than aga children. conclusion: it could be concluded that 79% of sga children had the risk of deciduous tooth enamel defect with hypocalcification as the most. key words: enamel defect, small gestational age, intrauterine growth restriction abstrak latar belakang: defek email dapat terjadi karena faktor genetik dan lingkungan sistemik yang terjadi saat prenatal. adanya malnutrisi prenatal dapat mengakibatkan kelahiran bayi dengan kecil masa kehamilan. kecil masa kehamilan (kmk) adalah bayi dengan berat badan lahir di bawah -2sd nilai normal atau dibawah persentil 10 kurva pertumbuhan intra uterin lubchenco. kondisi ini terjadi sebagai akibat intra uterine growth restriction (iugr) yang mengakibatkan defek perkembangan organ tubuh, termasuk gigi sulung karena masa kritis pertumbuhan dan perkembangan gigi sulung terjadi pada periode prenatal. tujuan: tujuan penelitian ini adalah untuk mengetahui insidensi defek email gigi sulung pada anak kmk dan mengetahui besarnya risiko kmk untuk terjadinya defek email pada gigi sulungnya. metode: disain penelitian adalah epidemiologi dengan consecutive admission. sample terdiri dari 153 anak kmk berusia 9–48 bulan. diperoleh 59 tipe simetri dan 94 tipe asimetri dengan indeks ponderal. sebagai kontrol diperiksa 390 anak sesuai masa kehamilan (smk) berusia 4–48 bulan. pemeriksaan intra oral dilakukan untuk melihat ada tidaknya defek. tipe defek adalah hipoplasia dan hipokalsifikasi. uji chi-kuadrat digunakan untuk menguji risiko relatif defek email pada anak kmk dan smk. hasil: hasil penelitian menunjukkan insidensi defek email pada anak kmk sebesar 86,92% dan pada anak dengan sesuai masa kehamilan (smk) sebesar 23,08%, dengan jumlah defek terbanyak adalah hipokalsifikasi sebanyak 66,60%. dengan p < 0,05 anak kmk berisiko 79% untuk mengalami defek email gigi sulung. kesimpulan: dari penelitian ini disimpulkan bahwa anak kmk berisiko 79% mengalami defek email gigi sulung dengan tipe defek terbanyak adalah hipokalsifikasi. kata kunci: defek email, kecil masa kehamilan, intra uterine growth restriction correspondence: willyanti s syarif, c/o: fakultas kedokteran gigi universitas padjajaran. jl. sekeloa selatan i bandung, indonesia. e-mail: willyantir@yahoo.com 92 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 91-96 introduction enamel is a structure that cannot be remodeled. it means that if there is any defect occurred on the enamel, it will be considered as a permanent. genetic and environmental factors, either systemic or local, can actually cause enamel defect at the stages of histodifferentiation, morphodifferentiation, aposition, and classification during the first, second, or third trimester of prenatal period with hypoplasia or hypocalcification depended on the time the defect occured.1–6 enamel defect caused by systemic factor, usually effect entire teeth, meanwhile the one caused by local factor does not effect entire, but unilaterally. meanwhile, environmental factor in prenatal period disrupting the growth and development of deciduous teeth is intra uterine growth restriction (iugr) causing small for gestational age (sga) baby. the defect then can easily cause caries because of the accumulation of plague. if this defect occurred on deciduous teeth is not prompthy treated, it might cause early teeth extracted soon. this condition then affects their chewing function and aesthetics, so later it can cause malnutrition that can disrupt not only the growth and development of jaws, but also the development of psychology and health in general, causing the disruption of later growth and development processes entirely.2,7 sga babies, furthermore, are those whose birth weight was below the -2 sd normal value or 10th percentile on the intra-uterine lubchenco curve, and those whose birth are premature (<37 weeks of pregnancy), normal, or even mature (more than its appropriate months of pregnancy).8–11 sga is actually caused by disturbances that occurred during the development of intrauterine growth restriction (iugr), which is the restriction of prenatal development in wombs.12–14 there are two types of sga, symmetric type with disturbances occurred at the beginning of the first trimester of pregnancy and asymmetric type with problems occurred at the second or third trimester.8–12,15 the recorded incidence of sga babies in dr. hasan sadikin hospital in 2005, were about 7.6–10%, meanwhile in usa it was known about 3–10% of all births.16,17 sga is actually caused by intrauterine growth restriction (iugr) that can be caused by maternal factors during pregnancy, placenta.2 maternal factors involves the mothers' age above 35 years old or at teenage period; the physical appearance of mothers that were short and thin; the none or slow increasing of mother's weight during the third trimester can cause malnutrition. other problems are vascular disease, severe infection during pregnancy, erythematous lupus syndrome, antiphospholipid syndrome, anemia, severity, lack of health service during pregnancy, nuliparity, smoking habit, alcohol consuming, cocain consuming, living in plateaus, and low social-economy status. furthermore, there are prenatal factors such as genetic abnormality and chromosome abnormality, abnormality of infant's placenta and tumor.8–12,15 besides that, there are also defects in the development of many organs, including the development of teeth that can be caused by iugr since prenatal period is considered as critical period for the development of deciduous teeth.1–4 in addition, previous studies relating enamel defect with low birth weight babies (lbwb) and premature birth show that children with premature birth and lbwb have higher risk of oral abnormality like enamel hypoplasia, hypocalsification, dental discoloration, abnormal dental structure, palatal groove, and delayed dental eruption.1,18–19 the prevalence of enamel defect in deciduous teeth, about 20–100%, even is suffered by children with the history of premature birth and lbwb.1–7,20–28 besides the retardation of deciduous tooth eruption, sga children also suffer enamel defect.5 it indicates that the growth of deciduous teeth in sga children are disrupted. therefore, this study was aimed to analyze the incidence and risk of enamel defect in sga children. material and method the subject of this study were 9–48 months-old children born in dr. hasan sadikin hospital bandung with the history of small for gestational age (sga). on the other side, as control group were 4–48 months appropriate for gestational age (aga) children, but not suffering caries. it means that the age range in sga subjects is different from that in aga subjects since unlike aga children, sga children suffer delayed eruption.15 the inclusion criteria, are thechildren must be 9–48 months old classified as sga subjects, and must be 4–48 months old classified as aga children; that the data of mothers and children must be completed; and that children must have abnormal genetics or syndrome as exclusion criterion. enamel defect then is determined by whether there is hypoplasia or hypocalsification or not. meanwhile, types of sga were determined by measuring the ponderal index with the following formulation: 3 100 heightbirth xweightbirth − − intraoral examination then was conducted through several stages: at first, the inform consent was fulfilled; secondly, dental examination was conducted with enough lighting like lamp mirror, and then teeth were cleaned and dried with cotton. hypoplasia actually can be determined if pit and fissure can be seen, and if there is also partially lost enamel. meanwhile, hypocalcification actually can be determined by examining whether enamel can be penetrated by light or not, oral examinations is done three times with 3 month interval; thirdly, next structured interview is conducted by matching with the birth history; fourtly, the obtained data then was tabulated into dummy table. chisquare test was finally used to analyze the difference of risk in sga children and in aga ones. 93syarif: enamel defect of deciduous teeth result the number of sga children as patients of dr. hasan sadikin hospital in bandung were about 184 children, 13 of whom were dead and 18 were not identified for the address, so only 153 of them were listed as subject of this study, 94 of whom were classified into asymmetric sga, while 59 children were classified in symmetric sga at the age 9–48 months. on the other side, 390 aga children at the age of 4–48 months were listed as the control group. the incidence of enamel defect in sga children is about 86.92%, higher than that in aga children (23.08%), meanwhile that of non enamel defect in sga children is about 13.07%. similarly, the incidence of enamel defect in sga children (86.92%) is higher than that in aga children (23.08%) since those sga children have suffered intra-uterine growth restriction (iugr) that eventually cause several developmental defects of teeth, like enamel defect (figure 1). moreover, shows that the highest incidence of enamel defect types in sga children is hypocalcification (66.60%), meanwhile hypoplasia is about 4.60%, and non defect is about 13.10%. on the other side, in aga children hypocalcification is only about 23.10%, non defect is about 76.90%, and hypoplasia is 0% (figure 2). thus, it indicates that the hypocalsification incidence of enamel defect in aga children (23.10%) is lower than that in sga children since hypocalcification in aga children is caused only by local factors. meanwhile, that the higher incidence of hypocalcification in sga children (66.60%), and the lower of hypoplasia (4.60%) shows that the enamel defect suffered by those sga children is not severe because of the lower incidence of hypoplasia, considered as the most severe type of enamel defect, only about 4.5%. relative risk rasio (rrr) in sga children is about 3.79 indicating that the risk of enamel defect in sga children is about 3.79 times high or aboutabout 79% (with the formulation p= r 1+r , so it becomes p= 3.79 1+3.79 = 0.7912 = 79%). then, using chi square test, it is found out that p<0.001 indicating that sga children has bigger risk of enamel defect. in addition, that the incidence of symmetric sga is about 100%, meanwhile that of asymmetric sga is about 78.72%, and that of non enamel defect is 0%. the reason for all of those symmetric sga subjects suffer enamel defect is because those symmetric subjects suffer the problem earlier, at the beginning of the first trimester, than those asymmetric ones at the second or third trimester (figure 3). similarly, it can be seen also at table 2 at which the risk of enamel defect in those symmetric subjects is higher than in those asymmetric ones. the biggest incidence of enamel defect types in asymmetric sga children is hypocalcification. the reason why hypocalcification occurs more commonly in asymmetric subjects is because the number of asymmetric subjects is higher than that of symmetric ones. meanwhile, there are no asymmetric sga children suffering hypoplasia. table �. the relative risk of enamel defect in sga children compared with that in aga children type of subject enamel defect total defect non defect n % n % n % sga 133 86.93 20 13.07 153 100,00 aga 90 23.08 300 76.92 390 100,00 total 224 319 543 133 90 rr: + = 0.87 + 0.23 = 3.79; c2: 185.11; p<0.001 153 390 23.08 76.92 86.92 13.07 0 20 40 60 80 100 sga aga subject pe rc en ta ge (% ) defect non defect figure �. incidence of enamel defect in sga and aga children. 94 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 91-96 table ��. the relative risks of enamel defect in symmetric and asymmetric sga children compared with those in aga children sga types rr p symmetric 4.35 <0.001 asymmetric 3.39 <0.001 the risk of enamel defect in symmetric sga children is about 4.35 times high or about 81% (4.35:5.35), meanwhile in asymmetric sga children is about 3.39 times high or about 77% (3.39:4.39) (table 2). it means that symmetric type has higher risk than asymmetric one, about 4.35 times as high as the other one. discussion the incidence of enamel defect in sga children is higher than that in aga children since sga children have problems with the growth and development of their deciduous teeth, one of which is iugr. enamel defect actually consists of hypoplasia and hypocalcification. nevertheless, hypocalcification is the most commonly suffered defect. the reason is because the majority of children studied in this study are classified into asymmetric sga (61.44%) without suffering severe complication. however, those sga children can actually suffer enamel defect, which is usually local enamel defect caused by local factors like trauma. trauma involves stressing of a small group of ameloblast, so the growth of teeth is disrupted, and furthers it causes local defects.10 besides, trauma can also be caused by the use of endotracheal intubation tools in neonatal babies with asphyxia, which is distress of breathing, because of birth delivery factors. nevertheless, manifestation of enamel defect caused by local factors does not affect the teeth entirely, only unilaterally. unlike local factors, systemic factors, such as prenatal malnutrition, prenatal or postnatal infection, involve the higher number of ameloblast, so the defects do not affect the teeth locally, but entirely and bilaterally.21,29,30 besides that, prenatal development is also depended on genetic materials of children (50%), and both intrauterine environment and genetics of mothers (50%). thus, aga children might still suffer/get enamel defects even in lower severity compared to those in sga children.31 the relative risk ratio in sga children is 3.79 times as high as that in aga children. it indicates that the risk of enamel defect in sga children is 3.79 times higher, 4.60 66.60 15.70 13.10 0 23.10 0 76.90 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 hypoplasia hypocalsification hypoplasia + hypocalsification normal persentase (%) types of defect sga aga figure ��. incidence of enamel defect in sga and aga children based on types of defect (hypoplasia, hypocalcification). figure ��. incidence of enamel defect based on types of sga. 0 20 40 60 80 100 120 symmetric sga asymmetric sga types of sga pe rc en ta ge (% ) non defect defect hypocalcification 95syarif: enamel defect of deciduous teeth about 79%, than that in aga children. it means that sga children have problems with the development of their deciduous teeth more commonly.5 this condition is caused by intrauterin growth restriction (iugr) causing sga which can stimulate any defects in many organs, like teeth. this condition is also supported by the fact that the critical development of deciduous teeth occurs at some prenatal phases, like histodifferentiation (9–10 weeks), morphodifferentiation (11–12 weeks), aposition, and calcification (12–16 weeks). as a consequence, abnormal dental structure then might occur at the end of bell stage (<16 weeks) which might later cause any disruption problems during the growth of dental enamel, such as hypoplasia. besides that, if the disruption occurs at calcification phase (>16 weeks), hypocalcifacation will occur.21 therefore, based on the fact that the incidence of enamel defect in sga children with p<0.05 is higher than that in aga children (figure 1), rr of sga children is 3.79 times, and the score of dde in sga children > that in aga children, it indicates that the risk of enamel defect in sga children is higher than that in aga children. in addition, figure 3 shows that the incidence of enamel defect in asymmetric sga children is 78.70%, while that in symmetric sga children is about 100%. the figure also shows that there are 21.28% of asymmetric sga children who do not suffer enamel defect. it means that the percentage of enamel defect in symmetric sga children is higher than that in asymmetric ones. the reason is those who were classified into symmetric type were all suffer problems at age above >8 weeks, whereas below 8 weeks it might cause major defect or even death. it can also be seen from the fact that there were no asymmetric sga children suffering the defect. it might be caused by the fact that symmetric sga children suffered the defect at the beginning of prenatal period (the first trisemester), while asymmetric sga children suffered it furtherly. thus, the highest incidence of hypocalcification about 61.44% is in sga children since it occurred in the middle of the second or third trimester at which the development of deciduous teeth is in the phase of calcification, so any problems at that period can cause hypocalsification.3,32,33 similarly, figure 3 shows that the incidence of enamel defect in symmetric sga children is higher than that in asymmetric sga ones. table 2 also indicates that rrr of symmetric sga children is 4.35 times higher, meanwhile rrr of asymmetric sga is only about 3.39. it means that symmetric sga children have higher risk than the asymmetric sga ones. the reason is because the problems during embryonic period (the first trimester) stimulate worse impacts than those during fetal period (the second and third trimesters).27,34 the reason is because during embryonic period (2–8 weeks) fetuses are more sensitive to problems. during this period the proliferation of cells is actually getting more active, indicating that the increasing number of cells is higher than those of the size of cells. but, the problems occurred during this embryonic period could also decrease the number of cells. unlike this embryonic period, during fetal period, at the age of 16 weeks until delivery (the second and third trimesters), the sensitivity of fetuses against the problems decreased.31 this condition is also supported by the opinions of some experts who stated that symmetric sga children have more severe defect, than asymmetric sga ones.8-11 moreover, it is also known that non enamel defect can be found only in asymmetric sga children. this is because when the growth of intrauterine is disrupted at the third trimester, the process of calcification is almost finished, and consequently, enamel defect does not occur.12,29 as a conclusion, firstly, it is found out that sga children have higher risk of enamel defect, about 79%, than the aga children. secondly, it is also found out that symmetric type of sga has higher risk than asymmetric one. finally, it is found out that enamel hypocalcification is the most commonly found defect. references 1. seow wk, humphrys c, tudehope di. increased prevalence of developmental dental defects in low birth-weight, prematurely born children: a controlled study. pediat dent; 1987; 9(3): 221–5. available at: http://www.aapd.org/. accessed september 13, 2005. 2. mathewson rj, primosch re. fundamentals of pediatric dentistry. 3rd ed. chicago: quintessence publishing co. inc; 1995. p. 78–80. 3. pinkham jr. pediatric dentistry: infancy through adolescence. 3rd ed. philadelphia: wb saunders co; 2005. p. 61–4. 4. stewart re, barber tk, tr. index for use. in: eutmen kc, wie shy, editors. pediatric dentistry. st louis: cv mosby co; 1982. p. 87–109, 900–7, 1005. 5. sjarif w, oewen r. erupsi gigi sulung pada anak dengan kecil masa kehamilan. majalah kedokteran gigi (dental journal) 2005; edisi khusus pin ikga i: 14–7. 6. maroney di. dental problems related to prematurity. 2001. available at: www.comeunity.com. accessed august 7, 2007. 7. welburry rr. pediatric dentistry. 2nd ed. new york: oxford university; 2001. p. 294, 394–5. 8. klauss mh, fanaroff aa. care of the high risk neonate. 4th ed. philadelphia: wb. saunders co; 2003. p. 69–83. 9. gomella tl, cunningham d, eyal fg, zenk ke. neonatology, management, procedures, on-call problems, diseases, and drugs. 5th ed. new york: mc graw hill; 2004. p. 21–8, 469. 10. anderson ms, hay ww. intrauterine growth restriction and the small for gestational age infant. in: avery gb, flecther ma, mac donald mg, editors. neonatologi, pathophysiology and management of the newborn. 5th ed. philadelphia: lippincott william & wilkins; 1999. p. 411–7. 11. hay ww, thureen p, anderson ms. intrauterine growth restriction. neoreviews 2001; 2: 119–28. available at: http://neoreviews. aappublications.org/cgi/content/full/neoreviews;2/6/e139. accessedaccessed oktober 27, 2008. 12. battaglia f, lubchenco l. a practical classification of newborn infants by weight and gestational age. j pediatr 1967; 71: 159. 13. laskaris g. color atlas of oral diseases in children and adolescents. stuttgart: thieme; 2000. p. 20-3. 14. seow wk. effect of preterm birth on oral growth and development. australian dental journal 1997; 42(2): 85–91. available at http:// www.aapd.org/. accessed september 27, 2005. 15. cunningham fg, leveno kj, bloom sl, hauth jc, gilstrap l, wenstrom kd. williams obstetrics. 22nd ed. new york: mcgraw hill; 2005. p. 744–64. 16. alkalay al, graham jr jm, pomerance jj. evaluation of neonatesevaluation of neonates born with intrauterin growth retardation: review and practice guidelines. j perinatol 1998; 18: 142–51. 96 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 91-96 17. perinatologi rsup dr. hasan sadikin: laporan tahunan perinatologi 2005. 18. seow wk, perham s, young wg, daley t. dilaceration of a primary maxillary incisor associated with neonatal laryngoscopy. pediat dent 1990; 12(5): 321–4. 19. grahnen h, larsson pg. enamel defects in the deciduous dentition of prematurely born children. odontol revy 1958; 9: 193–204. 20. avery jk, chiego dj. essentials of oral histology and embryology: a clinical approach. 3rd ed. st louis: mosby inc; 2006. p. 63–75. 21. balogh mb, fehrenbach mj. dental embryology, histology, and anatomy. 2nd ed. st louis: elsevier saunders; 2006. p. 63–79. 22. funakoshi y, kushida y, hieda t. dental observation of low birth weight infants. pediatric dentistry. 1981; 3(1): 21–5. 23. mellander m, noren jg, freden h, kjellmer i. mineralization defects in deciduous teeth of lowbirth weight infants. acta pediatr scand 1982; 71(5): 727–33. 24. seow wk. oral complications of premature birth. aust dent j. 1986; 31: 23–9. 25. seow wk. a study of the development of the permanent dentition in very low birthweight children. pediat dent 1996;18(5): 379–84. 26. drummond bk, ryan s, o'sullivan ea, congdon p, curzon mej. enamel defect of the primary dentition and osteopenia of prematuriry. pediat dent. 1992; 14(2): 119–21. available at: http://www.aapd.org/. accessed september 30, 2005. 27. lai py, seow wk, tudehope di, rogers y. enamel hypoplasia andenamel hypoplasia and dental caries in very-low birthweight children. a case-controlled, longitudinal study. pediat dent. 1997; 19: 42–9. available at: http:// www.aapd.org/. accessed september 13, 2005. 28. simmer jp. dental enamel formation and its impact on clinical dentistry. j dent education. 2001 65(9): 896-904. 29. eastman dl. dental outcomes of preterm infants. nbin 2003; 3(3): 93–8. available at: http://www.medscape.com/viewarticle/461574. accessed augusts 8, 2006. 30. jorgenson rj, yost c. etiology of enamel dysplasia. journal of pedodontics, summer. 1982; 315–467. 31. fisk nm, smith rp. fetal growth restriction; small for gestational age. in: chamberlain g, steer p, editors. turnbull's obstetrics. 3rd ed. london: churchill livingstone; 2001. p. 197–209. 32. stewart re, witkop cj, bixler d. the dentition. in: stewart re, barber tk, troutman kc, wei shy, editors. pediatric dentistry, scientific foundation and clinical practice. st. louis: cv mosby co; 1982. p. 87–94. 33. mcdonald re, avery dr. dentistry for the child and adolescent. 6th ed. st louis: cv. mosby year-book inc; 1994. p. 53–9. vol 38-no4-2005-isi.pmd 159 problem in the surgical correction of long-face with vertical open bite coen pramono d department of oral and maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract long-face cases usually need both treatment of orthodontic and surgery. the problem appearing in the correction of long-face might be able to be related with some difficult factors such as the crowded teeth and excessive vertical height. a class iii malocclusion and excessive open bite can be also followed in long face. this situation might worsen the facial aesthetic condition and increase the difficulty in orthodontic treatment. the orthodontic approach is oriented toward positioning the teeth pre-surgically to facilitate the surgical plan. the form of mandible which has grown in the downward direction in the area of mandible angle makes an extreme vertical open bite. the maxilla is usually presented with a maxillary hypolasia. double-jaw surgery was done as the correction of the lower jaw alone would produce a flattened face appearance and difficulty in repositioning the mandible to achieve a good facial performance. several cephalometric points were measured to observe the facial situation progress after surgery. two cases of longface are reported, and the same surgical treatments were performed and showed different results. key words: long face surgical correction, excessive vertical hight, occlusal plane angle korespondensi (correspondence): coen pramono d, c/o: bagian ilmu bedah mulut, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction tremendous advances in the area of orthognathic surgery for the treatment of dentofacial deformities have been made since the 1970s. fonseca1 reported the increased number of patients require surgical correction with an assumption 580.000 individuals in united states have a severe class iii malocclusion, with some 12,000 cases being added each year. trends in class iii surgical treatment indicate that fewer isolated mandibular setbacks, predominant procedure in the 1970s, are being done for correction of this type of malocclusion.1 a recent study has reported an increased number of two-jaw and maxillary advancement cases for correction of class iii dentofacial deformities.2 proffit et al.3 in 1990 reported approximately 220,000 individuals with long-face problem severe enough to warrant surgery, with some added to the population annually. a long-face patient can be described as skeletal class i rotated to class ii or as skeletal class iii rotated to class i. the primary distinguishing characteristic of a long face is a large total face height that is manifested almost entirely in elongation of the lower third, leading to disproportion between facial height and width. a major component of the problem is nearly always an inferior rotation of the posterior maxilla. as the face height increases and the maxilla palatal plane rotates down posteriorly and posterior teeth move down, the mandible tends to rotate downward and backward. for this reason, the vertical disproportion also affects anteroposterior (ap) jaw relationship.1 the growth of the mandible rotates down and back during the development of long face condition and this rotation therefore separates the incisors vertically, creating an open bite tendency. in long face, a skeletal correction is necessary to be performed as the malocclusion existed due to two factors as teeth mal-alignment and abnormality of bone growth pattern both in upper and lower jaws. preorthodontic treatment before surgery is obligatory as this treatment sequence has an objective to align each tooth position both in mandible and maxilla and to parallel the curve of spee in both jaws. these steps are considered to be highly important as it has a main purpose to achieve jaws occlusion during surgical reposition. correction of the lower incisors which has tipping lingually as the result of the resting lip pressure caused by mandibular rotation during the development of long-face condition also necessary to anticipate the backward movement of the mandible posteriorly during the mandibular setback which may lead the lower incisors in the more lingual position. the goal of the surgical treatment is to improve the maxilla and mandible in appropriate proportion situation to build a class i occlusion and relative facial balance with some parameters used, such as a normal difference between sna and snb angle known as anb, maxillary and mandibular height, facial convexity and occlusal plane angle as indicators of the relative antero-posterior position of the jaws as well as to reduce the vertical height. mandible reposition after the sagittal split osteotomy is done according to the calculated reposition of the upper 160 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 159–168 jaw after a lefort i osteotomy procedure. a precise and harmonious position of the maxilla would take as a key to achieve a relative harmonious facial balance. in such cases of a hyploplastic mandible appearance which could be shown after the mandible setback, a genioplasty is necessary to be considered to correct the mandibular angle and performance. two cases of long-face are reported, both cases presented with a class iii malocclusion, high vertical face height and extreme vertical open bite accompanied the clinical symptoms. difficulty on chewing, unaesthetic face, mouth breathing, and temporo-mandibular joints pain are the problems presented by patients during they appear to seek for disharmony correction. both cases were treated surgically with lefort i osteotomies as proposed by bell4 for repositioning the maxilla and a sagittal split osteotomies. combination of technique from dal pont5 and obwegesser6 were performed in both mandible for upward rotation and posterior setback. the purpose of this study is to examine the effect of surgical treatment in long-face cases with skeletal class iii malocclusion and extreme open bite. cases two cases of long-face with extreme vertical open bite visited my private practice and asked for facial corrections are reported. one case as shown in case 1 was already reported previously and some of the data presented in case 1 was taken from previous publication,7 and added with a cephalometric analysis method according to fonseca1 to compare another result of surgical correction existed in both cases, which had nearly same clinical symptoms. this article focuses on the result of surgical handling of longface cases existed with extreme vertical open bite and treated with the same surgical technique but shown with different result. the cases presented in 20 year-old (case 1) and 32 year-old (case 2) males with long-face who had undergone skeletal surgical correction are reported. these patients were sent by a general practice dentist asked for surgical correction. both two cases were then sent to an orthodontist for presurgical orthodontic treatment and the surgeries were performed six months after the presurgical orthodontic treatment. both cases had nearly the same chief complaints as inability to chew due to their severe open bite, unaesthetic faces, large mandibles, inability to close their mouths, and a mouth breathers. the patients considered that their facial appearance as a priority to be corrected. extra oral examination of both cases revealed of longface with hypotonic upper lips resulting in inability of mouths closure and a significant gummy smile presented in case 1 (figure 1a, b, c; 2a, b, c). cephalometry and clinical evaluation found with long-face cases accompanied with skeletal class iii malocclusions, vertical maxillary excesses and severe anterior open bites with maxillary hypoplasia. the surgical treatment decided were lefort i osteotomies for maxillary advancement with horizontal and vertical reduction. bilateral sagittal split osteotomies (bssro) was planned for a mandibular setback. a variety of cephalometric measurement methods were used to evaluate the patient's profile. a combination of ricket's,8 steiner's,9 down's,9 delaire's10 and legan's11 analyses were used to assess the relative relationship of the maxilla and in addition to the relationship of the maxillary and mandibular teeth. other examinations were done pre and post operatively by measuring the other cephalometric references point using: a) delaire hard tissue analysis with reference planes fm to clp. with a line perpendicular to this line, done by measuring the upper facial height from n to ans and the lower facial height from ans to me. the distance between ans and n should be 45% of total facial height and the measurement from ans to me along this line should be 55% of total facial height: 1) upper facial height (n-ans in mm), 2) lower facial height (ans-me in mm). b) legan soft tissue analysis included the ratio distances from g to sn and from sn to me, according to reference plan 7 degree above the sn plane. the ratio of this distance should be 1 to 1, g-sn (mm) should be equal to sn-me' (mm). the facial analyses as proposed by fonseca1 were also used in this observation, including the maxillary and mandibular depth, the bony chin position which was evaluated in the antero-posterior (ap) dimension by using sella-nasion (sn)-pogonion with normal range, 72–88 degrees; mean 80 degrees, sn-b point with normal range 72–88 degrees; mean, 79 degree. y growth axis with normal range of 53 to 66 degrees; mean 59 degrees. case 1: in this 32 year old male the surgical treatment was decided with lefort i osteotomy for maxillary advancement with horizontal and vertical reduction. a bssro was planned for mandibular correction. fifteen milimeter discrepancy between maxilla and mandible was noted in this case therefore correction of 5 mm in the maxilla forward and 10 mm mandibular setback were planned. chin correction of 7 mm forward was presumed to be necessary since the surgery predicted would reduce a large amount of the snb angle value post-operatively and it would interfere with the aesthetic appearance as the chin would loss its prominently, but the patient refused this procedure. case 2: in this 20 year old male a surgical treatment using lefort i osteotomy for maxillary advancement and horizontal and vertical reduction and a bssro for mandibular correction were used. twelve milimeter discrepancy between maxilla and mandible was noted in this case, therefore compensated correction of 5 mm in the maxilla forward and 7 mm mandibular setback were planned. a chin correction was presumed unnecessary to 161pramono: problem in surgical correction of long-face be performed as the patient had a relative extreme norm of snb angle, therefore after a mandibular setback the chin situation was predicted still in a compromised proportion. cases management both cases had received both pre surgical orthodontic and surgery, which le fort i and sagittal split osteotomies were performed for maxillary advancement and mandible setback and upward rotation. on a cast models pre operative simulations were done for predictive results on cast model. surgical templates were prepared in two steps individual occlusal splints for maxillary advancement and mandibular setback. the surgical results in case 1 and 2 were analyzed on their pre and post operative vertical relationships on their cephalometric photographs. in each patient, pre and post operative radiographs were taken in their centric relation with lips repose and were traced on 0.003-inch acetate tracing paper. the result of pre and post operative measurements on both patients presented as shown in table 1 to 4. by ricket's analysis as shown in table 1 and 3, the facial axis decreased from 101 to 95 degrees in case 1, and in case 2 increased from 85 to 87 degrees. the facial angle decreased in case 1 from 98 to 84 degrees, and in case 2 found increased from 83.5 to 88 degrees. the lower facial height had decrease in case 1 from 42 to 34 degrees and in case 2 no significant changed was found from 25 to 26 degree post operatively. the mandibular arch angle increased from 29 to 40 degrees in case 1 and decreased from 75 to 59 degree. the mandibular plane angle in case 1 showed with no change, but in case 2 had increased from 33 to 40 degrees. the total facial height increase from 48 to 50 degrees in case 1, and significant decreased found in case 2 from 122.5 to 84 degrees. the inclination of occlusal plane increased from 18 to 20 degrees in case 1 and decrease from 24 to 23 degrees in case 2. the result of measurement by delaire's analysis showed, the n-ans in case 1 had slightly increased from 5.0 mm to 5.5 mm and in case 2 decreased from 5.6 to 5.1 mm. the ans to me had decreased in case 1 from 9.8 to 8.7 mm. measurement of facial soft tissue height proportion which should be 1 to 1 in proportion, showed that the g to sn remain stable from 8.8 to 8.7 mm pre operatively and and from sn to me' decreased from 9.3 to 8.7 mm in case 1. in case 2 the g to sn had decreased from 9.7 to 7.8 and the sn to me' decrease from 10.0 to 8.1 mm. the table 2 and 4 show the result of measurement by steiner's analysis, some changes had noted as of the increased of sna from 70 to 78 degrees in case 1 and in case 2 found that sna had decreased from 101 to 94 degrees. the snb changed from 81 to 77 degrees in case 1 and in case 2, the snb decreased form 100 to 93 degrees. the anb had improved in case 1 from –11 to 2 degrees, and in case 2 only slightly had observed from 1.5 degrees pre operatively to 1 degree pos-operatively. the relation between upper and lower central incisors changed from 145 to 134 degrees in case 1, and in case 2 increased from 125 to 130 degrees. in down's analysis the facial angle (fh-np) had decreased from 88.5 to 88 degrees in case 1 and in case 2 increased from 85 to 90 degrees. the facial convexity (nap) decreased in both cases, in case 1 a significant decreased was noted from –23 degrees to + 1 degree, and in case, decreased from 2 degrees to 0 degree. the sellanasion (sn) to pogonion (pog) angle in case 1 decrease from 100 to 90 degrees and in case 2 no change on this angle was observed both pre-and postoperatively, constant from 94 to 94 degrees. the mandibular plane angle (fhgoga) decreased from 45 to 35 degrees in case 1 as well as had found in case 2 from 55 to 41 degrees. the y-fh axis increased in case 1 from 64 to 66 degrees and different result found in case 2 which found decreased from 74 to 60 degrees. the central incisors angle decreased from 142 to 138 degrees in case 1 and in case 2 increased from 123 to 132 degrees. the sn-b point angle decreased from 81 to 77 degrees in case 1, and in case 2 showed had decreased from 100 to 95 degrees. in fonseca's analysis of the maxillary depth (fh-na) increased in both cases, in case 1 had increased from 78 to 90 degrees, and in case 2, from 84 to 90 degrees. a different situation was observed in the mandibular depth as in case 1 which had a stable mandibular depth pre-and postoperatively in 88 degrees, but in case 2, the mandibular depth had increased from 86 to 90 degrees. the chin position in case 1 (figure 1d, e) shows less prominent post operatively as the pogonion (pog) point is located far behind the nasion (ns)-a line, which it should be located on this line or slightly anterior from this line. the surgical intervention in case 2 resulted a different situation as shown in case 1 as the pogonion (pog) located in a line of ns-a (figure 2d, e). discussion excessive face height was noted as clinical problem long before anything substantial could be done about it. as wrote by profit et al.3 that primary distinguishing characteristic of the long-face is a large total facial height that is manifest almost entirely in elongation of the lower third, leading to disproportions between facial height and width. in respect to facial proportion, the surgery had successfully changed the patients profile dramatically, showed with the changes of some cephalometric values, which had shifted nearly into relative normal values in facial axis, facial angle, lower facial height, sna, snb, anb, relation between upper and lower incisors, facial convexity (nap), a-b plane angle (ab-np), mandibular plane angle (fh-gogn), y-fh axis and sn-b point. 162 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 159–168 the facial angle, facial axis, and the sna and snb had changed the facial situation into relative good facial performance in both cases, both in its cephalometric values and clinical appearance. the facial convexity (nap) should be considered important as this value reflected the facial situation whether in concave or convex appearance. in both cases the nap had decreased, in case 1 from –23 degrees to +1 degree, and in case 2 decreased from +3 degrees to 1 degree represented an acceptable lateral facial appearances. the a-b plane angle (ab-np) also represented the facial lateral views, the surgery in case 1 had increased this angle from –14 to +3.5 degrees and in case 2 slightly changed had noted from 2 degrees to 0 degree. the surgery resulted positive facial appearances in both cases showed by the increase of these angles from convex to concave faces. the relation between lower incisors to upper incisors in both cases showed within the normal values as shown in case 1 increased from 123 to 132 degrees and in case 2 decreased from 145 to 134 degrees, the incisors procumbency in both cases had entered a normal range, and presented good inter-arch relationships. the anb had significant changed in case 1 from –11 degrees to +2 showed an improvement in the relationships between the maxilla and the mandible in remarkable situation. in case 2 the anb had changed from 0 to +1 degree means preoperative mandible position was in the rotated down position as usually showed in long face cases therefore the position of the b point was not extremely away from the a point. by maxillary advancement mandible rotation, the anb was not changed much. problem occurs in case 1, which the facial axis had shifted to 95 from 101 degrees. the decrease in facial axis angle represented posterior rotation of the mandible or as a result of backward movement of the symphysis. it can also be shown in the value of inclination of occlusal plane angle which had increased from 18 to 20 degrees in case 1 represented the maxillo-mandibular complex had rotated in clockwise rotation therefore the patient had lost his chin prominently. this situation sometimes can not be avoided since the mandible had rotated following the position of the maxilla. the downward movement of the symphysis can be avoided by reducing the degree of the clockwise rotation and change the direction with more upward movement of the anterior and posterior part of the maxilla during its reposition with the occlusal line keeps parallel to the champer's line. an extreme posterior upward movement should be considered, as this movement allow the mandible turn in more downward position. the situation given in case 2 was divers, the facial axis remains stable post-operatively represented the mandible had moved backward and upward and slightly upward rotated in the anterior teeth during a maxillo-mandibular reposition, shown by the decrease of the inclination of the occlusal plane therefore the chin situation in case 2 gave more prominent facial feature then as given in case 1. this assumption supported by the data given by the facial angle (fh-npog), in case 1 dropped from 98 to 84, the same situation also shown in the snb angle which had dropped from 81 to 77 degrees. the snb value in case 1 exhibited a strong decreased angle from 81 to 77 degrees in comparing to the sna situation which had increase significantly from 70 to 78 degrees. the surgical intervention in case 1 resulted slightly facial disharmony situation as the pog point is located far behind the ns-a line. therefore in case 1 further surgery of chin correction presumed to be necessary. in case 2 although the snb showed decreased in its norm but the chin clinical appearance was found undisturbed as the po point is located in the acceptable line of ns-a, and this situation might be existed due to the large value of snb shown preoperatively. correction of dentofacial deformities often requires double-jaw surgery to achieve a high quality functional and aesthetic result. cephalometric and clinical interrelationship should be highly considered in the diagnosis and treatment planning procedure for preparing in correction of dentofacial deformities. one of the surgical treatment goals is to correct the height of the occlusal plane angles. since the occlusal plane has significant influence on function and aesthetic, particularly when double-jaw surgery is performed. the occlusal plane angle is the angle formed by frankfort horizontal (fh) and a line tangent to the cusp tip of the lower premolar and the buccal grove of the second molar. an increased occlusal plane angle is usually reflected in an increased mandibular plane angle and a decreased occlusal plane angle correlates with the decreased of mandibular plane angle. traditional management in doublejaw surgery, regardless of steepness of the pre-surgical occlusal plane, either maintains the pre-surgical occlusal plane angulation, establishes the occlusal plane by auto rotation of the mandible, or selectively increases the occlusal plane relative to fh to improve stability.1 in both cases the occlusal plane angles were found high preoperatively, 30 degrees in case 1 and 39 degrees in case 2. the surgical treatment had decreased both values to 24 and 27 degrees respectively. a decreased occlusal planes usually correlates with a decreased of the mandibular plane angles, as it is shown in case 1, had decreased from 48 to 45 degrees as well as shown in case 2 which had decreased from 56 to 41 degrees. the maxillary and mandibular depth are two angle would be corrected, as a small values in the maxillary depth would be shown clinically with a hypoplastic maxilla and a small angle of the mandibular depth would be shown with a hypoplastic mandible. in both cases the surgery had corrected the maxillary depth pleasantly, increased into normal values, as shown in case 1 which had increased from 78 to 90 degrees and in case 2 from 84 to 90 degrees. the mandibular depth as shown in case 1 presented a stable value in 88 degrees, and in case 2 a better situation showed with the increase of its norm from 85 to 89 degrees. 163pramono: problem in surgical correction of long-face the mandibular depth had shown in case 1 presented a normal norm according to fonseca,1 but it is not clinically satisfying while the patient showed with lost of his mandible prominent, therefore a genioplasty actually necessary to be performed. the mandibular setback had changed the facial angle (fh-npog) norm too extreme in comparing with the result as shown in the facial axis (angle banptmgn lines), 84 to 95 degrees. another cause suspected in case 1 is the angulation of the occlusal plane angle which had slightly increased from 18 to 20 degrees, means the mandible had rotated into downward in direction during the maxillo-mandibular rotation, therefore this surgical rotation had decreased the chin prominently. the y axis is defined to be the direction of down growth and forward facial growth. the surgical treatment showed had reduced in some degrees, in case 1 from 68 to 66 degrees and in case 2 from 76 to 66 degrees represented the mandible had rotated backward and upward following the position of the maxilla, therefore the previous long face appearance obviously disappeared. the position of the mandible related to sella tursicanasion line showed by the sn-b point angle. in case 1 the sn-b point angle had decreased from 81 to 77 degrees this situation had brought the chin into retrograde mandible condition therefore a chin advancement should be performed to compensate this low degrees of he sn-b point angle. in case 2 a high degrees of this angle was found, and the mandibular setback had decreased this angle from 100 to 97 degrees, which shown above normal norm, but clinically the situation is still acceptable. according to legan's soft tissue analysis, in both cases the distance from g to sn and fro sn to me should be 1:1,10 the surgery had successfully achieved this objective. by delaire's analysis, the total hard tissue facial height achieved a good facial height proportion. the occlusal plane angles play as an important role in double-jaw surgery as it is related with significant aesthetic influence as this angle related to the facial type changes. theoretically the relation of the occlusal plane angle to the facial aesthetic can be explain as wrote by fonseca,1 in low and occlusal plane (lop) would produce a brachephalic face type, therefore surgical intervention to increase in occlusal plane angulation may be indicated. in high occlusal plane angle (hop) would produce a dolichocephalic facial type, the surgical correction may be indicated, which include a counterclockwise rotation of maxillomandibular complex. the lop and hop can be shown illustratively in figure3a and figure 3b. in both cases the surgical interventions were failed to decreases the value of occlusal plane angle in entering of a normal ranges but a clinical acceptable face still can be seen in both cases. in case 2 a better facial situation is seen as the surgery in case 2 was mentioned carefully to the value of the facial proportion between upper and lower face and in case 1 the surgery had brought the occlusal plane angle in slightly increased, shown with the chin had rotated in downward direction. in long-face correction, a genioplasty should always be considered as it might be needed to compensate the result of mandibular set back and the downward mandibular rotation. it concluded that in treating of long-face cases, a preoperative planning should be made by considering the facial proportion as proposed by legan,10 as it is sensitive in resulting of unbalance face due to over correction of the height of facial vertical dimension. changing of the long-face case directly into short face should not be done as a drastic changing of the facial height would be resulted a compressed face. a proportional acceptable face would be more acceptable as an extreme vertical reduction which only based on the mathematical calculation might produce a disproportion dimension in the lower third of the face area. in mention to the facial proportion, the upper and lower vertical height proportion changing should be made with consideration during the surgical procedure to ovoid a facial disproportion. the theory given by fonseca1 for lop and hop cases should be considered as it is related to the result of the surgery in aesthetic result. an alternative method of maxilla reposition after a le fort i osteotomy can be done by moving the maxilla forward as predicted pre-surgically and repotioned in the upward direction. the posterior part of the maxilla should not too strong upward repositioned, but should move the whole maxilla vertically with the occlusal line keeps parallel to a champer's line. the position of the four upper incisors teeth and upper lip also be used as guidance in positioning the four upper incisor teeth. this technique might be used to ovoid the chin rotated too downward as might given when a clockwise rotation movement is used and too strong rotated during the maxillomandibular complex movement. 164 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 159–168 figure 1a. pre-operative facial appearance. figure 1b. pre-operative cephalometric x-ray. figure 1c. pre-operative occlusion. figure 1d. post operative prifile taken one year after surgery. figure 1e. cephalometric after surgery. 165pramono: problem in surgical correction of long-face figure 2a. pre-operative facial profile. figure 2b. pre-operative panoramic radiograph. figure 2c. pre-operative occlusion. figure 2d. post operative profile taken fourteen days after surgery. figure 2e. post operative panoramic view. 166 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 159–168 ricket’s, fonseca’s, delaire’s and legan’s cephalometric measurements for case 1 table 1 mean presurgical post surgical facial axis (angle ban-ptmgn lines) 90° ± 3° 101° 95° facial angle (angle fh-npog, anatomic porion) 88° ± 3° 98° 84° mandibular plane (angle fh-goga) 25° ± 5° 44° 44° lower facial height (angle anxi-xipm) 47° ± 4° 42° 34° mandible arch (angle dcxi-xipm) 26° ± 4° 29° 40° total facial height 48° 50° inclination of occlusal plane 8° ± 4° 18° 20° maxillary-depth (fh-na) 90° ± 3° 78° 90° mandibular dept (fh-nb) 88° ± 3° 88° 88° n-ans – 5.0 mm 5.5 mm ans-me – 9.8 mm 8.7 mm g-sn – 8.8 mm 8.7 mm sn-me1 – 9.3 mm 8.7 mm table 1. the sum of ricket's fonseca's, delaire's and legan' cephalometric measurement for case 1 steiners cephalometric measurements for case 1 table 2 mean caucasoid interval caucasoid mean surabaya interval surabaya presurgical post surgical sna 82° 78°–86° 84.3° 79°–89° 70° 78° snb 80° 76°–84° 81.4° 74°–89° 81° 77° anb 2° 0°–4° 3° – –11° ± 2° relation between 1 – __ 1 131° 120°–150° 119.7° 105°–133° 145° 134° downs cephalometric measurements for case 1 facial angle (fh–np) 82° 78°–86° 84.3° – 88° 88° facial convexity (nap) 0° –8.5– +10° 6.1° – –23° +1° a–b plane angle (ab–np) –4.6° –9°–0° –4.6° – –14° +3.5° sella–nasion (sn)–pogonion – 72°–80° – – 100° 90° mandibular plane angle (fh–goga) 21.9° 17°–28° 27.5° – 45° 35° sn – bpoint 80° 72°–88° – – 81° 77° y– fh axis 59° 53°–66° 65.5° – 68° 66° 1 – __ 1 angle 135.4° 130°–150° – – 145° 134° table 2. the sum of steiner's and down's cephalometric measurements for case 17 167pramono: problem in surgical correction of long-face ricket’s, fonseca’s, delaire’s and legan’s cephalometric measurements for case 1 table 3 mean presurgical post surgical facial axis (angle ban-ptmgn lines) 90° ± 3° 85° 88° facial angle (angle fh-npog, anatomic porion) 87.3° ± 3° 83.5° 88° mandibular plane (angle fh-goga) 26° ± 4° 33° 40° lower face height (angle anxi-xipm) 47° ± 4° 25° 26° mandible arch (angle dcxi-xipm) 26° ± 4° 75° 59° total facial height – 122.5° 84° inclination of occlusal plane – 24° 23° maxillary-depth (fh-na) 90° ± 3° 84° 90° mandibular dept (fh-nb) 88° ± 3° 86° 90° n – ans – 5.6 mm 5.1 mm ans – me – 8.4 mm 7.9 mm g – sn – 9.7 mm 7.8 mm sn –me1 – 10.0 mm 8.1 mm table 3. the sum of ricket's, fonseca's, delaire's and legan's cephalomentric measurement for case 2 steiner’s cephalometric measurements for case 2 table 4 mean caucasoid interval caucasoid mean surabaya interval surabaya presurgical postsurgical sna 82° 78°–86° 84.3° 79°–89° 101° 94° snb 80° 76°–84° 81.4° 74°–89° 100° 93° anb 2° 0°–4° 3° – 1° +1° relation between 1__ 1 131° 120°–150° 119.7° 105°-133° 125° 130° down’s cephalometric measurements for case 2 facial angle (fh-np) 87.8° 82°–85° 84.8° – 85° 90° facial convexity (nap) 0° –8.5-+10° 6.1° – 3° 1° mandibular plane (angle fh-goga) 21.9° 17 o–28o 27.5° 55° 41o y–fh axis 59° 53°–66° 76° 66o 1__ 1 angle 135.4° 130°–150° – – 123° 132° sn–b point 80° 72°–88° – – 100o 93° sella-nasion (sn)-pogonion 76° 72°–80° – – 100o 94° y-fh axis 59.5° 53°–66° 65.5° – 74° 60° table 4. the sum of steiner's and down's cephalometric measurements for case 2 figure 3a. in surgical increase of the occlusal plane the chin rotates posteriorly relative to incisor tips, and the perinasal area advance. the posterior facial height decreases, the maxillary incisor angulation decreases, and the mandibular incisor increases.1 168 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 159–168 figure 3b. the surgical decrease of the occlusal plane results in increased projection of the chin, decreases prominence of the perinasal areas, increased maxillary incisor angulation, decreased mandibular incisor angulation, and increased oropharyngeal airway.1 references 1. fonseca jf. oral and maxillofacial surgery orthognathic surgery. philadelphia: saunders an imprint of elsevier; 2000. p. 3–23. 2. bailey lj, proffit wr, white wr. trends in surgical treatment of class iii skeletal relationships. j int adult orthod orthognath surg 1995; 10:10. 3. profit wr, white rp. surgical orthodontic treatment. in: profit wr, white rp (eds). long face problems. st louis: mosby; 1991. p. 381–427. 4. bell wh. lefort i osteotomy for correction of maxillary deformities. j oral surg 1975; 33: 412–25. 5. dal pont. retromolar osteotomy for correction of prognatism. j oral surg 1961; 19:42–7. 6. obwegesser hl. indication for surgical correction of mandibular deformity by sagittal splitting technique. br j oral surg 1964; 57–65. 7. coen pramono, jusuf sjamsudin. the effect of bimaxillary surgeries on facial proportions after surgical correction in skeletal class iii patients. dental journal faculty of dentistry airlangga university 2004 april; 37(2):52–60. 8. enacar a, taner tu, manav o. effect of singleor double jaw surgery on vertical dimension in skeletal class iii patients. int j adult orthod orthognath 2001; 16:30–5. 9. moyers re. handbook of orthodontic for the student and general practioner. 3rd ed. chicago: year book medical publisher incorporated; 1975. p. 387–425. 10. delaire j, schendel sa, tulasne jf. an architectural and structural craniofacial analysis. j oral surg 1981; 52:226–38. 11. legan hl, burstone cj. soft tissue cephalometric analysis for orthognathic surgery. j oral surg 1980; 38(7):744–51. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 44 no 3 sept 2011.indd contents page printed by: airlangga university press. (046/03.12/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 44 number 3 september 2011 issn 1978 3728 1. the role of probiotic on alveolar bone resorption desi sandra sari, zahara meilawaty, and m. nurul amin .......................................................... 117–121 2. dental measurements of deuteromalayid javanese students of the faculty of dentistry airlangga university myrtati dyah artaria and bambang soegeng herijadi ................................................................ 122–126 3. recent pharmacological management of oral bleeding in hemophilic patient monica widyawati setiawan .......................................................................................................... 127–131 4. treatment of lingual traumatic ulcer accompanied with fungal infections sella and mochamad fahlevi rizal ................................................................................................. 132–136 5. the effectiveness of nigella sativa seed extract in inhibiting candida albicans on heat cured acrylic resin hanoem eh, imam b, and kartika purnama pranoto ................................................................ 137–140 6. efficacy of various topical agents to prevent enamel demineralization priska lestari hendrawan, erwin siregar, and krisnawati ........................................................ 141–144 7. threshold value of enamel mineral solubility and dental erosion after consuming acidic soft drinks muhammad ilyas ............................................................................................................................. 145–149 8. anterior makeover on fractured teeth by simple composite resin restoration eric priyo prasetyo .......................................................................................................................... 150–153 9. management of horizontal crown fracture caused by traumatic injury with endorestoration treatment nanik zubaidah .............................................................................................................................. 154–158 10. sensitivity difference of streptococcus viridans on 35% piper betle linn extract and 10% povidone iodine towards recurrent apthous stomatitis maharani laillyza apriasari, bagus soebadi, and hening tuti hendarti ................................. 159–163 11. odontoblast layer structure alteration as a response to carious lesions tetiana haniastuti ........................................................................................................................... 164–168 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 190 volume 47, number 4, december 2014 indirect pulp capping in primary molar using glass ionomer cements murtia metalita, udijanto tedjosasongko, and prawati nuraini department of pediatric dentistry faculty of dental medicine universitas airlangga surabaya – indonesia abstract background: indirect pulp capping in primary teeth, however, is more rarely conducted than permanent teeth, since it thought to have low impact and most suggestion is for taking caries lesion aggressively on primary teeth. purpose: the study was aimed to evaluate the subjective complaint, clinical symptom, and radiographic appearance of indirect pulp capping treatment using glass ionomers cements in primary molar. methods: sixteen children in range of age 6 to 8 years old, who visited clinic of pediatric dentistry universitas airlangga dental hospital, surabaya indonesia, were the subject of study. they had one occlusal dental caries on one side of maxillary or mandibular primary molar with the diagnose of pulpitis reversible. the experimental group, had indirect pulp capping treatment with glass ionomer cements (gc fuji vii®), while the control group, had indirect pulp capping treatment with calcium hydroxide (metapaste). each group was filled with gc fuji ix® as permanent restoration. after one week, one month, and three months later, the observations were made on subjective complaint, clinical symptom, and radiographic appearance. results: the results showed no subjective complaint such as pain or problem on mastication; no negative clinical symptoms such as pain on palpation, gingivitis or periodontitis, and abnormal tooth mobility; no negative radiographic appearance such as pathological apical radioluscency, internal or external resorbtion, and change of ligament periodontal widthafter the treatment. conclusion: the study suggested that indirect pulp capping treatment using glass ionomer cement materials on primary teeth might be considered to be the treatment choice. key words: indirect pulp capping, primary molar, glass ionomer cement, children abstrak latar belakang: indirect pulp capping pada gigi sulung lebih jarang dilakukan dibandingkan gigi permanen, karena dianggap memiliki dampak yang rendah dan sebagian besar menyarankan untuk mengambil lesi karies secara agresif pada gigi sulung. tujuan: penelitian ini bertujuan untuk mengevaluasi keluhan subjektif, gejala klinis, dan gambaran radiografi perawatan indirect pulp capping menggunakan glass ionomer pada gigi molar sulung. metode: enam belas anak berusia 6 sampai 8 tahun, yang mengunjungi klinik kedokteran gigi rumah sakit gigi dan mulut universitas airlangga, surabaya indonesia, adalah subjek penelitian ini. mereka punya satu karies gigi oklusal molar sulung pada satu sisi maksila atau mandibula dengan diagnosa pulpitis reversibel. pada kelompok eksperimen dilakukan perawatan indirect pulp capping dengan glass ionomer (gc fuji vii®), sedangkan kelompok kontrol, dilakukan perawatan indirect pulp capping dengan kalsium hidroksida (metapaste). setiap kelompok ditumpat dengan glassionomer untuk gigi posterior (gc fuji ix®) sebagai restorasi permanen. observasi dilakukan setelah satu minggu, satu bulan, dan tiga bulan kemudian, pengamatan dilakukan pada keluhan subjektif, gejala klinis, dan gambaran radiografi. hasil: hasil penelitian menunjukkan tidak ada keluhan subjektif seperti rasa sakit atau masalah pada pengunyahan; tidak ada gejala klinis negatif seperti rasa sakit pada palpasi, gingivitis atau periodontitis, dan mobilitas gigi abnormal; tidak ada gambaran radiografi negatif seperti radioluscency patologis apikal, resorbsi internal atau eksternal, dan pelebaran periodontal ligamen setelah perawatan. simpulan: penelitian ini menunjukkan bahwa perawatan indirect capping menggunakan glass ionomer pada gigi sulung dapat dipertimbangkan sebagai pilihan perawatan. kata kunci: indirect pulp capping, molar sulung, glass ionomer, anak research report 191metalita, et al.: indirect pulp capping in primary molar using glass ionomer cements correspondence: udijanto tedjosasongko, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: udijanto@gmail.com introduction treatment of dental caries and effort of maintaining oral health for each patient must be considered as the main goal for dentist. the lost of healthy dental tissue must be considered to be a serious problem. for the reason, it is important to treat each tooth accurately and carefully. one of the clinical treatments conducted in deep caries lesion is indirect pulp capping. the aim of this treatment is to recover from dental caries by taking the infected dentin and isolating the caries lesion from oral liquid by using restoration materials.1 this treatment, moreover, is chosen more in order to maintain pulp vitality which can easily be affected by caries, trauma, or other causes. furthermore, indirect pulp capping treatment can be conducted in teeth with dentin caries. the deepest caries lesion is left remain to prevent pulp tissue exposure. materials considered as base like calcium hydroxide, zinc oxide eugenol, and glass ionomer cements are set in cavity base or dentin in order to stimulate the process of recovery and reparation. indirect pulp capping in primary teeth, however, is more rarely conducted than the permanent teeth. some dentists think that indirect pulp capping has low impact and suggests taking caries aggressively on primary teeth. nevertheless, based on the result of the previous experiment, it was reported that the level of successfulness is very high, about 90-99%.2 in addition, according to hilton and summit, the deepest layer or soft dentin that has been demineralized is abandoned and does not need to be taken. the other clinical experiments reported that the abandoned soft dentin caries will not affect the healthy dentin, and in next ten years the progress of lesion of the caries will stop and will not grow further.3-5 glass ionomers cements have been long used in atraumatic restorative technique (art). part of infected teeth or dentin actually can be cleaned only with hand instruments. glass ionomers cements are set in cavity in order to repair soft dentin. the reason is because the materials can improve re-mineralization process in dentin. it has been examined and published in many literatures that dentin caries which covered with glass ionomer cements generate process of remineralization.6 glass ionomer cements as liner can reduce the taking of dental tissue in which lesion of caries is located, and the taking of dentin that has been demineralized. the reason is because the setting of glass ionomer cement materials as liner can cause soft dentin get remineralization.7 massara et al., reported that the use of glass ionomers cements can stimulate process of remineralization, thus, the materials recommended as liner in indirect pulp capping treatment.8 glass ionomers cements considered as ideal materials for covering cavity base, through the adhesion process of ion alteration in demineralized dentin, actually can prevent the formation of nutrition for bacteria and reduce colonization of bacteria which still left in cavity base. besides that, glass ionomer cements continually secrete fluoride that consider as anti cariogenics.9 the gic only take short time for application in dental cavity, therefore, glass ionomer cements give more advantage relating with the duration of treatment in child patients.10 the in vitro experiment, moreover, has also proven that glass ionomer cements (gc fuji vii) can secrete fluoride six times as much as glass ionomer cements.11 according to svanberg and forss the materials can be considered as antimicrobe by reducing the growth of streptococcus mutans in the surface of restoration.12 the basic component of glass ionomer cements (fuji vii), in addition, is strontium fluoroalumino-silicate glass. strontium producing radiopacity and fluorine formed by fluoride ion entering into matrix phase can improve the remineralization process of dental structure got caries and give effect of anti microbe when the cements are getting harder.13 glass ionomer cements (gc fuji vii), thus, are located in cavity base, demineralized dentin or infected layer. the aim is to remineralize dentin that has been demineralized. this technique actually has already been used in developing countries, especially for indirect pulp capping, but there is still not many further experiment about it.11 the study was aimed to examine the subjective, clinical, and radiologic progression of indirect pulp capping treatment using glass ionomers cements (fuji vii) in primary teeth. materials and methods there were sixteen child patients, both male and female children visiting clinic of pediatric dentistry universitas airlangga dental hospital surabaya. the criteria of subject were: male or female children in the range of age 6 to 8 years old: patients had lesion of dentin caries that was deep and closed to pulp in primary molar teeth either in upper or lower jaw with diagnose of pulpitis reversible; the successor permanent teeth had not erupted, there was only one tooth suffering with class i of caries in one quadrant, patients had positive response on thermal test, the depth of cavity was about 1 – 0.5 mm from pulp based on radiologic picture; and patients did not have any allergy with any materials contained in glass ionomer cements. after explained the procedures of the experiment and the advantage and disadvantage of indirect pulp capping treatment with glass ionomer cements and calcium hydroxide, patients were divided into two groups, each of which consisted of ten children. the first group, as the experimental group, received indirect pulp capping treatment with glass ionomer cements, while the second 192 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 190–193 one, as the control group, received indirect pulp capping treatment with calcium hydroxide. first, subjective examination based on anamnesis to patients together with their parents, clinical examination and radiologic examination were conducted before the treatment in order to get diagnose of the dental caries that met the criteria of sample. then, lesion of caries was carefully cleaned with high speed bur. but, the lesion closed to pulp was abandoned in order to prevent the pulp opened, irrigated with aquadest, and then dried with cotton pellet. afterwards, in the first group, the cavity base was covered with glass ionomer cements (gc fuji vii®), while in the second group that was covered with calcium hydroxide (metapaste). then, the cavity in each group was fully filled with glass ionomer cements (gc fuji ix®). the progression of the treatment then was determined by three aspects observed, which were subjective, clinical, and radiologic progressions. those aspects of consist of: subjective complaint e.g. there was no pain based on anamnesis to patients and their parents, and good chewing function; clinical condition e.g. there was no pain on percussion or pressure, no sign of periodontitis, and no abnormal teeth mobility; and there was no picture of radiolucency pathologies in periapical area, no picture of internal or external root resorption, and no abnormal periodontal ligament.14 results the observation and evaluation of the treatment result were conducted three times, which were one week, one month, and three months after indirect pulp capping treatment. all subjects (8 patients) showed the same condition at 3 recall visits. they had no complaint concerning the pain or mastication problem. on intra oral examination, there was no pain on percussion or pressure, no gingival inflammation, and no abnormal tooth mobility. from radiographic examination there was no sign of pathology radiolucency in periapical area, no internal or external root resorption, and no sign of periodontitis or abnormal periodontal ligaments. discussion the subjective conditions observed and evaluated in this study involved two aspects the first aspect observed was pain, and the second aspect observed was chewing function. the control visit results showed that based on subjective observation, there was no pain suffered by either the first group glass ionomer cements (fuji vii®) or the second group metapaste. the second aspect of subjective observation was chewing function. it also revealed that the chewing function of the first group (glass ionomer) and the second group (metapaste) was all had no problem. the result showed that the application of glass ionomer cements or metapaste as sub base did not cause any pulp inflammation. moreover, the condition of pulp diagnosed as reversible pulpitis did not develop into irreversible pulpitis, but the inflammation seems to stop or disappeared. it might due to the bacteria causing inflammation had been eliminated after the infected dentin was taken and the cavity base was given liner. glass ionomer cements and metapaste reported to have anti bakterial effect in eliminating cariogenic bacteria that made the cavity sterile.15 the dental materials that is used for indirect pulp capping treatment should have the ability to overcome the inflammation to reduce the bacteria by disturbing bacterial metabolism, thus, pulp can be repaired and recovered.16 furthermore, when calcium hydroxide is applied and connected into pulp tissue, it can maintain the pulp vitality, will not cause inflammation, and can stimulate the formation of mineralized tissue barrier.17 the main component of glass ionomer cements is calcium or strontium fluoroalumino-silicate glass. the fluoride enter ion into matrix phase that can improve remineralization process of dental structure. besides that, strontium can also give effect of anti microbe after those cements are getting harder.13 strontium produce remineralization through ion exchange adhesion process in dentin, moreover, it can prevent the formation of nutrition for bacteria, and can decrease the colonization of bacteria live in cavity base.9 the second examination conducted during control period was clinical examination. this clinical examination involved three aspects of observation and evaluation, which were: no pain on percussion and pressure, no gingival inflammation or peridontitis, and no abnormal dental mobility. it appeared that in the first, second, and third visit controls the first group (glass ionomer as liner), and the second group (metapaste as liner) had no pain on percussion or pressure (100%). based on the result it could be known that indirect pulp capping treatment using either glass ionomer cements or calcium hydroxide as liner, the dentin demineralization process could be inhibited. thus, the process of pulp inflammation would not continue due to the lost of bacterial toxins. indirect pulp capping was conducted by taking the outer layer of dentin carries which contained microorganisms. by limited the demineralization process of the deepest dentin caused by bacterial toxins and by put restorative materials in the cavity, it can stimulate pulp to regenerate and to form reparative dentin.18 the result showed that based on the clinical observation in the first control, one week after the treatment, both the first group (glass ionomer cements) and the second group (metapaste) had no gingival inflamation or periodontitis. the same conditions were observed at the second and the third controls. the last aspect observed was the abnormal tooth mobility. the first group and the second group had no abnormal tooth mobility until the third recall visits. it showed that there was no inflammation either in hard or soft dental tissue. the periodontitis is marked by the abnormal 193metalita, et al.: indirect pulp capping in primary molar using glass ionomer cements tooth mobility. if the factor causing pulpitis was eradicated, there would be small possibility that pulpits would continue to be periodontitis. the dentin would get remineralization when the source of its infection was eradicated. in this case, glass ionomer cements as liner was used as the combination of antibacterial barrier and adhesive seal against the entrance of bacteria that can prevent the reparation and recovery of pulp.19 calcium hydroxide is believed as the best medicament for stimulating the formation of hard tissue and for recovering the vital pulp and periapical tissue.20 the last observation and evaluation for determining the progress of indirect pulp capping treatment was radiographic examination. there were three aspects examined in radiology description, e.g. no pathologic radiolucency in apical areas, no internal and external root resorption, and no the widening of periodontal ligament. the study showed that based on the radiographic examination at all recall visits, the first group had no either the pathologic radiolucency in apical areas, the internal and external root resorption, or the widening of periodontal ligament. similar condition observed in the second group. ngo 6 showed that the releasing of aluminum and fluoride simultaneously from glass ionomer cements has played a role as anti bacteria. the success of calcium hydroxide as liner was related with the effect of calcium hydroxide and its effect on the tissue and bacteria. estrela stated that calcium hydroxide passed some tests for years ago which proven that it has two enzyme characteristics, which are (1) enzyme that can restrain the formation of bacteria, through hydroxyl ion working in cytoplasmic membrane of bacteria and causing effects of anti bacteria; and (2) enzyme that can activate tissue, such as alkaline phosphates, that have effects of mineralization.21 the glass ionomer cements or metapaste could decrease or even eliminate the activity of cariogenic microorganism, so the pulp inflammation can be decreased or even be eliminated. since the pulp inflammation or reversible pulpitis was decreased or eliminated, there was no sign of pulp inflammation in the radiographic, that could continue into the inflammation of periodontal tissue (periodontitis). the radiographic examination showed that there was no radiolucency in apical areas; no internal and external root resorption; and no widening periodontal ligament. the success of indirect pulp capping treatment can be indicated by no clinical inflammation symptoms or pathologic symptoms.22 however, the further examination must be conducted to examine the vitality of primary molar teeth after indirect pulp capping treatment. the study suggested that based on subjective, clinical and radiographic examination, indirect pulp capping treatment using glass ionomer cement materials (gc fuji vii®) on primary teeth might be considered to be the treatment choice. references 1. ranly dm, garcia-godoy f. current and potential pulp therapies for primary and young permanent teeth. j dent 2000; 28(3): 153-61. 2. farooq ns, coll ja, kuwabara a, shelton p. success rate of formocresol pulpotomy and indirect pulp therapy in the treatment of deep dentinal caries in primary teeth. pediatr dent 2000; 22(4): 278-86. 3. mertz-fairhurst ej, curtis jw jr, ergle jw, rueggeberg fa, adair sm. ultraconservative and cariostatic sealed restorations: results at year 10. j am dent assoc 1998; 129(1): 55-66. 4. ribeiro c, baratieri ln. a clinical, radiographic and scanning electron microscope evaluation of adhesive restorations on carious dentin in primary teeth. quintessence int 1999; 30: 591-9. 5. ricketts d. management of the deep carious lesion and the vital pulp dentine complex. br dent j 2001; 191(11): 606-10. 6. ngo h. ionic exchange between glass ionomers and demineralized dentine; a thesis submitted in fulfilment of the requirements for the degree of doctor philosophy, school of dentistry the university of adelaide; 2006. p. 1-174. 7. knight mg. minimal intervention dentistry. ada news bulletin 2003; 5: 30-2. 8. pinkham jr, camassimo ps, mctigue dj, fields hw, nowak aj. pediatric dentistry: infancy through adolescence. 4th ed. st. louis, missouri: elsevier saunders company; 2005. p. 375-93. 9. ngo h. minimal intervention: how to treat the advanced lesions?. dental asia 2004; 38-41. 10. cho s, cheng ac. a review of glass ionomer restorations in the primary dentition. j can dent assoc 1999; 65(9): 491-5. 11. ngo h, fraser m. remineralization of artificial carious dentine exposed to two glass ionomers. j dent res 2002; 81: 386. 12. ten cate jm, van duinen rnb. hypermineralization of dentinal lesions adjacent to glass ionomer cement restorations. j dent res 1995; 74(6): 1266-71. 13. prentice lh, tyas mj, burrow mf. the effect of particle size distribution on an experimental glass ionomer cement. dent mater 2005; 21: 505-10. 14. vij r, coll ja, shelton p, farooq ns. caries control and other variable associated with success of primary molar vital pulp therapy. pediatr dent 2004; 26(3): 214-20. 15. büyükgüral b, cehreli zc. effect of different adhesive protocols vs calcium hydroxide on primary tooth pulp with different remaining dentin thickness: 24 months results. clin oral investig 2008; 12(1): 91-6. 16. ranly dm, garcia-godoy f. current and potential pulp therapies for primary and young permanent teeth. j dent 2000; 28(3): 153-61. 17. queiroz am, assed s, leonardo mr, nelson-filho p, silva la. calcium hydroxide for pulp capping. j app oral sci 2005; 13(2): 126-30. 18. ingle b. endodontics. 5th ed. canada: elsevier; 2004. p. 861-8. 19. van noort r. introduction to dental materials. 2nd ed. philadelphia: cv mosby company; 2002. p. 168-70, 175-6. 20. fava lr, saunders wp. calcium hydroxide pastes: classification and clinical indications. int endod j 1999; 32(4): 257-82. 21. estrela c, sidney gb, bammann ll, felipe júnior o. mechanisms of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. braz dent j 1995; 6(2): 85-90. 22. al-zayer ma, straffon lh, feigal jr, welch kb. indirect pulp treatment of primary posterior teeth: a retrospective study. pediatr dent 2003; 25(1): 29-36. �0 vol. 42. no. 1 january–march 2009 patient safety oriented to improve patient retention in oral health services tri erri astoeti department of dental public health and preventive dentistry faculty of dentistry trisakti university jakarta indonesia abstract background: oral health service systems should be designed to promote patient health, protection, and must be in compliance with indonesian laws that help protect patients from misuse of personal information. patient safety is a new healthcare discipline that emphasizes the reporting, analysis, and prevention of medical or dental error that often lead to adverse healthcare events. purpose: to describe correlation that patient safety would improve patent retention in oral health. patient safety is an essential component of quality oral health care and dentist is encouraged to consider thoughtfully the environment in which they deliver dental care, while at the same time services and to implement practices that decrease a patient’s risk of injury or harm during the delivery of care. reviews: designing oral health care systems that focus on preventing errors is critical to assure patient safety. some possible sources of error in oral health services are miscommunication, failure to review the patient’s medical history, and lack of standardized records, abbreviations, and processes. conclusion: patient safety would support patient satisfaction; therefore oral health services can increase patient retention. key words: patient safety, patient retention correspondence: tri erri astoeti, c/o: bagian ilmu kesehatan gigi masyarakat, fakultas kedokteran gigi universitas trisakti. jl. kyai tapa, grogol jakarta. e-mail: erriastoeti@yahoo.com introduction the quality of health services is one of every person’s basic needs. therefore, the approaches to quality-oriented services on patient satisfaction would be the main strategy for oral health services to exist in the global tight competition. the efforts of meeting patient’s satisfaction aimed to increase patients retention in the oral health services.1 oral health service systems should be designed to promote patient health and protection and must be in compliance with the laws that help protect patients from misuse of personal information.2 consequently, oral health service has to emphasize in patient safety serviceoriented. a number of recent international studies have concluded that action is needed to reduce the number of adverse events that occur in the health sector. institute of medicine in 2000 has reported 44.000-98.000 patients died in the united states due to medical error in the central health care.3 in indonesia, 48 criminal and 160 civil cases was reported in 2004-2005,4 and is cases of dental malpractice was reported in 2005-2008 by indonesian dental association.5 indonesian law no. 29/2004 on medical practice states the legal rights and obligations of patients will be protected, while the potential risk of service can be set with various rights and obligations of oral health services, managers and dental professions.6 however, it is not easy to implement these rules without the empowerment of the effectiveness of facilities and resources, systems and procedures and professional implementation. patient safety patient safety is a system that makes secure service to patients in health services and prevents the occurrence of injury caused by medical/dental errors due to certain action or no action that should be taken.7 it is a new healthcare discipline that emphasizes the reporting, analysis, and literature review ��astoeti: patient safety oriented to improve patient retention prevention of medical or dental error that often lead to adverse healthcare events. patient safety as an essential component of quality oral health care encourage dental professions to consider thoughtfully the environment which they deliver oral care services and to implement practices that decrease a patient’s risk of injury or harm during the delivery of care. therefore, the dental profession has to commit providing safe dental care, which is necessary for ensuring good general health, and to minimize risks and establish an open patient safety culture, in which practitioners can learn from their own and others’ experiences. the goals of patient safety program should be to reduce the risk injury to patients caused by treatment and remove or minimize hazards that increase risk.8 in indonesia, patient safety has been set in law no. 29/2004 on medical practice in article ii regarding patient safety and medical practices. this article was aimed to provide protection to patients, maintain and improve the quality of health care given by medical or dental professions, and to provide legal certainty to community, patient, and medical or dental professions.6 dental error dental error that occurred in the oral health services are potential to cause patient injury and if failed to implement, including a plan of treatment or use inappropriate treatment planning in dental error, there are known terms of “adverse event” and “near miss”. adverse events are events that resulted patient injury that is unexpected due to implement an action or no action unpreventable adverse event is a result of complications that can not be prevented with current knowledge. near miss is an error due to implement an action or no action should be taken so that cause patient injury, but no serious injuries occurred because of luck and prevention. some examples that usually occurred are seating or treating the patient incorrectly, wrong site surgery, non-sterile instruments used in patient care, swallowing/aspiration of teeth or instruments or retained foreign body, in radiographs such as mounted up side down, incorrect view, incorrectly filed, equipment not properly maintained, and lack of documented treatment plan (figure 1).9 most common basic causes of medical/dental errors there are 8 most common basic causes of medical or dental error: 1) communication problems is the most common cause of the occurrence of medical/dental error such as failure of communication either verbal or written within the team, 2) inadequate flow information, when that is not enough important information when the patients were referred to other places, 3) human resources problems are error-based knowledge such as dental profession does not have the adequate knowledge to treat the patient, 4) patientrelated issues such as inappropriate patient identification, incomplete patient assessment, the failure to obtain inform consent, uneducated patient, 5) organizational transfer of knowledge such as lack of training or orientation and the level of knowledge for running tasks 6) staffing patterns/work-flow such as inadequate human resources, 7) technical failures such as tool/equipment failure (dental units, dental x-ray), symptoms/treatment failure and inadequate instruction, 8) inadequate policies and procedures such as no guidelines, poor documentation, no records or standard operating procedure (sop), important information not included when the patients were referred to other places.10 the benefit of patient safety application the benefit to apply patient safety programme in oral health service are increasing and developing safety culture; developing communication with patients; decreasing adverse event; decreasing clinical risk; reducing complaints and litigation; increasing quality of services; increasing the image of oral health services and public confidence, followed by increasing patient retention.7,11 customer retention the quality of oral health service is very important for the customer retention and it can be attempted through service differentiation and competitive advantages that will attract new customers.1 the quality of oral health service has a positive influence on the intensity of customers to return and recommend our service to others (“magic word of mouth”) and become the informal public relations. longterm relationship with the customer will generate profits, dental error preventable near miss no injury patient process of care (non error) unpreventable adverse event injury patient figure 1. dental error. �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 50-53 therefore when customers pay and receive good services they have received “good value”, would increase their loyalty to the oral health service providers. it was reported that 5% customer retention can increase profit to 125%.1 discussion the overall aim of any dental patient safety programme should be the prevention of the full spectrum of dental errors, from the treatment of the wrong tooth to serious adverse events such as death. this ultimately resides with the dental professional who provides the treatment; however, prevention is also the shared responsibility of the entire dental team. one of the most interesting aspects of the programme here is how to use the whole dental team to decrease the number of dental patient safety errors. the main focus is on the various aspects and practice of teamwork because it has been found that 60% of dental errors occur due to a lack of communication within the team.8 the risk of adverse events is present throughout that whole procedure, relating, for example, to diagnosis, faulty equipment, general safety of the practice, poor communication to the patient or other health professionals, inadequate infection control or waste management. reduction of adverse events and improvement of patient safety is most effectively achieved through prevention, and preventive action to reduce adverse events is in turn a facet of high quality healthcare. quality cannot be promoted through force or sanctions from outside. it must be ensured that new measures ostensibly to improve patient safety, which can often add to the bureaucratic burden in the dental practice, do not hinder dentists from spending sufficient time with each patient, as this is an important parameter of high quality. the dental profession seeks to promote quality in many ways, including providing for continuing professional development to keep skills up to date; establishing local study groups for dentists and dental practices to learn from each others’ experiences; developing systems for reporting adverse events or near misses; and ensuring compliance with infection control and waste management law.2 patient safety programme is incomplete without its promotional campaigns. first, simply encourage teams to take the time out to check the patient’s full name and date of birth when they arrive at the clinic and again as they are seated in the dental chair. this simple procedure has been the most effective in reducing the number of wrong patients and dental treatment and, due to its ease of use, is ideal for practices that have a high turnover of patients throughout the day. the use of this initiative by displaying ‘time out’ posters throughout the centers and this also has the added benefits of bringing the patient into the team and emphasizing the requirement for good communication.8 secondly we have addressed the importance of the role of the entire dental team in the prevention of errors by providing training in an evidence-based teamwork training system which is aimed at optimizing patient outcomes by improving communication and other teamwork skills amongst dental professionals. the training is systematic and is spread over three phases: 1) assessment; 2) planning, training and implementation; and 3) sustainability.8 the assessment phase is designed to be a two-way process and initially requires the practice themselves to recognize that there is a need; then they are visited to assess their suitability for this training. the second phase recommends that two important selections are made: a practice champion who will act as a team motivator plus a ‘change team’. this phase is dependent on the size of the practice and whether or not the practice is part of a chain or group system. if the practice is small and so, only has a few staff then the entire team is trained; if it is large or part of a group system then training is given to the change team, which should represent as broad a cross-section of the personnel as possible. with the aid of a small team of master trainers this change team then receives training, which ideally takes place over one and a half days, and is responsible for developing a detailed action plan. this action plan is taken back to the practice and then followed to ensure the full implementation of the systems methods and principles. lastly the sustainability phase is there to ensure that these new teamwork tools and behaviors have been implanted into daily practice and also monitors the ongoing effectiveness of the training to identify opportunities for continued improvement.8 in conclusion, patient safety should preferably for patient satisfaction; therefore oral health services can increase patient retention. there is no panacea for patient safety as it is impossible to remove all the risks associated with the practice of dentistry, especially unpredictable ones. good reporting and other initiatives can only act as an adjunct to the prevention of dental errors and cannot substitute good leadership, the acceptance of personal responsibility and a shared goal to achieve excellence each and every day ensuring patient safety as part of undergraduate and post-graduate dental training curricula is suggested to strengthen further patient safety culture in healthcare; encourage dentists to be actively aware of the various elements of their professional practice where patient safety can be compromised; encourage dentists and the rest of the dental team to participate in continuing professional development relating to patient safety, to keep knowledge and skills up to date; ensure that dentists have a knowledge of languages, particularly in order that they be able to communicate with patients and other professionals; ensure that patient data is safely stored and available to health professionals as and when required, in accordance with national laws; ensure official registration of qualifications of dentists; ensure transparency of the qualifications and ��astoeti: patient safety oriented to improve patient retention competences of all other members of the dental team, as required by national law; consider establishing “study groups” to provide a forum for local dentists to discuss experiences openly and learn from each other; introduce national systems for voluntarily and anonymously reporting adverse events, near misses and problems with medical devices, to enable all dentists to learn from their own and others’ experiences. references 1. zittel-palamara k, fabiano j, davis e, waldrop d, wysocki j, goldberg l. improving patient retention and access to oral health care: the cares program. journal dental education. 2005; 69(8):912–8. 2. council of european dentist. patient safety. 2008. p. 2. 3. institute of medicine. patient safety: achieving a new standard for care. 2003. 4. sampurna b. aspek medikolegal pelayanan medik masa kini dan kaitannya dengan manajemen risiko klinik. makalah. jakarta. 2005. 5. pengurus besar persatuan dokter gigi indonesia. majelis kode etikmajelis kode etik kedokteran gigi. 2008. 6. undang-undang republik indonesia nomor 29 tahun 2004 tentang praktik kedokteran. 2004.2004. 7. yahya a. fraud and patient safety. seminar pamjaki, jakarta. 2007. 8. fildes t. patient safety stateside. vital 5, 2008. p. 45–47. 9. clarke j, lerner j, marella w. the role for leaders of health care organizations in patient safety. american journal of medical quality 2007; 22(5):311–8. 10. agency for healthcare research and quality. ahrq publication. 2003. 11. the joint commission. national patient safety goals ambulatory care program. available at:http://www.jointcommission.org/ patientsafety/nationalpatientsafetygoals. accessed july 30, 2007. vol 38-no 1-2005 32 pengaruh ketebalan bahan dan lamanya waktu penyinaran terhadap kekerasan permukaan resin komposit sinar (effects of materials thickness and length of light exposure on the surface hardness light-cured composite resins) annette alexandra susanto mahasiswa ppdgs fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract light-cured resin composite is one of the most commonly used molding materials due to its easy-to-mold characteristic. nevertheless, care must be taken while treating this material, especially with respect to material thickness and how long it is exposed to light. failure to treat the material with these optimal parameters will result in undesired hardness. to determine the most favorable value of these two parameters, an experiment was done with 3 different material thicknesses, and 3 different exposure times. after the sample of the resin composite was removed from its molding, it was stored under humid condition for 24 hours. afterwards hardness test was done on the sample using micro vickers hardness tester from this experiment, the significant difference in hardness was obtained and the maximum hardness was evaluated from the resin composite sample with 2 mm thickness and 60 seconds light exposure. key words: light-cured, resin composite, treatments, hardness korespondensi (correspondence): annette alexandra susanto, mahasiswa ppdgs, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan bahan restorasi yang baik dan yang dapat mengembalikan estetik merupakan kebutuhan masyarakat dewasa ini. resin komposit sinar memiliki berbagai macam keuntungan seperti sifat yang baik dalam hal pemakaian, memiliki resistensi yang baik terhadap keadaan kelas iv, mempunyai daya absorpsi air yang rendah, melekat dengan mudah pada permukaan gigi, warna yang mudah disesuaikan karena translusensi cahaya yang rendah, dan mudah dimanipulasi. proses pengerasan resin komposit memerlukan alat visible light cure (vlc) atau sinar tampak.1 keuntungan dari vlc adalah proses pengerasan yang cepat, dalam, dan dapat diandalkan. dalam waktu 40 detik setiap periode dengan ketebalan bahan minimal 2,5–3 mm dan maksimal 4,5 mm, dapat dipastikan bahan akan mengeras, meskipun melalui lapisan enamel bagian labial atau lingual, stabilitas warna yang dihasilkan sangat sesuai. 1 bahan tumpatan sinar menunjukkan warna yang lebih stabil dibandingkan sistem self-cured (pengerasan secara kimiawi), dan proses pengerasan atau polimerisasi yang terkontrol.2 namun secara klinis ditemukan kelemahan resin komposit yaitu shrinkage dan menurunnya kekerasan. komposisi resin komposit terdiri dari monomer dasar resin bis-gma atau bowen’s, monomer pengencer seperti triethylene atau tetraethylene glycol dimethacrylate untuk kemudahan mengalir, monomer pengisi yang bersifat penguat seperti crystaline quartz, lithium aluminosilicate, barium aluminoborate silica glass, dan fused silica, bahan penggabung untuk mendapatkan ikatan adesif yang sangat stabil oleh bahan pengisi terhadap resin dapat meningkatkan kekuatan dan daya tahan dari komposit, bahan penghambat polimerisasi untuk membatasi terjadinya proses polimerisasi selama penyinaran, bahan pemula polimerisasi (initiator) dan yang terakhir adalah bahan pengaktif polimerisasi (activator).3,4 proses polimerisasi terjadi dalam tiga tahapan yaitu inisiasi dimana molekul besar terurai karena proses panas menjadi radikal bebas. proses pembebasan tersebut menggunakan sinar tampak yang dimulai dengan panjang gelombang 460–485 nm. tahap kedua adalah propagasi, pada tahap ini monomer yang diaktifkan akan saling berikatan sehingga tercapai polimer dengan jumlah monomer tertentu. tahap terakhir adalah terminasi dimana rantai membentuk molekul yang stabil. sebagai salah satu bahan tumpatan sifat penting yang diperlukan adalah kekerasan permukaan. untuk mengukur kekerasan tumpatan dapat dipergunakan beberapa alat seperti brinell, knoop, rockwell, dan vickers.3,5 resin komposit dalam pemakaian sehari-hari di bidang kedokteran gigi sering dipertanyakan kemampuannya dalam menggantikan sifat kekerasan amalgam. pada kenyataannya sering terjadi kegagalan pada kasus penumpatan gigi geligi posterior, sehingga banyak penelitian dilakukan untuk mencari penyebabnya.6 ada banyak cara yang dipakai untuk menanggulangi proses penyusutan dan meningkatkan kekerasan seperti: 33susanto: pengaruh ketebalan bahan menambah bonding agent, menambah lapisan daya tahan elastis, meningkatkan intensitas light curing, memakai teknik peletakan bahan resin komposit lapis demi lapis, menggunakan monomer low-shrinking dan memasukkan bahan fluoride pada monomer resin untuk mencegah terjadinya marginal gaps pada kavitas.7 penyinaran bahan resin komposit sedikitnya adalah 30–40 detik.7 hal ini diperlukan untuk mendapatkan polimerisasi yang maksimal. walaupun proses penyinaran atau polimerisasi oleh vlc sepenuhnya dikontrol oleh operator yang dalam hal ini dokter gigi, teknik penyinaran seperti posisi dan arah sinar, intensitas sinar, ketebalan bahan restorasi, dan lamanya waktu penyinaran, sering kurang dipahami. penyinaran yang kurang akan mengakibatkan mengerasnya lapisan luar saja dan menghasilkan lapisan yang tidak matang atau lunak pada bagian dasar. 5 hal inilah yang melatarbelakangi diadakannya penelitian tentang teknik penyinaran terhadap bahan resin komposit sinar. penyinaran yang tidak menyeluruh pada permukaan tumpatan resin komposit juga akan menyebabkan penyusutan, hal ini dihubungkan dengan berat molekuler dari monomer resin dan jumlah monomer yang berikatan menjadi polimer resin.8 intensitas sinar juga perlu diperhatikan, untuk itu ujung alat sinar harus diletakkan sedekat mungkin dengan permukaan tumpatan (1 mm) tanpa menyentuhnya. kekerasan bahan resin komposit juga ditentukan oleh ketebalan bahan.9 idealnya resin komposit sinar diletakkan sebagai bahan restorasi sekitar 2–2,5 mm, dengan demikian sinar dapat menembus masuk sampai lapisan yang paling bawah.5,7 tujuan dari penelitian ini adalah untuk mengetahui pengaruh ketebalan bahan dan lamanya waktu penyinaran terhadap kekerasan bahan resin komposit sinar untuk menjawab permasalahan diatas. penelitian juga diharapkan dapat menambah pengetahuan tentang bahan restorasi estetik yang dalam hal ini bahan resin komposit sinar, untuk mengetahui hubungan antara ketebalan bahan dan lamanya penyinaran terhadap kekerasan resin komposit sinar, serta untuk meningkatkan mutu kekuatan dan kekerasan bahan tumpat tersebut bahan dan metode jenis penelitian yang dilakukan adalah eksperimental laboratorik dan dilakukan di laboratorium uji material ilmu logam fakultas teknik metalurgi, universitas indonesia. populasi dari penelitian adalah seluruh merek bahan resin komposit sinar yang dijual di pasaran indonesia. setelah dilakukan pengundian secara random, maka hasil yang didapat adalah resin komposit dengan merek g. proses pembuatan sampel dilakukan di dalam ruangan tertutup ber-ac dengan suhu 25º c untuk menghindari sampel dari kotoran dan debu. cetakan disiapkan dari cincin plastik yang bagian bawahnya diberi celluloid strip, disiapkan dan dicetak di atas kaca (glass plate). pasta resin komposit sinar dimasukkan ke dalam cetakan dengan menggunakan plastis instrumen dan kemudian dilakukan penyinaran. sampel terbagi dalam 3 kelompok, tiap kelompok terdiri dari 6 sampel, yaitu: kelompok i adalah sampel resin komposit sinar dengan tebal 2 mm yang disinar selama 20, 40, dan 60 detik. kelompok ii adalah sampel resin komposit sinar dengan tebal 3 mm yang disinar selama 20, 40, dan 60 detik. kelompok iii adalah sampel resin komposit sinar dengan tebal 4 mm yang disinar selama 20, 40, dan 60 detik. alat sinar diletakkan pada suatu standar dimana ujung alat sinar berjarak 1 mm dan tegak lurus terhadap permukaan sampel. masing-masing sampel lalu disinar dan diberi tanda sesuai dengan kelompoknya. seluruh sampel lalu dilepas dari cetakan dan direndam dalam petri dish yang berisi cairan nacl. cairan ini berfungsi untuk menjaga kelembaban sesuai dengan keadaan di dalam mulut. setelah ditutup rapat, petri dish disimpan selama 24 jam dalam suhu 25° c. cara pengujian sampel dilakukan dengan cara sampel yang telah direndam dalam nacl dilepas dari cetakan lalu sampel diamplas terlebih dahulu, kemudian dibuatkan suatu lempeng berlubang dari akrilik sebagai tempat sampel sebelum diuji di meja obyek yang disebut mounting. mounting tersebut dijepit dengan alat penjepit pada meja obyek alat micro vickers hardness tester. selanjutnya sampel diatur supaya tepat di tengah lensa obyektif dan difokuskan dengan memutar pegangan untuk fokus searah dengan jarum jam. setelah dalam lensa okuler terlihat gambar dalam keadaan fokus sampel dipindah dengan menggeser pegangan penggeser ke arah kanan sehingga tepat berada di bawah diamond penetrator. kemudian tombol penetrator ditekan, diamond akan turun dengan lampu hijau menyala sebagai tanda. bila diamond penetrator telah menyentuh sampel, maka lampu merah akan menyala. tigapuluh detik kemudian penetrator akan naik, setelah lampu merah dan hijau padam, sampel digeser ke tempat semula, dan difokuskan kembali. hasil penetrasi akan menunjukkan gambaran belah ketupat pada lensa okuler, dimana panjang diagonalnya dapat diukur langsung dengan micrometer melalui lensa okuler. hasil pengukuran dua panjang diagonal tersebut kemudian diambil nilai rata-ratanya. setelah didapat panjang diagonal, maka dapat dihitung nilai kekerasan dari permukaan sampel. pada masing-masing sampel dilakukan 10 kali pengujian pada tempat yang berbeda, kemudian hasilnya dirata-rata. data dianalisa dengan cara uji twoway anova dengan replikasi dan gambaran deskriptif. hasil hasil penelitian secara laboratorik mengenai rerata dan standart deviasi (sd) kekerasan resin komposit sinar 34 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 32–35 dengan pengaruh tebal bahan terdiri dari 2 mm, 3 mm, dan 4 mm, sedangkan untuk kelompok lamanya waktu terdiri dari 20 detik, 40 detik dan 60 detik terlihat pada tabel 1. pengaruh perbedaan dari perlakuan tersebut diuji dengan perhitungan statistik two-way anova dengan replikasi. berdasarkan analisa anova dengan membandingkan ftabel dan fhitung, maka didapat hasil fhitung (271,7115805) lebih besar dari f tabel (2,48444), menyatakan ada perbedaan yang bermakna antara tebal bahan dan lamanya waktu penyinaran terhadap kekerasan permukaan bahan resin komposit sinar. uji anova juga dapat dianalisis dengan melihat nilai probabilitas (p-value) dimana p-value hasil perhitungan dari komputer adalah 4,48888e-46 yang lebih kecil dari 0,05, maka ada interaksi antara tebal bahan dan lamanya waktu penyinaran. pada analisa tebal bahan dengan membandingkan ftabel dan fhitung, maka didapat hasil fhitung (2270,011053) lebih besar dari ftabel (3,10930659). dengan demikian kekerasan permukaan bahan resin komposit sinar dipengaruhi oleh ketebalan bahan. dengan melihat nilai probabilitas (pvalue), didapatkan hasil p-value hasil perhitungan dari komputer adalah 7,44308e-72 yang lebih kecil dari 0,05 sehingga rata-rata kekerasan permukaan bahan resin komposit sinar berdasarkan ketebalan bahan memang berbeda secara nyata. pada analisa lamanya waktu penyinaran dengan membandingkan ftabel dan fhitung, maka didapat hasil fhitung (3501,465141) lebih besar dari ftabel (3,10930659). dengan demikian kekerasan permukaan bahan resin komposit sinar dipengaruhi oleh lamanya waktu penyinaran. dengan melihat nilai probabilitas (p-value), didapatkan hasil p-value hasil perhitungan dari komputer adalah 2,2779e-79 yang lebih kecil dari 0,05, maka ratarata kekerasan permukaan bahan resin komposit sinar berdasarkan lamanya waktu penyinaran memang berbeda. perbedaan kekerasan tersebut juga dapat dilihat pada gambaran deskriptif berdasarkan nilai rata-rata hasil pengujian dengan alat micro vickers hardness tester. kekerasan meningkat seiring dengan semakin lamanya waktu penyinaran, sebaliknya kekerasan menurun seiring dengan semakin tebalnya bahan resin komposit sinar. kekerasan maksimal pada kondisi tebal bahan 2 mm dan waktu penyinaran 60 detik. berdasarkan gambaran deskriptif tersebut, terlihat adanya penambahan tingkat kekerasan seiring dengan semakin lamanya waktu penyinaran (60 detik). namun dari segi ketebalan bahan, penyinaran dengan tebal bahan 4 mm mempunyai nilai kekerasan yang lebih rendah dibandingkan dengan tebal 2 mm dan 3 mm, walaupun pada penyinaran 20 detik antara ketebalan 2 mm dan 4 mm perbedaan nilai kekerasannya tidak terlalu bermakna. pada setiap penambahan lama waktu penyinaran didapatkan peningkatan kekerasan, sebaliknya pada penambahan tebal bahan terjadi penurunan kekerasan resin komposit sinar. kekerasan maksimal terjadi pada keadaan dengan tebal bahan 2 mm dan disinar selama 60 detik. idealnya suatu bahan resin komposit diletakkan sebagai bahan restorasi sekitar 2–2,5 mm, dengan demikian proses polimerisasi dapat berlangsung dengan maksimal.5,7 pembahasan berdasarkan hasil penelitian yang sudah didapat, terlihat bahwa ada perbedaan yang bermakna antara ketebalan bahan dan lamanya waktu penyinaran terhadap kekerasan bahan resin komposit sinar. dalam penelitian laboratorik ini diketahui bahwa ketebalan maksimal yang dapat dilakukan untuk mendapatkan kekerasan yang optimal adalah 3 mm. kekerasan bahan dengan ketebalan bahan yang melebihi 3 mm akan menurun walaupun dilakukan penyinaran dalam waktu yang cukup lama. dilihat dari segi penyinaran umumnya nilai kekerasan meningkat pada ketebalan 2 mm dan 3 mm dengan penyinaran 40–60 detik, tidak terjadi perbedaan yang cukup berarti, walaupun ada peningkatan kekerasan. beberapa hal penting yang dapat mempengaruhi hasil penelitian tersebut adalah intensitas sinar pada saat penyinaran yang rendah akan mempengaruhi nilai kekuatan dari sinar itu sendiri, hal ini berakibat langsung terhadap kekerasan bahan resin, dan menurunnya nilai intensitas sinar menyebabkan menurunnya nilai kekerasan tabel 1. hasil rata-rata uji kekerasan permukaan resin komposit sinar pada kelompok tebal bahan dan lamanya penyinaran 2 mm 3 mm 4 mm 20 detik 40 detik 60 detik 15.8655 ± 0.3915 39.7794 ± 0.8558 50.1087 ± 0.5868 28.9812 ± 0.4808 37.7464 ± 0.6229 49.060 ± 2.8368 13.7877 ± 0.3124 18.0982 ± 0.7397 30.2681 ± 0.451 gambar 1. hasil rata-rata uji kekerasan resin komposit sinar. 2 3 4 20 40 60 0 10 20 30 40 50 60 ke ke ra sa n re sin ko m p o sit t e b a l ba h a n (m m ) (d e tik) p e rb e d a a n k e k e ra s a n k o m p o s it 20 40 60 35susanto: pengaruh ketebalan bahan bahan tumpat resin komposit sinar. kedua adalah pengaruh kelembaban tangan operator di ujung instrumen pada saat meletakkan bahan resin komposit sinar ke dalam cetakan, hal ini menyebabkan polimerisasi tidak dapat berlangsung dengan baik dan nilai kekerasannya akan menurun. berdasarkan hasil data penelitian, kesimpulan yang dapat diambil adalah: ada pengaruh ketebalan bahan dan ada pengaruh lamanya waktu penyinaran resin komposit sinar terhadap kekerasan permukaan bahan; mutu kekerasan dan kekuatan bahan resin komposit sinar menurun seiring dengan semakin tebalnya bahan pada saat penumpatan jika tidak disertai dengan penambahan lamanya waktu penyinaran. hal ini terjadi karena polimerisasi bahan tidak dapat berlangsung dengan baik. berdasarkan data penelitian juga terlihat dengan jelas bahwa kekerasan maksimum didapatkan pada sampel resin komposit sinar dengan ketebalan 2 mm dan lama waktu penyinaran 60 detik. secara umum lama waktu penyinaran sebaiknya dilakukan berkisar antara 40–60 detik, sedangkan ketebalan bahan tidak lebih dari 3 mm pada satu kali penyinaran. saran dari penelitian ini adalah: hindari kontak bahanbahan yang mengandung vaselin atau sejenisnya terhadap bahan tumpat resin komposit sinar, karena hal ini akan mengakibatkan mudah terlepasnya tumpatan dari kavitas; penyinaran bahan tumpat resin komposit sinar secara lapis demi lapis dengan ketebalan bahan tidak lebih dari 3 mm setiap lapisnya. dengan demikian diharapkan polimerisasi oleh sinar dapat berlangsung secara menyeluruh; dan penambahan lamanya waktu penyinaran pada resin komposit sinar yang berwarna lebih gelap perlu dilakukan. daftar pustaka 1. pollack bf, blitzer m. composite and microfill resins. dental abstract 1984; 29: 201–3. 2. asmusen e. factors affecting color stability of restorative resins. acta odontal scand 1983; 41: 11–5, 22–7. 3. combe ec. notes on dental materials. 6th ed. london: churchill livingstone; 1992. p. 23, 45–7, 89–99, 105–14, 126–34, 153–66. 4. smith bgn, wright ps, brown d. the clinical handling of dental materials. bristol: wright; 1980. p. 155–61. 5. craig rg. chemistry, composition, and properties of composite resin. dental clinique of north am; 1993. p. 25, 219–33. 6. eick jd, robinson sj, byerley tj, chappelow cc. adhesives and nonshrinking dental resins of the future. quintessence int 1993; 24: 632–40. 7. bruce jc, hewlett rr, jo y, hobo h, sumiya, hornbrook d. comtemporary esthetic dentistry practice fundamentals. tokyo: quintessence; 1994. p. 60–99. 8. lai jh, johnson ae. measuring polymerization shrinkage of photoactivated restorative materials by a water-filled dilatometer. dent mater journal 1993; 9: 139–43. 9. shortall ac, harrington e, wilson hj. light curing unit effectiveness assessed by dental radiometers. j dent 1995; 23: 227–32. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 70 the role of partial denture in management of hypohidrotic ectodermal dysplasia tania saskianti1, seno pradopo1, prawati nuraini1, and michael josef kridanto kamadjaja2 1department of pediatric dentistry 2department of prosthodontics faculty of dentistry, airlangga university surabaya indonesia abstract ectodermal dysplasia is a rare hereditary disorder with a characteristic physiognomy. the ectodermal dysplasia constitutes a group of hereditary disorders whose clinical manifestation can be defects in ectodermal structures. the case of a 11-year-old child with hypohidrotic ectodermal dysplasia and partial anodontia is presented. affected children require extensive dental treatment to restore appearance and help the development of a positive self image. partial denture was provided to encourage a normal psychological development and to improve the function of the stomatognatic system. it is important for the patient and the dentist to understand that continued monitoring for dental problems is necessary. this paper had an objective to relate and discuss a case of hypohidrotic ectodermal dysplasia, with the approach of the influence of an esthetic rehabilitation and functional alternative in the improvement of the quality of life. key words: ectodermal dysplasia, children, partial denture correspondence: tania saskianti, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: taniapedo@yahoo.com introduction ectodermal dysplasia is part of a group of hereditary diseases that may affects the development of two or more ectodermal derived structures, for instance, hair, fingernails, teeth and skin. it is more frequent in boys and mostly case is recessive autosomal character, but it could be dominant autosomal or linked to chromosome x. the pathognomy of the face in ectodermal dysplasia individuals of both sexes is similar and could be characterized.1,2 the types of dysplasia commonly observed are the hypohidrotic and anhidrotic. the first has as principal characteristics such as scarce hair, dystrophic fingernails and dental anomalies, involvement of the sudoriparous and sebaceous glands are not presented. the most common type of their anomaly is hydrotic ectodermal dysplasia. the major clinical findings of the disease are sparse hair, dryness of the skin, irregularities in nails, hypodontia or anodontia, hpohydrosis and hypotrychosis. anodontia or hypodontia, abnormal crown form shown with clinical shape, and short root with large pulpal chamber both in deciduous and permanent teeth are some of oral findings presented in ectodermal dysplasia.3–5 multiple dental absences as well as reduced vertical face dimension and, as consequence, resulting in protuberant lips besides the facial aspect of senility because of the decrease of the vertical dimension. saddle-shape nose, malformed and protruding ears, wrinkles around eyes, pigmentation disturbances, disorders in sexual and mental development are the other features of this disease. additionally, disphonia and hoarseness of the voice may be determined due to the atropic pharyngeal and laryngeal mucosa.6 ectodermal dysplasia may also cause a physical and emotional problem. ectodermal dysplasia patients require dental treatment primarily because of other dental needs and not always for good appearance. restoration of good appearance is merely one of the outcomes of a successful treatment.7 oral rehabilitation is one of the possible means to improve the quality of life. in this way, the objective of the present article was to describe a case of an hypohydrotic type of ectodermal dysplasia, with the approach of the influence of an esthetic rehabilitation and functional alternative in the improvement of the quality of life. case in 1999, a three year-old male patient, was referred to the airlangga university, faculty of dentistry, surabaya, indonesia for examination and treatment of his disorder. on clinical examination, he exhibited some dental absences and presented with “small” teeth (figure 1). history of pregnancy and delivery were normal. no systemic disorders or medication were documented. family history revealed that the parents were healthy, however his brother and grandfather showed the similar disorder. the extra oral examination (figure 2) revealed the typical facial physiognomy of hypohidrotic ectodermal dysplasia with prominent forehead and ears, protuberant lips and a saddle nose. a diminished lower facial height 71saskianti, et al.: the role of partial denture in management of hypohidrotic ectodermal dysplasia figure 4. patient, 10 years old with hypohidrotic ectodermal dysplasia. figure 3. a clinical view of the upper and lower arch, notice the dental absences and conoid teeth. figure 1. panoramic x-ray on august 1999 showing the dental agenesis. figure 5. panoramic radiographic on december 2005 showing the dental agenesis. figure 2. removable partial denture. figure 6. removable partial denture, a frontal view of the patient occlusion. contributed to a senile facial expression. the intraoral examination revealed partial absence of primary teeth (figure 2). the tongue was relatively large, but no signs of macroglossia could be detected. after the permission was received from the parents for the presentation of the child, full history with clinical examination and radiologic findings were obtained. radiographic examination (figure 3 and 4) revealed the partial absence of permanent tooth germs. case management the initial treatment plan in 1999 was to prepare maxillary and mandibular acrylic removable partial denture, for the patient. considering that the patient are too young to receive extensive prosthodontic treatment. the partial denture was use to restore the function and aesthetics (figure 5). the teeth were preserved without the need for restoration and extractions. in the present case the amount of saliva was sufficient for the adhesion of the dentures. these prosthesis need to be changed periodically according to the bone growth and development. the patient was monitored to perform a proper and efficient hygiene of the prosthetic appliances. he was satisfied with his aesthetic. in 2005, the patient began to feel discomfort due to changes in hard tissue structures so that he subsequently rarely used his dentures. on clinical examination, the permanent central incisive and first molar has erupted (figure 3). the extra oral examination showed the characteristic of hypohidrotic type of ectodermal dysplasia as described above (figure 4). the panoramic radiographic showing the dental agenesis (figure 5). he had another removable partial denture (figure 6). at recall appointments, he complained of fatigue on his temporomandibular joint because of posterior open bite. occlusal grinding was taken on the incisal edges of mandibular incisive also to improve his mastication. 72 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 70-73 discussion although most infants begin tooth eruption by 6 months of age, there is tremendous variation. in general, if the infant is growing, gaining, and has normal other ectodermal structures like hair, skin, and nails, and there are no dysmorphic features, no evaluation is necessary other than reassurance. normal number of primary teeth is 20 and there are 32 permanent teeth. mandibular central incisors are often the first to erupt followed by the maxillary central and lateral incisors. there may be familial cases of absence of individual teeth and certain conditions may be involved with delayed or absent tooth development. some are: down syndrome, ectodermal dysplasia, hypothyroid or hypopituitary.8 ectodermal dysplasia is commonly diagnosed in childhood, subsequent to a feverish episode of unknown origin.9 in many cases the diagnosis has been made by a pediatric dentist because missing teeth or the delay in teething often starts to worry the parents and leads them to visit the pediatric dentist.10 a pediatric dentist should not hesitate to radiographically examine a patient whose teeth have not erupted by the appropriate age in order to exclude ectodermal dysplasia. the screening limit for the first tooth to erupt is 15 months.10 if that stage aplasia of several teeth is seen, the patient should be referred to a geneticist in a pediatric unit with a suspecsion of ectodermal dysplasia (ed) diagnosis. the diagnosis of ectodermal dysplasia is extremely easy when the patients presents with more characteristics and clear clinical forms. in this case report when these characteristics are less evident, the diagnosis and the classification of the patient, became a difficult task for the pediatric dentist. in ectodermal dysplasia it was possible to improve the physical condition of the child, as well as the esthetics; the chewing and phonetic functions (figure 7). the dental treatment was done not only to re-establish the function, but to improve the emotional and social aspect of the child as well. in patients with ectodermal dysplasia, tooth agenesis and its secondary effects on growth and development of the jaws is often the most significant clinical problem. it is necessary to use the prosthetic appliances to the reestablishment of aesthetics and function, normalize the vertical dimension and support the facial soft tissues also to improve speech. the most common treatment in cases of ectodermal dysplasia with partial anodontia is partial dentures as used in the present case as an early treatment strategy. this favorable treatment has been suggested by several author.11–18 the denture must be periodically modified as alveolar growth, erupting teeth and rotational jaw growth change both the alveolar, occlusal and basal dimensions.19 after the repair of the prosthesis, the patient able to enjoy a better quality diet because he could then chew better he presented a positive feedback regarding psycho-social living, thus improving the quality of life. the patient could develop the social relationship with his peers because his aesthetic appearance was improved, and the problem of hoarseness disappeared which helped him to improve psychologically. this alternative treatment was successful as it established his masticatory speech functions and improved his social living, hence quality of the life of the patient. future treatment will include modification or replacement of the dentures according to the observed skeletal growth. some authors,20 recommend the placement of implants in the adult phase for the treatment of patient with ectodermal dysplasia, although another author21 suggest the placement of implant in a 3 year-old child. another treatment for some reasons in young children, we prefer to apply crown and bridges if they have also a limited retention and stability, a fastened bone destruction of an already hypoplastic alveolar process and the middle of the upper jaw is covered and so it blocks the sutural growth.19 the use of certain kinds of dentures depending on the patient’s age, eliminates esthetics, psychological, functional and speech disorder issues.22 as long as there are no physical, psychological or social burdens, no further treatment is necessary.19 references 1. kupietzky a, houpt m. hypohidrotic ectodermal dysplasia: characteristics and treatment. quintessence int 1995; 26:285–91. 2. sabah e, çalıskan mk, aydyn z. üç olgu nedeniyle ektodermal displazinin endodontik ve protetik tedavisi. edfd 1988; 9(3):45–54. 3. vierucci s, baccetti t, tollaro i. dental and craniofacial findings in hypohydrotic ectodermal dysplasia during the primary dentition phase. j clin pediatr dent 1994; 18:291–7. 4. chitty ls, dennis n, baraitser m. hidrotic ectodermal dysplasia of hair, teeth, and nails: case reports and review. j med genet 1996; 33:707–10. 5. bonilla ed, guerra l, luna o. over denture prosthesis for oral rehabilitation of hypohidrotic ectodermal dysplasia: a case report. quintessence int 1995; 28:657–65. 6. itthagarun a, king nm. ectodermal dysplasia: a review and case report. quintessence int 1997; 28:595–602. 7. ali rıza alpöz. hydrotic ectodermal dysplasia (associated with numerous enclosed permanent teeth including a primary molar). tr j of medical sciences 1999; 29:75–9. © tübitak figure 7. a facial view after treatment. 73saskianti, et al.: the role of partial denture in management of hypohidrotic ectodermal dysplasia 8. creighton pr. common pediatric dental problems. pediatric clinics of north america. 1998; 45(6):1579–600. 9. daniella dv, ana beatriz ac, lucianne cm, joao af. alternativeriz ac, lucianne cm, joao af. alternativealternative rehabilitation treatment for a patient with ectodermal dysplasia j clin pediatr dent 2004; 28(2): 103–6. 10. pirinen s. therapia odontologica. in: meurman, murtomaa, lebell, autti, luukkanen, eds. helsinki: academica kustannus oy; 1996. p. 414. 11. borg p, midtgaard k. ectodermal dysplasia: report of four cases. j dent child 1977; 44:314. 12. shore sw. ectodermal dysplasia: a case report. j dent child 1970; 37:254–7. 13. boj jr, von arx jd, cortada m, et al. dentures for a 3-yr-old child with ectodermal dysplasia: case report. am j dent 1993; 6:165–7.am j dent 1993; 6:165–7. 14. cook, wa, kane fj, a family history of hereditary anhidrotic mesodermal-ectodermal dysplasia. j a d a 1968; 76:1032–37. 15. bergendal t, eckerdal o, hallonsten al, et al. osseointegrated implants in the oral habilitation of a boy with ectodermal dysplasia: a case report. internat dent j 1991; 41:149–56. 16. boj jr, duran j, cortada m, et al. cephalometric changes in a patient with ectodermal dysplasia alter placement of dentures. j clin pediatr dent 1993; 17:217–20. 17. till mj, marques ap. ectodermal dysplasia: treatment considerations and case reports. northwest dent 1992; 71:25–8. 18. belanger gk. early treatment considerations for oligodontia in ectodermal dysplasia: a case report. quintessence int 1994; 25:705–11. 19. mortier k, wackens g. ectodermal dysplasia anhidrotic. orphanet encyclopedia 2004 september. available at: http://www.orpha. net/data/patho/gb/uk-ectodermal-dysplasia-anhidrotic.pdf 20. ekstrand k, thompson n. ectodermal dysplasia with partial anodontia: prosthetic treatment with implant fixed prosthesis. j dent child 1988; 55:282–4. 21. guckes ad, mccarthy gr, brahim j. use of endosseous implants in a 3-year-old child with ectodermal dysplasia: case report and 5-year follow-up. pediatr dent 1997; 19:282–5. 22. pervin i, sina u, seda gh. surgical and prosthodontic treatmentsurgical and prosthodontic treatment alternatives for children and adolescents with ectodermal dysplasia: a clinical report. j prosthet dent 2002; 88:569–72. mkg vol 39 no 1 jan 2006 isi.pmd 19 poor oral hygiene as trigger of diabetes mellitus progressiveness sunarko setyawan physiology department school of medicine airlangga university surabaya indonesia abstract diabetes mellitus is a systemic disease with several major complications affecting both the quality and length of life. the disease is characterized by increasing susceptibility to infection that important risk factor for oral infection progressiveness; periodontitis, infection or lesions. infection progressiveness and inflammation can increase blood cytokines. the cytokines modulate cells up and down regulation moreover apoptosis or necrosis cells. the increasing of the blood cytokines that implicate in the process of pancreatic β-cell destruction is not fully understood. poor oral hygiene stimulate proinflammatory cytokines (such as: il-1, il6, tnf-alpha, etc.) and make chronic infection worse. il-1β and/or tnf-α plus ifn-γ induce β-cell apoptosis via the activation of β-cell gene networks under transcription controlling factors, such as nf-κβ (nuclear factor-κβ) and stat-1 (signal transducers and activators of transcription-1). others mechanism of the decreased β-cell function may activate cytokines stimulated macrophages. the presence of activated macrophages within pancreatic islets in insulin-dependent diabetes mellitus suggests an involvement of ß-cell death. this paper describes that poor oral hygiene are high predisposition on the diabetic progressiveness. key words: diabetes mellitus, cytokines, progressiveness correspondence: sunarko setyawan, c/o: bagian physiology, fakultas kedokteran universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction the oral cavity is a continuous source of infectious agents, and it often reflects progression of systemic pathologies or multiple disease. diabetes is worldwide problem. the disease characterized by an increased susceptibility to infection, poor wound healing, and increased morbidity and mortality associated with disease progression.1–4 diabetes is also recognized as an important risk factor for more severe and progressive periodontitis, infection or lesions resulting in the destruction of tissues and supporting bone that form the attachment around the tooth.1 the mechanistic studies have examined the potential effects of periodontal infection in the presence periodontal tissue destruction related to an altered inflammatory response.2,3,4 the connection between periodontal disease and diabetes based on information in the literature and to discuss proper management and referral of patients who have signs and symptoms of periodontal disease and other oral complications. process of infection and inflammation can increase cytokines secretion in blood. various oral complications of the booming cytokines on diabetes that implicated to the multiple organ diseases are unclear.3 therefore the implication in the process of pancreatic βc e l l d e s t r u c t i o n i s n o t c l e a r l y u n d e r s t o o d . proinflammatory secreted cytokines (il-1, il6, tnf-alpha, ect.) increase on poor oral hygiene. il-1ß and/or tnf-α plus ifn-γ induce β-cell apoptosis via the activation of β-cell gene networks under the control of the transcription factors nf-κb and stat-1.5,6 the others mechanism may be activated by macrophages, which its cytokine stimulated for necrotic process. the presence of activated macrophages within pancreatic islets in insulin-dependent diabetes mellitus suggests an involvement of β-cell death by necrosis of β-cell death.7 these investigations show which poor oral hygiene are high predisposition of diabetic progressiveness. oral diabetic base problems diabetes and periodontal disease are common chronic diseases observed in the u.s. population. these diseases are thought to be associated biologically, and a number of reviews and studies have proposed mechanisms to explain the relationship, including microvascular disease, changes in components of gingival crevicular fluid, changes in collagen metabolism, an altered host response, altered subgingival flora, genetic predisposition, and non enzymatic glycation.4 during infection in diabetes, inflammatory cytokines induce regulated changes in the host’s internal milieu. recent evidence indicates that these cytokines are constitutively produced, their production is increased by environmental stressors other than microbes, and they modulate "normal" physiological processes.8 concentrations of pro-inflammatory cytokines are increased in the intestinal mucosa infection. polymorphonuclear neutrophil granulocytes (pmn) are the most abundant cell type in intestinal lesions, but interleukin 10 (il-10) secretion is an important contra-inflammatory cytokine which induces down regulation of proinflammatory cytokines.9 20 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 19–23 diabetes mellitus is a systemic disease with several major complications affecting both the quality and length of life. these oral complications are periodontal disease (periodontitis), decrease function of t cells, b cells and phagocytes on the oral cavity poor wound healing periodontal.10 the infection then leads to formation of pockets between the teeth and gums signaling breakdown of the periodontal apparatus and bone.11,12,13 in these factors may also be possible for the oral infection to predispose to systemic disease and this predisposes to chronic inflammation and periodontitis exacerbation. these processes can increases cytokines in blood. these cytokines can produce an insulin resistance syndrome similar to that observed in diabetes and initiate destruction of pancreatic beta cells leading to development of diabetes. it may also be possible for chronic periodontitis to induce diabetes.3,4 diabetes can lead to disturbance of the salivary secretion that markedly increased dental caries, parotid gland enlargement, inflammation and fissuring of the lips (cheilitis), inflammation or ulcers of the tongue and buccal mucosa, oral candidiasis, salivary gland infection.14 cracking, fissuring of the oral mucosa, burning mouth syndrome, and white lesions of the oral mucosa are increased in frequency.15 chronic diabetes caused salivary hypo function. salivary hypo function may predispose for secondary oral mucosal diseases. in these patients the protective coating of saliva is reduced or absent, leaving the oral mucosa more vulnerable can result in infection of the dental pulp and tooth abscess.11,13,15 systemic problem of inflammation diabetes is chronic diseases and also recognized as an important risk factor for more severe and progressive infection and inflammation.1,15 e. coli administration significantly increased serum pro-inflammatory cytokines such as interleukin (il)-1β, il-6, and tumor necrosis factor-χ and brain il-1β levels beginning at the 6-h time point.16 cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. their physiologic role in inflammation and pathologic role in systemic inflammatory states are increasingly recognized. major anti-inflammatory cytokines include interleukin (il)-1 receptor antagonist, il-4, il-6, il-10, il-11, and il-13 are recognized.17 pro-inflammatory cytokines are il-6, tnf-α, il-1β, il-8, il-10 and il-1 in the tissues inflammation likes rheumatoid arthritis that whom levels of il-6, il-8, tnf-α, il-1β and il-10 were significantly elevated.18 oral infections, especially periodontitis, as a causal factor for systemic diseases12. infectious diseases and other sources of inflammation in lifetime exposure can decrease life quality of organs. in the cohorts across the life-span since 1751 in sweden represents inflammatory processes that persist from early age into adult life.19 in vitro studies of human monocytes with diabetes have shown a hyper responsive phenotype with over expression of pro-inflammatory mediators such as interleukin-1β (il-1β), tumor necrosis factor-α (tnf-α), and prostaglandin e2. in similar in vivo studies, patients with periodontitis and diabetes were found to have significantly higher levels of local inflammatory mediators compared to systemically healthy individuals with periodontal disease.20 il-1 may exert neurotoxic or neuroprotective actions. interleukin-1 (il-1) participates in diverse forms of brain damage including ischemia, brain trauma, and excitotoxic injury. administration of low doses of il-1 markedly exacerbates these forms of brain damage, whereas blocking il-1 release or actions reduces neuronal death. il-1 receptor antagonist (il-1ra) is also up regulated by brain damage (mainly by neurons) and acts as an endogenous inhibitor of neuron degeneration, presumably by blocking il-1 actions on its receptor that can damage to pancreas cells.5,21 concentrations of pro-inflammatory cytokines are increased in the intestinal mucosa infection. polymorphonuclear neutrophil granulocytes (pmn) are the most abundant cell type in intestinal lesions, but interleukin 10 (il-10) secretion is an important contra-inflammatory cytokine which induces down regulation of proinflammatory cytokines and plays a protective role in blood vessels during diabetes.8,22,23 il-10 is an immunosuppressive cytokine or induces down regulation of proinflammatory cytokines in the immune system. il-19 belongs to the il-10 family, which includes il-10, il-19, il-20, il-22. little is known about the biologic function and gene regulation of il-19. to understand the gene regulation of human il-19, we identified a human il-19 genomic clone and analyzed its promoter region. treatment of monocytes with mouse il-19 induced the production of il-6 and tnf-α. it also induced mouse monocyte apoptosis and the production of reactive oxygen species. the results indicated that mouse il-19 may have some important roles in inflammatory responses due to the regulation of il-6 and tnf-α and induces apoptosis.8,24 discussion modulation cytokine in pancreas damage pancreatic beta cells damages are caused by mechanism of (1) apoptotic (2) acidosis were increased inflammatory and necrotic. tissue acidosis is an important feature of inflammation. acid-sensing ion channels (asics) transcript levels were increased in inflammatory conditions in vivo. now, we have found that this increase is caused by the proinflammatory mediators ngf, serotonin, interleukin1, and bradykinin. il-1 is a proinflammatory cytokine that plays important roles in inflammation. however, the role of this cytokine under physiological conditions is not known completely. these observations suggest that il-1 plays an important role in lipid metabolism by regulating insulin levels and lipase activity under physiological conditions.25 interleukin-1ß appears to be among the most important inflammatory mediators, causing pancreatic islet dysfunction and apoptosis through the up-regulation of 21setyawan: poor oral hygiene as trigger of diabetes mellitus inducible nitric oxide (no) synthase.26 cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting beta-cell lines. it is well established that cytokines such as interleukin-1beta (il-1beta), tumor necrosis factor-alpha (tnf-alpha), and gamma-interferon (ifn-gamma) inhibit beta-cell function and are cytotoxic to human and rodent pancreatic islets in vitro.5 the indicate that il-1beta-stimulated jnk (c-jun nh2-terminal kinase) activity may be distinctly targeted to cytoplasmic and/or membrane compartments in clonal insulin-producing cells, and that jip (jnk-interacting protein) may serve to localize jnk activity to specific substrates.27 decreasing of intracellular jnk signaling and confer long-term protection to pancreatic beta-cells from il-1beta-induced apoptosis type 2 diabetes. advances in the molecular biology of insulin resistance and ß-cell dysfunction increasingly support a role for inflammatory mediators, particularly cytokines, and elements of the innate immune system in the pathogenesis of type 2 diabetes. cytokine production as a consequence of an infectious challenge could potentially contribute to insulin resistance in a number of ways, including 1) modification of insulin receptor substrate-1 by serine phosphorylation, 2) alteration of adipocyte function with increased production of free fatty acids, and 3) diminution of endothelial nitric oxide production.28,29 in fact, cytokine-induced mechanisms have been suggested to participate in the ß-cell damage. type 1 diabetes. type 1 diabetes is an organ-specific autoimmune disease characterized by the destruction of the ß-cells within the islets of langerhans. the elimination of ß-cells is caused by self-reactive t-cells that infiltrate the pancreatic islets (insulitis). controversy exists as to the events that initiate the activation of the islet-reactive tcells. however, many studies have established that apoptosis is the major mechanism by which ß-cells are destroyed. a possible role for macrophage defective phagocytosis in the pathogenesis of type 1 diabetes. the ß-cell autoimmune destructions are induced by interventions that block ß-cell destructive leukocytes (t cells and macrophages) and their cytotoxic products (e.g., cytokines, perforin, granzymes, fasl/fas). type 1 diabetes is an organ-specific autoimmune disease that is mediated by autoreactive t cells. it showed that administration of a soluble dimeric peptide "major histocompatibility complex (pmhc) class ii chimera (def). autoreactive cd4+ t cells via alteration of early t cell receptor signaling and stimulation of interleukin 10"secreting t regulatory type 1 cells in the pancreas. soluble dimeric pmhc class ii may be useful in the development of immunospecific therapies for type 1 diabetes. analysis of the autoimmune nature of type 1 diabetes has led to attempts at prevention by two major approaches. first, the anti-inflammatory cytokines interleukin 4 (il-4), il-10, il-11, il-13 and transforming growth factor-β (tgf-β), antibodies specific for the proinflammatory cytokines interferon-γ (ifn-γ) and tumor necrosis factor-α (tnf-α), tcr-cd3 complex, cd4 and cd8 coreceptors, cd25 activation marker or cd40 ligand (cd40l) and b7-2 costimulatory molecules. these reagents have a variety of side effects that call into question the ethics of applying them to asymptomatic humans. second, there is the possibility of using immunospecific therapy to induce tolerance in autoreactive t cells.30 macrophage interactions with apoptotic cells can suppress inflammatory responses, but cell death by apoptosis may triggered by inflammation. tlr ligands induced early and sustained secretion of tnf-α, macrophage-inflammatory protein (mip) 1α and mip-2 with later secretion of il-10, il-12, and tgf1; apoptotic cells alone stimulated late tgf-β1 secretion only. the combination of apoptotic cells and tlr ligands enhanced early secretion of tnf-α, mip-1α, and mip-2 and increased late tgf-β1 secretion, while suppressing late tnf-β, il-10, and il-12 by mechanisms which could nevertheless be overridden by ifn-γ.31 in addition to their well known immune and proinflammatory activities, ifns possess homeostatic functions that limit inflammation and tissue destruction in a variety of conditions. the mechanisms underlying the homeostatic actions of ifns are not well understood. ifns completely suppressed the activation of il-1 signal transduction pathways in macrophages. the mechanism of ifn-mediated inhibition of il-1 action and signaling was modulation of il-1r expression, which was also observed in vivo. ifn-γ-mediated down-regulation of il-1r type i expression was dependent on stat1, a transcription factor typically considered to be a key mediator of macrophage activation by ifns. these results identify cellular and molecular mechanisms that contribute to the homeostatic role of ifns in limiting inflammation and associated tissue destruction.32 other addition to apoptosis being the main mechanism by which ß cells are destroyed, β-cell apoptosis has been implicated in the initiation of type 1 diabetes mellitus through antigen crosspresentation mechanisms that lead to β-cell-specific t-cell activation. caspase-3 is the major effector caspase involved in apoptotic pathways. despite evidence supporting the importance of β-cell apoptosis in the pathogenesis of type 1 diabetes, the specific role of caspase-3 in this process is unknown.33 fas seems to be implicated in β-cell apoptosis via an intracellular death domain and proinflammatory cytokines can induce up-regulation of fas expression on β-cells, making them susceptible to apoptosis in the presence of agonistic anti-fas antibodies or interaction with fas-ligand (fasl, cd95l)-expressing t-cells. the role of fas in β-cell apoptosis is still under debate and has been challenged by several studies. in addition, up-regulation of several anti-apoptotic members of the bcl-2 family of proteins, such as bcl-2 and bcl-xl has been closely associated with increased resistance to apoptosis and potentially to diabetes susceptibility.34 diabetic progressiveness on the quality of oral hygiene periodontitis can cause factor for systemic diseases.12 infectious diseases and other sources of inflammation in oral are excessively, especially adult life.19 uncontrolled 22 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 19–23 diabetes and low of oral hygiene evoked chronic infection and diabetic progressiveness.3,4,11 infection progressiveness and inflammation can be fluctuation of blood cytokines.8,20 the blow up blood cytokines that implicated in the process of pancreatic β-cell destruction is not fully understood. secretion of proinflammatory cytokines (il-1, il6, tnfalpha, ect) increase on poor oral hygiene (infection and inflammation). il-1β and/or tnf-α plus ifn-β induce β-cell apoptosis via the activation of β-cell gene networks under the control of the transcription factors nf-κb and stat-1. 5,24,26,32,35 this argues against a unifying hypothesis for the mechanisms of β-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent ß-cell death in type 1 and type 2 diabetes.36 others investigation showed that combination of il-1β plus interferon-γ causes a time-dependent increase in apoptotic cells in the β-cells (figure 1). 6 the others mechanism of the decreased ß-cell function maybe activated macrophages. the presence of activated macrophages within pancreatic islets in insulin-dependent diabetes mellitus suggests an involvement of ß-cell death by necrosis of ß-cell death.37 these investigations show that poor oral hygiene are high predisposition of diabetic progressiveness. diabetes is chronic diseases and also recognized as an important risk factor for more severe and progressive infection and inflammation in oral. infection progressiveness and inflammation in oral can increase blood proinflammatory cytokines. incremental of the cytokines may play important roles in pancreatic ß-cells islet destruction. diabetic patient ought to preserve oral hygiene to avoid progressiveness these disease. references 1. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 2004;27:1047-53. 2. tsai c, hayes c, taylor gw. glycemic control of type 2 diabetes and severe periodontal disease in the us adult population. community dent oral epidemiol 2002;30:182-92. 3. southerland jh, taylor gw, offenbacher s. diabetes and periodontal infection: making the connection. clinical diabetes 2005; 23:171–8. 4. lacopino am periodontitis and diabetes interrelationships: role of inflammation. ann periodontol 2001; 6:125–37. 5. hoorens a, stangé g, pavlovic d, pipeleers d. distinction between interleukin-1–induced necrosis and apoptosis of islet cells. diabetes 2001; 50:551–7. 6. van de casteele m, kefas ba, ling z, heimberg h, pipeleers dg. specific expression of bax-ω n pancreatic β-cells is down-regulated by cytokines before the onset of apoptosis. endocrinology 2002; 143(1):320–6. 7. saldeen j. cytokines induce both necrosis and apoptosis via a common bcl-2-inhibitable pathway in rat insulin-producing cells. endocrinology 2000; 141(6):2003–10. 8. cannon jg. inflammatory cytokines in no pathological states. news in physiological sciences 2000; 15(6):298–303. 9. nikolaus s, bauditz j, gionchetti p, witt c, lochs h, schreiber s. increased secretion of pro-inflammatory cytokines by circulating polymorph nuclear neutrophils and regulation by interleukin 10 during intestinal inflammation. gut 1998; 42:470–6. 10. giannobile wv. c-telopeptide pyridinoline cross-links: sensitive indicators of periodontal tissue destruction. annals of the new york academy of sciences 1999; 878:404–12. 11. atkinson jc, o’connell a, aframian d. oral manifestations of primary immunological diseases. am dent assoc 2000; 131(3): 345–56. 12. taylor gw, burt ba, becker mp, genco rj, shlossman m. glycemic control and alveolar bone loss progression in type 2 diabetes. ann periodontol l 1998; 3:30–39. 13. li x, kolltveit km, tronstad l, ingar olsen i. systemic diseases caused by oral infection. clinical microbiology reviews 2000; 13(4):547–58. 14. bauroth k, charles ch, mankodi sm, simmons k, zhao q, kumar ld. the efficacy of an essential oil antiseptic mouth rinse vs. dental floss in controlling interproximal gingivitis: a comparative study. j am dent assoc 2003; 134:359–65. 15. geerlings se, hoepelman ai. immune dysfunction in patients with diabetes mellitus (dm). fems immunol med microbiol 1999; 26:259–65. 16. jensena jl, barkvoll p. clinical implications of the dry mouth: oral mucosal diseases. annals of the new york academy of sciences 1998; 842:156–62. 17. campisi jmk, hansen mk, o’connor ka, biedenkapp jc, watkins lr, maier sf, fleshner m. circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral e. coli challenge. j appl physiol 2003; 95:1873–82. 18. opal sm, depalo va. anti-inflammatory cytokines. chest 2000; 117:1162-72. il-1 il-6 tnf-α apoptosis & necrosis macrophage activated figure 1. mechanisms of β-cell apoptosis and necrosis. 23setyawan: poor oral hygiene as trigger of diabetes mellitus 19. nowlan ml, drewe e, bulsara h, esposito n, robins ra, tighe pj, powell rj, todd i. systemic cytokine levels and the effects of etanercept in tnf receptor-associated periodic syndrome (traps) involving a c33y mutation in tnfrsf1a. rheumatology 2006; 45(1):31–37. 20. finch ce, crimmins em. inflammatory exposure and historical changes in human life-spans. science 2004; 305(5691):1736–9. 21. salvi ge, beck jd, offenbacher s. pge2, il-1 beta, and tnf-alpha responses in diabetics as modifiers of periodontal disease expression. ann periodontol 1998; 3:40–50. 22. stuart m. the role of proand antiinflammatory cytokines in neurodegeneration. annals of the new york academy of sciences 2000; 917:84–93. 23. gunnett ga, heistad dd, berg dj, faraci fm. il-10 deficiency increases super oxide and endothelial dysfunction during inflammation. am j physiol heart circ physiol 2000; 279:1555–1562 24. gunnett ca, heistad dd, faraci fm. interleukin-10 protects nitric oxide–dependent relaxation during diabetes. role of super oxide. diabetes 2002; 51:1931–7. 25. liao yc, liang wg, chen fw, hsu jh, yang jj, chang ms. il-19 induces production of il-6 and tnf-α and results in cell apoptosis through tnf-α1. the journal of immunology 2002; 169:4288–97. 26. matsuki t, horai r, sudo k, iwakura y. il-1 plays an important role in lipid metabolism by regulating insulin levels under physiological conditions. the journal of experimental medicine 2002; 198(6):877–88. 27. moulin n, widmann c. islet-brain (ib)/jnk-interacting proteins (jips): future targets for the treatment of neurodegenerative diseases?. current neurovascular research 2004; 1(2):111–27. 28. barshes nr, wyllie s, goss ja. inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts. journal of leukocyte biology 2005; 77:587–97. 29. hotamisligi gs, peraldi p, budavari a, ellis r, white mf, spiegelman bm. irs-1-mediated inhibition of insulin receptor tyrosine kinase activity in tnf-alphaand obesity-induced insulin resistance. science 1996; 271:665–8. 30. kolb h, poulsen mt. an immune origin of type 2 diabetes? diabetologia 2005; 48:1038–50. 31. casares s, hurtado a, mcevoy rc, sarukhan a, von boehmer h, brumeanu td. down-regulation of diabetogenic cd4+ t cells by a soluble dimeric peptide – mhc. 2002. 32. lucas m, stuart lm, savill j, hulbert la. apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion. the journal of immunology 2003; 171:2610–5. 33. hu x, ho hh, lou o, hidak c, ivashkiv lb. homeostatic role of interferons conferred by inhibition of il-1-mediated inflammation and tissue destruction. the journal of immunology 2005; 175:131–8. 34. liadis n, murakami k, eweida m, elford ar, sheu l, gaisano hy, hakem r, ohashi ps, woo m. caspase-3-dependent ß-cell apoptosis in the initiation of autoimmune diabetes mellitus. molecular and cellular biology 2005; 25(9):3620–9. 35. gil mjm, diosdado am. assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines. biol proced online 2005; 7:162–71. 36. ohara-imaizumi m, cardozo ak, kikuta t, eizirik dl, nagamatsu s. the cytokine interleukin-1β reduces the docking and fusion of insulin granules in pancreatic β-cells, preferentially decreasing the first phase of exocytosis. j biol chem 2004; 279(40):41271–4. 37. cnop m, welsh n, jonas jc, jörns a, lenzen s, eizirik dl. mechanisms of pancreatic ß-cell death in type 1 and type 2 diabetes. diabetes 2005; 54:s97–s107. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 103 vol. 41. no. 3 july–september 2008 cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using mtt assay on culture cell line christian khoswanto, ester arijani and pratiwi soesilawati department of oral biology faculty of dentistry airlangga university surabaya indonesia abstract background: open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. the researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. even though citric acid is not strong acid but it can be very erosive to oral mucous. several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. purpose: this study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using mtt assay. method: this study is an experimental research using the post-test only control group design. six samples of each 40, 50 and 60% citric acid for citotoxicity test using mtt assay. the density of optic formazan indicated the number of living cells. all data were statistically analyzed by one way anova. result: the percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. conclusion: citric acid is non toxic and safe to be used as dentine conditioner. key words: cytotoxicity, citric acid, mtt assay correspondence: christian khoswanto, c/o: departemen biologi oral, jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: christian khoswanto@hotmail.com introduction the structure of natural tooth is limited structure, therefore it should be maintained and protected as long as possible, consequently, in selecting restorative material, some aspects are necessarily considered in order to obtain the most proper material.1 open dentin is always covered by smear layer therefore, before restoration is performed, cavity or tooth which has been prepared should be clean from dirt produced during preparation, bacteria produced by carries or should not be contaminated by environment.2 the researchers suggested that clean dentin surface would reach effective adhesion between resin and dentin material, therefore dentin conditioner is used to reach the condition.3 dentin conditioner is solution which is polished on dentin surface.4 the term of dentin conditioner can be meant as an effort to make or to prepare dentin surface to be able to accept dentin bonding. dentin conditioner usually consist of weak acid solution and design to modify or clean the smear layer on dentin surface which has been prepared to be able to function well as supporting material for dentin bonding. the material can also be capable to wet the dentin surface, it can decrease constrain of dentin surface. in this way dentin bonding material can penetrate more into dentin tubule and finally the condition of binding each other can be reached.5 dentin conditioner technique for glass ionomer cement bonding in tooth substrate is applied in dentistry field with the aim to increase adhesion between resin and tooth substrate.5 glass ionomer cement is widely applied in class v cavity on cervical tooth which is bordered by gingival, prior to cement application in the cavity, clean surface is conditioner. in vitro study on the surface of the third molar showed that the effect of 40, 50 and 60% citrate acid dentin conditioner was effective.6 citric acid is not strong acid but research report 104 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 103-106 it can be very erosive due to the capability to bind metal ion. the other researcher found that influence of citrate acid is necessarily observed if it has surface contact either in enamel or dentin. enamel surface which is polished by citric acid will be erosive. smear layer will disappear from dentin and open dentin tubule through the loss dentin perihedral.7 powis et al. 8 suggested that the effect of citric acid is necessarily observed if it gets into contact with the surface either polished enamel or dentin. enamel surface which is polished by citric acid will be erosive. optimal adhesion will not be reached using excessive amount of citric acid or citrate acid in excessive high concentration. the loss of smear layer and tubule opening can cause tooth demineralization.7 the requirement of dental product applied in dentistry are non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment.9 cytotoxicity test of a material can be done using enzymatic test mtt [3 –(4.5–dimethylthiazol–2yl); 2.5–diphenyltetrazolium bromide]. mtt test is used to measure the capability of living cell based on mitochondria activity of culture cell which can reduce yellow tetrazolium bromide into insoluble purple blue formazan endapan. the product of formazan is solved by solvent in order to be easily detected. in this way, the number of detected living cell using spectrophotometer as the result of mtt product. the darker the color of purple blue, the higher the absorbent score and the higher the number of living cell this test is widely used to measure quantitatively cellular proliferation or the number of living cell.10 the advantage of this test is the accuracy and sensitivity measurement because the spectrophotometer which is used can detest clearly metabolism alteration, can easily be manipulated, and available at the laboratory, can save time and energy and it does not use radioactive isotope. based on the above reasons, in this study mtt test was used to test the cytotoxicity of dental material using fibroblast culture cell of baby hamster kidney-21 (bhk-21) considering the origin of this cell is embryonic cell so it would be easily grow and repeated subculture is easily done, having stable character, sensitive and mutation does not occur.11 based on the above aspects, the problem arises whether there is different cytotoxic effect of 40, 50 and 60% citrate acid dentin conditioner on mtt test. the purpose of this study was to prove the cytotoxicity of 40, 50 and 60% citric acid as dentin conditioner using mtt assay. the advantage of this study would inform the safety of citric acid as dentin conditioner, especially for dentistry colleague before filling the cavity and citric acid can be alternative material for dentin conditioner. material and method this study is an experimental research using the posttest only control group design. fibroblast cell culture (baby hamster kidney-21) was used with the number of samples was 8 based on the estimation, material and tools were applied such as: incubator for cell culture, micro-plate reader, micropipet multi channel, roux bottle, yellow tips, laminar flow, mtt reagent (sigma), saline water, dimetilsulfoksid (dmso) solution, 40, 50 and 60% citric acid, cell culture of baby hamster kidney (bhk-21), culture media rosewell park memorial institute–1640 (rpmi-1640) 89%, 1% penstrep, 10% fetal bovine serum (fbs), 100 until/ml fungizone, phosphate buffer saline (pbs). cell culture of bhk-21 in cell-line form was embedded in roux bottle. after it was confluent, the culture was harvested using trypsine versene solution. the result was embedded in media rosewell park memorial institute 1640 containing 10% fetal bovine serum albumin incubated for 24 hours at 37° c. the cell was cultured in every microplate 96–well until confluent. every well contained cell and rpmi media with 2 × 105 cell/ml density in 50m then each well was given 20 m of 40, 50 and 60% citric acid. each group consisted of 8 samples. control cell was prepared as positive control, and it was considered the percentage of living cell was 100%. control media as negative control considered the percentage of living cells was 0%. the microplate was incubated at 37° c, then, removed from incubator, added by 5 mg/ml mtt in pbs 20 m for every well, incubated again for 4 hours. next, every well was added by 50 ml dmso. formazan which had been formed was solved by into the solvent and the absorption was measured by a reader using spectrometer with 595 nm ware length. to know the percentage member of living cell, the equation below was used.12 table 1. mean of formazan optic density, standard deviation, percentage of living cell concentration of citric acid number of sample mean of optic formazan density standard deviation % living cell 40% 50% 60% control cell control media 8 8 8 8 8 0.254 0.248 0.247 0.271 0.079 0.008 0.007 0.008 0.007 0.0072 95.14% 93.42% 93.14% 100% 0% 105khoswanto, et al.: cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner % living cell = tested group + media × 100 cell + media note: % living cell = percentage number of living cell after the test tested group = score of formazanoptic density of every sample after the test media = score of formazan optic density in control media cell = score of formazan optic density in control result the data tabulated, then, statistical analysis using one-way anova test with 5% significant rate was done and continued by tukey hsd test if there was significant difference. the mean of formazan optic density, standard deviation, percentage of living cell could be seen on table 1. in tested group has show significant decrease. percentage of living cell that is percentage of optic density of dehydrogenase mitochondria enzyme in cell culture of bhk-21 as well as in control media has shown in table 1. table 1 shows all distribution group is normal, continued by parametrix test of one way anova to know the difference in the group with 5% significant rate (table 2). table 2 shows there is significant difference in every citric acid group and control, therefore continued by tukey hsd seen on table 3. table 3. the result of tukey hsd test shows the difference of living cell percentage between control group and 40, 50 and 60% citric acid group control cell group 40% group 50% group 60% control cell group 40% group 50% group 60% s s s ns ns ns s for significant and ns for non significant discussion citric acid is weak organic acid, functions as natural preservative substance, cleansing material and anti oxidant, used to add sour taste in food and soft drink, found in fruit especially citrus and vegetable. lemon and lime have the highest content of citric acid which containing 8 % of the whole fruit weight.13 dentin conditioner is solution which is polished on dentin surface.4 the term of dentin conditioner can be meant as an effort to prepare dentin surface so that it can accept dentin bonding. dentin conditioner usually consist of weak acid solution such as citric acid and designed to clean smear layer on dentin surface which has been prepared so that it can function well as supporting material of dentin bonding. the method is previously wetting dentin surface before polishing by dentin bonding. then, conditioner is cleansed using water spraying and dried by air blowing. so, dentin conditioner is solution which is polished on dentin surface before being covered by dentin bonding.5 the adhesion mechanism has not been exactly exposed, probably, this material could be wet much better the dentin surface. in this way, dentin bonding material can penetrate deeply into dentinal tubule and then bound each other.4 in this study, citric acid was used as dentin conditioner material. citric acid would permanently demineralized dentin surface by erasing smear layer and the most superficial dentin. the acidity of dentin conditioner would open dentinal tubule and making tissue of collageneous fibre due to microporousity which is filled by water in intratubular and peritubular dentin.14,15 the use of demineralized material on dentin surface is to improve the bond strength between tooth and restorative material. there are some controversial opinions on dentin conditioner. currently, dentin conditioner of third generation is made of weak acid, one of them is citric acid. however, the safety of citric acid in high concentration is still doubtful, considering strong acid such as 37% phosphate acid has proven that dentin bonding containing strong acid is very dangerous for the vitality of pulp tissue. citric acid as dentin conditioner can wash smear layer by hydroxiapatite dentin reaction. citric acid will release hydrogen ion so dentin structure would be demineralized. citric acid will bind calcium ion substrate phosphate ion in hydroxiapatite structure.16 one of the requirement of dental product to be applied in oral cavity should be biocompatible such as nontoxic substance.9 in vitro study of cytotoxicity using mtt assay table 2. the anova result from fibroblast cell on citric acid and control cell variation source number square free level mean square f. account probability between group in group 0,201 0,002 4 35 0.05 0.001 838.848 0.001 total 0,203 39 106 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 103-106 on culture cell lines are used due to the advantages such as passage can be done 50-70 times, high rate cell growth, well maintained cell integrity, cell is capable of multiplying in suspension cell lines have been widely applied in toxicity test for dental material and medicine, such as cell of bhk21.17 the result using bhk-21 can be applied as accurate basic test.18 mtt is yellow soluble molecule which can be used to evaluate cellular enzymatic activity based on the capability of living cell to reduce mtt. the mechanism is yellow tetrazolium salt would be reduced in cell which has metabolic activity.19,20 mitochondria are the principle sites for synthesis of adenosine triphosphate, surrounded by two membranes, which are structurally and functionally distinc. mitochondria of living cell also has important role produces dehydrogenase.21-23 if dehydrogenase is not active due to cytotxicity effect, formazan will not be formed. the number of formed formazan is equal to enzymatic activity of living cell. the result of this study shows the living cell of 40% citrate acid = 95.14%, group 50% = 93.42%, group 60% = 93.14%. the result of hsd test shows significant difference between control group and citric acid group. decreasing number of living cell in each tested group shows cytotoxicity of citric acid basically due to the effect of acidity than the content of citrate acid it self. most common case of dead cell due to citric acid can be prevented by regulating ph of culture media to be 7.5. in the study using sodium citric in 47.6% mmol/l concentration can cause almost no dead cell effect.24 the percentage of living cell in every tested group is almost 100%, meaning dentin conditioner of 40, 50 and 60% citric acid is safe due to good biocompatibility limit for the number of living cell 92.3–100%.19 this score is higher compared to the limit used by telli et al.,25 suggested that the parameter of toxicity based on cd50 meaning that material which is thought to be toxic if the percentage of living cell is below 50%. the number of living cell after citrate acid application in this study shows citric acid is relatively safe and it is supported by other study indicates that this acid can cause less dentin resorption and less coagulation in blood and tissue compared to other acid materials, reaction on the pulp will not occur and can have reaction with hydroxiapatite dentin.16 the advantages of using citric acid are: cheap, easily applied and bought. however, the pulp is necessarily protected before the acid is applied. especially thin dentin in deep caries of the occurrence of channel on root surface. the conclusion is cytotoxicity test using mtt assay shows 40, 50 and 60% citric acid is not toxic and safe for dentin conditioner. further in vivo study is necessarily done to know completely the biocompatibility effect. citric acid concentration in clinical use is essentially observed considering the acid concentration as dentin conditioner is different from the concentration as material of root canal irrigation. references 1. nawangsari n. perbedaan sitotoksisitas bahan bonding self etch dan total etch. surabaya: karya tulis akhir; 2004. p. 1–2. 2. prijambodo sk. pengaruh pemberian smear layer pada permeabilitaspengaruh pemberian smear layer pada permeabilitas dentin. int dent 1996; 3:133. 3. nordenvale kj, brannstrom m. in vivo impregnation dentin tubules. j prost dent 1980; 76:254–9. 4. fortin d, swift ej, deneby ge, reinhardt jw. bond strength andbond strength and micro leakage of current dentin adhesives. dent material 1994; 10:253–8. 5. attal jp, asmussen e, degrange m. effect of surface energy of dentin. dent material 1994; 10:159–264. 6. pramita tp. pengaruh perbedaan konsentrasi asam sitrat sebagai dentin konditioner pada permukaan dentin. surabaya: karya tulis akhir; 2006. p. 4–14. 7. wahyu a. perbedaan kekuatan per1ekatan tank antara resin komposit dan dentin yang diulas dengan larutan asam laktat 500/0 dan larutan asam sitrat 50%. surabaya: skripsi; 2002. p. 2–3. 8. powis dr, folleras t, mearson sa, wilson ad. improved adhesion of glass ionomer cement to dentin and enamel. j dent res 1982; 61:1416–22. 9. noort vr. introduction to dental material. 2nd ed. london: cv mosby company; 2003. p. 3–5. 10. fernandez br, vetviaeka v. method in cellular immunology. boca raton, new york: crc press; 1995. p. 47–52. 11. freshney ir. culture of animal cells. 2culture of animal cells. 2nd ed. new york: alan r lissnew york: alan r liss inc; 1987 p. 227–45. 12. titien ha. pengaruh tegangan listrik dan lama penyinaran pada semen ionomeri gelas modifikasi resin terhadap kekerasan permukaan dann dan sitotoksisitas. tesis. surabaya: pasca sarjana universitas airlangga; 2002. p. 25–35. 13. wikipedia, encyclopedia. wikipedia@ is a registered trademark of the wikimedia foundation, inc. available at: http://en.wikipedia. org/wiki/citric acid. accessed april 2006. 14. perdigao j. the effect etching time on dentin demineralization. quintessence international 2001; 32:19–26. 15. bath mb, fehrenbach mj. dental embryology, histology and anatomy. 2nd ed. missouri: elsevier saunders; 2006. p. 192–5. 16. soetanto, dewi, caecillia. pemakaian asam sitrat pada perawatan periodontitis marginalis gigi molar dengan cervical enamel projection. surabaya: karya tuiis akhir; 2001. p. 15–20. 17. siregar f, hadijono bs. uji sitotoksisitas dengan esei mtt. jkgui 2000; 7:28–32. 18. craig rg, powers jm. restorative dental materials. 6th ed. london: mosby co; 2002. p. 135–40. 19. rubianto m. biokompatibilitas bahan allograft (human bone powder) dibandingkan dengan bahan alloplast (hydroxylapatite). kumpulan naskah temu ilmiah nasional i (timnas i) fkg unair 1998; p. 507–509. 20. junqueira lc, carneiro j, kelley ro. basic histology. 9basic histology. 9th ed. appleton and lange. 2006. p. 14. 21. nanci a. oral histology development structure and function. 6th ed. st louis: mosby; 2003. p. 341. 22. burns er, cave md. histology and cell biology. 2nd ed. philadelphia: mosby; 2007. p. 13. 23. sherwood l. fisiologi manusia. edisi 2. jakarta: penerbit bukujakarta: penerbit buku kedokteran egc; 2001. p. 26. 24. lan wc, lan wr, chan cp, hsieh cc, chang mc, jeng jr. thethe effect of extra cellular citric acid acidosis on the viability, cellular adhesion capacity and protein synthesis of cultured human gingival fibroblast. 1999. p. 25–35. 25. telli c, serper a, dogan al, guc d. evaluation of the citotoxycity of calcium phosphate root canal sealers by mtt assay. j endo 1999; 25:811–3. 67 volume 47, number 2, june 2014 the relationship determination between menarche and the peak of skeletal maturation using hand wrist and cervical vertebrae index endah mardiati,1 soemantry es,1 haroen er,2 thahar b1 and sutrisna b3 1departement ortodontics, faculty of dentistry universitas padjadjaran, bandung-indonesia 2departement physiology, faculty of dentistry universitas padjadjaran, bandung-indonesia 3faculty of public health, universitas indonesia, jakarta-indonesia abstract background: menarche and skeletal maturation indices are physiological maturation indicators that can be used to establish the maturation stage of individual patient in orthodontic treatment, especially in orthodontic growth modification and orthognatic surgery. purpose: the purpose of this study was to determine the relationship between menarche and the peak of skeletal maturation using hand-wrist and cervical vertebrae indexes. methods: this was an observational diagnostic research with 220 female of deutero-malay indonesian subjects aged 8-17 years from dental hospital faculty of dentistry universitas padjadjaran, and some privates orthodontic practice in bandung. all subjects had hand-wrist radiograph and lateral cephalogram. menarche data were collected through interview with the subjects and their parents. there were 89 subjects who already had menarche but only 84 of them remembered the month and year of their menarche. the stage of hand-wrist skeletal maturation was analyzed using fishman method and cervical vertebrae maturation was analyzed using baccetty et.al., method. results: the result indicates that the menarche age of indonesian deuteromalay subject were 12.47 ± 0.73 year. the youngest age of were 10.92 ± 0.0 year and the oldest were 13.83 ± 0.23 year. conclusion: menarche could be used as an indicator that the pubertal growth peak has been exceeded and to predict the end of the pubertal growth. this study showed that 0.49 years after mp3cap stage of hand-wrist skeletal maturation index and 0.69 years after cvms2 stage of cervical vertebrae skeletal maturation index, the subject of indonesian deutero-malay will have their menarche. key words: menarche, hand-wrist maturation index, vertebrae maturation index, indonesian deutero-malayid abstrak latar belakang: menarke dan indeks maturasi skeletal merupakan indikator maturasi fisologis yang dapat digunakan untuk menentukan tahap maturasi pasien pada perawatan ortodonti modifikasi pertumbuhan dan bedah ortognati. tujuan: tujuan penelitian ini adalah menentukan hubungan antara menarke dengan puncak pertumbuhan skeletal dengan menggunakan indikator maturasi handwrist dan vertebra servikal. metode: penelitian ini merupakan penelitian diagnostik obeservasional dengan 220 subjek perempuan umur 8-17 tahun yang datang ke rumah sakit gigi dan mulut universitas padjadjaran bandung. seluruh subjek penelitian mempunyai foto rontgen hand-wrist dan sefalogram lateral. data menarke diperoleh melalui wawancara kepada pasien dan orang tuanya. subjek yang telah mengalami menarke sebanyak 89 orang tetapi hanya 84 subjek yang ingat dengan tepat bulan dan tahunnya. tahap maturasi hand-wrist dianalisis dengan menggunakan metode fishman dan tahap maturasi vertebra servikal ditentukan dengan menggunakan metode baccety dkk. hasil: hasil penelitian menunjukkan bahwa rata-rata umur menarke terjadi pada umur 12.47 ± 0,73 tahun, dengan umur termuda pada10,92 ± 0,0 tahun dan tertua pada umur13,83 ± 0,23 tahun. simpulan: menarke dapat digunakan sebagai indikator untuk menentukan bahwa puncak pubertas telah terlampaui dan untuk memprediksi akhir pertumbuhan pubertal. studi ini research report 68 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 67–71 menunjukkan bahwa 0,49 tahun setelah mp3cap stage dari maturasi skeletal pergelangan tangan dan 0,69 tahun setelah cvms2 maturasi skeletal vertebra leher anak perempuan indonesia deutero melayu akan memasuki masa haid. kata kunci: menarke, indeks maturasi hand-wrist, indeks maturasi vertebra servikal, subjek indonesia ras deutero-malayid correspondence: endah mardiati, c/o: fakultas kedokteran gigi, universitas padjadjaran. jl. sekeloa selatan i no. 1 bandung 40132, indonesia. email: endah_mardiati@yahoo.com introduction growth and development is an important factor in the treatment of orthodontic growth modification and orthognatic surgery of skeletal class ii and class iii maloclusion. the ideal time for orthodontic growth modification treatment is during the active of pubertal growth, whereas ortognathic surgery is after the growth has been finished.1-3 active stage of pubertal growth, is more accurate if determined using physiological maturation indicators, such as handwrist or cervical vertebrae skeletal maturation index, dental maturation indicators and menarche as secondary sexual maturation indicator in female.4-6 in orthodontic treatment, cervical vertebrae maturation indicators can be observed directly on the lateral cephalogram that commonly used to determine the diagnosis of orthodontic treatment.6 some studies showed that hand-wrist maturation index has polimorphism and sexual dimorfism7,8 whereas cervical vertebrae maturation index still controversi. some studies indicates that cervical vertebrae maturation index is a valid and reliable method for determination the stages of pubertal growth while other studies is not valid. 9-11 under these conditions menarche can be use as maturation index for determining the pubertal growth stages in female. menarche is a physiological maturation indicator that easily identified through interview with patient or parents. some studies showed that menarche reached approximately one year after the peak of skeletal growth.4,11 the purpose of this study was to determine the relationship between menarche and the peak of skeletal maturation using hand-wrist and cervical vertebrae indexes of indonesian deutero-malay subjects. in indonesia, menarcheal data has been widely studied, but how it relates to the skeletal maturation is still unknown. materials and methods this study was a diagnostic observational study. the subjects were 220 girls aged 8-17 years from orthodontic clinic faculty of dentistry, universitas padjadjaran, and some private specialist orthodontic practice in bandung. eighty nine subjects had menarche but only 85 subjecs remember their menarche. the subjects of this study have hand-wrist radiograph and lateral cephalogram with inclusion criteria, such as: indonesian deutero-malay, healthy, not suffering from serious diseases or systemic diseases, does not suffer from defects in dento-craniofacial structures, never have experienced with trauma in the neck and hand-wrist. hand-wrist maturation index of fishman method and cervical vertebrae maturation index of baccety et al.,5 method was used to analyzed the skeletal maturation stage, while menarche data was obtained retrospectively by interviewing the subject and parents. subjects who did not remember their menarche were not included for analysis. 12 hand-wrist maturation indicators of fishman method consists 11 skeletal maturation indicator (smi), which is epiphysis width equal with diaphysis width (smi1smi3), ossification of sesamoid bone (smi4), capping of epiphysis to the diaphysis (smi5-7) and fusion of epiphysis and diaphysis (smi8-11) (figure 1). skeletal maturation indicator 1-4 shows the acceleration of pubertal growth, smi 5-7 shows the peak of pubertal growth and smi 8-11 figure 1. location of assessment indicator hand-wrist maturation of fishman method.13 maturity indicators 69mardiati, et al.: the relationship between menarche, hand-wrist and cervical vertebrae maturation indicator shows a decrease and followed by the cessation of skeletal growth.12 cervical vertebrae maturation of baccety method consists of 5 stages indicator, using the changes of the 3rd an 4th cervical vertebrae body that can be observed on lateral sefalogram (figure 2).5 cvms1 and cvms2 showed pubertal growth acceleration, cvms3 , showed peak pubertal growth and cvms 4-cvms5 showed a decline and cessation of pubertal growth. to determine the validity and reliability of measurements, hand-wrist and lateral cephalogram from 10 subjects was randomly selected. measurements were performed 3 times with two weeks of interval. there were no significant differences for the determination of the handwrist maturation stage (p=0.042) and for cervical vertebrae maturation stage (p=0.375). statistical analysis was performed using spss 17 to determine the mean and standard deviation of handwrist and cervical vertebrae maturation stages and the relationship with the menarche (p=0.05). results menarche is an indicator of physiological maturation that can be used to determine the stages of pubertal maturation through interviews with patient or parents. study showed that menarche occur one year after the peak of pubertal growth and indicate that skeletal growth has been decline and less effective for orthodontic growth modification treatment.6 the result of this study showed figure 2. five-stages of cervical vertebrae maturation..5 table 1. the age of hand-wrist maturation stages of pubertal growth (year) hand-wrists maturation stage pp3= mp3= mp5= s dp3 cap mp3 cap mp5 cap dp3 u pp3 u mp4 u ru 9.41 ± 0.14 9.72 ± 0.66 10.40 ± 0.74 10.84 ± 0.69 11.24 ± 0.58 11.82 ± 0.56 12.87 ± 0.55 13.01 ± 0.78 13.68 ± 0.83 14.66 ± 0.89 15.93 ± 0.63 table 2. the age of cervical vertebrae maturation stages of pubertal growth (year) cvms1 cvms2 cvms3 cvms4 cvms5 9.91 10.61 12.58 13.88 15.14 ± ± ± ± ± 0.89 0.98 1.28 1.40 1.15 table 3. the mean age of menarche (year) calendar age n average sd minimum maximum 10 – 10.9 2 10.92 0.00 10.92 10.92 11 – 11.9 29 11.56 0.26 11.08 11.92 12 – 12.9 35 12.47 0.26 12.00 12.92 13 – 13.9 18 13.31 0.23 13.00 13.83 total 84 12.300 0.73 10.92 13.83 70 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 67–71 that initial acceleration of pubertal growth, pp3= is reached at 9.41 ± 0.14 years. the peak of pubertal growth (mp3cap) indicator was reached at 11.82 ± 0.56 years (tabel 1), the peak of pubertal growth of cervical vertebrae cvms ii is at 10.62 ± 0.98 years and cvms iii at 12.58 ± 1.2 years, while the average of menarche was 12.30 years (table 2). one year after the peak of skeletal growth, female would have their menarche, and in this study, if menarche was associated with indicators of hand-wrist maturation and cervical vertebrae maturation indicator, showed that 0.47 years after mp3cap and 1.69 years after cvms2 or 0.49 years before cvms3, the subjects would have menarche.12 the mean of menarche shown in table 3. discussion identification of pubertal growth stage, to determine how much the growth has been taken, or how long it will be finished, very important especially in orthodontic growth modification treatment and orthognatic surgery. studies showed that the success of preventive and interseptive treatment in maxillo-mandibular disharmony, was in the begining until around the peak of pubertal growth, while orthogantic surgery was after pubertal growth has been completed.1,2 the result of this study showed that using hand-wrist skeletal maturation index, initial acceleration stage of pubertal growth (pp3) is at 9.41 ± 0.14 years and the peak of pubertal growt (mp3cap) was reached at 11.82 ± 0.56 year (table 1), while using cervical vertebrae maturation index the acceleration stage cvms1 is at 9.91 ± 0,89 year, and the peak of pubertal growth (cvms3) is 12.58 ± 1.28 year (table 2). the average of menarche was 12.30 years (table 3). if menarche was associated with skeletal maturation index showed that after 0.47 years of mp3cap stage and 1.69 years after cvms2 or 0.49 years before cvms3, the subjects would have menarche. the results of the study in taiwan, from 148 subjects, showed that the average age of menarche is 11.97 years, and the average of menarche occurs at the stage iv-v ntuh-smi and v, and between stages iii and iv.13 the clinical implication in orthodontic treatment, patient who has menarche, has been in a decline stage of pubertal growth, and interceptive orthodontic treatment for maxillomandibular disharmony using functional appliances would have insignificant result. by knowing menarche of a patient, it can be used to predict how long the pubertal growth will be completed so that orthognatic surgery can be done without the risk of relaps due to the growth. the result of this study indicate that the final stage of pubertal growth (ru stage) using hand-wrist maturation indicator will be achieved in 3.63 years after menarche, while the final stage of cervical vertebrae maturation indicator (cvms5) will achieved 2.84 years after menarche. menarche data in this study was obtained retrospectively, by interviews with subjects and their parents. most subjects remember the exact month and year using indicators of special events like her birthday, school holiday, fasting month or new year eve. some menarche data in indonesia, showed that rural children menarche occured at 11.46 ± 0.99 years while urban children occured at 11.87 ± 0.99 years.14 the research of batubara15 in seven provinces in indonesia, menarche occured from the age of 12.5 years to 13.6 year..in this study the mean of menarcheal age was 12.30 ± 073 year with the youngest was 10.92 years and the oldest was 13.83 years. in industrial country the age menarche in usa shows at the age of 12.5 years. in european countries the average menarche in turkish was estimated as 13.30 year, swedia 13,05 ±5.. and uk 13.06 ± 0.10 years.16-18 for some countries in asia, average menarche in taiwan occured at the age of 11.97 year, in japan at the age of 12.2 year, and in korea at the age of 13.10 ± 0.06 year. when the results of this study compared with several countries, the menarche of indonesian children is later than the average taiwanese children, or nearly the same as the japanese children, but earlier than korea, turkey, sweden, usa, uk and canada.14,19-21 this differences can be caused by many factors such as the sample size, genetic, geographic, racial and etnic.11 the conclusion of this research is that menarche in indonesia deutero-malay girls varies between the age of 10.92 ± 0.00 years to 13.31 ± 0.23 years with a mean of 12.30 ± 0.73 years. menarche is associated with skeletal maturation stage hand-wrist and cervical vertebrae maturation stage, 0.47 years after mp3cap stage (11.83 years) and 1.69 years after cvms2 or 0, 49 years before the stage cvms3 (12.79 years) the subjects will have their menarche. menarche can be used for determining the active growth stage of a girl and to predict the finished of pubertal growth. references 1. o’brien k, wright j, conboy f, appelbe p, davies l, connolly i, mitchell l, littlewood s, mandall n, lewis d, sandler j, hammond m, chadwick s, o’neill j, mcdade c, oskouei m, thiruvenkatachari b, read m, robinson s, birnie d, murray a, shaw i, harradine n, worthington h. early treatment for class ii division 1 malocclusion with the twin block appliance: a multi-center, randomized, controled trial. am j orthod dentofacial orthop 2009; 135(5): 573-9. 2. siara-olds nj, pangrazio-kulbersh v, berger j, bayirli b. longterm dento skeletal changes with bionator, herbs, twin block and mara functional appliances. angle orthod 2010; 80(1): 18-29. 3. faltin j, faltin m, bacetti t, franchi l, ghiozzi b, mcnamara jj. long term effectiveness and treatment timing for bionator therapy. angle orthod 2003; 73(3): 221-30. 4. fishman s. maturational patterns and prediction during adolescence. angle orthod 1987; 57(3): 178-93. 5. baccetti t, franchi t, mcnamara jj. an improved version of the cervical vertebral maturation (cvm) method for assessment of mandibular growth. angle orthod 2002; 72(4): 316-23. 6. demirjian a, buschang r, tanguay, patterson k. interrelationship among measures of somatic, skeletal, dental and seksual maturity. am j orthod 1985; 88(5): 433-8. 71mardiati, et al.: the relationship between menarche, hand-wrist and cervical vertebrae maturation indicator 7. flores-mir c, nebbe b, major pw. use of skeletal maturation based on hand-wrist radiographic analysis as a predictor of facial growth: a systematic review. angle orthod 2004;74: 118-124. 8. santiago rc, miranda lp, vitral rwf, fraga mr, bolognese am, maia lc. cervical vertebrae maturation as a biologic indicator of skeletal maturity. a systematic review. angle orthod 2012; 82(6): 1123-31. 9. soegiharto b, cunningham s, david r, moles. skeletal maturation in indonesian and white children assessed with hand-wrist and cervical vertebrae methods. am j orthod and dentofacial orthop 2008; 134(2): 217-26. 10. wong rwk, alkhal ha, rable bm. use of cervical vertebrae maturation to determine skeletal age. am j orthod dentofacial orthop 2009; 136(4): 484.e1-6. 11. gabr iel db, southa rd k, qian f, ma rshal sd, franciscus rg, southard t. cervical vertebrae maturation method: poor reproducibility. am j orthod dentofacial orthop 2009; 136(4): 478e1-478 e7. 12. fishman s. radiographic evaluation of skeletal maturation. angle orthod 1982; 52(2): 88-112. 13. lai eh, chang jz, jane ycc, tsai sj, liu jp, chen yj. relationship between age at menarche and skeletal maturation stages in taiwanese female orthodontic patients. j formos med assoc 2008; 107(7): 527-32. 14. darmawati d. perbandingan usia menarke daerah pedesaan dan perkotaan di tangerang. disssertation. yogyakarta: universitas gadjah mada; 2011. 15. batubara jrl. adolescent development. sari pediarti 2010; 12: 21-9. 16. anderson se, dallal ge, must a. relative weight and race influence average age at menarche: results from two nationally representative surveys of us girls studied 25 years apart. pediatrics 2003; 111(4 pt 1): 844–50. 17. tuğba a, i̇smet k. menarcheal age in turkey: secular trend and socio-demographic correlates. annals of human biology 2011; 38(3): 345-53. 18. lindgren g. height, weight and menarche in swedish urban school children in relation to socio-economic and regional factors. ann hum biol 1976; 3(6): 501-28. 19. hosokawa m, imazeki s, mizunuma h, kubota t, hayashi k. secular trends in age at menarche and time to establish reguler menstrual cycling in japanese women born between 1930 and 1985. bmc womens health 2012; 12: 19 20. cho gj, park ht, shin jh, hur jy, kim yt, kim sh, lee kw, kim t. age at menarche in a korean population: secular trends and influencing factors. eur j pediatr 2010; 169(1): 89-94. 21. mardiati e, soemantri es, haroen er, thahar b, sutisna b. umur vertebra servikal dan tahap maturasi fisiologis untuk prediksi per tumbuhan puber tal anak indonesia ras deutero-malayu. disssertation. yogyakarta: universitas padjadjaran 2010. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 84 fungal inhibitory effect of citrus limon peel essential oil on candida albicans iwan hernawan, desiana radithia, priyo hadi, and diah savitri ernawati department of oral medicine faculty of dental medicine, universitas airlangga surabaya -indonesia abstract background: oral candidiasis is an opportunistic infections due to candida albicans that often found in people with hiv/aids. anti-fungi, polyne and azole, are used in the treatment of oral candidiasis, but often cause persistence and recurrence. citrus limon peel contains terpenoids capable of inhibiting the synthesis of ergosterol, a component of the fungal cell wall that helps to maintain cell membrane permeability. essential oil derived from citrus limon peel, thus, is expected to inhibit the growth of candida albicans. purpose: this research was aimed to know how essential oil derived from citrus limon peel can inhibit the growth of candida albicans. method: this research was a laboratory experimental research carried out in three phases. first, essential oil was made with cold pressing method, and then the concentration of 100% was diluted to 50%, 12.5%, 6.25%, 3.125%, 1.56% and 0.78%. a test was conducted on the culture of candida albicans in sabouraud broth, accompanied by control (+) and (-). second, the dilution of essential oil was conducted to alter the concentration with inhibitory power, from the strongest one to the weakest one, and then it was tested on the culture of candida albicans. third, spreading was carried out from liquid culture to agar media in order to measure the number of colonies. result: candida albicans did not grow on media with 100% essential oil treatment, but it grew on media with 50% essential oil treatment. in the second phase, dilution of 100%, 90%, 80%, 70%, 60% and 50% was conducted. the growth of candida albicans was found on the treatment media of 60% and 50%. on the agar media, the growth occurred in the cultured medium treated with 70%. conclusion: the minimum inhibitory power of essential oil derived from citrus limon peel against candida albicans was in the concentration of 80%. essential oil derived from citrus limon peel has antifungal effect and potential as a therapeutic agent for oral candidiasis. keywords: oral candidiasis; hiv/ aids; candida albicans; citrus limon; essential oil correspondence: iwan hernawan, c/o: departemen ilmu penyakit mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: hernawan500@gmail.com research report introduction candida albicans infection is an opportunistic infection mostly found in oral mucosa. candida albicans is a normal flora of the oral cavity that can be changed into pathogens in patients who are in immunocompromised condition then leading to an infection, known as candidiasis. oral candidiasis occurs in 50-90% of hiv/aids cases, and 90% of patients infected with hiv suffer from this infection.1 citrus limon essential oil, cedro oil, is obtained by extracting the peel of citrus limon. the oil can be extracted from the peel of citrus limon in three ways, namely: distillation, pressure (cold pressing) and solvent extraction. citrus limon essential oil contains more than 90% terpenoids potentially powerful as anti-fungi. terpenoids (the original turpentine of “terpinen”) is a hydrocarbon with the chemical structure of c10h16. this compound is actually contained in oil derived from plant products, serving to give aroma in the plant products.2 terpenoids is also found in a variety of traditional herbal medicine. various studies report that this compound has pharmacological properties, such as antibacterial and antineoplastic, as well as other various pharmacological functions. terpenoids plays a role in creating aroma in eucalyptus, giving flavor in dental journal (majalah kedokteran gigi) 2015 june; 48(2): 84–88 85 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 85hernawan, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 84–88 table 1. the growth of candida albicans in broth media during the first phase of the treatment tube no. concentration colony growth 1 100 % citrus limon essential oil ( ) 2 50 % citrus limon essential oil ( + ) 3 25 % citrus limon essential oil ( + ) 4 12.5 % citrus limon essential oil ( + ) 5 6.25 % citrus limon essential oil ( + ) 6 3.125 % citrus limon essential oil ( + ) 7 1.5625 % citrus limon essential oil ( + ) 8 0.78125 % citrus limon essential oil ( + ) 9 positive control ( + ) 10 negative control ( ) table 2. the growth of candida albicans in broth media during the second phase of the treatment tube no. concentration colony growth 1 100 % citrus limon essential oil ( ) 2 90 % citrus limon essential oil ( ) 3 80 % citrus limon essential oil ( ) 4 70 % citrus limon essential oil ( ) 5 60 % citrus limon essential oil ( + ) 6 50 % citrus limon essential oil ( + ) 7 positive control ( + ) 8 negative control ( ) cinnamon, cloves and ginger, as well as producing yellow color in sunflower and red color in tomatoes. steroids and sterols are biological products derived from terpenoid precursors. terpenoids when combined with protein in the cell membrane is known as isoprenylation.3 ergosterol is sterol found in fungi. ergosterol is also known as a component of fungal cell membranes. ergosterol is only found in fungal cell membranes, and does not exist in other living things. thus, it can be concluded that enzymes involved in the synthesis of ergosterol could be the main target of antifungal medicine.4 therefore, this study was aimed to observe the inhibitory power of essential oil derived from citrus limon peel against the growth of candida albicans. as a result, this research is expected to become useful base in the development of alternative medicines for oral candidiasis with herbal ingredients grown in indonesia. materials and methods this research was a laboratory experimental research with a post-test only control group design. extraction of essential oil derived from citrus limon was carried out with cold pressing method. a total of 0.5 kg of citrus limon peels produced essential oil 10 ml with a concentration of 100%. the essential oil with the concentration of 100% was then diluted into a concentration of 50%, 12.5%, 6.25%, 3.125%, 1.56%, and 0.78%. dilution was carried out to obtain a concentration of 90%, 80%, 70% and 60%. the extraction and dilution were conducted in the laboratory of phytochemistry, faculty of pharmacy, universitas airlangga. candida albicans specimens were obtained from oral candidiasis lesions in hiv/aids patients hospitalized in the inpatient care unit of intermediates infectious diseases in dr. soetomo hospital. the samples were 20-45 years old male or female patients diagnosed with hiv/ aids as well as oral candidiasis. candida culture was performed on sabouraud broth, then incubated for 48 hours at a temperature of 37º c in the laboratory of microbiology, faculty of dentistry, universitas airlangga, and identified in the balai besar laboratorium kesehatan surabaya with sugar fermentation test method, germ tube test, and chlamydospores test on cornmeal agar. after pure culture was obtained, and it was randomly divided for treatment with the provision of essential oil with different concentrations. results in the first phase, the effect of citrus limon essential oil with the concentration of 50%, 12.5%, 6.25%, 3.125%, 1.56% and 0.78% against candida albicans cultured in sabouraud broth was observed to know the sterility of the essential oil. candida albicans culture without treatment was as the positive control, while candida albicans culture with citrus limon essential oil treatment with the concentration of 100% was as the negative control. on the tube treated with citrus limon essential oil with the concentration of 100% (as the negative control), the turbidity in the broth media increased compared to that in the positive control. it indicates that there was no candida albicans growth. on the tubes treated with citrus limon essential oil with the concentration of 50%, 25%, 12.5%, 6.25%, 3.12%, 1.562% and 0.78%, the turbidity in the broth media increased. it means that there were candida albicans grown (table 1). in this phase, the minimum inhibitory power of citrus limon essential oil against the growth of candida albicans was still not determined since the range between 100% and 50% was too wide. therefore, this research continued to the second phase, in which the dilution of citrus limon essential oil was conducted by using 100% essential oil and corn oil as diluent to obtain a concentration of 100%, 90%, 80%, 70%, 60% and 50%. the results of the test on candida albicans cultures during this phase were represented in table 2. based on the data in table 2, there was no candida albicans growth in the culture tubes treated with citrus limon essential oil with dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 86 hernawan, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 84–88 table 3. the growth of candida albicans colonies in agar media no. group number of colonies 1 100 % citrus limon essential oil 2 90 % citrus limon essential oil 0 3 80 % citrus limon essential oil 0 4 70 % citrus limon essential oil 7 5 60 % citrus limon essential oil 12.5 6 50 % citrus limon essential oil 7 positive control 76 8 negative control 0 the concentration of 100%, 90%, 80% and 70%. but, there were candida albicans growth in the culture tubes treated with citrus limon essential oil with the concentration of 60% and 50%. this research then continued to the third phase. in this phase, spreading was conducted on the cultures in the sabouraud dextrose agar media from the treatment tube 2 to 5 by giving citrus limon essential oil with the concentration of 90%, 80%, 70% and 60%, and then colonies grown were measured. the average number of candida albicans colonies was getting increased as the concentration of citrus limon essential oil was getting decreased. the growth of candida albicans emerged in sabouraud dextrose agar medium treated with 70% citrus limon essential oil (7 colonies). similarly, there were candida albicans colonies grown in the medium treated with 60% citrus limon essential oil (table 3). it can be said that the greater the concentration of citrus limon essential oil is, the lower the number of colonies formed. there was no candida albicans grown in the agar medium 3, in which the spreading of the tube was treated with 80% citrus limon essential oil, as well as in the agar medium 2 treated with 90% citrus limon essential oil (table 3). discussion oral candidiasis is a common opportunistic infection in patients with hiv-positive diagnosis. oral candidiasis emerges as erythematous and pseudomembranous in hard palate, soft palate, tongue and buccal mucosa, as well as angular cheilitis. oral candidiasis occurs in 50-90% of hiv/ aids cases, and an estimated 90% of hivinfected patients also suffers from this infection during the progression of their disease.1,5 mucosal infection is generally not a directly life-threatening disease, but when microorganism can manage to penetrate the epithelial and gain access to the blood circulation, candidemia can trigger to fungal manifestations of fungal infection in kidneys, heart and brain.6 citrus limon is widely grown in indonesia, china and india. in indonesia, citrus limon is known as lemon. the fruit is widely cultivated in farm fields in batu (malang, east java). lemon has also been widely used for culinary and non-culinary purposes throughout the world. lemon even is also used for treatment, ie as aromatherapy, antioxidants and antiseptics. lemon is often used to reduce headaches and migraine, as well as for rheumatism and arthritis treatments. citrus limon peel juice has also been widely used as a substance mixed with warm water to be used as a mouthwash and to relieve pain due to injuries in the oral cavity.7 essential oil extracted from citrus limon is also known as cedro oil. the oil has a pungent smell, a refreshing pale yellowish green color and low viscosity. the essential oil derived from citrus limon peels is a byproduct of the industrial processing of lemons, so citrus limon essential oil is a relatively inexpensive material easily obtained. citrus limon essential oil contains various compositions that are useful in body protection, such as limonene, βpinene and ɣ-terpinene serving as antifungi.8 there are three ways of making essential oil, namely distillation, expression and extraction. to make citrus limon essential oil derived from its peels, cold pressing method is recommended. the fresh peels of citrus limon contain a lot of liquid so that essential oil can be produced by cold pressure method. the advantage of the cold pressure extraction method is that it will not damage thermolabile materials contained. thus, the cold pressure extraction method was chosen to make citrus limon essential oil in this research since the method is easy, which is by cold squeezing process. cold squeezing process is to squeeze the peels of citrus limon with a squeeze over juice filter, which results flow into a sterile tube.9 to determine the antifungal potential of citrus limon essential oil, the growth of candida albicans must be observed. candida is an opportunistic organism in oral cavity, causing disease in a healthy host, but the infection can only occur in individuals whose body resistance decreases. candida genus is comprised of more than 200 species, including pathogens such as c. albicans, c. dubliensis, c. tropicalis, c. parapsilosis, c. kefyr, c. guilliermondii and c. krusei. candida albicans is a species commonly found on the entire surface of oral cavity, especially on tongue and palate. it is also often found on the surface of the maxillary denture designed in such a way to make negative pressure on the palate mucosa, thereby inhibiting saliva containing immune components enter into the space, resulting in candida albicans growth. candida albicans is often found in the upper jaw of the healthy oral cavity after using maxillary denture for 12 months. candida albicans is also considered as the most pathogenic candida species. candida albicans infection is found in 66% of the healthy maxillary denture users.10-12 oral candidiasis in patients with hiv-aids is caused by the decreasing of immune system due to the depletion 87 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 87hernawan, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 84–88 of cd4+ leading to a failure in stemming the pathogenicity of candida albicans. oral candidiasis can occur from the early phase after seroconversion, asymptomatic chronic phase to the advanced phase when it reaches the stage of aids. an estimated 90% of hiv-aids patients is exposed to oral candidiasis during the progression of the disease, so the infection is closely related to hiv infection.1,5 in many countries that have not been able to distribute highly active anti-retroviral therapy (haart) evenly, such as in indonesia, mucosal candidiasis is still the highest cause of morbidity in aids patients.5,13 sabouraud broth and dextrose agar media were used as media for candida growth in this research. these media have acidic concentration (ph 5.6), so the growth of bacteria that are not acid resistant will be inhibited. in sabouraud media, the growth of candida can be observed after incubation for 24-48 hours.14 based on the results of the observation on the growth of candida albicans colonies treated with eight different concentrations of citrus limon essential oil and on the two control groups respectively replicated six times, there was no candida albicans grown in the treatment group with 100% citrus limon essential oil. candida albicans growth only occurred in the treatment group with 50% citrus limon essential oil (table 1). the wide ranges between these two concentrations underlay this research to continue into the next phase to determine the minimum inhibitory power of candida between the concentration of 100% and 50%. in the second stage, dilution was conducted to obtain the concentration of 90%, 80%, 70% and 60% (table 2). the results of the second phase (table 2) indicate that there were differences of the growth of candida albicans cultures treated differently. in the groups treated with citrus limon essential oil with the concentration of 100%, 90% and 80%, there was no candida albicans growth, while in the groups treated with citrus limon essential oil with the concentration of 70%, 60% and 50%, there were candida albicans growth. furthermore, to clarify the growth of candida albicans, culturing of candida albicans was conducted on sabouraud dextrose agar media by spreading, and then the number of candida albicans colonies grown was measured (table 3). essential oil contains chemical elements containing hydrocarbon consisting of several compounds, such as terpenoids (isoprenoid), non terpenoids, c13 norterpenoid, phenylpropanoid, ester, lactone, phtalide and isoyhiocynate. citrus limon essential oil contains antifungal classified into the class of terpenoids, namely limonene and terpinene as monocyclic monoterpinene, and β-pinene as bicyclic monoterpinene. therefore, the provision of terpenoids will inhibit the synthesis of ergosterol, a component of fungal cell membranes, which plays an important role in regulating cell membrane permeability that keep the liquid inside the fungal cells and other activities, such as enzymes associated with the fungal cells. as a result, the disruption of the synthesis of ergosterol can make the permeability of cell membranes increased so that the liquid will come out of the cells and the activities of the fungal cells will be inhibited. the provision of terpenoids with high concentration even can cause cellular activities stopped and the cells dead.15 the results showed that the increasing of the concentration of citrus limon essential oil could decrease the number of candida albicans colonies. in this research, 80% citrus limon essential oil expressed the minimum inhibitory power against candida albicans. minimum fungicidal concentration (mfcs) for amphotericin-b and iatraconazole in this research were better than mfcs in other researches, for amphotericin-b with the incubation time of 48 and 72 hours (86.4 to 87.7%) and for iatraconazole (91.4 to 93.8%).16 finally, it can be concluded that the mfc of citrus limon essential oil to be antifungal is 80%. it means that citrus limon essential oil has antifungal properties against candida albicans, so the oil can potentially be developed as a therapeutic agent for oral candidiasis that affects many patients with hiv / aids. therefore, further researches are suggested to conduct a toxicity test on the essential oil if it will be developed as a therapeutic agent. references 1. scully cs. oral and maxillofacial medicine. 3rd ed. edinburgh: churchill livingstone elsevier; 2013. p. 254-63. 2. gershenzon j, dudareva n. the function of terpene natural product in the natural world. nat chem biol 2007; 3(7): 408-14. 3. harrewijn p, oosten am. natural terpedoids as messengers-a multidisciplinary study of their production. biological function and practical applications. 2012; p. 94-107 4. williams d, lewis m. pathogenesis and treatment of oral candidosis. j oral microbiol 2011; 3. doi: 10.3402/jom.v3i0.5771. 5. maurya v, srivastava a, mishra j, gaind r, marak rsk, tripathi ak, singah m, venkatesh v. oropharyngeal candidiasis and candida colonization in hiv positive patients in northern india. j infect dev crtries 2013; 7(8): 608-13. 6. shomam s, marr ka. invasive fungal infections in solid organ transplant recipients. future microbiology 2012; 7(5) 639-55. 7. dhanavade mj, jalkute cb, ghosh js, sonawane kd. study antimicrobial activity of lemon (citrus lemon l.) peel extract. british journal of pharmacology and toxicology 2011; 2(3): 119-22. 8. voo ss, grimes hd, lange bm. assessing the biosynthetic capabilities of secretory gland in citrus peel. plant pysiol 2012; 159(1): 81-94. 9. gӧk a, kirbașlar fg. comparison of lemon oil composition after using different extraction methods. journal of essential oil research 2015; 27(1): 17-22. 10. bodey gp. overview. in: de pauw be, bodey gp, eds. serious candida infections: diagnosis, treatment, and prevention, selected readings: focus on fluconazole. volume ii. new york: pfizer international pharmaceuticals, reprinted by permission of the american j medicine; 1996. p. 170-6. 11. marsh pd, martin mv. oral microbiology. 4th ed. edinburgh: churchill livingstone elsevier; 2009. p. 24-44, 166-79. 12. dignani mc, solomkin js, anaissie ej. candida. in: anaissie ej, mcginnis mr, pfaller ma, eds. clinical mycology. 2nd ed. edinburg: churchill livingstone, elsevier, inc; 2009. p. 197-218. 13. devitt e, powderly wg. candida in hiv infection. in: volberding, pa editor. 2008. global hiv/aids medicine. philadelphia: saunders elsevier; 2008. p. 365-73. 14. mc. ginnis mr. current topics in medical mycology vol. 02. new york: springer-verlag; 2012. p. 163-5. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 88 hernawan, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 84–88 15. rao a, zhang y, muend s, rao r. mechanism of antifungal activity of terpenoid phenol resembles calcium stress and inhibition of the tor pathway. antimicrob agents chemother 2010; 54(12): 50629. 16. carrillo-muñoz aj, quindós g, del valle o, hernández-molina jm, santos p. antifungi activity of amphotericin b and iatraconazole against filamentous fungi: comparison of the sensititre yeast, one and nccls m38-a reference methods. j chemother 2004; 16(5): 486-73. � three dimensional changes in maxillary complete dentures immersed in water for seven days after polymerization shinsuke sadamori1, toshiya ishii1, taizo hamada1, and arifzan razak2 1 department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, hiroshima, japan 2 department of prosthodontics, the faculty of dentistry, airlangga university, surabaya, indonesia abstract the purpose of this study was to investigate the three dimensional changes in the fitting surface and artificial teeth of maxillary complete dentures which were fabricated using two different polymerizing processes: heat polymerization (hp) and microwave polymerization (mp), after immersion in water for seven days. the amount of distortion in the molar region of the alveolar ridge was significantly different between hp and mp. however, the overall distortion of the dentures polymerized using both methods was similar. the distortion due to immersion in water for seven days compensated for the polymerization distortion, but the amount of distortion was very slight. key words: maxillary complete denture, three dimensional change after polymerization, microwave polymerizing correspondence: dr shinsuke sadamori, department of prosthetic dentistry, graduate school of biomedical sciences, hiroshima university, 1-2-3 kasumi, minami-ku, hiroshima city, 734-8553, japan, tel: +81-82-257-5681, fax: +81-82-257-5684, e-mail: tsada@hiroshima-u.ac.jp introduction the provision of conventional complete dentures is the most realistic treatment for most edentulous older people, although other alternative treatments can be offered such as overdentures or implant retained dentures.1 generally, acrylic dentures should be washed in soap and warm water after polishing, and then stored in an antiseptic solution in a sealed polythene bag until supplied to the patient.2 acrylic polymers undergo dimensional changes that result in shrinkage and expansion,3 and are influenced by water uptake. changes in linear dimension, warpage, and water uptake of acrylic resin denture bases are influenced by the processing method and the thickness.4 overall dimensional denture base changes are the result of localized changes. in the clinic, the storage of polished dentures in water before they are supplied to the patient would be at most around one week. in our previous study,5 we used a three-coordinate measuring machine to measure various distortions of denture specimens fabricated using two different polymerization processes. the aim of the present study was to measure and compare three dimensional changes in the fitting surface and artificial teeth of maxillary complete dentures fabricated with two different polymerization processes after immersion in water for seven days. materials and methods specimen preparation denture specimens were prepared according to the method used in our previous study.5 to fabricate theto fabricate the working cast, a die of an edentulous maxillary arch (402u, nisshin, kyoto, japan) was duplicated using silicone elastomer (gc, tokyo, japan). a master cast was made of dental stone (new fujirock, gc, tokyo, japan) according to the manufacturer’s recommendation. the measuring points on the master cast consisted of 12 points on the alveolar ridge related to each second molar and first premolar tooth and the midway point of the incisor teeth, and on the denture flange in the molar, premolar and anterior regions. in addition, we set three datum-points (reference points) to set the coordinate system in the palate. the measuring points were set with steel balls with a diameter of 2 mm, and the datum-points were steel balls with a diameter of 4 mm set on the model using self-curing resin. the master cast was duplicated and six master casts were made. a wax denture was made on one of the six working casts. one thickness (approximately 1.5 mm) of base plate wax (paraffin wax, gc, tokyo, japan) was adapted to the working cast and the artificial teeth (gc duradent: anterior teeth c3, posterior teeth 30m, gc, tokyo, japan) were positioned in the usual manner. the core of the wax denture was made with dental stone and silicon impression material. after the working casts were measured, three steel balls 4 mm in diameter � dent. j. (maj. ked. gigi), vol. 41. no. 1 january–march 2008: 1–4 were set in the appointed positions as datum-points, and the wax dentures were made from the core. the measurement points were prepared on mesiolingual cusps of the right and left second molar tooth, the lingual cusps of the right and left first premolar tooth, and the mesial edge of the right incisor with a diamond point 1.8 mm in diameter (diamond point fg regular 340, shofu, kyoto, japan). polymerizing process two acrylic resins were used: bio resin (shofu, kyoto, japan) and acron mc (gc, tokyo, japan) (table 1). the bio resin was mixed using 4.5 ml liquid to 10 g powder. the polymerizing process followed japan industrial standard’s (jis) recommendation: an initial 90 minutes at 70° c followed by 30 minutes at 100° c (hp). the acron mc was mixed using 4.3 ml liquid to 10 g powder. specimens (mp) were processed for 3 minutes in a 500 w microwave oven (em-m 535 t, sanyo electric, osaka, japan). ten standardized denture specimens were fabricated: five using the conventional technique and five using the microwave technique. after polymerization, flasks were allowed to cool at room temperature for over 12 hours and deflasked. table 1 shows the polymerization process used. measuring method dimensional change was measured using a threecoordinate measuring machine (tristation, tst 600-fc, nikon corp., tokyo, japan) graduated to an accuracy of 0.5 µm or less at 20° c with a ball stylus measuring 0.5 mm in diameter.5 the measurements were performed on denture specimens after deflasking and immersion for seven days in water. results figures 1, 2, and 3 show the dimensional changes after seven days immersion in water for both the fitting surface and the polishing surface. the cross point of the x and y axes of the reference plane is the center of the three reference points, and this point is the reference point for measurement in this study (figure 1). hp mp occlusal view fitting surface view reference point ： 10mm： 10mm figure 1. dimensional change after immersion in water for seven days. the cross point of the axes indicates the center of the three reference points. arrows indicate the direction of the change. a line from the reference point shows distortion after deflasking. another thick line shows distortion from the position of the reference point at deflasking to its position after immersion in water for seven days (figure 2). lateral view hp mp posterior view hp mp distortion after polymerization distortion after immersion in water for seven days ： 10mm： 10mm figure 2. dimensional change after immersion inwater for seven days. the cross point of the axes indicates the center of the three reference points. distortion of the fitting surface the distortion of the fitting surface in both hp and mp dentures was very small and occurred from the center of the denture to the outside in the horizontal plane (figure 1). table 1. materials and polymerization process used in this study processing method brand polymerizing cycle processing technique powder-to-liquid ratio (g/ml) manufacturer heat polymerizing (hp) bio resin 90 min at 70° c 30 min at 100° c hot water bath 10/4.5 shofu kyoto, japan microwave polymerizing (mp) acron mc 3 min microwave 500 w 10/4.3 gc tokyo, japan �sadamori: three dimentional changes in maxillary complete dentures the distortion of the fitting surface in both hp and mp dentures occurred in a vertical direction to the occlusal plane (figure 2). the amount of distortion of the reference point near the origin was less than for the distant reference point. distortion of the fitting surface of the alveolar ridge beyond the border of the palate occurred in a vertical direction towards the occlusal plane. the distortion of hp dentures was similar to that of mp dentures. there was a significant difference in the amount of distortion in the molar region of the alveolar ridge between hp and mp (figure 4). hp mp hp mp lateral view posterior view ： 10mm： 10mm figure 3. dimensional change after immersion in water for seven days (artificial teeth). the cross point of the axes indicates the center of the three reference points. molar premolar anterior molar premolar anterior flange alveolar ridge center border palate molar premolar anterior hp mp figure 4. amount of distortion. distortion of the artificial teeth the horizontal distortion of the artificial teeth was similar to that on the fitting surface (figure 1). the distortion of artificial teeth occurred in a vertical direction towards the occlusal plane, and the amount of distortion of the reference points near the origin was less than for the distant reference points. there was no significant difference in the amount of distortion between hp and mp dentures. the distortion of hp in a vertical direction tended to be larger than that of mp (figure 3), and the distortion of mp in a horizontal direction tended to be larger than that of hp (figure 1). discussion our previous study4 indicated that the linear dimensional expansion and the amount of water sorption in thick resin specimens was greater than in thin samples. the linear dimension of each specimen tended to decrease after each day of storage in water. the linear dimensions of 1-mm hp specimens were similar from 1 day to 90 days in water. however, the 3-mm and 5-mm specimens increased in linear dimensions up until 60 days. the linear expansion and water sorption for specimens of the same thickness tended to be greater for microwave-activated resins than for heat-polymerized samples. the flanges of denture specimens shifted towards the buccal side, and the alveolar ridge of the fitting surface shifted towards the occlusal plane. the artificial teeth shifted towards the origin. this distortion would be the result of the release of stress after deflasking.6 in the clinical situation, it is common for dentures to be immersed in water for up to seven days. the results of this study indicate that the distortion of maxillary complete dentures fabricated by both polymerization methods following immersion in water for seven days compensated for the distortion that occurred during the polymerization process. however the amount of the distortion was very slight. these results are consistent with takahashi’s results.7 the linear dimensions of each specimen (hp and mp) tended to decrease after one day of storage in water. this additional shrinkage may be due to the release of a considerable amount of elastic stress.6 however, the expansion of resin specimens after one week of storage in water varied. the 1-mm specimens had similar linear dimensions from 1 day to 90 days in water. however, the 3-mm and 5-mm specimens increased in linear dimensions up until 60 days.4 the overall dimensional changes in maxillary complete dentures are the result of localized changes. dentists should therefore follow-up the condition of dentures until about 60 days after insertion into the mouth. it concluded that the overall distortion of maxillary complete dentures polymerized by both methods was similar. the distortion caused by immersion in water for seven days compensated for the distortion that occurred during polymerization, but the amount of the distortion was very slight. � dent. j. (maj. ked. gigi), vol. 41. no. 1 january–march 2008: 1–4 acknowledgement this study was supported in part by a grant-in-aid for scientific research (16390617) from japan society for the promotion of science (jsps). references 1. anastassiadou v, heath mb. the effect of denture quality attributes on satisfaction and eating difficulties. gerodontology 2006; 23:23–32. 2. bates jf, huggett r, stafford gd. removable denture construction.removable denture construction. 3rd ed. butterworth-heinemann ltd; 1991. p. 114–5.butterworth-heinemann ltd; 1991. p. 114–5. 3. skinnner ew, cooper en. physical properties of denture resins, i: curing shrinkage and water sorption. j am dent assoc 1943; 30:1845–52. 4. sadamori s, ishii t, hamada t. influence of thickness on the linear dimensional change, warpage, and water uptake of a denture base resin. int j prosthodont 1997; 10:35–43. 5. sadamori s, ishii t, hamada t, razak a. a comparison of three dimensional change in maxillary complete dentures between conventional heat polymerizing and microwave polymerizing techniques. dent j 2007; 40:6–10. 6. winkler s, ortman hr, morris hf, plezia ra. processing changes in complete dentures constructed from pour resins. j am dent assoc 1971; 82:349–53. 7. takahashi y. three dimensional changes of the denture base of the complete denture following polymerization. j jpn prosthodont 1990; 34:136–48. 90 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 90–94 research report the effects of topical application of red pomegranate (punica granatum linn) extract gel on the healing process of traumatic ulcers in wistar rats sri hernawati,1 yonanda az zikra,1 and dwi warna aju fatmawati2 1department of oral medicine 2department conservative dentistry faculty of dentistry, universitas jember jember – indonesia abstract background: trauma-induced ulcers constitute one of the painful abnormalities affecting the oral cavity about which numerous individuals complain. the prevalence of traumatic ulcers (15-30% of cases) is relatively high compared to other oral lesions. unfortunately, the use of anti-inflammatory drugs classified as steroids can lead to contraindications and serious side effects. hence, the use of natural ingredients represents an alternative treatment. one such ingredient is red pomegranate containing bioactive elements acting as anti-inflammatory, antibacterial, antimicrobial, and antioxidant agents which accelerate the healing process in traumatic ulcers. purpose: the aim of this study was to determine the effects of the topical application of red pomegranate (punica granatum linn) extract on the healing process in traumatic ulcers in wistar rats. methods: 24 male rats, aged 2-3 months, were divided into six groups, namely; two control groups (pc: triamcinolone acetonide and nc: sterile aquades) and four treatment groups (red pomegranate extract gel at respective concentrations of 12.5%, 25%, 50% and 75%). a traumatic ulcer was subsequently created using a flame-shaped burnisher tip 2mm in diameter and heated for ±15 seconds. the diameter traumatic ulcer of each research subject was measured and observed until it healed within the remedial parameters determined by means of a unc-15 periodontal probe which had been placed on it. the data obtained was analyzed using kruskal-wallis and mann-whitney tests. results: topically applied red pomegranate extract gel can reduce the width of the ulcerated area, while also accelerating traumatic ulcer healing. there was a significant difference in the healing time between the group using red pomegranate extract gel at a concentration of 75% and the positive control group using 0.1% triamcinolone acetonide with p=0.44 (p<0.05). conclusion: red pomegranate extract gel possesses the ability to accelerate the healing process in traumatic ulcers. the most effective concentration of red pomegranate extract gel at accelerating the healing process in traumatic ulcers is 75%. keywords: anti-inflammatory; antioxidant; red pomegranate; traumatic ulcer correspondence: sri hernawati, department of oral medicine, faculty of dentistry, universitas jember, jl. kalimantan 37, jember 68121, indonesia. e-mail: srihernawati.drg5@yahoo.com introduction an ulcer constitutes a pathological condition characterized by loss of the epithelium layer due to surface excavation having penetrated deeper than the epithelial tissue. traumatic ulcers are caused by thermal, mechanical, chemical and electrical trauma1 and usually found on nonkeratinous surfaces such as buccal/labial mucosa, tongue, lips and hard or soft palate.2 traumatic ulcers in the oral cavity require between 7 and 14 days to heal.3 in general, their treatment involves eliminating local causes through the topical administration of drugs such as corticosteroids to reduce inflammation and the use of antiseptic mouthwash containing 0.2% chlorhexidine gluconate or benzydamine hydrochloride.1 the administration of topical corticosteroids in the form of ointments, gels, or elixirs has traditionally been considered the most effective treatment in the management dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p90–94 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p90-94 mailto:srihernawati.drg5@yahoo.com http://e-journal.unair.ac.id/index.php/mkg 91hernawati, et al.//dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 90–94 of pain and to accelerate the recovery time of oral ulcers.1 one topical corticosteroid for the treatment of oral mucosal inflammation is 0.1% triamcinolone acetonide which according to skidmore-roth,4 demonstrates contraindications with regard to the treatment of fungal, viral or bacterial infections of the mouth and throat. in other words, the use of corticosteroids during active infections can suppress the body’s immune system.4 one side effect of topical corticosteroids on the oral mucosa is to promote the growth of candida spp. in the oral cavity potentially resulting in candidiasis. moreover, 0.1% triamcinolone acetonide produces other side effects such as a burning sensation, itching, irritation, dryness, peeling, perioral dermatitis, allergies and maceration, secondary infection and atrophy of the oral mucosa.5 due to these contraindications and the serious side effects of steroidal anti-inflammatory drugs, numerous treatments have recently begun to utilize natural ingredients including supplements and herbal medicines as pain and inflammation relievers. the use of natural ingredients as medicines rarely causes negative side effects compared to drugs manufactured from synthetic materials.6 one such natural ingredient is red pomegranate (punica granatum linn) containing bioactive ingredients which act as antiinflammatory, antibacterial, antimicrobial and antioxidant agents.7 pomegranate also contains polyphenol compounds that function as antioxidants in preventing several diseases. the antioxidant and anti-inflammatory properties of pomegranate are assumed to be caused by its high polyphenol content, such as ellagic acid (ea) in free or bound forms, gallotannins and anthocyanins, and other flavonoids. pomegranate extract is also assumed to be more effective than the administration of only one active ingredient since it constitutes a combination of several active substances that form a synergistic, mutually formulating formula.7 pomegranate has, in fact, been used as a medicinal plant since the time of ancient egypt.7 various studies of red pomegranate extract at concentrations of 12.5%, 25%, 50% and 75% have been carried out in vitro,8 although further studies need to be conducted in vivo. consequently, this study aims to determine the potential for pomegranate extract to cure traumatic ulcers. materials and methods the study constituted experimental laboratory research using pretest and posttest control group design and was conducted at the experimental animal laboratory, faculty of dentistry, universitas jember and the laboratory of liquid and semisolid technology, pharmaceutical division, faculty of pharmacy, universitas jember. this research involving the use of animal subjects received ethical approval from the faculty of dentistry, universitas jember (no.133/un25.8/kepk/dl/2018). before the procedure for making traumatic ulcers was performed, ketamine hydrochloride was injected into the thigh muscles of the subjects. a 15-40 mg/kg dose of general anesthesia was administered prior to a traumatic ulcer being created using a 2 mm diameter flame-shaped burnisher tip that had been heated in a bunsen burner flame for ±15 seconds before being applied to the lower lip mucosa of the subjects for one second. the depth of the application was that of the tip diameter of the burnisher that had been indicted with a marker. observation of the traumatic ulcers formed was subsequently conducted on days 1, 3, 5 and 7 until they had healed.9,10 the ulcers were then measured by placing a straight periodontal probe (unc-15) in the vertical and horizontal sides as well as one of the diagonal sides.10 the ulcer healing parameters consisted of the following: the absence of a yellowish/grayish ulcer, the similarity between the color of the ulcerated mucosa and that of normal mucous, being flush with the oral mucosal surface and complete healing of the lesion. the 24 rats with traumatic ulcers were divided into six groups, namely; four treatment groups each of which had been administered with pomegranate extract at respective concentrations of 12.5%, 25%, 50% and 75%, a positive control (pc) and a negative control (nc). each group contained four subjects which were isolated before the application by means of cotton buds of the designated material, namely; red pomegranate extract gel at varying concentrations for the treatment groups, the application of 0.1% triamcinolone acetonide for the positive control group and the administration of distilled water for the negative control group. each ingredient was topically applied by means of a micro brush twice a day (at intervals of no more than 12 hours).10 the data resulting from the observation focused on the recovery time within the traumatic ulcer healing process based on the percentage of the total number of traumatic ulcers healed and their surface areas. traumatic area was calculated by measuring the diameter of the ulcer from the day of its formation to the day that it was healed using a periodontal probe unc-15. for the purposes of this study, the ulcer healing parameters consisted of the following: absence of a yellowish/grayish ulcer, the similarity between the color of the ulcerated mucosa with that of normal mucous, being flush with the oral mucosal surface and complete healing of the lesion.2 the data produced focused on the number of days required for the healing of traumatic ulcers in each group after analysis by means of a saphiro wilk normality test and a homogeneity test. this data was subsequently analyzed using a non-parametric kruskalwallis test and a mann-whitney test. results the results indicated that the 10-day application of red pomegranate gel extract at concentrations of 12.5%, 25%, 50% and 75%, 0.1% triamcinolone acetonide, together dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p90–94 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p90-94 http://e-journal.unair.ac.id/index.php/mkg 92 hernawati, et al.//dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 90–94 with distilled water decreased the dimensions of the ulcers. according to the calculation results, the mean width of the ulcer area in each group decreased (table 1), while the percentage of ulcer recovery increased across all groups. the highest percentage was recorded by the group which had been administered with red pomegranate extract gel at a concentration of 75% (figure 1). moreover, the kruskalwallis nonparametric test results indicated differences in recovery time between the 12.5% group, the 25% group, the 50% group, the 75% group, the pc group and the nc group (p<0.05). there was also a significant difference in recovery time between the group which had been administered with 75% pomegranate extract gel and the positive control group with 0.1% triamcinolone acetonide (p <0.05) (table 2). discussion the study results revealed that the topical application of red pomegranate gel extract affected the healing process in the traumatic ulcers of the wistar rat subjects. although the recovery time varied between groups, the healing process stages did not. these comprised the yellowish white surface and the emergence of a peripheral erythema zone, the gradual whitening of the lesion surface, a decrease in lesion size, the lightening of both lesion surface and mucus color and the disappearance of the lesion.2 the contrasting recovery time between treatment groups using red pomegranate extract gel were due to differences in its concentration. moreover, the results also showed that the treatment group treated with red pomegranate gel extract at a concentration of 75% demonstrated a shorter table 1. the mean width and recovery time of traumatic ulcers groups the mean width of ulcers (mm) the mean of recovery timed1 d3 d5 d7 d8 d9 d10 group 12.5% 7.413 5.388 1.734 0.556 0.298 0.196 0 9.3 group 25% 9.412 4.533 1.254 0.708 0.196 0 8.5 group 50% 8.311 4.369 0.843 0.196 0 6.8 group 75% 9.604 2.508 0.508 0 6.5 pc group 13.154 5.617 2.781 0.785 0.349 0 8.3 nc group 12.767 4.352 2.258 0.9 0.6 0.298 0 9.8 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% d 1 d 3 d 5 d 7 d 8 d 9 d 1 0 pe rc en ta ge o f r ec ov er y timing of observation (day) g12.5 g25 g50 g75 pc nc figure 1. the percentage of traumatic ulcer recovery time. table 2. mann-whitney test results groups group 12.5% group 25% group 50% group 75% pc group nc group group 12.5% .096 .017* .015* .098 .186 group 25% .036* .017* .752 .032* group 50% .617 .099 .017* group 75% .044* .015* pc group .034* nc group note: *significant difference. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p90–94 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p90-94 http://e-journal.unair.ac.id/index.php/mkg 93hernawati, et al.//dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 90–94 recovery time than the positive control group to which 0.1% triamcinolone acetonide had been administered. this contrast was due to red pomegranate possessing anti-inflammatory, antibacterial, antiviral, antifungal and antioxidant characteristics,7 while 0.1% triamcinolone acetonide demonstrates anti-inflammatory, antipruritic and hypo-allergic qualities.11 the treatment group to which 75% red pomegranate extract gel was administered ranked highest in terms of accelerating the healing process in traumatic ulcers present in the oral mucosa. the more rapid healing process of traumatic ulcers in the group with the red pomegranate extract gel was due to the bioactive components contained, one such component being ellagic acid which plays an antiinflammatory role. pomegranate is known to be capable of inhibiting the activation of nuclear factor kappa beta (nf-kβ), cyclooxygenase (cox) and lipoxygenase (lox), thereby limiting the number of inflammatory cells migrating to the injured tissue. as a result, the inflammatory reaction will be shorter and the proliferation ability of transforming growth factor beta (tgf-β) uninhibited. this process triggers the immediate activation of the proliferation phase.7,12 ellagic acid is also known to be a polyphenol which demonstrates the ability not only to regulate the fibrosis process by reducing the excessive levels of collagen, the expression of transforming growth factor beta 1 (tgf-β1) and the amount of alpha-smooth muscle actin (α-sma) in tissues with chronic lesions, but also to decrease the production of reactive oxygen species (ros).7 another function of ellagic acid is to protect cell damage due to free radicals. this ability synergistically increases when ellagic acid is combined with another element within pomegranate, a natural dye called anthocyanidin, which also functions as a powerful antioxidant.7 anthocyanidin is an antioxidant that has been proven to improve blood vessel function, thereby accelerating the healing process.7 flavonoids can also stabilize reactive oxygen compounds which potentially reduce damage caused by free radicals. as an antioxidant, flavonoids promote anti-inflammatory activity.13 the results of this study revealed that the treatment group administered with 50% pomegranate extract gel was ranked second to the 75% concentration group. however, there was no significant difference in average recovery time between the treatment group given 50% pomegranate extract gel and the group administered with 75% pomegranate extract gel. this is because the consistency of the gel material, semisolid and slightly viscous, is almost identical. therefore, there is little difference between 50% pomegranate extract gel and 75% pomegranate extract gel in terms of the absorption of active substances present in the mucosa affected by traumatic ulcers. of all the treatment groups to which red pomegranate extract gel was administered, the group with 12.5% red pomegranate extract gel had the lowest recovery time. this is because the consistency of pomegranate extract gel at concentrations of 75% and 50% is very thick compared to that at a concentration of 25%. the 25% concentration pomegranate extract gel is light brown in color indicating that the composition of the extract is extremely restricted compared to gel at the higher concentration. the results of the study showed that the positive control group given 0.1% triamcinolone acetonide ranked third. the results also showed that the recovery time of the positive control group was more rapid than that of the negative control group with distilled water. similarly, a previous study argued that the average recovery time of traumatic ulcers treated with 0.1% triamcinolone acetonide was shorter, at 5.3 days, than that treated only with distilled water (8.2 days).14 the administration of distilled water in the study aimed to establish how traumatic ulcers are healed without the aid of active substances. the average recovery time of traumatic ulcers was 9.8 (9 days 22 hours 16 minutes) which is in accordance with the theory that traumatic ulcers can heal themselves within a period of 7-14 days.3 several factors affect the normal wound healing process which are divided into two groups, namely; local factors and systemic factors. one of the systemic factors that can influence healing is the provision of corticosteroid drugs, namely; 0.1% triamcinolone acetonide. corticosteroid drugs inhibit mediators and cellular immune response during inflammation by stimulating the formation of proteins (lipocortin) that inhibit phospholipase a2. this prevents activation of the arachidonic acid cascade and prostaglandin secretion which, in turn, reduces the number of lymphocytes and monocytes in the periphery and inhibits the ability of plasminogen activators (pas) to convert plasminogen into the plasmin which plays a role in breaking down kininogen to kinin and functions as a vasodilator.10 cmc-na can be used as a gelling agent in red pomegranate extract gel preparations due to its high degree of stability under both acidic and basic conditions (ph 2-10). in addition, cmc-na has several advantages, including its ability to produce gels that are neutral, colorless, stable, tasteless, its strong resistance to microbial attack and its consistent viscosity.15 the results indicated significant differences between each of the treatment groups, administered with red pomegranate extract gel at concentrations of 25%, 50% and 75%, and the negative control group. they also confirmed the ability of such gel to accelerate the healing process in traumatic ulcers. moreover, there was a significant difference in recovery time between the group which received red pomegranate extract gel at the concentration of 75% and those groups administered with the gel at concentrations of 12.5% and 25%, as well as the positive and negative control groups. a significant difference in recovery time also existed between the group given red pomegranate extract gel at a concentration of 50% and those groups receiving such gel at concentrations of 12.5% and 25%, in addition to the positive and negative control groups. this means that the more concentrated the extract, the higher the quantity of drugs absorbed and the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p90–94 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p90-94 http://e-journal.unair.ac.id/index.php/mkg 94 hernawati, et al.//dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 90–94 greater the acceleration of the healing process in traumatic ulcers.15 the optimum concentration of red pomegranate extract gel for accelerating the healing process in traumatic ulcers is 75%. acknowledgements we would like to express our sincere gratitude to universitas jember for its financial support and particularly to both the chancellor and its head of the research institute and community service. references 1. sunarjo l, hendari r, rimbyastuti h. manfaat xanthone terhadap kesembuhan ulkus rongga mulut dilihat dari jumlah sel pmn dan fibroblast. odonto dent j. 2015; 2(2): 14–21. 2. langlais rp, miller cs, nield-gehrig js. atlas berwarna: lesi mulut yang sering ditemukan. 4th ed. jakarta: egc; 2014. p. 172–3. 3. langkir a, pangemanan dhc, mintjelungan cn. gambaran lesi traumatik mukosa mulut pada lansia pengguna gigi tiruan sebagian lepasan di panti werda kabupaten minahasa. e-gigi. 2015; 3: 1–8. 4. skidmore-roth l. mosby’s 2014 nursing drug reference. 27th ed. missouri: mosby elsevier; 2014. p. 40–3. 5. budi dtw. efek salep ekstrak herba meniran (phyllanthus niruri l.) dibandingkan dengan triamcinolone acetonide 0.1% terhadap proses penyembuhan luka sayat mukosa rongga mulut tikus wistar jantan secara histopatologi. thesis. bandung: universitas kristen maranatha; 2017. p. 1–3. 6. kshitiz p, shipra z, rani s, jayanti s. anti-cariogenic effects of polyphenol plant products-a review. int j res ayurveda pharm. 2011; 2(3): 736–42. 7. hernawati s. ekstrak buah delima sebagai alternatif terapi recurrent apthous stomatitis (ras). stomatognaic j kedokt gigi. 2015; 12: 20–5. 8. setiadhi r, sufiawati i, zakiawati d, nur’aeny n, hidayat w, firman dr. evaluation of antibacterial activity and acute toxicity of pomegranate (punica granatum l.) seed ethanolic extracts in swiss webster mice. j dentomaxillofacial sci. 2017; 2(2): 119–23. 9. mujayanto r. pengaruh zinc sulfat 1% topikal terhadap jumlah makrofag ulkus traumatikus mulut tikus wistar diabetes. thesis. surabaya: universitas airlangga; 2016. p. 30–40. 10. mara rm. pengaruh ekstrak propolis terhadap penyembuhan ulkus traumatik pada mukosa oral. thesis. banda aceh: universitas syiah kuala; 2014. p. 40–54. 11. drugs.com. kenalog orabase fda prescribing information, side effects and uses. 2018. available from: https://www.drugs.com/pro/ kenalog-orabase.html. accessed 2018 jul 18. 12. pramono a, mayasari lo. efektifitas pemberian ekstrak gel belimbing manis (averrhoa carambola linn) terhadap kesembuhan ulkus traumatikus studi in vivo terhadap mukosa tikus (strain wistar). in: prosiding seminar nasional publikasi hasil-hasil penelitian dan pengabdian masyarakat. semarang: universitas muhammadiyah semarang; 2017. p. 39–44. 13. zhang y, krueger d, durst r, lee r, wang d, seeram n, heber d. international multidimensional authenticity specification (imas) algorithm for detection of commercial pomegranate juice adulteration. j agric food chem. 2009; 57(6): 2550–7. 14. rahmina p. pengaruh ekstrak daun pacar kuku (lawsonia inermis) 7,5% terhadap penyembuhan ulkus traumatik pada mukosa oral (penelitian ada tikus odel). thesis. banda aceh: universitas syiah kuala; 2013. p. 62. 15. istiana s. formulasi sediaan gel basis cmc-na ekstrak etanol daun cocor bebek (kalanchoe pinnata (lmk.) pers.) sebagai penyembuh luka bakar pada kelinci. thesis. surakarta: universitas muhammadiyah surakarta; 2016. p. 1–12. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p90–94 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p90-94 https://www.drugs.com/pro/ http://e-journal.unair.ac.id/index.php/mkg vol 38-no 1-2005 4 pengaruh peningkatan konsentrasi sukrosa dalam diet terhadap kadar kalsium gigi tikus wistar (the effect of increasing sucrose concentration in diet toward the content of calcium in tooth of wistar rats) christian khoswanto dan istiati soehardjo bagian biologi oral fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract sweet represents the quality of taste pleased by human beings since the birth. however, if the consumption of sucrose in the diet is excessive, it can change the calcium balance of the body. the aim of this study was to know the effect from the increased sucrose concentration toward the content of calcium in tooth of wistar’s rats. the experiment used 21 days-age of male wistar rats with body weight from 45 to50 grams. they were divided into four groups, and each group consisted of 8 rats. group i got 15% sucrose diet, group ii 30%, group iii 43% and group iv as a standard diet. six weeks after treatment, these rats were anesthetized with ether and killed then by decapitation. the lower incisor was separated from jaw, the mass of each fraction was weighted. atomic absorption spectrophotometer (aas) in mg/g was used to determine the concentration of calcium in wistar’s tooth. one way anava test indicated that there were significant differences between group of treatment and the content of calcium in tooth (p < 0.05). tukeyhsd (honestly significance difference) test indicated that there was a significant difference at group diet of sucrose 43% in tooth. it was concluded that the increased concentration of sucrose in diet could affect the decreased content of calcium in tooth of wistar rats. key words: sucrose, calcium korespondensi (correspondence): christian khoswanto, bagian biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pilihan dari berbagai rasa (manis, pahit, asin dan asam), maka rasa manis akan selalu menjadi pilihan utama.2 di indonesia umumnya 70–80%, bahkan lebih dari 80% dari seluruh energi untuk keperluan tubuh berasal dari karbohidrat. semakin rendah tingkat ekonomi, maka semakin tinggi persentasi energi yang berasal dari karbohidrat, karena energi dari karbohidrat termasuk yang paling murah. masyarakat yang mengalami tingkat kemajuan ekonomi, akan menunjukkan pergeseran asupan dari karbohidrat ke arah lemak dan protein. di negara dengan masyarakat yang mempunyai tingkat ekonomi tinggi, jumlah energi dalam makanan yang berasal dari karbohidrat dapat mencapai 40–50%.2 sukrosa merupakan karbohidrat yang tersusun dari dua satuan monosakarida yang dipersatukan oleh suatu hubungan glikosida dari karbon satu ke suatu oh satuan lain. sukrosa dicerna oleh sistem pencernaan jauh lebih cepat dibanding polisakarida atau kompleks karbohidrat. diet sukrosa dilaporkan dapat menyebabkan hiperkalsiuria pada manusia.3-5 gangguan pertumbuhan dentin yang diakibatkan oleh efek diet tinggi sukrosa berupa terbentuknya penipisan lapisan dentin.6 telah dilaporkan bahwa konsumsi sukrosa dengan kadar yang tinggi dapat menyebabkan kerapuhan dari tulang femur dan tulang tibia pendahuluan berbagai macam kelainan dapat terjadi di dalam rongga mulut dengan faktor etiologi yang berbeda. faktor etiologi dapat berasal dari luar dan dalam tubuh, baik bersifat lokal, maupun sistemik serta herediter. kelainan di dalam rongga mulut dapat terjadi pada setiap bagian antara lain, mukosa, jaringan penyangga dan gigi. salah satu kelainan di rongga mulut yang melibatkan gigi dapat mengenai lapisan enamel atau dentin. gangguan dapat terjadi baik pada saat proses pembentukan maupun pertumbuhan gigi, dan bersifat individual. gangguan pada pertumbuhan dapat menyebabkan gigi menjadi sensitif terhadap berbagai rangsangan. berbagai kelainan yang dapat terjadi dalam proses pertumbuhan gigi meliputi: amelogenesis imperfecta, dentinogenesis imperfecta, osteogenesis imperfecta, dan hipokalsifikasi gigi.1 kebutuhan tubuh terhadap karbohidrat diperlukan karena fungsinya sebagai penghasil energi. kalori yang diperlukan oleh tubuh terutama dihasilkan dari sintesis karbohidrat, lemak, dan protein. karbohidrat disakarida berfungsi sebagai pemanis dalam makanan. rasa manis merupakan salah satu kualitas kecapan yang disukai manusia sejak lahir. jika seorang bayi atau anak kecil diberi 5khoswanto: pengaruh peningkatan konsentrasi sukrosa dari hewan coba.7 gigi merupakan suatu jaringan yang menyerupai tulang, baik dalam komposisi, asal embriologi, sifat kimia maupun sifat fisiknya.8 garam gigi, seperti juga pada tulang, mengandung hidroksiapatit yang bergabung dengan karbonat yang telah diabsorpsi dan berbagai ikatan ionik bersama-sama dengan bahan kristal yang keras. tulang dan gigi terdiri atas matriks organik yang diperkuat oleh endapan garam kalsium, sebagai komposisi utama bahan anorganik. garam kristal yang diendapkan dalam tulang dan gigi dikenal sebagai hidroksiapatit, dengan kandungan terbanyak adalah kalsium.9 pada kondisi terjadi penurunan konsentrasi kalsium cairan ekstraseluler di bawah normal, maka sistem endokrin bekerja untuk mempertahankan homeostasis kalsium. pelepasan hormon paratiroid untuk mempertahankan konsentrasi kalsium, bekerja langsung pada tulang dan gigi dengan cara meningkatkan resorpsi garam tulang dan gigi, hal ini menyebabkan terjadi pelepasan sejumlah kalsium ke dalam cairan ekstraseluler untuk mengembalikan kadar kalsium kembali normal. bila keadaan ini terjadi secara terus menerus, maka komposisi konsentrasi kalsium pada gigi dan tulang akan terganggu. oleh karena itu jika konsentrasi kalsium terus menerus rendah dalam cairan ekstraseluler, dalam jangka waktu yang panjang akan menimbulkan gangguan pada gigi dan tulang.9,10 berdasarkan uraian di atas timbul permasalahan apakah peningkatan konsentrasi sukrosa dalam diet akan menyebabkan pengurangan kadar kalsium gigi tikus wistar. tujuan penelitian ini untuk mengetahui pengaruh dari peningkatan konsentrasi sukrosa dalam diet terhadap kadar kalsium gigi tikus wistar. bahan dan metode subyek penelitian terdiri dari 32 ekor tikus wistar jantan berusia 21 hari, berat badan 45–50 gram, yang dibagi menjadi 4 kelompok, masing-masing kelompok terdiri dari 8 ekor tikus wistar. kelompok i, merupakan kelompok perlakuan dengan diet sukrosa 15%, kelompok ii mendapat sukrosa 30%, kelompok iii mendapat sukrosa 43% dan kelompok iv merupakan kelompok kontrol dengan diet standar pakan wistar. enam minggu kemudian tikus wistar didekapitasi, setelah sebelumnya di anestesi terlebih dahulu dengan ether. rahang bawah diambil, kemudian dipisahkan dan dibersihkan. gigi insisif rahang bawah yang bebas karies dipisahkan dari tulang. masing-masing fraksi ditimbang beratnya dengan timbangan digital. gigi didestruksi dengan asam kuat, dan fraksi yang diperoleh kemudian dilarutkan dalam asam nitrat dan perklorat dengan perbandingan 5 : 2 sebanyak 5 ml, setelah itu diencerkan dengan aquades sampai 50 ml, larutan dibiarkan selama 24 jam untuk menghilangkan senyawa organik dan sekaligus melepas ikatan unsur dalam senyawa yang berada dalam sediaan, sampai sempurna. sediaan yang di dapat diukur absorpsinya dengan metode spektrofotometer serapan atom pada panjang gelombang yang sesuai, lebar celah dan api pembakar. secara bersamaan diukur pula absorpsi dari larutan baku, kurva kalibrasi dibuat dari zat baku caco3. penetapan kadar kalsium gigi tikus wistar dilakukan dengan spektrofotometer serapan atom (ssa) varian spectr aa 55 dalam mg/g berat sampel. selanjutnya setelah didapat kadar kalsium gigi, dicari rata-rata dan standart deviasi dari masing-masing kelompok. data yang diperoleh kemudian dilakukan uji statistik dengan anova, jika terdapat perbedaan bermakna maka dilanjutkan dengan uji tukeyhonestly significance difference (hsd) untuk mengetahui beda kemaknaan dari masing-masing kelompok. hasil dari penelitian yang telah dilakukan tentang pengaruh peningkatan konsentrasi sukrosa dalam diet terhadap kadar kalsium gigi tikus wistar, telah diperoleh data dari masingmasing kelompok. rerata dan standart deviasi kadar kalsium gigi dapat dilihat pada tabel 1. tabel 1. rerata dan standart deviasi kadar kalsium gigi (mg/g) x/sd kelompok i n = 8 kelompok ii n = 8 kelompok iii n = 8 kelompok iv n = 8 x 116,30688 115,35475 113,7200 116,26650 sd 1,296981 1,083159 1,181164 1,053456 keterangan: x = rata-rata kadar kalsium gigi; sd = standar deviasi dari hasil uji distribusi, semua kelompok sampel berdistribusi normal. hasil uji anova satu arah (tabel 2) untuk kadar kalsium gigi didapatkan nilai fratio = 8,750 dan p = 0,001 (p < 0,05). artinya paling tidak terdapat satu perbedaan bermakna di antara kelompok diet sukrosa. untuk menentukan letak perbedaan tersebut, maka dilanjutkan dengan uji statistik tuckey-hsd pada tabel 3. tabel 2. hasil uji anova kadar kalsium gigi sv db jk rk f s antar kelompok dalam kelompok total 3 28 31 35,177 37,522 72,699 11,726 1,340 8,750 0,001 keterangan: uji anova terdapat perbedaan signifikan bila p < 0,05. sv = sumber variasi, db = derajat bebas, jk = jumlah kuadrat, rk= rerata kuadrat, f = harga f hasil analisis, s = p = probabilitas tabel 3. hasil uji tukey-hsd beda kemaknaan kadar kalsium gigi masing-masing kelompok kelompok k i k ii k iii k iv k i k ii k iii ns * * ns ns * keterangan: ns = non significant, (*) = significant 6 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 4–7 tabel 3 menunjukkan adanya perbedaan bermakna (p < 0,05) antara k i dan k iii, k ii dan k iii, serta k iii dan k iv. hal ini menunjukkan bahwa peningkatan konsentrasi sukrosa 43% menyebabkan penurunan signifikan kadar kalsium gigi. pembahasan gigi mempunyai fungsi untuk memotong, menggiling dan mencampur makanan yang dimakan selain fungsi estetik. untuk dapat melaksanakan fungsi ini diperlukan gigi dan tulang rahang serta otot yang sehat, sehingga dapat menjalankan fungsinya dengan baik dalam jangka waktu yang lama. gigi mempunyai kekuatan oklusi bagian depan sebesar 50–100 pon dan bagian belakang 150–200 pon. mineral kalsium yang terdapat di dalam gigi menyebabkan mereka tahan terhadap daya kompresi, sedang serat kolagen menyebabkannya sangat tahan terhadap tegangan yang mungkin timbul sewaktu gigi berhubungan dengan benda padat. mineral yang terdapat pada gigi, seperti pada tulang, mengandung hidroksiapatit yang akan bergabung dengan karbonat setelah diabsorpsi dalam berbagai ikatan ionik bersama-sama dengan bahan kristal yang keras, juga secara konstan akan diendapkan mineral baru, sedangkan mineral yang lama akan direabsorpsi dari gigi, seperti yang terjadi pada tulang.9 pada penelitian ini semua tikus wistar diberikan makanan dan minuman secara ad libitum sehingga tidak ada kekurangan energi yang terjadi, karena wistar mengkonsumsi makanan sesuai dengan energi yang dibutuhkan. tikus wistar diberikan perlakuan setelah selesai disapih, yakni pada usia 21 hari. pada usia tersebut merupakan masa pertumbuhan yang cepat pada tulang dan gigi.11,12 beberapa peneliti berpendapat bahwa pembentukan dentin primer dapat dihambat oleh efek toksik proses metabolisme bakteri pada karies gigi. pengurangan ketebalan pada pembentukan dentin ini memungkinkan terjadinya pengurangan mineral pada gigi, mengingat proses pengendapan mineralisasi gigi sudah mulai terjadi pada pembentukan dentin.13,14 hasil pengamatan pada penelitian ini, pengurangan mineral pada gigi juga terjadi pada gigi yang bebas dari karies. pada penelitian peningkatan konsentrasi sukrosa 15%, 30%, dan 43% menunjukkan pengurangan kadar kalsium yang signifikan pada diet sukrosa 43% pada gigi tikus wistar. oleh karena itu dapat dikatakan bahwa ada pengaruh efek sistemik yang berperan terhadap pengurangan kadar kalsium pada gigi, namun mekanismenya belum dapat dipastikan secara jelas. beberapa peneliti beranggapan bahwa pengaruh yang ditimbulkan oleh sukrosa tersebut terjadi melalui perubahan ekskresi kalsium di urine baik pada manusia maupun hewan coba. diduga kehilangan kalsium tersebut disebabkan oleh pengurangan pada reabsorbsi tubulus ginjal akibat meningkatnya insulin. efek yang ditimbulkan insulin terhadap ekskresi kalsium pada urine mempunyai hubungan yang linier, di mana semakin tinggi insulin dalam plasma, semakin banyak ekskresi kalsium yang terdapat pada urine.3-6 respons sekresi insulin terhadap kenaikan konsentrasi glukosa darah menyebabkan timbul mekanisme untuk pengaturan konsentrasi glukosa darah, hal ini menyebabkan terjadi pengurangan konsentrasi glukosa darah sehingga kembali ke nilai normal. sekresi insulin dapat bertambah sampai sepuluh kali lipat bila konsentrasi glukosa dalam darah meningkat dua kali lipat dari kadar normal puasa, jika hal tersebut berlangsung terus menerus dapat menyebabkan hiperinsulinemia dan resistensi insulin. 9,15,16 resistensi insulin tersebut akan mempengaruhi reabsorpsi tubulus ginjal, yaitu terjadi gangguan pada ginjal dengan akibat pengurangan reabsorbsi kalsium yang menyebabkan peningkatan ekskresi kalsium pada urine. efek insulin tersebut merupakan efek langsung yang menghambat mekanisme transport kalsium atpase pada ginjal. kalsium atpase berada pada basal lateral membran dari ginjal yang terlibat pada proses transport kalsium. peningkatan kalsium di urine yang tinggi yang disebabkan oleh efek insulin akibat peningkatan konsentrasi sukrosa dalam diet menimbulkan risiko kehilangan kalsium dari gigi semakin tinggi. melalui mekanisme umpan balik dimana pada keadaan terjadi penurunan kadar kalsium cairan ekstraseluler, maka sistem endokrin akan bekerja untuk mempertahankan homeostasis kalsium. keadaan ini ditandai dengan pelepasan hormon paratiroid yang meningkat kemudian akan bekerja secara langsung pada gigi dan tulang dengan meningkatkan resorpsi kalsium, yaitu melalui cara mengabsorpsi kalsium pada gigi dan tulang, karena mineral gigi dan tulang merupakan tempat deposit kalsium dalam bentuk hidroksiapatit dengan kadar kalsium yang tinggi.4-6 menurut terada et al.17 kadar glukosa yang tinggi akibat diet tinggi sukrosa, menunjukkan efek langsung dalam menghambat pertumbuhan osteoblas, pada kultur osteoblas yang terpapar glukosa selama tujuh hari menunjukkan hambatan pada pertumbuhan sel osteoblas. diduga glukosa menyebabkan penurunan jumlah putrescine, yang merupakan produk dari ornitin decarboxylase (odc). ornitin decarboxylase (odc) merupakan enzim yang mempunyai peran penting dalam pertumbuhan sel. efek merugikan sukrosa dalam diet dengan kadar 43% dalam waktu 6 minggu pada jaringan yang mengalami mineralisasi, ditunjukkan dengan pengurangan kadar kalsium yang signifikan pada gigi tikus wistar pada masa pertumbuhan. berdasarkan hasil penelitian ini, dapat disimpulkan bahwa peningkatan konsentrasi sukrosa dalam diet dapat menyebabkan penurunan kadar kalsium gigi tikus wistar. oleh karena itu peneliti beranggapan bahwa pada proses mineralisasi gigi, yang terkait dengan pengurangan kadar kalsium merupakan fenomena yang juga dipengaruhi oleh pola diet seseorang. 7khoswanto: pengaruh peningkatan konsentrasi sukrosa daftar pustaka 1. schuurs ahb. patologi gigi geligi. yogyakarta: gajah mada university press; 1988. p. 102–15. 2. sediaoetama ad. ilmu gizi untuk mahasiswa dan profesi. 4th ed. jakarta: dian rakyat; 2000. p. 41–6. 3. berthelay s, hillier sy, nguyen nu, henriet mt, dumoulin g, haton d. relations between oral glucose load and urinary elimination calcium and phosphorus in healthy men with normal body weight. nephrologie 1984; 5: 205–7. 4. holl mg, allen lh. sucrose ingestion, insulin response and mineral metabolism in humans. j nutr 1987; 117: 1229–33. 5. nguyen nu, dumoulin g, henriet mt, regnard j. effects of i.v. insulin bolus on urinary calcium and oxalate excretion in healthy subjects. horm metab res 1998; 30: 222–6. 6. huumonen s, tjaderhane l, larmas m. greater concentration of dietary sucrose decrease dentin formation and increase the area of dentinal caries in growing rats. j nutr 1997; 127: 2226–30. 7. tjaderhane l, larmas ma. high sucrose diet decrease the mechanical sterngth of bones in growing rats. j nutr 1998; 128: 1807–10. 8. vincent p. fundamental of oral histology and embriology. 2nd ed. philadelphia: lea and febiger; 1988. p. 165–70. 9. guyton ac, hall je. buku ajar fisiologi kedokteran. 9th ed. jakarta: buku kedokteran egc; 1997. p. 1038, 1241–53. 10. greenspan fs, baxter jd. endokrinologi dasar dan klinik. 4th ed. jakarta: buku kedokteran egc; 2000. p. 290–9, 355–60. 11. dampster dw, lindsay r. pathogenesis of osteoporosis. lancet 1993; 341: 797–801. 12. heaney rp. nutritional factors in osteoporosis. annu rev nutr 1993; 13: 287–316. 13. kortelainen s, larmas m. effect of low and high fluoride levels on rat dental caries simultaneous dentine apposition. arch oral biol 1990; 35: 229–34. 14. hietala el, tjaderhane l, larmas m. dentin caries recording with schiff’s reagent, fluorescence and backsttered electron image. j dent res 1993; 72: 1588–92. 15. grimditch gk, barnard rj, sternlicht e, whitson rh, kaplan sa. effect of diet on insulin binding and glucose transport in rat sarcolemma vesicles. am j physiol 1987; 252: 420–5. 16. linder mc. nutritional biochemistry and metabolism. appleton and lange co; 1991. p. 21–7. 17. terada m, inaba m, yano y, hasuma t, nishizawa y, morii h, otani s. growth-inhibitory effect of a high glucose concentration on 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 41 no 4 oct-dec 2008.indd 199 15% edta, 67 5% tetracyline hydrochloride, 67 acrylic resin plate, 84 acrylic resin, 25 all ceramic crown, 62 aloe vera, 74 allergic symptoms, 155 allergy, 43 alveolar bone, 21 amalgam, 30 amniotic membrane, 123 angiogenesis, 88 anti proliferation, 5 antimicrobial effect, 39 ascending ramus, 10 fixator, 10 reposition, 10 asseointegration, 56 baduy, 188 bitter taste, 95 bone plating, 77 bootstrap, 190 bracket placement, 173 ca(oh)2, 35 calcium hydroxide, 39, 88, 182 cancer, 5 candida albicans, 25 caries, 190 chemical hazard, 30 citric acid, 103 cloning, 123 membrane, 123 c-myc, 194 collagen, 74 complex aesthetic, 179 connecting implant to natural tooth, 56 crown preparation, 62 cytotoxicity, 67, 103, 107 delayed placement, 173 dendrophtoespecies, 5 dental caries, 100 pulp,142 therapy, 147 direct pulp capping, 35 ectodermal dysplasia, 70 children, 70 partial denture, 70 essential oil, 147 eugenia polyantha wight, 147 exposure duration, 91 fibroblast, 67 flavonoid, 147 gingival immunity, 43 subject index volume 41 gustducin, 95 herb medicine, 5 herpes zoster, 132 hydrogen peroxide, 107, 186 hyperbaric oxygen, 151 il-1, 95 immunohistochemistry, 194 immunologicqal, 167 index, 43 inflammation, 88, 95 innate immunity, 142 intracoronal teeth bleaching agent, 186 invasive treatment, 137 lactobacillus sp, 53 major and minor ras, 47 malar prominence correction, 77 malformed teeth, 179 malondialdehyde, 151 management, 160 mandible joint angle, 10 prothesis, 10 reconstruction, 10 maxillary complete denture, 1 medical problem, 160 mercury, 30 microhybrid composite, 164 microwave polimerizing, 1 mtt assay, 91, 103 myeloma, 5 necrosis, 35 noni fruit juice, 84 occlusion rehabilitation, 77 odontoblas, 142 oncogene, 194 oral candidosis, 132 manifestation, 160 orega new sealer, 110 orthodontic tooth movement, 21 periapical lesions, 137 periodontal disease, 167 persistent oroantral fistula, 128 polycrystalline composite, 164 porcelain fused to metal crown, 179 preclinical simulation, 62 preventive dentistry, 100 primary teeth, 53 probiotic therapy, 100 prophylaxis, 160 psidium guajava linn, 25 pulp perforation, 182 rasp21, 194 200 reactionary dentin, 15 recombinant, 123 reparative dentin, 15 root canal irrigation, 107 saliva, 53, 151 scaling, 155 secretory leukocyte protease inhibitor, 123 self etch dentin bonding, 91 setting time, 110 skills, 62 smokers, 194 sodium peroxide, 186 splint, 179 streptococcus viridans, 39 mutans, 53 subgingival plaque, 155 bacteria, 114 sundanese, 118 surface roghness, 164 systemic diseases, 167 tamarindus indica extract, 107 tanine, 147 temperature and storage, 110 tertiary dentinogenesis, 15 tetracycline gel 0.5 0.7%, 114 tgf-β1, 35, 88, 182 three dimensional change after polymerization, 1 toll-like receptors, 47 tooth remodelling, 21 toxicity, 91 transforming growth factor beta,15 transversal strength, 25, 84 treatment of maxillary sinusitis, 128 two-way relationship, 167 type i collagen, 182 upper integument lip, 118 vacuolization, 35 various orthodontic cases, 173 veillonella sp, 100 water, 186 wound healing, 123 zygomatico maxillary fracture, 77 201 ardan, rachman, 118 arijanti, ester, 74 berniyanti, titiek, 30 budi, ananta tantri, 186 djamil, melanie sadono, 167 ernawati, diah savitri, 47 febriastuti, 179 haniastuti, tetiana, 15, 142 harijanti, kus, 132 indiani, sri redjeki, 84 juniarti, devi eka, 67 kamadjadja, david b, 77, 128 michael josef kridanto, 56 khoswanto, christian, 103 kintawati, silvi, 194 kriswandini, indah listiana, 100 kunarti, sri, 35, 88, 182 lazuardi, mochamad, 5 lestari, sri, 91 mulawarmanti, dian, 151 author’s index volume 41 munadziroh, elly, 123 naini, amiyatun, 25 oewen, roosje rosita, 160 peeters, harry huiz, 137 pradopo, seno, 43, 53 pramono, coen d, 10 prasetyo, eric priyo, 164 pudyani, pinandi sri, 21 sadamori, shinzuke, 1 saskianti, tania, 70 setiawati, ernie maduratna, 114 sumono, agus, 147 sunariani, jenny, 95 sunarko, bambang, 110 tarib, natasya ahmad, 62 utomo, haryono, 155 wigati, chandra, 173 wulandari, erawati, 107 yani, ristya widi endah, 190 zubaidah, nanik, 39 202 thanks to editors in duty of dental journal (majalah kedokteran gigi) volume 41 number 1 january–march 2008: 1. prof. dr. a.g.m. tielens (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands) 2. dr. leslie ang (restorative dentistry – national dental centre of singapore) 3. widowati witjaksono, drg., ph.d. (periodontic – university science malaysia, malaysia) 4. dr. m. suharsini, drg, m.s., sp. kga. (pediatric dentistry – indonesia university) 5. dr. nugrohowati, drg, m.kes. (conservative dentistry – prof. dr. moestopo university) 6. achmad gunadi, drg, m.s., ph.d. (prostodontic – jember university) volume 41 number 2 april–june 2008: 1. prof. nairn hutchinson fulton wilson, msc. ph.d., fds. (conservative dentistry – university of guy’s dental school, london) 2. prof. w.j. spitzer, dmd., md. (head department of cranio & oral – maxillofacial surgery hamburg university, germany) 3. prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a)endrajana, drg.,ms.,sp. bm. (oral maxillofacial surgery airlangga university) 4. achmad harijadi, drg.,ms.,sp.bm (oral maxillofacial surgery – airlangga university) 5. sudarjani gunawan, drg.,ms.,sp.kg. (conservative dentistry – airlangga university) volume 41 number 3 july–september 2008: 1. prof. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery – university science malaysia, malaysia) 2. prof. taizo hamada, d.d.s., ph.d. (prostodontic – university of hiroshima, japan) 3. prof. yukio kato, d.d.s., ph.d. (oral bio chemistry – university of hiroshima, japan) 4. sudarjani gunawan, drg.,ms.,sp.kg. (conservative dentistry – airlangga university) volume 41 number 4 october–december 2008: 1. prof. lakshman samaranayake (oral microbiology – the university of hongkong) 2. kok van kessel (medical microbiology and infections disease – erasmus university medical centre, rotterdam, the netherlands) 3. sudarjani gunawan, drg.,ms.,sp.kg. (conservative dentistry – airlangga university) 4. endanus harijanto, drg.,m.kes. (dental material – airlangga university) dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections: • title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces). • name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed. • abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract. • introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future. • materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method. • result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used. • d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary. • acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 42 no 2 april 2009.indd 76 vol. 42. no. 2 april–june 2009 case report aesthethic and masticatory rehabilitation on post mandibular resection with combination of hollow obturator and hybrid prosthesis arif rachman1, eha djulaeha2 and utari kresnoadi2 1 chief of ladokgi prosthodontic department, yos sudarso, makasar-indonesia 2 department of prosthodontic, faculty of dentistry airlangga university, surabaya-indonesia abstract background: replacing tooth lost caused by caries, periodontal disease, trauma and neoplasm including ameloblastoma which requires mandibular resection is important. purpose: the aim of the study to rehabilitation of post mandibular resection with combination of hollow obturator and hybrid prosthesis. case: a patient 25 years old, male, for having prosthesis to cover defect due to post right mandibular resection. case management: in this presented case, mandibular plate was applied due to spreading defect with losing almost a half body of mandible (class ii modification 2 according to cantor and curtis classification). the design of therapy was mandibular obturator using hybrid prosthesis (removable partial denture metal frame and fixed splint crown with precision attachment) with hollow obturator. the application was based on several advantages: good aesthetic performance, retention, stability, lighter weight and equal share of vertical load for teeth on non surgical site. the result of control i, ii, iii, showed that aesthetic performance, masticatory function, speech and swallowing were in good condition. conclusion: the design of mandibular obturator using hybrid denture with hollow obturator could rehabilitate aesthetic performance, masticatory function, speech and swallowing for patient with post mandibular resection. key words: mandibular resection, hollow obturator, hybrid prosthesis correspondence: utari kresnoadi, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: utari.kresnoadi@yahoo.com introduction tooth loss can be caused by several factors such as caries, periodontal disease and trauma. a partial or complete tooth replacement is required to regain masticatory and lingual function, to improve aesthetic and sustain tissue health.1 tooth loss is also contributed by another factor i.e neoplasm including ameloblastoma. neoplasia is uncontrolled process of abnormal cell growth resulting in neoplasm (tumor).2 ameloblatoma is local and benign adontogenic ephithelium neoplasm which has wide spectrum of histological pattern as an initial odontogenic. among odontogenic cyst and tumor, 1% is ameloblastoma and over 80% occurs in lower jaw, mostly in molar and ramus region. eversole4 suggested that ameloblastoma possibly stems from epithelial rest in malassez, reduced by enamel epithelium covering impacted tooth or gingival epithelium. losing a part of mandible might be caused by trauma, congenital defect, osteoradionecrosis, and surgical intervention by removing tissue of malignant neoplasm. a patient losing a part of the mandible would result in lacking of all stomatognatic system due to partial glossectomy and pharyngectomy so that the oropharyngeal will not function well.5 it is frequently found that ameloblastoma involves the bone of mandible, metastasis might occur resulting in mandible resection, consequently defect would occur due to surgery treatment and special care using obturator would be necessarily performed.6 laney7 suggested some reasons to determine the necessity of obturator application, such as: bad prognosis, 77rachman: aesthethic and masticatory rehabilitation general condition which contra indicated to total anesthesia during surgical performance, major defect which would contribute difficulty to perform reconstructive surgical treatment in addition to chemotherapy which would worsen the treatment outcome. obturator in the form of metal frame denture and hollow obturator, made after recovery period as permanent obturator. the principles of obturator design should be stable, retentive and light.8 obturator is not a permanent restoration due to the function and the accuracy are affected by the alteration of supporting and adjacent tissue. hollow obturator is an obturator with cavity (hollow) in the middle part to make the obturator light so the weight of obturator will not contribute to pressure rotation and dislogging of the remaining abutment teeth.9 the process of hollow obturator is more difficult compared to ordinary obturator. impression procedure with double impression has been successfully done in the process of hollow obturator. grossman10 suggested that mandibular obturator is a method to rehabilitate masticatory, speech, swallowing and aesthetic for patient post mandibulectomy. obturator is supported by resin bonded extra coronal resilient attachment as buccal retention. prosthetic treatment with resin bonded metal casting modified in abutment contour, was splinted to primary abutment in order to add the support, stability and retention on mandibular obturator. based on the above explanation, resin bonded extra coronal resilient attachment could be done on mandibullar defect by considering the patient’s systematic positive point of view. hybrid prosthesis is a complex removable partial denture (rpd) with some bridges and some removable parts and indicated if the fixed denture is unable to replace all the damaged tissue caused by widely damaged tissue due to trauma or required obturator.11 precision attachment is a fabricated tool consisting of male (patrix) and female (matrix) component forming precise attachment, separated one another, and could be independently removed by the patient and functioning as a retainer for rpd or combined fixed splint crown (hybrid prosthesis). in practice, precision attachment is applied on permanent and removable partial denture, over denture and implant denture. the types of precision attachment based on location are as follow: intracoronal attachment and extracoronal attachment,12 intraradicular attachment and extraradicular attachment. extracoronal attachment is located in the outer part of crown countour. the male part is in the abutment tooth and the female part is in the removable partial denture. in extracoronal attachment, the effect of masticatory pressure in the length of abutment tooth is higher than intra coronal attachment. if the location of attachment is closer to cervical part of abutment tooth, the stability would be higher. the advantage of using extracoronal attachment is minimal tooth reduction so tooth devitalization procedure could be avoided, having an ability to sustain normal contour of crown and insertion direction is easier.12 the patient should be recommended to keep his oral hygiene well to avoid irritation of periodontal tissue due to debris entrapment and the formation of calculus. this study discussed about the rehabilitation of patient with post mandibular resection to regain aesthetic performance, masticatory function, speech and swallowing by applying mandibular hollow obturator using hybrid prosthesis. case a 25 year old male patient came to dental hospital, faculty of dentistry airlangga university, referred from dr. soetomo general hospital for having prosthesis to cover the defect due to post right mandibular resection. three months prior to surgical treatment, the patient came to a hospital in banyuwangi due to increased swelling of right lower jaw which disturbed the aesthetic and mastication, then the patient was referred to dr. soetomo hospital. the surgical treatment was performed on june 8, 2005 at oral and maxillofacial surgery department, dr. soetomo general hospital, with the diagnosis of benign tumor in mandible figure 1. pre-operative patient’s profile (side view). figure 2. pre-operative patient’s profile (front view). 78 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 76−81 (ameloblastoma). the patient’s profile appearance, intra oral condition and radiographic finding prior to surgical treatment shown in figure 1, 2 and 3. chemotherapy and radiotherapy were not post operatively performed. on extraoral examination, the eyes, nose, lips, and were normal, the face was asymmetrical and oval. on intra oral examination: defect on right mandible was indicated as class ii modification 2 according to cantor and curtis.5 the lost tooth were: 43, 44, 45, 46, 47, right mandibular resection ranging from teeth 43 to mandible angulus. dental calculus was slightly found in almost all region, occlusion was still presented. the relation of posterior left teeth was cusp to marginal ridge 25 with 35, cusp to fossa 26 with 36 and 27 with 27. overjet and overbite were respectively 3 mm, dynamic occlusion: unilateral balance occlusion, premolar and molar right vestibulum were deep, low lingual frenum, right mandibular flat ridge, ridge relation with transversal plane ≥ 80°, normal front, flat torus mandibular, exotosis was not presented, right and left retromylohyoid were deep. patient’s post operative profile could be seen on figures 4, 5 and 6. on radiographic examination: mandibular plate was seen from right angle of mandible to 35. based on the above condition, clinical diagnosis was esthablished: post resection of right mandible due to ameloblastoma removal; defect classification: class ii modification 2 according to cantor and curtis5 and chronic gingivitis marginalis due to dental calculus on the upper and lower jaw. in this case, mandibular obturator using hybride prothesis (a complex removable partial denture with metal frame, and fixed denture with precision attachment) with hollow obturator was planned. fixed splint on 31, 32, 41, 42 with lingual milled, extracoronal attachment type archor on 42. double akers was used on 35 and 36, with acrylic teeth. case management in this presented case, mandibular obturator using hybrid prosthesis with hollow obturator was planned. the initial treatment was done by removing dental calculus followed by making of the upper and lower jaw impression using alginate. more amount of alginate material was given on the impression tray of right lower jaw due to deep defect so that the deepest region of the defect could be recorded. impression was filled with type 2 hard gypsum to get anatomical model while the base was made of plaster. anatomical model of lower jaw was covered by red wax limited to obturator outline and prosthesis. self curing figure 3. pre-operative radiograph. figure 6. post operative radiograph. figure 4. post operative patient’s profile (front view). figure 5. post operative patient’s profile (side view). 79rachman: aesthethic and masticatory rehabilitation acrylic was placed on the surface of red wax to form individual tray. temporary crown of anatomical model was made for 31, 32, 41, 41, by duplicating anatomical model, preparation was done on the duplicating model. next, pontic was made from sheet wax and impression was taken with alginate material. vaseline was given on the surface of duplicating model and the impression was given self curing acrylic no 4. after setting, it was removed and polished. crown preparation was done on 31, 32, 41, 42 for fixed splint crown. local anesthesia was previously performed before crown preparation in which it should be done not more than 2 mm considering dentine thickness, so that it would not involved the pulp. the next step, individual tray was tried in patient’s mouth and adjustment would be done if necessary. gingi master material was given on the surrounding cervical 31, 32, 41, 42. it was left for 1-2 minute to achieve detailed cervical edge of functional mold. impression was done with double impression i.e putty and light body while muscle trimming was performed. after the material was hardened, the tray was removed from patient’s mouth. the impression was filled up with type iii hard gypsum to form working model. maxillary and mandibulary working model was sent to dental laboratory to make fixed denture with precision attachment on 42. figure 7. extracoronal attachment on 42. on the following visit, adjustment of fixed denture and precision attachment on 42 was done (figure 7). after well adjusted, impression was made using polyvinyl siloxane material in individual tray, then the result was sent to dental laboratory for further process of metal frame. trial was also performed to know whether convenience was achieved between metal frame and the precision attachment (figure 8). glass ionomer cement type i (fuji i, gc japan) was used for cementing the fixed denture. bite rim was made from hard sheet wax supported by 0.7 mm wire on the metal frame. before bite adjustment, the patient was previously trained to open and close his mouth to achieve appropriate occlusion due to horizontal deviation which frequently occurred on the right lower jaw. bite rim was soften, then put in the patient’s mouth to obtain appropriate occlusion, and then returned to working model. fixation was done on upper and lower jaw working model of lower jaw. after that maxillary and mandibulary working model were fixated. prior to tooth alignment in the articulator, choice of tooth color was approved, followed by trial of wax mandibular obturator. evaluation was done to determine whether the condition and the occlusion appropriate for the patient. evaluation was done using mirror and the patient was involved during the process. working model of lower jaw as well as mandibular obturator were removed from the articulator and implanted in the cuvet. hollow obturator technic was done during acrylic processing by giving the space in order to reduce its weight. after finishing and polishing process were performed (figure 9), mandibular obturator was adjusted in the patient’s mouth. the evaluation was done on the aesthetic performance, occlusion, retention, stability and the possibility of pain. selective grinding was performed in the patient’s mouth cavity using articulating paper. premature contact was reduced to obtain good occlusion and equal tooth contact. after selective grinding process was completed, mandibular obturator was inserted into the patient’s mouth. the patient did not complain any pain, however, he still felt awkward with the new condition but quite satisfied with the aesthetic performance (figure 10). next, the patient was informed how to wear and to clean it. in the first 24 hours, mandibular obturator was recommended not to be used for eating but only for adaptation and must be worn during sleeping. the patient was also advised to have a regular control (three times) i.e: control i (one day after figure 9. (a) acrylic on model (sagital view), (b) acrylic on model (front view), (c and d) rpd acrylic with hollow obturator. figure 8. (a) trial fitting of metal frame and fixed denture with metal frame front view and (b) side view. c d 80 dent. j. (maj. ked. gigi), vol. 42. no. 2 april–june 2009: 76−81 insertion), control ii (7 days after insertion) and control iii (one month after insertion). control i: mandibular obturator had been applied and the patient did not feel any pain. on intra oral examination occlusion and mucosal disorder was not found, no significant complaint was presented, the stability was good even the patent felt satisfied with the new aesthetic performance. control ii: no complaint was presented, good occlusion stability shown on intra oral examination no lesion was found during prosthesis wear. the patient was recommended to use mandibular obturator during meal time, consuming soft food and mastication was done usingt left lower jaw to reduce the load on the right side. control iii: mandibular obturator had been applied consuming fairly solid food and right lower jaw was carefully used. no complaint was presented. on intra oral examination good occlusion and stability shown, lesion due to prosthesis wear was not found. the patient was advised to have a regular control in every 6 month period for follow up. discussion tooth loss due to dental caries, periodontal disease, trauma and neoplasm including ameloblastoma requires replacement to achieve regaining of masticatory and speech function, aesthetic as well as to maintain tissue health.1 in general, surgery is needed for patient with ameloblastoma by performing mandibular resection though it is based on the type of ameloblastoma.14 many ways of rehabilitation on post mandibular resection are done based on the size of the resection. the use of implant is the choice of optimal treatment and it can contribute to functional and aesthetic regain for the patient.15 the removal of ameloblastoma would involve large defect requiring almost half of the mandible from the ramus up to the symphisis of mandible. mandibular plate is post operatively used to connect surgical and non surgical part of the mandible, therefore vertical loading force should be well considered especially in prosthesis design. the outcome of surgical treatment shows good recovery, no significant disorder and good prognosis. the therapy of this case is mandibular obturator using hybrid prosthesis (metal frame denture and fixed splint crown with precision attachment) with hollow obturator. the main reason in choosing this therapy is the advantage for the patient especially considering the number of tooth loss, resecting half body of mandible from surgical and nonsurgical site connected by mandibular plate, therefore it can contribute more aesthetic, retention, stability, lighter weight of prosthesis and equal share of vertical load on the remaining teeth on non surgical site. the advantage of using hybrid prosthesis is that the patient can independently wear or remove the prosthesis so it can be easily cleaned, consequently, the health of oral cavity can be well maintained. equal vertical load on mandibular plate resulting stable position of the plate can be maintained and removed from its position or fracture on non surgical site can be avoided. the application of precision attachment include a component of male (patrix) and female (matrix) forming precise connection, separating one another, removable and functioning as a retainer on rpd or combination of hybrid prothesis. precision attachment can contribute good additional retention and can equally share vertical load on the remaining natural teeth. the application of hollow obturator can reduce the heavy load of mandibular obturator which might cause the occurrence of pressure, rotation and dislogging of the remaining abutment teeth.16 three important factors which would determine the success of maxillofacial prosthesis are the creativity of the operator, the technical mastering and the material in which those three factors would support a prosthodontist to express his maximal talent.17 the patient does not show concave appearance on the right buccal, therefore, right and left buccal are symmetrical and it would give psychological support to make the patient more confident in social life. the patient recommended to keep his oral cavity clean especially the extra coronal precision attachment of fixed denture and metal frame figure 10. (a) patient’s profile after insertion (front view), (b) patient’s profile after insertion (side view). 81rachman: aesthethic and masticatory rehabilitation denture. to observe the general condition of oral cavity as well as the prosthesis the patient is suggested to have regular control in every 6 months period. it is concluded that mandibular obturator design using hybrid prosthesis (rpd metal frame and fixed splint crown with precision attachment) with hollow obturator is applicable to rehabilitate masticatory function, esthetic, speech and swallowing in patient post mandibular resection. references 1. osborne j, lammie ga. partial denture. 5th ed. oxford, london, edinburgh, boston palo aito, melbourne: blackweel scientific pub; 1979. p. 20–40. 2. hosa practice test: medical spelling world list. available at: http:// www. mmachs. meridian school.org/hosa/meridian%20spelling% 20(ms).doc. accessed june 25, 2006. 3. marquette university school of dentistry. oral & maxillofacial pathology, ameloblastoma. available at: http://www.dental.mv.edv/ oral path/lesions/ameloblastoma.htm. accessed july 20, 2006. 4. eversole lr. ameloblastomas with pronounced desmoplasmia. j oral maxillofac surgery 1984; 42:735–40. 5. mc givney gp, castleberry dj. mc cracken’s: removable partial prosthodontics. 9th ed st louis, toronto, princenton: the cv mosby company; 1995. p. 476–88. 6. josef mk. penatalaksanaan pemakaian resilient denture liner dalam pembuatan hollow obturator (kasus post hemimaxillextomy). karya tulis akhir program pendidikan dokter gigi spesialis universitas airlangga. 2005. p. 1–9. 7. laney wr. mc. cracken’s removable partial prosthosontics: maxillofacial applications of removable partial prosthodontics. 7th ed. st. louis, toronto, princeton: the cv mosby company; 1985. p. 443–59. 8. boediono i, soeprapto. hollow acryclic resin obturator. pekan ilmiah kedokteran gigi terapan nasional. kongres nasional ke vii pabmi, 1998. p. 101–4. 9. kobayashi m, oki m, ozawa s, inoue t, mukohyama, takato t, ohyama t, taniguchi h. vibration analysis of obturator prostheses with different bulb height designs. j med dent sci 2002; 49:121–8. 10. grossman y, madjar d. resin-bounded attachments for maxillary obturator retention: a clinical report. j prosthet dent 2004; 93(5): 229–32. 11. watt dm, mac gregor ar. designing partial dentures. 1st ed. edinburg: johns wright & sons ltd. published ynder the wright imprint; 1984. p. 183–7. 12. staubli mdt, bagley as. attachment & implants references manual. 2nd ed. attachment international inc. 2002; p. 1–10. 13. preiskel hw. precision attachment in dentistry. 2nd ed. st. louis, toronto, princeton: the cv. mosby company; 1973. p. 41–111. 14. neville bw. oral and maxillofacial pathology. 2 nd ed. south carolina: saunders; 1984. p. 611–9. 15. oelgeisser d. rehabilition of irradiated mandible after mandibular resection using implant/tooth-supported fixed prosthesis: a clinical report. j prosthet dent 2004; 67:310–4. 16. aramany ma. basic principles of obturator design foe partialy edentoulous patient. part i: clasification. j prosthet dent 1978; 40: 554–7. 17. rahn ao, boucher lj. maxillofacial prosthetics-principles and concept. 2nd ed. philadelphia: wb saunders co; 1970. p. 39–83. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice issn 1978 3728volume 47, number 3, september 2014 editorial board of dental journal (majalah kedokteran gigi) sk: 059/un3.1.2/2014 january 2nd– december 31st, 2014 patron: dean of faculty of dental medicine universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 47, number 3, september 2014: endanus harijanto, drg., m.kes. (dental material – universitas airlangga) dr. intan nirwana, drg., m.kes. (dental material – universitas airlangga) dr. theresia indah budhy, drg., m.kes. (oral pathology and maxillofacial – universitas airlangga) dr. retno indrawati, drg., m.si (oral biology – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 206708019-204225738 contents page printed by: airlangga university press. (rk 002/01.15/aup-b5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 47, number 3, september 2014 issn 1978 3728 1. pengaruh chitosan belangkas (tachypleus gigas) nanopartikel terhadap celah antara berbagai jenis semen ionomer kaca dengan dentin (the effect of horseshoe crab (tachypleus gigas) dderived nanoparticle chitosan on interface between various glass ionomers and dentin) henny sutrisman, trimurni abidin dan harry agusnar ............................................................ 121–125 2. modulation of fgf2 after topical application of stichopus hermanii gel on traumatic ulcer in wistar rats rima parwati sari, endahwahjuningsih dan isidora karsini soeweondo ................................ 126–129 3. perbandingan sitotoksisitas tiga jenis algyrogel terhadap sel fibroblas (citotoxicity comparation of three types of algyrogel on fibroblast cells) stefany elan saktiyawardani, hardono jaya lauson, anugerah pekerti astamurtiningrum, mahadna aulia rahmah, pramana pananja putra dan juni handajani .................................. 130–134 4. pengaruh lama pemberian aspirin pada ekspresi protein ki-67 dan ketebalan epitel mukosa rongga mulut tikus wistar jantan (the effect of aspirin administration period on ki-67 expression protein and oral epithelial mucosal thickness in male wistar mice) dian yosi arinawati, heni susilowati dan supriatno ................................................................... 135–140 5. characterization of lactoferrin in gingival crevicular fluid of chronic periodontitis patient sisca meida wati, istiati and pratiwi soesilawati ......................................................................... 141–145 6. oral health knowledge among parents of autistic child in bandung-indonesia yetty herdiyati nonong, arlette setiawan, fellani danasra dewi and cugati navaneetha .... 146–152 7. efektifitas siwak (salvadora persica) dan pasta gigi siwak terhadap akumulasi plak gigi pada anakanak (effectiveness of siwak (salvadora persica) and siwak toothpaste on dental plaque accumulation in children) indra bramanti, iwa sutardjo rs, navilatul ula, dan muhammmad isa .................................. 153–157 8. penatalaksanaan impaksi caninus permanen rahang atas dengan surgical exposure (the management of impacted permanent canine with surgical exposure) syeh brata wijaya dan rinaldi budi utomo ................................................................................. 158–163 9. daya hambat xylitol dan nistation terhadap pertumbuhan candida albicans (in vitro) (inhibition effect of xylitol and nistatin combination on candida albicans growth (in vitro)) sarah kartimah djajusman, udijanto tedjosasongko, dan irmawati ...................................... 164–167 10. pelepasan ion nikel dan kromium kawat australia dan stainless steel dalam saliva buatan (the release of nickel and chromium ions from australian wire and stainless steel in artificial saliva) nolista indah rasyid, pinandi sri pudyani dan jcp heryumani .............................................. 168–172 11. perubahan suhu transisi kaca dan massa resin akrilik heat cured akibat kelembaban dan lama penyimpanan (changes in glass transition temperature and heat cured acrylic resin mass due to moisture and storage time) sherman salim ................................................................................................................................. 173–177 issn 1978 3728volume 46, number 4, december 2013 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dental medicine universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 46 number 4 december 2013: prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) dr. theresia indah budhy, drg., m.kes (oral pathology and maxillofacial – universitas airlangga) dr. indah listiana kriswandini, drg., m.kes (oral biology – universitas airlangga) dr. ernie maduratna setiawatie, drg., m.kes., sp.perio (periodontic – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 206708019-204225738 contents page printed by: airlangga university press. (rk 234/07.14/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 46, number 4, december 2013 issn 1978 3728 1. ekspresi cox-2 setelah pemberian ekstrak etanolik kulit manggis (garcinia mangostana linn) pada tikus wistar (cox-2 expression after mangosteen rind (garcinia mangostana linn) etanolic extract administration in wistar rats) rendra chriestedy prasetya, tetiana haniastuti, dan nunuk purwanti ................................... 173–178 2. profil jaringan lunak wajah kasus borderline maloklusi klas i pada perawatan ortodonti dengan dan tanpa pencabutan gigi (facial soft tissue profile on borderline class i malocclusion in orthodontic treatment with or without teeth extraction) pinandi sri pudyani dan yenni hanimastuti ................................................................................. 179–184 3. minyak ikan lemuru (sardinella longicep) menurunkan apoptosis osteoblas pada tulang alveolaris tikus wistar (fish oil of lemuru (sardinella longicep) reduced the osteoblast apoptosis in wistar rat alveolar bone) didin erma indahyani ..................................................................................................................... 185–189 4. respon inflamasi pulpa gigi tikus sprague dawley setelah aplikasi bahan etsa ethylene diamine tetraacetic acid 19% dan asam fosfat 37% (dental pulp inflammatory response of sprague dawley rats after etching application of 19% ethylene diamine tetraacetic acid and 37% phosphoric acid) nadie fatimatuzzahro, tetiana haniastuti dan juni handajani ................................................. 190–195 5. efek ekstrak daun singkong (manihot utilissima) terhadap ekspresi cox-2 pada monosit yang dipapar lps e.coli (the effect of manihot utilissima extracts on cox-2 expression of monocytes induced by lps e. coli) zahara meilawaty ............................................................................................................................ 196–201 6. the effect of cpp-acp containing fluoride on streptococcus mutans adhesion and enamel roughness yulita kristanti, widya asmara, siti sunarintyas, and juni handajani .................................... 202–206 7. sifat fisik hidroksiapatit sintesis kalsit sebagai bahan pengisi pada sealer saluran akar resin epoxy (physical properties of calcite synthesized hydroxyapatite as the filler of epoxy-resin-based root canal sealer) ema mulyawati, marsetyawan hnes, siti sunarintyas, dan juni handajani .......................... 207.–212 8. prevotella intermedia and porphyromonas gingivalis in dental caries with periapical granuloma risya cilmiaty, afiono agung prasetyo,khilyat ulin nur zaini, mandojo rukmo, suhartono taat putra and widya asmara ................................................................................... 213–217 9. antifungal effect of sticophus hermanii and holothuria atra extract and its cytotoxicity on gingivaderived mesenchymal stem cell kristanti parisihni and syamsulina revianti ................................................................................ 218–223 10. korelasi indeks morfologi wajah dengan sudut interinsisal dan tinggi wajah secara sefalometri (cephalometric correlation of facial morphology index with interincisal angle and facial height) pricillia priska sianita k dan verenna ......................................................................................... 224–228 11. orthodontic-surgical treatment of a severe class iii malocclusion pakpahan evie lamtiur ................................................................................................................ 229–234 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 6161 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 61–65 research report the correlation between ph and flow rate of salivary smokers related to nicotine levels labelled on cigarettes dewi saputri,1 abdillah imron nasution,2 mutiara rizki wardani surbakti,2 and basri a. gani 2 1department of periodontics 2department of oral biology faculty of dentistry, universitas syiah kuala banda aceh indonesia abstract background: saliva is a biological fluid in oral cavity that plays a role in maintaining the environmental balance and oral commensal. nicotine of cigarettes has been reported as a predisposing factor for changing of ph and salivary flow rate, thereby changing in biological salivary components. purpose: this study aimed to analyze the correlation between salivary ph and salivary flow rate in smokers with nicotine levels labeled on cigarettes. methods: purposive sampling was conducted involving 40 male smokers. before participating, they filled a questionnaire related to the history of their smoking habit. using a spitting method for 5 minutes their saliva was collected. results: result of pearson correlation test showed that there was a significant correlation between smoking intensity and salivary flow rate of those smokers (r = -0.486 and p<0.001). the results also indicated that there was a significant correlation between smoking intensity and salivary ph (r = -0.376 and p<0.017). on the other hand, there was no significant correlation between nicotine levels levels labeled on cigarettes with salivary ph of those smokers (r = -0.107, p>0.512). there was no correlation between nicotine levels labeled on cigarettes and salivary ph of those smokers (r = -0.216, p>0.181). nevertheless, there was a significant correlation between salivary flow rate and salivary ph of those smokers (r= 0.686, p<0.00,). conclusion: there is a strong correlation between the intensity of smoking with salivary flow rate and its ph. however, there is no correlation between nicotine levels labeled on cigarettes and both salivary flow rate as well as salivary ph. keywords: intensity of smoking; nicotine levels; salivary flow rate; salivary ph correspondence: dewi saputri, department of periodontics, faculty of dentistry, universitas syiah kuala, banda aceh darussalam, 23111, indonesia. e-mail: dewisaputri_emir@yahoo.co.id introduction smoking is one of the major health problems in the world, especially in developing countries.1 based on world health organization (who) data in 2012, there are 1 billion smokers in the world with a global smoking prevalence of 21%, 790 million of whom are from countries with low and middle-income economies, including indonesia on the fourth rank with the largest number of smokers in the world after china, russia and america.2 the age of 13-17 is also known to be a transition period to be active smokers in indonesia.3 smoking is an attempt to burn tobacco, inhale, and suck back smoke containing harmful substances, such as nicotine using both cigarettes and cigars.4 oral cavity is a part of the body mostly exposed to cigarette smoke.5 the exposure to cigarette smoke then can affect saliva, biological liquid, functioning to maintain the balance of the oral cavity.6 nicotine in cigarette smoke will be absorbed through the lungs and mucous membranes, then circulated through the bloodstream, and distributed to the brain and tissue in all organs of the body.7 nicotine circulating to the bloodstream can affect the blood vascularization to the salivary glands, resulting in decreased function and morphology of glandula.8 nicotine even reaches the brain within 10-20 seconds.9 nicotine then can work on certain cholinergic receptors in the brain that affect central nervous system activity triggering changes in salivary secretion.10,11 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p61-65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p61-65 62 saputri, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 61–65 the changes in salivary secretion can affect salivary flow rate in the group of smokers.10 the salivary flow rate is a modulator of salivary acidity (ph), thus, if the salivary flow rate is small, a small amount of bicarbonate then will be produced, resulting in low salivary ph.12 consequently, salivary flow rate and salivary ph can be considered as factors that play an important role in maintaining oral health.13 in addition, the changes in salivary ph and salivary flow rate are influenced by the duration of smoking and the level of nicotine labeled on the cigarette.14 the smoking intensity is derived from the average number of cigarettes smoked daily multiplied with smoking duration in a year.1 the concentration of nicotine in the salivary gland of smokers is different due to both the number of cigarettes smoked per day as well as the level of nicotine contained the cigarettes.15 therefore, this study aimed to analyze the ph profile and salivary flow rate in association with the labeled nicotine level. materials and methods this study has passed ethical clearance no. 031/ke/ fkg/2016 from the faculty of dentistry, universitas syiah kuala, indonesia. this study used cross-sectional design. in this research, risk factors were smoking frequency and nicotine levels labeled on cigarettes related to salivary ph and salivary flow rate. there were forty subjects in this study, consisted of active male smokers who smoke at least one cigarette per day. those research subjects were determined by using a purposive sampling technique.16 data of those smokers’ profile were taken by using an interview approach with referenced questions that had been prepared by the researchers. furthermore, saliva of those smokers was collected without stimulation at 09.00-12.00 wib. those subjects then were asked not to eat, drink, and brush their teeth 60 minutes before taking saliva. those subjects were also asked to sit on an upright back with the head slightly bowed, but facing forward, and their right hand holding a measuring cup.11 saliva then was collected using spitting method i.e saliva was collected in the mouth with closed lips. afterwards, it was spitted out into the measuring cup every 1 minute for 5 minutes. during collecting saliva, those subjects were not allowed to speak, to move their tongue and to swallow. salivary flow rate was calculated by dividing the collected salivary volume with the time used to collect saliva.17,18 salivary ph then was measured using a ph meter. the electrode tip of the ph meter detector was washed with deionized water (ion free water), dried, and then calibrated to a standard ph value (7.0). meanwhile, the electrode tip was dipped into prepared saliva. the values of the salivary ph tested then were displayed on the screen. each repetition of another salivary ph examination, the electrode tip of the ph meter had to be calibrated to the standard ph.18 the correlation between salivary ph and salivary flow rate was analyzed using a pearson test. the correlation strength analysis was interpreted as a follow: 0.00-0.199 (very weak), 0.20-0.399 (weak), 0.40-0.5999 (medium), 0.60-0.799 (strong), 0.80 to 1.000 (very strong).19 results tables 1, 2, 3, 4, and 5 generally illustrate the correlation between the age of the research subjects and the distribution of the research subjects based on nicotine levels labeled on cigarettes with respect to salivary ph and salivary flow rates. table 1 shows the distribution of the research subjects by age. table 2 describes the distribution of the research subjects based on nicotine levels labeled. table 2 indicates that twenty-six research subjects (65%) had the highest nicotine levels, 1 mg/trunk. table 3, illustrates that there were twenty-nine research subjects (72.5%) with a salivary table 1. distribution of the research subjects by age age (years) number of the research subjects (n) percentage (%) 17-25 26-35 36-45 46-55 9 10 11 10 22.5 25.0 27.5 25.0 total 40 100 table 2. distribution of the research subjects based on nicotine levels labeled on cigarettes nicotine levels labeled on cigarettes (mg) number of the research subjects (n) percentage (%) 1 1.1 1.8 2.2 2.3 2.5 26 4 1 1 7 1 65.0 10.0 2.5 2.5 17.5 2.5 total 40 100 table 3. distribution of the research subjects based on salivary flow rates salivary flow rates (ml/minute) number of the research subjects (n) percentage (%) 0.1-0.25 0.25-0.35 >0.35 29 11 0 72.5 27.5 0 total 40 100 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p61-65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p61-65 6363saputri, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 61–65 flow rate of 0.1-0.25 ml/min (good), while eleven research subjects (27.5%) had a salivary flow rate of 0.25-0.35 ml/ min (medium). none of the research subjects experienced a salivary flow rate of > 0.35 ml/min (bad). table 4 shows the distribution of the research subjects based on the salivary ph. in table 4, there were twenty-seven research subjects (67.5%) with a salivary ph of <6.7, while eleven research subjects (27.5%) had a salivary ph of 6.7-7.4 and, none of the research subjects had a salivary ph of >7.4. results of the statistical tests then showed that there was a significant correlation between salivary flow rate and salivary ph of those smokers (r= 0.686, p<0.00). result of pearson correlation test showed that there was a significant correlation between smoking intensity and salivary flow rate of those smokers (r = -0.486 and p<0.001). the results also indicated that there was a significant correlation between smoking intensity and salivary ph (r = -0.376 and p<0.017). there was a significant correlation between the intensity of smoking and changes in both salivary flow rate and salivary ph (p<0.01) (table 5). on the other hand, there was no significant correlation between nicotine levels levels labeled on cigarettes with salivary ph of those smokers (r = -0.107, p>0.512). there was no correlation between nicotine levels labeled on cigarettes and salivary ph of those smokers (r = -0.216, p>0.181). discussion oral cavity and salivary liquid are important parts of the mouth mostly exposed to cigarette smoke. on the other hand, nicotine is considered as a predispose factor to structural and functional changes of salivary glands that may interfere with salivary flow rate and salivary ph.20 based on the researchers’ analysis, the prevalence of smoking behavior increased by age. this correlates with nicotine contained in cigarettes as an addictive substance that can cause dependence and make cigarettes as a daily necessity at adult age (36-45 years), reaching 27% (table 1). the highest prevalence of smoking behavior was in the age group of 35-45 years, and then decreased in the age group of 45-64 year due to the increased awareness of the danger of smoking.21 the insignificant correlation between nicotine levels labeled on cigarettes and salivary flow rate had very weak correlation strength of 1.1%. it means that 98.9% of them were influenced by other factors. similarly, there was no significant correlation between nicotine levels labeled on cigarettes and salivary ph with weak correlation strength of 4.7%. this indicates that 95.3% of them were affected by other factors as well (table 5). this is because the number of research subjects who consumed cigarettes with a variety of nicotine levels was not controlled due to the random sampling. another assumption is that nicotine can cause stimulation and sedation in the central nervous system depending on the amount of exposure and the duration of exposure.22 each brand of cigarettes has a different nicotine level that causes the different levels of nicotine consumed by everyone even though the number of smoked cigarettes is the same.23 nicotine can work on certain cholinergic receptors in the brain that affect nerve activity triggering changes in salivary ph and salivary flow rates.10 clove cigarettes have a strong role to decrease salivary ph more than non-clove cigarettes.24 in general, nicotine from cigarette smoke had no effect on salivary flow rate (table 3). it means that the values of the salivary flow rate obtained were still in good and medium categories with a negative correlation (r) of -0.486 (table 5) and a correlation coefficient (r2) of 0.236. in other words, the correlation between smoking intensity and salivary flow rate was only 23.6% with a significance probability of <0.01. the results of this research were in line with a research conducted by dyasanoor that showing that the more cigarettes consumed daily for long period can generate a greater risk of decreased salivary flow rate.25 a table 4. distribution of the research subjects based on salivary ph salivary ph number of the research subjects (n) percentage (%) <6.7 6.7-7.4 >7.4 27 13 0 67.5 32.5 0 total 40 100 table 5. the correlation analysis between the intensity of smoking, the levels of nicotine labeled on cigarettes, salivary flow rate, and salivary ph correlation coefficient of correlation (r) coefficient of determination (r2) p values smoking intensity salivary flow rate -0.486* 0.236 0.001** smoking intensity salivary ph -0,.376* 0.142 0.017** nicotine levels labeled on cigarettes salivary flow rate -0.107 0.011 0.512 nicotine levels labeled on cigarettes salivary ph -0.216 0.047 0.181 salivary flow rate salivary ph 0.686* 0.470 0.000** * r significant at 0.01 level 2-tail; ** significant (p<0.01) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p61-65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p61-65 64 saputri, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 61–65 steady heat that blows continuously into the oral cavity also may cause changes in blood flow and a decrease in salivary secretion.26 this is because smoking habits, involving a large number of cigarette intakes per day over a long period of time, can lead to a decrease in sensitivity to oral receptors, resulting in a decrease in salivary reflex.11 consuming 10-15 cigarettes per day for more than 6 months may lower salivary flow rate into 0.20 ml/min (low category).12 a significant correlation between smoking duration and salivary flow rate, as a result, both stimulated and non-stimulated salivary flow rates decreased as the smoking duration increased, but the decreased salivary flow rates was not significant compared to the number of cigarettes consumed daily.27 furthermore, table 4 shows that there were twentyseven research subjects had a salivary ph of <6.7 (67.5%), while eleven research subjects had a salivary ph of 6.7-7.4 (27.5%). the results also showed that there was a negative significant correlation between smoking intensity and salivary ph (r = -0.376) with a correlation coefficient (r2) of 0.142 and a significance correlation (p) of <0.01 (table 5). it suggests that 85.8% of the salivary ph detected from the research subjects was influenced by other factors, such as type of food consumed which is rich of carbohydrates. consequently, the salivary ph will decrease since carbohydrates contained can be utilized by acidogenic bacteria for fermentation, and yield as the product of the bacterial fermentation is acidic, making the oral cavity become acidic.28 in addition, changes in salivary ph may also be affected by changes in bicarbonate structures in saliva and biological rhythms.29 as a result, changes in salivary ph in smokers is usually triggered by changes in electrolytes and ions in saliva, especially bicarbonate structures.10 in addition, smokers who consumed 10-15 cigarettes per day over 6 months had acidic salivary ph of 6.3.12 the average salivary ph in smokers was lower at 6.75 (± 0.11) than in non-smokers with an average salivary ph of 7 (± 0.28), however, there was no significant correlation.30 salivary flow rate is actually considered as a modulator of salivary acidity (ph).10 in this research, there was a positive and significant correlation between salivary flow rate and salivary ph with a correlation strength of 47%. generally, the high salivary flow rate was followed by the low salivary ph of 47% (table 5). the increased salivary secretion can lead to an increase in the number and composition of salivary contents, such as bicarbonate which can increase salivary ph.14 changes in salivary flow rate and salivary ph are actually not only influenced by smoking habits and nicotine levels labeled on cigarettes, but also greatly affected by age, drug consumption, disturbed general state, stress level, circadian rhythm, alcohol consumption and others.31 in conclusion, there is a correlation between smoking intensity and both salivary ph as well as salivary flow rate in smokers. there is also a correlation between nicotine levels labeled on cigarettes and salivary flow rate. however, there was no correlation between nicotine levels labeled on cigarettes and salivary ph. references 1. rad m, kakoie s, brojeni fn, pourdamghan n. effect of long-term smoking on whole-mouth salivary flow rate and oral health. j dent res dent clin dent prospects. 2010; 4(4): 110–4. 2. ng m, freeman mk, fleming td, robinson m, dwyer-lindgren l, thomson b, wollum a, sanman e, wulf s, lopez ad, murray cjl, gakidou e. smoking prevalence and cigarette consumption in 187 countries, 1980-2012. jama. 2014; 311(2): 183–92. 3. ng n, weinehall l, ohman a. “if i don”t smoke, i’m not a real man’--indonesian teenage boys’ views about smoking. health educ res. 2007; 22(6): 794–804. 4. audrain-mcgovern j, benowitz nl. cigarette smoking, nicotine, and body weight. clin pharmacol ther. 2011; 90(1): 164–8. 5. warnakulasuriya s, dietrich t, bornstein mm, casals peidró e, preshaw pm, walter c, wennström jl, bergström j. oral health risks of tobacco use and effects of cessation. int dent j. 2010; 60(1): 7–30. 6. kurku h, kacmaz m, kisa u, dogan o, caglayan o. acute and chronic impact of smoking on salivary and serum total antioxidant capacity. j pak med assoc. 2015; 65(2): 164–9. 7. naik p, fofaria n, prasad s, sajja rk, weksler b, couraud p-o, romero i a, cucullo l. oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe? bmc neurosci. 2014; 15(1): 1–14. 8. arslan e, samanci b, samanci sb, caypinar b, sengezer t, deveci e, seker u. effects of nicotine on the submandibular gland in rats. anal quant cytopathol histopathol. 2015; 37(5): 317–21. 9. hukkanen j, jacob p, benowitz nl. metabolism and disposition kinetics of nicotine. pharmacol rev. 2005; 57(1): 79–115. 10. rudzińiski r. effect of tobacco smoking on the course and degree of advancement inflammation in periodontal tissue. ann acad med stetin. 2010; 56(2): 97–105. 11. khan gj, javed m, ishaq m. effect of smoking on salivary flow rate. gomal j med sci. 2010; 8(2): 221–4. 12. singh m, ingle na, kaur n, yadav p, ingle e. effect of long-term smoking on salivary flow rate and salivary ph. j indian assoc public heal dent. 2015; 13(1): 11–3. 13. gani ba, soraya c, sunnati s, nasution ai, zikri n, rahadianur r. the ph changes of artificial saliva after interaction with oral of artificial saliva after interaction with oral micropathogen. dent j (maj ked gigi). 2012; 45(4): 234–8. 14. rooban t, mishra g, elizabeth j, ranganathan k, saraswathi tr. effect of habitual arecanut chewing on resting whole mouth salivary flow rate and ph. indian j med sci. 2006; 60(3): 95–105. 15. asha v, dhanya m. immunochromatographic assessment of salivary cotinine and its correlation with nicotine dependence in tobacco chewers. j cancer prev. 2015; 20(2): 159–63. 16. palinkas la, horwitz sm, green ca, wisdom jp, duan n, hoagwood k. purposeful sampling for qualitative data collection and analysis in mixed method implementation research. adm policy ment health. 2015; 42(5): 533–44. 17. wong dt. salivary diagnostics. new delhi: wiley-blackwell; 2008. p. 39-42. 18. topkas e, keith p, dimeski g, cooper-white j, punyadeera c. evaluation of saliva collection devices for the analysis of proteins. clin chim acta. 2012; 413(13–14): 1066–70. 19. islas-granillo h, borges-yañez sa, medina-solís ce, galan-vidal ca, navarrete-hernández jj, escoffié-ramirez m, maupomé g. salivary parameters (salivary flow, ph and buffering capacity) in stimulated saliva of mexican elders 60 years old and older. west indian med j. 2014; 63(7): 758–65. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p61-65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p61-65 6565saputri, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 61–65 20. greenberg ms, glick m, ship ja. burket’s oral medicine. 11th ed. sciences-new york. hamilton: bc decker inc; 2008. p. 191-2, 366-8. 21. moosazadeh m. meta-analysis of prevalence of smoking in 15-64year-old population of west of iran. int j prev med. 2013; 4(10): 1108–14. 22. quik m, mallela a, chin m, mcintosh jm, perez xa, bordia t. nicotine-mediated improvement in l-dopa-induced dyskinesias in mptp-lesioned monkeys is dependent on dopamine nerve terminal function. neurobiol dis. 2013; 50(1): 30–41. 23. goniewicz ml, hajek p, mcrobbie h. nicotine content of electronic cigarettes, its release in vapour and its consistency across batches: regulatory implications. addiction. 2014; 109(3): 500–7. 24. agnihotri r, gaur s. implications of tobacco smoking on the oral health of older adults. geriatr gerontol int. 2014; 14(3): 526–40. 25. dyasanoor s, saddu sc. association of xerostomia and assessment of salivary flow using modified schirmer test among smokers and healthy individuals: a preliminutesary study. j clin diagnostic res. 2014; 8(1): 211–3. 26. herawati h, sunariani j. the effect of nicotine on the periodontal tissue. indonesian j trop infect dis. 2010; 1(3): 151–4. 27. petrušić n, posavac m, sabol i, mravak-stipetić m. the effect of tobacco smoking on salivation. acta stomatol croat. 2015; 49(4): 309–15. 28. khemiss m, ben khelifa m, ben saad h. preliminary findings on the correlation of saliva ph, buffering capacity, flow rate and consistency in relation to waterpipe tobacco smoking. libyan j med. 2017; 12(1): 1–7. 29. rojas-morales t, navas r, viera n, alvarez cj, chaparro n. ph and salivary sodium bicarbonate in cancer patients: correlation with seric concentration. med oral patol oral cir bucal. 2008; 13(7): e456-9. 30. grover n, sharma j, sengupta s, singh s, singh n, kaur h. longterm effect of tobacco on unstimulated salivary ph. j oral maxillofac pathol. 2016; 20(1): 16–9. 31. kanwar a, sah k, grover n, chandra s, singh rr. long-term effect of tobacco on resting whole mouth salivary flow rate and ph: an institutional based comparative study. eur j gen dent. 2013; 2(3): 296–9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p61-65 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p61-65 vol 38-no4-2005-isi.pmd 173 the efficacy of clorhexidine 0.2% after scaling in marginal gingivitis m shahrohisham* and widowati witjaksono** ** student of school of dental sciences university science malaysia ** department of periodontic, school of dental sciences university science malaysia and faculty of dentistry airlangga university abstract thirty male subjects aged around 20-30 years old, with complete anterior teeth, no inter proximal caries and good general health conditions were selected by using simple random sampling. the anterior teeth were divided into 2 segments right side for 11, 12, 13 and left side for 21, 22, and 23. these two sites were treated by different way for comparing two treatment variables, between scaling therapy alone and scaling therapy with adjunction of chlorhexidine (chx) 0.2%. gingival index (gi) score was used to determine the gingivitis status of the subjects. clinical experiment showed that the duration of healing process in subjects treated with scaling with adjunction of chx 0.2% was faster than scaling therapy alone group. however, in the statistical analysis, there was no significant difference because of several factors. in conclusion, it was found that chx 0.2% had the efficacy to enhance the healing process in the marginal gingivitis patient that was treated by scaling therapy, but further study using chx 0.2% with longer duration time should be done to achieve more pronounce effect key words: chlorhexidine, marginal gingivitis, chemical plaque control agent correspondence: widowati, department of periodontic, school of dental sciences university science malaysia, health campus 16150 k. kerian, malaysia. introduction gingivitis is inflammation of the gingiva in the absence of clinical attachment loss.1 it is a reversible disease.2 this disease was sub-classified based upon etiology, clinical presentation and associated complicating factors. marginal gingivitis is one of the classifications of gingivitis. it is gingivitis involve the gingival margin and may include a portion of the contiguous attached gingiva.3,4 it occur widely in most populations affecting both children and adult. adolescents have a higher prevalence of gingivitis than pre-pubertal children or adult due to increase in sex hormone during pubertal effects and the composition of the sub gingival micro flora. the most common form of gingivitis is plaque associated gingivitis in which a buildup of bacteria plaque irritates the gingiva, resulting in redness, swelling and pain.2 there is study found that many patients lack the motivation or skills to attain and maintain a plaque free state for significant period time.2 many patient with gingivitis have calculus and the ability to remove bacterial plaque, an acceptable therapeutic result for these individuals is usually obtained when personal plaque control measures are performed in conjunction with professional removal of plaque, calculus and other local factors. scaling and root planning procedures are using hand or ultrasonic instruments accomplish removal of dental calculus. these procedures are to remove plaque and calculus to reduce the number of oral bacteria below the threshold level capable of initiating inflammation.5 the use of chemical plaque control agents to assist in the reduction of bacterial plaque was beneficial for the prevention and treatment of gingivitis in some patients. if properly used, the addition of chemical plaque control agents to a gingivitis treatment methode for patient with deficient plaque control will likely results in reduction of gingivitis. one of the agents is chx. it has been test extensively and has been shown to be the most effective chemical plaque control and the most available agent. there was study found that chx containing rinses to be more effective then phenolic and plant alkaloid rinses.6 the efficacy of chx as an anti-plaque agent is dose dependent in the range of 0.03% to 0.2% chx and is an active agent against a wide range of gram-positive and gram-negative microorganisms and fungi by altered the bacteria cell wall.6 the aim of this study is to evaluate the efficacy of using chx 0.2% after scaling therapy in marginal gingivitis. materials and method patients seeking at dental clinic hospital university sains malaysia (husm) were selected and invited to participate in this study. only male volunteers aged around 20 to 30 years old with complete anterior teeth and no inter proximal caries were taken. those with systemic disease and not taken standard oral hygiene instruction were excluded. women were excluded because they tend to more hormonal imbalance, which affect the result of this study. informed consent was obtained from all volunteers. 174 maj. ked. gigi. (dent. j.), vol. 38. no. 4 oktober–desember 2005: 173–175 a clinical experiment study design was used for data collection. the gingivitis status per visit was recorded. gingivitis status was determined by using gingival index (gi) from loe and silnes 1967.7 the anterior teeth were divided into 2 segments, right side for 11, 12, 13 and left side for 21, 22, 23. after whole mouth scaling and prophylaxis were done, the right side was applied by chx 0.2% two times per day in the morning and night by using cotton pellets starting on the same day, while the other side without the adjunction of chx 0.2%. they should continue applying chx 0.2% everyday during the observation time. every patient had to attend the appointment on 1st, 3rd, 5th and 7th day to re-evaluate. independent t-test was used to analyze the data. results the reduction of gingival inflammation after scaling therapy could be interpreted in the table 1 as follows: day 0 (before treatment) mean score was 1.50 (moderate gingivitis), mean score on 1st day (after therapy) was 1.40, 3rd day was 1.20, 5th day was 1.00 and 7th day was 0.80 (mild gingivitis). as for the reduction of gingival inflammation after scaling therapy followed by chx 0.2% application could be interpreted as follows: before treatment (day 0) mean score was 1.83 (moderate gingivitis), mean score on 1st day (after therapy) was 1.29, 3rd day was 0.91, 5th day was 0.67 and 7th day was 0.41 (mild gingivitis). these results showed that there were reduction in the severity of gingivitis from moderate to mild gingivitis both in patient that was treated by scaling therapy alone and scaling followed by chx 0.2% application. this table also showed that all p values were less than 0.05, which means there was a significant decrease of gi scores by both therapies. in detail, the efficacy of both therapies can be seen from the figure 1. the differences between scaling therapy alone and scaling therapy with followed by chx 0.2% application can be seen in table 2. the reduction of inflammation was obvious seen on the 1st, 3rd, 5th and 7th days which show that the mean gi score of scaling therapy with followed by chx 0.2% was smaller than the mean in the scaling therapy alone. however, on the other hand, both therapies had p value more than 0.05, which means, there were no significant different in healing process between scaling therapy alone and scaling by adjunction of chx 0.2%. discussion in this study, observations were done on the 1st day, 3rd day, 5th day, and 7th day or sevent day. the first day (1st day) examinations were to observe the chx 0.2% effect in the scaling therapy, while the rest of the days (3rd day, 5th day and 7th day) were to examine the whole healing process. this statement was supported by one study which was stated that the healing process occurs in 7th–10th days.8 from the result study and statistical analysis, it showed that with scaling therapy, the gi score was reduce, which was also mentioned 5 according to them, scaling and root planning can reduce the severity of gingivitis and pocket formation. besides that, frequent scaling could increase the duration of healing.9 antiseptic mouth wash usage can prevent plaque formation, which contained of microorganisms, which can cause caries formation and periodontal diseases, like gingivitis. this study also showed decrease in gi score in sample who was treated by scaling therapy with chx.10 from this clinical investigation it was found that the duration of healing process in the samples that were treated by scaling with adjunction of chx 0.2% is faster than scaling therapy alone group. however, in statistical analysis there was no significant different, may be because of several factors such as patients were not compliance to chx, wrong technique during application of the agent and short duration of observation. the conclusion it was found that chx 0.2% has the efficacy to enhance the healing process in marginal gingivitis patient that was scaling, however further study about using chx 0.2% agent with longer duration time should be done to achieve more pronounce result. day therapy 0 1 st 3rd 5th 7th mean (sd) mean (sd) mean (sd) mean (sd) mean (sd) scaling 1.50 (0.39) 1.40 (0.40) 1.20 (0.41) 1.00 (0.35) 0.80 (0.32) p value 0.03 0.01 0.01 0.01 scaling + chx 0.2% 1.83 (1.78) 1.29 (0.39) 0.91 (0.39) 0.67 (0.30) 0.41 (0.23) p value 0.03 0.03 0.03 0.03 table 1. gingival index (gi) score in healing process of gingivitis after therapy independent t-test 175shahrohisham and witjaksono: the efficacy of clorhexidine references 1. the american academy of periodontology. plaque induced gingivitis. j peridontol 2000 may; 71(suplement):851-52. available from url: http:www//perio.org/resources-products/posppr3-2.html. 2. lindhe j. clinical periodontology and implant dentistry. 4th ed. in: karring t, niklaus p, lang, editors. oxford: blackwell publishing co; 2003. p. 200–3. 3. james stephen md. gingivitis. e medicine. available from: url: http://www.emedicine.com/emerg/topic 217.htm. accessed december 20, 2004. 4. gary c. armitage. development of a classification system for periodontal diseases and conditions. ann periodontol 1999 december; 4(1):1–5. 5. martu s, mocanu c, popovici cg. comparison on the effects of chlorhexidine in subgingival applocation in the treatment of adult periodontitis. rev med chir soc med nat lasi. 2000 apr-june; 104(2):125–30. 6. jacob horwitz, eli e, machtei, micha peled, dov laufer. amine fluoride/stannous fluoride and chlorhexidine mouthwashes as adjuncts to surgical periodontal therapy: a comparative study. j of periodontol 2000 oct; 71(10):1601–6. 7. newman, takei, carranza. carranza's clinical periodontology. 9th ed. philadelphia usa: wb saunders co; 2002. p. 80. 8. haffajee ad, socransky ss, goodson gm. subgingival temperature (i) relation to baseline clinical parameters. j clin periodontology 1992; (19):401–8. 9. esther m wilkins. clinical practice of the dental hygienist. 9th ed. a wolters kluwer co. 2005. p. 647–50. 10. pai mr, acharya ld, udupa n. the effect of two different dental gels and a mouthwash on plaque and gingival scores: a six-week clinical study. int dent j 2004 aug; 54(4):219–23. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 1 3 5 7 day m ea n s caling alone s caling and chlorhexidine day therapy 0 1st 5th 7th mean (sd) mean (sd) mean (sd) mean (sd) scaling 1.50 (0.39) 1.40 (0.40) 1.00 (0.35) 0.80 (0.32) scaling + chx 0.2% 1.83 (1.78) 1.29 (0.39) 0.67 (0.30) 0.41 (0.23) p value 0.27 0.55 0.23 0.09 table 1. gingival index (gi) score in healing process of gingivitis after therapy paired t-test figure 1. gingival index after therapies. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 150 the effect of 25% mauli banana stem extract gel to increase the epithel thickness of wound healing process in oral mucosa maharani laillyza apriasari,1 ariska endariantari,1 and ika kustiyah oktaviyanti2 1department of oral medicine, faculty of dentistry, universitas lambung mangkurat 2department of anatomy pathology, faculty of medicine, universitas lambung mangkurat banjarmasin indonesia abstract background: mauli banana is a special plant of south borneo that can be used as alternatif medicine for wound healing wound healing. recent studies showed that mauli banana stem contained some compound such as flavonoid, saponin, and tannin that had antibacterial and antiinflamation effect, and can accelerate the wound healing. purpose: this study was aimed to know the effect of 25% mauli banana extract gel to the epithel thickness of wound healing process in oral mucosa. method: it was the real experimental with post test only control group design. it used 36 sprague dawley rats that divided into 3 groups: the negative control group by giving aquadest, the positive control group by giving drug contain aloe vera, and the treatment group by giving 25% ethanol extract of mauli banana stem. biopsy was done on day 3, 5, 7 and the preparat was made to measure the thickness of oral mucosa epithel by image j software. result: the result showed that 25% ethanol extract of mauli banana stem can increased the thickness of oral mucosa epithel on third day (51.26 µm), fifth days (108.49 µm), and seventh day (170.66 µm). the top thickness of mucosa epithel was on the seventh day. two-ways anova and post hoc lsd (p<0.05) showed the significant different between aquadest and 25% ethanol extract of mauli banana stem. 25% ethanol extract of mauli banana stem and drug contains aloe vera are the aqual of meaningfull. conclusion: 25% ethanol extract of mauli banana can increase the epithelial thickness of wound healing procces in oral mucosa. keywords: epithel; mauli banana stem extract; oral mucosa; wound healing correspondence: maharani laillyza apriasari, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas lambung mangkurat. jl. veteran 128 b, banjarmasin, indonesia. e-mail: maharaniroxy@gmail.com research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 151–154 introduction over the last few decades the role of medicinal plants as a main ingredient in health preservation and management of disease receive a great attention. the treatment by using medicinal plants is increasingly favored because it has generally less side effects than the chemical drugs.1 one of the traditional materials that is used for the treatment is banana. banana have a lot of varieties, including mauli banana which is a typical banana of south borneo.2 the stem of mauli banana is empirically known having an efficacy as a medicinal plant which can accelerates the wound healing process. the people of hulu sungai utara, south kalimantan often use mauli banana stems to accelerate the skin wound healing.3,4 mauli banana as a medicinal plant which is used as an alternative medicine for healing wounds are containing some type of phytochemical compounds, such as saponin, tannin, flavonoid, ascorbic acid, lycopene and β carotene. the function of tannin as an antibacterial can reduce inflammation and increase the epithelium forming and cause the vasoconstriction effect on blood vessels. it is containing flavonoid as antioxidant, antibacterial, anti-inflammatory and analgesic. the flavonoid itself can increase the process of inflammation and inducing the epithel formation.5,6 the study of apriasari et al.,7 showed that the ethanol of 25% mauli banana stem extract is bacteriosid and fungisid. the extract of 100% mauli banana stems can accelerate the back wound healing process of mice by increasing of macrophages.4 the phytochemical content of flavonoids and tannin in the banana stems has immunostimulatory effects dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p150-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p150-153 151151apriasari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 150–153 to enhance the activity and the number of macrophages.8,9 the functions of activated macrophages are secreting the cytokin producing the growth factor. the growth factors play a role in the formation of new cells that are important for wound healing and epithelium formation.9,10 base on apriasari et al.,11 mauli banana stem extract could increase the number of microphage in wound healing on day 3. another study showed that application of mauli banana stem extract can heal the wounds without leaving a scar tissue and the healing speed took place as similar as the application of aloe vera.10 the processes of wound healing include the inflammatory phase, proliferation, and remodelling. in the inflammatory phase there will be a process of phagocytosis and inflammatory reactions that take place within a few minutes. the proliferative phase begins approximately 4 days after the injury and finishes up on 3-4 weeks or more. this phase is characterized by reepithelization, fibroblast proliferation, and angiogenesis. the reepithelization stage of wound healing is very important because it serves to restore the integrity of the oral mucosa. the faster the reepithelization the sooner the wound is closed so the wound healing is also becoming fast. the previous study suggests that the process of epithel formation is reaching the peak on day 7. the next phase is the remodeling which is the final phase in the process of wound healing.12,14 based on the background, the mauli banana stem has several contents of active compound; tannin and flavonoids which are very important in increasing the wound healing process. information of the use mauli banana stem extract as an ingredient to accelerate the wound healing process is fewer compared to aloe vera that already widely known by the society. this study was aimed to determine the effect of 25% mauli banana extract gel to increase the epithel thickness of wound healing process in oral mucosa. materials and methods the experimental animals in this study were 2-3 month male white rats (rattus norvegicus) sprague dawley, 200-250 g body weight, with active movement and in a good condition. this research was purely experimental with post test design and control group. the sampling technique was done at randomly sampling, as many as 36 rats were divided into 3 groups: group 1 (negative control) was given distilled water, group 2 (positive control) given a medicine which is containing aloe vera gel, and group 3 (treatment) was given ethanol of 25% mauli banana extract gel. the observation of this research was done on the day 3, 5 and 7. the fetching of mauli banana stem was conducted in the pertanian pembangunan negeri school, banjarbaru. the making of mauli banana stem extract was done at the faculty of mathematics, universitas lambung mangkurat by maceration method with ethanol, followed by the ethanol-free test. the next step was the making of carbopol gel in the water, and followed by adding the prophylene glycol. the next phase was adding hydroxypropyl cellulose medium (hpmc) and mauli banana stem extract until reached a concentration of 25% in a gel form. the research procedure on day 1 for all groups was done by inhalation anesthetic with diethyl ether, and then conducted by making incision wound on the left buccal mucosa 10 mm throughout and 1mm in depth. the blood that comes out during the manufacture of wound was sterilely cleaned and dried. in that area, performed water application for negative control group, while, the medicine with aloe vera was given for positive control group, and 25% mauli banana extract gel for the treatment group. each treatment was carried for two times a day at 8.00 and 16.00 wita. a decapitation for all groups was done on day 3, 5, and 7, followed by biopsy of the wound. the next stage was making blood smear with staining of hematoxylin eosin (he). the blood smear was observed using olympus light microscope with a magnification of 100x a single field of view. the epithel thickness was measured by using the software image j. the epithel area in the visual field was divided into 10 areas and separated by the measuring line. the measurement of epithel thickness was performed on each measuring line then summed and calculated the average value that was a measurement for a single subject research. the obtained data were tested for its normality by the shapiro-wilk and levene’s test of homogenity. if the data were normally distributed and homogeneous (p>0.05), it would be analyzed by two-way anova with a confidence level of 95%, followed by lsd post hoc test. results in table 1, it showed that in the negative control group (distilled water), positive control group (aloe vera) and 25% mauli banana extract gel group there was an enhancement of epithel thickness on day 3th, 5th, and 7th. for each group, the lowest epithel thickness was on the 3rd day and the highest epithel thickness was on the 7th day. the increasing of epithel thickness in the negative control group was lower than the positive control group and the 25% mauli banana extract gel (figure 1). the results of the analysis with two-way anova based on the time variables of observation obtained a significant value 0.000 (p<0.05), which means that there were significant differences for each group. table 1. the average of reepithelizations thickness for each treatment group day the average of thickness reepithelizations (µm) ± sd control negative control positive mauli extract 3 33.70 ± 6.58 59.67 ± 6.70 51.26 ± 8.44 5 74.00 ± 6.98 106.90 ± 5.57 108.49 ± 9.29 7 115.55 ± 9.22 176.72 ± 7.60 170.66 ± 2.81 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p150-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p150-153 152 apriasari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 150–153 the post hoc lsd test showed that there was no significant difference among the group of 25% mauli banana extract gel and a negative control group, p=0.000 (p<0.05). there was no significant difference p=0.000 (p<0.05) between positive control group and negative control group. positive control group and 25% ethanol extract of mauli banana stem showed p=0.213 (p>0.05). there was no significant difference. between 25% ethanol extract of mauli banana stem had the same effect with medicines with extracts of aloe vera, which is both were able to increase the thickness of the epithelium in oral mucosa wound healing. discussion the phytochemical content which is owned by mauli banana stem extract and patent medicines with aloe vera can accelerate the wound healing process. the mauli banana stem gel extract is containing some type of phytochemical compounds, such as antibacterial such as tannins which reduces inflammation and increase the process of reepithelization and cause the vasoconstriction effect on blood vessels. the content of flavonoids as anti -inflammatory helps to accelerate the inflammatory process and may induce reepithelization so the wound healing is getting faster.12 it is supported by a previous study conducted by prasetyo6 which stated that some of the content of phytochemical compounds such as flavonoids and tannins in the extract gel of ambon banana stem can accelerate the wound healing process with several activities. those activities include affecting the inflammatory cells, increasing the reepithelization, the process of angiogenesis, and the formation of connective tissue in the skin so, it can be used as an alternative wound closures. when the injury occurred, there would be continuity losing or damaging the epithel tissue. the broken epithel tissue is going to have a reepithelization process which is a process of epithel cell reparation so, the wound will be closed. reepithelization is a stage where wound will be healed; it includes migration, mitosis, and differentiation of epithelial cells. these stages will restore the lost mucosal integrity. reepithelization will occur through the movement of epithel cells from the network edge to the network breaks free. the faster the reepithelization, the sooner the structure of the oral mucosa epithelium reaches normal circumstances.6,12 a few hours after injury that was leading to tissue destruction, the epithel cells of the wound edges slowly began to migrate to the injured area. the wound edge of epithelium composed of basal cells detached from its base and became loose knot and then enlarged and moved to fill the wound surface. the epithelium began to closely migrate to the edge of the wound within 24 hours after injury. it was caused by an inflammatory phase, a process of phagocytosis and cleaning the debris, so the reepithelization process is less.16,17 on the 3rd day, the epithel thickness was continuing increase. in table 1 there was a difference between the increasing of epithelium gel thickness of the mauli banana stem extract with negative control. the thickness of the epithelium in the group of 25% mauli banana extract gel was higher than the negative control. this was caused by the wound healing has already entered the stage of proliferation on the 3rd day. the phagocytosis process of strange particles by inflammatory cells in 25% ethanol extract of mauli banana stem was more rapidly, thus it made the proliferation of epithel cell was getting faster anyway. the epithel cells near the wound area were rapidly divided and migrated as intersect movement one another. the migrated epithel cells were getting change of its shape into a more columnar and increasing its mitotic activity.15,16 on the 5th day the thickness of the epithelium was increased. the inflammatory phase had begun to decline and epithel layer was thickened. the increment of epithel thickness was influenced by several growth factors that helped in increasing and stimulating the migration and epithel cells mitosis. those factors that affect reepithelization are fibroblast growth factor (fgf), platelet derived growth factor (pdgf), transforming growth factor-α (tgf-α), and epidermal growth factor (egf).15 on the 7th day, the epithel thickness was in the highest position, at that time the inflammatory phase started to stop and a lot of fibroblast were migrating to the wound area and the collagen and fibroblasts were being attached to the edges of the wound, so that the epithelium can be a b c figure 1. the thickness of epithelial mucous on seventh days by 100 x magnification. (a) negative control; (b) positif control; (c) the extract of mauli banana stem. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p150-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p150-153 153153apriasari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 150–153 thickened, especially on day 7-14.12,18 the epithel thickness had appeared on day 1 and then increased on day-3 and more increased on day 7, since the formation of epithel was started from the wound forming until the wound healing process has stopped and the wound was closed. 10 the increasing of epithel thickness might indicate the healing process can take place more quickly and the inflammatory processes became more shortly.19 the measurement results of epithel thickness in figure 1 showed the positive control group and the group 25% ethanol extract of mauli banana stem had the same thickness. the epithel thickness of the negative control group was thinner than group of 25% ethanol extract of mauli banana stem. this was due to the containing flavonoid and tannin in the 25% mauli banana extract. tannin as antibacterials can minimize the microorganism’s infections and suppressed the infections which can inhibit the reepithelization process. the containing of phytochemical flavonoid accelerated wound contraction and reepithelization.20 the flavonoid had immunostimulatory effects to increase the phagocytosis activation by macrophages and increase the inflammation process.8,9 the increment of macrophage affected the growth factor production which contributed the process of proliferation thus help to accelerate the reepithelization process. this result is similar with the process of wound healing in mice using gel extract of ambon banana stems.6 reepithelization in mauli banana stems group was higher than the negative controls group until the 7th day, however there was no difference between positive control group and gel extract of mauli banana stems group. it was the same as effect of ambon banana stems extract.10,12 compared to the positive control group, the group of 25% mauli banana extract gel had the same epithelium thickness. this was due to a patent medicine with aloe vera that also stimulated the wound healing by stimulating the anti-inflammatory and increasing the inflammatory cells activity. it was helping the reepithelization phase.20 the epithel thickness on negative control group was thinner than 25% ethanol extract of mauli banana stem and a positive control, since the aquades is sterile water and does not have the active ingredient. the absence of active ingredients was causing many microorganisms and damaged cells that should be cleaned up, so the healing process goes slowly.12,22,23 after the epithel got the maximum thickness, the mitotic activity of epithel cells would decrease and the already formed tissue began to enter maturation phase. 21,22 based on the results, it can be concluded that 25% mauli banana extract gel can increase the epithel thickness in the wound healing process in oral mucosa. references 1. ghosh ak, sourav b, bhabatosh h, nishith rb. an overview on different variety of musa species: importance and its enormous pharmacological action. ijpi’s journal of pharmacognosy and herbal formulations 2011; 1(2): 25-7. 2. yulianty m, eny dp, badruzsaufari. analisis kariotipe pisang mauli. bioscientiae, 2006; 3(2): 103-9. 3. septianoor h, apriasari ml, carabelly an. uji efektivitas antifungi ekstrak metanol batang pisang mauli (musa sp) terhadap candida albicans. jurnal pdgi 2013; 62(1): 7-10 4. apriasari ml. potensi batang pisang mauli (musa acuminata) sebagai obat topikal pada penyembuhan luka mulut. banjarmasin: grafika wangi kalimantan; 2015. p. 1-70. 5. apriasari ml, iskandar, suhartono e. kandungan ekstrak metanol batang pisang mauli (musa sp) 100%. international journal of bioscince, biochemistry and bioinformatics, 2014; 4(2): 110-5. 6. prasetyo bf, wientarsih i, pontjo b. aktivitas sediaan salep ekstrak batang pohon pisang ambon (musa paradisiaca var sapientum) dalam proses penyembuhan luka pada mencit (mus musculus albnus). majalah obat tradisional, 2010; 15(3): 121-37. 7. apriasari ml, puspitasari d, dachlan yp, ernawati ds, adhani r. the evaluation of mauli banana stem extract in bioavailability analyzes and clinical mucosa wound healing. australian journal of medical science , 2015; 3: 1-7. 8. mukherjee pk, nema nk, bhadra s, mukherjee d, braga fc, matsabisa. immunomodulatory leads from medicinal plants. indian journal of traditional knowledge, 2014; 13(2): 235-56. 9. yilmaz n, nisbet o, nisbet c, ceylan g, hosgor f, dede od. biochemical evaluation of the therapeutic effectiveness of honey in oral mucosal ulcer. bosnian journal of basic medical sciences 2009; 9(4): 291-5. 10. agarwal pk, singh a, gaurav k, goel s, khanna hd, goel rk. evaluation of wound healing activity of extracts of plantain banana (musa sapientum var. paradisiacal) in rats. indian journal of experimental biology, 2009; 47: 32-40. 11. apriasari ml, carabelly an, aprilia gf. efektifitas ekstrak metanol batang pisang mauli 100% pada penyembuhan luka punggung mencit (mus muculus) ditinjau dari jumlah sel radang. dentofasial jurnal kedokteran gigi 2014; 13(1): 33-7. 12. kun l, diao y, zhang h, wang s, zhang z, yu b, huang s, yang h. tannin extracts from immature fruits of terminalia chebula fructus retz promote cutaneous wound healing in rats. bio medical center complementary and alternative medicine 2011; 86(11): 1-9. 13. apriasari m.l, iskandar, suhartono e. antibacterial activity and total flavanoid of mauli banana stem. international proceedings of chemical, biological and environmental engineering (ipcbee) , 2015; (83): 153-6. 14. ackermann mr. pathologic basis veterinary disease. missouri: mosby elsevier; 2007. p. 111. 15. majewska i, darmach eg. proangiogenic activity of plant extracts in accelerating wound healing: a new face of old phytomedicines. acta biochimica polonica 2011; 58(4): 449–60. 16. kumar v, cotran rs, robbns sl. buku ajar patologis penyakit. edisi 7. jakarta: egc; 2010. p. 65-80. 17. guo s, dipietro la. factors affecting wound healing. j dent res 2010; 89(3): 219-29. 18. sharma y, jeyablan g, singh r, semwal a. a review : current aspect of wound healing agents from medicinal plants. journal of medicinal plants studies, 2013; (1)3: 1-11. 19. larjava h. oral wound healing; cell biology and clinical management. west sussex uk: wiley blackwell; 2012. p. 43. 20. brancato sk, albina je. wound macrophages as key regulators of repair origin, phenotype, and function. am j pathol, 2011; (178)1: 19-25. 21. yadav kch, kumar jr, basha si, deshmukh gr, gujjula r, santhamma b. wound healing activity of topical application of aloe vera gel in experimental animal models. international journal of pharma and bio science, 2012; 3(2): 69-71. 22. novianty ra, bernadetta ec, goeno s. effect of allicin for reepithelization during healing in mouth ulcer model. the indonesian j dent res, 2011; 1(2): 87-94. 23. juniantito, vetnizah, bayu fp. aktivitas sediaan gel dari ekstrak lidah buaya (aloe barbadensis mil.) pada proses persembuhan luka mencit (mus musculus albinus). j pert indon, 2006; 11(1): 18-22. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p150-153 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p150-153 194 volume 47, number 4, december 2014 microleakage of conventional, resin-modified, and nano-ionomer glass ionomer cement as primary teeth filling material dita madyarani, prawati nuraini, and irmawati department of pediatric dentistry faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: glass ionomer cements are one of many dental materials that widely used in pediatric dentistry due to their advantage of fluoride release and chemical bond to tooth structure. adherence of the filling material to the cavity walls is one of the most important characteristic that need to be examined its effect on microleakage. purpose: this study was conducted to examine the microleakage of nano-ionomer glass ionomer cement compared with the conventional and resin-modified glass ionomer cements. methods: standard class v cavities sized 3 mm x 2 mm x 2 mm were made on a total of 21 extracted maxillary primary canine teeth and restored with the conventional, resin-modified, dan nano-ionomer glass ionomer cements. all the teeth were immersed in a 2% methylene blue dye for 4 hours. the depth of dye penetration was assessed using digital microscope after sectioning the teeth labio-palatally. the results were statistically analyzed using kruskal-wallis test. results: all type of glass ionomer material showed microleakage. conventional glass ionomer cement demonstrated the least microleakage with mean score 1.29. the resin-modified glass ionomer cements (mean score 1.57) and nano-ionomer glass ionomer cement (mean score 2.57). conclusion: the conventional glassionomer, resin modified glassionomer, and nano-ionomer glassionomer showed micro leakage as filling material in primary teeth cavity. the micro leakage among three types was not significant difference. all three material were comparable in performance and can be used for filling material but still needs a coating material to fill the microleakage. key words: glass ionomer, micro leakage, primary teeth abstrak latar belakang: semen ionomer kaca adalah salah satu dari banyak bahan gigi yang banyak digunakan dalam praktek kedokteran gigi anak karena bahan tersebut merilis fluoride dan berikatan kimia dengan struktur gigi. perlekatan bahan tumpatan pada dinding kavitas adalah salah satu karakteristik paling penting yang perlu diteliti efeknya terhadap kebocoran mikro. tujuan: penelitian ini dilakukan untuk meneliti kebocoran mikro nano-ionomer glass ionomer dibandingkan dengan glass ionomer konvensional dan resin-modified. metode: standard kelas v kavitas berukuran 3 mm x 2 mm x 2 mm dibuat pada total 21 gigi kaninus sulung rahang atas hasil pencabutan dan ditumpat dengan glass ionomer tipe konvensional, resin-modified, dan nano-ionomer. kemudian semua gigi direndam dalam 2% metilen biru selama 4 jam. setelah gigi dibelah labio-palatal kedalaman penetrasi pewarna dinilai menggunakan mikroskop digital. hasil dianalisis secara statistik menggunakan uji kruskal-wallis. hasil: semua jenis bahan glass ionomer menunjukkan kebocoran mikro. glass ionomer tipe konvensional menunjukkan kebocoran mikro terendah dengan rata-rata skor 1,29; glass ionomer tipe resin-modified (rata-rata 1,57) dan glass ionomer tipe nano-ionomer (rata-rata skor 2.57). hasil uji statistik menunjukkan kebocoran pada tepi ketiga bahan tersebut tidak berbeda secara signifikan. simpulan: bahan tumpatan glass ionomer tipe konvensional, resin-modified, dan nano-ionomer, ketiganya menunjukkan kebocoran tepi tumpatan yang tidak berbeda signifikan. ketiga bahan yang sebanding dalam performance dan dapat digunakan untuk bahan tumpatan tapi masih membutuhkan bahan pelapis untuk mengisi kebocoran mikro yang terjadi. kata kunci: glass ionomer, kebocoran mikro, gigi sulung research report 195madyarani, et al.: microleakage of conventional, resin-modified, and nano-ionomer glass ionomer cement correspondence: dita madyarani, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction restoring carious teeth is one of the major treatment needs in pediatric dentistry. micro leakage has been recognized as the major clinical problem in developing an ideal direct filling dental restoration.1,2 microleakage is defined as the chemically undetectable passage of bacteria, fluids, molecules or ions between the cavity walls and restorative materials.3 microleakage may be the precursor of secondary caries, may promote tooth discoloration, staining of restorative margins, an adverse pulpal response, post operative sensitivity, and even hasten the breakdown of certain filling materials.1,2 the thickness of enamel rods in primary teeth are low compared with permanent teeth which leads to a greater possibilities of pulpal response if microleakage was created.1,2 glass ionomer cements has some physical and chemical properties to make it an excellent dental restorative materials for pediatric patients. they provide a slow release of fluoride that produces a cariostatic action, chemically bind to enamel and dentin, thereby reducing the need for the retentive cavity preparation, and are compatible with pulpal tissue.1,4 conventional glass ionomer cements has been already used since 1970-ies, and development continued to enhance some of their physical abilities such as prolonged setting time, rough surface texture and opaqueness, and led to the introduction of resin-modified glass ionomer cement at 1990-ies.4 to overcome the needs of surface strength, at 2008 a nano-ionomer was found, which has the same basic material with resin-modified, but added with nanofiller and nanocluster technology.5 all of these three glass ionomer materials were usable in present, but the development was not in microleakage performance. the study was designed in-vitro and aimed to examine the microleakage of nano-ionomer glass ionomer cement compared with the conventional and resin-modified glass ionomer cements in primary teeth cavity. materials and methods a total of 21 non-carious extracted primary canine with no or minimal root resorbtion were selected for the study. surface debridement of all teeth was performed and the teeth were stored in normal saline at 4o c temperature until further use. the teeth were randomly divided into three groups of 7 teeth each as follows, group a: cavities filled with nano-ionomer glass ionomer cement; group b: cavities filled with resin-modified glass ionomer cement; group c: cavities filled with conventional glass ionomer cement. a class v cavity of size was prepared on the labio-cervical surface of each tooth with no mechanical retention, using inverted diamond bur in a contra angle high-speed air motor hand piece with water coolant. for group a, nano-glassionomer (ketac primer® 3m espe, usa) was applied to the walls of the cavity for 15 s using microbrush, dried with an air syringe for 10 s and light cured for 10 s. an equal amount of two pastes was dispensed on a paper pad and mixed for less than 20 s. the cavity was restored; excess material removed and condensed using celluloid strip and light cured for 20 s. for group b, resin modified glassionomer (fuji ii lc® gc japan), dentin conditioner was applied to the walls of the cavity for 20 s using microbrush, rinsed with water spray and blotted dry with cotton pellet or air syringe. the capsule filled with restorative material was mixed using triturator for 10 s, then applied into cavity using capsule applier, and excess material removed using plastic filling covered by cocoa butter, and condensed using celluloid strip, and light cured for 20 s. for group c, conventional glassionomer (fuji ix gp® gc japan), the dentin was conditioned, rinsed and dried as above. cement was mixed and restored as above without light curing, and let the cement to set for about 2.5 minutes. all the teeth then stored in normal saline at room temperature for 24 hours (table 1). the apices of all the teeth were sealed with red wax and the entire tooth surface were sealed with two coats of nail table 1. details of the materials investigated in the study grup material commercial name manufacturer packaging combination mixing grup a nano-ionomer gic ketac n100 3m espe, usa clicker dispenser paste-paste manual grup b resin-modified gic fuji ii lc gc, japan capsule powder-liquid triturator grup c conventional gic fuji ix gp gc, japan capsule powder-liquid triturator 196 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 194–197 varnish except for an area approximately 1 mm from the periphery of the restoration. all the teeth were immersed in 2% methylene blue dye for 4 hours. after removal from the dye solution and nail varnish, the teeth then allowed to dry. they were sectioned labio-palatally through the center of the restoration using a carborundum disk. the specimens were then studied under a digital microscope with a magnification of 25x to measure the depth of the dye penetration on the cavity walls of the teeth. the scoring was done as described by tyas and burrow as follows (figure 1). 4 scoring for microleakage was carried out independently by one examiner for 3 times each specimen in order to escalate the validity. the scoring was performed independently by one examiner using the largest score for each specimen. data analysis was done using kruskal wallis, with spss package 18.0. results measurement with the kruskal wallis test was found there was no significant score differences’ between all three glass ionomer materials. the microleakage scores are given in table 2. the difference in microleakage was found in both incisal and gingival wall, but there were no figure 1. diagrammatic representation of the cavity showing walls (a) and scoring (b). notice: score 0 = no leakage; score 1 = less than and up to one-third the depth of the cavity preparation penetrated by the dye; score 2 = more than one-third and up to two-third of the depth of the cavity preparation penetrated by the dye; score 3 = more than two-third up to the junction of the axial and incisal or gingival wall but not including the axial wall; score 4 = dye penetration including the a b table 2 microleakage scores of the three materials used no. sampel group a nano-ionomer gic group b resin-modified gic group c conventional gic 1 4 1 1 2 1 1 1 3 2 4 1 4 1 1 1 5 4 0 1 6 2 2 2 7 4 1 2 table 3. intergroup comparisons of mean microleakage group mean n std. deviation p-value* notes a = nano-ionomer gic 2.57 7 1.397 0.119 not significant b = resin-modified gic 1.57 7 1.134 c = conventional gic 1.29 7 0.488 total 1.81 21 1.167 significant differences between both walls. thus, the wall with maximum scores was considered for the study. the mean score for group anano-ionomer gic was 2.57, group bresin-modified gic was 1.57, and 1.29 for group cconventional gic (table 3). within group c, the microleakage scores shown the least variation compared to group a and b. score 0 was found only in group b, and score 4 found mostly in group a. microleakage in group c did not exceed score 2. intergroup comparison showed that there was no significant differences in microleakage between group a, b and c with p = 0.119. 197madyarani, et al.: microleakage of conventional, resin-modified, and nano-ionomer glass ionomer cement discussion microleakage is used as a measure to evaluate the performance of the restorative materials. this in vitro study was carried out to evaluate and compare the microleakage of nano-ionomer glass ionomer cement, with conventional and resin-modified glass ionomer cement. methylene blue dye was used to assess the microleakage as this was the simplest and fastest method. this method was also used by some researcher to evaluate the microleakage.6,7 the result demonstrates that none of the three glass ionomer cements was free from microleakage, but nano-ionomer glass ionomer cement showed the most microleakage with a mean score 2.57, and conventional glass ionomer cement showed the least microleakage with mean score 1.29. but there were no significant differences found between those three glass ionomer materials (p = 0.119). manipulation and application process become the main concern when microleakage found in a restorative treatment. the present study showed that nano-ionomer glass ionomer cement with manual manipulation and application showed more microleakage than conventional and resin-modified glass ionomer cement. this could be caused by low condensation when filling the material into cavity, different with the conventional and resin-modified glass ionomer cement that use the capsule applicator to put the material into the cavity. unfilled space in a cavity can lead to microleakage.8 the main difference in this glass ionomer cements is the basic component. resin-modified and nano-ionomer glass ionomer cement contain resin that needs a metarcrylates polymerization to set into a restorative material. when polymerization took place, shrinkage happened in cements material, and can produce a micro gap between cavity wall and filling margins.8,9 nano-ionomer glass ionomer cement contains resin more than the others, and it leads to more microleakage found in this filling material. this showed that all resin-based glass ionomer material showed more microleakage than conventional glass ionomer cement. this finding is in accordance with previously reported in vitro study of microleakage of glass ionomer restorations.6 the coefficient thermal expansion of conventional glass ionomer cement is similar to that of adjacent tooth structure, which could be a reason for less microleakage compared with other two glass ionomer cements contains resin that have higher coefficient thermal expansion than tooth structure.9 this can be the possible explanation for less microleakage in conventional glass ionomer cement compared with resin-modified and nano-ionomer glass ionomer cements. another reason for differences in microleakage might be due to differences in maturation of setting reaction. conventional glass ionomer cement sets faster and is of higher viscosity because if finer glass particles, anhydrous polyacrylic acids of high molecular weight and a high powder to liquid mixing ratio.9,10 the result revealed that although all the materials showed microleakage, conventional glass ionomer cement showed slightly less microleakage than resin-modified and nano-ionomer glass ionomer cement. the study suggested that the marginal sealing ability of nano-ionomer, resinmodified and conventional glass ionomer cement were comparable based on the mean score and percentage score of dye penetration, but conventional glass ionomer cement showed slightly better results, though the findings were statistically not significant. all of the glass ionomer material were comparable in performance and can be considered to be materials safe for usage in pediatric dentistry, as long we use a coating layer on the restoration and adjacent tooth structure to fill the micro gaps between filling material and cavity walls that can lead to microleakage. references 1. mcdonald a d. dentistry for the child and adolescent. 9th ed. mosby publication; 2010. p. 333-7, 342-3. 2. pinkham cf, mctigue n. pediatric dentistry: infancy through adolescent. 4th ed. missouri: elsevier saunders; 2005. p. 328-31, 364-66. 3. mosby’s medical dictionary. 8th ed. elsevier; 2009. p. 262. 4. tyas mj, burrow mf. adhesive restoratif materials: a review. australian dent j 2004; 49(3): 112-21. 5. ketac nano: technical product profile. 2011. available from: www.3mespe.com. accessed february 22, 2012. 6. masih s, thomas am, koshy g, joshi jl. comparative evaluation of the microleakage of two modified glass ionomer cements on primary molars. an in-vivo study. j indian soc pedod prev dent 2011; 29(2): 135-9. 7. upadhyay s, rao a. nano ionomer: evaluation of microleakage. j indian society of pedodontics and preventive dentistry 2011; 29(1): 20-4. 8. curtis r, watson t. dental biomaterials: imaging, testing, and modeling. cambridge, england: woodhead publishing ltd; 2008. p. 171-82. 9. schmalz g, arenholt-bindslev d. biocompatibility of dental materials. germany: springer; 2009. p. 149-56. 10. lohbauer u. dental glass ionomer cements as permanent filling material? – properties, limitation, and future trends. materials 2010; 3: 76-96. contents page printed by: airlangga university press. (165/11.08/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail:aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 41 number 3 july-september 2008 issn 1978 3728 dental journal majalah kedokteran gigi 1. cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using mtt assay on culture cell line christian khoswanto, ester arijani and pratiwi soesilawati ...................................................... 103–106 2. cytotoxicity of 5% tamarindus indica extract and 3% hydrogen peroxide as root canal irrigation erawati wulandari .......................................................................................................................... 107–109 3. effect of various temperature and storage duration on setting time of orega sealer bambang sunarko ........................................................................................................................... 110–113 4. the effectiveness of 0.5–0.7% tetracycline gel to reduced subgingival plaque bacteria ernie maduratna setiawati ............................................................................................................. 114–117 5. the profile of upper integument lip of baduy and the nearby living sundanese in south banten, west java, indonesia rachman ardan .............................................................................................................................. 118–122 6. isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene elly munadziroh ............................................................................................................................. 123–127 7. the role of proper treatment of maxillary sinusitis in the healing of persistent oroantral fistula david b. kamadjaja ........................................................................................................................ 128–131 8. the management of oral candidosis in diabetic patient with maxillary herpes zoster kus harijanti, dwi setyaningtyas, and isidora ks ...................................................................... 132–136 9. non-invasive endodontic treatment of large periapical lesions harry huiz peeters .......................................................................................................................... 137–141 10. potential role of odontoblasts in the innate immune response of the dental pulp tetiana haniastuti ........................................................................................................................... 142–146 11. the use of bay leaf (eugenia polyantha wight) in dentistry agus sumono and agustin wulan sd ............................................................................................ 147–150 217217 dental journal (majalah kedokteran gigi) 2020 december; 53(4): 217–222 research report oral health profile of the elderly people in the pandalungan community amandia dewi permana shita,1 zahreni hamzah,1 zahara meilawaty,1 tecky indriana,1 ari tri wanodyo handayani2 and dyah indartin setyowati3 1department of biomedical science, 2department of dental public health, 3department of oral medicine, faculty of dentistry, universitas jember, jember – indonesia abstract background: the pandalungan community is a unique community established through the assimilation of two dominant cultures: the javanese and madurese. both of these communities created a community with a new culture called the pandalungan community culture. the people of this community live in coastal, rural and urban areas. generally, research on the uniqueness in the oral health behaviour of the pandalungan community has not been conducted since the oral health practices of the pandalungan community are considered to be the same as that of the javanese community. purpose: in order to develop programmes for oral health prevention, this research aims at comparing the oral health profiles of the elderly (classified as per age) living in the rural and urban areas in the jember regency. methods: the research employs a cross-sectional approach. the subjects of the research were selected on the basis of the total number of elderly people who attended the monthly meetings of the karang werda (those not willing to participate in the study were excluded). the study was conducted by organising extensive interviews, performing observations and intraoral examinations. each group was classified into three subgroups on the basis of age: pre-elderly, elderly and high-risk elderly. the intraoral examination conducted included the oral hygiene index-simplified (ohi-s), the number of teeth missing, the depth of the pocket and the number of all functional tooth units (all-ftu). results: the oral health profile of people in the rural community was poor when compared to the oral health profile of people living in the urban community (by accounting for nearly all the variables in the examination). conclusion: the oral health profile of the elderly people in the pandalungan community was poor. adequate prevention and care are essential to maintain the oral health of people in the pandalungan community. keywords: elderly; javanese; madurese; oral health; pandalungan correspondence: zahreni hamzah, department of biomedical science, faculty of dentistry, universitas jember. jl. kalimantan 37, jember 68121, indonesia. e-mail: zahreni.fkg@unej.ac.id introduction the pandalungan community was established with the combination of two dominant cultures: javanese and madurese cultures.1 both cultures have different characteristics. the javanese community is widely known as a community in which people speak softly and politely and live in harmony.2,3 the madurese community is known for being more religious and tough on defending dignity, even resorting to violence to resolve problems.4 both of these communities mingled and established a new culture called the pandalungan community culture. administratively, the pandalungan community lives in the eastern part of the east java province, which includes districts and cities, such as pasuruan, probolinggo, lumajang, jember, bondowoso, situbondo and north and south banyuwangi.1 some people live in coastal, urban and rural areas. many people from this community lack education and have inadequate financial assistance. in general, the elderly in the rural areas work as farm workers, gardeners and fishermen,5 while those who live in the urban areas work in many sectors.1,6,7 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 mailto:zahreni.fkg@unej.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 218 shita et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 217–222 cultural behaviour has important implications for human health. the role of culture in a social system is shared among individuals and groups by sharing knowledge, beliefs and/or different practices between group members.8 thus, occasionally, a new behaviour is formed. several aspects can affect the speed and change the way a new behaviour is formed, such as socioeconomic status, gender, religion and moral values. these factors play a role in changing dynamically the behavioural patterns pertaining to health among community members.9–12 culture plays a crucial role in promoting health among the elderly. foster and anderson13 state that culture might influence the health of an individual through many ways, including (1) influence through traditions (2) ethnocentric attitude (an attitude that regards one’s own culture as the best), (3) values and norms in the community that influence and set out what is considered as the best behaviour, (4) pride in the group’s status, (5) the influence of values that are inherited by the members in a community as part of socialisation, (6) fatalistic attitude (an attitude where members do not seek immediate help or treatment and instead give up) and (7) the consequences of innovation on healthy behaviour.13 changing the dental and oral health practices of a community is difficult. additionally, what makes the change even more difficult is that these behaviours are tied to a culture that has existed for a long time. annually, there has been an increase in the number of elderly people in indonesia. the percentage of elderly in the jember district in 2010 was 10.85%. in 2020, this percentage reached 14.30%.14 if a large number of elderly people are not taken care of properly, it will have an impact through an increase in the morbidity rate and an increase in the cost of health care services (individual, family and government funds). on the basis of this reasoning, the elderly would burden those who are young.15,16 although age restrictions are placed in research and health planning, there is no general agreement about age limits.17 generalised age thresholds are used as an indicator for deciding old-age thresholds and also making biological age assumptions. however, until now, there has been no definite agreement in any country to mark an old-age threshold. this is, presumably, because the development of old age is not always proportional to biological age.18 therefore, in this study, we have employed some modifications to decide on the age limit among the elderly in indonesia.17,18 the elderly were classified into the young elderly (middle age, ages between 45 and 59 years), elderly (ages between 60 and 74 years), old elderly (ages between 75 and 90 years) and the very old elderly (over 90 years of age).19,20 dental problems in the elderly are different from those found in other ages.21 usually, elderly people have poor oral health status. oral health cannot be separated from the overall health of the body. poor oral health may cause difficulty during chewing and lead to nutritional disturbances (which leads to diseases all over the body).22–24 this research focused on the study of oral health status among the elderly in the pandalungan community. the examination was conducted using the modified oral hygiene index-simplified (ohi-s),25 the depth of the pocket,26,27 the number of teeth missing17 and the number of allftu.13 these important indicators influence the function of mastication.28 in order to develop programmes for oral health prevention and care, this research aims at comparing the oral health profiles of the elderly (classified as per age) who live in the rural and urban areas of the jember regency. the findings from the study will provide basic data for providing optimal oral healthcare (based on the special needs of the elderly community). materials and methods the research employs a cross-sectional descriptive approach to verify the oral health profile and treatment needed for elderly people in the rural and urban areas of the pandalungan community. the area where the elderly people lived was randomly assigned to reduce bias. the research subjects arrived at on the basis of the total number of elderly people who attended the monthly meetings of the karang werda (a platform to support the welfare needs and accommodate the activities of the elderly). those who were not willing to participate in the study were excluded. the study was conducted by organising extensive interviews and performing observations and intraoral examinations. the elderly in the pandalungan community were classified based on the location where they lived: rural or urban areas in the jember district. each group was further classified into three subgroups on the basis of age: pre-elderly, elderly and high-risk elderly.16 the rural areas comprised the puger-grenden village and the sucopangepok village (n = 90). the urban areas comprised the kaliwates sub-district and sumbersari (n = 78). the elderly respondents in each area were arrived at by using a total sampling technique in which all of the elderly who attended the karang werda meetings willingly took part in the research and were categorised into three sub-groups based on their ages: (1) pre-elderly, ages between 45 and 59 years old (n = 66); (2) elderly, ages between 60 and 74 years old (n = 87); and (3) elderly at high risk ≥ 75 years old (n = 15). all of the respondents completed the informed consent form. ethical clearance was carried out by the ethics commission of the faculty of dentistry at the university of jember (number 924/un25.8/kepk/dl/2019). the respondents for this research were interviewed by employing the interview guidelines provided by the center of environment, ageing and health 2018.21 the interviews were conducted to examine oral hygiene habits, including tooth brushing frequency and methods as well as other procedures related to the risk of tooth loss. oral health practices (guided by oral health surveys from the world health organization, 201316) were examined, such as dental and oral hygiene using ohi-s based on the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 219shita et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 217–222 greene–vermillion index,25 pocket depth,26,27 the number of teeth lost17 and the number of all-ftu.28 teeth in the elderly were measured by examining 28 teeth in their mouth cavities and by removing the wisdom molar teeth. the measurement of ohi-s was obtained by the addition of index debris and calculus covering the tooth’s surface (with some modifications, such as tooth loss, remaining tooth, edentulous and extruded tooth). considering the criteria for ohi-s, a good score of oral hygiene ranged between 0.0–1.2, a moderate score of oral hygiene ranged between 1.3–3.0, and a poor score of oral hygiene ranged between 3.0–6.0.15 the criteria for a healthy pocket depth was ≤3 mm, medium criteria was 4–5 mm and heavy criteria was ≥6 mm.28 all-ftu estimations were based on the total number of functional tooth units (ftu), defined as a similar, natural tooth pair and/or the opposite of a replaced tooth (anterior and posterior) that could be supported by an implant, dental bridge pontics or removable prosthetics.28 the total ftu was divided into six categories: natural to natural teeth (ntftu), natural teeth to fixed prosthetic (nf-ftu), natural teeth to removable prosthetic (nr-ftu), fixed prosthetic to fixed prosthetic (ff-ftu), fixed prosthetic to removable prosthetic (fr-ftu) and removable prosthetic to removable prosthetic (rr-ftu). this estimation did not include the third molar teeth, a tooth with wide coronal destruction, tooth loss and a tooth that had contact with a non-similar tooth (the latter three aforementioned categories of tooth were categorised as non-functional). the molar tooth was considered as two units, and, thus, right–left was eight units. the premolar and anterior teeth were considered as one unit. the premolar right–left was considered as four units. therefore, the total number of ftu in the mouth cavity was 18 units/intact dentition.28 data analysis was conducted using a statistical product and service solutions (spss, version 22) (ibm, new york, usa). the data was tabulated and tested for homogeneity using the levene test, followed by a t-test to determine differences in oral health profiles between the elderly people (based on age group) residing in the urban and rural areas of the pandalungan community. results the data presented in figure 1 pertains to respondents who willingly joined the research. the percentage of female and male respondents was 83.93% and 16.07%, respectively. based on this data, it can be observed that the karang werda monthly meetings were predominantly attended by elderly women. additional, the elderly in rural areas had a poor ohi-s score (table 1), pocket depth (table 2), tooth loss (table 3) and all-ftu (table 4) when compared to the elderly in urban areas (considering all ages). the result of the t-tests revealed a significant difference, especially in the pocket depth and all-ftu among the elderly (ages between 60 and 74 years) in urban and rural areas (p < 0.05). 11.90% 27.40% 7.14% 27.38% 24.40% 1.78% pra elderly-urban elderly-urban high risk elderly-urban pra elderly-urban elderly-rural high risk elderly-rural figure 1. characteristics among the elderly respondents in the pandalungan community. table 1. average ohi-s among the elderly in the rural and urban areas in the pandalungan community. elderly groups urban rural p-value n x ± sd n x ± sd pre-elderly 20 1.56 ± 1.45 46 1.70 ± 1.30 0.720 elderly 46 0.94 ± 5.15 41 2.67 ± 1.75 0.189 high-risk elderly 12 1.28 ± 0.93 3 1.67 ± 1.53 0.900 total 78 90 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 220 shita et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 217–222 discussion the elderly women in the pandalungan community (rural and urban areas) were more active in joining the karang werda monthly meetings when compared to the elderly men. this is because the number of elderly women was higher than the number of elderly men.29 mamai-homata et al.30 stated that women are more concerned about their health than men. hamzah et al.31 also declared that a person who is concerned about their health adopts healthy behaviour. elderly men were more inclined to passively partake in activities conducted during the karang werda monthly meetings; the reason for this could be due to a smaller percentage of elderly men attending the karang werda monthly meetings. the elderly people in the rural areas had a higher ohi-s score, indicating that their oral hygiene was poor when compared to the elderly from the urban areas (considering all ages). however, no significant difference was observed in the ohi-s scores among the elderly in the rural and urban areas. the reason for this could be due to the improved dental facilities and better dental information provided in the urban areas when compared to the rural areas. based on the interviews, pre-elderly people in rural areas brushed their teeth twice a day and used mouthwash; however, there was no improvement in their oral hygiene. an assumption that could be made from this finding is that their toothbrushing technique was not effective. in addition, some elderly and high-risk elderly people mentioned that they did not brush their teeth, as they encountered pain while brushing (because of the high number of tooth loss). these concerns of the elderly and high-risk elderly are supported by a previous study that found that the elderly people who had a high number of missing teeth had difficulties in cleaning the remaining tooth root.32 furthermore, on the basis of the findings from the interview, these two groups (elderly and high-risk elderly) said that they only rinsed their mouth when performing wudu (an islamic procedure for washing parts of the body prior to salah) and had been following instructions handed over to them by their parents for years. from these findings, it is essential to devise a suitable and novel method for cleaning the oral cavities in the elderly who have a lot of missing teeth. with the passage of time, the culture in the pandalungan community is undergoing both social and cultural changes in families and societies due to improvements and changes in the educational, economic and health sectors, as evidenced by the practices followed by the present generation of family members in both rural and urban areas. the social and cultural values that have been maintained in the pandalungan community are also undergoing more positive changes,33 particularly in the urban areas. these findings are indicated in the t-test results of oral hygiene among the elderly people (between 60 and 74 years of age in the urban areas). table 2. average pocket depth among the elderly in the rural and urban areas in the pandalungan community. elderly groups urban rural p-value n x ± sd n x ± sd pre-elderly 20 0.80 ± 0.89 46 0.91 ± 0.83 0.900 elderly 46 0.59 ± 0.65 41 1.02 ± 0.69 0.007* high-risk elderly 12 0.55 ± 0.69 3 0.33 ± 0.58 0.660 total 78 90 * significant different table 3. average of tooth loss among the elderly in the rural and urban areas in the pandalungan community elderly groups urban rural p-value n x ± sd n x ± sd pre-elderly 20 3.50 ± 2.50 46 4.37 ± 5.17 0.536 elderly 46 4.67 ± 4.11 41 6.07 ± 6.38 0.216 high-risk elderly 12 9.36 ± 7.67 3 15.67 ± 7.77 0.005* total 78 90 * significant different table 4. average of all-ftu among the elderly in the rural and urban areas in the pandalungan community elderly groups urban rural p-value n x ± sd n x ± sd pre-elderly 20 8.74 ± 3.77 46 8.00 ± 5.73 0.558 elderly 46 7.59 ± 5.15 41 5.12 ± 4.38 0.023* high-risk elderly 12 4.91 ± 5.65 3 2.00 ± 2.89 0.320 total 78 90 * significant different dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 221shita et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 217–222 as per the findings in the periodontal pocket depth examination (table 2), the elderly people in the rural areas had a significantly deeper pocket depth compared to those in the urban areas. razak et al.32 explained that an increase in age has a bearing on the duration of the periodontal tissue that is exposed to the dentogingival bacterial plaque (which indicates a history of individual oral cumulative). accumulation of bacterial plaque can cause mild to moderate alveolar bone resorption, resulting in deeper periodontal pockets, tooth mobility and can eventually lead to tooth loss.34 table 3 indicates that tooth loss is directly proportional to age among elderly people. a higher incidence of tooth loss is observed in rural areas than in urban areas. these findings may be related to the data shown in tables 1 and 2, which indicates poor oral hygiene and deeper periodontal pockets. the results of the t-test indicate there is no significant difference in the pre-elderly and elderly groups in both rural and urban areas. the average number of tooth loss in rural areas is significantly more (twice than the average number of tooth loss in urban areas). a higher number of tooth loss leads to progressive changes in the structure and function of the oral cavity, including masticatory efficiency, and can thus affect the general health.21–23,32,33 with regards to tooth loss, elderly people can undergo a reduction in all-ftu, which plays an important role in mastication. based on the measurements carried out on allftu, the number of all-ftu among the elderly is smaller in rural areas than in urban areas (considering all ages) (table 4). the findings reveal that the number of teeth used for mastication decrease, which leads to a decline in masticatory efficiency. these findings correspond with a study conducted by shinsho35, who revealed that a minimum of 20 healthy, natural teeth are required for avoiding masticatory difficulty among the elderly people. table 4 indicates that there is no significant difference in measurement of all-ftu in the pre-elderly and highrisk elderly in both rural and urban areas, while there is a significant difference in the elderly people. this difference could be attributed to a high number of the crown, or tooth loss or changes in tooth position. to obtain a higher number of all-ftu, dental filling and tooth replacement with a dental fix or a removable denture is required.32 elderly people who live in rural areas, usually, prefer to opt for the services of an illegal dental practice, offering a direct, partial or a full denture and a door to door service (despite the quality of the treatment being poor and not in accordance with health standards) than visit a licensed dentist. the elderly also do not need to stand in queues to obtain treatment, which is more convenient. based on the above results, it appears that the dental and oral health of the elderly in the pandalungan community is still poor. findings from researches conducted in several countries indicate that the dental health services among the elderly are still inadequate, especially the elderly who lack education and are facing socio-economic limitations.36 the elderly, usually, possess physical disabilities that make it difficult for them to brush their teeth thoroughly and effectively.32,37 the elderly also face difficulties in grasping the handle of a conventional toothbrush. the findings from this study present a big challenge for dental care among the elderly, especially the elderly, in the pandalungan community. the elderly need support to practise oral health procedures that take into account the actual oral health of the elderly. self-care related to oral hygiene for the elderly is challenging and includes multiple factors, including cultural values that have long influenced the health and behaviour of the elderly. therefore, the data pertaining to the oral health profile of an elderly person must be accessed in order to develop future plans for dental and oral health services among the elderly. this study has not identified all the factors that could affect the ability of the elderly in the pandalungan community to carry out comprehensive oral care. acknowledgements we would like to express our gratitude to the rector of the university of jember and the chair of lembaga penelitian dan pengabdian kepada masyarakat (lp2m) (the institute for research and community services), who provided financial assistance and facilitated this research. we would also like to express our gratitude to all agencies that supported this research as well as to all respondents who agreed to take part in our interviews and observations. references 1. sutarto a. sekilas tentang masyarakat pandalungan (overview of the pandalungan community). in: jelajah budaya 2006. yogyakarta: balai kajian sejarah dan nilai tradisional yogyakarta; 2006. p. 1–7. 2. nadar fx. the prominent characteristics of javanese culture and their reflections in language use. humaniora. 2007; 19(2): 168–74. 3. akhda nt. tolerance as the essential key for javanese society in preserving the traditional cultures. dinika acad j islam stud. 2017; 2(2): 199–210. 4. dharmawan a, aji gg, mutiah. madurese cultural communication approach. j phys conf ser. 2018; 953: 012195. 5. samudji s, haryono a, sofyan a. the values of nationalism and behavioral conducts in madurese local wisdom (mlw). int j soc sci humanit invent. 2017; 4(9): 4001–10. 6. sair a. etika masyarakat pandhalungan dalam merajut kebhinekaan (agama). j sosiol pendidik humanis. 2019; 4(1): 47–58. 7. zoebazary mi. orang pandalungan: penganyam kebudayaan di tapal kuda. jember: paguyupan pandhalungan jember; 2017. p. 233. 8. jia y, gao j, dai j, zheng p, fu h. associations between health culture, health behaviors, and health-related outcomes: a crosssectional study. plos one. 2017; 12(7): e0178644. 9. kashima y. how can you capture cultural dynamics? front psychol. 2014; 5(sep): 995. 10. suranto aw. komunikasi sosial budaya. yogyakarta: graha ilmu; 2010. p. 265. 11. samova r la, por ter r e, mcdaniel er. komunikasi lintas budaya: communication between culture. 7th ed. jakarta: salemba humanika; 2014. p. 540. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 222 shita et al./dent. j. (majalah kedokteran gigi) 2020 december; 53(4): 217–222 12. hernandez m, gibb jk. culture, behavior and health. evol med public heal. 2020; 2020(1): 12–3. 13. foster gm, anderson bg. medical anthropology. new york: mcgraw-hill college; 1978. p. 354. 14. parwoto, agustin t. proyeksi penduduk kabupaten/kota provinsi jawa timur 2010-2020. jakarta: badan pusat statistik; 2015. p. 1–165. 15. kowal p, dowd je. definition of an older or elderly person proposed working definition of an older person in africa for the mds project. research on ageing in africa. geneva: world health organization; 2001. p. 1–4. 16. world health organization. oral health surveys: basic methods. geneva: world health organization; 2013. p. 1–125. 17. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cjl, marcenes w. global burden of severe tooth loss: a systematic review and meta-analysis. j dent res. 2014; 93(7 suppl): 20s-28s. 18. freund am, smith j. content and function of the self-definition in old and very old age. journals gerontol psychol sci. 1999; 54b(1): 55–67. 19. lee sb, oh jh, park jh, choi sp, wee jh. differences in youngestold, middle-old, and oldest-old patients who visit the emergency department. clin exp emerg med. 2018; 5(4): 249–55. 20. ouchi y, rakugi h, arai h, akishita m, ito h, toba k, kai i. redefining the elderly as aged 75 years and older: proposal from the joint committee of japan gerontological society and the japan geriatrics society. geriatr gerontol int. 2017; 17(7): 1045–7. 21. hamzah z. analysis of the use of the dmf-t index for the elderly. jember; 2018. 22. hamzah z, indriana t, indahyani de, barid i. sistem stomatognati (pengunyahan, penelanan dan bicara). yogyakarta: deepublish; 2020. p. 235. 23. zhao c, wong l, zhu q, yang h. prevalence and correlates of chronic diseases in an elderly population: a community-based survey in haikou. plos one. 2018; 13(6): e0199006. 24. pangrazzi l, naismith e, miggitsch c, carmona arana ja, keller m, grubeck-loebenstein b, weinberger b. the impact of body mass index on adaptive immune cells in the human bone marrow. immun ageing. 2020; 17(1): 15. 25. greene jc, vermillion jr. the simplified oral hygiene index. j am dent assoc. 1964; 68(1): 7–13. 26. h i rotom i t, yosh i ha ra a, ya no m, a ndo y, m iya za k i h. longitudinal study on periodontal conditions in healthy elderly people in japan. community dent oral epidemiol. 2002; 30(6): 409–17. 27. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cjl, marcenes w. global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. j dent res. 2014; 93(11): 1045–53. 28. hsu kj, yen yy, lan sj, wu ym, chen cm, lee he. relationship between remaining teeth and self-rated chewing ability among population aged 45 years or older in kaohsiung city, taiwan. kaohsiung j med sci. 2011; 27(10): 457–65. 29. maryani h, kristiana l. pemodelan angka harapan hidup (ahh) laki-laki dan perempuan di indonesia tahun 2016 (modeling life expectancy for men and women in indonesia 2016). bul penelit sist kesehat. 2018; 21(2): 71–81. 30. mamai-homata e, koletsi-kounari h, margaritis v. gender differences in oral health status and behavior of greek dental students: a meta-analysis of 1981, 2000, and 2010 data. j int soc prev community dent. 2016; 6(1): 60–8. 31. hamzah sr, suandi t, ismail m, muda z. association of the personal factors of culture, attitude and motivation with health behavior among adolescents in malaysia. int j adolesc youth. 2019; 24(2): 149–59. 32. razak pa, richard kmj, thankachan rp, hafiz kaa, kumar kn, sameer km. geriatric oral health: a review article. j int oral heal. 2014; 6(6): 110–6. 33. smith a, macentee mi, beattie bl, brondani m, bryant r, graf p, hornby k, kobayashi k, wong st. the influence of culture on the oral health-related beliefs and behaviours of elderly chinese immigrants: a meta-synthesis of the literature. j cross cult gerontol. 2013; 28(1): 27–47. 34. gholami m, pakdaman a, virtanen ji. common perceptions of periodontal health and illness among adults: a qualitative study. isrn dent. 2012; 2012: 1–6. 35. shinsho f. new strategy for better geriatric oral health in japan: 80/20 movement and healthy japan 21. int dent j. 2001; 51(3 suppl): 200–6. 36. jo e-d, kim e-s, hong h-k, han g-s. effects of professional toothbrushing and instruction in the elderly: a randomized trial. j dent hyg sci. 2018; 18(5): 305–11. 37. grönbeck lindén i, hägglin c, gahnberg l, andersson p. factors affecting older persons’ ability to manage oral hygiene: a qualitative study. jdr clin transl res. 2017; 2(3): 223–32. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i4.p217–222 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i4.p217-222 74 the use of 90% aloe vera freeze drying as the modulator of collagen density in extraction socket of incicivus cavia cobaya ester arijani and christian khoswanto department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract wound healing is basically a complex process in which cellular and matrix act in concern to re-establish the integrity of injury tissues. this process can be simplified to be healing process consisted of haemostatic, inflammation, cell proliferation and tissue remodeling. the aimed of this research was to know the influence of freeze drying 90% aloe vera application as mandible collagen density modulator in extraction socket of incisive cavia cobaya. this research was done using post test only control groups design and cavia cobaya as the sample. six samples of each control group and 90% aloe vera group applied to test each collagen density for three days and seven days. then, the data was analyzed statistically using mann whitney with 5% significance rate. the result of the study indicates that administering 90% aloe vera can accelerate the growth of collagen density in healing process of extraction socket. the conclusion is 90% aloe vera can modulate the density of collageneous fiber in socket of extraction incicivus tooth wound of cavia cobaya. key words: collagen, aloe vera correspondence: ester arijani, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: christiankhoswanto@hotmail.com introduction anti inflammation medicine has been widely available in the market, however, the price is still relatively expensive. this matter really stimulates researchers to use traditional herb as alternative medicine in which the material is easily obtained and the price is affordable. one of traditional plants which is widely used is aloe vera known as lidah buaya. based on the result of reported study that aloe vera can function as anti inflammation, antibacterial, antivirus, antifungi, antiallergy, increasing immunity, accelerating wound healing process by increasing cell regeneration and lowering blood sugar level.l,2 aloe vera has been widely used in dentistry due to its multiple benefits. the use of aloe vera as a tooth paste helps recover mandible inflammation and lower coloring due to cigarette smoking.3 in addition, product which consists of aloe vera can be used for gingivitis treatment, pain due to denture use and other oral disturbances. aloe vera can also directly be used for mandible inflammation treatment by giving fresh resin to mouth sore, supporting wound healing and functioning as pain relief.4 observation result on wound of baby mice was given aloe vera resin would close twice faster than the wound of baby mice without being given medication. microscopic observation on tissue of wound area showed wound closing and union of skin tissue faster in baby mice given aloe vera resin. regeneration of epithelial cells will occur faster and also the formation of new blood vessel as well as the number of phagocyte cell is higher. aloe vera consists of glucomannan and giberrelins which function to stimulate fibroblast to proliferate faster in wound area and to accelerate wound healing with epithelial cell proliferation as well as to prevent infection which could inhibit wound healing.5 wound healing basically is complicated process in which cellular and matrix component functions to regain the integrity of damage tissue, this process can be simplified to be healing process which consists of homeostasis, inflammation, cell proliferation and tissue remodeling. during the process, some protein structure would develop especially for rigidity elasticity and strength, which depend on the adjacent environment and functional need that is collagen. collagen can be found in all region of the body such as dermist, plain muscle, bone, cartilage and basal membrane. collagen is protein which is mostly found in human body in which 30 % of total weight collagen consists of 33.5% glicine amino acid, 12% proline, 10% proline hydroxide and other amino acids. the purpose of this study was to know the effect of freeze drying 90% aloe vera application as mandible collagen density modulator in of incicivus cavia cobaya. materials and methods this research was an experimental laboratory study using post-test only control groups design. the tools and materials were forceps, special elevator, scissor, syringe 75arijani and khoswanto: the use of 90% aloe vera freeze drying as the modulator 2.5 cc, pinset, cover glass, object glass, microtom, heater, balance, sterile aquadest, 90% aloe vera, either, absolute alcohol, 70%, 80%, 90%, 95%, 99% alcohol, formalin solution, paraffin, xylol, ma staining, nitrate acid. general anesthetic was done on cavia cobaya which was fulfilled the requirement using 10% either in special box. right lower incicivus tooth was cleaned from the food debris by water spraying then it was dried. the incicivus tooth was carefully extracted using needle holder and elevator (all the tools had been sterilized before being used) done in the same direction to prevent the tooth root from fracture, and the tooth was perfectly extracted, then the socket was irrigated by sterile aquadest. aloe vera was given orally by inserting into the syringe, dropped into the socket until completely filled and put it into the wound in post extracted region and sutured. the execution was done using 10% either in lethal dosis on day 3 and 7. the mandible was taken by releasing from the angulus mandible and removed. the specimen was staining using mallory azan to see the collagen fibers in light microscopy using 400× magnificent. the data from collagen density, were tabulated and statistical analysis was done using mann whitney test with 5% significance rate. result on the study of 24 samples of cavia cobaya were classified into control groups and aloe vera groups which applied 90% aloe vera gel in the socket which was used for incicivus tooth extraction by making histological preparation taken on day 3 and day 7. table 1 shows the result from histological preparation based on collagen appearance from socket incisivus cavia cobaya. table 2 shows the mean and standard deviation of collageneous fiber density. the table shows the increase of collageneous fiber density on day 3 and 7 either in control and 90% aloe vera groups. table 2. mean and standard deviation of collageneous fiber density time number control 90% aloe vera x ± sd x ± sd day 3 day 7 6 6 0,83 ± 0,41 1,50 ± 0,51 1,33 ± 0,51 2,66 ± 0,51 table 3. the significant difference of collageneous fiber density time assym. sig. (2-tailed) day 3 day 7 0.092 0.011 table 3 shows the number of collageneous fiber on day 3 did not show significant difference from control group p = 0.092 while day 7 showed significant difference in the wound given 90% aloe vera from the wound without 90% aloe vera (control group) p = 0.01 discussion collagen is a protein which is most frequently found in human body, covering 30% of its drying weight. the main amino acid that composed collagen are glicine, proline and hydroxilicine. collageneous fiber is long fine structure with diameter varies between 20–90 nm.6 the result of histological preparation showed the increase number of collageneous fiber on day 3 and day 7 either in control groups or tested groups. day 3 and day 7 were selected to be the indications based on the consideration that on day 3 collageneous fiber was formed and fibroblast proliferation occurred, while on day 7 collagen had been accumulated.7 the comparison of collageneous fiber score number showed higher of table 1. the data of collagen appearance on day 3 and 7 day group control group aloe vera group group score group score day 3 control 3.1 control 3.2 control 3.3 control 3.4 control 3.5 control 3.6 1 1 1 1 1 1 aloe vera 3.1 aloe vera 3.2 aloe vera 3.3 aloe vera 3.4 aloe vera 3.5 aloe vera 3.6 1 1 2 1 1 2 day 7 control 7.1 control 7.2 control 7.3 control 7.4 control 7.5 control 7.6 1 2 2 1 1 2 aloe vera 7.1 aloe vera 7.2 aloe vera 7.3 aloe vera 7.4 aloe vera 7.5 aloe vera 7.6 2 3 3 3 3 3 76 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 74-76 collageneous fiber in control groups than tested groups either on day 3 or day 7 (table 2). the result of data analysis showed significant difference between control group and treatment group. the result suggested no significant difference was found between control group and aloe vera group in which both groups on day 3, it is possible due to wound healing process on day 3 the formation of new collageneous fiber just started so the effect given by 90% aloe vera tested group to stimulate collageneous fiber formation had not occurred. while between control group and tested group in which each group on day 7 showed significant difference due to the effect of 90% aloe vera given to tested group had already functioned. significant difference in the number of collageneous fiber in control group and tested group was due to aloe vera content which functioned to stimulate wound healing process. aloe vera functions to stimulate the growth of new fibroblast cell and to accelerate wound healing due to the substance content such as glucomannan which is polysaccharide complex mostly consisting of sugar mannose in which could stimulate fibroblast to proliferate faster in wound region. gibberelins is growth hormone which could stimulate fibroblast proliferation and accelerate protein sinthesis.6 aloe vera consist of vitamin a, b, b1, b2, b3, b6, c, d, e niachinamida, cholin, folate acid which are important for growth and improvement of damaged tissue.8 a study suggests that vitamin given orally could increase the formation of collageneous fiber.9 amylase, catalase, celluse, oxidase, lipase, protease enzyme regulate chemical process in the human body and accelerate wound healing and regeneration process. aloe emodin in aloe vera is useful to support recovery and improvement of damaged tissue and also relieve the pain. antraquinones has the effect of anti inflammation, analgesic and anti microbe. saponine has the effect of anti septic, anti microbe, anti fungi and anti virus. amino acid in aloe vera is important for growth and tissue proliferation.6,10,11 antimicrobial and anti septic substance in aloe vera are useful to prevent infection which is one of the most influence local factors in wound healing process. healing varies according to the turnover times of the tissue.12 the occurrence of infection can inhibit the wound healing. in addition to various useful contents for medical treatment, aloe vera also has harmful and dangerous content for health. the yellow resin containing antraquinone is a laxative irritant producing that can harm enzymatic system in gastric wall and contribute toxic effect in human body and that was it is not applied on this study. aloe vera had been peeled up and washed until it was free from yellow resin before the process and freeze drying method were done. the conclusion is 90% aloe vera can modulate the density of collageneous fiber in socket of extraction incicivus tooth wound of cavia cobaya. references 1. wolfe b. the handbook of aloe vera. new mexico: albuquerque; 2001. p. 116–7. 2. furnawanthi i. khasiat dan manfaat lidah buaya. bogor. 2002. p. 25–30. 3. trelia b. daya anti bakteri pada beberapa konsentrasi dan kadar hambat tumbuh minimal aloe vera. dentika dental journal 2002; 7(1): 58–66. 4. hembing hm. tanaman berkhasiat obat di indonesia. jakarta: pustakajakarta: pustaka kartini; 1992. p. 622–7, 1070.1992. p. 622–7, 1070. 5. davis r. biological activity of aloe vera. available at: http://www. aloevera and polysacharadis.com. accessed april 2006. 6. junqueira lc, carneiro j, kelley ro. basic histology. 9basic histology. 9th ed. appleton and lange. 2006. p. 91–120. 7. vinay k, et al. robbins pathologic basic of disease. 6th ed. toronto: wb saunders company; 1999. p. 98–9, 102–11. 8. santoso hb. lidah buaya juga untuk kanker. rubrik media medis senior; 2000. p. 28. 9. kathleen a. effect of retinoic acid and airspace development and lung collagen in hyperoxia-exposed newborn rats. paediatric research 2000; 48:434–44. 10. sullatia gg, dkk. farmakologi dan terapi. edisi ke-4. jakarta: bagianjakarta: bagian farmakologi fakultas kedokteran universitas indonesia; 2001. p. 220–7.–7.7. 11. nanci a. oral histology development structure and function. 6th ed. st louis: mosby; 2003. p. 410. 12. bath mb, fehrenbach mj. dental embryology, histology and anatomy. 2nd ed. missouri: elsevier saunders; 2006. p. 149. vol 44 no 3 sept 2011.indd dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3–5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 97 vol. 43. no. 2 june 2010 pediat dent. 1992; 14(2): 119–21. available at: http://www.aapd.org/. accessed september 30, 2005. 27. lai py, seow wk, tudehope di, rogers y. enamel hypoplasia andenamel hypoplasia and dental caries in very-low birthweight children. a case-controlled, longitudinal study. pediat dent. 1997; 19: 42–9. available at: http:// www.aapd.org/. accessed september 13, 2005. 28. simmer jp. dental enamel formation and its impact on clinical dentistry. j dent education. 2001 65(9): 896-904. 29. eastman dl. dental outcomes of preterm infants. nbin 2003; 3(3): 93–8. available at: http://www.medscape.com/viewarticle/461574. accessed augusts 8, 2006. 30. jorgenson rj, yost c. etiology of enamel dysplasia. journal of pedodontics, summer. 1982; 315–467. 31. fisk nm, smith rp. fetal growth restriction; small for gestational age. in: chamberlain g, steer p, editors. turnbull's obstetrics. 3rd ed. london: churchill livingstone; 2001. p. 197–209. 32. stewart re, witkop cj, bixler d. the dentition. in: stewart re, barber tk, troutman kc, wei shy, editors. pediatric dentistry, scientific foundation and clinical practice. st. louis: cv mosby co; 1982. p. 87–94. 33. mcdonald re, avery dr. dentistry for the child and adolescent. 6th ed. st louis: cv. mosby year-book inc; 1994. p. 53–9. case report integrated orofacial therapy in chronic rhinosinusitis management for children with sleep bruxism haryono utomo dental clinic faculty of dentistry, airlangga university surabaya – indonesia abstract background: the prevalence of rhinosinusitis was 20% in ambulatory patients and was mostly affected by viral infections and allergy. if conservative treatments of rhinosinusitis failed, surgical procedure is an alternative choice. previous case report revealed that the rhinosinusitis symptoms were successfully relieved by the "assisted drainage" therapy only. nevertheless, this therapy was less successful in children with sleep bruxism (sb). purpose: to report an integrated orofacial therapy for management of rhinosinusitis children with sleep bruxism (sb) which consisted of the assisted drainage, night-guard and masseter muscle massage therapies. case: two boys who suffered from rhinosinusitis with bruxism were unsuccessfully treated with conventional treatment. case management: patients was subjected to the assisted drainage therapy that was scaling and root planning combined with gingival massage, and masseter muscle massage; night guard was worn in night sleep. they successfully relieved the rhinosinusitis symptoms. conclusion: based on the successful result, this integrated therapy could be suggested as an adjuvant in rhinosinusitis management. key words: assisted drainage, night guard, rhinosinusitis, children, bruxism abstrak latar belakang: prevalensi rinosinusitis adalah 20% pasien rawat jalan dan umumnya disebabkan oleh infeksi virus dan alergi. apabila terapi konservatif rinosinusitis mengalami kegagalan maka pilihan terakhir adalah operasi. pada laporan kasus yang ada telah terjadi perbaikan gejala rinosinusitis setelah dilakukan terapi “assisted drainage” saja. akan tetapi, terapi ini kurang berhasil pada anak dengan sleep bruxism (sb). tujuan: melaporkan suatu terapi orofasial terintegrasi untuk tatalaksana rinosinusitis pada anak dengan sleep bruxism (sb) yang terdiri dari terapi assisted drainage, night guard dan masase otot masseter. kasus: dua anak laki-laki yang menderita rinosinusitis dengan bruxism telah mengalami kegagalan pada perawatan konsvensional. tatalaksana kasus: pasien dilakukan terapi assisted drainage yang adalah scaling dan root planning yang dikombinasikan dengan masase gingiva dan masase otot masseter; sedangkan nightguard dipakai saat tidur malam. terapi ini berhasil mengurangi gejala rinosinusitis. kesimpulan: berdasarkan keberhasilan terapi, terapi terintegrasi ini dapat digunakan sebagai ajuvan dalam tatalaksana sinusitis. kata kunci: assisted drainage, night guard, rinosinusitis, anak, bruksism correspondence: haryono utomo, c/o: rumah sakit gigi dan mulut, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: dhoetomo@indo.net.id introduction the term of rhinosinusitis was proposed by the american academy of otolaryngology–head and neck surgery in 1996, to substitute the term of sinusitis. this term was considered to describe the pathologic process more accurately than sinusitis only. the prevalence of rhinosinusitis was 20% in ambulatory patients which was mostly affected by viral infections and allergic reactions, and self recovered without the use of antibiotics.1 however, contradictory, to the usa there were 13 million antibiotics were prescribed for rhinosinusitis.2 conservative treatments of rhinosinusitis are decongestants, corticosteroids, antibiotics and diathermy. 98 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 97-101 if it failed, surgical procedures such as adenoidectomy had to be done.3 it was also believed that rhinosinusitis also has relationship to dental infection, mostly dental caries.4 moreover, some case reports which related to children revealed the possibility of chronic gingivitis as an etiology of rhinosinusitis symptoms via the "neurogenic switching" mechanism.5,6 according to several studies the possible involvement of stressful conditions either to rhinosinusitis and allergic patients7 or to sleep bruxism (sb)8,9 had been studied and revealed its positive correlations. however, the mechanism of sb as a trigger of rhinosinusitis symptoms is not clearly understood. sleep bruxism and clenching are related with parafunctional muscle activities of masticatory muscles during sleeping, particularly the pterygoid muscles (grinding) and temporalis muscles (clenching).8 the "night guard" or occlusal splint therapy is a treatment of choice for sb, and considered more effective than cognitivebehavioral treatment. nevertheless, the effectiveness of night-guard therapy in children is still questionable due to the possibility of their poor compliance.8,9 the possible correlation of rhinosinusitis with chronic gingivitis had been proven by dental plaque control therapy and the "assisted drainage" therapy (adt).5,6 this intra-oral approach had been effectively reduced both rhinosinusitis and chronic gingivitis symptoms. nevertheless, they had less satifactory result in children with sb such as in this case report. the objective of this case report was to reported an integrated therapy for management of rhinosinusitis children with sb which consisted of the assisted drainage, night-guard and masseter muscle massage therapies. case case #1 a nine years old boy, suffering from rhinosinusitis symptoms for years; however, the worst symptoms were started one year before. he was diagnosed to have bilateral maxillary rhinosinusitis by an otolaryngologist confirmed with water's projection radiograph. he was brought to our private dental clinic because his brother who also suffered from rhinitis and epistaxis had recovered from the similar symptoms after dental plaque control therapy in our clinic. his main complaints were nasal congestion, hearing impairment, sleep disturbance, fatigue, forgetfulness, difficult to concentrate and headache. almost every month he was suffered from febris (39-40° c). treatments which had been done by the otolaryngologist, was mostly antibiotics and nasal decongestants. sometimes it was also accompanied by diathermy. the result was not satisfactory; he still suffered from rhinosinusitis symptoms almost every month. extraorally, the patient appeared fatigue, and there was a small amount of thin nasal discharge in the nostrils. intra-oral examination showed moderate dental plaque in every region, and abundant dental plaque was seen in the upper posterior regions. inflamed gingiva was also seen in several regions, especially the #16 #55 and #65 #26, on which pseudopockets and subgingival pockets were detected. the patient had neither caries nor filling. case management at the first visit, it was noticed that the patient speak loudly and in high pitch voice. her mother said that other than rhinosinusitis symptoms he had a kind of hearing impairment so everybody had to speak louder to communicate with him. the first thing to be done was explaining the connection between dental plaque and rhinosinusitis to motivate the parents and patient to follow the dental health education (dhe) for home maintenance. in order to evaluate and convince either the patient or his parent for the effective result of the treatment, before treatment a specific test that was proposed termed as the "paper blowing test" was performed. the test was done by blowing a piece of paper or tissue (approximately 3x7 cm) with one nostril. other nostril and mouth had to be closed tightly. in a congested nose, patient had to blow with hard effort to move the tissue paper. treatment was initiated with prophylactic procedure using rotating brush, pumice and contra-angled-low speedhandpiece. the pseudopockets and interdental spaces were irrigated with hexetidine 0.1%; after one minute the assisted drainage therapy (adt) was done. the adt was a procedure developed for removing subgingival plaque within the pseudopockets of the chronic gingivitis area which concomitantly drained the inflammatory mediators using a sickle shaped sealer. the sealer was moved slowly forward and backward with gentle pressure until bleeding comes (figure 1). the patient was told to raise his left hand if pain felt. interestingly, the adt which performed in chronic gingivitis does not elicit pain. in this case, coincident with other chronic gingivitis cases, the dark red blood oozed from the interdental spaces and pseudopockets, especially in the upper left and right posterior teeth. figure1. the“assisted drainage” therapy figure 2-a. masseter muscle trigger points (1) and the referred area (2) (adapted from alexander, 2008) figure 2-b. masseter muscle massage figure 3. maxillary nerve-shenopalatine ganglion connection (gray’s anatomy online, 2006) figure 4. soft night guard for children with short teeth (extended buccal flange 2 1 sphenopalatine ganglion figure �. the "assisted drainage" therapy. 99utomo: integrated orofacial theraphy in chronic rhinosinusitis management the outer and inner "cheek" masticatory muscle (laymen's term of the masseter muscles) regularly (2–3 times/day) for five minutes for helping reduce the muscle spasm (figure 2b). figure1. the“assisted drainage” therapy figure 2-a. masseter muscle trigger points (1) and the referred area (2) (adapted from alexander, 2008) figure 2-b. masseter muscle massage figure 3. maxillary nerve-shenopalatine ganglion connection (gray’s anatomy online, 2006) figure 4. soft night guard for children with short teeth (extended buccal flange 2 1 sphenopalatine ganglion figure ��b. masseter muscle massage. the next visit, one week later, the rhinosinusitis symptoms were completely disappeared which was confirmed by the otolaryngologist who treated him earlier. at that time, spasm and trigger points in the masseter muscles were disappeared, so did the sb one month later. evaluations were done two monthly, the last evaluation was in april 2008, about one year after the first visit. during that time, severe rhinosinusitis symptoms were not recurrent, only mild rhinitis occured when he was very tired. he also grew as an healthy, active, bright and easy to concentrate children. case #2 an eight years old boy was diagnosed suffered from maxillary rhinosinusitis by an otolaryngologist, and the symptoms had been suffered for more than 3 years. the chief complaints were runny nose, nasal congestion and headache. he was brought by her mother to our private dental clink after got informed that there was a connection between rhinosinusitis and oral hygiene. he had been treated by several otolaryngologists and pediatricians either in indonesia or an abroad. the most prescribed medications were mostly antibiotics, antihistamines and nasal decongestants. sometimes he also sent to have diathermy therapy by a physiotherapist, but severe symptoms still recurrent if the medications were stopped. before visiting the dental practitioner, the previous otolaryngologist suggested to do surgery (adenoidectomy) to reduce the rhinosinusitis symptoms. nevertheless, the parent refused and still look for another opinions. during that time he had to consume antibiotics and other rhinosinusitis medications. at a glance, the boy looked unhealthy, skinny, irritable, hyperactive and uncooperative. he always scratch his nose and face, it seems that he feel his nose itching all the time (the" allergic salute" symptoms). extra oral inspection showed that his eyes were watery and also had blackened and swollen lower eyelids, which were common symptoms in rhinosinusitis. after the adt was done bilaterally, approximately three minutes later, the patient was conducting the paper blowing test once more. at that time he could move the paper by blowing with one nostril with almost no effort and also did with his another nostril. the parent said that before the dental procedures, despite many medications and diathermy, the patients could not breathe easily through the nose. afterwards, the patient and his parent were taught for dental health education and prescribed hexetidine 0.1% mouthwash. he was scheduled for the next visit in a week time. on the second visit the patient looked more cheerful. the parent said that all the symptoms related to rhinosinusitis (i.e. nasal congestion, headache) had already disappeared. interestingly, that on the second visit the patient did not speak loudly, he also speak in a considered normal voice pitch. intra-orally, oral hygiene was good, the chronic gingivitis symptoms also disappeared and so did the pseudopockets. patient was instructed to maintain oral hygiene and visit the dental practitioner every month for evaluation. a month later it was found that the adt had poorer result than previous visit because the rhinosinusitis symptoms recurrent, including headache. after taking comprehensive medical and dental history, especially for the persistent headache symptom, it was revealed that according his mother the patient always grinds his teeth every night. this condition was confirmed by palpating the affected masticator muscles (the pterygoid and temporal muscles), which had spasm and also painful by tender pressure (trigger point). since the pterygoid muscles were difficult to palpate, okeson20 suggested palpating its referral pain region (trigger points) in the outer and inner masseter muscles (figure 2a). figure1. the“assisted drainage” therapy figure 2-a. masseter muscle trigger points (1) and the referred area (2) (adapted from alexander, 2008) figure 2-b. masseter muscle massage figure 3. maxillary nerve-shenopalatine ganglion connection (gray’s anatomy online, 2006) figure 4. soft night guard for children with short teeth (extended buccal flange 2 1 sphenopalatine ganglion figure ��a. masseter muscle trigger points (1) and the referred area (2).17 in order to reduce muscle spasm caused by sleep bruxism (sb), maxillary impression had been taken for the night-guard fabrication. it was made from silicon sheet and should be worn every night when sleeping, it should be worn for several hours at daytime for adaptation. his parent was also taught to massage the trigger points on 100 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 97-101 intraorally, he had a moderate dental plaque accumulation and chronic gingivitis. inflamed gingiva was also seen in several regions, especially the #16, #55, and #65, #26, on which pseudopockets were detected. the patient had neither caries nor filling. at masticatory muscles palpation, muscle spasm and trigger points were detected. case management at the first visit, after detected the bruxism habit based on the muscle examination, and confirmed by his mother; he received to the regular protocol for the "sleep bruxismrelated rhinosinusitis" (a proposed term) management similar to case#1 that was the integrated orofacial therapy. coincident with case #1, even though the management was more difficult because of his uncooperative behavior, approximately three minutes after adt, the patients could breathe easily through his nostrils. night-guard therapy was also recommended for this patient, and his mother also told to massage the masseter muscles everyday. the patient was scheduled for evaluation in one week time. at second visit, extra oral examination showed that his eyes and lower eyelids became normal. runny nose and nasal congestion also disappeared, but the headache still persisted a little bit. he also became more cooperative and did not scratch his face and nose. at this occasion, he was instructed to begin wearing the night-guard. one month later, sb disappeared, so did the headache. cross-examination of this successful integrated orofacial therapy, for rhinosinusitis management was conducted by an otolaryngologist. evaluation was done two monthly, the latest evaluation was in may 2008, approximately seven months after the first visit; severe rhinosinusitis symptoms did not reappear, only sneezing in the morning when the weather was cold. discussion rhinosinusitis may affect everyone due to infancy, since the maxillary sinuses have already developed in the third month of fetal life, followed by the ethmoid sinuses.1 as rhinosinusitis mostly accompanied by headache, especially migraine headache, or vice versa, diagnosis of the main etiology could be difficult.10 consequently, it will become more difficult if happen to infants or young children. several mechanisms were also proposed related to the interrelationship between rhinosinusitis, migraine headache and allergy that are: 1) autonomic symptoms caused by parasympathetic cranial activation, and 2) neurogenic and immunogenic interaction or "neurogenic switching" mechanism which were confirmed by bellamy et al.11 nevertheless, recent publications also included stress as a contributing factor in allergic rhinosinusitis due to its ability to stimulate nasal autonomic symptoms.7,11 therefore, the interrelationship between stress, allergy, rhinosinusitis and migraine headache is possible. nasal congestion is the most common symptom in rhinosinusitis, which related to turbinate dysfunction. the autonomic nervous system provides the general innervation to the nose, with the parasympathetic nerves supplying the resting tone and controlling secretions. the parasympathetic ganglion within the nasal region is the sphenopalatine ganglion (spg) (figure 3).13 according to the neurogenic switching mechanism, the trigger of parasympathetic stimulation could be initiated from chronic gingivitis.14 this phenomenon was confirmed by a study in allergic wistar rats which stimulated with intragingival injection of porphyromonas gingivalis lipopolysaccharide.15 instant resolution of rhinosinusitis symptoms after removal of the subgingival plaque and drainage of inflammatory mediators was suspected due to rapid decrease of the neurogenic switching mechanism, which also confirmed by adt in allergic wistar rats that was done by utomo in 2009.15 however, in this case report, the adt alone was insufficient for completely eliminate rhinosinusitis symptoms. the idea to find the etiology of the persistent headache was to correlate it with masticatory muscle spasm. it was in accordance with janal et al.16 that sb caused masticatory muscle spasm which triggers headache. in this case, masseter muscles massage was considered advantageous to reduce muscle spasm.17 figure1. the“assisted drainage” therapy figure 2-a. masseter muscle trigger points (1) and the referred area (2) (adapted from alexander, 2008) figure 2-b. masseter muscle massage figure 3. maxillary nerve-shenopalatine ganglion connection (gray’s anatomy online, 2006) figure 4. soft night guard for children with short teeth (extended buccal flange 2 1 sphenopalatine ganglion figure ��. m a x i l l a r y n e r v e s p h e n o p a l a t i n e g a n g l i o n connection.19 it was suggested by boyd13 that sb was able to stimulate autonomic symptoms via the otic ganglion, a parasympathetic ganglion that related with mandibular nerve. it was sensitized by the parafunctional activity of the masticatory muscles which were innervated by the mandibular nerve. this suggestion was agreed by green18 who also proposed that parasympathetic activation may lead to autonomic symptoms such as rhinosinusitis. thus, chronic gingivitis and sb are proposed to have the propensity to sensitize the parasympathetic ganglions, either to the spg or the otic which facilitate the recurrence of the rhinosinusitis symptoms.19 although dental practitioners are considered to be incompetent in diagnosing rhinosinusitis symptoms; however, patient's information abot the past medical history which had been done by otolaryngologist was considered 101utomo: integrated orofacial theraphy in chronic rhinosinusitis management valid. nevertheless, it is uneasy to improve parents' and children's trust to dental practitioner competency in treating rhinosinusitis. therefore, a simple but effective treatment evaluation method should be created, such as the "paper blowing test". ideally, it was suggested that the treatment of sb using night guard or occlusal splint should follow rules regarding to the type of sb i.e. severe grinder or mild grinder.13 in addition, according to okeson20 the ideal occlusal spint or night guard for sb should be fabricated from hard clear acrylic. however, relevant to widmalm,21 soft night guards were preferred by some patients because their cushioning effect. therefore, considering that children are: a) possibly careless i.e. unintentionally break the hard night guard; b) have poorer motivation and compliance; c) not easily adapted to pressure to the teeth which more pronounced in hard night guard; and d) still have erupting permanent teeth and short primary teeth that which could not easily adapted by hard night guard and lack of retentive area. thus, soft night guard is preferred to be used by children. moreover, for increasing retention in short teeth, soft night guard flange could be extended to the vestibule, which mimicking full denture with minimal discomfort (figure 4). figure1. the“assisted drainage” therapy figure 2-a. masseter muscle trigger points (1) and the referred area (2) (adapted from alexander, 2008) figure 2-b. masseter muscle massage figure 3. maxillary nerve-shenopalatine ganglion connection (gray’s anatomy online, 2006) figure 4. soft night guard for children with short teeth (extended buccal flange 2 1 sphenopalatine ganglion figure �. soft night guard for children with short teeth (extended buccal flange. rhinosinusitis in this case report is proposed as "sleep bruxism-related rhinosinusitis" which related to stressful condition. since recent stressful environment (i.e. intense school assignments, extracurricular activities and lack of leisure time) which may affect children also increasing; thus, it is possible that the prevalence could be increasing. moreover, because sb is more pronounced in children then decreased relevant with age;8 hence, health professional who treat children, either medical or dental should be aware of this phenomenon. it concluded that regarding to the successful treatment procedures in this case report, the integrated orofacial therapy in rhinosinusitis management which consisted of the combination of the adt, night guard and masseter muscle massage therapies is considered effective. nevertheless, for further improvement and perfection of this method, conducting collaborate researches between dental and medical practitioners are mandatory. this teamwork is also beneficial for avoiding unnecessary prolonged medications or therapy, especially surgery which were considered harmful to the patient. references 1. kentjono wa. rhinosinusitis: etiology and pathophysiology. ear, nose and throat continuing education iv. 2004. p. 1–3. 2. behrman re, kliegman rm, jenson hb. nelson textbook of pediatrics. 17th ed. philadelphia: saunders; 2004. p. 760, 1832. 3. felisati g, ramadan h. rhinosinusitis in children: the role of surgery. pediatr allergy immunol 2007; 18(suppl. 18): 68–70 4. mylonakis e, bajracharya h. chronic sinusitis. available online at url: http://www.emedicine.com/med/topic2556.htm. accessed may 10, 2008. 5. utomo h. the sphenopalatine ganglion sensitization by periodontal inflamma-tion: a possible etiology for headache and sinusitis in children. majalah kedokteran gigi fkg universitas airlangga. 2006; 39(2): 66–71. 6. utomo h, pradopo s. practical dental approach in children's rhinosinusitis management. j dentistry 2006; 13(3): 133–6. 7. mçsges r, klimek l. today_s allergic rhinitis patients are different: new factors that may play a role. allergy 2007; 62: 969–75. 8. lavigne gj, kato t, kolta a, sessle bj. neurobiological mechanisms involved in sleep bruxism. crit rev oral biol med 2003; 14(1): 30–46. 9. ommerborn ma, schneider c, giraki m, schäfer r, handschel j, franz m, raab wh. effects of an occlusal splint compared to cognitive-behavioral treatment on sleep bruxism activity. eur j oral sci 2007; 115: 7–14. 10. cady rk, schreiber cp. sinus headache or migraine.neurology 2002; 58: s10–4. 11. bellamy jl, cady rk, durham pl. salivary levels of cgrp and vip in rhinosinusitis and migraine patients. headache 2006; 46: 24–33. 12. bousquet j, khaltaev n, cruz aa, denburg j, fokkens wj. allergic rhinitis and its impact on asthma (aria) 2008: review article. allergy 63 2008; (suppl. 86): 8–160. 13. boyd j. pathophysiology of migraine and rationale for a targeted approach and prevention. available online at url http://www. migraineprevention.com/index/html. accessed february 15, 2006. 14. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol. 2004; 15(2): 82–98. 15. utomo h. immunoneuromodulatory mechanism of the assisted drainage therapy in allergic rats induced by porphyromonas gingivalis lipopolysaccharide. dissertation. postgraduate program airlangga university. 2009. p. 111–5. 16. janal mn, raphael kg, klausner j, teaford m. the role of toothgrinding in the maintenance of myofascial face pain: a test of alternate models. am ac pain med 2007; 8(6): 486–96. 17. alexander d. masseter muscle,massage therapy, tmj, tooth pain, sinus pain. available online at url http://www.massagetherapypractice. com/text/ 1166848766515-0626/pc/1164930096301-0119/ anatomy-&-technique.htm. accessed may 10, 2008. 18. green mw. diagnosing and treating migraine: low tech diagnosis, high tech treatment. available online at url http://www.ama-assn. org/ama1/pub/ upload/ mm/31/24pres-green.pdf. accessed february 20, 2006. 19. gray's anatomy of the human body. the trigeminal nerve. available online at url. http://education.yahoo.com/reference/gray. accessed february 10, 2006. 20. okeson jp. bell's orofacial pain.6th ed. carol stream: quintessence pub. 2005. p. 52–3. 21. widmalm se. bite splints in general dental practice. available online at url http://sitemaker.umich.edu/widmalm/files/bsconstruc.pdf. accessed may 5, 2008. 107 vol. 41. no. 3 july–september 2008 cytotoxicity of 5% tamarindus indica extract and 3% hydrogen peroxide as root canal irrigation erawati wulandari department of conservative dentistry faculty of dentistry jember university jember indonesia abstract background: preparation of root canal is an important stage in endodontic treatment. during conducting preparation, it is always be followed with root canal irrigation that has aim to clean root canal from necrotic tissue remains, grind down dentin powder, micro organism, wet the root canal to make preparation process of root canal easier, and solute root canal content at area that can not be reached by equipment. flesh of tamarindus indica (pulpa tamarindorum) is used as traditional medicine and it contains vitamin c (antioxidant), protein, fat, glucose, etc. previous research shows that 5% tamarindus indica extract can clean smear layer but it is more cytotoxicity to cell line bhk–21 than sterilized aquabides. purpose: this research is to compare cytotoxicity between 5% tamarindus this research is to compare cytotoxicity between 5% tamarindus indica extract with 3% h2o2 as root canal irrigation material. method: four teen culture cell line bhk 21 divides into 2 groups. group four teen culture cell line bhk 21 divides into 2 groups. group 1 is treated with 3% h2o2 and group 2 is treated with 5% tamarindus indica extract, for about 2.5 minutes in every group. then, living and death cell percentage is measured. data is analyzed with independent t test with significant level of 0.05%. result: the research the research showed that death cell in group 1 was 29.3% and in group 2 was 21.1%. there was a significant different (p < 0.05) between group 1 and group 2. conclusion: cytotoxicity of 5% tamarindus indica extract to the cell line bhk–21 is lower than 3% h cytotoxicity of 5% tamarindus indica extract to the cell line bhk–21 is lower than 3% h2o2. key words: cytotoxicity, tamarindus indica extract, hydrogen peroxide, root canal irrigation correspondence: erawati wulandari, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas jember. jln. kalimantan no. 37 jember 68121, indonesia. email: bundayona@yahoo.co.id introduction the main purpose of root canal treatment is to prevent more serious teeth damage spreading by losing microorganism and necrotic tissue inside root canal. one of important treatment step of root canal treatment is preparation that is followed by root canal irrigation. the purpose of root canal irrigation is to clean root canal from necrotic tissue remain, grind down dentin powder, microorganism, wet the root canal to make root canal preparation process easier, and solute root canal content in area that can not be reached by equipment.1.2 one of root canal irrigation requirement is low periapical tissue cytotoxicity.3 this is considering that root canal irrigation solution can permeate to periapical especially at mandibular teeth with necrotic pulp in case with open foramen apical, if it contacts with periapical tissue and causing periapical complication.3 koulaozidou et al.,4 stated that if irrigation material comes out from teeth apex it will cause periapex tissue irritation and damaging. root canal irrigation material used is 3% hydrogen peroxide (h2o2). if it contacts with organic material, it will produce nascent oxygen, that is white foam formed from oxidation result of the material and has removed out debris from the root canal mechanically. nascent oxygen is free radical that can damage membrane and organelle from cell. concerning about it, toxic effect from material used in endodontic treatment is very important to be paid attention. exact root canal irrigation material must be chosen carefully because treatment failure is sometimes caused by improper material choosing. research report 108 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 107-109 many kinds of plant growing in indonesia can be used for medication purposes. one of them is tamarindus indica. the flesh (pulpa tamarindorum) can be used as scurvy medication. chemical component of the flesh is vitamin c that is determined as antioxidant, citric acid, tartrat acid, protein, carbohydrate, fat, water, etc.5,6 dharmayanti7 stated that 20%, 15%, 10% and 5% of tamarindus indica concentration had antibacteria of streptococcus viridans. previous research showed that 5% tamarindus indica extract as root canal irrigation could clean smear layer of root canal wall. material that is used in oral cavity must be non toxic. to measure toxicity from a material to cell can be done by cytotoxicity test with culture cell method by using baby hamster kidney-21 (bhk-21). it is used because the cell has the same characteristic with fibroblast of periapical tissue.8 previous research showed that cytotoxicity of 5% tamarindus indica extract to cell line bhk–21 is higher than sterilized aquabides, but it has not known yet the cytotoxicity difference between 5% tamarindus indica extract with 3% h2o2. based on the background that has been mention above, the research is done to compare cytotoxicity between 5% tamarindus indica extract with 3% h2o2 as root canal irrigation to cell line bhk–21. from this research hopefully can be used as therapy development base in endodontic field, whereas it can be found alternative material of root canal irrigation in cleaning root canal. material and method research materials are 3% h2o2, 5% tamarindus indica extract, sterilized aquabides, culture cell line bhk–21 (stock), 10% bovine serum, phosphate buffer saline (pbs), 0,25% versene tripsine solution, tryphan blue, eagle media, and neutralized salt solution. research equipment are roux bottle, incubator, weighing balance (triple beam balance, ohause), tweezers, mortal and pastel to make the flesh smooth, magnetic stirring to mix the flesh with sterilized aquabides, centrifugal (kokusan), 0.45 µm millipore filter (sartorius) to filter 5% tamarindus indica extract, pipettes, microplate 24-well, laminar flow, flight microscope (olympus ch 40), hemositometer to measure living and death cell. parental cell bhk–21 (stock) is being revival by putting it into the roux bottle and is given eagle media, 10%bovine serum, 0.02 ml streptomycin, 0.06% fungison. then it is kept in 37o c incubator until confluent. eagle media is removed and washed with 15 ml pbs twice to remove serum remain inside the bottle. after that, trypsinase is conducted with 0.25% versene trypsine as much as 1 ml. if the cell has already been separated, it must be put into microplate 24-well, each of them is 120 cell/ml. then, new eagle media and 10% bovine serum will be added in every well, and then it is put into 37o c incubator until confluent. the next step is preparing test material (5% tamarindus indica extract). material test preparation is 5 gram flesh of tamarindus indica, then it is added with sterilized aquabides until the volume is 100 cc and it is stirred by using magnetic stirring so that the flesh will easily soluble, it is centrifuged (2500 rpm) for 15 minutes to separate the deposit from its water. tamarindus indica extract is taken over and filtered by using millipore 0.45 µm filter so that 5% tamarindus indica extract can be obtained. toxicity test procedure is fibroblast cell bhk–21 in microplate 24 well were divided into 2 groups. at the first group, cell inside the microplate is washed with pbs, and it is given 0.5 cc of 3% h2o2, and let it for about 2.5 minutes. after that, 3% h2o2 is removed, washed with pbs and it is given 0.1 cc versene trypsine. then, wait until the cell is shed. after that, it was given eagle media + 0.9 cc serum and stirred with inhale procedure and sprayed repeatedly by using micropipette until the cell is separated. cell inside microplate is taken at about 0.025 cc and then added with 0.025 tryphan blue then it is dropped into hemositometer. this is done to conduct cell counting under the microscope. at the second group, the same procedure is done, but material that is used is 5% tamarindus indica extract. all procedure is conducted inside laminary flow with the purpose to stabilize research condition in sterilized condition. counting procedure of cell that is living (bright) and death (dark) cell in the box from hemositometer view field is counting then put into bird and forrester (1981) formula so that it can be got cell death percentage. formula: percentage of death cell = death cell × 100% living cell + death cell the higher value of death cell percentage so the higher cytotoxicity of material test. result this data shows the death cell percentage counting from cytotoxicity test with die exclusion test method by using tryphan blue (table 1). table 1. death cell percentage of from 3% h2o2 and 5% tamarindus indica extract group group sample large average (%) deviation standard significant level 3% h2o2 5% eaaj 7 7 29.3 21.1 2.17 1.83 0.00 normality test is done using kolmogorov smirnov test. after normal distribution data is gotten, the independent t test with parametric statistic analysis was done. based on homogeneity test (levene’s test), homogenous data is 109wulandari: cytotoxicity of 5% tamarindus indica extract and 3% hydrogen peroxide got and independent t test shows that there is significantly difference between group 1 and 2 (p < 0.05). discussion death cell percentage of cell that treated with 3% h2o2 is higher than that were inhibited with 5% tamarindus indica extract. hydrogen peroxide is determined as free radical that can damage cell membrane. the cell membrane contains doubled unsaturated fatty acid that is responsive to free radical. if it is broken, it will cause membrane structure and function change and the death of the cell.9 according to kumar et al.,10 there is chain autokalisis reaction if free radical attack doubled bounding at doubled unsaturated fatty acid from membrane fat, it will create unstable peroxide and reactive that cause membrane, organelle and cellular damage. the damage of membrane will cause osmotic balance lost of protein, enzyme, co enzyme and ribonucleic acid, it will cause the death of cell. free radical is also formed as side product of oxidation or cell burning that is lasted at cell metabolism. its function is to prevent from damaging caused by virus, bacteria, and also other materials. free radical over production will attack the cell in the same way as attacking other strange thing and it will cause destructive. if imbalance condition between free radical formation and antioxidant happened, it will cause oxidative stress so that it will cause cell damaging. imbalance will happened because lack of antioxidant and over production of free radical.10,11 this is estimated has caused group cell damaging with the given of 3% h2o2 that is higher than 5% tamarindus indica extract. it is because 3% h2o2 is included on free radical and it does not contain antioxidant. group with 5% tamarindus indica extract has lower death percentage than group with 3% h2o2. this is because tamarindus indica extract containt vitamin c, an antioxidant to free radical. antioxidant has function to change free radical to be less effect molecule, catch free radical compound and also prevent chain reaction of free radical formation. this can be done by accepting or give electron to bind with free radical electron that is not paired.9,10,12 vitamin c is also used as co enzyme in cell metabolism process and has important role in cell regeneration.13 tamarindus indica contains glucose, fat, and protein that is used for glutathione peroxides (gsh) activity that is determined as endogen antioxidant. if gsh does not working properly, the balancing process of free radical will be uncontrolled.10 cell death can be caused by low ph, so that it will cause injury to the cell. contact time as much as 2.5 minutes is determined have big role in the death of cell in group 1 and 2. cellular respond to injury stimuli is depend on injury type, injury period and serious condition. in certain boundary, cell can repair the damage that is caused by injury, and if stimuli are stopped, so the cell will be back to normal. persistent injury or more will because cell go across threshold boundary to injury irreversible and will cause cell death.10 it concluded that cytotoxicity of 5% tamarindus indica extract as root irrigation material to fibroblast cell line bhk-21 is lower than 3% h2o2. references 1. cohen s, burns rc. pathways of the pulp. 8th ed. mosby inc; 2002. p. 235, 244–47. 2. vianna me, gomes bpfa, berber vb, zaia aa, ferraz ccr, de souza-filho fj. in vitro evaluation of the antimicrobial activity ofin vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. oral surg oral med oral pathol oral radiol endod 2004; 97:79–84. 3. chang yc, huang fm, tai kw, chou my. the effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. oral surg oral med oral pathol oral radiol endod 2001; 92:446–50. 4. koulaouzidou fa, margelos j, beltes p, kortsaris ah. cytotoxic effect of different concentrations of neutral and alkaline edta solutions used as root canal irrigant. j endod 1999; 1:21–3.j endod 1999; 1:21–3. 5. wijayakusuma w. tanaman berkhasiat obat di indonesia. jilid 3.5. wijayakusuma w. tanaman berkhasiat obat di indonesia. jilid 3. pustaka kartini; 1997. p. 26–9. 6. verheij ewm, coronel re. sumber daya nabati asia tenggara 2, buah-buahan yang dapat dimakan. prosea: gramedia pustaka utama; 1997. 7. dharmayanti aw. kemampuan larutan buah asam jawa (tamarindus indica l) dalam menghambat pertumbuhan streptococcus viridans. skripsi. jember: fkg universitas jember; 2003. 8. freshney ri. culture of animal cells. a manual of basic technique. 4th ed. new york: wiley-liss inc; 2000. p. 330–7. 9. karyadi e. antioksidan, resep sehat dan umur panjang. available from http:/www.indomedia.com/intisari/1997/juni/antioks.html. accessed march 18, 2006. 10. kumar, abbas, fausto. robbins and cotran pathologic basis of disease. 7th ed. elsevier saunders; 2005. p. 16–8. 11. sauriasari r. mengenal dan menangkal radikal bebas. artikel iptek 22 januari. available from http://www.beritaiptek.com/zberitaberitaiptek/2006/01/22. accessed march 18, 2006. 12. best b. antioxidant molecules. in general antioxidant actions. available from http://www.benbest.com/nutrceut/antioxidants.html. accessed june 21, 2006. 13. harijanti k. peranan vitamin c dalam kesehatan jaringan lunak rongga mulut. majalah kedokteran gigi 1996; 29(3):59–62. 7171 dental journal (majalah kedokteran gigi) 2017 june; 50(2): 71–75 research report increase of collagen in diabetes-related traumatic ulcers after the application of liquid smoke coconut shell meircurius dwi condro surboyo,1 ira arundina,2 and retno pudji rahayu3 1master program in dental health science 2department of oral biology 3department of oral pathology and maxillofacial faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: traumatic ulcers in patients with diabetes mellitus may experience delayed healing despite their diabetic condition being controlled. liquid smoke coconut shell containing phenolic compounds can potentially accelerate the healing process. one healing process indicator is the increased number of fibroblasts, another being the increased amount of collagen. purpose: this study aimed to analyze the amount of collagen in traumatic ulcers in diabetics after application of liquid smoke coconut shell. methods: alloxan was induced in twenty-four male wistar rats as models of diabetes mellitus. a traumatic 10 mm ulcer was made along the labial fornix incisive inferior with a round, stainless steel blade before liquid smoke coconut shell and benzydamine hydrochloride (as the control) was administrated once a day. a biopsy of the labial fornix incisive inferior was subsequently performed after the topical application for 5 and 7 days. histological assessment was conducted to analyze the amount of collagen by means of masson trichome staining. results: histologically, the topical application of liquid smoke coconut shell for 5 days significantly increased the amount of collagen, higher than that of benzydamine hydrochloride as the control (p=0.006) (p<0.05). meanwhile, the topical application of liquid smoke coconut shell for 7 days made the concentration of collagen no significantly different from that of benzydamine hydrochloride as the control (p=0.156) (p>0.05). conclusion: liquid smoke coconut shell applied for 5 days increase the amount of collagen in traumatic ulcers in diabetic patients. keywords: liquid smoke coconut shell; traumatic ulcer; diabetes mellitus; collagen correspondence: ira arundina, department of oral biology, faculty of dental medicine, universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: arundinafkg@yahoo.com. introduction diabetes mellitus is prone to the onset of both traumatic and aphtous ulcers in the oral mucosa. its prevalence shows that 24.2% of ulcerated lesions (traumatic and aphtous ulcers), were found in the oral mucosa in diabetics, while 5.4% of patients with diabetes mellitus presented traumatic ulcers.1,2 traumatic ulcers in people suffering from diabetes mellitus may experience delayed healing despite their diabetic condition being under control.3 the main complain resulting from a traumatic ulcer is that of pain caused by the loss of the epithelial layer within the oral cavity. this results in the opening of nerve endings in the lamina propria, producing pain.4 the topical application often employed is in the form of mouthwash which serves as a supportive therapy. benzydamine hydrochloride is a nonsteroid antiinflammation drug (nsaid) possessing analgesic, anti-inflammatory, and antimicrobial properties that is available in the form of a mouthwash. it is appropriate for ulcer therapy with predisposing diabetes and its working mechanism may be associated with the inhibition of prostaglandin and inflammatory cytokine.5 nevertheless, there is a specific case in which the provision of topical therapy cannot promote the healing process.3 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p71-75 mailto:arundinafkg@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p71-75 72 surboyo, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 71–75 liquid smoke is a condensate that is indirectly burned at high temperatures and which can be produced from coconut shell.6,7 people in indonesia do not only employ liquid smoke coconut shell as a natural preservative for various fish and its products, such as tuna, stingray, tofu and meatballs, but have also traditionally resorted to its use as a means of reducing pain and accelerating the healing process in cases of skin burns.8-11 liquid smoke coconut shell has been proven to have an analgesic effect. previous research employing a writhing reflex method with 0.1% acetic acid induction confirmed that liquid smoke coconut shell is able to reduce this reflex in animal subjects due to acetic acid.12 the analgesic effect is related to the phenolic compound contained in liquid smoke coconut shell.13 in cases of a diabetes mellitus condition, reactive oxygen species (ros) and pro-inflammatory cytokines, such as tumor necrosis factor α (tnf-α), are increasingly formed. these interfere with the proliferation of fibroblasts by stimulating a fibroblast apoptotic process.14 liquid smoke coconut shell can, in addition, accelerate healing by increasing the number of fibroblast cells in wounds.11 phenolic compounds in liquid smoke can bind to ros, thus indicating reductions in tnf-α formation,15 resulting in deceased stimulation of the fibroblast apoptotic process.16 fibroblasts represent the main element within collagen synthesis. an increased number of fibroblasts can stimulate the synthesis of collagen which plays an important role in tissue regeneration.17 this study aimed to analyze the increase in the amount of collagen in traumatic ulcer after the application of liquid smoke coconut shell in subjects suffering from diabetes. materials and methods the shells used, identified as a species derived from cocos nucifera l at conversion plant in purwodadi, were derived from 5-6 month old coconuts obtained from a local market in surabaya. liquid smoke was obtained through a pyrolysis process conducted at 4000 c18 with a heating rate of 3.330 c for 4.5 hours. liquid smoke coconut shell was made at the research and development center of forest engineering and forest product processing (pustekolah). coconut shells weighing 5 kg were cleaned and dried before being placed into a pyrolysis tool. the liquid smoke produced in this process was 51.18% and was then settled for 48 hours prior to filtering with whatman 52 (watman 52, 110 mm circle, cat no 1452, ge healthcare life science, singapore). the purification process was conducted by means of distillation at 1200c19 to produce 84% liquid smoke. this research constituted a laboratory-based experimental investigation incorporating a post-test only-control group design and involving twenty-four, 2 month-old, male wistar rats weighing 120–160gr. they were then intraperitoneally induced with alloxan monohydrate (alloxan monohydrate a7413, sigma aldrich., st.louise, mo, usa) at a dose of 150 mg/kg to stimulate a diabetic condition.20 preparation of alloxan was subsequently performed by dissolving 0.9 grams of alloxan monohydrate into 6ml of pbs to produce a concentrate of 150 mg/ml.21 the rats did not receive food or water for more than 12 hours overnight before the induction of alloxan occurred. the development of diabetes mellitus in these animals was confirmed seventy-two hours after the alloxan induction had taken place by a fasting glucose level of >200 mg/dl using glucodrtm (agm-2100, allmedicus, korea).22 after the animals had been confirmed as presenting the condition of diabetes mellitus (fasting glucose >200mg/ dl), a traumatic ulcer sized 10mm was created along the labial fornix incisive inferior, using a round stainless steel blade.23,24 prior to this traumatic ulcer being made, the animals were anesthetized using a ketamine/xylazine cocktail.25 the traumatic ulcer was confirmed after 24 hours with the clinical appearance of a yellowish-white ulcer with a reddish edge. at this point in the process, therapeutic topical applications of liquid smoke coconut shell and benzydamine hydrochloride 1.25% (tantum verde™, soho, jakarta, indonesia) were performed on the traumatic ulcers of the twenty-four animals with the following distribution: a) six animals were treated with the topical application of liquid smoke coconut shell once a day for 5 days at a dose of 20μl/20gr weight; b) six animals were treated with the topical application of benzydamine hydrochloride once a day for 5 days at a dose of 20μl/20gr weight; c) six animals were treated with the topical application of liquid smoke coconut shell once a day for 7 days at a dose of 20μl/20gr weight; d) six animals were treated with the topical application of benzydamine hydrochloride once a day for 7 days at a dose of 20μl/20gr weight. the rats were terminated, their labial fornix incisive inferior tissue was subjected to biopsy before a histological examination was performed using masson trichome (mt) staining to determine the amount of collagen present. the density of collagen expressed is a percentage (%) calculated by dividing the area of collagens (μm2) observed by the total area of measurement using a light microscope (nikon h600l microscope; nikon, japan) at a magnification of 200x (ds fi2 300mp digital camera; nikon, japan, software digital imaging by nikkon image system, nikon, japan). the result obtained was the mean plus standard deviation (sd) value. an independent t-test then was performed to determine the differences in the amount of collagen present in each group (p <0.05). results figure 1 depicts collagen present in traumatic ulcers in the labial fornix incisive inferior after the topical application of liquid smoke coconut shell and benzydamine hydrochloride for 5 and 7 days. the blue area indicates dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p71-75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p71-75 7373surboyo, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 71–75 11 figure 1. the pictures of collagen on the traumatic ulcers using masson trichome (mt) staining at a magnification of 200x (collagen appears as blue area, pointed by arrow). (a) the topical application of benzydamine hydrochloride for 5 days (the number of collagen was 68.8%); (b) the topical application of benzydamine hydrochloride for 7 days (the number of collagen was 76.2%); (c) the topical application of liquid smoke coconut shell for 5 days (the number of collagen was 83.4%); (d) the topical application of liquid smoke coconut shell for 7 days (the number of collagen was 81.6%). a b c d 11 figure 1. the pictures of collagen on the traumatic ulcers using masson trichome (mt) staining at a magnification of 200x (collagen appears as blue area, pointed by arrow). (a) the topical application of benzydamine hydrochloride for 5 days (the number of collagen was 68.8%); (b) the topical application of benzydamine hydrochloride for 7 days (the number of collagen was 76.2%); (c) the topical application of liquid smoke coconut shell for 5 days (the number of collagen was 83.4%); (d) the topical application of liquid smoke coconut shell for 7 days (the number of collagen was 81.6%). a b c d 11 figure 1. the pictures of collagen on the traumatic ulcers using masson trichome (mt) staining at a magnification of 200x (collagen appears as blue area, pointed by arrow). (a) the topical application of benzydamine hydrochloride for 5 days (the number of collagen was 68.8%); (b) the topical application of benzydamine hydrochloride for 7 days (the number of collagen was 76.2%); (c) the topical application of liquid smoke coconut shell for 5 days (the number of collagen was 83.4%); (d) the topical application of liquid smoke coconut shell for 7 days (the number of collagen was 81.6%). a b c d 11 figure 1. the pictures of collagen on the traumatic ulcers using masson trichome (mt) staining at a magnification of 200x (collagen appears as blue area, pointed by arrow). (a) the topical application of benzydamine hydrochloride for 5 days (the number of collagen was 68.8%); (b) the topical application of benzydamine hydrochloride for 7 days (the number of collagen was 76.2%); (c) the topical application of liquid smoke coconut shell for 5 days (the number of collagen was 83.4%); (d) the topical application of liquid smoke coconut shell for 7 days (the number of collagen was 81.6%). a b c d figure 1. the pictures of collagen on the traumatic ulcers using masson trichome (mt) staining at a magnification of 200x (collagen appears as blue area, pointed by arrow). (a) the topical application of benzydamine hydrochloride for 5 days (the number of collagen was 68.8%); (b) the topical application of benzydamine hydrochloride for 7 days (the number of collagen was 76.2%); (c) the topical application of liquid smoke coconut shell for 5 days (the number of collagen was 83.4%); (d) the topical application of liquid smoke coconut shell for 7 days (the number of collagen was 81.6%). the presence of collagen. the highest concentration of collagen was obtained after the topical application of liquid smoke coconut shell for 5 days. meanwhile, the lowest amount of collagen was found after the topical application of benzydamine hydrochloride for 5 days. in general, the topical application of liquid smoke coconut shell-generated collagen was higher than for the topical application of benzydamine hydrochloride (figure 2). furthermore, results of the kolmogorov smirnov test show that data in each observation group during the 5 and 7-day periods were normally distributed. an independent t-test was subsequently conducted to determine the difference between the topical application of liquid smoke coconut shell and that of benzydamine hydrochloride over 5 and 7 days. figure 2. the mean number of collagen on the traumatic ulcers in each treatment gro up. * 100 90 80 70 60 50 40 30 20 10 0 5 days the number of collagen 7 days benzydamine hydrochloride liquid smoke coconut shell meanwhile, the amount of collagen after the topical application of liquid smoke coconut shell for a period of 7 days did not differ to any meaningful degree from that of benzydamine hydrochloride over 7 days (p=0.156). discussion the main complaint arising from instances of ulcers in the oral cavity is that of the resulting pain. benzydamine hydrochloride is a nonsteroid anti inflammation drug (nsaid) whose analgesic, anti-inflammatory, and antimicrobial effects are superior to those of chlorhexidine.26 it is considered to be an appropriate option as far as the control and reduction of pain in the case of oral mucositis is concerned.27 the mechanism of benzydamine hydrochloride is inhibitive to prostaglandins and inflammatory cytokines.5 for this reason, this particular chemical was used for the purposes of the research described here due to its analgesic the results of the independent t-test indicated that the amount of collagen after the topical application of liquid smoke coconut shell for 5 days was significantly higher than that of benzydamine hydrochloride over 5 days (p=0.006). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p71-75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p71-75 74 surboyo, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 71–75 effects which are derived from coconut shell.12 the liquid smoke coconut shell test on animals using acetic acid induction which leads to writhing reflex (a stretching or twisting movement due to contraction of the research sample animals’ abdominal muscles) is one of methods used for peripheral analgesic screening, local peritoneal cell response, and prostaglandin pathway.28 the analgesic effect is related to cyclooxigenase inhibition of the tissue resulting in a decrease in pge2 production. this working mechanism is the same as that of nsaid.29 in reality, the healing process of traumatic ulcers consists of three stages, namely inflammation, proliferation, and remodeling. the systemic condition of diabetes mellitus can affect the healing process.30 traumatic ulcers in cases of diabetes mellitus can precipitate a delayed healing process due to a prolonged inflammatory process marked by an increased pro-inflammatory cytokine, tnfα.31,32 tnf-α is pro-inflammatory cytokine leading to inflammation and which demonstrates a close correlation with diabetes mellitus.33 increased tnf-α does not only occur at a systemic level, but also in the body’s tissues. a previous piece of research shows that traumatic ulcers in alloxan-induced diabetic rats can significantly improve tnf-α expression.31 the topical application of liquid smoke is capable of reducing the production of tnf-α.15 the mechanism by which it does so is the inhibiting of reactive oxygen species (ros), and signaling nfқb for the production of tnf-α.15,34 liquid smoke coconut shell contains a highly effective phenolic compound, namely; 2-methoxyphenols (guaiacol) at a proportion of 21.71%, phenol at 14.87%, and 4-ethyl-2-methoxyphenol (emp) at 3.97%.35 these three compounds demonstrate antioxidant properties. ros is a compound of free radicals derived from oxygen due to the high metabolism of glucose oxidation in the forms of superoxide (o2), hydroxyl (oh), and peroxide (h2o2).36 superoxide (o2) is one such free radical that plays a role in the activation of nfκb signaling to produce tnf-α. 36 in addition, the mechanism of liquid smoke coconut shell in inhibiting the formation of ros and tnf-α is related to the role of a phenolic compound. this forms part of a hydroxyl group (-oh) directly attached to the aromatic hydrocarbon ring that constitutes a biologically active side of the phenolic compound.37 phenol is highly reactive to the binding of ros, while also possessing strong antioxidant properties which can inhibit the modulation of inflammatory mediator formation, such cytokine.34 the resistance mechanism of these free radicals consists of binding superoxide radicals (o2-) to the hydroxyl group (-oh) of phenol so that nfκb activation decreases thereby inhibiting the production of tnf-α.34,38 the proliferative stage of the healing process is, moreover, characterized by the proliferation of fibroblast and collagen synthesis.30 the formation of granulation tissue at the proliferative stage of the wound healing process triggers fibroblasts to reduce collagenase so that the degradation process decreases. furthermore, at this stage, the fibroblasts also undergo apoptosis resulting in the granulation tissue being replaced by collagen.17 in cases of a hyperglycemic or diabetic condition, an increase in inflammatory mediators and advanced glycation end products (ages) occurs. ages are formed during the pathogenic process of diabetes, binding to the receptor of ages (rage). this bond can produce the formation of ros in order to stimulate apoptotic processes through the activation mechanism of the pro-apoptotic transcription factor (foxo1) in fibroblasts.14,39-41 the topical application of liquid smoke over both 5 and 7-day periods produced a higher concentration of collagen than that resulting from benzydamine hydrochloride application during the same period (figure 2). however, only the topical application of liquid smoke coconut shell for 5 days significantly improved the amount of collagen to a level higher than that of benzydamine hydrochloride (p=0.006) (p<0.05). the mechanism of increased collagen is associated with the inhibition of ros and tnf-α formation as well a higher number of fibroblasts. previous research shows that the topical application of liquid smoke coconut shell can decrease ros formation and tnf-α production, as well as potentially increasing the number of fibroblast cells.14,15 fibroblast constitutes the main element in collagen synthesis. the correlation between increased collagen and the higher number of fibroblasts is possibly due to a decreased apoptosis of fibroblast cells. other past research into the condition of diabetes mellitus suggests that ros and tnf-α plays a role in the stimulation of foxo1 and caspase-3 producing an increase in the process of fibroblast apoptosis.14,16 topical application of liquid smoke coconut shell over 7 days showed no significant difference in the formation of new collagen compared to that of benzydamine hydrochloride (p=0.156) (p>0.05). however, data showed that the number of collagen after the application of liquid smoke coconut shell was higher than for benzydamine hydrochloride (figure 2). this condition can be explained by the liquid smoke coconut shell being capable of accelerating the proliferative phase (showing a higher concentration of collagen after application for 5 days) by accelerating the inflammatory phase in traumatic diabetic ulcer sites compared to benzydamine hydrochloride. as a result, in order to analyze and confirm how liquid smoke coconut shell affects the formation of collagen through a decreased inflammation process (tnf-α) and fibroblast apoptosis due to activation of foxo1 and caspase-3, further research is required. it can be concluded that the topical application of liquid smoke coconut shell over 5 days increase the concentration of collagen in traumatic diabetes mellitus-related ulcers. references 1. silva mfa, barbosa kgn, pereira jv, bento pm, godoy gp, gomes dq de c. prevalence of oral mucosal lesions among patients with diabetes mellitus types 1 and 2. an bras dermatol. 2015; 90(1): 49–53. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p71-75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p71-75 7575surboyo, et al./dent. j. (majalah kedokteran gigi) 2017 june; 50(2): 71–75 2. vasconcelos bc, novaes m, sandrini fal, maranhão filho aw, coimbra ls. prevalence of oral mucosa lesions in diabetic patients: a preliminary study. braz j otorhinolaryngol. 2008; 74(3): 423–8. 3. tripathi r, tripathi k. management of non healing oral ulcer in diabetic patient using tropical application of epidermal growth factor : a case report. sch acad j biosci. 2015; 3(8): 640–3. 4. ali sa, saudi hi. an exper t system for the diagnosis and management of oral ulcers. tanta dent j. 2014; 11(1): 42–6. 5. parashar a. mouthwashes and their use in different oral conditions. sch j dent sci. 2015; 2(2b): 186–91. 6. wagiman fx, ardiansyah a, witjaksono. activity of coconutshell liquid smoke as an insecticide on the rice brown planthopper (nilaparvata lugens). arpn j agric biol sci. 2014; 9(9): 293–6. 7. zuraida i, sukarno, budijanto s. antibacterial activity of coconut shell liquid smoke (cs-ls) and its application on fish ball preservation. int food res j. 2011; 18(1): 405–10. 8. saloko s, darmadji p, setiaji b, pranoto y. antioxidative and antimicrobial activities of liquid smoke nanocapsules using chitosan and maltodextrin and its application on tuna fish preservation. food biosci. 2014; 7: 71–9. 9. swastawati f, susanto e, cahyono b, trilaksono wa. sensory evaluation and chemical characteristics of smoked stingray (dasyatis blekeery) processed by using two different liquid smoke. int j biosci biochem bioinformatic. 2012; 2(3): 212–6. 10. purba r, suseno sh, izaki af, muttaqin s. application of liquid smoke and chitosan as natural preservatives for tofu and meatballs. int j appl sci technol. 2014; 4(2): 212–7. 11. tarawan vm, mantilidewi ki, dhini im, radhiyanti pt, sutedja e. coconut shell liquid smoke promotes burn wound healing. j evid based complementary altern med. 2016; 22(3): 436–40. 12. surboyo mdc, tantiana t, arundina i. analgesic effect of coconut shell (cocos nucifera l) liquid smoke on mice. dent j (maj ked gigi). 2012; 45(3): 156–60. 13. murakami y, hirata a, ito s, shoji m, tanaka s, yasui t, machino m, fujisawa s. re-evaluation of cyclooxygenase-2-inhibiting activity of vanillin and guaiacol in macrophages stimulated with lipopolysaccharide. anticancer res. 2007; 27(2): 801–7. 14. sun k, wang w, wang c, lao g, liu d, mai l, yan l, yang c, ren m. ages trigger autophagy in diabetic skin tissues and fibroblasts. biochem biophys res commun. 2016; 471(3): 355–60. 15. yang jy, kang my, nam sh, friedman m. antidiabetic effects of rice hull smoke extract in alloxan-induced diabetic mice. j agric food chem. 2012; 60(1): 87–94. 16. siqueira mf, li j, chehab l, desta t, chino t, krothpali n, behl y, alikhani m, yang j, braasch c, graves dt. impaired wound healing in mouse models of diabetes is mediated by tnf-α dysregulation and associated with enhanced activation of forkhead box o1 (foxo1). diabetologia. 2010; 53(2): 378–88. 17. tracy le, minasian r a., caterson ej. extracellular matrix and dermal fibroblast function in the healing wound. adv wound care. 2016; 5(3): 119–36. 18. lombok jz, setiaji b, trisunaryati w, wijaya k. effect of pyrolisis temperature and distillation on character of coconut shell liquid smoke. asian j sci technol. 2014; 5(6): 320–5. 19. desniorita d, maryam m. the effect of adding liquid smoke powder to shelf life of sauce. int j adv sci eng inf technol. 2015; 5(6): 457–9. 20. radenković m, stojanović m, prostran m. experimental diabetes induced by alloxan and streptozotocin: the current state of the art. j pharmacol toxicol methods. 2016; 78: 13–31. 21. bako hy, mohammad js, waziri pm, bulus t, gwarzo my, zubairu mm. lipid profile of alloxan-induced diabetic wistar rats treated with methanolic extract of adansonia digitata fruit pulp. sci world j. 2014; 9(2): 19–24. 22. asgary s, rafieian-kopaei m, shamsi f, najafi s, sahebkar a. biochemical and histopathological study of the anti-hyperglycemic and anti-hyperlipidemic effects of cornelian cherry (cornus mas l.) in alloxan-induced diabetic rats. j complement integr med. 2014; 11(2): 63–9. 23. hitomi s, ono k, miyano k, ota y, uezono y, matoba m, kuramitsu s, yamaguchi k, matsuo k, seta y, harano n, inenaga k. novel methods of applying direct chemical and mechanical stimulation to the oral mucosa for traditional behavioral pain assays in conscious rats. j neurosci methods. 2015; 239: 162–9. 24. kılıç ç, güleç peker eg, acartürk f, kılıçaslan sms, çoşkun cevher ş. investigation of the effects of local glutathione and chitosan administration on incisional oral mucosal wound healing in rabbits. colloids surfaces b biointerfaces. 2013; 112: 499–507. 25. he s, atkinson c, qiao f, chen x, tomlinson s. ketamine-xylazineacepromazine compared with isoflurane for anesthesia during liver transplantation in rodents. j am assoc lab anim sci. 2010; 49(1): 45–51. 26. sesha n h, sha navas s, ashwini s. effective evaluation of benzydamine hydrochloride as a mouth wash in subjects with plaque induced gingival inflammation. int j oral heal dent. 2016; 2(3): 161–70. 27. roopashri g, jayanthi k, guruprasad r. efficacy of benzydamine hydrochloride, chlorhexidine, and povidone iodine in the treatment of oral mucositis among patients undergoing radiotherapy in head and neck malignancies: a drug trail. contemp clin dent. 2011; 2(1): 8–12. 28. ezeja mi, ezeigbo ii, madubuike kg. analgesic activity of the methanolic seed extract of buchholzia coriacea. res j pharm, biol chem sci. 2011; 2(1): 187–93. 29. vinoth prabhu v, nalini g, chidambaranathan n, sudarshan kisan s. evaluation of anti inflammatory and analgesic activity of tridax procumbens linn against formalin, acetic acid and cfa induced pain models. int j pharm pharm sci. 2011; 3(2): 126–30. 30. xu f, zhang c, graves dt. abnormal cell responses and role of tnf-α in impaired diabetic wound healing. biomed res int. 2013; 2013: 1–9. 31. brizeno lac, assreuy ams, alves apnn, sousa fb, de b. silva pg, de sousa scom, lascane nas, evangelista js-am, mota mrl. delayed healing of oral mucosa in a diabetic rat model: implication of tnf-α, il-1β and fgf-2. life sci. 2016; 155: 36–47. 32. yamano s, kuo wp, sukotjo c. downregulated gene expression of tgf-βs in diabetic oral wound healing. j cranio-maxillofacial surg. 2013; 41(2): e42-8. 33. donath my, dalmas é, sauter ns, böni-schnetzler m. inflammation in obesity and diabetes: islet dysfunction and therapeutic opportunity. cell metab. 2013; 17(6): 860–72. 34. costa g, francisco v, lopes mc, cruz mt, batista mt. intracellular signaling pathways modulated by phenolic compounds: application for new anti-inflammatory drugs discovery. curr med chem. 2012; 19(18): 2876–900. 35. budijanto s, hasbullah r, prabawati s, setyadjit, sukarno, zuraida i. identifikasi dan uji keamanan asap cair tempurung kelapa untuk produk pangan. j penelitian pascapanen pertanian. 2008; 5(1): 32–40. 36. matough fa, budin sb, hamid za, alwahaibi n, mohamed j. the role of oxidative stress and antioxidants in diabetic complications. sultan qaboos univ med j. 2012; 12(1): 5–18. 37. gülçin i, beydemir ş. phenolic compounds as antioxidants: carbonic anhydrase isoenzymes inhibitors. mini rev med chem. 2013; 13(3): 408–30. 38. sikorski ze, sinkiewicz i. smoking | traditional. in: dikeman m, devine c, editors. encyclopedia of meat sciences. 2nd ed. elsevier; 2014. p. 321–7. 39. erejuwa oo. oxidative stress in diabetes mellitus: is there a role for hypoglycemic drugs and/or antioxidants? in: lushchak vi, gospodaryov d v., editors. oxidative stress and diseases. intech; 2012. p. 217–46. 40. garcia-bailo b, el-sohemy a, haddad ps, arora p, benzaied f, karmali m, badawi a. vitamins d, c, and e in the prevention of type 2 diabetes mellitus: modulation of inflammation and oxidative stress. biol targets ther. 2011; 5: 7–19. 41. desta t, li j, chino t, graves dt. altered fibroblast proliferation and apoptosis in diabetic gingival wounds. j dent res. 2010; 89(6): 609–14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i2.p71-75 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i2.p71-75 dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 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english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusionocclusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. mkg vol 39 no 1 jan 2006 isi.pmd 24 inhibition effect of calcium hydroxide point and chlorhexidine point on root canal bacteria of necrosis teeth andry leonard je*, achmad sudirman** and karlina samadi ** ** dental practician ** department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract calcium hydroxide point and chlorhexidine point are new drugs for eliminating bacteria in the root canal. the points slowly and controly realease calcium hydroxide and chlorhexidine into root canal. the purpose of the study was to determined the effectivity of calcium hydroxide point (calcium hydroxide plus point) and chlorhexidine point in eleminating the root canal bacteria of nescrosis teeth. in this study 14 subjects were divided into 2 groups. the first group was treated with calcium hydroxide point and the second was treated with chlorhexidine poin. the bacteriological sampling were measured with spectrofotometry. the paired t test analysis (before and after) showed significant difference between the first and second group. the independent t test which analysed the effectivity of both groups had not showed significant difference. although there was no significant difference in statistical test, the result of second group eliminate more bacteria than the first group. the present finding indicated that the use of chlorhexidine point was better than calcium hydroxide point in seven days period. the conclusion is chlorhexidine point and calcium hydroxide point as root canal medicament effectively eliminate root canal bacteria of necrosis teeth. key words: calcium hydroxide plus point, chlorhexidine point, necrosis teeth correspondence: andry leonard je, c/o: dental practician. jln praban no. 10 surabaya. introduction the diseases of the pulp tissue and periapical lesion are caused by pathogen bacteria infection. the main bacteria causing the infection have not yet been known. it has been said that infection in the root canal is infection of polimicroba.1 if the pulp tissue infected, the root canal treatment should be done. root canal treatment could be done by taking all the infected pulp tissue in the pulp chamber and the root canal.2 the treatment will show a good result if the whole infected pulp tissue has taken out by root canal preparation, appropriate sterilization has been done, and the root canal has been filled appropriately.1 the use of medicament for sterilization in root canal treatment plays an important role, since the elimination of bacteria and its substrate is the main factor of a success root canal treatment. the bacteria elimination could be achieved by chemo-mechanical preparation and the use of antibacterial medicament. brystrom and sundqvist3 showed that there are proliferation of the remaining bacteria in the root canal after the chemomechanical preparation. it is an established procedure to use calcium hydroxide [ca(oh)2] as a therapeutic component to achieve sterilization of infected root canals. studies has been conducted for a long time to examine the antibacterial effect of calcium hydroxide and chlorhexidine on root canal microorganism.3 calcium hydroxide was initially introduced as pulp capping material by hermann in 1930 and since then its use in the field of endodontic has been increasing. now the use of calcium hydroxide is one of recommended material for root canal sterilization.4 calcium hydroxide is effective for root canal treatment since it could fill whole pulp chamber and the hydroxide ion diffuse through dentine tubulus and root canal ramification entering the area of bacteria and its product. calcium hydroxide could prevent re-infection by disturbing the supply of bacteria nutrition, since its ph affects the cell membrane and protein structure of bacteria.5 another root canal medication is chlorhexidine, a cationic material with wide spectrum antibacterial. the characteristic of cationic material is it makes easier contact with anion on the surface of bacteria and changes its integrity. potassium ion is the first substance which can been seen when the sitoplasma membrane is damaged. the change of the permeability of sitoplasma membrane could cause precipitation of sitoplasma protein, changed of the osmotic cell balance, disturbing growth metabolism, splitting cells, blocking the atp membrane, and prevent anaerobic process.6 now gutta percha which contain root canal medication is invented. five percent chlorhexidine diasetat material is added into solid gutta percha as chlorhexidine point. one of its brand is active point produced by roeko, which is available in sizes 15–80. the chlorhexidine point releases the diasetat chlorhexidine slowly from gutta percha matrix. the surface of gutta percha matrix releases much 25leonard et al: inhibition effect of calcium hydroxide point chlorhexidine, while the inside of gutta percha releases chlorhexidine slower. the affinity of chlorhexidine on dentin makes its concentration remain stable for some times. calcium hydroxide point also has been produced by roeko (calcium hydroxide plus point), which contain 52% calcium hydroxide, 42% gutta percha, sodium chloride surfactant, and coloring agent. these two materials are used for root canal medications. the benefits of these materials are no mixing procedure, easy to apply, left no residue, and the root canal could be filled from periapical to coronal. the purpose of the study was to examined the inhibition effect of the calcium hydroxide point and chlorhexidine point on root canal bacteria. the clinician could choose the best root canal medication by knowing the ability of the calcium hydroxide point and the chlorhexidine point in eliminating root canal bacteria. materials and methods the maxillary anterior teeth from patients aged 18–45 years who visited airlangga university dental hospital, were choose as the samples. the teeth were diagnosed as pulp necrosis due to dental caries with no periapical lesion, and had straight single root canal. the informed consent from the patients were taken prior to the study. the samples were divided into 2 groups, group one used calcium hydroxide point material (1 piece), and group two used chlorhexidine point material (1 piece). each group consisted of 7 samples. aseptic procedures were done to all the tools, materials, and teeth before the treatment. the teeth were isolated using rubber dam and the cavity entrance was made using a high speed bur. after that the necrotic pulp tissue was taken by extirpation needle. files needle no 10 was inserted to pulp chamber until 1 mm prior root apex, and diagnostic wire photo was taken to measure the work length of root canal preparation. the tooth was preparated using file type k with conventional technique. each time changing the file number, the irrigation was carried out using 30% of h2o2 (2.5 cc) and aquabidest (2.5 cc) with light pressure. the root preparation was continued until file no. 70. each file type k no. 10–70 was used only for 3 teeth samples. after root preparation finished, the root canal was dried up using 6 pieces sterile paper points. a new sterile paper point inserted into the root canal and kept it for one minute. then, it was put into the culture media brain heart infusion (bhi) broth and incubated in 37 °c for 24 hours, and the bacteria number was counted using spectrofotometri. the root canal was sterilized using a point of calcium hydroxide no. 60. the remaining points were cut with excavator 2 mm prior the orifice, covered by cotton pellet and temporary filling. the patients were recalled 7 days later. at the recalled visit (7 days later), the patients were checked whether the temporary fillings still good. the teeth were isolated using rubber dam and temporary filling were opened. the root canal medications were taken out, followed by irrigation of 3% h2o2 and aquabidest of 2.5 cc with light pressure. then it were dried up with 6 pieces sterile paper point. another a new sterile paper point was put into the root canal and kept for one minute. then it was put into the culture media bhi broth and incubated in 37 °c for 24 hours. the bacteria counting were done using spectrofotometri. the teeth were sterilized again by putting the root canal medications, covered by cotton pellet and temporary filling. reaction tube containing sterile bhi broth was placed inside spectrophotometry, and wave length was arranged at 600 nm and calibrated to 0. the bhi broth which contained 24 hours incubated bacterial breeding was shaked until homogenous for 3 minutes with vibrax stirring tool. afterwards, it was moved to another reaction tube without paper point, placed again inside the spectrophotometry, so that the bacterial density value was shown with digital numbers. the higher the result, the more bacteria in the bhi broth. data was analyzed with paired t test before and after treatment, and independent t test for calcium hydroxide point and chlorhexidine point. results table 1 showed that after treatment with calcium hydroxide point and chlorhexidine point, the number of bacteria decreased. acquired data was homogeneous, normal, tested with one sample kolmogorov-smirnov test. to analyze the difference between before and after treatment with calcium hydroxide point and chlorhexidine point, paired t test was used with p < 0.05. this analysis showed a significant difference between before and after treatment with calcium hydroxide point and chlorhexidine point. to determine the difference of two materials, independent t test was utilized. table 2 showed no significant difference between the breeding result of calcium hydroxide point and chlorhexidine point. table 1. the mean, standard deviation and paired t test on the turbidity value of the bacteria before and after giving calcium hydroxide point and chlorhexidine point x ± sd group n before after p calcium hydroxide point chlorhexidine point 7 7 0.745 ± 0.071 0.795 ± 0.079 0.303 ± 0.033 0.196 ± 0.018 0.001 0.001 26 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 24–27 tabel 2. the result of statistical test using independent t test the difference of the turbidity value of the bacteria before and after between the group of hydroxide calcium point and chlorhexidine point group clorhexidine point calcium hydroxide point p x ± sd 0.599 ± 0.069 0.442 ± 0.045 0.081 discussion from statistical paired t test result, the bacterial turbidity value before and after treatment with calcium hydroxide point and chlorhexidine point showed a significant difference (p < 0.05). this finding proved that both calcium hydroxide point and chlorhexidine point had antibacterial effect. treatment with both substances could reduce bacterial count inside root canal which previously had been biomechanically prepared. from bacterial count, a difference of bacterial turbidity value before and after calcium hydroxide point treatment was 0.442 ± 0.045, while the difference in chlorhexidine point treatment was 0.599 ± 0.069. the independent t test had shown that there was no significant difference for the difference of bacterial turbidity value between calcium hydroxide point and chlorhexidine point (p = 0.081 which was more than 0.05). although there was no significant difference between two substances, chlorhexidine point eliminated more bacteria than calcium hydroxide. the ability to eliminate more bacteria was possibly caused by chlorhexidine point as a strong kation, interacted with negative content bacterial cell wall influencing tissue osmotic pressure, causing contraction of cell membrane and later died. chlorhexidine effect entered bacterial cytoplasma causing precipitation of cytoplasma content so that mitochondria did not produce energy anymore, lysis, disturbing glycolytic enzyme which eventually decreasing acid production, and then bacteria died. in low concentration it was bacteriastatic, and in high concentration it gave bactericidal effect. in gutta percha form, it was a slow released device (srd) which possessed high ability to enter dentinal tubuli in relatively short time to give maximal antibacterial effect until 500 μm, and in dentinal tubuli bacteria were not found.7 chlorhexidine was capable to inhibit the growth of enterococcus faecalis, capable to penetrate the biofilm layer produced by enterococcus faecalis colony.8 gutta percha had a basic substance which enabled it to be used as root canal sterilization medicine containing chlorhexidine, it possessed iso standard measurement and radio-opaque in nature. soon after root canal preparation and irrigation was done, gutta percha was inserted into root canal. drying root canal was not necessary since gutta percha could still release chlorhexidine in wet condition.7 accurate point application matching with work length was crucial to give antibacterial effect along root canal and avoiding excessive substance get to periradicular tissue. taking out gutta percha after 7 days application was easy, no remnats on root canal wall (as in paste form), which later on could disturb the bonding of root canal filling material.7 a study by lin et al.9 demonstrated chlorhexidine had a better antibacterial effect compared to calcium hydroxide. gomes et al.10 stated that although calcium hydroxide had antibacterial effect, in his research calcium hydroxide could not kill and eradicate enterococcus faecalis at any given time, meanwhile, chlorhexidine in 2% gel form, was able to perform antibacterial activities towards enterococcus faecalis after 1,2,7, and 15 days. many researchers affirmed that chlorhexidine was significantly better towards enterococcus faecalis and candida albicans compared to calcium hydroxide. but not all researches agreed with the statement above as lyne et al.8 wrote that both chlorhexidine and calcium hydroxide were equally effective. hydroxide ions released from calcium hydroxide had the ability to enter into teeth tissue and have affinity with dentin, direct contact with bacteria inside dentinal tubuli. on the other hand, ions inside gutta percha, released, and formed interaction inside root canal, so that the concentration increased to eliminate root canal bacteria. one mechanism to explain antibacterial activity of calcium hydroxide was the absorption of carbondioxide (co2) inside root canal, where the gas was essential for bacteria. if calcium hydroxide absorbed carbondioxide, then co2 – depending – bacteria could not endure living.10 it could be concluded from this research that the application of chlorhexidine point into root canal was able to eliminate bacteria growth more than the application of calcium hydroxide point even after root canal preparation. although from statistical test there was no significant difference between both medications, chlorhexidine point and calcium hydroxide point are good materials, both are applicable for root canal sterilization due to easy application and effective antibacterial effect supporting root canal treatment. further research to examine more detail the effect of both materials should be done. references 1. walton re, torabinajed m. prinsip dan praktek ilmu endodonsi. edisi ke-2. jakarta: penerbit buku kedokteran egc; 1998. p. 360–78. 2. cohen c, burn rc. pathways of the pulp. 8th ed. st louis: mosby; 2002. p. 231–91. 3. podbielksi a, spahr a, haller b. additive antimicrobial activity of calcium hydroxide and chlorhexidine on common endodontic bacterial pathogen. j endod 2003 may; 29(5):340–5. 4. evans md, baumgartner c, khemaleelakul s, xia t. efficacy of calsium hydroxide: chlorhexidine paste as an intracanal medication in bovine dentin. j endod 2003 may; 29(5):338–9. 5. spangberg lsw. intracanal medication. in: ingle ji, bakland l, editors. endodontics. 4th ed. baltimore: williams & wilkins; 1994. p. 627–40. 27leonard et al: inhibition effect of calcium hydroxide point 6. wuerch rmw, apicella mj, mines p, yancich pj, pashley dh. effect of 2% chlorhexidine gel as an intracanal medication on the apical seal of the root canal system. j endod 2004 nov; 30(11): 788–91. 7. lin s, zuckerman o, weiss ei, mazor y, fuss z. antibacterial efficacy of a new chlorhexidine slow release device to desinfect dentinal tubules. j endod 2003 jun; 29(6):416–8 8. suzuki k. antimicrobial effect of calcium hydroxide on bacteria isolated from infected root canals. dentistry in japan 1999; 35:43–7. 9. lin y, mickel ak, chogle s. effectiveness of selected materials againts enterococcus faecalis: part 3. the antibacterial effect of calcium hydroxide and chlorhexidine on enterococcus faecalis. j endod 2003 sep; 29(9):565–6. 10. gomes bp, souza sf, ferraz cc, teixeira fb, zaia aa, valdrighi l, souza–filho fj. effectiveness of 2% chlorhexidine gel and calcium hydroxide against enterococcus faecalis in bovine root dentine in vitro. int endod j 2003 apr; 36(4):267–75. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice �� vol. 42. no. 1 january–march 2009 esthetic rehabilitation of crowded and protruded anterior dentition cecilia g. j. lunardhi and eric priyo prasetyo department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: recent trends put esthetic rehabilitation as a demanding treatment in order to correct malpositioned anterior dentition. this is enhanced by the patient’s background, especially careers that require prime appearance for the public. purpose: to describe that even though there are many treatment alternatives and procedures, esthetic rehabilitation on crowded and protruded anterior dentition using endodontic treatment, cast posts and all ceramic crowns, can improve patient’s appearance. case: this article presents a case report on esthetic rehabilitation of crowded and protruded anterior dentition. treatment was done due to patient’s refusal in receiving orthodontic treatment. the patient requested esthetic rehabilitation as an expectation for faster and instant esthetic result. case management: endodontic treatment was done to the involved dentition prior to the final restoration. cast posts and all ceramic crowns were used as final restoration to correct the crowded and protruded anterior teeth. conclusion: esthetic rehabilitation can be done successfully on crowded and protruded anterior dentition. instant result could be achieved by this treatment. this is supported by the fact that dentists should be aware of not only choosing the right treatment and materials but also patient’s expectations and conditions. key words: esthetic rehabilitation, cast post, all ceramic crown correspondence: cecilia g.j. lunardhi, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: ep_prasetyo@yahoo.com introduction esthetic rehabilitation becomes more popular and widely known in today’s dynamic society. dentists as clinicians must have logical diagnostic approach when planning an esthetic rehabilitation therefore creating esthetic dental appearance as expected by their patients.1 in order to achieve optimal esthetic, dentists must really create natural appearance as natural dentition in the right arch, proper inclination and alignment to the adjacent teeth. considering esthetic, the best material of choice for matching the natural state of a complex human dentition as in indirect anterior restoration is ceramic for the highly desirable properties in color stability, translucency, light transmission, and biocompatibility.2–4 in choosing the right materials, dentists should consider objective and subjective factors as well as patient’s expectations, while this surely require a thorough understanding about the art and science of esthetics thus making it a significant challenge to dentists.5 in somein some cases, in order to correct malpositioned teeth to be in the right alignment requires decaputation of partial or all of tooth crown and restore it with indirect post, core and crown restoration. regarding this, endodontic treatments are needed to be performed over the involved dentition, although these teeth are normally intact and in vital condition. therefore, several important considerations in determining the post-endodontic restorations are needed and based on the protection and conservation of the remaining tooth structure to reduce pressure over teeth in restorative aspect, esthetic condition, inclination, and to achieve the natural tooth morphology.6 this article presents a case report on esthetic rehabilitation of crowded and protruded anterior dentition using endodontic treatment, cast posts, and all ceramic crowns as final esthetic restorations. case report ��lunardhi: esthetic rehabilitation of crowded case a forty-year old female patient came with crowded and protruded anterior dentition (figure 1). this patient worked as a public relation representative in a private company. in the first appointment, anamnesis and clinical observation were done. from anamnesis, it was found that the patient had refused orthodontic treatment since it required more time and discomfort during treatment. on clinical examination, it was found that the patient was in good health. after thorough explanations, the patient approved and consented about conservative esthetic rehabilitation procedure through conventional endodontic treatment. the restoration planning was determined using cast post and all ceramic crowns. alginate impression was done to produce the study model and temporary crowns as provisional restoration. figure 1. patient’s anterior teeth condition before treatment. case management after anamnesis, clinical examination, thorough explanation, and patient’s consent about esthetic rehabilitation treatment procedure, on the second visit, one visit endodontic treatment was done on four upper anterior teeth. local infiltration anesthesia was applied on 12, 11, 21, and 22 (citoject heraeus). after local diagnostic wire photo was done to tooth number 12, 11, 21, and 22, endodontic treatment was performed in crown down pressureless technique (universal protaper dentsply) for the cleaning and shaping of all four root canals according to the manufacturer’s sequence and working lengths (central incisors 22 mm, lateral incisors 21 mm) of each root canal. sample irrigation was done with sodium hypochlorite (chlorcid ultradent) between preparation sequences. trial photo was taken to confirm the preparation and sealing of gutta point obturation (figure 2). obturation was done according to working lengths using single cone technique with f2 gutta point (protaper dentsply) and obturation paste (top seal dentsply), then obturation photo was taken (figure 3). after the endodontic treatment was accomplished, teeth number 12, 11, 21, and 22 were decaputated and prepared for post and core. after decapitation and post preparation, double impression was done (panasil kettenbach) as a mould to fabricate the ni-cr post and core. bite registration record was taken, and then the impression was sent to dental laboratory with a written detailed laboratory prescription. the first temporary provisional acrylic crowns (tempron gc) with post and core were cemented (figure 4) using temporary cement (freegenol gc). figure 2. gutta point trial photo. figure 3. obturation photo. on third visit, before the temporary restoration was removed, alginate impression (chroma heraeus) was taken as mould for further making of direct provisional crown from acrylic (tempron gc). there was neither patient complaint nor pain reported during one week after endodontic treatment although analgesic was not administered. after the cast posts and cores from dental laboratory were available, the temporary restoration was �� dent. j. (maj. ked. gigi), vol. 42. no. 1 january–march 2009: 46-49 removed and cleaned. cast posts and cores from the lab was cemented permanently (figure 5) using luting cement (fuji i gc). double impression (panasil kettenbach) and bite registration record (panasil kettenbach) were done for the second time as a guide to make the final all ceramic crowns (figure 6 & 7). remains of the impression materials were cleaned and the teeth were prepared for temporary provisional crowns cementation (freegenol gc). the second temporary provisional crowns (tempron gc) were cemented, the impression result was sent back to the dental laboratory for all ceramic crowns production along with a detailed laboratory prescription. for color mapping, a vita shade guide of a3 was selected, as well as explicit details about what to be done regarding the anatomical morphology, normal anterior alignments, and occlusion. figure 7. bite registration record for the final restoration. on the fourth visit, all ceramic crowns were available and were put on the model (figure 8). these crowns were cemented one-by-one using resin cement (calibra dentsply). the excess from cementation was cleaned before full set with hand instrument and contacts of each crown were checked using dental floss (figure 9). occlusion and contact showed a fit state. the final result showed better teeth alignment compared to the initial condition prior to esthetic treatment hence changing the appearance and finally increase patient’s self esteem (figure 10). patient follow ups were done 6 months and 1 year after treatment, there were no complaint and the patient was satisfied with the result. figure 8. all ceramic crowns on model. figure 4. cementation of first temporary posts and crowns. figure 5. cementation of cast posts and cores. figure 6. double impression for final restoration. ��lunardhi: esthetic rehabilitation of crowded figure 9. all ceramic crowns in place. figure 10. final esthetic result. discussion esthetic rehabilitation need patient’s objective and subjective considerations, because beside the high cost of treatment, it involves removal of natural tooth structures and vitality, moreover it also requires good cooperation and understanding between the patient and dentist as operator. in this case, the ideal treatment of choice to correct the crowded and protruded anterior dentition should be orthodontic treatment, but the patient refused and in the other hand, demanded a more instant esthetic result in a relatively short time. as an alternative, the patient opt the conservative esthetic rehabilitation on her crowded and protruded anterior dentition. this case showed that crowded and protruded anterior dentition could be overcome with esthetic rehabilitation using endodontic treatment, cast posts, and all ceramic crowns in relatively short time which was only four treatment visits within one week (7 days), excluding the two follow-up visits afterwards. cast posts were used in this case to fix the extremely protruded anterior dentition into their natural inclinations, where the use of fiber posts were notably impossible, although esthetically, fiber post has much better properties to be done on cases of normal or slightly mal-positioned tooth inclination. endodontic treatment (pulpectomy) was done on four upper anterior teeth under consideration of extreme mal-alignment, therefore crowns and posts as intra canal retentions are needed to correct the position of those anterior dentition, with regard that endodontic treatment has high success rate, about 90% or even more in pulpectomy cases.6,7 this is surely must be supported with proper techniques and quality post-endodontic restorations. all ceramic crowns were chosen for better esthetics.2–5 the use of all ceramic crowns has been increasing in strength and popularity. this is supported with the newly developed materials such as zirconia and the invention of cad/cam technology.8,9 final cementations were done using resin cements because crown breakage rate has been high when cemented with traditional dental cements.10 cementation of the restoration is probably the most strict procedure, therefore dentists should follow manufacturer’s instruction to ensure long lasting restorations. the patient was advised to seriously care for the restored teeth, regularly do dental check-ups, and avoid overload teeth contacts as these would harm the restorations and teeth underneath. in conclusion, esthetic rehabilitation can be done successfully on crowded and protruded anterior dentition. endodontic treatment, cast posts, and all ceramic crowns were used to improve patient’s appearance where instant result could be achieved by this treatment. this is also supported by the fact that dentists should be aware of not only choosing the right treatment and materials but also patient’s expectations and conditions. the growing popularity and demand for esthetic rehabilitation will in fact encourage dentists to be able to provide the supply as well as educating patients about realistic expectations for the corresponding esthetic restorations. since there are many different philosophies and technologies that can be applied to esthetic rehabilitation cases, dentists must enrich themselves with thorough understanding about recent technologies and materials, and with that would come a greater ease in providing esthetic services with satisfactory results for the patients. references 1. spear fm, kokich vg, mathews dp. interdisciplinary management of anterior dental esthetics. j am dent assoc 2006; 137(2):160–9. 2. powers jm, sakaguchi rl. craig’s restorative dental materials. 12th ed. philadelphia: elsevier inc; 2006. p. 454–6. 3. roulet jf. indirect aesthetic restorations. journal of advances in aesthetic and restorative dentistry 2003; 5:15–9. 4. shillingburg ht, hobo s, whitsett ld, jacobi r, brackett se. fundamentals of fixed prosthodontics. 3rd ed. chicago: quintessence publishing; 1997. p. 433–6. 5. prasetyo ep. esthetic management for anterior teeth: a case report. jakarta: apdc publishing; 2007. p. 123. 6. summitt jb, robbins jm, hilton tj, schwartz rs. fundamentals of operative dentistry: a contemporary approach. 3rd ed. chicago: quintessence publishing; 2006. p. 571–84. 7. shabahang s. state of the art and science of endodontics. j am dent assoc 2005; 136(1): 41–52. 8. finke pm. the all ceramic system of the future. journal of advances in aesthetic and restorative dentistry 2003; 7: 25–6. 9. robeson tm, heyman ho, swift ej. sturdevant’s art and science of operative dentistry. 5th ed. philadelphia: mosby inc; 2006. p. 610–1. 10. christensen gj. the state of the art in esthetic restorative dentistry. j am dent assoc 1997; 128(9):1315–7. mkg vol 41 no 4 oct-dec 2008.indd 151 vol. 41. no. 4 october–december 2008 research report effect of oxygen hyperbaric therapy on malondialdehyde levels in saliva of periodontitis patients with type 2 diabetes mellitus dian mulawarmanti1 and widyastuti2 1 department of biochemistry 2 department of periodontology faculty of dentistry hang tuah university surabaya indonesia abstract background: lipid peroxidation (lpo) has implication in pathogenesis of several pathological disorders including periodontitis. malondialdehyde (mda) is end products of upid peroxidation. hyperbaric oxygen therapy (hbot) involves the administration of 100% oxygen under atmosphere pressure and has been used as an adjuvant therapy, while saliva is a diagnostic tool for many oral and systemic diseases. purpose: the aim of this study was to examine the effect of hbot on malondialdehyde in saliva to measure lipid peroxidation in periodontitis patients with type 2 diabetes mellitus (dm). methods: eight regulated type 2 dm subjects were compared to ten unregulated periodontitis patients type 2 dm (n = 18). pre hbot and after 10 days hbot with 2.4 ata dose, unstimulated whole saliva samples from study subjects were collected, centrifuged at 3000 g for 15 minutes and were then stored at −80° c until analyzed. the mda level was determined with 2-thiobarbituric acid by a colorimetric method at 532 nm. results: data showed that regulated type 2 dm had lower level of mda (3.08 ± 0.62 ug/mol) compared with unregulated periodontallytype 2 dm subjects (5.88 ± 1.04 ug/mol) (p>0.05). mda levels were significantly lower after hbot in regulated dm (2.30 ± 0.46 ug/mol) compared with unregulated periodontally type 2 dm (4.09 ± 0.77ug/mol) (p < 0.05). the regulated dm subjects and post hbot showed mda levels lower than the periodontally-unregulated group significantly. conclusion: the saliva of periodontitis patients with unregulated type 2 dm showed more lipid peroxidation than regulated dm type 2. hbot decreased mda levels in regulated and unregulated type 2 dm with periodontitis. key words: hyperbaric oxygen, malondialdehyde, saliva correspondence: dian mulawarmanti, bagian biokimia, fakultas kedokteran gigi universitas hang tuah. jl. abdul rachman hakim no. 150 surabaya, indonesia. e-mail: dianmulawarmanti@yahoo.com introduction periodontal disease has been reported as the sixth complication of diabetes, along with neuropathy, nephropathy, retinopathy, and microand macrovascular diseases. periodontal infection and gingival inflammation, a number of other oral complications have often been reported in patients with diabetes. these include xerostomia, dental caries, candida infection, burning mouth syndrome, lichen planus, and poor wound healing.1 many studies have been published describing the bidirectional interrelationship exhibited by diabetes and periodontal disease. studies have provided evidence that control of periodontal infection has an impact on improvement of glycemic control evidenced by a decrease in demand for insulin and decreased hemoglobin a1c levels.2,3 diabetes increases the risk of developing periodontitis. epidemiologic research supports an increased prevalence and severity of attachment loss and bone loss in adults with diabetes. subjects with type 2 diabetes had approximately threefold increased odds of having periodontitis compared with subjects without diabetes. diabetes also may increase the risk of experiencing continued periodontal destruction.1,3 hyperbaric oxygen therapy (hbot) is a mode of medical treatment in which the patient is entirely enclosed 152 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 151−154 in a pressure chamber breathing 100% oxygen at a pressure greater than one atmosphere.4,5 hyperbaric oxygen therapy (hbot) has been successfully used for the treatment of a variety of clinical conditions related to hypoxia, including acute carbon monoxide intoxication, air embolism, soft tissue infections, radiation necrosis and impaired wound healing,5 but the effects of hbot on oxidant/antioxidant metabolism are controversial and its effects on periodontitis with diabetes are not known. reactive oxygen species (ros) are derived from a variety of sources, such as the xanthine oxidase system, activated neutrophils, the electron transport chain of mitochondria, and the arachidonic acid pathway. since free radicals have very short half-life, the clinical assessment of oxidative stress in vivo is based on the measurement of different stable oxidized products of modified lipids, proteins, carbohydrates and nucleic acids.6,7 free radicals can be defined as molecules or molecular fragments with an unpaired electron which imparts certain characteristics to the free radicals such as reactivity.4,8 reactive free radicals are able to produce chemical modifications and to damage proteins, lipids, carbohydrates and nucleotides in the tissues.5,9 it’s known that free oxygen radicals are probably mediators for tissue damage in periodontal disease.3 reactive free radicals may damage cells by initiation of lipid peroxidation that causes profound alteration in the structural integrity and functions of cell membranes. free radical induced lipid peroxidation has been implicated in the pathogenesis of several pathological disorder.5,6,9 malondialdehyde (mda), one of the most widely used biomarkers of oxidative stress, is produced enzymatically by the breakdown of unstable hydroperoxides during peroxidation of unsaturated fatty acyl moieties and used as a stable index of free radical attack on membrane phospholipids.6 the concentration of lipid peroxidation product, malondialdehyde (mda), is most widely used.7 saliva is a diagnostic tool for many oral and systemic disease. the detection of salivary mda level may provide additional advantages in elucidating the pathogenesis of periodontal disease.2 there is increasing evidence about the ability of hbot to induce cellular protection in a similar manner with other protective oxidative stress mechanisms.7 repeated hbot exposure significantly attenuated the inflammatory mediators, free radicals, and mortality in endotoxic rats.10 these protective effects of hbot may be related to the fact that reactive oxygen species can trigger a wide variety of cellular mechanisms by functioning as signal molecules.3,10 the aim of this study was to examine the effects of hbot on the levels of mda in noninsulin dependent diabetic patients with periodontitis who were exposed to hyperbaric oxygen for the treatment of periodontitis. materials and methods lipid peroxidation products malondialdehyde (mda) was analyzed in patients of periodontitis with diabetic. eighteen type 2 dm patients with periodontitis who received hbot were included in the study. eight periodontitis with regulated type 2 dm patients (hba1c ≤ 6,5) were compared to ten periodontitis with unregulated type 2 dm patients (hba1c ≥ 7 ) who received hbot. local ethics committee approved the study protocol and all study subjects gave their informed consents. patients were followed by the same physician responsible for diabetes control, wound care and antibiotic therapy according to the clinical and laboratory findings, and were given a diet depending on their metabolic needs without vitamin supplementation. all diagnostic tests were evaluated for diagnosing periodontitis disease. hyperbaric oxygen therapy (hbot) was carried out in a multiplace hyperbaric chamber once a day for ten days. the treatment protocol was inhalation of 3 × 30 min periods of 100% oxygen at a pressure of 2.4 ata, interspersed with 5 min periods of air breathing.11 samples were taken before hbot with dose 2.4 ata 3 × 30 minutes and after the exit from the chamber, on the day of 10th hbo sessions. unstimulated whole saliva samples (2.5 ml) were collected from each patient in standard sterile vacuum tubes. saliva samples were immediately centrifuged at 3000 g for 15 min and were then stored at –70° c until analyzed. the measurement lipid peroxidation products of malondialdehyde (mda) levels were analyzed by modification thiobarbiturat acid substance (tbars) condense with two equivalents of thiobarbituric acid to give a fluorescent red derivative that can be assayed spectrophotometrically.2 the pair t-test was used to compare both of groups. all hypothesis tests were two-tailed with statistical significance assessed at the p value < 0.05 level with 95% confidence intervals (ci). the data was expressed as the mean ± sem. statistical computations were calculated using spss 14 for windows software (spss inc, chicago, il, usa). result statistic analysis showed that the regulated type 2 dm had lower level of mda (4,09 ± 0,77ug/mol) compared with unregulated periodontallytype 2 dm subjects (5,88 ± 1,04 ug/mol) (p > 0.05). mda levels were significantly lower after hbot in regulated dm (2,30 ± 0,46 ug/mol) compared with unregulated periodontally type 2 dm (3,08 ± 0,62 ug/mol) with p < 0.05. the regulated dm subjects and post hbot showed mda lower levels than the periodontally-unregulated group significantly. 153mulawarmanti: effect of oxygen hyperbaric therapy table 1. the salivary mda levels of patients with regulated and unregulated periodontally type 2 dm subjects mda levels pre hbot post hbot regulated dm 4,09 ± 0,77ug/mol 2,30 ± 0,46 ug/mol unregulated dm 5,88 ± 1,04 ug/mol 3,08 ± 0,62 ug/mol discussion hyperglycemia stimulates the production of advanced glycolysated end products, enhances the polyol pathway, and activates protein kinase c, which may lead to increased oxidative stress.12 in the pathogenesis of diabetic complications, there is an increasing evidence for the role of oxidative stress, which is manifested by enhancing lipid peroxidation, superoxides,9 nitric oxide,12 increased protein, and dna damage.9 abnormal nitric oxide (no) synthesis has been implicated in the pathogenesis of both periodontal disease and diabetes mellitus. in diabetic patients, increased inducible no synthase in inflammed gingiva correlated with no in gingival crevicular fluid.12 nitric oxide, a toxic free radical with multiple biological functions, including inhibition of neutrophil chemotaxis, adhesion to endothelium, and upregulation of tumor necrosis factor alpha, is generated by oxidative deamination of larginine by nitric oxide synthase (nos).12,13 the inducible form of nos (inos) is rapidly and durably expressed by inflammatory cells in response to bacteria or their products, such as lipopolysaccharide (lps). small amounts of no induced by constitutive nos are considered beneficial, whereas excess inos-induced no can mediate cell and tissue injury. periodontal diseases are chronic inflammatory infections associated with gram-negative bacteria, including porphyromonas gingivalis, prevotella intermedia, and actinobacillus actinomycetemcomitans, which stimulate macrophages to generate no. moreover, no is increased in inflammed gingival tissue, and a selective inos inhibitor can prevent bone destruction in ligature-induced rodent periodontitis.12 inducible nos, independent of calcium, catalyzes g e n e r a t i o n o f l a r g e a m o u n t s o f n o ( n a n o m o l a r concentrations) over extended periods (hours or days) in response to inflammatory stimuli such as cytokines and lipopolysacharides. nitric oxide synthase (nos) inhibition stabilizes oxygen to stimulate and prevent peroxidation during no generation.13 during oxidant stress superoxide is readily generated. under basal condition, nitric oxide undergoes a rapid biradical reaction with superoxide anions to form peroxynitrite this reaction, and hence the formation of peroxynitrite is augmented in inflammatory conditions. no is the only currently known biological molecule produced in high enough concentrations to react fast enough with superoxide to outcompete endogenous superoxide dismutase.8,13 peroxynitrite interacts with a number of biotargets, such as heme containing proteins where the iron is in its ferrous state, peroxidases, seleno-proteins such as glutathione dna-binding transcription factors. in contrast to mostly beneficial and cytoprotective effects of no, the generation of peroxynitrite has mainly been attributed with cytotoxic effects. in vivo, when thiol-containing agents (glutathione, albumin, cysteine) are available to convert the peroxynitrite anion to nitrosothiols and related products, it may exhibit protective properties.8,12,13 radicals attack other biomolecules such as dna, protein, and most commonly lipids and in doing so generate new radicals. in the presence of metal ions the interaction between lipid peroxides and hydrogen peroxide can lead to a metal catalyzed fenton reaction and this could form strong oxidizing agents capable of propagating lipid peroxidation. this leads to the production of toxic metabolites like aldehydes malondialdehydes (mda). these products of peroxidation can increase vascular permeability, produce edema inflammation, promote cell death and can alter the functions of membrane proteins like receptors, ion channels and enzymes.2,11 lipid peroxidation has been shown to cause a cell damage and profound alteration in structural intergrity.5,9 mda, one of the most widely used biomarkers of oxidative stress. elevated mda level have been shown in periodontitis.2 hyperbaric oxygen (hbo) treatment in human can caused dna damaged in lymphocyte, but dna damage was found only after the first hbo exposure and not after further treatment. speit et al14 demonstrate increased levels of heme oxygenase-1 (ho-1) in lymphocyte after hbot. rothfuss et al.15 studies also provided evidence for a functional involvement of the inducible enzyme heme oxygenase-1 (ho-1) in this adaptive protection. the induction of ho-1 and increased sequestration of iron could explain why the cells are protected after hbot. its suggest the increased of heme oxygenase-1 might be involved in the adaptive protection after hbot. the activity of ho-1 leads to degradation of the pro-oxidant heme and to accumulation of the antioxidant bilirubin. bilirubin, a metabolite of heme degradation is in it self a potent antioxidant. induction of ferritin synthesis as a result of iron removal from the degradation of heme by ho-1. the deleterious effects of reactive oxygen species such as h2o2 are dependent on the presence of iron. intracellular free iron can react with h2o2 and increase the toxic hydroxyl radical via fenton reaction. due to the release of free iron during the catalysis of heme by ho-1, ferritin may be released and restrict iron from participation of the fenton reaction. various in vivo studies with animals and in vitro studies with mammalian cell lines have indicated the involvement of ho-1 in the resistance to oxygen toxicity.14,15 hyperbaric oxygen (hbo) induced cellular protection in a similar manner with other protective oxidative stress mechanisms. these protective effects of hbo may be 154 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 151−154 related to the fact that reactive oxygen species can trigger a wide variety of cellular mechanisms by functioning as signal molecules.9,14 hbo treatment in human leads to the induction of adaptive response that protects cells against the induction of dna damage by the second hbot.4,10,15 the reactive oxygen species generated by hyperbaric oxygen (hbo) triggers the increase of antioxidant enzyme activities regulation, thereby induces tolerance against ischemia in the tissues.4,14 the body contains a number of protective antioxidant mechanisms, whose specific role is to remove harmful oxidants or to repair cell damage caused by reactive oxygen species.15,16 the increase in concentration and partial pressure of oxygen during hbo therapy provides more oxygenation in the whole body.5 the increased tissue oxygen enhances the growth of fibroblast, formation of collagen, angiogenesis, and phagocytic capabilities of the hypoxic leukocytes, so it has beneficial effects on wound healing.5,17 diabetic patients have significant defects of antioxidant defense elements, and the generation of reactive oxygen species is one of the major determinants of diabetic complications.8 the current findings in diabetic patients with periodontitis indicated the upregulation of antioxidant enzymes by hbo therapy.18,19 although the activities of antioxidant defense enzymes were not measured in this study, there were evidences to confirm this hypothesis. hbo improves the oxygen delivery to the tissues, accelerates the rate of healing, and also has anti-infectious properties against various microorganisms.5,14,16,20 this research showed mda level decrease after hbot . it has been proved by randomized, controlled clinical trials that hbot is effective in diabetic periodontitis. it is concluded that hbo 2.4 ata with 3 × 30 minutes dose triggers and upregulates the defense mechanisms against oxidative stress. hbot decreased mda levels in regulated and unregulated type 2 dm with periodontitis. increased oxygenation of tissues due to hbo therapy may also activate other endogenous factors that prevent hazardous effects of the disease itself. saliva of periodontitis patients with unregulated type 2 dm showed more lipid peroxidation than regulated type 2 dm. the findings of our study suggest that hbo 2.4 ata 3 × 30 minutes for 10 days has beneficial effects on the treatment of periodontitis in diabetes and this effect may occur through the antioxidant systems. references 1. southerland jh, taylor g, offenbacher ws. diabetes and periodontal infection: making the connection. clinical diabetes 2005; 23(4): 171–8. 2. rai b. salivary lipid peroxidation product malondialdehyde in periodontal diseases. the internet journal of laboratory medicine 2007; (2). available from http\\www.drbalwantraissct @rediffmail. com. accessed august 2, 2008. 3. mealey bi, rethman mp. periodontal disease and diabetes mellitus. dent today 2003; 22(4): 107–13. 4. özden ta, uzun h, bohloli m, toklu as, paksoy m, simsek g, et al. the effects of hyperbaric oxygen treatment on oxidant and antioxidants levels during liver regeneration in rats. the tohoku journal of experimental medicine 2004; 203(4): 253–65. 5. mathieu d, linke jc, wattel f. non-healing wounds. in: mathieu d, editor. handbook on hyperbaric medicine. netherlands: springer; 2006. p. 401–27. 6. gürdöl f, cimit m, yidoan yo, körpinar , yalçinkay s, koçah h. early and late effects of hyperbaric oxygen treatment on oxidative stress parameters in diabetic patients. physiol. res 2008; 57: 41–7. 7. yogaratnam jz, laden g, madden la, seymour am, guvendik l, cowen m, et al. hyperbaric oxygen: a new drug in myocardial revascularization and protection? cardiovasc revasc med 2006; 7: 146–54. 8. elayan im, axley mj, prasad pv. ahlers st, auker cr. effect of hyperbaric oxygen treatment on nitric oxide and oxygen free radicals in rat brain. j. neurophysiol. 2000; 83: 2022–9. 9. rothfuss a, speit g. investigations on the mechanism of hyperbaric oxygen (hbo)-induced adaptive protection against oxidative stress mutation research/fundamental and molecular mechanisms of mutagenesis 2002; 508(issues1-2): 157–65. 10. lin hc, wan fj, wu cc, tung cs, wu th. hyperbaric oxygen protects against lipopolysaccharide-stimulated oxidative stress and mortality in rats. eur j pharmacol 2005; 508: 249–54. 11. davi g, falco a, patrono c. lipid peroxidation in diabetes mellitus. antioxid redox signal 2005; 7: 256–68. 12. skaleric u, gaspirc b, cartney nm, masera a, wahl sm. proinflammatory and antimicrobial nitric oxide in gingival fluid of diabetic patients with periodontal disease. infect immun 2006; 74(12):7010–3. 13. hardy p, dumont i, bhattacharya,m, hou x, lachapelle p, varma, dr, et al. oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy . review. cardiovascular research 2000; 47: 489–509. 14. speit g, dennog c, eichhorn u, rothfuss a, kaina b. induction of heme oxygenase-1 and adaptive protection against the induction of dna damage after hyperbaric oxygen treatment. carcinogenesis 2000; 21(10): 1795–9. 15. rothfuss a, radermacher p, speit g. involvement of hemeoxygenase-1 (ho-1) in the adaptive protection of human lymphocytes after hyperbaric oxygen (hbo) treatment. carcinogenesis 2001; 22(12): 1979–85. 16. rothfuss a, dennog c, speit g: adaptive protection against the induction of oxidative dna damage after hyperbaric oxygen treatment. carcinogenesis 1998; 19: 1913–7. 17. panjamurthy k, manoharan s, ramachandran cr. lipid peroxidation and antioxidant status in patients with periodontitis. cell mol biol lett 2005; 10:255-64. 18. lie h, xiong l, lao n, chen s, xu n, zhu z. hyperbaric oxygen preconditioning induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes in rabbits. j cereb blood flow metab 2006; 26: 666–74. 19. gregoveric p, lynch gs, williams da. hyperbaric oxygen modulates antioxidant enzyme activity in rat skeletal muscles. eur j appl physiol 2001; 86: 24–7. 20. benson rm, minter lm, osborne ba, granowitz ev. hyperbaric oxygen inhibits-induced proinflammatory cytokine synthesis by human blood derived monocyte-macrophages. clin exp immunol 2003; 134(1): 57–62. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 72 volume 47, number 2, june 2014 maturasi dan erupsi gigi permanen pada anak periode gigi pergantian (the maturition and eruption of permanent teeth in mixed dentition children) sri kuswandari departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas gadjah mada yogyakarta indonesia abstract background: tooth eruption might used as predictor of child age when the chronologic age is unknown. beside the dental maturity, tooth eruption is also influenced by some factors, such as caries and tooth extraction. purpose: the aim of this research was to examine the relationship of maturity and eruption of permanent teeth with chronologic age in mixed dentition period children. methods: the subjects were patients of prof. soedomo dental hospital pediatric dental clinic, consist of 38 boys and 39 girls in the aged of 6-12 years. the tooth eruption data was taken by counting the permanent teeth in intra oral examination. the dental maturity was assessed by dermijian method from dental panoramic radiology. the data were statistical analyzed by regresion-correlation and t-test program of spss 16.0 for windows. results: maturation of permanent teeth in each of age group was more advanced in girls than boys. however, only groups of 7, 8 and 11 years were showed significant different (p<0.05), while for tooth eruption there was no significant difference (p>0.05). the coefficient correlation between tooth eruption, chronologic age and dental maturation scores were relative high, between 0.75–0.86 (p<0.01). the contribution of chronologic age and dental maturation to predict tooth eruption (r2) were 58–73.6%. the dermijian method predicted age 0.83 years higher. conclusion: there were close relationship between chronologic age, dermijian method dental maturity, and eruption of permanen tooth, and could be used as predictor for eruption of permanent teeth in the mixed dentition period children. the dermijian method predicted 0.83 years older than the chronologic age. key words: dermijian method, dental maturity, tooth eruption, chronologic age, mixed dentition period abstrak latar belakang: erupsi gigi sering digunakan untuk memperkirakan umur anak. selain maturasi gigi, erupsi gigi juga dipengaruhi oleh faktor, seperti karies dan pencabutan gigi. tujuan: penelitian ini bertujuan untuk meneliti hubungan antara maturasi dan erupsi gigi permanen dengan umur pada anak periode gigi bercampur. metode: subjek terdiri atas 38 anak laki-laki dan 39 anak perempuan berumur 5,98–11,90 tahun pasien klinik ilmu kedokteran gigi anak rumah sakit gigi dan mulut prof. soedomo, fakultas kedokteran gigi universitas gadjah mada. penghitungan jumlah gigi permanen yang telah erupsi dilakukan dengan pemeriksaan klinis. penentuan maturitas gigi metode dermijian dilakukan dengan rontgen panoramik. analisis statistik dengan regresi korelasi dan uji t program spss 16,0 for windows. hasil: maturasi gigi permanen pada anak perempuan tiap kelompok umur lebih tinggi dibandingkan pada anak laki-laki, tetapi hanya pada kelompok umur 7, 8 dan 11 menunjukkan perbedaan bermakna (p<0,05). tidak ada perbedaan bermakna (p>0,05) erupsi gigi antara laki-laki dan perempuan. koefisisen korelasi antar erupsi gigi, umur kronologis, skor maturasi gigi cukup tinggi, yaitu 0,75–0,86 (p<0,01). kontribusi umur kronologis dan maturasi gigi dalam memprediksi erupsi gigi (r2) berkisar research report 73kuswandari: maturasi dan erupsi gigi permanen pada anak periode gigi pergantian 58–73,6%. metode dermijian memprediksi umur 0,83 tahun lebih tinggi. simpulan: antara umur kronologis, maturasi gigi metode dermijian dan erupsi gigi berhubungan erat, dan merupakan prediktor yang baik bagi erupsi gigi pada anak periode gigi bercampur. metode dermijian memprediksi 0,83 tahun lebih tinggi dibandingkan umur kronologis. kata kunci: metode dermijian, maturitas gigi, erupsi gigi, umur anak, periode gigi bercampur korespondensi (correspondence): sri kuswandari, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281, indonesia. e-mail: kuswandarisri@gmail.com pendahuluan pertumbuhan seorang anak diperlukan adanya patokan atau standar normal dalam penilaian klinis, agar umur fisiologis sistem jaringan bisa dibandingkan dengan umur kronologis.1 pada umur kronologis yang sama anakanak bisa menunjukkan tahap perkembangan biologis yang berbeda. perawatan gigi pada anak yang masih dalam periode tumbuh kembang diperlukan perhatian khusus, oleh karena itu seorang dokter gigi anak perlu memiliki pengetahuan tentang perkembangan, khususnya perkembangan gigi, serta variasinya.2 dengan adanya variasi maturasi fisik yang bersifat individual tersebut, maka oleh sir edwin saunders diusulkan penggunaan erupsi gigi permanen untuk menentukan umur anak antara 7–14 tahun yang tidak diketahui tanggal lahirnya.3 erupsi gigi sering dipergunakan dalam ilmu forensik untuk memperkirakan umur anak dalam ilmu kedokteran gigi erupsi gigi juga digunakan untuk menilai maturasi gigi atau dental age secara klinis. dental age digunakan oleh dokter gigi antara lain untuk menentukan waktu yang tepat untuk memulai perawatan orthodontik tertentu dan menentukan perawatan bagi gigi desidui. penilaian dental age pada dasarnya dilakukan dengan dua cara, yaitu berdasarkan status gigi yang telah muncul di dalam rongga mulut (erupsi), misalnya metode hellman 4 dan barnet;5 dan berdasarkan tahap pembentukan gigi yang tampak pada gambaran foto rontgen, misalnya metode dermijian,6 metode nolla,7 haavikko.3 penilaian dental age berdasarkan erupsi gigi lebih praktis, tidak memerlukan foto rontgen, sehingga pasien tidak perlu terpapar radiasi. kekurangannya adalah erupsi gigi sangat dipengaruhi oleh faktor lingkungan, seperti karies gigi, ekstraksi gigi yang terlalu awal, ketersediaan ruang untuk erupsi, adanya ankilosis dan hanya bisa digunakan untuk anak pada periode gigi bercampur. metode foto rontgen ada dua macam, yaitu berdasarkan ukuran gigi yang terlihat pada gambaran rontgen orthopantomograf (opg)8 dan berdasarkan mineralisasi atau kalsifikasi yang dapat diamati dari gambaran rontgen opg juga.6 saat ini yang paling sering digunakan adalah metode berdasarkan kalsifikasi yang dibagi dalam tahap perkembangan yang dapat dilihat pada foto rontgen. studi histologi dan radiografi menunjukkan bahwa saat yang tepat onset kalsifikasi tonjol gigi tidak bisa ditentukan dengan foto rontgen, karena ukurannya yang mikroskopis. akan tetapi apabila telah terjadi kalsifikasi akan bisa terlihat sebagai bentuk kerucut (٨), sehingga saat onset kalsifikasi gigi bisa ditentukan berdasarkan foto rontgen.9 tujuan penelitian ini adalah untuk meneliti hubungan maturasi dan erupsi gigi permanen dengan umur kronologis pada anak periode gigi bercampur. bahan dan metode subjek penelitian terdiri atas 38 anak laki-laki dan 39 anak perempuan pasien klinik kedokteran gigi anak rsgm prof soedomo, fakultas kedokteran gigi universitas gadjah mada antara bulan agustus–oktober 2013. kriteria subjek adalah, pasien (orang tua/wali) menyetujui untuk menjadi subjek penelitian dengan menandatangani inform consent, sedang dalam periode gigi bercampur, tidak menderita kelainan tumbuh kembang, tidak ada riwayat menderita penyakit yang menahun yang berpengaruh pada tumbuh kembang, minimal termasuk kategori status gizi sedang berdasarkan standar whonchs. keterangan kelaikan etik penelitian (ethichal clearance) telah diperoleh dari tim etik penelitian fakultas kedokteran gigi universitas gadjah mada. pengambilan foto radiografi orthopantomograf (opg) digital dilakukan dengan pesawat dental x-ray panora deluxe dengan spesifikasi 70-80 kvp, 12ma, 12s sesuai standar operasional yang berlaku di instalasi radiologi rumah sakit gigi dan mulut prof soedomo. analisis tingkat maturasi gigi berdasarkan metode dermijian6 dengan menggunakan foto opg dilakukan oleh seorang peneliti (penulis). cara pemberian skor adalah sebagai berikut, foto opg digital dilihat dengan menggunakan program microsoft office picture manager pada perbesaran 100%. skor diberikan pada gigi-gigi permanen bawah kiri (kecuali molar tiga). setiap gigi diberi grade berdasarkan tingkatan kalsifikasi, yaitu dari a sampai h. grade a apabila mulai tampak adanya tanda kalsifikasi, sampai dengan grade h apabila sudah terjadi penutupan apeks akar. skor diberikan berdasarkan tahapan (grade) kalsifikasi dari tiap tipe gigi. tingkat maturasi gigi dinilai berdasarkan jumlah skor ketujuh gigi permanen rahang bawah kiri. data erupsi gigi diperoleh dengan menghitung jumlah gigi permanen yang telah muncul di dalam rongga mulut. umur kronologis dihitung dengan program microsoft 74 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 72–76 excel, berdasarkan umur subjek dalam tahun yang dihitung berdasarkan jumlah hari dari tanggal lahir sampai saat dilakukan pemeriksaan foto rontgen dibagi 364. analisis data dilakukan secara terpisah antara subjek laki-laki dan perempuan. pengamatan maupun pemberian skor dilakukan oleh seorang peneliti (penulis). uji reproduksibilitas pengukuran (intra-examiner reliability) dilakukan dengan melakukan analisis ulang 5 foto opg pada selang waktu satu minggu. penghitungan kappa cohen diperoleh koefisien korelasi antara 0,82 sampai 0,85 antara pengukuran pertama dengan yang ke dua, yang berarti termasuk excelent agreement (>0,75). analisis statistik deskriptif, uji t independent maupun berpasangan, uji regresi dan korelasi pearson product moment digunakan program spss 16,0 for windows. hasil subjek penelitian berumur antara 5,98-11,90 tahun, dengan rerata 8,86 ± 1,56 tahun. rerata (x) dan simpangan baku (sb) skor maturasi gigi metode dermijian dan jumlah gigi permanen erupsi pada subjek laki-laki dan perempuan, serta uji-t menurut kelompok umur (tabel 1) menunjukkan bahwa pada umumnya skor maturasi gigi pada subjek perempuan lebih tinggi, dibandingkan subjek laki-laki, tetapi perbedaan bermakna (p < 0,05) hanya pada kelompok umur tujuh, delapan dan sebelas tahun. tidak ditemukan perbedaan bermakna (p > 0,05) data erupsi gigi antara subjek laki-laki dan perempuan pada semua kelompok umur. uji regresi dan korelasi product moment dari pearson antar variabel prediksi dan prediktor (tabel 2) menunjukkan nilai koefisien korelasi yang tinggi dan bermakna (p < 0,05), yaitu antara 0,76 sampai 0,86. prediksi erupsi gigi (y) berdasarkan umur kronologis (x) bermakna (p < 0,05) dengan koefisien determinan (r2) sebesar 73,6% pada subjek laki-laki dan 64,8% pada subjek perempuan. maturasi gigi mempunyai r2 sebesar 70,6% pada subjek laki-laki dan 58,4% pada subjek perempuan terhadap erupsi gigi. tabel 1. rerata (x) dan simpangan baku (sb) skor maturitas gigi permanen metode dermijian dan jumlah gigi permanen erupsi, serta uji-t antara subjek laki-laki dan perempuan tiap kelompok umur kelompok umur variabel laki-laki perempuan t p n x ± sb n x ± sb 6 skor 6 62,45 ± 15,21 4 68,78 ± 10,70 -0,72 0,50 erupsi 6,83 ± 3,25 6,25 ± 3,86 0,26 0,80 7 skor 7 72,21 ± 6,40 6 81,85 ± 5,88 -2,52* 0,03 erupsi 7,86 ± 2,73 10,33± 0,82 -2,13 0,06 8 skor 10 79,92 ± 9,18 9 88,86 ± 3,50 -2,74* 0,01 erupsi 11,60 ± 3,34 12,56 ± 2,19 -0,73 0,48 9 skor 7 89,57± 4,32 8 92,40 ± 3,95 -1,33 0,21 erupsi 14,29 ± 2,43 14,88 ± 4,05 -0,34 0,74 10 skor 5 89,86 ± 4,75 6 93,75 ± 3,20 -1,62 0,14 erupsi 15,00 ± 3,74 15,67 ± 4,23 -0,27 0,79 11 skor 3 94,30 ± 0,53 6 96,77 ± 0,92 -4,23* 0,00 erupsi 22,33 ± 4,04 19,83 ± 4,49 0,81 0,45 keterangan: n: jumlah subjek; *: signifikan p<0,05; skor: skor maturasi gigi metode dermijian; erupsi: jumlah gigi permanen yang telah erupsi tabel 2. hasil analisis korelasi dan regresi linier antar variabel pada subjek laki-laki dan perempuan variabel laki-laki (n=38) perempuan (n=39) y x r r2 (%) regresi r r2 (%) regresi erupsi umur 0,86* 73,6 3,06x – 14,65 0,81* 64,8 2,76x – 11,23 maturasi gigi 0,84* 70,6 0,37x – 17,77 0,76* 58,4 0,48x – 27,98 maturasi gigi umur 0,81* 66,2 6,54x + 22,74 0,82* 66,7 4,5x + 47,25 keterangan: y : variabel prediksi x: variabel prediktor; r : koefisien korelasi r2 : koefisien determinan; regresi : persamaam regresi * : bermakna p<0,05 75kuswandari: maturasi dan erupsi gigi permanen pada anak periode gigi pergantian metode dermijian terlalu tinggi dalam memprediksi umur kronologis (tabel 3), pada uji t-berpasangan menunjukkan perbedaan sangat bermakna (p < 0,01). rerata selisih antara umur prediksi dermijian dan umur kronologis masing-masing 0,71 tahun pada subjek laki-laki dan 0,95 tahun pada subjek perempuan. pembahasan foto rontgen gigi selain digunakan untuk mendukung diagnosis dan rencana perawatan, sering juga digunakan untuk menilai tingkat perkembangan maturasi gigi dan memperkirakan umur anak. penilaian perkembangan gigi dengan radiografi telah digunakan lebih dari 60 tahun dan mampu menggambarkan tahap-tahap secara berurutan terjadinya mineralisasi pada gigi geligi individu. radiografi bisa digunakan untuk studi cross sectional maupun longitudinal pada subjek manusia hidup untuk penilaian dental age, akan tetapi tidak bisa untuk mendeteksi tahap awal perkembangan gigi pada tahun awal kehidupan, karena resolusi radiografi tidak bisa membedakan pertumbuhan gigi secara mikroskopis.10 metode dermijian merupakan salah satu metode penilaian maturasi gigi yang paling sering digunakan, karena relatif memiliki reliabilitas tinggi, hanya menggunakan 8 tahap perkembangan dan mempunyai kriteria diskripsi paling detail menggunakan panjang relatif mahkota dan akar gigi.4,11 hasil penelitian ini menunjukkan skor maturasi gigi pada subjek perempuan lebih tinggi dibandingkan subjek laki-laki (tabel 1), meskipun perbedaan bermakna hanya ditemukan pada kelompok umur tujuh, delapan dan sebelas tahun. hal ini menunjukkan, pertumbuhan gigi seperti juga pertumbuhan skeletal, yaitu puncak pertumbuhan (growth spurt) pada anak perempuan terjadi lebih awal dibandingkan pada anak laki-laki.12 jumlah gigi permanen yang telah erupsi (eupsi gigi) tidak menunjukkan adanya perbedaan bermakna (p > 0,05) antara laki-laki dan perempuan. hal ini bisa mengindikasikan bahwa meskipun erupsi gigi dipengaruhi oleh maturasi gigi (tabel 2), tetapi faktor-faktor lain juga sangat berpengaruh, misalnya adanya prematur loss.13 antara umur kronologis dengan maturasi gigi dan erupsi gigi terdapat hubungan yang sangat erat (tabel 2). kontribusi umur kronologis dan maturasi gigi pada prediksi terjadinya erupsi gigi masing-masing lebih dari 50%, pada subjek laki-laki lebih tinggi daripada subjek tabel 3. rerata dan simpangan baku (sb) umur kronologis sebenarnya, umur prediksi metode dermijian dan perbedaan antara kedua variabel dengan uji t-pair serta koefisien korelasi kelompok subjek n umur prediksi selisih t-pair test r mean ± sb mean ± sb mean ± sb laki-laki 38 8,66 ± 1,59 9,37 ± 1,61 -0,71 ± 0,95 -4,64** 0,82** perempuan 39 9,07 ± 1,53 10,03 ± 1,72 -0,95 ± 0,90 -6,58** 0,85** total 77 8,87 ± 1,57 9,71 ± 1,69 -0,83 ± 0,93 -7,89** 0,84** keterangan: n jumlah subjek ** signifikan p<0,01 r koefisien korelasi product moment perempuan. erupsi gigi dipengaruhi oleh beberapa faktor, baik yang langsung maupun tidak langsung. resorpsi dan aposisi tulang, karakteristik vaskularisasi periodontal dan perkembangan akar merupakan faktor yang langsung berhubungan dengan erupsi gigi,14 sedangkan faktor tidak langsung misalnya nutrisi, ekstraksi dan sebagainya. erupsi gigi peramanen yang terlalu awal (premature eruption) bisa terjadi akibat gigi desidui yang digantikan mengalami karies yang parah sehingga merusak tulang di koronal gigi permanen pengganti, akibatnya gigi permanen erupsi terlalu awal meskipun akar baru terbentuk kurang dari 50%. adanya variasi individual yang luas dalam umur kronologis dan erupsi gigi membuat rata-rata umur erupsi gigi tidak reliabel untuk prediksi individu. indeks pertumbuhan juga bervariasi dalam populasi, karena genetik dan lingkungan bisa berpengaruh terhadap waktu kalsifikasi.15 erupsi gigi adalah proses berkesinambungan yang menggerakkan gigi mulai dari dental crypt menuju garis oklusi dan mempertahankannya dalam oklusi. gigi cenderung muncul dalam rongga mulut setelah pembentukan akar mencapai 50%.14 prediksi umur metode dermijian lebih tinggi (overestimasi) 0,71 tahun pada subjek laki-laki dan 0,95 tahun pada subjek perempuan (rerata 0,83 ± 0,93 tahun) dibandingkan umur kronologis (umur sebenarnya), namun memiliki koefisien korelasi yang cukup tinggi, yaitu masingmasing 0,82 pada laki-laki dan 0,85 pada perempuan. ini menunjukkan bahwa metode dermijian bisa diandalkan untuk digunakan prediksi umur pasien di klinik kedokteran gigi anak prof soedomo reproduksibilitas cukup tinggi. nilai prediksi yang terlalu tinggi bisa diperbaiki dengan melakukan modifikasi disesuaikan dengan target populasi. overestimasi penggunaan metode dermiian juga ditemukan pada studi terhadap anak-anak china selatan umur 3–16 tahun di hongkong, terdapat perbedaan sebesar sebesar 0,62 tahun pada anak laki-laki dan 0,36 pada anak perempuan.16 studi pada anak-anak jepang umur 3–16 tahun menunjukkan perbedaan antara -0,76–1,22 pada anak laki-laki dan -0,46–1,00 pada anak perempuan.4 studi pada populasi anak-anak iran umur 3,5–13,5 tahun menunjukkan metode dermijian overestimasi 0,15 tahun pada anak laki-laki dan 0,21 tahun pada anak perempuan, namun secara klinis cukup baik digunakan untuk populasi anak-anak iran.17 secara teori adanya overestimasi prediksi umur metode dermijian dapat disebabkan perbedaan subjek penelitian dalam ras (genetik) dan periode atau masa penelitian. 76 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 72–76 metode dermijian dibangun berdasarkan populasi anakanak canada kulit putih keturunan perancis sebelum tahun 1971.6 jadi sudah lebih dari 40 tahun yang lalu. studi yang dilakukan oleh nadler18 menunjukkan bahwa anak-anak periode 1990-an mengalami maturasi gigi yang lebih awal dibandingkan anak-anak periode 1970-an. hal ini dibuktikan dengan penelitiannya tentang maturasi gigi dengan subjek gigi kaninus (43) yang mengalami maturasi lebih awal 1,21 tahun pada anak laki-laki dan 1,52 pada anak perempuan, atau rata-rata 1,40 tahun. gejala maturasi yang lebih awal dibandingkan generasi sebelumnya bukan hanya terjadi terhadap perkembangan gigi, tetapi juga pada tanda kelamin sekunder. studi cross sectional pada anakanak di amerika serikat menunjukkan bahwa anak-anak perempuan mengalami tanda-tanda pubertas yang lebih awal dibandingkan standar yang terdapat di textbook, anak afro-amerika mengalami pubertas antara 89 tahun, sedangkan pada anak-anak caucasid sekitar 10 tahun.19 disimpulkan bahwa antara umur kronologis, maturasi gigi metode dermijian dan erupsi gigi berhubungan erat, dan merupakan prediktor yang baik bagi erupsi gigi pada anak periode gigi bercampur. metode dermijian memprediksi 0,83 tahun lebih tinggi dibandingkan umur kronologis. penilaian dental age metode dermijian cukup dapat diandalkan, akan tetapi perlu adanya modifikasi atau penyesuaian bila digunakan untuk populasi anak-anak indonesia. ucapan terima kasih terima kasih kepada dekan fakultas kedokteran gigi universitas gadjah mada atas dana penelitian melalui dana masyarakat fakultas kedokteran gigi universitas gadjah mada 2013 dan drg. iyop ropika yang telah membantu pengumpulan rontgen foto. daftar pustaka 1. smith h. standar of human tooth formation and dental age assesment. in: advances in dental anthropology. wiley-liss, inc; 1991. p. 143-68. 2. daito m, kawahara s, tanaka m, imai g, nishihara g, hieda t. calcification of the permanent anterior teeth observed in panoramic radiograph. j osaka dent univ 1990; 24(1): 63-85. 3. parekh s. dental age assessment–developing standards for uk subjects. thesis. london uk: division of craniofacial development, ucl easman dental institute; 2011. p. 21-22. 4. agurto g h, satake t, maeda t, akimoto y. dental age in japanese children a modified dermijian method. ped dent j 2009; 19(1): 828. 5. barnett em. pediatric occlusal therapy. 1st ed. saint louis: the cv mosby co; 1974. p. 9-48. 6. demirjian a, goldstein h, tanner j m. a new system of dental age assessment. hum biol 1973; 45: 221-7. 7. burdi ar, moyers re. development of the dentition and occlusion. in: moyers re, editor. handbook of orthodontic. 4th ed. chicago: yearbook medical publishing inc; 1988. p. 111-7. 8. mӧrnstad h, staaf v, welander u. age estimation with aid of tooth development : a new method based on objective measurements. scand j dent res 1994; 102(3): 137-43. 9. gleiser i, hunt ee. the permanent mandibular first molar: its calcification, eruption and decay. am j phys antrop 1955; 13(2): 253-83. 10. reid dj, dean mc. variation in modern human enamel formation times. j hum evol 2006; 50(3): 329-46. 11. maber m, liversidge hm, hector mp. accuracy of age estimation of radiographic methods using developing teeth. forensic sci int 2006; 159(suppl): s68–73. 12. fishman ls. maturational pattern and prediction during adolescence. angle orthod 1987; 57(3): 178-93. 13. sleichter cg. the influence of premature loss of deciduous molars and the eruption of their successors. angle orthod 1963; 33(4): 279–83. 14. dale m. interceptive guidance of occlusion with emphasis on diagnosis. in: graber tm, vanarsdall rlj, eds. orthodontics: current principles and technique. st louis: mosby co; 2005. p. 422–39. 15. proffit wr. concept of growth and development. in: proffit wr, fields hw, server dm, editors. contemporary orthodontics. 4th ed. st louis: elsevier – mosby co; 2007. p. 21-6. 16. jaya raman j, k ing n m, rober t gj, wong hm. dental age assessment: are dermijians appropriate for southern chinese children?. j forensic odontostomatol 2011; 29(2): 22-8. 17. bagherian a, sadeghi m. assessment of dental maturity of children aged 3.5 to 13.5 years using the dermijian method in iranian populatiion. j oral sci 2011; 53(1): 37– 42. 18. nadler gl. earlier dental maturation: fact or fiction. angle orthod 1998; 68(6): 535–8. 19. herman-giddens me, slora ej, wasserman rc. secondary sexual characteristics and menses in young girls seen in office practice: a study from the pediatric research in office setting network. pediatric 1977; 88: 505-12. vol 38-no 1-2005 36 perubahan warna lempeng resin akrilik yang direndam dalam larutan desinfektan sodium hipoklorit dan klorhexidin (the color changes of acrylic resins denture base material which are immersed in sodium hypochlorite and chlorhexidine) david* dan elly munadziroh** * mahasiswa ppdgs ** bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract one of the acrylic resins properties is the water absorption including color fluids and chemically fluids that affect on the color changes of the acrylic resins. this laboratory experiments studied sodium hypochlorite and chlorhexidine effect on the color changes of acrylic denture base resins material. the study was conducted by immersing heat cured acrylic plate samples of 26 mm of diameter and 0.4 mm of thickness in sodium hypochlorite for 10; 70 and 140 minutes and chlorhexidine for 15; 105 and 210 minutes. seven samples were used for each experiment. an optical spectrometer bpx-47 type photo cell and a digital microvoltage were used for the color changes observation. the statistical test used were t-test, one-way anova and lsd with 0.05 significance degree level. the results of the studied showed that the color of acrylic resins denture base plate changed after immersion in sodium hypochlorite for 70 and 140 minutes and chlorhexidine for 105 and 210 minute of immersion. key words: acrylic resins, chlorhexidine, color change, sodium hypochlorite korespondensi (correspondence): elly munadziroh, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan bahan dasar basis gigi tiruan yang sering dipakai adalah resin akrilik polimetil metakrilat jenis heat cured. resin akrilik dipakai sebagai basis gigi tiruan oleh karena bahan ini memiliki sifat tidak toksik, tidak iritasi, tidak larut dalam cairan mulut, estetik baik, mudah dimanipulasi, reparasinya mudah dan perubahan dimensinya kecil.1 kekurangan dari resin akrilik yaitu mudah patah bila jatuh pada permukaaan yang keras atau akibat kelelahan bahan karena lama pemakaian serta mengalami perubahan warna setelah beberapa waktu dipakai dalam mulut.2 salah satu cara untuk merawat gigi tiruan adalah dengan merendam dalam pembersih gigi tiruan yang mengandung larutan desinfektan. berbagai bentuk pembersih gigi tiruan yang beredar dipasaran antara lain ada yang berbentuk pasta, tablet, cairan dan lain-lain. prosedur pemakaiannya harus disesuaikan dengan petunjuk pabrik. lempeng resin akrilik yang direndam pembersih gigi tiruan dalam jangka waktu yang terus–menerus dapat terjadi perubahan warna. sodium hipoklorit sebagai desinfektan dapat mengurangi mikroorganisme yang melekat pada gigi tiruan,3 sedangkan bahan desinfektan sebagai bahan pembersih seperti klorhexidin glukonat atau salisilat dapat mengurangi plak pada gigi.4 selain kedua bahan di atas, ada juga bahan tradisional seperti daun sirih, daun saga, daun semanggi yang dapat dipakai sebagai bahan desinfektan.5 sodium hipoklorit termasuk golongan halogenated yang oxygenating. sodium hipoklorit dalam larutan membentuk hypochlorus acid (hocl) dan oxychloride (ocl).6 desinfektan ini adalah larutan yang berbahan dasar klorin (cl2), larutan ini merupakan desinfektan derajat tinggi (high level disinfectants) karena sangat aktif pada semua bakteri, virus, jamur, parasit, dan beberapa spora. bahan tersebut bekerja cepat atau fast acting, sangat efektif melawan hepatitis b virus (hbv) dan human immunodeficiency virus (hiv).7 pemakaian sodium hipoklorit sebagai desinfektan dengan konsentrasi 0,5% untuk merendam gigi tiruan dianjurkan 10 menit tiap hari.8 selain sodium hipoklorit, dalam bidang kedokteran gigi ada suatu bahan yaitu klorhexidin glukonat yang dipakai sebagai dental gel, obat kumur, bahan pembersih gigi tiruan. sebagai dental gel dipakai konsentrasi 1% sedangkan sebagai obat kumur dipakai konsentrasi 0,2%. klorhexidin merupakan derivat bis-biquanite yang efektif dan mempunyai spektrum luas, bekerja cepat dan toksisitasnya rendah.9 bahan ini digunakan dalam bentuk yang bervariasi, misalnya klorhexidin asetat atau glukonat yang merupakan antiseptik yang bersifat bakterisidal atau bakteriostatik terhadap bakteri gram positif dan gram negatif. selain itu klorhexidin juga menghambat virus dan aktif melawan jamur, tetapi tidak aktif melawan spora bakteri pada suhu kamar. pemakaian klorhexidin sebagai desinfektan untuk merendam gigi tiruan dianjurkan 15 menit tiap hari.10 37david: perubahan warna lempeng bahan resin akrilik mempunyai salah satu sifat yaitu menyerap air secara perlahan-lahan dalam jangka waktu tertentu, dengan mekanisme penyerapan melalui difusi molekul air sesuai hukum difusi.11 terjadinya penyerapan zat warna cairan dalam resin akrilik merupakan salah satu faktor penyebab perubahan warna pada resin akrilik.12 bahan kimia seperti alkohol, kloroform, zat warna buatan atau asli, dan karbonat dapat menyebabkan perubahan warna pada resin akrilik.13 salah satu cara untuk mengamati perubahan warna yang terjadi adalah dengan menggunakan rangkaian alat spectrometer optic, foto sel type bpy-47, dan mikrovolt digital yang dapat mengukur besarnya intensitas cahaya yang diserap oleh suatu benda. benda yang memantulkan suatu gelombang cahaya tertentu akan kelihatan (berwarna) seperti cahaya yang dipantulkan,14 misalnya benda yang berwarna hitam menyerap semua warna sedangkan benda berwarna putih akan memantulkan semua sinar yang datang. untuk mengetahui besarnya perbedaan intensitas cahaya tersebut maka dapat melihat nilai yang tertera pada alat voltmeter. sodium hipoklorit dan klorhexidin dipakai sebagai bahan desinfeksi oleh sebagian pemakai gigi tiruan. oleh karena pemakaiannya dalam jangka waktu yang terusmenerus maka timbul permasalahan apakah terjadi perubahan warna pada lempeng resin akrilik yang direndam dalam sodium hipoklorit dan klorhexidin. tujuan dari penelitian ini untuk mengetahui perubahan warna lempeng resin akrilik yang direndam dalam sodium hipoklorit dan klorhexidin. bahan dan metode jenis penelitian ini adalah penelitian eksperimental laboratorik. pengukuran perubahan warna dilakukan di laboratorium fisika optik fakultas matematika dan ilmu pengetahuan alam universitas airlangga. pembuatan lempeng akrilik dilakukan di laboratorium ilmu material dan teknik kedokteran gigi fakultas kedokteran gigi universitas airlangga. penelitian ini dilakukan pada tahun 2003. sampel yang dipakai berbentuk silinder berdiameter 26 mm dan tebal 0,4 mm,3 yang terbuat dari bahan resin akrilik jenis heat cured (merk qc). sampel untuk masingmasing kelompok berjumlah 7 buah.15 pembuatan sampel dari lempeng akrilik adalah sebagai berikut, kuvet disiapkan dengan terlebih dahulu mengulasinya dengan vaselin. gip keras (merk giludur) diaduk dengan perbandingan bubuk dan air 100 gr : 30 ml (sesuai petunjuk pabrik) diisikan dalam kuvet. selanjutnya master model dari logam kuningan bentuk silinder dengan diameter 26 mm dan tebal 0,4 mm,3 yang telah diulasi vaselin diletakkan di atas adonan gip dengan posisi mendatar. setelah gip pada kuvet bagian bawah mengeras, permukaan atas gip dan master model diulasi vaselin. lalu kuvet lawan dipasang dan dituangi adonan gip keras sambil diletakkan di atas vibrator. kemudian kuvet ditutup dan dipres, ditunggu sampai gip mengeras. setelah gip mengeras, kuvet dibuka dan master model dikeluarkan. cetakan dibersihkan serta diulasi selapis bahan separasi could mould seal dengan menggunakan kuas dan ditunggu sampai kering. bahan resin akrilik heat cured (merk qc) dengan perbandingan bubuk : cairan = 23 mg : 10 ml (sesuai petunjuk pabrik) dimasukkan dalam pot porselen dan diaduk, pot ditutup. setelah mencapai dough stage adonan dimasukkan dalam cetakan dan kuvet lawan ditutupkan, lalu ditekan dengan pres kemudian kuvet dibuka dan kelebihan akrilik diambil dengan menggunakan pisau model. selanjutnya kuvet lawan ditutupkan dan ditekan dengan pres kembali sampai tekanan 22 kg/cm2 hg. penekanan dengan pres pada kuvet diulang sebanyak dua kali sampai tidak ada kelebihan akrilik, lalu ditekan dengan pres dan klem kemudian siap digodok. tempat penggodokan diisi air sampai di atas kuvet. proses pemanasan dengan menaikkan suhu dari suhu kamar sampai 100° c selama 20 menit. setelah kuvet dingin kemudian dibuka lalu lempeng akrilik dikeluarkan, kelebihan akrilik dibuang dan dihaluskan dengan kertas gosok no. 0 di bawah aliran air. lempeng akrilik yang sudah halus dan tidak porus kemudian dikeringkan. selanjutnya sampel direndam dalam tempat toples kaca dengan cara digantung, yang berisi aquadestilata selama 2 × 24 jam.16 kemudian diberi perlakuan, dengan membagi 3 kelompok, yaitu: kelompok i (kontrol) lempeng uji direndam dalam aquadestilata; kelompok ii lempeng uji direndam dalam sodium hipoklorit 0,5% dengan cara menambahkan aquadestilata ke dalam bahan pemutih naclo (bayclin) dengan perbandingan 1 : 10 untuk mendapatkan konsentrasi 0,5% naclo;17 kelompok iii lempeng uji direndam dalam klorheksidin 0,2% (minosep). lama perendaman plat resin akrilik heat cured dalam sodium hipoklorit 0,5% 10 menit, 70 menit, 140 menit dan klorheksidin 0,2% 15 menit, 105 menit, 210 menit. sebelum melakukan pengukuran, sampel dibersihkan dengan menggunakan sikat gigi halus, kemudian dibilas dengan air dan dikeringkan.3 lempeng akrilik selanjutnya diletakkan pada alat pengukur dan dilakukan pengukuran melalui sinar yang datang dari lampu gas natrium diperkecil ukuran berkas cahayanya memakai celah (kisi) dari spectrometer optic. kemudian berkas cahaya tersebut dijatuhkan pada sampel dan dilakukan pengukuran perbedaan intensitas cahaya yang datang pada sampel serta intensitas cahaya yang keluar dari sampel dengan melihat nilai pada voltmeter. pengukuran dengan foto sel type bpy-47 dan mikrovolt digital, dengan satuan lux (lumen/ m2) dan skala 10–2. mikrovolt digital sangat sensitif terhadap cahaya dan penggunaan skala 10–2 pada penelitian ini sesuai dengan tempat penelitian dilakukan. dengan demikian dapat diketahui besarnya perbedaan intensitas cahaya yang diserap sampel dengan intensitas cahaya yang dipantulkan sampel dengan melihat nilai yang tertera dalam mikrovolt digital.14 data dikumpulkan dan 38 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 36–40 ditabulasi, kemudian dianalisis dengan t-test dan uji oneway anova dengan derajat kemaknaan 0,05. hasil hasil nilai rerata dan standart deviasi dari perubahan warna lempeng akrilik heat cured pada kelompok perlakuan dan kontrol dengan satuan lux dapat dilihat pada tabel 1 dan tabel 2. tabel 1. nilai rerata dan standart deviasi dari perubahan warna lempeng akrilik heat cured setelah direndam dalam akuadestilata dan sodium hipoklorit 0,5% (lux). tabel 3. hasil pooled t-test terhadap perubahan warna lempeng akrilik yang direndam dalam akuadestilata dan sodium hipoklorit 0,5% selama 10 menit, 70 menit, dan 140 menit. kelompok akuades tilata 10 menit akuades tilata 70 menit akuades tilata 140 menit sodium hipoklorit 10 menit t = 0.408 p = 0.691 sodium hipoklorit 70 menit t = 14.811 p = 0.001* sodium hipoklorit 140 menit t = 32.887 p = 0.001* keterangan: * = ada perbedaaan bermakna tabel 4. hasil pooled t-test terhadap perubahan warna lempeng akrilik yang direndam dalam akuadestilata dan klorhexidin 0,2% selama 15 menit, 105 menit, dan 210 menit kelompok akuades tilata 10 menit akuades tilata 70 menit akuades tilata 140 menit klorhexidin 15 menit t = 0.718 p = 0.487 klorhexidin 105 menit t = 41.319 p = 0.001* klorhexidin 210 menit t = 86.983 p = 0.001* keterangan: * = ada perbedaaan bermakna hasil uji pada kelompok kontrol akuadestilata 15 menit kelompok klorhexidin 15 menit lebih besar dari 0,05 yang berarti tidak ada perbedaan yang bermakna. sedangkan kelompok akuadestilata 105 menit dan 210 menit dengan kelompok klorhexidin 105 menit dan 210 menit mempunyai nilai p lebih kecil dari 0,05 yang berarti ada perbedaan yang bermakna. uji one-way anova digunakan untuk mengetahui adanya perbedaan perubahan warna lempeng akrilik antara lempeng akrilik yang direndam dalam sodium hipoklorit 0,5%. hasil uji anova tersebut didapatkan nilai p lebih kecil dari nilai 0,05 yang berarti ada perbedaan yang bermakna antar kelompok perlakuan yang diuji. untuk mengetahui lebih lanjut letak perbedaan tersebut, maka dilanjutkan dengan uji lsd pada tabel 5. tabel 5. hasil uji lsd perubahan warna lempeng akrilik yang direndam dalam sodium hipoklorit 0,5% selama 10 menit, 70 menit, dan 140 menit kelompok 10 menit 70 menit 140 menit 140 menit 70 menit 0,001 0,001 0,001 perlakuan (menit) n rerata standart deviasi akuadestilata 10 sodium hipoklorit 10 akuadestilata 70 sodium hipoklorit 70 akuadestilata 140 sodium hipoklorit 140 7 7 7 7 7 7 9.78e-04 9.70e-04 9.88e-04 5.58e-04 9.88e-04 3.33e-04 3.146e-05 4.119e-05 2.178e-05 7.367e-05 2.178e-05 4.805e-05 tabel 2. nilai rerata dan standart deviasi dari perubahan warna lempeng akrilik heat cured setelah direndam dalam akuadestilata dan klorheksidin 0,2% (lux). perlakuan (menit) n rerata standart deviasi akuadestilata 15 klorheksidin 15 akuadestilata 105 klorheksidin 105 akuadestilata 210 klorheksidin 210 7 7 7 7 7 7 9.85e-04 9.77e-04 9.88e-04 2.61e-04 9.88e-04 1.05e-04 2.378e-05 1.918e-05 2.178e-05 4.113e-05 2.178e-05 1.571e-05 uji normalitas dilakukan dengan menggunakan uji kolmogorov smirnov. asumsi homogenitas dilakukan dengan levene test. didapatkan hasil bahwa semua kelompok mempunyai nilai p > 0,05, yang berarti sampel berasal dari populasi yang berdistribusi normal dan homogen. untuk membandingkan hasil masing-masing kelompok sodium hipoklorit dan kelompok kontrol maka digunakan uji t pada tabel 3. hasil uji pada kelompok kontrol akuadestilata 10 menit dengan kelompok sodium hipoklorit 10 menit nilai p lebih besar dari 0,05 yang berarti tidak ada perbedaan yang bermakna. sedangkan pada kelompok akuadestilata 70 menit dan 140 menit dengan kelompok sodium hipoklorit 70 menit dan 140 menit mempunyai nilai p lebih kecil dari 0,05 yang berarti ada perbedaan yang bermakna.untuk membandingkan hasil masing-masing kelompok klorhexidin 0,2% dan kelompok kontrol maka digunakan uji t pada tabel 4. 39david: perubahan warna lempeng pada tabel 5 terlihat nilai p yang lebih kecil dari 0,05 berarti ada perbedaan yang bermakna antar masing-masing kelompok sampel. untuk mengetahui adanya perbedaan perubahan warna lempeng akrilik antara lempeng akrilik yang direndam dalam klorhexidin 0,2% maka dilakukan uji one-way anova. hasil uji anova tersebut didapatkan nilai p lebih kecil dari nilai 0,05 yang berarti ada perbedaan yang bermakna antar kelompok perlakuan yang diuji. untuk mengetahui lebih lanjut letak perbedaan tersebut, maka dilanjutkan dengan uji lsd pada tabel 6. tabel 6. hasil uji lsd perubahan warna lempeng akrilik yang direndam dalam klorhexidin 0,2% selama 15 menit, 105 menit, dan 210 menit kelompok 15 menit 105 menit 210 menit 210 menit 105 menit 0,001 0,001 0,001 pada tabel 6 terlihat nilai p yang lebih kecil dari 0,05 berarti ada perbedaan yang bermakna antar masing-masing kelompok sampel. pembahasan sodium hipoklorit dan klorhexidin dipakai untuk merendam gigi tiruan resin akrilik karena berfungsi sebagai desinfektan dan kedua bahan tersebut mudah didapatkan. sodium hipoklorit dan klorhexidin mengandung klor. lama perendaman sodium hipoklorit 0,5% sebagai desinfektan yang dianjurkan adalah 10 menit tiap hari,8 sedangkan untuk klorhexidin 0,2% adalah 15 menit tiap hari.10 pada penelitian ini dilakukan perendaman lempeng akrilik dengan bahan sodium hipoklorit selama 10 menit, 70 menit, 140 menit karena diasumsikan identik dengan awal perendaman gigi tiruan selama 1 hari, 7 hari, dan 14 hari. untuk perendaman dalam bahan klorhexidin dilakukan selama 15 menit, 105 menit, 210 menit karena diasumsikan identik dengan awal perendaman gigi tiruan selama 1 hari, 7 hari, dan 14 hari. prinsip pengukuran pada percobaan ini adalah dengan menggunakan perbedaan intensitas cahaya, dalam hal ini diasumsikan sebanding dengan nilai voltmeter. hal ini didapatkan dari adanya gerak elektron dari katode ke anode akibat ada perbedaan intensitas cahaya pada efek foto listrik. adanya pergerakan elektron tersebut dapat diketahui dari tegangan listriknya (volt). bila cahaya yang dipantulkan lebih banyak dari cahaya yang diteruskan, maka nilai pada voltmeter menurun. warna merah pada resin akrilik yang dilihat itu merupakan pemantulan spektrum warna merah, sedangkan warna lain diteruskan. bila warna tersebut menjadi lebih muda atau mengarah ke warna putih, berarti lebih banyak spektrum yang dipantulkan daripada yang diteruskan, sehingga nilai pada voltmeter menjadi turun. semakin lama perendaman dalam sodium hipoklorit dan klorhexidin tenyata pigmen warna lempeng akrilik semakin memudar sehingga perubahan warna yang terjadi semakin besar. hal ini dapat terlihat pada nilai rerata yang semakin lama semakin menurun dengan bertambahnya lama perendaman, maka dapat terlihat nilai voltmeter yang semakin menurun, spektrum cahaya yang dipantulkan lebih banyak daripada yang diteruskan. sodium hipoklorit merupakan desinfektan tinggi karena sangat aktif pada semua bakteri, virus, fungi, parasit dan beberapa spora. desinfektan ini merupakan larutan yang mengandung klorin.7 kelompok perendaman dalam 10 menit sodium hipoklorit dibandingkan kelompok kontrol perendaman dalam akuadestilata 10 menit dapat dilihat bahwa tidak terdapat perbedaan yang bermakna. hal ini kemungkinan karena waktu kontak yang tidak terlalu lama, sehingga pengaruh klorin yang terkandung dalam sodium hipoklorit belum menyebabkan perubahan warna yang berarti pada lempeng akrilik. pada perlakuan kelompok perendaman dalam sodium hipoklorit 70 menit dan kelompok perendaman dalam sodium hipoklorit 140 menit dibandingkan kelompok kontrol perendaman dalam akuadestilata 70 menit, dan kelompok kontrol perendaman dalam akuadestilata 140 menit didapatkan bahwa hasilnya terlihat adanya perbedaan yang bermakna. berdasarkan pengujian yang telah dilakukan ini berarti bahwa terjadi perubahan warna lempeng akrilik. perendaman lempeng akrilik dalam sodium hipoklorit kemungkinan menyebabkan adanya perubahan dalam matrix interstitial pada struktur permukaan sehingga terjadi efek pemutihan dan terjadi perubahan warna lempeng akrilik.18 klorin selain sebagai desinfektan juga dipakai sebagai bahan pemutih pakaian dan untuk menghilangkan noda pakaian sehingga klorin mempunyai kemampuan untuk memudarkan warna. kelompok perendaman dalam klorhexidin 15 menit dibandingkan kelompok kontrol perendaman dalam akuadestilata 15 menit dapat terlihat bahwa tidak terdapat perbedaan yang bermakna yang berarti tidak terjadi perubahan warna yang berarti. hal ini kemungkinan karena waktu kontak yang tidak terlalu lama sehingga pengaruh klor belum menampakkan perubahan warna. pada kelompok perendaman dalam klorhexidin 105 menit dan kelompok perendaman dalam klorhexidin 210 menit dibandingkan kelompok kontrol perendaman dalam akuadestilata 105 menit dan kelompok kontrol perendaman dalam akuadestilata 210 menit ternyata terdapat perbedaan yang bermakna yang berarti terjadi perubahan warna, hal ini terjadi akibat interaksi kation dan anion dari klor yang terkandung dalam klorhexidin dengan akrilik sehingga zat warna akrilik memudar. 19 perubahan warna lempeng akrilik dapat disebabkan oleh karena kemampuan penyerapan cairan pada bahan dan lingkungan sekitar tempat anasir gigi tiruan, sehingga zat yang terserap dapat bereaksi dengan unsur dalam resin akrilik. 12 bahan seperti zat warna, kloroform dapat 40 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 36–40 menyebabkan terjadinya perubahan warna lempeng akrilik.13 sodium hipoklorit yang mengandung klorin dapat menghilangkan stain, menghilangkan komponen organik dari deposit tartar. klorin bereaksi langsung dengan menghambat formasi dari kalkulus dengan menghilangkan organik matriks plak, tetapi klorin ini meyebabkan pemutihan.6 larutan ini bereaksi dengan bahan dasar dari lempeng akrilik. selain itu efek pemutihan permukaan lempeng akrilik dapat terjadi jika sering direndam dalam larutan yang keras yang mengandung kadar klorin tinggi. perubahan warna lempeng akrilik tidak hanya disebabkan oleh karena perendaman dalam larutan desinfektan saja tetapi juga karena faktor macam makanan dan minuman sehari-hari yang dikonsumsi oleh pemakai gigitiruan misalnya teh, kopi, minuman yang mengandung cola menyebabkan warna lempeng akrilik menjadi tambah gelap.20 hal ini karena adanya akumulasi penempelan pigmen warna pada permukaan dan absorsi perlekatan partikel yang masuk ke bagian liang renik resin akrilik, sehingga warna yang diserap lebih banyak daripada warna yang dipantulkan. pada keadaan ini nilai rerata semakin besar dengan bertambahnya waktu. hal ini berbeda pada perendaman dalam sodium hipolorit dan klorhexidin karena tidak terjadi akumulasi noda pada permukaan ataupun liang renik melainkan karena reaksi klorin atau klor dengan lempeng akrilik kemudian terjadi efek pemutihan sehingga warna akrilik menjadi lebih muda. dari pembahasan di atas dapat disimpulkan bahwa sodium hipoklorit 0,5% dapat menyebabkan perubahan warna resin akrilik setelah perendaman selama 70 menit, sedangkan klorhexidin 0,2% dapat menyebabkan perubahan warna resin akrilik setelah perendaman selama 105 menit. semakin lama perendaman dalam sodium hipoklorit dan klorhexidin ternyata pigmen warna lempeng akrilik semakin memudar sehingga perubahan warna yang terjadi semakin besar. daftar pustaka 1. combe ec. notes on dental material. 6th ed. edinburg: churchill livingstone; 1992. p. 26–161. 2. billmeyer fw. textbook of polimer science. 3rd ed. new york: internasional john wiley and sons; 1984. p. 409–11. 3. mcneme sj, von gonten as, woolsey gd. effects of laboratory disinfecting agents on color stability of denture acrylic resins. j prosthet dent 1991; 66: 132–6. 4. budtz-jorgensen e. materials and methods for cleansing dentures. j prosthet dent 1979; 42: 619–23. 5. djulaeha eha. khasiat infusa daun kacapiring sebagai obat kumur terhadap keberadaan candida albicans. jurnal kedokteran gigi 1999; 156–9. 6. martindale. the extra pharmacopoea. 28th ed. london: the pharmaceutical press; 1982. p. 554–6, 564–5. 7. nike hendrijantini. cara dan bahan pembersih untuk menghambat pertumbuhan candida albicans pada gigi tiruan akrilik. jurnal kedokteran gigi 1998; 291–6. 8. nike hendrijantini. pengaruh konsentrasi larutan sodium hypochloride sebagai desinfektan gigi tiruan resin akrilik terhadap candida albicans. jurnal kedokteran gigi 1996; 30: 73–7. 9. hennesey td. some antibacterial properties of chlorhexidine. j perodont res 1973; 8: 61–7 10. putra m sukarsyah. pengenceran bahan disinfektan untuk sanitasi gigi tiruan secara optimal. majalah ilmiah kedokteran gigi fkg usakti 1999; 416–21. 11. philips rw. science of dental material. 9th ed. philadelphia: wb saunder; 1991. p. 23–26, 177–213. 12. crispin bj, caputo aa. colour stability of temporary restorative materials. j prosthet dent 1979; 42: 27–33. 13. horn hr. practical consideration for succesful crown and bridge therapy. 9th ed. philadelphia: wb saunders company; 1976. p. 117–25. 14. pudjianto. karakterisasi detektor cahaya fotosel. surabaya: petunjuk praktikum fisika optika, fmipa universitas airlangga; 1996. h. 16-20. 15. hulley sb, cummings sr. designing clinical research: an epidemiologic approach. baltimore: williams & wilkins; 1988. p. 139–50. 16. american dental association (ada). guide to dental materials and device. 7th ed. chicago; 1974. p. 219–29. 17. bell ja, brockmann sl, feil p, nad sackuvich da. the effectiviness of two desinfectans on denture base acrylic resin with anorganic load. j prosthet dent 1989; 61: 580–3. 18. nike hendrijantini. sodium hipoklorid dan struktur permukaan resin akrilik. jurnal kedokteran gigi 2002; 136–9. 19. widyastuti budhianto. peranan chlorhexidine dalam pemeliharaan gigi tiruan. tesis. surabaya: universitas airlangga; 1982. 20. hanoem ek. perubahan warna resin akrilik heat cured dan cold cured karena minuman coca-cola. tesis. surabaya: universitas airlangga; 2001. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38 no 3 2005 115 reversibilitas kalsifikasi tulang akibat kekurangan protein pre dan post natal (reversibility of bone calcification on pre and post natal protein deficiency) pinandi sri pudyani bagian ortodonsia fakultas kedokteran gigi universitas gadjah mada yogyakarta indonesia abstract the growth and development play an important role in orthodontics mainly in bone, because it can determine the maturity of the bone. bone maturity evaluation is very important in orthodontic treatment, because there are many individual variations in growth and development such as time, duration and velocity of the growth. nutritional status during pregnancy and infant period will influence the growth and the development of bone. protein diet is an important factor, which will determine the optimal calcification during bone growth and development stages. bone calcification, in orthodontics, can be used to estimate the bone maturity for diagnosis and treatment planning. the purpose of this study was to recognize ones ability to surpass calcium and phosphor deficiency because of pre and postnatal protein deficiency. there were three groups of samples of rattus norvegicus rats. the first group was the control group with standard diet, the second was the infant group with pre and postnatal protein deficiency, and the third group was young rat at weaning age with pre and postnatal protein deficiency supplemented with enough protein in the diet. bone calcification stage was analyzed: 1) histologically by measuring epiphyseal width on right femur; 2) by measuring calcium and phosphor concentration on left femur with spectrophotometry atomic absorption and spectroscopy ultra light visible. the data were analyzed by one way anova continued by t test. the result showed that: 1) there was significant (p < 0.01) epiphyseal width difference between group i and ii, i and iii (p < 0,01) but there was not significant difference between group ii & iii (p > 0.05); 2) there was significant calcium and phosphor concentration on bone between group i, ii and iii (p < 0.01). it was concluded that bone calcification damage because of pre and post natal protein deficiency was an irreversible process. protein supplement after bone calcification could not restore the condition. key words: calcification, protein, reversibility korespondensi (correspondence): pinandi sri pudyani, bagian ortodonsia, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. ii, sekip utara yogyakarta 55281, indonesia. pendahuluan faktor tumbuh kembang memegang peranan penting dalam bidang ortodonsi, terutama tumbuh kembang tulang oleh karena akan menentukan status kematangan tulang. evaluasi kematangan tulang sangat penting dalam rencana dan perawatan ortodonsi oleh karena terdapat variasi individual dalam waktu, durasi dan kecepatan pertumbuhan.1,2 perkiraan potensi pertumbuhan penting diketahui pada perawatan dengan penarikan ekstra oral, penggunaan alat ortodonsi fungsional dan tindakan ortodonsi bedah. perkiraan baik waktu maupun jumlah aktif pertumbuhan, khususnya kompleks kraniofasial akan sangat berguna bagi ahli ortodonsi.3,4 tulang selain berguna untuk menetapkan kematangan tulang, dalam bidang ortodonsia sangat penting peranannya oleh karena kualitas tulang sangat menentukan keberhasilan pergerakan gigi. kualitas tulang ditentukan oleh banyaknya kalsifikasi tulang.5 kematangan tulang dapat ditentukan dari banyaknya kalsifikasi tulang. kalsifikasi tulang pada dasarnya adalah pengendapan mineral terutama kalsium dan fosfor ke dalam matriks organik tulang.1 terdapat beberapa metode untuk mengukur kematangan tulang, yaitu: 1) radiografi tulang tangan dan telapak tangan, salah satunya adalah dengan tanner white house 2 (tw2). banyaknya kalsifikasi pada tulang akan menyebabkan gambaran radiopaque yang menandai pemunculan tulang karpal dan tulang telapak tangan kemudian dibandingkan dengan atlas standar pertumbuhan tulang; 2) penggunaan sefalometri radiografi vertebra servikal dengan mengukur pemunculan lempeng epifisis dari prosesus odontoid servikal; 3) pengukuran densitas tulang dengan beberapa metode diantaranya adalah dxa (x-ray absorptiometry)6 dan fotodensitometri;7 4) secara histologis dengan mengukur lebar lempeng epifisis pada tulang panjang, misalnya: femur, radius, dan ulna. secara histologis ketebalan lempeng epifisis menunjukkan potensi tumbuh kembang tulang, dengan demikian dapat digunakan untuk mengukur kualitas dan kematangan tulang. 2 pertumbuhan memanjang pada tulang panjang disebabkan oleh adanya proliferasi pada zona tenang dan zona proliferasi lempeng 116 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 115–119 epifisis. pada akhir pertumbuhan kartilago pada epifisis tulang seluruhnya akan diganti tulang sehingga epifisis bersatu dengan diafisis (fusi) yang ditandai dengan terbentuknya garis epifiseal;8 5) kematangan tulang juga dapat ditentukan dengan mengukur kadar mineral tulang diantaranya kalsium dan fosfor dengan metode spektroskopi serapan atom dan spektrofotometri ultra light visible, oleh karena banyaknya mineral tulang menentukan banyaknya kalsifikasi tulang.9 variasi individual dalam tumbuh kembang anak disebabkan oleh karena tumbuh kembang dipengaruhi oleh faktor lingkungan, hormonal, diantaranya hormon pertumbuhan dan genetik.10-13 nutrisi termasuk salah satu faktor lingkungan yang berpengaruh pada tumbuh kembang tulang sejak prenatal. pembentukan tulang terjadi secara berkesinambungan. nutrien, diantaranya protein dapat mempengaruhi pertumbuhan tulang dengan jalan menghambat diferensiasi seluler, merubah kecepatan sintesis unsur pokok matriks tulang yaitu protein kolagen dan non kolagen yang masing-masing mempunyai peranan spesifik pada pembentukan tulang.10–12 terdapat dua metabolisme utama dalam pembentukan tulang yang rentan terhadap kekurangan nutrien, diantaranya adalah protein, yaitu: proses sintesis protein untuk membentuk matriks organik tulang yang terdiri dari jaringan kolagen dan non kolagen protein. sintesis protein yang normal diperlukan untuk perkembangan jaringan lunak dan keras diantaranya tulang. kekurangan protein akan menyebabkan perubahan pada timbunan asam amino, hal tersebut mengakibatkan hambatan reaksi sintesis protein sehingga menimbulkan hambatan juga dalam pembentukan matriks organik tulang. 12,14 proses berikutnya adalah kalsifikasi tulang, pada tahap ini mineral diantaranya kalsium dan fosfor diendapkan dalam matriks tulang.14,15 jika terdapat hambatan dalam pembentukan matriks organik, maka akan ada hambatan juga dalam proses kalsifikasi tulang sehingga terjadi penurunan kadar mineral tulang, diantaranya kalsium dan fosfor tulang.16 beberapa penelitian telah dilakukan untuk meneliti pengaruh kekurangan protein terhadap metabolisme mineral dan kepadatan tulang pada anak tikus masa pertumbuhan. kelompok perlakukan diberikan diet protein 5% selama 4, 6 dan 8 minggu, pada kelompok kontrol diberikan protein 18%. hasil penelitian menunjukkan meskipun didapatkan pengurangan dimensi skeletal pada ketiga kelompok perlakuan, tetapi tidak terdapat perbedaan pada kepadatan tulang. disimpulkan bahwa pengurangan diet protein menyebabkan kelambatan pertumbuhan, tetapi kepadatan tulang tetap dipertahankan jika masih ada pengurangan ekskresi kalsium melalui urin.17 likimani et al.18 meneliti tentang pengaruh kekurangan protein dalam makanan terhadap metabolisme mineral dan kepadatan tulang. hasilnya ialah diet protein 10 mg/kg berat badan/hari menyebabkan pengurangan kandungan mineral tulang terutama pada ujung proksimal yang terutama terdiri dari tulang trabekula. gangguan perkembangan, baik berasal dari faktor genetik, virus ataupun kelainan nutrisi berpengaruh kuat pada berbagai tahap perkembangan tulang. beberapa sel atau sekelompok sel kemungkinan lebih peka dari sel yang lain selama siklus kehidupan. tahap peka ini kemungkinan bersifat sementara, tetapi rangkaian kelainan yang parah dapat mempengaruhi kemampuan pembentukan struktur jaringan yang normal.12 pada permulaan pertumbuhan, terjadi pembelahan sel yang cepat (hiperplasi). organ tubuh mengalami beberapa periode hiperplasi pada pertumbuhan yang melibatkan aktivitas metabolik seluler yang cepat. pada periode ini jika terjadi penyakit yang mengganggu replikasi dna dapat menyebabkan hambatan pertumbuhan yang menetap (ireversibel) oleh karena jaringan tidak dapat menamah jumlah sel.14,15 berdasar pernyataan tersebut, maka ingin diteliti daya reversibilitas kalsifikasi tulang akibat kekurangan protein pre dan post natal dengan memberikan makanan standar dengan cukup protein dari umur sapih (30 hari) sampai umur dewasa (56 hari) pada anak tikus dengan kekurangan protein pre dan post natal. daya reversibilitas dilihat dengan membandingkan lebar epifisis, kadar kalsium dan fosfor tulang pada tikus dengan tambahan pakan standar tersebut dengan tikus normal usia dewasa. jika tidak ada perbedaan yang bermakna antara lebar lempeng epifisis, kadar kalsium dan fosfor di antara tikus tersebut, maka hambatan kalsifikasi tulang bersifat sementara (reversibel). tujuan penelitian adalah untuk mengetahui daya reversibilitas kalsifikasi tulang akibat kekurangan protein pre dan post natal, yang diukur dengan membandingkan lebar lempeng epifisis, kadar kalsium dan fosfor tulang pada tikus dengan kekurangan protein pre dan post natal dengan tikus yang mendapat tambahan pakan cukup protein dari umur sapih sampai dewasa. bahan dan metode subyek penelitian terdiri dari 30 ekor anak tikus rattus norvegicus. kelompok i adalah 10 anak tikus yang berasal dari induk tikus yang diberi pakan standar sejak bunting dan melahirkan, sedang anak tikus setelah usia 30 hari (sapih) tetap diberi pakan standar sampai umur 56 hari, kelompok ini merupakan kelompok kontrol dan kelompok perlakuan terdiri dari kelompok ii dan iii. kelompok ii (10 ekor) anak tikus dari induk yang sejak bunting diberi pakan rendah protein (10%) sampai melahirkan, kemudian anak tikus yang dilahirkan diberikan pakan rendah protein (4%) sampai umur 56 hari, kelompok ke iii adalah untuk mengetahui daya reversibilitas kalsifikasi tulang. kelompok iii yaitu 10 ekor anak tikus yang berasal dari induk yang diberi pakan dengan rendah protein setelah umur 30 hari (disapih) diberikan pakan standar dengan protein 25% sampai umur 56 hari. susunan bahan pakan rendah protein (4%) adalah kasein 4%, sukrosa 48,5%, tepung jagung 30%, selulosa 117pudyani: reversibilitas kalsifikasi tulang 8%, minyak jagung 5%, vitamin dan campuran mineral 4,5%. pakan setandar terdiri dari kasein 25%, sukrosa 30,5%, tepung jagung 30%, selulosa 5%, minyak jagung 5%, vitamin dan campuran mineral 4,5%.19 untuk mengetahui pengaruh kekurangan protein pre dan post natal terhadap daya reversibilitas kalsifikasi tulang, maka: 1) diukur lebar epifisis tulang femur kanan pada kelompok penelitian. lebar epifisis tulang diukur secara histologis dengan pengecatan haematoxylin eosin, dihitung dalam mikron; 2) diukur kadar kalsium dan fosfor tulang femur kiri untuk mengetahui kepadatan tulang, oleh karena kuantitas dan kualitas kepadatan tulang akan menentukan kematangan tulang. kadar kalsium tulang diukur dengan metode spektroskopi serapan atom (ssa) dan kadar fosfor tulang femur dengan spektrofotometri ultra light visible. dalam μgr/100gr berat sampel. analisis data dilakukan dengan one-way anova dan t test. hasil rerata dan standar deviasi lebar epifisis, kadar kalsium dan fosfor tulang pada kelompok i, ii dan iii dapat dilihat pada tabel 1. hasil analisis dengan menggunakan one-way anova didapatkan bahwa kekurangan pre dan post natal berpengaruh terhadap lebar epifisis, kadar kalsium dan fosfor tulang (p < 0,01). hasil uji t didapatkan adanya perbedaan yang bermakna (p < 0,01) lebar epifisis tulang di antara kelompok i dengan ii dan kelompok i dengan iii, sedangkan antara kelompok ii dan iii tidak berbeda bermakna (p > 0,05). dari hasil uji t didapatkan perbedaan yang bermakna (p < 0,01) kadar kalsium dan fosfor tulang di antara kelompok i, ii dan iii (tabel 3 dan 4). pembahasan kekurangan protein pre dan post natal menyebabkan hambatan kalsifikasi tulang, hal ini dapat dibuktikan dengan: 1) masih lebarnya lempeng epifisis pada kelompok ii yaitu kelompok dengan kekurangan protein pre dan post natal, yaitu: 294,00 ± 29,51 dibandingkan dengan pada kelompok dewasa normal (kelompok i) yaitu 143,397 ± 2,47; 2) adanya penurunan kadar kalsium tulang pada kelompok ii yaitu 10,715 ± 1,240, kadar kalsium tulang pada kelompok dewasa normal (i): 30,357 ± 0,35; 3) adanya penurunan kadar fosfor tulang pada kelompok ii, yaitu: 3,861 ± 0,570 dibanding dengan kadar fosfor tulang pada kelompok dewasa (i), yaitu: 10,540 ± 1,810. untuk mengetahui daya reversibilitas kalsifikasi tulang akibat kurang protein pre dan post natal, maka dibuat kelompok penelitian iii, yaitu setelah umur sapih (30 hari), hewan coba diberi pakan standar sampai umur dewasa (56 hari). hasil penelitian menunjukkan bahwa penambahan pakan standar tidak dapat memperbaiki hambatan kalsifikasi tulang yang telah terjadi, hal ini dapat dilihat dari masih lebarnya lempeng epifisis pada kelompok iii, yaitu: 332,5 ± 50,190 dibandingkan kelompok i (kelompok dewasa normal) yaitu: 143,297 ± 2,47. dari pengukuran lebar epifisis, dapat dikatakan sama sekali tidak ada perbaikan hambatan kalsifikasi tulang oleh karena lebar lempeng epifisis setelah penambahan pakan standar pada umur sapih sampai umur dewasa tidak berbeda bermakna (p > 0,05) dengan lebar epifisis pada kelompok dengan kekurangan protein pre dan postnatal. hambatan kalsifikasi tulang juga dapat dilihat dari kurangnya kadar kalsium tulang pada kelompok iii. dari uji t didapatkan perbedaan yang bermakna (p < 0,01) kadar kalsium tulang antara kelompok i (kelompok dengan pakan standar) yaitu: 30,357 ± 0,350 dengan kadar kalsium tulang kelompok iii, yaitu: 14,40 ± 2,500. dari hasil tersebut dapat dilihat bahwa penambahan pakan standar pada umur sapih sampai dewasa tidak dapat memperbaiki penurunan kadar kalsium yang telah terjadi oleh karena kekurangan protein pre dan post natal. perbaikan hambatan kalsifikasi tulang tidak terjadi, hal ini dapat dilihat juga dari kurangnya kadar fosfor tulang pada kelompok iii. dari uji t didapatkan perbedaan yang bermakna (p < 0,01) kadar fosfor tulang antara kelompok i dan kelompok iii. kadar fosfor tulang kelompok i: 10,540 ± 1,810 sedang pada kelompok iii: 5,908 ± 2,050. dari hasil tersebut dapat dinyatakan bahwa penambahan pakan pada umur sapih sampai dewasa tidak dapat memperbaiki penurunan kadar fosfor tulang yang telah terjadi oleh karena kekurangan protein pre dan post natal. tabel 1. rerata dan standar deviasi lebar epifisis tulang rerata dan standar deviasi kelompok lebar epifisis tulang (mikron) kadar kalsium tulang (μgr/100 gr berat sampel) kadar fosfor tulang (μgr/100 gr berat sampel) i ii iii 143,397 ± 2,47 294,00 ± 29,510 332,5 ± 50,190 30,357 ± 0,35 10,715 ± 1,240 14,40 ± 2,500 10,540 ± 1,810 3,861 ± 0,570 5,908 ± 2,050 keterangan: kelompok i = induk dengan pakan standar, anak dengan pakan standar kelompok ii = induk dengan pakan rendah protein, anak dengan pakan rendah protein kelompok iii = induk dengan pakan rendah protein, anak umur sapih dengan pakan standar 118 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 115–119 tabel 2. uji t lebar epifisis tulang antar kelompok kelompok i ii iii i ii < 0,01* < 0,01* > 0,05 keterangan: * = berbeda bermakna tabel 3. uji t kadar kalsium tulang antar kelompok kelompok i ii iii i ii < 0,01* < 0,01* < 0,01* keterangan: * = berbeda bermakna tabel 4. uji t kadar fosfor tulang antara kelompok kelompok i ii iii i ii < 0,01* < 0,01* < 0,01* dari hasil pengukuran lebar lempeng epifisis, kadar kalsium dan kadar fosfor tulang kelompok iii, disimpulkan bahwa penambahan pakan standar pada umur sapih sampai dewasa tidak dapat memperbaiki hambatan kalsifikasi tulang yang telah terjadi akibat kekurangan protein pre dan post natal, sehingga hambatan bersifat ireversibel. hal tersebut dapat diterangkan bahwa tumbuh kembang yang dimulai pada periode embrional merupakan proses yang sangat kompleks dan memerlukan rangkaian metabolisme yang baik.12 meskipun faktor genetik memegang peranan khusus pada pertumbuhan, tetapi beberapa faktor lingkungan termasuk nutrisi merupakan hal utama yang penting untuk tercapainya hasil optimal perkembangan. oleh karena nutrisi mengandung berbagai nutrien yang merupakan bahan bakar dalam metabolisme tubuh.11,13 kuantitas dan kualitas yang tepat dari nutrisi ibu dibutuhkan selama kehamilan untuk mendukung pembelahan sel, diferensiasi dan replikasi sel untuk pertumbuhan dan persiapan jaringan pada waktu menyusui. protein merupakan nutrien yang sangat penting dalam masa kehamilan untuk tercapainya perkembangan optimal anak termasuk tulang.20–23 protein yang cukup tetap dibutuhkan anak sesudah kelahiran untuk mendukung tumbuh kembang yang optimal. kekurangan bahan tersebut pada masa pertumbuhan akan menghambat pertumbuhan.20–22 protein berfungsi untuk membentuk matriks organik tulang, sehingga kekurangan protein sejak prenatal akan menghambat pembentukan matriks organik. pada proses kalsifikasi tulang, mineral diantaranya kalsium dan fosfor dideposisikan ke dalam matriks organik, salah satu fungsi protein dalam hubungannya dengan kalsium adalah bahwa plasma kalsium (40%) terikat dengan protein sebagai timbunan.15 dengan banyaknya persentase plasma kalsium yang terikat dengan protein, dapat diartikan protein sangat penting untuk pengikatan kalsium. kekurangan protein akan menyebabkan hambatan metabolisme kalsium. hal ini juga dapat dibuktikan dari hasil penelitian ini, yaitu terdapat penurunan yang bermakna kadar kalsium tulang. hambatan pembentukan matriks organik oleh karena kekurangan protein akan menyebabkan berkurangnya deposisi mineral terutama kalsium dan fosfor dalam matriks tersebut, sehingga menyebabkan penurunan kadar kalsium dan fosfor tulang. hal ini terbukti dengan penurunan kadar kalsium, fosfor tulang pada kelompok dengan kekurangan protein pre dan post natal. penurunan kadar kalsium dan fosfor tulang menyebabkan hambatan kalsifikasi tulang, hal tersebut dapat dibuktikan dengan masih lebarnya lempeng epifisis pada kelompok ii. matriks tulang merupakan komponen organik, terutama terdiri dari kolagen tipe i yang dapat memberikan daya rentang dan komponen anorganik terutama hidroksi apatit yang dapat memberikan kekakuan terhadap tekanan.23 penelitian pada anak tikus dengan diet tanpa protein selama 30–50 hari mengakibatkan banyak sekali pengurangan pada kekuatan pembengkokan dan kekakuan tulang. perubahan tersebut berhubungan dengan parahnya kerusakan dalam jumlah dan atau susunan arsitektur materi tulang, yaitu: volume, rasio dinding dan lumen, pengurangan jumlah kalsium dan elastisitas jaringan tulang.18 kalsifikasi tulang akan menentukan kualitas tulang dengan demikian akan menentukan kematangan tulang, oleh karena kematangan tulang ditentukan oleh jumlah deposisi mineral dalam matriks tulang. penentuan kematangan dan evaluasi potensi pertumbuhan penting dalam bidang ortodonsia, oleh karena selama pertumbuhan setiap tulang mengalami perubahan berurutan yang relatif konsisten untuk setiap tulang pada individu. variasi dalam waktu perubahan tulang terjadi oleh karena tiap individu mempunyai jadwal biologik tersendiri.1,24,25 terdapat hubungan antara kematangan tubuh yang dapat diketahui dari menstruasi, kematangan tulang, kematangan gigi dan pertumbuhan fasial. kelambatan dalam perkembangan tulang akan menyebabkan kelambatan pola pertumbuhan fasial. pertumbuhan fasial maksimal dicapai dengan tercapainya tinggi badan maksimal. kematangan tulang dapat dilihat dari: 1) pemunculan tulang karpal dan tulang ulnar pada gambaran radiografi bertepatan dengan pertumbuhan tinggi badan maksimal; 2) terjadinya fusi dari diafisis dan epifisis tulang radius;26 dan 3) pemunculan lempeng epifisis dari prosesus odontoid pada vertebra servikal. hal tersebut merupakan metode yang tepat untuk penilaian kematangan mandibula pada individu tanpa harus menambah paparan sinar x, oleh karena dapat dilihat dari sefalogram. ketepatan dalam menentukan kecepatan pertumbuhan mandibula sangat membantu dalam menentukan waktu perawatan kasus hambatan pertumbuhan mandibula,24 juga dalam penilaian stabilitas oklusal setelah perawatan, oleh karena potensi 119pudyani: reversibilitas kalsifikasi tulang pertumbuhan mandibula berhubungan erat dengan kematangan tubuh.27 penelitian ini menunjukkan bahwa terjadi hambatan fusi diafisis dan epifisis tulang femur, hal ini dapat diketahui dari masih lebarnya lempeng epifisis pada tikus dewasa (kelompok ii), dan penambahan diet standar pada usia sapih sampai dewasa tidak dapat memperbaiki hambatan pertumbuhan yang telah terjadi (kelompok iii). hal tersebut disebabkan kurangnya deposisi mineral, yaitu kalsium dan fosfor ke dalam matriks tulang. deposisi mineral ke dalam matriks tulang akan menentukan densitas tulang dan hal ini akan mempengaruhi tumbuh kembang dan kematangan tulang. pada penelitian dengan metode dxa (x-ray absorptio metry) didapatkan adanya korelasi positif antara densitas mineral tulang dengan panjang ramus mandibula.6 menurut paulen8 pertumbuhan memanjang pada tulang panjang disebabkan oleh adanya proliferasi pada zona tenang dan zona proliferasi lempeng epifisis. pada akhir pertumbuhan kartilago pada epifisis tulang seluruhnya akan diganti tulang sehingga epifisis bersatu dengan diafisis (fusi) ditandai dengan terbentuknya garis epifiseal. pada penelitian ini pada kelompok ii (kelompok dengan kekurangan protein pre-post natal) dan kelompok iii (kelompok dengan penambahan makanan standar sejak umur sapih sampai dewasa untuk mengukur daya reversibilitas tulang) belum terbentuk garis epifiseal, hal tersebut menunjukkan adanya hambatan kalsifikasi tulang. dari gambaran histologis lempeng epifisis pada kelompok ii dan iii dapat diketahui banyaknya tulang trabekular dan pengurangan tulang kortikal. hasil penelitian menunjukkan adanya perbedaan yang bermakna (p < 0,01) lebar epifisis, kadar kalsium dan fosfor tulang pada kelompok kontrol (i) dan kelompok iii (perlakuan, dengan penambahan pakan standar setelah umur sapih sampai dewasa). hal ini menunjukkan bahwa masih terjadi hambatan kalsifikasi tulang akibat kekurangan protein pre dan post natal, akibatnya akan menyebabkan hambatan dalam pembentukan sel osteoklas, oleh karena kekurangan protein dapat menghambat diferensiasi seluler dan menghambat proses sintesis unsur pokok matriks, selanjutnya akan terjadi penurunan kadar mineral tulang.12 disimpulkan bahwa pemberian pakan standar setelah umur sapih sampai umur dewasa pada anak tikus yang berasal dari induk kurang protein tidak dapat memperbaiki kalsifikasi tulang, sehingga hambatan kalsifikasi tulang bersifat ireversibel. perlu dilakukan penelitian lebih lanjut tentang pengaruh protein terhadap tumbuh kembang jaringan lunak fasial serta pengaruh nutrien lain, misal: magnesium, seng, yodium terhadap tumbuh kembang tulang. daftar pustaka 1. hazel b, farman g. skeletal maturation evaluation using cervical vertebral. am j orthod dentofac orthop 1995; 107(1):58–66. 2. mito t, sato k, mitani h. cervical vertebral bone age in girls. am j orthod dentofac orthop 2002; 122(4):380–5. 3. moore rn, moyer ba, dubois lm. skeletal maturation and craniofacial growth. am j orthod 1990; 98(1):33–40. 4. lewis ab. comparisons between dental and skeletal ages. angle orthod 1991; 61(2):87–92. 5. noxon s, king g, gu g, hung g. osteoclast clearance from periodontal tissue during orthodontics tooth movement. am j orthod dentofac orthop 2001; 120(5):466–76. 6. maki k, sato k, nishioka t, marmoto a, naito m, kimura m. bone mineral density in the radius measured by the dxa method and evolution of the morphology of the mandibule. dent in japan 2000; 36:102–4. 7. mayama h. a study of mandibular bone mineral content and bone age in young patient with congenitally missing permanent teeth. dent in japan 2004; 40:71–5. 8. paulen de. basic histology, examination and board review. 1st ed. appleton & lange, a publishing division of prentice hall; 1990. p. 70–6. 9. narsito. metode pengukuran spektroskopi serapan atom dan spektrofotometri ultra light visibel. jogjakarta: laboratorium kimia dan fisika pusat universitas gadjah mada. 1992. 10. arvytas, mg. early eruption of deciduous and permanent teeth: a case report. am j orthod 1974; 66:189–96. 11. carraza f, marcoudes e, sperroto o. commentary of growth and body compotition in childhood. in: bruner o, carraza f, gracey m, nichols b, senterre j, editors. clinical nutrition of the young child, new york: raven press; 1985. p. 85–9. 12. roughead zk, kunkel me. effect of diet on bone matrix constituents. j am nutr 1991; 10(3):242–6. 13. rabie abm, hagg u. factors regulating mandibular condylar growth. am j orthod dentofac orthop 2003; 122(4):401–9. 14. roth g, calmes r. oral biology. 1st ed. st. louis, toronto: the cv mosby company; 1981. p. 173–96. 15. mac gillivary nh. disorders of growth and development. in: felig p, baxter jd, broadus ae, frohman l, editors. endocrinology and metabolism.. philadelphia: mc graw hill book company; 1985. p. 105–10. 16. kimura m, nishudo i, tofani i, kojima y. effect of calcium and zink on endochondral ossification in mandibular condyle of growing rats. dent in japan 2004; 40:106–14. 17. orwoll e, ware m, stribska l, bikle d, sanchez i, anton m, hougfeng li. effect of dietary protein on mineral metabolism and bone mineral density. am j chin nutr 1992; 56:314–9. 18. likimani s, whitford gm, kunkel ne. the effect of protein deficiency and fluoride on bone mineral content of rat fibia. calcief tissue int 1992; 50(2):157–64. 19. anonim. animal requirement of laboratory animals. 3rd ed. washington dc: national academy at sciences; 1978. 20. navia jm. nutrition in dental development and disease. in: winick m, editor. nutrition pre and post natal. new york: m plenum press; 1979. p. 105–10. 21. mercoff j. association of fetal growth with maternal nutrition. in: falkner jm, tanner jm, editors. human growth, a comprehensive treatise. new york: plenum press; 1986. p. 333–78. 22. jellife db, jellife efp. nutrition and growth. 1st ed. new york: plenum press; 1979. p. 31–45. 23. bulkwater ja, cooper rr. bone structure and function. instr course lect 1987; 36:27–48. 24. franchi l, bacceti t, mc. namara j. mandibular growth as related to cervical vertebrae maturation and body height. am j orthod dentofac orthop 2000; 118(3):335–40. 25. suda n, suzuki mi, herose k, hiyama s, suzuki s, kuroda. effective treatment plan for maxillary protraction: is the bone age useful to determine the treatment plan. am j orthod dentofac orthop 2000; 118(1):55–62. 26. revelo b, fishman l. maturational evaluation of ossification of the mid palatal suture. am j orthod dentofac orthop 1994; 105(3):288– 92. 27. sato k, mito t, milani h. an accurate method of predicting mandibular growth potensial based on bone maturity. am j orthod dentofac orthop 2001; 120(3):286–90. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no 1-2005 8 perlekatan koloni streptococcus mutans pada permukaan resin komposit sinar tampak (the adherence of streptococcus mutans colony to surface visible light composite resins) ajeng anggraeni, anita yuliati, dan intan nirwana bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract visible light composite resins was used to restore anterior and posterior teeth, and it is always covered by saliva pellicle. s. mutans can adhere to all of the surface of oral cavity and visible light composite resins. the aim of this study was to know the amount of s. mutans colony adherence to visible light composite resins surface. the sample of 5 mm diameter and 3 mm in thickness was immersed in saliva for one hour, than the samples were put into bacteria suspension, incubated for 24 hours at 37° c. the amount of s. mutans was determined by direct count using microscope. the data were statistically analyzed by using t test. the result showed a significance difference of s. mutans colony between hybrid and micro fill visible light composite resins. the conclusion was that the amount of s. mutans adherence on the surface of hybrid was higher than the micro fill visible light composite resins. key words: hybrid and micro fill visible light composite resins, adherence s. mutans korespondensi (correspondence): ajeng anggraeni, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. dalam rongga mulut seseorang mengandung berbagai macam spesies bakteri yang bersifat komensal. di antara bakteri tersebut adalah streptococcus mutans (s. mutans) yang bersifat kariogenik dan merupakan penyebab utama karies gigi. salah satu ciri dari bakteri ini adalah mempunyai kemampuan menempel pada semua lokasi permukaan habitatnya dalam rongga mulut, sehingga tidak menutup kemungkinan adanya bakteri yang melekat pada permukaan restorasi resin komposit sinar tampak dalam rongga mulut.5 aktivitas perlekatan s. mutans terhadap host melalui reseptornya dalam hal ini adalah pelikel saliva, karena pelikel saliva mempunyai beberapa macam reseptor untuk perlekatan s. mutans, dikatakan juga pelikel saliva merupakan mediator tempat melekatnya bakteri rongga mulut pada permukaan gigi dan restorasi.6 dengan demikian yang menjadikan permasalahan dalam penelitian ini adalah apakah ada perbedaan jumlah koloni s. mutans yang melekat pada permukaan resin komposit sinar tampak jenis mikrofil dan hibrid. penelitian ini bertujuan untuk mengetahui perbedaan jumlah koloni s. mutans yang melekat pada permukaan resin komposit sinar tampak jenis mikrofil dan hibrid. dengan diketahuinya perbedaan jumlah koloni s. mutans yang melekat pada permukaan resin komposit sinar tampak jenis mikrofil dan hibrid, maka dapat dijadikan sebagai bahan pertimbangan dalam memilih material untuk suatu restorasi dengan harapan dapat mengurangi terjadinya karies sekunder. pendahuluan kemajuan yang sangat menonjol di bidang restorasi gigi pada saat ini ditandai dengan dikembangkannya material resin komposit yang banyak digunakan sebagai material restorasi untuk kavitas klas iii, iv, v dan kavitas klas i yang tidak menerima beban kunyah yang besar.1 berdasarkan sistim aktivasi, ada dua macam resin komposit yaitu yang beraktivasi secara kimia dan sinar tampak. saat ini, resin komposit sebagai material restorasi yang beraktivasi dengan sinar tampak sangat populer penggunaannya. keunggulan material ini mempunyai estetik yang lebih baik dibanding restorasi lain, waktu kerja dan kontur restorasi dapat diatur oleh operator dan tidak diperlukan pengadukan sehingga masalah terperangkapnya udara dalam resin komposit dapat dihindari.2 berdasarkan ukuran dan jenis material pengisi, resin komposit dibagi menjadi 3 macam yaitu resin komposit konvensional, mikrofil, dan hibrid. resin komposit konvensional mempunyai ukuran partikel pengisi paling besar yaitu 5–25 μm, sedangkan resin komposit mikrofil mempunyai ukuran partikel pengisi paling kecil yaitu 0,04–0,06 μm. resin komposit hibrid berisi campuran antara partikel pengisi besar dan kecil yaitu dengan ukuran 0,04–15 μm.3 adanya perbedaan ukuran partikel pengisi ini akan mempengaruhi kekasaran permukaan resin komposit sinar tampak.4 9anggraeni dkk: perlekatan koloni streptococcus mutans bahan dan metode jenis penelitian adalah eksperimental laboratorik. penelitian ini dilakukan di bagian mikrobiologi fakultas kedokteran gigi universitas airlangga. pelaksanaan penelitian dimulai agustus sampai september 2003. bahan yang digunakan: resin komposit sinar tampak jenis mikrofil (heliomolar, vivadent) dan hibrid (tetric ceram, vivadent), kultur s. mutans, media cair brain heart infusion (bhi), media padat tripticase yeast cystein (tyc) dan larutan phosphat buffer saline (pbs). alat yang dipergunakan: teflon bentuk silindris ukuran 5 × 3 mm,7 sonde, spatula plastik, glass slab, anak timbangan 1 kg, kaca pembesar, celluloid strip, mikropipet, cawan petri, tabung reaksi, inkubator (precision), vorteks, kuring unit sinar tampak (litex). pembuatan sampel dari resin komposit sinar tampak adalah sebagai berikut, resin komposit yang beraktivasi dengan sinar tampak yang belum mengeras disiapkan sebagai sampel. pada bagian bawah dari cetakan sampel diberi celluloid strip dan diletakkan di atas glass slab. pembuatan sampel ini dilakukan selapis demi selapis setelah cetakan sampel penuh dengan resin komposit lalu bagian atas dari sampel diberi celluloid strip, kemudian ditekan dengan glass slab dan diberi beban 1 kg. glass slab dan beban diambil, selanjutnya permukaan sampel disinari dengan sinar tampak selama 40 detik sesuai dengan aturan pabrik. jarak antara ujung tip dari alat kuring unit dengan sampel adalah 2 mm, tegak lurus pada permukaan sampel.8 jumlah sampel pada setiap kelompok 10 buah. bakteri s. mutans yang akan dipakai dalam penelitian diambil dari stok s. mutans di bagian mikrobiologi fakultas kedokteran gigi universitas airlangga dengan cara sebagai berikut, diambil satu mikroorganisme s. mutans ditanam pada media tyc agar, penanaman dilakukan pada suasana anaerob pada suhu 37° c selama 48 jam. selanjutnya s. mutans yang tumbuh pada media tyc diambil sebanyak 5 koloni dan ditanam ulang pada media bhi sebanyak 5 ml dan diinkubasi pada suhu 37° c selama 24 jam. hasil pertumbuhan s. mutans yang diperoleh sesuai dengan standart mc farlan 3 (jumlah koloni 3 × 108) kemudian dari hasil tersebut dilakukan pengenceran sebanyak 10–3 dan diambil 0,1 ml, kultur ini yang digunakan untuk penelitian. saliva dikumpulkan dari individu yang tidak mempunyai karies pada giginya. subyek disuruh meludah sebanyak 5 ml dan ditampung dalam wadah steril. saliva yang terkumpul disentrifugasi dengan kecepatan 1000 rpm selama 20 menit pada suhu 4° c, selanjutnya supernatan diambil dan disaring menggunakan filter selulose.9 pengujian sampel dilakukan dengan cara, sampel disterilkan terlebih dahulu dalam autoclave selama 30 menit pada suhu 121° c, kemudian direndam dalam saliva selama 1 jam pada suhu kamar. setelah itu, sampel diambil dan dibilas dengan larutan pbs, dimasukkan ke dalam kultur s. mutans pada media cair bhi sebanyak 3 ml lalu diinkubasi selama 24 jam pada suhu 37° c. setelah 24 jam sampel diambil dan dimasukkan kembali ke dalam media bhi steril, vorteks selama 1 menit untuk melepas s. mutans yang melekat pada permukaan sampel.9 selanjutnya media yang mengandung kultur s. mutans dilakukan pengenceran sampai 10 –4, karena pada pengenceran kurang dari 10–4 didapatkan koloni s. mutans yang sangat banyak sehingga mempersulit dalam perhitungannya. tahap berikutnya media bhi diambil sebanyak 0,1 ml dimasukkan ke media padat tyc pada cawan petri, diratakan dengan menggunakan spreader lalu diinkubasi suhu 37° c selama 2 × 24 jam. s. mutans yang tumbuh pada media padat tyc dihitung dengan cara membagi permukaan media padat tyc menjadi 8 bagian, lalu dihitung jumlah koloni s. mutans yang tumbuh pada tiap bagian tersebut. hasil dari penjumlahan pada permukaan media padat tyc merupakan jumlah seluruh koloni s. mutans dalam cawan. hasil rerata dan standart deviasi dari jumlah koloni s. mutans yang melekat pada permukaan resin komposit sinar tampak jenis hibrid dan mikrofil tampak pada tabel 1. tabel 1. rerata dan standart deviasi jumlah koloni s. mutans yang melekat pada permukaan resin komposit sinar tampak (cfu/ml) jenis resin komposit sinar tampak rerata ± standart deviasi hibrid mikrofil 196,7 ± 35,765 78,8 ± 27,624 pada perhitungan statistik uji homogenitas terlihat nilai dari lavene test adalah p = 0,222 (p > 0,05), maka kedua kelompok tersebut memiliki varians yang sama. dari kolmogorov-smirnov test didapatkan nilai p = 0,848 (p > 0,05), maka kedua kelompok tersebut mempunyai distribusi yang normal. pada tabel 1 menunjukkan bahwa nilai rerata jumlah koloni s. mutans pada permukaan resin komposit sinar tampak jenis hibrid lebih banyak daripada jenis mikrofil. hasil uji t dari jumlah koloni s. mutans pada permukaan resin komposit sinar tampak didapatkan nilai p = 0,01 (p < 0,05). hasil ini menunjukkan bahwa ada perbedaan bermakna antara jumlah koloni s. mutans yang menempel pada permukaan resin komposit sinar tampak jenis hibrid dengan mikrofil. pembahasan kekasaran permukaan resin komposit dipengaruhi oleh ukuran partikel dan kandungan material pengisi dalam komposit.10 pada komposit jenis hibrid mempunyai ukuran partikel pengisi sebesar 0,04–15 μm dengan kandungan 10 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 8–11 material pengisi sekitar 60–70%. pada komposit mikrofil mempunyai ukuran partikel pengisi paling kecil yaitu 0,04– 0,06 μm dengan kandungan material pengisi sekitar 35–50%.3,4 material pengisi resin komposit dengan ukuran yang kecil dan halus yaitu kurang dari 1μm akan memperbaiki sifat fisik terutama daya tahan terhadap abrasi dan dapat mengurangi kekasaran permukaan resin komposit.11 bila kandungan material pengisi pada resin komposit ditingkatkan jumlahnya, akan meningkatkan pula kerapuhan, kekasaran dan modulus elastik.12 adanya kekasaran permukaan inilah yang merupakan faktor retensi bagi bakteri untuk melekat pada permukaan resin komposit dan meningkatkan akumulasi plak gigi. keadaan tersebut terlihat pada hasil penelitian ini, jumlah koloni s. mutans yang melekat pada resin komposit sinar tampak jenis hibrid berbeda bermakna dengan jenis mikrofil s. mutans yang melekat pada resin komposit jenis hibrid (196,7 cfu/ml) lebih banyak daripada mikrofil (78,8 cfu/ml) seperti tampak pada tabel 1. hal ini kemungkinan disebabkan karena adanya perbedaan kekasaran permukaan komposit sinar tampak jenis mikrofil dan hibrid. ukuran partikel material pengisi komposit jenis mikrofil lebih kecil daripada hibrid, sehingga permukaan resin komposit jenis mikrofil lebih halus daripada hibrid.10 hal ini mengakibatkan tidak banyak s. mutans yang melekat pada permukaan resin komposit mikrofil. ditinjau dari jumlah kandungan material pengisi antara kedua jenis resin komposit tersebut semakin rendah kandungan material pengisi semakin halus permukaan resin komposit sehingga semakin rendah s. mutans yang melekat pada permukaan resin komposit. material restorasi dalam rongga mulut suatu resin komposit sinar tampak selalu diselimuti oleh lapisan yang disebut dengan pelikel gigi yang berasal dari saliva. lapisan pelikel ini terbentuk adanya absorbsi yang selektif dari partikel saliva yang mengandung glikoprotein saliva. pelikel inilah yang merupakan reseptor dari beberapa bakteri dalam rongga mulut termasuk s. mutans yang oleh para peneliti telah ditetapkan sebagai bakteri penyebab karies.13 s. mutans akan berikatan dengan pelikel melalui suatu adesin yang ada pada bakteri, akhirnya bakteri ini dapat berkolonisasi, berkembang biak dan menghasilkan asam. ditunjang dengan kemampuan s. mutans yang dapat melekat pada berbagai macam permukaan pada rongga mulut, misalnya pada daerah fisur permukaan gigi dan permukaan bahan restorasi yang kasar.14 peneliti lain juga menyimpulkan bahwa pelikel saliva secara signifikan berperan pada permulaan perlekatan bakteri streptokokus rongga mulut pada breket ortodontik.15 mekanisme perlekatan bakteri tersebut ada dua tahap. pada tahap pertama bakteri melekat pada suatu permukaan di dalam rongga mulut dengan perantara pelikel. tahap kedua bakteri tersebut berkembang biak sehingga pelikel berubah membentuk plak.13 pelikel gigi merupakan mediator melekatnya bakteri rongga mulut pada permukaan restorasi. 6 proses perlekatannya dimulai dari adanya interaksi antara bakteri dengan pelikel. mekanisme interaksi tersebut dipengaruhi oleh kekuatan elektrostatik, hidrofobik, komponen organik dan multiple binding sites. bakteri yang melekat pada permukaan bahan restorasi karena adanya interaksi elektrostatik atau melalui calcium bridging, yaitu ion ca2+ dalam saliva akan menjembatani dan mengikat permukaan sel bakteri dan pelikel gigi yang bermuatan negatif. interaksi hidrofobik didasari oleh kontak yang rapat antara molekul pada pelikel dengan permukaan bakteri. komponen organik s. mutans dengan mempergunakan enzim glycosyltransferase (gtf) dan non-enzym glucanbinding protein untuk mensintesis polisakarida ekstraseluler dan membentuk suatu glukan yang bersifat lengket. glukan merupakan tempat perlekatan, sehingga keduanya dapat membantu perlekatan s. mutans pada permukaan gigi, sedangkan perlekatan bakteri melalui multiple binding site karena adanya interaksi lectinlike, yaitu protein yang terdapat pada permukaan bakteri s. mutans akan bereaksi dengan high molecular weight salivary glycoproteins dan mengadsorbsi hidroksiapatit enamel sehingga terjadi interaksi antara bakteri dengan pelikel gigi.13 satou16 berpendapat, keberadaan koloni s. mutans memperlihatkan korelasi yang positif dengan zeta-potential material restorasi. hal ini menandakan bahwa mekanisme interaksi elektrostatik adalah hal yang penting dalam perlekatan suatu bakteri. sudut kontak dan zeta-potential perlekatan sel bakteri pada permukaan keramik porselen, resin komposit dan enamel gigi manusia yang dilapisi saliva lebih kecil daripada yang tidak dilapisi saliva.17 peneliti lain juga berpendapat tentang mekanisme perlekatan s. mutans pada pelikel yang tergantung adanya sukrose dalam rongga mulut. perlekatan s. mutans yang tidak disertai adanya substrat sukrose, masih tetap dapat berlangsung, walaupun tidak sempurna, seperti bila terdapat sukrose. terdapatnya sukrose menyebabkan perlekatan s. mutans dengan pelikel bersifat irreversible, karena terjadi kohesi antar s. mutans sehingga memudahkan terjadinya agregasi dari s. mutans lainnya.5 pendapat peneliti lain, dianjurkan memberi glazing agent untuk mengurangi kekasaran permukaan suatu restorasi resin komposit. glazing agent terdiri dari bis-gma dilute solution yang merupakan bagian dari matriks resin dari komposit itu sendiri.1 karena dengan permukaan yang halus dapat meminimalkan terbentuknya akumulasi plak, iritasi ginggiva, estetik yang jelek dan perubahan warna.10 dengan demikian permukaan restorasi resin komposit yang halus akan mengurangi perlekatan koloni s. mutans atau bakteri lain yang ada di dalam rongga mulut, sehingga mengurangi insiden terjadinya karies sekunder.18 karena terjadinya proses perlekatan pada permukaan host merupakan langkah awal terjadinya patogenesis dari bakteri ini yang menyebabkan terjadinya karies gigi. kesimpulan dari hasil penelitian jumlah koloni s. mutans yang menempel pada permukaan resin komposit sinar tampak jenis hibrid lebih banyak daripada jenis mikrofil. 11anggraeni dkk: perlekatan koloni streptococcus mutans daftar pustaka 1. craig rg, powers jm. restorative dental material. 11st ed. st louis, london, philadelphia, sydney, toronto: mosby a harcourt health sciences company; 2002. p. 232–51. 2. combe eg. notes on dental material. 6th ed. edinburgh, london, madrid, melbourne, new york, tokyo: churchill livingstone; 1992. p. 89–95. 3. anusavice kj. phillips’ science of dental material. 11th ed. usa: elsevier science; 2003. p. 399–441. 4. mc cabe jf, walls awg. applied dental materials. 8th ed. united kingdom: the blackwell science ltd; 2000. p. 169–88. 5. tanzer jm. microbiology of dental caries. in: contemporary oral microbiology and immunology. st louis, missouri: mosby year book; 1992. p. 377–422. 6. edgerton m, levine ml. biocompatibility: its future in prosthodontis research. j prosthet dent 1993; 69: 406–15. 7. sharifah farihah, asti meizarini, anita yuliati. variasi ketebalan celluloid strip terhadap kekerasan permukaan resin komposit sinar tampak. majalah kedokteran gigi 2001; 34(4): 753–5. 8. sturdevant cm, roberson tm, heymann ho, sturdevant jr. the art and science of operative dentistry. 3rd ed. st. louis, baltimore, berlin, boston, chicago, london, new york, philadelphia, sydney, tokyo, toronto: mosby company; 1995. p. 253–576. 9. rostiny. perbedaan proses kuring lempeng resin akrilik heat cured terhadap kekerasan permukaan dan perlekatan koloni streptococcus mutans. majalah kedokteran gigi 2003; 36(3): 102–5. 10. filho hn, d’azevedo mtfs, nagem hd, marsola fp. surface roughness of composite resins after finishing and polishing. braz dent j 2003; 14: 37–41. 11. jacobsen ph. the current status of composite restoration materials. brit dent j 1981; 140: 167–73. 12. ferracane jl, berge hx, condon jr. in vitro aging of dental composites in water-effect of degree of conversion, filler volume, and filler matrix coupling. biomed mater res j 1998; 42: 465–72. 13. nisengard rj and newman mg. oral microbiology and imunologi. 2nd ed. united states of america: wb saunders co; 1994. p. 320–5. 14. smith dj. ontogeny of immune mechanism in oral cavity. in contemporary oral microbiology and immunology. st louis, missouri: mosby year book; 1992. p. 513–23. 15. ahn sj, kho hs, lee sw, nahm ds. roles of salivary proteins in the adherence of oral streptocci to various orthodontic brackets. j dent res 2002; 6: 411–5. 16. satou j, fukunaga a, satou n, shintani h, okuda k. streptococcal adherence on various retorative materials. j dent res 1988; 67: 588–91. 17. yoshida y, wakasa k, kajie y, takahashi h, urabe h, satou n, shintani h, yamaki m. adherent bacteria cells in five dental materials: sonication effect. journal of material science materials in medicine 1998; 6: 117–20. 18. pedrini d, gaetti-jardim junior e, vasconceloss ac. retention of oral microorganisms on conventional and resin-modified glassionomer cements. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 89 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 89 expression analysis of cd63 in salivary neutrophils and the increased level of streptococcus mutans in severe early childhood caries muhammad luthfi department of oral biology faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: severe early childhood caries (s-ecc) and decay exfoliation filling teeth (def-t) >6 is a destructive disease that afflicts teeth, including maxillary anterior teeth. in indonesia, the prevalence of this disease is still high, for instance in semarang 2007, the rate reached 90.5% in urban areas and 95.9% in rural areas for early childhood caries which is caused by streptococcus mutans (s. mutans). neutrophils are effector cells of innate immunity which become the main component of the very first line of defense against microbes. purpose: this study analyzed the effect caused by the change of cd63 expression on the surface of salivary neutrophils and the increased level of s. mutans in s-ecc. method: this study employs observational analytic and cross sectional approach by using t test analysis technique for forty cases of early childhood that had been divided into two groups, first group of twenty children positively diagnosed as s-ecc and second group of twenty children negatively diagnosed as the control group. the sample’s result of gargling with 1.5% nacl was used for neutrophils isolation and analysis function of salivary neutrophils phagocytosis by using flow cytometry test, while the sample of saliva was used to isolate s. mutans and calculate the level of s. mutans. result: the expression of cd63+ salivary neutrophils in s-ecc was lower (2.32% ± 0.57) than in caries-free (2.67% ± 0.46), while the level of s. mutans showed that the level was not higher than in s-ecc (9.78 ± 2.22)x105 cfu/ml compared to in caries-free (5.13 ± 1.86)x105 cfu/ml. conclusion: the low expression of cd63 in salivary neutrophils can lead to the increased level of s. mutans in s-ecc. keywords: salivary neutrophils; streptococcus mutans; s-ecc correspondence: muhammad luthfi, c/o: departemen biologi oral, fakultas kedokteran gigi universitas airlangga. jl. mayjen prof. dr. moestopo 47 surabaya 60132, indonesia. e-mail: m.luthfi7@yahoo.com introduction early childhood caries (ecc) is caries experienced by younger children and is a serious problem in all over the world, particularly in developing countries.1 the prevalence of dental caries occurred to children from minority ethnics in china is very high, such as the prevalence in zhuang, bonan, dai, dongxiang, korea, tibet.2 if ecc is not treated seriously, it can thrive and cause dental caries on the entire teeth in a short period of time which is known as severe early childhood caries (s-ecc), it will affect the physical and mental health3 and it will increase the risk for the subsequent caries on permanent teeth4 that will not be able to handle by a mere restorative treatment. the classical etiology of ecc involves bacteria, diet, and host affected by the interaction of sociological and environmental factors, while the existence of cariogenic microbes, the frequency of consuming foods and drinks, oral hygiene, educational level of parents, family income, knowledge of oral health and the child’s behavior is proven to be the main cause in ecc.4 in the recent years, the role of neutrophils has changed dramatically in which neutrophils become the main component of the first line of defense against microbes.4 research report dental journal (majalah kedokteran gigi) 2015 june; 48(2): 89–93 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 90 luthfi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 89–93 neutrophils do not only act as microbes exterminator with phagocytosis, releasing reactive oxygen species (ros) and antimicrobial peptides but also as a regulator to the activation of immune response.5 neutrophils also evidently produce cytokine, chemokine and growth factor until become the main contributor in the pro-inflammatory cytokine production on the infected areas.6 the important function of neutrophils in exterminating microbial pathogens is phagocytosis, which significantly more effective due to the opsonization process by antibody and complement on the microbial surface. phagocytosis on microbes can generate oxidative burst process to produce reactive oxygen species (ros) with degranulation of cytoplasmic granules in phagosome contains antimicrobial peptides and proteases comprised microbes.7 azurophilic granule (primary granule) contains antimicrobial proteins such as defensin, elastase, cathepsin dan proteinase-3 and also contains cd63 in its membrane. streptococcus mutans (s. mutans), the bacteria that cause caries, can activate neutrophils host until it produces antimicrobial peptide (amps) in the form of human neutrophil peptide (hnp) 1-3. besides functioning as antimicrobial, it also acts as chemoattractant and immunomodulatory. hnp 1-3 amps function as natural antibiotics which give the first line of defense with wide spectrum of various bacteria.8 various preventions of dental caries have been done, for example by brushing the teeth properly, fluoridation by topical application, and vaccines manufacturing that still has not shown any expected results until today.9 therefore, this study was aimed to analyze the change of expression of cd63 salivary neutrophils as the effector cells of innate immunity towards the increased level of s. mutans in s-ecc. materials and methods the sample of this study was obtained from saliva and gargling result with 1.5% nacl of kindergarten children aged 4 to 6 years old in surabaya. examination of dental caries was done in advance by measuring the def-t index, and then the subjects were divided into two groups: cariesfree group and s-ecc group with def-t index higher than 6. before the sample was taken, the questionnaire were distributed and the inform consent were signed by the parents respectively. the sample was taken from the saliva without stimulation as much as 2 ml by using expectorate within the falcon tube 5 ml during school hours between 08.00 up to 10.00 a.m. to determine the level of s. mutans and 5 minutes later the children were instructed to gargle with 1.5% nacl which then accommodated in the 50 ml falcon tube to determine the expression of cd63 on the surface of salivary neutrophils. the sampling was done by the researcher and the trained personnel using standard protocol. the subjects of this study were not allowed to eat, drink, chew gum, or brush their teeth for 60 minutes before the sampling. after the sample was collected, it was frozen at -80°c to be analyzed.10 s. mutans isolation was done by taking saliva sample from preschool children identified either as severe caries (def-t >6) or caries-free performed in the following instructions: biochemical isolation and characterization from s. mutans. saliva sample then were diluted in brain heart broth (bhi), after incubated for 24 hours, sample was planted on gelatin medium triptone yeast cystein (tyc). the colonies assumed as s. mutans then were sub-cultured to be biochemically tested by using mannitol fermentation, raffinose, sorbitol, salicin, esculin and arginine. isolates were identified as s. mutans when it is positive to mannitol fermentation, raffinose, sorbitol, salicin, esculin and is negative to arginine and subsequently is confirmed by gram staining and negative catalase test. isolates of s. mutans were stored at -80°c.10 based on its morphology, all colonies of s. mutans on gelatin tyc were calculated using the formula: number of colonies x dilution factor x 50 (1 ml volume) = cfu/ml with minimum detection level 1 x 103 cfu/ml. profile measurement of neutrophil cells employed cd63 antibody which marks the active neutrophils. cd63 within primary granule membrane were expressed on the surface of neutrophils membrane because of azurophilic granule fusion and plasma membrane which increased due to the stimuli given to neutrophil cells in which the signs can be measured with flow cytometry using the method modified by bjornsson.11 the sample used was the result of gargling with 1.5% nacl which its neutrophils had been isolated. the suspension of isolated neutrophil cells then was inserted into microtube filled by 500µl pbs. the cell suspension was then centrifuged at 2500rpm speed, for 5 minutes at 4° c temperature. the pellets obtained were subsequently stained with extracellular antibodies 50µl (biolegend antihuman α-cd63pe), and biolegend α-pi pe conjugated with antibody ratio: pbs is 1:200. cells that have been added with antibody were then stored at 4° c temperature for 30 minutes. the suspension cells were then added with 1ml pbs and were centrifuged at 2500rpm speed, for 5 minutes at 4° c temperature. biolegend cytofix cytoperm was then added to the pellets as much as 100µl and was homogenized until it well blended. the incubation was done subsequently at 4° c temperature without light for 20 minutes. after incubation, cells were then added with 1ml biolegend washperm once and subsequently were centrifuged at 2500 rpm speed, for 5 minutes at 4°c temperature. the obtained pellets were then coupled with intracellular antibodies, including bd antihuman α-cd64 percp conjugated, and then suspension cells were inserted into cuvette flow cytometer, and then added with pbs as much as 300µl, and mounted on nozzle bd facs calibur to do running with flow cytometer machine. the sample was then analyzed by flow cytometry (facs calibur flow cytometry, bd bio sciences, san jose, ca). 91 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 91luthfi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 89–93 neutrophil gate was identified by density and size with side angle light scatter and then continued with forward angle light scatter. this compensation was achieved by employing fitc and pe labeled with individual antibodies. the result was shown as mean fluorescence intensity (mfi). facs calibur of becton dickinson with cell quest software program was used for the analysis. results the result of s. mutans number calculation from secc group and caries-free group using colony counter and the result of activated salivary neutrophils (cd64+) analysis showed that cd63+ was expressed in ecc-free and s-ecc. the calculation result of the number of s. mutans saliva from triptone yeast cystein gelatin medium using colony counter tested by t 2 independent samples showed the significance value smaller than α. this means that there were significant differences in the s. mutans numbers between the two groups. based on the mean value, it was known that the number of s. mutans in caries-free children (5.14 x105 ±1.86 x 105 cfu/ml) was significantly lower than in children with severe caries (9.78 x 105 ±2.23 x 105 cfu /ml) (table 1 and figure 1). the result of analysis using flow cytometry activated salivary neutrophils that express cd63+ after given comparative test using t 2, independent sample showed that the significant value was lower than α. this means that there was a significant difference in cd63+ expression between the two groups. based on the mean value, it was confirmed that salivary neutrophils that expresses cd63+ in ecc-free was higher (2.67% ± 0.46) than in s-ecc (2.32% ± 0.57) (table 2 and figure 2). discussion ecc is a multifactorial disease that occurs as a result of a series of interactions between vulnerable hosts, cariogenic bacteria, cariogenic diet and behavior. dental caries is not caused by exogenous bacteria, but is caused by the irregularities in ecology so that commensal oral bacteria become pathogenic after the disruption of the immune system and homeostasis of the body which later develop into dental caries. the important role in the homeostasis of the oral cavity and the prevention of dental caries depends on the content of immune component in saliva.12 s. mutans have integral role as the etiology regarding to the occurrence of ecc which is an infectious and contagious disease,13 so that s. mutans is considered an important predictor as cariogenic bacteria because it is acidogenic (able to produce acid) and aciduric (able to survive in acidic environment).14 in saliva, neutrophils are the most prominent first line of defense from the immune cells for defense against microbial pathogens. the importance of neutrophils in host immune system in patients with neutropenia or defect in neutrophils function leads to the tendency for serious infection to happen.15 neutrophils recruitment process, transmigration, phagocytosis, and activation are highly coordinated to prevent or eliminate infection in human. in the infected area, neutrophils bind and engulf microbes through a process known as phagocytosis. neutrophils recognize table 1. the mean and standard deviation of the number of s. mutans in saliva calculated by colony counter in s-ecc and cariesfree (105 cfu/ml) groups n mean ± standard deviation 95% ci p value caries-free 20 95.13 ± 1.86 394.789,36–632.210,64 p < 0.0001 s-ecc 20 9.78 ± 2.22 834.661,22 –1.119.338,78 (p < α) table 2. the mean and standard deviation of activated salivary neutrophils (cd64+) that express cd63+ in ecc-free and s-ecc (%) groups n mean ± standard deviation 95% ci p value caries-free 20 2.67±0.46 2.37 – 2.96 p < 0.040 s-ecc 20 2.32±0.57 1.96 – 2.68 (p < α) 12 1,200,000.00 1,000,000.00 800,000.00 600,000.00 400,000.00 200,000.00 0.00 free caries s-ecc figure 1. the mean and standard deviation of s. mutans numbers on saliva calculated by colony counter in s-ecc and caries-free (cfu/ml). 513500 t he m ea n of s . m ut an s nu m be rs (c fu /m l) 977000 figure 1. the mean and standard deviation of s. mutans numbers on saliva calculated by colony counter in s-ecc and caries-free (105 cfu/ml). 12 10 8 6 4 2 0 9.8 5.14 t he m ea n of s . m u ta n s nu m be rs ( 10 5 c f u /m l) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 92 luthfi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 89–93 the bound-surface or free molecule secreted by bacteria, including peptidoglycan, lipoprotein, lipoteichoic acid (lta), lipopolysaccharide (lps), cpg containing dna, and flagellin. this pathogenic molecule is known as pathogen associated molecular pattern (pamps), interacts directly to a number of pathogen recognition receptors (prrs) which is expressed on the surface of cells, including toll like receptors (tlrs).16 s. mutans as the main etiology agent of ecc because it has several mechanisms to colonize on the tooth surface and under particular condition to be cariogenic species which signifies the highest within the biofilm environment of oral cavity;17 thus, indicating the existence of causal link between dental caries and the high number of s. mutans. several studies suggested that the development of dental caries is preceded by an increase in colonization of s. mutans,12 whereas the other researchers said that the increased level of s. mutans in saliva is an indication of the increased risk of dental caries.18,19 phagocytosis is a process which is mediated by active receptor, the internalization of cell to the microbes and is subsequently followed by the rearrangement of cytoskeletal, the enlargement of neutrophil plasma membrane around the microbes and the formation of membrane-bound vacuoles called phagosome. in phagosome neutrophils release a variety of antimicrobial proteins and intracellular enzymes that function to kill microbes. primary granules (azurophilic) contains many antimicrobial compounds, such as myeloperoxidase (mpo), defensin like human neutrophil peptide 1-3 (hnp 1-3), lysozyme, azurocidin, and serine proteinase elastase, cathepsin g, proteinase 3, esterase n. azurophilic granules are the one that associated with phagocytic vesicles which then release the content in phagosome which contains phagocytized microbes.20 neutrophil proteins in the primary granules (azurophilic) are alarmins which is a molecule that can activate antigen precenting cells (apc) and stimulate innate dan adaptive immunity responses.21 based on the results of this study (table 1) of salivary neutrophils suggested that the expression level of cd63+ in s-ecc was lower than the expression level of cd63+ in caries-free children with the average value in s-ecc is lower (2.32% ± 0.57) than the expression level of cd63+ in caries-free children (2.67% ± 0.46). there is a chance of the low expression level of cd63 in salivary neutrophils in s-ecc is caused by s. mutans which have been internalized by neutrophils through a phagocytosis process that mediated through fcαr (cd89) or cr1 (cd35) may be able to develop three strategies of defense system to avoid intracellular killing, firstly, escaping out of phagosome, secondly, blocking the fusion of phagosome-lisosome, and thirdly, using a mechanism that allows survival in phagolysosomes. there is also a chance of the low expression level of cd63 in salivary neutrophils in s-ecc is caused by the deficiency of proteins elastase and cathepsin g.22 less active neutrophils will release fewer neutrophil extracellular traps (nets) that work to kill extracellular microbes because it contains lactoferrin, cathepsin and enzymes which are highly toxic for microbes. in addition, nets also facilitate the phagocytosis process.23,24 s. mutans level in s-ecc is higher than s. mutans level in caries-free maybe because of the pathogenic s. mutans is not optimal by removed. it can be concluded that the low expression of cd63 in salivary neutrophils can lead to cause the increased level of s. mutans in s-ecc. references 1. bagramian ra, garcia-godoy f, volpe ar. the global increase in dental caries. a pending public health crisis. am j dent 2009; 22(1): 3–8. 2. zhang s, liu j, lo ec, chu ch. dental caries status of dai preschool children in yunnan province, china. bmc oral health 2013; 13: 68. 3. isaksson h, alm a, koch g, birkhed d, wendt lk. caries prevalence in swedish 20-year-olds in relation to their previous caries experience. caries res 2013; 47(3): 234–42. 4. wigen ti, wang nj. caries and background factors in norwegian and immigrant 5-year-old children. community dent oral epidemiol 2010; 38(1): 19–28. 5. nathan c. neutrophils and immunity: challenges and opportunities. nat review immunol 2006; 6(3): 173-82. 6. mantovani a, cassatella ma, costantini c, jaillon s. neutrophils in the activation and regulation of innate and adaptive immunity. nat rev immunol 2011; 11(8): 519-31. 7. kobayashi sd, voyich jm, burlak c, deleo fr. neutrophils in the innate immune response. arch immunol ther exp. 2005; 53(6): 505-17. 13 3 2.5 2 1.5 1 0.5 0 free caries s-ecc 2.67 2.32 figure 2 . the mean and standard deviation of activated salivary neutrophils (cd64+) that express cd63+ in ecc-free and s-ecc (%). a b figure 3. activated salivary neutrophils (cd64+) that expresses cd63+ detected by flow cytometry in ecc-frees (a) and in s-ecc (b). b. 2.32% a. 2.67% t he m ea n of c d 64 -c d 63 figure 2. the mean and standard deviation of activated salivary neutrophils (cd64+) that express cd63+ in ecc-free and s-ecc (%). 13 3 2.5 2 1.5 1 0.5 0 free caries s-ecc 2.67 2.32 figure 2 . the mean and standard deviation of activated salivary neutrophils (cd64+) that express cd63+ in ecc-free and s-ecc (%). a b figure 3. activated salivary neutrophils (cd64+) that expresses cd63+ detected by flow cytometry in ecc-frees (a) and in s-ecc (b). b. 2.32% a. 2.67% t he m ea n of c d 64 -c d 63 figure 3. activated salivary neutrophils (cd64+) that expresses cd63+ detected by flow cytometry in ecc-frees (a) and in s-ecc (b). 93 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 93luthfi/dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 89–93 8. yang d, biragyn a, hoover dm, lubkowski j, oppenheim jj. multiple roles of antimicrobial defensins, cathelicidins, and eosinophilderived neurotoxin in host defense. annu rev immunol 2004; 22: 181–215. 9. trigg te, wright pj, armour af, williamson pe, junaidi a, martin gb, doyle ag, walsh j. use of a gnrh analogue implant to produce reversible longterm suppression of reproductive function in male and female domestic dogs. j reprod fertil suppl. 2001; 57: 25561. 10. phattarataratip, e. the role of salivary antimicrobial peptides in shaping streptococcus mutans ecology. annu rev microbiol 2010; 57: 677-701. 11. borjesson dl, kobayashi sd, whitney ar, voyich jm, argue cm, deleo fr. insights into pathogen immune evasion mechanisms: a naplasma phagocy toph i lu m fa i ls to i nduce a n apoptosis differentiation program in human neutrophils. j immunol 2005; 174(10): 6364–72. 12. tanzer jm, livingston j, thompson am. the microbiology of primary dental caries in humans. j dent educ 2001; 65(10): 102837. 13. thakur as, acharya s, singhal d, rewal n, mahajan n, kotwal b. a comparative study of mutans streptococci and lactobacilli in mothers and children with early childhood caries (ecc), severe early childhood caries (s-ecc) and caries free group in a low income population. oral health dent manag 2014; 13(4): 1091-5. 14. tinanoff n, reisine s. update on early childhood caries since the surgeon general’s report. acad pediatr 2009; 9(6): 396-403. 15. rosenzweig sd, holland sm. phagocyte immunodeficiencies and their infections. j allergy clin immunol 2004; 113(4): 620–6. 16. akira s, takeda k. toll-like receptor signalling. nat rev immunol 2004; 4(7): 499–511. 17. ramamurthy ph, swamy hs, bennete f, rohini m, nagarathnamma t. relationship between severe-early childhood caries, salivary mutans streptococci, and lactobacilli in preschool children on low socioeconomic status in bengaluru city. j indian soc pedod prev dent 2014; 32(1): 44-7. 18. beighton d. the complex oral microflora of high-risk individuals and groups and its role in the caries process. community dent oral epidemiol 2005; 33(4): 248–55. 19. corby pm, lyons-weiler j, bretz wa, hart tc, aas ja, boumenna t, goss j, corby al, junior hm, weyant rj, paster bj. microbial risk indicators of early childhood caries. j clin microbiol. 2005; 43(11): 5753–9. 20. skubitz km. neutrophilic leukocytes. in: greer jp, eds. wintrobe’s clinical hematology. 12th ed. philadelphia: lippincott williams and wilkins; 2009. p. 170-213. 21. kobayashi sd, deleo fr. role of neutrophils in innate immunity: a systems biology-level approach. wiley interdiscip rev syst biol med 2009; 1(3): 309–33. 22. urban cf, lourido s, zychlinsky a. how do microbes evade neutrophil killing?. cell microbiol 2006); 8(11): 1687–96. 23. phillipson m, kubes p. the neutrophil in vascular inflammation. nat med 2011; 17(11): 1381–90. 24. papayannopoulos v, zychlinsky a. nets: a new strategy for using old weapons. trends immunol 2009; 30(11): 513–21. guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 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(008/01.12/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 44 number 2 june 2011 issn 1978 3728 1. the effectivity of toothpick tooth brushing method on plaque control chiquita prahasanti, iwan ruhadi, and agus sobar mulyana ................................................... 59–62 2. effects of different saliva ph on hybrid composite resin surface roughness nirawati pribadi and adioro soetojo ............................................................................................ 63–66 3. endothelial cell cultured on ha/tcp/chitosan scaffold for bone tissue engineering bachtiar ew, amir lr, abbas b, and utami s ............................................................................ 67–71 4. elderly nutritional status effection salivary anticandidal capacity against candida albicans ria puspitawati, nurtami soedarsono, elisabeth a putri, anissha d putri, and boy m bachtiar ................................................................................................................................ 72–76 5. saliva as a future potential predictor for various periodontal diseases zahreni-hamzah .............................................................................................................................. 77–81 6. relieving idiopathic dental pain without drugs haryono utomo and m. rulianto ................................................................................................... 82–87 7. effectivity of 0.15% benzydamine on radiation-induced oral mucositis in nasopharynx carcinoma remita adya prasetyo ..................................................................................................................... 88–92 8. constraints on the performance of school-based dental program in yogyakarta, indonesia: a qualitative study rosa amalia, niken widyanti, johan w. groothoff, and rob m.h. schaub ........................... 93–100 9. orthodontic treatment with skeletal anchorage system arya brahmanta and jusuf sjamsudin .......................................................................................... 101–105 10. the activity of stichopus hermanii extract on triglyceride serum level in periodontitis rima parwati sari and syamsulina revianti ................................................................................. 106–110 11. effect of soybean extract after tooth extraction on osteoblast numbers rosa sharon suhono, coen pramono, and djodi asmara ........................................................... 111–116 dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. editor 105105 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 105–109 research report the role of cox-2, caspase-1 and il-17 in pericoronitis-related inflammation due to lower third molar impaction adi prayitno department of dentistry faculty of medicine, universitas sebelas maret surakarta – indonesia abstract background: inflammation of the pericorona due to lower third molar impaction (ltmi) is often diagnosed as pericoronitis. expression of cyclooxigenase-2 (cox-2) and caspase-1 may be induced by lipopolysacharide (lps) and cause pyroptosis with minimal inflammation. when lps activates toll-like receptor (tlr-4), nod-like receptors containing domain pyrin 3 (nlrp3) inflammasome will activate the release of pro-caspase-1 to caspase-1, followed by the secretion of interleukin (il)-1β. il-1β and il-23 which induces cd4+ tcells (th17) to produce il-17 as a pro-inflammation cytokine. purpose: this study aimed to identify the respective roles of cox2, caspase-1 and il-17 in pericoronitis inflammation of the pericorona due to ltmi. methods: frozen section samples were produced through ltmi pericorona tissue biopsy using material provided by the dental and oral clinic at muwardi hospital, surakarta. the paraffin block produced was subsequently cut using a clean microtome with the resulting thin slices being placed on an object glass coated with polylysine. a diagnosis of pericoronitis was subsequently made by a pathologist. immunohistochemical staining for cox-2, caspase-1 and il-17 was carried out by indirect tyramide signal amplification (tsa) method. photos were obtained by means of 100x, 200x, 400x and 1000x objective lensed microscopes to qualitatively assess the above mentioned protein expressions. t-test was conducted in order to establish the difference in expression between the control group and pericoronitis due to ltmi. results: the presence of a brownish yellow color indicated the expression of cox-2, caspase-1 and il-17 in pericorona epithelial cells which visible expression categorized as moderate (30-70%). the mean expression of cox-2, caspase-1 and il-17 was categorized as mild and there was no significant difference between the expression of the three proteins. conclusion: cox-2, caspase-1 and il-17 play an important role in the phyroptosis signal of ltmi pericoronitis in cases of low inflammation. keywords: caspase-1; cox-2, il-17; inflammation; pericoronitis correspondence: adi prayitno, department of dentistry, faculty of medicine, universitas sebelas maret. jl. ir. sutami 36a, surakarta 57126, indonesia. e-mail: adiprayitno@staff.uns.ac.id. introduction severe inflammation of the gums can result in damage to the soft tissue around the teeth.1,2 pericoronitis is a polymicrobial infection of the soft tissues surrrounding the crown of a partially or incompletely erupted tooth. pericoronitis generally occurs due to third molar impaction. the prevalence of pericoronitis, which constitutes an inflammatory process, is estimated to be between 8% and 59%.3,4 consequently, the condition needs to be appropriately treated immediately .5 during pericoronitis, the inflammatory process is initiated by competent immuno cells present in tissues throughout the body. several pro-inflammatory mediators, including: interleukin (il), tumor necrosis factor and tumor growth factor, have been reported as forming part of the pericoronitis mechanism,6–8 these mediators also comprise cyclooxigenase (cox)-2, caspase-1 and il-17. these cox-2 and endogenous and exogenous prostaglandin-2 (pge-2) molecules are expressed by macrophage cells and triggered by the presence of lipopolysacarida (lps) on bacterial cell surfaces. it has dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p105–109 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p105-109 106 adi prayitno/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 105–109 been stated that the stimulating effects of endogenous and exogenous pge-2 leads to the camp-pka-akapdependent pathway resulting in the suppression of nuclear factor kappa beta (nf-κβ) expression, thereby continuing to suppress cox-2 expression.9 in a depressed state, due the presence of pathogenic bacteria (lps), cells will program their own death by expressing caspase.10 caspases are expressed by both immune and nonimmune cells and function as inactive zymogens consisting of the carboxy effector-terminal protease domain and the pro-domain amino-terminal referred to as the caspaseassociated recruitment domain (card).11 caspase-1 actively converts pro il-1β and pro il-18 into its active form and initiates an inflammatory response. at the same time, gasdermin d will become active and lead to pyroptosis.12–14 when lps binds to toll-like receptor-4 (tlr-4), atp signaling will continue in the nod receptor protein and be forwarded to an inflammasome containing pyrin domain 3 (nlpr-3). with active nlpr-3, caspase-1 will be released and change to caspase-1. furthermore, pro il-1β will be changed to il-1β by caspase-1. in addition, il-23 will also be expressed, subsequently functioning as a paracrine for th-17 cells in the production of il-17.12–14 il-17 is an important cytokine which is known to regulate various immunocompetent cells such as macrophages, neutrophils and/or epithelial cells during several pathological processes.15 il-17a, il-17c and il17f also play a role in triggering tissue repair and epithelial cell responses to extracellular bacteria.16 in another study, it was found that il-17 responses would have implications for inflammatory events and cause tissue damage.17,18 the aim of this study is to identify the correlations of cox-2, caspase-1 and il-17 to the role of inflammation in pericoronitis resulting from lower third molar impaction (ltmi). materials and methods ethical clearance was issued by the ethical commission of research, faculty of medicine, universitas sebelas maret, muwardi hospital, surakarta (no. ec-98/viii/2008). frozen sections were manufactured during a ltmi pericorona tissue biopsy performed at the dental and oral clinic of muwardi hospital, surakarta. the paraffin block produced was then cut using a clean microtome machine. thin slices were placed on object glass previously coated with polylysine. a diagnosis of pericoronitis was arrived at by a pathologist. the immunohistochemical stain for cox-2, caspase-1 and il-17 with monoclonal antibody (santa cruz biotech, amersham pharmacia biotech) at 1:500 was carried out by indirect tyramide signal amplification (tsa) method (nen life science products, renaissence).19,20 by using 100x, 200x, 400x and 1000x objective lensed microscopes (nikkon), photos were obtained to qualitatively assess the abovementioned protein-protein expressions. t-test was conducted in order to establish the difference in expression between the control group and pericoronitis due to ltmi. results figure 1 shows the immunohistochemical staining using anti-cox-2, caspase-1 and il-17 monoclonal antibodies. a brownish yellow color indicates the expression of cox-2, caspase-1 and il-17 in pericorona epithelial cells (arrows). visible expression is categorized as moderate (30-70%). table 1 contains the data relating to imunohistochemical staining which shows the expression of cox-2, caspase-1 and il-17 in pericorona epithelial cells as the percentage of the power of expression (positive cells expressed from 40x 100x 400x 1000x cox-2 caspase-1 il-17 figure 1. comparison of the expression of cox-2, caspase-1 and il-17-produced pericorona epithelial cells. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p105–109 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p105-109 107adi prayitno/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 105–109 one view as 100 cells). visible expression is categorized as moderate (30-70%). the data analyzed with a pair t-test is shown in table 2–5. from this analysis, it can be concluded that the expression of cox-2, caspase-1 and il-17 proteins are difference beetwen control/healthy and pericoronitis. and the expression of cox-2, caspase-1 and il-17 as a pro-inflammation cytokine was low (30%-70%) due to cox-2–pge-2 feedback system through suppressed nf-κβ proteins (as a central integritor for proteins expression). discussion inflammation is stimulated by chemical mediators released by injured cells for the purpose of blocking the spread of infection and initiating healing of damaged tissue. inflammation is strictly regulated by the body where inadequate inflammatory processes can cause damage or persistent infection, while excessive inflammation potentially results in chronic or systemic inflammatory disease.21–24 table 1. comparison of the expression of cox-2, caspase-1 and il-17-produced pericorona epithelial cells expression (%) cox-2 38.94 casp-1 41.05 il-17 33.68 table 5. paired sample t-test of cox-2–caspase1, cox-2–il-17 and caspase1–il-17 paired differences t df sig. (2-tailed)mean std. deviation std. error mean 95% confidence interval of the difference lower upper pair 1 cox-2–casp-1 -.21053 1.35724 .31137 -.86470 .44364 -.676 18 .508 pair 2 cox-2–il-17 .42105 1.16980 .26837 -.14277 .98488 1.569 18 .134 pair 3 casp-1–il-17 .63158 1.01163 .23208 .14399 1.11917 2.721 18 .014 table 3. paired samples statistics mean n std. deviation std. error mean pair 1 cox-2 25.0000 38 15.46574 2.50887 pair 2 casp-1 26.3158 38 16.13642 2.61767 pair 3 il-17 22.6316 38 13.54357 2.19706 table 2. paired samples correlations n correlation sig. pair 1 cox-2 38 .914 .000 pair 2 casp-1 38 .926 .000 pair 3 il-17 38 .906 .000 table 4. paired sample test paired differences t df sig. (2-tailed)mean std. deviation std. error mean 95% confidence interval of the difference lower upper pair 1 cox-2 23.50000 15.00405 2.43398 -28.43171 -18.56829 -9.655 37 .000 pair 2 casp-1 24.81579 15.66862 2.54179 -29.96594 -19.66564 -9.763 37 .000 pair 3 il-17 21.13158 13.08635 2.12289 -25.43295 -16.83020 -9.954 37 .000 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p105–109 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p105-109 108 adi prayitno/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 105–109 cox)converts arachidonic acid into prostaglandin-h2. two forms of cox have been identified, namely; cox-1 which is expressed constitutively and cox-2 which is expressed due to growth factors, oncogenes, cytokines and endotoxins.25,26 lps is a component of the cell wall of gram-negative bacteria whose extreme sensitivity activates the inflammatory response via tlr-4. macrophages activated by lps show high cox-2 expression.27 caspase-1 is a component of inflammasome which is released when inflammation is active. caspases-4, -5 and -11 also activate inflammatory nlrp3 in response to lps. finally, caspases-4, -5 and -11 are also referred to as caspase-1 activators to promote caspase-1 division.28–32 the maturation of pro il-1β and pro il-18 to il-1β and il-18 promoted by caspase-4 has also been proposed,33,34 but further studies are required to confirm this result. activation of caspases-4, -5 and -11 has also been shown to lead to pyroptosis.35 biochemically, it has been revealed that caspases-4, -5 and -11 will directly trigger the formation of a gasdermin d substrate that leads to the event of pyroptosis by activating non-canonical nlrp3 inflammation.36 caspase-1 also triggers gas d mirror to mediate pyroptosis by canonical inflammation.37–39 in infectious diseases, expression of caspases-1 and -11 regulates the protective response by releasing il-1β and il-18 in specific contexts. it can be argued that caspase-1 activation and il-18 release through nlrp3 inflammation contribute to colorectal cancer protection.40–42 simultaneously, il-23, il-1β and il-18 will induce the expression of il-17a by th17 lymphocyte cells, γδ t cells and inkt cells.43–46 the direct effect of il-17a on other cells remains to be explored. to test the hypothesis that il-18 and il-1β can stimulate il-17a secretion in cells has been demonstrated in mice.47 the majority of pathological conditions include pyroptosis which can, therefore, be used to identify an infection, hereditary auto-inflammation syndrome and inflammatory bowel disease.48–51 in a mouse subject suffering from septic shock, the presence of pyroptosis is probably the crucial determinant of mortality resulting from excessive lps.52 in conclusion, cox-2, caspase-1 and il-17 play a significant part in pyroptosis signaling of pericoronitis ltmi with low inflammation. references 1. darveau rp. periodontitis: a polymicrobial disruption of host homeostasis. nat rev microbiol. 2010; 8(7): 481–90. 2. mccoy jm. complications of retention: pathology associated with retained third molars. atlas oral maxillofac surg clin. 2012; 20(2): 177–95. 3. kadaryati l, indiarti is. perawatan perikoronitis regio molar satu kanan bawah pada anak laki-laki usia 6 tahun. j dent indones. 2008; 14(2): 127–31. 4. drugs details. pericoronitis – definition, reasons, sign and symptoms, contagious, pain management and prevention. 2018. available from: https://drugsdetails.com/pericoronitis-definition-reasons-sign-andsymptoms-contagious-pain-management-and-prevention/. accessed 2018 sep 15. 5. opie el. inflammation and immunity. j immunol. 1929; 17(4): 329–42. 6. bergsbaken t, fink sl, cookson bt. pyroptosis: host cell death and inflammation. nat rev microbiol. 2009; 7(2): 99–109. 7. messer js. the cellular autophagy/apoptosis checkpoint during inflammation. cell mol life sci. 2017; 74(7): 1281–96. 8. new ton k , di x it v m. sig na l i ng i n i n nat e i m mu n it y a nd inf lammation. cold spring harb perspect biol. 2012; 4(3): a006049. 9. lee j-h, jung n-h, lee b-h, kim s-h, jun jh. suppression of heme oxygenase-1 by prostaglandin e2-protein kinase a-akinase anchoring protein signaling is central for augmented cyclooxygenase-2 expression in lipopolysaccharide-stimulated raw 264.7 macrophages. allergy asthma immunol res. 2013; 5(5): 329. 10. nurmi k, virkanen j, rajamäki k, niemi k, kovanen pt, eklund kk. ethanol inhibits activation of nlrp3 and aim2 inflammasomes in human macrophages--a novel anti-inflammatory action of alcohol. catapano a, editor. plos one. 2013; 8(11): e78537. 11. creagh em. caspase crosstalk: integration of apoptotic and innate immune signalling pathways. trends immunol. 2014; 35(12): 631–40. 12. wynick c, petes c, tigert a, gee k. lipopolysaccharide-mediated induction of concurrent il-1β and il-23 expression in thp-1 cells exhibits differential requirements for caspase-1 and cathepsin b activity. j interf cytokine res. 2016; 36(8): 477–87. 13. mckenzie bs, kastelein ra, cua dj. understanding the il-23–il17 immune pathway. trends immunol. 2006; 27(1): 17–23. 14. gaffen sl, jain r, garg a v, cua dj. the il-23-il-17 immune axis: from mechanisms to therapeutic testing. nat rev immunol. 2014; 14(9): 585–600. 15. onishi rm, gaffen sl. interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease. immunology. 2010; 129(3): 311–21. 16. pappu r, rutz s, ouyang w. regulation of epithelial immunity by il-17 family cytokines. trends immunol. 2012; 33(7): 343–9. 17. iwakura y, ishigame h, saijo s, nakae s. functional specialization of interleukin-17 family members. immunity. 2011; 34(2): 149–62. 18. kasahara di, kim hy, williams as, verbout ng, tran j, si h, wurmbrand ap, jastrab j, hug c, umetsu dt, shore sa. pulmonary inflammation induced by subacute ozone is augmented in adiponectin-deficient mice: role of il-17a. j immunol. 2012; 188(9): 4558–67. 19. kim sh, shin yk, lee km, lee js, yun jh, lee sm. an improved protocol of biotinylated tyramine-based immunohistochemistry m inim izing nonspecific background staining. j histochem cytochem. 2003; 51(1): 129–32. 20. leica biosystems. an animated guide to immunohistochemistry. ihc & ish advanced staining guide. 2013. available from: https:// www.leicabiosystem.com. accessed 2018 sep 15. 21. sencimen m, saygun i, gulses a, bal v, acikel ch, kubar a. evaluation of periodontal pathogens of the mandibular third molar pericoronitis by using real time pcr. int dent j. 2014; 64(4): 200–5. 22. sixou j-l, magaud c, jolivet-gougeon a, cormier m, bonnauremallet m. evaluation of the mandibular third molar pericoronitis flora and its susceptibility to different antibiotics prescribed in france. j clin microbiol. 2003; 41(12): 5794–7. 23. sabra sm, soliman mm. the prevalence of impacted mandibular wisdom with associated physical signs and microbial infections among under graduate girls at taif university, ksa. world appl sci j. 2013; 21(1): 21–9. 24. kityamuwesi r, muwaz l, kasangaki a, kajumbula h, rwenyonyi c. characteristics of pyogenic odontogenic infection in patients attending mulago hospital, uganda: a cross-sectional study. bmc microbiol. 2015; 15(1): 46. 25. hwang d. modulation of the expression of cyclooxygenase-2 by fatty acids mediated through toll-like receptor 4-derived signaling pathways. faseb j. 2001; 15(14): 2556–64. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p105–109 https://drugsdetails.com/pericoronitis-definition-reasons-sign-andsymptoms-contagious-pain-management-and-prevention/ https:// http://www.leicabiosystem.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p105-109 109adi prayitno/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 105–109 26. h a r r i s r e . c yc l o ox yg e n a s e -2 (c ox-2) b l o c k a d e i n t h e chemoprevention of cancers of the colon, breast, prostate, and lung. inflammopharmacology. 2009; 17(2): 55–67. 27. ren w, hu l, hua f, jin j, wang y, zhu l. myeloid differentiation protein 2 silencing decreases lps-induced cytokine production and tlr4/myd88 pathway activity in alveolar macrophages. immunol lett. 2011; 141(1): 94–101. 28. martinon f, burns k, tschopp j. the inflammasome: a molecular platform triggering activation of inf lammatory caspases and processing of proil-beta. mol cell. 2002; 10(2): 417–26. 29. kayagaki n, warming s, lamkanfi m, walle l vande, louie s, dong j, newton k, qu y, liu j, heldens s, zhang j, lee wp, roosegirma m, dixit vm. non-canonical inflammasome activation targets caspase-11. nature. 2011; 479(7371): 117–21. 30. rathinam vak, vanaja sk, waggoner l, sokolovska a, becker c, stuart lm, leong jm, fitzgerald ka. trif licenses caspase11-dependent nlrp3 inflammasome activation by gram-negative bacteria. cell. 2012; 150(3): 606–19. 31. broz p, ruby t, belhocine k, bouley dm, kayagaki n, dixit vm, monack dm. caspase-11 increases susceptibility to salmonella infection in the absence of caspase-1. nature. 2012; 490(7419): 288–91. 32. gurung p, malireddi rks, anand pk, demon d, walle l vande, liu z, vogel p, lamkanfi m, kanneganti t-d. toll or interleukin-1 receptor (tir) domain-containing adaptor inducing interferon-β (trif)-mediated caspase-11 protease production integrates tolllike receptor 4 (tlr4) proteinand nlrp3 inflammasome-mediated host defense against enteropathogens. j biol chem. 2012; 287(41): 34474–83. 33. fassy f, krebs o, rey h, komara b, gillard c, capdevila c, yea c, faucheu c, blanchet a-m, miossec c, diu-hercend a. enzymatic activity of two caspases related to interleukin-1beta-converting enzyme. eur j biochem. 1998; 253(1): 76–83. 34. knodler la, crowley sm, sham hp, yang h, wrande m, ma c, ernst rk, steele-mortimer o, celli j, vallance ba. noncanonical inf lammasome activation of caspase4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens. cell host microbe. 2014; 16(2): 249–56. 35. casson cn, copenhaver am, zwack ee, nguyen ht, strowig t, javdan b, bradley wp, fung tc, flavell ra, brodsky ie, shin s. caspase-11 activation in response to bacterial secretion systems that access the host cytosol. isberg rr, editor. plos pathog. 2013; 9(6): e1003400. 36. lukens jr, vogel p, johnson gr, kelliher ma, iwakura y, lamkanfi m, kanneganti t-d. rip1-driven autoinflammation targets il-1α independently of inflammasomes and rip3. nature. 2013; 498(7453): 224–7. 37. kayagaki n, stowe ib, lee bl, o’rourke k, anderson k, warming s, cuellar t, haley b, roose-girma m, phung qt, liu ps, lill jr, li h, wu j, kummerfeld s, zhang j, lee wp, snipas sj, salvesen gs, morris lx, fitzgerald l, zhang y, bertram em, goodnow cc, dixit vm. caspase-11 cleaves gasdermin d for non-canonical inflammasome signalling. nature. 2015; 526(7575): 666–71. 38. shi j, zhao y, wang k, shi x, wang y, huang h, zhuang y, cai t, wang f, shao f. cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death. nature. 2015; 526(7575): 660–5. 39. agard nj, maltby d, wells ja. inflammatory stimuli regulate caspase substrate profiles. mol cell proteomics. 2010; 9(5): 880– 93. 40. zaki mh, vogel p, body-malapel m, lamkanfi m, kanneganti t-d. il-18 production downstream of the nlrp3 inflammasome confers protection against colorectal tumor formation. j immunol. 2010; 185(8): 4912–20. 41. allen ic, tekippe em, woodford r-mt, uronis jm, holl ek, rogers ab, herfarth hh, jobin c, ting jp-y. the nlrp3 inflammasome functions as a negative regulator of tumorigenesis during colitisassociated cancer. j exp med. 2010; 207(5): 1045–56. 42. dupaul-chicoine j, a rabzadeh a, dagenais m, douglas t, champagne c, morizot a, rodrigue-gervais ig, breton v, colpitts sl, beauchemin n, saleh m. the nlrp3 inflammasome suppresses colorectal cancer metastatic growth in the liver by promoting natural killer cell tumoricidal activity. immunity. 2015; 43(4): 751–63. 43. lalor sj, dungan ls, sutton ce, basdeo sa, fletcher jm, mills khg. caspase-1-processed cytokines il-1beta and il-18 promote il-17 production by gammadelta and cd4 t cells that mediate autoimmunity. j immunol. 2011; 186(10): 5738–48. 44. dungan ls, mills khg. caspase-1-processed il-1 family cytokines play a vital role in driving innate il-17. cytokine. 2011; 56(1): 126–32. 45. doisne j-m, soulard v, bécourt c, amniai l, henrot p, havenardaughton c, blanchet c, zitvogel l, ryffel b, cavaillon j-m, marie jc, couillin i, benlagha k. cutting edge: crucial role of il-1 and il-23 in the innate il-17 response of peripheral lymph node nk1.1 invariant nkt cells to bacteria. j immunol. 2011; 186(2): 662–6. 46. campillo-gimenez l, cumont m-c, fay m, kared h, monceaux v, diop o, müller-trutwin m, hurtrel b, lévy y, zaunders j, dy m, leite-de-moraes mc, elbim c, estaquier j. aids progression is associated with the emergence of il-17-producing cells early after simian immunodeficiency virus infection. j immunol. 2010; 184(2): 984–92. 47. li t, lewallen m, chen s, yu w, zhang n, xie t. multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells. cell res. 2013; 23(6): 788–802. 48. fink sl, cookson bt. pyroptosis and host cell death responses during salmonella infection. cell microbiol. 2007; 9(11): 2562– 70. 49. shao w, yeretssian g, doiron k, hussain sn, saleh m. the caspase-1 digestome identifies the glycolysis pathway as a target during infection and septic shock. j biol chem. 2007; 282(50): 36321–9. 50. simon a, van der meer jwm. pathogenesis of familial periodic fever syndromes or hereditary autoinflammatory syndromes. am j physiol integr comp physiol. 2007; 292(1): r86–98. 51. siegmund b, lehr ha, fantuzzi g, dinarello ca. il-1β-converting enzyme (caspase-1) in intestinal inflammation. proc natl acad sci usa. 2001; 98(23): 13249–54. 52. li p, allen h, banerjee s, franklin s, herzog l, johnston c, mcdowell j, paskind m, rodman l, salfeld j, towne e, tracey d, wardwell s, wei f-y, wong w, kamen r, seshadri t. mice deficient in il-1β-converting enzyme are defective in production of mature il-1β and resistant to endotoxic shock. cell. 1995; 80(3): 401–11. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p105–109 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p105-109 contents page printed by: airlangga university press. (034/02.10/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 43 number 1 march 2010 issn 1978 3728 dental journal majalah kedokteran gigi 1. relationship between trauma mechanism and etiology on mandibular fracture patterns fakhrurrazi ...................................................................................................................................... 1–5 2. noma management in a child with systemic lupus erythematosus irna sufiawati, asri arum sari, budi setiabudiawan, and rahmat gunadi ............................. 6–10 3. tissue engineered bone as an alternative for repairing bone defects evy eida vitria and benny s. latif ................................................................................................ 11–16 4. mozart effect on dental anxiety in 6–12 year old children arlette suzy setiawan, hilma zidnia, and inne suherna sasmita .............................................. 17–20 5. the comparison of minocycline oral-rinse and gel on pocket depth eka fitria augustina ....................................................................................................................... 21–25 6. human-leukocyte antigen typing in javanese patients with recurrent aphthous stomatitis diah savitri ernawati, bagus soebadi, and desiana radithia ..................................................... 26–30 7. mengkudu (morinda citrifolia linn.) gel affect on post-extraction fibroblast acceleration christian khoswanto ....................................................................................................................... 31–34 8. special considerations for orthodontic treatment in patients with root resorption haru s. anggani .............................................................................................................................. 35–39 9. various curing methods on transverse strength of acrylic resin sherman salim ................................................................................................................................ 40–43 10. the frequency of bottle feeding as the main factor of baby bottle tooth decay syndrome mochamad fahlevi rizal, heriandi sutadi, boy m bachtiar, and endang w bachtiar ........... 44–48 historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base subject index volume 46 19% edta, 190 37% phosphoric acid, 190 8-oxo-dg, 119 acyclovir, 30 adhesion, 202 aesthetic component iotn, 97 alginate, 5 extraction, 65 allergic, 85 amelogenin, 113 expression, 135 anterior bite splint, 23 anti inflammatory gel, 30 antisense p45skp2 gene, 18 antropometry, 167 apoptosis, 18, 185 asymmetrical, 55 baby bottle nipple, 75 bactericidal, 9 bcl-2, 35 bite marks, 107 bone resorption, 185 borderline cases, 179 calbindin-28kda, 113 calcite synthesized hydroxyapatite, 207 calcium, 113 cassava leaves, 196 cbct, 61 chelex,107 chemical element, 65 children, 158, 167 chitosan gel, 152 citotoxicity, 9, 218 citrus aurantifolia swingle, 50 class i maloclussion, 179 class iii dentofacial deformity, 229 communication performances, 1 compressive strength, 101 contact angle, 207 cox-2, 196 cpp-acpf, 39, 202 cyclooxygenase-2, 173 cytotoxicity, 221 deciduous tooth, 61 demand, 97 density of pulp chamber, 61 dental caries, 61, 158, 213 dental health services, 1 dental pulp, 130 , 190 denture cleanser, 75 desensitizing agent, 204 diagnose, 146 differentiation, 140 dry heat oven, 71 eflae, 9 ellagic acid, 35, 148 enamel defect, 55 enamel matrix density, 113 enterococcus faecalis, 14, 45 environmental carcinogen, 140 epstein-barr virus, 140 esthetic line, 92 extraction, 181 facial morphology index, 224 height, 224 profile, 92 soft tissue profile, 179 type, 224 fiber composite post, 162 film thickness, 207 fish oil, 185 fluoride, 124, 202 fluorosis, 113 functional groups, 65 garcinia mangostana linn, 173 gene, 18 gentian violet 1%, 75 gestational diabetes mellitus, 135 gic bonding, 39 gingival, 218 ulcer, 152 halitosis, 50 hemolysin, 45 heteroplasmy, 130 holothuria atra, 228 hybrid composite resin , 101 identification, 224 inducible nitric oxide synthase, 14 inflammation, 190 interincisal angle, 224 intra-uterine growth restriction, 55 invasion, 18 javanesse population, 92 lateral cephalometric, 167 lightbox, 101 lipopolysaccharide, 85 local muscle soreness, 23 locus vwa and th01, 107 lps, 196 lstr 3mix-mp, 80 macrophage, 152 malignant cells, 35, 148 malocclusion, 97 mangosteen rind etanolic extract, 173 manihot utilissima, 196 mental retardation, 167 mesenchymal stem cell, 218 metal crown, 162 methyl mercaptan, 50 micro hardness, 207 micronucleus, 119 molar band, 71 molecular biomarker, 140 monocytes, 196 morphology, 65 mtdna, 130 n-3 pufa, 185 nasopharyngeal carcinoma, 140 necrotic pulp, 213 neutrophyl, 152 non invasive, 146 teeth extraction, 179 obesity, 158 oral thrush, 75 orthodontic treatment, 97, 229 orthognatic surgery, 229 osteoblast, 185 p53 wild, 154 patients expectation, 1 periapical granuloma, 213 periapical lesions, 14 periodontal disesase, 185 periodontitis, 173 periodontopathic bacteria, 85 peroxide alkaline, 75 personal identification, 130 physical properties, 207 polimerization, 5 pomegranate fruit extract, 35 pomegranate, 154 porphyromonas gingivalis, 50, 213 post curing, 101 prevotella intermedia, 213 primary teeth, 80 primer, 45 pulp necroses, 80 ponica granatum l (pgl), 148 rabbit, 119 radiography panoramic exposure, 119 root canal, 45 sealer, 207 saliva, 45 salivary leptin, 158 sensitive, 146 setting time, 5 shear strength, 39 small for gestational age, 55 soft palate cancer cell, 18 putty, 23 sprague dawley rats, 190 specific, 140 spectrophotometry, 124 steam autoclave, 71 sterilization, 71 sticophus hermanii, 218 streptococcus mutans, 202 symmetrical, 55 teeth extraction, 179 toothpaste, 124 transmission, 85 unidentified angular ulceration, 30 virulence, 45 water temperature, 5 whole morphology facial index, 228 wild p53, 148 wistar rats, 135, 173 wound healing, 152 young permanent molar, 162 authors index volume 46 adistya, tasya, 152 amtha, rahmi, 30 apriasari, maharani laillyza, 75 aryanto, mirza, 101 atzmaryanni, elfrida, 158 ayu n.p.a, dewa, 45 bramantoro, taufan, 1 budipramana, melisa, 39 cilmiaty, risya, 213 dewi, nurdiana, 135 elianora, dewi, 167 fatimatuzzahro, nadie, 190 febri k, ardyni, 130 fitriyani, nadiya, 92 hastuti, susanti pudji, 124 herdiyati y, 61 hernawati, sri, 35, 148 indahyani, didin erma, 185 indrani, decky j, 5, 65 kaolinni, wees, 97 kristanti, yulita, 202 laksono, harry, 23 lamtiur, pakpahan evie, 229 meilawaty, zahara, 196 mulyawati, ema, 207 parisihni, kristanti, 218 prasetya, rendra chriestedy, 173 pratiwi, theresia dhearine, 162 pudyani, pinandi sri, 179 saskianti, tania, 80 shantiningsih, rurie ratna, 125 sianita k, pricillia priska, 224 sjarif, willyanti soewondo, 55 sudiono, janti, 9, 140 supriatno, 18 sutrisno, imelda kristina, 107 tridianti, anggia, 71 utomo, haryono, 85 wahluyo, soegeng, 113 yuanita, tamara, 14 yusinta, anindya prima, 50 thanks to editor dental journal (majalah kedokteran gigi) volume 46 number 1 march 2013 : 1. dr. retno indrawati, drg., msi. (oral biology – universitas airlangga) 2. dr. indah listiana, drg., m.kes. (oral biology – universitas airlangga) 3. wisnu setyari, drg., m.kes. (oral biology – universitas airlangga) 4. dr. ira arundina, drg., m.kes (oral biology – universitas airlangga) volume 46 number 2 june 2013 : 1. prof. dr. pinandi sri pudyani, drg., s.u., sp.ort (orthodontics – universitas gadjah mada) 2. prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) 3. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) 4. dr. ib. narmada, drg., sp.ort(k) (orthodontics – universitas airlangga) 5. kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) volume 46 number 3 september 2013 : 1. prof. dr. drg. iwa sutardjo rus sudarso, su., sp.kga(k) (pediatrics dentistry – universitas gadjah mada) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentstry – universitas airlangga) volume 46 number 4 december 2013 : 1. prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) 2. prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) 3. dr. theresia indah budhy, drg., m.kes (oral pathology and maxillofacial – universitas airlangga) 4. dr. indah listiana kriswandini, drg., m.kes (oral biology – universitas airlangga) guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 137 vol. 41. no. 3 july–september 2008 non-invasive endodontic treatment of large periapical lesions harry huiz peeters private practitioner bandung indonesia abstract background: in most cases of large periapical radiolucent lesions of pulpal origin, we often encounter a dilemmatic situation, such as whether to either treat these cases endodontically or surgically. development of techniques, instruments and root medicaments as well as the tendency toward minimally invasive treatment, all support dentists to treat those cases using the minimal invasive principle (i.e. endodontically instead of surgically). purpose: the purpose of this paper is to report and discuss the managing of periapical lesions by endodontic no invasive treatment. case management: the patient with large periapical lesions were treated with noninvasive endodontic treatment. after 6 months, patients in this report were asymptomatic and radiolucencies had disappeared. when the root canal treatment is done according to accepted clinical principles and under aseptic condition, including cleaning, shaping, abturating as well as proper diagnosis, the healing process of the infected area will occur. conclusion: some lesions, however, may not be treated conservatively and may require surgical treatment for total elimination of the lesions. key words: invasive treatment, periapical lesions correspondence: harry huiz peeters, private practitioner, cihampelas 41 bandung, indonesia. email: h2huiz@cbn.net.id introduction clinical classification of periapical lesions is divided into three clinical groups i.e: asymptomatic apical periodontitis, asymptomatic apical periodontitis and apical abscess. periapical lesions commonly occur due to irritants and are initiated by neglected dental caries. the irritants could be microorganism, virus, mechanic, thermal, and chemical.1 the effect of the intervention of microorganisms and their by-products into the root canals of untreated teeth may cause this intervention process to move apically, which leads to antigen-antibody reaction, finally, periapical lesions may occur.1 in this study, the presented cases were limited only to periapical lesions, such as: periradicular granuloma, periradicular cyst, condensing osteitis, apical abscess,1 which can be managed by endodontic non invasive treatment, and to non retreatment cases. in addition, some lesions, such as: true cysts, extraradicular infection lesions, fungi, foreign body reactions, can be treated through invasive treatment.2,3 the development of instruments, medicaments and techniques of root canal treatment allows periapical lesions to be perfectly treated by endodontic non invasive treatment. endodontic treatment should be conducted in accordance with good clinical principles such as establishing correct diagnosis as well as using the accepted chemico-mechanical debridement process in asepsis conditions. after clinical procedures have been done correctly, hermetical permanent restoration has the important role in preserving the endodontically treated teeth over an extended period. hermetical restorations are able to protect the treated root canal from the invasion of microorganisms.4,5,6 case case 1: a 23-year-old female visited the clinic with a chief complaint of having a lump at the palatum region. extraorally, no impairment was found. medical history was noncontributory. intraorally, a purulent nodule was found at the right palatum. tooth #12 (right maxillary lateral incisor) was tender to percussion and palpation. a large composite filling and secondary carries were found. tooth #12 was non-responsive to vitality test. radiographic examination showed a large radiolucent with a sharp margin around the apex of #12. the apex showed slightly curved, case report 138 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 137-141 discontinuity of periodontal ligament and lamina-dura. the periodontal condition was excellent, with no gingivitis and absence of pocket. the tooth was diagnosed as a chronic apical periodontitis. first of all, therapy was done by removing the whole remaining restorative material and carries; subsequently a new hermetic restoration was built up to form a new seal, preventing invasion of microorganisms. after obtaining infiltration anesthesia surrounding the gingiva by using 2% lidocaine solution (this procedure to avoid pain at gingival region when clamp was applied) the tooth was isolated under rubber dam. md-cleanser (17% edta = ethylenediaminetetraacetic) and 5% naocl were applied consecutively as irrigants before inserting endodontic files into the canal. this has been proved to be a valuable action in penetrating small spaces by removing debris. a size k file (0.8) was used to identify the canal. an apex locator was used to verify the working length. individual canal instrumentation was performed sequentially with k files using a crown-down pressureless technique to a master apical size of 50. copious chemical irrigation was performed with 17% edta and recapitulation was always done to verify the working-length during chemico-mechanical debridement. prior to obturation, a final irrigation with 2.5% sodium hypochlorite was used. ultrasonic tips were used to activate the irrigants in the entire canal for 2 minutes, subsequently, 2% chlorhexidine was used as a neutralizing agent. the root canal was dried with sterile paper point and obturation was performed using warm vertical compaction of gutta-percha and ah-26 as a root canal sealer. excess of gutta-percha cones were cut at the level of the root canal orifice and vertical compaction was applied immediately with iso size of 40 finger plugger after being heated by system-b. this action will ensure better homogeneity of gutta-percha which in turn will fill canal irregularties as well as accessory canals. a coronal temporary restoration of glass-ionomer was placed and a postoperative radiograph was taken in order to assess the quality of obturation. the following week, periapical radiographs were exposed for examination with the following results: patient asymptomatic, the tooth was nonresponsive to percussion and palpation. a final coronal permanent restoration of composite was placed. the patient was recalled 12 months postoperatively for clinical and radiographic examination. at that time, the patient was symptom-free. the clinical appearance of the area was satisfactory. the radiographic examination revealed that a great part of the lesion had been covered with healthy bone (figure 1). case 2: a 35-year-old female patient visited the clinic with a chief complaint of having sinus tract at left maxillary lateral incisor (22) buccally. extraorally, there was no evidence of swelling and medical history was noncontributory. intraorally, a sinus tract was present buccally. the tooth was non-responsive to palpation, percussion as well as vitality test. the periodontal condition was excellent, with no gingivitis and absence of pocket. radiographic examination showed an evidence of large radiolucency with discontinuity of periodontal ligament and lamina-dura. at first, therapy was done by removing the whole remaining restorative material and carries; subsequently a new hermetic restoration was built up to form a new seal, preventing invasion of microorganisms. after obtaining infiltration anesthesia surrounding the gingiva by using 2% lidocaine solution (this procedure to avoid pain at gingival region when clamp was applied) the tooth was isolated under rubber dam. md-cleanser (17% edta = ethylenediaminetetraacetic) and 5% naocl were applied consecutively as irrigants before inserting endodontic files into the canal. this has been proved as a valuable action in penetrating small spaces by removing debris. a size k file of 0.8 was the initial instrument of figure 1. radiographs immediately after completing the root canal therapy and at the 12-months follow up. a) radiographic examination after root canal treatment and b) after 12 month a b 139peeters: non-invasive endodontic treatment of large periapical lesions choice to negotiate the canal. an apex locator was used to verify the working length. individual canal instrumentation was performed sequentially with k files using a crowndown pressureless technique to a master apical size of 50. copious chemical irrigation was performed with 17% edta and recapitulation was always done to verify the working-length during chemico-mechanical debridement. prior to obturation, a final irrigation with 2,5% sodium hypochlorite was used. ultrasonic activation was used to activate the irrigants in the entire canal for 2 minutes, subsequently, 2% chlorhexidine was used as a neutralizing agent. the root canal was dried with sterile paper-points and obturation was performed using warm vertical compaction of gutta-percha and ah-26 as a root canal sealer. excess of gutta-percha cones were cut at the level of the root canal orifice and vertical compaction was applied immediately with iso size of 40 finger plugger after being heated by system-b. this action will ensure better homogeneity of gutta-percha which in turn will fill canal irregularties as well as accessory canals. a coronal temporary restoration of glass-ionomer was placed and a postoperative radiograph was taken in order to assess the quality of obturation. the following week, periapical radiographs were exposed for examination, with the following results, patient asymptomatic, the tooth was nonresponsive to percussion and palpation. a final coronal permanent restoration of composite was placed. the patient was recalled 12 months postoperatively for clinical and radiographic examination. at 12 months, the tooth remained symptom-free. the clinical appearance of the area was satisfactory. the radiographic examination showed almost complete osseous repair of preoperative periradicular pathosis (figure 2). discussion periapical lesions can commonly be treated through non invasive root canal treatment. the treatment consists of a series of mechanical and chemical procedures and is initiated by opening the occlusal with a dental bur to obtain the pulp chamber. the entire process is performed under rubber dam isolation, subsequently followed by chemicomechanical debridement. copious irrigants are used after each instrumentation. periapical lesions can occur as a result of interaction when the host responds to invasion of microorganisms and their by-products egress into the root canal. in 1697 anthonie van leeuwenhoek was the first person who could see bacteria from root canals using one of the first generation of microscopes.7 definitive pathological mechanisms of the periapical lesion are still unclear. initially, it was assumed that polymicrobial infection induced the human body defenses. the pathogenesis study of periapical lesions has been conducted in various animal models, such as: rats, dogs, and rabbits and monkeys. however, the result of the study should not be applied directly to humans owing to different responses between microbial flora and the human condition.7 the response of host defenses against microorganisms and their by-products causes various histological and clinical images,7 such as: acute and chronic periapical inflammation, chronic suppurative periapical inflamation, acute periapical abscess/cellulitis, periapical osteomyelitis, periapical osteosclerosis, granulomas and cysts. qualitative analysis has not been able to identify all of the microbial in figure 2. radiographs show immediately after completing the root canal therapy and twelve-month radiograph showing almost complete healing of periradicular radiolucency. a) radiographic examination after root canal treatment and b) after 1 yearradiographic examination after root canal treatment and b) after 1 year a b 140 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 137-141 the root canal systems of infected teeth, whereas microbial have been found in every infected tooth so far around hundreds of types.7 in 1965 kakehashi et al. published a classic experiment, which revealed that exposure of the pulp in rats with normal microbial flora produced pulpal necrosis and periradicular lesions developed. however, only minimal pathological changes occurred in germ-free rats when the pulp was exposed, furthermore a reparative bridge was formed. implication of this experiment shows that periapical lesions can repair themselves owing to the absence of microorganisms and their by-products. this experiment obviously showed that microorganisms are the predominant irritants of the dental and the periodontal tissues. this study reported that periapical lesions can improve by themselves when the microorganisms and their by-products are absent.8 extra -radicular infections, true cysts and foreign body reactions can only be successfully treated by corrective surgical treatment, whereas periapical lesions that heal by fibrous scar tissue do not require any treatment.9 based on the fact that periapical lesions may develop due to the interaction between human defense mechanisms against microorganisms and their by–products, the formation of lesions depends upon this interaction. whereas the recovery process of periapical lesions may occur when the microorganisms and their by-products are absent in the contaminated regions. this allows macrophages to invade the zones of infection and contamination in order to remove debris and dead cells. subsequently, osteoblasts, fibroblasts and new in-growing nerve fibers as well as blood vessels will proliferate into the infected zones. finally, the recovery process will occur, starting from the outer part of lesions to the inner part of the lesions until a normal periodontal ligament is formed. provided the periodontal tissues and cells are not irreversibly damaged then the recovery process will form cementum over the apical foramen which will completely isolate the entire root canal system from poor environments. the insufficient removal of inflammation may lead to an incomplete recovery process, whilst any delay in the recovery process is determined by treatment procedures and materials used.7 even though the precise mechanisms of the recovery of periapical lesions are still controversial, all inflammatory periapical lesions should be initially treated with non surgical treatment then by surgical treatment. adequate endodontic treatment will induce the recovery process around periapical tissues that follow the same recovery principles as that of connective tissues elsewhere in the body.10 when the root canal treatments are done according to accepted cleaning and shaping procedures, and are able to eliminate the entire microorganisms, necrotic tissues as well as the remnants of organic tissues from the infected root, the success rate is generally high.8 accepted endodontic treatments may induce a favorable healing process. recently, a study reported that programmed cell death has an important role in the entire healing process.10 after treating the root canal properly, the process of chemical debridement and removal of microorganisms will be continued by sealing a chemical agent in the canal hermetically over a period of time, to eliminate and to prevent microorganisms recolonizing the root canal system. previously, a culture test used to be done, but in modern root canal treatment the culture tests are not always conducted, although there are some who still practice it. as long as clinical symptoms, inflammatory signs and radiolucencies are absent, then the root canal system is ready to fill three dimensionally. the purpose of root canal obturating is to fill the root canal system with inactive materials (such as guttapercha and a sealer) to isolate from either periapex tissues or oral environment. the hermetical permanent restorations have an important role in providing definitive coronal seal preventing re-infection of the root canal system. it will entomb the residue of microorganisms in the canal; finally, the microorganisms cannot survive. the definitive measure of root canal treatment is the recovery of periapical lesions, since the purpose of the treatment is to eliminate the lesions. this measure could take up to 4 years or more for reevaluation by taking a series of radiographs until normal tissues are restored. in the fact, even the utmost sophisticated methods of root canal treatment are still unable to remove the entire microorganisms and their by-products from root canals and tubulus dentin, therefore, the obturation process becomes the second opportunity to entomb all the remaining microorganisms and their by-products in the root canal so that they are unable to escape. theoretically, a proper obturation will ensure microorganisms cannot survive, but in fact there is no perfect filling materials available in the market, consequently, communication still occurs between the root canal and the oral environment.11 to avoid that communication, a hermetical coronal restoration is needed in order to prevent re-invasion of either new microorganisms or nutrients into the root canals through the unsealed zone, if this is not done then within a couple of days new microorganisms or nutrients will reach the periapical tissues, resulting in the remaining microorganism becoming more active in their growth, and finally will develop a chronic periapical inflammation. clinicians must keep in mind, therefore, that the root canal system is not only pipe-like with open ends but also a complex series of canals with accessories, lateral canals, furcations and containing milliards of tubulus dentin. the root canal system should be filled three dimensionally and the final hermetical coronal restorations are needed.8 evaluation of successful and unsuccessful root canal treatments still remains controversial. nevertheless, when the teeth are clinically asymptomatic and radiographic images show improvement, the conclusion can be drawn that the root canal treatment is successful clinically, although the alteration of tissue structures has histologically and microscopically occurred.1 the main goal of this case study is to report successful treatments of the two cases owing to asepsis conditions, accepted clinical procedures 141peeters: non-invasive endodontic treatment of large periapical lesions and final hermetical coronal restorations that allow the periapical tissue to improve completely, regardless the size of the lesions and nonsurgical treatments. surgery is recommended after non invasive root canal treatment is unsuccessfully performed. acknowledgement the author would like to thank bill lawrie (english teacher) for his assistance and review of this paper. references 1. ingle. endodontics. 5th ed. hamilton: elsevier; 2002. p. 175–7. 2. nair pn, sjogren u, krey g, sundqvist g. therapy-resistant foreign body giant cell granuloma at the periapex of a root-filled human tooth. j endod 1990; 16:589–95. 3. nair pn, sjogren u, krey g, kahnberg ke, sundqvist g. intraradicular bacteriaand fungi in root-filled,asymptomatic human teeth with therapy-resistant periapical lesions:a long-term light and electron microscopic follow up study. j endod 1990; 16:580–8. 4. nair pn, sjogren u, figdor d, sundqvist g. persistent periapical radiolucencies of root-filled human teeth, failed endodontic treatments, and periapical scars. oral surg oral med oral pathol oral radiol endod 1999; 87:617–27. 5. yusuf h. the significance of the presence of foreign material periapically as a cause of failure of root treatment. oral surg oral med oral pathol 1982; 54:566–74. 6. bystrom a, happonen rp, sjogren u, sundqvist g. healing of periapical lesions of pulpless teeth after endodontic treatment with controlled asepsis. endod dent traumatol 1987; 3:58–63. 7. stock c, walker r, gulabivala k. endodontics. 3rd ed. st louis : mosby; 2004. p. 29–55. 8. cohen s, hargreaves km. pathways of the pulp. 9th ed. st. louis: mosby; 2006. p. 542. 9. yan mt. the management of perapical lesions in endodontically treated teeth. aus j endodon 2006; 32:2–15. 10. kim s, pecora g, rubinstein ra. color atlas of microsurgery in endodontics. toronto: wb saunders; 2001. p. 13. 11. lin l, geroge tjh, paul ar. proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing. j endod 2007; 33:908–15. 12. gutmann j, dumsha tc, lovdahl pe. problem solving in endodontics.problem solving in endodontics. 4th ed. st. louis: mosby; 2006. p. 3 127127 research report dental journal (majalah kedokteran gigi) 2017 september; 50(3): 127–130 comparison of esthetic smile perceptions among male and female indonesian dental students relating to the buccal corridors of a smile astriana nurfitrah, c. christnawati, and ananto ali alhasyimi department of orthodontics faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: a smile constitutes a form of measurement as to whether or not an orthodontic treatment has proved successful. a smile is said to be ideal if a balance exists between the shape of the face and teeth. one benchmark used to assess the quality of an ideal smile is that of buccal corridors. these are formed of the black space between the lateral edge of maxillary posterior teeth and the corner of the lip which appears during the action of smiling. evaluating the contrasting perceptions of male and female smiles based on buccal corridor aspects is considered important to identifying the specific qualities an ideal smile. purpose: the purpose of this study was to determine the difference between the perceptions of an ideal smile held by indonesian dental students of both genders based on buccal corridors. methods: a total of 36 dental students, equally divided between male and female students and ranging in age from 18-21 years old, were enrolled in this study. the smiles of all subjects were photographed from the front for later assessment by the subjects themselves. assessment was undertaken twice, with a two-week interval between the first and second, by comparing subjects’ photographs with reference pictures of buccal corridors. data gathered were analyzed by using kappa-statistic and u-mann whitney. results: the results indicated that all the subjects showed a good level of coincidence in their analysis (κ=0.76). statistical analysis showed that the score of 0.123 (p>0.05) was shown in u-mann whitney. conclusion: indonesian male and female dental students have the same perception of an aesthetic smile with regard to its buccal corridor. keywords: buccal corridors; ideal smile; perception correspondence: ananto ali alhasyimi, department of orthodontics, faculty of dentistry, universitas gadjah mada. jl. denta sekip utara, bulaksumur, yogyakarta 55281, indonesia. e-mail: anantoali@ugm.ac.id introduction the main objective of orthodontic treatment is to correct malocclusion in order to achieve appropriate occlusion and optimum dentofacial function.1 although the principal goal remains the restoration of oral health and function, the importance of facial esthetics and their psychological impact is increasing to the point of their becoming a necessity.2 the perception of facial esthetics plays a significant role in a person’s decision to seek orthodontic treatment and, furthermore, contributes greatly to facial attractiveness. a smile represents a facial expression which communicates feeling, friendship or a desire to reward an individual’s achievements.3 the perfect smile is said to exist when there is harmony and balance between the shape of the face and teeth.4 a smile constitutes one of the main criteria for patients when measuring the success of orthodontic treatment. forming an ideal smile requires analysis and evaluation of the face, lips, gingival tissue, shape and color of the teeth and the combination of these components. the components of a smile considered to be important include: buccal corridors, the extent of incisor and gingival display and the existence of a midline and diastema.5 buccal corridors constitute an important aspect to be considered when measuring a smile and can be defined as the dark area or black space (lateral negative space) between the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p127-130 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p127-130 mailto:anantoali@ugm.ac.id 128 nurfitrah, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 127–130 lateral edge of the maxillary posterior teeth and the corner of the mouth which appears when someone smiles (figure 1).6 buccal corridors occur in the dark background inside the mouth depending on the shape and width of the upper tooth curve and the facial muscles which determine the width of an individual’s smile.7 buccal corridors disappear when the lips are in a closed position since their existence are the products of facial and perioral muscular activity.8 the assessment of a smile can be completed by evaluating photographs of buccal corridors and will be conducted using a range of six classifications of buccal corridors, including: extra-broad (0% buccal corridors), broad (5% buccal corridors), medium-broad (10% buccal corridors), medium (15% buccal corridors), medium-narrow (20% buccal corridors) and narrow (25% buccal corridors) (figure 1).8 a broad smile with a minimum of buccal corridors (0% buccal corridors) possesses greater aesthetic value than a narrow smile with wide buccal corridors.3,9 perception is a process through which one chooses, organizes and interprets the stimuli which are accepted as making up a picture representing their world. this process is mostly influenced by consciousness, memory, mind, and language which involve individual interpretation of a specific object. thus, each individual will have a different perception, although these perceptions deal with the same object.10 research conducted at a dental school in brazil showed that as far as perceptions of a positive aesthetic smile are concerned, women feel less satisfied with their smiles compared to men.11 women tend to think more that the aesthetic appearance of their teeth is important than do men.12 this is influenced by many factors which differ from one another and which influence individuals in contrasting ways according to their age, gender, marital status, social and economy condition, education, profession, family, friends, culture, and the mass media. similarly, younger individuals pay more attention to the aesthetic appearance of their teeth than the elders.11 previous research revealed a difference with regard to confidence in that men are more self-confident than women.13 from the previous research, it could be said that further investigation needs to be conducted into how the relationship between the aesthetic charm of a smile, tooth size and form, lip curve, gingiva form, and the display of buccal corridors compares to individual perceptions of a smile.11 this present study was carried out to compare indonesian male and female dental students’ perceptions of esthetic smiles based on buccal corridors. materials and methods thirty six photographs were obtained from 36 subjects (consisting 18 males and 18 females) for use in this study. the subjects consisted of current, 18 to 21 year old dental students of the faculty of dentistry, universitas gadjah mada, indonesia who had never undergone orthodontic treatment, had angle class i relation, no craniofacial anomalies or missing teeth and no evident asymmetry. this research has already been passed by the eligible ethics sub-committee of the universitas gadjah mada ethics commission and assigned the number 00958/kkep/fkgug/ec/2017. this research project employed the use of a digital camera (canon, eos 700d (18.0 megapixels, iso 200, tokyo, japan), a printer (hp® deskjet ink advantage 2135, usa), and a laptop (hp® notebook series, usa). afterward, explanations of the research procedure were given to subjects who, subsequently, signed the consent form, thereby confirming their agreement. the data were in the form of photographs taken of the subjects using a tripod-mounted camera, with an object-to-lens distance of 8 figure 1. series reference images illustrating the range of buccal corridors (blue area): (classification 1) extra broad (0% buccal corridor), (classification 2) broad (5% buccal corridor), (classification 3) medium-broad (10% buccal corridor), (classification 4) medium (15% buccal corridor), (classification 5) medium-narrow (20% buccal corridor), and (classification 6) narrow (25% buccal corridor).5 figure 2. photoshoot technique. figure 1. series reference images illustrating the range of buccal corridors (blue area): (classification 1) extra broad (0% buccal corridor), (classification 2) broad (5% buccal corridor), (classification 3) medium-broad (10% buccal corridor), (classification 4) medium (15% buccal corridor), (classification 5) medium-narrow (20% buccal corridor), and (classification 6) narrow (25% buccal corridor).5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p127-130 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p127-130 129129nurfitrah, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 127–130 30 inches (91 cm). the camera was set at iso 200 in auto focus mode. subjects were positioned in an upright seated position, with an unsmiling face and instructed to look through a point at their eye level during the image capture in order to ensure natural head posture (figure 2). subjects were instructed to say “cheese” in order to ensure that they showed their teeth for two seconds, while looking straight at the camera. assessment of the photograph was undertaken after the result had been printed. the assessment was conducted by trained and calibrated examiners (who also act as subjects in a process of self-assessment) when they were both healthy and emotionally stable. before completing the selfassessment, examiners were given an explanation of the criteria of smiles based on buccal corridors and then given training. each examiner assessed the photographs from all previous research by comparing them to six reference pictures of buccal corridors (figure 1). the examiners were instructed to choose one of six reference pictures, which they thought similar to their own and focus on the region of interest (figure 3). the results of the examiners’ assessments were returned via the assessment form and consisted of awarding a score from the buccal corridor classification reference 1, 2, 3, 4, 5, or 6 on each subject. the examiners assessed the photograph twice with an interval of two weeks between assessments. this approach aimed to minimize potential bias in the observation by reducing the effect of examiner subjectivity and focusing the study on the effect of the experience.14 the average score taken after the final assessment was subsequently analyzed by means of kappa analysis statistics to identify the level of agreement between examiners. a mann-whitney u test was employed to know whether any difference between male and female perceptions existed regarding their buccal corridors (table 1). results the results provided by the subjects formed two sets of assessment data, perception assessments i and ii, relating to their perceptions of an ideal smile based on buccal corridors. the kappa statistic was employed in order to establish the reliability between intra-examiner and interexaminer results of assessment i and ii, calculated as the number of agreement scores divided by the total number of scores. all examiners demonstrated an extremely high level of agreement in both their intra-examiner and interexaminer analysis (κ = 0.76). the result confirms that there was no difference between the first and the second assessment. thereafter, a u mann-whitney test was conducted to establish whether there was any contrast between male and female perceptions with regard to their buccal corridors. the u mann-whitney test value was 0.123, meaning that there was no male-female difference (table 1). this result, in turn, implies that no difference exists between the perceptions of members of the two genders. based on the contents of table 1, the responses of males were similar to females in that their assessment of subjects’ smiles in classification 5 was 30.56%, while that of their female counterparts was 33.3%. discussion the kappa statistic results indicate that there was no difference between perception assessments i and ii. these 8 figure 1. series reference images illustrating the range of buccal corridors (blue area): (classification 1) extra broad (0% buccal corridor), (classification 2) broad (5% buccal corridor), (classification 3) medium-broad (10% buccal corridor), (classification 4) medium (15% buccal corridor), (classification 5) medium-narrow (20% buccal corridor), and (classification 6) narrow (25% buccal corridor).5 figure 2. photoshoot technique. figure 2. photoshoot technique. table 1. percentage on value of buccal corridors for each group and and results of the u mann-whitney test comparing 2 groups tested classification of buccal corridors number of choice ± percentage of classification of buccal corridors (%) p -value male female 1 2 (5.56) 1 (2.78) 0.1232 2 (5.56) 2 (5.56) 3 4 (11.11) 4 (11.11) 4 8 (22.2) 9 (25) 5 11 (30.56) 12 (33.3) 6 9 (25) 8 (22.2) total 36 (100) 36 (100) notes: 1) extrabroad buccal corridor; 2) broad buccal corridor; 3) medium-broad buccal corridor; 4) medium buccal corridor; 5) medium-narrow buccal corridor; 6) narrow buccal corridor. 9 figure 3. regio of interest: (a) the number of maxillary teeth; (b) outer commissure; (c) buccal corridor (blue area). table 1. percentage on value of buccal corridors for each group and and results of the u mannwhitney test comparing 2 groups tested classification of buccal corridors number of choice ± percentage of classification of buccal corridors (%) p-value male female 1 2 (5.56) 1 (2.78) 0.123 2 2 (5.56) 2 (5.56) 3 4 (11.11) 4 (11.11) 4 8 (22.2) 9 (25) 5 11 (30.56) 12 (33.3) 6 9 (25) 8 (22.2) a c b c figure 3. regio of interest: (a) the number of maxillary teeth; (b) outer commissure; (c) buccal corridor (blue area). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p127-130 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p127-130 130 nurfitrah, et al./dent. j. (majalah kedokteran gigi) 2017 september; 50(3): 127–130 might have been influenced by several factors including: the sharing information about buccal corridors and their classification in the form of audio (utterances) and visual (reference) buccal corridors by the researchers. before the subjects provided assessment i and assessment ii they shared a common educational background in that they were all students of the faculty of dentistry. this is in line with the statement that perceptions can be influenced by an object that has previously been encountered. words, colors, shapes, and location can be easily remembered and familiarity may with an object that has been seen or heard.15 results of the frequency calculation based on the percentage on each classification of buccal corridors, together with the mode of each group, confirm that males give more weight to classification 5 and less to classification 1. those two groups (male and female) appear to have the same mode of perception with regard to classification 5. this is supported by the theory stating that males and females tend to have the same interest in the similar smile (the same mode in classification 5).16 age and gender are not influenced by one perception in assessing the size of buccal corridors. males and females have the same opinion about the aesthetic factors which influence an attractive smile, related to buccal corridors but females tend to be more sensitive to changes in those factors.17 another theory also argues that males and females have the same interest in a similar smile based on the same mode. therefore, it can be argued that gender does not appear to influence the perception of buccal corridors assessment.18 another piece of research into the perception of smiles based on teeth and face displays revealed that males are less critical than females when assessing a photograph. this probably occurs because printed photographs of the subjects’ smile display buccal corridors indistinctly due to the inferior quality of photographic techniques related to brightness and photoshoot.17 prior research on buccal corridors indicates that the broader a smile on buccal corridors, the greater its aesthetic quality compared to a narrower one.9 the results of this research are different to that conducted by faculty of dentistry, universitas gadjah mada students into perceptions of an ideal smile based on buccal corridors. this research shows that dentistry students of both genders choose classification for those buccal corridors considered to be better for one’s smile. this demonstrates the need to classify the buccal corridors which suit indonesian society. the transversal dimension is one of the basic aspects of a smile in relation to buccal corridors which can be assigned to one of six classifications: 1 (extrabroad); 2 (broad); 3 (medium-broad); 4 (medium); 5 (medium-narrow), and 6 (narrow).5 there was no difference between males and females’ perception of the ideal buccal corridor-based smile which was influenced by several factors, one of them being background knowledge. the research subjects were faculty of dentistry students enrolled on dental anatomy and orthodontics courses during the years 2014 and 2015. this factor might influence the similarity of one subject’s perception to that of another. individual perception of smile is based on education, gender, friend, and profession.11 moreover, one’s perception can be influenced by the social environment.15 all research subjects were current faculty of dentistry students in the sense that they inhabited the same social environment and, as a result, the perception of male and female subjects were similar. it can be concluded that indonesian male and female dental students have the same perception of an aesthetic smile with regard to its buccal corridors. references 1. alhasyimi aa, sunarintyas s, soesatyo mh. pengaruh implantasi subkutan logam kobalt kromium sebagai bahan alternatif mini screw orthodontics terhadap reaksi jaringan kelinci albino. maj ked gigi ind. 2015; 1(1): 94-101. 2. avriliyanti f, suparwitri s, alhasyimi aa. rinsing effect of 60% bay leaf (syzygium polyanthum wight) aqueous decoction in inhibiting the accumulation of dental plaque during fixed orthodontic treatment. dent j (maj ked gigi). 2017; 50(1): 1–5. 3. monica ga. gambaran senyum pasien pasca perawatan ortodonsia (kajian foto frontal). indonesia j dent. 2007; 14(2): 136–45. 4. davis nc. smile design. dent clin north am. 2007; 51: 299–318. 5. ioi h, nakata s, counts al. effects of buccal corridors on smile esthetics in japanese. angle orthod. 2009; 79(4): 628–33. 6. branco ncc, janson g, de freitas mr, morais j. width of buccal and posterior corridors: differences between cases treated with asymmetric and symmetric extractions. dental press j orthod. 2012; 17(5): 138–44. 7. nascimento dc, dos santos êr, machado awl, bittencourt mav. influence of buccal corridor dimension on smile esthetics. dental press j orthod. 2012; 17(5): 145–50. 8. janson g, branco nc, fernandes tmf, sathler r, garib d, lauris jrp. influence of orthodontic treatment, midline position, buccal corridor and smile arc on smile attractiveness. angle orthod. 2011; 81(1): 153–61. 9. zaib f, waheed-ul-hameed. effect of buccal corridors width on smile esthetics. pak orthod j. 2009: 1–5. 10. cardoso-leite p, gorea a. on the perceptual/motor dissociation: a review of concepts, theory, experimental paradigms and data interpretations. seeing perceiving. 2010; 23(2): 89–151. 11. da silva gc, de castilhos ed, masotti as, rodrigues-junior ar. dental esthetic self-perception of brazilian dental students. rsbo. 2012; 9(4): 375–81. 12. samorodnitzky-naveh gr, geiger sb, levin l. patients’ satisfaction with dental esthetics. j am dent assoc. 2007; 138: 805–8. 13. ring p, neyse l, david-barett t, schmidt u. gender differences in performance predictions: evidence from the cognitive reflection test. front psychol. 2016; 7: 1–7. 14. tortopodis d, hatzikyriakos a, kokoti m, menexes g, tsiggos n. evaluation of the relationship between subjects’ perception and professional assessment of esthetic treatment needs. j esthet restor dent. 2007; 19:154–163. 15. kristjánsson á, campana g. where perception meets memory: a review of repetition priming in visual search tasks. atten, percept, psychophys. 2010; 72(1): 5–18. 16. tikku t, khanna r, maurya r, ahmad n. role of buccal corridor in smile esthetics and its correlation with underlying skeletal and dental structures. indian j dent res. 2012; 23: 187–94. 17. martin aj, buschang ph, boley jc, taylor rw, mckinney tw. the impact of buccal corridors on smile attractiveness. eur j orthod. 2007; 29: 530–7. 18. oshagh m, zarif nh, bahramnia f. evaluation of the effect of buccal corridor size on smile attractiveness. eur j esthet dent. 2010; 5(4): 370–80. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v50.i3.p127-130 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v50.i3.p127-130 165165 case report dental journal (majalah kedokteran gigi) 2015 december; 48(4): 165–169 challenges in the management of oral ulceration in elderly patients nanan nur’aeny department of oral medicine faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: oral ulceration can be experienced by anyone, including those who are elderly. various trigger factors can occur in elderly patient, but the main thing to consider is the degenerative factors that affect the occurrence of some medical problems. handling oral ulceration in elderly patients should be done carefully and holistically otherwise the improvement is only temporary and can reappear or even be worse. purpose: in this paper we will discuss two different case reports of elderly female patients and both having some oral ulceration. cases: first case of recurrent oral ulceration experienced by 58 years old patient, and second case is concerning a 77 years old patient with chronic oral ulceration and also having some medical problems. aphthous like ulcers (alu) is a diagnosis for recurrent oral ulceration associated with systemic condition, and usually started after adolescent age. elderly or geriatric condition itself is a special condition that contribute to the degree of a disease. cases management: both patients given non pharmacology and pharmacology therapies. the non pharmacology therapy includes communication, information, and education, also oral hygiene instruction. steroid as anti-inflammatory drugs had an important role in healing process, beside other medication. conclusion: oral ulceration in elderly patients with or without a medical problems becomes a challenging thing to handle due to the complexity of their condition. as a dentist we have more careful to arrange the treatment plans for elderly patients when combine with some therapy related systemic disease. keywords: ulceration; oral mucosa; elderly correspondence: nanan nur’aeny, c/o: departemen penyakit mulut, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan no. 1 bandung 40132, indonesia. e-mail: nanan.nuraeny@fkg.unpad.ac.id introduction oral ulceration can be experienced by anyone, it does not depend on age or gender. various trigger factors such as trauma due to bitting, or exposed to something sharp, food allergies, or microorganism infection often associated with the emergence of acute, chronic, or recurrent oral ulceration.1,2 the history of medical problems such as diabetes mellitus, hypertension, or others that suffered by patients who have oral ulceration should also be noted because it could be interrelated or even aggravate the condition of oral ulceration. age classification varied between countries and over time, but as far back as 1875, in britain, the friendly societies act, defined the term old age as any age after 50. nowadays, in most developed world countries, the chronological age of 65 years as a definition of elderly or older person, but the united nation agreed begin 60 years to refer to the older population.3 after reaching the age of 40 years, people experience a progressive decline in homeostatic control and in the ability to respond to stress and change.4 in this paper we will discuss two case reports of elderly female patients who had oral ulceration. first case of recurrent oral ulceration experienced by 58 years old female patient and second case is concerning a 77 years old female patient with chronic oral ulceration. diagnosis at first upheld based on history and clinical examination, but further investigation will be plan, including blood testing. treatment plans was given to both patients includes nonpharmacological and pharmacological therapies. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p165-169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p165-169 166 nur’aeny/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 165–169 cases patient 1: a married woman, 58 years old, came with complaints of pain in her mouth since a year ago. patient had oral ulcer since came back from saudi arabia for umrah. she aware that the ulceration disappear and arise with tightly frequency. on average each ulcer occurs within 2 weeks, and then followed by a new ulcer. ulceration usually occur on the tongue, both labial mucosa, and both buccal mucosa which caused swollen. this time she complaint pain of the right side inner cheek and tongue. patient had seek treatment to many doctors and dentists, but complaints still occur. she did not know what caused the complaints, but she realized that there were somethings disturb her minds. patient feel desperate to face this condition. there is no abnormalities on extraoral examination, and the intraoral examination showed there are some ulcers in the right buccal mucosa. clinically there are 3 ovoid ulcers, approximately 3 mm of diameter, with yellowish in center and erythema in the border, the right buccal mucosa also look swollen. on the dorsum of tongue there are thick white layer (figure 1). according to world health organization system of tooth nomenclature, the dental status in this patient showed some missing teeth in 37, 36, 45, 46 tooth region, and some dental caries found in 16, 17, and 47 tooth region, but no dental fillings was found. almost all tooth region covered with stain, plaque, and calculus. working 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) figure 1. the initial condition. (personal documentation) 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) a b figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) b 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) c 9 figure 1. the initial condition. figure 2. oral conditions in 1 month later (ulcers healed). (personal documentation) (personal documentation) figure 3. patient came with more severe condition. a) coated tongue, b) ulcer spread on oral mucosa. (personal documentation) figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) a figure 4. condition of patient after 1 month, a) ulcer on left buccal mucosa, b) ulcer on dorsum of the tongue, c) ulcer on ventral of the tongue. (personal documentation) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p165-169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p165-169 167167nur’aeny/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 165–169 diagnosis for the oral lesion was made as suspected aphtous like ulcers (alu) for the ulcers in right buccal mucosa and suspected as acute pseudomembranous candidiasis for the tongue lesion. patient is advised to get back in 1 week, but she get back after 1 month. the ulcers healed, but she complaint of soreness on the tongue while brushing (figure 2). at this second visit, patient brought the result for the laboratory test. there were rbc 12,4 g/dl, wbc 8.500/ mm3, pcv 39%, pplatelet 391.000/mm3, esr 100 mm/h, basophil 0, eosinophil 1, neutrophil stab 3, neutrophil segmented 64 lymphocyte 28, monocyte 4. on the next visit (3rd visit), patient cannot comes within suggested time, because she has a problem due to a long distance between the residence and hospital. patient came with more severe condition than the earlier one. after she did not take any medicines, the complaint reappeared (figure 3). patient aware that the oral condition become severe triggered by the food, she was eating salt fish, although it has been advised since the beginning to not consume foods that contain preservatives. latest condition after 1 month later (6th visit), patient showed significant improvement (figure 4). patient 2: a woman, 77 years old, came with complaint of pain on the edge of the tongue that has suffered since 2 months ago. patients have been treated on several general practitioners and also specialists in internal medicine, but still not healed. patient said that she had a history of heart disease, hypertension, and diabetes mellitus. she is still taking medicines related to her condition until now. extraoral examination did not found any abnormalities. intraoral examination showed the dental status according who system, for entire teeth in the upper jaw and lower left were residual roots, and patient has weared dentures in the upper jaw. in 46 and 47 tooth region there are missing teeth. some dental filling are found in 26 and 48 tooth region. the oral lesion occurred as an ulcer, size approximately 1 cm on ventrolateral of the tongue or at 36 and 37 tooth region, ulcer surrounded by white areas, and indurated margin (figure 5). diagnosis was made as suspected traumatic ulcer due to residual roots of 36 and 37. after one week (2nd visit), patient came and showed good improvement, less indurated of the margin, and less pain. (figure 6a). a week later (3rd visit), also found more good improvement, the lesion was getting smaller, clinically appear as fissure (figure 6b). at this time, patient was still not dare to undergo any tooth extractions (36 and 37 teeth region), which has been recommended, but finally after she get more explanation about the possibility to recurrent the same oral ulceration, patient has willingness to get her tooth extraction. at 4th visit, the healing process became faster and the lesions were healed perfectly after eliminate the factors that cause trauma on the tongue (figure 7). cases management patient 1: patient was given non-pharmacological and pharmacological therapy. non-pharmacological therapies include communication, information, and education for the patient to avoid foods that were spicy, hot, contain preservatives, and suggestion to increase the consumption of water, fruits and vegetables. she was also received the oral hygiene instruction (ohi) consist of tooth brushing method and the technique of scrapping the tongue. pharmacological therapy at the initial visit include prescribing triamcinolone acetonide paste in ora base given topically, chlorhexidine mouthwash, multivitamins, folic acid, vitamin b12 administered orally. patient also asked to do the routine hematology test. then in second visit, patient was given prescribing of antifungal, which was nystatin oral suspension 4 times 1 ml a day, chlorhexidine mouthwash, immunomodulator, multivitamin, and suggestion to consume substitution foods. in 3rd visit, additional non pharmacology therapy was suggestion to avoid toothpaste 10 figure 5. indurated ulcer on ventrolateral of the tongue. (personal documentation) figure 6. healing ulcer after a) 1 week, and b) two weeks of treatment. (personal documentation) figure 7. ulcer healed perfectly after tooth extraction (36 and 37) (personal documentation.) figure 5. indurated ulcer on ventrolateral of the tongue. (personal documentation) 10 figure 5. indurated ulcer on ventrolateral of the tongue. (personal documentation) figure 6. healing ulcer after a) 1 week, and b) two weeks of treatment. (personal documentation) figure 7. ulcer healed perfectly after tooth extraction (36 and 37) (personal documentation.) b 10 figure 5. indurated ulcer on ventrolateral of the tongue. (personal documentation) figure 6. healing ulcer after a) 1 week, and b) two weeks of treatment. (personal documentation) figure 7. ulcer healed perfectly after tooth extraction (36 and 37) (personal documentation.) a figure 6. healing ulcer after a) 1 week, and b) two weeks of treatment. (personal documentation) 10 figure 5. indurated ulcer on ventrolateral of the tongue. (personal documentation) figure 6. healing ulcer after a) 1 week, and b) two weeks of treatment. (personal documentation) figure 7. ulcer healed perfectly after tooth extraction (36 and 37) (personal documentation.) figure 7. ulcer healed perfectly after tooth extraction (36 and 37). (personal documentation) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p165-169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p165-169 168 nur’aeny/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 165–169 that contain detergent. for medication, patient was added prednisone for rinsed and swallowed twice a day, each time 2 tablets were dissolved within 4 spoons of water. other drugs were still continued. in next visit (4th visit), patient suggested to stop prednisone and anti fungal, but still advised to continue the consumption of multivitamins, folic acid, vitamin b12, and the use of antiseptic, chlorhexidine daily at the same dose with previous. at the 5th visit, there were two small ulcers reappeared, so patient re-advised to use the topical triamcinolone to be applied into ulcers three times a day, and other medications were still continued. at the last visit, patient showed good improvement, but the patient still advised to consume the multivitamins, and folic acid to maintain the immune system, and to do all things as the non pharmacology therapy throughout her life. patient 2: non pharmacology therapy was as the same as patient 1, but the pharmacology therapies are quite different. this patient was given prednisone orally since the first visit, with the doses for three tablets (15 mg) in the morning and three tablets (15 mg) in the afternoon for a week. patient was also advised to applied triamcinolone acetonide paste in orabase to the lesion three times a day, after used antiseptic rinsed three times a day, and given multivitamin one tablet a day. for the next treatment at 2nd visit, patient was given tappered dose of prednisone tablet for two tablets (10 mg) in the morning and two tablets (10 mg) in the afternoon for a week, others drugs were still continued. patient was also suggested to have tooth extraction in 36 and 37 teeth region. at the 3rd visit, the condition was improved, prednisone dose was tappered into one tablet (5 mg) in the morning and one tablet (5 mg) in the afternoon for a week, others drugs were still continued, until the 4th visit, except the dose of prednisone which was planned to be stop, one tablet (5 mg) as alternate dose for a week. discussion aging can cause physiological changes in oral cavity.5 during the aging process, oral mucosa loses much of its efficacy, getting predisposed to oral lesions. elderly mostly related with some systemic condition due to their physiological changes, an several systemic factors not only influences the patient’s ability to maintain oral hygiene and promote the oral health, but also can actually be related to the occurrence of certain oral diseases or condition and among those are the intake of drugs.6,7 though impairments are not life threatening, they affect a person’s quality of life.7 handling both the patients in this case reports requires slightly different attention with the younger patients because the complexity of factors owned by elderly patients. they are more susceptible to oral conditions due to age-related systemic diseases, functional changes, pharmacotherapy, and cognitive impairment.8,9 in patient 1, a 58 years old woman, the recurrent ulceration was diagnosed as an aphthous like ulcer (alu), due to many suspected causes factors such as food preservative, microorganism, and emotional stress. the frequency of ulceration is quite often after she came back from umrah a year ago, this can be as a trigger factor to decreased the immune system due to her exhausted condition and different weather. she also said that she often had emotional stress due to some problems from the job and families . psychological factors may be an important factor as some patients notice that their ulcers become worse in periods of illness, stress or extreme fatigue. some form of stress management counselling might be considered in some of these cases.2 this condition was diagnosed more as alu than other similar clinical feature which was recurrent aphthous stomatitis (ras), due to late occured (above 40 years old), and related with systemic condition. the blood test showed normal values except for the increased esr value. esr stands for erythrocyte sedimentation rate that indirectly measures how much inflammation is in the body and is used often as a nonspecific measure in monitoring disease activity.10 patient did not have any complaints in other parts of body, so the increased level of esr was assumed due to the oral inflammation, clinically as oral ulceration and suspected oral candidiasis. oral candidiasis occured in this patient related to more decreased of immune system due to lack of nutrition, especially as protein-energy malnutrition that often happened in elderly patient. malnutrition may in turn lead to poor tissue healing and predispose to ill-health.11 this patient given suggestion to consume a subtitution food contains high protein, such as peptisol® / ensure® to raised an adequate immune system. a compromised nutritional status, in turn can further undermine the integrity of the oral cavity are closely interrelated, diet and nutrition should be considered as an integral part of the oral health assessment and management of the elderly.7 multivitamins and immunomodulator given in this patient also increased the immune system because in the recurrent ulceration often occur hematologic deficiency including serum iron, folic acid, or vitamin b12.12 vitamin b12 can influence the production of rbc so the blood supply can be support by the consumption this supplement. patient complaints pain when scrapped with tongue scrapper, this was said when patient came at 2nd visit. the sign and symptom of oral candidiasis appeared as creamy, white plaques on the tongue and when scrapped, it leaved a red, painful ulcerated surface exposed.6 began at the 2nd month of therapies, patient taking nystatin as anti fungal with 1 ml dose 4 times a day for a week. nystatin is one of fungal polyenes and used as the first line antifungal agent for oral candidiasis without systemic candida infections.13 patient was also suggested to scrapped tongue gently to desquamated the layer of debris, and also to create hygiene environment of the oral mucosa. this activities of maintaining oral hygiene includes teeth brushing twice a day and scrapping the tongue can improve the oral health. after healed from the oral candidiasis patient then dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p165-169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p165-169 169169nur’aeny/dent. j. (majalah kedokteran gigi) 2015 december; 48(4): 165–169 suggested to rinsed often with chlorhexidine gluconate 0.2% as an antiseptic mouthwash 2-3 times daily after teeth brushing. patient was very pleasant with her condition after rinsed with this medicine, and sometimes she increased the schedule of rinsed because she feel comfotable after used it. a decline in protective barrier function of the oral mucosa could expose the aging host to myriads of pathogens and chemicals that enter the oral cavity during daily activities.7 during the treatment she got two times recurrency of ulceration triggered by food includes its ingredients and preservatives, such as salt fish, coconut milk, and spicy food. this condition maybe related to hypersensitivity reaction of food after contact to oral mucosa. from the first time of therapy, patient often did not follow the instruction, she still consume various kind of food that could trigger oral ulceration, and the patient is not disciplined in following the treatment schedule, so often withdrawal eventually led to reappeared the ulceration. patient 2 is a woman, 77 years old who have traumatic lesion on lateral of tongue due to friction from the sharp part of residual teeth. thinner and smooth oral mucosa and also a decreased rate of wound healing often found with age.7 this condition made oral mucosa is more fragile when exposed to something sharp and in this patient was occured as a chronic ulcerative lesion. this patient also has history of mild hypertension, cardiovascular disease and diabetes mellitus. according to her age it was concluded that her immune system was not as good as her immune system when she was younger. this medical problems didn’t involve with oral lesion. no oral complication have been associated with the hypertension itself.4 diabetic condition often associated with dry mouth and poor wound healing,4 although this patient didn’t complaint of dry mouth, but this condition may aggravate the friction into the oral mucosa. patients also experienced some hard times after her husband passed away a few months ago. patient was given steroid systemic as the adequat drugs to help decreased the inflammatory reaction, beside that patient also referred to oral surgeon to have teeth extracted as the causes of the ulcerative due to her medical problems. non pharmacology therapy also play important role in healing process, and this patient have a good compliance to follow the therapy. in conclusion, oral ulceration in elderly patients with or without a medical problems becomes a challenging thing to handle due to the complexity of their condition. as a dentist we have more careful to arrange the treatment plans for elderly patients when combine with some related systemic disease therapy. references 1. scully s. oral and maxillofacial medicine. the basis of diagnosis and treatment. 2nd ed. 2. edinburgh: churchill livingstone elsevier; 2008. p. 131-9. 3. talacko aa, gordon ak, aldred mj. the patient with recurrent oral ulceration. aust dent j 2010; 55:(1 suppl): 14–22. 4. department of economic and social affairs population division. world population ageing. new york: united nation; 2013. p. 1-114. 5. little jw, falace da, miller cs, rhodus nl. dental management of the medically compromised patient. 7th ed. st louis: mosby elsevier; 2008. p. 35-50, 212-35. 6. pardis s, taheri mm, fani mm. oral and maxillofacial lesions in an elderly population in shiraz, iran. avicenna j dent res 2014; 6(1): 1-4. 7. jayakarann tg. the effect of drugs in the oral cavity-a review. j pharm sci and res 2014; 6(2): 89-96. 8. razak pa, richard kmj, thankachan rp, hafiz kaa, kumar kn, sameer km. geriatric oral health : a review article. j int oral health 2014; 6(6): 110-6. 9. ko-yeh c, katz ms, saunders mj. geriatric dentistry: integral component to geriatric patient care. taiwan geriatrics & gerontology 2008; 3(3): 182-92. 10. al dress am. oral and perioral physiological changes with ageing. pakistan oral & dental journal 2010; 30(1): 26-30. 11. vennapusa b, cruz ldl, shah h, michalski v, zhang qy. erythrocyte sedimentation rate (esr) measured by the streck esr auto plus is higher than with the sediplast westergren method. am j clin pathol 2011; 135: 386-90. 12. scully c. medical problems in dentistry. 6th ed. edinburg: churcill livingstone elsevier; 2010. p. 573-6. 13. woo sb, greenberg ms. ulcerative, vesicular, and bullous lession. in: burket’s oral medicine. 11th ed. hamilton: bc decker inc; 2008. p. 57-60. 14. lamont rj, burne ra, lantz ms, leblanc dj. oral microbiology and immunology. washington: asm press; 2006. p. 333-45. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i4.p165-169 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i4.p165-169 110 vol. 41. no. 3 july–september 2008 effect of various temperature and storage duration on setting time of orega sealer bambang sunarko department of conservative dentistry faculty of dentistry airlangga university surabaya – indonesia abstract background: choosing the right rigid material and root canal paste are crucial in the success of root canal obturation. n choosing the right rigid material and root canal paste are crucial in the success of root canal obturation. n2 is a root canal paste containing formaldehyde, which is toxic and carcinogenic. whilst zinc oxide, resorcin, eugenol, glycerin, and hydrochloric acid, abbreviated as orega, are considered a safer root canal paste. in order to perform good obturation, root canal paste’s setting time plays an important role. this is connected with how long and in what temperature the paste’s substances are stored. purpose: this experiment was performed to find out the effect of various temperature and storage duration on the setting time ofthis experiment was performed to find out the effect of various temperature and storage duration on the setting time of orega sealer. method: orega and n orega and n2 sealers were used as samples. eighty sealer samples were produced for both sealers providing 10 samples foe each testing category. each of these samples were stored in 27°c room temperature, 4°c refrigerator temperature, and put into storage for the duration of 0, 1, 2, and 3 months. after these treatments, the samples were tested and analyzed. result: datadata collected were analyzed by two-way anova, showing no significant difference of the setting time among temperature and storage duration (p> 0.05). conclusion: temperature and storage duration do not affect the setting time of orega root canal paste. temperature and storage duration do not affect the setting time of orega root canal paste. key words: orega new sealer, setting time, temperature and storage correspondence: bambang sunarko, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction there are two types of root canal treatments, pulpectomy and intracanal endodontic. pulpectomy is root canal treatment followed by removing of either healthy or unhealthy vital pulp tissue. pulpectomy requires local anesthesia to relief the pain during treatment. intracanal endodontic is a root canal treatment performed in non vital teeth and called pulp cavity debridement. the main treatment of pulpectomy and intracanal endodontics are preparation, sterilization and obturation (root canal filling).1 root canal preparation cleans pulp tissue, bacteria, infected dentinal tissue and widen root canals to make sterilization and obturation easy. root canal sterilization releases root canals and pulp chambers from micro organisms. the purpose of root canal filling is to close and fill root canals tightly especially in one third of apical regions. solid and soft root canal filler materials are needed to fill the root canal. silver point, acrylic point, amalgam and guttap point are solid materials. root canal paste is the soft material, which is used to fill apical loopholes, additional root canals and ramifications.2 theoretically, root canal paste could manage root canal treatment either on vital or non vital teeth with or without periapical impairment, however in reality, the treatment evaluation still has various results. the formulation of root canal paste is created in consideration that root canal paste will not contain formaldehyde, has setting time equal the working time of root canal filling and safe. formaldehyde is a gas material, soluble either in water or fluid at body temperature, relatively stable, toxic, but carcinogenic if used in high concentrations; therefore it is not recommended.3,4 the material discuss in this study has proven to be appropriate in setting time and working time, effective and safe.5 the formulation of root canal paste consist of zinc oxide powder research report 111sunarko: effect of various temperature and storage duration figure 1. laterel view (lv) and front view (fv) of the setting time instrument (iso 4823. 1984). note: a : gillmore nedle, b : load, c : sliding place for gillmore nedle, d : c support, e : d support, f : buttom of the instrument, g : scale, h : indicator and s = sample. and solutions containing resorcin, eugenol, glycerin, and hydrochloride acid. this mixture identified by layer chromatography and called orega. this material is easily obtained in indonesia, cheaper than n2 containing formaldehyde which was usually used in faculty of dentistry airlangga university since 1997. in one visit root canal treatment, spad root canal paste was used. this material has resin content and mutagenic.6 powder and liquid mixing is always done in manipulation of orega and n2. the most determining factor is setting time which has measured since initial mixing until the hardening period that shown by gillmore time indicator set in zero position.7 root canal paste was stored in room temperature (27° c) or in refrigerator (4° c). other component which could be affected by different temperature were materials containing zinc oxide. at lower temperature zinc oxide would easily bind oxygen from the air, oxidation would occur and changed into zinc dioxide. both materials observed consist of zinc oxide, therefore, they are both strongly affected by temperature and storage. the purpose of this study is to find the effect of temperature and storage on setting time of orega sealer. it is hoped the achieved root canal paste which has been proven to be effective on temperature and storage will to support better treatment result. material and method the composition of orega root canal paste are 0.2 gr zno powder, liquid mixture; 0.03 gr : resorcin, 0.032 gr : eugenol, 0.042 gr: glycerin, 0.025 gr hydrochloride acid. the composition of n2 root canal paste is: paraformaldehyde, bismuth salt, zno, eugenol, rose oil and the other percentage is not mentioned by manufacturer agsa japan co. ltd. the applied instrument is: gillmore indicator, glass lab, cement spatula, time recorder (seiko japan), plastic ring, refrigerator. the sample test used the instrument schematically shown on figure 1. sample preparation, fabrication of orega root canal paste using thin layer chromatography to obtain formulation of orega root canal paste fulfilling setting time, methanol-acetone were used as eluent with ratio 1:1, chloroform-methanol 2:1, methanol-water1:2, acetonewater 1:1. methanol has boiling point 64.6° c/760 torr, acetone is 56.5° c/ 760 torr, chloroform is 61.3°c/760 torr and water has boiling point 100° c/760 torr.8,9,10 the test was coroucted seventeen times to achieve the composition appropriate to the composition of orega root canal paste i.e: 0.2 gr zinc oxide; 0.03gr resorcin fluid; 0.032 gr eugenol, 0.042 gr glycerin; 0.025gr hydrochloride acid. setting time test on samples were classified into two groups: orega or n2 root canal paste at 27° c and 4° c and storage is 0, 1, 2, 3 months. the procedure: powder and liquid of orega and n2 root canal paste with ratio between powder and liquid volume: 60 gr : 60 ml was stirred, using cement spatula and put into ten plastic rings on glass plate. sample(s) put at the bottom of the instrument (f) gillmore indicator was pressed on the sample surface repeatedly on different places, setting time was considered complete after gillmore indicator could not penetrate sample surface. this procedure was conducted 10 times to examine setting time of orega and n2 root canal paste at room temperature and refrigerator in 0, 1, 2, 3 months. result the mean setting time in 0 month at 27° c for orega root canal paste is 3610.8 second and n2 is 5419.7 score, indicate that: setting time in 0 month at 27° c is faster compared to n2, while setting time in 0 month at 4° c lv 112 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 110-113 table 1. mean and standard deviation of orega and n2 paste setting time (second) rcp temperature n month 0 x + sd month 1 x + sd month 2 x + sd month 3 x + sd orega n2 27° c 4° c 27° c 4° c 10 10 10 10 3610.8 + 1.3166 3610.7 + 2.4518 5419.7 + 6.2370 5419.6 + 6.9314 3610.9 + 1.6633 3614.8 + 2.2509 5419.8 + 8.4696 5421.6 + 8.3160 3611.1 + 1.1972 3614.8 + 1.7512 5420.0 + 7.9722 5427.2 + 9.1141 3611.4 + 0.9661 3622.9 + 2.1833 5420.1 + 7.9722 5434.6 + 9.1141 note: rcp = root canal paste; x = mean; sd = standard deviation orega was 3610.7 score and n2 is 5419.6 second, setting time in 0 month at 4° c orega is faster compared to n2. the mean setting time in the 1st month at 27° c: orega is faster than n2, while setting time in the 1 st month at 4° c, orega is 3614.8 second and n2 was 5421.6 second, showing month 1, at 4° c setting time, orega is faster than n2. the mean setting time in the 2nd month at 27° c, orega: 3611.1 second and n2: 5420 second showing orega setting time at 27° c is faster compared to n2, while setting time in the 2 nd month at refrigerator temperature, orega setting time: 3614.8 second and n2: 54127.2 second, showing setting time in the 2nd month at 4° c, orega is faster compared to n2. the mean setting time in the 3rd month at 27° c orega: 3611,4 second and n2: 5420.1 second showing orega setting time in the 3 rd month at 4° c is faster compared to n2 while setting time in month 3 at refrigerator temperature orega: 3622.9 second n2: 5434.6 second showing orega setting time is faster than to n2. two way anova test was performed to know the effect of temperature and storage on setting time of orega root canal paste, showing that there was no significant difference between temperature and storage in the setting time of the root canal paste (p>0.05). discussion formula of orega root canal paste is similar to the criteria of root canal paste, resorcin, eugenol, glycerin, with exact amount bound by zinc oxide. resorcin less than 0.030 gram. the hardness is 30 newton, hydrochloride acid is less than 0.025 the setting time is 50000 second. the use gillmore test is applied in setting time based on the appropriate standard. in this study, the method of factorial experiment with perfect random design was used and in this method three factors are considered to affect the accuracy of variable i.e.: temperature, storage, the type of root canal paste. room temperature (27° c) and refrigerator temperature (4° c) were the temperature factor which is the normal temperature for the existence of a material. zinc oxide is a component material which could be affected by different temperature. both materials which would be compared: orega and n2 containing zinc dioxide therefore temperature could be reasonably applied as definitive variable. storage is a definitive variable correlated with the storage temperature, it is assumed that the longer the material is kept at a certain temperature, the more the chemical binding would be affected if the material is not stable. orega root canal paste consist of zno powder and liquid containing resorcin, eugenol, glycerin and hydrochloride acid; eugenol was mixed with resorcin in an untransparent bottle, glycerin was mixed with hydrochloride acid placed in an untransparent bottle. n2root canal paste consists of powder and liquid containing paraformaldehyde, bismuth salt, zinc oxide, eugenol, reso oil and so on. data analysis found that there was no effect of temperature and storage on setting time of the orega and n2 new sealer. mean setting time of orega new sealer compared to n2 found that orega new sealer setting time is faster. however, there is still enough time to do root canal filling, so it fulfills to the requirement of root canal paste11,12 due to the effect of hydrochloride acid on setting time is 5000 second, 250 gr the setting time is 5 second. the requirement of root canal paste and material in medical field should be biocompatible.13,14,15 the setting of root canal paste is highly correlated with the following antibacterial trait, completeness of apex closing and reaction with periapical tissue, while restorative material correlated with the strength on abrasion during clinical use.16,17 the conclusion is that the temperature and storage will not affect the setting time of orega root canal paste. references 1. cohen sc, burns rc. pathways of pulp. 6th ed. st louis: cv mosby co; 1994. p. 219–26, 230–3, 264–66. 2. combe ec. notes on dental material. 8th ed. edinburg, london, new york: churchill livingstone; 1996. p. 7–13. 3. haddad lm, winchester jf. clinical management of poisoning and drug overdose. philadelphia, london, toronto, mexico city, rio de jainero, sydney, tokyo: wb saunders co; 1990. p. 537–41. 4. world health organization. formaldehyde: environmental health criteria. geneva: who; 1989. p. 14–135. 5. reynolds jef. martindale: the extra pharmacopoeia. 31st ed. london: royal pharmaceutical society; 1996. p. 311–29, 576, 712, 1705. 6. schweikl h, schmalz g, stimmelmayr h, bey b. mutagenicity of ah26 in an in vitro mammalian cell mutation assay. j endod 1995; 21:407–10. 7. american dental association. guide to materials and devices. 8guide to materials and devices. 8th ed. chicago: illionis; 1984. p. 189–96. 113sunarko: effect of various temperature and storage duration 8. johnson el, stevenson r. 1972. basic liquid chromatography: dasar kromatografi cair. padmawinata k, editor. bandung: penerbit itb; 1978. p. 1–2. 9. yost rw, ettre ls, conlon rd. practical liquid chromatographypractical liquid chromatography an introduction. 1st ed. england: perkin elmer ltd; 1980. p. 6, 72–116. 10. sastrohamidjojo h. kromatografi. edisi i. yogyakarta: universitas gadjah mada 1991. p. 1–3. 11. nicholls e. endodontics. 1st ed. bristol: john wright ltd; 1977. p. 33–63, 72–87. 12. grossman li, oliet s, del rio ce. endodontic practice. 11endodontic practice. 11th ed. philadelphia: lea and febriger; 1988. p. 102–14, 126–31, 179–225. 13. craig, rg, powers, jm. restorative dental material. 11th ed. london, usa: cv mosby inc; 2002. p. 50–53, 56–57 14. palma, rg, matson e, ramos, rp. microhardness of estheticmicrohardness of esthetic restorative materials at different depths. materials research 2002; 6(1): 85–90. 15. van noort r. introduction to dental material. 2nd ed. st. louis: cv mosby company; 2003. p. 124–35. 16. anusavice kj. phillips science of dental materials. 11st ed. philadelphia: saunders; 2003. p. 471–7. 17. sukaton. perbedaan lama penyinaran glass ionomer cement systemlass ionomer cement system dual cure terhadap kekerasan permukaan. karya tulis akhir.karya tulis akhir. surabaya. 2007. p. 1–48 198 volume 47, number 4, december 2014 daya antibakteri penambahan propolis pada zinc oxide eugenol dan zinc oxide terhadap kuman campur gigi molar sulung non vital (the antibacterial effect of propolis additional to zinc oxide eugenol and zinc oxide on polybacteria of necrotic primary molar) yemy ameliana, herawati, dan seno pradopo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya-indonesia abstract background: materials commonly used for root canal filling of primary teeth is zinc oxide eugenol. eugenol has some disadvantages that can irritate the periapical tissues, has the risk of disturbing the growth and development of permanent tooth buds, and has a narrow antibacterial spectrum. studies showed that propolis at concentration of 20 % has antibacterial activity against staphylococcus aureus. purpose: the purpose of this study was to examine the antimicrobial activity of root canal pastes with the additional of propolis additional to zinc oxide eugenol (zoep) and to zinc oxide (zop). methods: polybacteria cultures collected from root canals of necrotic primary molar from 5 children patients who received root canal treatment. the bacteria were grown in bhi broth, and inoculated into muller hinton agar media. the agar plates was divided into 3 areas, and one well was made at each area. the first well filled with zoe as a control, second well filled with zoep and the third well filled with zop, then incubated for 24 hour at 370 c. antimicrobial activity was determined by measuring the diameters of inhibition zones of polybacteria growth. the data were statistically analyzed by independent t-test. results: the pasta mixture of zinc oxide propolis had the strongest antibacterial activity against polybacteria of necrotic primary molar, followed by zinc oxide eugenol propolis paste, and zinc oxide eugenol paste. there were significant differences of inhibition zones between zoe, zoep and zop (p<0,05). conclusion: the study suggested that the additional of propolis to zinc oxide paste could increase the antimicrobial effect against root canal polybacteria of necrotic primary molar. key words: antimicrobial activity , propolis, zinc oxide, eugenol abstrak latar belakang: bahan yang sering digunakan untuk pengisian saluran akar gigi sulung adalah zinc oxide eugenol. eugenol memiliki beberapa kekurangan yaitu dapat mengiritasi jaringan periapikal, beresiko mengganggu pertumbuhan dan perkembangan benih gigi permanen pengganti, serta memiliki spektrum antibakteri yang sempit. penelitian menunjukkan propolis dengan konsentrasi 20% memiliki daya antibakteri terhadap bakteri staphylococcus aureus tujuan: penelitian ini bertujuan meneliti efek aktivitas antimikroba pasta saluran akar dengan penambahan propolis pada zinc oxide eugenol (zoep) dan pada zinc oxide (zop) metode: kultur bakteri campur diperoleh dari saluran akar gigi molar sulung 5 pasien anak yang sedang dirawat saluran akar. bakteri ditumbuhkan dalam bhi broth dan diinokulasi ke muller hinton agar media. plate agar dibagi menjadi 3 bagian, dan setiap bagian dibuat satu sumuran. sumuran pertama diisi dengan zoe sebagai kontrol, sumuran kedua diisi dengan zoep dan sumuran ketiga diisi dengan zop, kemudian diinkubasi selama 24 jam, pada 37°c. daya antimikroba ditentukan dengan mengukur diameter zona hambatan pertumbuhan bakteri campur. data dianalisis statistik dengan t-test independent. hasil: pasta campuran zinc oxide propolis memiliki daya antibakteri terhadap kuman campur gigi molar sulung non vital paling kuat, diikuti pasta zinc oxide eugenol propolis, dan pasta research report 199ameliana, et al.: daya antibakteri penambahan propolis pada zinc oxide eugenol dan zinc oxide zinc oxide eugenol. terdapat perbedaan zona hambat yang signifikan diantara zoe, zoep dan zop (p<0.05). simpulan: penelitian ini menunjukkan bahwa tambahan propolis pada pasta zinc oxide dapat meningkatkan efek antimikroba terhadap polybacteria saluran akar dari molar sulung yang nekrotik. kata kunci: aktivitas antimikroba, propolis, zinc oxide, eugenol korespondensi (correspondence): yemy ameliana, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: herawati@gmail.com pendahuluan karies gigi masih merupakan penyakit gigi dan mulut yang paling umum dijumpai pada anak-anak di indonesia. karies pada gigi molar sulung cepat meluas dan menyebabkan terbukanya pulpa. pulpa yang terbuka menjadi jalan masuk mikroorganisme yang dapat menyebabkan inflamasi, dan bila berlanjut mengakibatkan pulpa menjadi non vital.1 usaha untuk mempertahankan gigi sulung non vital sampai gigi pengganti erupsi ialah dengan perawatan pulpektomi.2 tujuan perawatan pulpektomi yaitu mengontrol sepsis pada pulpa dan jaringan periradikular, sehingga gigi sulung dapat dipertahankan dalam keadaan non patologis sampai gigi pengganti siap tumbuh.3 pulpektomi meliputi tiga tahap, yaitu preparasi, sterilisasi, dan pengisian saluran akar. preparasi dan sterilisasi saluran akar dilakukan untuk mengeliminasi semua bakteri patogen pada pulpa dan saluran akar yang terinfeksi, akan tetapi dalam studi klinis bakteri-bakteri patogen masih dapat ditemukan bertahan dalam saluran akar yang nantinya berperan dalam kegagalan perawatan pulpektomi karena sisa-sisa mikroorganisme di dalam saluran akar dapat tumbuh dan menimbulkan infeksi dikemudian hari. oleh sebab itu diperlukan bahan pengisi saluran akar yang memiliki daya antibakteri untuk membunuh bakteri yang masih bertahan.4 bahan yang sering digunakan untuk pengisian saluran akar gigi sulung adalah zinc oxide eugenol. eugenol bersifat analgesik ringan, dan antibakteri. eugenol memiliki beberapa kerugian yaitu dapat mengiritasi jaringan periapikal, mengakibatkan nekrosis pada tulang dan sementum, ada resiko mengganggu pertumbuhan dan perkembangan benih gigi permanen pengganti, serta hanya memiliki spektrum antibakteri yang kecil. beberapa penelitian klinis yang dilakukan pada hewan dan manusia menunjukkan bahwa tingkat keberhasilan perawatan pulpektomi gigi sulung dengan zinc oxide eugenol sebagai bahan pengisi saluran akar adalah sebesar 65-95%. untuk meningkatkan keberhasilan perawatan pulpektomi gigi sulung, dicari suatu bahan tambahan yang dapat dicampurkan pada zinc oxide eugenol, atau bahan alternatif yang memiliki sifat-sifat yang lebih baik dari zinc oxide eugenol. pada tahun 1990 penelitian yang dilakukan di rusia, merekomendasikan propolis sebagai bahan pengisian saluran akar gigi karena memiliki efek antibakteri, anti inflamasi, anestetik dan merangsang regenerasi struktur tulang.5-7 penelitian secara in vitro menunjukkan propolis dengan konsentrasi 20% memiliki daya antibakteri terhadap bakteri staphylococcus aureus, merupakan salah satu bakteri yang terdapat dalam saluran akar gigi non vital.8 berdasarkan penelitian tersebut, penulis melakukan penelitian pendahuluan untuk mengetahui daya antibakteri konsentrasi propolis yang akan ditambahkan pada bahan zinc oxide, terhadap kuman campur gigi sulung non vital, dengan membandingkan propolis konsentrasi 20%, 30% dan 40%. hasilnya menunjukkan bahwa penambahan propolis dengan konsentrasi 40% pada zinc oxide memiliki daya antibakteri yang paling besar. selanjutnya toksisitas bahan ini diuji, dan didapatkan hasil bahwa penambahan propolis dengan konsentrasi 40% pada zinc oxide dan zinc oxide eugenol tidak toksik terhadap kultur sel fibroblas.9 propolis dan eugenol sama-sama memiliki efek antibakteri, tetapi belum diketahui bahan mana yang memiliki daya antibakteri yang lebih besar. dan apabila zinc oxide eugenol ditambahkan propolis apakah dapat meningkatkan daya antibakteri bahan tersebut. tujuan penelitian ini adalah untuk meneliti daya antibakteri setelah penambahan propolis pada zinc oxide eugenol dan zinc oxide terhadap kuman campur gigi molar sulung non vital. bahan dan metode penelitian ini merupakan penelitian analitik eksperimental laboratoris dengan mengambil bakteri campur dari gigi molar sulung pertama atau kedua, rahang atas atau rahang bawah non vital 5 pasien anak yang datang ke rumah sakit gigi dan mulut departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga dengan kriteria: (a) gigi belum pernah dilakukan perawatan; (b) rencana perawatan: pulpektomi lengkap; (c) penderita belum mendapat pengobatan dengan antibiotik. pengambilan bakteri campur menggunakan papper point steril nomor 15 yang dimasukkan ke dalam saluran akar sesuai panjang gigi dengan menggunakan pinset steril, dibiarkan selama 1 menit. bakteri ditumbuhkan pada brain hearth infusion broth (bhib), diinkubasi selama 24 jam pada suhu 370 c. dilakukan penipisan bakteri sesuai standar mc farland ½. bakteri yang telah ditipiskan, diusapkan secara merata pada media muller hinton agar (mha) dalam cawan 200 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 198–201 petri, setelah itu dibuat 3 sumuran dengan cara meletakkan cincin platinum yang berukuran 5 mm dan tinggi 3 mm dari permukaan dengan pinset steril. pasta zinc oxide eugenol dimasukkan pada sumuran pertama, menggunakan excavator, sumuran kedua diisi dengan pasta campuran zinc oxide eugenol + propolis 40%, sumuran ketiga diisi pasta campuran zinc oxide + propolis 40%, kemudian diinkubasi selama 24 jam dalam inkubator pada suhu 37°c. daya antibakteri dapat diketahui dari zona hambat yang terjadi pada semua sampel. pengukuran zona hambat menggunakan alat jangka sorong, dengan cara mengambil dua garis saling tegak lurus melalui titik pusat lubang sumuran, garis ketiga diambil diantara kedua garis tersebut membentuk sudut 450. untuk mendapatkan ketepatan, pengukuran dilakukan sebanyak tiga kali pada tempat yang berbeda, kemudian diambil rata-ratanya. semakin besar diameter zona hambat, semakin besar daya antibakteri bahan yang diuji. data yang diperoleh, dianalisa secara statistik menggunakan independent t-test. hasil terdapat perbedaan yang bermakna nilai diameter zona hambat antar masing-masing kelompok bahan uji (p<0,05), dimana zinc oxide propolis memiliki diameter zona hambat yang paling besar, diikuti oleh zinc oxide eugenol propolis, dan zinc oxide eugenol memiliki diameter zona hambat yang paling kecil. (tabel 1 dan 2) pembahasan daya antibakteri pasta campuran zinc oxide propolis lebih besar dibanding pasta zinc oxide eugenol disebabkan mekanisme kerja propolis dalam membunuh bakteri lebih lengkap dibanding eugenol. bahan aktif penghambat pertumbuhan bakteri oleh propolis adalah flavonoid (pino-cembrin dan galangin). sebagai antibakteri flavonoid bekerja dengan menghambat perkembangan mikroorganisme karena mampu membentuk senyawa kompleks dengan protein melalui ikatan hidrogen. mekanisme kerjanya dengan mendenaturasi molekulmolekul protein dan asam nukleat yang menyebabkan koagulasi dan pembekuan protein yang akhirnya akan terjadi gangguan metabolisme dan fungsi fisiologis bakteri. jika metabolisme bakteri terganggu maka kebutuhan energi tidak tercukupi sehingga mengakibatkan rusaknya sel bakteri secara permanen yang pada akhirnya menyebabkan kematian bakteri.8 flavonoid menyebabkan terjadinya kerusakan permeabilitas dinding sel bakteri, mikrosom, dan lisosom sebagai interaksi flavonoid dengan dna bakteri. flavonoid mampu melepaskan energi transduksi terhadap membran sitoplasma bakteri, selain itu juga menghambat motilitas bakteri. gugus hidroksil yang terdapat pada struktur senyawa flavonoid menyebabkan perubahan komponen organik dan transpor nutrisi yang akhirnya akan mengakibatkan timbulnya efek toksik terhadap bakteri.10 propolis memiliki aktivitas antibakteri mirip dengan antibiotik, yaitu mencegah pembelahan sel, juga merusak sitoplasma dan dinding sel bakteri, menghambat sintesis protein bakteri dengan cara menghambat polimerase dna-dependant rna.7 eugenol memiliki tegangan permukaan yang lebih tinggi dari bakteri sehingga dapat berkontak dengan bakteri, bahan ini dapat membunuh bakteri dengan cara merusak membran sitoplasma bakteri, mengakibatkan peningkatan permeabilitas membran sel bakteri. selanjutnya cairan di dalam membran sel bakteri keluar sehingga bakteri lisis.5 daya antibakteri pasta campuran zinc oxide propolis yang lebih besar dibanding pasta campuran zinc oxide eugenol propolis disebabkan oleh kemampuan zinc oxide propolis dalam berdifusi ke dalam media kultur lebih tinggi daripada zinc oxide eugenol propolis dan zinc oxide eugenol, dapat dilihat pada media agar, dimana terdapat warna kuning kecokelatan disekeliling sumuran bahan zinc oxide propolis. bahan zinc oxide eugenol bila ditambahkan dengan larutan lain akan mempengaruhi reaksi kimia antara bubuk zinc oxide dan cairan eugenol, mengakibatkan waktu pengerasan bahan lebih cepat,11 sehingga kandungan air dalam pasta campuran zinc oxide eugenol propolis sangat rendah. hal ini membuat kemampuan difusi pasta campuran zinc oxide eugenol propolis ke dalam media kultur rendah. daya antibakteri zinc oxide propolis yang paling besar pada penelitian ini, menunjukkan adanya kemungkinan zinc tabel 1. rerata dan standar deviasi nilai diameter zona hambat pada kelompok penelitian kelompok n rerata standar deviasi zinc oxide eugenol 5 11,9880 0,54792 zinc oxide eugenol + propolis 40% 5 14,6760 1,32124 zinc oxide + propolis 40% 5 24,4200 3,46900 tabel 2. uji beda nilai diameter zona hambat antara masingmasing kelompok penelitian menggunakan uji independent t-test zinc oxide eugenol zinc oxide eugenol + propolis zinc oxide + propolis zinc oxide eugenol 0,007* 0,001* zinc oxide eugenol + propolis 0,002* zinc oxide + propolis keterangan: * = ada perbedaan bermakna (p<0,05) 201ameliana, et al.: daya antibakteri penambahan propolis pada zinc oxide eugenol dan zinc oxide oxide dan propolis memiliki efek sinergis dalam membunuh bakteri. bahan ini dapat digunakan sebagai bahan alternatif pengisi saluran akar gigi sulung pada perawatan pulpektomi, karena memiliki daya antibakteri yang lebih besar dibanding zinc oxide eugenol, tetapi diperlukan penelitian lebih lanjut secara in vivo. penelitian ini menunjukkan bahwa tambahan propolis pada pasta zinc oxide dapat meningkatkan efek antimikroba terhadap polybacteria saluran akar dari molar sulung yang nekrotik. daftar pustaka 1. widhianti, i, suwelo, is. perawatan saluran akar satu kali kunjungan pada gigi insisivus sulung non vital. jurnal kedokteran gigi universitas indonesia 2003; (edisi khusus): 693-698. 2. welbury r, duggal m, hosey mt. paediatric dentistry. 3rd ed. oxford university press; 2005. p. 169-72. 3. sidharta w. pemikiran rasional perawatan saluran akar gigi. jurnal kedokteran gigi universitas indonesia 2003; (edisi khusus): 68892. 4. kayaoglu, g, er ten, h, alacam, t, orstavik d. shor t term antibacterial activity of root canal sealers towards e. faecalis. international endodontic j 2005; 38(7): 483. 5. piva f, faraco junior im, estrela c. antimicrobial activity of different root canal filling paste used in deciduous teeth. j materials reserch 2008; 11(2): 171-3. 6. chawla hs, setia s, gupta n, gauba k, goyal a. evaluation of a mixture of zinc oxide, calcium hydroxide, and sodium fluoride as a new root canal filling material for primary teeth. the indian social pedodontic preventive dentistry j 2008; 53-7. 7. wander p. health from the hive: apllications of propolis in dentistry. dm jan clin 2005; 50-1. 8. dwiandiari hp, widjijono, sastromihardjo w. pengaruh konsentrasi propolis terhadap staphylococcus aureus. indonesian j dent 2006; 13(3): 160-3. 9. pratiwi i. toksisitas penambahan propolis pada pasta zinc oxide eugenol dan pasta zinc oxide terhadap sel fibroblast. surabaya. karya tulis akhir. surabaya: universitas airlangga; 2010. h. 37. 10. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). majalah kedokteran gigi (dent j) 2005; 8(35): 135-41. 11. roberson t m, hyma n ho, swif t e j. dent a l mater ia l. i n: studervant’s art & science of operative dentistry. 4th edition. st. louis: mosby; 2002. p. 173. 154 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 155–159 experimental comparative study and fracture resistance simulation with irrigation solution of 0.2% chitosan, 2.5% naocl and 17% edta ernani,1 trimurni abidin,1 and indra2 1department of conservative dentistry, faculty of dentistry universitas sumatera utara 2department of mechanical engineering, faculty of engineering universitas sumatera utara medan-indonesia abstract background: preparation in endodontic need irrigation materials as root canal debridement and disinfectant. however, irrigation materials is one of the factors that influence the tendency of fracture. purpose: this study was aimed to see the resistance and fracture distribution if teeth irrigated with high molecular horseshoe crab chitosan at 0.2% concentration, 2.5% naocl solution and 17% edta solution in endodontic treatment with finite element method (fem) simulation study and experimental studies. method: endodontic treatment performed on 28 maxillary premolars with group a: irrigation solution of 17% edta and 2.5% naocl solution; group b: 2.5% naocl irrigation solution; group c: 2.5% naocl irrigation solution and 0.2% chitosan solution; group d: 0.2% chitosan solution irrigation. final restoration was done using prefabricated glass fiber post. cementation of post using resin cement then restored with direct composite resin restorations. pressure test was performed with a universal testing machine with a speed of 0.5 mm/min until fracture occurred. a three dimensional finite element analysis was performed for total deformation, equivalent (von-mises) stress, and equivalent elastic strains. result: anova test showed significant differences in fracture resistance (p<0.05) in stroke difference between four groups. based on the results of the analysis of post-hoc bonferroni test and lsd, fracture resistance was significantly different between group a (1038.4±201.6) with group c (1515.6±243.3). kruskal-wallis test showed no significant difference in the distribution of fractures among the four groups (p>0.05). statistical analysis showed no significant difference (p>0.05) between the results of experiment and fem analysis results using the t-test. conclusion: the results of this study demonstrated that there was effect of the use of high molecular 0.2% chitosan as a combined irrigation with naocl, but did not affect the fracture pattern distribution of endodontically treated teeth both experimentally and fem analysis test. keywords: irrigation solution; chitosan solution; fracture resistance; finite element method correspondence: ernani, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas sumatera utara. jl. alumni no. 2 kampus usu padang bulan medan 20155, indonesia. e-mail: khairu_nisa2@yahoo.com introduction the post-endodontic teeth has a higher fracture risk than the vital teeth. the strength of post-endodontic teeth is directly proportional to the remaining healthy teeth structure and if the teeth structure is lost, the teeth fracture potential will be increasing.1 the cause of fractures in post endodontic teeth is multifactorial which are iatrogenic and noniatrogenic.2 some chemicals for endodontic irrigation are causing the changes in the chemical composition of dentin.3 endodontically trated teeth should have a good prognosis so it can function as a support to the final restoration.1,2 chitosan as natural polysaccharide after cellulose that is obtained through deacetylation of chitin have biocompatible properties, bioadhesion and nontoxic for human cells.4 trimurni et al. cit. pretty et al. 5 showed that chitosan blangkas (tachypleus gigas) with degrees 84.20% deasetilisasi and a molecular weight of 893 000 mv. is proven stimulate the dentinogenesis when it used as an dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p154-158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p154-158 155155ernani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 154–158 ingredient of pulp capping. chitosan liquid in hydrochloric acid produces dentin surface without the smear layer and contains collagen fiber6 while silva et al. showed that 0.2 % of chitosan is able to lift the smear layer compared to 15% of edta and 10% of citric acid.6 chitosan solution has a chelating properties which cause erosion of the dentine, yet it safe for intertubular dentin.4 mechanical destructive tests such as fracture test is important to analyze the nature of biomechanics and dental restorative materials that will be studied when given the heavy load. this test has limited capacity to explain the stress-strain relationships on dental restorations complex.7 the development of computer technology has increased the use of finite element method (fem) in various fields of science, especially in the dentistry. fem program can calculates the stress, strain and deformation in the three dimension view.8 an analysis of fem also obtains some informations such as distribution of internal pressure as compare to the experimental study. this study was aimed to find out the differences between naocl irrigation material that was combined with edta, and naocl that was combined with irrigation chitosan blangkas and the chitosan blangkas itself against the fracture resistance and fracture distribution after endodontic treatment using the experimental test and fem analysis test. materials and methodos twenty-eight of maxillary premolar that had been extracted for orthodontic purposes was suited to the inclusion criteria. the samples of this study were divided into 4 groups; (a) group a: teeth that were irrigated with 17% of edta and 2.5% of naocl solution; (b) group b: teeth that were irrigated with 2.5% of naocl solution; (c) group c: teeth that were irrigated with a solution of naocl 2.5% and 0.2% of chitosan solution, (d) group d: teeth that were irrigated with 0.2% of chitosan solution. the dental samples were planted on the gypsum then the pulp was opened by endo access bur (high speed) and root canal preparation was done by rotary instrument (protaper file), crown down system with x-smart endomotor (denstply, switzerland) using irrigation solution according to the test group treatment. the irrigation itself was using irrigation needle -shaped one side-vented size 30g (max-i-probe®, dentsply, switzerland). root canal was dried by paperpoint and then canal obturation was done. fiberglass post was put into a root canal using self adhesif brezee resin cement (pentron, usa), then followed by 20 second light cure. the teeth was restored by composite resin and polishing was done by enhance bur. six samples of teeth was released from the gypsum block and immersed in artificial saliva for 24 hours for thermocycling process.9 six teeth were planted on self curing acrylic in a cut of 10 ml spuit pieces. the process of pressure test was done to determine the strength of fracture resistance according to american society for testing and material (astm e1434-00, 2006). the samples were placed in acrylic base and tested with pressing test (torsee’s universal testing machine, japan). the samples were pressed from the occlusal side of the teeth to the axis of teeth (zero degrees). constant and slow pressure (not in the form of shock\/sudden pound) was given with direction speed of 0.5 mm/min until the fracture happened. the emphasis tool (zig) made by metal, sized 5 x 5 x 0.3 cm, flattened shape with rounded edges. the load invoice was immediately recorded right after the fracture of sample in newton (n), then fracture distribution also observed and recorded based on the location of the fracture. the fem test of this study was ansys program 14 by entering the data of modulus elasticity, poison ratio, density of each test material which is the preliminary decision stage (table 1).10 the next step was processing table 1. mechanical character of fem material test10 material modulus elastisitas poison ratio density g/cm3 email 41.0 gpa 0.30 ν 2.97 dentin 18.6 gpa 0.31ν 2.14 fiber glass post 33 gpa 0.28 ν 2.5 resin composite 18.9 gpa 0.24 ν 2.09 9 9 table 1. mechanical character of fem material test10 material modulus elastisitas poison ratio density g/cm3 email 41.0 gpa 0.30ν 2.97 dentin 18.6 gpa 0.31ν 2.14 fiber glass post 33 gpa 0.28 ν 2.5 resin composite 18.9 gpa 0.24 ν 2.09 figure 1. the stage of fem analysis process of post endodontic maxillary premolar with glass fiberpost. figure 2. fracture patterns of repairable (a) location of fractures in the crown and (b) location of fractures in the crown and post. 9 9 table 1. mechanical character of fem material test10 material modulus elastisitas poison ratio density g/cm3 email 41.0 gpa 0.30ν 2.97 dentin 18.6 gpa 0.31ν 2.14 fiber glass post 33 gpa 0.28 ν 2.5 resin composite 18.9 gpa 0.24 ν 2.09 figure 1. the stage of fem analysis process of post endodontic maxillary premolar with glass fiberpost. figure 2. fracture patterns of repairable (a) location of fractures in the crown and (b) location of fractures in the crown and post. figure 1. the stage of fem analysis process of post endodontic maxillary premolar with glass fiberpost. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p154-158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p154-158 156 ernani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 154–158 stages which were model designing of the teeth, defining feature for each object material, performing meshing, load and support. after that, it was done with solve the model stage so that it will produce a post processing stage which contains a review of results and checked the validity of the results of the solution (figure 1). results the result of fracture resistance test showed the differences of fracture stress in n for each treatment group. based on the statistical test results one way anova, obtained p value = 0.019 (p<0.05), which showed there was force difference between those four groups and based on analysis test result of post–hoc bonferroni, lsd and tukey hsd obtained the difference fracture resistance which significantly different between group a (17% edta + naocl 2.5%) and group c (naocl 2.5% + 0.2% chitosan) with mean value of the highest fracture resistance in group c was 1515.6 n. the occurred fracture distribution can be analyzed after the endurance test of the fracture by analyzing the location of the fractures in the whole sample. the location of the fracture pattern was divided into two categories; repairable when fractures occur in the crown, post and crown, and cervical (figure 2). irreparable for the fracture patterns in the middle root teeth, horizontal and vertical cracks until the root of the teeth (figure 3).11 10 10 figure 3. fracture patterns of irreparable (a) location of fractures in the middle of the root and (b) location of vertical cracks along the root. figure 4. the result of total deformation of fem test a) 17% of edta + naocl 2.5%; b) naocl 2.5%; c) naocl 2.5 % + 0.2 % of chitosan; d) 0.2 % chitosan. figure 4. the result of total deformation of fem test a) 17% of edta + naocl 2.5%; b) naocl 2.5%; c) naocl 2.5 % + 0.2 % of chitosan; d) 0.2% chitosan. 9 9 table 1. mechanical character of fem material test10 material modulus elastisitas poison ratio density g/cm3 email 41.0 gpa 0.30ν 2.97 dentin 18.6 gpa 0.31ν 2.14 fiber glass post 33 gpa 0.28 ν 2.5 resin composite 18.9 gpa 0.24 ν 2.09 figure 1. the stage of fem analysis process of post endodontic maxillary premolar with glass fiberpost. figure 2. fracture patterns of repairable (a) location of fractures in the crown and (b) location of fractures in the crown and post. figure 2. fracture patterns of repairable (a) location of fractures in the crown and (b) location of fractures in the crown and post. 10 10 figure 3. fracture patterns of irreparable (a) location of fractures in the middle of the root and (b) location of vertical cracks along the root. figure 4. the result of total deformation of fem test a) 17% of edta + naocl 2.5%; b) naocl 2.5%; c) naocl 2.5 % + 0.2 % of chitosan; d) 0.2 % chitosan. figure 3. fracture patterns of irreparable (a) location of fractures in the middle of the root and (b) location of vertical cracks along the root. the result of kruskal -wallis test on the fracture pattern observation, data obtained value p=0.392 (p>0.05) indicating that there was no difference in the fractures distribution among the four groups or there was no difference of fracture pattern between irrepairable and repairable. the results of fem analysis showed: total deformation, equivalent (von-misses) stress, equivalent elastic strain for dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p154-158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p154-158 157157ernani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 154–158 each treatment group. fracture distribution of the fem analysis can be clearly seen by looking at the post process in equivalent (von-misses) stress based on the pattern of colours (red, orange, yellow, green, and blue). red is the maximum points, while the blue colour is a minimum point. fem analysis test, can be used for describing the simulation of strain and predicting the stress concentration area, which is a trigger point of fracture.7,8 the total value of highest deformation (red on the occlusal area) in this study was group c (naocl and chitosan) 2.0355 x 105 m and the lowest was group a (edta and naocl) 1.6634 x 105 m (figure 4). the blue colour was the minimum point, which is the area that did not have a deformation change that occurred in the root area. the result of fem equivalent (von-misses) stress at maximum point was sequentially listed from low to high as follows: group c 2.147 x 108 pa; group d 2.076 x 108 pa; group b 1.828 x 108 pa; group a 1.720x 108 pa. discussion endodontically treated teeth have a higher fracture risk than the vital teeth. the postendodontic teeth strength is directly proportional to the remaining healthy teeth structure and if the teeth structure is lost, the teeth fracture potential will increase. the cause of fractures in post endodontic teeth is multifactorial which are iatrogenic and noniatrogenic.2 from the perspective of biomechanics, fracture is a highly complex process which involves the formation and growth of micro crack and macro cracks. micro crack can grows by the time and increases the concentrations of stress and tensile stress which is producing a microscopic plastic deformation in the end of that pressure concentration that leads on fracture on teeth structure12. the result of sem examination showed that the micro crack can spread (propagation crack) for about 200 µm starting from the top of micro cracks. sem features showed that the crack pattern was starting from microstructures namely tubules. although, tubular did not have a big influence on the process of fracture or fracture pattern but, the cracks that occur continuously in peritubulus dentin can initiate the growth of the crack (sub crack). sub crack initiated the main crack. some chemicals used for endodontic irrigation are causing the changes in the chemical composition of dentin.3 endodontically treated teeth should have a good prognosis so it can function as a support to the final restoration.1,2 chitosan as natural polysaccharide after cellulose that is obtained through deacetylation of chitin has biocompatible properties, bioadhesi and nontoxic for human cells. furthermore, chitosan presents with biocompatibility, chelating capacity and also antimicrobial effects against a broad range of gram-positive and gram-negative bacteria as well as fungi.4 chitosan blangkas (tachypleus gigas) with deasetilisasi degrees of 84.20% and a molecular weight of 893 000 mv is proven the viability of pulp cell.5 chitosan blangkas is able to form a solid coagulum (sub base membrane) which is facilitating the attachment of pulp cells such as dentinoblast. chitosan blangkas is able to form glucosamine d when contacts to the pulp tissue that is easily accessing the cell proliferation so that the dentin genesis process occurs. chitosan treatment improves the resistance of the dentinal surface to degradation by collagenase.13 silva et al. show that 0.2% of chitosan is able to lift the smear layer compared to 15% of edta and 10% of citric acid.7 pimenta et al. stated that the chitosan solution has a chelating property which causes erosion of the dentine, yet it safe for intertubular dentin.4 the result of this study indicated that after the fracture resistance test, it statistically showed the significant difference in all four groups, especially in group a (17% of edta and naocl 2.5%) and group c (naocl 2.5% and 0.2% of chitosan). this result indicated that chitosan that was used as irrigation influenced the fracture resistance. chitosan was able to inhibit the formation and growth of micro cracks and macro cracks since 0.2% of chitosan opened the dentinal tubules without changing the intertubular dentin as compared to the other group test (15% of edta and 10% of citric acid), so that caused a little erosion of dentin.6 a test group (17% of edta and naocl 2.5%) was a test group with the lowest fracture resistance since naocl 2.5% was able to affect the composition and structure of dentin thus, affecting the mechanical properties of dentin due to degradation of the organic components of dentin. the changes of physical properties of dentin occur because the changes of organic and inorganic phase dentin so, that the dentine surface was rougher on the canal wall due to the demineralised dentin which causes loss of mechanical strength. the reduction of organic matter after naocl irrigation can lead mechanical changes. irrigation of 2.5% and 6% naocl for 5, 10, and 20 minutes was decreasing the microhardness dentin for about 500 µm.14 the changes of dentin in the mechanical characteristics and biomechanical response variations caused the post-endodontic teeth becomes fracture.2 the combination of edta and naocl caused a progressive dissolution of dentine extending to peritubular and intertubular area.7 edta solution can dissolved the dentin tissue by reacting with inorganic compounds. in this study, dentin was irrigated with 17% of edta for 10 ml for 1 and 10 minutes and followed by naocl 5% for 10 ml. in 1 minute group of edta smear layer was effectively removed, where as in the 10 minutes group of edta there was too much demineralization in the area peritubular and intertubular dentin.14 sayin et al.3 proved that the use of edta, either alone or in combination with naocl, significanly reduces the microhardness of dentin root. naocl actually impede the attachment of dentin with the resin-based material. insertion of fiber post needs a resin cement. naocl is a strong oxidizing material, it can alters the surface characteristics of root canal dentin becomes oxygenated. the residue of free oxygen affects the dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p154-158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p154-158 158 ernani, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 154–158 interfacial polymerization. the gap that occurs between the luting cement channel walls which can lead to micro cracks along the post rooms that affect the shape of the pattern of fracture occurrence.14 fracture distribution showed that there was no significant difference in the fracture pattern p=0.2 (p>0.05) between the treatment groups. the reason of this result was the use of glass prefabricated fiber post system that has a modulus of elasticity which is similar to the dentin so, that possibly distributing the evenly pressure while the teeth were receiving loads of mastication. the characteristics of fiber post had a similarity to resin luting materials, core materials and dentin so, that the concept of a monoblock system which produces a homogeneous pressure distribution was created. the concept of monoblock system showed the ability of fiber material as the bondable material which is strengthen the teeth after being treated.15 the development of computer technology has increased the use of fem in various fields of science, especially in dentistry. fem program can calculates the stress, strain and deformation in the three dimension view.7 an analysis of fem also obtains some information such as distribution of internal pressure as compare to the experimental study. fem analysis is very useful for indicating the pressure distribution and researching the new material to reduce the failure risk and fracture of restorative material and teeth structure. fem analyzes the changes of strain distribution materials of teeth structure after the placement of the post, core, and final restoration.9 fracture resistance of pure titanium post is almost the same and the best stress analysis (fem test) while compared to the commercial post.9 in addition, the fem analysis more easily compare the biomechanical response even with the addition of various test parameters, such as fem which analyzes the distribution of strain on the premolars with different contact point that analyzes different stress distribution in the postendodontic teeth along with other different posts.16,17 the result of statistical analysis of this study showed that there was no difference between the experimental test’s results and the results of fem analysis with p=0.642 (p>0.05) by t-test. the fem analysis were also showed that 0.2% chitosan solution as an alternative irrigation material affected the pressure distribution. this result was caused by the ability of chitosan as an antibacterial, chelating agent can remove the smear layer, less debris extrusion, not dentin eroding, capable for shaping the collagen fiber and has a d-glucose so it has the ability for reinforcing the dentin with the canal materials, thus increasing system adhesive.13 references 1. michael cm, husein a, wan bakar wz, sulaiman e. fracture resistance of endodontically treated teeth: an in vitro study. archives of orofacial sciences 2010; 5(2): 36-41. 2. kishen a. mechanisms and risk factors for fracture predilection in endodontically treated teeth. endodontic topics 2006; 13: 57–83. 3. sayin tc, serper a, cehreli zc, otlu hg. the effect of edta, egta, edtac, and tetracycline-hcl with and without subsequent naocl treatment on the microhardness of root canal dentin. oral surg oral med oral pathol oral radiol endod j 2007; 104(3): 41824. 4. pimenta ja, zaparolli d, pecora jd, cruz fam. chitosan: effect of a new chelating agent on the microhardness of root dentin. braz dent j 2012; 23(3): 212-7. 5. pretty fss, trimurni a, harry a. characteristic evaluation of rice husk ash with chitosan high molecule nanoparticle as dentinogenesis material. dent. j. (maj. ked. gigi), 2014; 47(2), 63–6 6. silva pv, guedes df, nakadi fv. pecora jd, cruz-filho am. timedependent effects of chitosan on dentin structures. braz dent j (2012) 23(4): 357-61 7. adıgüzel o, yigit os, bahsi e, yavuz i. finite element analysis of endodontically treated teeth restored with different posts under thermal and mechanical loading. idr 2011; 1(3): 75-80. 8. yamamoto et, pagani c, silva eg, noritomi py, uehara ay, kemmoku dt. finite element analysis and fracture resistance testing of a new intraradicular post. j appl oral sci 2012; 20(4): 427-34. 9. yazici ar, celik c, ozqunaltay g. microleakage of different resin composite types. quintessence int 2004; 35(10): 790-4. 10. o’brien wj. dental materials and their selection. 4th ed. michigan: quintessence; 2008. p. 327-54. 11. torabi k, fattahi f. fracture resistance of endodontically treated teeth restored by different frc post: an in vitro study. indian j dent res 2009; 20(3): 282-7. 12. nalla rk, kinney jh, ritchie ro. on the fracture of human dentin: is it stressor strain controlled?. j biomed mater res a 2003; 67(2): 484-95. 13. shrestha a, friedman s, kishen a. photodynamically crosslinked and chitosan-incorporated dentin collagen. j dent res 2011;90:1346135 14. slutzky-goldberg i, maree m, liberman r, heling i. effect of sodium hypoclorite on dentin microhardness. j endod 2004; 30(12): 880-2. 15. tay fr, pashley dh. monoblock in root canal-a hypothetical or a tangible goal. j endod 2007; 33(4): 391-8. 16. lin ts, huang tt, wu jh. the effect of designs and material of the post on endodontically treated premolar using finite element analysis. j medical and biological engineering 2009; 30(2): 79-83. 17. borcic j, antonic r, urek mm, petricevic n, nola-fuchs, catic a, smojver i. 3-d stress analysis in first maxillary premolar. coll antropol 2007; 31(4): 1025-9. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p154-158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p154-158 77 management of zygomatic-maxillary fracture (the principles of diagnosis and surgical treatment with a case illustration) david b. kamadjaja and coen pramono d department of oral maxillofacial surgery faculty of dentistry airlangga university surabaya indonesia abstract mechanical trauma to the face may cause complex fracture of the zygoma and the maxilla. the characteristic clinical signs of zygomatic bone fracture include flattening of the cheek, infraorbital nerve paraesthesia, diplopia, and trismus, whereas maxillary fracture may typically cause flattening of the midface and malocclusion. the diagnosis of zygomatic and maxillary fracture should be established with thorough clinical examination and careful radiologic evaluation so that a three-dimensional view of the fractured bones can be obtained. this is essential in order to plan a proper surgical treatment to reconstruct the face in terms of functions and aesthetic. a standard surgical protocol should also be followed in performing the surgical reconstruction of the zygoma and the maxilla. a case of delayed bilateral fracture of zygoma and maxilla is presented here to give illustration on how the principle of diagnosis and surgical treatment of complex zygomatico-maxillary fracture are applied. key words: zygomatico-maxillary fracture, occlusion rehabilitation, malar prominence correction, bone plating correspondence: david b. kamadjaja, c/o: departmen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: davidbk@sby.dnet.net.id introduction trauma to the midface may cause fractures affecting the maxilla, the zygoma, and the nasoorbital ethmoid complex. depending on the mechanism of injury, it is not uncommon that fracture of the midface involve both the zygoma and the maxillary bone.1,2 the zygomatic bone or zygoma is a strong buttress of the lateral portion of the middle third of the facial skeleton lying between the zygomatic processes to the frontal bone and the maxilla. due to its prominent position, it is frequently fractured, alone or along with other bones of the midface. direct blows usually first strike on the most prominent part which is the malar eminence. this causes disruption at the relatively weaker part which are the zygomatic arch, the frontal process, and the zygomaticomaxillary suture.3 the clinical signs and symptoms are related to displacement or rotation of the fragments which include enophthalmos, hypothalmos, proptosis, diplopia, trismus, mallar flattening, and hypoesthesia.1–5 a number of classifications for zygomatic fractures have been developed based on the anatomy and displacement of the fracture. the most widely used classification of zygomatic fracture are those proposed by dingman and rowe and killey’s.5 these classifications indicate that comminuted fractures and those demonstrating lateral displacement of the zygomatic complex, are unstable after closed reduction (figure 1). maxillary fracture is defined as the separation of parts or the entire tooth-bearing part of the maxilla from the residual midface or the neurocranium. among facial bone fractures, maxillary fractures are less fequent than nasal bone fractures, mandibular fractures, and zygoma fractures. current studies report an incidence of 14% of all facial fractures.6 the clinical symptoms of maxillary fracture may vary depending on the level at which the maxillary bone figure 1. the fracture scheme of rowe and killey indicates the fractures that are more stable following closed reduction. those that are alterally displaced and/or comminuted are less stable if treated by closed reduction.5 78 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 77-83 fractures. the common symptoms include: swelling of the facial soft tissues, retrusion of the midface, bleeding from the nose, and in many cases, unilateral or bilateral orbital hematomas. careful palpation may reveal bony steps and tenderness on the orbital rings and over the crista zygomaticoalveolaris, diplopia, sensory disturbance of the infraorbital nerves, and occlusal problems, mostly in the form of anterior open bite and premature contact in the molar region.1,3 the diagnosis of fracture of zygoma and maxilla can usually be made with thorough clinical examination and adequate radiological evaluation. plain radiograph commonly used in midfacial fracture is occipito-mental or water’s view which can clearly demonstrate the bone discontinuity in the zygomaticomaxillary buttress and the inferior orbital rim. the submentovertex view more clearly detects fractures of the zygomatic arch. ct scan and its three-dimensional applications is indicated for visualization of the orbit if the orbital portion of the zygomatic fracture is suspected. once the diagnosis is established a surgical treatment plan can be made. the current principle of treatment of maxillofacial fracture is open reduction and rigid fixation and in a complex fracture of the zygoma and the midface a certain surgical plan should be followed in order to have good result.5 a case of complex zygomatic-maxillary fracture is presented below to give illustration on how the principles of diagnosis and surgical treatment discussed above are applied. case a forty-year old male patient came to department of oral and maxillofacial surgery, faculty of dentistry, airlangga university, surabaya with chief complaint of inability in chewing after having accident in a liner where he was working 8 days previously. he felt from height and sustained injury on his face. shortly after the accident there was large swelling over his face on both sides and he noticed bloody discharge from his nose. he also complained that his upper jaw was moving whenever he tried to chew. on clinical presentation, his right malar prominence was obviously depressed, and right periorbital ecchymosis and subconjunctival haemorrhage were still seen. few stitches were noted over a 3 cm-long laceration in right supraorbital region (figure 2). eye ball movement were normal in all directions on both sides. bony step was felt on the right frontozygomatic suture and paraesthesia found over the infraorbital region bilaterally. intra orally, floating maxilla was clearly detected, malocclusion was noted showing anterior and posterior open bite on both sides and the absence of normal interdigitation of the upper and figure 2. the molar prominence on the right side was obviously depressed giving the appearance of flat cheek. figure 3. anterior and posterior open bite were noted on both sides with the absence of normal interdigitation between upper and lower teeth (left). maximum mouth opening showing acceptable interincisal distance of 27 mm (right). 79kamadjaja and pramono: management of zygomatic-maxillary fracture lower dentition (figure 3). however, he showed acceptable mouth opening with maximum interincisal distance of 27 mm (figure 2), but slight restriction and discomfort were reported by the patient during maximum opening. plain imagings available at his first presentation were postero-anterior and lateral skull x-ray, and occipitomental view which showed clearly displaced fracture on the right frontozygomatic suture and on the pterygomaxillary buttress bilaterally, minimally displaced fracture on the left frontozygomatic suture, and seemingly fracture right inferior orbital rim (figure 4). orthopantomogram was figure 4. pa skull x-ray showing severely displaced fracture at the right frontozygomatic suture and minimally displaced fracture at the left frontozygomatic suture (left), (b) water’s projection clearly showing bone displacement in zygomatico-maxillary buttress on both sides and at the right frontozygomatic suture, and seemingly fracture inferior orbital rim on the right side. subsequently made which revealed that the mandible was intact and no abnormality detected. the diagnosis made was displaced fracture of the right zygomatic complex and bilateral le fort i fracture of the maxilla. the planned surgery to reconstruct the face was open reduction and internal fixation using miniplates and screws to fix the right zygoma followed by fixation of the maxilla on both sides, and exploration of the right coronoid process of the mandible to exclude any restriction to the movement of coronoid process which may be secondary to zygomatic arch fracture. case management some preparations was made two days prior to the surgery which were dental scaling to the upper and lower teeth and placement of intermaxillary fixation using arch bars and elastic rings. acceptable occlusion was readily achieved upon completion of the intermaxillary fixation. the right eye brow incision was made which revealed displaced fracture on the right fronto-zygomatic suture. right infra orbital stepped incision was subsequently made to expose the inferior orbital rim. it was found that the right inferior orbital rim was intact, no fracture line was detected. the right zygoma was subsequently reduced using two periosteal elevators applied underneath the malar bone and the frontal process of the zygoma to move the fragment laterally and anteriorly. with this maneuver the zygoma was successfully repositioned without much effort as no callus formation had been formed. the fracture site at the right fronto-zygomatic region was fixed with one titanium miniplate and four screws, whereas the right le fort i fracture was fixed with two miniplates placed at the right zygomatico-maxillary buttress and nasomaxillary buttress respectively (figure 6). the le fort i fracture on the left side was subsequently reduced with the same elevator. the reduction was successfully accomplished more easily compared to the the surgery was done under general anesthesia using halothane via nasal intubation. it was initiated by making vestibular incision from the region of upper first molar to the same region on the opposite side to expose the maxilla up to malar prominence, pterygoid, and nasomaxillary regions. displaced fractures were found to run horizontally at a high le fort i level on both sides of the maxilla. the right zygoma was found to be severely displaced medially and posteriorly, whereas the left zygoma was relatively stable. the infraorbital neurovascular bundles on both sides were involved in the fracture site but seemed intact. (figure 5). 80 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 77-83 figure 5. displaced fracture of the right maxilla were found to run horizontally at a high le fort i level which cause the body of the right zygoma to be severely displaced medially and posteriorly, infraorbital neurovascular bundle involved in the fracture line and compressed by the displaced bone fragments (left); high le fort i fracture line on the left maxilla with minimally displaced left zygoma, the infraorbital neurovascular bundle also involved in the fracture line. figure 6. following complete reduction of the fracture bones, the right zygoma is fixed first to the stable frontal bone, followed by fixation of the maxilla on both sides to the stable zygoma. fracture at right frontozygomatic bone fixed with one stem of titanium miniplate and four screws. (left); le fort i fracture of the right maxilla fixed with two stem of miniplates placed at the right zygomatic buttress laterally and nasomaxillary buttress medially (center); two stem of miniplates placed in the same fashion on the left maxilla (right). right side. the fixation was then made using two stemmed titanium miniplates placed along the left zygomaticomaxillary and nasomaxillary buttresses (figure 7). the bony fracture at the left frontozygomatic area was not reduced as it was only minimally displaced and the left zygomatic bone as a whole was relatively stable. the intermaxillary fixation was released to check the stability of the occlusion and the range of motion of the figure 7. review 7 days after the surgery showing remnant of facial swelling on the right cheek obscuring the prominence of the right malar bone (left); good and stable occlusion (right). 81kamadjaja and pramono: management of zygomatic-maxillary fracture mandible. a digital exploration was then made to the right temporal region which revealed that the coronoid process movement was not restricted indicating that the zygomatic arch was not displaced medially. the operation wound on the skin of the right eye brow was closed with 5/0 prolene suture. the infraorbital region was closed in two layers in which the periosteum and the orbicularis oculi muscle were stitched back with 4/0 vycril suture and the overlying skin with 5/0 prolene, whereas the intraoral wound was closed using 4/0 vycril. post operatively large facial swelling was noted especially on the right side. he stayed at the hospital for three days during which time he was put on a soft diet. the oral hygiene was maintained with chlorhexidine mouth wash. intravenous ceftriaxone injection and metronidazole drip was given with the dosage of 2 g/day and 1.5 g/day respectively for four post-operative days. upon discharge, clindamycin 300 mg 3 times a day was prescribed and the patient was advised to have non-chewing diet for one month. post operative review done 7 days post operatively showed that the facial swelling was still present but the malar prominence on the right side seemed acceptable (figure 6). the paraesthesia over the skin of the infraorbital region was still noted but the sensation has somewhat improved. the mouth opening remained the same as that measured prior to the surgery and the maxilla as well as the occlusion were stable. post operative postero-anterior skull x-ray was made to show the orientation of the miniplates along the midfacial buttresses (figure 8). at one month postoperative review, the patient did not complain of any pain nor difficulty in eating, and he was very satisfied with his appearance. the sensation over his infraorbital skin on both sides was coming back to a certain degree. clinically, although remnant of swelling over the infraorbital region was still present the face appeared simetrical with nice malar prominence on both sides. intraorally, the the maxilla was stable and so was the occlusion. the patient’s mouth opening showed normal maximum interincisal distance of 35 mm (figure 9). discussion fracture of the zygoma complex has a characteristic clinical sign of flattening of the normal prominence in the malar area, especially in zygomatic complex injuries. it is figure 8. post operative pa skull x-ray showing orientation of the miniplates along the midfacial buttresses. figure 9. one month postoperative review. the face looks simetrical with nice malar prominence on the right side (left); the patient’s mouth opening showing normal maximum interincisal distance of 35 mm (right). 82 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 77-83 reported in 70 to 86 percent of cases.7 in the case reported herein, flattening of the right malar prominence was obvious, although the patient did not really notice it when presented to our clinic. it is most probably due to the fact that flattening may be difficult to discern soon after injury if the facial edema is still present.8 the other characteristic signs of zygoma fracture which mandate surgery are diplopia, trismus, and paraesthesia of the infraorbital skin. diplopia is usually caused by fracture of the orbital floor which result in entrapment of the periorbital fat and the subsequent tathering of the extraocular muscles. trismus which occured after zygoma complex fracture is usually caused by medially displaced fracture of the zygomatic arch which impinge on coronoid process of the mandible resulting in restriction of the mandibular movement. the infraorbital nerve paraesthesia is also commonly found in zygomatic complex fracture as the nerve is involved in the fracture site in the form of tear or compression by the surrounding tissue.8 out of the above signs only infraorbital paraesthesia which was clearly present in the current case. during surgery it was found that the infraorbital nerve on both sides were involved in the fracture sites but macroscopically they seemed intact. the findings indicate that neuropraxia may have happened to the nerves due to compression by the fracture bone around them. the pre operative mouth opening of 27 mm might indicate that there was some restriction to the mandible movement. since zygomatic fracture was not suspected in this case, this phenomenon may be secondary to swelling of the soft tissue deep to the fractured zygomatic complex on the right side which has caused some compression on the right coronoid process of the mandible. clinical review one month postoperatively which showed a normal mouth opening of 35 mm seems to support our hypothesis. the diagnosis of bilateral le fort i fracture of the maxilla in the case presented here was established based on clinical and radiologic findings. as he came to us eight days after the accident some of the clinical signs and symptoms of maxilla fracture might have disappeared. the only clinical signs of le fort i fracture exhibited by the patient were anterior and posterior open bite and floating of the maxilla. the radiologic findings that support le fort i fracture is occipito-mental view x-ray which showed bone discontinuity at the region of zygomaticmaxillary buttress bilaterally the midface is a complex three-dimensional structures, therefore recreating the facial functions and aesthetics would ideally requires a precise three-dimensional view of all the traumatized structures. in vew of this, ct scan and its three-dimensional application is essential in establishing the final diagnosis before surgical treatment is planned.9 however, a number of midfacial and zygomatic fracture cases in our department are not provided with ct scan as this type of imaging is still considered relatively expensive in this part of the country and this is the case with this patient. therefore the surgical reconstruction was planned based mainly on the clinical judgment and the plain imagings which are postero-anterior skull, occipitomental view, and orthopantomogram. in complex fracture of the zygoma and le fort i fracture of the maxilla, the surgical plan follows a certain principle that the treatment should begin with reconstruction of the load-bearing structures of the facial skeleton starting peripherally and moving centrally and that the building up of the face begins by establishing the anteroposterior dimension by reconstructing the outer facial frame, starting from the stable posterior regions and continuing toward the midline.5 it is suggested that open reduction and fixation of zygomatic arch and frontozygomatic fracture using miniplates results in the establishment of an outer facial frame with the correct anteroposterior projection and transverse facial width. the inner facial frame composed of nasoethmoid, inferior orbital rims, and upper maxilla is now reconstructed within this outer facial frame by building from the nasofrontal region above and the inferior orbital rim below. the lower facial frame is reconstructed last by establishing accurate intermaxillary fixation in combination with reduction and fixation of the four medial and lateral anterior maxillary buttresses using miniplate and screws.12 the zygomatic bone in the reported case sustained fracture in the frontozygomatic and zygomaticomaxillary buttresses. although neither submentovertex view nor ct scan was made prior to the surgery we assumed that zygomatic arch was intact, judged by the clinical presentation of the lateral part of the face and the absensce of trismus. therefore, fixing the frontozygomatic buttress alone was sufficient to achieve a stable outer facial frame. after fixation of the right frontozygomatic buttress with miniplates has been done the bilateral le fort i fracture was subsequently reconstructed based on the stable zygoma. the reconstruction of the maxilla should be initiated only when a proper occlusion has been established and this can be achieved only when the mandible is stable. the sequencing of treatment of complex facial trauma should follow the rule that mandible should be stabilized first before maxillary stabilization as the maxilla can be built on an exact occlusion in intermaxillary fixation as key and fixpoint.10 as our patient’s mandible was intact fixed occlusion can be achieved by placing intermaxillary fixation using arch bar and elastic rings two days prior to the surgery. the intermaxillary fixation was kept in place until the day of the surgery. this procedure is very useful because it could reduce the length of the surgery in the operating theatre. the current method of treatment in maxillofacial, including midface, fracture is open reduction and internal fixation with miniplates and screws which offers stable reduction of the fracture fragments. this would consequently allow for early mobilization of the jaw thus early and optimal recovery of function and esthetic.11 in order to fix the maxilla to its lateral and superior structures the miniplates were placed laterally in the pterygomaxillary 83kamadjaja and pramono: management of zygomatic-maxillary fracture buttress and medially in the nasomaxillary buttress since these buttresses are the pillars consisting of thicker bone that transmits the chewing forces to the supporting regions of the skull.12,13 the midface fractures has tendency to quick and spontaneous healing especially in non-displaced fractures where the maxilla is immobile or only slightly mobile. however, it may be disadvantageous in such clinical situation as when the trauma team needs several days to stabilize the patient and the maxillofacial surgeon cannot intervene early enough, the fractures may have begun to consolidate in displaced positions. fourteen days is the upper limit of primary facial reconstruction in midfacial fractures.6 it is suggested that reduction and fixation up to 3 weeks after trauma produces satisfactory results. after this time, bone healing and resorption begin to take place, and this period has been referred to as a gray time zone.13 this applies to the case presented here. as the bony fracture in the zygoma and the maxilla were still eight days old then it is reasonable that bone consolidation was not found during the surgery and the displaced zygomatic complex could be reduced without much difficulty. references 1. peterson l. contemporary oral and maxillofacial surgery. 2nd ed. st. louis: mosby; 1993. p. 597. 2. gruss j, van wyck l, phillips jh, antonyshyn o. the importance of the zygomatic arch in complex midfacial fracture repair and correction of posttraumatic orbitozygomatic deformities. plast reconstr surg 1990; 85:878–89. 3. kaastad e, freng a. zygomatic-maxillary fractures. j craniomaxillofac surg 1989; 17:210. 4. carr mr, mathog rh. early and delayed repair of orbitozygomatic complex fractures. j oral maxillofac surg 1997; 55:253–8. 5. booth pw, schendel sa, hausamen je. maxillofacial surgery. 2nd ed. st. louis, missouri: churchill livingstone; 2007. p. 104–19, 120–54. 6. gassner r, tuli t, hachl o. craniomaxillofacial trauma: a 10-year review of 9,543 cases with 21,067 injuries. j craniomaxillofac surg 2003; 31(1):51–61. 7. ellis e., el-attar a, moos kf. an analysis of 2,067 cases of zygomatico-orbital fracture. j oral maxillofac surg 1985; 43:428. 8. fonseca rj, walker vw. bett nj, barber hd. oral and maxillofacial trauma. 2nd ed. philadelphia: wb saunders co; 1997. p. 571–652. 9. manson pn, clark n, robertson b. comprehensive management of pan-facial fractures. j craniomaxillofac trauma 1995; 1: 43–56. 10. terheyden h, harle f. surgical management of maxillary fractures. in: booth pw, schendel sa, hausamen je, editors. maxillofacial surgery. 2nd ed. chapter 6. st. louis, missouri: churchill livingstone; 2007. p. 114–5. 11. prein j. manual of internal fixation of cranio facial skeleton. berlin: springer-verlag; 1998. p. 12. 12. manson pn, hoopes je, su ct. structural pillars of the facial skeleton: an approach to the management of le fort fractures. plast reconstr surg 1980; 66:54–7. 13. whitaker la, yaremchuk mj. secondary reconstruction of posttraumatic orbital deformities. ann plast surg 1990; 25:440. vol 51 no 3 jul sep 2018_pus.indd 104104 research report antibacterial potential of ocimum sanctum oils in relation to enterococcus faecalis atcc 29212 diani prisinda,1 ame suciati setiawan,2 and fajar fatriadi3 1,3department of conservative dentistry 2department of oral biology faculty of dentistry, universitas padjadjaran bandung indonesia abstract background: enterococcus faecalis (e. faecalis) is a gram positive cocci present in the root canal due to the failure of endodontic treatment and pulp tissue necrosis. the ideal root canal medicine offers biocompatible properties, ease of cleaning, absence of tooth staining and non-disruption of the root canal filling process. basil (ocimum sanctum) is one of the herbs widely used in salads which produces anti-bacterial, anti-fungal and anti-viral effects. the antibacterial effect of basil results from the eugenol which represents a main component demonstrating antibacterial properties. basil essential oil has an antibacterial effect on both gram positive and gram negative bacteria. purpose: this study aimed to determine whether the essential oils contained in basil leaves offer any antibacterial potential with regard to the growth of e. faecalis atcc 29212. methods: the research was experimental in nature incorporating a simple random sampling technique. in this study, groups of active substance compounds contained in basil leaves were extracted by distillation in order to obtain the essential oil. preparation of the test solution involved essence of basil leaf oil at concentrations of 5,000 ppm, 10,000 ppm and 20,000 ppm in methanol solvent. a phytochemical test of basil was subsequently conducted in order to identify the content of the compound. the bacteria in this study was tested utilizing a disc diffusion method (kirby and bauer test) by measuring the diameter of the clear zone (clear zone) which is indicative of the bacterial growth inhibition response of antibacterial compounds in the extract. results: the results of the research into the phytochemical test showed that basil contains phenolic flavonoids, triterpenoids, saponins, tannins which produce a negative result on steroids. the results of this study showed that the basil essential oil inhibition zone present in the e. faecalis growth had a diameter of 11.70 mm at a concentration of 20,000 ppm. this concentration therefore proved most effective in relation to e. faecalis than other concentrations. conclusion: it can be concluded that essential oils of basil leaves demonstrate anti-bacterial inhibitory properties with regard to e. faecalis. keywords: ocimum sanctum; anti-bacterial activities; enterococcus faecalis atcc 29212 correspondence: diani prisinda, department of conservative dentistry, faculty of dentistry universitas padjadjaran bandung. jl. sekeloa selatan no. 1 bandung 40132, indonesia. e-mail: diani.prisinda@fkg.unpad.ac.id introduction the success of endodontic therapy depends on the number of infection-causing microorganisms that can be eradicated from the root canal. the reduction in the number of such microorganisms can be achieved by root canal preparation, irrigation, administration of sterilizing medication and filling material. the administering of irrigation agents and drugs to the root canal plays an important role in reducing the amount of infected tissue and eliminating microorganisms from the root canal.1 enterococcus faecalis (e. faecalis) is a gram positive cocci bacterium that appears in the root canal arising from the failure of endodontic treatment and is contained in the necrotic pulp tissue.2 e. faecalis can tolerate a high alkaline atmosphere and is capable of entering the dentin tubules. this causes e. faecalis to become resistant and difficult to remove by means of root canal medications, such as calcium hydroxide.2 ideal root canal medications have biocompatible properties, are easy to clean, do not stain teeth and do not impede the root canal filling process.3 increased bacterial resistance to synthetic medications dental journal (majalah kedokteran gigi) 2018 september; 51(3): 104–107 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p104–107 http://dx.doi.org/10.20473/j.djmkg.v51.i3.p104-107 http://e-journal.unair.ac.id/index.php/mkg 105 prisinda, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 104–107 induces researchers to resort to herbs, one being basil, as root canal medicines with antibacterial effects. basil (ocimum sanctum) produces anti-bacterial, anti-fungal and anti-virus effects.3 the antibacterial effect of basil results from its essential oil content,4 a main component of which is eugenol that demonstrates antibacterial properties3 and plays a major role in those of basil leaves. basil leaves containing essential oil are effective against both gram positive and gram negative bacteria.3 the essential oil is effective in inhibiting the growth of gram-positive cocci bacteria at a minimum inhibitory concentration (mic) of 0.5 to 32 μl/ml and minimum bactericidal concentration of 8-32 μl/ml, with the exception of e. faecalis. e. faecalis can be inhibited by a concentration of >32 μl/ml.3 gram negative bacteria can be inhibited by basil essential oil of a lower concentration from 0.25 to 4 μl/ml and a higher minimum bactericidal concentration of 0.5-64 μl/ml.4 basil essential oil has an antibacterial effect on both gram positive and gram negative bacteria. research into the various stages of basil leaf development shows that, at the vegetative stage, basil leaves have an antibacterial effect on e. faecalis bacteria with a minimum antibacterial concentration (mbc) of > 64 μl/ml and mic of 32 μl/ml. the tip of the leaf stage exhibits an antibacterial effect against e. faecalis with mbc of 8 μl/ml and mic of 4 μl/ml. the full leaf stage shows an antibacterial effect against e. faecalis with mbc of 32 μl/ml and mic of 16 μl/ml.4 these results confirm the antibacterial effects of basil leaves essential oils at various leaf development stages against e. faecalis, with the minimum concentration being lowest for the tip of basil leaf. the aim of this research was to determine the antibacterial effects of basil leaf essential oil on e. faecalis. material and methods in this study, a group of active compound substances contained in the leaves of basil (ocimum sanctum) was distilled to obtain basil leaf essential oil. a phytochemical test was subsequently performed to determine the content of the compound. an antibacterial activity test of basil leaf essential oil againts e. faecalis was conducted by diffusion. the basil leaves were obtained from a plantation in lembang, west bandung regency, west java and later confirmed by the biology department of the mathematics and science faculty, universitas padjadjaran bandung. the bacteria studied were e. faecalis atcc 29212 because this is the same bacteria as that present in the root canal. the research methodology adopted constituted a laboratory experiment. a simple random sampling technique was used in selecting basil leaves on the assumption that each section of the population has an equal opportunity of being selected as part of the sample. in this study, a group of active compound substances contained in basil leaves were extracted by distillation to obtain its essential oil which was subsequently dissolved with methanol to produce concentrations of 5,000 ppm, 10,000 ppm and 20,000 ppm. in order to detect antibacterial activity, a diffusion method was implemented which involved measuring the inhibition zone produced by essential basil leaves. a phytochemical test was performed on the basil leaves at the biology department of the mathematics and science faculty, universitas padjadjaran, bandung to determine the compound content. the phytochemical tests include: phenolic tests, flavonoid tests, saponin tests, terpenoid tests, steroid tests and tannin tests. their test procedures consisted of phenolic testing which involved a basil leaf sample being inserted into the test tube before 5% fecl3 reagent was added. if the result proved positive, the phenol would appear purple blue. the testing of f lavonoids was carried out with basil leaf samples being inserted into the test tube before three drops of 2n h2so4 and ± 0.25 g of magnesium powder were added. any changes were subsequently observed and recorded. when an orange solution had formed, the positive test sample contained a flavanoid compound to which 2n h2so4 reagent was added. if an orange-colored solution was formed, the positive test sample contained f lavanoid compounds. when 10% naoh reagent was added to the sample and an old orange solution had formed, the positive test sample contained flavanoid compounds. the next step involved testing for steroids and triterpenoids. basil leaf samples were placed on the drop plate before a lieberman-burchard test was conducted during which two drops of anhydrous acetic acid, one drop of concentrated h2so4 and 2 ml diethyl ether were added. if a change in color to brown-red occurred, the positive test samples contained terpenoid. saponin testing involved basil leaf samples being inserted into the test tube and the reagent hcl + h2o then added. if the foam was stable for 3-10 minutes, the positive sample contained saponins. tanin testing constitutes another of the phytochemical testing procedures. basil leaf samples were added to the tube followed by 1% fecl3 reagent. if the result iwas positive, the tannin would appear purple blue in colour. antibacterial properties were determined by means of disc diffusion method (kirby bauer test). prior to the conduct of the test, the bacteria were rejuvenated by growing them in the muller hinton broth liquid medium for 24-48 hours at an agitation rate of 150 rpm at 37°c. if turbidity in the liquid medium met the standard 0.5 mc farland, confirming that the turbidity concentration of the bacteria was equal to 108 cfu/ ml, it could be tested. the bacteria growing in the liquid medium were extracted and transferred to a solid medium of blood agar and grown in an incubator in 5% co2 for 24-48 hours. a disk diffusion method (kirby and bauer tests) was adopted to determine antibacterial agent activity. discs containing antibacterial agents were placed on the agar medium that had been seeded with bacteria that would diffuse within it. the clear area indicated the inhibition dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p104–107 http://dx.doi.org/10.20473/j.djmkg.v51.i3.p104-107 http://e-journal.unair.ac.id/index.php/mkg 106prisinda, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 104–107 of bacterial growth by antibacterial agents on the surface of the agar medium. a disk containing basil leaf essential oil was placed in the media (mueller hinton broth) within which e. faecalis bacteria had been grown. two batches of media sample was produced for each concentration. the negative control was methanol, while the positive control consisted of chlorhexidine glucoronate 0.2%. a clear zone around the discs confirmed the antibacterial effect of basil leaf essential oil. the diameter of the clear zone around the discs was measured using a caliper. the bacterial test comprised several stages: preparing the e. faecalis, sterilizing 50 ml of nutrient agar (mueller hinton) media in erlenmeyer, adding the e. faecalis bacteria to the homogenized agar media, pouring it into a sterile petri dish and allowing it to solidify. in order to determine the antibacterial potential, a disc diffusion method was utilised in this study by measuring the diameter of the clear zone by means of a paper disc. the paper disc was divided into five groups: three respectively containing 5,000 ppm, 10,000 ppm and 20,0 0 0 ppm of basil lea f essence, one conta in ing 1,000 ppm of chlorhexidine as a positive control, and the final one containing methanol as a negative control which was added to first petri dish. the same procedure was followed with the second petri dish. both petri dishes were incubated at 37°c for 24-48 hours, with the results interpreted and the diameter of each sample concentration measured. the effects of bacterial growth inhibition in an antibacterial compound is shown in the sample. data was obtained by measuring the diameter of the clear zone which appeared as a clear area around the disc where substances promoting antibacterial activity had diffused. results phytochemical testing of basil leaves was carried out in this study to determine the content of the compounds contained within. in the phytochemical test, positive results for the phenolic group compound content such as flavonoids, triterpenoids, saponins, tannins and a negative result for steroids were obtained (table 1). essential oil of basil leaves at concentrations of 5,000 ppm, 10,000 ppm and 20,000 ppm execute activities characterized by the formation of an inhibition zone against bacteria e. faecalis atcc 29 212. a chlorhexidine concentration of 1,000 ppm generates antibacterial activity, while the solvent sample (methanol) creates an e. faecalis atcc 29 212 bacteria inhibition zone (figure 1). the three concentrations of active basil leaf essential oils can actively inhibit the growth of e. faecalis bacteria at concentrations of 20,000 ppm, 10,000 ppm and 5,000 ppm; while chlorhexidine gluconate, as an active positive control, is active at a concentration of 1,000 ppm. methanol also acts as a negative solvent control (table 2). the results of the basil leaf sample inhibition zone calculation confirmed the inhibition diameter at a concentration of 20,000 (11.70 mm) ppm which was greater than at concentrations of 5,000 (8.90 mm) and 10,000 (9.25 mm) ppm (table 2). ba figure 1 potential antibacterial test essential oil. ocimum sanctum to e. faecalis, a) first test; b) second test. table 1. phytochemical test resultmethodno. metabolit reagent 5% feclphenolic1. 3 + flavonoid2. a. reagent hcl concentrated + mg b. reagent 2n h2so4 c. reagent 10% naoh + + + steroid3. reagent lieberman-burchard – 4. triterpenoid + reagent hcl + hsaponin5. 2 +o reagent 1% fecltanin6. 3 + table 2. potential antibacterial test essential oil ocimum sanctum to e. faecalis atcc 29212 no. sample & concentrate (ppm) diameter of inhibition (mm) average (mm) result active11.7010.90 12.50esential oil (20.000)1 active9.259.409.10esential oil (10.000)2 active8.909.008.80esential oil (5.000)3 active9.139.109.15negatif control: metanol4 5 positif control: active14.4515.1013.80chlorhexidine (1.000) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p104–107 http://dx.doi.org/10.20473/j.djmkg.v51.i3.p104-107 http://e-journal.unair.ac.id/index.php/mkg 107 prisinda, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 104–107 discussion basil is a herb known to have antibacterial effects against some strains of bacteria5 which depends on the content of its leaves. the phytochemical test results produced within this study show that basil contains phenols, flavonoids, triterpenoids, saponins and tannins. these results are in accordance with those of phytochemical research which confirmed eugenol (minyal volatile), ursolic acid (triterpenoids) and rosmarinik acid (phenylpropanoid) to be active components contained in basil leaves. the phytochemical test conducted on its leaves by means of methanol extract indicated that basil contains steroids, alkaloids and tannins.6 the phytochemical content, consisting of steroids, alkaloids, flavonoids, tannins and phenols, appears to combat microorganisms.7 the main content of basil leaves is eugenol whose phenols damage the cell wall of the plasma membrane and protein membranes of bacteria.8 the hydrophobic properties of eugenol are paramount in producing the antibacterial effect of separating the fat tissue and mitochondria in the cell membrane of bacteria and altering structures to improve the penetration of eugenol into the cell membrane.8 the phytochemical test results of basil leaves in this study also confirmed the presence of tannin which, in medicinal plants, may indicate their antibacterial properties. tannin forms an irreversible compound with prolineric protein that can inhibit bacterial cell protein synthesis.7 the results of this study indicate that basil leaf essential oils have potentially inhibitive or antibacterial effects on e. faecalis bacteria, possibly due to their phenol content, specifically eugenol.3,8,9 e. faecalis constitutes gram positive facultative cocci that are generally present in the root canal. the essential oils taken from several parts of the basil plant can produce an anti-bacterial effect against gram positive bacteria cocci in almost all parts of the plant the leaves, stem and bud.4 other research has been conducted to distinguish the essential oil of basil leaves from that of other plants.4 the results of these studies confirmed the antibacterial effects of essential oils of basil leaves on e. faecalis, while proposing that the antibacterial effect of the essential oils of basil leaves is due to their linoleic acid, linolenic acid and eugenol content. the working mechanism of essential oils is thought to inhibit the replication of dna, thereby causing the eradication of the bacteria. research findings can be enhanced when the inhibitory effect on e. faecalis bacteria due to the use of essential oils of basil leaves is evident. the manufacture of essential oils in this study used a methanol solution, while methanol was also used as a negative control. however, this investigation confirmed the use of methanol to have an antibacterial effect on e. faecalis, a fact which, in turn, probably enhances the antibacterial effect of basil leaf essential oil on e. faecalis. the positive control consisted of chlorhexidine which is an antiseptic widely used to sterilize the root canal. the antibacterial effect of chlorhexidine shows reasonably effective inhibition of e. faecalis because it can penetrate the dentine tubules.2 from the results above, ocimum sanctum can be seen to have chemical properties that produce an antibacterial effect. this research proved that ocimum sanctum has antibacterial potential with regard to e. faecalis, although it is less than that of chlorhexidine. nevertheless, ocimum sanctum can be used as an alternative material for root canal sterilization, although further research in this area is necessary. it can be concluded that the essential oils of basil leaves have an anti-bacterial inhibitory effect on e. faecalis. acknowledgement the authors would like to thank the ministry of research, technology and higher education, republic of indonesia for its funding of this investigation and all researchers for their contributions to this investigation. references estrela c, sydney gb, figueiredo jap, estrela crda. antibacterial1. efficacy of intracanal medicaments on bacterial biofilm: a critical review. j appl oral sci. 2009; 17: 1–7. mozayeni ma, haeri a, dianat o, jafari ar. antimicrobial effects2. of four intracanal medicaments on enterococcus faecalis: an in vitro study. iran endod j. 2014; 9(3): 195–8. mishra n, logani a, shah n, sood s, singh s, narang i. preliminary3. ex-vivo and an animal model evaluation of ocimum sanctum’s essential oil extract for its antibacterial and anti-inflammatory properties. oral health dent manag. 2013; 12(3): 174–9. saharkhiz mj, kamyab aa, kazerani nk, zomorodian k, pakshir4. k, rahimi mj. chemical compositions and antimicrobial activities of ocimum sanctum l. essential oils at different harvest stages. jundishapur j microbiol. 2014; 8: 1–7. mediratta pk, sharma kk. effect of essential oil of the leaves and5. fixed oil of the seeds of ocimum sanctum immune response. j med aromat plant sci. 2000; 22: 694–700. singh ar, bajaj vk, sekhawat ps, singh k. phytochemical estima-6. tion and antimicrobial activity of aqueous and methanolic extract of ocimum sanctum l. j nat prod plant resour. 2013; 3: 51–8. bonjar ghs, aghigi s, nik ak. antibacterial and antifungal survey7. in plants used in indigenous herbal-medicine of south east regions of iran. j biol sci. 2004; 4(3): 405–12. kong x, liu x, li j, yang y. advances in pharmacological research8. of eugenol. curr opin complement altern med. 2014; 1(1): 8–11. mishra p, mishra s. study of antibacterial activity of ocimum9. sanctum extract against gram positive and gram negative bacteria. vol. 6, american journal of food technology. 2011. p. 336–41. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p104–107 http://dx.doi.org/10.20473/j.djmkg.v51.i3.p104-107 http://e-journal.unair.ac.id/index.php/mkg 77 volume 47, number 2, june 2014 surgical exposure dan perawatan ortodontik pada impaksi gigi insisif sentral rahang atas (surgical exposure and orthodontic treatment on labially impacted maxillary central incisor) bingah fitri melati, teguh budi wibowo, dan betadion rizki departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya – indonesia abstract background: as a clinician we have to concern for an unerupted teeth especially in mixed dentition. eruption failure can also be caused by early loss of deciduous teeth. purpose: to report a case of unerupted maxillary central incisor caused by early loss of deciduous teeth due to trauma and the combination of excisional and orthodontic treatment. case: a 8-years-old girl in mixed dentition phase came to universitas airlangga dental hospital with chief complaint of unerupted right maxillary central incisor while the left central incisor and both lateral incisor had erupted already. she had trauma when she was 1 year old and loss mostly her primary maxillary central incisors. an intraoral examination revealed lack of space in #11 region with root retained of #51, bulge was palpated in vestibulum and periapical radiograph showed that a delayed eruption upper central incisor without presence of disturbance. case management: the exposure of the tooth was under local anesthesia a year after the orthodontic performed to make enough space for traction the tooth. a button was placed at palatal and used elastic strait to traction the tooth. after 3 months, bracket placed at labial to positioning until leveled and aligned with adjacent teeth. conclusion: a simple excisional and orthodontic treatment were succesfully treated the labially impacted teeth. key words: impacted maxillary, central incisor, premature loss, trauma, excision, orthodontic treatment abstrak latar belakang: sebagai seorang klinisi kita harus memperhatikan apabila terdapat gigi yang belum erupsi terutama pada fase gigi pergantian. kegagalan erupsi gigi juga dapat disebabkan karena tanggal premature gigi sulung. tujuan: melaporkan kasus impaksi gigi insisif sentral rahang atas yang disebabkan kehilangan premature gigi sulung karena trauma dengan kombinasi eksisi sederhana dan perawatan ortodontik. kasus: anak perempuan usia 8 tahun pada fase gigi pergantian datang ke rumah sakit gigi dan mulut universitas airlangga dengan keluhan gigi insisif sentral kanan rahang atasnya (#11) belum erupsi meskipun gigi insisif sentral kiri #21 dan kedua insisif lateralnya #22 sudah erupsi. pasien tersebut pernah terjatuh saat masih usia 1 tahun dan hampir kehilangan seluruh gigi sulung insisif sentral rahang atasnya. pada pemeriksaan klinis tampak ruang yang sempit pada region #11 dan terdapat sisa akar gigi #51, jaringan keras teraba pada palpasi daerah vestibulum dan pemeriksaan radiografi periapikal tampak impaksi gigi insisif sentral rahang atas tanpa adanya penghalang. tatalaksana kasus: exposure gigi dilakukan dibawah anestesi lokal 1 tahun setelah perawatan ortodontik untuk membuka space bagi #11. button diletakkan di palatal gigi 11 dan digunakan elastic strait untuk case report 78 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 77–81 traksi gigi tersebut. setelah 4 bulan bracket dipasang untuk memposisikan gigi pada lengkung yang benar. simpulan: teknik eksisi sederhana dan perawatan ortodontik berhasil merawat gigi impaksi yang terletak di labial. kata kunci: impaksi gigi, insisif sentral, kehilangan prematur, trauma, eksisi, perawatan ortodontik korespondensi (correspondence): bingah fitri melati, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan keterlambatan waktu antara eksfoliasi gigi sulung dengan erupsi gigi permanen bisa berhubungan dengan suatu kelainan yang disebut dengan impaksi gigi. impaksi gigi insisif rahang bukan merupakan kasus yang sering ditemui, namun perawatannya cukup menantang karena gigi tersebut berkaitan erat dengan estetik.1 keterlambatan erupsi gigi insisif rahang atas perlu mendapat perhatian apabila ditemukan keadaan sebagai berikut: (1) gigi senama kontralateral telah lebih dulu erupsi ≥ 6 bulan; (2) gigi insisif rahang bawah telah erupsi lebih dari 1 tahun; (3) terjadi deviasi pola erupsi gigi, misalnya gigi insisif kedua telah lebih dulu erupsi.2 trauma gigi pada fase gigi sulung sering terjadi, frekuensinya mencapai 4-30%. gigi yang paling sering terkena dampak dari trauma dental pada anak-anak adalah gigi insisif sentral rahang atas. anal-anak usia 1-4 tahun masih memiliki kontrol motorik yang rendah dan sering terjatuh pada saat melakukan aktifitas seperti bermain atau olah raga, sehingga prevalensi trauma dental pada anak usia tersebut cukup tinggi. gangguan pertumbuhan pada gigi permanen yang disebabkan trauma pada gigi sulung dapat terjadi sekitar 12-69%. tingkat keparahan yang disebabkan dampak dari trauma gigi sulung tergantung dari usia anak saat terjadinya trauma, seberapa besar resorbsi akar pada gigi sulung, tahap pembentukan gigi permanen saat terjadinya trauma, dan tipe kerusakan gigi pada gigi sulung sebagai dampak dari trauma. semakin dini usia anak saat terjadinya trauma maka semakin tinggi tingkat keparahan yang dapat terjadi pada gigi permanen.3 terdapat dua pilihan perawtaan pada gigi impaksi, yaitu pencabutan gigi atau membuka akses erupsi gigi dengan cara surgical lalu memindahkannya kedalam lengkung rahang secara ortodontik.4 perawatan gigi impaksi dengan membuka akses dengan surgical tahapannya sebagai berikut: (1) membuka ruang untuk gigi impaksi; (2) menghilangkan seluruh hambatan baik jaringan lunak maupun jaringan keras pada saat surgical; (3) meletakkan attachment pada saat surgical atau beberapa saat setelah surgical; (4) menuntun proses erupsi alami dengan memberikan traksi secara langsung dan (5) memposisikannya dengan benar.5 terdapat dua teknik untuk membuka akses gigi dengan surgical, yaitu open eruption technique dan closed eruption technique. pada open eruption technique, setelah menghilangkan jaringan yang menghalangi seperti mukoperiosteum, tulang alveolar dan dental sac hingga mahkota gigi terlihat, luka dibiarkan terbuka dan penyembuhan diharapkan dari epitalisasi jaringan yang telah diambil. tetapi pada closed eruption technique, flap mukoperiosteal dibuat lebih luas dan dikembalikan dengan menjahit dan menutup daerah operasi.6 jika posisi gigi impaksi terletak di labial, teknik ekcisional uncovering dapat dilakukan dengan memperhatikan faktor-faktor sebagai berikut: (1) posisi mahkota dilihat dari akses labio-lingual; (2) letak gigi dari mucogingival junction dan (3) cukup tidaknya jaringan gingiva yang mendukung.7 laporan kasus ini melaporkan perawatan kasus impaksi gigi insisif sentral rahang atas kanan yang disebabkan karena trauma sehingga terjadi kehilangan gigi prematur dengan kombinasi eksisi sederhana dan perawatan ortodontik. kasus anak perempuan usia 8 tahun, datang diantar orangtuanya ke klinik ilmu kedokteran gigi anak rumah sakit gigi dan mulut fakultas kedokteran gigi universitas airlangga dengan keluhan gigi depan kanan rahang atas tidak kunjung tumbuh sedangkan gigi depan kiri nya sudah tumbuh sempurna. dari hasil anamnesa pasien tersebut pernah mengalami trauma jatuh saat usianya 1 tahun dan kehilangan sebagian gigi sulung depan kanan rahang atas. kesehatan umum pasien normal dan tidak ada riwayat medis tata laksana kasus. tatalaksana kasus pada pemeriksaan ekstra oral tidak ditemukan asimetris wajah dan tidak ada pembesara kelenjar submandibularis. pada pemeriksaan intraoral terdapat sisa akar gigi #51, teraba jaringan keras pada palpasi daerah mucogingival junction. relasi molar satu permanen adalah klas 1 angle. susuan gigi anterior bawah sedikit berjejal. status kebersihan mulut sedang (gambar 1a, b, c, d, dan e). dari hasil radiografik periapikal terlihat gigi #11 impaksi dengan posisi vertikal dan rotasi dan tidak ada jaringan keras lain didaerah koronal yang menghalangi diagnose gigi #11 impaksi e.c kekurangan tempat karena kehilangan prematur gigi sulung (gambar 2). setelah dilakukan perawatan pendahuluan seperti pencabutan, penambalan dan aplikasi sealant, dilakukan perawatan 79melati, et al.,: surgical exposure dan perawatan ortodontik ortodontik dengan pemasangan bracket pada gigi yang telah erupsi dan molar band pada gigi #16 dan #26. dan untuk membuka ruang di regio #11 menggunakan open coil spring. pembukaan ruang dan observasi dilakukan selama ± 1 tahun, namun gigi #11 belum erupsi secara spontan. keadaan tersebut merupakan indikasi dilakukan surgical exposure pada gigi #11 yang impaksi. exposure gigi #11 dimulai dengan prosedur asepsis daerah kerja ekstra oral menggunakan kapas dan alkohol 70% dan intra oral dengan kapas dan povidone iodine 10%. selanjutnya pengulasan anestesi topikal pada daerah mucogingival junction untuk kemudian dilakukan anestesi lokal dengan syringe. setelah efek anestesi tercapai, dilakukan flap dengan menggunakan scalpel dan pembukaan flap dengan rasparatorium, tampak bagian palatal dari mahkota gigi 11 dengan rotasi ke arah mesiolabial. dalam kasus ini tidak ada pengambilan jaringan tulang. setelah bagian palatal dari gigi 11 dibersihkan dari gambar 1. foto intra oral sebelum perawatan. (a) tampak depan; (b) regio rahang atas; (c) regio rahang bawah; (d) tampak sisi kanan; (e) tampak sisi kiri. permanen adalah klas 1 angle. susuan gigi anterior bawah sedikit berjejal. status kebersihan mulut sedang (gambar 1a, b, c, d, dan e). gambar 1. foto intra oral sebelum perawatan. dari hasil radiografik periapikal terlihat gigi #11 impaksi dengan posisi vertikal dan rotasi dan tidak ada jaringan keras lain didaerah koronal yang menghalangi diagnose gigi #11 impaksi e.c kekurangan tempat karena kehilangan prematur gigi sulung (gambar 2). setelah dilakukan perawatan pendahuluan seperti pencabutan, penambalan dan aplikasi sealant, dilakukan perawatan ortodontik dengan pemasangan bracket pada gigi yang telah erupsi dan molar band pada gigi #16 dan #26. dan untuk membuka ruang di regio #11 menggunakan open coil spring. gambar 2. foto periapikal setelah dilakukan pembukaan ruang di region 11. gambar 2. foto periapikal setelah dilakukan pembukaan ruang di region #11. permanen adalah klas 1 angle. susuan gigi anterior bawah sedikit berjejal. status kebersihan mulut sedang (gambar 1a, b, c, d, dan e). gambar 1. foto intra oral sebelum perawatan. dari hasil radiografik periapikal terlihat gigi #11 impaksi dengan posisi vertikal dan rotasi dan tidak ada jaringan keras lain didaerah koronal yang menghalangi diagnose gigi #11 impaksi e.c kekurangan tempat karena kehilangan prematur gigi sulung (gambar 2). setelah dilakukan perawatan pendahuluan seperti pencabutan, penambalan dan aplikasi sealant, dilakukan perawatan ortodontik dengan pemasangan bracket pada gigi yang telah erupsi dan molar band pada gigi #16 dan #26. dan untuk membuka ruang di regio #11 menggunakan open coil spring. gambar 2. foto periapikal setelah dilakukan pembukaan ruang di region 11. jaringan lunak dan darah, diletakkan button dan ligature wire yang dipilin sepanjang ±15 mm dengan ujungnya berbentuk bulat (pigtail).5 ujung dari pigtail diikat ke arch wire dengan menggunakan elastic strait lalu flap ditutup dengan periodontal pack (gambar 3). gambar 3. pemasangan button pada palatal gigi #11. gambar 4. keadaan klinis post surgical. a b c permanen adalah klas 1 angle. susuan gigi anterior bawah sedikit berjejal. status kebersihan mulut sedang (gambar 1a, b, c, d, dan e). gambar 1. foto intra oral sebelum perawatan. dari hasil radiografik periapikal terlihat gigi #11 impaksi dengan posisi vertikal dan rotasi dan tidak ada jaringan keras lain didaerah koronal yang menghalangi diagnose gigi #11 impaksi e.c kekurangan tempat karena kehilangan prematur gigi sulung (gambar 2). setelah dilakukan perawatan pendahuluan seperti pencabutan, penambalan dan aplikasi sealant, dilakukan perawatan ortodontik dengan pemasangan bracket pada gigi yang telah erupsi dan molar band pada gigi #16 dan #26. dan untuk membuka ruang di regio #11 menggunakan open coil spring. gambar 2. foto periapikal setelah dilakukan pembukaan ruang di region 11. d e 80 dent. j. (maj. ked. gigi), volume 47, number 2, june 2014: 77–81 pemerikasaan yang dilakukan pada kontrol hari pertama berupa anamnesa dan pemeriksaan klinis. pasien mengeluh sakit pada daerah operasi. periodontal pack, ligature wire dan elastic strait masih terpasang baik, kemerahan pada gingival sekitar dan dilakukan irigasi aquadest untuk menghilangkan debris. pelepasan periodontal pack dilakukan hari ketiga post surgical. traksi gigi #11 menggunakan elastic strait yang diikat pada steinless steel arch wire 0,14 modifikasi dan diganti tiap 2 minggu sekali (gambar 4, 5). pada bulan keempat gigi sudah erupsi dan semakin mendekati oklusal tetapi masih rotasi, kemudian button dipindahkan ke disto-labial gigi #11. setelah rotasi berkurang, button dilepas dan diganti dengan bracket. penggantian nikel titanium arch wire dilakukan secara bertahap dimulai dari diameter 0,12, 0,14 dan 0,16 (gambar 6). pada bulan ke tigabelas posisi gigi #11 sudah pada lengkung yang benar. traksi gigi #11 sudah selesai. karena pasien masih dalam fase gigi pergantian, perawatan ortodontik dilanjutkan sampai gigi permanen erupsi semua (gambar 7, 8, dan 9). pembahasan impaksi gigi adalah suatu kondisi gigi tidak erupsi atau erupsi sebagian karena terhalang oleh gigi lain, tulang atau jaringan ikat sehingga gigi tidak dapat erupsi sempurna sebagaimana mestinya.8 kehilangan prematur gigi sulung dapat menyebabkan pemendekan lengkung rahang sehinggga tidak ada cukup ruang untuk gigi permanen erupsi sehingga dapat menyebabkan gigi tersebut impaksi. kehilangan prematur gigi sulung juga bisa mengakibatkan terbentuknya masa hipreplastik jaringan ikat yang terbentuk didalam tulang alveolus sehingga dapat menghalangi erupsi gigi dan berkontribusi menyebabkan impaksi pada gigi permanen insisif rahang atas.9 dari hasil anamnesa pasien pernah mengalami trauma saat berumur 1 tahun, menurut literatur semakin dini usia anak saat terjadinya trauma maka semakin tinggi pula tingkat keparahan gangguan gambar 5. keadaan klinis saat periodontal pack dilepas. gambar 6. traksi gigi #11 dengan elastic strait yang diikat pada wire ss 0,14 modifikasi. gambar 7. gigi #11 sudah erupsi dengan rotasi mesiolabial. gambar 8. keadaan klinis bulan ke tujuh post surgical. gambar 9. perawatan gigi #11 selesai. 81melati, et al.,: surgical exposure dan perawatan ortodontik pertumbuhan pada gigi permanennya.3 kondisi gigi #11 yang impaksi kemungkinan juga disebabkan karena trauma sehingga terjadi perubahan morfologi akar gigi menjadi bengkok dan posisi mahkota gigi yang rotasi. surgical exposure dan traksi ortodontik pada gigi #11 dilakukan agar waktu yang dibutuhkan oleh gigi tersebut erupsi sempurna tidak lagi memakan waktu yang lama. erupsi secara spontan gigi impaksi kebanyakan terjadi setelah pembukaan ruang secara presurgical ortodontik, namun hal tersebut tidak terjadi pada kasus ini. hal ini kemungkinan disebabkan karena adanya perubahan morfologi pada akar (sedikit bengkok) dan arah tumbuh dari gigi impaksi tersebut. akar dari gigi yang impaksi tersebut baru tampak secara radiografik setelah perawatan selesai. pada saat operasi, setelah dilakukan flap pada jaringan gingival dan pembukaan flap dengan rasparatorium, mahkota dari gigi yang impaksi sudah terlihat tanpa tertutup jaringan tulang. maka dari itu tidak ada pengurangan jaringan tulang dan exposure dilakukan dengan eksisi sederhana.7 setelah itu daerah operasi ditutup dengan periodontal pack. teknik closed eruption technique dipilih karena beberapa penelitian menunjukkan bahwa gigi yang tidak ditutupi dengan flap akan mengakibatkan kerusakan jaringan gingival lebih banyak dan kurang estetik.10 penggunaan piranti cekat memiliki beberapa keuntungan yang dapat dimanfaatkan untuk kasus ini, seperti mudahnya kontrol pembukaan akses dan kekuatan tarik alat dapat diatur dibandingkan dengan alat ortodontik lepasan. keuntungan lain dari alat ortodontik cekat adalah lebih nyaman, pasien tidak harus kooperatif, kontrol pergerakan alat lebih baik, dan memungkinkan pergerakan ketiga arah.10 dengan alat ortodontik cekat membantu traksi gigi impaksi mendapatkan posisi di lengkung rahang yang baik. pada kasus ini etiologi impaksi gigi insisif sentral rahang atas adalah adanya pemendekan lengkung karena kehilangan preamatur gigi sulung dan adanya gangguan pertumbuhan gigi permanen menyebabkan perubahan morfologi akar dan posisi mahkota yang disebabkan adanya trauma saat proses pembentukan gigi permanen. posisi gigi impaksi yang terletak di labial memungkinkan penggunaan teknik eksisi sederhana untuk kemudian diberi attachment tanpa melakukan pengurangan jaringan tulang. perawatan diawali dengan membuka ruang kemudian dilanjutkan dengan surgical exposure dan traksi ortodontik dengan button dan ligature wire yang diikat ke arch wire. rangkaian perawatan ini memberikan hasil yang baik dan memuaskan. gigi #11 telah erupsi pada bulan keempat dan total waktu yang diperlukan gigi tersebut berada pada lengkung yang benar adalah tigabelas bulan. laporan kasus ini menunjukkan bahwa teknik eksisi sederhana dan perawatan ortodontik berhasil merawat gigi impaksi yang terletak di labial. daftar pustaka 1. smailiene d, sidlauskas a, bucinskiene j. impaction of the central maxillary incisor associated with supernumerary teeth. baltic dental and maxillofacial j 2006; 8: 103-7. 2. hitchen ad. the impacted maxillary incisor. dent pract dent rec 1970; 20: 423–33. 3. lips ar. treatment of an impacted maxillary central incisor in a mixed dentition. braz j dent traumatology 2011; 2(2): 71-4. 4. crawford lb. impacted maxillary central incisor in mixed dentition treatment. am j orthod dentofac orthop 1997; 112(1): 1-7. 5. becker. early treatment for impacted maxillary incisor. international symposium of early orthodontic treatment, 2002. p. 8-10. 6. huber kl, suri l, taneja p. eruption disturbances of the maxillary incisors: a literature review. j clin pediatr dent 2008; 32(3): 221–30. 7. kokich vg. surgical and orthodontic management of impacted maxillary canines. am j orthod dentofacial orthop 2004; 126: 278-83. 8. zwenner tj. boucher’s clinical dental terminology. a glossary of accepted term in all disciplines of dentistry. 3rd ed. st louis: mosby; 1982. 9. ferneini em, hutton ce, bennet jd. oral surgery for the pediatric patient. mcdonald and avery’s. dentistry for the child and adolescent. 9th ed. mosby; 2011. 10. proffit wr. contemporary orthodontics. 4th ed. mosby elsevier; 2007. 210 vol. 43. no. 4 december 2010 interleukin-1b expression on periodontitis patients in surabaya chiquita prahasanti department of periodontics faculty of dentistry, airlangga university surabaya indonesia abstract background: periodontal disease, commonly known as periodontitis is an infectious disease which has multifactorial etiologic factors. it may affect everybody in any ages with no gender nor sex predilection and usually can be detected under routine clinical examination. this disease is a manifestation of local factors, host factor and environmental factors, resulting in periodontal tissue damage which may cause tooth mobility and tooth loss. interleukin-1 is a pro-inflammatory protein which functions primarily as inflammatory mediator in host innate immune responses. il-1 is a regulator, affecting many biological activities including proliferation, development, homeostasis, regeneration, repair and inflammation which contribute to tissue damage and alveolar bone resorption. purpose: this research was aimed to reveal the basic pathogenesis of periodontitis and could determine the future definitive treatment for patients with periodontitis. methods: data were obtained from 40 patients with aggressive periodontitis and 40 patients with chronic periodontitis. samples were collected from periodontal tissue patients and protein expression of il-1b was performed with immunohistochemistry. results: most female patient suffer aggressive periodontitis and chronic periodontitis. the datas were analyzed with t-test. the t values result was -8623, significance 0.001, with a = 5%, which indicated there was significant difference in il-1b expression between aggressive and chronic periodontitis. the box plot diagram showed marked difference in distribution of protein expression of il-1b between patients with aggressive periodontitis and chronic periodontitis. with a regression equation, it might be concluded that the protein expression of il-1b might affect the incidence of aggressive periodontitis and chronic periodontitis. the or value was calculated for 0.746 (sign.= 0.001), which indicate each increment of one unit protein expression of il-1b will lead the risk for aggressive periodontitis 0.746 times higher or if the protein expression of respondents increased one unit, the risk of chronic il-1b periodontitis may be 1.34 times higher. conclusion: this study elucidated that the elevation proteins expression of il-1b in patients with chronic periodontitis demonstrated this cytokine as an indicator of inflammation. key words: aggressive periodontitis, chronic periodontitis, interleukin-1b abstrak latar belakang: penyakit periodontal yang biasa dikenal dengan periodontitis adalah penyakit infeksi, yang disebabkan oleh berbagai faktor, dapat menyerang setiap orang tanpa membedakan usia dan gender serta mudah ditemukan pada pemeriksaan klinis oleh seorang dokter gigi. penyakit ini merupakan manifestasi dari interaksi antara faktor lokal dengan faktor lingkungan, yang berakibat pada kerusakan jaringan periodontal, dapat mengakibatkan terjadinya kegoyangan gigi hingga tanggalnya gigi. interleukin-1 merupakan protein pro-inflamatori dengan fungsi utama sebagai mediator respon inflamasi pejamu pada sistem imunitas innate. interleukin-1 merupakan regulator, dimana memainkan peranan pada sejumlah aktivitas biologic termasuk proliferasi, pengembangan, homeostasis, regenerasi, repair dan keradangan berperan pada kerusakan jaringan ikat serta resorpsi tulang alveolar. tujuan: penelitian ini bertujuan untuk menentukan dasar patogenesa periodontitis dan dapat digunakan sebagai dasar perawatan penderita periodontitis pada masa mendatang. metode: data penelitian didapat dari 40 penderita dengan periodontitis agresif dan 40 penderita periodontitis kronis. sampel berasal dari jaringan yang mengalami kelainan periodontal dan uji ekspresi protein il-1b dilakukan secara imunohistokimia. hasil: penderita yang mengalami kelainan pada penelitian ini sebagian besar adalah perempuan baik periodontitis agresif maupun periodontitis kronis. uji statistik yang digunakan adalah uji-t diperoleh nilai t sebesar -8.623 dan signifikansi 0.001, dengan a = 5% maka terdapat perbedaan bermakna ekspresi protein il-1b antara penderita periodontitis agresif dan penderita periodontitis kronis. diagram box plot memperlihatkan sebaran ekspresi protein il-1b antara penderita periodontitis agresif dan penderita periodontitis kronis yang tampak sangat jauh berbeda. ekspresi protein il-1b berpengaruh pada kejadian research report 211prahasanti: interleukin-1b expression penderita periodontitis agresif dan penderita periodontitis kronis, dengan bentuk persamaan regresi. nilai estimasi or untuk variabel ekspresi protein il-1b adalah 0,746 (sign. = 0,00). artinya, jika ekspresi protein il-1b responden bertambah satu satuan, maka risiko terjadinya periodontitis agresif menjadi 0,746 kali atau jika ekspresi protein il-1b responden bertambah satu satuan, maka risiko terjadinya periodontitis kronis menjadi 1,34 kali. kesimpulan: ekspresi protein il-1b yang meningkat pada penderita periodontitis kronis menujukkan bahwa sitokinini merupakan indikator pada keadaan keradangan. kata kunci: periodontitis agresif, periodontitis kronis, interleukin-1b correspondence: chiquita prahasanti, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: chiquita_prahasanti@yahoo.com. telp. (031) 5933069. introduction periodontal disease, commonly known as periodontitis, is an infectious disease with multifactorial etiologic factors which may cause of periodontal tissue destruction. periodontitis is a multifactorial disease with bacterial infection as its main etiologic factor, the incidence and severity of illness were determined by the host response to infection, but until now, clinical diagnosis often remains unclear so that treatment often gives unsatisfactory results, which may indicate the necessity of exploration in molecular level. as a marker of active inflammation, the interleukin-1 (il-1) plays important role in immunological processes of inflammatory response. interleukin-1 is a pro-inflammatory protein produced by macrophages and endothelial cells platelets which functions mainly as a mediator of inflammatory responses in host innate immunity system. il-1 is a multifunctional cytokine which influences most of the inflammatory cell types, is a cytokine that is produced during inflammatory processes and involved in connective tissue destruction from the early phase. il-1 may function as a regulator, which plays a role in a number of biological activities including proliferation, development, homeostasis, regeneration, repair and inflammation.1,2 interleukin 1 exists in two forms, namely il-1a and il-1b. il-1b is a potent inflammatory cytokine involved in many important cellular functions, such as proliferation, activation, and differentiation and is an important component of the innate immune response moreover it; also induces the chemotactic of leukocytes by stimulating the induction of il-8 and activating neutrophils for phagocytosis and degranulation. it is also found that il-1b not only modulates the inflammation in gingival epithelial cells but also regulates the production of other inflammatory cytokines including il-8.3 il-1b is a potent substance that has catabolic effect on bone 10 times higher compared to il-1b. the biological impact of il-1 depends on the amount of cytokines that are released by the body. at low levels, it functions as a local inflammatory mediators, while in high level, il-1 may enter into the circulation and accelerates the endocrine activity.4-6 interleukin-1 may function as a regulator, which plays a role in a number of biological activities including proliferation, development, homeostasis, regeneration, repair and inflammation.1,2 il-1b stimulate various cell types to produce connective tissue catabolic and mediators of bone resorption such as il-1, il-6, tnfa, pge2 and matrix metalloproteinase. 7 il-1b and tumor necrosis factor (tnf) are cytokine that possess the ability to cause bone destruction.8,9 based on those previous studies above, identification of il-1b may be used as a risk factor for periodontitis. this research was aimed to reveal the basic pathogenesis of periodontitis and could determine the future definitive treatment of patients with periodontitis. materials and methods this research was an analytical observational study, case control study design in patients who experienced aggressive and chronic periodontitis. complete medical and dental histories were taken from all subjects. none of subjects had a history of systemic disease and had received antibiotics or other medication or periodontal treatment within the past 4 months. informed consent was obtained from the patients and the protocol was approved by the ethical committee of faculty of dentistry airlangga university. tissue samples were taken from periodontal tissue that was affected by periodontitis. the population of this research was patients who came to dental hospital of faculty of dentistry airlangga university for 10 month, and had been diagnosed with aggressive periodentitis (ap) or chronic periodontitis (cp). the patients were diagnosed according to clinical and radiographic criteria as ap (n = 40) and cp (n = 40). the parameters were shown as mean ± standard deviation and analyzed by t test. the expression of il-1b was detected by immunohistochemistry method. il-1b was detected with biotin-labeled antibodies and visualized with dab-deminobenzidine. 212 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 210–214 results the data that were eligible for analysis consisted of 40 patients with aggressive periodontitis and 40 patients with chronic periodontitis. the following diagram showed patient distribution with aggressive periodontitis and chronic periodontitis during the study which was distinguished by gender (figure 1). 0 5 10 15 20 25 ap cp man woman figure �. frequency distribution column, based on gender, aggressive and chronic periodontitis (ap and cp). patients who had periodontal disease, either aggressive periodontitis or chronic periodontitis, in this study were mostly women. this situation could be seen from 22 samples (55%) of patients with aggressive periodontitis and 24 samples (60%) of patients with chronic periodontitis were women (table 1). table �. descriptive value of protein expression of il-1b, people with aggressive periodontitis (ap) and chronic periodontitis (cp) in surabaya periodontitis n mean standard deviation protein expression il-1b ap 40 9.32 6.12 cp 40 20 4.87 protein expression of il-1b patient with aggressive periodontitis and patient with chronic periodontitis could be seen in table 1. the following were data values based on examination of 40 patients with aggressive periodontitis and 40 patients with chronic periodontitis. the independent samples t-test with variances homogeneous data was performed to see the difference in protein expression of il-1b between the patient with aggressive periodontitis and chronic periodontitis. the t-test values were obtained at -8623 and significance of 0.00. if a value was set on 5%, there was significant difference in protein expression of il-1b patients with aggressive periodontitis and chronic periodontitis patients. the average of protein expression in patients with aggressive periodontitis was 9.32, while in patients with chronic periodontitis was 20. patient with chronic periodontitis had significantly higher il-1b compared to patients with aggressive periodontitis (figure 2). figure �. expression of il-1b protein (arrow) in periodontal tissue by immunohistochemistry staining with peroxidase dab and 400× magnification. the difference of protein expression may be easily explained with the box plot diagrams expression of il-1b (figure 3). the box plots clearly demonstrated the data distribution of protein expression between patients with aggressive periodontitis and chronic periodontitis. 4040n = periodontitis periodontitis kronisperiodontitis agresi il -1 be ta 40 30 20 10 0 38 figure ��. box plots of il-1b protein expression. box plot diagram demonstrated the difference of protein expression of il-1 between patients with aggressive and chronic periodontitis. with a regression equation, it might be concluded that the protein expression of il-1b might affect the incidence aggressive periodontitis and chronic periodontitis. the or value for protein expression of il-1b was estimated 0.746 (sign = 0.001). it meant each increment of one unit protein expression of il-1b, the risk for aggressive periodontitis was 0.746 times higher or if the protein expression of respondents increased one unit, the risk of chronic periodontitis may be 1.34 times higher. ap cp aggressive periodontitis chronic perindontitis 213prahasanti: interleukin-1b expression discussion epithelial cells are one of the first lines of defense against pathogens, and although these cells are not as specialized as professional phagocytes in dealing with pathogens, they may play a sentinel role. the cells may use the extracellular secretion of il-1b to induce other neighboring epithelial cells in autocrine manner to help amplify the release of inflammatory, chemokine, and antimicrobial molecules. based on sample characteristics, it seemed that there was no gender predilection, because men and women had the same representation. however, this study showed that periodontitis was more likely to be found in women, possibly because womenvisit dentists more often than men and other possibilities were the age of puberty was earlier, hormonal changes during menstruation and pregnancy might affect the host and might also aggravate the periodontitis. it was corresponded with the previous studies from bret et al.,10 and guzeldemir et al.,1 which found that most of the study sample were woman. the il-1 is one of cytokine that was functioned in periodontal tissues, this kind of interleukin is more dominant as il-1b whichhas various pro-inflammatory capabilities, produced during inflammation and involved from the early stage of connective tissue damage and was considered as an important role in the pathogenesis of periodontitis. il-1b secretion is induced soon after microbial invasion, there for it could be hypothesized that il-1b might play an important role in the induction of other inflammatory cytokines.3 il-1b stimulates various cells types to produce connective tissue and catabolic mediators of bone resorption such as il-6, tnfa, pge2 and matrix metalloproteinase. level of cytokines which was secreted in response to the bacteria may explain individual response differences in sensitivity to and severity of the periodontal disease.7,11 the il-1b expression is associated with the severity of inflammatory diseases including periodontitis, which then would quickly encode pro-inflammatory proteins by transcription factors like nuclear factor-kb (nf-kb).12 this research demonstrated an increase in protein expression of il-1b in chronic periodontitis group compared with aggressive periodontitis, which was corresponded with the research conducted by gursoy et al.,13 who also found that levels of il-1b were high in the periodontitis group compared with healthy control samples. levels of il-1b on periodontal tissues might increase according to the severity of illness, which was related to the presence of periodontal pathogen.3 it shall be noted that gingival inflammation may affect the secretion of il-1b. research conducted by toker et al.,6 found that il-1b was significantly elevated in periodontal tissue and gingival fluid of the inflamed side compared to the healthy side. in vivo and in vitro studies had shown that in patients with periodontitis and other infectious diseases, il-1b which was produced was involved in the inflammatory process and aimed to eliminate the periodontal pathogen from host.14 high level of il-1 will stimulate the secretion of cd4 + t-cells which will increase the secretion of specific antibodies. il-1 also will stimulate the production of il-2 through the th1 cell, which in turn will stimulate cd8 + t cells, and suppress the activation of polyclonal b-cell and the production of antigen-specific antibodies. t-helper type 1 (th1), th2 and monocyte derived cytokines in gingival tissue and gingival crevicular fluid (gfc) was involved in periodontal inflammation. the il-1b (cytokine) imbalance may affect the periodontal supporting bone and collagen tissue destruction in patients with periodontitis. interleukin 1b may suppress il-10 production in periodontal ligament cells, which mean that there is interaction between il-1b and il-10, and the dynamic interaction between proinflammatory and anti-inflammatory cytokines plays an important role in the pathogenesis of periodontal defects. cytokines determines the local defense against bacterial endotoxins and maintain homeostasis in periodontal tissues.15,16 periodontopathogen bacteria that cause periodontitis produce lipopolysaccharide, which was potential in stimulating host response and contributing to tissue damage. it also stimulated macrophages to release il-1b and tnf. this cytokine had the ability to stimulate bone destruction.9,17 while neutrophils and the other cells that serve as protection were not working properly and at the end would cause bone destruction.18 as a multifunctional pro-inflammatory mediator with a crucial role in the regulation of inflammatory reactions, the observation that il-1b can act on large number of cells, like fibroblast, chondrocytes, bone cells, neutrophils and lymphocyte suggests that periodontal destruction and repair in periodontitis may in part be associated with this cytokine. it may be concluded that il-1b as a marker of active inflammation will contribute in pathological inflammatory response and periodontitis, as an active inflammation, will elevates the level of protein expression. the results of this study may be can be used as a further reference on therapeutic modality to actively control the inflammation and tissue damage in various diseases. references 1. guzeldemir e, gunhan m, ozcelik o,tastan h. interleukin-1 and tumor necrosis factor-a gene polymorphisms in turkish patients with localized aggressive periodontitis. j of oral science 2008; 50(2): 151–9. 2. chen h, wilkins lm, aziz n, cannings c, wyllie dh, bingle c, rogus j, beck jd, offenbacher s, cork mj, kolpin mr, hsieh cm, kornman ks, duff gw. single nucleotide polymorphisms in the human interleukin-1b gene affect transcription according to haplotype context. human molecular genetic 2006; 15(4): 519–29. 3. eskan me, benakanakere mr, rose bg, zhang p, zhao j, statthopoulou p, fujioka d,kinane df. interleukin-1b modulates pro-inflammatory cytokine production in human epithelial cells. infection and immunity 2008; 76(5): 2080–9. 4. kresno sb. imunologi: diagnosis dan prosedur laboratorium. edisi keempat. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 2007. p. 63–82. 214 dent. j. (maj. ked. gigi), vol. 43. no. 4 december 2010: 210–214 5. arab hr, afshari jt, radvar m, moeintaghavi a, sargolzaei n, rigi a, shirkhani m. il-1b+3954 genetic polymorphism association with generalized aggressive periodontitis in khorasaniran province. j med sci 2007; 7(2): 222–7. 6. toker h, poyraz o, eren k. effect of periodontal treatment on il-1b, il-1ra, dan il10 levels in gingival crevicular fluid in patients with aggressive periodontitis. j clin periodontol 2008; 35: 507–13. 7. boch ja,wara-aswapati n, auron pe. interleukin 1 signal transduction current concepts and relevance to periodontitis. j dent res 2001; 80(2): 400–7. 8. nishihara t, koseki t. microbial etiology of periodontitis. periodontol 2000; 2004; 36 14–26. 9. takahashi n, kobayashi m, takaki t, takano k, miyata m, okamatsu y, hasegawa k, nishihara t, yamamoto m. actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts. oral microbiol immunol 2008; 23: 259–64. 10. brett pm, zygogianni p, griffiths gs, tomaz m, parkar m, d'aiuto f, tonetti m. functional gene polymorphisms in aggressive and chronic periodontitis. j dent res 2005; 84(12): 1149–53. 11. lopez nj, jara l,valenzuela cy. association of interleukin-1 polymorphisms with periodontal disease. j periodontol 2005; 76: 234–43. 12. tipton da, pond dm. effect of nuclear factor-kb inhibition on interleukin-1b-stimulated matrix metalloproteinase-3 production by gingival fibroblasts from a patient with aggressive periodontitis. presented at the annual meeting of the american academy of periodontology, san diego; 2008. p. 187–96. 13. gursoy uk, kononen e, uitto vj, pussien pj, hyvarinen k, suominen-taipale l, knuuttila m. salivary interleukin-1b concentration and presence of multiple pathogens in periodontitis. j clinperiodontol 2009; 36: 922–7. 14. gorska r, gregorek h, kowalski j, laskus-perendyk a, syczewaka m, madalinski k. relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum sample from patient with chronic periodontitis. j clin periodontal 2003; 30: 1046–52. 15. n a k a m u r a t , n i t t a h , i s h i k a w a i . e f f e c t o f l o w d o s e actinobacillus actinomycetemcomitans lipopolysaccharide pretreatment on cytokine productionby human whole blood. j periodont res 2004; 39: 129–35. 16. ren l, jiang zq, fu y, leung wk, jin lj. the interplay of lipopolysaccharide–binding protein and cytokines in periodontal health and disease. j clin periodontol 2009; 36: 619–26. 17. nishihara t, koseki t. microbial etiology of periodontitis. periodontol 2000, 2004; 36: 14–26. 18. agrawal aa, kapley a, yeltiwar rk, purohit hj. assessment of single nucleotide polymorphism at il-1a+4845 and il-1b+3954 as genetic susceptibility test for chronic periodontitis in maharashtrian ethnicity. j periodontol 2006; 77(9): 1515–21. 102 vol. 43. no. 2 june 2010 research report the apical leakage of mineral trioxide aggregate as the retrograde filling material with various mixing agents ema mulyawati department of conservative dentistry faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: mineral trioxide aggregate (mta) is relatively considered as a new material in endodontic. it even has been used as retrograde filling material due to its biocompatibility, antibacterial effect, sealing ability and anti-moist effect. some materials have been used as mixing agent to achieve an appropiate setting of mta. purpose: the aim of this study is to investigate the effect of the mixing agents of mta towards the apical leakage when they are used together as retrograde filling materials. method: the samples of this research consist of 30 human extracted upper central incisors. first, the crown of each tooth is sectioned. the root canals are prepared by using the conventional technique and then are obturated with gutta percha. after cutting the root apex, 2 mm from apical, class 1 cavities are prepared by using fissure bur with the depth of 3 mm. the samples then are divided into 3 groups with 10 teeth for each. group i uses aquabidest as mixing agent of mta (mta-aquabidest), group ii uses saline (mta-saline), while group iii uses 0.12% chlorhexidine (mta-chlorhexidine). the apex of each group then is filled with the mixing mta determined already. afterwards, clearing method is used to evaluate the apical leakage. the apical leakage actually is determined by measuring the depth of methylene blue penetration with stereomicroscope. the statictical analyses of the linear dye penetration then are performed with analysis of varians anova. result: the dye penetration for both mta-aquadest and mta-saline groups indicates the lowest penetration, and there is even a significant difference compared with mta-0.12% chlorhexidine group (p<0.005). conclusion: it can be concluded that aquabidest and saline as mixing agents of mta produce less apical leakage compared with 0.12% chlorhexidine. key words: apical leakage, retrograde filling, mta, saline, chlorhexidine abstrak latar belakang: mineral trioxide aggregate (mta) merupakan bahan yang relatif baru dalam bidang endodontik. bahan tersebut diindikasikan sebagai bahan pengisi retrograd karena bersifat biokompatibel, antibakteri, kerapatannya bagus dan tidak terpengaruh kelembaban.untuk mendapatkan settingnya, beberapa bahan telah digunakan sebagai bahan pencampur mta. tujuan: penelitian ini bertujuan untuk mengetahui pengaruh bahan pencampur mta sebagai bahan retrograd terhadap kebocoran apikal. metode: bahan penelitian berupa 30 gigi insisivus sentral atas bekas cabutan. mahkota gigi dipotong dan saluran akar dipreparasi menggunakan teknik konvensional dan diobturasi dengan guta perca. akar dipotong dengan jarak 2 mm dari apeks dan dibuat preparasi kavitas kelas i menggunakan bur fisura dengan kedalaman 3 mm pada ujung akar tersebut. akar gigi tersebut dibagi dalam 3 kelompok masing-masing 10. kelompok i menggunakan akuabides sebagai bahan pencampur mta (mta-akuades), kelompok ii menggunakan salin (mta-salin) dan kelompok ii menggunakan chlorhexidine 0,12% (mta-chlorhexidine). ujung akar kemudian diisi campuran mta sesuai kelompoknya. evaluasi kebocoran apikal menggunakan teknik clearing. kebocoran apikal ditentukan dengan mengukurkebocoran apikal ditentukan dengan mengukur kedalaman penetrasi larutan biru metilen menggunakan mikroskopstereo. hasil pengukuran dianalisis menggunakan analisis varian (anova). hasil: penetrasi warna pada kelompok mta-akuades maupun mta-salin menunjukkan hasil yang paling kecil dan kedua kelompok tersebut berbeda secara signifikan dengan kelompok mta-chlorhexidine 0,12% (p<0,005). kesimpulan: bahan pencampur akuades dan salin menghasilkan mta dengan kebocoran apikal yang lebih kecil dibandingkan chlorhexidine. kata kunci: kebocoran apikal, pengisian retrograd, mta, salin, chlorhexidine 103mulyawati: the apical leakage of mineral trioxide correspondence: ema mulyawati, c/o: department of conservative dentistry, faculty of dentistry, gadjah mada university. jl denta. sekip utara. jogjakarta 55281, indonesia. e-mail: aandwinuryanto@yahoo.com materials.1 in a research, stowe even has already concluded that mta mixed with chlorhexidine can not only cause the increasing of its compressive strength, but can also cause the increasing of its antibacterial effect. the concentration of chlorhexidine used as mta mixing agent is 0.12%.1 the sealing ability of mta, furthermore, is likely derived from its natural hydrofilic and small expansion when located in a moist environment.11 the reason is because in moist environments, the further hydration of mta powder will increase its compressive strength and make the apical leakage lower.17 the success of root canal treatment either filled in orthograde or in retrograde is mainly determined by the sealing ability of apical filling quality. the easiest way to evaluate the apical sealing ability is by measuring the leakage rate. the smaller the apical leakage happens, the better sealing ability is. therefore, this study aims to determine the effect of mixing agents of mta as retrograde filling materials towards the apical leakage. material and method in this research, the samples are 30 human upper central incisors maxillary which have been extracted . however, the teeth should have the following criteria, such as the roots of those teeth are not curved, the roots are fully developed, and the root canal can be accessed by the maximum files until the number 25. firstly, the roots of teeth are cut with a distance of 14 mm from the apex of the teeth, and then are prepared by using conventional techniques with files up to number 40 with the working length 13 mm. every turn of the file, the root canals are irrigated by 1 cc of 2.5% naocl and saline, and then they are dried with paper points. before filling the root canals, they must be coated by paste (endomethasone) using lentulo which means that the root canals are filled with lateral condensation of gutta-percha point. the gutta-percha number 40 (master cone) which has been marked based on the working length is coated with root canal paste on one third of the apical, and then it is immediately inserted into the root canal. insert the main spreader between the guttapercha point and the root canal wall, and then press into the apical direction until it reaches 2 mm from the apex. the remained space then is filled with the additional guttapercha point until the root canal is full. the gutta-percha point is then cut based on the length work by using hot plastic instrument, while its corona is covered with zinc phosphate cement. those roots of the teeth are then stored in the 37o c incubator for 24 hours. later, the apex in the apical area is cut horizontally perpendicular to the axis of the tooth by using a fissure bur at high speed with a distance of 2 mm from the apical tip. the cut apex is then prepared with retrograde cavity type introduction one way to overcome the failure of root canal treatment is by conducting endodontic surgery, which is by cutting the apex of the tooth and then by putting the retrograde filling materials at the tip of the apex.1,2 mineral trioxide aggregate (mta) is a new retrograde filling material developed for a variety of endodontic treatments, such as for pulp capping, root perforation, apexification, and retrograde filling materials.3-6 nowadays, mta is widely used as a retrograde filling material due to its biocompatibility, antibacterial effect, sealing ability, and anti-moist effect. though mta still has some weaknesses one of which is that mta needs moisture to reach its settings, this condition is precisely appropriate to make mta to be used as retrograde filler since it is always in a moist environment, in the periapical area.1,2,7-9 nevertheless, mta setting time will expire after 28 days.10 mta, moreover, consists of hydrophilic particles composed of tricalcium silicates, tricalcium aluminates, tricalcium oxide, and silicate oxide as well as its additional material, bismuth oxide, used to add its radiopacity. the nature of the hydrophilic particles is what causes the mta requires moisture to its setting.11 as a result, the commonly mixing agent of mta powder is aquabidest with the ratio 3: 1. after being mixed with aquabidest, mta then will form a mixture that looks like wet sand, and later it will form an amorphous specific crystal structure with granules-shaped appearance.12 it means that the nature of mta are influenced by particle size, powder-liquid ratio, temperature, and humidity.11 the bonding of mta on dentin is possibly derived from its natural hydrophilic properties and its small expansion when located in moist environments. to obtain mta mixed with better properties, some researchers even use other materials besides aquabidest, such as saline and chorhexidine. in the reaction between the mta and saline, mta actually react with water molecules only, whereas nacl contained in saline does not come to react.13 the reason is because of the differences in the nature electronegativity among negative ions contained in the mta powder. 12 therefore, the result of the reaction between mta and water will form hydrated colloidal gel. since saline is considered as a hypertonic solution, the reaction between saline and colloidal will cause the water molecules that exist in the colloidal attracted to the saline solution, as a consequence, the colloidal gel becomes harder.13 in other words, the less water molecules contained in a material, the less porosity will be created.14 it may be concluded that mta mixed with saline has greater compressive strength than mta mixed with aquabidest. 15 in addition, chlorhexidine actually is considered as disinfectant that is widely used in endodontic treatment, such as being used as root canal irrigation and sterilization 104 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 103-106 class i by using a fissure bur with 1.5 mm in diameter and 3 mm in depth. then, those 30 teeth are randomly divided into 3 groups, each of which consists of 10 teeth. group i is filled with mta and aqubidest, group ii is filled with mta and saline, while group iii is filled with mta and chlorhexidine. in group i, for instance, mta powder is mixed with aquabidest with a ratio of powder: aquabidest 3: 1 (1 gram of powder mta: 0.33 grams aquabidest). next, stir it on the cup by using mta mixer until it becomes homogeneous. after that, it is inserted into the tip of the root which has been prepared in group i by using the micro apical placement (map) until it is full. then, after it is flattened, the cotton that has already been soaked in a solution of phosphate-buffered saline (pbs) is immediately used to cover the entire surface of mta as well as the tip of the tooth root. the reason is because pbs solution can be used to simulate the moisture in the periapical area.18 like in group i, in group ii, in which mta is mixed with saline, and in group iii, in which mta is mixed with chlorhexidine, moreover, the similar procedures is also conducted. to guarantee the condition is always moist, the application of wet cotton wool soaked in pbs solution on the surface of the tooth root tip must be conducted every day. besides that, the cotton wool must always be replaced with the new one every day for 28 days. after 28 days, the cotton that covers the tip of the tooth root is removed, and followed by apical leakage testing. the apical leakage testing actually uses the method of clearing. the entire of the outer surface of the teeth is coated with adhesive wax and closed again with nail polish, except in the surface area of retrograde. the whole of the subjects of this research is then soaked in 2% methylene blue for 48 hours. afterwards, they are washed with running water for 15 minutes, and the adhesive wax and the nail polish covering them are then removed by using le crown mess. furthermore, those teeth are demineralized by soaking them in 5% hno3 for 72 hours which should be replaced every 24 hours. the next stage, those teeth are dehydrated in 96% alcohol for 48 hours which also must be replaced every 24 hours. the last stage is conducting clearing in methyl salicylate until the penetration of the color of ink can be observed visually.19 the observation is conducted with a stereo microscope for measuring the most long penetration of the ink color (in units of mm), from the cut surface of the root tip to the coronal tooth, in order to obtain the number of apical leakage. the result data are finally analyzed by using one-way anova test followed by t-test. result the mean of the apical leakage in each treatment groups can be seen in table 1. to know the significance of differences among the three treatment groups, one-way anova test is used with the significance level of 95%. through anova tests, it is known that p<0.005, which means that there are differences in apical leakage among those three treatment groups using mta with three different mixing agents, aquabidest, saline, and chlorhexidine. moreover, to know which pair of those three treatment groups is really different, t-test is then conducted. the result of t-test among those three groups can be seen in table 2. based on the result, it is known that the apical leakage of the retrograde filling by using mta, as raw material, mixed with chlorhexidine has the largest apical leakage compared with the other two treatment groups using saline or aquabidest as the mixing agents of mta. meanwhile, the retrograde filling by using mta mixed with saline had the same apical leakage as that by using mta mixed with aquabidest. discussion mta is a relatively new material developed in the field of endodontics, especially as a retrograde filling material. as a retrograde filling material, mta has the best sealing ability compared with other retrograde materials previously used, such as amalgam, composite resin, reinforced eugenol oxide zinc cement, and glass ionomer cement. however, some mixing material agents, aquabidest and chlorhexidine, are still used to improve the nature of the mta.1–4 in this research, during the hardening phase mta materials are always applied with cotton soaked in pbs. this condition is the the simulation of periapical tissue condition.18 in wet condition those mta materials will continually release calcium and hydroxyl ions. as a result, calcium that released will react with phosphate contained in pbs solution, and then will form amorphous calcium phosphate. next, the formed calcium phosphate will be hydrolyzed in order to form carbonate apatite type b which is considered as hydroxyapatite. the hydroxyapatite then will fill the gap between those retrograde filling materials and the root canal wall.18 nevertheless, all of the samples in this research have apical leakages shown by the methylene blue dye penetration. no retrograde filling material can perfectly adapt to the tooth structure so that there will always be a gap between the materials of retrograde and tooth structure.20 the gap occurred is mainly caused by the condition in which the coefficient of thermal expansion of those tooth retrograde filling materials do not fit with the coefficient of thermal expansion of the tooth structure.21 based on the result of t-test it is known that there is no difference in the apical leakage of the retrograde filling process using mta mixed with saline and that mixed with aquabidest, meanwhile the apical leakage of the retrograde filling process using mta mixed with 0.12% chlorhexidine is known as the largest apical leakage compared with the other two mixing material agents. actually, the reaction of mta with aquabidest is almost the same as the reaction of aquabidest with portland cement in which the hardening process occurs through three stages.10 in the first stage, the reaction of hydration derived from tricalcium aluminate forms hydrated colloidal gel occurred after 24 hours. in the 105mulyawati: the apical leakage of mineral trioxide of calcium released by mta with phosphate contained in the pbs solution. this hydroxyapatite even is proven to be able to close the gap of mta-aquabidest as good as the one of mta-saline. the group with the mixture of mta-0.12 chlorhexidine still has the greater apical leakage than the ones with the mixture of mta and the mixing material agents, either aquabidest or saline, although like in those two groups with the mixture of mta and either aquabidest or saline, the hydroxyapatite occurs in the gap between teeth in the group with the mixture of mta-0.12% chlorhexidine. chlorhexidine is classified into an inorganic compound. thus, if it is combined with the organic compounds of mta (calcium, aluminum, silicate), it will form a complex bond.23 the complex bond is among aluminum elements of tricalcium silicate, tricalcium aluminate and tricalcium oxide with chlorhexidine, and among silicate elements of tricalcium silicate and silicate oxide. silicate oxide actually can not only make the hardening process uneasily occur, but can also make a bad adhesion between the mixture of mta-chlorhexidine and those teeth. calcium binding to chlorhexidine makes the mixture of mta-chlorhexidine more fragile since the calcium in mta is continually taken by chlorhexidine. the fragility of this material affects the apical leakage occurred. thus, the numbers of the apical leakage occurred on the mixture of mtachlorhexide is considered as the highest one compared with that occurred on the mixture of mta-saline-aquabidest. although hydroxyapatite which will enhance the sealing ability of mta is formed in the gap between all of those three different mixing agents of mta and those teeth, but the factor of the mta-chlorhexidine mixture play bigger role. based on the result of this research, it can be concluded that among three types of the mta mixing material agents, saline and aquabidest is considered as the best mta mixing materials. references 1. gutmann jl, dumsha tc, lovdahl pe. problem solving in endodontics. 4th ed. st louis: mosby; 2006. p. 142–55. 2. torabinejad m, mcdonald nj. endodontic surgery. in: torabinejad m, walton re, eds. endodontic principles and practice. 4th ed. china: saunders elseiver; 2009. p. 357–90. 3. coneglian pza, orosco fa, bramante cm, moraes ig, garcia rb, bernardile n. in vitro sealing ability of white and gray mineral trioxide aggregate (mta) and white potland cement used as apical plugs. j appl oral sci 2007; 15(3): 181–5. 4. torabinejad m, chivian n. clinical applications of mineral trioxide agregate. j endod 1999; 25(3): 197. 5. shabahang s, torabinejad m, boyne pp, abedi h, mcmillan p. a comparative study of root-end induction using osteogenic protein, calcium hydroxide and mineral trioxide aggregate in dogs. j endod 1999; 25(1): 1–5. 6. economides n, pantlidou o, kokkas a, tziafas d. short term periradicular tissue response to mineral trioxide aggregate (mta) as root end filling material. int endod j 2003; 36(1): 44–8. 7. fellipe wt, racha mjc. the effect of mineral trioxide agregate on the apexification and periapical healing of teeth with incomplete root formation. int endod j 2006; 39(1): 2–9. second stage, between the first day and the seventh day, the tricalcium silicate and the tricalcium aluminate reacting with water form ca(oh)2 form aluminum hydroxide and amorphous calcium silicate. in the third stage, between the seventh day and the twenty eighth day, the reaction is getting slow in which hydrated calcium silicate progressively forms hydrated silicate gel, and then ca(oh)2 will be spread on the gel giving strength to the cement that has already hardened, as a result, it makes the cement is getting harder.12 however, the hardening reaction of mta with saline is actually equal to that with aquabidest. when mta is mixed with saline, only water molecules reacts with mta powders, whereas nacl contained in saline solution does not participate in the reaction. saline is actually considered as a hypertonic solution, thus, the mixing of mta powders and saline can be affected by the hypertonic nature of saline. as a result, the hypertonic nature of saline causes water molecules contained in the powder mixture of mta-saline pulled outside.12,13 in other words, the less water molecules contained in a material, the less the resulting porosity. the hypertonic nature of saline actually causes the mixture of mta-saline less centered than the mixture of mta-aquabidest.15 thus, the less porosity occurs in the materials, the smaller the leaks occur.14 in this research it is known that the numbers of the apical leakages occurred in the treatment group with the mixture of mta-aquabidest is the same as those occurred in the treatment group with the mixture of mtasaline. the reason could be more because of the role of closing the gap between the retrograde filling materials and the dentin of the root canal dentin than because of the difference of the internal nature between mta materials and the mixing agents, either aquabidest or saline. moreover, the hydroxyapatite is actually formed from the hydrolysis process of calcium phosphate derived from the reaction table �. the mean of the apical leakage in the retrograde filling by using mta together with other mixing agents, aquabidest, saline, and 0.12% chlorhexidine (mm) the mixing agents of mta number of samples x standard deviation aquabidest 10 1.200 0.133 salin 10 1.040 0.217 chlorhexidine 10 2.560 0.241 table ��. the result of t-test on the apical leakage of the retrograde filling by using mta with three different mixing agents, aquabidest, saline, and 0.12% chlorhexidine mixing agents aquabidest saline chlorhexidine aquabidest 1.766>0.005 saline 16.778<0.005 chlorhexidine 15.021<0.005 p<0.005: significance 106 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 103-106 8. bonson s, jeansonne bg, lallier te. root end filling materials afterroot end filling materials after fibroblast differentiation. j dental research 2004; 83: 408–13. 9. ribeiro da, matsumoto ma, duarte mah, salvadori dmf. ex vivo biocompatibility test of reguler and white forms of mta. int end journal 2006; 39: 26–30. 10. dammaschake t, gerth huv, zuchner h, schafer e. chemical and physical surface and bulk material characterization of white prooroot mta and two portland cements. dental material 2005; 21: 731–38. 11. torabinejad m, hong cu, mcdonald f, ford trp. physical and chemical properties of a new root-end filling material. j endod 1995; 21(7): 349. 12. nandini s, ballal s, kandaswamy d. influences of glass-ionomer cement on the interface and setting reaction of mineral trioxide agregate when used as a furcal repair material using laser raman spectropic analysis. j endod 2007; 33(2): 167–72. 13. brady, james e.(1989) kimia universitas asas dan struktur. edisi 5. jakarta: binarupa aksara; 1999. p. 62–69. 14. anusavice, kenneth j. 1996. buku ajar ilmu bahan kedokteran gigi. edisi 10. jakarta: egc. 2004. p. 65–76. 15. kogan p, he j, glickman gm, watanabe i. the effect of various additives on setting properties of mta. j endod 2006; 32(6): 569. 16. stowe tj, sedgley cm, stowe b, fenno jc. the effect of chlorhexidine (0.12%) on the antimicrobial properties of tooth-colored pro root mineral trioxide agregate. j endod 2004; 30(6): 429–31. 17. sarkar kn, caicedo r, ritwik p, moiseyeva r, kawashima i. physicochemical basis of the biologic properties of mineral trioxide aggregate. j endod 2005; 31(2): 97–100. 18. martin lc, monticelli f, brackett ww, loushine rj, rockman ra, ferrari m, pashley dh, tay fr. sealing properties of mineral trioxide aggregate. j endod 2007; 33(3): 272. 19. martin lc, luque cmf, rodriguez gp, gijon rv, mondelo nf. a comparative study of apical leakage of endomethasone, top seal and roeko seal aealer cements. j endod 2002; 28(6): 423–26. 20. vasudev sk, goel br, tyagi s. root end filling materials-a review. endodontology 2003; 15: 12–18. 21. tredwin cj, stokes a, moles dr. influence of flowable liner and margin location on microleakage of conventioanl and packable class ii resin composites. oper dent 2005; 30(1): 32–38. 22. depkes ri. farmakope indonesia. edisi 5. jakarta: direktorat jendral pengawasan obat dan makanan; 1995. p. 157–63. 23. rowe rc, sheskey pj, owen sc. handbook of pharmaceutical excipients. 5th ed. london: pharmaceutical press; 2006. p. 163–7. vol 38-no 1-2005 41 peran musik sebagai fasilitas dalam praktek dokter gigi untuk mengurangi kecemasan pasien (the role of music as a dental practice facility in reducing patient’s anxiety) eric priyo prasetyo mahasiswa ppdgs fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract the people generally tend to associate a dental practice with a strange clinical atmosphere and a range of strange looking instruments, and furthermore pain during dental treatments. this condition more or less raises an anxiety to the patient and affects the patient’s regular dental attendance. since anxiety has a significant role to the perception of pain, many attempts to make the patients relax are therefore needed to be done. along with the advanced era, the development of science and technology affects the world of dental practice. dentists are encouraged to provide better dental services and treat their patients holistically. in order to fulfill this, additional facilities such as music are needed. it was found that patients who listened to the preferred music before, during, and after their dental treatment tended to have the lower rate of anxiety. this decreased anxiety was the result of greatly increased feelings of relaxation and calmness, since music could dramatically influence physiological and psychological processes. music based on preference also provided most patients a non-threatening and pleasurable experience. recently, music as part of dental practice has been widely used in america and europe. this article is purposed to inform that music as an additional facility plays an important role in reducing the patient’s anxiety to provide better dental care and patient management. key words: music, dental practice facility, patient’s anxiety korespondensi ( correspondence): eric priyo prasetyo, mahasiswa ppdgs, fakultas kedokteran gigi universitas airlangga. jl. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. tel/fax: (62-31)5039478. e-mail: ep_prasetyo@yahoo.com. pendahuluan masyarakat awam pada umumnya cenderung memberi kesan bahwa praktek dokter gigi memiliki suasana dan peralatan yang asing, dan terlebih lagi berhubungan dengan rasa nyeri.1,2 hal ini menyebabkan pasien menjadi cemas sehingga mempengaruhi kunjungan rutin pasien untuk berobat ke dokter gigi.3 para peneliti memperkirakan bahwa antara 50% hingga 80% dari seluruh kasus penyakit yang terjadi berkaitan secara langsung dengan kecemasan sebagai etiologi.1,4 karena kecemasan memiliki peranan yang sangat penting dalam persepsi pasien tentang rasa nyeri, maka diperlukan usaha untuk mengurangi kecemasan dan membuat pasien menjadi rileks. seiring dengan kemajuan jaman, ilmu pengetahuan dan teknologi yang selalu berkembang memberi dampak dalam dunia praktek dokter gigi. dokter gigi semakin dituntut untuk memberikan pelayanan pada pasiennya secara holistik (menyeluruh) meliputi fisik dan psikis,1 hal ini menuntut diupayakannya berbagai macam fasilitas untuk memenuhi keinginan tersebut, salah satunya yaitu dengan tersedianya fasilitas musik bagi pasien dalam praktek dokter gigi. musik dengan potensinya dalam mempengaruhi baik proses fisiologis dan psikologis menjadi fasilitas yang penting dalam praktek untuk mengatasi kecemasan. musik yang sesuai dengan selera pasien mempengaruhi sistem limbik dan saraf otonom, menciptakan suasana rileks, aman dan menyenangkan sehingga merangsang pusat rasa ganjaran dan pelepasan substrat kimia (gamma amino butyric acid (gaba), enkephalin, dan beta endorphin) yang akan mengeliminasi neurotransmitter rasa nyeri maupun kecemasan sehingga menciptakan ketenangan dan memperbaiki suasana hati (mood) pasien. saat ini di amerika serikat dan eropa fasilitas musik semakin menjadi bagian dalam praktek dokter gigi.1,4 adapun tujuan dari pembuatan artikel ini adalah untuk memberikan masukan bahwa musik sebagai fasilitas memiliki peran dalam mengurangi kecemasan pasien. kecemasan pasien kecemasan merupakan faktor psikologis afektif yang mempengaruhi persepsi rasa nyeri. pada banyak kasus nyeri akut seperti pulpitis, kecemasan banyak berhubungan dengan meningkatnya kejadian rasa nyeri,3 yakni tidak hanya menurunkan ambang rasa nyeri pasien tetapi juga pada kenyataannya mengakibatkan persepsi yang seharusnya tidak nyeri menjadi nyeri, bahkan di bawah kondisi yang berbeda, seorang pasien dapat menunjukkan reaksi yang berbeda walau rangsangannya sama. kecemasan pasien memberikan efek negatif terhadap prosedur perawatan yang akan dilakukan.5 kecemasan 42 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 41–44 dalam praktek dokter gigi merupakan halangan yang sering mempengaruhi perilaku pasien dalam perawatan.3 telah diketahui bahwa banyak pasien menjadi cemas sebelum dan sesudah perawatan.6 pasien yang menunggu perawatan pada umumnya cemas,7 dan kecemasan dapat ditingkatkan oleh persepsi pasien tentang ruang praktek sebagai lingkungan yang mengancam, tentang perawat, cahaya, bunyi, dan bahasa teknis yang asing bagi pasien.1 menunggu perawatan pada kenyataannya lebih traumatik daripada perawatan itu sendiri.7 hal yang menyebabkan tingginya tingkat stress dan kecemasan dari keseluruhan situasi praktek dokter gigi, adalah prosedur bedah mulut dan perawatan endodontik.2,8,9 oleh karena itu pasien yang datang untuk perawatan endodontik kemungkinan besar cemas dan mengalami rasa nyeri selama perawatan. kecemasan dapat menyebabkan pasien mengeluh nyeri walau tidak didapatkan adanya dasar patofisiologis, misalnya melakukan preparasi pada gigi dengan pulpa nonvital, kadang pasien tetap mengeluh nyeri walaupun telah dilakukan anestesi lokal. situasi ini berhubungan erat dengan ketakutan pasien terhadap perawatan dokter gigi, karena rasa nyeri memiliki sifat subyektif, sehingga tidak dapat dibedakan antara nyeri karena alasan psikologis dan nyeri karena reaksi jaringan, karena pasien menganggap keduanya sebagai rasa nyeri.3 musik musik dan bidang kedokteran memiliki hubungan sejarah yang erat dan panjang. sejak jaman yunani kuno musik digunakan sebagai sarana untuk meringankan penyakit dan membantu pasien dalam mengatasi emosi yang menyakitkan seperti kecemasan, kesedihan, dan kemarahan.4 para ahli filsafat, sejarah, dan ilmuwan dari jaman dahulu hingga sekarang banyak menulis dan menyatakan bahwa musik memiliki sifat terapeutik.10 musik dikenal melalui penelitian sebagai fasilitas perangsang relaksasi non farmasi yang aman, murah, dan efektif.5 musik memiliki peran signifikan dalam merawat pasien dengan kecemasan. para peneliti mengatakan bahwa musik mampu menurunkan gejala psikosomatik seperti kecemasan dengan jalan mempengaruhi proses fisiologis dan psikologis sehingga mampu membuat pasien mengalami keadaan yang aman dan menyenangkan, tetapi musik tidak seperti obat karena musik tidak memiliki potensi untuk menyebabkan ketergantungan.10 musik yang digunakan sejak lama untuk mencapai kenyamanan dan relaksasi telah diajukan sebagai salah satu cara untuk menurunkan kecemasan psikologis dan perilaku individual yang menunggu perawatan.4 efek positif musik dalam mengurangi kecemasan ditentukan oleh respons tiap individu pasien terhadap musik yang didengarnya, sehingga dalam hal ini selera masing-masing pasien memegang peranan yang penting. pada umumnya musik klasik popular dengan alunan rileks adalah pilihan yang sering digunakan. pasien juga dapat diminta membawa sendiri atau memilih jenis musik yang disukainya.1 pembahasan pasien yang tegang dan cemas lebih banyak merasakan nyeri selama perawatan dibandingkan pasien yang rileks karena kecemasan menciptakan harapan akan rasa nyeri,3 oleh karena itu pasien dengan kecemasan yang datang untuk perawatan dengan ingatan akan rasa nyeri yang sebelumnya pernah dialami cenderung membayangkan timbulnya rasa nyeri selama perawatan, sehingga pasien tersebut menyaring secara selektif setiap informasi sebelum perawatan dan memusatkan perhatian pada setiap rangsangan yang menyerupai atau berhubungan dengan rasa nyeri.2 sebagai contoh, tekanan pada gigi yang sangat ringan sekalipun dapat dipersepsikan sebagai rasa nyeri dan mengawali timbulnya reaksi rasa nyeri. perangsangan yang disebabkan oleh kecemasan juga dapat meningkatkan aktivitas saraf simpatik dan ketegangan otot sehingga menyebabkan rasa nyeri tambahan.11 kecemasan pre-operative memiliki sifat subyektif, dan secara sadar perasaan tentang kecemasan serta ketegangan yang disertai perangsangan sistem saraf otonom menyebabkan peningkatan tekanan darah, denyut jantung, dan tingkat respirasi.12,13 hal ini sangat berbahaya karena tingginya denyut jantung dan tekanan darah memperberat sistem kardiovaskuler dan meningkatkan kebutuhan akan oksigen dan kerja jantung.11 kondisi pasien yang diliputi kecemasan akan memperkuat rangsang nyeri yang diterimanya karena kecemasan menyebabkan zat penghambat rasa nyeri tidak disekresikan. dengan adanya musik sebagai fasilitas dalam praktek dokter gigi maka tingkat kecemasan pasien dapat dikurangi sehingga timbul perasaan tenang dan rileks, dan dapat mengurangi rasa nyeri. musik sebagai gelombang suara diterima dan dikumpulkan oleh daun telinga masuk ke dalam meatus akustikus eksternus hingga membrana timpani. oleh membrana timpani bersama rantai osikule dengan aksi hidrolik dan mengungkit, energi bunyi diperbesar menjadi 25–30 kali (rata-rata 27 kali) untuk menggerakkan medium cair perilimf dan endolimf. setelah itu getaran diteruskan hingga organ korti dalam kokhlea dimana getaran akan diubah dari sistem konduksi ke sistim saraf melalui nervus auditorius (n. viii) sebagai impuls elektris.14 impuls elektris musik masuk melalui serabut saraf dari ganglion spiralis corti menuju ke nukleus koklearis dorsalis dan ventralis yang terletak pada bagian atas medulla.13 pada titik ini semua sinap serabut dan neuron tingkat dua diteruskan terutama ke sisi yang berlawanan dari batang otak dan berakhir di nukleus olivarius superior. setelah melalui nukleus olivarius superior, penjalaran impuls pendengaran berlanjut ke atas melalui lemniskus lateralis kemudian berlanjut ke kolikulus inferior, tempat semua atau hampir semua serabut ini berakhir. setelah itu impuls berjalan ke nukleus genikulata medial, tempat semua serabut bersinap, dan akhirnya berlanjut melalui radiasio auditorius ke korteks auditorius, yang terutama terletak pada girus superior lobus temporalis.12 43prasetyo: peran musik sebagai fasilitas dari korteks auditorius yang terdapat pada korteks serebri area 41, jaras berlanjut ke sistem limbik, melalui cincin korteks serebral yang disebut korteks limbik. korteks yang mengelilingi struktur subkortikal limbik ini berfungsi sebagai zona transisional yang dilewati sinyal yang dijalarkan dari sisi korteks ke dalam sistem limbik dan juga ke arah yang berlawanan. dari korteks limbik, jaras pendengaran dilanjutkan ke hipokampus, tempat salah satu ujung hipokampus berbatasan dengan nuklei amigdaloid.13 amigdala yang merupakan area perilaku kesadaran yang bekerja pada tingkat bawah sadar, menerima sinyal dari korteks limbik lalu menjalarkannya ke hipotalamus. di hipotalamus yang merupakan pengaturan sebagian fungsi vegetatif dan fungsi endokrin tubuh seperti halnya banyak aspek perilaku emosional, jaras pendengaran diteruskan ke formatio retikularis sebagai penyalur impuls menuju serat saraf otonom. serat saraf tersebut mempunyai dua sistem saraf yaitu sistem saraf simpatis dan sistem saraf parasimpatis.12 kedua sistem saraf ini mempengaruhi kontraksi dan relaksasi organ-organ. relaksasi dapat merangsang pusat rasa ganjaran sehingga timbul ketenangan. sebagai ejektor dari rasa rileks dan ketenangan yang timbul, midbrain akan mengeluarkan gamma amino butyric acid (gaba), enkephalin, beta endorphin. zat tersebut dapat menimbulkan efek analgesia yang akan mengeliminasi neurotransmitter rasa nyeri pada pusat persepsi dan interpretasi sensorik somatik otak.13 musik dalam hal ini berfungsi sebagai sebuah intervensi untuk mengurangi tingkat kecemasan pasien dalam berbagai situasi klinis. musik juga terbukti memperbaiki suasana hati (mood) pasien post-operative.15 musik mengurangi kecemasan fisiologis pada individu yang siap menjalani perawatan dan tercatat adanya penurunan tekanan darah sistolik dan diastolik pasien.4 pemberian fasilitas musik menunjukkan penurunan denyut jantung,15 tingkat respirasi,7 dan kebutuhan oksigen pada pasien dalam ruang praktek. 16 musik juga dapat menimbulkan efek neuroendokrin yang berguna bagi pasien. musik dapat meningkatkan suatu respons seperti endorfin, yang dapat mempengaruhi suasana hati yang dapat menurunkan kecemasan pasien.6 dalam penataan praktek, musik dapat membantu pasien untuk rileks sebelum dan selama prosedur pemicu kecemasan, perawatan dan terapi yang berhubungan dengan kanker.7,17 dikatakan juga bahwa musik dapat menenangkan bayi dan anak-anak.18 musik mampu mengurangi persepsi dan pengalaman nyeri dan meningkatkan toleransi terhadap nyeri akut dan kronis.3 musik mengalihkan pasien dari rasa nyeri, memecah siklus kecemasan dan ketakutan yang meningkatkan reaksi nyeri, dan memindahkan perhatian pada sensasi yang menyenangkan. hal ini didukung oleh pelepasan endorfin yang menghasilkan efek paliatif.8 musik juga dapat memperbaiki suasana hati (mood) yang tertekan dan dapat menurunkan kecemasan yang sifatnya kronis maupun situasional.15,19 musik dapat membantu konsentrasi, memecahkan masalah, dan membantu fungsi kognitif.4,20 penelitian di jepang menunjukkan bahwa musik dapat menurunkan frekwensi respirasi ireguler secara signifikan.21 musik dikatakan sebagai ansiolitik (relaxing agent) yang efektif.5 peneliti menyimpulkan bahwa musik memberikan keuntungan bagi pasien rawat jalan.7 pasien yang diberi fasilitas musik juga menunjukkan penurunan kadar kortisol dalam saliva dan setelah satu jam penurunannya relatif sama dengan pasien yang tidak sedang dalam perawatan. hal ini menunjukkan bahwa fasilitas musik memiliki efek menguntungkan yang signifikan dalam menanggulangi kecemasan untuk pasien yang diberikan informasi tentang perawatan yang akan dilakukan.22 kesimpulan yang dapat diambil dari telaah pustaka ini yaitu fasilitas musik dalam praktek dokter gigi memiliki peranan yang signifikan dalam menurunkan kecemasan pasien. disarankan agar fasilitas musik yang sederhana, murah, dan efektif ini disediakan untuk setiap pasien dalam ruang praktek agar tercipta suasana rileks, sehingga kecemasan pasien menjadi berkurang, namun disarankan juga agar pasien diberi kebebasan dalam memilih musik sesuai dengan seleranya sehingga efek terapeutik musik dapat tercapai, dengan demikian dokter gigi dapat meningkatkan pelayanannya kepada pasien. ucapan terima kasih penulis ingin mengucapkan terima kasih kepada dr. r. darmawan setijanto, drg., m.kes. yang telah banyak memberikan bimbingan dan motivasi, juga kepada maria gunawan, drg. yang telah memberikan ide dalam penyusunan artikel ini. daftar pustaka 1. varley p. complementary therapies in dental practice. 1st ed. sydney: elsevier australia; 1997. p. 6–28. 2. cohen s, burns rc. pathways of the pulp. 8th ed. mississippi: mosby inc; 2004. p. 20–3. 3. bergenholtz g. textbook of endodontology. copenhagen: blackwell pub professional; 2003. p. 57–65. 4. alexander m. the charms of music: step-by-step prescription for patients. ncmj 2001; 62(2): 91–4. 5. palakanis kc, denobile jw, sweeney wb, blankenship cl. effect of music therapy on state anxiety in patients undergoing flexible sigmoidoscopy. dis colon rectum 1994; 37(5): 478–81. 6. winter mj, paskin s, baker t. music reduces stress and anxiety of patients in the surgical holding area. j post anesth nurs 1994; 9(6): 340–3. 7. augustin p, hains a. effect of music on ambulatory surgery patients’ postoperative anxiety. aorn j 1996; 63: 650–8. 44 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 41–44 8. brand hs, gortzak rath, palmer-bouva ccr, abraham re, abraham-inpijn l. cardiovascular and neuroendocrine responses during acute stress induced by different types of dental treatment. int dent j 1995; 45: 45–8. 9. eli i, bar-tal y, fuss z, silberg a. effect of intended treatment on anxiety and on reaction to electric pulp stimulation in dental patients. j endodont 1997; 23: 694–7. 10. kayumov l. personalzed ‘brain music’ helps sleep. bbc newshealth 2002; 2198316: 1–3. 11. haun m. effect of music on anxiety of women awaiting breast biopsy. behavioral medicine 2001; 3: 1–8. 12. guyton ac, hall je. buku ajar fisiologi kedokteran. edisi 9. irawati setiawan, dkk jakarta: egc; 1997. p. 827–38, 929–42. 13. ganong wf. buku ajar fisiologi kedokteran. edisi 17. widjajakusumah m djauhari, dkk. jakarta: egc; 1998. p. 165–78, 187–8, 218–25, 255–6. 14. herawati s, rukmini s. buku ajar ilmu penyakit telinga hidung tenggorok. surabaya: unair; 2002. p. 17. 15. barnason s, zimmerman l, nieveen j. the effects of music interventions on anxiety in the patient after coronary artery bypass grafting. heart lung 1995; 24(2): 124–32. 16. white jm. effects of relaxing music on cardiac autonomic balance and anxiety after acute myocardial infarction. am j crit care 1999; 8: 220–30. 17. dubois jm, bartter t, pratter mr. music improves patient comfort level during outpatient bronchoscopy. chest 1995; 108: 129–30. 18. pratt rr, abel hh, skidmore j. the effects of neuro feedback training with background music on eeg patterns of add and adhd children. int j arts med 1995; 4: 24–31. 19. field t. maternal depression effects on infants and early interventions. prev med 1998; 27: 200–3. 20. rauscher f, shaw g, ky k. music and spatial task performance. nature. 1993; 365: 611. 21. ishii c, hagihara s, minamisawa r. effects of music on reducing pain associated with a compulsory posture. nihon karyo kagakkaishi 1993; 13(1): 20–7. 22. miluk-kolasa b, obminski 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice editorial board of dental journal (majalah kedokteran gigi) sk: 2847/h3.1.2/kd/2009 june 1st, 2009 june 1st, 2011 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. dr. m. rubianto, drg, m.s., sp. perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d.,fds. (conservative dentistry university of guy's dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery university science malaysia, malaysia); prof. widowati witjaksono, dds, ph.d. (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds.,ph.d. (pediatric – university of hiroshima, japan); prof. dr. a.g. m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg, m.s., sp. kga. (pediatric dentistry indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic jember university) managing editors: prof. dr. arifzan razak, drg, msc, sp.pros. (prosthodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp.kg. (conservative dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg., su., sp.bm. (oral and maxillofacial surgery – airlangga university); prof. dr. mieke sylvia m.a.r., drg, m.s., sp.ort. (orthodontic – airlangga university); prof. dr. istiati, drg, m.s. (oral biology – airlangga university); prof. dr. anita yuliati, drg, m.kes. (dental material – airlangga university); seno pradopo, drg, s.u., ph.d. sp.kga. (pediatric dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d., sp.ort. (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes. (dental public health – airlangga university); dr. elly munadziroh, drg., ms. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); udijanto tedjosasongko, drg, ph.d., sp.kga. (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes. (oral medicine – airlangga university); dr. diah savitri ernawati, drg., m.si. (oral medicine – airlangga university); bagus soebadi, drg, mhped. sp.pm (oral medicine – airlangga university); endang pudjirochani, drg, m.s., sp.pros. (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes. (microbiology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); ira widjiastuti, drg, m.kes. sp.kg. (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms. (orthodontic – airlangga university); devi rianti, drg, m.kes. (dental material – airlangga university); chiquita prahasanti, drg., sp.perio. (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros. (prosthodontic – airlangga university); an'nisaa chusida, drg., m.kes. (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg. (conservative dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 42 no. 3 july–september 2009: prof. yukio kato, d.d.s., ph.d. (oral bio chemistry-university of hiroshima, japan) prof. kozai katsuyuki, dds., ph.d. (pediatric – university of hiroshima, japan) prof. dr. a.g. m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands) r. helal soekartono, drg., m.kes. (dental material – airlangga university) sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) dr. retno indrawati, drg., msi. (oral biology – airlangga university) dr. ernie maduratna setiawatie, drg., m.kes., sp.perio. (periodontic – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 83/dikti/kep/2009 issn 1978 3728 dental journal majalah kedokteran gigi volume 42 number 3 july september 2009 design cover photo by r. pudyanto hari pribadi, drg. contents page printed by: airlangga university press. (159/08.09/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 42 number 3 july september 2009 issn 1978 3728 dental journal majalah kedokteran gigi 1. treatment of recurrent aphthous stomatitis major with metronidazole and ciprofloxacin m. jusri and nurdiana ..................................................................................................................... 109–113 2. mechanical properties of carving wax with various ca-bentolite filter composition widjijono, purwanto agustiono, and dyah irnawati ................................................................... 114–117 3. immunoglobulin-g level on aggressive periodontitis patients treated with clindamycin agung krismariono ......................................................................................................................... 118–122 4. color stability of visible light cured composite resin after soft drink immersion alizatul khairani hasan, siti sunarintyas, and dyah irnawati .................................................. 123–125 5. storage duration effect on deformation recovery of repacked alginates siti sunarintyas and dyah irnawati ............................................................................................... 126–129 6. calprotectin mrna (mrp8/mrp14) expression in neutrophils of periodontitis patients with type 2 diabetes mellitus ahmad syaify, marsetyawan hnes, sudibyo, and suryono ....................................................... 130–133 7. the influence of xylitol containing toothpaste on plaque formation inhibition on fixed bridge hamim fithrony, eha djulaeha and michel soedjono ................................................................. 134–136 8. functional relationship of room temperature and setting time of alginate impression material dyah irnawati and siti sunarintyas ............................................................................................... 137–140 9. the role of msx1 and pax9 in pathogenetic mechanisms of tooth agenesis yani corvianindya rahayu and dyah setyorini ........................................................................... 141–146 10. the use of holmium-yttrium aluminum garnet laser as pit and fissure cleaner armasastra bahar ............................................................................................................................ 147–150 11. allergic asthma in children: inherited, transmitted or both? (the transmission of periodontopathic bacteria concept) seno pradopo and haryono utomo ................................................................................................ 151–156 vol 38 no 3 2005 120 breket titanium (titanium bracket) sianiwati goenharto dan achmad sjafei bagian ortodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract there has been a considerable discussion in the literature about corrosion and sensitivity to the nickel present in stainless steel brackets. titanium has been heralded as a material totally compatible in the oral environment and superior in structural integrity compared to stainless steel. many current applications in dentistry and medicine have made titanium an obvious choice for a possible substitute material. titanium based brackets have shown excellent corrosion resistance and possessed good biocompatibility. evaluation of titanium brackets for orthodontic therapy showed that titanium brackets were comparable to stainless steel brackets in passive and active configuration. study about metallographic structure, hardness, bond strength to enamel substrate, etc. showed that titanium brackets exhibited a potential for clinical application. it was concluded that titanium brackets were suitable substitute for stainless steel brackets. key words: titanium, brackets korespondensi (correspondence): sianiwati goenharto, bagian ortodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan teknik perlekatan breket secara langsung pada enamel gigi telah menjadi prosedur rutin dalam perawatan ortodontik dengan peranti cekat. teknik ini telah diterima dengan baik karena relatif mudah dilakukan, efisien dan meningkatkan estetik apabila dibandingkan dengan teknik bonding.1 salah satu komponen penting dalam peranti ortodontik cekat adalah breket. dari awal pemakaiannya sampai sekarang, breket yang dipakai juga terus dikembangkan baik dari segi bahan dasar pembuatnya ataupun desainnya. desain berkembang untuk meningkatkan penampilan dengan memperkecil ukurannya,2 sampai dengan jenis terbaru yaitu self ligating bracket.3 breket yang mula-mula dipakai adalah yang berbahan dasar logam. karena perawatan ortodontik dengan peranti cekat banyak dilakukan pada penderita dewasa yang menuntut estetika tinggi, dikembangkan breket estetik. mula-mula dipakai bahan dasar plastik (misalnya: breket polikarbonat), akan tetapi penggunaannya kurang karena sifatnya yang kurang menguntungkan. selanjutnya, pada tahun 1980-an tersedia breket estetik yang terbuat dari single crystal sapphire dan alumina polikristal. keduanya berbahan dasar sama yaitu al2o3. selain itu juga ada breket dari zirconia polikristalin (zro2), yang dilaporkan mempunyai toughness terbesar di antara semua keramik. sayangnya bahan di atas menghambat mekanika sliding dan bermasalah pada waktu proses pelepasan perlekatan (debonding). breket dari single crystal sapphire juga menunjukkan specular highlight dan pada awal perkembangannya selama gerakan torsi sayapnya cenderung mudah patah, dan saat dilepas sering menyebabkan enamel gigi juga ikut lepas. beberapa breket jenis polikristalin menunjukkan warna yang kurang bagus. breket jenis keramik apabila dipasang pada insisif atau kaninus rahang bawah dapat membuat abrasi gigi rahang atas antagonisnya.4 meskipun secara estetik kurang baik, breket logam masih mempunyai banyak keunggulan baik dalam sifat mekanik maupun fisik apabila dibandingkan dengan breket estetik, sehingga masih merupakan breket yang paling banyak digunakan. breket logam yang dipakai umumnya dari bahan baja nirkarat. bahan ini mengandung nikel yang dapat bersifat sebagai alergen. reaksi alergi yang pernah dilaporkan bervariasi, yaitu dari edema lidah, bibir, mouth lining sampai dengan anafilaksis. 5 potensi logam menyebabkan reaksi alergi berhubungan dengan pola dan modus korosi, yang diikuti pelepasan ion-ion logam seperti nikel ke dalam rongga mulut. hal ini tidak hanya tergantung pada komposisi logam, tetapi juga suhu dan ph lingkungan.6 titanium merupakan logam pilihan untuk penderita yang dicurigai sensitif terhadap logam. selama beberapa dekade, implan titanium telah dipakai dengan keberhasilan yang memuaskan pada penderita dengan reaksi alergi yang parah.7 oleh karena itu, keberadaan breket titanium dapat menjadi alternatif untuk melakukan perawatan pada penderita yang hipersensitif. 121goenharto dan sjafei: breket titanium titanium titanium adalah unsur terbanyak ke sembilan di kerak bumi dan terdistribusi secara luas. karena afinitasnya yang besar terhadap oksigen dan unsur lain, titanium tidak terdapat dalam bentuk logam statis di alam, tetapi dalam bentuk mineral yang stabil. bentuk umum mineral titanium adalah ilmenite dan rutile dalam bentuk titanium dioksida.8 unsur titanium mula-mula ditemukan oleh reverend william gregor pada tahun 1790, tetapi baru pada tahun 1910 bentuk pure titanium pertama kali diproduksi, dan bahkan sampai sekarang titanium masih sangat mahal apabila dibandingkan dengan logam lain, misalnya baja nirkarat. secara klinis, ada dua bentuk titanium,9 yang pertama adalah dalam bentuk titanium murni (cpti). titanium murni adalah logam putih, lustrous dengan sifat densitas rendah, kekuatan tinggi dan daya tahan terhadap korosi yang sangat baik. bentuk kedua adalah alloy titanium-6% alumunium4% vanadium. alloy ini mempunyai kekuatan yang lebih besar dari titanium murni. alloy dipakai dalam industri kapal terbang, militer oleh karena densitasnya yang rendah, kekuatan tarik yang besar (500 mpa) dan tahan terhadap temperatur tinggi.8,9 kesuksesan penggunaan titanium secara klinis sehubungan dengan sifat mekaniknya yang baik, daya tahan terhadap korosi dan biokompabilitas yang sangat baik. toksisitas titanium sangat rendah dan ditoleransi baik oleh tulang maupun jaringan lunak. percobaan pada hewan menunjukkan tidak adanya perubahan sel sehubungan dengan implan titanium. konsentrasi unsur logam yang meningkat dapat dipantau pada jaringan penyangga melalui analisis spektrofotometri, meskipun demikian secara klinik tidak ditemui efek negatip. adanya laporan tentang warna jaringan penyangga yang menjadi lebih gelap karena cpti, mungkin disebabkan karena kekerasan yang rendah dan daya tahan terhadap abrasi yang rendah pada logam bukan alloy.7 titanium merupakan logam yang paling tahan korosi. logam ini sangat reaktif, dan sifat ini sangat menguntungkan, karena oksida yang terbentuk pada permukaan (tio2) sangat stabil dan mempunyai passivating effect terhadap logam.9 absorbsi titanium dari saluran pencernaan makanan sangat jelek. logam titanium yang dipakai sebagai implan sangat baik ditoleransi oleh jaringan. komposisi titanium lain seperti titanium dioksida, salisilat dan tannate telah digunakan dalam pembuatan kosmetik, obat-obatan dan produk makanan lain tanpa adanya laporan dampak negatifnya.8 beberapa penderita mungkin alergi terhadap logam sewaktu berkontak dengan kulit. terdapat bukti bahwa bahan tahan korosi seperti baja nirkarat dan alloy krom kobalt memproduksi sejumlah kecil produk korosi yang mungkin menyebabkan reaksi alergi. kasus alergi menyangkut baja nirkarat dan alloy krom kobalt yang telah dilaporkan, menunjukkan bahwa unsur di dalam alloy seperti kobalt, nikel atau bahkan kromium bisa merupakan bahan penyebab sensitivitas. ion logam juga membentuk komponen penyebab kulit menjadi sensitif. titanium tidak menyebabkan hipersensitifitas, demikian juga dengan alloy titanium-6% alumunium4% vanadium. alloy titanium adalah satu-satunya alloy yang tidak mengandung sensitizing elements.7 breket titanium dengan memakai titanium, alloy titanium atau sejenisnya, breket ortodontik dapat dibuat lebih ringan dan kuat daripada breket konvensional semacam baja nirkarat, plastik, dan bahkan keramik. breket ini mempunyai daya tahan terhadap korosi dan biokompatibilas yang sangat baik. perlakuan permukaan termasuk: nitriding, diamond coating, pre-oxidation atau shot-peening pada permukaan dasar slot breket mengurangi koefisien geser terhadap kawat ortodontik. kekuatan perlekatan geser dapat ditingkatkan dengan shot-peening, ion beam etching atau etsa permukaan gigi.10 tidak semua pabrik membuat breket dari titanium. masing-masing pabrik membuat juga dengan karakteristik sendiri-sendiri, sehingga terdapat perbedaan yang bermakna pada komposisi, struktur mikro dan kekerasannya.11 pemeriksaan metalografik terhadap breket orthos2 (ormco, glendora, ca, usa) menunjukkan bahwa breket ini terdiri dari dua bagian yang disatukan melalui laser welding dengan jarak antarmuka dasar sayap yang besar; sedangkan breket rematitan (dentaurum, ispringen, germany) merupakan satu potongan logam saja. unsur dasar breket baik orthos2 maupun rematitan adalah titanium, tetapi aluminium (al) dan vanadium (v) juga ditemukan sebagai komponen sayap orthos2. hasil uji kekerasan vickers adalah sebagai berikut: orthos2 (sayap): 371 +/– 22, rematitan (sayap): 272 +/– 4, rematitan (dasar breket): 271 +/– 16, orthos2 (dasar breket): 165 +/– 2. deguchi et al.12 membuat breket ortodontik dengan metal powder injection molding with sintering, dengan lekukan pada dasar breket berbentuk seperti bola, oval, dan grooved. kekuatan perlekatan geser maksimum untuk tiap tipe adalah berturut-turut 11.1 kgf, 7.6 kgf, and 18.5 kgf. kekuatan perlekatan breket titanium ternyata ekuivalen dengan breket baja nirkarat yang biasa dipakai. dikatakan bahwa breket titanium yang dibuat dengan cara ini dapat digunakan untuk aplikasi klinik. breket baja nirkarat dan breket titanium ternyata tidak berbeda bermakna dalam konfigurasi pasif13 maupun aktif.14 kesamaan breket ini terjadi karena seperti ditunjukkan oleh spektroskopi electron untuk analisis kimia (esca), permukaan masing-masing bahan terdiri atas lapisan pasif. unsur kimia lapisan pasif baja nirkarat terdiri atas cr2o3 atau variasi crxo4 (dimana 0 < x < 2) dan titanium terdiri atas tio2 (rutile) dari pemeriksaan esca diketahui bahwa lapisan pasif titanium sangat tipis yaitu hanya 200 sampai 300 å. pada konfigurasi aktif, breket menerima stres dengan level yang tinggi. konfigurasi aktif terjadi saat tidak ada celah antara breket dan kawat busur. 122 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 120–123 pembahasan breket merupakan bagian penting dalam perawatan ortodontik dengan peranti cekat karena breket mentransmisikan kekuatan dari kawat ke jaringan periodontal, sehingga dapat terjadi pergerakan gigi. breket yang umum dipakai adalah dari bahan baja nirkarat, yang mengandung unsur nikel. penelitian menunjukkan adanya kenaikan kadar nikel dalam saliva dan serum secara bermakna setelah insersi peranti cekat ortodontik. nikel merupakan logam yang sering menyebabkan dermatitis kontak dalam bidang ortodontik dengan kasus reaksi alergi lebih banyak dilaporkan daripada karena logam lain. sekali hipersensitifitas terjadi, semua permukaan mukosa rongga mulut dapat terlibat. sensitifitas meningkat dengan adanya iritasi mekanik, skin maceration atau luka pada mukosa mulut, yang semuanya dapat terjadi selama perawatan ortodontik. sampai saat ini sensitifitas penderita terhadap nikel melalui perawatan ortodontik rutin telah banyak menjadi perhatian. meskipun breket baja nirkarat mempunyai kadar nikel yang relatif rendah yaitu 6%, breket bebas nikel yang dapat menjadi alternatif pilihan adalah: breket keramik (dari alumina polikristal, single crystal sapphire atau zirconia), breket polikarbonat (dari polimer plastik), breket lapis emas dan breket titanium.6 penampilan yang transparan dan translusen dari single crystal sapphire dan alumina polikristal membuat bahan ini secara estetik menyenangkan, tetapi bahan ini bersifat abrasif apabila beradu dengan enamel. estetika breket polimer juga bagus, tetapi mempunyai problem umum yaitu kurang kuat dan kaku.13 selama mekanika pergeseran gigi, faktor daya tahan geser merupakan faktor yang penting, dan harus dikontrol agar aplikasi kekuatan ringan yang optimal dapat dilakukan. daya tahan geser yang lebih tinggi memerlukan peningkatan besarnya kekuatan ortodontik untuk mengatasi frictional resistance agar didapatkan sisa kekuatan yang cukup untuk menggerakkan gigi secara optimal. dari berbagai bahan yang diteliti, breket baja nirkarat lebih disukai karena nilai gaya geser yang rendah. meskipun demikian, karena mengandung nikel, baja nirkarat dapat menyebabkan reaksi hipersensitivitas dan korosi pada lingkungan mulut. selain itu dapat terjadi distorsi gambaran ct dan mri karena adanya alloy baja nirkarat. untuk mengatasi hal ini dikembangkan breket dari titanium. titanium telah dikenal sebagai bahan yang sangat kompatibel dalam lingkungan mulut dan mempunyai integritas struktural yang lebih baik dari baja nirkarat. meskipun breket titanium tidak mengandung nikel, alloy ini cenderung menyebabkan galling, fretting dan mempunyai nilai gaya geser tinggi, sehingga membuat mekanika pergeseran (sliding) lebih sulit.15 kapur et al.16 membandingkan breket baja nirkarat dan breket titanium yang dikeluarkan oleh pabrik yang sama yaitu dentaurum. lebar mesiodistal breket titanium ternyata 0,20 mm lebih besar daripada breket baja nirkarat. hal ini menyebabkan kontak permukaan pada antar muka breket-kawat lebih besar, demikian juga regangan modul elastomerik untuk memegang kawat pada slot breket. breket titanium juga mempunyai struktur kimia dan kekerasan yang berbeda apabila dibandingkan dengan baja nirkarat. sifat mekanik titanium yang diinginkan pada penggunaan di bidang ortodontik adalah kekakuan yang rendah, elastisitas tinggi, dan mempunyai shape memory effect. sifat ini menyebabkan dapatnya memasang kawat full size selama perawatan, tetapi membiarkan breket berubah bentuk secara elastik dan menimbulkan reaksi terhadap kontrol tiga dimensi pergerakan gigi dengan kawat rektangular. breket titanium mempunyai stabilitas dimensi tinggi sebagai hasil sifat logam yang baik. dari studi ini dikatakan bahwa breket baja nirkarat menunjukkan nilai gaya geser statik dan kinetik lebih tinggi apabila ukuran kawat meningkat. sedangkan breket titanium menunjukkan gaya geser lebih rendah. meskipun demikian kusy et al.,13 telah melakukan evaluasi terhadap breket titanium dalam kondisi pasif. ternyata pada pengukuran friksional, breket titanium sebanding dengan breket baja nirkarat baik dalam kondisi kering maupun basah. penelitian tentang perbandingan transmisi beban dan deformasi breket antara breket titanium dan baja nirkarat juga telah dilakukan.15 hasil studi menunjukkan bahwa pada interval torsi 15° dan 30° transmisi beban breket titanium lebih tinggi dari breket baja nirkarat sedangkan pada torsi 45° lebih rendah. hal ini dapat diinterpretasikan bahwa pada interval torsi yang lebih rendah, gaya torsi breket titanium lebih tinggi, akan tetapi pada interval torsi 45° titanium telah mencapai titik deformasi elastik sehingga mempunyai nilai beban yang lebih rendah bila dibandingkan dengan breket baja nirkarat. hal ini ditunjang oleh sifat baja nirkarat yang lebih kaku. breket titanium mungkin lebih efektif meneruskan gaya torsi dalam jumlah kecil. breket titanium lebih reaktif pada interval torsi 45° dan memproduksi nilai beban lebih rendah karena dilepaskannya energi simpanan sebagai hasil fenomena rebound. evaluasi tentang perubahan bentuk breket setelah aplikasi gaya torsi menunjukkan bahwa pelebaran slot breket titanium lebih kecil secara bermakna. stabilitas struktural breket titanium pada aplikasi gaya torsi lebih baik dari baja nirkarat.15 pengukuran kekuatan fatigue pada breket titanium (6,78 +/– 0,53 mpa) ternyata tidak berbeda bermakna dengan breket baja nirkarat (5,97 +/– 0,37 mpa). kekuatan fatigue pada breket keramik (9,60 +/– 0,44 mpa) secara bermakna lebih tinggi dari breket titanium maupun baja nirkarat (p < 0,05). kekuatan perlekatan geser breket titanium (8,66 +/– 1,37 mpa) tidaklah berbeda dengan 123goenharto dan sjafei: breket titanium breket baja nirkarat (9,43 +/– 1,55 mpa), tetapi lebih tinggi secara bermakna dari breket keramik (12,06 +/– 2,17 mpa; p < 0,05). kekuatan perlekatan relatif lebih rendah daripada kekuatan fatigue.17 breket titanium sampai saat ini masih cukup sulit didapatkan di pasaran indonesia dan harganya relatif jauh lebih mahal bila dibandingkan dengan breket baja nirkarat. meskipun demikian dari sifat yang telah dibahas di atas maka dapat disimpulkan bahwa breket titanium dapat merupakan alternatif pengganti breket baja nirkarat, khususnya bila merawat pasien dengan riwayat hipersensitifitas. daftar pustaka 1. kocadereli i, canay s, akca k. tensile bond strength of ceramic orthodontic brackets bonded to porcelain surfaces. am j orhod dentofac orthop 2001; 119:617–20. 2. bishara se. textbook of orthodontics. philadelphia, london, new york: wb saunders company; 2001. p. 187–9. 3. harradine nwt. self-ligating brackets: where are we now? j of orthod 2003; 30:262–73. 4. kusy rp. orthodontic biomaterials: from the past to the present. angle orthod 2002; 72:501–12. 5. kusy rp. the future of orthodontic materials: the long term view. am j orhod dentofac orthop 1998; 113:91–5. 6. rahilly g, price. n. nickel allergy and orthodontics. j of orthod 2003; 30:171–4. 7. williams d. concise encyclopedia of medical & dental materials. new york: pergamon press; 1990. p. 360–4. 8. the international programme on chemical safety (ipcs). titanium. geneva: world health organization. 1982; p. 14–49. 9. van noort r. introduction to dental materials. 2nd ed. edinburg: mosby; 2002. p. 228–9. 10. sachdeva rcl, oshida y. orthodontic bracket. 1992. available at url: http://www.shotpeener.com/library/spc/1992070.htm. accessed january 17, 2005. 11. zinelis s, annousaki o, eliades t, makou m. metallographic structure and hardness of titanium orthodontic brackets. j orofac orthop 2003; 64(6):426–33. 12. deguchi t, ito m, obata a, koh y, yamagishi t, oshida y. trial production of titanium orthodontic brackets fabricated by metal injection molding (mim) with sintering. j dent res 1996; 75:1491–6. 13. kusy rp, whitley jq, ambrose ww, newman jg. evaluation of titanium brackets for orthodontic treatment: part i–the passive configuration. am j orhod dentofac orthop 1998; 114:558–72. 14. kusy rp, o’ grady pw. evaluation of titanium brackets for orthodontic treatment: part ii – the active configuration. am j orhod dentofac orthop 2000; 118:675–84. 15. kapur r, sinha pk, nanda rs. comparison of load transmission and bracket deformation between titanium and stainless steel brackets. am j orhod dentofac orthop 1999; 116:275–8. 16. kapur r, sinha pk, nanda rs. comparison of frictional resistance in titanium and stainless steel brackets. am j orhod dentofac orthop 1999; 116:271–4. 17. buxton, cm. the effect of bracket material on fatigue strength of the orthodontic bond. orthodontic thesis for m.s. degree, oregon health & science university, april 2004. available at url: http:// www.ohsu.edu/sod/ortho/grads2004.html. accessed march 7, 2005. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no 1-2005 12 faktor pendorong motivasi orang tua merawatkan gigi anak di klinik fakultas kedokteran gigi unair (stimulating factor of parents' motivation to take their children's dental health for treatment in the faculty of dentistry airlangga university) dita anggriana dan musyrifah mahasiswa ppdgs fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract children dental health is very beneficial for children's growth parent’s motivation for taking their children dental for treatment before more serious dental damage can help to decrease the prevalence of children's dental damage, especially for patients who came to pedodontia clinic in the faculty of dentistry airlangga university. this study aimed to know the parents' motivation to take their children dental for treatment in pedodontia clinic in the faculty of dentistry airlangga university by giving questionnaire to 42 patient’s parents. the result of this study suggested that clinic’s facilities (scored: 2.86) as the stimulating factor had the biggest influence in motivating patient’s parents. key words: parents motivation, children dental health korespondensi (correspondence): dita anggriana, mahasiswa ppdgs, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: d_anggriana @ yahoo.com pendahuluan manusia bertingkah laku tertentu karena didorong oleh keinginan untuk mencapai suatu tujuan yang berguna bagi kehidupannya.1 faktor pendorong ini muncul dari sejumlah kebutuhan dasar yang terdapat di dalam dirinya untuk berperilaku tertentu.2 motivasi merupakan keadaan psikologi yang merangsang dan memberi arah terhadap aktivitas manusia.3 motivasi untuk meningkatkan perilaku kesehatan dipengaruhi oleh faktor intern (dari diri sendiri) dan faktor ekstern (faktor lingkungan).4 perawatan kesehatan gigi anak secara dini sangat berguna bagi kesehatan gigi anak yang masih dalam taraf tumbuh kembang. keberhasilan suatu perawatan di bidang kesehatan gigi anak ditentukan oleh banyak hal antara lain, adanya bimbingan orang tua terhadap anak yang dipengaruhi oleh motivasi orang tua dalam berperilaku sehat. perilaku mempunyai peranan yang sangat besar terhadap status kesehatan individu, kelompok maupun masyarakat.5 adanya motivasi orang tua untuk merawat gigi anaknya sebelum terjadi kerusakan gigi yang lebih parah dapat membantu menurunkan prevalensi kerusakan gigi anak, khususnya penderita yang datang ke klinik pedodontia fakultas kedokteran gigi unair. dari penelitian terdahulu tentang prevalensi kerusakan gigi anak di klinik pedodontia fakultas kedokteran gigi unair didapatkan 9 sampai 10 gigi anak tersebut terserang karies. berdasarkan uraian tersebut di atas timbul permasalahan motivasi apa yang menjadi pendorong orang tua untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair. adapun tujuan penelitian ini adalah untuk memperoleh gambaran tentang faktor pendorong motivasi orang tua untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair. bahan dan metode penelitian ini termasuk penelitian observasional dengan populasi semua orang tua penderita yang datang ke klinik pedodontia fakultas kedokteran gigi unair. sampel yang diambil sebanyak 42 orang tua penderita yang datang pertama kali untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair dalam bulan september–november 1998. pada orang tua penderita tersebut diminta untuk mengisi sendiri faktor pendorong motivasi orang tua penderita yaitu tujuan orang tua datang ke klinik, pengetahuan orang tua penderita, perawatan di klinik, sikap dan pelayanan dokter muda dan karyawan, fasilitas klinik, jarak dan biaya. orang tua penderita juga diminta untuk mengisi tingkat pendidikan orang tua dan jenis pekerjaan orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair. 13anggriana: faktor pendorong motivasi orang tua pengisian kuesioner dilakukan di klinik pedodontia fakultas kedokteran gigi unair. dari data kuesioner semua orang tua penderita yang menjadi sampel dikumpulkan kemudian ditabulasi dan digunakan teknik analisa korelasi dengan uji statistik spearman’s rho untuk data continuous dengan distribusi tidak normal (bersambungan), teknik analisa tabulasi silang dengan uji statistik chi square test untuk data diskrit (kategorial). dari data diskrit, variabel penelitian dibagi dalam kategori yang ditentukan atas dasar tabel frekuensi. kemudian test reliabilitas dengan metode test–retest. hasil dari pengisian kuesioner kemudian dibuat tabel dan hasilnya seperti berikut ini. tabel 1. gambaran faktor pendorong motivasi orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair tahun 1998. faktor pendorong n rata-rata skor tujuan orang tua datang ke klinik pengetahuan orang tua penderita perawatan di klinik sikap dan pelayanan dokter muda dan karyawan fasilitas jarak biaya 42 42 42 42 42 42 42 2,65 2,40 2,56 2,78 2,86 2,47 1.67 pada tabel 1 terlihat fasilitas sebagai faktor pendorong terbesar motivasi orang tua penderita untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair. tabel 2. rerata total skor motivasi orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair tahun 1998. n nilai skor terendah nilai skor tertinggi rata-rata total skor total skor motivasi 42 40 58 50,69 didapat mean (rata-rata) dari total skor motivasi penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair = 50,69 digolongkan dalam kelompok orang tua penderita dengan motivasi tinggi dan bila skor kurang dari 50,69 digolongkan dalam kelompok orang tua dengan motivasi rendah. dari rerata total skor maka dapat diketahui orang tua penderita yang mempunyai motivasi tinggi dan motivasi rendah. tabel 3. tabel frekuensi orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair berdasarkan tingkat pendidikan orang tua tahun 1998. skor tingkat pendidikan motivasi rendah motivasi tinggi jumlah buta huruf sd smp sma pt (perguruan tinggi) 2 3 1 11 3 – 1 1 15 5 2 (4,7%) 4 (9,5%) 2 (4,7%) 26 (61,9%) 8 (19,1%) jumlah 20 22 42 (100%) pada tabel 3 terlihat pendidikan sma paling banyak menggunakan pelayanan kesehatan di klinik pedodontia fakultas kedokteran gigi unair. dari hasil perhitungan statistik dengan uji spearman’s rho didapatkan bahwa hubungan antara motivasi orang tua penderita dengan tingkat pendidikan orang tua penderita yang datang untuk merawatkan gigi anak ke klinik pedodontia fakultas kedokteran gigi unair sangat lemah sekali ≈ tidak ada hubungan dengan angka correlation coefficient = 0,15, jadi semakin mendekati angka nol maka hubungan antara variabel tersebut juga semakin lemah. tabel 4. tabel frekuensi orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair berdasarkan jenis pekerjaan orang tua penderita tahun 1998. skor jenis pekerjaan motivasi rendah motivasi tinggi jumlah ibu rumah tangga swasta pegawai negeri 11 6 3 12 9 1 23 (54,7%) 15 (35,7%) 4 (9,55) jumlah 20 22 42 (100%) pada tabel 4 menunjukkan ibu rumah tangga paling banyak menggunakan pelayanan kesehatan di klinik pedodontia fakultas kedokteran gigi unair. dari hasil perhitungan statistik dengan uji chi square didapatkan bahwa hubungan antara motivasi orang tua penderita dengan jenis pekerjaan orang tua penderita yang datang untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair sangat lemah sekali ≈ tidak ada hubungan dengan angka contingency coefficient = 0,19. 14 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 12–15 pembahasan tabel 1 menunjukkan bahwa fasilitas klinik sebagai faktor pendorong yang mempunyai pengaruh terbesar (skor: 2,86) dalam memotivasi orang tua penderita yang datang ke klinik pedodontia fakultas kedokteran gigi unair. hal ini dapat terlihat dari banyaknya tanggapan responden bahwa fasilitas yang ada sangat mencukupi baik alat dan tempat praktek yang bersih dan teratur, hal ini kemungkinan disebabkan karena fasilitas yang tersedia di klinik fakultas kedokteran gigi unair merupakan tempat pendidikan para dokter gigi muda yang memberikan perawatan relatif ideal. lain halnya dalam masalah biaya perawatan (skor: 1,67) dan pengetahuan orang tua penderita (skor: 2,40) yang sebagian besar orang tua penderita menginginkan biaya perawatan yang murah. dari hasil ini juga terlihat masih adanya kendala dan pertimbangan tertentu soal biaya bagi orang tua yang ingin merawatkan anaknya.3 dari faktor biaya dan pengetahuan penderita ini merupakan salah satu penyebab mengapa sebagian besar orang tua tidak merawatkan kesehatan gigi anaknya sedini mungkin. dari tabel 2 dapat diketahui kelompok motivasi tinggi dan rendah dari orang tua penderita yang datang ke klinik pedodontia fakultas kedokteran gigi unair. dengan menghitung rerata total skor motivasi didapatkan rerata total skor (mean) motivasi orang tua penderita = 50,69. bila mean > 50,69 dikatakan sebagai golongan motivasi tinggi dan bila mean < 50,69 dikatakan sebagai golongan motivasi rendah. dalam tabel 3 terlihat bahwa orang tua penderita yang berpendidikan sekolah menengah atas (sma) dan perguruan tinggi (pt) ternyata paling banyak menggunakan pelayanan kesehatan di klinik pedodontia fakultas kedokteran gigi unair dengan persentase sma 61,91% dan pt 19,05%. hal ini kemungkinan disebabkan karena pendidikan sma dan pt mempunyai jenjang pendidikan yang lebih tinggi dibandingkan dengan pendidikan yang lain.6 dan seseorang yang mempunyai pendidikan lebih tinggi akan lebih cepat menerima dirinya sebagai orang sakit bila mengalami suatu gejala tertentu dan lebih cepat mencari pertolongan dokter dibanding pendidikan lainnya. pada tabel 4 terlihat bahwa dari 42 sampel total yang diambil ternyata orang tua penderita dari kelompok jenis pekerjaan sebagai ibu rumah tangga paling banyak menggunakan pelayanan kesehatan di klinik pedodontia fakultas kedokteran gigi unair dibanding swasta dan pegawai negeri dengan persentase kelompok ibu rumah tangga 54,70%, swasta 35,71% dan pegawai negeri 9,53%. hal ini kemungkinan disebabkan karena jam buka klinik pada pagi hari sampai siang hari sehingga ibu rumah tangga punya kesempatan lebih banyak dibanding kelompok lainnya yang bekerja pagi hari sampai sore hari. dari uji spearman’s rho diketahui bahwa hubungan antara tingkat pendidikan dan motivasi orang tua penderita memiliki hubungan yang lemah sekali dengan corelation coeffisien 0,15. jadi semakin tinggi tingkat pendidikan orang tua penderita belum tentu memiliki motivasi yang lebih tinggi untuk merawatkan gigi anaknya ke klinik pedodontia fakultas kedokteran gigi unair, demikian pula belum tentu orang tua penderita dengan tingkat pendidikan rendah mempunyai motivasi yang lebih rendah pula. jenis pekerjaan orang tua penderita sangat bermotivasi dikelompokkan dalam 3 kelompok yaitu pegawai negeri, swasta dan ibu rumah tangga. ketiga jenis pekerjaan tersebut tidak menunjukkan suatu tingkat sosial ekonomi orang tua penderita tetapi sebagai data nominal yang tidak menunjukkan tingkat tertentu. dari uji chi square diketahui bahwa hubungan antara jenis pekerjaan dan motivasi orang tua penderita memiliki hubungan yang lemah sekali dengan contingen coefficient = 0,19. jadi dari jenis pekerjaan orang tua penderita tidak bisa ditentukan motivasi orang tua penderita itu tinggi atau rendah, karena masih ada faktor internal dan eksternal yang mempengaruhi motivasi orang tua dalam merawatkan gigi anaknya. tidak adanya hubungan antara motivasi dengan tingkat pendidikan dan jenis pekerjaan orang tua penderita oleh adanya faktor pendorong dan penghambat yang mempengaruhi motivasi orang tua penderita (faktor internal dan eksternal). faktor pendorong ini muncul dari sistem kebutuhan yang terdapat di dalam diri orang tua penderita. dalam diri manusia terdapat sejumlah kebutuhan dasar yang menggerakkannya untuk berperilaku tertentu dan hirarki kebutuhan manusia dapat dipakai untuk menjelaskan motivasinya. kebutuhan manusia terdiri dari lima macam kebutuhan pokok yaitu kebutuhan fisiologi, kebutuhan rasa aman, kebutuhan sosial, kebutuhan untuk dihargai, dan kebutuhan untuk dapat mengaktualisasikan diri dengan seluruh potensi yang ingin dikembangkannya. kelima kebutuhan ini terikat dalam sebuah sistem dengan hirarki tertentu, dan kebutuhan dalam hirarki yang terendah adalah kebutuhan fisik, yang meliputi kebutuhan dasar manusia untuk menjaga agar tetap hidup (misalnya makan, minum, rumah, dan lain-lain). jika kebutuhan ini belum terpenuhi, maka manusia berusaha untuk memenuhi kebutuhan ini dan kebutuhan yang lain menempati hirarki yang lebih rendah. jika kebutuhan fisiologi ini terpenuhi maka kebutuhan berikutnya yaitu kebutuhan rasa aman akan menjadi dominan. kebutuhan rasa aman adalah kebutuhan akan perlindungan dari kesakitan, perlindungan dari ketidakmampuan ekonomi, keselamatan keluarga, dan lain-lain. dan jika kebutuhan rasa aman secara relatif sudah terpenuhi maka kebutuhan hirarki berikutnya lebih menonjol. dengan melihat urutan kebutuhan yang menempati kedudukan semakin tinggi maka sebelum kebutuhan yang lebih rendah terpenuhi maka kekuatan desakan kebutuhan yang lebih tinggi akan terbatas dorongannya tetapi hal tersebut tidak mutlak bila kebutuhan tersebut hadir bersamaan. 7 perilaku sehat dimotivasi oleh rangsangan yang ada di sekeliling ataupun lingkungan seseorang, maka tindakan seseorang maupun keputusan untuk melakukan sesuatu 15anggriana: faktor pendorong motivasi orang tua perilaku tertentu dipengaruhi lingkungan yang dihadapi pada saat ini. perilaku kesehatan dipengaruhi oleh 3 faktor yaitu: 1) faktor predisposisi yang terwujud dalam pengetahuan, sikap, kepercayaan, keyakinan, nilai dari seseorang, 2) faktor pendukung yang terwujud dalam lingkungan fisik, dan 3) faktor pendorong yang terwujud dalam sikap dan perilaku prtugas kesehatan.8 sedangkan yang menyebabkan seseorang itu berperilaku karena adanya 4 alasan pokok yakni pemikiran dan perasaan, orang penting sebagai referensi, sumber daya, dan kebudayaan.9 perilaku manusia tidak dapat berdiri sendiri tetapi selalu berkaitan dengan faktor lain, karena perilaku tersebut merupakan suatu yang kompleks dan merupakan resultante dari berbagai aspek internal maupun eksternal, psikologi maupun fisik.10 dari hasil penelitian tersebut di atas, dapat di tarik kesimpulan bahwa faktor pendorong terbesar yang dapat memotivasi orang tua penderita untuk merawat gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair yaitu fasilitas di klinik pedodontia fakultas kedokteran gigi unair. faktor pendorong lain yang memotivasi orang tua untuk merawatkan gigi anaknya di klinik pedodontia fakultas kedokteran gigi unair yaitu sikap dan pelayanan dokter gigi muda dan karyawan, tujuan orang tua penderita datang ke klinik pedodontia, perawatan di klinik pedodontia, jarak, pengetahuan penderita, tentang kesehatan gigi anak dan biaya. perlu dilakukan penelitian lebih lanjut mengenai hubungan sebab akibat antara motivasi orang tua penderita dengan tingkat sosial ekonomi orang tua penderita. selain itu fakultas kedokteran gigi unair perlu meningkatkan pelayanan kesehatan gigi anak dan meningkatkan pemahaman mahasiswa sebagai calon dokter gigi dalam memotivasi orang tua penderita untuk merawat kesehatan gigi anak, khususnya tentang pengetahuan kesehatan gigi anak dan biaya perawatan di klinik pedodontia fakultas kedokteran gigi unair. daftar pustaka 1. notoatmodjo s. pengantar pendidikan kesehatan dan ilmu perilaku kesehatan republik indonesia. yogyakarta: andi offset; 1993. h. 33. 2. farozin m, fathiyah n k. pemahaman tingkah laku. cetakan ke-1. jakarta: pt. rineka cipta; 2004. h. 16–20. 3. langgulung m. teori-teori kesehatan mental. jakarta: pustaka al-husna; 1986. h. 52–6. 4. ahmadi a. psikologi umum. cetakan ke-3. jakarta: pt. rineka cipta; 2003. h. 113–5. 5. kartono k. hygiene mental. cetakan ke-7. bandung: pt. mandar maju; 2000. h. 36–40. 6. singgih g. psikologi perawatan. jakarta: pt. bpk gunung mulia; 2003; h. 15. 7. monks f j, knoers, haditono sr. psikologi perkembangan. pengantar dalam berbagai bagiannya. cetakan ke-14. yogyakarta: gajahmada university press; 2003. h. 11–30. 8. alwisol. psikologi kepribadian. cetakan ke-2. jakarta: pt rineka cipta; 2004. h. 251–61. 9. hartono. psikologi, sosiologi, antropologi dan pendidikan kesehatan masyarakat. horison majalah medika 1985; v(11): 12. 10. soemanto w. psikologi pendidikan. landasan kerja pemimpin pendidikan. cetakan ke-4. jakarta. pt. rineka 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 41 no 4 oct-dec 2008.indd 155 vol. 41. no. 4 october–december 2008 case report reducing allergic symptoms through eliminating subgingival plaque haryono utomo1, chiquita prahasanti2, and iwan ruhadi2 1 dental clinic 2 department of periodontology faculty of dentistry airlangga university surabaya indonesia abstract background: elimination of subgingival plaque for prevention and treatment of periodontal diseases through scaling is a routine procedure. it is also well-known that periodontal disease is related to systemic diseases. nevertheless, the idea how scaling procedures also able to reduce allergic symptoms i.e. eczema and asthma, is not easily accepted, because it is contradictory to the “hygiene hypothesis”. however, since allergic symptoms also depend on variable factors such as genetic, environmental and infection factors; every possible effort to eliminate or avoid from these factors had to be considered. subgingival plaque is a source of infection, especially the gram-negative bacteria that produced endotoxin (lipopolysaccharides, lps), a potential stimulator of immunocompetent cells, which may also related to allergy, such as mast cells and basophils. in addition, it also triggers the “neurogenic switching” mechanism which may be initiated from chronic gingivitis. objective: this case report may explain the possible connection between subgingival plaque and allergy based on evidence-based cases. case: two adult siblings who suffered from chronic gingivitis also showed different manifestations of allergy that were allergic dermatitis and asthma for years. they were also undergone unsuccessful medical treatment for years. oral and topical corticosteroids were taken for dermatitis and inhalation for asthma. case management: patients were conducted deep scaling procedures, allergic symptoms gradually diminished in days even though without usual medications. conclusion: concerning to the effectiveness of scaling procedures which concomitantly eliminate subgingival plaque in allergic patients, it concluded that this concept is logical. nevertheless, further verification and collaborated study with allergic expert should be done. key words: allergic symptoms, scaling, subgingival plaque correspondence: haryono utomo, dental clinic, faculty of dentistry, airlangga university. mayjen prof. dr. moestopo 47. surabaya. e-mail: dhoetomo@indo.net.id indonesia. telp. (031) 5053195. introduction allergic diseases may manifest in variety of symptoms i.e. rhinitis, asthma and eczema. additionally, a strong association between sensitization and symptoms of allergic diseases is exists. children with asthma, eczema or rhinitis are more likely to be sensitized to one or more allergens than those without these diseases. the “allergic march” alludes to the natural history of allergic diseases in childhood whereby eczema and sensitization to ingested allergens in infants and toddlers often improve by preschool age, when asthma and rhinitis become more prevalent, along with sensitization to aeroallergens.1 children who are sensitized to any allergens early in life have an increased risk of subsequently developing wheeze, airway hyper-responsiveness or rhinitis. there is also evidence that the prevalence of allergic sensitization increases progressively during school years and early adulthood, then often decreases throughout this time.1 nevertheless, it is interesting that in this case report, allergic symptoms which already reduce with age, but in middle adulthood (in the 30s), it recurrent and became more severe. additionally, even though they were sibling, it manifests in different symptoms, one has asthma and the other eczematous dermatitis (eczema). the oral focal infection theory had been studied almost for 100 years, and had been experienced the “fall” and “rise” era. it had been disregarded since 1930s after failures of symptoms relieving following extractions of suspected teeth. nevertheless, it was “reborn” in the 1990s by taubmann et al. who begin with the scientific research related to the focal infection theory.2 li et al.3 156 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 157−159 also contributed new theories about oral focal infection and revealed the importance lipopolysaccharides (lps), the endotoxin of gram-negative bacteria. gram-negative bacteria are abundant in the subgingival plaque. elimination of subgingival plaque reduce the risk of several disease i.e. stroke and diabetes mellitus. however, the exact mechanism how elimination of focal infection may reduce allergic symptoms is not clearly understood. several case reports revealed that elimination of oral focal infection relief allergic asthma symptoms in children4 or adult.5 focal infection may originate from pulpal and/or periodontal infection. periodontal infection is caused by subgingival plaque. furthermore, a collaborated clinical research conducted by dental practitioner and pediatrician revealed that elimination of dental plaque reducing allergic asthma symptoms.6 nevertheless, researches regarding the connection of focal infection and asthma in adult mostly were epidemiological studies and did not support this concept. friedrich et al. study revealed that adult periodontitis had inverse association with respiratory allergy.7 additionally, arbes et al study showed that higher immunoglobulin g (igg) to porphyromonas gingivalis were significantly associated with lower prevalences of asthma, wheeze, and hay fever.8 these literatures supports the ”hygiene hypothesis”. hygiene hypothesis stated in laymen’s term that “cleanliness makes people more allergic”.7,8 therefore, elimination of focal infection that caused by subgingival plaque for reducing allergic symptoms is not easily accepted by medical personnel. thus, explanation of the possible etiopathogenesis which may lead to the development of a new concept regarding the connection between subgingival plaque and allergy should be reviewed. the objective of this case report is to elucidate the possible etiopathogenesis of subgingival plaque-related allergy based on evidence-based cases which may beneficial for the development of a new concept and researches. through researches, this concept will be made clear and established. case case 1: male, 36 years old, lived in surabaya came to the dental clinic after informed by his relative about the possible connection between oral infection and asthma. he suffered from allergic asthma when he was 6, and after had swimming lesson, the symptoms disappeared. his parents are allergic, especially to dairy product (i.e. milk). when he was in the late 20s, he worked in bandung for 6 years, and asthmatic symptoms recurrent. latest medication was bronchodilator (inhaler), which at the time was used until 8 puffs/day; this was considered the severe persistent asthma according to the classification of asthma. extraorally, he looked tired and slow. intra-oral examination showed abundant calculus and severe chronic gingivitis with pseudopockets in 18 17 16 and 26 27 28. he said that when brushing his teeth, the upper gingiva was easily to bleed. case 2: male, 34 years old, his younger brother came to the dental clinic after told by her brother that his asthmatic symptoms were disappeared. he had been suffered from eczematous dermatitis (eczema) for seven years in both feet; nevertheless, the left foot had more severe symptoms after moving from surabaya to banjarmasin. when he was a child he suffered from otitis media and had been conducted paracentesis for 3 times. eczema was first treated with oral corticosteroid for years; unfortunately it caused osteoporosis, then it was stopped. subsequently, it was substituted with topical corticosteroid, nevertheless the symptoms still very annoying. extra oral examination looked normal. intra orally, he had only mild chronic gingivitis. the 48 and 38 had pseudopockets, nevertheless 38 was buccoversion and had deeper lingual and distal pseudopockets. case management at the first visit, the management of these patients was merely deep scaling; before scaling procedures, the patients were told to rinse with hexetidine 0.1% for 30 second. deep scaling was done with piezoelectric scaler followed by hand instrument, a thin universal scaler, which focused on the chronic gingivitis and pseudopockets area. during deep scaling, especially after scaling with hand instrument the gingival bleed easily. however it was beneficial for drainage of the pro-inflammatory mediators, thus reducing the inflammation. they were also prescribed hexetidine gargle 0.1% twice/day for daily maintenance, and were scheduled for evaluation one week later. at the second visit, the asthmatic patient was relieved from most of the symptoms, he only use inhaler once/day. two weeks later he moved to dubai, however when evaluated, her mother said that all of the asthmatic symptoms were disappeared. the second patient came two weeks after his first visit, he also felt that the symptoms were diminished. since the possible cause of eczema was oral focal infection from 48 38, especially 38 which had deeper pseudopocket, he was advised to checked regularly to a dental practitioner. however, for convenience, those teeth had to be conducted gingivectomy or to be extracted. the latest evaluation of the patients in this case report was approximately one year and 10 months later respectively. the asthmatic symptoms did not recurrent, so did the eczema in the right foot, however in the left foot may have a mild symptom if consuming dairy product (i.e. yoghurt). discussion the diversity of allergic symptoms throughout the life is likely, because according to allergic march, infants 157utomo, et al.: reducing allergic symptoms through elimination who had atopic dermatitis may have rhinitis or asthma in the following years. the etiopathogenesis of allergy is multifactorial i.e. genetics, environmental and allergens factors.1 however, it is still unclear why oral focal infection may involve in allergic development and symptoms. since oral focal infection usually related to toxins from oral pathogenic bacteria, the possible involvement of lps from gram-negative bacteria is possible. additionally, recent investigations showed that lps stimulation plays a synergistic role with antigen7 and increase the immunoglobulin e (ige) level.9 the oral infection theory revisited was conducted by several researchers such as taubman and slots in 1992, and li et al.3 nevertheless, in their researches, the main cause of systemic effects of oral focal infection was the immunological reaction of the host to bacteria and toxin.2 it was in accordance with pejcic et al.,10 in 2006 that oral focal infection can play a part in the creation of respiratory infections that are manifestated as sinusitis, tonsillitis, pneumonias, bronchial asthma etc. these diseases can be caused by the microorganisms from the oral cavity, following a direct inhalation from saliva and dental plaque, or by blood dissemination. there have also been numerous other descriptions of the mechanism where oral bacteria have been included in the pathogenesis of respiratory infections, i.e. porphyromonas gingivalis and actinomyces actinomycetemcomitans which can aspire into the lungs and cause infection (droplets infection); then the host`s and bacterial enzymes from the saliva can dissolve saliva pelicula on pathogens and allow them to adhere to the surface of mucous membrane; and also cytokines derived from the periodontal tissue can damage the respiratory epithelium by causing an infection via respiratory pathogens. damage of respiratory epithelium may lead to increase its sensitivity to respiratory allergens or stimulation.11,12 however, the possible involvement of oral focal infection in the etiopathogenesis of asthmatic symptoms is not merely immunological reaction. the subgingival biofilm which contains gram-negative bacteria releases lps which may induce immunogenic and neurogenic inflammation that is proposed termed as the “neurogenic switching mechanism” by meggs in 1993 which explain why local inflammation is able to propagate and stimulates inflammation in distant organs (figure 1).12 actually, according to lundy and linden13 this mechanism also occurs in periodontal disease via mast cell stimulation by lps which releases histamine, enzymes and cytokines. these enzymes and mediators then stimulate nerve endings to produce neuropeptides (i.e. substance p) that in turn induce mast cell activation. it is interesting that since allergy is an inherited disease, and in this case report the patients were sibling. coincidentally, it is well established that these periodontal pathogens are transmitted from parent to child (especially the mother in infant), from sibling to sibling, and between spouses. transmission is most likely through contact with saliva and the sharing of objects such as cups, spoons, and toothbrushes.14, 15 therefore, it is also interesting to conduct researches for investigating the connection between the transmission of periodontopathic bacteria and allergy that could be valuable to prevent allergic diseases. even though the previous explanation of the mechanism of asthmatic symptoms which related to the neurogenic switching mechanism could be happened in non-allergic asthma (non ige-mediated); the effects of lps from different strain of periodontopathic bacteria also play an important role in the development of allergy. according to kato et al.,16 induction of neonatal balb/c mice with low dose of lps from actinomyces actinomycetemcomitans (aalps) and porphyromonas gingivalis (pglps) gave different cytokines profile. the aalps produces t-helper1 (th1) profile cytokines, whereas pglps produce t-helper2 (th2) which related to allergy. in addition, ige level in the control mice were lower than mice induced by pglps. it is clear that the removal of subgingival plaque by deep scaling also reduce lps and pro-inflammatory mediators which may involved in the neurogenic switching mechanism and allergic reaction. nevertheless, according to friedrich et al.,7 this treatment could be contradictory for allergic diseases, because periodontitis patients were considered allergy-resistant and in accordance with the hygiene hypothesis. the plausible answer of this ambiguous question is that there are several types of periodontitis with different etiologies that are periodontitis-only, often with severe resorption (th1 disease), and periodontitis with gingivitis (th2 disease). the patients in these case report suffered from periodontitis with gingivitis, thus elimination of sub gingival plaque for reducing allergic symptoms was likely. figure 1. neurogenic switching. the site of inflammation is switched from the site of inoculation in both allergy and chemical sensitivity by pathways through the central nervous system (cns).12 158 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 157−159 another contradictory study which conducted by arbes et al.8 showed that higher igg to porphyromonas gingivalis was related to the reduced prevalence to allergy. it could be explained that lower concentration of pglps which existed in chronic gingivitis or chronic periodontitis with gingivitis stimulates the toll-like receptor2 (tlr2), which increase the production of th2 cytokines profile, i.e. interleukin-4 (il-4) which related to allergy. moreover, low pglps is antagonist to the tlr4 that resulted in less th1 cytokine production, the interferon γ (ifnγ). higher pglps concentration is related to tlr4 stimulation and more severe periodontitis.17,18 moreover, according to burt,19 periodontitis with gingivitis also have more prostaglandin e2 (pge2) in the pseudopocket than periodontitis-only; this resulted in the increase of sensory nerve ending sensitivity, or lowering of pain threshold, especially the trigeminal nerve.20 nevertheless, it is interesting that in chronic periodontal disease this condition do not manifest as pain. according to wadachi and hargreaves,21 it was the effect of pain receptor cleavage by proteases from periodontopathic bacteria. hence, the corresponding nerve is still in lower threshold which more easily stimulates by cold, capsaicin, glutamate etc.22 in the first case, patient moved to bandung, a city with more rainy weather, humid and colder climate. chronic stimulation of environmental factors to the asthmatics may result in airway remodeling that increase asthma severity.23 sensitization of the maxillary nerve, the second branch of the trigeminal nerve in the oral cavity which may propagate antidromically (opposite to the direction of regular impulse) to the nasal cavity is able to stimulate parasympathetic nerve directly via the sphenopalatine ganglion (spg),24 or indirectly via the trigeminal nucleus caudalis which acts as a relay center.25 since nasal cavity also innervated by the maxillary nerve; stimulation of this nerve can reflexively influence nasal engorgement, respiration rate, nasal secretion, and sneezing. since most trigeminal stimulants were lipid soluble, such as volatile chemicals; the stimulations were likely. stimulated trigeminal nerve and the sphenopalatine ganglion (spg) may referred to multiple chemical sensitivity syndrome (mcs) which initiated in the nasal cavity.26,27 the connection between periodontal disease and eczematous dermatitis is somewhat difficult to explain. the traditional etiopathogenesis of eczema is related to food allergy, especially in children.28 nevertheless, according to pejcic et al.,10 skin diseases could also related to oral focal infection, which most frequently occur as the consequence of transmission of microbes from dental foci are allergic diseases (urticaria, eczema etc.), lichen planus, acnae vulgaris, etc. it was in accordance with li et al.3 that oral microbes and toxins and also responsible for the release of histamine from mast cells or creation of circulating immune complex which resulted in skin problems. nevertheless, since in this patient the removal of subgingival biofilm in the pseudopockets especially in 38 also reduce eczematous symptoms in the left foot; the possible involvement of the neural system and not merely immunological was possible. the neurogenic switching mechanism is able to give an appropriate explanation. furthermore, according to the somatosensory system or the somatosensory homunculus of the brain, the skin sensitivity could be unilateral to the trigger area because it is located in the same somatosensory cortex (figure 2).29 this mechanism could be mimicking to the referral pain system i.e. temporomandibular pain is originated from shoulder pain (figure 3).24 figure 2. ‘sensory homunculus’, illustrating the projection of various body regions on the sensory cortex.29 for the concluding remarks, it is the important role of lps which is able to stimulate tlr2, thus shifting th1 cytokines profile to th2 locally, which may propagate to systemic and also stimulate the periodontal afferent nerve endings which initiates the “neurogenic switching” figure 3. pain impulse from the shoulder (source of pain) may perceive as pain in the site of pain, because they have adjacent converting neuron in the sensory cortex. this phenomenon is termed as heterotopic or referral pain.24 159utomo, et al.: reducing allergic symptoms through elimination mechanism. therefore, our concept that elimination of subgingival plaque may reduce allergic symptoms is logical. however, since these evidence-based cases are only examples from uncontrolled allergy by conventional medication, and that allergy is related to multifactorial etiologies; collaborated studies with medical personnel are mandatory. in addition, since transmission of periodontopathic bacteria also related to parent, sibling and spouses, other research could be done to investigate it possible correlation with the spread of allergic disease. references 1. almqvist c, li q, britton wj, kempwk as, xuan w, tovey e, marks gb. early predictors for developing allergic disease and asthma: examining separate steps in the ‘allergic march’. clin exp allergy 2007; 37: 1296–302. 2. slots j, taubman ma. systemic manifestations of oral infections. 1st ed. in: slots j, taubman ma, eds contemporary oral microbiology and immunology. st. louis: mosby; 1992. p. 500–23. 3. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microb rev 2000; 13(4): 547–58. 4. utomo h. relieving asthmatic symptoms through improving oral health: from imaginary to reality. pdgi journal special edition for the 23th pdgi congress 2008. p. 28-33. 5. utomo h. management of oral focal infection in patients with asthmatic symptoms. dent j (maj. ked. gigi) 2006; 39(3): 120–5. 6. wiyarni, sudiatmika iw, indrawati r, utomo h, endaryanto a, harsono a. changes in bacterial profiles after periodontal treatment associated with respiratory quality of asthmatic children. presented in the 4th asian congress of pediatrics infectious diseases jul 20, 2008, surabaya indonesia. 7. friedrich n, volzke h, schwahn c, kramer a, junger m, schafer t, et al. inverse association between periodontitis and respiratory allergies. clin exp allergy 2006; 36(4): 495–502. 8. arbes sj, sever ml, vaughn b, eric a, cohen ea, zeldin dc. oral pathogens and allergic disease: results from the third national health and nutrition examination survey. j allergy clin immunol 2006; 118(5): 1169–75. 9. jung yw, schoeb tr, weaver ct, chaplin dd. antigen and lipopolysaccharide plays synergistic roles in the effector phase of airway inflammation in mice. am j pathol 2006; 168: 1425–34. 10. pejcic a, pesevska s, grigorov i, bojovic m. periodontitis as a risk factor for general disorders. acta fac med naiss 2006; 23(2): 59–63. 11. scannapieco fa, bush rb, paju s. associations between periodontal disease and risk for nosocomial bacterial pneumonia and chronic obstructive pulmonary disease. a systematic review. ann periodontol 2003; 8(1): 54–69. 12. meggs wj. neurogenic inflammation and sensitivity to environmental chemicals. environ health perspect. 1993; 101: 234–8. 13. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol 2004; 15(2): 82–98. 14. darby i, curtis m. microbiology of periodontal disease in children and young adults. periodontology 2000. 2001; 26: 33–53. 15. lee y, straffon lh, welch kb, loesche wj. the transmission of anaerobic periodontopathic organisms. j dent res 2006, 85(2): 182–6. 16. kato t, kimizuka r, okuda k. changes of immunoresponse in balb/c mice neonatally treated with periodontopathic bacterial endotoxin. fems immunol med microbiol 2006; 47: 420–4. 17. stashenko p, goncalves rb, lipkin b, ficarelli a, sasaki h, camposneto a. th1 immune response promotes severe bone resorption caused by porphyromonas gingivalis. am j pathol 2007; 170(1): 203–13. 18. coats sr, pham tt, bainbridge bw, reife ra, darveau rp. md-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize escherichia coli lipopolysaccharide at the tlr4 signaling complex. j immunol 2005; 175: 4490-98. 19. burt b. epidemiology of periodontal disease. j periodontol 2005; 76: 1406–19. 20. kidd bl, urban la. mechanisms of inflammatory pain. brit j anaesth 2001; 87(1): 3–11. 21. wadachi r, hargreaves km. trigeminal nociceptors express tlr-4 and cd14: a mechanism for pain due to infection. j dent res 2006; 85(1): 49–53. 22. chih-feng t, baraniuk jn. upper airway neurogenic mechanisms. cur al clin immunol 2002; 2(1): 11–9. 23. fixman ed, stewart a, martin jg. basic mechanisms of development of airway structural changes in asthma. eur respir j 2007; 29: 379–9. 24. okeson jp. bell’s orofacial pain. 6th ed. carol stream: quintessenced pub; 2005. p. 52–3. 25. green mw. diagnosing and treating migraine: low tech diagnosis, high tech treatment. available online at : url http://www.ama-assn. org/ama1/pub/ upload/ mm/31/24pres-green.pdf. accessed february 20, 2006. 26. doty rl. intranasal trigeminal chemoreception: anatomy, physiology and psychophysics. in: doty rl editor. handbook of olfaction and gustation. 1st ed. new york: marcell dekker; 1995. p. 821–33. 27. klinghardt dk. the sphenopalatine ganglion (spg) and environmental sensitivity. lecture on 23rd annual international symposium on man and his environment. june 9-12, 2005. dallas texas. available online at url http://www.naturaltherapy.com. accessed march 20, 2006. 28. barnetson rsc, rogers m. childhood atopic eczema. bmj 2002; 324:1376-9. 29. sherwood l. fundamentals of physiology: a human perspective. 3rd ed. belmont: thomson brooks/cole; 2005. p. 120. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 41 no 4 oct-dec 2008.indd 179 vol. 41. no. 4 october–december 2008 case report complex aesthetic treatment on anterior maxillary teeth with malposition febriastuti department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract background: complex aesthetic treatment on anterior teeth involves more than one caries tooth with malformed shape and malposition. purpose: the purpose of this paper is to find the alternative treatment for anterior maxillary teeth with malposition. case: in this case, a 25 year-old man with a peg shaped teeth and caries on several teeth and malposition can be treated with complex aesthetic treatment. case management: endodontic pulpectomy treatment on anterior maxillary teeth and post construction with splint porcelain fused to metal crowns on 11, 12, and 21, 22 to correct the shape and position into normal position. conclusion: malformed and malpositioned teeth with caries can be treated with complex aesthetic treatment. key words: malformed teeth, complex aesthetic, splint, porcelain fused to metal crown correspondence: febriastuti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: febriastuti_drgkonservasi@yahoo.com introduction natural and convenient appearance constitutes an aesthetic shape are mostly desirable by everyone. teeth and face disorder will affect someone’s appearance and self confidence. aesthetic in dentistry is aimed to acquire better and natural contour, shape and texture on teeth surface.1 factors influencing aesthetic are teeth shape and proportions, color, teeth position dimension, including smile and lips lines position, teeth relation, midline to face and lips midline relation.2 esthetic problems on anterior teeth crowns are caused by color change, shape anomaly, atrision, caries, diastheme and fracture. these shall motivate someone to pay attention on his/her anterior teeth appearance, these could ambulate patients from one dentist to another to consult their dental treatment in order to get a natural appearance when smiling or talking. to get a good artificial look on the patient, a dentist should plan a good treatment with a prospect to achieve harmony among the teeth shape and color with the patient’s face to avhieve the desired aesthetic and cosmetic. in the treatment, a dentist will need the teeth impression mould to be used as diagnostic and working model. a working model is used to afford the management’s accuracy and success.3 some techniques for treatment have been developed to overcome the aesthetic problem of anterior teeth by composite resin restoration, composite direct veneer or porcelain indirect veneer and porcelain crown.4 in the case of anterior teeth which involve more than one caries teeth with shape anomaly and position change or teeth situation needs a complex aesthetic restorative treatment, i.e. a corrective restoration towards teeth anomaly or more than one teeth defects so they will be in the correct and normal arch. panoramic and local roentgen photos and study specimens to support diagnosis and treatment plan are required in undertaking the treatment. study specimens are also used to explain the patient about the treatment plan. this article is aimed to bring a solution/restoration treatment alternative in the case of anterior teeth with peg shape, caries and malposition. 180 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 179−181 case a 25 years old, male patient came to the conservative dentistry section of airlangga university dental hospital asking his caries to be treated and his peg shape and malformed teeth to be corrected. the maxillary anterior tooth had caries, never been treated and had no pain. figure 1. the teeth condition before treatment. in clinical observation, tooth number 11 was palatoversion and had caries media, vital, no pain on percussion and pressure, the treatment plan was pulpectomy and pin crown restoration. teeth number 12 and 22 were peg shaped and labioversion, no caries, vital teeth, no pain on percussion and pressure: vital. the treatment plan was pulpectomy and porcelain pin crown restoration. tooth number 21 was palatoversion, no caries, vital, no pain on percussion and pressure. the treatment plan was pulpectomy and porcelain pin crown restoration (figure 1). case management prior to the treatment application, teeth were first impressed and casted to provide the working model and study model. local and panoramic roentgen photo were conducted for diagnosis and treated planning purposes. after the new treatment planning for the crowns was done, the patient was illustrated about the treatment plan to be conducted using the study model. the working model was used for the fabrication of temporary crowns. the next step, cavity treatment was applied towards teeth number 11, 12, 21, 22 in a single visit (figure 2). then aseptic working area was performed using rubber dam application. cavity entrance, diagnostic wire photo and work length measurement were applied. afterwards, pulpal tissue extirpation, root canal space preparation weredone with conventional technique using k-file confirm to the working length were done. in every file change, the root canal space was irrigated with 3% h2o2 and sterile aquadest. then the trial gutta point and trial photo were applied. the root canals were filled with ah plus paste and gutta point filler material using the single cone technique. on teeth number 11, 12, 21 and 22 teeth pin crowns were made by reducing the gutta point as two thirds of the root canal space using peeso reamer and crown decaputation and then the double impression technique applied. bite record and temporary crown application with normal relation were made. the cores and pin crowns were made in the dental technology laboratory of airlangga university. figure 3. pin crowns installation. the cast post were inserted to teeth number 11, 12, 21 and 22 using type i glass ionomer cement, and then the crowns were prepared by scrapping the labial and lingual up to 1mm below the gingiva coincidently (figure 3). slicing to mesial part of tooth number 13 and 23 was done as the gap between 11 and 12 teeth, and 21 and 22 were narrow. those teeth crown preparation casted with double impression material. before it was casted, a tissue figure 2. radiographic photos on teeth after obturation. (a) tooth number 11, (b) tooth number 12, (c) tooth number 21, (d) tooth number 22. a b dc 181febriastuti: complex aesthetic treatment management using hemostat liquefied retraction thread to gingival was applied so the preparation result will be clearly visible on the cast product. antagonist teeth were casted using alginate materials, bite, and color matched. then temporary crown was inserted. on the next visit, porcelain fused to metal crown trial insertion were done after the color and shape were fitted. contact to antagonist teeth were also checked. porcelain fused to metal splint crowns were made on teeth number 11, 12, 21, and 22 teeth. as there was no more premature contact, the porcelain fused to metal crowns were permanently inserted using type i glass ionomer cement (figure 4). control was done in the next week and there was no pain after insertion. figure 4. after treatment. on the first control (a week after treatment) there was no pain complains, normal gingival color, no swelling, good radiographic photo showed mo periapical anomaly. on the second control (six months after insertion) there was no lamentation on anamnesis, on intraoral percussion and pressure examination, gingival mucosa was normal. discussion to improve teeth malposition by minimum modification, dental conservation also has an important role i.e. by complex aesthetic treatment. complex aesthetic treatment is a dental treatment by improving the shape, position and inclination which covers several teeth so better and natural contour, shape and teeth surface texture can be achieved.1 in this case the patient had no self confidence when he smiled therefore an aesthetic treatment was needed to improve the shape and position of his carious anterior teeth with malposition. prior to treatment, the dentist explained about the therapy using study model and panoramic photo, since communication and cooperation between patient and dentist are important keys achieve a successful treatment.5 in this case, a one-visit-pulpectomy root canal treatment with crown and post restoration was applied on vital teeth number 11, 12, 21 and 22 vital teeth was applied as to achieve good aesthetic an inclination improvement is needed. post type was chosen to correct the position, core dimension, and inclination.6 slicing the mesial part of teeth number 13 and 23 was done because the gaps between 11 with 12 and 21 with 22 were narrow. the purpose to apply slicing is to get enough space for teeth number 12 and 22 teeth in the dental arch. gingival margin retraction was needed in the preparation to cover the ceramic restoration to teeth transition so a good aesthetic restoration could be achieved, to get an entrance and to avoid soft tissue damage during the preparation procedure. gingival retraction prior to casting was done to open the gingival and facilitate the double impression materials to enter into the gingival and cast the marginal part accurately.4 the gingival retraction material used was alum cord with yellow color to facilitate the identification. the alum functioned as stiptic, obstructed local bleeding and at the same time compressed the gingival edge.7 the porcelain fused to metal crown fitted to the surrounding teeth in perception of color, dimension, shape, arch position, age and sex to get natural and artistic appearance.8 porcelain fused to metal splint crowns were applied to teeth number 11,12 and 21, 22 teeth, because the gaps were narrow. the treatment was applied in order to get the normal shape of teeth number 11, 12, 21 and 22, harmonic and fitted to the normal dental arch by endodontic treatment and post and porcelain fused to metal crown insertion. cooperation and understanding between patient and dentist is one of the success key of a therapy. it can be concluded that malposition and malformed teeth with caries can be restored by complex aesthetic treatment. references 1. fellippe la, baratieri ln. direct resin composite veneer: masking the dark prepared enamel surface. j quinteessence int 2000; 31: 557–62. 2. qualtrough aje, burke fjt. a look at dental esthetics. j quinteessence int 1994; 25(1):7–9. 3. goldstein re, haywood vb. esthetics in dentistry. 2nd ed. london: bc decker inc; 2002. p. 525–30. 4. dale bg, aschheim kw. esthetic dentistry a clinical approach to techniques and materials. philadelphia: lea and febiger; 1993. p. 101–12. 5. roger l. the challenge of esthetic dentistry and elective services. j am dent assoc 2005; 136:515–6. 6. mount gj, hume wr. preservation and restoration of tooth structure. mosby international ltd; 1998. p. 218–23. 7. baum l, phillips rw, lund mr. textbook of operative dentistry. 3rd ed. philadelphia: wb sounders company; 1995. p. 494–514. 8. soetanto s. perawatan estetika konservatif untuk menanggulangi cacat fasial gigi. maj. ked. gigi (dent j) 2001; 34(4):756–9. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 52 no 1 jan-mar 2019_new.indd 2727 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 27–31 research report the difference between porcelain and composite resin shear bond strength in the administration of 4% and 19.81% silane ira widjiastuti, dwina rahmawati junaedi, and ruslan effendy department of conservative dentistry faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: damage to porcelain restorations such as fractures requires a repair that can be performed either directly or indirectly. direct repair involves directly performing restoration of fractured porcelain with a composite resin application. this technique has more advantages than indirect repair because it requires no laboratory work and can be completed during a single visit. silane, on the other hand, has been widely used and is reported to increase porcelain and composite resin attachments during the direct repair process. purpose: this study aimed to determine the differences in shear bond strength between porcelain and composite resin during the administering of 4% and 19.81% silane. methods: 27 porcelain samples were divided into three groups, namely: group a 4% silane, group b 19.81% silane and group c no silane, prior to the application of composite resin. each sample was tested for shear bond strength by means of autograph and fracture analysis performed through stereomicroscope and scanning electron microscope tests. data analysis was subsequently performed using an anova test. results: there was a significant difference between the three groups with p=0.000 (p<0.005). the lowest bond strength was found in the group without silane, while the highest was in the group with 4% silane (p<0.005). conclusion: the use of 4% silane can produce the highest shear bond strength of porcelain and resin composite. keywords: porcelain repair; shear bond strength; silane correspondence: ira widjiastuti, department of conservative dentistry, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo 47, surabaya 60132, indonesia. e-mail: ira-w@fkg.unair.ac.id introduction porcelain restoration has been widely used as an indirect form of repair because of its benefits.1–3 nevertheless, it also suffers from certain drawbacks leading to restoration failure in the form of fractures.2,4 a previous investigation found that 5-10% of fractures occur in porcelain restorations which have been utilized for more than ten years, while 2.3-8% of fractures in porcelain are fused to metal restorations.5 the high risk of fracture explains the demand for repair of porcelain restorations. there are two types of repair, direct and indirect, relating to fractured porcelain restorations. direct repair offers more advantages than the indirect variety, since it requires less time, utilizes a less complicated technique and is more affordable.2,5 moreover, direct repair it will also produce a positive prognosis if the cause of the fracture constitutes a trauma to the anterior teeth. other prior research even shows that direct repair of porcelain with composites on anterior teeth has a higher success rate than that of posterior tooth restorations in addressing damage to the occlusal and proximal marginal ridge areas involving proximal contact.6,7 employing composite resins as porcelain fracture repair materials has been widely developed. the mechanism of composite attachment to porcelain consists of two forms, namely; micromechanical and chemical. micromechanical attachment using an adhesive system involves etching on porcelain with such agents as hydrofluoric acid. meanwhile, chemical attachment is conducted by introducing silane solution between the porcelain layer and the composite.8 silane, a coupling agent employed as a bonding material between organic and inorganic materials, is a bifunctional molecule consisting of functional and nonfunctional molecules. the functional molecule in silane can dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p27–31 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p27-31 28 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 27–31 polymerize with a functional group in an organic matrix of a composite resin. furthermore, silane can react with free radicals produced during composite resin polymerization, while also belonging to a degradable functional group that will polymerize the organic matrix of the resin. meanwhile, non-functional molecules in the alkoxy silane group can react with inorganic substrates in porcelain. in addition, silane contains silicone dioxide which can react with oh groups on the porcelain surface enabling it to form chemical attachments there.1,2,9 silane solution has also been widely employed as an adhesive material in porcelain fracture repair and is reported to increase the strength of the composite attachment to porcelain. a previous investigation comparing forms of porcelain fracture repair indicated that a group without silane has the lowest attachment strength compared to one with silane.8 one case report even argues that the direct repair of fractured porcelain restoration of anterior teeth with composites and silane additions can be well preserved for up to three years.6 in this research, silane products were used with ethanol solvent whose concentrations were 4% and 19.81%. a previous piece of research states that a relationship exists between the concentration of silane and the shear bond strength of the metal bracket to the porcelain surface. the highest shear bond strength is achieved by the use of silane at a concentration of 2.5% compared to that at concentrations of 5-15% and 15-20%.10 in addition, a low silane concentration has more beneficial properties than a high one because the autopolymerization process of silane becomes optimal in its solvent.11 thus, the higher the concentration of silane, the less perfect the chemical bonds formed since the formation of bridges between organic and inorganic components is inhibited.12 unfortunately, it is not yet known whether different silane concentrations have any effect on shear bond strength during porcelain repair. consequently, this research aims to determine the differences in shear bond strength between porcelain and composite resin using 4% and 19.81% silane. materials and methods this research used 27 samples in the form of cylindrical porcelain with a diameter of 4 mm and a height of 2.5 mm. the criteria applied to the porcelain samples employed included a flat, smooth and unglazed surface without cracks and/or porous sections.11 moreover, this research also used silane with ethanol solvent at concentrations of 4% and 19.81% determined according to the presence of silane products on the market. the samples were subsequently divided into three groups, namely: group a with 4% silane, group b with 19.81% silane and group c without silane. porcelain samples were inserted into acrylic molds and etched with 9% hydrofluoric acid (porcelain etch, ultradent, usa) for 90 seconds. thereafter, 4% silane (monobond plus, ivoclar, germany) was applied to group a and 19.81% silane (porcelain repair primer, ormco, north america) to group b for 60 seconds. bonding agent (adper single bond, 3m, usa) was then applied to all groups and illuminated by means of a light curing unit (led-e curing light, woodpecker) in accordance with factory rules. composite (estelite σ quick, tokuyama dental, japan) was then applied to porcelain samples using a layering technique. a shear bond strength test was performed using autograph (shimadzu, japan) with a cross head speed of 0.5 mm/minute. each sample was then placed on the plunger. the shear bond strength score shown on the tool was observed, especially when the sample experienced adhesive failure and recorded by a kn unit. the, sample interface was then checked using a stereomicroscope at 20x magnification and a scanning electron microscope (sem) at 50x and 150x magnification to determine the locations of the sample fractures. the results of the research were calculated to determine their mean value and standard deviation (table 1). a shapiro-wilk test was subsequently conducted to determine the normality of data distribution followed by a homogeneity test. in order to reveal differences, an anova test with a significance level of 0.05 and a tukey hsd test was then carried out. results according to the shapiro-wilk test results, the p value in all groups was more than 0.05 which indicated that the research data was normally distributed. a homogeneity test and levene test were, therefore, performed, the results of which showed a p value of 0.159 (p>0.05) indicating the homogeneity of the research data. an anova test was conducted in order to establish whether a difference in the research data existed,. the results showed a p value of 0.000 (p<0.005) indicating a significant contrast between the research groups. consequently, a tukey test was conducted to in order to identify differences between each pair within the research groups. the tukey test result values of p = 0.000 (p<0.005) indicated significant differences in silane at contrasting concentrations between the research groups (table 2). in other words, the concentration of silane demonstrated a significant difference from the shear bond strength. to find the location of the sample fractures, a stereomicroscope analysis was carried out followed by sem on the porcelain surface which involved selecting one sample randomly from each group. in the groups with 4% silane and 19.81% silane (figures 1, 2 and 3), fractures were in the form of mixed failure. this means that the adhesive failure occurred partially in composite resin and adhesive material. on the other hand, in the group without silane, a fracture occurred in the form of adhesive failure. this indicates that adhesive failure dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p27–31 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p27-31 29widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 27–31 k p k p a a b c figure 1. the pictures of porcelain surfaces under stereomicroscope with 20x magnification. arrows indicate porcelain and composite fracture areas. (note; p: porcelain, k: composite. a: adhesive (bonding)). a) 4% silane; b) 19.81% silane; c) without silane. k p p k a a b c figure 2. the pictures of porcelain surfaces under sem with 50x magnification. arrows indicate porcelain and composite fracture areas. (note: p: porcelain, k: composite. a: adhesive (bonding)). a) 4% silane; b) 19.81% silane; c) without silane. p k a k p a b c figure 3. the pictures of porcelain surfaces under sem with 150x magnification. arrows indicate porcelain and composite fracture areas. (note: p: porcelain, k: composite. a: adhesive (bonding)). a) 4% silane; b) 19.81% silane; c) without silane. table 1. the mean value and standard deviation of the shear bond strength of porcelain and composite resin using different silane concentrations (mpa). ntreatment groups x̄ sd 0.9857428.793394% silane 0.6412819.8178919.81% silane 0.5084016.91229without silane table 2. the results of tukey test on the shear bond strength of porcelain and composite resin. silane concentration without silane 19.81%4% 0.000without silane * 0.000* 0.0004% silane * 19.81% silane * significant difference p<0.05 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p27–31 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p27-31 30 widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 27–31 occurred in the adhesive material interface. moreover, it can also be said that the shear bond strength of porcelain and composite resin interface in the group without silane was not as great as that in the groups with silane. discussion the high risk of fracture in porcelain restoration requires a feasible repair treatment, one of which is direct repair which uses composite resin and has been widely applied in the field of dentistry with positive results. the attachment of composite resin to porcelain can occur both micromechanically and chemically. micromechanically, the attachment of composite resin to porcelain can occur through an etching process using hydrofluoric acid (hf) while, chemically, it involves administering silane solution before applying composite resin to the porcelain. in the direct porcelain repair process, silane also plays a role as an adhesive between two substances such as porcelain (inorganic) and composite (organic) materials in order to increase the shear bond strength between them. silane also has the potential to react with alkoxy groups which can be activated through a hydrolysis technique involving a reaction of silicone dioxide with the oh/ hydroxyl group on porcelain surfaces which can result in a chemical attachment (sior becomes sioh).9,12 in order to determine the effects of silane concentration contained in the product on the shear bond strength between porcelain and composite resin, this research used silane products at concentrations of 4% and 19.81%. the shear bond strength in the control group was the lowest since the surface of the porcelain was not treated with silane, but merely etched and bonded. this finding was in line with that of research conducted by newburg et al, comparing porcelain fracture repairs using silane and ones without silane. the results of this previous research suggested that the group without silane had the lowest shear bond strength compared to the groups with silane.8 according to a case report, the application of silane to porcelain repairs using composite resins can increase attachment by up to 25%.2 this signifies that silane has a higher adhesive strength. silane, given its function as a coupling agent for organic and inorganic substrates, must be salinized first through hydrolysis or an activation process and, subsequently, by condensation. following the hydrolysis process, siloxane oligomers will react with each other, forming a branch of hydrophobic siloxane (-si-o-si). this group can react with an inorganic matrix.11 meanwhile, the silane attachment with composite resin can occur through a reaction of silane with free radicals derived from polymerization of composite resin involving the formation of a c-c group.1,2,9 a concentration of silane which is too high can cause obstruction of bridges between organic and inorganic components and reduce attachment. meanwhile, low silane concentration can produce superior attachment since the auto-polymerization process of the silane molecule in the solvent becomes optimal. moreover, the low concentration of silane produces a thinner siloxane layer which can increase the attachment of composite and metal/porcelain resins.13,14 at low concentrations, silane also produces higher siloxane absorption during silane activation process than saline at high concentrations.15 in addition, the high concentrations of silane can interfere with oligomer formation in the process of siloxane formation in silane, thus compromising the adhesion of silanes to inorganic matrices.16 the two products employed in this research also contained solvent. the concentration of silane in a product is not considered to be the only major factor in the activation and condensation processes of silane. another factor affecting the processes is solvent whose concentration in silane products affects moisture and wettability. products with a lower concentration of silane and larger solvents will produce high humidity which facilitates penetration of composite resin into the porosity of the surface of the porcelain and increases the adhesion strength between porcelain and composite resin.15 hence, in this research the highest shear bond strength was found in silane at a concentration of 4%. based on the results of the stereomicroscope and sem tests, it can be argued that in the group without silane adhesive failure occurred only in the adhesive material. in contrast, in the groups with silane at concentrations of 4% and 19.81%, the adhesive failure occurred partially in both adhesive and cohesive materials (mixed failure). in other words, in the groups with silane at these concentrations, the failure occurred in the form of mixed failure; partly in the composite resin and partly in the adhesive/bonding material. this shows that the composite can bind well to the porosity of the surface of the porcelain. in contrast to the other groups, the failure in the control group (without silane) occurred only in the adhesive material. it can be argued that, unlike in the groups with silane, there was no damage to the composite resin in the control group. this indicates that silane can increase the attachment of composite resin and porcelain by forming optimal bonds between composites and porcelain. therefore, it can be concluded that silane at a concentration of 4%.can enhance the shear bond strength between porcelain and composite resin. references 1. blatz mb, sadan a, kern m. resin-ceramic bonding: a review of the literature. j prosthet dent. 2003; 89(3): 268–74. 2. ahmadzadeh a, ghasemi z, panahandeh n, golmohammadi f, kavyanie a. effect of silane on shear bond strength of two porcelain repair systems. j dent sch. 2016; 34: 9–18. 3. noerdin a. kemampuan lekat resin komposit pada reparasi restorasi porselen yang fraktur. j dent indonesia. 2000; 7: 96–106. 4. zhang y, sailer i, lawn br. fatigue of dental ceramics. j dent. 2013; 41(12): 1135–47. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p27–31 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p27-31 31widjiastuti, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 27–31 5. wady af, paleari ag, queiroz tp, margonar r. repair technique for fractured implant-supported metal-ceramic restorations: a clinical report. j oral implantol. 2014; 40(5): 589–92. 6. strassler he. repairing porcelain-metal restorations with composite resin. benco dental. 2012. p. 93–100. 7. ozcan m. fracture reasons in ceramic-fused-to-metal restorations. j oral rehabil. 2003; 30(3): 265–9. 8. newburg r, pameijer ch. composite resins bonded to porcelain with silane solution. j am dent assoc. 1978; 96(2): 288–91. 9. lung cyk, matinlinna jp. aspects of silane coupling agents and surface conditioning in dentistry: an overview. dent mater. 2012; 28(5): 467–77. 10. adisty d, krisnawati k, hoesin f. perbedaan kuat rekat geser braket metal terhadap permukaan porselen pada pemakaian tiga bahan silane coupling agent. thesis. depok: universitas indonesia; 2014. p. 56. 11. ozyoney g, yanıkoğlu f, tağtekin d, ozyoney n, oksüz m. shear bond strength of composite resin cements to ceramics. marmara dent j. 2013; 2(6): 61–6. 12. matinlinna jp, lassila lvj, ozcan m, yli-urpo a, vallittu pk. an introduction to silanes and their clinical applications in dentistry. int j prosthodont. 2004; 17(2): 155–64. 13. zanchi ch, ogliari fa, marques e silva r, lund rg, machado hh, prati c, carreño nlv, piva e. effect of the silane concentration on the selected properties of an experimental microfilled composite resin. appl adhes sci. 2015; 3: 1–9. 14. matinlinna jp, mittal kl. adhesion aspects in dentistry. boston: crc press; 2009. p. 148–9. 15. meng x, yoshida k, taira y, kamada k, luo x. effect of siloxane quantity and ph of silane coupling agents and contact angle of resin bonding agent on bond durability of resin cements to machinable ceramic. j adhes dent. 2011; 13: 71–8. 16. matisons jg. silanes and siloxanes as coupling agents to glass: a perspective. in: owen mj, dvornic pr, editors. silicone surface science. pennsylvania: springer; 2012. p. 281–98. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p27–31 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p27-31 p-issn: 1978-3728 e-issn: 2442-9740 volume 50, number 1, march 2017 editorial boards of dental journal (majalah kedokteran gigi) sk: 110/un3.1.2/2015 january 3rd – december 31st, 2017 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (department of conservative dentistry faculty of dental medicine, universitas airlangga editorial boards: roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors: priyawan rachmadi (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); ira widjisatuti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty of vocation, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric priyo prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); harmas yazid (department of oral surgery, faculty of dentistry, universitas padjadjaran, indonesia); prof. trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); al. supartinah santoso, (department of pediatric dentistry, faculty of dentistry, universitas padjajaran, indonesia); ekky soeriasoemantri (department of orthodontic, faculty of dentistry, universitas padjadjaran, indonesia); siti mardewi soerono akbar (department of conservative dentistry, faculty of dentistry, universitas indonesia, indonesia); widowati siswomihardjo (department of biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); melanie sadono djamil (department of biomedic, faculty of dentistry, universitas trisakti, indonesia); istiati (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); jenny sunariani (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); adi hapsoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); kus harijanti (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); retno puji rahayu (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); chiquita prahasanti (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); titiek berniyanti (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); david b. kamadjaja (department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 170/04.17/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 50, number 1, march 2017 p-issn: 1978-3728 e-issn: 2442-9740 1. rinsing effect of 60% bay leaf (syzygium polyanthum wight) aqueous decoction on the accumulation of dental plaque during fixed orthodontic treatment fitria avriliyanti, sri suparwitri, and ananto ali alhasyimi ..................................................... 1–5 2. the cleanliness differences of root canal walls after irrigated with east java propolis extract and sodium hypoclorite solutions tamara yuanita ............................................................................................................................... 6–9 3. effect of electrolyzed reduced water on malondialdehyde levels of wistar rats with chronic and aggresive periodontitis rini devijanti ridwan, wisnu setyari juliastuti, and darmawan setijanto .............................. 10–13 4. differences in tensile adhesion strength between hema and non-hema-based dentin bonding applied on superficial and deep dentin surface eresha melati kusuma wurdani, adioro soetojo, and devi eka juniarti ................................ 14–18 5. the correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in surabaya puspa dila rohmaniar, titiek berniyanti, and retno pudji rahayu ......................................... 19–22 6. socioeconomic characteristics of the parents and the risk prediction of early childhood caries wahyu aji wibowo, retno indrawati, and retno pudji rahayu ................................................ 23–27 7. anti-glucan effects of propolis ethanol extract on lactobacillus acidophillus ira widjiastuti, adioro soetojo, and febriastuti cahyani ........................................................... 28–31 8. correlation between working position of dentists and malondialdehyde concentration with musculoskeletal complaints hari wibowo, titiek berniyanti, and jenny sunariani ................................................................ 32–35 9. the potentiation of mangifera casturi bark extract on interleukin-1β and bone morphogenic protein-2 expressions during bone remodeling after tooth extraction bayu indra sukmana, theresia indah budhy, and iga wahju ardani .................................... 36–42 10. effects of citrus limon essential oil (citrus limon l.) on cytomorphometric changes of candida albicans rina prabajati, iwan hernawan, and hening tuti hendarti ...................................................... 43–48 11. correlation of cost, time, need, access, and competence with the public interest in installing dentures at non-professional dentist yayah sopianah, muhammad fiqih sabilillah, and ayyu fadilah ............................................. 49–53 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 94 the decrease of fibroblasts and fibroblast growth factor-2 expressions as a result of x-ray irradiation on the tooth extraction socket in rattus novergicus fatma yasmin mahdani,1 intan nirwana,2 and jenny sunariani3 1department of oral medicine 2department of dental materials 3department of oral biology faculty of dental medicine, universitas airlangga surabaya-indonesia abstract background: wound healing involves cellular, molecular, physiological, and biochemical processes as responses to tissue damage. for instance, when a failure during tooth extraction occurs, radiographic examination, x-rays, is required. x-rays as an enforcer diagnosis can damage dna chain, resulting in cell death and inhibition of wound healing process. purpose: this research aims to analyze fibroblasts cell number and fibroblast growth factor-2 (fgf-2) expressions during wound healing process after tooth extraction as a result of x-ray irradiation. methods: there were three research groups, each consisting of ten rats. incisor tooth extraction was performed on the left lower jaw, and then x-ray examination was conducted at certain irradiation doses, namely 0 msv, 0.08 msv, and 0.16 msv. those animals were sacrificed on day 3, and on day 7 after the extraction, histopathology and immunohistochemistry examinations were conducted to determine fibroblast cell number and fgf-2 expressions. data obtained were then analyzed by oneway anova and tukey hsd tests. results: the number of fibroblasts decreased significantly in the group with the irradiation dose of 0.16 msv applied on day 7 after the extraction (p <0.05). similarly, the number of fgf-2 expressions decreased significantly in the group with the irradiation dose of 0.16 msv applied on days 3 and 7 after the extraction (p <0.05). conclusion: x-ray irradiation at a dose of 0.16 msv can inhibit the healing process of tooth extraction wound due to the decreasing of fibroblasts cell number and fgf-2 expressions. keywords: wound healing; tooth extractions; x-rays; fibroblasts; fibroblast growth factor-2 correspondence: fatma yasmin mahdani, c/o: department of oral medicine, faculty of dental medicine, universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: fatmayasminmahdani@gmail.com introduction wound healing process is a body response to tissue damage. wound healing process of tooth extraction actually has the same principles with wound healing process in general. the wound healing process of tooth extraction is a complex pathophysiological process involving cell proliferation, cell migration, synthesis and deposition of extracellular matrix proteins, and tissue remodeling.1 there are four phases in the process of wound healing, namely haemostasis, inflammatory phase, proliferation phase, and remodelling phase.2,3,4 the prevalence of tooth extraction in indonesia in 2007 is quite high, namely 38.5%. the extraction action can cause complications, such as bleeding, infection, fracture, and dry socket.5,6 thus, evaluation needs to be conducted to determine further actions in case of failure or complications during extraction process. one of them is by conducting dental radiographic examination.7 dental radiographic examination is an examination aimed to get a picture of tooth socket by using x-ray irradiation. periapical radiographic can provide information about the location and size of roots left due to tooth fracture. however, this examination can cause negative effects, research report dental journal (majalah kedokteran gigi) 2015 june; 48(2): 94–99 95 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 95mahdani, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 94–99 such as mucosal epithelial cell damage and slow wound healing process. x-ray irradiation at a dose of 0.08 msv can increase apoptosis and necrosis of the oral mucosal epithelial cells.8 dose used in a periapical radiographic examination actually is 0.08 msv. this dose, unfortunately, can increase apoptosis and necrosis of oral mucosal cells.8 nevertheless, periapical radiographic examination may be conducted once more when a failure occurred during the previous manufacturing process of radiograph. as a result, irradiation dose derived from radiographic examination will increasingly transfer to the body. x-ray irradiation can damage dna chains, carbohydrates, proteins, and lipids, resulting in inhibition of cell cycle checkpoint, inactivation of cell proliferation, induction of apoptosis, and inhibition of cell cycle. 9,10,11 cells and tissues that play a role in healing process of tooth extraction wound are high radiosensitive, so mucosal epithelial cells, inflammatory cells, epithelial cells, and fibroblasts will get the direct effects of x-ray irradiation. consequently, the wound healing process will be inhibited.9,12 some growth factors, furthermore, also play a role in wound healing process. fibroblast growth factor (fgf) is a growth factor that has the potential effect in the repair and regeneration of tissue. fgf can be identified as a protein that can stimulate proliferation and deposition of fibroblasts, formation of granulation tissue, formation of new blood vessels, as well as reepithelialization and deposition of extracellular matrix proteins. some important fgfs required in wound healing process are fibroblast growth factor-2 (fgf-2), fibroblast growth factor-7 (fgf7) and fibroblast growth factor-10 (fgf-10).13,14 however, the effects of x-ray irradiation on the healing process of tooth extraction wound determined by fibroblast cell number and fgf-2 expression still have not been revealed. the reason is that fibroblasts and fgf-2 play a role in three of the four phases of wound healing, i.e. from the inflammatory phase to the last phase of tissue remodelling. fgf-2 are mainly produced by macrophages and endothelial cells from day 2 to 4, while the active proliferation of fibroblasts occurs from day 3 to 7 after tooth extraction.3 therefore, this research aims to analyze fibroblasts cell number and fgf-2 expressions on days 3 and 7 after the extraction as a result of x-ray irradiation with a dose of 0.08 msv and 0.16 msv. materials and methods thirty male wistar rats aged 8-10 weeks old and weighed 180-200 g were randomly divided into three groups, namely the control group, the treatment group 1 and the treatment group 2. each of the groups was consisted of ten animals. those animals were adapted at the laboratory of biochemistry, faculty of medicine, universitas airlangga, surabaya for 7 days. they were then put in cages placed in a room with quite airflow and light. the base of the cage was covered with husks thickened 2 cm and replaced every two days. those animals were intramuscularly induced with ketamine and diazepam. their mandibular incisors were extracted and exposed with x-ray irradiation. luxation and rotation were conducted on the teeth until the teeth were unstable. the cervical of the teeth was drilled (± 1 cm) as a marker of making tooth fracture. those teeth were then fractured. x-ray irradiation exposure was then given to the fractured tooth with 0.08 msv dose for the treatment group 1, and 0.16 msv dose for the treatment group 2. those teeth in the treatment groups were then extracted after exposed to x-ray irradiation. meanwhile, those teeth in the control group were extracted without irradiation. the mandible of those five experimental animals was cut under anaesthesia ether 10% on day 3, while the mandible of the other five animals was cut on day 7. tissue fixation was conducted in nbf 10% and decalcified into edta 10% to remove calcium from bone tissue. after the bone tissue has been softened, several processes were conducted, namely dehydration, clearing, impregnation, embedding in paraffin blocks, and cutting tissue. the results of these phases obtained were slide preparations placed on object glasses. hpa preparations were used for observing the number of fibroblast cells stained with he, while immunohistochemistry preparations were used for observing the fgf-2 expression using antifgf-2 (santa cruz, sc-79) and kit novolink, novocastra, re7230-k. the reading of the results was conducted by observing those preparations under the light microscope with a magnification of 400 times to see fibroblasts and 1,000 times to observe fgf-2. the entire examination used h600l nikon microscope equipped with a ds fi2 300 megapixel digital camera and nikkon image system as image processing software. data then were analyzed with statistical product and service solutions (spss) for windows version 17.0. finally, data were analyzed by one way anova test followed by tukey hsd test. results fibroblasts observed were found in the apical third of the tooth socket. fibroblast cells had certain criteria, such as large, flattened, and oval core cells covered with delicate nuclear membrane and purplish red branches. figure 1 shows fibroblast cells. the fibroblast cells in the control group, the treatment groups 1 and 2 on day 3 looked equally solid as shown in figure 1. meanwhile, fibroblast cells in the control group and in the treatment group 1 on day 7 looked more solid than in the treatment group 2. the highest mean of the number of fibroblasts on day 3 and 7 was found in the control group, while the lowest mean of the number of fibroblasts on day 3 and 7 was found in the treatment group 2. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 96 mahdani, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 94–99 kolmogorov-smirnov and levene tests were conducted on the data about the number of fibroblasts cells after x-ray irradiation on tooth extraction wound. the results showed that the distributions of data on days 3 and 7 were normal and homogeneous (p> 0.05). therefore, one way anova test was conducted. the results showed that there was no significant difference in the number of fibroblasts cells on day 3 between in the control group, in the treatment group 1 and in the treatment group 2 (p>0.05). however, there was a significant difference in the number of fibroblasts cells on day 7 among the groups (p<0.05). consequently, tukey hsd test was conducted to determine differences in the groups on day 7. the results showed that there was a significant difference between the control group and the treatment group 2, and between the treatment group 1 and the treatment group 2 (p<0.05). positive of fgf-2 expressions were shown in brown color. fgf-2 expressions were shown in figure 2. on day 3, the densest fgf-2 expression was found in the control group. on day 7, however, fgf-2 expressions in the control group and in the treatment group 1 seemed equally solid. fgf-2 expression in the treatment group 2 on day 7 was rarely found. it indicates that fgf-2 expression in this group was low. the highest mean of fgf-2 expression on day-3 was found in the control group, while the lowest mean was found in the treatment group 2. on the other hand, table 1. the mean and standard deviation of the number of fibroblast cells and fgf-2 expressions on days 3 and 7 group number of fibroblast cells fgf-2 expressions day 3 day 7 day 3 day 7 control 355.4 ± 109.07 435.4 ± 68.332 8.6 ± 1.673 7.0 ± 0.707 treatment 1 269.6 ± 69.547 374.2 ± 66.792 6.8 ± 1.789 7.0 ± 1.581 treatment 2 214.4 ± 74.942 203.8 ± 59.912 3.2 ± 0.837 1.8 ± 0.837 10 table 1. the mean and standard deviation of the number of fibroblast cells and fgf-2 expressions on days 3 and 7 number of fibroblast cells fgf-2 expressions group day 3 day 7 day 3 day 7 control 355.4 ± 109.07 435.4 ± 68.332 8.6 ± 1.673 7.0 ± 0.707 treatment 1 269.6 ± 69.547 374.2 ± 66.792 6.8 ± 1.789 7.0 ± 1.581 treatment 2 214.4 ± 74.942 203.8 ± 59.912 3.2 ± 0.837 1.8 ± 0.837 figure 1. fibroblasts as shown with arrows. fibroblasts, in the control group on day 3 (a), in the treatment group 1 on day 3 (b), and the treatment group 2 on day 3 (c), looked equally solid. fibroblasts, in the control group on day 7 (d) and in the treatment group 1 on day 7 (e) looked more solid than the treatment group 2 on day 7 (f). figure 1. fibroblasts as shown with arrows. fibroblasts, in the control group on day 3 (a), in the treatment group 1 on day 3 (b), and the treatment group 2 on day 3 (c), looked equally solid. fibroblasts, in the control group on day 7 (d) and in the treatment group 1 on day 7 (e) looked more solid than the treatment group 2 on day 7 (f). a d b e c f 97 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 97mahdani, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 94–99 fgf-2 expressions on day 7 in the control group were the same as in the treatment group 1, while the lowest one was found in the treatment group 2. discussion x-ray irradiation can ionize atoms or molecules of the body, especially water molecules composing 70% of the body’s components. the ionization makes free radicals formed become not stable and destructive.3,12 the sensitivity level of various cells and tissues to irradiation is actually very diverse. the sensitivity of irradiation is called as radio-sensitivity. rapidly dividing cells are very sensitive to irradiation, or so-called high radiosensitive, such as blood vessel cells (endothelial cells), white blood cells, bloodforming cells, mucosal epithelial cells, as well as forming sperm cells and egg cells. meanwhile, cells dividing when there is damage have mid-radiosensitive, such as fibroblast, salivary gland cells, liver parenchymal cells, kidney, and thyroid. cells that require a long maturation process has a low radiosensitive, such as muscle-forming cells, boneforming cells and nerve tissue-forming cells.12 the results of the research on the effect of x-ray on the extraction wound of the mandibular incisors of the wistar rats showed that there was no significant difference in the number of fibroblasts between in the control group, in the treatment group 1 and in the treatment group 2 on day 3 (p>0.05). it indicates that the initial response of fibroblast cells was good. in other words, the body can neutralize free radical damage caused by x-ray irradiation exposure at doses of 0.08 msv and 0.16 msv on day 3. thus, there was no difference in the number of fibroblasts significantly. meanwhile, the results of the research on day 7 showed that there was no significant difference in the number of fibroblasts between in the control group and in the treatment group 1 (p>0.05). it means that x-ray irradiation at a dose of 0.08 msv on day 7 did not decrease the number of fibroblasts. in other words, the body up to day 7 can still neutralize free radicals produced by x-ray exposure. therefore, the irradiation exposure at this dose cannot damage or increase fibroblast cell death. as a result, the 11 figure 2. fgf-2 expression as shown with arrows. fgf-2 expression, in the control group on day 3 (a) looked more solid than the treatment group 1 on day 3 (b) and the treatment group 2 on day 3 (c). fgf-2 expression, in the control group on day 7 (d) and the treatment group 1 on day 7 (e) looked equally solid. fgf-2 expression in the treatment group 2 on day 7 (f) is less observed. figure 2. fgf-2 expression as shown with arrows. fgf-2 expression, in the control group on day 3 (a) looked more solid than the treatment group 1 on day 3 (b) and the treatment group 2 on day 3 (c). fgf-2 expression, in the control group on day 7 (d) and the treatment group 1 on day 7 (e) looked equally solid. fgf-2 expression in the treatment group 2 on day 7 (f) is less observed. a b c d e f dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 98 mahdani, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 94–99 number of fibroblasts in this group was no significantly different from in the group without x-ray irradiation. the results showed that there were significant differences between the treatment group 2 and the control group, as well as between the treatment group 1 and the treatment group 2 on day 7 (p<0.05). it indicates that x-ray irradiation at a dose of 0.16 msv on day 7 decreased the number of fibroblasts since x-rays can generate quite high free radicals that can damage the chains of dna, proteins, carbohydrates, and fibroblast cell lipid, while the body is still not able to repair the damage. the body needs time to regulate damage resulting in inhibition of fibroblast proliferation.11 free radicals, furthermore, cause damage to dna, such as change and loss of bases, breakage of hydrogen bond among chains, cross-linking, and breaking strands of dna, both single-strand break (ssb) and double-strand break (dsb). dna and rna are the building blocks of genes and chromosomes controlling all the metabolic processes in the body. dna damage will cause a deviation in the metabolic process controlled by the defective gene. disruption to the dna then can be seen through cell death or genetic mutation. x-rays also interfere with the function of mitochondria of the cells, resulting in oxidation of carbohydrates, lipids and cell proteins, thereby disrupting the cycle of energy in the cells. damage can also be in the form of protein denaturation and coagulation. consequently, hydrogen bonds and disulphide bonds can be disconnected, so damaging secondary and tertiary structures that result in changes in protein activities.11,15 in addition, free radicals that are not neutralized by the body can lead to inactivation of cell proliferation, induction of cell apoptosis, checkpoint of cell cycle, as well as inhibiting cell cycle.10,11 therefore, the number of fibroblasts in tooth extraction wound after exposed to x-ray irradiation at a dose of 0. 16 msv was significantly different from the group without irradiation and the group with x-ray irradiation at a dose of 0.08 msv on day 7. saputra (2012), similarly, states that x-ray irradiation at doses of 0.08 msv, 0.16 msv and 0.24 msv can decrease the number of mucosa epithelial cells of the wistar rats on day 10 after exposure. the apoptosis of epithelial cells occurs because of the activation of the p53 protein occurred due to free radicals. the activation of p53 causes the induction of the cyclin-dependent kinase (cdk), thus retaining cell cycle on growth-1-synthesis (g1-s) phase and slowing down the repairing process of the damaged dna before replication and mitosis progresses. in other words, apoptosis in epithelial cells will increase as the dose of xray irradiation increase.8 x-ray irradiation can decrease the number of inflammatory cells, epithelial cells, endothelial cells, and fibroblasts proliferating from day 3 to day 9 after an injury. the decreasing of cell proliferation then can make granulation tissue formed be less. the imbalance of proliferation and the increasing of apoptosis can disturb the proliferative phase of wound healing process, so inhibit the whole wound healing process. the results also stated that on day 9, the size of the granulation tissue area in wistar rats during the normal wound healing process is at 80.32 + 2.11% area, so much bigger than in the wound irradiated with 1 mgy, which is 45.67 + 0.86% area. those results support the results of this research since the area of granulation tissue in the treatment group 2 on day 7 was smaller than the area of granulation tissue in the control group and in the treatment group 1.9 the results of this research showed that there was significant difference in the number of fgf-2 expressions on days 3 and 7 between in the control group and in the treatment group 2, and between in the treatment groups 1 and 2 (p<0.05). this indicates the body is able to repair the damage caused by irradiation exposure at a dose of 0.08 msv. in other words, the irradiation at this dose does not give any effect on the regulation of fgf-2 in the wound healing process, but decreases fgf-2 expressions. in addition, the treatment group 2 had the lowest number of fgf-2 expression, both on day 3 and day 7. this condition is caused by the decreasing of fgf-2 produced by macrophages and endothelial cells. endothelial cells that have a high radiosensitive character will get a direct effect when exposed to x-ray irradiation, such as inhibition of proliferation and increasing of apoptosis. the decreasing of the number and function of endothelial cells then can result in the decreasing of fgf-2 expression.12 macrophages are the results of monocyte differentiation in tissues with chronic inflammation. macrophages play an important role in the immune system. monocytes, on the other hand, are inflammatory cells that are hypersensitive or high radiosensitive. as a result, monocytes can be damaged when exposed to x-ray irradiation. x-rays lead to oxidative stress and trigger reactive oxygen species (ros) in the body. ros can easily penetrate walls and monocyte components, and can also damage monocyte dna. consequently, the number and function of macrophages derived from monocyte differentiation decrease.15 monocytes exposed to x-ray irradiation, furthermore, experience dsb induction and dna base modification. the dna damage is the originator of cell death due to dna damage response (ddr) controlled by atr-chk1atm-chk2-p53 pathway, leading to response of fas and activation of caspase-3, caspase-7, and caspase-8. apoptosis is an activity caused by the execution of ddr, and can result in the death of monocytes 3 hours after the irradiation exposure.15,16 during wound healing process, macrophages and endothelial cells are the major producers of fgf-2, which has the role of stimulating fibroblast proliferation and regenerating blood vessels as a supply of oxygen and nutrients to the cells. the decreasing of the number and function of macrophages and endothelial cells then will lead to the decreasing of fgf-2 and fibroblast proliferation, the formation of new blood vessel, the deposition and synthesis 99 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 99mahdani, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 94–99 of extracellular matrix, thus inhibiting the whole healing process.1,13,14 in conclusion, x-ray irradiation at a dose of 0.16 msv can inhibit the healing process of tooth extraction would due to the decreasing of fibroblasts cell number and fgf-2 expressions. references 1. barrientos s, stojadinovic o, golinko ms, brem h, tomic-canic m. growth factors and cytokines in wound healing. wound repair regen 2008; 16(5): 585-601. 2. enoch s, leaper dj. basic science of wound healing. inggris: elsevier ltd. surgery; 2007. p. 26. 3. nanci a. ten cate’s oral histology: development, structure and function. cina: mosby, elsevier; 2008. p. 64-74, 379-90. 4. koca kutlu a, cecen d, gurgen sg, sayin o, cetin f. a comparison study of growth factor expressing following treatment with transcutaneous electrical nerve stimulation, saline solution, povidone iodine, and lavender oir in wound healing. evid based complement alternat med 2013; 2013: 361832. 5. anonim. penyakit tidak menular: kesehatan gigi. riskesdas 2007: departemen kesehatan republik indonesia; 2008. p. 130-47. 6. fragiskos fd. oral surgery. yunani: springer; 2007. p. 74, 95-9. 7. suyatno f. aplikasi radiasi sinar-x di bidang kedokteran untuk menunjang kesehatan masyarakat. yogyakarta, indonesia. seminar nasional iv, sdm teknologi nuklir; 2008. issn: 1978-0176. 8. saputra d. apoptosis dan nekrosis sel mukosa rongga mulut akibat radiasi sinar-x dental radiografik. surabaya: universitas airlangga; 2012. p. 71-8. 9. liu x, liu jz, zhang e. impaired wound healing after local soft x-ray irradiation in rat skin: time course study of pathology, proliferation, cell cycle, and apoptosis. j trauma 2005; 59(3): 682-90. 10. grudzenski s, raths a, conrad s, rübe ce, löbrich m. inducible response required for repair of low dose irradiation damage in human fibroblast. proc natl acad sci usa 2010; 107(32): 14205-10. 11. a z z a m e i , jayg e r i n j p, p a i n d. io n i z i ng i r r a d i a t io n inducedmetabolic oxidative stress and prolonged cell injury. cancer lett 2012; 372(1-2): 48–60. 12. white sc, pharoah mj. oral radiology: principles and interpretation. 6th ed. missouri: mosby; 2008. p. 33-58. 13. schultz gd, wysocki, a. interaction between extracellular matrix and growth factors in wound healing. wound repair regen 2009; 17(2): 153-62. 14. yun yr, won je, jeon e, lee s, kang w, jo h, jang jh, shin us, kim hw. fibroblast growth factors: biology, function, and application for tissue regeneration. j tissue eng 2010; 2010: 218142. 15. bauer m, goldstein m, christmann m, becker h, heylmann d, kaina b. human monocytes are severely impaired in base and dna double-strand break repair that renders them vulnerable to oxidative stress. proc natl acad sci usa 2011; 108(52): 21105-10. 16. smith j, tho lm, xu n, gillespie da. the atm-chk2 and atrchk1 pathways in dna damage signaling and cancer. adv cancer res 2010; 108: 73-112. issn 1978 3728volume 46, number 3, september 2013 editorial board of dental journal (majalah kedokteran gigi) sk: 166/h3.1.2/kd/2013 january 2nd– december 31st, 2013 patron: dean of faculty of dental medicine universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 46 number 3 september 2013: prof. dr. drg. iwa sutardjo rus sudarso, s.u., sp.kga(k) (pediatric dentistry – universitas gadjah mada) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com; website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 205162250-204225738 contents page printed by: airlangga university press. (165/11.13/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 46, number 3, september 2013 issn 1978 3728 1. peran kalsium sebagai prevensi terjadinya hipoplasia enamel (the role of calcium on enamel hypoplasia prevention) soegeng wahluyo ............................................................................................................................. 113–118 2. korelasi antara jumlah mikronukleus dan ekspresi 8-oxo-dg akibat paparan radiografi panoramic (the correlation of micronucleus formation and 8-oxo-dg expression due to the panoramic radiography exposure) rurie ratna shantiningsih, suwaldi, indwiani astuti dan munakhir mudjosemedi ............... 119–123 3. determination of fluoride content in toothpaste using spectrophotometry susanti pudji hastuti, devinta lestari and yohanes martono .................................................... 124–129 4. analisis heteroplasmy dna mitokondria pulpa gigi pada identifikasi personal forensik (heteroplasmy analysis of dental pulp mitochondrial dna in forensic personal identification) ardyni febri k, retno pudji rahayu dan agung sosiawan ........................................................ 130–134 5. effect of gestational diabetes mellitus on the expression of amelogenin in rat offspring tooth germ nurdiana dewi, ahmad syaify and ivan arie wahyudi .............................................................. 135–139 6. dna epstein-barr virus (ebv) sebagai biomaker diagnosis karsinoma nasofaring (epstein-barr virus (ebv) dna as biomaker of nasopharyngeal carcinoma diagnosis) janti sudiono dan irma hassan ...................................................................................................... 140–147 7. efek ekstrak buah delima (punica granatum l) terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster (the pomegranate extracts (punica granatum l) effect on the wild p53 expression in oral mouth malignant cell of swiss webster strain mice) sri hernawati, fedik abdul rantam, i ketut sudiana dan retno pudji rahayu ..................... 148–151 8. the effect of chitosan gel concentration on neutrophyl and macrophage in gingival ulcer of sprague dawley rat tasya adistya, fajar kumalasari, anne handrini dewi and mayu winnie rachmawati ....... 152–157 9. kadar leptin saliva dan kejadian karies gigi anak obesitas (salivary leptin levels and caries incidence in obese children) elfrida atzmaryanni dan mochamad fahlevi rizal .................................................................... 158–161 10. restorasi mahkota logam dengan pasak fiber komposit pada molar permanen muda (metal crown restoration with fiber composite post in young permanent molar) theresia dhearine pratiwi dan mochamad fahlevi rizal ............................................................ 162–166 11. ukuran kranial dan indeks sefalik pada anak retardasi mental (cranial size and cephalic index of mentally retarded children) dewi elianora, iwa sutardjo dan bambang udji rianto ............................................................. 167–172 dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author’s responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain: • title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia. • name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia • abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia. • abstract in research reports should consists of “background:”, “purpose:”, “method:”, “result:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in case reports should consists of “background:”, “purpose:”, “case(s):”, “case management:” and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • abstract in literature reviews should consists of “background:”, “purpose:”, “reviews:”, and “conclusion:” typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract. • key words contain 3-5 words and / or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia. • correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed. • materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable. • results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by ��int (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. • case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations. • case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. guide for authors iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews. • introduction comprises the problem’s background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by: • discussion explains the meaning of the examination’s results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary. • acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add ”et al.”. the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, et al. occlusionocclusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d’un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher’s prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url:http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/ eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. v4.0. san diego: media scientific, 1998. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. historyitem_v1 trimandshift range: all pages trim: cut bottom edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc smaller 8.5039 bottom qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all pages trim: extend top edge by 8.50 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 both alldoc currentavdoc bigger 8.5039 top qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 54 1 historyitem_v1 trimandshift range: all odd numbered pages trim: cut right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc smaller 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all odd numbered pages trim: extend left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 odd alldoc currentavdoc bigger 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 52 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: cut left edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc smaller 2.8346 left qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historyitem_v1 trimandshift range: all even numbered pages trim: extend right edge by 2.83 points shift: none normalise (advanced option): 'original' 32 1 0 no 789 202 none up 0.0000 0.0000 even alldoc currentavdoc bigger 2.8346 right qite_quiteimposingplus2 quite imposing plus 2 2.0a quite imposing plus 2 1 0 54 53 27 1 historylist_v1 qi2base 114 vol. 41. no. 3 july–september 2008 the effectiveness of 0.5–0.7% tetracycline gel to reduced subgingival plaque bacteria ernie maduratna setiawati department of periodontic faculty of dentistry airlangga university surabaya indonesia abstract background: the tetracycline was an antimicrobial agent, that a broad spectrum. in addition to the antimicrobial effects, their the tetracycline was an antimicrobial agent, that a broad spectrum. in addition to the antimicrobial effects, their efficacy was also anticollagenase and removal of the smear layer on the root surface. purpose: the aim of the study was to evaluate the aim of the study was to evaluate effectiveness tetracycline gel 0.5–0.7% to reduction subgingival plaque bacteria. method: a laboratory experimental study was a laboratory experimental study was conducted to investigate the effectiveness tetracycline gel 0.5–0.7%. samples were divided into 5 groups with different concentration. the antimicrobial effect was performed using spectrophotometer. the statistical test was used one-way anova with significant difference 5% and subsequently tukey-hsd test. result: the study showed that tetracycline gel 0.5% has the highest antimicrobial the study showed that tetracycline gel 0.5% has the highest antimicrobial effect. conclusion: tetracycline gel with 0.5% concentration is effective in inhibiting the growth of subgingival plaque bacteria. tetracycline gel with 0.5% concentration is effective in inhibiting the growth of subgingival plaque bacteria. key words: subgingival plaque bacteria, tetracycline gel 0.5–0.7% correspondence: ernie maduratna setiawati, c/o: departemen periodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: setiawati_ernie@yahoo.co.id introduction periodontal disease is accepted as an infection of the periodontium since the primary etiologic factor is bacteria, which triggers the host immune response and tissue destruction.1 the most important in periodontitis therapy is plaque and calculus removal with scaling and root planning. however recurrence frequently occurs due to the invasion of periodontal pathogen in gingival epithelium, cementum and dentin tubulus which is unreachable by mechanical removal.2 it recently has been considered that mechanical treatment only is insufficient in the periodontal treatment strategy. antimicrobial therapy, especially is of importance as an adjunct to mechanical periodontal treatment.3 the most widely used antibiotics in the treatment of periodontal disease have been tetracycline since they show the highest concentrations in gingival crevicuar fluid (gcf) and are highly effective on actinobacillus actinomycetemcomitans (aa).4 in addition to the antimicrobial effects, their efficacy was also anticollagenase and removal of the smear layer on the root surface.5 regarding the side effects of systemic antibiotic treatments, local delivery system have been developed in the last quarter of the 20th century. many studies on clinical effects of both systemic and local tetracycline have been performed. in some of these studies, systemic and local tetracycline provided significant decreases even in probing depth, clinical attachment levels and improved clinical parameters.6 a number of local drug delivery devices have been proposed, including fibers, strips, films, gels, sponges and micro particles. goodson et al., 2004 developed ethyl vinyl-acetate hollow fibers loaded with tetracycline hcl, but the fibers being nonresorbable, have to be removed after 10 days. these delivery systems have a number of shortcomings, including limited duration of drug release, difficulty in application and poor retention in the periodontal pocket.7 tetracycline gel could be effective in the pocket for 48 hours to 14 days.8 hydrophilic gel is one of semisolid prepares with some advantages such easily and quickly applied, unsticky and comfort.9 currently, metronidazole gel 25% is sold in indonesia is metrodanizole gel 25%. metrodanizole can survive in gingival sulcular fluid only for 24 hours. metrodanizole is effective for obligate anaerobic bacteria and spirochaeta. as a choice for facultative anaerob bacteria are ampicillin, research report 115setiawati: the effectiveness of 0.5–0.7% tetracycline gel chloramphenicol, tetracycline, and clindamycin.10 the use of local antimicrobial to this time is at 10–25%. in high concentration, tetracycline is toxic towards gingival epithelial cells and fibroblasts, thus the choice for local antimicrobial material needs biocompatible concentration and able to function optimally in inhibition of mouth microorganism, especially subgingival bacteria. tetracycline is biocompatible to fibroblast cell at below 0.7% but the question whether biocompatible of tetracycline is effective to reduce the subgingival plaque bacteria. while the aim of this research was to obtain certain concentration of tetracycline gel that effective in inhibiting subgingival plaque bacteria. materials and methods the type of this research is experimental laboratory study with research design post test only control group. the sample size was 9 patients. this research is conducted in periodontics clinic and microbiology laboratory of faculty of dentistry, airlangga university. material used are mouth mirror, pinset, periodontal probe, spiritus brander, excavator, glass beaker, reaction tube, measuring glass, mortar and pestle, measuring pipette, electric scale, incubator, spectrophotometer. the material used is hydroxypopile methylcellulose, propylene glycol, aquadest, pure tetracycline hydrochloride, brain heart infusion (bhi) media, gas pack. subgingival plaque bacteria taken from patient’s pocket who visit periodontics clinic faculty of dentistry, airlangga university; with the periodontitis diagnosis criteria as follows: pocket depth 5–7 mm, had not consume any antibiotic for 6 months, had no systemic problems. nine patients that had already checked according to criteria, had their subgingival plaque extracted using sterile excavator and quickly inserted to brain heart infusion media in reaction tubes. after that it is incubated in the incubator for 48 hours to grow anaerobic bacteria. the research are divided to 5 different treatment groups, that are, groups added tetracycline gel with concentrations of 0.5%, 0.6%, 0.7%, groups with control positive and control negative are each replicated 7 times. samples are incubated for 24 hours, which then had the inhibition of subgingival plaque bacteria scaled using spectrophotometer uv-vis to see the growth of bacteria marked with the degree of optical density with the wavelength of 570 nm. to compare the effectivity of 0.5–0.7% tetracycline hydrochloride gel concentration towards the prevention of subgingival plaque bacteria, anova statistic test is used. result the inhibitory capacity of tetracycline towards subgingival plaque bacteria are examined according to the degree of optical density analyzed using spectrophotometer with the wavelength of 570 nm. according to the observed calculation, the result could be seen in table 1 and 2. table 1. the mean of optical density 0.5–0.7% tetracycline gel toward subgingival plaque bacteria treatment control – 0.5% 0.6% 0.7% control + mean 0.04 0.073 0.088 0.088 1.672 table 1, it can be seen that the average degree of optical density after the application of tetracycline gel at 0.5% is the lowest. while in positive control, the graphic rises sharply. to identify the difference between each treatments anova test is done, the result is p < 0,00, since p < 0,05 thus means that there is a significant difference of optical density degree between the application of tetracycline gels in varied concentrations. the next analysis used tukeyhsd examination with result as shown in table 2. table 2. the result of tukey-hsd of optical density 0.5–0.7% tetracycline gel toward subgingival plaque control – 0.5% 0.6% 0.7% control + control – 0.5% 0.6% 0.7% control + ------0,00* -----0,00* 0,03* ------0,00* 0.08* 1,0 -----0,00* 0,00* 0.00* 0,00* ------- * = significant figure 1. graphic of mean of optical density 0.5–0.7% tetracycline gel toward subgingival plaque bacteria. 116 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 114–117 discussion antibiotics are organic or synthetic substance which functions as a way to inhibit or kill bacteria. according to the american academy of periodontology, the administering of systemic antibiotic has significant meaning if given to certain types of periodontitis. those are acute periodontitis, refractory periodontitis, periimplantitis, acute periodontal infection with systemic manifestation or for prophylaxis.1 antibiotics are only administered, in case conventional treatments are not effective. while local antimicrobial material can be used as adjunction of scaling and root planning, especially in deep moderate periodontal pocket. due to unreachable in deep pockets therefore the result of plaque control cannot be predicted.11 tetracycline is one of the antibiotics choice for periodontitis cases. in aggressive periodontitis case, combination of antibiotics of amoxicillin-metronidazole or combinations of tetracycline-metronidazole is often used.4 tetracycline can be consumed systemically or locally. systemic tetracycline 250 mg 4 times a day, it is seen that the concentration reached in the gingival sulcular fluid is between 0 to 8 mg/ml.12 actinobacillus actinomycetemcomitans bacteria have minimal inhibitory concentration (mic) and minimal bactericidal concentration (mbc) more than 64 microgram/ml, while porphymonas gingivalis have mic 0.5 microgram/ml and mbc 16 microgram/ml.10 biofilm in plaque bacteria, its extra cellular components will inhibit the diffusion of antimicrobials components. therefore, subgingival plaque in the form of biofilm need antibiotics with 50 times the amount of concentration. with the local application of antimicrobial preparation, the concentration in sulcus gingival fluid can reach 100 times higher.12 the effectivity of antimicrobial preparation applied can be reached if the concentration in the pocket is enough and can inhibit the growth of subgingival plaque bacteria. previously research, it was concluded that tetracycline gel was biocompatible with fibroblast cells at concentration below 0.7%,5 but still not known its inhibition capacity towards subgingival plaque bacteria. in this research, inhibition capacity of tetracycline gel is examined with concentration between 0.5–0.7% towards subgingival plaque bacteria. from this research result, it is proven that tetracycline gel with 0.5% concentration is adequate for inhibition of subgingival plaque bacteria compared to the positive control. this proves that tetracycline gel could be applied locally in 0.5% concentration. it shows that the concentration inside the pocket can reach up to 5000 µg/ml. that concentration is far above the mic or mbc of periodontopathogenic bacteria, and adequately effective to inhibit subgingival plaque bacteria.10 the use of tetracycline gel with high concentration that is 10-35% will cause bitterness at application to the patient, and also toxic towards epithelial cell and gingival fibroblast. nevertheless, the use of tetracycline at 25% in the form of film strips is very difficult and takes a lot of time because it has to be placed below the interproximal contact point and tied securely with a thread.13 by applicating tetracycline as gel, it can be absorbed into the gingival connective tissue and penetrate the root surface, therefore increasing the antibacterial capacity towards periodontopathogenic bacteria.8 in addition, tetracycline also inhibits the activity of matrix metalloproteinase produced by pmn (mmp-8 and mmp-9) by binding ca2+ and zn2+ which is located at its active side. so it inhibits the resorption of alveolar bones and collagen degeneration.1 tetracycline also display a number of independent, pleiotropic activities on inflammatory and immune processes. they have been shown to specifically decrease level of inducible no synthesis, inhibit phospholipase a2 and cyclooxigenase-2 mediated prostaglandin synthesis.14 this wide array of biological activities has resulted in the widespread application of tetracycline – based therapy in inflammatory disorders such as periodontitis and rheumatoid arthritis. following the discovery of a striking variety of non antibiotic properties of tetracycline, the therapeutic indications for these drugs have been successfully extended in the past 15 years to periodontal disease and potential new applications are being explored for conditions as stroke, aortic aneurysm, parkinson’s disease and metastatic cancer. this is certainly in large part due to the tetracyclines’ability to inhibit the release of proinflammatory mediators, including no, tnf alpha and il-1 as well as the production and activity of enzymes directly involved in tissue destruction, such as matrix metalloproteinase and stromelysin. the application of antimicrobial preparation can only be done in certain periodontitis cases. systemic antibiotic therapy combined with mechanical therapy generally has been noted to be a beneficial therapeutic approach in refractory periodontitis previously unsuccessfully treated with solely conventional therapy. local antimicrobial preparations is suggested to be applied for chronic periodontitis cases after scaling and root planning 2 times a week for two weeks. it seems clear that mechanical treatment is essential for periodontal therapy. however, it has been noted that if the microbiological or clinical analysis indicate persistent pathogenic infection, so local antibiotic therapy should be considered. thus, the tetracycline gel formulation along with with scaling root and planning was effective in reducing gingival inflammation, bleeding on probing, pocket depth and increasing clinical attachment.8 in summary, our result suggest that tetracycline gel with 0,5% concentration is effective in inhibiting the growth of subgingival plaque bacteria. refferences 1. newman mg, takei n, klokkevold p, carranza f. clinical periodontology. 10th ed. wb saunders company; 2006. p. 168–81. 2. mombelli a, schmid b, rutar a, lang np. persistence patternspersistence patterns of p gingivalis, p intermedia and a actinomycetemcomitans after mechanical therapy of periodontal disease. j periodontol 2000; 71:14–21. 3. genco r, rose l, cohen d. periodontal medicine. hamilton, london: bc decker inc; 2000. p. 264–73. 117setiawati: the effectiveness of 0.5–0.7% tetracycline gel 4. prajitno sw. menyongsong perkembangan dan peran periodonsia dalam indonesia sehat 2010. journal kedokteran gigi indonesia 1999; 4:35–42. 5. maduratna e. biokompatibilitas gel tetrasiklin dan pengaruhnya terhadap terlepasnya lapisan smir pada permukaan akar. thesis. program pascasarjana universitas airlangga. 1999. 6. akalin f, taskin m, etikan i. a comparative evaluation of the clinical effects of systemic and local doxycycline in the treatment of chronic periodontitis. j of oral science 2004; 46:25–35. 7. manson j d, eley b m. outline of periodontics. 4th ed. somerset, oxford, auckland, boston: bath press, 2000; p. 227–70. 8. maheshwari m, miglani g, yamamura s. development of tetracycline-serratiopeptidase containing periodontal gel. aaps pharm scitech 2006; 7:3–10. 9. hendradi e. pelepasan difenhidramin hci dari beberapa basis sediaan topical melewati membrana selofan. laporan penelitian lembaga penelitian, universitas airlangga. 1997. 10. widowati. aktivitas antibiotik terhadap bakteri periodontopatogen. ceramah ilmiah. fkg unair. 1997. 11. mombelli a. the use of antibiotics in periodontal therapy. clinical periodontology and implant dentistry. 4th ed. munksgaard blackwell; 2003. p. 494–507. 12. sakellari d, goodson jm, socransky ss, kolokotronis a. concentration of 3 tetracycline in plasma, gingival crevice fluid and saliva. j clin periodont 2000; 27:53–60. 13. aimetti m, romano f, torta i, cirillo d, coposio p. debridement and local application of tetracycline loaded fibers in management of persistent periodontitis. j clin periodont 2004; 34:166–72. 14. kuzin i, snyder e, ugine g, lee s. tetracycline inhibit activated b cell function. int immun 2001; 12:921–31. 202 volume 47, number 4, december 2014 usia saat inisial akuisisi streptococcus mutans dan jumlah erupsi gigi sulung pada anak (initial acquisition age of mutans streptococci and number of erupted primary teeth in children) citra adinda, udijanto tedjosasongko, dan teguh budi wibowo departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: mutan streptococci (ms) are considered as major bacteria in human dental caries. previous experiments reported that ms needs permanent surface to make stable colonization in human oral mouth. transmission of ms occured directly or indirectly by salivary contact. the younger the child acquired ms the higher dental caries risk of the child. purpose: the purposes of this study was to determine the age and number of primary teeth erupted during ms initial acquisition in children. methods: the subjects were 30 infants aged 5 months old of simomulyo region, surabaya, east java, indonesia. monthly plaque samples were taken using sterile cotton bud and oral examination were done to check number of primary teeth for 6 months period. the ms isolate were isolated using bhi broth and tyc respectively. the ms identification was done by gram staining and colony morphology. number of erupted primary teeth was determined by counting the erupted teeth in each month. results: as the result 83% children acquired ms and 17% children remain free from ms. the mean age of initial acquisition was 7,76±0,96 months and the average number of erupted teeth was two teeth. conclusion: the study revealed that the mean age of ms initial acquisition in children was 7,76±0,96 month and the colonization of ms was found in children with average 2 primary teeth erupted. key words: initial acquisition, mutans streptococci, caries, tooth eruption, children abstrak latar belakang: streptococcus mutans (sm) merupakan bakteri utama penyebab karies gigi. percobaan sebelumnya melaporkan bahwa sm membutuhkan permukaan permanen untuk membuat kolonisasi stabil di rongga mulut. penularan sm terjadi secara langsung atau tidak langsung melalui kontak saliva. semakin muda anak mengakuisisi sm semakin tinggi resiko terjadinya karies pada anak tersebut. tujuan: penelitian ini bertujuan meneliti umur dan jumlah rata-rata gigi erupsi pada masa inisial akuisisi sm pada anak metode: subyek penelitian ini adalah 30 bayi berusia 5 bulan di wilayah simomulyo, surabaya, jawa timur, indonesia. sampel plak diambil setiap bulan dengan cotton bud steril. dilakukan pula pemeriksaan rongga mulut untuk memeriksa jumlah gigi sulung yang erupsi selama 6 periode 6 bulan. sm diisolasi menggunakan bhi broth dan tyc. identifikasi sm dilakukan dengan pewarnaan gram dan pemeriksaan morfologi koloni. jumlah gigi erupsi ditentukan dari penghitungan jumlah gigi sulung yang erupsi setiap bulannya. hasil: delapan puluh tiga persen anak-anak positif didapatkan sm dan 17% anak-anak tetap bebas dari sm. studi ini menunjukkan bahwa sm rata-rata usia awal akuisisi anak adalah 7,76 ± 0,96 bulan dan jumlah rata-rata gigi erupsi adalah 2 gigi. simpulan: penelitian ini menunjukkan bahwa usia rata-rata inisial akuisisi sm pada anak-anak adalah 7,76±0,96 bulan dan kolonisasi sm ditemukan pada anak-anak dengan rata-rata 2 gigi sulung telah erupsi. kata kunci: akuisisi awal, streptococcus mutans, karies, erupsi gigi, anak research report 203adinda, et al.: usia saat inisial akuisisi streptococcus mutans korespondensi (correspondence): udijanto tedjosasongko, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. email: udijanto@gmail.com pendahuluan karies gigi sebagai salah satu penyakit yang dapat ditularkan, saat ini masih merupakan masalah kesehatan yang utama di beberapa negara.1 prevalensi karies gigi di indonesia masih cukup tinggi. angraini d. menyatakan bahwa anak-anak di indonesia mempunyai resiko tinggi terkena karies atau gigi berlubang. usia 12 tahun merupakan persentase terbesar pada anak sd yang terkena karies dengan nilai def-t 2,21 ditahun 1995.2 penyebab karies dipengaruhi banyak faktor, yang terutama adalah infeksi streptococcus mutans (s. mutans) dan kebiasaan hidup.3 streptococcus mutans dan streptococcus sobrinus merupakan mikroorganisme yang berperan utama terjadinya karies gigi.4 saat proses kelahiran, secara umum dalam rongga mulut bayi kekurangan beberapa kolonisasi bakteri penting. namun segera setelah bayi lahir, terjadilah kolonisasi bakteri secara bertahap dari berbagai macam spesies pada permukaan epitel rongga mulut melalui kontaminasi dari makhluk hidup maupun benda mati disekitarnya.5 beberapa bulan kemudian, rongga mulut memproses kumpulan organisme mikrobiota yang mulai dapat terdeteksi. proses pembentukan ekologi yang utama dalam rongga mulut ini adalah saat terjadinya erupsi dari gigi sulung bayi yang berkisar pada usia 6 bulan.6 s. mutans dapat bertahan dalam rongga mulut dengan membentuk koloni pada permukaan mukosa dan hidup bebas dalam saliva bahkan berpoliferasi dan berkembangbiak, akan tetapi s. mutans akan tertelan bersama saliva.7 bakteri ini memerlukan adanya permukaan yang permanen untuk membentuk koloni yang stabil di rongga mulut. oleh karena itu s. mutans hanya dapat ditemukan setelah gigi erupsi, pemakaian obturator atau gigi tiruan.8,9 transmisi s. mutans terjadi melalui saliva, baik kontak langsung ataupun tidak langsung. kontak tidak langsung melalui media sendok, sikat gigi, pasta gigi, ataupun media lain yang terkontaminasi oleh saliva.10 jika terjadi kolonisasi yang tetap dan stabil dalam rongga mulut, maka terjadilah inisial akuisisi.4 inisial akuisisi s. mutans pada bayi juga dipengaruhi oleh beberapa faktor individu antara lain adalah jumlah s. mutans, status karies, berat bayi lahir yang kurang, penggunaan obturator, jumlah erupsi gigi, level antibodi ig a saliva yang rendah.11-13 penelitian yang dilakukan kohler et al.,14 menunjukkan bahwa usia anak pada inisial akuisisi mempengaruhi resiko terjadinya karies dikemudian hari. semakin muda usia anak mengakuisisi bakteri ini, maka semakin tinggi resiko anak tersebut mengalami karies. tingginya angka karies anak di indonesia, menunjukkan bahwa kemungkinan karena anak indonesia mengalami inisial akuisisi s. mutans pada usia yang sangat dini. namun hal ini masih memerlukan penelitian yang lebih jauh dan pada usia berapa anak indonesia pada umumnya mengakuisisi s. mutans. dari penelitian yang dilakukan oleh tedjosasongko et al.,9 usia rata-rata inisial akuisisi balita di puskesmas pucang sewu surabaya, indonesia adalah 9,7 bulan, sedangkan menurut penelitian yang dilakukan oleh maria pada tahun 2010, usia rata-rata inisial akuisisi balita di wilayah tropodo adalah 8,64 bulan. ini menunjukkan bahwa resiko karies pada subyek tersebut tinggi.15,16 penelitian ini dilakukan pada daerah simomulyo, surabaya. pemilihan daerah tersebut berdasarkan survey pendahuluan yang telah dilakukan. diketahui bahwa dari 50 anak balita di daerah simomulyo, surabaya sebanyak 35 anak balita (70%) telah mengalami karies. ini menunjukkan bahwa tingginya angka karies pada daerah tersebut. daerah simomulyo memiliki 2 posyandu yang aktif melakukan penyuluhan dan penimbangan pada bayi, dimana bayi peserta posyandu tersebut memenuhi kriteria dan jumlah sampel penelitian penulis. jumlah gigi yang telah erupsi dapat mempengaruhi kolonisasi s. mutans dimana gigi berfungsi sebagai tempat yang stabil dalam rongga mulut untuk berkolonisasi. semakin banyak gigi erupsi maka semakin luas permukaan gigi untuk kolonisasi bakteri sehingga resiko transmisi s. mutans pada anak akan semakin tinggi. menurut hanada,17 untuk terjadinya karies gigi, kolonisasi dari bakteri kariogenik diatas permukaan gigi mempunyai peranan yang cukup penting dalam inisial akuisisi s. mutans. studi ini bertujuan meneliti usia rata-rata serta jumlah erupsi gigi sulung bayi saat inisial akuisisi s. mutans di wilayah simomulyo, surabaya. bahan dan metode penelitian ini dilakukan pada 30 bayi berusia 5 bulan yang setiap bulan datang ke posyandu rw vi-viii di kelurahan simomulyo, surabaya selama 6 bulan. sampel diambil secara total sampling. keseluruhan sampel dilakukan pengambilan sampel plak setiap bulan sekali dengan menggunakan cotton tips sterile pada keseluruhan bagian mukosa mulutnya serta dilakukan pencatatan jumlah gigi sulung yang telah erupsi setiap bulan saat pengambilan sampel plak. sampel plak yang didapat kemudian diolah di laboratorium kemudian dilakukan identifikasi koloni s. mutans dengan pengecatan gram. analisa data sebelum dilakukan uji beda, distribusi data diuji dengan kolmogorov-smirnov test. untuk menguji beda variabel usia inisial akuisisi dan erupsi gigi sulung berdasarkan jenis kelamin pada responden pada inisial akusisi digunakan uji beda mann whitney. 204 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 202–205 hasil didapatkan hasil bahwa usia rata-rata inisial akuisisi s. mutans pada bayi di wilayah simomulyo, surabaya adalah 7,76 ± 0,97 bulan (tabel 1). jumlah rata-rata gigi sulung yang erupsi pada saat ditemukannya s.mutans adalah 2 gigi (tabel 2), sedangkan usia rata-rata erupsi gigi insisif pertama sulung rahang bawah adalah 7.28 ± 1.34 bulan (tabel 3). tabel 4 menunjukkan hasil uji beda variabel usia inisial akuisisi dan erupsi gigi sulung berdasarkan jenis kelamin pada responden yang mengalami inisial akusisi. pembahasan inisial akuisisi s. mutans dapat terjadi apabila ada transfer langsung s. mutans dari host terinfeksi ke host lain, atau secara tidak langsung melalui objek yang terkontaminasi saliva, seperti makanan dan air. masa yang paling riskan anak-anak tertular s. mutans adalah ketika gigi tumbuh yaitu antara 8 bulan sampai 3 tahun.3 beberapa penelitian yang menunjukkan bahwa usia inisial akuisisi bervariasi antara 7 hingga 36 bulan yaitu periode erupsi gigi sulung.18 penelitian yang dilakukan roeters et al.,19 menunjukkan bahwa usia anak pada inisial akuisisi s. mutans dapat menjadi faktor resiko yang paling mempengaruhi terjadinya karies dikemudian hari. semakin muda usia anak mengakuisisi bakteri ini, maka semakin tinggi resiko anak tersebut mengalami karies. dari penelitian ini 25 bayi diamati dari usia 5-11 bulan didapatkan usia rata-rata inisial akuisisi pada bayi tersebut adalah 7,76 bulan, sedangkan usia paling awal terpapar s. mutans adalah 6 bulan. dari hasil uji beda yang dilakukan didapatkan hasil perbedaan tidak bermakna usia inisial akuisisi s. mutans berdasarkan jenis kelamin dengan signifikasi lebih besar dari 0,05 (p = 0,102; p > 0,05). kesimpulan hasil usia rata-rata inisial akuisisi s. mutans pada penelitian ini menjadi lebih awal jika dibandingkan pada 2 penelitian sebelumnya yang dilakukan oleh tedjosasongko et al.9 yaitu 9,67 bulan dan pada penelitian yang dilakukan oleh maria pada tahun 2009 didapatkan usia rata-rata inisial akuisisi s. mutans yaitu 8,64 bulan. pada penelitian ini rata-rata usia erupsi gigi insisif sulung pertama rahang bawah adalah 7,28 bulan. usia rata-rata erupsi gigi insisif sulung pertama rahang bawah pada penelitian ini sedikit berbeda dengan mc. donald,20 gigi insisif sulung pertama rahang bawah tumbuh rata-rata pada usia 6 bulan. menurut hasil penelitian dari scuurs,21 tidak ditemukan perbedaan signifikan antara waktu erupsi gigi sulung pertama pada anak perempuan maupun lakilaki. hal ini memiliki kesamaan dengan hasil dari uji beda waktu erupsi gigi sulung yang dilakukan peneliti dimana menunjukkan hasil perbedaan tidak bermakna usia erupsi gigi sulung pertama berdasarkan jenis kelamin dengan signifikansi lebih besar dari 0,05 (p = 0,46; p > 0,05). ada tendensi erupsi gigi sulung pertama pada anak perempuan lebih cepat dibanding anak laki-laki, yaitu pada anak perempuan pada usia 9 bulan, sedangkan pada anak lakilaki pada usia 10 bulan.22 namun hal ini berbeda dengan hasil penelitian ini, dimana usia rata-rata erupsi pada anak tabel 1. usia rata-rata inisial akuisisi bayi laki-laki dan perempuan dari usia 5-11 bulan di wilayah simomulyo, surabaya no. kelompok n x (bulan) sd 1 laki-laki 12 7,0833 0,99620 2 perempuan 13 7,4615 0,87706 3 lakilaki dan perempuan 25 7,7600 0,96954 tabel 2. jumlah rata-rata erupsi gigi sulung saat inisial akuisisi bayi laki-laki dan perempuan pada saat inisial akuisisi dari usia 5-11 bulan di wilayah simomulyo, surabaya no. kelompok n x sd 1 laki-laki 12 2,5833 0,99620 2 perempuan 13 1,5385 1,12550 3 lakilaki dan perempuan 25 2,0400 1,17189 tabel 3. usia rata-rata erupsi gigi insisif sulung pertama rahang bawah bayi lak-laki dan perempuan dari usia 5-11 bulan di wilayah simomulyo, surabaya no. kelompok n x (bulan) sd 1 laki-laki 13 7,1667 1,26730 2 perempuan 17 7,3846 1,44559 3 lakilaki dan perempuan 30 7,2800 1,33915 tabel 4. hasil uji beda variabel usia inisial akuisisi dan erupsi gigi sulung berdasarkan jenis kelamin pada responden yang mengalami inisial akusisi variabel kelompok signifikansi uji beda mann whitney usia inisial laki-laki 0,102 perempuan usia erupsi laki-laki 0,646 perempuan 205adinda, et al.: usia saat inisial akuisisi streptococcus mutans laki-laki lebih cepat dibandingkan dengan anak perempuan (tabel 3). terjadinya variasi waktu erupsi gigi telah banyak dipelajari dan diteliti. berdasarkan hasil penelitian ini menunjukkan bahwa usia rata-rata inisial akuisisi s. mutans pada bayi di wilayah simomulyo, surabaya adalah 7,76 ± 0,96 bulan dengan 2 gigi sebagai jumlah rata-rata erupsi gigi sulung saat inisial akuisisi s. mutans. resiko terjadinya karies pada bayi-bayi tersebut diprediksi tinggi, sehingga perlu dilakukan tindakan pencegahan karies gigi. penelitian ini dapat menjadi penelitian dasar dalam melakukan penelitian berkelanjutan untuk melihat karakteristik dari tipe strain s. mutans ibu dan bayi serta mengetahui waktu terjadinya karies pada bayi, sehingga didapatkan data yang lebih menyeluruh untuk melihat pola transmisi s. mutans sebagai usaha untuk menghambat inisial akuisisi serta pertumbuhan s. mutans pada bayi. daftar pustaka 1. ra mosgomez fj, weint raub ja, ga nsk y sa, hoover ci, featherstone jd. bacterial, behavioral, and environmental factor associated with early childhood caries. j clin pediatr dent 2002 winter; 26(2): 165-73. 2. anggraini d. hubungan antara tingkat konsumsi karbohidrat dan frekuensi makan makanan kariogenik dengan kejadian penyakit karies gigi pada anak prasekolah di tk aba 52. semarang:unnes press; 2009. p. 3. 3. gronroos l. quantitative an qualitative characterization of mutans streptococci in saliva and the dentition. dissertation. institute of dentistry, university of helsinzki; 2000. p. 9-25. 4. klein mi, flório fm, pereira ac, höfling jf, gonçalves rb. longitudinal study of transmission, diversity, and stability of streptococcus mutans and streptococcus sobrinus genotype in brazilian nursery children. j clin microbiol 2004; 42(10): 4620-6. 5. murray pr, rosenthal ks, kobayashi gs, pfaller. commensal and pat hogen ic m ic robia l f lor a i n hu ma ns. i n: me d ica l microbiology. st. louis: mosby inc; 2002. p. 78-87. 6. richard j. oral microbiology and immunology. washington, dc: american society for microbiology; 2006. p. 90. 7. berkowitz rj. mutans streptococci: acquisition and transmisson. pediatr dent 2006; 28(2): 106-9. 8. berkowitz rj, jones p. mouth to mouth transmission of bacterium streptococcus mutans between mother and child. arch oral biol 1985; 30(4): 377-9. 9. tedjosasongko u, kozai k. initial acquisitionand transmission of mutans streptococci in children at day nursery. asdc j dent child 2002; 69(3): 284-8, 234-5. 10. alaluusua s. transmission of mutans streptococci. proc finn dent soc 1991; 87(4): 443-7. 11. milgrom p, riedy ca, weinstein p, tanner ac, manibusan l, bruss j. dental caries and its relationship to bacterial infection, hypoplasia, diet and oral hygiene in 6-to-36-month-oldchildren. community dent oral epidemiol 2000; 28(4): 295-306. 12. wan ak, seow wk, walsh lj, bird p, tudehope dl, purdie dm. association of streptococcus mutans infection and oral developmental nodules in pre-dentale infants. j dent res 2001; 80: 1945-48. 13. wan ak, seow wk, purdie dm, bird ps, walsh lj, tudehope di. a longitudinal study of streptococcus mutans colonization in infant after tooth eruption. j dent res 2003; 82(7): 504-8. 14. köhler b, andréen i, jonsson b. the earlier the colonization by mutans streptococci, the higher caries prevalence at 4 year of age. oral microbiol immunol 1988; 3(1): 14-7. 15. te djosa song ko u, nu r a i n i p, p r a dop o s. i n isia l a k u isisi streptococccus mutans pada anak balita di puskesmas pucang sewu surabaya. maj. ked. gigi kd (dent j) 2005; edisi khusus pin ikga i: 47-50. 16. noviantari hm. inisial akuisisi streptococcus mutans pada bayi dibawah usia satu tahun. karya tulis akhir. surabaya: program pendidikan dokter gigi spesialis universitas airlangga; 2010. 17. hanada n, kuramitsu hk. isolation and characterization of the streptococcus mutans gtfc gene, coding for synthesis of both soluble and insoluble glucan infect immun 1988; 56(8): 1999-2005. 18. caufield pw, cutter gr, dasanayake ap. initial acquisition of mutans streptococci by infant: evidence for a discrete window of infectivity. j dent res 1993; 72(1): 37-45. 19. roeters fj, van der hoeven js, burgersdijk rc, schaeken mj. lactobacilli, mutans streptococci, and dental caries: a longitudinal study in 2year-old children up to age of 5 years. caries res 1995; 29(4): 272-9. 20. mc. donald r, david ra. dentistry for child and adolescent. 8th ed. st. louis: mosby, 2004; p. 180-90. 21. scuurs ahb. patologi gigi geligi dan kelainan jaringan keras gigi. yogyakarta: gadjah mada university press; 1993. h. 116-34. 22. gupta a. emergence of primary teeth in sunsari district of eastern nepal. mc gill j medicine 2007; 10(1): 11-15. vol 38 no 3 2005 154 model kekuatan geser dan kekuatan tarik perlekatan copper alloy dengan resin akrilik setelah tin plating (tensile strength and shear strength models bonds in between copper alloy and acrylic resin after tin plating) endanus harijanto dan sri yogyarti bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract tooth crown restoration was made in a complex system consisting of several elements, namely tensile strength and shear strength bond between copper alloy and acrylic resin after tin plating. the aim of this exemination was to find a model representing connection between tensile strength and shear strength in between copper alloy with acrylic resin in statistic method. in conclusion, this exemination utilizing a strength model = 0.645 + 1.237 × tensile strength resulted shear strength exemination. on the other hand, the utilization of a strength = –0.506 + 0.808 × shear strength resulted tensile strength exemination. key words: model, tensile strength, shear strength, copper alloy, acrylic resin, tin plating korespondensi (correspondence): endanus harijanto, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan restorasi suatu mahkota gigi bertujuan untuk mengembalikan fungsi kunyah, estetik dan fungsi bicara. oleh karena itu pembuatan restorasi mahkota membutuhkan suatu bahan yang dapat menahan beban kunyah dan memenuhi syarat estetik. pada beberapa kasus, penggunaan copper alloy mempunyai warna keemasan yang tidak memenuhi persyaratan estetik tetapi mempunyai daya tahan beban kunyah yang cukup baik, sehingga perlu diperbaiki dengan memberi lapisan resin akrilik yang mempunyai warna seperti gigi asli. perlekatan antara logam dan resin akrilik sulit diperoleh oleh karena tidak ada ikatan kimia antara logam dan resin akrilik yang menyebabkan kebocoran tepi antara logam dan resin akrilik.1 dengan mempergunakan tin plating ikatan kimia antara copper alloy dan resin akrilik dapat terbentuk sehingga diharapkan kekuatan perlekatan dapat meningkat.2 perlekatan antara dua bahan atau bond strength merupakan suatu sistem komplek yang terdiri dari elemen kekuatan tarik dan kekuatan geser . perlekatan copper alloy dengan resin akrilik belum diketahui keterkaitan antara hasil uji tarik dan geser, oleh karena itu diperlukan model berupa pemetaan dari karakteristik sistem dan trasformasi karakteristik sistem ke dalam formula yang pada umumnya merupakan formula matematik. tujuan pemodelan di sini digunakan sebagai alat menjelaskan atau menggambarkan suatu fakta keterkaitan beberapa elemen, karena belum ada teori yang menerangkan hal tersebut. dengan adanya fakta keterkaitan antara elemen tersebut bermanfaat untuk efisiensi dalam melakukan uji dengan jalan estimasi nilai kekuatan suatu elemen berdasarkan hasil salah satu uji kekuatan elemen lainnya. untuk melakukan estimasi dipergunakan teknik regresi sebab mempunyai aplikasi yang luas, penerapannya lebih mudah dan pada prosedur statistik yang komplek mudah dipahami.3,4 menurut gaspersz4 proses pemodelan regresi pada dasarnya merupakan suatu proses yang bersifat iteratif, yang secara garis besar terdiri dari tiga tahap yaitu: spesifikasi atau identifikasi model, penentuan atau pendugaan nilai parameter model termasuk pemilihan model yang baik dan pengujian terhadap model. bahan dan metode model dibentuk berdasarkan data sekunder yang diperoleh dari hasil penelitian tentang kekuatan perlekatan tarik antara copper alloy terhadap resin akrilik dan dari hasil uji kekuatan perlekatan geser antara copper alloy dengan resin akrilik.5,6 data sekunder tersebut didistribusikan dalam tabel 1 dan tabel 2. data penelitian tersebut diperoleh dengan uji eksperimental yang dilakukan di laboratorium bagian ilmu material dan teknologi kedokteran gigi mempergunakan bahan uji copper alloy atau logam campur tembaga, resin akrilik tipe heat cured, dan menggunakan larutan sncl2 sebagai bahan tin plating. proses penelitian diawali dengan pembuatan spesimen uji yang terbuat dari copper alloy, bentuk spesimen disesuaikan dengan kebutuhan alat uji. 155harijanto dan yogyarti: model kekuatan geser dan kekuatan tarik spesimen kelompok penelitian tin plating digosok sebanyak 20 kali menggunakan kertas gosok aluminium oxide no. 2,5 dengan diberi beban 1 kg selanjutnya dilakukan tin plating dengan periode singkat yaitu menggunakan arus 6 volt dan 9 volt dengan pertimbangan periode singkat tersebut dapat mengoptimalkan prosedur electroplating 7, hasilnya dikeringkan dengan udara selama satu menit supaya terbentuk lapisan oxide film yang berasal dari timah yang terkena udara,8 selanjutnya diberi lapisan akrilik. pada kelompok kontrol spesimen uji yang terbuat dari copper alloy langsung diberi lapisan akrilik tanpa melewati proses plating. tabel 1. nilai rerata dan standar deviasi kekuatan perlekatan tarik antara logam campur tembaga terhadap resin akrilik (kgf/mm2) kelompok x sd kontrol plating 4,3181 21,4787 0,8252 1,5056 tabel 2. nilai rerata dan standar deviasi hasil uji kekuatan perlekatan geser antara copper alloy dengan resin akrilik (kgf/mm2) kelompok x sd kontrol plating 5,9666 27,2277 0,683 0,929 selanjutnya berdasarkan data kasar hasil uji dibuat model atau hubungan fungsional antara nilai uji kekuatan tarik dan nilai uji kekuatan geser. kedua nilai uji kekuatan tarik maupun geser bisa berperan sebagai variabel prediktor maupun variabel respon yang nantinya akan didapatkan dua model regresi sesuai dengan peran masingmasing yaitu regresi nilai kekuatan tarik atas nilai kekuatan geser maupun regresi nilai kekuatan geser atas nilai kekuatan tarik. proses pemodelan meliputi: 1) spesifikasi atau identifikasi model, ditentukan dengan melihat penebaran data ke dalam grafik untuk melihat hubungan apakah linier atau non linier, yang pada intinya untuk merumuskan model yang diperkirakan sesuai dengan perilaku sistem konkrit yang dipelajari; 2) penentuan atau pendugaan nilai parameter model termasuk pemilihan model yang terbaik (seleksi model) untuk menjelaskan sistem konkrit yang dipelajari. suatu model dianggap tepat apabila nilai dugaan bagi parameter telah dinyatakan stabil, hal ini berkaitan dengan besarnya ragam atau varian dari nilai dugaan bagi parameter; 3) pengujian terhadap model, merupakan suatu proses yang bersifat kritis, dimana ketepatan model yang telah dipilih dievaluasi kembali. analisis terhadap tingkat kesalahan model (misalnya melalui analisis residu) merupakan bagian penting dari proses ini. hasil identifikasi model dengan mempergunakan penebaran data antara nilai uji kekuatan tarik dan nilai uji kekuatan geser perlekatan antara copper alloy dan resin akrilik terlihat penebaran data terpola linier (gambar 1). g e s e r tarik 30 20 10 0 30 20 10 0 observ linear gambar 1. penebaran data nilai uji kekuatan tarik dan nilai uji kekuatan geser. penentuan atau pendugaan nilai parameter model regresi kekuatan geser atas kekuatan tarik pada tabel 3 diperoleh β0 = 0,645 dengan siginifikansi p > 0 dan β1 = 1,237 dengan signifikansi p < 0. berdasarkan uji anova pada tabel 4 diperoleh nilai pemilihan model regresi kekuatan geser atas kekuatan tarik yang terbaik dengan nilai signifikan (0,001). pendugaan nilai parameter model regresi kekuatan tarik atas kekuatan geser pada tabel 5 diperoleh β0 = -0,506 dengan siginifikansi p > 0 dan β1= 0,808 dengan signifikansi p < 0. berdasarkan uji anova pada tabel 6 diperoleh nilai pemilihan model regresi kekuatan tarik atas kekuatan geser terbaik dengan nilai signifikan (0,001). tabel 3. nilai koefisien model regresi kekuatan geser atas kekuatan tarik coefficientsa unstandardized coefficients model b std. error standardized coefficients t sig. constant ,645 ,325 1,987 ,118 kekuatan tarik 1,237 ,021 ,999 59,022 ,000 dependent variable: kekuatan geser tabel 4. uji anova model regresi kekuatan geser atas kekuatan tarik anovab model sum of squares df mean square f sig. regression 677,532 1 677,532 3483,651 ,000 residual ,778 4 ,194 total 678,310 5 1. predictors: (constant), kekuatan tarik 2. dependent variable: kekuatan geser 156 maj. ked. gigi. (dent. j.), vol. 38. no. 3 juli–september 2005: 154–157 tabel 5. nilai koefisien model regresi kekuatan tarik atas kekuatan geser coefficientsa unstandardized coefficients model b std. error standardized coefficients t sig. constant -,506 ,270 -1,878 ,134 kekuatan geser ,808 ,014 ,999 59,022 ,000 a dependent variable: kekuatan tarik tabel 6. uji anova model regresi kekuatan tarik atas kekuatan geser anovab model sum of squares df mean square f sig. regression 677,532 1 677,532 3483,651 ,000 residual ,778 4 ,194 total 678,310 5 a. predictors: (constant), kekuatan geser b. dependent variable: kekuatan tarik scatterplot dependent variable: kekuatan geser regression studentized residual 1,51,0,50,0-,5-1,0-1,5-2,0 kek uat an ge ser 30 20 10 0 gambar 2. penebaran data antara residu dengan variabel respon kekuatan geser. scatterplot dependent variable: kekuatan tarik regression studentized residual 2,01,51,0,50,0-,5-1,0-1,5 ke ku at an ta rik 30 20 10 0 gambar 3. penebaran data antara residu dengan variabel respons kekuatan tarik. uji ketepatan model dilakukan dengan melihat penebaran data atau plot antara variabel respons dengan residu (gambar 2), dimana penebaran antara residu dengan ramalan atau variabel respons model regresi kekuatan geser atas kekuatan tarik terlihat tidak terpola. pada gambar 3 penebaran antara residu dengan variabel respons model regresi kekuatan tarik atas kekuatan geser terlihat tidak terpola. pembahasan data sekunder eksperimental uji kekuatan tarik dan kekuatan geser antara copper alloy dengan resin akrilik menunjukkan adanya variasi perubahan kekuatan yang disebabkan oleh proses plating dimana terjadi peningkatan kekuatan setelah melewati proses plating dibanding tanpa plating. hal ini disebabkan hasil tin plating pada copper alloy setelah terjadi oksidasi mampu membentuk oxide film berupa tin oxide yang mengakibatkan meningkatkan kekuatan perlekatan antara resin akrilik dan logam.8–10 sesuai dengan pernyataan combe11 yang menyatakan bahwa apabila tin dilapiskan pada logam campur akan terbentuk lapisan oxide film yang akan bereaksi dengan resin dan memberikan suatu ikatan kimia. craig and powers12 juga menyatakan bahwa terbentuknya oxide pada permukaan logam campur telah terbukti berperan dalam menghasilkan perlekatan yang kuat. variasi peningkatan kekuatan terjadi pada uji kekuatan tarik maupun uji kekuatan geser, dimana hal ini menunjukkan adanya suatu perubahan yang sifatnya berpasangan yang memungkinkan untuk dilihat apakah pasangan data ini mempunyai hubungan yang fungsional. hubungan fungsional antara nilai kekuatan tarik dan nilai kekuatan geser berhasil diperoleh melalui metode statistika dengan jalan menentukan spesifikasi atau identifikasi model. dari penebaran data nilai kekuatan tarik dan nilai kekuatan geser dapat dilihat bahwa penebaran data mempunyai pola yang linier atau garis lurus (gambar 1), di sini disimpulkan bahwa model yang terbentuk merupakan model linier: y = β0 + β1x + ε y = variable respons x = faktor bertaraf kuantitatif β0 = parameter intersep β1 = pengaruh variabel bebas pada respons ε = galat (error) dengan pedoman bahwa model yang dibentuk merupakan model yang linier, dicari koefisien β0 dan β1 dengan mempergunakan metode kuadrat terkecil atau least squares method 3,13 yang hasilnya dapat dilihat pada tabel 3 dan 5 diperoleh: model i: kekuatan geser = 0,645 + 1,237 × kekuatan tarik; model ii: kekuatan tarik = –0,506 + 0,808 × kekuatan geser komponen β1 model i memberikan arti bahwa peningkatan satu unit kekuatan tarik akan meningkatkan 1,237 kekuatan geser dan komponen β1 model ii memberikan arti bahwa peningkatan satu unit kekuatan geser akan meningkatkan 0,808 kekuatan tarik. 157harijanto dan yogyarti: model kekuatan geser dan kekuatan tarik setelah melewati uji t komponen β1 model i dan model ii menunjukkan hasil yang signifikan (p < 0,05) berarti β1 mempunyai kontribusi yang berarti sebesar 99,99% terhadap nilai y sehingga dapat digunakan sebagai prediktor dan dapat dimasukkan ke dalam model. sedangkan arti komponen β0 mempunyai beberapa pengertian, pertama apabila komponen model i kekuatan tarik sama dengan nol akan diperoleh nilai rata-rata kekuatan geser sebesar 0,645 akibat pengaruh dari β0 dan pada model ii apabila kekuatan geser sama dengan nol akan diperoleh nilai rata-rata kekuatan tarik sebesar -0,506 akibat pengaruh dari β0. kondisi ini tidak mungkin berdasarkan pendapat dari anusavice menyatakan bahwa tensile strength, shear strength, compressive strength dan flexural strength masing-masing merupakan ukuran tekanan yang diperlukan untuk mematahkan suatu bahan. maka jika nilai kekuatan tarik maupun geser sama dengan nol berarti tanpa tekanan apapun perlekatan antara copper alloy dengan resin akrilik sudah terlepas atau tidak terjadi perlekatan sama sekali antara copper alloy dengan resin akrilik . kedua berdasarkan hasil uji perlekatan antara copper alloy dengan resin akrilik walaupun tanpa perlakuan atau plating sudah mempunyai nilai bukan nol yaitu 4,3181 kgf/mm2 untuk kekuatan tarik dan 5,9666 kgf/mm2 untuk kekuatan geser, kemungkinan hal ini terjadi sesuai dengan pendapat craig and powers12 bahwa logam mulia tahan terhadap oksidasi untuk memudahkan oksidasi harus ditambah elemen lain seperti indium atau timah. bahan yang dipergunakan dalam penelitian ini adalah copper alloy, bukan merupakan logam mulia sehingga dapat mengalami oksidasi sehingga terbentuk oxide film yang membentuk ikatan secara kimia dengan resin akrilik. oleh karena itu pengertian bahwa kekuatan geser maupun kekuatan tarik sama dengan nol dapat kita abaikan. ketiga walaupun uji “t” menunjukkan nilai signifikansi β0 pada model i dan model ii dengan hasil tidak signifikan (p > 0,05) yang berarti β0 tidak mempunyai kontribusi yang berarti terhadap y , namun keberadaan nilai β0 tetap dipertahankan oleh karena secara teori tidak mungkin uji kekuatan tarik dan kekuatan geser sama dengan nol dan di dalam penelitian ini tidak ada nilai nol yang masuk sebagai data prediktor. sesuai dengan pendapat gujarati d16 yang menyatakan bahwa secara umum orang harus menggunakan akal sehat dalam menafsirkan intersep (β0) karena jangkauan sampel nilai x tidak memasukkan nol sebagai satu dari nilai yang diamati. penentuan model yang kita peroleh sudah merupakan model yang terbaik dapat kita lihat pada tabel 4 dan tabel 6 setelah melewati uji anova menunjukkan bahwa model i dan model ii mempunyai nilai signifikansi p < 0,05 berarti model i dan model ii mempunyai nilai dugaan yang stabil berarti merupakan pilihan model yang terbaik. pengujian terhadap model yang telah dipilih dievaluasi kembali dalam hal ketepatan modelnya, dari gambar 2 dan gambar 3 yang menggambarkan penebaran data antara residu dengan variabel respon menunjukkan tebaran data yang tidak terpola, yang dimaksud residu adalah perbedaan antara nilai pengamatan dengan nilai ramalan. dengan penebaran data tidak terpola berarti tidak ada hubungan antara residu dengan nilai ramalan, berarti asumsi linieritas dan homogenitas varian model terpenuhi. dari penelitian ini dapat disimpulkan efisiensi diperoleh, dengan mempergunakan model kekuatan geser = 0,645 + 1,237 × kekuatan tarik didapatkan nilai estimasi kekuatan geser berdasarkan nilai kekuatan tarik. dan dengan mempergunakan model kekuatan tarik = – 0,506 + 0,808 × kekuatan geser didapatkan estimasi nilai kekuatan tarik berdasarkan nilai kekuatan geser. untuk penyempurnaan model perlu dilakukan penambahan variasi data berdasarkan perlakuan selain tin plating. daftar pustaka 1. imbery ta, evans db, koeppen rg. a new method of attaching gold occlusal surfaces to acrylic resin denture teeth. quintessence int 1993; 24:29–33. 2. mc. lean jw. the science and art of dental ceramic. the nature of dental ceramic and their clinical use. quintessence int 1979; 10:63–88. 3. kleinbaum dg, kupper ll, muller ke. applied regression analysis and other multivariable methods. 2nd ed. boston: pws-kent publishing co; 1992. p. 36, 41–4. 4. gaspersz v. teknik analisis dalam penelitian percobaan. jilid 2. bandung: tarsito; 1992. h. 37–9. 5. asmara e. pengaruh pemberian tin plating pada permukaan logam campur tembaga terhadap peningkatan kekuatan perlekatan tarik resin akrilik. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2002. h. 13–26. 6. dewi rk. peningkatan kekuatan perlekatan geser antara copper alloy dan resin akrilik setelah proses tin plating. skripsi. surabaya: fakultas kedokteran gigi universitas airlangga; 2002. h. 14–27. 7. van der veen h, krajenbrink t, bronsdijk b, van der poel f. resin bonding of tin electroplated precious metal fixed partial denture one-year clinical result. quintessence int 1986; 17:299–301. 8. kirk, othmer. encyclopedia of chemical technology. 2nd ed. usa: john willey & sons; 1977. p. 42–5. 9. guastaldi ac, lacefield wr, leinfelder kf, mondelli j. metallurgical evaluation of a copper alloy-based alloy for dental castings. quintessence int 1991; 22:647–52. 10. oscan m, pfeiffer p, nergiz i. a brief history and current status of metal and ceramic surface-conditioning concepts for resin bonding in dentistry. quintessence int 1998; 29: 713–24. 11. combe ec. notes on dental materials. 6th ed. edinburg, london, madrid, melbourne, new york, tokyo: churchill livingstone; 1992. p. 26–8, 157–61, 263. 12. craig rg, powers jm. restorative dental materials. 11st ed. st louis: mosby inc; 2002. p. 85, 578–9, 636. 13. box gep, hunter wg, hunter js. statistics for experimenters an introduction to design, data analysis and model building. new york, chichester, brisbane, toronto: john willey & sons; 1978. p. 473–7. 14. anusavice kj. science of dental materials. 11st ed. elsevier science. usa: saunders; 2003. p. 59. 15. gujarati d. ekonometrika dasar. edisi ke-4. jakarta: penerbit airlangga; 1995. h. 48. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious dise�se. �merg infect dis �seri�l online] 1995 j�n-m�r; 1�1��:�24 screens]. av�il��le from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. editor 84 the transversal strength of acrylic resin plate after being immersed soaking in noni fruit (morinda citrifolia linn.) juice sri redjeki indiani department of dental material faculty of dentistry, airlangga university surabaya indonesia abstract the disadvantages of acrylic resin plate are liquid absorption and porosity, allowing microorganisms to grow and multiply resulting in inflammation in the oral cavity. the juice of the noni fruit (morinda citrifolia linn.) contains active flavonoid and atsiri oil. flavonoid is a phenol substance that degrades acrylic resin plate in prolonged contact. the purpose of this study was to examine the transversal strength of acrylic resin plate after being immersed in noni fruit juice. an acrylic resin plate of 65 × 10 × 2.5 mm was immersed in 4%, 6%, 8%, 10%, and 12% concentrations of noni fruit juice and distilled water in a control group for 31, 46, and 61 days. the transversal strength of acrylic resin plate was tested using an autograph with a crosshead speed of 1/10mm/second; the distance for the two supporting parts was 50 mm. the data was analyzed by using a one–way anova test. there was no significant difference in the transversal strength of the plate after being soaked for 31, 46, and 61 days in 4%, 6%, 8%, 10%, and 12% concentrations of the juice. this study showed that the soaking of acrylic resin plate for 31, 46, and 61 days in 4%, 6%, 8%, 10%, and 12% concentrations of noni fruit juice does not decrease the transversal strength. key words: transversal strength, acrylic resin plate, noni fruit juice correspondence: sri redjeki indiani, c/o: departemen material dan kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47, surabaya 60132, indonesia. introduction nowadays, acrylic resin is widely applied as a denture base. however, acrylic resin has disadvantages such as its liquid absorbing ability and micro porosity, allowing food debris to accumulate. microorganisms can easily colonize and grow, especially microorganisms which can contribute to plaque formation.1 saliva has not only the function of lubricating, but also protecting, oral tissue and as medium for microorganisms to survive and to develop.2 saliva can form a pellicle on the surface of acrylic resin plate resulting in adhesion of microorganisms such as candida albicans (c. albicans).3 plaque accumulation in dentures is mostly found not only on the fitting surface location in the maxilla or mandible, but also on the polished side of the sublingual.4 there is significant correlation between bad denture hygiene and inflammation.5 an increase in the presence of candida albicans is usually followed by increased toxin production which can penetrate into the mucous membranes and result in inflammation. therefore, the acrylic resin based denture user should always pay particular attention to denture hygiene. in elderly patients with poor attention to oral hygiene, use of a mild antiseptic mouth rinse is recommended to reduce pathogenic microorganisms in the oral cavity such as candida albicans, and to prevent denture stomatitis. gargling is a mechanical activity and has an anti-microbial effect.6 noni fruit (morinda citrifolia linn.) is a tropical plant used for thousands of years in the traditional treatment of various diseases. it contains saponin, flavonoid, atsiri oil, and alkaloids, which have been found to be anti bacterial and anti-fungal,7 and specifically effective against escherichia coli, stapilococcus aureus, and pseudomonas aeroginosa.8 it is currently reported that noni fruit juice is used not only as a drink but also in gargling therapy.8,9 as a mouth rinse it should be used two or three times a day or whenever needed.9 this previous study reported that a growth of c. albicans colony did not occur when acrylic resin plate was immersed in over 12.5% noni fruit juice and incubated with c. albicans for twenty four hours in a solid media.10 this is the basis for establishing the concentration used in this study. the chemical composition of noni fruit consists of flavonoid, which is a phenol substance,11 a substance known to degrade acrylic resin denture when in prolonged contact.12 five percent phenol would weaken acrylic polymer bond and it would penetrate and soften the resin after 2 hours of immersion. the degradation of acrylic resin would decrease the mechanical character such as transverse strength.13 transverse strength is the resistance of the denture strength to pressure and pulling force during normal functioning. the duration of acrylic resin immersion in noni fruit juice is based on the assumption that the patient would 85indiani: the transversal strength of acrylic resin plate after being immersed soaking in noni fruit gargle two to four times per day, with an ideal duration of denture use being approximately a 4-years period.10,14 therefore in this study the acrylic resin plate was immersed for 61 days. the purpose of study were to examine the transversal strength of the acrylic resin plate after being immersed in noni fruit juice for some period of times and to determine the concentration of juice and duration of immersion which cause no effect on transversal strength of acrylic resin plate. materials and methods materials used in this study were hard gypsum (moldano, bayer), heat cured acrylic resin (qc, england), cold mould seal (detrey, england), sandpaper no. 300 and 600 (sail brand, china), the juice of the noni fruit, and distilled water. the tools used were a metal master, model 65 × 10 × 2.5 mm, a porcelain bowl, cuvet, press and curing unit, an autograph ag-10 te (shimadzu), and a light microscope. acrylic resin plate preparation: gypsum and water were mixed in a ratio of 100 gr of gypsum in 24ml of water (based on factory procedure). the mix was manually stirred for 12 seconds and then placed in a vacuum mixer for 30 seconds. the mix was placed into a cuvet, which was placed on the vibrator, and the gypsum paste was set for 60 minutes. the surface of gypsum was polished with vaseline, and the upper cuvet was filled with gypsum paste on the vibrator. after the gypsum was hardened, the cuvet was opened and the metal master model removed, cleaned in hot water. the next step was the application of an appropriate separating medium onto the walls of the mold cavity, allowing it to dry for 20 minutes (based on factory procedure). acrylic filling: the material of acrylic resin was mixed with the powder and the liquid based on factory procedure (11.5 mg powder and 5 cc of liquid) and was placed into a porcelain bowl, then vibrated. after the paste reached the consistency of dough, it was placed into the mold, which was polished by separation material. the cuvet was closed and pressed by a hydraulic press. the cuvet was slowly opened and excess acrylic was cut. then, the cuvet was closed and pressed again until the pressure reached 22 kg/cm2. next, the cuvet was moved to a clamp. curing process: the cuvet containing the acrylic was placed into the curing unit. the process was carried out at 100° c for 30 minutes (based on factory procedure). after the curing process was completed, the cuvet was cooled and then the specimen was removed from the cuvet. to smooth out the tested plate of acrylic resin it was held under running water and sanded using 300–600 sandpaper until the size reached 65 × 10 × 2.5 mm. finally, it was placed into a closed tube to avoid drying. the samples were classified into 6 groups, each group consisting of 16 samples. the fruit of the noni tree (morinda citrifolia linn.), which can be found in the sutorejo area of east java, indonesia should be ripe and stringent in odor, a transparent yellowish white in color, and with a soft, juicy texture, and with a diameter of not less than 7 cm. it must be washed and the outer part peeled, the juice squeezed and classified into 5 groups consisting of concentrations of 4%, 6%, 8%, 10%, 12% in amounts of 20 ml each. the acrylic resin plate was immersed in 50 ml of noni fruit juice in a transparent bottle and loosely closed for 31, 46, and 61 days in every concentration group. control groups were immersed in distilled water in the same duration. during the immersing process, the plate was positioned upright allowing the whole surface to be immersed. the immersion solution was changed everyday. after the immersion process was completed, the acrylic resin plate was washed in distilled water and dried. finally, it was tested for transverse strength. the test of transverse strength was done using an autograph ag-10 te with a cross head speed of 1/10 mm/ second. the distance between supports was 50 mm.15,16 the result was obtained using the equation: s = 2 bd2 3 ip kg/cm2 note: s : transversal strength (kg/cms : transversal strength (kg/cm2) l : length/distance supporting (cm) b : width of acrylic resin plate (cm) d : thickness of acrylic resin plate (cm) p : load (kg) the data was analyzed using a one-way anova test with significance rate of a 0.05. result the mean and standard deviation of transversal strength of acrylic resin plate soaked in 4%, 6%, 8%, 10%, and 12% concentrations of noni fruit juice and water for 31 and 46 and 61 days is shown in table 1. a kolmogorof smirnov test was carried out on the data to determine the normality of distribution. the result of every tested group showed p > 0.05, therefore all the data had normal distribution. a levene test was done to determine the data homogenity and the significant rate was 0.214 (p > 0.05) for the duration of immersion, meaning the data was homogenous. therefore, a one-way anova test was done and showed p > 0.05 (p = 1.000) so a significant difference was not found in the transverse strength of acrylic resin plate after being submersed in noni fruit juice between the control group (distilled water) and 4%, 6%, 8%, 10%, 12% concentrations of juice. a concentration group of p > 0.05 (p = 0.965) was obtained from a one-way anova test on transverse strength due to the duration of soaking; 86 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 84-87 no significant difference was found in transversal strength of acrylic resin plate immersed in the noni fruit juice for 31, 46, and 61 days. decrease of transversal strength of acrylic resin plate was not found after being immersed in noni fruit juice in 4%, 6%, 8%, 10%, 12% concentrations and distilled water or after being soaked for 31, 46, and 61 days. the result was supported by a topography test on the surface of acrylic resin plate using a light microscope with 500× magnification (figure 1 and 2). discussion the previous study proved that no growth of c.albicans colony was present in noni fruit juice with a concentration above 12.5% when incubated with c.albicans for 24 hours. the mouth rinse is an oral antiseptic to prevent microorganism growth.17 in order not to irriate the mucous membranes, the concentration of mouth rinse should be lower than 12,5%, classified into 5 groups according to the arithmetical progression of 4%, 6%, 8%, 10%, 12% with interval 2. based on ada specification no. 12, that solubility of dental plastic in water is not higher than 0.04 mg/cm2, acrylic resin solubility in water 0,02 mg/cm,1 the immersion process of acrylic resin plate in water was used as a control group. the immersion duration of acrylic resin in noni fruit juice is based on the assumption that a patient would gargle 2–4 times per day. it is estimated that each time gargling is done; noni fruit juice residue would be left in the oral cavity ± 15 minutes, so in one day the acrylic resin would be immersed for 60 minutes. the life of a denture is ideally 4 years, 14 which is equal to (1 × 365 × 4) hour = 1460 hours, an estimated 61 days. if the patient gargles three times per day, the soaking would be 46 days, and gargling twice per day the immersion would be 31 days if the control group were immersed in distilled water for the same duration. the mean of transversal strength of acrylic resin plate soaked in noni fruit juice (table 1) shows the immersion with higher concentration and longer duration will not decrease transverse strength. a one–way anova test, shows no significant difference in the transverse strength of the plate after being immersed in noni fruit juice in 4%, 6%, 8%, 10%, 12% concentrations and also in distilled water for 31 days, 46 days and 61 days, showing that decrease in transversal strength does not occur. acrylic resin is a long chain polymer of ester substance easily hydrolyzed and accelerated by acid and water. the acidity of the flavonoid content of noni fruit is not as strong as phenol.10 the data obtained from the photochemical laboratory of airlangga university pharmacy shows that the total flavonoid content of dry noni fruit extract is 0.0863%, for noni fruit juice is much lower when compared to dry noni fruit extract. consequently, if it is exposed to acrylic resin table 1. the mean and standard deviation of transversal strength of acrylic resin plate after to immersion in noni fruit juice and distilled water (n/m2) concentration 31 days of immersion 46 days of immersion 61 days of immersion x ± sd x ± sd x ± sd distilled water 4% juice 6% juice 8% juice 10% juice 12% juice 144.68 ± 9.34 143.78 ± 8.57 143.93 ± 11.03 144.38 ± 10.54 144.45 ± 8.01 144.16 ± 7.41 144.50 ± 9.61 144.44 ± 6.99 144.56 ± 9.91 144.82 ± 8.86 144.03 ± 9.36 144.19 ± 9.09 144.50 ± 12.77 144.73 ± 7.78 144.88 ± 6.57 144.11 ± 8.22 144.38 ± 9.80 144.73 ± 8.04 figure 1. topography on the surface of acrylic resin plate after immersion in distilled water for 61 days. degradation was not seen on the surface of the acrylic resin plate (500× magnification). figure 2. topography on the surface of acrylic resin plate after being soaked in 12% concentration of noni fruit juice for 61 days, degradation was not seen on the surface of the acrylic resin plate (500× magnification). 87indiani: the transversal strength of acrylic resin plate after being immersed soaking in noni fruit plate in relatively long duration, the hydrolysis reaction of polymethil methacrylate does not occur, so polymer chain bonding is not disturbed. therefore, degradation of acrylic resin does not occur and the transverse strength does not decrease due to the relatively good strength of acrylic resin against weak acid.1,19 the level of destruction due to acrylic immersion in disinfectant material depends on the immersion duration, the higher concentration of the disinfectant the worse the degradation will be.12 transverse strength is one physical parameter to examine the dental base strength in tolerating force during mastication. the test shows the specimen strength to sustain high force and give deflection the higher the fragility of material the lower the deflection and the risk of damaging the acrylic plate.13 the transverse strength of heat cured acrylic resin for dentures should be less than 50 n/mm. 2,18 the mean of transverse strength of acrylic resin plate immersed in distilled water for 61 days is 144.50 n/mm, 2 and the mean of transverse strength of acrylic resin plate immersed in the highest concentration of 12% noni fruit juice for 61 days is 144,73 n/mm2. the result is still above the standard which has been recommended.18 it can be seen with topographic imaging that the surface of acrylic resin using a light microscope with 500x magnification shows no degradation on the surface of the acrylic resin (figures1 and 2). the study shows that the immersion duration of acrylic resin plate for 31, 46, 61 days in 4%, 6%, 8%, 10%, 12% concentrations of noni fruit juice does not decrease the transversal strength. further study is necessarily to determine the total flavonoid content in noni fruit juice. references 1. craig rg, peyton fa. restorative dental material. 5th ed. st louis: the mosby co; 2002. p. 388–485. 2. sumarijah s. respon imun humoral terhadap streptococcus sanguis pada recurrent apthous stomatitis (ras). dissertation. surabaya: pasca sarjana unair; 1985. p. 9–20. 3. edgerton m, levine m. characterization of acquired denture pellicle from healthy and stomatitis patients. j prosthet dent 1992; 68:683–91. 4. siong bk, lim m. denture plaque distribution and effectiveness of a perborate– containing denture cleanser. quint int 1996; 27:341–5. 5. kulak y, arikan. an etiology of denture stomatitis. journal of marmara university dental faculty 1993; 4:307–14. 6. sweet jb, macynski aa. effect of antimicrobial mouth rinse on incidence of localized alveolitis and infection following mandibular third molar oral surgery. oral med oral phathol 1985; 59:24–6. 7. syamsuhidayat ss, hutapea jr. inventarisasi tanaman obat indonesia i. jakarta: balai penerbitan dan qs pengembangan kesehatan; 1991. p. 390–1. 8. goretti mw. sehat dengan mengkudu. 2th ed. jakarta indonesia: msf group; 2000. p. 3–17. 9. brown in. 53 ways to use noni fruit juice for your better health. a handbook of oral, topical and internal application and procedures. utah: pride publishing; 1998. p. 177–82. 10. indiani sr. efek perasan buah mengkudu sebagai perendam resin akrilik terhadap keberadaan candida albicans. majalah kedokteran gigi (dental journal) 2003; edisi khusus temu ilmiah nasional iii: 13–6. 11. pramono s. diktat kuliah tinjauan umum senyawa fenol nabati. yogyakarta: fakultas pascasarjana, universitas gajah mada; 1988. p. 34–9. 12. shen c, nikzad s, javid. the effect of glutaraldehyde base disinfectants of denture base resins. j prosthet dent 1989; 61(5): 583–8. 13. attin t, vattaschiki m, hellwig e. properties of resin modified glass–ionomer restorative material and two polyacid modified resin composite material. quint int 1996; 27:203–9. 14. american dental association (ada). guide to dental material and devices. 7th ed. chicago. 1974. p. 70–106. 15. robinson jg, mc cabe jf, storer r. the whitening of acrylic resin dentures: the role of denture cleansers. j br dent 1985; 19:247–50. 16. reitz pv, sanders jl, levin b. the curing of acrylic resins by microwave energy: physical properties. dent res quint int 1985; 8:547–51. 17. jawets e, melnick jl, adelberg ea. mikrobiologi untuk profesi kesehatan (review of medical microbiology) tonang h, editor. edisi 16. jakarta: egc; 1991. p. 382. 18. asad t, watkinson ac, huggett nr. the effect disinfection procedures on flexural property of denture base acrylic resins. j prosthet dent 1992; 68:191–5. 19. billmeyer fw. textbook of polymer science. 3rd ed. new york, chichester brisbane, toronto, singapore: a wiley-interscience publication. john wiley and sons; 1984. p. 11–6. 409–11. 154 applicability of moyers and tanaka-johnston analyses for the arab population of pekalongan, indonesia fani tuti handayani and rizki amalia hidayah school of dentistry, faculty of medicine, universitas jenderal soedirman, purwokerto – indonesia abstract background: the moyers and tanaka-johnston analyses, both of which were developed through research into the nordic-caucasoid population, constitute the most widely used non-radiographic mixed dentition analyses. application of these prediction methods to other populations has been extensively investigated, although their suitability to the arab population resident in indonesia has rarely been studied. purpose: this study aimed to investigate the reliability of moyers and tanaka-johnston analyses of the arab population of pekalongan, a city in central java, indonesia. methods: this research constituted an observational study incorporating a crosssectional design of 33 subjects who willingly signed an informed consent form. their actual tooth size was determined in accordance with the inclusion criteria and then compared with the predictive tooth size values of the moyers and tanaka-johnston analyses. results: comparison of mean values indicated that the predictive values of moyers 75% (ym) and tanaka-johnston (yt) were higher than the actual value (y). the reliability test result confirmed a value of k = 0.026 for the y-ym ratio; and k = 0.025 for yt-y ratio. the calculation results for the mandible indicated a k-value of 0.000 for both types of comparisons between ym-y and yt-y. the moyers 75% prediction for maxilla had a higher reliability value than that of tanaka-johnston. the shapiro-wilk normality test applied to y, ym, and yt in the maxilla and mandible indicated that data was normally distributed (sig > 0.05). a levene’s test of homogeneity was conducted and produced homogeneous data (sig > 0.05) with values of 0.333 for y, 0.516 for ym, and 1.000 for yt. an anova test showed y-ym and y-yt to have insignificant differences (p<0.05). conclusion: the moyers 75% and tanaka-johnston analyses were reliable because the values produced differed slightly from the actual values. this study concluded that moyers and tanaka-johnston analyses remain valid means of calculating mixed dentitions. keywords: arab population; mixed dentition analysis; moyers method; tanaka-johnston method correspondence: fani tutihandayani, school of dentistry, faculty of medicine, universitas jenderal soedirman, jl. dr. soeparno karangwangkal, purwokerto, jawa tengah 53123, indonesia. e-mail: fanitutihandayani@gmail.com introduction the mixed dentition period involves transition from primary to permanent teeth characterized by eruption of the first mandibular incisors or the first molars.1 this period has been widely employed by researchers to predict the size of future permanent teeth and the availability of the jaw arch space.1 early detection of conformity of the jaw arch space and tooth size can be a reference for orthodontic intervention and correction in the prevention and treatment of malocclusion.1,2 mixed dentition analysis methods can be categorized into three groups, namely; regression equations, radiographic examination, and a combination of both these methods.2 the most widely used mixed dentition analysis method, the moyers and tanaka-johnston analysis, is one that utilises the mesiodistal size regression equation of erupted teeth.3,4 this analytical tool was developed from measurements of the teeth of north american children (nordic-caucasoid).4 the applying of moyers prediction tables and tanakajohnston equations to other populations around the world can be adjusted by considering the results of tooth size dental journal (majalah kedokteran gigi) 2019 september; 52(3): 154–158 research report dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p154–158 mailto:fanitutihandayani@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p154-158 155handayani, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 154–158 analysis studies in these populations.3,5 the accuracy of the predicted size of teeth that will occupy the jaw arch is a determining factor in the early detection of malocclusion and formulation of an orthodontic treatment plan.4,5 the differing tooth size variations between one population and another causes the reliability of these two methods to be evaluated, especially in populations with typical jaw characteristics and tooth size.6,7 one population with distinctive characteristics in the jaw and dentition is that of the arabs8,9 who have settled in various parts of indonesia, especially on java, constituting the second largest immigrant population within the country. the largest arab population in central java province is resident in pekalongan, specifically a village consisting of three districts, namely; sugihwaras, klego, and poncol.9–11 analyses of the mixed dentition within this group of arab ethnicity have rarely been conducted. therefore, this study aimed to investigate the reliability of the moyers and tanaka-johnston model in conducting such an analysis. materials and methods this study was jointly approved by the health research ethics commitee of dr. moewardi general hospital and the school of medicine, sebelas maret university of surakarta (no:420/v/hrec/2016). research consisting of an observational study with cross-sectional design was conducted between july and december 2016. the inclusion criteria applied in the course of subject selection were as follows: arab residents of pekalongan aged between 13 and 14 years old. all permanent teeth (with the exception of the second and third molars) should be present in the oral cavity and fully erupted in both the maxillary and the mandibular arches. all teeth presented normal morphology without any extensive caries or restorations including the mesiodistal edge or interproximal fractures. children with congenital craniofacial anomalies or previous histories of orthodontic treatment were excluded from the study. the subjects were students of the ma’had islamiyah and al irsyad junior high schools. the selection of these two educational institutions was based on a preliminary study confirming them to be ones largely attended by 13-14 year old children of arab descent. the total subject population comprised 33 children, consisting of 12 males and 21 females. an impression was made of the maxilla and mandible of each subject prior in order to manufacture study models which were then analysed at the basic dental science laboratory of the school of dentistry, faculty of medicine, jenderal soedirman university. standard mixed dentition analysis procedures were implemented in accordance with the methods proposed by rao.1 first, measurement of the study model was completed by drying the positive moulds of the maxilla and mandible and cleaning the remnants (nodules) of casts to render them suitable for this purpose. the study models were subsequently coded on the basis of the data of the subject to which they related and subsequently measured, with each result being recorded in a log according to the data code of each individual. the first measurement was of the mesiodistal width of the four permanent mandibular incisors, followed by that of the mesiodistal width of canines and the first premolars and second premolars of the left and right maxilla and mandibles. measurement of the mesiodistal width of the teeth was performed by placing the two ends of a sliding calliper parallel to the dental axis. the measurement taken was checked according to a method suggested by lundstorm, whereby a single investigator takes all the measurements after carefully marking the maximum mesiodistal width on the relevant teeth and then re-measures a number of randomly selected casts.12 the results were recorded as preliminary data until all the study models had been measured. the results of this study include x and y values. x represents the measurement value of the four mandibular incisors that serve as predictors for the maxillary and c-p1p2 mandibular values within 75% moyers and tanakajohnston predictions. y constitutes the actual measurement of the maxillary and c-p1-p2 mandibular. in this study, the predictive value of the c-p1-p2 for the 75% moyers is referred to as ym, while the tanaka-johnston predictive value is known as yt. the first calculation involved predicted the number of canines, first premolar and second premolar dimensions, using the moyers table at 75% percentile using a predictor of the mesiodistal size of the four mandibular incisors. the second calculation was based on the actual count of the size of the canines, first premolar, and second premolar of the arab population in pekalongan which were determined as predictors using the moyers table at 75% percentile, to quantify the mesiodistal size of the four mandibular incisors. the third calculation was completed by measuring the number of canines, first premolars, and second premolars by means of the tanaka-johnston equation. the data obtained was analysed using the statistical product and service solutions (ibm spss software) version 20.0. the correlation between x and y was assessed using pearson’s correlation analysis. data is expressed as mean ± standard deviation (sd). a kappa (k) reliability test and a shapiro-wilk normality test (p>0.05) were both performed, while homogenity was assessed by means of a levene’s test (p>0.05). a comparison of the y-ym and y-yt analyzes was conducted using an anova test (p<0.05). results in this study, the mesiodistal size of four mandibular incisors (x) and the mesiodistal size of the c-p1-p2 teeth as measured (y) resulting in respective values of 0.656 and 0.613 for the maxilla and mandible. this value indicated a linear relationship between the size of four mandibular dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p154–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p154-158 156 handayani, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 154–158 incisors with that of the c-p1-p2 teeth. table 1 shows that the comparison between the average 75% moyers and tanaka-johnston predictive value was higher than the actual value. the mean and standard deviations y, ym, and yt are presented in table 1, while the comparison of y, ym and yt value distribution in the maxilla and mandible are contained in figure 1 and figure 2. a kappa (k) reliability test comparing the actual value (y) and 75% moyers predictive value or tanaka-johnston predictive value was performed. the calculation results of the maxilla showed that the comparison between the 75% moyers predictive value with the actual value of c-p1-p2 in this study was k = 0.026, while the comparison between the tanaka-johnston predictive value the actual value c-p1p2 was k = 0.025. the calculation results for the mandible showed a k-value = 0.000 for a comparison of both 75% moyers and tanaka-johnston predictive values with the actual value. these results indicated that the reliability of 75% moyers and tanaka-johnston prediction was sufficient for measurements of the maxilla but not for those relating to the mandible. the results of the shapiro-wilk normality test between y, ym, and yt relating to the maxilla and mandible presented in table 2 confirmed the data as normally table 1. mean and standard deviations of y, ym and yt jaw mean standard deviation y maxilla mandible 21.8024 20.6215 0.97175 0.88756 ym maxilla mandible 22.4641 21.7853 0.76534 0.84212 yt maxilla mandible 22.1812 21.6812 0.70415 0.70415 table 2. shapiro-wilk normality test jaw sig. y maxilla mandible 0.117 0.056 ym maxilla mandible 0.255 0.177 yt maxilla mandible 0.613 0.613 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 y y moyers y tanaka subjects y v al ue (m m ) figure 1. comparison of y, ym and yt value distribution in the maxilla. 18.00 19.00 20.00 21.00 22.00 23.00 24.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 y y moyers y tanaka y v al ue (m m ) subjects figure 2. comparison of y, ym and yt value distribution in the mandible. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p154–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p154-158 157handayani, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 154–158 distributed (sig>0.05). homogeneity was assessed using a levene’s test which produced data (sig > 0.05) with values of 0.333 for y, 0.516 for ym, and 1.000 for yt. the anova test results produced a p-value = 0.069 for y-ym and p = 0.051 for y-yt (p<0.05) which indicated insignificant differences in both comparisons. this study indicated a constant value of (a) = 11.684 for the maxilla and 11.9834 for the mandible. this value was higher than the predictive constant of 75% moyers and tanaka-johnston, while the regression coefficient of (b) was lower than the 75% moyers and tanaka-johnston prediction, which stood at 0.4525 for the maxilla and 0.3864 for the mandible.the following constitute the equations employed in this study for the purposes of prediction: maxilla with y = 11.6854 + 0.4525x and mandible with y = 11.9834 + 0.3864x. discussion the development of dento-craniofacial structures in arab population is unique compared to other races,8 with a tendency to protrusion of the incisors as they age. if left untreated at an early stage, they can cause malocclusion. it was revealed that arabs have a higher convex profile with reduced chin prominence, steeper mandibular plane angle, and greater bimaxillary protrusion.9 even within those saudi ethnic groups with well-balanced faces, there were some fundamental variations in the craniofacial structure of saudi arabs.13 malocclusion should be treated early during the mixed dentition period, by detecting a discrepancy in tooth size that occurs during that period.1 mixed dentition analysis methods can be categorised into three groups, namely; thoseutilising regression equations; those involving radiographic examination; and a combination of both methods.2 the most widely employed mixed dentition analysis method is one that utilises the mesiodistal size regression equation of erupted teeth, namely the moyers and tanaka-johnston analysis.3,4 in this study, the actual measurements of the c-p1p2 size of both the maxilla and mandible in the arab population proved to be lower than the predicted 75% moyers and tanaka-johnston values. the same result was also produced by previous studies conducted among the belgaum population in karnataka, india14 and the population of bangalore.15 similar results were produced by studies conducted amongst school children in mumbai.16 prediction of c-p1-p2 size in orthodontic treatment influences determination of the treatment plan that will be developed for the patient. consideration for skeletal expansion during growth and development is one of the treatment plans based on an analysis of space requirements which suggests that the c-p1-p2 size exceeds the available space. overestimation of the size of c-p1-p2 can result in excess space in the future leading to malocclusion. a comparison of the distribution of y, ym, and yt values for each jaw is contained in the following figures. figure 1 featuress a comparison of the distribution of y, ym, and yt maxillary values, while figure 2 shows a comparison for the mandible. the results of this study indicated the reliability of 75% moyers and tanaka-johnston prediction was sufficient for measurement of the maxilla, but inadequate for the mandible. 75% moyers prediction for maxilla had a higher reliability value than tanaka-johnston. these results were consistent with research conducted among the population of south india.17 however, different results were produced studies conducted among school children from medellin in colombia which found that the 75% moyers data was more accurate in predicting the mandible measurement value, whereas the tanaka-johnston predictive value was more accurate in predicting the maxilla measurement value.18 research conducted by thimmegowda et al. (2017), suggested that the original tanaka-johnston method of analysis had overestimated the local bangalore population and that, consequently, a new regression equation should be formulated. new regression equations and prediction tables were derived for males and females separately, which should be more conveniently employed chairside by the clinician.15 the results of that study were consistent with those of the research reported here, although the difference between the actual and predicted values of the tanakajohnston analysis were not statistically significant. even though the same results were found in the study conducted by asiry et al. (2014),19 that author stated that further work on a large representative sample from various arab populations around the world is required in order to draw a firm conclusion. differences in location can cause racial assimilation potentially leading to differences in physical features of the same race, due to physiological adaptation processes. the study conducted by galvão et al. (2013),6 stated that the moyers mixed dentition analysis should be employed judiciously because its accuracy regarding the probability level remained questionable. the difference between the actual and predicted values of the moyers 75% analysis in this study was not statistically significant. however, the reliability variations of moyers and tanaka-johnston prediction require numerous researchers to develop regression equations similar to those formulated for this study according to the populations studied.7 in this study, comparative tests could not be performed to assess the tendency of one analysis to be superior in predicting the c-p1-p2 value. however, the insignificant differences resulting from the interpretation of data in this study indicated that the moyers and tanaka-johnston analysis remains deployable in the calculation of mixed dentition analysis. in the study conducted by connor et al.,20 no significant differences between the sn-fh measurement of arabs and north american caucasians were identified. meiners asserted that the caucasian race encompassed all of the ancient and most of the modern native populations of europe, in addition to the aboriginal inhabitants of west dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p154–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p154-158 158 handayani, et al./dent. j. (majalah kedokteran gigi) 2019 september; 52(3): 154–158 asia (including the phoenicians, hebrews and arabs).21 rawlani et al. (2017),22 also stated that arabs have a tooth morphology which is classified as caucasoid. it can be concluded that a moyers 75% and tanaka-johnston analysis was sufficently reliable for mixed dentition analysis of the arab population of pekalongan. new regression equations and predictions based on the population studied remain prerequisites to the obtaining of more acurate predictive values. acknowledgement this research was funded by the lecturer research grant kindly provided by the institute of research and community service at jenderal soedirman university. references 1. rao a. principles and practice of pedodontics. 3rd ed. new delhi: jaypee brothers medical publishers; 2012. p. 97, 141–2, 145–6. 2. antonieta pm, marcelo c, juan r, gabriel b, claudia f. applicability of the moyers prediction tables at 75 % on mapuche-huilliche patients , chile. odontoestomatologia. 2014; 16(24): 13–8. 3. kaur a, singh r, mittal s, sharma s, bector a, awasthi s. evaluation and applicability of moyers mixed dentition arch analysis in himachal population. dent j adv stud. 2014; 2(2): 96–104. 4. thimmegowda u, sarvesh sg, shashikumar hc, kanchiswamy ln, shivananda dh, prabhakar ac. validity of moyers mixed dentition analysis and a new proposed regression equation as a predictor of width of unerupted canine and premolars in children. j clin diagnostic res. 2015; 9(8): zc01–6. 5. ajayi e. regression equations and probability tables for mixed dentition analysis in a nigerian population. j dent heal oral disord ther. 2014; 1(5). 6. galvão m de ab, dominguez gc, tormin st, akamine a, tortamano a, fantini sm de. applicability of moyers analysis in mixed dentition: a systematic review. dental press j orthod. 2013; 18(6): 100–5. 7. hashim h, al-hussain h-n, hashim ma. prediction of the size of unerupted permanent canines and premolars in a qatari sample. int j orthod rehabil. 2019; 10(1): 10. 8. hassan ah. cephalometric norms for the saudi children living in the western region of saudi arabia: a research report. head face med. 2005; 1: 5. 9. albarakati sf, baidas lf. orthognathic surgical norms for a sample of saudi adults: hard tissue measurements. saudi dent j. 2010; 22(3): 133–9. 10. van den berg lwc. hadramaut dan koloni arab di nusantara. 3rd ed. jakarta: inis; 1989. p. 95–100. 11. fachruddin c. orang arab di kota medan. j antropol sos budaya etnovisi. 2005; 1(3): 130–5. 12. hixon eh, oldfather re. estimation of the sizes of unerupted cuspid and bicuspid teeth. angle orthod. 1958; 28(4): 236–40. 13. al-jasser nm. cephalometric evaluation of craniofacial variation in normal saudi population according to steiner analysis. saudi med j. 2000; 21(8): 746–50. 14. vijayashree uh, naik vr. reliability of moyers and tanaka johnston mixed dentition analysis in school children of belgaum. indian j orthod dentofac res. 2016; 2(4): 166–71. 15. thimmegowda u, divyashree, niwlikar kb, khare v, prabhaka ac. applicability of tanaka jhonston method and prediction of mesiodistal width of canines and premolars in children. j clin diagnostic res. 2017; 11(6): zc16–9. 16. hambire c, sujan s. evaluation of validity of tanaka-johnston analysis in mumbai school children. contemp clin dent. 2015; 6(3): 337–40. 17. kamatham r, vanjari k, nuvvula s. applicability of moyers′ and tanaka-johnston′s mixed dentition analyses for predicting canine and premolar widths in south indian population a cross sectional study. j orofac sci. 2017; 9(1): 52. 18. botero pm, cuesta dp, agudelo s, hincapie c, ramã\-rez c. assessment of moyers and tanaka-johnston mixed dentition analyses for the prediction of mesiodistal diameters of unerupted canines and premolars. rev fac odontol univ antioquia. 2014; 25: 359–71. 19. asiry ma, albarakati sf, al-maflehi ns, sunqurah aa, almohrij mi. is tanaka-johnston mixed dentition analysis an applicable method for a saudi population? saudi med j. 2014; 35(9): 988–92. 20. connor am, moshiri f. orthognathic surgery norms for american black patients. am j orthod. 1985; 87(2): 119–34. 21. dana ca, ripley g. the new american cyclopaedia: a popular dictionary of general knowledge, volume 4. charleston: nabu press; 2010. p. 588. 22. rawlani s, rawlani s, bhowate r, chandak r, khubchandani m. racial characteristics of human teeth. int j forensic odontol. 2017; 2(1): 38. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i3.p154–158 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i3.p154-158 142 vol. 41. no. 3 july–september 2008 potential role of odontoblasts in the innate immune response of the dental pulp tetiana haniastuti oral biology department faculty of dentistry gadjah mada university yogyakarta indonesia abstract background: odontoblasts are the cells lining of tooth’s hard structure at the dentin-pulp border, which become the first cells encountered oral microorganisms entering dentin. however, they do not only form a physical barrier by producing dentin, but also provide an innate immune barrier for the tooth. purpose: the aim of this review was to discuss the potential role of odontoblasts in the innate immune response of the dental pulp. reviews: recent studies have proven that odontoblasts express toll-like receptors, and capable of producing chemokines (i.e. il-8, ccl2, cxcl2, and cxcl10), and cytokines (il-1β and tnf-α) following lipopolysacharide exposure. thereby odontoblasts are actively participating in the recruitment of immune cells in response to caries–derived bacterial products. furthermore, odontoblasts also produce antimicrobial peptides (hbd-1, hbd-2, and hbd-3), and transform growth factor β that induce antimicrobial and anti-inflammatory activities. conclusion: the presence of those innate immune molecules indicates that the nonspecific, natural, and rapidly acting defense may also be an important function of odontoblasts. key words: odontoblasts, dental pulp, innate immunity correspondence: tetiana haniastuti, c/o: bagian biologi oral, fakultas kedokteran gigi universitas gadjah mada. jln. denta no. 1, sekip utara yogyakarta 55281, indonesia. email: haniastuti@yahoo.com introduction cariogenic bacteria are the major cause of pulpal inflammation and infection. gram-positive bacteria (streptococcus and lactobacillus spp.) are common oral micro flora detected in shallow dental caries or the outer dentinal tubules of deep dental caries; while gram-negative bacteria (fusobacterium, phorphyromonas and prevotella spp.) are found in deep dental caries and infected root canals.1 enamel protects the underlying dentin and the connective tissue situated in the dental pulp. enamel demineralization caused by oral microorganism makes the enamel barrier is disrupted. oral bacteria and their substances might penetrate into the dentinal tubule and entering the pulp. the dental pulp injury caused by carious lesions is unusual, in that toxins reach the tissue well ahead of the bacteria that release them, eliciting the development of inflammatory and immune reactions in the dental pulp.2 several cell types contributing to innate and adaptive immunity are present in the tooth pulp such as lymphocytes, macrophages and dendritic cells.3 odontoblasts, the cells lining of tooth’s hard structure at the dentin-pulp border, are the first pulpal cells encountered oral dental pathogens from entering dentin. due to odontoblasts location is in the periphery of the dental pulp and their cellular extension into dentin, it is likely that odontoblasts also play an important role in innate immune responses of the dental pulp. this hypothesis is based on a paradigm which compares the dentin-pulp complex with the epidermis. although mesenchymal in origin, the odontoblasts are noted to be epithelial-like morphologically and functionally. the columnar, palisading presentation of the odontoblast layer in the pulp is reminiscent of columnar epithelium, as is the secretory capacity of the cell. the observation that dendritic cells reside within the odontoblast layer of the pulp is analogous with the epidermis, in which langerhans cells are known to reside in close association with keratinocytes.4 review article 143haniastuti: potential role of odontoblasts in the innate immune response it has long been unclear how the immune systems of the dental pulp works, particularly the role of odontoblasts the first pulpal cells encounter dental pathogens. this review discusses recent progress in understanding the potential role of odontoblasts in the innate immune response of the dental pulp. innate immune response the immune system is composed by two major subdivisions which are the innate or nonspecific immune system and the adaptive or specific immune system. the innate immune system is a primary defense mechanism against invading organisms, while the adaptive immune system acts as a second line of defense. both aspects of the immune system have humoral and cellular components by which they carry out their protective functions. in addition, there is interplay between these two systems, i.e. cells or components of the innate immune system influence the adaptive immune system and vice versa.5,6 the innate immune system is activated upon the initial invasion of microbes and does not require a period of time for induction. the basic protective strategy of an innate immune system is for the organism to constitutively produce generic receptors that recognize conserved patterns on different classes of pathogens to trigger an inflammatory response that limit pathogen invasion. these receptors include toll-like receptors (tlrs), lipopolysacharide (lps)-binding proteins, peptidoglycan recognition proteins, nucleotide-binding oligomerization domains, cd14, scavenger receptors, and c-type lectin which enable mammalian cells to differentially recognize highly conserved microbial structures and consequently mediate innate host responses.7 tlrs are members of an evolutionary conserved interleukin-1 (il-1) superfamily of transmembrane receptors that recognize pathogen-associated molecular patterns. tlrs constitute a major class of microbial recognition receptors. their activation regulates the production of antimicrobial peptides, cytokines, and chemokines as well as their receptors, thus, consequently controls leukocyte trafficking, t-cell function, and also recruitment and maturation of dendritic cells; thereby, providing a bridge between innate and adaptive immunity.5 recently 12 members of the tlr family have been identified in mammals.7 tlr2 alone or tlr2 heterodimerizing with either tlr1 or tlr6 is crucial for the recognition of gram-positive bacterial cell wall components, including lipoteichoic acid, lipopeptides, and peptidoglycan. tlr3 recognizes viral double-stranded (ds) rna, while tlr4 plays a major role in detecting lps, a characteristic component of the cell wall of gram-negative bacteria.5,7 the components of the innate immune response of the dental pulp to caries include outward flow of dentinal fluid and the deposition of intratubular immunoglobulins, odontoblasts, neuropeptides, innate immune cells, cytokines and chemokines.4 the onset of innate immune response in the dentin-pulp complex is difficult to specify because carious lesions usually progress slowly into the dental pulp. however, before actual carious exposure, the dental pulp beneath shallow caries is capable of evoking innate immune responses to slow down the bacterial invasion.8 the extremely rich innervation of the dental pulp can influence the immune response by either directly stimulating immunocompetent cells via neuropeptides or by increasing vascular permeability, which facilitates the delivery and accumulation of immune cells and macromolecules to the inflamed tissue.3 odontoblast cells odontoblasts are the cells responsible for the formation of dentin, the collagen-based mineralized tissue that forms the bulk of teeth. they are derived from ectomesenchymal cells, exhibit a tall columnar shape, and establish a continuous single layer with a clear epithelioid appearance.9 the odontoblasts are unique cells. whilst the cell body of other mineral forming cells is close to the cell process and stays within the calcified matrix, the cell processes of odontoblasts extend a considerable distance into the dentin matrix. in some cases, they may even extend all the way to the outer boundary of the dentin, while the cell body remains in the pulp at the inner boundary. in other words, the cell process extends some distance from its nutritional and controlling centre. the odontoblastic process is extremely fine and resides within dentinal tubules, which is like a capillary tube with a diameter that is much smaller than that of an erythrocyte.10 after dentinogenesis, odontoblasts are aligned along the periphery of the dental pulp, thus playing a role in the maintenance of tooth integrity owing to their capacity of depositing new layers of dentin throughout life. in addition, newly differentiated odontoblast-like cells may also form a layer of reparative dentin after some tissue injuries. odontoblasts synthesize and secrete all the matrix constituents and therefore, they exhibit well-developed synthesis organelles. the odontoblast layer is separated from the mineralized dentin by a 10-40 µm-thick layer of unmineralized matrix, the predentin, which is similar to the osteoid that separates the osteoblasts from the bone’s mineralized matrix.9 in addition to a role in forming dentin, odontoblasts may be involved in sensory transduction. the presence of tight, adhering and gap junctions may imply that these cells communicate with each other; and if one is affected, many others are also affected. gap junction exists between and among odontoblasts and nerve fibres, and they provide a pathway of low electrical resistance between and among the odontoblasts and nerve fibres. the hydrodynamic effects of fluid displacement within the dentinal tubules or the odontoblasts may activate mechanoreceptors of sensory nerve axons.11 odontoblasts are also implicated in the regulation of pulp blood flow and in the development of pulp inflammation. the enzyme nadph-diaphorase, involved 144 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 142-146 in the production of nitric oxide, is a potent vasodilator present in the odontoblasts. their capacity to synthesize the inflammatory mediator pgi2 has been demonstrated and this may excite nerves in the vicinity resulting in a brief hyperalgesia.12 the earliest signs of pulp reaction to insults (such as dental caries) are morphological changes and an overall reduction in the number and size of odontoblast cell bodies. the disruption in the underlying odontoblast cell layer occurs even before the appearance of inflammatory changes in the pulp. the nuclei of the cells may be aspirated into the dentinal tubules due to the outward flow of tubular fluid, or the cells may be irreversibly damaged which results in the release of tissue injury factors affecting neighbouring odontoblasts and underlying connective tissue. cells may undergo vacuolization, ballooning degeneration of mitochondria, and reduction in the number and size of the endoplasmic reticulum.13,14 discussion the initial step of an innate immune response is the detection of pathogens through specialized pattern recognition receptors present in the cell membrane of immune and no immune cells, among which tlrs are key participants.5,15 previous studies have proven that odontoblasts constitutively express tlr1, 2, 3, 4, 5, 6, and 9.16 this large range of tlrs expressed by odontoblasts appears comparable to what has been reported for cultured epithelial cells, including keratinocytes, intestinal epithelial cells, and gingival epithelial cells. thus, odontoblasts might be involved in the recognition of bacterial products such as triacetylated lipoprotein, lipoteichoic acid (lta), diacetylated lipoproteins, peptidoglycans, lps, flagellin, and unmethylated cpg motif-containing dna, and also of viral dsrna.17,18 dental caries is caused by gram-positive and gramnegative bacteria. odontoblasts express tlr2 and tlr4 on the cellular processes and cell surfaces, suggesting a capacity of odontoblasts to receive signals from gram-positive and gram negative bacteria in tooth decay (figure 1).19–22 as mentioned earlier, tlr2 is crucial for the recognition of gram-positive bacteria components, including lta, lipopeptides, and peptidoglycan. meanwhile, tlr4 is the predominant receptor for lps, a characteristic component of the cell wall of gram-negative bacteria.18,23 tlr activation initiates the effectors phase of the innate immune response, mainly through the activation of the nf-κb pathway.24 this includes the production of pro-inflammatory cytokines and chemokines that recruit and activate blood borne inflammatory cells.25,26 lpsmediated tlr4 activation increases production of proinflammatory cytokines il-1β and tnf-α in odontoblasts. those cytokines act on vascular endothelial cells at the site of infection to induce the expression of adhesion molecules that promote extravasations of phagocytes during inflammation.3 an in vitro study by levin et al.27 has revealed that odontoblasts are capable of producing il-8 following exposure to bacterial lps. il-8 is a chemokine which has numerous functions and is considered to be the primary regulatory molecule in the acute inflammatory response. it has been shown to be chemotactic for neutrophils, t lymphocytes, and basophils, to stimulate neutrophil degranulation and oxidative burst activity, and to stimulate histamine release from mast cells. in addition, il-8 has been shown to induce increased expression of the cell adhesion molecule mac-1 on neutrophils, which enhances the adhesion of neutrophils to vascular endothelium.28 in addition, unstimulated odontoblast cells also express several chemokine genes including ccl2, cxcl4, cxcl12, and cxcl14.29 following lta stimulation, ccl2, cxcl2, and cxcl10 genes and two corresponding proteins (ccl2 and cxcl10) are clearly up-regulated.16 ccl2 is a key inflammatory chemokine produced during microbial infection that attracts immature dc and also monocytes, activated t cells, nk cells, and basophils figure 1. the innate immune response of odontoblasts in responding to cariogenic bacteria. 145haniastuti: potential role of odontoblasts in the innate immune response through ccr1 and ccr 2, thereby facilitating their interaction with invading bacteria. furthermore, through up-regulation of cxcl2 and cxcl10 expression, odontoblasts are likely to contribute to the recruitment of neutrophils and lymphocytes, respectively, during infection.26 chemokines not only induce cell locomotion but also influence angiogenesis.30 among chemokines expressed by odontoblasts, ccl2, cxcl2, and cxcl12 are proangiogenic, whereas cxcl4, cxcl10, and cxcl14 are angiostatic.31 the production of angiostatic chemokines in the healthy dental pulp might be involved in the maintenance of blood vessels outside the odontoblast layer. during dental caries-induced inflammation, the number of capillaries is augmented in the pulp under the lesion, and some of them penetrate into the odontoblast layer.32 the expression of the proangiogenic chemokine, cxcl2, is strongly up-regulated in lta-stimulated odontoblasts, suggesting that cxcl2 might thus contribute to the increased vascularization by binding to cxcr2 that is highly expressed on endothelial cells.30 an in vitro study by bofero et al33 has revealed that odontoblast-like cells stimulated by lps up-regulate vascular endothelial growth factor (vegf) expression suggesting a novel role for odontoblasts in the regulation of pulpal angiogenesis. up-regulated vegf synthesis by odontoblasts stimulated with lps might increase the permeability of existing pulp blood vessels, thus facilitating the process of diapedesis of neutrophils, lymphocytes, and monocytes. furthermore, it might also recruit new blood vessels to the area closest to the carious lesion to enhance the access of the blood-derived antibodies and defense cells to protect the pulp tissue against bacterial challenge. previous studies21,34 have demonstrated that odontoblasts express human b-defensin (hbd)-1, hbd-2, and hbd3. defensins are a group of small (3-5kda), cationic, cysteine-rich b-sheet peptides which have a broad spectrum of antimicrobial activity and are involved in the innate host defense.35 expression of defensins correlates with inhibition of bacterial rna, dna, and protein synthesis, as well as with reduced bacterial viability.36 hbd-1 and hbd-3 display broad antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and adenovirus. hbd-2 has antimicrobial activity against streptococcus mutans37 and has a high antimycotic potency as well as being a chemoattractant for nk cells, memory cd4+ t cells, and immature dc.38 hbd-2 may initiate or enhance the cytokine-induced pro-inflammatory reaction of odontoblasts as well.39 in healthy pulps, transforming growth factor beta (tgf-b) is secreted by odontoblasts,40 and its expression is increased under carious lesions.41 generally, tgf-b has a proinflammatory function during the initial stages of inflammation, while having anti-inflammatory effects during the later stages. the proinflammatory functions of tgf-b include immune cell recruitment and induction of matrix metalloproteinase secretion.42 tgf-b stimulates accumulation of immature dendritic cells in odontoblast and subodontoblast layers of the pulp horn close to the lesion in locations strategic to encounter foreign antigens entering the dentinal tissue. after capture of foreign antigens at the dentin-pulp interface, dendritic cells migrate, while undergoing a process of maturation, via the afferent lymphatic to regional lymph nodes, to stimulate naïve tlymphocytes; thus, initiating a primary immune response. during the later stage of inflammation, tgf-β exhibits anti-inflammatory effects through repression of lymphocyte proliferation, tlr signaling, and antigen-presenting dendritic cell and macrophage activation.43 in conclusion, beside their function in forming and maintaining dentin, odontoblasts are also capable of recognizing and responding to microorganisms and thus, eliciting the immune cells. odontoblasts express tlrs, and are capable of producing chemokines (i.e. il-8, ccl2, cxcl2, and cxcl10), and cytokines (il-1β and tnf-α) following lipopolysacharide exposure; thereby, actively participating in the recruitment of immune cells in the response to caries–derived bacterial products. furthermore, they also produce antimicrobial peptides (hbd-1, hbd-2, and hbd-3) and tgf-b that induce antimicrobial and antiinflammatory activities, respectively. the presence of these innate immune molecules indicates that the nonspecific, natural, and rapidly acting defense may also be important function of odontoblasts. acknowledgment the author thanks dr. phides nunez for her help in the english writing of this paper. references 1. love rm. invasion of dentinal tubules by root canal bacteria. endod top 2004; 9:52–65. 2. love rm, jenkinson hf. invasion of dentinal tubules by oral bacteria. crit rev oral biol med 2002; 13:171–83. 3. hahn c, liewehr fr. innate immune responses of the dental pulp to caries. j endod 2007; 33:643–51. 4. jontell m, okiji t, dahlgren u, bergenholtz g. immune defense mechanisms of the dental pulp. crit rev oral biol med 1998; 9(2):179–200. 5. akira s, takeda k. toll-like receptor signaling. nat rev immunol 2004; 4:499–511. 6. cooper md, alder mn. the evolution of adaptive immune systems. cell 2006; 124:815–22. 7. akira s, uematsu s, takeuchi o. pathogen recognition and innate immunity. cell 2006; 124:783–801. 8. izumi t, kobayashi i, okamura k, sakai h. immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth. arch oral biol 1995; 40(7):609–14. 9. arana-chavez ve, massa lf. odontoblasts: the cells forming and maintaining dentine. int j biochem cell bio 2004; 36:1367–73. 10. holland gr. the odontoblast process: form and function. j dent res 1985; 64:499–514. 11. sasaki t, garant pr. structure and organization of odontoblasts. anat rec 1996; 245:249. 12. yu c, abbott pv. an overview of the dental pulp: its functions and responses to injury. aust dent j 2007; 52(1 suppl):s4–s16. 146 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 142-146 13. bjørndal l. dentin and pulp reactions to caries and operative treatment: biological variables affecting treatment outcome. endod top 2002; 3:123–36. 14. tziafas d. the future role of a molecular approach to pulp-dentinal regeneration. caries res 2004; 38:314–20. 15. iwasaki a. medzhitov r. toll-like receptor control of the adaptive immune responses. nat immunol 2004; 5:987–95. 16. durand sh, flacher v, romeas a, carrouel f, colomb, e, vincent c, et al. lipoteichoic acid increases tlr and functional chemokine expression while reducing dentin formation in in vitro differentiated human odontoblasts. j immunol 2006; 176:2880–7. 17. takeda kt, kaisho t, akira s. toll-like receptors. annu rev immunol 2003; 21:335–76. 18. medzhitov r, preston-hurlburt p, janeway ca. a human homologue of the drosophila toll protein signals activation of adaptive immunity. nature 1997; 388:394–7. 19. jiang h, zhang w, ren b, zeng j, ling j. expression of toll-like receptor 4 in normal human odontoblasts and dental pulp tissue. j endod 2006; 32:747–51. 20. bofero tm, shelburne ce, holland gr, hanks ct, nör je. tlr4 mediates lps-induced vegf expression in odontoblasts. j endod 2006; 32:951–5. 21. veerayutthwilai o, byers mr, pham t-tt, darveau rp, dale ba. differential regulation of immune responses by odontoblasts. oral microbiol immunol 2007; 22:5–13. 22. mutoh n, tani-ishii n, tsukinoki k, chieda k, watanabe k. expression of toll-like receptor 2 and 4 in dental pulp. j endod 2007; 33:1183–6. 23. dillon s, agrawal s, banerjee k, letterio j, denning tl, oswaldrichter k, et al. yeast zymosan, a stimulus for tlr2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. j clin invest 2006; 116:916–28. 24. li q, verma im. nf-κb regulation in the immune system. nat rev immunol 2002; 2:725–34. 25. yoshie o, imai t, nomiyama h. chemokines in immunity. adv immunol 2001; 78:57–110. 26. mantovani a, sica a, sozzani s, allavena p, vecchi a, locati m. the chemokine system in diverse forms of macrophage activation and polarization. trends immunol 2004; 25:677–86. 27. levin lg, rudd a, bletsa a, reisner h. expression of il-8 by cells of the odontoblast layer in vitro. eur j oral sci 1999; 107:131–7. 28. graves dt, jiang y. chemokines, a family of chemotactic cytokines. crit rev oral biol med 1995; 6:109–118. 29. staquet mj, duran sh, colomb e, romeas a, vincent c, bleicher f, et al. different roles of odontoblasts and fibroblasts in immunity. j dent res 2008; 87(3):256–61. 30. pober js, sessa wc. evolving functions of endothelial cells in inflammation. nat rev immunol 2007; 7:803–15. 31. belperio j, keane mp, burdick md, gomperts b, xue yy, hong k, et al. role of cxcr2/cxcr2 ligands in vascular remodeling during bronchiolitis obliterans syndrome. j clin invest 2005; 115:1150–62. 32. rodd hd, boissonade fm. immunocytochemical investigation of immune cells within human primary and permanent tooth pulp. int j paediatr dent 2006; 16:2–9. 33. bofero tm, mantellini mg, song w, hanks ct, nör je. effect of lipopolysacharides on vascular endothelial growth factor expression in mouse cells and macrophage. eur j oral sci 2003; 111:228–34. 34. dommisch h, winter j, açil y, dunsche a, tiemann m, jepsen s. human beta defensin (hbd-1, -2) expression in dental pulp. oral microbiol immunol 2005; 20:163–6. 35. lehrer ri. primate defensins. nat rev microbiol 2004; 2(9): 727–38. 36. mullard a. immune evasion: overcoming defensins. nat rev microbiol 2008; 6:415. 37. nishimura e, eto a, kato m, hashizume s, imai s, nisiziwa t, et al. oral streptococci exhibit diverse susceptibility to human betadefensin-2: antimicrobial effects of hbd-2 on oral streptococci. curr microbiol 2004; 48:85–7. 38. hover dm, wu z, tucker k, lu w, lubkowski j. antimicrobial characterization of human b−defensin 3 derivatives. antimicrob agents chemother 2003; 47(9):2804–9. 39. dommisch h, winter j, willebrand c, eberhard j, jepsen s. immune regulatory functions of human beta-defensin-2 in odontoblast-like cells. int endodont j 2007; 40:300–7. 40. lucchini m, romeas a, couble ml, bleicher f, magloire h, farges jc. tgf beta 1 signaling and stimulation of osteoadherin in human odontoblasts in vitro. connect tissue res 2002; 43:345–5. 41. sloan aj, perry h, matthews jb, smith aj. transforming growth factor beta isoform expression in mature human healthy and carious molar teeth. histochem j 2000; 32:247–52. 42. strobl h, knapp w. tgf-b1 regulation of dendritic cells. microbes infect 1999; 1:1283–90. 43. farges jc, romeas a, melin m, pin jj, lebecque s, lucchini m, et al. tgf-b1 induces accumulation of dendritic cells in the odontoblast layer. j dent res 2003; 82(8):652–6. volume 50, number 2, june 2017 the increased number of osteoblasts and capillaries in orthodontic tooth movement postadministration of robusta coffee extract · the influence of artificial salivary ph on nickel ion release and the surface morphology of stainless steel bracket-nickel-titanium archwire combinations · increase of collagen in diabetes-related traumatic ulcers after the application of liquid coconut shell smoke p-issn: 1978-3728 e-issn: 2442-9740 volume 50, number 2, june 2017 editorial boards of dental journal (majalah kedokteran gigi) sk: 275/un3.1.2/2017 january 3rd – december 31st, 2017 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors rostiny (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); markus budi rahardjo (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty of vocation, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); anis irmawati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); yuliati (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); trimurni abidin (department of conservative dentistry, faculty of dentistry, universitas sumatera utara, indonesia); al. supartinah santoso, (department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, indonesia); pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); siti mardewi soerono akbar (department of conservative dentistry, faculty of dentistry, universitas indonesia, indonesia); siti sunarintyas (department of biomaterials, faculty of dentistry, universitas gadjah mada, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia); moh. rubianto (department of periodontics, faculty of dental medicine, universitas airlangga, indonesia); jenny sunariani (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); sri kunarti (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); indeswati diyatri (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); mieke sylvia m.a.r (department of odontology forensic, faculty of dental medicine, universitas airlangga, indonesia); adi hapsoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); kus harijanti (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); i.b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); ira arundina (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); maretaningtias dwi ariani (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); taufan bramantoro (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia). administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 334/08.17/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 50, number 2, june 2017 p-issn: 1978-3728 e-issn: 2442-9740 1. the efficacy of sarang semut extract (myrmecodia pendens merr & perry) in inhibiting porphyromonas gingivalis biofilm formation zulfan m. alibasyah, ambrosius purba, budi setiabudiawan, hendra dian adhita, dikdik kurnia, and mieke h. satari ............................................................................................. 55–60 2. the correlation between ph and flow rate of salivary smokers related to nicotine levels labelled on cigarettes dewi saputri, abdillah imron nasution, mutiara rizki wardani surbakti, and basri a. gani ............................................................................................................................ 61–65 3. the effects of audio-video instruction in brushing teeth on the knowledge and attitude of young slow learners in cirebon regency yayah sopianah, muhammad fiqih sabilillah, and oedijani ..................................................... 66–70 4. increase of collagen in diabetes-related traumatic ulcers after the application of liquid smoke coconut shell meircurius dwi condro surboyo, ira arundina, and retno pudji rahayu .............................. 71–75 5. effects of strong bite force on the facial vertical dimension of pembarong performers christina, achmad sjafei, and ida bagus narmada ..................................................................... 76–79 6. the influence of artificial salivary ph on nickel ion release and the surface morphology of stainless steel bracket-nickel-titanium archwire combinations ida bagus narmada, natalya tanri sudarno, achmad sjafei, and yuli setiyorini .................. 80–85 7. scaffold combination of chitosan and collagen synthesized from chicken feet induces osteoblast and osteoprotegerin expression in bone healing process of mice saka winias, diah savitri ernawati, maretaningtias dwi ariani, and retno pudji rahayu .. 86–90 8. the increased number of osteoblasts and capillaries in orthodontic tooth movement postadministration of robusta coffee extract herniyati ........................................................................................................................................... 91–96 9. the inhibition of streptococcus mutans glucosyltransferase enzyme activity by mangosteen pericarp extract nirawati pribadi, yovita yonas, and widya saraswati ................................................................ 97–101 10. differences in surface roughness of nanohybrid composites immersed in varying concentrations of citric acid gabriela kevina alifen, adioro soetojo, and widya saraswati .................................................. 102–105 11. the effect of a combination of 12% spirulina and 20% chitosan on macrophage, pmn, and lymphocyte cell expressions in post extraction wound nike hendrijantini, rostiny, mefina kuntjoro, kevin young, bunga shafira, and yunita pratiwi .......................................................................................................................... 106–110 72 vol. 43. no. 2 june 2010 research report the effect of monofluorophosphate implant in white rat mothers towards the level of fluor in the incisors of their young babies (rattus-rattus) widjijono department of biomaterial faculty of dentistry, gadjah mada university yogyakarta indonesia abstract background: fluoride has been widely used in the prevention of dental caries for a long time. to prevent dental caries, fluoride must be induced in low amount at high frequency. inducing it through implantation process even make slow release of small concentration of fluoride. purpose: the aim of this research was to analyze whether the induction of monofluorophosphate (mfp) implant into the white rat mothers affects the level of fluoride in the incisors of their young babies. method: the objects of the research were twenty white rat mothers in two days of pregnancy which then were divided into four groups (n=5). first, those mothers have been induced with implant under their back skin until their born young babies in the age of 35 days (n=5). the level of fluoride in the incisors of those young babies then is measured with potentiometer. the obtained data were finally analyzed with one-way anova test and continued by with lsd test (p=0.05). result: the result of this research showed that the means of the fluoride level in the incisors of those babies divided into those four groups in series were about 11956.16±201.35 ppb (k), 27328.04±234.56 ppb (p1), 37267.21±248.86 ppb (p2), and 18103.50±267.11 ppb (p3). the result of anova test then showed that the induction of various mfp implant levels significantly affected the level of fluoride in the incisors of the babies. the mean differences among the treatment groups after being tested with lsd 0.05 were also significant. conclusion: the finding confirm that the significant increasing of the optimal fluoride retention in the incisors of white rat babies can be achieved with the induction of fluoride with mfp ions implant in about 52.98 mg. key words: mfp implant, fluoride, the incisors of white rats abstrak latar belakang: pencegahan karies gigi menggunakan senyawa fluor telah banyak dilakukan dan berlangsung dalam jangka waktu lama. pemberian fluor dalam jumlah rendah dan frekuensi tinggi merupakan pemenuhan kebutuhan pencegahan karies gigi. pemberian dengan cara implantasi memberikan keluaran fluor jumlah kecil dan waktu lama. tujuan: penelitian ini bertujuan untuk mengetahui apakah induk tikus yang diberi implan-mfp berpengaruh terhadap kandungan fluor gigiseri anak tikus. metode: subjek penelitian adalah 20 ekor induk tikus putih bunting 2 hari dibagi 4 kelompok (n=5). induk diberi implan pada bawah-kulit punggung hingga anak tikus lahir dan pada umur 35 hari (n=5). kandungan fluor pada gigi seri anak tikus diukur menggunakan potensiometer. data yang diperoleh dianalisis dengan anova 1 jalur dilanjutkan uji lsd (p=0,05). hasil: penelitian menunjukkan rerata fluor gigiseri anak tikus berturut-turut sebesar: 11956,16±201,35 ppb (k), 27328,04±234.56 ppb (p1), 37267,21±248.86 ppb (p2), dan 18103,50±267,11 ppb (p3). hasil: anava membuktikan bahwa ada pengaruh bermakna akibat variasi kadar mfp dalam implan terhadap kandungan fluor gigi anak tikus. beda rerata antar kelompok perlakuan diuji dengan lsd0,05 memperlihatkan perbedaan bermakna pada semua kelompok. kesimpulan: penelitian dapat disimpulkan bahwa kenaikan secara bermakna terhadap retensi fluor optimal dalam gigiseri tikus putih pada pemberian fluoridasi menggunakan implan dengan muatan mfp: 52,98 mg. kata kunci: implan-mfp, fluor, gigi seri tikus putih correspondence: widjijono, c/o: bagian biomaterial, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara yogyakarta 55281. e-mail: widji_bomat@yahoo.com 73widjijono: the effect of monofluorophosphate implant introduction the prevention of dental caries with chemical application using fluoride has already been done. however, the prevention of dental caries must be conducted in the long term process started from the formation of tooth until they get maturation. the prevention of dental caries even is supposed to be conducted minimally until the age of twelve. unfortunately, the prevention of dental caries using fluoride given orally cannot be well controlled since it can cause dental fluorosis. therefore, the alternative way as such implantation process is needed to control the application of fluoride especially to prevent fluorosis. the main usage effects of fluoride on body actually can be particularly seen in calcificated tissues since fluoride has narrow therapeutic window. fluoride at low concentration level can increase the crystalinity of teeth and bones, while at the high concentration level it can cause abnormality in calcificated tissues, such as fluorosis.1 induction of fluoride at low concentration with high frequency of application can prevent dental caries.2 in other words, the daily controlled of fluoride intake will effectively decrease dental caries and fluorosis risks.3 induction of fluoride through implantation will cause fluoride released in small amount and in long term. sodium-monofluorophosphate (natrium-monofosfat, mfp) considered as potential fluoride that can be used as anti-caries with low toxicity which is not only more effective than naf, but also can be absorbed faster without being affected by calcium ions.3 mfp is chosen because it has anti-caries, its toxicity about one third lower than that in naf,4 it still can be degraded by alkali or acid phosphate through hydrolysis process,5 and by the substitution of the structure of phosphate hydrogen on hydroxy apatite with monofluorophosphate ion.6 the dissolution of mfp will 20 times faster if the level of calcium ions is so high to make calcium-monofluorophosphate compound that easily dissolved.7 in recent years, various products of medicines modified with controlled fluoride release have actually been developed for several reasons. the first one is because of their affectivity and efficiency. another one is to solve the impracticality caused by the routine of the long term treatment. the various use of polymer in dental health has widely developed.8 the selection of polymer type, poly-dllactic acids (pla), in implant materials is aimed to obtain fluoride carriers through controlled releasing. besides that, pla in the form of monolith has also several characteristics, such as small degradation constant, bio-erosion, and low permeability.9 fluoride induced orally makes the dissolved fluoride be absorbed and diffused in a simple way through digestive system, especially gastro-intestinal tract.10 inducing through the implant of endoderm is directly absorbed and diffused into blood vessel. the reason is because fluoride induced orally must diffuse through first pass of metabolism channel. as a result, none of fluoride intake can be absorbed. on the other side, mfp implant induced in the mothers of young rats makes fluoride intake can easily be absorbed by those mothers and be diffused into blood. some of the fluoride diffused into those mothers’ blood then will be induced into placenta and will be functioned as the source of fluoride for their fetuses. placenta also functions as partial barrier when the amount of fluoride is suddenly increased. there is a direct correlation between the amount of serum in mothers and in the placenta of their fetuses. the amount of fluoride in placenta is 75% bigger than that in their mothers.11 the previous researches actually have already pointed out that fluoride in the form of mfp implant can be distributed homogeneously in the form of monolith (bar), and fluoride release can be controlled seven days after the implantation.12 the aim of this study was to analyze the amount of fluoride in the incisors of the white rat babies whose mothers were induced with various levels of mfp in the form of poly-dl-lactate acid implant. material and method the objects of the research are twenty wistar white rat mothers (rattus-rattus) in two days of pregnancy. the main materials used in this study, are natriummonofluorophosphat (mfp) (na@211, australia), polydl-lactate acid (poly science, usa) and formalin. mfp implant consists of mfp and pla with the ratio 20:80. in other words, mfp implant is made based on the ratio of mfp to poly-dl-lactate acid, 20:80 (b/b mg). the implant was made according to beck et al. method.13 for mfp implant (p1), there are two kinds of solution. solution i is made by dissolving 26.49 mg of mfp to 3 ml of methanol, meanwhile solution ii is made by dissolving 105.96 mg of pla to 7 ml of chloroform and methilen chloride mixed with the ratio 1:1. solution i and solution ii then are mixed together until they become homogenous. organic solvent then is steamed with dryer at temperature of 50° c and stirred until the dough becomes plastic. afterwards, the dough is poured into die and pressed by hydraulic pressure about 50 kgf/cm2 for 1 minute. similarly, implants (p2), (p3) and implant (k) are made of mfp which in series are about 52.98 mg, 264.9 mg and 0 mg (table 1). table �. implants k, p1, p2 and p3 with their mfp-pla level ratio implants mfp(mg) pla (mg) implant k 0 100 implant p1 26.49 105.96 implant p2 52.98 211.92 implant p3 264.9 1059.6 74 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 72-75 twenty white rat mothers in two days of pregnancy are divided into four groups, group 1 with controlled implant treatment without mfp (k), group 2 with implant p1, group 3 with implant p2, and group 4 with implant p3. implantation process then is conducted on the back skin of those mothers. that skin must be cleanly shaved before being disinfected. the incision of the skin of their back is conducted for about 0.5 cm long. after that, the blunt dissection is conducted along the implant, 1 cm from the incision edge. next, the implant is inserted into the cavity, and the wound of the incision then needs silk-thread stitches. those mothers then must be on normal ad libitum diet modified without fluoride. after those mothers delivered their babies, the incisors of their 35 day old babies must be extracted under anesthetic. the extracted teeth are measured by cutting them into small pieces, and they then are dissolved in 2.5 ml of 65% hno3. afterwards, 1 ml of the dental solution is taken as the sample which then is mixed with 9 ml of 0.5m hclo4 and 10 ml tisab ii. specific potentiometer of fluoride ions connected with digital tps (titralizer action electrode, calomel refference) is used to determine the level of fluoride based on the interpolation mechanism related with the potential difference measured with the standard solution curve. the data is analyzed by using one way variant analysis in order to analyze the effect of mfp implant variants towards the level of fluoride in the incisors of those white rat babies, meanwhile and the mean of the difference of fluoride level in the incisors among treatment groups is tested with lsd 0.05. result this research finds that the means of fluoride level in the incisors of those white rat babies is as the following table 2. table ��. the mean and standard deviation of fluoride level in the incisors of white rats groups mean± sd (ppb) k 11956.16 ± 201.35 p1 27328.04 ± 234.56 p2 37267.21 ± 248.86 p3 18103.50 ± 267.11 k: control, p1: sample with 26.96 mg mfp group, p2: sample with 52.98 mg mfp group, p3: sample with 264.9 mg mfp group graphically, it also shows that the increasing of fluoride is not linear, but at certain levels it can reach the optimum absorption level. at the increasing level above the optimum one, the amount of fluoride absorbed is decreased (figure 1). the level of fluoride in the incisors of those white rat babies classified into three treatment groups, p1, p2 and p3, induced with implant is gradually increased, in series about 2.3 times, 3.2 times, and 1.5 times bigger than the controlled ones. it means that the biggest increasing of the amount of fluoride occurs in the treatment group induced with implant p2 (52.98 mg of mfp). 0 5 0 0 0 10 0 0 0 15 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 3 5 0 0 0 4 0 0 0 0 m f p 0 m f p 2 6 m f p 5 8 m f p 2 6 4 figure �. graph of fluoride level in the incisor of thirty-five day old white rat babies (ppb). table ��. the result of lsd0.05 test between the controlled group and the treatment groups, p1, p2 and p3 groups k p1 p2 p3 k 15371.878* 25311.056* 6147.338* p1 9939.178* 9224.540* p2 19163.718* p3 note: *) significant difference (p<0.05) all of the treatment groups are significantly different from the controlled ones through the statistic analysis using spss 13.0 for window with one way anova, moreover, it is also known that f is about 10675.56, bigger than f in the table. this result indicates that the variants of mfp implant level significantly affect the level of fluoride in the incisors of those white rat babies. the difference of means among those treatment groups tested with lsd 0.05 also shows that there is significant difference in those treatment groups (table 3). discussion the use of mfp implant is aimed to obtain the lower and controlled fluoride release. polylactate-natrium monofluorophosphate implant in the bar shape even can be dispersed in homogeneous way. in other words, by using mfp implant the controlled fluoride release occurred seven days after implantation.12 the purpose of selecting pla is to obtain fluoride carriers with controlled disposal. pla in the form of monolith actually has several characteristics, such as: small degradation constant, bio-erosion, and low permeability.9 thus, if the reason of the use of mfp is because of anti-caries activities in mfp with its toxicity about one third lower than that in naf,3 it still can be degraded by alkali or acid phosphate 75widjijono: the effect of monofluorophosphate implant through hydrolysis process,5 and by the substitution of the structure of phosphate hydrogen on hydroxy apatite with monofluorophosphate ion.6 the average of fluoride contained on the incisors of those thirty-five day old white rat babies and the binding of fluoride ion on the teeth, actually depends on the level of fluoride in their blood plasma. the increasing of fluoride level in the certain availability of plasma will increase the amount of fluoride bound on teeth. the increasing of the availability in their blood plasma which is more than the optimal one can decrease the binding of dental fluor (figure 1). p2 implant has the average of plasma fluoride level, about 125.61± 23.30 ppb,12 which is not only appropriate with the level of blood fluoride, about 0.1–0.2 ppm,10 but is also close to the level of therapeutic-hypothetic in blood plasma of society with the level of consumed water about 1 ppm. moreover, the variants of fluoride level in their incisors caused by the increasing amount of fluoride that can also be explained by the opinion of fejerkov et al.,1 stating that fluoride has narrow therapeutic window mechanism, which means that at the low dosage its influence is not significant, but at the high dosage it can disrupt the growth of dental structure. fluoride with low dosage (such as in p1 implant), will increase dental crystalinity. it is possibly caused by the reaction of the substitution of isoionic f with hydroxyl structure in dental apatite which then can form apatite fluoride or hydroxy-fluor apatite. the forming of fluor apatite crystal is also supported by a research conducted by monjo et al.,15 stating that the inducing of implant with fluoride modification in bones can not only make fluoride modulate the forming of osteogenic marker, but also increase the density of bones located in interface part between implant and bones. the surface of implant modified with fluoride can increase osseointegration in the early stage of recovery.16 on the other hand, fluoride in the high dosage (such as in p3 implant) can cause disruption during the growth of enamel affecting in enamel organic and inorganic components which then causes either hypoplasia of permanent teeth10 or partial resistance in proteinase that has responsibility for breaking enamel protein. as a result, it can either decelerate protein disposal during maturation or cause the disruption of calcification or fluorosis.14 it means that the disruption in disposal process of protein substituted with mineral can relatively decrease the amount of fluoride released from the disrupted tissue. therefore, it can be concluded that the optimal resistance of fluoride in the incisors of white rat babies is significantly increased during fluoridation using implant with mfp ions, about 52.98 mg. references 1. fejerkov o, richards a, denbasten p. the effect of fluoride on the tooth mine-ralization. in: ekstrand j, burt ba, editors. fluoride in dentistry. copenhagen: munksgaard; 1996; p. 112–47. 2. hargreaves ja. water fluoridation and fluoride supplementation: consideration for future. j dent res 1990; 69(spec iss): 775–70. 3. cremer hd, buttner w. absorption of fluoride. in: fluoride and human health. geneva: who; 1970. p. 84–5. 4. white we. monofluorophosphate–its beginning. caries res 1983; 17(suppl 1): 2–8. 5. pearce eif. biochemistry of monofluorophosphate. caries res 1983; 17(suppl 1): 21–35. 6. ingram gs. the reaction of monofluorophosphate with apatite. caries res 1972; 6: 1–15. 7. ericsson y. monofluorophosphate physiology: general considerations. caries res 1983; 17(suppl 1): 46–55. 8. shargel l, yu abc. 1985. biofarmasetika dan farmakokinetika terapan. edisi 2. surabaya: penerbit universitas airlangga (aup); 1988. p. 454–56. 9. pitt cg, schindler a. the design of controlled drug delivery system based on biodegradable polymers. in: hafez ese, van os waa, editors. biodegradable and delivery system for contraception. boston: gk hall medical pub; 1980. p. 27. 10. cole as, eastoe je. biochemistry and oral biology. singapore: toppan co ltd; 1977. p. 123. 11. ekstrand j. fluoride metabolism. in: ekstrand j, burt ba, editors. fluoride in dentistry. copenhagen: munksgaard; 1996. p. 112–47. 12. widjijono. penggunaan implan polilaktat-natrium monofluorofosfat dengan kajian availabilitas fluor sediaan, biokompatilitas dan bioavailabilitas fluoride dalam darah dan gigi pada tikus putih. disertasi. surabaya: program pascasarjana universitas airlangga; 2001. p. 115. 13. beck lr, cowsar dr, lewis dh. systemic and local delivery of contraceptive steroids using biodegradable microcapsules. in: hafez ese, van os waa, editors biodegradable and delivery system for contraception. boston: gk hall medical pub; 1980. p. 63–82. 14. bawden jw, crenshaw ma, wright gt, legeros rz. consideration of possible biologic mechanism of fluorosis. j dent res 1995; 74(7): 1349–52. 15. monjo m, lamolle sf, lyngstadaas sp, ronold hj, ekkingsen je. in vivo expression of osteogenic marker and bone mineral density at the surface of fluoride-modified titanium implants. biomaterials 2008; 29(28): 3771–80. 16. berglundh t, abrahamsson i, albouy jp, linde j, bone healing at implants with a fluoride-modified surface: an experimental research in dogs. clinical oral implants research 2007; 18(2): 147–52. vol 51 no 3 jul sep 2018_pus.indd 138138 research report dental journal (majalah kedokteran gigi) 2018 september; 51(3): 138–142 effects of liquid ionic silver concentration on caspase-3 and p53mt expressions in the oral mucosal epithelium of wistar rats r. aries muharram,1 i. istiati2 and pratiwi soesilawati3 1 department of oral and maxillofacial surgery 2 department of oral and maxillofacial pathology 3 department of oral biology faculty of dental medicine, universitas airlangga, surabaya – indonesia abstract background: silver, especially oxidized silver, has been used as a medicine considered to have bactericidal properties. in the present day, ionic silver (ag+) is also used in the manufacture of cosmetics, socks, food containers, detergents, sprays and other products to prevent the spread of germs. unfortunately, ionic silver is assumed to be toxic not only to bacteria, but also to humans and the environment. therefore, it is essential to identify the optimum dosage of ionic silver considered safe by investigating the effects of ionic silver concentration on cell death through activation of mutant p-53 expression by caspase 3 in the oral epithelium. purpose: this research aimed to analyze the effects of concentrated liquid ionic silver (ag+) on caspase-3 and mutant p53 expression in the oral mucosal epithelium. methods: this research constituted a laboratory-based experimental study with posttest-only design. the research subjects consisted of 28 wistar rats, divided into four treatment groups, namely; kk (with aquadest), kp 1 (with 5% liquid ionic silver), kp 2 (with 10% liquid ionic silver) and kp 3 (with 15% liquid ionic silver). each rat was then treated orally with 0.5ml of liquid ionic silver at fixed concentrations twice a day for seven days. the wistar rats were then terminated and their tissue samples processed by means of histopathological and immunohistochemical staining examination. the monoclonal caspase-3 and mutant p53 expressions in each group were evaluated with the data being tabulated and analyzed statistically. results: mutant p53 expression was also found in the control group. moreover, the higher the concentration of liquid ionic silver, the greater the elevated caspase-3 and mutant p53 expressions. conclusion: the concentration of liquid ionic silver plays an important role in elevating caspase-3 and mutant p53 expressions. keywords: ionic silver (ag+); oral epithelium; caspase-3; mutant p53 expression correspondence: r. aries muharram, c/o: department of oral and maxillofacial surgery, faculty of dental medicine, universitas airlangga, jl. prof. dr. moestopo no. 47, surabaya, indonesia. email: aries-m@fkg.unair.ac.id; ies_id@yahoo.com introduction silver has long been considered a remarkable substance, having been used to prevent microorganism-related infections as long ago as the fifteenth century. historically, silver has also been effective in destroying almost all strains of microorganisms. research into silver, first conducted at the beginning of 4000 bc.1,2, confirmed it to be the third most widely used metal after gold and copper in ancient times. in recent years, silver has not only been widely used in a variety of medical procedures, but also for sanitation of public facilities such as baths, toilets, hand sanitizers, topical therapies outside the body, wound bandages, catheters as well as for oral antimicrobial medicine.1,2 ionic silver can immobilize enzymes during oxygen metabolism in viruses, fungi, bacteria and single-celled pathogenic microbes. within minutes, the pathogenic microbes can weaken and die before being removed from the body by the immune, lymphatic and elimination systems. unlike antibiotic therapy, which is generally harmful to animal enzymes, ionic silver tends to maintain the integrity of tissue cells. therefore, ionic silver is considered safe for humans, reptiles, plants and other living creatures.3 liquid dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p138–142 mailto:ies_id@yahoo.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p138-142 139 muharram, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 138–142 ionic silver (aquasil®) at a concentration of 15ppm is even widely marketed as a mouthwash. according to murphy and evans (2012), ionic silver can be employed during wound healing treatment.4 however, in vivo research suggests that ionic silver at concentrations of 10 ppm and 32 ppm cannot cause toxic effects to the lungs, liver, brain, circulatory system and reproductive system.3 ionic silver at concentrations of 10 ppm and 32 ppm in the blood vessels is also known to leave the number of erythrocytes, granulocytes or granulocytes unaffected. silver is actually contained in the blood vessels, largely in the form of ions. however, it remains unclear whether the ions circulate through the digestive system or through their attachment to blood components. moreover, ionic silver is not found in urine and has no effects on hydrogen peroxide production. on the other hand, other previous research revealed that when ionic silver diffuses into the body through the respiratory system, the ions will bind to pulmonary epithelial cells and macrophages with the result that cell function will be limited.3 according to research conducted by heydarnejad (2014), ionic silver also caused toxicity when its dose was increased to 10 ppm on day 7.5 researchers at herald university (2014), even argue that nano-silver can trigger the formation of free radicals in cells, a condition subsequently leading to changes in the shape and quantity of proteins. other researchers even posit that the excess production of free radicals in cells causes cancer and nerve disorders, such as alzheimer’s disease and parkinson’s disease.6 it has also been accepted that the higher the dose of ionic silver administered, the more oxidative stress increases resulting in metabolic disturbances in mitochondria. in addition, the more reactive oxygen species (ros) generated affects the cell cycle,1,2 leading to either apoptosis (programmed cell death) or necrosis (cell destruction). if such disruption occurs, in addition to producing apoptosis and necrosis in the cell cycle, cells can also undergo mutation.5 caspase-3 is the first signaling protein in the apoptosis system which subsequently triggers the activation of p53 in cells targeted to initiate the apoptosis process. conversely, mutant p53 expression is a symptom of function deviation from p53 often found in cancerous tissues, but sometimes also encountered in non-cancer patients. since ionic silver may precipitate changes in cells, turning them from normal to mutant, oral mucosal cells can be directly exposed to ionic silver due to oral consumption. ionic silver is assumed to cause local and systemic effects. as a result, this research aimed to reveal the systemic effects of ionic silver at a concentration of 15 ppm diffused through digestion in mutant p53 and caspase-3 expressions in the oral mucosal epithelium.3 in this research, the oral mucosal epithelial cells of wistar rats were used since these were considered equivalent to those of humans.7 materials and methods twenty-eight wistar rats (rattus norvegicus) aged 3-4 months, weighing 200 grams and pronounced healthy by veterinarians, were used as samples in this research. these subjects were obtained from the department of biochemical sciences laboratory, faculty of medicine, universitas airlangga. they were subsequently divided into four groups, namely three treatment groups given liquid ionic silver and a control group. the first treatment group was given 5 ppm of liquid ionic silver. the second 10 ppm of liquid ionic silver and, the third 15 ppm of liquid ionic silver. meanwhile, the control group was given distilled water. each of those groups was given 0.5 ml of liquid ionic silver at determined concentrations twice a day for seven days. after seven days, the subjects were sacrificed and their cheek mucosa were cut to 3x4 mm in size using a no.15 scalpel. the mucosal tissues were soaked in a fixative solution, 10% acetate buffered formalin, and then processed using an autotechnicon® tool. the fixed specimens were subsequently hydrated with ethanol, before a 60-minute clearing process was performed twice with the same material and for the same duration. subsequently, media infiltration (embedding process) was performed using paraffin wax (tissue prep, fisher sci., 56-570 c melting point). the last procedure was that of casting or blocking specimens whereby the mucosal epithelium specimens were planted in paraffin. thereafter, the tissues were observed using embedding rings and the paraffin blocks were stored at 40 c for 15 minutes to harden. hematoxylin and eosin staining were then carried out. an analysis of caspase-3 and mutant p53 expressions in cheek mucosal specimens in paraffin blocks was performed by means of an immunohistochemical staining technique using anti-caspase 3 monoclonal antibodies of the wistar rats (cleaved caspase-3 (asp175), signalstain® and cell signaling technology® (trademarks of cell signaling technology, inc.), mouse monoclonal antibodies, and antimutant p53 (p53 (do-7): sc-47698, santa cruz biotechnology, inc.). observation was subsequently performed using a light microscope at 40x magnification. the operational definition of mutant p53 protein and caspase-3 expressions was indicated by the presence of brown color in the basement membrane of the epithelial cells. the examiners consisted of a trio of anatomists and histologists. the mutant p53 protein and caspase-3 expressions were then calculated in 20 fields of view as recommended by pizem and cor (2003).8 tho three examiners conducted the examination and calculation of samples separately, writing the results of each calculation on the worksheet. the mean value per field of view was calculated before being analyzed statistically8,9 with normality and homogeneity tests (one-sample kolmogorov-smirnov test), followed by a one-way anova test with significance of α: 0.05. correlation and regression analysis (post hoc tukey’s hsd) was then conducted on each group. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p138–142 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p138-142 140muharram, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 138–142 results the research was conducted on four groups of male wistar rats, three groups being given silver ions for seven days and one control group. the procedure was replicated seven times with each group times to identify mutant p53 and caspase-3 expressions. caspase-3 and mutant p53 expressions were then examined by staining method as shown in figures 1 and 2. the mean number of caspase-3 and mutant p53 expressions rose with the increase in ag+ concentrations as depicted in figure 3. the immunohistochemical staining process using mouse antibody monoclonal anti-caspase 3 was performed on epithelial cells, the results of which are illustrated in figure 2. after the data of caspase-3 and mutant p53 expressions had been collated, normality analysis was performed by means of a kolmogorov-smirnov test. the results of the normality analysis revealed that the data of mutant p53 (p=0.180) and caspase-3 (p=0.743) expressions was normally distributed. a homogeneity test then was conducted, the results of which showed the p value of the group given 5 ppm of liquid ionic silver to be 1.0, p=1.0 for the group given 10 ppm of liquid ionic silver, and p=0.122 for the group given 15 ppm of liquid ionic silver. this indicated that the data obtained was homogeneous. thus, one-way anova and tukey hsd tests were carried out, the results of which are shown in tables 1 and 2. the results of the one-way anova test on caspase 3 expressions showed α value of 0.05, whilethose of the tukey hsd test then indicated a p value of 1.0 (p> 0.05) at all concentrations. this means that the data obtained was homogeneous. consequently, a one-way anovas was conducted as illustrated in table 1. at the next stage, a post hoc tukey hsd test was performed whose results indicated that the mean number of caspase-3 expressions in the control group was lower than those in the treatment groups. there were even significant differences in the mean numbers of the caspase-3 expressions between the control group and all treatment groups as well as between the treatment groups (p<0.05). moreover, based on the contents of the above table, the greater the concentration of liquid ionic silver, the higher figure 1. the results of oral immunohistochemistry staining on mutant p53 expression in oral epithelium exposed to ag+ at varying concentrations atn a magnification of 40x (orange arrows showing mutant p53 expression). note: control group (a), group given 5 ppm of liquid ionic silver (b), group given 10 ppm of liquid ionic silver (c), and group given 15 ppm of liquid ionic silver (d). figure 2. the results of oral immunohistochemistry staining on caspase 3 expression in oral epithelium exposed to ag+ at varying concentrations with a magnification of 40x (orange arrows showing caspase 3 expressions) note: control group (a), group given 5 ppm of liquid ionic silver (b), group given 10 ppm of liquid ionic silver (c), and group given 15 ppm of liquid ionic silver (d). table 1. examination results of caspase-3 expression groups control group treatment group i (5ppm) treatment group ii (10ppm) treatment group iii (15ppm) control group -14.000*-9.429*-4.143* treatment group i (5ppm) -9.857*-5.286*-4.143* treatment group ii (10ppm) -4.571*-5.286*-9.429* treatment group iii (15ppm) -4.571*-9.857*-14.000* * = significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p138–142 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p138-142 141 muharram, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 138–142 the mean number of caspase-3 expressions in the mucosal epithelium of wistar rats. this confirms that the number of cells experiencing apoptosis was increasing. a post hoc tukey hsd test was subsequently, carried out, the results of which showed that the mean number of mutant p53 expressions in the control group was lower than those in the treatment groups. in other words, there was a significant difference in the mean number of mutant p53 expressions between the control group and all treatment groups with a p value of 0.000 (p<0.05). however, there was no significant difference in the mean number of mutant p53 expressions between group ii (with 10 ppm of silver ions) and group iii (with 15 ppm of silver ions) with a p value of 0.122 (p>0.05). furthermore, based on the contents of the table above, the greater the concentration of liquid ionic silver, the higher the mean number of mutant p53 expression in the mucosal epithelium of wistar rats. discussion this research represented an in vivo study during which the mucosal epithelial cells of wistar rats were exposed to various concentrations (ppm) of liquid ionic silver for seven days. mutant p53 protein expressions were then used to detect abnormalities of wild-type p53. mutant p53 is a protective genome which the p53 test confirms the existence of two types. first, wild-type p53 is responsible for supporting damaged cells and directing them to the apoptotic pathway. second, mutant p53 is a special protein managing cells in the arrest phase of the cell cycle at both the g1 / s and g2 / m stages. in other words, mutant p53 plays a role in maintaining the cell cycle with the result that cell duplication does not occur. the expression of mutant p53 can also be considered as a sign that cells will be arrested in the subsequent cell cycle.10 treatment using liquid ionic silver, moreover, can cause changes in cell morphology, cell viability, metabolic activity and oxidative stress. liquid ionic silver which diffuses into cells can reduce atp cell contents causing mitochondrial damage and elevating reactive oxygen species (ros) production as doses increase. in mitochondria and cell nuclei, liquid ionic silver can trigger mitochondrial and dna damage. with the involvement of ros production initiated by silver ions, disruption to both the mitochondrial respiratory chain and atp synthesis will occur, eventually causing damage to dna. another process supporting the passage of liquid ionic silver into cells is that of endocytosis through which silver can penetrate the nucleus and harm dna. as a result, a number of researchers have evaluated the potential use of liquid ionic silver in cancer therapy. however, at certain concentrations and an exposure time in excess of seven days, mutant p53 expression can emerge, being considered a tumor marker.1,2 in this research, mutant p53 expressions also increased as the concentration of liquid ionic silver intensified. similarly, wong (2011) argues that the higher the concentration of liquid ionic silver, the greater the toxic effect.11 changes to the mucosal epithelial cell structure of wistar rats were highly visible in the stratum spinosum which is the thickest layer. mutant p53 expression can actually be found in normal cells, indicating that a small number already existed in wistar rats. this research found that, in the epithelial cells exposed to 5 ppm of liquid ionic silver, mutant p53 expressions emerged with intact basal structures. in the epithelial cells exposed to 10 ppm of liquid ionic silver, the number of mutant p53 expressions was higher than in those exposed to 5 ppm of liquid ionic silver with a stretching basal structure. furthermore, in the epithelial cells exposed to 15 ppm of liquid ionic silver, the number of mutant p53 expressions was highest with loose basal structures passing into the endothelium. consequently, it can be said that cells exposed to the high concentration of liquid ionic silver will become cancerous. researchers at the university of herald in 2014 also obtained the same results related to the appearance of mutant p53 expressions.6 on the other hand, caspase is an enzyme inducing natural cell death known as apoptosis. large quantities of caspase play a role in this process. the closest caspase triggering the apoptosis process is caspase-3 which was selected as a sign of apoptotic pathways in this research whose results revealed that caspase-3 expressions in each figure 3. p53mt and caspase-3 expression table 2. results of post hoc tukey hsd test on mutant p53 expression groups control group treatment group i (5ppm) treatment group ii (10ppm) treatment group iii (15ppm) control group -16.000*-14.429*-5.143* treatment group i (5ppm) -10.857*-9.286*-5.143* treatment group ii (10ppm) 1.571*-9.286*-14.429* treatment group iii (15ppm) 1.571*-10.857*-16.000* * = significant dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p138–142 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p138-142 142muharram, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 138–142 treatment group increased as the concentration of silver ions rose. similarly, alexander (2009) argues that ionic silver liquid therapy constitutes an extremely effective cure for infection in living creatures, since considerable numbers of microbes experience lysis after exposure to it. this suggests that higher concentration of liquid ionic silver can produce greater toxic effects.1 in this research, liquid ionic silver was not only found to be effective on microbes, but also induced a change in the epithelial cell structure of wistar rats. in normal epithelial cells, the number of caspase-3 expressions was limited, while in the epithelial cells exposed to 5 ppm of liquid ionic silver caspase-3 expressions emerged with intact basal structures. in the epithelial cells exposed to 10 ppm of liquid ionic silver, the number of caspase-3 expressions was higher than those exposed to 5 ppm of liquid ionic silver with a stretching basal structure. meanwhile, in the epithelial cells exposed to 15 ppm of liquid ionic silver, the number of caspase-3 expressions was the highest with loose basal structures passing into the endothelium. it can, therefore, be assumed that cells exposed to the high concentration of liquid ionic silver will become cancerous.6 finally, it can be concluded that the increase in liquid ionic silver concentration is in line with that of mutant p53 and caspase-3 expressions in the buccal mucosa epithelium of wistar rats. references alexander jw. history of the medical use of silver. surg infect1. (larchmt). 2009; 10(3): 289–92. panyala nr. silver or silver nanoparticles: a hazardous threat to the2. environment and human health. j appl biomed. 2008; 6: 117–29. hongbao m, deng-nan h, shen c. colloidal silver. j am sci. 2007;3. 3(3): 74–7. murphy ps, evans grd. advances in wound healing: a review of4. current wound healing products. plast surg int. 2012; 2012: 1–8. heydarnejad ms, yarmohammadi-samani p, dehkordi mm,5. shadkhast m, rahnama s. histopathological effects of nanosilver (ag-nps) in liver after dermal exposure during wound healing. nanomedicine j. 2014; 1(3): 191–7. university herald. exposure to nanosilver causes cancer, alzheimer’s6. and parkinson’s, study. 2014. available from: https://www.universityherald.com/articles/7867/20140303/ exposure-nanosilver-causescancer-alzheimer-s-parkinson-study.htm#ixzz3qj0isedx. accessed 2014 dec 20. kondo m, yamato m, takagi r, murakami d, namiki h, okano t.7. significantly different proliferative potential of oral mucosal epithelial cells between six animal species. j biomed mater res part a. 2014; 102(6): 1829–37. pizem j, cör a. detection of apoptotic cells in tumour paraffin sec-8. tions. radiol oncol. 2003; 37(4): 225–32. kumar v, abbas ak, fausto n, aster jc. robbins and cotran9. pathologic basis of disease. 8th ed. philadelphia: saunders; 2010. p. 1064-1077. 10. asharani p v, mun glk, hande mp, valiyaveettil s. cytotoxicity and genotoxicity of silver nanoparticles in human cells. acs nano. 2009; 3(2): 279–90. 11. wong rsy. apoptosis in cancer: from pathogenesis to treatment. j exp clin cancer res. 2011; 30: 1–14. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p138–142 https://www.universityherald.com/articles/7867/20140303/ http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p138-142 contents page printed by: airlangga university press. (012/01.10/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 42 number 4 october-december 2009 issn 1978 3728 dental journal majalah kedokteran gigi 1. immunopathological aspects of oral erythema multiforme maharani laillyza apriasari and retno pudji rahayu ................................................................ 159–163 2. cemento-ossifying fibroma of the jaw david b. kamadjaja ........................................................................................................................ 164–171 3. fibrous epulis associated with impacted lower right third molar ni putu mira sumarta and david b kamadjaja ........................................................................... 172–174 4. correction parameters in conventional dental radiography for dental implant barunawaty yunus .......................................................................................................................... 175–178 5. evaluation of seat and non-seat post preparation design using conventional and computational methods g. subrata, z. hasratiningsih, e. kurnikasari, and t. dirgantara ............................................ 179–184 6. detection of aggressive periodontitis by calprotectin expression desi sandra sari and suryono ......................................................................................................... 185–188 7. the ability of igy to recognize surface proteins of streptococcus mutans basri a. gani, santi chismirina, zinatul hayati, endang winiati b, boy m. bachtiar, and i. wayan t. wibawan ...................................................................................................................... 189–193 8. the management of over closured anterior teeth due to attrition eha djulaeha and sukaedi ............................................................................................................... 194–198 9. glucosyltransferase b/c expression in streptococcus mutans of rampant and caries-free children yetty herdiati h. nonong ............................................................................................................... 199–203 10. treatment results evaluation using the index of orthodontic treatment need thalca hamid ................................................................................................................................... 204–209 11. dental health economics and diagnosis related groups/casemix in indonesian dentistry ronnie rivany .................................................................................................................................. 210–215 mkg vol 41 no 4 oct-dec 2008.indd 160 vol. 41. no. 4 october–december 2008 review article the role of dentists on medically compromised children’s oral and dental prophylaxis in hospital roosje rosita oewen department of pediatric dentistry faculty of dentistry padjadjaran university bandung indonesia abstract background: one of dentist’s main roles is to coordinate the management of medically compromised children. the term of medically compromised refers to those children who have medical conditions which affect the dental treatment or manifest as a specific oral and dental problem. patient’s visit to special care for dentistry clinic dr. hasan sadikin hospital bandung showed a remarkable increase. from under 10 new visit in 2003, now july–december 2008 showed 81 new visit. purpose: this paper discusses several medical problems (cardiovascular, hematology, respiratory system, and genetic disorder) in children and the role of dentist in the treatment of those patients in the hospital. review: the increase of attention by all level to these medically compromised children in the hospital also increases the dentist’s role in supporting the prognosis of the disease and patient’s quality of life. the most important effort is oral and dental prophylaxis to prevent oral pathology which is caused by the manifestation of disease as well as the side effects of treatment. conclusion: it is, concluded that role of the dentist in managing these patients is giving preventive efforts and dental treatment that may be improve patient’s quality of life. the preventive effort and dental treatment is customize according to the patients condition. nevertheless, cooperation from the dentist and other professional is needed in treating these patients. key words: medical problem, oral manifestation, management, prophylaxis correspondence: roosje rosita oewen, c/o: bagian kedokteran gigi anak, fakultas kedokteran gigi universitas padjadjaran. jl. sekeloa selatan i bandung, indonesia. e-mail: arlettesuzy@yahoo.com introduction one of dentist’s main roles is to coordinate the management of special needs children. the term of special need refer to a child who has a medical condition that affects dental treatment or shows specific oral and dental manifestations. some countries called these patients as medically compromised children.1a survey to special needs children who came to special care for dentistry clinic dr. hasan sadikin hospital in bandung showed that these children have poor oral hygiene level. many general medical conditions may directly affect dental treatment and in several conditions are a consequence of dental disease, or even a dental treatment may cause an implication that leads to life threatening. increase in number of children who survive from complex medical disorder shows several abnormalities in their oral cavity. the remarkable decline in childhood mortality has led to increasing emphasis on maintaining and enhancing the quality of the child’s life and ensuring that children reach adult life as physically, intellectually, and emotionally healthy as possible. dental care can play an important part in enhancing this quality of life.2 according to the data from special care for dentistry dr. hasan sadikin hospital bandung, in 2003 there are under 10 new visits and increase to 81 new visits in july-december 2008.3 there are many systemic diseases that show oral manifestation.4 dentist should be able to recognize the manifestation so diagnoses and treatment plan can be done accurately. this paper discusses about several medical conditions in children and the dentist’s role in the treatment of those patients in the hospital. cardiovascular disorder heart diseases can be divided into two main groups, congenital heart disease and acquired heart disease.2 161oewen: the role of the dentist on medically almost every heart disease in children occurs congenitally with the prevalence of 8-10 in 1000 life birth.1,2,5 children with congenital heart disease are the most common medically compromised children seen by the dentist.1 from july–december 2008, there are 64 new visits of children with congenital heart disease to special care for dentistry dr. hasan sadikin hospital. they were refer from their pediatricians in order to have mouth preparation before heart surgery. etiology of congenital heart disease is rarely known and may be the combination of genetic and environment factors, including infection during the second month of pregnancy. several chromosome disorders such as down syndrome is related to a severe congenital heart disease.2 generally, congenital heart disease include ventricular septal defect (vsd), arterial septal defect (asd), patent ductus arteriosus (pda), and tetralogy of fallot (tof).1,2,5–7 acquired heart disease include myocarditis, infective endocarditis, and rheumatic fever. all of these diseases may cause death in children.2,5 the most important consideration in planning a dental treatment for children with cardiovascular disorder is to prevent the occurrence of dental disease. when a child is diagnosed having a heart disease, the child should immediately refer to a dentist to get a proper dental treatment and preventive efforts which include diet counseling, fluoridation, fissure sealant, and oral hygiene instruction. regular check up, clinically or radiographically for preventive efforts, is highly recommended.2,5 treatment of active dental disease should be done before heart surgery.2 invasive dental treatments, such as tooth extraction, scaling, and endodontic treatment may cause bacteriemia.5 pulpotomy is contraindicated due to the possibility to cause bacteriemia.1 if a patient is going to be treated with a treatment that may cause bacteriemia, antibiotic prophylaxis is needed to prevent the development of endocarditis.2 an antiseptic mouthwash such as chlorhexidine 0.2% can be given before dental procedure.1 during dental treatment, patient should be monitored by pulse oxymetry to evaluate his/her pulse and oxygen intake (figure 1). oxygen inhalation also needed if patient’s saturation is below 70%.6,7 children with congenital heart disease are included to a group of high risk caries, especially in primary dentition. however, there is an increase prevalence of enamel mineralization disturbance, such as enamel hypoplasia.1,5 if the child has a lot of caries, it is considerable to undergo the dental treatment under general anesthesia. this may reduce the antibiotic therapy and dental visit. dental treatment under general anesthesia needs coordination with pediatrician and anesthesiologist.1 hematology disorder in early childhood, many bleeding disorders have genetic background. common hematology disorders in children are thallasemia and leukemia.2 thallasemia is a blood disorder with the absence or lack of one of the globin chain from hemoglobin complex. normally, blood from a healthy adult contain hemoglobin a that include two chain of globin (hba, α2β2) and a minor amount of hemoglobin a2 (hba2, α2γ2). children also develop fetal hemoglobin (hbf,α2δ2). 1 dental implication of thallasemia is malformation of jaws. this was due to overgrowth in the maxilla and zygoma. class ii division 1 maloclusion is a common jaw disorder in thallasemia children. treatments for thallasemia are regular blood transfusion and the administration of desferrioxamine-an iron-chelating agent. blood transfusion may cause gingival discoloration (hemosiderosis) resulted from ferrum accumulation.1 bone involvement is the commonest clinical manifestation of thallasemia. these include the involvement of alveolar bone.9 dental treatment for patient with thallasemia needs an adequate medical history evaluation. consultation with child’s pediatrician or hematologist before initiating dental treatment is important. treatment that is highly recommended for those patients is preventive treatment and regular dental check up. dental treatment is preferable to be carried out after blood transfusion. the treatment is postponed if patient’s hemoglobin level is below 100 g/l.2 respiratory system disorder respiratory system disorder that commonly occurs in children is asthma. asthma is a diffuse obstructive lungs disease that may cause short winded, cough, and wheezing. this is related to hyperactivity of airway to any stimuli.2 children with asthma commonly receive medication with steroids. these drugs may cause extrinsic discoloration in tooth surface due to oral flora changes which results in a candidiasis. corticosteroid can also change oral cavity ph and reduces salivary flow resulting an increase in the possibility of dental erosion. children with asthma breathe figure 1. nine year-old boy with tetralogy of fallot being monitored during dental treatment.8 162 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 160−163 through mouth, may lead to the development of gingivitis and gingival enlargement in anterior part.1 dental treatment may cause emotional stress which can develop asthmatic attack. dental extraction or other treatments that need local anesthesia usually do not cause any trouble.2 generally, dental treatment for children with asthma is regular dental prophylaxis. child is ordered to wash their mouth after using steroid inhaler or other medication.1 genetic disorder child with genetic disorder usually visit a dentist with specific dental anomalies that is related to their condition or a medical problem that complicate dental treatment. not every child having genetic disorder come to the dentist. history taking can be simplified by making a simple family pedigree.1 generally, dental treatment for a child with genetic disorder is to overcome the oral complication and manifestation that related to the disorder. however, it is important for a dentist to recommend the parents or patient’s relative for genetic counseling. this is a process to make diagnostic assessment, information, and support to the family or individual who have the risk in developing genetic disorder.1 genetic disorder discussed in this paper is apert syndrome, which is a rare genetic disorder and characterized by specific abnormality of craniofacial and extremities structures. oral manifestations are prominent mandible, decline edge of mouth, cleft palate, dental malposition, crowding, delayed tooth eruption, thickened alveolar ridge, malocclusion and hard palate deformity termed byzantine arch deformity.10,11 apert syndrome is characterized by midface hypoplasia, syndactyly of the hands and feet, proptosis of eyes, steep and flat frontal bones, and premature union of cranial sutures. maxillary hypoplasia, deep palatal vault, anterior open bite, crowding of the dental arch, severely delayed tooth eruption, and dental malocclusion are the main oral manifestations of this syndrome.12 management of children with apert syndrome needs team approach that consist of craniofacial surgeon, neurosurgeon, oral surgeon tnt specialist, audiologist, speech pathologist, psychology, ophthalmologist, pedodontist, dan orthodontist.2,9 the main treatment method is surgery that is needed to correct craniofacial abnormalities and fused fingers and toes. beside surgery, the treatment also termed to correct upper respiratory tract, eyes deformities, or abnormalities in dental area.11 discussion many medical disorders may directly affect dental treatment and in several conditions it is a consequence of dental disease, thus it may leads to life threatening. children with medically compromised condition sometimes have to be treated in hospital. these may lead to a lack of dental care that resulted in complex oral manifestations. lack of knowledge from their parents about the importance of maintaining oral health may exacerbate the existing medical conditions. early professional’s intervention is very important in carrying examination, risk assessment, and giving information and tutorial, thus oral diseases can be prevented.5 congenital heart disease is more common occur in children than acquired heart diseases. many heart diseases need antibiotic prophylaxis before undergoing invasive dental treatment. another important aspect to be carried out is patient’s monitoring during dental treatment. these includes oxygen administration and observation of pulse and oxygen saturation with pulse oxymetri (figure 1).2 child with bleeding disorders, such as hemophilia, thrombocytopenia, and von willebrand’s disease, have to be checked their hematological status before carrying out dental treatment. hematologic replacement therapy figure 2. (a) patient’s face, (b) byzantine arch deformities.13 a b 163oewen: the role of the dentist on medically may be needed before operative treatment. bleeding disorder in form of anemia is in a risk of dental treatment under general anesthesia.2 the importance of adequate dental plaque control techniques in order to prevent inflammation, potential bleeding and infection in these patients is emphasized. the pediatric dentist must be aware of the clinical appearance of bleeding disorder in order to recognize the condition and successfully manage the patient.14 child with genetic disorder needs a special attention in their oral and dental care especially due to the complexity of the abnormalities in oral cavity. these include the abnormality of dentocraniofacial complex.9,10 dental treatment of these patient usually needs specific team approach consists of pedodontist, pediatrician, tnt specialist, anesthesiologist, and surgeon.11 increased number of children who survive from complex medical problem may be due to the increase of the advanced medical treatment and the management of oral manifestation and complication through oral and dental prophylaxis. role of the dentist in managing these patients is giving preventive efforts and dental treatments that may be improve patient’s quality of life. nevertheless, cooperation from the dentist and other professional is needed in treating these patients. in conclusion managing children with congenital heart disease, dentist may make intervention as early as possible after a child being diagnosed having a heart disease. with preventive efforts, the child will have a good oral conditions so they can undergo heart surgery. children with hematology disorder, such as thallasemia, have dental implication due to malformation of the jaw which lead to class ii division i maloclusion. the role of the dentist is to minimize this maloclusion so the child may have a “normal” appearance. these is also due to children with genetic disorder. the dentist’s role is to correct the abnormalities in dental area. children with respiratory system disorder have dental implication due to the drugs they used to treat their conditions. the role of the dentist in this children is to minimize the side effect of the drugs to their oral environment. references 1. cameron ac, widmer rp. handbook of pediatric dentistry. 2nd ed. sydney: cv mosby; 2003. p. 234–84. 2. welbury rp. paediatric dentistry. 2nd ed. new york: oxford university press; 2001. p. 369–90. 3. data kunjungan klinik special care for dentistry smf gigi dan mulut rumah sakit dr. hasan sadikin bandung tahun 2008. 4. long rg, hlousek l, doyle jl. oral manifestations of systemic diseases. ms journal october-november 1998; 54(5, 6): 309–15. 5. koch g, poulsen s. pediatric dentistry, a clinical approach. copenhagen: munksgaard; 2001. p. 445–61. 6. baraas f. penyakit jantung pada anak. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 1995. p. 140–51. 7. tetralogy of fallot. texas: texas heart institute. available from url: www.americanheart.org. accessed nopember 28, 2005. 8. pertiwi asp, sasmita is, nonong yh. oral and dental management in children with tetralogy of fallot. dental journal (majalah kedokteran gigi) 2007; 40(1): 43. 9. antonio a, irene r. bone involvement in sickle cell disease. british journal of haematology 2005 may; 129(4): 482–90. 10. campis lb. children with apert syndrome: developmental and psychologic consideration. clinics in plastic surgery april 1991; 18(2): 409–16. 11. wilkie ao. fgfs their receptors and human limb malformations: clinical and molecular correlations. american journal of medical genetic 2002; 112(3). available at : www.thecraniofacialcenter.org accessed november 28, 2005. 12. tosun g, sener y. apert syndrome with glucose-6 phosphatase dehydrogenase deficiency: a case report. international journal of pediatric dentistry 2006 april; 16(3): 218–21. 13. pertiwi asp, hidayat s. sindrom apert pada seorang anak laki-laki: tinjauan dari aspek gigi dan mulut. majalah kedokteran bandung 2004; 36(4): 163. 14. vaisman b, medina ac, ramirez g. dental treatment for children with chronic idiopathic thrombocytopaenia purpura. international journal of paediatric dentistry 2004 september; 14(5): 355–62. 15. little jw, falace da, miller cs, rhodus nl. dental management of the medically compromised patient. 6th ed. missouri: the mosby inc; 2002. p. 52–5, 147–60. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no 1-2005 45 perbedaan khasiat antibakteri bahan irigasi antara hidrogen peroksida 3% dan infusum daun sirih 20% terhadap bakteri mix (the comparison of the antibacterial effect of irrigation solutions hydrogen peroxyde 3% and piper betle folium infusum 20% to bacterial mix) dian agustin w bagian konservasi gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract piper betle folium was indonesian’s traditional medicinal herb. the most important element in its was phenol with has antibacterial effect. h2o2 3% was commonly used because readily available, can lift up debris from root canal preparation. the purpose of this study was to determine the antibacterial effect of two solutions: hydrogen peroxide 3% and piper betle folium infusum 20% to bacterial mix invitro. the twenty specimens are divided into two groups. the result is statistically analyzed using wilcoxon man whitney test. this study showed that piper betle folium infusum 20% had greater antibacterial effect than h2o2 3% solution. key words: piper betle folium, h2o2 3%, antibacterial effect korespondensi (correspondence): dian agustin w, bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan pada penelitian awal ditemukan beberapa spesies di antaranya streptococci, micrococci, dan sejumlah kecil bakteri anaerob pada infeksi saluran akar maupun penyakit periradikular. bakteri anaerob meliputi 90% dari bakteri penyebab infeksi saluran akar.1 berdasarkan temuan tersebut, ternyata penyebab infeksi saluran akar tidak hanya satu macam bakteri tetapi berbagai macam bakteri yang terlibat termasuk organisme anaerob seperti porphyromonas, bacterioides gingivalis, phorphyromonas bacterioides endodontalis, dan prevotella bacterioides buccae yang dinamakan bacterioides spesies. tahapan penting dalam perawatan saluran akar gigi yang terinfeksi adalah preparasi, sterilisasi dan pengisian.2 preparasi saluran akar gigi akan menunjang proses sterilisasi dan menghasilkan pengisian yang baik sehingga didapatkan hasil yang maksimal.3 pada tahap preparasi diperlukan bahan irigasi saluran akar yang bertujuan menghilangkan jaringan nekrotik, tumpukan serpihan dentin dan membasahi saluran akar gigi sehingga mempermudah dalam pelaksanaan preparasi serta pengurangan jumlah mikroorganisme di dalam saluran akar kemudian sisa bakteri dimatikan dengan obatobatan.3,4 bahan yang dapat digunakan untuk irigasi antara lain hidrogen peroksidase (h2o2) 3%, naocl, 3%, edta 15%, chlorhexidine, akuades. hidrogen peroksidase (h2o2) 3% merupakan salah satu bahan irigasi yang sering digunakan karena mudah didapat, dapat mengangkat kotoran dari hasil preparasi saluran akar. penggunaan larutan h2o2 3% diikuti dengan larutan irigasi lainnya misal akuades, karena sisa oksigen peroksida dalam saluran akar harus dinetralisir atau dihilangkan. oksigen yang terjadi akan menghasilkan gelembung udara kemudian akan membantu pengeluaran kotoran secara efektif. walaupun demikian sekarang metode ini tidak dianjurkan karena oksigen yang tersebut dapat terbawa keluar menuju jaringan periapikal dan menimbulkan empisema.4 hidrogen peroksidase (h2o2) 3% harus dibersihkan dari kavitas gigi sebelum kavitas ditutup, karena evaluasi oksigen setelah penutupan dapat mendorong kotoran dan mikroorganisme ke jaringan periapikal.5 semula dikatakan bahwa h2o2 3% dianggap dapat mengeluarkan debris karena mempunyai aksi nascent berbusa namun tidak terbukti karena ternyata peningkatan debridement tidak terjadi. hal ini disebabkan oleh terbatasnya daya antibakteri dari bahan ini.4 di indonesia terdapat tanaman sirih, yang khasiat daunnya telah banyak digunakan6,7,8 efek astringent bahan ini, telah diketahui sebagai obat kumur, tidak menimbulkan iritasi selaput lendir rongga mulut. pada konsentrasi 20% bekerja lebih baik terhadap streptococcus viridans.6,9,10 dalam daun sirih 100 gram terdapat kandungan:11 air 85,4 mg; protein 3,1 mg; karbohidrat 6,1 mg; serat 2,3 mg; yodium 3,4 mg; mineral 2,3 mg; kalsium 230 mg; fosfor 40 mg; besi ion 3,5 mg; karoten (vitamin a) 9600 iu, kalium nitrat 0,26–0,42 mg; tiamin 70 mg; riboflavin 30 mg; asam nikotinal 0,7 mg; vitamin c 5 mg; kanji 1,0–1,2%; gula non reduksi 0,6–2,5%; gula reduksi 46 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 45–47 1,4–3,2%. sedangkan minyak atsirinya terdiri dari: alilkatekol 2,7–4,6%; kadinen 6,7–9,1%; karvakol 2,2–4,8%; kariofilen 6,2–11,9%; kavibetol 0,0–1,2%; kavikol 5,1–8,2%; sineol 3,6–6,2%; eugenol 26,8– 42,5%; eugenol metil eter 26,8–15,58%; pirokatekin. penelitian ini bertujuan untuk mengetahui perbedaan khasiat antibakteri bahan irigasi antara h2o2 3% dan infusum daun sirih 20% terhadap bakteri mix. hasil penelitian ini diharapkan dapat meningkatkan manfaat daun sirih sebagai tanaman obat tradisionil. bahan dan metode rancangan penelitian yang digunakan adalah aksperimental laboratories. bahan yang digunakan dalam penelitian ini adalah infusum daun sirih 20% dan h2o2 3%. infusum daun sirih 20% didapatkan dari daun sirih yang segar dipotong-potong dengan diameter kurang lebih 0,5 cm, kemudian dikeringkan di bawah sinar matahari sampai berwarna kehitam-hitaman dan rapuh (mudah diremas). daun sirih yang telah kering dihaluskan dengan menggunakan blender untuk mendapatkan bubuk daun sirih. untuk mendapatkan infusum ambil 10 gram bubuk daun sirih ditambah akuades 100cc dididihkan selama 15 menit kemudian diambil 80cc dididihkan lagi sampai mendapat 40cc. sedangkan sediaan h2o2 3% didapatkan dari apotik yang telah tersedia dalam sediaan botol. untuk mendapatkan bakteri dalam media brain heart infusion broth (bhib), pertama masukkan paper poin steril ke dalam saluran akar gigi untuk mendapatkan bakteri mix dengan diagnosa nekrosis pulpa selama 1menit. paper poin yang telah dimasukkan dikeluarkan dan dimasukkan ke dalam tabung media yang mengandung bhib kemudian dimasukkan ke dalam inkubator selama 24 jam. blood agar yang telah jadi dibagi menjadi 4 zona, kemudian dilakukan pemberian bakteri yang telah dibiakkan di dalam bhib. selanjutnya diberi perlakuan penetesan h2o2 3% dan penetesan infusum daun sirih dengan menggunakan mikro pipet pada kertas serap masing-masing satu tetes (10 microliter) yang telah ditaruh di atas blood agar. kemudian dimasukkan ke dalam inkubator selama 48 jam. selanjutnya untuk mengetahui daya hambatnya dilakukan pengukuran zona inhibisi, zona hambatan tersebut ditandai dengan adanya daerah jernih di sekeliling kertas serap. makin besar zona hambatan menunjukkan bahwa khasiat antibakteri bahan tersebut makin kuat. cara pengukuran dengan menggunakan kaliper dengan mengukur panjang dan lebar kertas serap ditambah daerah jernih kemudian dibagi dua. hasil analisis data menggunakan uji wilcoxon man whitney test. melihat perbedaan antara diameter zona hambatan pertumbuhan bakteri mix oleh bahan irigasi h2o2 3% dan infusum daun sirih 20% (tabel. 1) tabel 1. rerata diameter zona hambatan pertumbuhan bakteri mix dengan hidrogen peroksida 3% dan infusum daun sirih 20% bahan n x (mm) sd p h2o2 3% 10 0,8 0,48 0,001* infusum daun sirih 20% 10 8,53 1,04 (< 0,05) keterangan: * berbeda bermakna dari hasil uji wilcoxon beda diameter zona hambatan pertumbuhan bakteri mix oleh bahan irigasi hidrogen peroksida 3% dan infusum daun sirih 20% diperoleh nilai p = 0,001 yang berarti terdapat perbedaan bermakna antara besar diameter zona hambatan bakteri mix oleh bahan irigasi hidrogen peroksida 3% dan infusum daun sirih 20%. pembahasan pada tahap preparasi diperlukan bahan irigasi, diharapkan semua kotoran yang berada di dalamnya akan ikut mengalir keluar bersama dengan cairan irigasi. bahan irigasi saluran akar sebaiknya bersifat antiseptik yaitu dapat merusak, dapat menghambat reproduksi atau metabolisme mikroba dan sekaligus menstrerilkan saluran akar.2,3,4adapun syarat bahan antiseptik saluran akar adalah mampu membunuh mikroba organisme, mempunyai efektifitas yang cepat mampu mengadakan penetrasi yang dalam, tetap efektif dengan adanya bahan organik, tidak merubah warna gigi, secara kimia bersifat stabil, tidak berbau dan tidak berasa, ekonomis.2,3,4 hasil penelitian menunjukkan bahwa diameter zona hambatan bakteri mix oleh infusum daun sirih 20% lebih besar dari hidrogen peroksida 3% dan berbeda bermakna (p < 0,05 ), berarti infusun daun sirih 20% mempunyai efek antibakteri lebih kuat dari hidrogen peroksida 3%. infusum daun sirih mengandung minyak atsiri yang di dalamnya terdapat senyawa phenol yang bersifat bakterisid.10 senyawa phenol apabila terjadi interaksi dengan dinding sel mikroorganisme akan terjadi denaturasi protein dan meningkatkan permeabilitas mikroorganisme.10 interaksi antar mikroorganisme mengakibatkan perubahan keseimbangan muatan dalam molekul protein, sehingga terjadi perubahan struktur protein dan menyebabkan terjadinya koagulasi. protein yang mengalami denaturasi dan koagulasi akan kehilangan aktivitas fisiologis sehingga tidak dapat berfungsi dengan baik. perubahan struktur protein pada dinding sel bakteri akan meningkatkan permeabilitas sel sehingga pertumbuhan sel akan terhambat dan kemudian sel menjadi rusak. selain itu senyawa kavikol memberikan bau yang khas pada daun sirih dan memiliki daya bunuh bakteri 5 kali lebih besar dari phenol. senyawa kariofilen bersifat antiseptik dan anestetik lokal, sedangkan senyawa eugenol bersifat antiseptik dan analgesik topikal. 47agustin: perbedaan khasiat antibakteri hidrogen peroksida merupakan larutan yang terbentuk dari reaksi asam sulfat dan barium peroksida.3 hidrogen peroksida 3% apabila berinteraksi dengan darah, pus, serum, air liur dan bahan organik lainnya akan menghasilkan h2o + onascent. efek tersebut mengangkat kotoran dalam saluran akar. berdasarkan penguraian senyawa h2o2 menjadi h2o + onascent. onascent yang timbul bersifat sementara, selanjutnya akan berubah menjadi o 2. gas oksigen yang terbentuk akan menghancurkan kuman anaerob beserta bahan yang dihasilkan.10 daya antibakteri hidrogen peroksida 3% kurang terhadap bakteri mix saluran akar karena sebagian besar bakteri dalam saluran akar bersifat fakultatif anaerob (streptococcus viridans) yang dapat hidup dengan atau tanpa adanya oksigen. oleh karena itu oksigen dari hidrogen peroksida 3% tersebut tidak dapat membunuh bakteri mix. dari hasil penelitian ini dapat ditarik kesimpulan bahwa khasiat antibakteri infusum daun sirih 20% lebih baikdari hidrogen peroksida 3% terhadap bakteri mix. diharapkan infusum daun sirih dapat dimanfaatkan sebagai bahan irigasi. daftar pustaka 1. burnett gw, schuster gs. oral microbiology and infection disease. london: william and wilkins; 1980. p. 62–66, 141–60. 2. cohen s, burns rc. pathway of the pulp. 5th ed. st louis: mosby co; 2002. p. 123–47. 3. grossman li, oliet s, del rio ce. endodontic practic. 12nd ed. philadelphia: lea and febiger; 1995. p. 196–262. 4. walton re, torabinejad m. prinsip dan praktik ilmu endodonsia. edisi ii. jakarta: buku kedokteran egc; 1996. hal. 262–81. 5. nicholls e. cleaning and preparation of the pulp cavity, endodontic. 3rd ed. 1988. p. 145–9. 6. www.lembaga.wima.ac.id/ippm/ppot/isi, 2004. 7. kartasaputra g. budidaya tanaman berkhasiat obat. edisi ii. jakarta: pt rineksa cipta; 1992. h. 25–26. 8. darwis sn. potensi sirih sebagai tanaman obat. jogjakarta: makalah dalam seminar sirih. kelompok kerja nasional tanaman obat indonesia. 1991. 9. indah ts, retno s, ristanto. pengaruh teknik penyimpanan daun sirih sebagai obat kumur terhadap akumulasi plak gigi dan pertumbuhan bakteri s sanguis. jogjakarta: laporan penelitian: ugm; 1990. 10. mieke hs, yanti m, razak u. usaha pemeriksaan daya anti mikroba dan ekstrak daun sirih terhadap beberapa bakteri. denpasar: konas xvi. pdgi; 1985. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 159159 vegf expression and new blood vessel after dental x-ray irradiation on fractured tooth extraction wound niluh ringga woroprobosari,1 jenny sunariani,2 and eha renwi astuti3 1department of dental radiology, faculty of dentistry, universitas islam sultan agung, semarang-indonesia 2department of oral biology, faculty of dental medicine, universitas airlangga, surabaya-indonesia 3department of dental radiology, faculty of dental medicine, universitas airlangga, surabaya-indonesia abstract background: dental x-ray has an important role in dentistry. complication case such as tooth fracture extraction requires this examination to determine the appropriate treatment measures. dental x-ray can also cause a negative impact to the body at cellular and even molecular level. purpose: the aim of this study was to evaluate the decrease of vascular endothelial growth factor (vegf) expression and new blood vessels number caused by dental x-ray irradiation on fractured tooth extraction wound on day 3 and 7 after extraction. method: we used 30 wistar rats which was randomly divided into 6 groups. each rat’s central insisive of left mandible was fractured and then extracted after or without x-ray irradiation. group ka and kb were control groups without irradiation. group p1 a and p1 b were treatment groups with 0.08 msv irradiation dose. group p2 a and p2 b were treatment groups with 0.16 msv irradiation dose. the subject from group ka, p1 a, and p2 a were sacrficed and sockets were collected at day 3. the subject from group kb, p1 b, and p2 b were sacrficed and sockets were collected at day 7. socket were processed and painted with hematoxylin eosin and immunohistochemistry, then observed with a microscope. data processing was performed with spss 16 through one way anova test and post hoc tukey test hs. result: the lowest means expression of vegf and the number of new blood vessels on the day 3 was found in p2 a group, and the highest found in the ka group. the lowest means expression of vegf and the number of new blood vessels on the day 7 was found in p2 b group, and the highest found in the kb group. conclusion: dental x-ray irradiation dose of 0.08 msv and 0.16 msv causes decrease of vegf expression and new blood vessels in the wound fractured tooth extraction in day 3 and day 7 post-extraction. keywords: x-ray irradiation; vegf; angiogenesis; socket healing correspondence: niluh ringga woroprobosari, departemen radiologi kedokteran gigi, fakultas kedokteran gigi universitas islam sultan agung. jl. kaligawe semarang, indonesia. e-mail: niluhringga@gmail.com introduction tooth extraction is a common action performed by a dentist. tooth extraction process can cause complications with a prevalence of 37.6% in indonesia. fractures are the most common complication encountered in indonesia with a prevalence of 30.4%.1 tooth extraction injures the dental tissues area where the tooth extracted, including sockets, mucosa, and blood vessels. this leads to the wound healing process by the body.2 angiogenesis is a process occured in the proliferation phase.3 proliferation phase occurs in the range of day 3 to day 5 post-injury. this process is characterized by the formation of new blood vessels around the wound.4 vascular endothelial growth factor (vegf) is a growth factor initiates the formation of new blood vessels through the formation mechanism of budding.5 dental x-ray plays an important role to support a diagnosis and to determine appropriate treatment, such as in the fracture extraction case.6 the common radiographs used to examine tooth extraction fractures is periapical radiographs.7 the use of dental x-ray irradiation in medical examination can also give negative impacts. soft x-ray irradiation at 50 rad, 100 rad, and 700 rad can slow down the wound healing process, inhibit cells proliferation, and activate cell apoptosis. soft x-ray irradiation also inhibits the cells cycle at day 3 up to day 5, which may be one of research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 160–165 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 160 woroprobosari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 159–164 the cellular mechanism to slow down the wound healing process.8 irradiation can inhibit the early inflammatory response by reducing the infiltration of macrophages and neutrophils. irradiation can also damage the blood vessels, resulting in the formation and maturation of granulation tissues are inhibited. fibroblasts are damaged and re-epitelisation process becomes slow cause the wound healing process requires more amount of time.9 dental x-ray periapical irradiation at 0.08 msv dose can cause apoptosis in mucosal cells sudah dapat menyebabkan apoptosis pada sel mukosa. irradiation dose 0.08 msv is the dosage used for periapical radiographic examination.10 researchers observed the expression of vegf and the number of new blood vessels after exposed to dental x-ray irradiation on the fracture wound of wistar rat tooth extraction. vegf expression was observed through immunohistochemistry examination, while the formation of new blood vessels was observed through histological preparation observation with hematoxylin eosin staining. tissues sampling was collected on the third day posttreatment since the beginning of proliferation phase, and on the seventh day post-treatment for a final extended period of proliferation phase if the wound healing process is inhibited.5 materials and methods this research is laboratory experiment with post-test only control group design.11 the sample was selected by using simple random sampling of the population that met the need of inclusion criteria. thirty male wistar rats were used for this research and were divided into 6 randomized groups in which each group consists of 5 male wistar rats. the control groups are ka and kb group with fracture of the left mandibular central incisors extraction without dental x-ray exposure. the treatment groups consist of p1 a and p1 b group with fracture of the left mandibular central incisors extraction and 0.08 msv dental x-ray exposure, and p2 a and p2 b group with fracture of the left mandibular central incisors extraction and 0.16 msv dental x-ray exposure. ka, p1 a, and p2 a groups were examined o the third day, while kb, p1 b, and p2 b groups were examined on the seventh day after extraction. the research subjects were adaptated first to the environment of laboratpry of biochemistry, faculty of medicine, universitas airlangga for 7 days with the treatment on the form of food, drink, and appropriate cage that meets the ethical standard of health research ethics and airworthiness committee of faculty of dental medicine, universitas airlangga. the fracture extraction were performed to the whole subject of research on the left mandibular central incisor, after being given intramuscular ketamine and diazepam anaesthesia. each rat was in supine position (abdomen above) and the tongue of the rat was held under a sterile gauze rolls. sonde was inserted in the depth of 2 mm into the gingival sulcus of the left mandibular central incisor and was moved around the sulcus to damage the periodontal ligament. the left mandibular central incisor was fractured by using diamond bur. the extraction of the remaining part of tooth fractures was resumed after dental x-ray exposure to the treatment group. the rats were fixed by using wire netting first so that they could not move when the exposure was given. cone beam periapical air photo was directed to the left mandibula of the rat. the extraction of the remaining fractures was performed after the dental x-ray irradiation was given. the research subjects were anaesthetized by using 10% ether solution. each rat was put in a glass box with a lid, then the entire wall of the inner box was sprayed with 10% ether and was closed again so that the steam can be fully inhaled by the rats. left mandible was taken and was immediately put in a container of nbf 10 fixated solution. mandibular tissues fixation was performed in 10% neutral buffered formalin (nbf). the next stage was decalcification so that the calcium content within the tissues becomes lost and facilitates the tissues processing to the next stage. soft bone tissues were dehydrating, clearing, and then embedding into paraffin blocks to be easily cut with microtome. the pieces were placed into waterbath by using sengkelit with 40-50°c temperature, until they stuck into object glass, and then were painted with hematoxylin staining eosin.12 vegf observation was performed by using monoclonal antobody vegf santa cruz, sc-152 and the kit novolink, novocastra, re7230-k. the painting process was performed according to the instructions of use. then the preparation was performed with mounting by using entelan.12 the results reading was done by observing the preparation under the light microscope with 1000x magnification. new blood vessels were observed by counting the number of lumens arterioles and venules around the sockets on the former extraction in one visual field quantitatively. vegf expression was calculated by examining the positive expression of the growth factor on the dark brown coloured macrophage cells, in which this interpretation was done quantitatively. the reading of vegf expression and new blood vessels were done five times visual field for each sample, before taking the mean value. the data were analyzed by spss 16 program through one way anova test and post hoc test tukey hs. results the mean and standard deviation value of vegf expression and the number of new blood vessels after the exposure of dental x-ray irradiation on fracture wound of wistar rat tooth extraction can be seen in table 1. figure 1 is a picture of vegf positive expression. the figure of vegf positive expressions are dark brown coloured and evenly circulated around blood vessel lumens in which can be observed in figure 1-a (control group a) and b (control group b). figure 1-c shows vegf expressions in group p1 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 161161woroprobosari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 159–164 figure 1. the figure of immunohistochemistry on vegf expression (circled and pointed by black small arrow  blood vessel lumens) with monoclonal antibody vegf anti-rats and paint material dab on sockets of wistar rats tooth extraction. the observation was performed by microscope with 1000x magnification. positive expression is shown by the coloured figure around the blood vessel lumens. (a) group ka; (b) group kb; (c) group p1 a; (d) group p1 b; (e) group p2 a; (f) group p2 b. figure 2. the figure of histological lumens of new blood vessels (circled and pointed by yellow small arrow  blood vessel lumens) with hematoxylin eosin painting. the observation was performed by microscope with 1000x magnification. (a) group ka; (b) group kb; (c) group p1 a; (d) group p1 b; (e) group p2 a; (f) group p2 b. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 162 woroprobosari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 159–164 a which are brown coloured and less than in figure a and b. the observation of group p1 b in figure 1-d resulting in the similar figure to figure 1-c. vegf expressions are brown coloured and the least one is seen in figure 1-e (group p2 a) and figure 1-f (group p2 b). the observation result of the new blood vessel can be seen in figure 2. figure 2-a shows a picture of the new and dense blood vessels in control group ka. the figure of dense blood vessels can also be seen in figure 2-b which is in control group kb. the density of blood vessels can be seen from the lumens density of blood vessels formed. group p1 a shows the rarer figure of blood vessel lumens in figure 2-c than in figure a and b. it is also shown in figure 2-d, that is in group p1 b. figure 2-e and f show the least density in group p2 a and p2 b, when are compared to the previous figures. the data were then processed by using spss 16.0 program for windows. first statistical test was conducted by using kolmogorov smirnov test to find out the distribution of the research data as a requirement prior to the one way anova test. the test results of all groups show that p value is greater thab 0.05 so it can be stated that all data were normally distributed. levene test was also conducted first to see the variance homogenity of the groups that will be compared. the results of levene test show that the values were above 0.05, so it can be stated that the variance of the entire data is homogeneous variant. the analysis was then followed by parametric test using one way anova test because the data had normally distributed and had homogeneous variances. the analysis was continued by post hoc test tukey hs to compare whether there are any differences between each researched group. discussion the mean values of each group showed that the greater the dose of dental x-ray irradiation is given, the lesser the vegf expression in the fracture sockets of tooth extraction on the day-3 and day-7. the decreased expression of vegf is caused by the biological effects of ionized radiation. the biological effects can occur without the interference of threshold radiation dose, namely stochastic effects. stochastic effects are often found in dna damage. dna damage can be directly or indirectly resulted from the formation of free radicals and water molecules.13 in the research conducted by saputra,10 the exposure of dental x-ray irradiation was given to the buccal mucosa of wistar rats. the result showed that there was an increase of apoptosis and necrosis cells which were proportional to the increased dose of dental x-ray irradiation as much as 0.8 msv, 0.16 msv, and 0.24 msv. those results correspond with the results in this research, that there is damage to the table 1. the mean value of vegf expression and new blood vessels on the day-3 and 7 group vegf expression mean ± sd the number of new blood vessels mean± sd day-3 (a) day-7 (b) day-3 (a) day-7 (b) k 5.80±1.09 6.60±1.52 455.00±57.41 420.80±90.47 p1 5.40±1.67 6.20±1.48 383.00 ±41.67 315.20±80.12 p2 3.00±1.23 2.60±1.14 276.20±67.11 192.00±35.34 notes: k= fractures of wistar rats tooth extraction without dental x-ray irradiation (control); p1= fractures of wistar rats tooth extraction with dental x-ray irradiation 0.08 msv; p2= fractures of wistar rats tooth extraction with dental x-ray irradiation 0.16 msv. table 2. the p value post hoc test tukey hs of vegf expression on the day-3 researched group ka p1 a p2 a ka 0.888 0.017 p1 a 0.888 0.039 p2 a 0.017 0.039 table 3. the p value post hoc test tukey hs of vegf expression on the day-7 researched group kb p1 b p2 b kb 0.893 0.002 p1 b 0.893 0.004 p2 b 0.002 0.004 table 4. the p value post hoc test tukey hs of new blood vessels on the day-3 researched group ka p1 a p2 a ka 0.150 0.001 p1 a 0.150 0.028 p2 a 0.001 0.028 table 5. the p value post hoc test tukey hs of new blood vessels on the day-7 researched group kb p1 b p2 b kb 0.095 0.001 p1 b 0.095 0.049 p2 b 0.001 0.049 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 163163woroprobosari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 159–164 body either cellular or molecular due to the exposure of dental x-ray irradiation even though only in small doses. the damage caused by the exposure to the dental x-ray irradiation is proportional to the increased doses given. macrophages, neutrophils, endothels, fibroblasts and smooth muscle cells are the cells that produce vegf. monocytes are the most damaged cells when experiencing oxidative stress compared to macrophages. the study showed that monocytes which were given oxidative stressor such as tert-buyl hydroperoxide (booh) 400µm did not express protein poly adp-ribose) polymerase 1 (parp-1) and x-ray repair cross-complimenting protein 1 (xrcc1). the study identified that the probable cause is the transcriptional down-regulation on monocytes. the absence of xrcc1 expression resulted in the decreased expression of lig iiiα (deoxyribonucleic acid ligase iiiα) for xrcc1 is a protein that stabilizes lig iii. protein deoxyribonucleic acid protein kinase catalytic subunit (dna-pkcs) that plays role in the double strand repairment is also not detected on monocytes.14 monocytes are more sensitive to oxidative agents than macrophages and neutrophils as monocytes are the progenitor cells that differentiate into macrophages and dendritic cells.15 energy absorption of radiation by water molecules within cells causes molecules excitation and ionizing. ions generated from the reaction will form free radicals within the body and can penetrate the lipid bilayer of the membranes and then lead to the cell damage.16 the decreasing of monocytes results in the decreasing of macrophages within sockets. in addition, the cells ability to repair themselves and adapt to the ionizing x-rays radiation will be limited. most of macrophage cells will fail to adapt dan will be exposed to the bilogical effects of the radiation, which whill lead to the cells death. macrophage cells that die will become debris and can inhibit the recovery of the other cells. such condition will cause the decreasing of vegf, as monocytes and macrophages are two types of cells involved in releasing vegf to initiate a series of angiogenesis processes. cell damage from dental x-ray irradiation does not only occur on monocytes alone. the number of cells on peritoneal macrophages culture of the rats decreases after exposed to x-ray irradiation at dose of 3 gy nd 6 gy.15 the damage on neutrophils, monocytes, and macrophages will result in the decreased expression of vegf, in accordance with the results of this study. the comparisons between group ka and p1 a, and group kb and p1 b have no significant difference in the statistical tests. however, the data averagely showed the difference of vegf expressions between the each group. the non-significant result may be caused by the biological effects resulted in dental x-ray irradiation at a dose of 0.08 msv which was minimum and could be neutralized by the body, so that the data still showed the decreased espression of vegf but did not appear statistically. the mean result of new blood vessels from each group shows that the gretaer the dose of dental x-ray irradiation is given, the lesser the number of new blood vessels formed around the sockets of fracture extraction on the day-3 and day-7.17 the decreased number of new blood vessels as the increased dose of radiation are also related to the decreased expression of vegf and the cells on the fracture sockets of tooth extraction. vegf is a cytokine that initiates the angiogenesis and vaskulogenesis processes. the decreased expression of vegf results in the inhibition of endothelial signaling process to move forming the bud. barriers bud formation lead to the formation of capillary network become blocked, so that there is a decrease in the number of new blood vessels in sockets.18 the biological effects of ionizing x-rays radiation also directly affect the cells cycle. soft x-ray irradiation at dose of 5.21 gy slows down the wound healing rate on the third until the ninth day. this is caused by the cells cycle disruption, in which the cells stop in phase g0/g1 and s, and becomes slow in phase g2/m. 8 cells cycle disruption results in the reduced proliferation and the increased apoptosis process, causing an imbalance in the proliferation stage. the decreased proliferation of endothelial cells and fibroblasts hamper the formation of new blood vessels which affects the next wound healing stage.15 there is no significant difference in the comparison between group ka and p1 a and between group kb and p1 b. however, the average number of new blood vessels still shows the differences where the mean value of group p1 a is smaller than the mean value of group ka, and the mean value of group p1 b is smaller than the mean value of group kb. this result may be caused by the biological effects on dental x-ray irradiation at dose of 0.08 msv that results in new blood vessels are still minimum, so they do not appear statistically compared to the group without dental x-ray irradiation. in conclusion, dental x-ray irradiation dose of 0.08 msv and 0.16 msv causes decrease of vegf expression and new blood vessels in the wound fractured tooth extraction in day 3 and day 7 post-extraction. references tim riskesdas. penyakit tidak menular: kesehatan gigi. riskesdas 1. 2007. departemen kesehatan republik indonesia; 2008. p. 13047. coulthard p, keith h, philip s, theaker ed. master dentistry: oral 2. and maxillofacial surgery, radiology, pathology and oral medicine. 2nd ed. edinburg: churchill livingstone; 2008. p. 241-5. monaco jl, lawrence wt. acute wound healing: an overview. clin 3. plastic surg 2003; 30: 1-12. mendonça rj. angiogenesis in wound healing. in: davies j, editor. 4. tissue regeneration from basic biology to clinical application. rijeka: intech; 2012. p. 93-98. bao p, kodra a, tomic-canic m, golinko ms, ehrlich hp, brem 5. h. the role of vascular endothelial growth factor in wound healing. j surg res 2009; 153(2): 347–58. williamson gf. best practices in intraoral digital radiography. 6. rdhmag 2011; 11(11): 79-89. peterson jl. oral and maxillofacial surgery. 47. th ed. st. louis: mosby co; 2003. p. 116-7. liu x, liu jz, zhang e, li p, zhou p, cheng tm8. . impaired wound healing after local soft x-ray irradiation in rat skin: time course dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 164 woroprobosari, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 159–164 study of pathology, proliferation, cell cycle, and apoptosis. j trauma. 2005; 59(3): 682-90. gu q, wang d, cui c, gao y. effects of radiation on wound healing. 9. j environ pathol toxicol oncol 1998; 17(2): 117-23. saputra d. apoptosis dan nekrosis sel mukosa rongga mulut akibat 10. radiasi sinar-x dental radiografik. surabaya: universitas airlangga; 2012. p. 61-78. notoatmodjo s. metodologi penelitian kesehatan. jakarta: pt. asdi 11. mahasatya; 2005. p. 167. survana sk, layton c, bancroft jd. bancroft’s theory and practice 12. of histological techniques. 7th ed. oxford: elsevier ltd; 2013. p. 70-139, 172-86, 316-52. alatas z. efek kesehatan pajanan radiasi dosis rendah. prosiding 13. seminar aspek keselamatan radiasi dan lingkungan pada industri non-nuklir. jakarta: batan; 2003. p. 28-38. bauer m, goldstein m, christmann m, becker h, heylmann d, 14. kaina b. human monocytes are severely impaired in base and dna double-strand break repair that renders them vulnerable to oxidative stress. pnas 2011; 108 (52): 21105-10. cunha a, lourenço a, cancela j, castelhano j, carvalho s, saiote 15. a, et al. effect of ionizing radiation on rat peritoneal macrophages. revista do detua 2007; 4(7): 818-21. azzam ei, jay-gerin j, pain d. ionizing radiation-induced metabolic 16. stress and prolonged cell injury. can let 2012; 327(2012): 48-60. kumar v, abbas ak, fausto n, aster j. robbins and cotran 17. pathologic basis of disease. 8th ed. philadelphia: saunders elsevier; 2010. p. 54-71, 102-04. lalani z, wong m, brey em, mikos ag, duke pj, miller mj, 18. et al. spatial and temporal localization of fgf-2 and vegf in healing tooth extraction sockets in a rabbit model. j oral maxillofac surg 2005; 63(10): 1500-07. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p159-164 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p159-164 49 vol. 43. no. 2 june 2010 case report erythema multiforme as the result of taking carbamazepine maharani laillyza apriasari� and m. jusri�� 1 faculty of dentistry, lambung mangkurat 2 department of oral medicine, faculty of dentistry, university of airlangga surabaya indonesia abstract background: erythema multiforme is an acute mucocutaneus disease which is caused by the hypersensitivity reaction. it is characterized by target lesions on the skin or ulcerative oral lesion. etiology of the disease is unknown, it is currently considered as immunologic disease. the triggering factors is the use of certain type of drugs like antibiotics, anticonvulsant, and nsaid. most of the dentists do not know about it is mechanism, so a lot of people consider it as a malpractice. purpose: this paper reported a case of a man, 46 years old which had ulcerative oral mucous, peeled and pain lips after taking carbamazepine drugs. case: the clinical diagnosis of this case was erythema multiforme because of the hypersensitivity reaction as the result of taking carbamazepine. case management: the final diagnosis based on anamnesis history of taking systemic drugs and clinical manifestation of erythema multiforme in the oral cavity. the drugs therapy that had been given were antihistamine, oral corticosteroid, gargle liquid contained of topical anesthetic, corticosteroid, and antibiotic. conclusion: in this case, it can be concluded that erythema multiforme appeared was triggered by taking carbamazepine as the drug of choice for trigeminal neuralgia therapy. these drugs can cause type iii hypersensitivity reaction. the final diagnosis based on anamnesis history of taking carbamazepine before lesions erupted and the characterized clinical manifestation. key words: erythema multiforme, carbamazepine, hypersensitivity abstrak latar belakang: erythema multiforme adalah penyakit mukokutaneus akut yang menyerang kulit dan mukosa sebagai akibat dari reaksi hipersensitivitas. secara karakteristik ditandai oleh lesi target pada kulit atau lesi ulserasi pada mukosa rongga mulut. etiologi penyakit ini belum jelas, diduga karena adanya reaksi imunologi. pencetusnya dikarenakan adanya pemakaian obat-obatan tertentu seperti antibiotik, antikonvulsan dan nsaid. banyak dokter gigi kurang memahami mekanisme timbulnya penyakit ini, sehingga oleh masyarakat dianggap sebagai malpraktek. tujuan: tulisan ini melaporkan kasus pasien pria berusia 46 tahun dengan keluhan sariawan dan bibir terkelupas dan sakit setelah sehari meminum obat karbamazepin. kasus: diagnosis klinis kasus ini adalah erythema multifome karena reaksi hipersensitivitas terhadap pemakaian obat karbamazepin. tatalaksana kasus: diagnosis ditegakkan berdasarkan anamnesis riwayat pemakaian obat sistemik dan manifestasi klinis dari erythema multiforme pada rongga mulut. pengobatan yangpengobatan yang diberikan adalah antihistamin, kortikosteroid oral, obat kumur dengan anastesi topikal, kortikosteroid topikal dan antibiotik topikal. kesimpulan: dapat disimpulkan bahwa erythema multiforme yang timbul pada kasus ini dipicu oleh pemakaian obat karbamazepin yang merupakan obat pilihan untuk terapi trigeminal neuralgia. obat ini menimbulkan efek samping reaksi hipersensitivitas tipe iii. diagnosis ditegakkan berdasarkan anamnesa riwayat pemakaian obat karbamazepin sebelum timbulnya lesi dan pemeriksaan klinis pada pasien. kata kunci: erythema multiforme, karbamazepin, hypersensitivitas correspondence: maharani laillyza apriasari, c/o: program studi kedokteran gigi fakultas kedokteran gigi universitas lambung mangkurat. jl. a. yani km 36 banjarbaru kalsel, indonesia. e-mail: rany.rakey@gmail.com 50 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 49-53 introduction erythema multiforme is an acute mucocutaneus disease which is caused by the hypersensitivity reaction. it is characterized by target lesions on the skin or ulcerative oral lesion. in the serious case, it is named steven johnson's syndrome. it attacks eyes, mouth, genital, and skin. 20–30% of erythema multiforme patients suffer it in oral mucous lesion as ruptured multiple vesicle and it leaves large erosion covered by white pseudo membrane.1–3 the disease always attack young people especially men and rarely attack children and old people.4 there are two kinds of erythema multiforme, they are minor type and major type. major type of erythema multiforme has serious degree condition which is called steven johnson's syndrome. it is an acute disease. it is started by the symptoms like fever, dizzy, and malaise. less then 24 hours, it appears explosive lesion on the skin and the mucosa. the lightest lesions on the skin are macula and papule with 0.5–2 cm diameter. lesion on the oral cavity is started by vesicle and bulla which is very easy to be ruptured. it has a specific clinical manifestation which can be fatal because of secondary infection and unbalance electrolyte liquid.3,5 etiology of the disease is unclear. hipersensitivitas reaction that appears as erythema multiforme can be triggered by taking various drugs like antibiotic, barbiturate, phenylbutazone and carbamazepine.1,2,6 carbamazepine is a drug of choice for trigeminal neuralgia. it also can be use to cure headache because of neuropathy pain, but the side effects of carbamazepine therapy are always happened. twenty five percents of all patient which have been given carbamazepine got the side effects like dizzy, vertigo, ataxia, diplopic, and blurred eyes. this drug can cause hypersensitivity like steven johnson's syndrome and dermatitis. steven johnson's syndrome is always happened relatively, so that the patient must be reminded for returning to the doctor if there is vesicle appeared on the skin or oral mucous.7,8 this paper reported the case of erythema multiforme in the oral cavity as a reaction of hypersensitivity caused by carbamazepine therapy. most of the dentists do not know about erythema multiforme mechanism, so a lot of people consider it as a malpractice. the clinical manifestation and anamnesis about history of this disease are very important, because it is related to the triggering factors that can make final diagnosis accurately so they can give the therapy as soon as possible. case a 46 years old male patient came to oral medicine department of faculty of dentistry airlangga university suffering arthralgia, fever and malaise. hands and foot feel numb. previously, the patient came with seriously pain on the right face then it was diagnosed as trigeminal neuralgia. patient had been given carbamazepine 100 mg which are taken 3 × 0.5 tablet/day. the next day after the patient had taken this drug for one day, there were oral ulcerative pain, peeled lips, and feeling wounded face. the patient had stopped taking the drugs, and then patient gave revanol on his peeled lips, while the right face was still painful. case management on 27 may 2008, extra oral examination showed the upper and lower lips of the patient had hyper pigmentation, painful, yellow and red crusted. intra oral examination could not be done, because of the pain (figure 1). figure �. visit 1: there was hyper pigmentation, yellow and red crusted on upper and lower lips. based on anamnesis and clinical examination, it could be concluded that the final diagnose was erythema multiforme. patient was given feksofenedin 120 mg 1×1/ day at night, prednisone 5 mg 3×2/day, benzydamin hcl gargle 2×1/day, zalf contain of hydrocortisone 125 gr, chemisitin 0.5 gr, lanolin, and vaseline was spread on his upper and lower lips 3×1/day after eating. the patient had been required to consume soft meals and liquid with high calorie and protein; like milk, fruit juice, and bread. patient was asked to come 3 days later. on 29 may 2008 (control 1), based on the anamnesis on the 3rd day, the pain of oral ulcerative were decreased. the patient had been able to eat. extra oral examination showed the crusted, peeled, and bleeding on the upper and lower lips. there was yellow crusted on the corner of the lips. intra oral examination showed that there were ulcer, multiply, 1.2 cm in diameter, irregular form, indurate barrier, and white pseudo membrane on the right cheek mucous. there was also the other ulcer with 5 mm diameter, irregular form, flat barrier, and white pseudo membrane rounded by red areas (figure 2 & 3). 51apriasari: erithema multiforme figure ��. visit 2: there were ulcers, multiply, irregular form, red barrier and white pseudo membrane. figure ��. visit 2: there were crusted, peeled and bleeding on upper and lower lips. the patient was asked to continue taking feksofenedin tablet 120 mg, benzydamin hcl gargle, prednisone 5 mg 3×1/day, zalf contain of hydrocortisone 125 gr, chemisitin 0.5 gr, lanolin, and vaseline was spread on his upper and lower lips 3×1/day after eat. patient was asked to come again 3 days later. on 5 june 2008 (control 2), based on anamnesis it could be shown that there was no pain on the right mucous cheek and lips. the patient was able to eat, but his serious pain on his right face was not cured, especially when he tried to move. during this week, the pain on his right face was more serious. extra oral examination showed the red crusted and peeled on the upper lip and the corner lips. intra oral examination showed irregular white cicatrix on the right buccal mucous (figure 4 & 5). the patient was asked to continue taking the drugs and sent to upf neurology rsud dr. sutomo to recover his trigeminal neuralgia, because the drug replacement was the responsible of the specialist of neurology. figure �. visit 3: there were red crusted and peeled on upper lip and corner lips. figure 5. visit 3: there were white cicatrix and irregular form. discussion erythema multiforme is an acute mucocutaneus disease which is caused by the hypersensitivity reaction. it is characterized by target lesions on the skin or ulcerative oral lesion. the diseases always happen to young people especially men and rarely attack children and old people.1–4 erythema multiforme is type iii hypersensitivity reaction as the result of antigen antibody complex increased which makes the vasculitis. specific factors of vasculitis in immune complex is caused by the hypersensitivity reaction as a result of the use of various drugs, microorganism, radiotherapy, systemic disease, and cancer.6 final diagnosis of erythema multiforme must be supported by accurate anamnesis and characteristic clinical signs. the specific clinical manifestation like target lesion, can also be found in the skin. if there is no target lesion, it will difficult to make the diagnose, and the differential diagnose is primary herpetic stomatitis.10 52 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 49-53 this case was about a patient with erythema multiforme in oral cavity without skin lesion. the lesion erupted in oral cavity after the patient had been taking carbamazepin as trigeminal neuralgia therapy. when the hypersensitivity reaction appeared, the patient stopped taking the drug, so the erythema multiforme reaction was not become serious and the skin lesion eruption can be a avoided.11 carbamazepin was the triggering factor of erythema multiforme. the lesion typically affect the oral mucosa, the lips, and bulbar conjunctivae. initial bullae rupture caused hemorrhagic pseudomembrane of the lips and made superficial oral ulcerates were need.12 hypersensitivity reactions occur as a result of an individual's immune system responding to an inappropriate stimulus, which may take the form of the drug-modified self-protein subsequent to drug bioactivation.13 based on immunopathology, erythema multiforme occured because of the type iii hypersensitivity reaction that makes immune complex reaction of antigen antibody. the pathogenesis of systemic immune complex disease can be divided into three phases: formation of antigen-antibody complex in the circulation, deposition of the immune complex in various tissues, and an inflammatory reaction at the sites of immune complex deposition.14 the first phase is initiated by the introduction of antigens, it is resulting in the formation of specific antibodies. in circulation, they make antigen antibody complex. if antigens are not removed by stopping the drug or replacing it with the other drugs. it will make the antigens stay longer in circulation. it makes deposition of the immune complex in various tissues and continues to form vasculitis.15,16 immune complex which leave the circulation can deposit in or out of the blood vessels and will make blood vessels permeability increased. this condition is signed by immune complex that is related to inflammatory cell through fc and c3b receptors. the receptor can trigger mast cell release and basophile that release various mediator of vasoactive and cytokine. complement can make cell lyses if immune complex have deposited in various tissues. vasoactive substance that is formed by mast cell and trombocyt can make vasodilatation, increased of vascular permeability, and inflammation. neutrophile will go out to eliminate immune complex, but when neutrophile was encircle by tissues, it will be difficult to eliminate immune complex, as a results of granular released by neutrophile that increase the tissue destruction.13,16 when immune complex condition is deposited in the tissues, it will form the reaction of inflammation at the 3rd phase. in this phase there are fever symptoms, urticaria, arthralgia, and lymphadenopathy.12,13,15 it happens in the first form of erythema multiforme, then followed by the clinical manifestation. prodromal symptoms were not as serious as virus infection disease. the main therapy of the patient was to stop taking carbamazepine, then the patient was given feksofenedin 120 mg 1×1/day in the night, prednisone 5 mg 3×2/day, benzydamin hcl gargle 2×1/day, zalf contain of hydrocortisone 125 gr, chemisitin 0.5 gr, lanolin, and vaselin was spread on his upper and lower lips 3×1/day after eat. feksofenedin is an antihistamine without sleepy effect. this 2nd generation antihistamine inhibit h1 receptor which is useful for curing hypersensitivity reaction where mast cell has released histamine. histamin is stimulated by the complement, and antigen antibody complex is formed on erythema multiforme.8,16 therapy for erythema multifome is giving oral corticosteroid. in the beginning, the patient takes prednisone 30 mg/day. in the simple case the patient can take prednisone 20–40 mg/day during 4–6 days. then dosage can be decreased through tapering off the dosage which is given not more than 2 weeks.7 action prevent the adrenal crisis, because this drug can disturb adrenal gland as the producer of natural steroid. prednisone is the oral corticosteroid with intermediate work (class 1 immunosuppressant). this drug is given before induction phase, is formed in the immune response of the body before antigen stimulation happened. the immunosuppressant effect of this drug can be reached through: inhibiting fagocytosis process and antigen process to be immunogenic antigen by macrophage, inhibiting antigen introduction by immunocompetent lymphoid cell, and destroying immunocompetent lymphoid cell.8 the other therapy is by using topical drugs in oral cavity. it can be given gargle contain of topical anesthetic and zalf contains of topical corticosteroid, antibiotic, lanolin and vaseline that was spread on his upper and lower lips. these drugs remove the inflammation, uncomfortable feeling, and prevent secondary infection.1,18 on the last visit, the patient was sent to upf neurology rsud dr. sutomo to recover his trigeminal neuralgia, because the drug replacement was the responsible of the specialist of neurology. several non-drug therapies have been recommended if pharmacologic treatment is unsuccessful. the most popular therapies were transcutaneous electrical nerve stimulation, lasers and several surgical approaches.1 it can be concluded that erythema multiforme can be triggered by the use of carbamazepine as the drug of choice of trigeminal neuralgia. this drug caused type iii hypersensitivity reaction. the final diagnosis based on anamnesis the history of taking carbamazepine before lesion eruption and the characteristics clinical manifestation. references 1. george l. treatment of oral disease: a concise textbook. stuttgart, germany: thieme; 2005. p. 66–7. 2. cawson ra, odell ew. cawson's essentials of oral pathology and oral medicine. 7th ed. london: churchill livingstone; 2002. p. 205–7. 3. regezi aj, sciubba jj, jordan ckr. oral pathology: clinical pathologic correlations. 4th ed. usa: saunders elsevier; 2003. p. 44–6. 4. langlais rp, miller cs. color atlas of common oral disease. 3rd ed. philadelphia, pennsylvania: lippincott williams & wilkins; 2003. p. 151. 53apriasari: erithema multiforme 5. david w, lowe dog, dagg hj, felix hd, scully c. textbook of general and oral medicine. london: churchill livingstone; 2001. p. 238–9. 6. glick m, greenberg sm. burket's: oral diagnosis and treatment. 10th ed. hamilton, ontario: bc decker inc; 2003. p. 208–11. 7. yagiela aj, dowd jf, neidle ae. pharmacology and therapeutics for dentistry. 5th ed. st louis, missouri: elsevier mosby; 2004. p. 392–3. 8. tim farmakologi fakultas kedokteran universitas indonesia. farmakologi dan terapi. edisi 5. jakarta: bagian farmakologi fakultas kedokteran universitas indonesia; 2007. p. 252, 495–8, 707. 9. field a, longman l. tyldesley's oral medicine. 5th ed. new york: oxford university press; 2004. p. 135–6. 10. george l. pocket atlas of oral disease. stuttgart, new york: thieme; 2006. p. 108–13. 11. shah kn, honig j paul, yan c albert. urticaria multiforme: a case series and review of acute annular urticarial hypersensitivity syndrome in children. journal watch dermatology 2007; 119(5): 117–83. 12. scully c, bagan jv. adverse drug reactions in the orofacial region. j oral biol med 2004; 15: 221. 13. kumar v, cotran sr, robbins ls. pathologic basis of disease. 7pathologic basis of disease. 7th ed. philadelphia, pennsylvania: elsevier saunders; 2005. p. 128–30. 14. naisbitt dj, britschgi m, guong g, farrell j, depta jph, chadwick dw, pichler wj, pirmohamed m, park bk. hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug– specific t cell clones. journal moleculerjournal moleculer pharmacology 2003; 63:732–41. 15. roeslan bo. imunologi kelainan di dalam rongga mulut. jakarta: abadi dhaya insani; 2000. p. 69–76, 86–9. 16. baratawidjaja kg. imunologi dasar. edisi 6. jakarta: fakultasjakarta: fakultas kedokteran universitas indonesia; 2004. p. 180–6. 17. zlotoff b, leggott j, doherty s. cutaneous reactions to drugs in children. journal of the american academy of pediatrics 2007; 120: 1082–96. 18. sen p, chua sh. a case of recurrent erythema multiforme and its therapeutic complication. j ann acad med singapore 2004; 33:793–6. mkg vol 39 no 1 jan 2006 isi.pmd 28 effects of irrigation solutions and calcium hydroxide dressing on root canal treatments of periapical lesions vita nirmala department of conservative dentistry faculty of dentistry gadjah mada university yogyakarta indonesia abstract the preparation of root canal in endodontic treatment plays an important role in treating non vital teeth with periapical lesion. some factors influence the success of root canal treatment in short and long terms are the irrigation of root canal using antiseptic solution and the use of root canal medicament. the aim of this literature study is to determined the effect of irrigation solution and calcium hydroxide dressing in root canal treatment of periapical lesions. the use of root canal medicament during the endodontic treatment could sterilized and decreased the number of pathogenic microorganism of root canal. an effective root canal irrigation solution must be able to dissolve organic and anorganic debris, lubricate endodontic instruments, disinfect microorganisms, non toxic and economical. the best irrigation solution has maximum antimicrobial effect with minimum toxicity. division of calcium hydroxide into calcium and hydroxyl ions is responsible for alkalinization of cavity, subsequently it makes the condition of cavity to be inappropriate for bacterial endotoxin in vitro as well as in vivo, and considered as the only clinically effective medicament in inactivating bacterial endotoxin. calcium hydroxide is the only medication which has the ability to clinically inactive bacterial endotoxin in vitro in vivo and accepted as the best of root canal medication. key words: root canal treatment, irrigation solutions, calcium hydroxide dressing, periapical lesion correspondence: vita nirmala, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas gadjah mada. jln. sekip utara, yogyakarta 55281, indonesia. telp. 0274-547130. introduction the preparation of root canal in endodontic treatment plays an important role in treating non vital teeth with periapical lesion. the bacteria are not only causing the periapical lesion but also play role in the defense mechanism of the lesion.1,2 the infected root canal must be taken using endodontic instruments and cleaned with antiseptic irrigation solution. the root canal medicaments are a part of root canal treatment and play an important part in the success of endodontic treatment. some factors influence the success of root canal treatment in short and long terms are the irrigation of root canal using antiseptic solution and the use of root canal medicament. the use of root canal medicament during the endodontic treatment could sterilized and decreased the number of pathogenic microorganism of root canal. georgopoulou et al.4 reported that root canal medicaments reduced or eliminated microorganism in root canal. root canal irrigation is one stage of endodontic treatment which is essential, neglecting it will cause treatment failure. the irrigation must be done after root canal preparation to remove the remaining infected pulp tissue and dentin. the cleaness of root canal is influenced by effective irrigation.3 the effectivity of irrigation depends on the quantity of irrigation solution, root canal diameter, and pulp condition. in pulpless teeth the irrigation solution not only entering the entire root canal but also penetrate into periapical.3 root canal medicament is still needed since the irrigation using sodium hypochlorite 5% and hydrogen peroxide 3% after root canal biomechanic preparation could not reduce or eliminate all root canal microorganism. chlorhexidine digluconate is recommended for root canal irrigation of infected teeth. it has antimicrobial effect and absorption of hard tissue in therapeutic level, which is called substantivity effect.4,5 root canal dressing is used in the teeth with periapical lesion for root canal disinfection after biomechanical preparation. for one visit obturation, it is recommended to use irrigation solution with bactericidal effect. calcium hydroxide has been used in conservative dentistry since nygren in 1838.2,6 in 1930, calcium hydroxide was used as pulp capping material for the first time but it was not been publicized.2,7 calcium hydroxide in the market is sold in various forms i.e. powder mixed with water, saline solution, methyl cellulose, glycerin and paste form. in paste form, there are calcium hydroxide with methyl cellulose (pulpadent), calcium hydroxide powder in ringer solution (calxyl), mixed paste (dycal), tube paste or syringe paste, and in point form (calcium hydroxide plus points) which recently available in dental market. the aim of this literature study is to determined the effect of irrigation solution and calcium hydroxide dressing in root canal treatment of periapical lesion. 29nirmala: effects of irrigation solutions root canal irrigation solution an effective root canal irrigation solution must be able to dissolve organic and anorganic debris, lubricate endodontic instruments, disinfect microorganisms, non toxic and economical.8 the best irrigation solution has maximum antimicrobial effect with minimum toxicity. anusavice9 stated that all dental materials must have oral biocompatibility. all dental materials must be harmless for pulp and soft tissue, have no systemic effect if it absorp or diffuse in body circulation, free allergen, and have no carcinogenic potential. the irrigation solution which common used in endodontic treatment has antiseptic effect on microorganisms in vitro and in vivo. the effectivity and toxicity of irrigation solution depend on the concentration, temperature and time.10,11 the irrigation solutions for root canal treatment are: halogen group chlorine (naocl 5%) has some effects i.e. lubricant, pulp tissue solvent, whitening, and strong antiseptic.3 chlorine also has an adverse effect, it can not dissolve anorganic debris, 1/3 apical region unreachable, corrosive on carbon steel endodontic instruments, and toxic. iodofor, an organic solution containing iodine. this material could be used for cleaning root canal, since it has low surface tension, antiseptic effect, less toxic than naocl, and not causing allergic reaction. the unwanted characteristic of iodofor is that its toxicity ten times greater than its antimicrobial effect, and irritates oral tissue, i.e. wescodyne, iodopax, iodine potassium iodide. detergent group the use of detergent in irrigation solution could make the root canal cleaner since it could dissolve the remaining lipid.12 the effectivity of detergent in cleaning root canal due to its active surface tension, binding the organism and organic debris, so that it could be disposed from root canal. the mechanism of its antibacterial effect is by way of disturbing cell membrane lipoprotein, but weaker than naocl.13,14 irrigation substance belonging to cationic detergent is quartenary ammonium compound group. although it has good cleansing effect, it is not an ideal irrigation solution due to its low antibacterial effect, can inhibit and prolong wound healing process.14,15 the cationic detergents are: edtac, zephiran, salvizol. irrigation solution belonging to anionic detergent (nonionic) among others are lauryl sulphate and soap. a combination of calcium hydroxide with lauryldiethylene-glycol-ether-sodium-sulphate 10% and 20% has antibacterial effect higher than calcium hydroxide solution on streptococcus faecalis, streptococcus salivarius, neisseria sp, iphteroid, staphylococcus aureus, lactobacillus sp, staphylococcus epidermidis, bacillus subtilis and candida albicans.15 chelating solution chelating solution is a material used to declassify narrow root canals. common solutions used are usually acidic type such as edta, citric acid,16 lactic acid, sulphuric acid, and tannic acid. other solutions are edtac, rc-prap salvizol,15 salvidont.17 calcium hydroxide dressing in inactivating bacterial endotoxin first reference,18 the use of calcium hydroxide in 1938, later was developed after hermann’s research,19 in 1920. calcium hydroxide has high alkali ph, very frequently used at dental clinics for direct pulp protection, pulpotomy of deciduous and permanent teeth, root canal dressing in permanent dental care with uncompleted rhizogenesis, root canal filling sealer, root perforation, dental resorption and intracanal antiseptic dressing.20 the application is much related to antibacterial activities,4,21,22 biocompatibility,5,22 hygroscopic, ability to reduce periapical tissue exudates, and its capacity to accelerate mineralization.23,24 it can dissolve necrotic tissue after biomechanics preparation, and it can act as bacterial substrate leading to stimulation of apical and periapical dental reparation with chronic lesion. among endodontic treatment dressings with non vital pulp and periapical lesion, calcium hydroxide is utilized for its proven bactericidal effect and its capacity to neutralize bacterial endotoxin. division of calcium hydroxide into calcium and hydroxyl ions is responsible for alkalinization of cavity, subsequently it makes the condition of cavity to be inappropriate for bacterial growth and proliferation. calen paste, one of calcium hydroxide paste with few cp (camphorated paramono chlorophenol), has produced excellent result in teeth with periapical lesion.20 recent spotlight in endodontic treatment is dental care with necrotic pulp and periapical lesion, for the fact that higher rate of treatment failure compared to treatment towards no periapical lesion. in dental with chronic periapical lesion, there is a bigger prevalence of anaerob bacteria hiding inside all root canal system (dentinal tubules, apical craters, cementum lacuna) including apical bacteria biofilm, due to the fact that these areas cannot be reached by instruments, therefore, intracanal dressing is recommended to help eliminate bacteria and to improve the possibility of successful treatment in dental clinics.23,25,26,27 according to leonardo et al.,28 teeth, with or without periapical lesion has different pathologic bacteria that requires different dental treatment. in cases with periapical lesion, it requires root canal dressing during inter session treatment, for the reason that the success of periapical lesion treatment is related directly to bacterial cleansing, product and subproduct of root canal system. root canal treatment procedure and medicament are not only bactericidal, but also creating the inactivation of bacterial endotoxin. 30 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2005: 28–31 lacked of information on intracanal dressing effect towards bacterial endotoxin residues which can attach to mineralization tissue,29 had made safavi and nicholas30 evaluated in vitro the effect of calcium hydroxide on bacterial endotoxin. they concluded that calcium hydroxide hydrolyzed high toxic lipid a molecule which was responsible for damaging endotoxin. in their further study, safavi and nicholas23 concluded that calcium hydroxide transformed lipid a to become fatty acid and amino sugar belonging to non toxic component. this result was renewed by barthel et al.31 and olsen et al.31 who reported calcium hydroxide ability to detoxicate bacterial endotoxin in vitro. in 2002, nelson-filho et al.26 did an in vivo research to evaluate radiographically, the effect of endotoxin with calcium hydroxide application in apical and periapical tissue of dog teeth. they observed that endotoxin caused periapical lesion after 30 days and that calcium hydroxide inactivated bacterial endotoxin. silva et al.20 analyzed histopathologically all dog teeth apical and periapical tissues, where it was filled with bacterial endotoxin and calcium hydroxide. they reported that endotoxin caused periapical lesion, and calcium hydroxide detoxicated endotoxin in vivo. jiang et al.32 also evaluated the direct effect of bacterial endotoxin on osteoclastogenesis and the capacity of calcium hydroxide to inhibit osteoclast forming, stimulated by endotoxin. they reported that calcium hydroxide significantly reduced osteoclast differentiation. this new finding has renewed the concept of root canal dressing, in pertaining to calcium hydroxide not only as the best medicament but fundamentally as the one and only medication which has the ability to inactivate endotoxin found in root canal system with necrotic pulp and chronic periapical lesion. discussion according to histopathology result, apical and periapical tissue healing is better in teeth with root canal dressing compared with group with direct obturation. the group applying root canal dressing produces tighter apical closure, creating non intens periapical inflammation reaction, emphasizing the importance of root canal dressing in endodontic treatment for necrotic pulp and periapical lesion. other study reported how essential root canal dressing for eliminating bacteria from root canal and apical surface areas.20 sodium hypochlorite is commonly used as root canal irrigation solution in dental treatment with necrotic pulp with or without periapical lesion. the high concentrated sodium hypochlorite solution has potent antimicrobial action. this sodium hypochlorite is recommended for dental care with periapical lesion.23 bacterial endotoxin is a component of gram-negative bacterial cell wall, found in all teeth with necrotic pulp and chronic periapical lesion, seen radiographically. bacterial endotoxin holds a fundamental role in genesis and periapical lesion endurance, caused by inflammation induction and bone resorption. among dressing for dental non vital pulp endodontic treatment with chronic periapical lesion, calcium hydroxide is used for its proven bactericidal effect and its capacity to neutralize bacterial endotoxin. division of calcium hydroxide into calcium and hydroxyl ions is responsible for alkalinization of cavity, subsequently it makes the condition of cavity to be inappropriate for bacterial growth and proliferation.23,24 root canal irrigation solutions must be able to dissolve organic and anorganic debris, lubricate endodontic instruments, disinfect microorganisms, non-toxic and economical. the most effective irrigation solution is one that has maximal antimicrobial capacity and minimal toxicity. the most effective irrigation solution is one that has maximal antimicrobial capacity and minimal toxicity. sodium hypochlorite is recommended for dental care with periapical lesion30 due to its higher effectivity compared to liquefied solution and the low surface tension is important for root canal cleansing. calcium hydroxide inactivated toxic effect of bacterial endotoxin in vitro and in vivo and accepted not only as the best medicament but fundamentally as the one and only medication which has the ability to clinically inactivate bacterial endotoxin. references 1. leonardo mr, almeida wa, da silva la, utrilla ls. histopathological observations of periapical repair in teeth with radiolucent areas submitted to two different methods of root canal treatment. j endod 1995 mar; 21(3):137–41. 2. martin dm, crabb hsm. calcium hydroxide in root canal therapy. brit dent j 1977; 277–83. 3. grossman li. ilmu endodontik dalam praktek. 11st ed. seymour oliet, carlos e, del rio. rafiah a, suryo s, editor. jakarta: penerbit egc; 1995. p. 248. 4. georgopoulou m, kotakiotis e, nakou m. in vitro evaluation of the effectiveness of calcium hydroxide and paramonochlorophenol on bacteria from the root canal. endod dent traumatol 1993; dec; 9(6):249–53. 5. holland r, souza v. tratamento conservador da polpa dental. in: leonardo mr, leal jm, editors. endodontia: tratamento de canals radiculares. san paulo: ed medica panamericana; 1998. p. 63–75. 6. delany gm, patterson, ss, miller ms, newton cw. the effect of chlorexidine gluconate irrigation on the root canal flora of freshly extracted necrotic teeth. oral surg oral med oral pathol 1982 may; 53(5):518–23. 7. sidharta w. perawatan saluran akar konvensional pada gigi non vital dengan kelainan periapikal lanjut menggunakan kalsium hidroksida (laporan kasus). j ked gigi ui 1997; edisi khusus kppikg xi: 4. 8. harty fj. endodontik klinis (clinical endodontics). yuwono l. edisi ke-3. jakarta: hipokrates; 1993. p. 128. 9. anusavice kj. philip’s science of dental materials. 10th ed. philadelphia: wb saunders company; 1996. p. 75–9. 10. kennedy wa, walker iii wa, gough rw. smear layer removal effects on apical leakage. j endod 1996; 12:21–7. 11. siswandono, soekardjo b. kimia medisinal. cetakan i. surabaya: airlangga university press; 1995. p. 247–8. 12. barbosa sv, spangberg lsw, almeida d. low surface tension calcium hydroxide solution is an effective antiseptic. int endod j 1994 jan; 27(1):6–10. 31nirmala: effects of irrigation solutions 13. kolstad r, white rr. disinfection and sterilization. in: willett np, white rr, rosen s, editors. essential dental microbiology. new jersey: prentice-hall int inc; 1995. p. 57–61. 14. weine fs. endodontic therapy. 3rd ed. st louis: the cv mosby co; 1985. p. 317–22. 15. spangberg l. intracanal medication. in: ingle jl, bakland lk, editors. endodontics. 4th ed. philadelphia: lea & febiger; 1994. p. 632–7. 16. yamaguchi m, yoshida k, suzuki r, nakamura h. root canal irrigation with citric acid solution. j endod 1996 jan; 22(1):27–9. 17. spangberg l, pascon ea, kaufman ay, safavi k. tissue irritating properties of bis-dequalinium acetat solutions for endodontic use. j endod 1988; 14:88–97. 18. nygreen ja. radgivare angaende basta sttet att varda och levara tandernas friskhet apud martin dm, crabb hsm. calcium hydroxide in root canal therapy. a review. br dent j 1977 may 3; 142(9): 277–83. 19. hermann bw. calcium hydroxide as mitted zin behandeed und fullen von wurzel. diss. wurzbrug, 1920 apud leonardo mr, leal jm. endodontia: tratamento de canais radiculares. sao paulo: panamericana. 1991. p. 1–18 20. silva lab, assed s, nelson-filho p. protecao direta da popa: como fazer e o que utilizar. in: atualizacao na clinica odontologica, sao paulo: artes medicas. 2002; 2:267–88 21. leonardo mr, silva lab, leonardo rt, utrilla ls, assed s. histological evaluation of therapy using a calcium hydroxide dressing for teeth with incompletely formed apices and periapical lesions. j endod 1993; 19(7):348–52. 22. leonardo mr, silva la, leonardo rt. devemos usar medicação intracanal no tratamento de dentes com necrose pulpar? in: odontologia integrada? atualização multidisciplinar para o clinico e o especialista. rio de janeiro: editora pedro primeiro ltda; 1999. p. 179–95. 23. safavi ke, nichols fc. effect of calcium hydroxide on bacterial lipopolysaccharide. j endod 1993 feb; 19(2):76–8. 24. silva lab. rizogenese incompleta-efeitos de diferentes pastas a base de hidroxido de calciona complementacao radicular e na reparacao periapical em dentes de caes-estudo histologico. arraquara; 1988 [dissertacao de mestrado. faculdade de odontologia da universidade estadual paulista]. 25. katebzadeh n, hupp j, trope m. histological periapical repair after obturation of infected root canals in dogs. j endod 1999 may; 25(5):364–8. 26. nelson-filho p, leonardo mr, silva la, assed s. radiographic evaluation of the effect of endotoxin (lps) plus calcium hydroxide on apical and periapical tissues of dogs. j endod 2002 oct; 28(10):694–6. 27. trope m, delano eo, orstavik d. endodontic treatment of teeth with apical periodontitis: single vs. multivisit treatment. j endod 1999 may; 25(5):345–50. 28. leonardo mr, silva lab, leonardo rt. tratamento de canal radicular em sessão ßnica: crença vs. ciência. in: feller, gorab r. atualização na clinica odontol^gica. sao paulo: artes medicas; 2000. p. 29–57. 29. dahlen g, magnusson bc, moller a. histological and histochemical study of the influence of lipopolysaccharide extracted from fusobacterium nucleatum on the periapical tissues in the monkey macaca fascicularis. archs oral biol 1981; 26:591–8. 30. barthel cr, levin lg, reisner hm, trope m. tnf-alpha release in monocytes after exposure to calcium hydroxide treated escherichia coli lps. int endod j 1997 may; 30(3):155–9. 31. olsen mh, difiore pm, dixit sn, veis a. the effects of calcium hydroxide inhibition on lps induced release of il-1b from human monocytes in whole blood. j endod 1999; 25:289. 32. jiang j, zuo j, chen sh, holliday ls. calcium hydroxide reduces lipopolysaccaharide-stimulated osteoclast formation. oral surg oral med oral pathol oral radiol endod. 2003 mar; 95(3):348–54. << 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. vol 52 no 1 jan-mar 2019_new.indd 8 dental journal (majalah kedokteran gigi) 2019 march; 52(1): 8–12 research report the treatment of gingival recession with coronally advanced flap with platelet-rich fibrin asti rosmala dewi,1 agus susanto,2 and yanti rusyanti2 1department of periodontics, faculty of dentistry, universitas sriwijaya, palembang – indonesia 2department of periodontics, faculty of dentistry, universitas padjadjaran, bandung – indonesia abstract background: of the various techniques developed for the treatment of gum recession, the current innovation in the use of platelet rich fibrin (prf) has been applied to the treatment of root-end closure procedures. purpose: this study analyzed the effect of the coronally advanced flap (caf) in combination with prf during treatment of gingival recession. methods: this research constituted an experimental study incorporating a split-mouth design which was conducted on eight participants (with 16 recession defects) who were divided into two groups consisting of the caf group with prf and another caf group without prf. clinical parameters consisting of gingival recession (gr), keratinized gingival width (kgw), recession width (rw) and clinical attachment level (cal) were measured both before and 21 days after surgery). results: the average differences between clinical parameters in the test group were as follows: gr (2.25 ± 0.27), kgw (1.81 ± 0.75), rw (3.44 ± 1.35), and cal (2.56 ± 0.50); while in the control group they were as follows: gr (2.00 ± 0.71), kgw (1.94 ± 0.78), rw (3.50 ± 1.91) and cal (2.00 ± 0.76). there were significant differences in the gr and cal between the test and control groups (p < 0.05). conclusion: a combination of the caf procedure and prf was proven to be more effective in covering gr and increasing cal. keywords: coronally advanced flap; gingival recession; platelet-rich fibrin correspondence: agus susanto, department of periodontics, faculty of dentistry, universitas padjadjaran, jl. raya bandung sumedang km.21, jatinangor, jawa barat 45363, indonesia. e-mail: agus.susanto@fkg.unpad.ac.id introduction gum recession constitutes a clinical condition during which the gingival margin is in a more apical location than the cementoenamel junction (cej), causing part of the radicular surface to be opened.1 gingival recession can be present in healthy periodontal tissue and appears as wedge shaped lesions on the buccal surface of the teeth, especially in association with hard toothbrush use, whereas in individuals with poor oral hygiene it can be present on any tooth surface. risk factors in gingival recession include: tooth malposition, inadequate oral hygiene, alveolar bone dehiscence, muscle attachment, frenulum traction, periodontal disease and restorative iatrogenic treatment.1,2 gingival recession often causes aesthetic problems, root caries and tooth abrasion, with patients usually complaining of hypersensitive teeth.1,2 various types of gingival recession treatments comprise: laterally positioned flaps (lpf), coronally advanced flap (caf) and connective tissue graft (ctg) with a success rate of 65% to 98%.3,4 the choice of technique depends on the extent of recession defect, the location of the required aesthetic improvement and the need for additional gingival tissue (graft). the caf technique is popular because of its simple procedure and excellent post-surgical healing.4 the first platelet rich fibrin (prf) was developed in france and utilized in the field of oral and maxillofacial surgery. prf was a second generation platelet concentrate, consisting of platelets and growth factors in the form of fibrin membranes derived from the blood of the patient which is free of various anticoagulants.5,6 prf improves wound healing and regeneration. multiple studies confirmed more rapid wound healing resulting from the use of prf.6 the prf fabrication process is easier, cheaper, more effective dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p8–12 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p8-12 9dewi, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 8–12 and does not require any bovine thrombin or anticoagulants, therefore, rendering regenerative membranes more easily formed. the use of prf in the field of periodontics has rendered it one of the most frequently used materials in regenerative surgical procedures.5,7 prf has a dense fibrin matrix with leukocytes, cytokines, glycoprotein structures as well as growth factors, for example transforming growth factor β1, plateletderived growth factor, vascular endothelial growth factor and glycoprotein such as thrombospondin-1.8 leukocytes concentrated in prf play an essential role in releasing growth factors, regulating immune response, inducing anti-infective activity and remodeling matrix during wound healing.5,8 combination techniques using autogenous or allograft and guided tissue regeneration membranes were subsequently developed to correct the mucogingival defect.9 this study was intended to analyze the effect of a combination of caf and prf in the treatment of gingival recession. materials and methods this research constituted experimental research incorporating split-mouth, pre-test and post-test designs. the study was conducted at the department of periodontics, faculty of dentistry, universitas padjadjaran using a protocol and research design previously approved by the health research ethics committee. all participants completed an informed helsinki declaration consent form containing information about the research protocol (no. 284/un6.c2.1.2/kepk/pn/2013). the total number of participants consisted of eight individuals (two men and six women) who attended the periodontics clinic chiefly complaining of hypersensitive teeth or gum recession. the criteria for inclusion as a research subject comprised: (1) males or females above the age of 18 and free of active periodontal disease. (2) class i miller gum recession, recession defects > 1 mm in the buccal/labial incisors and premolars. (3) the patient enjoyed sound systemic health free of any medical condition contraindicated for periodontal surgery. (4) the tooth was not endodontically treated and had not undergone buccal or interproximal restoration. (5) the patient was not using antibiotics, corticosteroid, chemotherapeutic, immune modulator or any other medication that could potentially modify oral tissue. (6) the patients were exclusively nonsmokers. the autologous prf for use in this study was extracted from the blood of the patients prior to surgery. all blood samples were inserted in the 10ml glass-coated plastic tube of a table centrifuge without an anticoagulant and subjected to centrifugation at 3,000 rpm for 10 minutes.5,10 a fibrin clot formed in the middle section, with a cellular plasma present in the upper part and red corpuscles in the lower section. the fibrin clot was subsequently extracted with sterile tweezers and prepared using the prf preparation kit in order to obtain the prf membrane.11 the length of time between blood collection and the centrifugation process is crucial to the clinical results and success of this procedure. protracted management of the blood centrifugation process will result in diffuse polymerization, leading to the formation of small blood clots with irregular consistency.5,8 the measurement of initial clinical parameters was performed prior to surgery. random selection of members of the test group (caf + prf) and control group (caf only) was made on the result of a coin toss and with the same operator working on both groups. the administering of oral antiseptic was performed using the 0.2 chlorhexidine digluconate, a local anesthetic containing 2% lidocaine hydrochloric acid with adrenaline by means of an infiltration technique. prior to incision, a blood sample was taken for preparation of prf membrane. an intrasulcular incision was made around the tooth with recession defect and two vertical incisions were made on each adjacent tooth with the interdental papilla being maintained (figure 1). full-thickness flap and split-thickness techniques were performed on the apical section to enable repositioning of the coronal flap without pressure. the papilla was epithelialized to produce a connective tissue bearing-scaling, while root planning was performed on the radicular surface by means of a curette. tetracycline hcl was applied for three minutes to the radicular surface which was then rinsed with water. in the test group (caf + prf), the prf membrane was positioned on the recession defect at the level of cej (figure 2). the margin of the gingival flap was repositioned on the enamel in both the test and control groups and held in that position with horizontal sling sutures and vertical incisions with interrupted sutures using non-resorbable threads (5-0 nylon, ailee®) (figure 3). the placement of periodontal packs was performed by means of non-eugenol dressing (coe-pack™, gc america inc., usa). after surgery, the patient was given 500mg of amoxicillin cap three times a day and 50mg of cataflam™ twice a day for four days. the patient was instructed to rinse his/her mouth with 0.2% cumulative chlorhexidine medication, with the periodontal dressing and sutures being opened on the 10th day. on the 21st postoperative day, the patient was examined for clinical parameters, including: gingival recession (gr), keratinised gingival width (kgw), recession width (rw) and clinical attachment level (cal). the gr is the distance from the cej to the gingival margin measured in millimeters using a periodontal probe (osung™ unc-15, korea). the examination was performed on the mid-buccal side of the treated tooth. the kgw is the distance from the gingival edge to the mgj measured by a probe on the mid-buccal side. the rw is the distance between points placed on the cej, at the mesial-most and distal-most ends. the cal is the distance from the cej border to the sulcus base. the values were represented in terms of their means ± standard deviation. the significance of the difference within and between groups before and after treatment was evaluated by means of a paired t-test and mann-whitney test. differences were considered statistically significant at p < 0.05. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p8–12 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p8-12 10 dewi, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 8–12 table 1. assessment of the average gr, kgw, rw and cal, before and after treatment in the test and control groups. clinical parameters test group mean ± sd control group mean ± sd p-value 0.5582.50 ± 0.752.75 ± 0.53gr baseline gr after 21 days 1.0000.5 ± 0.590.50 ± 0.70 (p = 0.000)*(p = 0.000)* 0.5134.37 ± 0.914.12 ± 0.51kgw baseline kgw after 21 days 0.1926.31 ± 0.535.93 ± 0.56 (p = 0.000)*(p = 0.000)* 0.8304.68 ± 1.284.56 ± 0.97rw baseline rw after 21 days 0.9271.18 ± 1.412.12 ± 1.27 (p = 0.000)*(p = 0.000)* 0.3573.43 ± 0.903.81 ± 0.65cal baseline cal after 21 days 0.5291.43 ± 0.561.25 ± 0.59 (p = 0.000)*(p = 0.000)* note: gr = gingival recession; kgw = keratinized gingival width; rw = recession width; cal = clinical attachment level table 2. differences of the average gr, kgw, rw and cal, before and after treatment in the test and control groups. variable test group mean ± sd control group mean ± sd p-value 0.05*2.00 ± 0.712.25 ± 0.27gr 0.371.94 ± 0.781.81 ± 0.75kgw 0.473.50 ± 1.913.44 ± 1.35rw 0.05*2.00 ± 0.762.56 ± 0.50cal note: gr = gingival recession; kgw = keratinized gingival width; rw = recession width; cal = clinical attachment level figure 1. incision design. figure 2. prf membrane on recession defects. figure 3. flap sutures. results in this study, the gender composition of the respondents was 75% male to 25% female. there were 16 sites of class i miller recession defects which were divided into two groups, namely; the test group (caf + prf) and the control group (caf only). the average rg, kgw, rw and cal showed significant differences between the pre-treatment situation and after 21 days of treatment in both groups (p = 0.000) (table 1). the average difference of the rg and cal in the test and control groups was significant (p < 0.05) (table 2), indicating that the test group presented superior closure in the radicular of the gingival recession than the control group. in other words, the use of prf played a significant role in achieving the closure of gum recession. improvement in the cal was also more marked dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p8–12 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p8-12 11dewi, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 8–12 in the test group than the control group. the kgw and rw parameters did not show significant differences between the two groups since application of prf did not affect the kgw and rw. discussion this study was intended to evaluate the effectiveness of prf with cap in the treatment of gum recession. the research subjects consisted of eight individuals aged between 29 and 39 years-old who presented identical bilateral miller’s class i gingival recession. the splitmouth design helped to avoid variations in characteristics between patients. caf is a predictable procedure to treat miller’s class i mucogingival defects. gingival thickness was the most significant factor associated with complete root coverage identified by analyzing factors such as gr, the kgw, rw and cal measured at the time that the baseline was established and 21 days after the operation had been conducted on the test and control groups. platelet-rich fibrin has been claimed to enhance soft tissue healing, promote initial stabilization and the revascularization of flaps and grafts in root coverage.12 platelets play an essential role in wound healing by accelerating it after periodontal treatment. the wound healing process begins with the formation of blood clots and platelet attachment. moreover, the substances released by platelets can stimulate tissue repair, angiogenesis, inflammation and immune responses. prf is rich in platelets, growth factors and cytokines, all of which have the potential to improve wound healing in both hard and soft tissue.13 prf stimulates the expression of phosphorylated extracellular signal-regulated protein kinase (p-erk) and production of osteoprotegerin (opg) which play important roles in osteoblast proliferation.14 frf fibrin matrix, in addition to directly stimulating the angiogenesis process, contains platelets, growth factors and cytokines that may enhance the healing potential of both bone and soft tissues.15 prf membranes release various growth factors such as platelet-derived growth factor (pdgf), transforming growth factor (tgf), vascular endothelial growth factor (vegf) and epidermal growth factor (egf) for between seven and 28 days. this indicates that the prf membrane stimulates the healing process during tissue re-modelling. natural fibrin matrix plays a direct role in increasing angiogenesis. fibrin constitutes a natural support to immunity which impedes the inflammatory process.6 in this study, the use of prf as a membrane during the caf procedure produced superior root coverage and a decrease in clinical attachment loss compared to the caf procedure alone. the results of this study were consistent with those of research conducted by padma et al. (2013),16 which posited that the addition of prf membrane with caf provides superior root coverage with the extra benefits of an increase in the cal and kgw six months after the operation. another investigation reported inferior recession coverage using prf membrane when compared with caf only in multiple recession defects.12 the average ppd reduction and cal gain in caf only-treated gingival recession defect were 0.35 mm and 2.60 mm respectively, while the difference was statistically significant (p < 0.05).17 improvement in cal occurred due to the recession coverage as a result of the coronal shift of attachment apparatus during caf procedures.18 in this study, the kgw and rw did not show significant differences between the caf with prf group and the caf-only group, a finding in contrast to the research that reported a long-term increase in the keratinized gingiva width associated with an apical shift of the mucogingival junction.19 this might be due to the evaluation not having been performed over a more extended period. the average recession coverage in this study was 80% using the caf-only procedure, whereas in the treatment with prf combination it was 81.8%. previous studies conducted by aroca et al.(2009),12 and jankovic et al.(2012),20 which reported root coverage as high as 80.7% and 88.7% respectively. the treatment of gum recession with the combination of prf resulted in perfect root coverage with satisfactory tissue contour and color. it also had several advantages, including: minimal bleeding, faster healing time and minimal postoperative pain.9 the use of prf was also employed during other periodontal treatment such as that relating to periodontal bone defects. various studies have indicated the effectiveness of prf in the treatment of periodontal defects, an improved clinical attachment and radiographic height elevation with treatment using prf.21 thorat et al. (2011), investigated the clinical and radiological effectiveness of autologous prf in the treatment of intrabony defect of chronic periodontitis patients. they reported a greater reduction in pocket depth, more gain in clinical attachment level and greater intrabony defect fill at sites treated with prf than those treated with open flap debridement alone.21 it evaluated the effectiveness of prf with demineralized freeze-dried bone allograft (dfdba) material in the treatment of intrabony defects. the study concluded that a combination of prf and dfdba produced superior results in reducing bleeding at the probing depth and clinical attachment level compared to dfdba alone in the treatment of intrabony defects.22 further studies with larger sample sizes and longer follow-up periods are required to determine the advantages of using prf in caf technique for root coverage. factors such as prf consistency and platelet concentration not being tested in this study may have affected the final clinical outcome. it is concluded that caf procedure is a predictable treatment for isolated miller’s class i recession defects. a combination of the caf procedure with prf was proven to be more effective in the coverage of the gr and in increasing the cal. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p8–12 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p8-12 12 dewi, et al./dent. j. (majalah kedokteran gigi) 2019 march; 52(1): 8–12 references 1. patel m, nixon pj, chan mfw-y. gingival recession: part 1. aetiology and non-surgical management. br dent j. 2011; 211(6): 251–4. 2. jati as, furquim lz, consolaro a. gingival recession: its causes and types, and the importance of orthodontic treatment. dental press j orthod. 2016; 21(3): 18–29. 3. cardaropoli d, tamagnone l, roffredo a, gaveglio l. treatment of gingival recession defects using coronally advanced flap with a porcine collagen matrix compared to coronally advanced flap with connective tissue graft: a randomized controlled clinical trial. j periodontol. 2012; 83(3): 321–8. 4. chambrone l, lima la, pustiglioni fe, chambrone la. systematic review of periodontal plastic surgery in the treatment of multiple recession-type defects. j can dent assoc. 2009; 75(3): 203a-f. 5. preeja c, arun s. platelet-rich fibrin: its role in periodontal regeneration. saudi j dent res. 2014; 5(2): 117–22. 6. shetty s, yadav n, mehta m, vaish s, v d. autologous platelet-rich fibrin: a boon to periodontal regeneration report of two cases. j dent spec. 2014; 2(2): 112–9. 7. keceli hg, ozkan sy, turkal ha, tozum tf. management of two-wall infra-bony defect with platelet rich fibrin and connective tissue graft combination: a case report. j dent probl solut. 2015; 2(2): 025–30. 8. dohan dm, choukroun j, diss a, dohan sl, dohan ajj, mouhyi j, gogly b. platelet-rich fibrin (prf): a second-generation platelet concentrate. part i: technological concepts and evolution. oral surgery, oral med oral pathol oral radiol endodontology. 2006; 101(3): e37–44. 9. a n il k uma r k , geet ha a, umasud ha ka r, ra ma k r ish na n t, vijayalakshmi r, pameela e. platelet-rich-fibrin: a novel root coverage approach. j indian soc periodontol. 2009; 13: 50–4. 10. naik b, karunakar p, jayadev m, marshal vr. role of platelet rich fibrin in wound healing: a critical review. j conserv dent. 2013; 16(4): 284–93. 11. dohan ehrenfest dm, del corso m, diss a, mouhyi j, charrier j-b. three-dimensional architecture and cell composition of a choukroun’s platelet-rich fibrin clot and membrane. j periodontol. 2010; 81(4): 546–55. 12. aroca s, keglevich t, barbieri b, gera i, etienne d. clinical evaluation of a modified coronally advanced f lap alone or in combination with a platelet-rich fibrin membrane for the treatment of adjacent multiple gingival recessions: a 6-month study. j periodontol. 2009; 80(2): 244–52. 13. rock l. potential of platelet rich fibrin in regenerative periodontal therapy: literature review. can j dent hyg. 2013; 47: 33–7. 14. chang y-c, wu k-c, zhao j-h. clinical application of platelet-rich fibrin as the sole grafting material in periodontal intrabony defects. j dent sci. 2011; 6(3): 181–8. 15. diss a, dohan dm, mouhyi j, mahler p. osteotome sinus floor elevation using choukroun’s platelet-rich fibrin as grafting material: a 1-year prospective pilot study with microthreaded implants. oral surgery, oral med oral pathol oral radiol endodontology. 2008; 105(5): 572–9. 16. padma r, shilpa a, kumar p, nagasri m, kumar c, sreedhar a. a split mouth randomized controlled study to evaluate the adjunctive effect of platelet-rich fibrin to coronally advanced flap in miller′s class-i and ii recession defects. j indian soc periodontol. 2013; 17(5): 631–6. 17. moka lr, boyapati r, m s, d ns, swarna c, putcha m. comparison of coronally advanced and semilunar coronally repositioned flap for the treatment of gingival recession. j clin diagn res. 2014; 8(6): zc04-8. 18. akkaya m, böke f. shallow localized gingival recession defects treated with modified coronally repositioned flap technique: a case series. eur j dent. 2013; 7(3): 368–72. 19. thamaraiselvan m, elavarasu s, thangakumaran s, gadagi j, arthie t. comparative clinical evaluation of coronally advanced flap with or without platelet rich fibrin membrane in the treatment of isolated gingival recession. j indian soc periodontol. 2015; 19(1): 66–71. 20. jankovic s, aleksic z, klokkevold p, lekovic v, dimitrijevic b, kenney eb, camargo p. use of platelet-rich fibrin membrane following treatment of gingival recession: a randomized clinical trial. int j periodontics restorative dent. 2012; 32(2): e41-50. 21. thorat m, pradeep ar, pallavi b. clinical effect of autologous platelet-rich fibrin in the treatment of intra-bony defects: a controlled clinical trial. j clin periodontol. 2011; 38(10): 925–32. 22. bansal c, bharti v. evaluation of efficacy of autologous plateletrich fibrin with demineralized-freeze dried bone allograft in the treatment of periodontal intrabony defects. j indian soc periodontol. 2013; 17(3): 361–6. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i1.p8–12 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i1.p8-12 vol 38-no 1-2005 16 kekuatan transversa resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda (the transverse strength of the hybrid acrylic resin after glass fiber reinforcement with different method) intan nirwana bagian ilmu material dan teknologi kesehatan gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract different types of fibers have been added to acrylic resin materials to improve their mechanical properties. the purpose of this study was to know the transverse strength of the hybrid acrylic resins after glass fiber reinforcement with difference method. this study used rectangular specimens of 65 mm in length, 10 mm in width and 2.5 mm in thickness. there were 3 groups consisting of 6 specimens each, hybrid acrylic resin without glass fiber (control), glass fibers dipped in methyl methacrylate monomer for 15 minutes before being reinforced into hybrid acrylic resin (first method), glass fibers reinforced into a mixture of polymer powder and monomer liquid after the hybrid acrylic resin was mixed directly (second method). all of the specimens were cured for 20 minutes at 100° c. transverse strength was measured using autograph. the statistical analyses using one way anova and lsd test showed that there were significant differences in transverse strength (p < 0.05) among the groups. the means of transverse strength were 94,94; 118,27; and 116,34 mpa. it meant that glass fibers reinforcement into hybrid acrylic resin enhanced their transverse strength compared with control. glass fiber reinforcement into hybrid acrylic resin with differenciate method didn’t enhance their transverse strength. key words: hybrid acrylic resins, transverse strength, glass fiber korespondensi (correspondence): intan nirwana, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan resin akrilik sampai saat ini masih merupakan pilihan untuk pembuatan basis gigi tiruan lepasan oleh karena harganya relatif murah, mudah direparasi, proses pembuatan gigi tiruan mudah dan menggunakan peralatan sederhana, warna stabil, dan mudah dipulas.1 resin akrilik hybrid yang berkembang saat ini juga sangat efektif, praktis, mempunyai dua aktivator yaitu kimia dan panas serta proses kuring cepat hanya 20 menit suhu 100° c.2,3 lama proses kuring tersebut sangat singkat dibandingkan dengan proses kuring resin akrilik terdahulu. selain itu resin akrilik dapat dilakukan proses kuring menggunakan gelombang mikro yang hanya memerlukan waktu 15 menit sehingga waktu kerja lebih efisien. kekurangan resin akrilik adalah mudah patah, dan patahnya basis gigi tiruan dapat terjadi di luar mulut yaitu jatuh pada tempat yang keras, sedangkan patah yang terjadi di dalam mulut dapat disebabkan oleh karena fatique maupun occlusal forces.4 menurut narva et al.,5 patahnya basis gigi tiruan dapat disebabkan oleh fitting dari gigi palsu tidak bagus, tidak adanya keseimbangan oklusi, fatique maupun jatuh. penelitian yang telah dilakukan untuk meningkatkan sifat mekanik resin akrilik yaitu dengan menambah fibers, carbon, aramid, glass dan metal wire,6-8 atau dengan menambahkan ultra high modulus polyethylene fibres.9,10 carbon dan aramid fiber dapat memperkuat polimetil metakrilat tetapi resin akrilik sukar di pulas dan estetik menjadi jelek.6 metode tradisional terdahulu menggunakan metal wire sebagai penguat basis gigi tiruan. resin akrilik yang mengandung glass fibers menunjukkan sifat mekanik yang lebih baik dibandingkan dengan resin akrilik tanpa penambahan glass fibers.11 persentase kandungan glass fiber yang makin meningkat menyebabkan perubahan dimensi dan absorpsi air menurun, 12 sedangkan penambahan glass fiber dengan proses kuring konvensional meningkatkan kekuatan transversa sampai 21,1%.13 pada penelitian lain, penambahan glass fiber menggunakan metode berbeda meningkatkan kandungan monomer sisa.14 kandungan monomer sisa yang tinggi berpotensi untuk menyebabkan iritasi jaringan mulut, inflamasi, dan alergi terutama daerah mukosa di bawah gigi tiruan.15,16 selain itu kandungan monomer sisa yang tinggi dapat mempengaruhi sifat fisik polimer yang dihasilkan karena monomer sisa akan bertindak sebagai plasticiser dan membuat resin akrilik menjadi fleksibel dan kekuatan menurun. salah satu metode penambahan glass fibers adalah merendam glass fibers tersebut dalam metil metakrilat selama 15 menit.12 hal tersebut menyebabkan kandungan monomer dalam resin akrilik lebih banyak dari 17nirwana: kekuatan transversa resin perbandingan polimer dan monomer yang telah ditentukan pabrik. metode lain adalah menambahkan glass fiber langsung dalam campuran polimer dan monomer yang baru diaduk, jadi viskositas campuran resin akrilik masih rendah.17 sampai saat ini belum ada laporan informasi tentang kekuatan transversa resin akrilik hybrid setelah penambahan glass fiber dengan kedua metode tersebut. berdasarkan hal tersebut di atas, maka perlu diteliti kekuatan transversa resin akrilik hybrid dengan kedua metode penambahan glass fiber yang berbeda tersebut. tujuan penelitian ini adalah untuk mengetahui kekuatan transversa resin akrilik hybrid dengan metode penambahan glass fiber yang berbeda yaitu dengan cara merendam glass fiber dalam metil metakrilat 15 menit terlebih dahulu, dan dengan cara menambahkan langsung (tanpa direndam) dalam campuran polimer dan monomer. manfaat penelitian ini adalah hasil penelitian ini diharapkan dapat memberi informasi tentang pemilihan metode penambahan glass fiber dalam resin akrilik hybrid yamg tepat, sehingga menghasilkan resin akrilik yang mempunyai kekuatan transversa tinggi. bahan dan metode bahan yang digunakan pada penelitian ini adalah resin akrilik hybrid (biocryl), gip keras, woven glass fiber (yakasu, japan), akuades. alat yang digunakan adalah model master kuningan dengan ukuran 65 × 10 × 2,5 mm, kuvet logam, timbangan digital, termometer, autograph (shimadzu, japan). cara kerja penelitian adalah sebagai berikut, gip keras dengan perbandingan 100 gram bubuk dan 24 ml air (sesuai petunjuk pabrik) diaduk dengan menggunakan spatel, kemudian diletakkan di atas vibrator dan dimasukkan ke dalam kuvet yang telah disiapkan di atas vibrator. model master kuningan diletakkan ditengah kuvet didiamkan sampai gip mengeras. setelah mengeras, permukaan gip diulasi vaselin, kuvet antagonis dipasang, diisi adonan gip di atas vibrator dan ditekan, dibiarkan sampai gip mengeras. kuvet dibuka, model master diambil, maka didapat cetakan model (mould), kemudian diolesi separator, tunggu sampai kering selama 10 menit. persiapan pembuatan sampel dengan penambahan glass fiber adalah sebagai berikut, glass fiber ukuran 63 mm × 8 mm ditimbang seberat 0,15 g kemudian direndam dalam metil metakrilat monomer sebanyak 10 ml selama 15 menit (metode 1). kemudian polimer dan monomer dengan perbandingan 4 g : 2 ml diaduk dalam pot porselin. setelah 5 menit adonan mencapai tahap dough, selanjutnya adonan dimasukkan ke dalam mould, setelah bagian tengahnya diletakkan glass fiber, yang telah direndam dalam metil metakrilat monomer. kuvet ditutup, sebelumnya resin akrilik ditutup dengan kertas selopan dan ditekan perlahan-lahan dengan press hidrolik. kuvet dibuka kembali, kelebihan dipotong kemudian kuvet ditutup kembali, dilakukan penekanan dengan tekanan 2200 psi atau 50 kg/cm2. prosedur diulang 3 kali. dibiarkan selama 15 menit (aturan pabrik). selanjutnya dilakukan proses kuring dengan suhu 100° c selama 20 menit. metode 2, polimer dan monomer yang baru diaduk dimasukkan ke dalam mould dan bagian tengahnya diletakkan glass fiber ukuran 63 mm × 8 mm (tanpa direndam lebih dulu). kuvet ditutup, yang sebelumnya resin akrilik ditutupi dengan kertas selopan. dilakukan penekanan dengan press hidrolik ditunggu selama 5 menit kemudian kuvet dibuka. kelebihan dipotong kemudian kuvet ditutup lagi. selanjutnya dilakukan penekanan dan proses kuring seperti pada metode 1. pada penelitian ini terdapat 3 kelompok: kelompok 1 resin akrilik hybrid tanpa glass fiber (kontrol), kelompok 2 penambahan glass fiber dalam resin akrilik hybrid yang sebelumnya direndam dalam metil metakrilat monomer selama 15 menit (metode 1), dan kelompok 3 penambahan glass fiber langsung dalam resin akrilik yang baru diaduk (metode 2). batang uji yang dihasilkan dihaluskan dengan kertas gosok ukuran 600 dibawah air mengalir sampai diperoleh ukuran yang ditetapkan. kemudian diberi tanda pada garis tengahnya dengan pensil. pengukuran kekuatan transversa dilakukan dengan menggunakan alat autograph (shimadzu) ag-10 te dengan kecepatan cross head 1/10 mm/detik. jarak antara kedua penyangga adalah 50 mm.18 sebelum dilakukan tes, sampel direndam dalam air suling dengan suhu 37° c ± 1° c selama 48 jam.19 batang uji yang telah diberi tanda diletakkan ditengah alat tekan supaya tekanan tertuju pada satu garis batang uji. kemudian mesin dihidupkan, pemberat alat turun menekan tepat pada tengah batang uji sampai terjadi patahnya batang uji dan secara otomatis alat berhenti dan pada monitor menunjukkan nilai yang didapat dari hasil uji. cara perhitungan kekuatan transversa digunakan rumus sebagai berikut:18 2 bd2 ip 3 s = keterangan: s = kekuatan transversa (kg/cm2); b = lebar batang uji (cm); i = jarak pendukung (cm); d = tebal batang uji (cm); p = beban (kg). pengukuran kekuatan transversa dilakukan di laboratorium dasar bersama universitas airlangga pada bulan september 2004. untuk mengetahui perbedaan kekuatan transversa resin akrilik hybrid dengan penambahan glass fiber dilakukan uji anova dilanjutkan dengan lsd. hasil perhitungan rerata dan standart deviasi kekuatan transversa pada kelompok 1, 2 dan 3 terlihat pada tabel 1. 18 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 16–19 tabel 1. nilai rerata, standart deviasi dan hasil uji anova kekuatan transversa resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda (mpa) kelompok n rerata (mpa) standart deviasi p 1 2 3 6 6 6 94,94 118,27 116,34 9,45 11,09 12,66 0,001 keterangan: kelompok 1 = resin akrilik hybrid tanpa penambahan glass fiber (kontrol) kelompok 2 = resin akrilik hybrid ditambah glass fiber dengan metode 1 kelompok 3 = resin akrilik hybrid ditambah glass fiber dengan metode 2 hasil anova satu arah menunjukkan ada perbedaan bermakna kekuatan transversa antara kontrol dan resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda (p < 0,05). untuk mengetahui perbedaan antar kelompok digunakan uji lsd. tabel 2. uji lsd kekuatan transversa (mpa) pada kelompok kontrol dan resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda kelompok 1 kelompok 2 kelompok 3 kelompok 1 kelompok 2 kelompok 3 * * keterangan: * = berbeda bermakna pada tabel 2 menunjukkan adanya perbedaan bermakna antara kelompok 1 dan 2, kelompok 1 dan 3. hal tersebut berarti resin akrilik hybrid dengan penambahan glass fiber menunjukkan perbedaan kekuatan transversa yang bermakna dibandingkan dengan kelompok kontrol. sedangkan penambahan glass fiber dengan metode berbeda menunjukkan perbedaan yang tidak bermakna. penambahan glass fiber menghasilkan kekuatan transversa lebih tinggi dari kelompok kontrol. sedangkan penambahan glass fiber dengan metode berbeda tidak berbeda kekuatan transversanya. pembahasan fiber dapat digunakan untuk memperkuat bahan polimer, hal ini sangat penting karena terjadi adesi optimal antara fibers dan matrik polimer.20 pada penelitian ini penambahan glass fibers pada kelompok 2 dan 3 menunjukkan kekuatan transversa masing-masing 118,27 mpa dan 116,34 mpa, keduanya lebih tinggi dibandingkan dengan kelompok kontrol yaitu 94,94 mpa (tanpa glass fibers). pada kelompok 2 glass fibers sebelumnya direndam dalam metil metakrilat monomer selama 15 menit, hal tersebut menyebabkan adesi antara fiber dan matrik polimer menjadi baik. menyatunya fibers dengan resin akrilik menyebabkan fibers melekat pada matrik polimer. perendaman glass fiber tersebut merupakan prasyarat untuk melekatnya fibers pada matrik polimer. dengan meresapnya monomer dalam fiber menyebabkan fiber tersebut melekat dengan baik pada matrik polimer sehingga meningkatkan kekuatan resin akrilik.16 demikian juga pada kelompok 3 yang menggunakan metode 2 yaitu penambahan glass fibers langsung setelah bubuk dan cairan resin akrilik dicampur, jadi kondisi campuran tersebut masih cair (viskositas rendah) sehingga semua fiber dapat seluruhnya terbasahi. dengan demikian adesi antara fibers dan matrik polimer juga menjadi baik, akibatnya kekuatan transversa tinggi. metode tersebut dapat meningkatkan juga fracture resistance dari plat resin akrilik yaitu dengan adanya bahan matrik polimer yang cukup untuk fiber tersebut dan untuk chemical bonding antar fiber, maka matrik polimer harus menutupi glass fiber secara rata.17 hasil penelitian ini juga didukung oleh penelitian sebelumnya yang dilakukan oleh cemal et al., 13 menunjukkan adanya peningkatan kekuatan transversa sebesar 50% setelah penambahan stick glass fibers/ unidirectional glass fibers (serat memanjang) dan 21,2% setelah penambahan stick net glass fiber/woven glass fibers (anyaman) pada resin akrilik heat cured dibandingkan dengan tanpa fibers. sedangkan pada penelitian ini kekuatan transversa menunjukkan peningkatan sebesar 24,5% pada kelompok 2 dan 22,5% pada kelompok 3 setelah penambahan woven glass fibers (anyaman) pada resin akrilik hybrid. hasil penelitian ini menunjukkan bahwa penambahan glass fiber pada resin akrilik hybrid sangat bermanfaat untuk meningkatkan kekuatan basis gigi tiruan. faktor lain yang mempengaruhi kekuatan resin akrilik adalah jumlah (konsentrasi) fibers dalam matrik polimer, selain adesi fibers pada polimer. efek adesi fibers pada matrik polimer merupakan aspek yang sangat penting secara klinis sebab adesi sangat mempengaruhi kekuatan.21 sedangkan efek penambahan glass fibers pada resin akrilik dengan persentase atau konsentrasi berbeda terhadap kekuatan transversa menunjukkan bahwa penambahan 1% glass fibers meningkatkan kekuatan transversa.22 pada penelitian ini dapat disimpulkan bahwa penambahan glass fiber pada resin akrilik hybrid dengan metode berbeda ternyata tidak berbeda kekuatan transversanya. daftar pustaka 1. anusavice kj. phillips science of dental materials. 11st ed. philadelphia: wb saunders co; 2003. p. 246–9. 2. kedjarune u. release of methylmethacrylate from heat cured and autopolymerized resins: cytotoxicity testing related to residual monomer. australian dental journal 1999; 44(1): 25–30. 3. craig rg. restorative dental materials. 11st ed. mosby-year book, inc; 2002. p. 655–8. 19nirwana: kekuatan transversa resin 4. polyzois gl, andrepoulos ag, lagouvardos pe. acrylic resin denture repair with adhesive resin and metal wires:effects of strength parameters. j prosthet dent 1996; 75: 381–7. 5. narva kk, vallittu pk, helenius h, yli-upro a. clinical survey of acrylic resin removable denture repairs with glass fiber reinforcement. int j prosthodont 2001; 14(3): 219–24. 6. larson wr, dixon dl, aquilino sa, clancy jm. the effect of carbon graphite fiber reinforcement on the strength of provisional crown and fixed partial denture resins. j prosthet dent 1991; 66: 216–20. 7. vallittu pk. dimensional accuracy and stability of poltmethyl methacrylate reinforced with metal wire or with continuous glass fiber. int j prostodont 1996; 75: 617–20. 8. solnit gs.the effect of methyl methecrylate reinforcement with silane-treated and untreated glass fibers. j prosthet dent 1991; 66: 310–4. 9. braden m, davi kwm, parker s. denture base poly (methyl methacrylate) reinforced with ultra-high modulus polyethylene fibres. br dent j 1988; 164; 109–13. 10. gutteridge dl. the effect of including ultra-high modulus polyethylene fibre on the impact strength of acrylic resin. br dent j 1988; 164: 177–80. 11. vallittu pk. effect of water storage on the flexural properties of e-glass and silica fiber acrylic resin composite. int j prosthodont 1998; 11: 340–50. 12. neset e, nur h, erdal s. water sorption and dimensional changes of denture base polymer reinforced with glass fiber in continuous unidirectional and woven form. int j prosthodont 2000; 13: 487–93. 13. cemal a, handan y, alper c. effect of glass fiber reinforcement on the flexural strength of different denture base resins. quintessence int 2002; 33: 457–63. 14. handan y, cemal a, alper c ahmet y. the effect of glass fiber reinforcement on the residual monomer content of two denture base resins. quintessence int 2003; 34: 148–53. 15. hensten, petterson a, yacobson n. perceived side effect of biomaterials in prosthetic dentistry. j prosthet dent 1991; 65: 138–44. 16. combe ec. notes on dental materials. 6th ed. new york: churchill livingstone; 1992. p. 158–60. 17. vallittu pk, lassila vp, lappalainen. acrylic resin fiber composite: the effect of fiber concentration on fracture resistance. j prosthet dent 1994; 71: 607–12. 18. goodacre c, swartz ml, viveros c, andres. coparison of microwave polymerized denture base resins. j prosthodontic 1990; 3: 249–55. 19. american dental association. guide to dental materials and devices. 7th ed. chicago: ada; 1974. p. 203–8. 20. uzun g, hersek n, tincer t. effect of five woven fiber reinforcements on the impact and transverse strength of a denture base resin. j prosthet dent 199; 81: 616–20. 21. miettinen vm, vallittu pk. release of a residual methyl methacrylate into water from glass fiber polymethyl methacrylate composite used in dentures. biomaterials 1997; 18: 181–5. 22. stipho hd. effect of a glass fiber reinforcement on some mechanical properties of autopolymerizing polymethyl methacrylate. j prosthet dent 1998; 79: 580–4. << /ascii85encodepages 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 42 no 2 april 2009.indd issn 1978 3728 dental journal ! nbkbmbi!lfeplufsbo!hjhj editorial board of dental journal (majalah kedokteran gigi) sk: 2847/h3.1.2/kd/2009 june 1st, 2009 − june 1st, 2011 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. dr. m. rubianto, drg, m.s., sp. perio. (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc. ph.d.,fds. (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery university science malaysia, malaysia); prof. widowati witjaksono, dds, ph.d. (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, d.d.s., ph.d. (prostodontic university of hiroshima, japan); prof. yukio kato, d.d.s., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds.,ph.d. (pediatric – university of hiroshima, japan); prof. dr. a.g. m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam , the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); dr. nugrohowati, drg, m.kes. (conservative dentistry prof. dr. moestopo (b) university); prof. dr. m. suharsini, drg, m.s., sp. kga. (pediatric dentistry indonesia university); achmad gunadi, drg, m.s., ph.d. (prostodontic jember university) managing editors: prof. dr. arifzan razak, drg, msc, sp.pros. (prosthodontic – airlangga university); prof. dr. latief mooduto, drg, m.s., sp.kg. (conservative dentistry – airlangga university); prof. r.m. coen pramono danudiningrat, drg., su., sp.bm. (oral and maxillofacial surgery – airlangga university); prof. dr. mieke sylvia m.a.r., drg, m.s., sp.ort. (orthodontic – airlangga university); prof. dr. istiati, drg, m.s. (oral biology – airlangga university); prof. dr. anita yuliati, drg, m.kes. (dental material – airlangga university); seno pradopo, drg, s.u., ph.d. sp.kga. (pediatric dentistry – airlangga university); thalca i. agusni, drg, mhped. ph.d., sp.ort. (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes. (dental public health – airlangga university); dr. elly munadziroh, drg., ms. (dental material – airlangga university); priyawan rachmadi, drg, ph.d. (dental material – airlangga university); udijanto tedjosasongko, drg, ph.d., sp.kga. (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes. (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes. (oral medicine – airlangga university); dr. diah savitri ernawati, drg., m.si. (oral medicine – airlangga university); bagus soebadi, drg, mhped. sp.pm (oral medicine – airlangga university); endang pudjirochani, drg, m.s., sp.pros. (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes. (microbiology – airlangga university); susy kristiani, drg., m.kes. (oral biology – airlangga university); ira widjiastuti, drg, m.kes. sp.kg. (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms. (orthodontic – airlangga university); devi rianti, drg, m.kes. (dental material – airlangga university); chiquita prahasanti, drg., sp.perio. (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros. (prosthodontic – airlangga university); annissa chusida, drg., m.kes. (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg. (conservative dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 42 no. 2 april–june 2009: prof. madya. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery university science malaysia, malaysia) prof. widowati witjaksono, dds, ph.d. (kulliyah of dentistry, international islamic university malaysia) prof. taizo hamada, d.d.s., ph.d. (prosthodontic university of hiroshima, japan) sudarjani gunawan, drg.,ms.,sp.kg. (conservative dentistry – airlangga university) prof. dr. trijoenadi widodo, drg.,ms.,sp.kg. (conservative dentistry – airlangga university) dr. theresia indah budhy, drg.,m.kes. (oral biology – airlangga university) hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478 / 5030255. fax. (031) 5039478 / 5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 48/dikti/kep/2006 volume 42 number 2 april-june 2009 design cover photo by r. pudyanto hari pribadi, drg. contents page printed by: airlangga university press. (097/06.09/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 42 number 2 april-june 2009 issn 1978 3728 dental journal ! nbkbmbi!lfeplufsbo!hjhj 1. diagnosis and management of crohn’s disease in retarded child rahmi amtha ................................................................................................................................... 55–59 2. pseudomembranous candidiasis in patient wearing full denture nurdiana and m. jusri ..................................................................................................................... 60–64 3. cooperation of patient as key factor to overcome oral habits magdalena lesmana ........................................................................................................................ 65–69 4. transformation analysis of oral epithelial dysplasia to carcinoma in-situ and squamous cell carcinoma by p53 expression and gene mutations mei syafriadi .................................................................................................................................... 70–75 5. aesthethic and masticatory rehabilitation on post mandibular resection with combination of hollow obturator and hybrid prosthesis arif rachman, eha djulaeha and utari kresnoadi ...................................................................... 76–81 6. the effect of watermelon frost on prostaglandin e2 (pge2) in inflamed pulp tissue (in vitro study) dennis and trimurni abidin ........................................................................................................... 82–85 7. shear bond strength between porcelain and nano filler composite resin with or without 9% hydrofluoric acid etching kun ismiyatin ................................................................................................................................... 86–89 8. screening of oral premalignant lesions in smokers using toluidine blue yanti leosari, sri hadiati and dewi agustina .............................................................................. 90–93 9. biocompatibility of acrylic resin after being soaked in sodium hypochlorite nike hendrijatini ............................................................................................................................. 94–98 10. the aesthetic treatment for anterior teeth with lost crown by endorestoration nanik zubaidah ............................................................................................................................... 99–103 11. prognostic value of molecular markers of oral pre-malignant and malignant lesions peter agus ......................................................................................................................................... 104–108 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 4� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 4� the effect of �,25-dihydroxyvitamin d� on msx2 gene expression during tooth and alveolar bone development intan ruspita department of prosthodontics faculty of dentistry, universitas gadjah mada yogyakarta-indonesia abstract background: 1,25-dihydroxyvitamin d3 has been proven to be able to control the formation and biomineralization of tissue through a regulatory gene. a previous research even showed that a cell responsible for the formation of the enamel, dentin and bone was the target of 1,25dihydroxivitamin d3. purpose: this research was aimed to determine the role of 1,25dihydroxyvitamin d3 in vivo in the development of teeth and alveolar bone tissue by analyzing msx2 gene expression as a gene marker responsible for the growth and development of enamel, dentin, tooth root and alveolar bone. methods: samples used for rt-pcr analysis were total rna of insisivus teeth and alveolar bone derived from mice. rt-pcr analysis was conducted by using primer-specific gene, msx2. primer gene, gapdh, was also used as an internal control. five hundred nanograms of total rna were used as a template for pcr. semi quantitative results of pcr were quantified by using imagej software. results: rt-pcr analysis showed that the expression level of msx2 was enhanced in the samples of teeth and alveolar bone treated with 1,25 dihydroxyvitamin d3. the increasing of msx2 expression significantly occurred in alveolar bone samples. conclusion: it can be concluded that 1,25 dihydroxyvitamin d3 could enhance msx2 expression as a marker of the development of teeth and alveolar bone tissue. therefore, 1,25-d3 dihydroxyvitamin is expected to be used as an agent to help the regeneration of teeth and bone tissue. keywords: 1,25-dihydroxyvitamin d3; msx2; tooth; alveolar bone correspondence: intan ruspita, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas gadjah mada. jl. denta i, sekip utara 55281, indonesia. e-mail: intanruspita@ugm.ac.id, intanruspita@gmail.com research report introduction the rapid development in the science of molecular biology has prompted scientists to innovate to make replacement for teeth/ alveolar bone lost with a biotechnological artificial teeth/ alveolar bone. the biotechnological artificial teeth/ alveolar bone is not only used to improve the growth of the entire teeth/ bone as a unit, but also to include biological restoration of each tooth components, including enamel, dentin, cementum, or dental pulp. clinically, the replacement of teeth by using titanium implant is effective to replace the missing teeth, but still has several deficiencies. for instance, dental implants can integrate directly to the bone through osteointegration process without the mediation of periodontal ligament (pdl), whereas pdl serves the function of sensory, absorption and distribution of the load generated during mastication process. pdl also plays an important role in the movement of teeth and in maintaining new homeostasis.1-3 technology that facilitates the regeneration of tooth must receive considerable attention, especially in prosthodontics.4-7 in this case, the conventional regenerative dentistry has been developing stem cell technology, scaffolds and growth factors to produce biotechnological artificial teeth. researches on molecular mechanisms underlying tooth regeneration also has recently been developed. several transcription factors including sp6 and msx2 have a role in promoting cell growth and also as an important modulator in the growth of teeth and cranial bones.8-9 a research report also showed that 1,25-dihydroxyvitamin d3 as a hormonal form of vitamin d has been proved to dental journal (majalah kedokteran gigi) 20�5 march; 48(�): 4�–4� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 44 ruspita/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 43–47 be able to control the formation and biomineralization of tissue through a regulatory gene.10 some new researches even show that 1,25-dihydroxyvitamin d3 was able to regulate the expression of several marker genes of tooth and bone growth. a cell responsible for the formation of enamel tissue, dentin and bone is the target of 1,25dihydroxyvitamin d3.11 this research was aimed to determine the role of 1,25dihydroxyvitamin d3 in the expression level of msx2 gene as a marker of tooth and bone growth. by using the basic sciences of experimental embryology, developmental biology and molecular biology, the researcher tried to study and contribute knowledge in tissue regeneration, especially tooth and alveolar bone tissue. materials and method experimental animals used were eighteen male and female white mice (balb/ c) aged 7-8 weeks. those animals then were divided into three control groups and three treatment groups, each consisting of three mice. those animals were tamed in cages and fed in ad libitum for one week before treatment. one male and three female mice were placed in one cage. if plug is found in female mice in the morning, it is confirmed that those female mice are pregnant aged 0.5 days. those pregnant female mice were moved into separate cages, administrated orally with 2ug/ kg/ day of 1,25-dihydroxyvitamin d3 for four weeks,14 and given food and drink in ad libitum. the incisors and the alveolar bones of female mice’s children (aged 7 days) was taken for isolation of total rna. total rna obtained were used as samples of rt-pcr. protocol of working procedures in this research was approved by the ethics committee of faculty of medicine, universitas gadjah mada, yogyakarta. those incisors or alveolar bones were included in liquid nitrogen and then transferred into rna stabilization reagent (invitrogene, ny, usa). those samples were homogenized with pastel and mortal followed by rna extraction using trizol kit. (invitrogen, ny, usa). total rna obtained were dissolved in rnase free water. total rna derived from teeth and alveolar bones were transcribed into cdna. 500ng of (1µl) cdna in 20 µl of pcr (invitrogen, ny, usa) reaction solution was used as a sample to be analyzed. each treatment was repeated three times. primers used are listed in table 1. (integrated dna technologies, ca, usa). results research on the effect of 1,25 dihydroxyvitamin d3 on the growth and development of teeth and alveolar bone tissue obtained can be seen in the following results. normalization results of semi-quantitative pcr on msx2 genes against gapdh derived from tooth samples of the control and treatment groups. the expressions of msx2 genes on tooth samples of the treatment groups treated with 1,25 dihydroxyvitamin d3 were more increased than those in the control groups. normalization results of semi-quantitative pcr on msx2 genes against gapdh derived from alveolar bone samples of the control and treatment groups. the expressions of msx2 genes on alveolar bone samples of the treatment groups treated with 1,25 dihydroxyvitamin d3 were more significantly increased than those in the control groups. r e s u l t s o f p c r a n a l y s i s s h o w e d t h a t 1 , 2 5 dihydroxyvitamin d3 can increase the msx2 gene expressions during the growth and development of dental tissues (figure 1) and alveolar bones (figure 2). to obtain a quantitative value of the thickness of pcr band, gel quantification analysis was conducted by using image j software (table 1 and 2). the quantification results of msx2 gene expressions obtained were then normalized with the results of internal control gene quantification, namely gapdh and presented in graphical form. the calculation results of msx2 gene normalization against gapdh showed that msx2 gene expressions were table 1. primer, sequence and condition of pcr primer sequence annealing extention cycle msx2 forward 5’-agacatatgagcccc accac-3’ 56oc 60 sec 30 msx2 reverse 5’-caaggctagaagctgggatg-3’ gapdh forward 5’gcaaagtggagattgttgccat-3’ 58.7oc 60 sec 20 gapdh reverse 5’-ccttgactgtgccgttgaattt-3’, table 2. results of the quantification of pcr band of msx2 and gapdh genes on teeth samples using image j software msx2 gapdh msx2/gapdh control 6411.104 12545.388 0.51103274 1,25 dihydroxyvit.d3 6660.912 11877.903 0.560781815 45 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 45ruspita/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 43–47 increased after the administration of 1,25 dihydroxyvitamin d3 on dental tissue and alveolar bone tissue compared to those in the control groups. the increasing of msx2 gene expressions on the growth and development of teeth indicated no significant change, but the increasing of msx2 gene expressions on the growth and development of alveolar bone tissue showed significantly change (figure 1 and 2). discussion vitamin d is known to have an important role in maintaining the homeostasis of calcium and phosphorus to promote bone mineralization and other tissue mineralization. 1,25 dihydroxyvitamin d3 is a kind of hormones in the active form of vitamin d that has anti-proliferative properties, pro-apoptotic properties and pro-differentiation of various body cell types. cyp27b1 enzyme has a role in changing 25-hydroxyvitamin d into the active form of 1,25 dihydroxyvitamin d3 able to promote the proliferation of osteoblasts. the metabolism of 25-hydroxy25d1hydroxylase (cyp27b1) on bone cells indicates a function on osteoblasts and osteoclasts. previous researches even 9 figure 1. results of semi-quantitative pcr analysis of msx2 and gapdh genes on the samples of teeth. note: the expressions of msx2 genes on the growth and development of tooth tissues in the treatment groups with 1,25 dihydroxyvitamin d3 indicates more improvement than the control groups. c: control, t: treatment, c(-): the negative control/ distillated water. l: leader. table 2. results of the quantification of pcr band using image j software figure 2. graph of normalization of msx2 gene values against gapdh gene values derived from tooth samples. a. b. msx2 gapdh msx2/gapdh control 6411.104 12545.388 0.51103274 1,25 dihydroxyvit.d3 6660.912 11877.903 0.560781815 c t c(-) tooth c(-) tooth t l l c gapdh msx2 control tulang alveolar p c(-) c l tulang alveolar c p c(-) l msx2 gapdh alveolar bone alveolar bone figure 1. semi-quantitative pcr analysis of msx2 and gapdh genes on the samples of teeth. the expressions of msx2 genes on the growth and development of tooth tissues in the treatment groups with 1,25 dihydroxyvitamin d3 indicates more improvement than the control groups. c: control, t: treatment, c(-): the negative control/ distillated water. l: leader. 1 figure 2. graph of normalization of msx2 gene values against gapdh gene values derived from tooth samples. figure 4. graph of normalization of msx2 gene values against gapdh gene values derived from alveolar bone samples. figure 2. graph of normalization of msx2 gene values against gapdh gene values derived from tooth samples. table 3. results of the quantification of pcr band of msx2 and gapdh genes on teeth samples of alveolar bone using image j software msx2 gapdh msx2/gapdh control 4729.84 5771.376 0.819534371 1,25 dihydroxyvit.d3 48746.1 1949.861 24.99978614 9 figure 1. results of semi-quantitative pcr analysis of msx2 and gapdh genes on the samples of teeth. note: the expressions of msx2 genes on the growth and development of tooth tissues in the treatment groups with 1,25 dihydroxyvitamin d3 indicates more improvement than the control groups. c: control, t: treatment, c(-): the negative control/ distillated water. l: leader. table 2. results of the quantification of pcr band using image j software figure 2. graph of normalization of msx2 gene values against gapdh gene values derived from tooth samples. a. b. msx2 gapdh msx2/gapdh control 6411.104 12545.388 0.51103274 1,25 dihydroxyvit.d3 6660.912 11877.903 0.560781815 c t c(-) tooth c(-) tooth t l l c gapdh msx2 control tulang alveolar p c(-) c l tulang alveolar c p c(-) l msx2 gapdh alveolar bone alveolar bone figure 3. semi-quantitative pcr analysis of msx2 and gapdh genes on the samples of alveolar bone. the expressions of msx2 genes on the growth and development of alveolar bone tissues in the treatment groups with 1.5 dihydroxyvitamin d3 indicates the more significant improvement than the control groups. c: control, t: treatment, c(-): the negative control/ distillated water. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 46 ruspita/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 43–47 proved that 1,25 dihydroxyvitamin d3 is able to modulate genes essential for bone and tooth growth.11 1,25 dihidroxyvitamin d3 can increase the mineralization of osteoblast mediators by stimulating the production of alp-positif vehicle matriks.13 on the other hand, 1,25 dihydroxyvitamin d3 also have opposite effects on mineralization process that can infiltrate body mineral tissues.14 the results of previous research even show 1,25 dihydroxyvitamin d3 can stimulated osteoclst formation in vitro.15 however, osteoprotegerin (opg), a member of the tnf receptor family can prevent the stimulation of 1,25 dihydroxyvitamin d3 from resorption. recent researches also reported that 1,25 dihydroxyvitamin d3 is able to regulate genes responsible for mineralization process.10 osteoblast, odontoblast and ameloblast cells responsible for bone and tooth mineralization process are known as the target of 1,25 dihydroxyvitamin d3. therefore, this research was aimed to report the effects of 1,25 dihydroxyvitamin d3 on the growth and development of teeth and bone tissues by analyzing one gen marker for the growth of teeth and bones, which is msx2. msx2 is known to be involved in the formation of cranial bone and tooth tissue. msx2 expression is widely distributed in craniofacial tissue. msx2 deficiency can cause defects in parietal foramina caused by lack of proliferation activity of calvaria cells.16 msx2 deficiency also causes defects in tooth mineralization tissues, such as enamel, dentin and cementum, as well as makes ameloblast cell polarization lower than normal. clinical appearance to the teeth is in the form of enamel or dentin tissue depletion associated with amelogenesis imperfecta or dentinogenesis imperfecta.17 b a s e d o n t h e r e s u l t s , i t i s k n o w n t h a t 1 , 2 5 dihidroxyvitamin d3 given orally to those animals could affect the expression of msx2 at the rna level that tested with the semi-quantitative pcr analysis method. figures 1 and 2 showed that msx2 gene expressions were increased in treatment groups given with 1,25 dihydroxyvitamin d3 during the growth and development of teeth and alveolar bone tissue. the increased msx2 gene expression on dental tissue proved that 1,25-dihydroxyvitamin d3 plays a role in helping the growth and development of dental tissues. msx2 has been known to be expressed in dental tissue during the growth and development of the teeth in embryonic period.18 disruption of msx2 expressions can cause growth abnormalities/ disability in the development of teeth. other researches analyzing the expressions of msx2 in mice also showed that mutant msx2 in newborn and adult can cause both abnormal formation of enamel, a very complex enamel disability associated with amelogenin and enamelin genes, and loss of ameloblast cells in tooth germs. the loss of ameloblast cells is actually caused by the decreasing of laminin 5 and cytokeratin 5 expressions relating to the bonds among the cells. on the other hand, other researches using msx2 over-expression technique show that the increasing of amelogenin genes is associated with the thickening dental email.19 physiological functions of msx2 on the alveolar bone and tooth development have also been analyzed using transgenic mice. in mice with mutant msx2, there will be changes in the morphology of the teeth and periodontal tissues. the highest expression of msx2 contained in the active site of bone modeling can be associated with tooth growth and dental root extension.17 similarly, the increasing of msx2 gene expression in alveolar bone growth indicates that 1,25-dihydroxyvitamin d3 can improve the regeneration of alveolar bone. another research even showed that msx2 expression is widely distributed on multiple craniofacial tissue structures. in addition, msx2 is also expressed in osteoclast cells, suggesting that msx2 also plays a role in the process of bone resorption.16 there are several clinical conditions that require the increasing of bone regeneration either locally or systemically. various methods are currently used to increase or accelerate bone repair. in dentistry, local injection of 1,25 dihydroxyvitamin d3 is able to increase bone formation in order to stabilize the teeth after teeth movements. in prosthodontics, the increased volume of alveolar bone is useful to facilitate dental implant placement and alveolar bone preparation in denture making. the increased volume of alveolar bone is also useful for the retention of the dentures. 1,25 dihydroxyvitmin d3 has also been reported to be able to increase bone osseointegration after installation of titanium dental implants. osseointegration is needed for successful dental implant installation in order to support prosthesis mounted on the implant, so it is not easily rocking. it can be concluded that 1,25-dihydroxyvitamin d3 can increase the expression of msx2 genes essential for the growth of bones and teeth. 1,25-dihydroxyvitamin d3, consequently, is expected to act as an agent that is able to regenerate mineralized tissue, such as enamel, dentin, cementum and bone. 1,25-dihydroxyvitamin d3 is also expected to be useful for the application in of clinical dental care. 1 figure 2. graph of normalization of msx2 gene values against gapdh gene values derived from tooth samples. figure 4. graph of normalization of msx2 gene values against gapdh gene values derived from alveolar bone samples. figure 4. graph of normalization of msx2 gene values against gapdh gene values derived from alveolar bone samples. 4� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 4�ruspita/dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 43–47 refferences 1. palaiologou a, stoute d, fan y, lallier te. altered cell motility and attachment with titanium surface modifications. j periodontol 2011; 83(1): 90-100. 2. siar ch, toh cg, romanos ge, ng kh. comparative assessment of the interfacial soft and hard tissues investing implants and natural teeth in the macaque mandible. clin oral investig 2015; 19(6): 135362. 3. masamitsu oshima m, inoue k, nakajima k, tachikawa t, yamazaki h, tomohide isobe t, sugawara a, ogawa m, tanaka c, saito m, kasugai s, takano-yamamoto t, inoue t, tezuka k, kuboki t, yamaguchi a, tsuji t. functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy. sci rep 2014; 13(4): 6044. 4. tsuji t, na kao k. dent a l regenerat ion t herapy: stem cel l transplantation and bioengineered tooth replacement. japanese dental science 2008; 44: 70-5. 5. ikeda e, morita r, nakao k, ishida k, nakamura t, yamamoto t, ogawa m, mizuno m, kasugai s, tsuji t. fully functional bioengineered tooth replacement as an organ replacement therapy. proc natl acad sci usa 2009; 106(32): 13475-80. 6. oshima m, tsuji t. functional tooth regenerative therapy: tooth tissue regeneration and whole tooth replacement. odontology 2014; 102(2):123-36. 7. egusa h, sanoyama w, nishimura m, atsuta i, akiyama k. stem cells in dentistry-part i: clinical application. j prosthodont res 2012; 56(3): 151-65. 8. ruspita i, miyoshi k, muto t, abe k, horiguchi t, noma t. sp6 down regulation of follistatin gene expression in ameloblast. j med invest 2008; 55(1-2): 87-98. 9. saadi i, das p, zhao m, raj l, ruspita i, xia y, papaioannou e, bei m. msx1 and tbx2 antagonistically regulate bmp4 expression during the bud-to-cap stage transtition in tooth development. development 2013; 140(13); 2697-702. 10. kawakami m, yamamoto t. local injection of 1,25-dihydroxyvitamin d3 enhanced bone formation for tooth stabilization after experimental tooth movement in rats. j bone miner metab 2004; 22(6): 541-6. 11. papagerakis p, macdougall m, berdal a. differential epitelial and mesenchymal regulation of tooth-specific matrix proteins expression by 1,25-dihydroxyvitamin d3 in vivo. connect tissue res 2002; 43(2-3): 372-5. 12. saito h, harada s. eldecalcitol replaces endogenous calcitriol but does not fully compensate for its action in vivo. j steroid biochem mol biol 2014; 144 pt a: 189-96. 13. woeckel vj, alves rd, swagemakers sm, eijken m, chiba h, van der eerden bc, van leeuwen jp. 1alpha,25-(oh)2d3 acts in the early phase of osteoblast differentiation to enhance mineralization via accelerated production of mature matrix vesicles. j cell physiol 2010; 225(2): 593–600. 14. yamaguchi m, weitzmann mn. high dose 1,25(oh)2d3 inhibits osteoblast mineralization in vitro. int j mol med 2012; 29(5): 9348. 15. lee sk, kalinowski j, jastrzebski s, lorenzo ja. 1,25(oh)2 vitamind3-stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced il-1 alpha and receptor activator of nf-kappa b ligand production in osteoblast. j immunol, 2002; 169(5):2374-80. 16. marijanovic i, kronenberg ms, ivkosic ie, lichtler ac. comparison of proliferation and differemtiation of calvarial osteoblast cultures derived from msx2 deficient and wilt type mice. colleg antrop 2009; 33(3): 919. 17. aioub m, lezot f, molla m, castaneda b, robert b, goubin g, nefussi jr, berdal a. msx2 -/transgenic mice develop compound amelogenesis imperfecta, dentinogenesis impefecta and periodental osteopetrosis. bone 2007; 41(5): 851-9. 18. zhang yd, chen z, song yq, liu c, chen yp. making a tooth: growth factors, transcription factors and stem cells. cell res 2005; 15(5): 301-16. 19. babajko s, de la dure-molla m, jedeon k, berdal a. msx2 in ameloblast cell fate and activity. front physiol 2015; 5: 510. 88 pulp tissue inflammation and angiogenesis after pulp capping with transforming growth factor b1 sri kunarti department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract in restorative dentistry the opportunity to develop biomemitic approaches has been signalled by the possible use of various biological macromolecules in direct pulp capping reparation. the presence of growth factors in dentin matrix and the putative role indicating odontoblast differentiation during embryogenesis has led to the examination on the effect of endogenous tgf-b1. tgf-b1 is one of the growth factors that plays an important role in pulp healing. the application of exogenous tgf-b1 in direct pulp capping treatment should be experimented in fibroblast tissue in-vivo to see the responses of inflammatory cells and development of new blood vessels. the increase in food supplies always occurs in the process of inflammation therefore the development of angiogenesis is required to fulfil the requirement. this in-vivo study done on orthodontic patients indicated for premolar extraction between 10–15 years of age. a class v cavity preparation was created in the buccal aspect 1 mm above gingival margin to pulp exposure. the cavity was slowly irrigated with saline solution and dried using a sterile small cotton pellet. the sterile absorbable collagen membrane was applied and soaked in 5 ml tgf-b1. it was covered by a teflon pledge to separate from glass ionomer cement restoration. evaluation was performed on day 7; 14; and 21. all samples were histopathologycally examined and data was statistically analysed using one way anova and dunnet t3.there were no inflammatory symptoms in clinical examination on both ca(oh)2 and tgf-b1, but they increased the infiltration of inflammatory cells on histopathological examination. there were no significant differences (p > 0.05) between ca(oh)2 and tgf-b1 in inflammation cell and significant differences (p < 0.05) in angiogenesis on day 7 and 14. there were no significant differences (p > 0.05) in inflammation cell with in tgf-b1 groups and significant differences (p < 0.05) with in ca(oh)2 groups on day 7 and 14. it is concluded tgf-b1 functions as direct capping medication has the same inflammatory response as ca(oh)2, however, tgf-b1 developed angiogenesis earlier than ca(oh)2. key words: calcium hydroxide, tgf-b1, inflammation, angiogenesis correspondence: sri kunarti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: attybp@yahoo.com introduction homeostatis condition in human’s tissue needs balanced interaction between cells and extra cellular matrix as protein supplier involving the role of various cytokines through specific receptor. disease will consequently occur if the balance between cells and extra cellular matrix is disturbed. the improvement of damaged tissue will manifest if the body successfully maintains the homeostatic condition from the effect of harmful environment.1 dentin perforation will initially cause several episodes such as: inflammation, synthesis of new collagen and the formation of dentinal bridge, finally, reparative dentin formation will be necessarily done.2,3 the inflammation is indicated by movement of fluid circulation, plasma protein and leukocyte to tissue responding to the presence of danger. inflammation is essential mechanism which is required to improve structure and tissue functional disturbance as well as to defend itself from the danger such as: tissue damage, microorganism invasion and foreign body disturbing the balance interaction between cells and extra cellular matrix (ecm). inflammatory response completely depends on blood vessel, cell as well as fluid circulating in blood vessel.4 the initial symptom of inflammation is indicated by secretion of various mediators such as transforming growth factor-b1 (tgf-b1) followed by activation of complement, coagulation system, inflammation cell and endothelial cell. immediately after the injury is present in dentin, it will be followed by recovery process by releasing various growth factors, cytokine and accumulative molecule from serum from blood vessel rupture and platelet degranulation.5 inflammation cell is also the source of various growth factor which are needed to initiate proliferation phase in tissue recovery. the formation of reparative dentin is completely influenced by inflammation process, cell proliferation, cell migration, angiogenesis and production of extra cellular matrix. tgf-b1 regulates all of the episodes including chemotaxis of inflammation cell, angiogenesis, deposition of extra cellular matrix and the formation of new tissue.6 concentration of tgf-b1 increases in dentin caries condition comparing to healthy dentin. tgf-b1 in reparative dentin matrix is higher than primary dentin 89kunarti: pulp tissue inflammation and angiogenesis after pulp capping matrix. the increase of intracellular expression of tgf-b1 and ecm in dentin carries is suspected that tgf-b1 has an essential role to response injury and tissue recovery.7 the parameter of inflammation response in the present study is the number of inflammation cells and synthesis of new blood vessels (angiogenesis) in 7, 14 and 21 days of recovery process. the purpose of the present study was to observe the response of inflammation cell and angiogenesis of tgf-b1and material with calcium hydroxide [ca(oh)2] as the comparation because up to the present the material of direct pulp capping. materials and methods the study was done on orthodontic patients indicated for premolar extraction. anaesthesia in buccal fold region was done using 0.6 ml xylestesin f. rubber dam, and saliva ejector were aplied. alcohol 70% were used to disinfect the preparation at buccocervical. preparation of the cavity on 1 mm buccal site above gingival margin, completed by intermittent bur with light pressure. preparation was done declining toward apical using no. 3 round bur in 1.5 mm diameter almost reaching the pulp, then no. 1 round bur in 0.5 mm diameter was used to penetrate thin dentin layer reaching perforation in pulpa coronalis. slow irrigation using 0.5 ml saline solution was performed in cavity region and dried by sterile cotton pellet. visible light of ca(oh)2 in tube (calcimol) was applied 1 mm on pulp for 40 seconds. in other groups, 20 ng/ml tgf-b1 caried by material collagen membrane3 was applied and completed by filling material of glass ionomer cement type 2. teflon pledged8 was given to avoid the possible reaction between glass ionomer cement type 2 and material of pulp capping. then extraction was classified into 3 extraction timing on day 7, 14 and 21 direct pulp capping. the preparation of histological preparat was began since the initial process of extraction. fixation using 10% formalin buffer for 48 hours followed by decalcification using alcl, formic acid, 37% hcl and aquadest continued by dehydration process to extract the water from the tissue and substituted by hardening media (paraffin) and clearance was done using xylene. after paraffin hardening, the tissue could be cut using microtome in 4 µm thickness. the next step, hematoxylin and eosin were stained on the resulting preparat. light microscope in 400 times magnification was used to count the number of inflammation cell (figure 1) and blood vessel (figure 2). table 1. the mean and standard deviation of inflammation in control group of ca(oh)2 and tgf-and tgf--b1 on day 7, 14, and 21 variable material 7 days 14 days 21 days mean sd mean sd mean sd inflammation ca(oh)2 tgf-b1 19.3750 21.6250 6.2321 3.8522 19.1250 19.8750 4.1897 3.0909 16.5000 18.5000 4.5981 2.0000 angiogenesis ca(oh)2 tgf-b1 24.3750 16.6250 1.7678 4.6579 20.1250 12.8750 2.7484 4.7461 17.8750 12.3750 7.0191 3.3354 sd: standar deviation figure 1. inflammation cell figure 2. angiogenesis ( ) result anova test was performed to know the different response between inflammation cell and angiogenesis in ca(oh)2 and tgf-b1. the result showed significant difference (p < 0.05) in control group, followed by lsd test to know the difference in the period of 7–14 days, 14–21 days, and 7–21 days (table 2). 90 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 88-90 table 2. lsd-test on inflammation cell and angiogenesis and ca(oh) 2 and tgf-b1 in the period 7–14 days, 14–21 days, and 7–21 days–21 days, and 7–21 days21 days, and 7–21 days–21 days21 days variable period of observation p value ca(oh)2 tgf-β1 inflammation cell 7–14 days 7–21 days 14–21 days 0.923 0.271 0.313 0.268 0.055 0.382 angiogenesis 7–14 days 7–21 days 14–21 days 0.009* 0.093 0.785 0.096 0.061 0.818 * = significant difference p of ca(oh)2 group showed significant difference in angiogenesis in comparison period of 7–14 days. the result of t-test between ca(oh)2 and tgf-b1 on day 7, 14 and 21 showed significant different in angiogenesis on day 7 and 14 (table 3). table 3. t-test inflammation cell and angiogenesis comparison of ca(oh)2 and tgf-b1 among of 7 days, 14 days and 21 days variable p value ca(oh)2 and tgf-β1 7 days 14 days 21 days inflammation cell angiogenesis 0.400 0.001* 0.690 0.002* 0.287 0.073 * = significant difference discussion in this study, there was no significant difference in infiltration of inflammation cell between ca(oh)2 and tgf-b1 on day 7, 14, and 21, however the mean showed that tgf-b1 was higher comparing to ca(oh)2 due to potential proinflammation character tgf-b1 such as: affecting leukocyte in the initial response of inflammation and strengthening inflammation response in low concentration by stimulating integrin expression, in addition, initiating chemotaxis process as well as increasing extravasation by enhancing useful enzyme in extravasation process. the concentration of tgf-b1 increased during inflammation process, by adding exogenic tgf-b1, thus, the concentration would be higher, monocyte/macrophage and lymphocyte would be activated by tgf-b1 to remove the pathogen. tgf-b1 has the most potential role in inflammation and immune response among tgf-b1 isoform. foreign molecules, tissue debris and accumulative leukocyte are no longer necessary prior to pathogen removal. subsequently the number of plasmin production would also lower and the concentration of tgf-b15 would be similar. in this study, the observation on day 7 and 14 showed significant difference in angiogenesis. the mean of tgf-b1 was lower compared to the mean ca(oh)2 due to the presence of angiogenesis/neovascularization prior to day 7 in tgf-b1 group. the initial recovery started on day 3rd prior to injury meanwhile the granulation tissue is completely vascular. capillary new blood vessel would penetrate into necrotic tissue in which inflammatory exudates is eradicated by macrophage. observation on day 7 showed the presence of collagen synthesis type i and simultaneously showed the decrease of capillary blood vessel. the proceeding recovery process would be followed by decrease of ratio between vascular tissue and fibroblast tissue and subsequently collagen production would elevate.9 tgf-b1 is one of the growth factors which would influence angiogenesis activity and it is important for vascular maturization and remodelling.10 tgf-b1 not only increase fibrous tissue deposition, migration of fibroblast proliferation but also collagen synthesis. based on the above explanation it is concluded that angiogenesis decrease before day 7 in tgf-b1 group compared to ca(oh)2 in which angiogenesis is higher. in the comparison between both materials the increase of inflammation cell is not present. references 1. werner s, grose r. regulation of wound healing by growth factors and cytokines. physiol rev 2003; 83:835–70. 2. trope m, chivian n, sigurdsson a. traumatic injuries. in: cohen s, burns rc, editors. pathways of the pulp. 8th ed. st louis: mosby inc; 2002. p. 560–72. 3. hu cc, zhang c, qian q, tatum nb. reparative dentin formation in rat molars after direct pulp capping with growth factors. j endod 1998; 24:744–51. 4. arenberg da, strieter rm. angiogenesis. in: gallin ji, snyderman r, eds. inflammation basic principles and clinical correlates. 3rd ed. philadelphia: lippincott williams & wilkins; 1999. p. 851–53. 5. wahl sm. transforming growth factor-b (tgf-b) in the resolution and repair of inflammation. in: gallin ji, snyderman r, eds. inflammation basic principles and clinical correlates. 3rd ed. philadelphia: lippincott williams & wilkins; 1999. p. 837–42. 6. about i, bottero mj, de denato p, camps j. human dentin productionhuman dentin production in vitro. exp cell res 2000; 258:33–41.2000; 258:33–41.258:33–41. 7. murray pe, windsor lj. analysis of pulpal reactions to restorative procedures, material, pulp capping, and future therapy. crit rev oral biol med 2002; 13:509–20. 8. kunarti s. stimulasi aktivitas fibroblas pulpa dengan pemberian tgf-b1 sebagai bahan perawatan direct pulp capping. disertasi. 2005. p. 91. 9. mcmahon rft, sloan p. inflammation. in: essentials of pathology forin: essentials of pathology for dentistry. 1st ed. edinburgh: churchill livingstone; 2000. p. 26–31. 10. cotran rs, kumar v, collin t. acute and chronic inflammation. in: robbins pathologic basis of disease. 6th ed. philadelphia: wb saunders company; 1999. p. 50–111. 206 volume 47, number 4, december 2014 kekerasan mikro enamel gigi permanen muda setelah aplikasi bahan pemutih gigi dan pasta remineralisasi (enamel micro hardness of young permanent tooth after bleaching and remineralization paste application) budianto liwang, irmawati, dan els budipramana departemen ilmu kedokteran gigi anak fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract background: studies showed that bleaching agent had demineralization effect to enamel, and encourage use of remineralization paste after bleaching treatment especially in young permanent tooth which in post-eruptive enamel maturation. purpose: the study were aimed to determine the bleaching agent effect on enamel surface micro hardness, and to determine the effect of remineralization paste application on enamel surface micro hardness of young permanent tooth after bleaching treatment. methods: fourteen young permanent teeth were placed in a block of resin with a window on the buccal surface enamel. the initial enamel surface hardness was measured using microvickers hardness tester. then the application of hydrogen peroxide bleaching materials 30% was done three times for 15 minutes and followed by surface hardness of enamel measurement. samples were divided into 2 groups; the first group was applied paste of hydroxy apatite + naf 1450ppm , and the second group was applied paste of cpp–acp + naf 900ppm. each paste was applied for 30 minutes for 7 days, then the enamel surface hardness of samples were measured. results: the enamel surface micro hardness decreased after bleaching from 333.09 ± 10.49 vhn to 299.15±5.70 vhn. micro hardness after application of hidroxy apatite + naf 1450ppm was 316.61±5.87 vhn and after application of cpp-acp + naf 900ppm was 319.94±3.25 vhn, however the micro hardness still lower than initial micro hardness. conclusion: tooth bleaching agent caused a decrease of enamel surface micro hardness in young permanent tooth. the use of remineralization paste enabled to increase the enamel surface micro hardness young permanent tooth. key words: bleaching, enamel, young permanent tooth, micro hardness, naf, cpp-acp abstrak latar belakang: penelitian-penelitian sebelumnya menunjukkan bahwa produk pemutih gigi memiliki efek demineralisasi enamel gigi, dan mendorong penggunaan pasta remineralisasi setelah pemutihan gigi terutama di gigi muda permanen yang enamelnya masih dalam proses maturasi pasca-erupsi. tujuan: penelitian ini bertujuan untuk meneliti kekerasan mikro permukaan email gigi permanen muda, dan efek aplikasi pasta remineralisasi setelah pemutihan gigi. metode: empat belas gigi permanen muda ditempatkan dalam blok resin dengan jendela pada enamel permukaan bukal dan dilakukan pengukuran kekerasan permukaan enamel sampel awal dengan menggunakan alat microvickers hardness tester. kemudian dilakukan aplikasi bahan bleaching hidrogen peroxide 30% sebanyak 3 kali masing-masing selama 15 menit. setelah aplikasi bahan bleaching, kekerasan permukaan enamel sampel diukur kembali. sampel dibagi 2 kelompok; kelompok pertama diaplikasi pasta remineralisasi hidroksi apatit + naf 1450ppm, dan kelompok kedua diaplikasi pasta cpp-acp + naf 900ppm. masing-masing pasta tersebut diaplikasikan selama 30 menit 7 hari berturut-turut. setelah aplikasi pasta remineralisasi, sampel diukur kembali kekerasan permukaan enamelnya. hasil: kekerasan mikro permukaan enamel menurun setelah aplikasi pemutih gigi, dari 333.09 ± 10.49 vhn ke 299,15 ± 5.70 vhn. kekerasan mikro setelah aplikasi hidroxy apatit + naf research report 207liwang, et al.: kekerasan mikro enamel gigi permanen muda 1450ppm adalah 316,61 ± 5.87 vhn dan setelah aplikasi cpp-acp + naf 900ppm adalah 319,94 ± 3,25 vhn, namun kekerasan mikro setelah aplikasi pasta remineralisasi masih lebih rendah dari kekerasan mikro awal. simpulan: bahan pemutih gigi menurunkan kekerasan mikro permukaan enamel gigi permanen muda secara signifikan. aplikasi pasta remineralisasi dapat meningkatkan kembali kekerasan mikro permukaan enamel gigi permanen muda. kata kunci: pemutihan gigi, enamel, gigi permanen muda, kekerasan mikro, naf, cpp-acp korespondensi (correspondence): budianto liwang, departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: she_ming2003@yahoo.com pendahuluan semakin meningkatnya kesadaran masyarakat akan pentingnya kesehatan gigi membuat adanya trend baru dalam ilmu kedokteran gigi. seorang dokter gigi saat ini tidak hanya dituntut untuk dapat menangani masalah karies dan pencegahannya tapi juga bagaimana cara untuk membuat orang dapat terlihat lebih menarik, hal ini dikenal sebagai esthetic dentistry yang salah satu perawatannya adalah pemutihan gigi.1 penampilan gigi sangat penting pada semua kalangan umur dan sering dihubungkan dengan kesehatan secara umum.2 estetik dentistry semakin diperkuat dengan semakin berkembangnya teknologi dalam komunikasi dan informasi sehingga informasi mengenai perawatan estetika terhadap gigi dapat menjangkau seluruh masyarakat, perawatan estetika gigi tidak hanya diperlukan oleh orang dewasa saja tetapi juga menjadi trend dikalangan pasien dengan umur yang tergolong remaja, sehingga diperlukan informasi yang lebih jelas mengenai pemutihan gigi.3 gigi yang mengalami perubahan warna dapat mempengaruhi psikologis anak pada usia remaja dan dapat menjadi indikasi perlunya tindakan pemutihan gigi (dental bleaching).4 dental bleaching adalah tindakan aplikasi bahan kimia pada gigi untuk mengoksidasi pigmentasi organik. warna gigi normal adalah warna translusen dan sedikit memperlihatkan warna dentin, tetapi warna gigi ini dapat dipengaruhi oleh beberapa faktor penyebab perwarnaan gigi baik ekstrinsik maupun intrinsik. secara ekstrinsik warna gigi dapat dipengaruhi oleh penyerapan zat warna oleh gigi dari makanan dan minuman yang dikonsumsi setelah gigi erupsi, dan secara intrinsik warna gigi dapat dipengaruhi oleh pencemaran pada dentin dan enamel oleh materi kromatogenik pada masa odontogenesis. bahan yang digunakan pada prosedur dental bleaching adalah bahan yang berasal dari golongan peroxida yaitu hidrogen peroxida (h2o2), dan carbamide peroxida, bahan ini dapat diaplikasikan oleh dokter gigi pada praktek dokter gigi ataupun oleh pasien sendiri di rumah dengan pengawasan dokter gigi, perbedaan aplikasi di praktek dokter gigi dan di rumah oleh pasien adalah pada persentase bahan aktif yang digunakan pada aplikasi di rumah menggunakan bahan aktif dengan persentase yang lebih kecil, dengan resiko yang lebih kecil tetapi penggunaan bahan yang lebih lama untuk mendapatkan hasil yang diinginkan.5 hidrogen peroksida (h2o2), mempunyai kemampuan menembus email dan dentin yang terkena diskolorisasi. penembusan ini terjadi karena berat molekul hidrogen peroksida yang rendah dan mempunyai kemampuan denaturasi protein sehingga dapat meningkatkan gerakan ion-ion melalui gigi. selain itu hidrogen peroksida merupakan bahan oksidasi dan reduksi berkekuatan tinggi. hidrogen peroksida melalui radikal bebas reaktif yang dihasilkannya dapat menghancurkan ikatan konjugasi pada molekul-molekul zat warna pada stain sehingga molekul tersebut menjadi lebih sedikit berpigmen dan menyebabkan efek pemutihan.6 meskipun metode bleaching sudah hampir menjadi prosedur standart dalam aplikasi praktek kedokteran gigi, tetapi tidak sedikit praktisi maupun pasien yang meragukan keamanan aplikasi bahan bleaching terhadap gigi, hal ini juga menjadi perhatian orang tua bagi pasien yang masih berumur remaja. efek samping bleaching yang sangat jelas terlihat adalah sensitivitas baik pada gigi maupun jaringan sekitar, serta permukaan gigi yang terasa lebih kasar, hal ini disebabkan karena bahan peroxida yang digunakan pada bleaching merupakan bahan yang bersifat hipertonis yang dapat menarik air dan menyebabkan dehidrasi pada struktur gigi, radikal bebas yang dilepaskan oleh bahan peroxida juga dapat masuk ke dalam ruang pulpa melalui tubuli dentin dan menyebabkan terjadinya pulpitis reversible pada gigi.5 beberapa penelitian juga menunjukkan terjadinya perubahan stuktur permukaan gigi, kekerasan permukaan dan hilangnya jaringan keras gigi menyerupai proses demineralisasi.7 efek samping bleaching sebagian besar hanya dilaporkan pada gigi permanen pasien dewasa saja tetapi penelitian pada pasien anak masih jarang ditemukan.3 gigi permanen pada anak yang masih dalam fase pergantian dikenal dengan sebutan gigi permanen muda dimana gigi tersebut masih memiliki ujung akar yang masih belum menutup sempurna dan pada gigi permanen muda gigi masih menjalani post-eruptive enamel maturation dan memiliki karakteristik tertentu yang mengakibatkan gigi lebih rentan dalam mengalami demineralisasi. enamel gigi permanen muda lebih porous, memiliki kandungan apatit karbonat yang lebih besar, dan memiliki komposisi mineral yang tidak murni yang menyebabkan kristal enamel lebih mudah larut pada keadaan demineralisasi.8 chen et al7 menunjukkan bahwa efek samping yang ditimbulkan oleh bahan bleaching dapat dihindari dengan 208 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 206–210 beberapa cara, salah satunya adalah dengan memodifikasi bahan bleaching dengan pemberian kandungan fluoride dan air pada bahan serta aplikasi bahan yang dapat mendorong terjadinya remineralisasi. remineralisasi merupakan proses pengembalian mineral yang hilang dari gigi akibat proses demineralisasi, remineralisasi pada gigi membutuhkan mineral kalsium, fosfat dan fluoride.9 bahan yang dapat mendorong remineralisasi yang banyak mendapatkan perhatian saat ini adalah casein phosphopeptide protein amorphous calcium phosphate (cpp-acp). bahan cppacp merupakan derivat susu sapi dengan kadar kalsium dan fosfat yang tinggi sehingga menjamin ketersediaan kalsium dan fosfat pada saat dibutuhkan dalam lingkungan mulut, menghambat demineralisasi enamel gigi, meningkatkan proses remineralisasi serta menjadikan suasana buffer di dalam mulut karena adanya pengaturan saturasi ion kalsium dan fosfat pada biofilm gigi dan saliva,10 terdapat produk baru yaitu cpp-acpf yaitu cpp-acp dengan penambahan 0.2% sodium fluoride yang dapat memperkuat mekanisme dalam menahan proses demineralisasi gigi.11 tujuan penelitian ini adalah untuk meneliti kekerasan mikro permukaan email gigi permanen muda, dan efek aplikasi pasta remineralisasi naf dan cpp-acp setelah pemutihan gigi. bahan dan metode penelitian ini merupakan penelitian experimental laboratories dengan menggunakan 14 elemen gigi premolar permanen muda post pencabutan untuk perawatan orthodonti dengan kriteria yang sudah ditentukan. setiap elemen gigi yang akan dipergunakan ditanam dalam balok resin dengan membuat jendela pada bagian bukal masingmasing sampel dan diberi nomor, kemudian dilakukan pengukuran kekerasan permukaan awal masing-masing sampel tersebut dengan menggunakan alat microvickers hardness tester. kekerasan permukaan awal dicatat kemudian dilakukan perlakuan pemberian bahan bleaching hidrogen peroxide 30% sebanyak 3 kali aplikasi masingmasing selama 15 menit. setelah aplikasi bahan bleaching, kekerasan permukaan sampel diukur kembali dengan menggunakan alat microvickers hardness tester untuk mendapatkan nilai kekerasan permukaan enamel setelah perlakuan bleaching. sampel kemudian dibagi menjadi 2 kelompok; kelompok pertama (no 1-7) diberi perlakuan bahan pasta remineralisasi dengan kandungan bahan pasta hidroksi apatit + naf 1450ppm, dan kelompok kedua (no 8-14) diberi perlakuan bahan pasta cpp-acp + naf 900ppm. masing-masing pasta tersebut diaplikasikan selama 30 menit 7 hari berturut-turut. setelah perlakuan pasta remineralisasi, sampel diukur kembali kekerasan permukaannya dengan menggunakan alat microvickers hardness tester. hasil diperoleh data kekerasan permukaan enamel gigi permanen muda pada kelompok awal yang tidak diberi perlakuan apapun adalah 333.09 ± 10.49 vhn, setelah mendapatkan terapi bleaching kekerasan permukaan enamel gigi permanen muda turun menjadi 299.15 ± 5.70 vhn, setelah mendapatkan perlakuan bahan remineralisasi hidroksi apatit + naf 1450ppm selama 7 hari didapatkan kekerasan permukaan naik menjadi 316.61 ± 5.87 vhn, sedangkan kekerasan permukaan enamel gigi permanen muda yang mendapat perlakuan bahan remineralisasi cppacp + naf 900ppm selama 7 hari didapatkan kekerasan permukaan naik menjadi 319.94 ± 3.25 vhn (tabel 1). data yang diperoleh dari setiap kelompok dilakukan pengukuran normalitas data dan pengujian hipotesis dengan menggunakan program spss 20 dan didapatkan bahwa terdapat perbedaan kekerasan yang signifikan antara nilai awal enamel gigi permanen muda terhadap nilai setelah perlakuan bleaching, terdapat kenaikan yang signifikan antara kekerasan enamel gigi setelah mendapatkan terapi pasta remineralisasi dan pasta remineralisasi yang menggunakan bahan cpp-acp + naf memiliki kenaikan yang lebih signifikan daripada pasta remineralisasi hidroksi apatit + naf 1450ppm (gambar 1). tabel 1. rerata kekerasan permukaan kelompok percobaan (satuan: vhn) perlakuan jumlah sampel mean ± standar deviasi awal 14 333.09 ± 10.49 bleaching 14 299.15 ± 5.70 perlakuan pasta hidroksi apatit + naf 1450ppm 7 316.61 ± 5.87 perlakuan cpp-acp + naf 900ppm 7 319.94 ± 3.25 gambar 1. diagram rerata kekerasan permukaan enamel gigi permanen muda (satuan: vhn). 209liwang, et al.: kekerasan mikro enamel gigi permanen muda pembahasan penelitian ini, dilakukan untuk menunjukkan efek terapi pemutihan gigi dengan menggunakan hidrogen peroxida 30%. terjadi penurunan kekerasan permukaan enamel gigi permanen muda, hal ini menunjukkan bahwa terapi pemutihan gigi menyebabkan perubahan struktur enamel gigi dan menyebabkan terjadinya kehilangan jaringan keras gigi, karena pada enamel gigi permanen muda lebih porous, memiliki kandungan apatit karbonat yang lebih besar, dan memiliki komposisi mineral yang tidak murni yang menyebabkan kristal enamel lebih mudah larut pada keadaan demineralisasi.8 penelitian yang dilakukan oleh potocnik et al.,12 terhadap bahan pemutih gigi menunjukkan bahwa terdapat perubahan pada struktur gigi menyerupai dengan terjadinya initial caries. menurut hegedus et al.,13 bahan pemutih gigi yang mengandung peroxida menyebabkan terjadinya perubahan pada struktur organik enamel, tidak hanya pada permukaan enamel tetapi juga pada struktur bagian dalam pada enamel, hal ini disebabkan karena hidrogen peroxida memiliki berat molekul yang ringan. radikal bebas oksigen bersifat tidak spesifik dan bereaksi pada struktur organik pada gigi.14 penurunan kekerasan permukaan dan perubahan struktur pada enamel gigi dihubungkan dengan terjadinya demineralisasi pada enamel gigi atau hilangnya mineral pada enamel gigi. setelah terapi pemutihan gigi, penurunan kekerasan permukaan gigi dapat segera dikembalikan dengan adanya fase remineralisasi pada gigi.7 demineralisasi dan remineralisasi pada gigi merupakan suatu proses keseimbangan yang berkesinambungan, saliva dengan kandungan mineral yang terdapat didalamnya merupakan agen remineralisasi alamiah yang diproduksi oleh tubuh,9 sedang penggunaan bahan lainnya adalah berupa topikal aplikasi fluoride ataupun bahan remineralisasi lain. perlakuan pasta remineralisasi pada gigi yang menjalani terapi pemutihan gigi pada penelitian ini menunjukkan terjadinya kenaikan kekerasan permukaan yang signifikan baik pada kelompok perlakuan cpp-acp + naf 900ppm maupun pada kelompok perlakuan hidroksi apatit + 1450ppm hal ini disebabkan karena fungsi dan kegunaan bahan tersebut sebagai agen remineralisasi dengan kandungan kalsium, fosfat dan fluoride yang sangat berperan penting dalam mendorong terjadinya remineralisasi pada gigi. proses remineralisasi pada gigi sangat bergantung pada ion kalsium dan fosfat serta dibantu oleh fluoride untuk membentuk lapisan baru pada lesi yang terjadi akibat demineralisasi.15 kandungan kalsium dan fosfat pada bahan remineralisasi ini berguna sebagai penyedia cadangan ion kalsium dan fosfat yang akan bekerja untuk menggantikan ion kalsium dan fosfat pada enamel gigi yang mengalami demineralisasi. penelitian ini menunjukkan bahwa pada kedua kelompok perlakuan pasta remineralisasi menunjukkan selisih kenaikan kekerasan permukaan enamel gigi yang memiliki perbedaan secara signifikan antara kelompok perlakuan cpp-acp + naf 900ppm dan hidroksi apatit + naf 1450ppm, hal ini dapat dijelaskan karena kandungan ion kalsium dan ion fosfat yang sangat berbeda antara kedua produk tersebut. produk cpp-acp + naf 900ppm memiliki kandungan kalsium terlarut sebesar 321.8 ± 2.6 µmol/g dan 245.7 ± 2.7 µmol/g16 sedangkan produk hidroksi apatit + naf 1450ppm memiliki kandungan kalsium dan fosfat terlarut ≤ 10 µmol/g, sehingga menyebabkan proses remineralisasi antara kedua produk tersebut memiliki kekerasan permukaan yang berbeda secara signifikan. meskipun pasta hidroksi apatit + naf 1450ppm memiliki efek yang lebih kecil dibandingkan cpp-acp + naf 900ppm tetapi kedua pasta remineralisasi ini dapat menaikkan kekerasan permukaan enamel gigi secara signifikan. pasta cpp-acp + naf 900ppm merupakan pasta remineralisasi yang berasal dari derivat susu sapi dan tidak dapat dipergunakan pada pasien yang alergi susu sapi sedang produk hidroksi apatit + naf 1450ppm tidak memiliki kandungan susu dan dapat dipergunakan pada pasien dengan alergi terhadap susu. penelitian ini menunjukkan bahwa bahan pemutih gigi (dental bleaching) menurunkan kekerasan mikro permukaan enamel gigi permanen muda secara signifikan; dan aplikasi pasta remineralisasi hidroksi apatit + naf 1450ppm dan cpp-acp + naf 900ppm dapat meningkatkan kembali kekerasan mikro permukaan enamel gigi permanen muda. daftar pustaka 1. frysh h. the chemistry of bleaching. in: goldstein re, garber da. complete dental bleaching. chicago: quintessence book; 1995. p. 25-32. 2. zekonis r, matis ba, cochran ma, al shetri se, eckert gj, carlson tj. clinical evaluation of in-office and at-home bleaching treatments. oper dent 2003; 28(2): 114-21. 3. american association of pediatric dentistry. policy on the use of dental bleaching for child and adolescent patients. council on clinical affairs reference manual 2009; 33(6): 67-9. 4. donly kj. the adolescent patient: special whitening challenges. compend contin educ dent 2003; 24(4a): 390-6. 5. rismanto dy, dewayani i, dharma rh. dental whitening. jakarta: dental lintas mediatama; 2005. 6. joiner a. the bleaching of teeth: a review of the literature. j dent 2006; 34(7): 412-9. 7. chen hp, chang ch, liu jk, chuang sf, yang jy. effect of fluorideide containing bleaching agents on enamel surface properties. j dent 2008; 36(9): 718-25. 8. palti dg, machado ma, silva sm, abdo rc, lima je. evaluation of superficial microhardness in dental enamel with different eruptive ages. braz oral res 2008; 22(4): 311-5. 9. margeas r. remineralization with a unique delivery system. inside dentistry 2006; 4(2): 86 210 dent. j. (maj. ked. gigi), volume 47, number 4, december 2014: 206–210 10. meirina g, eka c, rossi s. the role of casein phosphopetide amorphous calcium phosphate in caries prevention. journal pdgi august; 58-62. 11. aimutis wr. bioactive properties of milk protein focus on anticariogenic. j nutr 2004; 134(4): 989s-95s. 12. potocnik i, kosec l, gaspersic d. effect of 10% carbamide peroxide bleaching gel on enamel microhardness, microstructure and mineral content. j endod 2000; 26: 203–6. 13. hegedüs c, bistey t, flóra-nagy e, keszthelyi g, jenei a. an atomic force microscopy study on the effect of bleaching agents on enamel surface. j dent. 1999; 27(7): 509-15. 14. sasaki tr., arcanjo aj, florio fm, basting rt. micromorphology and microhardness of enamel after treatment with home-use bleaching agents containing 10% carbamide peroxide and 7,5% hydrogen peroxide. j appl oral sci 2009; 17(6): 611-6. 15. walsh lj. contemporary technologies for remineralization therapies: a review. international dentistry sa 2009; 11(6). 16. cai f, yuan y, reynolds c, reynolds ec. water soluble calcium, phosphate and fluorideide of various dental products. j dent res 2009; 89(spec iss b): 57. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 100 the effects of golden sea cucumber extract (stichopus hermanii) on the number of lymphocytes during the healing process of traumatic ulcer on wistar rat’s oral mucous ira arundina,1 yuliati,1 pratiwi soesilawati,1 dian w damaiyanti,2 and dania maharani1 1 department of oral biology, faculty of dental medicine, universitas airlangga 2 department of farmacology, faculty of dentistry, universitas hang tuah surabaya-indonesia abstract background: indonesia is a country with the world’s biggest potential and producer of sea cucumbers. golden sea cucumber contains glicosaminoglycans, such as heparan sulphate and chondroitin sulphate, which could have a positive implication on wound healing process. this acceleration of wound healing process could be observed through the increasing of lymphocytes on ulcus traumaticus. purpose: this study aims to analyze the effects of golden sea cucumber extract on the number of lymphocytes during the healing process of traumatic ulcer on wistar rat’s oral mucous. method: golden sea cucumber extrat was made with freeze-dried method, and then gel was prepared using peg 400 and peg 4000 solvent. twenty male rats with mucosal ulcus made were divided into a control group and three treatment groups with 20%, 40% and 80% golden sea cucumber extracts. all samples were euthanized on day 4 and then a preparation for histopathological examination was made to examine the number of lymphocytes. result: the biggest number of lymphocytes was found in the treatment group with 40% golden sea cucumber extract, while the lowest one was found in the control group. the results of one way anova test then showed a significant difference between the control group and the treatment groups. and, the results of tukey hsd showed a significant difference between the control group and the treatment group with 40% golden sea cucumber extract. conclusion: it can be concluded that 40% golden sea cucumber (stichopus hermanii) extract can increase the number of lymphocytes during the healing process of traumatic ulcer on wistar rat’s oral mucous. keywords: wound healing; traumatic ulcer; stichopus hermanii’s extract; glicosaminoglycans, lymphocytes correspondence: ira arundina, c/o: departemen biologi oral, fakultas kedokteran gigigi universitas airlangga. jl mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: arundinafkg@yahoo.com research report dental journal (majalah kedokteran gigi) 2015 june; 48(2): 100–103 introduction ulcer is a lesion eroding epithelial tissue with clear border. ulcer mostly found in several cases in communities is traumatic ulcer triggered by trauma and stomatitis aphtosa recurrent (sar) that can happen spontaneously and reccurently.1,2 traumatic ulcer on the mucous membranes of the oral cavity is a clinical appearance of inflammation indicated by an area with exudat and surrounded by connective tissue. inflammation is a process of destroying antigens and microorganisms moving into the body or a damaged tissue.3 inflammation process, moreover, involves the roles of blood vessel and inflammatory cells, such as leukocytes (netrophil, eosinophil, basophil, monocytes, and lymphocytes) and macrophage. lymphocytes have an important role in destroying micoorganisms moving into the body (antigen) during the process of inflammation.4 traumatic ulcer, furthermore, can be treated with certain medical therapies, namely topical corticosteroid, sodium bicarbonate with water, or mouthwash with antiseptics, such as 0.2% chlorhexidine gluconate or benzydamin hydrochloride. unfortunetely, the side effect of using chlorhexidine in a long term is the change of tooth colour.5 indonesia is a country with the biggest sea cucumber potential in the world. the production of sea cucumber in 101 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 101arundina, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 100–103 indonesia in 1994 was about 1.318.000 kg. the newest data showed that the production of sea cucumber was 42 ton during 2004.6 golden sea cucumber has been used in china and malaysia as traditional medicine and has been known as an effective tonic for wound and burn healing process.7 golden sea cucumber contains big bioactive materials, one of which is glycosaminoglycan. many previous researches show that glycosaminoglycans (gag) sulphate, such as chondroitin sulphate and heparan sulphate, have a positive effect on the wound healing process.8 moreover, lymphocytes act in the process of wound healing by realeasing lymphokines affecting the number of other inflammatory cells. one of the lymphokines produced is interleukin -2 (il-2). il-2 can bind with heparan sulfat that can help the proliferation process of t lymphocytes.9 t lymphocytes will secrete transforming growth factorβ (tgf-β). tgf-β functionates to stimulate fibroblast proliferation which plays a role on wound healing.10 many previous researches show that the water extract of golden sea cucumber could increase the number of fibroblast cells with optimal concentration of water extract from golden sea cucumber as much as 40% on the traumatic ulcer of wistar rats.11 as the biggest producer of sea cucumber, thus, indonesia should harness the potential. the acceleration of wound healing can be observed by the increasing of lymphocytes. therefore, this study aims to analyze the effects of water extract from golden sea cucumber with the concentrations of 20%, 40%, and 80% on the number of lymphocytes during the wound healing process of wistar rat. materials and method this study is a laboratory experimental research with randomized post test only control group design. this study was conducted with both variables after treatment using random sampling, and a negative control group as a comparison. this study used 20 male rats aged around 2-4 months old and weighed 200-300 grams as samples (rattus norvegicus) with certain criteria, such as normal lower lip mucous and no abnormalities in physical condition.12 ulcer was made by inhalation anesthesia. wistar rat’s lower lip mucous was wounded by no 4 burnisher with 2 mm diameter that had been heated for 1 minute, and it was then touched to wistar rat’s lip mucous for 1 second.11 the samples were divided into four treatment groups, namely the 1st treatment group with 80% golden sea cucumber extract, the 2nd treatment group with 40% golden sea cucumber extract, the 3rd treatment group with 20% golden sea cucumber extract, and the 4th group with aquadest as the control group. furthermore, the golden sea cucumber extract gel with 20%, 40%, and 80% concentrations was then applied one time to ulcer as much as 0.1 mg for each treatment group. afterward, those rats were sacrificed using ether in lethal doses on the 4th day. the lower lip mucous of those rats was cut until the corner of mouth including the ulcer part and the normal part, and then put in fixation solution, while the dead rats were burried.11 moreover, the number of lymphocytes was measured on histometric paraffin block preparation under compound light microscope with 400x magnification. it means that cell counting was conducted using graticulae dividing visual from light microscope to a certain size for easy reading and preventing cell duplication. data obtained were derived from the mean calculation in five visual areas of the graticulae.13 normality test with one-sample kolmogorov-smirnov test was then conducted on each sample. the results showed that the samples had a normal distribution (p>α=0.05). homogenitas test was also conducted. the results showed that the samples were homogen (p>α=0.05). next, anova test was conducted to examine the significance of the difference between groups. finally, tukey hsd test was conducted to examine the significance of the differences between one group with the others (p<α=0.05). results figure 1 shows lymphocytes on wistar rat’s traumatic ulcer during the histopathological preparation of the treatment groups. the red arrows shows lymphocytes. the mean and standard deviation of lymphocytes on each group can be seen in figure 2 and table 2. the biggest number of lymphocytes was found in the treatment group with 40% golden sea cucumber extract, while the lowest one was in the control group. moreover, the results of tukey hsd test showed that there was a figure 1. histological illustration of traumatic ulcer lymphocytes on wistar rat’s oral mucous. male wistar using he with 400x maginification. a) control group; b) 20% gold sea cucumber extract. c) 40% gold sea cucumber extract. illustration shows a significant rise of lymphocytes. d) 80% golden sea cucumber extract. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 102 arundina, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 100–103 significant difference between the treatment group with 40% golden sea cucumber extract and the control group. meawhile, there was no significant difference between the treatment groups with 20% and 80% golden sea cucumber extracts and the control group. the analysis of tukey hsd results can be seen in table 2. discussion like the previous researches, golden sea cucumber extract with the concentrations of 20%, 40%, and 80% was given to wistar rat’s lower lip mucous in this research. the previous researches show that the 40% golden sea cucumber extract is optimal to increase the number of fibroblasts in the healing process of traumatic ulcer.11 euthanasia was conducted on the 4th day to examine the number of lymphocytes after the administration of golden sea cucumber extract on the treatment groups compared to the control group expected to achieve the highest number from day 5 to day 7 during the wound healing process.14 the administration of golden sea cucumber extract, 8 figure 1. histological illustration of traumatic ulcer lymphocytes on wistar rat’s oral mucous. male wistar using he with 400x maginification. a) control group; b) 20% gold sea cucumber extract. c) 40% gold sea cucumber extract. illustration shows a significant rise of lymphocytes. d) 80% gold sea cucumber extract. figure 2. the graph of mean and standard deviation of wistar rat’s oral mucous lymphocytes. figure 2. the mean and standard deviation of wistar rat’s oral mucous lymphocytes. table 1. the mean and standard deviation of lymphocytes on day 4 group x ± sd control 4.6 ±1.51 20% 9.4 ± 1.14 40% 17.0±12.63 80% 7.5 ± 1.64 table 2. the results of tukey hsd analysis control 20% 40% 80% control 0.628 *0.028 0.869 20% 0.628 0.257 0.958 40% *0.028 0.257 0.095 80% 0.869 0.958 0.095 (*)= a significant difference/ significance (p<α=0.05) consequently, is expected to accelerate the wound healing process. research data showed that golden sea cucumber extract could increase the number of lymphocytes on the treatment groups compared to the control group. on the 4th day, the number of lymphocytes significantly increased. this indicates that the administration of golden sea cucumber extract could accelerate the wound healing process. moreover, the results of histological examination results showed that the biggest number of lymphocytes was found in the treatment group with 40% golden sea cucumber extract. 20% golden sea cucumber extract could not affect the number of lymphocytes, while 80% golden sea cucumber extract could decrease the number of lymphocytes. therefore, it indicates that the concentrations of golden sea cucumber extract can differently affect, so the dose should be given correctly.15 every drug has their own concentration rules. putranti’s research proves that 40% golden sea cucumber extract is the most optimal concentration to increase angiogenesis on the wistar rat’s traumatic ulcer healing process.16 the increasing of lymphocytes compared to control group because of glycosaminoglycans contained in golden sea cucumber extract. glycosaminoglycans, usually known as mucopolysaccharides, is a complex carbohydrate molecule which interacts with protein and involved in various physiological and pathological process. gag consists of two types, namely gag sulphate and gag non sulphate.17 examples of gag sulphate are chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, and heparin. chondroitin sulphate and heparan sulphate have positive effects on wound healing process.18 heparan sulphate located in organs and tissue is part of the extracellular matrix, such as colagen, fibronectin and laminin. various types of protein can bind with heparan sulphate, such as extracellular matrix, growth factor, cytokine and chemokine. heparan sulphate is involved in various physiological processes, such as proliferation, migration, differentiation, and interaction between cells. heparan sulphate is known for its substansial role in a variety of cell interactions, and its bond with various types of protein can put those proteins on the cell surface.19 the binding of cytokine with heparan sulphate can modulate the bioactivity of cytokine itself. heparan sulphate can bind with various types of cytokines such as il-2, il-3, il-4, il-5, il-6, il-7, il-8, il-10, interferon-γ (ifnγ), and fibroblast growth factor.20 lymphocytes is the centre of cell immunity system that has an important role in the inflammation process by destroying microorganisms entering the body (antigen) and forming immunity (antibody) in the form of immunoglobulin.4 the role of lymphocytes is to release lymphokines triggering the other inflammation of cell population. some of the lymphokines released, consequently, can stimulate the agregation of macrophage in wound healing process. t lymphocytes then secrete various 103 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 103arundina, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 100–103 types of cytokine, one of which is il-2. il-2 function to activate macrophage by stimulating the synthesis of ifnγ.21 t lymphocytes also produce tgf-β that function on the proliferation of fibroblast.10 some of the cytokines that can bind with heparan sulphate are il-2 and ifn-γ. a research shows that the binding of heparan sulphate with il-2 can trigger activation and proliferation of t lymphocytes. il-2 is an autocrine growth factor for t lymphocytes. il-2 is produced by lymphocytes. il-2 has a role for the growth factor of lymphocytes itself. on the other hand, a research shows that binding of heparan sulphate with ifn-γ can affect bioactivity of ifnγ by protecting from proteolytic degradation. it can also trigger ifn-γ to bind with it’s receptor. ifn-γ is derived from the stimulation of il-2. ifn-γ can activate macrophage. consequently, it is also known as macrophage activating factor.21 in addition, ifn-γ is considered as a major cytokine stimulant to activate monocytes and macrophage, as a result, the activated macrophage will activate lymphocytes and other cells that will work together in the inflammation process until the inflammation is gone.22 furthermore, heparan sulphate has a great effect on the immune response through the modulation of antigen presenting cell (apc). on of the apc, there are macrophages. when many macrophages are activated, they will release more cytokines, one of which is il-1. il-1 can contribute to the activation of lymphocytes.21 thus, the number of lymphocytes will increase. finally, it can be concluded that golden sea cucumber extract could increase lymphocytes, and 40% golden sea cucumber extract is the most optimal concentration in increasing lymphocytes during the process of wistar rat’s oral mucous traumatic ulcer healing. references 1. regezi ja, sciubba jj, jordan rck. oral pathology and clinical pathologic correlation. 5th ed. st. louis: elsevier; 2008; p. 21-4. 2. baratawidjaja kg. imunologi dasar. edisi 6. jakarta: balai penerbit fakultas kedokteran universitas indonesia; 2004; p. 35, 288, 568. 3. goldsby ra, kindt tj. kuby imunology. 5th ed. america: wh freeman and company; 2003. p. 32. 4. pinel j, naboulet c, weiss f, henkens h, grouzard v. essential drugs. practical guidelines 2013; 1-13. 5. arlyza is. teripang dan bahan aktifnya. oseana 2009; 34(1): 1-2. 6. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods—a review. mar drugs. 2011; 9(10): 1761-805. 7. masre sf, yip jw, sirajudeen kns, gazhali fc. wound healing activities of total sulfated glycosaminoglican (gag) from stichopus vastur and stichopus hermanni integumental tissue in rats. int j molec med and adv sci 2010; 6: 49-52. 8. miller jd, stevens et, smith dr, wight tn, wrenshall le. perlecan: a major il-2-binding proteoglycan in murine spleen. immunol cell biol. 2008; 86(2): 192-9. 9. peakman m, vergani d. basic and clinical immunology. usa: appleton and lange; 2009. p. 107-18. 10. damaiyanti dw. aplikasi ekstrak air teripang emas sebagai akselerator proliferasi fibroblast dan kolagen tipe i ulkus traumatikus rongga mulut tikus wistar. tesis. surabaya: fkg universitas airlangga; 2012. p. 45. 11. dewi m, wijaya i, wijayahadi n. efek ekstrak bawang putih (allium sativum) terhadap ekspresi insulin dan derajat insulitis pankreas tikus spraque-dawley jantan yang diinduksi streptozotocin. media medika indonesia 2011; 45(2): 105-12. 12. ashari y, istiati, arijani e. application of mengkudu leaf extract towards collagen fibers density increase on oral mucosa of guinea pig. oral biology dental journal 2012; 4(2). 13. monaco jl, lawrence wt. acute wound healing: an overview. clin plast surg. 2003; 30: 4. 14. mutschler e. dinamika obat. farmakologi dan toksikologi. edisi 5. bandung: itb; 2010. p. 176. 15. arundina i, soesilowati p, larasati ar. effect of sticophus hermanii extract to increase re-epithelitation in healing process of traumatic ulcer in wistar rat's oral mucous. oral biology dental journal 2013; 5(2): 41-6. 16. kimata k, habuchi o, habuchi h, watanabe h. knockout mice and proteoglikan. amsterdam: elsevier; 2007. p. 159-91. 17. zou xh, foong wc, cao t, bay bh, ouyang hw, yip gw. chondroitin sulfate in palatal wound healing. j dent res. 2004; 83(11): 880-5. 18. islam t, lindhart rj. chemistry biochemistry and pharmaceutical potentials oh glikosaminaglikan and related saccharide. in: chihuey w, editor. carbohydrate-based drug discovery. weinheim: wiley vch; 2003. p. 407-33. 19. varki a. six blind men and the elephant-the many faces of heparan sulfate. pnas 2002; 99(2): 1229-36. 20. abbas ak, licthman ah. cellular and molecular immunology. 5th ed. philadelphia: saunders; 2011. p. 146. 21. robbins rs, kumar v. basic patology. edisi 7. jakarta: egc; 2007. p. 195. vol 38 no 2-2005 49 respons imun humoral pada pulpitis (humoral immune response on pulpitis) trijoedani widodo bagian ilmu konservasi gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract pulpitis is an inflammation process on dental pulp tissue, and usually as the continuous of caries. the microorganism in the caries is a potential immunogenic triggering the immune respons, both humoral and celluler immune responses. the aim of this research is to explain the humoral immune response changes in the dental pulp tissues of pulpitis. this research was done on three group samples: irreversible pulpitis, reversible pulpitis and sound teeth as the control group. the result showed that there were three pulpitis immunopathologic patterns: the sound teeth immunopathologic pattern showing a low humoral immune response, in a low level of igg, iga and igm, the reversible pulpitis pattern showing that in a higher humoral immune response, igg and iga decreased but igm increased, the irreversible pulpitis pattern showing that igg and igm increased, but it couldn't be repaired although it has highly immunity, and it showed an unusually low level of iga. this low level of iga meant that irreversible pulpitis had a low mucosal immunity. key words: humoral immune response, pulpitis korespondensi (correspondence): trijoedani widodo, bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan pulpitis adalah proses radang pada jaringan pulpa gigi, yang pada umumnya merupakan kelanjutan dari proses karies.1,2,3 jaringan pulpa terletak di dalam jaringan keras gigi sehingga bila mengalami proses radang, secara klinik sulit untuk menentukan seberapa jauh proses radang tersebut terjadi.4 selama ini radang pulpa ditentukan dengan adanya keluhan rasa sakit yang sifatnya subyektif. secara patofisiologik, pulpitis dibagi menjadi pulpitis reversibel dan pulpitis ireversibel, karena yang penting dalam menentukan diagnosis pulpitis adalah jaringan pulpa tersebut masih dapat dipertahankan atau sudah tidak dapat dipertahankan lagi.5 klasifikasi pulpitis ini kemudian diikuti oleh penulis yang lain sampai saat ini.6-9 beberapa peneliti menemukan komponen imun di dalam jaringan pulpa yang mengalami proses radang, yang menunjukkan bahwa jaringan pulpa mempunyai kemampuan untuk melawan kuman yang berasal dari karies.10-13 proses karies merupakan proses patologik yang kronik yang dapat menimbulkan berbagai perubahan pada jaringan pulpa, di antaranya berupa respons imun. mikroorganisme yang terdapat pada jaringan gigi yang karies merupakan imunogen yang potensial untuk memicu terjadinya respons imun.14,15 respons imun ada dua macam yaitu respons imun humoral yang diperankan oleh sel b yang menghasilkan immunoglobulin, dan respons imun seluler yang diperankan oleh sel t.16,17 penelitian ini dilakukan untuk mengetahui adanya perubahan yang terjadi pada jaringan pulpa yang mengalami proses radang berupa respons imun humoral dengan menggunakan konsep imunopatologik dan pendekatan morfofungsi,18,19 yang dilakukan dengan menggunakan jaringan pulpa yang diambil dari gigi karies yang telah dicabut, dengan diagnosis pulpitis ireversibel, pulpitis reversibel dan gigi sehat sebagai pembanding. dengan mengamati perubahan yang terjadi pada jaringan pulpa berupa terjadinya immunoglobulin dalam jenis maupun komposisinya, dapat ditentukan tingkat dari proses radang yang terjadi. bahan dan metode jenis penelitian adalah observasional cross-sectional, karena perubahan respons imun yang diamati merupakan perubahan saat itu tanpa diadakan perlakuan. bahan yang digunakan sebagai sampel dalam penelitian ini adalah 40 jaringan pulpa yang diambil dari gigi karies yang baru dicabut, dari penderita laki-laki usia 16–40 tahun, dengan diagnosis pulpitis ireversibel, pulpitis reversibel, dan gigi sehat sebagai pembanding. sampel dibagi menjadi tiga kelompok yaitu: kelompok 1 dengan diagnosis pulpitis ireversibel (irp); kelompok 2 dengan diagnosis pulpitis reversibel (rp); dan kelompok 3 dari gigi sehat (normal) sebagai pembanding. variabel yang diamati adalah komponen imun humoral meliputi 50 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 49–51 igm, igg, dan iga. sebagai unit analisis adalah jaringan pulpa yang sudah diproses dengan menggunakan teknik penanaman jaringan fiksasi dan ditanam di dalam bahan tanam parafin. kemudian dilakukan pewarnaan sesuai dengan immunoglobulin yang akan diamati, yaitu dengan menggunakan immu stain kit.20 sediaan diamati di bawah mikroskop cahaya, dan data dari hasil pengamatan tersebut dilakukan uji manova untuk melihat apakah ada perbedaan perubahan respons imun humoral pada kelompok pulpitis ireversibel, pulpitis reversibel dan gigi sehat. kemudian untuk melihat seberapa besar perbedaan antar kelompok, dilakukan uji diskriminan untuk mendapatkan harga kontribusi diskriminan. setelah didapatkan harga kontribusi diskriminan, dapat dibuat pola imunopatologik dari ketiga kelompok tersebut yang dapat digunakan untuk menjelaskan perubahan respons imun yang terjadi. hasil hasil pengamatan terhadap komponen imun igm, igg, dan iga pada jaringan pulpa dari ketiga kelompok sampel yang telah diproses dengan teknik jaringan fiksasi dan dilakukan uji manova terhadap data yang terkumpul dapat dilihat pada tabel 1. tabel 1. harga rata-rata dan standart deviasi igm, igg dan iga yang ditemukan pada kelompok irp, rp, dan normal irp rp sehat variabel mean sd mean sd mean sd igm 2,215 1,733 0,746 0,805 0,108 0,133 igg 2,882 1,662 0,970 1,147 0,291 0,666 iga 2,078 1,166 0,800 0,943 0,63 0,250 keterangan: mean = harga rata-rata setiap variabel kelompok irp, rp, dan normal; sd = standar deviasi setiap variabel kelompok irp, rp, dan normal; irp = kelompok pulpitis ireversibel; rp = kelompok pulpitis reversibel; normal = kelompok gigi sehat. manova yang menguji perbedaan antar kelompok menghasilkan p = 0,001 < 0,05 yang berarti ada perbedaan yang signifikan antara kelompok irp, rp dan normal. untuk menentukan seberapa besar perbedaan komponen imun yang ada pada masing-masing kelompok dilakukan analisis diskriminan untuk mendapatkan harga kontribusi diskriminan masing-masing kelompok. dengan harga kontribusi diskriminan yang dihasilkan dapat dibuat pola untuk memudahkan dalam menafsirkan seberapa besar perbedaan antar kelompok pulpitis ireversibel, pulpitis reversibel dan gigi sehat. harga kontribusi diskriminan antar kelompok dapat dilihat pada tabel 2. tabel 2. harga kontribusi diskriminan komponen respons imun humoral irp rp normal variabel mean sd mean sd mean sd igm 3,212 2,514 0,840 0,907 0,103 0,411 igg 2,081 1,200 –0,191 0,226 –0,106 0,242 iga –0,717 0,504 –0,484 0,571 –0,079 0,315 keterangan: mean = harga rata-rata kontribusi diskriminan setiap komponen; sd = standar deviasi kontribusi diskriminan setiap komponen; irp = kelompok gigi dengan diagnosis pulpitis ireversibel; rp = kelompok gigi dengan diagnosis pulpitis reversibel; normal = kelompok gigi sehat. igg igm iga normal rp irp -1 -0,5 0 0,5 1 1,5 2 2,5 3 3,5 normal rp irp gambar 1. pola respons imun humoral pada kelompok pulpitis ireversibel, pulpitis reversibel, dan kelompok gigi sehat (irp, rp, normal). pada pola respons imun humoral pada gambar 1 di atas dapat dijelaskan bahwa pada kelompok normal terlihat igm sudah timbul walaupun tidak tinggi (0,103) dan diikuti igg (–0,106) dan iga (–0,079) yang juga rendah. pada kelompok pulpitis reversibel terlihat igm meningkat (0,840), sedang igg (–0,191) dan iga (–0,484) masih tetap rendah. pada kelompok pulpitis irreversibel terlihat igm (3,212) dan igg (2,081) meningkat tinggi, sedangkan iga (–0,717) semakin menurun. pembahasan hasil pengamatan dari kelompok gigi sehat didapatkan igm tampak sudah timbul walaupun tidak tinggi, yang diikuti dengan igg dan iga yang lebih rendah bila dibandingkan dengan igm. temuan ini menunjukkan bahwa pada jaringan pulpa pada gigi sehatpun sudah bisa terjadi perubahan respons imun walaupun rendah, yang menunjukkan adanya respons terhadap adanya rangsang berupa penggunaan gigi secara fungsional yang dapat 51widodo: respons imun humoral pada pulpitis merupakan rangsang pada jaringan pulpa walaupun ringan. hal ini sesuai dengan pendapat bahwa sejak lapisan enamel terluka, walaupun ringan sudah dapat menimbulkan perubahan pada jaringan pulpa yang ada di bawahnya.3 pada kelompok pulpitis reversibel terlihat igm meningkat, igg dan iga tetap rendah. hal ini menunjukkan bahwa reaksi imun pada pulpitis reversibel masih rendah, tetapi masih lebih tinggi bila dibandingkan dengan gigi sehat, dan menunjukkan adanya unsur protektif. kondisi ini dapat dikatakan bahwa pada pulpitis reversibel terjadi dua kemungkinan, yaitu pertama terjadi proses radang yang baru mulai terjadi. kemungkinan kedua, proses radang sudah masuk dalam stadium kronik yang menuju ke arah kesembuhan. secara umum kondisi komponen imun humoral yang terlihat pada kelompok pulpitis reversibel hampir sama dengan kelompok gigi sehat, maka pada gigi dengan diagnosis pulpitis reversibel jaringan pulpa yang sudah mengalami proses radang, masih mungkin untuk disembuhkan. hal ini sesuai dengan pendapat beberapa penulis yang menyatakan bahwa gigi dengan diagnosis pulpitis reversibel, jaringan pulpa yang sudah mengalami proses radang tidak perlu dibuang dengan perawatan pulpektomi, tetapi masih dapat disembuhkan dengan perawatan pulp capping.4-9 pada kelompok pulpitis ireversibel terlihat igg dan igm meningkat tinggi, namun iga menurun sekali yang menunjukkan bahwa ketahanan mukosalnya rendah. tingginya igg dan igm menunjukkan adanya ketahanan jaringan pulpa yang tinggi terhadap mikroorganisme. reaksi imunitas yang tinggi dari pulpitis ireversibel seharusnya diikuti dengan terjadinya kesembuhan, namun kenyataan pulpitis ireversibel tidak dapat sembuh kembali, bahkan dikatakan bahwa pulpitis ireversibel sering kali mudah berkembang menjadi nekrosis.21 hal ini terjadi karena jaringan pulpa yang berada di dalam ruang pulpa yang sempit, dan menerima sirkulasi darah hanya melalui pembuluh darah yang masuk ke dalam jaringan pulpa melalui foramen apikal yang sempit pula, sehingga pulpitis ireversibel mudah berkembang menjadi nekrosis pulpa. perawatan yang tepat untuk gigi dengan diagnosis pulpitis ireversibel adalah pulpektomi yaitu perawatan endodontik dengan membuang jaringan pulpa yang telah mengalami proses radang tersebut. dari penelitian ini dapat disimpulkan bahwa sudah terjadi respons imun humoral pada jaringan pulpa gigi sehat walaupun rendah, pada pulpitis reversibel juga rendah tetapi masih lebih tinggi bila dibandingkan dengan gigi sehat, dan tampak ada reaksi protektif. respons imun humoral pada pulpitis ireversibel terlihat tinggi yang ditandai dengan meningkatnya igg dan igm, yang seharusnya diikuti dengan proses kesembuhan, tetapi kenyataan radang pulpa tidak dapat sembuh kembali bahkan sering kali berkembang menjadi nekrosis pulpa. iga yang sangat rendah menunjukkan adanya respons imun mukosa pada pulpitis ireversibel rendah. untuk mengetahui perubahan yang terjadi pada jaringan pulpa secara lebih jelas dan rinci, dan untuk menjawab mengapa radang pulpa pada pulpitis ireversibel tidak dapat sembuh kembali, masih perlu dilakukan penelitian terhadap komponen imun yang lain yang ada di dalam jaringan pulpa yang mengalami proses radang dengan melihat adanya perubahan respons imun seluler yang diperankan oleh limfosit t. daftar pustaka 1. roitt im, lehner t. immunology of oral disease. boston: blackwell scientific publications; 1981. p. 363–7. 2. lawler w, achmed a, hume wj. 1987. buku pintar patologi untuk kedokteran gigi. agus djaya. jakarta: ecg; 1992. h. 9–36. 3. kim s, trowbridge ho. pulp reaction to caries and dental procedures. in: cohen s, burns rc. pathway of the pulp. 3rd ed. london: cv mosby co; 2000. p. 5–35. 4. cohen s. diagnostic procedures. in: cohen s, burns rc. pathway of the pulp. 3rd ed. london: cv mosby co; 2000. p. 5–35. 5. smulson mh. classification and diagnosis of pulpal pathosis. the dental clinics of north america 1984; 28: 699. 6. grossman li, oliet s, del rio ce. endodontic practice. 11st ed. philadelphia: lea and febriger; 1988. p. 1-18, 36–49, 59–77. 7. weine fs. endodontic therapy. 4th ed. london: cv mosby co; 1989. p. 74–153. 8. simon jhs, walton re, pashley dh, dowden we, bakland lk. pulpal pathology. in: ingle jl, bakland lk. endodontics. 4th ed. philadelphia: lea and febriger; 1994. p. 320–433. 9. widodo t. klasifikasi radang pulpa dan indikasi perawatannya. dentofasial. jurnal kedokteran gigi 2003; edisi khusus (1): 256–60. 10. honjo h, tsubakimoto k, utsumi n, tsusui m. localization of plasma protein in the human dental pulp. j dent res1970; 49: 888. 11. pulver wh, taubman ma, smith dj. immune components in normal and inflamed human dental pulp. arch oral biol 1977; 22: 103. 12. hahn cl, falkler wa. antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries. j endod 1987; 18: 28–31. 13. jontel m, gunraj mn, bergenholtz g. immunocompetent cells in the normal dental pulp. j dent res 1987; 66: 1149–53. 14. morse dr. immunologic aspects of pulpal periapical disease. a rivew. oral surg 1977; 43: 436–51. 15. trowbridge ho. pulp histology and phisiology. in: cohen s, burns rc. pathway of the pulp. 3rd ed. london: the cv mosby co; 1998. p. 323–78. 16. stites dp, terr al, parslow tg. basic and clinical immunology. 8th ed. london: appleton and lange; 1994. p. 40–79. 17. petterson rc, watts a. pulp responses to two strains of bacteria isolated from human carious dentine (l. plantarum) (ntct 1406) and s. mutans (ntct 10919). int endod j 1992; 25: 134–41. 18. widodo t. analisis perubahan imunopatologik pada pulpitis reversibel dan ireversibel untuk memperbaiki diagnosis atas dasar imunopatogenesis pulpitis. disertasi. surabaya: pascasarjana unair; 1997. 19. widodo t. perubahan imunopatologik pada pulpitis. surabaya: kumpulan ceramah ilmiah kongres nasional patobiologi; 2000. h.18. 20. widodo t. pembuatan sediaan mikrokopik jaringan pulpa pada kasus pulpitis. majalah kedokteran gigi 2001; 34: 18–22. 21. ingle jl, bakland lk. endodontics. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 5757 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 57–60 case report the assistance of er,cr:ysgg laser in pulp injury related to anterior teeth trauma gülşah balan,1 harry huiz peeters,2 and serap akyüz1 1 department of pedodontics, faculty of dentistry, university of marmara, istanbul – turkey 2 laser research center, bandung – indonesia abstract background: erbium chromium or erbium lasers constitute preferred instruments for the preparation of that section of tooth adjacent to the pulp chamber rather than high-speed drills, especially in cases of dental injury. their advantages can support modified operations in achieving optimum recovery and avoiding complications related to the healing process. purpose: the aim of the study was to describe another modality of the assistance of erbium, chromium:yattrium-scandium-gallium-garnet (er,cr:ysgg) laser in traumarelated pulp injury affecting the anterior teeth. case: the results of laser treatment applied to four pulp injuries of three children were analyzed in this case report. case management: er,cr:ysgg laser-assisted cavity preparation was performed without resort to a local anesthetic, the cavities being sealed with mineral trioxide aggregate (mta). none of the cases demonstrated symptomatic or peri-radicular pathology during clinical or radiographic examinations. conclusion: the results contained in this case report support the application in pediatric dentistry of a er,cr:ysgg laser to pulpotomy cavities as part of the treatment of traumatic pulpal injuries to permanent incisors. keywords: dental trauma; er,cr:ysgg; pulp therapy; mta; permanent incisors correspondence: gülşah balan, department of pedodontics, faculty of dentistry, marmara university, basibuyuk campus, 34854, istanbul, turkey. email: gulsahbalan@marun.edu.tr introduction the majority of dental trauma affects the anterior teeth due to the high level of activity among school age children.1 pulp therapies play an important role in maintaining pulp vitality and avoiding impeded root development or root resorption in young permanent teeth. dental lasers constitute efficient tools within the essentialal therapy required to lower the risk of complications such as inflammation and infection.2 oral laser applications also offer the prospect of painless procedures that address the concerns which children may harbor regarding the operation.3 compared to other forms of laser, erbium chromium or erbium lasers produce superior tooth preparation results. however, the heat generated during the procedure may cause irreversible damage to pulp tissue as well as pain and discomfort to the patient. erbium, chromium:yttriumscandium-gallium-garnet (er,cr:ysgg) laser, emits light at a wavelength of 2780 nm, is effective in cutting hard and soft dental tissues due to its water-mediated ablation mechanism.4 moreover, a hydrokinetic system serves to minimize the mechanical damage to the structure of teeth without any thermal risk.5-6 this photo-thermal interaction within tissue ablation produces a therapeutic effect by removing organic material and the smear layer, while also ensuring a decontaminated operation site. pulpal inflammation following acute injury is due to interrupted neurovascular supply7 or localized increase in intestinal fluid pressure, rather than bacterial invasion.8 as far as is known, laser irradiation is effective in wound control by minimizing hematoma formation in soft tissue.9 it is assumed that er,cr:ysgg laser represents the optimum choice for essential pulp therapy (including pulp capping and pulpotomy) in the treatment of traumatized teeth due to its overcoming of deficiencies in the procedure and reduction of complications during the healing process. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p57–60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p57-60 58 balan, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 57–60 cases the patients constituting the subjects of this report were treated at the department of pedodontics, faculty of dentistry, marmara university, istanbul, turkey. all cases presented anterior teeth fracture with trauma-related pulp injury. no extraoral impairment was clinically identified and no pocket was located intraorally and the patient demonstrating mixed dentation with well-articulated occlusion. radiographic examination indicated no other abnormalities such as jaw or condylar head fractures in any of the subjects. clinical views and periapical radiographs of traumatized upper incisors are shown in figure 1. case i: a healthy boy aged 8 years 9 months attended the clinic chiefly complaining of anterior fractures to teeth #11 and #21 due to an accident at school five hours earlier. from a clinical perspective, an oblique crown fracture line was observed in tooth #21 and a horizontal fracture adjacent to the pulp chamber without exposure in tooth #11 (figure 1a). radiographic examination of the teeth indicated complete root development without apex formation (figure 1b). figure 2a and b represents the pictures of case management steps of case i. case ii: a healthy girl aged 9 years 11 months visited the clinic seven hours after an incident at school chiefly complaining of fractured anterior upper incisors. during clinical examination, it was observed that teeth #11 and #21 had an exposed pulp zone less than 2 mm in diameter, accompanied by severe dentinal caries on all upper incisors and significantly poor oral hygiene (figure 1c). radiographic examination confirmed complete root formation and an oblique fracture line adjacent to the pulp chamber in both teeth #11 and #21(figure 1d). figures 2c and d demonstrate stages in the case management, relate to case ii. case iii: a boy aged 9 years 5 months visited the clinic 16 hours after an accident on the front porch of his house. during clinical examination, a crown fracture with pulp injury and an exposed area less than 2 mm in depth was observed in tooth #21 (figure 1e). radiographic examination further indicated an open apex with 3/4 incomplete root formation and an oblique fracture line extending below the gingival margin adjacent to the pulp chamber (figure 1f). figure 2e and f represents the stages in case management, relate to case iii. case management on the same visit day, a pulpotomy was performed on the teeth which had been determined as vital after a cold test. the method involved two stages: laser-assisted cavity preparation of the exposed dentin and pulp zone followed by sealing of the cavity with mineral trioxide aggregate b a c d e f figure 1. a) clinical view of the crown fractures and b) the periapical radiograph of case i. c) clinical examination of the upper incisors and d) the periapical radiograph shows decayed and fractured teeth of case ii. e) clinical examination of the crown fractures and f) radiograph of #21 showing an open apex with 3/4 root development of case iii. b a d c f e figure 2. a) heamorrhage seen after intentional cavity preparation and b) mta placement for #11 of case i. c) preparation of the cavity with laser beam and d) mta application for #11 and #21 of case ii. e) haemorrhage seen after cavity preparation and f) mta application on #21 of case iii. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p57–60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p57-60 59balan, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 57–60 (mta) which precedes the constructing a restoration of the missing part. figure 2 represents the clinical pictures of case management steps. therapy was initiated by means of a cotton-roll isolation without resort to local anesthesia. a modified partial pulpotomy was conducted with the assistance of a 2780 nm er,cr:ysgg (waterlase md, biolase, usa). the laser in question operated at 1 w-2.5 w and 20 hz in 140 µs pulse duration (h mode), with a mgg6 sapphire tip (6 mm long, 600 micron) directed with 90% water and 80% air. the tip was applied to the surface at a 45-degree angle in order to ease the running stream and avoid misplacement of the infected dental parts into the pulp chamber. the dentine border of the exposed zone was ablated to a depth of 1 mm below the fracture until the cavity formed extended to represent a barrier (figure 2a,c, and e). white-mta (angelus, londrina, pr, brazil) was applied to the cavity after hemostasis had been achieved (figure 2b,d, and f) and covered a thin layer of light-cured glass ionomer cement liner (ionoseal, voco, germany). a transitional restoration (ketac molar, 3m, usa) was implemented as an emergency measure. occlusion (bite) was not increased and dental splints were not required. the crown restorations were completed by means of resin composite fillings completed during subsequent visits. over the course of the following months, panoramic and periapical radiographs were exposed for examination and vitality tests were performed with the following results: absence of symptoms and pathology, crown discoloration and the tooth being vital and non-responsive to percussion and palpation. the patient was recalled at intervals of one, three and six months during the subsequent two years for clinical and radiographic examination, irrespective of his/ her not having subsequently presented symptoms. in case i, because the fracture line was flat and extremely close to the vital structure it proved necessary to prepare a cavity capable of accommodating the biomaterial. moreover, in the opinion of the authors, the trauma may have caused inflammation and precipitated a localized increase in both intestinal fluid pressure and blood flow in the low-compliant pulp that could induce pulp necrosis.8 an intentional partial removal of hard tissue mode dentine was concurrently performed with a partial pulpotomy resulting in a haemorrhage and no attempt was made to set other lasering parameters for pulp vaporization in soft tissue mode. follow-up on the condition of the tooth was conducted for two years without any subsequent pathology. however, tooth #21 experienced both enamel and dentine fracture without exposure and required root canal treatment after three months due to its presenting symptoms of pulpitis (figure 3a and b). in cases ii and iii, the teeth already possessed exposure sites, although these had been not fully opened which resulted in pulp vaporization. therefore, it was the intention of the authors to prepare cavities in the hard tissue, all of which were filled with mta to produce a hermetic seal. the blood in the pulp was considered the liquid source necessary to complete setting of the biomaterial. the teeth in case ii were monitored for more than 18 months (figure 4a and b). in case iii, the immature tooth referred to in the report showed evidence of continuous root development and apex formation throughout the monitoring period without presenting pathologic symptoms (figure 5a and b). discussion in cases of traumatic dental injuries, pulp therapy is planned according to the time between trauma and therapy, the size of pulpal exposure and the type of bleeding. if the pulp examined is considered vital, a pulpotomy procedure involving partial removal (2 mm) of the pulp tissue using a sterile diamond bur housed in a high speed hand piece a b figure 3. a) clinical and b) radiological view of the central incisors of case i. #11 shows completed apex formation after a two-year follow-up period, and root canal filling of #21 is shown in the radiologic picture. a b figure 4. a) clinical and b) radiological evidence of #11 and #21 of case ii indicate no periapical or pulpal pathology after a b figure 5. a) clinical and b) radiological pictures of #11 of case iii, show completed root development and apex formation without pathology, reviewed over a period of almost two years. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p57–60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p57-60 60 balan, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 57–60 followed by the application of a pulp dressing prior to restoration is usually performed.10 furthermore, a partial pulpotomy might be advisable in cases of young patients with complicated trauma involving exposure larger than 1 mm or/and occurring within 48 hours of therapy.6-10 because the treatment includes surgical removal of soft tissue and preparation of the hard dental structure, the research investigated laser-assisted partial pulpotomy as an atraumatic and aseptic technique which represents an alternative to conventional procedures. the majority of the clinical studies reported in the literature highlight the laser coagulation procedure relating to mechanically-exposed pulp during the removal of caries. however, there is limited data on er,cr:ysgg-assisted pulp therapy applied to traumatized permanent teeth with the result that it can be confused with the pulpotomy procedure employed on primary teeth. olivi et al. (2015) have recently argued that er,cr:ysgg laser applications for vital pulp therapy involved initial removal of deep dentin caries (150 mj, 15-20 hz, short pulse duration (with water/air spray) usually followed by indirect pulp capping practice which includes decontamination of dentin melting at lower energies (25 mj, 10 hz and short pulse duration (with low water and air spray). direct capping involves another treatment strategy intended to protect pulp from the irritants at as early a point as possible. laser-assisted pulp capping differs from the practice of dentin melting which coagulates the pulp tissue by means of 10-25 mj, 10 hz with short pulse duration and a 600 mm diameter tip in defocused mode and requires a hermetic seal of the exposed area. a partial pulpotomy performed on permanent teeth involves a similar exposure of pulp for deep dentin caries, but differs with regard to the practice of vaporization of the pulp tissue (150 mj, 10 hz, short pulse duration with active water/air spray) for traumatic injuries.6 although laser irradiation increases the alkaline phosphatase activity of osteoblasts responsible for the production of dentinal hard tissue,11 pulp therapy demands a non-degradable and bioactive cement such as mta, portland cement, or biodentine, to avoid potential complications during the healing process. one histological study highlighted the role of the capping material and mta+laser in producing superior results from the perspective of preserving pulp vitality and preventing necrosis than mta alone or ca(oh)2 with a laser. 12 the patients reported no pain during the cavity operation, although they did experience mild sensitivity to the tactile stimulus during the barrier placement. the findings of this case report concur with those of previous studies that er,cr:ysgg laser ablation simplifies clinical procedures, while contributing to effective behavior management in children by reducing discomfort and inducing a pain threshold.2 furthermore, such treatment positively affects the healing of these teeth over a period of 18-22 months. no pathological symptoms or crown discoloration occurred during the follow-up period. the fact that no root resorption was observed in any cases which constituted a positive result of the investigation. although the current report is based on a limited sample of participants, the findings suggest that er,cr:ysgg laser applications in pulpotomy practice constitute extremely delicate surgical operations during which both hard and soft dental tissue should be protected. in conclusion, this case report was intended to assess the importance of laser-assisted pulpotomy in dental trauma. as with all dental applications, accurate diagnosis and an appropriate treatment plan play significant roles in the successful management of traumatic injuries. therefore, being aware of medical advances, such as those concerning the application of lasers and the range of available treatment, is crucial. references 1. bastone eb, freer tj, mcnamara jr. epidemiology of dental trauma: a review of the literature. aust dent j. 2000;45(1):2-9. 2. nazemisalman b, farsadeghi m, sokhansanj m. types of lasers and their applications in pediatric dentistry. j lasers med sci. 2015;6(3):96-101. 3. arapostathis k. laser-assisted pediatric dentistry. lasers in dentistry—current concepts: springer; 2017. p. 231-43. 4. eversole lr, rizoiu i, kimmel ai. pulpal response to cavity prepa ration by a n erbium, ch rom ium:ysg g laser-powered hydrokinetic system. j am dent assoc. 1997;128(8):1099-106. 5. komabayashi t, ebihara a, aoki a. the use of lasers for direct pulp capping. j oral sci. 2015;57(4):277-86. 6. olivi g, genovese md. laser applications for vital pulp therapy. in: olivi g, olivi m, editors. lasers in restorative dentistry: a practical guide. berlin, heidelberg: springer; 2015. p. 223-47. 7. andreasen fm, kahler b. pulpal response after acute dental injury in the permanent dentition: clinical implications—a review. j endod. 2015;41(3):299-308. 8. heyeraas kj, berggreen e. interstitial fluid pressure in normal and inflamed pulp. crit rev oral biol med. 1999;10(3):328-36. 9. suzuk i m, kato c, kawashima s, shinkai k. clinical and histological study on direct pulp capping with co2 laser irradiation in human teeth. oper dent. 2019;44(4):336-47. 10. bimstein e, rotstein i. cvek pulpotomy – revisited. dental traumatology. 2016;32(6):438-42. 11. ohbayashi e, matsushima k, hosoya s, abiko y, yamazaki m. stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts. j endod. 1999;25(1):30-3. 12. hasheminia sm, feizi g, razavi sm, feizianfard m, gutknecht n, mir m. a comparative study of three treatment methods of direct pulp capping in canine teeth of cats: a histologic evaluation. lasers med sci. 2010;25(1):9-15. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p57–60 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p57-60 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa 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research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into 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... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dental medicine, universitas airlangga editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (+6231) 5039478 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg i wish to subscribe dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) name/nama: .......................................................................... date of birth/tanggal lahir: .................................................... job title/pekerjaan: ................................................................ institution/institusi: .................................................................. 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........................................................................................... address/alamat: ...................................................................................... .................................................................................................................. country/negara: ...................................................................... telp.: ....................................................................................... fax.: ........................................................................................ e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month 1 year surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " mkg vol 41 no 4 oct-dec 2008.indd 182 vol. 41. no. 4 october–december 2008 research report stimulation of type i collagen activity in healing of pulp perforation sri kunarti department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract background: tgf-β1 is a connective tissue stimulant, potential regulator for tissue repair, and promoter in wound healing. the healing of pulp perforation is decided by quantity and quality of new collagen deposition. tgf-β1 upregulates collagen transcription. however, after several weeks production of type i collagen synthesis is stopped and enzymatic degradation of collagen matrix will occur. purpose: observe synthesis type i collagen during the process of pulp perforation healing in 7, 14, and 21 days after being treated using tgf-β1. methods: this research in vivo from orthodontic patients indicated for premolar extraction, between ages 10–15 years. a class v cavity preparation was created in the buccal aspect 1 mm above gingival margin until pulp exposure. cavity was irrigated slowly with saline solution and dried with a sterile small cotton pellet. the sterile absorbable collagen membrane used, soaked with 5 μl of tgf-β1. it was covered by teflon pledge to separate from glass ionomer cement as restoration. evaluated on day 7th, 14th, and 21st. all samples were extracted and prepared for immunohystochemical examination. result: data were analyzed by t-test. there was significant difference in synthesis type i collagen compared between 7–14 days and 7–21 days after treatment by tgf-β1 also ca(oh)2. there was significant difference compared between tgf-β1 and ca(oh)2 in 14–21 days after treatment (means tgf-β1 > ca(oh)2). conclusion: elevation of synthesis type 1 collagen by tgf-β1 in 20 mg/ml. key words: type i collagen, tgf-β1, calcium hydroxide, pulp perforation correspondence: sri kunarti, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: attybp@yahoo.com introduction remarkable vascularized granulation tissue is shown in the initial recovery process followed by fibroblast proliferation and decreasing proportion of vascularized and fibroblast tissue of collagen formation. granulation tissue is gradually replaced by more elastic and regular extra cellular matrix containing most component of type i collagen. during transitional period from granulation tissue to scar, collagen remodeling depends on the balance between synthesis catabolism and further deposition.1,2 epithelial tissue is not found in pulp structure, therefore, there is no epithelial response in pulp recovery process but the response of fibroblast tissue is similar to both tissues and collagen as the product of scar tissue formation and continued by mineralization to form reparative dentin.3 collagen family is at least consisting of 30 different gens which producing 19 types of collagen. mesenchim cell and its derivates (fibroblast, odontoblast, and cementoblast) are the major collagen producers. the other types of cell (epithel, endothel and muscle) also synthesize collagen although in little amount and few types. type i, ii, iii, iv, and xi collagen are combined in extra cellular to form febrile. fibroblast tissue is mostly containing type i collagen, then, type iii (reticular fibroblast) and type v is usually found between type i and iii expected to regulate fibrile’s diameter. type i collagen is the most dominant collagen in dentine (86%), while type i and ii collagen could be found in pulp. type i collagen is synthesized and secreted by odontoblast, then, penetrates into dentine matrix, while fibroblast produces type i and iii collagen in pulp.3,4 the information on the application of exogenous tgfβ1 to stimulate reparative dentinogenesis is still not much 183kunarti: stimulation type i collagen activity available. the combination of tgf-β1 has the capability to stimulate reactive response if it is administered directly on the odontoblast layer. however, only part of dentine matrix containing tgf-β1 shows positive response if it is applied in imperforated cavity.5 the application of exogenous tgf-β1 is expected as a booster on endogenous tgf-β1 to increase the existing character such as: to stimulate fibroblast proliferation, to regulate cell attachment, to increase transcription of new collagen, to trigger inactive cell in g0 phase entering g1 active phase, to overcome healing disturbance.6 the regulation of bioactivity of tgfβ1 in the inside part of dentin matrix is important due to the presence of tgf-β1 inside dentine matrix is capable to influence cell behavior surrounding dentine-pulp complex.7 tgf-β1 is secreted as latent precursor molecule which could be activated by various factors. approximately half of tgfβ1 is isolated from dentine matrix has been reported to be active form although the presence of other molecules inside ecm modulating the capability and activity of tgf-β1. for examples: proteoglycan and betaglycan can stimulate tgf-β1 and make the appearance easier in cell surface, while decorine can bind and inhibit the activity of growth factor.7 the aim of this study is to observe synthesis type i collagen during the process of pulp perforation healing in 7, 14, and 21 days after being treated using tgf-β1. materials and methods first premolar extraction was done for orthodontic treatment prepared for the sample of this study and fulfilling the requirement of having vital condition, non caries and intact, aged 10-20 years old, having positive result of cold test using ethyl chloride, percussion and compression test is negative, clinically healthy and having good general condition. the total number of samples was 48 classified into tgf-β1 and ca(oh)2 groups which were divided into three subgroups. extraction was performed 7 days after treatment in subgroup 1, 14 days after treatment in subgroup 2 and 21 days after treatment in subgroup 3. firstly, anesthesia was done in buccal fold using 0.6 ml xylestesin f, rubber dam and saliva suction were applied and continued by tooth disinfected using 70% alcohol in buccocervical region. cavity preparation on buccal side was done 1 mm above the gingival margin, intermittent bur with light pressure was used, preparation was slantingly done toward apical using round bur no 3 with 1.5 mm diameter until closed to pulp, then, followed by using round bur no 1 with 0.5 mm to penetrate thin dentine layer until perforation reached pulp region. cavity was slowly irrigated using 0.5 ml saline solution7 and dried by sterile cotton pellet. tgf-β1 group was treated using 20 mg/ml concentration of tgfβ1 applied by dropping 5 μl in 1 mm2 absorbable collagen membrane as carrier (done in laminar flow hood) put into eppendorf tube and kept in nitrogen liquid at –80° c. the membrane was applied on the perforation, covered by teflon pledged and filled by restorative material type ii glass ionomer cement then varnish. for ca(oh)2 group (visible light group), ca(oh)2 was applied in 1 mm diameter tube and visible light cured tool was prepared. 1mm ca(oh)2 was administered in the bottom of cavity and illuminated for 40 sec in the shortest distant and covered by teflon pledged, filled type ii glass ionomer cement and polished by varnish. the preparation of immunohystochemistry initiated since tooth extraction was done. fixation was performed using 10% buffer formalin for 48 hours followed by decalcification applying alcl, formine acid, 37% hcl and aquadest. further process was dehydration to extract the water from the tissue and replaced by paraffin, continued by clearance using xylene. after the paraffin was hardened, the tissue could be cut using microtome in 4 μm thickness. deparafin and rehydration were subsequently performed: slide was soaked in xylol for 5 minutes and in absolute ethanol for 5 minutes. followed by being soaked in 95% ethanol for 5 minutes and it was all done twice; then, washed by flowing aquadest minimally for 30 sec. slide was then soaked in 0.01 m citrate buffer and put into the microwave for 30 minutes at 60° c. trypsine was done in 15 minutes at room temperature with 0.025% (w/v) trypsin (1 : 250) in phosphate buffer saline (pbs). the sample was washed using pbs than incubated in 3% hydrogen peroxide for 5 minutes to eliminate endogenous peroxide, than, washed by pbs. primary antibody type 1 collagen monoclonal antibody was diluted by antibody diluent (containing 0.05 m tris-hcl buffer ph 7.2–7.6, 1% bovine serum albumin) with ratio 1 : 200. incubation was done for 12 hours, next, it was washed by pbs. secondary antibody biotinylated link human anti rabbit was dropped, incubated for 10 minutes, was wash by pbs. streptavidin hrp was dropped and incubated for 10 minutes, washed by pbs. subtrate-chromogen solution: dab chromogen (0.05% 3-3’-diaminobenzidine) diluted by substrate buffer with ratio 1 : 50, incubated for 10 minutes. counterstained using haematoxyline mayer for 1–2 minutes until the color was blue, washed by flowing water and dried. it was given entelan, and covered by cover glass. light microscope was applied and the evaluation was done by two observers. result the region which would be evaluated was dark brown observed and magnified 40 times of the perforation region located between two dentin walls using graticule. the number of graticules in perforation region was observed and the evaluation was done 400 times magnification in all sub groups. figure 1-a and 1-b showing the sample of type i collagen synthesis 14 days and 21 days after being given tgf-β1. the mean and standard deviation of each group resulting from immunohystochemistry study done in 7, 14, and 21 days (table 1). 184 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 182−185 the main of type 1 collagen in 7, 14 and 21 days after treatment showing tgf-β1 was higher compared to ca(oh)2 group. either tgf-β1 group indicating the elevation of mean in 7, 14, 21 days after treatment using kolmogorov-smirnov statistical test showed normal distribution (p>0.05) in which fulfilling the requirement of parametric test. the result of homogeneity test using levene test in tgf-β1 and ca(oh)2 groups as well as between tgf-β1 groups (p<0.05). anova test showed there were significant difference between ca(oh)2 groups, and also between tgf-tgf-β1 groups. dunnet t3 test and t-test performed between tgf-β1 as well as between ca(oh)2 groups (table 2). t-test was also done comparing between tgf-β1 and ca(oh)2 groups (table 3). table 2. level of significance showed in 7–14 days, 14–21 days and 7-21 days between tgf-β1 and ca(oh)2 groups variable period of time p tgf-β1 ca(oh)2 type 1 collagen 7–14 days 0.000* 0.000* 7–21 days 0.001* 0.000* 14–21 days 0.866 0.572 note: * = significant difference significant difference of p value in tgf-β1 group of type i collagen comparing between 7–14 days and 7–21 days while significant difference of p value in ca(oh)2 group of type collagen comparing between 7-14 days and 7–21 days. table 3. level of significance in comparison between tgf-β1 and ca(oh)2 groups in 7, 14, 21 days variable p value tgf-β1 ca(oh)2 groups 7 days 14 days 21 days type i collagen 0.057 0.047* 0.011* note: * = significant difference there was no significant difference in p value between tgf-β1 and ca(oh)2 in period of 7 days (p > 0.05) while significant difference was found in 14-21 days (p < 0.05). discussion in this study, the role of tgf-β1 was clearly seen in synthesis type i collagen. formation of type i collagen in perforation region occurred in the whole samples of tgf-β1 on day 7, comparing to ca(oh)2 group, significant difference was found in tgf-β1 group in comparison between 7–14 days and 7–21 days while significant difference was not found in 14–21 days. this study also showed progressive elevation on day 7 until day 14 meanwhile a little elevation was shown on day 14 until day 21. due to the lower concentration of tgf-β1 on wound healing.1 soon after the wound occurred in an hour, the concentration of tgf-β1 actively increased until 9 times higher, in which it was the peak concentration of tgf-β1, then, decreasing in 24 hour period however it was still higher figure 1. (a) pulp tissue with the elevation of type i collagen synthesis, 14 days after being given tgf-β1 (400× magnification); (b) pulp tissue with the elevation of type i collagen synthesis, 21 days after being given tgfβ1 (400× magnification). table 1. the mean and standard deviation of tgf-β1 and ca(oh)2 groups on day 7 th, 14th, 21st after treatment variable material 7days 14 days 21 days mean s.d mean s.d mean s.d type collagen tgf-β1 6.6250 2.3867 27.2500 8.1372 30.6250 11.1604 ca(oh)2 3.3750 3.6621 18.8750 7.2592 17.1250 6.7493 185kunarti: stimulation type i collagen activity than normal condition. the second peak concentration would occur 5 days after the occurrence of wound and 14 days the wound the active concentration of tgf-β1 would return to normal. the addition of exogenous tgf-β1 would increase the total number of active tgf-β1 resulting in more remarkable effect. during healing process, some kinds of cells including macrophage, fibroblast, endothelial cell, keratinocyte would migrate to the wound region in which they would produce and active tgf-β1. the second peak concentration of active tgf-β1 which is present on day 5 would contribute the elevation of wound healing.8 tgf-β1 is stimulant of fibroblast tissue cell, a potent regulator as well as a promoter in wound healing, in addition tgf-β1 has unique character and stimulates ecm deposition by increasing matrix protein synthesis and decreasing matrix degradation in which both conditions could be achieved by decreasing protease synthesis and increasing protease inhibitor synthesis. tgf-β1 also increased synthesis integrin receptor, therefore there is an increase interaction between cells and ecm.9 the healing of perforation wound would be determined by the number and the quality of new collagen deposition. after some weeks synthesis collagen is stopped and enzymatic degradation of collagen matrix would occur, this condition would contribute the balance of collagen formation. synthesis and lysis are strictly controlled by cytokine and growth factor. tgf-β1 plays the role of increasing new collagen transcription and the production of collagen would be lowered by tissue inhibitors of metalloproteinase (timp). the balance between deposition and degradation of collagen would determine the integrity and the strength of tissue.10 mechanism of collagen degradation would occur through enzyme secretion done by cell and extra cellular matrix molecule or collagen fibril selection and intra cellular degradation by fibroblast.3 triple helix collagen is really resistant against proteolytic reaction. matrix metalloproteinase (mmp) family is proteolytic enzyme is capable of degrading collagen and other macromolecule matrix to be small extra cellular peptide. mmp is synthesized and secreted by fibroblast, inflammation cell and other cells. a number of cells secrete mmp inhibitor in active condition, then, through proteolytic process becoming active. intracellular degradation is most important mechanism in physiologic condition and remodeling collagen fibroblast tissue.4 the application of topical tgf-β1 would increase breaking strength which could be clearly seen in ecm especially synthesis and deposition collagen.1 the formation of collagen occurs on day 3 and more extensive after day 7.11 well arranged fibres, collagen formation, mesenchim cell and fibroblast would develop to be cell-rich layer. cell would proliferate and differentiate to be preodontoblast and columnar shape odontoblast.3,12 tgf-β1would directly stimulate matrix production including to enhance collagen synthesis, fibronectin and other matrix molecule. tgf-β1 indirectly regulates fibroblast proliferation.1 in the previous study13 it was reported that angiogenesis occurred before day 7 of tgf-β1 application meaning the increase of vascularization was faster consequently synthesis type 1 collagen occurring faster than ca(oh)2 group. as it has been understood that lowering the number of capillar will be running simultaneously with continuing healing process, decreasing the ratio between vascullarized and fibroblast tissue, followed by the elevation of collagen production.14 further process initiated by the formation of reparative dentinogenesis consisting of odontoblast like cell layer is formed in connection to superficial calcification and tubule matrix which has been mineralized secreted in predentin like pattern. many studies have proved that early formation of fibrodentin with osteotypic appearance in trauma affected region15 frequently happens in the mechanism of wound healing. it is concluded that tgf-β1 administration will increase synthesis type i collagen. references 1. chettibi s, ferguson mwj. wound repair: an overview. in: gallin ji, snyderman r, eds. inflammation basic principles and clinical correlates. 3rd ed. philadelphia: lippincott williams & wilkins; 1999. p. 865–77. 2. mcmahon rft, sloan p. inflammation. in: essentials of pathology for dentistry. 1st ed. edinburgh: churchill livingstone; 2000. p. 26–31. 3. nanci a. repair and regeneration of oral tissue. in: ten cate’s oral histology: development, structure and function. 6th ed. st louis: mosby; 2003. p. 397–415. 4. trownbridge ho, kim s, suda h. structure, and function of the dentin and pulp complex. in: cohen s, burns rc, eds. pathways of the pulp. 8th ed. st louis: mosby inc; 2002. p. 411–447. 5. sloan aj, smith aj. stimulation of the dentine-pulp complex of rat incisor teeth by transforming growth factor-β isoform 1–3 in vitro. archs oral biol 1999; 44: 149–56. 6. wahl sm. transforming growth factor-β (tgf-β) in the resolution and repair of infl ammation. in: gallin ji, snyderman r, eds. infl ammation basic principles and clinical correlates. 3rd ed. philadelphia: lippincott williams & wilkins; 1999. p. 837–42. 7. smith aj, matthews jb, hall rc. transforming growth factor-β1 (tgf-β1) in dentin matrix: ligand activation and receptor expression. eur j oral sci 1998; 106(suppl 1): 179–84. 8. yang l, qiu cx, ludlow a, ferguson mwj, brunner g. active transforming growth factor-β in wound repair. am j path 1999; 154: 105-11. 9. krakauer t, vilcek j, oppenheim jj. proinfl ammatory cytokines tnf and il-1 families, chemokines, tgf-β and others. in: paul we, editor. fundamental immunology. 4th ed. philadelphia: lippincott-raven; 1999. p. 800–3. 10. efron dt, witte mb, barbul a. wound healing: physiology, clinical progress, growth factor and the secret of the fetus. in: baue ae, faist e, fry de, eds. multiple organ failure pathophysiology, prevention and therapy. springer; 2000. p. 553–7. 11. kitasako y, shibata s, pereira pn, tagami j. short-term dentin bridging of mechanically-exposed pulps capped with adhesive resin systems. oper dent 2000; 25: 155–62. 12. stanley hr. pulp capping: conserving the dental pulp-can it be done? is it worth it? oral surg oral med oral pathol 1989; 68: 628–39. 13. kunarti s. pulp tissue infl ammation and angiogenesis after direct pulp capping with transforming growth factor β1. dental journal (majalah kedokteran gigi) 2008l; 41(2): 92–5. 14. mcmahon rft, sloan p. inflammation. in: essentials of pathology for dentistry. 1st ed. edinburgh: churchill livingstone; 2000. p. 26–31. 15. tziafas d. the future role of a molecular approach to pulp-dentinal regeneration. caries res 2004; 38: 314–20. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 147 vol. 41. no. 3 july–september 2008 the use of bay leaf (eugenia polyantha wight) in dentistry agus sumono1 and agustin wulan sd2 1department of dental material 2 department of biomedic faculty of dentistry jember university jember indonesia abstract background: bay leaf or eugenia polyantha wight is a species that has several chemical properties. bay leaf consists of tanine, bay leaf or eugenia polyantha wight is a species that has several chemical properties. bay leaf consists of tanine, flavonoid, essensial oil, including citric acid and eugenol. however, only few reports were published about the use of bay leaf in dentistry. purpose: the aim of this article is to describe the chemical properties of eugenia polyantha wight that are widely used in the aim of this article is to describe the chemical properties of eugenia polyantha wight that are widely used in dentistry. reviews: the chemical properties of eugenia polyantha wight have analgesic, antibacterial, and anti-inflammatory effect, the chemical properties of eugenia polyantha wight have analgesic, antibacterial, and anti-inflammatory effect, so they can be used as an alternative dental therapy. these properties can be used as a basic of therapy or as a basic ingredients of treatment. conclusion: eugenia polyantha wight has some useful pharmacologic activities that are useful in dentistry. we suggest eugenia polyantha wight has some useful pharmacologic activities that are useful in dentistry. we suggest this article can be used as a basic knowledge for dental researchers. key words: eugenia polyantha wight, essensial oil, tanine, flavonoid, dental therapy correspondence: agus sumono, c/o: bagian ilmu material teknologi kedokteran gigi, fakultas kedokteran gigi universitas jember. jln. kalimantan 37 jember 68121, indonesia. email: goesteen_wulan@yahoo.com, telp. (0331) 333536. introduction bay leaf or eugenia polyantha wight has been known since long time ago as a species that can be used for therapy. the use of bay leaf has been developed medically, as an alternative medical plant.1 bay leaf has a lot of chemical properties that is useful in medical, even as basic materials in dentistry. empirically, eugenia polyantha wight can be used for hypertension, diabetic, diarrhea, gastritis, drunks, and skin diseases. the plant also has other benefits such as diuretic and analgesic effect.2 eugenia polyantha wight has many chemical properties. the chemical properties of eugenia polyantha wight are basic matters that are used in almost every section in dentistry, as basic matters for therapy and treatment.2 the aim of this article is to describe the chemical properties of bay leaf and the benefits of its chemical properties in dentistry. we suggest this article can be used by dentists, as a basic knowledge for future researches about bay leaf in dentistry. the morphology of bay leaf bay trees mostly grown in the forest, but they may be planted in the garden. bay trees can be found in lowlands until 1.400 meters above sea level.3 bay trees may height about 25 meters, have large straight root, round trunk and smooth surface.4 bay trees have small, white, and fragrant flowers. bay leaf (figure 1) has 2.5–8 centimeters long leaf figure 1. bay leaf.5 review article 148 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 147-150 with flat margins, the tip is blunt and the base of the leaf stretch along length and thight.2 chinese therapy books describe that bay leaf has fragrant smell and has astringent to the taste.1 taxonomy of bay leaf scientifically, bay leaf is named eugenia polyantha wight and the synonyms are eugenia lucidula miq and syzyqium polyanthum wight.6 the taxonomical chart put this plant in the spermatophyte division, pinophyta subdivision, coniferopsida class, eugenia family, myricales genus, and eugenia polyanthum (wight) walp species.7 in some region or provinces in indonesia, bay leaf is known as meselangan (sumatra), ubar serai (malay), salam (java, sunda, madura), gowok (sunda), manting (java) or kastolam (kangean).2 benefit of bay leaf bay leaf can be used not only as spices for cooking purposes, but it can also be used as medicine. its roots and fruits extract have the ability to neutralize hang over caused by too much alcohol consumption. beside those two utilities, bay leaf extract is usually used to stop diarrhea, gastritis, diabetes mellitus, itchy, astringent, and scabies.8 it is also stated that bay leaf has lower side effect compared to synthetic drugs. to be consumed as drugs, we extract bay leaf by boiling it, while as ointment, bay leaf is crashed and applied on the affected skin.3 bay leaf can also be used to treat patients with high uric acid. new researches describe that infuse of bay leaf in 0.5 mg doze can increase the excretion of uric acid in urine of wistar male rat.9 recurrent apthous stomatitis (ras) is an ulcer cause by oral mucous membrane inflammation that is often found. ras causes pain and difficulties in eating, speaking, and tooth cleaning. the common treatment is giving topical antibacterial, antiseptic, and anti-inflammatory medicines,9 but the infuse of eugenia polyantha wight can be used to treat that disease. chemical contents of bay leaf in some study, eugenia polyantha wight has a lot of chemical properties. the chemical properties consist of tanines, flavonoid and essential oils (0.05%), including citric acid and eugenol.1 tanine is a liquid glycoside derived from polypeptide and ester polymer which can be hydrolyzed by the secretion of bile (3, 4, 5–trinidrokside benzoic acid) and glucose.10 tanine or tanat acid isolated from some part of plants can be found in market. it is cream colored powder, amorf, astringent taste and aromatic.11 tanine is used as astringent for gastrointestinal tract or skin. tanine as an astrigent that can make precipitation of the cell membrane protein and also have a little penetration activity, so it can influence the permeability of cell membrane.12 tanine can also be used as anti diarrhea. tanine can inhibit topoisomerase enzyme i and ii, viral transcriptase in 0.01µg/ ml concentration.11 pharmacologic and physiologic effects of tanine are derived from its activity to form complex compound. the form of its complex is based on the formation of hydrogen chain and hydrofobic interaction between tanine and protein.11 tanine is an active compound that has antibacterial activity.13 antimicrobial activity of tanine depends on the ability of this compound to inhibit the activity of some enzyme selectively or its ability to inhibit inter ligan chain in some receptor.11 eugenia polyantha wight can be used in periodontics, like in the periodontitis case. periodontitis treatment are mechanically (scalling, curretage, or gingivectomy) and chemically (oral rinse).14 tanine in the bay leaf functions as astringent.6 astringent is a medicine which has the ability to decrease the mucous matrix. it also cause protein precipitation on the cell surface which have low permeability.13 whereas, tanine is one of active matters of eugenia polyantha wight and part of phenol group,15 that can inhibit the growth of bacteria by precipitation and denaturation of bacteria protein.16 the structure of tanines can be seen in figure 2. flavonoid is a genetic term used for aromatic heterocyclic oxygen compound which is derived from 2 – phenilbenzopiran or its 2, 3 – dehydro.10 flavonoid is one of natural phenolic compound present in most plant. it is found in seeds and fruits. it is synthesized in small amount about 0.5–1.5% and can be found in almost every part of a plant.18 antusianin (anthocyannis) is a subgroup of flavonoid, responsible to give yellow, red and blue pigment. flavonoid is classified based on the level of oxidation level into catechin, leukoanthocianidine, flavanol, flavon and antocianidine.10 flavonoid coumpound in the form of aglikon in the intestine is absorbed along with the secretion of the bile through the ephitelia into the vacular system. through the vena porta, most of the flavonoids are headed to the liver as the primary organ for metabolism. metabolism process can also take place in the big intestine as well as in the renal organ. some of the biological function will increase when flavonoid is absorbed. those functions include the figure 2. structure of tanines.17 149sumono and wulan: the use of bay leaf (eugenia polyantha wight) in dentistry protein sinthesys process, cell differentation, proliferation and angiogenesis.19 in vivo and in vitro studies showed that flavonoid has biological and pharmacologic activities, including antibacterial activity.19 flavonoid in eugenia polyantha wight has an antiinflammatory effect and can support vascular wall, therefore the bleeding can be stopped. the mechanism of flavonoid as anti-inflammatory is through prostaglandine synthesis inhibiting and proline hydroxylation stimulating.19 the structure of flavonoid can be seen in figure 3. essential oil is mainly consists of terpenoid compound with atomic carbon framework of five.10 the characteristics of essential oil are highly evaporized in room temperature without decomposition, bitter, sweet smell in accordance with plant that produce it and soluble in organic solvent but not water soluble. another compound that form essential oil including phenilpropane biosynthetic is phenol compound such as eugenol, khavikol and khavibetol.21 essential oil in some plants have biological activity as antibacterial and antifungal, so essential oil can be used as food preservatives and natural antimicrobial.22 essential oil has antiseptic and antioxidant activity. essential oil also has activity to inhibit the growth of some bacteria and fungi.23 the structure of essential oil can be seen in figure 4. discussion recently, a lot of exploitation on natural sources is used as alternative therapy. because the side effects are less than synthetic drugs, one of the natural sources used is bay tree or eugenia polyantha wight. all part of eugenia polyantha wight can be used as alternative medical therapy, including the leaves, roots, and barks. the chemical properties of eugenia polyantha wight consist of tanine, flavonoid, essensial oil, including citric acid and eugenol.1 eugenia polyantha wight can be used in conservative and endodontics such as pulpitis treatment. pulpitis is an inflammatory of the pulp that is caused by bacterial invasion. acute pulpitis treatment is relief of pain by eugenol application or prescribing the medicines. in reversible pulpitis, the treatment is filling or reducing the amount of streptococcus mutans. streptococcus mutans is one of the bacteria that caused caries.25 recent studies showed that eugenia polyantha wight can reduce streptococcus sp colony in samples who rinsed with 100%, 75% and 50% eugenia polyantha wight solution. because it contains tanine, flovonoid and essential oil, that has antibacterial effect.25 eugenol or clove oil is an acidic essential oil that is used as pulp irritant and mild antiseptic.26 infuse of eugenia polyantha wight can be used as oral rinse for relief of pain or analgesic, because eugenol can inhibit interdental neuron impulse.27 the use of eugenia polyantha wight in prosthodontics is as a denture cleanser. soft deposits from food easily adhere on denture. these soft deposits must be removed or cleaned regularly, to prevent bacteria and fungi from growing. denture cleanser must be bactericidal and fungicidal.28 forty percent, 60%, and 80% extract of eugenia polyantha wight can inhibit candida albicans growth in acrylic resin denture base.29 flavonoid has antibacterial properties because it has the ability to interact directly with the dna of the bacteria.19 the basic structure of the dna itself has an important role in the transcription and duplication process, therefore, every compound that has the ability to disturb the stability of the double helix dna structure will be able to affect all the growth process and metabolism of the bacteria.19 those interaction will result in the damage of the permeability of the bacteria cell wall, microsome and lysosome. in addition, flavonoid is also capable in producing transduction energy that will affect the cytoplasm of the bacteria and slow down its motility. it is known that the hydroxyl ion present in flavonoid can chemically change the organic compound and nutrition transportation thus cause toxicity effect to the bacteria cell.18 astringent oil inhibits the growth of the candida albicans by protein and nucleic acid denaturizing. protein denaturizing process involve the changes in the protein molecular constituent stability and causes protein structural changes and allows coagulation process to happen. the protein that undergoes the denaturizing process will lose its physiological activity and incapable to function well. the changes occur in the protein and cell wall will increase the cell permeability. the damage and the increase in the cell permeability will wreck the cell.21 flavonoid in eugenia polyantha wight can be used as analgesic. flavonoid in honey can reduce cytokine (il1 and tnfa) produced by macrophage and expression figure 3. structure of flavonoid.20 figure 4. the structure of essential oil (eugenol).24 150 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 147-150 receptor of cytokine, so pain and tissue destruction can be reduced. flavonoid can increase mitogenesis process, cell interaction and adhesion that have a role in ephitelization process.30 beside that, flavonoid in eugenia polyantha wight can be used in oral surgery. flavonoid can accelerate post extraction healing process. flavonoid can increase proliferation of fibroblast and collagen production. flavonoid can also reduce pain after tooth extraction, by inhibiting prostaglandin synthesis.19 the conclusion of this article is eugenia polyantha wight has many pharmacological activities that are useful in dentistry. references 1. winarto wp, tim karyasari. memanfaatkan bumbu dapur untuk mengatasi aneka penyakit. jakarta: agromedia pustaka; 2004. p. 50. 2. utami p, tim lentera. tanaman obat untuk mengatasi rematik dan asam urat. jakarta: agromedia pustaka; 2005. p. 57–8.jakarta: agromedia pustaka; 2005. p. 57–8.2005. p. 57–8. 3. dalimartha s. salam (syzyqium polyanthum wight). 2005. available at: http://www.pdpersi.pdpersi.co.id. accessed april 29, 2008. 4. wijayakusuma h. rempah, rimpang dan umbi. jakarta: milenia populer; 2002. p 17–9. 5. anonim. 2000. daun salam sebagai obat alternatif asam urat. http://www.indonesia/intisari.htm. accessed february 2006. 6. dalimartha s. atlas tumbuhan obat indonesia. jilid i/cetakan 1. jakarta: trubus agriwidya indonesia; 1999. p. 137. 7. katzer g. gernot katzer’s spice dictionary. 2000. available at:2000. available at: http://www.ang.kfunigrans.ac.at/’katzer/genericframe.html. (online). accessed april 28, 2008. 8. wijayakusuma h. tanaman berkhasiat obat di indonesia. jilid ii.jilid ii. jakarta: pustaka kartini; 1995. p. 55–7. 9. apriono dk, dwi waf, agustin wsd. kemampuan infusum daun salam (eugenia polyantha walp.) dalam meningkatkan ekskresi asam urat pada tikus wistar jantan. laporan penelitian dipa. lembagalaporan penelitian dipa. lembaga penelitian universitas jember, jember. 2008. 10. dorland. 1985. kamus kedokteran dorland. tim penerjemah egc dari dorland, illustrated medical dictionary. jakarta: egc; 1996. p. 673, 746, 931. 11. mahtuti, erni y. pengaruh daya antimikroba asam tanat terhadap perumbuhan bakteri salmonella typhii secara in vitro. penelitian eksperimental laboratoris. tesis master dari jiptunair. 2004. available at: http://adln.lib.unair.ac.id/gophp?id=jiptunair-gdl-s22004-mahturiern. accessed april 30, 2008.accessed april 30, 2008. 12. rahardjo mb. kemampuan alium sativum linn dan kaempferia galanga dalam menghambat pertumbuhan streptococcus mutans. majalah kedokteran gigi fkg unair 1996; 818–23. 13. arif a, udin s. obat lokal. dalam farmakologi dan terapi. edisi 4. jakarta: gaya baru. 393–415. 14. goldman hm, cohen dw. periodontal therapy. 5th ed. st. louis: mosby co; 1973. p. 355–7. 15. robinson t. 1990. kandungan organik tumbuhan tinggi. edisi ke–6. koasih padmawinata, editor. bandung: penerbit itb; 1995. p. 71–196. 16. brooks gf, butel js, morse sa. 1998. mikrobiologi kedokteran.mikrobiologi kedokteran. edisi ke-1. jakarta: salemba medika; 2001. p. 79–84. 17. senbuerch vp. 2003. basic structure of tanine. http://biologie.unihamburg.de/b-online/e.26/11.htm. accessed 30 april 2008. 18. sabir a. aktivitas antibakteri flavonoid propolis trigona sp terhadap bakteri streptococcus mutans (in vitro). maj ked gigi (dentalmaj ked gigi (dental journal) 2005; 38(3): 75–9. 19. sabir a. pemanfaatan flavonoid di bidang kedokteran gigi. maj ked gigi (dental journal) 2003; edisi khusus temu ilmiah nasional iii:81–7. 20. anonim. what is flavonoid. arita laboratorium departement of computa biology university of tokyo. http://www.metabolosom. jp/software/flavonoidviewer/. accessed april 30, 2008. 21. wahyuningtyas e. pengaruh minyak atsiri zingiber purpurea terhadap pertumbuhan candida albicans serta kekuatan transversa plat dasar gigi tiruan resin visible light cured dan resin akrilik. karya tulis ilmiah yogyakarta. program pendidikan dokter gigi spesialis fakultas kedokteran gigi universitas gadjah mada; 1998. p. 36–7. 22. yuharmen, yum eryanti, nurbalatif. uji aktivitas minyak atsiri dan ekstrak metanol lengkuas (alpinia galaga). available at: http://www. unri.ac.id/jurnal/jurnal_natur/vol4(2)/yuharmen.pdf. accessed april 30, 2008. 23. djauleha f. khasiat infusa daun kaca piring sebagai obat kumur terhadap keberadaan candida albicans. maj ked gigi 1999; 32(4): 32 –6. 24. young dg. the chemistry of essential oils. http://www.atasehorsecare. com/viewcategory/32. accessed april 30, 2008. 25. sumono a, dwi warna af. kemampuan larutan daun salam (eugenia polyantha wight) dalam menurunkan jumlah koloni bakteri streptococcus sp. laporan penelitian beasiswa unggulan depdiknas; 2007. 26. vanable de, laurence rl. using dental materials. new jersey: pearson education inc; 2004. p. 227–30. 27. grossman li, oliet s, del rio ce. 1995. ilmu endodontik dalam praktek. edisi ke 11. abyono r, editor. jakarta: penerbit bukuabyono r, editor. jakarta: penerbit buku kedokteran egc; 1998. p. 71–7, 249. 28. combe ec. sari dental material. slamet tarigan, editor. jakarta: balaislamet tarigan, editor. jakarta: balai pustaka; 1990. p. 377–8. 29. shelly dem. penagruh berbagai konsentrasi perasan daun salam (eugenia polyantha wight) sebagai bahan pembersih gigi tiruan terhadap jumlah candida albicans pada lempeng resin akrilik. skripsi. jember: fakultas kedokteran gigi universitas jember; 2006. p. 30–5. 30. ernawati ds. madu sebagai terapi alternatif stomatitis aftosa rekuren (sar). maj ked gigi (dental journal) 2001; 34:473–5. 118 vol. 41. no. 3 july–september 2008 the profile of upper integument lip of baduy and the nearby living sundanese in south banten, west java, indonesia rachman ardan department of prosthodontics faculty of dentistry padjadjaran university bandung indonesia abstract background: based on the two migration theory and on cultural anthropology, the baduy is classified as protomalay and thebased on the two migration theory and on cultural anthropology, the baduy is classified as protomalay and the sundanese as deuteromalay. historically and socioculturally the baduy is part of the nearby living sundanese (ns) who has isolated themselves and settled down in kanekes. linguistics and archaeology could not tell whether the culture’s spread was due to a source population’s migrating or to a destination population’s copying the technology and language. craniofacial anthropometry could resolve this because people’s physiognomy does not change rapidly due to mere migration. purpose: the ob�ective of this study is to the ob�ective of this study is to determine whether the people of baduy are protomalay or deuteromalay based on profile of upper integument lips (puil), which is the size of the angle formed by (sn-ls) line to frankfurt horizontal plane. angle size < 90º = procheili, 90º = orthocheili, and > 90º = opisthocheili. lip prominence is strongly influenced by racial and ethnical characteristics, its form is determined especially by genetic factor. method: sub�ect sample consisted of 43 inner baduys (ib)�� 92 outer baduys (ob)�� and 135 ns of south banten were sub�ect sample consisted of 43 inner baduys (ib)�� 92 outer baduys (ob)�� and 135 ns of south banten were measured using fasiogoniometer to determine puil. average value data was determined and analyzed by testing equal variances to compare two variances between groups. result: the result of this study showed that ib, ob as well as ns are mild procheili (angle the result of this study showed that ib, ob as well as ns are mild procheili (angle size = 50o–69.9o). puil of ib and ob compared to ns are slightly different, however, between ib and ob are similar. conclusion: based on the characteristic of puil, ib, ob, as well as ns, are classified as mongoloid subrace, and physically should be classified as deuteromalay. key words: upper integument lip, baduy, sundanese correspondence: rachman ardan, c/o: bagian ilmu prostodonsia, fakultas kedokteran gigi universitas padjadjaran. kompleks unpad-jatinangor-jalan raya bandung sumedang km 21 tlp. (022)7794120, fax (022)7794121 kabupaten sumedang, jawa barat, indonesia. home: jl.dr.setiabudhi no.78, bandung 40141. e-mail: rach7544@yahoo.com introduction the baduy is an isolated tribal ethnic group living in an isolated inland area in kanekes village, leuwidamar district, lebak sub-province, south banten, west java province, indonesia (figure 1 and 2). their taboos and prohibition order has separated this group from the nearby living sundanese (ns).1-3 based on the two migration theory about the origin of the indonesian people,4,5 and on cultural anthropology, the baduy is classified as protomalay and the sundanese is deuteromalay. historical and socio-cultural (language, history, culture) of the baduy is part of the ns who has isolated themselves and settled down in kanekes.6 in 16th centuries in northen banten there were three kingdoms: wahanten girang; pulasari; and panimbang which are under the power of susuhunan pakuan pajajaran.6,7 negara kertabumi manuscript from cirebon reported that king of banten and his adherent were evacuated to pakuan when banten was attacked by moslem army alliance of demak and cirebon in 1527 ac,8 some of ruling family member evacuated to southland, looking for protection in the upper coarse area of ciujung river.6 the baduy society consists of two groups. those are the inner baduy (ib), which is the nucleus of the baduy, and the outer baduy (ob) as the complement. the kinship research report 119ardan: the profile of upper integument lip of baduy tribe figure 2. the map of kanekes village (baduy). inner baduy (blue colour); outer baduy (grey colour), and nearby living sundanese (white colour).1 system and marriage system of the baduy is endogamy. infringement against this prohibition order will cause an ib sent into exile to ob, and an ob expelled out of kanekes.1–3,9 lip is part of the mouth and its outer form is influenced by anterior part of alveolar process and anterior teeth. on the other hand, its form depends on its race. lips consist of two parts, the integument lip and mucous membrane lip. integument lip is part of the skin of the face that closes over orbicularis oris muscle, which edge continues to become skin of the cheek and base of the nose. in medial part of upper lip there is philtrum or sulcus nasolabialis, which is a deep channel that began from nose downwards. the end part between upper and lower mucous membrane lip is called angulus oris or cheilion. in many situations, the whole part of the mouth is ranging from looking like bulbar to looking like snout. this characteristic can be found in black race. if curve profile of upper integument lip is vertical to the nose-ear line (fhp=oae=ohr-augen-ebene)10 called orthocheili, if heel up to posterior called opisthocheili, and procheili if heel to anterior. curve profile of integument lip may form straight line, concave, or convex.11 like facial divergence, lip prominence is strongly influenced by racial and ethnical characteristics. whites of northern european and middle-eastern origin normally have more prominent lip and incisor than their northern cousins. greater degrees of lip and incisor prominence normally occur in individuals of asians and african descent. the puil in mongoloid is mild procheili, in negroid extreme procheili.11 lip posture and incisor prominence are evaluated by viewing the profile with the patient’s lips relaxed.12 lips development in boys were larger than in girls, and attainment of adult dimensions was faster in the upper than in the lower.13 the lips and the other soft tissues of the face sag downward with aging, and become progressively thinner with less vermillion display. environmental influences during growth and development of the face, jaws, and teeth consist largely of pressures related to the physiologic activity. a relationship between anatomical form and physiological function is apparent in animals. over evolutionary time, adaptations in the jaws and figure 1. the map of west java (in 1992).1 n village kanekes 120 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 118-122 figure 3. fasiogoniometer used in measurement. two bars which can slide to each other represent frankfurt horizontal plane (one tragion point and both of orbitale points). the base of the top end are paralleled to the measured surface. the scale of top end are and lower end are determine the size of the angle. table 1. sample distribution population target population sample men women men & woman ib ob ns 61 662 835 24 52 75 19 40 60 43 92 135 s 1558 151 119 270 dental apparatus are prominent in the fossil record. formfunction relationship at this level is controlled genetically. a careful examination of the facial profile yields the same information, though in less detail for the underlying skeletal relationships, as that obtained from analysis of lateral cephalometric radiographs.12 phylogeography craniofacial anthropometry is used to identify and track major long–distance migrations that bands of humans undertook, especially in prehistoric time. linguistics can follow the movement of languages and archaeology can follow the movement of artifact styles, but neither can tell whether a culture’s spread was due to a source population’s physically migrating or to a destination population’s simply copying the technology and learning the language. craniofacial anthropometry helped resolve this because a people physiognomy does not change rapidly due to mere migration.14 the facial appearance also would clearly help in the objective assessment of treatment quality, and be useful in monitoring growth changes,15 and disease diagnosis which have important facial feature.16 in forensic dentistry race determination in skeletal remains traditionally focuses on craniofacial characteristic, and mandible and dentition reflect racial characteristic.17 the objective of this study is to determine whether people of baduy are protomalay or deuteromalay based on profile of upper integument lips (puil) which is the size of the angle formed by the profile of the upper integument lip to frankfurt horizontal plane. the puil is part of the lip, and its form conform with the race, and it is useable in comparing groups of people.11 materials and methods the research method was a descriptive analytic survey, held in south banten area in 1992, in leuwidamar district and bojongmanik district, sub-province lebak, west java, indonesia to be precise. the 270 people randomly sampled from populations of inner baduy (ib), outer baduy (ob), and nearby living sundanese (ns) from surrounding area, are: aged between 17–30 years, no facial defect that will cause measurement error, and descendant of at least two generations of the population group (ib, ob, or ns) (table 1), all were measured by using a self-designed instrument called fasiogoniometer (figure 3),18 which is especially designed to measure the angle between each part of the face and the frankfurt horizontal plane (fhp). the design of this instrument is simple and it is very easy to use, and also very practical and portable, because it can be detached into four parts. the material of this instrument is rigid, light, and strong. reliability test in stability of measurement is good.18 some points of orientation were used in measurement. profile line of the upper integument lip is the line that passes through labrale superiores (ls) and subnasion (sn). frankfurt horizontal plane is the plane that passes tragion (tr) and orbitale (or). we used one tragion point and two orbitale point. by using fasiogoniometer we can determine the size of the angle formed by the profile line of the upper integument lip (sn-ls) with the frankfurt horizontal plane (fhp). average value data was determined and analyzed by testing equal variances to compare two variances value between groups. result the measurement was conducted to 270 randomly sampled subjects consisted of 43 inner baduy (ib), 92 outer baduy (ob), and 135 nearby living sundanese (ns). the criteria of subject samples are: aged between 17–30 years; no facial defect that will cause measurement error; descendant of at least two generations of the populations (ib, ob, or ns (table 1)). all of the subjects in each population group are procheili, after which divided into three categories (classes): extreme procheili (value = 30o–49.9o); mild procheili (value = 50o–69.9o); and less procheili (value = 70o–89.9o) (table 2). profile of upper integument lip data was obtained by measurement at the site, and was analyzed to compare variability value difference of two variances between groups (table 3). 121ardan: the profile of upper integument lip of baduy tribe discussion the value of puil (α) = 90o indicates that the inclination of upper integument lip is vertical, and if the value of the puil is less than 90o indicates that the inclination of upper integuments lip inclines to anterior (procheili). the mean value of puil in ib = 63.70o, ob = 61.73o, and in ns = 67.64o, therefore, the puil in ib, ob, and ns is procheili (table 2). prognatism of the alveolus caused procheili. soelarko19 who studied the position of the tip of dentes canini in upper jaw revealed that in indonesian the position of the tip of dentes canini in upper jaw is always anterior to orbital plane. according to snell, profile of maxilla in caucasoid is nasal orthognatism with alveolar mesognatism, in mongoloid is nasal orthognatism with alveolar prognatism, and in negroid is nasal mesognatism with alveolar hyperprognatism. the result of this study is similar with the study of soemardi20 that indonesian people have maxillary and mandibular alveolar prognatism. negroid is extreme procheili, mongoloid is mild procheli, and caucasoid is orthocheili.11 according to garna1,3 and danasasmita6 the baduy was the intermixture between local population with evacuated population which came to south banten from north banten when the moslems of demak and cirebon conquested north banten in 1527 ac.8 jacob4 based on findings of human fossils, concluded that at mesolitical period (+ 11.000 years past) melanesian feature was dominant physically, whereas at neolitical period (+ 4.000 years past) mongoloid feature (deuteromalay) was dominant physically, especially in west indonesia. if banten was under the power of susuhunan pajajaran, attacked by alliance of moslem army of demak and cirebon, mongoloid feature (deuteromalay) should have been dominant. the puil of ib, ob, as well as ns are mild procheili (mongoloid feature). so ib, ob, and ns are deuteromalay. on the other hand, socioculturally (language, history, culture) the baduy is part of the ns who has isolated themselves and settled down in kanekes. the language of ib and ob are sundanese, and they confess themselves as “sunda wiwitan” (early sundanese).1-3,6,9 the puil difference between ns and ib as well as between ns and ob resulted from endogamy marriage pattern, and genetic flow to outside (infringement against prohibition order will cause an ib sent into exile to ob, and an ob expelled out of kanekes.8,12 the ns itself is still open to genetic flow from outside. the result of this study showed that ib, ob and also ns are mild procheili (69.8 %; 71.7 %; and 51.9 %). based on the characteristic of puil, ib, ob, as well as ns are mongoloid subrace, and physically should be classified as deuteromalay. the result of this research also strengthens the opinion that, depending on neolithic fossil finding, history, cultures, the people of baduy (ib and ob) are classified as deuteromalay. references 1. garna jk. orang baduy. bangi, selangor, malaysia: penerbitbangi, selangor, malaysia: penerbit university kebangsaan; 1987. p. 35–41, 60–79, 110–5. 2. garna jk. budaya sunda: melintasi waktu menantang masa depan. bandung: lembaga penelitian unpad dan judistira foundation; 2008. p. 79–96. 3. garna jk. tangtu telu jaro tujuh: kajian struktural masyarakat baduy di banten selatan jawa barat. dissertation. bangi, selangor, malaysia: university kebangsaan; 1988. p. 34–57. 4. jacob t. some problems pertaining to the racial history of the indonesian region [proeftschrift]. utrecht: rijkunversiteit; 1967. p. 128–46. 5. fischer c. south east asia: a social economic and political geography. london: metheun; 1967. p. 238–63. 6. danasasmita s, djatisunda a, djunaedi, u. masyarakat kanakes. bandung: bappeda d.t. i jabar; 1983. p. 10–12, 57–66. 7. ambary hm. catatan singkat kepurbakalaan lama. analisiscatatan singkat kepurbakalaan lama. analisis kebudayaan; 1981. i(1):117–27. 8. iskandar y, erawan m. sejarah budaya jawa barat. bandung: penerbitbandung: penerbit geger sunten; 1990. p. 16–25. 9. berthe l. aines et cadets l’alliance et la hierarchie chez les baduj (java occidentale). garna jk, editor. bandung : fakultas pascasarjanagarna jk, editor. bandung : fakultas pascasarjana unpad; 1989. p. 1–33. 10. martin r, knußman r. anthropologie. handbuch der vergleichendenhandbuch der vergleichenden biologie des menschen. band 1. teil. stuttgart: gustav fischer verlag; 1988. p. 129–60. 11. martin r. lehrburch der antropologie: in sytematischer darstelung: somatologie. 4th ed. jena: verlag von gustav fischer; 1928.omatologie. 4th ed. jena: verlag von gustav fischer; 1928. p. 234–65. ib, ob and ns are procheili. in each population group, the mild procheili is the most represented, the second is the less procheili, and the last is the extreme procheili (very rare) (table 2). table 2. percentage of puil in each population group inner baduy (ib), outer baduy (ob) and nearby living sundanese (ns). classification ib % ob % ns % n = 43 n = 92 n = 135 extreme procheili (30o–49.9o ) mild procheili (50o–69.9o ) less procheili (70o–89.9o) 2.3 69.8 27.9 9.8 71.7 18.5 0.7 51.9 47.4 t o t a l 100.0 100.0 100.0 table 3. testing equal variances of mean values of puil between inner baduy (ib), outer baduy (ob) and nearby living sundanese (ns) ib ob ns n x sd = = = 43 63,70 7,6 92 61,73 9,2 135 67,64 7,7 f (calculated) = 14,75 p < 0,001* (ib – ns) ; (ob – ns) n = sample size; x = mean value; sd = standard deviation; * = very significant 122 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 118-122 12. proffit wr, fields hw. contemporary orthodontics. 4th ed. st. louis: mosby inc; 2000. p. 47–50, 119–22, 145–7, 174–86, 219, 333.33. 13. ferrario vf, sforza c, schmitz jh, ciusa v, colombo a. normal growth and development of lips: a 3-dimensional study from 6 years to adulthood using a geometric model. j anat 2000; 196. p. 415–23: [9 screens]. available from: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=1468077. accessed august 20, 2008. 14. wikipedia craniofacial anthropometry. san diego: wikipedia, the free encyclopedia available from: http://en.wilkipedia.org/w/index. php?title=craniofacial_anthropometry&printable=yes. accessed september 11, 2008. 15. edler r, agarwal p, wertheim d., greenhill d. the use of anthropometric proportion indices in the measurement of facial attractiveness. eur j orthod. 2006 jan 1328(2006):274–81. [8 screens]. available from: http://ajo.oxfordjournals.org/cgi/content/ full/28/3/274; accessed august 20, 2008. 16. brinkman jc, vedder a, hollak c, richfeld l, mehta a, orteu k, et al. three-dimensional face shape in fabry disease. eur j hum genet 2007; 15:535-42: [8 screens]. available from: http://www.google. com/search?hl=en&rlz=1t4adbf_enid242id243&q=journal+of +anthropometry:+lips+profile&start3. accessed august 20, 2008. 17. stimson pg, mertz ca. forensic dentistry. washington dc: crcforensic dentistry. washington dc: crc press; 1997. p. 1–14 18. ardan r. fasiogoniometer (pengembangan instrumen pengukur sudut kemiringan muka terhadap fhp). kongres nasional dokter gigi 1992; semarang: universitas diponegoro; 1992. 19. soelarko rm. beberapa pengukuran cephalometrik pada tengkorak-beberapa pengukuran cephalometrik pada tengkoraktengkorak indonesia sebagai dasar bagi norma-norma prothetik bangsa indonesia. bandung: angkasa; 1979. p. 133–64 20. soemardi r. frekuensi anomali pada anak-anak sekolah rakjat di jogjakarta. jakarta: balai pustaka; 1957. p. 21–5. 217 subject index volume 42 9% hydrofluoric acid, 86 aa serotype b crude toxin, 41 acrylic resin, 94 adhesion, 189 aesthetic, 99 component, 204 aggressive periodontitis, 118, 185 alginates, 126, 137 all ceramic crown, 46 ankylosis prevention, 25 anterior teeth, 99 tongue thrust swallow (atts), 65 aphthous stomatitis, 6 apoptosis, 41 artemisia vulgaris l., 37 attrition, 194 australian refined--drg, 210 autoimmune, 159 biocompatibility, 94 ca-bentonite, 114 calprotectin, 185 mrna, 130 candida albicans, 60 carcinoma in-situ, 70 cariogenic bacteria, 199 carving wax, 114 cast post, 46 cell culture, 94 cementifying fibroma, 164 child, 55 chitosan, 15 chronic gingivitis, 6 cigarette, 90 ciprofloxacin, 109 cleaning methods, 147 clindamycin, 118 color stability, 123 composite resin, 123 crohn’s disease, 55 c-shaped orifices, 12 deformation recovery, 126 dental health, 1 operating microscope, 12 implant, 175 caries, 189 cost analysis, 210 health component, 204 radiography, 175 denture, 60 diagnosis, 55 related groups (drg’s) and casemix, 210 disinfectant, 94 dysplasia, 70 endorestoration, 99 epulis, 172 esthetic rehabilitation, 46 etching, 86 excision, 172 eythema multiforme, 159 fibrous epulis, 172 finite element method (femm), 179 fixed bridge, 134 genetic regulation, 141 gingival crevicular fluids, 185 glucosyltransferase, 199 gsh, 41 hardness, 114 hollow obturator, 76 hybrid prosthesis, 76 hypersensitivity, 159 igy, 189 immersion durations, 123 immunoglobulin-g, 118 impacted molar, 172 ina-drg’s, 210 ina-dental cost analysis, 210 index of orthodontic treatment need, 204 inflammation, 82 initial setting time, 137 kayu sugi, 21 laser, 147 major ulcer, 109 management, 55 mandibular resection, 76 measurement, 1 melting point, 114 metronidazole, 109 minocycline, 41 msx1, 141 mtt assay, 15 mutation, 70 nano filler composite, 86 neuroimmunobiological approach, 6 n-hexane: ethyl acetate fraction, 37 occlusal disorder, 31 radiography, 175 splint, 31 oral cancer, 104 mucosa, 90 218 pre-cancer, 104 squamous cells carcinoma, 37 orthodontic treatment, 6, 25 ossifying fibroma, 164 over closure, 194 p53, 70 pain, 82 panoramic radiography, 175 pathogenetic mechanisms, 141 patient acceptance, 65 awareness, 65 comfortable, 65 cooperation, 65 retention, 50 safety, 50 pax9, 141 pericapical radiography, 175 periodontitis, 130 pit and fissure, 147 plaque, 134 formation, 21 polimerization, 94 poor oral hygiene, 60 porcelain, 86 post preparation design, 179 prognostic, 104 prostaglandin-e2, 82 recurrent aphthous stomatitis, 109 replantation protocol, 25 room temperature, 137 root fracture resistance, 179 shear bond strength, 86 smokers, 90 soft drink, 123 squamous cell carcinoma, 70 storage time, 126 streptococcus mutans, 189, 199 stress distribution, 179 surface proteins, 189 surgical margin, 104 temporomandibular joint, 31 thermal expansion, 114 thrush, 60 toluidine blue, 90 tooth paste, 21 agenesis, 141 toxicity, 15 transformation method, 1 tumor markers, 104 tumor's behaviors, 164 type2 diabetes mellitus, 130 ultrasonic tips, 12 watermelon frost, 82 xylitol, 134 219 author's index volume 42 agus, peter, 104 amtha, rahmi, 55 apriasari, maharani laillyza, 159 ariani, maretaningtias dwi, 15 arundina, ira, 37 astoeti, tri erri, 50 bahar, armasastra, 147 dahlan, agus, 31 dennis, 82 djulaeha, eha, 194 fithrony, hamim, 134 g. subrata, 179 gani, basri a, 189 hamid, thalca, 204 hapsoro, adi, 1 hasan, alizatul khairani, 123 hendrijatini, nike, 94 irnawati, dyah, 137 ismiyatin, kun, 86 kamadjaja, david b, 164 krismariono, agung, 118 leosari, yanti, 90 lesmana, magdalena, 65 lunardhi, cecilia gj, 46 m. jusri, 109 nonong, yetty herdiati h, 199 nugraeni, yuli, 25 nurdiana, 60 peeters, harry huiz, 12 prahasanti, chiquita, 6 rachman, arif, 76 rahayu, yani corvianindya, 141 rivany, ronnie, 210 sari, desi sandra, 185 setiawati, ernie maduratna, 41 sumarta, ni putu mira, 172 sunarintyas, siti, 126 syafriadi, mei, 70 syaify, ahmad, 130 widjijono, 114 widowati w, 21 yunus, barunawaty, 175 zubaidah, nanik, 99 thanks to editors in duty of dental journal (majalah kedokteran gigi) volume 42 number 1 janury–march 2009: 1. prof. nairn hutchinson fulton wilson, msc. ph.d.,fds. (conservative dentistry university of guy’s dental school, london) 2. prof. w.j. spitzer, dmd., md. (head department of cranio & oral maxillofacial surgery hamburg university, germany) 3. prof. edward c. combe. m.sc. ph.d. d.d.sc. (biomaterial – minnesota university, u.s.a) 4. sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) 5. endanus harijanto, drg., m.kes. (dental material – airlangga university 6. dr. peter agus, drg., sp.bm. (oral maxillofacial surgery airlangga university) volume 42 number 2 april–june 2009: 1. prof. madya. h. ab. rani samsudin d.d.s., fdsrc, am. (oral and maxillofacial surgery university science malaysia, malaysia) 2. prof. widowati witjaksono, dds, ph.d. (kulliyah of dentistry, international islamic university malaysia) 3. prof. taizo hamada, d.d.s., ph.d. (prosthodontic university of hiroshima, japan) 4. sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) 5. prof. dr. trijoenadi widodo, drg., ms., sp.kg. (conservative dentistry – airlangga university) 6. dr. theresia indah budhy, drg., m.kes. (oral biology – airlangga university) 7. hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) volume 42 number 3 july–september 2009: 1. prof. yukio kato, d.d.s., ph.d. (oral bio chemistry-university of hiroshima, japan) 2. prof. kozai katsuyuki, d.d.s., ph.d. (pediatric – university of hiroshima, japan) 3. prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands) 4. r. helal soekartono, drg., m.kes. (dental material – airlangga university) 5. sudarjani gunawan, drg., ms., sp.kg. (conservative dentistry – airlangga university) 6. dr. retno indrawati, drg., msi. (oral biology – airlangga university) 7. dr. ernie maduratna setiawatie, drg., m.kes., sp.perio. (periodontic – airlangga university) volume 42 number 4 october–december 2009: 1. endrajana, drg., ms., sp.bm (oral & maxillofacial surgery airlangga university) 2. dr. retno indrawati, drg., msi (oral biology – airlangga university) 3. hendrik setiabudi, drg., m.kes. (oral biology – airlangga university) section should include the conclusion of the reported work and suggestion for further studies if necessary.  acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. these notes to authors dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections:  title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces).  name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed.  abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract.  introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future.  materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method.  result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used.  d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. issn 1978 3728volume 46 number 2 june 2013 editorial board of dental journal (majalah kedokteran gigi) sk: 166/h3.1.2/kd/2013 january 2nd– december 31st, 2013 patron: dean of faculty of dental medicine universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 46 number 2 june 2013: prof. dr. pinandi sri pudyani, drg., s.u., sp.ort (orthodontics – universitas gadjah mada) prof. dr. mandojo rukmo, drg., msc., sp.kg(k) (conservative dentistry – universitas airlangga) prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga) dr. ib. narmada, drg., sp.ort(k) (orthodontics – universitas airlangga) kus harijanti, drg., ms., sp.pm (oral medicine – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from www.fotolia.com invoice number: 2051 62250-204 225738 contents page printed by: airlangga university press. (165/11.13/aup-b5e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 46 number 2 june 2013 issn 1978 3728 1. enamel defect of primary dentition in sga children in relation to onset time of intrauterine growth disturbance willyanti soewondo sjarif ............................................................................................................. 55–60 2. gambaran densitas kamar pulpa gigi sulung menggunakan cone beam ct-3d (description of pulp chamber density in deciduous teeth using cone beam ct-3d) herdiyati y, epsilawati l, oscandar f dan nurianingsih r ....................................................... 61–64 3. a study of extraction and characterization of alginates obtained from brown macroalgae sargassum duplicatum and sargassum crassifolium from indonesia decky j. indrani and emil budianto .............................................................................................. 65–70 4. effectiveness of various sterilization methods of contaminated post-fitted molar band anggia tridianti, krisnawati and nia ayu ismaniati ................................................................... 71–74 5. peroxide alkaline for cleansing the baby bottle nipple to prevent oral thrush relaps maharani laillyza apriasari .......................................................................................................... 75–79 6. treatment of non-vital primary molar using lesion sterilization and tissue repair (lstr 3mixmp) tania saskianti, udijanto tedjosasongko and irmawati ............................................................. 80–84 7. new concept in allergy: non-allergic rats becomes allergic after induced by porphyromonas gingivalis lipopolysaccharide haryono utomo ................................................................................................................................ 85–91 8. garis estetik menurut ricketts pada mahasiswa fakultas kedokteran gigi universitas airlangga (ricketts esthetic line of dental student of universitas airlangga) nadiya fitriyani, i.g.a.wahju ardani dan elly rusdiana .......................................................... 92–96 9. dental student’s perception to aesthetic component of iotn and demand for orthodontic treatment wees kaolinni, thalca hamid and ervina r. winoto .................................................................. 97–100 10. compressive strength resin komposit hybrid post curing dengan light emitting diode menggunakan tiga ukuran lightbox yang berbeda (compressive strength of hybrid composites resin with post curing light emitting diode using three different sizes of lightbox) mirza aryanto, milly armilia dan dudi aripin ............................................................................ 101–106 11. identifikasi bite marks dengan ekstraksi dna metode chelex (bite marks identification with chelex methods in dna extraction) imelda kristina sutrisno, ira arundina dan agung sosiawan ................................................... 107–112 guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms 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done. iii. contents in literature reviews the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for electronic publications: 1. morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan–mar; 1(1): [24 screens]. available from: url: http://www.cdc.gov/ ncidoc/eid/eid.htm. accessed december 25, 1999. 2. yu f. management of thumbs duplication. emerg infect dis (on line) http://www.cdc.gov/ncidod/eid/eid.html. 1997. 3. surgery, edward g. media scientific, producer. 4th ed. san diego: media scientific, 1998. p. 4 citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. 76 vol. 43. no. 2 june 2010 early removal of odontoma resulting in spontaneous eruption of the impacted teeth achmad harijadi department of oral and maxillofacial surgery faculty of dentistry, airlangga university surabaya indonesia abstract background: compound odontomas in the anterior maxilla during mixed dentition frequently cause obstruction to the eruption pathway of permanent upper anterior teeth. removal of the odontomas may or may not lead to spontaneous eruption of the impacted teeth depending on the age when the surgery is performed, the size of the lesion, and the stage of tooth development of the involved teeth. purpose: this paper attempts to emphasize the importance of early removal of compound odontoma to enable spontaneous eruption of the affected teeth. case: a case of odontoma in the anterior maxilla causing failure of eruption and delayed root formation of upper right permanent lateral incisor and canine in a 10-year-old male patient is presented. case management: the odontoma was surgically removed under general anesthesia and histology result confirmed the diagnosis of compound odontoma. a three-year post surgical follow up showed spontaneous eruption as well as continued root formation of the two affected teeth. conclusion: removal of odontoma may lead to spontaneous eruption of the affected teeth if their root development are not completed. key words: odontoma, delayed root formation, spontaneous eruption abstrak latar belakang: compound odontoma pada daerah anterior maksila pada masa geligi pergantian sering menyebabkan hambatan erupsi pada gigi permanen penggantinya. kemungkinan terjadinya erupsi spontan gigi permanen yang impaksi setelah pengambilan odontoma tergantung dari: umur penderita pada saat dilakukan operasi, ukuran lesi odontoma dan tahapan perkembangan gigi permanen yang terlibat. tujuan: laporan kasus ini ingin menekankan pentingnya pengambilan compound odontoma sedini mungkin untuk memberi kesempatan tumbuhnya gigi permanen yang terlibat dan erupsi secara spontan. kasus: ditampilkan sebuah kasus compound odontoma pada regio maksila anterior menyebabkan gagalnya erupsi dan terlambatnya pembentukan akar gigi insisif kedua dan kaninus permanent rahang atas kanan pada penderita anak laki-laki berumur 10 tahun. tatalaksana kasus: dilakukan eksisi odontoma dengan pembiusan umum dan pemeriksaan histopatologi menunjukkan lesi tersebut adalah compound odontoma. dalam kurun waktu 3 tahun pasca pembedahan kedua gigi permanen yang impaksi dapat erupsi sempurna secara spontan disertai dengan berlanjutnya proses pembentukan akar. kesimpulan: pengambilan odontoma dapat menyebabkan erupsi secara spontan gigi permanen yang impaksi dengan syarat proses pembentukan akar gigi yang bersangkutan belum selesai. kata kunci: odontoma, delayed root formation, erupsi spontan correspondence: achmad harijadi, c/o: departemen bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: hariadioesodo@yahoo.com case report 77harijadi: early removal of odontoma resulting introduction odontoma is a pathologic entity known as hamartoma of odontogenic origin, malformation tumor, representing 22% of the odontogenic tumors.1 odontomas are the most common benign odontogenic tumors of epithelial and mesenchymal origin. they are composed of mature enamel, dentin, and pulp and may be termed as "compound" or "complex", depending on the extent of morphodifferentiation or on their resemblance to normal teeth.2 compound odontomas are usually located in the anterior part of the mouth, either over the crowns of unerupted teeth or between the roots of erupted ones. radiographically, the lesions are usually unilocular, containing multiple radiopaque structures that resemble miniature of teeth which may vary from as few as 2 to 3 miniature tooth-like structures or as many as 20 to 30.2 complex odontomas, on the other hand, are commonly found in the posterior part of the mandible over impacted posterior teeth and can attain sizes up to severals centimeters.3 they appear as a solid unilocular, radiopaque mass exhibiting some nodularity and are surrounded by a thin radiolucent zone and a distinct line of cortication, and there is no individual toothlike structures.1,3 compound odotomas are clinically presented as an asymptomatic, slow growing lesion, and in many cases they do not cause any swelling or facial asymmetry. therefore, odontomas may grow undetected until diagnostic radiographs are made in cases where one or few anterior teeth fail to erupt. surgical exposure and elimination of mechanical obstruction is frequently the treatment of choice and spontaneous eruption can then be expected.4 a compound odontoma in the anterior part of maxilla which has caused obstacle to normal eruption of two anterior permanent teeth is presented. surgical excision of the tumor and the post operative follow-ups showed that there was no tumor recurrence and, in addition, it has gradually led to spontaneous eruption of the affected permanent teeth. case a 10 years old male patient came to a private clinic referred by his dentist for follow up treatment. the patient's parents complained that the unerupted upper right permanent lateral incisor and canine which had caused unacceptable appearance. clinical examination showed that there was no facial asymmetry. intra oral examination showed mixed dentition with very good oral hygiene, the upper right permanent lateral incisor and canine were missing with the space being occupied by upper right primary lateral incisor and canine (figure1). panoramic x-ray showed radiopaque lesion with tooth-like structure appearance displacing and causing impaction of the right upper permanent lateral incisor and canine (figure 2). based on clinical and radiographic presentation the lesion was diagnosed as a compound odontoma. case management the case was treated with extraction of the upper lateral primary lateral incisor and canine followed by surgical excision of the odontoma under general anesthesia. the lesion was found relatively large and attached to the adjacent teeth therefore it was cut into few pieces to facilitate its removal while minimizing the risk of damage to the adjacent structures. upon excision of the lesion it was found many tooth-like structures with various forms and sizes (figure 3). histologic examination of the specimen showed that the lesion contained calcified matrix of bone tissue without the presence of odontogenic cells, no evidence of malignancy was found (figure 4). the clinical appearance and histologic result of the lesion are consistent with those of compound odontoma. five weeks after the surgery the patient came back without any complaint. the wound had healed completely. figure �. intraoral view showing the non-exfoliating primary upper right lateral incisor and canine. figure ��. panoramic radiograph showing the intact primary upper lateral incisor and canine and the compound odontoma obstructing the eruption pathway of permanent upper lateral incisor and canine. 78 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 76-80 panoramic radiograph showed that the upper right permanent lateral incisor had slightly moved in disto-incisal direction, and the upper right canine in mesial direction, both towards the space previously occupied by the lesion (figure 5). figure �. microscopic view of the specimen showing calcified matrix of bone tissue without the presence of odontogenic cells. figure 5. panoramic radiograph six weeks after surgery showing the developing upper lateral incisor and canine start moving down towards the normal eruption pathway. fifteen months post operative review showed that the upper right permanent lateral incisor was clinically almost fully erupted while the canine was not visible. panoramic radiograph showed that the upper right permanent lateral incisor was almost fully erupted with the root undergoing apex formation. the upper right permanent canine was on its way of eruption with the root being longer, compared with that prior to surgery, indicating an active root formation (figure 6). figure 6. clinical photograph fifteen months after surgery showing the erupting permanent upper lateral incisor (left); panoramic radiograph fifteen months after surgery showing the fully erupted upper right permanent lateral incisor and canine on its erupting way (right). figure 7. clinical photograph 3 years after surgery showing the fully erupted and well aligned permanent upper lateral incisor and canine (left); panoramic radiograph showing the fully erupted upper right permanent lateral incisor and canine which have not achieved complete root formation compared to their counterpart teeth (right). figure ��. the specimen taken out from the operation site showing numerous miniature tooth-like structures indicating compound type of odontoma, together with 79harijadi: early removal of odontoma resulting the final review was made three years after the surgery. clinically, both upper right permanent lateral incisor and canine were fully erupted and in good alignment. panoramic radiograph showed that the roots of the upper right permanent lateral incisor and canine were much longer and almost attained their complete root formation, but their apices were not closed yet (figure 7). discussion odontoma are the most frequent benign odontogenic tumor in the oral pathology. they are generally asymptomatic and constitute casual findings in the course of routine radiological studies, particularly in the second and third decades of life.5 there are two types of odontomas: complex and compound odontomas, the latter being twice as frequent as the former. compound odontomas show a predilection for the anterior sector of maxilla, while complex odontomas are typically found in the posterior mandibular region.2,6 this is in accordance with the odontoma in this case as it is found in the anterior maxilla and appeared as small tooth-like structures. therefore it is classified as compound odontoma. in this presented case the reason for which the patient's parents sought dental consult was the retention of two upper anterior primary teeth in addition to delayed eruption of two upper anterior permanent teeth, while the contralateral teeth had already well erupted. the cause of the problem could be revealed only after panoramic radiograph had been made which showed an odontoma in the right anterior maxilla causing displacement of the two unerupted anterior permanent teeth. this condition is appropriate with the previous studies, showing delayed tooth eruption1,7 the upper left permanent lateral incisor exhibited normal tooth development whereas the upper left canine was showed to have faster tooth development and eruption time. normally, upper canines should appear intra orally between 11 and 13 years of age and complete their roots formation between 14 and 15 years of age.8-10 however, the upper left canine in this case had fully erupted clinically, reached occlusion and attained three quarter of its root length at the age of ten, which is one to three years ahead of its normal chronology. interestingly and surprisingly, the two unerupted anterior maxillary teeth on the right side showed very much delayed root formation compared with those of the contralateral teeth. it seems logic to say that there should be some relation between the failure of tooth eruption and delayed tooth development in this presented case. one retrospective study seems to support this relation as the result showed that impaction of wisdom teeth can delay the root development.11 although the exact pathophysiology of this phenomenon is not understood yet, the difference in growth pattern, tooth development and eruption time of permanent dentition are common findings among different individuals and even in the same individuals. the interval from crown completion and the beginning of eruption until the tooth is in full occlusion is approximately 5 years for permanent teeth.6,9 the delayed root formation of the impacted upper right permanent lateral incisor and canine is considered advantageous in this case as it is believed to have important role in the spontaneous eruption of those affected teeth following removal of the odontoma. although the exact mechanism has not been clearly understood, the role of incomplete root formation in spontaneous eruption of impacted teeth has been well documented. in a large clinical retrospective study of 140 intruded teeth, it was concluded that in the case of immature root development the best treatment would be observation with anticipation of spontaneous re-eruption.12 in a retrospective study on the eruption of teeth associated with dentigerous cyst treated with marsupialization, hyomoto et al.13 found that eruption potential was closely related to root formation, so that teeth with incomplete root formation had good potential to erupt, whereas those with complete root formation hadless potential to erupt. the spontaneous eruption of the upper right canine in this case into its final position within 3 years duration without any intervention is considered unusual based on the fact that it has been severely displaced away from its correct position by the presence of large odontoma. this phenomenon is contradictory to the result of a study by ashkenazi et al.14 in which spontaneous eruption of the impacted teeth were significantly impeded if there is higher distraction of the apex of the impacted tooth relative to its estimated correct position and if the obstacle are in the form of tuberculated supernumeraries and odontomas. in their study most of the impacted teeth obstructed by odontomas had to be pulled out with orthodontic appliances into their final position. the presence of compound odontoma during mixed dentition in the above case has created obstacle to the eruption pathway of two permanent anterior maxillary teeth and appeared to have seized their tooth development, i.e. root formation. after removal of the odontoma, the eruption process as well as the root formation of those teeth were continued until they came into occlusion and functioned normally. it is concluded that removal of odontoma may lead to spontaneous eruption of the affected teeth if their root development are not completed yet. references 1. shafer wg, hine mk, levy bm. a textbook of oral pathology. 5th ed. philadelphia: wb saunders co; 2005. p. 305–17. 2. chong hl, gyeong ju p. complex and compound odontomas are clinico-pathological entities, basic and applaied pathology. departement of oral and maxillofacial pathology, dental college, dankook university, chungnam korea. 2008. p. 30–3. 3. sapp jp, eversole lr, wysocki gp. cotemporary oral and maxillofacial pathology. 2nd ed. st. louis: mosby; 2004. p. 156–8. 4. sanadettin d, mustafa g, özkan m.compound odontoma associated with maxillary impacted permanent central incisor tooth: a case report. the internet journal of dental science 2007; 5(2). 5. sciubba jj, fantasia je, khan lb. tumors and cysts of the jaw. washington: armed forces institute of pathology; 2001. p. 117–20. 80 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 76-80 6. steven db. compound and complex odontoma. usa: emory university school of dentistry, atlanta, ga; 2005. p. 501–5. 7. barnes l, evenson jw, reicart p, sidransky d. orld health organization classification of tumors. pathology and genetics of head and neck tumors. lyon: iarc press; 2005. p. 310–1. 8. avery jk, steele pf. oral development and histology. 3rd ed. new york: thieme med. publ; 2002. p. 108–22. 9. mcdonald r, avery dr, dean ja. dentistry for the child and adolescence. 8th ed. st. louis: mosby; 2004. p. 175–9. 10. pinkham jr, casamassimo p, fields hu, metique dj, maia a. pediatric dentistry. infancy though adolescent. 4th ed. st louis missouri: elsevier saunders; 2005. p. 190–3. 11. reinhard ef, ulbricht c, von maydell ab. the influence of wisdom tooth impaction on root formation. ann anat 2003; 185(5): 481–92. 12. andreasen jo, andreasen fm, andersson l. textbook and color atlas of traumatic injuries to the teeth. 4th ed. victoria: willey-blackwell; 2007. p. 432–3. 13. hyomoto, shear m, speight p. cysts of the oral and maxillofacial region. 4th ed. oxford, uk: blackwell munksgaard; 2007. p. 75. 14. ashkenazi, m, greenberg bp, chodik g, rakocz m. post operative prognosis of unerupted teeth after removal of supernumerary teeth or odontomas. am j orthod dentofac orthop 2007; 131(5): 614–9. mkg vol 39 no 1 jan 2006 isi.pmd 32 the effect of caffeine on osteoblast proliferation after tooth extraction in wistar rats budi yuwono department of oral surgery faculty of dentistry jember university jember indonesia abstract caffeine is the most well-known substance which consumed by most people daily. behind its popularity as favorable drinks and food, this substance also known can inhibit the post extraction wound healing by decreasing the proliferation of osteoblast cells through the increase of intracellular cyclic adenosine mono phosphate (camp). the objective of this study was done to observe the effect of caffeine intake toward the number of osteoblast cells during the wound healing of post dental extraction in wistar’s rats. this study was an experimental laboratory research and the post test-only control group design was used for the statistical evaluation. the samples used were 24 healthy 3 months old male wistar’s rats, with approximately 200 grams of body weight and devided into 4 groups. three groups were taken and represented as a treated group (p) and the rest of one group was used as a control group (ko). caffeine diet with a dosage of 3.78 mg/100 ml grams of body weight/cc was given for 7 days in group p1, p2 for 14 days, and 21 days in group p3 and the diet was given orally using an oral sonde. teeth extractions of the right first molar in the lower jaw were done in all groups according to the interval time had been scheduled. seven days of post-extraction time was waiting in all groups before the sample being decapitated for further histological examination in the post extracted sites. a hematoxillin and eosin staining was used and the number of osteoblast cells were counted under light microscopy with 400 times magnification. one-way anova and least significant difference (lsd) test were used for the statistical evaluation. the result of the study shown a significant decrease of the number of osteoblast cells in caffeine consumed group of 7, 14, and 21 days observed (p < 0.05). this study conclude that the duration time of caffeine consumed had been interfered significantly with the osteoblast cell proliferation during the wound healing after teeth extractions in wistar’s rats. key words: caffeine, osteoblast, healing correspondence: budi yuwono, c/o: bagian ilmu bedah mulut, fakultas kedokteran gigi universitas jember. jln. kalimantan i no. 58 jember 68121, indonesia. introduction caffeine is the most well-known substance which is consumed by approximately 80% people in the world. we find this substance is in coffee, tea, chocolate, coke, ice cream, yogurt, and orange juice. caffeine is widely known as a nerve stimulant and it is assumed to enhance intelligence.1 although caffeine is popular as favorable drinks and food that consumed by children and adults daily, caffeine can inhibit wound healing process. based on our experience, we found many patients with decreasing of wound healing after dental extraction. this phenomenon urged us to study further the role of caffeine in wound healing process. kamagata et al.2 found that caffeine inhibit osteoblast proliferation (in vitro) through the increasing of cyclic adenine mono phosphate (camp) as the cause of intracellular phosphodiesterase inhibition. furthermore, it was reported that caffeine have a role in bone metabolism. wound healing process in lacerated tissue is initially responded by an inflammation. the first change is in the vascular stage by vasodilatating, extravasating and exudating the wounded area. the release of prostaglandin e2 (pge2) as the inflammation mediator in wound area stimulate production of colagenase which trigger degradation of collagen and other potent factor for bone resorption, and also increase osteoclastic activity.3 kamagata et al.2 reported that the increased pge2 alone could inhibit osteoblast proliferation through intracellular camp increment (in vitro). the released of pge2 in wound area that accompanied by certain doses of caffeine intake, could inhibit wound healing. kamagata et al.2 had proved that the combination effect of caffeine and pge2 could inhibit osteoblast proliferation significantly (in vitro). more over, it was reported that caffeine intake could increase pge2 production and 0.1mm/ml caffeine could enhance pge2 production. based on the explanation that caffeine intake at certain doses and pge2 can inhibit osteoblast proliferation and increase osteoclast activity in bone resorption and remodeling mechanism, it is assumed that caffeine is influential towards the osteoblast number of post tooth extraction. 33yuwono: the effect of caffeine the aim of this study is to evaluate the effect of caffeine on the number of osteoblasts after tooth extraction wound healing. hopefully this study may develop the post tooth extraction wound healing theory, give information about substances which can inhibit the wound healing process and also give information to patients who have high risk to osteoporosis, diabetes mellitus, old age, stress and pmn dysfunction caused by excessive caffein intake. materials and methods this study was an experimental laboratory research with a random post test-only-control-group-design. the samples were 24 healthy 3-month-old male wistar’s rat, with ± 200 grams of body weight and divided into 4 groups (6 rats each). one group was control group with no caffeine treated. three groups (p1, p2, and p3) were treated with 3.78 mg/100gr of body weight/cc per oral caffeine (merc, germany). each group was placed in a different cage and some food ad libitum were given. in control group: the right molar in the lower jaw was extracted and was let live for 7 days. caffeine was given for 7 days in group p1, p2 for 14 days and 21 days in group 3, then the right molar in the lower jaw was extracted and was let live for 7 days. to obtain the right lower jaw, all samples were mortified with ether anesthetic. each right lower jaw was soaked to 10% formaldeyde to prepare an object/preparat of right lower jaw bone with the routine procedure, decalcification, dehydration and embedding. a sagittal section was prepared showing post extraction dental socket. the sample was 7 section micrometer, painted and colored with a hematoxillin & eosin routine dye, then with a light microscope of 400x enlargement, the osteoblast cells were counted in the post extraction socket. the data was analyzed with one-way anova and least significant difference (lsd) tests. the research was conducted at the biochemical laboratory, tropical disease center-airlangga university and at the pathologic laboratory of dr.soetomo general hospital in may until july 2005. results the average number of osteoblasts in post-extraction wound healing could be seen in figure 1. there was an average difference of the number of osteoblast in p1 (7 days), p2 (14 days) and p3 (21 days) as well as in the control group. to determined the differences of the osteoblast numbers in 4 groups, a statistical test one-way anova was performed. the result have shown a significant difference in group of control, p1 , p2, and p3 (p < 0.05). to find out the difference of osteoblas numbers among groups, lsd test with margin of error (α = 0.05) was performed. the result showed a significant decrease of the number of osteoblasts in all of the group (p < 0.05) as seen on table 1. table 1. lsd analysis of inter group sample with degree of error 0.05 control 7 days caffeine 14 days caffeine 21 days caffeine control 7 days caffeine 14 days caffeine 21 days caffeine p = 0.001* p = 0.001* p = 0.038* p = 0.001* p = 0.001* p = 0.011* explanation: * = significant with α = 0.05 discussion based on many theories presented previously, it revealed that caffeine intake could decrease osteoblast cells activity due to the increased of pge2 which was stimulated by the enhancement of intracellular camp. caffeine will be absorbed by intestines, distributed to all body parts including the nerve-circuit. the nerve stimulant mechanism by caffeine was still unclear, but it was found that the methyl compound of caffeine substructure detached itself when interacted with cell membrane. the methyl compound diffused between two membrane layers, adding methyl component to its fat, resulting a change in surface tension. the decreased of surface tension made the membrane damped by water and soluble substance easily, until the ions could easy to diffuse and push ion formation effectively. it caused a more active intersynapse exitation. the active exitation is the electrical simulator for the release of central neurotransmitter like gamma amino butirate acid (gaba) or choline acethyl which is make strong post synaptic respond to stimulate the central nervous system actively.4 the stimulation of the central nervous system trigger the cell surface to release a2 phospholipase enzyme. the a2 phospholipase pushed ahead the release of arachidonic acid from the lipid membrane and converse it to pge2 cyclic derivative through the work of cyclo-oxygenase. the pge2 give a certain effect to the body i.e. inhibiting electrolyte resorption on proximal tubulus and make the entering water was not re-absorbed, thus causing a constant excretion quantity, yielding a tendency to diuresis.5 54.4 47.33 43.33 38.33 0 10 20 30 40 50 60 o st eo b la st co u nt control caff. 7 days caff. 14 days caff. 21 days figure 1. block diagram of the average osteoblast number in the control group, 7 days caffeine, 14 days caffeine and 21 days caffeine. 34 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 32–34 lerner and mellstrom6 reported that caffeine has a role on bone resorption in relation to calsium. caffeine could raise the spontaneous detachment of calsium mineral from neonatal mouse calvarian bone (in vitro). a certain dose of caffeine could trigger the raise of calsium detachment from bone, which could stimulate bone resorption action through camp cyclase adenilate system. the increased of pge2 not only bring a diuretic effect, it also directly influence osteoclastic differentiation through a raising activity of mononucleat precursor to become mature osteoclast. beside this activity, pge2 also increase mature osteoclast activity to re-absorb bone and to form extracellular matrix.7 this effect was related to the increased of camp inside the bone through activated cyclase adenilate that increased the number, activity and mobilization of osteoclast.8 high pge2 level also decrease osteoblast differentiation, by specific receptor which is raise phosphodiesterase enzyme activity in osteoblast. phosphodiesterase enzyme stimulated camp degradation, lowering camp content, resulting a decrease in osteoblast activity.7 basically, pge2 influenced bone changes to become more effective. the increase of pge2 level by caffeine that influenced the escalation of osteoclasts activity will stimulate bone resorption. this statement was in accord with kamagata et al.,2 that 0.1mm/ml caffeine could already increase pge2 production that inhibit osteoblast activity. pge2 also activated osteoclast which was crucial in bone resorption. beside its popularity as favorable drinks and food, caffeine was also assumed to inhibit wound healing process. caffeine inhibited osteoblast cell proliferation (in vitro), through the increased level of camp caused by intracellular phosphodiesterase.2 the increased camp level inhibited osteoblast proliferation. moreover, it was reported that caffeine influence bone metabolism. wound healing process in lacerated tissue is initially responded by an inflammation. the first change is in the vascular stage by vasodilatating, extravasating and exudating the wounded area. the release of prostaglandin e2 as the inflammation mediator in wound area, could stimulate the production of collagenase enzyme production which could trigger collagen dehydration, other potent factor for bone resorption and could increase osteoclastic activity.3 kamagata et al.2 reported that the increased pge2 alone inhibit osteoblast proliferation through intracellular camp increment (in vitro). the released pge2 in wound area accompanied by certain doses of caffeine intake could inhibit wound healing. a study done by kamagata et al.2 had proved that the combination effect of caffeine and pge2 could inhibit osteoblast proliferation (in vitro). significantly moreover, it was reported that caffeine intake could increase pge 2 and 0.1 mm/ml caffeine could already increase pge2 production. grounded on the result of this study (in vitro), it has been proven that caffeine can decline significantly the number of osteoblast cell (p < 0.05) on 3 groups of 7, 14, and 21 days caffeine diet. the longer the caffeine intake take place, the more decreased of osteoblast cell numbers. this fact strengthen the assumption that caffeine both in vitro and in vivo is involved in bone mechanism, resorption, apposition on wound healing process. this research demonstrated that chronic caffeine intake has a risk towards the decline of osteoblast cells. the duration time of caffeine consumed had been interfered significantly with the osteoblast cell proliferation during the wound healing after teeth extractions in wistar’s rats. references 1. fen s. kafein sebuah penenang semu. jakarta: penerbit pustaka utama; 1993. p. 78–9. 2. kamagata y, mitsuhiro c, gina, saltzman mj. combined effects of pge2 and caffeine on alveolar resorption. j periodontology 1999 march; 15:234–38. 3. nakano k, ohishi m, ogawa y, ohba t, kido j. protaglandin e2 inhibit alveolar bone resorption in experimental periodontitis in hamsters. j periodontology 1998 march; 34:108–11. 4. ungaro, feas jkrn, marry gr, casey b. the effect of caffeine and alcohol on hepatic cellular. j medical & health 1997 july; 34:65–9. 5. guyton. textbook of human psychology. london: mosby-year book inc; 1995. p. 711–15. 6. lerner uh, mellstrom d. caffeine has the capacity to stimulate calcium release in organ culture of neonatal mouse calvaria. university of umea, sweden. calcif tissue int; 1992. p. 424-28. 7. schwatz zj, goultschin dd, dean, byan bd. mechanism of alveolar bone destructions of periodontitis. j endodontic 1997 december; 22:677–80. 8. offenbecher s, heasman, collins jg. modulation of host pge2 secretions as a determinant of periodontal disease expression. j periodontology 1993 august; 64:432–44. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 51 no 4 okt-des 2018.indd 189 application of pomegranate (punica granatum linn.) fruit extract for accelerating post tooth extraction wound healing intan nirwana department of dental materials science and technology faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: trauma occurring during tooth extraction can cause complications such as bleeding, infection, fracture and dry socket and constitutes an inflammatory response trigger. pomegranate (punica granatum linn.) extract, which contains large amounts of punicallagin and ellagic acid, possesses various qualities, including; anti-inflammatory, anti-bacterial and anti-oxidant. pomegranate extract can inhibit proinflammatory cytokine production, while also suppressing inflammation response thereby accelerating wound healing. purpose: this study aimed to analyze the effect of pomegranate extract application to the tooth extraction wounds of cavia cobaya (c. cobaya) on the expression of fibroblast growth factor-2 (fgf-2) and transforming growth factor β (tgf-β) on the fourth day of the wound-healing process. methods: this study used 12 c. cobaya, divided into two groups, namely; control and treatment. the subjects were anesthetized, before their lower left central incisor was extracted and the entire socket filled with cmc-na 3% in members of the control group and pomegranate extract in those of the treatment group. the twelve c. cobaya were sacrificed on day 4, their lower jaw subsequently being removed and decalcified for approximately 30 days. the mandibula tissue was stained using a immunohistochemical technique. fgf-2 and tgf-β were used to evaluate the healing process in the extracted tooth socket. differences in the expression of fgf-2 and tgf-β were evaluated statistically by means of a t-test. results: this study indicated a significant difference between the control and the treatment groups (p<0.05). the treatment group members whose sockets were filled with pomegranate extract showed high fgf-2 and tgf-β expression. conclusion: this study confirmed that the administration of pomegranate extract to post-extraction tooth wounds of c. cobaya increases the expression of fgf-2 and tgf-β on day 4, thereby accelerating the wound healing process. keywords: fgf-2; post extraction wound; punica granatum linn; tgf-β correspondence: intan nirwana, department of dental materials science and technology, faculty of dental medicine, universitas airlangga. jl. mayjend prof. dr. moestopo 47 surabaya 60123, indonesia. e-mail: intannirwana@ymail.com research report introduction trauma can occur during tooth extraction resulting in complications such as bleeding, infection, fracture and dry socket.1 trauma is one of the trigger factors in the inflammatory response which is a stimulation-precipitated immune mechanism which protects against various threats by maintaining tissue homeostasis.2 periodontal tissue damage caused by tooth extraction also results from inflammation.3 the response is triggered by tissue damage in the body and the condition activates macrophages and cytokine synthesis which involves pro-inflammatory activity, including interleukin-1 (il-1), interleukin-6 (il-6), interleukin-8 (il-8) and tumor necrosis factor α (tnfα).4 it is essential to control inflammation due to its potential negative effects on the surrounding tissue. the healing process generally consists of three stages, namely; inflammation, proliferation and repair. the control of inflammation will enable the next healing stage to occur.5 research into various types of plants regarded as having medicinal properties beneficial to human health has been widely conducted. one such plant is pomegranate (punica granatum linn), almost all parts of which have traditionally been used and are believed to promote medicinal activities.6 pomegranate extract possesses anti-inflammatory, dental journal (majalah kedokteran gigi) 2018 december; 51(4): 189–193 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p189–193 mailto:intannirwana@ymail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p189-193 190nirwana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): anti-bacterial, anti-oxidant, anti-cancer and anti-fungal properties,7 with the result that they have been the subject of a considerable volume of research. the phytochemical and pharmacological content of pomegranate, including punicalagin and ellagic acid, can prevent and treat a wide variety of diseases. ellagic acid promotes anti-inflammatory activity, namely that of reducing il-6 by inhibiting nuclear factor kappa beta (nf-kb).8,9 pomegranates possess several therapeutic properties applied through a variety of mechanisms and several studies have been conducted into their antioxidant, anticancer and anti-inflammatory roles.9 it is anticipated that the application of pomegranate extract to tooth extraction sites will accelerate healing due to its antibacterial, anti-inflammatory and anti-oxidant properties. at the healing stage, fibroblast growth factor-2 (fgf-2) and transforming growth factor-β (tgf-β) will stimulate fibroblasts to synthesize type i collagen which is a marker of the healing process. increased growth factors such as fgf-2, which has the effect of tissue regeneration, play an important role in the wound healing process. tgf-β proteins represent cytokines that control cellular processes, including: cell proliferation, differentiation, adhesion, angiogenesis, apoptosis and immunity maintenance.10,11 tgf-β is a multifunctional cytokine which acts as a key regulator of ecm formation and remodeling. increased tgfβ-1 present in injury sites during the healing process promotes signal tissue regeneration.12 the purpose of this study was to analyze the stimulating effect of pomegranate extract on cavia cobaya (c. cobaya) tooth extraction wounds by accelerating the healing process through the expression of fgf-2 and tgf-β1 on the fourth day. an increase in the expression of fgf-2 and tgf-β was observed. materials and methods this study involved the use of wistar rats as subjects and received approval from the ethical committee of the faculty of dental medicine, universitas airlangga, confirmed by ethical clearance certificate number: 53/ hrecc.fodm/5/2015. the material employed consisted of standardized pomegranate extract powder, at a concentration of 2.5% 13, and containing 40% ellagic acid (xi’an biof bio-technology co., ltd china.), while the gel base material was 3% sodium carboxy methyl cellulose (cmc-na) (merck). the immunohistochemical staining materials were monoclonal antibodies to fgf-2 (abcam, ab92337) and tgf-β (abcam, ab179695). this study used 12 c. cobaya aged 2.5 months and weighing 150-200 grams as experimental subjects which were acclimatized for a week prior to treatment. the subjects were divided into two groups, namely; the control group (c) administered with 3% cmc-na gel, and the treatment group (t) whose members received pomegranate extract gel. 3% cmc-na gel was produced from three grams of cmc-na powder which was gradually added to 100 ml of water and agitated until homogeneous. 2.5% pomegranate extract gel was produced from 2.5 grams of pomegranate extract powder added to 97.5 grams of 3% cmc-na gel. the c. cobaya were anesthetized using a combination of ketamine hcl and diazepam (1:1 cc with a dose of 1 ml/kg bm administered intramuscularly).14 extraction of the lower incisors of the subjects was performed using a needle holder and sterile elevator in direct motion (to lingual direction). in the treatment group, the resulting tooth sockets of the subjects were filled with 2.5% pomegranate extract gel, while in the case of the control group 0.1 ml of 3% cmc-na gel was inserted into each socket with an insulin syringe. extraction wounds were sutured using non-absorbable threads. the control and treatment group subjects were sacrificed on the fourth day post-treatment, the mandible being extracted following removal of the mandibular angles. the mandibular extracted from the subjects was soaked in buffer formalin for 24 hours, subsequently decalcified with 10% ethylene diamine tetra acetic acid (edta) for approximately 30 days to soften the mandibular bone tissue, thereby enabling it to be disected into small, rectangular pieces. embedding was performed by means of paraffin solution at a temperature of 56-59 ºc. a paraffin block was cut to a thickness of 5 μm before immunohistochemical tissue staining was conducted by using monoclonal anti fgf-2 antibodies to observe fgf-2 expression cells and anti tgf-β monoclonal antibodies to observe tgf-β expression cells. observations with light microscopes and cameras (olympus) were carried out at 400x magnification. the expression of fgf-2 and tgf-β was measured by counting the number of cells (macrophages) expressing fgf-2 and tgf-β. data obtained from the results of the study were analyzed using a t-test to determine the differences in expression of fgf-2 and tgf-β between the control and treatment groups with a significance of 0.05. results fgf-2 expression cells in the control group (cmc-na) and the treatment group (pomegranate extract) are shown in figure 1, while tgf-β expression cells in the control group (cmc-na) and treatment group (pomegranate extract) can be seen in figure 2. the mean and standard deviation of fgf-2 and tgf-β expression is presented in table 1. the difference between control and treatment groups can be seen in table 2. the t-test for the expression of fgf-2 and tgf-β demonstrated significant differences (p<0.05) between the control and the treatment groups. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p189–193 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p189-193 191 nirwana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 189–193 a b figure 1. post-extraction fgf-2 expression on day 4 after. a) in the control group; b) in the treatment group (400x magnification, arrow indicates immunoreactive cells, highlighted in brown). a b figure 2. post-extraction tgf-β expression on day 4. a) in the control group; b) in the treatment group (400x magnification, arrow indicates immunoreactive cells, highlighted in brown). figure 3. fgf-2 and tgf-β expression of control and treatment groups. table 1. the mean and standard deviation of fgf-2 and tgf-β expression in the control and treatment groups ngroup tgf-fgf-2 β sdmeanmean sd 1.2117.671.9414.836control 2.99410.832.13712.176treatment table 2. t-test of fgf-2 and tgf-β expression in the control and treatment groups sigt valueexpressiongroup 0.0006.223fgf-2control treatment tgf-control treatment β 0.0372.401 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p189–193 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p189-193 192nirwana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): discussion nf-kb is a key transcription factor for macrophages and required for the induction of a number of inflammatory genes, including those that encode tnf-α, il-1β, il-6 and cyclooxygenase-2.15 trauma will cause nf-kb translocation to the cell nucleus, an event triggering the transcription process for the formation of various proinflammatory cytokines. the release of pro-inflammatory cytokines tnf-α, il-1β, il-6 and cyclooxygenase-2 induces changes in the tissue referred to as inflammation.16 the application of pomegranate extract high in ellagic acid to post-tooth extraction wounds can inhibit nf-kb activity. such inhibition of nf-kb activation can occur by means of three mechanisms, namely; blocking the incoming stimulating signal (e.g. binding ligand to the receptor) resulting in an imperfect signal effect; interfering with the cytoplasm stage of the nf-kb activation pathway by inhibiting activation of ikb kinase (ikk) or ikb (inhibitory protein kappa b) degradation; and, lastly, inhibiting nuclear nf-kb activity which inhibits translocation into the nucleus and hinders nf-kb-dna binding.17 the obstacle of nfkb translocation into the nucleus causes inflammation to be appropriately regulated in order that the production of fgf-2 and tgf-β by macrophages is increased. it has been proven in this study that an increase in fgf-2 and tgf-β expression occurs in post-extraction wounds with the result that healing is accelerated because fgf-2 and tgf-β stimulate the formation of granulation tissue.18 in addition to fgf-2 and tgf-β, macrophages secrete vascular endothelial growth factor (vegf) which modulates endothelial cell proliferation and causes angiogenesis.19 together with il-10, tgf-β is a strong anti-inflammatory cytokine that actively reduces pro-inflammatory tnfα, il1β and il-2 among other proteins. tgf-β also plays an important role in tissue regeneration, cell proliferation and cell differentiation,20 while fgf-2 is an effective growth factor indicator with the potential to affect tissue repair and regeneration.21 fgf-2 is mainly produced by macrophages on day 2, while active fibroblasts proliferate from day 3.22 tgf-β was released immediately after the trauma in the inflammatory phase and increased in the proliferation phase of the day 3.23 therefore, studies of fgf-2 and tgf-β in post-extraction wounds were carried out on the fourth day after treatment. analysis of the results of this study confirmed that the number of cells expressing fgf-2 and tgf-β was higher in post-extraction wounds after the administering of standardized pomegranate extract containing 40% ellagic acid compared to the control group. this is because ellagic acid possesses anti-inflammatory qualities which reduce inducible nitric oxide synthase (inos), cyclooxygenase (cox-2), tnf-α and il-6 by suppressing nf-kb activation, thereby enabling the effective control of inflammation in cavia cobaya9. this, in turn, not only leads to a decrease in the transcription process of pro-inflammatory cytokines, but also activate genes that produce anti-inflammatory cytokines, namely: il-10, fgf and tgfβ. il-10 inhibits pro-inflammatory cytokines, while fgf stimulates fibroblast cell proliferation. as a result of this stimulation, fibroblast cells secrete tgfβ-1 which affects fibroblast cell proliferation in an autocrine manner and causes increased collagen synthesis which is indicative of a healing process24. this study is supported by other research findings confirming that the ellagic acid content of pomegranate fruit extract can also reduce proinflammatory cytokine activity in the sockets of diabetic mice.13 fgf-2 is mitogenic for several cell types found in wound sites, including fibroblasts, which contribute significantly to the wound healing response. this hypothesis has been corroborated by other research indicating that the application of local fgf-2 stimulates tissue repair.21 previous studies revealed that, after tooth extraction, fgf-2 decreased in untreated diabetic rats compared to those treated with ellagic acid and that a correlation existed between the decrease in fgf-2 expression and wound healing disorder25. fgf-2 expression decreased during wound healing in healing-impaired genetically diabetic subjects compared to control group subjects. the expression of fgf-2 was found to be present in both undamaged and wounded skin, while it increased after the skin had been lacerated. in this study, fgf-2 was identified as a woundregulated protein. the expression of fgf-2 was found to increase after injury in healthy subjects, but not in diabetic ones.26 in conclusion, the administration of pomegranate extract to post-extraction wounds of c. cobaya increased the expression of fgf-2 and tgf-β on day 4 and accelerated the wound-healing process. references 1. lodi g, figini l, sardella a, carrassi a, del fabbro m, furness s. antibiotics to prevent complications following tooth extractions. cochrane database syst rev. 2012; 11: 1–62. 2. maskrey bh, megson il, whitfield pd, rossi ag. mechanisms of resolution of inflammation: a focus on cardiovascular disease. arterioscler thromb vasc biol. 2011; 31(5): 1001–6. 3. brunicardi fc, andeson dk, billiar tr, dunn dl, hunter jg, matthews jb, pollock re. schwartz’s principles of surgery. 9th ed. new york: mcgraw-hill medical; 2010. p. 210-9. 4. dunster jl. the macrophage and its role in inflammation and tissue repair: mathematical and systems biology approaches. wiley interdiscip rev syst biol med. 2016; 8: 87–99. 5. wynn ta, vannella km. macrophages in tissue repair, regeneration, and fibrosis. immunity. 2016; 44(3): 450–62. 6. sohrab g, nasrollahzadeh j, zand h, amiri z, tohidi m, kimiagar m. effects of pomegranate juice consumption on inflammatory markers in patients with type 2 diabetes: a randomized, placebocontrolled trial. j res med sci. 2014; 19(3): 215–20. 7. forouzanfar f, afkhami goli a, asadpour e, ghorbani a, sadeghnia hr. protective effect of punica granatum l. against serum/glucose deprivation-induced pc12 cells injury. evidence-based complement altern med. 2013; 2013: 1–9. 8. naik kk, thangavel s, alam a. cytotoxicity and anti-inflammatory activity of flavonoid derivatives targeting nf-kappab. recent pat inflamm allergy drug discov. 2017; 10(2): 119–32. 9. umesalma s, sudhandiran g. differential inhibitory effects of the polyphenol ellagic acid on inflammatory mediators nf-κb, inos, cox-2, tnf-α, and il-6 in 1,2-dimethylhydrazine-induced dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p189–193 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p189-193 193 nirwana, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 189–193 rat colon carcinogenesis. basic clin pharmacol toxicol. 2010; 107(2): 650–5. 10. massagué j. tgfβ signalling in context. nat rev mol cell biol. 2012; 13(10): 616–30. 11. kubiczkova l, sedlarikova l, hajek r, sevcikova s. tgf-β – an excellent servant but a bad master. j transl med. 2012; 10: 1–24. 12. edwards jp, thornton am, shevach em. release of active tgfβ1 from the latent tgf-β1/garp complex on t regulatory cells is mediated by integrin β8. j immunol. 2014; 193(6): 2843–9. 13. nirwana i. the activity of pomegranate extract as a pulp capping material on il-6, il-10, tgfβ-1, mmp-1 and type i collagen expression on rat teeth mechanically exposured. dissertation. surabaya: universitas airlangga; 2012. 14. flecknell pa. laboratory animal anaesthesia. 3rd ed. cambridge: elsevier academic press; 2009. p. 204-10. 15. wang n, liang h, zen k. molecular mechanisms that influence the macrophage m1-m2 polarization balance. front immunol. 2014; 5: 1–9. 16. liu t, zhang l, joo d, sun s-c. nf-κb signaling in inflammation. signal transduct target ther. 2017; 2: 1–9. 17. gupta sc, sundaram c, reuter s, aggarwal bb. inhibiting nf-κb activation by small molecules as a therapeutic strategy. biochim biophys acta gene regul mech. 2010; 1799(10–12): 775–87. 18. behm b, babilas p, landthaler m, schreml s. cytokines, chemokines and growth factors in wound healing. j eur acad dermatology venereol. 2012; 26(7): 812–20. 19. gonzalez ac de o, costa tf, andrade z de a, medrado arap. wound healing a literature review. an bras dermatol. 2016; 91(5): 614–20. 20. travis ma, sheppard d. tgf-β activation and function in immunity. annu rev immunol. 2014; 32(1): 51–82. 21. yun y-r, won je, jeon e, lee s, kang w, jo h, jang j-h, shin us, kim h-w. fibroblast growth factors: biology, function, and application for tissue regeneration. day r, editor. j tissue eng. 2010; 2010: 1–18. 22. nanci a, ten cate ar (arnold r. ten cate’s oral histology: development, structure, and function. 7th ed. china: elsevier; 2009. p. 64-74; 379-90. 23. sinno h, prakash s. complements and the wound healing cascade: an updated review. plast surg int. 2013; 2013: 1–7. 24. chao c, mong m, chan k, yin m. anti-glycative and antiinflammatory effects of caffeic acid and ellagic acid in kidney of diabetic mice. mol nutr food res. 2010; 54(3): 388–95. 25. al-obaidi mmj, al-bayaty fh, al batran r, hussaini j, khor gh. impact of ellagic acid in bone formation after tooth extraction: an experimental study on diabetic rats. sci world j. 2014; 2014: 1–14. 26. peplow pv., baxter gd. gene expression and release of growth factors during delayed wound healing: a review of studies in diabetic animals and possible combined laser phototherapy and growth factor treatment to enhance healing. photomed laser surg. 2012; 30(11): 617–36. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p189–193 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p189-193 76 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 76–80 research report the combination of carbonate hydroxyapatite and human β-defensin 3 to enhance collagen fibre density in periodontitis sprague dawley rats ika andriani,1,2 edy meiyanto,3 s. suryono4 and ika dewi ana5 1graduate program, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 2department of periodontology, school of dentistry, faculty of medical and health sciences, universitas muhammadiyah yogyakarta, yogyakarta, indonesia 3cancer chemoprevention research center, faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia 4department of periodontology, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia 5department of dental biomedical sciences, faculty of dentistry, universitas gadjah mada, yogyakarta, indonesia abstract background: carbonate hydroxyapatite (cha) is used as a scaffold to repair bone resorption. alveolar bone resorption in periodontitis caused by an infection requires the presence of an antibacterial to support bone regeneration. human β-defensin 3 (hbd3) is an antimicrobial peptide. the local application of the hbd3 antimicrobial is beneficial to inhibiting drug resistance and protecting tissue regeneration against invasive bacteria. purpose: this study aims to investigate the effect of the administration of a combination of cha with hbd3 on the collagen density of periodontitis rats (sprague dawley). methods: this study was a true experimental study with a post-test control group design. thirty-two sprague dawley animal models were randomly blind selected and placed under anaesthetic, then a 2-mm silk ligature was attached as a ligation to the mandibular incisors for 14 days in order to generate periodontitis. the study subjects were divided into two groups, the group with cha and cha-loaded hbd3 (cha + hbd3) implantation. on days 7, 14, 21 and 28, four rats were taken randomly from each group for decapitation, followed by histological processing and examination with trichrome mallory staining. the data was analysed using the kruskal–wallis test (p<0.05). results: an increase in collagen density during the healing process was found. there was a significant difference between cha and cha+hbd (p=0.004 and p=0.008; p<0.05) in collagen density between the groups. conclusion: the combination of cha and hbd3 can enhance the collagen density in periodontitis sprague dawley rats, compared to cha-only groups. keywords: carbonate hydroxyapatite; collagen; human β-defensin 3; periodontitis correspondence: ika dewi ana, department of dental biomedical sciences, faculty of dentistry, universitas gadjah mada, jl. denta no. 1, sekip utara, yogyakarta 55281, indonesia. email: ikadewiana@ugm.ac.id introduction periodontitis is a periodontal tissue infection caused by a polymicrobial infection characterised by the destruction of the supporting tissue of the teeth.1 one of the damaged periodontal tissues is the alveolar bone, which thereby requires surgical treatment and bone grafting to facilitate the formation of new bone tissue.2 bone grafts consist of three types, namely autografts, allografts, xenografts and synthetic grafts such as hydroxyapatite. this material has been widely approved as a biocompatible scaffold, promoting osteoconduction of bone healing. it can be absorbed by the body.3 hydroxyapatite includes a synthetic calcium phosphate material which is widely used as a bone graft. it has a similar chemical composition as the bone mineral matrix, producing superior biocompatibility, osteoconduction and osteointegration.4 apatite is found in teeth and bone, and is 3-5% composed of carbonate ions, an arrangement of bone components called carbonate apatite (co3ap).5 carbonate hydroxyapatite (cha) is a biomimetic, biocompatible, biodegradable, osteoconductive bone dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p76–80 mailto:ikadewiana@ugm.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p76-80 77andriani et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 76–80 graft that promotes bone tissue without fibrotic tissue formation and stimulates osteogenesis.6,7 based on previous studies, the biological activity of synthetic cha is better than synthetic hydroxyapatite because the incorporation of carbonates in hydroxyapatite causes an increase in solubility and decreases crystallisation.6 cha bone grafts can also be combined with various antibiotics, for example, metronidazole.8 the use of local and systemic antimicrobial therapy in extensive periodontal treatment is important as adjunctive therapy to prevent the growth and development of pathogenic bacteria.9 human β-defensin3 (hbd3) is an antimicrobial cationic peptide with an immunomodulatory effect on innate and adaptive immune responses. the local application of an hbd3 antimicrobial is beneficial for protecting regenerated tissue against invasive bacteria that causes infection and for reducing the need for conventional antibiotics that cause drug resistance.10 compared with other human defensins, hbd3 can reduce the formation of osteoclasts and reduce alveolar bone loss.11 it functions as an osteogenic promoter through anti-inflammatory effects in the micro-inflammatory environment.12 the combination of tissue techniques or synthetic bone grafts and antimicrobial therapy has strong potential for more successful tissue repair because infection is always a major risk factor during tissue regeneration.11 periodontitis therapy with bone grafts and the addition of local antimicrobial therapy increases bone regeneration and bone repair.12,13 collagen is the main structural component of the extracellular matrix as a positive indication of the healing development.14 stages of bone formation start from the synthesis of type 1 collagen, collagen secretion, microfibril formation, fibrils and collagen fibers, maturation, the collagen matrix and the formation of hydroxyapatite crystals. all of these elements are under the influence of osteoblasts.15 the purpose of this study was to investigate the effect of the administration of a combination of cha loaded with hbd3 in collagen density on sprague dawley rat with periodontitis condition. materials and methods all the procedures of the in vivo experiment were approved by the ethical committee of the faculty of dentistry, universitas gadjah mada, no.001511/kkep ugm/ec/2018. the cha was prepared by the research laboratory of the faculty of dentistry, universitas gadjah mada, as described in the previous study.16 the membrane was prepared by a chemical mixture between carbonate hydroxyapatite and type b gelatin (mw 99 x 103) from a bovine source (nitta gelatin, osaka, japan) and was carried out in sodium citrate solution at 37°c. the ratio of gelatin and cha was of 7:3 (w.t %) respectively. the mixture was stirred continuously until a homogeneous solution was formed. phosphate acid was dripped in the homogeneous solution and stored for 2 hours. after 2 hours, the homogeneous solution was dropped with 2 n hcl until the ph level reached 7.4. the gelatin solution containing cha which has been chemically mixed was molded in 1gr/2025 mm2 and was then stored in a freezer at 4°c overnight to dry and to form a membrane. when the membrane was dried, it was cross-linked with dehydrothermal at 140°c for 72 hours in a vacuum oven (yamato adp 200, yamato scientific co., ltd, tokyo, japan) and the gelatin cha membrane was stored in an aluminium foil wrap at room temperature.16 hbd3 is an antimicrobial bd-3, a human recombinant srp4524-20ug lyophilized solid (sigma aldrich, saint louis, mo, usa). the 10 μl of hbd3 with a concentration of 12.5 μg/l was loaded into 2 mg of membrane cha.8 this research is a study using 32 healthy and active sprague dawley male rats, weighing between 300 and 400 grams, and aged 3 to 4 months. experimental animals, sprague dawley rats, were adapted in a clean cage for 7 days. the rats were housed in each cage and maintained under a 12-hour light/dark cycle at a temperature of 23˚c and a relative humidity of 50%, with access to standard rat chow pellets and water ad libitum. the sprague sawley rats were anaesthetised with ketamine (0.1 ml) and xylazine (0.1 ml) by an intramuscular injection in the thigh (dose 6–12 mg/kg). after the experimental animals entered the anaesthetic stage, a 2 mm silk ligature was attached as a ligation to the mandibular incisors for 14 days in order to generate periodontitis (figure 1).17 the study subjects were divided into two groups, the group with cha and the group with a cha-loaded hbd3 (cha + hbd3) implantation of the periodontitis in the mandibular incisor region. on days 7, 14, 21 and 28, four rats were taken randomly from each group for decapitation, followed by histological processing and an examination with trichrome mallory staining and 40x× magnification with an optical microscope olympus bx 51 (olympus, tokyo, japan). the criteria for evaluating the collagen fibers were set according to tandelilin et al.18 as depicted in table 1 and figure 2. collagen density was observed via the blind method. each sample was prepared using a different code by a person who was not involved in the study. figure 1. day 14 ligation in sprague dawley rats. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p76-80 78 andriani et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 76–80 the data was expressed as the mean ± stan dard deviation. spss 20.0 software (ibm corp., armonk, ny, usa) was used for all the statistical analyses. after checking the data normality through the shapiro–wilk test (p <0.05), when detects not normality data, the significant difference was calculated using the kruskal-wallis test (p <0.05). results means and standard deviations of the collagen density cha and cha-loaded hbd3 (cha+hbd3) in each group was based on observation days (figure 3). the shapiro–wilk test (p<0.05) showed data normality. there were two data groups that were not normally distributed: the 14-day cha treatment group and 28-day cha treatment group. on observation, days 7,14, 21 and 28 showed that the density of collagen fibres in the implantation area of the cha + hbd3 group was higher than the cha-only group (figure 4). the variance analysed using the kruskal–wallis test showed significant differences (p <0.05) in collagen density between the treatment groups using cha and the chaloaded hbd3 (table 2). discussion the results on the 7th day showed that cha and cha loaded with hbd had formed collagen fibres of alveolar bone in the sprague dawley periodontitis rats.19 collagen 22 26.25 27.25 30.5 26.5 33.5 34.25 36 0 5 10 15 20 25 30 35 40 day 7 day 14 day 21 day 28 co lla ge n de ns ity cha cha + hbd3 figure 3. the increasing pattern of the collagen density fibre based on observation days between the cha group and the cha + hbd3 group. table 2. results of the kruskal–wallis test on the mean and standard deviation of collagen density in cha and cha + hbd3 groups implantation time cha cha + hbd3 day 7 22.00 ± 1.414 26.50 ± 1.291 day 14 26.25 ± 0.500 33.50 ± 1.291 day 21 27.25 ± 0.957 34.25 ± 1.258 day 28 30.50 ± 0.577 36. 00 ± 0.816 asymp. sig. (p) 0.004* 0.008* *p < 0.05 figure 2. collagen density score, score 1 (a), score 2 (b) and score 3 (c). table 1. scoring for collagen density used in the study score 1 collagen fibre density is less than 50% with less dense tissue structure, vascularisation, mononuclear cells, and many cells can be found. 2 collagen fibre density is more than 50% with more dense tissue structure, less inflammatory reaction. 3 avascular and acellular collagen fibrous density. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p76-80 79andriani et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 76–80 cha + hbd 3 cha day 7 day 14 day 21 day 28 figure 4. histological findings of collagen density (black arrow) on observation days between the cha and the cha + hbd3 group by trichrome mallory staining (magnification, 40x×). is a major protein that forms the extracellular matrix component, which is needed in the wound healing process and also needed in the formation of bone matrices.20 a successful parameter in the wound healing process and bone formation process is the presence of collagen.19 on the 7th day, the transition phase of the blood vessel granulation tissue had been formed, with beginnings of fibroblast and fibrin tissue. macrophages and cytokine regulation produce platelet-derived growth factors (pdgf), interferon gamma (ifn γ), fibroblast growth factors (fgf) and transforming growth factors (tgf-β) as growth factors that result in the reduction of fibroblasts to proliferate and migrate, and to produce extracellular fibres such as collagen, elastin fibres, and reticular fibres that needed for cutting processes. cha and hbd3 membranes have the same properties that affect the proliferation of fibroblasts.21,22 fibroblast cells play an important role in collagen synthesis, depositing and renovating connective tissue and regenerating bone tissue. collagen synthesis increases in the damaged area21 and results in the complication on day 7 relating to both cha and cha + hbd3. on days 14 to 28 after the implantation, there was an increase in collagen fibre and collagen synthesis. the results of this study were not consistent with the previous theory stating that the formation of collagen fibres begins on day 3 and peaks on day 7, then decreases between days 14 and 21.20 however, the results of this study are in line with research by ardhiyanto and siswomihardjo23 that shows that after the implantation of hydroxyapatite on the 14th day, the amount of collagen continues to increase until the 28th day. this condition is caused by the cha providing the right microenvironment to repair bone resorption of tissue24 and to eliminate the inflammation.25 thus, collagen becomes more dense and alveolar bone regeneration occurs. on days 21 to 28, a bone remodelling process occurs that regulates the balance of bone formation and resorption. the purpose of the remodelling phase that occurs from day 21 to one year after the injury is to complete tissue repair and to restore tissue integrity.21,26 the results of the study on all days showed that the periodontal regeneration of the cha + hbd3 group was higher than the cha group, characterised by thicker and denser collagen in the cha + hbd3 group. this might happen because of the interaction between the cha, which has high osteoconductive properties, so it could stimulate new bone growth5 and the hbd3 that has antimicrobial properties, promoting regeneration in the inflammatory environment. moreover, cha + hbd3 also increases cellular activity, provides cells that stimulate differentiation of the extracellular matrix, which synthesises the development of new tissue to increase tissue osteogenic differentiation and periodontal regeneration.21,26,27 the addition of hbd3 to the cha membrane is necessary because pathogenic microbes that adhere to the tooth surface and contaminate periodontal lesions are confounding factors in the regeneration of periodontal tissue. infection control could be carried out to optimise the regeneration process.28 the antibacterial scaffold (cha + hbd3) makes the extracellular matrix situation in the damaged tissue change quickly in order to accelerate the healing process and the regeneration of alveolar bone. limitation in the research lies in the limited hbd3 material and the long delivery process. in conclusion, a combination cha and hbd3 could enhance the collagen density in the periodontitis animal model compared to the cha group only. it is suggested for the future research to investigate the variables in a longer time frame, approximately three months, and to investigate more of the osteoblast and osteoclast variables in order to see alveolar bone regeneration. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p76-80 80 andriani et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 76–80 acknowledgment the authors thank the ministry of research, technology, and higher education indonesia for the budi-dn scholarship with the grant number fr-412019166048. references 1. silva n, abusleme l, bravo d, dutzan n, garcia-sesnich j, vernal r, hernández m, gamonal j. host response mechanisms in periodontal diseases. j appl oral sci. 2015; 23(3): 329–55. 2. blackwood ka, bock n, dargaville tr, ann woodruff m. scaffolds for growth factor delivery as applied to bone tissue engineering. int j polym sci. 2012; 2012: 1–25. 3. valiense h, fernandes gvo, moura b, calasans-maia j, alves a, rossi am, granjeiro jm, calasans-maia m. effect of carbonateapatite on bone repair in non-critical size defect of rat calvaria. key eng mater. 2011; 493–494: 258–62. 4. wang y-f, wang c-y, wan p, wang s-g, wang x-m. comparison of bone regeneration in alveolar bone of dogs on mineralized collagen grafts with two composition ratios of nano-hydroxyapatite and collagen. regen biomater. 2016; 3(1): 33. 5. zakaria mn, cahyanto a. an introduction to carbonate apatite as a biocompatible material in dentistry. in: pertemuan ilmiah tahunan 8, prodi kedokteran gigi unjani. bandung; 2016. p. 80–4. 6. ana id, matsuya s, ishikawa k. engineering of carbonate apatite bone substitute based on composition-transformation of gypsum and calcium hydroxide. engineering. 2010; 2(5): 344–52. 7. rahyussalim aj, supriadi s, marsetio af, pribadi pm, suharno b. the potential of carbonate apatite as an alternative bone substitute material. med j indones. 2019; 28(1): 92–7. 8. ardhani r, setyaningsih, hafiyyah oa, ana id. preparation of carbonated apatite membrane as metronidazole delivery system for periodontal application. key eng mater. 2016; 696: 250–8. 9. herrera d, matesanz p, bascones-martínez a, sanz m. local and systemic antimicrobial therapy in periodontics. j evid based dent pract. 2012; 12: 50–60. 10. zhu m, miao b, zhu j, wa ng h, zhou z. expression a nd antimicrobial character of cells transfected with human β-defensin-3 against periodontitis-associated microbiota in vitro. mol med rep. 2017; 16(3): 2455–60. 11. cui d, lyu j, li h, lei l, bian t, li l, yan f. human β-defensin 3 inhibits periodontitis development by suppressing inflammatory responses in macrophages. mol immunol. 2017; 91: 65–74. 12. zhu m, miao b, zhu j, wang h, zhou z. transplantation of periodontal ligament cell sheets expressing human β-defensin-3 promotes anti-inflammation in a canine model of periodontitis. mol med rep. 2017; 16(5): 7459–67. 13. zhu l, chuanchang d, wei l, yilin c, jiasheng d. enhanced healing of goat femur-defect using bmp7 gene-modified bmscs and load-bearing tissue-engineered bone. j orthop res. 2010; 28(3): 412–8. 14. nayak bs, kanhai j, milne dm, pereira lp, swanston wh. experimental evaluation of ethanolic extract of carapa guianensis l. leaf for its wound healing activity using three wound models. evidence-based complement altern med. 2011; 2011: 1–6. 15. o’brien fj. biomaterials & scaffolds for tissue engineering. mater today. 2011; 14(3): 88–95. 16. patriati a, ardhani r, pranowo hd, putra egr, ana id. the effect of freeze-thaw treatment to the properties of gelatin-carbonated hydroxypatite membrane for nerve regeneration scaffold. key eng mater. 2016; 696: 129–44. 17. ionel a, lucaciu o, moga m, buhatel d, ilea a, tabaran f, catoi c, berce c, toader s, campian rs. periodontal disease induced in wistar rats experimental study. hum vet med. 2015; 7(2): 90–5. 18. tandelilin rt. augmentation of demineralized bone matrix posttooth extraction increases the density of gingival collagen fiber of rabbit mandible. indones j dent res. 2010; 1(1): 9–16. 19. agarwal pk, singh a, gaurav k, goel s, khanna hd, goel rk. evaluation of wound healing activity of extracts of plantain banana (musa sapientum var. paradisiaca) in rats. indian j exp biol. 2009; 47(1): 32–40. 20. guo s, dipietro la. factors affecting wound healing. j dent res. 2010; 89(3): 219–29. 21. caetano gf, fronza m, leite mn, gomes a, frade mac. comparison of collagen content in skin wounds evaluated by biochemical assay and by computer-aided histomorphometric analysis. pharm biol. 2016; 54(11): 2555–9. 22. cañedo-dorantes l, cañedo-ayala m. skin acute wound healing: a comprehensive review. int j inflam. 2019; 2019: 1–15. 23. a rd h iya nto h b, siswom i ha rdjo w. ju m la h osteoblas d a n kepadatan kolagen tipe 1 pada proses penyembuhan tulang pasca implantasi hidroksiapatit sintesis kalsit (pt. omya, surabaya, jawa timur) (studi in-vivo pada tulang tikus sprague dawley). thesis. yogyakarta: universitas gadjah mada; 2013. 24. wahyudi ia, nurwadji lm. the effect of non freeze-dried hydrogelcha on fibroblast proliferation. j dent indones. 2015; 21(3): 89–93. 25. fu n, meng z, jiao t, luo x, tang z, zhu b, sui l, cai x. p34hb electrospun fibres promote bone regeneration in vivo. cell prolif. 2019; 52(3): 1–10. 26. wang h, watanabe h, ogita m, ichinose s, izumi y. effect of human beta-defensin-3 on the proliferation of fibroblasts on periodontally involved root surfaces. peptides. 2011; 32(5): 888–94. 27. zhou j, zhang y, li l, fu h, yang w, yan f. human β-defensin 3-combined gold nanoparticles for enhancement of osteogenic differentiation of human periodontal ligament cells in inflammatory microenvironments. int j nanomedicine. 2018; 13: 555–67. 28. chen fm, zhang j, zhang m, an y, chen f, wu zf. a review on endogenous regenerative technology in periodontal regenerative medicine. biomaterials. 2010; 31(31): 7892–927. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p76-80 54 vol. 43. no. 2 june 2010 research report tensile bond strength of hydroxyethyl methacrylate dentin bonding agent on dentin surface at various drying techniques kun ismiyatin department of conservative dentistry faculty of dentistry, airlangga university surabaya indonesia abstract background: there are several dentin surface drying techniques to provide a perfect resin penetration on dentin. there are two techniques which will be compared in this study. the first technique was by rubbing dentin surface gently using cotton pellet twice, this technique is called blot dry technique. the second technique is by air blowing dentin surface for one second and continued by rubbing dentin surface gently using moist cotton. purpose: this experiment was aimed to examine the best dentin surface drying techniques after 37% phosphoric acid etching to obtain the optimum tensile bond strength between hydroxyethyl methacrylate (hema) and dentin surface. method: bovine teeth was prepared flat to obtain the dentin surface and than was etched using 37% phosphoric acid for 15 seconds. after etching the dentin was cleaned using 20 cc plain water and dried with blot dry techniques (group i), or dried with air blow for one second (group ii), or dried with air blow for one second, and continued with rubbing gently using moist cotton pellet (group iii), and without any drying as control group (group iv). after these drying, the dentin surfaces were applied with resin dentin bonding agent and put into plunger facing the composite mould. the antagonist plunger was filled with composite resin. after 24 hours, therefore bond strength was measured using autograph. result: data obtained was analyzed using one-way anova with 95% confidence level and continued with lsd test on p0.05. the result showed that the highest tensile bond strength was on group i, while the lowest on group iv. group ii and iv, iii and iv, ii and iii did not show signigicant difference (p>0.05). conclusion: dentin surface drying techniques through gentle rubbing using cotton pellet twice (blot dry technique) gave the greatest tensile bond strength. key words: tensile bond strength, hydroxyethyl methacrylate dentin bonding agent, dentin surface drying technique abstrak latar belakang masalah: tehnik pengeringan permukaan dentin agar resin dapat penetrasi dengan sempurna adalah dengan cara pengusapan secara halus sebanyak dua kali menggunakan bulatan kapas, yang disebut blot dry technique, dengan semprotan udara selama 1 detik atau dengan semprotan udara selama 1 detik yang dilanjutkan dengan pengusapan secara halus menggunakan bulatan kapas basah yang diperas dan kelebihan air pada kapas diserap dengan kertas hisap. tujuan: penelitian ini bertujuan untuk mengetahui tehnik pengeringan permukaan dentin yang terbaik setelah etsa dengan asam phosphat 37% untuk mendapatkan kekuatan perlekatan tarik yang optimum antara hema dentine bonding agent dan permukaan dentin dengan menggunakan alat ukur autograph. metode: permukaan dentin gigi sapi diasah rata, kemudian di etsa dengan asam phosphat 37% selama 15 detik. permukaan dentin dicuci dengan 20 cc air dan kemudian dikeringkan dengan cara blot dry technique (kel. i), dengan semprotan udara selama 1 detik (kel. ii) atau semprotan udara selama 1 detik dan dilanjutkan dengan pengusapan secara halus menggunakan bulatan kapas basah yang diperas dan kelebihan air pada kapas diserap dengan kertas hisap (kel. iii) dan tanpa pengeringan permukaan dentin sebagai kontrol (kel. iv). selanjutnya permukaan dentin diulasi dengan resin bonding dan diletakkan kedalam plunger dengan permukaan menghadap permukaan komposit. setelah 24 jam dilakukan pengukuran kekuatan tarik dengan menggunakan alat ukur autograph. hasil: analisis data menggunakan uji anova satu arah dengan derajat kepercayaan 95% dan dilanjutkan dengan test lsd pada p≤0,05. dari hasil penelitian didapatkan kekuatan perlekatan tarik yang paling tinggi pada kelompok i, sedangkan yang paling rendah pada kelompok iv. didapatkan perbedaan bermakna pada kelompok i, ii, iii, dan iv. pada kelompok ii dan iv, iii, dan iv, ii, dan iii tidak 55ismiyatin: tensile bond strength didapatkan perbedaan bermakna (p>0,05). kesimpulan: tehnik pengeringan permukaan dentin dengan cara pengusapan secara halus menggunakan bulatan kapas sebanyak 2 kali menghasilkan kekuatan perlekatan tarik terbesar. kata kunci: kekuatan perlekatan tarik, hema dentine bonding agent, tehnik pengeringan permukaan dentin. correspondence: kun ismiyatin, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend.jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: ismiyatinkun@yahoo.com tensile strength.8 the best condition of the dentin surface is moist in order to obtain the maximum bond between resin bonding agent and dentin collagen. dentin moist means the condition related with water content contained in the dentin surface. the humidity developed around the dentin collagen will influence the physical and chemical nature so that it will affect the collagen bond towards resin agent. water content in dentin can be measured from the ratio between water weight in dentin with the total of dentin weight and water content in it (%). to reach maximal bonding between dentin bonding agent on dentin surface, it is important that collagen fibril should be in permeable condition/active.1,2 permeable collagen fibril is strongly influenced by the moist surrounding dentin surface. the dentin surface drying techniques to enable a perfect resin penetration, some studies reported that dentin surface drying by rubbing gently using a cotton pellet for two times (blot dry technique), or with air blow for 1 second or with air blow for 1 second, and then followed by gently rubbing it with a cotton pellet which has been wetted by water, the excess of water was removed by squeezing and put on blotting paper. the purpose of this study was to examine the best dentin surface drying techniques after acid etching with 37% phosphoric acid in 15 second to obtain optimum tensile strength of hydroxyethyl methacrylate (hema) dentin bonding agent to bovine dentin surface. material and method bovine's incisives were taken from slaughter house of pegirian, surabaya. in this experiment, a newly pulled out incisive from the bovine tooth which was vital, caries free, unscratched, with no chipped part, no abrasion that had been checked under a light microscope is used. the tooth was then cleaned, soaked in physiologic solution and kept in a refrigerator in 4° c. after that the tooth was cut under flowing water using a diamond disk and then with a fissure diamond drill so that the dentin was open. the dentin surface was smoothed by number 400 and number 1000 silicon scrub papers, cleaned under 20 cc water sprayer, the dentin surface part was covered with an adhesive tape which has been given 3 mm hole and was stocked right to the middle of the dentin surface, and then it was planted into a hard gypsum cylinder block which was then etched with 37% phosphoric acid using a cotton pellet in 15 seconds and at last it was washed with 20 cc aquadest from an injection syringe. introduction one of the phase of teeth restoration using composite resin is the application of dentin bonding agent on the surface of dentin that functions to join composite restoration with tooth tissues so that the dentin bonding strength increase. the application of dentin bonding using total etching system is by acid etching on enamel and dentin surfaces previously followed by washing and drying and then application of resin dentin bonding agent. the purpose of acid etching is to open the dentin collagen fibril tissues so that resin agent could bind physically and chemically with fibril collagen. other researchers said that the purpose of washing is to remove salts which are formed by the reaction between acid etching agent and enamel and dentin minerals. after the washing, drying up is done to remove which is meant to remove the left over of washing water after acid etching.1 other researchers stated that the procedure of dentin surface drying is different from the one of the enamel surface, for that reason the surface of dentin must not too dry or too wet. if the surface is too dry, the hydrogen bond will be cut off so that the dentin collagen may collapse, shrink, and the collagen amino cluster will be covered among the structure of secondary collagen. this condition will results in difficulty for the resin agent to penetrate into the remaining cavity and it will cause the absence of mechanical retention between resin bonding agent and dentin collagen. if the surface of the dentin is too wet, there will be a lot of water molecules surrounding the dentin collagen so that a bond between hydrogen and water with carbonyl resin bonding cluster may be developed resulting in the ability to bond with dentin collagen amino cluster.2 the success of the fusing of dentin bonding agent to the dentin surface depends on monomer viscosity, types and concentration of monomer, acid etching application as conditioner, temperature and humidity around the fibril collagen.2,3,4 the studies found out that in 33%, 50%, 75% and 100% humidity, the tensile strength between dentin bonding agent and dentin teeth gets increased following the decrease of humidity.5 in vitro study on dentin humidity of 30%, 50%, 65%, 80% and 95% showed that tensile strength of bonding agent on dentin will increase following the decrease of the humidity around dentin.6 whereas, the study in humidity of 50%, 80%, and 95% proves that tensile strength dentin bonding will decrease with regard to the increase of humidity.7 other research study, proved that 70% humidity is the best one, because it develops the maximum bond between the carbonyl hema cluster and dentin collagen whereas 60% humidity gives the maximum 56 dent. j. (maj. ked. gigi), vol. 43. no. 2 june 2010: 54-57 the experiment samples were made more than 40 samples and then 40 samples which comply with the criteria were randomly taken, 10 samples for each experiment group. group i: the dentin surface was dried with blot dry technique,9 group ii: dentin surface was dried by air blow for 1 second, group iii: the dentin surface was dried by air blow for 1 second, and then followed by gently rubbing it with a cotton pellet which had been wetted by water, then the excess water removed by squeezing and put on blotting paper, and group iv: the control group, with no dentin surface drying. after the dentin surface drying process had been done, the dentin surface was covered with total etched dentin bonding agents, prime and bond. it was left that way for 30 seconds, sprayed using air blower, then was lighted up with light cured for 20 seconds (as instructed by the factory). after having been given treatment the cylinder block was put into a plunger, with its surface was made facing to the composite mold. the opponent plunger was filled with resin composite on the dentin bonding and top of it was given a celluloid strip and a thin glass, next a weight of 1000 gram was put on it for one minute, and then the left over was removed using a scalpel, and after that it was light cured for 40 seconds with a light curing unit (factory direction is followed). after the composite application had been finished, the specimen was taken out from the composite placing equipment aid and the molding ring was released. then the sample that had been ready was placed into a plastic tube containing aquadest for 24 hours. after 24 hours the sample tensile strength was tested using an autograph measuring equipment (shimadzu, japan) with the cross head speed of 10 mm/minute, range: 5, load cell capacity 5 kn/500 kgf. the result was a tensile strength in kgf where 1 kgf = 9.81 n. 1 mpa = n/mm2 width of experimental dentin surface = πr2 = 7.1 mm2. the data was analyzed with one-way anova at 95% confidence level continued with lsd test on p≤0.05. result the tensile bond strength, mean, and standard deviation of hema dentin bonding agent on bovine dentin surface at various dentin surface drying techniques can be seen in table 1. in order to find out if the distribution of the tensile strength data was nomal and homogenious, the kolmogorovsmirnov test was administered, resulting p>0.05 which means that the data has a normal distribution. to examine if the data was homogenous, the levene test is administered. the difference of all samples in this experiment were analyzed with one-way anova at 95% confidence level. the result showed that tensile bond strength of hema dentin bonding agent to bovine dentin collagen with various dentin surface drying techniques significantly different (p<0,05). to determine the difference of each samples continued with lsd test on p0.05 (table 2). the result showed that experiment group i was compared with experiment group ii, iii and iv, it will have p<0.05, meaning that there was a significant difference. the tensile bond strength of resin bonding to dentin at group ii and iv, iii and iv, ii and iii was not signigicantly different (p>0.05).10 tabel �. mean and standard deviation of tensile bond strength between hema dentin bonding agent and bovine dentin surface at various dentin surface drying techniques (mpa) drying technique n x sd group i 10 17.9021 2.6853 group ii 10 9.5432 1.4314 group iii 10 11.6121 1.7418 group iv (control) 10 7.3214 1.0982 description: x : mean of tensile bond strength, n : the number of samples, sd : standard deviation group i : the dentin surface was dried with blot dry technique group ii : the dentin surface was dried with air blow for one second. group iii : the dentin surface was dried with air blow for one second, followed by rubbing it softly using a cotton pellet which has been wetted with water, the excess water was removed by squeezed and put on blot paper. group iv : no drying was done on dentin surface (control group). table ��. lsd of the tensile bond strength between hema dentin bonding agent on dentin surface at various dentin surface drying techniques drying technique groupi group ii group iii group iv (control) group i xxx + + + group ii + xxx – – group iii + – xxx – group iv + – – xxx description: +: significant difference, -: not significant discussion the tensile bond strength between hema dentin bonding agent on dentin surface is derived because there are chemical bonding and mechanical retention. mechanically, because there is a penetration of hema into inter-fibril nano space, dentin tubules, dentin peritubules, dentin intertubules which later polymerized. in addition, physical bonding occurs because of van der waals tensile bond between both agents. whereas this chemically happens because of the interactions between carbonyl ester of hema from the 57ismiyatin: tensile bond strength bonding agent with amino of collagen dentin which produce amide groups [c(o)nh]. this bonding is strong due to its covalent characteristics called as inter-atomic primer bond. microscopically, collagen is not found in the entire dentin surface, where as mechanical affixing envolves the whole dentin surface so that the mehanical bonding is more dominant than the chemical bonding.11 dentin is living tissue which contain approximately 60% inorganic components (hydroxyapatite), 30% organic component and 10% water. those organic component are 90% collagen and 10% non collagen. most of those collagens are type i and few of them are type v. the tensile strength in this experiment is the adhering strength between resin bonding agent (hema) and bovine dentin surface which was flat. in this research the dentin bovine was used because it contains type i collagen, type v collagen, non collageus protein and proteoglicane and others. the tensile strength of resin bonding on the human dentin surface is a little bit different from the surface of bovine teeth. other researcher stated that resin bonding strength on human dentin is higher than on bovine teeth, this is due to the amount of bovine dentin apatite mineral is less than the one of human teeth.2 in this research, the influence of various dentin surface drying techniques to tensile bond strength of resin bonding agent to dentin can be seen in table 1. group i showed the highest tensile bond strength, which is 17.9 mpa. the result of this study goes along well with other researchers which states that the highest value of tensile strength is earned in 60% moisture. because of dentin surface drying technique was the same with what was done by other research, the acquired dentin surface moisture which was probably ± 60%.8 the result of this research was also appropriate with the research of other researchers, which stated that 60% moisture is the best moisture since the number of water molecules is ideal enough to re-expand fibril collagen so that the resin will penetrate easily into nano space among fibril and chemically there will be strong interactions between resin and collagen. resin agent contains aceton which easily evaporates so that the resin dentin bonding that reacts with fibril collagen would form hybrid dentin layer which was believed to be the main strength of resin attachment with dentin.12,13 the higher tensile bond strength happend because the efficacy of monomer infiltration is totally at demineralized dentin layer. on dentin surface drying techniques that was applied on group ii, group iii, and without drying process on the dentin surface on group iv, it was found that this drying process might probably result in bigger number of water molecules than that of blot dry technique as in group i. as the number of water molecules was higher than those in blot dry technique, the capability of acetone to drive away water and then evaporate around the dentin collagen was also low, so that the resin bonding conjugation with fibril collagen will decrease too.3,12,14 at blot dry technique, the acetone ability to chase water was higher, and in this condition bonding resin will immediately penetrate to collagen. acetone concentration influences the thickness of resin bonding layer and tensile bond strength.12 higher moisture will reduce the decrease of hema concentration in nano space among fibrils, it means there was a decrease in the mechanism retention between hema resin and dentin collagen and there was a decrease in the chemical bond between hema and collagen, as when there are a lot of water molecules around dentin collagen, the hydrogen bond between water and collagen amino may happen and this will prevent the bonding agent from conjugate with collagen.2 while the drying using air blow may cause trauma on the soft fibril collagen so that the fibril collagen will collapse. due to the collapse of fibril collagen, so it was difficult for resin bonding to bind with dentin collagen, resulting in the decrease of its tensile strength.11 it can be concluded the drying technique of the dentin surface by rubbing it gently using a dry cotton pellet twice (blot dry technique) will result in the highest tensile strength. as suggestion, further research is needed to examine the tensile bond strength of hema dentin bonding agent with dentin surface at various ph of hema, hema concentration, and acetone concentration. references 1. anusavice kj. phillip's science of dental materials. 11th ed. philadelphia, london, toronto: wb saunders co; 2003. p. 21–395. 2. nakabayashi np, pashley dh. hybridization of dental hartd tissues. 1st ed. chicago il: quintess publ co, ltd; 1998. p. 1–107. 3. perdigao j, lopes m. the effect of etching time on dentin demineralization. restorative dent 2001; 32: 19–26. 4. breschi l, gobbi p, marzotti g, falconi m. high resolution sem evaluation of dentin etched with maleic and citric acid. dent mat 2002; 18: 26–35. 5. werner jf, tani c. effect of relative humidity on bond strength of self-etching adhesive to dentin. j adhes dent 2002; 4(4): 277–82. 6. besnault c, attal jp. influence of simulated oral environmental on dentin bond strength of two adhesive systems. am j dent 2001; 14(6): 367–72. 7. chiba y, miyasaki m, rikuta a, moore bk. influence of environmental conditions on dentin bond strengths of one application adhesive system. oper dent 2004; 29(5): 554–9. 8. soetojo a. tensile bond strength of hydroxyethyl methacrylate (hema) bonding agent to bovine dentin surface at various humidity. dent j (maj.ked.gigi) 2006; 39(2): 59–62. 9. van meerbeek b, yoshida y, lambrechts p, vanherle g. a tem study at two water based adhesive systems bonded to dry and wet dentin. j dent res 1998; 77(1): 50–9. 10. wayne dw. biostatistics: a foundation for analysis in the health science. 5th ed. singapore: john willey & sons; 1991. p. 155. 11. craig rg, powers jm, wataha jc. dental materials. properties and manipulation. 8th ed. baltimore, boston, carlsbad: mosby inc; 2002. p. 57–78. 12. cho bh, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the micro tensile bond strength. dent mat 2004; 20: 107–15. 13. suzuki k, nishiyama n, nemoto k. effect of n-methacryloil amino acid application on hybrid layer formation at the interface of intertubuler dentin. j dent res 1998; 77: 1881–8. 14. swift ej, wilder ad, may kn, waddell sl. shear bond strength of one-bottle dentin adhesive using multiple applications. oper dent 1997; 22(10): 194–9. vol 51 no 3 jul sep 2018_pus.indd 129 the effect of avocado leaf extract (persea americana mill.) on the fibroblast cells of post-extraction dental sockets in wistar rats christian khoswanto, wisnu setyari juliastuti, and karina awanis adla department of oral biology, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: tooth extraction, a common practice among the dental profession, causes trauma to the blood vessels during the wound healing process. the acceleration of wound healing, within which fibroblasts play an important role, is influenced by nutrition. avocado leaves contain a variety of chemicals, including flavonoid compounds, tannins, katekat, kuinon, saponin and steroids/triterpenoid. avocado leaves also contain glycosides, cyanogenic, alkaloids and phenols which function as anti-inflammatory, antibacterial and antioxidant agents. this avocado leaf content could be used as an alternative medicine to accelerate the wound healing process in posttooth extraction sockets. purpose: to determine the role of avocado leaves (persea americana mill) in accelerating fibroblast cells proliferation in tooth socket post-extraction. methods: the sample was divided into four groups, a control group and three treatment groups. the treatment groups used avocado leaf extract and 3% cmc na solution which was inserted into the tooth sockets of wistar rats. both the control and treatment groups had their mandibula decapitated with all the required specimens being prepared on the 3rd and 7th days of the experiment. mandibular decapitation and tooth extraction socket were prepared by hpa (histology pathology anatomy) with hematoxylin eosin (he) staining. the fibroblast proliferation was analyzed by means of a light microscope at 400x magnification. the obtained data was analyzed using a t-test. result: the t-test obtained a significance value 0.001 (p <0.05) between the control and treatment groups. the number of fibroblast cells increased in the group treated on the third day and decreased in the group treated on the seventh day. conclusion: avocado leaf extract (persea americana mill.) accelerates proliferation of fibroblast cells in wistar rats post-tooth extraction. keywords: avocado leaf extract; wound healing; fibroblast correspondence: christian khoswanto, department of oral biology, faculty of dental medicine, universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132. e-mail: christiankhoswanto@hotmail.com dental journal (majalah kedokteran gigi) 2018 september; 51(3): 129–132 research report introduction within the dental profession, one of the most common procedures performed is tooth extraction which may cause trauma to the blood vessels. after trauma occurs to the blood vessels, the hemostasis process, involving blood clotting on the walls of damaged blood vessels in order to prevent bleeding, commences. the process of postextraction wound healing can occasionally cause infections, possibly even leading to complications.1–4 patients require appropriate post-extraction management in order to reduce the possibility of complications and accelerate blood clotting, thereby promoting wound healing after extraction. the wound healing process itself is relatively complex, consisting of various processes and assisted by many cells, one of them being fibroblasts. fibroblasts are cells found in connective tissue responsible for the phagocytosis of bacteria. tgf-β (transforming growth factor β) and pdgf (plateletderived growth factor) stimulate fibroblast structures to become miofibroblasts located at the edges of ecm which promote wound closure in tissues. fibroblasts will appear in the wound area after three days with the number of fibroblast cells peaking on the seventh day after trauma.5–8 the avocado plant possesses the benefits of traditional remedies9 since almost all its constituent parts possess properties akin to those of such medicines. the leaves, fruit and seeds all have a high nutrient content. avocado dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p129–132 mailto:christiankhoswanto@hotmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p129-132 130khoswanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 129–132 leaves contain a variety of chemicals, including: flavonoid compounds, tannins, katekat, kuinon, saponin, steroids/ triterpenoids, glycosides, cyanogenic compounds, alkaloids and phenols.7–9 the aim of this study was to determine the effect of avocado leaf extract on fibroblast proliferation rates and inflamation indicators. materials and methods this study used rodent subjects to evaluate wound healing activity indicated by fibroblast proliferation. approval by the ethical board was granted (304/ hrecc. fodm/xii/2017). this study used a post-test only control group design with 24 male wistar rat subjects, 150-200 grams in weight and aged 2-3 months which were allowed to freely consume pellet food for one week. the sample was divided into four groups, a control group (n=6) and the treatment groups (n=6). in the control group, the subjects were given a 3% cmc na solution to synchronize the physiological state of their bodies which had no negative effect on their tissues or organs. the treatment groups had avocado leaf extract and 3% cmc na solution as a 0.1cc solvent inserted into their tooth sockets. both the control and treatment groups had their mandibula decapitated with all the required specimens being prepared on the 3rd and 7th days of the experiment period. fresh avocado leaves were obtained from and identified at upt materia medica, kota batu, east java. the leaves were washed thoroughly, dried and liquified in a blender with 96% ethanol solvent, placed in a tightly sealed jar for 24 hours and agitated in a digital agitator at 50 rpm. the resulting liquid extract was filtered by being passed through a cloth, inserted in an erlenmeyer tube and subsequently evaporated in a rotary evaporator for 90 minutes and stored in a freezer until required. a general anesthetic was administered to the subjects by means of chloroform inhalation. tooth extraction was performed on the left mandibular incisor using pliers after which irrigation was carried out using sterile aquades to remove the remaining debris. in order to stop postextraction bleeding, a sterile cotton roll was applied to the resulting socket. the treatment protocol adopted was that advocated by krinke whereby, following removal of the teeth and discontinuation of bleeding from the sockets, the subjects were treated.10 the treatment group was selected to have its mandibula decapitated and made into preparations on the 3rd and 7th days. decapitation of the mandible in the treatment group and preparation on the 3rd and 7th day were performed because fibroblasts appeared in the wound area three days after the trauma before peaking after seven days. on the 3rd and 7th days, a mandibular retrieval procedure was performed by anesthetizing the subjects in a glass gas chamber filled with 10% chloroform. the members of each group had their mandibula decapitated and appropriately disposed of. the decapitated mandibles were made into tissue preparation, before being stained with he (haematoxylin eeosin) and observed. histopathologic observation was performed by counting the number of fibroblasts under a light microscope at 400x magnification. data was analyzed by means of a one-way anova test with a 5% significance rate and subsequently with an lsd test to establish whether a significant difference existed.11,12 results the results in table 1 show that after a 3-day experimental period the number of fibroblasts in the treatment group had increased compared to that in the control group (figures 1 & 2). conversely, after seven days the number of fibroblast cells in the treatment group was lower than that in the control group (figures 3 & 4). table 1 shows the extent of fibroblast proliferation on day 3 was significantly different in the wounds in the treatment group and the control group, while on day 7 no such significant difference was observed between the two groups. discussion tooth extraction will result in a wound which then undergoes a healing process consisting of a series of complex processes involving a number of cells, cytokines, growth factors and extracellular components that play a role in repairing damage to the hard tissue and soft tissue.2 the wound healing process is influenced by several factors including: bacterial infections, damage to the tissue, necrosis, hematoma (tissue bleeding), excessive movement of injured tissue, low blood supply and drug administration.8,13,14 the injured tissue rapidly experiences an acute inflammatory reaction. the inflammatory phase precedes healing and wound immobilization. the instantaneous acute inflammatory phase is characterized by the exudation of plasma proteins and neutrophils. the chronic inflammatory phase is characterized by the presence of chronic inflammatory cells (macrophages, lymphocytes, and plasma cells).7 in this study, it was observed that the table 1. mean amount of fibroblast in the treatment group and control group group x±sd day 3 x±sd day 7 k1 3.00b 14.33±0.89 b±0.51 k2 20.33a±1.75 10.16b±0.75 note: different superscript showed significance difference (p < 0.001) k1: control group k2: treatment group dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p129–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p129-132 131 khoswanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 129–132 proliferation phase begins on the third day and can last for several weeks.2 in the proliferation phase, neutrophil cells digest bacteria, then release intracellular enzymes into the surrounding matrix before expiring. monocytes will move from the blood capillaries into the ecm, transforming into macrophages which are then mediated by the inflammatory mediator tgf β. tgf β activates fibroblast cells and stimulates collagen deposition by increasing collagen synthesis. with the synthesis of collagen by fibroblasts, the formation of the epithelial layer will be enhanced by regulating the balance between it and the granulation tissue.10,15,16 as a result, the mucous epithelium and collagen layer will form.2 acceleration of the wound healing process can be confirmed by the presence of several indicators, one of which is the number of fibroblasts. fibroblasts are key to the proliferative phase of wound healing, such as destroying fibrin clot, forming collagen, elastin, glycosaminoglycan and proteoglycans induced by tgf-β to form a new extracellular matrix to close the wound and affect the reepithelization process in the wound.10 thus, as indicated in this study, the more fibroblasts appear in the socket sample, the more rapid the wound healing process might be.2 this study showed that on the third day an increase in the number of fibroblast cells occurred due to active substances such as flavonoids contained in the avocado leaves (persea americana mill) that have an antiinflammatory effect through inhibition of cyclooxygenase and lipoxygenase. in this manner, they are able to limit the number of inflammatory cells that migrate to the wound area. flavonoids play an important role in maintaining permeability and increasing capillary vascular resistance. therefore, flavonoids are present in pathological conditions such as disruption to the permeability of the blood vessel walls. flavonoids and phenol substances in avocado leaves accelerate wound healing through antioxidant mechanisms which inhibit the activity of free radicals to donate hydrogen atoms and bond to unstable free radicals that can cause damage to cell membranes and impede cell functioning. the existence of this bond will render free radicals more stable, thereby reducing damage to cell membranes and enabling the proliferation phase to proceed more rapidly. this reduces the duration of the inflammatory reaction, induces earlier tgf-β proliferation and results in the production of fibroblasts. in addition, avocado leaves also contain tannins which are active substances that increase the formation of fibroblast cells and capillary blood vessels, d figure 1. fibroblasts hpa in control group; 3rd day (400x magnification) figure 2. fibroblasts after the application of avocado leaves extract for 3 days (400x magnification) figure 3. fibroblasts from the control group; 7th day (400x magnification) figure 4. fibroblasts after the application of avocado leaves extract for 7 days (400x magnification) dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p129–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p129-132 132khoswanto, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 129–132 causing growth factor to stimulate the proliferation of fibroblast cells.1,13,17 other content of avocado leaves (persea americana mill) includes saponin, another active substance, which increases monocyte proliferation and can augment the number of macrophages that will secrete growth factors such as egf, fgf, pdgf and tgf-β. these, in turn, can stimulate the migration to and proliferation of fibroblasts in the wound area in order to more rapidly synthesize collagen.1,15 this study showed a decrease in the number of fibroblast cells on the seventh day. due to a significant increase in fibroblast cell production on day 3, fibroblasts are sufficient to synthesize collagen. this has the result that, on day 7, the number of fibroblast cells decreases as they are transformed into myofibroblasts located on the ecm margins of wound tissue closure.10,18 this study showed that avocado leaves (persea americana mill) topically applied to the post-extraction socket were capable of accelerating the amount of fibroblast present in the wound healing process in wistar rat tooth sockets on day 3. references ardiana t, kusuma apk, firdausy md. efektivitas pemberian gel1. binahong (adredera cordifolia) 5% terhadap jumlah sel fibroblast pada soket pasca pencabutan gigi marmut (cavia cobaya). odonto dent j. 2015; 2: 64–70. barrientos s, stojadinovic o, golinko ms, brem h, tomic-canic2. m. growth factors and cytokines in wound healing. wound repair regen. 2008; 16(5): 585–601. diegelmann rf, evans mc. wound healing: an overview of acute,3. fibrotic and delayed healing. front biosci. 2004; 9: 283–9. guo s, dipietro la. factors affecting wound healing. j dent res.4. 2010; 89(3): 219–29. guyton ac, hall je. guyton dan hall buku ajar fisiologi kedokteran.5. 11th ed. jakarta: egc; 2008. p. 480-1. hargreaves km, goodis he. seltzer and bender’s dental pulp.6. chicago: quintessence publishing; 2002. p. 13, 63-4. hartono fa, prabowo pb, revianti s. aplikasi gel kitosan berat7. molekul tinggi dan rendah terhadap ketebalan epitel mukosa pada proses penyembuhan luka pencabutan gigi. dent j kedokt gigi. 2015; 9: 1–10. ibsen oac, phelan ja. oral pathology for the dental hygienist. 58. th ed. philadelphia: elsevier saunders; 2009. p. 88-91. irawati nav. antihypertensive effects of avocado leaves extract9. (persea americana mill). majority. 2015; 4: 44–8. 10. khoswanto c, soehardjo i. the effect of binahong gel (anredera cordifolia (ten.) steenis) in accelerating the escalation expression of hif-1α and fgf-2. j int dent med res. 2018; 11(1): 303–7. 11. arciero j, swigon d. equation-based models of wound healing and collective cell migration. in: complex systems and computational biology approaches to acute inflammation. new york: springer; 2013. p. 185–207. 12. krinke g. the laboratory rat. new york: academic press; 2000. p. 45-50, 295-6. 13. neagos d, mitran v, chiracu g, ciubar r, iancu c, stan c, cimpean a, iordachescu d. skin wound healing in a free floating fibroblast poplated collagen lattice model. rom j biophys. 2006; 16(3): 157–68. 14. nijveldt rj, van nood e, van hoorn de, boelens pg, van norren k, van leeuwen pa. flavonoids : a review of probable mechanism of action and potential applications. am j clin nutr. 2001; 74(4): 418–25. 15. orsted hl, keast d, forest-lalande l, françoise m. basic principles of wound healing an understanding of the basic physiology of wound healing provides. wound care canada. 2012; 9(2): 4–13. 16. putra atw, ade w, hamidy my. tingkat kepadatan fibroblas pada luka sayat mencit dengan pemberian gel lidah buaya (aloe chinensis baker). 2013; : 1–8. 17. rowe rc, sheskey pj, owen sc, american pharmacists association. handbook of pharmaceutical excipients. 6th ed. usa: pharmaceutical press; 2009. p. 918. 18. sheikh aa, sayyed z, siddiqui ar, pratapwar as, sheakh ss. wound healing activity of sesbania grandiflora linn flower ethanolic extract using excision and incision wound model in wistar rats. int j pharmtech res. 2011; 3(2): 895–8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p129–132 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p129-132 mkg vol 41 no 4 oct-dec 2008.indd 164 vol. 41. no. 4 october–december 2008 research report the surface roughness difference between microhybrid and polycrystalline composites after polishing eric priyo prasetyo, karlina samadi, and cecilia gerda juliani lunardhi department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract background: one of the success criteria for a composite resin restorative material is the surface roughness that can be achieved through polishing. considering that there are so many types of composite resin materials on the market, including polycrystalline composites, information on this type of composite’s surface roughness is needed. purpose: the aim of this laboratory experiment was to compare the surface roughness difference between microhybrid and polycrystalline composite after polishing. methods: in order to obtain this, a laboratory experiment was done. four groups of composites were produced, the first two groups consist of microhybrid composite and the second two groups consist of polycrystalline composite. two groups with the same material were treated with two different treatments as follows: the first group was not finished (the surface is under celluloid matrix), the second group was finished and polished. after these treatments, each sample’s surface was measured using surface roughness measuring instrument and then the results: were analyzed statistically using independent t-test (α = 0.05). conclusion: the result showed that after polishing, the surface roughness of polycrystalline composite is lower than that of microhybrid composite. key words: surface roughness, microhybrid composite, polycrystalline composite correspondence: eric priyo prasetyo, c/o: departemen ilmu konservasi gigi, fakultas kedokteran gigi universitas airlangga, jl. mayjend. prof. dr. moestopo no. 47, surabaya 60132, indonesia. e-mail: ep_prasetyo@yahoo.com introduction in the mastication system, teeth have a major function. today, beside this function, patients tend to correlate teeth with esthetics on how to make their teeth look better.1,2 in conservative dentistry, this is surely connected with the right choice of supporting dental instruments and materials. considering this, currently there are many esthetic-based dental instruments and materials developed in the market.3, 4 in relation with esthetics, to get a natural dental restoration, a restorative material should bear a surface roughness and gloss equal to natural enamel.5 based on clinical observations, composite-resin restorations usually change in color and prone to secondary caries. these are caused by massive plaque and food pigments deposition caused by rough restoration surfaces as a result from incomplete finishing and polishing.6 according to previous studies against microhybrid composite-resins, finishing and polishing with various instruments and methods have not satisfactorily provided an acceptable natural smooth surface.7 this makes the availability of better composite-resin types and polishing instruments in high demand.5–8 composite resins are considered as one of highly acceptable esthetic materials because of their wide varieties of natural shade selection.9 the most widely used composite resin is the microhybrid type because of its wide range of applications, but this type of composite-resin has softer matrix with irregular filler shape and size, resulting a higher inter-filler space, therefore finishing and polishing would abrade the softer matrix and leave the un-abraded harder filler, hence resulting a rough surfaced restoration. a new composite resin type that has been introduced lately is the polycrystalline composite-resin, containing phenolic-epoxyne (pex) liquid crystal matrix and glassceram silica fillers. 165prasetyo, et al.: the surface roughnes difference between microhybrid this polycrystalline composite resin was introduced to have better physical properties compared to regular composites and the use is clinically multi purpose.10 this composite resin contains nano-sized particles and has tight filler-matrix structure in nano-cluster, resulting in high consistency and density, therefore finishing and polishing might not leave filler un-abraded and resulting a smooth surface, since finishing and polishing is an integral part that must be done in making such restorations. polycrystalline composite resin is a new product in the market and there are limited experiments and references regarding this type of composite resin, therefore more information about this composite resin is needed. considering that there are so many types of composite resins available in the market, dentists as clinicians need information to choose the right materials which have better properties, and applicable in their daily practice. according this background, information about the surface roughness between microhybrid and polycrystalline composite resin after polishing is needed. the purpose of this article is to know the surface roughness difference between microhybrid and polycrystalline composite resins after polishing. materials and methods materials used in this experiment were esthetx (dentsply, usa), diamond lite (drm research laboratories inc., usa), sharpcut fine finishing diamond bur no.503 ef (dentsply, usa), astropol (ivoclarvivadent, liechtenstein), cotton pellet, distilled aqua, glue, and marker (bic, france). whereas the instruments used in this experiment were curing light qhl 75 (dentsply, usa), e type low speed contra angle (nsk nakanishi inc., japan), micromotor (nsk nakanishi inc., japan), dental unit (belmont, japan), pana air high speed contra angle (nsk nakanishi inc., japan), jar and tweezers (kohler, germany), scale and timer (tanita, japan), celluloid matrix (svenska dentorama, sweden), glass slab and acrylic mould (3m, usa), plastic filling instrument (dentsply, usa), and surface roughness measuring instrument (hahn&kolb tesa, germany). the samples prepared for this experiment were discshaped composite resins in 8 mm diameter and 3 mm thickness.11 in order to produce the composite resin samples according to the certain criteria, 8 mm diameter holes were made in a 3 mm thick acrylic sheet. this acrylic sheet was then used as mould. composite resins were screwed out from the syringe packaging to make 28 composite resin samples. fourteen samples were prepared for microhybrid (m) group and fourteen others were prepared for polycrystalline (p) group. these composite resins were applied to the mould using plastic filling instrument. after the mould has been filled thoroughly with composite resins, the upper part of the mould were covered with celluloid matrix and glass slab and then weighted with 1 kilograms of weigh for 60 seconds. the remaining excess of composite resins was cleaned. these composite resin samples were then cured with curing light for 20 seconds as directed by the manufacturer. after curing, these composite resin samples were taken out from the mould. before finishing and polishing procedure, seven composite resin samples of each group were taken and called m0 for samples with microhybrid composites and p0 for samples with polycrystalline composites. on the remaining seven composite resin samples of each group according to the grouping, initial finishing were done using fine finishing diamond bur with 30.000 rpm speed, 50 grams of pressure (these actions were performed under the help of a digital scale), and simultaneous one-direction movements for 10 seconds (5 movements). after that, polishing were done using astropol each with 10,000 rpm speed, 50 grams of pressure (these actions were also performed under the help of a digital scale), and simultaneous one-direction movements for 60 seconds (30 movements). these treated groups (finished and polished) were taken and called m1 for samples with microhybrid composites and p1 for samples with polycrystalline composites. each composite resin sample were coded according to grouping and were immersed in a jar containing distilled aqua and stored in an incubator at 37° c temperature. twenty-four hours afterwards these samples were measured using surface roughness measuring instrument. the surface roughness of each sample’s flat surface was measured using surface roughness measuring instrument with 100 times magnification and 2 mm range. result based on the result of 28 samples using surface roughness measuring instrument, several series of data were recorded and tested for the normality using one-sample kolmogorov-smirnov test. further statistical analysis was performed using independent t-test with 95% level of accuracy between sample groups. the independent t-test results between sample groups could be seen on table 1. table 1. statistical analysis result of the surface roughness between microhybrid and polycrystalline before initial finishing and after polishing sample group mean ± standard deviation significance m0 0,6571 ± 0,0976 p = 0,789 (> 0,05) not significantp0 0,6429 ± 0,0976 m1 0,9286 ± 0,0951 p = 0,007 (< 0,05) significantp1 0,7857 ± 0,0690 m0 0,6571 ± 0,0976 p = 0,000 (< 0,05) significantm1 0,9286 ± 0,0951 p0 0,6429 ± 0,0976 p = 0,008 (< 0,05) significantp1 0,7857 ± 0,0690 166 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 164–166 according to table 1, independent t-test between m0 and p0 groups show no significant difference (p > 0.05). independent t-test between m1 and p1, m0 and m1, p0 and p1 groups showed significant differences (p < 0.05). discussion based on the result on this experiment involving microhybrid and polycrystalline composite resin samples, it was known that the surface roughness between microhybrid and polycrystalline before initial finishing has no significant difference in surface roughness, this indicate that both microhybrid and polycrystalline has equal surface roughness. this could happen since the surface of both microhybrid and polycrystalline composite resins follow the smoothness of celluloid matrix which was originally smooth. this condition appeared on the smooth-glossy surface of each corresponding samples, this was supported by the mean measurement result which was the lowest among all other groups. this phenomenon was following the basic nature of composite resins. there was significant difference on the sample groups between the surfaces of microhybrid and polycrystalline composite resins after polishing. the greater mean on the surface roughness of microhybrid composite resin showed that after polishing, microhybrid composite resins has rougher surfaces, and otherwise, the polycrystalline composite resins has smoother surfaces. the rough surface of microhybrid composite were caused by prominent composite resin filler particles which was hard and could not be abraded by abrasive particles of the finishing instrument during polishing procedure.6 the smoother polycrystalline composite resin surface was caused by the smoother composite resin filler particles, regular shaped and high density filler particle composition. the abraded matrix surface during polishing procedure was equal to the abraded filler particles, therefore producing a lower surface roughness.10 the experiment on sample groups between microhybrid and polycrystalline composite resin’s surfaces before initial finishing and after polishing were significant, with average mean surface roughness higher than before initial finishing (surface under the celluloid matrix) means that the surface roughness under the celluloid matrix was the smoothest surface compared to all other groups. this phenomenon revealed that polishing modifies the surface of composite resins.12 the modification was resulted from the abrasion of matrix and filler particles sequentially by coarser to smoother abrasive particles of the polishing instrument.13 furthermore, this was happened because the smoother matrix would be abraded first and afterwards smoother the surface achieved by the previous polishing instrument, repeatedly until the last step of polishing by the smoothest abrasive particles of the polishing instrument producing the lowest surface roughness. polishing caused the surface of composite resins both microhybrid and polycrystalline coarser if compared to the surface under celluloid matrix surface. however polishing is always an important step in making any restorations because the end result would still smoother if compared to without polishing at all after initial finishing, in consideration that initial finishing is needed to take away excess or residue of composite resins and to form the appropriate dental anatomy during the restoration-making procedure. therefore finishing and polishing are clinically integral steps, thus must be carried out one after another in making every composite resin restoration. references 1. spear fm, kokich vg, mathews dp. interdisciplinary management of anterior dental esthetics. journal of the american dental association. 2006; 137:160-9. 2. robison b. extreme makeover: practice edition. mouth 2004; 24(4): 5. 3. lacy am. single visit, indirect, esthetic composite resin restorations for anterior teeth. advances in aesthetic and restorative dentistry 2001; 5–10. 4. chantaramungkorn m. creativity with direct composite resin: anatomic and color stratification technique. advances in aesthetic and restorative dentistry 2003; 5–7. 5. willems g, lambrechts p, braem m, vanherle g. composite resins in the 21st century. quintessence international 1993; 24(9): 641–58. 6. setcos jc, tarim b, suzuki s. surface finish produced on resin composites by new polishing systems. quintessence international 1999; 30(3): 169–73. 7. prasetyo ep, lunardhi cgj, sukaton. the effectiveness of pogo tm one step diamond micro polisher as a composite resin restoration polishing instrument. dental journal (amajalah kedokteran gigi) 2003; 36(4): 125–8. 8. krejci i, lutz f, boretti r. resin composite polishing-filling the gaps. quintessence international 1999; 30(7): 490–5. 9. nash rw, lowe ra. recreating nature using today’s composite materials. apdn havas medimedia 2001; 58: 4–12. 10. megantara pu. diamondcrown-chairside: the elite of dental restoratives. drm research laboratories, inc. 2005. 11. neme al, frazier kb, roeder lb, debner tl. effect of prophylactic polishing protocols on the surface roughness of esthetic restorative materials. operative dentistry 2002; 27: 100–4. 12. vichi a, ferrari m, davidson cl. color and opacity variations in three different resin-based composite products after water aging. journal of the academy of dental materials 2004; 20: 530–4. 13. ivoclar-vivadent clinical. astropol finishing, polishing, and high gloss polishing system: instructions for use. ivoclar vivadent ag. 2003. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice mkg vol 42 no 2 april 2009.indd dental journal (majalah kedokteran gigi) publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, inovative thinking in dentistry. they also support scientific advancement, education and dental practice. manuscript should be written in english or in indonesian. authors should follow the manuscript preparation guidelines. i. research reports preparation guidelines the text of research report should be devided into the following sections: • title, should be brief, specific and informative. include a short title (not exceeding 40 letters and spaces). • name of author(s), should include full names of authors, address to which proofs are to be sent, name and address of the departement(s) to which the work should be attributed. • abstract, concise description (not more than 250 words) of the background, purpose, methods, results and conclusions required. key words (3–5 words) should be provided below the abstract. • introduction, comprises the problem’s background, its formulation and purpose of the work and prospect for the future. • materials and methods, containing clarification on used materials and schema of experiments. method to be explained as possible in order to enable others examiners to undertake retrial if necessary. reference should be given to the unknown method. • result, should be presented in logical sequence with the minimum number of tables and illustrations n e c e s s a r y f o r s u m m a r i z i n g o n l y i m p o r t a n t observations. the vertical and horizontal line in the table should be made at the least to simplify of view. mathematical equations, should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers, should be separated by point (.) for english-written-manuscript, and be separated by comma (,) for indonesian-written manuscript. tables, illustration, and photographs should be cited in the text in consecutive order. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. explain in footnotes all nonstandard abbreviations that are used. • d i s c u s s i o n , e x p l a i n i n g t h e m e a n i n g o f t h e examination’s results, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work and suggestion for further studies if necessary. • acknowledgements, to all research contributors, if any, should be stated in brief at the manuscript, prior to references. • references, should be arranged according to the vancouver system. references must be identified in the text by the superscript arabic numerals and numbered in consecutive order as they are mentioned in the text. the reference list should appear at the end of the articles in numeric sequence. examples: 1) grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20:355–6. 2) cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. 3) morse ss. factors in the emergence of infectious disease. emerg infect dis [serial online] 1995 jan-mar; 1(1):[24 screens]. available from: url:http://www/ cdc/gov/ncidoc/eid/eid.htm. accessed december 25, 1999. 4) bennett gl, horuk r. iodination of chemokines for use receptor binding analysis. in: horuk r, editor. chemoking receptors. new york: academic press; 1997. p. 134–48. 5) amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 6) salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. ii. reviews article preparation guidelines the text of literature reviews should be devided into the following sections: title, name of author(s), abstract, introduction, overview, discussion that ended by conclusion & suggestion, references. iii. case reports preparation guidelines the text of case reports should be devided into the following sections: title, name of author(s), abstract, introduction, case(s), case management(s) that completed with photograph/descriptive illustrations, discussion that ended by conclusion & suggestion, references. photographs could be clear or glossy. color or black and white photographs must be submitted for both illustrations and graphs. photographs should be prepared with the minimum size of 125 × 195 mm. the manuscript should be submitted in a floppy disc or compact disc and be typed using ms word program. three notes to authors editor -editor legible photocopies or an original plus two legible copies of manuscript which are typed double space with wide margins on good quality a4 white paper (210 × 297 mm) should be enclosed. the length of article should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. the editor reserves the right to edit manuscript, fit articles into available, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscript and their accompanying illustration become the permanent property of publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from publisher. all datas, opinion or statement appear on the manuscript are the sole responsibility of the contributor. accordingly, the publisher, the editorial board, and their respective employees of the dental journal accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinion, or statement. ethical clearance should be attached on research report and case report article. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 50, number 3, september 2017 deoxypyridinoline and mineral levels in gingival crevicular fluid as disorder indicators of menopausal women with periodontal disease · dental care for children with autism spectrum disorder · potency of garcinia mangostana l peel extract combined with demineralized freeze bovine bone xenograft on il-1β expression, osteoblasts, and osteoclasts in alveolar bone p-issn: 1978-3728 e-issn: 2442-9740 volume 50, number 3, september 2017 editorial boards of dental journal (majalah kedokteran gigi) sk: 275/un3.1.2/2017 may 5th – december 31st, 2017 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (department of pediatric dentistry faculty of dental medicine, universitas airlangga) editorial boards roeland jozef gentil de moor (department of restorative dentistry and endodontology, dental school, ghent university, belgium); cortino sukotjo (university of illinois at chicago college of dentistry, department of restorative dentistry, chicago, united states); guang hong (liaison center for innovative dentistry, graduate school of dentistry, tohoku university, japan); kenji yoshida (department of oral and maxillofacial surgery, school of dentistry, aichi gakuin university, nisshin, japan); miguel rodrigues martins (co-worker aachen dental laser center, rwth aachen university, aachen, germany); sajee sattayut (department of oral surgery, faculty of dentistry, khon kaen university, khon kaen, thailand); samir nammour (department of dental science, faculty of medicine, university of liege, belgium); reza fekrazad (laser reseach center in medical science, dental faculty, aja university of medical science, tehran, iran); hong sai loh (department of oral and maxillofacial surgery, faculty of dentistry, national university of singapore, singapore); widowati witjaksono (kulliyah of dentistry, international islamic university malaysia, malaysia); hamid nurrohman (missouri school of dentistry & oral health a.t. still university 800 w. jefferson st. kirksville, missouri, usa, united states); harry huiz peeters (laser research center, bandung, indonesia); rahmi amtha (department of oral medicine, faculty of dentistry, universitas trisakti, indonesia); elza ibrahim auerkari (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); r. darmawan setijanto (department of dental public health, faculty of dental medicine, universitas airlangga, indonesia); anita yuliati (department of dental material, faculty of dental medicine, universitas airlangga, indonesia). managing editors rostiny (department of prosthodontics, faculty of dental medicine, universitas airlangga, indonesia); sianiwati goenharto (faculty of vocation, universitas airlangga, indonesia); ketut suardita (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); hendrik setia budi (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia). assistant editors eric prasetyo (department of conservative dentistry, faculty of dental medicine, universitas airlangga, indonesia); saka winias (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia). peer-reviewers harmas yazid yusuf (department of oral surgery, faculty of dentistry, universitas padjadjaran, indonesia); boy m. bachtiar (department of oral biology, faculty of dentistry, universitas indonesia, indonesia); pinandi sri pudyani (department of orthodontics, faculty of dentistry, universitas gadjah mada, indonesia); al. supartinah santoso, (department of pediatric dentistry, faculty of dentistry, universitas gadjah mada, indonesia); mieke sylvia m.a.r (department of odontology forensic, faculty of dental medicine, universitas airlangga, indonesia); jenny sunariani (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); els sunarsih budipramana (department of pediatric dentistry faculty of dental medicine, universitas airlangga); chiquita prahasanti (department of periodontic, faculty of dental medicine, universitas airlangga, indonesia); i.b. narmada (department of orthodontics, faculty of dental medicine, universitas airlangga, indonesia); theresia indah budhy (department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); wisnu setyari (department of oral biology, faculty of dental medicine, universitas airlangga, indonesia); david kamadjaja (department of oral surgery and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); haryono utomo (dental hospital, faculty of dental medicine, universitas airlangga, indonesia); ernie maduratna s. (department of periodontic, faculty of dental medicine, universitas airlangga, indonesia); agung krismariono (department of periodontic, faculty of dental medicine, universitas airlangga, indonesia); ni putu mira s. (de-department of oral surgery and maxillofacial, faculty of dental medicine, universitas airlangga, indonesia); nurina f. ayuningtyas (department of oral medicine, faculty of dental medicine, universitas airlangga, indonesia); administrative assistant: novi dian prastiwi (faculty of dental medicine, universitas airlangga); abdullah mas’udy (faculty of dental medicine, universitas airlangga) editorial address: faculty of dental medicine universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (+6231) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 32a/e/kpt/2017 cover photo purchased from: www.fotolia.com invoice number: 208879494-206415982 contents page printed by: airlangga university press. (rk 334/08.17/aup-b1e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aup.unair@gmail.com volume 50, number 3, september 2017 p-issn: 1978-3728 e-issn: 2442-9740 1. correlation between reactive oxygen species and oral conditions in elderly individuals with hypertension: a preliminary study nanan nur’aeny, wahyu hidayat, and indah suasani wahyuni ................................................ 111–115 2. automation of gender determination in human canines using artificial intelligence f. fidya and bayu priyambadha..................................................................................................... 116–120 3. fgf-2 expression and the amount of fibroblast in the incised wounds of rattus norvegicus rats induced with mauli banana (musa acuminata) stem extract didit aspriyanto, intan nirwana, and hendrik setia budi .......................................................... 121–126 4. comparison of esthetic smile perceptions among male and female indonesian dental students relating to the buccal corridors of a smile astriana nurfitrah, c. christnawati, and ananto ali alhasyimi......................... ...................... 127–130 5. deoxypyridinoline and mineral levels in gingival crevicular fluid as disorder indicators of menopausal women with periodontal disease agustin wulan suci dharmayanti and banun kusumawardani ................................................. 131–137 6. grafting effectiveness of anadara granosa shell combined with sardinella longiseps gel on the number of osteoblast-osteoclast cells eddy hermanto, rima parwati sari, asri cahyadita dwi imaniar, and kevin anggoro ........ 138–143 7. a comparison of class i malocclusion treatment outcomes with and without extractions using an abo grading system for dental casts and radiographs bunga ayub rukiah, amalia oeripto, and nurhayati harahap ................................................. 144–148 8. rankl expressions in preservation of surgical tooh extraction treated with moringa (moringa oleifera) leaf extract and demineralized freeze bovine bone xenograft s. soekobagiono, adrian alfiandy, and agus dahlan ................................................................. 149–153 9. gingival enlargement as oral manifestation in acute myeloid leukemia patient sandra olivia kuswandani, yuniarti soeroso, and sri lelyati c. masulili .............................. 154–159 10. dental care for children with autism spectrum disorder amrita widyagarini and margaretha suharsini ........................................................................... 160–165 11. potency of garcinia mangostana l peel extract combined with demineralized freeze-dried bovine bone xenograft on il-1β expression, osteoblasts, and osteoclasts in alveolar bone imam safari azhar, utari kresnoadi, and retno pudji rahayu................................................. 166–170 vol 38-no 1-2005 20 sitotoksisitas bahan restorasi cyanoacrylate pada variasi perbandingan powder dan liquid menggunakan mtt assay (cytotoxicity of the cyanoacrylate restoration material with variation of powder and liquid ratio by using mtt assay) asti meizarini bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract the requirements for dental material include not toxic, not irritant, no carcinogenic potential, nor cause an allergic response with the use in oral cavity. the cyanoacrylate restoration material has certain substance that can be toxic. because of the ratio amount of powder and liquid is not known, it can lead the restoration more toxic. the purpose of this study was to know the cytotoxicity of the cyanoacrylate restoration material with different variation of powder and liquid ratio using mtt assay. six cylinder samples of 5 mm in diameter and 2 mm in thickness were used for each group of 1:1.00; 1:0.75; 1:0.50 powder and liquid ratio of cyanoacrylate restoration materials. each of samples was immersed in eppendorf micro tubes consisting of media culture. after 24 hour, the immersion of media culture was used to investigate the cytotoxic effect to bhk-21 cell lines by mtt assay method. the density of optic formazan indicated the amount of living cells. all data were statistically analyzed by one-way anova and hsd. the results showed that the percentage of living cells amount of powder and liquid ratio 1:1.00; 1:0.75; 1:0.50 were 98.59%; 95.76%; 94.92% respectively. there was a significant difference between 1:1.00 and 1:0.50 group ratio. the conclusion was that the cytotoxicity between 1:1.00 and 1:0.50 powder and liquid ratio of cyanoacrylate restoration materials in this study decreased. key words: cytotoxicity, cyanoacrylate restoration material, powder and liquid ratio, mtt assay korespondensi (correspondence): asti meizarini, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan bahan restorasi cyanoacrylate adalah bahan kedokteran gigi yang indikasinya dapat dipakai untuk berbagai macam kegunaan dalam bidang restorasi, antara lain untuk menumpat, memperbaiki facing porselen yang pecah, veneer, splinting gigi geligi. bahan restorasi cyanoacrylate terdiri dari campuran powder polymethyl methacrylate dan liquid cyanoacrylate ditambah aselerator methyl methacrylate untuk mengeraskan. waktu prosesing sangat tergantung pada jumlah powder yang digunakan. jumlah powder yang dicampur ke liquid dikatakan tidak berpengaruh pada kekerasan akhir. kontra indikasi dan kelainan yang ditimbulkan belum diketahui. 1 perbandingan takaran powder dan liquid tidak disebutkan. powder dan aselerator pada bahan restorasi cyanoacrylate mempunyai kandungan yang sama dengan bahan restorasi resin akrilik. resin akrilik sebagai bahan restorasi adalah hasil polimerisasi dari powder polymethyl methacrylate dan cairan monomer methyl methacrylate. di bidang kedokteran gigi, pemakaian restorasi resin akrilik secara direct jenis ini telah ditinggalkan, karena mempunyai banyak kekurangan diantaranya efek monomer sisa dan terjadi penyusutan yang besar oleh karena proses polimerisasi.2 methyl methacrylate dapat juga mengiritasi pulpa dengan cara berdifusi melalui tubuli dentin.3 cyanoacrylate dapat juga digunakan sebagai tissue adhesive (pelekat jaringan). adanya air atau cairan tubuh menyebabkan ethyl-2-cyanoacrylate berpolimerisasi dengan cepat membentuk lapisan tipis yang dapat melekatkan tepi jaringan atau kulit dengan erat, 2 menit setelah setting. empat alasan penunjang dipakainya bahan ethyl-2-cyanoacrylate sebagai pelekat jaringan, adalah karena mempunyai efek hemostatik baik, bakteriostatik dan sebagai bakterisidal, kemampuan polimer melekat dengan erat pada jaringan hidup dan mendapatkan estetik yang baik selama proses penyembuhan luka.4 sebaliknya, ada pendapat lain yang menyebutkan bahwa penggunaan cyanoacrylate terbatas karena dapat menyebabkan degradasi dalam sistem biologis dan terjadi iritasi lokal.5 ethyl-2-cyanoacrylate bila kontak dengan alkohol, amine atau air dapat menyebabkan polimerisasi dan hasil degradasinya termasuk formaldehyde, dekomposisi termalnya dapat termasuk hydrogen cyanide, oksida dari karbon dan nitrogen.6 salah satu syarat bahan yang digunakan dalam bidang kedokteran gigi seharusnya tidak toksik, tidak mengiritasi 21meizarini: sitotoksisitas bahan restorasi cyanoacrylate dan harus mempunyai sifat biokompatibilitas atau bahan yang diproduksi tidak boleh mempunyai efek yang merugikan terhadap lingkungan biologis, baik lokal maupun sistemik.7 uji sitotoksisitas adalah bagian dari evaluasi bahan kedokteran gigi dan diperlukan untuk prosedur screening standart. salah satu metode untuk menilai sitotoksisitas suatu bahan adalah dengan uji enzimatik menggunakan pereaksi 3-(4,5-dimethylthiazol2-yl) 2,5-diphenyl tetrazolium bromide (mtt).8 dasar uji enzimatik mtt adalah dengan mengukur kemampuan sel hidup berdasarkan aktivitas mitokondria dari kultur sel. uji ini banyak digunakan untuk mengukur proliferasi seluler secara kuantitatif atau untuk mengukur jumlah sel yang hidup. berdasarkan hal tersebut di atas, maka timbul permasalahan apakah pemakaian perbandingan takaran powder dan liquid bahan restorasi cyanoacrylate yang berbeda akan mempunyai efek sitotoksisitas yang berbeda pula bila diuji mengunakan uji mtt (mtt assay). tujuan penelitian ini untuk mengetahui sitotoksisitas bahan restorasi cyanoacrylate akibat perbedaan perbandingan takaran powder dan liquid dengan menggunakan mtt assay. manfaatnya untuk memberi informasi ilmiah kepada dokter gigi dan masyarakat mengenai sitotoksisitas bahan restorasi cyanoacrylate, khususnya pengaruh jumlah powder dan liquid bahan cyanoacrylate yang digunakan terhadap sitotoksisitasnya, karena tidak adanya perbandingan atau takaran powder dan liquid yang jelas, dikhawatirkan menyebabkan bahan restorasi menjadi toksik. bahan dan metode jenis penelitian eksperimental laboratoris, rancangan penelitian post test only controle group. penelitian dilakukan di laboratorium imunokimia bioteknologi, pusat antar universitas, universitas gajah mada yogyakarta pada bulan agustusseptember tahun 2004. bahan yang digunakan pada penelitian ini adalah bahan restorasi cyanoacrylate merek cyano veneer (meyerhaake-germany) terdiri dari powder berisi polymethylmethacrylate; fast/retarder berisi ethyl-2cyanoacrylate; quick berisi methylmethacrylate, ethylen glycoldimethacrylate dan n,n-dimethyl-p-toluidin (brosur), alkohol 70%, kultur cell line baby hamster kidney (bhk-21), media kultur berisi rosewell park memorial institute-1640 (rpmi-1640) 89%; penstrep 1%; fetal bovine serum (fbs) 10%; fungizone 100 unit/ml, pereaksi mtt, phosphat buffer saline (pbs), dan sodium dodecyl sulphate (sds). alat yang digunakan adalah plat kuningan untuk fiksasi, cetakan sampel diameter dalam 5 mm dan tinggi 2 mm, stop watch, timbangan digital, kuas, semen stopper, sonde, pisau model, plastic filling instrument, matrix strips, anak timbangan 500 gr, filter millipore 0,2 mm, flask, eppendorf, microplate, pipet mikro, pipet pasteur, spektrofotometer. pembuatan sampel adalah sebagai berikut, cetakan sampel berbentuk silinder, diameter dalam 5 mm dan tebal 2 mm,9 terbuat dari teflon, di fiksasi dengan plat kuningan. daerah kerja dibersihkan dengan alkohol 70% kemudian dikeringkan. bagian dasar daerah kerja di ulas selapis tipis fast menggunakan kuas. powder dicampur fast dengan perbandingan takaran sesuai dengan kelompok masingmasing 1 : 1,00; 1 : 0,75; 1 : 0,50. bahan campuran di aplikasikan ke dalam cetakan sampel dengan plastic filling instrument. bagian atas cetakan diberi matrix strips, ditekan dengan plat kuningan yang diberi beban seberat 500 gr selama 30 detik. beban, plat dan matrix diambil, setelah itu diberi setetes aselerator quick, kemudian diulas 1 lapis fast. setelah sampel mengeras dilepas dari cetakan, sehingga diperoleh sampel berbentuk silinder. untuk setiap kelompok dipakai 6 sampel. tahap pengujian sampel dilakukan dengan cara memasukkan sampel ke dalam eppendorf yang berisi media kultur 200 μl, direndam selama 24 jam dalam suhu ruang dan dikelompokkan sesuai dengan kelompok sampel. setelah 24 jam, sampel diambil, dilakukan sterilisasi bahan perendam dengan filter millipore ukuran 0,2 mm.10 hasil filter ditampung dalam eppendorf, siap digunakan untuk uji sitotoksisitas.11 kultur sel bhk-21 dipersiapkan sesuai petunjuk pada laboratorium biotek-pau dan microplate dengan 96 sumuran. setiap sumuran pada microplate diisi 100 μl sel dengan kepadatan 2 × 105. larutan bekas perendaman sampel cyanoacrylate yang telah di filter, ditambahkan ke dalam tiap sumuran sebanyak 50 μl, sesuai dengan kelompok sampel. disiapkan pula kontrol sel sebagai kontrol positif berisi sel dalam media kultur, dianggap persentase sel hidup 100% dan kontrol media sebagai kontrol negatif berisi media kultur saja, dianggap persentase sel hidup 0%. microplate di inkubasi 20 jam pada suhu 37° c, kemudian dikeluarkan dari alat inkubator, ditambahkan pereaksi mtt 5 mg/ml dalam pbs sebanyak 25 μl untuk setiap sumuran. di inkubasi kembali selama 4 jam. setelah inkubasi selesai ditambahkan larutan sds 10% sebanyak 80 μl pada setiap sumuran. tahap selanjutnya di sentrifuse 30 rpm selama 5 menit. nilai densitas optik formazan di hitung dengan spektrofotometer panjang gelombang 540 nm.8,12 untuk mengetahui persentase jumlah sel hidup dilakukan dengan memakai rumus:13 % sel hidup = 100% mediasel mediaperlakuan × + + keterangan: % sel hidup = persentase jumlah sel hidup setelah pengujian perlakuan = nilai densitas optik formazan pada setiap sampel setelah pengujian media = nilai densitas optik formazan pada kontrol media sel = nilai densitas optik formazan pada kontrol sel 22 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 20–24 data yang diperoleh ditabulasi, kemudian dilakukan analisis statistik menggunakan anova satu arah dengan taraf kemaknaan 5% dan dilanjutkan dengan tukey high significant difference (hsd). hasil nilai rerata densitas optik formazan, standart deviasi, persentase sel hidup bahan restorasi cyanoacrylate dapat dilihat pada tabel 1. tampak bahwa pada kelompok perlakuan, menunjukkan rerata nilai densitas optik formazan bahan restorasi cyanoacrylate yang semakin menurun sesuai dengan berkurangnya jumlah fast (ethyl-2cyanoacrylate). persentase sel hidup, yaitu persentase densitas optik ensim mitokondrial dehidrogenase pada kultul sel bhk-21 juga terjadi penurunan. probabilitas normalitas pada kolmogorov smirnov test menunjukkan semua kelompok mempunyai distribusi normal, karena didapatkan probabilitas normalitas lebih besar dari 0,05 (p > 0,05). setelah diketahui semua kelompok mempunyai distribusi normal, maka untuk mengetahui adanya perbedaan, dilakukan uji parametrik anova satu arah dengan taraf kemaknaan 0,05%. hasilnya dapat dilihat pada tabel 2. untuk mengetahui kelompok mana yang berbeda bermakna, maka dilakukan uji hsd pada α = 0,05 yang dapat dilihat pada tabel 3. kelompok perlakuan yang bermakna adalah yang mempunyai signifikansi kurang dari 0,05 (p < 0,05). tabel 1. nilai rerata densitas optik formazan, standart deviasi, persentase sel hidup dari uji toksisitas bahan restorasi cyanoacrylate dan probabilitas normalitas rasio powder:fast jumlah sampel rerata densitas optik formazan standart deviasi % sel hidup probabilitas normalitas kelompok i p:f 1 : 1,00 6 0,267 0,005 98,59% 0,408 kelompok ii p:f 1 : 0,75 6 0,257 0,003 95,76% 0,408 kelompok iii p:f 1 : 0,50 6 0,254 0,009 94,92% 0.613 kontrol sel (+) 6 0,272 0,009 100% 0,436 kontrol media (–) 6 0,082 0,002 0% 0,746 tabel 2. anova satu arah dari uji toksisitas bahan restorasi cyanoacrylate sumber variasi jumlah kuadrat derajat bebas rerata kuadrat f hitung probabilitas antar kelompok 0,157 4 0,03934 971,128 0,001 dalam kelompok 0,001013 25 0,00004051 total 0,158 29 tabel 3. uji hsd persentase sel hidup antar kelompok perlakuan dan kontrol sel kontrol sel kelompok i p:f 1 : 1,00 kelompok ii p:f 1 : 0,75 kelompok iii p:f 1 : 0,50 kontrol sel -- kelompok i p:f 1 : 1,00 tb (0,657) -- kelompok ii p:f 1 : 0,75 b tb (0,079) -- kelompok iii p:f 1 : 0,50 b b tb (0,923) -- keterangan : b = bermakna tb = tidak bermakna 23meizarini: sitotoksisitas bahan restorasi cyanoacrylate dari hasil uji hsd terlihat ada perbedaan persentase jumlah sel hidup antara kelompok i dengan iii dan antara kelompok kontrol sel dengan kelompok ii dan iii. pembahasan cyanoacrylate yang terkandung dalam bahan restorasi cyano veneer adalah ethyl-2-cyanoacrylate. pemakaian bahan restorasi cyano veneer merupakan kombinasi antara polymethyl methacrylate (powder) dengan ethyl-2cyanoacrylate(fast), kemudian diulas cairan methyl methacrylate(quick) dan terakhir ulasan ethyl-2cyanoacrylate(fast) lagi.1 pada pencampuran polymethyl methacrylate dengan cyanoacrylate terjadi kopolimerisasi, setelah monomer methyl methacrylate diulas pada permukaannya baru terjadi polimerisasi.14 pengaplikasian cairan quick yang mengandung monomer methyl methacrylate di atas campuran polymethyl methacrylate dengan ethyl-2-cyanoacrylate menimbulkan keraguan, apakah tidak ada reaksi lanjutan monomer yang bersifat toksik. salah satu persyaratan bahan kedokteran gigi untuk dapat diaplikasikan pada rongga mulut, harus bersifat biokompatibel, antara lain tidak mengandung substansi toksik.2,7 untuk membuktikannya, maka dilakukan uji sitotoksisitas secara in vitro pada kultur sel bhk-21 menggunakan mtt assay. kultur cell lines digunakan karena mempunyai keuntungan, yaitu pasase dapat dilakukan 50–70 kali, kecepatan pertumbuhan sel tinggi, integritas sel tetap terjaga dan sel mampu bermultiplikasi dalam suspensi. cell lines telah banyak digunakan untuk menguji toksisitas bahan dan obat-obatan di bidang kedokteran gigi, antara lain sel bhk-21 yang berasal dari fibroblas ginjal bayi hamster. sel fibroblas merupakan sel terpenting pada komponen pulpa, ligamen periodontal dan gingiva.8,15 hasil uji dengan menggunakan bhk-21 dapat dipakai sebagai dasar pengujian yang akurat.16 mtt adalah molekul larut berwarna kuning, yang dapat digunakan untuk menilai aktifitas ensimatik selular, didasarkan pada kemampuan sel hidup untuk mereduksi garam mtt. mekanismenya adalah garam tetrazolium berwarna kuning tersebut akan direduksi di dalam sel yang mempunyai aktifitas metabolik. mitokondria dari sel hidup yang berperan penting dalam hal ini, adalah yang menghasilkan dehidrogenase. bila dehidrogenase tidak aktif karena efek sitotoksik, maka formazan tidak akan terbentuk. jumlah formazan yang terbentuk, proporsional dengan aktifitas ensimatik sel hidup.13,17 perbandingan takaran powder dan liquid bahan restorasi cyanoacrylate yang dipakai dalam penelitian ini, didapatkan sesuai hasil penelitian pendahuluan dengan kriteria konsistensi hasil pengadukan polymethyl methacrylate dengan cyanoacrylate cukup kental dan punya kemampuan mengalir, sehingga dapat diaplikasikan ke kavitas dengan baik.14 berdasarkan tabel 2, adanya perbedaan persentase jumlah sel hidup antara kelompok i dengan iii, antara kelompok kontrol sel dengan kelompok ii dan iii, kemungkinan disebabkan karena kopolimerisasi tidak sempurna. pada kelompok i jumlah powder dan liquid fast sama banyak, ethyl-2-cyanoacrylate (liquid fast) sebagai bahan adesif, dapat membasahi dan mengikat polymethyl methacrylate (powder) dengan baik, ditandai dengan pengadukan campuran ini cukup mudah dan cepat homogen. pada kelompok ii dan iii, semakin sedikit jumlah ethyl-2-cyanoacrylate yang dipakai, sedang jumlah polymethyl methacrylate sama banyak dengan kelompok i, kemungkinan menyebabkan liquid ethyl-2cyanoacrylate tidak dapat mengikat semua powder polymethyl methacrylate yang ada. hal ini dapat dilihat pada pengadukan campuran powder dan liquid fast perbandingan 1:0,50 lebih sulit homogen dan hasil campuran lebih kental. pengaplikasian cairan quick yang mengandung monomer methyl methacrylate di atas campuran polymethyl methacrylate yang tidak terikat sempurna, kemungkinan menyebabkan terjadinya polimerisasi tersendiri antara polymethyl methacrylate dengan monomer methyl methacrylate. seperti telah diketahui bahwa polimerisasi polymethyl methacrylate dan monomer methyl methacrylate merupakan suatu proses yang pada kenyataan tidak pernah dapat berlangsung sempurna, sehingga pada akhir polimerisasi selalu terdapat sejumlah monomer yang tidak bereaksi menjadi polimer. monomer sisa selalu ada pada resin yang mengandung polymethyl methacrylate dan methyl methacrylate.2 aplikasi selanjutnya, ulasan 1 lapis fast yang mengandung ethyl-2-cyanoacrylate sebagai bahan pengisi dan pengeras, kemungkinan hal ini yang dapat menghambat bila ada pelepasan monomer ke permukaan restorasi, sesuai dengan pendapat yang menyatakan ethyl2-cyanoacrylate (c6h7no2) bereaksi cepat dengan air membentuk polimer padat.6 namun meskipun hasil akhir sampel diulas ethyl-2-cyanoacrylate, sehingga bila ada monomer sisa, akan tertutup ethyl-2-cyanoacrylate, ternyata pada hasil penelitian ada perbedaan nilai densitas optik formazan yang cenderung menurun sesuai dengan penurunan jumlah ethyl-2-cyanoacrylate pada campuran powder dan liquid fast. hal ini kemungkinan dapat terjadi disebabkan (poly)methyl methacrylate yang tidak bereaksi sempurna masih bisa menyerap air melalui proses imbibisi, 2 sehingga merembes ke permukaan dan menyebabkan kematian sejumlah sel, meskipun tidak banyak. hasil penelitian ini didapatkan sel hidup kelompok i = 98,59%, kelompok ii = 95,76%, kelompok iii = 94,9%. hasil uji anova didapatkan p < 0,05 berarti ada perbedaan bermakna yang disebabkan variasi perbandingan takaran powder dan liquid bahan restorasi cyanoacrylate. persentase jumlah sel hidup semua kelompok masih mendekati 100%, yang berarti restorasi cyanoacrylate belum dapat dikatakan toksik, karena masih di atas patokan toksisitas atau biokompatibilitas sesuai dengan pendapat telli et al.11 dan rubianto16 yang dipakai untuk penelitian ini. rubianto16 menggunakan patokan biokompatibilitas 24 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 20–24 yang baik adalah jumlah sel hidup 92,3%–100%. nilai ini lebih tinggi dibandingkan patokan telli et al.11 yang pada penelitiannya menyatakan bahwa parameter toksisitas berdasarkan cd50, artinya suatu bahan dikatakan toksik apabila persentase sel hidup setelah terpapar bahan tersebut, kurang dari 50%. masih banyaknya sel hidup dalam penelitian ini, dapat juga disebabkan karena ethyl2-cyanoacrylate tidak mengalami degradasi atau dekomposisi termal. pendapat yang menyatakan bahwa bahan ethyl-2-cyanoacrylate cukup aman,4 dalam penelitian ini terbukti, meskipun dalam penelitiannya dipakai ethyl-2-cyanoacrylate dalam bentuk cairan sebagai bahan adesif jaringan dan tidak dicampur dengan (poly)methyl methacrylate. kesimpulannya ada penurunan sitotoksisitas pada restorasi cyanoacrylate dalam penelitian ini antara perbandingan takaran powder dan liquid 1 : 1,00 dengan 1 : 0,50. uji sitotoksisitas adalah uji awal untuk biokompatibilitas suatu bahan. untuk memastikan toksisitas bahan restorasi cyanoacrylate, disarankan penelitian lebih lanjut mengingat banyak faktor yang harus dipertimbangkan pada pemakaian bahan di dalam mulut, misalnya perbedaan keasaman makanan dan minuman yang dikonsumsi sehari-hari dan juga uji lanjutan untuk mengetahui efek biokompatibilitas secara keseluruhan. ucapan terima kasih pada kesempatan ini ucapan terima kasih disampaikan kepada ketua lembaga penelitian universitas airlanggga yang telah memberikan dana dik suplemen tahun 2004 universitas airlangga untuk penelitian ini. daftar pustaka 1. compendium cyano veneer. application examples and processing instructions. meyer-haake medizin-und dentalhandels gmbh. brosur. 2. anusavice kj. science of dental materials. 11st ed. elsevier science usa saunders; 2003. p. 166, 171–4, 400, 734–5. 3. o’brien wj dan ryge g. an outline of dental materials and their selection. philadelphia: wb saunders co; 1978. p. 78–9, 83, 86, 89, 94. 4. lehmann rr. ethyl-2-cyanoacrylate as a tissue adhesive for external use. universität münster. d-48129 münster. e-mail:lehmr@uni-muenster.de,dr.r.r.lehmann@t-online.de. 1997. accessed augustus 27, 1997. 5. american dental association (ada). dentist’s desk reference: materials, instruments and equipment. 2nd ed. chicago: american dental association; 1983. p. 116, 122. 6. concise international chemical assesment (cicad) document 36. 2001. methyl-ethyl cyanoacrylate. (online). available from: htpp:// www.inchem. org/documents/cicads/cicads/cicad 36.htm. accessed april 21, 2003. 7. van noort r. introduction to dental material. 2nd ed. london: cv mosby company; 2003. p. 3–5. 8. fazwishni s dan hadijono bs. uji sitotoksisitas dengan esei mtt. jkgui. 2000; 7: 28–32. 9. marais jt, dann heimer mf, germis hpj, borman jw. dept of cured of light cured composite resin with light curing units of difference intencity. j dent assoc 1997; 52: 403–7. 10. maat s. sterilisasi dan disinfeksi. ceramah sehari penyucihamaan (sterilisasi) sarana pelayanan kesehatan. patologi klinik rsud dr.soetomo surabaya, 2001; h. 14. 11. telli c, serper a, dogan al, guc d. evaluation of the cytotoxicity of calcium phosphate root canal sealers by mtt assay. j endodon 1999; 25: 811–3. 12. kasugai s, hasegawa n and ogura h. application of the mtt colorimetric assay to measure cytotoxic effect of phenolic compound on established rat pulp cells. j dent res 1991; 70: 127–30. 13. titien ha. pengaruh tegangan listrik dan lama penyinaran pada semen ionomeri gelas modifikasi resin terhadap kekerasan permukaan dan sitotoksis. tesis. surabaya: pasca sarjana universitas airlangga; 2002. 14. neihart tr. cyanoacrylate veneer facing: an alternate approach. j prosthet dent 1984; 56: 777–9. 15. freshney, ri. culture of animal cells. a manual of basic technique. 2nd ed. new york: alan r liss inc; 1987. p. 9, 71, 128, 239. 16. rubianto m. biokompatibilitas bahan allograft (human bone powder) dibandingkan dengan bahan alloplast (hydroxylapatite). kumpulan naskah temu ilmiah nasional i (timnas i) fkg unair, 1998; h. 507–9. 17. craig rg, powers jm. restorative dental materials. 6th ed. london: mosby co; 2002. p. 135–40. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 135135 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 135–138 apoptosis of rattus novergicus gingival fibroblasts caused by silver nano-particles gel exposure kharinna widowati,1 titiek berniyanti,2 and retno pudji rahayu3 1 department of oral medicine, faculty of dentistry, universitas hangtuah 2 department of dental public health, faculty of dental medicine, universitas airlangga 3 department of oral pathology and maxillofacial, faculty of dental medicine, universitas airlangga surabaya – indonesia abstract background: the use of silver nanoparticle are growing, especially in medical science. it’s used in many concentration. in dentistry, it’s used to decrease halitosis, periodontal diseases, and wound healing. it can affect the viability of the cells, give bad effects to the human’s health and environment if used in a long duration and in certain concentration. purpose: the purpose of this study was to learn the apoptosis of gingival fibroblasts in rattus novergicus which is exposed with 15 µg/ml silver nano-particle gel by the expression of caspase-3. method: this study used 9 male wistar rats and were divided into 3 groups. sample in group a were cut (hurt) in the oral gingiva and exposed to ag-np gel 15 µg/ml for 3 days. after 3 days, they were sacrificed and cut the gingival fibroblasts off 3x4 cm size with scalpel. samples in group b were cut in the oral gingiva and exposed to ag-np gel 15 µg/ml for 5 days. after 5 days they were sacrificed and the gingival fibroblasts off 3 x 4 cm with a scalpel. samples in group c were cut in the oral gingiva and exposed to none for 3 days then cut the gingival fibroblasts off 3 x 4 cm size with scalpel. the expressions of caspase-3 in the apoptotic and wound healing process were analyzed by immunohistochemical test. this data was analyzed by using the t-test method. result: mean expression numbers of caspase-3 in the group a=5.67; group b=11.33; and group c (control)=18.67. t-test sign.number of group a and c=0.009; group b & c=0.000. conclusion: the exposure of 15 µg/ml silver gel nanoparticle to gingival fibroblasts of rattus novergicus reduces the expressions of caspase-3 in the day-3 and day-5 post exposure. the amounts of cell death through the apoptotic pathway which were analyzed by the expressions of caspase-3 will decrease too. keywords: apoptotic; silver; nano-particles; caspase-3 correspondence: kharinna widowati, c/o: departemen ilmu penyakit mulut, fakultas kedokteran gigi universitas hangtuah. jl. arif rahman hakim 150 surabaya 60111, indonesia. e-mail: kharinna.widowati@gmail.com introduction the use of silver reminds us of the luxury culture in greek, roman, and egyptian. silver is well-known type of metal and ranks number third after gold and silver copper in those nations.1 silvers are generally used for water containers and the other liquid materials to keep the cleanliness and sterility.2 silver use has developed very fast for health purpose. the macedonians use silver to prevent infection after surgery. aside from being antiseptic, hippocrates used the preparation of silver nitrate for ulcer treatments, compound fractures, and a good wound healing supporting material.1 avicenna use silver nitrate to purify blood, to prevent heart palpitation, and to handle respiratory diseases. in 1880, doctor carl siegmund franz crede, a german obstetrician, became the first person to use eye drops made from 1% silver nitrate to prevent ophthalmia neonatorium (gonorrheal ophthalmia) on babies.3 anti-bacterial function of silver nano-particles are also utilized by the dentists as mouthwash therapy for periodontal diseases, to reduce bad breath, to help wound healing process and to prevent infection in tooth extraction and surgery.4 the use of silver nano-particles progresses rapidly in the field of nanobiotechnology; however, silver nano-particles also have negative consequences to human and environment dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p135-138 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p135-138 136 widowati, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 135–138 for prolonged use or in uncontrollable concentration.5 silver particels have a nano size because they can easily fit into cells. therefore, if silver nano-particles are used continuously with uncontrollable concentrations, they can lead to cells death and affect human biological system.6 several studies have been conducted to examine the effect of silver nano-particles use on rats liver cells. the result showed that the use of silver nano-particles with 5-10 µg/ml concentration can affect the decline of mitochondrial function and the integrity of liver cell membranes after 24 hours incubation.7 the other in vitro studies, 24-48 hours exposure of silver nano-particles with 10-25 µg/ml concentration on human lung cells fibroblasts can stimulate the release of pro-inflammatory cytokines as the oxidative stress level and reactive oxygen species (ros) production are proliferated that can potentially damage dna cells.8 this implies that some information about the other effects caused by the use of silver nano-particles with concentration circulated in the market are needed so that people will be more vigilant in using silver nano-particles. based on the data above, we conducted a deeper research by using gingival fibroblasts cells of rattus novergicus as the subject test because their tissues are similar to human gingival fibroblasts cells that potentially get exposed directly when the application process of silver nano-particles was done topically. the purpose of this study was to examine the death of rattus novergicus gingival fibroblasts cells through apoptosis by analyzing caspase-3 expression. materials and mthods this study is experimental laboratory with post test only control group design. this study was conducted in the laboratory of biochemistry and pathology, faculty of medicine universitas airlangga, and institute of tropical disease (itd) universitas airlangga surabaya. this study used male rattus novergicus, age 3–4 months, weight ± 200 grams, and declared healthy on physical examination by veterinarian. materials tested were silver nano-particles from aquasil brand produced by nanonasb pars company in gel form with 15 µg/ml concentration. nine rattus novergicus were divided into three groups. the incision was made on each group in the anterior mandibular gingiva, extending from interdental gingiva of the mandibular central insisiv downwardly along for ≥ 5 mm in length and 2 mm in depth. after the incision, 15 µg/ml silver nano-particles gel was topically applied into group a as much as ± 1 ml and then was followed by suturing process on the wound to prevent silver nano-particles gel leaked out from the wound area, and decapitation on the day 3 after exposure process of silver nano-particles gel application. the same steps including slicing process, gel application, and suturing process are also performed in group b, followed by decapitation on the day 5 after exposure process of silver nano-particles gel application. the incision of group c was made without followed by the application process of silver nano-particles gel 15 µg/ml, and then continued by suturing process and decapitation on the day 3 after the slicing process and suturing (control). decapitation control group was only given on the day 3 because in the normal condition of healing process (without any supporting materials for healing process), is estimated that the cells death through apoptosis will appear 24-72 hours after the incident lesion.10 for the treatment groups, in addition to decapitation on day 3, they were also given decapitation on day 5 as the healing process could occur faster or slower than in normal condition due to the silver nano-particles exposure. the examination of caspase-3 was conducted by using immunohistochemistry test, and then was observed by using light microscope with 400x magnification. comparative test independent t-test was used to examine the significant difference between treatment groups and control groups. results table 1 shows the results of 15 µg/ml silver nanoparticles gel exposure on caspase-3 expression of fibroblasts research (table 1). the highest average of caspase-3 expressions were found in control group. the following bar chart shows the mean value of caspase-3 expressions of fibroblasts in treatment group on the day-3, day-5 (figure 1). figure 2 shows the result of immunohistochemical examination. thick and long brown colour lines pointed by the small arrows depict the form of fibroblast cells indicating caspase-3 expression. on the day-3, caspase-3 table 1. the mean and standard deviation of caspase-3 expression in fibroblasts on day-3 and day-5 day-3 day-5 group group name mean (x) + standard deviation (sd) p group name mean (x) + standard deviation (sd) p treatment a 5.67 + 0.577 0.009 b 11.33 + 0.577 0.000 control c 18.67 + 1.155 notes: * there are significant difference (p<0,05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p135-138 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p135-138 137137widowati, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 135–138 expression on fibroblasts of control group (c) appeared to be denser than of treatment group (a). caspase-3 expression on fibroblasts of treatment group b (day-5) appeared to be denser than of treatment group a (day-3). t-test examination was performed after the data were normally distributed by using kolmogorov-smirnov test and homogeneous by using levene test. the result of the comparative test by using t-test on a-c groups showed that the number of expressions of caspase-3 in fibroblasts have significant difference with significance value p=0.009. while the result of comparative test by using t-test on b-c groups also have significant difference with significance value p=0.000 (p<0.05). discussion the research of 15 µg/ml silver nano-particles gel exposure on the expression number of caspase-3 in fibroblasts showed the significant difference of the mean value between treatment group and control group. caspase-3 expressions in fibroblasts of treatment group and macrophage are natural immune cells triggered the supression of pro-inflammatory cytokines release and various systems such as the complement system and acute phase response as the increased oxidative. macrophage cells as antigen presenting cell (apc) has mhc class ii molecules. through mhc class ii, b cells will receive antigen, the antigen is presented to the cells surface to activate t helper cells which then will secrete proinflammatory cytokines. the declination of mitochondrial function resulted by cells stress also triggers the activation of gene p-53 as pro-apoptosis gene in mitochondria.11 the activation of gene p-53 followed by the inactivation of protein bcl-2 and the increased production of bax will affect the permeability of mitochondrial membrane that can release cytocrom c. the released cytocrom c will be bound by apoptosis activating factor (apaf-1) and then will form apoptosom. apoptosom will activate caspase 9 (initial caspase activated by the released cytocrom c), caspase-9 will activate caspase-3 which acts as apoptosis executioner.12 the number of expressed caspase-3 indicates the number of cells undergoing apoptosis. caspase 7 figure 1. bar chart of mean value of caspase-3 expressions in fibroblasts on the day-3 and day-5. notes: a) treatment group on the day-3; b) treatment group on the day-5; c) control group. figure 2. overview of caspase 3 expression in fibroblasts of each group. fibroblasts are shown by the small arrows. notes: a) treatment group onthe day-3; b) treatment group on the day-5; c) control group. a b c mean figure 1. bar chart of mean value of caspase-3 expressions in fibroblasts on the day-3 (a) and day-5 (b), and control (c) 7 figure 1. bar chart of mean value of caspase-3 expressions in fibroblasts on the day-3 and day-5. notes: a) treatment group on the day-3; b) treatment group on the day-5; c) control group. figure 2. overview of caspase 3 expression in fibroblasts of each group. fibroblasts are shown by the small arrows. notes: a) treatment group onthe day-3; b) treatment group on the day-5; c) control group. a b c mean figure 2. overview of caspase 3 expression in fibroblasts of each group. fibroblasts are shown by the small arrows. notes: a) treatment group onthe day-3; b) treatment group on the day-5; c) control group. are lesser than of control group (0 µg/ml). the significant difference of caspase-3 expressions were also found between treatment group a (on the day-3) and b (on the day-5). this suggests that the exposure of 15 µg/ml silver nano-particles gel is able to supress the expression of caspase-3 in gingival fibroblasts cells that suffered injury of incision. typical sign of cells undergoing apoptosis is the expression of caspase-3 (caspase executioner) of the cells. the supression of caspase-3 expression on gingival fibroblasts cells indicates the reduced number of cells undergoing apoptosis. exposure to 15 µg/ml silver nano-particles gel on injury (cuts) were able to suppress the activation of the innate immune response which is the beginning of an inflammatory mechanism in the immune cells activation, the complement system, the identification and the removal of foreign substances, as well as the activation of the adaptive immune system. phagocytes cells such as polymorphonuclear neutrophils, monocytes, dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p135-138 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p135-138 138 widowati, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 135–138 expression will appear in the next 6–9 hours ao that the estimated death of cells will appear 24–72 hours after incident lesion.10 it is concluded that the exposure of 15 µg/ml silver nanoparticles gel to gel rattus novergicus gingival fibroblasts decline caspase-3 expression. this suggests that apoptosis of the observed cells through caspase-3 expression is also decreasing. acknowledgement gratitude and appreciations are addressed to all staffs of the laboratory of pathology anatomy faculty of dentistry airlangga university, the laboratory of biochemistry faculty of medicine airlangga university, and institute of tropical disease airlangga university. references 1. hill j. colloidal silver: medical uses, toxicology, and manufacture. 3rd ed. yelm, wa., usa: clear springs press; 2009. p. 68-87. 2. elzey s, grassion vh. agglomeration, isolation, and dissolution of commercially manufactured silver nano-particles in aqueous environments. j nanopart res 2009; 12(5): 1945-58. 3. vermeulen h, van hattem jm, versloot nm, ubink dt. topical silver for treating infected wounds. cochrane database systematic reviews 2007; issue 1. art. no. cd005486. 4. arora s, jain j, rajwade jm, paknikar km. cellular responses induced by silver nano-particles :in vitro studies. toxicol lett 2008; 179(2): 93–100. 5. yoon ky, hoon bj, park jh, hwang j. susceptibility constants of escherichia coli and bacillus subtilis to silver and copper nanoparticles. sci total environ 2007; 373(2-3): 572-5. 6. kumar v, ramzi sc, stanley lr. buku ajar patologi robbins. edisi 7. vol. 1. jakarta: penerbit buku kedokteran egc; 2007. p. 130-59. 7. hussain sm, hess kl, gearhart jm, geiss kt, schlager jj. in-vitro toxicity of nano-particles in brl 3a rat liver cells. toxicol in-vitro 2005; 19: 975-83. 8. asharani pv, low kah mun g, hande mp, valiyaveettil s. cytotoxicity and genotoxicity of silver nano-particles in lung cells. acs nano 2009; 3(2): 279-90. 9. vandenabeele p. more than one way to die : apoptosis, necrosis and reactive oxygen damage. oncogene 2006. 18(54): 7719-30. 10. dirnagl u, iadecola c, moskowitz ma. pathobiology of ischaemic stroke: an integrated view. trends neurosci 2005; 22(9): 391–97. 11. wong ml, strenberg em. immunological assays for understanding neuroimmune interactions. arch neurol 2000. 57(7): 948-52. 12. amantea d, nappi g, bernardi g, bagetta g, corasaniti mt. postischemic brain damage: pathophysiology and role of inflammatory mediators. febs journal 2008; 276(1): 13–26. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p135-138 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p135-138 vol 38 no 2-2005 52 epulis and pyogenic granuloma with occlusal interference widowati witjaksono* and ban tawfeek shareff al ani** * department of periodontic, school of dental sciences university science malaysia and faculty of dentistry airlangga university ** department of oral pathology school of dental sciences, university science malaysia abstract in dental clinic of hospital university science malaysia (husm), there were cases with localized gingival enlargement (lge) in the oral cavity with occlusal interference. in this study, three cases were observed. they were a 13 yearold female with fibrous lge around 31 and 32 with occlusal interference in protrusive movement due to x bite, a 15 year – old female with pyogenic granuloma near 11 & 21 with occlusal interference due to deep bite; and a 24 – year – old female who was eight months in pregnancy with pyogenic granuloma on the 34-35 and severe generalized pregnancy gingivitis with occlusal interference in centric occlusion and lateral movement. clinical and histopathological diagnosis of the first case showed fibrous epulis, whereas the second and third cases disclosed pyogenic granuloma. chronic trauma of the gingiva due to occlusal interference was assumed to be the cause of those lge in case 1 and 2, while in case 3 poor oral hygiene and chronic trauma were assumed to be the etiologic factors. key words: localized gingival enlargement, fibrous epulis, pyogenic granuloma, occlusal interference correspondence: widowati, department of periodontic, school of dental sciences university science malaysia, health campus 16150 k. kerian, k. bharu, kelantan, malaysia. introduction the term epulis means tumor or a lump localized on the gum. the best describes as chronic inflammatory hyperplasias. the tumor can be fibrous epulis, pyogenic granuloma and giant-cell granuloma.1 fibrous epulis usually arises from an interdental papilla in a firm, pink nodule or varying shape. fibrous epulis usually associate with a source of chronic irritation such as traumatic occlusion, calculus or the rough edge of a restoration. histopathologically the lesion consists of hyperplastic connective tissue, can be ulcerated and covered by stratified squamous epithelium.1–3 pyogenic granuloma usually arises from the interdental papilla. it appears as an elevated, pedunculated or sessile mass with a smooth or lobulated surface. it is deep red or reddish-purple in colour, and the surface maybe ulcerated. it also has tendency to bleed, either spontaneously or on provocation with slight trauma. it may develop rapidly to a variable size and then remain stable for an indefinite period.2,4 the lesion appears as a result from local irritation, but in some cases there may be a hormonal conditioning factors, such as in the lesions occurring in pregnancy and at puberty.1–4 case some cases of localized gingival enlargement (lge) with occlusal disharmony often come to dental clinic hospital university science malaysia (husm). three cases of lge in the patients with occlusal interference will be discussed in this article case 1 management a 13–year–old female was referred from a general practitioner to klinik pakar perubatan (kpp) of dental clinic husm. her main complaint was non-painful anoying of gingival swelling on the lower left anterior region. gingiva was starting to lump since 6 months ago. extra orally, no abnormality was detected. intra oral examination, there was a gingival swelling between teeth 31 and 32, pink and fibrous consistency (1a), its diameter was around 7 mm, with fair oral hygiene.5 tooth 31, mesial pocket 3 mm, distal 8 mm, buccal 2 mm, and lingual 3 mm respectively, whereas tooth 32 mesial pocket 8 mm, distal 3 mm, buccal 4 mm, and lingual 5 mm with 1 degree of mobility. occlusal relationship in the anterior region was mild cross bite or x bite. figure 1. a) before treatment; b) 4 weeks after treatment; c) histological feature. 53witjaksono: epulis and pyogenic granuloma there was occlussal interference on 31 and 32 in the protrusive movement, no bone loss was detectable in the radiograph. in the initial therapy, scaling and prophylaxis must be done, and the patient had to perform daily home plaque control. after oral hygiene was satisfying, then we proceeds to adjust the occlusal on particular teeth. later, surgical excision was done from this lge. four weeks after the surgical intervention, intra oral examinations revealed that particular gingivae were inflammation free and exhibit a generally physioloc morphology. a very slight recurrence of gingival hyperplasia can be detected between 31 and 32 papillae (1b). histopathologically the lesion consist of accumulation of fibrous connective tissue (1c). the diagnosis based on clinical examination and histopathological view is fibrous epulis. case 2 management a 15-year-old female came to kpp of dental clinic husm with the main complaint of gingival swelling in the maxillary anterior region. she had used an orthodontic appliance and it was removed one week before her attendance to dental clinic. gingiva had starting to lump approximately one year ago and gradually increasing it's size. there was no abnormality detected on the extra oral examination. from the intra oral examination, there was a gingival swelling around teeth 11 and 21, it was red and fluctuant consistency (2a), its diameter around 7 mm, with fair oral hygiene.5 occlusal relationship in the anterior region was deep bite, with occlusal interference in protrusive movement in the central incisive region. pocket depth 3 mm around tooth 11. tooth 12, mesial pocket 3 mm, distal 3 mm, buccal 5 mm, and lingual 3 mm. figure 2. a) before treatment; b) 4 weeks after treatment; c) the histologic view. similarly, in this case scaling and prophylaxis must be done, and the patient had to perform daily home plaque control. after oral hygiene was satisfying, then proceeds to adjust the occlusal on particular teeth. later, surgical excision was done from this lge. four weeks after the excision of the lesion, the gingivae were totally inflammation-free and exhibit a generally physiologic morphology (2b). radiographic examination showed no widening of periodontal space and no bone destruction. histopathologically the excised lesion showed a loose granulation tissue filled with blood (2c). clinical and histopathological investigations assumed that its diagnosed as pyogenic granuloma. case 3 management a 24-year-old female was eight months pregnant. the patient presented to kpp of dental clinic husm with a complaint of biting the swollen gum on the left side of her mouth, and profuse bleeding after brushing her teeth. according to the history, present illness, the onset of gum bleeding was long time ago whereas the lumpness on the gum had started in the first three semester. from extra oral examination, no abnormality was detected. intra orally revealed a severe generalized gingivitis as well as the swollen fluctuant localized growth between 34 and 35 with 7 × 6 × 3 mm in size (3a), and poor oral hygiene.5 occlusal relationship in the anterior region is normal class i, but there was an open bite in the lateral side. figure 3. a) before treatment (from lateral and anterior site); b) the radiograph of 34 and 35; c) the histologic section of gingivae; d) three months after gingivoplasty (two months post partum). there was occlusal interference in protrusive and lateral movements. pocket depth was 7 mm around teeth 34 and 35, otherwise generally 4 mm. radiographic examination depicts some horizontal loss of the crestal compact bone of the interdental septa (3b). this case have similarity with other two cases (case 1 and 2), however it was take longer time to decide surgical intervention. during the pregnancy, motivation, repeated oral hygiene instruction, plaque and calculus removal and adjustment of particular teeth were done. in this case the redundant tissue have to be removed by gingivoplasty also during pregnancy, because the epulis was ulcerated (3a left) since the patient's maxillary teeth bite into the tissue during mastication. the histologic section (of gingiva, not the epulis) showed normal oral epithelium, a relatively mild inflammatory infiltrate and widely dilated vessels (3c). 54 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 52–55 the patient suggested to come again after given birth. two months post partum/and three months after gingivoplasty the gingivae look pale pink in colour, the margin is knife edged and scalloped showing physiological morphology (3d). the patient suggested to come again after breast feeding is terminated for re-evaluation and further treatment planning (for restorative work). from the clinical and pathological examinations, it was assumed as pyogenic granuloma near 34 and 35 with severe generalized pregnancy gingivitis. discussion gingival enlargement may result from chronic or acute inflammatory changes. the former is by far the more common, and these cases similarly have the same h i s t o r y o f c h r o n i c e x p e r i e n c e d . i n a d d i t i o n , inflammatory enlargements commonly are a secondary complication to any of other types of enlargement, creating a combined gingival enlargement. in these cases it is important to understand the double etiology and treat them adequately. these three localized gingival enlargement is caused by prolonged exposure to dental plaque. factors that favor plaque accumulation and retention6 include poor oral hygiene, as well irritation by anatomic abnormalities such as occlusal interference in these cases and improper orthodontic appliances in case 2. based on the etiological factors, a thorough removal of dental plaque must be performed initially before proceeds to surgical therapy or excision of the gingiva. many research, clinical trials and project in different geographical and settings have confirmed that effective removal of dental plaque is essential to dental and periodontal health throughout life.7 we also encourage patients to do plaque control by removing dental plaque and prevent its accumulation on the teeth and adjacent gingival surfaces by perform tooth brushing and flossing. daily removal of dental plaque lead to resolutions of the gingival inflammation in just a few days,8 and carefully performed daily home plaque control, has been demonstrated to reduce supragingival plaque, decrease the total number of microorganisms in moderately deep pockets, and greatly reduce the number of subgingival sites with porphyromonas gingivalis, which is a significant periodontal pathogen.9 after all prophylaxis/plaque control have been done, the next step was to adjust the probable irritating factors. thinking about local irritating factor, is important to understand what kind of irritating factor presented in these cases. as stated before these three cases have similar irritating factors that favor plaque accumulation that is, occlusal interference. interference that was happened during occlusion and produces such injury is called a traumatic occlusion.10 the dentition may be anatomically and aesthetically acceptable but functionally injurious. although trauma from occlusion is reversible under such conditions, it does not always correct itself, nor is it therefore always temporary and of limited clinical significance. the injurious force must be relieved for repair to occur.11 we have done occlusal adjustment to remove such traumatic occlusion based on the conclusion that occlusal adjustment resulted in a more favorable attachment level.12 finally after we have controlled all of the etiological and the irritating factors, the whole lesions had been carefully excised with care, placed in formal saline fixative and sent for histological confirmation of the diagnosis. lack of care in these respects can lead to recurrence of the lesion.1 these three cases great possibility that there may be a hormonal conditioning factor such as in the lesions occurring in the 24 year-old-female with 8 months pregnant and the other two cases with puberty 13 and 15 years-old-female. during pregnancy there is an increase in levels of both progesterone and estrogen, which, by the end of the third trimester, reach 10 and 30 times of the levels during the menstrual cycle, respectively.13 these hormonal changes induce changes in vascular permeability leading to gingival edema and an increased inflammatory respons to dental plaque. the subgingival microbiota may also undergo changes, including an increase in prevotella intermedia.14,15 this condition is not observed clinically in every pregnant women, however, with poor oral hygiene like in this case a pronounced enlargement can be developed. concerning the other two cases, the oral hygiene of the patient is only on the average or not too bad like in the pregnancy case, but showing an increases in gingival inflammation. according with nakagawa et al.16 there is statistically significant increases in gingival inflammation and in the proportions of p. intermedia and prevotella nigrescens in puberty gingivitis. based on these three cases, it can be concluded that clinical periodontal therapy must be includes an understanding of our role in the total health and well-being of our patients. female patients in there cases may present with periodontal and systemic considerations that alter conventional therapy. the cyclic nature of the female sex hormones often reflect in the gingival tissues as the initial signs and symptoms. medical histories and dialogues should include deep investigation of the individual patient's problem and needs. questioning should reflect hormonal stability and medications associated with regulation. patients should be educated regarding the profound effect the sex hormones may play on periodontal and oral tissues as well as the consistent need for home and office removal of local irritants. further informative study, regarding specific management and etiology of sex hormon-mediated infections will enhance our ability to provide quality care to our patients. 55witjaksono: epulis and pyogenic granuloma acknowledgement these cases were presented in the 9th national conference on medical sciences 22nd–23rd may 2004 in kelantan, malaysia. we are grateful to prof. ab. rani samsudin, dean school of dental sciences for his encouragement to our participation in the conference. references 1. manson, eley.outline of periodontics. 4th ed. new york: wright; 2000. p. 364–5. 2. sapp jp, eversole lr, wysocki gp. contemporary oral and maxillofacial pathology. toronto; 1997. p. 278–81, 305–7. 3. regezi aj, sciubba j. clinical pathologic correlations. 2nd ed. philadelphia: wb saunders co; 1985. p. 194–6. 4. buchner a, calderon s, ramon y. localized hyperplastic lesion of gingivae: a clinicopathological study of 302 lesion. j periodontol 1997; 48(2): 125. 5. greene jc, vermillion jr. oral hygiene index: a method for classifying oral hygiene status. j am dent assoc 1960; 61: 172. 6. thomason jm, seymour ra, ellis js, et al. determinants of gingival overgrowth severity in organ transplant patients. j clin periodontol 1996; 23: 628. 7. lang np, attstrom r, loe h, et al. proceedings of the european workshop on mechanical plaque control. chicago: quintessence; 1998. p. 54 8. loe h, theilade e, jensen sb. experimental gingivitis in man. j periodontol 1965; 36: 177. 9. hellstrom m-k, ramberg p, krok l, et al. the effects of supragingival plaque control on subgingival microflora in human periodontitis. j clin periodontol 1996; 23: 934. 10. bhaskar sn, orban b. experimental occlusal trauma. j periodontol 1995; 26: 270. 11. polson am, meitner sw, zander ha. trauma and progression of marginal periodontitis in squirrel monkeys iv. reversibility of bone loss due to trauma alone and trauma superimposed upon periodontitis. j periodont res 1976; 11: 290. 12. burgett fg, ramfjord sp, nissle rr, et al. a randomized trial of occlusal adjustment in the treatment of periodontitis patients. j clin periodontol 1992; 19: 381. 13. amar s, chung km. influence of hormonal variation on the periodontium in women. periodontology 2000; 1994. 6: 79. 14. kornman ks, loesche wj. the subgingival microbial flora during pregnancy. j periodont res 1980; 15: 111. 15. raber-durlacher je, van steenbergen tjm, van der velde u, et al. experimental gingivitis during pregnancy and post partum: clinical, endocrinological and microbiological aspects. j clin periodontol 1994; 21: 549. 16. nakagawa s, fuji h, machida y, et al. a longitudinal study from prepuberty to puberty of gingivitis. correlation between the occurrence of prevotella intermedia and sex hormones. j clin periodontol 1994; 21(10): 658. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 53 the colony number of streptococcus mutans and lactobacillus in saliva of dental caries and free caries children seno pradopo department of paediatric dentistry faculty of dentistry, airlangga university surabaya indonesia abstract streptococcus mutans (s. mutans) are regarded as the main initiator microorganism of caries, with lactobacilli participating on caries progression, due to its carcinogenic capacity. the purpose of this study was to examine the number of s. mutans and lactobacillus sp in children with in children’s saliva with dental caries and free caries. twenty children attending the paediatric dental clinic in airlangga university participated in our study. their age ranged from 1–14 years old. subject was divided into two groups, which were study group consisting of 10 children with 3–5 dmft/dmft and control group with 10 caries free children. subjects were examined and their caries number was recorded using who index. stimulated saliva was collected from each subject for bacterial assessment. colony counting of s. mutans and lactobacillus sp count in each saliva sample group were done. the study showed that subject with 3–5 dmft/dmft had higher number of s. mutans and lactobacillus sp than caries free. key words: streptococcus. mutans, lactobacillus sp, saliva, primary teeth correspondence: seno pradopo, c/o: departemen ilmu kedokteran gigi anak, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: pradopo_seno@yahoo.com introduction in the last decade, oral and dental health have remarkably increased, however, the prevalence of dental caries in children as clinical problem remains significantly high. dental caries is one of pathological conditions mostly suffered by indonesian children. in indonesia the prevalence of dental caries in children of 10 years old is 80%. dental caries is an infectious disease initiated by progressive demineralization in hard tissue on the crown surface and the tooth root. several interrelated factors such as: the tooth and saliva (host), microorganism, substrate and time are involved in the process of dental caries.1 streptococcus mutans (s. mutans) and lactobacillus sp are the active microorganisms contributing dental caries. the latest study suggests that the essential role of s. mutans is in the initial process of caries while lactobacillus sp is correlated with the active caries episode.2 s. mutans is the main bacteria contributing dental caries.3 in free caries patients, low number of s. mutans is found as normal flora in oral cavity, on the contrary it is dominant in plaque of patients with active multiple caries lesions. the critical period of s. mutans colonization occurs at the age of 19–31 months in which it is considered the window of infectivity.4 however, the evidence showed that s. mutans could be found soon after tooth eruption, even in babies of 6 months old who do not have any teeth yet.5,6 the study conducted on children either with high caries or free caries revealed that children with good oral condition have low number of lactobacillus and children with active caries indicate the presence of bacteria with higher adhesion capacity and agglutination process.7 the objective of this study was to examine the number of s. mutans and lactobacillus sp in saliva of caries children and free caries children. material and method twenty children (boys and girls) attending the paediatric dental clinic in airlangga university surabaya participated in our study. subject was divided into two groups, which were study group consisting of 10 children with 3-5 dmft/dmft and control group with 10 caries free children. both groups were classified into four groups consisted of 5 samples. the criteria for inclusion of the samples in this study were as follows: aged 1–14 years with primary dentition, mixed dentition, or permanent dentition, 3–5 dmft/dmft for study group and free caries for control group. the criteria for exclusion were if the children are taking antibiotic, syrup form medicine or suspension with high carbohydrate content one month before the saliva was taken. sample of saliva was taken through stimulating salivary gland by chewing parafilm, and collected in a 15 ml tube. the samples was suspended in vial containing 1 ml saline, and incubated at 37° c for two hours. the saliva sample was shaken for 30 seconds using vortex mix, and then aliquots of 25 µml plaque sample was inoculated in both agar media. mitis saliva agar media was used to culture s. mutans and 54 dent. j. (maj. ked. gigi), vol. 41. no. 2 april–june 2008: 53–55 rogosa media to culture lactobacillus sp. after the agar media were incubated in anaerobe condition at 37° c for 48 hours, colony counting was done.8 the number of s. mutans and lactobacillus sp were statistically analyzed using kolmogorov smirnov test and the variable was shown by the mean value. independent t-test was applied to analyze the different number of s. mutans and lactobacillus sp in saliva. result the mean and deviation standard of number of s. mutans and lactobacillus sp in both groups are shown in table 1. it showed that the mean number of s. mutans is significantly higher than lactobacillus sp. in free caries group, the number of s. mutans is lower compared to 3–5 dmft/dmft groups. the number of lactobacillus sp is also higher compared to free caries groups. table 1. the mean colony number of s. mutans and lactobacillus sp microorganism type of caries n x sd s. mutants free caries 5 246 34.2 3–5 dmft/dmft 5 575.8 40.1 lactobacillus sp free caries 5 55 4.47 3–5 dmft/dmft 5 117.2 25.44 statistical analysis using kolmogorov-smirnov test was done followed by test on the different number of s. mutans and lactobacillus sp in free caries groups and 3–5 dmft/dmft group. the result of kolmogorov smirnov test is shown in table 2. table 2. the result of kolmogorov-smirnov test on number of s. mutans and lactobacillus sp microorganism type of caries kolmogrov smirnov test s. mutants free caries p = 0.987 3–5 dmft/dmft p = 0.917 lactobacillus sp free caries p = 1 3–5 dmft/dmft p = 1 the result of kolmogorov-smirnov test in all groups p > 0.05 is shown in table 2 indicating all groups have normal data distribution. statistical analysis using independent t test was applied to determine the different number of s. mutans between free caries and 3–5 dmft/dmft group, p = 0.001 (p > 0.05) indicating significant difference in the number of s. mutans between those two groups. statistical analysis using independent t test was also applied to determine the different number of lactobacillus sp between free caries and 3–5 dmft/dmft group, p = 0.001 (p > 0.05) indicating significant difference in the number of lactobacillus sp between those two groups. statistical analysis using independent t test was applied to determine the different number of s. mutans and lactobacillus sp in 3–5 dmft/dmft group p = 0.001 (p < 0.05) indicating significant difference in s. mutans number and lactobacillus sp group of 3–5 dmft/dmft. discussion s. mutans frequently found in dental plaque are regarded as the main initiator microorganism of dental caries. however, micro flora found in saliva similar to those in any surface of oral cavity, thus the method of colony count using saliva sample could represent bacterial in dental plaque.9 transmission and s. mutans colonization in oral cavity are important factors to prevent dental caries considering s. mutans has a potential role in caries initial formation but in further lactobacillus sp would play role.3 in this study, the number of s. mutans was significantly higher than the number of lactobacillus sp found in twenty children aged 1–14 years either with 3–5 dmft/dmft or caries free. the number of s. mutans and lactobacillus sp in 3–5 dmft/dmft children showed significant correlation (p < 0.001). this study suggested children with high number of s. mutans and lactobacillus sp would also indicate to have high number of caries. the lower the number of bacteria, the lower the caries number. the result of this study support the previous study on preschool children which found positive correlation between the number of s. mutans and dental caries.10–13 previous researcher reported14 similar result, patients with active caries having higher number of s. mutans and lactobacillus sp compared to caries free patients. some researchers also reported the positive correlation between the number of lactobacillus sp and the severity of caries.15–16 brambilla et al.17 stated that lactobacillus sp is predisposition factor for caries due to the capability of bacteria to interact with microorganism during colonization. brambilla et al.17 also reported that there are positive correlation between the number of lactobacillus sp in 3–5 dmft/dmft in 21% children aged 9–13 years old. van houte3 suggested high number of lactobacillus sp in dental caries but not in caries free, showed that lactobacillus sp play role more on caries process than initial process. number of s. mutans in oral cavity closely related with the caries severity and the prediction of caries risk in the future.18,19 s. mutans virulence in dental caries depends on the host, the quality and the duration of bacterial adhesion in the plaque. s. mutans adhesion could be accelerated by purified salivary agglutinin which interacts with agglutinin receptor (high-molecular-weight surface protein) and presence of s. sanguis in oral cavity. suspensions is obtained from micro bacterial interaction producing bacterial aggregation 55pradopo: the colony number of streptococcus mutants and lactobacillus in saliva to destroy the enamel.20 in which agglutinin also involved in initial process adhesion of s. mutans in salivary pellicle. purified agglutinin could bind hydroxyapatite to support s. mutans adhesion.21 the latest data shows agglutinin could bind bacteria in dental surface simultaneously to support s. mutans adhesion. in this study, saliva was used as the sample. lactobacillus is bacteria with salivary component receptor while s. mutans receptor mostly found in dental plaque. however, all groups indicating to have higher number s. mutans compared with lactobacillus is due to the capability of s. mutans more rapidly to multiply and to produce acid compared with lactobacillus (aciduric and acidogenic).22 the study showed that s. mutans and lactobacillus sp were significantly higher in 3–5 dmft/dmft compared with free caries group. detection of s. mutans and lactobacillus sp number is important to be done in children in order to be able to evaluate and to prevent dental caries. references 1. widjiastuti i. peran agglutinin saliva sebagai mediator perlekatan bakteri streptococcus. mutans pada penderita bebas karies dan karies gigi. research report jiptunair. 1999. 2. van houte j. microbial predictors of caries risk. adv dent res 1993; 7:87–96. 3. van houte j. bacterial specificity in the aetiology of dental caries. int dent j 1980; 30:305–26. 4. caufield pw, cuttter gr, dasanayake ap. initial acquisition of mutans streptococci by infants: evidence for a discrete window of infectivity. j dent res 1993; 72(1):37–45. 5. wan ak, seow wk, walsh lj, bird ps, tudehope di, purdie dm. association of streptococcus. mutans infection and oral developmental nodules in pre-dentate infants. j dent res 2001; 80(a):1945–8. 6. wan ak, seow wk, purdoe dm, bird ps, walsh lj, tudehope di. oral colonization of streptococcus. mutans in six-month-old predentata infants. j dent res 2001; 80(b):2060–5. 7. aguilera galavis la, premoli g, gonzales a, rodriguez ra. caries risk in children: determined by numbers of mutans streptococci and lactobacillus. j clin pediatr dent 2005; 29(4):329–33. 8. khrisnakumar r, singh s, subba reddy vv, davangere. comparison of numbers of mutans streptococci and lactobacilli in children with nursing bottle caries, rampant caries, healthy children with 3-5 dmft/ dmft and healthy caries free children. j indian soc pedo prev den 2002; 20:1:1–5. 9. axelsson p. diagnosis and risk prediction of dental caries. volume 2. quintessence publishing co; 2000. p. 91–150. 10. klock b, krasse b. microbial and salivary conditions in 9-12 year-old children. scand j dent res 1977; 85:56–63. 11. bretz wa, djahjanh c, almeida rs, hujoel pp, loesche wj. relationship of microbial and salivary parameters with dental caries in brazilian pre-school children. community dent oral epidemiol 1992; 20:261–4. 12. thibodeau ea, o’sullivan dm. salivary mutans streptococci and incidence of caries in pre-school children. caries res 1995; 29:148–53. 13. zoitopoulos l, brailsford sr, gelbier s, ludford rw, marchant sh, beighton d. dental caries and caries-associated microorganisms in the saliva and plaque of 3-and 4-year-old afro-caribbean and caucasian children in south london. arch oral biol 1996; 41:1011–8. 14. ismiyatin k. hubungan efektivitas buffer saliva dengan intensitas karies. research report jiptunair. 2004.research report jiptunair. 2004. 15. bratthall d. the global epidemiology of mutans streptococci. in: johnson nw, editor. risk markers for oral diseases. volume 1. dental caries. cambridge: cambridge univ pr; 1991. p. 287–312. 16. loesche wj. role of streptococcus. mutans in human dental decay. microbiol rev 1986; 50(4):353–80. 17. brambilla e, twetman s, felloni a, cagetti mg, canegallo l, garcia-godoy f, strohmenger l. salivary mutans streptococcus and lactobacilli in 9 and 13 year old italian schoolchildren and the relationship to oral health. 1999. 3(7):10. 18. van houte j. role of microorganisms in caries aetiology. j dent res 1994; 73:672–81. 19. kohler b, pettersson bm, bratthall d. streptococcus mutans in plaque and saliva and the development of caries. scand j dent res 1981;1981; 89:19–25. 20. mandel id. non immunologic aspects of caries resistance. j. dentj. dent res 1987; 55:c22–c31. 21. lee sf, progulske-fox a, erdos gw, piacentini da, ayakawagy, crowley pj, bleiweis as. construction and characterization ofconstruction and characterization of isogenic mutans of streptococcus. mutans deficient in major surface protein antigen p1 (i/ii). infect immun 1989; 57:3306–13.1989; 57:3306–13.57:3306–13. 22. kriswandini il. penentuan adesin dan reseptor streptococcus mutans yang berperan dalam patogenesis karies gigi. disertasi. surabaya: universitas airlangga; 2004. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 48 the effects of ultrasonic scaling duration and replication on caspase-� expression of sprague dawley rat's pulp cells archadian nuryanti, marsetyawan hne soesatyo, dewi agustina, and siti sunarintyas graduate student of faculty of dentistry, universitas gadjah mada yogyakarta – indonesia abstract background: ultrasonic scaling has been used commonly for stain and calculus removal in dental clinic for over 60years. previous researches even had proved that ultrasonic scaling may give effects on the surface of tooth root. ultrasonic wave exposure for 20 seconds or more can increase caspase-3 activity as an indicator of increased apoptotic cells associated with tissue damage. purpose: this research was aimed to investigate the effects of ultrasonic scaling duration and replication on caspace-3 expression in dental pulp cells. methods: the samples of this research were 54 male sprague dawley rats aged 2 months old divided into 2 groups, each of which consisted of 27 mice. the first group was induced with stain, while the second group was not. each group was divided into 3 subgroups for ultrasonic scaling 1, 3, and 5 times. each subgroup was divided into 3 sub-subgroups for duration procedure of 15, 30 and 60 seconds respectively. during scaling process, those rats were anesthetized using 0.1 ml of ketamine and 0.1 ml of xylol added to 2 ml of distilled water injected intramuscularly into their right thigh as much as 0.4 ml. scaling was done on buccal surface of right first maxillary molar from cervical to occlusal. the teeth were decalcified and embedded in paraffin, then their sagittal plane was cut for thickness of 3µm and painted with immunohystochemistry for detecting caspace-3 expression of cell within dental pulp. results: the results showed that the duration and replication of ultrasonic scaling procedures affected on the expression of caspace-3 cells as analyzed with univariate analisis of variance test (p<0.05). conclusion: it can be concluded that duration and replication of ultrasonic scaling procedure on teeth with and without stain enhauced the expression of caspace-3 in dental pulp cells. keywords: duration; replication; ultrasonic scaling; pulp cells; caspace-3 correspondence: archadian nuryanti c/o: departemen biomedika, fakultas kedokteran gigi universitas gadjah mada, jl. denta no., sekip utara yogyakarta 55281, indonesia. e-mail: archadian_fkgugm@yahoo.com research report introduction ultrasound means sound that can not be heard by human ear at frequency above 20,000 hz. in dentistry, ultrasonic waves are used for professional tooth cleaning, root canal cleaning, bone implant and surgical procedures.1 ultrasonic scaler can also be used for biofilm, stain and calculus removal of tooth surface.2 piezoelectric scaler moving back and forth 28000-36000 times even can be used to clean tooth surface.3 if ultrasonic waves expose on liver cells of rats for 20 seconds or more, the activity of caspase-3 as an indicator of increased apoptosis associated with tissue damage.4 ultrasonic waves can cause sonification. sonification mechanism that occurs in ultrasonic scaling procedure even can cause acoustic microstreaming mechanism, turbulence flow, cavitation, and microjet formation resulting cavitation bubbles collapsed and making shock waves.4 if this mechanism occurs on tooth surface with stain, the stain will be detached from the tooth surface causing partial removal of organic and inorganic materials from the tooth surface triggering to tooth surface damage. microstreaming flow around the air bubbles generated by ultrasonic waves can get into bloodstream, and penetrate cell membrane through sonoporation molecular mechanisms.5 ultrasonic waves at a frequency of 20 khz-2 mhz even can raise small air dental journal (majalah kedokteran gigi) 20�5 march; 48(�): 48–52 4� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 4�nuryanti, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 48–52 bubbles resulting in cell injury.6 it means that duration and cavitation of ultrasound exposure can affect on activity of cells exposure.7 the increasing of caspase-3 expression after ultrasound wave exposure can become an indicator of the increasing of apoptosis cells.8 this research was aimed to evaluate the effects of ultrasonic scaling duration and replication on caspase-3 expression in dental pulp cells of teeth with and without stain. materials and method this research is a pra-experimental research since there were manipulation, repeatable measurements and control group, but without randomization approach.9 this research used 54 male sprague dawley rats aged two months old, divided into two groups each of which consisted of 27 rats. the first group of mice were given a drink with a mixed solution of tea and coffee. the solution of tea was made of 2 grams of black tea powder in pouch packaging brewed into 100 ml of boiled water. meanwhile, the solution of coffee was made of 2 grams of ground coffee without pulp brewed into 100 ml of boiled water. both solutions were mixed and waited 10 minutes to be drunk by those rats for 21 days. on the other hand, the second group of mice were given a drink with water. each group of rats was divided into 3 subgroups with ultrasonic scaling 1, 3, and 5 times for each consisted of 9 rats. each subgroup then was divided into 3 sub-subgroups each of which consisted of 3 rats with ultrasonic scaling duration of 15, 30 and 60 seconds respectively. during scaling process, rats were anesthetized using 0.1 ml of ketamine and 0.1 ml of xylol added to 2 ml of distilled water injected intramuscularly into their right thigh as much as 0.4 ml. scaling was done on the buccal surface of their right first maxillary molar, starting from cervical line to occlusal surface, using supra-gingival stain tip. tip was used on the tooth surface without pressure. the position of the tip was parallel to the axis teeth. in addition, the volume of cooling water used was about 20 ml/ sec. the engine power used was moderate, and scaling process was done by one people. the procedure was then repeated started from the induction of stain into rats to scaling process 3 and 5 times with 21-day pause. after scaling, the rats were decapitated, and their teeth were extracted. the decalcified teeth then were embedded in paraffin, and their sagittal plane was cut for thickness of 3µm. those were fixed on object glasses. deparaffinization was conducted using xylol for 3 x 5 minutes, absolute alcohol, 96% alcohol, 70% alcohol respectively for 5 minutes, and dehydration was performed. the slides were washed using running water, and then washed by distilled water. samples were incubated with 3% h2o2 for 15 minutes and washed with running water, and later washed with distilled water. cox2 retrieval then was conducted using both citrate buffer with ph 6 and decloac for 40 minutes at 95° c. after that, the samples were chilled for about 30 minutes. those samples then were washed 2x times using pbs for 3-5 minutes, and incubated with blocking serum or normal serum for 15 minutes. the samples were drained and cleaned, then were incubated with anti-caspase-3 antibody for 1 hour, and washed 2x times using pbs for 3-5 minutes. the samples were incubated again with secondary antibody for 20 minutes, and then washed 2x times using pbs for 3-5 minutes. the samples were incubated again with trikkie avidin hrp for 10 minutes, and then washed 2x times using pbs for 3-5 minutes. afterwards, the samples were dropped with chromogen dab (1:50), then left for 2 minutes, and washed with water. counterstain then was performed with hematoxylin mayer for 2 minutes, and washed with water. those samples were dipped in alcohol with concentration from 70%, 96%, to 100%, and also xylol, followed with mounting, and then observed using a microscope at 400x magnification.10 results data of the effects of ultrasonic scaling duration and replicatin on the expression of caspase-3 in the dental pulp cells with and without stain were presented in table 1. the expression of caspase-3 in the dental pulp cells with and without stain for once scaling with a duration of 15 seconds showed relatively equal numbers. the expression of caspase-3 cells in the teeth with a stain was increased as the increasing of duration and number of replication of scaling. in contrast, the expression of caspase-3 cells in the teeth without stain was incresed domninantly due to the increasing of the duration of scaling. the expression of caspase-3 cells for once scaling with a duration of 60 seconds was higher than those for 3 and 5 times with the same scaling duration. leverne test then was performed for homogeneity of the data. the results obtained was p = 0.344, which means that the data were homogeneous and qualified to be analyzed with univariate analisis of variance test. based on the results of univariate analisis of variance test (table 2), duration and repetition of scaling either individually or communaly could affect significantly the expression of caspase-3 in the dental pulp cells with and without stain (p <0.05). it means that there were significant effects of ultrasonic scaling duration and replication on the number of cells expressing caspase-3 in the dental pulp cells with and without stain. based on lsd test, it is also known that there were 9 types of relations without significant difference of the average number of caspase-3 expression (p>0.05). lsd test showed that the expression of caspase-3 cells in the group induced with stain for once scaling with a duration of 15 seconds was not significantly different from that in the group without stain with the same scaling duration. similarly, the expression of caspase-3 cells in the group induced with stain for three times of scaling with a duration dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 50 nuryanti, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 48–52 of 30 seconds had almost the same number to the group induced with stain for five times of scaling with a duration of 60 seconds. the effects of ultrasonic scaling duration and replication on the expression of caspase-3 in the dental pulp cells with and without stain can be seen in figures 1 and 2. figure 1 shows that the increasing of the duration and replication of ultrasonic scaling could increase the expression of caspase3 in dental pulp cells with stain. figure 2 shows that the increasing of the duration and replication of ultrasonic scaling could increase the expression of caspase-3 in dental pulp cells without stain. discussion data in tables 1 and 2 and in figures 1 and 2 show the effects of ultrasonic scaling duration and repetition on the expression of caspase-3 in the dental pulp cells with and without stain. this is consistent with the explanation that the increase in temperature of ultrasonic scaling can damage the pulp and disrupt circulatory system resulting in dental pulpitis or necrosis.11 the effects caused by ultrasonic wave exposure to tissue is thermal, mechanical (cavitation and microstreaming), and chemical (sonochemistry). clinical effect is depending on the application technique used.12 the tip of ultrasonic scaler, actualy can get vibrations, and the movements are influenced by the design of the scaler. the movements of the tip then can trigger cavitation and microstreaming derived from the cooling water of the ultrasonic scaler.13 therefore, it can be said that the longer the exposure to ultrasonic waves is, the longer the cavitation and acoustic microstreaming mechanisms are. similarly, some in vitro researches also show that the duration of the ultrasonic scaling can affect on tooth surface damage.3 the damage of tooth surface then can lead to the increasing of cavitation and acoustic microstreaming mechanisms until air bubbles are collapsed and a shock wave is emerged. however, ultrasonic equipment is still expected to be used for dental pulp treatment because it can cause more minimal damage than using bur with high speed.14 mechanism of ultrasonic scaler actually can clean tooth surface from materials due to chipping mechanism of scaler probe oscillating.15 this mechanism usually occurs in areas with limited view and excessive scaling duration.16 thus, over-instrumentation of ultrasonic scaling must not occur since it can lead to damage, from tooth surface to dental pulp cells. table 1. the average number of caspase-3 expression in dental pulp cells replication and duration (second) average±standard deviation of teeth with stain average±standard deviation of teeth without stain 1x 15 1x 30 1x 60 3x 15 3x 30 3x 60 5x 15 5x 30 5x 60 14.60± 2.30 20.20± 3.11 33.00± 1.88 29.60± 4.72 51.60± 4.51 69.40± 5.18 43.20± 1.92 66.00± 4.36 80.40± 5.03 14.00 ± 2.88 23.00± 3.00 72.60± 4.77 44.20± 2.78 44.60± 2.07 52.60± 3.91 31.00± 1.87 40.00± 3.53 50.40± 3.65 table 2. the results of univariate analysis of variance on the caspace-3 expression in dental pulp cells source f sig. intercept tooth stain (1) replication (2) duration (3) (1) and (2) (1) and (3) (1), (2) and (3) 169346.844 344.178 8651.356 13987.089 5086.422 463.089 3719.511 0.000 0.000 0.000 0.000 0.000 0.000 0.000 12 figure 1. graph of caspase-3 expression in dental pulp cells with stain. figure 2. graph of caspase-3 expression in dental pulp cells without stain. the number of replication scalling t he n um be r o f c as pa se 3 seconds seconds seconds t he n um be r o f c as pa se 3 the number of replication scalling seconds seconds seconds seconds figure 1. caspase-3 expression in dental pulp cells with stain. 12 figure 1. graph of caspase-3 expression in dental pulp cells with stain. figure 2. graph of caspase-3 expression in dental pulp cells without stain. the number of replication scalling t he n um be r o f c as pa se 3 seconds seconds seconds t he n um be r o f c as pa se 3 the number of replication scalling seconds seconds seconds seconds figure 2. caspase-3 expression in dental pulp cells without stain. 5� dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 5�nuryanti, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 48–52 in this research, the increasing of caspase-3 expression of dental pulp cell occurred as the increasing of duration and replication of scaling. cell injury occur when the cooling water of ultrasonic scaler flows into the dental pulp chamber.17 the changes of dental pulp cells, consequently, were detected in this research by the increasing expressing caspase-3of dental pulp cell. the increasing of tooth temperature after ultrasonic scaling also cause damage in dentin and dental pulp tissue.18 over-instrumentation lead to tooth root damage, so the teeth will become more sensitive, thus, the longer the duration of ultrasonic scaling is, the higher the negative effects will be obtained.19 in other words, certain effects of ulrasonic wave exposure to dental cells can be considered as normal, lysis, apoptosis or necrosis, depending on the duration of ulrasonic wave exposure.11 ultrasonic scaling procedures can also cause cavitation and acoustic microstreaming mechanisms, shock waves and free radical bubbles.21 these mechanisms then can lead to the changing of bubbles in the tissues both in size and number. air bubbles will increasingly enlarge until they reaches maximum size and eventually burst, and the mechanisms occur repeatedly until the ultrasonic wave exposure is stopped.22 therefore, it is suspected as the cause change of the dental pulp cells resulting the increasing of caspase-3 expression. the results of this research show that the number of caspase-3 expression in dental pulp cells after once scaling with a duration of 15 seconds on the tooth surface induced with stain was 14.60 ± 2.30, while that on the surface without stain was 14.00 ± 2.88. in general, it can be said that the number of caspase-3 expression in the group of teeth with stain was higher than in the group without stain. the number of caspase-3 expression in dental pulp cells after five times of scaling with a duration of 60 seconds in the group of teeth with stain was 80.40 ± 5.03, while that in the group without stain was 50.40 ± 3.65. it indicates that ultrasonic wave exposure to the surface of the teeth with and without stain cause similar mechanisms in the dental pulp cells since the enamel and dentin are porous, so the tissues in the pulp chamber can be affected by the complex mechanism. one of indicators of apoptosis cells is caspase-3 expression.23 physiologically, apoptosis will be increased when deciduous teeth become permanent ones.19 the results of univariate analysis of variance that duration and replication ultrasonic scaling individually or jointly significantly influenced on the number of caspase-3 expression in dental pulp cells with and without stain (p<0.05). it is suspected due to the complex mechanisms of the ultrasonic waves, including vibration, acoustic microstreaming, cavitation, and bubble rupture in the dental pulp chamber. these mechanisms can explain how the cooling water enters the pulp chamber and causes air bubbles in the pulp tissue and in the blood vessels of the pulp. in normal condition, air bubbles are small, but with ultrasonic scaling, the volume and number of air bubbles become excessive. after the size of the bubbles is maximum, the continuing ultrasonic wave exposure will cause the bubbles brust. as a results, the bubbles around the cells will damage, leading to tooth pain of patients during ultrasonic scaling. a mechanism that arises later is that the body create a balance with the increased number of apoptosis cells as one of indicators of the increased number of caspase-3 expression in dental pulp cells. during ultrasonic scaling procedure, enamel structure consisting of organic and inorganic elements will also get impact pressure from the tip of the scaler to the tip of the tooth surface. the vibration of tip scaler and spray cooling can remove stain from the tooth surface. during scaling, cavitation and acoustic microstreaming mechanisms also occurs, as a result, the cooling water can enter the dental pulp chamber due to the nature of the porous enamel and dentin. these mechanisms occur because of exposure to the cell resulting in changes of the cell membrane, followed by membrane permeability, morphology and activity changes of cell membrane.23 it takes a duration of 1-1.5 minutes for cleaning stain on the tooth surface using ultrasonic scaler.24 the longer the duration is, the higher the negative effects will be obtained 25 and the higher the permeability of cytoplasm will occur.26 it can be said that the ultrasonic figure 3. the expressions of caspase-3 in dental pulp cells with stain as follow: a) 1x of scaling for 15 seconds; b) 3x of scaling for 30 seconds; c) 5x of scaling for 60 seconds. the arrows show the expressions of caspase-3 with ihc staining, dark brown among light purple tissues. 13 figure 3. the expressions of caspase-3 in dental pulp cells with stain as follow: a) 1x of scaling for 15 seconds; b) 3x of scaling for 30 seconds; c) 5x of scaling for 60 seconds. the arrows show the expressions of caspase-3 with ihc staining, dark brown among light purple tissues. a b c 13 figure 3. the expressions of caspase-3 in dental pulp cells with stain as follow: a) 1x of scaling for 15 seconds; b) 3x of scaling for 30 seconds; c) 5x of scaling for 60 seconds. the arrows show the expressions of caspase-3 with ihc staining, dark brown among light purple tissues. a b c dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 52 nuryanti, et al./dent. j. (majalah kedokteran gigi) 2015 march; 48(1): 48–52 waves can alter the number and nature of the cell nucleus in apoptosis process.27 the results of lsd test, indicate that the number of caspase-3 expression in dental pulp cells the group of teeth without stain was not significantly different from the other groups of teeth without stain, compared with the group of teeth with stain. it is because the tooth surface without stain will make the tip directly contact with the enamel surface, but because stain is porous, air bubbles can get into the pulp chamber during the ultrasonic scaling. sonification can trigger acoustic microstreaming, flow turbulence, microjet and shock wave leading to rupture of air bubbles.2 this mechanism is suspected to be cause of damage to the surface of the organic structures and the dental pulp cells. sonoporation mechanism is used as a drug delivery by penetrating cell membranes.5 nevertheless, this mechanism can give negative effects, such as pulpitis or dental pulp necrosis.8 at the time of the evaluation of the number of caspase-3 expression, there was the increasing of the number of small-sized bubbles which were more numerous and larger size as the increasing of the number of scaling replication and duration. most preparations even showed an increase in the number and size of air bubbles in the blood vessels leading to the rupture of the blood vessels. this condition can indicate the occurrence of pulpitis or necrosis mechanism after the scaling. it then can be understood that one of the effects of ultrasonic waves on a tissues are the increasing of local temperature due to mechanical pressure passing the tissue. ultrasonic pressure resulting in small bubbles on a live tissue can lead to distortion of the cell membrane influenced by flux and activity of ions.20 the changes of bubble size that occur periodically even can cause expansion, contraction and death for the tissue. these side effects can be temporary or permanent. the effects of ultrasonic waves can also be reversible and irreversible disrupting cell membranes through cavitation mechanism. it can be concluded that ultrasonic scaling duration and replication on teeth with and without stain can improve the number of caspase-3 expression in dental pulp cells. refferences 1. sapna n, vandana kn. ultrasound in periodontics. ijda 2010; 2(3): 274-77. 2. lea gc, felver b, landini g, walmsley ad. ultrasonic scaler oscilation and tooth-surface defects. j dent res 2009; 88(3): 22934. 3. chaterjee a, baiju cs, bose s, shetty ss, wilso r. hand vs ultrasonic instrumentation: a review. j dent sc and oaral rehab 2012; 8-12. 4. klima j. aplication of ultrasound in electrochemistry, an overview of mechanisms design of experimental arrangement. ultras 2010; 51(2): 2002-9. 5. manasseh r, babanin av, ooi a, riboux g, riso f. interfacial dynamics and frequencies of passive bubble-acoustic emission. ica 2010; 1-8. 6. zao j, wei w, meng-lu q. cellular damage and apoptosis along with changes in nf-kappa b expression were induced with contrast agent enhanced ultrasound in gastric cancer cells and hepatoma cells. cancer cell int 2012; 12: 8-12. 7. pellicer b, herraiz s, taboas e, felipo v, simon c, pellicer. ultrasound biofect in rats: quantification of cellular of cellular damage in the fetal liver pulsed doppler imaging. ultras obs gyn 2011; 37(6): 643-8. 8. o’berien wd, deng cx, harris gr, herman ba, merrit cr, shanghvi n, zachary jf. the risk of exposure to diagnostic ultrasound in postnalat subject. j ultras med 2008; 27: 517-35. 9. er ta mba ng n. desa in da n implema ntasi r iset eksper imen. yogyakarta: upp stim ykpn; 2013; 2: 75-105. 10. mcclusky lm, barnhoorn iej, van dyk jc, bornman ms. testicular apoptosis in feral clarias gariepinus using tunel and cleaved caspase3 immunohistochemistry. eco and env saf 2008; 71: 41-6. 11. arabaci t, çiçek y, çanakçi cf. sonic and ultrasonics scaler in periodontal treatment: a review. int j of dent 2007; 5(1): 2-12. 12. bains vk, mohan r, bains r. aplication of ultrasound in periodontics: part i. j indian soc periodont 2008; 12(2): 29-33. 13. walmsley ad. ultrasonic in dentistry. physics proced 2015; 63: 201-7. 14. lee ms, chen yl, huang ps, chiang yc, chang hh, wu j, lin cp. effects of ultrasonic and high-speed air-driven devices on pulp-dentin reactions: an animal study. j of dent sci 2013; 9: 3959363. 15. lea sc, felver b, landini g, walmsley ad. ultrasonic scaler oscilation and tooth-surface defects. j dent res 2009; 88(3): 22934. 16. ribeiro fv, casarin rcv, nociti junior fh, sallum ea, sallum aw, casati mz. comparative in vitro study of root roughness after instrumentation with ultrasonic and diamond tip sonic scaler. j appl oral sci 2006; 14(2): 124-9. 17. santos, hm, lodeiro c, capelo-martinez jl. the power of ultrasound, ultrasound in chemistr y, analytical application. weinheim: willey-vch, verlag gmbh & co.kgaa; 2009. p. 5367. 18. ahire m, patil k, ali fm, prasant mc. ultrasonic dental scaler: associated hazards and management 2013; 1(25): 1-4. 19. hodges ko. components of optimal ultrasonic scaling. j of professional excellence 2014; 12(2): 22-7. 20. brendan j, o’daly, morris e, gavin gp. high-energy low-frequency ultrasound: a review of tissue dissection and ablation in medicine and surgery. j of mat proc tech 2008; (8): 38-58. 21. donikov a, bouakaz a, theoretical investigation of shear stress generated by contrast microbuble on cell membrane as a mechanism for sonoporation. j acoust soc am 2010; 128(1): 10-9. 22. wells jd. electroporation and ultrasound enhauced non-viral gene delivery in vitro and in vivo. cell biology and toxicology 2010; 26(1): 21-9. 23. hutchenson jd, schlider rk, prausnits mr. saving cells from ultrasound-induced apoptosis: quantification of cell deth and uptake following sonification and effects on targeting calcium chelating. ultrasound med bio 2010; 36(6): 1008-21. 24. cosyn l, miremadi sr, schaubroeck d, lang np, de moor rgj, de bruyn. clinical effects of an essential solution used as coolant during ultrasonic debridement. int j of dent hygiene 2013; 11: 628. 25. hodges ko. components of optimal ultrasonic scaling. j of prof excell 2014; 12(2): 22-4, 26-7. 26. sirsi sr, borden ma. advanced in ultrasound mediated gene therapy using microbubble contras agents. theranostics 2012; 2(12): 120822. 27. vald v, berghea f, libianu s, bolanescu a, bojinca v, constantinescu c, abobului m, predeteanu d, ionescu i. ultrasound inrheumatoid arthritis volar versus dorsal sybovitis evaluation and scoring. bmc musc sce dys 2011; 12: 124. issn 1978 3728 editorial board of dental journal (majalah kedokteran gigi) sk: 52/h3.1.2/kd/2011 may 2nd, 2011 – may 2nd, 2013 patron: dean of faculty of dentistry airlangga university advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg.,ph.d., sp.kg. (conservative dentistry – airlangga university) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm (oral and maxillofacial surgery – airlangga university); prof. dr. m. rubianto, drg., ms., sp.perio (periodontic – airlangga university); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic university of hiroshima, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontica – airlangga university); prof. dr. latief mooduto, drg., m.s., sp.kg (conservative dentistry – airlangga university); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort (orthodontic – airlangga university); prof. dr. istiati soehardjo, drg., ms (oral biology – airlangga university); prof. dr. anita yuliati, drg., m.kes (dental material – airlangga university); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – airlangga university); prof. dr. diah savitri ernawati, drg., m.si (oral medicine – airlangga university); thalca i. agusni, drg., mhped., ph.d., sp.ort (orthodontic – airlangga university); dr. r. darmawan setijanto, drg., m.kes (dental public health – airlangga university); dr. elly munadziroh, drg., ms (dental material – airlangga university); priyawan rachmadi, drg., ph.d (dental material – airlangga university); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – airlangga university); dr. retno pudji rahayu, drg., m.kes (oral biology – airlangga university); dr. eha renwi astuti, drg., m.kes (dental radiology – airlangga university); bagus soebadi, drg., mhped (oral medicine – airlangga university); endang pudjirochani, drg., ms., sp.pros (prosthodontic – airlangga university); markus budi rahardjo, drg., m.kes (oral biology – airlangga university); susy kristiani, drg., m.kes (oral biology – airlangga university); ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – airlangga university); sianiwati goenharto, drg., ms (orthodontic – airlangga university); devi rianti, drg., m.kes (dental material – airlangga university); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – airlangga university); rostiny, drg., m.kes., sp.pros (prosthodontic – airlangga university); an’nissa chusida, drg., m.kes (oral biology – airlangga university); eric priyo prasetyo, drg., sp.kg (conservative dentistry – airlangga university); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – airlangga university); hendrik setiabudi, drg., m.kes (oral biology – airlangga university); otty ratna wahyuni, drg., m.kes (dental radiology – airlangga university); anis irmawati, drg., m.kes (oral biology – airlangga university); yuliati, drg., m.kes (oral biology – airlangga university); retno palupi, drg., m.kes (dental public health – airlangga university); eka augustina, drg., sp.perio (periodontica – airlangga university); febriastuti, drg., sp.kg (conservative dentistry – airlangga university); mega m. puteri, drg., sp.kga (pediatric dentistry – airlangga university) administrative assistant: novi dian prastiwi (faculty of dentistry – airlangga university) thanks to editor in duty of dental journal (majalah kedokteran gigi) vol. 44 no. 4 december 2011: sudarjani gunawan, drg., ms., sp.kg (conservative dentistry – airlangga university)(conservative dentistry – airlangga university) dr. theresia indah budhy, drg., m.kes (oral biology – airlangga university) dr. retno indrawati r, drg., msi (oral biology – airlangga university) dr. daniel haryono utomo, drg., sp.ort (dental clinic faculty of dentistry, airlangga university) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id www.dentj.fkg.unair.ac.id accredited no. 83/dikti/kep/2009 volume 44 number 4 december 2011 design cover photo by setyabudi, drg., mars., sp.kg contents page printed by: airlangga university press. (047/03.12/aup-b5e). kampus c unair, jln. mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id. ijin penerbit: no. 0787/sk/dir. pk/sit/1969. accredited no. 48/dikti/kep/2006. volume 44 number 4 december 2011 issn 1978 3728 1. betel leaf toothpastes inhibit dental plaque formation on fixed orthodontic patients rizka amelia mayasari, sianiwati goenharto, and ahmad sjafei ............................................ 169–172 2. in vivo characterization of polymer based dental cements widiyanti p and siswanto ................................................................................................................ 173–176 3. oral rinse as a potential method to culture candida isolate from aids patients desiana radithia, hening t. hendarti, and bagus soebadi ......................................................... 177–180 4. in vitro effect of q-switched nd:yag laser exposure on morphology, hydroxyapatite composition and microhardness properties of human dentin retna apsari, siswanto, anita yuliati, and noriah bidin ............................................................ 181–186 5. the effectiveness of mimba oil (azadirachta indica a. juss) spray disinfectant on alginate impression hanoem eh, wahjuni w, and dinda dewi artini ........................................................................ 187–191 6. the effect of spirulina gel on fibroblast cell number after wound healing fitria rahmitasari, wisnu setyari j, and ester arijani rachmat .............................................. 192–195 7. the difference of acrylic resin residual monomer levels with various polymerization method sherman salim ................................................................................................................................. 196–199 8. stimulation of osteoblast activity by induction of aloe vera and xenograft combination utari kresnoadi and retno pudji rahayu ..................................................................................... 200–204 9. the difference of dental anxiety in children based on frequency of dental appointment mia giri astri, eka chemiawan, and eriska riyanti ................................................................... 205–209 10. volatile sulphur compounds elimination: a new insight in periodontal treatment ernie maduratna setiawatie and rikko hudyono ........................................................................ 210–214 11. piperin and piplartin as natural oral anticancer drug berlian bidarisugma, mar’atus sholikhah, sarah usman balbeid, and anis irmawati ........... 215–219 91 the effect of exposure duration of self etch dentin bonding on the toxicity of human gingival fibroblast of cell culture sri lestari department of conservative dentistry faculty of dentistry, jember university jember indonesia abstract self etch dentin bonding created to make light easily activate the application of composite resin on tooth surface. the monomer content has acid effect that could irritate tooth pulp. the purpose of this study was to evaluate the effect of light exposure duration of self etch dentin bonding on toxicity of human gingival fibroblast of cell culture by mtt assay. self etch dentin bonding was used as on experimental unit and the sample was exposed by visible light curing in different duration: 10, 20, 30 seconds and immerged in artificial saliva in ph 7 for 24 hours. 100 µl artificial saliva was exposed to human gingival fibroblast of cell culture 20.000 cells/100 µl rpmi for 24 hours. toxicity was evaluated by mtt assay, optical density was measured using 550 nm spectrophotometer. the data was analyzed using kruskal wallis in 5% degree of significance. the result showed that increasing exposure duration (10, 20, 30 seconds) of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture. it is concluded that 30 seconds-exposure of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture. key words: exposure duration, self etch dentin bonding, toxicity, mtt assay correspondence: sri lestari, c/o: bagian ilmu konservasi gigi, fakultas kedokteran gigi universitas jember. jln. kalimantan no. 37 jember 68121, indonesia. telp. (0331) 333536 introduction the use of filler with similar color of natural tooth has high esthetical value and it is increasingly applied due to the increasing knowledge and awareness in oral health. the filler material is composed of visible light composite resin. before finally filling is performed, the application procedure is considerably complicated, requiring acid etching technique for tooth surface adhesion and bonding application. totally etching technique is performed using phosphate acid and to remove residual acid, irrigation is done. etching technique is applied to support the adhesion process of composite resin on tooth surface while bonding material should be previously applied. this episode would have an impact on the duration of existing fuller material on tooth surface. the material of dentin bonding-generating vi (self etch type) is created to simplify the procedure of bonding application and it is adhesive material with one step system which would enable a dentist to perform simultaneously etching, priming and bonding.1 self etch technique does not require acid etching phase and prolonged irrigation therefore it could be time saving for clinical application. bonding material self etch type is containing monomer with acid ph, polymer and adhesive resin. the application without irrigation would possibly cover the acid material and long duration contact with dentin is really vital.2 self etch type is achieved by increasing the concentration of acid resin monomer. water is an important additional material to ionize monomer to enable adhesive material to have optimal function, while. etching should be performed on smear layer as well as on the dentin, consequently, hybridized smear layer and hybrid layer would be formed on dentin. one of the advantages of self etch type is not to remove smear layer during etching, priming and bonding process, but to modify, therefore, smear plug is formed to decrease hydrophilic flow through dentinal tubule.1 residual monomer is a substance which is unreacted at the end of polymerization.4 residual monomer and acid in dentin bonding applied on tooth tissue would immediately come into contact. they would be released into the adjacent soft tissue or pulp tissue resulting the death of cells. resin based restorative dental material would release the substance having biological viability. the application of most sophisticated method to detect the substance is unable adequately to detect biological property correlated with the concentration.5 primary component which is mostly released from urethane dimethacrylate (udma) composite resin is monomer, then followed by small number of 1.6 hexane dimethacrylate, chamfer quinone.6 resin based restorative material shows cytotoxicity effect and in-vitro change of cell function, while, high performance chromatography (hplc) is incapable to detect clearly the number of released substance.5 when the polymerization process is completed, the component existed in tooth resin would be immediately eluated into the environment resulting metabolic change in various cells.7 exposure duration is one of the factors affecting polymerization of resin monomer originating from bonding material and composite resin. effective exposure 92 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 91-94 duration for composite resin is 20 to 60 seconds with 2 mm restoration thickness.8 the 10 second of exposured duration is recommended by the manufacture for self etch dentin bonding. the increase of exposured duration would cause more adequate polymerization of adhesive resin composed of bonding material self etch type resulting low residual monomer. prolonged exposure duration whether it would terminate the process of acid etching in tooth hard tissue contacting to bonding material is still unknown. the impact on physical and chemical property as well as on adjacent soft tissue occurring in the process of acid etching without irrigation followed by polymerization is also unknown. the impact on soft tissue in-vitro test could be identified by toxicity occurring in human gingival fibroblast of primary culture cell. the use of human gingival fibroblast of primary culture cell because it has been considered closed to natural condition compared to animal fibroblast it culture cell or line cell and it has different sensitivity. toxicity test using mtt assay is a method which is considered sensitive and easy to be done to detect living cell compared to other methods. mtt assay is based on the capability of living cell to reduce mtt salt. the principle is to break down ring of mtt tetrazolium [3-(4.5 dimethilthiazol-z-yl)-2.5-diphenyl tetrazolium bromide)] through dehydrogenase in active mitochondria, producing unsoluble blue formazan.9 formazan product can be counted by dissolving and measuring optical density of solution outcome.8 blue color reaction is used as an indicator of living cell measured as mtt product using spectrophotometer with 570 nm wave length.9,10 based on the above explanation, a study is necessarily conducted on the effect of exposure duration of bonding material self etch type in toxicity of human gingival fibroblast of primary culture cell using mtt assay. the advantage of the outcome is to understand the impact of exposure duration and toxicity on human gingival fibroblast. materials and methods the study was performed by experimental laboratory. method and self etch type as the sample. the artificial saliva was composed of 36 gr nacl, 1.69 gr kcl, 0.95 cacl2, 0.85 gr nahco3 and 400 cc destilled water. the devices applied were light curing (latex), micropipette, flask, conical, petridish, syringe, 96 well micro plate, laminar flow, co2 incubator, spectrophotometer, sheaher, centrifuge, and counter. the sample was classified into 3 groups according to exposure duration: 10, 20, 30 seconds, each group was consist of 7 samples. material of self etch type dentin bonding was mixed equally 1:1, homogenously stirred using brush. 10 µl mixture was taken by micro point and poured sterile aluminium foil, followed by curing in individual group for 10, 20, 30 seconds and soaked in tube containing 1500 µl artificial saliva for 24 hours, the mixture was removed after the period was over. the next process, the artificial saliva which was used to soak the self etch type dentin bonding (eluate) would be applied as the sample of the study. 100 µl of each controlled group was taken for toxicity test. the preparation process of human gingival fibroblast of primary culture was performed by obtaining gingival tissue taken from extracted gingival of healthy tooth. next, the gingival tissue was washed using povidon iodine, and irrigated by phosphate buffer saune (pbs). gingival tissue was repeatedly washed (3 times) by pbs composed of penicillin and streptomycin in petridish and cut into pieces using sterile scissors. next, it was placed in another petridish filled by rpmi culture media added by fungi zone or gentamycin. the petridish was covered and shaken to make the gingival tissue equally spread in petridish. the incubation was done for 3 days in 5% co2 incubator at 37° c, the process was done in sterile environment of laminar flow. culture media was changed once in 3 days until the cell growth would reach 50% of petridish surface or confluent in which the subculture would be possibly conducted. toxicity test was performed using 96 hole microplate in which in every hole would accommodate 20.000 human gingival fibroblast of cell culture and 10100 µl rpmi. in negative controlled group consisted of cell + rpmi media, while, cell + rpmi media + artificial saliva in positive controlled group. controlled group included the sample consisted of 100 µl artificial saliva which had been exposed by eluate of self etch type dentin bonding in different exposure duration (10, 20, 30 seconds) in every hole. microplate was incubated for 24 hours (or 20 hours) in 5% co2 incubator then removed out and added by 5 mg/ml mtt and 100 µl pbs in every hole. the incubation was continued for 4 to 5 hours, the supernatant was disposed, meanwhile, 200 µl dimethyl sulfoxide (dmso) was dropped in every hole, waited for 5 minutes, sheaher was done in 30 rpm for 30 minutes the supernatant was removed then it was washed twice using 100 µl pbs. the next, microplate was inserted into spectrophotometer in 550 nm wave length and color absorbance could be identified (formazan optical density = optical density). result the mean of optical density, the percentage of living cell and primary culture toxicity of human gingival fibroblast cell exposed by eluate of self etch dentin bonding type in artificial saliva could be shown in table 1. low toxicity was shown by high optical density, on the contrary high toxicity was shown by low optical density. the higher the number of living cell, the higher the amount of color absorbance would occur (high od) showing low toxicity. toxicity scoring was identified by od value in controlled group cell + media was 100% (estimated toxicity value = 0) cell toxicity was consecutively graded 93lestari: the effect of exposure duration of self etch dentin bonding on the toxicity according to exposure duration: 10, 20, 30 seconds. prior to statistical analysis. homogeneity test was done using levene test showed value of significance 0.001 (p < 0.005), the meaning is in homogeneous data. the next followed by non parametric test using kruskal wallis. the result could be shown on table 2. table 2. kruskal wallis statistical test analysing toxicity of human gingival fibroblast of cell culture exposed eluate of self etch dentin bonding exposure duration chi square df a symp sig 8.440 6 208 the result of kruskal wallis test showed no significant difference was found in the group exposed by eluate of bonding dentin self etch in exposure duration 10, 20, 30 seconds (p > 0.001) indicating that toxicity of human gingival fibroblast of cell culture was not affected by the increase of exposure duration of self etch type dentin bonding. discussion the result of the study showed there was no significant difference in toxicity among the groups with exposure in different duration. the highest toxicity was found in bonding dentine self etch type for 10 second exposure due to unpolymerized monomer which was much higher comparing to 20 seconds and 30 seconds exposure. lack exposure decreased energy which functioned to activate polymerization process therefore the degree of polymerization would also decrease4 and the decrease of light energy would also simultaneously contribute the decrease of photon number which was useful to activate free radical formation.12 consequently the decrease of free radical would make the process of polymerization less maximal resulting in the increase of residual monomer. residual monomer would be instantly released into fluid environment producing the response on the contact site of dentin bonding.7 the finding is also supported by the idea that cytotoxity occurs due to the release of unpolymerizes main component from the material on air resistant layer. inadequate polymerization process might make high concentration of residual monomer.13 the release of residual monomer would be more and higher in bonding dentin self etch type which has high concentration residual monomer, because bonding dentin self etch type with low exposure duration possibly will have high residual monomer therefore the release would come into contact with the adjacent fluid tissue is high resulting higher value of toxicity compared to 20, 30 second-exposure duration. in this case, it is in accordance with the previous study stated that inadequate polymerized monomer would release 50% after 3 hours being soaked in oral fluid and 85–100% after 24 hours.14 in this study, the existing residual monomer could be released into artificial saliva affecting soft tissue and as a matter of fact the residual monomer was toxic to human gingival fibroblast. it was revealed in bonding dentin for 10 secondexposure that od 0.206, living cell 26.7%, toxicity 73.3% (cd > 50%), it was evidently toxic. the toxicity of residual monomer was also similar to the previous study suggested that dental material would be toxic in the condition of cd > 50%.15 the analysis using kruskal wallis showed no significant difference suggesting the cytotoxicity was most possible due to the acid content in bonding dentin as well as residual monomer resulted from incomplete polymerization process.1,16 the occurrence of many dead cells because of acid monomer (ph < 1) content in bonding dentin self etch type.1 the elevation of cell membrane permeability was also contributed by the monomer, as a result, internal plasma membrane was exposed by the acid material, therefore, cell inflammation would eventually exhibit the synthesis process and dead cell would occur. low ph might also cause protein denaturation that was the damage of covalent intra molecular binding disulfide and ionic binding, hydrophobic and hydrogen binding.16 another reason suggested that bonding dentin self etch type was consisted of hema which was used to increase the strength of bonding and dentin with 130.14 molecule weight could be completely soluble in water. high concentration supported by hema rapid release in self etch dentin table 1. the mean of optical density, the percentage of living cell and the toxicity of human gingival fibroblast of cell culture exposed by eluate of self etch type dentin bonding 10 seconds 20 seconds 30 seconds od lc dc od lc dc od lc dc cell + media (controlled) cell + media + artificial saliva (controlled) eluate of bonding dentin in 100 µl artificial saliva 0.772 0.767 0.206 100% 99.3% 26.7% 0% 0.76% 73.3% 0.772 0.767 0.437 100% 99.3% 56.6% 0% 0.76% 43.4% 0.772 0.767 0.465 100% 99.3% 60.2% 0% 0.76% 39.8% note: od = optical density, lc = living cell, do = death cell > 50% 94 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 91-94 bonding.17 in addition, the cause of toxicity was the activity of material which was not neutralized by calcium ion of the teeth as it was clinically applied in-vivo study. this would differ between the application on tooth with in-vitro study without application on tooth tissue. performing in vitro toxicity test on cell culture could be the evidence of initial biocompatibility of dental material. in this study, primary culture of human gingival fibroblast cell was applied in order to achieve closely similar to natural clinical condition. because human gingival fibroblast cell is the most important cell and the biggest component of pulp and periodontal ligament, in this study primary culture of gingival tissue was applied. the strength of primary culture of human gingival fibroblast cell is having sensitive response compared to cell line culture. the weakness is primary culture having short life span and difficult to do repeated subculture therefore it requires procedure of new culture process for application. based on the result of the study, it could be concluded that 10, 20, 30 seconds light exposure of dentin bonding self etch type on human gingival fibroblast cell reduces toxicity. references 1. wei sh, tay fr. xeno iii and self etch bonding. clinical suplement 2003; update (mei–agustus,14): 1–5. 2. van meerbek b. adhesion to enamel and dentin current status and future challenges. operative dentistry 2003; 215–35. 3. pashley dh, tay fr. aggressiveness of contemporary self etching adhesives part ii: etching effects on unground enamel. dental material 2001; 17:430–44. 4. siswomihardjo w. poliester ebp 2421 sebagai alternatif bahan basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1999. p. 80–5. 5. wataha jc, rueggerberg fa, lapp ca, lewis jb. in vitro cytotoxicity of resin containing restorative materials after aging in artificial saliva. clinic oral invest 1999; 3:144–9. 6. mohsen nm, craig rg, hanks ct. cytotoxity of urethane dimethacrylate composites before and after aging and leaching. j biomed mater res 1998; 39(2):252–60. 7. lefebvre ca, knorenschild ki, schuster gs. cytotoxicity of evaluates from light-polymerized based resin. j prosth dent 1994; 72:644–50. 8. craig rg, powers jm. restorative dental materials. 11th ed. st. louis: cv mosby co; 2002. p. 136–7. 9. kasugai s, hasegawa n, ogura h. application of the mtt colorimetric assay to measure cytotoxic effects of phenolic compounds on established rat dental pulp cells. j dent res 1991; 70:27–30. 10. fazwishni s, hadijono bs. uji sitotoksisitas dengan esei mtt. j keduji sitotoksisitas dengan esei mtt. j kedj ked gigi universitas indonesia 2000; 7:28–32. 11. kawaguchi m, takahashi y, fukushima t, habu t. effect of light exposure duration on the amount of leachable monomers from lightactivated reline materials. j prosth dent 1996; 75:183–7. 12 annusavice jk. phillip’s science of dental materials. 10th ed. philadelphia, london, toronto, montreal, sydney, tokyo: wb saunder’s co; 1996. p. 69–298. 13. pearson gj, longman cm. water sorption and solubility of resinbased material following inadequate polymerization by a visiblelight curing system. j oral rehab 1989; 16:57–61. 14. hanks ct, strawn se, wataha jc, craig rg. cytotoxic effects of resin components on cultured mammalian fibroblast. j dent rest 1991; 70(11):1450–5. 15. telli c, serper a, dogen al, guc d. evaluation of the cytotoxity of calcium phosphate root canal sealers by mtt assay. journal endodontics. 1999; 25:811–3. 16. erawati w, latief m, theresia ibs. uji sitotoksisitas ekstrak air asam jawa 5% terhadap cell line bhk 21. indonesian journal of dentistry 2007; 14(1):18–21. 17. vajrabhaya l, pasasuk a, harnirattisai c. cytotoxicity evaluation of single component dentin bonding agents. operative dentistry 2003;operative dentistry 2003; 20:442–4.–4.4. mkg vol 41 no 4 oct-dec 2008.indd 186 vol. 41. no. 4 october–december 2008 review article the combination of sodium perborate and water as intracoronal teeth bleaching agent ananta tantri budi department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract background: the color change on post-endodontic treated teeth can be overcome by intracoronal tooth bleaching using walking bleach. some agents used in walking bleach are combination of sodium peroxide and hydrogen peroxide, and combination of sodium perborate and water. purpose: the objective of this review is to provide information and consideration of using safe and effective bleaching agents in the field of dentistry. reviews: on one side, the use of sodium perborate and water combination does not cause the reduction of dentin hardness, enamel decay, and root resorbtion. on the other side, the use of sodium perborate and 30% hydrogen peroxide combination indicates that it takes longer time in yielding the proper color of teeth. conclusion: the use of sodium perborate and water combination as bleaching agents is effective and safe. key words: intracoronal teeth bleaching agents, sodium peroxide, hydrogen peroxide, water correspondence: ananta tantri budi, c/o: departemen konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. introduction efforts for tooth bleaching by endodontic treatment has been undergone for a long time. the tooth bleaching in pulp chamber is called intracoronal tooth bleaching.1 the bleaching agents which are commonly used are sodium perborate and hydrogen peroxide. since 1924, sodium perborate and 30% hydrogen combination have been used. the combination of sodium perborate and 30% hydrogen peroxide placed in pulp chamber are known as walking bleach technique. in further development, the combination of sodium perborate and water is applied.2 hydrogen peroxide 30% known as superoxol may be used with or without another agent. hydrogen peroxide 30% must be used cautiously because it can cause skin and mucosal membrane burn like chemical burn.3 the use of sodium perborate combined with hydrogen 30% is effective for tooth bleaching. as hydrogen peroxide 30% has some cautions, the use of sodium perborate and hydrogen peroxide 30% may cause unexpected complication. for example, it increases dentin permeability that can cause the decrease of dentin hardness, and the enamel decay of external root resorbtion.2 kaneko2 recommends the combination of sodium perborate and water as bleaching agents. this combination may not obliterate oral mucosa. timpawet et al.,3 in his research claimed that combination of sodium perborate and water is the best intracoronal tooth bleaching agent. based on this background, the writer needs to explain the use of sodium perborate and water as effective and safe intracoronal tooth bleaching agents. it is expected that this writing can provide information and consideration of using sodium perborate and water in the field of dentistry. sodium perborate sodium perborate is called perborate acid or metaperborate. in the form of white powder containing nabo3, is water soluble, stable in dry and cool air. it decomposes and releases oxygen in hot and humid air. usually, it is available in granular the form which has to be ground into powder before use.4 sodium perborate is available in two forms: tetrahydrate and monohydrate. sodium perborate tetrahydrate is gained 187budi: the combination of sodium perborate and water by adding hydrogen peroxide into sodium metaborate solution in 20° c. sodium metaborate can be formulated from reaction of boraxspentahydrate and sodium hydroxy. compared to sodium perborate tetrahydrate, sodium perborate monohydrate has more advantages. it has more oxygen, more stable in heating process, and more soluble in the water.5 independently, sodium perborate in the form of crystal is difficult to be applied, but if it is combined with hydrogen peroxide 30% or water, it can be changed into pasta.1 sodium perborate can react with sodium tetraborate, hydrogen peroxide, and sodium hydroxide. sodium perborate rapidly releases oxygen at temperatures above 60° c. to activate sodium perborate at lower temperature (40–60° c), sodium perborate can be blended with appropriate activator such as tetra-asetildiamin (tead).6 superoxol blended with sodium perborate will turn out to be pasta containing sodium metaborate, water, and oxygen. the reaction of sodium perborate mixed with water will be as the following nabo3 + h2o2 nabo2 + h2o + on. even though the combination of hydrogen peroxide and sodium perborate is effective in tooth bleaching, both bleaching agents–hydrogen peroxide and sodium perborate–can cause unexpected complications. they can enhance dentin permeability, cause the micro leakage on restoration, and trigger the occurrence of external root resorbtion.3 hydrogen peroxide thenad7,8 in 1818 reported that hydrogen peroxide was made from decomposing berium peroxide with sulphate acid and phosphate acid. the hydrogen peroxide is sheer, colorless, viscous, lighter than water, more water soluble, durable for its oxidator and able to produce boundless radical which is very reactive. hydrogen peroxide can dissolve because of light. therefore, it must be stored in cool place and no direct high contact. hydrogen peroxide can dissolve into water and reactive oxygen, as the following reaction: 2 h2o2 2 h2o + o2 + energy it will dissolve and be ionized as the following reaction: nabo3 + h2o nabo3 + h2o2 (alkali) h2o + on the presence of ionized sodium perborate will increase the effectiveness of unbound radical production. hydrogen peroxide can release unbound radical, perhydroxyl anion, or combination of both. the reaction is as the following: h2o2 h + ooh h2o2 ho + oh when perhydroxyl anion is released by h2o2, the reaction will be: h2o2 + h + + ooh– then, h2o2 obtains unbound radical and anion in the acid and alkali solution, as the following reaction: hoo– + oh– o2 – + h2o hoo– o– + h+ these formed compounds tend to be drawn by double bond (=) alkenes by forming unstable peroxide and ultimately forming colorless alcohol, and become water soluble. hydrogen peroxide can also increase dental tissue permeability, so it helps inward or outward ion diffusion. the reasons are because the mass of hydrogen peroxide is heavier than that of the molecules, and its ability of protein denaturation can also increase the diffusion process.9 the reaction between hydrogen peroxide and alcohol, ketone, carboxylic acid, and phosphate can cause fire and explosion. hydrogen peroxide with concentration more than 50% is corrosive, can cause irritation on eyes, mucosa membrane, and skin. if this hydrogen peroxide solution is swallowed accidentally, it can be decomposed into excessive gas which can cause internal bleeding. in some cases, it can cause lung irritation because of inhaling hydrogen peroxide which concentration is more than 10%.7 superoxol is a solution which is composed of 30% hydrogen peroxide and 100% distilled water. the distilled water has a molecular mass of 34.01 g/mol.10 this agent is pure, colorless, unstable, odourless liquid which must be saved into a light proof bottle, and kept away from heat since it is explosive. it is also better to save it in closed place since it can decompose easily. superoxol is more stable and easily applicable. it can be applied by itself or can be mixed with sodium perborate as paste in walking bleach technique.1 however, superoxol must be carefully used since it can cause wound like chemical burn in skin and mucosa membrane. the reason is because superoxol contains oxygen twice as much as sodium perborate, so it is reactive and can cause soft tissue burns.2 tooth bleaching mechanism tooth bleaching mechanism consists of oxygen releasing action, mechanical cleansing actions, oxidation, and reduction. the bleaching process can occur if there is an alteration in ph, temperature, and light of the peroxide agent in order to produce active oxygen as oxidants. peroxide as oxidator agent has oxidants which have single electrons as strong and unstable electrophile, so those oxidants can force other organic molecule in order to be stable and produce other oxidants. since electrophile has only one electron in its chemical structure, it then tries to get pairs of electrons in order to be stable. those unstable electrons will be released 188 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 186−189 and then accepted by enamel. finally, during oxidation process those electrons will be oxidized by organic agents causing the change of the tooth colour.11 the oxidants of peroxide are perhydroxyl and oxygenise. perhydroxyl is a strong oxidant while oxygenase is a weak oxidant. the formation of perhydroxyl can be increased by increasing ph of peroxide from 9.9 to 10.9, and then this buffer process can also improve the effect of tooth bleaching. oxygenase as a weak oxidant has also a role in tooth bleaching. oxygenase can react with hydroxyapatite molecules in teeth, which reaction is as below: ca10(po4)6(oh)2 + on 10 cao + 3 p2o5 +h2o hydroxyapatite white colour oxygenase reaction with hydroxyapatite in teeth can cause cao deposit. this cao deposit can create white colour in teeth.12 discussion the bleaching agents used for tooth bleaching after endodontic treatment are the combination of sodium perborate and 30% hydrogen peroxide, and sodium peroxide and water. the combination of each agent has weaknesses and strengths. the good bleaching agent must have strong penetration power to penetrate organic agents inside dentin tubules and enamel interprismatic chambers without devastating the crown.13 enamel and dentin erosion occurs when ph is less than 4; meanwhile other researchers show that ph 4.6–7.4 can influence the structure of enamel and dentin even though it generally gives minor influence.14,15 pecora et al.16 did a research by using 36 first incisive tooth elements in upper jaw to analyze the effect of the bleaching agents on the hardness of dentin. the combination of sodium perborate and water resulted the least dentin hardness reduction compared to the other combination. the combination of 10% carbamide peroxide and 30% hydrogen peroxide resulted the highest dentin hardness reduction; meanwhile the combination of sodium perborate and hydrogen peroxide caused medium dentin hardness reduction. some researchers indicate that intracoronal bleaching agents can cause cervical root resorption. cervical root resorption is a progressive root resorption which is from the dental epithel, generally in dental cervical area.17 this condition is caused by the acidic condition which can make hydroxyl apatite soluble so that there is demineralization of hard tissue components which then causes cervical root resorption.18 the ability of the bleaching agents can alter the acid ph around tooth produced by the penetration of the bleaching agents. the use of intracoronal bleaching agents in pulp chamber can diffuse through dentin tubule into cervical of periodontal ligament. by accumulating into cervical area at the edge of dentinali, it can influence periodonsium tissue during the concurrence between enamel and cementum, containing bacterial colonies which can cause root resorption.10 the result of in vitro study shows that ph of dentin and cementum can become more acidic after the occupying of the combination of sodium perborate and 30% hydrogen perborate in pulp chamber. polymorphonuclear leukocyte and osteoclast activities are increased in acid condition, as a result, it can produce hydroxyl apatite solution and stimulate demineralizing hard tissue components and also prevents the formation of new hard tissue, causing cervical root resorption.18 the alkaline condition of periodontal tissue in cervical is not good for osteocla activities so that it can prevent the devastating and dematerializing of hard tissue because of the procedures of intracoronal tooth bleaching. the character of sodium perborate is alkaline; meanwhile the character of hydrogen peroxide is acid. thus, if both agents are combined, it can produce an agent which is alkaline.10 bleaching with increase the ph of post endodontic treated teeth. it shows that ph tends to increase as long as the bleaching agents are decomposed. the increasing ph is indicated by the alteration of 30% hydrogen peroxide, which is acid, to become oxygen and water.10 this difference is caused by of the different bleaching agents and method used. this alteration of ph depends on the bleaching agents used as well as the amount of 30% hydrogen peroxide which can create acid condition if it is in higher amount. this condition shows that the combination of sodium peroxide and 30% hydrogen peroxide can become acid or alkaline rely upon the amount of 30% hydrogen peroxide which is added. the combination of sodium perborate and water produces alkaline condition so that it can not cause root resorption. according to some researchers, there is no root resorption in tooth which have been treated with intracoronal bleaching therapy by using the combination agents of sodium perborate and water. the colour of the teeth is also the same as the colour of those which have been bleached by using 30% hydrogen peroxide. based on the discussion above, it can be concluded that intracoronal bleaching therapy by combining sodium perborate and water has the same result in tooth bleaching as by combining sodium perborate and 30% h2o2, without causing root resorption and resorbing the dentin tissue. references 1. halim f. bleaching setelah perawatan saluran akar. majalah ilmu kedokteran gigi fakultas kedokteran gigi universitas trisakti 2001; 16(43): 22–26. 2. kaneko j, ivone s, sano h. bleaching effect of sodium perborat on discolored pulp less teeth in vitro 1-year study. j endod 2000; 26: 25–8. 3. timpawat s, et al. effect of bleaching agent on bonding to pulp chamber dentine. international endodontic journal 2005; 38(3): 571–5. 189budi: the combination of sodium perborate and water 4. wikipedia encyclopedia. sodium peroxides. available at: http:// en.wikipedia.org/wiki. .2006. accessed february 16, 2008. 5. syangyucem. expert on peroxides. 2005. available at: http://www. chemword.com/sodium perborate.htm. accessed november 3, 2007. 6. wikipedia encyclopedia. sodium perborate. 2006. availlable at: http://en.wikipedia.org/wiki. accessed february 16, 2008. 7. wikipedia encyclopedia. hydrogen peroxides. available at: http://. wikipedia.org/wiki. 2006. accessed february 16, 2008. 8. usman m. pemutih gigi yang berubah warna akibat trauma. majalah ilmu kedokteran gigi fakultas kedokteran gigi universitas trisakti 2002; edisi khusus foril:76–80. 9. kanzil l, santoso r. efek samping pemakaian bahan pemutih gigi terhadap tumpatan amalgam. majalah ilmu kedokteran gigi 2002; 17(49): 99–104. 10. diatri nr. pengaruh kerusakan sementum terhadap penetrasi bahan pemutih gigi hydrogen peroksida 30% dikombinasi dengan sodium perboratke daerahsekitarakar gigi. majalah ilmu kedokteran gigi indonesia 2005; iv(7): 139–42. 11. yudha dr, maya id, robert d. dental whitening. dental lintas meditama. 2005. p. 9–25. 12. tarigan, rosinta. perawatan pulpa gigi (endodonti). jakarta: widya medika; 1996. p. 205. 13. sudarjani gh. pencegahan perubahan warna kembali setelah perawatan pemutihan gigi. majalah kedokteran gigi 1999; 32(1): 23–5. 14. touti b, miara p, nathason r. treatment of tooth discoloration pada esthetic dentistry and ceramic restoration. london: martin dunitz; 1999. p. 81–116. 15. suprastiwi e. penggunaan karbamid peroksida sebegai bahan pemutih. indonesian journal of dentistry 2005; 12(3): 139–45. 16. pecora jd, cruz-filho am, desousa-nero md, silva rg. in vitro action of various bleaching agent on the micro hardness of human dentin. 1999. available at: http// www.forp.usp.br/ restouradora/ trabalas/action.htm. accessed january 22, 2007. 17. martin t. cervical root resorbstion. journal american dental assosiation 1997; 128: 56–9. 18. ne rf, witherspan de, gutman jl. tooth resorbtion. quitessence international 1999; 30(1): 9–25. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice p-issn: 1978-3728 e-issn: 2442-9740 volume 48, number 1, march 2015 editorial board of dental journal (majalah kedokteran gigi) sk: 04/un3.1.2/2015 january 2nd– december 31st, 2015 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: ketut suardita, drg., ph.d., sp.kg (conservative dentistry-universitas airlangga) editorial boards: prof. widowati witjaksono, dds, ph.d (kulliyah of dentistry, international islamic university malaysia); prof. boy m. bachtiar, drg., ms., ph.d (oral biology – universitas indonesia); prof. heriandi sutadi, drg., sp.kga., ph.d(k) (pediatric dentistry – universitas indonesia); prof. tri murni abidin, drg., m.kes., sp.kg(k) (conservative dentistry – universitas sumatera); prof. dr. iwa sutardjo rus sudarso, drg., su., sp.kga(k) (pediatric dentistry – universitas gadjah mada); prof. dr. drg. regina titi christinawati, m.sc (oral biology – universitas gadjah mada); prof. dr. latief mooduto, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. adioro soetojo, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. sri kunarti, drg., ms., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlanggasia); dr. chiquita prahasanti, drg., sp.perio(k) (periodontics – universitas airlangga); dr. indah listiana kriswandini, drg., m.kes. (oral biology – universitas airlangga); dr. retno indrawati, drg., msi. (oral biology – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral pathology and maxillofacial – universitas airlangga); dr. agung krismariono, drg., m.kes., sp.perio (periodontics – universitas airlangga); dr. ira arundina, drg., m.kes. (oral biology – universitas airlangga); wisnu setyari, drg., m.kes (oral biology – universitas airlangga). managing editors: dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); udijanto tedjosasongko, drg., ph.d., sp.kga (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); ketut suardita, drg., ph.d., sp.kg (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontics – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); eka fitria augustina, drg., m.kes., sp.perio (periodontics – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga). administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/ 5030255. fax. (031) 5039478/ 5020256 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg accredited no. 56/dikti/kep/2012 cover photo purchased from: www.fotolia.com invoice number: 206803056-204225738 preface dental journal (majalah kedokteran gigi) is proud to be one of few certified scientific journal by the indonesian higher education authority. we have been publishing since 1968. we are supported by indonesian and international editors. quality is always maintained in order to provide our readers with update scientific information. with the spirit and vision to be an internationally indexed journal, dental journal (majalah kedokteran gigi) is published in english and now available both print and online. our full journal articles can be accessed online since 2005. in 2015 we change our design layout, and update our guide for authors. we also open our online journal portal to begin processing online journal submission. we understand that there are problems and challenges to become internationally recognized and indexed but we believe with optimism and hard work, our solid team will succeed. we would also like to acknowledge generous supports from the dean of faculty of dental medicine universitas airlangga, airlangga journal development team, and national scientific journal reviewer board of the indonesian ministry of education. best regards, chief editor ketut suardita, drg., ph.d., sp.kg. contents page printed by: airlangga university press. (rk 163/06.15/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 48, number 1, march 2015 p-issn: 1978-3728 e-issn: 2442-9740 1. trans-adapted, reliability, and validity of children fear survey schedule-dental subscale in bahasa indonesia arlette suzy, julian amriwijaya and efi fitriana ........................................................................ 1–6 2. effect of casein phosphopeptide-amorphous calcium phosphate on the flexural strength of enameldentin complex following extracoronal bleaching diatri nari ratih and hendargo agung pribadi ........................................................................... 7–11 3. the application of lesion sterilization and tissue repair 3mix-mp for treating rat's dental pulp tissue raditya nugroho, ananta tantri budi, and sri kunarti ............................................................. 12–15 4. genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast bhk-21 cell cultures ceples dian kartika w.p, sri kunarti, and ari subiyanto ......................................................... 16–21 5. the effect of silanated and impregnated fiber on the tensile strength of e-glass fiber reinforced composite retainer niswati fathmah rosyida, siti sunarintyas, and pinandi sri pudyani ...................................... 22–25 6. the effect of nanochitosan hydrogel membrane on absorbtion of nickel, inhibition of streptococcus mutans and candida albicans andi triawan, pinandi sri pudyani, soesatyo marsetyawan hne, and sismindari ................ 26–30 7. the increasing of beta-defensin-2 level in saliva after probiotic lactobacillus reuteri administration tuti kusumaningsih ........................................................................................................................ 31–34 8. the decreasing of nfκb level in gingival junctional epithelium of rat exposed to porphyromonas gingivalis with application of 1% curcumin on gingival sulcus agung krismariono ......................................................................................................................... 35–38 9. the influence of adhesin protein from aggregatibacter actinomycetemcomitans on il-8 and mmp-8 titre in aggressive periodontitis rini devijanti ridwan .................................................................................................................... 39–42 10. the effect of 1,25-dihydroxyvitamin d3 on msx2 gene expression during tooth and alveolar bone development intan ruspita .................................................................................................................................... 43–47 11. the effects of ultrasonic scaling duration and replication on caspase-3 expression of sprague dawley rat's pulp cells archadian nuryanti, marsetyawan hne soesatyo, dewi agustina, and siti sunarintyas ...... 48–52 issn 1978 3728volume 47, number 2, june 2014 editorial board of dental journal (majalah kedokteran gigi) sk: 059/un3.1.2/2014 january 2nd– december 31st, 2014 patron: dean of faculty of dental medicine, universitas airlangga advisors: vice dean i, vice dean ii, vice dean iii chief editor: udijanto tedjosasongko, drg., ph.d., sp.kga(k) (pediatric dentistry – universitas airlangga) editorial boards: prof. r.m. coen pramono d, drg., su., sp.,bm(k) (oral and maxillofacial surgery – universitas airlangga); prof. dr. m. rubianto, drg., ms., sp.perio(k) (periodontic – universitas airlangga); prof. nairn hutchinson fulton wilson, msc., ph.d., fds (conservative dentistry university of guy’s dental school, london); prof. w.j. spitzer, dmd., md (head department of cranio & oral maxillofacial surgery hamburg university, germany); prof. edward c. combe, msc., ph.d., ddsc (biomaterial – minnesota university, u.s.a); prof. madya. h. ab. rani samsudin, dds., fdsrc, am (oral and maxillofacial surgery university sains, malaysia); prof. widowati witjaksono, dds., ph.d (kulliyah of dentistry, international islamic university malaysia); prof. taizo hamada, dds., ph.d (prosthodontic tohoku university, japan); prof. yukio kato, dds., ph.d. (oral bio chemistry-university of hiroshima, japan); prof. kozai katsuyuki, dds., ph.d (pediatric dentistry – university of hiroshima, japan); prof. dr. a.g.m. tielens (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); prof. lakshman samaranayake (oral microbiology – the university of hongkong); kok van kesel (medical microbiology – university and infections diseases – erasmus university medical centre, rotterdam, the netherlands); dr. leslie ang (restorative dentistry – national dental centre of singapore); prof. dr. m. suharsini, drg., ms., sp.kga (pediatric dentistry universitas indonesia); achmad gunadi, drg., ms., ph.d (prosthodontic universitas negeri jember) managing editors: prof. dr. arifzan razak, drg., msc., sp.pros (prosthodontics – universitas airlangga); prof. dr. latief mooduto, drg., m.s., sp.kg(k) (conservative dentistry – universitas airlangga); prof. dr. mieke sylvia m.a.r., drg., ms., sp.ort(k) (orthodontic – universitas airlangga); prof. dr. istiati soehardjo, drg., ms (oral biology – universitas airlangga); prof. dr. anita yuliati, drg., m.kes (dental material – universitas airlangga); prof. seno pradopo, drg, su., ph.d., sp.kga (pediatric dentistry – universitas airlangga); prof. dr. diah savitri ernawati, drg., m.si., sp.pm(k) (oral medicine – universitas airlangga); prof. thalca i. agusni, drg., mhped., ph.d., sp.ort(k) (orthodontic – universitas airlangga); dr. r. darmawan setijanto, drg., m.kes (dental public health – universitas airlangga); dr. elly munadziroh, drg., ms (dental material – universitas airlangga); priyawan rachmadi, drg., ph.d (dental material – universitas airlangga); dr. retno pudji rahayu, drg., m.kes (oral biology – universitas airlangga); dr. eha renwi astuti, drg., m.kes (dental radiology – universitas airlangga); bagus soebadi, drg., mhped., sp.pm (oral medicine – universitas airlangga); endang pudjirochani, drg., ms., sp.pros (prosthodontic – universitas airlangga); markus budi rahardjo, drg., m.kes (oral biology – universitas airlangga); dr. susy kristiani, drg., m.kes (oral biology – universitas airlangga); dr. ira widjiastuti, drg., m.kes., sp.kg (conservative dentistry – universitas airlangga); ketut suardita, drg., ph.d., sp.kg. (conservative dentistry – universitas airlangga); sianiwati goenharto, drg., ms (orthodontic – universitas airlangga); devi rianti, drg., m.kes (dental material – universitas airlangga); dr. chiquita prahasanti, drg., sp.perio(k) (periodontic – universitas airlangga); rostiny, drg., m.kes., sp.pros(k) (prosthodontic – universitas airlangga); an’nissa chusida, drg., m.kes (oral biology – universitas airlangga); eric priyo prasetyo, drg., sp.kg (conservative dentistry – universitas airlangga); els sunarsih budipramana, drg., ms., sp.kga(k) (pediatric dentistry – universitas airlangga); dr. hendrik setiabudi, drg., m.kes (oral biology – universitas airlangga); otty ratna wahyuni, drg., m.kes (dental radiology – universitas airlangga); anis irmawati, drg., m.kes (oral biology – universitas airlangga); yuliati, drg., m.kes (oral biology – universitas airlangga); retno palupi, drg., m.kes (dental public health – universitas airlangga); eka augustina, drg., sp.perio (periodontica – universitas airlangga); febriastuti, drg., sp.kg (conservative dentistry – universitas airlangga); mega m. puteri, drg., sp.kga (pediatric dentistry – universitas airlangga) administrative assistant: novi dian prastiwi (faculty of dental medicine – universitas airlangga) thanks to editor in duty of dental journal (majalah kedokteran gigi) volume 47, number 2, june 2014: prof. dr. hj. roosje rosita oewen, drg., sp.kga (pediatric dentistry – universitas padjadjaran) prof. dr. al supartinah, drg., su., sp.kga (pediatric dentistry – universitas gadjah mada) prof. dr. drg. regina titi christinawati, m.sc (oral biology – universitas gadjah mada) maretaningtias dwi ariani, drg., m.kes., ph.d (prosthodontics – universitas airlangga) editorial address c/o: fakultas kedokteran gigi universitas airlangga jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp. (062-31) 5039478/5030255. fax. (031) 5039478/5020256 e-mail: dental_journal@yahoo.com website: www.journal.unair.ac.id accredited no. 56/dikti/kep/2012 cover photo purchased from: www.folia.com invoice number: 206708019-204225738 contents page printed by: airlangga university press. (rk 427/12.14/aup-a45e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, telp./fax. (031) 5992248. e-mail: aupsby@rad.net.id; aup.unair@gmail.com accredited no. 56/dikti/kep/2012. volume 47, number 2, june 2014 issn 1978 3728 1. evaluasi karakteristik abu sekam padi dengan kitosan molekul tinggi nanopartikel sebagai bahan dentinogenesis (characteristic evaluation of rice husk ash with chitosan high molecule nanoparticle as dentinogenesis material) pretty farida sinta silalahi, trimurni abidin dan harry agusnar ............................................ 63–66 2. the relationship determination between menarche and the peak of skeletal maturation using hand wrist and cervical vertebrae index endah mardiati, soemantry es, haroen er, thahar b and sutrisna b ................................... 67–71 3. maturasi dan erupsi gigi permanen pada anak periode gigi pergantian (the maturition and eruption of permanent teeth in mixed dentition children) sri kuswandari ................................................................................................................................ 72–76 4. surgical exposure dan perawatan ortodontik pada impaksi gigi insisif sentral rahang atas (surgical exposure and orthodontic treatment on labially impacted maxillary central incisor) bingah fitri melati, teguh budi wibowo, dan betadion rizki .................................................. 77–81 5. penurunan jumlah streptococcus mutans pada saliva anak dengan ortodonti cekat setelah konsumsi yoghurt (reduction of salivary mutans streptococci in children with fixed orthodontic appliance after yoghurt consumption) dewi anggreani bibi, udijanto tedjosasongko, dan irmawati ................................................... 82–86 6. aplikasi teori belajar sosial dalam penatalaksanaan rasa takut dan cemasan anak pada perawatan gigi (application of social learning theory in the management of children dental fear and anxiety) arlette suzy setiawan ...................................................................................................................... 87–91 7. koreksi dimensi vertikal oklusal dengan modifikasi restorasi mahkota logam pada kasus severe early childhood caries (correcting occlusal vertical dimension using modified stainless steel crown restoration in severe early childhood caries case) amrita widyagarini dan sarworini b budiardjo ........................................................................ 92–97 8. koreksi gigitan terbalik posterior dan anterior dengan alat cekat rapid maxillary expansion dan elastik intermaksila (correction of posterior and anterior crossbite using fixed orthodontic appliance with rapid maxillary expansion and intermaxillary elastic) retno dewati, teguh budi wibowo, dan masyithah .................................................................... 98–102 9. paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance biofilm aggregatibacter actinomycetemcomitans (iron exposure to specific protein expression of extracellular polymeric substance of aggregatibacter actinomycetemcomitans biofilm) marchella hendrayanti w dan indah listiana k ......................................................................... 103–109 10. topical applications effect of casein phospho peptide-amorphous calcium phosphate and sodium fluoride on salivary mutans streptococci in children fajriani and aini dwi handini ........................................................................................................ 110–114 11. karakterisasi stem cell pulpa gigi sulung dengan modifikasi enzim tripsin (the characterization of stem cells from human exfoliated deciduous teeth using trypsin enzym) tri wijayanti puspitasari, tania saskianti dan udijanto tedjosasongko .................................. 115–119 111111 dental journal (majalah kedokteran gigi) 2020 june; 53(2): 111–114 research report comparison of thrombocyte counts during the post-oral administration of aspirin and the holothuria scabra ethanol extract in wistar rats (rattus norvegicus) dian mulawarmanti and rima parwati sari department of oral biology, faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: long bleeding time is a risk factor in dental treatment, especially in patients who consume aspirin or other antithrombotic drugs. holothuria scabra (h. scabra) are mostly echinodermata and have been studied in indonesia; they contain omega-3 and glycosaminoglycans, with an influence of an antithrombotic drug. purpose: this study aimed to investigate the thrombocyte counts during the post-administration of aspirin and the h. scabra extract in wistar rats (rattus norvegicus). methods: this study was true experimental with a post-test control group design. the sample consisting of 30 healthy male wistar rats (r. norvegicus) with a bodyweight of 150–250 g was divided into three groups (n = 10). the rats in group 1 were given sodium carboxymethyl cellulose (na cmc). the rats in group 2 were given aspirin, and the rats in group 3 were given the h. scabra ethanol extract with a 25 mg/200 g dose as per their body weight (bw). oral administration was given for seven days. the rats’ blood was taken on the eighth day. the amount of thrombocyte was measured using wright’s stain methods. the analysis of variance (anova) and the least significant difference (lsd) tests were conducted for data analysis (p < 0.05). results: the thrombocyte counts (179.00 ± 10.56) in aspirin administration were lower than those in h. scabra (265.00 ± 18.54) and control groups (334.17 ± 13.9), with a significant difference between the groups (p = 0.0001; p < 0.05). conclusion: this study indicates that the oral administration of aspirin and h. scabra decreases thrombocyte counts, whereas the administration of aspirin reduces thrombocyte counts to levels lower than those in h. scabra in wistar rats (r. norvegicus). keywords: antithrombotic; aspirin; glycosaminoglycans; holothuria scabra correspondence: rima parwati sari, department of oral biology, faculty of dentistry, universitas hang tuah, jl. arif rahman hakim no. 150, surabaya 60111, indonesia. e-mail: rima.parwatisari@hangtuah.ac.id introduction atherosclerosis is a leading cause of vascular disease throughout the world with manifestations of ischemic heart disease, stroke and peripheral arterial disease. in 2010, there were 665 people per 100,000 population with ischemic heart disease in central asia per year.1 based on the results of the 2018 basic health research report, the prevalence of heart disease in indonesia was 1.5%.2 lipoproteins that form in blood vessels lead to plaque formation in specific locations of the arteries through inflammation, necrosis, fibrosis and intimal calcification. the plaque causes coronary thrombosis due to acute rupture, which can result in partial or total blockage of the affected arteries.3 dismissal of the thrombus will become an embolism and obstruct the arterial system in pulmonary embolism and brain, for example.3,4 one of the medicines used widely in the treatment of atherosclerosis is aspirin, which functions as a longterm antithrombotic option for oral administration for the secondary prevention of myocardial infarction.5,6 aspirin in small doses (100 mg per day) shows an antithrombocyte activity by irreversibly inhibiting thrombocyte by preventing thromboxane a2 (txa2) synthesis, which damages thrombocyte secretion and aggregation.4,7 problems arise in the administration of aspirin in patients dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p111–114 mailto:rima.parwatisari@hangtuah.ac.id http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p111-114 112 mulawarmanti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 111–114 with coronary heart disease, which involves bleeding that occurs in the gastrointestinal tract and intracranial due to long-term use of low-dose aspirin.8 in the field of dentistry, bleeding complications can occur after surgery/tooth extraction, disrupting the healing process.6,9,10 therefore, before surgery and/or the extraction of teeth, it is vital to carry out a laboratory examination, which includes a thrombocyte count.11 sea cucumber (phylum echinodermata) has been widely used as medicine in several countries, such as china and korea. in indonesia, sea cucumbers are one of the leading marine products that have begun to be commercially cultivated in several regions because of their high economic value. however, there has been limited investigation into their benefits in the health sector.12 one type of sea cucumber that has been widely cultivated is the sandfish (holothuria scabra). this biota is commonly found across indonesia’s coasts, with straightforward breeding. the results of previous studies have shown that the content of sea cucumbers include lectins, sterols, saponins (triterpen glycosides), proteins, collagen, mucopolysaccharides, glycosaminoglycans (gags), chondroitin sulfate, amino acids, fatty acids, vitamin a, vitamin c, riboflavin, niacin, carotenoids, minerals, polyphenols, flavonoids and superoxide dismutase (sod). the content of these substances can be used as a source of protein and considered as an anti-inflammatory, anticoagulant, anti-cholesterol and antithrombotic agent; it can also accelerate the process of wound healing.13 some sea cucumber content such as gag sulfate, dermatan sulfate and heparin are important anticoagulants that inhibit clot formation through interaction with antithrombin and heparin cofactors ii.14 these substances have a mechanism that is synergistically useful in the treatment of acute coronary syndrome (acs).15 this is used as the basis for selecting sandfish as an antithrombotic that can be used as a substitute for aspirin. the purpose of this study is to compare the administration of aspirin with the h. scabra extract, which is given orally to the thrombocyte count in the white rat strain of wistar (rattus norvegicus). materials and methods this research is an experimental laboratory with a randomised, completed research design. this study was approved by the experimental animal ethics from the health research ethics commission (kepk) of the faculty of dentistry, universitas hang tuah, surabaya (ec/082/kepk-fkguht/xii/2019). the parameter of this study involved several thrombocytes taken from blood preparations from mice that had been treated. thirty research samples were randomly divided into three groups. the sample used had the following inclusion criteria: white rats (r. novergicus), male wistar strain aged 2–3 months, bodyweight 150–200 g. the selection of experimental animals was made based on their fur, eyes and physical conditions, as well as their randomized group design. monitoring and recording were conducted to determine whether there existed any side effects or the experimental unit experienced any pain to be removed from the sample. acclimatisation was carried out before and during the study, which involved monitoring environmental conditions, foods and drinks.16 the h. scabra extract was obtained from a beach in east kalimantan (borneo island), indonesia. it was transported in freezing conditions; its internal organs were removed, and it was washed thoroughly. using a blender mixed with distilled water, it was chopped into a ratio of 1:2. the drying process was performed using the freeze–drying method. the freeze–drying results in the form of soft, dry preparations were mashed with mortar and pestle and sifted using a mesh 50.17 sodium carboxymethylcellulose was added to ease transfer to the stomach of the rat using the oral gavage. research on r. novergicus began with acclimatisation for seven days in a laboratory environment. before being treated, the wistar rats fasted for about 18 hours, albeit they were given drinking water. sick rats were excluded from the study. the white rats were divided into three groups. group 1 consisted of the control group that was given na cmc. group 2 included the treatment group and was given an aspirin dose of 1.8 mg/200 g as per bw, which was based on the conversion of an aspirin dose to rats (200 g) as an antithrombotic of 100 mg/day. group 3 consisted of the treatment group and was given the h. scabra extract at a dose of 25 mg/200 g as per bw, which was based on previous research on the benefits of h. scabra on thrombocyte counts.18 the treatment was carried out for seven days by giving a single dose. after blood sampling, the animal was terminated with cervical dislocation. thrombocyte count examination was performed on the eighth day, using the wright wipe method. this method was carried out by adding 0.1 g of wright powder dissolved with 60 ml methanol to 0.1 g of wright reagent (merck® paint no. 1.01383.0500, darmstadt, germany). afterwards, it was stored in glass bottles and kept in a closed cupboard to avoid sunlight. the solution was used after 10 days of storage. in addition to wright staining, the use of a buffer solution was needed to fix the wright smear with a ph of 6.4. the wright smear procedure was performed by dripping the wright solution onto the preparation until all smears were inundated. furthermore, the buffer solution was dripped until all swabs were flooded and left for 5–12 min. the smear was rinsed with water, and the back of the dirty smear was cleaned of the remaining dye. preparations of peripheral blood smear were left to dry in the air.18 observations were made by two people with five visual fields using a light microscope (olympus® cx21, japan) with 400× magnification. using the statistical package for social sciences version 23 (ibm® 2015, new york, united states), the one-way analysis of variance (anova) was performed (p < 0.05), followed by the post-hoc least significant difference (lsd) test (p < 0.05). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p111–114 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p111-114 113mulawarmanti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 111–114 results the histological section of the thrombocyte in each group can be seen in figure 1. the mean and standard deviation of the thrombocyte counts in each group describe the differences between the groups (figure 2). the application of sandfish extract (h. scabra) at a dose of 25 mg/200 g as per bw reduced the thrombocyte counts (265.00 ± 18.54) compared with the control group (334.17 ± 13.93). however, it did not reduce the thrombocyte counts similar to the aspirin group (179.00 ± 10.56). the anova test identified a significant difference in the mean of the thrombocyte counts. the lsd test showed that the results were significantly different (p < 0.05) between groups k and a (p = 0.001; p < 0.05), group k with hs group 25 (p = 0.001; p < 0.05), and group a with hs group 25 (p = 0.001; p < 0.05). these findings illustrate a significant difference between the administration of aspirin and h. scabra. discussion this study found that compared with the administration of aspirin, which can prevent bleeding after antithrombotic/ anticoagulant medication, some thrombocytes did not decrease significantly. normally, thrombocytes are associated with the initiation of the coagulation process, whereby their reaction to damage to blood vessels becomes the main target in haemostasis. thrombocyte hyperactive reaction triggers side effects in coronary artery disease, which results in thrombosis.4 the antithrombocyte effect of aspirin involves inhibiting the synthesis of thromboxane a2 (txa2) from arachidonic acid in thrombocytes because of the irreversible acetylation process and inhibition of cyclooxygenase – an essential enzyme in the synthesis of prostaglandins and thromboxane a2.19 the presence of these obstacles causes a decrease in the number of thrombocytes present in the blood. this is indicated by the significant difference between the control group and the aspirin-administered group, where the thrombocytes in the latter group have thrombocyte counts that are much lower than those in the controls. sea cucumbers contain gags and omega-3s, which play a vital role in the thrombogenic process.13 one of the gags that plays a role in antithrombotics is dermatan sulfate. through the formation of complex covalent bonds with heparin-ii (hcii) cofactors, dermatan sulfate selectively inhibits thrombin action, thereby preventing vascular thrombosis.20 heparin/heparan sulfate contained in gags consists of 20–100 units of n-acetate d-glucosamine α disaccharide associated with glucuronic acid. the molecular mechanism of heparin/heparan sulfate as an anticoagulant can bind and increase the inhibitory activity of plasma protein antithrombin against several serine proteases from the coagulation system, the most important of which are factor iia (thrombin), xa and ixa.21 although the anticoagulant mechanism is more dominant, heparin/heparan sulfate also has antithrombotic properties. antithrombotic mechanisms occur with the release of tissue factor pathway inhibitors (tfpi) associated with the molecular weight and sulfate content of heparin.22 the gags found in the echinoderm cannot help in concluding that antithrombotic properties are strong. some studies summarised by pavão23 show that heparin-like polymers in ascidians and molluscs are different from low anticoagulant ability, as well as significant antithrombotic and anti-inflammatory activities; however, they do not cause bleeding. figure 1. histological section of thrombocytes (green arrows) during post-administration of aspirin (a), the holothuria scabra extract (hs) and control in wistar rats. staining of wright’s methods. observation using a light microscope at 400x× magnification. figure 2. mean and standard deviation of the thrombocyte counts in each group. *significance difference: p = 0.05. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p111–114 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p111-114 114 mulawarmanti et al./dent. j. (majalah kedokteran gigi) 2020 june; 53(2): 111–114 clinical results in humans show that omega-3 from marine biota can function as antithrombotic. omega-3 fatty acids in marine life containing eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) consumption 2–4 g/day and cause optimal anti-atherosclerotic, anti-inflammatory and antithrombotic effects.24 dha has a quicker start in inhibiting thrombocyte aggregation induced by adenine diphosphate (adp). however, both epa and dha are incorporated into thrombocyte phospholipids by inhibiting the formation of arachidonic acid (aa), which can help in reducing thrombocyte aggregation by lowering thrombocyte procoagulant metabolites arising from aa synthesis. epa also competes with aa in the cyclooxygenase pathway, which directly and indirectly reduces the formation of txa2 pro-aggregatory aa metabolites.24 this was proved in studies with thrombocyte counts in h. scabra administration compared with the control group. a previous study mentioned that the presence of a barrier in txa2 and thrombocyte aggregation triggers bleeding.19 in their multinational study in the united states, akintoye et al.25 conducted a placebo-controlled trial involving 1,516 patients, who were given perioperative fish oil (epa-dha), 8–10 g/day for 2–5 days before surgery and then 2 g/day postoperatively. compared with the placebo, the administration of fish oil did not show any bleeding.25 akintoye et al.’s study is in line with this study’s results whereby the administration of the h. scabra ethanol extract containing epa–dha was not proved to increase perioperative bleeding; conversely, it even reduced the amount of blood transfusion. high omega-3-pufa administration is associated with a lower risk of bleeding. conclusions from this study indicate that the oral administration of aspirin and h. scabra decreases thrombocyte counts, whereas the administration of aspirin reduces thrombocyte counts to lower levels than those in h. scabra in wistar rats (r. novergicus). references 1. herrington w, lacey b, sherliker p, armitage j, lewington s. epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. circ res. 2016; 118(4): 535–46. 2. badan penelitian dan pengembangan kesehatan. hasil utama r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 66–71. 3. bentzon jf, otsuka f, virmani r, falk e. mechanisms of plaque formation and rupture. circ res. 2014; 114(12): 1852–66. 4. stone j, hangge p, albadawi h, wallace a, shamoun f, knuttien mg, naidu s, oklu r. deep vein thrombosis: pathogenesis, diagnosis, and medical management. cardiovasc diagn ther. 2017; 7(suppl 3): s276–84. 5. sikka p, bindra vk. newer antithrombotic drugs. indian j crit care med. 2010; 14(4): 188–95. 6. vitria ee. evaluasi dan penatalaksanaan pasien medicallycompromised di tempat praktek gigi evaluation and management of m e d ic a l ly c om p r om i s e d p a t ie nt i n d e nt a l p r a c t ic e. j dentomaxillofacial sci. 2011; 10(1): 47. 7. madao da, mongan ae, manoppo f. hubungan antara lama penggunaan aspirin dengan nilai agregasi trombosit pada pasien hipertensi di rsup prof. dr. r. d. kandou manado. j e-biomedik. 2014; 2(2): 545–50. 8. rodríguez lag, martín-pérez m, hennekens ch, rothwell pm, lanas a. bleeding risk with long-term low-dose aspirin: a systematic review of observational studies. plos one. 2016; 11(8): 1–20. 9. darawade d, desai k, hasan b, kumar s, mansata a. influence of aspirin on post-extraction bleeding a clinical study. j int soc prev community dent. 2014; 4(4): 63. 10. scottish dental clinical effectiveness programme. management of dental patients taking anticoagulants or antiplatelet drugs. dundee: dundee dental education centre; 2015. p. 1–52. 11. goswami a, bora a, kundu g, ghosh s, goswami a. bleeding disorders in dental practice: a diagnostic overview. j int clin dent res organ. 2014; 6(2): 143–50. 12. h a r t a t i r , w id i a n i n g s i h w, d j u n a e d i a . u lt r a s t r u k t u r alimentary canal teripang holothuria scabra dan holothuria atra (echinodermata : holothuroidea). bul oseanografi mar. 2016; 5(1): 86–96. 13. bordbar s, anwar f, saari n. high-value components and bioactives from sea cucumbers for functional foods--a review. mar drugs. 2011; 9(10): 1761–805. 14. sobczak ais, pitt sj, stewart aj. glycosaminoglycan neutralization in coagulation control. arterioscler thromb vasc biol. 2018; 38(6): 1258–70. 15. perh i mpu na n dok ter sp esia l is k a rd iovask u la r i ndonesia. pedoman tatalaksana sindrom koroner akut. 3rd ed. jakarta: centra communications; 2015. p. 88. 16. sert np du, hurst v, ahluwalia a, alam s, avey mt, baker m, browne wj, clark a, cuthill ic, dirnagl u, emerson m, garner p, holgate st, howells dw, karp na, lidster k, maccallum cj, macleod m, petersen o, rawle f, reynolds p, rooney k, sena es, silberberg sd, steckler t, würbel h. the arrive guidelines 2019: updated guidelines for reporting animal research. biorxiv. 2019; (july): 1–19. 17. sari rp, sudjarwo sa, rahayu rp, prananingrum w, revianti s, kurniawan h, bachmid af. the effects of anadara granosa shellstichopus hermanni on bfgf expressions and blood vessel counts in the bone defect healing process of wistar rats. dent j (majalah kedokt gigi). 2017; 50(4): 194–8. 18. mckenzie sb, williams l. clinical laboratory hematology. 3rd ed. new jersey: pearson education inc; 2014. p. 44–152. 19. smith sa. antithrombotic therapy. top companion anim med. 2012; 27(2): 88–94. 20. tollefsen dm. vascular dermatan sulfate and heparin cofactor ii. prog mol biol transl sci. 2010; 93: 351–72. 21. tripathi ckm, banga j, mishra v. microbial heparin/heparan sulphate lyases: potential and applications. appl microbiol biotechnol. 2012; 94(2): 307–21. 22. gray e, hogwood j, mulloy b. the anticoagulant and antithrombotic mechanisms of heparin. in: lever r, mulloy b, page cp, editors. heparin a century of progress. berlin, heidelberg: springer-verlag; 2012. p. 43–61. 23. pavão msg. glycosaminoglycans analogs from marine invertebrates: structure, biological effects, and potential as new therapeutics. front cell infect microbiol. 2014; 4: 1–6. 24. dinicolantonio jj, okeefe j. importance of maintaining a low omega-6/omega-3 ratio for reducing platelet aggregation, coagulation and thrombosis. open hear. 2019; 6(1): 1–4. 25. akintoye e, sethi p, harris ws, thompson pa, marchioli r, tavazzi l, latini r, pretorius m, brown nj, libby p, mozaffarian d. fish oil and perioperative bleeding. circ cardiovasc qual outcomes. 2018; 11(11): 1–10. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v53.i2.p111–114 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v53.i2.p111-114 123 vol. 41. no. 3 july–september 2008 isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene elly munadziroh department of dental material and technology faculty of dentistry airlangga university surabaya indonesia abstract background: secretory leukocyte protease inhibitor (slpi) has been found to facilitate epithelialization, maintain a normal secretory leukocyte protease inhibitor (slpi) has been found to facilitate epithelialization, maintain a normal epithelial phenotype, reduce inflammation, secrete growth factors such as il-4, il-6, il-10, egf, fgf, tgf, hgfand 2-microbulin. slpi is serine protease inhibitor, which found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, amniotic fluid and amniotic membrane. impaired healing states are characterized by excessive proteolysis and oftenbacterial infection, leading to the hypothesis that slpi may have a role in the healing process in oral inflammation and contributes to tissue repair in oral mucosa. the oral wound healing response is impaired in the slpi sufficient mice since matrix synthesis and collagen deposition delayed. the ob�ective of this research is to isolate and identify the amniotic membrane of java race slpi gene. methods: slpi rna was isolated from java race amniotic membrane and the cdna was amplified by polymerase chain reaction slpi rna was isolated from java race amniotic membrane and the cdna was amplified by polymerase chain reaction (pcr). result: through sequence analyses, slpi cdna was 530 nucleotide in length with a predicted molecular mass about 12 kda. through sequence analyses, slpi cdna was 530 nucleotide in length with a predicted molecular mass about 12 kda. the nucleotide sequence showed that human slpi from sample was 98% identical with human slpi from gene bank. pcr analysis revealed that the mrna of slpi was highly expressed in the amniotic membrane from java race sample. conclusion: it is demonstrated it is demonstrated that human slpi are highly conserved in sequence content as compared to the human slpi from gene. key words: secretory leukocyte protease inhibitor, amniotic membrane, cloning, recombinant, wound healing correspondence: elly munadziroh, c/o: departemen ilmu material kedokteran gigi. jln. mayjend. prof. moestopo no. 47 surabaya 60132, indonesia. email: emunadziroh@yahoo.com introduction gingival recession occurs at all ages varied from 65 years old or higher (88%) and 18–64 years old (50%).1 surgical treatment of gingival recession cause fairly large muco gingival injury due to gingival retraction toward coronal. tension will lead to excessive thin gingival and laceration.2 in the oral cavity, bacterial free environment is difficult to achieve. further complication would occur if immediate treatment is not carried out since it would be easily to be contaminated by bacteria, leading to cellulites, abscess or osteomyelitis. various method have been developed to achieve injury recovery.3,4 the present concept of treatment and recovery has been developed simultaneously with dental biomaterial progress. biological material is currently considered to be applied due to the supporting component of treatment and recovery. one of the bio material expected to be able to accelerate injury recovery is amniotic membrane that effective in burn injury treatment as well as in ophthalmology.5,6,7 secretory leukocyte protease inhibitor (slpi) has been found to be an active material of amniotic membrane expected to be influential to injury recovery. slpi is one of the inhibitors of serine in addition to a1antitrypsin (a1-pi) and leafin. the main target of slpi is to inhibit serine protease-the human netrophil protease (hne) including chemotripsyn super-family member of serine protease expressed in monosite and mast sell especially netrophil. the function of this intracellular enzyme is to degrade foreign extra cellular matrix (ecm), remodeling damaged tissue and facilitating netrophil migration into other tissue.8 research report 124 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 123-127 additional function of slpi is to inhibit leukocyte proteinase, to maintain immunity against infection, regulating leukocyte function, producing matrix and improving tissue. slpi also facilitates several functions: inhibiting protease, controlling leukocyte activity, reducing tgf-beta, anti inflammatory, anti bacteria, anti retroviral (human immunodeficiency virus). another function of slpi are to control synthesis of intracellular enzyme, and to suppress mmp production, to prevent the formation of scar tissue.9 the study was conducted to analyze the character of amniotic membrane slpi molecular protein and cloning of amniotic membrane slpi protein to achieve amniotic membrane slpi protein recombinant in large amount which would be beneficial as biomaterial candidate to achieve fast recovery of gingival injury. material and method identification of amniotic membrane slpi gene used the following materials: amniotic membrane, proteinase k, rnase, rna later, tbe buffer, ethidhium bromide, bromophenol blue, marker x 174x174 rf dna/hae iii fragments rnaa later, rt buffer, ribonuclease, random primer, reverse transcriptase, 2% agarose, ethidium bromide, loading dye, primer (sense): 5’ act cct gcc ttc acc atg aa 3’ and primer (anti sense) 5’ att cga tca act gga ctt 3’ (invitogen). 100 bp dna marker (fermentas), distilled water, tth buffer reverse, cdna mineral oil, enzyme tth dna polymerase, binding buffer (pb), washing buffer (buffer pe), 200 µl buffer pe, elution buffer (eb), sodium acetate, 100% alcohol, 70% alcohol, pure dna, template suppression reagent. amplification, purification and sequencing of amniotic membrane slpi gene using the following materials: amniotic membrane slpi, primer : 5’ cac cat gaa gtc cag cgg cct ctt cc 3’ and 5’ agc ttt cac agg gga aac gca ggat 3’, rneasy mini kit (qiagen), 100 pb dna marker, nuclease free water (mp bio), ethidhium bromide (mp bio), agarose (fermentas), 6x loading dye solution (fermentas), tris bufferc edta (tbe) buffer 10× ilt (mp bio), gel extraction purification kit (qiagen), low melting agar (ssigma), dna sequencing kit. rna isolation method as the following: 3 × 3 mm (30 µl) fresh amniotic membrane sample was put into rna later and kept at 40° c in refrigerator until requested. 30 µl amnion membrane sample was added to 300 µl buffer rlt followed by 590 µl distilled water into 20µl eppendorf tube, then, 10 µl proteinase k was added. the mixture was incubated at 55° c for min. next, centrifugation was done in 3 min at 10.000 rpm at 20–25° c. 1.5 µl other eppendorf was filled with 900 µl supernatant added 450 µl (1/2 vol) ethanol 100% mixture of sample was done using pipette into rna tube, centrifugation at 10.000 rpm, the lower part of the solution was evacuated. centrifugation was repeated for 15 second at 10.000 rpm, lower part of the solution evacuated added by 700 µl buffer rw, centrifuged for 15 second at 10.000 rpm. new collective tube was filled by 500 µl buffer rpe, following by 2 minute 10.000 rpm centrifugation, repeated to throw out the lower part of the solution. 1.5 ml eppendorf tube was filled by 50 µl rneasy free water, then centrifugation was done was done in 1 minute at 10.000 rpm. the result of the procedure was rna continued by cdna isolation. isolation of cdna using the following procedures: 10µl rna was incubated at 65° c for 3 minute, added by 4µl (5x) buffer rt, 4 µl dntps mix, 0.5µl ribonuclease, 0.5µl random primer, 0.5 µl reverse transcriptase (19.5 µl). the mixture was incubated at 42° c for 1 hour. electrophoresis was performed to identify whether cdna was formed in 2% agarose gel containing ethidium bromide. 5 µl cdna was added by 2 µl loading dye and put into well agarose, then, it was run in 100 volt for ± 30 minute, followed by detection with uv-transilluminator and documented by polaroid camera. polymerase chain reaction (pcr) was done in the following procedure: 70 µl distilled water mixed with 9 µl buffer reaction tth, 8 µl dntp mix, 1µl primer reverse cat tcg atc aac tgg cac tt and 1 µl primer forward act cct gcc ttc acc atg aa 1 µl cdna (total 99 µl), next, added by 102 µl mineral oil denaturation was done at 72° c for 30 second and 94° c for 5 minutes, continued by adding 1 µl tth dna polymerase, then inserte d into pcr machine. initially denaturation was performed at 940 c for 40 second, annealing at 52° c for 40 second, extension was done at 72° c for 40 second, extra extension at 72° c for 10mm. pcr was done in 35 cycles analysis of pcr product with electrophoresis agarose gel. the result of pcr could be seen through electrophoresis in 2% agarose gel containing ethidhium bromide. 5 µl cdna added by 2 µl loading dye put into well agarose. the mixture was run in 100 volt for 30 min. next, detection was conducted by uv-transuminator, then, the result was documented using polaroid camera. purification of pcr product was performed using purification kit (qiagen). 100µl pcr product was added by 500 µl buffer binding (pb) and mixed to release mineral oil. the mixture was moved into column, nest step, centrifugation was performed at 14.000 rpm at room temperature for 1minute. the solution in the lower part was removed and rinsed by 750 µl washing buffer, the mixture was centrifuged at 14.000 rpm at room temperature for 1 minute, the fluid was thrown out, added by 200µl buffer pb centrifugation was performed again at 14.000 rpm at room temperature for 1 minute. pure dna which was purified put into eppendorf, added by 50 µl buffer elution (b.e) and it was left for 5 minute. centrifugation was done at 14.000 rpm at room temperature for 1 min. electrophoresis was done to identify the result of dna purification in 2% agarose gel containing ethidhium bromide. 5 µl pcr product had been purified and mixed 125munadziroh: isolation and identification of java race amniotic membrane with 2 µl loading dye into well agarose. next, it was run in 100 volt for about 30 minute, detected using uvtransluminator finally documented by polaroid camera. sequencing process is 5 µl sodium acetate 3m was added into sample, followed by 50 µl alcohol 100% and the eppendorf was agitated. the mixture was than incubated at room temperature for 15 minute and continued by centrifugation at 15.000 rpm at 40 c in 15 minute. supernatant was evacuated again, and drying process in vacuum 15 min. the result was ready to kept or directly sequenced.25 µl template suppression reagent (tsr) was added into pure dna and dried at 950 c for 5 minute, then, put into the ice for 10 minute, moved into 0.5 ml (500µl) tube, finally placed into sequencing machine for one hour (10 nucleotide for 1 minute). amplification, purification and sequencing gen encoding slpi amniotic membrane: gen encoding slpi could be amplified after rna isolation was previously done from amniotic membrane using rneasy mini kit. 50µl sample was put into eppendorf tube (1.5 ml), added by 550 µl b –me/rlt and 600 µl ethanol 70% into the sample, then, mixed by agitating the tube. 600µl solution was moved into rneasy spin column and kept in collective tube 2ml, followed by centrifugation at 12.000 rpm for15sec. the lower part of the solution was removed and the remain was repeated. 700 µl rw1 solution was added into rneasy column followed by centrifugation at 12.000 rpm for 15 sec. and the lower part of the solution was removed. rneasy column was moved to a new tube (2 ml), added by 500µl rpe buffer and centrifuged at 12.000 rpm for 15 sec. the lower part of the solution was removed and repeated. rneasy column was moved into another tube then centrifuged at 13.000 rpm for 2 minute (to release the ethanol). rneasy column was moved again into 15ml tube, next, added by 30 µl rneasy free water in the middle part of the column, centrifugation was done at 12.000 rpm for 2 minutes. reverse transcriptase to obtain cdna product was done on the lower part of the solution containing collective rna. this present study applied two step pcr method and primer was designed to fulfill the criteria of gen insertion in pet 101/d-topo by adding cacc prior to atg (at initial gen) i.e. primer (sense ): 5’ cac cat gaa gtc cag cgg cct ctt cc 3’ and primer (anti sense)5’ agc ttt cac agg gga aac gca gga t 3’. amplification was done using reagent mixture consisting of : 70µl distillated water mixed with 9 µl reaction buffer tth, 8 µl dntp mix, 1µl primer forward and 1µl primer reverse, 10µl cdna (total 99 µl) and added by mineral oil. the process was done according to the following stages: predenaturation at at 72° c for sec. and 94° c for 5 min. the mixture was then added by 1µl tth dna polymerase enzyme, put into pcr machine with denaturation process at 94° c for 40 sec. followed by annealing at 52° c for 40 sec. and extension at 72° c for 40 sec, next final step extra extension at 72° c for 10 sec. pcr was done in 35 cycles. the result of pcr could be seen by electrophoresis in 2% gel agarose containing ethidium bromide, 5µl cdna added by 2µl loading dye put into well agarose and operated in 100 volt approximately 30 min, continued by uv-transluminator detection, and documentation was taken using polaroid camera. preparation for sequencing and cloning, the pcr product should be previously purified. the product was run in 1.5% low melting agar, dna fragment was cut from gel agarose with scalpel. under uv light, gel cutting containing dna fragment added by 3 volume of qg buffer in 1 volume gel (100 mg ± 100 µl) the ext step, incubation was performed at 50° c for 10 min until all gel was dissolved and a mixture of gel and vertex was done in tube every 2–3 minute during the incubation. after all gel was dissolved, the color of the solution would be yellow similar to qg buffer prior to dissolving gel. 1 volume of isopropanol was mixed with the sample, then, qia quick spin was placed column in 2 ml collective tube and sample was added in column, then it was centrifuged at 13.000 rpm for 1mm. the lower part of the solution was removed, then, column was put back into tube. 750µl pe buffer was added in column, followed by centrifugation at 13.000 rpm for 1 min. the solution in the lower part of centrifugation was thrown out and centrifugation was performed again at 13.000 rpm for 1 min. column was placed in 1 ml tube of new micro centrifuge. next, it was added by 50 µl eb buffer (10 mm tris-cl, ph 8.5) or h2o, centrifugation was done at 13.000 rpm for 1min. a part of dna could be sequenced and the remaining was cloned. result identification of encoding slpi gene was initiated by obtaining dna of slpi, sample preparation of amniotic membrane was previously done to extract rna, than, cdna slpi was achieved. amplification was done using two step pcr (qiagen) method with primer sense: 5’act ctt gcc ttc acc atgaa 3’ and 5’att cga tca act gga ctt 3’, producing fragment of dna 530 bp. visualization of pcr product was performed using agarose figure 1. electrophoresis of pcr product to identify slpi gene. m: marker × 174 rf dna/hae iii fragments; 1: slpi. 126 dent. j. (maj. ked. gigi), vol. 41. no. 3 july–september 2008: 123-127 electrophoresis supported by uv-transilluminator and documented by polaroid camera, as shown on figure 1. in this present study identification of amniotic membrane slpi gene of pcr product should be done through purification prior to sequencing and using qiaquuick pcr purification kit (qiagen), followed by uv-transluminator detection, documented using polaroid camera (figure 2). amplification of sequence of amniotic membrane slpi gene encoding protein with primer reserve cat tcg atc aac tgg cac tt and primer forward act cct gcc ttc acc atg aa (zhang et al, 2001) producing about 530 bp amplicont as shown on figure 3. amplification of amniotic membrane slpi cdna is required for analysis of amniotic membrane slpi gene using two-step pcr method i.e rna isolation method was previously done and continued by cdna isolation. cdna amplification was done in thermal cycler. primer was designed in accordance with the criteria of gene insertion on pet 101/d-topo adding cacc prior to atg (at the early gene) presented on figure 4. figure 3. sequensing of slpi gene using forward primer. figure 4. primer design for slpi gene amplification. figure 5. the result of electrophoresis slpi gen pcr product with topo primer: m: marker × 174 rf dna/hae iii fragment, 1: slpi figure 6. result of slpi gene purification. m: marker × 174 rf dna/hae iii fragment, 1: slpi 5' cacc-atgaagtccagcggcctcttcc 3' 5' agctttcacaggggaaacgcaggat 3' figure 2. electrophoresis of purified pcr product to identify slpi gene. m: marker × 174 rf dna/hae iii fragments; 1: slpi. amplification of slpi gen producing dna fragment in ± 530 bp positions can be shown on figure 5. pcr products of amniotic membrane were purified using topo primer prior to sequencing.. band contaminants was found during visualization of pcr product through electrophoresis, therefore prior to purification, running of pcr product was done using 1.5% lomelting agar. under uv light, the required band was cut and mixed with qg buffer containing ph indicator. optimal binding between dna and silica gel requested ph ≤ 7.5 and would produce yellow appearance, while, purple appearance would occur, ph was too high. after dna was bound with the membrane continued by dna rinsing process by releasing unnecessary primer and other contaminants. pe buffer additional containing ethanol would make contaminants. flow out from matrix column through centrifugation stage. dna bound with membrane eluated by adding elution buffer exactly in the middle of membrane and partly purified dna from electrophoresis to identify the existence of purification, followed by uv-transluminator detection, finally, documented by polaroid camera shown on figure 6. 127munadziroh: isolation and identification of java race amniotic membrane discussion the existence of amniotic membrane spli gene was detected using pcr method to obtain amplification of dna segment limited by synthetic oligonucleotide (primer). t h i s s t u d y u s e d s p e c i f i c p r i m e r o f h u m a n s l p i ( 5 ’ a c t c c t g c c t t c a c c a t g a a 3 ’ / 5 ’-attcgatcaactggactt-3’). the result of amplification obtained 530 bp dna. primer selection based on the study done by zhang9 that successfully isolated slpi gene from human amniotic fluid by rt-pcrmethod obtaining 570 bp slpi gene.10 homology analysis of gene bank shows human homology slpi indicating 98–100% homology, based on this indication therefore primer was applied. some studies showed slpi gene has various size. the size of slpi in parotid gland is 580 bp,11 in cervical mucus is 570 bp, and in endometrial epithelial cell is 451 bp.9 various size of slpi according to gene bank shown on table 1. alignment was done on some sequence of nucleotide obtained from gene bank showed similar homology and it is proven that the identify of amniotic membrane slpi gene is similar to human slpi registered in gene bank. amplification of slpi gene was conducted using pcr technique i.e in vitro enzymatic method producing specific dna in a large amount and in very short time through the stage of denaturation, annealing and extension at different temperature. prior to detection of slpi existence, isolation of dna genome was initially conducted by extracting cdna and pcr method was applied to achieve amplification of a certain dna segment limited by synthetic of 2 oligonucleotide called primer. this technique was applied due to the ability to purify dna polymerase and to synthesize chemically dna oligo nucleotide therefore; it would be possible to conduct cloning in specific dna sequence that is amniotic membrane slpi gen. zhang8 suggested the production of human amniotic membrane slpi gene and regulating concentration of amniotic fluid through pcr amplification stated that the length of nucleotide of amniotic membrane slpi gene is 570 pb. however, sequence data of amniotic membrane slpi nucleotide is not found at gene bank. it was reported that expressed slpi in parotic gland is 399 pb, while, in this study, slpi gene is 530 pb.1 if the alignment between sequence result of this study and the result of study done by stedler,9 98% homology was found. if the alignment was done only cds and the alignment found 100% it shows slpi gene has the same conserve area but the gene size is not the same. the conclusion of this study, human slpi are highly conserved in sequence content as compared to the human slpi from gene. references 1. kassab mm, cohen re. the etyology and prevalence of gingival recession. j am dent asso 2003; 134(2):220–25. 2. nurman mg, takei hh, carranza fa. carranza’s clinical periodontology. 9th ed. philadelphia: wb saunders company; 2002. p. 651–61. 3. goldman lmd, ausiello dmd. 2004. cecil textbook of medicine. 22nd ed. philadelphia: wb saunders co; p. 208–33. 4. senthil k, wong peng foo, david lj. what is new in wound healing ?. turk j med sci 2004; 34: 147–60. 5. ley-chavez e, martinez-pardo me, roman r, oliveros-lozano fj, canchola-martinez. application of biological dressings from radiosterilized amnios with cobalt 60 and serologic studies on the handling of burns in pediatric patients. ann transplant 2003; 8(4): 46–9. 6. heinz c, eckstein a, steuhl kp, meller d. amniotic membrane transplantation for reconstruction of corneal ulcer in graves ophthalmopathy. journal of cornea 2004; 23(5):524–26. 7. takahiro n, makoto y, helen r. sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. investigative ophthalmology and visual science 2004; 45:93–9. 8. fitch pm, roghanian a, howie sem, sallenave jm. human neutrophil elastase inhibitors in innate and adaptive immunity. biohem soc trans 2006; 34:279–82. 9. zhang daying, rosalia cm, simmen, frank j. michel, ge zhao, dustin vale-cruz, et al. secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes. journal of biological chemistry 2002; 277 (33): 29999–30009. 10. zhang qing, koichiro shimoya, akihiro moriyama, kaoru yamanaka, atsuko nakajima, toshikatsu nobunaga, et al. production of secretory leukocyte protease inhibitor by human amniotic membranes and regulation of its concentration in amniotic fluid. molecular human reproduction 2001; 7(6):573–79. 11. stedler g, m t brewer, thompson rc. isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. nucleic acids res 1986; 14 (20):7883–96. table 1. gene bank’s list of slpi size no accession number gen size (pb) source of slpi 1 2 3 4 5 6 7 8 9 10 x04502 m74444 bc020708 nm003064 cs106326 x04470 x04503 dq891365 dq894545 af114471 2657 1472 625 598 594 594 573 439 439 399 parotid tissue epithelium cell endometrial cell cervical uterus parotid tissue synthesis synthesis intestinal epithelium cell 76 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 76–80 case report the management of herpes labialis, oral thrush and angular cheilitis in cases of oral diabetes maharani laillyza apriasari department of oral medicine faculty of dentistry, universitas lambung mangkurat banjarmasin – indonesia abstract background: as reported in several studies, prolonged or uncontrolled cases of diabetes mellitus (dm) may induce a more extreme inflammatory response. mucosal lesions can be observed in patients who present this systemic condition. purpose: the purpose of this study was to elaborate the management of herpes labialis, oral thrush and angular cheilitis as oral manifestations of diabetes. case: a 49-year-old male complained of having experienced painful lip ulceration for the preceding seven days. the anamnesis of the patient revealed that, prior to lip ulceration, he had experienced high fever and malaise in addition to frequent urination at night and a tingling sensation in the toes and fingertips on waking. case management: the patient was referred to undergo blood count, fasting blood glucose and oral glucose tolerance tests. the blood test result confirmed the patient to be suffering from dm and anemia. the immunocompromised condition of the patient prompted the occurrence of herpes labialis, oral thrush and angular cheilitis. conclusion: multidisciplinary treatment for herpes labialis resulting in oral candidiasis in dm patients is indispensable. concurrent infection with hsv and candidiasis necessitates a more prolonged healing process. consequently, it is a essential to treat the hyperglycemia which induces an immunocompromised state in diabetic patients. keywords: angular cheilitis; diabetes mellitus; herpes labialis; management; oral thrush correspondence: maharani laillyza apriasari, department of oral medicine, faculty of dentistry universitas lambung mangkurat, jl. veteran 128b banjarmasin, indonesia. e-mail: maharaniroxy@gmail.com introduction diabetes mellitus (dm) constitutes a chronic disorder induced by the inability of the body to produce insulin or to produce it in adequate quantities. characterized by a high blood glucose level, dm is classified into two types where the first (type 1) is induced by genetic and autoimmune factors, while the second (type 2) is due to lifestyle.1 the basic health survey of 2018 revealed an increased prevalence of dm from 6.9% to 8.5% in indonesia during the preceding five years. this resulted in indonesia is ranking sixth in the world in terms of the occurrence of dm.2 prolonged or uncontrolled dm may cause a stronger inflammatory response as reported in previous studies.3 hyperglycemia demonstrates a direct interrelation with the development of inflammatory conditions by enhancing pro-inflammatory cytokine expression such as il-6, and tnf-α. dm is also notable as an inflammatory disease in which the production of reactive oxygen species (ros) is intensified.3,4 an increase of ros in the inflammatory stage of the diabetic healing process will lead to inevitable results such as a prolonged healing process in the oral mucosa.4,5 pathological symptoms in the soft tissues, including; a reduction in salivary flow, xerostomia and taste disorders may often be identified in patients suffering from dm. mucosal lesions resulting from fungal infection (oral candidiasis and angular cheilitis), bacterial infection (gingivitis and periodontitis), viral infection (herpes labialis and herpes zoster) and other lesions (oral lichen planus, lichenoid reaction, recurrent aphthous stomatitis) can also frequently be observed. 6,7 this case report seeks to elaborate the management of herpes labialis, oral thrush and angular cheilitis as dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p76–80 mailto:maharaniroxy@gmail.com http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p76-80 77maharani laillyza apriasari/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 76–80 oral manifestations in diabetic patients. treatment of the symptoms of oral diabetes requires significant time and a collaborative approach from the internist to refine the blood glucose level. in order to achieve successful results, the two conditions should be controlled and managed concurrently. case a 49-year-old male patient presented painful lower lip ulceration the first symptoms of which had appeared seven days previously. three days prior to lip ulceration, high fever and malaise were indicated by the anamnesis results of the patient who reported pain while eating, speaking and tooth-brushing. this individual had merely selfadministered paracetamol and applied honey once a day to reduce the extreme discomfort. a history of consistently experiencing tingling sensations in the toes and fingertips on awakening from sleep were highlighted together with the need to urinate more than three times during the night. case management during the initial consultation (day 1), an extraoral examination revealed painful bilateral palpable submandibula lymph nodes firm in consistency. the lower lip presented symptoms including a painful erythematous crust, erosion and ulceration (figure 1). intraoral examination illustrated painless erythematous erosion on the lower lip mucosa. the provisional diagnosis was one of herpes labialis due to the appearance of the lips and coated lesion on the tongue (figure 2). the drugs prescribed for a period of seven days comprised aloclair to be gargled and applied topically to the lower lip three times a day, two 400 mg caplets of acyclovir to be taken three times a day, one 400 mg caplet of ibuprofen to be taken three times a day and one caplet of neurobion forte to be taken once a day. excessive urination occurring more than three times at night, a tingling sensation in the toes and fingertips invariably present on awakening from sleep, together with malaise constituted requisite symptoms to the conducting of a complete blood count and fasting blood glucose and oral glucose tolerance tests at a clinical pathology laboratory. during the second consultation (day 6), anamnesis revealed that drugs had been regularly consumed and a test was conducted at a clinical pathology laboratory. a complete blood count produced a result of haemoglobine 7.4 g/dl (13-17.5 g/dl), erythrocyte 3.53 million/ul (4.5-6 million/ul), leukocyte 11.000/ul (4700-10500/ ul), hematocryte 23.5% (40-50%), trombocytes 815000 (150000-350000/ul), lymphocytes 8.6% (25-40%), neutrophils 80.8% (33-66%), mcv 66.6 fl (80-97 fl), mch 21.0 pg (27-32 pg), mchc 31.5 g/dl (32-40 g/dl) and rdw-cv 16.8% (11.5-14.7%). a fasting blood glucose test produced a result of 170 mg/dl (70-110 dl), while an oral glucose tolerance test one of 140 mg/dl (<125 mg/dl). based on these clinical pathology laboratory test results, the patient was diagnosed with dm and anemia which were subsequently referred to an internist. elimination of lower lip pain resulted from anamnesis. however, the patient reported having to frequently scratch his lower lip in order to relieve the irritation experienced. extraoral examination indicated painless palpable submandibular lymph nodes of a firm consistency. meanwhile, the lower lip region was diagnosed as suffering from erythematous erosion, drying crust and itchiness (figure 3), although the tongue was no longer coated (figure 4). recommended therapies comprised aloclair to be applied three times a day, two 400 mg caplets of acyclovir three times a day, one capsule of sangobion once a day and one 10 mg cetirizine tablet per day. all the drugs mentioned above were prescribed for a period of seven days. during the third consultation (day 13) with the internist, the patient was prescribed one 500mg metformin tablet to be taken daily for 30 days. the anamnesis result confirmed the elimination of lip pain and itchiness, although soreness and a burning sensation on the dorsal of the tongue was reported by the patient. extraoral examination demonstrated normal submandibular lymph nodes. although no erosion of the figure 1. painful, itchy, erythematous ulceration, erosion and crust on the lower lip. figure 2. presentation of a painless, scrapable, brownish white plaque lesion on the tongue. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p76-80 78 maharani laillyza apriasari/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 76–80 lips was observed, painful white erosion and fissures were present on the lateral commissures (figure 5). intraoral examination confirmed the presence of a painful, burning, scrapable, white yellowish plaque lesion on the tongue (figure 6). herpes labialis therapy was terminated and the patient was subsequently diagnosed with oral thrush and angular cheilitis, the prescribed drugs for which consisted of 1 ml nystatin oral suspension applied three times a day and one capsule of sangobion once a day. all drugs were prescribed for a period of seven days and the patient was instructed to clear plaque from the surface of the tongue and the commissures of the lips prior to the application of nystatin. the patient was not allowed to eat, drink or gargle for 30 minutes after application. figure 4. normal condition of the tongue with no presentation of coated tongue. figure 5. painful white erosions and fissures at the commissures of the mouth. lips exhibiting the absence of erosive lesion. figure 6. tongue presenting a painful, scrapable, burning, yellowish white plaque lesion. figure 7. lower lip presenting itchy and painful erythematous erosion, both lateral commissures of the mouth and tongue exhibiting normal condition. figure 8. normal presentation of lower lip devoid of erosion, pain or itchiness. figure 3. presentation of itchy erythematous ulceration and drying crust on the lower lip. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p76-80 79maharani laillyza apriasari/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 76–80 during the fourth consultation (day 23), no pain or burning sensation was reported during the most recent anamnesis. however, the patient complained of itchy and painful ulceration on his lower lip, despite the medication regime prescribed by the doctor having been fully adhered to. extraoral examination confirmed both normal and painless bilateral commissures of the mouth combined with itchy painful erythematous erosion on the lower lip and mucosa. intraoral examination confirmed a normal tongue devoid of plaque (figure 7). the patient was diagnosed with herpes labialis, prescribed drugs including two caplets containing 400mg of acyclovir three times a day, one 10mg tablet of cetirizine once a day and instructed to gargle with aloclair three times a day. all drugs were prescribed for a period of seven days. during the fifth consultation (day 30), anamnesis confirmed that the patient had observed a regular intake of drugs. extraoral examination of the lower lip confirmed it to be normal without the presence of erosion, pain or itchiness (figure 8) and the patient was declared to have been healed. discussion the patient complained of lower lip ulceration and fever which had commenced three days prior to the appearance of the lesion and was subsequently diagnosed as herpes labialis. a painless scrapable yellowish white plaque on the surface of the tongue had concurrently developed resulting in a diagnosis of coated tongue. this constituted a differential diagnosis of oral thrush since no pain and burning sensation accompanied the coated tongue lesion.8 in this case, a history of a burning sensation and pain reported by the patient, allied with clinical examination results indicating the presence of white plaque which could be scraped off leaving a reddish area, oral thrush was recorded. another patient experienced malaise, a tingling sensation in the toes and fingertips accompanied by frequent urination at night leading to a diagnosis of dm and was referred for a complete blood count test, fasting blood glucose test and oral glucose tolerance test at a clinical pathology laboratory. the results of a clinical pathology laboratory test confirmed the patient to be suffering from dm and he was subsequently prescribed with metformin. this drug is categorized within the guanidine group which may enhance sensitivity to insulin, inhibit glucose formation in the liver, and reduce low density lipoprotein (ldl) and the level of triglyceride. its ability to suppress appetite renders it the first drug of choice. metformin is commonly prescribed in cases of an initial diagnosis of dm.1,9 in the case described here, the patient was suffering from both dm and anemia. these conditions are induced by an increase in proinflammatory cytokine expression among diabetic patients with anemia compared to those suffering only from dm. anemic individuals are characterized by increased interleukin-6 (il-6) production and b cell activity which enhances the link between il-6 and antierythropoietic action. furthermore, patients with dm and anemia demonstrate high levels of c-reactive protein and ultra-sensible ferritin. in contrast, low iron levels are present in those patients where the increase in ferritin is interconnected with the chronic inflammatory process evident in cases of diabetes.3,9 anemia can cause degradation of cellular immunity, reduced bactericidal activity in polymorphonuclear leukocytes, inadequate antibody response and abnormality in epithelial tissues. this condition can reduce the activity of mitochondrial enzymes due to oxygen and nutrition transport being disrupted. it leads to the inhibition of epithelial cell differentiation and growth. the terminal differentiation process in the epithelial cells of the stratum corneum will be impeded resulting in loss of normal keratinization and atrophy. as a result, the oral mucosa tissue will become thinner and easily ulcerated.10 in anticipation of the development of anemia, the patient was prescribed with sangobion, a supplement containing 250 mg ferrous (fe) gluconate, 0.2 mg mangan sulfate, 0.2 mg copper sulfate, 50 mg vitamin c, 1 mg folic acid, and vitamin b12. fe gluconate is an iron compound crucial to the energy metabolism process. at the same time, the presence of mangan sulfate and copper sulfate as iron transporting compounds is essential, while vitamin c assists the absorption of iron by the intestines to be subsequently transported by blood serum around the circulatory system. moreover, vitamin b12 and folic acid act as pivotal cofactors in blood cell deoxyribonucleic acid (dna) synthesis.11 the patient initially complained of lower lip ulceration accompanied by fever which was diagnosed as herpes labialis. he was prescribed acyclovir which can prevent virus replication through three mechanisms: phosphorylation of acyclovir to phosphate derivates within the cell via viral thymidine kinase, inhibition of dna polymerase by acyclovir activation, and termination of chain elongation by eliminating cyclic sugar from acyclovir triphosphate.12 acceleration of the wound healing process and prevention of secondary infection in herpes labialis were managed by prescribing topical alloclair to be gargled and topical application to the lower lip region. due to its aloevera extract content, aloclair can accelerate wound healing through stimulation of and increase in anti-inflammatory cell activity which enhances the reepithelization process.13 acyclovir intake was consistently managed by the patient during a period of two weeks. an antiviral drug was administered to resolve the herpes infection resulting from the immunocompromised condition of the patient who was eventually declared to have been cured of herpes labialis. nevertheless, this positive development was followed by the occurrence of oral thrush on the surface of the tongue dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p76-80 80 maharani laillyza apriasari/dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 76–80 and angular cheilitis on the lateral vermilion border of the lips. in order to manage these conditions, the patient was subsequently prescribed nystatin oral suspension. considered the most common therapy in dentistry, the application of topical nystatin plays an essential role in oral and systemic candidiasis prophylaxis among immunocompromised patients. this drug is primarily recommended for the treatment of oral candidiasis because of its high efficacy, low cost and less serious side effects. the colony of candida species adheres to the oral mucosa via epithelial cells, germ tube formation and the hydrophobicity of cell surfaces. absorption of topical drugs by the oral mucosa is crucial to the elimination of hyphae. nystatin, one of the topical drugs used in candidiasis treatment, can be prescribed for between one and six weeks.14,15 following administration of nystatin during a period of ten days, the patient was cured of oral thrush and angular cheilitis. however, the recurrence of herpes labialis induced extreme irritation in the lower lip which was managed by the patient immediately being prescribed a combination of cetirizine and acyclovir. several studies posit that viral infection may induce exacerbation of acute urticaria as indicted by the appearance of a herpes simplex lesion. the chronic urticaria presented by the patient was instigated through reccurent herpes simplex infection.16 reversible dysfunction of t lymphocyte can occur as a result of a hyperglicemic condition. several studies have identified the seropositivity of the herpes simplex virus (hsv) as a risk factor for diabetes due to chronic inflammation or immune activation. hyperglycemia can potentially induce t-cell inhibition against hsv, thereby enabling viral escape from immune control. viruses are exclusively localized in the neuron. the paramount importance of herpes virus latency located in the ganglion is related to reactivation and clinical manifestations. dna will be maintainedin a heterochromatin state rendering gene expression silenced. despite this, latency associated transcripts (lats) are produced which increase the efficiency of latency establishment and reactivation because of their ability to promote both neuronal apoptosis and lytic gene expression inhibition. lats may impede superinfection by other strains of the same herpes virus or by other herpes viruses.17,18 this case presents a recurrent infection of herpes labialis coinciding with oral thrush induced by the presence of candida biofilm which may act as a persistent reservoir not only for fungal cells, but also infectious viruses. these viruses might be preserved and protected within the biofilm. representing an additional health risk factor, candida biofilm is reported as entrapping viral particles, thereby protecting the virus from conventional antiviral treatment. anti-candida effector function was inhibited by hsv infection. it was protected by candida biofilm that encompassed hsv inactivation resulting from acyclovir treatment.18,19 it can be concluded from the foregoing discussion that multidisciplinary management of herpes labialis co-occuring with oral candidiasis in dm patients is indispensable. concurrent infection of hsv and candidiasis in diabetic indivuduals will require a more protracted wound healing process. therefore, stabilizing the hyperglycemic level which induces an immunocompromised condition in dm patients is pivotal. references 1. putra rjs, achmad a, rachma h. kejadian efek samping potensial terapi obat anti diabetes pasien diabetes melitus berdasarkan algoritma naranjo. pharm j indones. 2017; 2(2): 45–50. 2. badan penelitian dan pengembangan kesehatan. hasil utama r iskesdas 2018. ja ka r ta: kementer ian kesehatan republik indonesia; 2018. p. 66–71. 3. barbieri j, fontela pc, winkelmann er, zimmermann cep, sandri yp, mallet ekv, frizzo mn. anemia in patients with type 2 diabetes mellitus. anemia. 2015; 2015: 1–7. 4. apriasari ml, ainah y, febrianty e, carabelly an. antioxidant effect of channa micropeltes in diabetic wound of oral mucosa. int j pharmacol. 2019; 15(1): 137–43. 5. apriasari ml, puspitasar d. effect of channa micropeltes for increasing lymphocyte and fibroblast cells in diabetic wound healing. j med sci. 2018; 18(4): 205–10. 6. al-maskari ay, al-maskari my, al-sudairy s. oral manifestations and complications of diabetes mellitus: a review. sultan qaboos univ med j. 2011; 11(2): 179–86. 7. indurkar ms, maurya as, indurkar s. oral manifestations of diabetes. clin diabetes. 2016; 34(1): 54–7. 8. apriasari ml, baharuddin em. penyakit infeksi rongga mulut. surakarta: yuma pustaka; 2012. p. 6–8, 11–3. 9. almasdy d, sari dp, suhatri s, darwin d, kurniasih n. evaluasi penggunaan obat antidiabetik pada pasien diabetes melitus tipe-2 di suatu rumah sakit pemerintah kota padang – sumatera barat. j sains farm klin. 2015; 2(1): 104–10. 10. hatta i, firdaus iwak, apriasari ml. the prevalence of oral mucosa disease of gusti hasan aman dental hospital in banjarmasin, south kalimantan. dentino j kedokt gigi. 2018; 2(2): 211–4. 11. apriasari ml, tuti h. stomatitis aftosa rekuren oleh karena anemia. j dentofasial. 2010; 9(1): 39–46. 12. apriasari ml. methisoprinol as an immunomodulator for treating infectious mononucleosis. dent j (majalah kedokt gigi). 2016; 49: 1–4. 13. apriasari ml, endariantari a, oktaviyanti ik. the effect of 25% mauli banana stem extract gel to increase the epithel thickness of wound healing process in oral mucosa. dent j (majalah kedokt gigi). 2015; 48(3): 150–3. 14. apriasari ml. peroxide alkaline for cleansing the baby bottle nipple to prevent oral thrush relaps. dent j (majalah kedokt gigi). 2013; 46(2): 75–9. 15. lyu x, zhao c, hua h, yan z. efficacy of nystatin for the treatment of oral candidiasis: a systematic review and meta-analysis. drug des devel ther. 2016; 10: 1161–71. 16. zawar v, godse k, sankalecha s. chronic urticaria associated with recurrent genital herpes simplex infection and success of antiviral therapy — a report of two cases. int j infect dis. 2010; 14(6): e514–7. 17. mcdonald p, krishnan-natesan s. concurrent reactivation of vzv and hsv-2 in a patient with uncontrolled diabetes mellitus: a case report. eur j med case reports. 2017; 1(3): 108–13. 18. ascione c, sala a, mazaheri-tehrani e, paulone s, palmieri b, blasi e, cermelli c. herpes simplex virus-1 entrapped in candida albicans biofilm displays decreased sensitivity to antivirals and uva1 laser treatment. ann clin microbiol antimicrob. 2017; 16(1): 1–8. 19. mohammadi f, javaheri mr, nekoeian s, dehghan p. identification of candida species in the oral cavity of diabetic patients. curr med mycol. 2016; 2(2): 1–7. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p76–80 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v52.i2.p76-80 vol 51 no 3 jul sep 2018_pus.indd 124124 research report dental journal (majalah kedokteran gigi) 2018 september; 51(3): 124–128 potential immunomodulatory activity of phyllanthus niruri aqueous extract on macrophage infected with streptococcus sanguinis suryani hutomo,1 denise utami putri,2,3 yanti ivana suryanto,4 and heni susilowati5 1department of microbiology, faculty of medicine, universitas kristen duta wacana 2doctoral program, faculty of medicine, universitas gadjah mada 3international phd program in medicine, taipei medical university, taipei, taiwan 4department of physiology, faculty of medicine, universitas kristen duta wacana 5department of oral biology, faculty of dentistry, universitas gadjah mada yogyakarta indonesia abstract background: streptococcus sanguinis is an oral commensal bacterium commonly found in periodontal lesions and deep abscesses that are usually dominated by anaerobic bacteria. as an important causative agent of systemic diseases, and with the increasingly numerous cases of antimicrobial resistance, some means of modulating the immune response to bacterial infection is thus necessary. phyllanthus niruri linn is widely used as a medicinal herb to both prevent and treat disease and demonstrates immunomodulatory properties. purpose: this study aimed to observe the potential for aqueous extract of phylanthus niruri to induce macrophage proliferation and no production following s. sanguinis infection. methods: macrophages were isolated from the peripheral blood of healthy subjects, stimulated with p. niruri aqueous extract in graded doses and infected with s. sanguinis atcc 10556 bacterial suspension. cell proliferation and nitric oxide release was observed at 24 and 48 hours to determine macrophage activities. results: no production and cell proliferation started to increase upon 50 and 100μg/ml p niruri respective stimulation. statistical analysis using one-way anova demonstrated a significant difference of cell proliferation after stimulation with p. niruri aqueous extract at various doses (p<0.05). conclusion: p. niruri aqueous extract induced macrophage proliferation and no secretion upon s sanguinis infection, showing potential antibacterial and immunomodulatory activities. at the same concentrations, no production and macrophage were higher at 48 hours than at 24 hours. keywords: streptococcus sanguinis; macrophage; phyllanthus niruri; medicinal plant; oral bacteria correspondence: suryani hutomo, department of microbiology, faculty of medicine, universitas kristen duta wacana, yogyakarta 55224, indonesia, email: suryanihutomo_drg@yahoo.com; introduction streptococcus sanguinis is an oral commensal bacterium belonging to the viridans streptococci group and one of the initial species to colonize tooth surfaces. its role in oral disease is uncertain, but this species is often implicated in infective endocarditis and is, in fact, the most frequently involved.1 regular dental procedures can sometimes carry the risk of exposure to oral microorganisms in the circulatory system. these bacteria, either naturally commensal or pathogenic, may induce unexpected infection and inflammation, including infective endocarditis. poor oral hygiene is another predisposing factor in bacterial endocarditis, although the bacteria can also enter the body through the daily diet. infection is more likely in the presence of an abnormal heart valve such as that caused by congenital heart disease.2,3 upon exposure to a microbial antigen, macrophage of the immune system serves as one of the initial defenders of the host. macrophages play an important role in the immune system, not only as phagocytic cells, but also as antigen-presenting cells. as phagocytic cells, they function by direct engulfment and secretion of chemokines including reactive oxygen species (ros) and nitric oxide dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p124–128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p124-128 125 hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 124–128 (no) that destroy bacteria, viruses and parasites. they also process the engulfed antigen and introduce it into t cells, while simultaneously secreting pro-inflammatory cytokines which, together, will induce activation of the cellular immune response cascade, recruitment of other macrophages and further clearance of pathogen.4–6 in an immunocompetent subject, the macrophage functions, further activating immunity, resulting in eradication of pathogens. however, in some patients with impairment of the immune system, prophylactic measures are often necessary to minimize the risk of systemic infection. in fact, prophylactic interventions are not always effective since previous studies have reported multiple cases of resistance, including that of streptococcus viridans to both penicillin and fluoroquinolone.7,8 therefore, the application of antibiotic agents should be minimized and every attempt to modulate the immune response to harmful pathogens be made. phyllanthus niruri linn, is a tropical plant commonly found in south east asia, india, china and the usa. in indonesia, known as meniran, it is widely used as a medicinal herb to both prevent and treat diseases. plants from the genus phyllanthus have been shown to have anti-bacterial and anti-viral effects, including against staphylococcus aureus and streptococcus agalactiae.9–11 in addition, extract of p. niruri has been observed to induce macrophage activity by increasing phagocytosis and no in mice macrophage infected with salmonella typhii12 and in the macrophages of tuberculosis patients.13 despite numerous studies in elucidating the medicinal use of p. niruri, little is known about its antibacterial potential in relation to human commensal bacteria, as well as its effect on human macrophage. therefore, in this study, human macrophages from the peripheral blood of healthy subjects were extracted in order to observe the effect of p. niruri aqueous extract on macrophage proliferation and no production on s. sanguinis infection. from this study, it was expected to gain new insights into the potential use of p. niruri as an immunomodulatory agent in the management of infections caused by s. sanguinis. materials and methods streptococcus sanguinis was prepared as in our previous study.14 the bacteria was grown in brain-heart infusion broth (bhi; difco inc., detroit, mi) under microaerobic conditions at 37°c. a bacterial stock suspension was prepared at a concentration of 1.5x108 colony forming units (cfu). dried phyllanthus niruri leaves were obtained and confirmed by a botanical expert at the herbal manufacturing company, cv merapi farma, yogyakarta. the dried leaves were made into an aqueous extract by means of a maceration technique at the integrated research and testing laboratory, universitas gadjah mada, yogyakarta. extract in paste form was dissolved in dimethyl sulfoxide (dmso) solution to form a stock which was then diluted with culture medium to yield concentrations of 25, 50, 100, 200 and 400 μg/ml peripheral blood mononuclear cells (pbmc) were isolated from the peripheral venous blood of three healthy subjects. mononuclear cells were isolated by ficollgradient density separation, as described above.13 after centrifugation, the pbmc layer containing lymphocytes and monocytes weas transferred to a rpmi 1640 (sigma) culture medium with 10% fbs, 2% penstrep, and 0.5% fungizone. pbmc numbers were calculated using a 1:20 dilution of tryphane blue dye. the cells were seeded into a 24-well plate with coverslips at a density of 5x104 cells/ well and incubated at 37°c in 5% co2 for maturation. the culture media were changed every three days. after six days, mature macrophages were present as adherent cells on the cover slips ready for further studies. macrophage cells were cultured with 0, 25, 50, 100, and 200 μg/ml of p. niruri aqueous extract for four hours and stimulated with 1.5x108 cfu (0.5 mc farland) s. sanguinis. this culture was then incubated for either 24 hours (group 1) or 48 hours (group 2) for time dependent study. macrophage proliferation was observed under a phase contrast microscope after hematoxylin-eosin (he) staining and calculated based on the number of cells visible in ten observation fields. a kolmogorov-smirnov normality statistic test was performed, followed by a levene homogenity statistic test. the difference between the groups of macrophage proliferation was established by the conducting of a one-way anova test at a significance level of 0.05. a no release assay was performed using a previously described modified griess method by green.15 griess reagent was made by adding 0.1% n-(1-napthyl) ethylenediamine dihydrochloride (ned, sigma) dissolved in sterile water and 1% sulfanilamide (sigma) dissolved in 5% phosphoric acid mixed in equal volume. for standard nitric, nano2 was dissolved in sterile water (2mm stock), while graded dilution of 0-200 μm was undertaken by dilution of the standard nitric with culture medium. 100 μl of griess reagent was distributed into a 96-well plate and 100 μl of supernatant from the macrophage culture or graded doses of nitric standard was added to the wells. the control medium was used as a blank. the absorbance of the pink colour was measured at 550 nm using a microplate reader (thermo scientific, rockford, illionis, usa) after 15 minutes of incubation at room temperature. for further analysis, a standard curve was made based on simple linear regression from standard nano2 reading. no concentration was quantified based on the standard curve in μm units. results the increase in no production occurred at concentrations of 50 μg/ml, 100 μg/ml, 200 μg/ml, and 400 μg/ml of p. niruri supplementation in both groups, characterized by an increased optical density (od) value in both groups at dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p124–128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p124-128 126hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 124–128 24 hours and 48 hours (table 1). a modified griess assay was used to calculate no concentration in the culture supernatant. a regression line equation from standard nitric absorbance was obtained (r2: 0.9975) and the means of od values were plotted on the curve (figure 1). upon s. sanguinis stimulation, control samples produced 19.70 μm of no at 24 hours (group 1) and 20.74 μm at 48 hours (group 2). with 25 μg/ml p. niruri supplementation, no concentrations were lower in both groups (19.35 and 20.51 μm for group 1 and group 2, respectively) compared to the controls. however, with the addition of 50, 100, 200 and 400 μg/ml of p. niruri there was a corresponding increase in no concentration observed in both groups. the highest no concentration was recorded with the addition of 400μg/ml p. niruri, 53.77 for group 1 and 74.23 μm for group 2. at the same concentrations, no production was higher in group 2 than in group 1. this study also demonstrated that p. niruri extract has a proliferative effect on macrophages reflected in an increase in the number of macrophage cells per field of view. proliferation occurs from a concentration of 100 μg ml as shown in table 2. cell proliferation increased with greater extract concentration and exposure time. similar to no production, cell proliferation was greater with 48 hours of treatment than with 24 hours (figure 2). the data was analyzed using a one-way anova test at a significance level of 0.05 and there was significant difference between groups (p=0.000). table 1. mean and standard deviation of optical density (od) of nitric oxide production of p. niruri supplementation at various concentrations time exposure concentration (μg/ml) 40020010050250 0.223+0.0020.097+0.0040.085+0.00010.078+0.00010.075+0.0030.076+0.000124 h 0.311+0.0030.215+0.0030.096+0.00010.086+0.0020.08+0.00010.081+0.00348 h table 2. mean and standard deviation of the number of macrophage cells upon p. niruri supplementation at various concentrations and time exposures time exposure concentration (μg/ml) 4002001000 24 h 2+1.83 4+0.67 5+0.82 6.4+0.97 48 h 2.3+2.0 7.8+1.23 12+1.89 17+2.05 figure 2. macrophage proliferation (40x magnification). untreated cells at 24 h (a) and 48h (b), no visible proliferation. macrophages with 100 μg/ml p. niruri extract and s.sanguinis at 48h (d) showed greater macrophage proliferation compared with 24 h exposure (c). similarly, 200 μg/ml p. niruri at 48h (f) there was greater proliferation compared with 200 μg/ml 24h (e). likewise, 400 μg/ml p. niruri at 48h (h) resulted in greater macrophage proliferation than at 24h exposure (g). figure 1. standard curve and plotting of nitric oxide concentration dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p124–128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p124-128 127 hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 124–128 discussion before the development of specific immune response, bacterial infection is predominantly countered by circulating macrophages and dendritic cells (dcs). bacteria ingested by macrophages are internalized in a phagosome where they can be killed by a number of mechanisms, including that of reactive nitrogen intermediates (rnis), particularly no. rois alone have been shown to be insufficient to kill mycobacteria inside macrophage, but they enhance killing by rnis.16,17 macrophages also produce cytokines such as tnf-α and il-12, which subsequently induce nk-cells to secrete ifn-ɣ. tnf-α and ifn-ɣ stimulated more il12 production from the infected macrophages, creating a positive feedback loop. moreover, these cytokines also play important functions in the activation of cellular immune response mediated by t cells, as well as further recruitment of macrophages to the infection site.16,17 our study observed induced macrophage proliferation upon stimulation with p. niruri aqueous extract. it indicates that this substance has mitogenic ability in macrophages. in line with our findings, eze et al used methanol extract of p niruri and observed an increase in mobilization and proliferation of polymorphonuclear (pmn) neutrophils in rodent peritoneal fluid.18 another study conducted by amin et al also reported induced proliferation of pbmc after p. niruri aqueous extract stimulation.9 no, a substance that results from catalyzing inducible nitric oxide synthase, is an enzyme in the phagolysosomes that are activated during phagocytosis. other enzymes that are also activated during phagocytosis: inducible nitric oxide synthase, phagocyte oxidase and lysosomal proteases, referred to as ros, destroy ingested pathogen. the increases in no and ros production reflects an intensification of phagocytic processes by macrophages.6 in our study, the no release assay demonstrated that no production upon s. sanguinis infection gradually increased beginning at 50 μg/ml of p. niruri supplementation in both groups, and the increase observed was both doseand time-dependent. this suggests that p. niruri aqueous extract may modulate an increase in phagocytic activity upon s. sanguinis infection in macrophages. a similar dose response phenomenon was observed in a study conducted by putri et al.13 using macrophage cells from tuberculosis patients. the results of this study are also consistent with one conducted by nworu et al.19 which reported that treatment with p. niruri extract in animal model macrophages significantly enhance the activation and function of these cells, such as phagocytosis, lysosomal enzyme activity and tnf-α release, which also modulate nitric oxide release from macrophages. while the increase in no secretion may reflect both macrophage phagocytic activity and the destruction of intracellular bacterial, these observed phenomena can also be the result of higher macrophage numbers due to increased proliferation. nevertheless, it was demonstrated that the aqueous extract of p niruri did subsequently exhibit toxicity upon macrophages. in conclusion, this study concludes that p. niruri aqueous extracts showed an ability to potentiate macrophage responses to s. sanguinis infection by inducing proliferation and no production in doseand time-dependent ways, with the highest effect observed at a concentration of 400 μg/ml at 48 hours. this study adds to the existing knowledge of the antibacterial properties of p niruri, that may not only act directly against bacteria, but also induce strong immune protection against them. further studies are required to establish the effects of p niruri stimulation to other types of immune cells including the subsequently activated cellular immune cells: t and b cells, upon bacterial infection. acknowledgement the authors would like to thank the faculty of medicine, universitas kristen duta wacana, yogyakarta, indonesia for the grant which funded the research reported here. references ge x, kitten t, chen z, lee sp, munro cl, xu p. identification of1. streptococcus sanguinis genes required for biofilm formation and examination of their role in endocarditis virulence. infect immun. 2008; 76(6): 2551–9. lockhart pb, brennan mt, sasser hc, fox pc, paster bj, bahrani-2. mougeot fk. bacteremia associated with toothbrushing and dental extraction. circulation. 2008; 117(24): 3118–25. westling k. viridans group streptococci septicaemia and endocardi-3. tis. thesis. stockholm: karolinska institutet; 2005. p. 9-31. bah a, vergne i. macrophage autophagy and bacterial infections.4. front immunol. 2017; 8: 1–9. 5. vazquez-torres a, stevanin t, jones-carson j, castor m, read rc, fang fc. analysis of nitric oxide-dependent antimicrobial actions in macrophages and mice. methods enzymol. 2008; 437: 521–38. abbas ak, lichtman ah. basic immunology : functions and disor-6. ders of the immune system. 3rd ed. philadelphia: saunders; 2011. p. 312. masuda k, nemoto h, nakano k, naka s, nomura r, ooshima t.7. amoxicillin-resistant oral streptococci identified in dental plaque specimens from healthy japanese adults. j cardiol. 2012; 59(3): 285–90. sahasrabhojane p, galloway-peña j, velazquez l, saldaña m, horst-8. mann n, tarrand j, shelburne sa. species-level assessment of the molecular basis of fluoroquinolone resistance among viridans group streptococci causing bacteraemia in cancer patients. int j antimicrob agents. 2014; 43(6): 558–62. amin za, abdulla ma, ali hm, alshawsh ma, qadir sw. assess-9. ment of in vitro antioxidant, antibacterial and immune activation potentials of aqueous and ethanol extracts of phyllanthus niruri. j sci food agric. 2012; 92(9): 1874–7. 10. ibrahim d, hong ls, kuppan n. antimicrobial activity of crude methanolic extract from phyllanthus niruri. nat prod commun. 2013; 8(4): 493–6. 11. liu s, wei w, shi k, cao x, zhou m, liu z. in vitro and in vivo anti-hepatitis b virus activities of the lignan niranthin isolated from phyllanthus niruri l. j ethnopharmacol. 2014; 155(2): 1061–7. 12. ibnul a. uji komparasi aktivitas fagositosis makrofag dan produksi nitrit oksida pada mencit balb/c akibat perlakuan ekstrak meniran hijau (phyllanthus niruri) dan meniran merah (phyllanthus urinaria) yang diinfeksi bakteri salmonella typhi. thesis. surakarta: universitas sebelas maret; 2012. 13. putri du, rintiswati n, soesatyo mh, haryana sm. immune modulation properties of herbal plant leaves: phyllanthus niruri aqueous dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p124–128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p124-128 128hutomo, et al./dent. j. (majalah kedokteran gigi) 2018 sept; 51(3): 124–128 extract on immune cells of tuberculosis patient in vitro study. nat prod res. 2018; 32(4): 463–7. 14. hutomo s, susilowati h, agustina d, asmara w. analysis of antistreptococcus sanguinis igy ability to inhibit streptococcus sanguinis adherence. dent j (maj ked gigi). 2018; 51: 33–6. 15. green lc, wagner da, glogowski j, skipper pl, wishnok js, tannenbaum sr. analysis of nitrate, nitrite, and [15n]nitrate in biological fluids. anal biochem. 1982; 126: 131–8. 16. weiss g, schaible ue. macrophage defense mechanisms against intracellular bacteria. immunol rev. 2015; 264(1): 182–203. 17. myers jt, tsang aw, swanson ja. localized reactive oxygen and nitrogen intermediates inhibit escape of listeria monocytogenes from vacuoles in activated macrophages. j immunol. 2003; 171(10): 5447–53. 18. eze co, nworu cs, esimone co, okore vc. immunomodulatory activities of methanol extract of the whole aerial part of phyllantus niruri l. j pharmacogn phyther. 2014; 6(4): 41–6. 19. nworu cs, akah pa, okoye fbc, esimone co. aqueous extract of phyllanthus niruri (euphorbiaceae) enhances the phenotypic and functional maturation of bone marrow-derived dendritic cells and their antigen-presentation function. immunopharmacol immunotoxicol. 2010; 32(3): 393–401. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i3.p124–128 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i3.p124-128 vol 51 no 4 okt-des 2018.indd 205 dental journal (majalah kedokteran gigi) 2018 december; 51(4): 205–209 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg the effects of acanthus ilicifolius chloroform extract on tlr-2 expression of macrophages in oral candidiasis dwi andriani and agni febrina pargaputri department of oral biology faculty of dentistry, universitas hang tuah surabaya – indonesia abstract background: immunosuppressed conditions due to long-term corticosteroid and tetracycline consumption are susceptible to fungal invasion, especially by candida albicans (c. albicans), that requires treatment of oral candidiasis. toll like receptor-2 (tlr-2) plays a role in candida recognition. nystatin is regularly employed for oral candidiasis, but produces certain side-effects. chloroform extract of acanthus ilicifolius (a. ilicifolius) leaves represents both a potent inhibitor of c. albicans growth and an antioxidant. purpose: this study aimed to compare the effect of a. ilicifolius leaf chloroform extract and nystatin treatment on tlr-2 expression in oral candidiasis immunosupressed models. methods: this study constitutes a true experimental investigation incorporating a post test-only control group design. 20 healthy male rattus novergicus (wistar), aged 12 weeks and with an average weight of 250g, were immunosuppressed through oral administration of dexamethasoneand tetracycline for a period of 21 days before being induced with c. albicans (atcc-10231) 6 x 108 for two weeks. the subjects were divided into five groups (n=4/group): healthy (h), no-treatment(p), nystatin treatment(n), a. ilicifollius (8%) treatment (ai-1) and a. ilicifollius (16%) treatment (ai-2). the subjects were treated for 14 days, with their tongue being subsequently biopsied. tlr-2 expression was subjected to immunohistochemical examination, observed under a microscope (400x magnification) and statistically analyzed (one-way anova, lsd-test, p<0.05). results: tlr-2 expression of p (6.25 ± 2.5), n (11.25 ± 0.96), ai-1 (13.00 ± 1.15), ai-2 (12.75 ± 1.7) was higher than h (1.75 ± 0.5). significant differences existed between n to p, n, ai-1, ai-2; p to n, ai-1 and ai-2 (p<0.05). no significant differences were present between n, ai-1 and ai-2 (p < 0.05). conclusion: a. ilicifolius extract can increase expression of tlr-2 in oral candidiasis-immunosuppressed models. a. ilicifolius extract produces the same effect in increasing tlr-2 expression when compared to nystatin. keywords: achanthus ilicifolius; candida albicans; immunosupressed; oral candidiasis; tlr-2 correspondence: dwi andriani, department of oral biology, faculty of dentistry, universitas hang tuah. jl. arief rahman hakim 150 surabaya 60111, indonesia. e-mail dwi.andriani@hangtuah.ac.id research report introduction infection in humans can be either bacterial or fungal in nature. although often a commensal organism in humans, several fungi can prove to be pathogenic under certain conditions. fungal infections can occur in immunocompromised patients suffering from trauma, hiv infection, immunosuppression and neutropenia and in whom the normally protective bacterial microflora is disrupted.1 prolonged use of systemic drugs, for example broad-spectrum antibiotics, immune-suppressants and drugs with xerostomic side-effects, alter the local oral flora or disrupt the mucosal surface or reduce salivary flow, thereby creating a favorable environment for candida to grow and cause oral infection.2 drug induced oral candidiasis has been reported in asthma patients treated with steroid inhalers for approximately three months.3 the most common fungal infection affecting the oral cavity is oral candidiasis approximately 60% of the cases of which are caused by candida albicans (c. albicans) infection.4 according to research conducted in surabaya during 2007, following candida isolation in aids patients suffering from oral candidiasis with clinical features of acute pseudomembrane candidiasis, acute erythematosus doi: 10.20473/j.djmkg.v51.i4.p205–209 http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p205-209 206andriani, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 205–209 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p205–209 candidiasis, chronic hyperplastic candidiasis, acute erythematosus candidiasis and perleche, c. albicans species was found in approximately 35.29% of cases, while non-c. albicans species were detected in the other 64.71%.5 neutrophils and macrophages constitute the immune cell types involved in immune mechanism defense against fungal infections. they recognize candida through pattern recognition receptors (prrs) which interact with specific molecules called pathogen-associated molecular patterns (pamps) exposed on the surface of the candida.6 recognition of fungal cells occurs by means of prr, dectin-1 (-1,3 glucan) and several other prrs involved in identifying different cell wall polysaccharides of this pathogen. these include toll like receptors including: (tlr) 2 which recognizes phospholipomannan of c. albicans, tlr4 which recognizes o-mannan of c. albicans and mannose receptor (mr) which recognizes n-mannan of c. albicans 7. these cells release cytokines and chemokines to further modulate the immune response for mucosal protection.8,9 activation of tlrs, includingtlr-2, not only induces inflammatory responses, but also the development of antigen-specific adaptive immunity against fungal infection. nystatin is a drug which, in addition to amphotericin b, miconazole, itraconazole and fluconazole, is commonly used in oral candidiasis therapy. it produces several sideeffects including: unpleasant tastes in the mouth, vomiting, diarrhea, nausea, anorexia, abdominal pain, thrush and headaches.10 acanthus ilicifolius (a. ilicifolius) is a mangrove plant of the acanthaceae family native to tropical regions in asia and africa employed by communities in malaysia, india and thailand to treat rheumatism, neuralgia, wounds resulting from poisoned arrows, coughs, asthma, influenza and dermatitis.11,12 a. ilicifolius leaf extract contains several active phytochemical components such as proteins, resins, steroids, tannins, glycosides, reducing sugars, saponins, sterols, terpenoids, phenols, cardioglycosides and catachols.11 this plant also has the ability to act as an antimicrobial.13 chloroform and n-hexane extracts from a. ilicifolius leaves have a powerful inhibitive effect on bacillus subtilis, staphylococcus aureus, c. albicans, aspergilus fumigatus and aspergilus niger.12 the chloroform extract from a. ilicifolius leaves at a dose of 400 mg/kgbw has been reported to have an anti-ulcerative effect in mice, while one of 500 mg/kgbw has been demonstrated to be a practical anti-carcinogenic.14,15 the chloroform extract of a. ilicifolius leaves has an anti-candida effect and produces the highest inhibitory zone compared with other extraction methods.16 chloroform extract of a. ilicifolius leaf 1% is effective in inhibiting c. albicans through thermoplastic nylon soaking. the administration of chloroform extract a. ilicifolius 8 mg/ml may increase il-17 in immunosuppressed subjects suffering from oral candidiasis.17,18 the purpose of this study was to compare the effect of a. ilicifolius chloroform extract and nystatin on tlr-2 expression immunosuppressed models as an alternative medicine derived from natural materials for oral candidiasis. materials and methods this research represented a true experiment incorporating post test-only control group design. it employed 20 healthy, 12-week old, male, rattus novergicus (wistar), each 250 grams in weight, which were divided into five groups (n=4/group). group-1(h): healthy/normal subject group, group-2 (p): subjects induced with c. albicans and 0.1% cmcna, group-3 (n): subjects induced with c. albicans and treated with nystatin topical, group a. ilicifollius 8% treatment (ai-1): subjects induced with c. albicans and treated with 8% a. ilicifolius chloroform extract topically, groupa. ilicifollius 16% treatment (ai-2): subjects induced with c. albicans and treated topically with 16% a. ilicifolius chloroform extract. the method of animal subject induction in this study was based on chami’s et al. research, with several modifications. immunosuppression was achieved by orally administering dexamethasone 0.5 mg/day and 1% tetracycline /day for 21 days. between the 7th and 21st days the subjects were induced with 0.1 cc of c. albicans (atcc-10231) 6 x 108, applied to their tongues with a sterile cotton bud three times a week for two weeks.19 a. ilicifolius chloroform extract was produced by dehydrating leaves in the open air for two days. 10 grams of dried a. ilicifolius leaves were subsequently mixed with 200 ml of chloroform using a mortal and pastle, covered and allowed to stand for five hours. the solvent was removed and filtered with whatman’s no. 1 filter paper before being evaporated at low pressure by using a buchi rotavapor r-200 at 45°c. the chloroform extract was then stored in a refrigerator for future use.20 treatment using a. ilicifolius chloroform extract 8% (group ai-1), 16% (group ai-2) and nystatin as control groups was performed by applying 0.5 cc of the material to the surface of the tongue at the same time on 14 consecutive days, after which the subjects in each group were sacrificed and biopsed. tlr-2 expression was examined using a immunohistochemical staining method and then observed by means of a light microscope at 400x magnification. the statistical analysis used both one-way anova and lsd statistical tests to determine the significance of differences between groups. results the expression of tlr-2 in an oral candidiasis immunosuppressed model derived from the tongue of a subject is shown in figure 1. figure 2 indicates that the healthy/ normal group (h) had the lowest expression of tlr-2 (1.75 + 0.5) compared to p (6.25 ± 2.5), n (11.25 ± 0.96), ai-1 (13.00 ± 1.15) and ai-2 (12.75 ± 1.7), while http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p205-209 207 andriani, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 205–209 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p205–209 figure 1. tlr-2 expression of macrophage in the tongue of rattus novergicus (wistar) (black arrow). a: healthy/ normal group (h), b: oral candidiasis group (p), c: group of subjects induced by c. albicans and treated with nystatin topical (n), d: group of subjects induced by c. albicans and treated with a. ilicifolius chloroform extract topically (ai) (400x magnification). figure 2. means of tlr-2 expression of macrophage. this graphic represents the mean + standard deviation in each group of tlr-2 expression of macrophage after treatment with nystatin (n) and a. ilicifolius 8% (ai-1) and a. ilicifolius 16% (ai-2) compared with healthy subjects (h) and those receiving no treatment (p). there were significant differences between the healthy/normal group (h) and p, n, ai-1 and ai-2; p compared to n, ai-1 and ai-2 (p < 0.05). while there were no significant differences between n compared to ai-1 and ai-2, and ai-1 compared to ai-2 (p > 0.05). (*p < 0.05 vs h and p). http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p205-209 208andriani, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 205–209 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p205–209 the highest expression of tlr-2 appeared in the group of subjects which had been induced with c. albicans and treated topically with 8% a. ilicifolius chloroform extract (ai-1) compared to other groups. a one-way anova statistical test was employed which indicated the significant differences between groups (p<0.05). a subsequent lsd statistical test confirmed significant differences between the healthy/normal group (h) compared to groups p (0.018), n (0.017), ai-1 (0.017) and ai-2 (0.018) (p<0.05). significant differences also existed in p compared to n (0.020), ai-1 (0.019) and ai-2 (0.021) (p<0.05), while there was no significant differences between n and ai-1 (0.063) or ai-2 (0.18) and ai-1 compared to ai-2 (0.766) (p>0.05). discussion immunosuppressive conditions and long-term antibiotic consumption represent predisposing factors that can lead to oral candidiasis. in this study, subjects were immunosuppressed through the administering of dexamethasone and combined with tetracycline, the latter of which is intended to cleanse the oral cavity of bacteria, thereby enabling c. albicans to proliferate without contamination by other bacteria. the tongues of subjects were used as a site of inoculation, treatment and sampling. in this study, the level of tlr-2 expression, the nystatin treatment, 8% a. ilicifolius treatment, and 16% a. ilicifolius treatment, was found to be lower in the healthy group when compared to the candidiasis group. the untreated candidiasis also possessed lower tlr-2 expression compared to that of the treatment groups. immunosuppressive factors (catecholamines and steroids) induced by stress may also down-regulate the expression of tlrs. a number of studies have reported that tlrs are up-regulated in inflammatory conditions and downregulated in immunosuppressive conditions.21 therefore, it is speculated that immunosuppressed conditions may have down-regulated the expression of tlr-2 in this study. there was no significant difference between the treatment group of nystatin, the treatment of 8% a. ilicifolius and the treatment of 16% a. ilicifolius (p < 0.05). this indicates that nystatin therapy involving both 8% a. ilicifolius, and 16% a. ilicifolius administered to subjects with immunosuppressed candidiasis demonstrated the same ability to increase tlr-2 expression which was possibly due to the phytochemical content of the extract. chloroform leaf extract of a. ilicifolius contains phenol, flavanoid and tannin, which promote both antimicrobial and antioxidant activity.21 one study reported that the pentacyclic triterpenoid (isolated chloroform extract) present has the potential to restore vascular disorders associated with hypertension, obesity, diabetes, atherosclerosis. it could also be used in cancer therapy, as anti-ulcer drugs, as well as for the prevention and treatment of metabolic diseases.12,22 previous studies of both nystatin and a. ilicifollius extract (8% and 16%) found that the latter were similar to nystatin in increasing the amount of il-17 expression in oral candidiasis immunosuppressed conditions models.18 this indicates that the capture of tlr-2 leads to a decrease in the number of candida and increased pro-inflammatory cytokines. in this study, the concentration of 8% a. ilicifolius extract produced the effect on the increase of proinflammatory cytokines via tlr-2 pathway. upregulation of tlrs may lead to an inflammatory response and protective function against infection, while downregulation of tlrs may suppress inflammation and facilitate subsequent infection.23 another study reported an increase in the capture capability of tlr-2 in mice afflicted with candidiasis which was associated with decreased tnf and chemokine production.24 tlr-2 can enhance the ability of macrophages in c. albicans-induced mice25, while its absence can reduce neutrophil chemotaxis and antifungal mechanisms.26 tlr-2 binds phospholipomannans from c. albicans in the presence of galectin-3 and β-mannosides to induce a macrophage pro-inflammatory response.27 tlr-2 in combination with dectin-1 can also bind the β-glucan fungus. inhibition of tlr-2 in human mononuclear cells can cause a 4050% reduction in the production of proinflammatory cytokines in vitro. a decrease in the amount of tlr-2 may escalate susceptibility to infection by c. albicans.28 tlr activation, in particular that of tlr-2, can suppress defense immunity against c. albicans through il-10 induction and t-regulatory cells.29 tlr signals through the myd88 pathway leading to activation of mapk and induces the translocation of nuclear factor kappa b to the nucleus which promotes the transcription and synthesis of proinflammatory cytokines.30 in conclusion, a. ilicifolius chloroform extract can increase expression of tlr-2 in oral candidiasisimmunosupressed models. a. ilicifolius chloroform extract produces the same effect by increasing tlr-2 expression compared with nystatin. further research is required regarding the function of acanthus in this model and its role in the mechanism of oral candidiasis. references 1. van de veerdonk fl, kullberg b-j, netea mg. pathogenesis of invasive candidiasis. curr opin crit care. 2010; 16(5): 453–9. 2. rao pk. oral candidiasis – a review. sch j med. 2012; 2(2): 26–30. 3. wahyuli hn, suyoso s, prakoeswa crs. manifestasi klinis dan identifikasi spesies penyebab kandidiasis oral pada pasien hiv/ aids rsud dr. soetomo surabaya. berk ilmu penyakit kulit dan kelamin. 2010; 22: 11–6. 4. martins n, ferreira icfr, barros l, silva s, henriques m. candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. mycopathologia. 2014; 177(5–6): 223–40. http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p205-209 209 andriani, et al./dent. j. (majalah kedokteran gigi) 2018 december; 51(4): 205–209 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v51.i4.p205–209 5. acharya s, lohe vk, bhowate rr. diagnosis and management of pseudomembranous candidiasis. j otolaryngol res. 2017; 8(3): 1–4. 6. mogensen th. pathogen recognition and inflammatory signaling in innate immune defenses. clin microbiol rev. 2009; 22(2): 240–73. 7. netea mg, gow nar, munro ca, bates s, collins c, ferwerda g, hobson rp, bertram g, hughes hb, jansen t, jacobs l, buurman et, gijzen k, williams dl, torensma r, mckinnon a, maccallum dm, odds fc, va n der meer j w m, brown a j p, kullberg bj. immune sensing of candida albicans requires cooperative recognition of mannans and glucans by lectin and toll-like receptors. j clin invest. 2006; 116(6): 1642–50. 8. moyes dl, naglik jr. mucosal immunity and candida albicans infection. clin dev immunol. 2011; 2011: 1–9. 9. gow nar, netea mg. medical mycology and fungal immunology: new research perspectives addressing a major world health challenge. philos trans r soc lond b biol sci. 2016; 371: 1–10. 10. lyu x, zhao c, hua h, yan z. efficacy of nystatin for the treatment of oral candidiasis: a systematic review and meta-analysis. drug des devel ther. 2016; 10: 1161–71. 11. ganesh s, vennila jj. phytochemical analysis of acanthus ilicifolius and avicennia officinalis by gc-ms. res j phytochem. 2011; 5: 60–5. 12. singh d, aeri v. phytochemical and pharmacological potential of acanthus ilicifolius. j pharm bioallied sci. 2013; 5: 17–20. 13. firdaus m, prihanto aa, nurdiani r. antioxidant and cytotoxic activity of acanthus ilicifolius flower. asian pac j trop biomed. 2013; 3: 17–21. 14. mani senthil kumar kt, gorain b, roy dk, zothanpuia, samanta sk, pal m, biswas p, roy a, adhikari d, karmakar s, sen t. antiinflammatory activity of acanthus ilicifolius. j ethnopharmacol. 2008; 120: 7–12. 15. almagrami aa, alshawsh ma, saif-ali r, shwter a, salem sd, abdulla ma. evaluation of chemopreventive effects of acanthus ilicifolius against azoxymethane-induced aberrant crypt foci in the rat colon. mccormick dl, editor. plos one. 2014; 9(5): 1–12. 16. govindasamy c, arulpriya m. antimicrobial activity of acanthus ilicifolius: skin infection pathogens. asian pacific j trop dis. 2013; 3(3): 180–3. 17. prabowo a, teguh pb, andriani d. perbedaan efektivitas ekstrak daun mangrove acanthus ilicifolius dengan sodium bikarbonat 5% terhadap penurunan jumlah koloni candida albicans pada perendaman nilon termoplastik. dent j kedokt gigi. 2015; 9(2): 198–208. 18. andriani d, revianti s. il-17 expression in oral-candidiasisimmunosuppressed-models treated with acanthus ilicifolius extracts. in: 2017 south east asian division meeting. taipei, taiwan: international association for dental research; 2017. p. 10–3. 19. subha ts, gnanamani a. candida biofilm per fusion using active fractions of acorus calamus. j anim plant sci. 2009; 4(2): 363– 71. 20. thirunavukkarasu p, ramanathan t, ramkumar l. hemolytic and anti microbial effect in the leaves of acanthus ilicifolius. j pharmacol toxicol. 2011; 6(2): 196–200. 21. sofia s, merlee teresa m v. isolation of bioactive compounds ompounds by gc-ms and biological iological potentials of acanthus ilicifolius, l. int res j biol sci. 2017; 6(6): 7–19. 22. furtado najc, pirson l, edelberg h, miranda lm, loira-pastoriza c, preat v, larondelle y, andré cm. pentacyclic triterpene bioavailability: an overview of in vitro and in vivo studies. molecules. 2017; 22(3): 1–24. 23. matsumura n, takeyama y, ueda t, yasuda t, shinzeki m, sawa h, nakajima t, kuroda y. decreased expression of toll-like receptor 2 and 4 on macrophages in experimental severe acute pancreatitis. kobe j med sci. 2007; 53(5): 219–27. 24. villamón e, gozalbo d, roig p, o’connor je, fradelizi d, gil ml. toll-like receptor-2 is essential in murine defenses against candida albicans infections. microbes infect. 2004; 6: 1–7. 25. blasi e, mucci a, neglia r, pezzini f, colombari b, radzioch d, cossarizza a, lugli e, volpini g, giudice g del, peppoloni s. biological importance of the two toll-like receptors, tlr2 and tlr4, in macrophage response to infection with candida albicans. fems immunol med microbiol. 2005; 44: 69–79. 26. tessarolli v, gasparoto th, lima hr, figueira ea, garlet tp, torres sa, garlet gp, da silva js, campanelli ap. absence of tlr2 influences survival of neutrophils after infection with candida albicans. med mycol. 2010; 48: 129–40. 27. jouault t, el abed-el behi m, martínez-esparza m, breuilh l, trinel p-a, chamaillard m, trottein f, poulain d. specific recognition of candida albicans by macrophages requires galectin-3 to discriminate saccharomyces cerevisiae and needs association with tlr2 for signaling. j immunol. 2006; 177(7): 4679–87. 28. netea mg, brown gd, kullberg bj, gow nar. an integrated model of the recognition of candida albicans by the innate immune system. nat rev microbiol. 2008; 6: 67–78. 29. netea mg, sutmuller r, hermann c, van der graaf caa, van der meer jwm, van krieken jh, hartung t, adema g, kullberg bj. toll-like receptor 2 suppresses immunity against candida albicans through induction of il-10 and regulatory t cells. j immunol. 2004; 172(6): 3712–8. 30. franco lh, fleuri aka, pellison nc, quirino gfs, horta c v., de carvalho rvh, oliveira sc, zamboni ds. autophagy downstream of endosomal toll-like receptor signaling in macrophages is a key mechanism for resistance to leishmania major infection. j biol chem. 2017; 292(32): 13087–96. http://e-journal.unair.ac.id/index.php/mkg http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v51.i4.p205-209 mkg vol 39 no 1 jan 2006 isi.pmd 35 hybrid layer difference between sixth and seventh generation bonding agent grace syavira suryabrata department of conservative dentistry faculty of dentistry prof. dr. moestopo (b) university jakarta selatan indonesia abstract since etching is completed at the same stage as priming and bonding, when applying the sixth and seventh generation bonding, the exposed smear layers are constantly surrounded by primer and bonding and cannot collapse. the smear layer and the depth of penetration of resin bonding in dentinal tubules are completely integrated into hybrid layer. the purpose of this laboratory research was to study the penetration depth of two self etching adhesive. fourteen samples of human extracted teeth were divided into two groups. each groups consisted of seven samples, each of them was treated with sixth generation bonding agent and the other was treated with seventh generation bonding agent. the results disclosed that the penetration into dentinal tubules of seventh generation bonding agent was deeper than sixth generation bonding agent. conclusion: bond strength will improve due to the increasing of penetration depth of resin bonding in dentinal tubules. key words: bonding agent, penetration depth correspondence: grace syavira suryabrata, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas prof. dr. moestopo (b). jln. bintaro permai raya no. 3 jakarta selatan, indonesia. introduction dental adhesive system is used to bind bonding resin to email and dentin. bonding resin substance has developed through generations with changes in chemistries mechanism, packaging/number of bottles, application technique, clinical effectivenes and also in sensitivity technique.1,2 bonding resin is used to cover dentin so that pulp is protected from the movement of oral fluid and contaminating agent. functionally, bonding resin is regarded as an artificial email.3 several factors influence bonding, among other dentin demineralization, temperature, collagen, dentinal pain, biocompatibility of bonding agent, penetration ability in smear layer to form hybrid layer. mineralized dentin causing monomer’s inability to diffuse to substrate, therefore dentinal surface has to be etched first.3 etching’s function is to form surface texture, so that it can strengthened the bond between restoration agent and bonding resin.4 excessive etching on dentin caused deep demineralization which hampering resin can not completely enter into the collagen fibers at the bottom of demineralized dentin.5 phosphoric acid that remove the smear layer on total etching will cause the collapse of demineralized intertubules dentin matrix if it is dried with air,3 whether excessively on sufficiently.6 bonding to dentin with phosphoric acid etching will dramatically increase dentinal permeability causing the raise of flow-out fluid to dentinal surface. fluid is influential to adhesion because resin has a hydrophobic nature, not adhesive to hydrophilic substrate even after resin tag has been formed inside dentinal tubules.6 bonding agent composition has been developed to form dentinal hybrid and resin tag hybrid.3 bonding to dentin occurs mechanically or chemically.5 several researchers stated that chemical bonding gives little influence on bonding dentin resin. it is because bonding dentin resin is generated from a micromechanical reaction result of polymer chain molecules with collagen fibers, called hybrid layer or hybrid zone.3 similar process happened on demineralized dentin, there was no possibility of a chemical reaction between bonding and hydroxi-apatite because there was no calcium or phosphate, it consisted only of collagen fibers and water.6 effort to increase the strength of adhesion is indicated by remove the smear layer with acid, it actually occurs with demineralizing dentin surface under the smear layer.7 nowadays, bonding system without omitting the smear layer but dissolving, is called self-etching adhesives, so that it will make simpler clinical application procedure. bonding’s work method in dentin surface demineralizing process happened at the same time with the penetration of bonding resin according to demineralization depth.8 this method happens at the sixth and seventh bonding generations. hybrid layer thickness is formed between 2–3 μm. smear layer occurred during email or dentin preparation with the thickness according to the roughness of the bur used.7 on hybrid layer with network collagen fiber characteristic, it will form interfiber space almost like the shag carpet on dentin surface, appeared in tubule walls and hybridation of lateral tubules.8 36 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 35–38 the sixth generation bonding resin with one stage mechanism composed of two components. etching depth and bonding resin penetration depth is identical, forming chemical and mechanical bond. mechanical bond occurs between bonding resin and dentin. chemically bonding occurs between calcium hydroxide – apatite and matrix because of phosphoric ester with low ph as dentin solvent and make an adhesion to dentin. a more simple packaging i.e. two components in one blister is often presenting problem by imperfect mixing causing decreased adhesion strength. due to the simple application, dentists give less attention to the working manual from the manufacturer. the strength of adhesion will be doubled if applied twice in 15 seconds.5 seventh generation bonding, with one stage mechanism consisted of one component. the process of sixth and seventh bonding generation is similar but the seventh generation has one component, thus avoiding any mistake in mixing. the strength of adhesion to dentin is very good, and three times application subsequently in 30 seconds has enabled deeper resin penetration into dentinal tubules.9 bonding resin bond to dentin depends on the penetration of bonding resin into the smear layer and on the depth of bonding resin penetration into the dentinal tubules. the deeper the penetration of bonding resin into dentinal tubules, the better bonding resin retention to dentin will be. the purpose of this research was to identify the different of penetration ability from the of sixth and seventh bonding generations into dentinal tubules. the benefit of this study is to fully understand the penetration ability of sixth and seventh generation bonding resin into dentinal tubules. materials and methods materials used in this research was 14 caries-free and post-extraction of left or right upper jaw first premolar teeth, , two bonding agent types: sixth generation (adper prompt l-pop made by 3m espe usa) and seventh generation (ibond made by heraeus kulzer gmbh and co kg germany), and acrylic self curing platinum. the tools used in this study was metal diamond disc typed 7016 (carlo italy), scanning electron microscope (sem) made in uk, platinum coater tool (polaron sputter coater), rough polisher machine, smooth polisher machine (buehler polisher ecomet iii grinder), light curing instrument (litex usa), bonding brusher, micrometer, vacuum set, and dental pinset. extracted teeth were rinsed with water, dried with gauzed cloth, occlusal surface was cleaned with metal diamond disc around 3mm from central fissure. the surface was cut, dampened with flowing water and blowed gently with low pressure air (less than 2 atm) until the dental surface was wet and shiny. afterwards, bonding agent was applicated with new brush for every sample following manual instruction of each substance. seventh generation bonding agent needed 20 seconds light curing polimerization and sixth generation needed 10 seconds. then, the tooth was cut at bucco palatal side, and cut again to mesio distal direction. the tooth was rinsed with aquades and dried. two casts, 1 inch diameter was pasted at its bottom with adhesive laque band, until closely tied. the tooth was removed with pinset and the exposed surface was pasted to the base of the dental cast. acrylic self curing was inserted to the dental cast until the tooth was soaked with 9 mm thickness. moreover, a small label was drowned inside liquefied resin to unite with resin and to be read easily from sample side. after 1 hour, resin hardened and dental cast was opened. acrylic was then polished until it reached 7 mm thickness, the exposed surface was again polished until smooth using alumina and nylon cloth. the sample was coated with platinum using platinum coater and in the end was vacuumed (figure 1). the samples were observed with scanning electron microscope (sem), with 5.90 kilo electron volt resolution. after hybrid layer and the length of bonding resin penetration into dentinal tubules were seen, it was photographed, the result was printed, magnified by 3000 times. the length of bonding resin tag to dentinal tubules was measured with micrometer directly from sem monitor screen. sem examination was intended to observed the penetration depth of bonding resin agent into dentinal tubules from dentinal surface. results the measurement of bonding resin penetration depth in dentinal tubules on 2 treatment groups has shown in table 1. figure 1. dental samples implanted in acrylic, coated with platinum and vacuumed. 37suryabrata: hybrid layer difference table 1. average value and standard deviation of bonding resin penetration into dentinal tubules (μm) group sample number mean standard deviation 1 2 7 7 12.451 9.471 2.51 2.07 note: group 1: applicated with seventh bonding generation group 2: applicated with sixth bonding generation to identify the different result of treated groups, statistical independent t test (p = 0.05) was utilized. the result showed that t test observed probability was 0.032 meaning there was a significant difference between 2 groups, first group with sixth generation bonding resin (adper prompt l-pop) and second group with seventh generation bonding resin (ibond). sem examination demonstrated the depth of seventh generation bonding resin agent was longer than the sixth generation (figure 2 and 3). discussion the adhesive mechanism of both bonding agent groups was to maintain the smear layer, dissolved and exploited as hybrid layer formed between dentin and resin; not resin and neither dentin, but hybrid from both substances. bonding will cover collagen fibers and move on penetrating into dentinal tubules to form resin tag.1 the different packaging and application technique of both groups i.e.: seventh generation bonding was packed in one bottle without premixed while sixth generation bonding was packed in two components subsequently needed premixing. seventh generation bonding was apply to dentin for three times in 30 second, whereas the sixth generation bonding was treated once in 15 seconds.10,11 the benefit of self-etching adhesive which was a combination of etching, primer and adhesive in one stage, was in lessening work time and mistakes of any work stage.12 the bond of two bonding groups occurred through monomer as the result of released protons by phosphoric acid 4 meta, estherized by hema. at the time of etching process, methacrylate polymerized with ca++, ph inclined slowly, causing decreased acidity and etching process will stop.11 the depth of etching was similar with resin penetration depth.10 the length of bonding resin penetration into dentinal tubules was influenced by the length of etching acid reaction from acid ph until it became normal ph inside dentinal tubules. the longer the reaction time, the deeper the bonding resin penetration into dentinal tubules. the deeper the penetration, the better bonding resin retention to dentin. seventh bonding generation needed 30 seconds to etching acid reaction as mentioned in the manufacturer’s manual, while the sixth generation needed only 15 seconds. the result of independent t test was a significant difference of bonding resin penetration depth between seventh and sixth generation (p < 0.05). the average value of the first group bonding penetration depth (seventh generation) was 12.451 μm and the second group (sixth generation) was 9.471 μm. therefore, based on the average penetration depth value, seventh bonding generation was deeper than sixth generation, or tag resin from seventh generation was longer than the sixth generation. it was illustrated as follows (figure 4). tag resin length dentinal tubules hybrid layer hybrid layer figure 3. seventh generation bonding penetration length (sem picture). figure 2. sixth generation bonding penetration length (sem picture). figure 4. hybrid layer of dentinal surface with bonding-dentin resin inter surface in the form of hybridizing plaited collagen fibers and hybridizing in dentinal tubules. 38 maj. ked. gigi. (dent. j.), vol. 39. no. 1 january–march 2006: 35–38 from independent t test, the average length of tag resin of the second group was longer than the first group. the longer tag resin of hybrid layer will produce better hybrid layer retention. it can be concluded that the seventh generation bonding resin hybrid layer has better retention into dentin compared to the sixth generation bonding resin. references 1. dunn jr. ibond: the seventh – generation, one – bottle dental bonding agent. j com 2003 feb; 24(2):14–18. 2. bowen rl, harjenhoff wa. development of an adhesive bonding system. operative dentistry 1992; 5(supplement):75–80. 3. nakabayashi n, pasley dh. hibridization of dental hard tissues. tokyo, chicago, berlin, london, paris, barcelona, sao paulo, moscow, prague and warsaw: quintessence publ co ltd 1998; p. 14–17, 37, 92–93. 4. allbeury j. new development prompt a rethink on self etching adhesives. special report dental practice march–april 2004; 114–9. 5. baier re. principles of adhesion. operative dentistry 1992; 5(supplement):1–9. 6. roberson tm, heymann ho, swift ej. sturdevant’s art and science of operative dentistry. 4th ed. st louis, london, philadelphia, sydney, toronto: cv mosby co; 2002. p. 177–85. 7. oliviera ssa, pugach mk, hilton jf, watanabe lg, marshall sj, marshall jr gw. the influence of the dentin smear layer on adhesion: self-etching primer vs total etch system. dental materials 2003; 19(8):758–67. 8. summits jb. fundamental of operative dentistry. 2nd ed. chicago, berlin, tokyo, paris, london, sao paulo, barcelona, moscow, prague and warsaw: quintessence pub co inc; 2001. p. 196–8, 208–11. 9. pitel m. bonding general conditions. quintessence publ co inc. office online publ. ed. j adhes dent 2003; 20(8):1–22. 10. ibond info. available at: www.ibond-info.com. accessed november 28, 2004. 11. technical product profile. adper prompt l-pop and adper prompt. special report 3 m espe. 2004; 1–21. 12. tay fr, pashley dh. aggressiveness of contemporary self etching systems. i: depth of penetration beyond dentin smear layers. j den mat 2001 august; 17:296–308. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice dfdbbx guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak,1 matsuo hamada,2 ninuk hartati,3 and harold whitfield4 1 department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia 2 department of prosthodontics school of dentistry hiroshima university japan 3 department of dental public health faculty of dentistry airlangga university surabaya indonesia 4 department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  keywords contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dental medicine, universitas airlangga editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (+6231) 5039478 e-mail: dental_journal@yahoo.com; website: www.e-journal.unair.ac.id/index.php/mkg i wish to subscribe dental journal (majalah kedokteran gigi) saya ingin berlangganan dental journal (majalah kedokteran gigi) name/nama: .......................................................................... date of birth/tanggal lahir: .................................................... job title/pekerjaan: ................................................................ institution/institusi: .................................................................. address/alamat surat: ............................................................ ................................................................................................. ................................................................................................. country/negara: ................................................ telp.: ................................................................. fax.: .................................................................. e-mail: ............................................................... date/tanggal: ......................................................................... signature/tanda tangan: ....................................................... for costumers only untuk pelanggan mail to: dental journal (majalah kedokteran gigi) jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132 or fax to: (+6231) 5039478 change of mailling address perubahan alamat surat name/nama: ........................................................................................... address/alamat: ...................................................................................... .................................................................................................................. country/negara: ...................................................................... telp.: ....................................................................................... fax.: ........................................................................................ e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month 1 year surabaya q rp 200.000,00 q rp 400.000,00 java island (pulau jawa) q rp 250.000,00 q rp 500.000,00 outside java island (luar pulau jawa) q rp 300.000,00 q rp 600.000,00 other countries (negara lain) q us $ 30 q us $ 60 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 142-00-1495197-3 name of bank : bank mandiri name of beneficiary : ketut suardita " guide for authors dental journal (majalah kedokteran gigi) only publishes original articles on all aspects of dentistry and dental related disciplines. articles are considered for publication with the condition that they have not been published or submitted for publication elsewhere. articles can be classified as research reports, case reports and literature reviews that keep the readers informed of current issues, innovative cases and reviews in dentistry. they should also support scientific advancement, education and dental practice development. manuscripts will be published in english therefore it is the author's responsibility to ensure the submitted manuscript to be provided in appropriate english. the language used in manuscript must be non numeral, and free of mistypes. the length of manuscript must be proportional. the manuscript must be submitted in soft copy file via cd or e-mail. manuscript should be typed using ms word program. the font used should be times new roman, sizing 14 pt for the title, and 12 pt for others. headlines are typed in bold, while latin names are typed in italics. three legible copies of the manuscript which are typed in double space with wide margins on good quality a4 white paper (210 × 297 mm) should also be enclosed. the length of article should not below 10 pages and should not exceed 12 pages. the left, right, top, and bottom margin should be 2.5 cm or 1 inch length. authors should also follow the manuscript preparation guidelines. all research reports, case reports, and literature reviews must contain:  title should be brief, specific and informative. the title must contain maximum 10 words (not exceeding 40 letters and spaces) with capital letter on the first word of the title. the title must be provided in english and bahasa indonesia.  name of author(s) should include full names of author(s), address to which proofs are to be sent, name and address of the department(s) to which the work should be attributed and appointed accordingly with asterisk (*) symbol. example: jamal bin razak*, matsuo hamada**, ninuk hartati***, and harold whitfield**** * department of oral and maxillofacial surgery faculty of dentistry university of malaya malaysia ** department of prosthodontics school of dentistry hiroshima university japan *** department of dental public health faculty of dentistry airlangga university surabaya indonesia **** department of endodontics school of dental and health sciences the university of melbourne australia  abstract should be structured with concise description (contains not more than 250 words, formatted in 1 space, and done in one paragraph). the abstract must be provided in english and bahasa indonesia.  abstract in research reports should consists of "background:", "purpose:", "method:", "result:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in case reports should consists of "background:", "purpose:", "case(s):", "case management:" and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  abstract in literature reviews should consists of "background:", "purpose:", "reviews:", and "conclusion:" typed in bold within one paragraph. footnotes, references, and abbreviations are not used in the abstract.  key words contain 3-5 words and/or phrases and must be provided below the abstract. the key words must be provided in english and bahasa indonesia.  correspondence should contain separated by semicolons (;) details of the author in charge with detailed mailing address and e-mail. correspondence is followed by the following sections according to type of article (research reports, case reports, or literature reviews) as follows: i. contents in research reports: the research reports should contain the following sections: introduction, materials and methods, and results.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and exposures to be discussed.  materials and methods contain clear description on used materials and scheme of experiments as well as methods in order to enable other examiners to undertake retrial or duplication and validity checked if necessary. reference should be given to the unknown method. research ethics on animal and human subjects must be stated accordingly, if applicable.  results should be presented accurately and concisely in logical sequence with the minimum number of tables and illustrations necessary for summarizing only important observations. tables must be made in horizontal (without vertical line separation) for simple viewing. mathematical equations should be clearly stated. when mathematical symbols are not available on the typewriter, hand written symbols with soft lead pencil could be used. decimal numbers should be separated by point (.). tables, illustration, and photographs should be cited in the text in consecutive order and provided separately from the texts of manuscript. the titles and detailed explanations of the figures belong in the legends for illustrations (figures, graphs) not on the illustrations themselves. all non-standard abbreviations that are used must be explained in footnotes. ii. contents in case reports: the case reports should contain the following sections: introduction, case(s), and case management.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future.  case(s) contain clear and detailed description on presented case(s) including anamnesis and clinical examinations.  case(s) management should be presented accurately and concisely in chronological sequence supported with figures and detailed descriptions on what was done. iii. contents in literature reviews literature reviews are reviews provided by expert in its field with verified supporting credentials. the literature reviews should contain the following sections: introduction, and overviews.  introduction comprises the problem's background, its formulation and purpose of the work or case or review and prospect for the future. introduction in literature reviews are followed by headline topics and overviews to be discussed. all research reports, case reports, and literature reviews must be followed by:  discussion explains the meaning of the examination's results, not repeating the result, in what way the reported result can solve the problems, differences and equalities with previous study and development possibilities. this section should include the conclusion of the reported work or case and suggestion for further studies if necessary.  acknowledgements to all research contributors, if any, should be stated in brief at the manuscript, prior to references.  references should be arranged according to the vancouver system. references must be numbered consecutively in the order in which they are first mentioned in the text, and listed at the end of the text in numeric, not alphabetical order. identify references in text, tables, and legends by arabic numerals in superscript. references must be valid and taken within the last 10 years of publication, containing at least 70% of primary references (from journals, thesis, dissertations, and patent documents). unpublished sources, such as manuscripts in preparation and personal communications are not acceptable as references. only sources cited in the text should appear in the reference list. list all authors when four or fewer authors are involved; when there are more than four authors, list the first three authors and add "et al.". the name of authors must be written in consistency. the number and volume of journals must be included. edition, publisher, city, and page numbers of textbooks must be included. internet downloaded references must include the time of access and web address. any abbreviation of journals must comply with dental index and medic index. citation format for journal articles: 1. donnelly pv, miller c, ciardullo t, occlusion and its role in esthetics. j esthetic dentistry. 1996; 8: 111–8. 2. grimes ew. a use of freeze-dried bone in endodontics. j endod. 1994; 20: 355–6. 3. bilhaut. guerison d'un pounce par un noueau procede o p e r a t o i r e . c o n g r e s f r a n c a i s d e c h i r u r g i e 1 9 9 8 ; 4: 576–580. citation format for textbooks: 1. hickey jc, zarb ga. boucher's prosthodontic treatment for edentulous patients. 9th ed. st. louis: mosby; 1985. p. 452–9. 2. cohen s, burns rc. pathways of the pulp. 5th ed. st louis: mosby co; 1994. p. 123–47. citation format for proceedings: 1. perry ch, lu f, namavar f, kalkhoran nm, soref ra. radical styloid. proceedings of the 10 th international congress of clinic; new york, usa. amsterdam: elsevier; 1991. 2. favier jj, camel d. enforcement of data in medical information. in: lun kl, editor. proceedings of the eight international conference on medicine; york, uk, 1986 sept 8–10. citation format for thesis and dissertations: 1. ramos r. preventive health amendments. phd thesis. college van dekanen. university of twente. the netherland, 1992. 2. amerongen avn, michels lfe, roukema pa, veerman eci. 1986. ludah dan kelenjar ludah arti bagi kesehatan gigi. rafiah arbyono dan sutatmi suryo. yogyakarta: gadjah mada university press; 1992. p. 1–42. 3. salim s. pengaruh humiditas dan waktu penyimpanan serta cara curing terhadap sifat fisik, kimia dan mekanik akrilik basis gigi tiruan. disertasi. surabaya: pascasarjana universitas airlangga; 1995. p. 8–21. citation format for patents: 1. yamagishi h, hiroe a, nishio h, miki k, tawada y. methods procedures of hand surgery. us patent no. 5264710, 1993 nov 23. all figures, illustrations and photos must be relevant, informative, concise, and referred if any, provided in file (jpg, tiff, etc.). non-file photos should be printed in clear glossy paper. the size should meet with the minimum measurement of 125 × 195 mm. the maximum number of figures, illustrations, photos, and tables in research report and literature review is 4 (four). the maximum number of figures, illustrations, photos, and tables in case report is 8 (eight). all figures, illustrations and photos must be separated from the texts of manuscript. written permission must be obtained for material that has been published in copyrighted material; this includes tables, figures, and quoted text that exceeds 150 words. signed patient release forms are required for photographs of identifiable persons. a copy of all permissions and patient release forms must accompany the manuscript. the editor reserves the right to edit the manuscript, fit articles into space availability, and ensure conciseness, clarity, and stylistic consistency. all accepted manuscripts and their accompanying illustrations will become the permanent property of the publisher, and may not be published elsewhere in full or in part, in print or electronically, without written permission from the publisher. all data, opinions or statements appeared on the manuscript are the sole responsibility of the author(s). accordingly, the publisher, the editorial board, and their respective employees of the dental journal will accept no responsibility or liability whatsoever for the consequences of any such inaccurate or misleading data, opinions, or statements. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... d e ta c h h e re ( p o to n g d i s in i) subscription order form formulir berlangganan dental journal majalah kedokteran gigi faculty of dentistry airlangga university editorial address c/o: jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia telp./fax.: (062-31) 5039478 e-mail: 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....................................................................................... fax.: ........................................................................................ e-mail: ..................................................................................... date/tanggal: .......................................................................................... signature/tanda tangan: ........................................................................ international subscription – include shipping [please tick (ü)] country issue* 6 month � year 2 years surabaya q rp80.000,00 q rp160.000,00 q rp320.000,00 java island (pulau jawa) q rp90.000,00 q rp180.000,00 q rp360.000,00 outside java island (luar pulau jawa) q rp100.000,00 q rp200.000,00 q rp400.000,00 other countries (negara lain) q us $ 27 q us $ 54 q us $ 108 * quarterly publication (terbit 4 kali setahun) i am paying this magazine by: [please tick (ü)] saya membayar majalah ini dengan: [beri tanda (ü] q bank draft/cheque q money-order/wesel q transfer to: q others/lainnya (please specify/sebutkan): ....................... ........................................................................................... acount no : 033-01-11343-16-0 name of bank : bank niaga cabang dharmahusada name of beneficiary : drg. sianiwati goenharto " 139139 case report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 139–143 autogenous tooth transplantation: an alternative to replace extracted tooth david b. kamadjaja department of oral and maxillofacial surgery faculty of dental medicine, universitas airlangga surabaya indonesia abstract background: the gold standard treatment to replace missing tooth is dental implants, however, in certain cases, such as in young patients its placement is contraindicated. autogenous tooth transplantation, which has been widely done in scandinavian countries for many years, may become a good alternative to overcome this problem. purpose: this article attempted to provide information about the indication, treatment planning, surgical technique and the successful result of autogenous tooth transplantation. case: a fifteen year old male patient presented with large caries and periapical disease of his lower left first molar, which was partially erupted and the roots was not fully formed in radiograph. case management: autogenous tooth transplantation procedure was performed consisting of extraction of #36, odontectomy of #38 followed by its implantation to socket #36 and fixation of the transplanted tooth to the adjacent teeth. post operative evaluation was done on regular basis within 18 months period. there was no complaint, the tooth was clinically stable and no evidence of periodontal problem. serial radiographs showed healing of alveolar bone and periodontal tissue, and the complete root formation was evident by 18 months post operatively. conclusion: autogenous tooth transplantation is a potential alternative to replace extracted tooth. provided that the case be properly planned and operation carefully performed, successful result of this treatment can be achieved. keywords: autogenous tooth transplantation; extracted tooth; incompletely formed root correspondence: david b. kamadjaja, c/o: departemen ilmu bedah mulut dan maksilofasial, fakultas kedokteran gigi universitas airlangga. jl. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: davidbk65@gmail.com introduction replacement of lost or extracted tooth can be done by several ways, either with removable denture or fixed prosthesis such as bridge work and dental implant-supported crown. the fast development in osseointegrated dental implant system has made it the most desirable treatment option in replacing lost teeth because of its predictable and long-term results. however, dental implants should not be used in young patients whose alveolar bone are still actively growing because it may cause infraocclusion, poor esthetic result and interdental gaps with the adjacent teeth later in life.1 another alternative implant treatment is, therefore, required to be used in patients with growing alveolar bone. one of the treatment options suitable for such individuals is autogenous tooth transplantation autogenous tooth transplantation is transplantation of buried, partially erupted or fully erupted tooth from one location to another in the same individual.2 donor teeth are transplanted from donor to recipient sites which can be post extraction sockets or surgically prepared sites. tooth transplantation is frequently indicated for children or adolesence to replace fractured or missing incisives due to trauma or defective molars due to large caries.3 autogenous tooth transplantation consists of few steps, i.e. minimally traumatic tooth extraction, surgically removed donor tooth followed by its transplantation into the recipient socket. this requires that strict case selection, type of donor and recipient sites, and rigorous surgical procedure be met to achieve successful result.4 the objective of this article is to provide information regarding the potential succesful treatment with autogenous tooth transplantation dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p139-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p139-143 140 kamadjaja/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 139–143 if it is properly planned and carefully performed. this article presented successful treatment using autogenous tooth autotransplantation to replace extracted tooth in adolescence. case a fifteen year old male patient came to the author’s private dental clinic with chief complaint of his lower left molar having large cavity since one year ago causing discomfort during eating. there was no history of dental treatment. intra oral examination showed that #36 presented with large occlusal caries with furcation perforation which was found to be non-vital. there was no sign of acute periodontal or periapical infection. tooth #37 was partially erupted while #38 was not clinically seen (figure 1). panoramic x-ray showed radiolucency of alveolar bone in bifurcation and periapical region of #36. tooth #38 was partially erupted and impacted mesioangularly to #37 as well as the overlying bone, while the roots were seen to be incompletely formed (figure 2). the parents of the patient were explained of the poor condition of tooth #36 and the poor prognosis of root canal treatment and restoration of the tooth, therefore tooth extraction would be the treatment of choice. they were also informed that tooth #38 was impacted and nonfunctional which most likely needed surgical removal in the future to avoid damage to tooth #37 structure. they were offered treatment with autogenoous tooth transplantation, i.e. extraction of #36, surgical removal of impacted #38 followed by transplantation to #36 socket. realizing the advantages of this treatment the parents signed the informed consent. case management clinical and radiographic evaluation were done prior to the surgery to measure the crown dimension of donor tooth as well as the recipient site. the donor tooth crown width was 1.5 mm lower than the width of the recipient site which was the space between distal wall of tooth #35 and mesial wall of #37. the result suggested that tooth #38 was a good candidate for donor tooth. the procedure of tooth transplantation was done under local anesthesia. after desinfection with povidone iodine solution and mandibular block anesthesia with 2% lidocaine tooth #36 was extracted. the extraction was done with split technique to minimize trauma to the adjacent bone and gingiva. upon complete delivery of the two segments, thorough curettage was done to remove granulation tissue in the socket, which then irigated with 0.9% saline solution and covered with sterile moist gauze. the next step was odontectomy of tooth #38. after trapezoidal incision and flap reflection were made judicious amount of bone surrounding the crown of #38 was removed using round figure 1. intra oral situation preoperatively. tooth #36 presented with large caries with darkening of the crown, #37 was cllinically partially erupted while #38 was totally unerupted. figure 2. panoramic x-ray pre-operatively. tooth #36 was seen to have deep caries and radioluscency at furcation and periapical area, tooth #38 was partially erupted and impacted mesioangularly against #37 with the roots being incompletely formed. figure 3. transplantation of donor tooth in the recipient socket. after transplantation the interdental papillas at #36 region and the odontectomy flap were sutured with 3-0 black silk suture, followed by fixation of the donor tooth to the adjacent teeth. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p139-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p139-143 141141kamadjaja/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 139–143 bur down to the level of cemento-enamel junction. a great care was taken during bone removal with drilling so that it did not cause any injury to the surface of the root’s cementum. the dental follicle was carefully detached from the adjacent fibrous tissue using amalgam carver. the tooth was then delivered out from its socket with bein elevator and wrapped in moist sterile gauze to keep it from unexpected drying. preparation of socket #36 was made by removing the blood clot and irigating with sterile saline solution. the donor tooth was then placed in the recipient socket by holding its crown with extraction forceps. there was not much of manipulation required to implant the donor tooth as the crown and root dimension of the donor tooth were smaller than the that of the recipient site. the tooth was placed slightly infraposition so that it did not contact with the opposing tooth and then the post-odontectomy flap and interdental papillas at the recipient site were sutured with black silk sutures (figure 3). the tooth was then fixed with glassionomer cement to the adjacent teeth and periapical x-ray taken immediately afterwards (figure 4). figure 4. periapical x-ray immediate after surgery. the mesiodistal dimension of donor crown which was smaller than the recipient site facilitated transplantation procedure and minimized manipulation of the donor tooth and recipient socket. it was fixed to the adajent teeth with glassionomer cement in an under-occlusion position. figure 5. three months post-operative review. the donor tooth was in good alignment showing normal marginal gingiva without any sign of inflammation (left); the donor tooth was in contact with the opposing teeth indicating spontaneous vertical movement associated with clinical eruption (right). figure 6. post operative periapical x-ray. at 9 month review, post extraction socket had been replaced with normal trabecular bone, the roots of donor tooth was seen to have fully formed but the distal apical foramen was still wide indicating incomplete root formation, lamina dura was evident but its density still uneven (left); at 18 month review both roots had fully formed, lamina dura was clearly defined with even density indicating normal bone and periodontal structure (right). dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p139-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p139-143 142 kamadjaja/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 139–143 antibiotic, nsaid, analgesic and antiseptic mouth rinse were presecribed. the patient was instructed to take soft diet and avoid chewing with the left side for 2 weeks. post operative review was done on weekly basis for the first month followed by monthly for the next six months. the fixation cement and sutures were removed 2 weeks after surgery. there was no complaint, slight mobility was perceived with finger palpation and the pulp test indicated vital pulp although the vitality level was lower compared to that of the adjacent teeth. at one month review, there was no detected mobility and the pulp test value revealed compared to the previous one. at three months review, the tooth was stable, gingival margin was normal and no periodontal pocket detected (figure 5), the pulp test indicated higher vitality level compared to the one previously recorded. periapical radiograph taken at nine months check up showed root development in which the mesial apex seemed to form completely, while the distal apex has not fully formed. the alveolar bone surrounding the roots showed normal density and trabecular pattern, but lamina dura were not clearly defined. (figure 6). at 18 months post surgery, the clinical parameters were normal and the pulp test indicated that the pulp of the transplanted tooth was still vital and the recorded vitality value was similar to that of the adjacent teeth. radiographically, the trabecular bone was normal, both root apices had fully formed, and the lamina dura along the roots was clearly seen with even radiopacity (figure 6). discussion procedure of autogenous tooth transplantation had been applied for more than three decades especially in scandinavian countries during which time success rate was reported as low as 59% to 76% in 5-10 years of observation period.5 interestingly, the success rate was reported to increase dramatically in the past two decades. andreasen et al. reported 95% success rate over 13 years of study, lundberg dan isaksson achieved 94% in 5 years, kugelberg et al. reported 94% success rate during 4 years of observation, and cohen showed 98-99% success rate over 5 years.6 autogenous tooth transplantation was commonly used to replace permanent first molars as these are the first permanent teeth to erupt and most frequently damaged.2 autogenous tooth transplantation can also be considered as treatment of choice in such cases as agenesis of premolars or lateral incisive, tooth loss due to trauma, ectopic canine, root resorption, and root fractures. this procedure is indicated in young individuals whose alveolar bone are still undergoing growth and development which make fixed prosthesis such as dental implants unsuitable. this was best done to replace freshly extracted tooth because delay of the treatment might cause resorption of the alveolar process leading to decrease in the amount of alveolar bone available for the donor tooth. 7 in order to achieve best result of autogenous tooth transplantation case selection is very important. with this regards, one should consider that donor teeth do not have fully formed root, mesio-distal dimension of donor teeth should be at least the same or smaller than the extracted teeth, there should not be any acute periapical or periodontal infection of the recipient socket.7,8 it was suggested that if partially erupted wisdom teeth are to become donor, the best case would be when their occlusal surface were already at the level of cervical part of second molars so that risk of injury to the donor teeth can be minimized.4 in the author’s experience, transplantation of wisdom tooth germ was not advisable as preserving dental follicle and, especially, dental papilla were extremely difficult during delivery of the germ leading to failure of subsequent root formation. the patient in this case met the above criteria, as he was classified as adolescence, the tooth #38 used as the donor tooth showed incompletely formed root and its dimension was smaller than the extracted tooth and there was no active inflammation at the periapical of recipient site. besides, the occlusal surface of the donor tooth was above the level of cervical part of the adjacent tooth which made odontectomy procedure relatively uncomplicated. apart from good planning, meticulous surgical procedure should be done to achieve successful result comprising of atraumatic tooth extraction and donor tooth removal and judicious manipulation of the socket as well as the donor tooth. atraumatic extraction should be able to maintain as much bone and soft tissue integrity in the recipient socket as possible.3,4,7,9 atraumatic extraction in this case was performed by splitting the tooth longitudinally with bur followed by removal of the root segments one after the other preserving the interdental alveolar crest. the atraumatic donor tooth removal in this case was performed by removing adequate amount of the overlying bone under copious saline irigation to preserve the periodontal ligament and dental follicle of the donor tooth as much as possible during delivery. preservation of viable periodontal ligament cells was also applied by always holding the tooth on its crown surface and had it wrapped in moist gauze at all times during socket manipulation procedure and by keeping the extra oral time at minimum time as suggested by several authors.3,7 the success criteria for autogenous tooth transplantation are healing of the periodontal ligament (pdl), healing of gingival tissue and alveolar bone, healing of the pulp and continuation of root development.1 all the abovementioned parameters for successful result were foubnd in this case evidenced by clinical and radiographic examination. the most important determinant of success in this procedure was the stage of root development and viability of periodontal ligament cells of donor tooth.2,10 root with open apex would facilitate revascularization to the pulp canal which was very critical to the viability of the pulp tissue. studies showed that following traumatic injuries to the pulp tissue, various growth factors incorporated in blood clot and dentin played important role in the cell proliferation inside dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p139-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p139-143 143143kamadjaja/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 139–143 the root canal space.11 with regard to the apical opening, revascularization seems to be more predictable when apical diameter is greater than 1.0 mm and is unlikely to occur in apical opning narrower than 0.3 mm.1,12 damage to pdl cells may cause bony ankylosis in which case external root resorption was inevitable leading to tooth loss.9,10 failure of autogenous tooth transplantation were usually caused by inflammatory root resorption and replacement root resorption or ankylosis. inflammatory resorption was resorption of the root dentin caused by injury to the innermost layer of the pdl and cementum eliciting a deep osteoclastic attack on the root surface exposing dentinal tubules. when the tubules communicate with the bacteria from the necrotic pulp, activation of the resorption process further continues.4 replacement resorption or ankylosis is the result of extensive injury to the innermost layer of pdl and cementum in which healing initiated from the adjacent bone hence forming bone ankylosis. being the integral part of the bone the tooth subsequently undergoes remodelling cycle leading to resorption of the tooth overtime. 4 inflammatory resorption usually commenced from four week after transplantation, while ankylosis between root cementum and the adjacent bone usually occured after 4 months after the procedure.5 these two types of root resorption were not found in the reported case until 18 months post operatively. this was most probably due to vitality of the pulp tissue and periodontal ligament cells of the transplanted tooth supported by the radiographic evidence of fully formed lamina dura. authors suggested that few parameters can be used to assess the health of transplanted tooth, i.e. free from complaint of pain, tooth mobility, gingivitis, periodontal pocket, and any form of root resorption.8 in the case presented here, result of the assessment of all parameters above and the normal pulp vitality indicated that the transplanted tooth was healthy, and therefore it was considered as successful treatment. the conclusion of this case report was that autogenous tooth transplantation was a potential method to replace an extracted tooth. the success of this treatment depended highly on good case selection and meticulous surgical procedure which should be done according to rigorous criteria. references 1. park jh, tai k, hayashi d. tooth autotransplantation a treatment option: a review. j clin pediatr dent 2011; 35(2): 129-36. 2. tirali re, sar c, ates u, kizilkaya m, cehreli sb. autotransplantation of a supernumerary tooth to replace a misaligned incisor with abnormal dimensions and morphology: 2-year follow-up. case reports in dentistry article id 146343, 2013. 5 pages http://dx.doi. org/10.1155/2013/146343. 3. miranda rb, de almeida erl, david lg, miranda tb. autogenous tooth transplantation: a case report. braz j dent traumatol 2009; 1(2): 45-9. 4. andreasen jo. atlas of replantation and transplantation of teeth. philadelphia: wb saunders co; 1992. p. 208-21 5. kvint s, lindsten r, magnusson a, nilsson p, bjerk lin k. autotransplantation of teeth in 215 patients. a follow-up study. angle orthodontist 2010; 80(3): 446-51. 6. nimcenko t, grazvydas o, varinauskas v, bramanti e, signorino f, cicciù m. tooth auto-transplantation as an alternative treatment option: a literature review. dent res j (isfahan) 2013; 10(1):1–6. 7. howlader m m r, beg um s, naula k ha d. autogeous toot h transplantation: a case report and review of literature. j bangl coll physc surg 2006; 24(2): 79-85. 8. andreasen jo, andersson l, andreasen fm. textbook and color atlas of traumatic injuries to the teeth. wiley-blackwell. 2007. p. 740-59. 9. clokie cml, yau dm, chano l. autogenous tooth transplantation: an alternative to dental implant placement?. j can dent assoc 2001; 76: 92-6. 10. sunil kc, mohan b, lakhsiminarayanan l. autogenous tooth transplantation–two clinical cases. endodontology 2001; 13: 51-6. 11. wang q, lin xi, lin zy, liu gx, shan xl. expression of vascular endothelial growth factor in dental pulp of immature and mature permanent teeth in human. shanghai kou qiang yi zue 2007; 16(3): 285-9. 12. andreasen jo. pulp and periodontal tissue repair–regeneration or tissue metaplasia after dental trauma. a review. dent traumatol 2012; 28: 19-24. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p139-143 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p139-143 56 combination of natural teeth and osseointegrated implants as prosthesis abutments in a posterior cantilever bridge michael josef kridanto kamadjaja department of prosthodontic faculty of dentistry, airlangga university surabaya indonesia abstract dental implants have been used for several decades. patients of all ages have chosen dental implants to replace a single tooth or several teeth or to support partial or full dentures. this paper reports two cases of patients treated with dental implant as alternative to replace the missing teeth and connected with natural tooth as abutments in a fixed restoration with distal cantilever bridge. the underlining reasons that we decided to make such kind fixed prostheses are because of clinically imposible to put the implant on certain area and the patients asked for prostheses as optimum as possible, so the mastication function could return to the homeostasis condition. the benefit of these treatments are that prostheses could be made as optimum as possible with a more economic price, so the patients feel quite satisfied. the result shows that a few years after the treatments finished there is no any disadvantageous effect of connecting teeth to implants as abutments in fixed partial dentures and there is no sign of a harmful effect to the opposing teeth either. key words: connecting implant to natural tooth, osseointegration correspondence: michael josef kridanto kamadjaja, c/o: departemen prostodonsia, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo no. 47 surabaya 60132, indonesia. e-mail: josef_310563@yahoo.com introduction a dental implant is an artificial tooth root that places into your jaw to hold a replacement tooth or bridge. dental implants are an ideal option for people in good general oral health who have lost a tooth or teeth due to periodontal disease, an injury, or some other reason. dental implants are so natural-looking and feeling, you may forget you ever lost a tooth dental implants are teeth that can look and feel just like your own! under proper conditions, such as good and diligent patient maintenance, implants can last a lifetime. long-term studies continue to show improving success rates for implants.1 implant treatment generally is a three-part process that takes several months. in the first step, the dentist surgically places the implant in the jaw, with the top of the implant slightly above the top of the bone. a screw is inserted into the implant to prevent gum tissue and other debris from entering. the gum then is secured over the implant. the implant will remain covered for approximately three to six months while it fuses with the bone, a process called “osseointegration”. there may be some swelling, tenderness or both for a few days after the surgery, so pain medication usually is prescribed to alleviate the discomfort. a diet of soft foods, cold foods and warm soup often is recommended during the healing process. in the second step, the implant is uncovered and the dentist attaches an extension, called a post, to the implant. the gum tissue is allowed to heal around the post. some implants require a second surgical procedure in which a post is attached to connect the replacement teeth. with other implants, the implant and post are a single unit placed in the mouth during the initial surgery. once healed, the implant and post can serve as the foundation for the new tooth. in the third and final step, the dentist makes a crown, which has a size, shape, colour and fit that will blend with your other teeth. once completed, the crown is attached to the implant post.2 endosteal implant is the most commonly used type of implant. the various types include screws, cylinders or blades surgically placed into the jawbone. each implant holds one or more prosthetic teeth. root form implants are the closest is shape and size to the natural tooth root. they are commonly used in wide, deep bone to provide a base for replacement of one, several or a complete arch of teeth. this type of implant is generally used as an alternative for patients with bridges or removable dentures.1,2 some cases were done by connecting an implant with a natural tooth as abutments in a fixed restoration.3,4,5 this opinion is still remain pro and contra between the dentists. some of them are agree of connecting implant to natural tooth, but the others are still disagree. the purpose of this case report is to show the connection of implant to natural tooth as abutments in a fixed restoration with posterior cantilever pontic for certain conditions. cases two cases of patients treated with dental implant as alternative to replace the missing teeth are reported. those 57kamadjaja: combination of natural teeth and osseointegrated implants two patients are a-48-year old female patient presented in case 1 and a-63-year old male patient presented in case 2. both patients asked for dental implant as the refused for removable prostheses after their teeth being extracted. in both cases the author used different type of implant abutment, 2-pieces implant abutment for case no 1 and 1-piece abutment for case no. 2. in case no 1 we used oraltronic implant and in case no. 2 we used q-implant (trinon). both of them were osseointegrated implant, where we waited four to six months before complete the procedure with the placement of a porcelain bridge. informed consent had been aproved by the patients before the treatments began. case management case 1: a-48-year old female patient came to the dental practice with chief complaints of pain in chewing at the left lower side. it had been starting since 3 days ago and she had not taken any medicine yet. clinical and panoramic examinations revealed that tooth 37 was broken and should be extracted (figure 1). the patient did not want use a removable prostheses and she asked for dental implant treatment and bridge denture rehabilitation after the tooth being extracted. porcelain bridge 35, 36, 37 was separated and tooth 37 was extracted, a deep curettage was done to clean the socket from cyst and the gums closed with several stiches. one week after that the stiches were opened and the patient didn’t feel anything. patient came back five months after the extraction and brought panoramic photo also. the region 37 did not look to have enough density (figure 2a & c), so we decided to put the implant fixture in region 36. after application of anesthetic, we did several of incisions and bone preparations before the implant being placed. a pitt –easy bio-oss implant from oraltronics with 3.75 mm diameter and 12 mm length was inserted in region 36 (figure 2b & d) and the gums are closed with several stitches. the patient was covered with antibiotic, anti inflammation and analgesic drug during the healing process.2 continuance of normal oral hygiene with chlorhexidine mouth rinses would help to prevent wound infection. up to three weeks showed no clinical symptoms and inflammatory process. tooth 34 that suffered from gangraen pulpae was treated with root canal treatment and strengthened with metal post in it. we ordered the patient to come back in the next 4 months. figure 1. panoramic radiography view: tooth 37 was broken. figure 2. a) intraoral condition in region 37 before implant placement; b) a pitt –easy bio-oss implant from oraltronics with 3.75 mm diameter and 12 mm length; c) region 37 does not look have enough density; d) implant fixture was inserted in region 36. a c b d 58 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 56-61 when the patient came back, we opened the cover screw and we changed with healing screw (figure 3a). a week after that we changed the healing screw with the abutment post and a porcelain cantilever bridge was done from teeth 34, 35, 36, 37 (figure 3b–f). panoramic radiograph six months after the treatment finished showed that there was nothing wrong with the treatment (figure 3g). up till now it has been 4 years after the treatment finished and there was nothing wrong with the treatment. figure 3. a) healing screw was attached to the implant fixture; b) healing screw was opened; c) abutment post was attached to the implant fixture; d) teeth 34, 35 and abutment post were prepared; e) their relation to the opposing teeth; f) cantilever porcelain bridge from 34, 35, 36, 37 was inserted; g) panoramic radiography view: cantilever porcelain bridge from 34, 35, 36, 37. case 2: a-66-year old male patient came to the dental practice with chief complaint of difficulty in chewing at the left side. clinical examination showed that teeth 24 till 27 had been extracted (figure 4a & b). the patient had a removable partial denture, but he did not feel comfortable with it, so he asked for a fix artificial dentures. he agreed for having dental implant treatment and bridge denture rehabilitation after application of anesthetic by using a tissue punch with 3 mm diameter we made a hole on the gum. pilot drill was used to make an optimal implant direction until 12 mm depth was reached and continued with shaping drill with selected diameter and length. the implant was gently taken from the sterile cover and inserted into the prepared bone cavern with the insertion wrench. it might be helpful to use the hand wheel or hand wrench. one q-implant from trinon a b c d e f g 59kamadjaja: combination of natural teeth and osseointegrated implants with 3.5 mm diameter, standard neck heights (4 mm) and 12 mm length were inserted in region 25 (figure 4c). the q-implant were constructed with a self-cutting thread and inserted by using minimal force could achieved a high primary stability.6 tooth 23 was prepared and temporary bridge from 23, 24, 25 was placed. the patient was covered with antibiotic, anti inflammation and analgesic drug during the healing process. clinical symptoms as pain, implants mobility and sign of inflammatory process were not presented. chlorhexidine mouth rinses was also prescribed for preventing wound infection and maintaining the normal oral hygiene.2 during the osseointegration period, the patient did not allow to use the left side of his teeth for chewing food. four months after the implant placement, cantilever porcelain bridge was made from teeth 23, 24, 25, 26 (figure 4d–f). we ordered the patient to come back figure 4. a) intraoral condition before implant placement; b) panoramic radiographic view : region 24, 25, 26, 27; c) q-implant from trinon with 3,5 mm diameter, standard neck heights (4 mm) and 12 mm length were inserted in region 25; d) cantilever porcelain bridge in region 23, 24, 25, 26 was inserted; e) interocclusal relation after the porcelain bridge was inserted; f) panoramic radiographic view: cantilever porcelain bridge from 23, 24, 25, 26. every six months for routine check up and up till now it has been two years after the treatment finished and there was nothing wrong with the treatment. discussion two cases of dental implant placement were reported. in case 1 we used two phase concept of implant placement which the abutment post was engaged by using screw a b c d e f 60 dent. j. (maj. ked. gigi), vol. 41. no. 2 april-june 2008: 56-61 type and in case 2 we used one phase concept of implant placement. in case 1, which we used two phase concept of implant placement, we needed more implant process than case 2, which used one phase concept of implant placement. both cases were delayed loading which waited 4 to 8 months before providing the tooth restoration. in case 1 the implant placement was done by making several incisions, but in case 2 we did it without making any incision (flapless). implant placement with flapless could reduced the bleeding of the blood and wound healing could be achieved in a short time, but if you did not sure about the bone condition you better did it with any flap, because we could do bone management better than flapless. dental implants are among the most successful procedures in dentistry. there is no guarantee that an implant procedure will be successful, but studies have shown a five-year success rate of 95% for lower jaw implants and 90% for upper jaw implants. the success rate for upper jaw implants is slightly lower because the upper jaw (especially the posterior section) is less dense than the lower jaw, making successful implantation and osseointegration potentially more difficult to achieve. lower posterior implantation has the highest success rate for all dental implants.7 two cases of patients treated with dental implant as alternative to replace the missing teeth that connected with natural teeth are reported. connecting dental implants and natural teeth are still become pro and contra between the dentists. the reason why dentist did not like splinting implants with natural teeth is for one major reason. dentist believed that because the natural tooth has a periodontal membrane surrounding it with a certain degree of mobility, although this movement is very small. many dentist believe because a dental implant is osseo-integrated and there is no periodontal membrane around it, so the dental implant will fail because the movement of the natural tooth will cause the implant to move slightly. it is speculated that this movement will cause the dental implant to fail.8 according to purely theoretical considerations, the splinting of osseointegrated (functionally ankylosed) implants to natural teeth that are suspended by a periodontal membrane with a certain degree of mobility is not rational. because the two types of attachment are basically different it is possible that while functioning, the involved implant abutment is the primary recipient of the load, comparable to a cantilever bridge abutment. whether or not this has any effect on the prognosis is still unclear at the present time.3,4,5 in general, it seems advisable to avoid connecting natural teeth to implant abutments whenever possible. naert et al.9 said that more bone is lost around implants which are rigidly connected to teeth than freestanding ones. over the period from 0 to 15 years, there was significantly more marginal bone loss (0.7 mm) in tooth-implant connected versus freestanding prostheses. kindberg et al.10 confirms that treatments with periodontal healthy teeth and implants splinted together in rigid one-piece superstructures show excellent longterm follow-up results. srinivasan and padmanabhan11 concluded that it is indeed beneficial to connect natural tooth to implants in a fixed partial restoration and that the type of connector advocated is a rigid one. also conclusive is the fact that periodontally compromised teeth can be integrated in the restoration in combination with an implant as a conservative treatment option. the use of non rigid connectors in any situation may be erroneous. takeda12 did a 10 year study and presented his findings, “the harmony of iti implants and natural teeth”. in summary his observations were that “connection distance had a significant effect on changes to teeth, i.e., intrusion or cement washout”. this seems to suggest that if you have to connect teeth to implants the distance between the two should be at least 10 mm or so. i have had no problems when following this rule. clinical study by radnai et al.13 found that there is no disadvantageous effect of connecting teeth to implants as abutments by fixed partial dentures was found. there was no sign of a harmful effect of the implant to the opposing teeth either. the tooth-to-implant bridges function in their biological environment without affecting it adversely. in two cases above, cantilever porcelain bridges were made as fixed restorations because the patients asked that the restoration could reach the optimum distal point, so they could chew the food properly. the underlining reasons that we decided to make a posterior cantilever bridge that supported by tooth-implant abutments are because of clinically imposible to put the implant on certain area and the patients asked for prostheses as optimum as possible, so the mastication function could return to the homeostasis condition. the benefit of this kind of treatment is that prostheses could be made as optimum as possible with a more economic price, so the patients feel quite satisfied and up till now there are not any problems with these two cases. the treatment lacks of these treatments are combining tooth and implant as abutments of prostheses are still debated between dentists and fixed prostheses with cantilever pontic are not preferred by the dentists, because they are afraid that there would be any harmful effect to the abutments. distal cantilever pontic, based upon the unfavourable leverage and negative experiences reported in the literature for similarly configured conventional prostheses, this variation should be avoided whenever possible.14 the occlusal surface area of the pontic is generally decreased by making the pontic smaller than the original tooth, so the abutments did not receive too much load. the dimensions of the bridge are defined by ante’s law: “the root surface area of the abutment teeth has to equal or surpass that of the teeth being replaced with pontics”.15 jan et al.16 from his study failed to demonstrate that the presence of cantilever extensions in an fixed partial dentures (fpd) had an effect on peri-implant bone loss, but smoking had a significant influence on peri-implant bone level change on the fpd level. 61kamadjaja: combination of natural teeth and osseointegrated implants romeo et al.17 from his study that performed on a sample of 38 partially edentulous patients treated with 49 partial cantilever fixed prostheses and supported by 100 implants said that seven years after loading cantilever prostheses, the overall cumulative implant survival rate (ocsr) was 97% and the prostheses success rate was 98%. mesial cantilever prostheses registered a lower success rate (97.1%) than distal cantilever prostheses (100%). furthermore, a better prognosis was not observed when the opposite dentition of the prostheses comprised natural teeth, or fixed prostheses on natural teeth, when compared with the cases in which opposite teeth were implant-supported fixed prostheses. in spite of all that statements, it was concluded that combining implants to natural teeth as abutment of fixed prostheses with cantilever pontic although are still remained pro and contra among the dentists, but that kind of treatment should be considered as a viable prosthetic option. suppose we should do it, some conditions must be fulfilled, e.g periodontally healthy teeth, rigid connection between tooth and implant, successful implantation and osseointegration could be achieved properly. references 1. world centre for dental implantology. copyright 2001. available at http://www.implant types.htm. accessed december 23, 2007. 2. floyd p, palmer r, barrett v. treatment planning for implant restorations. british dental journal 1999 sept; 187(6):25. 3. schroeder a, sutter f, buser d, krekeler g. oral implantology. basics, iti hollow cylinder system. 2nd ed. new york: thieme medical publisher, inc; 1996. p. 243–6. 4. astrand p, borg k, gunne j, olsson m. combination of natural teeth and osseointegrated implants as prosthesis abutments: a 2-year longitudinal study. int j oral max-fac implants 1991; 6:305–12. 5. gunne j, astrand p, ahlen k, borg h, olsson m. implants in partially edentulous patients. a longitudinal study of bridges supported by both implants and natural teeth. clin oral impl res 1992; 3:49–56. 6. concept trinon implant.htm. accessed january 12, 2008. 7. johnstone g. dental implants. available at http://dental implants -a complete consumer guide.htm. accessed december 23, 2007. 8. pd/ “connecting-implants-and-natural-teeth ?”/ available at: www. osseonews.com//#comment4002.htm. accessed january 10, 2008. 9. naert ie, duyck ja, hosny mm, quirynen m, van steenberghe d. freestanding and tooth-implant connected prostheses in the treatment of partially edentulous patients part ii: an up to 15-years radiographic evaluation. clin oral implants res 2001 jun; 12(3):245–51. 10. kindberg h, gunne j, kronström m. tooth and implant supported prostheses: a retrospective clinical follow-up to 8 years. int j prosthodont 2001 november-december; 14(6):575–81. 11. srinivascan m, padmanabhan t. implant prosthodontic: an in-vitro photoelastic stress analysis. journal of indian prosthodontic society 2005; 12. takeda t. the harmony of iti implants and natural teeth. available at: www.osseonews.com/dental implant discussion / dental implants connecting natural teeth.htm. accessed january 10, 2008. 13. radnai m, fazekas a, vajdovich i, kostinek d, fogorv sz. clinical study of tooth-to-implant supported fixed partial dentures. dental implant research 1998 july; 91(7):195–202. 14. strub jr, linter h, marinello cp. the rehabilitation of partially edentulous cases with cantilever bridge bridges: a retrospective study. int j periodont restorat dent 1989; 9:364. 15. shillingburg ht, hobo s, whitsett ld, jacobi r, brackett se. fundamentals of fixed prosthodontics. 3rd ed. quintessence publishing co, inc 1997; 92. 16. jan w, zurdo j, karlsson s, annika e, kerstin g, lindhe j. bone level change at implant-supported fixed partial dentures with and without cantilever extension after 5 years in function. j of clin periodontology december 2004; 31(12):1077–83. 17. romeo e, lops d, margutti e, ghisolfi m, chiapasco m, vogel g. implant-supported fixed cantilever prostheses in partially edentulous arches. a seven–year prospective study. clin oral implants res 2003; 14:303–11. vol 38 no 2-2005 56 sitotoksisitas resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda (cytotoxicity of the hybrid acrylic resin after glass fiber reinforcement with difference method) intan nirwana dan r. helal soekartono bagian ilmu material dan teknologi kedokteran gigi fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract glass fiber reinforcement of the hybrid acrylic resin with difference method can enhance residual monomer content of the material; it can cause cytotoxic effect on fibroblast cells. the purpose of this study was to know the cytotoxicity of hybrid acrylic resins after glass fiber reinforcement with difference method on the cultured fibroblasts. the squared specimens of 10 mm in length, 10 mm in width and 1.5 mm in thickness were cured for 20 minutes at 100° c. the fibroblast cells were grown in eagle's minimum essential medium to be 2 × 105 cells/ml, then the cells were added to the samples in the plates and incubated at 37° c. after 48 hours, the cytotoxic effect was determined by direct cell number count using microscope and a hemocytometer. the statistical analyses using one way anova and lsd test showed that there were significant difference in cell viability (p < 0.05) among the groups. the means percentage of cell viability were 90.00%, 99.,11%, 98.66%, it could be concluded that glass fiber reinforcement into hybrid acrylic resin with either first method or second method was not toxic. key words: hybrid acrylic resins, cytotoxicity, glass fiber korespondensi (correspondence): intan nirwana, bagian ilmu material dan teknologi kedokteran gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan resin akrilik hybrid yang berkembang saat ini sangat efektif, praktis, dan mempunyai dua aktivator yaitu kimia dan panas serta proses kuring cepat hanya 20 menit suhu 100° c.1,2 waktu tersebut sangat singkat dibandingkan dengan proses kuring resin akrilik terdahulu, sehingga waktu kerja lebih efisien. kekurangan resin akrilik adalah mudah patah dan patahnya basis gigi tiruan dapat terjadi di luar mulut yaitu jatuh pada tempat yang keras, sedangkan patah yang terjadi di dalam mulut dapat disebabkan oleh karena fatique maupun occlusal forces.3 patahnya basis gigi tiruan dapat disebabkan oleh fitting dari gigi palsu tidak baik, tidak adanya keseimbangan oklusi, dan fatique maupun jatuh.4 untuk meningkatkan sifat mekanik resin akrilik yaitu dengan menambah fibers, carbon, aramid, glass dan metal wire5,6,7 atau dengan menambahkan ultra high modulus polyethylene fibers.8,9 carbon dan aramid fiber dapat memperkuat polimetil metakrilat tetapi resin akrilik sukar dipulas dan estetik menjadi jelek.5 metode tradisional terdahulu menggunakan metal wire sebagai penguat basis gigi tiruan. resin akrilik yang mengandung glass fiber menunjukkan sifat mekanik yang lebih baik dibandingkan dengan resin akrilik tanpa penambahan glass fiber.10 penambahan glass fiber dengan menggunakan metode berbeda menunjukkan kandungan monomer sisa yang meningkat,11 tentunya monomer sisa yang terlepas juga meningkat akhirnya akan mempengaruhi sitotoksisitas resin akrilik terhadap sel fibroblas secara in vitro.12 kandungan monomer sisa yang tinggi berpotensi untuk menyebabkan iritasi jaringan mulut, inflamasi, alergi terutama daerah mukosa di bawah gigi tiruan.13,14 selain itu kandungan monomer sisa yang tinggi dapat mempengaruhi sifat fisik polimer yang dihasilkan karena monomer sisa akan bertindak sebagai plasticiser dan membuat resin akrilik menjadi fleksibel dan kekuatan menurun. salah satu metode penambahan glass fiber adalah merendam glass fiber tersebut dalam metil metakrilat selama 15 menit (metode 1). hal tersebut menyebabkan kandungan monomer dalam resin akrilik lebih banyak dari perbandingan polimer dan monomer yang telah ditentukan pabrik. metode lain adalah menambahkan glass fiber langsung dalam campuran polimer dan monomer yang baru diaduk, jadi viskositas campuran resin akrilik masih rendah (metode 2). beberapa metode penambahan glass fiber yang digunakan oleh peneliti terdahulu tersebut perlu dipertimbangkan karena masih mempunyai kekurangan, sedangkan faktor yang mempengaruhi jumlah monomer 57nirwana: sitotoksisitas resin akrilik hybrid sisa dalam resin akrilik adalah perbandingan antara bubuk atau likuid bahan resin akrilik, proses kuring, dan jenis polimerisasi.1 resin akrilik dalam pemakaiannya sebagai gigi tiruan memang tidak diimplantasikan ke dalam jaringan, tetapi karena pemakaiannya di dalam rongga mulut cukup lama, maka kemungkinan kontak dengan mukosa rongga mulut sangat besar, sehingga persyaratan biokompatibilitas sangat mutlak diperlukan. sitotoksisitas merupakan uji tahap awal dari uji biokompatibilitas dan bahan kedokteran gigi harus memenuhi syarat biokompatibilitas yang dapat diterima oleh tubuh atau host atau dengan kata lain tidak membahayakan penderita.15 jadi idealnya bahan yang diletakkan dalam mulut disyaratkan tidak toksik, tidak iritan, tidak karsinogenik dan tidak menimbulkan alergi.2,15 sampai saat ini belum didapatkan informasi tentang uji sitotoksisitas resin akrilik hybrid setelah penambahan glass fiber. berdasarkan hal-hal tersebut di atas maka perlu diteliti sitotoksisitas resin akrilik hybrid dengan kedua metode penambahan glass fiber yang berbeda tersebut. tujuan penelitian ini adalah untuk mengetahui sitotoksisitas resin akrilik hybrid dengan metode penambahan glass fiber yang berbeda yaitu dengan cara merendam glass fiber dalam metil metakrilat 15 menit terlebih dahulu, dan dengan cara menambahkan langsung (tanpa direndam) dalam campuran polimer dan monomer. hasil penelitian ini diharapkan memberikan informasi tentang sitotoksisitas resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda, sehingga dapat dipilih metode penambahan glass fiber yang aman dan menghasilkan resin akrilik yang tidak toksik. bahan dan metode bahan yang digunakan pada penelitian ini adalah resin akrilik hybrid (biocryl), gips keras, glass fiber (yakasu, japan), sel bhk 21, media eagle's, bouvine serum, larutan pbs, larutan trypsine versene, tripan biru. alat yang digunakan adalah model master kuningan dengan ukuran 10 × 10 × 2,5 mm, kuvet logam, timbangan digital, termometer, pipet mikro, laminar flow cabinet, mikroskop, inkubator, hemositometer berskala, plate kaca. cara kerja penelitian adalah sebagai berikut: gips keras dengan perbandingan 100 gram gips dan 24 ml air (sesuai petunjuk pabrik) diaduk dengan menggunakan spatel, kemudian diletakkan di atas vibrator dan dimasukkan ke dalam kuvet yang telah disiapkan di atas vibrator. model master kuningan diletakkan ditengah kuvet didiamkan sampai gips mengeras. setelah mengeras, permukaan gips diulasi vaselin, kuvet antagonis dipasang, diisi adonan gips di atas vibrator dan ditekan, dibiarkan sampai gips mengeras. kuvet dibuka, model master diambil, maka didapat cetakan model (mould), kemudian diolesi separator, tunggu sampai kering selama 10 menit. persiapan pembuatan sampel dengan penambahan glass fiber adalah sebagai berikut, glass fiber ukuran 8 × 8 mm ditimbang sebanyak 0,25 gr, kemudian direndam dalam metil metakrilat monomer sebanyak 3 ml selama 15 menit (metode 1), kemudian polimer dan monomer dengan perbandingan 4 g : 2 ml diaduk dalam pot porselin. setelah 5 menit adonan mencapai tahap dough, selanjutnya adonan dimasukkan ke dalam mould, yang bagian tengahnya diletakkan glass fiber yang telah direndam dalam metil metakrilat monomer. kuvet ditutup sebelumnya resin akrilik ditutup dengan kertas selopan dan ditekan perlahanlahan dengan press hidrolik. kuvet dibuka kembali, kelebihan dipotong kemudian kuvet ditutup kembali, dilakukan penekanan dengan tekanan 2200 psi atau 50 kg/cm2, prosedur diulang 3 kali, dibiarkan selama 15 menit (aturan pabrik). kuvet yang berisi resin akrilik dengan penambahan glass fiber dilakukan proses kuring dengan suhu 100° c selama 20 menit. sedangkan pada metode 2, polimer dan monomer yang baru diaduk dimasukkan ke dalam mould yang ditengahnya diletakkan glass fiber, kuvet ditutup yang sebelumnya resin akrilik ditutupi dengan kertas selopan. dilakukan penekanan dengan press hidrolik ditunggu selama 5 menit kemudian kuvet dibuka, kelebihan dipotong kemudian kuvet ditutup lagi selanjutnya dilakukan penekanan dan proses kuring seperti pada metode 1 di atas. pada penelitian ini terdapat 3 kelompok: 1) resin akrilik hybrid tanpa glass fiber (kontrol); 2) penambahan glass fiber dalam resin akrilik hybrid yang sebelumnya direndam dalam metil metakrilat monomer selama 15 menit (metode 1); 3) penambahan glass fiber langsung dalam resin akrilik yang baru diaduk (metode 2). uji sitotoksisitas dilakukan di pusvetma dan tahapannya adalah sebagai berikut: persiapan kultur cell line bhk-21 clone 21: kultur bhk-21 dalam bentuk monolayer ditanam dalam roux besdar. setelah confluent (penuh), kultur dipanen dengan menggunakan larutan trypsine versene. hasil panenan dicampur kembali dalam media yang mengandung 10% bouvine serum dan dibuat kepadatan 2 × 10 5 sel/ml, kemudian sel tersebut dipindahkan dalam roux kecil. selanjutnya sel siap untuk digunakan dalam pengujian sampel. persiapan sampel: sampel terlebih dahulu ditempelkan menggunakan silicone grease pada dasar plate, disterilkan dengan ultra violet selama 15 menit kemudian ditambahkan sel yang telah mempunyai kepadatan 2 × 105 sel/ml dengan menambahkan fetal bouvine serum 10%, selanjutnya dimasukkan ke dalam inkubator co2 selama 48 jam dengan suhu 37° c, diamati pertumbuhan sel di sekitar sampel. perhitungan jumlah pertumbuhan sel diamati selama 48 jam. persiapan perhitungan sel setelah 48 jam dalam inkubator: setelah 48 jam kultur sel dikeluarkan dari inkubator, kemudian media kultur sel dibuang, sel dicuci dengan phosphate buffer saline sebanyak 2 kali agar sisasisa media benar-benar hilang. selanjutnya dilakukan tripsinasi dengan menambahkan trypsine versene guna merontokkan sel dari dinding plate dengan cara ditunggu 58 maj. ked. gigi. (dent. j.), vol. 38. no. 2 april–juni 2005: 56–59 beberapa saat (5 menit) kemudian diberi lagi fetal bouvine serum 10% agar diperoleh suatu suspensi sel (kepadatan sel 2 × 105 sel/ml). cara perhitungan: diambil sebanyak 0,1 cc (sel dengan media serum) ditambahkan 0,9 cc cairan tripan biru, kemudian sel dihitung dengan alat hemositometer (0,0025 mm2). alat tersebut terdiri dari 9 kotak, sehingga hasil yang diperoleh berupa jumlah ratarata sel hidup dan mati dari ke sembilan kotak tersebut. sel yang hidup ditandai dengan tidak terserapnya warna biru (warna terang), sedangkan sel mati menyerap warna biru. metode yang digunakan untuk menghitung sel yang hidup adalah sebagai berikut:16 jumlah sel hidup × 100% jumlah seluruh sel (hidup dan mati) hasil hasil yang diperoleh pada penelitian tentang sitotoksisitas resin akrilik hybrid setelah penambahan glass fiber dengan metode berbeda adalah sebagai berikut: perhitungan sel hidup pada kelompok 1, 2 dan 3 diperoleh hasil rata-rata dan simpang baku yang terlihat pada tabel 1. tabel 1. nilai rata-rata, simpang baku dan hasil uji anova jumlah sel hidup pada kelompok 1, 2 dan 3 kelompok n rata-rata (%) simpang baku p 1 6 90,00 0,86 0,001 2 6 99,11 0,92 3 6 98,66 0,86 keterangan: kelompok 1 = resin akrilik tanpa glass fiber (kontrol); kelompok 2 = resin akrilik ditambah glass fiber dengan metode 1; kelompok 3 = resin akrilik ditambah glass fiber dengan metode 2. hasil anova satu arah menunjukkan ada perbedaan yang bermakna dari jumlah sel hidup antara kontrol dan resin akrilik ditambah glass fiber dengan metode 1 dan 2 (p < 0,05). untuk mengetahui perbedaan antar kelompok digunakan uji honestly significance difference (hsd). tabel 2. uji hsd jumlah sel hidup (%) pada resin akrilik tanpa glass fiber dan resin akrilik ditambah glass fiber dengan metode 1 dan 2 kelompok 1 kelompok 2 kelompok 3 kelompok 1 * * kelompok 2 kelompok 3 keterangan: * = berbeda bermakna pada tabel 2 menunjukkan adanya perbedaan yang bermakna antara kelompok 1 dan 2, kelompok 1 dan 3. pembahasan pada penggunaan gigi tiruan, salah satu bagian menempel pada gingiva yang sebagian besar terdiri dari sel fibroblas. oleh karena itu pada penelitian ini dilakukan uji sitotoksisitas terhadap kultur sel fibroblas. sel fibroblas yang digunakan berasal dari baby hamster kidney (bhk 21) oleh karena sel fibroblas bhk 21 mudah tumbuh dan mudah di sub kultur. kultur sel terbaik berasal dari sel embrionik atau sel jaringan muda.17 secara umum glass fiber kemungkinan bersifat sitotoksik apabila glass fiber kontak dengan media agar. apabila jumlah glass fiber menyebabkan perubahan yang dapat mengakibatkan efek sitotoksik, maka jumlah fibers yang meningkat mengakibatkan efek sitotoksisitas lebih tinggi.18 pada penelitian ini, setelah dilakukan uji sitotoksisitas yang merupakan tahap awal uji biokompatibilitas menunjukkan persentase sel hidup 90,00% pada resin akrilik hybrid tanpa glass fiber (kontrol), sedangkan resin akrilik hybrid setelah penambahan glass fiber dengan metode 1 dan 2 masing-masing 99,11% dan 98,66%. pada penelitian ini menunjukkan adanya perbedaan bermakna persentase sel hidup pada resin akrilik hybrid setelah penambahan glass fiber dengan metode 1 dan 2 dibandingkan dengan kontrol (p < 0,05). pada plat akrilik setelah penambahan glass fiber dengan metode 1 (kelompok 2) menunjukkan persentase sel hidup lebih besar (99,11%) dibandingkan dengan kontrol (90,00%). secara teori adanya glass fiber yang direndam dalam monomer metil metakrilat (metode 1) berarti monomer lebih banyak dari yang seharusnya menurut ketentuan pabrik. asumsinya monomer sisa menjadi lebih tinggi dan akibatnya efek sitotoksik tinggi. hal tersebut tidak terjadi pada penelitian ini yang terbukti persentase tingginya jumlah sel hidup (99,11%). kemungkinan hal ini disebabkan karena sebagian metil metakrilat bereaksi dengan glass fiber, dan pada saat dilakukan penekanan dengan press hidrolik pada waktu packing akrilik, metil metakrilat monomer yang terserap oleh glass fiber keluar dari glass fiber tampak pada gips sekitar akrilik basah oleh metil metakrilat. oleh karena itu monomer sisa dalam resin akrilik menjadi rendah dan akibatnya persentase sel hidup menjadi tinggi. jadi perendaman glass fiber sebelum packing akrilik tidak meningkatkan kandungan monomer sisa dalam resin akrilik. penelitian terdahulu pada resin akrilik hybrid tanpa penambahan glass fiber menunjukkan kandungan monomer sisa 1,9% 19 dan persentase sel hidup 90,00%. hal tersebut berarti kandungan monomer sisa pada kelompok 2 kemungkinan lebih rendah dari 1,9% terbukti persentase sel hidup meningkat yaitu 99,11%. 59nirwana: sitotoksisitas resin akrilik hybrid pada kelompok 3 penambahan glass fiber dengan metode 2 juga menunjukkan persentase sel hidup yang tinggi yaitu 98,66% dibandingkan dengan kontrol. dapat dijelaskan bahwa perbandingan bubuk dan cairan resin akrilik sesuai ketentuan pabrik. pada saat penambahan glass fiber, viskositas campuran bubuk dan cairan cukup rendah sehingga semua glass fiber dapat seluruhnya terbasahi oleh campuran tersebut. 20 hal tersebut menyebabkan metil metakrilat monomer sebagian bereaksi dengan glass fiber sehingga kemungkinan kandungan monomer sisa dalam plat akrilik juga rendah akibatnya persentase sel hidup tinggi, sedangkan pada kelompok kontrol tanpa glass fiber menunjukkan persentase sel hidup lebih rendah, kemungkinan kandungan monomer sisa dalam plat akrilik lebih tinggi walaupun perbandingan bubuk dan cairan resin akrilik sesuai dengan ketentuan pabrik. hal tersebut dikarenakan tidak adanya glass fiber. pada kelompok 2 dan 3 menunjukkan perbedaan persentase sel hidup yang tidak bermakna oleh karena kandungan monomer sisa dalam plat resin akrilik pada kedua kelompok tersebut kemungkinan sama rendahnya (< 1,9%) walaupun penambahan glass fiber dengan metode berbeda. kandungan monomer sisa tidak boleh melampaui 2,2% untuk resin akrilik heat cured dan 4,5% untuk self cured.21 parameter toksisitas adalah berdasarkan cd50, yang artinya suatu bahan dikatakan toksik apabila persentase sel hidup setelah terpapar bahan tersebut kurang dari 50%.22 pada penelitian ini dapat disimpulkan bahwa resin akrilik hybrid setelah penambahan glass fiber dengan metode 1 maupun metode 2 adalah tidak toksik. daftar pustaka 1. kedjarune u. release of methylmethacrylate from heat cured and autopolymerized resins: cytotoxicity testing related to residual monomer. australian dental journal 1999; 44(1): 25–30. 2. craig rg. restorative dental materials. 11st ed. mosby-year book. inc; 2002. p. 655–58. 3. polyzois gl, andrepoulos ag, lagouvardos pe. acrylic resin denture repair with adhesive resin and metal wires: effects of strength parameters. j prosthet dent 1996; 75: 381–87. 4. beyli ms, fraunhover ja. an analysis of causes of fracture of acrylic resin dentures. j prosthet dent 1981; 46: 238–41. 5. larson wr, dixon dl, aquilino sa, clancy jm. the effect of carbon graphite fiber reinforcement on the strength of provisional crown and fixed partial denture resins. j prosthet dent 1991; 66: 216–20. 6. vallittu pk. dimensional accuracy and stability of poltmethyl methacrylate reinforced with metal wire or with continuous glass fiber. 1996. 75: 617–20. 7. solnit gs. the effect of methyl methecrylate reinforcement with silane-treated and untreated glass fiber. j prosthet dent 1991; 66: 310–14. 8. braden m, davi kwm, parker s. denture base poly (methyl methacrylate) reinforced with ultra-high modulus polyethylene fibres. br dent j 1988; 164: 109–13. 9. gutteridge dl. the effect of including ultra-high modulus polyethylene fibre on the impact strength of acrylic resin. br dent j 1988; 164: 177–80. 10. vallittu pk. some aspects of the tensile strength of unidirectional glass fiber-polymethyl methacrylate composite used in dentures. j oral rehabil 1998; 25: 100–05. 11. handan y, cemal a alper c, ahmet y. the effect of glass fiber reinforcement on the residual monomer content of two denture base resins. quintessence int 2003; 34: 148–53. 12. lefebvre ca. cytotoxicity of eluates from lightpolymerized denture base resins. j prosthet dent 1994; 72: 644–50. 13. hensten, petterson a and yacobson n. perceived side effect of biomaterials in prosthetic dentistry. j prosthet dent. 1991; 65: p. 138–44. 14. combe ec. notes on dental materials. 6th ed. new york: churchill livingstone; 1992. p. 158–60. 15. anusavice kj. phillips science of dental materials. 10th ed. philadelphia: wb saunders company; 1996. p. 246–49. 16. bird br, forrester ft. basic laboratory techniques in cell culture. us. department of health and human services. public health service. centers for disease control; 1981. p. 33–43. 17. freshney ir. culture of animal cells. 2nd ed. new york: alan r liss inc; 1987; p. 227–45. 18. vallittu pk. in vitro cytotoxicity of fibre-polymethyl methacrylate composite used in dentures. j oral rehabilitation 1999; 26: 666– 71. 19. intan n. kandungan monomer sisa dalam resin akrilik rapid heat cured dengan proses kuring berbeda. majalah kedokteran gigi 2001; 34(3): 119–21. 20. vallittu pk, lassila vp, lappalainen. acrylic resin fiber composite: the effect offiber concentration on fracture resistance. j prosthet dent 1994; 71: 607–12. 21. international standards organization. denture base polymers (iso/ dis 1567). geneva; 1998. p. 1–27. 22. telli c, serper a, dogan al, gue d. evaluation of the cytotoxicity of calcium phosphate root canal sealers by mtt assay. j endodon 1999; 25: 811–13. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no 1-2005 25 peranan sorbitol dalam mempertahankan kestabilan ph saliva pada proses pencegahan karies (the role of sorbitol in maintaining saliva’s ph to prevent caries process) diana soesilo,* rinna erlyawati santoso,* dan indeswati diyatri** * mahasiswa ppdgs ** bagian biologi oral fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract people in indonesia often consume food containing sucrose. if the sucrose consumed is in a large amount, it will decrease saliva’s ph and soon teeth destruction will happen. to avoid it, it is necessary to change sucrose consumption habit into another kind of sugar, namely sorbitol. sorbitol is preferred to use, because it is cheaper and easier to get. sorbitol is made from cassava, which is plentifully grown in indonesia. sorbitol is not good media for bacteria to grow. because sorbitol has a diol, so it’s difficult to catalyst by glucosyltransferase enzyme, which is produced by bacteria streptococcus mutans. the conclusion is that sorbitol is difficult to be fermented by streptococcus mutans so it will not decrease saliva’s ph. key words: sorbitol, saliva’s ph, caries korespondensi (correspondence): diana soesilo, mahasiswa ppdgs, fakultas kedokteran gigi universitas airlangga. jln. mayjen. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan glukosa merupakan bagian utama diet penduduk di indonesia. selain sebagai makanan pokok, gula juga dikonsumsi sebagai makanan ringan atau camilan seperti yang terdapat dalam permen, wafer, kue, biskuit, dan dalam minuman ringan.1 menurut penelitian di posyandu di wilayah dki jakarta tahun 1993, diperoleh data bahwa sekitar 96,7% ibu membelikan jajan makanan manis kepada anaknya dan hanya 3,3% yang membelikan jajan yang mengandung protein.2 jenis gula yang paling banyak digunakan adalah sukrosa.3 konsumsi sukrosa dalam jumlah besar dapat menurunkan kapasitas buffer saliva sehingga mampu meningkatkan insiden terjadinya karies.4 manifestasi sukrosa dalam kehidupan sehari-hari adalah dalam bentuk gula putih. sukrosa banyak dikonsumsi orang karena rasa manisnya enak, bahan dasarnya mudah diperoleh, dan biaya produksinya cukup murah. tapi ternyata menurut penelitian, sukrosa yang menaikkan indikasi karies paling besar. hal ini disebabkan karena sintesa ekstra sel sukrosa lebih cepat daripada gula lainnya seperti glukosa, fruktosa, dan laktosa sehingga cepat diubah oleh mikroorganisme dalam rongga mulut menjadi asam.3 oleh karena itu, dicari suatu solusi untuk mengurangi jumlah konsumsi sukrosa yaitu menggantikannya dengan gula alkohol. gula alkohol adalah gula yang komposisi kimianya terdiri dari tiga atau lebih kelompok hidroksil. bentuk gula alkohol antara lain sorbitol, xylitol, manitol, dulcitol, dan inositol.5 di indonesia, sorbitol lebih banyak digunakan daripada jenis gula alkohol yang lainnya karena bahan dasar pembuatannya lebih mudah diperoleh dan harganya lebih murah yaitu dari tepung tapioka.6 adapun tujuan pembuatan makalah ini adalah untuk memberikan informasi bahwa sorbitol dalam mempertahankan ph saliva sehingga dapat mencegah proses karies. manfaat pembuatan makalah ini adalah untuk memberikan tambahan informasi tentang sorbitol sebagai alternatif pemanis pengganti sukrosa kepada masyarakat, sehingga masyarakat menjadi semakin selektif dalam memilih makanan kecil atau camilan yang akan dikonsumsinya. karies karies gigi terjadi pada semua penduduk di seluruh dunia tanpa memandang golongan usia, termasuk penduduk indonesia.7 berdasarkan survey kesehatan gigi yang dilakukan oleh direktorat kesehatan gigi republik indonesia pada tahun 1994, prevalensi karies gigi pada anak usia 14 tahun sebesar 73,2% dengan indeks dmf-t sebesar 2,69. hasil ini menunjukkan bahwa karies gigi merupakan masalah kesehatan gigi dan mulut yang dominan di negara kita.2 karies merupakan suatu penyakit jaringan keras gigi yaitu email, dentin, dan sementum yang disebabkan oleh aktivitas suatu jasad renik dalam suatu karbohidrat yang dapat diragikan.7 terdapat empat faktor utama yang berperan dalam proses terjadinya karies, yaitu host, mikroorganisme, substrat, dan waktu.8 faktor-faktor tersebut bekerja bersama dan saling mendukung satu sama lain. bakteri plak akan memfermentasikan karbohidrat (misalnya sukrosa) dan 26 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 25–28 menghasilkan asam, sehingga menyebabkan ph plak akan turun dalam waktu 1–3 menit sampai ph 4,5–-5,0.9 kemudian ph akan kembali normal pada ph sekitar 7 dalam 30–60 menit, dan jika penurunan ph plak ini terjadi secara terus menerus maka akan menyebabkan demineralisasi pada permukaan gigi. kondisi asam seperti ini sangat disukai oleh sterptococcus mutans dan lactobacillus sp, yang merupakan mikroorganisme penyebab utama dalam proses terjadinya karies.10 menurut penelitian streptococcus mutans berperan dalam permulaan (initition) terjadinya karies gigi, sedangkan lactobacillus sp, berperan pada proses perkembangan dan kelanjutan karies.11 pertama kali akan terlihat white spot pada permukaan enamel kemudian proses ini berjalan secara perlahan sehingga lesi kecil tersebut berkembang, dan dengan adanya destruksi bahan organik, kerusakan berlanjut pada dentin disertai kematian odontoblast.8 saliva saliva merupakan cairan mulut yang kompleks terdiri dari campuran sekresi kelenjar saliva mayor dan minor yang ada dalam rongga mulut. saliva sebagian besar yaitu sekitar 90 persennya dihasilkan saat makan yang merupakan reaksi atas rangsangan yang berupa pengecapan dan pengunyahan makanan.8 saliva membantu pencernaan dan penelanan makanan, di samping itu juga untuk mempertahankan integritas gigi, lidah, dan membrana mukosa mulut. di dalam mulut, saliva adalah unsur penting yang dapat melindungi gigi terhadap pengaruh dari luar, maupun dari dalam rongga mulut itu sendiri. makanan yang kita makan dapat menyebabkan ludah kita bersifat asam maupun basa. peran lingkungan saliva terhadap proses karies tergantung dari komposisi, viskositas, dan mikroorganisme pada saliva.12 secara teori saliva dapat mempengaruhi proses terjadinya karies dalam berbagai cara, antara lain aliran saliva dapat menurunkan akumulasi plak pada permukaan gigi dan juga menaikkan tingkat pembersihan karbohidrat dari rongga mulut. selain itu, difusi komponen saliva seperti kalsium, fosfat, ion oh–, dan fluor ke dalam plak dapat menurunkan kelarutan email dan meningkatkan remineralisasi gigi. saliva juga mampu melakukan aktivitas antibakterial karena mengandung beberapa komponen yang antara lain adalah lisosim, sistem laktoperoksidase-isitiosianat, laktoferin, dan imunoglobulin ludah.4 derajat keasaman ph dan kapasitas buffer saliva ditentukan oleh susunan kuantitatif dan kualitatif elektrolit di dalam saliva terutama ditentukan oleh susunan bikarbonat, karena susunan bikarbonat sangat konstan dalam saliva dan berasal dari kelenjar saliva. derajat keasaman saliva dalam keadaan normal antara 5,6–7,0 dengan rata-rata ph 6,7. beberapa faktor yang menyebabkan terjadinya perubahan ada ph saliva antara lain18 rata-rata kecepatan aliran saliva, mikroorganisme rongga mulut, dan kapasitas buffer saliva. derajat keasaman (ph) saliva optimum untuk pertumbuhan bakteri 6,5–7,5 dan apabila rongga mulut ph-nya rendah antara 4,5–5,5 akan memudahkan pertumbuhan kuman asidogenik seperti streptococcus mutans dan lactobacillus.12 sorbitol konsumsi sukrosa sebagai pemanis makanan sekarang mulai digantikan dan dikurangi penggunaanya.13 bahan pengganti gula harus memenuhi persyaratan yaitu harus mempunyai rasa manis, tidak toksik, tidak mahal, tidak bisa diragikan oleh bakteri plak gigi, berkalori, di samping itu juga harus dapat dikerjakan secara industrial. dari semua persyaratan tersebut, maka bahan pengganti gula yang baik adalah yang berasal dari golongan gula alkohol.14 sorbitol merupakan bahan pengganti gula dari golongan gula alkohol yang paling banyak digunakan, terutama di indonesia.6 di indonesia sorbitol (c6h14o6) paling banyak digunakan sebagai pemanis pengganti gula karena bahan dasarnya mudah diperoleh dan harganya murah.15 di indonesia, sorbitol diproduksi dari tepung umbi tanaman singkong (manihot utillissima pohl) yang termasuk keluarga euphoribiaceae.16 selain itu sorbitol juga dapat ditemui pada alga merah bostrychia scorpiodes yang mengandung 13,6% sorbitol. tanaman berri dari spesies sorbus americana mengandung 10% sorbitol. famili rosaceae seperti buah pir, apel, ceri, prune, peach, dan aprikot juga mengandung sorbitol.5 sorbitol juga diproduksi dalam jaringan tubuh manusia yang merupakan hasil katalisasi dari d-glukosa oleh enzim aldose reductase, yang mengubah struktur aldehid (cho) dalam molekul glukosa menjadi alkohol (ch2oh).14 sorbitol dapat digunakan sebagai pengganti sukrosa pada penderita penyakit diabetes. nilai kalori makanan yang mengandung sorbitol sama tinggi dengan gula, tapi rasa manisnya kira-kira hanya 60 persen rasa manis sukrosa.3 kerugian sorbitol adalah bila dipakai dalam jumlah yang berlebihan dapat menyebabkan terjadinya diare. sorbitol merupakan gula yang diabsorbsi sangat sedikit oleh usus halus, sehingga sorbitol akan langsung masuk ke usus besar dan dapat menunjang terjadinya diare dan perut kembung.10 sorbitol (c6h14o6) berasal dari golongan gula alkohol.17 gula alkohol merupakan hasil reduksi dari glukosa di mana semua atom oksigen dalam molekul gula alkohol yang sederhana terdapat dalam bentuk kelompok hidroksil, sinonim dengan polyhidric alcohol (polyols). polyols dapat dibagi menjadi dua yaitu polyols asiklik dan polyols siklik. sorbitol termasuk dalam kelompok polyols asiklik dengan enam rantai karbon.5 rumus kimia sorbitol dapat dilihat pada gambar 1. 27soesilo, dkk: peranan sorbitol h2c⎯oh ⏐ ch⎯oh ⏐ oh⎯c⎯h ⏐ hc⎯oh ⏐ hc⎯oh ⏐ h2c⎯oh gambar 1. rumus kimia sorbitol.18 pembahasan sorbitol baik digunakan sebagai pemanis pengganti sukrosa karena mempunyai keuntungan, antara lain tidak bersifat kariogenik.19 menurut penelitian edgar dan geddes20 dengan melakukan penelitian pada dua kelompok sampel. di mana sampel yang pertama diminta untuk mengunyah permen karet dengan pemanis sukrosa dan kelompok sampel kedua mengunyah permen karet dengan pemanis sorbitol. setelah 5 menit diukur ph saliva dari masing-masing kelompok sampel, ternyata diperoleh hasil bahwa kelompok pertama ph salivanya turun menjadi 4 sedang kelompok kedua ph-nya masih sekitar 7. sorbitol termasuk dalam golongan gula alkohol yang mempunyai keunikan, yaitu gula alkohol tidak mempunyai gugus karbonil dalam rantainya.21 fakta ini membuat gula alkohol kurang reaktif secara kimiawi daripada gula yang mempunyai ikatan aldosa dan ketosa sehingga kurang berpartisipasi dalam pembentukan asam pada plak gigi. 22 untuk memfermentasi substrat dan menghasilkan asam, normalnya terdapat keseimbangan secara stoikiometri antara jumlah atom-atom karbon, oksigen, dan hidrogen.23 gula alkohol mempunyai dua tambahan atom hidrogen sehingga strukturnya menjadi (ch2o)n.2h. sedangkan struktur kimia karbohidrat pada umumnya adalah (ch2o)n. pada gambar 2 terlihat bahwa pada rumus kimia sorbitol, terdapat ujung diol (bagian atas dan bawah rumus kimia sorbitol ditutup oleh ion oh-).18 dengan adanya tambahan dua atom hidrogen dan ujung diol tersebut, maka sulit bagi enzim glukosiltransferase yang terdapat pada dinding sel streptococcus mutans memecah rantai gula alkohol menjadi asam laktat, asam asetat, dan asam format. 18 dalam tubuh sorbitol dapat dikatalisis oleh enzim sorbitol dehidrogenase untuk selanjutnya menjadi fruktosa, tapi fruktosa yang dihasilkan oleh sorbitol tidak dapat melewati siklus asam piruvat.5 pada hasil akhirnya sorbitol tidak memproduksi asam laktat, asam format, dan etanol, sehingga tidak dapat menyebabkan ph saliva menjadi asam.24 menurut penelitian kecepatan dari proses fermentasi sorbitol amat lambat bila dibandingkan dengan sukrosa dan glukosa,25 sehingga asam yang terbentuk dapat dinetralisir oleh kapasitas buffer dari saliva. berdasarkan penelitian houwink6 seperti yang terlihat pada gambar 2, sorbitol baru dapat difermentasikan oleh streptococcus mutans setelah dikonsumsi lebih dari 60 menit. gambar 2. perubahan ph dalam plak setelah konsumsi gula (glukosa) dan bahan pengganti gula (xylitol dan sorbitol).6 pada penggunaan sorbitol yang efektif, maka sorbitol akan melewati jalur metabolisme seperti yang tertera di gambar 3.18 sorbitol akan diuraikan oleh enzim sorbitol dehidrogenase bukan oleh enzim glukosiltransferase. sehingga sorbitol akan melalui jalur lipogenesis bukan glukolisis. sorbitol akan dikonversikan menjadi lemak, sehingga tidak efektif jika menggunakan sorbitol untuk program diet.8 sorbitol (c6h14o6) fruktosa fruktosa 1-p dhap plus glyceraldehide gliserol lipogenesis sorbitol dehidrogenase aldolase fruktokinase alkohol dehidrogenase gambar 3. skema jalur metabolisme sorbitol.18 pratiwi dkk.26 pada tahun 2001 melakukan penelitian dengan mengamati pertumbuhan streptococcus mutans yang diambil dari sampel saliva 30 orang responden yang diberi perlakuan sebanyak dua kali yaitu mengunyah permen yang mangandung sorbitol dan sukrosa. penelitian dilakukan dengan menghitung jumlah colony forming units (cfu) streptococcus mutans dari sampel saliva responden saat mengunyah permen yang mengandung sorbitol dan sukrosa, yang dibiakkan pada pada media padat trypticase yeast extract sucrose with bacitracin (ty20sb).12 dari hasil penelitian tersebut diperoleh hasil bahwa cfu streptococcus mutans pada pemakaian gula sorbitol sesudah perlakuan terlihat adanya penurunan baik pada minggu kedua maupun minggu ketiga bila dibandingkan dengan sebelumnya.25 hal ini menunjukkan bahwa sorbitol bukan merupakan media yang baik bagi pertumbuhan bakteri. 28 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 25–28 konsumsi sorbitol yang efektif adalah di bawah 60 menit dengan jumlah maksimum yang direkomendasikan adalah 150 mg sorbitol per kilogram berat badan setiap harinya. bila konsumsi berlebihan akan menyebabkan timbulnya diare.27 agar efektivitasnya optimal sebaiknya permen karet sorbitol dikunyah selama 20 menit saja, jadi pemakaian sorbitol sebagai bahan pemanis pengganti sukrosa yang bersifat non kariogenik masih tetap dianjurkan.15 oleh karena itu sorbitol paling baik digunakan sebagai pemanis pada permen karet. mengkonsumsi 6–7 gram sorbitol dalam bentuk permen karet setiap harinya mempunyai suatu efek kuratif terhadap permulaan karies.6 penggunaan permen karet dapat berfungsi untuk merangsang sekresi air liur serta meningkatkan kecepatan sekresi saliva, jadi berguna sebagai pembersih mulut dari sisa makanan karbohidrat yang mudah difermentasi oleh mikroorganisme rongga mulut. juga pembersihan asam yang terbentuk akibat proses glikolisis karbohidrat oleh mikoorganisme asidogenik, karena kecepatan yang tinggi dari saliva akan mengalir di atas plak. 20 selain itu dengan bertambahnya sekresi saliva akan menyebabkan peningkatan kapasitas buffer saliva sehingga dapat menetralkan ph plak yang asam, karena bertambahnya ion bikarbonat (hco3–) yang berperan dalam kapasitas buffer saliva. bertambahnya aliran saliva akan meningkatkan kadar urea, amoniak (nh3), kalsium (ca2+), fosfat (hpo42+), natrium (na+) yang merupakan sumber alkalinitas saliva sehingga dapat menaikkan ph plak yang turun akibat proses glikolisis karbohidrat.15 akibat pertambahan ion kalsium di dalam saliva, maka proses remineralisasi email akan meningkat. hal ini disebabkan sorbitol dapat membentuk senyawa kompleks dengan kalsium yang terdapat di dalam saliva, dan senyawa yang terbentuk ini lebih stabil daripada senyawa kompleks kalsium dengan sukrosa atau glukosa, sehingga proses difusi kalsium ke dalam plak lebih cepat dalam bentuk senyawa kompleks daripada dalam bentuk ion kalsium. proses difusi senyawa kompleks kalsium dengan sorbitol lebih cepat karena senyawa kompleks ini larut dalam air. stimulasi saliva oleh permen karet akan menambah jumlah dan konsentrasi ion-ion ca2+, po43–, f–, dan oh– yang merupakan komponen mineral gigi. 20 sorbitol mempunyai kelebihan, yaitu tidak mempunyai gugus karbonil dalam rantainya. sorbitol kurang reaktif dan tidak menyebabkan pembentukan asam pada plak gigi. kesimpulan dari penulisan makalah ini adalah sorbitol bukan merupakan media yang baik bagi pertumbuhan bakteri dan tidak menurunkan ph saliva,28 sehingga saliva tetap bertahan atau stabil dalam ph tertentu. daftar pustaka 1. sabir a. peranan bahan pemanis dan bahan pengganti gula dalam mencegah karies gigi. surabaya. majalah kedokteran gigi fkg unair 2001 agustus; 34(3a): 291–6. 2. yuyus r, magdarina da, sintawati f. karies gigi pada anak balita di 5 wilayah dki tahun 1993. jakarta: cermin dunia kedokteran no. 134; 2002. h. 1–5. 3. harris no, christen ag. primary preventive dentistry. 4th ed. connectitut. appleton and lange; 1995. p. 342–8. 4. amerongen a, van nieuw. ludah dan kelenjar ludah. yogyakarta: gadjah mada university press; 1991. h. 1–42, 157–71. 5. goldberg i. functional foods. new york: chapmann hall; 1994. p. 219–37. 6. houwink b. ilmu kedokteran gigi pencegahan. yogyakarta: gadjah mada university press; 1993. h. 88–91, 190–3. 7. tarigan r. karies gigi. cetakan 3. jakarta: hipokrates; 1993. h. 17–35. 8. kidd eam, bechal sj. dasar-dasar karies penyakit dan penanggulangannya. cetakan 2. jakarta: egc; 1992. h. 66–96. 9. suwelo is. karies gigi pada anak dengan pelbagai faktor etiologi. jakarta: egc; 1992. h. 23–7. 10. kusumaningsih t. hubungan antara indeks keparahan karies dengan jumlah lactobacillus sp. di dalam saliva anak taman kanak-kanak. majalah kedokteran gigi fkg unair okt-des 1999; 32(4): 291–6. 11. willet np, white rr, rose s. essential dental microbiology. connectitut. prentice-hall; 1991. p. 346–54. 12. nolte wa. oral microbiology with basic microbiology and immunology. 4th ed. saint louis: mosby; 1982. p. 287–9, 304–5, 309–10, 336–8. 13. panjaitan m. berbagai jenis gula untuk penderita diabetes melitus dan pengaruhnya terhadap karies gigi. majalah kedokteran gigi fkg unair juli-sept 1998; 31(3): 102–6. 14. garrow js, james wpt. human nutrition and dietetics. 9th ed. singapore: longman singapore; 1993. p. 40–1, 340–1, 570–7. 15. kanzil lb, santoso r. efek peningkatan ph plak dan potensial remineralisasi dari beberapa pemanis dalam permen karet sesudah makan karbohidrat. majalah ilmiah kedokteran gigi fkg usakti 1999; 2(edisi khusus forum ilmiah vi): 47–50. 16. krisnowati. pengganti gula indosorb ts-35 produksi pt. sorini corporation. konsep laporan wisata kerja pt. sorini corporation; 1997. h. 1–6. 17. almatsier s. prinsip dasar ilmu gizi. jakarta: gramedia pustaka utama; 1994. h. 30–4. 18. linder mc. nutritional biochemistry and metabolism. 2nd ed. connectitut. appleton and lange; 1991. p. 35–40. 19. mahan lk, arlin m. krause’s food, nutrition, and diet therapy. 8th ed. philadelphia: wb saunders; 1996. p. 29–33. 20. edgar wm, geddes dam. chewing gum and dental health. british dental journal 168; 1990. p. 173–7. 21. devlin tm. textbook of biochemistry with clinical correlations. 3rd ed. new york: wiley-liss; 1993. p. 311, 941–2. 22. assev s, rolla g. does the presence of xylitol in sorbitol-containing chewing gum affect the adaptation to sorbitol dental plaque? scandinavia dental journal 102; 1994. p. 281–3. 23. matthews ck, van holde ke. biochemistry. california: the benjamin/cummings; 1990. p. 213–5. 24. murray kr, granner kd, mayes ap, rodwell wv. biokimia harper. edisi 24. jakarta: egc; 1994. h. 217–20. 25. manning rh, edgar wm. ph changes in plaque after eating snacks and meals, and their modification by chewing sugared-or sugar free gum. british dental journal 174; 1993. 241–5. 26. pratiwi t, heriandi s, mangundjadja s, apriati y. pengaruh sorbitol dalam permen terhadap populasi streptokokus mutans di saliva. majalah kedokteran gigi fkg unair agustus 2001; 34(3a): 620–3. 27. williams rad, elliott jc. basic and applied dental biochemistry. 2nd ed. london: churchill livingstone; 1989. p. 307–16, 370–80, 410–29. 28. roeslan bo, sudjana mr. pola ph air liur setelah mengunyah permen karet dengan pemanis sorbitol dan pemanis sukrosa. majalah ilmiah kedokteran gigi fkg usakti 1996; 1 (edisi khusus forum ilmiah v): 477–82. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 9595 dental journal (majalah kedokteran gigi) 2019 june; 52(2): 95–99 research report effects of glycerin application on the hardness of nanofilled composite immersed in tamarind soft drinks titis mustikaningsih handayani,1 raditya nugroho,1 lusi hidayati,2 dwi warna aju fatmawati,1 and agus sumono2 1department of conservative dentistry 2department of dental material faculty of dentistry, universitas jember jember – indonesia abstract background: loss of tooth structure is a consideration in the performance of restorative treatment involving nanofilled composite resins. material polymerization factors and water absorption can affect the hardness of composite resins. imperfect polymerization producing an oxygen inhibited layer (oil) and causing water absorption can even compromise the hardness of nanofilled composite resins. tamarind soft drink, on the other hand, has an acidic ph that compromises the hardness of nanofilled composite resins. purpose: this study aimed to reveal the effects of glycerin application on the hardness of nanofilled composite resins immersed in tamarind soft drinks. methods: the research constituted a laboratory experiment using 24 nanofilled composite resin samples with diameters of 5mm or 2mm, divided into six groups, namely: group g, group g as 60, group g as 120, group tg, group tg as 60, and group tg as 120. glycerin was applied to the surfaces of three groups before curing, while the other three groups were not treated with glycerin. finishing was subsequently conducted on all samples using a highspeed handpiece and superfine finishing bur, before they were polished with a low speed handpiece. the samples were then divided into specific groups, namely: a group with a 120-minute immersion time, a group with a 60-minute immersion time, and a group which was not immersed and maintained at a temperature of 37oc. each sample was tested at three points using a vickers hardness tester (vht). results: the results showed that the groups with glycerin had a higher hardness level than those groups. in addition, the non-immersed groups had a higher hardness level than those groups which were immersed. the one-way anova test results confirmed that there was a statistically significant difference (p<0.05) between all groups. conclusion: the application of glycerin to nanofilled composite resins immersed in tamarind soft drinks can increase their hardness levels. keywords: glycerin; hardness of nanofilled composite resin; tamarind soft drink correspondence: raditya nugroho, department of conservative dentistry, faculty of dentistry, universitas jember, jl. kalimantan 37 jember 68121, indonesia. e-mail: ranugtab@gmail.com introduction loss of tooth structure due to erosion, enamel abrasion and dental caries is one factor leading to restorative treatment.1 one restoration material frequently used to replace the function of missing tooth structures is composite resin which offers the advantages of promoting attractive aesthetics of the anterior teeth and the greater abrasive resistance of the posterior teeth.2,3 composite resin comprises three main components, namely: matrix resin, filler and silane coupling agent. matrix resin consists of bisphenol a-glycidyl methacrylate (bis-gma), urethane dimethacrylate (udma) and tryehtyleneglycol dimethacrylate (tegdma). composite resin filler, ranging from traditional composite resins (macrofillers), microfillers, flowables, packables, hybrids to nanofillers, particularly with regard to its particle size has been improved. nanofillers are composed of smooth particles with the result that the restoration possesses a smooth surface and is aesthetically attractive.4 however, nanofillers suffer from certain disadvantages, one of which is their higher hydrophilic properties compared to other larger types affecting the water absorption of composite resin.5 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p95–99 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p95-99 mailto:ranugtab@gmail.com http://e-journal.unair.ac.id/index.php/mkg 96 handayani, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 95–99 composite resin can experience changes in its mechanical properties due to oral conditions, such as salivary ph and food intake.6 one such vatable property is hardness7 which can, consequently, be considered a measure of the resistance to wear of restoration materials since it can influence mechanical friction during mastication and tooth brushing.8 the properties of composite resin potentially affecting its hardness include: water absorption, composite resin hardness, irradiation distance and material polymerization.8 the polymerization of composite resin materials can be disrupted when their surfaces are exposed to air. the disturbance causes obstruction of the polymerization process, producing an oxygen inhibition layer (oil) which can then affect the prognosis of composite restorations because it reduces surface hardness, durability and marginal adaptation. to minimize the occurrence of oil, glycerin inhibitors can be employed during the curing process. glycerin is stable in a medium of atmospheric oxygen because, when exposed to air, the glycerin will be in equilibrium with the water vapor (relative humidity) in the surrounding atmosphere. therefore, glycerin bonds and the surrounding objects will not experience changes at normal temperatures. hence, glycerin can be used as a barrier to prevent the formation of oil on occlusal surfaces or those of composite resins that are difficult to access.9 another factor affecting the hardness of composite resins is the absorption of water present in the food and beverage consumed daily by patients and in direct contact with tooth surfaces.10 soft drinks constitute one popular beverage consumed by the indonesian public with annual per capita consumption within the country amounting to 33 liters.11 such drinks are non-alcoholic processed liquids containing food ingredients or other additives, both natural and synthetic, which are packaged ready for consumption.12 java tamarind, on the other hand, contains several kinds of acid, including citric acid, in addition to antioxidant compounds which donate h atoms from their phenolic groups, thereby promoting increased antioxidant activity. since they contain a variety of healthy ingredients, the consumption of tamarind-derived soft drinks has increased in popularity.13 such beverages are considered to be soft drinks with a ph of 3.7.12 this level of acidity causes greater micromorphological damage to the composite resin.10 consumption of acidic drinks can also dissolve the composite resin since it contains numerous h ions capable of continuously eroding the composite resin material. this subsequently leads to the degradation of the composite resin component.7 moreover, it also affects its users by compromising surface hardness8 through the process of polymerization triggered by glycerin and water absorption of composite resins. this study aimed to reveal the effects of glycerin application on the hardness of nanofilled composite resins immersed in tamarind soft drinks. materials and methods this research constituted an experimental laboratory study featuring a post-test only control group design. there were six groups, namely: group g to which glycerin had been applied; group g as 60 to which glycerin had been applied and which was immersed for 60 minutes; group g as 120 to which glycerin had been applied and which was immersed for 120 minutes; group tg which was glycerin-free; group tg as 60 to which no glycerin had been applied and which was immersed for 60 minutes; and group tg as 120 which was glycerin-free and which was immersed for 120 minutes. each of the groups consisted of four samples. the immersion time was determined by calculating the duration of the contact between nanofilled composite resins and drinks consumed in each package/day (one minute) for 30 days. the estimated total amount of time required when consuming drinks is one month and two months, which in this study were converted to 30 minutes and 60 minutes.7 the number of samples for each group was based on federer’s formula: (n-1) (t-1) ≥15. consequently, with t (number of groups) being 6, the number of samples in each treatment group was four. nanofilled composite resin (filtextm 3m espe z 350 xt) was produced with a plastic 5 mm x 2 mm sized ring mold that had been inserted into a brass disc and subsequently applied using plastic filling instruments and condensed using a cement stopper. 0.5 ml of glycerin was applied with a microbrush during each treatment before curing (f led-b, woodpecker, usa) was performed for 20 seconds at a distance of 0 mm, forming a plane perpendicular to the resin surface. finishing was carried out using a highspeed handpiece (s max m, nsk, usa) and superfine finishing bur (314 c 850, edenta, switzerland). the samples were then polished using a polishing kit (cw 351 4, ra 0309 tobuom, china).with a low speed handpiece (type ex, nsk, usa) at 15,000 rpm for one minute in the same direction to obtain the same pressure on each side. immersion in 20 ml of tamarind soft drink (ultra jaya tbk, indonesia) contained in glass beakers was conducted, all samples being inserted using tweezers with the entire upper surface of each sample being immersed. the glass beakers were then covered with aluminum foil. following immersion 60 or 120 minutes in duration or no immersion, the samples were incubated at a temperature of 37oc.7 the hardness of each sample was subsequently measured at three points using a vickers hardness tester (vht, 402mvd, wilson®, usa). the points were determined from the center of the composite resin before being shifted 1mm to the right and left and indented on the section focused on the lens. indenting was performed at a pressure of 100 gf for 15 seconds. hardness testing on each sample was conducted on the upper surface of each sample. the pressure exerted by the indenter centered on the three points positioned in line and focused on the observation lens. the hardness results were determined dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p95–99 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p95-99 http://e-journal.unair.ac.id/index.php/mkg 97handayani et. al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 95–99 after the application of vertical and horizontal pressure through the observation lens and calculated as an average per group.14 the normality of the data was analyzed using a shapirowilk test, while a levene’s test assessed its homogeneity producing a significance level of p>0.05. since the data was normally and homogeneously distributed, it was analyzed using a one-way anova test. an lsd test with a significance level of p>0.05 was subsequently conducted to observe differences between the groups. results the results of the research showed that the average vickers hardness number (vhn) across all groups with glycerin was higher than that in those without glycerin. moreover, in all groups with time variations it decreased as the duration of immersion became more extended (table 1). this research indicated differences in the average vhn between all treatment groups (table 2). the highest vhn average occurred in the group with glycerin (g), while the lowest average was recorded by the group without glycerin (tg as 120) with an immersion time of 120 minutes. the results indicated differences in the average vhn between the treatment groups featuring glycerin application and those without. the groups with glycerin application (g, g as 60 and g as 120) had a higher vhn than those without (t, tg as 60 and tg as 120). there were also differences in the average vhn between the groups with glycerin application and those groups without after the same immersion time (table 3). the results of this research revealed differences in the average vhc between the treatment groups with differing immersion times (without immersion, 60-minute immersion, and 120-minute immersion). the group with a 120-minute immersion time recorded the lowest average vhn with the result that it recorded the highest difference in the average vhn compared to the group without immersion. moreover, the difference in the average vhn of the 60-minute immersion group and that of the 120-minute immersion group indicated that there was a decrease in the average vhn based on the duration of immersion. the data obtained was analyzed by means of a shapiro-wilk normality test the results of which showed a significance value greater than 0.05, thereby indicating that the data was normally distributed. the results of a subsequent levene’s test indicated a significance value of 0.058 (p>0.05) and confirmed the homogeneity of the data. table 4 shows that a one-way anova parametric test produced a significance result of 0.000 (p<0.05) indicating differences between the test groups. subsequent multiple post-hoc comparison tests followed by an lsd test produced a significance result of 0.000 (p<0.05) across all groups with the exception of groups g as 120 and tg as 60 (0.002). therefore, significant differences can be said to have existed between treatment groups. table 1. the average of vhn in all groups groups the average of vhn g 98.12 ±0.46 g as 60 67.34 ±0.85 g as 120 61.10 ±1.39 tg 72.24 ±0.91 tg as 60 63.72 ±1.21 tg as 120 54.38 ±0.9 table 2. differences in the average of vhn based on glycerin application treatment groups difference in the average of vhn g tg 25.88 g as 60 tg as 60 3.62 g as 120 tg as 120 6.72 table 3. differences in the average of vhn based on immersion treatment groups difference in the average of vhn differences in the average of vhn between immersion times glycerin immersion for 60 minutes 30.78 6.24 immersion for 120 minutes 37.02 without glycerin immersion for 60 minutes 8.52 9.4 immersion for 120 minutes 17.92 table 4. one-way anova parametric test results sum of squares df between groups 4648.555 5 within groups 18.098 18 total 4666.653 23 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p95–99 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p95-99 http://e-journal.unair.ac.id/index.php/mkg 98 handayani, et al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 95–99 discussion the results showed that the groups using composite resins without glycerin application, but with varied immersion times, had lower vhn than the groups using composite resin with glycerin application and varied immersion times. the average vhn values in the group with a 120-minute immersion time was lower than in the 60-minute immersion group and in the non-immersion groups due to the nature of the effect of the polymerization process on surface hardness and water absorption of composite resins. table 2 indicates that the reduction in the surface hardness of the glycerin-free groups was influenced by several factors including the degree of polymerization conversion and the presence of oil layers. the degree of conversion resulting from the polymerization process is the percentage of carbon metal methacrylate double bonds capable of binding to free radicals and producing a single bond which forms a polymer chain. up to 50-70% of c = c covalent bonds can be converted to produce approximately 30-50% of metal methacrylate which do not undergo initiation with free radicals.2 this is also supported by the glycerin-free treatment with the result that free radicals bind with oxygen to produce peroxyl radicals:15 r • + o2 = roo • free radicals + oxygen = peroxyl radicals free radicals that bind to oxygen reduce the number of free radicals that should bind to the covalent c = c bond in metal methacrylate.2 the results of monomers that are not bound to free radicals forming oil when combined with the remainder of the unconverted methacrylate monomer can induce changes in the surface hardness of the composite resin.15 during this research, a droplet of glycerin was applied to the surface of the composite resins. its consistency did not harden during curing and had a transparent color which did not affect the irradiation distance and intensity of the led light during polymerization. the application of glycerin is intended to prevent the bond between free radicals and oxygen in order to increase the surface hardness of composite resins.15 other factors affecting polymerization of composites include: exposure time, exposure distance and composite resin thickness.8 all groups were irradiated for 20 seconds as recommended with a irradiation distance of 0 mm in the perpendicular position and with the same thickness of 2 mm. these factors were used as dependent variables applied to all samples in order that they did not affect the hardness of the composite resin. the hardness of nanofilled composite resins is also influenced by water absorption.8 the specific resin used in this research was filtex tm z350 xt transluscent shade which contains bis-gma, udma, tegdma and bis-ema resin. tegdma constitutes one of the comonomers possessing the largest hydrophilic properties at 69.51 µg/mm3. udma monomers also contain the element o which is electronegative with the result that it tends to attract oh groups which release h+ from water.16 this research employed a combination of non-agglomerated and non-aggregated fillers which included silica 20 nm in size and zirconia 4-11 nm size. non-aggregated fillers are ones that do not undergo central collection in a specific area with the result that they possess large surfaces.2 the surface of zirconia filler is porous which facilitates the absorption of water by the composite resins.17 water absorption in composite resin subsequently causes degradation of composite resins which involves the loss of chemical structure in composites such as bis-gma. this is due to various factors including hydrolysis and water-related environmental influences.6 in the composite resin groups involving immersion in tamarind drinks, the surface hardness was lower than that in the groups without immersion in tamarind drinks. this indicates that food and beverages consumed can affect the hardness of composite resins. tamarind soft drinks have an acidic ph of approximately 3.7. low ph drinks (3-6) damage the resin surface. hence, the ph value can be an indicator determining h+ ion content since at low ph levels it will be higher.5 h+ ions are absorbed into the matrix and react with the ester group of dimethacrylate monomers, forming carboxylic acids and alcohol molecules. dimacrylate monomers that bind to h+ ions break down the double bond of the resin monomer into a single bond and produce oh-. this causes expansion of the material, softening of the matrix and enlargement processes.17,18 in addition, water absorption caused by immersion also results in degradation of siloxane bonds (bonds between silanol groups on the surface of silica and silane coupling agents) through hydrolysis reaction. water in contact with the surface of silica particles can break the bond of siloxane and subsequently trigger a bond between the particles of the filler material which can increase weight loss in the composite resin. consequently, this affects the bond between the filler and the resin matrix causing it to become unstable.19 bonds are released and the material becomes porous. the release of this filler then causes numerous small gaps in the composite resin with the result that it becomes less dense, thereby reducing its mechanical properties, namely its hardness. one of the acidic elements within the composition of soft drinks derived from tamarind is citric acid containing electropositive h+ ions which are readily attracted to the double bond o with electropositive properties. this condition can promote degradation of the bis-gma monomer and siloxane bonds by a similar mechanism to that produced by hydrolysis. this process can subsequently cause reduced hardness in composite resins. it can be argued that the longer the immersion time, the lower the average vhn. the consumption of acidic drinks, such as soft drinks and fruit juices, can actually reduce the hardness of the composite resin due to surface dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p95–99 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p95-99 http://e-journal.unair.ac.id/index.php/mkg 99handayani et. al./dent. j. (majalah kedokteran gigi) 2019 june; 52(2): 95–99 damage caused by material they contain.20 immersion of composite resin for 60 minutes and 120 minutes resulted in a gradual decrease in hardness depending on the length of the immersion time. the decrease in the average vhn was due to water absorption requiring a significant period to reach equilibrium. consequently, the lowest hardness value was registered by the group with an immersion time of 120 minutes, but without the application of glycerin.9,14 it can be concluded that the application of glycerin is capable of increasing the hardness of composite resins immersed in tamarind soft drinks. references 1. van noort r. introduction to dental materials. 3rd ed. amsterdam: mosby/elsevier; 2007. p. 127–43. 2. sakaguchi rl, powers jm. craig’s restorative dental materials. 13th ed. philadelphia: mosby elsevier; 2012. p. 162–94. 3. tuncer d, karaman e, firat e. does the temperature of beverages affect the surface roughness, hardness, and color stability of a composite resin? eur j dent. 2013; 7(2): 165–71. 4. anusavice kj, phillips rw, shen c, rawls hr. phillips’ science of dental materials. 12th ed. budiman ja, purwoko s, translators. jakarta: egc; 2013. p. 275–306. 5. valinoti ac, neves bg, da silva em, maia lc. surface degradation of composite resins by acidic medicines and ph-cycling. j appl oral sci. 2008; 16(4): 257–65. 6. rinastiti m, özcan m, siswomihardjo w, busscher hj. effects of surface conditioning on repair bond strengths of non-aged and aged microhybrid, nanohybrid, and nanofilled composite resins. clin oral investig. 2011; 15(5): 625–33. 7. sitanggang p, tambunan e, wuisan j. uji kekerasan komposit terhadap rendaman buah jeruk nipis (citrus aurantifolia). j e-gigi. 2015; 3(1): 229–34. 8. kafalia rf, firdausy md, nurhapsari a. pengaruh jus jeruk dan minuman berkarbonasi terhadap kekerasan permukaan resin komposit. odonto dent j. 2017; 4(1): 38–43. 9. park h-h, lee i-b. effect of glycerin on the surface hardness of composites after curing. j korean acad conserv dent. 2011; 36(6): 483–9. 10. yanikoğlu n, duymuş zy, yilmaz b. effects of different solutions on the surface hardness of composite resin materials. dent mater j. 2009; 28(3): 344–51. 11. rosyada h, ardiansyah bg. analisis fisibilitas pengenaan cukai atas minuman berpemanis (sugar-sweetened beverages). kaji ekon keuang. 2017; 1(3): 229–41. 12. cahyadi w. analisis & aspek kesehatan bahan tambahan pangan. 2nd ed. jakarta: bumi aksara; 2009. p. 134. 13. andari es, wulandari e, robin dmc. efek larutan kopi robusta terhadap kekuatan tekan resin komposit nanofiller. stomatognatic (jkg unej). 2014; 11(1): 6–11. 14. ikhsan n. perbedaan kekerasan permukaan bahan restorasi resin komposit nanofiller yang direndam dalam minuman ringan berkarbonasi dan minuman beralkohol. thesis. padang: universitas andalas; 2016. p. 59–60. 15. strnad g, kovacs m, andras e, beresescu l. effect of curing, finishing and polishing techniques on microhardness of composite restorative materials. procedia technol. 2015; 19: 233–8. 16. ren y-f, feng l, serban d, malmstrom hs. effects of common beverage colorants on color stability of dental composite resins: the utility of a thermocycling stain challenge model in vitro. j dent. 2012; 40: e48–56. 17. rahim tnat, mohamad d, md akil h, ab rahman i. water sorption characteristics of restorative dental composites immersed in acidic drinks. dent mater. 2012; 28(6): e63–70. 18. drummond jl. degradation, fatigue, and failure of resin dental composite materials. j dent res. 2008; 87(8): 710–9. 19. handayani dp, puspitasari d, dewi n. efek perendaman rebusan daun sirih merah (piper crocatum) terhadap kekerasan permukaan resin komposit. maj kedokt gigi indones. 2016; 2(2): 60–5. 20. hengtrakool c, kukiattrakoon b, kedjarune-leggat u. effect of nat u r a l ly a cid ic agent s on m ic roha rd ness a nd su r fa c e micromorphology of restorative materials. eur j dent. 2011; 5(1): 89–100. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 32a/e/kpt/2017. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v52.i2.p95–99 http://dx.doi.org/10.20473/j.djmkg.v52.i2.p95-99 http://e-journal.unair.ac.id/index.php/mkg mkg vol 41 no 4 oct-dec 2008.indd 167 vol. 41. no. 4 october–december 2008 review article the relation of periodontal diseases to systemic diseases melanie sadono djamil1 and boedi oetomo roeslan2 1biochemistry and oral biology department, faculty of dentistry, trisakti university 2lecturer, immunopathology and molecular biology, postrgaduate program, trisakti university abstract background: the relationship between systemic disorders and periodontal disease has been studied extensively. with few exceptions, it is more accurate to consider systemic diseases to be contributing factors in the pathogenesis of periodontal disease rather than the primary etiologic factors. the development of periodontal disease cannot be separated from the weakening of immunologic and immunopathological responses. periodontal disease may enhance susceptibility to certain systemic diseases in several ways. lipopolysaccharide (lps) and gram-positive bacteria in the biofilm and proinflammatory cytokines produced from inflamed periodontal tissues may enter the circulation system causing the development of certain systemic diseases. on the other hand, through immunologic mediators, certain systemic disease may enhance susceptibility to periodontal disease caused by the decrease of immune responses and the increase of proinflammatory cytokines. purpose: the purpose of this article is to review the immunologic aspect of two way relationship between systemic diseases and periodontal diseases. review: this review studied the relationship between general health status, systemic diseases, and periodontal diseases through immunopathological responses and the weakening of the immune system in the periodontal tissue. conclusion: there is a two-way relationship between periodontal diseases and systemic diseases. key words: periodontal disease, systemic diseases, immunological, two-way relationship correspondence: melanie sadono djamil, biochemistry and oral biology department, faculty of dentistry, trisakti university. fakultas kedokteran gigi universitas trisakti. kyai tapa, grogol 260. jakarta, indonesia. e-mail: melaniehendriaty@yahoo.com introduction the rising of human life expectancy has made periodontal diseases and their treatment become more complex. advanced aging and systemic diseases such as diabetes mellitus and cardiovascular diseases often lead to complications in periodontal tissue. similarly, patients using medication on a continual basis, such as steroids, anti-coagulants or immunosuppressives often rise manifestations in the periodontal tissue or complications when carrying out actions for treatment. with regard to the increase of systemic diseases experienced by today’s population, early identification needs to be done about the potential medical risks related to periodontal diseases and the success of treatment.1 the relationship between general health status, systemic diseases, and periodontal diseases has been studied intensively. with few exceptions, it has been shown that the contribution of systemic diseases to the pathogenesis of periodontal diseases is more significant than primary etiologic factors.2 the development of periodontal diseases cannot be separated from the immunopathological responses and the weakening of the immune system in the periodontal tissue.3 periodontal diseases increase the susceptibility to systemic diseases through various ways. lipopolysaccharide (lps) and gram-positive bacteria in the biofilm and periodontal tissue proinflammatory cytokines may enter the circulation leading to certain systemic diseases such as cardiovascular diseases.4 on the other hand, through immunologic mediators, some systemic diseases may increase the susceptibility to periodontal diseases or even make them worse. this is generally caused by the declining immune response due to the systemic diseases, while some systemic diseases result in periodontal complications or vice-versa can be explained immunologically. the article focuses on diabetes mellitus and cardiovascular diseases, in consideration that these two systemic diseases are commonly found in today’s modern society. 168 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 167−172 immune response of periodontal diseases the trigger of periodontal diseases is the gram-negative bacteria on the surface of the roots of teeth, known as biofilm. lipopolysacharide (lps) in the membrane of gram-negative bacteria and other compounds increase the access to gingival tissue, at first, and give rise to immunoinflammation that causes the production of a high level of proinflammatory cytokines which then induce the metaloproteinase matrix resulting in the destruction of gingival connective tissue and periodontal ligaments. beside that, prostaglandin as a mediator bone will be produced.5 the components of the immune response to periodontal diseases include salivary iga whose function is to eliminate attachment of bacteria colonization on the surface of teeth and mucous membrane. the neutrophil, antibodies, and complements act as bactericides, while lymphocyte, macrophage, and lymphokine act as tissue damager, as well as the immunoregulatory system that controls the immune response.4 the hypersensivity reactions that are similar in periodontal diseases are type i – anafilaxis, type ii – cytotoxic, and type iii – immune complex.6 at the onset of periodontal diseases, adhesion and aggregation of bacteria is impeded by antibodies and complements in the fluids of the gaps in the gums at the same time dissolve it so that a reduction in the amount of bacteria occurs. when the host immune response is insufficient, the products of the following bacterial invasion will continue. in this situation, the destruction of bacteria is mediated by antibody-complements as well as chemotaxis and phagocytosis effects, especially by neutrophil polymorphonuclear (pmn) leukocytes. the damage of periodontal tissue is caused by the type ii hypersensivity reaction, which is mediated by antibodies, cellular immune response, and tissue factor activities, such as collagenase. when the immune response is adequate, repair and fibrosis occur as a result from the fibroblast activity.3 the role of pmn neutrophil in the immune response of periodontal diseases is very important. it functions as the maintenance center for the integrity of the periodontal ligaments.7 in malignant periodontal disease, the capacity of pmn neutrophil seems to decline in controlling the periodontal pathogen.8 neutrophil abnormalities, including chemotactic damage, caused deficiency in adhesiveness, and the lack of specific granules as destroyers and neutralizers of microorganisms and their products, will result in malignant periodontal disease. in addition to being a bacteria destroyer through phagocytosis, macrophage is also important in the defence system against the development of periodontal diseases.7 cytokines secretes macrophage which not only function in destroying the target cells, but also has a side effect on the host cells.9 the cytokine and other biochemical compounds that are secreted by the macrophage in response to the endotoxin stimulation, the immune-complex, or lymphokine, include interleukin-1 (il-1), il-6, il-8, il10, tumor necrosis factor-α(tnf-α), stimulator factors, impediments, and growth, as well as prostaglandin and cyclic adenosine monophosphate (camp). collagenase macrophage has a significant role in the destruction of collagen in periodontal diseases. other cells which play important role in immune respons of periodontal diseases are lymphocyte-t, lymphocyte-b as a precursor of plasma cells, natural killer (nk) and killers (k) cells, and mast cells7 that secrete active pharmacological compounds such as histamines, slow-reacting substances of anaphylaxis (srs-a), heparin, eosinophil chemotactic factor of anaphylaxis (ecf-a), and bradycynine.3,7 the important humoral immune response mediators in periodontal diseases are antibodies, especially igg, and complements. however, potential mediators in inflammation including arachidonate acid derivatives, particularly prostaglandin e2 (pge2), and cytokines, particularly il-1, il-2, il-4, il-6, and tnf-α. all of these humoral mediators can be used as signs for diagnosis of periodontal diseases.7 cytokines are presumed to play an important role in periodontal pathologic changes. the level of il-1 in gingival tissue and the gum-gap fluids would declined after periodontal treatment,7 followed by increasing fibroblast procollagen, prostaglandin e2 (pge2), and bone resorption activity.4 il-2 which stimulates macrophage activity also rises in periodontitis. the same applied to il-4 as an activator of b cell proliferation and differentiation, growth of t cells, the function of macrophage, and growth of mast cells. the induction of antibody production, il-6, increases its level in gum inflammation and plays a role in bone resorption.10 the ability of leukocytes to adhere to endothelium cells will rise because of the tnf-α induction and also the phagocytosis and its chemotaxis. the effect of tnf-α on leukocytes and its induction against macrophage plays a role in vascular changes as occurs in periodontal diseases.3 cytotoxicity of tissue cells can be caused by the direct interaction of lymphocyte with the target cells with specific antigens on their surface. the antigens will be responsed by the lymphocyte, which is generally very specifically sensitized, the cytotoxic effect of the host cell-lymphocyte interaction is usually not specific. therefore, it is estimated that the persistence of the tooth plaque antigen deposits in the periodontal tissue is assisted by the formation of cells that produce lymphotoxin and/or directly because of the lymphocytoxity. this incidence can result in tissue damage in cases of periodontal abnormality.7 in this paradigm, periodontal disease represents a well-regulated response to protracted bacterial infection directed by the inflammatory cells of the host immune system (figure 1). 169djamil and roeslan: the relation of periodontal diseases neutrophil abnormalities neutrophil or pmn leukocytes play a very important role in the defence mechanism against bacterial infection. the production abnormalities and functions of neutrophil will increase the susceptibility to bacterial infection. in the body’s defence system, neutrophil is the main phagocyte against extracellular bacteria. a person with neutrophil abnormalities, both quantitative (neutropenia) and qualitative (adhesion, chemotaxis, microbicidal activity), often had experiences with the periodontal diseases.11 a pmn decrease often results in serious periodontal disease12 and this has implications for its treatment process. several periodontal diseases that are related to neutrophil can be seen table 1. cases showing neutrophil as a key protective cell against periodontal infection support the concept that any disturbance to the functions of neutrophil is a risk factor in the development of periodontal diseases.9 in immunology, the function of neutrophil response is like a two-edged sword: the main role not only acts as protection to the host tissue, but also as a proinflammation cell that can cause tissue damage. therefore, neutrophil dysfunction is often related to periodontal diseases, usually through the weakening of host resistance to periodontal pathogens. diabetes mellitus the relationships between diabetes mellitus and the prevalence and seriousness of periodontal diseases have long been studied. increased periodontopathic bacteria, the dysfunction of neutrophil, increased cytokine mediators, and changes in connective tissue in diabetes mellitus have contributions to the seriousness of periodontal diseases. increasing periodontopathic bacteria colonies are mostly caused by weak body’s defence mechanisms as a complement of hyperglycemia.13 the glucose level in diabetes mellitus saliva rose evidently.14 these conditions will amplify the growth of microorganisms. the dysfunction of neutrophil in diabetes mellitus usually increases the susceptibility to periodontitis.9 chemotaxis disturbances and phagocytosis also occur in diabetes mellitus.13 diabetes mellitus depends on insulin because of the auto-immune reaction, the means being through genes that are related to the hla-dr3, hladr4, and hla-drq regions. it is very interesting to note that these regions are also related to forms of progressive periodontal diseases. the disturbance of synthetic collagen together with the cellular response failure to injured tissue in diabetes mellitus can result in the slow healing of wounds.13 the two-way relationship between periodontal diseases and diabetes mellitus has been developed through a hypothetical model. the connective tissue damage in periodontal diseases is the result of the interaction of the bacteria and their products with the mononuclear phagocyte and fi broblast. this interaction will trigger infl ammation mediator activity and local secretions, especially il-1β, pge2, tnf-α, and il-6. the biological mechanism that causes diabetes mellitus to contract serious periodontal diseases is mediated by the accumulation of advanced glycation end product (age). this product is formed because glycation of protein non-enzymatically as a result of hyperglycemia. mononuclear phagocyte will take the age through the receptor advanced glycation end product (rage) or macrophage scavenger receptor (msr). as a result, the mononuclear phagocyte is stimulated to proliferate and induce free radicals and proinfl ammatory cytokines. the free radicals will directly damage the tissue, while the cytokines will activate other cell infl ammation resulting in tissue damage.15 in the condition of hyperglycemia, the persistence of the proinfl ammation figure 1. scheme of the host bacterial interaction in periodontal diseases.5 table 1. periodontal abnormalities related to neutrophil3 neutrophil damage related to perio dontal disease abnormalities periodontal abnormalities with neutrophil damage diabetes mellitus acute necrotizing ulcerative gingivitis papillon-lefevre syndrome local juvenile periodontitis down’s syndrome prepuberty periodontitis chediak-higashi syndrome rapidly progressing periodontitis granulocytosis due to medication refractory periodontitis cyclic neutropenia 170 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 167−172 of cells that is activated will increase its adhesiveness as a result of the cytokine stimulation, which will further worsen periodontal tissue damage.13 periodontopathic microorganism parts such as lipopolysaccharide (endotoxin), lypoteicoat, short chain fatty acids, and proteinase will activate the synthesis and secretion of especially il-1β, pge2, tnf-α, and il-6 by mononuclear phagocyte. tnf-α in particular will induce insulin resistance and reduce its action. consequently, the condition of serious hyperglycemia will occur with result that protein will be glycated and there will be an accumulation of age protein. the binding of age by rage will induce an expressive regulation of cytokine and oxidative pressure. in addition, a hydrolase secretion and metaloproteinase matrix will occur that will worsen the periodontal tissue damage.13, 15 cardiovascular diseases epidemiological studies show that one of the factors that is biologically potential as a cause of vascular disease is infection in the oral cavity have shown that there is a relationship between the bad state of oral health and cardiovascular diseases without being affected by other factors.1 in the research fi ndings, that there was a relationship between infections in the mouth and arteriosclerosis.16 this has been backed up by many research reporting the relationship between periodontal and cardiovascular diseases. a long-term study over ten years, has shown that periodontal disease could be used as a predictor of cardiovascular disease.17 periodontal disease is an additional cardiovascular disease risk factor, acting as a predispositionary factor because periodontal diseases are a low degree chronic infection.18 infections are considered to be risk factors for arteriogenesis and thromboembolism. periodontal infections and a host response that is not good will lead to chronic infl ammation. an in vitro thrombosis model showed that certain plaque bacteria such as s. sanguis and p. gingivalis can induce platelet aggregation.19 during periodontitis, plaque bacteria, particularly p. gingivalis,5 will invade through the blood, which then infects the vascular endothelium resulting in arteriosclerosis which is a risk factor in the occurrence of ischemia and myocardial infarct. those two disorders can also be cause by thromboembolism. in vitro, plaque bacteria, including s. sanguis and p. gingivalis, can induce platelet aggregation. intravenous infusion of s. sanguis in rabbits caused changes in the electrocardiogram, heartbeat, blood pressure, and heart contractions. these changes are consistent with what happens in myocardial infarct.20 meurman et al. (2004) developed a hypothetical model on the relationship between periodontal diseases and arteriosclerosis, heart attack diseases and stroke. from one individual to another, differences were found in responses to bacterial infections. the differences were found in the t cells and the capacity to secrete monocyte. some individuals will respond to lps with an infl ammation response that is refl ected through release of infl ammation mediators such as il-1β, pge2 and tnf-α at a high level. individuals with a hyperinfl ammatory monocyte phenotype (m∅+) will secrete these mediators 3–10 times the normal monocyte phenotype. m∅+ is found in patients with early-onset periodontitis, refractory periodontitis and diabetes mellitus depending on insulin. the relationship of m∅+ with periodontal infections and arteriosclerosis is mediated by cells that are one channel with monocyte and proinfl ammatory cytokine that have a critical role in starting and increasing the formation of ateroma and periodontal diseases. the m∅+ exists because of genetical factors and environmental changes. hyper-responsive monocyte to lps has been mapped to be found in hla-dr3/4 or –dq areas that are also the regions, which can increase the susceptibility to diabetes mellitus depending on insulin. diet that can induce an increase in low density lipoprotein (ldl), such as fat, also affect the monocyte response to lps resulting in increasing secretion of proinfl ammatory cytokine that can destroy tissue. through this mechanism, an increase in the seriousness of periodontal and cardiovascular diseases will occur.21 hormonal imbalance puberty, menstruation, pregnancy, and use of contraception pills show the existence of changes in the composition of microfl ora in the oral cavity, both quantitatively and qualitatively.22 at the same time as the decline in the immune response to periodontal pathogens, periodontal diseases will develop. during menstruation, periodic granulocytic leukopenia is found that results in changes in the gingiva. in addition to increased levels of systemic estradiol and progesterone during pregnancy, pressure on the lymphocyte-t response result in increased anaerobic fl ora23 that will activate cell infl ammation. hematological abnormalities hematological abnormalities that cause declines in host immune response will increase bacterial invasions in plaque to periodontal tissue. this is seen in leukemia sufferers because of the occurrence of granulocytopenia.23 this is the same as what happens in immunodefi ciency in cellular, humoral, combinations of cellular and humoral aspects as well as complements with various manifestations.4 the most evident manifestation is in periodontal tissue in cases of acquired immune defi ciency syndrome (aids).23 use of medication cyclosporine, an immunosuppressive that is effective for preventing rejection in kidney, heart or liver transplants,8 has the side effect of selectively depressing the lymphocytet subpopulation and influencing the production of lymphokine, il-1 and il-2.6 phenacetin, which is used in the control of epilepsy, suppresses serum iga.8 control of malignancy with chemotherapeutic substances has direct effects at the cellular level and not indirectly through myelo-immunosuppression of hematopoietic and lymphoid tissue.6 secondary immunodefi ciency can also occur in 171djamil and roeslan: the relation of periodontal diseases the use of various types of medicine, such as phenacetin, chloramphenicol, or anti-thyroid and anti-convulsion medicines.8 acute periodontal infection in users of these medicines occurs because of exacerbation of chronic conditions due to the increased periodontopathic bacteria during episodes of granulocytopenia. stress resorption of the alveolar bone, degeneration of periodontal ligaments, decline of osteoblast activity, the formation of periodontal pockets, and the slow healing of connective tissue and bones can be related to conditions of stress. a weak infl ammation response and low periodonsium resistance accompanied by ischemia will increase the invasion of periodontopathic bacteria.6 in states of chronic stress the siga level declines, and in states of acute stress the level rises. this is possibly caused by the increased cortisol in saliva during stress.24 in addition, the rising cortisol will affect the immune response by reducing the chemotactic and phagocytic pmn response.6 discussion until now, the general opinion is that systemic diseases will result in periodontal diseases. however, the new paradigm on the relationship of periodontal and systemic diseases is that there is a two-way relationship, even that periodontal diseases may cause and worsen diabetes mellitus and cardiovascular diseases. it could be said that the majority of periodontal diseases are related to the immune response and therefore every systemic disease that has implications for a decline in host immune response has a connection with periodontal disease. besides the decline in the phagocytic amount and functions, both neutrophil and mononuclear phagocyte, proinfl ammatory cytokines, such as il-1β, pge2, tnf-α, and il-6 that are secreted by mononuclear phagocyte, plays a big role as mediators of periodontal disease with diabetes mellitus13 and cardiovascular diseases.21 the old paradigm showed that periodontal diseases are a manifestation of diabetes mellitus, but the hypothetical model recently developed13 shows that periodontal diseases increase the insulin resistance so that hyperglycemia occurs and diabetes mellitus arises or is worsened. the seriousness of diabetes mellitus increases the serious of periodontal diseases. this two-way relationship is mediated by the age protein. the macrophage of diabetes mellitus sufferers will bind the age so that it activates the synthesis and secretion of local proinfl ammatory cytokines that lead to the destruction of connective tissue and bone resorption in periodontal diseases. simultaneously, periodontal infection induces a chronic condition of insulin resistance resulting in hyperglycemia. the presence of the m∅+ phenotype will place certain individuals in the risk position of contracting arteriosclerosis/cardiovascular diseases and periodontal diseases.25 the critical control roles of the activity channel, lps with microorganism and cytokine mediators, are very important in the process of the occurrence of periodontal diseases and arteriosclerosis as well as thromboembolism which is cause by infection. in addition to genetical factors, diet can exacerbate the hyperinflammatory monocyte phenotype that contributes to the occurrence of arteriosclerosis and periodontal diseases. periodontal infections have a direct contribution on the pathogenesis of arteriosclerosis and thrombosis through continuous stimulation of lps and proinflammatory cytokines. periodontal pathogens themselves can also be a factor of the etiology of thromboembolism that can cause arteriosclerosis which is a risk factor in the occurrence of ischemia and myocardial infarct.21 the relationships between hormonal imbalances, hematological abnormalities, medication, and stress and periodontal diseases are more caused by pressure on the host immune response. hormones and certain medicines, immunodeficiency, or stress will reduce the host’s endurance so that the normal fl ora in the oral cavity will change, both quantitatively and qualitatively. the result, the development of periodontal diseases cannot be held off any longer. based on the matters discussed, it can be seen that periodonsium tissue health cannot be separated from general health. chronic infections in the oral cavity, particularly periodontal infections, have a wide impact. periodontal diseases are particularly connected to the seriousness of diabetes mellitus and the increased incidence of cardiovascular diseases. the problem that remains is how dentists can convince the patients, community, and other health personnel about the relationships between periodontal diseases and diabetes mellitus and cardiovascular diseases. in conlusion, there is a two-way relationship between periodontal diseases and diabetes mellitus: the latter causes the former and vice-versa, and the mediator is the age protein. the two-way relationship between periodontal and cardiovascular diseases occurs because of the similarity of pathogenesis in the critical control path of lps activity with microorganism create cytokine mediators. in both the relationships of periodontal diseases with diabetes mellitus and with cardiovascular diseases, the head of the spear is the proinfl ammatory cytokines. the occurrence of periodontal diseases is because of hormonal imbalances, hematological abnormalities, medication, and stress is more caused by pressure on the host immune response. references 1. boehm tk, scannapieco fa. the epidemiology, concequences and management of periodontal disease in older adults. j am dent assoc 2008 mar; 139(3): 252–3. 2. grollmus nzc, chavez mmc, donat sfj. periodontal disease associated to systemic genetic disorders. med oral patol oral cir bucal 2007; 1(12): 3. 172 dent. j. (maj. ked. gigi), vol. 41. no. 4 october–december 2008: 167−172 3. roeslan bo. immunologi. kelainan di dalam rongga mulut. jakarta: abadi dhaya insani; 2000. p. 51–55. 4. samaranayake l. essential microbiology for dentistry. 3rd ed. united kingdom: churchill livingstone. elsevier; 2006. p. 149-52; 275–83. 5. kinder haake s, nisengard rj, newman mg, miyasaki kt. microbial interaction with the host in periodontal diseases. in: newman mg, takei hh., carranza fa, han tj, flemming tf, goodman sf, cooney jp, chang tl, editors. carranza’s, clinical periodontology. 9th ed. philadelphia: saunders; 2006. p. 137-46. 6. chung hy, lu hc, chen wl, lu ct, yang yh, tsai cc. immunoglobulin g profiles in different forms of periodontitis. j periondontal res 2003; 38(5): 471–6. 7. nisengerad rc, newman mg, sanz m. host response: basic concepts. in: carranza fe, newman mg, editors. clinical periodontology. 9th ed. philadelphia: saunders; 2006. p. 111–20. 8. lehner t. immunology of oral diseases. 4th ed. london: blackwell sci; 2000. p. 48–50, 58, 63–4. 9. al-zahrani ms, kayal ra, bissada nf. periodontitis and cardiovascular disease: a review of shared risk factors and new findings supporting a causality hypothesis. quintessence int 2006; 37(1): 11–8. 10. takahashi k, nishimura f, kurihara m, iwamoto y, takashiba s, miyata t, murayama y. subgingival microflora and antibody responses against periodontal bacteria of young japanese patients with type 1 diabetes mellitus. j int acad periodontol 2001; 3(4): 104–11. 11. chaves es, jeffcoat mk, ryerson cc, snyder b. persistent bacterial colonization of porphyromonas gingivalis, prevotella intermedia, and actinobacillus actinomycetemcomitans in periodontitis and its association with alveolar bone loss after 6 months of therapy. clin periodontol 2000; 27(12): 897–903. 12. trindade sc, gomes-filho is, meyer rj, vale vc, pugliese l, and freire sm. serum antibody levels against porphyromonas gingivalis extract and its chromatographic fraction in chronic and aggressive periodontitis. j int acad periodontol 2008; 10(2): 50–8. 13. perrino ma. diabetes and periodontal disease: an example of an oral/systemic relationship. ny state dent j 2007; 3(5): 38–41. 14. roeslan, bo, kartini, febrianti i. kadar glukosa dan aseton di dalam saliva penderita diabetes melitus. j.k.g.i. pdgi. 2002; 52:26-30. 15. mustapha iz, debrey s, oladubu m, ugarte r. markers of systemic bacterial exposure in periodontal disease and cardiovascular disease risk: a systematic review and meta-analysis. j periodontol 2007; 78(12): 2289–302. 16. mattilla kj, pussinen pj, paju s. dental infections and cardiovascular diseases: a review. j periodontol 2005; 76(11 suppl):2085-815. 17. skagamas m, breen tl, leroith d. update on diabetes mellitus: prevention, treatment, and association with oral diseases. oral dis 2008; 14(2): 105–14. 18. booth v, solakoglu o, bavisha n, curtis ma. serum igg1 and igg2 antibody responses to porphyromonas gingivalis in patients with periodontitis. oral microbiol immunol 2006; 21(2): 93–9. 19. guo s, takahashi k, kokeguchi s, akashiba s, kinane df, murayama y. antibody responses against porphyromonas gingivalis infection in patients with early-onset periodontitis. j clin periodontol 2000; 27(10): 769–77. 20. deliargyris p, madianos w, kadoma i, marron s, smith j, beck s, offenbacher. periodontal disease in patients with acute myocardial infarction: prevalence and contribution to elevated c-reactive protein levels. american heart journal 2004; 147(6): 1005–1009 e. 21. meurman jh, sanz m, sok-ja j. oral health, atherosclerosis, and cardiovascular disease. crit rev oral biol med 2004; 15(6): 403–13. 22. li zhang, huanxin meng, qiyan li, hongshan zhao, li xu, zhibin chen, dong shi, xianghui feng. estrogen receptor-� gene polymorphisms in patients with periodontitis. j period res 2004; 39(5): 362–6. 23. antman em, braunwald e. acute myocardial infarction in principles of internal medicine. 15th ed. mc graw hill, usa: harrisons churchill livingstone. elsevier, uk; 2001. p. 1378–9. 24. lee sr, kwon hk, song kb, choi yh. dental caries and salivary immunoglobulin a in down syndrome children. j paediatr child health 2004; 40: 530–3. 25. scannapieco fa, bush rb, paju s. associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, andstroke. a systemic review. ann periodontol 2003; 8: 38–53. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vol 38-no 1-2005 29 kekuatan perlekatan geser semen ionomer kaca terhadap dentin dan nicr alloy (shear bond strenght of glass ionomer cement in dentin and nicr alloy) mira leonita* dan r. iskandar** * mahasiswa ppdgs ** bagian prostodonsia fakultas kedokteran gigi universitas airlangga surabaya indonesia abstract glass ionomer cements were used broadly in restorative dentistry. that’s why researchers always try to invent new form of glass ionomer cement. the newest invention was the paste-paste formulation. shear bond strenght of powder-liquid glass ionomer cement and paste-paste glass ionomer cement in dentin and nicr alloy was tested to 4 groups of samples. each group consisted contain 6 samples that were shaped into cylinder with 4 mm of diameter and 5 mm of height. group a was dentin with powder-liquid glass ionomer cement, group b was dentin with paste-paste glass ionomer cement, group c was alloy with powder-liquid glass ionomer cement, and group d was alloy with paste-paste glass ionomer cement. each sample in each group was tested with autograph. the datas were analyzed statistically using t-test with level of signficance 0.05. the result showed that powder-liquid glass ionomer cement shear bond strenght was 211 n and paste-paste glass ionomer cement was 166.92 n. that showed that powder-liquid glass ionomer cement had a better shear bond strenght. key words: bond strenght, glass ionomer cement, dentin, alloy korespondensi (correspondense): mira leonita, mahasiswa ppdgs, fakultas kedokteran gigi universitas airlangga. jln. prof. dr. moestopo no. 47 surabaya 60132, indonesia. pendahuluan apabila seseorang menderita kehilangan satu atau beberapa gigi, maka seyogyanya gigi yang hilang itu segera diganti dengan suatu gigi tiruan. salah satu alternatif adalah dengan memberi perawatan dengan gigi tiruan tetap (gtt) atau yang biasa disebut gigi tiruan jembatan. tujuan pembuatan gtt adalah untuk memulihkan daya kunyah (masticating efficiency) yang menjadi berkurang, untuk memperbaiki kondisi estetik, untuk mencegah terjadinya pergeseran gigi sebelahmenyebelahnya ke tempat gigi yang hilang, untuk memelihara atau mempertahankan kesehatan gusi dan untuk memulihkan fungsi fonetik (pengucapan).1 dalam proses perawatan, gtt perlu dilekatkan ke gigi penyangga di sebelah-menyebelahnya secara tetap dengan bantuan semen bahan gigi. macam bahan yang dipakai dalam konstruksi gtt yang sering diaplikasikan di klinik fakultas kedokteran gigi universitas airlangga adalah bahan porselen bertaut logam (porselen fused to metal). bagian logam tersebut yang merupakan bagian inti dari suatu konstruksi gtt yang akan berhadapan dengan permukaan dentin dari gigi penyangga. dalam proses penyemenan suatu gtt terhadap gigi penyangga, perlekatan terjadi antara bahan semen dengan permukaan jaringan dentin dari gigi penyangga dan permukaan lapisan logam dari konstruksi gtt. menyadari bahwa semen merupakan bagian yang paling lemah dalam konstruksi gtt, maka secara klinis manipulasinya harus dilakukan dengan teliti dan rapi sesuai dengan aturan pakai.1 semen untuk gtt yang sering digunakan adalah semen dari bahan ionomer kaca. semen ionomer kaca dapat dibagi menjadi dua tipe yaitu tipe 1 digunakan sebagai perekat bahan restorasi (luting semen) dan tipe 2 sebagai bahan restorasi. perlekatan semen ionomer kaca didasarkan pada kemampuannya untuk berikatan secara adhesi terhadap dentin, enamel, dan logam.2 mekanisme perlekatan semen ionomer kaca pada gigi yaitu oleh karena adanya pertukaran ion kalsium dalam dentin gigi dengan ion karboksilat dalam semen.3 selain berlekatan dengan dentin, dalam sebuah konstruksi gtt semen juga berlekatan dengan logam yang merupakan bagian dari gtt tersebut. perlekatan antara semen dengan logam ini lebih merupakan suatu perlekatan mekanik yang diperoleh dari kekasaran permukaan dalam gtt.1 logam yang digunakan pada konstruksi gtt adalah nicr alloy, yang merupakan logam campur yang terdiri dari ni, cr, si, bo, c, mn, dan fe. pemilihan logam nicr didasarkan pada sifatnya yang tahan korosi, ringan, dan keras.4 saat ini, di pasaran terdapat semen ionomer kaca yang berbentuk dua buah pasta disamping semen ionomer kaca yang berbentuk bubuk-cairan. peneliti tertarik untuk 30 maj. ked. gigi. (dent. j.), vol. 38. no. 1 januari 2005: 29–31 mengetahui bagaimana kekuatan perlekatan antara semen ionomer kaca bubuk-cairan dan semen ionomer kaca pastapasta dalam pemakaiannya sebagai semen tetap pada aplikasi gtt. dalam mengukur kekuatan perlekatan tersebut yaitu antara semen dengan dentin atau logam akan dipakai tes kekuatan geser, karena bahan restorasi harus melawan berbagai bentuk gaya selama pengunyahan dan salah satunya adalah geseran.5 di samping itu, berdasarkan brosur belum pernah dilakukan penelitian mengenai kekuatan perlekatan geser dari kedua semen ini. sehubungan dengan hal tersebut di atas, peneliti tertarik untuk mengetahui bagaimana kekuatan perlekatan dari semen ionomer kaca bubuk-cairan dan semen ionomer kaca pasta-pasta terhadap dentin dari gigi penyangga dan logam dari gtt. penelitian ini dimaksudkan untuk mengetahui besarnya kekuatan perlekatan dari semen ionomer kaca bubuk-cairan dan semen ionomer kaca pastapasta terhadap dentin dari gigi penyangga dan logam dari gtt, sehingga melalui penelitian ini dapat diketahui semen mana yang lebih baik perlekatannya terhadap dentin dari gigi penyangga dan logam dari gtt. bahan dan metode jenis penelitian ini adalah eksperimental laboratorik, adapun bahan yang digunakan adalah gigi insisiv permanen rahang atas (ra) bekas pencabutan sebanyak 12 buah dimana mahkota gigi dipisahkan dari akarnya kemudian mahkota gigi dipreparasi hingga bagian dentin terbuka, logam campur nicr berdiameter 4 mm sebanyak 12 buah, semen ionomer kaca bubuk-cairan (ketac cem μ), semen ionomer kaca pasta-pasta (fuji cem). sedangkan alat yang dipakai dalam penelitian ini antara lain: straight handpiece dan carborundum disk, alat timbangan, cetakan diameter 4 mm panjang 5 mm, kaca datar setebal 0,5 cm, spatula semen, cylinder plunger,tabung penyangga, alat tarik (autograph merk shimadzu). penelitian ini dilakukan di laboratorium bersama universitas airlangga pada tanggal 7 agustus 2003 pukul 16.00–21.00. dalam penelitian ini terdapat 4 kelompok percobaan dengan masing-masing besar sampel 6 buah: kelompok gigi yang dilekati dengan semen ketac cem μ, kelompok gigi yang dilekati dengan semen fuji cem, kelompok alloy yang dilekati dengan semen ketac cem μ, kelompok alloy yang dilekati dengan semen fuji cem. sampel dipotong sesuai dengan bentuk cetakan yaitu dengan diameter 4 mm, ditanam pada cetakan dengan akrilik jenis self cured sebagai bahan fiksasi dengan permukaan labial menghadap ke atas setinggi separuh cetakan, sehingga permukaan gigi berada di tengah-tengah cetakan. kemudian pada cetakan tersebut diisi dengan adonan semen. setelah semen dicampur dan lalu dimasukkan ke dalam cetakan secara perlahan sehingga tidak terjadi porositas pada semen. setelah adonan masuk seluruhnya di atas adonan diberi plastik yang berfungsi sebagai tempat untuk menekan adonan semen sehingga permukaan gigi seluruhnya kontak dengan semen dan menghasilkan satu cetakan yang kompak dan utuh. setelah itu diatasnya diberi beban sebesar 1 kg. setelah setting, sampel dilepas dari cetakan.6 gambar 1. skema peletakan sampel dalam alat. pengukuran kekuatan geser dilakukan dengan cara meletakkan sampel pada suatu alat sehingga posisi sampel berada di tempat yang tepat, artinya bidang yang akan digeser tepat pada permukaan gigi atau logam yang dilekati semen. sesudah alat tersebut siap maka alat tersebut diletakkan pada alat autograph. dengan autograph ini angka kekuatan geser tiap sampel dapat dilihat lalu dicatat. setelah data dari keempat kelompok diperoleh, maka data dianalisis dengan uji statistik student t test dengan kemaknaan 0,05. hasil dari hasil penelitian yang telah dilakukan tentang kekuatan perlekatan antara semen ionomer kaca bubukcairan dan semen ionomer kaca pasta-pasta terhadap dentin dan nicr alloy, diperoleh nilai rata-rata dan simpangan baku dari kekuatan geser masing-masing kelompok seperti yang tercantum dalam tabel 1. tabel 1. nilai rata-rata dan simpangan baku kekuatan perlekatan geser semen ketac cem μ dan semen fuji cem terhadap dentin dan nicr alloy (n) perlakuan jumlah sampel rata-rata kekuatan geser standar deviasi ketac cem + dentin fuji cem + dentin ketac cem + nicr fuji cem + nicr 6 6 6 6 211 166,92 128,33 106,08 3,03 6,04 3,83 3,10 untuk mengetahui ada atau tidaknya perbedaan kekuatan perlekatan semen ketac cem μ dengan semen fuji cem pada dentin gigi penyangga dan nicr alloy pada gtt dilakukan analisis statistik dengan menggunakan uji t-test. hasil uji t-test antara kekuatan perlekatan geser semen ketac cem μ dengan semen fuji cem terhadap dentin gigi 31leonita: kekuatan perlekatan geser semen ionomer kaca penyangga maupun nicr alloy didapatkan taraf signifikan p < 0,05. hal ini menunjukkan bahwa ada perbedaan yang bermakna antara kekuatan perlekatan semen ketac cem μ dengan semen fuji cem terhadap dentin gigi penyangga dan nicr alloy. pembahasan dari penelitian yang telah dilakukan mengenai kekuatan perlekatan semen ionomer kaca bubuk-cairan dan semen ionomer kaca pasta-pasta terhadap permukaan dentin dan nicr alloy dalam suatu konstruksi gigi tiruan tetap, peneliti menggunakan tes kekuatan geser untuk mengetahui kekuatan perlekatan antara semen terhadap dentin maupun nicr alloy. hal ini dilakukan oleh karena gaya geseran merupakan salah satu unsur tekanan yang menunjang terwujudnya tekanan kunyah yang diterima gtt di dalam rongga mulut.5 di samping itu, berdasarkan brosur dari kedua semen belum pernah dilakukan penelitian mengenai kekuatan gesernya.6,7 tes kekuatan geser dilakukan setelah 24 jam mengingat bahwa setelah 24 jam dapat dianggap semen telah benar setting. oleh karena itu, pada penyemenan gtt penderita tidak diperkenankan memfungsikan gtt dalam waktu 24 jam pertama.8 dari hasil penelitian tersebut menunjukkan bahwa nilai rata-rata kekuatan geser semen ionomer kaca bubuk-cairan terhadap dentin yaitu 211 newton, sedangkan nilai rata-rata dari semen ionomer kaca pasta-pasta yaitu 166,92 newton. hal ini menunjukkan bahwa kekuatan perlekatan semen ionomer kaca bubuk-cairan terhadap dentin lebih besar daripada semen ionomer kaca pastapasta terhadap dentin. pada uji kekuatan geser semen ionomer kaca bubukcairan terhadap nicr alloy diperoleh nilai rata-rata sebesar 128,33 newton. sedangkan kekuatan geser dari semen ionomer kaca pasta-pasta sebesar 106,08 newton. hal ini menunjukkan bahwa kekuatan perlekatan semen ionomer kaca bubuk-cairan terhadap nicr alloy lebih besar daripada semen ionomer kaca pasta-pasta. berdasarkan data tersebut dapat disimpulkan bahwa ada perbedaan yang bermakna, di mana kekuatan perlekatan semen ionomer kaca bubuk-cairan terhadap dentin maupun nicr alloy lebih tinggi dari kekuatan perlekatan semen ionomer kaca pasta-pasta terhadap dentin maupun nicr alloy dengan p < 0,05. semen ionomer kaca bubuk-cairan dan semen ionomer kaca pasta-pasta merupakan golongan yang sama yaitu semen ionomer kaca. perbedaan antara keduanya adalah pada bentuknya semen ionomer kaca bubuk-cairan berbentuk bubuk-cairan sedangkan semen ionomer kaca pasta-pasta berbentuk pasta. terjadinya perbedaan kekuatan perlekatan tersebut mungkin dikarenakan adanya perbedaan komposisi kedua semen tersebut, dimana pada semen ionomer kaca bubukcairan terdapat asam tartaric yang dapat meningkatkan stabilitas material.4 selain itu dari brosur yang dimiliki oleh tiap semen, compressive strength semen ionomer kaca bubuk-cairan sebesar 141 ± 14 mpa sedangkan compressive stregth semen ionomer kaca pasta-pasta sebesar 122 mpa. hal ini menunjukkan bahwa compressive stregth semen ionomer kaca bubuk-cairan lebih besar daripada semen ionomer kaca pasta-pasta.6,7 dari data klinis kedua semen juga diperoleh bahwa semen pasta-pasta memiliki flow dan ketebalan lapisan film yang lebih kecil daripada semen bubuk-cairan. flow yang baik memang menguntungkan dalam ketepatan peletakkan gtt akan tetapi juga menyebabkan berkurangnya kekuatan perlekatannya.6,7 adanya butiran granular pada semen bubuk-cairan juga menambah kekuatan perlekatannya, dimana butiran granular tersebut berperan sebagai filler yang akan mengisi ikatan adesi semen dan gigi.6 filler tersebut akan mengisi kekasaran permukaan dentin maupun alloy yang timbul oleh karena proses adesi. setelah semen pada kedua permukaan mengeras, filler akan bertindak sebagai kunci yang memegang erat permukaan tersebut.1 dari penelitian ini dapat disimpulkan bahwa kekuatan perlekatan semen ionomer kaca bubuk-cairan pada permukaan dentin maupun logam nicr alloy dalam konstruksi gtt lebih besar daripada kekuatan perlekatan semen ionomer kaca pasta-pasta. perlu kiranya dilakukan penelitian yang lebih lanjut mengenai perlekatan semen pada permukaan dentin maupun permukaan logam pada bentuk kekasaran permukaan yang berbeda dalam konstruksi gtt. daftar pustaka 1. martanto p. teori dan praktek ilmu mahkota dan jembatan. 2nd ed. jakarta: erlangga; 1985. p. 289–90. 2. ernst cp. clinical case presentation: all ceramic crown cementation using ketac cem m glass ionomer cement. 3m espe; 2002. 3. phillips rw. science of dental materials. 10th ed. philadelphia, london, toronto, montreal, sydney, tokyo: wb saunders co; 2003. p. 452–76. 4. craig rg, powers jm. restorative dental materials. 11st ed. st louis: mosby co; 2002. p. 480–8, 594, 614–16. 5. combe eg. notes on dental material. 6th ed. edinburgh, london, melbourne, and new york: churchill livingstone; 1992. p. 116–21, 148–51. 6. soekartono r helal. pengaruh penambahan logam campur amalgam ke dalam bubuk ionomer gelas (shear strength). surabaya: penelitian laboratoris, fakultas kedokteran gigi universitas airlangga; 1995. h. 14. 7. 3m espe. 3m espe ketac cem glass ionomer cement technical product profile. 3m espe; 2002. 8. gc. glass ionomer cements & bonding agents–fuji cem. available from: www.gceurope.com. accessed 2000. 9. horn hr. practical consideration for succesful crown and bridge therapy. 1st ed. philadelphia, london: wb saunders co; 1976. p. 37–9. << /ascii85encodepages false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 144 research report dental journal (majalah kedokteran gigi) 2015 september; 48(3): 144–150 the effect of ethyl acetate fraction of citrus limon peel on mesenchymal cell proliferation and polybacterial growth astrid marinna,1 priyo hadi,2 and desiana radithia2 1 dinas kesehatan kota tarakan, kalimantan utara indonesia 2 department of oral medicine, faculty of dental medicine, universitas airlangga, surabaya indonesia abstract background: oral diseases remain to be global health problem. the common therapy involved the use of modern medicines with their various side effects. citrus limon are potentials as anti-inflammatory, anti-fungal, anti-oxidant, anti-viral and anti-bacterial. purpose: the purpose of this study was to determine the effect of ethyl acetate fraction of citrus limon peel extract on human gingival mesenchymal cell proliferation and palm commensal polybacterial growth. method: this study was experimental study with post test only control group design. citrus limon peel extracted and partitioned in order to obtain ethyl acetate fraction of 3.125%, 2.75%, 2.375%, 2%, and 1.5625%. toxicity test was performed after 24 hours using the mtt assays. cell viability was measured by optical density formazonand read by elisa reader 620 nm. results: all treatment groups showed less than 60% cell viability. the highest cell was 19.36 (1.5625% concentration) and the lowest was 12.65 (3.125% concentration). the highest anti-bacterial inhibition value was 8.9125 mm (3.125% concentration) and the lowest was 6.0625 mm (1.5625% concentration). conclusion: the higher concentration of ethyl acetate fraction citrus limon peel extract, the higher toxicity and inhibitory properties against commensal palm polybacteria. keywords: citrus limon; toxicity; gingival mesenchymal cell; palm polybacteria correspondence: astrid marinna, dinas kesehatan kota tarakan. jl. kalimantan no. 1. kota tarakan kalimantan utara, indonesia. e-mail: astridmarinna@gmail.com introduction oral diseases remain to be global health problem. the common theraphy using modern medicines is preferrable in oral diseases treatment to give either immediate or nonimmediate side effect, or accumulated side effect. most of chemical medicines we consumed are international products which make their price high.1 indonesia is a rich source of herbs to produce conventional herbal medicines. the addictive effect of using chemical medicines can be taken away by using herbal medicines. herbal medicines are more effective than chemical medicines because they are affordable and are relatively safer than modern medicines. however, conventional medicines can also give negative effect if they are used in inaccurate way. the dose of convenional medicines is often empiric, without a clear prescription. some studies had been conducted to advance the use of conventional medicines, including: cellular level study (prominent potential test with toxicity test), guinea pig level study, human level study and human in a big scale level study (multicenter). citrus limon, also known as citron, is one of herbal medicine which is quite popular for its various merits. the most usable part of citrus limon is its fruit. the fruit is good for health for increasing immune system, preventing bacteria and free radicals. ascorbic acid in lemon functions as anti-inflammatory and accelerating the recovery process, whereas citrus limon peel is not used.2 the peel of citrus lemon contains many chemical compounds polymethoxylated flavones, such as flavonoid glycosides, coumarins, β dan γsitosterol, terpenoid glycosides and volatile oils which have several significant bioactivities and rarely found in the other herbs. polymethoxylated flavones dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 145145marinna, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 144–149 in citrus limon give many benefits for biological activities including anti-bacterial, anti-inflammatory, anti-fungal, anti-diabetic, anti-oxidant and anti-viral.2 the ingredients are required to be non-toxic, nonirritating and have biocompability, means that the produced ingredients must not harm either local or systemic biological environtment.3 toxicity test must be absolutely conducted for conventional or herbal medicines before they are broadly circulated in the market to detect the toxic effect directly (in vitro) by culturing cell lines.4 the observation on mesenchymal cell viablity during culturing process can be used to indicate concentration effect and exposure time of a substance, including cytotoxic effect. mesenchymal cell is multipotent cell which can be differentiated into several cells including fibroblast, due to its pureness and sensitivity when it is used as toxicity test.5 before and after contaminated by cytotoxin, mesenchymal cell viability which is shown by inactive cells percentage can be used as measured parameter, to find out cytotoxic effect of an ingredient.5-7 if the ingredient is toxic on mesenchymal cell, it cannot be used as medicine; however, that ingredient can be used to control microbes population on the skin body through anti-bacterial potential and as extra oral antiseptic ingredient through anti-fungal. the purpose of antiseptic is to hinder the growth of bacteria. one of antiseptic is hand cleanser antiseptic or hand sanitizer. hand sanitizer products are generally made from chemical substances consist of alcohol and triclosan. however, the use of alcohol as hand antiseptic has deficiencies, such as ineffectiveness use on wounded skin, inflammable, dryness and skin irritation on repetitive use.8 this study was aimed to determine the ethyl acetate fraction of citrus limon peel extract effect on human gingival mesenchymal cell proliferation and commensal palm polybacterial growth. materials and methods citrus limon peel polar compounds (ethyl acetate fraction) and the screening of phytochemical citrus limon peel active compounds (ethyl acetate fraction) were made in the laboratory of pharmacology and phytochemical, faculty of pharmacy, universitas widya mandala surabaya. extract experimental test with mtt assay by using human mesenchymal cell culture was conducted in institute of tropical desease, universitas airlangga surabaya. resistance capacity experimental test was conducted in the laboratory of microbiology, faculty of dentistry, universitas airlangga surabaya. ethyl acetate fraction toxicity of citrus limon peel extract on human gingival mesenchymal cell was using mtt method (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. mesenchymal cell was cultured into line cell. in splinting line cell, dulbeco modification of eagle’s (dmem) medium and fetal bovine serum (fbs) was required. compound contents of flavonoid class that often researched was flavonoid glicosides. compounds generated from flavonoid glicosides include hesperidin, luteolin, and quercitin2 the compounds contained in ethyl acetate fraction in this research material were then analyzed in badan penelitian dan konsultasi industri (bpki), which contained: hesperidin 3.61%, luteolin 1.82%, quercetin 2.16%, volatile oil 1.88%, lemonene 1.36%, and citric acid 0.51%. in this research, 5 treatments were given with concentration of ethyl acetate fraction for each was 1.5625%, 2%, 2.375%, 2.75% and 3.125%. mtt method test (3-4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is an enzymatic test to measure the active cells ability based on the mitochondrial activities from cultured cells. the measurement was performed by using spectrophotometer at the wave length of 620 nm, that resulted in numerical data optical density. the scale of optical density was the value of colour concentration from the cultured cells. the more concentrated the colour resulted, the higher the value of optical density. the percentage number of human gingival mesenchymal cells that still active after the sprinkling of citrus limon peel extract was counted by active cells percentage formula:3 % active cells = od treatment-od media x 100 % od cells control od media notes : % active cells : percentage of total live cell after test; od treatment : optical density value of mesenchymal cells for each sample after the reading result of elisa reader test; od media : optical density value of mesenchymal cells on media control; od cells control : optical density value of mesenchymal cells on control cells. it is toxic if the number of the active mesenchymal cells after the test is less than 60% or the inactive mesenchymal cells after the test is greater than 50%.3 anti-microbial effects on palm can be examined through the positive polybacteria inhibitory if the inhibition zone is in the form of clear zone surround the disc paper (figure 1). positive control employed in this research was phenol compounds 5%. the population of bacteria were taken from the palm where each palm must be brushed together before checked so that the bacterial content in the palms will be homogeneous, and then the palms were swabbed using table 1. the classification of inhibitory responses to the microbila growth diameter of the inhibition zone inhibitory growth responses < 5 mm weak 5 – 10 mm moderate 10 – 20 mm strong > 20 mm very strong dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 146 marinna, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 144–149 the sterile cotton soaked by 0.9% nacl, stroked in the opposite direction with the palm line, and then the cotton was caressed and reproduced using brain heart infusions (bhi) media. the ability of the researched substance in inhibiting the growth of bacteria was determined by the scale of inhibition zone that was the area around the disc paper where the growth of polybacteria was not found. the classification of inhibitory responses to the microbial growth on anti-microbial compunds can be seen in the table 1. results the data were analyzed using active cells percentage formula3 and the obtained results can be seen in table 1. the result from the cells viability calculation (active cells percentage). each concentration from ethyl acetate fraction within citrus limon peel extract inhibit mesenchymal cells proliferation with the value of cells viability less than 60%, which means toxic. statistically, it can be concluded that the higher the concentration of ethyl acetate fraction of citrus limon peel extract, the higher the toxicity. based on table 2, it can be stated that the highest mean value of optical density formazon of ethyl acetate fraction of citrus limon peel extract is in concentration 1.5625%, that is 19.36109. the greatest standard deviation is in concentration 3.125%, that is 5.238175. this concludes that the higher the ethyl acetate fraction concentration of citrus limon peel extract, the smaller the value of optical density formazon which means the more mesenchymal cells will die. table 3 shows the inhibitory value of ethyl acetate fraction of citrus limon peel extract on commensal palm polybacteria, in the group 3.125% has the highest value (8.9125). visually, it can be stated that the higher concentration, the greater the inhibitory value of commensal palm polybacteria; however, it needs to be proved statistically through normality test, homogenity test, and comparison test intra inhibitory groups on commensal palm polybacteria towards ethyl acetate fraction of citrus limon peel extract. statistically it can be concluded that the higher the concentration of ethyl acetate fraction of citrus limon peel extract, the higher the toxicity and the inhibitory of commensal palm polybacteria, which is in accordance with the hypothesis. discussion the compounds of ethyl acetate fraction of citrus limon peel extract have anti-fungal ability with minimum inhibitory concentration (mic) value is 1.5625% and minimum fungal concentration (mfc) value is 3.125%.10 those materials can be used as mouthwash ingredients. conditions where a material can be used as medicine are non-toxic, non-irritating, and biocompatible, means the produced materials do not harm biological environment, either locally or systemically.1 it underlies this research to find out the toxicity of ethyl acetate fraction of citrus limon peel extract on human gingival mesenchymal cells by using mtt method (3-4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay. the national toxicity program (ntp) is stated that the recommended dose of limonene on rats is no more than 1650 mg/kg body weight per day, and if it is used more than the recommended dose will likely result in the increase of renal tubuli tumor as the activation of pro-oxidant that cause nephropathy.10 limonene within ethyl acetate extract in this research contains 1.36% table 2. the mean value of optical density formazon and the standard deviation of ethyl acetate fraction of citrus limon peel extract concentration of ethyl acetate fraction (%) mean value (%) optical density formazon ethyl acetate fraction ± standard deviation 1.5625% 19.36 ± 3.7 2% 17.26 ± 5.1 2.375% 14.46 ± 4.7 2.75% 12.64 ± 5.4 3.125% 17.12 ± 5.2 table 3. the mean value of inhibitory and standard deviation of commensal palm polybacteria on ethyl acetate fraction of citrus limon peel extract ethyl acetate fraction concentration the mean of polybacteria inhibitory (mm) ± standard deviation 1.5625% 6.06 ± 0.0 2% 6.53 ± 0.0 2.375% 7.03 ± 0.0 2.75% 7.55 ± 0.0 3.125% 8.91 ± 0.0 control + 8.02 ± 0.0 14 figure 1. the diameter measurement of anti-bacterial inhibitory zone.6 notes: a = diameter of paper disc (6 mm); b = diameter of the formed inhibitory zone (mm); c = the area of bacterial growth. figure 1. thawing process of gingival fibroblast cells. a) gingival fibroblasts cells seen under the microscope micro inverter b) gingiva fibroblast cells in log phase (fibroblasts look solid). figure 1. the diameter measurement of anti-bacterial inhibitory zone.6 notes: a = diameter of paper disc (6 mm); b = diameter of the formed inhibitory zone (mm); c = the area of bacterial growth. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 147147marinna, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 144–149 concentration which equals to 12240 mg/kg, exceeding the recommended dose of limonene. in several studies comparing cancer cells to normal cells, oxidative stress related to oncogenic transformation, metabolic activity, and reactive oxygen species (ros) is increasing. the increased ros in cancer cells will stimulate cell proliferation, gene mutation, and cellular sensitivity on anti-cancer agents. ros is a reactive molecule that chemically contains oxygen, namely superoxide anion (o2-), hydrogen peroxide (h2o2) and hydrogen radical (oh-). ros plays important role in pathogenesis including cancers, ageing, and the other degenerative diseases. mitochondrial dna codes protein which is the important component of energy formation pathway within mitochondria, namely oxidative phosphorylation (oxphos) that produces ros to create adenosine-5’triphosphate (atp) product through mitochondrial respiratory chain and initiate the cell death.12 dna cell damage caused by toxic compunds can induce cells to initiate apoptosis process. for instance, the genome damage within nucleus, there is parp-1 enzyme triggered the apoptosis. this enzyme has important role to keep genome integrity, even though the excessive activation can waste atp, that can change the process of cell death into necrosis (unprogrammed cell death).13 citric acid can cause hypercalemia, hypotension and tachycardia if exceeding the dose of 530g/l. citric acid functions as anti-oksidan to inhibit the oxidative stress.14 oxidative stress is caused by the balance disruption between ros as metabolism product within normal cells which is pro-oxidant on enzymes and anti-oxidative co-factor. this imbalance condition is caused either by the excessive production of ros h2o2, o2 and oh, or the shortage of ros release due to oxidant defense mechanism.14 the function of the anti-oxidant may increase the mesenchymal cell proliferation, as the dose of the extract in this research was 0.51% which equals to 4.131g/l. the use of quercetyne over the recommended dose 135mg/kg can cause toxicity, while for the fraction in this study the concentration used was 19440 mg/kg, the toxic effect was as pro-oxidant that caused the chain formation of ros seperti superoxide and hydrogen peroxide (h2o2), the pro-oxidant activity will damage the fat, protein and dna.16 that condition will also damage the local organ cells and this necrosis networking process can even inhibit the mesenchymal cells proliferation13. hesperidin functions to inhibit the enzyme phospholipase and lipoxygenase, the histamine release from the mast cells, and lipid peroxidase forming free-radicals;17 however, a bioactive material compound can cause toxicity if it is used in a high dose,18 the recommended dose of hesperidin is 2 g/kg19 in this extract 3.61% equals to 32.49 g/kg, therefore it is assumed to be the cause of cell death and inhibit the proliferation of mesenchymal cells. luteolin functions to inhibit the nf-κβ activity, so that pro-inflammatory cytokines can be blocked20 and inhibit the inflammatory medium such as nitride oxide resulted by liposacharide (lps), and inhibit il-5 activity, where il-5 is chemotactic factor that stimulates eosinophils which plays role in inflammatory condition due to allergy.21 the recommended dose of luteolin is not more than 1-2 mg/kg.21 in this extract the concentration was 1.82% that equals to 16.38 or 16380 mg/kg, while according to the other studies, it is stated that the toxic dose of luteolin is over 411 mg/kg i.p and 592 mg/kg i.m.21 toxic dose of luteolin emerges may be because of the concentration change of alpha-tocopherol that affects the absorption of beta-carotene that will disrupt the functions of liver, eyesight, blood vessels regeneration process in eyes, and also increase the cancer risk.22 the compounds of ethyl acetate fraction in this research, limonene, quercitin, hesperidin and luteolin inhibit the proliferation of mesenchymal cells, only citric acid can increase the proliferation of mesenchymal cells, so that the most of ethyl acetate fraction in this research is inhibiting the proliferation of mesenchymal cells rather than increasing. ethyl acetate fraction of citrus limon peel extract in this research was toxic even in the lowest concentration, and the higher the concentration, the higher the toxicity. based on the evidence found in this study, ethyl acetate fraction of citrus limon peel is toxic to be used as intra-oral medicines. to take the benefit of it, the further research was conducted, that is as herbal hand antiseptic materials. the lsd results showed that significance value is (p)<0.05, it states that there was a significant difference between each inhibitory group on commensal palm polybacteria. the significance was caused by the activity of phenols compund, volatile liquid, and citric acid that inhibit the cell wall biosynthesis and increase the cytoplasmic membranes permeability and disrupt the bacterial protein synthesis if the concentration of ethyl acetate fraction of the lime peel extract is higher. concentra of 3.125% is the maximum concentration that can kill greater number of bacteria than positive control. phenols are used as positive control for it was the basic compund in the desinfectan test and has a larger work mechanism. phenols can harm cell walls and cell membranes, coagulates proteins, damage atp ase damage sulphohydril from protein, and damage dna so that it is effective to kill bacteria.23 citrus limon peel contains volatile liquid that can inhibit the aero-bacterial growth, that is the anti-bacterial compunds limonene, linalool and mirsen that work by damaging the bacterial membrane cells.anti-microbial activities may be occurred as their ability in affecting extracellular proteins and forming cell walls of the permeability of bacterial cell wall membranes.24 limonene is a hydrocarbon compound containing terpene cluster, a pale coloured liquid, and has a strong lime smell. limonene content has anti-microbial ability by harming bacterial membranes. it damages the integrity of cytoplasmic membranes that act as selective permeability barriers, bring active transportation, and control internal cells composition.25 the damaged cytoplasmic membranes can cause cell membranes permeability lessen and the substance transportation into and out of cells becomes uncontrollable. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 148 marinna, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 144–149 the substances within cells, such as enzyme organic ions, amino acids, and nutritions can come out of cells. if the enzymes come out of cells along with substances like water and nutrition, they can cause the inhibition of metabolism in which lead to the reduced atp that required for cellular growth and reproduction, and then the bacterial cell growth become inhibited and died. flavonoid compound can damage the cytoplasmic membranes and leak the important metabolites and activate bacterial enzyme systems. this damage may leak nucleotides and amino acids preventing the active substances to enter the cells, this causes the membrane of bacterial cell walls undergo lysis. on the damaged cytoplasmic membranes, h+ ions from phenols compound and its derivative (flavonoid) will attack the polar cluster (phospate cluster) so that the phospholipide will strand into glycerols, carboxylic acids and phosporic acids. this causes phospholipides cannot maintain the cytoplasmic membrane form resulting in the leakage of the cytoplasmic membrane that the bacterial growth will be inhibited and eventually died.26 volatile liquid is chemically composed of the blend of steroid compound and the other compound that acts as antibacterial by disrupting the formation process of membranes or cell walls that they are imperfectly formed.2 the mechanism may be occured by disrupting the constituent component of peptidoglycan within bacterial cells that causing the layer of the cell wall is not fully formed. the disruption of peptidoglycan synthesis resulted in the imperfect cell formation due to the absence of peptidoglycan and the cell wall is only involving the cell membranes. the base of bacterial cell wall is peptidoglycan layers. peptidoglycan is constituted by n-acetyl glucosamine and n-acetyl muramat acid, bonded by 1.4-glycoside. in n-acetyl muramat acid, there are short amino acid chains: alanine, glutamic, diaminopimelat, lysine and alanine, bonded by peptide chain. the role of peptide chain is to link one chain to another. the mechanism of bacterial wall damage occurs because the assembling process of bacterial cell wall initiated by the formation of peptide chains that form crossed bridge of peptides which integrate glycan chains from peptidoglycan to the other chains to form the perfect assembling of the cell wall. this causes the bacterial cells will easily undergo lysis, either physically or osmotically and causes the death of cell.26 flavonoid is the largest phenols compound in the universe.9 phenols compound interacts with the bacterial cells through absorption process involving hydrogen bonds. on the low level, phenol protein complex formed in a frail bond and will immediately be apart, followed by phenols penetration into cells resulting in precipitation and protein denaturation. the protein denaturation of bacterial cell wall will cause the fragility that the cell wall is easily penetrated by the other active bacterostatics substances. if the denaturated protein is enzyme protein, the enzyme will not work causing the metabolism and nutrition absorption process disrupted. on the high level, phenols cause protein coagulation and the lysis of membrane cells.8 h+ ions from phenols compound and its derivative (flavonoid) will attack the polar cluster (phospate cluster) so that the phospholipide will break down into glycerols, carboxylic acids and phosporic acids. this causes phospholipides cannot maintain the cytoplasmic membrane form resulting in the leakage of the cytoplasmic membrane that the bacterial growth will be retarded and eventually died.26 it can be concluded that ethyl acetate fraction within citrus limon peel at concentration of 1.5625%, 2%, 2.375%, 2.75%, and 3.125% is toxic on human gingival mesenchymal cells, so it is not recommended to be developed into intra oral medicines; however, it has a very effective inhibitory power on commensal palm polybacteria, which had proven that in the lowest concentration it can inhibit the bacterial growth. the higher the concentration, the higher the inhibitory power resulted. references 1. katno p. tingkat manfaat dan keamanan tanaman obat dan obat tradisional. tawangmangu: balai besar litbang tanaman obat dan obat tradisional, badan litbangkes, depkes ri; 2008. p. 1-4. 2. dhanavade mj, jakulte cb, ghosh js, sonawane kd. study antimicrobial activity of lemon (citrus lemon l peel extract. british j pharmacology and toxicology 2011; 2(3): 119-22. 3. freshney ri. culture animal cell: a manual of basic technique and specialized application 6th. new jersey: john wiley and sons; 2010. p. 18, 111-4, 187-206, 365-77. 4. hassan rafiee mehr. evaluation of cytotoxic effect of zinc on raji cell-line by mtt assay. iranian journal of toxicology 2011; 4(4): 390-6. 5. jan barfoot, donald bruce, graeme laurie, nina bauer, janet paterson and mary bownes. stem cell: science and ethics. stemcell-resourse 3rd. edinburgh: bbsrc; 2016. p. 1-56. 6. sananta p. uji toksisitas fresh frozen tendon graft dan freeze dried tendon graft terhadap mesenchymal stem cell. thesis. surabaya: universitas airlangga; 2011. 7. hendrawan rd, putranti na, falah mn. anti bacterial and cytotoxicity of guava (psiudum guava l.) leaves extract against streptococcus mutans. asia pasific dental students j 2012; 3(1): 90-8. 8. pramita f. formulasi sediaan gel antiseptic ekstrak methanol (polygonum minus huds). jurnal mahasiswa farmasi fakultas kedokteran dan ilmu kesehatan masyarakat untan 2013; 3(1): 1-9. 9. indriani n. aktivitas antibakteri daun senggugu (cleodendronserratum [l] spr.). j pharmacology 2007; 32: 7-10. 10. sidharta y. daya anti jamur flavonoid kulit buah lemon terhadap oral candidiasis. tesis. surabaya: ilmu penyakit mulut fakultas kedokteran gigi universitas airlangga 2014. 11. sun j. d-limonene: safety and clinical applications. alternative medicine review 2007; 12(3): 259-64. 12. lilik maslachah, rahmi sugihartuti dan rahma kurniasanti. the inhibition of vitamin e (αtocopherol) antioxidant to superoxide radical reactive oxygen species (o2-) production on the white rat (rattus norvegicus) stressed by an electric shock. media kedokteran hewan 2008; 24(1): 21-6 13. rode hj, eisel d. apoptosis. cytotoxicity and cell proliferation. 4th ed. germany: roche diagnostics gmbh; 2008. p. 2-16, 116-30. 14. hall ah, rumack bh. citric acid. national library of medicine hsdb data base information system micromedexinc englewood co edition expires nov. ccis 2014; 162. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 149149marinna, et al/dent. j. (majalah kedokteran gigi) 2015 september; 48(3): 144–149 15. petersen. the global burden of oral desease and risk of oral health. buletin of who 2005; 8(3): 631-8. 16. harwood m, nikiel b, borzelleca j, flamm gw, williams gm, lines tc. a critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotic/carcinogenic properties. food and chemical toxicology 2007; 45: 2179-205. 17. kuntic v, beboric j, antunovic ih, markovic su. evaluating the bioactive effects of flavonoid hesperidin – a new literature data survey. vojnosanit pregl 2014; 71(1): 60–5. 18. european food safety authority (efsa). scientific opinion on the re-evaluation of lutein preparations other than lutein with high concentrations of total saponified carotenoids at levels of at least 80%. efsa panel on food additives and nutrient sources added to food (ans). efsa journal 2011; 9(5): 2144. 19. bigoniya p, singh k. ulcer protective potential of standarized hesperidin, a citrusflavonoid isolated from citrus sinensis. rev bras farmacogn 2014; 2(4): 330-40. 20. lawrence t. the nuclear factor nf-kb pathway in inflammation. cold spring harbor laboratory press 2009; all rights reserved; doi: 10.1101/cshperspect.a. 21. kalariya nm, ramana kv, srivastava sk, van kuijk. carotenoid derived aldehydes-induced oxidative stress causes apoptotic cell death in human retinal pigment epithelial cells. experimental eye research. fjgm 2007; 86: 70-80. 22. european food safety authority (efsa). statement on the safety assessment of the exposure to lutein preparations based on new data on the use levels of lutein. efsa panel on food additives and nutrient sources added to food (ans). efsa journal 2012; 10(3): 2589. 23. waluyo l. mikrobiologi umum. edisi revisi. malang: universitas muhammadiyah malang press; 2007. p. 14, 40, 122-3. 24. bansode ds, chavan md. studies an antimicrobial activity and photocemical analysis of citrus fruit juices against selected enteric pathogens. international research journal of pharmacy 2012; 3(1): 122-6. 25. gattuso g, barrreca d, gargiulli c, leuzzi u, caristi c. flavonoid composition of citrus juices. molecules 2007; 12: 1641-73. 26. retnowati y, bialangi n, posangi nw. pertumbuhan bakteri staphylococcus aureus pada media yang diekspos dengan infus daun sambiloto (andrographispaniculata). saintek 2011; 6(2): 7-8. dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg doi: 10.20473/j.djmkg.v48.i3.p144-149 http://e-journal.unair.ac.id/index.php/mkg http://dx.doi.org/10.20473/j.djmkg.v48.i3.p144-149 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 74 micronucleus frequency in exfoliated buccal cells from hairdresser who expose to hair products koh hui yee, alma linggar jonarta, and regina tc. tandelilin department of oral biology faculty of dentistry, universitas gadjah mada yogyakarta-indonesia abstract background: hairdresser is one of the fastest growing occupations in today’s society. hairdresser help styling, cutting, colouring, perming, curling, straightening hair and various treatment to customer. somehow, hairdresser are constantly exposed to chemical substances such as aromatic amines, hydrogen peroxide, thioglycolic acid, formaldehyde in hair products which can cause damage to human’s genome. micronucleus is one of the effective biomarker for processes associated with the induction of dna damage. purpose: the aim of this study was to determine the micronucleus frequencies in buccal mucosa epithelial cells of hairdresser who were exposed to chemical of hair products. method: this study was conducted on twenty female subjects, who were divided into 2 groups: exposed and non-exposed (control) group. all subjects recruited were working in the same beauty salon. buccal cells were obtained from each individual by using cytobrush. the cells were stained with modified feulgen-ronssenback method and counting of micronucleus per 1000 cell was done under light microscope. the data were analyzed using independent t-test and one-way anova (p<0.05). result: the result showed a significant difference in micronucleus frequency between 2 groups. there were a significantly increase of micronucleus frequency in hairdressers and increase of micronucleus frequency with the longer duration of exposure. conclusion: it concluded that the chemical substances of hair products had affected the micronucleus frequency of the epithelial cells in buccal mucosa of hairdressers. keywords: hairdresser; micronuclei; genome correspondence: regina tc. tandelilin, departemen biologi oral, fakultas kedokteran gigi universitas gadjah mada. jl. denta i sekip utara yogyakarta 55281, indonesia. e-mail: regina30mei@yahoo.com research report introduction hairdressers or hairstylist represent a large and fast growing group of professionals. this is due to people of today’s society always want to look good and feel proud to be considered as fashionable or fashion and style conscious. hairdressers and hairstylists help provide hair care services for those who want to improve their appearance. they offer a wide array of services such as styling, cutting, straightening, curling, and coloring hair. the growing popularity of specialized hairstyling services have contributed to job growth in this occupation. hairdressers are exposed daily to a great variety of chemical substances through inhalation, absorption or accidentally ingested.1 these professionals are exposed to several thousands of chemicals contained in colourants, bleaches, shampoos and hair conditioners. they may also exposed to volatile solvents, propellants and aerosols from hairsprays such as formaldehyde, methacrylates and nitrosamines.2 hairdressers and their clients are chronically exposed to variety of chemical and mechanical hair treatment that contain a high number of potentially harmful chemicals, including acetone, benzaldehyde, valeraldehyde,3 lead acetate, p-phenylenediamine, hydrogen peroxide or bisthmuth citrate.4 toxic substances such as thioglycolic acid (tga), which can absorb through the skin and cause damage to organs and animal systems. tga caused a disorder in the reproductive cycle of rats and dental journal (majalah kedokteran gigi) 2015 june; 48(2): 74–79 75 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 75yee, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 74–79 increased the frequency of micronuclei in bone marrow cells.5 the oral epithelial cells represent a target site for earlier genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion. buccal mucosa cells are the first barrier which are capable of metabolizing carcinogens to reactive products. it is known that chronic exposure of oral mucosa to toxic substances leads to keratinisation with synthesis of keratine bodies.6 in recent study, excess risk for cancer of the upper aerodigestive tract, lungs, colon, cervical and pancreatic were identified in hairdressers.5 there are several biomarkers for studying individuals exposed to known or potential genetic agent. chromosome alterations, such as chromosomal aberrations (cas), sister chromatid exchanges (sces), and micronuclei, which are directly visible as changes in chromosome structure. among these biomarkers, micronucleus appears to be the most suitable for epidemiological studies because it is simple and can reflect changes due to early-stage carcinogenesis.7 micronuclei originate from chromosomal region lagging or regularly migrating at anaphase. they can derive from acentric chromatid or chromosome fragments produced by chromosome breakage, or from entire chromatids lagging on account of spindle disturbances. in the course of telophase these chromosome regions are included in the daughter cells where they can fuse with the main nucleus or can form one or more smaller secondary nuclei.8 it has been suggested that micronucleus plays an important role in the occurrence of genetic instability and cancer.9 the increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers due to daily exposure to harmful chemical substances.1 besides exposure of chemical substances in hair products, genomic damage is produced by lifestyle such as alcohol consumption, smoking habits, tobacco chewing, intake of drugs and stress. others risk factors are medical procedure for instance radiation and chemicals, micronutrient deficiency, and genetic factors .10 ultraviolet light is an environment factor that causes skin cancer. ultraviolet irradiation may modify dna base pairs, and produces a reactive oxygen species, which directs cells toward carcinogenesis.11 the climate of yogyakarta is generally tropical and receive a great amount of sunlight and ultraviolet irradiation since yogyakarta is located at the latitudinal and longitudinal of 7 47 s and 110 22 e.12 hormones that can affect micronucleus are estrogen and progesterone. both of these hormones can affect the metabolism and the biochemical system of cells and may induce chromosomal damage.13micronucleus frequency was higher in women than men because of the influence of sex hormones. evaluation of micronuclei in epithelial cells of the buccal cavity of the mouth is a method of detection of dna damage that is non-invasive in humans. cells observed were cells exfoliated using a cytobrush, carried by staining with feulgen-rosssenbeck modified method and observation under a microscope by counting the frequency of micronuclei in epithelial cells.10 the aim of this study was to determine the effect of exposure to harmful chemical substances in hair product to the micronucleus frequency of buccal mucosa epithelial cells of hairdressers. this study can be used as a pre-research for further investigating research that will be used as a tool for early detection of diseases of the mouth, particularly on the risk of oral cancer due to exposure to harmful chemical substances in hair products. materials and methods a total of 10 female hairdressers were included in this study. a control group composed of 10 female not occupationally exposed as hairdresser was selected. this study case stated after received a letter of ethical clearance issued by the ethics and advocacy unit of the faculty of dentistry, universitas gadjah mada (no. 510/kkep/fkgugm/ec/2013) as well as permission letter from faculty to conduct study case in salon and laboratory of histology. criteria for subject requited were female hairdressers who exposed to chemical substances in hair products for minimal 2 years, age 18-30, and habits such as alcohol consumption, oral contraceptive and systemic disease were completely omitted. subject who fulfilled the inclusion criteria and did not have any of the exclusion criteria, were given informed consent forms to sign under ethical clearance as an agreement to take part in this study. before they signed, explanation about the procedures of the study, their role as a participant and the content of the informed consent form, was given to them. informed consent form was given to each subject who satisfied the criteria of this study. subjects were asked to rinse to avoid contamination of debris in the oral cavity and to remove exfoliated dead cells. buccal cells were obtained from each individual by gentle brushing of the buccal mucosa with a cytobrush damped with 0.09% nacl. epithelial swabbing was done by rotating cytobrush at least 360° on the buccal mucosa. every subject was swab with the same type of cytobrush with the similar brushing force to attain exfoliated buccal cells. after swabbing the buccal mucosa, cells that had been attached to the cytobrush smeared on glass object by rotating oppose to the direction of rotation on the buccal mucosa. the slides then fixed in a fixation solution (3:1, methanolacetic) which has been prepared earlier. staining was performed with modified feulgen-rossenbeckmethod.14 specimens were immersed in a solution of 5m hcl at room temperature for 15 minutes, then washed with distilled water for 10-15 minutes. the first staining using schiff’s reagent for 90 minutes and followed by staining with fast green counterstain 1% for one minute. identification is then performed with a light microscope magnification of 200 times and a computer monitor with a magnification dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 76 yee, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 74–79 of 100times. a total of at least individual 1000 cells were screened per subject. buccal cells were collected and analyzed to a standard protocol described by titenko-holland et al.15 only cells that were not smeared clumped or overlapping and that contained intact nuclei were included in the analysis. micronucleus was identified according to certain characteristics. the micronucleus should be less than 1/3 diameter of the main nucleus. micronucleus is smooth oval or round shape and has the same colour, texture and refraction as the main nucleus. the micronucleus is clearly separated from the main nucleus.15 cells were observed at 200x magnification with a light microscope to determine the presence of micronucleus cells. micronucleus performed in dark green with modified feulgen-rossenbeck staining method. number of identified micronucleus was recorded using handy counter. micronucleus frequencies in units per 1000 cells were recorded. the data obtained were analyzed by using one-way anova, followed by post hoc lsd test to compare duration of exposure among exposure group and also analyzed using independent sample t-test for exposed group and control group. results micronucleus is round or oval in shape. they are not linked or connected to the main nuclei. the scoring is done according to the criteria which were based on the morphological features of micronucleus cells. in this study, it was found that the diameter of micronuclei varies between 1/16 and 1/3 of the mean diameter of the nuclei and located within cellular cytoplasm (figure 1). surprisingly, it was also found that a cell can contain more than one micronucleus as shown in figure 2. micronucleus frequency of exposed group and the control group were expressed as the number of cell that contain micronucleus per 1000 cells. the mean of micronucleus frequency of non-exposed and exposed group were showed in figure 3. there was an increase of micronucleus frequency in the exposed group (hairdressers). shapiro-wilk normality test showed significance values exposed group and control group, respectively for 0.528 and 0.393. this showed that both groups had normal distribution of data. levene’s test showed a p value of 0.01 which indicated that the data did not show homogeneity of variances (alternative hypothesis accepted). the data were further analyzed and the significance of the mean difference between the two groups was determined by using independent samplet-test test. table 1 shows the summary of the results of independent samplet-test. table 1 showed significant differences between the mean micronucleus frequency of exposed group compared to the control group. this indicated that exposure to chemical substances in hair product can increase the frequency of micronuclei in oral buccal epithelial cell. the data of exposed group then analyzed further to investigate if the duration of exposure will cause an effect in the increase of micronucleus frequency. the summary result of one-way anova test among exposure group was showed in table 2. the results of table 2 showed significant differences in the value of micronucleus frequency exposure duration between groups. this indicated that the increase of micronucleus frequency is directly proportional to the 11 figure 1. cells that contain micronucleus (arrow) under magnification of 200. figure 2. a cell with two micronuclei (arrow) under magnification of 400. figure 1. cells that contain micronucleus (arrow) under magnification of 200. 11 figure 1. cells that contain micronucleus (arrow) under magnification of 200. figure 2. a cell with two micronuclei (arrow) under magnification of 400. figure 2. a cell with two micronuclei (arrow) under magnification of 400. 12 figure 3. the micronuclei frequency of buccal epithelial cells base on group. table 1. result of independent sample t-test (p<0.05) group micronucleus frequencies t df sig. mean diff std. error ci(95%) exposed 10.06 18 0.02** 17.60 1.74 13.92421.434 non-exposed tabel 2. summary results of one-way anova test among exposure duration subgroup (p <0.05) duration of exposure mean and standard deviation of micronucleus f sig. < 5 years 16.00±2.0 36.347 0.00** 6-10 years 21.50±2.1 >10 years 26.80±1.5 tabel 3. summary results of post hoc lsd test duration of exposure between exposed subgroup (p<0.05) 0 5 10 15 20 25 groups fr eq ue nv y of m ic ro nu cl es pe r 1 00 0 ce lls figure 3. the micronuclei frequency of buccal epithelial cells base on group. 77 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 77yee, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 74–79 duration of exposure. this result shows there was an increase in the frequency of micronuclei in subjects who work as hairdresser with a longer duration. furthermore, lsd post hoc test was used to determine the significance of differences between groups micronucleus frequency duration of exposure and the results was summarized in table 3. the result revealed a significant increase in the frequency of micronuclei with the increase of duration of exposure. discussion hairdressers and their clients are chronically exposed to chemical and mechanical hair treatments that contain a high number of potentially harmful chemicals. many of these chemicals are either known or suspected allergens, carcinogenic or mutagens.16 the micronucleus test in exfoliated cells of buccal mucosa is a potentially excellent biomarker to detect genome damage.10 hairdressers have a significant increase in micronucleus frequency compared to control group. this result in line with the study of galiotte et al.16 that stated the increase in genetic damage could be associated in part with the occupational conditions to which hairdressers are constantly exposed to potentially hazardous chemical products. there was an increase of micronucleus frequency with the duration of exposure. this result was supported by the study of jeffrey that stated the dna damage, mutagenic and genotoxic effect increase with the duration of exposure to chemical composition in hair product.17 the increase length of time exposed to harmful substances will increase the risk for adverse health effects. chemical substances found in hair product include amines, resorcinol, hydrogen peroxide, ammonium, formaldehyde. hair dyes contain a variety of chemical agents, some which are considered proven, probable or possible human carcinogens.18 many permanent and semipermanent dyes used by hairdressers were shown to contain aromatic amine derivates such as 2-amino-4 nitrophenol, 2amino-5 nitrophenol and 1,4-diamino-2-nitrobenzene, many of which are mutagenic.3 aromatic amines used in oxidative hair dyes were identified as mutagenic or carcinogenic in rodents.14 ammonium releaser such as ammonium chloride or ammonium phosphate are used in the process of hair dye in order to improve the hair penetration so that hair strand could open up and absorb color. formaldehyde is substance used in hair straightening solutions and have been found carcinogeic by the internatioal agency for research on cancer.3 protective equipments are also used to avoid or reduce irritation due to exposure of hazardous substances in working environment.22 in this study, we found that the micronucleus frequency were increase, this phenomena could caused by the indiscipline behavior of the hairdresser. some of the subjects in this study admitted their improper usage of protective equipment. they do not adjust their face masks to fully cover up their nose and mouth during hair dying or straightening process. besides that, a few of the subjects do not change their gloves during hair dying process when the gloves were torn. some of the regular customers of the salon told that the hairdresser actually did not even put on any gloves or face mask. many of the hairdressers also told that protective equipments are inconvenient and uncomfortable to put on. personal protective equipment (ppe) protocols is one of the most crucial elements to protect worker’s health in any workplace. policy and protective protocol in the salon were not followed and carried out by most of the hairdresser. improper usage of protective equipment will increase risk of toxic exposure. the oral cavity is the first barrier of the inhalation or ingestion of carcinogens and may play a role in metabolism to a reactive substance.23 buccal mucosa is very sensitive to toxic exposure and readily to form micronucleus.24 table 1. result of independent sample t-test (p<0.05) group micronucleus frequencies t df sig. mean diff std. error ci(95%) exposed 10.06 18 0.02** 17.60 1.74 13.924-21.434 non-exposed tabel 2. summary results of one-way anova test among exposure duration subgroup (p <0.05) duration of exposure mean and standard deviation of micronucleus f sig. < 5 years 16.00±2.0 36.347 0.00** 6-10 years 21.50±2.1 >10 years 26.80±1.5 tabel 3. summary results of post hoc lsd test duration of exposure between exposed subgroup (p<0.05) 5 years 6-10 years >10 years <5 years 0.011** 0.000** 6-10 years 0.011** 0.008** >10 years 0.000** 0.008** dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 78 yee, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 74–79 inhalation and dermal absorption are considered the most frequent routes of exposure to chemicals agent among hairdressers.21 personal protective equipment (ppe) may be the most practical and effective way of minimizing genotoxic risk. examples of personal protective equipment in hair salons are gloves, aprons, protective masks and eyes protection. micronucleus is an effective biomarker of disease and process is associated with the induction of dna or genome damage. the scoring is done based on the number of cell that contain micronucleus but not the number of micronucleus in each cell. this scoring was done in this study for cell that has more than 1 micronucleus as well as the cell that contains 2 micronuclei as shown in figure 5. this study in line with the study of pawitan, in which stated that the diameters of micronuclei are almost 1/3 of the mean diameter of the nuclei and must be located within cellular cytoplasm. a cell can contain 1-3 micronuclei. there can be more than one micronucleus forming when more genetic damage has happened due to more chromosomal breakage in anaphase.25 micronucleus is genotoxicity biomarkers in human erythrocytes, lymphocytes, reticulocytes, and buccal mucosa cells.26 according to takkouche, hairdresser has a higher risk of cancer than the general population. the risk increase is substantial for lung, larynx, bladder cancer and multiple myeloma. an increased risk was observed for cancer of pancreas, aerodigestive tract, cervix, in situ of the skin and colorectal adenocarcinoma in hairdresser. 18 in this study, micronucleus also found in non exposed group. this is because a normal healthy person also contain about 1-9 micronucleus per 1000 cells of buccal mucosa epithelial cell.27 micronucleus frequency was higher in women than men because of the influence of sex hormones.1 hormones that can affect micronucleus are estrogen and progesterone. both of these hormones can affect the metabolism and the biochemical system of cells and may induce chromosomal damage.13 hence, only female were chosen as the inclusive criteria of the subject. the increase in micronucleus frequency in females can be accounted for by the greater tendency of the x chromosome to be lost as an micronucleus relative to other chromosome and females have two copies of chromosome compared to only one in male. thus subject selected were all female and age range between 18-35.28,29 other than exposure of harmful chemical substance, smoking, alcohol consumption, stress, pharmacotherapy and intake of drugs are risk factor of micronucleus.10 in this study, subjects selected were those who without smoking habit and alcohol consumption. cigarette contains several carcinogens. these materials activate in different tissues, which cause the dna adducts products. the time influence on dna adducts has controversial results.30 it has been shown that the synergic effect of cigarette smoking and alcohol consumption is more than 5.5 fold.31 none of the subjects were alcohol consumers, so the role of confounding factors especially consumption of alcohol has omitted completely. hairdresser should be aware of the increase of micronucleus frequency since forming of micronuclei in associated with the induction of dna or genome damage.26 in conclusion, chemical substances of hair products had affected the micronucleus frequency of the epithelial cells in buccal mucosa of hairdressers. acknowledgement we would like to extend my gratitude to all staffs of oral biology department, faculty of dentistry, universitas gadjah mada, for their contributions in the fruitful discussions before and during doing this research project. references 1. rickes ln, alvarengo mc, souza tm, graciaus, martino-roth mg. increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers: genet. mol res 2010; 9(3): 1921-8. 2. ferreira a.p., occupational health hazards of female hairdressers in jacarepagua, rio de janeiro, brazil. j environ occup sci 2013; 2(10): 27-32 3. durgam s, formaldehyde exposures urin brazilian blowout hair smoothing treatment at a hair saloohio. health harzard evaluation report. 2011: 0014.3147. 4. cho ja, oh e, lee e, sul d. effects of hair dyeing on dna damage in human lymphocytes. j ac health2003; 45: 376-81. 5. gan hf, meng xs, song ch. a survey of health effects in a human population exposed to permenant-waving solution containing thioglycolicacis. job occp health 2003; 45: 400-4. 6. orellana-busto ai, espinoza-sanrander il, franco-martinez me, lobos-james n, ortega-pinto av. evaluation of keratinization and agnors count in exfoloative cytology of normal oral mucosa from smokers and non-smokers. med oral 2004; 9: 197-203. 7. norppa h. cytogenetic markers of susceptibility: influence of polymorphic carcinogen-metabolizing enzymes. environ health perspect 1997; 105: 829–35. 8. sarto f, finotto s, giacomelli l, mazzotti d, tomanin r, levis ag. the micronucleus assay in exfoliated cells of the human buccal mucosa. mutagenesis 1987; 2: 11-7. 9. ia r ma rcova i g. micronuclei frequency in per ipheral blood lymphocytes of cancer patients: a meta-analysis. mutat res 2008; 659(3): 274–83. 10. holland, nina, claudia b, micheline kv, stefano b, zeiger e, knasmueller s, fenech m. the micronucleus assay in human buccal cells as a tool for biomonitoring dna damage: the humn project perspective on current status and knowledge gaps. mut res 2008; 16(7): 16-32. 11. hiroshi s, keizo s. carcinogenic risk factors. prevention and early detection for cancer. jmaj 2001; 44(6): 245-9. 12. daerah yogyakarta in figure 2013. statistics of d.i. yogyakarta province. 2013: 34563.13.12. 13. herrera la, montero rl, allan ja, wolf cr. sexual differentiation and regulation of cytochrome p-450 cyp2c7. biochim biophys acta1992; 1118(43): 99-106. 14. casartelli g, monteghirfo m, bonatti s, scala s, toma s, margarino g, abbondadolo a. staining of micronuclei in squamous epithelial cells of human oral mucosa. anal quant cytol histol 1997; 19(8): 475-81. 15. titenko-holland n, moore le, smith mt. measurement and characterization of micronuclei in exfoliated human cells by fluorenscences in situ hybridization with a centromeric probe. mutat res 1994; 312: 39-50. 79 dental journal (majalah kedokteran gigi) p-issn: 1978-3728; e-issn: 2442-9740. accredited no. 56/dikti/kep./2012. open access under cc-by-sa license. available at http://e-journal.unair.ac.id/index.php/mkg 79yee, et al./dent. j. (majalah kedokteran gigi) 2015 june; 48(2): 74–79 16. galiotte mp, kohler p, mussi g, gilka j, gattas f. assessment of occipational genotoxic risk among brazilian hairdressers. ann occup hyg 52 (7): 645-51, 2008. 17. jeffrey ka. side effect of drugs annual 30: a worldwide yearly survey of new data and trends in adverse drug reaction, elsevier, oxford, united kingdom. 2008, 18. czene k, tiikkaja s, hemminki k. cancer risks in hairdressers: assessment of carcinogenicity of hair dyes and gels. int j canc 2003; 105: 108-12. 19. yu lq, jiang sf, leng sg. early genetics effects on worker occupationally exposed to formaldehyde. zhonghua yu fang yi xueza 2005; 39(6): 392-5. 20. hamdouk m, abdelraheem m, taha a, cristina d, checherita i a, alexandru c. the association between prolonged occupational exposure to paraphenylenediamine (hair-dye) and renal impairment. arab j nephrol trasplant 2011, 4(1): 21-5. 21. wong r, chien h, luh d. correlation between chemical-safety knowledge and personal attitudes among taiwanese hairdressing students. am j ind med 2005; 47: 45-53. 22. dontas s, geeorgiadou e, koukoulaki t. occupational health and safety in the sector: hazardous substances. europen agensy for health and safety at work. european union, luxembourg, europen agency; 2014. p. 4. 23. majer bj, laky b, knasmuller s, kassie f. use of micronucleus assay with exfoliated epithelial cells as a biomarker for monitoring individuals at elevated risk of genetic damage and in chemoprevention trials. mut res 2005; 489: 147-72. 24. huang y, hou h, yi q, zhang y, chen d, jiang xia y, fenech m, shi q. the fate of micronucleated cells post x-irradiation detected by live cell imaging. mutat res 2011; 10: 629-38. 25. pawitan ja. peran histologi untuk meningkatkan kesehatan masyarakat. uji mikronukleus untuk mendeteksi adanya aberasi kromosom. med j indon 2005; 12: 213-6. 26. heddle ja, cimmino mc, hayashi m, romafna f, shelby d, tucker jd, vanpary p, macgregor jt. micronuclei as an index of cytogenetic damage: past, present, and future. environ mol mut 1991; 18: 277-91. 27. fenech m, morley aa. measurement of micronuclei in human lymphocytes. mutat res 1985; 9(148): 29-36. 28. fenech m, bonassi s. the effect of age, gender, diet and lifestyle on dna damage measured using micronucleus frequency in human peripheral blood lymphocytes. mutagenesis 2011; 26(1): 43–9. 29. norppa h, falck gc. what do human micronuclei contain?. mutagenesis 2003; 18: 221-33. 30. phillips dh. smoking-related dna and protein adducts in human tissues. carcinogenesis 2002; 23: 1979-2004. 31. stich hf, rosin mp. quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells. int j cancer 1983; 31: 305-8.